Application to sublingual tablets and desensitization therapies

Abstract

The present invention relates to the field of pharmaceutical preparations, in particular to sublingual tablets and their application in hyposensitization therapy. [Solution] The sublingual tablet of the present invention is a low-dose allergen sublingual tablet, containing a freeze-dried powder of an allergen protein, as well as lactose, mannitol, low-substituted hydroxypropyl cellulose, low-moisture microcrystalline cellulose, and magnesium stearate. The allergen protein sublingual tablet of the present invention is prepared by freeze-drying the freeze-dried powder of the allergen protein to produce tablets using direct powder compression molding. The low-dose sublingual tablet of the present invention is excellent in various parameters such as appearance, hardness, tablet weight difference, disintegration time, swallowing sensation, and content uniformity, and particularly has significant advantages in disintegration time, content uniformity, stability, and manufacturing process, making it worthy of industrial application.

Description

Technical Field

[0005]

[0001] The present invention relates to the field of pharmaceutical preparations, and particularly to sublingual tablets and their application to desensitization therapy drugs.

Background Art

[0002] Allergen-specific immunotherapy is currently the only "causal therapy" aimed at treating allergic diseases through immunomodulation. This aims to increase the patient's tolerance to allergens by gradually increasing the dosage of allergens, reduce the symptoms caused by exposure to the allergens, and ultimately achieve tolerance and even immune tolerance.

[0003] The main administration methods of allergen-specific immunotherapy include subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT). Allergen-specific immunotherapy has a long treatment period (2 - 3 years), requires multiple injections in subcutaneous injection, has low patient compliance, and is prone to side effects such as severe systemic anaphylactic shock. In contrast, sublingual administration has fewer side effects, high safety, and also improves patient compliance.

[0004] Currently, there are mainly two dosage forms for sublingual administration of allergens: sublingual solution and sublingual tablets. Sublingual tablets refer to tablets that are placed under the tongue and quickly dissolve or rapidly disintegrate in saliva during use, and the drug is absorbed through the oral mucosa under the tongue to exert a systemic effect. Compared with sublingual solution, sublingual tablets are convenient to take and carry, easy to control the dosage, can ensure the standardization of drug dosage, have high patient compliance, and also have excellent stability.

[0005] Developing formulations for allergen sublingual tablets is more difficult than developing standard sublingual tablets. First, the active ingredient in allergen sublingual tablets is a protein, which is unstable to moisture and heat. Currently, common methods for manufacturing sublingual tablets include granulation and compression (including wet and dry granulation), blister freeze-drying, and direct tablet compression molding. Of these, wet granulation and compression requires granulation, drying, and sizing, making it unsuitable for heat- and moisture-sensitive drugs with high solubility. Dry granulation and compression requires compression to create interparticle bonding forces, and high pressure can easily lead to problems such as changes in crystal structure and decreased activity. Commercially available allergen sublingual tablets, such as ODACTRA® from ALK, use blister freeze-drying. This method requires a dedicated manufacturing line, such as a liquid nitrogen freezing tunnel, which has high demands on manufacturing equipment, resulting in high equipment costs, and related technologies are not yet mature in Japan. Overall, direct powder compression molding eliminates the wet granulation process and the need for a drying process, making it suitable for drugs that are unstable to moisture and heat. It also reduces the overall number of steps, saving time and energy. However, direct powder compression molding has drawbacks, including poor powder flowability, large variations in tablet weight, tablet breakage, and reduced content uniformity. The issue of content uniformity becomes particularly pronounced when the active ingredient content in the tablet is low.

[0006] Next, compared to regular sublingual tablets, allergen sublingual tablets have an extremely low active ingredient content, with the active ingredient accounting for less than 2% of the tablet weight, and in some cases even less than 1%. Such extremely low levels of active ingredient lead to problems with content uniformity and stability. Chinese patent CN108524454A discloses a method for producing low-dose drug compositions using a double-screw technology apparatus. Chinese patent CN1531423A discloses a method for producing low-dose drugs using a high-shear granulation method. Similarly, the production of low-dose drug formulations requires additional manufacturing equipment. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] China patent CN108524454A [Patent Document 2] China patent CN1531423A [Overview of the project] [Problems that the invention aims to solve]

[0008] This invention addresses the shortcomings of the prior art by providing low-dose sublingual tablets applicable to various active ingredients and their application to desensitization therapies, thereby resolving issues of content uniformity and stability of low-dose sublingual tablets, as well as the complexity of manufacturing equipment and processes. [Means for solving the problem]

[0009] The present invention provides a type of sublingual tablet. The sublingual tablet contains a protein raw material, lactose, mannitol, low-substituted hydroxypropyl cellulose, low-moisture microcrystalline cellulose, and magnesium stearate, with the mass percentages of each component being 0-15.6%, 0-83.4%, 0-83.4%, 4-6%, 10-15%, and 0.4-0.6%, respectively.

[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%, respectively.

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

[0013] There are many types of allergens, which can be broadly divided into two categories: inhalant allergens and food allergens. Common inhalant allergens include house dust mites, powdery house dust mites, cockroaches, cat and dog dander, mold, and pollen, while common food allergens include milk, peanuts, and eggs. The allergen proteins may be obtained through natural extraction or recombinant expression. The major allergen proteins of house dust mites (Der p) are Der p 1 and Der p 2, and the major allergen proteins of powdery house dust mites (Der f) are Der f 1 and Der f 2. The major allergen proteins of Artemisia annua pollen are Art a 1 and Art a 3, and the major allergen proteins of Artemisia vulgaris pollen are Art v 1 and Art v 3. Because the sublingual tablets of the present invention have an extremely low content of active ingredients, despite differences in the physicochemical properties of different active ingredients, the data from the examples demonstrates that differences in active ingredients do not affect the performance of the final sublingual tablets, such as hardness, tablet weight variation, disintegration time, content uniformity, and stability. Therefore, the formulation of the sublingual tablets of the present invention is applicable to a variety of low-dose active ingredients and is not limited to the specific active ingredients listed in the examples.

