Application of ginsenoside Rg1 in preparation of lung-targeted slow-release lung injury protection medicine

Abstract

The invention discloses an application of ginsenoside Rg1 in preparation of a lung-targeted slow-release lung injury protection medicine. The drug-loaded liposome provided by the invention is obtained by encapsulating a drug with a lipid membrane material, the drug is ginsenoside Rg1, the lipid membrane material is composed of phospholipid and cholesterol, and the phospholipid is a mixture of dipalmitoyl phosphatidylcholine and distearyl phosphatidyl ethanolamine-polyethylene glycol. The ginsenoside Rg1 can promote the medicine to be slowly released and enriched in the lung for a long time through aerosol inhalation, is suitable for prevention and treatment of radiation-induced lung injury, and has important clinical value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of ginsenoside Rg1 in preparing lung-targeted sustained-release lung injury protection drugs. Background Art

[0002] Radiation-induced lung injury (RILI) is the most common serious complication of radiotherapy for patients with thoracic tumors. Radiation may cause DNA damage, massive release of cytokines and recruitment of cytokine inflammatory infiltration, leading to radiation pneumonitis (RP). The body will further produce radiation pulmonary fibrosis (RPF) in the process of repairing the damage. Among them, the incidence of radiation pneumonitis of grade 3 and above is 10% to 20%, and the mortality rate is as high as 50%. There is currently no specific drug for RILI. Clinically, symptomatic drug treatment is mainly used to relieve symptoms, and severe patients are combined with oxygen and bed rest. Amifostine is the only radioprotectant approved by the FDA for clinical use so far, but it has serious adverse reactions such as hypotension and severe nausea, and its tolerance to intravenous administration is poor, so its actual clinical application is very limited. In addition, nintedanib and pirfenidone are often used to improve RPF, and glucocorticoids such as prednisone are often used to relieve the clinical symptoms of RP patients, but they are usually accompanied by adverse reactions such as central obesity, hypertension, diabetes, osteoporosis, and immune system suppression, and are prone to relapse after treatment.

[0003] Traditional Chinese medicine has the unique advantages of multi-target action, treating both the root cause and the symptom, and high safety, and is particularly suitable for the effective prevention and treatment of chronic diseases and difficult and complicated diseases. Ginsenosides derived from ginseng and Panax notoginseng of the Araliaceae family are a class of tetracyclic triterpenoid compounds, which are known to have a variety of pharmacological activities that are beneficial to health, such as anti-inflammatory, antioxidant, and immunomodulatory.

[0004]

[0005] Through a series of studies, it was found that among the numerous ginsenoside compounds, ginsenoside Rg1 has significant anti-inflammatory and anti-fibrosis activities, and has a high natural abundance and a wide range of sources, which may be suitable for the prevention and treatment of RILI. However, ginsenoside Rg1 is easily degraded by intestinal bacteria and is quickly eliminated by metabolism, resulting in low systemic bioavailability. Even if it is possible to be used for the prevention and treatment of RILI, it is difficult to achieve drug application. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides the use of ginsenoside Rg1 in preparing lung-targeted sustained-release lung injury protection drugs, and the lung injury drug application is a radiation lung injury drug application, rather than other lung injury drug applications.

[0007] The technical solution is as follows:

[0008] Application of ginsenoside Rg1 in the preparation of lung-targeted sustained-release lung injury protection drugs, wherein the lung injury drug application is a radiation lung injury drug application;

[0009] Furthermore, ginsenoside Rg1 liposomes are obtained by encapsulating drugs in lipid membrane materials, wherein the drug is ginsenoside Rg1, and the lipid membrane materials are phospholipids and cholesterol.

[0010] In a preferred embodiment, the mass ratio of the drug to the lipid membrane material is 1:3 to 12.3;

[0011] Wherein, the phospholipids include dipalmitoylphosphatidylcholine and distearoylphosphatidylethanolamine-polyethylene glycol, and, in terms of molar fractions,

[0012] Cholesterol 20-37.5 parts,

[0013] 57.5-75 parts of dipalmitoylphosphatidylcholine,

[0014] Distearoylphosphatidylethanolamine-polyethylene glycol 5 parts.

