Solid dispersions of 3,5,7-trihydroxyflavone derivatives

Abstract

The application belongs to the field of pharmaceutical chemistry, and particularly relates to a solid dispersion of 3,5,7-trihydroxy flavone derivatives, a preparation method and application thereof. The 3,5,7-trihydroxy flavone derivatives and PEG are firstly ground to form a solid dispersion, and then hot melt extrusion is carried out, the hot melt temperature of the 3,5,7-trihydroxy flavone derivatives and PEG is reduced by controlling specific hot melt extrusion conditions, the stability of the drug is ensured, and the obtained solid dispersion can achieve a rapid dissolution effect in a dissolution medium, and the bioavailability is correspondingly improved.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically to solid dispersions of 3,5,7-trihydroxyflavonoid derivatives, their preparation methods, and uses. Background Technology

[0002] Literature reports that 3,5,7-trihydroxyflavonoid derivatives (Formula 1) have an effective inhibitory effect on PDE5A1, with an IC50 very close to that of sildenafil (IC50 75 vs. 74 nM); its selectivity for phosphodiesterase-6 (PDE6) and cyclic adenosine monophosphate phosphodiesterase (cAMP-PDE) is higher than that of sildenafil; this compound has strong potential for development.

[0003]

[0004] During the research and development process, it was discovered that the compound is a water-insoluble crystal, slightly soluble in glacial acetic acid, very slightly soluble in methanol, insoluble in water, and has very low bioavailability.

[0005] For poorly soluble drugs, solid dispersions prepared using water-soluble carriers not only maintain a high degree of drug dispersion but also exhibit good wetting properties. This is significant in improving drug solubility, accelerating dissolution rates, and thus enhancing bioavailability. Solid dispersions (SDs) refer to a dispersion system in which a drug is highly dispersed in a solid carrier, existing in solid form. The drug particle size in the carrier is between 0.001 and 0.1 mm, primarily used to accelerate and increase the dissolution of poorly soluble drugs, thereby improving their bioavailability. For example, Southwest Pharmaceutical Factory No. 3 used a melt method with PEG6000 as a carrier to prepare griseofulvin droplets. The results showed that the solid dispersion was almost completely absorbed within 2 hours of oral administration, while the micronized tablets were absorbed only 44.3% within 30-80 hours. The absorption of griseofulvin dispersions with a drug-carrier ratio of 1:10-1:5 was more than twice that of the micronized tablets. Ozkan et al. prepared an etordosulfuric acid-PEG immediate-release solid dispersion, and the results showed that the solvent-based SD had better dissolution performance than the melt-based product. PEG6000 was selected as the best choice, showing the best drug dissolution with PEG6000, with over 60% dissolving within 10 minutes. The amorphous form remained unchanged in SD after 9 months of storage. Betageri et al. found that glibenclamide-PEG solid dispersions prepared by solvent-freeze-drying released the drug faster than melt-processed products, significantly increasing the dissolution rate of glibenclamide. Summary of the Invention

[0006] To address the technical problem of low bioavailability of the target compound, the inventors initially considered preparing it as a solid dispersion. Those skilled in the art know that PEG has low toxicity, is easily absorbed in the gastrointestinal tract, does not interfere with drug content analysis, and can significantly increase the drug's dissolution rate and bioavailability, making it the most commonly used PEG. To increase the dissolution rate and bioavailability of the compound of Formula 1, the inventors attempted to prepare a solid dispersion using existing technology, first mixing the compound of Formula 1 with PEG6000 via a solvent method. However, they found that this did not significantly improve the dissolution effect.

[0007] The inventors continued to try using the grinding method, with the ratio of Formula 1 compound to PEG6000 at 1:9, to prepare a solid dispersion. Verification tests showed that its dissolution rate was improved, but not significantly.

