CN114788813A

CN114788813A - Andrographolide freeze-dried liposome preparation for aerosol inhalation and preparation method and application thereof

Info

Publication number: CN114788813A
Application number: CN202110100563.8A
Authority: CN
Inventors: 刘珂; 唐星; 叶祖光; 张广平
Original assignee: Shandong Biyuan Biomedical Co ltd
Current assignee: Shandong Biyuan Biomedical Co ltd
Priority date: 2021-01-26
Filing date: 2021-01-26
Publication date: 2022-07-26

Abstract

The invention relates to an andrographolide freeze-dried liposome preparation for aerosol inhalation for treating respiratory tract infection and a preparation method thereof, and the freeze-dried liposome preparation is characterized by comprising the following components in parts by weight: 100-200mg of andrographolide, 1045-2090mg of soybean lecithin, 67-134mg of cholesterol, 1325-2651mg of freeze-drying supporting agent mannitol and 883-1767mg of sucrose. The preparation prepared by the invention has good stability, stable membrane drug loading, controllable and very small particle size and is suitable for atomization drug delivery. Meanwhile, the freeze-dried liposome preparation can greatly cover the bitter taste of the medicine and reduce the irritation of the medicine. The liposome can solve the problems of poor absorption, low bioavailability, difficult effective delivery and the like of andrographolide, and obviously improve the curative effect of andrographolide on treating respiratory tract infection.

Description

Andrographolide freeze-dried liposome preparation for aerosol inhalation and preparation method and application thereof

The technical field is as follows:

the invention relates to the technical field of medicines, and in particular relates to an andrographolide freeze-dried liposome preparation for aerosol inhalation as well as a preparation method and application thereof.

The background art comprises the following steps:

respiratory tract infection is the most common infectious disease in clinic and has the characteristic of repeated attack. Respiratory tract infections are common in clinic and generally have two types: wherein, the upper respiratory tract infection mainly refers to acute inflammation between the nasal cavity and the throat; while the lower respiratory tract infection mainly refers to bronchitis, pneumonia, chronic bronchitis, bronchiectasis and the like. The vast majority of upper respiratory infections are due to viral invasion, followed by bacterial infection. Lower respiratory tract infection is mainly caused by mycoplasma and chlamydia, as well as bacterial and viral infections. At present, the first choice for treating respiratory tract infection is to determine pathogens and then perform targeted antibiotic treatment. However, due to the widespread use of antibiotics, resistance occurs in many respiratory infected patients. Therefore, there is a need to develop new, highly effective, non-drug resistant anti-inflammatory agents.

Andrographolide is a labdane diterpenoid compound extracted from andrographis paniculata of acanthaceae, is one of effective components of traditional Chinese medicine andrographis paniculata, has the effects of clearing away heat and toxic materials, cooling blood and relieving swelling, has special curative effects on various infectious diseases, and is known as natural antibiotic medicine. The chemical structural formula is as follows:

andrographolide can inhibit the expression of some pro-inflammatory proteins, the proteins play a role of nuclear factor (NF-kB) connection sites in genes, and the andrographolide plays an anti-inflammatory role by inhibiting the connection of NF-kB and DNA, so that the expression of pro-inflammatory proteins such as COX-2 is reduced. On the one hand, andrographolide can reduce CD caused by chemotactic peptide (fMLP) _11b And CD ₁₈₊ Thereby down regulating mac-1 high expression on the surface of the neutrophil; on the other hand, the inhibitor inhibits the generation of Reactive Oxygen Species (ROS) and the up-regulation of integrin Mac1 mediated by human neutrophil Protein Kinase C (PKC), thereby reducing the adhesion and wall-penetrating process of neutrophils and preventing the neutrophils from migrating to an inflammation site. The traditional Chinese medicine composition is clinically used for treating upper respiratory tract infection, acute and chronic bronchitis, bacillary dysentery, meningitis, otitis media, periodontitis, pneumonia, endometritis, cold, fever and the like.

Andrographolide is extremely bitter in taste, can cause vomiting due to the extremely bitter taste in common oral administration, has poor oral absorption effect and low in-vivo bioavailability, and the oral bioavailability is only 2.67 percent, thereby greatly influencing the clinical effect. Hydrophilic groups are introduced to the molecular structure to enhance water solubility, so that the injection route administration is realized, and the varieties applied to clinic at present comprise potassium dehydroandrographolide succinate, potassium sodium dehydroandroan drographolide succinate, and the like. However, research shows that the chemical stability and safety effectiveness of the structural modification products are not equal to those of parent compounds, certain anaphylactic reaction and toxic and side effects often occur in the clinical use process, and adverse reactions such as anaphylactic shock, thrombocytopenia and the like gradually occur.

Patent CN1568970 discloses an andrographolide drop pill and its preparation method, which solves the problem of low dissolution of andrographolide tablet due to strong hydrophobicity of andrographolide component, but some common chemical synthetic adjuvants of drop pill are not in food additive catalog of China, and the drop pill has poor taste, less matrix available for drop pill, and is only suitable for drug with small dosage. The large-scale production and use of the medicine are seriously influenced by the defects of small medicine loading amount and the like.

