CN109453123B - A kind of compretin derivatives freeze-dried powder injection and preparation method thereof - Google Patents
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Abstract
The invention discloses a combretastatin derivative freeze-dried powder injection and a preparation method thereof. The freeze-dried powder injection is composed of a combretastatin derivative phospholipid compound, a stabilizer and a freeze-drying protective agent, and the preparation method comprises the following steps: preparing a combretastatin derivative phospholipid complex; homogenizing under high pressure; and (5) freeze drying. The lyophilized powder for injection is dissolved in 5% glucose or 0.9% normal saline injection for use, and can be used for intravenous injection, instillation or direct oral administration for treating non-small cell lung cancer, hepatocarcinoma, and colon cancer. The freeze-dried powder injection prepared by the invention has high medicine-to-lipid ratio and small toxic and side effects, can improve the hydrophilicity and lipophilicity of the combretastatin derivatives, has certain slow release effect and passive targeting, can improve the oral bioavailability, reduce the intravenous administration dosage, has the advantages of toxicity attenuation and synergy and good re-solubility, and the preparation method has simple process and is easy for industrial production.
Description
Technical Field
The invention relates to the technical field of pharmaceutical preparations and preparation thereof, in particular to a combretastatin derivative freeze-dried powder injection and a preparation method thereof.
Background
Combretastatin (Combretastatin) is a cis-stilbene natural product separated from the bark of African bush-willow, wherein Combretastatin A4 (hereinafter abbreviated as CA4) is a compound with the strongest cytotoxicity and the simplest structure in all structures, and has the basic structure of a 3,4, 5-trimethoxy-substituted benzene ring and a 3-hydroxy-4-methoxy-substituted benzene ring which are connected by cis-carbon double bond connecting arms. The antitumor mechanism of CA4 mainly is to inhibit tubulin polymerization, induce apoptosis and act on antitumor blood vessel, the action target is similar to colchicine, and the activity is obviously superior to colchicine.
However, CA4 has poor water solubility and lipid solubility, and its cis-stilbene structure is more active but unstable, so that currently, 3 structural modifications are mainly performed: modifying an A ring structure; modifying the structure of the B ring; and modifying carbon-carbon double bond bridging bond structure. The CA4 derivative with stronger activity is obtained by structural modification, has low onset dosage and less toxic and side effects when in use, and has huge clinical application prospect. The CA4 derivative has a mother nucleus structure as follows:
wherein: a is N, O or S; r3 is preferably selected from a group having a structure capable of forming van der Waals' force, hydrogen bond, or the like with a phospholipid, such as a phenyl group, a hydroxyl group, or an amino group. According to the patent CN201110422678, the preferable active structures are 4- (3, 5-dimethoxy) -5- (4-methoxyphenyl) oxazole with a structure 1, 2-phenyl-4- (3,4, 5-trimethoxyphenyl) -5- (4-pyridyl) thiazole with a structure 2, and 3:4- (3, 5-dimethoxyphenyl) -5- (3-hydroxy-4-methoxyphenyl) imidazole with a structure 3.
However, the water solubility of CA4 derivatives is still very poor, which limits their clinical applications, and therefore designing and preparing water-soluble prodrug derivatives thereof is the current focus of research. The studies on the CA4 derivatives reported in the literature at home and abroad up to now are mostly limited to the design and synthesis of phosphate prodrugs thereof. The phosphate prodrug CA4 phosphate (combretastatin A4 phosphate, CA4P) was designed and synthesized by Oxigene, USA, and has been in clinical trial. Although CA4P improves the water solubility of CA4, it cannot be stably stored in aqueous solution, so that it needs to be prepared into freeze-dried powder for storage, and the freeze-dried powder for storage has the following defects: firstly, the CA4P freeze-dried powder is easy to degrade after redissolving and has poor stability; ② the phosphate has poor fat solubility, is not easy to enter cells to exert the drug effect and has high effective dose; the toxic and side effects in vivo are large, the degradation and quick release can be realized after intravenous administration, the elimination is quick, and the sustained-release effect is not realized; fourthly, the medicine is limited to be administrated in the vein, and has no convenience and compliance of oral administration.