[0008] The inventors then considered the melt method for preparing solid dispersions. The melt method involves mixing and melting the drug with a carrier material, rapidly cooling it into a solid, and then placing the solid at a certain temperature to make it brittle. This method is suitable for heat-stable drugs and carrier materials that are poorly soluble in organic solvents and have low melting points, such as PEG, citric acid, and sugars. Since the melting point of compound 1 is above 200°C, the melt method easily leads to thermal decomposition of both the drug and the carrier for drugs with such high melting points. Therefore, this method is not suitable for achieving the desired effect. However, the inventors, with a try-it-out attitude, wanted to understand how much the melting point of the mixture of PEG and compound 1 differed from that of compound 1 itself. The inventors unexpectedly discovered that by first grinding the compound of Formula 1 and PEG6000 in a ratio of 1:(10-12) to form a solid dispersion 1, and then performing hot melt extrusion, the hot melt temperature of the compound of Formula 1 and PEG could be reduced to 120-150°C by controlling specific hot melt extrusion conditions, thus ensuring the stability of the drug. The resulting solid dispersion could achieve rapid dissolution in the dissolution medium, thereby improving its bioavailability.

[0009] The specific plan is as follows:

[0010] The dispersion contains a compound of formula 1 and PEG;

[0011] Specifically, the dispersion comprises the following components in the following weight ratios:

[0012] One part of compound 1

[0013] PEG 10-14 parts

[0014] The preferred composition includes the following components in the following weight ratios:

[0015] One part of compound 1

[0016] PEG 10-12 parts

[0017] Further preferred are components comprising the following weight ratios:

[0018] One part of compound 1

[0019] PEG 11 parts

[0020] The molecular weight of the PEG is 2000-20000;

[0021] The specific preparation steps are as follows:

[0022] 1) Pulverize the raw material compound 1 and PEG separately and pass them through a 50-100 mesh sieve. Mix them evenly in proportion and then grind them into a solid dispersion.

[0023] 2) Set the extrusion temperature of the twin-screw extruder to 120-150℃. After the temperature reaches the set value, start the screw and add the solid dispersion from step 1) into the extruder. After melting and extrusion, it is finally extruded in strip form.

[0024] 3) After cooling the strip extrudate, crush it and sieve it to obtain hot melt extruded solid dispersion particles of compound 1.

[0025] Ethanol is preferably added during grinding in step 1); the amount of ethanol is 0.01-1 part; preferably 0.01-0.1 part.

[0026] The present invention also provides a solid dosage form, specifically, the target solid dispersion powder obtained in step 3) is directly packaged as granules or powders, or processed into capsules, tablets, coated onto microspheres by lamination, or coated on the surface of the preparation to form the corresponding solid dosage form.

[0027] This invention employs differential scanning calorimetry (DSC) to investigate the hot-melt extruded solid dispersion of compound 1. The DSC results show that the endothermic peak of compound 1 has disappeared.

[0028] Compared with traditional processes, the present invention has the following outstanding advantages:

[0029] 1. The melting point of the compound of Formula 1 is above 200°C. In this invention, the inventors creatively used pre-grinding followed by hot melt extrusion in the hot melt extrusion of the compound of Formula 1, and used PEG as a solubilizer and plasticizer, which achieved unexpected results. The hot melt temperature can be reduced to below 150°C, which not only increases the dissolution of the drug and reduces energy consumption, but also ensures the stability of the drug.

[0030] 2. The choice of the present invention can not only reduce the hot melt extrusion temperature and facilitate the extrusion process, but also obtain a sufficiently strong screw shearing action, thereby effectively improving the dispersion state of the drug in the carrier; at the same time, it can also play a solubilizing role, improve the bioavailability of poorly soluble drugs, reduce the dosage, and reduce adverse drug reactions.

[0031] 3. The preparation process provided by this invention is simple, consumes little energy, leaves no solvent residue, and does not introduce other impurities throughout the process, making it easy to achieve continuous large-scale production. Attached Figure Description

[0032] Appendix Figure 1 Differential scanning calorimetry (DSC) spectra of the solid dispersion of compound 1 (a), the active pharmaceutical ingredient of compound 1 (b), and the hot melt extruded solid dispersion of compound 1 (c) prepared in Example 1, compared with those of Example 1. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the following description is only for explaining the present invention and does not limit its content. Unless otherwise specified, the content of each component used below is a weight percentage.

