Aerosol inhalation preparation for treating chronic obstructive pulmonary disease and asthma and preparation method thereof

Abstract

The invention belongs to the new technical field of pharmacy, and relates to an aerosol inhalation preparation for treating chronic obstructive pulmonary disease and asthma and a preparation method of the aerosol inhalation preparation. In particular to an amino tetraphenyl porphyrin photosensitizer (D4) aerosol inhalation solution preparation with high sensitivity and light sensing. According to the invention, an amino tetraphenyl porphyrin photosensitizer (D4) is used as an active ingredient for treating chronic obstructive pulmonary disease and asthma, and the prepared D4 aerosol inhalation preparation comprises the following components: 200-400 [mu] g / ml of a small molecule compound D4 liquid medicine, 0.1-0.6% w / w of acrylic polymer Carbopol, 3-25% w / w of poloxamer 407, 3-25% w / w of poloxamer 188, a proper amount of an isotonic agent, a pH regulator and purified water. The D4 aerosol inhalation preparation prepared by the invention has a remarkable treatment effect on chronic obstructive pulmonary disease and asthma after being excited by 650nm laser. After treatment, alveolar spacing is reduced, inflammatory infiltration is reduced, airway change is relieved, and the thicknesses of airway walls, smooth muscle layers and basilar membrane layers are reduced. The method is efficient, low in toxicity, high in targeting, high in atomization efficiency and short in treatment period, the preparation method is simple, convenient and reliable, quality control standards can be conveniently formulated in production, and quality monitoring and large-scale production of drugs are facilitated.

Description

Technical Field

[0001] The present invention belongs to the new pharmaceutical technology field, and relates to an atomized inhalation preparation for treating chronic obstructive pulmonary disease and asthma and a preparation method thereof, and specifically relates to an atomized inhalation solution preparation of a porphyrin photosensitizer (D4) with high photosensitivity. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) and asthma (AS) are common respiratory diseases. Their main symptoms include fever, headache, nasal congestion, runny nose, sore throat, cough, etc.

[0003] The pathogenesis of COPD is not yet fully understood. Oxidative stress, inflammatory response, protease / antiprotease imbalance and other factors lead to tissue remodeling, which in turn leads to chronic respiratory symptoms and airflow limitation. Recent studies have shown that autoimmune regulatory mechanisms, genetic risk factors and lung development-related factors may also play an important role in the occurrence and development of COPD. Currently, the treatment of COPD and asthma includes drug therapy and non-drug therapy: stable drug therapy is the basis for long-term management of patients with COPD and asthma, among which inhalation therapy is the preferred treatment method; non-drug interventions mainly include smoking cessation, vaccination and pulmonary rehabilitation.

[0004] Pulmonary inhalation drug therapy is a non-invasive drug delivery method through the throat and bronchi. Compared with oral administration, inhaled local administration can prevent the drug from being damaged before entering the blood circulation, thereby improving the bioavailability of the drug. This method has better efficacy and safety. The main bronchodilators include β2 receptor agonists, anticholinergic drugs, theophylline drugs and antibacterial drugs, which can be selected according to the effects of the drugs and the patient's treatment response. However, theophylline preparations may cause arrhythmias or worsen existing arrhythmias, and patients may experience symptoms such as palpitations; they should be used with caution in patients with heart failure, liver and kidney dysfunction, or persistent fever. Hormonal drugs cannot be used for a long time, and antibacterial drugs are prone to adverse consequences such as drug resistance and reduced efficacy. Therefore, it is necessary to continue to find and develop new treatments to alleviate the current adverse effects.

[0005] Aminotetraphenylporphyrin photosensitizers have unique and significant advantages in inhaled drugs. Current studies have shown that this type of photosensitizer has antibacterial and anti-inflammatory effects, and has shown good efficacy in the treatment of respiratory diseases such as acute pneumonia. At the same time, the preparation method is simple, convenient and reliable, which is conducive to drug quality control and large-scale production. Aminotetraphenylporphyrin photosensitizers may become an effective strategy for photodynamic therapy of chronic obstructive pulmonary disease and asthma through pulmonary atomization. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a porphyrin photosensitizer (D4) atomized inhalation solution preparation with high light sensitivity, which acts locally on the lungs through oral and nasal inhalation, in view of the above problems existing in the current treatment of chronic obstructive pulmonary disease and asthma.

