CN113827580A - A kind of fenugreek inhalation medicament and preparation and preparation method and application thereof - Google Patents
A kind of fenugreek inhalation medicament and preparation and preparation method and application thereof Download PDFInfo
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- CN113827580A CN113827580A CN202110706534.6A CN202110706534A CN113827580A CN 113827580 A CN113827580 A CN 113827580A CN 202110706534 A CN202110706534 A CN 202110706534A CN 113827580 A CN113827580 A CN 113827580A
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Abstract
本发明公开了一种千金藤素吸入药剂和制剂及其制备方法与应用，该药剂包括千金藤素和药剂学上可接受的辅料形成的组合物。该吸入药剂可通过吸入的给药方式用于预防和/或治疗新型冠状病毒，其生物利用度高、快速释药、快速起效。The invention discloses a fenugreek inhalation medicament and preparation as well as a preparation method and application thereof. The medicament comprises a composition formed by fenugreek and pharmaceutically acceptable auxiliary materials. The inhaled medicament can be used for the prevention and/or treatment of novel coronavirus by inhalation administration, and has high bioavailability, rapid drug release, and rapid onset of action.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a cepharanthine inhalation medicament, a cepharanthine inhalation preparation, a preparation method thereof and application thereof in preparing a novel coronavirus prevention and/or treatment medicine.
Background
Cepharanthine is a bisbenzylisoquinoline alkaloid, and is extracted from Stephania cepharantha of Menispermaceae in 1934 by separation. Cepharanthine has antiinflammatory, antibacterial, and immunity regulating effects. In recent years, many researchers find that the medicine also has the functions of stimulating the reticuloendothelial system, activating hematopoietic tissues, promoting the proliferation of bone marrow tissues and the like, and has great effect on clinically preventing and treating the leucopenia of tumor patients caused by radiotherapy or chemotherapy at present.
Cepharanthine has been approved as an active ingredient of drugs for the treatment of various diseases, with a history of use of up to 40 years. Cepharanthine has acute toxicity: oral-rat LD50 was 2000 mg/kg; oral administration, mouse LD50 1900mg/kg, intraperitoneal injection of cepharanthine free base, mouse LD50 260 mg/kg. It can be seen that the high safety of cepharanthine has been well verified for a long time.
At present, the stephanine preparation clinically applied in China is mostly an oral tablet, has the problem of low dissolution rate after being taken, and also has the problem of poor absorption due to low bioavailability (less than 6 percent) caused by liver and intestine first pass effect, thereby influencing the exertion of drug effect and finally influencing the treatment effect.
Therefore, it is one of the problems to be solved in the art to provide a new cepharanthine dosage form to improve its bioavailability.
Disclosure of Invention
The invention aims to overcome the technical defects in the prior art, and provides a cepharanthine inhalation medicament which can improve the bioavailability of the cepharanthine inhalation medicament, wherein the cepharanthine inhalation medicament comprises a composition formed by cepharanthine and pharmaceutically acceptable auxiliary materials, the composition is cepharanthine micro powder or a solution containing the cepharanthine micro powder, and the particle size of the cepharanthine micro powder is 0.1-25 mu m.
The adjuvants include disintegrating agent (such as sodium carbonate and citric acid), etc.
The cepharanthine micro powder is obtained by drying cepharanthine microspheres or cepharanthine nanometer suspension, or directly pulverizing cepharanthine.
The cepharanthine microspheres are prepared by dripping cepharanthine-polysorbate microemulsion (the cepharanthine-polysorbate microemulsion is prepared by stearic acid dispersed with cepharanthine and polysorbate) into cold water; or
The cepharanthine nanosuspension is prepared by dissolving cepharanthine and poloxamer (F127) in ethanol, evaporating to dryness, and adding water.
The cepharanthine may be cepharanthine salt.
The cepharanthine salt includes, but is not limited to, one or more of cepharanthine hydrochloride, cepharanthine sulfate, cepharanthine acetate, cepharanthine sulfonate, etc.
The cepharanthine micropowder and adjuvants are in the form of capsule, vesicle or multi-dose storage.
In a second aspect, the present invention provides a method for preparing the above cepharanthine inhalation medicament, comprising the following steps:
the first method is as follows: preparing cepharanthine, stearic acid and polysorbate into cepharanthine-polysorbate microemulsion, dripping the microemulsion into cold water to form microspheres, and drying the microspheres to obtain cepharanthine micropowder (preferably spray drying method); or
The second method comprises the following steps: dissolving cepharanthine and adjuvants to obtain cepharanthine nanometer suspension, and drying to obtain cepharanthine micropowder (preferably spray drying method); specifically, the cepharanthine nanosuspension is prepared by mixing cepharanthine, poloxamer (F127) and citric acid, dissolving in ethanol, evaporating to dryness, adding sodium bicarbonate, and adding water to obtain cepharanthine nanosuspension; or
The third method comprises the following steps: pulverizing cepharanthine directly to obtain cepharanthine micropowder with particle size distribution of 0.1-25 μm; or
The method is as follows: dispersing or dissolving the cepharanthine micropowder obtained in any one of the first to third modes in a solvent to form a suspension or solution.
In a third aspect, the invention provides a cepharanthine inhalation formulation comprising the above cepharanthine inhalation formulation and an inhalation device comprising a dosing system, an aerosolization system, a powder degradation system and an aerosol inlet introduction system to enable a patient to inhale the cepharanthine inhalation formulation in the form of a dry powder or solution into the lungs.
The inhalant comprises a dry powder inhalant preparation, a solution inhalant preparation and an atomized inhalant preparation; alternatively, the inhalation formulation comprises a single-dose type, a multi-dose type, a depot type.
In a fourth aspect, the present invention provides the use of the cepharanthine inhalation formulation or the cepharanthine inhalation formulation in the manufacture of an anti-coronavirus medicament.
