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Clinical Cancer Research Vol. 11, 6699-6704, September 15, 2005
© 2005 American Association for Cancer Research

Cancer Therapy: Preclinical

Effects of Ketoconazole on Glucuronidation by UDP-Glucuronosyltransferase Enzymes

Wei Peng Yong1, Jacqueline Ramirez2, Federico Innocenti1,2,3 and Mark J. Ratain1,2,3

Authors' Affiliations: 1 Committee on Clinical Pharmacology and Pharmacogenomics, 2 Department of Medicine, and 3 Cancer Research Center, The University of Chicago, Chicago, Illinois

Requests for reprints: Mark J. Ratain, The University of Chicago, 5841 South Maryland Avenue, MC2115, Chicago, IL 60637. Phone: 773-702-4400; Fax: 773-702-3969; E-mail: mratain{at}medicine.bsd.uchicago.edu.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Ketoconazole has been shown to inhibit the glucuronidation of the UGT2B7 substrates zidovudine and lorazepam. Its effect on UGT1A substrates is unclear. A recent study found that coadministration of irinotecan and ketoconazole led to a significant increase in the formation of SN-38 (7-ethyl-10-hydroxycamptothecine), an UGT1A substrate. This study investigates whether ketoconazole contributes to the increase in SN-38 formation by inhibiting SN-38 glucuronidation.

Experimental Design: SN-38 glucuronidation activities were determined by measuring the rate of SN-38 glucuronide (SN-38G) formation using pooled human liver microsomes and cDNA-expressed UGT1A isoforms (1A1, 1A7 and 1A9) in the presence of ketoconazole. Indinavir, a known UGT1A1 inhibitor, was used as a positive control. SN-38G formation was measured by high-performance liquid chromatograph.

Results: Ketoconazole competitively inhibited SN-38 glucuronidation. Among the UGT1A isoforms screened, ketoconazole showed the highest inhibitory effect on UGT1A1 and UGT1A9. The Ki values were 3.3 ± 0.8 µmol/L for UGT1A1 and 31.9 ± 3.3 µmol/L for UGT1A9.

Conclusions: These results show that ketoconazole is a potent UGT1A1 inhibitor, which seems the basis for increased exposure to SN-38 when coadministered with irinotecan.

Irinotecan was approved for the first-line treatment of metastatic colorectal cancer in combination with 5-flurouracil and leucovorin following the positive results of two large randomized control trials showing improvement in treatment survival (1, 2). Irinotecan has a relatively complex metabolic pathway. It is converted to its active metabolite, SN-38 (7-ethyl-10-hydroxycamptothecin), predominantly by carboxylesterase 2 in the liver (3, 4). Irinotecan undergoes oxidative biotransformation by CYP3A4 and CYP3A5 to several inactive metabolites (5, 6). SN-38, in turn, is detoxified primarily by UGT1A1 to SN-38 glucuronide (SN-38G; refs. 7–10). UGT1A9 may also have a minor role in the hepatic clearance of SN-38, whereas UGT1A7 may be important in the extrahepatic formation of SN-38G (9, 10). Functional polymorphisms in UGT1A1 have been associated with reduced catalytic efficiency and patients with UGT1A1*28 and –3156G>A variants are more likely to experience treatment related diarrhea or leucopenia when treated with irinotecan (11, 12).

Ketoconazole is a widely used antifungal agent well known for its inhibitory effect on CYP3A4 and the ABCB1 transporter (13, 14). A recent study by Kehrer et al. showed that coadministration of irinotecan and ketoconazole led to a significant increase in the relative formation of SN-38 (15). Such increase was attributed to a reduction in irinotecan clearance secondary to the inhibitory effect of ketoconazole on CYP3A enzymes, although a reduction of irinotecan clearance was not observed in the study. An alternative hypothesis would be that ketoconazole inhibited SN-38 glucuronidation resulting in an increase in SN-38 AUC.

