Atomization preparation based on canine parvovirus specific nano antibody

Abstract

The invention relates to the technical field of medicines, and particularly discloses an atomized preparation based on a canine parvovirus specific nano-antibody, which comprises a nano-antibody which is screened from an alpaca source immune antibody library and is specifically combined with canine parvovirus VP2 protein; a mucous membrane penetration enhancer: 0.1-0.5% (w / v) of a chitosan-citric acid derivative; the protease inhibitor compound is a composition of 0.05% (w / v) aprotinin and 0.02% (w / v) alpha-antitrypsin; the freeze-drying protection matrix is a glassy state forming agent composed of trehalose (5-8% w / v) and cane sugar (2-4% w / v) according to the proportion of 3: 1-1: 1; a targeting activator: 1-3 mM of intestinal alkaline phosphatase response type phosphate bond masking peptide; through the design of a delivery system, targeted coverage is realized by precisely regulating and controlling particle size distribution, so that 1-3-micron-grade particles are efficiently deposited in deep respiratory tract and intestinal lesion areas, and 5-8-micron-grade particles form a slow release library in oropharynx, so that the drug residence time is remarkably prolonged.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to an aerosol preparation based on canine parvovirus-specific nanoantibodies. Background Art

[0002] At present, the prevention and treatment of canine parvovirus mainly relies on two major strategies: antibody therapy and vaccine prevention. Among antibody therapy, hyperimmune serum and monoclonal antibodies are the core treatment methods:

[0003] Hyperimmune serum preparations are made by immunizing healthy dogs to obtain hyperimmune plasma, and immunoglobulins are extracted through steps such as step-by-step salting out with saturated ammonium sulfate, desalting and purification. They are mainly used for subcutaneous or intramuscular injection to neutralize viruses through passive immunization; monoclonal antibodies are produced on a large scale using hybridoma technology or bioreactors; vaccine technologies include inactivated vaccines, live attenuated vaccines and virus-like particle vaccines; in addition, combination therapy is gradually becoming a trend.

[0004] The existing technology still has the following key defects:

[0005] The Fc fragment of mouse-derived monoclonal antibodies contains heterologous components, and repeated use can easily induce dogs to produce anti-mouse antibodies, resulting in decreased efficacy or allergic reactions; live attenuated vaccines have the risk of reversion to virulence, while inactivated vaccines require multiple booster immunizations and lack cross-protection against mutant strains; antibodies have a large molecular weight and are difficult to penetrate the intestinal mucosal barrier and intracellular infection targets, resulting in low virus neutralization efficiency; antibodies are prone to aggregation and inactivation during traditional nebulization administration, and their affinity is reduced. Summary of the Invention

[0006] The object of the present invention is to provide an aerosolized preparation based on canine parvovirus-specific nanobodies to solve the problems raised in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The aerosolized formulation based on canine parvovirus-specific nanoantibodies contains the following components:

[0009] Nanobodies that specifically bind to canine parvovirus VP2 protein, screened from an alpaca-derived immune antibody library;

[0010] Mucosal penetration enhancer: 0.1-0.5% (w / v) chitosan-citric acid derivative;

[0011] Protease inhibitor complex: a combination of 0.05% (w / v) aprotinin and 0.02% (w / v) α-antitrypsin;

[0012] Lyophilized protective matrix: a glass former composed of trehalose (5-8% w / v) and sucrose (2-4% w / v) in a ratio of 3:1 to 1:1;

[0013] Targeted activator: 1-3 mM intestinal alkaline phosphatase-responsive phosphate-masked peptide.

[0014] Preferably, the nanobody is obtained by the following screening method:

[0015] (i) using a conformationally locked VP2 trimer as an antigen;

[0016] (ii) Using microfluidic single B cell sorting technology to bind with a dissociation constant ≤ 10 -9 Positive clone enrichment strategy for M.