[0034] Example 1

[0035] Prepare hot-melt extruded solid dispersions of Formula 1 compounds with the following formulations:

[0036] 100g of compound of formula 1

[0037] PEG2000 1000g

[0038] The above components were pulverized through a 60-mesh sieve, mixed evenly, and 5 mL of ethanol was added. Then, the mixture was ground into a solid dispersion. The extruder temperature was set at 130°C. After the temperature reached the set value and stabilized, the obtained solid dispersion was added at a uniform rate. The temperature was raised to 145°C and completely melted. The mixture was extruded to obtain strip-shaped material. After cooling to room temperature, the material was pulverized through a 40-mesh sieve to obtain hot-melt extruded solid dispersion particles of compound 1.

[0039] Example 2

[0040] 100g of compound 1

[0041] PEG6000 1400g

[0042] Preparation process: The above components are pulverized through a 60-mesh sieve, mixed evenly, 5 mL of ethanol is added, and then ground into a solid dispersion. The extruder temperature is set at 120℃. After the temperature rises to the set value and stabilizes, the obtained solid dispersion is added at a uniform rate, the temperature is raised to 140℃, and it is completely melted. The strip-shaped material is extruded, cooled, and pulverized through a 40-mesh sieve to obtain hot melt extruded solid dispersion particles of compound 1.

[0043] Example 3

[0044] 100g of compound 1

[0045] PEG4000 1200g

[0046] Preparation process: The above components are pulverized through a 60-mesh sieve, mixed evenly, 5 mL of ethanol is added, and then ground into a solid dispersion. The extruder temperature is set at 130℃. After the temperature rises to the set value and stabilizes, the obtained solid dispersion is added at a uniform rate. The temperature is raised to 140℃ and completely melted. Strips are extruded, cooled to room temperature, and pulverized through a 40-mesh sieve to obtain hot melt extruded solid dispersion particles of compound 1.

[0047] Example 4

[0048] 100g of compound of formula 1

[0049] PEG4000 1100g

[0050] Preparation process: The above components are pulverized through a 60-mesh sieve, mixed evenly, 5 mL of ethanol is added, and then ground into a solid dispersion. The extruder temperature is set at 130℃. After the temperature rises to the set value and stabilizes, the obtained solid dispersion is added at a uniform rate. The temperature is raised to 140℃ and completely melted. Strips are extruded, cooled to room temperature, and pulverized through a 40-mesh sieve to obtain hot melt extruded solid dispersion particles of compound 1.

[0051] Example 5

[0052] 100g of compound of formula 1

[0053] PEG10000 1000g

[0054] Preparation process: The above components are pulverized through a 60-mesh sieve, mixed evenly, 5 mL of ethanol is added, and then ground into a solid dispersion. The extruder temperature is set at 140℃. After the temperature rises to the set value and stabilizes, the obtained solid dispersion is added at a uniform rate. The temperature is raised to 150℃ to completely melt the solid dispersion. The solid dispersion is then extruded to obtain strip-shaped material. After cooling to room temperature, the solid dispersion is pulverized through a 40-mesh sieve to obtain hot-melt extruded solid dispersion particles of compound 1.

[0055] Comparative Example 1

[0056] 100g of compound of formula 1

[0057] Soluplus 1100g

[0058] The above components are pulverized through a 60-mesh sieve and mixed evenly to prepare a physical mixture.

[0059] Comparative Example 2

[0060] 100g of compound of formula 1

[0061] PEG4000 900g

[0062] Preparation process: The above components are pulverized through a 60-mesh sieve, mixed evenly, 5 mL of ethanol is added, and then ground into a solid dispersion. The extruder temperature is set at 140℃. After the temperature rises to the set value and stabilizes, the obtained solid dispersion is added at a uniform rate. The temperature is raised to 190℃ to completely melt the solid dispersion. The solid dispersion is then extruded to obtain strip-shaped material. After cooling to room temperature, the solid dispersion is pulverized through a 40-mesh sieve to obtain hot melt extruded solid dispersion particles of compound 1.

