Penfrenone sustained-release microsphere inhalation dry powder as well as preparation method and application of pirfrenone sustained-release microsphere inhalation dry powder

Abstract

The invention belongs to the field of pharmaceutical preparations, and particularly relates to a preparation method of pirnenone sustained-release microsphere inhalation powder inhalation and application of the pirnenone sustained-release microsphere inhalation powder inhalation in treatment of idiopathic pulmonary fibrosis. Chitosan is subjected to mPEG modification to serve as a carrier material, the carrier material and pirfenidone are prepared into the drug-loaded microspheres through an electrostatic spraying technology, and the prescription and the preparation process for preparing the drug-loaded microspheres are optimized. The prepared PFND-mPEG-CS-MS has better encapsulation efficiency, drug loading capacity and in-vitro release behavior, and the characterization of a sample shows that the powder chemical properties of the PFND-mPEG-CS-MS meet the requirements of inhalation powder aerosols. In-vitro cell experiments show that the blank carrier microspheres have good biocompatibility, and the drug-loaded microspheres have high cell inhibition rate. Animal in-vivo experiment results show that the PFND-mPEG-CS-MS has remarkable significance in prolonging the half-life period of the medicine, improving the bioavailability of the medicine, improving the treatment effect and reducing the hepatotoxicity.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical preparations, and specifically relates to a preparation method of a pirfenidone sustained-release microsphere inhalation powder aerosol and an application of the same in the treatment of idiopathic pulmonary fibrosis. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is a type of interstitial lung disease, which is a rare disease. It is mainly manifested by fibrosis of the lung interstitial and alveolar epithelial cells during inflammatory damage and self-repair. The causes of idiopathic pulmonary fibrosis include viral and bacterial infections, improper use of drugs, the influence of dust environment, etc. The main clinical manifestations are dry cough and dyspnea, and the patient's respiratory function will continue to deteriorate as the disease progresses.

[0003] Pirfenidone (CAS: 53179-13-8) is a small molecule pyridone compound with the chemical name 5-methyl-1-phenyl-2-(1H)-pyridone. It is a cytokine inhibitor that inhibits TGF-β-induced Smad3 / 9 phosphorylation by regulating the Wnt / GSK-3β / β-catenin and TGF-β1 / Smad2 / 3 signaling pathways, downregulates TGF-β cytokine levels, and reduces the mRNA and protein levels of CollagenI, CollagenⅡ, α-SMA, and fibronectin, thereby reducing the expression of other cytokines that promote the progression of IPF, reducing the immune response caused by inflammatory factors, avoiding tissue damage and the subsequent tissue self-repair and IPF progression, reducing ECM deposition, preventing abnormal proliferation of HLF, and weakening the expression and synthesis of CollagenⅠ and CollagenⅡ.

[0004] In 2014, the FDA approved pirfenidone capsules and tablets, which are one of the first two drugs in the world approved for the treatment of IPF. Currently, the only drug on the market in China is pirfenidone capsules (Aisiri Beijing Contini Pharmaceutical Co., Ltd.) and pirfenidone tablets (Beijing Kain Technology Co., Ltd.). Due to dosage form restrictions, it can only be used in the form of gastrointestinal administration, and pirfenidone must be taken with three meals to prevent gastrointestinal adverse reactions. The first-pass effect of the liver also leads to low drug bioavailability, large dosage and hepatotoxicity, maintenance dose of 600 mg / time, multiple administration of the maximum tolerated dose of 600 mg / time, low drug bioavailability and safety, and a single dosage form limits the clinical application of the drug. Patent CN103271886A discloses a pirfenidone tablet and a preparation method thereof for the treatment of pulmonary fibrosis. However, the oral administration dosage is large, the onset is slow, and it can cause liver and kidney toxicity and gastrointestinal discomfort, with great side effects. Patent CN110742875 discloses a pirfenidone solution preparation for inhalation and its preparation method and application, which are used to prevent and treat mild and moderate idiopathic pulmonary fibrosis, liver fibrosis, renal fibrosis and other diseases. However, the inhaled pirfenidone solution needs to be inhaled into the respiratory system in the form of atomization by an ultrasonic nebulizer or an air compression nebulizer, and there are too many additives in the solution, which is not conducive to the safety of the drug, and the patient's compliance is also low. In view of this, it is necessary to develop a preparation that can be directly administered through the lungs to reduce the drug dosage, enhance the local efficacy, improve the patient's compliance, reduce toxic side effects and have high bioavailability to treat idiopathic pulmonary fibrosis. Summary of the invention

