Application of synephrine dry powder inhalant in preparing medicine for treating acute lung injury

Abstract

The present invention discloses the use of synephrine dry powder inhalant in the preparation of a medicament for treating acute lung injury. By formulating synephrine into a dry powder inhalant (DPI) dosage form, the present invention overcomes the problem of low bioavailability of synephrine. After pulmonary administration, it directly and actively targets the lungs, increases the blood drug concentration in the lungs, reduces the distribution of the drug in other tissues, thereby improving the drug efficacy and reducing systemic side effects. At the same time, the dry powder inhalation method avoids the first-pass effect and is beneficial to the stability of the drug, and the non-invasive administration route increases the compliance of patients during treatment. The present invention provides a new idea for the research of pulmonary drug delivery systems and has broad clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and particularly relates to the use of synephrine dry powder inhalant in the preparation of a medicament for treating acute lung injury. Background Art

[0002] Acute lung injury (ALI) or its more severe clinical manifestation - acute respiratory distress syndrome (ARDS) is an acute inflammatory lung disease with relatively high annual incidence and mortality. It is a common critical illness with a fatality rate higher than 40%, seriously threatening the lives of patients. Acute lung injury is an acute respiratory insufficiency caused by the injury of alveolar epithelial cells and capillary endothelial cells. ALI is induced by a variety of direct or indirect problems. Direct injuries include pneumonia, aspiration of gastric contents, inhalation of harmful gases or lung contusion. Indirect injuries are lung injuries caused by other severe diseases such as sepsis, pancreatitis, multiple traumas or severe systemic infections. Among them, sepsis is the most common cause of human acute respiratory distress syndrome. At present, clinical treatment methods cannot completely cure acute lung injury, but can only control the development of the disease and reduce the pain and injury of patients. The main clinical treatment methods include mechanical ventilation, inhalation of vasodilators and the use of highly anti-inflammatory glucocorticoids to treat acute lung injury.

[0003] Synephrine is a natural component of fruits of plants in the genus Citrus of the family Rutaceae, such as Fructus Aurantii Immaturus, Fructus Aurantii, Pericarpium Citri Reticulatae, Pericarpium Citri Reticulatae Viride, etc. It has the highest content in the immature fruits of bitter orange and is the main active ingredient in extracts of Fructus Aurantii Immaturus, bitter orange, etc., belonging to ephedrine alkaloids. The similarity between synephrine and endogenous adrenergic receptor agonists adrenaline and noradrenaline lies in the common basic structure - phenethylamine. Due to the similar structure of synephrine and sympathomimetic amines, it can bind to and activate different α- and β-adrenergic receptors, thereby producing different physiological and pharmacological effects. The pharmacological effects mediated by β-adrenergic receptors can inhibit the expression of pro-inflammatory cytokines in acute lung injury and can reduce the level of oxidative stress to play a therapeutic role.

[0004] Synephrine is a white powder with a bitter taste. It has both phenolic hydroxyl groups and secondary amino groups in its structure, has high solubility in water or alcohol solvents, has general thermal stability, can react with acids or bases to form salts, has amphoteric properties in the chemical sense, and is unstable and easily oxidized under strong acids, strong bases or high temperatures. After oral administration, the absorption amount in the lungs is relatively low, and the bioavailability is limited. Higher doses and more frequent administrations are required, resulting in low patient compliance.

[0005] Therefore, how to improve the stability, targeting and bioavailability of synephrine drugs is an urgent problem to be solved. Summary of the Invention

[0006] Aiming at the deficiencies of the above-mentioned existing technologies, the purpose of the present invention is to provide an application of synephrine dry powder inhalant in the preparation of a drug for treating acute lung injury. By formulating synephrine into a dry powder inhalant (DPI) dosage form and adopting the pulmonary inhalation administration method, the present invention not only avoids the first-pass effect and is beneficial to the stability of the drug, but also can significantly improve the targeting and bioavailability of the drug.

[0007] The present invention is realized through the following technical solutions:

[0008] The present invention provides an application of synephrine dry powder inhalant in the preparation of a drug for treating acute lung injury.

[0009] Preferably, the aerodynamic particle size of the synephrine dry powder inhalant is 1 - 5 μm. The atomization performance of the dry powder inhalant is related to the size of the aerodynamic diameter of the particles. Generally, particles with a diameter less than 0.5 μm are easily excreted from the body with breathing, and if greater than 10 μm, they are easily deposited in the upper respiratory tract. Therefore, to achieve good distribution in the deep lung region, the optimal aerodynamic diameter of the inhaled particles is 1 - 5 μm.

[0010] Preferably, the fine particle fraction (FPF) of the synephrine dry powder inhalant is 28 - 33%, the emptying rate is 94 - 98%, the inhalable fraction is 60 - 70%, and the in vitro pulmonary effective deposition rate is 18 - 20%. The synephrine dry powder inhalant prepared by the present invention has suitable physical and chemical properties such as an appropriate emptying rate and fine particle fraction, meets the requirements of pulmonary inhalation administration, and can be effectively deposited in the lungs.

[0011] The preparation method of the synephrine dry powder inhalant described in the present invention includes the following steps:

[0012] (1) Dissolve the synephrine raw material in a good solvent to form a good solvent phase A, and use n-hexane as an anti-solvent phase B;

[0013] (2) Drop the good solvent phase A into the anti-solvent phase B under stirring to obtain a uniform suspension;

[0014] (3) Centrifuge the suspension obtained in step (2), dry the precipitate, and obtain the synephrine dry powder inhalant.

[0015] Based on the solubility of synephrine raw material in solvents with different polarities, the present invention adopts the anti-solvent recrystallization method. Without the need for other carriers and excipients, synephrine forms a dry powder inhalant with uniform particle size, good morphology, and stable chemical properties during the recrystallization process. Compared with the traditional preparation method, the method of the present invention has a shorter reaction time, is more convenient to operate, environmentally friendly, and does not require the use of materials toxic to humans or the environment, providing a new way for the industrial scale-up production of dry powder inhalants by the anti-solvent recrystallization method.

[0016] In step (1), the good solvent is one or more of water, absolute ethanol, acetonitrile, isopropanol, or n-butanol, and more preferably absolute ethanol.