[0063] Comparative Example 3

[0064] 100g of compound of formula 1

[0065] PEG4000 1100g

[0066] Preparation process: Crush the above components through a 60-mesh sieve, mix them evenly, set the extruder temperature to 150℃, and after the temperature rises to the set value and stabilizes, add the physical mixture at a uniform speed, heat until 182℃ to melt, extrude to obtain strip-shaped material, cool, crush through a 40-mesh sieve, and obtain hot melt extruded solid dispersion particles of compound 1.

[0067] Comparative Example 4

[0068] 100g of compound of formula 1

[0069] Soluplus 1100g

[0070] Preparation process: The above components are pulverized through a 60-mesh sieve, mixed evenly, 5 mL of ethanol is added, and then ground into a solid dispersion. The extruder temperature is set at 140℃. After the temperature rises to the set value and stabilizes, the obtained solid dispersion is added at a uniform rate. The temperature is raised to 190℃ to completely melt the solid dispersion. The solid dispersion is then extruded to obtain strip-shaped material. After cooling to room temperature, the solid dispersion is pulverized through a 40-mesh sieve to obtain hot melt extruded solid dispersion particles of compound 1.

[0071] Comparative Example 5

[0072] 100g of compound of formula 1

[0073] PEG4000 100g

[0074] Preparation process: The above components are pulverized through a 60-mesh sieve, mixed evenly, 5 mL of ethanol is added, and then ground into a solid dispersion. The extruder temperature is set at 160℃. After the temperature rises to the set value and stabilizes, the obtained solid dispersion is added at a uniform rate. The temperature is raised to 210℃ to completely melt the solid dispersion. The solid dispersion is then extruded to obtain strip-shaped material. After cooling to room temperature, the solid dispersion is pulverized through a 40-mesh sieve to obtain hot-melt extruded solid dispersion particles of compound 1.

[0075] Comparative Example 6

[0076] 100g of compound of formula 1

[0077] PEG4000 100g

[0078] The above components are pulverized through a 60-mesh sieve and mixed evenly to prepare a physical mixture.

[0079] Verification Example 1

[0080] Differential scanning calorimetry (DSC) experiments were performed on the active pharmaceutical ingredient of Formula 1, the hot-melt extruded solid dispersion of Formula 1 prepared in Example 1, and the product prepared in Comparative Example 2. The corresponding spectra are shown in [reference needed]. Figure 1 Differential scanning calorimetry (DSC) method: Weigh 10 mg of sample into an aluminum pan, using an alumina crucible as a reference, and heat in a nitrogen stream within the range of 20℃ to 300℃ at a rate of 10℃ / min. -1 The rate of temperature increase scanning.

[0081] Depend on Figure 1 It can be seen that: the spectral lines of the active pharmaceutical ingredient of Formula 1 contain an obvious endothermic peak of Formula 1; this peak is significantly reduced in the physical mixture obtained in Comparative Example 2, but still exists; this peak completely disappears in the spectral lines of the hot melt extruded solid dispersion of Formula 1 prepared in Example 4; the drug Formula 1 compound in the hot melt extruded solid dispersion of Formula 1 obtained by the technical solution of the present invention is more uniformly dispersed in the carrier material.

[0082] Verification Example 2

[0083] Dissolution determination: Accurately weigh appropriate amounts of the active pharmaceutical ingredient, physical mixture, and extruded powder (equivalent to approximately 10 mg of drug). Following Method II, Appendix XC, Part II of the 2010 Chinese Pharmacopoeia, using 900 mL of purified water as the dissolution medium and a rotation speed of 75 r / min, collect 5 mL of solution at 5, 10, 15, 20, 30, 45, and 60 min, simultaneously replenishing with the same volume and temperature of dissolution medium. Filter the solution through a 0.45 μm microporous membrane to obtain the test solution. Separately, accurately weigh an appropriate amount of the compound of Formula 1 reference standard and prepare an 11 μg / mL reference solution. Perform high-performance liquid chromatography (HPLC) (Appendix VD, Part II of the 2010 Chinese Pharmacopoeia), using octadecyl-bonded silica gel as the stationary phase and 20% acetonitrile-water as the mobile phase, with a detection wavelength of 250 nm. Accurately inject 10 μL into the HPLC system and record the chromatogram. Calculate the cumulative drug dissolution amount based on peak area using the external standard method. Dissolution experiments were conducted on the active pharmaceutical ingredient of Formula 1, physical mixtures, imported tablets (Xarelto 10mg, batch number: BXG3651), hot melt extruded solid dispersion particles of Formula 1 obtained in Examples 1-5 and Comparative Examples 1-6, and the raw material of Formula 1. The corresponding data are shown in Table 1.

