divergence of antiangiogenic activity and hepatotoxicity of … · hepatotoxicity of different...

Cancer Therapy: Preclinical

Divergence of Antiangiogenic Activity andHepatotoxicity of Different Stereoisomers ofItraconazoleJoong Sup Shim1,2, Ruo-Jing Li1, Namandje N. Bumpus1,3, Sarah A. Head1,Kalyan Kumar Pasunooti1, Eun Ju Yang2, Junfang Lv2,Wei Shi1,4, and Jun O. Liu1,5

Abstract

Purpose: Itraconazole is a triazole antifungal drug that hasrecently been found to inhibit angiogenesis. Itraconazole is arelatively well-tolerated drug but shows hepatotoxicity in a smallsubset of patients. Itraconazole contains three chiral centers andthe commercial itraconazole is composed of four cis-stereoi-somers (named IT-A, IT-B, IT-C, and IT-D). We sought to deter-mine whether the stereoisomers of itraconazole might differ intheir antiangiogenic activity and hepatotoxicity.

Experimental Design: We assessed in vitro antiangiogenicactivity of itraconazole and each stereoisomer using humanumbilical vein endothelial cell (HUVEC) proliferation and tubeformation assays. We also determined their hepatotoxicity usingprimary human hepatocytes in vitro and a mouse model in vivo.Mouse Matrigel plug and tumor xenograft models were used toevaluate in vivo antiangiogenic and antitumor activities of thestereoisomers.

Results: Of the four stereoisomers contained in commercialitraconazole, we found that IT-A (2S,4R,20R) and IT-C(2S,4R,20S) were more potent for inhibition of angiogenesisthan IT-B (2R,4S,20R) and IT-D (2R,4S,20S). Interestingly, IT-Aand IT-B were more hepatotoxic than IT-C and IT-D. In mousemodels, IT-C showed more potent antiangiogenic/antitumoractivity with lower hepatotoxicity compared with itraconazoleand IT-A.

Conclusions: These results demonstrate the segregation ofinfluence of stereochemistry at different positions of itraconazoleon its antiangiogenic activity and hepatotoxicity, with the 2 and 4positions affecting the former and the 20 position affecting thelatter. They also suggest that IT-C may be superior to the racemicmixture of itraconazole as an anticancer drug candidate dueto its lower hepatotoxicity and improved antiangiogenic activity.Clin Cancer Res; 22(11); 2709–20. �2016 AACR.

IntroductionAngiogenesis, the formation of new blood vessels from pre-

existing vasculature, has been shown to play a critical role in bothnormal physiologic and pathologic processes. It is essential dur-ing development and wound healing, and is tightly regulated byendogenous pro- and antiangiogenic factors. It has also beenimplicated in a number of diseases including cancer, rheumatoidarthritis and macular degeneration (1–3). Since the angiogenesishypothesis was first proposed by Judah Folkman in 1971 (4), anumber of angiogenesis inhibitors have been discovered anddeveloped into antiangiogenic drugs (5–7). A monoclonal anti-

body against VEGF, bevacizumab, was first approved by the FDAin 2004 for the treatment of metastatic colon cancer in combi-nation with standard chemotherapy (8). Subsequently, severalsmall-molecule angiogenesis inhibitors have been entered theclinic, including sorafenib (Nexavar), sunitinib (Sutent), andpazopanib (Votrient). However, most of these small-moleculeantiangiogenic drugs are kinase inhibitors that lack specificity orlead to a high frequency of drug resistance (9–11), necessitatingthe development of small-molecule angiogenesis inhibitors withnovel mechanisms of action.

In an effort to accelerate drug discovery and development, weassembled the Johns Hopkins Drug Library over a decade ago andscreened it for new antiangiogenic activity among existing drugsusing a proliferation assay with primary human umbilical veinendothelial cells (HUVEC). Among the most interesting hits wasthe antifungal drug itraconazole (12). Interestingly, itraconazolewas also found to be an inhibitor of the hedgehog signalingpathway in a separate screen, rendering itraconazole a novelanticancer drug candidate capable of inhibiting the growth ofboth tumor vasculature and tumor cells themselves (13). A seriesof tests of itraconazole in preclinical angiogenesis and cancermodels confirmed the antiangiogenic and anticancer activity ofitraconazole in vivo (12–14). On the basis of these promisingpreclinical results, itraconazole entered multiple Phase II humanclinical studies for treating different types of cancer. (http://www.clinicaltrials.gov/). Positive clinical results have been reported foradvanced lung cancer, metastatic prostate cancer, and basal cellcarcinoma (15–17).Moreover, itraconazolewas found to increase

1DepartmentofPharmacologyandMolecularSciences, JohnsHopkinsUniversity School ofMedicine, Baltimore,Maryland. 2Facultyof HealthSciences, University of Macau,Taipa, Macau SAR, China. 3Departmentof Medicine, Johns Hopkins University School of Medicine, Baltimore,Maryland. 4Department of Chemistry and Biochemistry, University ofArkansas, Fayetteville, Arkansas. 5Department of Oncology, JohnsHopkins University School of Medicine, Baltimore, Maryland.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

J.S. Shim and R.-J. Li contributed equally to this article.

CorrespondingAuthor: JunO. Liu, JohnsHopkins University School ofMedicine,Room 516, Hunterian Building, 725 North Wolfe Street, Baltimore, MD 21205.Phone: 410-955-4619; Fax: 410-955-4520; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-15-1888

�2016 American Association for Cancer Research.

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progression or overall survival among late-stage ovarian, triple-negative breast and metastatic pancreatic cancer patients uponretrospective analysis of previous clinical data (18–21). Together,these encouraging results strongly suggest that itraconazole is apromising new anticancer drug lead.

