阅读说明：本技术 一种含螨变应原的舌下温敏凝胶及其制备方法 (Sublingual temperature-sensitive gel containing mite allergen and preparation method thereof ) 是由 不公告发明人 于 2020-04-30 设计创作，主要内容包括：本发明公开了一种含螨变应原的舌下温敏凝胶,由螨变应原、阳离子单体和温敏凝胶材料组成,其中阳离子单体和温敏凝胶材料的含量比为(1:2)～(1:1)。本发明提供的含螨变应原的舌下温敏凝胶,在普通含螨变应原舌下滴剂基础上提出舌下温敏凝胶剂,延续了舌下滴剂给药方便优点的同时,延长了疫苗在舌下的滞留时间,提高药效；缩短了温敏凝胶的胶凝时间,降低给药过程中吞咽造成的损失,减少了给药量；克服温敏凝胶吸附蛋白质的温敏性,提高凝胶在液体状态时对蛋白质的吸附,进而减少给药量。(The invention discloses a sublingual temperature-sensitive gel containing a mite allergen, which consists of the mite allergen, a cationic monomer and a temperature-sensitive gel material, wherein the content ratio of the cationic monomer to the temperature-sensitive gel material is (1:2) - (1: 1). The sublingual temperature-sensitive gel containing the mite allergen provided by the invention is a sublingual temperature-sensitive gel based on common sublingual drops containing the mite allergen, and has the advantages of convenience in administration of the sublingual drops, prolonged residence time of vaccines under the tongue and improved drug effect; the gelling time of the temperature-sensitive gel is shortened, the loss caused by swallowing in the administration process is reduced, and the administration amount is reduced; overcomes the temperature sensitivity of temperature sensitive gel to adsorb protein, improves the adsorption of the gel to the protein in a liquid state, and further reduces the dosage.)

1. A sublingual temperature-sensitive gel containing a mite allergen is characterized by consisting of the mite allergen, a cationic monomer and a temperature-sensitive gel material, wherein the content ratio of the cationic monomer to the temperature-sensitive gel material is (1:2) - (1: 1).

2. The sublingual temperature-sensitive gel containing mite allergen of claim 1, wherein the content ratio of the cationic monomer to the temperature-sensitive gel material is 1: 2.

3. The sublingual temperature-sensitive gel containing mite allergen of claim 1 or 2, wherein the cationic monomer is carboxymethyl chitosan.

4. The sublingual temperature-sensitive gel containing mite allergen of claim 1 or 2, wherein the temperature-sensitive gel material is one of poloxamer and poly (N-isopropylacrylamide).

5. A method for preparing the sublingual temperature-sensitive gel containing the mite allergen according to any one of claims 1-4, which comprises the following steps: preparing a temperature-sensitive gel material and a cationic monomer into a 1mol/L mixed aqueous solution, adjusting the pH to 7.2, adding an initiator, introducing N₂Polymerizing to obtain a copolymer; adding a cross-linking agent, dialyzing in water, and refining to obtain hydrogel of the copolymer; soaking the hydrogel in distilled water for 1 week, slowly adding mite allergen under magnetic stirring at 4 deg.C, and mixing to obtain the final product.

Technical Field

The invention relates to the technical field of administration of mite allergen, in particular to sublingual temperature-sensitive gel containing mite allergen and a preparation method thereof.

Background

Allergy is considered as a major health problem in the world by the World Health Organization (WHO) at present, the incidence of allergy is high, and about l/3 of people reported in the literature suffer from allergic diseases in the lifetime. WHO data show that 22-25% of people worldwide suffer from allergic diseases at present, the speed of the people is increased by 23 times every 10 years, and at present, two hundred million people suffer from the diseases only in China. Allergy and anti-allergy are therefore serious problems facing modern people that cannot be avoided. The allergens causing allergic reactions mainly include dust mites, pollen, dust, catkin, animal dander, etc., and mite allergens are among the most intensely distributed allergens worldwide.