[0017] Preferably, in step (1), the concentration of the good solvent phase A is 10 - 20 mg / mL.

[0018] Preferably, in step (2), the stirring speed is 600 - 1200 rpm, and the stirring time is 2 - 10 h.

[0019] Preferably, in step (2), the volume ratio of the good solvent phase A to the anti-solvent phase B is 1:8 - 12.

[0020] Preferably, in step (3), the drying method is constant temperature drying at 30 - 40 °C.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] The present invention provides a new application of synephrine dry powder inhalant in the preparation of drugs for treating acute lung injury. By formulating synephrine into a dry powder inhalant (DPI) dosage form, the problem of low bioavailability of synephrine is overcome. After pulmonary administration, it directly and actively targets the lungs, increases the blood drug concentration in the lungs, reduces the distribution of the drug in other tissues, thereby improving the drug efficacy and reducing systemic side effects. At the same time, the dry powder inhalation method avoids the first-pass effect and is beneficial to the stability of the drug, and the non-invasive administration route increases the compliance of patients in treatment. The present invention provides a new idea for the research of pulmonary drug delivery systems and has broad clinical application prospects. Description of the Drawings

[0023] Figure 1 Shows the effect of stirring speed on the particle size of synephrine dry powder inhalant;

[0024] Figure 2 Shows the effect of stirring time on the particle size of synephrine dry powder inhalant;

[0025] Figure 3 Is the scanning electron microscope image of synephrine dry powder inhalant;

[0026] Figure 4 Is the particle size distribution diagram of synephrine dry powder inhalant;

[0027] Figure 5 are the PXRD patterns of synephrine raw material and dry powder inhaler;

[0028] Figure 6 are the DSC curves of synephrine raw material and dry powder inhaler;

[0029] Figure 7 are the FTIR spectra of synephrine raw material and dry powder inhaler;

[0030] Figure 8 are the moisture adsorption curves of synephrine raw material and dry powder inhaler;

[0031] Figure 9 are the in vitro deposition distribution maps of synephrine raw material and dry powder inhaler;

[0032] Figure 10 is the effect of SYN-DPI on the survival rate of RAW264.7 cells;

[0033] Figure 11 are the effects of SYN and SYN-DPI on the expression of pro-inflammatory cytokines in the LPS-induced RAW264.7 inflammation model;

[0034] Figure 12 are the effects of SYN and SYN-DPI on the wet / dry weight ratio of lung tissue in the LPS-induced rat ALI model;

[0035] Figure 13 are the pathological HE and Masson section diagrams of lung tissue in the LPS-induced rat ALI model by SYN and SYN-DPI;

[0036] Figure 14 is the effect of SYN-DPI on the total protein content in BALF of ALI rats;

[0037] Figure 15 are the effects of SYN and SYN-DPI on serum inflammatory factors in the LPS-induced rat ALI model;

[0038] Figure 16 are the effects of SYN and SYN-DPI on the oxidative stress level in the LPS-induced rat ALI model;

[0039] Figure 17 are the effects of immunohistochemical detection on the protein expressions of NF-κB, Nrf2 and p38 MAPK in lung tissue of ALI rats in different groups;

[0040] Figure 18 is the average plasma drug concentration-time curve of synephrine after different administration methods in rats. Specific implementation manners

[0041] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. The described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0042] Example 1: Preparation of synephrine dry powder inhaler

[0043] Dissolve 1.00 g of excess synephrine raw material in 50 mL of absolute ethanol. After ultrasonic treatment (300 W, 40 kHz) for 15 min, centrifuge and take the supernatant to obtain a saturated synephrine alcohol solution. Under stirring conditions, add the saturated synephrine alcohol solution dropwise to the n-hexane solution at a volume ratio of 3:30 (mL:mL). Start timing the stirring time from when the saturated synephrine alcohol solution is completely dropped into the n-hexane. After stirring, centrifuge, discard the supernatant solution, and place the precipitate in a blast drying oven at 40 °C for 24 h to obtain the synephrine dry powder inhaler.

[0044] I. Screening of the preparation process of synephrine dry powder inhaler (SYN-DPI)

[0045] 1 Reagents (see Table 1):

[0046] Table 1

[0047]

[0048] 2 Methods

[0049] 2.1 Determination of synephrine solubility by high performance liquid chromatography

[0050] 2.1.1 Liquid chromatography conditions

[0051] Chromatographic column: Agilent C18 (250 mm × 4.6 mm, 5 μm) chromatographic column, mobile phase: 0.1% formic acid (A) - acetonitrile (B), detection wavelength: 273 nm, flow rate: 1 mL / min, column temperature: 30 °C, injection volume: 10 μL, isocratic elution: 1 - 10 min, 97% A.

[0052] 2.1.2 Determination of synephrine raw material solubility in different solvents

[0053] Weigh an excess of synephrine raw material and place it in an EP tube. Add 1.00 mL of water, absolute ethanol, isopropanol, ethyl acetate, n-butanol, n-octanol, acetonitrile, and n-hexane solution respectively. After ultrasonic treatment (300 W, 40 kHz) until the drug no longer dissolves, centrifuge at 6000 r·min -1Centrifuge for 15 min, filter through a 0.22-μm microporous membrane. Dilute the continuous filtrate with methanol and inject for analysis under HPLC conditions to calculate the solubility of synephrine in different solvents.

[0054] 2.2 Investigation of stirring speed

[0055] First, dissolve 1.00 g of excess synephrine raw material in 50 mL of absolute ethanol. After ultrasonic treatment (300 W, 40 kHz) for 15 min, centrifuge and take the supernatant to obtain a saturated synephrine alcohol solution. Add the saturated synephrine alcohol solution drop by drop to the n-hexane solution according to a volume ratio of 3:30 (mL:mL), and stir under the technological conditions of rotation speeds (600, 900, 1200 rpm). After stirring, centrifuge, discard the supernatant, and place the precipitate in a blast drying oven at 40 °C for drying for 24 h to obtain the synephrine dry powder inhaler.

[0056] 2.3 Investigation of stirring time

[0057] First, dissolve 1.00 g of excess synephrine raw material in 50 mL of absolute ethanol. After ultrasonic treatment (300 W, 40 kHz) for 15 min, centrifuge and take the supernatant to obtain a saturated synephrine alcohol solution. Add the saturated synephrine alcohol solution drop by drop to the n-hexane solution according to a volume ratio of 3:30 (mL:mL), and set the stirring times to (0.5, 1, 1.5, 2, 6 h) respectively. After stirring, centrifuge, discard the supernatant, and place the precipitate in a blast drying oven at 40 °C for drying for 24 h to obtain the synephrine dry powder inhaler.

[0058] 2.4 Investigation of particle size

[0059] [[ID=ID=18]]Take an appropriate amount of synephrine raw material and synephrine dry powder inhaler, and use a laser particle size analyzer to measure their particle size distributions by the dry dispersion method.

[0060] 3 Results and analysis

[0061] 3.1 Solubility of synephrine raw material in different solvents

[0062] The solubility of synephrine raw material in different solvents is shown in Table 2. The results show that the solubility of synephrine in absolute ethanol and water is relatively large, the solubility in n-octanol and ethyl acetate is relatively small, and it is almost insoluble in n-hexane. According to the principle of antisolvent precipitation, that is, it has high solubility in a good solvent and poor solubility in an antisolvent, and considering the ease of removing residual solvents after purification, absolute ethanol is finally selected as the good solvent and n-hexane as the antisolvent to prepare the synephrine dry powder inhaler.

[0063] Table 2

[0064]

[0065] 3.2 Effect of stirring speed on particle size distribution of synephrine dry powder inhaler

[0066] Effect of stirring speed on particle size of synephrine dry powder inhaler Figure 1 As shown in the figure, compared with the API, increasing the rotation speed can reduce the particle size of synephrine powder. Under the rotation speed of 600-1200 rpm, the cumulative distribution of synephrine in the particle size range of 1-5 μm increased from 12.29% to 32.74%.

[0067] 3.3 Effect of stirring time on particle size distribution of synephrine dry powder inhaler

[0068] Effect of stirring time on particle size of synephrine dry powder inhaler Figure 2 As shown in the figure, prolonging the stirring time can effectively reduce the particle size of synephrine dry powder inhaler. When the stirring time is 6 h, the cumulative distribution of the particle size range of 1-5 μm is the largest at 70.40%.

[0069] 3.4 Particle size distribution

[0070] The cumulative and frequency distribution of synephrine at 600 rpm for 6 hours is as follows: Figure 4 As shown, it can be seen that after anti-solvent precipitation, the distribution of synephrine dry powder inhaler in the particle size range of 1-5 μm is significantly increased, and the particle size at D50 is less than 5 μm, which belongs to the appropriate inhalation particle size range, which is conducive to the effective deposition of the dry powder inhaler in the lungs.

[0071] 2. Characterization and Quality Evaluation of SYN-DPI

[0072] Taking SYN-DPI preparation as the investigation target, SEM, PXRD, DSC and FTIR were used to characterize SYN-DPI and evaluate the powder properties of dry powder inhaler to explore whether it meets the requirements of dry powder inhaler and investigate the in vitro deposition of dry powder inhaler.

[0073] (1) Scanning electron microscope (SEM)

[0074] The synephrine dry powder inhaler was evenly pasted on a conductive tape. After gold spraying, the sample was placed under a SEM and observed under the conditions of a vacuum degree of 5×10-4 Pa, an electron beam operating voltage of 10 kV, a working distance of approximately 10 mm, and a magnification of 1000-10000.

[0075] The morphology of SYN-DPI was observed using scanning electron microscopy. Figure 3 As shown, synephrine dry powder inhaler is in the form of tablets or blocks and is uniform in size.

[0076] (2) Investigation by powder X-ray diffraction (PXRD)

[0077] Weigh appropriate amounts of synephrine raw material and dry powder inhaler respectively, grind them into uniform powders, make flat sample slides, and detect them under the conditions of copper target, high voltage intensity of 40 kV, tube current of 100 mA, scanning range of 10° - 80° (2θ), and scanning speed of 0.2° / min, and draw the PXRD pattern.

[0078] The PXRD results of synephrine raw material and dry powder inhaler are as Figure 5 shown. It can be judged from Figure 5 that both the synephrine raw material and the dry powder inhaler are in crystal structure. The synephrine raw material has strong diffraction peaks at 12.22°, 15.13°, 17.35°, 18.13°, 20.27°, 23.59°, 26.53°, 27.23°, 28.42°, 31.47°. The dry powder inhaler of synephrine has strong diffraction peaks at 12.12°, 15.12°, 17.35°, 18.09°, 20.25°, 23.61°, 26.47°, 27.21°, 28.40°, 31.51°. Compared with the synephrine raw material, the positions of the characteristic diffraction peaks of the dry powder inhaler have not changed, indicating that the crystal structure of synephrine has not changed after anti-solvent precipitation.

[0079] (3) Investigation by differential scanning calorimetry (DSC)

[0080] Precisely weigh 5.41 mg of synephrine raw material and 2.23 mg of dry powder inhaler and place them in an aluminum sample pan. Use a stamping die to tightly seal the aluminum crucible and put it into the sample cell, and at the same time put a reference crucible. Set the purge gas as high-purity nitrogen with a flow rate of 40 mL / min to maintain a dry N2 environment. After the system is stable, set the initial temperature to 20°C, and perform a programmed temperature increase detection at a heating rate of 20.0°C / min within the range of 20 - 350°C, and draw the DSC curve of the sample.

[0081] The DSC results of synephrine raw material and dry powder inhaler are as Figure 6 shown. The synephrine raw material shows an exothermic peak at 175°C and a broad exothermic peak at 179°C and 233 - 242°C. While the dry powder inhaler of synephrine has an exothermic peak at 175.44°C and a small exothermic peak at 186°C, indicating that the crystal form of the synephrine dry powder inhaler obtained after anti-solvent precipitation has not changed, and the broad melting endothermic peak at 233 - 242°C has disappeared. It is speculated that the anti-solvent precipitation method can not only prepare dry powder inhalers but also has a purification effect.

