CN106236773B - Water-soluble realgar solid dispersion and preparation method and application thereof - Google Patents
Water-soluble realgar solid dispersion and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a water-soluble realgar solid dispersion, a preparation method and application thereof, wherein the composition comprises 1 weight part of realgar and 1-20 weight parts of macromolecule; 0-5 parts by weight of a surfactant. The realgar solid dispersion provided by the invention solves the problem of low bioavailability caused by the fact that realgar is insoluble in water, effectively improves the dissolution rate and the accumulated dissolution rate of realgar in water, improves the bioavailability of realgar, and prolongs the blood arsenic circulation time, thereby enhancing the anti-leukemia drug effect. The preparation and processing process is simple, the production efficiency is high, and the preparation process of the solid dispersion can be directly linked with the subsequent crushing or shaping process. In clinical application, the dosage of realgar can be effectively reduced, and particularly in the intensive treatment period and the consolidation treatment period, the taking frequency and dosage can be reduced, so that the compliance of patients can be improved, the risk of health caused by toxic and side effects of long-term large-dosage administration can be reduced, and the treatment cost can be obviously reduced.
Description
Technical Field
The invention relates to the field of medicines, and in particular relates to a water-soluble realgar solid dispersion as well as a preparation method and application thereof.
Background
Realgar contains arsenic disulfide (As) As main ingredient2S2) The clinical use of realgar in traditional Chinese medicine is over two thousand years old. The Chinese patent medicines commonly used in clinic at present, such as bezoar detoxicating tablet, Angong bezoar pill, Liushen pill, Zijin pill, toad venom pill, etc., all contain realgar.
Research finds that the realgar applied to Chronic myelocytic Leukemia (also called Chronic myelocytic Leukemia, CML) can lead the complete remission rate of patients to reach 72 percent, and the complete remission rate of the early juvenile myelocytic Leukemia (APL) treated by the Realgar-containing formulated preparation compound Huangdai tablet (Leukemia tablet) reaches 98.3 percent and has no obvious bone marrow suppression phenomenon. Compared with the first-line chemotherapy drugs commonly used at present, such as Imatinib Mesylate (IM), all-trans retinoic acid (ATRA) and the like, the realgar has wider application range and better curative effect on APL and CML, particularly patients in accelerated phase (CML-AP) and terminal phase (CML-BP) of CML. Realgar can also be used for treating Acute Lymphoblastic Leukemia (ALL) with positive expression of medullary system, and has good patient tolerance, slight adverse reaction after long-term use, and arsenic trioxide (As)2O3) First-line drugs such as Imatinib Mesylate (IM), cytarabine and trans-retinoic acid have no cross-resistance.
However, the important problem faced by the clinical use of realgar is that the realgar is difficult to dissolve in water solution, is not only insoluble in water, but also insoluble in hydrochloric acid, so that the bioavailability is low, and the bioavailability of oral administration is only 4%, so that the clinical dose is large (about 330mg/d-1080 mg/d). In the traditional Chinese medicine processing method, the processing and preparation of realgar are mainly used for removing impurity minerals and reducing toxic impurity arsenic (As)2O3) For the main purpose, the bioavailability of realgar cannot be obviously improved. Therefore, the active ingredient As of the realgar is improved2S2The dissolution rate in water, thereby improving the bioavailability of the realgar and having very important significance for reducing the clinical dosage of the realgar and improving the curative effect of the realgar.
