Advertisement
Research ArticleArticle

Comparison of the Drug-Drug Interactions Potential of Erlotinib and Gefitinib via Inhibition of UDP-Glucuronosyltransferases

Yong Liu, Jacqueline Ramírez, Larry House and Mark J. Ratain
Drug Metabolism and Disposition January 2010, 38 (1) 32-39; DOI: https://doi.org/10.1124/dmd.109.029660

Abstract

We aimed to investigate and compare the effects of erlotinib and gefitinib on UDP-glucuronosyltransferase (UGT) activities and to quantitatively evaluate their drug-drug interaction (DDI) potential due to UGT inhibition. The inhibitory effects of erlotinib and gefitinib on UGTs were determined using high-performance liquid chromatography by measuring the formation rates for 4-methylumbelliferone (4-MU) glucuronide, imipramine N-glucuronide, and bilirubin glucuronides using recombinant human UGT isoforms and human liver microsomes (HLMs) in the absence or presence of erlotinib and gefitinib. Inhibition kinetic studies were conducted. Area under the curve (AUC) ratios were used to predict the risk of potential DDI in vivo. Erlotinib exhibited selective potent competitive inhibition against 4-MU glucuronidation by UGT1A1, and gefitinib demonstrated a wide range of inhibition against UGT-mediated 4-MU glucuronidation, particularly against UGT1A1, UGT1A7, UGT1A9, and UGT2B7. Erlotinib also exerted potent mixed inhibition against bilirubin glucuronidation in HLMs. We estimated that coadministration of erlotinib at 100 mg/day or higher doses may result in at least a 30% increase in the AUC of drugs predominantly cleared by UGT1A1. Thus, the coadministration of erlotinib with drugs primarily cleared by UGT1A1 may result in potential DDI. In contrast, gefitinib is unlikely to cause a clinically significant DDI through inhibition of glucuronidation.

Erlotinib and gefitinib are potent, reversible, and selective inhibitors of the tyrosine kinase of the epidermal growth factor receptor. Both drugs have been approved for the treatment of patients with non–small-cell lung cancer, and erlotinib is also indicated for pancreatic cancer. In addition, phase II trials have suggested activity for these agents in a number of other solid tumors (Sequist and Lynch, 2008). Gefitinib and erlotinib share a common chemical backbone structure and exhibit similar oral bioavailability and disposition characteristics in humans after oral administration (Siegel-Lakhai et al., 2005).

Drug-drug interactions (DDI) have received increasing attention over the past few decades. Several DDIs were reported to be associated with erlotinib and gefitinib. Coadministration of erlotinib has been reported to enhance the carboplatin exposure (Patnaik et al., 2006) and increase the serum concentration of phenytoin (Grenader et al., 2007). Drug interactions were also observed in two patients who received both gefitinib and warfarin simultaneously, which resulted in an enhancement of the warfarin effect (Onoda et al., 2005). Combination of gefitinib with capecitabine and radiation in patients with pancreatic and rectal cancer (Czito et al., 2006), as well as the combination of gefitinib with 5-fluorouracil, leucovorin, and irinotecan in patients with colorectal cancer (Veronese et al., 2005), are associated with excessive toxicity, suggesting an interaction at a pharmacokinetic or pharmacodynamic level.

Metabolizing enzyme-based DDI constitute the major proportion of clinically important DDI. Gefitinib and erlotinib are eliminated predominantly by oxidation by cytochrome P450 enzymes. Both drugs are metabolized primarily by CYP3A4 and CYP3A5. In addition, CYP1A2 is considerably involved in erlotinib metabolism, and CYP2D6 is involved in gefitinib metabolism (Li et al., 2007). Therefore, both drugs may alter the systemic exposure of some cytochromes P450 substrates, implying a potential DDI. However, it is unknown whether DDI by erlotinib and gefitinib are associated with other metabolizing enzymes.

UDP-glucuronosyltransferase (UGT) enzymes catalyze the conjugation of various endogenous substances and exogenous compounds. The human UGT superfamily comprises two families (UGT1 and UGT2). At least 19 human UGTs have been identified to date based on sequence homologies (Mackenzie et al., 2005). UGT-catalyzed glucuronidation reactions are responsible for the metabolism of approximately 35% of all drugs metabolized by phase II enzymes. Approximately one-seventh of the drugs prescribed in the United States in 2002 are cleared by UGTs (Williams et al., 2004). UGT-mediated drug interactions can potentially occur for many drugs. In fact, several significant DDI have been clinically observed. Erlotinib has been suggested to be an inhibitor of UGT1A1 [European Medicines Agency (EMEA), http://www.emea.europa.eu/humandocs/PDFs/EPAR/tarceva/H-618-PI-en.pdf]. However, the effects of erlotinib and gefitinib on UGT activity have not been fully characterized. Understanding the effects of erlotinib and gefitinib on UGT activities is important to ensure their safe administration and develop new coadministration therapies with both drugs.

The aim of this study was to investigate and compare the effects of erlotinib and gefitinib on the activities of human UGTs. Using a panel of recombinant human UGT isoforms, we found potent inhibition of erlotinib against UGT1A1. On the other hand, gefitinib demonstrated a wide range of inhibition against not only UGT1A1 but also UGT1A7, UGT1A9, and UGT2B7. The effects of erlotinib and gefitinib on bilirubin glucuronidation were investigated in human liver microsomes (HLMs). The potential for DDI in vivo was also quantitatively predicted and compared by using area under the curve (AUC) ratios.

Materials and Methods

Chemicals.