[0005] To solve the above problems, the present invention provides a method for preparing and using a pirfenidone sustained-release microsphere inhalation powder. The present invention adopts electrostatic spray technology to prepare the pirfenidone drug microsphere inhalation powder, and modifies the microsphere carrier to avoid phagocytosis of macrophages, providing a new preparation method for such sustained-release microsphere preparations. Compared with traditional preparations such as tablets and capsules, it can directly reach the lungs, reduce drug loss, enhance local efficacy, prolong drug half-life, improve bioavailability, reduce dosage, and reduce organ toxicity and side effects. Compared with sprays and aerosols, powder sprays do not require the pressure of a manual pump and can avoid the use of propellants. Patients actively inhale atomized drugs, which improves the patient's medication compliance and safety.

[0006] The technical solution provided by the present invention is as follows:

[0007] A pirfenidone sustained-release microsphere inhalation powder is prepared from the following raw materials in parts by mass: 0.22-0.25 parts of pirfenidone raw material, 1-3 parts of carrier material, 0.2-0.8 parts of polyethylene oxide and 4-20 parts of acetic acid.

[0008] The present invention also provides a method for synthesizing the above-mentioned pirfenidone sustained-release microsphere inhalation powder aerosol carrier material mPEG-CS, which is achieved by the following technical scheme:

[0009] (1) Weigh 1 g of chitosan and dissolve it in 100 mL of 1% acetic acid solution to prepare a 10 mg / mL solution for later use. The chitosan has a molecular weight of 20,000 to 30,000 and a deacetylation degree of 90% to 95%.

[0010] (2) Weigh 0.1 g of polyethylene glycol monomethyl ether carboxyl (mPEG-COOH) and dissolve it in 1% acetic acid solution. Add 0.0240 g of EDCI and 0.0144 g of NHS and stir at 20°C for 3 h for activation.

[0011] (3) Add chitosan solution to the activation solution in a volume ratio of 20:1; adjust the pH to 5.0 with NaOH and stir at 20°C for 48 hours. After the reaction is completed, evaporate the solution to about 50 mL, which is about 1 / 2 of the original volume; transfer it to a 8000-14000Da dialysis bag and dialyze it with pure water for 24 hours. After the dialysis is completed, transfer the solution to a watch glass and freeze-dry it for 24 hours to obtain a white solid product mPEG-CS.

[0012] The present invention also provides a prescription for the above-mentioned pirfenidone sustained-release microsphere inhalation powder, which is achieved by the following technical scheme:

[0013] In the above-mentioned sustained-release microsphere inhalation powder, the pirfenidone sustained-release microsphere inhalation powder is prepared by the following prescription: 0.24% pirfenidone raw material, 1%-3% carrier material, 0.2%-0.8% polyethylene oxide and 4%-20% acetic acid aqueous solution.

[0014] Among them, the content of pirfenidone raw material, carrier material, polyethylene oxide is the proportion (w / v%) of it in the acetic acid aqueous solution, and the content of the acetic acid aqueous solution is the proportion (v / v%) of acetic acid in water.

[0015] Furthermore, the carrier material is a polyethylene glycol-modified chitosan (mPEG-CS) carrier material mPEG-CS.

[0016] The present invention also provides a method for preparing the above-mentioned pirfenidone sustained-release microsphere inhalation powder, which comprises the following steps: dissolving the pirfenidone raw material, carrier material and polyethylene oxide in an acetic acid aqueous solution, stirring magnetically until completely dissolved and standing for a period of time so that there are no bubbles in the mixed solution; and treating the mixed solution with electrostatic spraying to obtain the pirfenidone sustained-release microsphere inhalation powder.

[0017] Furthermore, the preparation method of the carrier material includes: dissolving chitosan in an acetic acid solution to prepare a chitosan solution; dissolving polyethylene glycol monomethyl ether carboxyl in an acetic acid solution, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to obtain an activation solution; dripping the chitosan solution into the activation solution, adjusting the pH and fully stirring the reaction, after the reaction is completed, rotary evaporating the solution and dialyzing it, and freeze-drying it to obtain the carrier material mPEG-CS.

[0018] Further, the chitosan solution was dripped into the activation solution, the pH was adjusted to 5.0 with NaOH, and stirred at 20°C for 48 hours. After the reaction was completed, the solution was rotary evaporated and transferred into a 8000-14000Da dialysis bag and dialyzed with pure water for 24 hours. After the dialysis, the solution was transferred to a watch dish and freeze-dried for 24 hours to obtain the carrier material mPEG-CS.