Realgar developed at presentThe technology of the new formulation mainly comprises the following steps: (1) solvent grafting method; (2) liposome encapsulation; (3) jet milling; (4) ball milling method. However, the processing methods have disadvantages. For example, the solvent bonding method has a problem of organic solvent residue; the liposome encapsulation method and the airflow pulverization method have low production efficiency and are not suitable for large-scale industrial production. The ball milling method is a preparation method of nanometer Realgar, and has related patent literature reports (publication No. CN1288723A), and the method can prepare nanometer Realgar with particle diameter of 1-100nm, and can improve antitumor activity to a certain extent, but the nanometer Realgar prepared by the method has highly toxic byproduct As2O3The content of the compound is obviously increased, the acute toxicity is obviously increased, and the medication safety is seriously influenced; in addition, the specific surface area is increased, the surface energy is increased, the stability of the nano realgar particles is poor, and oxidation (the oxidation product is As) can occur along with the prolonging of the storage time2O3) And agglomeration phenomena, thereby affecting As2S2Dissolution and bioavailability. Aiming at the problem that long-term storage of nano realgar can cause As2O3The content increase and particle agglomeration phenomena are caused, researchers mix the nanometer realgar with the macromolecule by melting through direct heating, or disperse the nanometer realgar into the macromolecule solution by using a solvent method, and prepare the solid dispersion through cooling or solvent volatilization, and the agglomeration and the oxidation in storage of the nanometer realgar are prevented by using the macromolecule material (patent application publication No. CN 1478486A). However, the process must prepare the realgar and then mix the realgar with the polymer, which cannot solve the problem of increase of toxic by-products in the preparation process of the realgar. In addition, the production of the method can not be completed in one step, and the nanometer realgar particles are only mixed in the polymer matrix by a common stirring process and exist in an agglomeration mode (Guotang. solid dispersion technology is used for researching the influence of the nanometer realgar on the stability and in-vitro dissolution [ J]Chinese traditional medicine J2013, 38(17): 2782-7).
Disclosure of Invention
The invention aims to provide a water-soluble realgar solid dispersion with obviously improved bioavailability aiming at the technical defects in the prior art, which is prepared from raw materials comprising 1 part by weight of realgar, 1-20 parts by weight of polymer and 0-5 parts by weight of surfactant.
The realgar is water-refined realgar with a particle size of 20-75 μm.
The polymer is selected from one or more of the following substances: polyvinyl alcohol polyethylene glycol Copolymer (Macrogol Poly (vinyl alcohol) Grafted Copolymer), polyvinylpyrrolidone (PVP), crosslinked polyvinylpyrrolidone (PVPP), polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft Copolymer (Soluplus), vinyl polyvinylpyrrolidone-vinyl acetate Copolymer (PVP-VA), and poloxamer.
The surfactant may be a nonionic surfactant or an ionic surfactant.
The surfactant is selected from one or more of the following substances: polysorbate 20, polysorbate 60, polysorbate 80, sodium lauryl sulfate, polyethylene glycol cetostearyl ether and sodium cetostearyl sulfate.
The active ingredient is As2S2The macromolecule is a dispersion matrix, the solid dispersion can be rapidly dissolved in an aqueous solution to form a stable orange yellow colloidal solution, wherein the hydration diameter range of realgar colloidal particles is between 100 and 1000nm, the average hydration diameter is 400 and 700nm, and As in the water-soluble realgar solid dispersion2S2The cumulative dissolution rate in the artificial gastric juice is 9 to 25 percent.
The invention also aims to provide a method for preparing the water-soluble realgar solid dispersion, which comprises the steps of mixing the raw materials of the water-soluble realgar solid dispersion, adding the mixture into a double-screw extruder for hot melt extrusion, and cooling the extrudate at room temperature to obtain the water-soluble realgar solid dispersion.
The temperature in the double-screw extruder is 50-200 ℃, and the screw rotating speed is 5-100 r/min.
The invention also aims to provide the water-soluble realgar solid dispersion prepared by the method, wherein the active ingredient is As2S2The polymer is a dispersion matrix, and the solid dispersion can be quickly dissolved in an aqueous solution to form stable orange yellow glueThe hydrated diameter of the realgar colloidal particles is between 100-1000 nm, the average hydrated diameter is 400-700nm, and As in the water-soluble realgar solid dispersion2S2The cumulative dissolution rate in the artificial gastric juice is 9 to 25 percent.
The invention also aims to provide application of the water-soluble realgar solid dispersion in preparation of drugs for treating acute and chronic myelogenous leukemia, wherein the leukemia is Acute Promyelocytic Leukemia (APL) and Chronic Myelogenous Leukemia (CML).
The realgar solid dispersion is a water-soluble solid dispersion, solves the problem of low bioavailability of realgar caused by the fact that the realgar is insoluble in water, effectively improves the dissolution speed and the cumulative dissolution of the realgar in water, improves the bioavailability of the realgar, prolongs the circulation time of blood arsenic, and enhances the anti-leukemia efficacy of the realgar.
