CN111000989A - Anthrax rPA dry powder inhalant and application thereof - Google Patents
Anthrax rPA dry powder inhalant and application thereof Download PDFInfo
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- CN111000989A CN111000989A CN202010004098.3A CN202010004098A CN111000989A CN 111000989 A CN111000989 A CN 111000989A CN 202010004098 A CN202010004098 A CN 202010004098A CN 111000989 A CN111000989 A CN 111000989A
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- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A61K9/0073—Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
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- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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Abstract
The invention discloses an anthrax rPA dry powder inhalant and application thereof. The anthrax rPA vaccine dry powder inhalant prepared by the invention is immunized by a lung delivery inhalation way, can enable a mouse to generate better immune reaction, generates very high IgG antibody titer, has a certain effect on the infection and the progress of bacillus anthracis, and can effectively resist the killing effect of anthrax toxin.
Description
Technical Field
The invention relates to an anthrax rPA dry powder inhalant and application thereof.
Background
Anthrax caused by Bacillus Anthracis (BA) is a potent zoonosis. The anthrax spores have strong resistance to a series of adverse environments such as physicochemical properties and the like, and are difficult to be eliminated, so that the anthrax spores can become the first-choice pathogenic microorganisms of biological warfare agents. There are two main pathogenic factors of bacillus anthracis, the capsule and the anthrax toxin. Anthrax toxin is composed mainly of Protective Antigen (PA), Lethal Factor (LF) and Edema Factor (EF). PA is the main factor of the pathogenic and immune functions of Bacillus anthracis and is one of the important protective immunogens for human vaccines. At present, most anthrax rPA vaccines are used as immunization routes in subcutaneous injection or nasal drip and other modes, the antibody titer after immunization is generally low, and the immune protection effect is not ideal.
Disclosure of Invention
The invention aims to provide an anthrax rPA dry powder inhalant and application thereof.
In a first aspect, the present invention provides a method of preparing a dry powder inhaler, comprising the steps of: (A) preparing a liquid preparation; (B) carrying out spray freeze drying treatment on the liquid preparation prepared in the step (A) to obtain the dry powder inhalant;
the liquid preparation contains the following solutes in percentage by mass: 0.1% of anthrax rPA protein, 0.05-0.15% of CpG0.5-1.5% of mannitol, 0.5-1.5% of inositol, 0.4-0.6% of leucine and 0.04-0.06% of poloxamer.
The anthrax rPA protein may be specifically a product of List Biological laboratories inc, cat # 171E.
The solvent of the liquid preparation is water.
In the embodiment of the invention, the liquid preparation specifically contains the following solutes in percentage by mass: 0.1% of anthrax rPA protein, 0.1% of CpG, 1% of mannitol, 1% of inositol, 0.5% of leucine and 0.05% of poloxamer.
In the above method, the step (B) includes the following steps (B1) to (B3):
(B1) pretreating the liquid preparation prepared in the step (A) for 1-3h at 4 ℃;
(B2) after step (B1) is completed, spraying the liquid formulation to liquid nitrogen using a two-fluid spray head; the distance between the spray head and the liquid level of the liquid nitrogen is 9-11 cm; the air pressure of the air pump is 0.1-0.2 MPa during spraying;
(B3) after completion of step (B2), the ice crystals are vacuum freeze dried along with a small amount of remaining liquid nitrogen.
In the step (B1), the pretreatment time may be 2 hours.
In the step (B2), the distance between the shower head and the liquid level of the liquid nitrogen may be 10 cm.
In the step (B2), the air pressure of the air pump during spraying may be 0.15 MPa.
In the step (B3), the freeze-drying time may be 36 hours.
In a second aspect, the invention provides a dry powder inhaler prepared by the method of the first aspect.
In a third aspect, the invention protects the use of the dry powder inhaler described in the second aspect, which is any one of the following (a1) - (a 4):
(a1) as a bacillus anthracis vaccine;
(a2) preparing a bacillus anthracis vaccine;
(a3) preparing a product for preventing and/or treating bacillus anthracis infection;
(a4) preparing a product for resisting damage of anthrax lethal toxin.