Erlotinib (OSI-774) was purchased from Biaffin GmbH and Co. KG (Kassel, Germany). Gefitinib and indinavir were purchased from Toronto Research Chemicals, Inc. (North York, ON, Canada). Bilirubin, 4-methylumbelliferone (4-MU), 4-methylumbelliferone-β-d-glucuronide (4-MUG), β-glucuronidase (from Escherichia coli), alamethicin (from Trichoderma viride), Tris-HCl, imipramine, p-nitrophenol, androsterone, diclofenac, phenylbutazone, ascorbic acid, hecogenin, 7-hydroxycoumarin, and uridine 5′-diphosphoglucuronic acid (UDPGA) (trisodium salt) were purchased from Sigma-Aldrich (St. Louis, MO). All other reagents were of high-performance liquid chromatography (HPLC) grade or of the highest grade commercially available.

Human Liver Microsomes and Recombinant Human UGTs.

Pooled HLMs and a panel of recombinant human UGT Supersomes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17) expressed in baculovirus-infected insect cells were purchased from BD Gentest (Woburn, MA). Pooled HLMs were derived from 22 donors (90% white, 5% Hispanic, and 5% African-American; 15 men and 7 women). The median age was 48 years with a range of 10 to 70.

Inhibition of 4-MU Glucuronidation Assay.

4-MU, a nonselective substrate of UGTs, was used as probe substrate for all UGTs except UGT1A4. Incubations with each individual enzyme were conducted by using conditions described previously (Uchaipichat et al., 2004), with a slight modification. A typical incubation mixture (in a total volume of 200 μl) contained recombinant UGTs (final concentration: 0.25, 0.15, 0.05, 0.05, 0.05, 0.05, 0.05, 0.5, 0.25, 0.75, and 0.5 mg/ml for UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17, respectively), 5 mM UDPGA, 5 mM MgCl2, 50 mM Tris-HCl buffer (pH 7.4), 50 μg/mg protein alamethicin, and 4-MU in the absence or presence of different concentrations of inhibitors. Incubations with 4-MU were performed at the concentration corresponding to the apparent Km or S50 value reported for each isoform (110, 1200, 110, 15, 750, 30, 80, 1200, 350, 250, and 2000 μM 4-MU for UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17, respectively) (Uchaipichat et al., 2006b). Known UGT inhibitors were used as positive controls: diclofenac for UGT1A1, UGT1A6, UGT1A7, and UGT1A9; androsterone for UGT1A3, UGT2B7, and UGT2B15; and phenylbutazone for UGT1A8 and UGT1A10, respectively (Uchaipichat et al., 2004, 2006a). There is no positive control reported available for UGT2B4 and UGT2B17. The negative controls are incubations without UDPGA. These tested chemicals and inhibitors were dissolved in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide in the incubation system was 1% (v/v). Alamethicin was dissolved in 90:10 incubation buffer/ethanol, and the final concentration of ethanol was less than 0.1%. Microsomes were preincubated with alamethicin on ice for 15 min before incubation. There was a 5-min preincubation step at 37°C before the reaction was started by addition of UDPGA. Incubation times were 120 min for UGT1A1, UGT1A10, UGT2B4, UGT2B15, and UGT2B17, 75 min for UGT1A3, and 30 min for UGT1A6, UGT1A7, UGT1A8, and UGT1A9. The reactions were quenched by adding 100 μl of acetonitrile and internal standard (7-hydroxycoumarin, 100 μM). Rate of product formation for each isoform was linear with respect to protein concentration and incubation time. The incubation mixtures were then centrifuged at 20,500g for 15 min to obtain the supernatant. Aliquots (20 μl) were then analyzed by HPLC (Hitachi High Technologies America, Schaumburg, IL). Chromatographic separation was achieved by using a C18 column (3.9 × 300 mm i.d., 10-μm particle size) (Sigma-Aldrich) at a flow rate of 1 ml/min and UV detection at 316 nm. The mobile phase consisted of 10 mM KH2PO4, pH 2.7 (A), and acetonitrile (B). The following gradient was applied at a flow rate of 1 ml/min: 0 to 4 min 80% A and 20% B; 4.1 to 8 min 50% A and 50% B; 8.1 to 12 min, 30% A and 70% B. The metabolites were quantified by using a standard curve made by combining 4-MUG stock and incubation buffer and processing as described above. All experiments were performed in two independent experiments in duplicate.

Inhibition of Imipramine N-Glucuronidation Assay.

Imipramine was used as probe substrate for UGT1A4. Imipramine N-glucuronidation activity was determined as published previously (Nakajima et al., 2002). Imipramine was incubated in the absence or presence of different concentrations of inhibitors. Hecogenin, a known UGT1A4 inhibitor, was used as positive control. Incubation was performed for 90 min by using a protein concentration of 0.5 mg protein/ml recombinant UGT1A4 or pooled HLMs. HPLC separation was achieved by using a C18 column (3.9 × 300 mm i.d., 10 μm) (Sigma-Aldrich) at a flow rate of 1 ml/min and UV detection at 254 nm. The mobile phase consisted of 30:70 acetonitrile: 10 mM KH2KO4 buffer (pH 2.6) (v:v). For the quantification of imipramine N-glucuronide, it was assumed that the glucuronide of imipramine had the same molar absorbance as its aglycone, because no imipramine monoglucuronide standards were available. The quantification of the glucuronide was accomplished by using a standard curve for imipramine. All experiments were performed in two independent experiments in duplicate.

Inhibition of Bilirubin Glucuronidation Assay.