[0019] Furthermore, the concentration of chitosan in the chitosan solution is 10 mg / mL.

[0020] Furthermore, the concentration of the acetic acid solution is 1%.

[0021] Furthermore, the mass ratio of polyethylene glycol monomethyl ether carboxyl, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 8-12:2-3:1-2, preferably 1:0.24:0.144.

[0022] Furthermore, the mass ratio of the pirfenidone API to the carrier is 1:8.33.

[0023] Furthermore, the electrospraying process parameters are: voltage 12.5-17.5 kV, spraying speed 0.05-0.1 mm / min, receiving distance 15-20 cm, and nozzle inner diameter 0.34-0.51 mm.

[0024] Furthermore, the preparation method comprises:

[0025] (1) Weigh the prescribed amount of pirfenidone API, carrier material mPEG-CS and spinning aid PEO, dissolve them in acetic acid aqueous solution, stir them magnetically at 37°C until they are completely dissolved and let them stand for a period of time so that there are no bubbles in the electrospray liquid.

[0026] (2) Use a syringe to absorb the electrospray solution, install the nozzle, and load it on the electrostatic sprayer. Set the electrospray process parameters (voltage, spray speed, receiving distance, temperature and humidity) and collect the microsphere powder aerosol. The preparation process parameters of the preparation include 23-21G model (inner diameter 0.34-0.51mm) nozzle, voltage

[0027] +12.5kv (-2.5kV) - +17.5kV (-2.5kV), spray speed 0.05-0.1mm / min, receiving distance 15-20cm, temperature 37℃, humidity 20%.

[0028] The Carr index Cp of PFND-mPEG-CS-MS is less than 20%, the aerodynamic diameter da is less than 3 μm, the lung deposition rate PDR is greater than 40%, and the emptying rate ER is greater than 90%.

[0029] The present invention also provides the use of the pirfenidone sustained-release microsphere inhalation powder in the preparation of a pulmonary administration preparation that reduces or eliminates liver toxicity.

[0030] The present invention also provides the use of the PFND-mPEG-CS-MS in treating bleomycin-induced idiopathic pulmonary fibrosis in rats.

[0031] Beneficial Effects

[0032] The present invention prepares pirfenidone as an inhalation powder for the first time. Compared with traditional preparations such as tablets and capsules, it can directly reach the lungs, reduce drug loss, enhance local efficacy, prolong drug half-life, improve bioavailability, reduce dosage, and reduce organ toxicity and side effects.

[0033] Compared with sprays and aerosols, inhalation powders avoid the use of propellants and preservatives, are safer for the human body, and do not require the use of solvents. They are of great significance for maintaining the activity and stability of drugs, and provide a new formulation improvement idea for drugs for the treatment of respiratory diseases.

[0034] Pirfenidone inhalation powder was prepared by electrostatic spray technology, and the traditional carrier was optimized to make drug-loaded microspheres, providing a new preparation method for this type of sustained-release microsphere preparation.

[0035] The present invention uses natural polymer material chitosan as a carrier material, which has excellent biocompatibility and can be naturally degraded. In order to avoid phagocytosis of macrophages in the body, the present invention also uses mPEG with a long circulation effect in the body to modify chitosan. Compared with magnesium stearate alone as a carrier material, the carrier material of the present invention plays a role in prolonging the drug action time in the body and improving bioavailability.

[0036] After multiple tests, it was found that the final preferred mass ratio of the pirfenidone drug particles to the carrier magnesium stearate was 1:5.47. In the subsequent atomization experiment of the powder inhaler, the maximum product rate of the particles in the lungs was only 28.9%; while the final mass ratio of the pirfenidone drug particles to the carrier in the present invention was 1:8.33. In the subsequent atomization experiment of the powder inhaler, the deposition rate of the powder inhaler particles of the present invention in the lungs reached 48.72%. It is proved that the higher ratio of the carrier to the drug in the present invention has a better particle lung deposition rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a SEM morphology image of the pirfenidone sustained-release microsphere inhalation powder prepared by the optimal prescription in the example;

[0038] Figure 2 This is a diagram of the fixation method of rats in Example 12;

[0039] Figure 3 The in vivo drug-time curves of PFND-mPEG-CS-MS and PFND in Example 12;

[0040] Figure 4 The ALT and AST levels in the lungs of rats in each group in Example 13;

[0041] Figure 5 is a graph showing the hydroxyproline content in the lungs of rats in each group in Example 13;

[0042] Figure 6 HE and Masson staining images of rat lungs in Example 13;

