The main content of preclinical toxicology research is the safety evaluation of drugs. Whether the drug is safe and effective is the determining factor for the success of drug development. In the entire process of drug development, toxicity (safety) is an important factor in terminating drug development. one of the reasons. The purpose is to study the relationship between drug exposure and toxic reactions in animal models, explain possible toxic target organs and toxic reactions, predict human safety, and provide a reliable toxicokinetic basis for later (human) clinical trials.
The main specific contents of the research are:
①Acute toxicity test;
② long-term toxicity test;
③ Genotoxicity test;
④ Reproductive toxicity test;
⑤ carcinogenic toxicity test.
1) Acute toxicity test (also known as single-dose toxicity test)
I. Definition: It refers to the study of the toxicity of the drug within a certain period of time after a single or multiple administration of the drug within 24 hours. The core of the test is to observe the toxicity of the animal after administration.
II. Significance:
①It plays an important role in initially clarifying the toxic effects of drugs and understanding their toxic target organs.
② It has a direct reference for the dose design of long-term toxicity tests.
③ It is of reference significance for the selection of the initial dose of later human clinical trials.
III. Animal requirements: At least two mammals, rodent and non-rodent, are used, usually healthy adult animals of two sexes, half male and half male.
IV. Test method: According to the characteristics of the tested drug, the appropriate dose should be selected through preliminary experiments. In principle, the dose should include the dose with no toxic reaction to the dose with severe toxicity or the maximum dose. Commonly used The test methods include approximate lethal dose method, maximum dose method; maximum tolerated dose method; fixed dose method; median lethal dose method; cumulative dose method (pyramid method) and so on.
V. Test content:
① After administration, collect blood or tissue for analysis according to the experimental design, and study the relationship between exposure and time.
②The experimental animals are generally observed continuously for at least 14 days to observe the symptoms of toxic reactions and the time of appearance, recovery and death. Common observation indications include (1) clinical symptoms (animal appearance; behavior; diet; response to stimulation; secretions; excrement) (2) Death (time and moribund reaction) (3) Body weight change (observation period before and after administration)
③ After the animals were euthanized, gross dissection and histopathological examination were performed to determine the toxic target organs.
2) Long-term toxicity test (also known as repeated dose toxicity test)
I. Definition: It refers to the observation of the toxic reactions produced by animals after repeated administration. It is the most comprehensive non-clinical safety evaluation, the most informative toxicology test and the most meaningful for clinical guidance. (Because of the long test period and high cost, in order to reduce blindness, it is usually carried out after the pharmacological test and acute toxicity test or pre-experiment, and the test drug is confirmed to have further research value)
II. Significance:
①Predict the possible clinical adverse reactions caused by drugs, and provide reference for clinical adverse reaction monitoring and prevention and rescue.
② Determine the toxic target organs and target tissues of repeated drug administration.
③ Determine the dose level at which no clinical adverse reactions are observed (NOAEL), extrapolate the results to the human body, calculate the initial dose of the first-in-human clinical trial (FIH), and provide a safe dose range for subsequent clinical trials.
III. Animal requirements: rodent and non-rodent mammals are usually used, usually rats and beagle dogs or monkeys, normal, healthy and sexually mature animals are generally selected, and disease animal models are used for testing if necessary.
IV. Experimental Design:
① During the administration period, at least three dose groups, low, middle and high, and a vehicle or excipient control group are set. equivalent dose. Moderate dose combined toxicity mechanism and characteristics are in between.
②Set a recovery period. The length of the recovery period is determined according to the pharmacokinetic characteristics and target organ toxicity and recovery. Generally, it should not be less than 4 weeks. After the introduction of the drug, some animals will continue to observe the recovery period to understand the toxicity of the reaction. Reversible and possible late toxicity.
③ The route of administration should generally be consistent with the intended clinical route.
④ The duration of the long-term toxicity test is generally determined according to the clinical administration course. For example, when the clinical administration course is 2 weeks, a 1-month animal long-term toxicity test is required to support the drug marketing application. If the administration course is from 2 weeks to 1 month , a 3-month animal long-term toxicity test is required.