Although the precise mechanism for the antiangiogenic andanti-hedgehog activities remains unknown, preliminary evidencesuggests that the mode of action of itraconazole is distinct fromany other known inhibitors of angiogenesis or the hedgehogsignaling pathway. Early on, we ruled out the cholesterol biosyn-thetic enzyme, lanosterol 14a-demethylase, which mediates theantifungal activity of itraconazole, as the relevant target for itsantiangiogenic activity (12). Subsequently, we serendipitouslydiscovered that itraconazole blocks the traffic of cholesterol andlikely lipids out of the lysosome, causing a phenotype similar toNiemann-Pick Disease Type C (NPC) in endothelial cells.We alsofound that itraconazole is a potent inhibitor of the mTOR path-way in endothelial cells (22). Importantly, the blockade of cho-lesterol trafficking through the endolysosome can only partiallyexplain the inhibition of mTOR by itraconazole, suggesting thatthere exist unidentified molecular target(s) for itraconazole. Sim-ilarly, the mechanism underlying the inhibition of hedgehogsignaling also remains unclear, even though it has been shownto block the translocation of Smoothened from intracellularvesicles into the primary cilium (13). Using a number of syntheticanalogs of itraconazole, we explored the structure-activity rela-tionship of itraconazole for its twodistinct activities and foundnocorrelation between the two, further suggesting that the antian-giogenic and anti-hedgehog activity of itraconazole are likely to bemediated by different molecular mechanisms (23).

Itraconazole has been widely used to treat fungal infections.Although itraconazole is known to be relatively well tolerated,about 7% of patients who received the drug experience hepato-toxicity (24). In addition to lanosterol 14 alpha-demethylase(25), itraconazole is also known to be a potent inhibitor ofhuman liver cytochrome P450 3A4 (CYP3A4), accounting for itsextensive drug–drug interactions with other medications (26).It is believed that the effect of itraconazole on CYP3A4 is respon-sible for the human hepatotoxicity (27). The commercial itraco-nazole consists of four cis-stereoisomers of itraconazole, includ-ing IT-A (2S,4R,20R), IT-B (2R,4S,20R), IT-C (2S,4R,20S), and IT-D

(2R,4S,20S). All four isomers are known to be ligands ofCYP3A4 (28). However, only IT-B and IT-D among the fourisomers were metabolized by CYP3A4 (28). To assess theantiangiogenic activity of the different stereoisomers, we pre-viously synthesized each of cis-stereoisomers and determinedtheir effects on both fungi and endothelial cells (29). In thisstudy, we conducted a large-scale purification of each stereo-isomer from the racemic itraconazole mixture using the pre-parative supercritical fluid chromatography (SFC) and obtainedgram quantities of each purified stereoisomer. Using the puri-fied stereoisomers, we compared the antiangiogenic activityand hepatotoxicity of each stereoisomer of itraconazole and theracemic itraconazole both in vitro and in vivo. We found that IT-A and IT-C are more potent at inhibiting angiogenesis com-pared to the others, whereas IT-C and IT-D are less hepatotoxiccompared to IT-A, IT-B and racemic itraconazole. Together,these results suggest that IT-C is the most potent antiangiogenicand anticancer agent with the least hepatotoxicity among thefour stereoisomers of itraconazole.

Materials and MethodsCells

HUVECs (Lonza) were grown in endothelial cell growthmedium-2 (EGM-2) using the EGM-2 bullet kit (Lonza) permanufacturer's instructions. HUVEC phenotype was verified bymorphological observation throughout serial passage by themanufacturer. Human pericytes (placental derived) were pur-chased from Zen-Bio (Research Triangle Park, NC) and weregrown in the complete pericyte growth medium (Zen-Bio).HCC1954 human breast cancer and CWR22Rv1 human pros-tate cancer cells were grown in RPMI-1640 medium containing10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA) and1% antibiotic (penicillin and streptomycin) solution (Invitro-gen). The cells were maintained in a humidified incubator at37�C adjusted to 5% CO2.

ReagentsRacemic itraconazole and filipin were purchased from Sigma-

Aldrich (St Louis, MO). Four individual cis-stereoisomers ofitraconazole (IT-A, IT-B, IT-C, and IT-D) were obtained usingpreparative supercritical fluid chromatography (SFC) separationof commercial itraconazole, which was performed by WuXiAppTec. Quality control of each purified stereoisomer was con-ducted by chiral HPLC and optical rotation using previouslysynthesized stereoisomer samples as standards (29). Low-densitylipoprotein (LDL) from human plasma was purchased fromFisher Scientific.

HUVEC proliferation assayHUVEC (5,000 cells/well) were seeded in 96-well plates con-

taining 0.2 mL of EGM-2 medium and allowed to adhere at 37�Cfor 24 hours. The cells were then treated with various concentra-tions of each compound for 24 hours. Cells were pulsed with0.5 mCi [3H]-thymidine (PerkinElmer) for 16 hours, before thecells were treated with 1� trypsin-EDTA (Invitrogen). Thedetached cells were harvested onto FilterMat A glass fiber filters(Wallac) using a Harvester 96 cell harvester (Tomtec). [3H]-thymidine counts were determined using aMicroBeta plate reader(PerkinElmer). Each treatment was done in triplicate and theexperiments were conducted four times independently. The IC50

Translational Relevance

Itraconazole, a known antifungal drug, was found to pos-sess anti-angiogenic and anti-hedgehog activity. However, itsclinical development has been limited in part by its hepato-toxicity. We determined the antiangiogenic activity and hep-atotoxicity of each of the four individual cis-stereoisomers ofitraconazole. We found that one particular stereoisomer, the2S4R20S stereoisomer, designated IT-C, has more potent anti-angiogenic and anticancer activity with less hepatotoxicity,both in vitro and in animal models, in comparison with theracemic mixture of itraconazole. These results revealed for thefirst time that stereochemistry of itraconazole has differentialeffects on its antiangiogenic activity and hepatotoxicity, sug-gesting that IT-C is likely to be superior to itraconazole forfuture clinical development as an anticancer and anti-angio-genic drug.

Shim et al.

Clin Cancer Res; 22(11) June 1, 2016 Clinical Cancer Research2710




values of each compound and their 95%confidence intervalswerecalculated using the GraphPad Prism 5.0 software (GraphPadSoftware).

Western blot analysisWhole cell lysates were prepared by adding 2� Laemmli buffer

to the cells [4% SDS, 20% glycerol, 10% 2-mercaptoethanol,0.004% bromophenol blue, 0.125 M Tris-HCl (pH 6.8)] andboiling for 5minutes. Proteins were then separated by SDS-PAGEand transferred to nitrocellulose membranes (Bio-Rad). Afterblocking at room temperature for 1 hour, membranes wereincubated at 4�Covernight with the primary antibodies includinganti-VEGFR2 (Cell Signaling Technology), anti–phospho-S6K(Cell Signaling Technology), anti-S6K (Santa Cruz Biotechnolo-gy), and anti-GAPDH (Santa Cruz Biotechnology) antibodies,followed by incubation with horseradish peroxidase–conjugatedanti-mouse or anti-rabbit IgG at room temperature for 1 hour.Antibody–protein complexes were detected using enhancedchemiluminescence (ECL) immunoblotting detection reagent(GE Healthcare).

