UGT Inhibition

UGT Enzyme Inhibition Studies

UDP glucuronosyltransferase (UGT) enzymes are responsible for glucuronidation of their substrates (exogenous or endogenous compounds) via covalent linkage (conjugation) with UDP-glucuronic acid (UDPGA) to convert them into more polar glucuronides. Glucuronidation, a Phase II metabolic process, facilitates the excretion of xenobiotics, or their metabolites generated during phase I reactions, in urine and bile, and generally results in detoxification. A limited number of drug glucuronides, however, still possess biological activity. UGTs constitute a family of membrane-bound microsomal enzymes which differ in terms of catalytic function, regulation, and tissue expression. To date, 17 human UGTs have been identified and classified into two separate families (UGT1 and UGT2) based on the sequence identity of the encoded proteins. Of these, to date, the main UGTs identified to play a role in the metabolism of certain drugs are [1]:

UGT1A1
UGT1A3
UGT1A4
UGT1A6
UGT1A9
UGT2B7
UGT2B15

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 [2].

In case formation of glucuronide metabolites is observed for an orally administered drug, it may be important to assess the impact of first pass metabolism, including glucuronidation, on its bioavailability. Substrates to metabolic enzymes can also be their inhibitors, but drugs that are not undergoing glucuronidation may still interfere with UGT function, potentially leading to side effects or drug interactions. Assessment of UGT-mediated drug-drug interactions in vitro is therefore required by regulatory agencies. The most recently released regulatory draft, the ICH M12 Harmonized Guideline for Drug Interaction Studies [3] calls for 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.

Assay description and technical considerations

Our in vitro UGT Enzyme Inhibition assays were set up following the recommendations of the International Council for Harmonisation (ICH) [3] in two different assay systems:

Recombinant UGT enzymes or;
human liver microsomes,

using (relatively) selective probe substrates. While microsomes more closely mimic physiological liver functions when it comes to drug metabolism and glucuronide formation, the use of recombinant UGTs makes it possible to identify the involvement of each UGT individually.

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Generally, both systems are accepted for UGT Enzyme Inhibition assessment by regulatory agencies, however, there may be some overlap in UGT substrate specificity, and for this reason, it is particularly recommended to address UGT2B15 inhibition using recombinant enzymes instead of microsomes.

To obtain a concentration-dependent inhibition curve, and – where possible – determine an IC₅₀ value, Test Articles (TA) are typically studied at least over a 500-fold concentration range. For each UGT enzyme studied, a specific probe substrate is pre-incubated with either the recombinant enzymes or human liver microsomes (HLM) on ice for 15 minutes. Alamethicin is added as a pore-forming agent to facilitate compound uptake and access to metabolizing enzymes in both systems. Following pre-incubation, UDPGA (the co-factor of the UGT enzymes) and the Test Article (TA) are added to initiate the reaction, followed by a second incubation to allow metabolite formation. Glucuronide metabolites specifically formed by each single UGT enzyme are quantified by HPLC-MS. To confirm enzyme functionality, control inhibitors are also tested in parallel with the TA(s) in each experiment. Probe substrates, their metabolites and the inhibitors used for each UGT are outlined in Table 1. Because of the overlapping selectivity of the substrates, it is not practical to examine the inhibition of UGT2B15 in human liver microsomes (see Table 1.).

UGT enzymes	Probe substrates	Metabolites	Inhibitors
UGT1A1	ß-Estradiol	ß-Estradiol-17-ß-D-glucuronide	Silybin
UGT1A3	CDCA	CDCA glucuronide	Troglitazone
UGT1A4	Trifluoperazine	Trifluoperazine glucuronide	Itraconazole
UGT1A6	1-Naphthol	1-Naphthol glucuronide	Troglitazone
UGT1A9	Propofol	Propofol glucuronide	Niflumic acid
UGT2B7	Zidovudine	Zidovudine glucuronide	Diclofenac
UGT2B15	4-Methylumbelliferone	4-MU glucuronide	Amitriptyline

Table 1. Probe substrates, their metabolites and control inhibitors of the examined UGT enzymes.

Figure 1. UGT Inhibition curves. A) Inhibition of CDCA glucuronidation by Troglitazone via UGT1A3 in HLM (in orange) or using recombinant rUGT1A3 (in blue). B) Inhibition of 1-Naphthol glucuronidation by Troglitazone in HLM (orange curve) or using rUGT1A6 (blue curve).

Human liver microsomes used in our UGT enzyme inhibition assays are pooled from 150 mixed-gender donors to make sure the results are representative for the general population. Recombinant UGT enzymes are prepared from baculovirus-transfected insect cells and display high levels of catalytic activities.

UGT inhibition data is presented on a relative scale with 100 % defined as specific metabolite formation in the presence of only the solvent with no TA added, considered a no-inhibition condition. Results are plotted as inhibition of metabolite formation [relative activity (%)] versus TA concentration (μM or μg/mL), and in case inhibition occurs, a decrease in enzyme activity and thus product formation can be observed. In case a curve can be fitted based on enzyme kinetics, and at least 50% inhibition is reached, the IC₅₀ (µM) value can be determined, defined as the TA concentration required to inhibit maximal activity by 50%. IC₅₀ values for UGT inhibitors are usually in good correlation across systems (except for UGT2B15, where only recombinant enzymes are used), with results obtained using recombinant enzymes usually yielding slightly lower IC₅₀ values as in this setup, no other metabolizing processes are present, and TAs can readily interact with the UGTs.

References

[1] Miners JO, Rowland A, Novak JJ, Lapham K, Goosen TC. Evidence-based strategies for the characterisation of human drug and chemical glucuronidation in vitro and UDP-glucuronosyltransferase reaction phenotyping. Pharmacol Ther. 2021 Feb;218:107689. doi: 10.1016/j.pharmthera.2020.107689. Epub 2020 Sep 25. PMID: 32980440.

[2] Rowland A, Miners JO, Mackenzie PI. The UDP-glucuronosyltransferases: their role in drug metabolism and detoxification. Int J Biochem Cell Biol. 2013 Jun;45(6):1121-32. doi: 10.1016/j.biocel.2013.02.019. Epub 2013 Mar 7. PMID: 23500526.

[3] International Council For Harmonisation of technical requirements for pharmaceuticals for human use, ICH HARMONISED GUIDELINE DRUG INTERACTION STUDIES M12, Draft version, Endorsed on 24 May 2022.