(1. Center for Drug Evaluation of the State Food and Drug Administration, Beijing 100038; 2. Laboratory of Pharmacology and Toxicology, Shanghai Family Planning Research Institute, China Fertility Regulation Drug Toxicology Testing Center, Shanghai 200032; 3. National Population and Planning Key Laboratory of Family Planning Medicines of the Fertility Committee, Shanghai 200032; 4. Institute of Reproduction and Development, Fudan University, Shanghai 200031)
The 2005 edition of the “Technical Guidelines for Long-term Toxicity Testing of Traditional Chinese Medicines and Natural Medicines” and the “Technical Guidelines for Toxicity Research on Repeated Administration of Drugs” refers to Chinese medicinal materials that are included in the “Administrative Measures for Toxic Drugs for Medical Use” of the State Council. That is, stone stone, frost, mercury, raw horse money, Sichuan black abalone, grass black, white aconite, raw aconite, raw pinellia, raw southern star, raw croton, spotted worm, red worm, red worm, gansui ､German wolf venom, garcinia yellow, lavender, celestial fairy, mutton flower, wormwood on the snow, red rose, white jiangdan, toad crisp, gold flower, red powder, light powder and realgar. In addition, where is The medicinal materials with toxic effects or compound prescriptions containing obvious toxic ingredients discovered in recent years are treated as toxic medicinal materials.
The toxicity of traditional Chinese medicine has received attention from all parties. According to the current new drug technical review requirements, if the prescription contains toxic medicinal materials and an obvious toxic reaction occurs in an animal toxicology study, it is generally required to supplement the repetition of the second animal Dosing toxicity test, and it is recommended to conduct accompanying toxicokinetic studies on the toxic components of measurable toxic medicinal materials. Explore the pharmacokinetic characteristics of the toxic components in the body, whether they are fully exposed in the body, and whether they are similar to the toxic symptoms of the tested animals. Time-effect relationship and dose-effect relationship, etc., provide more information for the human pharmacokinetic study of the planned phase I clinical trial. With the gradual improvement of drug registration management methods and extensive and in-depth research on basic research, Chinese medicine and natural medicine The requirements for safety evaluation research will gradually increase, and focus on the evaluation of its safety, provide non-clinical safety evaluation data for risk control of clinical trials and provide reference for the formulation of clinical programs.
Although the quality controllability of the standards for toxic medicinal materials of traditional Chinese medicines included in the current pharmacopoeia has been improved compared with the past, it is still not comprehensive enough, and the methods used for pharmaceutical quality control are not suitable for non-clinical and clinical studies of Chinese pharmacokinetics/toxicokinetics in vivo Detection and analysis of the concentration of the tested drug. At present, most of the toxic components of traditional Chinese medicine have not been subjected to pharmacokinetic/toxicokinetic studies, and even the testing and analysis of the test substances are not mandatory, and most of the toxic components have no relevant reference materials available. Therefore, it is recommended to encourage and strengthen basic scientific research on toxic Chinese medicinal materials and their toxic components, guide the establishment and verification of pharmacokinetic/toxicokinetic methodologies for toxic components of toxic medicinal materials, and improve Chinese medicines containing toxic medicinal materials and their compound prescriptions. Risk control of preparations.
The main toxic components of toxic medicinal materials and their main toxic effects are shown in Table 1. In recent years, there have been more and more reports on the toxicological research of toxic medicinal materials, and relevant detection and analysis of the toxic ingredients contained.
Bufogin has a variety of pharmacological activities, but its toxic effect is great. Libufagin and Cinobufagin are its main active components and also its main toxic components. Liu Dong et al. established bbufalin and bufolin in rabbit plasma. Liquid chromatography of lipobufastin and used in the study of its pharmacokinetics.