[0043] Figure 7 This is a statistical diagram of the positive area of ​​collagen fibers in rat lungs in Example 13;

[0044] Figure 8 This is the immunofluorescence image of Collagen Ⅰ, TGF-β, and α-SMA in the lungs of rats in Example 13;

[0045] Fig. 9 This is a statistical diagram of the immunofluorescence positive areas of Collagen Ⅰ, TGF-β, and α-SMA in the lungs of rats in Example 13;

[0046] Fig.10 This is the HE staining image of other organs of rats in Example 13. DETAILED DESCRIPTION

[0047] The preparation process and the substances or the amounts of the substances used in the following pharmaceutical preparation examples are not limited to the textual description, and all methods involving the pharmaceutical preparation provided by the present invention belong to the protection scope of the present invention.

[0048] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0049] Example 1: Preparation of mPEG-CS, a carrier material for sustained-release microsphere inhalation powder of pirfenidone

[0050] (1) Weigh 1 g of chitosan and dissolve it in 100 mL of 1% acetic acid solution to prepare a 10 mg / mL solution for later use.

[0051] (2) Weigh 0.1 g of polyethylene glycol monomethyl ether carboxyl (mPEG-COOH) and dissolve it in 1% acetic acid solution, add 0.0240 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 0.0144 g of N-hydroxysuccinimide (NHS) and stir at 20°C for 3 h for activation.

[0052] (3) The chitosan solution was dripped into the activation solution, the pH was adjusted to 5.0 with NaOH, and stirred at 20°C for 48 hours. After the reaction was completed, the solution was rotary evaporated to about 50 mL remaining, transferred into a 8000-14000Da dialysis bag and dialyzed with pure water for 24 hours. After the dialysis was completed, the solution was transferred to a watch glass and freeze-dried for 24 hours to obtain a white solid product mPEG-CS.

[0053] Comparative Example 1: Patent CN109414433 A discloses a pharmaceutical composition for inhalation, using lactose as a drug carrier of pirfenidone, and generating a molecular aggregate by compounding particles and the carrier.

[0054] Comparison between Example 1 and Comparative Example 1: Due to the difference in drug carriers in Example 1 and Comparative Example 1, the preparation in Example 1 has a safer and longer-lasting effect. Chitosan is a cationic polysaccharide contained in shrimp and crab shells, has excellent biocompatibility, can be naturally degraded, has cell affinity, and has antibacterial, anti-inflammatory and hemostatic properties. In order to avoid the body's phagocytosis of chitosan, the present invention also uses a hydrophilic material PEG to modify chitosan, which plays a role in prolonging the drug's action time in the body and improving bioavailability. At the same time, the drug and the carrier in the present invention are mixed by a simple physical method, which is easy to operate.

[0055] Example 2-10: Preparation of pirfenidone sustained-release microsphere inhalation powder.

[0056] (1) Weigh the prescribed amount of pirfenidone API, carrier material mPEG-CS and spinning aid PEO, dissolve them in acetic acid aqueous solution, stir magnetically at 37°C until completely dissolved and let stand for a period of time to eliminate bubbles in the electrospray liquid.

[0057] (2) Use a syringe to absorb the electrospray solution, install the nozzle, and load it on the electrostatic sprayer. Set the electrospray process parameters (voltage, spray speed, receiving distance, temperature and humidity) and collect the microsphere powder aerosol.

[0058] The specific amounts of the carrier material mPEG-CS, the spinning aid polyethylene oxide, and the acetic acid aqueous solution in the prescriptions of Examples 2-10 are shown in Table 1.

[0059] Table 1: Comparative Experimental Design and Results in Examples 2-10

[0060]

[0061]

[0062] Note: For the above-mentioned carrier materials, the percentage of the spinning aid is the percentage of the spinning aid in the acetic acid aqueous solution (w / v, %); the percentage of the acetic acid aqueous solution is the percentage of acetic acid in water (v / v, %).

[0063] The electrospray process parameters were further determined based on the dosage in the prescription of Example 8.

[0064] The electrospraying process parameters (voltage, spraying speed, receiving distance, nozzle inner diameter) in Examples 11-19 refer to Table 2.