[0084] Table 1. Drug dissolution curve data

[0085]

[0086] As can be seen from Table 1: Examples 1-5 of the present invention exhibit rapid dissolution; Comparative Example 1, which uses physically mixed PEG with a high melting temperature, results in a dispersion with slow dissolution; Comparative Example 2, which does not use the proportion of PEG specified in the present invention as a solid dispersion carrier, also shows slow dissolution; Comparative Example 3, which does not undergo grinding treatment before hot melting, results in unsatisfactory dissolution; Comparative Example 4, which uses Soluplus instead of PEG, does not yield good results; The compound in Formula 1 exhibits the worst dissolution when measured solely by the raw material due to its poor solubility. The dissolution test results further verify the superiority of the present invention.

[0087] Example 6: Effect of the solid dispersion of the present invention on bone marrow suppression induced by chemotherapy in tumor-bearing mice.

[0088] 1. Model preparation and grouping for drug administration: Sixty Balb / C mice were selected, weighed, and randomly divided into 5 groups of 12 mice each. The grouping and drug administration are as follows:

[0089] Normal group: Administered an equal volume of sodium carboxymethyl cellulose;

[0090] Blank control group: Administered an equal volume of sodium carboxymethyl cellulose;

[0091] Formula I compound group: 0.5 mg / kg of Formula I compound (dissolved in sodium carboxymethyl cellulose) was administered by gavage;

[0092] Comparative Example 6 group: 0.5 mg / kg of the formulation of Comparative Example 6 was administered by gavage;

[0093] Example 1 group: 0.5 mg / kg of the preparation of Example 1 was administered by gavage;

[0094] Intraperitoneal injection of 50 mg / kg cyclophosphamide daily for one week significantly reduced platelet count in mice. The above-mentioned drug was administered to each treatment group one week prior to the start of the modeling process.

[0095] Each treatment group was administered the drug once daily and fed normally. After 3 weeks of continuous administration, mice were anesthetized, blood was collected, and the number of white blood cells and platelets was measured to investigate the effect of the solid dispersion of the present invention on white blood cells and platelets.

[0096] 2. Experimental Results

[0097] Through the effects of the solid dispersion of the present invention on cyclophosphamide-induced white blood cells and platelets in mice in this embodiment (Table 2), it was found that the solid dispersion of the present invention can alleviate the bone marrow suppression caused by chemotherapy.

[0098] Compared with the model control group, the total number of white blood cells and the total number of platelets in the solid dispersion group of the present invention were increased and the difference was significant.

[0099] Compared with the compound group of Formula I, the total number of white blood cells and the total number of platelets in the solid dispersion group of the present invention are significantly increased.

[0100] Compared with the control group 6, the total number of white blood cells and the total number of platelets in the solid dispersion group of the present invention were significantly higher.

[0101] Table 2. Effects of the solid dispersion of the present invention on cyclophosphamide-induced leukopenia and thrombocytopenia in mice.

[0102]

[0103] Compared with the blank control group, $ p<0.05, $$ p<0.01;

[0104] Compared with the group of compounds of formula I, # p<0.05;

[0105] Compared with the 6th group of comparative examples, ＊ p<0.05.

