Drug Metabolism and Pharmacokinetics (DMPK): Understanding Dosage, Toxicity, and Therapeutic Index
Drug metabolism and pharmacokinetics, or commonly referred to as DMPK, is a scientific discipline within drug discovery, dealing with safety and efficacy evaluation of drug candidates before entering clinical trials. DMPK studies come along with absorption, distribution, metabolism, excretion, and toxicity analysis (ADMET) of drug candidates. The DMPK discipline is the basis for optimizing compounds so that bioavailability, drug-drug interactions (DDI), and related risks to a drug compound can be evaluated. Assessment of DMPK profiles minimizes the rate of attrition of drug candidates and increases the efficiency of drug discovery overall.
The objective of DMPK studies is to evaluate multiple properties of drugs, such as clearance, distribution volume, half-life, bioavailability, drug-drug interaction, and metabolic profile. Evaluation of CYP, other drug enzymes, transporters, and inhibition and induction minimize the rate of potential DDI liabilities and reduce attrition for lack of efficacy and poor safety profiles. Another benefit of DMPK studies during drug discovery and development is that it allows chemists and analysts to achieve reasonable structural modifications such that pharmacokinetic properties and efficacy of the drug can be improved.
DMPK Assays: Plasma Protein Binding, Microsomal Stability, CYP450 Inhibition, etc
DMPK studies offer an understanding of the dose regimen, toxicity level, therapeutic index, PK/PD relationships, and other PK parameters. DMPK studies during drug discovery and development utilize two to four animal doses for testing the generic formulation of a compound.
Typically, the oral dose level is 10mg/kg, and the intravenous dose level is 1mg/kg. Either single Compound Dosing (Discrete Dosing), or high-throughput Cassette Dosing (N–in–one) method can be implemented to obtain the pharmacokinetic data. For both dosing regimens, the blood samples are collected at specified time points and analyzed by techniques such as LC-MS/MS or ELISA Assay.
Here’s a list of several industry-standard DMPK assays performed during IND-enabling studies:
In Vitro Toxicity Assay
The in vitro toxicity assays are developed to asses toxic mechanisms corresponding to a drug compound. These mechanisms may include metabolite induction, reactivity, cytotoxicity, and mutagenicity. These DMPK studies allow evaluating the toxic effects of drug candidates ahead of the animal studies. In vitro toxicity assays include:
- Mutagenicity/Genotoxicity
- hERG block assay
- Ames assay
- Cytotoxicity
- Glutathione trapping assay
Blood Plasma Partitioning Assay
DMPK pharmacokinetics obtained from plasma can be misleading, owing to the preferential drug binding to Red Blood Cells. The blood to plasma ratio presents relevant and informative data about drug distribution in blood and provides investigators a better understanding of the drug pharmacokinetic behavior in animal or clinical ADME studies.
Plasma Protein Binding Assay
This type of assay provides information about the unbound fraction of drug in plasma. Usually, it is performed using an equilibrium dialysis method. Upon reaching the equilibrium between the compound spiked plasma sample and buffer system, a value of the fraction of a compound unbound to proteins (fu) can be calculated.
In Vitro Microsomal Stability Assay
During in vitro microsomal stability assay, the test sample dissolved in DMSO is incubated with microsomal fraction in the presence of a co-factor at 37°C. The aliquots are then removed at a particular time and analyzed by techniques, such as LC-MS/MS MS and ELISA Assay. Upon drug depletion over time, clearance rate can be estimated.
CYP Inhibition Assay
CYP enzymes are primary targets in the assessment of drug-drug interactions leading to potentially toxic effects. For general DMPK assay, the CYP isozyme isoform or combination, industry accepted substrate compound, and the test compound at several concentrations are incubated together. The difference in the formation of metabolite for each given substrate/CYP enzyme can be calculated along with IC50 value.
CYP Induction Assay
The induction of CYP increases the drug metabolizing enzyme’s activity through activation of nuclear receptors leading to induction of their gene expression. This results in a reduction of the therapeutic concentration of the drug compound, which may become the cause of reduced drug efficacy or increased risk of toxicity.
The mechanism of CYP induction is assessed with nuclear receptors:
- Aryl Hydrocarbon Receptor (AhR)
- Constitutive Androstane Receptor (CAR)
- Pregnane X Receptor (PXR)