V. Test content:
① After administration, collect blood or tissue for analysis according to the experimental design, and study the relationship between exposure and time.
②Observe the symptoms of toxic reactions. Common observation indications include (1) clinical symptoms (animal appearance; behavior; diet; response to stimulation; secretions; excretions) and changes in body weight (observation period before and after administration), Record each index in detail to detect toxic reactions as early as possible and reflect the relationship between changes in index parameters and test duration.
③ After the administration, the animals in the main experimental group were systematically dissected, the main organs were weighed, and the histopathological examination was carried out to issue a complete pathological examination report.
3) Reproductive toxicity test
I. Definition: Refers to the effects of the research drug on the reproductive function and development of mammals, including Section I: general reproductive toxicity test, Section II: teratogenic sensitivity test and Section III: perinatal toxicity test.
Section I: General reproductive toxicity test: It is to study the toxicity test of drugs on fertility and early embryonic development from conception before mating to implantation to evaluate the toxicity and interference effects of drugs on animal reproduction.
Section II: Teratogenic Sensitivity Phase Test: refers to the evaluation of the effects of drugs on the development of pregnant animals, embryos and fetuses from embryo implantation to embryonic major organ formation and pregnancy termination in pregnant animals.
Section III: Perinatal toxicity test: refers to the fetus from birth to weaning and then to sexual maturity, evaluating the enhanced toxicity of pregnant animals compared with non-pregnancy, the death of offspring before and after birth, developmental and functional defects.
II. Purpose: To predict the possible adverse effects of drugs on germ cells and parental reproductive functions such as conception, pregnancy, childbirth and lactation, as well as adverse effects on offspring embryo-fetal development and postnatal development.
III. Animal requirements: usually use the same species as other toxicology tests, which is conducive to comparison with other results. Generally, at least one animal is used. Rodents are commonly used in rats, and non-rodents are commonly used in rabbits. The selected animals must be Young, sexually mature, females are nulliparous. (The requirements for experimental animals are close to humans, small in size, fast in reproduction, cheap in price, short in gestation period, and large in litter size)
IV. Experimental Design:
① The test generally consists of three dose groups and a solvent control group.
②The principle of administration is consistent with the intended clinical use.
③ Usually there are no less than 20 pregnant mice.
④The high dose should have some mild maternal toxicity or the maximum tolerated dose, and the low dose should be the dose level at which no clinical adverse reactions were observed (NOAEL).
⑤Dosing period:
For I-segment males 4-10 weeks before mating to the end of mating, females were dosed from 2 weeks before dosing until embryo implantation (GD6-GD7).
For segment II, female mouse and female rabbits were implanted in embryos to form major embryonic organs (GD6-GD18).
For segment III, from embryonic major organ formation to the end of lactation (GD15-PND18).
V. Test content:
① After administration, collect blood or tissue for analysis according to the experimental design, and study the relationship between exposure and time.
②On the basis of counting the reaction, death, body weight, drinking water and diet after administration, the fertility rate, sperm count, motility and deformity rate of male mice were counted, and the conception rate, pregnancy rate, implantation mortality, litter birth rate, etc. of female mice were counted. , dead babies, the number of deformities and the survival, growth and development of their offspring.
③Usually, before the start of clinical research, it is necessary to provide the research data of stage I and stage II of reproductive toxicity, and provide the research data of stage III in the marketing application.
4) Genotoxicity test
In vivo and in vitro assays for the detection of test substances that directly or indirectly induce genetic damage through different mechanisms, mainly used to predict the carcinogenicity of test substances. The requirements for the carcinogenicity test of new drugs in the Drug Registration Management Measures:
(1) The structure of the new drug is related to known carcinogens, and the metabolites are similar to known carcinogens;
(2) New drugs that have cytotoxic effects or can make certain organs and tissue cells abnormally active in the long-term toxicity test, as well as new drugs with positive mutagenic results, must submit carcinogenic test data.
Because carcinogenicity testing is time-consuming and animal-resource-intensive, carcinogenicity testing, usually using rats or mice, is only performed when it is truly necessary to study the potential carcinogenicity resulting from drug exposure in humans by long-term animal dosing.
