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Cancer Therapy: Preclinical

Activated X-Receptor Is a Target for Ketoconazole and Its Analogs Hongwei Wang,1, 2 Haiyan Huang,1, 2 Hao Li,1, 2 Denise G. Teotico,8 Michael Sinz,5 Sharyn D. Baker,6 Jeffrey Staudinger,7 Ganjam Kalpana,1,4 Matthew R. Redinbo,8 and Sridhar Mani1, 2 ,4

Abstract Purpose: Variations in biotransformation and elimination of microtubule-binding drugs are a major cause of unpredictable side effects during cancer therapy. Because the orphan receptor, pregnenolone X-receptor (PXR), coordinately regulates the expression of metabolizing and transport , controlling this process could improve therapeutic outcome. Experimental Design: RNA-, protein-, and transcription-based assays in multiple cell lines derived from hepatocytes and PXR wild-type and null mouse studies were employed to show the effects of ketoconazole and its analogues on -activated PXR-mediated gene transcription and translation. Results: The transcriptional activation of genes regulating biotransformation and transport by the liganded human nuclear xenobiotic receptor, PXR, was inhibited by the commonly used anti- fungal ketoconazole and related analogs. Mutations at the AF-2 surface of the human PXR ligand-binding domain indicate that ketoconazole may interact with specific residues outside the ligand-binding pocket. Furthermore, in contrast to that observed in PXR (+/+) mice, genetic loss of PXR results in increased (preserved) blood levels of paclitaxel. Conclusions: These studies show that some azole compounds repress the coordinated activa- tion of genes involved in by blocking PXR activation. Because loss of PXR maintains blood levels of paclitaxel upon chronic dosing, ketoconazole analogues may also serve to preserve paclitaxel blood levels on chronic dosing of drugs. Our observations may facilitate new strategies to improve the clinical efficacy of drugs and to reduce therapeutic side effects.

The erratic of microtubule-binding anticancer drugs Therefore, approaches to control or ‘‘unify’’ metabolism that can be directly linked in part to the consequences of CYP450- include both biotransformation and transporter function could directed drug biotransformation and transporter-dependent result in uniform and , as elimination (1–4). A recent randomized prospective clinical well as increased efficacy and predictability of drug treatment. study has shown that CYP3A activity-guided dosing We and others (9, 10) have previously shown that micro- results in more uniform drug pharmacokinetics and less tubule-binding drugs activate the pregnenolone X-receptor, PXR. than traditional body surface area–based dosing (5). For other The nuclear xenobiotic receptor PXR and, to a lesser extent, other drugs belonging to this class, inducing or inhibiting either adopted orphan nuclear receptors (e.g., constitutive CYP450 or multidrug resistance-1(MDR1)function has receptor, CAR) control genes encoding drug-metabolizing resulted in altered drug metabolism, leading to distinct enzymes and transporters at the level of transcription (11, 12). pharmacokinetic and pharmacodynamic sequelae (6–8). In mice, paclitaxel activates PXR, induces expression of CYP3A4 and MDR1 gene products, and accelerates its own metabolism, resulting in lowering of blood levels with chronic dosing (13, 14). Human PXR activation mediated by in cell culture 1 2 Authors’ Affiliations: Albert Einstein Cancer Center, Departments of Medicine, induces CYP3A4 (responsible for 3-hydroxy-paclitaxel metabo- 3Pathology, and 4Molecular Genetics, Albert Einstein College of Medicine, Bronx, New York; 5Bristol Myers Squibb Co. Wallingford, Connecticut; 6Johns Hopkins lite), CYP2C8 (responsible for 6-hydroxy-paclitaxel metabolite), University, Baltimore, Maryland; 7University of Kansas, Lawrence, Kansas; and MDR1, and MRP-2 (responsible for biliary and 8University of North Carolina at Chapel Hill, Chapel Hill, North Carolina intestinal cell efflux of paclitaxel and its metabolites); together, Received 6/29/06; revised 12/29/06; accepted 1/18/07. this results in altered paclitaxel pharmacokinetics through Grant support: Damon Runyon Cancer Research Foundation grant CI: 15-02 metabolic pathways (10, 15, 16). Biotransforming enzymes and (S. Mani). The costs of publication of this article were defrayed in part by the payment of page transporters are regulated by PXR such that substrates and/or charges. This article must therefore be hereby marked advertisement in accordance metabolites of CYP3A4 are coordinately exported in and with 18 U.S.C. Section 1734 solely to indicate this fact. bowel tissues by the MDR1gene product, and overall metabolic Requests for reprints: Sridhar Mani, Albert Einstein Cancer Center, Albert homeostasis is achieved. Drug-drug interactions with regard to Einstein College of Medicine, 1300 Morris ParkAvenue, Chanin 302D-1, Bronx, NY 10461. Phone: 718-430-2871;Fax: 718-904-2892; E-mail: [email protected]. paclitaxel therapy can occur by the direct inhibition of the F 2007 American Association for Cancer Research. activities of CYP450 enzymes. Drug-drug interactions occur at doi:10.1158/1078-0432.CCR-06-1592 the level of PXR, where xenobiotics can function as activating

