Andrographolide nanocrystalline suspension as well as preparation and application thereof

Abstract

The invention relates to an andrographolide nanocrystal suspension, preparation thereof and application of the andrographolide nanocrystal suspension in preparation of an andrographolide nanocrystal dry powder inhaler with an anti-inflammatory effect. The andrographolide nanocrystal suspension contains a) an active component andrographolide nanocrystal; b) a stabilizer; wherein the content of andrographolide in the suspension is 0.1%-30%, and the content of the stabilizer is 0.1%-5%. According to the andrographolide nanocrystal dry powder inhalant and the preparation method thereof, andrographolide nanocrystals are prepared into micro powder by adopting a spray drying technology, and auxiliary material addition and a preparation process are optimized, so that the andrographolide nanocrystal dry powder inhalant with good inhalation performance is successfully prepared and directly acts on the respiratory tract through a pulmonary drug delivery system, the administration dosage is reduced, and the inhalation effect is improved. Higher lung local concentration is obtained, the first-pass effect of the liver is avoided, meanwhile, the efflux effect of p-glycoprotein is avoided as much as possible, and then the bioavailability is improved; the technical problem that the bioavailability is poor due to the fact that andrographolide is excreted through p-glycoprotein and rapidly absorbed and metabolized in the intestinal tract is solved.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical preparations, and in particular relates to an andrographolide nanocrystal suspension and a preparation method thereof, and an application of the andrographolide nanocrystal dry powder inhaler having anti-inflammatory effects. Background Art

[0002] Andrographolide (AG) is a diterpene lactone compound extracted from Andrographis paniculata. This compound is white square prism or flaky crystals and is the main active ingredient of the natural plant Andrographis paniculata. It dissolves in boiling ethanol, slightly soluble in methanol or ethanol, very slightly soluble in chloroform, and almost insoluble in water. Andrographolide has the effects of removing heat and detoxification, anti-inflammatory and analgesic, and has special therapeutic effects on bacterial and viral upper respiratory tract infections and dysentery. Therefore, andrographolide is known as a natural antibiotic drug, providing patients with an effective treatment option. The efficacy of andrographolide is not limited to the relief of common diseases. It also has multiple biological activities such as antioxidant, anti-inflammatory, and anti-tumor. These characteristics make andrographolide have a wide range of application prospects in the medical field and provide new ideas for drug research and development. In addition, as people's pursuit of health and quality of life continues to improve, the demand for natural medicines is also increasing. As a natural antibiotic, andrographolide has broad market prospects. Therefore, strengthening research on its dosage form, efficacy, and pharmacological mechanism is of great significance for promoting the development and application of andrographolide.

[0003] According to previous studies, the oral bioavailability of AG in rats was 1.19% and the absolute bioavailability was 2.67%. The oral bioavailability of a drug depends on many factors, among which the physicochemical properties of the drug and the biochemical state of the liver and intestinal epithelial cells are particularly important. Studies have shown that the biotransformation of AG is very fast and can be excreted through p-glycoprotein, which is one of the reasons for its poor bioavailability. P-glycoprotein is a transmembrane transport protein that is mainly expressed in hepatocytes, renal proximal tubular epithelial cells, intestinal epithelial cells, brain capillary epithelial cells, and placental epithelial cells. It is responsible for protecting the body from damage by foreign harmful substances and exporting mediating substances. In clinical practice, andrographolide has been developed into oral preparations such as dispersible tablets, pills, and capsules for the treatment of bacterial and viral upper respiratory tract infections. However, it faces problems such as poor water solubility, p-glycoprotein efflux, and low bioavailability. Therefore, researchers have modified its structure to improve its water solubility and developed injection preparations such as Chuanhuning, Yanhuning, and Xiyanping. However, these injection preparations have large dosages, obvious toxic side effects, and damage to the patient's body. In order to overcome the existing defects, it is possible to consider improving the dosage form of AG in order to obtain better drug efficacy.

[0004] Drug nanocrystals are a special type of drug preparation with a particle size of less than 1 μm. They only use the stabilizing effect of a small amount of surfactants or polymer materials, and suspend drug molecules in a dispersion medium by mechanical crushing or controlling the crystal precipitation process to form a drug nanodispersion system. This system can be either crystalline or amorphous. Usually, drug nanocrystals often exist in the form of crystalline dispersed particles. When they exist in liquid form, they are called drug nanocrystal suspensions. Compared with other nanoformulations, the advantages of drug nanocrystals are mainly reflected in the following aspects: 1. High safety: Since drug nanocrystals do not use carriers for delivery, they do not contain a large amount of surfactants and carrier materials, and do not produce material metabolites. 2. High bioavailability: Through micronization, drug particles are reduced to nanometer level, the specific surface area of ​​poorly soluble drugs is increased, solubility and dissolution rate are improved, and there is greater surface adhesion, which prolongs the drug's action time in the mucus layer. 3. Improve drugability: Nanocrystal technology is suitable for Class II and IV drugs in the Biopharmaceutical Classification System (BCS), especially the former, which greatly improves the drugability of poorly soluble drugs. 4. High drug loading: In addition to the necessary stabilizers, no other excipients are required in the product. The drug itself is used as the delivery system, and the drug loading capacity is high. Drug nanocrystals can be used as both the final dosage form and the intermediate dosage form, and can be further processed into tablets, capsules, inhalation preparations, etc., and can be administered orally, by injection, pulmonary inhalation, etc.

