A detection model for allergic reactions to Qingkailing injection.

Abstract

This invention provides a detection model for allergic reactions to Qingkailing injection. The model comprises seven modules: a mouse ear blue staining area percentage testing and data storage module; a mouse ear blue staining area scoring module; a mouse ear blue staining area result judgment module; a mouse ear tissue Evans blue (EB) exudate quantitative testing and data storage module; a mouse ear tissue Evans blue exudate calculation module; a mouse ear tissue Evans blue exudate result judgment module; and a result output module. The model provides clear evaluation and analysis methods and result judgment criteria, exhibiting a high detection reaction rate. At a dosage of 10 ml / kg, the reaction rate of Qingkailing injection reaches 100%, and the EB exudate amount is 6-10 times that of the negative control group. Furthermore, the method is simple to operate, less time-consuming, and has good repeatability, accurately and truthfully reflecting whether Qingkailing injection causes allergic reactions. This model has significant value and importance for the quality control, process research, and clinical application safety of Qingkailing injection.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to an allergy detection model for Qingkailing injection. Background Technology

[0002] Qingkailing Injection is mainly prepared from a formula of medicinal materials including Gardenia jasminoides, Isatis indigotica, Lonicera japonica, baicalin, cholic acid, deoxycholic acid, mother-of-pearl, and buffalo horn using modern production processes. There are clinical reports of anaphylactoid reactions to Qingkailing Injection; therefore, exploring detection methods for anaphylactoid reactions to Qingkailing Injection is of great significance for guiding clinical medication.

[0003] The article "A Discussion on Emergency Testing Methods for Allergic Reactions to Qingkailing Injection" by Yu Xiaoyan, published in *Northern Pharmacy*, Vol. 12, 2015, explores emergency testing methods for allergic reactions to Qingkailing injection. It employs an in vivo allergic reaction test in ICR mice combined with an in vitro RBL-2H3 cell β-aminohexosidase and histamine release rate test as the emergency testing method for allergic reactions to Qingkailing injection. However, this method requires simultaneous detection of the absorbance of the supernatant and lysis buffer during in vitro testing, making the experimental operation and data processing cumbersome and unsuitable for large-scale sample testing.

[0004] Our team previously studied a preclinical evaluation method for drug-induced allergic reactions (Liang Aihua, 2012, China Journal of Traditional Chinese Medicine, Research on Preclinical Evaluation Methods for Drug-Induced Allergic Reactions (I) - Establishment and Validation of a Mouse-Induced Allergic Reaction Evaluation Method). This method uses a single intravenous injection of mice containing 0.4% Evans blue (EB) as the allergy-like evaluation index to assess intravenous injection-induced allergic reactions. However, this method showed significant differences in evaluation for different injectable drugs. According to the research data, this method showed strong responses to Shuanghuanglian injection and Shengmai injection; however, the response rate to Qingkailing injection was very poor (only 40%, see Table 3 in the literature). At a dosage concentration of 12 ml / kg, the response rate (response rate = number of animals with blue ear staining / number of animals in the group × 100%) of Qingkailing injection was 40%, and the EB exudation data was also unsatisfactory, with the EB exudation only slightly more than double that of the saline group. The detection results of this method for Qingkailing injection do not meet clinical needs.

[0005] Therefore, researching and exploring a more efficient, time-saving, high-response, and more accurate and stable detection scheme for allergic reactions to Qingkailing injection is of great value and significance for the quality control, process research, and clinical application safety of Qingkailing injection. Summary of the Invention

[0006] The technical problem to be solved by this invention is to address the shortcomings of the detection technology for allergic reactions to Qingkailing injection, and to provide a detection model and method for allergic reactions to Qingkailing injection.

[0007] The present invention achieves the above-mentioned objectives through the following technical solutions:

[0008] A detection model for allergic reactions to Qingkailing injection includes seven modules:

[0009] (1) Module 1: Mouse ear blue staining area ratio test and data storage module;

[0010] This module includes: an injection module, an ear blue staining area percentage statistics module, and a data storage module;

[0011] The injection module includes injectable reagents: Qingkailing injection, Evans blue, histamine, and physiological saline;

[0012] The ear blue staining area percentage statistics module is used to statistically analyze the proportion of blue staining area in mouse ears to the auricle area after injection, and obtain the blue staining area percentage data of each group of mice; the data of each group is stored in the data storage module.

[0013] (2) Module 2: Mouse ear blue staining area scoring module;

[0014] This module includes the scoring criteria shown in Table 1;

[0015] This module is used to receive the data from Module 1 above, analyze it according to the scoring criteria shown in Table 1, and provide scoring data;

[0016] Table 1 Scoring criteria for blue staining area (S) of auricle

[0017]

[0018] (3) Module 3: Mouse ear blue staining area result judgment module;

[0019] This module includes the criteria for judging the results based on the scoring data from Module 2; the criteria are as follows:

[0020] Qingkailing injection group: If no blue staining is observed in any animal, the result is negative;

[0021] Qingkailing injection group: If ≤30% of animals have a blue staining score ≥1 and all scores <2, the result is negative;

[0022] Qingkailing injection group: If more than 30% of the animals have a blue staining score ≥1, but less than 10% of the animals have a score ≥3, the result is considered suspicious.

[0023] Qingkailing injection group: If more than 10% of the animals have a blue staining score ≥3, the result is positive.

[0024] If the result is questionable, the test should be repeated; if the result of the retest is still questionable or negative, it is judged as negative and no further test is needed; if the result of the retest is positive, it is tested once more, and the result of two consistent results out of three tests is taken as the final judgment.