Ketoconazole has also been shown to inhibit the glucuronidation of UGT2B7 substrates zidovudine and lorazepam in in vitro studies (16–18). A recent study by Satoh et al. also showed that ketoconazole inhibited UGT1A1-mediated glucuronidation of estradiol using human liver microsomes (19). In this study, we investigated the inhibitory effects of ketoconazole on SN-38 glucuronidation in vitro using pooled human liver microsomes, cDNA-expressed isoforms of UGT1A, and human hepatocytes.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and reagents. Irinotecan and SN-38 were kindly provided by Dr. Kiyoshi Terada (Yakult Honsha Co., Ltd., Japan). Indinavir was obtained from Merck & Co., Inc. (Whitehouse Station, NJ). Fluconazole was obtained from Pfizer (New York, NY). Radiolabeled uridine diphosphoglucuronic acid (14C-UDPGA) was purchased from ICN Biomedicals, Inc. (Irvine, CA). Ketoconazole, cyclosporine A, bilirubin, L-{alpha}-phosphatidylcholine, saccharolactone (D-saccharic acid 1,4-lactone), camptothecin, dihydrocarbamazepine, and UDPGA were all obtained from Sigma-Aldrich (St. Louis, MO). All other reagents and high-performance liquid chromatograph solvents were of the highest quality commercially available.

Human hepatocytes in a 12-well collagen culture plate, from a single donor, were purchased from In Vitro Technologies (Baltimore, MD). Human recombinant uridine diphosphate glucoronosyltransferases (UGT1A1, UGT1A7, and UGT1A9) produced using baculovirus (Autographa californica)–transfected insect cells (BTI-TN-5B1-4) were purchased from Gentest (Woburn, MA).

Human liver microsome preparation. Human liver samples from 10 separate donors were obtained from the Liver Tissue Procurement and Distribution System (NIH NO1-DK-9-2310, Pittsburgh, PA). Microsomes were prepared by differential centrifugation methods (20). Total protein content in microsomes was determined by the Bradford method using bovine serum albumin as the standard (21).

In vitro studies with microsomes and cDNA-expressed UGT1A isoforms. The IC50 values for inhibition of SN-38 glucuronidation by ketoconazole and indinavir, a known UGT1A1 inhibitor (22), were determined using human liver microsomes. The concentrations of ketoconazole and indinavir ranged from 100 nmol/L to 500 µmol/L and from 100 nmol/L to 1 mmol/L, respectively. The incubation mixture (200 µL) contained SN-38 (5 µmol/L), MgCl2 (10 mmol/L), Tris-HCl buffer at pH 7.4 (25 µmol/L), and microsomal protein (1 mg/mL) with appropriate inhibitors. After preincubation at 37°C for 10 minutes, the reaction was started with the addition of UDPGA (5 mmol/L). Reaction mixtures were incubated for 30 minutes, after which 400 µL of cold methanol were added to stop the reaction. Fifty microliters of 2 µmol/L irinotecan were added as internal standard. After centrifugation at 13,000 x g for 30 minutes (4°C), the supernatant was analyzed by high-performance liquid chromatograph. The rate of SN-38 glucuronidation was linear with respect to incubation time up to 90 minutes and microsomal protein concentrations up to 2 mg/mL (8).

To select UGT1A isoforms for additional inhibition studies, the inhibition of SN-38 glucuronidation by UGT1A1, UGT1A7, and UGT1A9 was investigated using ketoconazole and indinavir at concentrations ranging from 100 nmol/L to 500 µmol/L. The Ki of ketoconazole and indinavir was determined for UGT1A1 and UGT1A9. The range of SN-38 concentrations used was 1 to 500 µmol/L and both ketoconazole and indinavir were used at 10, 25, and 50 µmol/L. The incubation conditions were similar to human liver microsome studies but cDNA-expressed UGT1A isoforms (1 mg/mL) were used instead.

The inhibitory effects of ketoconazole and indinavir on UGT1A1, UGT1A7, and UGT1A9 were compared with cyclosporine A and fluconazole using a single concentration of inhibitor (10 µmol/L for both ketoconazole and indinavir, 1 µmol/L for cyclosporine A, and 32.6 µmol/L for fluconazole). The concentrations chosen were comparable with plasma concentrations achieved at the usual clinical dosages (15, 23–25). Ketoconazole and cyclosporine A were dissolved in 100% methanol and diluted in 25 µmol/L Tris-HCl buffer to the appropriate final concentration containing 0.1% methanol, whereas both indinavir and fluconazole were reconstituted in 25 µmol/L Tris-HCl buffer. No inhibition of SN-38 glucuronidation was observed in control experiments containing 0.1% methanol but without the inhibitor. All the experiments using human liver microsomes and cDNA-expressed UGT1 isoforms were done in duplicate.

