Sublingual tablets and their application in hyposensitization therapy

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]

[0001] The present invention relates to the field of pharmaceutical formulations, in particular to sublingual tablets and their application in hyposensitization therapy. [Background technology]

[0002] Allergen-specific immunotherapy is currently the only "causal therapy" that achieves the goal of treating allergic diseases through immunomodulation. It aims to increase a patient's tolerance to an allergen by gradually increasing the dose of the allergen, thereby reducing the symptoms caused by exposure to that allergen, and ultimately achieving resistance 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 subcutaneous injections, has low patient compliance, and is prone to side effects such as severe systemic anaphylactic shock. In contrast, sublingual administration has fewer side effects, is safer, and improves patient compliance.

[0004] Currently, there are two main dosage forms for sublingual administration of allergens: sublingual liquid and sublingual tablets. Sublingual tablets are tablets that are placed under the tongue and quickly dissolve, or disintegrate in saliva when used, allowing the drug to be absorbed through the sublingual oral mucosa and exert its systemic effect. Compared to sublingual liquid, sublingual tablets are more convenient to take and carry, easier to control the dosage, can ensure standardized drug dosage, and have higher patient compliance. In addition, sublingual tablets have better stability.

[0005] Compared to standard sublingual tablets, developing a formulation for allergen sublingual tablets is more challenging. First, the active ingredient in allergen sublingual tablets is a protein, which is unstable to moisture and heat. Currently, common manufacturing methods for sublingual tablets include granulation and compression (including wet granulation and dry granulation), blister freeze-drying, and direct tablet compression. Among these, wet granulation and compression requires granulation, drying, and sieving, making it unsuitable for heat- and moisture-sensitive or highly soluble drugs. Dry granulation and compression requires compression force to create interparticle bonds, which can lead to problems such as changes in crystalline structure and reduced activity due to high pressure. Commercially available allergen sublingual tablets, such as ALK's ODACTRA®, use the blister freeze-drying method, which requires dedicated production lines such as liquid nitrogen freezing tunnels, placing high demands on manufacturing equipment and high costs. The related technology is not yet mature in China. Overall, direct powder compression molding eliminates the need for wet granulation and a drying process, making it suitable for drugs that are unstable to moisture or heat, reducing the overall process time and energy. However, direct powder compression molding has drawbacks such as poor powder flowability, large tablet weight variations, tablet cracking, and poor content uniformity. In particular, when the content of the active ingredient in the tablet is low, the content uniformity problem caused by the direct powder compression molding process becomes more pronounced.

[0006] Second, compared with standard sublingual tablets, the active ingredient content of allergen sublingual tablets is extremely low, accounting for less than 2% of the tablet weight, and in some cases even less than 1%. Such extremely low active ingredient content can 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 device. 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 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention addresses the shortcomings of the prior art, provides a low-dose sublingual tablet applicable to various active ingredients and its application to a hyposensitization therapeutic drug, and solves the problems of content uniformity and stability of low-dose sublingual tablets, as well as the complexity of manufacturing equipment and manufacturing process. [Means for solving the problem]

[0009] The present invention provides a sublingual tablet comprising 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 material is an allergenic protein 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 to 15.6%, 50 to 83.4%, 0 to 33.4%, 4 to 6%, 10 to 15%, and 0.4 to 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 tablet weight are 0.276 to 15.6%, 59.3 to 76.48%, 5 to 13.4%, 4 to 6%, 10 to 12.5%, and 0.4 to 0.6%, respectively.

[0013] There are many types of allergens, which can be divided into two main categories: inhalant allergens and food allergens. Common inhalant allergens include Dermatophagoides pteronyssinus, Dermatophagoides farinae, cockroaches, cat and dog dander, mold, and pollen, while common food allergens include milk, peanuts, and eggs. The allergen proteins can be naturally extracted or recombinantly expressed. The major allergen proteins of Dermatophagoides pteronyssinus (Der p) are Der p 1 and Der p 2, and the major allergen proteins of Dermatophagoides farinae (Der f) are Der f 1 and Der f 2. The major allergenic proteins of Artemisia annua pollen are Art a 1 and Art a 3, and the major allergenic 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 in the examples demonstrate that the 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 various low-dose active ingredients and is not limited to the specific active ingredients listed in the examples.

