Elements of in vivo pharmacokinetic studies in animals


pharmacokinetic studies

1. Dosage selection

In vivo pharmacokinetic studies in animals should set at least three dose groups, the high dose should preferably be close to the maximum tolerated dose, and the medium and low doses should be selected according to the upper and lower limits of the effective dose in animals. The main purpose is to investigate whether the in vivo kinetics of the drug is linear or non-linear within the tested dose range, so as to facilitate the interpretation of the findings in pharmacodynamics and toxicology studies.

2. Experimental animals

Adult and healthy animals are generally used. Commonly used animals are mice, rats, rabbits, guinea pigs, dogs, miniature pigs and monkeys.
The general principles of animal selection are as follows:
– Preferred animals: as consistent as possible with pharmacodynamic and toxicological studies;
– Try to test in a awake state, and it is best to sample multiple times from the same animal for kinetic studies;
-Innovative drugs should use two or more animals, one of which is a rodent; the other is a non-rodent (such as dogs, miniature pigs or monkeys, etc.). For other drugs, an animal can be used, and non-rodents are recommended;
– Oral administration should not use herbivorous animals such as rabbits.

3. Route of administration

The route and mode of administration used should be as consistent as possible with clinical medication. Commonly used routes of administration are oral administration (oral, gavage), subcutaneous injection, intraperitoneal injection and intravenous injection. In addition, there are administration methods such as intracerebral administration, intrarectal administration, and transdermal administration.

4. Research projects

– plasma concentration-time curve
– Binding to plasma proteins
– Effects on the activity of drug metabolizing enzymes

5. Plasma drug concentration-time curve

(1) Number of tested animals
– Each sampling point of the blood drug concentration-time curve is not less than 5 data, preferably multiple sampling from the same animal;
– If the data of multiple animals together form a blood drug concentration-time curve, the number of animals should be increased accordingly to reflect the influence of individual differences on the test results;
– It is recommended to use half male and female test animals. If a significant gender difference in kinetics is found, the number of animals should be increased in order to recognize the gender difference in the pharmacokinetics of the test substance. For single-sex medication, the gender consistent with the clinical medication can be selected.

(2) Sampling point
– Before administration, blood should be collected as a blank sample to observe the matrix effect of plasma;
– In order to obtain a complete plasma concentration-time curve, the design of sampling time points should take into account the absorption phase, equilibrium phase (near peak concentration) and elimination phase of the drug. Generally, at least 2-3 sampling points are required in the absorption phase. For drugs administered extravascularly with rapid absorption, the first point should be avoided as much as possible, which is the peak concentration (Cmax); at least 3 sampling points are required near Cmax; 4-6 sampling points are required;
– The entire sampling time should last for at least 3-5 half-lives, or until the plasma concentration is 1/10-1/20 of Cmax;
– In order to ensure the best sampling point, it is recommended to select 2-3 animals for pre-test before the formal test, and then review and revise the originally designed sampling point according to the results of the pre-test.

(3) way of administration
– Oral administration: Generally, food should be fasted for more than 12 hours before administration to exclude the influence of food on drug absorption. In addition, in the test, attention should be paid to the fasting time after administration according to the specific situation to avoid the data fluctuation and the influence of food. Generally, water and food can be given 3-4 hours after oral administration;
-Injection administration: diet and drinking water have little effect on the drug in the blood.

(4) Pharmacokinetic parameters
According to the blood drug concentration-time data of each test animal measured in the test, the main pharmacokinetic parameters of the test substance were obtained. For intravenous administration, parameters such as t1/2 (elimination half-life), Vd (apparent volume of distribution), AUC (area under the plasma concentration-time curve), and CL (clearance rate) should be provided; for extravascular administration, except for In addition to the above parameters, parameters such as Cmax and Tmax (time to peak) should be provided to reflect the law of drug absorption. In addition, providing statistical moment parameters, such as MRT (mean residence time), AUC(0-t) and AUC(0-∞), etc., are also meaningful for describing the pharmacokinetics of drugs.

(5) Pharmacokinetic study of multiple doses
When the drug will be used multiple times in clinical practice, the pharmacokinetic characteristics of multiple doses should be clarified. According to the research purpose, the steady-state concentration (Css) of the drug after multiple administrations, the fluctuation coefficient (DF) of the drug trough and peak concentration, and the existence of drug accumulation and/or drug-enzyme induction should be investigated.