The invention content is as follows:

in view of the defects of the prior art, the invention aims to provide a novel pharmaceutical formulation taking andrographolide as a main active ingredient, the andrographolide freeze-dried liposome preparation is used for atomization and inhalation, the preparation is dissolved by a proper solvent such as saline before use, is used together with an atomizer, is directly inhaled through the mouth and the nose, can be aggregated into a higher concentration in the lung, further can achieve the aim of whole body treatment, avoids the first-pass effect of the liver and the damage and degradation of the gastrointestinal tract, greatly reduces the metabolic process of the medicine by the liver and the kidney, and greatly reduces the organ damage of a patient. The invention develops the medicine into a liposome atomization inhalation preparation, solves the problem of low solubility in water, and improves the bioavailability of the medicine; after freeze-drying, the andrographolide compound has a better bitter taste masking mechanism and smaller irritation, fills the blank of the current domestic market, and provides a novel safe and effective andrographolide administration preparation and an administration mode.

Liposomes are vesicles composed of phospholipid bilayers, and drugs can be encapsulated in the lipid membranes depending on the nature. The liposome is composed of biodegradable substances (phospholipid and cholesterol), and has no toxicity to human body and good histocompatibility. The liposome has strong affinity with cell membrane, and can increase the ability of the encapsulated drug to penetrate cell membrane. The aerosol inhalation preparation is prepared by dispersing an aqueous solution or suspension of a medicament into liquid drops or solid particles suspended in gas through an atomizer, and depositing the liquid drops or the solid particles in respiratory tracts and/or lungs by means of inhalation, and is widely used for treating various respiratory tract diseases, particularly children and old patients suffering from asthma, and is not suitable for other inhalation preparations. When used for treating respiratory system diseases, the andrographolide freeze-dried liposome for aerosol inhalation can play a role in targeting positioning, can reduce adverse reactions, and has good safety and small toxic and side effects.

Generally, the andrographolide liposome aerosol inhalation preparation can be prepared by the following steps:

(1) preparation of andrographolide liposome

(2) Preparing andrographolide liposome atomizing inhalating preparation

The andrographolide liposome is prepared by a method selected from the group consisting of film dispersion, reverse phase evaporation, multiple emulsion, melting, injection, lyophilization, surfactant treatment, centrifugation, proliposome, pressure extrusion, and calcium fusion, preferably by film dispersion, reverse phase evaporation, injection, and pressure extrusion, more preferably by film dispersion. The preparation method can be designed and operated by a professional technician by referring to relevant professional books and literatures. If the liposome is prepared by thin film dispersion method, andrographolide and phospholipid etc. membrane materials are dissolved in organic solvent together, put into a flask, and subjected to rotary evaporation under reduced pressure to obtain a layer of thin film, and then added with water or appropriate buffer solution, and subjected to oscillation and ultrasound to form uniform suspension.

The lipid contained in the andrographolide liposome is preferably selected from lecithin, soybean phospholipid, cholesterol, dilauryl phthalein phosphatidylcholine, distearyl phthalein phosphatidylcholine, distearoyl phosphatidylglycerol (DSPG), distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000), dipalmitoyl phosphatidylcholine (DPPC), and dilauroyl phosphatidylcholine (DLPC), more preferably from egg yolk lecithin, soybean lecithin, cholesterol, and dipalmitoyl phosphatidylcholine, and most preferably from soybean lecithin and cholesterol.

When the andrographolide liposome is prepared from soybean lecithin and cholesterol, the molar ratio of the soybean lecithin to the cholesterol is selected from 100: 1-1: 2, preferably 20: 1-1: 1, and more preferably 10: 1-2: 1.

The amount of andrographolide and the amount of auxiliary materials in the andrographolide liposome are not limited, and the preferable medicine-lipid ratio of the andrographolide content in the andrographolide liposome is 1:4.8 as long as the preparation requirements and the treatment requirements of andrographolide aerosol inhalation are met.

The experiment adopts a film dispersion-ultrasonic method to prepare the andrographolide liposome. Dissolving soybean lecithin S-100 and cholesterol in 250mL round-bottom flask, adding chloroform/methanol mixed solvent (2:1, v: v) to dissolve completely, adding andrographolide, dissolving completely, rotary steaming (45 deg.C, 100rpm, -0.1MPa), and rotating to bottom to form uniform yellowA colored film; adding prepared buffer salt solution (precisely called 1.3609g KH) ₂ PO ₄ 0.28g of NaOH, 200ml of deionized water is added, the PH value is 7.2 by a PH meter), the mixture is expanded for 1 hour, and then ultrasonic treatment is carried out for 30 s; the microfluidics were cycled 6 times (25000psi), using Malvern to measure particle size; the samples were dispensed into 20mL vials.

The freeze-dried liposome preparation for aerosol inhalation provided by the invention is packaged in single dose, and the use process is convenient; can greatly reduce the microbial pollution and waste in the using process, and avoids the defects of repeated measuring, repeated diluting and preparation and easy microorganism breeding caused by multiple doses and large package by adopting the dosage of single administration. The invention provides a novel preparation which is lack in the prior art, has accurate medicinal dosage, high and stable medicine quality and safe and simple clinical application and a preparation method thereof.

Different types and concentrations of protectants have different mechanisms and effects of protecting liposomes during lyophilization. Commonly used lyoprotectants are: saccharides such as glucose, lactose, sucrose, maltose, and trehalose; polyhydric alcohols such as mannitol, sorbitol, and glycerol; surfactants such as polyoxyethylene sorbitan, monooleate (Tween, 80); glycine, glutamic acid, arginine, and the like; other protective agents such as antioxidants and buffers are also included. More preferably from mannitol and sucrose, which can be used as a protective agent to better prevent the aggregation of the liposome, so that the freeze-dried product has good appearance, particle form and stability, and the drug leakage is obviously reduced.

