CN113116832B

CN113116832B - Influenza vaccine temperature-sensitive gel freeze-dried product for nasal mucosa administration

Info

Publication number: CN113116832B
Application number: CN201911419813.3A
Authority: CN
Inventors: 张庶民; 周荔葆; 马凤森; 杨文腰; 廖辉; 修雪亮; 吴铮; 刘苗苗; 徐国标; 王一平
Original assignee: Liaoning Chengda Biotechnology Co ltd
Current assignee: Liaoning Chengda Biotechnology Co ltd
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2023-09-08
Anticipated expiration: 2039-12-31
Also published as: CN113116832A

Abstract

The invention discloses a freeze-dried influenza vaccine Wen Minning gel product which comprises an influenza vaccine and a freeze-drying protective agent, wherein the freeze-drying protective agent is a combination of sucrose and mannitol. The influenza vaccine Wen Minning jelly freeze-dried product prepared by selecting the freeze-dried protective agent with specific types and contents and screening different solvents has reversible thermal gelation, and can ensure the medicine stability of the vaccine in long-term storage.

Description

Influenza vaccine temperature-sensitive gel freeze-dried product for nasal mucosa administration

Technical Field

The invention relates to the technical field of administration of influenza vaccines, in particular to a nasal mucosa administration influenza vaccine Wen Minning jelly freeze-dried product.

Background

Currently, influenza vaccines are approved for sale as injectables, mainly inducing a strong systemic immune response. However, most pathogens infect their hosts preferentially through mucosal surfaces of the gastrointestinal tract, respiratory tract, genitourinary tract, or eye, and conventional systemic vaccine delivery to infectious diseases using injection methods cannot induce a strong mucosal immune response. The vaccine delivered through the nasal mucosa can induce not only systemic IgG antibody response but also mucosal IgA antibody response. The nasal mucosa provides a large surface area (about 150-160cm 2) due to the presence of about 400 microvilli per cell, the large surface area of the nasal epithelium allowing for efficient absorption through the nasal mucosa. And gastrointestinal tract damage can be avoided when drugs and vaccines are administered nasally. In addition, the nasal mucosa has a relatively low enzymatic activity, protects the vaccine from enzymatic degradation in the nasal cavity, and allows smaller doses. The antigen dose delivered through the nasal cavity can be reduced by a factor of 4 compared to oral vaccination. The nasal cavity has a high density of dendritic cells that can mediate strong systemic and mucosal immune responses against antigens and pathogens that invade the body through the upper respiratory tract. It is worth mentioning that nasal mucosa contains M cells compared with oral mucosa, periocular mucosa and the like, and is more favorable for capturing and presenting antigen.

However, the absorption amount of drugs by the nasal mucosa is limited, and how to improve the bioavailability of the administration of drugs by the nasal mucosa is a technical problem which must be solved when researching the administration of vaccines by the nasal mucosa. Among the methods for improving the bioavailability of drugs in nasal mucosa, changing the dosage form of the drug to improve the drug adhesion and increase the residence time of the drug on the mucosa is a relatively simple and effective method. The invention provides a gel based on a common dissolving agent to increase the viscosity of influenza vaccine mucous membrane. Since the temperature-sensitive gel is liquid below the phase transition temperature, it is gel above the phase transition temperature. The administration is liquid, and gel on mucous membrane can enhance antigen exposure and contact with mucous membrane tissue. The invention provides a temperature-sensitive gel formulation for convenient administration while improving viscosity.

The temperature-sensitive gel is a water agent (liquid type) at normal temperature, and the water agent has the defects of difficult preservation and easy activity reduction, so that the development limit of the temperature-sensitive gel on application is quite large.

From the above, how to provide an influenza vaccine temperature-sensitive gel with good stability and high activity is a key point for developing a nasal mucosa delivery influenza vaccine preparation.

Usually, auxiliary materials such as a stabilizer, a preservative, a surfactant, an antioxidant and the like are added to solve the defects of poor stability and low activity of the temperature-sensitive gel, and the technical proposal has the defects that: (1) because the finished product is prepared into the dosage form of water aqua, the influence of the water aqua on the activity of the medicine is not fundamentally solved; (2) the addition of preservatives and antioxidants is prone to causing patient recalcitrance in patients administered with influenza vaccine activity.