In a fourth aspect, the invention provides a bacillus anthracis vaccine, the active ingredient of which is the dry powder inhalant described in the second aspect.
The vaccine is a pulmonary delivery vaccine.
In a fifth aspect, the invention provides a product for the prophylaxis and/or treatment of a bacillus anthracis infection, the active ingredient of which is a dry powder inhaler as described in the second aspect.
In a sixth aspect, the invention provides a kit for the prevention and/or treatment of infection by bacillus anthracis comprising a dry powder inhaler as described in the second aspect and a dry powder aerosol lung delivery device.
In a seventh aspect, the present invention protects the liquid formulation described in any one of the first aspects.
In an eighth aspect, the present invention protects the use of the liquid formulation of the seventh aspect in the manufacture of a product; the product is used as any one of the following (a1) - (a 4):
(a1) as a bacillus anthracis vaccine;
(a2) preparing a bacillus anthracis vaccine;
(a3) preparing a product for preventing and/or treating bacillus anthracis infection;
(a4) preparing a product for resisting damage of anthrax lethal toxin.
The product may in particular be a dry powder inhaler.
Any of the above bacillus anthracis specifically can be a bacillus anthracis Sterne strain.
The novel anthrax rPA dry powder inhalant with good physical properties, biological activity and immunological activity is prepared, the single yield is about 160-190 mg, and the single yield is about 33%. The bioactivity of the dry powder inhalant is kept intact through formulation evaluation; the particles are in a typical porous loose ball shape and have good air compliance; the aerodynamic mass median diameter is 2.53 +/-0.32 mu m and accords with the range of the particle size section of the inhalable particles at the deep part of the lung; the water content is 8.58%, the drying degree is good, but the water absorption is fast, air and water are required to be isolated for storage, and the inoculation is fast carried out after the water absorption is started.
The dry powder inhalant has the advantages of stable performance, long-term storage, independence on cold chain transportation and the like compared with a liquid preparation, and the inhalation immunization has high efficiency, compliance and safety compared with the conventional injection inoculation, so the anthrax rPA vaccine dry powder inhalant prepared by the invention can greatly improve the rapid self-inoculation capability of large-scale crowds in normal combat through the inhalation immunization, can solve the vaccine transportation and storage problems in special environments such as field camps, extreme temperatures and the like, and is a biological control vaccine preparation with good application prospect.
The anthrax rPA vaccine dry powder inhalant prepared by the invention is immunized by a lung delivery inhalation way, can enable a mouse to generate better immune reaction, generates very high IgG antibody titer, has a certain effect on the infection and the progress of bacillus anthracis, and can effectively resist the killing effect of anthrax toxin.
Drawings
Figure 1 is a schematic diagram of a dry powder inhaler preparation process.
FIG. 2 shows the results of the bioactivity assay in example 2.
FIG. 3 shows the results of the immunogenicity test in example 2.
FIG. 4 shows the results of water content measurement in example 2.
Fig. 5 is a moisture absorption curve in example 2.
FIG. 6 shows the results of SEM observation in example 2.
FIG. 7 is a graph showing the distribution of the total particle size in example 2.
FIG. 8 is the survival curve of the mouse in example 3.
FIG. 9 shows serum IgG antibody titers of immunized BALB/c mice in example 3.
FIG. 10 shows lung homogenate IgG antibody titers of immunized BALB/c mice in example 3.
FIG. 11 shows serum IgG antibody titers of the immunized DBA/2 mice in example 3.
FIG. 12 shows lung homogenate IgG antibody titers from immunized DBA/2 mice in example 3.
FIG. 13 shows the statistics of cell viability in example 3.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Anthrax rPA protein: list Biological laboratories inc, cat #: 171E.
Anthrax lethal toxin lethal factor LF: list Biological laboratories inc, cat #: 169L.
