UGT2B10 is the Major UDP-Glucuronosyltransferase 2B Isoform Involved in the Metabolism of Lamotrigine and is Implicated in the Drug-Drug Interaction with Valproic Acid.

Lamotrigine is a phenyltriazine anticonvulsant that is primarily metabolized by phase II UDP-glucuronosyltransferases (UGT) to a quaternary N2-glucuronide, which accounts for ~ 90% of the excreted dose in humans. While there is consensus that UGT1A4 plays a predominant role in the formation of the N2-glucuronide, there is compelling evidence in the literature to suggest that the metabolism of lamotrigine is catalyzed by another UGT isoform. However, the exact identity of the UGT isoform that contribute to the formation of this glucuronide remains uncertain. In this study, we harnessed a robust reaction phenotyping strategy to delineate the identities and its associated fraction metabolized (fm) of the UGTs involved in lamotrigine N2-glucuronidation. Foremost, human recombinant UGT mapping experiments revealed that the N2-glucuronide is catalyzed by multiple UGT isoforms. (i.e., UGT1A1, 1A3, 1A4, 1A9, 2B4, 2B7, and 2B10). Thereafter, scaling the apparent intrinsic clearances obtained from the enzyme kinetic experiments with our in-house liver-derived relative expression factors (REF) and relative activity factors (RAF) revealed that, in addition to UGT1A4, UGT2B10 was involved in the N2-glucuronidation of lamotrigine. This was further confirmed via chemical inhibition in human liver microsomes with the UGT1A4-selective inhibitor hecogenin and the UGT2B10-selective inhibitor desloratadine. By integrating various orthogonal approaches (i.e., REF- and RAF-scaling, and chemical inhibition), we quantitatively determined that the fm for UGT1A4 and UGT2B10 ranged from 0.42 - 0.64 and 0.32 - 0.57, respectively. Finally, we also provided nascent evidence that the pharmacokinetic interaction between lamotrigine and valproic acid likely arose from the in vivo inhibition of its UGT2B10-mediated pathway.

Clotrimazole Identified as a Selective UGT2B4 Inhibitor Using Canagliflozin-2'-O-Glucuronide Formation as a Selective UGT2B4 Probe Reaction.

UGT2B4 is a highly expressed drug-metabolizing enzyme in the liver contributing to the glucuronidation of several drugs. To enable quantitatively assessing UGT2B4 contribution toward metabolic clearance, a potent and selective UGT2B4 inhibitor that can be used for reaction phenotyping was sought. Initially, a canagliflozin-2'-O-glucuronyl transferase activity assay was developed in recombinant UGT2B4 and human liver microsomes (HLM) [±2% bovine serum albumin (BSA)]. Canagliflozin-2'-O-glucuronidation (C2OG) substrate concentration at half-maximal velocity value in recombinant UGT2B4 and HLM were similar. C2OG formation intrinsic clearance was five- to seven-fold higher in incubations containing 2% BSA, suggesting UGT2B4 susceptibility to the inhibitory unsaturated long-chain fatty acids released during the incubation. Monitoring for C2OG formation, 180 compounds were evaluated for UGT2B4 inhibition potency in the presence and absence of 2% BSA. Compounds that exhibited an apparent UGT2B4 IC50 of < 1 µM in HLM with 2% BSA were evaluated for inhibition of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, UGT2B10, UGT2B15, and UGT2B17 catalytic activities to establish selectivity suitable for supporting UGT reaction phenotyping. In this study, clotrimazole was identified as a potent UGT2B4 inhibitor (HLM apparent IC50 of 11 to 35 nM ± 2% BSA). Moreover, clotrimazole exhibited selectivity for UGT2B4 inhibition (>24-fold) over the other UGT enzymes evaluated. Additionally, during this study it was discovered that the previously described UGT2B7 inhibitors 16α- and 16ß-phenyllongifolol also inhibit UGT2B4. Clotrimazole, a potent and selective UGT2B4 inhibitor, will prove essential during UGT reaction phenotyping. SIGNIFICANCE STATEMENT: To mechanistically evaluate drug interactions, it is essential to understand the contribution of individual enzymes to the metabolic clearance of a drug. The present study describes the development of a UGT2B4 activity assay that enabled the discovery of the highly selective and potent UGT2B4 inhibitor clotrimazole. Clotrimazole can be used in UGT reaction phenotyping studies to estimate fractional contribution of UGT2B4.