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

[0015] A second aspect of the present invention relates to the freeze-drying process. The buffer components and protein concentrations of the freeze-drying solution suitable for a particular protein can be determined through a finite number of experiments based on conventional methods. The buffer components selected in the specific examples are illustrative and do not limit the buffer components used for freeze-drying allergen proteins. For example, if the active ingredients are Der p 2 and Der f 2, PB and mannitol may be selected as buffer components. If the active ingredients are Der p 1, Der f 1, Art a 1, Art a 3, Art v 1, and Art v 3, citrate-sodium citrate and mannitol may be selected as buffer components. The protein concentration may be selected from the range of 0.03 to 1.0 mg / mL. Furthermore, gelatin may or may not be added to the freeze-drying solution of the allergen protein. If the stability of the allergen protein is poor, adding gelatin is advantageous in maintaining the stability of the allergen protein sublingual tablets.

[0016] A general freeze-drying process is as follows: The solution containing the allergen protein is replaced with an appropriate buffer system and pH conditions, quantified to the appropriate concentration, and then obtained as a freeze-dried product through three steps: pre-freezing, primary drying, and analytical drying. A freeze-drying process suitable for a particular protein can be determined by screening using conventional methods. Production Examples 12-15 illustrate freeze-drying processes for different allergen proteins; these processes are illustrative and do not limit the freeze-drying process for allergen proteins. For example, in Production Examples 12, 16, and 20, different freeze-drying processes are applied to mite group 2 proteins, and the performance of the final tablets is almost identical. The pre-freezing step is performed in two stages. In the first stage, the pre-freezing temperature is set to -12 to -15°C and maintained for 60 to 90 minutes after reaching that temperature. In the second stage, the pre-freezing temperature is set to -45 to -50°C and maintained for 120 to 800 minutes after reaching that temperature. The temperature for the primary drying is set to -10 to -20°C and maintained for 840 to 960 minutes after reaching that temperature. The vacuum level is 0.18 to 0.2 mbar. The temperature for the analytical drying is set to 20 to 25°C and maintained for 480 to 1500 minutes after reaching that temperature. The vacuum level is 0.18 to 0.2 mbar.

[0017] A third aspect of the present invention relates to a direct powder compression molding process for allergen sublingual tablets, which does not require complex processes or equipment. In the direct powder compression molding method, tablets are obtained by directly compressing a mixture of the active drug and additives without going through a granulation process. The process steps of this method mainly include crushing, sieving, mixing, adding and remixing of the main drug and additives, and compression. Through many years of research, the inventors have found that the resulting low-dose sublingual tablet formulation not only has ideal hardness, tablet weight variation, disintegration time, content uniformity, and stability, but is also very suitable for the direct powder compression molding method, overcoming the above-mentioned problems that exist in conventional direct powder compression molding methods.

[0018] The sublingual tablets of the present invention are excellent in various parameters such as physical properties (smooth surface and uniform white tablets in color tone), hardness (appropriate hardness of 15 to 60 N, which does not have a great impact on the packaging and transportation of the tablets), tablet weight variation, disintegration time, taste during administration, and content uniformity. In particular, there are outstanding advantages in terms of disintegration time, content uniformity, stability, and manufacturing process.

[0019] 1. The sublingual tablets of the present invention can disintegrate rapidly under the tongue and dissolve with almost no foreign body sensation. The active ingredient is absorbed through the oral mucosa under the tongue to exert a systemic effect. However, if the disintegration time is too long, it is likely to be swallowed, and some of the active ingredients may not be completely absorbed as expected via the sublingual mucosa, which may cause inaccuracy in the dosage.

[0020] 2. The sublingual tablets of the present invention have good uniformity in allergen content and can avoid the problem of content non-uniformity that occurs when the content of the active ingredient in the tablets is low. Therefore, it is suitable for sublingual allergen tablets with a low content of the active ingredient.

[0021] 3. The sublingual tablets of the present invention not only have high stability at room temperature but also have high stability under severe environmental conditions and can be applied to various bioactive drugs.

[0022] 4. The low-dose sublingual tablets of the present invention achieve the above excellent comprehensive performance, but the manufacturing process is simple, applicable to the powder direct compression molding method, the process is convenient, and there are no additional requirements for production equipment.

Embodiments for Carrying Out the Invention

[0023] Examples of production and comparative examples will be given below for explanation, but the present invention is not limited thereto. Production Examples 1 to 11 are production examples and comparative examples of blank sublingual tablets containing no active ingredient. For the obtained tablets, measurement of tablet hardness, tablet weight variation, disintegration time, and sensory tests were carried out. Production Examples 12 to 26 are production examples of low-dose sublingual tablets containing an active ingredient. For the obtained tablets, in addition to measuring tablet hardness, tablet weight variation, disintegration time, content uniformity, and sensory evaluation, factor tests such as long-term storage, high temperature, light irradiation, and high humidity are carried out on the sublingual tablets containing the active ingredient, and the total protein content and purity are measured to evaluate stability.

