TAS-116, a Highly Selective Inhibitor of Heat Shock Protein 90a … · Small Molecule Therapeutics TAS-116, a Highly Selective Inhibitor of Heat Shock Protein 90a and b, Demonstrates

Small Molecule Therapeutics

TAS-116, a Highly Selective Inhibitor of HeatShock Protein 90a and b, Demonstrates PotentAntitumor Activity and Minimal Ocular Toxicity inPreclinical ModelsShuichi Ohkubo1, Yasuo Kodama1, Hiromi Muraoka1, Hiroko Hitotsumachi2,Chihoko Yoshimura1, Makoto Kitade1, Akihiro Hashimoto1, Kenjiro Ito1, Akira Gomori1,Koichi Takahashi1, Yoshihiro Shibata1, Akira Kanoh1, and Kazuhiko Yonekura1

Abstract

The molecular chaperone HSP90 plays a crucial role in cancercell growth and survival by stabilizing cancer-related proteins. Anumber of HSP90 inhibitors have been developed clinically forcancer therapy; however, potential off-target and/or HSP90-related toxicities have proved problematic. The 4-(1H-pyrazolo[3,4-b]pyridine-1-yl)benzamide TAS-116 is a selectiveinhibitor of cytosolic HSP90a and b that does not inhibit HSP90paralogs such as endoplasmic reticulum GRP94 or mitochon-drial TRAP1. Oral administration of TAS-116 led to tumorshrinkage in human tumor xenograft mouse models accompa-nied by depletion of multiple HSP90 clients, demonstrating thatthe inhibition of HSP90a and b alone was sufficient to exertantitumor activity in certain tumor models. One of the mostnotable HSP90-related adverse events universally observed todiffering degrees in the clinical setting is visual disturbance. A

two-week administration of the isoxazole resorcinol NVP-AUY922, an HSP90 inhibitor, caused marked degeneration anddisarrangement of the outer nuclear layer of the retina andinduced photoreceptor cell death in rats. In contrast, TAS-116did not produce detectable photoreceptor injury in rats, prob-ably due to its lower distribution in retinal tissue. Importantly, ina rat model, the antitumor activity of TAS-116 was accompaniedby a higher distribution of the compound in subcutaneouslyxenografted NCI-H1975 non–small cell lung carcinoma tumorsthan in retina. Moreover, TAS-116 showed activity against ortho-topically transplanted NCI-H1975 lung tumors. Together, thesedata suggest that TAS-116 has a potential to maximize antitumoractivity while minimizing adverse effects such as visual distur-bances that are observed with other compounds of this class.MolCancer Ther; 14(1); 1–9. �2014 AACR.

IntroductionThe molecular chaperone heat shock protein 90 (HSP90) is a

highly conserved protein that regulates the activity and stabilityof a diverse range of "client" proteins (1, 2). HSP90 clientsinclude many of the cancer-related proteins necessary for tumordevelopment, including receptor tyrosine kinases, signal trans-ducers, cell-cycle regulators, and transcriptional factors (1–3).HSP90 is reported to be specifically overexpressed (4, 5) and toexist as activated multi-chaperone complexes in cancer cells andcancer tissues (6, 7); indeed, it has been proposed that cancersare highly "addicted" to HSP90 for their survival and prolifer-ation (1). HSP90 has therefore emerged as an attractive target

for cancer interventions, and many HSP90 inhibitors have beendeveloped (8).

Though HSP90 inhibitors have been shown in different pre-clinical models to exert their potent antitumor activities bydestabilizing HSP90 clients (9–13), they have also been shownto have limited single-agent activity in the clinical setting(3, 8, 14). One explanation for this discrepancy may be that thelevels of drug exposure reached in patients are insufficient toeffectively inhibit tumor cell HSP90 because of undesirable off-target and/or HSP90-related adverse events. For example, livertoxicity was a major limitation in the clinical development of thefirst-generation ansamycin derivatives tanespimycin (17-AAG)and alvespimycin (17-DMAG; ref. 15). It is possible that thisexcess liver toxicity is related to the quinone structure of ansa-mycin (16). The second generation of HSP90 inhibitors are basedon a diverse variety of chemical scaffolds and exhibit less livertoxicity (11, 13). However, these compounds cause other dose-limiting toxicities, some of which are difficult to manage. Forexample, neurological toxicities such as syncope and dizzinesswere seen in a phase I trial of the purine analogue BIIB021 (17),although there is no evidence to support this to be target orpurine-scaffold related. In clinical trials of otherHSP90 inhibitors,the most commonly observed HSP90-related adverse events havebeen visual disorders such as night blindness, photopsia, blurredvision, and visual impairment, though the frequency and degreevaried among the compounds (18–21). For example, 43% of

