An aerosolized inhalation formulation for the treatment of chronic obstructive pulmonary disease and asthma and a method of making the same

Abstract

This invention belongs to the field of new pharmaceutical technology and relates to a nebulized inhalation formulation for treating chronic obstructive pulmonary disease (COPD) and asthma, and its preparation method. Specifically, it relates to a nebulized inhalation solution formulation of an aminotetraphenylporphyrin photosensitizer (D4) with high light sensitivity. This invention uses an aminotetraphenylporphyrin photosensitizer (D4) as the active ingredient for treating COPD and asthma. The prepared D4 nebulized inhalation formulation comprises the following components: 200-400 μg / ml of the small molecule compound D4 solution, 0.1%-0.6% w / w acrylic polymer carbopol / 3%-25% w / w poloxamer 407 / 3%-25% w / w poloxamer 188, appropriate amounts of isotonic agent, pH adjuster, and purified water. The D4 nebulized inhalation formulation prepared by this invention, after excitation with a 650nm laser, shows significant therapeutic effects on COPD and asthma. After treatment, the alveolar septa narrowed, inflammatory infiltration decreased, airway changes were alleviated, and the thickness of the airway wall, smooth muscle layer, and basement membrane layer decreased. This method is highly effective, low in toxicity, highly targeted, has high nebulization efficiency, a short treatment cycle, and the preparation method is simple, convenient, and reliable, facilitating the establishment of quality control standards in production and promoting drug quality monitoring and large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of new pharmaceutical technology and relates to a nebulized inhalation preparation for the treatment of chronic obstructive pulmonary disease and asthma and its preparation method. Specifically, it relates to a nebulized inhalation solution preparation of a porphyrin photosensitizer (D4) with high light sensitivity. Background Technology

[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, and cough.

[0003] The pathogenesis of COPD is not fully understood. Multiple factors, including oxidative stress, inflammatory responses, and protease / antiprotease imbalance, lead to tissue remodeling, resulting in chronic respiratory symptoms and airflow limitation. Recent studies suggest that autoimmune regulatory mechanisms, genetic risk factors, and lung development-related factors may also play important roles in the occurrence and development of COPD. Current treatments for COPD and asthma include pharmacological and non-pharmacological therapies: stabilizing drug therapy is the foundation of long-term management for patients with COPD and asthma, with inhaled therapy being the preferred treatment; non-pharmacological interventions mainly include smoking cessation, vaccination, and pulmonary rehabilitation.

[0004] Inhaled pulmonary drug therapy is a non-invasive method of drug delivery through the throat and bronchi. Compared with oral administration, inhalation prevents drug damage before it enters the bloodstream, thus improving bioavailability and resulting in better efficacy and safety. Major bronchodilators include β2-receptor agonists, anticholinergics, theophylline, and antibiotics, which are selected based on their effects and the patient's treatment response. However, theophylline and similar preparations may cause arrhythmias or worsen existing arrhythmias, potentially leading to palpitations; caution should be exercised in patients with heart failure, liver or kidney dysfunction, or persistent fever. Hormonal drugs cannot be used long-term, and antibiotics are prone to drug resistance and reduced efficacy. Therefore, it is necessary to continue searching for and developing new treatment methods to mitigate these adverse effects.

[0005] Aminotetraphenylporphyrin photosensitizers possess unique and significant advantages in inhaled medications. Current research indicates that these photosensitizers exhibit antibacterial and anti-inflammatory effects, demonstrating good efficacy in treating respiratory diseases such as acute pneumonia. Furthermore, their preparation methods are simple, convenient, and reliable, facilitating quality control and large-scale production. Photodynamic therapy using aminotetraphenylporphyrin photosensitizers via pulmonary nebulization may become an effective strategy for treating chronic obstructive pulmonary disease and asthma. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to address the above-mentioned problems in the current treatment of chronic obstructive pulmonary disease and asthma, and to provide a nebulized inhalation solution of a porphyrin photosensitizer (D4) with high light sensitivity, which acts locally on the lungs through inhalation via the mouth and nose.

[0007] The technical solution of the present invention is a nebulized inhalation formulation for treating chronic obstructive pulmonary disease and asthma, comprising the following components: 200-400 μg / ml small molecule compound D4 solution, 0.1%-0.6% w / w acrylic polymer carbopol / 3%-25% w / w poloxamer 407 / 3%-25% w / w poloxamer 188, appropriate amounts of isotonic agent, pH adjuster and purified water, wherein the pH value is adjustable within the range of 5-10.