[0005] In the past few decades, the burden of various respiratory diseases (such as lung cancer, pneumonia, tuberculosis, asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, etc.) has risen sharply, and there is an urgent need to develop efficient and safe inhalation therapies. The pulmonary drug delivery system refers to a drug delivery system in which the drug enters the respiratory tract directly through a special drug delivery device to exert local or systemic therapeutic effects. Compared with systemic (such as intravenous or oral) administration, it is the most direct and effective route of administration for the treatment of lung diseases. In recent years, dry powder inhalers (DPI) have gradually become the focus of pulmonary drug delivery systems. Dry powder inhalers are solid micronized APIs alone or mixed with suitable carriers, in the form of capsules, blisters or multi-dose reservoirs, using a special dry powder inhalation device, and the patient actively inhales the aerosolized drug into the lungs, which can exert local or systemic therapeutic effects. Because the preparation is in powder form, the active drug is relatively stable compared with aerosol preparations. DPI can achieve small-dose administration, reduce the incidence of local and systemic side effects, and significantly improve the consistency of delivered doses. There are no coordination issues between the patient's inhalation and the powder flow from the device, the drug is activated by the patient's airflow, and no energy source is required, making it popular. DPI has been used to deliver a variety of active pharmaceutical ingredients, such as antibiotics, asthma treatments, anti-tuberculosis drugs, insulin, proteins and peptides, and vaccines.

[0006] It is worth noting that in order to achieve effective lung deposition, the aerodynamic diameter (Da) of the inhaled drug must be controlled to 1-5 μm, and the aerodynamic diameter is affected by the shape, size and density of the particles. Therefore, excellent carriers and optimized spray drying processes are key factors affecting the inhalation performance of andrographolide dry powder inhalers. Summary of the invention

[0007] In view of the deficiencies in the prior art, the purpose of the present invention is to provide an andrographolide nanocrystal suspension and its preparation and application in the preparation of andrographolide nanocrystal dry powder inhaler with anti-inflammatory effect. It meets the requirements of the 2020 edition of the Chinese Pharmacopoeia for dry powder inhalers, and has been proven to have good anti-inflammatory effects and good practical application value.

[0008] To achieve the above purpose, the present invention adopts the following technical solution:

[0009] The invention discloses an andrographolide nanocrystal suspension, which contains a) an active ingredient andrographolide nanocrystals and b) a stabilizer. The andrographolide content in the suspension is 0.1% to 30% (w / v), and the stabilizer content is 0.1% to 5% (w / v).

[0010] The suspension comprises active ingredient andrographolide nanocrystals, a stabilizer and water; wherein the content of andrographolide in the suspension is 1% to 10% (w / v) and the content of the stabilizer is 0.25% to 2% (w / v).

[0011] The particle size of andrographolide nanocrystals in the andrographolide nanocrystal suspension is 1 to 1000 nm, and the dispersibility index is 0.000 to 1.000. The preferred particle size is 10 to 600 nm, and the dispersibility index is 0.000 to 0.400; the more preferred particle size is 10 to 350 nm, and the dispersibility index is 0.000 to 0.200.

[0012] The stabilizer is poloxamer 188 and / or poloxamer 407.

[0013] A method for preparing an andrographolide nanocrystal suspension, wherein the andrographolide raw material drug after air flow pulverization is subjected to wet medium grinding to obtain andrographolide nanocrystals with a particle size of 1 to 1000 nm and a dispersibility index of 0.000 to 1.000, and then the andrographolide nanocrystals are mixed with a solution containing a stabilizer to obtain an andrographolide nanocrystal suspension

[0014] Wherein, the andrographolide nanocrystals are prepared by bottom-up and top-down techniques, the former including precipitation method and emulsification method, and the latter including high-pressure homogenization method and medium grinding method; preferably, the method selects high-pressure homogenization method and medium grinding method, and the more preferred method is medium grinding method.

[0015] An application of the andrographolide nanocrystal suspension, and an application of the nanocrystal suspension in the preparation of andrographolide nanocrystal dry powder inhaler.

[0016] An andrographolide nanocrystal dry powder inhaler, the preparation contains the andrographolide nanocrystal suspension and a diluent; wherein the content of andrographolide nanocrystals is 1% to 60% (w / w), and the amount of the diluent is 1% to 99% (w / w). Preferably, the content of andrographolide nanocrystals is 1% to 60% (w / w), and the amount of the diluent is 20% to 80% (w / w).

