CN115645381B

CN115645381B - Levalmol hydrochloride aerosol inhalation solution sustained release agent and preparation method thereof

Info

Publication number: CN115645381B
Application number: CN202211287768.2A
Authority: CN
Inventors: 刘艳华
Original assignee: Shenzhen Daphne Pharmaceutical Co ltd
Current assignee: Shenzhen Daphne Pharmaceutical Co ltd
Priority date: 2022-10-20
Filing date: 2022-10-20
Publication date: 2023-05-30
Anticipated expiration: 2042-10-20
Also published as: CN115645381A

Abstract

The invention discloses a levalbuterol hydrochloride aerosol inhalation solution sustained release agent and a preparation method thereof, mainly comprising the following components: the preparation method comprises the steps of compound nano-particles of levalbuterol hydrochloride, an isotonic agent, a pH regulator and a solvent. The levalbuterol hydrochloride composite nano-particle is obtained by complexing a composite network of iron and tannic acid and the encapsulated levalbuterol hydrochloride dimer, has long-term and excellent stability, can not be released prematurely, has good biocompatibility, and can also meet the current loading requirements of medicines. Inhalation administration can improve patient compliance and also alleviate or avoid some adverse drug reactions.

Description

Levalmol hydrochloride aerosol inhalation solution sustained release agent and preparation method thereof

Technical Field

The invention relates to the technical field of pharmaceutical preparations, in particular to a levalbuterol hydrochloride aerosol inhalation solution sustained release agent and a preparation method thereof.

Background

Salbutamol (Albuterol) is an adrenergic beta receptor agonist, which can bind to beta 2 receptor on human respiratory smooth muscle, increase the concentration of cyclic adenosine monophosphate (cAMP) in cells, and the cAMP further activates protein kinase A to phosphorylate muscle protein, reduce the concentration of calcium ions in cells and relax bronchus smooth muscle. Increased cAMP concentrations also inhibit the release of mast cell inflammatory mediators. Salbutamol is a racemate, wherein only the levorotatory body has the effect of dilating bronchial smooth muscle, and the dextrorotatory body has no effect, but can be combined with beta receptor to generate side effects such as headache, dizziness, palpitation, finger tremor and the like. The levosalbutamol is a single optical isomer of the salbutamol, the drug effect is 80 times of that of the salbutamol, the side effect is reduced, the curative effect is further improved, the same curative effect can be produced by only 1/4 of the dosage of the raceme of the levosalbutamol, and the effect is better than that of the raceme at 1/2 of the dosage. In Chronic Obstructive Pulmonary Disease (COPD), the pro-inflammatory response of levalbuterol is weaker than albuterol. The traditional Chinese medicine has obvious bronchiectasis effect, and is the first choice medicine for clinically treating bronchial asthma, asthmatic bronchitis, bronchospasm of emphysema patients, acute asthma attack and the like at present.

Levalbuterol currently relieves asthma by oral ingestion, intravenous injection and aerosol inhalation. The average retention time of the whole body of the levalbuterol is 3 hours, the elimination half-life period is 3-4 hours when the levalbuterol is taken orally, the bioavailability is about 30-50% when the levalbuterol is taken orally, and the bioavailability is reduced by 14.8%. The biological half-life of levalbuterol is short, so frequent administration is required to maintain bronchodilatory effect, and in order to use levalbuterol for treating nocturnal asthma, development of a sustained-release long-acting levalbuterol dosage form has been receiving extensive attention, and many researches have focused on developing a sustained-release long-acting oral levalbuterol form. The atomized inhalation has the advantages of faster curative effect, strong targeting, avoidance of first pass effect, less administration dosage, capability of reducing toxic and side effects of the drug on other tissues of the whole body, and is the safest and effective method for treating asthma at present, and is proposed by WHO global asthma prevention and treatment as the first choice therapy. Inhaled aerosolized β2 receptor agonists have been used in clinical treatment of severe acute bronchial asthma attacks, however, inhaled albuterol also requires frequent dosing due to the faster pharmacokinetic processes. The medicine takes effect 5-15 min after inhalation, the bronchiectasis time reaches 6 hours, the routine inhalation amount of adults is 0.25-0.5 mg/time, and the medicine is taken as required. The asthma attack has circadian rhythm, the best time of the lung function condition is 4 pm, and the time of the lung function condition is the weakest in the early morning, in order to ensure that the anti-asthma medicine still has effective dose to relieve asthma symptoms in the patient when the asthma attack is in the early morning, the development of the long-acting anti-asthma medicine which can be taken in the evening before day and can exert the medicine effect until the next morning is necessary, and the long-acting inhaled beta 2 receptor agonist (such as salmeterol, formoterol) can effectively control night asthma which is insufficient to control the anti-inflammatory medicine. In addition, sustained release theophylline and controlled release oral β2 receptor agonists (such as oral controlled release salbutamol and terbutaline) are also effective. Studies have shown that long-acting inhaled β2 receptor agonists are superior to other long-acting bronchodilators. Long-acting inhaled β2 receptor agonists are the first option for the treatment of nocturnal asthma in addition to anti-inflammatory drugs and other standard control measures such as environmental quality control. Because the mode of aerosol inhalation of the levosalbutamol is superior to oral intake, the development of a long-acting preparation of the aerosol inhalation of the levosalbutamol is also necessary, and the long-acting preparation of the aerosol inhalation of the levosalbutamol can not only exert the advantages of aerosol inhalation, but also be beneficial to the aerosol inhalation of the levosalbutamol for treating night asthma. The levalbuterol needs a proper carrier for pulmonary delivery, so that the levalbuterol inhaled by atomization not only can play a role in timely relieving asthma, but also can continuously exist at an action part for a long time and play a drug effect through the slow release effect of the carrier.