[0008] The active ingredient in the nebulized inhalation formulation is a D4 small molecule compound.

[0009] The pharmaceutical polymeric excipients used in the nebulized inhalation formulation of this invention are acrylic polymers carbomer / poloxam 407 / poloxam 188.

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

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

[0012] The pharmaceutical excipients used in the nebulized inhalation formulation of the present invention include isotonic agents and pH adjusters.

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

[0014] The pH adjuster described in this invention is selected from one or more of citric acid, sodium citrate, lactic acid, sodium lactate, sodium hydroxide, or potassium hydroxide, wherein the pH value of one of the nebulized inhalation preparations for treating chronic obstructive pulmonary disease and asthma is adjusted to a range of 5-10.

[0015] The administration method used in this invention is local administration. The nebulized inhalation preparation acts directly on the lungs through inhalation via the mouth and nose, can accumulate to a high concentration in the lungs, enter the respiratory and circulatory 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 a nebulized inhalation formulation for treating chronic obstructive pulmonary disease and asthma, comprising the following preparation process: preparing a mixture of 0.1%-0.6% w / w acrylic polymer carbopol / 3%-25% w / w poloxamer 407 / 3%-25% w / w poloxamer 188 containing 200-400 μg / ml small molecule compound solution, and mixing thoroughly; adding an appropriate amount of isotonic agent and purified water to the above homogenate, dissolving it evenly by ultrasonication, and then adding an appropriate amount of pH adjuster; filtering with a 0.22-micron filter membrane, and storing in a sterile glass bottle.

[0017] The beneficial effects of this invention are as follows:

[0018] Compared with existing technologies, the D4 nebulized inhalation formulation prepared in this invention, after being excited by a 650 nm laser, exhibits significant therapeutic effects on chronic obstructive pulmonary disease and asthma. Nebulized inhalation greatly reduces the metabolic process of drug concentration by the liver and kidneys, significantly minimizing liver and kidney damage in patients. This method is highly efficient, low in toxicity, highly targeted, has high nebulization efficiency, a short treatment cycle, and the preparation method is simple, convenient, and reliable. It facilitates the establishment of quality control standards in production, which is beneficial for drug quality monitoring and large-scale production. Attached Figure Description

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

[0020] Figure 2 Image of cell staining in BALF of COPD and asthma rats;

[0021] Figure 3 The graph shows the statistical analysis of TNF-α content in the BALF of rats in the COPD and asthma groups. Detailed Implementation Plan

[0022] The invention will be further illustrated below through implementation, with the aim of providing a better understanding of the invention rather than limiting its scope of protection:

[0023] Example 1: Preparation of Nebulized Inhalation Solution

[0024] A mixture of 0.1%-0.6% w / w acrylic polymer carbopol, 3%-25% w / w poloxamer 407, and 3%-25% w / w poloxamer 188 containing 200-400 μg / ml of small molecule compound was prepared and thoroughly mixed. An appropriate amount of isotonic agent and purified water was added to the homogenate, and the mixture was sonicated until homogeneous. Then, an appropriate amount of pH adjuster was added. The mixture was filtered through a 0.22-micron filter membrane and stored in sterile glass bottles. The pH and viscosity were examined, and droplets were atomized using an atomizer to examine particle size and adhesion, assessing the likelihood of formulation formation. Results showed that each carbopol 940 formulation was a purple solution, while the carbopol 971P formulation was a clear purple solution. The 0.4% and 0.6% w / w carbomer 940 formulations and the 0.4% and 0.6% carbomer 971P formulations had high viscosity and low pH, which caused blockage when sprayed as droplets by nebulizers, and thus could not be formulated into ideal inhalation formulations. The 0.1% and 0.2% carbomer 940 formulations and the 0.1% and 0.2% carbomer 971P formulations had moderate viscosity and pH that was more suitable for pulmonary administration, and could be sprayed as droplets by nebulizers.

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

[0026] Rat feeding

[0027] Male SD rats, SPF grade, weighing 230-250 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd. After being introduced and tested according to requirements, the rats were housed in separate cages with free access to food and water. The room temperature was controlled at 23±2℃, humidity at 50±10%, and a 12 / 12 h light / dark cycle was maintained. After 5 days of acclimatization observation, the animals were weighed for experiments. All animal experimental procedures were approved by the Laboratory 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, then bring the volume to 2.5 ml to obtain a 5% NaOH solution.