In addition, although the method for directly preparing the medicament into novel preparations such as liposome, solid dispersed nanoparticles, polymeric micelles and the like can improve the medicament, the method has the problems of low medicament loading rate, unstable encapsulation rate and the like, the corresponding freeze-dried powder injection has similar problems, and the preparation process, the using amount of auxiliary materials, the safety of the auxiliary materials, the storage and the like are insufficient.
In the prior patent, the invention related to the combretastatin derivative phospholipid complex is not found.
Disclosure of Invention
The invention aims to solve the defects of the combretastatin derivative freeze-dried powder injection and provides a combretastatin derivative phospholipid complex.
The invention also aims to provide a combretastatin derivative phospholipid complex freeze-dried powder injection and a preparation method thereof.
The purpose of the invention can be realized by the following technical scheme:
a combretastatin derivative phospholipid complex is composed of a combretastatin derivative and phospholipid, wherein the molar ratio of the combretastatin derivative to the phospholipid is 2: 1-1: 10, and preferably 1: 3.
Wherein the combretastatin derivatives are imidazole, oxazole and thiazole combretastatin A4 derivatives of which the A ring contains 4-substituted 3, 5-dimethoxyphenyl, the compound preferably has a structure capable of forming van der Waals' force, hydrogen bond, or the like with a phospholipid, such as a phenyl group, a hydroxyl group, or an amino group, and preferably has 4- (3, 5-dimethoxy) -5- (4-methoxyphenyl) oxazole, 2-phenyl-4- (3,4, 5-trimethoxyphenyl) -5- (4-pyridyl) thiazole, 4- (3, 5-dimethoxyphenyl) -5- (3-hydroxy-4-methoxyphenyl) imidazole, and more preferably has 4- (3, 5-dimethoxyphenyl) -5- (3-hydroxy-4-methoxyphenyl) imidazole.
Wherein the phospholipid is selected from phospholipids of natural origin and phospholipids of synthetic origin; the natural phospholipid is preferably soybean lecithin, yolk lecithin, cephalin, sphingomyelin, and serine phospholipid; the synthetic-derived phospholipid is preferably a hydrogenated phospholipid, PMPC, DPPC, DMPC, DPPE, DSPE, DPPAS, and structurally modified analogs thereof.
The combretastatin derivative phospholipid complex disclosed by the invention is applied to preparation of a combretastatin derivative phospholipid complex freeze-dried powder injection.
A combretastatin derivative phospholipid compound freeze-dried powder injection is composed of the combretastatin derivative phospholipid compound, a stabilizer and a freeze-drying protective agent.
Wherein the stabilizer is selected from poloxamer 188, polysorbate 80, polysorbate 20 or phospholipids and any combination thereof.
Wherein the lyoprotectant is selected from mannitol, lactose, glucose, trehalose, sorbitol or sucrose and any combination thereof.
A preparation method of the combretastatin derivative freeze-dried powder injection comprises the following steps:
(1) dissolving the combretastatin derivatives and phospholipid in an organic solvent (a), controlling the reaction concentration to be 0.5-20 mg/mL, and standing and compounding at the temperature of 0-100 ℃ for more than 0.5 h;
(2) removing the organic solvent (a) used for compounding by adopting a reduced pressure evaporation and vacuum drying method to obtain a combretastatin derivative phospholipid compound solid;
(3) dispersing combretastatin derivative phospholipid complex into water for injection dissolved with stabilizer and freeze-drying protective agent, homogenizing under high pressure, passing through membrane, and freeze-drying; wherein, the freeze-drying solution comprises, by weight, 0.1% -2.0% of combretastatin phospholipid complex, 0-10% of stabilizer and 2-10% of freeze-drying protective agent.
The reaction solvent (a) is preferably selected from one or more of aromatic hydrocarbon, halogen derivative, cyclic ether, dichloromethane, trichloromethane, acetone and ethanol, and is preferably ethanol.
Wherein the reaction concentration is preferably 3mg/mL, the reaction temperature is preferably 25 ℃, and the complexing time is preferably 12 h.
The combretastatin derivative phospholipid compound freeze-dried powder injection prepared by the method can be re-dissolved in 5% glucose or 0.9% normal saline injection, the particle size after re-dissolution is preferably 180.7 +/-23.9 nm, and the PDI is 0.108 +/-0.021.
The combretastatin derivative phospholipid compound freeze-dried powder injection is dissolved in 5 percent glucose or 0.9 percent normal saline injection for use, and can be used for intravenous injection, drip injection or direct oral administration to treat non-small cell lung cancer, liver cancer and colon cancer.