[0024] The specific measurement methods are as follows. 1. Hardness measurement Use a hardness measurement device dedicated for hardness measurement (CJY-2C type hardness meter manufactured by TianDa TianFa) to measure the hardness of the tablets. Extract 10 tablets for measurement and record the average value and RSD value as the displayed amount.

[0025] 2. Tablet weight variation measurement According to the tablet weight variation measurement method in the General Principles of the Chinese Pharmacopoeia 2020 Edition 0101, take 20 tablets of the test sample, accurately measure the total weight, calculate the average tablet weight, and then accurately measure the weight of each tablet. Compare the weight of each tablet with the displayed amount, and the number of tablets exceeding the weight variation limit shall not exceed 2 tablets, and no single tablet shall exceed 1 times the limit (when the displayed amount or the average tablet weight is 0.3 g or less, the weight variation limit is ±7.5%).

[0026] 3. Disintegration time measurement According to the disintegration time limit measurement method in the General Principles of the Chinese Pharmacopoeia 2020 Edition 0921, use a disintegration tester (ZB-1E type manufactured by TianDa TianFa), immerse it in a 1000 ml beaker containing water at 37 ± 1 °C, and take 6 tablets of the test sample for measurement. Confirm that each tablet completely disintegrates and dissolves within 5 minutes.

[0027] 4. Moisture measurement of freeze-dried powder Refer to the first method (Karl Fischer method) coulometric titration of the moisture measurement method in the General Principles of the Chinese Pharmacopoeia 2020 Edition 0832.

[0028] 5. Sensory evaluation Six healthy adults were selected to take a sublingual tablet by placing it under their tongue, and their perception of the tablet's texture during administration was evaluated. The evaluation criteria mainly included the presence or absence of dryness or discomfort, the presence or absence of a gritty texture, the appropriateness of the sweetness, and the time of disintegration in the mouth. After the test, the sublingual tablet was spat out and the participants rinsed their mouths with water.

[0029] 6. Compatibility testing of additives A suitable amount of lyophilized active drug powder is weighed into a vial, and additives of different proportions are added to each vial. Microcrystalline cellulose, low-moisture microcrystalline cellulose, filler, and low-substituted hydroxypropyl cellulose are added in amounts five times that of the lyophilized powder, while the lubricant is added in the same amount as the lyophilized powder. The compatibility of the lyophilized active drug powder with each additive is confirmed by conducting accelerated, high-humidity, and light irradiation tests and measuring the purity.

[0030] 7. Accelerated testing Place the test sample in a vial of the appropriate size and seal it. Place the vial in a stability test chamber (KBF-P-720 type, Binder) set to accelerated conditions (40±2℃, 75±5%RH), remove it after the specified time, and perform the measurement.

[0031] 8. High-temperature test Place the test sample in an appropriately sized vial and seal it. Place the vial in a stability test chamber (KBF-P-720 type, Binder) set to high temperature conditions (60±2℃), remove it after the specified time, and perform the measurement.

[0032] 9. High humidity test Place the test sample in an appropriately sized vial and leave it open. Place the vial in a stability test chamber (KBF-720 type, Binder) set to high humidity conditions (25±2℃, 75±5%RH), remove it after the specified time, and perform the measurement.

[0033] 10. Light irradiation test Place the test sample in an appropriately sized vial and seal it. Place the vial horizontally in a stability test chamber (KBF-P-720 type, Binder) set to light irradiation conditions (25±2℃, 60±5%RH, 4500±500lx), remove it after the specified time, and perform the measurement.

[0034] 11, Purity measurement Der p 1 and Der f 1 are measured according to the size exclusion chromatography method in the 2020 edition of the Chinese Pharmacopoeia General Rules 0514, and Der p 2 and Der f 2 are measured according to the reversed-phase chromatography method in the 2020 edition of the Chinese Pharmacopoeia General Rules 0512.

[0035] 12. Measurement of protein content and uniformity in freeze-dried powder Der p 1 and Der f 1 measure the protein content and uniformity (RSD value) of lyophilized powder from different parts of the same batch, based on the size exclusion chromatography method described in the 2020 edition of the Chinese Pharmacopoeia General Rules 0514, while Der p 2 and Der f 2 measure the reversed-phase chromatography method described in the 2020 edition of the Chinese Pharmacopoeia General Rules 0512.

[0036] 13. Measurement of the content and titer of major allergens Protein content is measured using the double antibody sandwich ELISA method. The content of the major mite group 1 allergen refers to the sum of the contents of Der p 1 and Der f 1, and the content of the major mite group 2 allergen refers to the sum of the contents of Der p 2 and Der f 2. The content of the major allergen of Artemisia princeps pollen group 1 refers to the sum of the contents of Art a 1 and Art v 1, and the content of the major allergen of Artemisia princeps pollen group 3 refers to the sum of the contents of Art a 3 and Art v 3.

[0037] The titer is measured using the suppression ELISA method. Recombinant protein samples are diluted in a series of dilution steps and then incubated with serum pooling serum. The incubated mixture is added to an ELISA plate pre-coated with the recombinant protein to establish a linear relationship of "suppression rate - dilution factor". The biological activity required to reduce IgE in serum pooling serum by 50% is defined as 100 BU / ml, and the biological activity of the recombinant protein is calculated. To more clearly reflect the trend of activity change, the obtained biological activity is calculated as a percentage based on the theoretically indicated amount, and the measured value is expressed as measured activity / theoretically indicated amount × 100%.