[0017] The diluent is selected from sugars or sugar alcohols and amino acid acid carriers, the sugar carrier is one or more of lactose, sucrose, glucose, trehalose, and β-cyclodextrin; the sugar alcohol carrier is one or more of mannitol, sorbitol, and xylitol; the amino acid carrier is one or more of alanine, valine, leucine, isoleucine, phenylalanine, glycine, threonine, aspartic acid, lysine, and arginine.

[0018] Preferred diluents are mannitol and leucine

[0019] When the diluents are mannitol and leucine, the mass ratio of mannitol to leucine is 1:(1-10) or (1-10):1; preferably, the mass ratio of mannitol to leucine is 1:(1-5); and more preferably, the mass ratio of mannitol to leucine is 1:1.

[0020] A method for preparing the andrographolide nanocrystal dry powder inhaler,

[0021] Step 1: preparing andrographolide nanocrystal suspension;

[0022] Step 2: dissolving the diluent in the andrographolide nanocrystal suspension prepared in step 1, and spray drying.

[0023] The andrographolide nanocrystal dry powder inhalation is carried out in a spray dryer, the inlet temperature of the spray dryer is 100-180°C, the feed rate is 0.3-6mL / min, the gas flow rate is 335-1005L / h, and the cyclone strength is 80%-100%. The more preferred inlet temperature of the spray dryer is 160°C, the feed rate is 2.4mL / min, the gas flow rate is 837L / h, and the cyclone strength is 100%. The andrographolide nanocrystal dry powder inhalation has an aerodynamic particle size of 0.5-10μm and a span of 0-6; the preferred aerodynamic particle size is 1-5μm and the span is 0-4; the more preferred aerodynamic particle size is 1.5-3μm; the span is 0-2.

[0024] Compared with the prior art, the present invention has the following positive and beneficial effects:

[0025] 1. The present invention prepares the water-insoluble drug AG into a nanocrystalline suspension (AG-NC) by a medium grinding method, thereby increasing the solubility and improving the drugability, solving the problem of low solubility of AG, and providing a better intermediate for the later preparation of DPI; on the other hand, this technology and the spray drying technology for preparing DPI complement each other, wherein there is a small amount of P188 in AG-NC. The present invention uses poloxamer 188 as a stabilizer, which is an amphiphilic substance with high safety. As a stabilizer, it can reduce the free energy of the nanocrystalline system, slow down the aggregation rate of nanocrystals, provide stability, and increase the solubility of andrographolide; at the same time, it also acts as an antistatic agent to solve the problem that the inhaled powder will show static electricity in winter, increase the fluidity of the particles, and improve the atomization performance of the particles; in addition, the spray drying technology used can solidify the AG-NC suspension, which is more conducive to the stability of AG-NC.

[0026] 2. The present invention adopts spray drying technology to make andrographolide nanocrystals into micropowder, and optimizes the addition of auxiliary materials and the preparation process, so as to successfully prepare andrographolide nanocrystal dry powder inhaler with good inhalation performance, and directly acts on the respiratory tract through the pulmonary drug delivery system, thereby reducing the dosage and obtaining a higher local lung concentration, avoiding the first-pass effect of the liver while avoiding the p-glycoprotein efflux effect as much as possible, thereby improving its bioavailability; and solves the technical problem that andrographolide is excreted through p-glycoprotein and quickly absorbed and metabolized in the intestine, resulting in poor bioavailability. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 : is the particle size and Zeta potential diagram of andrographolide nanocrystals, wherein A is the particle size distribution diagram of andrographolide nanocrystals, and B is the Zeta potential diagram of andrographolide nanocrystals;

[0028] Figure 21 is the particle size and distribution diagram of andrographolide nanocrystal dry powder inhaler, wherein 1 is the particle size and distribution diagram measured for the first time, 2 is the particle size and distribution diagram measured for the second time, and 3 is the particle size and distribution diagram measured for the third time;

[0029] Figure 3 This is the DSC phase transition characteristic diagram of andrographolide nanocrystal dry powder inhalation;

[0030] Figure 4 This is the XRD phase change characteristic diagram of andrographolide nanocrystal dry powder inhalation;

[0031] Figure 5 This is the morphological characteristic diagram of andrographolide nanocrystal dry powder inhalation;

[0032] Figure 6 It is a graph of TNF-a and IL-6 levels in rat bronchoalveolar lavage fluid in the anti-inflammatory effect experiment, where A is the level of TNF-a and B is the level of IL-6;

[0033] Figure 7 These are H&E stained pathological sections of lung tissues of rats in the normal group and model group in the anti-inflammatory effect experiment, where A is the normal group and B is the model group;

[0034] Figure 8 These are H&E stained pathological sections of lung tissue of rats in the andrographolide nanocrystal dry powder inhalation treatment group and the andrographolide capsule treatment group in the time-dependent anti-inflammatory effect experiment. DETAILED DESCRIPTION

[0035] The following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0036] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. In addition, it should also be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates that there are features, steps, operations, parts and / or combinations thereof.

[0037] The experimental methods in the following examples without specifying specific conditions all adopt conventional techniques in the art or follow the conditions recommended by the manufacturers; the reagents or instruments used without specifying the manufacturers are all conventional products that can be obtained commercially.