[0106] Example 7: Solid dispersion of the present invention 60 Effect of Co-irradiated mice on blood cell count

[0107] 1. Model preparation and grouping for drug administration: Eighty Kunming mice were selected. Except for the normal control group (n=10), all other groups of mice were irradiated with 4 Gy.60 Co radiation was administered via a single whole-body irradiation session with an absorbed dose of 4 Gy at a rate of 0.88 Gy / min. Whole blood cell counts were determined by collecting blood samples from the orbital vein on days 3, 7, and 10 post-irradiation. A white blood cell count below 3.0 × 10⁻⁶ was considered acceptable. 9 / L or platelet count less than 500×10 9 Mice with a weight of / L were removed, and the remaining mice were used for experiments.

[0108] Mice that met the experimental requirements after irradiation were randomly divided into groups of 10 each, and each group was treated or administered drugs as follows.

[0109] Normal group: Administered an equal volume of sodium carboxymethyl cellulose;

[0110] Blank control group: Administered an equal volume of sodium carboxymethyl cellulose;

[0111] Formula I compounds: Intraperitoneal injection of 0.1 mg / kg of Formula I compound (dissolved in sodium carboxymethyl cellulose);

[0112] Comparative Example 6 group: Intraperitoneal injection of 0.1 mg / kg of the formulation of Comparative Example 6;

[0113] Example 1 group: Intraperitoneal injection of 0.1 mg / kg of the preparation of Example 1;

[0114] Each treatment group was administered the drug once daily and fed normally. After 10 consecutive days of administration, mice were anesthetized, blood was collected, and the number of white blood cells and platelets was measured to investigate the effect of the solid dispersion of the present invention on white blood cells and platelets.

[0115] 2. Experimental Results

[0116] Through this embodiment, the solid dispersion of the present invention is used to accept... 60 The effects of Co radiation on white blood cells and platelets in mice (Table 3) showed that the solid dispersion of the present invention could alleviate bone marrow suppression caused by radiotherapy.

[0117] Compared with the model control group, the total number of white blood cells and the total number of platelets in the solid dispersion group of the present invention were increased and the difference was significant.

[0118] Compared with the compound group of Formula I, the total number of white blood cells and the total number of platelets in the solid dispersion group of the present invention are significantly increased.

[0119] Compared with the control group 6, the total number of white blood cells and the total number of platelets in the solid dispersion group of the present invention were significantly higher.

[0120] Table 3 Solid dispersions 60 Effect of Co-irradiated mice on blood cell count

[0121]

[0122] Compared with the blank control group, $ p<0.05, $$ p<0.01;

[0123] Compared with the group of compounds of formula I, # p<0.05;

[0124] Compared with the 6th group of comparative examples, ＊ p<0.05.

[0125] Example 8: Inhibitory effect of the solid dispersion of the present invention on airway smooth muscle

[0126] Airway remodeling is an important pathological feature of bronchial asthma, and airway smooth muscle cells (ASMCs) are the main effector cells causing airway remodeling. Compared with normal individuals, patients with severe asthma have significantly increased ASMC levels, a phenomenon mainly caused by smooth muscle cell proliferation.

[0127] 1.1 Materials

[0128] 150–200 g SD rats; DM EM medium (Gibico); fetal bovine serum (Hangzhou Sijiqing Co., Ltd.); trypsin (Sigma).

[0129] 1.2 Experimental Methods

[0130] 1.2.1 Culture of rat ASMCs

[0131] Following the method described in relevant literature: Under aseptic conditions, the trachea was longitudinally dissected, the outer membrane was carefully peeled off, and the inner membrane was gently scraped away. The tracheal segment was carefully cut into small tissue blocks (1mm × 1mm × 1mm) using ophthalmic scissors. These blocks were then placed on the bottom of a 5cm × 5cm culture flask, arranged at equal intervals. 2mL of DMEM (high glucose) medium containing 25% fetal bovine serum was added, ensuring the culture medium did not contact the tissue blocks. The culture flask was placed bottom-up and incubated at 37°C with 5% carbon dioxide for approximately 3 hours to allow the tissue blocks to nearly dry. The culture flask was then gently inverted so that the culture medium just covered the surface of the tissue blocks. After 3 days of semi-open, absolutely static culture, the culture medium was added to 5mL. The medium was completely changed after 6 days, and thereafter changed every 3 days. After approximately 7 days, the cells reached confluence and were passaged. Cells from passages 4-5 were used in the experiment. The cultured rat ASMCs were identified morphologically.