[0014] Preferably, the mite allergen protein raw material is a mixture of one or more of Der p 1 raw material, Der p 2 raw material, Der f 1 raw material, and Der f 2 raw material. The phlox orbicularis pollen allergen protein raw material is a mixture of one or more of 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 allergen protein.

[0015] The second aspect of the present invention relates to a freeze-drying process. The buffer components and protein concentration of a freeze-drying solution suitable for a particular protein can be determined through limited experiments using conventional methods. The buffer components selected in the specific examples are merely illustrative and do not limit the buffer components used in freeze-drying an allergen protein. For example, when the active ingredient is Der p 2 or Der f 2, PB and mannitol may be selected as the buffer components. When the active ingredient is Der p 1, Der f 1, Art a 1, Art a 3, Art v 1, or Art v 3, citric acid-sodium citrate and mannitol may be selected as the 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 allergen protein freeze-drying solution. When the allergen protein is unstable, adding gelatin is advantageous for maintaining the stability of the allergen protein sublingual tablet.

[0016] The general freeze-drying process is as follows: A solution containing an allergen protein is replaced with an appropriate buffer system and pH conditions, and then quantified to the appropriate concentration. The freeze-dried product is then obtained through three steps: pre-freezing, primary drying, and analytical drying. The appropriate freeze-drying process for a specific protein can be determined by screening using conventional methods. In Production Examples 12 to 15, freeze-drying processes for different allergen proteins are illustrated. These processes are for illustrative purposes only and are not intended to limit the freeze-drying process for allergen proteins. For example, in Production Examples 12, 16, and 20, different freeze-drying processes were applied to mite group 2 protein, and the final tablet performance was nearly 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 after reaching this temperature, it is maintained for 60 to 90 minutes. In the second stage, the pre-freezing temperature is set to -45 to -50°C, and after reaching this temperature, it is maintained for 120 to 800 minutes. The temperature for the primary drying is set to -10 to -20°C, and after reaching this temperature, it is maintained for 840 to 960 minutes. The degree of vacuum is 0.18 to 0.2 mbar. The temperature for the analytical drying is set to 20 to 25°C, and after reaching this temperature, it is maintained for 480 to 1500 minutes. The degree of vacuum is 0.18 to 0.2 mbar.

[0017] The 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. Direct powder compression molding involves directly compressing a mixture of an active drug and additives to obtain tablets without a granulation process. The process steps mainly include grinding the active drug and additives, sieving, mixing, adding a lubricant and remixing, and compressing. Through years of research, the inventors have found that the resulting low-dose sublingual tablet formulation not only has ideal hardness, tablet weight variance, disintegration time, content uniformity, and stability, but is also highly suitable for direct powder compression molding, overcoming the above-mentioned challenges of conventional direct powder compression molding.

[0018] The allergen sublingual tablets of the present invention are excellent in various parameters, such as properties (white tablets with a smooth surface and uniform color), hardness (moderate hardness of 15 to 60 N, which does not significantly affect tablet packaging or transportation), tablet weight variation, disintegration time, swallowing feel, and content uniformity. In particular, they have significant advantages in terms of disintegration time, content uniformity, stability, and manufacturing process.

[0019] 1. The sublingual tablets of the present invention disintegrate quickly under the tongue and dissolve with almost no foreign body sensation. Sublingual tablets allow the active ingredient to be absorbed through the sublingual oral mucosa to exert a systemic effect, but if the disintegration time is too long, they may be easily swallowed, and some of the active ingredient may not be completely absorbed via the sublingual mucosa as expected, which may result in inaccurate dosing.

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

[0021] 3. The sublingual tablet of the present invention not only has high stability at room temperature but also has high stability under harsh environmental conditions, making it applicable to a variety of bioactive drugs.

[0022] 4. The low-dose sublingual tablets of the present invention achieve the above-mentioned excellent comprehensive performance, but the manufacturing process is simple, applicable to direct powder compression molding, the process is simple, and no additional requirements are placed on production equipment. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention will be explained below with reference to production examples and comparative examples, but is not limited thereto. Preparation Examples 1 to 11 are preparation examples and comparative examples of blank sublingual tablets containing no active ingredient. The obtained tablets were subjected to measurements of tablet hardness, tablet weight difference, and disintegration time, as well as a sensory test. Production Examples 12 to 26 are examples of the production of low-dose sublingual tablets containing an active ingredient. The obtained tablets were subjected to measurements of tablet hardness, tablet weight difference, disintegration time, and content uniformity, as well as sensory testing. In addition, tests on factors affecting sublingual tablets containing an active ingredient, such as long-term storage, high temperature, light exposure, and high humidity, were also conducted, and the total protein content and purity were measured to evaluate stability.