[0025] (4) Module 4: Quantitative test and data storage of Evans blue exudate in mouse ear tissue;

[0026] This module includes: an extraction module, a filtration module, an absorbance testing module, and a data storage module;

[0027] The extraction module includes a formamide solution containing 0.2-1.0% (preferably 0.5% KOH) for extracting Evans blue from mouse ear tissue, yielding an extract.

[0028] The filtration module is used to filter the extract to obtain the filtrate;

[0029] The absorbance testing module is used to test the absorbance data of the filtrate. The obtained data is stored in the data storage module and output to module five.

[0030] (5) Module 5: Calculation module for Evans blue exudate in mouse ear tissue;

[0031] This module includes the Evans Blue standard curve;

[0032] This module receives absorbance data from module four and calculates the Evans blue exudate in the mouse ear tissue of each group based on the Evans blue standard curve.

[0033] (6) Module Six: Module for Judging the Evans Blue Exudate in Mouse Ear Tissue;

[0034] This module receives Evans blue exudate data from each group of mouse ear tissues from Module 5. If the Evans blue (EB) exudate in the Qingkailing injection group is significantly higher than that in the negative control group, it is judged as suspicious or positive. The specific judgment criteria are as follows:

[0035] Increase <50%, negative;

[0036] The increase ranged from 50-75%, and the statistical difference was P<0.05, which is suspicious;

[0037] Increased by 75%-100%, weakly positive;

[0038] An increase of >100% indicates a positive result;

[0039] (7) Module 7: Results Output Module;

[0040] This module is used to comprehensively analyze the results of Modules Three and Six according to the following criteria, and to give and output the final judgment result:

[0041] If both the results of Module 3 and Module 6 are negative, then the final result is judged as negative.

[0042] If the results of Module 3 and / or Module 6 are suspicious, then the final result is judged to be suspicious.

[0043] If the results of Module 3 and / or Module 6 are positive, then the final result is judged as positive.

[0044] The final judgment result is the combined assessment of the auricle blue staining area and the Evans blue (EB) exudate amount.

[0045] Preferably, in Module 1 of the above model, the source requirements for each group of data are as follows:

[0046] ① Data on the percentage of blue-stained area in mouse ears in the Qingkailing injection group, source requirements and test methods: Qingkailing injection and 0.6-1.0% (preferably 0.8%) Evans blue solution were mixed at a volume ratio of 1:1 and administered intravenously to mice. The percentage of blue-stained area in mouse ears was observed within 30 minutes after the injection.

[0047] ② Data on the percentage of blue-stained area in the ears of mice in the histamine-positive control group, source requirements and test methods: Mix 0.5-2.0 mg / ml (preferably 1.0 mg / ml) histamine with 0.6-1.0% (preferably 0.8%) Evans blue solution at a volume ratio of 1:1, and administer the mixture to mice intravenously. Observe the percentage of blue-stained area in the ears of mice within 30 minutes after the mixture is administered.

[0048] ③ Data on the percentage of blue-stained area in the ears of mice in the saline negative control group. Source requirements and test methods: After mixing saline with 0.6-1.0% (preferably 0.8%) Evans blue solution at a volume ratio of 1:1, the mice were intravenously injected with the mixture, and the percentage of blue-stained area in the ears of the mice was observed within 30 minutes.

[0049] Preferably, Qingkailing injection is diluted with physiological saline at a volume ratio of 1:(0-1) before use. The concentrations of Qingkailing injection and histamine mentioned herein refer to the drug concentrations before mixing with the EB solution.

[0050] The stock solution is diluted with physiological saline at a volume ratio of 1:0 and then used as the stock solution. When the stock solution is mixed with EB at a ratio of 1:1 and then injected, the actual concentration of the drug is 1 / 2 of the stock solution. The drug is administered intravenously at a dose of 20 ml / kg of mouse body weight, so the actual dosage is 10 ml / kg.

[0051] The original solution and physiological saline were diluted at a volume ratio of 1:1, and then mixed with EB at a ratio of 1:1 before injection. The actual concentration of the drug was 1 / 4 of the original solution. The drug was administered intravenously at a dose of 20 ml / kg of mouse body weight, and the actual dosage was 5 ml / kg.

[0052] Preferably, in the above model, the absorbance testing module is an enzyme-linked immunosorbent assay (ELISA) reader or an ultraviolet spectrophotometer, with wavelengths including 610 nm.

[0053] Preferably, in the above model, the method for constructing the Evans blue standard curve is as follows: using formamide containing 0.5% KOH as a solvent, Evans blue is prepared into solutions of 0, 1, 2, 4, 6, 8, and 10 μg / ml, with two replicates for each concentration, and 1 ml is taken from each well. The absorbance is measured at a wavelength of 610 nm; with absorbance as X and EB concentration as Y, a standard curve is plotted.

[0054] Preferably, in the method of using the model, during the data acquisition process of Module 1, the intravenous injection dose for mice is 20 ml / kg of mouse body weight.

[0055] Preferably, in the method of using the model, the mouse is a KM mouse or an ICR mouse.

[0056] Preferably, in the method of using the model, the mouse is male.

[0057] Preferably, in the method of using the model, the weight of the mouse is 23-28g, more preferably 26-28g.

[0058] Preferably, in the method of using the model, the injection experiment is performed after the mice have been acclimatized for 1 day.

[0059] Preferably, in the method of using the model, the injection speed is: maintaining a uniform speed to complete the injection within 30 seconds.

[0060] Preferably, in the method of using the model, physiological saline is used as the solvent for the injection.

[0061] Based on the above model, the present invention provides a method for using the above model, namely a method for detecting allergic reactions to Qingkailing injection, the specific operation of which is described in Example 1.

[0062] The technical solution of the present invention has the following beneficial effects:

[0063] This invention provides a detection model and method for allergic reactions to Qingkailing injection, which can accurately and realistically reflect whether Qingkailing injection causes allergic reactions. According to experimental data, at a dosage concentration of 10 ml / kg, the amount of EB exudation of Qingkailing injection is 6-10 times that of the negative control group (2.51 vs. 0.24, 1.43 vs. 0.24), and the reaction rate can reach 100%.