[0007] The technical solution of the present invention is an aerosol inhalation preparation for treating chronic obstructive pulmonary disease and asthma, which comprises the following components: 200-400 μg / ml small molecule compound D4 liquid, 0.1%-0.6% w / w acrylic acid polymer Carbopol / 3%-25% w / w Poloxamer 407 / 3%-25% w / w Poloxamer 188, appropriate amount of isotonic agent, pH regulator and purified water, wherein the adjustment range of the pH value is 5-10.

[0008] The effective active ingredient of the aerosol inhalation preparation is a D4 small molecule compound.

[0009] The pharmaceutical polymer excipients selected for the atomized inhalation preparation of the present invention are acrylic acid polymer carbomer / poloxamer 407 / poloxamer 188.

[0010] The acrylic acid polymer carbomer described in the present invention is selected from one or more of Carbomer 940, Carbomer 941, Carbomer 971P and Carbomer 974P.

[0011] Preferably, the acrylic acid polymer carbomer has a concentration of 0.1%-0.6% w / w.

[0012] The pharmaceutical excipients selected for the atomized inhalation preparation of the present invention include isotonic agents and pH regulators.

[0013] As a preferred embodiment, the isotonic agent is selected from a mixture of one or more of sodium chloride, potassium chloride, glucose and the like.

[0014] The pH regulator of the present invention is selected from a mixture of one or more of citric acid, sodium citrate, lactic acid, sodium lactate, sodium hydroxide or potassium hydroxide, wherein the pH value of an aerosol inhalation preparation for treating chronic obstructive pulmonary disease and asthma is adjusted to a range of 5-10.

[0015] The administration method used in the present invention is local administration. The aerosol inhalation preparation acts directly on the lungs through oral and nasal inhalation, can accumulate into a higher concentration in the lungs, enter the respiratory and blood circulation systems and then act on the whole body, greatly reducing liver and kidney damage.

[0016] Another object of the present invention is to provide a method for preparing an aerosol inhalation preparation for treating chronic obstructive pulmonary disease and asthma, comprising the following preparation process: preparing 0.1%-0.6% w / w acrylic acid polymer Carbopol / 3%-25% w / w poloxamer 407 / 3%-25% w / w poloxamer 188 containing 200-400 μg / ml small molecule compound liquid, and mixing them thoroughly; adding an appropriate amount of isotonic agent and purified water to the above homogenate, and then adding an appropriate amount of pH regulator after ultrasonic dissolution; filtering with a 0.22 micron filter membrane and storing in a sterilized glass bottle.

[0017] The beneficial effects of the present invention are:

[0018] Compared with the prior art, the D4 atomized inhalation preparation prepared by the present invention has a significant therapeutic effect on chronic obstructive pulmonary disease and asthma after 650nm laser excitation, and atomized inhalation greatly reduces the metabolic process of drug concentration by the liver and kidney, greatly reducing liver and kidney damage to patients. The method is efficient and low toxic, has strong targeting, high atomization efficiency, and a short treatment cycle. The preparation method is simple, convenient and reliable, which is convenient for formulating quality control standards in production, and is conducive to quality monitoring and large-scale production of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 H&E staining of pathological sections of lung tissues of rats in COPD and asthma groups;

[0020] Figure 2 The images are of cell staining in BALF of rats in COPD and asthma groups;

[0021] Figure 3 This is a statistical analysis of TNF-a content in BALF of rats in the COPD and asthma groups. Specific implementation methods

[0022] The present invention is further described below by way of implementation, the purpose of which is only to better understand the content of the present invention but not to limit the protection scope of the present invention:

[0023] Example 1 Preparation of atomized inhalation liquid medicine

[0024] Prepare 0.1%-0.6% w / w acrylic acid polymer Carbopol / 3%-25% w / w poloxamer 407 / 3%-25% w / w poloxamer 188 containing 200-400 μg / ml small molecule compound solution, mix thoroughly; add appropriate amount of isotonic agent and purified water to the above homogenate, ultrasonically dissolve evenly, and then add appropriate amount of pH regulator; filter with 0.22 micron filter membrane, and store in sterilized glass bottle. Inspect its pH and viscosity, and spray droplets with a nebulizer to examine the particle size and adhesion degree, and evaluate the possibility of forming a preparation. The results show that each Carbomer 940 preparation is a purple solution, and the Carbomer 971P preparation is a purple clear solution. 0.4%, 0.6% w / w Carbomer 940 preparations and 0.4%, 0.6% Carbomer 971P preparations had high viscosity and low pH, and clogging occurred when spraying droplets with a nebulizer, and they failed to be made into ideal inhalation preparations; 0.1%, 0.2% Carbomer 940 preparations and 0.1%, 0.2% Carbomer 971P preparations had moderate viscosity and a pH that was more suitable for pulmonary administration, and droplets could be sprayed with a nebulizer.

[0025] Example 2 In vivo pharmacodynamic study of photodynamic therapy

[0026] Rat husbandry

[0027] SD rats, male, SPF grade, weighing 230-250g, were purchased from Beijing Huafukang Biotechnology Co., Ltd. Rats were introduced and inspected as required, and then housed in cages with free access to food and water. The room temperature was controlled at 23±2℃, humidity was 50±10%, and the light-dark cycle was 12 / 12h. After the animals were adapted for 5 days, they were weighed and the experiment was carried out. All animal experimental operations have been approved by the Experimental Animal Ethics Committee of the Institute of Biomedical Engineering, Chinese Academy of Medical Sciences.

[0028] Reagent preparation

[0029] Sodium hydroxide solution

[0030] Weigh 0.125 g of NaOH, add ddH2O and stir until completely dissolved, and dilute to 2.5 ml to obtain a 5% NaOH solution.

[0031] Aluminum Hydroxide Gel

[0032] Weigh 0.61g Al2(SO4)·18H2O, add ddH2O and stir until completely dissolved, dilute to 6.25ml to obtain 5% Al2(SO4) solution, slowly add 2.5ml prepared 5% NaOH solution under constant stirring, centrifuge at 4000rpm for 15min, and discard the supernatant. Wash the precipitate twice with 10ml of normal saline, centrifuge at 4000rpm for 10min, and dilute to 6.25ml with normal saline to obtain aluminum hydroxide gel, which is ready for use.

[0033] OVA solution

[0034] Weigh 3.0 mg, 12.0 mg and 16.0 mg of OVA respectively, stir with physiological saline until completely dissolved, make up to 10 ml, and obtain final concentrations of 0.30 mg / ml, 1.20 mg / ml and 1.60 mg / ml OVA solutions respectively, and store at 4°C for later use.

[0035] LPS solution

[0036] Weigh 3.0 mg, 4.0 mg and 6.0 mg LPS respectively, stir with physiological saline until completely dissolved, make up to 10.0 ml, and obtain final concentrations of 0.30 mg / ml, 0.40 mg / ml and 0.60 mg / ml LPS solutions respectively, and store at 4°C for later use.

[0037] Establishment and treatment of COPD and asthma rat models

[0038] COPD Group

[0039] Eighteen SD rats were weighed and randomly divided into six groups, with six rats in each group.

[0040] COPD group: On the 15th and 28th days of the experiment, the rats were anesthetized and 200 μL of 0.6 mg / ml LPS solution was instilled into the trachea. On the 1st to 14th days, 16th to 27th days and 29th to 62nd days, the rats were placed in a homemade small animal smoke inhalation box (temperature 25±3℃, oxygen>18.5%, smoke concentration 250-350ppm) to prepare the COPD model, 60 minutes each time, once a day. Blank control group: intraperitoneal injection and tracheal instillation of the same volume of normal saline as other groups.

[0041] Asthma Group

[0042] On the 1st, 7th and 14th days of the experiment, rats were intraperitoneally injected with 1 ml of the mixture (200 μL of 0.30 mg / ml OVA solution + 800 μL of aluminum hydroxide gel) for sensitization and immune boosting. On the 26th to 33rd days, 2% OVA saline solution was prepared and each rat was challenged by inhaling 1 ml per day through an ultrasonic nebulizer, once a day for 7 consecutive days. Blank control group: intraperitoneal injection, nebulization inhalation and other groups were given the same volume of saline.