[0038] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments. The preparation process and process parameters of the following pharmaceutical preparation embodiments and the substances used in the preparation or the amount of the substances used in the preparation are not limited to the textual description, and all preparation methods containing the pharmaceutical composition provided by the present invention belong to the protection scope of the present invention.

[0039] The present invention aims to solve the problem of poor water solubility of AG and combines the advantages of drug nanocrystals to first prepare it into AG nanocrystal (Nanocrystals, NC) suspension (AG-NC) to increase its specific surface area, improve solubility, and enhance the drugability of AG. In addition, a better intermediate (andrographolide nanocrystal suspension (AG-NC)) is provided for preparing dry powder inhalers. Secondly, in view of the problem that AG is rapidly absorbed and metabolized in the intestine due to its efflux through p-glycoprotein, it is designed as dry powder inhalers (DPI) to directly act on the respiratory tract through a pulmonary drug delivery system, thereby reducing the dosage and obtaining a higher local lung concentration, avoiding the first-pass effect of the liver and weakening the efflux effect of p-glycoprotein as much as possible.

[0040] Example 1: Screening of stabilizers

[0041] In order to explore the effects of different stabilizers on the stability and particle size of andrographolide nanocrystals, the present invention carried out Examples 1-1 to 1-7. The specific contents of Examples 1-1 to 1-7 are as follows.

[0042] Example 1-1:

[0043] A method for preparing andrographolide nanocrystal suspension, the specific steps are as follows:

[0044] (1) The andrographolide raw material is micronized by air flow milling, and the andrographolide after micronization has a D10 of 0.86 μm, a D50 of 3.29 μm, a D90 of 9.44 μm, and a D[4,3] of 4.46 μm;

[0045] (2) Weigh 0.5 g of the above-mentioned micronized andrographolide and add 20 mL of water (containing a stabilizer, the final concentration of the stabilizer in water is 0.5% sodium dodecyl sulfate (w / v))

[0046] In the medium, high-speed shearing was performed at 15000 rpm in a small homogenizer for 5 min to obtain a primary suspension of andrographolide;

[0047] (3) Add the above primary suspension into the grinding tank of a ball mill (FRITSCH Instrument Company, Germany), and then add 80g of zirconium oxide beads. The mixture was ground at a speed of 400 r / min for 2 h to obtain andrographolide nanocrystal suspension.

[0048] The contents of Examples 1-2 to 1-7 are basically the same as those of Example 1-1, except that different stabilizers are used at the same concentration; among them, the stabilizers used in Examples 1-2 to 1-7 are: lecithin, vitamin E polyethylene glycol succinate, poloxamer 188, poloxamer 407, polyvinylpyrrolidone K30, and hydroxypropyl methylcellulose, respectively.

[0049] The particle size and PDI of the andrographolide nanocrystal suspension prepared in Examples 1-1 to 1-7 were tested (using a Malvern laser particle size analyzer). The test results are shown in Table 1.

[0050] Example Stabilizer Average particle size (nm) PDI Example 1-1 Sodium Lauryl Sulfate 578.5 0.276 Example 1-2 Lecithin 489.5 0.260 Examples 1-3 Vitamin E polyethylene glycol succinate 769.3 0.306 Examples 1-4 Poloxamer 188 363.5 0.186 Examples 1-5 Poloxamer 407 368.2 0.186 Examples 1-6 Polyvinylpyrrolidone K30 689.3 0.274 Examples 1-7 Hydroxypropyl methylcellulose 425.3 0.207

[0051] As shown in Table 1, the particle size and PDI of the andrographolide nanocrystal suspension with P188 and P407 as stabilizers are the smallest. In order to distinguish P188 from P407, the prepared nanosuspension was left to stand for 7 days. It was found that the nanocrystals with P407 as stabilizer agglomerated and the crystals turned black, and stratification occurred, while the nanocrystals with P188 as stabilizer did not agglomerate significantly and remained a uniform suspension. Therefore, P188 was selected as the stabilizer for the andrographolide nanocrystal suspension.

[0052] Example 2: Investigation of Stabilizer Concentration

[0053] In order to explore the effect of stabilizer concentration on the particle size and PDI of andrographolide nanocrystal suspension, the present invention carried out Examples 2-1 to 2-4. The specific contents of Examples 2-1 to 2-4 are as follows.

[0054] Example 2-1:

[0055] A method for preparing andrographolide nanocrystal suspension, the specific steps are as follows:

[0056] (1) Weigh 0.5 g of the above-mentioned micronized andrographolide, add it to 20 mL of water (containing 0.25% poloxamer 188 (w / v)), and high-speed shear for 5 min at 15000 rpm in a small homogenizer to obtain a primary suspension of andrographolide;

[0057] (2) Add the above primary suspension into the grinding tank of the ball mill, and then add zirconium oxide beads The mixture was ground at a speed of 400 r / min for 2 h to obtain andrographolide nanocrystal suspension.