[0024] The specific measurement method is as follows. 1. Hardness measurement The hardness of the tablets is measured using a dedicated hardness measurement device (Tendaitenha CJY-2C hardness tester). Ten tablets are extracted and measured, and the average value and RSD value are recorded as the displayed amount.

[0025] 2. Tablet weight difference measurement According to the 2020 Chinese Pharmacopoeia General Rules 0101, Tablet Weight Difference Measurement Method, 20 test tablets were taken, the total weight was precisely measured, the average tablet weight was calculated, and then the weight of each tablet was precisely measured. The weight of each tablet was compared with the labeled amount, and no more than two tablets exceeded the weight difference limit, and no single tablet exceeded the limit by more than one (if the labeled amount or average tablet weight is 0.3g or less, the weight difference limit is ±7.5%).

[0026] 3. Disintegration time measurement According to the 2020 edition of the Chinese Pharmacopoeia General Provisions 0921, disintegration time limit measurement method, a disintegration tester (Tianda Tianfa ZB-1E type) was used to measure the disintegration time limit by immersing six test tablets in a 1000ml beaker containing water at 37±1℃. Each tablet was confirmed to completely disintegrate and dissolve within 5 minutes.

[0027] 4. Moisture measurement of freeze-dried powder Refer to the 2020 edition of the Chinese Pharmacopoeia General Rules 0832, Method 1 for Determination of Moisture (Karl Fischer Method) Coulomb Titration.

[0028] 5. Sensory test Six healthy adults were selected to take the sublingual tablets under their tongues and evaluate their texture upon ingestion. Evaluation items included the presence or absence of dryness or discomfort, the presence or absence of a sandy texture, the appropriateness of sweetness, and the oral disintegration time. After the test, the sublingual tablets were spit out and gargled with water.

[0029] 6. Compatibility test of additives The appropriate amount of lyophilized powder of the active drug is weighed into a vial and various ratios of additives are added. Microcrystalline cellulose, low-moisture microcrystalline cellulose, filler, and low-substituted hydroxypropyl cellulose are added in amounts five times the amount of the lyophilized powder, and lubricant is added in an amount equal to the amount of the lyophilized powder. Accelerated, high-humidity, and light irradiation tests are conducted to measure purity and confirm the compatibility of the active drug lyophilized powder with each additive.

[0030] 7. Accelerated testing Test samples are placed in vials of appropriate size and sealed, and the vials are placed in a stability test chamber (KBF-P-720, Binder) set at accelerated conditions (40±2°C, 75±5% RH), and removed at the specified time points for measurement.

[0031] 8. High temperature test The test sample is placed in a vial of appropriate size and sealed. The vial is placed in a stability test chamber (KBF-P-720, Binder) set at a high temperature (60±2°C), and is removed at the specified time for measurement.

[0032] 9. High humidity test The test sample is placed in an open vial of appropriate size, placed in a stability test chamber (KBF-720, Binder) set at high humidity conditions (25±2°C, 75±5% RH), and removed at the specified time for measurement.

[0033] 10. Light irradiation test The test sample is placed in a vial of appropriate size and sealed. The vial is placed horizontally in a stability test chamber (KBF-P-720, Binder) set under light irradiation conditions (25±2°C, 60±5% RH, 4500±500 lx), and removed at the specified time for measurement.

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

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

[0036] 13. Content and titer measurement of major allergens Protein content was measured using a double-antibody sandwich ELISA. The content of mite group 1 major allergens refers to the sum of Der p 1 and Der f 1, and the content of mite group 2 major allergens refers to the sum of Der p 2 and Der f 2. The content of Artemisia angustifolia / Artemisia pollen group 1 major allergens refers to the sum of Artemisia angustifolia / Artemisia pollen group 3 major allergens refers to the sum of Artemisia angustifolia / Artemisia pollen group 3 major allergens refers to the sum of Artemisia angustifolia / Artemisia pollen group 3 allergens.