CpG, which is called Class B CpG oligonucleotide, has immune activation function, is an immunostimulant for human or mouse TLR9, and is a typical mucosal immune adjuvant. The CpG used in the examples is Invivogen company under catalog number tlrl-2006-5, which has the following website links: https:// www.invivogen.com/odn 2006.
Anthrax Sterne strain: reference documents: chitlaru T, Israeli M, Rotem S, et al. A novel loved avian influenza Vaccine based on an acapsular, Bacillus antrhris, Sterne strain with events in the same, htrA, lef, and, cya, genes [ J ]. Vaccine,2017: S0264410X17303390; the public can be obtained from the military medical research institute of military science institute of the national people liberation military.
Mouse macrophage j774a.1: reference documents: raso G M, Meli R, Carlo G D, et al, inhibition of indole nitrile oxide synthases and cyclooxygenase-2expression by flavone in macrophage J774A.1[ J ]. Life Sciences,2001,68(8): 921-931); the public can be obtained from the military medical research institute of military science institute of the national people liberation military.
PA21 (human anti-PA neutralizing antibody IgG): reference documents: peterson JW, Comter J E, Noffsinger DM, et al, human Monoclonal Anti-diagnostic Antibody complex protectsRabbits and Is synthetic with Circuit in detecting Mice and Guineapins against Infection antibiotic [ J ]. Infection and Immunity,2006,74(2):1016 and 1024; the public can be obtained from the military medical research institute of military science institute of the national people liberation military.
Mannitol: sigma, M1902-500G.
Inositol: sigma, I7508-50G.
Leucine: sigma, L8912-25G.
Poloxamer: sigma, P5556-100 ml.
A handheld liquid aerosol lung delivery device: beijing Huilong and science and technology Co.
A handheld dry powder aerosol lung delivery device: beijing Huilong and science and technology Co.
Example 1 preparation of anthrax rPA Dry powder inhaler
First, preparation of dry powder inhalant before spraying
1. Anthrax rPA dry powder inhalant sample before spraying
The anthrax rPA dry powder inhaler pre-spray sample (pH 7.2) consists of solute and solvent; the solutes and their concentrations in the sample before spraying were: 0.1 percent (mass percentage content) of anthrax rPA protein, 0.1 percent (mass percentage content) of CpG, 1 percent (mass percentage content) of mannitol, 1 percent (mass percentage content) of inositol, 0.5 percent (mass percentage content) of leucine and 0.05 percent (mass percentage content) of poloxamer; the solvent is deionized water.
2. CpG Dry powder Pre-spray sample
CpG dry powder pre-spray sample (pH 7.2) consisted of solute and solvent; the solutes and their concentrations in the sample before spraying were: CpG 0.1% (mass percentage content), mannitol 1% (mass percentage content), inositol 1% (mass percentage content), leucine 0.5% (mass percentage content), poloxamer 0.05% (mass percentage content); the solvent is deionized water.
Preparation of dry powder inhalant
The schematic diagram of the preparation process of the dry powder inhalant is shown in figure 1.
And (3) respectively operating 20ml of the anthrax rPA dry powder inhalant sample prepared in the step one and the CpG dry powder inhalant sample before spraying according to the following steps to obtain the anthrax rPA dry powder inhalant and the CpG dry powder inhalant.
1. The sample was ice-cooled at 4 ℃ for 2h before spraying.
2. After the step 1 is completed, 20ml of the precooled sample is transferred into a 20ml syringe, a two-fluid nozzle is connected, and the sample is directly sprayed into liquid nitrogen (the liquid nitrogen is placed in a stainless steel basin, the diameter of the bottom of the stainless steel basin is about 16cm, the height of the bottom of the stainless steel basin is about 13cm, the volume of the liquid nitrogen is about 2L) (shown in figure 1A), stirring is carried out all the time in the spraying process, the distance between the nozzle and the liquid level of the liquid nitrogen is about 10cm, and the air pressure of an air pump is set to be 0.15. The sprayed atomized liquid drops are rapidly frozen into ice crystals under the action of liquid nitrogen, and the spraying time is recorded for 5 min. Transferring the ice crystals together with a small amount of residual liquid nitrogen into a stainless steel cup (FIG. 1B), sealing a gauze layer at the opening, and drying in a vacuum freeze dryer for 36h (FIG. 1C).
