CN106546705B - Test method for in-vitro release of liposome drug - Google Patents
Test method for in-vitro release of liposome drug Download PDFInfo
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Abstract
The invention relates to a test method of liposome drugs, in particular to a test method for simulating the drug release process of liposome drugs after entering the body in vitro. The invention fills liposome drug solution and air into the container together, and simulates the dynamic action of in vivo blood circulation on the drug by utilizing the oscillation or rotation of the container, so that the air in the system is dispersed to form bubbles in the motion and physically collides with the liposome, thereby destroying the lipid membrane and achieving the effective release of the drug. All the operating parameters including temperature, rotating speed, volume ratio of liposome medicine to air in the container, etc. are controllable, so that it is convenient to regulate release rate, and can implement effective release of medicine in short time. Ion exchange resin can be added to adsorb released free medicine, so as to achieve the effect of simulating the state of the leakage groove in vivo.
Description
Technical Field
The invention relates to a test method of liposome drugs, in particular to a test method for simulating the drug release process of liposome drugs after entering the body in vitro.
Background
Since the discovery of phospholipid bilayer membranes in the 90's of the 20 th century, the study of liposomes as drug delivery vehicles has been under-developed. Liposomes refer to the microvesicles formed by encapsulating a drug within a lipid bilayer. The phospholipid molecules are inserted into water from the hydrophilic head part of the molecule in the water, and the hydrophobic tail part extends to the air to form the spherical liposome with a lipid bilayer structure, wherein the particle size is usually 25-1000 nm.
The double-layer membrane structure of the liposome is similar to a biological membrane, and has good biocompatibility; the liposome-encapsulated drug has the advantages of targeting property, long-acting property, low toxicity and good encapsulation protection. Thus, liposomes are used for the delivery of a variety of active drugs to improve the blood circulation time after the drug enters the body, increasing accumulation at the target site. The function of the liposome can be changed by changing the particle size, the lipid formulation composition and the surface characteristics of the lipid membrane. Different lipid materials can be used for preparing various functional liposomes such as long-circulating liposome, thermosensitive liposome, pH sensitive liposome, immunoliposome and the like, thereby achieving the purpose of special treatment.
Therefore, in order to rationally design a liposome drug delivery system, detailed studies on the in vivo and in vitro release behaviors of drugs are required, and the drug release process after the liposome drugs enter the body is tested by in vitro simulation.
However, the only test methods available today have serious shortcomings. Taking doxorubicin liposome as an example, the in vitro release investigation method in the FDA guideline only gives two experimental parameters of medium pH and temperature, and simulates the in vivo physiological environment by a large volume of buffer or a buffer containing human plasma. However, because doxorubicin exists in liposome in a precipitated form of doxorubicin sulfate, the solubility is very low, so that the release rate is too slow to meet the requirement of rapid evaluation in a quality control system.
The Low Frequency Ultrasound (LFUS) method adopted by the Avi Schroeder and Yechezkel Barenholz can release 80% of drugs within a short time (3 minutes), but the low frequency ultrasound can cause transient pore-like defects of a lipid membrane, so that the damage to the liposome is severe, and the released liposome has no preparation concept and cannot simulate the physiological environment in vivo. Atsuko Hioki and Yoshie Maitani use Bovine Serum Albumin (BSA) added under high temperature (50 ℃) condition to accelerate drug release; koji Nakamura and Hiroaki Kasukawa evaluate drug release behavior by adding ammonium ions at different concentrations to liposomes prepared by a pH gradient method, and distinguish different lipid components of the drug-loaded liposomes. Although the method can complete the release in a short time, the release curve is quite unstable and has poor repeatability, and the method cannot be used as a standard for quality evaluation.
Jennifer a. shabbits and Lawrence d. mayer investigated the drug release rate of multilamellar vesicles "precipitated" in an excess of 100-fold environment of large unilamellar vesicles (LUVs 100nm) to mimic the large membrane pool in blood cells and tissues. Although this method can obtain the true attributes of liposomal drug retention in vivo, accurate mass assessment is still difficult to achieve due to the variability of MLVs.
In conclusion, the existing in vitro release test method for liposome drugs is difficult to balance the drug release rate with in vivo and in vitro correlation and repeatability.