1Tsukuba Research Center, Taiho Pharmaceutical Co., Ltd., Tsukuba,Ibaraki, Japan. 2TokushimaResearchCenter,TaihoPharmaceuticalCo.,Ltd., Kawauchi-cho, Tokushima, Japan.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Shuichi Ohkubo, Taiho Pharmaceutical Co., Ltd., 3Okubo, Tsukuba, Ibaraki 300-2611, Japan. Phone: 81-29-865-4527; Fax: 81-29-865-2170; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-14-0219

�2014 American Association for Cancer Research.

MolecularCancerTherapeutics

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patients suffered visual disturbances in a phase I trial of NVP-AUY922 (21). Though HSP90-related visual disorders, whichhave included grade 3 events, are reversible (21), these adverseevents interferewith obtaining sufficient drug exposure because ofdrug interruption or discontinuation, or dose reduction.

In this report, we describe our preclinical characterization ofTAS-116, a novel, selective HSP90a and b inhibitor, and showthat, due to limited exposure of nontarget eye tissues and greaterexposure of target tumor tissues to the compound, TAS-116 has afavorable therapeutic index.

Materials and MethodsChemical compounds

The HSP90 inhibitors TAS-116 (Fig. 1A), NVP-AUY922,BIIB021, SNX-2112, SNX-5422, and ganetespib were synthesizedat Taiho Pharmaceutical, Co. Ltd., and 17-AAG and 17-DMAGwere purchased from Biotrend Chemikalien GmbH and LC Lab-oratories, respectively. Fluorescein isothiocyanate–labeled gelda-namycin (geldanamycin-FITC) was purchased from Enzo LifeSciences International, Inc.

AntibodiesThe following antibodies were purchased from Cell Signaling

Technology, Inc.: anti-phospho-HER2 (Tyr1221/1222; #2243);anti-HER2 (#2165); anti–phospho-HER3 (Tyr1289; #4791);anti–phospho-p44/42 MAPK (Thr202/Tyr204; #4370); anti-p44/42 MAPK (#9102); anti–phospho-AKT (Ser473; #4060); anti-AKT(#4691); anti–phospho-S6 Ribosomal Protein (Ser235/236;#4858); anti-S6 Ribosomal Protein (#2217); anti-DDR1(#3917); anti-HSP70 (#4876); and anti-GAPDH (#2118). Anti-HER3 (sc-285)was purchased fromSanta Cruz Biotechnology, Inc.Anti-FLT3 (MAB8121) was purchased from R&D Systems Inc.

Cell lines and cell cultureAll cell lines were purchased from American Type Culture

Collection in 2008 and 2009, and reauthenticated by means ofshort tandem repeat–based DNA profiling in 2012. Cells werecultured inMcCoy's 5Amedium (HCT116), RPMI 1640medium(NCI-N87 and NCI-H1975), or Iscove's modified Dulbecco'smedium (MV-4-11) supplemented with 10% FBS.

HSP90 affinity determinationRecombinant human HSP90AA1 (HSP90a), HSP90AB1

(HSP90b), and TRAP1 were purchased from Enzo Life SciencesInternational, Inc., and HSP90B1 (GRP94) was purchased fromATGen Co., Ltd. The interactions of TAS-116 with proteins fromthe HSP90 protein family were determined by means of a fluo-rescence polarization competitive binding assay with geldanamy-cin-FITC (22, 23). Each compound was incubated with recom-binant HSP90a, HSP90b, GRP94, or TRAP1 for 2 hours. Gelda-namycin-FITC was added and the mixture was incubated for afurther 5 hours. The concentration of each compound required toreduce by 50% the amount of recombinant proteins bound togeldanamycin-FITC (IC50) was determined and Ki values werecalculated from the IC50 values with the Cheng–Prusoff equation(23, 24).