Strychnine and brucine are the main toxic components, which are highly toxic. They are generally used as medicine after processing. Strychnine and brucine are the main toxic components, accounting for 45% to 50% of the total alkaloids of strychnine. There are related reports on the content detection of strychnine and strychnine, the toxic components of the seeds, including the use of high performance liquid chromatography to control the content of related alkaloids in pharmaceutical preparations and the blood drugs for pharmacokinetic studies in animal experiments. Concentration detection. In addition, this research unit conducted a Bea-gle canine vaginal administration repeated dosing toxicity test on a traditional Chinese medicine compound preparation containing strychnine, because it is different from the traditional way of administration of strychnine, during the test process Concomitant toxicokinetic studies were used to analyze the blood concentration of its toxic components by liquid chromatography-mass spectrometry (LC-MS/MS). The results found that the content of this toxic component in the blood was extremely low. The results of kinetics research can quantify its metabolism in the body.
Studies have found that many toxic medicinal materials have good medicinal effects. In order to change the situation of “talking about poisoning and discoloration”, it is necessary to conduct in-depth research from the basic research level and provide detailed test data to fully understand the test substance, especially the toxic ingredients. The exposure of traditional Chinese medicines in the body, to obtain more safety evaluation information of the test substance, whether it is the protection of the safety of the subjects or the evaluation of the supervision and management agency, it can be used to analyze its toxic components from a scientific point of view. The basic situation in the body provides more objective information.
Toxicokinetics is a marginal branch involving pharmacokinetics and toxicology research. It uses the principles and methods of pharmacokinetics to quantitatively study the absorption, distribution, metabolism, and metabolism of drugs in animals at toxic doses. Excretion process and characteristics, and then to explore the regularity of the occurrence and development of drug toxicity. The purpose of toxicokinetic research is to know the degree and duration of systemic exposure of the test substance at different dose levels in the toxicity test, and to predict the test substance in the human body Potential risks during exposure. The focus of toxicokinetic research is to interpret the results of toxicity tests and predict human safety, rather than simply describing the characteristics of the basic pharmacokinetic parameters of the test substance. Toxicokinetics has become a non-clinical toxicity test One of the important research content.
Recently, the State Council’s “Opinions on Reforming the Drug and Medical Device Review and Approval System” (Guo Fa [2015] No. 44) and the State Food and Drug Administration “Announcement on Several Policies on Drug Registration Review and Approval” ([2015] No. 230 No.) After the promulgation, the Drug Evaluation Center of the State Food and Drug Administration pays more attention to the importance of drug safety in the review of new drugs. There is no need to “talk about toxic discoloration” in new drug development and drug review, as long as the content of the drug is fully studied. The toxicity of the test substance of toxic medicinal materials includes the exploratory study of the toxic components associated with toxicokinetics, so that the toxic effect and mechanism of the test substance have a more comprehensive understanding, which is convenient for clinical research and post-marketing supervision. Promote further drug development under controllable conditions.
Traditional Chinese medicine has its particularity due to many factors. At present, it is not mandatory to carry out toxicokinetic studies in non-clinical safety evaluation. However, the establishment, verification and verification of pharmacokinetic/toxicokinetic methodologies of toxic ingredients of toxic medicinal materials are not mandatory. Application is conducive to more scientific research on drug safety, quantitative evaluation of drug absorption, distribution, metabolism and excretion in the body, and can fully understand the dose-effect relationship and time-effect relationship between the toxicity of the test substance and the toxic ingredients ｡On the basis of obtaining more toxic effects and mechanism information, clinical trial research can reduce the risk to a minimum, and carry out indicator monitoring and risk control more scientifically. With the gradual deepening and improvement of the basic level of drug research and development, Technical requirements related to Chinese medicine will be gradually revised and research requirements will be improved.
In the safety evaluation research of Chinese medicine compound preparations, the content of toxic ingredients related to toxic medicinal materials can be detected, which can more clearly understand the absorption and metabolism of toxic ingredients in the body, and explore the toxic reactions that occur in the toxicity test research and the dose. Relevance. The toxicokinetic studies of the toxic ingredients of toxic medicinal materials can generally be carried out in a single-dose toxicity test, repeated-dose toxicity test, and reproductive toxicity test research.
