Contact Us

Small Molecule Drug Development:
Characterization of Biopharmaceutical Properties

Bioanalytical Method Development

In vitro ADME

PK Studies and Sample Analysis

 


 

Bioanalytical Method Development

LC-MS is the primary tool for bioanalysis and metabolite profiling; however, a number of factors contribute to the effectiveness of its selectivity, specificity and sensitivity, including sample preparation and chromatographic separation. Sample preparation is critical, needed to remove interfering matrices and also to concentrate the samples to improve the LOD. Chromatographic separation is critical for selectivity, sensitivity, accuracy and throughput. Often these factors must be balanced to achieve the requisite separation.

 

Bioanalytical method development typically includes several activities. For quantitative bioanalysis, one of a few known compounds must be monitored, and generally the throughput is very high because of the large number of samples. In contrast, metabolite identification requires detection and structural elucidation in a variety of matrices, and the number of samples is far fewer. However, data analysis and interpretation for metabolite identification require significant time. Therefore, the bioanalytical strategies differ depending upon the purpose of the method.

 

In vitro ADME

Assessment of Adsorption, Distribution, Metabolism and Excretion (ADME) properties of possible candidates at an early stage is critical for selection of compounds with suitable pharmaceutical properties and for identification of liabilities inherent with any given route of administration. The ADME screens assess the metabolic stability and recovery of compounds in specific biological conditions, and the overall metabolite profile.

 

In vivo metabolic pathways may complicate the interpretation of preliminary toxicology and pharmacokinetic studies of a potential investigational drug. Therefore, Wolfe Laboratories employs many in vitro models to evaluate the extent, location and resulting metabolites of investigational new drugs following incubation with the appropriate enzymatic systems. The variety of in vitro assays available at Wolfe Laboratories allows a stage-appropriate assessment of metabolic liabilities throughout the entire development process, ranging from simple microsomal incubations through enzymatic induction in intact hepatocytes. As many ADME properties are species-dependent, performing thorough screening across the relevant species early on is critical to program success. Wolfe Laboratories performs the in vitro ADME tests summarized in the following table.

 

Summary of in vitro ADME tests performed by Wolfe Laboratories

Caco-2 permeability (A-B ± BSA, B-A)

Plasma half-life

Caco-2 P-gp substrate assessment

Plasma stability

CYP450 stability1

Protein binding

CYP450 stability/half-life1

RBC Partition

CYP450 inhibition1

TDI human2,3

CYP450 IC501

Microsomal or S9 stability

Human hepatocyte stability

Microsomal or S9 half-life

Human hepatocyte half-life

Metabolite identification

CYP450 Induction

 

1Recombinant human (1A2, 2B6, 2C9, 2C19, 2D6, 3A4)

2Recombinant human (1A2, 2C9, 2C19, 2D6, 3A4)

3Microsomes (1A, 2C9, 2C19, 2D6, 3A)

 

Plasma Stability

Compounds that are not stable in plasma can lack efficacy in vivo. Conversely, for pro-drugs, plasma instability can be a desired trait in order to release the active compound. In this case, Wolfe Laboratories can monitor the appearance of active compound in the assay.

 

Red Blood Cell Partitioning

Knowledge of the RBC to plasma ratio can result in a more accurate understanding of the pharmacokinetic (PK) profile, as PK parameters obtained from the analysis of plasma may overestimate the actual blood clearance.

 

Plasma Protein Binding

The binding of test compounds to plasma proteins is an important parameter for metabolism and pharmacokinetic studies and impacts drug distribution. If the drug is highly bound to plasma proteins, the amount of drug available to tissues is reduced, and subsequently the efficacy may be significantly diminished. Determination of the level of protein binding may be helpful in correlating with in vivo efficacy and for allometric scaling between species. Additionally, the determination of the specific proteins involved in the binding can provide insight into interindividual variability and drug-drug interaction potential.

 

Cytochrome P450 (CYP450) Profiling

Wolfe Laboratories performs three types of individual CYP450 isoform phenotyping of test compounds to provide important information regarding metabolism, including the effect of test compounds on CYP450 activity. These phenotyping tests include the assessment of test compound metabolism by individual recombinant CYP450 isoforms, the inhibition of test compound metabolism in liver tissue and the CYP450 induction of metabolic activity of hepatocytes.

 

Hepatocyte and Microsomal Stability

As part of in vitro ADME screening, Wolfe Laboratories uses hepatocytes, liver microsomes and S9 incubations from humans and animals to determine the potential metabolic stability of test compounds. Wolfe Laboratories performs intrinsic clearance measurement, an assessment of compound metabolic clearance in vitro, using hepatocytes across a range of test compound concentrates to identify the enzyme kinetic constant values of Km and Vmax that describe the reaction kinetics.

 

Evaluation of In vitro Intestinal Permeability and Efflux using Caco-2

Wolfe Laboratories analyzes in vitro permeability and efflux using the immortalized colorectal adenocarcinoma cell line, Caco-2. The confluent monolayer of Caco-2 cells serves as a physical and biochemical barrier between the two wells permitting selective transfer and transport of molecules, analogous to the small intestinal epithelium. A P‑gp inhibitor in the system characterizes potential involvement of P‑glycoprotein (P-gp) in any observed efflux.

 

PK Studies and Sample Analysis

Rodent and Non-Rodent Studies

Following formulation selection, a third party vendor or a researcher at the clients’ vivarium doses rodents and draws plasma samples. After completing the bioanalysis of the samples, Wolfe Laboratories estimates PK parameters from the plasma concentration data using non-compartmental analysis.