[0132] 1.2.2 Detection of ASMC proliferation in rats using the CCK-8 assay

[0133] Fourth-generation rat ASMCs were cultured and prepared into a single-cell suspension, at a concentration of 1×10⁻⁶ cells / cells. 4Cells were seeded per well in a 96-well plate and cultured at 37°C with 5% carbon dioxide for 24 hours. When the cells reached a confluent state, culture medium containing 0.1% fetal bovine serum was added (to arrest cell growth in the G0 phase) and the cells were cultured for another 24 hours.

[0134] The culture medium was replaced with one containing 1% fetal bovine serum, and the cells were randomly divided into:

[0135] Control group: DMEM was added to each well only;

[0136] Group of compounds of formula I, with a concentration of 1×10⁻⁶. -6 mol / L;

[0137] Comparative Example 6: The concentration of Comparative Example 6 was 1×10⁶. -6 mol / L;

[0138] Example 1 group: The concentration of Example 1 is 1×10 -6 mol / L;

[0139] Each group was configured with 5 replicates, and a control group (containing no cells, but with the same DMEM concentration as the corresponding group) was also included in each group. After culturing at 37℃ and 5% CO2 for 48 h, 10 μL of CCK-8 reagent was added to each well, and the cells were cultured for another 4 h. The OD value of each well was measured at 450 nm. The cell growth inhibition rate of each group was calculated using the formula: Cell growth inhibition rate = 1 - [(OD value of each drug-treated group - OD value of the corresponding control group) / (OD value of the control group - OD value of the corresponding control group)] × 100%.

[0140] 2 Results

[0141] 2.1 Identification of ASMC

[0142] Observation with an inverted phase contrast microscope revealed that ASMCs were spindle-shaped before merging, and after merging, cells in some areas were arranged in bundles, exhibiting a typical "peak and valley" shape.

[0143] 2.2 Effects on ASMC proliferation

[0144] After the solid dispersion of the present invention was applied to ASMC for 48 hours, the OD value of the cells in the solid dispersion group of the present invention was significantly reduced compared with that of the compound group of Formula I, and the difference was statistically significant.

[0145] Compared with the cells in Comparative Example 6, the inhibition rates of all groups of cells treated with the compounds of the present invention were significantly increased, with statistically significant differences. (See Tables 4 and 5)

[0146] Table 4. OD values ​​of solid dispersions after 48 hours of treatment with ASMC

[0147]

[0148] Compared with the normal control group, $ p < 0.05, $$ p < 0.01;

[0149] Compared with the group of Compound I, # p < 0.05;

[0150] Compared with the group of Comparative Example 6, ＊ p < 0.05.

[0151] Table 5 Inhibition rate of solid dispersion on ASMC for 48 h

[0152]

[0153] Compared with the group of Compound I, $ p < 0.05, $$ p < 0.01.

[0154] Compared with the group of Comparative Example 6, ＊ p < 0.05.

[0155] Example 9 Determination of the bioavailability of the solid dispersion of the present invention

[0156] 1. Animal grouping and administration

[0157] Thirty-six Wistar rats (270 ± 30) g, half male and half female, were provided by the Experimental Animal Center of Shandong Xinsdailai Pharmaceutical Co., Ltd., production license number: SCXK (Lu) 20060019. They were raised under the conditions of temperature 20 - 22°C, relative humidity 45% - 65%, and light / dark 12 h / 12 h, with free diet and water.

