Sublingual tablet and application of sublingual tablet in desensitization treatment medicine

Abstract

The invention relates to a sublingual tablet and application of the sublingual tablet in a desensitization treatment medicine. The sublingual tablet provided by the invention is a low-dose allergen sublingual tablet. The sublingual tablet contains allergen protein freeze-dried powder, and the balance of lactose, mannitol, low-substituted hydroxypropyl cellulose, low-moisture microcrystalline cellulose and magnesium stearate. According to the allergen protein sublingual tablet disclosed by the invention, allergen protein freeze-dried powder is obtained by adopting a freeze-drying process, and the tablet is obtained by adopting a direct powder tabletting process. The low-dose sublingual tablet has excellent parameters including characters, hardness, tablet weight difference, disintegration time limit, taste and content uniformity, especially has outstanding advantages in the aspects of disintegration time limit, content uniformity, stability and preparation process, and has industrial application prospects.

Description

A sublingual tablet and its application in desensitization treatment medicine Technical Field

[0001] The present invention relates to the field of pharmaceutical preparations, and in particular to a sublingual tablet and application thereof in desensitization treatment medicine. Background Art

[0002] Allergen-specific immunotherapy is currently the only "cause-directed" treatment for allergic diseases that uses immune regulation to achieve its therapeutic goal. By administering gradually increasing doses of the allergen, it aims to increase the patient's tolerance to the allergen, alleviate symptoms caused by exposure to the allergen, and ultimately achieve tolerance and even immune tolerance.

[0003] The main routes of administration for allergen-specific immunotherapy include subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT). Allergen-specific immunotherapy has a long course of treatment (2-3 years), requiring multiple subcutaneous injections. Patient compliance is poor and it can easily cause various adverse reactions, such as severe anaphylactic shock. Compared to subcutaneous administration, sublingual administration offers fewer toxicities and side effects, greater safety, and improved compliance.

[0004] Currently, sublingual allergen administration primarily consists of two dosage forms: sublingual drops and sublingual tablets. Sublingual tablets are designed to dissolve rapidly when placed under the tongue or disintegrate rapidly in saliva upon use. The drug is absorbed through the sublingual oral mucosa to exert systemic effects. Compared to sublingual drops, sublingual tablets are easier to take and carry, with easier dosage control, ensuring standardized dosage, and resulting in higher patient compliance. Sublingual tablets also offer superior stability.

[0005] Compared with ordinary sublingual tablets, the formulation and development of allergen sublingual tablets is more difficult. First of all, the active ingredient of allergen sublingual tablets is protein, which is unstable in moisture and heat. At present, the preparation of ordinary sublingual tablets includes granulation and tableting method (including wet granulation and dry granulation), blister freeze-drying method and direct compression method. Among them, the wet granulation and tableting method requires granulation, drying and whole granulation, and is not suitable for heat-sensitive, moisture-sensitive and highly soluble drugs. The dry granulation and tableting method requires compression force to generate binding force between particles, and high pressure can easily cause problems such as crystal transformation and reduced activity. Allergen sublingual tablets that have been on the market, such as ALK's The blister freeze-drying method is used, which has high requirements for production equipment and requires the use of special production lines such as liquid nitrogen freezing tubes. The equipment cost is high, and the relevant domestic technology is not yet mature. In general, the direct powder compression method eliminates the wet granulation step and avoids the drying process. It is more suitable for drugs that are unstable to moisture and heat, and the overall process is less, saving time and energy. However, the direct powder compression process has some shortcomings, such as poor powder fluidity, large differences in tablet weight, easy to cause cracking, poor content uniformity and other problems. Especially when the content of active ingredients in the tablets is low, the problem of poor content uniformity in the direct powder compression process is more prominent.

[0006] Secondly, compared to ordinary sublingual tablets, the active ingredient content of allergen sublingual tablets is extremely low, accounting for less than 2% of the tablet weight, or even 1% or less. The extremely low active ingredient content raises issues such as content uniformity and stability. Chinese patent CN108524454A discloses the use of twin-screw technology equipment to prepare low-dose pharmaceutical compositions; Chinese patent CN1531423A uses a high-shear granulation method to prepare low-dose drugs. Similarly, low-dose drug preparations place additional requirements on production equipment.

[0007] Summary of the Invention

[0008] In response to the defects of the prior art, the present invention provides a low-dose sublingual tablet suitable for multiple active ingredients and its application in desensitization treatment drugs, while solving the problems of poor content uniformity and stability of low-dose sublingual tablets, as well as complex production equipment and processes.

[0009] The present invention provides a sublingual tablet, which comprises a protein raw material, lactose, mannitol, low-substituted hydroxypropyl cellulose, low-moisture microcrystalline cellulose, and magnesium stearate, wherein the mass percentage of each component is 0%-15.6%, 0%-83.4%, 0-83.4%, 4%-6%, 10%-15%, and 0.4%-0.6%.

[0010] Preferably, the protein raw material is an allergen protein raw material.

[0011] Preferably, the mass percentages of the protein raw material, lactose, mannitol, low-substituted hydroxypropyl cellulose, low-moisture microcrystalline cellulose, and magnesium stearate are 0%-15.6%, 50%-83.4%, 0%-33.4%, 4%-6%, 10%-15%, and 0.4%-0.6%.

[0012] Preferably, the protein raw material, lactose, mannitol, low-substituted hydroxypropyl cellulose, low-moisture microcrystalline cellulose, and magnesium stearate account for 0.276%-15.6%, 59.3%-76.48%, 5%-13.4%, 4%-6%, 10%-12.5%, and 0.4%-0.6% of the tablet weight, respectively.

