Key Considerations in Toxicokinetic

Toxicokinetics (TK), as an interdisciplinary field combining pharmacokinetics and toxicology, has now become an integral part of the nonclinical research for drugs. It plays a vital role in the nonclinical (preclinical) safety assessment in drug development. By quantitatively studying the absorption, distribution, metabolism, and excretion of a drug at toxic doses, TK helps to explore the occurrence and progression of drug toxicity, serving as an essential basis in the drug development process. Therefore, in investigating TK, selecting the correct experimental parameters and methods is crucial to obtaining reliable data that establish effective links between animal experiments and human safety. This article briefly explores TK studies from aspects such as dose selection, matrix selection, choice of animal models, and sample collection.

Selection of Experimental Models

In TK research, the selection of animal models needs to comprehensively consider multiple factors to ensure the reliability of the study results, generally including the following key points:

• Metabolic similarity: Ideally, the experimental animals should have a metabolism of the test substance similar to that of humans. For specific drugs, species that express receptors similar to human ones may be preferred.
• Applicability of disease models: Choose disease models relevant to human diseases based on the therapeutic target of the drug.
• Sensitivity: The selected animals should be sensitive to the test substance, enabling the detection of the drug's effects and potential toxicity.
• Animal species: For innovative drugs, especially small-molecule drugs, trials should typically involve at least two types of mammals, usually one rodent and one non-rodent species.
• Gender and age differences: Toxicity studies usually employ animals of both genders to evaluate gender differences in toxicity. Additionally, factors such as the age and pregnancy sensitivity of the animals should be considered.

Dose Selection

Three dose groups, low, medium and high, as well as a solvent control group and, if necessary, a blank and/or positive control group, are usually set up. Dose settings are based on the following principles.

• Low dose choice: It should be a no-toxic-effect level, where the exposure of the test animals to the drug or its metabolites is equivalent to or slightly exceeds the maximum expected exposure in humans.
• Medium dose choice: It usually represents an appropriate multiple of the low dose or a fraction of the high dose, used to examine the dose-response relationship in toxicity.
• High dose choice: The high dose should fully expose the animals and produce significant toxic reactions, or reach the maximum feasible dose (MFD), or achieve systemic exposure equal to 50 times the clinical systemic exposure (based on AUC). However, it should not lead to excessive mortality to ensure the validity of the study results.
• Consideration of non-linear kinetics: Special attention is needed to the association of non-linear kinetics with toxic responses in toxicity studies when increasing doses lead to such kinetics. Non-linear kinetics do not mean that doses cannot be escalated or that new toxic responses will not appear.
• Species differences and metabolic characteristics: When selecting doses, consider the physicochemical properties, bioavailability, interspecies metabolic differences, and the pharmacological activity and toxicological effects of metabolites.

Matrix Selection

Understanding the exposure of a chemical substance in the body is key to toxicokinetics research, and the selected matrix should accurately reflect the systemic exposure level for an accurate evaluation of toxic effects. For most drugs, blood is a commonly used matrix. Typically, plasma (or serum) is preferred over blood due to its homogeneous nature. However, blood is more suitable for monitoring drugs that bind to or sequester in erythrocytes and/or exhibit non-linear (e.g., temperature or concentration-dependent) blood-to-plasma distribution. Additionally, specific organs or tissues, such as liver tissue, bile, and urine, may serve as important study matrices for certain drugs with specific targets.

Sample Collection

• Sampling time: Sampling time points should be sufficiently frequent to ensure adequate exposure estimation but not so frequent as to interfere with the normal conduct of the toxicity study or cause excessive physiological stress to the test animals. The number of sampling points should be rationalized based on kinetic data from early studies, pilot, or dose-range studies.
• Sampling volume: The volume should be adequate to meet the detection and quantification limits required by the analysis method to ensure accurate measurement of the drug or toxicant concentration.
• Sampling scheme: TK samples can be collected from the main study animals or dedicated satellite TK animals. In toxicity studies, fewer time points and minimal repeat samples are typically used, collected via serial or sparse sampling schemes.

Analytical Methods

• Specificity and accuracy: Analytical methods used in toxicokinetics research must possess high specificity for the analyte to ensure the determination of only the target analyte without matrix interference. The methods must also exhibit sufficient accuracy and precision to ensure the reliability of the measurement results.
• Detection limit and quantification range: The detection limit of the analytical method should meet the expected concentration range in toxicokinetics research to ensure accurate determination of analyte concentrations in samples.

Reference

1. Li W, Picard F. Toxicokinetics in preclinical drug development of small-molecule new chemical entities. Biomed Chromatogr. 2023. 37(7):e5553.

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