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

Abstract

The invention discloses an application of a synephrine dry powder inhalant in preparation of a medicine for treating acute lung injury. According to the invention, the synephrine is prepared into a dry powder inhaler (DPI) dosage form, so that the problem of low bioavailability of the synephrine is overcome, and the synephrine is directly and actively targeted to the lung after being administered to the lung, so that the plasma concentration of the lung is increased, and the distribution of the medicine in other tissues is reduced, thereby improving the curative effect of the medicine and reducing the side effects of the whole body. Meanwhile, the first-pass effect is avoided by a dry powder inhalation mode, so that the stability of the medicine is facilitated, and the treatment compliance of a patient is improved by a non-invasive administration way. The invention provides a new thought for the research of a pulmonary drug delivery system, and has a wide clinical application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to application of a synephrine dry powder inhaler in preparing a medicine for treating acute lung injury. Background Art

[0002] Acute lung injury (ALI) or its more serious clinical manifestation, acute respiratory distress syndrome (ARDS), is an acute inflammatory lung disease with a high annual morbidity and mortality. It is a common critical illness with a mortality rate of more than 40%, which seriously threatens the patient's life. Acute lung injury is acute respiratory insufficiency caused by damage to alveolar epithelial cells and capillary endothelial cells. ALI is caused by a variety of direct or indirect problems. Direct damage includes pneumonia, aspiration of gastric contents, inhalation of harmful gases or lung contusion. Indirect damage is lung damage caused by other serious diseases such as sepsis, pancreatitis, multiple trauma or severe systemic infection. Sepsis is the most common cause of acute respiratory distress syndrome in humans. At present, clinical treatment methods cannot completely cure acute lung injury, but can only control the progression of the disease and reduce the pain and injury of patients. Clinical treatment methods mainly include mechanical ventilation, inhaled vasodilators and the use of strong anti-inflammatory glucocorticoids to treat acute lung injury.

[0003] Synephrine is a natural component of the fruits of Citrus aurantium, Citrus aurantium fructus, dried orange peel, and green peel of the Rutaceae family. The highest content is found in young fruits of sour orange. It is the main active ingredient in extracts of Citrus aurantium, bitter orange, etc. It belongs to the ephedrine alkaloid class. The similarity between synephrine and endogenous adrenergic receptor agonists adrenaline and norepinephrine lies in the common basic structure - phenylethylamine. Because synephrine is similar in structure to sympathomimetic amines, it can produce different physiological and pharmacological effects by binding to and activating different α- and β-adrenergic receptors. The pharmacological effects mediated by β-adrenergic receptors can inhibit the expression of proinflammatory cytokines in acute lung injury and 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 and secondary amino groups in its structure. It is highly soluble in water or alcohol solvents, has average thermal stability, can react with acids or bases to form salts, has chemically amphoteric properties, is unstable and easily oxidized in strong acids, strong bases or high temperatures. After oral administration, the amount absorbed by the lungs is low and the bioavailability is limited, requiring higher doses and more frequent administration, and patient compliance is low.

[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] In view of the deficiencies of the above-mentioned prior art, the object of the present invention is to provide an application of a synephrine dry powder inhaler in the preparation of a drug for treating acute lung injury. The present invention prepares synephrine into a dry powder inhaler (DPI) dosage form and adopts a pulmonary inhalation administration method, which 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 achieved through the following technical solutions:

[0008] The invention provides application of synephrine dry powder inhalation in preparing medicine for treating acute lung injury.

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

[0010] Preferably, the fine particle fraction (FPF) of the synephrine dry powder inhaler is 28-33%, the emptying rate is 94-98%, the inhalable fraction is 60-70%, and the in vitro lung effective deposition rate is 18-20%. The synephrine dry powder inhaler prepared by the present invention has suitable physical and chemical properties such as 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 inhaler of the present invention comprises the following steps:

[0012] (1) dissolving the synephrine API in a good solvent as the good solvent phase A, and using n-hexane as the anti-solvent phase B;

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

[0014] (3) centrifuging the suspension obtained in step (2), and drying the precipitate to obtain a synephrine dry powder inhaler.

[0015] The present invention adopts an anti-solvent recrystallization method based on the solubility of synephrine raw materials in different polar solvents, and does not require other carriers and excipients, so that synephrine forms a synephrine dry powder inhaler 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 easier to operate, is green and environmentally friendly, does not require the use of materials that are toxic to humans or the environment, and provides a new way for the industrialized production of dry powder inhalers by anti-solvent recrystallization method.