The traditional oral dosage form has large dosage, can not directly act on the affected part, needs to enter the circulatory system to act on the affected part through the first pass effect of the liver, has slower effect, and limits the medication of patients to a certain extent. The injection needs to act on the affected part through a circulatory system, so that the medicine is distributed more in other tissues, the toxic and side effects of the medicine are increased, and meanwhile, the problem of pain at the administration part and poor patient compliance exists in the administration of the injection. The aerosol inhalation solution preparation can be used as a respiratory tract or lung administration preparation, can directly act on a patient part, can improve the administration concentration of the respiratory tract or the lung, has quick response, avoids the first pass effect of the liver, reduces the administration dosage of the medicine, improves the bioavailability of the medicine, reduces the distribution of the medicine in other tissues, and reduces side effects. The pulmonary inhalation preparation refers to a preparation which can exert systemic or local effects through deep respiratory tract, cavity tract, mucosa and the like after being administered by a special device. Such dosage forms currently in clinical use mainly include metered dose inhalation aerosols (metered dose inhaler, MDI), dry powder inhalants (dry powder inhaler, DPI) and aerosol inhalants (nebulizer). The aerosol inhalation is an administration method for preparing the medicine into tiny fog particles or fog drops (the diameter is generally smaller than 5 mu m) to suspend in the gas by utilizing the negative pressure space of the aerosol inhaler, so that the medicine is humidified and inhaled into the respiratory tract to achieve the effects of moistening mucous membrane, diminishing inflammation, eliminating phlegm, relieving spasm, relieving asthma and the like. The drug is delivered through the lung in a non-invasive way, not only can provide local lung effect, but also has higher system bioavailability, can avoid first pass metabolism, can exert the treatment effect more quickly, and the alveoli provide huge available surface area.

Pulmonary inhalation administration is another more attractive route of administration in addition to oral, injectable administration. From the action time, the medicine can be rapidly eliminated in the lung and has short half-life. Clinical studies on pulmonary drug delivery currently show that due to the unique physiological characteristics of the lung (large blood flow and abundant capillaries), multiple drug delivery is mostly required and patient compliance is poor. Therefore, the development of a lung sustained-release preparation capable of improving the curative effect of the drug and improving the use compliance of the drug is needed. The preparation can avoid the clearance of lung macrophages, and a nano preparation which resides in the lung for a plurality of weeks (the mechanism of ensuring that the nano preparation is not exhaled is inhaled after the nano particles are agglomerated to a micron-sized agglomeration body) is a very potential lung slow release carrier. The nano-carrier for pulmonary administration mainly comprises nano-liposome, nano-micelle, solid lipid nanoparticle, microsphere, nanocrystal, nanoemulsion and the like.

Patent CN110898039 a discloses a levalbuterol hydrochloride solution preparation for inhalation and a preparation method thereof. A levalbuterol hydrochloride solution formulation for inhalation comprising: levalbuterol hydrochloride, and/or hydrates thereof; osmotic pressure regulator; a pH regulator; the stabilizer and the solvent the levosalbutamol hydrochloride solution preparation for inhalation has the advantages of high stability, reliable quality, good safety, long storage time, no need of nitrogen filling, simplified preparation process, reduced requirements on production equipment, simplicity, easy operation, low production cost and easy industrialized production.