The Chinese patent publication No. CN 1164185A discloses a freeze-dried preparation of an aqueous pharmaceutical composition capable of providing reversible thermal gelation, which is prepared by freeze-drying an aqueous composition with reversible thermal gelation properties, wherein the freeze-dried preparation consists of an effective amount of a medicament, methylcellulose, citric acid and polyethylene glycol. The technical scheme has the following defects: (1) the provided freeze-dried preparation object is a small molecular chemical drug, whether large molecular biological drugs of influenza vaccines can be used or not is determined, and the measured biological activity is uncertain; (2) the uncertainty exists on whether the adjuvant related in the literature can still have reversible thermal gelation in vaccine medicines; (3) the freeze-dried product after long-term storage has uncertainty as to whether the product can be reversibly thermally gelled or not;

it is well known that vaccine formulations are prepared differently from chemical formulations and that additives such as adjuvants, stabilizers and the like must be added. The components and the like in the additives can influence the hydrophilicity and hydrophobicity of the thermosensitive polymer, and further influence the thermosensitive property such as the temperature of the thermosensitive gel phase transition temperature. (Zhu Ya. Study of the effect of chain flexibility on the performance of thermosensitive polymers and hydrogels [ D ] 2015.). The applicant can know through preliminary experiments that the addition of the adjuvant and the vaccine stabilizer in the influenza vaccine can have a great influence on the temperature-sensitive property of the temperature-sensitive gel.

In summary, how to apply the influenza vaccine Wen Minning jelly freeze-dried product for nasal mucosa administration has the advantages of reversible thermal gelation and capability of ensuring the drug stability of the vaccine in long-term storage, and is a technical problem which needs to be solved by the people in the field.

Disclosure of Invention

The invention aims to solve the technical problems of providing a freeze-dried influenza vaccine Wen Minning gel, which solves the defects in the prior art by selecting proper freeze-drying protective agent types and contents, proper solvent for re-dissolution and the like.

For this purpose, the invention adopts the following technical scheme:

an influenza vaccine Wen Minning jelly freeze-dried product comprises an influenza vaccine and a freeze-drying protective agent, wherein the freeze-drying protective agent is a combination of sucrose and mannitol.

Preferably, the influenza vaccine Wen Minning jelly freeze-dried product consists of influenza vaccine and Wen Minning jelly solids; wherein, wen Minning gel solids comprise a freeze-drying protective agent, a gel material, a gel additive and a phosphate buffer matrix, and the mass ratio of the freeze-drying protective agent to the Wen Minning gel solids is 12 percent.

Preferably, the temperature-sensitive gel solid is reconstituted by a solvent, the solvent comprising a vaccine adjuvant and a solvent, wherein the vaccine adjuvant is a combination of polyinosinic and zymosan.

Preferably, the content ratio of polyinosinic to zymosan in the vaccine adjuvant is 1:2.

Preferably, the content ratio of the freeze-dried powder to the solvent is 2:3.

In summary, compared with the prior art, the invention has the beneficial effects that:

the influenza vaccine Wen Minning jelly freeze-dried product prepared by selecting the freeze-dried protective agent with specific types and contents and screening different solvents has reversible thermal gelation, and can ensure the medicine stability of the vaccine in long-term storage.

Detailed Description

The following are specific examples of the present invention, and the technical solutions of the present invention are further described, but the scope of the present invention is not limited to these examples. All changes and equivalents that do not depart from the gist of the invention are intended to be within the scope of the invention.

Example 1 preparation of influenza vaccine Wen Minning jelly lyophilized product and vehicle

The influenza vaccine Wen Minning jelly freeze-dried product to be prepared by the invention consists of influenza vaccine and Wen Minning jelly solids, wherein the component content of the temperature-sensitive jelly solids is shown in table 1:

TABLE 1 influenza vaccine temperature sensitive gel solids content

Gel material (wt%)	Gel additive (wt%)	Freeze-drying protective agent (wt%)	Phosphate buffered matrix (wt%)
				80	2.6	Pending	Pending

The solvent to be prepared by the invention consists of a vaccine adjuvant and a solvent.

Preparation of influenza vaccine temperature-sensitive gel: dissolving the CS and the freeze-drying protective agent in a prescription amount in a proper amount of PBS, slowly adding the P407 and the P188 in the prescription amount under magnetic stirring at 4 ℃, swelling for 12-48 hours at 4 ℃, taking out, and adding the PBS for volume fixing. Slowly adding one third of the gel volume of the influenza vaccine stock solution into the prepared temperature-sensitive gel solution under magnetic stirring at the temperature of 4 ℃.

Preparation of influenza vaccine Wen Minning jelly freeze-dried product: the sample is filled in penicillin bottles with the specification of 1mL per bottle. After half-tamponading, the mixture is transferred to a freeze dryer for freeze drying. Preserving the pre-freezing temperature at-50 ℃ for 3 hours, then rapidly heating to-25 ℃, preserving the heat for 42 hours to obtain white powder, and placing the white powder into a refrigerator at 4 ℃ for standby.

The preparation method of the solvent comprises the following steps: accurately weighing the prescribed amount of adjuvant (polyinosinic acid and zymosan) and dissolving in PBS (pH=7.2) to obtain a solution containing 0.3wt% of adjuvant as a solvent.