3. After completion of step 2, dry powder samples were collected into 5ml vials with rubber stoppers (FIG. 1D) to obtain dry powder, which was stored hermetically at-20 ℃.
The preparation is carried out for 5 times according to the steps, and the yield is calculated.
The statistical results of the yields of anthrax rPA dry powder inhalants are shown in table 1. The yield was 33.11. + -. 1.84%.
TABLE 1
Example 2 characterization of anthrax rPA Dry powder inhaler
First, biological activity detection
1. The anthrax rPA dry powder inhalant prepared in example 1 is taken and SDS-PAGE is adopted to detect the target protein. Anthrax rPA protein was used as a positive control.
The results are shown in FIG. 2. In FIG. 2, the dry powder is the detection result of anthrax rPA dry powder inhalant; the liquid is the detection result of anthrax rPA protein. The results show that the protein content of the anthrax rPA protein is consistent before and after the anthrax rPA protein is prepared into dry powder, and the anthrax rPA protein is not degraded.
2. By adopting an ELISA method and referring to the specification operation of an ELISA auxiliary kit (product number: 1030011) of Shenzhen Dake as bioengineering Limited company, anthrax rPA protein (rPA liquid) and anthrax rPA dry powder complex solution (PBS solution) are respectively adopted for coating, the antibody titer is measured, and the anthrax rPA dry powder titer is verified.
The results are shown in FIG. 3. The results show that the immunogenicity of the rPA protein after being made into dry powder is hardly affected.
Second, measuring water content and moisture absorption curve
And measuring by adopting a thermogravimetric analyzer to obtain the residual moisture content.
The moisture absorption curve measurement method is as follows:
1. the electronic balance is placed in a constant temperature and humidity box, the humidity is adjusted to 60%, and the balance is carried out at 25 ℃ (about 1 h).
2. And (3) after the step 1 is finished, taking a small plate, removing the cover of the small plate, weighing the small plate on an electronic balance, and peeling the small plate.
3. After step 2 is completed, a certain amount of the anthrax rPA dry powder inhalant prepared in example 1 is placed in a small plate, quickly placed in an electronic balance, the mass of the added anthrax rPA dry powder inhalant is recorded, the adjustment is performed to zero, and the increase in mass is recorded every 30 min. The experimental testing can be stopped when the mass no longer increases for 3 consecutive measurements.
The water content measurement results are shown in fig. 4. The moisture content of anthrax rPA dry powder inhalant is about 8.58%.
The moisture absorption curve is shown in fig. 5. The anthrax rPA dry powder inhaler mass change is shown in table 2.
TABLE 2
Thirdly, observing the particle shape and the particle size distribution
And (3) observing the dry powder particles through a scanning electron microscope, taking a plurality of visual fields, observing the particle morphology, and estimating the particle size distribution of the single particles.
The results are shown in FIG. 6 (3000 XSEM image on the left and 100 XSEM image on the right). The anthrax rPA vaccine dry powder inhalant has uniform particle size and good dispersibility.
Fourth, aerodynamic parameter detection
And (3) opening a small aerosol settlement evaluation cabin (the diameter of the bottom in the cabin is 0.76m, the height is 0.73m), and adjusting to a circulating air mode. Starting a mass concentration detector TSI8530, monitoring the mass concentration of the aerosol particles in the cabin in real time, and sampling the flow at 3L/min until the mass concentration of the aerosol particles in the cabin is reduced to 0.005mg/m3. Closing the circulating air in the cabin and opening the bottom in the cabin for placementThe small power fan. And opening a sampling port of the evaluation cabin, and generating dry powder aerosol into the cabin by using a small animal-suitable handheld biological dry powder aerosol lung delivery device, wherein the generation amount is 5 mg/time and is only 1 time. And (4) after the dry powder aerosol occurs, uniformly mixing for 30s by using a fan, and closing the fan. A laser particle size analyzer TSI3321 is used, the sampling flow is 5L/min, and the sampling time is 5 min. The test indexes comprise total particle number, total particle concentration, total particle size distribution, total particle aerodynamic number median diameter, total particle aerodynamic mass median diameter and the like of a single sampling.