In vitro metabolism studies with human hepatocytes. Human hepatocytes from a single donor were used to evaluate the effect of ketoconazole on the formation of SN-38 and SN-38G using irinotecan as substrate. Hepatocytes were preincubated with ketoconazole (final concentration of 1.6 and 16 µmol/L, 0.1% methanol) in hepatocyte maintenance medium (Cambrex Bioscience Walkersville, Inc., Walkersville, MD) at 37°C. After 15 minutes, the medium was aspirated and replaced with medium containing irinotecan (10 or 100 µmol/L) and ketoconazole (1.6 and 16 µmol/L) in hepatocyte maintenance media at 37°C. After 45 minutes of incubation, the media were removed to stop the reaction. The concentrations of SN-38 and SN-38G in the media were analyzed by high-performance liquid chromatograph as described below. The incubation time selected was based on our previous optimization study with human liver microsomes.

High-performance liquid chromatograph analysis of SN-38G formation. The SN-38 glucuronidation assay was adapted from a previously described method (8). Twenty-microliter aliquots were injected into a high-performance liquid chromatograph assay system coupled to a fluorescence detector (Hitachi Instruments, Inc., San Jose, CA). A µBondapak C18 column (3.9 x 300 mm, 10 µm, 125 Å; Waters Corp., Milford, MA) with a Novapak guard column (4 µm, 60 Å, Waters) was used with fluorescence detection using an excitation frequency of 355 nm and an emission frequency of 515 nm. The mobile phase A consisted of 8:4:88 acetonitrile/tetrahydrofuran/0.9 mmol/L 1-heptanesulfonic acid in 50 mmol/L potassium phosphate buffer (pH 4.0) and mobile phase B was a mix of 35:65 acetonitrile/5 mmol/L 1-heptanesulfonic acid in 50 mmol/L potassium phosphate buffer (pH 4.0). Elution was done at the flow rate of 0.9 mL/min using the following gradient: 0 to 7 minutes, 100% A; 7 to 25 minutes, 100% B; and 25 to 35 minutes, 100% A. Irinotecan was used as the internal standard in studies involving human liver microsomes and recombinant UGTs, whereas camptothecin was used in the experiments with hepatocytes. The retention times for SN-38G, irinotecan, camptothecin, and SN-38 were 13.3, 15.7, 17.1, and 17.8 minutes, respectively.

Bilirubin glucuronidation assay. The bilirubin glucuronidation assay was based on a previously described method with slight modification using 14C-UDPGA and an ethyl acetate extraction to separate glucuronides from unreacted UDPGA (26). Ketoconazole (10 µmol/L) was added to examine its effect on bilirubin glucuronidation. Briefly, human liver microsomes (1 mg/mL) were incubated with bilirubin (340 µmol/L), L-{alpha}-phosphatidylcholine (0.75 mg/mL), saccharolactone (8.5 mmol/L), MgCl2 (10 mmol/L), and 14C-UDPGA (0.05 µCi) in a 0.5 mol/L Tris-HCl buffer (pH 7.4) for 60 minutes at 37°C. The reaction was stopped with 100 µL of cold 1% Triton X-100 in 0.7 mol/L glycine/HCl buffer (pH 2.2). Radiolabeled bilirubin glucuronides were extracted into 1 mL ethyl acetate. Three hundred–microliter aliquots were measured by liquid scintillation counting (Tri-Carb 4530; Packard, Meriden, CT).