Disclosure of Invention
The invention aims to provide a method for testing in-vitro release of liposome drugs, which can highly simulate the physiological environment of in-vivo drug release, has controllable operation parameters, can greatly improve the drug release rate and has high repeatability.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for testing the in vitro release of a liposomal drug, said liposomal drug being in the form of a solution, the method comprising the steps of:
(1) filling the liposome medicine and air into a container according to a preset volume ratio at a preset temperature, and closing the container;
(2) -subjecting the container to a movement with a back and forth displacement in a vertical direction;
(3) samples were taken at desired time points and either the undelivered drug concentration or the released drug concentration in the liposomal drug was determined and the drug release rate was calculated.
Further, the liposome drug is liposome anthracycline drug, liposome vincristine or liposome amphotericin B, preferably liposome adriamycin.
Further, the movement in the step (2) is rotation around a non-vertical rotation axis, and the container is strip-shaped and is installed in a manner of being radially perpendicular to the rotation axis.
Further, the rotation motion of the step (2) is completed by using a molecular hybridization instrument, the rotating shaft is horizontal, the container is a centrifugal tube, and the rotating speed is 15-50 rpm.
Further, in the step (1), the volume ratio is 1 (0.1-5); the preset temperature is 30-60 ℃.
Further, in the step (1), the predetermined volume ratio of the liposome drug to air may be defined as that no less than 25% of the volume of air still exists in the container.
Further, in the step (1), a predetermined amount of ion exchange resin for adsorbing the released free drug is added to the container, the predetermined amount being not less than an amount required for adsorbing all of the drug encapsulated in the liposome drug; the concentration of undelivered drug in the liposomal drug is determined in step (3).
Further, when the drug encapsulated in the liposomal drug is selected from the group consisting of anthracyclines, vincristine, and amphotericin B; the ion exchange resin is a cation exchange resin.
Compared with the prior art, the method has the following advantages:
(1) the dynamic action of in-vivo blood circulation on the medicine is simulated through the movement of the container, so that the release behavior of the medicine after entering the body can be better simulated, and the release process is closer to the real situation of in-vivo release;
(2) all the operation parameters including temperature, rotating speed, volume ratio of liposome medicine and air in the container are controllable, so that the release rate can be conveniently regulated, and effective release of the medicine can be realized in a short time;
(3) the release process can be suspended at any time, the drug release after restart is not influenced, and the sampling is convenient;
(3) the experimental repeatability is much higher than that of various methods in the prior art;
(4) if the ion exchange resin is added into the container, the released free medicine can be immediately adsorbed by the resin, the effect of simulating the in-vivo leakage groove state is achieved, the concentration of the residual unreleased medicine can be rapidly measured, and the experimental efficiency is greatly improved.
In conclusion, the invention obtains the balance between the drug release rate and the in vivo and in vitro correlation and repeatability, and is a method which can comprehensively meet the requirements of various aspects of in vitro release experiments.
Drawings
FIG. 1: in examples 1-3, drug release profiles at different temperatures;
FIG. 2: drug release profiles at different drug volumes in examples 4, 2, 5.
FIG. 3: in examples 6-8, drug release profiles were obtained for different drug volumes.
FIG. 4: drug release profiles at different rotational speeds in examples 2, 9, 10.
FIG. 5: in examples 11 to 14, the drug release profiles were determined at different amounts of cationic adsorbent resin added.
FIG. 6: the repeatability test of the invention: doxorubicin release rate at different time points for different samples.
Detailed Description
The basic principle of the invention is to simulate the dynamic action of in vivo blood circulation on the drug by the motion of the container, and the air in the system is dispersed in the motion to form air bubbles which physically collide with the liposome, thereby destroying the lipid membrane and achieving the effective release of the drug.
In terms of experimental temperature, the phase transition temperature of phospholipid is preferably about 52 ℃, and preferably 40-50 ℃. Although the higher the temperature, the better the fluidity of the lipid membrane and the faster the drug release rate, the excessively high temperature may destroy the liposome itself, namely, the release is not said. Since the release rate of the drug in this method can be adjusted not only by temperature but also by the rotation speed or air ratio, it is not necessary to select an excessively extreme temperature unless it is specifically tested for a temperature gradient, and 30 degrees or less, 60 degrees or more can be selected.