The cepharanthine inhalant or its inhalant can be used for preparing medicines for resisting squama Manis coronavirus xCoV or SARS-COV-2 virus or novel coronavirus 2019-nCoV.
In a fifth aspect, the present invention provides a medicament for the prophylaxis and/or treatment of coronavirus, comprising the above cepharanthine inhalation agent; preferably, the coronavirus includes, but is not limited to, pangolin coronavirus xCoV and/or SARS-COV-2 virus and/or novel coronavirus 2019-nCoV. The composition is a dry powder composition or a solution composition.
The cepharanthine is used as a raw material and is prepared into a micro powder inhalation medicament after being matched with pharmaceutically acceptable auxiliary materials, and the micro powder inhalation medicament is used for treating respiratory tract infection and can increase the local medicament concentration of a focus. The preparation can be made into inhalation preparation for preventing and/or treating novel coronavirus by inhalation administration, and has the advantages of rapid release, rapid action, and high bioavailability (up to 64%).
The cepharanthine inhalation preparation provided by the invention is convenient to carry and easy to operate; the medicine in a dry powder state has better stability at room temperature and is not easily polluted by microorganisms. When in administration, the medicine powder enters the respiratory tract by the spontaneous respiration of the patient and reaches the lung, so as to realize the purpose of positioning administration.
Drawings
FIG. 1 is a graph showing the time course of the concentration of cepharanthine in plasma of KM mice after inhalation of 1mg/kg of the inhaled dose of cepharanthine.
FIG. 2 is a graph showing the mean plasma concentration as a function of time in SD rats after 1mg/kg inhalation and intravenous administration of cepharanthine inhalation.
FIG. 3 is a graph showing the time course of local drug concentration in lungs of SD rats after inhalation of 1mg/kg stephanine.
Figure 4 shows a morphogram of Vero E6 cells 72 hours after infection with cepharanthine added to a final concentration of 10 μ M (micromole per liter) and xCoV with a multiplicity of infection of 0.01.
Figure 5 shows a morphological map of Vero E6 cells 72 hours after infection with ceratin added to a final concentration of 10 μ M (micromole per liter) and xCoV with a multiplicity of infection of 0.01.
Figure 6 shows a morphogram of Vero E6 cells 72 hours after infection with mefloquine hydrochloride added to a final concentration of 10 μ M (micromole per liter) and xCoV with a multiplicity of infection of 0.01.
FIG. 7 shows the inhibition of xCoV by three compounds, 10. mu.M cepharanthine inhibits xCoV viral replication by 15393-fold, 10. mu.M ceratin inhibits xCoV viral replication by 5053-fold, and 10. mu.M mefloquine hydrochloride inhibits xCoV viral replication by 31-fold.
FIG. 8 shows the median Effective Concentration (EC) of cepharanthine versus xCoV50) Half the Cytotoxic Concentration (CC) against Vero E6 cells at 0.9851. mu.M50) 39.32 μ M, and a Selection Index (SI) of 39.91.
FIG. 9 shows the median Effective Concentration (EC) of ceratin to xCoV50) Half the Cytotoxic Concentration (CC) against Vero E6 cells at 1.908. mu.M50) 6.227 μ M, and a Selection Index (SI) of 3.290.
FIG. 10 shows the median Effective Concentration (EC) of mefloquine hydrochloride on xCoV50) Half the Cytotoxic Concentration (CC) against Vero E6 cells at 2.728. mu.M50) 10.08 μ M and a Selection Index (SI) of 3.695。
FIG. 11 shows the results of the Time-of-Addition test of cepharanthine on xCoV.
FIG. 12 shows the results of the Time-of-Addition test of ceratin on xCoV.
FIG. 13 shows the results of the Time-of-Addition test of mefloquine hydrochloride on xCoV.
FIG. 14 shows transcriptome analysis results of cepharanthine against xCoV replication.
Detailed Description
The surface area of the alveoli in the lung is large, and the surface is distributed with abundant capillaries. The pulmonary inhalation drug preparation can not only ensure that the drug is intensively distributed in the lung and quickly exert the drug effect, but also avoid the first pass effect of the liver and the intestine and improve the bioavailability, is recommended by the world health organization as the first choice preparation for respiratory diseases, and is particularly suitable for quickly treating acute pulmonary diseases.
Due to the poor water solubility of cepharanthine, a common dosage form of cepharanthine as a drug is a tablet, and the administration mode is oral. The patent application with the publication number of CN102475680A discloses a stephanine hydrochloride injection, which is prepared by taking stephanine hydrochloride as a raw material to obtain a water-soluble injection. However, the patient compliance of the injection is poor, professional administration is required, and the drug cannot be locally distributed at the focal site, which is not suitable for the treatment of pulmonary diseases, especially acute pulmonary diseases.
Experiments show that the inhalation can improve the blood concentration of the lung and respiratory tract for preventing and treating the novel coronavirus.
The invention provides a cepharanthine inhalation medicament, which is a dry powder composition or a solution composition prepared from cepharanthine and pharmaceutically acceptable auxiliary materials. On the basis, the cepharanthine dry powder inhalation preparation is prepared by taking the dry powder composition as a raw material, or the cepharanthine aerosol inhalation preparation is prepared by taking the solution composition as a raw material.
A Dry Powder Inhalation (DPI) is a preparation which is prepared by preparing cepharanthine and pharmaceutically acceptable auxiliary materials into cepharanthine micro Powder (with the particle size of 0.1-25 μm), then forming capsules, vesicles or multi-dose storage forms with a carrier, placing the mixture into a Dry Powder Inhalation device, and inhaling atomized medicine into the lung when a patient breathes autonomously.