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

(1) at present, there is no andrographolide preparation for children, old people or other patients with dysphagia or injection difficulty, the aerosol inhalation preparation can meet the requirement of effective aerosol particles, can be removed through respiratory tract, can not be deposited in lung, reduces incidence rate of lung infection, and can not stimulate or damage lung tissue;

(2) the liposome is dispersed in saline water for atomization inhalation after freeze-drying, so that the bitter taste of the medicine can be greatly covered, the irritation of the medicine is reduced, the bioavailability of the medicine is improved, and the compliance of a patient is improved;

(3) the liposome is prepared into the freeze-dried powder, and nitrogen is filled for protection in the whole preparation and freeze-drying processes, so that the problems of oxidative discoloration, hydrolysis, drug leakage and the like caused by long-term placement of the liposome or change of environmental temperature and the like are avoided, and the stability is improved;

(4) after redissolution, the liposome is rapidly and uniformly dispersed, and compared with the liposome before freeze-drying, the particle size and the entrapment rate are not obviously changed, so that the bioavailability is high and the curative effect is good;

(5) the invention is inhalation type, the medicine directly reaches the target organ lung, thereby avoiding the first pass effect of the liver and the damage and degradation of the gastrointestinal tract;

(6) the andrographolide liposome prepared by the invention has stable membrane drug loading, and the particle size is controllable and very small. The drug did not leak even with pressurization and disruption, indicating that the drug was carried in the membrane rather than the internal aqueous phase.

(7) The andrographolide liposome prepared by the invention can obviously reduce the number of neutrophils in lung lavage fluid of a model animal after aerosol inhalation administration, and inhibit lung tissue inflammation of the model animal.

Obviously, according to the above-mentioned contents of the present invention, other forms of modification, substitution or variation can be made without departing from the basic technical idea of the present invention according to the common technical knowledge and conventional means in the field.

Drawings

Fig. 1 is a transmission electron microscope photograph of andrographolide freeze-dried liposomes prepared in example 1 of the present invention before freeze-drying.

FIG. 2 is a transmission electron micrograph of andrographolide freeze-dried liposomes reconstituted in example 1.

Figure 3 is a distribution diagram of andrographolide freeze-dried liposome nebulized particles.

FIG. 4 is a pathological picture of blank group, model group, positive group (dexamethasone injection), Xiyanping injection, andrographolide dripping pill group, andrographolide atomization group for 10min, and andrographolide atomization group for 20 min.

FIG. 5 is a general experimental setup for microdialysis pharmacokinetic determination in anesthetized rats.

FIG. 6 is a graph of concentration versus time following pulmonary and femoral intravenous injection of Xiyanping in rats.

Figure 7 is a concentration-time curve of andrographolide liposomes administered by pulmonary and femoral intravenous injection in rats.

The specific implementation method comprises the following steps:

100ml of the andrographolide freeze-dried liposome solution is prepared from the following raw materials: andrographolide 231mg, sterol substance 155mg, phospholipid 2415.2mg, and the balance PBS buffer.

In the experiment, the andrographolide liposome is prepared by adopting a film dispersion-ultrasonic method

Example 1:

the andrographolide freeze-dried liposome is prepared by mixing an andrographolide liposome solution with a freeze-drying protective agent and then freeze-drying the mixture; 100ml of the liposome solution is prepared from the following raw materials: andrographolide 115.5mg, sterol substance 77.5mg, phospholipid 1207.6mg, and the balance PBS buffer solution. The freeze-drying protective agent is mannitol and sucrose, wherein the dosage of the mannitol is 3.0g of mannitol per 100ml of liposome suspension, and the dosage of the sucrose is 2.2g of sucrose per 100ml of liposome suspension; the phospholipid is S-100 soybean lecithin; the sterol substance is cholesterol.

The preparation method of the andrographolide freeze-dried liposome comprises the following steps:

(1) 1207.6mg of S-100 soybean lecithin and 77.5mg of cholesterol are dissolved in a 250mL round-bottom flask, chloroform/methanol mixed solvent (2:1, v: v) is added to fully dissolve the components, 115.5mg of andrographolide is added, and after complete dissolution, rotary evaporation is carried out (45 ℃, 100rpm, -0.1MPa) until a uniform yellow film is formed at the bottom.

(2) 80ml of prepared buffer saline solution (precisely 1.3609g KH) is added into the step (1) ₂ PO ₄ 0.28g NaOH, 200ml deionized water, pH 7.2 with a pH meter), swelling for 1h, and post-sonication for 30 s.

(3) The resulting sample was cycled 6 times using a microfluidizer at a homogenization pressure of 25000psi and then particle size was measured using malvern. The sample is divided into 20mL penicillin bottles.

Example 2

This example is the same as example 1, except that: the phospholipid is one of yolk lecithin E80, soybean lecithin, natural phospholipid (injection grade) PC-98T, and dipalmitoyl phosphatidylcholine (DPPC). One wherein the molecular ratio of drug to said phospholipid is between 1:4 and 1: 20; the freeze-drying protective agent is one or more than two of sucrose, glucose, mannitol, lactose and trehalose.