[0064] Moreover, this method is simple to operate, time-consuming, has good repeatability, and has important value and significance for the quality control, process research of Qingkailing injection, and the safety of clinical application. Detailed implementation manners

[0065] The following takes specific examples to further illustrate the present invention, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0066] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0067] The experimental reagents are all commercially available: normal saline for injection, formamide (analytical pure), 75% alcohol, Evans blue (EB), positive drug histamine (for injection), potassium hydroxide.

[0068] Experimental animals: KM mice, ICR, C57, BALB / C mice, all from Beijing Vital River Laboratory Animal Technology Co., Ltd., license number: SCXK (Beijing) 2016-0011.

[0069] Example 1 Establishment of the detection method for anaphylactoid reaction of Qingkailing injection

[0070] I. Grouping

[0071] Weigh the KM mice and record the weight of each mouse. Randomly group the mice according to their weights, with 10 animals in each group. At least the following groups should be included:

[0072] (1) Qingkailing injection group:

[0073] It is divided into 3 concentration groups. The high concentration is the stock solution (the actual administration dose is 10 ml / kg), the medium concentration is the stock solution diluted with normal saline at a volume ratio of 1:1 (the actual administration dose is 5 ml / kg), and the low concentration is the stock solution diluted with normal saline at a volume ratio of 1:3 (the actual administration dose is 2.5 ml / kg);

[0074] (2) Positive control group: Histamine 1.0 mg / ml (the actual administration dose is 10 mg / kg);

[0075] (3) Negative control group: Normal saline.

[0076] After grouping, label the cages with tags.

[0077] II. Treatment: Intravenous injection

[0078] Qingkailing injection: After each concentration group of Qingkailing injection is mixed with 0.8% EB solution at a volume ratio of 1:1, it is intravenously injected according to 20 ml / kg of the mouse weight.

[0079] Positive control group: 1.0 mg / ml histamine was mixed with 0.8% EB solution at a volume ratio of 1:1 and injected intravenously at a dose of 20 ml / kg of mouse body weight.

[0080] Negative control group: physiological saline and 0.8% EB solution were mixed at a volume ratio of 1:1 and injected intravenously at a dose of 20 ml / kg of mouse body weight.

[0081] III. Observation and Recording of Allergic Reactions

[0082] Immediately after intravenous administration, the appearance of blue staining in both auricles of the mice was observed. The animals were sacrificed 30 minutes after administration, and auricle tissue was collected to test the amount of EB exudate.

[0083] 1. Area of ​​blue staining on the auricle:

[0084] Record the size of the blue-stained area of ​​each ear for each animal, such as 1 / 8, 1 / 4, 1 / 2, 3 / 4 or the entire auricle area; the number of animals with blue-stained auricles and the number of blue-stained ears in each group;

[0085] 2. Quantitative determination of EB exudate in auricular tissue:

[0086] Three methods can be used for measurement:

[0087] ① Determination of EB exudate in whole animal ear tissue: Ears were cut near the base of the auricle (note that the cutting position must be consistent for each animal; due to the rich blood supply at the base of the auricle, inconsistent cutting positions may lead to human error due to varying amounts of blood introduced. Therefore, it is best to cut the auricle from approximately 2-3 mm away from the fold between the auricle and the brain in each mouse). Two ears from each mouse were chopped and placed in the same test tube. 2 ml of a 0.5% KOH formamide solution was added and the mixture was soaked at room temperature for 24 hours until the EB in the ear tissue was completely extracted into the formamide solution (the ear tissue turned white, without any blue residue). The tissue was filtered out using a 120-mesh sieve. 1 ml of the filtrate was added to a 48-well microplate with good light transmittance, and the absorbance was measured at 610 nm using a microplate reader. The EB dye exudate amount (μg) was calculated based on the EB standard curve.

[0088] ② Determination of EB exudate in a single ear: The method is the same as above, except that EB is extracted and tested separately on the left and right sides.

[0089] ③ EB detection per unit area: Ear patches are punched using a 9mm punch, weighed, and then the tissue is minced. EB is extracted and detected using the same method as described above. Note: The location of the ear patch should be consistent for each animal. Additionally, because the distribution of vascular exudate in the auricle may differ among mice after an allergic reaction, this method may increase inter-animal variability.

[0090] IV. Plotting the EB Standard Curve

[0091] Using formamide containing 0.5% KOH as a solvent, EB was prepared into solutions of 0, 1, 2, 4, 6, 8, and 10 μg / ml. Each concentration was prepared in duplicate, with 1 ml taken from each well, and the absorbance was measured at a wavelength of 610 nm. A standard curve was plotted with absorbance as X and EB concentration as Y.

[0092] V. Results Analysis

[0093] (1) Scoring of blue staining area of ​​auricle and assessment of allergy-like symptoms:

[0094] Score each animal according to Table 1:

[0095] Table 1 Scoring Method for Blue Staining Area (S) of Auricle

[0096]

[0097] (2) EB exudate:

[0098] The amount of EB exudate (μg / ml) for each mouse was calculated based on the EB standard curve.

[0099] VI. Result Judgment

[0100] The following are acceptable standards for the test. If the following standards are not met, it is recommended that the test be repeated:

[0101] 1. Negative control group: The number of animals with a blue staining score ≥1 should not exceed 20%. There should be no animals with a score of 2 or higher.

