CN114010805B - Stabilizer-containing microsphere, preparation method and application thereof - Google Patents
Stabilizer-containing microsphere, preparation method and application thereof Download PDFInfo
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- CN114010805B CN114010805B CN202111191290.9A CN202111191290A CN114010805B CN 114010805 B CN114010805 B CN 114010805B CN 202111191290 A CN202111191290 A CN 202111191290A CN 114010805 B CN114010805 B CN 114010805B
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
- A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres
Abstract
The invention provides a microsphere containing a stabilizer, which is suitable for wrapping a medicament or inert gas, and comprises a microsphere carrier material, an osmotic pressure regulator, a stabilizer, a medicament or inert gas, wherein the stabilizer comprises a mixture of mannitol, chitosan and polymethyl methacrylate, the mass volume percentage (%, w/v) of the mixture is respectively 2% -3%, 0.2% -1.5% and 0.2% -1.5%, and the stabilizer is added, so that the problems of common easy layering and extremely non-uniform microsphere concentration in a microsphere suspension can be solved, the stability of the microsphere is improved, and the microsphere is not easy to break, thereby improving the stability of a microsphere preparation and being suitable for industrial production of the microsphere.
Description
Technical Field
The invention belongs to the field of medicine and chemistry, and particularly relates to a stabilizer-containing microsphere, a preparation method and application thereof.
Background
The microspheres are spherical entities with the particle size of several microns to several hundred microns, which are prepared by coating or dispersing medicines and the like in a biodegradable material matrix. Has the advantages of reducing administration frequency, improving compliance, reducing toxic and side effects, improving curative effect, reducing blood concentration fluctuation, improving medication safety and the like, and is widely applied to sustained and controlled release injections, implants and the like. For example, those that are currently on the market
(leuprorelin microspheres for injection), Sandostatin
(octreotide acetate microspheres for injection) and the like.
One type of microspheres can be loaded with inert gas and is mainly appliedIn the field of ultrasonic contrast, the characteristic that liquid containing bubbles has strong scattering on ultrasonic waves is utilized, and an ultrasonic contrast agent is clinically injected into a human blood vessel to enhance an ultrasonic Doppler signal of blood flow and improve the definition and resolution of an ultrasonic image. At present, the second generation ultrasonic contrast agent is a thin and soft bubble with an outer membrane mainly wrapping high-density inert gas, and has long stabilization time and good vibration and echo characteristics. Inert gases commonly used, e.g. already on the market
Perfluoropropane (perfluoropropane human serum albumin microspheres for injection),
(sulfur hexafluoride microbubbles for injection), and the like.
Microspheres, especially gas microsphere products (microbubbles), are similar to the structure of a sphere wall, gas in the sphere and solution, and the solution can wait for subsequent preparation to be a freeze-dried powder injection after production and configuration, and then the freeze-dried powder injection can be re-dissolved into suspension for injection into a human body when in use. The problem relates to a long-standing quality problem, namely that the stability of microspheres in a microbubble suspension (preparation tank) in industrial production is poor, firstly, the solution is layered (generally divided into an upper layer, a middle layer and a lower layer, the concentration of the microspheres obtained is different according to different sampling positions), and secondly, the microspheres in the solution are cracked (the microspheres are cracked after being placed at room temperature for 10 min), so that the quality stability of the solution of the microspheres is further reduced.
In order to avoid the problem of instability of the gas microspheres, the continuous production mode described in patent CN110101878A is generally adopted in the prior art, that is, the gas microspheres are not allowed to stand, and the microspheres are prepared and immediately filled, but the problems of high linkage requirement, low capacity, high loss, low yield and the like of the whole production line exist. Patent CN101130094A describes another way, microspheres are left standing overnight, after unstable microspheres disappear and layering is stable, the microspheres are remixed for filling, but the process is complicated and time-consuming, and the concentration and uniformity of the mixed product cannot be guaranteed, which is not suitable for industrialization.
The two schemes have the defects of long time consumption, material waste and the like, and the problem of microsphere stability cannot be fundamentally solved, namely, after the microspheres are prepared, the microsphere solution does not delaminate within a certain time, such as 2 hours (the time is enough to ensure the completion of the subsequent filling process), and the microsphere breakage rate is very low.
Therefore, when microspheres are prepared, the formula of the microspheres is adjusted, and after the inert gas is blended, a stable microsphere suspension is obtained, so that the stability of the solution is maintained.
Disclosure of Invention
In order to solve the technical problem, the invention provides a stabilizer-containing microsphere which comprises a solution and a fluoride inert gas, wherein the solution consists of the following components in percentage by mass (percent, w/v):
2% -10% of microsphere carrier material, wherein the microsphere carrier material is one or a mixture of silk fibroin, zein and albumin;
2% -10% of a stabilizer, wherein the stabilizer is a mixture of mannitol, chitosan and polymethyl methacrylate;
0.2-20% of osmotic pressure regulator, wherein the osmotic pressure regulator is one or a mixture of sodium chloride and glucose;
the balance of water;
the fluoride inert gas is dispersed in the solution in a ratio of 40-140 g: 1000ml, the fluoride inert gas is one or a mixture of sulfur hexafluoride, perfluoropropane and perfluorobutane.
Because the product is usually an injection, the injection amount of a human body is small (about 5 ml), so that the osmotic pressure is relatively loose to adjust, and the design can float up and down in an isotonic range and even can be higher than an isotonic condition.