[0013] There are many types of allergens, mainly divided into two categories: inhalation allergens and food allergens. Common inhalation allergens include house dust mites, house dust mites, cockroaches, cat and dog dander, mold, pollen, etc., and common food allergens include milk, peanuts, eggs, etc. The allergen protein can be naturally extracted or recombinantly expressed. The main allergenic proteins of the house dust mite (Dermatophagoides pteronyssinus, Der p) are Der p1 and Der p2, and the main allergenic proteins of the house dust mite (Dermatophagoides farinae, Der f) are Der f1 and Der f2. The main allergenic proteins of Artemisia annua pollen are Art a1 and Art a3, and the main allergenic proteins of Artemisia vulgaris pollen are Art v1 and Art v3. Since the content of active ingredients in the sublingual tablets of the present invention is extremely low, although the physical and chemical properties of different active ingredients are different, the data in the examples prove that different active ingredients do not affect the hardness, tablet weight difference, disintegration time, content uniformity, stability and other properties of the final sublingual tablets. Therefore, the sublingual tablet prescription of the present invention is applicable to a variety of low-dose active ingredients, and is not only applicable to the specific active ingredients listed in the examples.

[0014] Preferably, the dust mite allergen protein raw material is composed of one or a mixture of two or more of Der p 1, Der p 2, Der f 1, and Der f 2. The Artemisia annua / Artemisia vulgaris pollen allergen protein raw material is composed of one or a mixture of two or more of Art a 1, Art a 3, Art v 1, and Art v 3. Preferably, the allergen protein raw material is freeze-dried allergen protein powder.

[0015] The second aspect of the present invention relates to a freeze-drying process. The buffer composition and protein concentration of a freeze-drying solution suitable for a specific protein can be determined through limited trials according to conventional methods. The buffer composition selected in the specific examples is merely illustrative and does not limit the buffer composition used for freeze-drying of allergen proteins. For example, when the active ingredient is Der p 2 or Der f 2, the buffer composition can be PB and mannitol; when the active ingredient is Der p 1, Der f 1, Art a 1, Art a 3, Art v 1, or Art v 3, the buffer composition can be citric acid-sodium citrate and mannitol. The protein concentration can be selected to be 0.03-1.0 mg / mL. Furthermore, the freeze-dried allergen protein solution may or may not contain gelatin. When the allergen protein is less stable, the addition of gelatin may be more beneficial in maintaining the stability of the allergen protein sublingual tablet.

[0016] The general freeze-drying process is to replace the solution containing the allergen protein with an appropriate buffer system and pH conditions, and quantify it to an appropriate concentration. The solution undergoes three steps: pre-freezing, primary drying, and desorption drying to obtain a freeze-dried product. Conventional methods can be used to screen for freeze-drying processes suitable for specific proteins. Preparation Examples 12 to 15 list freeze-drying processes for different allergen proteins. The listed processes are only examples and do not limit the freeze-drying processes of allergen proteins. For example, different freeze-drying processes were used for dust mite type II protein in Preparation Examples 12, 16, and 20, and the final tablet performance was essentially the same. The pre-freezing step is divided into two stages. The pre-freezing temperature in the first stage is set to -12°C to -15°C, and the temperature is maintained for 60 to 90 minutes after reaching this temperature. The pre-freezing temperature in the second stage is set to -45°C to -50°C, and the temperature is maintained for 120 to 800 minutes after reaching this temperature. The primary drying temperature is set to -10°C to -20°C, and the temperature is maintained for 840 to 960 minutes after reaching this temperature. The vacuum degree is 0.18 to 0.2 mbar. The desorption drying temperature is set to 20° C. to 25° C., and after reaching this temperature, the temperature is maintained for 480-1500 minutes, and the vacuum degree is 0.18-0.2 mbar.

[0017] A third aspect of the present invention relates to a powder direct compression process for allergen sublingual tablets, which does not require complex processes and equipment. The powder direct compression method bypasses the granulation process and directly compresses the mixture of active drug and excipients into tablets. The process steps primarily include pulverizing the active drug and excipients, screening, mixing, adding a lubricant, remixing, and tableting. After extensive research, the inventors have developed a low-dose sublingual tablet formulation that not only exhibits ideal hardness, tablet weight variation, disintegration time, content uniformity, and stability, but is also highly suitable for direct powder compression, overcoming the aforementioned problems associated with conventional direct powder compression.

[0018] The allergen sublingual tablets of the present invention exhibit superior properties across various parameters, including appearance (white tablets with a smooth surface and uniform color), hardness (moderate hardness of 15-60N, with no significant impact on tablet packaging and transportation), tablet weight variation, disintegration time, mouthfeel, and content uniformity. Disintegration time, content uniformity, stability, and manufacturing process are particularly advantageous: 1. The sublingual tablets of the present invention rapidly disintegrate under the tongue and melt until no noticeable foreign body sensation is present. Sublingual tablets absorb the active ingredient through the sublingual oral mucosa for systemic effects. Prolonged disintegration times can easily lead to swallowing, and some active ingredients may not be fully absorbed through the intended sublingual mucosa, resulting in inaccurate dosing. 2. The sublingual tablets of the present invention exhibit excellent allergen content uniformity, avoiding the problem of uneven allergen content that can occur when tablets contain low levels of active ingredient, making them suitable for allergen sublingual tablets with low active ingredient content. 3. The sublingual tablets of the present invention are not only stable at room temperature but also highly stable under harsh environments, making them suitable for use with a variety of biologically active drugs. 3. The low-dose sublingual tablets of the present invention achieve the above-mentioned excellent comprehensive performance, but the preparation process is simple, and the powder direct tableting method is applicable, the steps are simple, and there are no additional requirements for production equipment. DETAILED DESCRIPTION

[0019] The following will provide preparation examples and comparative examples for detailed description, but the present invention is not limited thereto.

[0020] Preparation Examples 1 to 11 are preparation examples and comparative examples of blank sublingual tablets containing no active ingredient. The resulting tablets were subjected to tablet hardness, tablet weight variation, disintegration time, and sensory tests.