The combretastatin derivative phospholipid compound freeze-dried powder injection provided by the invention has the following advantages:
1. the preparation process is simple, the toxicity of the introduced organic solvent is low, and the method is suitable for industrial production;
2. compared with liposome, the same mole number can load more CA4 derivatives, the drug loading is high, and the phospholipid dosage is reduced;
3. association of phospholipid molecules with specific CA4 derivative molecules reduces the leakage rate of the drug;
4. the phospholipid structure has amphipathy, compared with the CA4 derivative, the hydrophilicity and lipophilicity of the CA4 derivative phospholipid complex can be obviously improved, and the lipid membrane can improve the uptake of the drug by cells;
5. the CA4 derivative phospholipid complex is orally administered, and compared with the CA4 derivative phosphate, the oral bioavailability is improved;
6. compared with CA4 derivative phosphate, the CA4 derivative phospholipid compound is good in stability in the preparation and storage processes, has a certain slow release effect and pH response, and the nanoparticles have a certain passive targeting effect, so that the administration dosage can be reduced, the drug effect can be improved, and the defect of high administration dosage of the CA4 derivative phosphate is overcome;
7. the CA4 derivative phospholipid complex can obviously reduce the irritation of oral administration of CA4 derivative and intravenous injection of CA4 derivative phosphate.
Drawings
FIG. 1: and (3) measuring in vitro release of the combretastatin derivative phospholipid complex freeze-dried powder injection.
FIG. 2: and (3) measuring the cellular uptake of the combretastatin derivative phospholipid complex freeze-dried powder injection.
FIG. 3: and (3) determining cytotoxicity of the combretastatin derivative phospholipid complex freeze-dried powder injection.
FIG. 4: and (3) determining the oral bioavailability of the combretastatin derivative phospholipid complex.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are illustrative of the invention and are not intended to limit the scope of the invention.
Example 1
Precisely weighing the combretastatin derivative (4- (3, 5-dimethoxy) -5- (4-methoxyphenyl) oxazole) and soybean lecithin in a molar mass ratio of 2:1 into a 50mL round-bottom flask, adding 5mL of acetone, controlling the concentration of the medicine to be 20mg/mL, stirring until the medicine is completely dissolved, standing and compounding for 1h at the temperature of 30 ℃, vacuumizing and drying to obtain the combretastatin derivative phospholipid compound, wherein the determined compounding rate is 50%.
Example 2
Precisely weighing combretastatin derivatives (2-phenyl-4- (3,4, 5-trimethoxyphenyl) -5- (4-pyridyl) thiazole) and soybean lecithin in a molar mass ratio of 2:1 into a 50mL round-bottom flask, adding 200mL of acetone, controlling the drug concentration to be 0.5mg/mL, stirring until the drugs are completely dissolved, standing and compounding for 2 hours at 35 ℃, vacuumizing and drying to obtain the combretastatin derivative phospholipid compound, and determining the compounding rate to be 72 percent
Example 3
Precisely weighing the combretastatin derivative (4- (3, 5-dimethoxyphenyl) -5- (3-hydroxy-4-methoxyphenyl) imidazole) and the egg yolk lecithin in a molar mass ratio of 1:1 into a 50mL round-bottom flask, controlling the drug concentration to be 3mg/mL, adding 30mL of benzene, stirring until the benzene is completely dissolved, standing and compounding for 2h at 40 ℃, vacuumizing and drying to obtain the combretastatin derivative phospholipid compound, wherein the determined compounding rate is 62%.
Example 4
Precisely weighing 1:2 combretastatin derivatives (4- (3, 5-dimethoxyphenyl) -5- (3-hydroxy-4-methoxyphenyl) imidazole) and egg yolk lecithin in a molar mass ratio of 1:2 into a 50mL round-bottom flask, controlling the drug concentration to be 2mg/mL, adding 40mL of ethanol, stirring until the drugs are completely dissolved, standing and compounding for 2h at 50 ℃, vacuumizing and drying to obtain the combretastatin derivative phospholipid compound, wherein the determined compounding rate is 62%.