The movement of the container must be provided with a vertical displacement, otherwise the air floating above the solution cannot be made to collide against the liposomes in the solution. The simplest movement to achieve mixing is shaking or rotating up and down. In practical operation, it is most convenient to rotate around a horizontal axis by using a molecular hybridization instrument, and the centrifugal tubes need to be vertically arranged around the rotating shaft in a manner of radially and vertically perpendicular to the rotating shaft. The rotating mode enables the air to be better dispersed, and the simulation of the blood circulation in the body is more real.
Meanwhile, the released free drug can be adsorbed by the ion exchange resin, and the concentration of the residual drug can be conveniently and directly measured by adsorbing the released free drug by the ion exchange resin.
The cation resin can adsorb anthracycline (daunorubicin, adriamycin, aclarubicin, epirubicin, idarubicin, valrubicin, mitoxantrone), vincristine, amphotericin B, etc., wherein the strong acid type cation exchange resin has strong acidic reactive group such as sulfonic group (-SO)3H) Can widely adsorb all cations; the weak acid type cation exchange resin has a weak reactive group such as carboxyl (-COOH group), and only cations such as Ca in the weak base can be exchanged2 +、Mg2 +. The anion exchange resin is mainly strong base type anion exchange resin, and can exchange and adsorb all anions.
If the ion exchange resin is added before the experiment begins, the effect of simulating the state of the leakage groove in the body can be achieved; if ion exchange resin is added after the experiment is finished and the sample is taken, only the function of adsorbing free drugs is achieved, and the operation of the sample before detection is simplified.
The following examples further illustrate specific embodiments of the present invention. The examples examined the following 5 parameters: the type of centrifuge tube, temperature, rotational speed, volume ratio of drug solution to air, and the amount of ion exchange resin added, on the release rate of the drug.
Example 1: 5ml centrifuge tube-resin with 37 ℃ temperature, 25rpm rotation speed and 2 ml-10% of medicine volume
(1) Preparing a liposome adriamycin solution:
placing Phosphate Buffered Saline (PBS) with pH of 6.0 in an ultrasonic cleaning machine, and ultrasonically cleaning for 20min to remove gas; the liposome doxorubicin 1.25ml to 50ml volumetric flasks were pipetted using a pipette, and the initial drug concentration was calculated using the above PBS to make a volume of 40-fold dilution.
(2) Pretreatment with cation exchange resin
Adding cation exchange resin into the chromatographic column, draining water naturally, and blowing and beating with ear washing ball until no continuous liquid flows out. (3) In vitro Release assay
a. Adding 2ml of liposome adriamycin diluent and 0.2g (w/v ═ 10%) of cation exchange resin into a 5ml EPPENDOF centrifuge tube;
b. setting the temperature of the molecular hybridization instrument to be 37 ℃ and the rotating speed to be 0rpm, and preheating;
c. fixing the centrifugal tube on a hybridization tube frame of the molecular hybridization instrument in a manner of being vertical to the horizontal rotating shaft in the radial direction;
d. the temperature of the molecular hybridization apparatus was set at 37 ℃ and the rotation speed was set at 25rpm, and the rotation was started and timed.
e. Sampling at the time points of 2h, 4h and 6h, and obtaining a sample by using one centrifugal tube. The residual drug concentration in the sample supernatant was determined by HPLC.
f. The drug release rate (1-residual drug concentration/initial drug concentration) × 100%, the experimental results are given in the following table:
table 1, release rates of doxorubicin at different time points of example 1
Example 2: 5ml centrifuge tube-resin with 49 ℃ temperature, 25rpm rotation speed and 2 ml-10% of medicine volume
The specific procedure is as in example 1, with parameter settings such as title and temperature change. The results of the experiment are shown in the following table:
table 2, release rates of doxorubicin at different time points of example 2
Example 3: resin with 5ml centrifuge tube temperature of 52 ℃, rotation speed of 25rpm and medicine volume of 2 ml-10%
The specific procedure is as in example 1, with parameter settings such as title and temperature change. The results of the experiment are shown in the following table:
table 3, release rates of doxorubicin at different time points of example 3
FIG. 1 is a graph showing the results of examples 1 to 3, showing the release profiles of the drugs at different temperatures under otherwise identical conditions (5ml centrifuge tube, 2ml drug volume, 25rpm rotation, addition of 10% w/v cationic adsorption resin). It can be seen that temperature affects the release rate of the adriamycin drug, and that the release is relatively fast with increasing temperature.