[0021] Preparation Examples 12-26 describe the preparation of low-dose sublingual tablets containing the active ingredient. In addition to testing for tablet hardness, tablet weight variation, disintegration time, content uniformity, and sensory testing, the resulting tablets were also tested for factors affecting long-term exposure, high temperature, light, and high humidity. Stability was assessed by testing the total protein content and purity of the tablets.

[0022] The specific detection methods are as follows:

[0023] 1. Hardness test

[0024] Tablet hardness was tested using a dedicated instrument for hardness measurement (CJY-2C hardness tester, Tianda Tianfa). Ten tablets were randomly sampled for testing, and the average value and RSD value were indicated.

[0025] 2. Tablet weight difference detection

[0026] According to the 2020 edition of the Chinese Pharmacopoeia General Chapter 0101 Tablet Weight Variation Test Method: Take 20 tablets of the test sample, accurately weigh the total weight, calculate the average tablet weight, and then accurately weigh each tablet separately. No more than two tablets may exceed the weight difference limit compared to the labeled weight, and no single tablet may exceed the limit by more than 100%. (For tablets with a labeled weight or average tablet weight of less than 0.3g, the weight difference limit is ±7.5%).

[0027] 3. Disintegration time limit detection

[0028] According to the disintegration time test method of the 2020 edition of the Chinese Pharmacopoeia General Chapter 0921: use a disintegration instrument (ZB-1E model, Tianda Tianfa), immerse it in a 1000ml beaker filled with water at a temperature of 37℃±1℃, take 6 tablets of the test sample, and test them. Each tablet should completely disintegrate and dissolve within 5 minutes.

[0029] 4. Moisture detection of freeze-dried powder

[0030] Refer to the 2020 edition of the Chinese Pharmacopoeia General Chapter 0832 Water Determination Method 1 (Fischer Method) Coulometric Titration

[0031] 5. Sensory test

[0032] Six healthy adults were selected to administer the sublingual tablet. The oral texture was evaluated, primarily for dryness or discomfort, grittiness, sweetness, and oral disintegration time. The tablet was then spit out and the mouth was rinsed with water.

[0033] 6. Excipient compatibility test

[0034] Appropriate amounts of active drug lyophilized powder were weighed into vials, and excipients in different proportions were added, including microcrystalline cellulose, low-moisture microcrystalline cellulose, fillers, and low-substituted hydroxypropyl cellulose at 5 times the amount of lyophilized powder, and lubricant at an equal proportion of lyophilized powder. Accelerated, high-humidity, and light tests were performed, and purity tests were performed to confirm the compatibility of the active drug lyophilized powder with each excipient.

[0035] 7. Accelerated testing

[0036] The test sample was placed in a vial of appropriate size and sealed. The vial was placed in a stability test chamber (KBF-P-720, Binder) under accelerated parameters (40±2°C, 75%±5%RH). The sample was taken out at the corresponding time point for testing.

[0037] 8. High temperature test

[0038] The sample to be tested was placed in a vial of appropriate size and sealed. The vial was placed in a stability test chamber (KBF-P-720, Binder) under high temperature (60±2°C) parameters. The sample was taken out at the corresponding time point for testing.

[0039] 9. High humidity test

[0040] The test sample was placed in an open vial of a suitable size, and the vial was placed in a stability test chamber (KBF-720, Binder) under high humidity (25±2°C, 75%±5%RH) parameters. The vial was taken out at the corresponding time point for testing.

[0041] 10. Lighting test

[0042] The test sample was placed in a sealed vial of appropriate size. The vial was placed horizontally in a stability test chamber (KBF-P-720, Binder) under the following lighting parameters: 25±2°C, 60%±5%RH, 4500±500lx. The sample was taken out at the corresponding time point for testing.

[0043] 11. Purity testing

[0044] Der p1 and Der f1 are in accordance with the 2020 edition of the Chinese Pharmacopoeia General Chapter 0514 Size Exclusion Chromatography. Der p2 and Der f2 are in accordance with the 2020 edition of the Chinese Pharmacopoeia General Chapter 0512 Reversed Phase Chromatography.

[0045] 12. Freeze-dried powder protein content and uniformity testing

[0046] Der p1 and Der f1 were tested according to the size exclusion chromatography method in accordance with the 2020 edition of the Chinese Pharmacopoeia General Chapter 0514. Der p2 and Der f2 were tested according to the reverse phase chromatography method in accordance with the 2020 edition of the Chinese Pharmacopoeia General Chapter 0512. The protein content and homogeneity (RSD value) of the freeze-dried powders from different sites in the same batch were tested.

[0047] 13. Detection of content and potency of major allergens

[0048] Protein content was determined using a double antibody sandwich ELISA method. The main allergen content of dust mite type I refers to the sum of Der p1 and Der f1, and the main allergen content of dust mite type II refers to the sum of Der p2 and Der f2. The main allergen content of Artemisia annua / Artemisia vulgaris pollen type I refers to the sum of Art a1 and Art v1, and the main allergen content of Artemisia annua / Artemisia vulgaris pollen type III refers to the sum of Art a3 and Art v3.

[0049] The potency was tested using the inhibition ELISA method. The recombinant protein sample was serially diluted and incubated with serum from the serum pool. The incubated mixture was added to an ELISA plate pre-coated with the recombinant protein. A linear relationship of "inhibition rate-dilution factor" was established, and the biological activity required to reduce serum IgE from the serum pool by 50% was stipulated to be 100 BU / ml. The biological activity of the recombinant protein was calculated. To better reflect the trend of activity changes, the obtained biological activity was calculated as a percentage of the theoretical labeled amount. The test value is reflected as actual detection value of activity / theoretical labeled amount*100%.