[0043] Treatment begins after modeling is completed. Prepare 5 ml of atomized drug solution (weight is about 230 g) in a centrifuge tube in a dark place, and inhale with a nebulizer for 10-30 minutes to allow the drug to be evenly distributed. 1-2 hours after administration, turn on the semiconductor laser 7407, the specific wavelength is 650nm, insert a 0.7mm optical fiber, adjust the power of the optical fiber light output to 10-40mW, take a 0.7mm optical fiber and insert it into the trachea from the glottis, control the depth of optical fiber insertion, and the insertion depth is 8-9cm. Preheat for 30 minutes before the experiment. 1-2 hours after administration, insert the optical fiber and illuminate for 10-30 minutes. Treat once every other day, and a total of 3-6 treatments are required. After the treatment is completed, the animals should be protected from light.

[0044] After 5 days of recovery from treatment, the animals were sacrificed, and samples were collected for subsequent experimental testing and analysis.

[0045] Collecting samples

[0046] Blood was collected from the abdominal aorta, and then the rats were killed by exsanguination. The blood was completely drained, and then the chest cavity was opened, the lungs were separated, and the right 4 lobes of the rat lung were clamped with hemostatic forceps. The trachea was separated, a notch was cut, and a hose was inserted. The left lung was repeatedly lavaged 3 times with 3ml of 4℃ normal saline, and each time was kept for 5s. The alveolar lavage fluid (BALF) was recovered in a 1ml centrifuge tube, centrifuged at 1500rpm and 4℃ for 10min, and the supernatant was taken. The total protein content in the lavage fluid was determined by the Coomassie Brilliant Blue method. The right lung was divided as follows: the connective tissue of the right anterior lobe was removed and the water was absorbed with filter paper, which was used for the determination of the lung coefficient; the paraffin section of the right middle lobe was prepared, immersed in 4% paraformaldehyde for fixation, and used for pathological detection; the right third lobe was immersed in RNA preservation solution and stored at 4℃ overnight and then transferred to -80℃; the right fourth lobe was completely immersed in liquid nitrogen for preservation, and used for qPCR and Western blot experiments.

[0047] Collection of serum and lung lavage fluid and classification and counting of inflammatory cells

[0048] By performing Wright-Giemsa staining on BALF and analyzing the inflammatory cells and immune cells in the blood for cell counting, many lung diseases (sarcoidosis, allergic alveolitis, idiopathic pulmonary fibrosis, and lung malignant tumors, etc.) can be identified, diagnosed, and the therapeutic effect observed.

[0049] Morphological specimen processing

[0050] (1) Fixation: Cover the right lung lobe with gauze, fix it with a paper clip, and place it in a 4% paraformaldehyde solution for fixation. Make sure the specimen is completely immersed in the solution to avoid floating on the liquid surface.

[0051] (2) After fixation for 24 hours, remove the tissue and place the right lung lobe into a dehydration box in a fume hood.

[0052] (3) Gradient alcohol dehydration: 70% alcohol → 70% alcohol → 80% alcohol → 80% alcohol → 90% alcohol → 95% alcohol → anhydrous ethanol 1 → anhydrous ethanol II → xylene → xylene → paraffin → paraffin.

[0053] (4) Embedding: The wax-impregnated tissue is embedded in an embedding machine. After the wax melts, it is spread into the embedding frame. Before it solidifies, the left lung lobe is placed and marked. Then it is placed in a 0° freezing table to cool. After the wax solidifies, the wax block is separated from the embedding frame.

[0054] (5) Sectioning: Set the slice thickness to 5 μm on the slicer. Let the slice float on the surface of 40°C warm water on the slicer. After the tissue is flattened, pick it up with a slide and put it into a 60°C oven for baking. After it is dry, take it out and store it at room temperature for later use.