In the present invention, the components are all w/v, and one or more of sodium chloride and glucose can be selected according to the osmotic pressure regulator, wherein the concentration of sodium chloride is tend to be equal to that of physiological saline, while the concentration of glucose is also tend to be an isotonic solution, usually 5% w/v, and more than 5% can be used in special cases, and the concentration of glucose injection at present comprises 5%, 10%, 20%, and 50% (w/v), therefore, the glucose concentration is recommended to be 5% -20%, and more preferably 5% -10%.
In the invention, the concentration of sodium chloride should be considered for screening, and since the concentration of the normal saline is 0.9%, according to the osmotic pressure research, the concentration range of NaCl of the injection is preferably 0.5% -1% (w/v) considering the concentration of other components, and the concentration belongs to the physiologically acceptable range.
The iso-osmosis is 285-310 mOsmol/kg according to the Chinese pharmacopoeia, and the iso-osmosis meets the following regulations except other regulations: 260-500 mOsmol/kg of intravenous injection, wherein the osmotic pressure of the prescription meets the requirements of pharmacopoeia on intravenous injection administration injection under the condition of high concentration of each component.
The inert gas is mixed at a ratio of 40-140 g: the proportion of 1L is dispersed in the liquid, which is enough to ensure that the solution obtains enough inert gas, thereby generating the microbubble suspension which meets the standard, and the excessive proportion causes the waste of the inert gas.
In the formula, the content of each component is in a normal dosage, even lower than the normal dosage of the component, and the osmotic pressure measurement is carried out, so that the injection belongs to the physiologically acceptable range in the field of the current injection.
In addition, the microsphere carrier material is adjusted to 2-10%, which is beneficial to better carrying gas, and the reduction of the concentration of the microsphere carrier material can reduce the gas carrying capacity.
Further, the microsphere comprises a solution and a fluoride inert gas, wherein the solution consists of the following components in percentage by mass (percent, w/v):
2% -8% of microsphere carrier material, wherein the microsphere carrier material is one or a mixture of silk fibroin, zein and albumin;
2% -6% of a stabilizer, wherein the stabilizer is a mixture of mannitol, chitosan and polymethyl methacrylate;
0.5% -15% of osmotic pressure regulator, wherein the osmotic pressure regulator is a mixture of sodium chloride and glucose;
the balance of water;
the fluoride inert gas is dispersed in the solution in a ratio of 40-140 g: 1000 ml.
Further, the microsphere comprises a solution and a fluoride inert gas, wherein the solution consists of the following components in percentage by mass (percent, w/v):
2% -5% of microsphere carrier material, wherein the microsphere carrier material is one or a mixture of silk fibroin, zein and albumin;
2% -5% of a stabilizer, wherein the stabilizer is a mixture of mannitol, chitosan and polymethyl methacrylate;
the sodium chloride accounts for 0.5 to 1 percent;
the glucose is 5% -10%;
the balance of water;
the fluoride inert gas is dispersed in the solution, and the proportion is 40-140 g: 1000 ml.
Further, provided is a microsphere, which comprises the following components in percentage by mass volume (%, w/w):
human serum albumin 4%; 10% of glucose; 0.54% of sodium chloride; 2.2% of mannitol; chitosan 0.5% (molecular weight 19000 daltons); polymethyl methacrylate 0.5% (molecular weight 15000 daltons); water is adjusted to 100% to give a solution, fluoride inert gas is added at a rate of 40-140 g: adding the solution according to the proportion of 1L to obtain the suspension of the microspheres.
In the present invention, a general technical problem in the art is solved, and the stability of the gas-mixed microsphere suspension during the preparation process greatly affects the product quality and the use.
According to the design concept in the field, it is generally better to have fewer components, therefore, in the research and development process of the applicant, the existing process adopts experimental example 1, a conventional formula, wherein the carrier protein is human serum albumin, and glucose and sodium chloride are added simultaneously, so as to simulate the environment of human isotonic solution, when perfluoropropane is added for carrying gas, a long-term neglected problem is found, namely, the obtained microbubbles are particularly fragile, for example, broken, or fused among microbubbles, the breaking is quickly caused after room temperature prevention, the concentration of the microspheres is also reduced, layering occurs, the concentration of the microspheres RSD of the upper layer, the middle layer and the lower layer reaches 20-80%, and when the difference of the concentration of the microspheres of the upper layer, the middle layer and the lower layer is increased, stable subpackaging cannot be directly caused, which is not allowed by the quality requirement after filling. Wherein the microsphere solution causes the particle size of the microspheres to increase along with the change of the particle size of the microspheres.
In order to solve the technical problems, the invention adopts a higher standard quality control system, wherein the concentration and the diameter of the microspheres are as follows: the concentration of microspheres is not less than 1 × 109Per mL (microsphere concentration of the existing commercial products is usually 0.8X 10)9Less than/mL); the average microsphere diameter should be 2.0-5.0. mu.m, preferably 2.5-4.0. mu.m, more preferably 2.50-3.50. mu.m.
On one hand, the average inner diameter of the human capillary is less than 8 μm, and the microspheres with overlarge particle sizes cannot freely move in the capillary to interfere the blood flow; on the other hand, if the particle size of the microspheres is too small, the ultrasonic reflection capability is poor, and the ultrasonic image is not clear, so that the particle size of the microspheres should be controlled within a certain range.
Due to various reasons affecting the breakage of the microspheres, it is uncertain what is the reason, probably due to the toughness, pressure and the like of the microspheres. The inventors need to try to improve this problem by adding a stabilizer material with safety. Through a series of screening, the inventor discovers a special stabilizer, and the stabilizer is a combination of mannitol, chitosan and polymethyl methacrylate, and the addition of the stabilizer is unexpectedly found to improve the toughness of a microsphere wall material, keep the microsphere shape, improve the viscosity of the microsphere and avoid the sedimentation of the microsphere. Preferably, in the microsphere solution (suspension), the ratio of mannitol: chitosan: the mass volume percentage of the polymethyl methacrylate is respectively 2-5 percent, 0.1-2 percent, preferably 2-3 percent, 0.2-1.5 percent and 0.2-1.5 percent. More preferably 2% to 3%, 0.2% to 1.0%, and still more preferably 2% to 3%: 0.5% -1.0%: 0.5 to 1.0 percent.