[0017] Preferably, the freeze-dried protective matrix adopts a gradient annealing process during the freeze-drying process:

[0018] Pre-freezing stage: -40℃ for 2 hours;

[0019] Primary drying: heating to -25°C at 0.5°C / min, maintaining vacuum ≤20Pa;

[0020] Secondary drying: Raise the temperature to 30°C at 0.3°C / min and maintain for 12 hours.

[0021] Preferably, the aerosol particles generated by atomization after reconstitution of the preparation have a bimodal particle size distribution:

[0022] Main peak: 1.0-3.0 μm (accounting for ≥65%) for deep respiratory tract deposition

[0023] Secondary peak: 5.0-8.0 μm (accounting for 15-25%) is used for oropharyngeal sustained-release delivery.

[0024] Preferably, the targeted activator is specifically dephosphorylated under the action of intestinal alkaline phosphatase, exposing the cell-penetrating peptide domain at the C-terminus.

[0025] An atomization drug delivery device for any of the above-mentioned preparations, characterized by comprising:

[0026] (a) Vibrating mesh atomizer, mesh diameter 3.0 ± 0.2 μm;

[0027] (b) Intelligent temperature control system to maintain the liquid temperature at 15±2℃;

[0028] (c) Respiratory synchronization trigger module, which starts the nebulization pulse 200ms before the inspiratory phase.

[0029] A method for treating canine parvoviral enteritis, comprising:

[0030] The above lyophilized powder was reconstituted with 2 ml of sterile citric acid buffer (pH 5.8) and administered by atomization through the above device twice a day, each time delivering an aerosol containing 50-80 μg of nanoantibodies, and the treatment cycle was 3-5 days.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) Through delivery system design and precise control of particle size distribution, targeted coverage is achieved, so that 1-3 μm particles are efficiently deposited in deep respiratory tract and intestinal lesions, while 5-8 μm particles form a sustained-release reservoir in the oropharynx, significantly extending the drug residence time.

[0033] (2) Through the synergistic effect of enzyme-responsive activators and mucus penetration enhancers, the intestinal mucus barrier and intracellular delivery obstacles are overcome, allowing the nanoantibodies to accurately reach the virus-infected target cells and exert a highly effective neutralizing effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a process flow chart of the present invention. DETAILED DESCRIPTION

[0035] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, 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 efforts are within the scope of protection of the present invention.

[0036] Example 1: Nanobody Screening and Preparation

[0037] See also Figure 1 As shown, the preparation method of the aerosol preparation based on canine parvovirus-specific nanoantibodies comprises:

[0038] Step 1: Antigen Engineering

[0039] Alanine at position 300 of the canine parvovirus VP2 protein was mutated to proline, and valine at position 307 to tyrosine by site-directed mutagenesis. The modified gene was cloned into the pFastBac vector, and the trimer protein was expressed in Sf9 insect cells. After purification by nickel affinity chromatography, size exclusion chromatography showed that the trimer had a purity of 98.7%, and dynamic light scattering determined the hydrated particle size to be 28.3±1.2 nm.

[0040] Step 2: Nanobody Screening

[0041] After immunization of alpacas, peripheral blood lymphocytes were extracted and a phage display library (3.5×10^9 capacity) was constructed. B cells that bind to VP2 trimers were screened using a microfluidic single-cell sorting platform (Fluidigm C1), and a high-affinity clone, Nb-VP2-7D3, was obtained. Surface plasmon resonance analysis showed that the dissociation constant of the antibody for VP2 binding was 0.78 nM, and the apparent binding rate was 3.2×10^5 M -1 s -1 Virus neutralization test confirmed that the concentration that inhibited 50% of virus plaque formation was 12.3ng / mL;

