UGT Inhibition, Induction and Phenotyping Assays

Evaluating Drug Interactions with UGT Enzymes

Understanding how a drug interacts with drug metabolizing enzymes is essential for understanding its behavior in the body and assessing the associated drug-drug interaction risk. UDP glucuronosyltransferase (UGT) enzymes are the second most common enzyme family involved in the metabolism of marketed drugs, following CYP450s.^1,2 Therefore, assessing a new drug candidate’s interaction with UGT enzymes is often necessary in the drug development process.

UGTs catalyze the glucuronidation of their substrates, either exogenous or endogenous compounds by attaching them to UDP-glucuronic acid (UDP-GlcA). This conjugation makes the compounds more polar and thus more water-soluble, facilitating their excretion in urine and bile. While glucuronidation, a Phase II metabolic process, generally detoxifies compounds (for example, xenobiotics or their Phase I metabolites), some glucuronide metabolites retain biological activity.

The UGT family consists of membrane-bound microsomal enzymes that differ in terms of catalytic function, regulation, and tissue expression. These enzymes are predominantly expressed in the liver but are also found in the kidney, GI tract, lungs, prostate, mammary glands, skin, brain, spleen, and nasal mucosa.³ Currently, 17 human UGTs have been identified and categorized into two families (UGT1 and UGT2) based on the sequence identity of the encoded proteins. Of these, 7-9 main UGTs have been identified to play a role in drug metabolism.⁴

In case the formation of glucuronide metabolites is observed for an orally administered drug, it is essential to identify the responsible UGT enzymes. Assessment of the impact of first-pass metabolism, including this glucuronidation, on the drug’s bioavailability is also essential. Substrates to metabolic enzymes can also act as their inhibitors, and drugs that don’t undergo glucuronidation themselves may still impact UGT function (via UGT inhibition or induction), leading to potential side effects or drug interactions. Regulatory agencies, including the ICH M12 Harmonized Guideline for Drug Interaction studies, now require in vitro UGT inhibition testing not only for compounds that undergo glucuronidation themselves but also in case a new molecule is expected to be co-administered with known UGT substrate drugs.⁵

Regulatory Requirements for UGT-mediated DDI

Drug-drug interactions (DDI) via UGT inhibition or induction can pose significant risks to drug safety. This is exemplified by the withdrawal of several drugs from the market, or their use has been restricted due to the significant impact of the DDI on drug exposure relative to the associated therapeutic window. The ICH M12 guidelines for drug-drug interaction assessment, while not mandatory, recommend assessing interactions with UGT enzymes as part of drug development.⁵ A test compound may be an inhibitor, inducer, or substrate of these enzymes. These interactions, potentially leading to DDI risk, are usually assessed from the hit-to-lead stage up to the development stages before clinical trials.

The ICH M12 guidelines emphasize the need to identify metabolic enzymes involved in ≥ 25% drug elimination. Consequently, if glucuronidation is a major elimination pathway, the individual UGT enzymes involved should be identified in UGT substrate assays. If a candidate drug’s major elimination route is via glucuronidation, testing its potential for UGT inhibition is also recommended. For UGT non-substrate drugs, UGT inhibition assessment is still relevant if they will be administered alongside a drug that is cleared via glucuronidation to rule out UGT-mediated DDI and related complications later in development. While UGT induction in DDIs remains less understood, the ICH M12 guidelines mention that these enzymes are regulated via the Pregnane X Receptor (PXR), a mechanism shared with several other metabolic enzymes and transporters that may be involved in DDI.

Specifications of our UGT phenotyping, UGT inhibition, and UGT induction assays are described below.

UGT Assay Availability

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UGT Phenotyping Assay

UGT phenotyping is essential for identifying which UGT enzymes are involved if a drug candidate undergoes glucuronidation, as identified in metabolite profiling experiments. UGTs are the second most common enzyme family involved in the metabolic clearance of known small-molecule drugs after CYP450 enzymes. Of the several known UGT families, drug glucuronidation is almost exclusively catalyzed by the UGT1 and UGT2 families.⁶ UGT1A1, UGT1A3, UGT1A4, UGT1A9, or UGT2B7 are among the most common enzymes responsible for metabolizing drugs, with the UGT2B7 being most often involved.7 However, additional UGTs with more limited substrate specificities, e.g., UGT2B15, may still be involved in the clearance of certain drugs.⁷

The metabolic clearance of a drug depends on the activity and abundance of the relevant drug-metabolizing enzymes. Changes in the clearance rate may lead to systemic exposure outside of the therapeutic window, causing changes in efficacy or introducing toxicity. Genetic variance, environmental factors, and/or DDIs can affect the activity and expression of these enzymes. Drugs metabolized by the same enzyme can affect each other’s clearance rate, resulting in altered pharmacokinetics and potential changes in efficacy and effect. Regulatory drug-drug interaction assessment guidelines⁵ call for the identification of enzymes that contribute to ≥ 25% of drug elimination in case of considerable metabolic clearance (based on metabolic stability and mass balance studies).

UGT phenotyping is typically conducted using two complementary experimental approaches:

• Chemical inhibition: Assesses the effects of specific UGT enzyme inhibitors targeting individual UGTs on the drug candidates’ metabolism in human liver microsomes. If the recovery of the drug candidate is increased upon inhibition, the enzyme in question is considered involved in the metabolism.
• Recombinant enzyme: Uses recombinant cDNA expressed UGT enzymes to directly observe the enzyme’s impact on drug metabolism by following depletion of the candidate.

The UGT enzymes responsible for catalyzing the drug candidate’s metabolism can be most reliably identified using output from both experiments.