In the preparation process, the polymer is used as a dispersing matrix, and optionally added with a surfactant, and the polymer and the water-refined realgar are processed by Hot Melt Extrusion (HME) to obtain the realgar solid dispersion. Compared with the traditional solid dispersion preparation technology (a melting method or a solvent method), the method provided by the invention has the advantage that the dissolution rate and the bioavailability of the insoluble drug realgar are obviously improved in the development of the insoluble drug realgar. In addition, the method for preparing the solid dispersion has high integration degree, the raw material realgar does not need to be pretreated and can be directly mixed with the polymer in the extruder, the processing process is simple, the production efficiency is high, and the preparation process of the solid dispersion can be directly linked with the subsequent processing process. The water-refined realgar used for preparing the solid dispersion is a medicament recorded in the first part of Chinese pharmacopoeia, the high polymer and the surfactant both accord with the standards of pharmaceutical excipients in the second part of Chinese pharmacopoeia, and the water solubility, the bioavailability and the anti-leukemia activity of the prepared realgar solid dispersion are all obviously improved, so that the realgar solid dispersion has industrial potential.
In clinical application, the realgar solid dispersion can be used as a new oral dosage form of realgar medicine, the dosage of realgar can be effectively reduced due to the improvement of bioavailability, particularly, the taking frequency and dosage can be reduced in an enhanced treatment period and a consolidated treatment period, the compliance of a patient can be improved, the risk of health caused by toxic and side effects of long-term large-dosage taking can be reduced, the treatment cost can be obviously reduced, the burden of the patient and a national medical insurance system can be reduced, and good social benefits can be generated.
Drawings
FIG. 1 is a comparative graph of water dispersion of Feishui Realgar and the product of example 1;
FIG. 2 shows As in simulated gastric fluid of Realgar, example 2, example 7 and example 102S2Cumulative dissolution profile of (a);
FIG. 3 is a graph showing the change of arsenic content in cells of Feihua and the product of example 4 after incubation with human chronic myelogenous leukemia cell line K562 cells for different periods of time;
FIG. 4 is an X-ray diffraction (XRD) spectrum of Realgar and the product of example 5;
FIG. 5 is a Scanning Electron Microscope (SEM) image of the cross-section of Feihua and the product of example 5;
FIG. 6 is a graph showing dynamic light scattering of particle size distribution of Soluplus and the product of example 6 in an aqueous phase;
FIG. 7 is a Scanning Electron Microscope (SEM) image of Feishui Realgar and Realgar particles in the aqueous dispersion of the product of example 6;
FIG. 8 is a graph showing the variation of arsenic concentration in blood in rats within 72 hours after oral administration of Realgar and the product of example 8;
FIG. 9 is a graph showing the effect of Feihua and the product of example 9 on the proliferation of human chronic myelogenous leukemia cell line K562 cells at different incubation times;
FIG. 10 is a graph showing the effect of different incubation times of Feihua and the product of example 9 on the proliferation of human acute promyelocytic leukemia cell line HL-60.
Detailed Description
The Hot Melt Extrusion (HME) technology was first applied to the plastic processing industry as a new Solid dispersion technology (Solid Dispersions), and was introduced into the field of drug development in the last 80 th century, based on the principle that a drug and a polymer as a dispersion matrix are added into an extruder, and drug particles are crushed and cut down by a huge shearing force generated when a screw in the extruder rotates, so that a drug Solid dispersion is obtained. Compared with the traditional solid dispersion preparation method, the HME technology has the following advantages: (1) organic solvent is not used in the preparation process, so that the problem of organic solvent residue in the final product is avoided; (2) the preparation process is directly linked with the downstream processing process (such as crushing or forming), the integral operation degree is high, the production efficiency is high, the reproducibility is good, the economy is high, and the product quality is easy to control; (3) in the preparation process, the medicine is more uniformly and tightly dispersed in the polymer matrix through the shearing and extrusion effects provided by the rotation of the screw; (4) the macromolecule can protect the effective components of the medicine from being oxidized in the preparation process.