Filipin stainingHUVECwere seeded at 3� 104 cells per well in a Nunc Lab-Tek

II Chamber (8) Slide (Thermo Fisher Scientific) and allowed toadhere overnight. Following drug treatment for 24 hours, cellswere fixed in 4% paraformaldehyde in PBS (pH 7.4) for 20minutes and then washed with PBS three times. The slides wereincubated with PBS containing filipin (at a final concentration of50 mg/mL) in the dark for 1 hour at room temperature. The cellswere then washed with PBS three times, mounted with Immu-mount (Thermo Fisher Scientific), and stored in the dark beforeimaging with a Zeiss 510 Meta confocal microscope (Carl Zeiss).

HUVEC tube formation assayThe tube formation assay, a model for assessment of angio-

genesis in vitro, was conducted as previously described (30).Briefly, a 96-well plate was coated with Matrigel (BD Biosciences)by adding 50 mL of ice-coldMatrigel solution perwell followed byincubation at 37�C for 1 hour. HUVEC were then seeded on theMatrigel-coated wells (2� 104 cells/well). Cells were treated withcompound alone or in combinationwith LDL and then incubatedin a CO2 incubator for 16 hours. For the posttreatment experi-ments, compounds were added to the cells 6 hours after seedingon Matrigel and the incubation was continued for 18 hours.The cells were washed carefully with PBS once, and Calcein-AM(BD Biosciences) solution in PBS (2 mmol/L final concentration)was added. After incubation at 37�C for an additional 30minutes,the cells were washed with PBS and the fluorescence-labeledHUVEC tubes were observed under the Nikon Eclipse TS100fluorescence microscope (485-nm excitation and 520-nm emis-sion) at magnification �100. Each treatment was done in tripli-cate and the experiments were conducted three times indepen-dently. The total tube length, size, and number of junctions fromthe fluorescence images were quantified using the AngioQuantv1.33 software (The MathWorks).

HUVEC-pericyte co-culture tube formationGrowing pericytes in a 6-cm cell culture dish were labeled with

Calcein-AM for 30 minutes before the tube formation experi-ments. Ibidim-Slide angiogenesis chamber (Ibidi)was coatedwithMatrigel by adding 10 mL of ice-cold Matrigel solution per well

followed by incubation at 37�C for 1 hour. HUVEC (1.5 � 104

cells/well) and pericytes (500 cells/well) were mixed at a ratio of30:1 in complete EGM-2 media, seeded on the Matrigel-coatedwells and then treated with compounds. After 20 hours of incu-bation, the co-culture tube formation was analyzed under a ZeissAxioObserver Z1 fluorescence microscope with ApoTome appa-ratus (Carl Zeiss) at magnification �50. The total tube lengths ofthe fluorescence-labeled pericytes were quantified using theAngioQuant v1.33 software.

Determination of hepatotoxicity in primary humanhepatocytes

Fresh primary human hepatocytes (6-well plates) with Matri-gel overlay from three donors were obtained from XenoTech,LLC. Ages and genders of donors were as follows: 22-year-oldmale, 54-year-old male, and 63-year-old male. Hepatocyteviability from each donor was at least 74%. Upon receiptof the hepatocytes, the shipping medium was replaced withWilliams' Medium E supplemented with 10% FBS (Invitrogen),penicillin–streptomycin (Sigma-Aldrich), and L-glutamine(Invitrogen). The hepatocytes were then incubated overnightat 37�C in a 5% CO2 humidified environment. Following theovernight incubation the medium was replaced with freshWilliam's Medium E supplemented as described above andthe hepatocytes were incubated with DMSO (vehicle control,0.1% final volume), racemic itraconazole (2 mmol/L or10 mmol/L) as well as stereoisomers denoted as IT-A, IT-B,IT-C, and IT-D at a final concentration of either 2 or 10 mmol/Lfor 72 hours. Following incubation, to monitor hepatocytefunction, the medium was removed and alanine aminotransa-minase (ALT) levels were measured using an alanine amino-transaminase activity assay kit purchased from BioVision Inc.Cell viability following treatment with itraconazole and stereo-isomers was determined using the Cell Death Detection ELISAKit according to the manufacturer's instructions (Roche Diag-nostics Corporation) as we have reported previously (31).Intracellular glutathione (GSH) was measured using HPLC byquantification of the 5-thio-2-nitrobenzoic acid formed follow-ing the addition of Ellman's reagent to hepatocyte lysates aspreviously described (32).

Determination of hepatotoxicity in miceFemalemice (BALB/c, AnNCr) ages 6 to8weekswerepurchased

from the National Cancer Institute (Frederick, MD) and treated inaccordance with Johns Hopkins Animal Care andUse Committeeprocedures. Mice (n ¼ 5/group) were treated intraperitoneally (i.p.) with vehicle (saline with 5% DMSO, 5% PEG500, and 5%tween-80), 60 mg/kg itraconazole, 60 mg/kg IT-A, and 60 mg/kgIT-C once daily for 10 days. Mice were sacrificed and livers fromeach group were isolated, photographed, and fixed in 10% neu-tral-buffered formalin (Sigma-Aldrich). Fixed liver tissues wereembedded in paraffin, processed for histology sections andstained with hematoxylin and eosin (H&E).