[0082] (4) Investigation by Fourier transform infrared spectroscopy (FTIR)

[0083] Weigh about 2.00 mg of synephrine raw material drug and dry powder inhalant respectively, and 200 mg of KBr powder. Grind them in a mortar, put the powder into the die of a tablet press, combine the die and press the tablet. Take out the pressed transparent tablet and put it into the sample transmission measurement rack, and scan it within the wavelength range of 3500 - 500 cm-1, with a resolution of 0.09 cm-1 and 16 scanning times. Scan with potassium bromide as the background.

[0084] The FTIR of synephrine raw material drug and dry powder inhalant is as Figure 7 shown. It can be seen from Figure 7 that there are characteristic absorptions at 3043 cm -1 (υC-H), 1604 cm -1 (ζC-H), 1558 cm -1 (ζC=C), 1340 cm -1 (ζO-H, N-H), 1249 cm -1 (υC=O) for both synephrine raw material drug and dry powder inhalant, indicating that both the synephrine raw material drug and the DPI micropowder are crystals and the crystal form has not changed after anti-solvent precipitation.

[0085] (5) Hygroscopicity investigation

[0086] The hygroscopicity of synephrine raw material drug and dry powder inhalant was analyzed by a dynamic vapor sorption (DVS) instrument. The experimental temperature was set at room temperature of 25 °C, the carrier gas was nitrogen, and the total gas flow rate was 200 mL / min. Weigh the sample and put it on the balance tray of the dynamic vapor sorption instrument, set the relative humidity (RH) change program according to the requirements, and set the humidity equilibrium time at the same time.

[0087] The moisture adsorption curves of synephrine raw material drug and dry powder inhalant are as Figure 8As shown. The results showed that under the condition of 90% RH, the maximum moisture adsorption of synephrine raw material medicine was 0.12%. According to the "Guidelines for Hygroscopicity Test of Drugs" in Part IV of the Chinese Pharmacopoeia (2020 Edition), it belongs to the sample with no or almost no hygroscopicity (the weight gain due to hygroscopicity is less than 0.2%). For the synephrine dry powder inhaler under the condition of 90% RH, the percentage of weight gain due to moisture absorption was 0.30%, belonging to the sample with slightly hygroscopicity (the weight gain due to hygroscopicity is less than 2% but not less than 0.2%). This may be because the obtained micronized particles have a smaller particle size, a larger contact surface area with the outside world, and a faster moisture diffusion rate, so the hygroscopicity is stronger. At the same time, under the conventional storage conditions (60% RH) of synephrine raw material medicine and dry powder inhaler, the percentage of weight gain due to moisture absorption is less than 0.2%, which can be considered as basically non-hygroscopic, meeting the requirements for the production, transportation and storage of dry powder inhalers, suitable for the pulmonary drug delivery system, and will not cause problems such as particle aggregation and deposition when encountering the moisture in the respiratory system during the delivery process.

[0088] (6) Determination of the deposition rate of effective parts in vitro

[0089] The next generation pharmaceutical impactor (NGI) was used to investigate the in vitro pulmonary deposition of the synephrine dry powder inhaler sample. Connect the various components of the instrument in sequence, including the device of NGI, adapter, artificial larynx, pre-separator and collection plate. After installation, check the airtightness and uniformity of the device, and the gas flow rate is 75 L / min. Under this gas flow rate, the cut-off Da values of each stage collection plate are: S1 = 7.145 μm, S2 = 3.971 μm, S3 = 2.522 μm, S4 = 1.495 μm, S5 = 0.835 μm, S6 = 0.481 μm, S7 = 0.293 μm, MOC = 0.104 μm.

[0090] The specific method is as follows: 10 mg of synephrine micronized powder and raw material medicine were respectively loaded into No. 3 hydroxypropyl methylcellulose capsules, and loaded into a single-capsule inhalation device. After connecting the NGI, the measurement was carried out, and 5 capsules were inhaled each time. The inhalation flow rate was 75 L / min, and the inhalation duration was 3.2 s. The drug powder deposited at each stage of NGI was collected with ultrapure water, and the content of synephrine was determined under ultraviolet conditions, and the fine particle fraction (FPF) and respirable fraction (RF) were calculated, etc.

[0091] The deposition of synephrine raw material medicine and dry powder inhaler at each level of NGI is as Figure 9As shown. The results showed that after anti-solvent precipitation, the evacuation rates of synephrine API and dry powder inhaler were 90.98% and 95.51% respectively, the fine particle fractions were 2.69% and 31.39% respectively, the respirable fractions were 41.26% and 65.41% respectively, and the in vitro pulmonary effective deposition rates were 2.25% and 19.27% respectively, meeting the requirements of dry powder inhalation.

[0092] Example 2: In vitro and in vivo pharmacodynamic study of synephrine dry powder inhaler (SYN-DPI) on acute lung injury

[0093] First, the cytotoxicity of SYN-DPI was explored in vitro. A cell inflammation model was constructed with lipopolysaccharide, and the in vitro pharmacodynamics of SYN-DPI was preliminarily evaluated by enzyme-linked immunosorbent assay; then, a rat acute lung injury model was constructed by intratracheal instillation of lipopolysaccharide. The therapeutic effect of SYN-DPI on acute lung injury was evaluated by detecting rat pulmonary edema, observing lung tissue pathological sections, and measuring the expression of oxidative factors and inflammatory factors in lung tissue homogenates and sera. The related protein pathways of SYN-DPI in the treatment of ALI were analyzed by immunohistochemistry of lung tissue.

[0094] 1 Experimental cells and animals

[0095] In this experiment, mouse mononuclear macrophages (abbreviated as RAW264.7 cells) were selected as cells, and SPF-grade male SD rats (body weight 200 - 220 g) were used as experimental animals. They were adaptively fed for 7 days. 12 hours before the experiment, they could drink water but needed to fast.

[0096] 2 Experimental methods

[0097] 2.1 Cell culture

[0098] 2.1.1 Cryopreservation of RAW264.7 cells

[0099] Cells in the logarithmic growth phase with good status were selected, the old culture medium was removed, cell cryopreservation solution was prepared, the cell concentration was adjusted to 5×106 cells / mL, subpackaged into cryopreservation tubes, placed in a programmable cooling box, and taken out after 24 hours and cryopreserved in a liquid nitrogen tank.