Bilirubin glucuronidation activity was determined with a slight modification of a previously published method (Udomuksorn et al., 2007). Bilirubin was incubated in the absence or presence of different concentrations of erlotinib or gefitinib (0–100 μM). Indinavir, a known inhibitor of bilirubin glucuronidation, was used as positive control. Reactions were performed for 20 min in 0.5 mg/ml pooled HLMs. HPLC separation was achieved by using a SymmetryShield RP8 column (3.0 × 150 mm i.d., 5-μm particle size) (Waters, Milford, MA) with a flow rate of 1 ml/min and UV detection at 450 nm. Initial mobile-phase composition was 78% 100 mM ammonium acetate buffer at pH 4.85 (mobile phase A) and 22% acetonitrile (mobile phase B). The proportion of acetonitrile was increased to 44% over 20 min, then further increased to 90% over 1 min, and then held constant for 7 min before returning to the starting composition. Authentic standards of each of the bilirubin glucuronides were not available for use, so a bilirubin standard curve (0.1–5 μM) was used to quantify glucuronide formation.

Inhibition Kinetics Analysis.

Inhibition constant (Ki) values were determined by using various concentrations of 4-MU or bilirubin in the presence or absence of erlotinib and gefitinib. Ki values were calculated by nonlinear regression by using the equations for competitive inhibition (eq. 1), noncompetitive inhibition (eq. 2), or mixed inhibition (eq. 3) (Copeland, 2000): Embedded Image Embedded Image Embedded Image where v is the velocity of the reaction; S and I are the substrate and inhibitor concentrations, respectively; Ki is the inhibition constant describing the affinity of the inhibitor for the enzyme; and Km is the substrate concentration at half of the maximum velocity (Vmax) of the reaction. The type of inhibition was determined from the fitting of data to the enzyme inhibition models. Goodness of fit to kinetic and inhibition models was assessed from the F statistic, r2 values, parameter S.E. estimates, and 95% confidence intervals. Kinetic constants are reported as the mean ± S.E. of the parameter estimate. IC50 values (concentration of inhibitor that reduces enzyme activity by 50%) were determined by the Data Analysis Toolbox of Microsoft Excel software (Microsoft Inc., Redmond, WA).

Predicted Concentrations of Erlotinib and Gefitinib at UGT Catalytic Sites.

The concentrations of erlotinib and gefitinib in humans were estimated according to the equations for the average systemic plasma concentration after repeated oral administration ([I]av) (eq. 4), the maximum systemic plasma concentration after repeated oral administration ([I]max) (eq. 5), the maximum unbound systemic plasma concentration after repeated oral administration ([I]max,u) (eq. 6), the maximum hepatic input concentration ([I]in) (eq. 7), and the maximum unbound hepatic input concentration ([I]in,u) (eq. 8) (Ito et al., 2004): Embedded Image Embedded Image Embedded Image Embedded Image Embedded Image where D and τ represent dose and dosing interval of inhibitors used in the in vivo interaction study, respectively; k is the elimination rate constant; ka is the absorption rate constant; Fa is the fraction absorbed from the gut into the portal vein; Qh is the hepatic blood flow rate; and fu is the unbound fraction. The values of ka, Fa, and fu for erlotinib and gefitinib were obtained from the literature (EMEA, http://www.emea.europa.eu/humandocs/PDFs/EPAR/tarceva/H-618-PI-en.pdf; Siegel-Lakhai et al., 2005; Frohna et al., 2006; Li et al., 2006; Rudin et al., 2008; Schaiquevich et al., 2008). Qh was assumed to be 1610 ml/min (Ito et al., 2004).

Calculation of AUCi/AUC Ratio.

The magnitudes of inhibitory interactions of erlotinib and gefitinib were estimated as the ratio of the area under the plasma concentration-time curve in the presence and absence of the inhibitor (AUCi/AUC). This ratio was predicted by using the formula (eq. 9) for oral administration of a high hepatic clearance drug (Ito et al., 2004): Embedded Image where AUCi and AUC are the AUC in the presence and absence of inhibitor, respectively; Ki is inhibitor constant (obtained from in vitro experiments); fm is the fraction metabolized by the inhibited enzyme; and [I] is the inhibitor concentration at the enzyme active site.

In view of the general assumption that only unbound drug is available for interaction with the enzyme active site, and the consideration that the aim of DDI research is to exclude the highest risk, in this study we used the maximum unbound hepatic input concentration ([I]in,u) as the inhibitor concentration at the active site of the UGTs, with the exception of UGT1A7. Because UGT1A7 is present only in the esophagus, stomach, and lung (Strassburg et al., 1997), we used the maximum unbound systemic plasma concentration after repeated oral administration ([I]max,u) as the inhibitor concentration at the enzyme active site in extrahepatic tissues. Because the fraction metabolized by the UGT isoforms inhibited by erlotinib or gefitinib (fm) of the coadministered drug is unknown, we arbitrarily selected 0.1 to 1 to calculate the AUCi/AUC ratio.

Results

Inhibition of UGT Activities by Erlotinib and Gefitinib in Recombinant Human UGTs.

The IC50 values of the inhibitors of each UGT isoform were comparable with previously published data (Uchaipichat et al., 2004, 2006a). No glucuronidation of erlotinib and gefitinib was found in the course of the incubations.

As shown as Fig. 1, erlotinib (100 μM) inhibited UGT1A1 activity, reducing 4-MU glucuronidation by 88.3% (P < 0.01). The inhibition by erlotinib was also observed against UGT1A3, UGT2B7, UGT1A9, UGT1A7, and UGT2B15, reducing 4-MU glucuronidation activities by 42.3, 32.8, 31.9, 27.4, and 18.1% at 100 μM, respectively.

Fig. 1.
Fig. 1.