[0015] The mass ratio of the drug to the membrane material is 1:3 to 12.3, preferably 1:4;

[0016] The molar ratio of phospholipid to cholesterol in the membrane material is 4:1 to 5:3, preferably 7:3;

[0017] The membrane material phospholipids are composed of dipalmitoylphosphatidylcholine and distearoylphosphatidylethanolamine-polyethylene glycol and are prepared according to a molar ratio of 65:5.

[0018] The particle size of the ginsenoside Rg1 lung-targeted liposome prepared by the present invention is (180-480) nm, and the Zeta potential is -(18.08-21.55) mV;

[0019] Furthermore, experiments show that the ginsenoside Rg1 of the present invention can be used to prepare lung-targeted sustained-release lung injury drugs, specifically drugs for radiation-induced lung injury, rather than other lung injuries. Ginsenoside Rg1 exists in the form of drug liposomes and has lung targeting and sustained-release properties. Moreover, the drug-loaded liposomes, i.e., ginsenoside Rg1 liposomes, achieve targeted drug delivery through the administration route of atomized inhalation, promoting the release and enrichment of drugs in the lungs. Based on the above application, the drug-loaded liposomes of the present invention achieve targeted drug delivery through the administration route of atomized inhalation.

[0020] The present invention further provides a method for preparing ginsenoside Rg1 liposomes, comprising the following steps:

[0021] (1) adding the drug and lipid membrane material to an organic solvent at a feed ratio of 6.7 mg of the drug and lipid membrane material: 1 mL of the organic solvent, and removing the organic solvent by vacuum rotary evaporation after they are completely dissolved until a uniform lipid film is formed, wherein the organic solvent is selected from any one or more of methanol, ethanol, and chloroform;

[0022] (2) adding a hydrating medium to the lipid film obtained in step (1) until it is dissolved, and then swelling and hydrating the film at 30 to 60° C. for 30 to 70 minutes, and ultrasonicating the film for 10 to 30 minutes to prepare a drug-loaded liposome solution having a ginsenoside Rg1 concentration of 0.6 to 2 mg / mL;

[0023] The hydration medium is any one or more of ultrapure water, physiological saline, and PBS buffer solution;

[0024] (3) adding a lyophilization protectant to the drug-loaded liposome solution obtained in step (2) at a feeding ratio of 50 to 200 mg of a lyophilization protectant per 1 mL of the drug-loaded liposome solution, wherein the lyophilization protectant is any one or more of trehalose, mannitol, lactose, and sucrose;

[0025] The mixture was dispersed evenly, pre-frozen overnight at -20°C, and then freeze-dried overnight to obtain the ginsenoside Rg1 liposome freeze-dried powder.

[0026] Preferably, the solvent is methanol.

[0027] Preferably, the hydration medium is ultrapure water.

[0028] Preferably, the hydration temperature in step (2) is selected from 30 to 60°C; most preferably, it is 45°C.

[0029] Preferably, the hydration time in step (2) is selected from 30 to 70 minutes; most preferably, it is 50 minutes.

[0030] Preferably, the ultrasonic time in step (2) is selected from 10 to 30 min; most preferably, it is 15 min.

[0031] Preferably, the lyoprotectant in step (3) is selected from mannitol, trehalose, lactose, and sucrose; trehalose is most preferred.

[0032] Preferably, the ratio (g / mL) of the lyophilization protective agent trehalose to the liposome suspension in step (3) is 1:(20-5), most preferably 1:10.

[0033] Preferably, the freeze-drying process in step (3) is to pre-freeze overnight at -20°C and freeze-dry for 12 hours to obtain ginsenoside Rg1 liposome freeze-dried powder.

[0034] The present invention adopts the above technical solution, which has the following technical effects compared with the prior art:

[0035] The ginsenoside Rg1 lung-targeted liposomes of the present invention are spherical liposomes of uniform size. The drug-loaded liposomes of the present invention are made into freeze-dried powder, which can promote the long-term slow release and enrichment of drugs in the lungs through atomization inhalation, and are suitable for the prevention and treatment of radiation lung injury, and have important clinical value.