[0100] 2.1.2 Resuscitation of RAW264.7 cells

[0101] The constant temperature water bath was heated to 37°C, the cryopreserved cells were quickly placed in the water bath and shaken constantly to melt them as soon as possible. After melting, they were aspirated into a centrifuge tube, and more than 10 times the volume of fresh culture medium was added. Centrifuged at 1250 rpm for 5 minutes, the supernatant was aspirated and discarded, and resuspended with DMEM culture medium containing 10% FBS and inoculated into a culture dish.

[0102] 2.1.3 Culture of RAW264.7 cells

[0103] The cells were cultured in DMEM medium containing 10% FBS in a 5% CO2 incubator at 37°C.

[0104] 2.2 In vitro anti-inflammatory experimental method of SYN-DPI

[0105] 2.2.1 Cytotoxicity experiment

[0106] The CCK-8 method was used to study the inhibitory effect of different concentrations of SYN-DPI on the survival of RAW264.7 cells. The RAW264.7 cell suspension in the logarithmic growth phase was inoculated into a 96-well plate, 100 μL per well, and cultured in a cell incubator at 5% CO2 and 37°C for 24 h. The blank group was added with fresh medium, and the experimental groups were added with the test drugs at different concentrations. Six replicates were set for each group, 100 μL per well, and then continued to be placed in the cell incubator for incubation for 24 h. The remaining liquid was aspirated, 10 μL of CCK-8 reagent was added to each well, the culture plate was placed in the incubator for continued incubation for 1 - 2 h, and the absorbance was measured at a wavelength of 450 nm with an enzyme-labeled instrument.

[0107]

[0108] 2.2.2 Establishment of cell inflammation model

[0109] RAW264.7 cells in the logarithmic growth phase were seeded into a 96-well plate at 100 μL per well and continued to be cultured at 37°C and 5% CO2 for 24 h. The supernatant was aspirated and discarded, 100 μL of medium containing an appropriate concentration of synephrine was added to each well for pretreatment for 1 h, and then the medium containing LPS was added. The cells were co-incubated with the test substance for 24 h.

[0110] 2.2.3 Determination of cell inflammatory factors

[0111] According to the results of the CCK-8 experiment, RAW264.7 cells in the logarithmic growth phase were inoculated into a 96-well plate. The cells were divided into a Control group, a LPS group, a LPS + SYN group, and a LPS + SYN-DPI group. A cell inflammation model was established. After culturing for 24 h, the supernatant of each well was collected and centrifuged at 3500 rpm for 10 min. The supernatant was collected and the operation was carried out strictly according to the instructions of the ELISA kit. The absorbance value was measured at 450 nm with an enzyme-labeled instrument and the contents of IL-1β, IL-6, TNF-α, and NO were calculated.

[0112] 2.3 In vivo experimental method for SYN-DPI to improve the LPS-induced rat ALI model

[0113] 2.3.1 Construction of the LPS-induced rat ALI model

[0114] After one week of adaptive feeding, male Sprague-Dawley (SD) rats were used to establish an acute lung injury model by intratracheal instillation of LPS. The specific modeling steps were as follows: First, the rats were anesthetized with isoflurane, and then fixed in the supine position for intratracheal administration to the lungs. LPS (8 mg / kg) was atomized into the alveoli, and then the microsprayer was removed. The animals were placed in a vertical position and gently rotated for 30 seconds to evenly distribute LPS throughout the lungs.

[0115] 2.3.2 Grouping and administration of rats with acute lung injury

[0116] Referring to relevant literature on synephrine, the maximum human administration dose of synephrine is 100 mg. According to the clinical equivalent dose, the administration dose for the intragastric administration group of rats was 9 mg / kg, and the low-dose inhalation administration group was 4.5 mg / kg. The administration conditions for each group are shown in Table 3.

[0117] Table 3

[0118]

[0119]

[0120] 2.3.3 Collection of peripheral blood

[0121] Six hours after administration, the rats were anesthetized by intraperitoneal injection of sodium pentobarbital, and blood was taken from the abdominal aorta into a sterile blood collection tube. After gently shaking, it was left to stand, centrifuged at 3500 rpm for 15 minutes, and the serum was separated and stored in a -80°C refrigerator for detection of serum inflammatory factor levels.

[0122] 2.3.4 Collection of bronchoalveolar lavage fluid

[0123] For the rats that had completed the blood collection operation, after the rats' hearts stopped beating, the rats were fixed in the supine position on the operating table. The skin of the chest and abdomen, the sternum and surrounding soft tissues of the rats were successively cut open with tissue scissors to expose the lung tissues including the main bronchus under the glottis. The right lung was ligated. After cutting a "reverse V-shaped" small opening on the trachea with surgical scissors, a syringe was carefully inserted through the incision for lavage. The left lung was perfused with normal saline through the trachea 3 times, with 1.5 mL slowly injected and withdrawn each time, and the alveolar lavage fluid was collected.

[0124] 2.3.5 Collection of lung tissues

[0125] After the perfusion of the left lung of the rats was completed, the left and right lungs were taken, washed and dried with normal saline. The lower lobe of the left lung was used for determination of the wet / dry ratio of the rat lung tissues, the upper lobe of the right lung was temporarily stored in liquid nitrogen and transferred to a -80°C refrigerator for detection of oxidation factors, and the remaining right lung was stored in 4% paraformaldehyde for histopathological examination and immunohistochemical analysis.

[0126] 2.3.6 Determination of wet / dry ratio of lung tissues

[0127] Take the lower left lobe of the rat's lung, remove the surface blood and stains, dry the surface moisture and weigh it, which is recorded as the wet weight. Then place it in an oven at 60 °C for 24 h of drying, and weigh it again, which is recorded as the dry weight. Evaluate the degree of pulmonary edema by calculating the lung wet / dry weight ratio.

[0128] Lung wet-to-dry weight ratio = wet weight of the left lung / dry weight of the left lung.