[0031] Aluminum hydroxide gel

[0032] Weigh 0.61 g Al2(SO4)·18H2O, add ddH2O and stir until completely dissolved. Make up to 6.25 ml to obtain a 5% Al2(SO4) solution. Slowly add 2.5 ml of prepared 5% NaOH solution while stirring continuously. Centrifuge at 4000 rpm for 15 min and discard the supernatant. Wash the precipitate twice with 10 ml of physiological saline, centrifuge at 4000 rpm for 10 min, and make up to 6.25 ml with physiological saline to obtain aluminum hydroxide gel, ready for immediate use.

[0033] OVA solution

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

[0035] LPS solution

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

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

[0038] COPD group

[0039] Thirty-six SD rats were weighed and then randomly divided into six groups of six rats each.

[0040] COPD group: On days 15 and 28 of the experiment, after anesthesia, rats were instilled with 200 μL of 0.6 mg / ml LPS solution via tracheal instillation. On days 1-14, 16-27, and 29-62, rats were placed in a self-made small animal smoke inhalation chamber (temperature 25±3℃, oxygen >18.5%, smoke concentration 250-350 ppm) to establish a COPD model, 60 min each time, once daily. Blank control group: Rats received intraperitoneal injection and tracheal instillation of the same volume of physiological saline as the other groups.

[0041] Asthma group

[0042] On days 1, 7, and 14 of the experiment, rats were sensitized and immune-boosted by intraperitoneal injection of 1 ml of a mixture (200 μL of 0.30 mg / ml OVA solution + 800 μL of aluminum hydroxide gel). From days 26 to 33, a 2% OVA saline solution was prepared and administered to each rat via ultrasonic nebulizer, with 1 ml inhaled daily for 7 consecutive days. The blank control group received the same volume of saline solution as the other groups via intraperitoneal injection and nebulized inhalation.

[0043] Treatment begins after model establishment. Prepare 5 ml (approximately 230g in weight) of nebulized medication in a centrifuge tube, avoiding light. Inhale the medication via nebulizer for 10-30 minutes to ensure even distribution. 1-2 hours after administration, turn on the 7407 semiconductor laser at a specific wavelength of 650 nm. Insert a 0.7 mm optical fiber, adjusting the fiber's output power to 10-40 mW. Insert the 0.7 mm fiber into the trachea at the glottis, controlling the insertion depth to 8-9 cm. Preheat the laser for 30 minutes before the experiment. 1-2 hours after administration, irradiate the animal with light for 10-30 minutes after inserting the fiber. Treat every other day, for a total of 3-6 treatments. After treatment, protect the animal from light.

[0044] Five days after treatment and recovery, the animals were euthanized, samples were collected, and subsequent experimental testing and analysis were conducted.

[0045] Sample collection

[0046] Blood was collected from the abdominal aorta, and the rat was then euthanized by exsanguination. The blood was completely drained, and the thoracic cavity was opened. The lungs were separated, and the right four lobes of the lung were clamped with hemostatic forceps. The trachea was separated, a notch was cut, and a soft tube was inserted. The left lung was repeatedly irrigated three times with 3 ml of 4°C physiological saline, each time for 5 seconds. All bronchoalveolar lavage fluid (BALF) was collected into a 1 ml centrifuge tube and centrifuged at 1500 rpm, 4°C for 10 min. The supernatant was collected, and the total protein content in the lavage fluid was determined using the Coomassie Brilliant Blue method. The right lungs were allocated as follows: the right anterior lobe lung, after removing connective tissue and absorbing water with filter paper, was used for lung coefficient determination; the right middle lobe lung was prepared into paraffin sections, fixed in 4% paraformaldehyde, and used for pathological examination; the right third lobe lung was immersed in RNA preservation solution and stored at 4°C overnight, then transferred to -80°C; the right fourth lobe lung was completely immersed in liquid nitrogen for preservation and used for qPCR and Western blot experiments.

[0047] Collection of serum and bronchoalveolar lavage fluid and differential counting of inflammatory cells

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

[0049] Morphological specimen processing

[0050] (1) Fixation: Cover the right lung lobe with gauze, fix it with paper clips, and then fix it in 4% paraformaldehyde solution. Note that the specimen should be completely immersed and avoid floating on the surface of the liquid.

