A stability-enhanced sublingual drop of dermatophagoides farinae allergen and a preparation process thereof

Abstract

The application belongs to the technical field of medicine, and discloses a dust mite allergen sublingual drop with enhanced stability and a preparation process thereof. The preparation process comprises the following steps: culturing pure dust mites in an immunoregulatory special culture medium, performing gradient vibration screening on the dust mites, compounding active components, performing CO2 supercritical extraction degreasing, performing high-pressure nanometer homogenization wall-breaking extraction, performing tangential flow deep purification and concentration, and embedding the allergen sublingual drop with high activity and high mucous membrane permeability by using hydroxypropyl-beta-cyclodextrin to obtain the allergen sublingual drop. The preparation process can significantly improve the extraction efficiency of the allergen, improve allergic rhinitis, and realize long-term stable reservation of the activity of the allergen.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to allergen immunotherapy agents and their preparation processes, particularly to a stable sublingual drop for house dust mite allergens and its preparation process. Background Technology

[0002] Allergic diseases are prevalent globally, and house dust mites, as a major inhaled allergen, induce abnormal immune responses that are a core mechanism in the development of diseases such as allergic rhinitis. Sublingual immunotherapy has become the preferred clinical treatment due to its significant advantages. However, as a core formulation, house dust mite allergen sublingual drops need to maintain allergen activity and structural stability throughout the entire process while ensuring delivery efficiency. Existing formulations suffer from multiple technical challenges, including low allergen extraction efficiency, poor activity stability, and room for improvement in therapeutic efficacy. Therefore, developing house dust mite allergen sublingual drops and their preparation process that achieve the above-mentioned core requirements through process optimization and formulation structural innovation has become a key issue that urgently needs to be addressed in this field, and is of great significance for promoting the upgrading of sublingual immunotherapy technology for allergic diseases. Summary of the Invention

[0003] In view of this, the present invention provides a stable sublingual drop of house dust mite allergen and its preparation process. The preparation process involves culturing pure house dust mites in an immunomodulatory special culture medium, followed by gradient vibration sieving to form active components, CO2 supercritical extraction for defatting, high-pressure nano-homogenization extraction, tangential flow deep purification and concentration, and finally encapsulation with hydroxypropyl-β-cyclodextrin to obtain a highly active sublingual drop of allergen. This process can significantly improve the allergen extraction efficiency, improve allergic rhinitis, and simultaneously achieve long-term stable retention of allergen activity.

[0004] Firstly, this invention provides a process for preparing a stable sublingual drop for house dust mite allergens, comprising the following steps: S1. Culture and harvesting of house dust mites: Pure house dust mites are cultured on a large scale for 28-35 days in a culture medium with a specific composition using a sterile bottle with ventilation. During this period, the mites are turned over for 10 minutes every 7 days. The culture is terminated when the mite density in the culture medium is ≥5000 mites / g and the excrement accounts for ≥40% of the total mite count, and all cultures are harvested. S2. Gradient Vibrating Sieving and Compounding of Active Components: The harvested culture is fed into a vibrating sieve machine and sieved through 40-mesh, 120-mesh, and 200-mesh standard sieves in three stages. The material above the 40-mesh sieve is residual culture medium, and the material between the 120-200-mesh sieve is mainly fragments of the insect body and eggs (mainly containing inactive chitinous exoskeleton). To reduce interference from non-specific impurities and improve the purity of the preparation, both are removed. The material above the 120-mesh sieve (i.e., the material between the 40-120-mesh sieve, mainly intact insect bodies) and the material below the 200-mesh sieve (i.e., the material passing through the 200-mesh sieve, mainly culture metabolites, such as excrement and fine particles) are collected separately. To balance the various active components, the above two parts are precisely compounded and mixed evenly to obtain an allergen raw material with clearly defined components and controllable activity. Through extensive experimental research, this invention has found that allergens derived from insect bodies and allergens derived from metabolites have complementary effects in the immune regulation mechanism. After optimization through single-factor experiments, when the above two parts are compounded at a mass ratio of 1:2 (i.e., 120-mesh sieve material: 200-mesh sieve material = 2:4), the obtained raw materials show the best synergistic effect in inducing immune tolerance. Deviations from this ratio (such as 1:1 or 5:1) all lead to a significant decrease in efficacy.

[0005] S3. Inspection of raw materials for dust mite allergens: ① The moisture content must be ≤8% using a moisture analyzer; ② The total proportion of the target components (insect bodies, fragments and excrement) must be ≥90% confirmed by microscopic examination, and the culture medium residue must be ≤5%; ③ The sensory indicators should meet the requirements of light brown color, typical mite culture odor, no mold, no clumping, and no foreign impurities. Only after all indicators are qualified can the raw materials be put into the subsequent defatting and extraction processes. S4. CO2 supercritical extraction degreasing pretreatment: The qualified dust mite allergen raw material is degreased by supercritical CO2 extraction for 130-150 minutes to obtain low-fat raw material. S5. Extraction and Primary Clarification of Allergens: The pretreated low-fat raw material was added to a composite buffer extraction solution at 4°C and homogenized three times using a high-pressure nano-homogenizer at 300-400 bar pressure for gentle cell disruption. After disruption, extraction was carried out under nitrogen protection with stirring at 150-200 rpm for 8 hours. After extraction, the mixture was centrifuged at 4°C and 12,000 rpm for 40 minutes, and the supernatant was collected. The supernatant was then sequentially filtered through 1.2 μm, 0.45 μm, and 0.22 μm PES membranes to remove microorganisms and fine particles, yielding a clear and sterile crude allergen extract. S6. Tangential flow deep purification and concentration: The crude allergen extract is transferred to a tangential flow system and filtered using a 100kDa PES ultrafiltration membrane at 4°C, inlet pressure 0.12MPa, and transmembrane pressure 0.06MPa to remove macromolecular impurities. The permeate is collected and replaced with a 5kDa PES membrane, and deep purification and concentration are performed under the same conditions. The permeate is discarded, and the retentate is collected to obtain a highly purified allergen concentrate. S7. Allergen protein encapsulation and liquid formulation stabilization: Take the above allergen concentrate, add hydroxypropyl-β-cyclodextrin (HP-β-CD), and stir at 200 r / min for 4 h at 25℃ in the dark to encapsulate the allergen protein; add sorbitol to a final concentration of 2.0% (w / v) as a stabilizer and taste modifier, and add poloxamer 188 to a final concentration of 0.05% (w / v) as a surfactant, continue stirring for 1 h, and after mixing evenly, the dust mite allergen sublingual drops are obtained.