Wherein the molecular weight of the chitosan is 5000-40000 daltons, and the molecular weight of the polymethyl methacrylate is 4000-20000 daltons.
Further preferably, the molecular weight of the chitosan is 5000-20000 daltons, and the molecular weight of the polymethyl methacrylate is 4000-20000 daltons. Preferably, the molecular weight range of the polymethyl methacrylate is 4000-16000 daltons, more preferably, the molecular weight of the chitosan is 18000-20000 daltons, and the molecular weight range of the polymethyl methacrylate is 14000-16000 daltons.
In the test, the molecular weight of the chitosan and the polymethyl methacrylate is improved, and the quality of the obtained microbubbles is better.
Further, the microspheres are prepared into suspension, the suspension is placed for 120min at room temperature, the reduction of the concentration of the microspheres is less than or equal to 10 percent compared with the initial value, and the concentration of the microspheres is not less than 1 multiplied by 109Per mL; the particle size of the microspheres is 1.0-5.0 μm, preferably 2-3.5 μm, further 2.5-3.5 μm, and the concentration RSD of the microspheres sampled on the upper layer, the middle layer and the lower layer of the sample is less than or equal to 10%; the initial values are all parameters measured for the first time after the microspheres are stabilized from the sound vibration chamber, such as within 0-5 min.
The upper, middle and lower layers are subject to a subjective fuzzy definition, but exist objectively due to limitations in the height of industrial production tanks, such as 2-10m high production tanks, for example, the upper layer is located 1/5 from the top of the sample tank, the middle layer is located 1/2 from the top of the sample tank, and the lower layer is located 4/5 from the top of the sample tank, and this expression is by way of example only and can be imagined, understood and implemented by those skilled in the art and adjusted within suitable ranges.
The invention further provides a preparation method of the microsphere, wherein the preparation method comprises the following steps:
mixing and stirring a microsphere carrier material, glucose, an osmotic pressure regulator, a stabilizer and water to prepare a solution, and then mixing the solution and fluoride inert gas or a medicament in a sound vibration process to obtain the microsphere. The material is prepared by a sound vibration process (the sound vibration process belongs to the prior art in the field, for example, the invention adopts a sound vibration instrument (XL2020), American Heat System company, the sound vibration power is 30-80W, and the sound vibration temperature is 50-72 ℃).
Specifically, the microsphere carrier material, glucose, mannitol, sodium chloride, chitosan, polymethyl methacrylate and water are mixed and stirred at a low speed of 100-. The fluoride inert gas and the solution respectively and continuously enter the sound vibration chamber to prepare the microsphere suspension.
Further, the invention provides an application of the microsphere containing the stabilizing agent in preparing a contrast agent.
The microsphere suspension prepared is preferably bottled and freeze-dried to prepare freeze-dried powder, and when the microsphere suspension is used, a corresponding solution, such as an isotonic glucose solution or physiological saline, is used for redissolving (the change of related osmotic pressure is negligible), and then injection operation is performed, and the method belongs to the conventional operation in the field.
According to the invention, the prepared microsphere suspension is placed at room temperature for 120min, and the concentration of the microspheres is measured by a Coulter counter. Compared with the prior art, the invention provides the high-stability microspheres, mannitol, chitosan and polymethyl methacrylate are added, in fact, the situation that mannitol, chitosan (within a physiologically acceptable concentration range) and PMMA (within a physiologically acceptable range) are used alone cannot be improved, and even if the mannitol, chitosan and PMMA are combined, the effect is poor. Therefore, the three simultaneous adding conditions are few practices in the field, belong to unconventional practices, and simultaneously produce good effects,
the difference is that the three components of mannitol, chitosan and PMMA play different roles, and the specific action mechanism is not clear, and the three components have no suggestion and description of synergistic action due to less related researches in the field.
The technical effects achieved are as follows:
1. the applicant sets 120min at room temperature as a boundary point, and not only gives sufficient time for subsequent perfusion and subpackaging, but also gives sufficient time for the prepared microbubble suspension, so that the change of the microbubble suspension is facilitated, and the stability, the particle size and other parameters of the microbubble suspension are examined.
The microsphere suspension of the invention has good stability and no delamination phenomenon after being placed for 120min at room temperature. Compared with sampling at 5min, the microsphere concentration value has no obvious change (the change value is less than or equal to 10 percent and is as low as 3.3 percent at least, see example 1) at 120min, and the microsphere concentration is more than or equal to 1.0 multiplied by 109one/mL (the optimal prescription reaches 1.721 multiplied by 10)9See example 1), the microsphere concentration RSD of the upper, middle and lower layers is less than or equal to 10% (the optimal prescription reaches 6.2%, see example 1).
The concentration and the particle size of the microspheres ensure the stability of the gas carrying capacity, the change value is low, the quantity difference of the microspheres which are subpackaged into each bottle is very small, the stability of the microspheres provided by the invention is good, the batch production and the continuous filling process are favorably carried out, and the yield and the productivity of the microspheres are higher.
2. The raw materials have good biocompatibility and do not contain surfactant components which are easy to cause immediate-type anaphylactic reaction. The type and the proportion of the microsphere stabilizer are optimized, the concentration of the microsphere is obviously improved, and the stability and the effectiveness of the microsphere are ensured.