Clin Cancer Res 2007;13(8) April 15, 2007 2488 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. Ketoconazole, Nuclear Receptors, and Drug Metabolism (e.g., the constituent of St. John’s wort) and supplemented with 10% fetal bovine serum, only passage 4 induce the metabolism of paclitaxel, thereby significantly was used for transfection. LS174T (both from ATCC) were lowering its blood levels and therapeutic efficacy (17). maintained in MEM supplemented with 10% fetal bovine serum. Consequently, we have shown that activated orphan nuclear Where indicated, the media were supplemented with charcoal- receptors controlling drug metabolism may be inactivated by adsorbed fetal bovine serum (Hyclone, Logan, UT). Cos-1cells ketoconazole, a known inhibitor of CYP450 and UGT1A (African green monkey cells; ATCC) were propagated in activity. We have shown that high doses of ketoco- DMEM supplemented with 10% fetal bovine serum. Human nazole inhibit not only enzyme activity but also the hepatocyte(s) were purchased from In Vitro Technologies, Inc. transcription of genes regulated by the orphan receptors (Baltimore, MD) and maintained in InVitroGrow HI medium. PXR, CAR, the liver X-receptor (LXR), and the farnesoid X- Fa2N-4 cells are SV40 large T antigen immortalized human receptor (FXR), by limiting associations with the trans- hepatocytes and represent another in vitro model for assessment criptional coactivator receptor coactivator-1(SRC-1; of hepatocyte function (XenoTech, Lenexa, KS). They were ref. 18). We have proposed that at clinically achievable propagated as per manufacturer’s instructions. concentrations, less toxic analogues of ketoconazole can be Cell survival [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- employed to potently block nuclear receptor-driven transcrip- zolium bromide] assay. Aliquots of 2 to 5 103 cells were plated tion and, thus, can be used to modulate the coordinated in 96-well plates in triplicate. Twelve hours later, the cells were activation of numerous genes involved in drug metabolism treated with serial dilutions of each drug. Following incubation and efflux. Because the majority of anticancer drugs undergo for 48 h, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- metabolism by multiple enzymes and efflux transporters, lium bromide assay was done as previously described (9). transcriptional inhibition of nuclear receptor activity would Semiquantitative RT-PCR and real-time RT-PCR. Total RNA be expected to generate relatively broad control of drug was isolated using the Qiagen RNeasy Mini kit (Qiagen, Inc., metabolism pathways, which could improve anticancer drug Valencia, CA). To eliminate amplification of contaminating pharmacokinetics and pharmacodynamics. chromosomal DNA in the real-time quantification, the isolated In this study, we extend our findings of ketoconazole action total RNA was treated with RNase-free DNase (Promega, on PXR-mediated gene transcription in multiple in vitro Madison, WI), following the supplier’s instructions. Reverse hepatocyte and colon cancer cell line models. Furthermore, transcription was done using Superscript first strand synthesis we provide novel evidence that closely related analogues of system (Invitrogen, Carlsbad, CA). For semiquantitative PCR, ketoconazole, including enilconazole and , provide one-tenth the reverse transcription (RT) reaction products were a preliminary base to evaluate structure-activity relationships. subjected to PCR amplification for 35 cycles in a multiplex We have also shown that human PXR (hPXR) mutants can be format. Equal volumes of PCR product were loaded into each designed that preserve ligand binding and activation of the well of a 1% ethidium bromide–stained agarose gel as receptor, but are immune to the inhibitory effects of described previously (19). Real-time PCR for cDNA quantifi- ketoconazole. Such mutants indicate that the activation cation was done using TaqMan universal PCR master mix and function 2 (AF-2) region on the surface of the hPXR ligand- TaqMan probes, using VIC as the 5¶ reporter fluorochrome and binding domain is critical for binding to ketoconazole, tetramethylrhodamine (TAMRA) as the 3¶ quencher fluoro- suggesting that the azole compounds directly block coactivator chrome. Simultaneous quantification of the 18S RNA using binding to the AF-2 site. Finally, we show that inhibiting PXR- a kit from ABI Systems (Foster City, CA) allowed for nor- mediated metabolism of paclitaxel reduces variability of malization between samples. A standard curve for CYP3A4 paclitaxel kinetics in mice. We conclude that specific inhibitors cDNA was constructed to ensure linearity in the concentration of PXR may prevent adverse drug effects and unanticipated range studied. The CYP3A4 forward primer sequence was PXR-mediated drug metabolism. 5¶-cattcctcatcccaattcttgaagt-3¶, the CYP3A4 reverser primer sequence was 5¶-ccactcggtgcttttgtgtatct-3¶, and the CYP3A4 probe sequence was 5¶-VIC-cgaggcgactttctttcatcctttttacagattttc- Experimental Procedures TAMRA-3¶, all spanning exon junctions, thus preventing amplification of genomic DNA. Amplification was detected Plasmids and reagents. The CYP3A4 luciferase reporter and analyzed using the ABI Prism 7700 sequence detector with plasmid (10466 to +53) and PAR-2 in pcDNA3.1 was obtained SDS 2.1analysis software (Applied Biosystems, Foster City, from Dr. Jonas Uppenberg (Biovitrum, Stockholm, Sweden). CA). The relative fold increase in mRNA in samples compared Gal4DB-hPXR-LBD, pCMX-hPXR, and Tk-M H100x4-Luc were with controls was calculated using the comparative CT method. provided by Dr. Ronald Evans (Salk Institute, La Jolla, CA). Transient transcription and mammalian one-hybrid and two- Cremophor EL, ketoconazole, and rifampicin was obtained form hybrid assays. Cells were split onto 24-well plates at densities Sigma Chemical Co. (St. Louis, MO). The Gal4 and VP16 of 2 to 8 104 cells per well the day before transfection. plasmids were obtained from Dr. Ronald Evans and Dr. Bruce Transfections were done using the LipofectAMINE reagent Blumberg (University of California–Irvine, Irvine, CA). Clinical- (Invitrogen) according to the manufacturer’s instructions and grade paclitaxel was obtained from The Albert Einstein College as previously described (9). of Medicine Pharmacy, Bronx, NY. All drugs (except paclitaxel Paclitaxel pharmacology studies in C57BL/6 mice. Adult for in vivo use, which was formulated in Cremophor EL) were female wild-type PXR (+/+) and PXR-null (/)were dissolved in 100% DMSO and stored at 20jC. The final maintained on standard laboratory chow and were allowed food concentration of DMSO was V0.2% in all experiments. and water ad libitum (23). To show the in vivo significance of PXR Cell culture. HepG2 cells [American Type Culture Collection activation as it relates to paclitaxel metabolism, thirty-six 8-week- (ATCC), Manassas, VA] were maintained in RPMI 1640 old C57BL/6 PXR (+/+) and PXR (/) female mice received