In vivo Matrigel plug angiogenesis assayFemale mice (BALB/c, AnNCr) ages 6 to 8 weeks were used for

the Matrigel plug angiogenesis assay. Mice (n ¼ 5/group) werepretreated i.p. with vehicle (saline with 5% DMSO, 5% PEG500,and 5% tween-80), itraconazole (20 and 50mg/kg), IT-A (20 and50 mg/kg), and IT-C (20 and 50 mg/kg) once daily for 2 daysbefore Matrigel implantation. Matrigel (0.5 mL/injection) con-taining 200 ng/mL VEGF and 500 ng/mL basic fibroblast

Antiangiogenic Activity and Toxicity of Itraconazole Isomers

www.aacrjournals.org Clin Cancer Res; 22(11) June 1, 2016 2711




growth factor (bFGF) was implanted subcutaneously into mice.The drug treatment was continued once daily for an additional8 days. Mice were sacrificed and whole Matrigel plugs withmouse skin were excised, fixed in 10% neutral-buffered forma-lin, embedded in paraffin. For staining infiltrated cells inthe Matrigel, the fixed Matrigel plugs were processed for his-tochemical staining using Masson trichrome staining, whichstains the Matrigel blue and the infiltrated cells and vessels red.For staining functional vasculatures, the fixed Matrigel plugswere stained with anti-CD31 (BD Pharmingen), a blood vesselmarker, and anti-NG2 (Abcam), a pericyte marker, to assessMatrigel plug angiogenesis. A cross-section of the Matrigel plugwas photographed at �100 magnification under a NikonEclipse TS100 microscope and CD31-positive blood vesselsand NG2-stained cells were counted per field for quantificationof angiogenesis.

In vivo tumor xenograft assayFemale athymic nude mice (BALB/c, nu/nu-NCr) ages 6 to 8

weeks were purchased from the National Cancer Institute (NCI)-Frederick and treated in accordance with Johns Hopkins AnimalCare and Use Committee procedures. HCC1954 human breastcancer cells (2 � 106) were implanted subcutaneously into themice. After tumors became palpable, themice (n¼ 7/group) weretreated i.p. with vehicle (saline with 5%DMSO, 5% PEG500, and5% tween-80), itraconazole (25 and 50mg/kg), and IT-C (25 and50mg/kg) once daily for 30 days. For CWR22Rv1 humanprostatecancer xenografts, male athymic nude mice (BALB/c, nu/nu-NCr)ages 6 to 8 weeks (NCI-Frederick) were used for tumor cellimplantation (2 � 106 cells/injection). The tumor-bearing mice(n¼ 6/group) were treated i.p. with vehicle, itraconazole (20 and60mg/kg) and IT-C (20 and 60mg/kg) once daily for 24 days. Thetumor volume was measured periodically using a vernier caliperand calculated according to the modified ellipsoid formula:tumor volume (mm3) ¼ (width)2 � (length) � p/6. After treat-ment, the mice were sacrificed and whole tumor tissues from theHCC1954 xenograft were excised. Tumor tissues were then fixedin 10% neutral-buffered formalin, embedded in paraffin andsliced to obtain 5-mm sections. The blood vessels in the tumortissue sections were stained with an anti-CD31 antibody (BDPharmingen), a blood vessel marker, to assess tumor angiogen-

esis. A cross-section of the tumor tissue was photographed atmagnification �100 under the Nikon Eclipse TS100 microscopeand blood vessels stained with anti-CD31 antibody were countedper field for the quantification of tumor angiogenesis. The lumensizes of the blood vessels were also quantified using the Zen imageanalysis tool (Carl Zeiss).

Statistical analysisStatistical analysis of the data was performed using Graph-

Pad Prism version 5.0. Statistical significance, determined bythe two-tailed Student t test (between two groups) and two-tailed single sample t test (test group vs. hypothetical controlvalue), is indicated throughout by the following: �, P � 0.05and ��, P � 0.01.

ResultsRacemic itraconazole and its stereoisomers inhibited HUVECproliferation with different potencies

Racemic itraconazole and each of the four purified cis-stereo-isomers were tested for HUVEC proliferation by measuring theability of the cells to incorporate [3H]-thymidine into DNA.Itraconazole dose-dependently inhibited the thymidine incorpo-ration of HUVEC with an IC50 value of 137 nmol/L (Table 1).Under the same assay conditions, the 2S4R stereoisomers, includ-ing IT-A and IT-C, showed more potent inhibition againstHUVEC proliferation compared with itraconazole with IC50

values of 103 and 98 nmol/L, respectively. However, the 2R4Sstereoisomers including IT-B and IT-D were less potent thanitraconazole with IC50 values of 223 and 199 nmol/L, respec-tively. Collectively, 2S4R stereoisomers were roughly two timesmore potent than 2R4S stereoisomers in HUVEC proliferation.A one-to-one combination of IT-A and IT-C or IT-B and IT-Dshowed no synergy in HUVEC proliferation, suggesting thatthey might share the same mechanism of action to inhibitHUVEC proliferation (Table 1).

Itraconazole stereoisomers inhibited VEGFR2 glycosylation,mTOR activity, and cholesterol trafficking in HUVEC

We next determined whether all stereoisomers still sharea common mechanism of action to inhibit endothelial cell

Table 1. Chemical structures of itraconazole cis-stereoisomers and their antiproliferative activities against HUVEC

Compound Stereochemistry IC50 (nmol/L) 95% CI (nmol/L) Inhibitory indexa Pb

Itraconazole Racemic 137.8 127.6–148.9 1 —

IT-A 2S4R20R 103.6 91.3–117.5 0.75 0.042IT-B 2R4S20R 223.1 199.4–249.6 1.62 0.001IT-C 2S4R20S 98.3 86.8–111.4 0.71 0.003IT-D 2R4S20S 199.5 182.6–218.0 1.45 0.005IT-A þ IT-C (1:1) 101.2 91.4–112.0 0.73 0.013IT-B þ IT-D (1:1) 211.0 187.4–237.5 1.53 0.009

NOTE: Mean IC50 values and their 95% confidence intervals (CI) from four independent experiments are shown.aInhibitory index represents the ratio of the IC50 value of stereoisomers or mixtures to racemic itraconazole.bP values of IC50s for stereoisomers versus racemic itraconazole.