[0006] Preferably, the sublingual drops containing dust mite allergen prepared according to the present invention have the effect of improving allergic rhinitis.

[0007] Preferred method for obtaining and identifying purebred house dust mites: Environmental samples are collected, and single mites are isolated using a 40-80 mesh sieve and a stereomicroscope. Several pairs (at least 10 pairs) of morphologically intact adult male and female mites are selected, with each pair containing both a male and a female mite. The selected mites are placed in a basal culture medium for preliminary culture. After culture, the mites are taken and first subjected to morphological identification to confirm that the morphological purity of the house dust mites is 100%, meaning that each selected pair of mites is a house dust mite and there is no contamination with other mite species. Subsequently, genomic DNA is extracted, and PCR amplification and sequencing are performed on mite-specific gene fragments (such as the ITS2 gene fragment). The sequencing results are compared with a database to confirm that the sequence similarity is ≥95%. Mites meeting the above dual criteria are confirmed as purebred house dust mites. The basic culture medium consisted of 88.95% standard flour, 10% yeast powder FM888, 1% compound vitamins (compound vitamins are composed of the following components by weight: 25 parts vitamin B1, 10 parts vitamin B2, 50 parts vitamin B6, 5 parts vitamin B9, 3 parts vitamin B12, and 7 parts vitamin D3), and 0.05% tricalcium phosphate (anti-caking agent).

[0008] A further preferred method for collecting environmental samples is to use a vacuum cleaner to remove the powder from the outer surface of a flour bag that has been stored for 3 months.

[0009] Further preferred method: In order to meet the inoculation requirements for large-scale production, the above-identified purebred house dust mites (e.g., 10 pairs or more) are placed in a basal culture medium for expansion culture to obtain a purebred house dust mite population, which serves as the inoculation source of purebred house dust mites in step S1 of this application.

[0010] Preferred: The specific culture medium formulation (by weight) in step S1 of this invention comprises the following components: 76-85% standard flour (medium gluten flour), 8-12% yeast powder (FM888 yeast powder), 3-5% soybean peptone, 1.5-2.5% Jerusalem artichoke polysaccharide, 0.4-0.8% β-glucan, 0.2-0.5% glutamine, 0.5-1.0% potassium dihydrogen phosphate, 0.5-1.0% magnesium sulfate, 0.5-1.5% compound vitamins (compound vitamins are composed of the following by weight: 25 parts vitamin B1, 10 parts vitamin B2, 50 parts vitamin B6, 5 parts vitamin B9, 3 parts vitamin B12, and 7 parts vitamin D3), 0.01-0.05% tricalcium phosphate, and 0.05-0.2% sodium butyrate. This culture medium is a dry powder, kept moist by adding 2-5% water, and sterilized by irradiation before use.

[0011] A further preferred embodiment: The specific culture medium formulation (by w / w) in step S1 of the present invention comprises the following components: 81.05% standard flour, 10.0% yeast powder, 3.6% soybean peptone, 1.8% Jerusalem artichoke polysaccharide, 0.6% β-glucan, 0.3% glutamine, 0.8% potassium dihydrogen phosphate, 0.7% magnesium sulfate, 1.0% compound vitamins (compound vitamins are composed of the following by weight: 25 parts vitamin B1, 10 parts vitamin B2, 50 parts vitamin B6, 5 parts vitamin B9, 3 parts vitamin B12, and 7 parts vitamin D3), 0.05% tricalcium phosphate, and 0.1% sodium butyrate.

[0012] Preferably, the inoculation amount of pure-cultured house dust mites in step S1 of the present invention is 8-10% (w / w) of the culture medium mass.

[0013] Preferably, in step S1 of the present invention, the culture environment of the culture room for culturing dust mites is controlled at a temperature of 25-27°C and a relative humidity of 70-75%.

[0014] Preferably, in step S1 of the present invention, sterile air with a humidity of 70% is introduced at a rate of 0.3-0.5 L / min.

[0015] Preferably, in step S2 of the present invention, the mixing ratio of the material over the 120-mesh sieve and the material under the 200-mesh sieve is 2:4.

[0016] Preferably, the parameters for supercritical CO2 extraction in step S4 of the present invention are: temperature 35-37℃, pressure 20-25MPa, and CO2 flow rate 20-22L / h.

[0017] Preferably, the composite buffer extraction solution in step S5 of the present invention is a 10mM phosphate buffer containing 0.1mM disodium ethylenediaminetetraacetate and 2mM reduced glutathione, with a pH of 8.0±0.2. Disodium ethylenediaminetetraacetate can specifically inhibit the activity of metalloproteinases, and reduced glutathione can maintain the reducing environment of the system to prevent the oxidation of allergen proteins. The two work synergistically to construct a stable extraction system for allergens, maximizing the preservation of their biological activity.

[0018] Preferably, the pretreated low-fat dried raw material in step S5 of the present invention is mixed with the composite buffer extract at 4°C at a ratio of 1:30 (w / v).

[0019] Preferably, in step S7 of the present invention, the solid-liquid ratio of hydroxypropyl-β-cyclodextrin to allergen concentrate is 1:12 (w / v).

[0020] Secondly, the present invention provides a stable sublingual drop for house dust mite allergen prepared by the aforementioned process. The house dust mite allergen sublingual drop provided by the present invention can significantly improve allergen extraction efficiency, alleviate allergic rhinitis, and simultaneously achieve long-term stable retention of allergen activity.

[0021] The present invention has the following beneficial effects: 1. Improve the extraction efficiency of total protein from house dust mite allergens. Through the synergistic effect of key processes, promote the efficient release of intracellular allergen proteins while reducing protein degradation and aggregation loss during the extraction process.

[0022] 2. Enhances the treatment effect of allergic rhinitis, significantly relieves nasal symptoms caused by allergies, helps the body build immune tolerance, and has outstanding desensitization treatment effect.