[0050] 14. Content uniformity test

[0051] The protein content of the tablets was tested using the immunospot assay, and the content uniformity was calculated according to the 2020 edition of the Chinese Pharmacopoeia General Chapter 0941 Content Uniformity Test Method. According to General Chapter 0941, for sublingual tablets, the L value should be 15. This means that the standard deviation S of the content test results for 10 test tablets and the absolute value A of the difference between the labeled amount and the mean should be calculated. If A + 2.2S is less than 15, the content uniformity is considered acceptable.

[0052] Example 1: Formulation Study 1 (Selection of Disintegrant)

[0053] Table 1 Ingredient ratio of Preparation Example 1

[0054] The lactose used in this application is from Germany Meijile, model 100; mannitol comes from Roquette, France, specification model 100SD; low-substituted hydroxypropyl cellulose specification model LH-21, hydroxypropoxy content 10.0%-12.9%, purchased from Huzhou Zhanwang Pharmaceutical Co., Ltd., low-substituted hydroxypropyl cellulose containing 5.0%-16.0% hydroxypropoxy calculated on a dry basis can be used in this application without affecting tablet performance; low-moisture microcrystalline cellulose specification model VIVAPUR PH112, drying loss value ≤1.5%, purchased from Shanghai Fenghong Pharmaceutical Excipients Technology Co., Ltd.

[0055] Preparation Example 1: Using the amounts listed in Table 1, a stepwise addition method was used, with lactose being the starting material. 5% lactose, mannitol, 5% lactose, low-substituted hydroxypropyl cellulose, 5% lactose, low-moisture microcrystalline cellulose, and the remaining 85% lactose were added and mixed. Finally, magnesium stearate was added and mixed. Tablets were compressed using a small single-punch tablet press, with a core hardness of 20-60 N and a tablet weight of 50 mg.

[0056] Control groups 1-3: The low-substituted hydroxypropyl cellulose in Preparation Example 1 was replaced with hydroxypropyl cellulose (SSL-L), cross-linked sodium carboxymethyl cellulose, and sodium carboxymethyl starch. The types and amounts of other excipients remained unchanged. They were respectively used as Control Groups 1-3, and the preparation method was the same as that of Preparation Example 1.

[0057] The experimental results showed that when low-substituted hydroxypropyl cellulose was used, the RSD value of sublingual tablet hardness and tablet weight were small, the oral disintegration time was short, the oral sensory experience was not dry or gritty, and the sweetness was moderate. When other disintegrants were used, the disintegration time was too long, and even if the type and amount of other excipients in the prescription were adjusted, tablets with the above-mentioned excellent comprehensive performance could not be obtained. The specific results are as follows:

[0058] Table 2 Experimental results of Preparation Example 1 and Control Groups 1-3

[0059] Example 2: Prescription Study 2 (Selection of Filler and Its Dosage)

[0060] Table 3 Ingredient ratio of Preparation Examples 2-5

[0061] According to the composition ratio in Table 3, the sublingual tablets of Preparation Examples 2 to 5 were prepared. In Preparation Example 2, all the mannitol in Preparation Example 1 was replaced with lactose. In Preparation Examples 3 to 5, part or all of the lactose in Preparation Example 1 was replaced with mannitol. Other contents were the same as in Preparation Example 1.

[0062] The specific results are shown in Table 4: the lactose ratios in Preparation Examples 1-5 are approximately 70.0%, 83.4%, 50.0%, 27.4%, and 0%, respectively, and the sublingual tablet hardness RSD value, tablet weight difference, oral disintegration time, and oral sensory parameters are all relatively good. Among them, Preparation Example 1 is the best, with the shortest disintegration time, followed by Preparation Example 3, and Preparation Example 2 is relatively good. The disintegration time of Preparation Examples 4 and 5 is relatively long, and the taste is slightly poor. Therefore, the combination of lactose and mannitol is the best, and with the increase in the proportion of lactose added, the hardness RSD value and tablet weight difference are relatively small, the oral disintegration time is short, the oral sensory feeling is not dry or gritty, and the sweetness is moderate.

[0063] Table 4 Experimental results of Preparation Examples 2-5

[0064] Example 3: Formulation Study 3 (Low-Moisture Microcrystalline Cellulose / Microcrystalline Cellulose, Selection of Lubricant)

[0065] Table 5: Ingredient ratio of Preparation Example 6-7

[0066] The low-moisture microcrystalline cellulose in Preparation Example 1 was replaced with microcrystalline cellulose to obtain Preparation Example 6, and the preparation method was the same as that of Preparation Example 1.

[0067] The magnesium stearate in Preparation Example 1 was replaced with behenyl glyceride to prepare Preparation Example 7, and the preparation method was the same as that of Preparation Example 1.

[0068] Control group 4: According to the composition ratio in Table 5, control group 4 was prepared without using low-moisture microcrystalline cellulose or microcrystalline cellulose and mannitol. The preparation method was the same as that of Preparation Example 1.

[0069] The specific results are shown in Table 6, indicating that the use of microcrystalline cellulose and behenyl glyceride resulted in superior sublingual tablet hardness RSD values, tablet weight variation, oral disintegration time, and oral sensory parameters (however, in the excipient compatibility study of Preparation Examples 11-14, microcrystalline cellulose and behenyl glyceride were not compatible with the protein allergen active ingredient). Control group 4, however, experienced severe powder leakage during tableting, indicating that the powder direct compression process was unsuitable.

[0070] Table 6 Experimental results of Preparation Examples 6-7 and Control Group 4

[0071] Example 4: Prescription Study 4 (Excipient Ratio Study)

[0072] Table 7 Ingredient ratio of Preparation Examples 8-11

[0073] According to the composition ratio in Table 7, the sublingual tablets of Preparation Examples 8 to 11 and Control Groups 5 and 6 were prepared using the same preparation method as that of Preparation Example 1.