Shim et al.





proliferation by assessing three previously identified activitiesof itraconazole, that is, inhibition of VEGFR2 glycosylation,mTOR, and cholesterol trafficking (NPC phenotype). Itracona-zole inhibited VEGFR2 glycosylation in HUVEC as evidencedby the dose-dependent shift of high-molecular weight, hyper-glycosylated form of VEGFR2 to the low-molecular weight,hypo-glycosylated form. All four stereoisomers inhibitedVEGFR2 glycosylation with slight different potencies. All fourstereoisomers as well as racemic itraconazole dose-dependentlyinhibited mTOR activity as measured by S6 kinase (S6K)phosphorylation, a downstream target of mTOR. It was appar-ent that IT-A and IT-C (2S4R series) were more potent than IT-Band IT-D (2R4S series) for the inhibition of VEGFR2 glycosyl-ation and mTOR activity (Fig. 1A). Next, we determined theeffects of itraconazole and the stereoisomers on cholesteroltrafficking in HUVEC. Cellular cholesterol was fluorescentlylabeled by filipin and the cholesterol distribution was observedunder a confocal microscope. HUVEC were treated with eithervehicle (DMSO) or 50 nmol/L of itraconazole or stereoisomers.The concentration of 50 nmol/L that we used here was thelowest concentration of itraconazole that is sufficient to inducean NPC phenotype. At this concentration, IT-A and IT-C as wellas itraconazole showed an NPC phenotype as judgedby the accumulation of cholesterol fluorescence in the peri-nuclear region of HUVEC. However, IT-B and IT-D failed toshow NPC phenotype at this concentration (Fig. 1B). At con-centrations over 100 nmol/L, IT-B and IT-D were able to inducean NPC phenotype in HUVEC (data not shown). Thus, forinhibition of cholesterol trafficking, mTOR activity andVEGFR2 glycosylation, 2S4R series were more potent than2R4S series, which correlated with the relative potency of thestereoisomers against HUVEC proliferation.

Inhibition of angiogenesis by itraconazole stereoisomers isdependent on cholesterol

We next used an in vitro tube formation assay to assess theactivity of the stereoisomers. Upon seeding on top of solidifiedMatrigel, individual endothelial cells migrate and elongate toform tubule-like networks, reminiscent of new blood vesselformation (Fig. 1C). Treatment of HUVEC with 3 mmol/Litraconazole, IT-A and IT-C strongly inhibited HUVEC tubeformation (Supplementary Fig. S1A and S1B). However, treat-ment with IT-B and IT-D showed little or no inhibition ofHUVEC tube formation at the same concentration (Supple-mentary Fig. S1A and S1B), again demonstrating that IT-A andIT-C are more potent than the other two stereoisomers. Tosee whether the compounds are able to inhibit pre-formedtubes of HUVEC, itraconazole and stereoisomers were added6 hours after cell seeding on Matrigel and the incubationwas continued for an additional 18 hours. At a higher concen-tration (6 mmol/L), itraconazole and the two potent stereoi-somers (IT-A and IT-C) significantly inhibited the pre-formedtubes of HUVEC, again verifying that IT-A and IT-C are morepotent anti-angiogenic agents than IT-B and IT-D (Supplemen-tary Fig. S2A and S2B). As the antiproliferative activity ofitraconazole was shown to be dependent on cholesterol traf-ficking (22), we conducted a rescue experiment of angiogenesisinhibition by itraconazole and stereoisomers with LDL. Treat-ment of HUVEC with LDL slightly enhanced the tube formationof HUVEC. When HUVEC was treated with itraconazole orstereoisomers together with LDL, the inhibition of tube for-

mation was completely reversed to the control level (Fig. 1Cand D). Furthermore, inhibition of VEGFR2 glycosylation byitraconazole or stereoisomers was also completely reversed byLDL (Fig. 1E and F). These results indicated that, similar toitraconazole, the antiangiogenic activity of the different stereo-isomers is dependent on their ability to inhibit cholesteroltrafficking.

Racemic itraconazole and its stereoisomers inhibitedHUVEC-pericyte co-culture tube formation

Pericytes are vascular-supporting cells that interact withendothelial cells and play an important role in initiation ofangiogenesis by guiding the endothelial cell sprouting andstabilizing the vessels (33). Therefore, pericytes, together withendothelial cells, have been recognized as a putative target incancer treatment (34). To examine the effects of racemic itra-conazole and its stereoisomers on pericyte angiogenesis, weperformed pericyte alone and pericyte-HUVEC co-culture tubeformation experiments. Under our experimental conditions,and as shown in other reports, pericytes alone could not formtubular networks on Matrigel (data not shown; ref. 35). Wetherefore used HUVEC-pericyte co-culture tube formation at aratio of 30:1 of HUVEC and pericytes, respectively. At a con-centration of 3 mmol/L, racemic itraconazole, IT-A and IT-Csignificantly inhibited pericyte tube networks as well as overallco-culture tube formation on Matrigel (Fig. 2A and B). Incontrast, IT-B and IT-D showed negligible effects on co-culturetube formation. These results indicated that racemic itracona-zole and the two stereoisomers IT-A and IT-C inhibited angio-genesis in the presence of pericytes.

Itraconazole stereoisomers possess distincthepatotoxicity profiles

Having shown that the 2S4R pair of itraconazole stereoi-somers are more potent than 2R4S pair in all in vitro angio-genesis-related assays, we wondered whether the stereochem-istry had a similar influence on the relative hepatotoxicity ofthe different stereoisomers. Using freshly prepared humanprimary hepatocytes, we determined three representative mar-kers of hepatotoxicity, including hepatocyte death, intracellularGSH depletion, and alanine transaminase (ALT) release. Asexpected, incubation with 2 and 10 mmol/L itraconazoleincreased primary human hepatocyte death 1.6- and 1.8-fold(P � 0.01), respectively, as compared with vehicle control(Fig. 3A). Similarly, itraconazole effects on GSH depletion(76%; P � 0.01) and ALT levels in the cell culture medium(92.5 IU/L; P � 0.01) were consistent with the hepatotoxicity(Fig. 3B and C). Interestingly, IT-A and IT-B (both have a 20Rstereochemistry) appeared to have slightly higher hepatotox-icity than racemic itraconazole as judged by induction of celldeath by 2.2- (P � 0.01) and 2.0-fold (P � 0.01), respectively,at a final concentration of 10 mmol/L as well as increases inGSH depletion [70% (P � 0.01) and 67% (P � 0.01),respectively] and extracellular ALT levels [117 IU/L (P �0.01) and 128 IU/L (P � 0.01), respectively]. In contrast, IT-C and IT-D (both have a 20S stereochemistry) did not showappreciable hepatotoxicity, as judged by cell death, hepaticGSH levels or the concentration of ALT in the culture medium(Fig. 3A–C). These results were quite unexpected as the hep-atotoxicity profile is totally different from the antiangiogenicprofile of stereoisomers. Thus, the stereochemistry at the C2






and C4 chiral centers of itraconazole appeared to be importantfor the antiangiogenic activity whereas that of the C20 chiralcenter influences hepatotoxicity (the chemical structure ofitraconazole is shown in Table 1).