The results of the measurement were as follows:
total number of particles per sample: 659125, respectively;
total particle concentration: 144/cm 3;
the total particle size distribution diagram is shown in fig. 7;
total particle aerodynamic number median diameter: 1.07 mu m;
total particle aerodynamic mass median diameter: 2.03 mu m;
the aerodynamic mass median diameter measurements are shown in table 3:
TABLE 3
Example 3 immunogenicity and immunoprotection evaluation of anthrax rPA Dry powder
First, animal immunity experiment
Experimental animals: BALB/c mice and DBA/2 mice, 6-8 weeks old, Beijing Wintolite laboratory animals Co.
Experimental animals were divided into five groups (50 animals per group) as follows;
rPA dry powder inhaler group: mice were vaccinated with a handheld dry powder aerosol lung delivery device, 0.5mg rpa dry powder inhaler/mouse, 0.3ml of air impact (using a 1ml syringe), 2 impacts consecutively. The dry powder was delivered via lung completely into the mouse lungs and the mice died without non-specificity. Performed at week 0 (primary), week 3 (secondary), and week 6 (tertiary).
PBS resuspension group of rPA dry powder inhalant: 50 μ l/stick (prepared as 0.5mg of rPA dry powder dissolved in 50 μ l of normal saline) were delivered using a handheld liquid aerosol lung delivery device. Performed at week 0 (primary), week 3 (secondary), and week 6 (tertiary).
CpG dry powder inhaler group: mice were vaccinated with 0.5mg cpg dry powder/mouse, 0.3ml of air impact (using a 1ml syringe), 2 consecutive impacts using a small animal-adapted handheld dry powder aerosol lung delivery device. The dry powder was delivered via lung completely into the mouse lungs and the mice died without non-specificity. Performed at week 0 (primary), week 3 (secondary), and week 6 (tertiary).
PBS heavy suspension group of CpG dry powder inhalant: 50 μ l/stick (prepared as 0.5mg of CpG dry powder dissolved in 50 μ l of normal saline) were delivered using a handheld liquid aerosol lung delivery device. Performed at week 0 (primary), week 3 (secondary), and week 6 (tertiary).
PBS control group: 50 μ l/PBS was delivered using a handheld liquid aerosol lung delivery device. Performed at week 0 (primary), week 3 (secondary), and week 6 (tertiary).
At 9 weeks, spores of the anthrax Sterne strain are suspended in 0.05% (mass percent) poloxamer aqueous solution and inserted into the lung through a liquid aerosol trachea for delivery; the live spore toxin-counteracting dose of BALB./c mice is 5 multiplied by 106CFU/mouse (lung delivery route about 50 × LD50), DBA/2 mice 2.5 × 104CFU/mouse (pulmonary delivery route about 5 × LD 50). Observations were recorded 14 days after challenge and the experiment was terminated at week 11.
1. Survival curve of mouse
After challenge, 10 mice were randomly selected from each group, and at time points of 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, … … 14 days after challenge, the time points were used to record death status of the mice, and a survival curve of the mice was drawn.
The results are shown in FIG. 8. In FIG. 8, the upper panel shows the results of BALB/c mouse experiment, and the lower panel shows the results of DBA/2 mouse experiment. The result shows that the death time of the mice immunized by the rPA dry powder inhalant complex solution and the rPA dry powder inhalant is delayed, and the protection force is higher than that of the CpG control group and the PBS control group. The rPA vaccine dry powder inhalant can have a certain effect on resisting infection and development of anthrax spores after a mouse is immunized by pulmonary delivery.