Patent CN111228243 a discloses a tobramycin liposome for aerosol inhalation, which consists of the following components: tobramycin 0.1-15.0%, phospholipid 0.5-36.0%, stabilizer 0.05-20.0%, charge modifier 0.01-10.0%, antioxidant 0.01-5.0%, organic phase medium 5.0-50.0%, and water phase medium for the rest; compared with oral administration, the tobramycin liposome for aerosol inhalation can directly transfer the medicine to the respiratory tract, has quick absorption and quick action, and can improve the medicine concentration of the respiratory tract; the bioavailability and stability are high, and the safety is good; the preparation process is simple, easy to prepare and reasonable, and the performance is stable, thereby creating conditions for realizing industrial productization.

Generally, there are two strategies for constructing drug delivery systems. The traditional method is to wrap the hydrophobic drug by means of hydrophobic interactions, electrostatic interactions, hydrogen bonding, pi-pi stacking and the like. Because of the simplicity of operation, this method is widely used in constructing drug delivery systems and in therapy. However, this relatively weak physical interaction may lead to premature release of the drug delivery system in the blood circulation in the body following administration. At the same time, it is also limited by low drug loading. Another strategy is to chemically couple the drug directly to the carrier to prepare prodrug nanoparticles. The prodrug nanoparticle has the advantages of high drug loading, low toxic and side effects, good stability, reduction of undesirable premature release and the like. Compared with liposome, the microsphere has more stable physical and chemical behaviors in vivo and in vitro, can achieve slow release effect, has better pharmacological activity, and is beneficial to delivering drugs to the lung. Therefore, the application of the nano levosalbutamol hydrochloride particles to the aerosol inhalation solution sustained release preparation is developed, the drug effect is greatly improved, and the drug release time and the stability are further improved.

Disclosure of Invention

In view of the above-mentioned drawbacks in the prior art, the present invention aims to provide a levalbuterol hydrochloride aerosol inhalation solution sustained release agent with longer efficacy and more stability and a preparation method thereof.

Pulmonary administration is a non-invasive mode of administration. Compared with oral administration, the lung has large specific absorption surface area, the alveolar epithelial cell layer is thin (0.1-0.2 μm), the drug permeation distance is short, small molecules can be rapidly absorbed by the body, and the bioavailability is high. In addition, the metabolic enzyme activity of the lung is low, and the degradation effect on protein and polypeptide medicines is small. Compared with injection administration, the lung administration is more convenient, and the patient compliance is high. The lung administration mode can directly deliver the medicine to lung tissues, is particularly suitable for treating lung diseases, and can reduce systemic toxic and side effects. Many current pulmonary modes of administration improve drug biology by encapsulating the drug to produce nanoparticle delivery systems; the preparation has the defects of low utilization rate, serious side effects and the like, but the application of the preparation is still severely limited due to the reasons of complex preparation, poor stability, low drug loading, premature release of the drug and the like. The present inventors have greatly improved the above drawbacks by synthesizing a drug delivery system for metal phenolic networks, which has good stability, drug loading and biocompatibility, and which is synthesized mainly by: firstly preparing the levosalbutamol hydrochloride dimer through oxidation-reduction reaction, then encapsulating the levosalbutamol hydrochloride dimer by using the prepared modified poly (D, L-lactic acid-ethylene glycol) polymer, enhancing the water solubility and improving the stability, and then complexing the complex network of iron and tannic acid and the encapsulated levosalbutamol hydrochloride dimer to obtain the levosalbutamol hydrochloride composite nano-particles. The nanoparticle has long-term and excellent stability, does not release prematurely, has good biocompatibility, and can also meet the current loading requirements.

The technical scheme of the invention is as follows:

the levosalbutamol hydrochloride aerosol inhalation solution sustained release agent mainly comprises the following components in percentage by mass: 0.05 to 0.06 percent of levalbuterol hydrochloride composite nano-particles, 0.2 to 0.9 percent of isotonic agent, 0.001 to 0.005 percent of pH regulator and the balance of solvent.

The preparation method of the levalbuterol hydrochloride composite nano-particles comprises the following steps:

x1, weighing 6-7 g of levosalbutamol hydrochloride, adding the levosalbutamol hydrochloride into 60-70 mL of dichloromethane, stirring for 1-2 h, adding 0.02-0.03 g of 4-dimethylaminopyridine, 0.02-0.03 g of 3, 3-dithiodipropionic acid, 0.5-0.6 g of N, N' -dicyclohexylcarbodiimide, heating to 40-45 ℃ for reflux, stirring for 2-3 d, filtering after the reaction is finished, and drying a filter cake to obtain levosalbutamol hydrochloride dimer;