[0058] The contents of Examples 2-2 to 2-4 are basically the same as those of Example 2-1, except that different stabilizer concentrations are used. The stabilizer concentrations used in Examples 2-1 to 2-4 are 0.25%, 0.5%,

[0059] 0.75%, 1%.

[0060] The particle size and PDI of the andrographolide nanocrystal suspension prepared in Example 2-1 to Example 2-4 were tested, and the test results are shown in Table 2.

[0061] Example Stabilizer concentration (w / v) Average particle size (nm) PDI Example 2-1 0.25％ 742.1 0.462 Example 2-2 0.5％ 363.5 0.186 Example 2-3 0.75％ 349.0 0.172 Embodiment 2-4 1％ 318.4 0.168

[0062] It can be seen from Table 2 that with the increase of stabilizer concentration, the particle size and PDI tend to decrease. However, considering the later preparation of dry powder inhaler, P188 has a low melting point and excessive dosage will affect spray drying. Therefore, the final concentration of P188 is determined to be 0.5%.

[0063] Example 3: Investigation of grinding time

[0064] In order to explore the effect of stabilizer concentration on the particle size and PDI of andrographolide nanocrystal suspension, the present invention carried out Examples 3-1 to 3-4. The specific contents of Examples 3-1 to 3-4 are as follows.

[0065] Example 3-1:

[0066] A method for preparing andrographolide nanocrystal suspension, the specific steps are as follows:

[0067] (1) Weigh 0.5 g of the above-mentioned micronized andrographolide, add it to 20 mL of water (containing 0.5% poloxamer 188), and high-speed shear it in a small homogenizer at 15,000 rpm for 5 minutes to obtain a primary suspension of andrographolide;

[0068] (2) Add the above primary suspension into the grinding tank of the ball mill, and then add zirconium oxide beads The mixture was ground at a speed of 400 r / min for 0.5 h to obtain andrographolide nanocrystal suspension.

[0069] The contents of Example 3-2 to Example 3-4 are basically the same as Example 3-1, except that the grinding time of the ball mill is different; among them, the grinding time used in Example 3-1 to Example 3-4 is 0.5h, 1h, 2h, and 3h, respectively.

[0070] The particle size and PDI of the andrographolide nanocrystal suspension prepared in Example 3-1 to Example 3-4 were tested, and the test results are shown in Table 3.

[0071] Example Grinding time Average particle size (nm) PDI Example 3-1 0.5h 903.5 1.000 Example 3-2 1h 308.3 0.158 Example 3-3 2h 314.3 0.172 Embodiment 3-4 3h 320.3 0.197

[0072] As shown in Table 3, the longer the grinding time, the smaller the particle size and PDI, and finally tend to be stable. Considering energy saving and consumption reduction and improving preparation efficiency, the final grinding time is 1h. The particle size and Zeta potential of Andrographis paniculata nanocrystals prepared with a grinding time of 1h are shown in Figure 1 As shown, the results showed that the particle size was 308.3 nm and the Zeta potential was -23.5 mV.

[0073] Example 4: Screening of Carbohydrate and Sugar Alcohol Carriers

[0074] In order to screen a suitable diluent for andrographolide nanocrystal dry powder inhalation, the present invention carried out Examples 4-1 to 4-6. The specific contents of Examples 4-1 to 4-6 are as follows.

[0075] Example 4-1:

[0076] A method for preparing andrographolide nanocrystal dry powder inhalation, the specific steps are as follows:

[0077] (1) preparing andrographolide nanocrystal suspension according to Example 3-2;

[0078] (2) The above-mentioned andrographolide nanocrystal suspension is appropriately diluted, and the prescribed amount of α-lactose monohydrate and leucine are weighed and dissolved in the suspension, and a suspension having a solid content of 6.2% (w / v) is prepared according to the ratio of andrographolide: α-lactose monohydrate: leucine in the andrographolide nanocrystal suspension of 0.5:1.67:0.83 (w / w / w), and spray-dried to obtain andrographolide nanocrystal dry powder inhaler. The preliminary spray drying process is: inlet temperature 120°C, feed rate 3mL / min, gas flow rate 586L / h, cyclone intensity 100%.

[0079] The contents of Examples 4-2 to 4-6 are basically the same as those of Example 4-1, except that different sugar and sugar alcohol carriers are added; among them, the sugar and sugar alcohol carriers added in Examples 4-2 to 4-6 are anhydrous lactose, mannitol, trehalose, glucose, and sorbitol, respectively.

[0080] For the andrographolide nanocrystalline dry powder inhalation prepared in Examples 4-1 to 4-6, the yield, particle size and its distribution, span, Da were calculated as evaluation indicators to investigate the effect of the above carriers on the properties of andrographolide nanocrystalline dry powder inhalation. The evaluation results are shown in Table 4.

[0081]

[0082] As shown in Table 4, the formulations with trehalose and mannitol as carriers have higher yields and smaller particle sizes, and the aerodynamic particle sizes are close to the requirements for lung inhalation particle sizes. During the sample storage process, it was found that the samples with trehalose added had serious agglomeration, which is related to the high hygroscopicity of trehalose. Therefore, mannitol was selected as the sugar alcohol carrier for the above-mentioned dry powder inhaler.