[0074] The specific results are shown in Table 8. The sublingual tablets of Preparation Examples 8-11 exhibited excellent RSD values ​​for hardness, tablet weight variation, oral disintegration time, and oral sensory parameters. Control Groups 5 and 6 exhibited significant tablet weight variation and prolonged disintegration times, so the excipient ratios used in Control Groups 5 and 6 were not suitable.

[0075] Table 8 Experimental results of Preparation Examples 8-11

[0076] Example 5: Study on the freeze-drying process of recombinant dust mite type 2 allergen and preparation of protein sublingual tablets

[0077] 1) Freeze-drying process research

[0078] Condition 1: The recombinant Der f 2 protein solution was replaced with a 50 mM PB + 3% mannitol, pH 7.0 buffer system. The protein concentration was determined using a BCA protein concentration assay kit to a value of 1.0 mg / ml. The solution was then poured into a lyophilization plate with the sample liquid level controlled at ≤1 cm. Lyophilization was performed using a vacuum freeze drying oven (LY0-21SPOIP, Tofflon) according to the parameters in Tables 9-12 (lyophilization processes 1-4) and Table 15 (lyophilization process control 1) to prepare a lyophilized powder.

[0079] Condition 2: The recombinant Der f 2 protein solution was replaced with 5 mM PB + 1% mannitol + 1.5 mg / ml 150LB gelatin (bovine gelatin, purchased from Baotou Dongbao Biotechnology Co., Ltd.), pH 7.0 buffer system. The protein concentration was determined to be 0.03 ± 0.015 mg / ml, and the solution was poured into a freeze-drying tray. The sample liquid level was controlled to be ≤ 1 cm. The solution was freeze-dried using a vacuum freeze drying oven according to the parameters in Tables 13, 14, 16, and 17 (freeze-drying processes 5 and 6, and freeze-drying process controls 2 and 3) to prepare freeze-dried powder. The gelatin in this example was bovine-derived and purchased from Baotou Dongbao Biotechnology Co., Ltd. It was verified that gelatin of different sources and specifications had no significant difference in moisture, protein recovery, uniformity, and appearance of the freeze-dried powder obtained by the freeze-drying process.

[0080] The set time refers to the time it takes for the freeze dryer to reach the set temperature, and the duration refers to the time the freeze dryer continues to maintain the set temperature after reaching the set temperature.

[0081] Table 9 Freeze-drying process 1

[0082] Table 10 Freeze-drying process 2

[0083] Table 11 Freeze-drying process 3

[0084] Table 12 Freeze-drying process 4

[0085] Table 13 Freeze-drying process 5

[0086] Table 14 Freeze-drying process 6

[0087] Table 15 Freeze-drying process comparison 1

[0088] Table 16 Freeze-drying process comparison 2

[0089] Table 17 Freeze-drying process comparison 3

[0090] The specific results obtained by different freeze-drying processes are shown in Table 18. Freeze-drying processes 1-4 all had moisture content ≤3.0% and uniformity ≤1%, indicating that the processes were applicable. Among them, freeze-drying process 4 was the best, with the best indicators. Freeze-drying processes 5-6 all had moisture content ≤3.5% and uniformity ≤1%, indicating that the processes were applicable, with all indicators being excellent. Freeze-drying process controls 1 and 2 had relatively high moisture content, indicating that the freeze-drying process was not applicable. Freeze-drying process control 3 had unsatisfactory appearance, indicating that the freeze-drying process was not applicable.

[0091] Table 18 Test results of freeze-dried powder obtained by different freeze-drying processes

[0092] 2) Excipient compatibility study

[0093] The freeze-dried powder obtained in freeze-drying process 4 was ground and pulverized, passed through a 30-mesh sieve, and combined according to Table 19. The compatibility of the excipients was studied through accelerated, light, and high-humidity tests.

[0094] Table 19 Der f2 compatibility study ratio

[0095] The excipient compatibility test results are shown in Table 20. Lactose, mannitol, low-moisture microcrystalline cellulose, low-substituted hydroxypropyl cellulose, magnesium stearate, and behenyl glyceride (magnesium stearate was slightly superior to behenyl glyceride) showed relatively good compatibility with the recombinant Der f 2 protein freeze-dried powder, while sorbitol, microcrystalline cellulose, croscarmellose sodium, and sodium starch glycolate showed poor compatibility with the recombinant Der f 2 protein freeze-dried powder. The freeze-dried powders obtained using freeze-drying processes 1-6 were found to have comparable compatibility with the aforementioned excipients.

[0096] Table 20 Der f2 compatibility study results

[0097] 3) Preparation of protein sublingual tablets

[0098] The lyophilized powder was prepared according to condition 1 and lyophilization process 4. According to the results of the excipient compatibility study and the prescription composition in Table 21, Preparation Example 12 was prepared.

[0099] Table 21 Preparation Example 12 Der f2 Tablet Ratio

[0100] According to the mixing amounts listed in Table 21, using the progressive addition method, lactose was added layer by layer, followed by 5% lactose, lyophilized powder, 5% lactose, mannitol, 5% lactose, low-substituted hydroxypropyl cellulose, 5% lactose, low-moisture microcrystalline cellulose, and the remaining lactose. Finally, magnesium stearate was added and mixed. Sublingual tablets were produced using a small single-punch tablet press, with a core hardness of 20-60 N and a tablet weight of 50 mg.

[0101] Testing has shown that the lyophilization process for recombinant Der f 2 protein is also applicable to recombinant Der p 2 protein. Excipient compatibility studies have shown consistent results with those for recombinant Der f 2 protein. A recombinant Der p 2 protein solution (without gelatin) was lyophilized to obtain a lyophilized powder, which was then directly compressed into sublingual tablets (Preparation Example 13). The preparation methods for the lyophilized powder and sublingual tablets were the same as those for Preparation Example 12.