The water-soluble realgar solid dispersion of the invention takes realgar which is a traditional Chinese medicine raw material as an active ingredient, takes amphiphilic macromolecules as a dispersion matrix, and has the composition of 1 weight part of water-ground realgar (namely realgar which can be directly used in the clinical practice of traditional Chinese medicine and has the particle size of 20-75 mu m); 1-20 parts by weight of a polymer as a dispersion matrix; 0-5 parts by weight of a surfactant.
The polymer used as the dispersion matrix comprises one or more of the following polymers: polyvinyl alcohol polyethylene glycol Copolymer (Macrogol Poly (vinyl alcohol) Grafted Copolymer, Kollicoat IR), polyvinylpyrrolidone (PVP, Kollidon), cross-linked polyvinylpyrrolidone (PVPP, Kollidon CL), vinyl-polyvinylpyrrolidone-vinylacetate Copolymer (PVP-VA, Kollidon VA), polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft Copolymer (Soluplus), poloxamer.
The surfactant can be a nonionic surfactant or an ionic surfactant, and specifically can be any one of the following compounds: polysorbate 20, polysorbate 60, polysorbate 80, sodium dodecyl sulfate, polyethylene glycol cetostearyl ether and sodium cetostearyl sulfate.
According to the existing disclosed solid dispersion, no technical report aiming at improving the water solubility and the water accumulated dissolution of the water-refined realgar by directly taking the traditional Chinese medicine raw material namely the water-refined realgar as a raw material or an active ingredient so as to improve the bioavailability and the drug effect of the water-refined realgar is available.
The invention relates to a method for preparing water-soluble realgar solid dispersion, which comprises the steps of adding traditional Chinese medicine raw materials of water-flying realgar and amphiphilic macromolecules into a double-screw extruder (Haake MiniLab II (Thermo Fisher Scientific, Karlsruhe, Germany) for hot melt extrusion, converting the water-flying realgar from large crystal particles into small microcrystal particles through shearing force and friction force generated by a screw in the mixing process, and uniformly dispersing the small microcrystal particles in macromolecules to obtain the realgar solid dispersion, wherein after the realgar solid dispersion prepared by the method is subjected to water, realgar particles in the dispersion can be dissolved out along with the dissolution of the macromolecules to form realgar particles coated by the macromolecules in the water, so that the prepared water-soluble realgar solid dispersion can be uniformly and stably dispersed in an aqueous phase, therefore, the dissolution and bioavailability of the realgar in the aqueous phase are greatly improved, and the leukemia and solid tumor resistant treatment effects are improved, the method directly uses the realgar raw material, does not need to carry out nano processing on the realgar in advance, and does not generate highly toxic by-products generated in the nano processing process.
The present invention will be described more specifically and further illustrated with reference to the accompanying drawings and specific examples, which are not intended to limit the invention in any way. Any variations of the embodiments of the present invention that would occur to one skilled in the art and which are made in the light of the present disclosure will be within the scope of the present invention.
Examples
The water-soluble realgar solid dispersions of examples 1-10 were prepared by the following preparation method, only adjusting the composition of the raw materials and the preparation process parameters, specifically adjusting the composition and the preparation process parameters as shown in table 1 (mentioned in table 1). The preparation method comprises the following steps: weighing a certain weight of fluvial realgar and macromolecules (and surfactant), firstly stirring and mixing the components in a container to obtain a material, and then adding the material into an extruder. The temperature in the extruder is set to be 50-200 ℃, and the rotating speed of the screw is 5-100 r/min. Extruding the materials through a die hole of a machine head, and cooling at room temperature to obtain an orange solid product.
Table 1 raw material composition and preparation process parameters of the water-soluble realgar solid dispersion of the present invention
Examples of the experiments
The comparative example 1 in the experimental example is bulk drug of water-refined realgar.
The experimental procedures and data of some examples are listed in the following experimental examples, and the same experiment is performed in other examples, and the experimental results are the same as those of the listed examples.