Western blot analysisCells were lysed in lysis buffer [M-PER Mammalian Protein

Extraction Reagent (Thermo Fisher Scientific Inc.) supplementedwith cOmplete, Mini, Protease Inhibitor Cocktail (Roche AppliedScience) and PhosSTOP Phosphatase Inhibitor Cocktail (RocheApplied Science)]. Proteins were separated by SDS-PAGE andtransferred to polyvinylidene fluoride membranes (Bio-Rad Lab-oratories, Inc.). Membranes were blocked with Blocking One orBlocking One P blocking reagent (Nacalai Tesque, Inc.) andprobed with the appropriate primary antibodies. Themembraneswere then incubated with horseradish peroxidase–linked second-ary antibodies (Cell Signaling Technology, Inc.), and proteinswere visualized by means of luminol-based enhanced chemilu-minescence (Thermo Fisher Scientific Inc.). Luminescent imageswere capturedwith an LAS-3000 imaging system (Fuji Photo FilmCo., Ltd.).

Proximity ligation assayCells were grown on Lab-Tek II Chambered Coverglass

(Thermo Fisher Scientific Inc.), and then treated with TAS-116or 17-AAG for 8 hours. The cells were then fixed in 4% parafor-maldehyde and blocked in Duolink II solution (Sigma-AldrichCo. LLC). The slides were incubated with anti-LRP6 rabbit(ab134146; Abcam plc) and anti-GRP94 mouse (AM09037PU-S; Acris Antibodies, Inc.) antibodies, followed by incubation with

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Figure 1.Effects of TAS-116 on the levels of HSP90-related proteins and GRP94/LRP6 complex formation in HCT116 cells. A, chemical structure of TAS-116, 3-ethyl-4-[3-(1-methylethyl)-4-[4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl]-1H-pyrazolo[3,4-b]pyridin-1-yl]benzamide. B, dose-dependent change of HSP90-relatedproteins in HCT116 cells treatedwith TAS-116 or 17-AAG for 8 hours. Western blotting was performed by using 10 mg of cell lysate. C, in situ proximity ligation assay inHCT116 cells treated with 3 mmol/L of TAS-116 or 1 mmol/L of 17-AAG for 8 hours. Primarymouse and rabbit antibodies against GRP94 and LRP6were combined withsecondary proximity ligation assay probes (Sigma-Aldrich Co., LLC.). Interaction events are visible as red dots. Nuclei were stained blue. Scale bar, 20 mm.

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Duolink PLA Rabbit MINUS and PLA Mouse PLUS proximityprobes (Sigma-Aldrich Co. LLC). The proximity ligation assaywas performed by using a Duolink detection reagent kit (Sigma-Aldrich Co. LLC) according to the manufacturer's instructions.Nuclei were visualized by means of Hoechst 33342 (NacalaiTesque, Inc.) staining. Stained cells were imaged by using an LSM510 confocalmicroscopewith aPlan-Apochromat 20�/0.8 objec-tive (Carl Zeiss AG) and the LSM Image Browser software.

In vivo efficacy studiesCancer cells were subcutaneously implanted into six-week-old

male BALB/c nude mice (Clea Japan, Inc.) or six-week-old maleF344 nude rats (Clea Japan, Inc.) and allowed to grow. Five or sixanimals were assigned to each group for each experiment, andTAS-116 formulated in 0.5% (w/v) hydroxypropyl methylcellu-lose (Metolose 60SH-50; Shin-Etsu Chemical Co., Ltd.) in H2Owas administered orally every day at 3.6 to 14.0 mg/kg/day.Tumor volume was calculated with the formula [length �(width)2]/2. Statistical significance was calculated by using eitherDunnett or Student t test to assess the difference in tumor volumesbetween control (either vehicle or untreated) and TAS-116–trea-ted groups. P < 0.05 was considered statistically significant. Forpharmacodynamic analysis, tumors were harvested at the indi-cated time points after administration of TAS-116. The excisedtumors were homogenized in lysing matrix D (MP Biomedicals,LLC) containing lysis buffer, and lysates were prepared.