weighing 0.63-0.65 g of D, L-lactic acid, 0.27-0.30 g of ethylene glycol, 0.1-0.15 g of maleic acid, adding into an ampoule bottle, adding 0.0.3-0.04 g of benzoyl peroxide, 0.01-0.02 g of stannous iso-octoate, vacuumizing the ampoule bottle, drying at 30-35 ℃ for 10-12 h, performing melt polymerization at 140-150 ℃, reacting for 20-24 h, dissolving the mixture into 20-30 mL of chloroform after the reaction is finished, adding into 100-200 mL of ethanol, precipitating, filtering, and vacuum drying a filter cake to obtain maleic anhydride modified polylactic acid;

x3, dissolving 1-1.1 g of the maleic anhydride modified polylactic acid in the step X2 in 5-10 mL of chloroform, slowly dropwise adding 0.2-0.3 g of 1, 4-butanediamine, stirring for 10-20 min at 5-10 ℃, heating to room temperature, stirring for 30-40 min, adding the mixture into 50-100 mL of ethanol after the reaction is finished, stirring, precipitating, filtering, washing a filter cake with water until the pH value is=7-8, and then drying at 35-40 ℃ for 20-24 h to obtain a modified poly (D, L-lactic acid-ethylene glycol) polymer;

x4, weighing 1.5-1.8 g of levosalbutamol hydrochloride dimer, adding 0.125-0.15 g of modified poly (D, L-lactic acid-ethylene glycol) polymer into 5-6 mL of dimethyl sulfoxide, stirring and dissolving, adding 45-54 mL of water, performing ultrasonic dispersion for 1-2 h, continuously adding 2-2.4 mg of tannic acid, 0.5-0.6 mg of ferric chloride, performing ultrasonic dispersion for 1-2 h, centrifuging at 8000-10000 rpm, obtaining lower-layer sediment, and drying to obtain the levosalbutamol hydrochloride composite nano-particles.

The invention also provides a preparation method of the levalbuterol hydrochloride aerosol inhalation solution sustained release agent, which comprises the following steps:

s1, adding 50-80% of the total amount of solvent into a liquid distributor, adding the prescription dose of levalbuterol hydrochloride composite nano particles and an isotonic agent, and stirring until the solvent is completely dissolved;

s2, adding a pH regulator to regulate the pH;

s3, adding the solvent to the full amount, and stirring to uniformly mix the solvent;

s4, performing primary filtration by using a 0.45 mu m filter membrane, performing fine filtration by using a 0.22 mu m filter membrane, wherein the primary filtration and the fine filtration are sterile filtration, and filling the sterile filtration into an ampoule with the filling amount of 3mL.

Further, the solvent is purified water, water for injection or sterile water for injection.

Further, the total amount of water is 3 to 4L.

Further, the prescription amount of the levalbuterol hydrochloride composite nano-particles is 4-5 g.

Further, the isotonic agent is one of sodium chloride, magnesium chloride and calcium chloride.

Further, the adding amount of the isotonic agent is 27-30 g.

Further, in the step S1, the stirring temperature of the solvent is controlled to be 15-35 ℃.

Further, the pH regulator is one of hydrochloric acid, sulfuric acid, lactic acid, malic acid, acetic acid, phosphoric acid and citric acid.

Further, the pH value adjusted in the step S2 is 4.5 to 5.5.

Preferably, the pH regulator is one of sulfuric acid or hydrochloric acid.

Compared with the prior art, the invention has the beneficial effects that:

(1) The inventor greatly improves the existing defects by synthesizing a metal phenolic network drug delivery system, and the drug delivery system of the levalbuterol hydrochloride composite nano-particles has good stability, drug loading capacity and biocompatibility;

(2) Compared with oral administration, the medicine can be directly transferred to respiratory tract by using an aerosol inhalation technology, has the advantages of quick absorption, quick action, high concentration of the medicine in the respiratory tract, improved curative effect, minimized systemic exposure, reduced systemic toxic and side effects, avoidance of first pass effect of liver, high bioavailability and reduced medicine dosage; compared with injection administration, inhalation administration can improve patient compliance and also alleviate or avoid partial adverse drug reactions;

(3) The technological preparation process of the research is simple, easy and reasonable to prepare, and stable in performance, and creates conditions for realizing industrial productization.

Detailed Description

The technical scheme of the present invention will be described in detail by means of specific examples, which should be explicitly set forth for illustration, but should not be construed as limiting the scope of the present invention.

The parameters of partial raw materials in the embodiment of the invention are as follows:

sulfuric acid, 0.1mol/L aqueous solution, product number: 68279, merck.

Levosalbutamol hydrochloride, product number: PHR3457, merck.

97% by weight of 3, 3-dithiodipropionic acid, product number: t30201 merck.

Phosphate buffer, ph=7.2-7.4, with armed pramipexole.

D, L-lactic acid with purity of 90 percent, shanghai Haohong biological medicine technology.

