Method for testing passive skin anaphylaxis of radiopharmaceuticals

Abstract

The invention relates to a radiopharmaceutical passive skin anaphylaxis test method, which comprises the following steps: animal grouping: dividing test animals into a positive control group, a negative control group and a drug group, and marking; preparing the medicine: respectively mixing the negative reference substance, the positive reference substance and the radiopharmaceutical with the adjuvant to obtain each group of mixed emulsion; preparing antibody serum: taking a plurality of test animals from each group, performing drug injection, sampling blood on the fourteenth day after the last sensitization, separating the serum, and diluting to obtain antibody serum with medium, high and low concentrations; passive sensitization: taking a plurality of normal test animals from each group, injecting corresponding antibody serum into each group, and carrying out passive sensitization; and excitation: after sensitization is carried out for 48 hours, injecting each group of solutions containing dye liquor into each group of test animals for excitation, measuring the size of blue spots, and determining that the test animals with the diameter greater than 5 mm are positive. The method can be used for safely, effectively, simply and conveniently carrying out preclinical evaluation research on the sensitization of various radiopharmaceuticals.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a method for passive skin allergy testing of radioactive drugs. Background Art

[0002] Allergic reaction is a common adverse reaction of drugs, which can cause local or systemic adverse symptoms in the skin system, respiratory tract, digestive tract, etc. In severe cases, wheezing, difficulty breathing, and systemic adverse symptoms such as hypotension and arrhythmia may occur, and may even induce anaphylactic shock or death.

[0003] Drugs can enter the local or systemic body fluid circulation through blood vessels, muscles, subcutaneous tissues, etc. to achieve diagnostic or therapeutic effects. According to the technical guidelines for drug irritation, allergy and hemolysis research, drugs (injections, etc.) that are usually administered locally to exert systemic effects need to be examined for type I (immediate allergic type) allergic reactions. Preclinical allergy evaluation test methods include active systemic allergy tests and passive skin allergy tests. Radioactive drugs are used as imaging diagnosis and effective treatment types for diseases such as tumors. Injections are the main preparation. Like non-radioactive drugs, whether they can cause type I allergic reactions in the body is an important part of preclinical safety evaluation. In addition, due to the particularity of radioactive drugs, the skin allergy test method established for them requires not only high accuracy and good repeatability, but also easy radiation protection during operation to fully ensure the radiation protection safety of operators. Summary of the invention

[0004] The purpose of the present invention is to overcome the defects in the prior art and provide a method for passive skin allergy test of radioactive drugs, which can safely, effectively and simply carry out preclinical evaluation research on the sensitization of various radioactive drugs.

[0005] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] A method for passive skin allergy testing of radiopharmaceuticals, comprising:

[0007] Animal grouping: The experimental animals were divided into a positive control group, a negative control group and a drug group and marked.

[0008] Preparation of drugs: negative control, positive control, radioactive drug and adjuvant were mixed evenly in equal volumes to obtain mixed emulsions of each group;

[0009] Preparation of antibody serum: Several test animals were selected from each group for drug injection. The test animals were divided into positive control group, negative control group and drug group, and injected with corresponding mixed emulsion for sensitization for several times. On the 14th day after the last sensitization, blood was collected from the abdominal aorta of the sensitized animals, serum was separated and diluted to obtain antibody serum of medium, high and low concentrations.

[0010] Passive sensitization: Take a number of normal test animals (non-sensitized) from each group, and inject the corresponding antibody serum into each group for passive sensitization; the injection volume is 0.1ml / point;

[0011] Stimulation: Add Evans blue dye solution to each group of solutions (negative control, positive control, radiopharmaceutical) to prepare a mixed solution; 48 hours after the passive sensitization of the test animals, inject the corresponding mixed solution into the tail vein of each group of test animals for stimulation, observe the symptoms of the animals after stimulation, anesthetize and kill the animals 30 minutes later, observe and measure the size of the blue spots in the inner layer of the skin, those with a diameter of blue spots greater than 5 mm are positive, and the diameter of irregular spots is half of the sum of the long and short diameters.

[0012] As a further technical solution, in the preparation of the drug, the mixing operation of the radioactive drug and the adjuvant is carried out behind a protective screen or in a glove box.

[0013] As a further technical solution, during the sensitization phase, experimental animals in the drug group were housed in single cages with lead plates or lead glass around the cages for protection;

[0014] As a further technical solution, the test animals are healthy male SPF-grade SD rats; the sensitization test is carried out after one week of adaptive feeding.

[0015] As a further technical solution, the feeding and management of rats: rats are kept in cages, with no more than 5 rats in each cage. The temperature in the feeding room is controlled at 20-25°C, and the humidity is controlled at 40-70%. The rat feeding cages and water bottles are disinfected regularly, and the bedding used is sterilized by high pressure.