The first experimental example: evaluation of Water solubility
32mg of the product of example 1 and 8mg of the powder of comparative example 1 (both containing As) were weighed out2S2The content is 8mg), respectively placing the red orpiment and the red orpiment in test tubes, adding 8mL of pure water, and carrying out ultrasonic treatment for 10min to obtain the water dispersion liquid of the example 1 and the water dispersion liquid of the comparative example 1, as shown in figure 1, after the product of the example 1 is subjected to ultrasonic treatment, the water dispersion liquid is orange yellow, which shows that a large amount of realgar in the product can be dissolved out and enter a water phase to form a stable water dispersion liquid; after the powder of comparative example 1 was sonicated in water, the realgar still precipitated at the bottom of the tube (indicated by the white arrow), and the water phase was still colorless and transparent, indicating that the realgar in comparative example 1 was difficult to enter the water phase.
The experimental results show that the product of example 1 can promote the dissolution and release of realgar in water, and can effectively improve the dissolution of realgar in water.
The water solubility experiments for all the examples in table 1 were substantially similar and gave orange yellow aqueous dispersions, which are not described in detail.
Experiment example two: as2S2Cumulative dissolution in simulated gastric fluid
100mg of the product of example 2, 200mg of the product of example 7, 80mg of the product of example 10 and 20mg of the powder of comparative example 1 (As contained in the above sample) were weighed out separately2S220mg) according to the method for determining dissolution rate by the small cup method in appendix III of the first part of Chinese pharmacopoeia (test condition of 37 ℃ water bath, stirring paddle rotation speed of 100r/min, dissolution medium of artificialThe gastric juice is prepared by a method which is self-prepared according to the method of appendix X A of the second part of Chinese pharmacopoeia), 2mL of liquid is absorbed (supplemented by 2mL of artificial gastric juice) at 5, 10, 20, 30, 45, 60, 90 and 120min after the drug administration, and the volume is fixed to 50mL by 2% dilute nitric acid after microwave digestion. Arsenic content was determined using an iCAP6300 inductively coupled plasma emission spectrometer. The results show that the powders of example 2, example 7, example 10 and comparative example 1 are As in artificial gastric juice2S2The cumulative elution amounts were 25%, 16%, 9% and 0.2%, respectively, and the results are shown in FIG. 2. Compared with the same As2S2Comparative example 1, product of each example As in simulated gastric fluid2S2The cumulative elution amount was greatly increased, wherein example 2 was increased 205 times as compared with comparative example 1.
As of the present invention2S2The accumulative dissolution amount in water is obviously superior to the reported method (Guotang. solid dispersion technology is used for researching the influence of the stability and in-vitro dissolution of the nano realgar [ J]The Chinese traditional medicine journal, 2013,38(17): 2782-7), shows that the realgar water-soluble solid dispersion prepared by HME technology has incomparable advantages and outstanding effects of other methods in the aspect of improving the dissolution amount of realgar in water.
The results of the simulated gastric fluid cumulative dissolution test in all the examples in Table 1 are substantially similar and will not be described in detail.
Experiment example three: determination of total arsenic content in K562 cells
The K562 cells were cultured in RPMI-1640 medium (Thermo corporation, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) at 37 ℃ in 5% CO2Culturing under the condition. Collecting logarithmic growth phase K562 cells by centrifugation, suspending K562 cells with medium, counting, and collecting 2.5 × 10 cells5cells/mL K562 cells were seeded in cell culture dishes at 12mL each. 40mg of the product of example 4 and 20mg of the powder of comparative example 1 (containing As) were each weighed out2S220mg each) was dissolved in 3mL of physiological saline to form an aqueous dispersion, and a certain amount of the aqueous dispersion was added to the medium to make As in the medium2S2The final concentration reached 20.8mg/L, after incubation for 6, 12, 24, 48 and 72h, the plates were incubatedCells were resuspended, centrifuged and harvested and after cell lysis the As content was determined using XSERIES model 2 ICP-MS.
As shown in fig. 3, the results show that realgar entering the aqueous phase is taken up by K562 cells. With the same As2S2Compared with the comparative example 1, the realgar in the product water dispersion liquid of the example 4 can be greatly absorbed by K562 cells, the peak value is reached in 12 hours, then the peak value is gradually reduced to 48 hours, and the content of As in the cells reaches a steady state; in contrast, in comparative example 1, realgar was difficult to be taken up by cells because realgar was difficult to enter the aqueous phase.