Example 5
Precisely weighing 1:4 combretastatin derivatives (4- (3, 5-dimethoxyphenyl) -5- (3-hydroxy-4-methoxyphenyl) imidazole) and cephalin in a molar mass ratio into a 50mL round-bottom flask, adding 35mL of chloroform, controlling the drug concentration to be 2.5mg/mL, stirring until the drugs are completely dissolved, standing and compounding at 20 ℃ for 12h, vacuumizing and drying to obtain the combretastatin derivative phospholipid complex, wherein the determined compounding rate is 53%.
Example 6
Precisely weighing 1:3 combretastatin derivatives (4- (3, 5-dimethoxyphenyl) -5- (3-hydroxy-4-methoxyphenyl) imidazole) and egg yolk lecithin in a molar mass ratio into a 50mL round-bottom flask, adding 30mL ethanol, controlling the drug concentration to be 3mg/mL, stirring to completely dissolve the drugs, standing and compounding at 25 ℃ for 12h, vacuumizing and drying to obtain the combretastatin derivative phospholipid compound, wherein the determined compounding rate is 97%.
Example 7
100mg of combretastatin derivative phospholipid complex (example 1), 188500 mg (5%) of Poloxamer and 200mg (2%) of glucose are weighed, dissolved and dispersed in 10mL of water for injection, and filled in a 10mL penicillin bottle after high-pressure homogenization, and freeze-dried powder is subjected to freeze drying and appearance collapse, and the particle size after redissolution is determined to be 242.1 +/-36.9 nm.
Example 8
Weighing 100mg of combretastatin derivative phospholipid complex (example 2), 2mg of Tween-80200 mg (2%) and 500mg of lactose (5%), dissolving and dispersing in 10mL of injection water, homogenizing under high pressure, filling in a 10mL penicillin bottle, freeze-drying to obtain fluffy lyophilized powder, and determining the particle size of the reconstituted powder to be 221.6 +/-45.6 nm.
Example 9
100mg of combretastatin derivative phospholipid complex (example 6), 188300 mg (3%) of Poloxamer and 500mg (5%) of mannitol are weighed, dissolved and dispersed in 10mL of injection water, and filled in a 10mL penicillin bottle after high-pressure homogenization, freeze-dried and freeze-dried powder is molded in appearance, and the particle size is 180.7 +/-23.9 nm after redissolution is determined.
Example 10
Determination of solubility and apparent oil-water partition coefficient
Measuring by adopting a shake flask method (GB/T21853-. 10mL of supernatant is taken, placed in ground triangular flasks respectively, 10mL of n-octanol is added respectively, shaken at 25 ℃ until the solution is balanced, and centrifuged at 2000 Xg for 20 min. And respectively transferring 2mL of the water phase and 2mL of the n-octyl alcohol phase into a 10mL measuring flask, performing constant volume by using methanol, and determining the solubility of the combretastatin derivatives and the combretastatin derivative phospholipid complex in an n-octanol-water system. See table 1. The result shows that the solubility of the combretastatin phospholipid complex in water is improved by about 8 times, and the LogP is improved by about 1.2 times.
TABLE 1 solubility of combretastatin derivatives and their phospholipid complexes in n-octanol water system
Example 11
In vitro release assay.
1mL of the combretastatin derivative phospholipid complex lyophilized powder injection (example 6) re-dissolved substances are respectively placed in a dialysis bag to meet the leakage groove condition, two ends of the dialysis bag are sealed by sealing clamps, and the dialysis bag is respectively placed in 200mL of release media, wherein the release media are PBS buffer solutions containing 1% Tween80 and having pH values of 7.4, 6.6 and 5.5, and the release conditions of the injection are examined under the stirring of 100rmp at 37 ℃. And (3) taking 2mL of release medium in the C118P group respectively for 5min, 10min, 15min, 0.5h, 1h, 2h, 4h, 6h, 8h and 12h and respectively for 0.5h, 1h, 2h, 4h, 6h, 8h and 12h, simultaneously supplementing an equal volume of fresh release medium at 37 ℃, enabling a sample to pass through a 0.22 mu m filter membrane, carrying out LC-MS sample injection, recording peak areas, calculating the cumulative release degree at each time point, and drawing a release curve. See fig. 1. The result shows that the in vitro release model of the combretastatin derivative phospholipid composite freeze-dried powder preparation accords with first-order kinetics of drug release under the conditions of pH7.4 and pH6.6, accords with zero-order kinetics under the condition of pH5.5, has obvious slow release effect and pH response phenomenon of drug release, and can predict that the drug release of the preparation in vivo has the slow release and tumor tissue enrichment capacity.