[0016] In step (1), the good solvent is one or more of water, anhydrous ethanol, acetonitrile, isopropanol or n-butanol, more preferably anhydrous 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 inhaler in the preparation of a drug for treating acute lung injury. By preparing synephrine into a dry powder inhaler (DPI) dosage form, the problem of low bioavailability of synephrine is overcome. After pulmonary administration, the drug is directly and actively targeted to the lungs, the blood drug concentration in the lungs is increased, and the distribution of the drug in other tissues is reduced, 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 conducive to the stability of the drug, and the non-invasive administration route increases the compliance of the patient's treatment. The present invention provides a new idea for the research of the pulmonary drug delivery system and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0025] Figure 3 This is a scanning electron micrograph of synephrine dry powder inhaler;

[0026] Figure 4 This is the particle size distribution diagram of synephrine dry powder inhaler;

[0027] Figure 5 PXRD patterns of synephrine API and dry powder inhaler;

[0028] Figure 6 This is the DSC curve of synephrine API and dry powder inhaler;

[0029] Figure 7 FTIR spectra of synephrine API and dry powder inhaler;

[0030] Figure 8 The moisture adsorption curves of synephrine API and dry powder inhaler are shown below;

[0031] Fig. 9 The in vitro deposition profiles of synephrine raw material and dry powder inhaler are shown;

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

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

[0034] Fig.12 The effects of SYN and SYN-DPI on the wet-to-dry weight ratio of lung tissue in the LPS-induced ALI model of rats;

[0035] Fig.13 HE and Masson sections of lung tissue pathology of SYN and SYN-DPI in LPS-induced ALI rat model;

[0036] Fig.14 The effect of SYN-DPI on the total protein content in BALF of ALI rats;

[0037] Fig.15 The effects of SYN and SYN-DPI on serum inflammatory factors in LPS-induced ALI model in rats;

[0038] Fig.16 The effects of SYN and SYN-DPI on the oxidative stress level in LPS-induced ALI model in rats;

[0039] Fig.17 To detect the expression of NF-κB, Nrf2 and p38 MAPK proteins in lung tissues of ALI rats in different groups by immunohistochemistry;

[0040] Fig.18 The mean plasma drug concentration-time curve of synephrine after different administration methods in rats. DETAILED DESCRIPTION

[0041] The technical scheme 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 part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0042] Example 1: Preparation of Synephrine Dry Powder Inhaler

[0043] An excess of 1.00 g of synephrine bulk drug was dissolved in 50 mL of anhydrous ethanol. After ultrasonication (300 W, 40 kHz) for 15 min, the supernatant was centrifuged to obtain a saturated alcohol solution of synephrine. The saturated alcohol solution of synephrine was added dropwise to the n-hexane solution at a volume ratio of 3:30 (mL:mL) under stirring. The stirring time was counted from the time when all the saturated alcohol solution of synephrine was added dropwise to the n-hexane. After the stirring was completed, the mixture was centrifuged, the supernatant solution was discarded, and the precipitate was placed in a 40°C forced air drying oven and dried for 24 h to obtain a synephrine dry powder inhaler.

[0044] 1. Preparation process screening 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 HPLC

[0050] 2.1.1 Liquid chromatography conditions

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

[0052] 2.1.2 Determination of the solubility of synephrine API in different solvents

[0053] Weigh an excess of synephrine raw material into an EP tube, add 1.00 mL of water, anhydrous ethanol, isopropanol, ethyl acetate, n-butanol, n-octanol, acetonitrile, and n-hexane solution, respectively, and sonicate (300 W, 40 kHz) until the drug no longer dissolves. -1Centrifuge for 15 minutes, filter through a 0.22 μm microporous membrane, dilute the filtrate with methanol and analyze it according to liquid chromatography conditions to calculate the solubility of synephrine in different solvents.

[0054] 2.2 Investigation of stirring speed

[0055] First, an excess of 1.00 g of synephrine raw material was dissolved in 50 mL of anhydrous ethanol. After ultrasonication (300 W, 40 kHz) for 15 min, the supernatant was centrifuged to obtain a saturated alcohol solution of synephrine. The saturated alcohol solution of synephrine was added dropwise to the n-hexane solution at a volume ratio of 3:30 (mL:mL), and stirred at the process conditions of rotation speeds (600, 900, and 1200 rpm). After stirring, the solution was centrifuged, the supernatant was discarded, and the precipitate was dried in a 40°C forced air drying oven for 24 h to obtain a synephrine dry powder inhaler.

[0056] 2.3 Investigation of mixing time

[0057] First, an excess of 1.00 g of synephrine raw material was dissolved in 50 mL of anhydrous ethanol. After ultrasonication (300 W, 40 kHz) for 15 min, the supernatant was centrifuged to obtain a saturated alcohol solution of synephrine. The saturated alcohol solution of synephrine was added dropwise to the n-hexane solution at a volume ratio of 3:30 (mL:mL). The stirring time was set to (0.5, 1, 1.5, 2, 6 h) respectively. After stirring, the solution was centrifuged and the supernatant was discarded. The precipitate was placed in a 40°C forced air drying oven and dried for 24 h to obtain a synephrine dry powder inhaler.