To establish an orthotopic lung tumor model, NCI-H1975was engineered to stably express luciferase (H1975-Luc) byusing a lentiviral vector (Life Technologies Corp.). H1975-Luccells (1 � 106 cells/mouse) were mixed with Matrigel (BD Bios-ciences) and directly transplanted into the left lobe of the eight-week-old BALB/c nude mouse lung. Six weeks after transplanta-tion, TAS-116 at 14.0 mg/kg/day was orally administered fivetimes a week for four weeks. Mice were intravenously adminis-tered D-luciferin potassium salt (Promega Corp.) at a dose of 150mg/kg, and then dorsal and ventral bioluminescence images weretaken with an IVIS Lumina II Imaging System (Perkin Elmer).Images were analyzed with the Living Image 3.1 software (PerkinElmer). All animal experiments were performedwith the approvalof the institutional animal care and use committee of TaihoPharmaceutical Co., Ltd. and carried out according to the guide-lines for animal experiments of Taiho Pharmaceutical Co., Ltd.

Histopathologic examination of rat eye tissuesTAS-116 was administered orally every day to six-week-old

male SpragueDawley (SD) rats (Charles River Laboratories Japan,Inc.) and six-week-old male Long–Evans (LE) rats (Institute forAnimal Reproduction) for two weeks. NVP-AUY922 formulatedin 5% (v/v) dimethyl sulfoxide and 5% (v/v) Tween 20 in salinewas administered intravenously to SD rats three times a week for

two weeks at 10.0 mg/kg/day. Eyes were harvested 24 hours afterthe final dose, fixed inDavidson solution for 48 hours, and storedin 10% (v/v) buffered formalin solution before routine paraffinembedding and sectioning. Eye sections underwent hematoxylinand eosin (HE) staining, and terminal deoxynucleotidyl transfer-ase dUTP nick end labeling (TUNEL; ApopTag Plus PeroxidaseIn Situ Apoptosis Detection Kit; Merck Millipore).

Quantitation of plasma, tumor, and retinal tissue exposure tothe test compounds

Test compounds were administered orally or intravenously toSD, LE, or tumor-bearing F344 nude rats. Plasma, retina, and/ortumor samples were collected according to the sampling scheduleafter dosing. Retina or tumor samples were homogenized in PBS.Plasma and homogenized samples were analyzed by means ofliquid chromatography with tandem mass spectrometry or high-performance liquid chromatography. The pharmacokinetic para-meters of TAS-116 inmice, rats, and dogswere calculated by usingnoncompartmental methods (25).

ResultsTAS-116 selectively inhibits HSP90a and b

TAS-116 (Fig. 1A) is a novel, small-molecule HSP90 inhibitorthat was discovered during a multiparameter lead optimizationcampaign to have high target-selectivity for certain HSP90 pro-teins (26). In the present study, TAS-116 inhibited geldanamycin–FITC binding to HSP90 proteins with Ki values of 34.7 nmol/L,21.3 nmol/L, >50,000 nmol/L, and >50,000 nmol/L for HSP90a,HSP90b, GRP94, and TRAP1, respectively (Table 1).OtherHSP90inhibitors, including 17-AAG, 17-DMAG,NVP-AUY922, BIIB021,and SNX-2112, also inhibited GRP94 and TRAP1 in additionto HSP90a and b, though generally these compounds inhibitedGRP94 andTRAP1 less than they didHSP90a andb. Furthermore,TAS-116 did not inhibit other ATPases such as HSP70 (IC50,>200 mmol/L; data not shown) or any of 48 different proteinkinases tested (IC50, all >30 mmol/L; Supplementary Table S1).