Detailed Description
The following preferred embodiments are merely illustrative of the technical solutions of the present invention and are not restrictive, and although the present invention has been described in detail with reference to the following preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the present invention as defined by the appended claims.
In the present invention, unless a solvent is specifically defined, all refer to an aqueous solution, and all refer to room temperature when the temperature is not defined.
Some examples of the present invention are described below, and the above-described contents of the present invention are further described in detail but it should not be understood that the scope of the above-described subject matter of the present invention is limited only to the following examples.
The stability of the microsphere preparation product can be improved by adding a stabilizer into a prescription reported by a document, and the stabilizer mainly adopted comprises the following components: saccharides, which enhance the stabilizer by promoting the formation of a hydration layer of the microspheres; the high molecular polymer improves the viscosity of the solution and avoids the sedimentation of the microspheres through the dispersion, suspension and emulsification effects, thereby enhancing the stability; the surfactant stabilizes the interface by inhibiting the aggregation of the microspheres, reducing the surface tension and increasing the electrostatic repulsion; the pH regulator slows down the degradation of the microspheres by regulating the external environment of the microspheres; amino acids, which enhance the stability of the microspheres by enhancing the hydrogen bond acting force; the low-concentration salt can form a complex with the microspheres, so that the secondary structure of the microspheres is more compact and stable, and the stability of the microspheres is enhanced. However, the test results show that the stability of the stabilizer to the fluoride gas microspheres is improved to a certain extent, and the stability problem cannot be completely solved, actually, the prior art does not give clear indications, and what kind of components can be used as a universal stabilizer, so that no clear guidelines exist, and the method belongs to the research blank in the field.
In view of this situation, the inventors of the present invention employed the basic recipe of example 1 (which is a commercially available product and has simple components) instead of the typical components, and tried screening, and selected the following screening methods:
in the technical document:
the used stabilizer screening ingredients illustrate that:
in the present invention, each component, such as (PMMA, chitosan) and the like, has only one common concentration for injection, such as 5-20% (w/v) of the common concentration for intravenous infusion of mannitol. The common concentration of chitosan for injection is 1.2% (w/v), LD50> 16g/kg (rat) and tween 80 is usually used in amounts below 2%, otherwise there is a risk of hemolysis. Other materials have no recommended dosage, therefore, in the invention, all component dosages comply with the parallel principle of the general screening method, but simultaneously the screening of different concentrations is compatible, and only the main experiment example is carried out due to excessive data.
As the inventor does not know, aiming at the plasma injection, particularly the kind of the stabilizer which should be used by the carrier gas material microbubble product, the screening of the representative additives is carried out, and the additives are divided into mannitol (sugar), PMMA (high molecular polymer, 4000-16000 Dalton), chitosan (sugar 5000-19000 Dalton), hyaluronic acid (high molecular polymer), Tween 80 (surfactant), sodium acetate (pH regulator), cysteine (amino acids), PVP k30 (high molecular polymer), and see experimental examples F1-F9.
Human serum albumin, oxtet amema pharmaceutical manufacturing ltd, for injection;
medical chitosan, Shanghai Bingsheng biological preparation Co., Ltd, 18000-20000 daltons (mainly 19000 daltons), 5000 daltons;
medical PMMA, French Dorkma, SG7, 14000-16000 daltons (mainly 15000 daltons); 4000 daltons, and other parameters, are readily available in the art.
Other ingredients belong to the medicinal ingredients commonly used in the art and can be obtained by those skilled in the art by referring to commercially available products.
The water in the invention refers to water for injection.
Referring to a prescription of a commercially available ultrasound contrast agent, a gas microsphere suspension is prepared as a control (F1, control group 1), representative saccharides, high molecular polymers, surfactants, pH regulators, amino acids and the like are individually used as stabilizers to be screened (see table 1), and referring to the literature and the results of compatibility experiments, the inventor of the previous experiment firstly screens the concentration of mannitol, which is 2-3% (w/v) and has an improvement effect on the stability of microspheres, and the effect is better, and 2-2.5% of the concentration is a preferred range, therefore, a parallel experiment is designed, and the same preparation method is adopted in each prescription, and the method comprises the following main steps: mixing and stirring a microsphere carrier material, an osmotic pressure regulator, a stabilizer and water to prepare a solution, then introducing the solution and fluoride inert gas into a sound vibration chamber together, and performing sound vibration preparation to obtain a microsphere suspension. The invention inspects three indexes of microsphere concentration, upper, middle and lower layers of RSD and microsphere grain diameter, the order of the evaluation indexes is that the upper, middle and lower layers of RSD is larger than the microsphere concentration change and larger than the microsphere grain diameter, namely, the stability and the uniformity are the first standard.
The stability of the microsphere suspension prepared in this example was evaluated by placing the microsphere suspension at room temperature (25 ℃) for 5min, 60min, and 120min at 1/5 (upper layer), 1/2 (middle layer), and 4/5 (lower layer) from the top of the sample tank, sampling at three points, measuring the concentration of microspheres using a coulter counter, and measuring the particle size of microspheres using a laser particle sizer.
TABLE 1 formulation of microspheres with Single component stabilizer composition
The results show that compared with the commercial microsphere preparation (formula F1), the mannitol (F2) has the best stabilizing effect in the stabilizer combination with a single component, can improve the stability of the microsphere concentration to a certain extent, partially relieves the delamination, and the microsphere concentration of a 60min sample is more than 1.0X 109one/mL, variation less than 10% (compared to 5 min), RSD less than 10%; the microsphere concentration of 120min sample is less than 1.0 × 109one/mL, variation greater than 10% (compared to 5 min), RSD greater than 20%. Still not meeting the requirements. The F3 chitosan group has very high reduction of microsphere concentration and RSD, and the F4 PMMA group has almost the same change trend of the microsphere concentration as F1. All groups had particle sizes slightly above 5 μm, highlighting that a single ingredient did not yield the desired microsphere product.
