CN113925832B - Allopregnanolone liposome and preparation method thereof - Google Patents
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Abstract
The invention discloses a preparation of allopregnanolone and a preparation method thereof. The preparation method is simple and easy to implement, the phospholipid has good biocompatibility, can be biologically degraded, has wide sources and low cost, and the liposome has no immunosuppression effect on human bodies and has low toxicity. The allopregnanolone is wrapped between phospholipid double layers of the liposome, so that the water solubility of the allopregnanolone is improved. The allopregnanolone of the invention has good physical stability and can be placed for a long time. The allopregnanolone preparation has simple and mature preparation method, and is convenient for industrial production. The preparation has slow release, achieves therapeutic effect and can improve the safety of administration.
Description
Technical Field
The invention belongs to the field of medicaments for treating postpartum depression, and particularly relates to allopregnanolone.
Background
Postpartum Depression (PPD), a significant and readily identifiable major depressive disorder, usually begins to occur in late gestation or 4 weeks after delivery, is the leading cause of postpartum suicide in women, is also a common complication of delivery in women, and has significant clinical manifestations including significant cognitive, affective and behavioral disturbances, severely affecting the developmental health of neonates. PPD is highly recurrent, with more than about half of the first onset showing a second onset within the next 5 years, with 1/3 of the patients having a second onset even within the 1 st year. With the full release of the 'two-child policy' in China, the incidence population of postpartum depression increases year by year, and the health of women and children in China is greatly endangered.
The traditional medicine for treating depression has poor overall treatment effect on PPD, long onset time and low cure rate.
Allopregnanolone (3 a,5 a-THP) is a novel allosteric regulator of GABAa receptor, can regulate the function of GABAa receptor positioned in nerve synapse and outside synapse, has the characteristics of quick effect, good tolerance and the like based on a brand new action mechanism, is expected to become a novel method for treating postpartum depression, and is a cholesterol derivative with poor water solubility. Therefore, increasing the solubility of the compound in water, increasing its bioavailability, would be likely to bring promise to post-partum depression patients.
Previous studies have shown that patients receiving allopregnanolone cyclodextrin inclusion complex treatment are at serious risk of overstrain or sudden loss of consciousness during the course of treatment, and thus new formulations are urgently needed to improve the safety of clinical administration.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a liposome of allopregnanolone and a preparation method thereof. The preparation method is simple and easy to implement, the phospholipid has good biocompatibility, can be biologically degraded, has wide sources and low cost, and the liposome has no immunosuppression effect on human bodies and has low toxicity. The allopregnanolone is wrapped between phospholipid double layers of the liposome, so that the water solubility of the allopregnanolone is improved. The allopregnanolone of the invention has good physical stability and can be placed for a long time. The allopregnanolone liposome has the advantages of simple and mature preparation method and convenient industrial production.
According to one aspect of the invention, a liposome of allopregnanolone is provided, which consists of the following raw materials in parts by weight, 1 part of allopregnanolone, 5-50 parts of phospholipid, 3-15 parts of cholesterol and 2-10 parts of emulsifier.
According to one aspect of the invention, a liposome of allopregnanolone is provided, which consists of the following raw materials in parts by weight, 1 part of allopregnanolone, 15-30 parts of phospholipid, 3-10 parts of cholesterol and 3-8 parts of emulsifier.
According to one aspect of the invention, there is provided a liposome of allopregnanolone, wherein allopregnanolone in the liposome is a monomer raw material, and the content of allopregnanolone raw material is more than 80%; the monomer raw material is synthetic monomer raw material or natural monomer raw material.
According to one aspect of the invention, a liposome of allopregnanolone is provided, wherein the content of allopregnanolone raw material in the liposome is 98%.
According to an aspect of the present invention, there is provided a liposome of allopregnanolone, wherein the phospholipid in the liposome is selected from one or more of natural extracted phospholipid, semisynthetic or fully synthetic phospholipid.
According to an aspect of the present invention, there is provided a liposome of allopregnanolone, wherein the phospholipid in the liposome is selected from one or more of soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, hydrogenated egg yolk lecithin or dimyristoyl lecithin amide.