[0037] The titer is measured using an inhibition ELISA method. The recombinant protein sample is diluted in a series of dilutions and then incubated with pooled serum. The incubated mixture is added to an ELISA plate pre-coated with the recombinant protein to establish a linear relationship between the "inhibition rate and dilution factor." The biological activity of the recombinant protein is calculated by defining the biological activity required to reduce IgE in pooled serum by 50% as 100 BU / ml. To more clearly reflect the change in activity, the obtained biological activity is calculated as a percentage based on the theoretical labeled amount, and the measured value is expressed as the actual activity value / theoretical labeled amount × 100%.

[0038] 14, Content uniformity measurement The protein content in the tablets was measured using the immunodot blot method, and the content uniformity was calculated in accordance with the 2020 Chinese Pharmacopoeia General Rules 0941 Content Uniformity Measurement Method. According to General Rules 0941, this product, as a sublingual tablet, has an L value of 15. Ten test tablets were sampled, and the standard difference S and the absolute value A of the difference between the labeled amount and the average were calculated. If A + 2.2S is less than 15, the content uniformity is deemed to meet the acceptable standard. [Example]

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

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

[0041] Manufacturing Example 1: Using the ingredients listed in Table 1, and following the sequential addition method, lactose was added sequentially as the raw 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, and finally magnesium stearate was added and mixed. Tablets were compressed using a small single-shot tablet compression machine, controlling the tablet hardness to 20-60 N and tablet weight to 50 mg.

[0042] Control groups 1 to 3: The low-substituted hydroxypropyl cellulose used in Production Example 1 was replaced with hydroxypropyl cellulose (SSL-L), cross-linked sodium carboxymethyl cellulose, and sodium carboxymethyl starch, and the types and amounts of other additives were kept unchanged. Control groups 1 to 3 were produced, respectively. 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 had small RSD values for hardness and small tablet weight variance, a short oral disintegration time, no dry or gritty feeling in the mouth, and a moderate sweetness. On the other hand, when other disintegrants were used, the disintegration time was too long, and even when the types and amounts of other additives in the formulation were adjusted, tablets with the above-mentioned excellent overall performance could not be obtained. Specific results are as follows:

[0044] [Table 2]

[0045] Example 2: Formulation Study 2 (Filler Selection and Amount Used) [Table 3]

[0046] Sublingual tablets of Production Examples 2 to 5 are manufactured based on the blending ratios shown in Table 3. In Production Example 2, all of the mannitol in Production Example 1 is replaced with lactose, and in Production Examples 3 to 5, the lactose in Production Example 1 is partially or completely replaced with mannitol. Other aspects are the same as in Production Example 1.

[0047] The specific results are shown in Table 4. In Production Examples 1 to 5, the lactose ratios were approximately 70.0%, 83.4%, 50.0%, 27.4%, and 0%, respectively, and the sublingual tablet hardness RSD, tablet weight difference, oral disintegration time, and oral texture parameters were all excellent. Production Example 1 was the best, with the shortest disintegration time. Production Example 3 was next best, and Production Example 2 was relatively good. Production Examples 4 and 5 had relatively long disintegration times and slightly inferior textures. Therefore, the combination of lactose and mannitol was optimal, and as the lactose ratio increased, the hardness RSD and tablet weight difference were relatively small, the oral disintegration time was short, the oral texture was not dry or gritty, and the sweetness was moderate.

[0048] [Table 4]

[0049] Example 3: Formulation Study 3 (Low Moisture Microcrystalline Cellulose / Microcrystalline Cellulose, Lubricant Selection) [Table 5]

[0050] The low-moisture microcrystalline cellulose in Production Example 1 was replaced with microcrystalline cellulose to give Production Example 6. The production method was the same as in Production Example 1. This is Production Example 7, except that magnesium stearate in Production Example 1 is replaced with glycerin behenate. The production method is the same as in Production Example 1. Control Group 4: Control Group 4 was produced according to the blending ratio shown in Table 5, without using low-moisture microcrystalline cellulose or microcrystalline cellulose and mannitol. The production method was the same as in Production Example 1.

[0051] The specific results are shown in Table 6. When microcrystalline cellulose and glycerol behenate were used, the RSD value of sublingual tablet hardness, tablet weight difference, oral disintegration time, and oral sensation were all excellent (however, the compatibility study of additives in Examples 11 to 14 showed that microcrystalline cellulose and glycerol behenate were not compatible with the protein allergen active ingredient). On the other hand, control group 4 experienced serious powder leakage during compression molding, making it unsuitable for direct powder compression molding.