At present, the common desensitization therapy is subcutaneous injection therapy, has quick response, but is easy to cause pain and cause intolerance of patients. Subcutaneous injection also easily causes local or systemic adverse reactions, which can lead to death seriously. And the treatment of sharp objects such as needles and the like is also a troublesome problem, and the sharp objects are placed at a proper position after being used for subsequent treatment, so that the cost is high and the cost is high. Sublingual specific immunotherapy (SLIT) is a specific immunotherapy method for gradually achieving immune tolerance by oral mucosal administration, and the dosage and concentration of a specific allergen vaccine are from low to high by enabling a patient to achieve a preset saturation dosage within 3-5 weeks and maintain for a period of time in a manner of Sublingual buccal administration, so that the immune system of the patient is stimulated to generate tolerance to the allergen, and the effect of immunotherapy is achieved. In 1986, Scadding GK firstly reports the success of sublingual buccal immunotherapy for the random double-blind experimental study of allergic rhinitis treatment, and SLIT is widely applied to the treatment of allergic rhinitis and allergic asthma as a replacement therapy of subcutaneous immunotherapy abroad in recent years.

Most of the existing common mite allergen sublingual vaccines are drops, the bioadhesion of the drops is weak, 1-1.5L of saliva is secreted in the oral cavity of a normal person every day, the drops used for the sublingual use can be flushed away by the saliva, the retention time of the mite allergen on the sublingual mucosa is shortened, and the treatment effect of the mite allergen sublingual drops is greatly reduced. The gel protects the antigen from enzymatic degradation and minimizes loss from the oral cavity, thereby maximizing absorption to provide adequate stimulation. Therefore, the invention provides the gel on the basis of the common drops to increase the viscosity of the mite allergen vaccine on the sublingual mucosa. However, a high viscosity gel increases the sublingual residence time of the vaccine, but has a problem that the dosage cannot be accurately determined when the vaccine is administered. The temperature-sensitive hydrogel is composed of special block copolymers, and has the characteristics of being liquid at low temperature, condensing when the temperature is raised to a certain temperature, and reversible change along with the temperature. Therefore, the invention aims to solve the technical problem of taking account of viscosity and quantitative administration by developing the sublingual temperature-sensitive gel preparation of the mite allergen vaccine.

Through retrieval, no literature report for developing mite allergen vaccines into sublingual temperature-sensitive gel preparations is found at present, and similar literatures are as follows:

chinese patent publication No. CN102014954A discloses a mucosal allergen-specific immunotherapy using seasonal allergens, and proposes a concept of developing pollen allergens into solid dosage forms (sublingual gel-containing preparations), but the patent has disadvantages in that: firstly, only the sublingual pollen allergen gel is provided, and the retention problem of the gel is still not provided with a solution; ② whether the gel can be quantitatively administered or not can not be known from the knowledge.

Chinese patent publication No. CN1840190A discloses a pharmaceutical composition for treating allergic diseases, which comprises dermatophagoides pteronyssinus allergen, pollen allergen and pharmaceutically acceptable carrier, and the composition can be developed into sublingual buccal preparation, but the technology has the disadvantages that: it is known from the preparation of sublingual buccal preparation of example 4 that the drops have the disadvantages of short residence time of sublingual mucosa and poor therapeutic effect of mite allergen.

White J A et al reported in the literature the development of polio vaccine as sublingual temperature sensitive gel, using a heat responsive gel (TRG) in combination with a bacterial heat labile toxin (DMLT) double mutant to enhance the immune effect of polio vaccine (White J A, Blum J S, Hosken N A, et al, serum and microbial antigens to inactive polar organic rear surface immunological immunization using a thermal responsive gel delivery system [ J ]. Human Vaccines & immunotherpeutics, 2014,10(12): 3611-. However, the technical disadvantages of the document are that: the adjuvant of the poliomyelitis vaccine is DmlT, so that the side effect is large, and the immune response level is low; ② the pH value of the temperature-sensitive gel is not researched, and uncertainty exists.