[0055] H&E staining to detect pathological changes in lung tissue

[0056] 1) Dewaxing to water: Place the rat lung tissue slices in a 60°C oven for 1 hour. After the wax is completely melted, place the slices in xylene 1, 2 for 15 minutes each, anhydrous ethanol 1, 2 → 95% alcohol 1, 2 → 90% alcohol → 80% alcohol → 70% alcohol → 60% alcohol → 50% alcohol for 2 minutes each, and gently rinse with tap water for 5 minutes.

[0057] 2) After 15 minutes of hematoxylin staining, wash away the excess stain with tap water.

[0058] 3) Differentiate with ethanol containing 1% hydrochloric acid for 30 seconds and wash with tap water for 10 minutes.

[0059] 4) Stain with 1% eosin solution for 10 min and wash off excess stain with tap water for 1 min.

[0060] 5) Gradient alcohol dehydration: 50% alcohol → 60% alcohol → 70% alcohol → 80% alcohol → 90% alcohol → 95% alcohol 1, II → 100% anhydrous ethanol 1, I for 2 min each. Xylene 1, II for 10 min each, and neutral gum for sealing.

[0061] 6) Observe the changes in lung tissue and bronchial wall, as well as the infiltration of inflammatory cells under an optical microscope.

[0062] ELISA method to detect the levels of inflammatory factors in rat BALF

[0063] The TNF-a content in lung BALF was determined according to the kit instructions provided by the supplier.

[0064] (1) Equilibrate all samples to be tested and ELISA test kits to room temperature (18-25°C).

[0065] (2) Determine the number of 96-well plates to be used in the experiment based on the number of samples (15, with 3 replicates for each sample).

[0066] (3) Prepare 50 ml of 1× Wash Buffer by mixing 5 ml of Wash Buffer concentrate (10×) with 45 ml of distilled water.

[0067] (4) Prepare an antibody mixture using antibody diluent, capture antibody and detection antibody: Mix 300 μL 10×Capture Antibody and 300 μL 10×Detector Antibody with 2.4 ml Antibody diluent to make 3 ml of antibody mixture and mix gently.

[0068] (5) Preparation of standard products:

[0069] ① Add 500 μL sample dilution saline to dissolve rat TNF-a recombinant protein lyophilized powder, fully dissolve and mix, gently mix at room temperature for 10 minutes to obtain a 2000 pg / ml standard solution.

[0070] ② Label 8 EP tubes as standard samples 1-8, and add 150 μL of sample diluted with saline into tubes 1-8.

[0071] ③ Use the standard solution to prepare a series of dilutions. Add 150 μL of the prepared 2000 pg / ml standard solution to tube 1 to prepare a 1000 pg / ml standard solution. Add 150 μL of the prepared 1000 pg / ml standard solution to tube 2 to prepare a 500 pg / ml standard solution. Add 150 μL of the prepared 500 pg / ml standard solution to tube 3 to prepare a 250 pg / ml standard solution. Prepare standard solutions of 125 pg / ml, 62.5 pg / ml, 31.3 pg / ml and 15.6 pg / ml in tubes 4, 5, 6 and 7 respectively. Tube 8 is the blank control.

[0072] (6) Sample addition: Add 50 μL of sample (BALF) or standard to the appropriate wells, and then add 50 μL of antibody mixture to each well. Be careful to operate gently and avoid creating bubbles.

[0073] (7) Incubation: Seal the plate with a film and incubate on a shaker set at 400 rpm / min at room temperature for 1 h.

[0074] (8) Washing: Discard the liquid in the well plate and wash each well with 350 μL 1× Wash Buffer for 3 times. After completion, turn the 96-well plate upside down on a clean paper towel to remove excess liquid.

[0075] (9) Color development: Add 100 μL of TMB matrix to each well and incubate in a shaker at 400 rpm for 10 min in the dark. The liquid in the well plate will turn blue.

[0076] (10) Stop: Add 100 μL of stop solution to each well and mix until the blue liquid turns yellow.

[0077] (11) Set the microplate reader to 450 nm to measure the OD value.

[0078] (12) Calculation: With OD value as the dependent variable y and standard concentration as the independent variable x, calculate the standard equation and then substitute the OD value of the measured sample into the equation to obtain the TNF-a concentration.