The inhalation preparation provided by the invention has the characteristics of targeting the pathological part with the medicine, high local medicine concentration (instead of exposing the whole human body to the high-concentration medicine), small toxic and side effect, high medicine utilization degree, quick response and the like. The dry powder inhalation device involved in the cepharanthine dry powder inhalation formulation comprises four basic functional components, namely: a dosing system, an aerosolization system, a powder degradation system, an aerosol inlet introduction system (available from shanghai yitai balance pharmaceutical co., ltd.). The cepharanthine dry powder inhalation preparation can be prepared into single dosage type, multi-dosage type and reservoir type to meet the requirements of different people and occasions. The cepharanthine used in the present invention can be prepared by any known method.
The present invention will be described more specifically and further illustrated with reference to specific examples, which are by no means intended to limit the scope of the present invention.
Example 1 preparation of Dry powder inhalation preparation
Weighing 1 part by weight of cepharanthine, sieving with a 200-mesh sieve, adding 20 parts by weight of stearic acid, and placing at 65 ℃ to fully dissolve the cepharanthine; adding ethanol solution of polysorbate 80 (mass ratio of polysorbate 80 to ethanol is 1: 4, and mass of polysorbate 80 is 1-3 times of that of cepharanthine), adding appropriate amount of distilled water, and vortexing for 1min to obtain cepharanthine-polysorbate microemulsion.
Dripping 65 deg.C cepharanthine-polysorbate microemulsion into 2 deg.C cold water at a speed of 1 drop/5 s under electromagnetic stirring (1000r/min), and stirring at2 deg.C for 15min to obtain cepharanthine microsphere. Drying the cepharanthine microspheres into cepharanthine micro powder by using a YC-015 experimental spray dryer.
And 4 parts by weight of 200-mesh lactose (used as a carrier of the cepharanthine micropowder) can also be added, and the mixture is fully and uniformly mixed and filled into a capsule of an inhaler to obtain the cepharanthine-polysorbate-lactose micropowder.
EXAMPLE 2 preparation of Dry powder inhalant
Weighing a proper amount of cepharanthine, poloxamer (F127) and citric acid according to the mass ratio of 1:1.5:3, dissolving the mixture in ethanol with a proper volume, uniformly mixing by vortex, drying by nitrogen at 37 ℃, further evaporating by using a rotary evaporator, adding sodium bicarbonate with the weight being 4 times that of the cepharanthine, uniformly mixing, finally adding a certain amount of water to form a nano suspension, and performing spray drying to obtain the cepharanthine micro powder. Wherein citric acid and sodium bicarbonate are used as disintegrating agents. When the preparation is prepared into a preparation for pulmonary administration, the disintegrating agent can be rapidly disintegrated under the action of mucus in respiratory tract after the micropowder enters the lung, micron-sized cepharanthine micropowder particles can be dispersed into nanoscale particles, and the nanoscale drug particles can avoid phagocytosis of macrophages due to small enough particle size, so that the drug effect exerting time of the drug in the lung is prolonged, the administration interval is further prolonged, and the administration times are reduced.
EXAMPLE 3 preparation of Dry powder inhalant
Pulverizing cepharanthine to particle size distribution of 0.1-25 μm with jet mill or rotor-stator colloid mill to obtain cepharanthine micropowder.
Example 4 preparation of Aerosol inhalation formulation
Atomizing inhalation preparation one: regulating physiological saline with acetic acid until pH values are 3.5, 3.0 and 2.5 respectively, using the prepared solution as a solvent, adding cepharanthine into the solvent, preparing cepharanthine solutions with cepharanthine concentrations of 5mg/mL, 10mg/mL and 50mg/mL respectively, after vortex oscillation, completely dissolving the cepharanthine, clarifying the solution, and putting the solution with the pH value of 4.0-5.0 into an inhalation device, thus obtaining the aerosol inhalation preparation.
Atomizing inhalation preparation two: the method comprises the steps of using sulfuric acid to adjust physiological saline to enable the pH value of the physiological saline to be 1.5, using the sulfuric acid as a solvent, adding cepharanthine into the solvent to prepare a cepharanthine solution with the concentration of 5mg/mL, carrying out vortex oscillation, completely dissolving the cepharanthine, clarifying the solution, enabling the pH value to be within the range of 4.5-5.0, and loading the solution into an inhalation device to obtain the aerosol inhalation preparation.
Stephanine is a bisbenzylisoquinoline alkaloid, has poor solubility in aqueous solution, and is alkaline, so the pH value of the solution is adjusted, the solubility of the solution is improved, acid radical ions in body fluid are selected for trial, and finally, the acetic acid and stephanine sulfate atomization inhalation preparation is successfully prepared.
Main drugs and reagents:
stephanine (cephaloranthine) standard, lot number: t0131-1, available from ceramic Biotechnology, Inc.; internal standard Rutin (Rutin), batch number: t0795-1, available from ceramic Biotechnology, Inc.
Acetic acid (analytically pure), trisodium citrate (analytically pure), purchased from pharmaceutical group chemical agents, ltd; physiological saline, purchased from Shandong Qidu pharmaceutical Co., Ltd; methanol, acetonitrile, formic acid, HPLC grade, purchased from seimer feishell science & technology (china) ltd; deionized water, prepared by a Milli-Q Advantage A10 water purifier (Millipore, USA).
Main apparatus and equipment:
pulmonary inhalation of the formulation device, Huilong, Beijing and science and technology Limited.
The experimental method comprises the following steps:
1. preparing a cepharanthine test medicine solution:
weighing a proper amount of cepharanthine standard substance, adding normal saline (containing acetic acid and having a pH value of 3.5) to prepare a test mother solution with a cepharanthine concentration of 5mg/mL, fully performing vortex oscillation until the solution is completely dissolved, and then diluting the solution with normal saline until the cepharanthine concentration is 0.15mg/mL and the pH value is within a range of 5.0-6.0, wherein the test mother solution is used as a test drug solution of cepharanthine in the experiment.
2. And (3) experimental operation:
KM mice, each half male and female, with a body weight of 28-32g, were purchased from Beijing Keyu animal breeding center (Experimental animal license number: SCXK (Jing): 2018-. The experiment was set up for blank and dosing.