[0016] As a further technical solution, the sensitized animals in the high-dose and low-dose groups during the sensitization period were housed in single cages and given free access to food and water;

[0017] As a further technical solution, sodium chloride injection was used as a negative control;

[0018] As a further technical solution, the positive control substance used 1% ovalbumin;

[0019] As a further technical solution, radiopharmaceuticals use high-technetium [ 99m Tc] sodium acid injection;

[0020] As a further technical solution, the adjuvant is aluminum hydroxide adjuvant;

[0021] As a further technical solution, after the experimental animals were grouped, picric acid was used to mark the rats.

[0022] As a further technical solution, the negative control substance, the positive control substance, the radioactive drug and the adjuvant are mixed using a multifunctional mixer.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention uses a passive skin allergy test to evaluate the skin allergy of radioactive drugs, and uses immune adjuvants to enhance immunogenicity, increase the titer of antibodies, and induce or enhance delayed hypersensitivity reactions. The application in the passive skin allergy test can enhance the body's immune response ability to the test substance.

[0025] 2. The present invention uses aluminum adjuvant as an immune adjuvant, which has the following advantages: 1) During the sensitization stage, the preparation method of the drug-adjuvant complex is simple and convenient, and can be completed in a glove box or behind a lead screen, which can effectively reduce the radiation dose of the operator; 2) The formation of granulomas and persistent ulcers at the injection site can be avoided.

[0026] 3. In the present invention, during the sensitization stage, the experimental animals in the drug group are housed individually in protective cages to avoid mutual irradiation between individuals in the laboratory.

[0027] In summary, the method of the present invention is simple to operate, the positive system is reliable, and the radioactive drug and the adjuvant can be fully mixed under shielding conditions, thereby reducing the radiation dose of personnel, and can fully stimulate the immune response mechanism of animals, and safely and effectively evaluate the allergic properties of radioactive injection preparations. It can be used for the study and evaluation of the passive skin allergy of radioactive drugs in preclinical settings. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the blue spot condition of the inner layer of the skin in the negative control group in one embodiment of the present invention;

[0029] Figure 2 This is the blue spot condition of the inner layer of the skin of the positive control group in one embodiment of the present invention;

[0030] Figure 3 This is the blue spot condition of the inner layer of the skin of the drug group in one embodiment of the present invention. DETAILED DESCRIPTION

[0031] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] In addition, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0033] Unless otherwise specified, the raw materials used in the present invention are commercially available.

[0034] Embodiment 1:

[0035] 1. Instruments and equipment

[0036] Multifunctional mixer, electronic digital caliper;

[0037] 2. Animals and Grouping

[0038] 18 healthy male SPF SD rats were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. (Production License No.: SCXK (Beijing) 2021-0006), weighing 180-220g. According to the weight of the animals, they were randomly divided into negative control group, positive control group and drug group, with 6 rats in each group, and the animal numbers were (M101-M106, M201-M206, M301-M306). Rats were kept in cages, with no more than 5 rats in each cage. The sensitized animals in the high-dose and low-dose groups during the sensitization period were kept in single cages, with free food and water. The feed was SPF rat and mouse maintenance feed produced by Beijing Keao Xieli Feed Co., Ltd., feed production license: Beijing Feed Certificate (2018) 06073, the temperature of the feeding room was controlled at 20-25℃, the humidity was controlled at 40-70%, the rat cages and drinking bottles were disinfected regularly, and the bedding used was sterilized by high pressure. After grouping, the rats were marked with picric acid. The animals were adaptively raised for 1 week before the experiment.

[0039] 3. Test methods

[0040] Drug preparation: negative control substance (sodium chloride injection), positive control substance (1% ovalbumin), radiopharmaceutical (pertechnetium [ 99m Tc] sodium iodide injection) was mixed with aluminum hydroxide adjuvant in equal volumes, placed in a multifunctional mixer, and mixed at 1500 rpm / min for 15-20 min. The mixing process was carried out behind a protective screen or in a glove box, and each group of solutions was finally made milky;

[0041] Preparation of antibody serum (sensitized serum): 2 rats were selected from each group, and the emulsions of each group were injected alternately into the muscles of the four limbs, once every other day, and sensitized 3 times. The animals in the drug group were kept in single cages, and the cages were protected by lead plates or lead glass on all sides. On the 14th day after the last sensitization, blood was collected from the abdominal aorta of the sensitized animals, and the serum was separated. The serum was diluted 2 times and 8 times to obtain antibody sera of medium, high and low concentrations (original antibody serum, 2-fold diluted antibody serum and 8-fold diluted antibody serum);