In Vitro Ciclopirox Glucuronidation in Liver Microsomes from Humans and Various Experimental Animals.

BACKGROUND AND OBJECTIVE: Ciclopirox is a widely used antifungal drug, redisposition of which has drawn increasing attentions due to multiple promising activities. The drug undergoes extensive glucuronidation, which acts as a major obstacle in the ongoing novel application and still remains poorly understood. The current study aims to phenotype ciclopirox glucuronidation pathway and as well to decipher the related species differences. METHODS: Ciclopirox glucuronidation was investigated in liver microsomes from humans (HLM) and various experimental animals. Assays with recombinant uridine diphosphate glucuronosyltransferases (UGTs), enzyme kinetic analyses and selective inhibitors were used to determine the role of individual UGTs in ciclopirox glucuronidation. RESULTS: HLM is highly active in ciclopirox glucuronidation with Michaelis-Menten constant (Km), maximum velocity (Vmax), and intrinsic clearance (CLint) values of 139 µM, 7.89 nmol/min/mg, and 56 µL/min/mg, respectively. UGT1A9 displays by far the highest activity, whereas several other isoforms (UGT1A6, UGT1A7, and UGT1A8) catalyze formation of traced glucuronides. Further kinetic analysis demonstrates that UGT1A9 has a closed Km value (167 µM) to HLM. UGT1A9 selective inhibitor (magnolol) can potently inhibit ciclopirox glucuronidation in HLM with the IC50 value of 0.12 µM. The reaction displays remarkable differences across liver microsomes from mice, rats, cynomolgus monkey, minipig, and beagle dog, with the CLint values in the range of 26-369 µL/min/mg. In addition, ciclopirox glucuronidation activities of experimental animals' liver microsomes were less sensitive to magnolol than that of HLM. CONCLUSIONS: Ciclopirox glucuronidation displays remarkable species differences with UGT1A9 as a dominant contributor in humans. It is suggested that the pharmacological or toxicological effects of ciclopirox may be UGT1A9 and species dependent.

Identification of the human Cytochrome P450 enzymes (P450s) responsible for metabolizing infigratinib to its pharmacologically active Metabolites, BHS697, and CQM157, and assessment of their in vitro inhibition of P450s and UDP-glucuronosyltransferases (UGTs).

Infigratinib, an oral FGFR inhibitor for advanced cholangiocarcinoma, yielded two active metabolites, BHS697 and CQM157, with similar receptor affinity. Our study characterized P450s that are responsible for the metabolism of infigratinib to its two major active metabolites, BHS697 and CQM157. In vitro inhibition of P450s and UGTs by infigratinib, BHS697 or CQM157 was further investigated. The unbound apparent Km values for metabolism of infigratinib to BHS697 by HLM, human recombinant CYP2C8, CYP2C19, CYP2D6 and CYP3A4 enzymes are 4.47, 0.65, 2.50, 30.6 and 2.08 µM, while Vmax values are 90.0 pmol/min/mg protein, 0.13, 0.027, 0.81, and 0.56 pmol/min/pmol protein, respectively. The unbound apparent Km value for metabolism of infigratinib to CQM157 by HLM is 0.049 µM, while the Vmax value is 0.32 pmol/min/mg protein respectively. In HLM, infigratinib displayed moderate inhibition of CYP3A4 and CYP2C19 and weak or negligible inhibition of other P450 isoforms. BHS697 exhibited weak inhibition of CYP2B6, CYP2C9, CYP2C19 and CYP3A4, and no inhibition of CYP2C8 and CYP2D6. CQM157 moderately inhibited CYP2C9 and CYP3A4, and weakly or negligibly inhibited other P450 isoforms. Regarding UGTs, infigratinib moderately inhibited UGT1A4 and weakly inhibited UGT1A1, respectively. BHS697 weakly inhibited UGT1A1. In contrast, CQM157 moderately inhibited both UGT1A1 and UGT1A4. Our findings provide novel insights into the metabolism of and potential DDIs implicating infigratinib.

