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UGT1A1/UGT1A3 Inhibition Assay

Uridine 5’-diphospho‐glucuronosyltransferases (UGTs) are a superfamily of enzymes which catalyse the glucuronidation reaction, a major phase II metabolism pathway. Approximately 15% of approved drugs have glucuronide metabolites1, highlighting the importance of UGT enzymes. UGT inhibition is less often observed than CYP inhibition, but is a clinically significant form of drug-drug interaction (DDI), and can lead to toxicity.

DDIs can alter the concentration and duration profile of a co-administered drug, leading to clinical ineffectiveness or adverse effects. The FDA and EMA recommend that all new drugs should be investigated to understand their potential for DDIs. UGT inhibition should be explored when glucuronidation is a predominant metabolic elimination pathway, when the glucuronidation is catalysed by a single enzyme, and when the therapeutic concentrations of the inhibitor are close to the Ki of the target UGT2. It is recommended to study the inhibitory effect of both parent molecule and major metabolites.

The inhibition of UGT1A1 and UGT1A3 can have important implications for drug-induced liver injury. UGT1A1 is the only enzyme responsible for the glucuronidation of bilirubin, which makes the compound less toxic and more readily excreted. The inhibition of this enzyme can therefore lead to inadequate clearance of bilirubin from the body, resulting in a condition known as hyperbilirubinemia4. Glucuronidation is also an important metabolic pathway for hepatic bile acids. UGT1A3 is the enzyme responsible for the glucuronidation of chenodeoxycholic acid (CDCA)3, a major bile acid. Due to its detergent properties, CDCA is toxic. CDCA-glucuronide is less active, and more polar than the parent molecule. Inhibition of UGT1A3 can lead to accumulation of CDCA, and subsequent hepatotoxicity.

Sygnature’s UGT inhibition assay measures the capacity of a compound to inhibit the glucuronidation of a probe substrate. The probe substrate and test compound are incubated at several concentrations at 37°C with recombinant UGT supersomes in the presence of UDPGA. Alamethicin is used to reduce latency, and MgCl2 is added to the assay buffer to increase UGT activity5.

Protocol

  UGT1A1 UGT1A3
Compound requirements 10 mM DMSO, 20µL 10 mM DMSO, 20µL
Substrate 10 μM β-estradiol 5 μM chenodeoxycholic acid (CDCA)
Test Article Concentrations 10-point IC50 in duplicate 10-point IC50 in duplicate
Incubation Time 60 min at 37°C 60 min at 37°C
Test System Recombinant UGT1A1 supersomes Recombinant UGT1A3 supersomes
Analysis Method UPLC-MS/MS UPLC-MS/MS
Controls Silibinin Ritonavir
Data Delivery IC50 IC50

Results

Figure 1. Inhibition of UGT1A1 and UGT1A3 by positive control inhibitors Silibinin and Ritonavir

 

 

Table 1. Example IC50 values generated in Sygnature’s UGT inhibition assay

UGT isoform Probe Substrate Control Inhibitor IC50
UGT1A1 β-estradiol Silibinin 1.66 ± 0.41
UGT1A3 CDCA Ritonavir 0.35 ± 0.24

 

About Us

The DMPK & Physical Sciences department at Sygnature Discovery is dedicated to understanding and optimising the absorption, distribution, metabolism and excretion of drug candidates by working in close partnership with clients and other departments within Sygnature to provide successful optimisation strategies.

We have extensive know-how and expertise to provide well validated, state-of-the-art assays and a comprehensive applied consultancy service for interpretation of the in vitro ADME and in vivo PK data.

Our corporate vision is to accelerate the discovery of new medicines, from the laboratory into development to treat patients.

Our DMPK mission is to deliver tailored DMPK expertise through innovation, quality and commitment.

 

REFERENCES

  1. Williams et al., 2004, Drug Metab. Dispos., 32:1201–1208
  2. Remmel et al., 2007, in Drug Metabolism in Drug Design and Development, (Zhang D et al., eds); pp 37-88, John Wiley & Sons, Inc
  3. Trottier et al., 2006, Hepatology, 44:1158-1170
  4. Chang et al., 2013, Mol. Pharm., 10:3067-3075
  5. Fisher et al., 2000, Drug Metab. Dispos., 28:560-566

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