2. Antibody titer detection
At week 0 (2 days before primary immunization), week 3 (2 days before secondary immunization), week 6 (2 days before tertiary immunization), and week 9 (2 days before challenge), 4 surviving mice were taken per group, and 5 specific antibodies (IgG: Abcan, cat # ab 6789; IgG 1: Abcan, cat # ab 97240; IgG2 a: Abcan, cat # ab 97245; IgM: Abcan, cat # ab 97230; IgA: Abcan, cat # ab97235) were detected in serum and lung homogenate.
The serum of the sample at week 0 and week 3 was diluted from 1:200 times, and the lung homogenate was diluted from 1:50 times;
the serum samples at week 6 and week 9 were diluted from 1:1600 fold and the lung homogenate was diluted from 1:50 fold.
Serum IgG antibody titers of immunized BALB/c mice are shown in FIG. 9.
The lung homogenate IgG antibody titers of immunized BALB/c mice are shown in FIG. 10.
Serum IgG antibody titers from immunized DBA/2 mice are shown in FIG. 11.
The lung homogenate IgG antibody titers of immunized DBA/2 mice are shown in FIG. 12.
The results show that after the second immunization of BALB/c mice, IgG antibodies begin to be detected in the blood and lung of rPA liquid and rPA dry powder immunization groups, and after the third immunization, the IgG antibody titer in the blood of the two groups reaches 2 x 105. Whereas none of the CpG control groups produced the corresponding antibody.
Similarly, the DBA/2 mice began to detect IgG antibodies in both the blood and lung of the rPA liquid and rPA dry powder immunized groups after the first immunization, and the IgG antibody titer in the blood of both groups reached 4X 10 after the third immunization5None of the CpG control groups produced the corresponding antibody above.
Second, analysis of neutralizing activity of anti-anthrax toxin PA serum
1. Culturing mouse macrophage J774A.1 to logarithmic growth phase, adjusting concentration to 5 × 105one/mL. The cells were plated in 96-well cell culture plates at 100. mu.l per well to allow each well to contain cellsAbout 5X 104And culturing until the cell density reaches 70%.
2. After the step 1 is completed, taking the cell culture plate, and performing the following grouping operation;
blank group: no treatment was performed.
rPA + LF group: add 10. mu.g/ml anthrax rPA protein and 10. mu.g/ml anthrax lethal toxin lethal factor LF per well.
PA21+ rPA + LF group: each well was loaded with 4. mu.g/mL PA21 (humanized anti-PA neutralizing antibody IgG), 10. mu.g/mL anthrax rPA protein and 10. mu.g/mL anthrax lethal toxin lethal factor LF.
Antiserum group 1: mu.g/ml anthrax rPA protein, 10. mu.g/ml anthrax lethal toxin lethal factor LF and two weeks after three immunizations obtained in the above procedure were added to each well (1: 10 and 1:100, respectively).
Antiserum group 2: mu.g/ml anthrax rPA protein, 10. mu.g/ml anthrax lethal toxin lethal factor LF and two weeks after three immunizations obtained in the above procedure were added to each well (1: 10 and 1:100, respectively).
Antiserum group 3: mu.g/ml anthrax rPA protein, 10. mu.g/ml anthrax lethal toxin lethal factor LF and two weeks after three immunizations obtained in the above procedure were added to each well (1: 10 and 1:100, respectively).
Antiserum group 4: mu.g/ml anthrax rPA protein, 10. mu.g/ml anthrax lethal toxin lethal factor LF and two weeks after three immunizations obtained in the above procedure were added to each well (1: 10 and 1:100, respectively).
3. After the step 2 is finished, continuing to culture for 3h, observing cell death under a microscope, adding a CCK-8 reagent, and continuing to culture for 2h, OD450The absorbance value is measured.
The cell survival rate of the untreated group was 100%, and the cell survival rate of each group was calculated.
The calculation formula is as follows: survival (%). percent mean OD of experimental group450Value/untreated group mean OD450Value X100%.
The results are shown in FIG. 13.
The result shows that when 10 mu g/ml anthrax lethal toxin LF is added, the cell killing rate reaches 70%, the cell survival rate of the treatment by adding PA21 antibody according to 4 mu g/ml reaches more than 80%, and the results show that the cell survival rate of the antiserum No. 1-4 reaches more than 98% when adding antiserum No. 1-4 according to 1:10 and 1:100 respectively. Indicating that it is effective against the damage of anthrax lethal toxin LF.
Claims (10)
1. A method of preparing a dry powder inhaler comprising the steps of: (A) preparing a liquid preparation; (B) carrying out spray freeze drying treatment on the liquid preparation prepared in the step (A) to obtain the dry powder inhalant;
the liquid preparation contains the following solutes in percentage by mass: 0.1% of anthrax rPA protein, 0.05-0.15% of CpG, 0.5-1.5% of mannitol, 0.5-1.5% of inositol, 0.4-0.6% of leucine and 0.04-0.06% of poloxamer.
2. The method of claim 1, wherein: the liquid preparation contains the following solutes in percentage by mass: 0.1% of anthrax rPA protein, 0.1% of CpG, 1% of mannitol, 1% of inositol, 0.5% of leucine and 0.05% of poloxamer.
3. The method of claim 1 or 2, wherein:
the step (B) includes the following steps (B1) - (B3):
(B1) pretreating the liquid preparation prepared in the step (A) for 1-3h at 4 ℃;
(B2) after step (B1) is completed, spraying the liquid formulation to liquid nitrogen using a two-fluid spray head; the distance between the spray head and the liquid level of the liquid nitrogen is specifically 9-11 cm; the air pressure of the air pump is 0.1-0.2 MPa during spraying;
(B3) after completion of step (B2), the ice crystals are vacuum freeze dried along with a small amount of remaining liquid nitrogen.
4. A dry powder inhaler prepared by the process of any one of claims 1 to 3.
5. The use of the dry powder inhaler according to claim 4, which is any one of the following (a1) - (a 4):
(a1) as a bacillus anthracis vaccine;
(a2) preparing a bacillus anthracis vaccine;
(a3) preparing a product for preventing and/or treating bacillus anthracis infection;
(a4) preparing a product for resisting damage of anthrax lethal toxin.
6. A Bacillus anthracis vaccine comprising as an active ingredient the dry powder inhaler as claimed in claim 4.
7. A product for the prevention and/or treatment of Bacillus anthracis infection comprising as active ingredient the dry powder inhaler according to claim 4.
8. A kit for the prevention and/or treatment of a bacillus anthracis infection comprising the dry powder inhaler of claim 4 and a dry powder aerosol lung delivery device.
9. The liquid preparation according to claim 1 or 2.
10. Use of the liquid formulation of claim 9 in the manufacture of a product; the product is used as any one of the following (a1) - (a 4):
(a1) as a bacillus anthracis vaccine;
(a2) preparing a bacillus anthracis vaccine;
(a3) preparing a product for preventing and/or treating bacillus anthracis infection;
(a4) preparing a product for resisting damage of anthrax lethal toxin.
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| EP1972347A1 (en) * | 2007-03-19 | 2008-09-24 | Becton, Dickinson and Company, Wagner, Jaconda | Stable vaccine powder formulations |
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| EP1972347A1 (en) * | 2007-03-19 | 2008-09-24 | Becton, Dickinson and Company, Wagner, Jaconda | Stable vaccine powder formulations |
| CN108434451A (en) * | 2018-04-23 | 2018-08-24 | 中国人民解放军军事科学院军事医学研究院 | A kind of immunologic adjuvant that lung's mucosal immune response level can be improved and its application |
| CN110327314A (en) * | 2019-07-23 | 2019-10-15 | 中国人民解放军军事科学院军事医学研究院 | A kind of botulinum toxin type A AHc subunit vaccine Foradil Aerolizer formoterol fumarate of aerosolizable |
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