In vitro inhibitory effects of selumetinib on activity of human UDP-glucuronosyltransferases and prediction of in vivo drug-drug interactions.

Selumetinib is an oral, effective, and selective tyrosine kinase inhibitor targeting mitogen-activated protein kinase 1 and 2 (MEK1/2), which is clinically active in multiple tumor types, such as neurofibromatosis type 1 (NF1), melanoma, gliomas and non-small cell lung cancer (NSCLC). The purpose of this article was to assess the effects of selumetinib on the activities of twelve human UDP-glucosyltransferases (UGTs) including UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17, and its potential for inducing clinical drug-drug interactions (DDIs). The results demonstrated that selumetinib potently inhibited the activity of UGT2B7 through the mechanism of mixed inhibition with the inhibition constant value of 5.79 ± 0.65 µM. Furthermore, the plasma concentration of UGT2B7 substrate as the co-administered drug was predicted to be increased by at least 84 % when patients took selumetinib 75 mg twice daily, suggesting a high potential to induce clinical DDIs. Selumetinib exhibited weak inhibitory effects on other human UGTs and was unlikely to trigger off UGTs-mediated DDIs except for UGT2B7. Therefore, the combination of selumetinib with the substrate drug of UGT2B7 requires additional attention to avoid adverse events in clinical treatment.

Icaritin exhibits potential drug-drug interactions through the inhibition of human UDP-glucuronosyltransferase in vitro.

Icaritin is a prenylflavonoid derivative of the genus Epimedium (Berberidaceae) and has a variety of pharmacological actions. Icaritin is approved by the National Medical Products Administration as an anticancer drug that exhibits efficacy and safety advantages in patients with hepatocellular carcinoma cells. This study aimed to evaluate the inhibitory effects of icaritin on UDP-glucuronosyltransferase (UGT) isoforms. 4-Methylumbelliferone (4-MU) was employed as a probe drug for all the tested UGT isoforms using in vitro human liver microsomes (HLM). The inhibition potentials of UGT1A1 and 1A9 in HLM were further tested by employing 17ß-estradiol (E2) and propofol (PRO) as probe substrates, respectively. The results showed that icaritin inhibits UGT1A1, 1A3, 1A4, 1A7, 1A8, 1A10, 2B7, and 2B15. Furthermore, icaritin exhibited a mixed inhibition of UGT1A1, 1A3, and 1A9, and the inhibition kinetic parameters (Ki) were calculated to be 3.538, 2.117, and 0.306 (µM), respectively. The inhibition of human liver microsomal UGT1A1 and 1A9 both followed mixed mechanism, with Ki values of 2.694 and 1.431 (µM). This study provides supporting information for understanding the drug-drug interaction (DDI) potential of the flavonoid icaritin and other UGT-metabolized drugs in clinical settings. In addition, the findings provide safety evidence for DDI when liver cancer patients receive a combination therapy including icaritin.

Avapritinib Carries the Risk of Drug Interaction via Inhibition of UDP-Glucuronyltransferase (UGT) 1A1.