The blank group is not administrated and is normally bred;
administration group: the administration dose is 1mg/kg based on the weight of cepharanthine, and the administration mode is pulmonary inhalation administration. The specific operation is as follows: after anesthetizing a mouse, fixing the mouse on an operation table, using a small animal laryngoscope and a lung liquid quantitative atomizer in a matching way, administering the cepharanthine test solution prepared in the experimental method part 1 of the experiment in an aerosol mode through an air pipe, taking blood from a posterior ocular venous plexus for 0min, 15min, 1h, 2h, 4h, 7h, 12h and 24h after administration, taking about 0.1mL of blood each time, anticoagulating sodium citrate, slightly mixing uniformly, centrifuging at 4000r/min for 10min, taking supernatant, and storing the supernatant at-20 ℃ as a blood plasma sample to be tested for later use.
3. Preparation of Standard and quality control samples
The cepharanthine test solution was diluted with 75% (v/v) acetonitrile in water to working solutions of different concentrations. Adding 10 μ L of working solution with different concentrations into 90 μ L of blank plasma matrix (blank mice are blood-taken, sodium citrate is anticoagulated, after mixing gently, centrifuging at 4000r/min for 10min, and supernatant is blank plasma matrix) to obtain final concentrations of 0.5, 1, 5, 10, 25, 50, 100 ng. mL-1The standard sample of (1). Quality control samples independent of the standard sample were prepared in the same manner at concentrations of 1.5 ng/mL-1(Low), 30 ng. mL-1Neutralization 75 ng/mL-1(high).
4. Biological sample pretreatment method
50 mul of prepared standard sample, quality control sample and plasma sample to be tested are respectively added with 150 mul of acetonitrile (containing 10ng/mL internal standard rutin) for precipitating protein, after vortex oscillation for 1min, centrifugation is carried out at 14000rpm for 20min, and 100 mul of supernatant is taken as a biological sample to be tested.
5. Detection method
Detection was performed as described in the following chromatographic and mass spectrometric conditions.
1) And chromatographic conditions:
the instrument comprises the following steps: ACQUITY UPLC I-Class (Waters, USA);
a chromatographic column: ACQUITY UPLC BEH C18(2.1 mm. times.50 mm, 1.7 μm);
column temperature: 45 ℃;
flow rate: 400 mu L/min;
sample introduction amount: 5 mu L of the solution;
temperature of the sample chamber: 10 ℃;
analysis time: 3.5 min;
mobile phase: phase A: water (containing 0.1% (v/v) formic acid), phase B: acetonitrile (containing 0.1% (v/v) formic acid); gradient elution conditions: see table 1;
TABLE 1 gradient elution conditions
| Time (min) | A(％) | B(％) | Time (min) | A(％) | B(％) |
| 0.0 | 90 | 10 | 2.0 | 10 | 90 |
| 0.5 | 90 | 10 | 2.0 | 90 | 10 |
| 1.0 | 10 | 90 | 3.5 | 90 | 10 |
2) And mass spectrum conditions:
the instrument comprises the following steps: xevo TQ-S triple quadrupole mass spectrometry (waters, usa);
mass spectrum conditions: see table 2;
TABLE 2 Cepharanthine and internal standard (rutin) Mass Spectrometry conditions
6. Determination of biological samples
When actual plasma samples are analyzed, a standard curve is established for each analysis batch (Run) to calculate the concentration of the analyte in the samples of the analysis batch. The standard curve is obtained by using the concentration of cepharanthine in plasma as an abscissa and the ratio of the peak area of cepharanthine to the peak area of an internal standard as an ordinate, and using a weighted least square method (W is 1/X)2) And (4) performing regression calculation, wherein the obtained linear regression equation is a standard curve. Substituting the peak area ratio of the cepharanthine to the internal standard in the biological sample to be detected into the standard curve to calculate the concentration of the cepharanthine in the biological sample to be detected.
7. Data analysis and statistics
The concentration of cepharanthine in the plasma of mice after administration was calculated from a standard curve established for each assay batch, and data was processed and plotted using the computer programs Microsoft Office Excel and Origin 7.5.
The mean drug concentration and Standard Deviation (SD) were retained to two significant figures after the decimal point.
8. Acceptance and rejection criteria for standard curve and quality control
The standard curve consists of at least 6 non-zero concentration points, the relative deviation of the lower limit of quantitation (LLOQ) from the indicated value cannot exceed 20%, and the relative deviation of the other concentration points on the standard curve except the lower limit of quantitation from the indicated value cannot exceed 15%.
The relative deviation of the low-concentration quality control sample cannot exceed 20%, and the relative deviation of the medium-concentration quality control sample and the high-concentration quality control sample cannot exceed 15%.
At least two thirds of the standard concentration points need to meet the above requirements, otherwise the standard curve is not acceptable.
9. Results of the experiment
Blood concentration of KM mouse after lung inhalation of cepharanthine
After 1mg/kg of cepharanthine is administered by pulmonary inhalation of KM mice, the drug concentration of cepharanthine in plasma is shown in Table 3, and the blood drug concentration-time curve is shown in FIG. 1.
TABLE 3 plasma concentration of Cepharanthine inhaled into the lungs of KM mice (ng. mL)-1)
The results in table 3 show that, after lung inhalation of cepharanthine, cepharanthine was detected in plasma at each time point in KM mice, and the blood concentration value was highest at the first time point (i.e. 15min after administration), indicating that local administration to the lung can exert therapeutic effect systemically.
The experimental method comprises the following steps:
1. preparation of cepharanthine test drug solution
Weighing a proper amount of stephania tetragonoloba standard substance, adding normal saline (containing acetic acid and having a pH value of 3.5) to prepare a test drug mother solution with the stephania tetragonoloba concentration of 5mg/mL, fully performing vortex oscillation until the solution is completely dissolved, and then diluting the solution to 1 mg/mL by using the normal saline, wherein the pH value is within a range of 5.0-5.5 to serve as the test drug solution of the stephania tetragonoloba in the experiment. 2. Experimental procedures SD rats, male, weighing 180-.
