Pharmacokinetics in suckling rats

Pharmacokinetics in suckling rats （ Complete drug verification within 48 hours ）

PK studies of suckling mice (usually refers to young mice ≤21 days after birth) are crucial to pediatric drug development. Their physiological characteristics (immature organ development and low metabolic enzyme activity) significantly affect the drug ADME process. The following are the key steps and precautions for the experiment.

I. Key points of experimental design

1. Animal selection and grouping

Age of suckling mice ：Choose the appropriate stage according to the research objectives:

Neonatal period : P0-P7 (simulating human premature infants).

Lactation :P7-P14 (period of rapid organ development).

Weaning period :P14-P21 (metabolic enzymes gradually mature).

Control group setting : Comparison of suckling mice and adult mice (≥8 weeks) of the same age to evaluate development-related PK differences.

II. Dosage regimen

Route of administration :

Dose calculation : Based on body surface area conversion (usually the dose for suckling mice = the dose for adult mice × 2-3 times).

3. Design of blood collection time point

Dense Sampling ​(Applicable to precious samples):

Time : 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h after administration (multiple groups of animals are required, with 1-2 time points in each group).

Blood collection volume ：≤10% total blood volume (total blood volume of suckling mice ≈ 7% body weight, e.g. blood sampled from a 10 g suckling mouse ≤70 μL).

Micro-sampling technology :

Tail vein microblood sampling (5-20 μL/time, using heparinized capillaries).

Retro-orbital venous plexus blood sampling (Only collect blood at the endpoint to avoid repeated operations).

2. Sample processing and analysis methods

1. Plasma/serum preparation

Anticoagulants : Heparin sodium (5-10 IU/mL blood) or EDTA-K2 (1-2 mg/mL blood).

Centrifugation conditions : 4°C, 3000×g, 10 min (rat erythrocytes are highly fragile, avoid excessive centrifugation).

2. Drug concentration detection

LC-MS/MS : The preferred method, with high sensitivity (can detect pg/mL level), suitable for trace samples.

ELISA : Suitable for large molecules (such as antibodies and protein drugs).

Precautions :

Matrix Effects : The plasma protein content of suckling mice is low, and the extraction method needs to be optimized (such as protein precipitation).

Hemolysis interference : Avoid using hemolyzed samples (free hemoglobin affects the LC-MS signal).

3. Metabolite Analysis

Liver microsome incubation ：In vitro evaluation of CYP450 enzyme activity (suckling rat liver microsomes must be prepared before use).

Incubation system : Liver microsomes (0.5 mg/mL) + NADPH (1 mM) + drug (1-10 μM), incubate at 37°C for 30 min.

Metabolic phenotyping : qPCR/WB detected the expression of development-related metabolic enzymes such as CYP3A4 and CYP2D6.

III. Calculation of key PK parameters

IV. Precautions and Ethical Standards

1. Experimental operation risk control

Body temperature maintenance : Sucking mice cannot keep warm on their own, so a heating pad (37°C) is required throughout the experiment to prevent hypothermia.

Separation of mother mice : The blood collection or drug administration time should be ≤30 minutes to avoid the mother mouse refusing milk.

Nutritional support : Glucose solution (5% concentration, 50 μL subcutaneously injected) was supplemented after the experiment.

2. Data analysis and correction

Weight normalization : PK parameters need to be corrected according to body weight or body surface area (the body weight of suckling mice increases by 10-15% per day).

Developmental stage association : Differences in enzyme activity at different ages (such as P7 vs P21) need to be analyzed separately.

3. Ethical Standards

Blood collection restrictions : Blood should be collected from the same suckling mouse ≤ 3 times, and the total blood volume should be ≤ 15% of the blood volume.

End point criteria : If severe stress occurs (such as rapid breathing, stagnation of activity), immediate euthanasia is required.

V. Sample data (taking oral administration as an example)​