Example 4: 5ml centrifuge tube-resin with 49 ℃ temperature, 25rpm rotation speed and 1 ml-10% of medicine volume
The procedure is as in example 2, with the parameters set as title and volume of drug changed. The results of the experiment are shown in the following table:
table 4, release rates of doxorubicin at different time points of example 4
Example 5: 5ml centrifuge tube-resin with 49 ℃ temperature, 25rpm rotation speed and 4 ml-10% of medicine volume
The procedure is as in example 4, with the parameters set as title and volume of drug changed. The results of the experiment are shown in the following table:
table 5, release rates of doxorubicin at different time points of example 5
FIG. 2 is a graph showing the results of examples 4, 2 and 5, showing the release profiles of the drug at different drug volumes (i.e., the volume ratio of drug to air) under otherwise identical conditions (5ml centrifuge tube, 49 ℃ C., 25rpm rotation, addition of 10% w/v cationic adsorption resin).
As clearly shown in the figure, the volume ratio of the liquid medicine to air significantly changed the release behavior of doxorubicin. The doxorubicin release rate slowed significantly as the drug volume increased from 1ml to 4 ml. This is because in this test method, the air dispersed in the solution is relied upon to break the lipid membrane, and therefore the release rate can be accurately manipulated by controlling the air volume ratio, and the operation is intuitive and simple.
Example 6: 5ml centrifuge tube-resin with 47 ℃ temperature, 25rpm rotation speed and 1.8 ml-10% of medicine volume
The specific procedure is the same as example 1, the parameters are set as the titles, and the sampling time points are 1h, 3h and 5 h. The results of the experiment are shown in the following table:
table 6, release rates of doxorubicin at different time points of example 6
Example 7: 5ml centrifuge tube-resin with 47 ℃ temperature, 25rpm rotation speed and 2.4 ml-10% of medicine volume
The procedure is as in example 6, with the parameters set as title and the volume of the drug slightly changed. The results of the experiment are shown in the following table:
table 7, release rates of doxorubicin at different time points of example 7
Example 8: resin with 5ml centrifuge tube temperature of 47 ℃, rotation speed of 25rpm and medicine volume of 3 ml-10%
The procedure is as in example 6, with the parameters set as title and the volume of the drug slightly changed. The results of the experiment are shown in the following table:
table 8, release rates of doxorubicin at different time points of example 8
FIG. 3 is a graph of the release profiles of drugs at different drug volumes (i.e., drug to air volume ratio) under otherwise identical conditions (5ml centrifuge tube, 47 deg.C, 25rpm rotation, addition of 10% w/v cationic adsorption resin) according to examples 6-8. Compared with the attached figure 2, the drug volume change range in the experiment group is small, and the obtained drug release curve has no obvious difference.
Example 9: resin with 5ml centrifuge tube temperature of 49 ℃, rotation speed of 15rpm and medicine volume of 2 ml-10%
The specific procedure is the same as in example 2, and the parameters are set as indicated by the title and the rotation speed is changed. The results of the experiment are shown in the following table:
table 9, release rates of doxorubicin at different time points of example 9
Example 10: 5ml centrifuge tube-resin with 49 ℃ temperature, 35rpm rotation speed and 2 ml-10% of medicine volume
The specific procedure is the same as in example 2, and the parameters are set as indicated by the title and the rotation speed is changed. The results of the experiment are shown in the following table:
table 10, release rates of doxorubicin at different time points of example 10
FIG. 4 is a graph of the release of drug from examples 2, 9, 10 at different rotational speeds under otherwise identical conditions (5ml centrifuge tube, temperature 49 ℃, drug volume 2ml, addition of 10% w/v cationic adsorption resin). It follows that the rotational speed also has a large influence on the drug release rate. The rotating speed is increased to directly accelerate the damage of the bubbles to the lipid membrane, and the drug release rate is obviously improved.
Example 11: 5ml centrifuge tube-resin with 49 ℃ temperature, 30rpm rotation speed and 1.8 ml-0% of medicine volume
The specific procedure is the same as example 1, the parameters are set as the titles, and the sampling time points are 1h, 3h and 5 h. However, no cation exchange resin was added prior to spinning, and after spinning was completed and sampling was performed, resin was added to the sample to adsorb free drug. The results of the experiment are shown in the following table:
table 11, release rates of doxorubicin at different time points of example 11
Example 12: 5ml centrifuge tube-resin with 49 ℃ temperature, 30rpm rotation speed and 1.8 ml-5% of medicine volume
The procedure is as in example 11, with the parameters set as under the heading, and the content of the cationic adsorbent resin added is varied. The results of the experiment are shown in the following table:
table 12, release rates of doxorubicin at various time points of example 12
Example 13: 5ml centrifuge tube-resin with 49 ℃ temperature, 30rpm rotation speed and 1.8 ml-10% of medicine volume
The procedure is as in example 11, with the parameters set as under the heading, and the content of the cationic adsorbent resin added is varied. The results of the experiment are shown in the following table:
table 13, release rates of doxorubicin at different time points of example 13
Example 14: 5ml centrifuge tube-resin with 49 ℃ temperature, 30rpm rotation speed and 1.8 ml-20% of medicine volume
The procedure is as in example 11, with the parameters set as under the heading, and the content of the cationic adsorbent resin added is varied. The results of the experiment are shown in the following table:
TABLE 14 Adriamycin Release Rate for different time points of example 14
FIG. 5 is a graph of the release profiles of drugs from examples 11-14, after the addition of cationic polymeric adsorbent resins of different masses under otherwise identical conditions (5ml centrifugation, 49 ℃ temperature, 30rpm rotation, 1.8ml drug volume). Wherein the amount of all resin, including the minimum amount of 5% (w/v), exceeds the amount required to adsorb all of the drug in the centrifuge tube. As can be seen from the figure, the addition of the resin had very little effect on the drug release.
Example 15: resin with the temperature of 47 ℃, the rotating speed of 40rpm and the medicine volume of 10ml to 20 percent in a 15ml centrifuge tube
The specific steps are the same as those of the embodiment 1, the parameters are set as titles, the models of the centrifugal tubes are changed, and the sampling time points are 1h and 3 h. The results of the experiment are shown in the following table:
table 15, release rates of doxorubicin at different time points of example 15
Example 16: resin with the temperature of 47 ℃, the rotating speed of 40rpm and the medicine volume of 11ml to 10 percent in a 15ml centrifuge tube
The procedure is as in example 15, with the parameters set as headings, slightly increased drug volume and reduced resin addition. The results of the experiment are shown in the following table:
table 16, release rates of doxorubicin at different time points of example 16
Example 17: resin with the temperature of 47 ℃, the rotating speed of 30rpm and the medicine volume of 10ml to 20 percent in a 15ml centrifuge tube
The specific procedure is as in example 15, with the parameters set as indicated by the heading and the speed of rotation being varied. The results of the experiment are shown in the following table:
table 17, release rates of doxorubicin at different time points of example 17
Example 18: resin with the temperature of 47 ℃, the rotating speed of 20rpm and the medicine volume of 10ml to 10 percent in a 15ml centrifuge tube
The specific procedure is as in example 15, with the parameters set as indicated by the heading and the speed of rotation being varied. The results of the experiment are shown in the following table:
table 18, release rates of doxorubicin at different time points of example 18
Data comparison with above-mentioned 15ml centrifuging tube and 5ml centrifuging tube finds that the centrifuging tube volume increases, is favorable to the medicine release. The drug to air volume ratio in a 15ml centrifuge tube is greater than 5ml centrifuge tube at similar temperatures and at the same rotational speed, and the time required to achieve drug release rates above 90% is shorter than the latter. Therefore, increasing the drug release rate by increasing the size of the centrifugal tube is also one of the effective means.
Example 19: 5ml centrifuge tube-resin with 49 ℃ temperature, 25rpm rotation speed and 2 ml-10% of medicine volume
The specific procedure is the same as in example 1, and the parameters are set as under the heading. Sampling time points are 1h, 2h, 4h, 6h and 8h, and 6 samples are taken at each time point to verify repeatability. The results of the experiment are shown in the following table:
adriamycin Release Rate for different samples of Table 19, example 19 at different time points
FIG. 6 is a comparison of the drug release profiles of the above samples. As can be seen from the figure, the method is very reproducible and, in particular, the stability of drug release is very good at longer time scales.
The influence of different movement modes on the experimental effect is examined through a plurality of groups of examples and comparative examples.
Example 20: 15ml centrifuge tube-temperature 47 ℃, rotation speed 60rpm, medicine volume 10ml-2g resin-vertical sample loading
Comparative example 1: 15ml centrifuge tube-temperature 47 ℃, rotation speed 60rpm, medicine volume 10ml-2g resin-horizontal sample loading
The procedure of the experiment was substantially the same as in example 1, with the parameters set as under the heading. The difference is that in the comparative example, the centrifuge tube was installed in a manner radially parallel to the axis of rotation. Meanwhile, the two sets of experiments adopt relatively large rotating speeds.
Sampling time points were 1h, 2h, 3h, 4h, 5h, and 2 samples were taken at each time point. The results of the experiment are shown in the following table:
table 20, example 20 and comparative example 1 doxorubicin release rates at different time points
It can be seen from the above table that when the rotation speed is high, the sample loading mode which is vertical to the rotation shaft in the radial direction is adopted, a large release ratio is achieved only for 1h, and the sample loading mode which is horizontal is slow. This is because, when rotating around a horizontal rotation axis, if the centrifugal tube is also horizontal, the amplitude of the relative displacement between the air and the solution in the vertical direction is not large, and the air easily leans against the inside of the tube wall due to the centrifugal force, making it difficult to form vigorous agitation. Therefore, the vertical sample loading mode is adopted, the releasing speed is high, the horizontal sample loading is slow, and the vertical sample loading is preferred from the perspective of experimental efficiency.
Comparative example 2: 1.5ml EP tube-temperature 47 ℃, oscillation frequency 1000rpm, drug volume 1ml-0.2g resin-oscillation
This comparative example 2 examined drug release in the oscillatory mode.
The basic experimental procedure was as in example 1, with the parameters set as under the heading. The difference lies in that a 1.5ml EP tube is adopted for sample loading, and a constant-temperature oscillation blending instrument is used for oscillation, wherein the oscillation frequency is 1000 rpm. Sampling time points are 1h, 2h and 3h, 4 samples are taken at each time point, the samples are centrifuged, supernatant is taken, and the content of the adriamycin is detected by HPLC. The results of the experiment are shown in the following table:
adriamycin Release Rate at different time points in Table 21, comparative example 2
As can be seen from the above table, at the same time point, the release rates of different samples have a large difference, which indicates that it is difficult to ensure the repeatability of the experiment by adopting the oscillating movement mode, and the controllability is low. If the oscillation frequency is lowered, effective release cannot be achieved in a short time.
Comparative example 3: 50ml of resin with the temperature of 47 ℃, the oscillation frequency of 150rpm and the medicine volume of 50ml to 5g
This comparative example 3 examined drug release in a shaking motion mode using another oscillator.
The basic experimental procedure was as in example 1, with the parameters set as under the heading. Except that a 50ml Schottky bottle was used for loading, and 50ml doxorubicin liposome diluent was added thereto. The shaking was performed using a constant temperature shaker oscillator with a shaking frequency of 150rpm, and 2 bottles of solution were simultaneously mounted on the shaker. Sampling time points are 1h, 2h, 3h, 4h, 5h and 6h, 0.5ml of supernatant is sampled every time, and the content of the adriamycin is detected by HPLC. The results of the experiment are shown in the following table:
adriamycin Release Rate at different time points in Table 22, comparative example 3
As can be seen from the above table, even if the same constant temperature shaking table is used, the difference of the drug release behaviors of the two groups of samples tested at the same time is still large. This is probably because the shaking table does not hold the sample well enough and the sample will shift after the shaking starts, which affects the repeatability of the experiment.
As can be seen from comparative examples 2 and 3, although the oscillation as a motion mode can satisfy the requirement of the drug experiment in terms of release efficiency, in practical operation, it is difficult to precisely control the oscillation by using common equipment in a laboratory, so that the motion mode has low practicability, unlike the rotation mode.
Comparative example 4: 50ml penicillin bottle-resin with 47 ℃ temperature, 450rpm stirring speed and 10ml-2g medicine volume
Comparative example 4 examined drug release in a constant temperature magnetic stirring motion.
Comparative example 4 the basic experimental procedure was the same as example 1 with the parameters set as under the title. Except that 50ml penicillin bottles are adopted for loading, and 10ml adriamycin liposome diluent is added into the penicillin bottles. Stirring 6 groups of samples simultaneously by using a six-hole water bath magnetic stirrer, wherein the stirring speed is 450rpm, the sampling time points are 1h, 2h, 3h, 4h, 5h and 6h, 0.5ml of supernatant is sampled every time, and the content of the adriamycin is detected by using HPLC. The results of the experiment are shown in the following table:
adriamycin Release Rate at different time points in Table 23, comparative example 4
As can be seen from the above table, even if the same stirrer is used, the release rates of the 6 samples tested simultaneously under the same conditions are still very different. It follows that the controllability of the movement pattern of the magnetic stirring is also very low, which is not suitable for the selection of this pattern.
Comparative example 5: resin with dissolving cup temperature of 45 ℃, stirring speed of 250rpm and medicine volume of 250ml-20g
Comparative example 5 a general-purpose instrument dissolution apparatus for dissolution experiments was used, and stirring was performed using a stirring paddle.
250ml of doxorubicin liposome diluent was added to each of 5 dissolution cups at the same time, and the set rotation speed was 250rpm (usually, the rotation speed adopted in the dissolution experiment was several tens of rpm). The sampling time points are 1h, 2h, 3h, 4h and 5 h. This comparative example 5 was sampled and tested for residual concentration of undelivered doxorubicin liposomes. The results of the experiment are shown in the following table:
residual concentration of doxorubicin liposomes at different time points in table 24 and comparative example 5
As can be seen from the above table, even if the temperature is set to 45 ℃ which is high, the release of doxorubicin still does not occur with a relatively vigorous stirring rate. Therefore, this method cannot be used for drug release experiments at all.
The scope of the invention is not to be limited by the specific embodiments described, which are intended as single illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Each of the above-mentioned references is incorporated herein by reference in its entirety.
Claims (8)
1. A method for testing the in vitro release of a liposomal drug, said liposomal drug being in the form of a solution, said method comprising the steps of:
(1) filling the liposome medicine and air into a container according to a preset volume ratio at a preset temperature, and closing the container;
(2) -subjecting the container to a movement with a back and forth displacement in a vertical direction;
(3) sampling at a desired time point and determining the concentration of undelivered drug or the concentration of released drug in the liposomal drug to calculate the rate of drug release;
the movement in the step (2) is rotation around a non-vertical rotation axis, and the container is strip-shaped and is installed in a manner of being radially vertical to the rotation axis;
the liposome drug and air are defined as not less than 25% of the volume of the container in terms of a predetermined volume ratio.
2. The test method according to claim 1, wherein the predetermined temperature is 30 to 60 ℃.
3. The method of claim 1, wherein the rotation of the rotating shaft is performed using a molecular hybridization apparatus, the rotating shaft is horizontal, the container is a centrifuge tube, and the rotation speed of the molecular hybridization apparatus is 15-50 rpm.
4. The test method according to claim 1, wherein in the step (1), a predetermined amount of ion exchange resin for adsorbing the released free drug is added to the container, the predetermined amount being not less than an amount required for adsorbing all of the drug encapsulated in the liposome drug.
5. The test method according to claim 4, wherein the ion exchange resin is a cation exchange resin.
6. The test method of claim 1, wherein the liposomal drug is a liposomal anthracycline, liposomal vincristine, or liposomal amphotericin B.
7. The test method according to claim 6, characterized in that the anthracycline is daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, or mitoxantrone.
8. The test method according to claim 1, characterized in that the liposomal drug is liposomal doxorubicin.
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| CN108007826B (en) * | 2016-11-02 | 2020-11-27 | 广东嘉博制药有限公司 | Method for measuring in-vitro accumulated release rate of long-acting liquid preparation |
| CN108318646A (en) * | 2018-03-27 | 2018-07-24 | 苏州桓晨医疗科技有限公司 | A kind of dissolution medium of aids drug release in vitro |
| CN110736813A (en) * | 2019-10-25 | 2020-01-31 | 广东嘉博制药有限公司 | Method for measuring in-vitro release rate of pharmaceutical preparations |
| CN110865160B (en) * | 2019-10-25 | 2022-05-03 | 广东嘉博制药有限公司 | Method for measuring in-vitro release degree of propofol fat emulsion |
| CN114324680B (en) * | 2021-12-14 | 2023-03-28 | 南京美新诺医药科技有限公司 | In vivo biological analysis method for liposome drug |
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