[0065] Table 2: Orthogonal experimental design and results in Examples 11-19

[0066] Serial number Voltage(kV) Spray speed (mm / min) Receiving distance(cm) Nozzle inner diameter (mm) Yield (%) Embodiment 11 12.5 0.05 15 0.34 35.7 Example 12 12.5 0.07 18 0.41 44.6 Example 13 12.5 0.1 20 0.51 35.9 Embodiment 14 15.5 0.05 18 0.51 50.5 Embodiment 15 15.5 0.07 20 0.34 48.6 Example 16 15.5 0.1 15 0.41 46.2 Embodiment 17 17.5 0.05 20 0.41 66.9 Embodiment 18 17.5 0.07 15 0.51 68.4 Embodiment 19 17.5 0.1 18 0.34 70.3

[0067] Comparative Example 2: Patent CN107080741 A discloses a sustained-release preparation of pirfenidone and a preparation method thereof. It is characterized in that pirfenidone is dispersed in a sustained-release matrix material. At the same time, one or more of a filler, a binder, a lubricant, and a wetting agent need to be added to prepare a traditional preparation. Traditional preparations usually cannot directly reach the lungs, increasing the loss of the drug.

[0068] Comparison between Example 2-19 and Comparative Example 2: Due to the difference in the drug dosage forms in Example 2-19 and Comparative Example 2, the preparation in Example 2-19 can directly reach the lungs, reduce drug loss, enhance local efficacy, prolong drug half-life, improve bioavailability, reduce dosage, reduce organ toxicity and side effects, and provide a new treatment plan combined with mechanical ventilation for bedridden critically ill patients.

[0069] Comparative Example 3: Patent CN115919813A discloses a pirfenidone inhalation powder and a preparation method. The pirfenidone inhalation powder was prepared by spray drying technology. The spray drying method uses high temperature to quickly evaporate and sublimate the liquid to obtain a dry powder, but high temperature is not conducive to the stability of the drug. It can be seen from the results that the surface of the particles prepared by spray drying technology is wrinkled. This structure increases the roughness of the particle surface, resulting in poor dry powder fluidity. In addition, the aerodynamic diameter of the particles prepared by this technology is relatively large (9.13μm) and the fine particle fraction is relatively low (15%).

[0070] Comparison between Example 2-19 and Comparative Example 3: Due to the difference in the preparation technology of the inhalation powder aerosol used in Example 2-19 and Comparative Example 3, the physicochemical properties of the inhalation powder aerosol of Example 2-19 are better than those of Comparative Example 3. The reason is that the preparation of drug-loaded targeted sustained-release microsphere powder by electrostatic spray self-assembly can well maintain the activity of the drug, does not change the drug crystal form, and is simple to operate; on the other hand, compared with other drug loading methods, electrostatic spray loading has less solvent residue and higher encapsulation capacity, and does not require secondary drying processes such as freeze drying, thus avoiding the defect of introducing new impurities such as protective agents; in addition, the particle size of the microspheres can be effectively controlled by adjusting the electrospray parameters. The particle size of the powder aerosol prepared by the present invention is between 1-5 μm, and the fine particle fraction is 48.72%. The prepared microspheres have good powder fluidity, and there is no need to introduce other additives into the preparation. It shows that the use of electrostatic spray technology is more convenient than the spray drying technology, and the obtained powder aerosol has better physicochemical properties.

[0071] Example 11: Powder properties experiment of PFND-mPEG-CS-MS

[0072] The powder properties experiment was conducted on the pirfenidone sustained-release microsphere inhalation powder prepared by the optimal prescription in the example.

[0073] (1) Morphology: Take a small amount of microsphere powder and spread it on the sample plate. After gold spraying, use SEM to observe the morphology of the microspheres, and observe the morphology of the microsphere powder aerosol at a certain magnification. Figure 1 The prepared microspheres are shown to be full spherical, with no collapse on the surface of the microspheres, no beads, tadpoles or other structures, good dispersibility, and relatively uniform particle size distribution.

[0074] (2) Powder density and fluidity: Weigh a certain mass (m) of PFND-mPEG-CS-MS powder sample and put it into a 5 mL measuring cylinder. Read and record the volume of the sample in the measuring cylinder at this time, which is the bulk volume (V b ), then regularly tap the cylinder wall and observe the volume change of the sample in the cylinder. When the volume of the sample in the cylinder no longer changes, record the volume at this time as the tapped volume (V t ). According to the formula: ρ b=m / V b , ρ bt =m / V t , Calculate the bulk density (ρ b ), tap density (ρ bt ) and compression (Carr index C p The bulk density, tap density and Carr index of the prepared PFND-mPEG-CS-MS powder were (0.162±0.015) g / cm 3 、(0.195±0.014)g / cm 3 and (17.074±2.163)%.

