Metabolism Assay Services

Metabolism is an important determinant of oral bioavailability, clearance, and half-life of drug products in vivo. The metabolic pathway of your drug also has important implications for drug-drug interactions.  Understanding these properties can help develop safer, more clinically effective and relevant products.

CYPs are a major class of drug metabolic enzyme

 

Metabolism is the enzymatic modification of drugs and other xenobiotics, usually to increase clearance. Metabolic breakdown of drugs occurs predominantly in the liver and may also occur in the intestine or other organs. Hepatic drug metabolism is controlled by a well-established set of enzymes including cytochrome p450s (CYPs) and uridine 5′-diphospho-glucuronosyltransferases (UGTs).

 

How a drug is metabolised by these enzymes has important consequences:

  • Enzymes transform the drug into one or more active or inactive metabolites
  • Drugs can induce expression of enzymes to perturb metabolic activity
  • Metabolic by-products may be harmful or alter the kinetics of the active drug
  • Metabolism of one drug may alter metabolic kinetics of other xenobiotics, known as drug-drug interactions (DDIs)

The USFDA guidance prescribes studies on enzyme inhibition, induction and phenotyping to determine enzyme responsible for metabolism (In Vitro Metabolism and Transporter Mediated Drug-Drug Interaction Studies, 2020).

The Absorption Systems Difference

Absorption Systems has extensive experience evaluating the potential interactions of compounds with drug transporter proteins. We have years of experience with in vitro transporter substrate and inhibitor potential assay development.  Our expertise spas co-medications, product profiles, development plans, clearance routes, chemical structures, and physicochemical properties.

 

FREQUENTLY ASKED QUESTIONS

Why is understanding drug-drug interactions important?

Evaluation of an new molecular entity’s (NME) drug-drug interaction potential is an integral part of drug development and regulatory review prior to market approval. Many metabolic routes of elimination, including most of those occurring through the cytochrome P450 family of enzymes, can be inhibited or induced by concomitant drug treatment. An NME must be characterized both in terms of the enzymatic pathway(s) by which it is metabolized and in terms of the effects it may have on the metabolism of other drugs. If positive, the latter is an example of a pharmacokinetic drug-drug interaction.

Observed changes arising from metabolic drug-drug interactions can be substantial — an order of magnitude or more decrease or increase in the blood or tissue concentrations of a drug or metabolite — and can include increased formation of toxic and/or active metabolites or elevated exposure to a toxic parent compound such that toxic levels are achieved. For this reason, in vitro metabolic assessment plays a key role in early drug discovery with ExpressPlus™ screening assays as well as in advanced stages of development.

How do you test for enzyme inhibition?

Absorption Systems offers comprehensive p450 enzyme inhibition panel testing (reversible and IC50 determination), as prescribed by the USFDA in their latest guidance.

*Note: Our tests are currently done only in liver microsomes

Assessment Enzymes/isoforms Test systems Concentration selection
Inhibition CYP enzymes CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP2E1, CYP4F2, CYP3A Human liver microsomes, recombinant enzymes, or hepatocytes Range of concentrations based on maximal unbound plasma (Imax,u) and luminal (Igut)* concentrations

 

How do you test for enzyme induction?

Certain drugs can induce the expression of CYP enzymes, increasing the possibility of DDIs. FDA recommended endpoint is the change in mRNA expression level of the target gene.

Our induction assays are based on primary hepatocyte cultures, that best reflect the biological response to the liver in vitro. 

Assessment CYP isoforms Test systems Concentration selection
Induction CYP1A2 (AhR),  CYP2B6 (CAR), CYP3A (PXR) Hepatocytes (fresh or cryopreserved) Concentrations based on maximal unbound plasma (Imax,u) and luminal (Igut)* concentrations

 

What is the importance of CYP phenotyping?

Drugs that exhibit a propensity for a particular enzymatic metabolism pathway can increase the likelihood of toxicity through DDIs. The diagram below summarises the standard experimental pathway for defining CYP phenotype. 

Assessment Enzymes/isoforms Test systems Concentration selection
Phenotyping


Phase I enzymes Major CYP isoforms CYP1A2,  CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A

Human liver microsomes

Liver S9 or other tissue fractions

Fresh or cryoprotected hepatocytes

Microsomes from cells expressing recombinant enzymes

Sufficiently low therapeutically relevant concentrations
Other enzyme CYP2A6, CYP2J2, CES, CYP4F2, CYP2E1, MAO, FMO, AO/XO
Phase II enzymes UGTs, SULT and NAT

FURTHER READING

To find out more information on our metabolic services, visit our dedicated pathway pages:

Are you interested in finding out the metabolic profile of your compound?