[0023] 3. Optimize the purity and activity of allergens, remove non-specific impurities and large molecular proteins, avoid additional inflammatory responses, and ensure the specific immunomodulatory efficacy of allergens.

[0024] 4. Ensures the long-term storage stability of sublingual drops, effectively delays the decay of total allergen activity and the degradation of core allergen components, and extends the shelf life of the formulation. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a comparison chart of the total protein extraction rate of dust mite allergens in Example 1 and Comparative Examples 1-2.

[0027] Figure 2 This is a comparison chart of the number of times mice rubbed their noses and the number of times mice sneezed in Examples 1-3 and Comparative Examples 3-9.

[0028] Figure 3 and Figure 4 Morphological images of dust mites. Detailed Implementation

[0029] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features described therein can be combined with each other.

[0030] Disodium EDTA was purchased from Jiangsu Puxin Biotechnology Co., Ltd.; reduced glutathione was purchased from Shaanxi Zhonghe Jiantai Bioengineering Co., Ltd.; hydroxypropyl-β-cyclodextrin was purchased from Peptaan Biotechnology (Shaanxi) Co., Ltd.; and poloxamer 188 was purchased from Guangzhou Weierda Industrial Co., Ltd.

[0031] In this embodiment of the invention, the method for obtaining and identifying purebred house dust mites is as follows: A flour bag stored for 3 months is vacuum-cleaned to remove the surface dust. Single mites are separated using a 40-mesh sieve and a stereomicroscope. Thirty or more pairs of morphologically intact male and female adult mites are selected, where each pair contains both a male and a female mite. The selected mites are placed in a basic culture medium (containing: 88.95% standard flour, 10% yeast powder FM888, 1% compound vitamins, 0.05% tricalcium phosphate, kept moist with 3% water, and sterilized by irradiation; the compound vitamins consist of: 25 parts vitamin B1, 10 parts vitamin B2, 50 parts vitamin B6, 5 parts vitamin B9, 3 parts vitamin B12, and 7 parts vitamin D3 by weight) for preliminary culture. The cultured mites are then subjected to morphological identification, as shown in the morphological images below. Figure 3 and Figure 4 As shown, the morphological purity of the house dust mite was confirmed to be 100%, meaning that each selected pair of mites was confirmed to be a house dust mite, without any contamination with other mite species. Subsequently, genomic DNA was extracted, and PCR amplification and sequencing were performed on mite-specific gene fragments (such as the ITS2 gene fragment). The sequencing results were compared with the database, and the sequence similarity was confirmed to be ≥95%. Mites that meet the above dual criteria are confirmed as purebred house dust mites.

[0032] To meet the inoculation volume requirements for large-scale production, the aforementioned qualified purebred house dust mites (e.g., 10 pairs or more) are placed in a basal culture medium for expansion culture to obtain a purebred house dust mite population. This population serves as the inoculation source for the purebred house dust mites in step S1 of this application embodiment. It should be noted that the "purebred house dust mites" inoculated in step S1 do not refer to a single pair of mites, but rather to a purebred house dust mite population obtained from the expansion culture of male and female adult mites that have passed both morphological and genetic identification, and inoculated at a specific quality ratio.

[0033] Example 1

[0034] This embodiment provides a preparation process for a sublingual drop of house dust mite allergen, the specific steps of which are as follows: S1. Cultivation and harvesting of house dust mites: Pure house dust mites were selected and cultured on a large scale in double-layered sterile glass culture tanks equipped with ventilation and stirring devices. The inoculation amount was 8% of the culture medium mass. The culture room environment was controlled at a temperature of 25℃ and a relative humidity of 70%. Sterile air was introduced at 0.3L / min to prevent CO2 accumulation. During the cultivation period, the mixture was stirred at 100r / min for 10min every 7 days to prevent clumping and promote uniform growth of mites. The culture was terminated when the mite density in the culture medium was ≥5000 mites / g and the excrement content was ≥40%, and all cultures were harvested. The specific culture medium formula (by w / w) was as follows: 81.05% standard flour, 10.0% yeast powder, 3.6% soybean peptone, 1.8% Jerusalem artichoke polysaccharide, 0.6% β-glucan, 0.3% glutamine, 0.8% potassium dihydrogen phosphate, 0.7% magnesium sulfate, 1.0% compound vitamins (compound vitamins are composed of the following by weight: 25 parts vitamin B1, 10 parts vitamin B2, 50 parts vitamin B6, 5 parts vitamin B9, 3 parts vitamin B12, 7 parts vitamin D3), 0.05% tricalcium phosphate, and 0.1% sodium butyrate. This culture medium was a dry powder, kept moist by adding 3% water, and sterilized by irradiation before use. S2. Gradient Vibrating Sieving and Compounding of Active Components: The harvested culture is fed into a vibrating sieve machine and sieved through 40-mesh, 120-mesh, and 200-mesh standard sieves in three stages. The material above the 40-mesh sieve is residual culture medium, and the material between the 120-200-mesh sieve is mainly fragments of the insect body and eggs, both of which are removed. The material above the 120-mesh sieve (i.e., the material between the 40-120-mesh sieve, mainly intact insect bodies) and the material below the 200-mesh sieve (mainly culture metabolites, such as excrement and fine particles) are collected separately. To balance the various active components, the above two parts are precisely compounded at a mass ratio of 2:4. After thorough mixing, a raw material with clearly defined components and controllable activity of allergen is obtained. S3. Inspection of raw materials for dust mite allergens: ① The moisture content must be ≤8% using a moisture analyzer; ② The total proportion of the target components (insect bodies, fragments and excrement) must be ≥90% confirmed by microscopic examination, and the culture medium residue must be ≤5%; ③ The sensory indicators should meet the requirements of light brown color, typical mite culture odor, no mold, no clumping, and no foreign impurities. Only after all indicators are qualified can the raw materials be put into the subsequent defatting and extraction processes. S4. CO2 Supercritical Extraction Degreasing Pretreatment: The qualified dust mite allergen raw material is degreased by supercritical CO2 extraction with the following parameters: temperature 35℃, pressure 20MPa, CO2 flow rate 20L / h, and time 130min to obtain low-fat raw material. S5. Extraction and Primary Clarification of Allergens: The pretreated low-fat raw material was mixed at a ratio of 1:30 (w / v) with a 4°C compound buffer extraction solution (10 mM phosphate buffer containing 0.1 mM disodium EDTA and 2 mM reduced glutathione, pH 8.0±0.2). The mixture was then homogenized three times at 300 bar using a high-pressure nano-homogenizer at 4°C for gentle cell disruption. After disruption, extraction was carried out under nitrogen protection with stirring at 150 rpm for 8 h. After extraction, the mixture was centrifuged at 4°C and 12,000 rpm for 40 min, and the supernatant was collected. The supernatant was then sequentially filtered through 1.2 μm, 0.45 μm, and 0.22 μm PES membranes to remove microorganisms and fine particles, yielding a clear and sterile crude allergen extract. S6. Tangential flow deep purification and concentration: The crude allergen extract is transferred to a tangential flow system and filtered using a 100kDa PES ultrafiltration membrane at 4°C, inlet pressure 0.12MPa, and transmembrane pressure 0.06MPa to remove macromolecular impurities. The permeate is collected and replaced with a 5kDa PES membrane, and deep purification and concentration are performed under the same conditions. The permeate is discarded, and the retentate is collected to obtain a highly purified allergen concentrate. S7. Allergen protein encapsulation and liquid formulation stabilization: Take the above allergen concentrate and add hydroxypropyl-β-cyclodextrin (HP-β-CD) at a solid-liquid ratio of 1:12 (w / v). Stir at 200 r / min for 4 h at 25℃ in the dark to encapsulate the allergen protein. Add sorbitol to a final concentration of 2.0% (w / v) as a stabilizer and taste modifier, and add poloxamer 188 to a final concentration of 0.05% (w / v) as a surfactant. Continue stirring for 1 h. After mixing evenly, the house dust mite allergen sublingual drops are obtained.