The experiment sets up a venous administration group and a pulmonary inhalation administration group, and the administration dose is 1mg/kg calculated by cepharanthine.
Group for intravenous administration: adopting a rat tail vein administration mode;
pulmonary inhalation dosing group: after rat anesthesia, fixing the rat on an operation table, using a laryngoscope and a lung liquid quantitative atomizer in a matching way, and administering the prepared stephanine test drug solution in the experimental method part 1 of the experiment in an aerosol mode through an air pipe, wherein the stephanine test drug solution is taken from the venous plexus behind the eyes after administration for 0min, 1min, 5min, 15min, 30min, 1h, 2h, 4h, 8h, 12h, 24h and 48h, about 0.2mL of blood is taken each time, sodium citrate is anticoagulated, after gentle mixing, the mixture is centrifuged at 4000r/min for 10min, and the supernatant is taken as a plasma sample to be detected and stored at-20 ℃ for later use.
3. Preparation of Standard and quality control samples
As in experiment 1.
4. Biological sample pretreatment method
As in experiment 1.
5. Detection method
As in experiment 1.
6. Determination of biological samples
As in experiment 1.
7. Data analysis and statistics
As in experiment 1.
8. Acceptance and rejection criteria for standard curves and quality control
As in experiment 1.
9. Results of the experiment
1) SD rat pulmonary inhalation and blood concentration after intravenous administration of cepharanthine
The plasma cepharanthine concentration of rats in the pulmonary inhalation group is shown in table 4, the plasma cepharanthine concentration of rats in the intravenous group is shown in table 5, and the mean plasma concentration-time curve is shown in fig. 2.
TABLE 4 plasma concentration (ng. mL) in rats of pulmonary inhalation administration group-1)
TABLE 5 blood concentration (ng. mL) in rats of intravenous administration group-1)
The data in tables 4 and 5 were obtained and used to calculate bioavailability. 2) Evaluation of bioavailability of cepharanthine inhalant in rat
The pharmacokinetic parameters of the drugs in the plasma of rats in the pulmonary inhalation administration group and the intravenous administration group are shown in tables 6-7, wherein ke is the elimination rate constant, t1/2To eliminate half-life, CmaxTo achieve peak blood levels, TmaxTime to peak, AUClastArea under the curve for drug time, Vz _ F _ obs is apparent volume of distribution, Cl _ F _ obs is clearance, MRTlastIs the average residence time.
TABLE 6 pharmacokinetic parameters of plasma of rats in pulmonary inhalation administration group
| | Rat | 1 | Rat | 2 | Rat | 3# | Mean value of | SD |
| ke(1/h) | 0.050 | 0.053 | 0.041 | 0.048 | 0.006 | |||
| t1/2(h) | 13.74 | 13.20 | 16.98 | 14.64 | 2.05 | |||
| Cmax(ng/ml) | 47.05 | 77.35 | 71.41 | 65.27 | 16.05 | |||
| Tmax(h) | 0.017 | 0.017 | 0.017 | 0.017 | 0.000 | |||
| AUClast(h·ng/ml) | 359.5 | 352.1 | 436.4 | 382.7 | 46.7 | |||
| Vz_F_obs(l/kg) | 48.7 | 50.0 | 44.3 | 47.7 | 3.0 | |||
| Cl_F_obs(l/h/kg) | 2.46 | 2.63 | 1.81 | 2.30 | 0.43 | |||
| MRTlast(h) | 28.2 | 26.0 | 37.0 | 30.4 | 5.8 |
TABLE 7 pharmacokinetic parameters of rat plasma in intravenous administration group
| | Rat | 4 | Rat | 5 | Rat | 6# | Rat 7# | Mean value of | SD |
| ke(1/h) | 0.036 | 0.028 | 0.041 | 0.047 | 0.038 | 0.008 | |||
| t1/2(h) | 19.39 | 25.08 | 16.85 | 14.76 | 19.02 | 4.46 | |||
| Cmax(ng/ml) | 237.55 | 132.50 | 118.48 | 106.75 | 148.82 | 60.08 | |||
| Tmax(h) | 0.017 | 0.017 | 0.017 | 0.017 | 0.017 | 0.00 | |||
| AUClast(h*ng/ml) | 784.3 | 576.7 | 468.2 | 560.5 | 597.4 | 133.4 | |||
| Vz_obs(l/kg) | 30913 | 53430 | 49208 | 36013 | 42391 | 10658 | |||
| Cl_obs(l/h/kg) | 1105 | 1477 | 2024 | 1691 | 1574 | 385 | |||
| MRTlast(h) | 21.9 | 19.0 | 16.0 | 18.6 | 18.9 | 2.4 |
As can be seen from the results in tables 6 and 7, the absolute bioavailability of the pulmonary inhalation group was calculated to be 64.05% based on the bioavailability of the intravenous administration group being 100%, which was improved by 10 times or more compared to the bioavailability of the oral administration group.
This experiment is substantially similar to experiment 2, with only the differences listed, and the unrecited portions all being the same as experiment 2:
2. experimental procedures
SD rats, male, weighing 180-.
Only the lung inhalation administration group is set in the experiment, and the administration dose is 1mg/kg calculated by cepharanthine.
Pulmonary inhalation dosing group: after anesthetizing the rat, the rat is fixed on an operation table, a laryngoscope and a lung liquid quantitative atomizer are used cooperatively, the cepharanthine test drug solution prepared in the experimental method part 1 of the experiment is administered in an aerosol mode through an air pipe, lung tissue samples are collected 5min, 1h, 10h and 24h after administration respectively, and the lung tissue samples are stored at the temperature of minus 20 ℃ for later use.