[0102] 2. Positive control group: The proportion of individuals with an indigo staining score ≥3 was ≥70%;

[0103] 3. Result Interpretation:

[0104] (1) Scoring based on the area of ​​blue staining on the auricle:

[0105] Drug group: If no blue staining is observed in any animal, the result is negative;

[0106] Drug group: If ≤30% of animals have a blue staining score ≥1 and all scores <2, the result is negative;

[0107] Drug group: If more than 30% of the animals have a blue staining score ≥1, but less than 10% of the animals have a score ≥3, the result is considered suspicious;

[0108] Drug group: If more than 10% of the animals have a blue staining score of ≥3, the result is positive.

[0109] If the result is questionable, the test must be repeated. If the result of the retest is also questionable or negative, it is judged as negative and no further testing is needed. If the result of the retest is positive, one more test is conducted, and the result of two consistent results out of three tests is taken as the final judgment.

[0110] (2) Determination of EB exudate

[0111] If the amount of EB exudate in the Qingkailing injection group is significantly higher than that in the negative control group, it is judged as suspicious or positive, meaning that there may be a risk of allergic reaction; the specific judgment criteria are as follows:

[0112] If the increase is less than 50%, the result is negative;

[0113] If the increase is 50-75% and the statistical difference is P<0.05, the result is suspicious.

[0114] If the increase is 75%-100%, the result is weakly positive;

[0115] If the increase is greater than 100%, the result is positive.

[0116] The final judgment result is based on the combined assessment of the auricular blue staining area and the amount of EB exudation.

[0117] Example 2 Experimental Data

[0118] Using the allergy detection model for Qingkailing injection provided by this invention, an allergy detection experiment for Qingkailing injection was conducted according to the specific usage method in Example 1. The experimental results are shown in Table 2.

[0119] The results showed that no blue staining reaction was observed in the auricles of mice after intravenous injection of the negative control solution, physiological saline, indicating that no false positive reactions were observed in this experiment.

[0120] After intravenous injection of histamine 10 mg / kg into the positive control mice, all animals showed increased vascular permeability in the auricle and more pronounced blue staining of the auricle. 20 out of 20 mice exhibited a positive or higher-level allergy-like reaction (≥3 points). Compared to the negative control group, the amount of EB exudate was significantly increased, indicating a strong allergy-like reaction, and thus no false negatives were observed in this experiment.

[0121] When the dosage of Qingkailing injection was 10 ml / kg, the number of ears in mice with positive or higher allergic reactions (≥3 points) in both batches 210805 and 210807 was 12 / 20. The blue staining scores of the ears in both groups were significantly higher than those in the negative control group (P < 0.001); moreover, the amount of EB exudate in both groups was significantly higher than that in the negative control group (P < 0.01, 0.05).

[0122] When the dosage was 5 ml / kg, the number of mice with positive or higher allergic reactions caused by Qingkailing injection in batches 210805 and 210807 were 2 / 20 and 4 / 20, respectively. The blue staining score of the auricle of mice in batch 210807 (P<0.05) and EB exudation (P<0.05) were significantly higher than those in the saline group.

[0123] After the dosage was further reduced to 2.5 ml / kg, neither batches 210805 nor 210807 induced blue staining of the auricle in mice, and the allergy-like reaction was negative.

[0124] Based on the above results, allergic-like reactions were more pronounced at high and medium concentrations.

[0125] Table 2. Blue staining of mouse auricles and EB exudate levels in each group ( n=10)

[0126]

[0127] Note: Compared with the control group, 1) P<0.05, 2) P<0.01, 3) P<0.001

[0128] According to experimental data, at a dosage concentration of 10 ml / kg, the amount of EB exudate from Qingkailing injection was 6-10 times that of the negative control group (2.51 vs. 0.24, 1.43 vs. 0.24). The reaction rate of both batches was 100%, and the experimental animals all showed varying degrees of blue staining of the auricle.

[0129] Example 3 Effect of reagents on ear tissue extraction volume

[0130] The effects of adding KOH and not adding KOH on the extract yield of ear tissue were studied using formamide or formamide containing 0.5% KOH as solvents, respectively.

[0131] 1. EB standard curve:

[0132] EB showed good linearity between concentration and absorbance in the range of 0-50 μg / ml. To verify the effect of adding KOH on the standard curve, two standard curve solutions were prepared:

[0133] (1) Using formamide as a solvent, prepare standard curve working solutions with final concentrations of 0, 0.5, 1, 2, 4, 6, 8, and 10 μg / ml from 0.8% EB solution. Perform two replicates for each concentration, taking 1 ml from each well, and measure the absorbance at 610 nm using an ELISA reader. Plot the standard curve.

[0134] Linear equation: y = 14.119x - 0.9072 (R 2 =0.9996)

[0135] (2) Using formamide containing 0.5% KOH as a solvent, prepare standard curve working solutions with final concentrations of 0, 0.5, 1, 2, 4, 6, 8, and 10 μg / ml from 0.8% EB solution. Perform two replicates for each concentration, taking 1 ml from each well, and measure the absorbance at 610 nm using an ELISA reader. Plot the standard curve.

[0136] Linear equation: y = 14.386x - 0.0418 (R) 2 =1)

[0137] 2. Effect of adding KOH versus not adding KOH on the amount of ear tissue extracted.

[0138] (1) 50 mice were randomly divided into 5 groups: negative control group, positive control group 1 and 2, and Qingkailing group 1 and 2.

[0139] (2) Dosage:

[0140] Negative control group: 0.4% EB solution (dissolved in physiological saline) was injected intravenously at a dose of 20 ml / kg of animal body weight.

[0141] Positive control groups 1 and 2: 1.0 mg / ml histamine was mixed with 0.8% EB solution at a ratio of 1:1 (volume) and then injected intravenously at a volume of 20 ml / kg of animal body weight.

[0142] Qingkailing Group 1 and 2: Qingkailing injection stock solution and 0.8% EB solution were mixed at a volume ratio of 1:1 (volume) and then injected intravenously at a dose of 20 ml / kg of mouse body weight.