[0035] Example 2

[0036] This embodiment provides a preparation process for a sublingual drop of house dust mite allergen, the specific steps of which are as follows: S1. Cultivation and harvesting of house dust mites: Pure house dust mites were selected and cultured on a large scale in double-layered sterile glass culture tanks equipped with ventilation and stirring devices. The inoculation amount was 9% of the culture medium mass. The culture room environment was controlled at a temperature of 26℃ and a relative humidity of 72%. Sterile air was introduced at 0.4L / min to prevent CO2 accumulation. During the cultivation period, the mixture was stirred at 100r / min for 10min every 7 days to prevent clumping and promote uniform growth of mites. The culture was terminated when the mite density in the culture medium was ≥5000 mites / g and the excrement content was ≥40%, and all cultures were harvested. The specific culture medium formula (by w / w) was as follows: 81.05% standard flour, 10.0% yeast powder, 3.6% soybean peptone, 1.8% Jerusalem artichoke polysaccharide, 0.6% β-glucan, 0.3% glutamine, 0.8% potassium dihydrogen phosphate, 0.7% magnesium sulfate, 1.0% compound vitamins (compound vitamins are composed of the following by weight: 25 parts vitamin B1, 10 parts vitamin B2, 50 parts vitamin B6, 5 parts vitamin B9, 3 parts vitamin B12, 7 parts vitamin D3), 0.05% tricalcium phosphate, and 0.1% sodium butyrate. This culture medium was a dry powder, kept moist by adding 3% water, and sterilized by irradiation before use. S2. Gradient Vibrating Sieving and Compounding of Active Components: The harvested culture is fed into a vibrating sieve machine and sieved through 40-mesh, 120-mesh, and 200-mesh standard sieves in three stages. The material above the 40-mesh sieve is residual culture medium, and the material between the 120-200-mesh sieve is mainly fragments of the insect body and eggs, both of which are removed. The material above the 120-mesh sieve (i.e., the material between the 40-120-mesh sieve, mainly intact insect bodies) and the material below the 200-mesh sieve (mainly culture metabolites, such as excrement and fine particles) are collected separately. To balance the various active components, the above two parts are precisely compounded at a mass ratio of 2:4. After thorough mixing, a raw material with clearly defined components and controllable activity of allergen is obtained. S3. Inspection of raw materials for dust mite allergens: ① The moisture content must be ≤8% using a moisture analyzer; ② The total proportion of the target components (insect bodies, fragments and excrement) must be ≥90% confirmed by microscopic examination, and the culture medium residue must be ≤5%; ③ The sensory indicators should meet the requirements of light brown color, typical mite culture odor, no mold, no clumping, and no foreign impurities. Only after all indicators are qualified can the raw materials be put into the subsequent defatting and extraction processes. S4. CO2 supercritical extraction degreasing pretreatment: The qualified dust mite allergen raw material was degreased by supercritical CO2 extraction with the following parameters: temperature 36℃, pressure 22MPa, CO2 flow rate 21L / h, and time 140min to obtain low-fat raw material. S5. Extraction and Primary Clarification of Allergens: The pretreated low-fat raw material was mixed at a ratio of 1:30 (w / v) with a 4°C compound buffer extraction solution (10 mM phosphate buffer containing 0.1 mM disodium EDTA and 2 mM reduced glutathione, pH 8.0±0.2). The mixture was then homogenized three times at 350 bar using a high-pressure nano-homogenizer at 4°C for gentle cell disruption. After disruption, extraction was carried out under nitrogen protection with stirring at 175 r / min for 8 h. After extraction, the mixture was centrifuged at 4°C and 12,000 rpm for 40 min, and the supernatant was collected. The supernatant was then sequentially filtered through 1.2 μm, 0.45 μm, and 0.22 μm PES membranes to remove microorganisms and fine particles, yielding a clear and sterile crude allergen extract. S6. Tangential flow deep purification and concentration: The crude allergen extract is transferred to a tangential flow system and filtered using a 100kDa PES ultrafiltration membrane at 4°C, inlet pressure 0.12MPa, and transmembrane pressure 0.06MPa to remove macromolecular impurities. The permeate is collected and replaced with a 5kDa PES membrane, and deep purification and concentration are performed under the same conditions. The permeate is discarded, and the retentate is collected to obtain a highly purified allergen concentrate. S7. Allergen protein encapsulation and liquid formulation stabilization: Take the above allergen concentrate and add hydroxypropyl-β-cyclodextrin (HP-β-CD) at a solid-liquid ratio of 1:12 (w / v). Stir at 200 r / min for 4 h at 25℃ in the dark to encapsulate the allergen protein. Add sorbitol to a final concentration of 2.0% (w / v) as a stabilizer and taste modifier, and add poloxamer 188 to a final concentration of 0.05% (w / v) as a surfactant. Continue stirring for 1 h. After mixing evenly, the house dust mite allergen sublingual drops are obtained.