The inhibition of erlotinib and gefitinib on recombinant UGT activities. 4-MU or imipramine were incubated with pooled HLMs (0.5 mg protein/ml) or recombinant UGTs (0.5 mg protein/ml) at 37°C in the absence and presence of erlotinib (100 μM) or gefitinib (100 μM), respectively. Data represent the mean of triplicate or quadruplicate determination.

Likewise, gefitinib had an inhibitory effect against UGT1A1 activity, reducing glucuronidation by 79.1% at 100 μM. However, it exhibited a slightly broader inhibition profile than erlotinib. At 100 μM, gefitinib inhibited the activities of UGT1A7, UGT1A9, and UGT2B7 by 61.6, 55.5, and 70.9%, respectively. The inhibition was also observed against UGT2B15 (47.9%), 1A4 (39.8%), and 1A3 (18.8%) at 100 μM. In addition, erlotinib and gefitinib exhibited a stimulation of UGT1A4 and UGT2B17 catalytic activity by 67.3 and 81.5% at 100 μM, respectively.

Inhibition Kinetic Analysis in Recombinant UGTs.

Kinetic experiments were performed to further characterize the inhibition of UGT activities by erlotinib and gefitinib. Erlotinib and gefitinib strongly inhibited the formation of 4-MUG by UGT1A1. The representative Lineweaver-Burk plots for the inhibition of 4-MUG formation by erlotinib and gefitinib (Figs. 2A and 3A) and analysis of the parameters of the enzyme inhibition model suggested that the inhibition types were competitive. Based on nonlinear regression analysis and Dixon plots presented in Figs. 2B and 3B, erlotinib and gefitinib showed competitive inhibition against the formation of 4-MUG with Ki of 0.64 ± 0.06 and 2.42 ± 0.31 μM in recombinant UGT1A1, respectively.

Fig. 2.
Fig. 2.

Representative Lineweaver-Burk plots (A) and Dixon plots (B) of the effects of erlotinib on 4-MU glucuronide formation in recombinant UGT1A1. Reactions were performed as described under Materials and Methods. All data points shown represent the mean of duplicate measurements.

Fig. 3.
Fig. 3.

Representative Lineweaver-Burk plots and Dixon plots of the effects of gefitinib on 4-MU glucuronide formation in recombinant UGT1A1 (A and B), UGT1A7 (C and D), UGT1A9 (E and F), and UGT2B7 (G and H). Reactions were performed as described under Materials and Methods. All data points shown represent the mean of duplicate measurements.

Gefitinib was found to be a strong competitive inhibitor of UGT1A7 with a Ki of 5.11 ± 0.43 μM (Fig. 3, C and D). It also exerted intermediate mixed inhibition against UGT1A9 with Ki of 1.41 ± 0.16 μM and αKi of 44.10 ± 1.55 μM (Fig. 3, E and F), as well as intermediate competitive inhibition against UGT2B7 with Ki of 39.48 ± 4.17 μM (Fig. 3, G and H).

Inhibition of Bilirubin Glucuronidation Activity by Erlotinib and Gefitinib in HLMs.

The kinetic studies were first performed by using pooled HLMs. The apparent kinetic parameters Km and Vmax of bilirubin glucuronidation were estimated to be 1.11 ± 0.25 μM and 460.20 ± 22.57 pmol/min/mg protein, respectively.

Inhibition experiments were then conducted in HLMs. The IC50 value of indinavir was 110.6 μM, which is comparable with previously published data (Zhang et al., 2005). Erlotinib exhibited potent inhibition against bilirubin glucuronidation with an IC50 of 4.19 ± 0.24 μM at a bilirubin concentration of 1 μM. Further kinetic experiments showed mixed inhibition by erlotinib. The Ki was 2.97 ± 1.09 μM, and αKi, a measure of the affinity of enzyme-substrate complex for I, was 7.78 μM. However, the effect of gefitinib was surprisingly found to be much weaker than that of erlotinib, and the IC50 was more than 100 μM (Fig. 4).

Fig. 4.
Fig. 4.

Kinetics of bilirubin glucuronidation in HLMs (A), the inhibition of erlotinib and gefitinib against bilirubin (1 μM) glucuronidation in HLMs (B), and representative Lineweaver-Burk plots and Dixon plots of the effects of erlotinib on bilirubin glucuronides formation in HLMs (C and D). Reactions were performed as described under Materials and Methods. All data points shown represent the mean of duplicate measurements.

The Calculated Concentrations of Erlotinib and Gefitinib in Blood.

The oral doses and pharmacokinetic parameters of erlotinib and gefitinib were obtained from previous publications (EMEA, http://www.emea.europa.eu/humandocs/PDFs/EPAR/tarceva/H-618-PI-en.pdf; Ito et al., 2004; Siegel-Lakhai et al., 2005; Frohna et al., 2006; Li et al., 2006; Rudin et al., 2008; Schaiquevich et al., 2008). The calculated blood concentrations of erlotinib and gefitinib after oral administration are listed in Table 1. Comparison of the calculated [I]max with the reported Cmax after administration of oral doses of erlotinib or gefitinib showed that the [I]max values fell within the reported concentration range, with the exception of the [I]max at 700 mg/day gefitinib, which is higher than expected.

View this table:
TABLE 1

Calculation of possible concentrations of erlotinib and gefitinib

Quantitative Prediction of DDI Risk (AUCi/AUC).

The magnitudes of the potential inhibitory interactions of erlotinib and gefitinib with UGTs were evaluated by estimating the ratio of the AUC in the presence and absence of the inhibitor (AUCi/AUC).