[0075] (3) Aerodynamic diameter: 10 mg of PFND-mPEG-CS-MS sample was accurately weighed and added to 10 mL of deionized water. The sample was vortexed for 5 min to disperse evenly. 0.2 mL of low molecular weight suspending agent glycerol was added and vortexed again for 3 min to keep the sample stably dispersed. The geometric diameter (d) was immediately measured using a NanoBrook 90Plus PALS particle size analyzer. g ), through the formula where ρ bt is the tap density of the powder (g / cm3), ρ0 is the standard density (1g / cm3), and x is the dynamic shape factor (x = 1 if the powder is spherical). Calculate the aerodynamic particle size (d a The geometric diameter d of PFND-mPEG-CS-MS was obtained by particle size measurement. g The aerodynamic particle size d of the microspheres was calculated to be (2.24±0.16) μm. a It is (0.99±0.07)μm.

[0076] (4) Hygroscopicity: Place a saturated NH4Cl solution under the dryer, and place a dry weighing bottle (outer diameter 50 mm, height 30 mm) in a constant temperature and humidity dryer. After 24 hours, take out the weighing bottle, weigh it and record it as m0. Weigh an appropriate amount of microsphere powder sample and evenly cover the bottom of the weighing bottle to a thickness of about 1 to 2 mm. Weigh it accurately and record the weight at this time as m1. Open the weighing bottle and put it into the dryer together with the bottle stopper. After 24 hours, cover the bottle stopper and take it out to weigh it and record it as m2. According to the formula Calculate the weight gain percentage (R w The results of the hygroscopicity test showed that the percentage of weight gain (R w ) is (4.61±0.72)%.

[0077] (5) Pulmonary deposition rate and emptying rate: NGI was used to measure the pulmonary deposition rate and emptying rate of microsphere powder samples. Accurately weigh 3 portions of 20 mg of microsphere powder sample and fill them into No. 3 capsule shells, and then load the capsules into the powder inhaler. After the inhalation is completed, remove the mouthpiece adapter and L-shaped artificial throat, and clean them with 100 mL of 1% acetic acid aqueous solution; remove the pre-separator and clean them with 50 mL of 1% acetic acid aqueous solution; open the impactor, remove the tray and the collection cup together, and clean the sample powders in the 1st and 8th level collection cups with 10 mL of 1% acetic acid aqueous solution, and clean the sample powders in the 2nd to 7th level collection cups with 20 mL of 1% acetic acid aqueous solution. Ultrasonicate each part of the collected liquid for 20 minutes to break the microspheres, filter the membrane, and use the in vitro analysis method to determine the content of each level of drug. And use the formula The pulmonary deposition rate (PDR) and emptying rate (ER) of the powder inhaler were calculated. According to the measurement results of NGI, the deposition rate of PFND-mPEG-CS-MS in the mouthpiece adapter and artificial throat (Ada&Lar) was 23.87%, the deposition rate in the pre-separator (Pre) was 11.56%, the deposition rate in the 1st level collection cup (S1) was 15.60%, and the deposition rate in the 2nd to 7th level collection cups (S2-S7) was (48.72±7.04)%. The emptying rate of the sample was 90%-95%.

[0078] Example 12: Pharmacokinetics of PFND-mPEG-CS-MS and API PFND

[0079] Pharmacokinetic experiments were conducted on PFND-mPEG-CS-MS and the raw material PFND, respectively. The experimental animals were 10 male SD rats (170-210g). The 10 rats were divided into two groups, with 5 rats in each group. The control group was the PFND raw material group, and the experimental group was the PFND-mPEG-CS-MS preparation group. The PFND raw material group was given oral administration at 12 mg / kg, and the PFND-mPEG-CS-MS preparation group was given 12 mg / kg by pulmonary administration. Pulmonary administration method: The rats were anesthetized by intraperitoneal injection of 20 mg / mL sodium pentobarbital solution (40 mg / kg). The rats were placed in the pulmonary cavity. Figure 2Fix the rat's tongue with forceps and pull it out of the mouth along the corner of the mouth to the upper left. Use a homemade opener with two ends to open the rat's mouth. Use a light source to find the rat's glottis. When the glottis opens, quickly insert the powder inhalation drug delivery device through the glottis into the trachea, and spray PFND-mPEG-CS-MS inhalation powder into the trachea. After administration, 0.5 mL of blood was collected from the orbits of the two groups at 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours respectively (the EP tube and capillary tube were soaked and dried with sodium heparin solution in advance). After blood collection, the rats were pressed to stop bleeding in time and supplemented with the corresponding volume of normal saline. According to the in vivo analysis method, the blood drug concentration in the rat was calculated and the drug-time curve was drawn. The DAS2.0-compartment model was used to fit the pharmacokinetic parameters, and the Wagner-Nelson equation was used to calculate the percentage of drug absorption in the body at each time point (Fa), and the in vivo and in vitro correlation was evaluated. Figure 3 As shown: (1) PFND reaches its peak concentration at 0.5h, and the drug is basically eliminated at 8h. PFND-mPEG-CS-MS reaches its peak concentration at 2h, and the blood drug concentration is more stable than PFND. The drug content can still be detected at 24h. (2)