Example 2 Effect of lyoprotectant species on influenza vaccine temperature sensitive gel

The mass ratio of the lyoprotectant in the preparation is set to be a certain value, and the performance influence of different lyoprotectants on the temperature-sensitive gel of the influenza vaccine is explored only by changing the types of the lyoprotectant.

The freeze-drying protective agent is screened by measuring the hemagglutinin content before and after freeze-drying, and the screening method is as follows: standard antigen was diluted by 0.9% sterile sodium chloride solution in prime (4/4), 3/4, 2/4, 1/4, one well for each dilution, 1 well for each dilution of vaccine sample, 12 μl per well, placed in a horizontal wet box and diffused at room temperature for at least 18 h. After sample addition, the gel plate was immersed in a 0.9% sterile sodium chloride solution for 60min, the gel was removed, placed on filter paper under horizontal conditions, and pressed with a weight. And (3) placing the thinned gel in an electric oven at 50-60 ℃ for baking for 5-10 min until the gel is completely dried, wetting with purified water, and slightly removing from the filter paper. And immersing the gel in 2% coomassie brilliant blue staining solution for staining for 3-5 min. After staining, the gel was immersed in a destaining solution until the gel background was clear. The diameter of the precipitation ring was measured with a caliper, and each ring was measured twice in the transverse and vertical directions, and the gel antigen content was calculated.

A satisfactory is considered when the hemagglutinin content after lyophilization is not less than 80% of the pre-lyophilization content.

The average results of the measurements are shown in Table 2.

TABLE 2 influence of the type of lyoprotectant on temperature sensitive gel

As can be seen from table 2: (1) the group of sucrose and mannitol and the group of sucrose and propylene glycol meet the requirements of hemagglutinin content after freeze-drying; (2) in the grouping meeting the requirements, the content of hemagglutinin is higher after the freeze-drying of the sucrose and mannitol group, namely the freeze-dried powder has better protection effect.

Thus, a lyoprotectant combination of sucrose + mannitol was selected.

Example 3 Effect of lyoprotectant content in gel solids on temperature sensitive gel of influenza vaccine

Based on the embodiment 2, only the dosage of the lyoprotectant is changed to explore the performance influence of different dosages of the lyoprotectant on the influenza vaccine temperature-sensitive gel.

The gel viscosity of the temperature-sensitive gel is measured by a viscometer, and the measurement method is as follows:

the prepared gel solution is placed in a beaker with the diameter not smaller than 70mm, and is subjected to constant-temperature water bath at 25 ℃ for 0.5h. And (3) measuring the viscosity of the sample by using an NDJ-1 viscometer, wherein the absolute viscosity of the solution is obtained by multiplying the reading indicated by a pointer on a dial plate by a specific coefficient on a coefficient table during measurement. Each group was assayed 3 times in parallel and the results averaged.

When the viscosity is between 200 and 300 mPas, the viscosity is considered satisfactory.

The temperature-sensitive gel phase transition temperature is measured by adopting a stirrer method, and the testing method comprises the following steps:

pouring 10g of prepared temperature-sensitive gel solution into a penicillin bottle, putting a magnetic stirrer into the penicillin bottle, inserting a precision thermometer with the precision of 0.1 ℃, completely immersing a water silver ball of the thermometer into the gel solution, putting the gel solution into a normal-temperature (< 25 ℃) water bath at the rotating speed of 300r/min, keeping the water temperature continuously and slowly rising, and heating at the speed of about 0.5 ℃/min. Finally, the temperature at which the stirrer stopped rotating was recorded, and the measured temperature was the gel temperature (LCST). Each sample was assayed 3 times in parallel and the results averaged.

When the phase transition temperature is between 33 ℃ and 34 ℃, the phase transition temperature is the same as the nasal cavity temperature, and is regarded as meeting the requirements.

The average results of the measurements are shown in Table 3.

TABLE 3 influence of the content of lyoprotectant in gel solids on temperature sensitive gel

Dose of lyoprotectant (wt%)	Gel viscosity (mPa. S)	Phase transition temperature (. Degree. C.)
			8	135	32.8
10	150	33.1
			12	205	33.5
14	265	34.5

As can be seen from table 3: (1) when the dosage of the freeze-drying protective agent is more than 10%, the gel viscosity meets the requirement; (2) when the dose of the lyoprotectant is less than 14%, the phase transition temperature meets the requirement.

Therefore, only when the amount of the lyoprotectant is 12%, both the gel viscosity and the phase transition temperature are satisfactory.

Example 4 Effect of different vehicles on influenza vaccine temperature sensitive gel upon reconstitution

Based on the embodiment 3, the type and the dosage of the freeze-drying protective agent are set to be a certain value, and the performance influence of the adjuvant and the solvent with different contents on the temperature-sensitive gel of the influenza vaccine is investigated by only changing the content ratio of the adjuvant and the solvent.