Experimental example four: XRD analysis of realgar crystals in water-soluble realgar solid dispersion
XRD measurements were carried out on a D8Foucs type diffractometer equipped with a Cu target X-ray tube at a tube pressure of 40kV, a tube flow of 40mA and a scanning range of 2 θ from 10 ° to 90 °, and XRD patterns of the product of example and the powder of comparative example 1 were determined, respectively, as represented by the pattern of example 5, and are shown in fig. 4.
XRD test results show that the XRD pattern of the powder of comparative example 1 shows typical As2S2The diffraction pattern of the crystal has a stable baseline, which indicates that the sample is a crystal material. In the XRD pattern of the product of example 5, As2S2The crystal diffraction peak shows the phenomena of reduced intensity, broadened intensity and missing of a plurality of crystal diffraction peaks, the base line of the range of 10 degrees to 15 degrees is raised, and obvious amorphous diffuse reflection peaks appear, which shows that the crystal structure part of the realgar particles in the embodiment is damaged, and the grain size is obviously reduced.
Experimental example five: analysis of dispersion state of Realgar particles in Water-soluble Realgar solid Dispersion
SEM test is carried out on a Quanta 200F field emission environment scanning electron microscope, wherein products of the examples are broken off at room temperature, and the cross section is subjected to gold spraying treatment; comparative example 1 powder was ultrasonically dispersed in water and dropped on a silicon wafer to be dried, and after the two test materials were subjected to surface gold spraying treatment, the surfaces thereof were scanned at an accelerating voltage of 15kV and photographed, as represented by the photograph of example 5, as shown in fig. 5.
SEM test results show that the powder of comparative example 1 has a large particle size, and the particle size ranges from 25 to 75 μm. The cross section of the product of example 5 is smooth and flat, and small realgar particles (shown by white arrows in fig. 5) dispersed in the polymer matrix can be seen sporadically, which indicates that the large crystal particles of the fludared realgar can be crushed and uniformly dispersed in the polymer matrix by the HEM method to obtain a realgar solid dispersion.
Experimental example six: DLS analysis of particle size distribution of colloidal particles of Realgar in aqueous dispersion of water-soluble Realgar solid dispersion
Experiment on the particle size distribution of realgar colloidal particles in solid dispersion: the product of example 1, the product of example 2, the product of example 3, the product of example 4, the product of example 6, the product of example 8 and the product of example 10 were weighed out separately in amounts of 8mg, 10mg, 26mg, 4mg, 30mg, 32mg and 6mg (As in the above-mentioned sample)2S2Same content), dissolved in 3mL of physiological saline. 1mL of the dispersion was pipetted into a cuvette and tested using a Nano ZS90 nanometer size Analyzer, the results of which are shown in Table 2.
Taking the product of example 6 as an example, DLS was used to analyze the particle distribution of realgar solid dispersion in different dissolution media: 30mg of the product of example 6 was weighed and dissolved in 3mL of dissolution medium (physiological saline, artificial gastric juice (prepared by the method of X A in appendix of the second part of the Chinese pharmacopoeia) or artificial intestinal juice (prepared by the method of the same as the artificial gastric juice)). 1mL of the above dispersion was pipetted into a cuvette and tested using a Nano ZS90 nanometer size Analyzer, the results of which are shown in FIG. 6.
TABLE 2 mean hydrated diameter of Realgar colloidal particles in aqueous dispersion of Water-soluble Realgar solid Dispersion
| Examples | Average hydrated diameter (nm) of Realgar particles |
| Example 1 | 657.1 |
| Example 2 | 546.0 |
| Example 3 | 446.1 |
| Example 4 | 689.9 |
| Example 6 | 508.0 |
| Example 8 | 400.0 |
| Example 10 | 601.0 |
The above results show that the realgar in the products of the examples can form colloidal particles with macromolecules to be stably dispersed, and the particles in the dispersion liquid have three size distribution peaks, taking example 6 as an example, wherein peak 1 is a micelle formed by the macromolecules in the solution; the peak 2 is formed by polymer coated realgar particles formed in aqueous dispersion, the hydration diameter range is between 100 and 1000nm, the average hydration diameter is 400 and 700nm, and the realgar particles are normally distributed; peak 3 is the peak of the distribution formed by a very small number of realgar particles with a diameter greater than 1 μm in the solution.