According to an aspect of the present invention, there is provided a first-step dissolution preparation method of a liposome of allopregnanolone, the preparation method comprising the steps of:
(1) Dissolving allopregnanolone and the emulsifier by using water for injection to obtain a water phase;
(2) Weighing phospholipid and cholesterol according to the proportion of claim 1, and dissolving the phospholipid and cholesterol in an organic solvent to obtain an organic phase;
(3) Mixing the aqueous phase and the organic phase, treating by adopting a reverse evaporation method, a film dispersion method or an ethanol injection method, and dispersing by adopting ultrasonic waves to obtain the allopregnanolone liposome.
According to an aspect of the present invention, there is provided a second-step dissolution preparation method of a liposome of allopregnanolone, the preparation method comprising the steps of:
(1) Dissolving the emulsifier by using water for injection to obtain a water phase;
(2) Weighing phospholipid, cholesterol and allopregnanolone according to the proportion of claim 1, and dissolving the phospholipid, the cholesterol and the allopregnanolone in an organic solvent to obtain an organic phase;
(3) Mixing the aqueous phase and the organic phase, treating by adopting a reverse evaporation method or a film dispersion method or an injection method, and dispersing by adopting ultrasonic to obtain the allopregnanolone liposome.
The technical scheme of the preparation method of the allopregnanolone liposome is as follows: the organic solvent is preferably one or more of chloroform, diethyl ether, dichloromethane, n-hexane, methanol and ethanol.
The allopregnanolone liposome disclosed by the invention can be applied to the preparation of medicaments for treating postpartum depression.
The invention wraps allopregnanolone between phospholipid double layers of liposome, improves water solubility, has good physical stability, and can be placed for a long time. The allopregnanolone liposome disclosed by the invention is simple and mature in preparation method, and is convenient for industrial production.
Detailed Description
The following examples are provided to further illustrate some, but not all, of the preferred embodiments of the present invention. Other embodiments of the invention, which are based on the invention, will be apparent to those skilled in the art without undue burden, and are within the scope of the invention.
Example 1 preparation of allopregnanolone liposome, which consists of the following raw materials in weight ratio,
the formula comprises the following components: allopregnanolone 1; phospholipid 16;
cholesterol 5; and an emulsifying agent 5.
The allopregnanolone is a synthetic monomer raw material, wherein the content of the allopregnanolone is 86%; the phospholipid is dimyristoyl lecithin amide; the emulsifier is Tween-80.
The preparation method comprises the following specific steps:
(1) Dissolving allopregnanolone and emulsifier with water for injection to obtain water phase;
(2) Weighing phospholipid and cholesterol according to the proportion, and dissolving the phospholipid and cholesterol in an organic solvent to obtain an organic phase;
(3) Mixing the aqueous phase and the organic phase, treating by adopting a reverse evaporation method, and dispersing by adopting ultrasonic to obtain the allopregnanolone liposome.
Example 2 preparation of allopregnanolone liposome, which consists of the following raw materials in weight ratio,
the formula comprises the following components: allopregnanolone 1; a phospholipid 19;
cholesterol 7; and an emulsifying agent 4.
The allopregnanolone is a synthetic monomer raw material, wherein the content of the allopregnanolone is 92%; the phospholipid is hydrogenated egg yolk lecithin; the emulsifier is N, N-dimethylformamide.
The preparation method comprises the following specific steps:
(1) Dissolving allopregnanolone and emulsifier with water for injection to obtain water phase;
(2) Weighing phospholipid and cholesterol according to the proportion, and dissolving the phospholipid and cholesterol in an organic solvent to obtain an organic phase;
(3) Mixing the aqueous phase and the organic phase, treating by adopting an ethanol injection method, and dispersing by ultrasonic to obtain the allopregnanolone liposome.
Example 3 preparation of allopregnanolone liposome, which consists of the following raw materials in weight ratio,
the formula comprises the following components: allopregnanolone 1; a phospholipid 19;
cholesterol 7; and an emulsifying agent 4.
The allopregnanolone is a synthetic monomer raw material, wherein the content of the allopregnanolone is 98%; the phospholipid is hydrogenated egg yolk lecithin; the emulsifier is polyethylene glycol-4000.