[0083] Example 5: Investigation of inlet temperature

[0084] According to the preparation method of Example 4-3, the feed rate is fixed at 3mL / min, the gas flow rate is 586L / h, the cyclone intensity is 100%, and the solid content of the drug solution is 6.2%. The effect of inlet temperature of 100°C, 110°C, 120°C, 130°C, 140°C, 150°C, and 160°C on spray drying is investigated. According to the method specified in the Chinese Pharmacopoeia (2020 edition), the FPF (fine particles fraction) of andrographolide nanocrystalline dry powder inhaler is tested. FPF refers to the effective fine particle dose that can be deposited in the lungs under standard flow rate conditions. According to the regulations, the FPF value can be effectively deposited in the lungs when it is greater than 12%.

[0085] The results are shown in Table 5. When the inlet temperature is 100°C, the yield is very low. When this temperature is set, the outlet temperature detected is around 55°C, which cannot completely dry the material, resulting in more material sticking to the wall. The yields of other groups are all above 80%, which is quite impressive. FPF increases with increasing temperature. When the temperature is 160°C, FPF is the largest, but the powder Da at this time is not ideal. However, the powder Da prepared at 120°C is close to the particle size requirement for lung inhalation, so 120°C is temporarily set as the inlet temperature to explore subsequent processes.

[0086] Table 5 Evaluation of powder properties prepared at different inlet temperatures of spray drying

[0087]

[0088]

[0089] Example 6: Investigation of feed rate

[0090] According to the preparation method of Example 4-3, the inlet temperature was fixed at 120°C, the gas flow rate was 586L / h, the cyclone intensity was 100%, and the solid content of the liquid medicine was 6.2%. The effects of feed rates of 1.8, 2.4, 3, 3.6, and 4.2mL / min on spray drying were investigated. The results are shown in Table 6. The yield decreases with the increase of the feed rate, and the nozzle is easily blocked when the feed rate is faster; the particle size span of the prepared powder is less than 2. When the feed rate is 2.4mL / min, the prepared powder has a smaller Da diameter and the highest FPF, and the density is relatively small, and the fluidity is relatively good. This fixed rate is the best rate.

[0091] Table 6 Evaluation of powder properties prepared at different feed rates during spray drying

[0092]

[0093] Example 7: Investigation of gas flow rate

[0094] According to the preparation method of Example 4-3, the inlet temperature was fixed at 120°C, the feed rate was 2.4mL / min, the cyclone intensity was 100%, and the solid content of the drug solution was 6.2%. The effect of gas flow rates of 502, 586, 670, 753, and 837L / h on spray drying was investigated. The results are shown in Table 7. The aerodynamic particle size decreases with the increase of gas flow rate. When the gas flow rate increases to 670L / h, Da meets the requirements of pulmonary inhalation, and the yield is as high as 98.7%. Therefore, this gas flow rate is temporarily set to explore the subsequent process.

[0095] Table 7 Evaluation of powder properties prepared at different feed rates of spray drying

[0096]

[0097] Example 8: Investigation of Cyclone Intensity

[0098] The cyclone intensity refers to the cyclone intensity in the cyclone separator of the spray dryer. According to the preparation method of Example 4-3, the inlet temperature is fixed at 120°C, the feed rate is 2.4mL / min, the gas flow rate is 670L / h, and the solid content of the liquid medicine is 6.2%. The effects of 100%, 90%, and 80% cyclone intensity on spray drying are investigated. The results are shown in Table 8. The cyclone intensity has little effect on the powder particle size. However, as the cyclone intensity decreases, the yield and FPF of the powder decrease, so the cyclone intensity is fixed at 100%.

[0099] Table 8 Evaluation of powder properties prepared by spray drying at different cyclone intensities.

[0100]

[0101] Example 9: Investigation of drug loading ratio

[0102] According to the preparation method of Example 4-3, the prescribed amount of mannitol and leucine were weighed, and the ratio of andrographolide nanocrystals (in terms of andrographolide) to the carrier was 2:1, 1:1, 3:1, 4:1, 5:1 (w / w), according to the inlet temperature of 120°C, the feed rate of 2.4mL / min, the gas flow rate of 670L / h, the cyclone intensity of 100%, and the solid content of the liquid medicine of 6.2%. Spray drying was performed to prepare five dry powder inhalers with different drug contents. The results are shown in Table 9. When the drug-carrying ratio increases, the yield decreases, Da increases, and FPF shows a trend of first decreasing and then decreasing. When the drug-carrying ratio is 1:5, Da meets the requirements of pulmonary inhalation and FPF is the largest. Considering comprehensively, it is determined that the ratio of andrographolide to the carrier is 1:5.