www.aacrjournals.org 2489 Clin Cancer Res 2007;13(8) April 15, 2007 Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. Cancer Therapy: Preclinical daily morning i.v. injection with paclitaxel at a dose of 10 mg/kg >90% as compared with cells treated only with paclitaxel or for five sequential days (20–22). Clinical-grade paclitaxel was rifampicin, respectively. formulated as described previously (9, 14). At each time point To determine if variations in human hepatocyte donors from the start of injection of paclitaxel on days 1and 5 ( t = could account for the changes induced by ketoconazole, we pretreatment, 0.25, 0.5, 2, 4, and 8 h), six mice per time point carried out quantitative RT-PCR to examine the effect of drugs were sacrificed, and all organs, feces, , and blood were on CYP450 levels. Human hepatocytes were isolated from three isolated for further analysis of paclitaxel and its metabolites. separate liver donors with no prior history of medication use or Statistical analysis was done with Analyze-It software (Leeds LS27 hepatitis (In Vitro Technologies). These hepatocytes were 7WZ, England) using the nonparametric Mann-Whitney U test. treated at the same time with either rifampicin, ketoconazole, HPLC with tandem mass spectrometric (LC-MS-MS) method for or both in combination, and CYP3A4 and CYP2B6 mRNA measurement of paclitaxel and its two major metabolites (6a- expression were quantitated using RT-PCR. Ketoconazole hydroxypaclitaxel and p-3-hydroxypaclitaxel) concentrations in inhibited rifampicin-mediated induction of CYP3A4 and mouse plasma and tissue. Total paclitaxel (bound and un- CYP2B6 genes in a concentration-dependent manner (data bound) and its metabolites were extracted from 100 AL of mouse not shown). Furthermore, the mean concentrations (IC50 F plasma (in this assay, the lower limit is f50 AL) with SD) of ketoconazole resulting in 50% decrease in rifampicin- 2 mL of a mixture of acetonitrile/n-butyl chloride (1:4, v/v), induced CYP3A4 and CYP2B6 expression were 13.4 F 1.12 and with 20 nmol/L docetaxel used as the internal standard. 9.7 F 4.18 Amol/L, respectively (Table 1). These results indicate Separation of paclitaxel, including the internal standard, was that ketoconazole inhibited the ligand-mediated activation of achieved on a Waters X-Terra C18 (50 2.1mm i.d., 3.5 Amol/L) CYP3A4 and CYP2B6 genes in primary hepatocytes in a donor- analytic column with a mobile phase consisting of acetonitrile independent manner. containing 0.1% formic (80%) and 2 mmol/L ammonium Effect of ketoconazole analogues on the rifampicin-mediated acetate containing 0.1% formic acid (20%) using isocratic flow at activation of hPXR. Ketoconazole belongs to the azole family of 0.2 mL/min and an overall run time of 5 min. The analytes of compounds, many of which have potent properties. interest were monitored by tandem mass spectrometry with The above findings indicated that whereas ketoconazole slightly electrospray positive ionization at transitions 808 ! 527.2, activates the basal level transcriptional activity of hPXR in the 870.4 ! 286, 870.4 ! 569, and 854.5 ! 