The preparation method comprises the following specific steps:
(1) Dissolving allopregnanolone and emulsifier with water for injection to obtain water phase;
(2) Weighing phospholipid and cholesterol according to the proportion, and dissolving the phospholipid and cholesterol in an organic solvent to obtain an organic phase;
(3) Mixing the aqueous phase and the organic phase, treating by adopting a film dispersion method, and dispersing by ultrasonic to obtain the allopregnanolone liposome.
Example 4 preparation of allopregnanolone liposome, which consists of the following raw materials in weight ratio,
the formula comprises the following components: allopregnanolone 1; phospholipid 26;
cholesterol 8; and an emulsifying agent 6.
The allopregnanolone is a synthetic monomer raw material, wherein the content of the allopregnanolone is 93%; the phospholipid is hydrogenated soybean lecithin; the emulsifier is Tween-80.
The preparation method comprises the following specific steps:
(1) Dissolving the emulsifier with water for injection to obtain water phase;
(2) Weighing phospholipid, cholesterol and allopregnanolone according to the proportion, and dissolving the phospholipid, the cholesterol and the allopregnanolone in an organic solvent to obtain an organic phase;
(3) Mixing the aqueous phase and the organic phase, treating by adopting an ethanol injection method, and dispersing by ultrasonic to obtain the allopregnanolone liposome.
Example 5 preparation of allopregnanolone liposome, it is composed of the following raw materials in weight ratio,
the formula comprises the following components: allopregnanolone 1; phospholipid 26;
cholesterol 5; and an emulsifying agent 6.
The allopregnanolone is a synthetic monomer raw material, wherein the content of the allopregnanolone is 92%; the phospholipid is soybean lecithin; the emulsifier is Tween-80.
The preparation method comprises the following specific steps:
(1) Dissolving the emulsifier with water for injection to obtain water phase;
(2) Weighing phospholipid, cholesterol and allopregnanolone according to the proportion, and dissolving the phospholipid, the cholesterol and the allopregnanolone in an organic solvent to obtain an organic phase;
(3) Mixing the aqueous phase and the organic phase, then treating by adopting a film dispersion method, and dispersing by ultrasonic to obtain the allopregnanolone liposome.
Example 6 preparation of allopregnanolone liposome, which consists of the following raw materials in weight ratio,
the formula comprises the following components: allopregnanolone 1; a phospholipid 21;
cholesterol 8; and an emulsifying agent 5.
The allopregnanolone is a synthetic monomer raw material, wherein the content of the allopregnanolone is 92%; the phospholipid is soybean lecithin; the emulsifier is poloxamer 188.
The preparation method comprises the following specific steps:
(1) Dissolving the emulsifier with water for injection to obtain water phase;
(2) Weighing phospholipid, cholesterol and allopregnanolone according to the proportion, and dissolving the phospholipid, the cholesterol and the allopregnanolone in an organic solvent to obtain an organic phase;
(3) Mixing the aqueous phase and the organic phase, treating by adopting a reverse evaporation method, and dispersing by adopting ultrasonic to obtain the allopregnanolone liposome.
The allopregnanolone liposome of any of the above examples 1-6 was passed through a microporous membrane of 0.22um, potted, filled with nitrogen gas, and sterilized to obtain an allopregnanolone liposome injection.
The invention carries out the following researches on allopregnanolone liposome injection:
experiment 1 allopregnanolone liposome injection hemolysis experiment
1.1 Experimental materials
Allopregnanolone injection with concentration of 0.56mg/ml; allopregnanolone liposome a (prepared according to example 1) 1.0mg/ml; allopregnanolone liposome B (prepared according to example 4) 1.0mg/ml.
1.2 Experimental methods
The allopregnanolone injection and allopregnanolone liposome A, B are respectively diluted into three concentrations of 0.05mg/ml, 0.1mg/ml and 0.15mg/ml by physiological saline, and are used as test samples for standby, and each sample is added as a repeated tube for hemolysis examination.