[0058] 2.4 Investigation of granularity

[0059] Take appropriate amounts of synephrine bulk drug and synephrine dry powder inhalation and measure their particle size distribution using a laser particle size analyzer through a dry dispersion method.

[0060] 3 Results and analysis

[0061] 3.1 Solubility of Synephrine API in Different Solvents

[0062] The solubility of synephrine API in different solvents is shown in Table 2. The results show that synephrine has high solubility in anhydrous ethanol and water, low solubility in n-octanol and ethyl acetate, and is almost insoluble in n-hexane. Based on the principle of anti-solvent precipitation, the solubility in good solvent is high and the solubility in anti-solvent is low, and combined with the difficulty of removing residual solvents after purification, anhydrous ethanol was finally selected as the good solvent and n-hexane as the anti-solvent to prepare 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, compared with the raw material, increasing the rotation speed can reduce the particle size of synephrine powder. Under the condition of 600-1200 rpm rotation speed, 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, extending the stirring time can effectively reduce the particle size of synephrine dry powder inhaler. When the stirring time is 6h, the maximum cumulative distribution of the particle size range of 1-5μm is 70.40%.

[0069] 3.4 Particle size distribution

[0070] The accumulation and frequency distribution of synephrine at 600 rpm and 6 h are 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 1-5μm is significantly increased, and the particle size at D50 is less than 5μm, which belongs to the appropriate inhalation particle size range, which is conducive to the effective deposition of 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 the conductive tape, and the sample was placed under the SEM after gold spraying. The sample was observed under the conditions of vacuum degree 5×10-4Pa, electron beam working voltage 10kV, working distance of about 10mm, and magnification 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 with uniform size.

[0076] (2) Powder X-ray diffraction (PXRD) investigation

[0077] Appropriate amounts of synephrine bulk drug and dry powder inhaler were weighed respectively, ground into uniform powder, and flat sample test pieces were prepared. The samples were tested 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 PXRD patterns were drawn.

[0078] Synephrine API and dry powder inhaler RXRD results are as follows Figure 5 As shown by Figure 5 It can be judged that both the synephrine API and the dry powder inhaler are crystalline structures. The synephrine API has strong diffraction peaks at 12.22°, 15.13°, 17.35°, 18.13°, 20.27°, 23.59°, 26.53°, 27.23°, 28.42°, and 31.47°, and the synephrine dry powder inhaler has strong diffraction peaks at 12.12°, 15.12°, 17.35°, 18.09°, 20.25°, 23.61°, 26.47°, 27.21°, 28.40°, and 31.51°. Compared with the synephrine API, the position of the characteristic diffraction peaks of the dry powder inhaler has not changed, indicating that the crystal structure of synephrine has not changed after anti-solvent precipitation.

[0079] (3) Differential scanning calorimetry (DSC) study

[0080] Accurately weigh 5.41 mg of synephrine API and 2.23 mg of dry powder inhaler and place them in an aluminum sample pan. Press and seal the aluminum crucible with a stamping mold and place it in the sample pool. Place a reference crucible at the same time. Set the purge atmosphere to high-purity nitrogen at a flow rate of 40 mL / min to maintain a dry N2 environment. After the system is stable, set the starting temperature to 20°C, and program the temperature rise test at a heating rate of 20.0°C / min within the range of 20-350°C to draw the sample DSC curve.

[0081] Synephrine API and dry powder inhaler DSC results are as follows Figure 6 As shown, the synephrine raw material has an exothermic peak at 175°C, and there is a broad exothermic peak at 179°C and 233-242°C, while the synephrine dry powder inhaler has an exothermic peak at 175.44°C and a small exothermic peak at 186°C, indicating that the crystalline 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 disappears. It is inferred that the anti-solvent precipitation method can not only prepare dry powder inhalers, but also has a purification effect.

[0082] (4) Fourier transform infrared spectroscopy (FTIR) investigation

[0083] About 2.00 mg of synephrine bulk drug and dry powder inhaler and 200 mg of KBr powder were weighed respectively, ground in a mortar, and the powder was placed in a tablet press mold. The mold was assembled and tablets were pressed. The pressed transparent tablets were taken out and loaded into a sample transmission measurement frame. Scanning was performed in the wavelength range of 3500-500 cm-1, with a resolution of 0.09 cm-1 and 16 scans, and the scanning was performed using potassium bromide as the background.