[0038] 14, Content uniformity measurement The protein content in the tablets is measured using immunodot blotting, and the content uniformity is calculated based on the method for measuring content uniformity in accordance with General Rule 0941 of the 2020 edition of the Chinese Pharmacopoeia. According to the provisions of General Rule 0941, this product is a sublingual tablet and has an L value of 15. That is, the standard difference S and the absolute value A of the difference between the stated amount and the mean value are calculated from the results of measuring the content of 10 tablets of the test sample, and if A + 2.2S is less than 15, it is judged that the content uniformity meets the acceptance criteria. [Examples]

[0039] Example 1: Formulation Study 1 (Selection of Disintegrant) [Table 1]

[0040] The lactose used in this application is FlowLac® 100 from MEGGLE GmbH in Germany, the mannitol is Mannitol 100SD from Roquette GmbH in France, and the low-substituted hydroxypropyl cellulose is variety LH-21 with a hydroxypropoxy group content of 10.0% to 12.9%, purchased from Huzhou Dianyan Pharmaceutical Co., Ltd. Low-substituted hydroxypropyl cellulose with a hydroxypropoxy group content of 5.0% to 16.0% (calculated on the dry product) is all usable in this invention and does not affect tablet performance. The low-moisture microcrystalline cellulose is variety VIVAPUR PH112 with a dry weight loss of ≤1.5%, purchased from Shanghai Fenghong Pharmaceutical Aid Technology Co., Ltd.

[0041] Manufacturing Example 1: Following the proportions listed in Table 1, and using the sequential addition method as a reference, lactose is added sequentially to the raw materials. 5% lactose, mannitol, 5% lactose, low-substituted hydroxypropyl cellulose, 5% lactose, low-moisture microcrystalline cellulose, and the remaining 85% lactose are added and mixed in sequence, and finally magnesium stearate is added and mixed. A small single-stroke tablet compressor is used to compress the tablets, controlling the hardness to 20-60N and the tablet weight to 50mg.

[0042] Control Groups 1-3: The low-substituted hydroxypropyl cellulose used in Production Example 1 was replaced with hydroxypropyl cellulose (SSL-L), cross-linked carboxymethylcellulose sodium, and carboxymethyl starch sodium, while the types and amounts of other additives remained unchanged. Each group was then prepared to form Control Groups 1-3. The production method was the same as in Production Example 1.

[0043] The experimental results showed that when low-substituted hydroxypropyl cellulose was used, the sublingual tablets exhibited smaller RSD values ​​and tablet weight differences, shorter oral disintegration time, a pleasant oral experience without dryness or grittiness, and a moderate sweetness. On the other hand, when other disintegrants were used, the disintegration time was too long, and even after adjusting the types and amounts of other additives in the formulation, tablets with the above-mentioned superior overall performance could not be obtained. The specific results are as follows.

[0044] [Table 2]

[0045] Example 2: Formulation Study II (Selection of Filler and its Usage Amount) [Table 3]

[0046] Based on the formulation ratios listed in Table 3, prepare the sublingual tablets of Production Examples 2-5. In Production Example 2, all of the mannitol in Production Example 1 is replaced with lactose, while in Production Examples 3-5, the lactose in Production Example 1 is partially or completely replaced with mannitol. Otherwise, the process is the same as in Production Example 1.

[0047] The specific results are shown in Table 4. In manufacturing examples 1-5, the lactose ratios were approximately 70.0%, 83.4%, 50.0%, 27.4%, and 0%, respectively, and all parameters such as sublingual tablet hardness RSD value, tablet weight difference, oral disintegration time, and oral texture were excellent. Manufacturing example 1 was the best, with the shortest disintegration time. Manufacturing example 3 was the next best, and manufacturing example 2 was relatively good. Manufacturing examples 4 and 5 had relatively long disintegration times and slightly inferior texture. Therefore, the combination of lactose and mannitol is optimal, and as the lactose addition ratio increases, the hardness RSD value and tablet weight difference become relatively small, the oral disintegration time shortens, the oral texture is free from dryness and grittiness, and the sweetness is moderate.

[0048] [Table 4]

[0049] Example 3: Formulation Study III (Selection of low-moisture microcrystalline cellulose / microcrystalline cellulose and lubricant) [Table 5]

[0050] In Production Example 1, the low-moisture microcrystalline cellulose is replaced with microcrystalline cellulose to obtain Production Example 6. The production method is the same as in Production Example 1. Replace the magnesium stearate in Production Example 1 with glyceryl behenate ester to obtain Production Example 7. The manufacturing method is the same as in Production Example 1. Control Group 4: Control Group 4 is prepared according to the mixing ratios listed in Table 5, without using low-moisture microcrystalline cellulose or microcrystalline cellulose and mannitol. The manufacturing method is the same as in Manufacturing Example 1.

[0051] The specific results are shown in Table 6. When using microcrystalline cellulose and behenate glycerol ester, all parameters such as sublingual tablet hardness RSD value, tablet weight difference, oral disintegration time, and oral administration feel were excellent (however, compatibility studies of additives in manufacturing examples 11-14 showed that microcrystalline cellulose and behenate glycerol ester did not have good compatibility with protein allergen active ingredients). On the other hand, in control group 4, a serious powder leakage phenomenon occurred during the compression molding process, making it unsuitable for direct powder compression molding processes.