Taking a rabbit, taking 10ml of heart blood, placing the heart blood into a conical flask, adding glass beads, stirring for 10min to remove fibrinogen, making the heart blood into defibrinated blood, transferring the defibrinated blood into glass test tubes, adding about 5ml of physiological saline into each test tube, uniformly mixing, centrifuging for 5min (2000 r/min), removing supernatant, adding 5ml of physiological saline, uniformly mixing, and centrifuging. Repeating the above steps for 2-3 times until the supernatant is colorless and transparent (not red). The obtained red blood cells are added with normal saline according to volume to prepare 2% red blood cell suspension for experiments.
Clean test tubes are taken and respectively numbered and then arranged on a test tube rack. Various test solutions were added as shown in table 1 below, with No. 1 being a negative control, no. 2 being a positive control, and one tube repeated for each sample. After mixing the test solutions, the mixture was immediately placed in an incubator at 37.+ -. 0.5 ℃ for incubation, and observation was started every 15min for 3 hours, and every 2-3 hours after 3 hours. After 12 hours, the supernatant (about 2.5 ml) of each test tube was collected, centrifuged at 1000r for 10min, and the supernatant was collected and read on a spectrophotometer for OD value of each tube, measurement wavelength was 540nm, and distilled water was zeroed.
TABLE 1
| Solution/test tube | Negative control tube | Positive control tube | Each detection sample tube |
| 2% erythrocyte suspension (ml) | 2.5 | 2.5 | 2.5 |
| Normal saline (ml) | 2.5 | - | 2.2 |
| Distilled water (ml) | - | 2.5 | - |
| Test article (ml) | - | - | 0.3 |
1.3 results observations and calculations
If the solution in the experiment is in clear red, the bottom of the tube has no red blood cell residue or a small amount of red blood cell residue, which indicates that hemolysis occurs; if the red blood cells are all sinking, the supernatant is colorless and transparent, indicating that no hemolysis occurs.
If there is a brownish red or reddish brown flocculent precipitate in the solution, the precipitate will not disperse after shaking, indicating that red blood cell aggregation occurs. If the phenomenon of erythrocyte aggregation occurs, whether the aggregation is true or false can be further judged as follows. If the aggregate can be uniformly dispersed after the test tube oscillates, or the aggregate is placed on a glass slide, two drops of 0.9% NaCl solution are dripped at the edge of the glass slide, and the glass slide is observed under a microscope, the aggregated red blood cells can be scattered to form pseudo-aggregate, and if the aggregate is not scattered by shaking or is not scattered on the glass slide to form true aggregate.
When the negative control tube has no hemolysis and coagulation, and the positive control tube has hemolysis, the test substance can be injected for use if the solution in the test tube does not have hemolysis and coagulation within 3 hours.
The hemolysis (%) of each tube was calculated using the following formula:
hemolysis ratio (%) = (ODt-ODnc)/(ODpc-ODnc) ×100%
Wherein ODt is the test tube absorbance; ODnc is the negative control tube absorbance; ODpc is positive control tube absorbance.
Reference evaluation criteria: a hemolysis rate > 5% indicates that hemolysis has occurred and is statistically processed.
The experiment shows that: the upper layer of the negative control tube is colorless and clear, and red blood cells sink completely and are not hemolyzed; the red blood cells of the positive control tube are completely dissolved, the color of the solution is dark red, and the solution is completely hemolyzed; the allopregnanolone liposome injection has the advantages that a 0.05mg/ml concentration tube is not hemolyzed, the upper layer of a 0.1mg/ml concentration test tube is clear, but the erythrocyte sinking is incomplete; the red cell fraction of the tube at 0.15mg/ml concentration was called "red" and the supernatant liquid had some hemolysis. The three concentration test tubes of allopregnanolone liposome are not hemolyzed. Spectrophotometry detection results are shown in
TABLE 2
| Group of | Concentration (mg/ml) | First tube hemolysis Rate (%) | Second tube hemolysis Rate (%) | Hemolysis (±) device |
| Allopregnanolone injection | 0.05 | 0.22 | 0.25 | - |
| Allopregnanolone injection | 0.1 | 2.05 | 3.34 | - |
| Allopregnanolone injection | 0.15 | 10.21 | 11.74 | + |
| Allopregnanolone liposome A | 0.05 | 0.08 | 0.13 | - |
| Allopregnanolone liposome A | 0.1 | 0.17 | 0.24 | - |
| Allopregnanolone liposome A | 0.15 | 0.25 | 0.34 | - |
| Allopregnanolone liposome B | 0.05 | 0.03 | 0.05 | - |
| Allopregnanolone liposome B | 0.1 | 0.10 | 0.13 | - |
| Allopregnanolone liposome B | 0.15 | 0.15 | 0.22 | - |
As can be seen from table 2: the safe concentration of allopregnanolone injection hemolysis is about 0.1mg/ml, and the two allopregnanolone liposomes still have no hemolysis phenomenon at the concentration of 0.15mg/ml, which indicates that the liposome preparation can improve the hemolysis of allopregnanolone to a certain extent.