Polylactic acid-glycolic acid copolymer (50:50), molecular weight: 10000-20000, purity not less than 99wt%, purchased from MedChemexpress.

Comparative example 1

A preparation method of a levalbuterol hydrochloride aerosol inhalation solution comprises the following steps:

s1, adding 2L of purified water into a liquid distributor, adding 0.73g of levosalbutamol hydrochloride and 27g of sodium chloride, and stirring until the mixture is completely dissolved;

s2, adding sulfuric acid to adjust the pH to 5.3;

s3, adding purified water to 3L, and stirring to uniformly mix the purified water;

Example 1

A preparation method of a levalbuterol hydrochloride aerosol inhalation solution sustained release agent comprises the following steps:

s1, adding 2L of purified water into a liquid distributor, adding 4g of levalbuterol hydrochloride composite nano particles and 27g of sodium chloride, and stirring until the nano particles are completely dissolved;

s2, adding sulfuric acid to adjust the pH to 5.3;

x1, weighing 6g of levosalbutamol hydrochloride, adding the levosalbutamol hydrochloride into 60mL of dichloromethane, stirring for 2 hours, adding 0.02g of 4-dimethylaminopyridine, 0.02g of 3, 3-dithiodipropionic acid and 0.5g of N, N' -dicyclohexylcarbodiimide, heating to 40 ℃ for reflux, stirring for 3 days, filtering after the reaction is finished, and drying a filter cake to obtain levosalbutamol hydrochloride dimer;

x2, weighing 0.65g of D, L-lactic acid, 0.30g of ethylene glycol and 0.15g of maleic acid, adding 0.03g of benzoyl peroxide and 0.01g of stannous iso-octoate into an ampoule bottle, vacuumizing the ampoule bottle, drying at 35 ℃ for 12 hours, performing melt polymerization at 140 ℃ for 24 hours, dissolving the mixture into 30mL of chloroform after the reaction is finished, adding into 100mL of ethanol, precipitating, filtering, and vacuum drying a filter cake to obtain maleic anhydride modified polylactic acid;

x3, dissolving 1g of the maleic anhydride modified polylactic acid in the step X2 in 5mL of chloroform, slowly dropwise adding 0.3g of 1, 4-butanediamine, stirring at 10 ℃ for 10min, heating to room temperature, stirring for 30min, adding the mixture into 100mL of ethanol after the reaction is finished, stirring, precipitating to generate, filtering, washing a filter cake with water to pH=7, and drying at 40 ℃ for 24h to obtain a modified poly (D, L-lactic acid-ethylene glycol) polymer;

x4 weighing 1.5g of levosalbutamol hydrochloride dimer, adding 0.125g of modified poly (D, L-lactic acid-ethylene glycol) polymer into 5mL of dimethyl sulfoxide, stirring and dissolving, adding 45mL of water, carrying out ultrasonic dispersion for 2 hours at 40kHz and 400W, continuing adding 2mg of tannic acid and 0.5mg of ferric chloride, carrying out ultrasonic dispersion for 2 hours at 40kHz and 400W, centrifuging at 10000rpm to obtain a lower precipitate, and drying at 30 ℃ for 4 hours to obtain the levosalbutamol hydrochloride composite nano-particles.

Example 2

s1, adding 2L of purified water into a liquid distributor, adding 5g of levalbuterol hydrochloride composite nano particles and 27g of sodium chloride, and stirring until the nano particles are completely dissolved;

s2, adding sulfuric acid to adjust the pH to 5.3;

x2, weighing 1.5g of levosalbutamol hydrochloride dimer, adding 0.125g of polylactic acid-glycolic acid copolymer into 5mL of dimethyl sulfoxide, stirring and dissolving, adding 45mL of water, carrying out ultrasonic dispersion for 2h at 40kHz and 400W, continuously adding 2mg of tannic acid and 0.5mg of ferric chloride, carrying out ultrasonic dispersion for 2h at 40kHz and 400W, centrifuging at 10000rpm to obtain a lower precipitate, and drying at 30 ℃ for 4h to obtain the levosalbutamol hydrochloride composite nano-particles.

Example 3

s1, adding 2L of purified water into a liquid distributor, adding 4g of levosalbutamol hydrochloride liposome particles and 27g of sodium chloride, and stirring until the mixture is completely dissolved;

s2, adding sulfuric acid to adjust the pH to 5.3;

The preparation method of the levosalbutamol hydrochloride liposome particles comprises the following steps:

x1, weighing 8g of lecithin, 24g of cholesterol, 4g of levosalbutamol hydrochloride, adding 100mL of acetone for dissolution, then rotationally evaporating a spin-drying solvent, and then adding phosphate buffer for hydration of 500mL to obtain a suspension;

x2 the suspension in the step X1 is subjected to ultrasonic treatment for 30min under ice bath condition at 40kHz and 400W, and is subjected to spray drying after being filtered by a microporous filter membrane with the thickness of 0.8 mu m, the temperature is controlled at 35 ℃, the pressure is 0.4MPa, and the drying is carried out for 24h, so as to obtain the levosalbutamol hydrochloride liposome particles.