3. Preparation of Standard and quality control samples
Similar to experiment 2, only the blank plasma matrix was replaced with the normal rat lung tissue matrix, and the preparation process of the normal rat lung tissue matrix was as follows: taking lung tissues of blank mice, adding deionized water with the weight 3 times that of the tissues, and homogenizing to obtain the normal rat lung tissue matrix.
4. Biological sample pretreatment method
Like experiment 2, only the plasma sample to be tested was replaced with the lung tissue sample, which was prepared as follows: taking lung tissue of a rat with a lung inhalation administration group, adding deionized water with the weight 3 times that of the tissue, and homogenizing to obtain a lung tissue sample.
9. Results of the experiment
After 1mg/kg of cepharanthine is inhaled into the lungs of SD rats, the local drug concentration in the lungs is shown in Table 8, and the mean plasma drug concentration-time curve is shown in FIG. 3.
TABLE 8 pulmonary inhalation dosing group rat pulmonary topical drug concentration (μ g/g)
| Time of | Rat | 1 | Rat | 2 | Rat | 3# | Mean value of | SD |
| 5min | 19.69 | 15.54 | 15.84 | 17.02 | 2.32 | |||
| 1h | 10.85 | 12.87 | 10.59 | 11.44 | 1.25 | |||
| 10h | 7.65 | 6.51 | 6.93 | 7.03 | 0.58 | |||
| 24h | 2.39 | 2.18 | 1.83 | 2.13 | 0.28 |
The results in Table 8 show that after SD rats inhale 1mg/kg of cepharanthine in the lung, cepharanthine can be detected in the lung tissue at each time point, the local drug concentration in the lung rapidly reaches the peak after the SD rats inhale administration, and the drug concentration is (17.02 +/-2.32 mug/g) in 5min and is longer in duration. The results show that the drug can directly act on the target organ after the drug is inhaled into the lung, the local drug concentration of the lung is high, the drug can be maintained for a long time, and the drug is suitable for treating lung diseases.
Regarding the inhibitory effect of cepharanthine on coronavirus, professor perna canaliculus of the inventor is demonstrated in patent application No. 202110172158.7, specifically see experiment four and experiment five.
Experiment four, the effect of cepharanthine in treating novel pneumovirus
1. Verification of anti-novel pneumovirus effect of cepharanthine by using SARS-CoV-2 highly homologous pangolin coronavirus xCoV
The pangolin coronavirus xCoV is a coronavirus xCoV separated from pangolin scales before professor group of Mytilus edulis of an inventor (the coronavirus xCoV is preserved in China general microbiological culture Collection center (address: Beijing city No.1 Xilu No. 3 of the North Chen ward area, institute of microbiology of China academy of sciences), and the preservation number is CGMCC No.19295) in 2 months and 14 days in 2020, the genetic relationship of the coronavirus xCoV and SARS-CoV-2 is greatly superior to SARS virus, and the homology of the coronavirus xCoV and SARS-CoV-2S protein is 92.5%.
Seeded 2.5X 10 in 96-well cell plates4Vero E6 cells were infected 24 hours later with xCoV with MOI 0.01 to Vero E6 cells, to which known drugs (cepharanthine, ceratin, mefloquine hydrochloride) were added to a final concentration of 10. mu.M, cytopathic effect was observed under a microscope at day 3, RNA was extracted from cells and supernatant from culture wells with no significant cytopathic effect, and virus replication was measured in cells and supernatant by qRT-PCR.
2. Viral RNA extraction and real-time quantitative RT-PCR (qRT-PCR)
AxyPrep was used according to manufacturer's instructionsTMHumoral virus DNA/RNA miniprep kit (Axygen, product number AP-MN-BF-VNA-250) and AxyPrepTMA multipurpose total RNA micro-preparation kit (Axygene, product number AP-MN-MS-RNA-250G) collects cell culture supernatant and Vero E6 cells for RNA extraction. Reverse transcription was performed using a Hifair II 1 chain cDNA synthesis kit with gDNase (Shanghai assist san Biotech Co., Ltd., product No. 11121ES60), and qPCR was performed using a Hieff-qPCR-SYBR-Green-Master Mix (Shanghai assist san Biotech Co., Ltd., product No. 11202ES08) or a two-step Taqman probe detection qRT-PCR system (Applied-Biosystem), and sequence information of primers used is shown in Table 1. After confirmation of sequencing, the PCR product was inserted into a T-vector by bevacizco biotechnology limited, beijing, ruffikco, to generate a standard plasmid. Standard curve is determined by serial dilution of plasmid (10)3-109) The number of copies of (a). qPCR amplification by SYBR-Green method: 95 ℃ for 5min, 40 cycles, 95 ℃ for 10s, 55 ℃ for 20s, 72 ℃ for 31 s.
The Taqman method: the data in FIG. 12 were analyzed using GraphPad-Prism 8 software at 50 ℃ for 2min, 95 ℃ for 10min, 40 cycles, 95 ℃ for 10s, and 60 ℃ for 1 min.
3.EC50And CC50Detection and Time-of-Addition assay
Experiments were performed with cepharanthine, ceratin, mefloquine hydrochloride, in Vero E6 cells infected with xCoV with MOI 0.01.