BACKGROUND: Avapritinib is the only drug for adult patients with PDGFRA exon 18 mutated unresectable or metastatic gastrointestinal stromal tumor (GIST). Although avapritinib has been approved by the FDA for four years, little is known about the risk of drug-drug interactions (DDIs) via UDP-glucuronyltransferases (UGTs) inhibition. OBJECTIVE: The aim of the present study was to systematically evaluate the inhibitory effects of avapritinib against UGTs and to quantitatively estimate its potential DDIs risk in vivo. METHODS: Recombinant human UGTs were employed to catalyze the glucuronidation of substrates in a range of concentrations of avapritinib. The kinetics analysis was performed to evaluate the inhibition types of avapritinib against UGTs. The quantitative prediction of DDIs was done using in vitro-in vivo extrapolation (IVIVE). RESULTS: Avapritinib had a potent competitive inhibitory effect on UGT1A1. Quantitative prediction results showed that avapritinib administered at clinical doses might result in a 14.85% increase in area under the curve (AUC) of drugs primarily cleared by UGT1A1. Moreover, the Rgut value was calculated to be 18.44. CONCLUSION: Avapritinib has the potential to cause intestinal DDIs via the inhibition of UGT1A1. Additional attention should be paid when avapritinib is coadministered with UGT1A1 substrates.

Inhibition of human UDP-glucuronosyltransferase enzyme by entrectinib: Implications for drug-drug interactions.

As a new type of oral tyrosine kinase inhibitor, entrectinib can act on multiple targets and exert efficacy and has been approved for the treatment of non-small cell lung cancer (NSCLC) and solid tumors. However, whether entrectinib affects the activities of recombinant human UDP-glucuronosyltransferases (UGTs) remains unclear. Herein, we aimed to investigate the inhibitory effects of entrectinib on human UGTs and to assess the potential risk of causing drug-drug interactions (DDIs) based on the inhibition against UGTs. High-performance liquid chromatography (HPLC) was used to evaluate the inhibitory effects of entrectinib on UGTs according to the product formation rate of UGT substrate with or without entrectinib, and the inhibition kinetics experiment was conducted to assess the inhibitory type of entrectinib on UGTs. Our results showed that entrectinib exhibited extensive inhibitory effects on most human UGTs, and especially inhibited the activities of UGT1A7, UGT1A8, and UGT2B15 with Ki (Inhibition constant) of lower than 5 µM (0.95-4.38 µM). Furthermore, the results from quantitative prediction research suggested that the combination of entrectinib at 600 mg/day with substrates primarily metabolized by hepatic UGT2B15 or intestinal UGT1A7 and UGT1A8 might cause clinical DDIs. Thus, special attention should be paid to avoid adverse reactions induced by DDIs when co-administration of entrectinib and drugs metabolized by UGTs.

Medicinal Chemistry and NMR Driven Discovery of Novel UDP-glucuronosyltransferase 1A Inhibitors That Overcome Therapeutic Resistance in Cells.

The UDP glucuronosyltransferases (UGT) deactivate many therapeutics via glucuronidation while being required for clearance of normal metabolites and xenobiotics. There are 19 UGT enzymes categorized into UGT1A and UGT2B families based on sequence conservation. This presents a challenge in terms of targeting specific UGTs to overcome drug resistance without eliciting overt toxicity. Here, we identified for the first time that UGT1A4 is highly elevated in acute myeloid leukemia (AML) patients and its reduction corresponded to objective clinical responses. To develop inhibitors to UGT1A4, we leveraged previous NMR-based fragment screening data against the C-terminal domain of UGT1A (UGT1A-C). NMR and medicinal chemistry strategies identified novel chemical matter based on fragment compounds with the capacity to bind ∼20 fold more tightly to UGT1A-C (Kd âˆ¼ 600 µM vs ∼30 µM). Some compounds differentially inhibited UGT1A4 versus UGT1A1 enzyme activity and restored drug sensitivity in resistant human cancer cells. NMR-based NOE experiments revealed these novel compounds recognised a region distal to the catalytic site suggestive of allosteric regulation. This binding region is poorly conserved between UGT1A and UGT2B C-terminal sequences, which otherwise exhibit high similarity. Consistently, these compounds did not bind to the C-terminal domain of UGT2B7 nor a triple mutant of UGT1A-C replaced with UGT2B7 residues in this region. Overall, we discovered a site on UGTs that can be leveraged to differentially target UGT1As and UGT2Bs, identified UGT1A4 as a therapeutic target, and found new chemical matter that binds the UGT1A C-terminus, inhibits glucuronidation and restores drug sensitivity.