[0037] Example 3

[0038] This embodiment provides a preparation process for a sublingual drop of house dust mite allergen, the specific steps of which are as follows: S1. Cultivation and harvesting of house dust mites: Pure house dust mites were selected and cultured on a large scale in double-layered sterile glass culture tanks equipped with ventilation and stirring devices. The inoculation amount was 10% of the culture medium mass. The culture room environment was controlled at a temperature of 27℃ and a relative humidity of 75%. Sterile air was introduced at 0.5L / min to prevent CO2 accumulation. During the cultivation period, the mixture was stirred at 100r / min for 10min every 7 days to prevent clumping and promote uniform growth of mites. The culture was terminated when the mite density in the culture medium was ≥5000 mites / g and the excrement content was ≥40%, and all cultures were harvested. The specific culture medium formula (by w / w) was as follows: 81.05% standard flour, 10.0% yeast powder, 3.6% soybean peptone, 1.8% Jerusalem artichoke polysaccharide, 0.6% β-glucan, 0.3% glutamine, 0.8% potassium dihydrogen phosphate, 0.7% magnesium sulfate, 1.0% compound vitamins (compound vitamins are composed of the following by weight: 25 parts vitamin B1, 10 parts vitamin B2, 50 parts vitamin B6, 5 parts vitamin B9, 3 parts vitamin B12, 7 parts vitamin D3), 0.05% tricalcium phosphate, and 0.1% sodium butyrate. This culture medium was a dry powder, kept moist by adding 3% water, and sterilized by irradiation before use. S2. Gradient Vibrating Sieving and Compounding of Active Components: The harvested culture is fed into a vibrating sieve machine and sieved through 40-mesh, 120-mesh, and 200-mesh standard sieves in three stages. The material above the 40-mesh sieve is residual culture medium, and the material between the 120-200-mesh sieve is mainly fragments of the insect body and eggs, both of which are removed. The material above the 120-mesh sieve (i.e., the material between the 40-120-mesh sieve, mainly intact insect bodies) and the material below the 200-mesh sieve (mainly culture metabolites, such as excrement and fine particles) are collected separately. To balance the various active components, the above two parts are precisely compounded at a mass ratio of 2:4. After thorough mixing, a raw material with clearly defined components and controllable activity of allergen is obtained. S3. Inspection of raw materials for dust mite allergens: ① The moisture content must be ≤8% using a moisture analyzer; ② The total proportion of the target components (insect bodies, fragments and excrement) must be ≥90% confirmed by microscopic examination, and the culture medium residue must be ≤5%; ③ The sensory indicators should meet the requirements of light brown color, typical mite culture odor, no mold, no clumping, and no foreign impurities. Only after all indicators are qualified can the raw materials be put into the subsequent defatting and extraction processes. S4. CO2 supercritical extraction degreasing pretreatment: The qualified dust mite allergen raw material was degreased by supercritical CO2 extraction with the following parameters: temperature 37℃, pressure 25MPa, CO2 flow rate 22L / h, and time 150min to obtain low-fat raw material. S5. Extraction and Primary Clarification of Allergens: The pretreated low-fat raw material was mixed at a ratio of 1:30 (w / v) with a 4°C compound buffer extraction solution (10 mM phosphate buffer containing 0.1 mM disodium EDTA and 2 mM reduced glutathione, pH 8.0±0.2). The mixture was then homogenized three times at 400 bar using a high-pressure nano-homogenizer at 4°C for gentle cell disruption. After disruption, extraction was carried out under nitrogen protection with stirring at 200 rpm for 8 h. After extraction, the mixture was centrifuged at 4°C and 12,000 rpm for 40 min, and the supernatant was collected. The supernatant was then sequentially filtered through 1.2 μm, 0.45 μm, and 0.22 μm PES membranes to remove microorganisms and fine particles, yielding a clear and sterile crude allergen extract. S6. Tangential flow deep purification and concentration: The crude allergen extract is transferred to a tangential flow system and filtered using a 100kDa PES ultrafiltration membrane at 4°C, inlet pressure 0.12MPa, and transmembrane pressure 0.06MPa to remove macromolecular impurities. The permeate is collected and replaced with a 5kDa PES membrane, and deep purification and concentration are performed under the same conditions. The permeate is discarded, and the retentate is collected to obtain a highly purified allergen concentrate. S7. Allergen protein encapsulation and liquid formulation stabilization: Take the above allergen concentrate and add hydroxypropyl-β-cyclodextrin (HP-β-CD) at a solid-liquid ratio of 1:12 (w / v). Stir at 200 r / min for 4 h at 25℃ in the dark to encapsulate the allergen protein. Add sorbitol to a final concentration of 2.0% (w / v) as a stabilizer and taste modifier, and add poloxamer 188 to a final concentration of 0.05% (w / v) as a surfactant. Continue stirring for 1 h. After mixing evenly, the house dust mite allergen sublingual drops are obtained.

[0039] Comparative Example 1: In step S5, the high-pressure nano-homogenization cell disruption process was removed. That is, after the dust mite culture and the composite extraction buffer were mixed evenly, the high-pressure nano-homogenization cell disruption process was no longer performed, and the extraction was carried out directly by stirring. The remaining processes were consistent with those in Example 1.

[0040] Comparative Example 2: In step S5, the complex extraction buffer was replaced with a conventional phosphate buffer (10 mM, pH 7.4, without disodium EDTA and reduced glutathione), and the rest of the process was the same as in Example 1.

[0041] Comparative Example 3: The direct allergen protein encapsulation and liquid formulation stabilization processes were omitted. That is, the highly purified allergen concentrate prepared in step 6 is the highly purified allergen concentrate. All other processes are consistent with those in Example 1.

[0042] Comparative Example 4: Step S6, tangential flow deep purification and concentration process, was omitted; all other processes remained the same as in Example 1.