[0143] (3) After 30 minutes, the outer ears of each group of animals were cut off, and the ear tissue of each animal was divided into left and right sides. After being minced, each was placed in a separate test tube. 1 ml of formamide was added to the left ear tissue, and 1 ml of formamide containing 0.5% KOH was added to the right ear tissue. The test tubes were sealed with sealing film. The negative control group, positive drug group 1, and Qingkailing group 1 were soaked at room temperature for 48 hours, while positive drug group 2 and Qingkailing group 2 were soaked at room temperature for 24 hours. The filtrate was filtered, and the filtrate was injected into a transparent flat-bottomed culture plate. The absorbance was detected using an ELISA reader. The EB extraction amount of ear tissue was calculated using the corresponding EB standard curve, and the effect of adding KOH and not adding KOH on the extraction amount of ear tissue was compared.

[0144] Table 3. Blue staining of mouse auricles in each treatment group ( n=10)

[0145]

[0146]

[0147] The results showed that, compared with formamide, the amount of EB exudation was significantly increased when formamide containing 0.5% KOH was used as a solvent, which could significantly shorten the treatment time.

[0148] It should also be noted that the concentration of KOH mentioned above was obtained through experiments. According to the experimental data, the concentration of KOH can be controlled within the range of 0.2-1.0%, with 0.5% KOH being preferred.

[0149] Example 4: Methodological Investigation of Other Factors

[0150] 1. Studies on sex differences in laboratory animals

[0151] This experiment used male and female mice to test allergy-like reactions to Qingkailing injection, and observed whether there were gender differences in the reactions, providing a basis for selecting the appropriate animal sex for product allergy testing.

[0152] In the control group, both female and male animals were injected with saline and EB. No blue staining of the auricles was observed in any of the animals, indicating that no false positive reactions occurred. Histamine, a positive control drug, induced a significant blue staining of the auricles in both female and male mice, with all mice of both sexes showing positive or higher-level reactions (≥3 points).

[0153] When Qingkailing injection was administered at a dose of 2.5 ml / kg, 16 out of 20 male mice showed a negative reaction (0 points); however, at the same dose, all female mice showed negative results. At a dose of 5 ml / kg, 2 out of 20 male mice showed a positive or higher reaction (≥3 points), while no female mice showed a positive or higher reaction. At a dose of 10 ml / kg, 10 out of 20 male mice and 4 out of 20 female mice showed positive or higher reactions. Furthermore, at all concentrations, male mice had higher auricular blue staining scores and higher EB exudate levels than female mice.

[0154] In conclusion, male mice are more sensitive to allergy-like reactions induced by Qingkailing injection. Therefore, it is recommended that male mice be used for allergy testing using Qingkailing injection.

[0155] 2. Comparison of differences in animal strains

[0156] Allergy-like reaction tests were conducted using mice of four strains: ICR, C57, KM, and BALB / C. The results showed that a dose of Qingkailing injection of 2.5 ml / kg did not induce significant blue staining of the auricle in ICR, C57, KM, and BALB / C mice; the number of ears with suspected or negative reactions (≤1 point) was 20 / 20. A dose of Qingkailing injection of 10 ml / kg resulted in 8 / 20, 0 / 20, 12 / 20, and 0 / 20 ears with positive or higher reactions (≥3 points) in ICR, C57, KM, and BALB / C mice, respectively; the blue staining score was ranked as follows: KM≈ICR>BALB / C>C57. Furthermore, the amount of EB exudate induced by a dose of Qingkailing injection of 10 ml / kg was ranked as follows: KM>ICR>BALB / C>C57.

[0157] In summary, compared with other mouse strains, KM mice are more sensitive to allergic reactions induced by Qingkailing, while ICR mice are second only to KM mice. Therefore, it is recommended to use KM and ICR mice when conducting allergy testing for Qingkailing injection.

[0158] 3. Investigation of the mixing method of sample and indicator EB

[0159] This experiment used separate injection and combined injection methods to observe whether the allergic reaction of Qingkailing was related to the indicator mixing method, providing a basis for selecting an appropriate indicator mixing method for product allergy testing.

[0160] When the dosage of Qingkailing injection was 2.5 ml / kg, the number of ears with suspicious or negative reactions after separate and combined injections was 20 / 20. When the dosage was 5 ml / kg, 3 / 20 ears showed positive or higher reactions (≥3 points) after separate injections, and 16 / 20 were suspicious or negative (≤1 point); after combined injections, 20 / 20 were suspicious or negative. When the dosage was 10 ml / kg, the number of ears with positive or higher reactions after separate and combined injections were 2 / 20 and 12 / 20, respectively. This suggests that combined injections resulted in higher auricular blue staining scores, indicating a more pronounced allergy-like reaction. Regarding EB exudate, at a dosage of 10 ml / kg, combined injections significantly increased EB exudate compared to separate injections (P < 0.01).

[0161] In conclusion, combined injection is more sensitive for detecting allergic reactions induced by Qingkailing, and the combined injection procedure is simpler during the test. Therefore, it is recommended to use the combined injection method when conducting allergy testing for Qingkailing injection.

[0162] 4. EB specificity test

[0163] Two negative control groups were set up, one with added EB and the other without, to examine the specificity of EB. The results showed that no blue staining of the auricle was observed after intravenous administration of either the negative control solution with or without EB, indicating that no false positive reactions were observed in this experiment. In the control group with added EB, the amount of EB exudate was significantly higher than that in the control group without EB (P < 0.01), suggesting that the mice were sensitive to EB. Therefore, when conducting allergic reaction experiments with Qingkailing injection in mice, saline solution with added EB should be used as a negative control.