Data analysis. Results were presented as mean values ± SE. All experiments were carried out in duplicate, except for the bilirubin assays, which were done in triplicate. Appropriate enzyme kinetic models were fitted to enzyme kinetic data by nonlinear regression analysis using GraphPad software version 4.01 (GraphPad Software, Inc., San Diego, CA) to derive Km and Vmax. Both the Michaelis-Menten (Eq. A) and the substrate activation models (Eq. B) were used, where [A] is defined as the concentration of substrate A and n is the number of binding sites:

(A)

(B)
The best fit was based on a number of criteria which included visual inspection of the data plots, the distribution of the residuals, the size of the sum of squared residuals, the SE of the estimates, and the F ratio test. The type of inhibition was evaluated using graphical analysis with Lineweaver-Burke plots. Initial Ki values were estimated using Dixon plots. A more accurate value of Ki was determined by fitting the kinetic data into a competitive inhibition model (Eq. C) using nonlinear regression analysis:

(C)


   Results
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 Materials and Methods
 Results
Discussion
 References
 
Effects of ketoconazole, indinavir, cyclosporine A, and fluconazole on SN-38G formation in pooled human liver microsomes. Ketoconazole and indinavir both inhibited SN-38 glucuronidation, with IC50 values of 11 and 94 µmol/L, respectively (Fig. 1). Cyclosporine A and fluconazole showed minimal inhibitory effect (3.2% and 6.3%, respectively) on the formation of SN-38G by human liver microsomes.



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  		Fig. 1. Effects of increasing concentrations of (A) ketoconazole and (B) indinavir on SN-38 glucuronidation in pooled human liver microsomes after 30 minutes of incubation. Points, means of a duplicate experiment. % Control UGT1A activity (SN-38 glucuronidation activity in samples treated with inhibitor/SN-38 glucuronidation activity in control samples).

 
Effects of ketoconazole, indinavir, cyclosporine A, and fluconazole on SN-38G formation in cDNA-expressed isoforms. The effects of ketoconazole and indinavir on SN-38 glucuronidation were tested on cDNA-expressed UGT1A1, UGT1A7, and UGT1A9 (Fig 2). Ketoconazole showed the highest inhibitory effect on UGT1A1 and UGT1A9. At the concentration of 10 µmol/L, it reduced SN-38 glucuronidation of both UGT isoforms by ~35%. Indinavir, at 10 µmol/L, inhibited all the UGT1A isoforms tested showing the highest inhibitory effect on UGT1A9.



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  		Fig. 2. Effects of (A) ketoconazole and (B) indinavir (concentration range of 100 nmol/L to 500 µmol/L for both ketoconazole and indinavir) on SN-38 glucuronidation by cDNA-expressed UGT1A1, UGT1A7, and UGT1A9.

 
As ketoconazole exhibited no inhibitory effect on UGT1A7 within the range of concentrations studied (up to 500 µmol/L), only UGT1A1 and UGT1A9 were selected for further kinetic studies. The kinetics of SN-38 glucuronidation by UGT1A9 fit a Michaelis-Menten model, whereas UGT1A1 exhibits a substrate activation model. The Km, Vmax, and Vmax/Km for UGT1A1 were 33.5 ± 2.8 µmol/L, 204.3 ± 7.4 pmol/min/mg protein, and 6.1 µL/min/mg protein, and for UGT1A9 were 39.6 ± 1.3 µmol/L, 29.6 ± 0.3 pmol/min/mg protein, and 0.75 µL/min/mg protein, respectively. A Lineweaver-Burke plot was used to evaluate the mechanism of inhibition of SN-38 glucuronidation by ketoconazole. Ketoconazole competitively inhibited SN-38 glucuronidation by UGT1A1 and UGT1A9 (Fig. 3). The Ki values were estimated to be 3.3 ± 1.1 µmol/L for UGT1A1 and 31.9 ± 4.7 µmol/L for UGT1A9. Representative Dixon plots for ketoconazole inhibition of SN-38 glucuronidation by UGT1A1 and UGT1A9 are shown in Fig. 4.



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  		Fig. 3. Lineweaver-Burke plots showing competitive inhibition of ketoconazole on SN-38 glucuronidation by UGT1A1 (A) and UGT1A9 (B).

 


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  		Fig. 4. Representative Dixon plots for ketoconazole and (A) UGT1A1 and (B) UGT1A9.

 
Cyclosporine A and fluconazole were screened for their effect on SN-38 glucuronidation by UGT1A1, UGT1A7, and UGT1A9. At 1 µmol/L, cyclosporine A showed a small (<20%) but demonstrable inhibitory effect on UGT1A7 and UGT1A9 activities (18% and 14%, respectively). Fluconazole, on the other hand, did not inhibit the UGT isoforms tested (data not shown).