[0102] Example 6: Study on the freeze-drying process of recombinant dust mite type 1 allergen and preparation of protein sublingual tablets

[0103] 1) Lyophilization buffer and protein concentration study

[0104] Condition 1: The recombinant Der f 1 protein solution was replaced with a 50 mM acetic acid-sodium acetate pH 5.0 + 3% mannitol buffer system, the protein concentration was determined to be 0.1 mg / ml, and lyophilized using a vacuum freeze drying oven (LY0-21SP0IP, Tofflon) to prepare lyophilized powder as control group 6.

[0105] Condition 2: The recombinant Der f 1 protein solution was replaced with a 50 mM acetic acid-sodium acetate pH 5.0 + 3% mannitol buffer system, and the protein concentration was determined to be 0.5 mg / ml. The protein was lyophilized using a vacuum freeze drying oven (LY0-21SP0IP, Tofflon) to prepare a lyophilized powder as control group 7.

[0106] Condition 3: The recombinant Der f 1 protein solution was replaced with a 50 mM citric acid-sodium citrate pH 5.0 + 3% mannitol buffer system, the protein concentration was determined to be 0.1 mg / ml, and the solution was freeze-dried in a vacuum freeze-drying oven to prepare a freeze-dried powder as the control group 8.

[0107] Condition 4: The recombinant Der f 1 protein solution was replaced with a 50 mM citric acid-sodium citrate pH 5.0 + 3% mannitol buffer system, and the protein concentration was determined to be 0.5 mg / ml. The protein was lyophilized in a vacuum freeze-drying oven to prepare lyophilized powder as the control group 9.

[0108] Condition 5: The recombinant Der f 1 protein solution was replaced with a 50 mM citric acid-sodium citrate pH 5.0 + 3% mannitol buffer system, the protein concentration was determined to be 1.0 mg / ml, and lyophilized using a vacuum freeze drying oven to prepare lyophilized powder as a control group 10.

[0109] Condition 6: The recombinant Der f 1 protein solution was replaced with a 5 mM citric acid-sodium citrate + 1% mannitol + 2 mg / ml 150 LB gelatin buffer system (pH 5.0). The protein concentration was determined to be 0.04 ± 0.02 mg / ml. The solution was then freeze-dried using a vacuum freeze-drying oven to prepare a lyophilized powder as control group 11. The gelatin in this example was bovine-derived and purchased from Baotou Dongbao Biological Co., Ltd.; gelatin of different sources and specifications was found to have no significant effect on the moisture content, protein recovery, homogeneity, and appearance of the resulting lyophilized powder.

[0110] After testing, it was found that the freeze-drying process of recombinant Der f 2 protein is also applicable to recombinant Der f 1 protein. The freeze-drying parameters of conditions 1-5 of this example are the same as those of freeze-drying process 4 of example 5, and the freeze-drying parameters of condition 6 are the same as those of freeze-drying process 6 of example 5.

[0111] Controls 6-11 were tested under accelerated conditions for 24 hours. The results are shown in Table 22. It was confirmed that Der f 1 exhibited poor lyophilization performance at low concentrations in an acetic acid-sodium acetate pH 5.0 + 3% mannitol system (controls 6 and 7), with high water content. However, when lyophilized in a 50mM citric acid-sodium citrate pH 5.0 + 3% mannitol buffer system (controls 8, 9, and 10), protein quantification at 0.5 mg / ml and 1.0 mg / ml was relatively good. The 0.5 mg / ml lyophilized powder exhibited the best parameters and accelerated stability, representing the optimal condition. Stability was also observed when lyophilized in a 5mM citric acid-sodium citrate pH 5.0 + 1% mannitol buffer system plus 2 mg / ml 150 LB gelatin (control 11).

[0112] Table 22 Der f1 freeze-drying system research results

[0113] 2) Excipient compatibility study

[0114] The freeze-dried powder of control group 9 was ground and pulverized, passed through a 30-mesh sieve, and combined according to Table 23. The compatibility of the excipients was studied through accelerated, light, and high humidity tests.

[0115] Table 23 Der f1 compatibility study ratio

[0116] The excipient compatibility testing results are as follows: lactose, mannitol, low-moisture microcrystalline cellulose, low-substituted hydroxypropyl cellulose, and magnesium stearate are relatively compatible with recombinant Der f 1 protein lyophilized powder. Furthermore, when the active ingredient is other allergen proteins, the compatibility of these excipients with protein lyophilized powder is excellent. Croscarmellose sodium and behenyl glyceride protein lyophilized powder have poor compatibility.

[0117] Table 24 Der f1 compatibility study results

[0118] 3) Preparation of protein sublingual tablets

[0119] The freeze-dried powder of control group 9 was used according to the prescription composition of Table 25 to prepare sublingual tablet Preparation Example 14. The preparation method of the sublingual tablet was the same as that of Preparation Example 12.

[0120] Table 25 Preparation Example 14 Der f1 Sublingual Tablet Ingredients Ratio

[0121] The results of the lyophilization process for recombinant Der f 1 protein were consistent with those for recombinant Der p 1 protein.

[0122] The recombinant Der p 1 protein solution was lyophilized (without gelatin) to obtain lyophilized powder, which was then directly compressed into sublingual tablets, which is Preparation Example 15. The preparation methods of the lyophilized powder and sublingual tablets are the same as those of Preparation Example 14.