[0164] 5. Investigation of the dose-effect relationship of positive control drugs

[0165] The positive control drug histamine was set up with three dosage groups, with administration doses of 20 mg / kg, 10 mg / kg and 5 mg / kg, and histamine solution concentrations of 2.0 mg / ml, 1.0 mg / ml and 0.5 mg / ml, respectively. After being mixed with 0.8% EB solution at a volume ratio of 1:1, the drugs were administered intravenously at a dose of 20 ml / kg of mouse body weight to investigate the dose-response relationship of the positive control drug.

[0166] No blue staining of the auricle was observed in mice after intravenous injection of the negative control saline, indicating that no false positives were observed in this experiment. After intravenous injection of histamine at 20 mg / kg, 10 mg / kg, and 5 mg / kg, varying degrees of increased auricular vascular permeability were observed in each group of mice. The positive and above-positive reactions (≥3 points) were 20 / 20, 18 / 20, and 18 / 20, respectively. The auricular blue staining score and EB exudate were significantly higher than those in the control group (P < 0.001), indicating that the histamine-induced allergic-like reaction in mice is dose-dependent; the higher the histamine concentration, the more pronounced the induced allergic-like reaction, demonstrating a certain dose-effect relationship. However, the blue staining scores and EB exudate were quite similar in the 20 mg / kg and 10 mg / kg groups. Considering cost savings, it is recommended that the dosage of the positive control histamine be set at 10 mg / kg when conducting allergy testing with Qingkailing injection.

[0167] 6. Effects of different animal body weights on allergic reactions to Qingkailing injection.

[0168] A pseudo-allergic reaction test was conducted using mice from four different weight ranges. The results showed that no blue staining of the auricles was observed in mice from any of the four weight ranges with saline solution, indicating no pseudo-allergic reaction occurred and suggesting that no false positives were observed in any of the weight ranges. The positive control drug histamine induced similar levels of pseudo-allergic reactions in mice weighing 23-25g, 26-28g, and 29g+. After administration of histamine, the number of ears with positive or higher-level reactions (≥3 points) in mice weighing 23-25g, 26-28g, and 29g+ was 20 / 20. However, in mice with very small weights (20-22g), the pseudo-allergic reaction was weaker, and the number of ears with positive or higher-level reactions was 16 / 20.

[0169] After intravenous administration of Qingkailing injection at 2.5 ml / kg, the number of ears with suspicious or negative reactions (≤1 point) in mice weighing 20-22g, 23-25g, 26-28g, and 29g+ were 20 / 20, 19 / 20, 20 / 20, and 20 / 20, respectively. After intravenous administration of Qingkailing injection at 10 ml / kg, the number of ears with positive reactions or higher (≥3 points) in mice weighing 20-22g, 23-25g, 26-28g, and 29g+ were 5 / 20, 11 / 20, 12 / 20, and 10 / 20, respectively. No significant differences were found in the blue staining scores of ears in mice of different weights using the same test sample; the scores in the 26-28g and 23-25g groups were higher than the other two groups. The results of EB exudation showed that after intravenous administration of Qingkailing injection at 10 ml / kg, EB exudation significantly increased in mice of different weight ranges. The amount of EB exudate in the auricles of mice weighing 26-28g was generally higher than that in other weight ranges, followed by the 23-25g group.

[0170] Therefore, it is recommended to use mice weighing 23-28g for allergy testing of Qingkailing injection. Practical suggestion: Considering the need for animal acclimatization, the animals will gain 1-3g in weight during allergy testing; therefore, mice weighing 22-24g can be ordered.

[0171] 7. Sensitivity study at different injection rates

[0172] An allergy-like reaction test was conducted by intravenously injecting Qingkailing injection solution at different injection rates. The results showed that no positive blue staining reaction was observed in the auricles of mice in the saline control group at any injection rate, indicating that no allergy-like reaction occurred and suggesting that no false positives were observed in the tests at different injection rates. Histamine, the positive control drug, induced significant blue staining in the ears of mice in all injection rate groups, and the blue staining scores were similar, indicating positive or higher allergy-like reactions (≥3 points) in all groups, suggesting that no false negatives were observed in this experiment.

[0173] When Qingkailing injection (2.5 ml / kg) was administered to mice via the tail vein at three different injection speeds, the number of ears with suspicious or negative reactions (≤1 point) in the 0-5s, 30s, and 60s groups was 20 / 20. Ear blue staining scores showed similar results in the 0-5s and 60s groups, with a slightly higher score than the 30s group. After intravenous administration of Qingkailing injection (10 ml / kg), the number of ears with positive or higher reactions (≥3 points) in the 0-5s, 30s, and 60s groups were 13 / 20, 8 / 20, and 12 / 20, respectively, with no significant difference in ear blue staining scores. EB exudation results showed that after intravenous administration of Qingkailing injection (10 ml / kg), EB exudation significantly increased in all groups at different injection speeds, but there was no significant difference in overall EB exudation. These results suggest that the allergic reactions to Qingkailing induced by different injection speeds are relatively similar, and the injection speed has little effect on the allergic reactions induced by Qingkailing. Therefore, considering the feasibility and repeatability of the experiment, it is recommended that the injection be completed within 30 seconds. Experimenters need to be trained in controlling the injection speed to maintain the uniformity of the injection speed.

[0174] 8. Sensitivity study of Qingkailing injection after different preparations and storage time

[0175] This experiment used a mouse model of allergy-like reactions to compare the differences in allergy-like reactions induced by Qingkailing injection after different storage times following preparation. The results showed that in the saline control group, no positive blue staining of the auricle was observed in mice after different storage times following preparation, indicating no allergy-like reactions occurred. This suggests that no false positive reactions were observed in the saline control group at different storage times after preparation. The positive control drug histamine induced significant blue staining of the auricle and a significant increase in EB exudate at different storage times after preparation, indicating that no false negative reactions were observed in the positive control drug histamine at different storage times after preparation.