[0042] Step 3: Preparation of lyophilized preparation

[0043] The solution was prepared according to the following formula: nanobody: 1.5 mg / mL; mucosal penetration enhancer: 0.3% (w / v) chitosan-citric acid derivative (deacetylation ≥ 90%); protease inhibitor complex: 0.05% (w / v) aprotinin + 0.02% (w / v) α-antitrypsin; lyoprotectant: 6% (w / v) trehalose + 3% (w / v) sucrose; targeting activator: 2 mM phosphorylated transmembrane peptide;

[0044] Perform gradient annealing and lyophilization:

[0045] Pre-freezing stage: maintain at -40℃ for 2 hours to form ice crystal skeleton;

[0046] Primary drying: heating at 0.5°C / min to -25°C, maintaining vacuum at 15Pa for 36 hours;

[0047] Secondary drying: heating to 30°C at 0.3°C / min and maintaining vacuum at 5 Pa for 12 hours.

[0048] Example 2: Characterization of atomization device performance and aerosol characteristics

[0049] The device configuration includes: piezoelectric vibrating screen (nickel-titanium alloy, pore size 3.0±0.2μm), semiconductor Peltier module, PID algorithm to maintain 15±0.5℃, differential pressure sensor to detect dog's inspiratory flow, trigger delay 185±15ms;

[0050] Aerosol characteristics test:

[0051] The lyophilized powder prepared in Example 1 was reconstituted with 2 mL of citric acid buffer (pH 5.8), and 1.5 mL was added to the nebulizer chamber. The nebulization time was set to 5 min, the airflow mode was set to a tidal volume of 300 mL, a frequency of 20 times / min, and the temperature control was set to 15±0.5°C. The test results are shown in the following table:

[0052]

[0053]

[0054] From the above, we can see that the particle size distribution has a significant bimodal feature.

[0055] Main peak: 1.0-3.0 μm (accounting for 68.4±2.3%), targeting the deep respiratory tract;

[0056] Secondary peak: 5.0-8.0 μm (accounting for 21.7±1.8%), covering the oropharynx.

[0057] Example 3: Experimental treatment of viral enteritis in beagle dogs

[0058] Twenty-four CPV antigen-positive beagle dogs aged 6-8 weeks (ELISA titer ≥1:320) were randomly divided into groups (n=6 / group): Group A received the formulation of Example 1 + the device of Example 2; Group B received intravenous injection of the same dose (60 μg) of the Nanobody; Group C received a blank nebulized formulation (without antibody); Group D received the commercial monoclonal antibody palivizumab (2 mg / kg intravenous injection);

[0059] The lyophilized powder was reconstituted with 2 mL of sterile citric acid buffer (pH 5.8) and loaded into the nebulizer. A single dose of 60 μg of the nanobody was administered twice daily for a 4-day treatment cycle. The device automatically triggered a 200 ms nebulizer pulse when it detected an inspiratory flow rate > 0.3 L / s. The efficacy evaluation (72-hour treatment data) is shown in the following table:

[0060] index Group A Group B Group D Fecal viral load (log) 3.1±0.4**▲ 4.8±0.3 5.7±0.5 Intestinal pathology score 1.2±0.3**▲▲ 2.9±0.4 3.8±0.6 Intestinal mucosal antibody concentration (μg / g) 28.7±3.2**▲ 6.2±0.9 N / A

[0061] ▲ indicates p < 0.01 compared with group B; pathological scoring standard: 0 = normal, 5 = extensive necrosis;

[0062] As can be seen from the above, immunofluorescence sections showed that the nanoantibodies in group A (labeled with red Cy5) penetrated the intestinal epithelial cells, with an intracellular distribution density of 18.3±2.7 particles / cell (group B was only attached to the mucosal surface), and the targeted activator had a dephosphorylation efficiency of 89.2±4.1% under the action of intestinal alkaline phosphatase.