[0043] Comparative Example 5: The specific culture medium in step S1 was modified, and the specific formula was as follows: 83.45% standard flour, 10.1% yeast powder, 3.6% soybean peptone, 0.3% glutamine, 0.8% potassium dihydrogen phosphate, 0.7% magnesium sulfate, 1.0% compound vitamins (specifically including: 0.25% vitamin B1, 0.1% vitamin B2, 0.5% vitamin B6, 0.05% vitamin B9, 0.03% vitamin B12, and 0.07% vitamin D3), and 0.05% tricalcium phosphate. The remaining processes were consistent with those in Example 1.

[0044] Comparative Example 6: The mass ratio of material between 40-120 mesh sieve and material under 200 mesh sieve in step S2 was adjusted to 1:5, and the rest of the process remained the same as in Example 1.

[0045] Comparative Example 7: The mass ratio of the material between the 40-120 mesh sieve and the material under the 200 mesh sieve in step S2 was adjusted to 1:1 (3:3), and the rest of the process remained the same as in Example 1.

[0046] Comparative Example 8: The mass ratio of material between 40-120 mesh sieve and material under 200 mesh sieve in step S2 was adjusted to 4:2, and the rest of the process remained the same as in Example 1.

[0047] Comparative Example 9: The mass ratio of material between 40-120 mesh sieve and material under 200 mesh sieve in step S2 was adjusted to 5:1, and the rest of the process remained the same as in Example 1.

[0048] Comparative Example 10: Except for the removal of the hydroxypropyl-β-cyclodextrin encapsulation process in step S7, the rest of the process is the same as in Example 1.

[0049] Comparative Example 11: Except for replacing the hydroxypropyl-β-cyclodextrin embedding in step S7 with cyclodextrin embedding, the rest of the process is the same as in Example 1.

[0050] Comparative Example 12: Except for the absence of poloxamer 188 in step S7, the rest of the process was the same as in Example 1.

[0051] Experiment 1: Verification of the total protein extraction rate of house dust mite allergens

[0052] Total protein concentration detection (BCA method): The crude allergen extracts obtained in step S5 of Example 1 and Comparative Examples 1-2 were used. A standard curve was plotted using the BCA method with BSA as the standard. The protein concentration of the samples was measured at 562 nm absorbance. The measurement was repeated three times. The protein concentration measured by the BCA method was multiplied by the volume of the extract to calculate the total protein mass in the crude extract. The experimental results are shown in Table 1 and... Figure 1 As shown.

[0053] Total protein extraction rate (%) = (Total protein mass in crude extract / Sample mass) × 100% The sample quality refers to the quality of the pretreated low-fat raw materials added in step S5 of Example 1 and Comparative Examples 1-2.

[0054] Table 1

[0055] Table 1 compares the total protein extraction rates of house dust mite allergens in Example 1 and Comparative Examples 1-2. The results show that the total protein extraction rate in Example 1 was significantly higher than that in Comparative Examples 1-2. This indicates that the present invention achieves efficient release of intracellular allergen proteins through the synergistic effect of high-pressure nano-homogenization and the composite extraction buffer. Simultaneously, the composite extraction buffer reduces protein loss through its dual protective effects of inhibiting endogenous protease activity and preventing protein oxidation, thus significantly improving the total protein extraction rate. Comparative Example 1, lacking the high-pressure nano-homogenization step, did not effectively disrupt the house dust mite cell structure, resulting in insufficient protein release. Comparative Example 2, using a conventional buffer without protective components, was prone to protein degradation and aggregation during extraction, and both had significantly lower extraction rates than Example 1. This fully demonstrates the indispensable core role of these two optimized process features in improving extraction efficiency.

[0056] Experiment 2: A Study of Allergic Rhinitis

[0057] BALB / c mice, weighing 18-22g, with a 50 / 50 male-to-female ratio, were used in the experimental experiments. The mice were housed in an environment with a room temperature of 22±2℃, relative humidity of 55±5%, and 12-hour light-dark cycles, and were acclimatized for one week with free access to food and water. 108 mice were randomly divided into a control group, a model group, Example 1-3 groups, and Comparative Examples 3-9 groups.

[0058] Allergic rhinitis (AR) model construction: The blank group mice were injected with and dripped with an equal volume of PBS solution as the control group. The other groups were constructed using the Derf1 (house dust mite group 1 allergen) induction method. The modeling process was divided into sensitization period and challenge period: ① Sensitization period: On the 1st, 8th and 15th day of the experiment, mice were injected intraperitoneally with 200 μL of PBS solution containing 25 μg Derf1 and 0.2 mg aluminum hydroxide adjuvant; ② Challenge period: From the 22nd to the 35th day of the experiment, mice were dripped into the nasal cavity daily with 20 μL of PBS solution containing 500 μg Derf1. With the continuous stimulation of the nasal mucosa by Derf1, mice were induced to produce AR-related symptoms, and the model was completed.

[0059] Dosage regimen: The dosing period was from day 28 to 35 of the experiment. Sublingual administration was performed 30 minutes before nasal stimulation each day. Examples 1-3 and Comparative Examples 3-9 were administered sublingual drops of the corresponding house dust mite allergen at a dose of 75 μg / kg (Note: Sublingual administration to mice required mild anesthesia; the medication was placed under the tongue and the head was kept lowered for a short time). The blank control group and the model group received an equal volume of 0.9% sodium chloride solution.

[0060] Symptom observation: Within 10 minutes of the last nasal administration of Derf1, blinded observers with standardized training evaluated the nasal symptoms of mice in real time under quiet, low-light conditions. The number of nasal scratches (a mouse rapidly rubbing its nose or face with its forepaws for ≥1 second was counted as one instance) and the number of sneezes (a mouse rapidly extending its head forward with a spitting sound was counted as one instance) were recorded. Detailed experimental data are shown in Table 2 and... Figure 2 .