[0129] 2.3.7 Hematoxylin-eosin (HE) staining

[0130] The lung tissues fixed in 4% paraformaldehyde were dehydrated, routinely embedded in paraffin, and made into 5-μm sections. Then, after dewaxing the sections, they were routinely stained with HE, dehydrated, and sealed. Observe the lung tissue structure and morphological characteristics of rats in each group under a microscope.

[0131] 2.3.8 Masson staining

[0132] First, dewax the sections routinely to water, draw a hydrophobic circle, then add Bouin's solution dropwise, cover it, and incubate it in an incubator at 37 °C. Then rinse it with running water until the yellow color on the section disappears. Then stain it with Celestine blue staining solution for 3 - 5 min, and then rinse it with running water. After that, stain it with Mayer's hematoxylin staining solution for 3 min, and then rinse it with running water. After that, differentiate it with acidic ethanol differentiating solution for a few seconds, and then rinse it thoroughly with running water. Then stain it with Ponceau fuchsin staining solution for 3 min, and rinse it slightly with distilled water. Finally, treat it with phosphotungstic acid solution for 5 - 10 min. After pouring off the above solution, stain it with aniline blue staining solution step by step for 5 min, and then treat it slightly with a weak acid solution. After completion, perform routine dehydration and transparency treatment with xylene. Finally, seal the sections with neutral gum, and thus complete the Masson trichrome staining.

[0133] 2.3.9 Determination of protein content in bronchoalveolar lavage fluid

[0134] In this study, the BCA method was used to determine the total protein content in BALF. Accurately weigh the tissue weight, add normal saline according to the weight-to-volume ratio. After centrifugation of the lung tissue homogenate, take the supernatant, and add the test solution successively according to the protein quantification kit instructions. Measure the absorbance value at 562 nm with an enzyme-labeled instrument and calculate the protein concentration in BALF.

[0135] 2.3.10 Detection of serum inflammatory factor levels

[0136] Rat serum was taken. According to the instructions of the ELISA kits for rat TNF-α, IL-6, IL-8, IL-1β, and IL-1α, the test solutions were added successively. The OD value was measured at 450 nm using an enzyme-labeled instrument. A standard curve was plotted with the concentrations of TNF-α, IL-8, IL-6, IL-1β, and IL-1α standards as the abscissa and the absorbance values as the ordinate. The concentrations of TNF-α, IL-6, IL-8, IL-1β, and IL-1α in the serum were calculated based on the standard curve.

[0137] 2.3.11 Detection of Oxidative Stress Indexes in Lung Tissue

[0138] A certain weight of rat lung tissue was accurately weighed. Samples for detecting the activities of SOD, CAT, GSH, and MDA were prepared according to different ratios of the weight of lung tissue to normal saline. After homogenization and centrifugation under ice-water bath conditions, the supernatant was taken for detection, and the operation was carried out strictly according to the instructions of the kit.

[0139] 2.3.12 Immunohistochemical Analysis of Lung Tissue

[0140] The lung tissue sections were dewaxed to transparency, rinsed with ultrapure water for standby, and then placed in a 0.01 M sodium citrate buffer solution for 15 min for antigen extraction. Subsequently, the sections were incubated in 3% hydrogen peroxide for 10 min to block the activity of endogenous peroxidase. After blocking, the sections were incubated overnight at 4°C with the primary antibodies Nrf2 / NF-κB / p38 MAPK. The sections incubated with the primary antibodies were drained, and then the secondary antibody was added to cover the lung tissue on the sections and incubated at room temperature for 1 h. The sections were washed with PBS, drained, and the prepared chromogenic agent was added. The sections were rinsed with ultrapure water to terminate the chromogenesis. The nuclei were counterstained with hematoxylin for 5 min and rinsed with ultrapure water. After differentiating with hematoxylin differentiating solution for 10 s, rinsing was carried out again, followed by blueing with hematoxylin blueing solution, rinsing with ultrapure water, and finally dehydrating to transparency with absolute ethanol and xylene and mounting with neutral gum. Images were observed and collected under a microscope.

[0141] 3 Experimental Results

[0142] 3.1 Cytotoxicity

[0143] The cytotoxicity results of SYN-DPI on RAW2,64.7 cells are as Figure 10As shown. The results showed that SYN-DPI did not cause any significant cytotoxicity within the concentration range of 50 - 400 μM and had little effect on the activity of RAW264.7 cells; a decrease in cell viability was observed at concentrations of 800 μM and 1000 μM (87.3 ± 1.6% and 77.8 ± 3.8% respectively compared with the control group), which had an adverse effect on the activity of RAW264.7 cells and showed significant differences (P < 0.001). Therefore, we selected a concentration range of 50 - 400 μM for the experiment in the following study.

[0144] 3.2 Effects of SYN-DPI on the expression of pro-inflammatory factors in the LPS-induced in vitro inflammation model

[0145] The effects of SYN-DPI on the expression of pro-inflammatory cytokines in the LPS-induced cell inflammation model are as Figure 11 shown. The results showed that, compared with the control group, a large amount of IL-1β, IL-6 and TNF-α were produced after stimulating RAW264.7 cells with LPS, showing significant differences, indicating that the cell inflammation model was successfully constructed. Taking IL-6 as an example, comparing the 100 μM SYN-DPI group with the 400 μM SYN group, there was no significant difference (P > 0.05), indicating that SYN-DPI could achieve a similar effect of inhibiting the expression of inflammatory factors as 400 μM SYN at a dose of 100 μM; the inhibition rate of SYN-DPI (400 μM) on the production of IL-6 was 45.17%, while the inhibition rate of an equal amount of SYN (400 μM) on the production of IL-6 was 23.32%, indicating that SYN-DPI had better anti-inflammatory potential. Nitric oxide (NO) is a reactive free radical involved in the inflammatory process. The inhibition rate of SYN (200 μM) on the production of NO was 16.08%, while the inhibition rate of SYN-DPI (200 μM) was 49.77%, showing a higher inhibitory effect than SYN. In short, both SYN and SYN-DPI could inhibit the production of NO, but the inhibitory effect of SYN-DPI was better. To sum up, within the scope of this study, compared with SYN, SYN-DPI had better anti-inflammatory effects.