Example 12
Cell pharmacodynamic assay
Cellular uptake: setting blank, combretastatin derivatives, combretastatin derivative phosphate and combretastatin derivativesLyophilized powder for injection (prepared in example 9) of phospholipid complex were divided into four groups. Logarithmic production phase of A549 cells at 5X 106And inoculating each well into a 6-well plate, completely culturing in a cell culture box at 37 ℃ for 24 hours to allow the cells to adhere to the wall, discarding the original culture medium, incubating for 15min by using a serum-free culture solution, adding 1500 mu L (containing 0.0375 mu L of DMSO) of serum-free culture medium into each well, diluting the culture medium to 25nM of effective drug concentration, adding 1500 mu L (containing 0.0375 mu L of DMSO) of serum-free culture medium into a control group, incubating at 37 ℃, and inspecting the relationship between the uptake rate and the uptake time. Incubate for various periods of time (1h, 2h, 3h), observe cell morphology and density, quickly discard media, add 4 ℃ PBS to stop cell uptake, and wash cells 3 times. Adding 200 mu L of cell lysate into ice bath, shaking for 30min to break cells, centrifuging for 15min at 12000 Xg, sucking 20 mu L of supernatant, quantifying BCA protein, taking 50 mu L of supernatant, adding 200 mu L of methanol, vortexing for 3min, centrifuging for 5min at 12000 Xg, taking 100uL of supernatant, and determining the drug content by LC-MS (all groups n is 3). . See fig. 2. The results show that the cell uptake of the combretastatin derivative phospholipid composite freeze-dried powder injection group is higher than that of the bulk drug and phosphate group, and the significance is achieved, so that the combretastatin derivative phospholipid composite freeze-dried powder injection is more easily taken up and utilized by cells due to the lipophilic property.
Cytotoxicity: taking A549 cells in logarithmic production phase at 1 × 105One well was inoculated into a 96-well plate, incubated at 37 ℃ for 24h in complete medium, the medium was removed, 100. mu.L of the corresponding group (drug concentration 2.5, 5, 10, 25, 50, 100nM) diluted in serum-free medium was added to each well, and after incubation at 37 ℃ for 24h, 20. mu.L of 5mg/mL MTT solution was added to each well. After incubation at 37 ℃ for 4h, the medium was discarded, 100. mu.L of DMSO was added, the mixture was shaken for 10min to dissolve formazan crystals, and the absorbance at 570nm was measured using a microplate reader to calculate the cell survival rate. The combretastatin derivative phospholipid complex has strong cytotoxicity. See fig. 3. The result shows that the combretastatin derivative phospholipid complex freeze-dried powder injection IC50The concentration of the compound is lower than that of the bulk drug and phosphate group in 24h and 48h, and the compound has significance, which indicates that the cytotoxicity of the combretastatin derivative phospholipid compound freeze-dried powder injection is stronger than that of the combretastatin derivative phosphoric acidThe salt freeze-dried powder injection can improve the curative effect of the combretastatin derivatives and reduce the clinical administration dose of the combretastatin derivatives.
Example 13
Oral bioavailability assay
18 rats were selected and randomly divided into a combretastatin derivative group, a general mixture group, and a combretastatin-based derivative phospholipid complex group (prepared in example 6). After fasting for 12h, the stomach is perfused with 50mg/kg, 0.25, 0.5, 1, 1.5, 2, 3,4, 6, 8, 12, 24 and 36h of whole blood is collected from fundus venous plexus after administration, the whole blood is 0.3mL and placed in a heparin sodium test tube, centrifugation is carried out at 10000rpm for 10min, and upper plasma is taken to detect the concentration of the combretastatin derivatives. See fig. 4. The results show that compared with the raw material medicines, the combretastatin derivative phospholipid compound group has the advantages that the oral bioavailability is improved by about 7 times, the half life is prolonged by about 5 times, and the defect that the combretastatin derivative phosphate medicines are only limited to intravenous administration is overcome by oral administration.
Claims (9)
1. A combretastatin derivative phospholipid complex is characterized by consisting of a combretastatin derivative and phospholipid, wherein the molar ratio of the combretastatin derivative to the phospholipid is 2: 1-1: 10; the combretastatin derivative is selected from 4- (3, 5-dimethoxyphenyl) -5- (4-methoxyphenyl) oxazole, 2-phenyl-4- (3,4, 5-trimethoxyphenyl) -5- (4-pyridyl) thiazole or 4- (3, 5-dimethoxyphenyl) -5- (3-hydroxy-4-methoxyphenyl) imidazole.