509.4 for paclitaxel, absence of a ligand, the compound drastically inhibits hPXR 6a-hydroxypaclitaxel, p-3-hydroxypaclitaxel, and docetaxel, transactivation in the presence of PXR agonists such as respectively. The lower limit of quantitation of paclitaxel and rifampicin. We used this property of ketoconazole to identify its metabolites in mouse plasma was 10 ng/mL, corresponding to additional members of the azole family that exhibit a similar 0.0117 Amol/L (accuracy, 99.8%). For interassay precision, activity; such information could be used to elucidate structure- quality control samples containing 40, 200, and 400 ng/mL activity relationships. We did transient transcription assays in (corresponding to 0.05, 0.23, and 0.47 Amol/L) were used, HepG2 cells using full-length hPXR and its cognate reporter in resulting in coefficients of variation of 7.3%, 6.3%, and 2.2% the presence or absence of various to determine their effect and accuracies of 99.1%, 98.5%, and 100%, respectively. For on hPXR-mediated activation (Fig. 1B). As before, rifampicin measurement of paclitaxel and its metabolites in mouse feces activated hPXR 2.7-fold over control (DMSO)-treated cells. The and bile, tissue concentrations were standardized by adding PBS antifungal drugs in the absence of rifampicin showed two to a final concentration of 200 mg/mL. Samples were homog- distinct properties. Although fluconazole, enilconazole, and enized on ice until a uniform homogenate was achieved. The ketoconazole showed weak stimulatory activity (Fig. 1B and C), tissue homogenate was diluted 1:9 in mouse plasma and 500 AL , nitrate, miconazole, and R063373 of the mixture was extracted with 5 mL of a mixture of showed inhibitory effects on the basal activity of hPXR. acetonitrile/n-butyl chloride (1:4, v/v). Paclitaxel and its Interestingly, only fluconazole, enilconazole, and ketoconazole metabolite concentrations in bile and feces were determined inhibited the activation of hPXR to basal level in the presence of from the calibration curve constructed in mouse plasma. paclitaxel. The remaining drugs had profound inhibitory effects on rifampicin-activated hPXR to below the baseline. It is important to note that these drugs alone also had similar effects Results on the basal activity of hPXR. Collectively, these results indicated that of the azole drugs tested, fluconazole and enilconazole Donor- and cell type–independent inhibition of ligand- simulated the action of ketoconazole on hPXR and can be mediated activation of CYP3A4 and CYP2B6 gene transcription termed ‘‘activating antagonists’’ because they exhibit weak by ketoconazole. We have previously shown that ketoconazole activities alone but clear antagonist actions in the inhibits hPXR activation (18). To generalize this result, we presence of an established PXR agonist. determined the effect of ketoconazole on xenobiotic-mediated Ketoconazole binds hPXR outside the ligand-binding pocket. transcription of CYP450s in another liver-derived cell line, We have previously shown, using a scintillation-proximity assay Fa2N-4 (Fig. 1A). Using real-time reverse transcription-PCR that measures the binding of ligands to His-tagged hPXR-LBD, (RT-PCR), we found that rifampicin and paclitaxel, both hPXR that the IC50 of the established PXR agonist rifampicin was agonists, significantly increased the expression of CYP3A4 8.8 Amol/L. In contrast, the IC50 for ketoconazole was 74.4 mRNA when compared with DMSO-treated cells. Treatment of Amol/L. Similarly, the Kb (estimated using the Cheng-Prusoff cells with ketoconazole resulted in only a slight increase in equation) for rifampicin and ketoconazole were 6.3 and 55.3 CYP3A mRNA expression levels relative to DMSO-treated cells. Amol/L, respectively (18). These data suggested that biological- When ketoconazole was combined with either paclitaxel or ly active 25 Amol/L concentrations of ketoconazole were rifampicin, however, the CYP3A4 expression was decreased by unlikely to compete with bona fide ligands for binding within