In vitro glucuronidation of bilirubin by pooled human liver microsomes in the presence of ketoconazole and indinavir. To confirm ketoconazole's inhibitory effect on UGT1A1, bilirubin glucuronidation was studied in human using human liver microsomes. Ketoconazole and indinavir inhibited the glucuronidation of bilirubin, with IC50 values of 53 and 69 µmol/L, respectively.

Effects of ketoconazole on irinotecan metabolism in human hepatocytes. The aim of this experiment was to assess effects of ketoconazole on the disposition of irinotecan in human hepatocytes, a system with intact UGTs, CYP3A, and transporters. Table 1 shows the formation of SN-38 and SN-38G at two different substrate concentrations. A reduction of SN-38G concentration in the medium was observed when ketoconazole was added to the reaction at both low and high substrate concentrations. At the lower substrate concentration tested (10 µmol/L irinotecan), the formation of SN-38G was reduced by 39% and 81%, with ketoconazole at 1.6 and 16 µmol/L, respectively. At high substrate concentration (100 µmol/L irinotecan), a reduction in SN-38G formation by 44% and 83% was seen in the presence of ketoconazole at 1.6 and 16 µmol/L, respectively. When normalized for SN-38 formation, the relative formation of SN-38G (SN-38G/SN-38 ratio) remained reduced. A similar picture was observed in cell lysates, with a reduction of SN-38G formation in the presence of ketoconazole (data not shown). A modest increase in SN-38 formation (39%) was observed only when ketoconazole (1.6 µmol/L) was incubated at the lower irinotecan concentration tested (10 µmol/L).


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  		Table 1. Effects of ketoconazole on the formation of SN-38 and SN-38G in human hepatocytes

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
We report that ketoconazole exhibits a significant inhibitory effect on SN-38 glucuronidation by UGT1A1 and UGT1A9 and our findings are consistent with those of Satoh et al. (19). Inhibition of UGTs could lead to clinically significant interactions for drugs with a narrow therapeutic index (27–30). Glucuronidation is an important conjugation pathway, which accounts for the majority of phase II biotransformations. It prevents the accumulation of potential toxic compounds by converting lipophilic substrates to more soluble hydrophilic glucuronides, allowing for excretion via urine or bile (31). The UGTs have a broad spectrum of substrates including endogenous compounds (e.g., thyroid hormones, hydroxyl estradiol, and bilirubin), drugs (e.g., epirubicin, propofol, opiates, and zidovudine), environmental pollutants, and carcinogenic chemicals (32, 33).

Our kinetic analysis suggests that ketoconazole inhibited the formation of SN-38G via competitive inhibition. To investigate if ketoconazole is a substrate for UGT1A1, we incubated ketoconazole with 14C-UDPGA in pooled human liver microsomes and did not detect the presence of radiolabeled glucuronide of ketoconazole (data not shown). Although glucuronidation is a minor elimination pathway for another azole, fluconazole (34), ketoconazole is not known to be a substrate for UGT enzymes. Recent studies have suggested possible interactions between CYP enzymes and UGTs (35–37). Taura et al. showed that CYP1A1 may function as a substrate transporter for UGTs and inhibition of CYP1A1 could interfere with glucuronidation activity. However, we have shown, by using cDNA-expressed UGT1As, that ketoconazole can inhibit SN-38 glucuronidation in the absence of CYP enzymes.

In human hepatocytes, we observed that SN-38G formation was reduced by ketoconazole. Increased formation of SN-38, however, was not observed at a higher ketoconazole concentration. Given that ketoconazole is a potent inhibitor of CYP3A4, UGT1A1, and UGT1A9, this observation was somewhat surprising and may indicate additional inhibitory effects of ketoconazole on irinotecan or SN-38 transport. We did not find a difference in the SN-38 and SN-38G concentration between reaction medium and cell lysates. Hence, the observation cannot be explained by the inhibition of efflux transporters of SN-38 at a higher ketoconazole dose. Ketoconazole is known to inhibit influx transporters including the organic anion-transporting polypeptides (38). At higher concentration, ketoconazole may have inhibited the influx of irinotecan, thereby offsetting the effect of CYP3A4 and UGT1A1 inhibition. Recently, OATP1B1 (SLCO1B1), OATP2B1 (SLCO2B1), and OATP1B3 (SLCO1B3) were evaluated for their transport activity for irinotecan and its metabolites (39). OATP1B1 was found to transport SN-38 but not irinotecan and SN-38G. At this point in time, influx transporters that are involved in irinotecan have not been identified.

