UGT Inhibition

Creative Bioarray provides customers with the UDP-glucuronosyltransferases (UGTs) enzymes inhibition test to help study the metabolic pathways of the tested drugs except for CYPs.

UGT Inhibition Assay Introduction

  • Why do we need the UGT inhibition assay?
    • Uridine 5'-diphosphate glucuronosyltransferases (UGTs) are a family of enzymes that perform Phase II conjugative metabolism (glucuronidation), which usually follows the Phase I oxidative metabolism performed by cytochrome P450 (CYP) enzymes or other oxidative metabolic steps (Soars et al. 2004).
    • UGT enzymes catalyze the attachment of a glucuronic acid moiety to varied drugs and other xenobiotics, also on endogenous compounds like bilirubin. This conjugation promotes their excretion.
    • Glucuronidation caused by UGT is a crucial pathway for drug metabolism in humans and other mammals.
    • Regulatory agencies began to require UGT inhibition studies as part of drug-drug interaction (DDI) to determine whether clinical DDI studies are needed.
  • Mechanism of UGT involving metabolism
    • Glucuronidation involves adding (or combining) glucuronic acid directly into the drug itself or the oxidative metabolite of the drug. The addition of glucuronide causes the conjugate to be more polar and ionize at physiological pH. These features facilitate excretion through the kidneys. The liver can also excrete glucuronic acid through bile.
    • UGT enzyme can catalyze the connection of glucuronic acid moiety to the hydroxyl, carboxyl, amino, or sulfhydryl group of the target compound.
    • If the activity of UGT is impaired, the result may increase the toxicity of the drug. Otherwise, UGT will dispose of these drugs. The direct action of drugs that inhibit UGT may result in impaired UGT activity.

The catalytic reaction of UGT(Brody, 2018).Figure 1. The catalytic reaction of UGT(Brody, 2018).

Brief Protocol

  • The known UGT substrates were incubated with recombinant UGT enzymes, alamethicin, uridine-5'-diphospho-α-d-glucuronic acid (UDPGA), and a series of test compound concentrations at 37°C.
  • The protein concentration and time point depend on the specific UGT and have been previously determined by protein linearity and time linearity studies. Characterized enzyme kinetics of every reaction has previously determined the substrate concentration. The range of test compound concentration depends on the plasma Cmax, the solubility of the test compound, and the degree of plasma protein binding.
  • At the end of the incubation, the formation of UGT isoform-specific metabolites was monitored by LC-MS/MS at each test compound concentration. The reduction in metabolite formation compared to the vehicle control is used to calculate the IC50 value (the concentration of test compound that produces 50% inhibition).

Quotation and Ordering

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References

  1. Brody, Tom. "Chapter 7 - Drug–Drug Interactions: Part One (Small Molecule Drugs)." FDA's Drug Review Process and the Package Label, edited by Tom Brody, Academic Press, 2018, pp. 255–335. ScienceDirect.
  2. Soars, Matthew G., et al. "AN ASSESSMENT OF UDP-GLUCURONOSYLTRANSFERASE INDUCTION USING PRIMARY HUMAN HEPATOCYTES." Drug Metabolism and Disposition, vol. 32, no. 1, Jan. 2004, pp. 140–48. DOI.org (Crossref).

For research use only. Not for any other purpose.