[0063] Example 4: Visualization of mucus penetration

[0064] Step 1: Collect intestinal mucus from dogs (obtained during surgery) and divide them into groups for treatment: Group A: mucus + PBS (control); Group B: mucus + 0.3% chitosan-citric acid; Group C: mucus + all ingredients of this preparation

[0065] Step 2: The samples were fixed with 2.5% glutaraldehyde at 4°C overnight, dehydrated with an ethanol gradient (30%-100%), critical point dried, and gold coated. The samples were observed using a Hitachi HT7800 electron microscope (accelerating voltage 80 kV). The results are shown in the following table:

[0066] Group Average pore diameter (nm) <![CDATA[Pore density (number / μm 2 )]]> Mucus layer thickness (μm) Group A (PBS) 52.3±6.7 8.2±1.1 42.5±3.8 Group B 138.6±15.2**▲ 19.7±2.3**▲ 38.1±3.2 Group C 273.5±28.4**▲▲ 42.8±4.6**▲▲ 35.7±2.9

[0067] ▲: p < 0.01 vs group A; ▲▲: p < 0.01 vs group B;

[0068] From the above, we can see that electron microscopy shows that chitosan-citric acid increases the mucus pore size by 5.2 times and the pore density by 4.2 times.

[0069] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. An aerosolized preparation based on canine parvovirus-specific nanoantibodies, characterized in that: Contains the following components: Nanobodies that specifically bind to canine parvovirus VP2 protein, screened from an alpaca-derived immune antibody library; Mucosal penetration enhancer: 0.1-0.5% (w / v) chitosan-citric acid derivative; Protease inhibitor complex: a combination of 0.05% (w / v) aprotinin and 0.02% (w / v) α-antitrypsin; Lyophilized protective matrix: a glass former composed of trehalose (5-8% w / v) and sucrose (2-4% w / v) in a ratio of 3:1 to 1:1; Targeted activator: 1-3 mM intestinal alkaline phosphatase-responsive phosphate-masked peptide.

2. The aerosolized preparation based on canine parvovirus-specific nanoantibodies according to claim 1, characterized in that: The nanobody is obtained by the following screening method: (i) using a conformationally locked VP2 trimer as an antigen; (ii) Using microfluidic single B cell sorting technology to bind with a dissociation constant ≤ 10 -9 Positive clone enrichment strategy for M.

3. The aerosolized preparation based on canine parvovirus-specific nanoantibodies according to claim 1, characterized in that The freeze-dried protective matrix adopts a gradient annealing process during the freeze-drying process: Pre-freezing stage: -40℃ for 2 hours; Primary drying: heating to -25°C at 0.5°C / min, maintaining vacuum ≤20Pa; Secondary drying: Raise the temperature to 30°C at 0.3°C / min and maintain for 12 hours.

4. The aerosolized preparation based on canine parvovirus-specific nanoantibodies according to claim 1, characterized in that The aerosol particles generated by atomization after reconstitution of the preparation have a bimodal particle size distribution: Main peak: 1.0-3.0 μm (accounting for ≥65%) for deep respiratory tract deposition Secondary peak: 5.0-8.0 μm (accounting for 15-25%) is used for oropharyngeal sustained-release delivery.

5. The aerosolized preparation based on canine parvovirus-specific nanoantibodies according to claim 1, characterized in that The targeted activator is specifically dephosphorylated under the action of intestinal alkaline phosphatase, exposing the cell-penetrating peptide domain at the C-terminus.

6. A nebulized drug delivery device for any one of claims 1 to 5, characterized in that: Include: (a) Vibrating mesh atomizer, mesh diameter 3.0 ± 0.2 μm; (b) Intelligent temperature control system to maintain the liquid temperature at 15±2℃; (c) Respiratory synchronization trigger module, which starts the nebulization pulse 200ms before the inspiratory phase.

7. A method for treating canine parvovirus enteritis, characterized in that include: The lyophilized powder according to claim 1 is reconstituted with 2 ml of sterile citric acid buffer (pH 5.8) and administered by atomization through the device according to claim 6, twice a day, each time delivering an aerosol containing 50-80 μg of nanoantibodies, and the treatment cycle is 3-5 days.

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