The calculated results were shown as AUC ratio isolines plotted against fm by UGT isoform and oral doses of erlotinib or gefitinib in Fig. 5. When the dose of erlotinib is more than 140 mg/day and the fm of coadministered drug metabolized by UGT1A1 is more than 0.9, the AUC of coadministered drug will increase more than 40%; when the dose is more than 110 mg/day and the fm is more than 0.8, or the dose is more than 100 mg/day and the fm is 1, AUC will increase more than 30%. However, for gefitinib, even when administered at the highest dose (700 mg/day) and an fm of 1, the AUC ratio is less than 1.3 for the substrates of each UGT isoform inhibited.

Fig. 5.
Fig. 5.

Isolines plots for relationship of AUC ratio against oral dose of erlotinib and fm by UGT1A1 (A), as well as the relationship of AUC ratio against oral dose of gefitinib and fm by UGT1A1 (B), UGT1A7 (C), UGT1A9 (D), or UGT2B7 (E) for DDI study.

In this study, we assumed the fm of bilirubin is 1, because UGT1A1 is the dominant isoform involved in bilirubin glucuronidation. When the oral dose of erlotinib is 150 mg/day, the AUC of bilirubin will increase more than 10%.

Discussion

Our data offer in vitro evidence that erlotinib is a potent competitive inhibitor of UGT1A1. UGT1A1 inhibition is also the mechanism by which atazanavir and indinavir cause hyperbilirubinemia (Zhang et al., 2005). Reduced glucuronidation rates are associated with the risk for severe toxicity during irinotecan treatment (Innocenti et al., 2004). Because the reduction in UGT1A1 activity varies depending on the substrate and incubation system (Udomuksorn et al., 2007), we also investigated the effects of erlotinib and gefitinib on bilirubin glucuronidation in HLMs in the current study. The inhibition potential is similar to that of atazanavir, but it is much more potent than that of indinavir. It is consistent with the observation that erlotinib can cause hyperbilirubinemia (Jakacki et al., 2008), similar to that observed with atazanavir and indinavir (Zhang et al., 2005). Therefore, inhibition of UGT1A1 activity by erlotinib can decrease the conjugation of bilirubin, which can be clinically significant.

UGT1A1 is also responsible for the metabolism of several other endogenous and exogenous substrates, including 15% drugs that have glucuronidation as a clearance mechanism of the top 200 drugs in the United States in 2002 (Williams et al., 2004). The inhibition of UGT1A1 is particularly important if a drug has a narrow therapeutic index, such as etoposide and irinotecan (Kawato et al., 1991; Wen et al., 2007). It is interesting to note that gefitinib exhibited very weak inhibition against bilirubin glucuronidation in HLMs, although it potently inhibited 4-MU glucuronidation in recombinant UGT1A1. This finding confirms the observation that the reduction of UGT1A1 activity might vary with the substrate (Udomuksorn et al., 2007), and it also offers new experimental evidence for the opinion that UGT1A1 has two or more binding sites for xenobiotics and endobiotics (Rios and Tephly, 2002).

In contrast with erlotinib, gefitinib exhibited competitive inhibition against not only UGT1A1 but also UGT1A7, UGT1A9, and UGT2B7. The latter enzyme is involved in the metabolism of 35% drugs involved in glucuronidation of the top 200 prescribed drugs in the United States in 2002 (Williams et al., 2004). UGT1A9 and UGT1A7 are also involved in the glucuronidation of a number of drugs (Kiang et al., 2005). In addition, UGT1A1, UGT1A9, and UGT2B7 are expressed in both human liver and some extrahepatic tissues including the gastrointestinal tract, whereas UGT1A7 is present only in the esophagus, stomach, and lung (Kiang et al., 2005). UGTs in the gastrointestinal tract may contribute significantly to the first-pass metabolism of orally administered drugs that undergo glucuronidation. Our results showed that gefitinib might affect the glucuronidation and first-pass metabolism of more orally administered drugs than erlotinib.

We observed an increase of UGT1A4 activity in the presence of erlotinib and UGT2B17 activity in the presence of gefitinib. Enzyme activation is not an uncommon event in enzymology; however, the underlying mechanisms are unclear. One possibility is the existence of the enzyme in multimeric form, so that the binding of one molecule to one subunit may increase the affinity of the other subunit(s) for another molecule. Williams et al. (2002) found that UGT1A1-catalyzed estradiol-3-glucuronidation is stimulated by 17α-ethynylestradiol, anthraflavic acid, and 2-amino-1-methyl- 6-phenylimidazo [4,5-b] pyridine in human liver microsomes. They also proved that the observed activation is not an artifact of the in vitro systems examined but true behaviors of UGT1A1 at least in vitro (Williams et al., 2002). However, further studies will need to be performed to evaluate whether this in vitro phenomenon also occurs in vivo.

The quantitative prediction of DDI risk indicated that the coadministration of erlotinib at clinical doses could result in a significant increase of AUC of drugs primarily cleared by UGT1A1, suggesting that erlotinib can induce clinically significant DDI with coadministered UGT1A1 substrates. It is noteworthy that our calculation is based only on the inhibition of hepatic UGT1A1 by erlotinib, and abundant extrahepatic UGT1A1 exists. In addition, the pharmacokinetic parameters used to calculate concentrations are mean values of the parameters reported, but interindividual variability is high. The diplotype of two polymorphic loci in the ABCG2 promoter involving −15622C/T and 1143C/T is associated with higher Cmax of erlotinib (Rudin et al., 2008). Furthermore, the bioavailability of erlotinib can be increased to almost 100% with the intake of food (Smith, 2005). Moreover, in vitro data tend to underestimate inhibition of drug glucuronidation in vivo (Uchaipichat et al., 2006a). As a result, the actual effects of erlotinib might be more potent than those calculated here, in particular, for some individuals with higher blood concentrations of erlotinib.