TABLE 2 stability data for microsphere formulations consisting of single component stabilizers
2. Experimental example of the combination of two different stabilizer Components
The preparation of microspheres is carried out according to the experimental example 1, on the basis of adding mannitol as a stabilizing agent, another stabilizing agent component is added to improve the stability of the microspheres, because the mannitol is added and the use concentration of other stabilizing agents does not have the guiding specification, the second stabilizing agent is selected to have low concentration (0.5 percent, and the selected stabilizing agents are all in low use concentration) to carry out parallel tests, and the sampling method and the stability evaluation method are the same as the experimental example 1.
TABLE 3 two component stabilizer composition microsphere formulation
The results show that when two components are used as stabilizers, the stability of the prepared microspheres is higher than that when mannitol is used alone. When the microspheres are placed at room temperature for 120min, the concentration RSD of the microspheres in the upper layer, the middle layer and the lower layer is more than 10 percent, the numerical value change of the concentration of the microspheres is more than 10 percent, and the requirement is still not met. In the two-component stabilizer combination, mannitol in combination with chitosan works best, and therefore the choice was further selected on the basis of this combination. Also the microsphere size was above the set preference criteria, 5 μm.
TABLE 4 stability data for microsphere formulations consisting of two component stabilizers
Under the same conditions, the second stable used higher concentration, 5% did not exist, and the corresponding data did not improve. The invention thus likewise does not achieve the desired effect with the use of two stabilizers.
3. Experimental example of combination of three stabilizer Components
Referring to Experimental example 1, microsphere preparation was carried out by adding another stabilizer component (starting at a low concentration of 0.5%) to increase the stability of the microspheres, based on the addition of mannitol-chitosan as a stabilizer. The sampling method and stability evaluation method were the same as in example 1.
TABLE 5 microsphere formulation consisting of three component stabilizers
The results showed that the combination of mannitol-chitosan-polymethylmethacrylate was 2.0: 0.5: when 0.5 is a stabilizer (F17), the content can be further improvedStability of the microspheres. When the mixture is placed at room temperature for 60min or 120min, the concentration RSD of the microspheres at the upper layer, the middle layer and the lower layer is less than 10 percent, the numerical value of the concentration of the microspheres is less than 10 percent, and the concentration of the microspheres is more than 1.0 multiplied by 109The particle/mL meets the requirement, and the particle size of the microsphere also meets the requirement.
TABLE 6 stability data for microsphere formulations consisting of three component stabilizers
According to the F18-22 prescription, Tween-80, sodium acetate buffer solution, cysteine, PVP30 and the like are added into other combinations, the microsphere concentration of the solution, the upper microsphere concentration, the middle microsphere concentration and the lower microsphere concentration RSD still do not meet the specification, so that only F17 in the combination of the three components meets the set standard and meets the production requirement.
4. Experimental examples of different proportions of the three stabilizer components
Further, the ratio between mannitol-chitosan-polymethylmethacrylate was screened to determine the formulation range of the stabilizer combination. The preparation of microspheres, the sampling method and the stability evaluation method were the same as those in example 1, with reference to example 1.
TABLE 7 composition of formula of microspheres with different proportions of three component stabilizers
The results show that the combination of mannitol: chitosan: polymethyl methacrylate 2-3%: 0.2-1.5%: 0.2-1.5%, the microsphere concentration RSD of the upper, middle and lower layers is less than 10%, the variation of the microsphere concentration value is less than 10%, and the microsphere concentration is more than 1.0 multiplied by 10% when the film is placed at room temperature for 60min or 120min9Per mL microsphere stability requirement. Preferably, when the prescription ratio is the same as F28, the microspheres are suspendedAfter the solution is placed for 120min, the concentration can still be maintained to be 1.584 multiplied by 109The concentration RSD value of the microspheres at the upper layer, the middle layer and the lower layer is 6.0 percent, the particle size of the microspheres is not obviously changed, the microspheres are far superior to the microspheres prepared according to the prior art (F1), and the concentration of the microspheres is 0.865 multiplied by 10 after the microspheres are placed for 120min9The microsphere has the preparation effect that the concentration RSD value of the microspheres at the upper layer, the middle layer and the lower layer is 71.8 percent.
Since F17-28, RSD value and microsphere concentration reduction value are not very different, so it is suitable for microsphere development, mannitol is safe concentration under 2% -3%, considering other component safety, chitosan concentration is recommended to be 0.5-1.0%, and polymethyl methacrylate is better from 0.5% -1.0%.
TABLE 8 stability data for microsphere formulations with three component stabilizers in different proportions
5. Examples of experiments with different support materials
The microspheres were prepared according to example 1 and the effect of the microsphere support material on stability was examined. The sampling method and stability evaluation method were the same as in example 1.
TABLE 9 composition of microsphere formulations for different support materials
The results show that the microsphere carrier material has no obvious influence on the stability of the microspheres. Preferably, the microsphere carrier material is 4% human serum albumin.
TABLE 10 stability data for microsphere formulations consisting of different support materials
6. Experimental examples of chitosan and polymethyl methacrylate with different molecular weights
The microspheres were prepared according to example 1, and the effect of chitosan and polymethyl methacrylate of different molecular weights on stability was examined. The sampling method and stability evaluation method were the same as in example 1.
TABLE 11 prescription of chitosan and polymethylmethacrylate microspheres of different molecular weights
The results show that the microspheres prepared from high molecular weight chitosan and polymethyl methacrylate have the best stability.
TABLE 12 stability data for different molecular weight chitosan and polymethylmethacrylate microsphere formulations
7. Examples of different fluoride inert gases or drugs
The preparation of microspheres was performed with reference to experimental example 1, and the effect of different fluoride-containing gases or drugs on stability was examined. The sampling method and stability evaluation method were the same as in example 1.
TABLE 13 microsphere formulations of different fluoride inert gases
The results show that different fluoride gases or drugs and their gas carrying capacity have no influence on the stability.
TABLE 14 stability data for microsphere formulations of different fluoride inert gases
Conclusion
As can be seen from the above examples, when no stabilizer is added, the prepared microspheres are extremely unstable, and the microspheres begin to be crushed after being placed at room temperature (25 ℃) for 10min, resulting in a significant decrease in the concentration of the microspheres by more than 10%; layering phenomenon occurs at the same time, and the concentration RSD of the microspheres at the upper layer, the middle layer and the lower layer is more than or equal to 10 percent; when one of mannitol, chitosan and polymethyl methacrylate is used as a stabilizer, the stability of the concentration of the microspheres can be improved to a certain extent, and the layering condition is partially relieved, but the requirements are still not met. On the basis, mannitol, chitosan and polymethyl methacrylate are preferably added in combination, so that the stability of the microspheres can be increased more remarkably. When the ratio of mannitol: chitosan: the proportion of polymethyl methacrylate is 2% -3%, 0.2% -1%, especially 2% -3%: 0.5-1.0%: when the content is 0.5-1.0 percent, the high-stability microspheres are obtained. The preferable formula carries fluorocarbon insoluble gases such as sulfur hexafluoride, perfluoropropane, perfluorobutane and the like, and when the preferable formula is placed for 120min at room temperature, the concentration value of the microspheres is not obviously changed (the change value is less than 10 percent) compared with the initial value, and the concentration of the microspheres is more than or equal to 1.0 x 109The microsphere concentration RSD of the upper, middle and lower layers is less than or equal to 10 percent.
The invention is suitable for microsphere ultrasonic contrast agents, is suitable for various inert gases and carrier materials, has good stabilizer, is suitable for temporary microsphere solution storage and standing in actual production, does not need to adopt continuous production with high specification requirements, and the produced product has high microsphere concentration, low particle size and uniform upper, middle and lower microsphere concentration distribution, ensures the quality stability of the subpackaged medicament and has excellent production significance.
Remarking:
mannitol is a saccharide and is commonly used in common lyophilized formulations as an excipient, but no document reports its use as a stabilizer in microsphere formulations; polymethyl methacrylate is rarely used as a wall material of microspheres, but is widely applied to the fields of cosmetics, painting and the like; the chitosan for injection is mainly used for repairing articular cartilage, and no report is provided for gas microbubbles. Also, mannitol, polymethylmethacrylate, or chitosan alone had no significant effect on microbubble stability. The inventor creatively combines the stabilizer chitosan and the polymethyl methacrylate, and the mannitol can greatly enhance the toughness of the microsphere wall material, keep the microsphere shape and be difficult to delaminate.
Security data:
in vitro hemolysis test is one of the evaluation indexes for checking the safety of intravenous injection preparation, taking example 1 as an example, a concentration gradient is set (number 1-5 test tubes are about 20 times of dosage for human body) according to the conversion of human body weight (60kg) and rabbit weight (1.5 kg). Therefore, 1 ear vein of the rabbit is taken to take blood, the blood is placed in a triangular conical bottle with glass beads, the same volume of physiological saline is added and the mixture is gently shaken, and the mixture is centrifuged at 2500rpm for 5min to remove pigment and protein, and the operation is repeated until the supernatant is colorless and transparent. Adding physiological saline to prepare 2% rabbit blood physiological saline (v/v) suspension. Taking 7 clean test tubes, numbering, adding corresponding volume of liquid as shown in table 1, adding microsphere suspension into No. 1-5 test tubes respectively, adding physiological saline into No. 6 test tubes and distilled water into No. 7 test tubes, shaking uniformly, placing the test tubes in a water bath at 37 ℃, observing the test tubes respectively, recording results of 0.5, 1, 2 and 3 hours, indicating hemolysis if the solution turns clear and red, and being unsuitable for intravenous injection if the microsphere suspension causes hemolysis within half an hour. No hemolysis was caused as a pass in 3 hours.
Judgment standard of hemolysis test result:
(1) total hemolysis: the solution is clear and red, and no red blood cells remain at the bottom of the tube;
(2) partial hemolysis: the solution is clear, red or brown, and a small amount of red blood cells remain at the bottom; microscopic examination of rare or deformed erythrocytes;
(3) non-hemolysis: the red blood cells are all sunk, and the supernatant is colorless and clear; the erythrocytes are not condensed by microscopic examination;
(4) erythrocyte agglutination: the solution has brown-red or red-brown flocculent precipitate, and does not disperse after shaking;
(5) when the negative control tube has no hemolysis or aggregation and the positive control tube has hemolysis, if the solution in the test tube has no hemolysis or aggregation within 3 hours, the test can be injected for use, and if the solution in the test tube has hemolysis or aggregation within 3 hours, the test can not be injected for use.
The experimental results are shown in table 1, and no hemolysis occurs when test tubes 1-5 are added with microsphere suspensions of different volumes within 3 hours, so that the microspheres have good safety and can be used for intravenous injection, and related hemolysis effect cannot be caused by prescriptions of other concentrations.