[0036] Experiments show that the ginsenoside Rg1 liposomes of the present invention have constant and lasting in vitro release and have obvious sustained-release properties. The ginsenoside Rg1 lung-targeted liposomes of the present invention have high bioavailability in vivo release and have obvious sustained-release effects.

[0037] The ginsenoside Rg1 lung-targeted liposomes of the present invention have obvious lung targeting in tissue distribution.

[0038] The ginsenoside Rg1 lung-targeted liposomes of the present invention have the effect of preventing and treating radiation-induced lung injury, but not other lung injuries. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a transmission electron microscope observation photograph of the ginsenoside Rg1 liposome provided by the present invention.

[0040] Figure 2 This is the in vitro drug release curve of the ginsenoside Rg1 liposome provided by the present invention.

[0041] Figure 3 The present invention provides the tissue distribution of the ginsenoside Rg1 liposome in rats.

[0042] Figure 4 The invention discloses the pharmacodynamic effect of the ginsenoside Rg1 liposomes provided by the invention on mice with radiation-induced lung injury. DETAILED DESCRIPTION

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0044] Unless otherwise specified, the materials and instruments used in the following examples are all available from commercial sources.

[0045] The present invention is described in detail and specifically by specific examples below to provide a better understanding of the present invention, but the following examples do not limit the scope of the present invention.

[0046] Example 1. Preparation and characterization of ginsenoside Rg1 liposomes

[0047] (1) Preparation: Take 8 mg of ginsenoside Rg1, 22 mg of dipalmitoylphosphatidylcholine, 5 mg of distearoylphosphatidylethanolamine-polyethylene glycol, and 5 mg of cholesterol, mix well, add 6 mL of methanol until fully dissolved, slowly evaporate under reduced pressure in a constant temperature water bath at 45°C to form a uniform lipid film and remove all organic solvents, add 5 mL of ultrapure water for 50 min to fully swell the lipid film, continue ultrasonic hydration for 15 min to obtain ginsenoside Rg1 liposomes; add trehalose (10%, v / w) as a freeze-drying protective agent to the solution after ultrasonic hydration, pre-freeze overnight at -20°C, and continue freeze-drying for 12 h to obtain ginsenoside Rg1 lung-targeted liposome freeze-dried powder.

[0048] (2) Determination of drug loading, encapsulation efficiency and yield: The beneficial drugs not encapsulated by liposomes were separated by dialysis, and the content of ginsenoside Rg1 was determined by high performance liquid chromatography. Quantitatively transfer 2 mL of liposomes into the pretreated dialysis bag (molecular weight cutoff 14000), place the dialysis bag in the dialysis solution (deionized water), stir on a magnetic stirrer to ensure the flow of the liquid, replace the dialysis solution every 2 hours, and dialyze for 12 hours. After the dialysis, completely transfer the liposome solution to a 5 mL volumetric flask and make up to volume with methanol. Determine the drug concentration before and after dialysis, and calculate various indicators according to the following formulas: drug loading (%) = M1 / M×100%; encapsulation efficiency (%) = M2 / M1×100%; yield (%) = M1 / M0×100%,

[0049] Wherein, M0 is the dosage of the drug, M is the total mass of the liposome, M1 is the total amount of the drug in the liposome, and M2 is the mass of the drug encapsulated in the liposome.

[0050] The drug loading, encapsulation efficiency and yield of different batches of liposomes are shown in Table 1.

[0051] Table 1 Drug loading, encapsulation efficiency and yield of liposomes

[0052]

[0053] (3) Appearance and morphology observation

[0054] The morphology of liposomes was observed using a JEM-1400 Flash transmission electron microscope. 1 μL of liposome solution was dropped onto a copper mesh, excess liquid was removed with filter paper, and 2% phosphotungstic acid solution (pH 7.0) was added for negative staining. Excess negative staining liquid was removed, and the mixture was naturally air-dried at room temperature to allow the liposome particles to concentrate and deposit on the copper mesh. The liposomes were then observed and photographed under a transmission electron microscope. Figure 1 As shown, the transmission electron microscopy observation results show that the drug-loaded liposomes prepared by the present invention are spherical nanoparticles with uniform particle size.