[0052] [Table 6]

[0053] Example 4: Formulation Study IV (Study on the blending ratio of additives) [Table 7]

[0054] Based on the formulation ratios listed in Table 7, sublingual tablets for manufacturing examples 8-11 and control groups 5 and 6 are prepared. The manufacturing method is the same as for manufacturing example 1.

[0055] The specific results are shown in Table 8. Sublingual tablets from manufacturing examples 8-11 showed superior performance in all parameters, including hardness RSD value, tablet weight variation, oral disintegration time, and oral administration feel. On the other hand, control groups 5 and 6 had large tablet weight variations and long disintegration times, making the additive formulation ratios for control groups 5 and 6 unsuitable.

[0056] [Table 8]

[0057] Example 5: Study of freeze-drying process for recombinant mite group 2 allergens and production of protein sublingual tablets 1) Research on freeze-drying processes Condition 1: The recombinant Der f 2 protein solution was replaced with a buffer system of 50 mM PB + 3% mannitol, pH 7.0. The protein concentration was measured using a BCA protein concentration measurement kit and adjusted to 1.0 mg / ml. The solution was then poured into a lyophilization tray, the sample level was controlled to 1 cm or less, and lyophilization was performed using a vacuum lyophilizer (LY0-21SP0IP model, Dongfu Long) according to the parameters in Tables 9-12 (lyophilization processes 1-4) and Table 15 (lyophilization process control 1) to produce lyophilized powder.

[0058] Condition 2: The recombinant Der f 2 protein solution was replaced with a buffer system of 5 mM PB + 1% mannitol + 1.5 mg / ml 150 LB gelatin (bovine gelatin, purchased from Baotou Dongbao Biotechnology Co., Ltd.), pH 7.0. The protein concentration was measured and adjusted to 0.03 ± 0.015 mg / ml. The solution was then poured into a freeze-drying tray, the sample level was controlled to 1 cm or less, and freeze-drying was performed using a vacuum freeze-dryer according to the parameters in Tables 13, 14, 16, and 17 (freeze-drying processes 5 and 6, freeze-drying process controls 2 and 3) to produce freeze-dried powder. The gelatin used in this example was bovine and purchased from Baotou Dongbao Biotechnology Co., Ltd. Verification results showed no significant differences in moisture content, protein recovery rate, uniformity, or appearance of the freeze-dried powder obtained through the freeze-drying process, even when using gelatin of different origins and specifications.

[0059] 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 maintains that temperature after it has reached it.

[0060] Table 9

[0061] Table 10

[0062] Table 11

[0063] Table 12

[0064] Table 13

[0065] Table 14

[0066] Table 15

[0067] Table 16

[0068] Table 17

[0069] Table 18 shows the specific measurement results for freeze-dried powders obtained using different freeze-drying processes. For freeze-drying processes 1-4, the moisture content was 3.0% or less in all cases, and the uniformity was 1% or less in all cases, indicating that all processes were applicable. Of these, freeze-drying process 4 performed best, with the highest index. For freeze-drying processes 5-6, the moisture content was 3.5% or less in all cases, and the uniformity was 1% or less in all cases, indicating that the processes were applicable and all indexes were excellent. On the other hand, freeze-drying process controls 1 and 2 had relatively high moisture content, making the freeze-drying process unsuitable. Furthermore, freeze-drying process control 3 had a poor appearance, making the freeze-drying process unsuitable.

[0070] [Table 18]

[0071] 2) Compatibility study of additives The freeze-dried powder obtained in freeze-drying process 4 is polished and ground, passed through a 30-mesh sieve, and blended according to Table 19. Compatibility studies of the additives are then conducted through accelerated, light irradiation, and high-humidity tests.

[0072] [Table 19]

[0073] The results of the additive compatibility study are shown in Table 20. Lactose, mannitol, low-moisture microcrystalline cellulose, low-substituted hydroxypropyl cellulose, magnesium stearate, and behenate glycerol ester (magnesium stearate was slightly better than behenate glycerol ester) showed relatively good compatibility with recombinant Der f 2 protein lyophilized powder. On the other hand, sorbitol, microcrystalline cellulose, cross-linked carboxymethylcellulose sodium, and carboxymethyl starch sodium did not show good compatibility with recombinant Der f 2 protein lyophilized powder. Verification revealed that the compatibility results between the lyophilized powders obtained in lyophilization processes 1-6 and these additives were almost identical.

[0074] [Table 20]

[0075] 3) Manufacturing of protein sublingual tablets Using the freeze-dried powder produced under Condition 1 and freeze-drying process 4, Production Example 12 was prepared according to the formulation composition shown in Table 21, based on the results of additive compatibility studies.

[0076] [Table 21]

[0077] Based on the formulation amounts listed in Table 21, and following the sequential addition method, lactose is added sequentially to the raw materials. 5% lactose, freeze-dried powder, 5% lactose, mannitol, 5% lactose, low-substituted hydroxypropyl cellulose, 5% lactose, low-moisture microcrystalline cellulose, and the remaining lactose are added and mixed in sequence, and finally magnesium stearate is added and mixed. Sublingual tablets are manufactured by compression molding using a small single-stroke tablet compression molding machine, controlling the tablet hardness to 20-60N and the tablet weight to 50mg.

[0078] Experimental results confirmed that the freeze-drying process for recombinant Der f 2 protein is also applicable to recombinant Der p 2 protein. Research results regarding additive compatibility were consistent. A recombinant Der p 2 protein solution was freeze-dried (without gelatin) to obtain freeze-dried powder, and then sublingual tablets were manufactured using a direct powder compression molding method, which is referred to as Production Example 13. The method for manufacturing the freeze-dried powder and sublingual tablets is the same as in Production Example 12.