[0084] FTIR of Synephrine API and Dry Powder Inhaler Figure 7 As shown by Figure 7 It can be seen that synephrine API and dry powder inhaler are at 3043cm -1 (υC-H), 1604cm -1 (ζC-H), 1558cm -1 (ζC=C), 1340cm -1 (ζO-H, NH), 1249cm -1 There are characteristic absorptions at (υC=O), indicating that both synephrine API and DPI micropowder are crystalline, and the crystal form does not change after anti-solvent precipitation.

[0085] (5) Hygroscopicity study

[0086] The hygroscopicity of synephrine API and dry powder inhaler was analyzed by dynamic vaporsorption (DVS). The experimental temperature was set to room temperature 25°C, the carrier gas was nitrogen, the total gas flow rate was 200 mL / min, the sample was weighed and placed on the balance tray of the dynamic vapor absorption instrument, and the relative humidity (RH) change program was set according to the requirements, and the humidity balance time was set at the same time.

[0087] The moisture adsorption curves of synephrine API and dry powder inhaler are as follows: Figure 8As shown. The results show that the maximum water adsorption of the sample of synephrine API under 90% RH is 0.12%. According to the "9103 Drug Hygroscopicity Test Guidelines" in Part IV of the 2020 edition of the "Chinese Pharmacopoeia", it belongs to a sample with no or almost no hygroscopicity (moisture weight gain is less than 0.2%). The hygroscopic weight gain percentage of synephrine dry powder inhaler under 90% RH is 0.30%, which is a slightly hygroscopic sample (moisture weight gain is less than 2% but not less than 0.2%). This may be due to the small particle size of the obtained micropowder, the large surface area in contact with the outside world, and the rapid diffusion of water, so the hygroscopicity is strong. At the same time, under the conventional storage conditions (60% RH) of synephrine API and dry powder inhaler, the hygroscopic weight gain percentage is less than 0.2%, which can be considered to be basically non-hygroscopic, meeting the requirements for the production, transportation and storage of dry powder inhalers, and is suitable for pulmonary drug delivery systems. When encountering moisture in the respiratory system during the delivery process, it will not cause problems such as particle agglomeration and deposition.

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

[0089] The next generation pharmaceutical impactor (NGI) was used to investigate the in vitro lung deposition of synephrine dry powder inhalation samples. The various components of the instrument were connected in sequence, including the NGI device, adapter, artificial throat, pre-separator and collection plate. After installation, the air tightness and uniformity of the device were checked, and the gas flow rate was 75L / min. At this gas flow rate, the cut-off Da of each collection plate were: S1 = 7.145μm, S2 = 3.971μm, S3 = 2.522μm, S4 = 1.495μm, S5 = 0.835μm, S6 = 0.481μm, S7 = 0.293μm, MOC = 0.104μm.

[0090] The specific method is as follows: 10 mg of synephrine powder and API were loaded into No. 3 hydroxypropyl methylcellulose capsules, and then loaded into a single capsule inhalation device. After connecting to NGI, the measurement was performed, and 5 capsules were inhaled each time. The inhalation flow rate was 75 L / min, and the inhalation time was 3.2 s. The drug powders deposited at each level of NGI were collected with ultrapure water, and the synephrine content was determined under ultraviolet conditions, and the fine particle fraction (FPF) and respirable particle ratio (RF) were calculated.

[0091] Deposition of synephrine API and dry powder inhaler at each level of NGI Fig. 9The results showed that after anti-solvent precipitation, the emptying 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 inhalable fractions were 41.26% and 65.41% respectively, and the in vitro lung effective deposition rates were 2.25% and 19.27% ​​respectively, which met the requirements for 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 investigated in vitro, a cellular inflammation model was constructed with lipopolysaccharide, and the in vitro efficacy of SYN-DPI was preliminarily evaluated by enzyme-linked immunosorbent assay. Then, a rat acute lung injury model was established by tracheal instillation of lipopolysaccharide, and 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 homogenate and serum. The relevant protein pathways of SYN-DPI in the treatment of ALI were analyzed in combination with lung tissue immunohistochemistry.

[0094] 1 Experimental cells and animals

[0095] The experimental cells used in this experiment were mouse mononuclear macrophages (abbreviated as RAW264.7 cells), and the experimental animals were SPF-grade male SD rats (weight 200-220g) and were adaptively fed for 7 days. Drinking water was allowed 12 hours before the experiment, but food was not allowed.

[0096] 2 Experimental methods

[0097] 2.1 Cell culture

[0098] 2.1.1 Cryopreservation of RAW264.7 cells

[0099] Select cells in good logarithmic growth phase, remove old culture medium, prepare cell freezing solution, adjust cell concentration to 5×106 cells / mL, dispense into freezing tubes, place in programmed cooling box, take out and freeze in liquid nitrogen tank after 24 hours.