[0055] (4) Particle size distribution and Zeta potential determination

[0056] Take 1mL of freshly prepared ginsenoside Rg1 liposome solution and use BeNano 90Zeta nanoparticle size and Zeta potential analyzer to measure the particle size distribution, polydispersity index (PDI) and Zeta potential. The measurement was repeated three times, and the results are shown in Table 2. The measurement results show that the average particle size of the prepared liposomes is ~300nm, the particle size distribution is uniform, the surface has more negative charges, so it is not easy to aggregate, and the physical stability is good.

[0057] Table 2 Particle size distribution, polydispersity coefficient and Zeta potential of liposomes (n=3)

[0058]

[0059] Example 2: Drug sustained release characteristics of ginsenoside Rg1 liposomes

[0060] (1) In vitro drug release curve

[0061] Weigh 8 mg of ginsenoside Rg1 raw material and the equivalent liposome freeze-dried powder, reconstitute with 2 mL of normal saline, place in a dialysis bag with a cutoff relative molecular mass of 3500, use 20 mL of newly prepared artificial lung fluid (pH 7.4) as the release medium, incubate at 37°C, take 0.5 mL of samples at different time points, and add isothermal and equal volume of simulated lung fluid at the same time. Add an equal volume of methanol to the incubated sample, vortex mix, sonicate, and determine the content of ginsenoside Rg1 by high performance liquid chromatography, calculate the cumulative release of the drug, and draw a release curve. Figure 2 .

[0062] The test results showed that the drug release of the liposome preparation in the artificial lung fluid incubation system was significantly lower than that of the unencapsulated ginsenoside Rg1 naked drug; the drug release reached stability after 8 hours of incubation, with a cumulative release of ~50%, indicating that ginsenoside Rg1 liposomes have a sustained drug release effect.

[0063] (2) In vivo drug metabolism kinetics

[0064] The experimental subjects of this embodiment are SPF male SD rats purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. The experimental rats were randomly divided into 3 groups (n=5), and the experiment was started after one week of adaptive feeding. Fasting for 12h before administration, but water is not forbidden. On the day of administration, 10% chloral hydrate anesthesia was used, and after anesthesia, ginsenoside Rg1 liposomes were orally gavaged with ginsenoside Rg1 solution, tail vein injection and pulmonary atomization inhalation (liquid quantitative nebulizer, Bojian Biotechnology Co., Ltd.), and the dosage was 10mg / kg in terms of ginsenoside Rg1. 500μL of blood was collected through the orbit at different times after administration, placed in an anticoagulant tube containing sodium heparin, centrifuged (4000rpm×5min), and the supernatant was taken and stored in a -80℃ refrigerator.

[0065] Take 40 μL of plasma, add 200 μL of precipitant (200 ng / mL methanol internal standard solution), vortex for 1 min, centrifuge (4 ° C, 12500 rpm) for 15 min, and take the supernatant for sampling. The plasma samples taken from rats after administration were processed using the analytical method verified by the methodology, and then entered into the AB4500 QTRAP-LC / MS / MS system for detection to determine the blood concentration of ginsenoside Rg1 at different times. The results of the drug concentration determination in plasma were processed using DAS2.0 software to calculate the pharmacokinetic parameters. The results are shown in Table 3.

[0066] The measurement results showed that in rats, the absolute bioavailability of ginsenoside Rg1 liposomes by oral and pulmonary inhalation were 1.9% and 40.9%, respectively; compared with oral administration, the bioavailability of ginsenoside Rg1 liposomes by pulmonary inhalation was significantly increased by more than 20 times; compared with intravenous injection, the mean residence time MRT of the drug in the body in the pulmonary inhalation group was significantly prolonged (p<0.01), and the apparent distribution volume Vz was significantly increased (p<0.01), indicating that ginsenoside Rg1 liposome preparation has significant sustained-release characteristics, and pulmonary inhalation significantly changes the tissue distribution characteristics of the drug in the body.

[0067] Table 3 Pharmacokinetic parameters of ginsenoside Rg1 in rats (n=5)

[0068]

[0069]

[0070] Note: Compared with the intravenous injection of Rg1-Lip group, **, p<0.01; ***, p<0.001.