The HSP90 selectivity of TAS-116 was further evaluated at thecellular level. Treatment ofHCT116 cellswith either 0.1mmol/L of17-AAG or 0.3 mmol/L of TAS-116 for 8 hours resulted in reducedlevels ofDDR1,which interacts withHSP90a (27), and inductionof HSP70, which is a surrogate marker of cytosolic HSP90 inhi-bition (3, 28), at comparable levels for both compounds (Fig. 1B),thereby demonstrating the compounds' cytosolic HSP90 inhib-itory effects. GRP94 interacts with low-density lipoprotein recep-tor-related protein 6 (LRP6) and has a role in chaperoning LRP6(29). Therefore, LRP6/GRP94 complex formation was evaluatedin HCT116 cells in situ by means of a proximity ligation assay inwhich PLA-probe–labeled LRP6 or GRP94 molecules in closeproximity were detected as red dots (30). LRP6/GRP complex

Table 1. Binding affinity of HSP90 inhibitors to proteins in the HSP90 protein family

Binding affinity of HSP90 family proteins (Ki, nmol/L)Compound HSP90a HSP90b GRP94 TRAP1

TAS-116 34.7 � 8.4 21.3 � 3.0 >50,000 >50,00017-AAG 10.0 � 1.3 6.6 � 0.3 19.9 � 4.2 968.4 � 405.017-DMAG 5.8 � 0.5 4.8 � 0.4 12.6 � 1.0 110.7 � 14.7NVP-AUY922 4.5 � 0.1 3.2 � 0.1 27.0 � 2.6 71.8 � 4.8BIIB021 6.1 � 0.9 5.5 � 0.3 117.6 � 23.7 101.9 � 5.1SNX-2112 11.5 � 1.8 9.0 � 0.5 720.2 � 22.1 533.1 � 58.0

NOTE: Data are presented as mean � SEM (n ¼ 9).

Preclinical Characterization of HSP90a/b Inhibitor TAS-116

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was observed in untreated cells; however, treatment with 1mmol/L of 17-AAG for 8 hours caused a reduction in the amountof LRP6/GRP94 complex detected (Fig. 1C) and slightly reducedthe level of LRP6 (Fig. 1B), which is dependent on GRP94 for itscell-surface expression (29). In contrast, 3 mmol/L of TAS-116 didnot markedly affect the LRP6–GRP94 interaction. However, TAS-116 was unable to be used at higher concentrations becauseHCT116 cells became detached from the glass slide at concentra-tions of TAS-116 above 3 mmol/L. Our data suggest that 3 mmol/Lof TAS-116 does not inhibit GRP94, whereas 0.3 mmol/L of TAS-116 inhibits HSP90a and b in HCT116 cells.

TAS-116 shows favorable pharmacokineticsPharmacokinetic profiling of TAS-116 in rodent andnonrodent

species showed that TAS-116 was orally absorbed and had abioavailability of almost 100% in mice, 69.0% in rats, and73.9% in dogswithout special formulation (Table 2). In addition,the inhibitory activity of TAS-116 on cytochrome P450 (CYP)enzymes was minimal; only a limited number of isoforms wereinhibited (e.g., IC50, 21.6 mg/mL and 18.5 mg/mL for CYP2C8 andCYP2C9, respectively; Supplementary Table S2) above the effec-tive concentration of TAS-116 (Cmax of TAS-116 at a dose of14.0 mg/kg in mice was 6.7 mg/mL; Fig. 2A). Thus, it may bepossible to combine TAS-116 with other anticancer agents with-out inducing clinically relevant drug–drug interactions.

TAS-116 exhibits potent antitumor activity accompanied bydepletion of HSP90 clients in human tumor xenograft models

The antitumor activity of TAS-116 was evaluated in a HER2-expressing NCI-N87 (31) human gastric cancer xenograft mousemodel. The favorable pharmacokinetic profile of TAS-116was reflected in its dose-dependent antitumor activity; the T/C(tumor volume of TAS-116–treatedmice vs. vehicle-treatedmice)was 47%, 21%, and 9% for doses of 3.6 mg/kg, 7.1 mg/kg, and14.0 mg/kg, respectively (Fig. 2A). Chronic administration of TAS-116 was tolerable, with the average weight loss in mice notexceeding 10% during the treatment period (Fig. 2B). In thepharmacodynamic analysis, TAS-116 (14.0mg/kg) downregulatedHER2,HER3, andAKTprotein levels, leading to inhibitionof PI3K/AKT and MAPK/ERK signaling in xenografted tumors (Fig. 2C).

We then determined the antitumor activity of TAS-116 in anFLT3-ITD–expressing human acute myeloid leukemia (MV-4-11;ref. 32) xenograft mouse model. Oral administration of TAS-116at a dosage of 14.0 mg/kg/day for 14 days led to tumor shrinkage(Fig. 2D), accompanied by depletion of FLT3-ITD (Fig. 2E) in thismodel.