[0051] (2) After 24 hours of fixation, the tissue was removed and the right lung lobe was placed into the dehydration box in the 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 paraffin-impregnated tissue is embedded in an embedding machine. After the paraffin melts, it is laid into the embedding frame. Before solidification, the left lung lobe is placed and labeled. Then it is placed on a 0°C freezing stage to cool. After the paraffin solidifies, the paraffin block is separated from the embedding frame.

[0054] (5) Sectioning: Set the microtome to a thickness of 5 μm, and let the sections float on the surface of 40℃ warm water in the slide spreader. After the tissue is flattened, pick it up with a glass slide and place it in a 60℃ oven to bake. After drying, remove it and store it at room temperature for later use.

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

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

[0057] 2) After staining with hematoxylin for 15 minutes, wash away excess staining solution with tap water.

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

[0059] 4) Stain with 1% eosin solution for 10 min, then wash away 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 I, II → 100% anhydrous ethanol I, I, 2 min each. Xylene I, II for clearing, 10 min each, then mount with neutral resin.

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

[0062] ELISA method for detecting inflammatory factor levels in rat BALF

[0063] The TNF-α content in lung BALF was measured according to the supplier's kit instructions.

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

[0065] (2) Determine the amount 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× WashBuffer 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 of 10×CaptureAntibody and 300 μL of 10×DetectorAntibody with 2.4 ml of Antibody diluent to make a 3 ml antibody mixture and mix gently.

[0068] (5) Preparation of standard products:

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

[0070] ② Label 8 EP tubes as standards 1-8, and add 150 μL of sample diluted with physiological saline to tubes 1-8.

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

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

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

[0074] (8) Washing the plate: Discard the liquid in the plate and wash each well with 350 μL of 1×Wash Buffer for a total of 3 times. After completion, invert the 96-well plate onto 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) Termination: Add 100 μL of termination solution to each well and mix well. 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 dependent variable y and standard concentration as 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] In the COPD control group, the lung tissue structure of rats was relatively intact, with clear alveolar walls and normal alveolar septa. In contrast, the alveolar septa of rats in the COPD group were significantly enlarged, the airway walls were thickened, and the alveolar walls were damaged in multiple places, with some showing severe inflammatory infiltration. After treatment, the alveolar septa and airway walls became smaller, and the inflammatory infiltration was also reduced. From a histomorphological perspective, the treatment group improved the abnormal lung tissue morphology caused by COPD.

[0081] In the asthma control group, the bronchial tissue structure of rats was normal, with no inflammatory cell infiltration around the airway and no thickening of the airway wall and smooth muscle. In the model group, a large number of inflammatory cells were observed around the airway, the airway lumen was significantly narrowed, and the thickness of the airway wall, smooth muscle layer and basement membrane layer was significantly increased. In the treatment group, the airway changes were all alleviated, with a small number of inflammatory cells infiltrating around the airway and a decrease in the thickness of the airway wall, smooth muscle layer and basement membrane layer.

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

[0083] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. The use of a nebulized inhalation formulation in the preparation of a medicament for treating chronic obstructive pulmonary disease and asthma, characterized in that, The nebulized inhalation formulation comprises the following components: 200-400 μg / ml small molecule compound D4 solution, 0.1%-0.6% w / w acrylic polymer carbomer, 3%-25% w / w poloxamer 407, 3%-25% w / w poloxamer 188, isotonic agent, pH adjuster, and purified water, wherein the pH value is adjustable within the range of 5-10; The small molecule compound D4 has the following structural formula: 。 2. The application according to claim 1, characterized in that, The carbomer is selected as one or more of carbomer 940, carbomer 941, carbomer 971P, and carbomer 974P.

3. The application according to claim 1, characterized in that, The isotonic agent is selected from one or more of sodium chloride, potassium chloride, and glucose.

4. The application according to claim 1, characterized in that: The pH adjuster is selected from one or more of citric acid, sodium citrate, lactic acid, sodium lactate, sodium hydroxide, or potassium hydroxide.

5. The method for preparing the nebulized inhalation formulation according to claim 1, characterized in that, The preparation process includes the following steps: preparing a solution containing 200-400 μg / ml of small molecule compound D4, 0.1%-0.6% w / w acrylic polymer carbomer, 3%-25% w / w poloxamer 407, and 3%-25% w / w poloxamer 188, mixing thoroughly to obtain a homogenate; adding an appropriate amount of isotonic agent and purified water to the above homogenate, dissolving it evenly by ultrasonication, and then adding an appropriate amount of pH adjuster; filtering with a 0.22-micron filter membrane and storing in a sterile glass bottle.

Images