[0103] Table 9 Evaluation of powder properties prepared at different drug loading ratios

[0104]

[0105] Example 10: Orthogonal experiment to optimize the preparation process

[0106] On the basis of single factor investigation, the two process parameters of feed rate and cyclone intensity were fixed, and four factors, m(mannitol):m(leucine) (A), liquid solid content (B), inlet temperature (C), and gas flow rate (D), were selected as the investigation objects. Three levels were selected for each factor. According to L9(3 4 ) Orthogonal table design experiment, factor level table is shown in Table 8. According to the yield, compression degree (measured by tap density instrument), Da and FPF results of andrographolide nanocrystal dry powder inhaler prepared by orthogonal experiment, the multi-index comprehensive scoring method (Z score comprehensive evaluation method) was used to comprehensively evaluate each level selected in the experiment. Formula and ∑Z i =∑Z i，h -∑Z i，l Calculate, X i is the result of a certain indicator in a certain group of experiments. is the average value of this indicator in 9 groups of experiments, S i is the standard deviation of the indicator for the 9 groups of experiments, Z i，h is the Zi value of the high-quality index, Z i，l The Zi value is the low-quality index. Within a certain range, among the four selected indexes, the yield and FPF are "high-quality" indexes, and the compression and Da are "low-quality" indexes. The Z value of "high-quality" is "added" and the Z value of "low-quality" is "subtracted". The larger the final result, the better the result.

[0107] Table 10 Orthogonal experimental factor level coding table

[0108]

[0109] The orthogonal experimental design and results of the spray drying method for preparing andrographolide nanocrystalline dry powder inhalation are shown in Table 11.

[0110] Table 11 Orthogonal experimental design and results

[0111]

[0112] The scores were analyzed with a range analysis, and the order of the influence of each factor on the properties of andrographolide nanocrystalline dry powder inhalation in the orthogonal experiment was obtained as follows: A (m (mannitol): m (leucine)) > B (solid content of the drug solution) > D (gas flow) > C (inlet temperature). From the above experimental results, it can be seen that the optimal prescription and spray drying conditions of andrographolide nanocrystalline dry powder inhalation are: A1B2C3D1, that is, the optimized prescription and process conditions are: m (mannitol): m (leucine) = 1:1, solid content of the drug solution 3.1%, inlet temperature 160 ° C, gas flow 837L / h.

[0113] Example 11: Preparation of andrographolide dry powder nanocrystal dry powder inhaler

[0114] According to the preparation method of Example 4-3 and the optimal preparation process optimized in Example 10, andrographolide nanocrystalline dry powder inhalation was prepared. The prepared andrographolide nanocrystalline dry powder inhalation had a moisture content of 1.839%, a bulk density of 0.21 g / cm3, and a tap density of 0.39 g / cm 3 , Carr's index is 37.2%, D[4,3] is 2.58μm, span is 2.49, aerodynamic particle size is 1.69μm, emptying rate is 95.64%, FPF is 67.8%, and all indicators of the obtained preparation can meet the requirements of pulmonary inhalation administration.

[0115] Application Example 1 Anti-inflammatory effect of andrographolide nanocrystal dry powder inhalation on rats with acute pneumonia

[0116] The purpose of this experiment to investigate the anti-inflammatory effect is to explore the relationship between local drug exposure in the lungs and local efficacy, so preventive administration is adopted for evaluation.

[0117] Materials: Andrographolide nanocrystal dry powder inhaler prepared according to Example 11; Andrographolide capsules (Jiuhui Pharmaceutical Co., Ltd.); 4% paraformaldehyde (Jijia Biotechnology Co., Ltd.); Lipopolysaccharide (Jitai Biotechnology Co., Ltd.); TNF-α ELISA kit, IL-6 ELISA kit (Enzyme-linked Biotechnology Co., Ltd.).

[0118] Methods: Thirty rats were randomly divided into 4 groups: normal group (healthy rats), model group (rats with acute pneumonia induced by tracheal instillation of lipopolysaccharide, without treatment), andrographolide nanocrystal dry powder inhalation treatment group, andrographolide capsule treatment group.

[0119] Three rats were taken in the normal group and physiological saline was given through the trachea. Three rats were taken in the model group, and rats were anesthetized by intraperitoneal injection of urethane, and the rat trachea was exposed. 2mg / kg lipopolysaccharide was instilled through the trachea. After 6h of instillation of lipopolysaccharide, the rats were killed by blood sampling from the peritoneal vein, the right lung was ligated, and the left alveolar lavage fluid of the rat was taken to detect the levels of two cytokines, TNF-α and IL-6, and the right lung tissue was taken for pathological sections. The andrographolide nanocrystal dry powder inhalation administration group (obtained in Example 11) took 12 rats, and 3 rats were taken at each administration time point. When the tracheal administration was 1h, 3h, 6h, and 12h, lipopolysaccharide was instilled in the trachea, and the subsequent operation was the same as the model group. The andrographolide capsule administration group (Jiuhui Pharmaceutical Co., Ltd.) took 12 rats, and 3 rats were taken at each administration time point. When the intragastric administration was 1h, 3h, 6h, and 12h, lipopolysaccharide was instilled in the trachea, and the subsequent operation was the same as the model group. The dosage of the two groups treated with the above drugs was 20 mg / kg.