Experiment 2 determination of the encapsulation efficiency and drug-loading amount of allopregnanolone liposome
2.1 determination of recovery of free allopregnanolone
9 centrifuge tubes of 2ml are taken and divided into 3 groups, 0.2ml of blank liposome is added respectively, 0.2ml, 0.6 ml and 1.0ml of allopregnanolone reference stock solution are added respectively, the solution is diluted to scale by methanol, after centrifugation is carried out for 30min at 15000r/min, 1ml of supernatant is measured precisely, the solution of allopregnanolone with mass concentration of 0.4, 1.2 and 2.0ug/ml is prepared by methanol, and sample injection measurement is carried out, and the average recovery rate of allopregnanolone is 99.98%, 100.2% and 100.1% respectively (RSD is 0.6%, 0.15% and 0.09, n=3 respectively), so that the separation of liposome and medicine by an ultracentrifugation method is feasible.
2.1 determination of encapsulation and drug loading
Ultracentrifugation was chosen to separate the drug encapsulation and drug loading: precisely sucking the allopregnanolone liposome injection (prepared according to example 2) 1mL, centrifuging at 15 r/min for 30min, discarding supernatant, taking out the lower precipitate, adding 1mL methanol for dissolution, and determining the mass concentration c of the liposome-encapsulated allopregnanolone by HPLC; and taking 1ml of allopregnanolone liposome injection, performing constant volume demulsification by using methanol, performing water bath ultrasonic treatment (frequency: 45 kHz, power: 150W) for 10min, performing centrifugation for 10min at 15000r/min, and determining the total mass concentration Ctotal of allopregnanolone in the liposome injection by an HPLC method. The encapsulation efficiency and drug-loading rate (allopregnanolone encapsulation efficiency=c/c total×100%; allopregnanolone drug-loading rate= [ allopregnanolone drug amount in liposome (/ drug amount in liposome+carrier mass) ]×100%) were calculated, and the results are shown in table 3
TABLE 3 Table 3
Experiment 3 investigation of stability of allopregnanolone Liposome
3.1 physical stability of liposomes
The examination was carried out using a centrifugal acceleration test. Placing 2. 2mL allopregnanolone liposome injection into a 10mL centrifuge tube, centrifuging at 3000 r/min for 15min, discarding the upper layer liquid of 1.4 mL, shaking the rest liquid uniformly, placing 0.4mL into a 10mL measuring flask with a microsampler, fixing volume with distilled water, and measuring turbidity A at 450 nm wavelength. The turbidity AO was measured by precisely measuring 0.4. 0.4mL of the non-centrifuged liposome sample, as described above. Centrifugal stability constant (KE) = -A0 x 100%. As a result, KE of 3 batches of samples is 7.7, 8.5 and 9.1 respectively, which shows that the physical stability of the system is better, and the prepared allopregnanolone liposome is provided with good physical stability.
3.2 Liposome leave-on stability
2 batches of allopregnanolone liposomes (prepared as in example 5) were placed at (25.+ -. 2) and (4.+ -. 2) ℃ respectively, sampled at 0, 15 and 30, d respectively, and their appearance was observed to determine the encapsulation efficiency. As a result, the encapsulation efficiency of allopregnanolone liposome was rapidly decreased after 30 d at (25.+ -. 2) ℃ and a small amount of particles were found to precipitate and aggregate by observation. The results of the encapsulation efficiency of the samples placed at (4.+ -. 2) ℃ are excellent, and the results of the observation that no sedimentation aggregation or the like of the particles is found are shown in Table 4.