Inhibition of cytochrome P450 enzymes and uridine 5'-diphospho-glucuronosyltransferases by vicagrel in human liver microsomes: A prediction of potential drug-drug interactions.

Vicagrel, an antiplatelet drug candidate targeting platelet P2Y12 receptor and has finished its phase II clinical trial. The inhibition of six major cytochrome P450 enzymes (P450) (CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and six UDP-glucuronosyltransferases (UGT) (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, and UGT2B7) by vicagrel was evaluated using pooled human liver microsomes and specific probe substrates. Physiology-based pharmacokinetic (PBPK) simulation was further applied to predict the in vivo drug-drug interaction (DDI) potential between vicagrel and bupropion as well as S-mephenytoin. The results suggested that vicagrel inhibited CYP2B6 and CYP2C19 potently with apparent IC50 values of 1.6 and 2.0 µM, respectively. In terms of mode of reversible inhibition, vicagrel exhibited mixed-type inhibition of CYP2B6-catalyzed bupropion hydroxylation and noncompetitive inhibition of CYP2C19-mediated S-mephenytoin 4'-hydroxylation with Ki values of 0.19 µM and 1.2 µM, respectively. Vicagrel displayed profound time-dependent inhibition towards CYP2B6 with maximal rate constant of inactivation (kinact) and half-maximal inactivator concentration (KI) values of 0.062 min-1 and 1.52 µM, respectively. No time-dependent inhibition by vicagrel was noted for CYP2C19. For UGT, negligible to moderate inhibition by vicagrel was observed with IC50 values of >50.0, >50.0, 28.2, 8.7, >50.0 and 28.2 µM for UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7, respectively. In terms of mode of reversible inhibition, vicagrel exhibited mixed-type inhibition of UGT1A6-catalyzed N-Acetylserotonin ß-D-glucuronidation with a Ki value of 5.6 µM. No time-dependent inhibition by vicagrel was noted for UGT1A6. PBPK simulation indicated that neither altered AUC nor Cmax of bupropion and S-mephenytoin was observed in the presence of vicagrel. Our study provides inhibitory constants for future DDI prediction between vicagrel and drug substrates of CYP2B6, CYP2C19 and UGT1A6. In addition, our simulation suggests the lack of clinically important DDI between vicagrel and bupropion or S-mephenytoin.

Mechanism of dasabuvir inhibition of acetaminophen glucuronidation.

OBJECTIVES: Acetaminophen (APAP) (paracetamol) is a widely used non-prescription drug for pain relief and antipyretic effects. The clearance of APAP is mainly through phase-2 biotransformation catalysed by UDP-glucuronosyl transferases (UGT). Dasabuvir is an anti-hepatitis C drug reported to inhibit several UGT isoforms. The study evaluated the in-vitro inhibitory capacity of dasabuvir versus APAP glucuronidation. METHODS: Procedures included human liver microsomal incubations with APAP and isoform-selective probe substrates. KEY FINDINGS: Dasabuvir inhibited APAP metabolism by a reversible, mixed-type (competitive and non-competitive) partial inhibition, with an inhibition constant Ki = 3.4 µM. The index constant 'a' was 6.7, indicating the relative contribution of competitive and non-competitive inhibition. The enzyme-inhibitor complex was still able to catalyse the reaction by 12% of the control capacity. Dasabuvir produced strong partial inhibition effect of UGT1A1 and UGT1A9 and relatively complete inhibition of UGT1A6. CONCLUSIONS: Consistent with previous reports, dasabuvir inhibits the activity of 3 UGT isoforms associated with APAP metabolism. In-vitro to in-vivo scaling by 2 different approaches showed identical results, predicting an increased AUC of APAP by a factor of 1.3-fold with coadministration of dasabuvir. Until the findings are confirmed in clinical drug interaction studies, APAP dosage should not exceed 3 g per day in dasabuvir-treated patients to avoid potentially hepatotoxic APAP exposures.