[0100] 2.1.2 Recovery of RAW264.7 cells

[0101] Heat the constant temperature water bath to 37°C, quickly put the frozen cells into the water bath, and shake them continuously to thaw them as quickly as possible. After thawing, suck them into a centrifuge tube, add more than 10 times the volume of fresh culture medium, centrifuge at 1250rpm for 5min, discard the supernatant, resuspend them in DMEM culture medium containing 10% FBS, and inoculate them into a culture dish.

[0102] 2.1.3 Cultivation of RAW264.7 cells

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

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

[0105] 2.2.1 Cytotoxicity assay

[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 in a 96-well plate, 100 μL per well, and cultured in a cell culture incubator at 5% CO2 and 37°C for 24 hours. Fresh culture medium was added to the blank group, and different concentrations of the drug to be tested were added to the experimental group. Six replicate wells were set up in each group, 100 μL per well, and the cells were placed in a cell culture incubator for 24 hours. 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 1-2 hours, and the absorbance was measured at a wavelength of 450 nm using an enzyme reader.

[0107]

[0108] 2.2.2 Establishment of cellular inflammation model

[0109] Take RAW264.7 cells in the logarithmic growth phase, seed each well in a 96-well plate at 100 μL, and continue to culture for 24 hours at 37°C and 5% CO2. Pipette the supernatant and discard it, add 100 μL of medium containing appropriate concentration of synephrine to each well for pretreatment for 1 hour, then add medium containing LPS, and incubate the cells with the test substance for 24 hours.

[0110] 2.2.3 Determination of cellular inflammatory factors

[0111] According to the results of CCK-8 experiment, RAW264.7 cells in logarithmic growth phase were inoculated into 96-well plates, and the cells were divided into Control group, LPS group, LPS+SYN group, and LPS+SYN-DPI group. A cell inflammation model was established, and the supernatant of each well was collected after 24 hours of culture and centrifuged at 3500rpm for 10 minutes. The supernatant was collected and operated strictly according to the instructions of ELISA kit. The absorbance value was measured at 450nm with a microplate reader and the contents of IL-1β, IL-6, TNF-α, and NO were calculated.

[0112] 2.3 Experimental methods of SYN-DPI improving LPS-induced ALI model in rats in vivo

[0113] 2.3.1 Construction of LPS-induced ALI model in rats

[0114] After one week of adaptive feeding, male SD rats were used to establish an acute lung injury model by tracheal instillation of LPS. The specific modeling steps are as follows: first, the rats were anesthetized with isoflurane, and then the rats were fixed in a supine position to facilitate intratracheal administration to the lungs, and LPS (8 mg / kg) was atomized in the alveoli, and then the micro-sprayer 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 drug administration of rats with acute lung injury

[0116] According to the literature on synephrine, the maximum human dose of synephrine is 100 mg. According to the clinical equivalent dose, the oral administration dose of rats was 9 mg / kg, and the low-dose inhalation administration dose was 4.5 mg / kg. The administration of each group is 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 collected from the abdominal aorta in a sterile blood collection tube. The blood was gently shaken and allowed to stand, and then centrifuged at 3500 rpm for 15 min. The serum was separated and stored in a -80°C refrigerator for the detection of serum inflammatory factor levels.

[0122] 2.3.4 Collection of bronchoalveolar lavage fluid

[0123] After the blood collection operation is completed, wait until the rat's heart stops beating, fix the rat in supine position on the operating table, use tissue scissors to cut the rat's chest and abdominal skin, sternum and surrounding soft tissue in turn, expose the lung tissue including the main bronchus under the glottis, ligate the right lung, use surgical scissors to cut a small "inverted V-shaped" incision on the trachea, carefully insert a syringe through the incision and perform lavage, and use normal saline to perfuse the left lung through the trachea 3 times, slowly injecting and withdrawing 1.5 mL each time, and collect the alveolar lavage fluid.

[0124] 2.3.5 Collection of lung tissue

[0125] After the perfusion of the left lung of the rat was completed, the left lung and right lung were washed and dried with normal saline. The lower lobe of the left lung was used to determine the wet-dry specific gravity of the rat lung tissue. The upper lobe of the right lung was temporarily stored in liquid nitrogen and transferred to a -80°C refrigerator for oxidative factor detection. The remaining right lung was stored in 4% paraformaldehyde for histopathological detection and immunohistochemical analysis.