TABLE 4 Table 4
It should be noted that the foregoing description of the preferred embodiments is merely illustrative of the technical concept and features of the present invention, and is not intended to limit the scope of the invention, as long as the scope of the invention is defined by the claims and their equivalents. All equivalent changes or modifications made in accordance with the spirit of the present invention should be construed to be included in the scope of the present invention.
Claims (8)
1. The allopregnanolone liposome is characterized by comprising, by weight, 1 part of allopregnanolone, 5-50 parts of phospholipids, 3-15 parts of cholesterol and 2-10 parts of emulsifying agents, wherein the emulsifying agents are tween-80, N-dimethylformamide, polyethylene glycol-4000 and poloxamer 188.
2. The allopregnanolone liposome according to claim 1, wherein the liposome comprises the following raw materials, by weight, 1 part of allopregnanolone, 15-30 parts of phospholipids, 3-10 parts of cholesterol and 3-8 parts of emulsifying agents.
3. The allopregnanolone liposome according to claim 1, wherein allopregnanolone is a monomeric material, and allopregnanolone is present in an amount of greater than 80%; the monomer raw material is synthetic monomer raw material or natural monomer raw material.
4. A allopregnanolone liposome according to claim 3, wherein the allopregnanolone material is 98%.
5. A liposome of allopregnanolone according to claim 3, wherein the phospholipid is selected from one or more of a naturally extracted phospholipid, a semisynthetic phospholipid, and a fully synthetic phospholipid.
6. The allopregnanolone liposome of claim 5, wherein the phospholipid is selected from one or more of soy lecithin, egg yolk lecithin, hydrogenated soy lecithin, hydrogenated egg yolk lecithin, and dimyristoyl lecithin amide.
7. The method for preparing the allopregnanolone liposome according to claim 1, which comprises the following steps:
(1) Dissolving allopregnanolone and the emulsifier by using water for injection to obtain a water phase;
(2) Weighing phospholipid and cholesterol according to the proportion of claim 1, and dissolving the phospholipid and cholesterol in an organic solvent to obtain an organic phase;
(3) Mixing the aqueous phase and the organic phase, treating by adopting a reverse evaporation method, a film dispersion method or an ethanol injection method, and dispersing by adopting ultrasonic waves to obtain the allopregnanolone liposome.
8. The method for preparing the allopregnanolone liposome according to claim 1, which comprises the following steps:
(1) Dissolving the emulsifier by using water for injection to obtain a water phase;
(2) Weighing phospholipid, cholesterol and allopregnanolone according to the proportion of claim 1, and dissolving the phospholipid, the cholesterol and the allopregnanolone in an organic solvent to obtain an organic phase;
(3) Mixing the aqueous phase and the organic phase, treating by adopting a reverse evaporation method or a film dispersion method or an injection method, and dispersing by adopting ultrasonic to obtain the allopregnanolone liposome.
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| CN102000028A (en) * | 2010-11-03 | 2011-04-06 | 江苏康缘药业股份有限公司 | Solamargine liposome, preparation method and usage thereof |
| WO2014031792A2 (en) * | 2012-08-21 | 2014-02-27 | Sage Therapeutics | Methods of treating epilepsy or tatus epilepticus |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102000028A (en) * | 2010-11-03 | 2011-04-06 | 江苏康缘药业股份有限公司 | Solamargine liposome, preparation method and usage thereof |
| WO2014031792A2 (en) * | 2012-08-21 | 2014-02-27 | Sage Therapeutics | Methods of treating epilepsy or tatus epilepticus |
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| Title |
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| "ON THE SIMILARITY OF HEMOLYSIS INDUCED BY PLANT SAPOGENINS AND BY NEUTRAL STEROIDS";RUTH SEGAL等;《Biochemical Pharmacology》;2163-2167 * |
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