[0042] Passive sensitization: On the day after the antibody serum was prepared, the remaining 4 animals in each group were injected intradermally into the hair removal areas on both sides of the back of the animals with the antibody serum of the corresponding group (original antibody serum, 2-fold diluted antibody serum and 8-fold diluted antibody serum) for passive sensitization; the injection volume was 0.1 ml / point;

[0043] Stimulation: Add Evans blue dye solution to each group of solutions (negative control, positive control, radiopharmaceutical) to prepare a mixed solution; 48 hours after the passive sensitization of the test animals, inject the corresponding mixed solution into the tail vein of each group of test animals for stimulation, observe the symptoms of the animals after stimulation, anesthetize and kill the animals 30 minutes later, observe and measure the size of the blue spots in the inner layer of the skin, those with a diameter of blue spots greater than 5 mm are positive, and the diameter of irregular spots is half of the sum of the long and short diameters.

[0044] 4. Test results

[0045] After stimulation, no blue spots were found in the inner layer of the skin of the 4 animals in the negative control group, which was negative. Blue spots appeared in the skin of the 4 animals in the positive control group at the injection points of the original serum, 2-fold and 8-fold diluted serum, and the average diameter of the blue spots was greater than 5mm, with a positive rate of 100%. 99m Tc] sodium phosphate injection) No abnormalities were observed in all animals after provocative administration, and no blue spots were observed at the intradermal injection points of all animals, and the results were negative. See Table 1 and Figure 1-3 .

[0046] Table 1 Average diameter of blue spots in passive skin allergy test

[0047]

[0048]

[0049] The above-described embodiments are only preferred embodiments of the present invention, and are not exhaustive of the feasible implementations of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

Claims

1. A method for passive skin allergy testing of radiopharmaceuticals, characterized in that: include: Animal grouping: The experimental animals were divided into a positive control group, a negative control group and a drug group and marked. Preparation of drugs: negative control, positive control, radioactive drug and adjuvant were mixed evenly in equal volumes to obtain mixed emulsions of each group; Preparation of antibody serum: Several test animals were selected from each group for drug injection. The test animals were divided into positive control group, negative control group and drug group, and injected with corresponding mixed emulsion for sensitization for several times. On the 14th day after the last sensitization, blood was collected from the abdominal aorta of the sensitized animals, serum was separated and diluted to obtain antibody serum of medium, high and low concentrations. Passive sensitization: Take a number of normal test animals from each group and inject the corresponding antibody serum into each group for passive sensitization; the injection volume is 0.1ml / point; Stimulation: Add Evans blue dye solution to each group of solutions to prepare a mixed solution; 48 hours after the test animals were passively sensitized, inject the corresponding mixed solution into the tail vein of each group of test animals for stimulation, observe the symptoms of the animals after stimulation, anesthetize and kill the animals 30 minutes later, observe and measure the size of the blue spots in the inner layer of the skin, those with a diameter of blue spots greater than 5mm are positive, and the diameter of irregular spots is half of the sum of the long diameter and the short diameter.

2. The method for passive skin allergy testing of radiopharmaceuticals according to claim 1, characterized in that: In the preparation of drugs, the mixing operation of radiopharmaceuticals and adjuvants is carried out behind a protective screen or in a glove box; During the sensitization stage, the experimental animals in the drug group were housed in single cages, and the cages were protected by lead plates or lead glass around them.

3. The method for passive skin allergy testing of radiopharmaceuticals according to claim 1, characterized in that: The experimental animals are healthy male SPF-grade SD rats; the sensitization test is performed after one week of adaptive feeding.

4. The method for passive skin allergy testing of radiopharmaceuticals according to claim 3, characterized in that: Rat feeding and management: Rats are kept in separate cages, with no more than 5 rats in each cage. The temperature in the feeding room is controlled at 20-25°C, and the humidity is controlled at 40-70%. Rat feeding cages and water bottles are disinfected regularly, and the bedding used is sterilized by high pressure.

5. The method for passive skin allergy testing of radiopharmaceuticals according to claim 4, characterized in that: During the sensitization period, the sensitized animals in the high-dose and low-dose groups were housed in single cages and were allowed to eat and drink freely.

6. The method for passive skin allergy testing of radiopharmaceuticals according to claim 1, characterized in that: Sodium chloride injection was used as negative control; The positive control used 1% ovalbumin; Radiopharmaceuticals using high-technetium 99m Tc] sodium acid injection; Aluminum hydroxide adjuvant was used as adjuvant.

7. The method for passive skin allergy testing of radiopharmaceuticals according to claim 1, characterized in that: After the experimental animals were divided into groups, the rats were marked with picric acid.

8. The method for passive skin allergy testing of radiopharmaceuticals according to claim 1, characterized in that: The negative control, positive control, radioactive drug and adjuvant are mixed using a multifunctional mixer.

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