Drug Metabolism (DMPK) Assays

MicroConstants performs industry-standard assays, custom drug metabolism research, and IND-enabling studies to assess drug-drug interaction potential, metabolic stability, metabolite profiling, and protein binding. Whether you are in discovery, lead optimization, or collecting data for regulatory submissions, we will work with you to define the level of research appropriate for your study. Project results from drug metabolism studies can be presented as a formal report suitable for IND submissions, or as informal reports such as raw data tables in Excel format.

MicroConstants specializes in performing the following drug metabolism
studies to support your preclinical discovery research:

Metabolic Stability Assays
Metabolite Profiling
Metabolite Identification
Reaction Phenotyping
CYP450 Inhibition Studies
UGT Inhibition Studies
CYP450 Induction Studies
Protein Binding Assays

Metabolic Stability Assays

Metabolic stability assays are helpful when trying to determine the potential half-life of a compound when dosed to animals or humans. We determine the stability of a test article in a variety of enzyme sources, including: hepatocytes, liver microsomal preparations, hepatic cytosol, hepatic mitochrondrial fraction, hepatic S9 fraction, and membrane preparations from recombinant bacteria or eukaryotic cells. The time points are customized for each project, and the deliverables include raw data, percent remaining, and half-life values.

Metabolite Profiling

We are able to generate and compare metabolite profiles to assist with species selection for toxicology studies. These studies are performed using accurate mass spectrometry (QTof Premier) to analyze samples from metabolic stability assays or in-life dosing studies. We search the data sets for potential metabolites (using Metabolynx XS), and compare the time profiles of parent compound loss and metabolite formation using enzyme sources from various animal species. Metabolite profiling studies can also be specifically designed to look for unique or disproportionate human metabolites.

Case Study: Rapid Turnaround of High-Quality Metabolic Profiling Studies with a UPLC/QTof and Metabolynx XS-Based Workflow (PDF)

Presentation: Adventures in Metabolite Profiling with an Accurate Mass QTof (PDF)

Metabolite Identification / Metabolite ID

We identify metabolites in profiling studies, or characterize impurities in your API, using accurate mass spectrometry (QTof Premier) with collision cell optimization. Samples are generated either in vivo or in vitro. A metabolite standard may also be supplied for comparison of the retention time and product ion spectra with those of the postulated metabolite.

Reaction Phenotyping / Enzyme Mapping

Reaction phenotyping, or enzyme mapping, determines the CYP enzymes that are involved in the metabolism of a compound. Three methods approved by the FDA may be used in this study: correlation analysis, specific chemical inhibition, and/or recombinant enzymes. The correlation analysis involves a bank of liver microsomes from at least 10 donors. We conduct a pilot study to determine the appropriate reaction conditions for each project. Recombinant UGT enzymes can be used to determine which isoforms are capable of metabolizing the test article.

Cytochrome P450 (CYP450) Inhibition Studies

CYP450 inhibition studies are used to investigate potential drug-drug interactions. These studies are conducted with human liver microsomes, FDA-accepted probe substrates, and control inhibitors. Both IC₅₀ and K_i values can be determined, and the pre-incubation of the test article with microsomes and NADPH are used to assess time-dependent inhibition. Alternatively, we can use recombinant CYP450 enzymes with fluorogenic probe substrates in screening assays.

UGT Inhibition Studies

Recombinant UGT enzymes are used to assess the IC₅₀ values of a test article with respect to the most common isoforms: 1A1, 1A4, 1A6, 1A9, 2B7, and 2B15.

Cytochrome P450 (CYP450) Induction Studies

Plateable cryopreserved hepatocytes from one or more donors are used to assess the potential of a compound to induce drug metabolizing enzymes and transporters. The receptors tested include AhR, PXR, and CAR.

Protein Binding Assays

Equilibrium dialysis, ultrafiltration, or ultracentrifugation are used to determine the extent of drug binding to plasma, or to proteins such as human serum albumin, or α1-acid glycoprotein. Multiple concentrations can be tested to obtain K_d estimates. We can also use in vivo samples to assess binding values. Click here to read more about protein binding assays.