[0061] Table 2

[0062] Figure 2 This is a comparison chart of the number of nasal rubbings and sneezes in mice in Examples 1-3 and Comparative Examples 3-9. The results showed that the number of sneezes and nasal rubbings in the control group were at normal levels, with no obvious nasal allergy symptoms. Compared with the control group, after Derf1 induction, both indicators in the model group mice increased significantly, and nasal allergy symptoms were obvious, indicating that the allergic rhinitis model was successfully established. Compared with the model group, the number of sneezes and nasal rubbings in mice in Examples 1-3 and Comparative Examples 3-9 groups were alleviated to varying degrees, with the improvement effect in Examples 1-3 groups being the most significant and the difference between the control and comparative groups being minimal. The above results indicate that the desensitization mechanism of the house dust mite allergen sublingual drops of the present invention stems from the synergistic effect of each step in the preparation process: a specific immunomodulatory culture medium provides immunomodulatory components to empower the growth of house dust mites, enabling the allergen to possess immunomodulatory potential; gradient vibration sieving and precise compounding balance the active components in the mites and metabolites, ensuring the specificity of the allergen; CO2 supercritical defatting reduces interference from lipid-soluble impurities, and high-pressure nano-homogenization combined with a composite buffer extract efficiently releases and protects the allergen protein from degradation; tangential flow deep purification removes non-specific impurities, avoiding the induction of additional inflammation; hydroxypropyl-β-cyclodextrin encapsulation combined with sorbitol and poloxamer 188 enhances the mucosal permeability and in vivo stability of the allergen, enabling it to efficiently penetrate the oral mucosal barrier, be recognized by the mucosal immune system, and induce specific immune tolerance in the body, inhibiting the release of allergy mediators, thereby relieving the symptoms of allergic rhinitis such as sneezing and nasal itching from the root cause.

[0063] Comparative Example 3: Without the allergen protein encapsulation and liquid formulation stabilization process, the allergen lacks the protection of encapsulation and stabilizers. It is easily degraded by enzymes in the oral mucosa, and the mucosa has poor permeability, making it difficult to be recognized by the immune system efficiently, thus making it difficult to induce sufficient immune tolerance and resulting in limited symptom relief. Comparative Example 4: The lack of tangential flow deep purification process cannot remove large molecular proteins and non-specific impurities in the crude allergen extract. These impurities can easily trigger additional inflammatory responses, interfere with desensitization, and result in poor symptom improvement. Comparative Example 5: Using a regular culture medium that does not contain Jerusalem artichoke polysaccharide, β-glucan, or sodium butyrate, the lack of immunomodulatory components for dust mites resulted in insufficient immunomodulatory efficacy of the allergens obtained from the culture, which could not synergistically induce the body to produce comprehensive immune tolerance, thus limiting the desensitization effect. Comparative Examples 6-9: The ratio of material between 40-120 mesh sieves (intact parasites) and material under 200 mesh sieves (metabolites) was adjusted, deviating from the optimal ratio of 2:4. This resulted in an imbalance of the allergen active components, which could not fully exert the synergistic desensitization effect of the parasites and metabolites. The therapeutic effects were all inferior to those of the Example Group.

[0064] Experiment 3: Stability Study

[0065] Experimental methods: Six samples of the sublingual drops prepared in Example 1 and Comparative Examples 10-12 were stored at 25±2℃. Samples were taken at 0 months, 6 months, 12 months, and 18 months, and tested according to the following methods: Allergen Der f 1 activity detection: The Der f 1 ELISA kit for house dust mite Der f 1 detection (purchased from Shanghai Bosheng Biotechnology Co., Ltd.) was used. The absorbance of Der f 1 in the sample was detected according to the kit instructions, and the Der f 1 activity retention rate was calculated (with 0% activity as 100%). The experimental results are shown in Table 3.

[0066] Allergen Der f2 activity detection: A commercially available house dust mite allergen Der f2 detection kit (purchased from Shanghai Bosheng Biotechnology Co., Ltd.) was used. The absorbance of Der f2 in the sample was measured according to the kit instructions, and the Der f2 activity retention rate was calculated (with 0% activity as 100%). The experimental results are shown in Table 3.

[0067] Allergen activity retention rate (%) = (A value of the sample to be tested / A value of the fresh sample at 0 months) × 100% Table 3

[0068] As shown in Table 3, Example 1 exhibited the best stability under long-term storage conditions at 25°C. The decay rates of allergen Derf1 and Derf2 activities were significantly lower than those of the comparative examples. The comparative examples, due to the lack of key stabilizing components or defects in the process, all showed significant activity decline. This advantage stems from the synergistic stabilizing system of hydroxypropyl-β-cyclodextrin (HP-β-CD) encapsulation and poloxamer 188 used in Example 1. HP-β-CD encapsulates the allergen protein through its unique hollow structure, isolating it from external destructive factors. Poloxamer 188 maintains the uniformity of protein dispersion. Combined with the optimized preparation process, this ensures efficient protein extraction and structural integrity, ultimately achieving stable retention of activity under long-term storage.

[0069] The technical effect of this invention stems from the precise synergistic effect of each process step. The possible mechanism is as follows: Inulin, β-glucan, and sodium butyrate in the immunomodulatory culture medium act as prebiotic analogs, optimizing the allergen spectrum in house dust mite metabolites, enabling allergens from the mites and excrement to produce complementary immunomodulatory effects; gradient vibration sieving and precise 2:4 compounding balance various active components, avoiding interference from non-specific impurities; CO2 supercritical defatting combined with high-pressure nano-homogenization and a composite buffer containing disodium EDTA / reduced glutathione achieves efficient and gentle release of intracellular allergen proteins and prevents oxidative degradation; tangential flow deep purification removes large molecular non-specific impurities; hydroxypropyl-β-cyclodextrin encapsulation combined with poloxamer 188 and sorbitol constructs multiple protective barriers, which not only improves sublingual mucosal permeability but also ensures the spatial conformational stability of allergen proteins during storage, ultimately synergistically inducing the body to produce more efficient specific immune tolerance.

[0070] Compared with existing technologies that commonly use sublingual drops to prepare house dust mite allergens (such as traditional culture medium combined with acetone defatting and conventional extraction stabilization methods), this invention cultivates pure house dust mites in a specialized immunomodulatory culture medium and combines this with an innovative process chain including gradient vibration sieving for precise compounding of active components, CO2 supercritical extraction defatting, high-pressure nano-homogenization and gentle cell wall disruption extraction, tangential flow deep purification and concentration, and hydroxypropyl-β-cyclodextrin encapsulation stabilization. This achieves a synergistic improvement in allergen extraction efficiency, purity, mucosal permeability, and long-term activity, thereby obtaining a high-activity immune tolerance induction effect and room temperature storage stability that is difficult to achieve with existing technologies. This provides a superior sublingual immunotherapy option for house dust mite-induced allergic rhinitis and other diseases.