Methylophiopogonanone A is a naturally occurring broad-spectrum inhibitor against human UDP-glucuronosyltransferases: Inhibition behaviours and implication in herb-drug interactions.

Methylophiopogonanone A (MOA) is an abundant homoisoflavonoid in the Chinese herb Ophiopogonis Radix. Recent investigations revealed that MOA inhibited several human cytochrome P450 enzymes (CYPs) and stimulated OATP1B1. However, the inhibitory effects of MOA on phase II drug-metabolizing enzymes, such as human UDP-glucuronosyltransferases (hUGTs), have not been well investigated. Herein, the inhibition potentials of MOA on hUGTs were assessed. The results clearly demonstrated that MOA dose-dependently inhibited all tested hUGTs including UGT1A1 (IC50 = 1.23 µM), one of the most important detoxification enzymes in humans. Further investigations showed that MOA strongly inhibited UGT1A1-catalysed NHPH-O-glucuronidation in a range of biological settings including hUGT1A1, human liver microsomes (HLM) and HeLa cells overexpressing UGT1A1. Inhibition kinetic analyses demonstrated that MOA competitively inhibited UGT1A1-catalysed NHPH-O-glucuronidation in both hUGT1A1 and HLM, with Ki values of 0.52 and 1.22 µM, respectively. Collectively, our findings expanded knowledge of the interactions between MOA and human drug-metabolizing enzymes, which would be very helpful for guiding the use of MOA-related herbal products in clinical settings.

Inhibition of UDP-Glucuronosyltransferase Enzymes by Major Cannabinoids and Their Metabolites.

The UDP-glucuronosyltransferase (UGT) family of enzymes play a central role in the metabolism and detoxification of a wide range of endogenous and exogenous compounds. UGTs exhibit a high degree of structural similarity and display overlapping substrate specificity, often making estimations of potential drug-drug interactions difficult to fully elucidate. One such interaction yet to be examined may be occurring between UGTs and cannabinoids, as the legalization of recreational and medicinal cannabis and subsequent co-usage of cannabis and therapeutic drugs increases in the United States and internationally. In the present study, the inhibition potential of the major cannabinoids Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN), as well as their major metabolites, was determined in microsomes isolated from HEK293 cells overexpressing individual recombinant UGTs and in microsomes from human liver and kidney specimens. The highest inhibition was seen by CBD against the glucuronidation activity of UGTs 1A9, 2B4, 1A6, and 2B7, with binding-corrected IC50 values of 0.12 ± 0.020 µM, 0.22 ± 0.045 µM, 0.40 ± 0.10 µM, and 0.82 ± 0.15 µM, respectively. Strong inhibition of UGT1A9 was also demonstrated by THC and CBN, with binding-corrected IC50 values of 0.45 ± 0.12 µM and 0.51 ± 0.063 µM, respectively. Strong inhibition of UGT2B7 was also observed for THC and CBN; no or weak inhibition was observed with cannabinoid metabolites. This inhibition of UGT activity suggests that in addition to playing an important role in drug-drug interactions, cannabinoid exposure may have important implications in patients with impaired hepatic or kidney function. SIGNIFICANCE STATEMENT: Major cannabinoids found in the plasma of cannabis users inhibit several UDP-glucuronosyltransferase (UGT) enzymes, including UGT1A6, UGT1A9, UGT2B4, and UGT2B7. This study is the first to show the potential of cannabinoids and their metabolites to inhibit all the major kidney UGTs as well as the two most abundant UGTs present in liver. This study suggests that as all three major kidney UGTs are inhibited by cannabinoids, greater drug-drug interaction effects might be observed from co-use of cannabinods and therapeutics that are cleared renally.

A Molecular-Splicing Strategy for Constructing a Near-Infrared Fluorescent Probe for UDP-Glucuronosyltransferase 1A1.