When conducting this research, we need to explore the question of what ingredients need to be tested. Chinese medicine is different from chemical drugs and biological products, and has certain uniqueness and complexity. In particular, the detection and analysis of Chinese medicine compound preparations has always been a recognized problem. In medicine The detection and analysis of all ingredients in safety research is neither scientific nor realistic, and specific issues need to be analyzed in detail.
For example, during the review process, there is a case of a Chinese medicine compound variety. Although it is derived from the Zuchuan secret recipe and has a large amount of early clinical use experience, the prescription contains many toxic medicinal materials. During the pre-clinical safety evaluation research process, it was discovered Both SD rats and Beagle dogs of compound preparations showed weight loss in repeated administration toxicity tests. Most of the dead animals were male animals. The review concluded that gender differences in toxicity should be considered. In addition, in the repeated administration toxicity test of Beagle dogs, Vomiting in the test animal may cause inaccurate dosage, and the obtained dose value without observable damaging effect is too low, the safety window is narrow, and it is difficult to ensure the safety of clinical medication. Pathological examination found that it is harmful to the digestive tract of Beagle dogs. Definite damage effect, 2 dogs died, the cause may be due to vomiting, diarrhea or even intestinal mucosal necrosis, resulting in dehydration, electrolyte flocculation, and acid-base imbalance. The review recommended the use of sensitive animals (such as cynomolgus monkeys). Carry out repeated administration toxicity test; this variety contains many toxic medicinal materials, which has potential safety hazards. In safety research, we should focus on the relationship between toxicity performance and blood content of toxic components in traditional Chinese medicine, especially toxic medicinal materials. It is recommended to choose several flavors of toxicity Toxic components in medicinal materials, carry out test substance content analysis and toxicokinetics studies in order to better reveal the toxic dose-response relationship. In addition, encourage the development of research on the interaction of multiple toxic components and explore the interaction of multiple toxic components in the body Attenuation and control of toxicity. When performing toxicokinetics in sensitive animals of the above species, it is necessary to consult relevant toxic medicinal materials to analyze the toxic components that may cause long-term vomiting of animals, even intestinal mucosal necrosis, and animal death, and focus on monitoring and research. And conduct accompanying toxicokinetic studies on toxic ingredients known in the prescription and available for reference substances.
At present, more and more attention is paid to the research on the reproductive toxicity of new drugs and the influence of the offspring, and the reproductive toxicokinetics research is carried out to explore the relevant mechanism of the reproductive toxicity test results from the quantitative indicators. The reproductive toxicity toxicokinetics study includes the specific time Exposure assessment of the mother, embryo, fetus or newborn. Reproductive toxicity test accompanied by toxicokinetics can deeply study whether the test substance will pass through the placental barrier after being metabolized in the mother’s body. The blood test barrier and the blood milk barrier are paired Is there a direct correlation between drug exposure and sperm testing indicators and other reproductive toxicity?
This research unit has carried out related research on the toxicokinetics of a test substance in the reproductive toxicity test, and the established research method has been applied to the toxicokinetic research content of the new drug preclinical safety evaluation study. The test has set up a control group. ､The low, medium and high-dose groups of the test substance were used to carry out the SD rat reproductive toxicity study, and the satellite group was used to carry out the concomitant toxicokinetics study. The first dose and after the last dose, both before and after the dose Approximately 0.4 mL of orbital venous plexus blood was collected at different time points, added to an EP tube containing an appropriate amount of heparin, left standing for 1 hour, and centrifuged at 1100×g for 10 minutes, and the plasma separator was taken from the EP tube and stored in the refrigerator at -80°C, using LC-MS /MS method for detection and analysis. In addition, in the toxicokinetics study of fertility and early embryonic developmental toxicity (reproductive stage I), the concentration of semen (liquid) drug, testicular tissue drug concentration, and sperm analysis indicators need to be carried out. Detection and research to investigate the passage of drugs through the blood testis barrier; in the embryo-fetal developmental toxicity (reproductive stage II) test accompanied by toxicokinetic studies, after the animal is dissected, the maternal blood, amniotic fluid, placental blood, placental tissue and fetus are taken The organization is waiting to test the drug concentration to investigate the placental barrier system; in the perinatal toxicity (reproductive stage III) test accompanied by toxicokinetic studies, it is necessary to determine the blood drug concentration of the parent, the milk (juice) drug concentration and the blood drug of the offspring Concentration testing is carried out to investigate the penetration of the drug through the blood and milk barrier.