[0079] Example 6: Study of freeze-drying process for recombinant mite group 1 allergens and production of protein sublingual tablets 1) Study of lyophilized buffer and protein concentration Condition 1: The recombinant Der f 1 protein solution was replaced with a buffer system of 50 mM acetate-sodium acetate pH 5.0 + 3% mannitol, the protein concentration was measured and adjusted to 0.1 mg / ml, and then freeze-dried using a vacuum freeze-dryer (LY0-21SP0IP type, Tofuryu). The resulting freeze-dried powder was designated as control group 6.

[0080] Condition 2: The recombinant Der f 1 protein solution was replaced with a buffer system of 50 mM acetate-sodium acetate pH 5.0 + 3% mannitol, the protein concentration was measured and adjusted to 0.5 mg / ml, and then freeze-dried using a vacuum freeze-dryer (LY0-21SP0IP type, Tofuryu). The resulting freeze-dried powder was designated as control group 7.

[0081] Condition 3: The recombinant Der f 1 protein solution was replaced with a buffer system of 50 mM citrate-sodium citrate pH 5.0 + 3% mannitol, the protein concentration was measured and adjusted to 0.1 mg / ml, and then freeze-dried using a vacuum freeze-dryer. The resulting freeze-dried powder was designated as control group 8.

[0082] Condition 4: The recombinant Der f 1 protein solution was replaced with a buffer system of 50 mM citrate-sodium citrate pH 5.0 + 3% mannitol, the protein concentration was measured and adjusted to 0.5 mg / ml, and then freeze-dried using a vacuum freeze-dryer. The resulting freeze-dried powder was designated as control group 9.

[0083] Condition 5: The recombinant Der f 1 protein solution was replaced with a buffer system of 50 mM citrate-sodium citrate pH 5.0 + 3% mannitol, the protein concentration was measured and adjusted to 1.0 mg / ml, and then freeze-dried using a vacuum freeze-dryer. The resulting freeze-dried powder was designated as control group 10.

[0084] Condition 6: The recombinant Der f 1 protein solution was replaced with a buffer system of 5 mM citrate-sodium citrate + 1% mannitol + 2 mg / ml 150 LB gelatin, pH 5.0. The protein concentration was measured and adjusted to 0.04 ± 0.02 mg / ml. Freeze-drying was then performed using a vacuum freeze-dryer, and the resulting freeze-dried powder was designated as control group 11. The gelatin used in this example was bovine-derived and purchased from Baotou Dongbao Biotechnology Co., Ltd. Verification results showed that even when using gelatin of different origins and specifications, there were no significant differences in moisture content, protein recovery rate, uniformity, or appearance of the freeze-dried powder obtained through the freeze-drying process.

[0085] The experimental results confirmed that the freeze-drying process for recombinant Der f 2 protein is also applicable to recombinant Der f 1 protein. The freeze-drying parameters for conditions 1-5 in this example are the same as those for freeze-drying process 4 in Example 5, and the freeze-drying parameters for condition 6 are the same as those for freeze-drying process 6 in Example 5.

[0086] Table 22 shows the results of measurements taken after leaving control groups 6-11 under accelerated conditions for 24 hours. Der f 1 showed poor performance in low-concentration freeze-drying under an acetate-sodium acetate pH 5.0 + 3% mannitol system (control groups 6, 7), and high moisture content was confirmed. Freeze-drying under a 50 mM citrate-sodium citrate pH 5.0 + 3% mannitol buffer system (control groups 8, 9, 10) showed relatively good results when protein was quantified at 0.5 mg / ml and 1.0 mg / ml. Among these, the freeze-dried powder obtained at 0.5 mg / ml showed the best performance in all parameters and accelerated stability, confirming that this was the optimal condition. Freeze-drying under a 5 mM citrate-sodium citrate pH 5.0 + 1% mannitol buffer system + 2 mg / ml 150 LB gelatin system (control group 11) showed good stability.

[0087] [Table 22]

[0088] 2) Compatibility study of additives The freeze-dried powder of control group 9 was polished and ground, passed through a 30-mesh sieve, and formulated according to Table 23. Compatibility studies of the additives were then conducted through accelerated heating, light irradiation, and high humidity tests.

[0089] [Table 23]

[0090] The results of the compatibility study of the additives are as follows: Lactose, mannitol, low-moisture microcrystalline cellulose, low-substituted hydroxypropyl cellulose, and magnesium stearate showed relatively good compatibility with recombinant Der f 1 protein freeze-dried powder. Furthermore, even when the active ingredient was a different allergen protein, the compatibility between the above additives and the protein freeze-dried powder was good. On the other hand, cross-linked carboxymethylcellulose sodium and behenate glycerol ester did not show good compatibility with the protein freeze-dried powder.

[0091] [Table 24]

[0092] 3) Manufacturing of protein sublingual tablets Using the freeze-dried powder of control group 9, sublingual tablets of manufacturing example 14 are prepared based on the formulation composition in Table 25. The method for manufacturing the sublingual tablets is the same as in manufacturing example 12.

[0093] [Table 25]

[0094] The experimental results confirmed that the freeze-drying process for recombinant Der f 1 protein is also applicable to recombinant Der p 1 protein. Furthermore, the research results regarding the compatibility of additives were consistent. A recombinant Der p 1 protein solution was freeze-dried (without gelatin) to obtain a freeze-dried powder, and then sublingual tablets were manufactured by a direct powder compression molding method, which is referred to as Production Example 15. The method for manufacturing the freeze-dried powder and sublingual tablets is the same as in Production Example 14.