[0052] [Table 6]

[0053] Example 4: Formulation Study 4 (Additive Mixing Ratio Study) [Table 7]

[0054] Based on the blending ratios shown in Table 7, sublingual tablets of Production Examples 8 to 11 and Control Groups 5 and 6 were manufactured. The manufacturing method was the same as in Production Example 1.

[0055] The specific results are shown in Table 8. The sublingual tablets of Preparation Examples 8 to 11 were excellent in all parameters of hardness RSD value, tablet weight difference, oral disintegration time, and oral intake sensation. On the other hand, the tablet weight difference was large and the disintegration time was long in Control Groups 5 and 6, so the excipient blending ratios of Control Groups 5 and 6 could not be applied.

[0056] [Table 8]

[0057] Example 5: Freeze-drying process study of recombinant mite group 2 allergen and preparation of protein sublingual tablets 1) Freeze-drying process research Condition 1: The recombinant Der f 2 protein solution was substituted 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 protein was then poured into a freeze-drying tray. The sample liquid level was controlled to be less than 1 cm. Freeze-drying was performed using a vacuum freeze dryer (LY0-21SP0IP, Tofuryu) according to the parameters in Tables 9-12 (freeze-drying processes 1-4) and Table 15 (freeze-drying process control 1) to produce a freeze-dried powder.

[0058] Condition 2: The recombinant Der f 2 protein solution was substituted 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 liquid level was controlled to less than 1 cm. 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, and freeze-drying control processes 2 and 3) to produce freeze-dried powder. The gelatin used in this example was bovine and purchased from Baotou Dongbao Biotechnology Co., Ltd. Testing showed that the freeze-dried powders obtained by the freeze-drying process did not differ significantly in moisture content, protein recovery, uniformity, or appearance, regardless of the gelatin used, regardless of the origin or specifications.

[0059] The set time refers to the time it takes the freeze dryer to reach the set temperature, and the duration refers to the time it takes the freeze dryer to maintain the set temperature after reaching 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] The specific measurement results for the freeze-dried powders obtained using different freeze-drying processes are shown in Table 18. For freeze-drying processes 1 to 4, the moisture content was all below 3.0% and the uniformity was all below 1%, making all processes suitable. Among these, freeze-drying process 4 was the best, with all indicators being the best. For freeze-drying processes 5 and 6, the moisture content was all below 3.5% and the uniformity was all below 1%, making all processes suitable and excellent. On the other hand, for freeze-drying process controls 1 and 2, the moisture content was relatively high, making the freeze-drying process inapplicable. Furthermore, for freeze-drying process control 3, the appearance was poor, making the freeze-drying process inapplicable.

[0070] [Table 18]

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

[0072] [Table 19]

[0073] The results of the excipient compatibility study are shown in Table 20. Lactose, mannitol, low-moisture microcrystalline cellulose, low-substituted hydroxypropyl cellulose, magnesium stearate, and glyceryl behenate (magnesium stearate being slightly better than glyceryl behenate) were relatively compatible with the lyophilized recombinant Der f 2 protein powder. On the other hand, sorbitol, microcrystalline cellulose, cross-linked sodium carboxymethylcellulose, and sodium carboxymethyl starch were less compatible with the lyophilized recombinant Der f 2 protein powder. The results of the validation study showed that the compatibility of these excipients with the lyophilized powders obtained by freeze-drying processes 1 to 6 was nearly 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, Preparation Example 12 was produced according to the formulation in Table 21 based on the results of the excipient compatibility study.

[0076] [Table 21]

[0077] Using the blending amounts listed in Table 21 and the sequential addition method, lactose is added sequentially as the raw material. 5% lactose, lyophilized powder, 5% lactose, mannitol, 5% lactose, low-substituted hydroxypropyl cellulose, 5% lactose, low-moisture microcrystalline cellulose, and the remaining lactose are added and mixed, and finally magnesium stearate is added and mixed. Sublingual tablets are produced by compressing the mixture using a small single-shot tablet compression molding machine, controlling the tablet hardness to 20-60 N and the tablet weight to 50 mg.

[0078] Experimental results confirmed that the lyophilization process for recombinant Der f 2 protein can also be applied to recombinant Der p 2 protein. Research results on excipient compatibility were consistent. The recombinant Der p 2 protein solution was lyophilized (without gelatin) to obtain a lyophilized powder, which was then prepared into sublingual tablets by direct powder compression molding, as Preparation Example 13. The preparation methods for the lyophilized powder and sublingual tablets were the same as those in Preparation Example 12.