Test example 1

In vitro release test is carried out on the levalbuterol hydrochloride aerosol inhalation solution sustained release agent prepared in the comparative example and the example, the specific experimental method is that the prepared levalbuterol hydrochloride aerosol inhalation solution sustained release agent is put in a dialysis bag and put in a PBS (pH=7.4) solution with the temperature of 37 ℃, and the release amount of the levalbuterol hydrochloride is sampled and detected at a specific time point, so that the sustained release effect of the levalbuterol hydrochloride after encapsulation is examined. Three batches were taken in parallel for each group, and the drug loading of each batch was measured, and the final results were averaged. Taking out 2mL of 3 batches of prepared levalbuterol hydrochloride aerosol inhalation solution sustained release agents respectively, respectively placing the two batches of prepared levalbuterol hydrochloride aerosol inhalation solution sustained release agents in 3 clean and leak-free dialysis bags, binding the two ends of the dialysis bags by ropes, and carefully checking whether the leakage phenomenon exists. 50mL of PBS buffer (pH=7.40) was precisely measured in volumetric flasks and placed in 3 clean 100mL flasks, respectively, and heated in a water bath on a hot plate of a magnetic stirrer, set at 37℃and a rotational speed of 50rpm. After the PBS buffer in the bottle had been kept at 37℃the dialysis bags were placed in the bottles, respectively, and the timing was started immediately, 1mL of sample in the bottle was taken at the following time points (0.5 h, 2h, 8h, 35h, 120h, 192 h) and 1mL of PBS was added. 3 batches of release samples were centrifuged at 13000rpm for 5 minutes and 800. Mu.L of supernatant was taken for detection by liquid chromatograph, the detected release being the cumulative release after this time.

The specific test results are shown in Table 1.

TABLE 1 in vitro Release test

In vitro release test studies were performed to verify the effect of the hypothesis that levosalbutamol hydrochloride after encapsulation could produce sustained release under in vitro physiological conditions. As can be seen from the results of the in vitro release test, the levosalbutamol hydrochloride in comparative example 1 is not encapsulated, 90.8% of the levosalbutamol hydrochloride is released in 8 hours, and the levosalbutamol hydrochloride encapsulates in examples 1 to 3 are completely released, and the levosalbutamol hydrochloride composite nanoparticle in example 1 has the best sustained-release effect, which is probably due to the tight combination of tannic acid molecules and iron ions to form a composite network, and the levosalbutamol hydrochloride dimer is complexed in the composite network, so that the sustained-release effect is enhanced, the active ingredient can maintain a stable and long-acting sustained-release effect, and the stability of the complexing structure further ensures the sustained-release effect.

Test example 2

The storage stability test was performed on the levalbuterol hydrochloride aerosol inhalation solution sustained release agent prepared in the comparative example and the example, and 5 batches of samples were taken in parallel for each group, three of which were stored at 4 ℃ for 10 days, 20 days, 30 days, and the other two batches were stored at 25 ℃ for 10 days, 20 days, respectively. After expiration of storage under different temperature conditions, 100 μl was sampled for detection of encapsulation efficiency. The specific test results are shown in Table 2.

Table 2 storage stability test results table

As can be seen from the storage stability test, the stability of examples 1 and 2 in examples 1 to 3 is obviously better than that in example 3, the encapsulation of the levosalbutamol hydrochloride liposome in example 3 is more stable under the low temperature condition, the encapsulation rate can still ensure a certain encapsulation rate after long-term preservation, but phospholipid and cholesterol encapsulated on the surface of the liposome are likely to be degraded when the temperature is higher, so that effective substances in the liposome leak, and in example 1, the prepared modified poly (D, L-lactic acid-ethylene glycol) polymer is used for encapsulating active substances, the water solubility is enhanced, meanwhile, the stability is improved, and the tight combination of tannic acid molecules and iron ions forms a composite network, and the levosalbutamol hydrochloride dimer is better in stability and can be ensured not to leak the effective components under the condition of higher temperature.