Comparing the test results of the product of example 6 in different dissolution media, it can be seen that the relative amount of the polymer coated realgar particles formed in the artificial gastric juice by the product of example 6 is the largest, and then in the artificial intestinal juice and the physiological saline, which indicates that the realgar contained in the product of example 6 can be rapidly dissolved in the physiological dissolution media, especially under the acidic condition in the stomach, the dissolution speed is the fastest. Comparative example 1 the powder was not stably dispersed in an aqueous solution and could not be detected by DLS method, so the DLS experimental data of comparative example 1 could not be provided.
Example seven: SEM analysis of Realgar colloidal particles in aqueous Dispersion of Water-soluble Realgar solid Dispersion
SEM analysis experiment: 30.0mg of the product of example and 2mg of the powder of comparative example 1 (As in the above sample) were weighed out2S2The content is 2mg), respectively dissolved in 3mL of physiological saline. Taking the water dispersion 10 of the two medicines5Centrifuging for 4h at r/min, precipitating Realgar particles in water phase, dripping on silicon wafer, air drying, spraying gold on surface, and performing SEM analysis (the SEM analysis uses the same electron microscope model and test conditions as those in experiment example five); the results are shown in FIG. 7, which is representative of the example 6 picture. The SEM results show that the particle size of the realgar particles dissolved in water by the product of example 6 is significantly smaller than that of comparative example 1, indicating that the HME process of the present invention can effectively reduce the particle size of the realgar particles to submicron level.
Experimental example eight: bioavailability assay for single oral administration in rats
SD rats (supplied by Beijing Wittiaxle laboratory animal technology Co., Ltd.) were orally administered with 144 mg/rat of the product of example 8 and 9 mg/rat of the powder of comparative example 1 (As in the above-mentioned sample)2S2All the contents are 9mg), 200 μ L of peripheral blood is collected at the intraorbital canthus vein at 0.5, 1, 2, 4, 8, 12, 24, 36, 48 and 72h after administration, and the arsenic content in the blood sample is detected by AFS 8230 atomic fluorescence spectrometer after digestion (the detection method is according to HJ 694-.
Compared with comparative example 1, the arsenic content in peripheral blood of rats can be significantly increased by 4.41 times after oral administration in example 8, and the circulation time of arsenic in peripheral blood can be prolonged, so that the arsenic content in blood can be kept at a higher level (70 hours) for a long time, while the arsenic in blood is substantially completely metabolized within 30 hours after oral administration in rats in comparative example 1, as shown in fig. 8.
Other examples have the same bioavailability as example 8 and are not described in detail.
Example nine: killing experiment on leukemia cell line
25.0mg of the product of example 9 and 2.5mg of the powder of comparative example 1 (As in the above sample)2S2The contents are all 2.5mg) are respectively dissolved in 3mL of physiological saline to prepare aqueous dispersion, As2S2The concentration was 833.3 mg/L. Adding a certain amount of the two aqueous dispersions into the culture medium respectively to enable the As in the K562 cell culture system2S2The concentration of (A) is 166.7mg/L, so that As in an HL-60 cell culture system2S2The concentration of (B) was 41.7 mg/L. After incubation for a certain time, adding a CCK-8 reagent into the culture system, and continuously culturing for 1h in an incubator, wherein the concentration of the CCK-8 reagent is 20 mu L/hole; then, the 96-well cell culture plate is taken out, centrifuged, 100 μ L/well of the supernatant is absorbed, absorbance (OD) values at 450nm and 630nm are detected by a microplate reader, and the number of living cells in the culture system (obtained by comparison with the drawn living cell number-absorbance curve) is calculated after background absorption is subtracted from the data. The control group is provided with no tested medicine, and only the solvent physiological saline with the same amount as that of the experimental group is added to eliminate the influence of the solvent on the cell growth.
The results showed that K562 cells and HL-60 cells exhibited exponential growth in the saline-untreated control group, whereas both comparative example 1 and example 9 inhibited proliferation of K562 cells and HL-60 cells, compared to the same As2S2The product of example 9 can obviously improve the growth inhibition effect of realgar on a chronic myelocytic leukemia cell line K562 (shown in figure 9) and an acute myelocytic leukemia cell line HL-60 (shown in figure 10) in the concentration of comparative example 1.