[0079] Example 6: Freeze-drying process study of recombinant mite group 1 allergen and preparation of protein sublingual tablets 1) Study of freeze-drying buffer and protein concentration Condition 1: The recombinant Der f 1 protein solution was replaced with a buffer system of 50 mM acetic acid-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, 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 acetic acid-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, 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 citric acid-sodium citrate pH 5.0 + 3% mannitol, the protein concentration was measured and adjusted to 0.1 mg / ml, and then the solution was 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 citric acid-sodium citrate pH 5.0 + 3% mannitol, the protein concentration was measured and adjusted to 0.5 mg / ml, and then the solution was 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 citric acid-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 substituted with a buffer system of 5 mM citric acid, sodium citrate, 1% mannitol, and 2 mg / ml 150 LB gelatin, pH 5.0. The protein concentration was measured and adjusted to 0.04 ± 0.02 mg / ml. The solution was then lyophilized using a vacuum freeze dryer. The resulting lyophilized powder was designated Control Group 11. The gelatin used in this example was bovine-derived and purchased from Baotou Dongbao Biotechnology Co., Ltd. Testing showed that the lyophilized powders obtained from the lyophilization process did not differ significantly in terms of moisture content, protein recovery rate, uniformity, or appearance, even when gelatin of different origins and specifications was used.

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

[0086] The results of measurements of control groups 6–11 after 24 hours under accelerated conditions are shown in Table 22. For Der f 1, low-concentration lyophilization using an acetic acid-sodium acetate pH 5.0 + 3% mannitol system (control groups 6 and 7) was ineffective, confirming high moisture content. Lyophilization using a 50 mM citric acid-sodium citrate pH 5.0 + 3% mannitol buffer system (control groups 8, 9, and 10) showed relatively good results at protein concentrations of 0.5 mg / ml and 1.0 mg / ml. Of these, the lyophilized powder obtained at 0.5 mg / ml showed the best results in terms of each parameter and accelerated stability, confirming the optimal conditions. Lyophilization using a 5 mM citric acid-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 is ground and passed through a 30-mesh sieve, and formulated according to Table 23. Compatibility studies of the additives are carried out through accelerated, light-irradiation, and high-humidity tests.

[0089] [Table 23]

[0090] The results of the compatibility study of excipients are as follows: Lactose, mannitol, low-moisture microcrystalline cellulose, low-substituted hydroxypropyl cellulose, and magnesium stearate were relatively compatible with recombinant Der f 1 protein lyophilized powder. Furthermore, even when the active ingredient was another allergen protein, the compatibility of the above excipients with the lyophilized protein powder was good. On the other hand, cross-linked sodium carboxymethylcellulose and glycerol behenate were not compatible with the lyophilized protein powder.

[0091] [Table 24]

[0092] 3) Manufacturing of protein sublingual tablets Using the freeze-dried powder of Control Group 9, sublingual tablets of Preparation Example 14 are prepared based on the formulation in Table 25. The method for preparing the sublingual tablets is the same as that of Preparation Example 12.

[0093] [Table 25]

[0094] The experimental results confirmed that the freeze-drying process for recombinant Der f 1 protein could also be applied to recombinant Der p 1 protein, and the results of studies on excipient compatibility were consistent. The recombinant Der p 1 protein solution was freeze-dried (gelatin-free) to obtain a freeze-dried powder, which was then prepared into sublingual tablets by direct powder compression molding, as Preparation Example 15. The methods for preparing the freeze-dried powder and sublingual tablets were the same as those in Preparation Example 14.

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

[0096] Recombinant Der f 2 and Der p 1 were each freeze-dried. The freeze-drying method for recombinant Der f 2 was the same as in Example 5 under Condition 1 and freeze-drying process 2, and the freeze-drying method for recombinant Der p 1 was the same as in Example 6 under Condition 4 and freeze-drying process 4. The resulting freeze-dried powders were each pulverized and passed through a 30-mesh sieve, and the protein content was measured. In Example 16, sublingual tablets containing two proteins were produced based on the formulations listed in Table 26. In this example, lactose was used as a raw material to which 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 were added and mixed in sequence, and finally magnesium stearate was added and mixed. A small single-shot tablet compression molding machine is used to perform compression molding, controlling the tablet hardness to 20 to 60 N and the tablet weight to 50 mg.