The solvent preparation method is as follows: accurately weighing polyinosinic and zymosan with prescription amount, dissolving in PBS solution (pH=7.2) to obtain solution containing 0.3wt% adjuvant as solvent.

The method for testing the viscosity of the temperature-sensitive gel is the same as in example 3; the method for testing the temperature-sensitive gel phase transition temperature is the same as in example 3.

The activity of influenza vaccines was evaluated by measuring antibody titers in the serum of immunized mice as follows:

SPF-class BALB/c mice (females, 6-8 weeks old, body weight 14-16 g) were randomly grouped, 5 in each group. After anesthetizing the mice, experiments were performed at a dose of 20 μl of gel vaccine containing 2 μg of influenza virus. The injection group was injected with the same dose of influenza virus. Immunization was performed once at 0d and 14d, respectively, and twice in total. At 14d after the last immunization, blood was collected from the tail vein of the mice, serum was separated, and antibody titer was measured by using a hemagglutination inhibition method.

An antibody titer >4 was considered positive, as satisfactory.

The average results of the measurements are shown in Table 4.

TABLE 4 influence of different vehicles on influenza vaccine temperature-sensitive gel during reconstitution

As can be seen from table 4: (1) the higher the yeast polysaccharide content in the adjuvant, the higher the gel viscosity and the higher the phase transition temperature; (2) the smaller the solvent dosage during the reconstitution, the higher the gel viscosity and the higher the phase transition temperature; (3) antibody titers were positive only when the ratio of polyinosinic to yeast polysaccharide was 1:2.

In summary, when the ratio of polyinosinic to zymosan is 1:2 and the ratio of lyophilized powder to vehicle is 2:3, both the gel property and the antibody titer meet the requirements.

Example 5 influenza vaccine with different storage modes

Acceleration stabilization experiment: the temperature is 25+/-2 ℃ and the relative humidity is 60+/-10 percent, and the hemagglutinin content of each of 2 influenza-loaded vaccines is respectively measured by sampling and storing for 0 month, 1 month, 2 months, 3 months and 6 months.

The method for measuring the hemagglutinin content was the same as in example 2.

The average results of the measurements are shown in Table 5.

TABLE 5 influenza vaccine potency assay for different storage modes

As can be seen from table 5: after the gel group influenza vaccine and the freeze-dried group influenza vaccine are stored for 6 months under the same environmental condition, the titer of the gel group influenza vaccine is reduced far more than that of the freeze-dried group.

Thus, lyophilization preservation is beneficial in reducing influenza vaccine potency decline.

Example 6 gel Property Change under Long term storage

Acceleration stabilization experiment: the gel was reconstituted in the same manner by sampling for 0 month, 1 month, 2 months, 3 months, and 6 months at 25 ℃ + -2deg.C and relative humidity 60% + -10%, respectively, and the gel properties were measured.

The method for testing the viscosity of the temperature-sensitive gel is the same as in example 3.

The method for testing the temperature-sensitive gel phase transition temperature is the same as in example 3.

The average results of the measurements are shown in Table 6.

TABLE 6 gel property change over time storage

As can be seen from table 6, the temperature-sensitive gel phase transition temperature and gel viscosity formed after the Wen Minning gel lyophilized powder is redissolved still meet the requirement of long-term storage. Therefore, the Wen Minning gel freeze-dried powder has good stability under long-term storage.

Claims

1. The influenza vaccine Wen Minning jelly freeze-dried product is characterized by comprising influenza vaccine and Wen Minning jelly solids, wherein the temperature-sensitive jelly solids comprise a freeze-drying protective agent and a vaccine adjuvant, the mass ratio of the freeze-drying protective agent in the Wen Minning jelly solids is 12%, and the freeze-drying protective agent is the combination of sucrose and mannitol; the vaccine adjuvant is the combination of polyinosinic cell and zymosan, and the content ratio of the polyinosinic cell to the zymosan is 1:2.

2. The influenza vaccine Wen Minning jelly freeze-dried product of claim 1, wherein the temperature-sensitive gel solid further comprises a gel material and a gel additive.

3. The influenza vaccine Wen Minning jelly freeze-dried product according to claim 1, wherein the temperature-sensitive gel solid is reconstituted by a vehicle comprising a vaccine adjuvant and a solvent.

4. The influenza vaccine Wen Minning jelly dried product according to claim 3, wherein the ratio of the influenza vaccine Wen Minning jelly dried product to the vehicle is 2:3.

5. The influenza vaccine Wen Minning jelly freeze-dried product according to claim 2, wherein the gel material comprises 80% of Wen Minning jelly solids by mass.

6. The influenza vaccine Wen Minning jelly freeze-dried product according to claim 2 or 5, wherein the gel additive comprises 2.6% of Wen Minning jelly solids by mass.