[0071] Example 3: Lung targeting properties of ginsenoside Rg1 liposomes

[0072] The experimental subjects of this embodiment are SPF male SD rats purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. After one week of adaptive feeding, the rats were randomly divided into two groups. They were fasted for 12 hours before administration, but not water. Before administration, 10% chloral hydrate was used for anesthesia, and ginsenoside Rg1 liposomes were injected intravenously and ginsenoside Rg1 liposomes were inhaled by atomization, once a day, for 3 consecutive days, and the dosage of each administration was 10 mg / kg in terms of ginsenoside Rg1. Blood, heart, liver, spleen, lung, and kidney were taken at different times after the last administration. The blood sample was placed in an anticoagulant tube containing sodium heparin, centrifuged (4000rpm×5min), and the supernatant was taken to obtain plasma; the connective tissue of each organ was removed, the blood stains were washed off, the moisture on the organ was blotted off, ultrapure water was added as a homogenization medium at a ratio of 1:5 (v / w), and the mixture was placed in a high-throughput homogenizer for thorough homogenization. The homogenate was centrifuged (12500rpm×15min), the supernatant was taken, and stored in a -80℃ refrigerator for testing. 40μL of plasma or tissue homogenate was taken, 200μL of internal standard methanol solution (200ng / mL) was added, vortexed for 1min, centrifuged (4℃, 12500rpm×15min), and the supernatant was taken for LC / MS / MS analysis (AB4500 QTRAP) to determine the drug concentration of ginsenoside Rg1 in the sample. The results are shown in Figure 3 .

[0073] The experiment showed that the drug distribution and its changing trend of ginsenoside Rg1 liposomes in plasma, heart, liver, spleen and kidney were similar in two different administration methods, and the drug concentration of the intravenous injection group was significantly higher than that of the nebulized inhalation group, but the drug concentration of the nebulized inhalation group in the lung was significantly higher than that of the intravenous injection group, showing obvious lung tissue targeted distribution characteristics. The results show that the ginsenoside Rg1 liposomes provided by the present invention have lung targeting when administered by nebulized inhalation, which is beneficial to improve the therapeutic effect of lung diseases.

[0074] Example 4: Pharmacological effects of ginsenoside Rg1 liposomes on radiation-induced lung injury

[0075] The experimental subjects of this embodiment are C57BL / 6 female mice, 8 weeks old, purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. After one week of adaptive feeding, the mice were randomly divided into 5 groups (n=6). Among them, the low-dose group and high-dose group of ginsenoside Rg1 were given Rg1-Lip solution by pulmonary atomization inhalation, with 15 mg / kg and 45 mg / kg of ginsenoside Rg1 respectively, once a day for 3 consecutive days; the positive control group was given 50 mg / kg of amifostine injection by intraperitoneal injection at the same time, and the model group was given an equal volume of normal saline by intraperitoneal injection at the same time, and the normal group was not treated. After the end of the drug treatment, all animals underwent a single thoracic (bilateral lung) irradiation with X-rays, with a radiation dose of 15 Gy, a dose rate of 2.792 Gy / min, a source-skin distance of 50 cm, and an energy of 259 KV. After two weeks of irradiation treatment, the eyeballs were removed to collect blood and the animals were killed. Lung tissues were taken, and H&E and Masson staining analyses of histopathological sections were performed. The results are shown in the table below. Figure 4 .

[0076] The results of H&E staining pathological sections showed that the blank group had clear alveolar structure, thin and smooth alveolar wall, intact capillaries, and no exudate in the alveolar cavity; the model group had collapsed and atelectasis, damaged capillary wall, thickened alveolar septum, cellulose-like exudate in some alveolar cavities, and inflammatory cell infiltration around the bronchioles. The results of Masson staining showed that the blank group had intact alveolar structure, no blue collagen deposition, and no obvious proliferation of fibrous tissue; the model group had a large number of blue-stained collagen fibers around the bronchioles, which were deposited in bands, and the alveolar cavity was significantly reduced. The results of pathological analysis showed that radiation irradiation caused severe lung tissue damage in experimental animals, and drug treatment could effectively improve lung tissue pathological damage. Rg1-Lip could improve the inflammatory response and fibrosis in the lung tissue of model animals in a dose-dependent manner through pulmonary aerosol inhalation treatment, and the effect of high dose was significantly better than that of positive control drugs.