[0095] Example 7: Production of a sublingual tablet containing two types of gelatin-free proteins (Der p 1 and Der f 2)

[0096] Recombinant Der f 2 and Der p 1 are freeze-dried separately. The freeze-drying method for recombinant Der f 2 is the same as in Condition 1 and freeze-drying process 2 of Example 5, and the freeze-drying method for recombinant Der p 1 is the same as in Condition 4 and freeze-drying process 4 of Example 6. The obtained freeze-dried powders are each ground and passed through a 30-mesh sieve to measure the protein content. Production Example 16 produces sublingual tablets containing two types of protein based on the formulations listed in Table 26. In this production example, lactose is added sequentially to the raw materials, and 5% lactose, recombinant Der f 2 freeze-dried powder, 5% lactose, recombinant Der p 1 freeze-dried powder, 5% lactose, mannitol, 5% lactose, low-substituted hydroxypropyl cellulose, 5% lactose, low-moisture microcrystalline cellulose, and the remaining lactose are added and mixed in order, and finally magnesium stearate is added and mixed. A small, single-stroke tablet compression molding machine is used to compress and mold the tablets, controlling their hardness to 20-60N and their weight to 50mg.

[0097] [Table 26]

[0098] Example 8: Production of sublingual tablets containing two types of proteins (Der p 1, Der f 2) with different types of gelatin. Recombinant Der f 2 and Der p 1 are freeze-dried separately. The method for producing recombinant Der f 2 is the same as in Example 5 under conditions 2 and freeze-drying process 6, and the method for producing recombinant Der p 1 is the same as in Example 6 under conditions 6 and freeze-drying process 5. The resulting freeze-dried powders are each pulverized, passed through a 30-mesh sieve, and the protein content is measured. Production Examples 17-19 use 180 LB gelatin, 240 LB gelatin, and 245 LB gelatin, respectively, to produce sublingual tablets containing different gelatin based on the formulations listed in Table 27. The 180 LB and 240 LB gelatin are bovine-derived gelatin purchased from Luosailoming Gelatin Co., Ltd., and the 245 LB gelatin is porcine-derived gelatin purchased from Jiali Gelatin Co., Ltd. The production method is the same as in Example 7.

[0099] [Table 27]

[0100] Example 9: Production of sublingual tablets containing four types of gelatin-free proteins (Der p 1, Der p 2, Der f 1, Der f 2) Recombinant Der f 2, Der p 2, Der f 1, and Der p 1 are each freeze-dried. The freeze-drying method for recombinant Der f 2 and Der p 2 is the same as in Condition 1 and freeze-drying process 3 of Example 5, and the freeze-drying method for recombinant Der f 1 and Der p 1 is the same as in Condition 4 and freeze-drying process 4 of Example 6. The resulting freeze-dried powders are each pulverized, passed through a 30-mesh sieve, and the protein content is measured. Production Examples 20-25 produce sublingual tablets containing four types of protein, based on the formulations described in Tables 28-33. Among these, the production methods for Production Examples 20 and 21 are the same as in Example 7, and the production methods for Production Examples 22-25 are as follows. Specifically, using a raw material to which lactose is added sequentially, a three-dimensional motion mixer is used to sequentially add and mix 5% lactose, low-substituted hydroxypropyl cellulose, 5% lactose, a mixed powder of recombinant Der f 2 lyophilized powder, recombinant Der f 1 lyophilized powder, recombinant Der p 2 lyophilized powder, and recombinant Derp 1 lyophilized powder, 5% lactose, low-moisture microcrystalline cellulose, mannitol, and the remaining lactose, and finally magnesium stearate is added and mixed. A rotary tablet compression molding machine is used to compress the tablets, controlling the hardness to 20-60N and the weight to 50mg. Here, the order in which the four types of lyophilized powders are added can be arbitrarily changed, and the addition ratios can be adjusted.

[0101] [Table 28]

[0102] [Table 29]

[0103] [Table 30]

[0104] [Table 31]

[0105] [Table 32]

[0106] [Table 33]

[0107] Example 10: Production of sublingual tablets containing four types of proteins (Der p 1, Der p 2, Der f 1, Der f 2) including different types of gelatin. Recombinant Der f 2, Der p 2, Der f 1, and Der p 1 are freeze-dried, respectively. The manufacturing method for recombinant Der f 2 and Der p 2 is the same as in Example 5 under conditions 2 and freeze-drying process 6, and the manufacturing method for recombinant Der f 1 and Der p 1 is the same as in Example 6 under conditions 6 and freeze-drying process 5. The resulting freeze-dried powders are each pulverized, passed through a 30-mesh sieve, and the protein content is measured. Manufacturing Examples 26-28 use 150 LB gelatin, 180 LB gelatin, and 240 LB gelatin, respectively, to produce sublingual tablets based on the formulations listed in Table 34. Manufacturing Examples 29-31 produce sublingual tablets based on the formulations listed in Tables 35-37, respectively. The manufacturing method is the same as in Example 7.