Compared with erlotinib, gefitinib may significantly affect a drug that is metabolized by a single UGT and is a high-capacity, low-affinity substrate. However, the majority of UGT substrates are glucuronidated by multiple UGT enzymes (Williams et al., 2004), thereby making it difficult to determine whether interactions arising from multiple UGTs inhibition by gefitinib exist when using in vitro inhibition data.

It is noteworthy that although there are a few studies predicting the magnitude of inhibitory drug interactions in vivo (Zhang et al., 2005; Uchaipichat et al., 2006b), the extrapolation from in vitro data to in vivo drug interactions should be taken with caution. Protein binding, active uptake, and efflux transporters in tissues may affect the estimation of unbound drug concentrations at the interaction site. In addition, because the lipid composition varies between membranes from different sources and is a determinant of membrane fluidity, thickness, shape, surface curvature, and the ability to form lipid rafts, it is conceivable that the kinetic properties of single recombinant UGT may be different (Miners et al., 2006). Another potential problem is that the enzymatically generated glucuronidated products are eliminated in vivo, but this does not occur in an in vitro incubation system, and the accumulation of products in the reaction medium may inhibit enzyme activity (Kiang et al., 2005). Furthermore, many studies have indicated UGTs are oligomeric enzymes. Although there are some evidences and experimental data showing that hetero-oligomerization could not be the major reason or basis for the complex substrate specificity of most UGTs (Finel and Kurkela, 2008), hetero-oligomerization might somewhat affect substrate specificity or the region selectivity of the tested enzyme. Moreover, UGT1A1, UGT1A7, UGT1A9, and UGT2B7 are also expressed in gastrointestinal tract, and drug-drug interactions via inhibition of these UGTs may also take place in the small intestine. The concentrations of substrates in the intestine may differ from the concentrations used here to predict the AUC ratio.

The present findings shed light on the mechanisms underlying clinically significant DDI associated with erlotinib, and they also provide the basis for further clinical studies investigating the DDI potential between tyrosine kinase inhibitors with UGT substrates, such as etoposide and SN-38 (the active metabolite of irinotecan).

Acknowledgments.

We thank Dr. R. Stephanie Huang for valuable suggestions.

Footnotes

  • This work was supported by the National Institutes of Health National Institute of General Medical Sciences [Grant UO1-GM61393] (Pharmacogenetics of Anticancer Agents Research Group).

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

    doi:10.1124/dmd.109.029660

  • DDI
    drug-drug interactions
    UGT
    UDP-glucuronosyltransferase
    HLMs
    human liver microsomes
    AUC
    area under the curve
    OSI-774
    N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine
    4-MU
    4-methylumbelliferone
    4-MUG
    4-methylumbelliferone-d-glucuronide
    UDPGA
    uridine 5′-diphosphoglucuronic acid
    HPLC
    high-performance liquid chromatography.

    • Received July 22, 2009.
    • Accepted October 21, 2009.