EC50And (3) detection: inoculating Vero E6 cells to a 24-hole cell culture plate, and performing a test when the cell density reaches 60-80%; the drug was diluted to 200. mu.M and then diluted in a two-fold gradient to 0.39. mu.M. After the Vero E6 cells are changed, the drug solution and the virus suspension are diluted 1:1 and then added to the cells. The final concentrations of the test drugs were 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M, 1.56. mu.M, 0.78. mu.M, 0.39. mu.M, 0.195. mu.M, and 0. mu.M, respectively. 5% CO at 37 ℃2Culturing for 60-72h, observing CPE, extracting cell nucleic acid for qPCR detection, and performing data analysis by GraphPad-Prism 8 software to calculate EC50。
CC50And (3) detection: CC Using Cell-Titer-Blue method50Detection of (3). Vero E6 cells were seeded into 96-well cell culture plates and tested at cell densities of 60% -80%. The medicine is diluted twice in proportion, and the diluted medicine is added after the liquid of the Vero E6 cell is changed. The final concentrations of the test drugs were 100. mu.M, 50. mu.M, 25. mu.M, 12.5. mu.M, 6.25. mu.M, 3.125. mu.M, 1.56. mu.M, 0.78. mu.M, 0.39. mu.M, 0.195. mu.M, and 0. mu.M, respectively. 5% CO at 37 ℃2Culturing for 48h, adding 20 μ l Cell-Titer-Blue into each well, detecting 593nm luminescence intensity at 0min, 30min, 60min and 120min, respectively, and performing data analysis with GraphPad-Prism 8 software to calculate CC50。
SI is CC50Divided by EC50And (6) calculating.
Time-of-Addition detection: vero E6 cells were seeded into 24-well cell culture plates and tested at cell densities of 60% -80%. The test drug concentration was selected to be 6.25. mu.M. The experimental method of the 'full time course': adding the mixture of medicine and virus, 5% CO at 37 deg.C2Changing the liquid after culturing for 2h, and adding the medicine-virus mixed liquid; "Pre-entry" protocol: adding the mixture of medicine and virus, 5% CO at 37 deg.C2Changing the culture solution after 2h of culture, and adding a pure culture medium; "post-entry" protocol: adding pure culture medium, 5% CO at 37 deg.C2Culturing for 2 hr, changing the solution, adding medicine-virus mixtureAnd (4) liquid. 5% CO at 37 ℃2And (5) continuing to culture for 60-72h, observing CPE, extracting cell nucleic acid, carrying out qPCR detection, and carrying out data analysis by using GraphPad-Prism 8 software.
The results obtained show that no significant cytopathic effect was seen in virus-infected cell culture wells to which cepharanthine (FIG. 4), ceratin (FIG. 5) and mefloquine hydrochloride (FIG. 6) were added at a final concentration of 10. mu.M. The cepharanthine, the ceratin and the mefloquine hydrochloride are strongly suggested to be potential strong xCoV infection cell inhibitors.
Further detection by real-time quantitative PCR technique found that 10 micromoles per liter of cepharanthine, ceratin, mefloquine hydrochloride inhibited viral replication 15393-fold, 5053-fold, 31-fold, respectively, 72 hours after xCoV infection of cells at a multiplicity of 0.01, compared to a control with 0.1% DMSO alone (all compounds dissolved in DMSO, thus 0.1% DMSO concentration in each cell culture well after drug addition) (fig. 7). The results of this experiment have been repeated 5 times and all can be repeated.
EC50、CC50Results with SI showed that cepharanthine inhibited the virus dose (EC) half of the Vero E6 cells50) Is 0.21 μm, and has significant inhibitory effect in vitro experiment. Compared with the results of in vitro experiments of other anti-neocorona medicaments, the EC of the Reidcciclovir and the chloroquine in Vero E6 cell in vitro experiments can be found50Respectively 0.77 mu M and 1.13 mu M, and the EC50 of another broad-spectrum antiviral drug method Pilatavir is higher and reaches 61.88 mu M. EC (EC)50、 CC50The results with SI also show that the inhibition of xCoV by cepharanthine (figure 8), ceratin (figure 9), mefloquine hydrochloride (figure 10) appears concentration-dependent. In addition, cepharanthine (fig. 11), ceratin (fig. 12), mefloquine hydrochloride (fig. 13) all exert a virus-inhibitory effect after xCoV entered the cell.
Specifically, fig. 11 shows the Time-of-Addition test results of cepharanthine on xCoV, indicating that cepharanthine exerts an inhibitory effect after xCoV enters cells, but cannot inhibit xCoV's invasion. FIG. 12 shows the results of the Time-of-Addition test of ceratin on xCoV, indicating that ceratin exerts an inhibitory effect upon entry of xCoV into cells, but cannot inhibit the entry of xCoV. FIG. 13 shows the results of the Time-of-Addition test of mefloquine hydrochloride on xCoV, indicating that mefloquine hydrochloride exerts an inhibitory effect after xCoV enters cells, but cannot inhibit the entrance of xCoV.
Experimental five, transcriptomics analysis of cepharanthine against xCoV infection
Cepharanthine (CEP) was tested at a concentration of 6.25 μ M and Vero E6 cells were infected with xCoV with an MOI of 0.01. Four groups were set up for the experiment: vero, Vero + Virus, Vero + CEP, Vero + Virus + CEP. After 72h incubation, cell samples were collected and RNA extraction was performed using TRIzol, rRNA was removed using the QIAseq FastSelect-rRNA HMR Kit (Qiagen, product No. 334387), and NEBNext Ultra was usedTMRNA Library Prep Kit for Illumina (NEB, product No. E7770L) A mRNA sequencing Library was created and RNA sequencing (RNA-seq) was performed using an Illumina Hiseq 2500 sequencing system (Annuodda Biotechnology Co., Ltd.).
FastQC (http:// www.bioinformatics.babraham.ac.uk/projects/FastQC /) tool and FASTX _ trimmers in the FASTX toolkit were used to remove low quality data and linker sequences; mapping the trimmed RNA-seq sequence to a reference green monkey genome ChlSab1.1 (GCA _000409795.2) using HISAT2 (v2.1.0); deletion of double-ended data repeats using SAMtools (v 1.5); counting each different gene using HTseq; use of DESeq2 to identify differentially expressed genes between different experimental groups; calculating the False Discovery Rate (FDR) by adjusting the P value by using a Benjamini-Hochberg method; genes with FDR q values <0.05 and | Log2 (fold change) | >1 were considered differentially expressed genes; and (4) drawing a volcanic chart by using a ggplot2 software package of the R language.