Example 3

The andrographolide freeze-dried liposome is prepared by mixing an andrographolide liposome solution with a freeze-drying protective agent and then freeze-drying the mixture; 100ml of the liposome solution is prepared from the following raw materials: andrographolide 5.75mg, sterol substance 6.34mg, phospholipid 100mg, and PBS buffer solution in balance. The lyoprotectant is mannitol and sucrose, wherein the dosage of mannitol is 3.0g of mannitol per 100ml of liposome suspension, and the dosage of sucrose is 2.2g of sucrose per 10ml of liposome suspension; the phospholipid is yolk lecithin E80; the sterol substance is cholesterol.

(1) yolk lecithin E80100mg and cholesterol 6.34mg were dissolved in a 250mL round-bottomed flask, followed by addition of chloroform/methanol mixed solvent (2:1, v: v) to dissolve the mixture thoroughly, addition of andrographolide 5.75mg, complete dissolution, rotary evaporation (45 ℃, 100rpm, -0.1MPa) to form a uniform yellow film on the bottom.

(2) 100ml of prepared buffer saline solution (precisely 1.3609g KH) is added into the step (1) ₂ PO ₄ 0.28g NaOH, 200ml deionized water, pH 7.2 with a pH meter), swelling for 1h, and post-sonication for 30 s.

(3) The resulting sample was cycled 6 times using a microfluidizer at a homogenization pressure of 25000psi and then particle size was measured using malvern. The samples were dispensed into 20mL vials.

Example 4

This example is the same as example 3, except that: the phospholipid is one of yolk lecithin E80, soybean lecithin, natural phospholipid (injection grade) PC-98T, and dipalmitoyl phosphatidylcholine (DPPC). One wherein the molecular ratio of drug to said phospholipid is between 1:4 and 1: 20; the freeze-drying protective agent is one or more than two of sucrose, glucose, mannitol, lactose and trehalose.

Example 5

The andrographolide freeze-dried liposome is prepared by mixing an andrographolide liposome solution with a freeze-drying protective agent and then freeze-drying the mixture; 20ml of the liposome solution is prepared from the following raw materials: andrographolide 5.79mg, sterol substance 6.37mg, phospholipid 100mg, and the balance PBS buffer solution. The freeze-drying protective agent is mannitol and sucrose, wherein the dosage of the mannitol is 300mg of mannitol for each 10ml of liposome suspension, and the dosage of the sucrose is 204mg of sucrose for each 10ml of liposome suspension; the phospholipid is S-100 soybean lecithin; the steroid is cholesterol.

(1) soybean lecithin S100100mg and 6.37mg of cholesterol were dissolved in a 250mL round-bottom flask, chloroform/methanol mixed solvent (2:1, v: v) was added to dissolve the mixture completely, 5.75mg of andrographolide was added, and after complete dissolution, the mixture was evaporated by rotary evaporation (45 ℃, 100rpm, -0.1MPa) until a uniform yellow film was formed on the bottom.

(2) 10ml of prepared buffer salt solution (precisely 1.3609g KH) is added into the step (1) ₂ PO ₄ 0.28g NaOH, 200ml deionized water, pH 7.2 with a pH meter), swelling for 1h, and post-sonication for 30 s.

Example 6

This example is the same as example 3, except that: the phospholipid is one of egg yolk lecithin E80, soybean lecithin, natural phospholipid (injection grade) PC-98T and dipalmitoyl phosphatidylcholine (DPPC). One wherein the molecular ratio of drug to said phospholipid is between 1:4 and 1: 20; the freeze-drying protective agent is one or more than two of sucrose, glucose, mannitol, lactose and trehalose.

Example 7

The andrographolide freeze-dried liposome is prepared by mixing an andrographolide liposome solution with a freeze-drying protective agent and then freeze-drying the mixture; 100ml of the liposome solution is prepared from the following raw materials: 50mg of andrographolide, 110mg of sterol substances, 500mg of phospholipid and the balance of PBS buffer solution. The freeze-drying protective agent is mannitol and sucrose, wherein the dosage of the mannitol is 3.0g of mannitol per 100ml of liposome suspension, and the dosage of the sucrose is 2.2g of sucrose per 100ml of liposome suspension; the phospholipid is natural phospholipid PC 98T; the sterol substance is cholesterol.

(1) PC98T500mg and cholesterol 110mg were dissolved in a 250mL round-bottomed flask, followed by addition of chloroform/methanol mixed solvent (2:1, v: v) to dissolve the mixture sufficiently, addition of 50mg of andrographolide, and rotary evaporation (45 ℃, 100rpm, -0.1MPa) after complete dissolution to form a uniform yellow film on the bottom.

(2) 100ml of prepared buffer salt solution (precisely 1.3609g KH) is added into the step (1) ₂ PO ₄ 0.28g NaOH, 200ml deionized water, pH 7.2 with a pH meter), swelling for 1h, and post-sonication for 30 s.

Example 8: freeze-drying process

Taking a proper amount of the liposome suspension into a penicillin bottle, adding a freeze-drying protective agent (mannitol: sucrose ═ 2:1), cooling from room temperature to-40 ℃ for 2 hours, and pre-freezing at-40 ℃ for 4 hours. Vacuumizing to 350mTorr and maintaining for 30 minutes, raising the temperature to-20 ℃ for 3 hours after 2 hours, raising the temperature to-10 ℃ for 3 hours after 1 hour, raising the temperature to 0 ℃ for 3 hours after 2 hours, raising the temperature to 20 ℃ for 2 hours after 2 hours, and storing at 4 ℃ for later use.