The reproductive toxicity test is accompanied by the establishment of toxicokinetics, which can study the metabolism of the test substance in the body, and can also conduct more in-depth research on the blood testis barrier, placental barrier, and blood breast barrier, which will be more comprehensive before the new drug is officially launched. Evaluate its potential risks carefully. Although there are the above classifications of drug reproductive toxicity test accompanied by toxicokinetics, in fact, it can be regarded as a complete drug reproductive toxicity test accompanied by three coherent parts of toxicokinetic research, but the focus is not the same. The establishment of the method must be standardized and rigorous, but also scientific and targeted. According to the purpose of the research, timely attention should be paid to the passage of the drug through the blood testis barrier, placental barrier, and blood milk barrier. This is related to whether the test substance is correct. Important evaluation measures for the toxic effects of offspring, providing information and early warning for further clinical trials of test substances.
More and more scientific researches have found that the classic blood drug concentration research methods cannot explain the drug effect of the test substance in some specific tissues such as tumors and brain tissues. The multi-quantity-effect relationship is not obvious so that it is difficult to predict the actual clinical application. The pharmacodynamic effect of pharmacokinetics. For pharmacokinetics/pharmacodynamics irrelevant issues, it is necessary to deepen the classic pharmacokinetic research from simple blood drug concentration to more scientific cellular/subcellular level cellular pharmacokinetics research.
For example, in the research of prostate cancer therapeutic drugs, the cytoplasm, cell membrane/organelles, nucleus and cytoskeleton of PC-3M cells are studied for the relevant effective and toxic components, and their cell pharmacokinetics/toxicokinetics. The establishment of PC-3M cell/subcellular level in vitro pharmacokinetic/toxicokinetic technology and models can provide new pharmacodynamic and safety evaluation tools for the discovery of androgen-independent anti-prostate cancer drugs, focusing on the drug resistance of related drugs Sexual mechanism of action.
Toxic medicinal materials of traditional Chinese medicine include not only the 28 major toxic medicinal materials referred to in the “Administrative Measures for Toxic Drugs for Medical Use” promulgated by the State Council in 1988, but also include the relevant regulations and statutory standards for medicinal materials that have been promulgated as marked as toxic (highly toxic). )､Poisonous medicinal materials, whose sources are such as various editions of the Chinese Pharmacopoeia, ministerial standards, local medicinal materials standards, etc. In addition, it should also include modern toxic Chinese medicines, such as Chinese medicines with serious adverse reactions detected according to modern clinical monitoring. Others Toxic medicinal materials can also refer to related methods for pharmacokinetic/toxicokinetic research. As the internationalization of Chinese medicine is becoming more and more urgent, going abroad also needs to comply with relevant international regulations. On the one hand, as a traditional Chinese medicine country, it should guide the formulation of international rules. On the other hand, on the basis of a comprehensive evaluation of the safety of Chinese medicine compound preparations, the pharmacodynamic/pharmacokinetic (toxicity) kinetics research method can be used to more clearly study the mechanism of action of the drug and the monitoring of the content of toxic components, which is a more scientific study of Chinese medicine. The clinical application of ､ lays a solid scientific foundation for the international arena, and makes greater contributions to human health.
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