Kehrer et al. reported that modulation by ketoconazole resulted in 87% reduction in the relative formation of 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxycamptothecin, an oxidative metabolite of irinotecan, and an increase in the relative exposure to SN-38 (15). Similar to ketoconazole, coadministration of cyclosporine A with irinotecan resulted in an overall increase in irinotecan and SN-38 AUC (40–42). However, in contrast to ketoconazole, no change in the relative formation of SN-38 (ratio of SN-38 AUC to irinotecan AUC) was observed suggesting that the overall increase in SN-38 is the result of the decreased irinotecan clearance from CYP3A4 inhibition. The change in relative formation of SN-38 depends on two critical processes: the formation of SN-38 by carboxylesterase 2 and elimination of SN-38 via SN-38 glucuronidation and biliary excretion. Our finding supports the conclusion that the change in the relative formation of SN-38 was primarily due to UGT1A1 inhibition by ketoconazole. In addition, when microsomes were incubated with irinotecan and ketoconazole, we did not see an increase in the formation of SN-38 (data not shown) lending further support that the change in relative SN-38 formation was caused by inhibition of SN38 glucuronidation.

With inhibition of SN-38 glucuronidation, one should expect a decrease in the relative formation of SN-38G (ratio of SN-38G AUC to SN-38 AUC). But in Kehrer et al.'s study, the relative formation of SN-38G was unchanged. It is unclear as to what could have accounted for the discrepancy. The relative formation of SN-38G may not be a good estimate of SN-38 glucuronidation activity, because it may be affected by a change in the biliary excretion of SN-38G. The clinical observation may be a reflection of the complex interaction among transporters, UGT, and CYP enzymes influenced by the relative concentration of ketoconazole and irinotecan. Additional, in vivo studies on transporters may shed light to drug-drug interaction involving ketoconazole.

In conclusion, we have shown that commonly known CYP inhibitors like ketoconazole and cyclosporine A can inhibit different UGT1A isoforms and may in part contribute to the disposition of SN-38, although the relative contribution compared with CYP3A4 and transporters inhibition is not clear. CYP3A4 and ABCB1 have been previously shown to have overlapping substrate specificity. In addition, substrates of CYP3A4 or ABCB1 frequently undergo phase II conjugation by UGT1A as well, including drugs commonly used by cancer patients (e.g., irinotecan, etoposide, and indinavir; refs. 43, 44). Previously reported drug-drug interactions involving ketoconazole may warrant reinterpretation in light of our findings, which may reflect a component of its effect on glucuronidation, as well as oxidation and transport. Given the widespread use of ketoconazole in cancer patients as antifungal and for treatment of prostate cancer, our study provides a strong rationale for additional clinical studies especially for drugs that are predominantly metabolized by UGT1A1 and/or UGT1A9.


   Acknowledgments
 
We thank Andrea Yoder Graber, Larry House, and Snezana Mirkov for their assistance in the laboratory; Tae-Won Kim for his invaluable advice; and the Liver Tissue Procurement and Distribution System (NIH contract N01-DK-9-2310) and the Cooperative Human Tissue Network for providing the human liver tissues for the study.


   Footnotes
 
Grant support: William F. O'Connor Foundation (M.J. Ratain) and Agency for Science, Technology, and Research (W.P. Yong).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: All authors have read the article and conflict of interest statement and approved their submission for publication; the work is original and has not been published and is not being considered for publication elsewhere, in whole or in part, in any language, except as an abstract.

None of the authors have financial or personal relationships that could potentially be perceived as influencing the described research.

Received 3/30/05; revised 6/ 2/05; accepted 6/21/05.


   References
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 Discussion
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