We next assessed the hepatotoxicity of the stereoisomers inmice. Femalemice were administeredwith vehicle or itraconazolestereoisomers (60 mg/kg) i.p. once daily for 10 days. After drugtreatment, we first determined the ALT levels in the blood samples

pS6K

S6K

VEGFR2

IT-A ( IT-B (mol/L) IT-C (mol/L) IT-D (mol/L) mol/L)A

DMSO IT-DIT-CIT-BIT-AITRA

B

ITRA ( mol/L)

DMSO

LDL

IT-CIT-AITRA

LDL+IT-CLDL+IT-ALDL+ITRA

VEGFR2

GAPDH

VEGFR2

GAPDH

FE

DC

Figure 1.Itraconazole and stereoisomers inhibit VEGFR2 glycosylation, mTOR activity, cholesterol trafficking, and tube formation in endothelial cells. A, HUVECwere treatedwith itraconazole (ITRA) and its stereoisomers at various concentrations (0, 0.125, 0.25, 0.5, and 1 mmol/L) for 24 hours. Whole-cell lysates were subjected toWestern blot analysis using antibodies specific for VEGFR2, phospho-S6K (pS6K), and total S6K. In the VEGFR2 Western blots, the top and the bottom bandscorrespond to hyperglycosylated and hypoglycosylated forms, respectively. B, HUVECwere treated with 50 nmol/L of itraconazole and stereoisomers for 24 hours.The cells were stained with filipin and the fluorescent images were captured under a confocal microscope. C, HUVEC were seeded on Matrigel and treated withitraconazole and stereoisomers at the concentration of 3 mmol/L and LDL (50 mg/mL) for 16 hours. Cells were then stained with Calcein-AM and the tube formationwas observed under a fluorescent microscope. D, total tube lengths, sizes and number of junctions from the fluorescence images were quantified using theAngioQuant software and plotted using GraphPad Prism. Data, mean � SE of three independent experiments. �� , P � 0.01 for drug alone versus drug þ LDL.E and F, HUVEC were treated with indicated concentrations of itraconazole and stereoisomers with or without LDL (50 mg/mL) for 24 hours. Western blot analysiswas done using antibodies specific for VEGFR2 and GAPDH as a loading control.

Shim et al.





of the mice, but found no significant change in blood ALT levelsfrom themice treated with stereoisomers (data not shown). Next,livers from each mouse were isolated and processed for H&Estaining. The livers from vehicle or IT-C–treated mice appearednormal, whereas those from IT-A–treated mice showed abnor-malities, that is, white colored and quite sticky to surroundingtissues. In comparison, livers in mice treated with itraconazolelooked partially abnormal (data not shown). From the H&Estaining of the liver sections, we found that IT-A caused severefatty liver in the mice (Fig. 3D). Mice treated with racemicitraconazole had partial fatty liver, whereas livers from thosetreated with vehicle or IT-C were normal (Fig. 3D). Once again,IT-C showed less hepatotoxicity in mice than either IT-A or theracemic itraconazole, consistent with the results from humanhepatocytes.

IT-C inhibited angiogenesis in vivoWe next determined and compared the in vivo antiangiogenic

activity of IT-A and IT-C along with racemic itraconazole as acontrol. We excluded the 2R4S series (IT-B and IT-D) from theseexperiments because they had lower antiangiogenic activitythan 2S4R series. Female mice were pre-treated with IT-A, IT-C, or itraconazole at the doses of 20 and 50 mg/kg for eachcompound for 2 days. The mice were then implanted subcu-taneously with Matrigel plugs containing angiogenic factors,including bFGF and VEGF. Treatment with vehicle or com-pounds was continued once daily for additional 8 days. Micewere sacrificed and Matrigel plugs were isolated from the micefor the immunohistologic analysis of blood vessel infiltrationinto the Matrigel using anti-CD31 (endothelial cell marker)and anti-NG2 (pericyte marker) antibodies. In addition to theantibody staining, entire population of cells infiltrated into the

Matrigel plugs was analyzed using Masson's trichrome staining(Supplementary Fig. S3A and S3B). Compared with the vehiclecontrol, itraconazole inhibited the number of CD31-positiveblood vessels infiltrated into the Matrigel by 19% and 46%at the dosage of 20 and 50 mg/kg, respectively (Fig. 4A and B).IT-A also inhibited the CD31-positive blood vessels by 19%and 48%. IT-C showed more significant inhibition on CD31-positive Matrigel angiogenesis, with 41% and 60% inhibition at20 and 50 mg/kg, respectively (Fig. 4A and B). Similar resultswere observed in NG2-positive pericyte staining. IT-C exhibitedthe strongest inhibition on the infiltration and vessel formationby NG2-positive pericytes, with 47% and 70% inhibition at 20and 50 mg/kg, respectively (Fig. 4C and D). These resultssuggested that IT-C is more active than IT-A for the antiangio-genic activity.

IT-C inhibited tumor growth and tumor-inducedangiogenesis in mice

Taking into consideration both the antiangiogenic activity andhepatotoxicity, IT-C was deemed superior to all other stereoi-somers due to a combination of its higher antiangiogenic potencyand lack of hepatotoxicity in both human cells andmice. We thusassessed the antitumor activity of IT-C in two tumor xenograftmodels (breast cancer and prostate cancer) in parallel with race-mic itraconazole. We also took the opportunity to determine theeffect of IT-C on tumor-induced angiogenesis. For the breastcancer xenograft model, female athymic nude mice bearingHCC1954 cells were treated i.p. with vehicle, itraconazole, orIT-C at 25 and 50 mg/kg for each compound, once daily, for30 days. Racemic itraconazole at 50 mg/kg significantly inhibitedthe growth of HCC1954 xenografts compared with the vehiclealone (Fig. 5A). Tumor volume was reduced by 40% at day 28 of

DMSO ITRA IT-A IT-B IT-C IT-D

Brig

htfie

ldP

eric

yte

Mer

ge

A

B

Figure 2.Itraconazole and stereoisomers inhibitthe tube formation of HUVEC-pericyteco-culture. A, pericytes were labeledwith Calcein-AM for 30 minutes beforebeing harvested for tube formation.HUVEC and pericyte mixture (30:1ratio) were treated with compounds at3 mmol/L, seeded on Matrigel andallowed to form tube networks for20 hours. Total cells (HUVEC andpericytes) are shown in the brightfieldimages and pericytes are shown in thegreen fluorescent images; scale bar,200 mm. B, total tube length from thegreen fluorescence images werequantified as the pericyte tubeformation using the AngioQuantsoftware and plotted using GraphpadPrism. Data, mean � SE of threeindependent experiments. � , P � 0.05;�� , P � 0.01 for control versus drug.