Gct format files (including Vero vs. Vero + Virus, Vero + Virus vs. Vero + Virus + CEP) are used as the process files. The gene set includes (1) regulation of heat shock response mediated by heat shock factor 1(HSF1), regulation of cellular pyrogenicity, HSF1 dependent transactivation, HYPOXIA, defense response to viruses, HIF1 targets, adipocyte differentiation and autophagy, available from the MSigDB, KEGG and Reactome databases, (2) up/down regulation genes of viruses, which are differentially expressed genes in the RNA-seq data described above with FDR q values <0.05 and | Log2 (fold change) | > 1. The Normalized Enrichment Score (NES) value and FDR value were obtained from the genome enrichment P values calculated for 1000 permutations using Signal2Noise mode run GSEA4.0.3 (https:// www.gsea-msigdb. org/gsea/index. jsp). The visualization heatmap is drawn by the R software package of GENE-E. And a heatmap is drawn from the MSigDB, KEGG, and Reactome databases to display the selected gene set by pathway patterns.
Gene Ontology (GO) analysis was performed on the genes described above that achieved differential expression using the Metascape tool (https:// Metascape. org). Pathways with P values <0.05 were used as significantly enriched pathways, the most significantly enriched pathway being demonstrated using a bubble map created by R package ggplot2, and the Cytoscape in the Metascape website was used to map the interaction network and protein-protein interaction (PPI) network for each important enriched pathway. And each given gene list was analyzed in detail PPI enrichment using BioGrid and OmniPath.
Transcriptome sequencing analysis suggests that stephanine exerts an anti-coronavirus effect by reversing most deregulated genes and pathways in infected cells, primarily by interfering with cellular stress responses, including the endoplasmic reticulum stress/unfolded protein response and the HSF 1-mediated heat shock response.
The above experiment verifies the effect of cepharanthine by using pangolin coronavirus xCoV, and in view of the homologous relationship between xCoV and SARS-COV-2, it can be inferred that cepharanthine has the same effect on coronavirus, such as novel coronavirus 2019-nCoV. The inhalation administration mode can improve the local medicine concentration, is favorable for improving the bioavailability, and further can deduce that the cepharanthine inhalation preparation can be applied to the preparation of the medicine or the medicine for preventing and/or treating the pangolin coronavirus xCoV.
Claims (10)
1. A cepharanthine inhalation medicament is characterized by comprising a composition formed by cepharanthine and pharmaceutically acceptable auxiliary materials, wherein the composition is cepharanthine micro powder or a solution containing the cepharanthine micro powder, and the particle size of the cepharanthine micro powder is 0.1-25 mu m.
2. The cepharanthine inhalation agent of claim 1, wherein said excipients comprise disintegrants (such as sodium carbonate and citric acid) and the like.
3. The cepharanthine inhalation formulation of claim 1 or 2, wherein the cepharanthine micropowder is obtained by drying a cepharanthine microsphere or a cepharanthine nanosuspension, or by directly pulverizing cepharanthine.
4. The cepharanthine inhalation agent of claim 3, wherein the cepharanthine microspheres are prepared by dropping cepharanthine-polysorbate microemulsion (prepared from stearic acid and polysorbates in which cepharanthine is dispersed) into cold water; or
The cepharanthine nanosuspension is prepared by dissolving cepharanthine and poloxamer (F127) in ethanol, evaporating to dryness, and adding water.
5. A process for the preparation of an inhaled cepharanthine as claimed in any of claims 1 to 4, comprising the following steps:
the first method is as follows: preparing cepharanthine, stearic acid and polysorbate into cepharanthine-polysorbate microemulsion, dripping the microemulsion into cold water to form microspheres, and drying the microspheres to obtain cepharanthine micropowder (preferably spray drying method); or
The second method comprises the following steps: dissolving cepharanthine and adjuvants to obtain cepharanthine nanometer suspension, and drying to obtain cepharanthine micropowder (preferably spray drying method); specifically, the cepharanthine nanosuspension is prepared by mixing cepharanthine, poloxamer (F127) and citric acid, dissolving in ethanol, evaporating to dryness, adding sodium bicarbonate, and adding water to obtain cepharanthine nanosuspension; or
The third method comprises the following steps: pulverizing cepharanthine directly to obtain cepharanthine micropowder with particle size distribution of 0.1-25 μm; or
The method is as follows: dispersing or dissolving the cepharanthine micropowder obtained in any one of the first to third modes in a solvent to form a suspension or solution.
6. An inhalation formulation of cepharanthine comprising an inhalation formulation of cepharanthine as claimed in any of claims 1 to 4, and an inhalation device comprising a dosing system, an aerosolization system, a powder degradation system and an aerosol inlet introduction system to allow a patient to inhale the said cepharanthine inhalation formulation in the form of a dry powder or solution into the lungs.
7. The cepharanthine inhalation formulation of claim 6, wherein said inhalants comprise dry powder inhalation formulations, solution inhalation formulations, and aerosolized inhalation formulations; alternatively, the inhalation formulation comprises a single-dose type, a multi-dose type, a depot type.
8. Use of an inhaled cepharanthine formulation according to any one of claims 1 to 4 or 6 or 7 in the manufacture of a medicament for the treatment of a coronavirus.
9. Use of an inhaled pharmaceutical preparation of cepharanthine as defined in any one of claims 1 to 4 or an inhaled formulation of cepharanthine as defined in claim 6 or 7 for the manufacture of a medicament against the pangolin coronavirus xCoV or SARS-COV-2 virus or a novel coronavirus 2019-nCoV.
10. A preventive and/or therapeutic agent for coronavirus, which comprises the cepharanthine inhalation agent of any one of claims 1 to 4; preferably, the coronavirus includes, but is not limited to, pangolin coronavirus xCoV and/or SARS-COV-2 virus and/or novel coronavirus 2019-nCoV.
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