The following detection tests were performed on the andrographolide lyophilized liposomes of the present invention:

appearance detection of andrographolide freeze-dried liposome

The andrographolide freeze-dried liposome is observed by naked eyes. The freeze-dried liposome is flat and smooth in appearance, is white powder, and has no phenomena of shrinkage and collapse. The andrographolide freeze-dried liposome is semitransparent milk white after redissolving.

Fig. 1 is a transmission electron microscope photograph of andrographolide freeze-dried liposomes prepared in example 1 of the present invention before freeze-drying, and fig. 2 is a transmission electron microscope photograph of andrographolide freeze-dried liposomes prepared in example 2 of the present invention after reconstitution. As can be seen from FIGS. 1 and 2, the andrographolide freeze-dried liposomes had round shape and uniform structure before and after freeze-drying and reconstitution.

Second, investigation of encapsulation efficiency of andrographolide freeze-dried liposome

Chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler, and methanol: the water is 45:55 as a mobile phase, and the detection wavelength is 223 nm. The amount of the sample was 20. mu.L, and the flow rate was 1 mL/min.

Preparing a test solution and determining: homogenizing, and making into 100 μ L product. 1.9mL of pH 7.2PBS was added. After mixing evenly, add 3KD ultrafilter tube, centrifuge for 30min under 3000 rpm. Centrifuging, taking down the liquid filtering membrane of the lower layer, and performing liquid phase measurement. The encapsulation efficiency was calculated as follows:

c to be measured (A to be measured) x C standard substance/A standard substance

m free ═ (C test × 2ml)/(100 μ L × 10) ^-3 ml/. mu.L). times.V Total

Encapsulation (%) - (M total-M free)/M total X100%

TABLE 1 measurement of encapsulation efficiency of andrographolide liposome

Thirdly, the particle meridian distribution of the andrographolide liposome for aerosol inhalation administration

(1) Experimental method

The atomizer is connected with the atomization compressor to ensure that the atomizer is not separated from the atomization compressor under the specific gas flow rate and pressure of the compressor, and the atomizer is filled with the medicine andrographolide liposome solution. The inlet of the laser diffractometer is connected, the outlet of the laser diffractometer is connected with an external filter, and the external filter is connected with a vacuum pump. The inlet was connected to a flow meter, the vacuum pump was turned on, and the inlet gas flow rate was regulated to stabilize to 15L/min (+ -5%). After adjusting the gas flow rate, the software (Spraytec software version 3.20, Malvern) was turned on the computer and the SOP set (test mode: continuous mode; refractive index of the particles: 1.33; dispersion medium: air; refractive index: 1.00) and then the light path background was started. And after the background test is qualified, tightly connecting the atomizer with the inlet end of the induction port. And starting a compressor to atomize and test the particle size of the drug particles, stopping the test when the concentration of the aerosol is rapidly reduced, and closing the atomizer. During the analysis, the particle size distribution (d10, d50, d90) and the laser intensity were continuously monitored.

(2) Results of the experiment

The results show that the atomized particles are: dv (10) ═ 1.088(μm), Dv (50) ═ 3.826(μm), Dv (90) ═ 9.074(μm), Span ═ 2.087, the results are shown in table 2, fig. 3.

TABLE 2 distribution of andrographolide liposomes after aerosol inhalation

In vivo anti-inflammatory experiment of andrographolide liposome aerosol inhalation administration

(1) Experimental Material

Wistar rats, male, 70, body weights of 170-190g, SPF grade.

The andrographolide liposome freeze-dried powder is dispersed into physiological saline before injection.

Xiyanping injection: produced by Jiangxi Qingfeng pharmaceutical industry Co., Ltd, batch number: 2019101803.

andrographolide drop pills: tianshili pharmaceutical group, Inc., lot number: 190606.

dexamethasone sodium phosphate injection: manufactured by Guizhou Tiandi pharmaceutical industry, Limited liability company, batch number: 19110802A.

Chloral hydrate: shanghai Michelin Biochemical technology Ltd, batch number: C10636049.

formaldehyde: manufactured by Fuchen chemical reagent, Inc., lot number: 20190920.

physiological saline: shijiazhuang four drugs, manufactured by limited company, lot number: 192110.

(2) experimental method

Wistar rats are adaptively fed for 5 days, and are randomly divided into 7 groups according to body weight, a blank group, a model group, a positive group (dexamethasone injection group), a Xiyanping injection group, an andrographolide dropping pill group, an andrographolide atomization group for 10min, and an andrographolide atomization group for 20min, wherein each group contains 10 rats. The dexamethasone injection is administered intravenously (3.6mg/kg), and the andrographolide liposome lyophilized powder is inhaled by atomization for 10min and 20 min. Except for the blank group, other groups are respectively inhaled with LPS solution (4mg/mL) for 15min for molding, the administration is carried out immediately after the molding, the administration is carried out continuously for three days, the fourth administration is carried out on each group in the fourth morning, namely 15h after the third molding, 1 hour after the administration (16 h after the molding), 10 percent chloral hydrate is used for anesthesia (0.35mL/100g), abdominal aorta is used for blood collection, the thoracic cavity is exposed, the right lobe is ligated, the left lung is irrigated with physiological saline, slowly injected, statically balanced for 30s, slowly extracted for 1 mL/time, irrigated for 2 times, and irrigated at the speed of 2mL/min (30 s/time), and the irrigating solution is combined. Wright-Giemsa staining was used to observe the content of inflammatory cells (macrophages, neutrophils) in lung lavage fluid. The left lung was fixed with 10% formaldehyde, stored at room temperature, and for further pathological examination, paraffin sections were prepared for histological examination.