[0126] 2.3.6 Determination of wet / dry specific gravity of lung tissue

[0127] The left lower lobe of the rat lung was removed, the blood stain on the surface was removed, the surface moisture was wiped off and weighed, recorded as the wet weight, and then placed in a 60℃ oven for drying for 24 hours and weighed as the dry weight. The degree of pulmonary edema was evaluated by calculating the lung wet / dry weight ratio.

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

[0129] 2.3.7 Hematoxylin-eosin (HE) staining

[0130] The lung tissues fixed in 4% paraformaldehyde were dehydrated, routinely paraffin-embedded, and made into 5 μm sections. The sections were then dewaxed, routinely stained with HE, dehydrated, and mounted. The structure and morphological characteristics of the lung tissues of the rats in each group were observed under a microscope.

[0131] 2.3.8 Masson staining

[0132] First, the slices were routinely dewaxed to water, and a hydrophobic circle was drawn. Then, Bouin solution was dripped, and the slices were covered and incubated in a 37°C incubator. Then, they were rinsed with running water until the yellow color on the slices disappeared. Then, the slices were dripped with lapis lazuli blue staining solution for 3 to 5 minutes, and then rinsed with running water. After that, Mayer hematoxylin staining solution was dripped for 3 minutes, and then rinsed with running water. After that, acidic ethanol differentiation solution was used for differentiation for a few seconds, and then rinsed with running water. Then, the slices were dripped with Ponceau magenta staining solution for 3 minutes, and then rinsed with distilled water. Finally, the slices were treated with phosphotungstic acid solution for 5 to 10 minutes, and the upper liquid was poured off and then the aniline blue staining solution was gradually dyed for 5 minutes, and then the slices were treated with weak acid solution. After completion, conventional dehydration was performed and xylene was used for transparency. Finally, neutral gum was used to seal the slices, and Masson trichrome staining was completed.

[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. The tissue weight was accurately weighed, and physiological saline was added according to the weight-to-volume ratio. The lung tissue homogenate was centrifuged and the supernatant was taken. The test solution was added in sequence according to the instructions of the protein quantification kit. The absorbance value was measured at 562 nm by an enzyme reader and the protein concentration in BALF was calculated.

[0135] 2.3.10 Detection of serum inflammatory factor levels

[0136] Rat serum was taken, and the test solution was added in sequence according to the instructions of the ELISA detection kits for rat TNF-α, IL-6, IL-8, IL-1β, and IL-1α, and the OD value was measured at 450nm by a microplate reader. A standard curve was drawn with the concentration of TNF-α, IL-8, IL-6, IL-1β, and IL-1α standard products as the abscissa and the absorbance value as the ordinate, and the concentrations of TNF-α, IL-6, IL-8, IL-1β, and IL-1α in serum were calculated according to the standard curve.

[0137] 2.3.11 Detection of oxidative stress indicators in lung tissue

[0138] Accurately weigh a certain weight of rat lung tissue, prepare samples for detecting SOD, CAT, GSH and MDA activities according to different ratios of lung tissue weight to physiological saline, homogenize and centrifuge in an ice water bath, take the supernatant for detection, and operate strictly according to the instructions of the kit.

[0139] 2.3.12 Immunohistochemical analysis of lung tissue

[0140] The lung tissue sections were dewaxed until transparent, rinsed with ultrapure water for standby use, and then placed in 0.01M sodium citrate buffer solution for 15 minutes for antigen extraction. Subsequently, the sections were incubated in 3% hydrogen peroxide for 10 minutes to block the activity of endogenous peroxidase. After blocking, the sections were incubated with the primary antibody Nrf2 / NF-κB / p38 MAPK at 4°C overnight, the sections after incubation with the primary antibody were dried, and then the secondary antibody was added to cover the lung tissue on the sections, incubated at room temperature for 1 hour, the sections were washed with PBS, dried, and the prepared color developer was added, and the sections were rinsed with ultrapure water to stop the color development, and the cell nuclei were counterstained with hematoxylin for 5 minutes and rinsed with ultrapure water. After differentiation for 10 seconds with hematoxylin differentiation solution, the sections were rinsed again, the hematoxylin blueing solution was blued, and the sections were rinsed with ultrapure water. Finally, the sections were dehydrated with anhydrous ethanol and xylene until transparent, and the sections were sealed with neutral gum. The sections were observed and images were collected under a microscope.

[0141] 3 Experimental results

[0142] 3.1 Cytotoxicity

[0143] The cytotoxicity results of SYN-DPI on RAW264.7 cells are shown in Fig.10The results showed that SYN-DPI did not cause any significant cytotoxicity in the concentration range of 50-400 μM, and had almost no effect on the activity of RAW264.7 cells; at concentrations of 800 μM and 1000 μM, a decrease in cell viability was observed (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, with significant differences (P<0.001), so we chose the concentration range of 50-400 μM for subsequent experiments.