It is well known that the major antigen Der P1 in mite allergens is most active at neutral pH (biota, wuquine, wangzheng. dermatophagoides farinae Der P1 research progress [ J ]. Chongqing medicine, 2007,36(2): 171-. That is, when developing sublingual temperature-sensitive gel of mite allergen, selecting temperature-sensitive gel with neutral pH and short gelation time is one of the key technical factors that must be considered.

In addition, the main antigen Der p1 in mite allergen is an anionic protease, and the adsorption amount in the temperature-sensitive gel changes along with the temperature change. The temperature-sensitive microgel does not absorb a protein in a large amount at a temperature not higher than the phase transition temperature (LCST) of the gel, but exhibits temperature sensitivity to protein and enzyme adsorption by a sudden increase in the amount absorbed near the LCST. Therefore, when developing a temperature-sensitive gel with neutral pH and short gel time, how to increase the adsorption force of the gel on mite allergen in a liquid state is the second key technical factor that must be considered.

Widely known researchers such as Heimeng and the like found that when a cationic polymer chitosan was used as a main material to prepare a dust mite nano vaccine, the dust mite nano vaccine was administered sublingually to asthmatic mice, and found that the dust mite chitosan nano vaccine had an immunotherapeutic effect on sensitized mice, and that the protein adsorption rate of chitosan reached 60% (Heimeng, Liu Shi Jiang, Guoshua, Zhouyiping. the therapeutic effect of sublingual administration of dust mite chitosan nano vaccine on asthmatic mice [ J ]. J. parasites and parasites diseases, 2011,29(1): 4-9). The technical disadvantages of this document are: firstly, chitosan can be dissolved only in an acid solution with the pH value lower than 6.2, the pH value of a gel solution can influence the rheological property, and the antigen Der p1 has the strongest activity in neutral pH, so that uncertainty exists in the situation that whether a cationic polymer can be used for developing sublingual temperature-sensitive gel of mite allergen; ② the retention of dust mite allergen on sublingual mucosa is not known. Therefore, uncertainty still exists in the existence of interaction and influence between the temperature-sensitive gel matrix material and the allergen, and the exploration and the verification are needed.

In summary, it is a technical problem that the skilled person in the art has not solved to select which material to develop the sublingual temperature-sensitive gel of the mite allergen so as to improve the sublingual residence time of the mite allergen, and still have good adsorbability below the phase transition temperature (LCST) of the gel.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a sublingual temperature-sensitive gel containing mite allergen and a preparation method thereof, and the defects of the prior art are overcome.

Therefore, the invention adopts the following technical scheme:

a sublingual temperature-sensitive gel containing a mite allergen consists of the mite allergen, a cationic monomer and a temperature-sensitive gel material, wherein the content ratio of the cationic monomer to the temperature-sensitive gel material is (1:2) - (1: 1).

Preferably, the content ratio of the cationic monomer to the temperature-sensitive gel material is 1: 2.

Preferably, the cationic monomer is carboxymethyl chitosan.

Preferably, the temperature-sensitive gel material is one of poloxamer and poly (N-isopropylacrylamide).

The invention also provides a preparation method of the sublingual temperature-sensitive gel containing the mite allergen, which comprises the following steps: preparing a temperature-sensitive gel material and a cationic monomer into a 1mol/L mixed aqueous solution, adjusting the pH to 7.2, adding an initiator, and introducing N2 for polymerization to obtain a copolymer; adding a cross-linking agent, dialyzing in water, and refining to obtain hydrogel of the copolymer; soaking the hydrogel in distilled water for 1 week, slowly adding mite allergen under magnetic stirring at 4 deg.C, and mixing to obtain the final product.

In the present invention, the mite allergen is a dust mite allergen, including but not limited to the antigen Der p 1.

Compared with the prior art, the sublingual temperature-sensitive gel containing the mite allergen has the following beneficial effects:

1) the sublingual temperature-sensitive gel is provided on the basis of the common sublingual drop containing the mite allergen, the advantages of convenience in administration of the sublingual drop are continued, meanwhile, the residence time of the vaccine under the tongue is prolonged, and the drug effect is improved; meanwhile, accurate quantitative administration is convenient;

2) the gelling time of the temperature-sensitive gel is shortened, the loss caused by swallowing in the administration process is reduced, and the administration amount is reduced;

3) overcomes the temperature sensitivity of temperature sensitive gel to adsorb protein, improves the adsorption of the gel to the protein in a liquid state, and further reduces the dosage.