(3) Results of the experiment

3.1 Total white blood cell count results in rat acute pneumonia alveolar lavage fluid show that: the total number of leukocytes was significantly increased in the model group compared to the blank control group (P <0.001), and significantly decreased in the alveolar lavage fluid in the positive control group compared to the model group (P < 0.01). The total number of leukocytes in the other groups decreased. The leukocyte classification result shows that the total number of the neutrophils in the model group is obviously increased (P is less than 0.01) compared with that in the blank control group, the number of the neutrophils in the alveolar lavage fluid can be obviously reduced (P is less than 0.05 or P is less than 0.01) in the positive control group, the Xiyanping intravenous injection group, the andrographolide atomization 10min group and the atomization 20min group, and the number of the neutrophils in the dripping pill group has a reduction trend. The results are shown in Table 3.

TABLE 3 Total number of alveolar lavage leukocytes and neutrophils in rats with acute pneumonia

Note: p <0.01, P <0.001 compared to the blank control group; comparison with model group # P <0.05, # # P < 0.01.

3.2 histopathological results show: the alveolar and bronchial epithelium of the blank control group was intact. In the model group, the lung can be changed by focal spots or diffuse alveolar epithelial degeneration necrosis with shedding, inflammatory cell infiltration (mainly neutrophils and monocytes) and the like. Among them, 11# and 13# animals had diffuse alveolar epithelium loss and severe, only individual animals (12# and 20 #) had mild focal suppurative necrosis with mild lesions and overall severe lung injury, indicating successful molding.

The incidence rates of epithelial cell necrosis and desquamation of the epithelium injury, the model group, the positive control group, the intravenous injection group, the CXL dripping pill group, the CXL atomization 10min group and the CXL atomization 20min group of the model group are respectively as follows: 8/10, 0/10, 9/10, 10/10, 8/10, 8/10. From the damage degree, 2 animals in the model group are severe alveolar epithelial necrosis and shedding, the positive control group does not have alveolar epithelial necrosis and shedding, the incidence rate of moderate alveolar epithelial necrosis and shedding in the model group, the intravenous injection group, the CXL dropping pill group, the CXL atomization 10min group and the CXL atomization 20min group is 6/10, 0/10, 0/10, 2/10 and 0/10, and compared with the model group, the damage degree of epithelial cells can be reduced in each treatment group, wherein the intravenous injection group, the CXL dropping pill group, the CXL atomization 10min group and the CXL atomization 20min group mainly have slight alveolar epithelial necrosis and shedding, and the treatment effect is equivalent. CXL atomization is slightly better than CXL atomization for 20min and intravenous injection.

Inflammatory cell infiltration. The neutrophil infiltration of the model group, the positive control group, the intravenous injection group, the CXL drop pill group, the CXL nebulization 10min group, and the CXL nebulization 20min group appeared in all the animal lungs, but in terms of extent, the neutrophil infiltration incidence of the model group was 10/10, which was moderate or more, and the other groups were mainly mild or less, and the incidence of moderate neutrophil infiltration was 8/10, 0/10, 0/10, 1/10, 0/10, 4/10. Therefore, the degree of inflammatory cell infiltration can be reduced to different degrees in the treatment groups, wherein the inflammatory cell infiltration in the intravenous injection group and the CXL atomization 10min group is slightly changed, the alveolar epithelial necrosis shedding of most animals is lower than a small degree, the treatment protection is better, and the treatment protection is equivalent to that of the positive control group.

Focal suppurative inflammation. The occurrence rates of focal suppurative necrosis of the model group, the intravenous injection group, the CXL dripping pill group, the CXL atomization 10min group and the CXL atomization 20min group are 2/10, 1/10, 3/10, 1/10 and 3/10, and the focal suppurative necrosis is not seen in the positive medicine group. The results are shown in Table 4, FIG. 4.

In conclusion, after the andrographolide is atomized, inhaled and administered, the number of neutrophils in lung lavage fluid of a model animal can be obviously reduced, the lung tissue inflammation of the model animal is inhibited, and the result indicates that the andrographolide has an obvious inhibiting effect on an LPS-induced pneumonia model. Compared with the indexes such as lung lesion degree and the like, such as epithelial injury, inflammatory cell infiltration, pyogenic inflammation and the like, the CXL atomization 20min group has slightly better treatment effect than other groups on the pathological change of neutral particle infiltration and is equivalent to the positive medicine group.