Clin Cancer Res 2007;13(8) April 15, 2007 2490 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. Ketoconazole, Nuclear Receptors, and Drug Metabolism the ligand-binding pocket. To firmly establish whether ketoco- The data showed a dose response for the effect of ketoconazole nazole competed with rifampicin for binding to the ligand- on the rifampicin-mediated activation of hPXR because the binding pocket or bound to a distinct region on hPXR, we did curves generated in the presence of the two agonist concen- transient transcription assays with full-length expression trations were essentially parallel (Fig. 2A). Note that we did test construct for hPXR and its cognate reporter. In these experi- ketoconazole at concentrations of 100 Amol/L; however, the ments, we used two concentrations of rifampicin (10 and 30 results were not reliable due to cytotoxicity. This behavior is Amol/L) to determine the profile of inhibition by ketoconazole. compatible with noncompetitive inhibition of rifampicin

Fig. 1. Select ketoconazole analogues abrogate ligand-mediated activation of hPXR. A, real-time RT-PCR analysis of mRNA isolated from immortalized Fa2N4 cells for CYP3A4 expression. The cells were treated with drugs for 48 h using the concentrations as indicated. B, transient transcription assays in HepG2 cells to study the effect of ketoconazole analogues on xenobiotic-mediated activation and/or repression of nuclear receptors. HepG2 cells were cotransfected with pCMX-hPXR, CYP3A4 luciferase reporter plasmid (10466 to +53), and pSV-h-galactosidase control vector for 24 h. Subsequently, the cells were treated with ketoconazole and its analogue(s) as indicated. Solid line (and *), the analogues that have similar potency as ketoconazole. C, these analogues are illustrated from top to bottom as fluconazole and enilconazole. Their structures are illustrated above; grey, the common chemical group ().