[0146] 3.3 Effects of SYN-DPI on the wet / dry ratio of rat lungs

[0147] The results of the wet / dry weight ratio of rat lungs are as Figure 12As shown in the figure. It can be seen from the figure that the wet / dry weight ratio of the lungs in the model group was significantly higher than that in the control group and the drug treatment group, with significant differences, indicating that the establishment of the LPS-induced acute lung injury rat model was successful. Compared with the model group, the wet / dry weight ratio of the lungs in the SYN and SYN-DPI groups of rats was significantly reduced, and the ability of the SYN-DPI group to improve pulmonary edema in rats was dose-dependent. Compared with the SYN group, there was no significant difference in the ability of the low-dose SYN-DPI group to reduce pulmonary edema (P>0.05), indicating that the low-dose SYN-DPI could achieve the same drug effect as SYN, and the ability of SYN-DPI to reduce pulmonary edema was stronger than that of SYN.

[0148] 3.4 Effects of SYN-DPI on pathological changes of lung tissues in the LPS-induced rat ALI model

[0149] The results of observing the pathological changes of lung tissues by HE and Masson staining are as Figure 13 shown. In the Control group, the structure of the bronchial ciliated columnar epithelium in the lung tissue was clear, and the alveolar cavity structure was intact. In the Model group, the alveolar cavity of the rats was significantly reduced, showing extremely severe protein and inflammatory cell infiltration, thickening of the alveolar wall, alveolar edema, and even pathological manifestations such as congestion and degeneration. The pathological damage of the lung tissue in the Dex group was significantly alleviated. There was no obvious difference in the pathological changes between the SYN group and the low-dose SYN-DPI group. Compared with the rats in the Model group, the inflammatory cells in the lung tissue of the rats in the low-dose SYN-DPI group were reduced. In the medium-dose group, the bronchial epithelium of the rats returned to flat, and there were still inflammatory cell infiltrations around the bronchi. In the high-dose group, the alveolar cavity of the rats recovered normal, and the inflammatory cells basically returned to normal, indicating that SYN-DPI could relieve the pathological damage of lung tissues caused by LPS, and showed a dose-dependence.

[0150] 3.5 Effects of SYN-DPI on the total protein content in BALF of ALI rats

[0151] The effects of SYN and SYN-DPI on the total protein content in BALF of ALI rats are as Figure 14 shown. It can be seen from the figure that compared with the blank group, the protein content in BALF of the model group increased significantly, with significant differences, indicating that the tissue space of the alveoli increased, and the acute lung injury model was successfully constructed. The protein content in the drug treatment group was significantly reduced compared with the LPS group, indicating that SYN-DPI and the positive drug Dex had similar therapeutic effects and could significantly improve the tissue space of the alveoli. By comparing the data analysis of the low, medium, and high-dose groups of SYN-DPI, it was found that the protein content in BALF was closely related to the drug dosage and showed a dose-dependence.

[0152] 3.6 Effects of SYN-DPI on serum inflammatory factors in LPS-induced rat ALI

[0153] The content results of IL-1α, IL-1β, IL-6, IL-8, and TNF-α in serum are as Figure 15 shown. Compared with the blank group, the secretion of inflammatory factors in the model group induced by LPS was significantly increased (P<0.0001). After treatment with SYN and SYN-DPI, compared with the model group, the expression levels of inflammatory factors in the SYN group and the SYN-DPI group and the Dex group were all significantly decreased (P<0.0001), indicating that both SYN and SYN-DPI can improve ALI and reduce the expression levels of inflammatory factors in serum, with obvious therapeutic effects. Taking TNF-α as an example, there was no significant difference between the low-dose group of SYN-DPI at 4.5 mg / kg and the SYN group at 9 mg / kg (P>0.05), indicating that the low-dose group of SYN-DPI can achieve a similar effect of inhibiting the expression of inflammatory factors as the SYN group. Analyzing the data of the SYN-DPI group also found that the secretion of inflammatory factors was dose-dependent. Increasing the dosage within the experimental concentration range could dose-dependently reduce the secretion of inflammatory factors and inhibit the inflammatory response induced by LPS in rats.

[0154] 3.7 Effects of SYN-DPI on oxidative factors in the lung tissue of rats with ALI induced by LPS

[0155] The effects of SYN and SYN-DPI on MDA, SOD, and MPO in the lung tissue of rats with ALI induced by LPS are as Figure 16 shown. The results showed that compared with the rats in the blank group, the levels of MDA and MPO were significantly increased and the activity of SOD was decreased after LPS stimulation in the model group, while SYN-DPI treatment could significantly reverse the effects of LPS on redox-related enzymes, reduce peroxidation metabolites, enhance the level of antioxidants, and maintain the oxidative stress balance.

[0156] 3.8 Effects of SYN-DPI on the protein expressions of NF-κB, Nrf2, and p38 MAPK in the lung tissue of rats with ALI

[0157] After immunohistochemical analysis and microscopic photography, the protein expressions of NF-κB, Nrf2, and p38 MAPK are as Figure 17As shown, the Image J software was used for semi - quantitative analysis of the expression of three proteins, NF - κB, Nrf2, and p38 MAPK, with the average optical density value as the measurement index. NF - κB and MAPK are two classic inflammatory signaling pathways, and the activated NF - κB and MAPK pathways can promote the production of pro - inflammatory mediators. Therefore, inhibiting the NF - κB and MAPK pathways helps to inhibit the production of pro - inflammatory cytokines. Therefore, in the LPS - induced ALI model, the effects of SYN - DPI on the NF - κB and MAPK signaling pathways were further detected. The results showed that LPS could significantly increase the expression of p38 MAPK protein, while after treatment with synephrine dry powder inhalation, the phosphorylation of the protein was significantly inhibited, indicating that synephrine can exert an anti - inflammatory effect through the MAPK signaling pathway. As a classic antioxidant pathway, LPS can block the Nrf2 signaling pathway and inhibit the expression of downstream antioxidant genes. As shown in the figure, compared with the blank group and the LPS - treated group, SYN - DPI treatment could effectively promote the nuclear transcription of Nrf2, indicating that SYN - DPI can effectively activate Nrf2.

