UGT1A1 Inhibition

UGT1A1 inhibition assay protocol

Substrate Estradiol Metabolite Estradiol 3-glucuronide Test Compound Concentration 0, 0.4, 1, 4, 10, 40 and 100μM (different concentrations available) Enzyme source Human UGT1A1 Supersomes™ Cofactors UDPGA Compound Requirements 50μL of 20mM solution Positive Control Silybin Analysis Method LC-MS/MS Data Delivery IC50 Standard error of IC50

Glucuronidation is a listed clearance mechanism for 1 in 10 of the top 200 prescribed drugs2

Data from the Cloe Select UGT1A1 inhibition assay

Time and protein linearity studies and enzyme kinetics studies were performed initially to set the final conditions for the IC₅₀ assessments. Known UGT1A1 inhibitors were assessed in the Cloe Select UGT1A1 Inhibition assay over 3 separate occasions.

Figure 1

Cloe Select UaGT1A1 Inhibition validation data

The effect of 5 known inhibitors (bromocriptine, indomethacin, ketoconazole, quercetin and silybin) on the 3-glucuronidation of estradiol was investigated on 3 separate occasions. The error bars represent the standard error of the IC₅₀ determination. The inhibitors show good consistency between different assay runs.

 

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Questions and answers on UGT1A1 inhibition

Why is glucuronidation an important clearance mechanism?

Uridine glucuronyl transferases (UGT) are a family of enzymes which play a major role in the Phase II metabolism of drugs. The importance of glucuronidation is highlighted by the fact that it is a listed clearance mechanism for 1 in 10 of the top 200 prescribed drugs².

Glucuronidation involves the addition (or conjugation) of glucuronic acid either directly to the drug itself or to an oxidative metabolite of the drug. The addition of a glucuronide results in conjugates that are more polar and ionized at physiological pH. These features facilitate excretion via the kidneys. Glucuronides may also be excreted by the liver through the bile.

Why is it important to investigate UGT1A1 Inhibition?

UGT1A1 is an important isoform of the UGT family of enzymes due to its role in the glucuronidation of the endogenous product, bilirubin, as well as estrogens and flavanoids. Certain individuals also exhibit a polymorphism in this isoform which may impair their ability to metabolise certain drugs. Inhibition of UGT1A1 enzyme has the potential to produce increased levels of bilirubin in the circulation which can lead to toxicological effects. Drug-drug interactions may be of greater significance in individuals who are carriers of the polymorphic variant of UGT1A1.

Please provide an overview of the Cloe Select UGT1A1 Inhibition assay.

For the UGT1A1 inhibition assay, the UGT1A1 substrate, estradiol, is incubated with cDNA-expressed human UGT1A1 Supersomes™, alamethicin, UDPGA and a range of test compound concentrations (typically 0.4 - 100μM) for 30 min at 37°C. At the end of the incubation, the formation of the metabolite, estradiol 3-glucuronide is monitored by LC-MS/MS at each of the test compound concentrations. A decrease in the formation of the metabolites compared to vehicle control is used to calculate an IC₅₀ value (test compound concentration which produces 50% inhibition). An example IC₅₀ profile is shown in Figure 2.

Figure 2

Graph displaying a typical inhibition profile.

 

What is the positive control inhibitor used in the Cloe Select UGT1A1 inhibition assay?

The selective UGT1A1 inhibitor, silybin, is used as the positive control inhibitor.

What is the concentration of the probe substrates relative to K_m?

The substrate concentration is equivalent to the K_m.

Why do you include UDPGA and alamethicin in the incubation?

UDPGA (UDP-glucuronic acid) is the conjugating agent required for formation of the glucuronide. In microsomal incubations, it is necessary to supplement with this cofactor for glucuronidation to occur. Due to the luminal location of the UGT’s within the endoplasmic reticulum of microsomal preparations, the passage of the water soluble cofactor UDPGA to the active site is challenging. In order to overcome this latency phenomenon, the pore forming agent, alamethicin, can be used to improve access to the active site. Alamethicin has the advantage that it does not impact on CYP activity, unlike some detergents which perform a similar function. Supersomes have a lower level of latency than mammalian tissue fractions.

Why do you use expressed enzyme rather than human liver microsomes to study UGT1A1 inhibition?

Estradiol 3-glucuronidation appears to be the most widely used reaction for studying UGT1A1. However, substrate specificity is not as well defined within the UGT isoforms as compared to the CYPs and although UGT1A1 is the main isoform involved in the 3-glucuronidation of estradiol, it has been shown, based on relative activity data, that UGT1A3 may account for up to one third of the formation of estradiol 3-glucuronide⁶. By using expressed enzyme, the metabolism of estradiol is not being complicated by any other metabolic reaction and is solely focusing on UGT1A1.

Can I further characterise the type of inhibition determined?

Following IC₅₀ determination we can then determine the K_i for the test compound against the appropriate isoform. This will give information as to the potency of the inhibition and the type of inhibition (competitive, non competitive, uncompetitive or mixed) and can be used to estimate the impact of any potential in vivo interactions.

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