[0123] Example 7: Preparation of a sublingual tablet containing Der p 1 and Der f 2 proteins without gelatin

[0124] Recombinant Der f2 and Der p1 were lyophilized separately, with the lyophilization method for recombinant Der f2 being the same as in Example 5, Condition 1 and lyophilization Process 2, and the lyophilization method for recombinant Der p1 being the same as in Example 6, Condition 4 and lyophilization Process 4. The resulting lyophilized powders were then pulverized, passed through a 30-mesh sieve, and assayed for protein content. Preparation Example 16: Sublingual tablets containing the two proteins were prepared according to the mixing amounts described in Table 26. Lactose was added layer by layer as the raw material, with 5% lactose, recombinant Der f2 lyophilized powder, 5% lactose, recombinant Der f1 lyophilized powder, 5% lactose, mannitol, 5% lactose, low-substituted hydroxypropyl cellulose, 5% lactose, low-moisture microcrystalline cellulose, and the remaining lactose being added and mixed. Finally, magnesium stearate was added and mixed. Tablets were compressed using a small single-punch tablet press, with a core hardness of 20-60N and a tablet weight of 50 mg.

[0125] Table 26 Preparation Example 16 Der p1, Der f2 two protein sublingual tablets composition ratio

[0126] Example 8: Preparation of Der p 1 and Der f 2 protein sublingual tablets containing different types of gelatin

[0127] Recombinant Der f2 and Der p1 were lyophilized separately. Recombinant Der f2 was prepared using the same method as in Example 5, Condition 2 and lyophilization Process 6. Recombinant Der p1 was prepared using the same method as in Example 6, Condition 6 and lyophilization Process 5. The resulting lyophilized powders were pulverized, passed through a 30-mesh sieve, and assayed for protein content. In Preparation Examples 17 to 19, sublingual tablets containing different types of gelatin were prepared using 180 LB gelatin, 240 LB gelatin, and 245 LB gelatin, respectively, in the proportions listed in Table 27. The 180 LB and 240 LB gelatin were bovine gelatin purchased from Rousselot Gelatin Co., Ltd., and the 245 LB gelatin was porcine gelatin purchased from Gelita Gelatin Co., Ltd. The preparation methods were the same as in Example 7.

[0128] Table 27 Ingredient ratio of Der p1 and Der f2 protein sublingual tablets in Preparation Examples 17 to 19

[0129] Example 9: Preparation of a sublingual tablet containing four proteins, Der p 1, Der p 2, Der f 1, and Der f 2, without gelatin

[0130] Recombinant Der f2, Der p2, Der f1, and Der p1 were lyophilized separately. The lyophilization method for recombinant Der f2 and Der p2 was the same as in Example 5, Condition 1 and Process 3. The lyophilization method for recombinant Der f1 and Der p1 was the same as in Example 6, Condition 4 and Process 4. The resulting lyophilized powders were pulverized, passed through a 30-mesh sieve, and assayed for protein content. Sublingual tablets containing the four protein preparations were prepared in Preparation Examples 20 to 25, respectively, according to the mixing amounts listed in Tables 28-33. The preparation methods for Preparation Examples 20 and 21 were the same as those for Example 7. For Preparation Examples 22-25, lactose was added layer by layer using a three-dimensional motion mixer. A mixture of 5% lactose, low-substituted hydroxypropylcellulose, 5% lactose, recombinant Der f2 lyophilized powder, recombinant Der f1 lyophilized powder, recombinant Der p2 lyophilized powder, and recombinant Der p1 lyophilized powder, 5% lactose, low-moisture microcrystalline cellulose, mannitol, and the remaining lactose were added and mixed. Finally, magnesium stearate was added and mixed. A rotary tablet press was used to control the tablet core hardness to 20-60 N and the tablet weight to 50 mg. The order of addition of the four lyophilized powders could be changed at will, and the addition ratio could be adjusted.

[0131] Table 28 Ingredient ratio of Preparation Example 20

[0132] Table 29 Ingredient ratio of Preparation Example 21

[0133] Table 30 Preparation Example 22 Ingredient Ratio

[0134] Table 31 Ingredient ratio of Preparation Example 23

[0135] Table 32 Ingredient ratio of Preparation Example 24

[0136] Table 33 Ingredient ratio of Preparation Example 25

[0137] Example 10: Preparation of four protein sublingual tablets containing different types of gelatin: Der p 1, Der p 2, Der f 1, and Der f 2

[0138] Recombinant Der f2, Der p2, Der f1, and Der p1 were lyophilized separately. The preparation methods for recombinant Der f2 and Der p2 were the same as those in Example 5, Condition 2 and lyophilization Process 6. The preparation methods for recombinant Der f1 and Der p1 were the same as those in Example 6, Condition 6 and lyophilization Process 5. The resulting lyophilized powders were pulverized, passed through a 30-mesh sieve, and assayed for protein content. Preparation Examples 26-28, respectively, used 150 LB of gelatin, 180 LB of gelatin, and 240 LB of gelatin, using the blending amounts listed in Table 34, to prepare sublingual tablets. Preparation Examples 29-31, respectively, used the blending amounts listed in Tables 35-37 to prepare sublingual tablets. The preparation methods were the same as in Example 7.

[0139] Table 34 Preparation Examples 26-28 Using Different Gelatin Lyophilized Protein Powders to Prepare Sublingual Tablets Ingredient Ratios

[0140] Table 35 Ingredient ratio of Preparation Example 29

[0141] Table 36 Ingredient ratio of Preparation Example 30

[0142] Table 37 Ingredient ratio of Preparation Example 31

[0143] Example 11: Preparation of four protein sublingual tablets containing different types of gelatin: Art a1, Art a3, Art v1, and Art v3

[0144] Art a1, Art a3, Art v1, and Art v3 were lyophilized separately, and the protein solutions were replaced with 10mM citric acid-sodium citrate + 1% mannitol + 3mg / ml 180LB gelatin, pH 5.0 buffer system. The protein concentration was determined to be 0.06±0.03mg / ml, and lyophilized using a vacuum freeze drying chamber to prepare lyophilized powders. The lyophilization process for Art a1 and Art v1 was the same as that for Example 5, lyophilization process 6. The preparation method for Art a3 and Art v3 was the same as that for Example 5, lyophilization process 5. The resulting lyophilized powders were pulverized, passed through a 30-mesh sieve, and tested for protein content. Preparation Examples 32 and 33 were each prepared using the mixing amounts described in Tables 38-39 for sublingual tablets. The preparation method was the same as that for Example 7.

[0145] Table 38 Ingredient ratio of Preparation Example 32

[0146] Table 39 Ingredient ratio of Preparation Example 33

[0147] Example 12: Testing of sublingual tablets prepared in Preparation Examples 12 to 31

[0148] Preparation Examples 12 to 33 active ingredients contain one protein, two proteins, and four proteins, respectively, with or without gelatin, or with gelatin from different sources. The hardness RSD value and weight difference of the obtained sublingual tablet are relatively small, the disintegration time is short, and the oral sensory experience is free of dryness, gravel, moderate sweetness, and excellent content uniformity. Preparation Examples 12-33 use a variety of different protein raw materials, and the proportion of protein raw materials in the tablet is relatively low, ranging from 0.276% (Preparation Example 21) to 13.3% (Preparation Examples 16, 26 to 31). They can all disintegrate rapidly under the tongue, and the oral disintegration time is within 90S (90 seconds). Protein raw material content is relatively low in this application's prescription, especially Preparation Example 12 protein raw materials only account for 0.74%, and Preparation Example 21 protein raw materials only account for 0.276% prescription, but all meet the requirement that the uniformity specified in the pharmacopoeia is less than 15. Some results are shown in Table 40.

[0149] In addition, since the protein content as the active ingredient in the sublingual tablet is relatively low, those skilled in the art can reasonably expect that when the active ingredient is other allergen proteins, the resulting sublingual tablet will still have excellent hardness RSD values, small tablet weight differences, short disintegration time, no dryness or grittiness when taken orally, moderate sweetness, and uniform content.

[0150] Table 40 Tablet test results of Preparation Examples 12 to 19

[0151] Long-term and accelerated tests were conducted on the sublingual tablets of Preparation Examples 16-19 containing two allergen proteins, and the content of type I major allergens and type II major allergens were tested. It was confirmed that the sublingual tablets of Preparation Examples 17-19 containing the auxiliary material gelatin in the preparation examples had better protein stability under high temperature conditions than the sublingual tablet of Preparation Example 16 without gelatin.

[0152] Table 41: Long-term storage at 25°C and accelerated test of tablets of Preparation Examples 16-19

[0153] Furthermore, the sublingual tablets of Preparation Examples 23, 24, and 26-33, containing four allergen proteins, were subjected to tests for factors such as 25°C, accelerated temperature, light exposure, and high humidity, and their main allergen content was measured. It was confirmed that the sublingual tablets of Preparation Examples 26-32, containing the excipient gelatin, exhibited superior protein stability under high temperature, light exposure, and high humidity conditions compared to the sublingual tablets of Preparation Examples 23 and 24, which did not contain gelatin. Preparation Examples 32 and 33 showed superior stability when stored at high temperatures of 25°C and 40°C for a long time. The results are shown in Tables 40 and 41.

[0154] Table 40 Test on long-term storage of dust mite tablets at 25°C with factors affecting acceleration, light and high humidity

[0155] Table 41 Study on the stability of Artemisia pollen tablets

[0156] In addition, the stability of different sublingual tablets containing single allergen proteins (Der p1, Der p2, Der f1, Der f2) with or without gelatin was investigated. It was also found that the sublingual tablets with gelatin were more stable than those without gelatin.

Claims

1. A sublingual tablet, characterized in that: The sublingual tablet contains protein raw material, lactose, mannitol, low-substituted hydroxypropyl cellulose, low-moisture microcrystalline cellulose, and magnesium stearate, and the mass percentage of each component is 0%-15.6%, 0%-83.4%, 0-83.4%, 4%-6%, 10%-15%, and 0.4%-0.6%.

2. The sublingual tablet according to claim 1, wherein the protein raw material is an allergen protein raw material.

3. The sublingual tablet according to claim 1, wherein the mass percentages of the protein raw material, lactose, mannitol, low-substituted hydroxypropyl cellulose, low-moisture microcrystalline cellulose, and magnesium stearate are 0%-15.6%, 50%-83.4%, 0%-33.4%, 4%-6%, 10%-15%, and 0.4%-0.6%.

4. The sublingual tablet according to claim 1, wherein the protein raw material, lactose, mannitol, low-substituted hydroxypropyl cellulose, low-moisture microcrystalline cellulose, and magnesium stearate account for 0.276%-15.6%, 59.3%-76.48%, 5%-13.4%, 4%-6%, 10%-12.5%, and 0.4%-0.6% of the tablet weight, respectively.

5. The sublingual tablet according to claim 1, wherein the protein is dust mite allergen protein, and is composed of one or a mixture of two or more of Der p 1 raw materials, Der p 2 raw materials, Der f 1 raw materials, and Der f 2 raw materials.

6. The sublingual tablet according to claim 1, wherein the protein is an Artemisia pollen allergen protein, and is composed of one or a mixture of two or more of Art a1 raw materials, Art a3 raw materials, Art v1 raw materials, and Art v3 raw materials.

7. The sublingual tablet according to any one of claims 1 to 6, wherein the protein raw material is a freeze-dried powder of allergen protein.

8. The sublingual tablet according to claim 7, wherein the lyophilized protein powder may or may not be added with gelatin.

9. The sublingual tablet according to claim 7, wherein: The buffer system used for protein freeze-drying contains citric acid-sodium citrate, mannitol; or PB, mannitol.

10. The sublingual tablet according to any one of claims 1 to 9, wherein: The protein freeze-dried powder used was prepared by the following method: The protein-containing solution is replaced with a suitable buffer system and pH conditions, and quantified to an appropriate concentration. After three steps of pre-freezing, primary drying, and analytical drying, a freeze-dried product is obtained.