[0158] Example 3: Pharmacokinetic Study of Synephrine Dry Powder Inhalant

[0159] To explore the changes in the drug concentration of SYN - DPI after absorption into the blood, this chapter intends to establish a simple, sensitive, and rapid LC - MS / MS method for determining the concentration of compound SYN in rat plasma, and strictly verify the methodology of this method according to the quantitative analysis of drugs in biological samples, explore the metabolic process of SYN in rats, and provide a basis for the subsequent research of SYN - DPI.

[0160] 1 Experimental Animals

[0161] SPF - grade male SD rats, weighing 180 - 220 g. All animals were raised under the same experimental conditions, with a temperature of (22 ± 1) °C, a relative humidity of (55 ± 5)%, a light / dark cycle of 12 h, and free access to food and water. They were adaptively raised for one week before the experiment.

[0162] 2 Experimental Methods

[0163] 2.1 Chromatographic Conditions

[0164] Chromatographic column: Hypersil GOLD C8 (100 mm × 2.1 mm; 3 μm); Mobile phase: Phase A: 0.1% formic acid in water, Phase B: acetonitrile; Flow rate: 0.3 mL / min; Column temperature: 40 °C; Injection volume: 2 μL; Gradient elution program: 0 - 1 min, 5% B; 1 - 4 min, 5% - 90% B; 4 - 5 min, 90% - 5% B.

[0165] 2.2 Mass spectrometry conditions

[0166] The mass spectrometry used the ESI positive ion scanning mode, which was a multiple reaction monitoring (MRM) detection mode. The collision gas was high-purity nitrogen; capillary voltage: 3.5 kV; nebulizer pressure: 45 psi; dryer temperature: 300 °C; drying gas flow rate: 5 L / min (N2); sheath gas temperature: 250 °C; sheath gas flow rate: 11 L / min (N2).

[0167] 2.3 Grouping and administration of experimental animals

[0168] The experimental animals were randomly divided into 3 groups: (1) tail vein injection group, (2) pulmonary inhalation group, and (3) gavage group, with 5 animals in each group. They were fasted for 12 h before administration and allowed free access to water.

[0169] 2.4 Collection of plasma samples

[0170] The rats were fasted but allowed free access to water 12 h before the experiment. The rats were weighed (to calculate the dosage) and numbered. After administration to the three groups of rats, 0.2 mL of blood was taken from the orbital vein at the time points of 2 min, 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, and 6 h, placed in an EP tube with heparin sodium, centrifuged at 3500 rpm for 15 min, and the upper-layer plasma was separated and stored in a -80 °C refrigerator.

[0171] 2.5 Detection of plasma samples

[0172] Precisely pipette 190 μL of rat plasma sample into an EP tube, add 10 μL of internal standard solution, vortex for 60 s, then add 400 μL of acetonitrile, vortex for 60 s to precipitate proteins, centrifuge at 10000 rpm for 15 min, take the supernatant, filter it through a 0.22 μm microporous filter membrane, and place it in a glass inner cannula for LC-MS / MS injection and detection analysis.

[0173] 3 Experimental results

[0174] The pharmacokinetic curve of synephrine is as Figure 18As shown. From the results, it can be seen that compared with the intragastric administration group, after administering the same dose of synephrine dry powder inhalant by pulmonary administration, the Cmax of the inhalation group was 845.52 ng / mL, the Cmax of the intragastric administration group was 301.76 ng / mL, and the inhalation group was 280.20% higher than the intragastric administration group; the half-life t1 / 2 of the inhalation group was 175.51 min, the half-life t1 / 2 of the intragastric administration group was 137.65 min, and the inhalation group was prolonged by 127.50% compared with the intragastric administration group; the AUC(0-t) of the inhalation group was 1168.24 ng / mL*h, the AUC(0-t) of the intragastric administration group was 876.07 ng / mL*h, and the inhalation group was 133.35% higher than the intragastric administration group. Compared with the injection group, after administering the same dose of synephrine micronized powder by pulmonary administration, the Cmax of the injection group was 1032.17 ng / mL, the inhalation group reached 81.92% of the injection group, and the intragastric administration group was 29.24% of the injection group; the half-life t1 / 2 of the injection group was 320 min, the inhalation group reached 54.84% of the injection group, and the intragastric administration group was 43.01% of the injection group; the AUC(0-∞) of the inhalation group was 1335.70 ng / mL*h, the AUC(0-∞) of the injection group was 1538.76 ng / mL*h, the inhalation group reached 86.80% of the injection group, while the intragastric administration group was only 64.48% of the injection group. The absolute bioavailability of the intragastric administration group was 32.24%, and the absolute bioavailability of the inhalation group was 86.80%. Therefore, according to the pharmacokinetic parameters of synephrine in different administration groups, inhalation administration can significantly improve its bioavailability.

Claims

1. Use of synephrine dry powder inhaler in the preparation of a drug for treating acute lung injury; The preparation method of the synephrine dry powder inhaler comprises the following steps: (1) Synephrine API was dissolved in a good solvent as the good solvent phase A, and n-hexane was used as the antisolvent phase B; (2) Add the good solvent phase A dropwise to the antisolvent phase B under stirring to obtain a uniform suspension; (3) centrifuging the suspension obtained in step (2), and drying the precipitate to obtain a synephrine dry powder inhaler; The good solvent is anhydrous ethanol; the stirring speed is 600-1200 rpm, and the stirring time is 2-10 h.

2. The use according to claim 1, characterized in that The aerodynamic particle size of the synephrine dry powder inhaler is 1-5 μm.

3. The use according to claim 1, characterized in that The synephrine dry powder inhaler has a fine particle fraction of 28-33%, an emptying rate of 94-98%, an inhalable fraction of 60-70%, and an in vitro lung effective deposition rate of 18-20%.

4. The use according to claim 1, characterized in that In step (1), the concentration of the good solvent phase A is 10-20 mg / mL.

5. The use according to claim 1, characterized in that In step (2), the volume ratio of the good solvent phase A to the anti-solvent phase B is 1:8-12.

6. The use according to claim 1, wherein In step (3), the drying method is constant temperature drying at 30-40 °C.

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