[0097] [Table 26]

[0098] Example 8: Preparation of sublingual tablets containing two proteins (Der p 1, Der f 2) with different types of gelatin Recombinant Der f 2 and Der p 1 were each freeze-dried. The production method for recombinant Der f 2 was the same as in Example 5 under Condition 2 and freeze-drying process 6, and the production method for recombinant Der p 1 was the same as in Example 6 under Condition 6 and freeze-drying process 5. The resulting freeze-dried powders were each pulverized and passed through a 30-mesh sieve to measure the protein content. In Examples 17 to 19, sublingual tablets containing different gelatins were prepared using 180 LB gelatin, 240 LB gelatin, and 245 LB gelatin, respectively, in the amounts listed in Table 27. The 180 LB and 240 LB gelatins were bovine gelatins purchased from Luosai Luoming Gelatin Co., Ltd., and the 245 LB gelatin was porcine gelatin purchased from Jialiida Gelatin Co., Ltd. The production method was the same as in Example 7.

[0099] [Table 27]

[0100] Example 9: Preparation of a sublingual tablet containing four proteins (Der p 1, Der p 2, Der f 1, Der f 2) without gelatin Recombinant Der f 2, Der p 2, Der f 1, and Der p 1 were each freeze-dried. The freeze-drying method for recombinant Der f 2 and Der p 2 was the same as in Example 5 under Condition 1 and freeze-drying process 3, and the freeze-drying method for recombinant Der f 1 and Der p 1 was the same as in Example 6 under Condition 4 and freeze-drying process 4. The resulting freeze-dried powders were each pulverized and passed through a 30-mesh sieve, and the protein content was measured. In Production Examples 20 to 25, sublingual tablets containing four types of proteins were produced based on the blend amounts listed in Tables 28 to 33. The production methods for Production Examples 20 and 21 were the same as in Example 7, and the production methods for Production Examples 22 to 25 were as follows. Specifically, lactose was added sequentially to the raw material using a three-dimensional motion mixer. 5% lactose, low-substituted hydroxypropyl cellulose, 5% lactose, a mixture 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 were added and mixed, and finally magnesium stearate was added and mixed. A rotary tablet compression molding machine was used to compress the tablets to a hardness of 20-60 N and a tablet weight of 50 mg. The order of addition of the four lyophilized powders could be varied as desired, and the addition ratios were adjustable.

[0101] [Table 28]

[0102] [Table 29]

[0103] [Table 30]

[0104] [Table 31]

[0105] [Table 32]

[0106] [Table 33]

[0107] Example 10: Preparation of sublingual tablets containing four proteins (Der p 1, Der p 2, Der f 1, Der f 2) with different types of gelatin Recombinant Der f 2, Der p 2, Der f 1, and Der p 1 were each freeze-dried. The production methods for recombinant Der f 2 and Der p 2 were the same as those in Example 5 under Condition 2 and freeze-drying process 6, and the production methods for recombinant Der f 1 and Der p 1 were the same as those in Example 6 under Condition 6 and freeze-drying process 5. The resulting freeze-dried powders were each pulverized, passed through a 30-mesh sieve, and the protein content was measured. In Production Examples 26 to 28, sublingual tablets were produced using 150 lb gelatin, 180 lb gelatin, and 240 lb gelatin, respectively, in the amounts listed in Table 34. In Production Examples 29 to 31, sublingual tablets were produced in the amounts listed in Tables 35 to 37. The production method was the same as in Example 7.

[0108] [Table 34]

[0109] [Table 35]

[0110] [Table 36]

[0111] [Table 37]

[0112] Example 11: Preparation of sublingual tablets containing four proteins (Art a 1, Art a 3, Art v 1, Art v 3) with different types of gelatin Art a 1, Art a 3, Art v 1, and Art v 3 were each lyophilized, and the protein solution was replaced with a buffer system of 10 mM citric acid-sodium citrate + 1% mannitol + 3 mg / ml 180 LB gelatin, pH 5.0. The protein concentration was measured and adjusted to 0.06 ± 0.03 mg / ml, and then lyophilized using a vacuum freeze dryer to produce lyophilized powders. The lyophilization process for Art a 1 and Art v 1 was the same as that for freeze-drying process 6 in Example 5, and the production method for Art a 3 and Art v 3 was the same as that for freeze-drying process 5 in Example 5. The resulting lyophilized powders were each pulverized and passed through a 30-mesh sieve to measure the protein content. In Production Examples 32 and 33, sublingual tablets were produced based on the formulations listed in Tables 38 and 39. The production method was the same as that in Example 7.