[0071] The above-described embodiments are merely illustrative of specific implementations of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of protection of the present invention. It should be noted that for those skilled in the art, any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the scope of protection of the present invention.

Claims

1. A preparation process for a stable sublingual drop of house dust mite allergen, characterized in that: The specific preparation process of the house dust mite allergen sublingual drops is as follows: S1. Culture and harvesting of house dust mites: Pure house dust mites are cultured on a large scale for 28-35 days in a culture medium with a specific composition using a sterile bottle with ventilation. During this period, the mites are turned over for 10 minutes every 7 days. When the mite density in the culture medium is ≥5000 mites / g and the excrement accounts for ≥40%, the culture is terminated and all cultures are harvested. S2. Gradient Vibrating Screening and Compounding of Active Components: The harvested culture is put into a vibrating screener and screened in three stages through standard sieves of 40 mesh, 120 mesh and 200 mesh. The material on the 40 mesh sieve is residual culture medium and is removed. The material on the 120 mesh sieve and the material under the 200 mesh sieve are collected separately. The two collected parts are compounded and mixed evenly to obtain an allergen raw material with controllable activity. S3. Inspection of raw materials for dust mite allergens: ① The moisture content must be ≤8% using a moisture analyzer; ② Microscopic examination confirms that the total proportion of mites and excrement is ≥90%, and the culture medium residue is ≤5%; ③ Sensory indicators should meet the requirements of light brown color, typical mite culture odor, no mold, no clumping, and no foreign impurities. Only after all indicators are qualified can the raw materials be put into the subsequent defatting and extraction processes. S4. CO2 supercritical extraction defatting pretreatment: The qualified dust mite allergen raw material is extracted with supercritical CO2 for 130-150 min to obtain low-fat raw material. S5. Extraction and primary clarification of allergens: The pretreated low-fat raw material was added to a composite buffer extraction solution at 4°C and homogenized three times at 300-400 bar using a high-pressure nano-homogenizer at 4°C for gentle cell disruption. After cell disruption, the mixture was extracted for 8 hours under nitrogen protection by stirring at 150-200 r / min. After extraction, the mixture was centrifuged at 4°C and 12,000 rpm for 40 minutes, and the supernatant was collected. The supernatant was then filtered sequentially through 1.2 μm, 0.45 μm, and 0.22 μm PES membranes to remove microorganisms and fine particles, resulting in a clear and sterile crude allergen extract. S6. Tangential flow deep purification and concentration: The crude allergen extract is transferred to a tangential flow system and filtered using a 100kDa PES ultrafiltration membrane at 4°C, inlet pressure 0.12MPa, and transmembrane pressure 0.06MPa to remove macromolecular impurities. The permeate was replaced with a 5kDa PES membrane, and deep purification and concentration were carried out under the same conditions. The permeate was discarded, and the retentate was collected to obtain a highly purified allergen concentrate. S7. Encapsulation of allergen proteins and stabilization of liquid formulations: Take the above allergen concentrate and add hydroxypropyl-β-cyclodextrin. Under light-protected conditions at 25°C, stir at 200 r / min for 4 h to encapsulate the allergen proteins. Continue to add sorbitol to a final concentration of 2.0% and poloxamer 188 to a final concentration of 0.05%. Continue stirring for 1 h until the mixture is homogeneous to obtain the house dust mite allergen sublingual drops.

2. The preparation process of a stable sublingual drop for house dust mite allergen according to claim 1, characterized in that: The specific culture medium formulation in step S1 contains the following components: 76-85% standard flour, 8-12% yeast powder, 3-5% soybean peptone, 1.5-2.5% Jerusalem artichoke polysaccharide, 0.4-0.8% β-glucan, 0.2-0.5% glutamine, 0.5-1.0% potassium dihydrogen phosphate, 0.5-1.0% magnesium sulfate, 0.5-1.5% compound vitamins, 0.01-0.05% tricalcium phosphate, and 0.05-0.2% sodium butyrate. This culture medium is a dry powder, kept moist by adding 2-5% water, and sterilized by irradiation before use.

3. The preparation process of a stable sublingual drop for house dust mite allergen according to claim 1, characterized in that: In step S1, the inoculation amount of purebred house dust mites is 8-10% of the culture medium mass.

4. The preparation process of a stable sublingual drop for house dust mite allergens according to claim 1, characterized in that: In step S1, the culture environment of the dust mites is controlled at a temperature of 25-27℃ and a relative humidity of 70-75%. In step S1, the dust mites are cultured by introducing sterile air with a humidity of 70% at a rate of 0.3-0.5 L / min.

5. The preparation process of a stable sublingual drop for house dust mite allergen according to claim 1, characterized in that: In step S2, the mixing ratio of the material from the 120-mesh sieve to the material from the 200-mesh sieve is 2:

4.

6. The preparation process of a stable sublingual drop for house dust mite allergen according to claim 1, characterized in that: The parameters for supercritical CO2 extraction in step S4 are: temperature 35-37℃, pressure 20-25MPa, and CO2 flow rate 20-22L / h.

7. The preparation process of a stable sublingual drop for house dust mite allergen according to claim 1, characterized in that: In step S5, the composite buffer extract is a 10mM phosphate buffer containing 0.1mM disodium ethylenediaminetetraacetate and 2mM reduced glutathione, with a pH of 8.0±0.

2.

8. The preparation process of a stable sublingual drop for house dust mite allergen according to claim 1, characterized in that: In step S5, the pretreated low-fat dried raw material is mixed with a 4°C compound buffer extract at a ratio of 1g:30mL.

9. The preparation process of a stable sublingual drop for house dust mite allergens according to claim 1, characterized in that: In step S7, hydroxypropyl-β-cyclodextrin and allergen concentrate are mixed at a ratio of 1g:12mL.

10. A stable sublingual drop for dust mite allergen prepared by the preparation process according to any one of claims 1-9.

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