TABLE 4 statistics of major lesions

Note: the lesions were classified into 4 grades using appropriate diagnostic terms according to their distribution, severity and morphological characteristics: mild, moderate, and severe, as indicated by +, +++, +++++ respectively

In vivo pharmacokinetics research of andrographolide liposome

(1) Experimental Material

Wistar rat, Shenyang pharmaceutical university laboratory animal center

The andrographolide liposome lyophilized powder is dispersed into normal saline before injection

Xiyanping injection: produced by Jiangxi Qingfeng pharmaceutical industry Co., Ltd, the batch number is: 2019101803

Xiyanping injection: production of Jiangxi Qingfeng pharmaceutical industry Co., Ltd

Microdialysis fluid (Ringer's solution): 147mM sodium chloride, 1.3mM calcium chloride, 4.0mM potassium chloride

Anticoagulated glucose solution (ACD): 3.5mM citric acid, 7.5mM sodium citrate and 13.6mM glucose

(2) Experimental methods

Implantation of the blood probe: rats were anesthetized by intraperitoneal injection of urethane (1.4 g/kg body weight) and given additional doses as necessary to maintain anesthesia throughout the experiment. Rats were fixed supine on the experimental plate and the normal body temperature (about 37 ℃) was maintained by infrared light illumination. The skin and muscle of the neck were peeled off, the left jugular vein of the rat was isolated, a small opening was opened on the upper side, and the probe was inserted about 1.5cm in the direction of the heart and ligated quickly to prevent the outflow of blood.

Implantation of a pulmonary probe: after anaesthesia, the rats were placed on their back, and after a small incision of 1.5cm at the midline of the neck, and the fascia was separated, a relatively large thyroid gland (300g about 1cm in diameter) was visible, which was easily detached from the surroundings and could not be cut to avoid bleeding. The anterior cervical muscle, i.e. the white, cricoid trachea, was then separated using a vascular clamp. Fixing trachea with one hand, opening a small opening with a diameter of 1mm in front with a sharp knife with the other hand, implanting the microdialysis probe into pulmonary mucosa layer of left lung lobe along left bronchus direction, and fixing the microdialysis probe exposed outside trachea on rat skin with adhesive tape.

Reverse dialysis determination of in vivo recovery of the probe: to be treatedAfter implantation of microdialysis probes into the jugular vein and lungs of rats, hemodialysis and pulmonary dialysis were perfused sequentially with 100,500 and 2500ng/ml andrographolide ACD solution and andrographolide Ringer solution (C) _perfusate ) Collecting microdialysis fluid samples under each perfusion flow concentration at a flow rate of 4 mug/min, collecting each concentration 3 times, and determining the drug concentration (C) in the microdialysis fluid _dialysis ). After the perfusate is replaced, the pump is started to flush for at least 30min, and then the next sample is collected. The in vivo recovery rate was calculated according to the following formula: r _inviv o＝1-C _dialysis /C _perfusate . The in vivo recovery rate of the blood microdialysis probe after correction is 5.57 percent, and the in vivo recovery rate of the pulmonary microdialysis probe is 5.88 percent.

Collecting samples: after the probe was implanted, the micro-syringe pump was started and blood and pulmonary microdialysis probes were perfused with the blank ACD and Ringer, respectively, at a flow rate of 3 μ g/min, and administration was started after at least 1.0h of equilibration. The lung of the rat is provided with andrographolide liposome solution, and the femoral vein of the rat is injected with Xiyanping injection, and the administration dosage is 90 mg/kg. The rats began to collect the dialysate immediately after pulmonary and femoral vein administration, once every 15min for 12 hours. The free andrographolide concentration (Cu) in blood or ELF was calculated by the following formula: cu ═ C _dialysis /R _invivo

(3) Results of the experiment

After the Xiyanping injection is injected into femoral vein of rat, the concentration value of free andrographolide in blood at different time is shown in Table 5, the concentration value of free andrographolide in ELF at different time is shown in Table 6, and the concentration time curve is shown in FIG 6.

TABLE 5 concentration values of free andrographolide in blood at different times of intravenous injection of Xiyanping injection

TABLE 6 concentration values of free andrographolide in ELF at different times of intravenous injection of Xiyanping injection

After the lung of the rat is administered with the andrographolide liposome aerosol inhalant, the concentration values of free andrographolide in blood at different times are shown in table 7, the concentration values of free andrographolide in ELF at different times are shown in table 8, and the concentration-time curve is shown in table 7.

TABLE 7 concentration values of free andrographolide in blood at different times of pulmonary administration of andrographolide liposomes

TABLE 8 concentration values of free andrographolide in ELF at different times of pulmonary administration of andrographolide liposomes

PLI＝(AUE _ELF /AUC _Blood ) _i.t /(AUE _ELF /AUC _Blood ) _i.v

A PLI value greater than 1 indicates pulmonary localization, and the greater the PLI value, the greater the pulmonary localization and the greater the local effect. As can be seen from the results, AUC of andrographolide liposomes after pulmonary administration _ELF 481.9mg.h/mL, AUC _blood 16.71mg.h/mL, and has significant difference compared with intravenous injection of Xiyanping (p)<0.05). This shows that after pulmonary administration, the concentration of andrographolide in ELF is increased, the PLI of andrographolide liposome aerosol inhalant is far greater than 1, and the lung localization is remarkable.

Pharmacokinetic parameters of andrographolide in blood and ELF were calculated using statistical moments and analysis of variance (One-Way ANOVA) was performed on each set of parameters using SPSS13.0 statistical software and compared using Newman-Keal post hoc commands. MRT represents the average retention time of andrographolide in lung mucus, and the andrographolide liposome aerosol inhalant remarkably increases the retention time of andrographolide in lung mucus, reduces the clearance rate and apparent distribution volume of andrographolide, further increases AUC and Cmax (p is less than 0.01) of unit dose, and has obvious sustained release effect.