treatment with 50 mg/kg itraconazole compared with the vehiclecontrol. Importantly, IT-C showed more pronounced inhibitionof the tumor xenograft growth than itraconazole at both 25 and50 mg/kg (Fig. 5B). It inhibited the tumor volume by 53% at day28 at 50 mg/kg. There were no apparent toxicities associated withitraconazole or IT-C based on assessment of mouse body weight(Supplementary Fig. S4). Similar results were observed in theCWR22Rv1 prostate cancer xenograft model. Treatment of maleathymic nude mice with either itraconazole (60 mg/kg) or IT-C(60mg/kg) significantly inhibited the tumor growth (Fig. 5C andD). To assess tumor-associated angiogenesis in the breast cancerxenografts, the tumor tissues were harvested from the mice andprocessed for immunohistochemical analysis of CD31, a bloodvessel marker. Compared with the vehicle, both racemic itraco-nazole and IT-C significantly inhibited angiogenesis in theHCC1954 xenografts (Fig. 5E and F), with IT-C beingmore potentthan itraconazole.

DiscussionAlthough itraconazole has been widely used to treat fungal

infections, several adverse effects have been reported. The mostfrequent side effects associated with itraconazole are gastrointes-tinal complaints including nausea, vomiting, diarrhea, flatulenceand constipation (36). Fortunately, these adverse effects aremostly transient and mild, and thus are tolerable to patients(37). However, rare but severe hepatoxicity associated with itra-conazole has also been reported. Up to 7% of patients receivingitraconazole have elevated ALT levels (24). Moreover, fatalitiesdue to hepatic failure have been reported in patients receivingitraconazole (38). Major efforts have been made to overcome theadverse effects of itraconazole on the liver. A pulse therapy ofitraconazole (typically, 400mg itraconazole twice daily for 1weekfollowed by a three-weeks holiday) was introduced in superficialfungal infections based on the pharmacokinetic (PK) propertiesof itraconazole (39). Upon cessation of drug intake, itraconazole

is rapidly cleared from systemic circulation (7–10 days), butpersists for 3 to 4 weeks in the stratum corneum and for up to6 to 12 months in the nail (40). This PK property of itraconazolemakes pulse therapy for onychomycosis and dermatomycosespossible, which could significantly reduce the hepatotoxicity-related adverse effects while showing therapeutic efficacies similarto continuous therapy (41). However, this strategy may notbe applicable for the cancer therapy, which requires a high doseof itraconazole in systemic circulation. Thus, the hepatotoxicityassociated with itraconazole still poses a significant challengefor the long-term, high-dose treatment of cancer patients withitraconazole.

Hepatotoxicity associated with itraconazole has long beenrecognized, but the mechanism underlying the liver toxicity ofitraconazole has remained obscure. Cytochrome P450, specifi-cally CYP3A4, is a main liver enzyme responsible for the metab-olism of itraconazole (42). Itraconazole also inhibits theenzyme activity of CYP3A4 (26). When rats were pretreatedwith phenobarbital, an inducer of cytochrome P450, hepato-toxicity induced by itraconazole was significantly reduced(27, 37). On the other hand, pretreatment of rats with SKF525A, an inhibitor of cytochrome P450, significantly enhanceditraconazole-induced hepatoxicity (27). These results suggestthat inhibition of cytochrome P450, probably CYP3A4, byitraconazole is, at least in part, responsible for itraconazole-induced hepatotoxicity. Commercial itraconazole currentlyused in the clinic is a racemic mixture of four cis-stereoisomers.It is interesting to note that CYP3A4 metabolizes itraconazolein a stereoselective manner. Only IT-B and IT-D are metabo-lized by CYP3A4 among the four stereoisomers. Although allfour stereoisomers are strong binders and inhibitors ofCYP3A4, IT-B, and IT-D have 2- to 4-fold stronger bindingaffinity for CYP3A4 and 2- to 4-fold lower IC50 values forinhibition of the enzymatic activity of CYP3A4 in comparisonwith IT-A and IT-C (28). In the present study, we confirmed thatIT-A and IT-C were more potent inhibitors of angiogenesis than

Figure 3.Itraconazole and each stereoisomer have distinct hepatotoxicity profiles. A to C, freshly prepared human primary hepatocyteswere incubatedwith itraconazole andstereoisomers at the indicated concentrations for 72 hours. Cell viability of the hepatocytes (A), intracellular glutathione levels from the hepatocyte lysates(B), and the alanine aminotransaminase (ALT) levels from the hepatocyte culture media (C) were measured as described in the Materials and Methods. � , P� 0.05;�� , P� 0.01 for 10 mmol/L versus 0 mmol/L (for A and B, two-tailed single sample t test). �� , P� 0.01 for drug versus DMSO control (for C, two-tailed Student t test).D, female BALB/cAnNCr mice were treated i.p. with itraconazole and stereoisomers at the dosage of 60 mg/kg for 10 days. Livers from each mouse wereisolated and processed for H&E staining. Representative images from five mice per group are shown; scale bar, 100 mm.

Shim et al.





IT-B and IT-D. In contrast, IT-A and IT-B were much morehepatotoxic than IT-C and IT-D both in mice and in humanhepatocytes. These results suggest that inhibition of CYP3A4by itraconazole or metabolism of itraconazole by CYP3A4may not be solely responsible for its hepatotoxicity. Oneinteresting possibility is that a cytochrome P450 enzymewith specificity for the C20 chiral center of itraconazole couldbe the main enzyme mediating the hepatotoxicity of itraco-nazole. In addition, the segregation of antiangiogenic activityand hepatotoxicity of the four stereoisomers of itraconazolesuggests that the cellular target protein(s) responsible forangiogenesis inhibition and hepatotoxicity might be different.Answers to these questions may become clear upon identifi-cation of the molecular targets mediating the different effectsof itraconazole.