[0108] [Table 34]

[0109] [Table 35]

[0110] [Table 36]

[0111] [Table 37]

[0112] Example 11: Production of sublingual tablets containing four types of proteins (Art a 1, Art a 3, Art v 1, Art v 3) including different types of gelatin. Art a 1, Art a 3, Art v 1, and Art v 3 are each freeze-dried, and their respective protein solutions are replaced with a buffer system of 10 mM citrate-sodium citrate + 1% mannitol + 3 mg / ml 180 LB gelatin, pH 5.0. The protein concentration is measured and adjusted to 0.06 ± 0.03 mg / ml, and then freeze-dried in a vacuum freeze-dryer to produce freeze-dried powder. The freeze-drying process for Art a 1 and Art v 1 is the same as freeze-drying process 6 in Example 5, and the manufacturing method for Art a 3 and Art v 3 is the same as freeze-drying process 5 in Example 5. The obtained freeze-dried powders are each pulverized and passed through a 30-mesh sieve to measure the protein content. Manufacturing Examples 32 and 33 are prepared as sublingual tablets based on the formulations described in Tables 38-39. The manufacturing method is the same as in Example 7.

[0113] [Table 38]

[0114] [Table 39]

[0115] Example 12: Measurement of sublingual tablets manufactured in manufacturing examples 12-31 In manufacturing examples 12-33, each containing one, two, or four types of protein as active ingredients, and regardless of whether gelatin was added or whether gelatin of a different origin was added, the resulting sublingual tablets were found to have small hardness RSD values, small tablet weight differences, short disintegration time, no dry or gritty sensation in the mouth, a moderate sweetness, and good content uniformity. Manufacturing examples 12-33 used multiple different protein raw materials, and the proportion of protein raw materials in the tablets was low, ranging from 0.276% (manufacturing example 21) to 13.3% (manufacturing examples 16, 26-31). In all cases, the tablets disintegrated rapidly under the tongue, and the oral disintegration time was confirmed to be within 90 seconds. In the formulations of this application, the protein raw material content is low in all cases, especially in manufacturing example 12, where the protein raw material is only 0.74%, and in manufacturing example 21, where the protein raw material is only 0.276%. However, in all cases, the uniformity requirement of 15 or less as stipulated in the pharmacopoeia is met. Some of the results are shown in Table 40.

[0116] Furthermore, because the protein content of the active ingredient in the sublingual tablet is low, those skilled in the art can predict that even if the active ingredient is another allergen protein, the resulting sublingual tablet will still have an excellent hardness RSD value, small tablet weight variation, a short disintegration time, no dryness or grittiness in the oral cavity, a moderate sweetness, and good uniformity of content.

[0117] [Table 40]

[0118] Long-term and accelerated testing was conducted on sublingual tablets of manufacturing examples 16-19, each containing two types of allergen proteins, and the content of major allergens in Group 1 and Group 2 was measured. As a result, it was confirmed that the sublingual tablets of manufacturing examples 17-19, which contained gelatin as an additive, exhibited superior protein stability under high-temperature conditions compared to the sublingual tablet of manufacturing example 16, which did not contain gelatin.

[0119] [Table 41]

[0120] Furthermore, sublingual tablets from manufacturing examples 23, 24, and 26-33, each containing four types of allergen proteins, were subjected to influence factor tests such as 25°C, accelerated sterilization, light irradiation, and high humidity, and the content of major allergens was measured. As a result, it was confirmed that the sublingual tablets from manufacturing examples 26-32, which contained gelatin as an additive, showed superior protein stability under high temperature, light irradiation, and high humidity conditions compared to the sublingual tablets from manufacturing examples 23 and 24, which did not contain gelatin. Manufacturing examples 32 and 33 showed good stability even when the sublingual tablets were stored for a long period at high temperatures of 25°C and 40°C. The results are shown in Tables 42 and 43 below.

[0121] [Table 42]

[0122] [Table 43]

[0123] Furthermore, stability evaluations were conducted on different sublingual tablets containing a single allergen protein (Der p 1, Der p 2, Der f 1, Der f 2), obtained with or without the addition of gelatin. The results similarly confirmed that the sublingual tablets with added gelatin were more stable than those without.

Claims

1. A sublingual tablet obtained by directly compressing a mixture of an active drug and an additive without granulation, It contains protein raw materials, lactose, mannitol, low-substituted hydroxypropyl cellulose with a hydroxypropoxy group content of 5.0-16.0% as a dried product, low-moisture microcrystalline cellulose with a drying loss of 1.5% or less, and magnesium stearate. A sublingual tablet characterized by having mass percentages of each component of 0.276-15.6%, 59.3-76.48%, 5-13.4%, 4-6%, 10-12.5%, and 0.4-0.6%, respectively.

2. A sublingual tablet according to claim 1, characterized in that the protein raw material is an allergen protein raw material.

3. A sublingual tablet according to claim 1, characterized in that the protein is a mite allergen protein and is composed of a mixture of one, two, or more of the following: Der p 1 raw material, Der p 2 raw material, Der f 1 raw material, and Der f 2 raw material.

4. A sublingual tablet according to claim 1, characterized in that the protein is an Artemisia pollen allergen protein and is composed of a mixture of one, two or more of the following: Art a 1 raw material, Art a 3 raw material, Art v 1 raw material, and Art v 3 raw material.

5. A sublingual tablet according to any one of claims 1 to 4, characterized in that the protein raw material is a freeze-dried powder of an allergen protein.

6. A sublingual tablet according to claim 5, characterized in that gelatin is added to the freeze-dried protein powder, or not.

7. A sublingual tablet according to claim 5, characterized in that the buffer system used for freeze-drying the protein comprises citrate-sodium citrate, mannitol, or phosphate buffer, mannitol.

Images