Test example 3

In-vivo efficacy experiments are carried out on the levalbuterol hydrochloride aerosol inhalation solution sustained release agent prepared in the comparative example and the example, the animals used in the experiments are ordinary albino guinea pigs which are purchased from animal experiment centers of university of south medical science, the male and female parts are half, the weight is 290+/-40 g, a proper amount of water and food are added to feed the animals in an environment with the temperature of 25+/-2 ℃ and the humidity of 60+/-5%, and the animals are alternately irradiated for 12 hours. Sequentially placing guinea pigs into a 4-liter sealed bell jar of a multifunctional cough and asthma inducing instrument, atomizing 0.6% histamine solution (prepared by physiological saline) into the sealed bell jar at an atomization speed of 0.15mL/min by using an ultrasonic atomizer for 15s, stopping atomization, and observing the asthma inducing latency of the guinea pigs, namely, the time from the start of atomization to the occurrence of asthma symptoms and dyspnea of the guinea pigs, which is the period from the occurrence of convulsion and fall, discarding the time when the latency exceeds 2min, and taking a free diet and drinking water by the screened guinea pigs, so that the animals to be tested are fasted one day before the experiment. The screened guinea pigs were weighed, the uratame solution was anesthetized by intraperitoneal injection at an amount of 1.25g/kg, after about half an hour of complete anesthesia, the abdomen of the guinea pigs were fixed with the limbs and head up, then the neck skin was carefully dissected, jugular vein cannulation (cannula required to be rinsed with 0.1% heparin sodium solution and filled in the tube) and tracheal cannulation were performed, three guinea pigs were taken per group, and all experimental results were averaged. The tracheal cannula extends out of the tracing box through a hole pipe with the same diameter on the wall of the tracing box and is connected with a flow head with the diameter of 1L for measuring the respiratory flow rate; and then an intrathoracic cannula is inserted between the fourth rib bone and the fifth rib bone of the right chest of the guinea pig, and the intrathoracic cannula is connected to a pressure transducer through a conduit from an opening at the side edge of the tracing box, so that the intrathoracic pressure, the respiratory flow rate change and the tidal volume can be recorded. And (3) 15 mug/mL of histamine is intravenously injected at a speed of 1mL/min to obtain 0.5mL of histamine, the physiological state of the animal is observed, at the moment, the animal can generate symptoms of dyspnea, the change of indexes such as small tidal volume, increased intrathoracic pressure, reduced respiratory flow rate, increased respiratory frequency and the like can be seen in the recorded parameters, after the intravenous stimulation is finished, the parameters are continuously recorded, and after the animal state is stable (about 15-30 min), the steps of administration and stimulation are started. Evacuating the flow head connected to the wall of the tracing box, connecting the spray head of the compression atomizer to connect the spray head with the trachea cannula, atomizing and inhaling 1mg/mL of levosalbutamol hydrochloride atomized inhalation solution slow release agent for 2min for animals, evacuating the drug administration device, reconnecting the flow head with the exported trachea cannula, after the animals are stabilized for about 4min, injecting 15 mug/mL of histamine for 0.5mL intravenously, observing the change of parameters, waiting for the stable state of the animals, and continuing recording the parameters. After 4 hours of administration, 15. Mu.g/mL histamine was intravenously injected 0.5mL, the change of the parameters was observed, the animal was waited for stable status, and the parameters were continuously recorded for about 5 minutes. The above values are calculated according to the airway resistance=the variation value of the intrathoracic pressure/the variation value of the respiratory flow rate, the dynamic lung compliance=the tidal volume/the variation value of the intrathoracic pressure, the above values after administration and before administration are subtracted and divided by the values before administration, and the variation rates of the two parameters are obtained, and the specific experimental results are shown in table 3.

TABLE 3 in vivo results of drug efficacy experiments

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The guinea pig asthma model is more suitable than the rat or mouse as a human airway pharmacological model; in this experiment, histamine phosphate was used to induce airway hyperresponsiveness in guinea pigs to establish an asthma model, which works on the principle of causing bronchosmooth muscle contraction by agonizing the H1 receptor. Guinea pigs are extremely sensitive to histamine, can be rapidly molded, and quickly recover normal breath after histamine stimulation is stopped, so that the guinea pigs are a commonly used rapid type bronchoconstriction model. The pathogenesis of the model is similar to that of human asthma, and the model belongs to type I allergy, and can well simulate the acute attack symptoms of human asthma, such as shortness of breath, wheezing, dyspnea and the like. The most main respiratory mechanics physiological indexes for determining lung function at present are as follows: airway resistance and dynamic lung compliance. Airway resistance refers to the pressure differential created per unit flow in the airway, expressed as the pressure differential created per minute ventilation. Dynamic lung compliance refers to the change in lung volume caused by a change in unit pressure, and represents the effect of changes in chest pressure on lung volume, the results of which can be used clinically to initially assess the patient's lung function. The combination of the two indexes can well reflect the ventilation function of the airway, compared with a blank control group, the control and the examples show obvious reduction of the rise rate of the airway resistance after administration, and the dynamic lung compliance reduction rate is obviously increased, which indicates that the asthma of guinea pigs is obviously relieved after atomization, but the asthma relieving effects of different groups are changed after the time is prolonged, the asthma relieving effect of the control example 1 is obviously poor, the example 1 is better, which is probably caused by the excellent stability and the long-acting slow release effect of the levalbuterol hydrochloride composite nano particles, and the experiment well proves that the prepared levalbuterol hydrochloride atomization inhalation solution slow release agent can last longer for relieving the asthma.