Detailed Description

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

Example 1 preparation of a sublingual temperature sensitive gel of mite allergen

Preparing a temperature-sensitive gel material and a cationic monomer into a 1mol/L mixed aqueous solution, adjusting the pH to 7.2, adding an initiator, introducing N₂After 2h of polymerization a copolymer was obtained. And (3) dialyzing the product in water for refining, and adding a cross-linking agent to obtain the copolymer hydrogel. The resulting hydrogel was soaked in distilled water for 1 week to remove unreacted monomers, crosslinkers and initiator molecules from the gel. Then, under the condition of magnetic stirring at 4 ℃, slowly adding the mite allergen, and uniformly mixing to obtain the finished product.

EXAMPLE 2 screening of temperature-sensitive gel materials

Assuming that the content ratio of the temperature-sensitive gel material to the cationic monomer is a certain value (such as 1:1), the temperature-sensitive gel material is one of poloxamer and poly (N-isopropylacrylamide) (PNIPAM), and the cationic monomer is one of chitosan, carboxymethyl chitosan, 2-diethylamino ethyl methacrylate and trimethyl-3-acrylamide chloride propyl amine; the influence of different types of temperature-sensitive gel materials and cationic monomers is researched by measuring the phase transition temperature and the mucous membrane retention.

In this example, the preparation method of the sublingual temperature-sensitive gel of the mite allergen is shown in example 1, and the measurement results are shown in table 1.

1. Phase transition temperature measurement method:

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 mercury ball of the thermometer into the gel solution, putting the gel solution into a water bath at normal temperature (<25 ℃), rotating at the speed of 300r/min, keeping the water temperature to continuously and slowly rise, and raising the temperature at the speed of about 0.5 ℃/min. Finally, the temperature at which the stirrer stops rotating is recorded, and the measured temperature is the phase transition temperature (LCST) of the gel. Each sample was assayed in duplicate 3 times and the results averaged.

When the phase transition temperature is close to the sublingual temperature of 36.3-37.2 ℃, the optimum phase transition temperature is considered to meet the test requirement.

2. Mucosal retention assay method:

and (3) placing the mixed solution of the temperature-sensitive gel and the mouse saliva in a small test tube in a constant-temperature water bath at 37 ℃, and then measuring the rheological property (namely viscoelasticity) of the mixed solution by using a magnetorheological instrument. A70 to 150 μm steel ball is carefully placed in a 2 to 5 μ l sample of the mucus and oscillated by an electromagnet at different drive frequencies to determine the displacement of the ball and its phase lag relative to the drive force, and a magnetorheological meter measures the viscoelasticity, the characteristic value of which is defined as logG (the vector sum of viscosity and elasticity).

logG is higher than 1.6, and the higher logG, the better the retention, considered to be in compliance with the test requirements.

3. The method for measuring the adsorption rate comprises the following steps:

after the gel adsorbs the mite-loaded allergen with the known total protein amount, the content of free protein in the supernatant is measured, and the adsorption rate is calculated by the following calculation formula: adsorption rate ═ (total protein-free protein)/total protein × 100%.

TABLE 1 temperature sensitive gel materials screening (mean + -SD, n ═ 3)

Temperature sensitive gel composition (cationic monomer + temperature sensitive gel material)	Phase transition temperature (. degree. C.)	logG*	Adsorption Rate (%)
				Chitosan + poloxamer	31.3±0.52	1.2	70±0.37
Carboxymethyl chitosan + poloxamer	32.2±0.53	1.3	71±0.47
				(2-diethylamino) ethyl methacrylate + poloxamer	31.4±0.15	1.4	67±0.88
Chloride trimethyl-3-acrylamidopropylamine + poloxamer	28.3±0.76	1.4	57±0.93
				Chitosan + PNIPAM	35.9±0.52	1.3	71±0.46
Carboxymethyl chitosan + PNIPAM	36.8±0.47	1.7	72±0.42
				2-diethylamino ethyl methacrylate + PNIPAM	34.5±0.47	1.6	65±0.45
Chlorinated trimethyl-3-acrylamidopropylamine + PNIPAM	32.9±0.51	1.7	55±0.67

