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.
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).
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:
