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Technical guidelines for non-clinical pharmacokinetic studies of drugs (excerpt)

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Technical guidelines for non-clinical pharmacokinetic studies of drugs (excerpt)

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One, two, three, four, five: slightly.
Sixth, appendix
(1) Basic requirements for biological sample analysis methods: slightly.
(2) Application of radioisotope labeling technology for non-clinical pharmacokinetic study of drugs
 In the development of new drugs, it is important to understand the changes in drug candidates in humans and animals used for toxicological and pharmacological studies. Therefore, various in vivo and in vitro pharmacokinetic tests must be performed at different stages of new drug development to elucidate the properties of drug candidate absorption, distribution, metabolism, and excretion (ADME). In particular, for metabolites present only in the human body, or at a steady state, the level of in vivo exposure is higher than 10% of all total exposures of drug-related substances and is much higher than the level of metabolites in any toxicological test animal species, There are potential safety hazards and metabolic product safety studies are needed. Although LC/MS technology has been widely used in these tests, radioisotope labeling technology is still widely used. Low-energy radioisotope (such as 14C, 3H) labeled compounds are used in pharmacokinetic studies. Because of their low background value, the detection is easy and sensitive, and the half-life is long. It is not necessary to correct the test results according to the radioactive half-life. The metabolites produced by drug candidates do not need to know their structure, and the non-ionized β-ray energy generated is extremely low without special protection. It has proved to be a safe and effective special technology, and the results are simple, clear and reliable. At present, there are no other alternatives in most cases. 
1. Application range of radioisotope labeling pharmacokinetic studies
Low-energy radioisotope labeling techniques can be used in a variety of ADME trials, such as: 1) performing overall and separate pharmacokinetic studies of prodrugs and metabolites to determine overall systemic exposure and bioavailability; 2) examining material balance and 3) Determining the spectrum of metabolites in blood and excretion, combined with chromatography and mass spectrometry to facilitate the identification of metabolites; 4) Determining the mechanism of clearance in vivo; 5) Performing in vitro pharmacokinetic tests of hepatocytes and liver microsomes A comprehensive spectrum of in vitro metabolites of humans and animals (eg, mice, rats, rabbits, dogs, monkeys, etc.) can be obtained, showing species differences, and selection of genus to help toxicological research; 6) Given the same radioisotope Different structures of compounds (such as drugs or metabolites) produce the same radioactivity, and radioactive metabolites can be used for qualitative and quantitative analysis of metabolites produced in the same animal's steady state, other animal species, and humans. Identification of human metabolic enzymes, early detection of high proportion of metabolites in human body, and provide basis for experimental design of drug interaction studies; 7) Weaving distribution data. The overall autoradiography results at different time points after administration in rats can also provide data for the calculation of clinical radioactive doses.
2. Selection of radioisotope labeling methods
The low-energy radioisotopes commonly used in the study of small molecule chemical ADMEs are carbon-14 (14C) and hydrazine (3H). 14C labeling is most commonly used, its biological background is low, biology has almost no isotope effect and affects metabolism, and isotope exchange is rare. The sensitivity is higher than 3H and it is easy to quantify. Metabolically stable sites should be selected when labeling carbon-14 compounds. In vivo testing The 3H label is used unless the 14C label is very difficult or not at all, and the dose is extremely low and requires a high specific activity. The 3H label is relatively simple and has higher specific activity than the 14C labeling compound, and is especially suitable for low-dose administration compounds or early biotransformation studies; similarly, the 代谢-labeled compound should also select metabolically stable sites as marker sites, and non-localization is not recommended. The water exchange mark method.
The radiochemical purity and chemical purity of the 14C and 3H labeled compounds should generally be ≥95%, and do not contain >1% of a single impurity.
3. Pharmacokinetic test of radioisotope labeled drugs
The radiopharmaceutical pharmacokinetic test is similar to the non-labeled drug pharmacokinetic test, such as dosage, route of administration, subject animal, and the like. In addition to the usual dose level (mg/kg), the dose is also required to provide a radioactive dose (μCi/kg). The formulation and administration route of the administration preparation should also be similar to the non-labeled pharmacokinetic test, and the special case should be explained. In order to reduce the experimental error, the weighing method is often used to determine the actual dose. Sample collection often includes samples of whole blood, plasma, urine, bile, feces, cage cleaning fluid, and tissue. The collection time of the blood sample can be determined according to the pharmacokinetic parameters of the drug, and the excretion is generally sampled for 7 to 10 days (for long half-life drugs, the sampling time should be appropriately extended), or the amount of radioactivity sampled to discharge exceeds the dose. 90% or 2 consecutive days of discharged radioactivity is less than 1% of the radioactive dose. When performing a material balance test on small animals (such as mice, rats, etc.), if the total radioactivity recovery rate is <90%, the total residual amount of cadaver should be determined. If necessary, the animal should be dissected to observe the main storage site and tissue of the drug. To prevent degradation of prototypical drugs and metabolites, the containers are placed in dry ice during the collection of urine and bile. Sample processing (such as centrifugation of liquid samples to solid impurities, plasma, feces, and tissue extraction) and analysis (such as the use of HPLC and online or offline radioactivity detectors to obtain radioactive metabolite profiles) should pay close attention to radioactivity recovery, generally total The recovery rate should be ≥ 85%. The percentage of plasma exposure of each metabolite obtained from the radioactive metabolite profile study and the percentage of dosing in the excretion should be selected to select the metabolites to be identified, and HPLC online or offline radioactivity detectors are used to help identify metabolites at work. monitor.
In addition to the contents of conventional pharmacokinetic studies, the radioactive ADME test report should also provide the labeling position, radiochemical purity, chemical purity, specific activity, etc. of the radioisotope labeled drug and the radiochemical stability data in the administered preparation. The results of the experiment should provide radioactivity recovery, and the identification of metabolites should provide mass spectrometry and associated data for online or offline radioactivity detectors.
(3) Several issues that need attention: slightly.