UDP-glucuronosyltransferase 1A1 (UGT1A1) is a vital metabolic enzyme responsible for the clearance of endogenous substances and drugs. Hitherto, the development of fluorescent probes for UGTs was severely restricted due to the poor isoform selectivity and on-off or blue-shifted fluorescence response. Herein, we established a novel "molecular-splicing" strategy to construct a highly selective near-infrared (NIR) fluorescent probe, HHC, for UGT1A1, which exhibited a NIR signal at 720 nm after UGT1A1 metabolism. HHC was then successfully used for the real-time imaging of endogenous UGT1A1 in living cells and animals and to monitor the bile excretion function. In summary, an isoform-specific NIR fluorescent probe has been developed for monitoring UGT1A1 activity in living systems, high-throughput screening of novel UGT1A1 inhibitors and visual evaluation of bile excretion function.

Potential Assessment of UGT2B17 Inhibition by Salicylic Acid in Human Supersomes In Vitro.

Glucuronidation is a Phase 2 metabolic pathway responsible for the metabolism and excretion of testosterone to a conjugate testosterone glucuronide. Bioavailability and the rate of anabolic steroid testosterone metabolism can be affected upon UGT glucuronidation enzyme alteration. However, there is a lack of information about the in vitro potential assessment of UGT2B17 inhibition by salicylic acid. The purpose of this study is to investigate if UGT2B17 enzyme activity is inhibited by salicylic acid. A UGT2B17 assay was developed and validated by HPLC using a C18 reversed phase column (SUPELCO 25 cm × 4.6 mm, 5 µm) at 246 nm using a gradient elution mobile phase system: (A) phosphate buffer (0.01 M) at pH = 3.8, (B) HPLC grade acetonitrile and (C) HPLC grade methanol. The UGT2B17 metabolite (testosterone glucuronide) was quantified using human UGT2B17 supersomes by a validated HPLC method. The type of inhibition was determined by Lineweaver-Burk plots. These were constructed from the in vitro inhibition of salicylic acid at different concentration levels. The UGT2B17 assay showed good linearity (R2 > 0.99), acceptable recovery and accuracy (80-120%), good reproducibility and acceptable inter and intra-assay precision (<15%), low detection (6.42 and 2.76 µM) and quantitation limit values (19.46 and 8.38 µM) for testosterone and testosterone glucuronide respectively, according to ICH guidelines. Testosterone and testosterone glucuronide were found to be stable up to 72 h in normal laboratory conditions. Our investigational study showed that salicylic acid uncompetitively inhibited UGT2B17 enzyme activity. Thus, drugs that are substrates for the UGT2B17 enzyme have negligible potential effect of causing interaction with salicylic acid in humans.

Reversible Mechanisms of Enzyme Inhibition and Resulting Clinical Significance.

Inhibition of a drug-metabolizing enzyme by the reversible interaction of a drug with the enzyme, thus decreasing the metabolism of another drug, is a major cause of clinically significant drug-drug interactions. This chapter defines the four reversible mechanisms of inhibition exhibited by drugs: competitive, noncompetitive, uncompetitive, and mixed competitive/noncompetitive. An in vitro procedure to determine the potential of a drug to be a reversible inhibitor is also provided. Finally, a number of examples of clinically significant drug-drug interactions resulting from reversible inhibition are described.

How Science Is Driving Regulatory Guidances.

This chapter provides regulatory perspectives on how to translate in vitro drug metabolism findings into in vivo drug-drug interaction (DDI) predictions and how this affects the decision of conducting in vivo DDI evaluation. The chapter delineates rationale and analyses that have supported the recommendations in the U.S. Food and Drug Administration (FDA) DDI guidances in terms of in vitro-in vivo extrapolation of cytochrome P450 (CYP) inhibition-mediated DDI potential for investigational new drugs and their metabolites as substrates or inhibitors. The chapter also describes the framework and considerations to assess UDP-glucuronosyltransferase (UGT) inhibition-mediated DDI potential for drugs as substrates or inhibitors. The limitations of decision criteria and further improvements needed are also discussed. Case examples are provided throughout the chapter to illustrate how decision criteria have been utilized to evaluate in vivo DDI potential from in vitro data.

Inhibition of human UDP-glucuronosyltransferase enzyme by Dabrafenib: Implications for drug-drug interactions.