[0080] The peak concentration of PFND-mPEG-CS-MS was about 1.2 μg / mL, which was half of the peak concentration of PFND (2.66 μg / mL). The half-life was 5.02 h, which was about three times the half-life of PFND (1.674 h). The area under the curve (AUC) t The AUC of PFND-mPEG-CS-MS and PFND increased from 6.375 h·μg / mL to 10.823 h·μg / mL. t The ratio was 169.76%, and the average retention time was extended from 2.711h to 10.659h. (3) According to the Wagner-Nelson equation, the 24h drug absorption percentage Fa in vivo was calculated to be 100.46%. The in vitro release percentage and in vivo absorption percentage of PFND-mPEG-CS-MS at each time point were regressed to obtain the linear regression equation Fa=1.0571x, R2=0.936, indicating that there is a good correlation between in vitro and in vivo.

[0081] Example 13: Pharmacodynamics experiment of PFND-mPEG-CS-MS and API PFND

[0082] Pharmacodynamic experiments were conducted on PFND-mPEG-CS-MS and the raw material PFND. The experimental animals were 24 male SD rats (170-210 g). The rats were divided into 4 groups, 6 rats in each group, namely:

[0083] Blank control group (Blank group), BLMS model group (Model group), pirfenidone positive control group (Control group) and PFND-mPEG-CS-MS inhalation powder test group (Test group); the blank group rats were not treated, and the BLMS model group rats were subjected to bleomycin (5mg / kg, 5mg / mL) lung modeling. After bleomycin (5mg / kg, 5mg / mL) modeling was performed in the pirfenidone positive control group rats, PFND raw material (3mg / kg, 5mg / mL) drug was administered to the lungs. After bleomycin (5mg / kg, 5mg / mL) modeling was performed in the PFND-mPEG-CS-MS inhalation powder test group rats, PFND-mPEG-MS-CS preparation (3mg / kg, 5mg / mL) drug was administered to the lungs. After 24 days of administration, all rats were anesthetized, 1mL of venous blood was taken from the eyeball, centrifuged at 3000rpm for 15min, and the upper serum was taken to measure liver function indicators ALT and AST. The rats were anesthetized and killed, and the heart, liver, spleen, lung, and kidney were removed and the surface of the organs was cleaned with saline. The right lung was taken to measure the hydroxyproline (Hyp) content, and the left lung and heart, liver, spleen, and kidney were fixed in 4% paraformaldehyde for HE, Masson pathological staining, and triple-label immunofluorescence. Figure 4 As shown: The normal ranges of ALT and AST levels in rat livers are 21.53-61.75U / L and 41.47-195.65U / L, respectively. The ALT and AST levels in the livers of rats in the blank group were within the normal range; the ALT and AST levels in the livers of rats in the modeling group were 63.70U / L and 203.36U / L, respectively, slightly higher than the normal range; the ALT and AST levels in the livers of rats in the control group were 81.24U / L and 244.04U / L, respectively, significantly higher than the normal range, and significantly different from the other three groups; the ALT and AST levels in the livers of rats in the experimental group were 63.20U / L and 196.26U / L, respectively, close to the normal level, and no significant difference from the blank group. Figure 5 As shown: the hydroxyproline content in the lungs of rats in the modeling group, control group, and experimental group increased to a certain extent compared with the blank group, and there was a significant difference between the modeling group and the control group; the hydroxyproline content in the control group and the experimental group decreased compared with the modeling group, and the hydroxyproline content in the lungs of rats in the experimental group was even lower, with a significant difference from the modeling group, and no significant difference from the blank group. Figure 6-7As shown: HE pathological staining results showed that the lungs of rats in the modeling group showed severe infiltration of inflammatory cells such as granulocytes, lymphocytes, and mononuclear macrophages (black arrows), and the alveolar structure had collapsed severely. The inflammatory cell infiltration in the lungs of rats in the control group and the experimental group was alleviated compared with the modeling group, and the alveolar morphology was maintained; Masson pathological staining results showed that there were a large number of blue collagen fibers deposited in the alveolar septa of rats in the modeling group, which was significantly different from the blank group. The blue collagen fibers in the control group and the experimental group were reduced, which was significantly different from the modeling group. The reduction of collagen fibers in the experimental group was more obvious, which was significantly different from the control group. Figure 8-9 As shown: Collagen I, TGF-β and α-SMA were overexpressed in rats in the IPF modeling group, which was significantly different from the blank group. Both oral administration of PFND and PFND-mPEG-CS-MS pulmonary inhalation administration decreased the fluorescence intensity, which was significantly different from the modeling group. However, at the same drug dose, PFND-mPEG-CS-MS pulmonary inhalation administration had a more obvious advantage in downregulating TGF-β cytokines and reducing the protein expression levels of Collagen I and α-SMA. Fig.10 As shown: the myocardial cells of the four groups of rats were arranged neatly, the white pulp and red pulp structures of the spleen, and the glomerular and tubular structures of the kidneys were clearly visible; the hepatocytes of the livers of the rats in the blank group, model group and experimental group were arranged neatly, radiating from the central vein to the surrounding areas, while the hepatocytes in the control group were arranged disorderly, and vacuolar degeneration of hepatocytes and lymphocyte infiltration in the portal area were observed (black arrows). It can be seen that oral gastrointestinal administration of PFND caused a certain degree of liver damage, while pulmonary inhalation administration of PFND-mPEG-CS-MS did not cause obvious liver damage.