[0079] Experimental Results

[0080] The lung tissue structure of rats in the COPD blank control group was relatively complete, with clear alveolar walls and normal alveolar septa; while the alveolar septa of rats in the COPD group were significantly enlarged, the airway walls were thickened, the alveolar walls were destroyed in many places, and some had severe inflammatory infiltration; after treatment, the alveolar septa and airway walls became smaller, and the inflammatory infiltration was also less. From the perspective of tissue morphology, the treatment group improved the abnormal lung tissue morphology caused by COPD.

[0081] The bronchial tissue structure of rats in the asthma control group was normal, there was no inflammatory cell infiltration around the airway, and the airway wall and smooth muscle were not thickened. A large number of inflammatory cells infiltrated around the airway of rats in the model group, the airway lumen was significantly narrowed, and the thickness of the airway wall, smooth muscle layer and basement membrane layer increased significantly; the airway changes in the treatment group were alleviated, a small amount of inflammatory cells infiltrated around the airway, and the thickness of the airway wall, smooth muscle layer and basement membrane layer decreased.

[0082] Compared with the control group, the amount of TNF-a in the BALF of rats after stimulation was significantly increased, while the content in both the COPD and asthma groups decreased after treatment.

[0083] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. An aerosol inhalation preparation for treating chronic obstructive pulmonary disease and asthma and a preparation method thereof, characterized in that: The solution preparation of D4 for nebulized inhalation comprises the following components: 200-400 μg / ml small molecule compound D4 liquid, 0.1%-0.6% w / w acrylic acid polymer Carbopol / 3%-25% w / w Poloxamer 407 / 3%-25% w / w Poloxamer 188, appropriate amount of isotonic agent, pH adjuster and purified water, wherein the pH value is adjusted in the range of 5-10.

2. The aerosol inhalation preparation for treating chronic obstructive pulmonary disease and asthma and its preparation method according to claim 1, characterized in that: The active ingredient of the aerosol inhalation preparation is a small molecule compound D4, which has the following structural formula.

3. The aerosol inhalation preparation for treating chronic obstructive pulmonary disease and asthma and its preparation method according to claim 1, characterized in that: The pharmaceutical polymer auxiliary material selected for the aerosol inhalation preparation is acrylic acid polymer Carbopol.

4. The acrylic acid polymer Carbopol according to claim 3, characterized in that: The carbomer is selected from one or more of carbomer 940, carbomer 941, carbomer 971P, and carbomer 974P.

5. The acrylic acid polymer Carbopol according to claim 3, characterized in that: The concentration of carbomer is 0.1%-0.6% w / w.

6. Poloxamer 407 and Poloxamer 188 according to claim 1, characterized in that: The concentration of poloxamer 407 is 3%-25% w / w; the concentration of poloxamer 188 is 3%-25% w / w.

7. The aerosol inhalation preparation for treating chronic obstructive pulmonary disease and asthma and its preparation method according to claim 1, characterized in that: The pharmaceutical excipients include isotonic agents and pH regulators.

8. The aerosol inhalation preparation for treating chronic obstructive pulmonary disease and asthma and its preparation method according to claim 1, characterized in that: The isotonic agent is selected from a mixture of one or more of sodium chloride, potassium chloride, glucose and the like.

9. The aerosol inhalation preparation for treating chronic obstructive pulmonary disease and asthma and its preparation method according to claim 1, characterized in that: The pH regulator is selected from a mixture of one or more of citric acid, sodium citrate, lactic acid, sodium lactate, sodium hydroxide or potassium hydroxide, wherein the pH value of an aerosol inhalation preparation for treating chronic obstructive pulmonary disease and asthma is adjusted to a range of 5-10.

10. The aerosol inhalation preparation for treating chronic obstructive pulmonary disease and asthma and its preparation method according to claim 1, characterized in that The preparation process includes the following steps: preparing 0.1%-0.6% w / w acrylic acid polymer Carbopol / 3%-25% w / w poloxamer 407 / 3%-25% w / w poloxamer 188 containing 200-400 μg / ml small molecule compound solution, and fully mixing; adding a proper amount of isotonic agent and purified water to the above homogenate, and then adding a proper amount of pH regulator after ultrasonic dissolution; filtering with a 0.22 micron filter membrane and storing in a sterilized glass bottle.

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