Claims

1. The levosalbutamol hydrochloride aerosol inhalation solution sustained release agent is characterized by comprising the following components in percentage by mass: 0.05-0.06% of levalbuterol hydrochloride composite nano particles, 0.2-0.9% of isotonic agent, 0.001-0.005% of pH regulator and the balance of solvent; the isotonic agent is sodium chloride; the pH regulator is sulfuric acid;

weighing 6-7 g of levosalbutamol hydrochloride by X1, adding the levosalbutamol hydrochloride into 60-70 mL of dichloromethane, stirring for 1-2 hours, adding 0.02-0.03 g of 4-dimethylaminopyridine, 0.02-0.03 g of 3, 3-dithiodipropionic acid, 0.5-0.6 g of N, N' -dicyclohexylcarbodiimide, heating to 40-45 ℃ for reflux, stirring for 2-3 days, filtering after the reaction is finished, and drying a filter cake to obtain levosalbutamol hydrochloride dimer;

weighing 0.63-0.65 g of D, L-lactic acid, 0.27-0.30 g of ethylene glycol, 0.1-0.15 g of maleic acid, adding 0.3-0.04 g of benzoyl peroxide and 0.01-0.02 g of stannous octoate into an ampoule bottle, vacuumizing the ampoule bottle, drying at 30-35 ℃ for 10-12 h, performing melt polymerization at 140-150 ℃ for 20-24 h, dissolving the mixture into 20-30 mL of chloroform after the reaction is finished, adding into 100-200 mL of ethanol, precipitating, filtering, and performing vacuum drying on a filter cake to obtain maleic anhydride modified polylactic acid;

x3, dissolving 1-1.1 g of the maleic anhydride modified polylactic acid in the step X2 in 5-10 mL of chloroform, slowly dropwise adding 0.2-0.3 g of 1, 4-butanediamine, stirring for 10-20 min at 5-10 ℃, heating to room temperature, stirring for 30-40 min, adding the mixture into 50-100 mL of ethanol after the reaction is finished, stirring, precipitating, filtering, washing a filter cake with water until the pH value is 7-8, and drying at 35-40 ℃ for 20-24 h to obtain a modified poly (D, L-lactic acid-ethylene glycol) polymer;

and X4, weighing 1.5g of levosalbutamol hydrochloride dimer, adding 0.125g of modified poly (D, L-lactic acid-ethylene glycol) polymer into 5-6 mL of dimethyl sulfoxide, stirring and dissolving, adding 45-54 mL of water, performing ultrasonic dispersion for 1-2 h, continuously adding 2mg of tannic acid and 0.5mg of ferric chloride, performing ultrasonic dispersion for 1-2 h, centrifuging at 8000-10000 rpm, obtaining lower-layer sediment, and drying to obtain the levosalbutamol hydrochloride composite nano particles.

2. A method for preparing the aerosol inhalation solution slow release formulation of claim 1, comprising the steps of:

s1, adding 50-80% of the total amount of solvent into a liquid dispenser, adding the prescription amount of levalbuterol hydrochloride composite nano particles and an isotonic agent, and stirring until the solvent is completely dissolved;

s2, adding a pH regulator to regulate the pH;

s4, performing primary filtration by using a 0.45 mu m filter membrane, performing fine filtration by using a 0.22 mu m filter membrane, wherein the primary filtration and the fine filtration are sterile filtration, and filling the sterile filtration into ampoule bottles with the filling amount of 3mL.

3. The method of claim 2, wherein the solvent is purified water, water for injection.

4. The method of claim 2, wherein the total amount of solvent is 3 to 4l.

5. The method of claim 2, wherein the prescription amount of the levalbuterol hydrochloride composite nanoparticle is 4-5 g.

6. The method of claim 2, wherein the isotonic agent is added in an amount of 27 to 30g.

7. The method according to claim 2, wherein the solvent stirring temperature in the step S1 is controlled to be 15-35 ℃; and the pH value adjusted in the step S2 is 4.5-5.5.