[0077] In summary, the experimental data show that the average particle size of the liposomes prepared by the present invention is ~300nm, the particle size distribution is uniform, the surface carries more negative charges, so it is not easy to aggregate, and the physical stability is good; further experiments show that the ginsenoside Rg1 liposomes of the present invention have obvious sustained-release effects; moreover, the present invention adopts a thin film dispersion-ultrasound method to prepare ginsenoside Rg1 liposomes, and the administration via the pulmonary atomization inhalation route has clear lung targeting, thereby obtaining the relative enrichment of the drug at the target site of the pharmacodynamic effect, and can achieve lung targeting by administration via the atomization inhalation route. In summary, the present invention prepares ginsenoside Rg1 liposomes, and exerts the preventive and therapeutic effect of radiation lung injury through pulmonary atomization inhalation. The liposome-encapsulated drug is directly delivered to the lungs via the inhalation route, achieving the enrichment and absorption of the drug at the treatment target site, while reducing the systemic exposure of the drug and reducing systemic toxicity. Therefore, the ginsenoside Rg1 liposomes of the present invention can be used to prepare lung-targeted sustained-release lung injury protection drugs, which are particularly suitable for radiation lung injury, rather than other lung injury drug applications.

[0078] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modification and substitution of the present invention is also within the scope of the present invention, therefore, the equalization and modification made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. Application of ginsenoside Rg1 in the preparation of lung-targeted sustained-release lung injury protection drugs, It is characterized in that The lung injury is radiation lung injury; Ginsenoside Rg1 liposomes are obtained by encapsulating drugs in lipid membrane materials, wherein the drug is ginsenoside Rg1, and the lipid membrane materials are phospholipids and cholesterol.

2. The use according to claim 1, It is characterized in that The mass ratio of the drug to the lipid membrane material of the ginsenoside Rg1 liposome is 1:3-12.3; The phospholipids in the lipid membrane material are dipalmitoylphosphatidylcholine and distearoylphosphatidylethanolamine-polyethylene glycol. Furthermore, in the lipid membrane material, according to the molar fraction, 20-37.5 parts of cholesterol, 57.5-75 parts of dipalmitoylphosphatidylcholine, and 5 parts of distearoylphosphatidylethanolamine-polyethylene glycol.

3. The use according to claim 1 or 2, Features The drug-loaded liposomes are administered by aerosol inhalation.

4. The method for preparing the ginsenoside Rg1 liposome according to claim 1, Features The following steps are involved: (1) Add the drug and lipid membrane material to the organic solvent at a feed ratio of 6.7 mg of drug and lipid membrane material: 1 mL of organic solvent, and remove the organic solvent by vacuum rotary evaporation after they are completely dissolved until a uniform lipid film is formed, wherein the organic solvent is selected from any one or more of methanol, ethanol, and chloroform; (2) adding a hydrating medium to the lipid film obtained in step (1) until dissolved, then swelling and hydrating at 30-60° C. for 30-70 min, and ultrasonicating for 10-30 min to prepare a drug-loaded liposome solution with a ginsenoside Rg1 concentration of 0.6-2 mg / mL; The hydration medium is any one or more of ultrapure water, physiological saline, and PBS buffer solution; (3) adding a lyoprotectant at a ratio of 50 to 200 mg per 1 mL of the drug-loaded liposome solution to the drug-loaded liposome solution obtained in step (2), wherein the lyoprotectant is any one or more of trehalose, mannitol, lactose, and sucrose; The mixture was dispersed evenly, pre-frozen overnight at -20°C, and then freeze-dried overnight to obtain the ginsenoside Rg1 liposome freeze-dried powder.

5. The preparation method according to claim 4, It is characterized in that The solvent is methanol.

6. The preparation method according to claim 4, It is characterized in that The hydration medium is ultrapure water.

7. The preparation method according to claim 4, It is characterized in that The freeze-drying protective agent is trehalose.

Images