2. The combretastatin derivative phospholipid complex of claim 1, wherein the combretastatin derivative is 4- (3, 5-dimethoxyphenyl) -5- (3-hydroxy-4-methoxyphenyl) imidazole.
3. The combretastatin-based derivative phospholipid complex of claim 1, wherein the phospholipid is selected from the group consisting of soybean lecithin, egg yolk lecithin, cephalin, sphingomyelin, and serine phospholipids.
4. Use of the combretastatin derivative phospholipid complex of any one of claims 1-3 in the preparation of a combretastatin derivative phospholipid complex lyophilized powder for injection.
5. A combretastatin derivative freeze-dried powder injection, which is characterized by comprising the combretastatin derivative phospholipid complex, a stabilizer and a freeze-drying protective agent according to any one of claims 1 to 3.
6. The combretastatin derivative freeze-dried powder injection of claim 5, wherein the stabilizer is one or more selected from poloxamer 188, polysorbate 80 and polysorbate 20.
7. The combretastatin derivative lyophilized powder for injection of claim 5, wherein the lyoprotectant is one or more selected from mannitol, lactose, glucose, trehalose, sorbitol and sucrose.
8. A preparation method of the combretastatin derivative freeze-dried powder injection of claim 5, which is characterized by comprising the following steps:
(1) dissolving the combretastatin derivatives and phospholipid in an organic solvent (a), controlling the reaction concentration to be 0.5-20 mg/mL, and standing and compounding at the temperature of 0-100 ℃ for more than 0.5 h;
(2) removing the organic solvent (a) used for compounding by adopting a reduced pressure evaporation and vacuum drying method to obtain a combretastatin derivative phospholipid compound solid;
(3) dispersing the combretastatin derivative phospholipid compound into water for injection in which a stabilizing agent and a freeze-drying protective agent are dissolved, homogenizing under high pressure, passing through a membrane, and freeze-drying to obtain the combretastatin derivative freeze-dried powder injection, wherein the freeze-dried solution comprises, by weight, 0.1-2.0% of the combretastatin phospholipid compound, 0-10% of the stabilizing agent, 2-10% of the freeze-drying protective agent, and the balance of the water for injection.
9. The method according to claim 8, wherein the organic solvent (a) is one or more selected from aromatic hydrocarbons, cyclic ethers, methylene chloride, chloroform, acetone, and ethanol.
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| JP5264478B2 (en) * | 2005-04-29 | 2013-08-14 | ユニバーシティー オブ ルイヴィル リサーチ ファウンデーション，インコーポレーテッド | Cell surface decoration with active substances |
| CN102190625B (en) * | 2010-03-18 | 2013-08-28 | 南京圣和药业有限公司 | Stilbene tumor targeted medicine Combretastatin A4 analogs |
| US20140010886A1 (en) * | 2011-04-07 | 2014-01-09 | Georgia Tech Research Corporation | Compositions comprising saccharide binding moieties and methods for targeted therapy |
| CN102863388B (en) * | 2011-07-05 | 2015-04-29 | 南京圣和药业股份有限公司 | Tumor targeted drug Combretastatin A4 derivatives |
| CN106580945B (en) * | 2015-10-14 | 2019-11-08 | 上海天氏利医药科技有限公司 | A kind of combretastatin A4 derivative and its preparation |
| CN105288648B (en) * | 2015-10-14 | 2018-11-06 | 东南大学 | A kind of phosphatide cpd of hydrophilic medicament, its pharmaceutical composition and application |
| CN105476966A (en) * | 2016-01-29 | 2016-04-13 | 中国药科大学 | Silibinin and phospholipid complex nano freeze-dried powder and preparation method thereof |
-
2018
- 2018-11-19 CN CN201811372794.9A patent/CN109453123B/en not_active Expired - Fee Related
- 2018-11-29 WO PCT/CN2018/118108 patent/WO2020103175A1/en active Application Filing
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| Publication number | Publication date |
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| CN109453123A (en) | 2019-03-12 |
| WO2020103175A1 (en) | 2020-05-28 |
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