Dabrafenib is a novel small molecule tyrosine kinase inhibitor (TKI) which is used to treat metastatic melanoma. The aim of this research was to survey the effects of dabrafenib on human UDP-glucuronosyltransferases (UGTs) and to evaluate the risk of drug-drug interactions (DDIs). The formation rates for 4-methylumbelliferone (4-MU) glucuronide and trifluoperazine-glucuronide in 12 recombinant human UGT isoforms with or without dabrafenib were measured and HPLC was used to investigate the inhibitory effects of dabrafenib on UGTs. Inhibition kinetic studies were also conducted. In vitro-in vivo extrapolation approaches were further used to predict the risk of DDI potentials of dabrafenib via inhibition of UGTs. Our data indicated that dabrafenib had a broad inhibitory effect on 4-MU glucuronidation by inhibiting the activities of UGTs, especially on UGT1A1, UGT1A7, UGT1A8, and UGT1A9, and dabrafenib could increase the area under the curve of co-administered drugs. Dabrafenib is a strong inhibitor of several UGTs and the co-administration of dabrafenib with drugs primarily metabolized by UGT1A1, 1A7, 1A8 or 1A9 may induce potential DDIs.

Comparison of the drug-drug interactions potential of ibrutinib and acalabrutinib via inhibition of UDP-glucuronosyltransferase.

Ibrutinib and acalabrutinib are two Bruton's tyrosine kinase (BTK) inhibitors which have gained Food and Drug Administration (FDA) approval for the treatment of various B cell malignancies. Herein, we investigated the effects of the two drugs on UDP-glucuronosyltransferase (UGT) activities to evaluate their potential risk for drug-drug interactions (DDIs) via UGT inhibition. Our data indicated that ibrutinib exerted broad inhibition on most of UGTs, including a potent competitive inhibition against UGT1A1 with a Ki value of 0.90 ± 0.03 µM, a noncompetitive inhibition against UGT1A3 and UGT1A7 with Ki values of 0.88 ± 0.03 µM and 2.52 ± 0.23 µM, respectively, while acalabrutinib only exhibited weak UGT inhibition towards all tested UGT isoforms. DDI risk prediction suggested that the inhibition against UGT1A1 and UGT1A3 by ibrutinib might bring a potential DDIs risk, while acalabrutinib was unlikely to trigger clinically significant UGT-mediated DDIs due to its weak effects. Our study raises an alarm bell about potential DDI risk associated with ibrutinib, however, the extrapolation from in vitro data to in vivo drug interactions should be taken with caution, and additional systemic study is needed.

In vitro inhibition of human UDP-glucuronosyltransferase (UGT) 1A1 by osimertinib, and prediction of in vivo drug-drug interactions.

Osimertinib is the only third-generation epidermal growth factor receptor tyrosine-kinase inhibitor (EGFR-TKI) approved by Food and Drug Administration (FDA). This study aimed to know the inhibitory effect of osimertinib on human UDP-glucosyltransferases (UGTs) and human liver microsomes (HLMs), as well as to identify its potential to cause drug-drug interaction (DDI) arising from the modulation of UGT activity. High inhibitory effect of osimertinib was shown towards UGT1A1, 1A3, 1A6, 1A7, 1A8, 1A10, 2B7 and 2B15. Especially, osimertinib exhibited competitive inhibition against UGT1A1 with a Ki,u of 0.87 ± 0.12 µM. It also noncompetitively inhibited SN-38 glucuronidation in pooled HLMs with a Ki,u of 3.32 ± 0.25 µM. Results from quantitative prediction study indicated that osimertinib administered at 80 mg/day may result in a 4.83 % increase in the AUC of drugs mainly metabolized by UGT1A1, implying low risk of DDI via liver metabolism. However, the ratios of [I]gut/Ki,u are much higher than 11 in HLMs and recombinant UGT1A1, indicating a risk for interaction in intestine. The effects of osimertinib on intestinal UGT should be paid more attention on to avoid unnecessary clinical DDI risks.