Although itraconazole was shown to elevate serum ALT levelsin rats, it did not increase the levels in mice in this study. Instead,itraconazole and IT-A induced moderate to severe fatty liver inmice. Rats and mice have shown different hepatotoxicityresponses to certain compounds, such as coumarin (43).Interstrain differences of hepatotoxicity responses have alsobeen observed in mice treated with cocaine and phenobarbital(44). This is thought to be due to the different liver metabolicenzymes present in different strains and animal species. Itra-conazole has shown similar hepatotoxicity profiles in humansand rats, suggesting that they respond similarly to this com-pound. In mice, the compound did not show ALT elevation,but induced fatty liver, suggesting that racemic itraconazoleand IT-A still have negative impact on mice liver but in a waydifferent from rats or humans. However, IT-C at doses up to 60

Figure 4.Itraconazole and the 2S4R series of stereoisomers inhibit Matrigel angiogenesis in vivo. A and C, female BALB/cAnNCrmice with Matrigel implantationwere treatedi.p. with itraconazole, IT-A and IT-C at dosages of 20 and 50 mg/kg for total of 10 days (2-day pre-treatment and 8-day post-treatment of Matrigel implantation).Matrigel plugs with mouse inner skin were isolated and processed for immunohistochemical (IHC) staining of CD31 (endothelial cell marker) and NG2(pericyte marker). Representative IHC images of CD31 staining (A) and NG2 staining (C) are shown. B and D, the number of CD31-positive vessels (B) andNG2-positive cells (D) were counted from the IHC samples and plotted using Graphpad Prism. Data, mean � SE of three independent IHC samples per group.� , P � 0.05; �� , P � 0.01 for each drug versus vehicle control; scale bar, 1.79 mm.






mg/kg did not cause apparent adverse effects on the mouseliver. In the experiments using human primary hepatocytes, IT-C showed no apparent signs of hepatotoxicity at concentra-tions up to 10 mmol/L for all three hepatotoxicity markers. Incontrast, IT-A was strongly hepatotoxic and racemic itracona-zole showed a medium degree of hepatotoxicity. Together,these results points to IT-C as the most promising candidatefor future clinical development with improved potency anddecreased hepatotoxicity in comparison with racemic itraco-nazole. That the C20 stereochemistry has a major influence onthe hepatotoxicity of itraconazole should also facilitate thedevelopment of next generations of itraconazole analogs asnew leads of antiangiogenic and anticancer drugs.

Several antiangiogenic agents, such as sunitinib and sorafenib,have successfully entered into the clinic as targeted cancer ther-apies. Although these agents have dramatically changed themanagement of cancer in patients these days, many clinicalchallenges still remain, including the huge financial burden topatients and the emergence of drug resistance that occurs withmost of the kinase inhibitor drugs. Development of new anti-angiogenic and anticancer agents from existing non-cancer drugs(the drug repositioning strategy) offers the possibility of reducingthe financial burden to patients (45). In addition, this approachcan also diversify molecular targets as exemplified with itracona-zole, which is not a kinase inhibitor. Development of agents withdiverse molecular targets provides better opportunities to find

Figure 5.Itraconazole and IT-C inhibit the tumor growth and angiogenesis in vivo. A and B, female athymic nude mice (BALB/c, nu/nu-NCr) bearing HCC1954 humanbreast cancer cells were treated with itraconazole (ITRA, A) or IT-C (B) for 30 days. C and D, male athymic nude mice (BALB/c, nu/nu-NCr) bearingCWR22Rv1 human prostate cancer cells were treated with itraconazole (ITRA, C) or IT-C (D) for 24 days. Tumor volume was measured periodically by a verniercaliper and plotted using GraphPad prism. Data, mean � SE of tumor volumes in each group (n¼ 6–7 mice). � , P � 0.05; ��, P� 0.01 for the drug versus vehiclecontrol at each time point. E, HCC1954 tumor xenograft tissues were processed for immunohistochemical (IHC) staining of a blood vessel marker, CD31.Representative images of CD31 staining of tumor sections from each group are shown; scale bar, 100 mm. F, the number of CD31-positive blood vessels werecounted from photos taken under a bright-field microscope and plotted using GraphPad Prism. The lumen size of blood vessels were measured usingthe Zen image analysis tool. Data, mean � SE of the number and the lumen size of blood vessels from the IHC images (n ¼ 5/group). �� , P � 0.01 for each drugversus vehicle control.

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drug combinations with standard chemotherapy or current tar-geted drugs to reduce the emergence of cancer drug resistance. Asevidenced from recent clinical investigations, itraconazole hasshown positive clinical outcome in patients with non–small celllung cancer, metastatic prostate cancer, and basal cell carcinoma.It also showed prolonged survival in patients with late-stageovarian, triple-negative breast, and metastatic pancreatic cancer.IT-C, as an equipotent isomer of itraconazole with reducedhepatotoxicity, will have a wider range of therapeutic windowthan racemic itraconazole, and thus is a promising lead for futuredevelopment as an antiangiogenic and anticancer drug.

Disclosure of Potential Conflicts of InterestJ.O. Liu has ownership interest (including patents) in Accelas Pharmaceu-

ticals and Hedgepath Pharmaceuticals. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: J.S. Shim, N.N. Bumpus, S.A. Head, J.O. LiuDevelopment of methodology: J.S. Shim, R.-J. Li, N.N. Bumpus

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J.S. Shim, R.-J. Li, N.N. Bumpus, E.J. Yang, J. LvAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.S. Shim, R.-J. Li, N.N. Bumpus, J.O. LiuWriting, review, and/or revision of the manuscript: J.S. Shim, R.-J. Li,N.N. Bumpus, S.A. Head, J.O. LiuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): K. Kumar, W. ShiStudy supervision: J.O. Liu

Grant SupportThis work was supported in part by R01CA184103, the Flight Attendant

Medical Research Institute (J.O. Liu), the Johns Hopkins Institute for Clinicaland Translational Research (ICTR), which is funded in part by grant numberUL1 TR 001079 and the PhRMA foundation fellowship in Pharmacology/Toxicology (to S.A. Head).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 7, 2015; revised December 11, 2015; accepted December 30,2015; published OnlineFirst January 22, 2016.

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Shim et al.




2016;22:2709-2720. Published OnlineFirst January 22, 2016.Clin Cancer Res Joong Sup Shim, Ruo-Jing Li, Namandje N. Bumpus, et al. Different Stereoisomers of ItraconazoleDivergence of Antiangiogenic Activity and Hepatotoxicity of

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