As can be seen from table 1, it is,

firstly, different kinds of cationic monomers are combined with different kinds of temperature-sensitive gel materials for use, and the adsorption capacity is improved to a certain extent;

and comprehensively considering the phase transition temperature and logG of the gel, and only when the cationic monomer is carboxymethyl chitosan and the temperature-sensitive gel material is PNIPAM, the prepared temperature-sensitive gel meets the test requirements.

Example 3 screening of content ratio of temperature-sensitive gel Material to cationic monomer

In this example, the influence of different content ratios of the temperature-sensitive gel material and the cationic monomer on the aforementioned indexes is studied by measuring the phase transition temperature, logG, the adsorption rate, and the gelation time.

In this example, the preparation method of the sublingual temperature-sensitive gel of the mite allergen is described in example 1; the method for measuring the phase transition temperature, the mucous membrane retention and the adsorption rate is shown in example 2; the results are shown in Table 2.

1. The gel time measuring 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, putting the penicillin bottle into a constant-temperature water bath at 25 ℃ for 20min, putting the penicillin bottle into a constant-temperature water bath at 37 ℃, starting timing instantly, and recording the time when the stirrer stops rotating as the gelling time. The temperature of the water bath is controlled between 36.8 ℃ and 37.2 ℃. Each group was assayed in duplicate 3 times and the results averaged.

And when the gel time is less than 5s, the gel is considered to meet the test requirement.

TABLE 2 influence of different content ratios of cationic monomer to gel material on temperature sensitive gel properties (mean + -SD, n ═ 3)

From table 2, it can be seen that, when the content ratio of carboxymethyl chitosan to PNIPAM is (1:2) to (1:1), the test requirements are met by comprehensively considering 4 influence factors of phase transition temperature, logG, adsorption rate and gelation time; considering the importance of viscoelasticity, the optimal prescription is selected when the content ratio of the carboxymethyl chitosan to the PNIPAM is 1: 2.

Example 4 determination of therapeutic Effect of mite allergen Sublingual temperature sensitive gel

Taking 4-6-week-old female BALB/c mice, and setting the following three experimental groups: a blank sublingual temperature-sensitive gel group, a mite allergen sublingual drop group and a mite allergen sublingual temperature-sensitive gel group. Each group was treated with 50. mu.g of dust mite allergen +2mg Al (OH) on days 0, 7 and 14₃Sensitized (negative control group using physiological saline) mice were injected intraperitoneally. After ether anesthesia of mice on day 28, three experimental groups were immunized by sublingual administration 18 times with 1d interval each time, and 50 μ is used after 1 week of the last immunizationThe mite allergen is excited by dropping into nose for 1 time per day and 7 times continuously. And (5) performing gas channel high-reaction detection after the last excitation for 24 hours. The mice were sacrificed 48h after the last nasal drip, blood was taken, serum was prepared by separation, and IgE levels in the serum were measured.

The results are shown in Table 3.

TABLE 3 IgE antibody level determination in mouse sera (mean + -SD, n ═ 10)

Note: the results of the pre-and post-treatment comparisons in the group,^aP<0.05; compared with the group of sublingual drops after treatment,^bP<0.05。

from table 3, it can be seen that:

comparing the mite allergen sublingual drop group and the mite allergen sublingual temperature-sensitive gel group in each group, and obviously reducing the IgE antibody level (P is less than 0.05) after treatment compared with before treatment;

secondly, compared with the treated mite allergen sublingual drop group, the IgE antibody level of the treated mite allergen sublingual temperature-sensitive gel group is obviously reduced (P is less than 0.05), and therefore, the treatment effect of the mite allergen sublingual temperature-sensitive gel preparation is better than that of the mite allergen sublingual drop preparation.