[0158] Gavage administration group of the compound of the present invention: Eighteen healthy Wistar rats that had fasted for 12 hours and had free access to water, half male and half female, were divided into 3 groups, the group of Compound I (gavaged with Compound I), the group of Comparative Example 6 (gavaged with the preparation of Comparative Example 6), and the group of Example 1 (gavaged with the preparation of Example 1). Each group was gavaged once, and the administration dose was 3 mg / kg. They were fasted 12 h before administration and had free access to water. Approximately 300 μL of blood was collected from the retroorbital venous plexus at 0 h (before administration), 0.083, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 h after administration. The blood was anticoagulated with heparin and centrifuged at 12000 rpm at 4°C for 5 min to separate plasma, which was stored in a -20°C freezer. They had free access to water during the experiment and ate 2 h after gavage.

[0159] The intravenous administration group of the compound of this invention: Eighteen healthy Wistar rats (half male and half female) that had been fasted for 12 hours and had free access to water were divided into three groups: the compound I group (injection of compound I), the comparative example 6 group (injection of the formulation of comparative example 6), and the example 1 group (injection of the formulation of example 1). All groups were administered the compound via tail vein injection at a dose of 3 mg / kg. Approximately 300 μL of blood was collected from the retro-orbital venous plexus before administration (0 h) and at 0.033, 0.083, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 h after administration. The blood was anticoagulated with heparin, centrifuged at 12000 rpm for 5 min at 4°C, and the plasma was separated and stored at -20°C. During the experiment, rats had free access to food and water.

[0160] 2. Plasma sample testing

[0161] All processed plasma samples were subjected to UPLC-MS / MS quantitative analysis to determine plasma drug concentration.

[0162] 3. Calculation of bioavailability

[0163] The measured blood drug concentration-time data were used to calculate pharmacokinetic parameters using DAS software (Drug and Statistics, compiled by Sun Ruiyuan et al., Chinese Society for Mathematical Pharmacology). The absolute bioavailability of the solid dispersion was calculated according to the formula, where t is the sampling time at which the final measurable drug concentration was obtained.

[0164]

[0165] 4. Absolute bioavailability of solid dispersions

[0166] Table 6 Bioavailability of Solid Dispersions

[0167]

[0168] As can be seen from the table above, the bioavailability of the solid dispersion of the present invention is significantly higher than that of the physical mixture of compound I and PEG, and it is well absorbed orally.

Claims

1. A solid dispersion, characterized in that, It includes compound of formula 1 and PEG; The solid dispersion comprises the following components in the indicated weight ratios: One part of compound 1 PEG 10-14 parts; The method for preparing the solid dispersion is characterized by the following specific preparation steps: 1) Pulverize the raw material compound of formula 1 and PEG separately and pass them through a 50-100 mesh sieve. Mix them evenly in proportion, and then add ethanol and grind them into a solid dispersion. 2) Set the extrusion temperature of the twin-screw extruder to 120-150℃. After the temperature reaches the set value, start the screw and add the solid dispersion from step 1) into the extruder. After melting and extrusion, it is finally extruded in strip form. 3) After cooling the strip extrudate, crush it and sieve it to obtain hot melt extruded solid dispersion particles of compound 1; The molecular weight of the PEG is 2000-20000.

2. The solid dispersion as described in claim 1, specifically, characterized in that, The solid dispersion comprises the following components in the following weight ratios: One part of compound 1 PEG 10-12 parts.

3. The solid dispersion as described in claim 1, specifically, characterized in that, The solid dispersion comprises the following components in the following weight ratios: One part of compound 1 PEG 11 parts.

4. The solid dispersion as described in claim 1, characterized in that, The amount of ethanol is 0.01-1 part.

5. The solid dispersion as described in claim 1, characterized in that, The amount of ethanol is 0.01-0.1 parts.

6. A pharmaceutical composition, characterized in that, It comprises the solid dispersion as described in any one of claims 1-5, and a pharmaceutically acceptable excipient.

7. A solid dosage form, characterized in that, Specifically, the solid dispersion described in any one of claims 1-5 or the pharmaceutical composition described in claim 6 may be directly packaged as granules or powders, or processed into capsules, tablets, coated onto microspheres by lamination, or coated onto the surface of the formulation to form the corresponding solid dosage form.

8. Use of the pharmaceutical composition of claim 6 or the solid dosage form of claim 7 in the preparation of a medicament for treating asthma and myelosuppression.

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