[0120] Results: From the perspective of cytokines, Figure 6 The levels of the two inflammatory factors in the rats in the normal group were lower; TNF-α and IL-6 in the rats in the model group increased significantly 6 hours after LPS administration, proving that the acute pneumonia model induced by LPS in rats was successfully established in this experiment; the two treatment groups had different degrees of anti-inflammatory effects in the lungs of rats at different time points of administration; the levels of TNF-α and IL-6 in the alveolar lavage fluid of rats in the andrographolide nanocrystal dry powder inhalation treatment group decreased in a time-dependent manner, and compared with the andrographolide capsule treatment group, due to direct administration to the lungs, it showed a good anti-inflammatory effect 1 hour after administration; however, the levels of TNF-α and IL-6 in the alveolar lavage fluid of rats in the andrographolide capsule treatment group did not decrease in a time-dependent manner, and the anti-inflammatory effect was not shown until 6 hours after administration.

[0121] From the pathological section, Figure 7 The lung tissue sections of the rats in the normal group were normal, with complete and clear lung tissue, thin alveolar septa, and no inflammatory exudate in the alveolar cavity; the lung tissue sections of the rats in the model group showed that the alveolar septa were significantly thickened, almost showing the phenomenon of "alveolar squeezing alveoli", with severe inflammatory infiltration, and the pink exudate in the alveolar septa increased significantly, which confirmed that the acute pneumonia model induced by lipopolysaccharide in rats was successfully established; Figure 8In the andrographolide nanocrystal dry powder inhalation treatment group, the alveolar septum of rats was thickened at the four time points of administration, with very little pink exudate in the alveolar septum, and the pulmonary artery wall was normal without rupture. It can be inferred that the homemade andrographolide nanocrystal dry powder inhalation has a better anti-inflammatory effect when administered by pulmonary inhalation; however, the alveolar septum of the andrographolide capsule treatment group was also thickened, with more pink exudate in the alveolar septum at 1h and 3h of administration, and less pink exudate in the alveolar septum at 6h and 12h of administration.

[0122] The above embodiments and experimental examples are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. An andrographolide nanocrystal suspension, characterized in that: The andrographolide nanocrystal suspension contains a) active ingredient andrographolide nanocrystals; and b) a stabilizer. The andrographolide content in the suspension is 0.1% to 30% (w / v), and the stabilizer content is 0.1% to 5% (w / v).

2. The andrographolide nanocrystal suspension according to claim 1, characterized in that: The suspension comprises active ingredient andrographolide nanocrystals, a stabilizer and water; wherein the content of andrographolide in the suspension is 1% to 10% (w / v) and the content of the stabilizer is 0.25% to 2% (w / v).

3. The andrographolide nanocrystal suspension according to claim 1 or 2, characterized in that: The particle size of andrographolide nanocrystals in the andrographolide nanocrystal suspension is 1-1000nm, and the dispersion index is 0.000-1.

000.

4. The andrographolide nanocrystal suspension according to claim 1 or 2, characterized in that: The stabilizer is poloxamer 188 and / or poloxamer 407.

5. A method for preparing the andrographolide nanocrystal suspension according to claim 1, characterized in that: The andrographolide raw material drug after air flow pulverization is subjected to wet medium grinding to obtain andrographolide nanocrystals with a particle size of 1 to 1000 nm and a dispersibility index of 0.000 to 1.000, and then the andrographolide nanocrystals are mixed with a solution containing a stabilizer to obtain an andrographolide nanocrystal suspension.

6. An application of the andrographolide nanocrystal suspension according to claim 1, characterized in that: The nanocrystal suspension is used in preparing andrographolide nanocrystal dry powder inhaler.

7. An andrographolide nanocrystal dry powder inhaler, characterized in that: The preparation contains the andrographolide nanocrystal suspension according to claim 1 and a diluent; wherein the content of andrographolide nanocrystals is 1% to 60% (w / w), and the amount of the diluent is 1% to 99% (w / w).

8. The andrographolide nanocrystal dry powder inhaler according to claim 7, characterized in that: The diluent is selected from sugars or sugar alcohols and amino acid acid carriers, the sugar carrier is one or more of lactose, sucrose, glucose, trehalose, and β-cyclodextrin; the sugar alcohol carrier is one or more of mannitol, sorbitol, and xylitol; the amino acid carrier is one or more of alanine, valine, leucine, isoleucine, phenylalanine, glycine, threonine, aspartic acid, lysine, and arginine.

9. A method for preparing the andrographolide nanocrystal dry powder inhaler according to claim 7, characterized in that: Step 1: preparing andrographolide nanocrystal suspension; Step 2: dissolving the diluent in the andrographolide nanocrystal suspension prepared in step 1, and spray drying.

10. The method for preparing the andrographolide nanocrystal dry powder inhaler according to claim 9, characterized in that: The andrographolide nanocrystal dry powder inhaler is prepared in a spray dryer, the inlet temperature of the spray dryer is 100-180°C, the feed rate is 0.3-6mL / min, the gas flow rate is 335-1005L / h, and the cyclone intensity is 80%-100%.

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