References

  1. ↵
    1. Bergman E,
    2. Forsell P,
    3. Persson EM,
    4. Knutson L,
    5. Dickinson P,
    6. Smith R,
    7. Swaisland H,
    8. Farmer MR,
    9. Cantarini MV,
    10. Lennernäs H
    (2007) Pharmacokinetics of gefitinib in humans: the influence of gastrointestinal factors. Int J Pharm 341:134–142.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Copeland RA
    (2000) Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis. Wiley-VCH, New York.
  3. ↵
    1. Czito BG,
    2. Willett CG,
    3. Bendell JC,
    4. Morse MA,
    5. Tyler DS,
    6. Fernando NH,
    7. Mantyh CR,
    8. Blobe GC,
    9. Honeycutt W,
    10. Yu D,
    11. et al
    . (2006) Increased toxicity with gefitinib, capecitabine, and radiation therapy in pancreatic and rectal cancer: phase I trial results. J Clin Oncol 24:656–662.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Finel M,
    2. Kurkela M
    (2008) The UDP-glucuronosyltransferases as oligomeric enzymes. Curr Drug Metab 9:70–76.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Frohna P,
    2. Lu J,
    3. Eppler S,
    4. Hamilton M,
    5. Wolf J,
    6. Rakhit A,
    7. Ling J,
    8. Kenkare-Mitra SR,
    9. Lum BL
    (2006) Evaluation of the absolute oral bioavailability and bioequivalence of erlotinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in a randomized, crossover study in healthy subjects. J Clin Pharmacol 46:282–290.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Grenader T,
    2. Gipps M,
    3. Shavit L,
    4. Gabizon A
    (2007) Significant drug interaction: phenytoin toxicity due to erlotinib. Lung Cancer 57:404–406.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Innocenti F,
    2. Undevia SD,
    3. Iyer L,
    4. Chen PX,
    5. Das S,
    6. Kocherginsky M,
    7. Karrison T,
    8. Janisch L,
    9. Ramírez J,
    10. Rudin CM,
    11. et al
    . (2004) Genetic variants in the UDP-glucuronosyltransferase 1A1 gene predict the risk of severe neutropenia of irinotecan. J Clin Oncol 22:1382–1388.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Ito K,
    2. Brown HS,
    3. Houston JB
    (2004) Database analyses for the prediction of in vivo drug-drug interactions from in vitro data. Br J Clin Pharmacol 57:473–486.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Jakacki RI,
    2. Hamilton M,
    3. Gilbertson RJ,
    4. Blaney SM,
    5. Tersak J,
    6. Krailo MD,
    7. Ingle AM,
    8. Voss SD,
    9. Dancey JE,
    10. Adamson PC
    (2008) Pediatric phase I and pharmacokinetic study of erlotinib followed by the combination of erlotinib and temozolomide: a Children's Oncology Group Phase I Consortium Study. J Clin Oncol 26:4921–4927.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Kawato Y,
    2. Aonuma M,
    3. Hirota Y,
    4. Kuga H,
    5. Sato K
    (1991) Intracellular roles of SN-38, a metabolite of the camptothecin derivative CPT-11, in the antitumor effect of CPT-11. Cancer Res 51:4187–4191.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Kiang TK,
    2. Ensom MH,
    3. Chang TK
    (2005) UDP-glucuronosyltransferases and clinical drug-drug interactions. Pharmacol Ther 106:97–132.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Li J,
    2. Brahmer J,
    3. Messersmith W,
    4. Hidalgo M,
    5. Baker SD
    (2006) Binding of gefitinib, an inhibitor of epidermal growth factor receptor-tyrosine kinase, to plasma proteins and blood cells: in vitro and in cancer patients. Invest New Drugs 24:291–297.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Li J,
    2. Zhao M,
    3. He P,
    4. Hidalgo M,
    5. Baker SD
    (2007) Differential metabolism of gefitinib and erlotinib by human cytochrome P450 enzymes. Clin Cancer Res 13:3731–3737.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Mackenzie PI,
    2. Bock KW,
    3. Burchell B,
    4. Guillemette C,
    5. Ikushiro S,
    6. Iyanagi T,
    7. Miners JO,
    8. Owens IS,
    9. Nebert DW
    (2005) Nomenclature update for the mammalian UDP glycosyltransferase (UGT) gene superfamily. Pharmacogenet Genomics 15:677–685.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Milton DT,
    2. Riely GJ,
    3. Azzoli CG,
    4. Gomez JE,
    5. Heelan RT,
    6. Kris MG,
    7. Krug LM,
    8. Pao W,
    9. Pizzo B,
    10. Rizvi NA,
    11. et al
    . (2007) Phase 1 trial of everolimus and gefitinib in patients with advanced nonsmall-cell lung cancer. Cancer 110:599–605.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Miners JO,
    2. Knights KM,
    3. Houston JB,
    4. Mackenzie PI
    (2006) In vitro-in vivo correlation for drugs and other compounds eliminated by glucuronidation in humans: pitfalls and promises. Biochem Pharmacol 71:1531–1539.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Nakajima M,
    2. Tanaka E,
    3. Kobayashi T,
    4. Ohashi N,
    5. Kume T,
    6. Yokoi T
    (2002) Imipramine N-glucuronidation in human liver microsomes: biphasic kinetics and characterization of UDP-glucuronosyltransferase isoforms. Drug Metab Dispos 30:636–642.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Onoda S,
    2. Mitsufuji H,
    3. Yanase N,
    4. Ryuge S,
    5. Kato E,
    6. Wada M,
    7. Ishii K,
    8. Hagiri S,
    9. Yamamoto M,
    10. Yokoba M,
    11. et al
    . (2005) Drug interaction between gefitinib and warfarin. Jpn J Clin Oncol 35:478–482.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Patnaik A,
    2. Wood D,
    3. Tolcher AW,
    4. Hamilton M,
    5. Kreisberg JI,
    6. Hammond LA,
    7. Schwartz G,
    8. Beeram M,
    9. Hidalgo M,
    10. Mita MM,
    11. et al
    . (2006) Phase I, pharmacokinetic, and biological study of erlotinib in combination with paclitaxel and carboplatin in patients with advanced solid tumors. Clin Cancer Res 12:7406–7413.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Ranson M,
    2. Hammond LA,
    3. Ferry D,
    4. Kris M,
    5. Tullo A,
    6. Murray PI,
    7. Miller V,
    8. Averbuch S,
    9. Ochs J,
    10. Morris C,
    11. et al
    . (2002) ZD1839, a selective oral epidermal growth factor receptor-tyrosine kinase inhibitor, is well tolerated and active in patients with solid, malignant tumors: results of a phase I trial. J Clin Oncol 20:2240–2250.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Rios GR,
    2. Tephly TR
    (2002) Inhibition and active sites of UDP-glucuronosyltransferases 2B7 and 1A1. Drug Metab Dispos 30:1364–1367.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Rudin CM,