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surface capable of coactivator binding (although not to wild- Table 1. Summary of the inhibitory effect of type levels; see Fig. 3B and C). Structural considerations also ketoconazole on rifampicin-mediated induction of suggest that the elimination of K277 may be responsible for the CYP3A4 and CYP2B6 in human hepatocytes loss of ketoconazole antagonism. Note that this residue is located close to the side chain of H697 in the nuclear receptor Hepatocyte Mean IC50 Mean IC50 donor (CYP3A4 mRNA) (CYP2B6 mRNA) (NR) box 2 of human SRC-1(Fig. 3C). Ketoconazole contains an azole ring that may mimic this histidine side chain and 113.005.75 2 12.53 9.26 compete for coactivator binding. This observation may explain 3 14.66 14.08 why the antagonism of PXR is agonist dependent: the ordering Mean 13.40 9.70 of the AF-2 surface and critically K277, enhanced by ligand SD 1.12 4.18 binding, is necessary for ketoconazole binding. A lysine is conserved in this position in all the NRs susceptible to NOTE: Three donor (as shown in Fig. 1B) were subjected ketoconazole antagonism (PXR, CAR, FXR, LXR), but is to drug treatment(s) over 48 h and total RNA isolated and replaced by a nonlysine in nuclear receptors that do not subjected to CYP3A4 and CYP2B6 real-time RT-PCR. This shows respond to this compound (e.g., Q in ERa). Taken together, the mean inhibitory concentration of ketoconazole required to inhibit rifampicin-induced CYP3A4 and CYP2B6 mRNA expression these data suggest that the AF-2 surface is the likely binding site by 50%. for ketoconazole. PXR is an important determinant of paclitaxel blood levels in mice. We and others have previously shown that paclitaxel is a ligand that activates both mouse and human PXR activation by ketoconazole and, hence, is consistent with the (9,18).InPXR(+/+)BALB/cmice,whenpaclitaxelis idea that ketoconazole doe not interact with the rifampicin- administered daily for 5 days i.v. at a dose of 20 mg/kg, the binding site. To corroborate these data, we did mammalian mean area under the curve (AUC) on day 5 compared with day two-hybrid assays using both full-length and ligand-binding 1is (86.2 F 5.5 Amol/L h versus 125.6 F 3.8 Amol/L h), mean domain fusions of hPXR with the VP16 transactivation domain, Cmax (32.6 F 6.8 Amol/L versus 79.2 F 5.8 Amol/L), and mean as previously described. Both assays clearly show that ketoco- t1/2b (3.52 F 0.46 h versus 3.13 F 0.18 h; ref. 14). To nazole inhibits the rifampicin-induced interaction between determine if paclitaxel is inducing its own clearance through hPXR and SRC-1(Fig. 2B and C). Importantly, the magnitude the activation of PXR and overexpressing cyp3a11 of the effect of ketoconazole on hPXR-SRC-1interaction is in mouse liver, we subjected C57BL/6 PXR (+/+) and PXR diminished when the NH2 terminus is truncated, which implies ( / ) mice to a 5-day course of paclitaxel injections i.v. at that the preservation of the NH2 terminus of hPXR is important a dose of 10 mg/kg. The formulation and schedule of for ketoconazole action. administration of paclitaxel were identical to the previously We hypothesized that ketoconazole impacts PXR transcrip- published report (9). In PXR (/)mice(n = 6 per time points tional regulation by binding at the AF-2 surface rather than in sampled at 0.25, 0.5, 2, 4, and 8 h after paclitaxel injection), the ligand-binding pocket. To test this hypothesis, several single using noncompartmental analysis, the mean F SD day 1and and double mutations were generated in the AF-2 region of PXR day 5 paclitaxel AUC0-t were 2,121 F 352 ng/mL h [coefficient (Fig. 3). Residues were replaced with corresponding amino of variation (CV), 16.6%] and 3,459 F 297 ng/mL h (CV, from the ERa steroid receptor that is not antagonized by 8.6%), respectively. Similarly, there was a 2.6-fold increase in ketoconazole. In each case, the single mutants lead to a loss of paclitaxel Ct =15¶ on day 5 versus day 1. In PXR (+/+) mice (n = PXR activity, apparently by introducing structural distortions in 6 per time points sampled at 0.25, 0.5, 2, 4, and 8 h after the AF-2 surface of the receptor (Fig. 3). A280W seems to paclitaxel injection), the mean F SD day 1and day 5 paclitaxel generate a clash with L428 of aAF, which would shift the AUC0-t were 2,925 F 924 (CV, 31.6%) and 1,288 F 138 (CV, position of this terminal a-helix (Fig. 3C) and prevent 10.7%), respectively (P < 0.05). Similarly, in contrast to PXR coactivator binding. The replacement of P268 with the (/) mice, there was a 2.2-fold decrease in paclitaxel Ct =15¶ nonproline residue H may similarly disrupt the AF-2 surface on day 5 versus day 1in PXR (+/+) mice (Table 2). These results by reducing the rigidity of the loop between a3¶ and a4, thus suggest that PXR activation is responsible for reduced accumu- altering the position of a4. T248E seems to introduce a clash lation of paclitaxel in blood, which may serve as one indicator with T422, which would likely shift aAF (up as shown in Fig. for drug resistance in mice. 3C) and lead to a loss in coactivator binding. Finally, replacement of K277 with Q would eliminate the capping of Discussion the electronegative helix dipole of aAF by the lysine, which may be critical to the proper positioning of aAF. The corresponding We have previously shown that micromolar concentrations helix in the steroid receptors are one to two turns longer and, of ketoconazole antagonize PXR-dependent gene transcription thus, cannot be capped by the equivalent side chain. (9, 14). In this study, we show that ketoconazole inhibits hPXR Significantly, however, the double-mutant T248E/K277Q in several cell types, and that ketoconazole analogues are form of PXR is active but is not susceptible to antagonism by readily available to dissect structure-activity relationships to ketoconazole (Fig. 3B and C). The recovery of receptor activity derive less toxic compounds that can inhibit PXR. At the is likely due to a synergistic combination of effects: T248E shifts molecular level, we reveal that ketoconazole’s effect is a direct the aAF up and perhaps stabilizes the electropositive NH2- consequence of binding to residues outside the ligand binding terminal helix dipole (Fig. 3C), but the shorter K277Q residue pocket of PXR and likely in the AF-2 region of the receptor. can accommodate the shift in aAF position to create an AF-2 These are new findings and define a pharmacophore for the