[0113] [Table 38]

[0114] [Table 39]

[0115] Example 12: Measurement of sublingual tablets produced in Production Examples 12 to 31 In Examples 12–33, the active ingredients contained one protein, two proteins, or four proteins, respectively. Regardless of whether gelatin was added or not, or whether gelatin of different origins was added, the resulting sublingual tablets exhibited small RSD values for hardness, small tablet weight variance, short disintegration times, no dry or gritty oral sensation, a moderate sweetness, and good content uniformity. Examples 12–33 used multiple different protein ingredients, with low tablet protein content ranging from 0.276% (Example 21) to 13.3% (Examples 16, 26–31). All tablets disintegrated rapidly under the tongue, with disintegration times of less than 90 seconds. The formulations in this application all contained low amounts of protein, particularly only 0.74% in Example 12 and 0.276% in Example 21. However, both met the Pharmacopoeia requirement of a uniformity of 15 or less. Some results are shown in Table 40.

[0116] Furthermore, because the content of the protein active ingredient in the sublingual tablets is low, those skilled in the art can predict that even if the active ingredient is another allergen protein, the resulting sublingual tablets will still have excellent hardness RSD values, small tablet weight variance, short disintegration time, no dry or gritty feeling in the mouth when taken, moderate sweetness, and good content uniformity.

[0117] [Table 40]

[0118] Long-term and accelerated tests were conducted on the sublingual tablets of Production Examples 16 to 19, which contain two types of allergen proteins, to measure the contents of the major allergens in Group 1 and Group 2. As a result, it was confirmed that the sublingual tablets of Production Examples 17 to 19, which contain gelatin as an additive, have superior protein stability under high temperature conditions to the sublingual tablet of Production Example 16, which does not contain gelatin.

[0119] [Table 41]

[0120] Furthermore, the sublingual tablets of Production Examples 23, 24, and 26-33, each containing four allergen proteins, were subjected to influence factor tests, including 25°C, accelerated storage, light exposure, and high humidity, to measure the content of major allergens. The results confirmed that the sublingual tablets of Production Examples 26-32, which contain gelatin as an additive, have superior protein stability under high temperature, light exposure, and high humidity conditions compared to the sublingual tablets of Production Examples 23 and 24, which do not contain gelatin. The sublingual tablets of Production Examples 32 and 33 also demonstrated good stability even when stored for long periods 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] Additionally, a stability evaluation was performed 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 added gelatin. The results also confirmed that the stability of sublingual tablets with added gelatin was superior to that of sublingual tablets without added gelatin.

Claims

1. A sublingual tablet comprising a protein raw material, lactose, mannitol, low-substituted hydroxypropyl cellulose, low-moisture microcrystalline cellulose, and magnesium stearate, wherein the mass percentages of each component are 0-15.6%, 0-83.4%, 0-83.4%, 4-6%, 10-15%, and 0.4-0.6%, respectively.

2. 2. The sublingual tablet according to claim 1, wherein the protein material is an allergen protein 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 to 15.6%, 50 to 83.4%, 0 to 33.4%, 4 to 6%, 10 to 15%, and 0.4 to 0.6%, respectively.

4. 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 relative to the tablet weight are 0.276 to 15.6%, 59.3 to 76.48%, 5 to 13.4%, 4 to 6%, 10 to 12.5%, and 0.4 to 0.6%, respectively.

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

6. 2. The sublingual tablet according to claim 1, wherein the protein is an Artemisia pollen allergen protein and is composed of a mixture of one, two or more of the following materials: Art a 1 material, Art a 3 material, Art v 1 material, and Art v 3 material.

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

8. 8. The sublingual tablet according to claim 7, characterized in that gelatin is added or not added to the freeze-dried powder of protein.

9. 8. The sublingual tablet according to claim 7, wherein the buffer system used for lyophilization of the protein comprises citric acid-sodium citrate, mannitol, or comprises PB, mannitol.

10. The sublingual tablet according to any one of claims 1 to 9, wherein the lyophilized protein powder used is produced by the following method: A sublingual tablet characterized in that a solution containing a protein is replaced with an appropriate buffer system and pH conditions, quantified to an appropriate concentration, and then subjected to three steps: pre-freezing, primary drying, and analytical drying to obtain a freeze-dried product.

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