[0176] After administration of Qingkailing injection at 2.5 ml / kg for different time periods (0 h, 2 h, and 4 h), the number of mouse ears eliciting negative and questionable reactions (≤1 point) were 20 / 20, 18 / 20, and 19 / 20, respectively. After administration of Qingkailing injection at 10 ml / kg for different time periods (0 h, 2 h, and 4 h), the number of mouse ears eliciting positive and above-positive reactions (≥3 points) were 20 / 20, 20 / 20, and 18 / 20, respectively. The blue staining scores of the ears at each time point were significantly higher than those of the saline group (P<0.001), but there were no significant differences among the groups. The results of EB exudate were consistent with the blue staining scores of the auricle. The EB exudate in the 10 ml / kg group was significantly higher than that in the saline group at all time points (P<0.001), but there were no significant differences among the groups. The results suggest that the allergic reactions caused by Qingkailing injection after preparation and storage for different times were quite similar. The storage time after preparation had little impact on the allergic reactions caused by Qingkailing. For convenience, it is recommended to prepare the solution immediately before use.

[0177] 9. Sensitivity assessment of different animals to adaptive feeding time

[0178] This experiment compared the effects of different acclimatization periods on allergic reactions induced by Qingkailing injection in mice. In the saline control group, no mice showed positive blue staining of the auricles after 1, 2, and 3 days of acclimatization, indicating no allergic reactions occurred. This suggests that no false positives were observed in the saline control group at different acclimatization periods. The positive control drug histamine induced significant blue staining of the auricles in mice after 1, 2, and 3 days of acclimatization. All mice showed positive or higher allergic reactions (≥3 points), and EB exudation values ​​were significantly increased, indicating that no false negatives were observed in the positive control group at different acclimatization periods.

[0179] When Qingkailing injection was administered at 2.5 ml / kg, there were no significant differences in ear blue staining scores and EB exudate volume between mice fed for 1, 2, and 3 days of acclimatization and those fed with saline. The number of ears with negative or suspicious reactions (≤1 point) were 14 / 20, 20 / 20, and 20 / 20, respectively. The ear blue staining score of mice fed for 1 day of acclimatization was slightly higher than that of mice fed for 2 and 3 days. When Qingkailing injection was administered at 10 ml / kg, the ear blue staining scores of mice fed for 1, 2, and 3 days of acclimatization were significantly higher than those of mice fed with saline (P < 0.001). The number of ears with positive or higher reactions (≥3 points) were 16 / 20, 11 / 20, and 12 / 20, respectively. The ear blue staining score of mice fed for 1 day of acclimatization was slightly higher than that of mice fed for 2 and 3 days, but the difference was not significant. Regarding EB exudate, except for mice fed for 2 days of acclimatization, the EB exudate volume was significantly higher than that of mice fed with saline after 1 and 3 days of acclimatization (P < 0.05). The results suggest that mice showed higher sensitivity to allergic reactions induced by Qingkailing injection after 1 day of acclimatization. Considering the feeding costs and the time required for the experiment, it is recommended that mice undergo an allergy test for Qingkailing injection after 1 day of acclimatization.

[0180] 10. Dose-effect relationship study of Qingkailing injection

[0181] This experiment used a previously established anaphylactic reaction methodology model to investigate the dose-effect relationship of Qingkailing injection. The results showed that no blue staining of the auricle was observed in any of the animals in the saline control group, indicating no false positive reactions. Histamine, the positive control drug, induced significant blue staining of the auricle, indicating a strong anaphylactic reaction, thus indicating no false negative reactions were observed in this experiment.

[0182] When Qingkailing injection was administered at 2.5 ml / kg, no blue staining of the auricle was observed in any animal. At a concentration of 1 / 4 of the undiluted solution, no positive or higher-level reactions were observed in mice; 2 / 20 ears showed weak positive reactions, and 18 / 20 showed questionable or negative reactions (≤1 point). At 10 ml / kg, 6 / 20 ears showed positive or higher-level reactions (≥3 points), 3 / 20 showed weak positive reactions, and 11 / 20 showed questionable or negative reactions. Furthermore, with increasing Qingkailing injection dosage (concentration), the blue staining score of the mouse ears and the amount of exudate (EB) significantly increased. In conclusion, the allergic-like reactions of Qingkailing injection have a clear dose-response relationship; higher concentrations produce significant reactions, but lowering the concentration may reduce the risk. This suggests that in clinical use, the drug concentration should be strictly controlled, and the administration rate should be minimized to reduce the instantaneous concentration in the body.

[0183] 11. Repeatability study of allergic reactions to Qingkailing injection

[0184] The experiment was repeated three times using the same batch number to evaluate the reproducibility of the methodology for detecting allergic reactions to Qingkailing injection. Results showed that the negative control group had negative reactions in all three experiments, and the positive control group had extremely strong positive reactions, with similar results. At a dosage of 2.5 ml / kg, three tail vein injections of Qingkailing injection resulted in 20 / 20, 20 / 20, and 20 / 20 mice showing suspected or negative allergic reactions (≤1 point). At a concentration of 1 / 4 of the original solution, 6 / 20, 1 / 20, and 5 / 20 mice showed weakly positive and positive allergic reactions (2-3 points). At a dosage of 10 ml / kg, 7 / 20, 8 / 20, and 11 / 20 mice showed positive and above allergic reactions (≥3 points). Furthermore, with increasing dosage (concentration), the blue staining score and EB exudate volume gradually increased in the three experiments. Although there were some differences among the groups at the same concentration, these differences were not statistically significant and were within an acceptable range. This indicates that the methodology for allergic reactions to Qingkailing injection established in this experiment has good reproducibility.