TABLE 1 in vitro hemolytic experiment
Note: tubes 1 to 5 were the test samples, tube 6 was the negative control tube, and tube 7 was the positive control tube (complete hemolysis control). Partial hemolysis is +; complete hemolysis was ++; is not hemolyzed to-.
All percentages in the examples, unless otherwise specified, refer to percent by mass volume (w/v).
Example 1
Human serum albumin 4% (oxter maima pharmaceutical products ltd, for injection); 10% of glucose; 0.54% of sodium chloride; 2.2% of mannitol; chitosan 0.5% (molecular weight 19000 daltons); polymethyl methacrylate 0.5% (molecular weight 15000 dalton), water was added and stirred to obtain a solution, perfluoropropane gas: the solution was 100 g: 1L, entering a sound vibration chamber, and carrying out sound vibration mixing to obtain the microsphere. The inert gas does not participate in the volume change of the solution and is dispersed in the liquid.
The configuration method comprises the following steps:
the preparation method comprises the steps of taking 40g of human serum albumin (solid raw materials or purchasing a high-concentration commercial albumin solution for injection, calculating to obtain the volume of the required solution), 100g of glucose, 5.4g of sodium chloride (solid or purchasing commercial normal saline, calculating to obtain the volume of the required solution), 22g of mannitol, 5g of chitosan and 5g of polymethacrylic acid, adding water to prepare 1L of solution, mixing and stirring at a low speed of 500r/min for 15min to obtain a physiologically acceptable injection solution, and filtering and collecting the solution through a 0.22 mu m two-stage sterilization filter. Perfluoropropane gas microspheres are prepared by adopting a sound vibration process. Perfluoropropane gas is filtered by a 0.22 mu m gas filter, 100g of perfluoropropane gas and solution respectively and continuously enter a sound vibration chamber, a sound vibration instrument (XL2020) is produced by Heat System company of America, the sound vibration power is 30-80W, and the sound vibration temperature is 50-72 ℃, so that microsphere suspension (microspheres) is obtained.
Example 2
Human serum albumin 4% (oxter maima pharmaceutical products ltd, for injection); 10% of glucose; 0.54% of sodium chloride; 2.2% of mannitol; chitosan 0.8% (molecular weight 19000 daltons); polymethyl methacrylate 0.2% (molecular weight 15000 dalton), water was added and stirred to obtain a solution, perfluoropropane gas: the solution was 100 g: 1L of the compound.
The preparation method is the same as example 1.
Example 3
Human serum albumin 4% (oxter maima pharmaceutical products ltd, for injection); 10% of glucose; 0.54% of sodium chloride; 2.2% of mannitol; chitosan 0.8% (molecular weight 5000 daltons); polymethyl methacrylate 0.2% (molecular weight 4000 dalton), water was added and stirred to obtain a solution, perfluoropropane gas: the solution was 100 g: 1L, water was added.
The preparation method is the same as example 1.
Example 4
Human serum albumin 4% (oxter maima pharmaceutical products ltd, for injection); 10% of glucose; 0.54% of sodium chloride; 2.2% of mannitol; chitosan 0.2% (molecular weight 19000 daltons); polymethyl methacrylate 0.8% (molecular weight 15000 dalton), water was added and stirred to obtain a solution, perfluoropropane gas: the solution was 100 g: 1L of the compound.
The preparation method is the same as example 1.
Example 5
Human serum albumin 4% (oxter maima pharmaceutical products ltd, for injection); 10% of glucose; 0.54% of sodium chloride; 2.2% of mannitol; chitosan 0.2% (molecular weight 5000 daltons); polymethyl methacrylate 0.8% (molecular weight 4000 dalton), water was added and stirred to obtain a solution, perfluoropropane gas: the solution was 100 g: 1L of the compound.
The preparation method is the same as example 1.
Example 6
Human serum albumin 4% (oxter maima pharmaceutical products ltd, for injection); 10% of glucose; 0.54% of sodium chloride; 2.2% of mannitol; chitosan 0.5% (molecular weight 19000 daltons); polymethyl methacrylate 0.5% (molecular weight 15000 dalton), water was added and stirred to obtain a solution, sulfur hexafluoride gas: the solution was 100 g: 1L, water is added. The inert gas does not participate in the volume change of the solution and can be dispersed in the liquid.
The preparation method is the same as example 1.
Example 7
Fibroin albumin 4% (sumamett biotechnology limited, low endotoxin); 10% of glucose; 0.54% of sodium chloride; 2.2% of mannitol; chitosan 0.5% (molecular weight 19000 daltons); polymethyl methacrylate 0.5% (molecular weight 15000 dalton), water was added and stirred to obtain a solution, perfluoropropane gas: the solution was 100 g: 1L of the compound. The inert gas does not participate in the volume change of the solution and can be dispersed in the liquid.
The preparation method is the same as example 1.
Claims (9)
1. A stabilizer-containing microsphere, characterized in that it consists of a solution and a fluoride-inert gas, the solution consisting of the following components in mass volume percent (w/v):
2% -10% of microsphere carrier material, wherein the microsphere carrier material is one or a mixture of silk fibroin, zein and albumin;
2% -10% of a stabilizer, wherein the stabilizer is a mixture of mannitol, chitosan and polymethyl methacrylate; the mannitol: chitosan: the mass volume percentage of the polymethyl methacrylate is respectively 2-5 percent, 0.1-2 percent and 0.1-2 percent; the molecular weight of the chitosan is 5000-20000 daltons, and the molecular weight range of the polymethyl methacrylate is 4000-20000 daltons;
0.2-20% of osmotic pressure regulator, wherein the osmotic pressure regulator is a mixture of sodium chloride and glucose;
the balance of water;
the fluoride inert gas is dispersed in the solution in a ratio of 40-140 g: 1000ml, the fluoride inert gas is one or a mixture of sulfur hexafluoride, perfluoropropane and perfluorobutane.