    2. Liu W,
    3. Desai A,
    4. Karrison T,
    5. Jiang X,
    6. Janisch L,
    7. Das S,
    8. Ramirez J,
    9. Poonkuzhali B,
    10. Schuetz E,
    11. et al
    . (2008) Pharmacogenomic and pharmacokinetic determinants of erlotinib toxicity. J Clin Oncol 26:1119–1127.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Schaiquevich P,
    2. Panetta JC,
    3. Throm S,
    4. Daw NC,
    5. Geyer JR,
    6. Furman WL,
    7. Stewart CF
    (2008) Population pharmacokinetic (PK) analysis of gefitinib in pediatric cancer patients (Meeting Abstracts). J Clin Oncol 26:2523.
    OpenUrl
  24. ↵
    1. Sequist LV,
    2. Lynch TJ
    (2008) EGFR tyrosine kinase inhibitors in lung cancer: an evolving story. Annu Rev Med 59:429–442.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Siegel-Lakhai WS,
    2. Beijnen JH,
    3. Schellens JH
    (2005) Current knowledge and future directions of the selective epidermal growth factor receptor inhibitors erlotinib (Tarceva) and gefitinib (Iressa). Oncologist 10:579–589.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Smith J
    (2005) Erlotinib: small-molecule targeted therapy in the treatment of non-small-cell lung cancer. Clin Ther 27:1513–1534.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Strassburg CP,
    2. Oldhafer K,
    3. Manns MP,
    4. Tukey RH
    (1997) Differential expression of the UGT1A locus in human liver, biliary, and gastric tissue: identification of UGT1A7 and UGT1A10 transcripts in extrahepatic tissue. Mol Pharmacol 52:212–220.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Swaisland HC,
    2. Cantarini MV,
    3. Fuhr R,
    4. Holt A
    (2006) Exploring the relationship between expression of cytochrome P450 enzymes and gefitinib pharmacokinetics. Clin Pharmacokinet 45:633–644.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Swaisland HC,
    2. Ranson M,
    3. Smith RP,
    4. Leadbetter J,
    5. Laight A,
    6. McKillop D,
    7. Wild MJ
    (2005) Pharmacokinetic drug interactions of gefitinib with rifampicin, itraconazole and metoprolol. Clin Pharmacokinet 44:1067–1081.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Uchaipichat V,
    2. Mackenzie PI,
    3. Elliot DJ,
    4. Miners JO
    (2006a) Selectivity of substrate (trifluoperazine) and inhibitor (amitriptyline, androsterone, canrenoic acid, hecogenin, phenylbutazone, quinidine, quinine, and sulfinpyrazone) “probes” for human UDP-glucuronosyltransferases. Drug Metab Dispos 34:449–456.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Uchaipichat V,
    2. Mackenzie PI,
    3. Guo XH,
    4. Gardner-Stephen D,
    5. Galetin A,
    6. Houston JB,
    7. Miners JO
    (2004) Human UDP-glucuronosyltransferases: isoform selectivity and kinetics of 4-methylumbelliferone and 1-naphthol glucuronidation, effects of organic solvents, and inhibition by diclofenac and probenecid. Drug Metab Dispos 32:413–423.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Uchaipichat V,
    2. Winner LK,
    3. Mackenzie PI,
    4. Elliot DJ,
    5. Williams JA,
    6. Miners JO
    (2006b) Quantitative prediction of in vivo inhibitory interactions involving glucuronidated drugs from in vitro data: the effect of fluconazole on zidovudine glucuronidation. Br J Clin Pharmacol 61:427–439.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Udomuksorn W,
    2. Elliot DJ,
    3. Lewis BC,
    4. Mackenzie PI,
    5. Yoovathaworn K,
    6. Miners JO
    (2007) Influence of mutations associated with Gilbert and Crigler-Najjar type II syndromes on the glucuronidation kinetics of bilirubin and other UDP-glucuronosyltransferase 1A substrates. Pharmacogenet Genomics 17:1017–1029.
    OpenUrlPubMed
  34. ↵
    1. Veronese ML,
    2. Sun W,
    3. Giantonio B,
    4. Berlin J,
    5. Shults J,
    6. Davis L,
    7. Haller DG,
    8. O'Dwyer PJ
    (2005) A phase II trial of gefitinib with 5-fluorouracil, leucovorin, and irinotecan in patients with colorectal cancer. Br J Cancer 92:1846–1849.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Wen Z,
    2. Tallman MN,
    3. Ali SY,
    4. Smith PC
    (2007) UDP-glucuronosyltransferase 1A1 is the principal enzyme responsible for etoposide glucuronidation in human liver and intestinal microsomes: structural characterization of phenolic and alcoholic glucuronides of etoposide and estimation of enzyme kinetics. Drug Metab Dispos 35:371–380.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Williams JA,
    2. Hyland R,
    3. Jones BC,
    4. Smith DA,
    5. Hurst S,
    6. Goosen TC,
    7. Peterkin V,
    8. Koup JR,
    9. Ball SE
    (2004) Drug-drug interactions for UDP-glucuronosyltransferase substrates: a pharmacokinetic explanation for typically observed low exposure (AUCi/AUC) ratios. Drug Metab Dispos 32:1201–1208.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    1. Williams JA,
    2. Ring BJ,
    3. Cantrell VE,
    4. Campanale K,
    5. Jones DR,
    6. Hall SD,
    7. Wrighton SA
    (2002) Differential modulation of UDP-glucuronosyltransferase 1A1 (UGT1A1)-catalyzed estradiol-3-glucuronidation by the addition of UGT1A1 substrates and other compounds to human liver microsomes. Drug Metab Dispos 30:1266–1273.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Yamamoto N,
    2. Horiike A,
    3. Fujisaka Y,
    4. Murakami H,
    5. Shimoyama T,
    6. Yamada Y,
    7. Tamura T
    (2008) Phase I dose-finding and pharmacokinetic study of the oral epidermal growth factor receptor tyrosine kinase inhibitor Ro50–8231 (erlotinib) in Japanese patients with solid tumors. Cancer Chemother Pharmacol 61:489–496.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Zhang D,
    2. Chando TJ,
    3. Everett DW,
    4. Patten CJ,
    5. Dehal SS,
    6. Humphreys WG
    (2005) In vitro inhibition of UDP glucuronosyltransferases by atazanavir and other HIV protease inhibitors and the relationship of this property to in vivo bilirubin glucuronidation. Drug Metab Dispos 33:1729–1739.
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Download PDF
Article Alerts
Email Article
Citation Tools

Research ArticleArticle

Comparison of the Drug-Drug Interactions Potential of Erlotinib and Gefitinib via Inhibition of UDP-Glucuronosyltransferases

Yong Liu, Jacqueline Ramírez, Larry House and Mark J. Ratain
Drug Metabolism and Disposition January 1, 2010, 38 (1) 32-39; DOI: https://doi.org/10.1124/dmd.109.029660
Reddit logo Twitter logo Facebook logo Mendeley logo

Jump to section

Advertisement