[0185] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A model for detecting Qingkailing injection allergy reaction, characterized in that, It includes seven modules: (1) Module 1: Mouse ear blue staining area ratio test and data storage module; This module includes: an injection module, an ear blue staining area percentage statistics module, and a data storage module; The injection module includes injectable reagents: Qingkailing injection, Evans blue, histamine, and physiological saline; The ear blue staining area percentage statistics module is used to statistically analyze the proportion of blue staining area in mouse ears to the auricle area after injection, and obtain the blue staining area percentage data of each group of mice; the data of each group is stored in the data storage module. (2) Module 2: Mouse ear blue staining area scoring module; This module includes the scoring criteria shown in Table 1; This module is used to receive the data from Module 1 above, analyze it according to the scoring criteria shown in Table 1, and provide scoring data; Table 1 Scoring criteria for blue staining area (S) of auricle (3) Module 3: Mouse ear blue staining area result judgment module; This module includes the criteria for judging the results based on the scoring data from Module 2; the criteria are as follows: Qingkailing injection group: If no blue staining is observed in any animal, the result is negative; Qingkailing injection group: If ≤30% of animals have a blue staining score ≥1 and all scores <2, the result is negative; Qingkailing injection group: If more than 30% of animals have a blue staining score ≥1, but less than 10% of animals have a score ≥3, the result is considered suspicious. Qingkailing injection group: If more than 10% of the animals have a blue staining score ≥3, the result is considered positive; If the result is questionable, the test should be repeated; if the result of the retest is still questionable or negative, it should be judged as negative; if the result of the retest is positive, it should be tested again, and the result of two consistent results out of three tests should be used as the final judgment. (4) Module 4: Quantitative test and data storage module for Evans blue exudate in mouse ear tissue; This module includes: an extraction module, a filtration module, an absorbance testing module, and a data storage module; The extraction module includes a formamide solution containing 0.2-1.0% KOH, used to extract Evans blue from mouse ear tissue, yielding an extract. The filtration module is used to filter the extract to obtain the filtrate; The absorbance testing module is used to test the absorbance data of the filtrate. The obtained data is stored in the data storage module and output to module five. (5) Module 5: Calculation module for Evans blue exudate in mouse ear tissue; This module includes the Evans Blue standard curve; This module receives absorbance data from module four and calculates the Evans blue exudate in the mouse ear tissue of each group based on the Evans blue standard curve. (6) Module Six: Module for Judging the Evans Blue Exudate Result in Mouse Ear Tissue; This module receives Evans blue exudate data from each group of mouse ear tissues from Module 5. If the Evans blue exudate in the Qingkailing injection group is significantly higher than that in the negative control group, it is judged as suspicious or positive. The specific judgment criteria are as follows: If the increase is less than 50%, the result is negative; If the increase is 50-75% and the statistical difference is P<0.05, the result is suspicious. If the increase is 75%-100%, the result is weakly positive; If the increase is greater than 100%, the result is positive; (7) Module 7: Result Output Module; This module integrates the results from Modules Three and Six according to the following criteria, and outputs the final judgment result: If both the results of Module 3 and Module 6 are negative, then the final result is judged as negative. If the results of Module 3 and / or Module 6 are suspicious, then the final result is judged to be suspicious. If the results of Module 3 and / or Module 6 are positive, then the final result is judged as positive.

2. The model of claim 1, wherein, In Module 1, the requirements for the source of each group of data are as follows: ① Data on the percentage of blue-stained area in the ears of mice in the Qingkailing injection group. Source requirements: Data on the percentage of blue-stained area in the ears of mice observed within 30 minutes after intravenous injection of Qingkailing injection and 0.6-1.0% Evans blue solution at a volume ratio of 1:

1. ② Data on the percentage of blue-stained area in the ears of mice in the histamine-positive control group. Source requirements: The percentage of blue-stained area in the ears of mice was observed within 30 minutes after intravenous injection of histamine and 0.6-1.0% Evans blue solution at a volume ratio of 1:

1. ③ Data on the percentage of blue-stained area in the ears of mice in the saline negative control group. Source requirements: The percentage of blue-stained area in the ears of mice was observed within 30 minutes after intravenous injection of saline and 0.6-1.0% Evans blue solution at a volume ratio of 1:

1.

3. The model of claim 2, wherein, The Qingkailing injection in Module 1① should be diluted with the original solution and physiological saline at a volume ratio of 1:(0-1) before use.

4. The model of claim 2 wherein, In Module 1, the concentration of Evans blue solution is 0.8%.

5. The model according to claim 2, characterized in that, In Module 1, the concentration of histamine is 0.5-2.0 mg / ml, and the solvent is physiological saline.

6. The model according to claim 1, characterized in that, The absorbance testing module is an enzyme-linked immunosorbent assay (ELISA) reader or a UV spectrophotometer, with wavelengths including 610 nm.

7. The model according to claim 1, characterized in that, The method for constructing the Evans blue standard curve is as follows: using formamide containing 0.5% KOH as a solvent, Evans blue is prepared into solutions of 0, 1, 2, 4, 6, 8, and 10 μg / ml. Two replicates are made for each concentration, and 1 ml is taken from each well. The absorbance is measured at a wavelength of 610 nm. The absorbance is X and the EB concentration is Y, and a standard curve is plotted.

8. The model according to claim 2, characterized in that, The intravenous injection dose for mice was 20 ml / kg of mouse body weight.

9. The model according to claim 2, characterized in that, The mice were either KM mice or ICR mice.

10. The model according to claim 9, characterized in that, The mice were male.

11. The model according to claim 9, characterized in that, The mice weighed 23-28g.

12. The model according to claim 2, characterized in that, After 1 day of acclimatization, the mice were subjected to an injection experiment.

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