Claims

1. A pirfenidone sustained-release microsphere inhalation powder, characterized in that: The pirfenidone sustained-release microsphere inhalation powder is prepared from the following raw materials in parts by weight: 0.22-0.25 parts of pirfenidone raw material, 1-3 parts of carrier material, 0.2-0.8 parts of polyethylene oxide and 4-20 parts of acetic acid.

2. The pirfenidone sustained-release microsphere inhalation powder according to claim 1, characterized in that: The carrier material is a chitosan carrier material mPEG-CS modified with polyethylene glycol.

3. The method for preparing the pirfenidone sustained-release microsphere inhalation powder according to claim 1, characterized in that: The preparation method comprises the following steps: dissolving the pirfenidone raw material, carrier material and polyethylene oxide in an acetic acid aqueous solution, stirring magnetically until completely dissolved and standing for a period of time so that there are no bubbles in the mixed solution; and treating the mixed solution with electrostatic spraying to obtain the pirfenidone sustained-release microsphere inhalation powder.

4. The method for preparing the pirfenidone sustained-release microsphere inhalation powder according to claim 1, characterized in that: The preparation method of the carrier material comprises: dissolving chitosan in an acetic acid solution to prepare a chitosan solution; dissolving polyethylene glycol monomethyl ether carboxyl in the acetic acid solution, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to obtain an activation solution; dripping the chitosan solution into the activation solution, adjusting the pH and fully stirring the reaction, and after the reaction is completed, rotary evaporating the solution, dialyzing it, and freeze-drying it to obtain the carrier material mPEG-CS.

5. The method for preparing the pirfenidone sustained-release microsphere inhalation powder according to claim 1, characterized in that: The chitosan solution was dripped into the activation solution, the pH was adjusted to 5.0 with NaOH, and stirred at 20°C for 48 hours. After the reaction was completed, the solution was rotary evaporated and transferred into a 8000-14000Da dialysis bag and dialyzed with pure water for 24 hours. After the dialysis was completed, the solution was transferred to a watch dish and freeze-dried for 24 hours to obtain the carrier material mPEG-CS.

6. The method for preparing the pirfenidone sustained-release microsphere inhalation powder according to claim 1, characterized in that: The concentration of chitosan in the chitosan solution is 10 mg / mL.

7. The method for preparing the pirfenidone sustained-release microsphere inhalation powder according to claim 1, characterized in that: The concentration of the acetic acid solution is 1%.

8. The method for preparing the pirfenidone sustained-release microsphere inhalation powder according to claim 1, characterized in that: The mass ratio of polyethylene glycol monomethyl ether carboxyl, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 8-12:2-3:1-2.

9. The method for preparing the pirfenidone sustained-release microsphere inhalation powder according to claim 1, characterized in that: The electrospraying process parameters are: voltage 12.5-17.5 kV, spraying speed 0.05-0.1 mm / min, receiving distance 15-20 cm, and nozzle inner diameter 0.34-0.51 mm.

10. Use of the pirfenidone sustained-release microsphere inhalation powder according to claim 1 or 2 in the preparation of a pulmonary administration preparation that reduces or eliminates hepatotoxicity.

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