Claims

1. An andrographolide freeze-dried liposome preparation for aerosol inhalation for treating inflammation, wherein 100ml of the liposome solution is prepared from the following raw materials: andrographolide 231mg, sterol substance 155mg, phospholipid 2415.2mg, cryoprotectant mannitol 3000mg, and sucrose 2041.6 mg.

2. The formulation of claim 1, wherein the lyophilized liposome formulation has a particle size ranging from 50-200 nm; preferably 50-100 nm; more preferably 50 nm; the phospholipid comprises natural phospholipid and synthetic phospholipid, wherein the natural phospholipid comprises egg yolk lecithin and soybean lecithin; the synthetic phospholipid comprises dilauryl phthalein phosphatidylcholine, distearyl phthalein phosphatidylcholine, distearoyl phosphatidylglycerol (DSPG), distearoyl phosphatidylcholine (DSPC), distearoyl phosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG 2000), dipalmitoyl phosphatidylcholine (DPPC), and Dilauroyl Lecithin (DLPC); preferably from egg yolk lecithin, soybean lecithin, dipalmitoylphosphatidylcholine, most preferably selected from soybean lecithin and dipalmitoylphosphatidylcholine; the sterol is cholesterol.

3. The formulation of claim 1, wherein; the medicine-lipid ratio of the andrographolide content in the andrographolide liposome is 1: 4.8; the molecular ratio of the soybean lecithin to cholesterol is 8:1, and the concentration of the soybean lecithin is 30 mg/mL.

4. The lyoprotectant of claim 1, comprising mannitol, sorbitol, xylitol, sucrose, lactose, maltose, glucose, fructose, sodium chloride, potassium chloride; mannitol and sucrose are preferred, and the mass ratio of mannitol to sucrose is 1: 1.5. The ratio of the total mass of the freeze-drying protective agent to the phospholipid cholesterol is 2:1, and the solid content of the freeze-drying protective agent is 10% -12%.

5. The formulation according to any one of claims 1 to 4, characterized in that it is prepared by a process comprising the steps of:

step one, respectively weighing 115.5mg of andrographolide, 77.5mg of cholesterol and S-1001207.6 mg of soybean lecithin, dispersing the weighed phospholipids and sterol substances in an organic solvent for complete dissolution, then adding andrographolide, and uniformly stirring to obtain an oil phase; the organic solvent is 40mL of chloroform and 20mL of methanol;

step two, carrying out rotary evaporation on the oil phase in the step one to remove the organic solvent in the oil phase, and carrying out rotary evaporation until the bottom of the oil phase is reached to form a uniform yellow film;

step three, preparing a phosphate buffer solution with the pH value of 7.2;

step four, completely hydrating the film in the step two by using a preheated phosphate buffer solution in the step three, and expanding for 1-2 hours to obtain primary emulsion;

fifthly, performing constant volume on the primary emulsion in the fourth step by using a phosphate buffer solution with the pH value of 6.5-7.5, and then homogenizing in a high-pressure homogenizer or performing ultrasonic treatment in an ultrasonic instrument to obtain a liposome solution;

step six, weighing 1531.2mg of mannitol and 1020.8mg of sucrose serving as cryoprotectants, dissolving the weighed cryoprotectants in the liposome solution obtained in the step five, subpackaging and freeze-drying to obtain the andrographolide freeze-dried liposome.

6. The method according to claim 5, wherein in the step five, the homogenization temperature is controlled to be 15-25 ℃, the homogenization pressure is 25000psi, and the number of homogenization cycles is 6; in the fifth step, the ultrasonic temperature is 15-25 ℃, the ultrasonic power is 200-600W, and the ultrasonic time is 30 s; the preparation of the phosphate buffer solution with the pH value of 7.2 comprises the following steps: 1.3609g of KH2PO4 and 0.28g of NaOH were precisely weighed and placed in a beaker, 200mL of deionized water was added to dissolve them sufficiently, and the pH was measured with a pH meter and adjusted to about 7.2.

7. The method of claim 5, wherein the lyophilized liposomes of andrographolide for aerosolization are prepared by cooling to-40 ℃ from room temperature for 2 hours and pre-freezing at-40 ℃ for 4 hours. Vacuumizing to 350mTorr and maintaining for 30 minutes, raising the temperature to-20 ℃ for 3 hours after 2 hours, raising the temperature to-10 ℃ for 3 hours after 1 hour, raising the temperature to 0 ℃ for 3 hours after 2 hours, and raising the temperature to 20 ℃ for 2 hours, thus obtaining the andrographolide freeze-dried liposome for atomization and inhalation, and storing the andrographolide freeze-dried liposome in an environment at 4 ℃ for later use.

8. The formulation according to any one of claims 1 to 4, wherein the formulation is reconstituted with saline before use and shaken up before inhalation.

9. Use of the lyophilized liposomes of andrographolide according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment of a disease associated with respiratory tract infections.

10. The use according to claim 9, wherein the disease associated with respiratory tract infections is one or both of upper respiratory tract infections and lower respiratory tract infections; the upper respiratory tract infection can be one or more of viral pharyngitis, laryngitis, herpangina, pharyngoconjunctival fever, influenza and bacterial pharynx-tonsillitis; the lower respiratory tract infection can be one or more of viral pneumonia and acute and chronic bronchitis.