Clin Cancer Res 2007;13(8) April 15, 2007 2492 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. Ketoconazole, Nuclear Receptors, and Drug Metabolism interaction of small molecular inhibitors outside the ligand- be beneficial to inhibit hPXR activation, thereby preventing binding pocket of the nuclear xenobiotic receptor PXR. rising xenobiotic concentrations from activating hPXR in an Preliminary structure-activity relationship studies with ketoco- autoregulatory loop. This strategy may yield more predict- nazole analogues suggest that the azole ring may be central to able steady-state levels of the drug upon chronic dosing. the inhibitory activity of these compounds. Finally, we show Although there are obvious benefits to inhibiting activated that PXR is a key regulator of paclitaxel kinetics, and that loss of hPXR, in the context of long-term therapeutics, there are risks PXR activity may prevent the decrease in paclitaxel AUC to this strategy that involve the effect of PXR on direct expression observed with repetitive dosing. of many different detoxifying genes. This could result in The clinical implications of our findings are significant. First, unexpected effects of other toxins and carcinogens ingested, as this strategy of inhibiting orphan nuclear receptor activation well as unexpected hypersensitivities to xenobiotics. Therefore, may be used to circumvent adverse drug interactions mediated inhibition of activated hPXR will likely have to be intermittently by xenobiotic activation of PXR. Many drugs in the human and cautiously approached. Third, there is growing literature on pharmacopeia activate hPXR, including some commonly used the consequences of hPXR activation as it relates to bone herbal remedies (24–29); thus, blocking the activation of PXR demineralization and the generation of osteomalacia (30, 31). may prevent drug-drug interactions. Second, as we have shown, Therefore, blocking unwanted PXR activation in this context, loss of PXR could be used to control drug metabolism to desired especially with other therapies like inhibitors that levels in an appropriate treatment setting. In addition, certain induce osteomalacia/osteoporosis, can be beneficial. Finally, xenobiotics (e.g., paclitaxel) may activate PXR and induce its although unproven, hPXR has been hypothesized to serve as a own clearance upon chronic dosing; however, proof of this hormone sensitivity factor in breast cancer (32). Unwanted phenomenon in humans is lacking (14). In such situations, activation may lead to reduced effectiveness of aromatase CYP450 enzymatic inhibitors will only serve to enhance inhibitors or receptor antagonists. In such situations, paclitaxel concentrations that further activate hPXR. It would it may be beneficial to inhibit tumor PXR activation.

Fig. 2. Ketoconazole inhibits ligand-mediated activation of hPXR and its interactions with SRC-1.Transient transfection assay in (A)CV-1cellstostudy the effect of ketoconazole on ligand concentration-dependent activation of hPXR. CV-1cells were cotransfected with pCMX-hPXR, CYP3A4 luciferase reporter plasmid (10466 to +53), and pSV-h-galactosidase control vector for 24 h. Subsequently, the cells were treated with drug(s) as indicated. The RLU/h-gal (relative luciferin light units normalized to h-galactosidase activity) reflects fold increase over untreated (no ketoconazole) controls. Mammalian two-hybrid studies in (B and C) CV-1cells to study the effect of ketoconazole on the interaction of hPXR-LBD and full-length clone with SRC-1. All transfections were normalized for transfection efficiency using pSV-h-galactosidase in the presence or absence of drug(s) as indicated (refer to Materials and Methods). The cells were harvested in equal aliquots at 24 h for luciferase and h-galactosidase assays (for details, see Materials and Methods). All experiments were done at least thrice in triplicates. Columns, mean; bars, SE.