2. The microsphere of claim 1, wherein the microsphere consists of a solution and a fluoride inert gas, and the solution consists of the following components in percentage by mass volume (w/v):
2% -8% of microsphere carrier material, wherein the microsphere carrier material is one or a mixture of silk fibroin, zein and albumin;
2% -6% of a stabilizer, wherein the stabilizer is a mixture of mannitol, chitosan and polymethyl methacrylate;
0.5-15% of osmotic pressure regulator, wherein the osmotic pressure regulator is a mixture of sodium chloride and glucose;
the balance of water;
the fluoride inert gas is dispersed in the solution in a ratio of 40-140 g: 1000 ml.
3. The microsphere of claim 1, wherein the microsphere consists of a solution and a fluoride inert gas, and the solution consists of the following components in percentage by mass volume (w/v):
2% -5% of microsphere carrier material, wherein the microsphere carrier material is one or a mixture of silk fibroin, zein and albumin;
2% -5% of a stabilizer, wherein the stabilizer is a mixture of mannitol, chitosan and polymethyl methacrylate;
the sodium chloride is 0.5 to 1 percent;
the glucose is 5% -10%;
the balance of water;
the fluoride inert gas is dispersed in the solution in a ratio of 40-140 g: 1000 ml.
4. The microsphere of claim 1, wherein the ratio of mannitol: chitosan: the mass volume percentage of the polymethyl methacrylate is respectively 2-3 percent, 0.2-1.5 percent and 0.2-1.5 percent.
5. The microsphere of claim 4, wherein the ratio of mannitol: chitosan: the mass volume ratio of the polymethyl methacrylate is 2-3 percent, 0.2-1.0 percent and 0.2-1.0 percent respectively.
6. The microsphere of claim 1, wherein the molecular weight of the chitosan is 18000 to 20000 daltons, and the molecular weight of the polymethyl methacrylate is 14000 to 16000 daltons.
7. The microspheres of claim 1, wherein the microspheres are prepared as a suspension and allowed to stand at room temperature for 120min, the reduction in microsphere concentration is less than or equal to 10% and the microsphere concentration is not less than 1.0 x 109Per mL; the particle size of the microspheres is 2.0-5.0 μm, and the concentration RSD of the microspheres sampled on the upper layer, the middle layer and the lower layer of the sample is less than or equal to 10%.
8. The method for preparing microspheres according to claim 1, wherein the method comprises:
mixing and stirring a microsphere carrier material, an osmotic pressure regulator, a stabilizer and water to prepare a solution, then introducing the solution and fluoride inert gas into a sound vibration chamber together, and carrying out sound vibration preparation to obtain the microsphere.
9. Use of microspheres according to claim 1 for the preparation of ultrasound contrast agents.
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Citations (5)
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| US5733572A (en) * | 1989-12-22 | 1998-03-31 | Imarx Pharmaceutical Corp. | Gas and gaseous precursor filled microspheres as topical and subcutaneous delivery vehicles |
| CN101130094A (en) * | 2007-09-06 | 2008-02-27 | 上海新兴医药股份有限公司 | Ultrasonic contrast medium and its preparing process |
| WO2008118133A2 (en) * | 2006-09-26 | 2008-10-02 | Trustees Of Tufts College | Silk microspheres for encapsulation and controlled release |
| CN106389389A (en) * | 2016-11-25 | 2017-02-15 | 陕西品达石化有限公司 | Preparation method of clindamycin loaded chitosan sustained-release microspheres |
| CN110101878A (en) * | 2019-06-12 | 2019-08-09 | 力品药业（厦门）有限公司 | A kind of stable and uniform gas micro freeze-drying acoustic contrast agent and preparation method thereof |
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2021
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5733572A (en) * | 1989-12-22 | 1998-03-31 | Imarx Pharmaceutical Corp. | Gas and gaseous precursor filled microspheres as topical and subcutaneous delivery vehicles |
| WO2008118133A2 (en) * | 2006-09-26 | 2008-10-02 | Trustees Of Tufts College | Silk microspheres for encapsulation and controlled release |
| CN101130094A (en) * | 2007-09-06 | 2008-02-27 | 上海新兴医药股份有限公司 | Ultrasonic contrast medium and its preparing process |
| CN106389389A (en) * | 2016-11-25 | 2017-02-15 | 陕西品达石化有限公司 | Preparation method of clindamycin loaded chitosan sustained-release microspheres |
| CN110101878A (en) * | 2019-06-12 | 2019-08-09 | 力品药业（厦门）有限公司 | A kind of stable and uniform gas micro freeze-drying acoustic contrast agent and preparation method thereof |
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| 超声对比剂的材料学特点及临床应用;李鉴峰等;《中国组织工程研究》;20121118;第16卷(第47期);第8885-8892页 * |
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Effective date of registration: 20230809 Address after: 361000 building 6, 2010 wengjiao West Road (Xiamen biomedical industrial park), Haicang District, Xiamen City, Fujian Province Patentee after: Lipin Pharmaceutical (Xiamen) Co.,Ltd. Patentee after: LP PHARMACEUTICAL (XIAMEN) CO.,LTD. Address before: 361022 the whole building of building 6, No. 2010, wengjiao West Road, Haicang District, Xiamen City, Fujian Province Patentee before: Lipin Pharmaceutical (Xiamen) Co.,Ltd. |