6. Absorption

– For new drugs to be administered orally, whole animal testing should be performed, as far as possible concurrently with intravascular administration testing, to provide absolute bioavailability. If necessary, in vivo or ex vivo intestinal absorption tests can be performed to characterize drug absorption;
– For other extravascularly administered drugs and certain drugs with altered dosage forms, absolute bioavailability should be provided as far as possible on a case-by-case basis.

7. Distribution

-It is more convenient to use rats or mice for tissue distribution test. After selecting a dose (usually an effective dose is appropriate) for administration, at least measure the concentration of the drug in the heart, liver, spleen, lung, kidney, gastrointestinal tract, gonad, brain, body fat, skeletal muscle and other tissues to understand the concentration of the drug in the tissues. The main distribution tissue in the body;
– Special attention should be paid to tissues and organs with high drug concentration and long accumulation time, as well as the distribution in the target organs of drug efficacy or toxicity (such as drugs affecting the hematopoietic system, the distribution in the bone marrow should be investigated);
– At least 5 animals should have data for each time point;
– Referring to the changing trend of the blood drug concentration-time curve, select at least 3 time points to represent the drug distribution of absorption phase, equilibrium phase and elimination phase respectively. If the drug concentration in a certain tissue is high, observation points should be added to further study the elimination of the drug in the tissue;
– For tissue distribution tests, attention must be paid to representativeness and consistency of sampling;
– In the tissue distribution test of isotope markers, parameters such as the radiochemical purity, labeling rate (specific activity), labeling position, and administration dose of the labeled drug should be provided; detailed methods used for radioactivity determination, such as analytical instruments, background counting, should be provided , counting efficiency, correction factor, sample preparation process, etc.; provide detailed procedures for biological experiments using radioactive tracers, as well as the correction equations for radioactive decay when biological samples are measured. Whole body autoradiographic images at different phases after administration are provided whenever possible.

8. Excretion

– Drug excretion in urine and feces: Generally, mice or rats are used, and the animals are placed in metabolic cages. After an effective dose is selected for administration, all samples of urine or feces are collected in sections at certain time intervals to determine the drug. concentration. The fecal samples were weighed after drying (different animal feces were different in dryness and wetness), homogenized according to a certain proportion, the total volume was recorded, and some samples were taken for drug content determination. Calculate the excretion rate and excretion amount of the drug through this route until the collected sample cannot detect the drug;
– Trial data from at least 5 animals per time point;
– Pre-administration urine and fecal samples should be taken, and with reference to the results of the pre-test, the time point for collecting samples after administration should be designed, including the entire process of drug excretion from urine or feces, peak excretion, and the end of excretion;
-Biliary excretion: Some drugs are excreted in the bile and reabsorbed in the intestine to form enterohepatic circulation, which can prolong the action time of the drug. Generally, rats are used for bile duct drainage under ether anesthesia, and the animals are awake after administration, and bile is collected in sections at appropriate time intervals for drug determination;
– Record the rate of drug excretion from feces, urine and bile and the total excretion (percentage of the total dose) to provide material balance data.

9. Binding to plasma proteins

– Binding of drugs to proteins will significantly affect the kinetics of drug distribution and elimination, and reduce the intensity of drug action at the target site. For drugs with a protein binding rate higher than 90%, it is recommended to carry out an in vitro drug competition binding test, that is, select a drug with a high protein binding rate that may be used in combination in clinical practice, and investigate the impact on the protein binding rate of the drug under study;
– A variety of methods can be used for the study drug binding to plasma protein, such as equilibrium dialysis, ultrafiltration, partition equilibrium, gel filtration, spectroscopy, etc.;
-According to the physicochemical properties of the drug and laboratory conditions, one method may be used to conduct plasma protein binding tests at at least 3 concentrations (including effective concentrations), and each concentration should be repeated at least three times to understand whether the plasma protein binding rate of the drug is Concentration-dependent.

10. Biotransformation

For innovative drugs, it is still necessary to understand the biotransformation in vivo, including the type of transformation, the main transformation pathway and the metabolic enzymes that may be involved. For new prodrugs, in addition to the study of their metabolic pathways and the structure of the main active metabolites, a systematic pharmacokinetic study of the original drug and active metabolites should be carried out.

11. Influence on the activity of drug metabolizing enzymes

For innovative drugs, the induction or inhibition of drug metabolizing enzymes, especially cytochrome P450 isoenzymes, should be observed. Changes in liver P450 enzyme activity after multiple doses of whole animals or in liver cells (preferably human liver cells) after repeated drug action can be observed to understand whether there is a potential metabolic interaction with the drug.

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