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Fig. 3. Ketoconazole does not abrogate ligand-activated mutant hPXR. Transient transfection assays in (A)HepG2and(B) NIH3T3 cells to study the effect of ketoconazole on ligand-activated hPXR and its mutants. Cells were cotransfected with pCMV-hPXR.2, CYP3A4 luciferase reporter plasmid (10466 to +53), and pSV-h-galactosidase control vector for 24 h. Subsequently, the cells were treated with drug(s) as indicated. All transfections were normalized for transfection efficiency using pSV-h-galactosidase in the presence or absence of drug(s) as indicated (refer to Materials and Methods).The cells were harvested in equal aliquots at 24 h for luciferase and h-galactosidase assays (for details, see Materials and Methods). All experiments were done at least thrice in triplicates. Columns, mean; bars, SE. C, close-up of the AF-2 surface of human PXR, as observed in the crystal structure of the LBD of this receptor bound to the NR box motif of SRC-1. Cyan, mutations examined in this paper; magenta, wild-type residues. Red, hydrogen bonding or polar interactions; gray, van derWaals clashes.

In summary, based on the data presented, we conclude that Finally, we show that the effect of ketoconazole on PXR-driven ketoconazole and selected analogues can antagonize activated metabolism is likely due to direct binding to a region on hPXR forms of the nuclear xenobiotic receptor PXR. The consequence that is distinct from the ligand-binding pocket. This observa- of loss of PXR activity on paclitaxel kinetics has been shown tion serves as a novel paradigm for inhibitor discovery targeting and suggests the clinical utility of PXR inhibitors. Because nuclear receptors. ketoconazole has pleiotropic effects on cellular targets and it The implications of our findings, specifically in terms of the has unpredictable kinetics (33–37), the optimal drug to inhibit control of drug metabolism, are far reaching. In cancer medicine, PXR would depend on defining the pharmacophore on PXR, targeted agents (e.g., epidermal growth factor receptor inhib- which could lead to the development of more specific drugs. itors) have surfaced as commonly used therapeutic options for

Table 2. Summary of paclitaxel concentrations in PXR (+/+) and PXR (/) mouse plasma

Mouse strain Day 1 Day 5

AUC0-t C t =15¶ AUC0-t C t =15¶

C57BL/6 PXR (+/+) 2,925 (924) 2,232 (629) 1,288 (138) 1,019 (829) C57BL/6 PXR / 2,121 (352) 1,899 (427) 3,459 (297) 5,105 (1,124)

NOTE: Six mice per time point [predose, and 0.25 (Ct =15¶), 0.5, 2, 4, and 8 h] from the end of paclitaxel dosing (10 mg/kg) were sacrificed either on day 1or day 5. The paclitaxel blood exposure (AUC 0-t) was calculated as described in Materials and Methods and Results. The values represent mean (SD) of plasma paclitaxel concentrations (ng/mL).

Clin Cancer Res 2007;13(8) April 15, 2007 2494 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. Ketoconazole, Nuclear Receptors, and Drug Metabolism patients with a variety of malignancies. These small-molecule unifying metabolism may lead to drug exposures that drugs are rapidly metabolized, and in fact, CYP3A4 is a major normalize toward the population mean. Fewer outliers would enzyme involved in their metabolism (e.g., Iressa, Tarceva, translate to less unpredictability in terms of drug pharmaco- ). Inducers of CYP3A4 (e.g., rifampicin) reduce blood kinetics-pharmacodynamics and perhaps to better compliance exposure to the parent drug often to subtherapeutic levels. and overall outcomes. For all these reasons, identifying small Because drug levels serve to identify both response [e.g., molecules that are themselves inert in terms of cellular toxicity imatinib dose and tumor response type, chronic myelogenous but are potent in blocking PXR-driven drug metabolism would leukemia (200 mg/day) versus gastrointestinal stromal tumors serve as a critical novel tool to apply to a wide variety of (400 mg/day)] and toxicity (e.g., imatinib steady-state levels therapeutic settings. and grade 3 edema), modifying drug levels from a single dose by controlling metabolism and unifying the pharmacokinet- Acknowledgments ics-pharmacodynamics would be beneficial. The same princi- The authors thankDr. Ronald M. Evans for helpful and insightful discussions. ples may apply to epidermal growth factor receptor inhibitors Dr. Kenny Ye from the Department of Epidemiology and Biostatistics of Albert with regard to response-toxicity relationships (e.g., rash- Einstein College of Medicine provided statistical support and advised on statistical predicting antitumor response). Finally, for cytotoxic drugs, methods used in this manuscript.

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www.aacrjournals.org 2495 Clin Cancer Res 2007;13(8) April 15, 2007 Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. Activated Pregnenolone X-Receptor Is a Target for Ketoconazole and Its Analogs

Hongwei Wang, Haiyan Huang, Hao Li, et al.

Clin Cancer Res 2007;13:2488-2495.

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