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

[0145] Effects of SYN-DPI on the expression of proinflammatory cytokines in the LPS-induced cellular inflammation model Fig.11 As shown. The results showed that compared with the control group, RAW264.7 cells stimulated with LPS produced a large amount of IL-1β, IL-6 and TNF-α, with significant differences, indicating that the cell inflammation model was successfully constructed. Taking IL-6 as an example, there was no significant difference between the 100μM SYN-DPI group and the 400μM SYN group (P>0.05), indicating that SYN-DPI at a dose of 100μM can achieve the same effect of inhibiting the expression of inflammatory factors as 400μM SYN; the inhibition rate of SYN-DPI (400μM) on IL-6 production was 45.17%, while the inhibition rate of the same amount of SYN (400μM) on IL-6 production was 23.32%, indicating that SYN-DPI has better anti-inflammatory potential. Nitric oxide (NO) is an active free radical involved in the inflammatory process. The inhibition rate of SYN (200 μM) on NO production 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 can inhibit the production of NO, but SYN-DPI has a better inhibitory effect. In summary, within the scope of this study, SYN-DPI has a better anti-inflammatory effect than SYN.

[0146] 3.3 Effect of SYN-DPI on dry-wet specific gravity of rat lung

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

[0148] 3.4 Effect of SYN-DPI on pathological changes of lung tissue in LPS-induced ALI rat model

[0149] HE and Masson staining were used to observe the pathological changes of lung tissue. Fig.13 As shown. The bronchial ciliated columnar epithelial structure of the lung tissue in the Control group was clear, and the alveolar cavity structure was intact. The alveolar cavity of the rats in the Model group was significantly reduced, showing extremely severe protein and inflammatory cell infiltration, alveolar wall thickening, alveolar edema, and even congestion, degeneration and other pathological manifestations. The pathological damage of the lung tissue of the rats in the Dex group was significantly alleviated. There was no significant difference in 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 decreased, the bronchial epithelium of the rats in the medium-dose group returned to flatness, and there was still inflammatory cell infiltration around the bronchi, and the alveolar cavity of the rats in the high-dose group returned to normal, and the inflammatory cells basically returned to normal, indicating that SYN-DPI can alleviate the pathological damage of the lung tissue caused by LPS, and it is dose-dependent.

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

[0151] Effects of SYN and SYN-DPI on the total protein content in BALF of ALI rats Fig.14 As shown in the figure, compared with the blank group, the protein content in the BALF of the model group increased significantly, with significant differences, indicating that the interstitial space of the alveoli increased and the acute lung injury model was successfully established. The protein content in the drug-treated 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 interstitial space of the alveoli. Comparison of the data analysis of the low, medium and high dose groups of SYN-DPI found that the protein content in BALF was closely related to the dose and was dose-dependent.

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

[0153] The results of serum IL-1α, IL-1β, IL-6, IL-8, and TNF-α are as follows Fig.15 As shown. Compared with the blank group, the secretion of inflammatory factors in the LPS-induced model group increased significantly (P<0.0001). After treatment with SYN and SYN-DPI, the expression of inflammatory factors in the SYN group and SYN-DPI group and the Dex group decreased significantly compared with the model group (P<0.0001), indicating that SYN and SYN-DPI can improve ALI and reduce the expression level of inflammatory factors in serum, and have obvious therapeutic effects. Taking TNF-α as an example, there was no significant difference between the low-dose group SYN-DPI of 4.5 mg / kg and the SYN group of 9 mg / kg (P>0.05), indicating that the low-dose group SYN-DPI can achieve the same inhibitory effect of inflammatory factor expression as the SYN group. Analysis of 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 can reduce the secretion of inflammatory factors in a dose-dependent manner, and has an inhibitory effect on the inflammatory response of rats induced by LPS.

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

[0155] Effects of SYN and SYN-DPI on MDA, SOD and MPO in lung tissue of rats with ALI induced by LPS Fig.16 The results showed that compared with the blank group, the model group significantly increased the levels of MDA and MPO and reduced the activity of SOD after LPS stimulation, while SYN-DPI treatment could significantly reverse the effect of LPS on redox-related enzymes, reduce peroxidation metabolites, enhance antioxidant levels, and maintain oxidative stress balance.