The results of the above experimental examples show that the water-soluble realgar solid dispersion prepared by the hot-melt extrusion technology can be quickly dissolved in the water phase, and the effective component As in the realgar can be improved2S2The dissolution amount is accumulated in water, the blood arsenic circulation time is prolonged, the bioavailability is improved, the growth inhibition effect of realgar on chronic myelocytic leukemia cells K562 and acute myelocytic leukemia HL-60 is improved, and the preparation method has the advantages of simple and convenient operation and high integration degree.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. The water-soluble realgar solid dispersion is characterized in that raw materials comprise 1 part by weight of realgar, 1-20 parts by weight of polymer and 0-5 parts by weight of surfactant, the raw materials are added into a double-screw extruder for hot melt extrusion after being mixed, and the water-soluble realgar solid dispersion is prepared after the extrudate is cooled at room temperature; the realgar is water-refined realgar with the particle size of 20-75 mu m;
the polymer is selected from one or more of the following substances: polyvinyl alcohol polyethylene glycol Copolymer (Macrogol Poly (vinyl alcohol) Grafted Copolymer), polyvinylpyrrolidone (PVP), crosslinked polyvinylpyrrolidone (PVPP), polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft Copolymer (Soluplus), and vinyl polyvinylpyrrolidone-vinyl acetate Copolymer (PVP-VA).
2. The water-soluble realgar solid dispersion according to claim 1, wherein the surfactant is a nonionic surfactant or an ionic surfactant.
3. The water-soluble realgar solid dispersion according to claim 2, wherein the surfactant is selected from one or more of the following substances: polysorbate 20, polysorbate 60, polysorbate 80, sodium lauryl sulfate, polyethylene glycol cetostearyl ether and sodium cetostearyl sulfate.
4. The water-soluble realgar solid dispersion according to any one of claims 1 to 3, wherein the active ingredient is As2S2The macromolecule is a dispersion matrix, the solid dispersion is quickly dissolved in the water solution to form stable orange yellow colloidal solution, wherein the hydration diameter range of realgar colloidal particles is between 100 and 1000nm, the average hydration diameter is 400 and 700nm, and As in the water-soluble realgar solid dispersion2S2Cumulative dissolution in artificial gastric juiceThe yield is 9% -25%.
5. A method for preparing water-soluble realgar solid dispersion is characterized in that the raw materials of the water-soluble realgar solid dispersion of any one of claims 1 to 4 are mixed and then added into a double-screw extruder for hot melt extrusion, and the extrudate is cooled at room temperature to obtain the water-soluble realgar solid dispersion.
6. The process as claimed in claim 5, wherein the temperature in the twin-screw extruder is from 50 to 200 ℃ and the screw speed is from 5 to 100 r/min.
7. The water-soluble realgar solid dispersion prepared by the method of claim 5 or 6, wherein the active ingredient is As2S2The macromolecule is a dispersion matrix, the solid dispersion can be rapidly dissolved in an aqueous solution to form a stable orange yellow colloidal solution, wherein the hydration diameter range of realgar colloidal particles is between 100 and 1000nm, the average hydration diameter is 400 and 700nm, and As in the water-soluble realgar solid dispersion2S2The cumulative dissolution rate in the artificial gastric juice is 9 to 25 percent.
8. The use of the water-soluble realgar solid dispersion according to any one of claims 1 to 4 for preparing anti-leukemia drugs, wherein the leukemia is Acute Promyelocytic Leukemia (APL) and Chronic Myelocytic Leukemia (CML).
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| Title |
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| "固体分散体技术对纳米雄黄稳定性及体外溶出的影响研究";郭腾等;《中国中药杂志》;20130930;第38卷(第17期);第2782-2787页 * |
| "难溶性药物固体分散体研究新进展";刘娱姗等;《药学进展》;20131231;第37卷(第4期);第166-173页 * |
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| 郭腾等."固体分散体技术对纳米雄黄稳定性及体外溶出的影响研究".《中国中药杂志》.2013,第38卷(第17期), * |
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