[0156] Effects of SYN-DPI on the expression of NF-κB, Nrf2 and p38 MAPK proteins in lung tissue of ALI rats

[0157] After immunohistochemical analysis and microscopic photography, the expression of NF-κB, Nrf2 and p38 MAPK proteins was Fig.17As shown. Image J software was used to semi-quantitatively analyze 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. Activated NF-κB and MAPK pathways can promote the production of proinflammatory mediators. Therefore, inhibiting NF-κB and MAPK pathways helps to inhibit the production of proinflammatory cytokines. Therefore, in the LPS-induced ALI model, the effect of SYN-DPI on NF-κB and MAPK signaling pathways was further detected. The results showed that LPS could significantly increase the expression of p38 MAPK protein, while synephrine dry powder inhalation treatment could significantly inhibit the phosphorylation of the protein, indicating that synephrine can exert anti-inflammatory effects 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 LPS-treated group, SYN-DPI treatment effectively promoted the nuclear transcription of Nrf2, indicating that SYN-DPI can effectively activate Nrf2.

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

[0159] In order to explore the changes in drug concentration of SYN-DPI after it is absorbed into the blood, this chapter intends to establish a simple, sensitive, and fast-response LC-MS / MS method for determining the concentration of the compound SYN in rat plasma. This method will be rigorously validated based on the quantitative analysis of drugs in biological samples, and the metabolic process of SYN in rats will be explored, providing a basis for subsequent research on SYN-DPI.

[0160] 1 Experimental animals

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

[0162] 2 Experimental methods

[0163] 2.1 Chromatographic conditions

[0164] Chromatographic column: Hypersil GOLD C8 (100mm×2.1mm; 3μm); mobile phase: phase A: 0.1% formic acid water, phase B: acetonitrile; flow rate: 0.3mL / min; column temperature: 40℃; injection volume: 2μL; gradient elution program: 0-1min, 5% B; 1-4min, 5%-90% B; 4-5min, 90%-5% B.

[0165] 2.2 Mass spectrometry conditions

[0166] The mass spectrometer used the ESI positive ion scanning mode, which was the 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 Experimental Animal Grouping and Dosing

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

[0169] 2.4 Collection of plasma samples

[0170] Rats were fasted but not allowed to drink water for 12 h before the experiment. The rats were weighed (to calculate the dosage) and numbered. After administration, 0.2 mL of blood was collected from the orbital vein of the three groups of rats at 2 min, 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, and 6 h, placed in EP with sodium heparin, centrifuged at 3500 rpm for 15 min, and the upper plasma was separated and stored in a -80°C refrigerator.

[0171] 2.5 Plasma sample testing

[0172] Accurately pipette 190 μL of rat plasma sample into an EP tube, add 10 μL of internal standard solution, vortex for 60 seconds, then add 400 μL of acetonitrile, vortex for 60 seconds to precipitate protein, centrifuge at 10,000 rpm for 15 minutes, take the supernatant and filter it through a 0.22 μm microporous filter membrane, place it into a glass inner tube, and inject it for detection and analysis by LC-MS / MS.

[0173] 3 Experimental results

[0174] Synephrine concentration-time curve Fig.18As shown. The results show that compared with the oral gavage group, after pulmonary administration of the same dose of synephrine dry powder inhalation, the Cmax of the inhalation group was 845.52 ng / mL, and the Cmax of the oral gavage group was 301.76 ng / mL, which was 280.20% higher than that of the oral gavage group; the half-life t1 / 2 of the inhalation group was 175.51 min, and the half-life t1 / 2 of the oral gavage group was 137.65 min, which was 127.50% longer than that of the oral gavage group; the AUC (0-t) of the inhalation group was 1168.24 ng / mL*h, and the AUC (0-t) of the oral gavage group was 876.07 ng / mL*h, which was 133.35% higher than that of the oral gavage group. Compared with the injection group, after pulmonary administration of the same dose of synephrine micropowder, the Cmax of the injection group was 1032.17 ng / mL, the inhalation group reached 81.92% of the injection group, and the oral gavage 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 oral gavage 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, and the oral gavage group was only 64.48% of the injection group. The absolute bioavailability of the oral gavage 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 dosing groups, inhalation administration can significantly improve its bioavailability.

Claims

1. Application of synephrine dry powder inhaler in the preparation of drugs for treating acute lung injury.

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: The preparation method of the synephrine dry powder inhaler comprises the following steps: (1) dissolving synephrine API in a good solvent as good solvent phase A, and n-hexane as anti-solvent phase B; (2) Adding the good solvent phase A dropwise into the anti-solvent 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.

5. The use according to claim 4, characterized in that: In step (1), the good solvent is one or more of water, anhydrous ethanol, acetonitrile, isopropanol or n-butanol, preferably anhydrous ethanol.

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

7. The use according to claim 4, characterized in that: In step (2), the stirring speed is 600-1200 rpm and the stirring time is 2-10 h.

8. The use according to claim 4, 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.

9. The use according to claim 4, characterized in that: In step (3), the drying method is constant temperature drying at 30-40 °C.

Images