TAS-116 shows antitumor activity without inducing eye injuryin rats

In clinical trials of HSP90 inhibitors, visual disturbance hasbeen a frequently observed adverse event. Indeed, ocular toxicity

has been seen even in preclinical models (33). It is possible thatocular toxicity is associatedwith the extent towhich eye tissues areexposed to the drug (33). Therefore, we evaluated the tissuedistribution profile and ocular toxicity of TAS-116 in rats bearingsubcutaneous human tumors (NCI-H1975). NVP-AUY922 wasused as the reference compound because visual impairment wasfrequently observed in a phase I study of this compound (21).After administration of NVP-AUY922 intravenously or TAS-116orally, plasma, retina, and tumor samples were collected and theconcentration of each compound in the tissues was determined.Whereas NVP-AUY922 was distributed more in retina than plas-ma and was detectable at 24 hours after administration, TAS-116was distributed less in retina than plasma and was eliminatedfrom the retina within 24 hours in tumor-bearing nude rats(Fig. 3A). TAS-116 was also distributed less in retina than inplasma after intravenous administration (data not shown), indi-cating the rapid establishment of a distribution equilibriumbetween plasma and retina.

In a histopathologic examination of eye tissues to evaluatethe ocular toxicity of the compounds, NVP-AUY922 wasadministered intravenously at 10.0 mg/kg/day three times aweek for two weeks and TAS-116 was administered orally at12.0 mg/kg/day every day in albino SD rats. NVP-AUY922caused marked degeneration and disarrangement of the outernuclear layer of the retina, and the numbers of TUNEL-positiveapoptotic cells increased according to the retinal distribution ofthe compound (Fig. 3B). In contrast, administration of TAS-116resulted in no histologic changes, no abnormalities in thephotoreceptor cell ratio, and no increases in the number ofTUNEL-positive cells in the outer nuclear layer of the retina inSD rats. Although retinal pigment epithelium has numerousfunctions in the maintenance of the integrity and function ofphotoreceptors (34), TAS-116 was less distributed in retinathan in plasma and did not cause photoreceptor injury inpigmented LE rats (data not shown).

TAS-116 showed a much higher distribution in tumorthan in retina in tumor-bearing rats (Fig. 3A). Importantly, atwo-week administration of TAS-116 at a dose of 12.0 mg/kg/day resulted in antitumor activity accompanied by depletionof HSP90 client, mutated-EGFR, leading to inhibition of PI3K/AKT and MAPK/ERK signaling in the NCI-H1975 rat xenograftmodel (Fig. 3C and D). Again, TAS-116 did not producedetectable photoreceptor injury in this model (data notshown). These data suggested that TAS-116 did not cause oculartoxicity at the effective dose in rats.

To further evaluate the tissue distribution profiles of otherstructurally unrelated HSP90 inhibitors, we also measuredplasma, retinal, and tumoral exposure to 17-DMAG, ganete-spib, and SNX-5422 in a subcutaneous xenograft rat model.Though all the HSP90 inhibitors tested, including NVP-AUY922 and TAS-116, were highly distributed and retained in

Table 2. Pharmacokinetic profiles of TAS-116 in rodent and nonrodent species

Species n Dose (mg/kg) tmax (h) Cmax (mg/mL) t1/2 (h) AUC0–24 (mg�h/mL) F (%)

Mouse 3 3.6 1.0 1.8 8.2 16.4 �1003 7.1 2.0 3.1 2.5 28.3 �1003 14.0 1.0 6.7 4.4 60.2 �100

Rat 4 4.0 1.4 1.4 2.2 8.4 69.0Dog 3 3.0 2.7 0.6 3.9 5.1 73.9

Abbreviations: AUC, area under the concentration-versus-time curve; Cmax, maximum plasma concentration; F, bioavailability; tmax, time ofmaximum concentration;t1/2, elimination half-life.

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tumor (Fig. 3A and Supplementary Fig. S1), as previouslyreported (9, 11, 35, 36), TAS-116 was more rapidly eliminatedfrom retina (t1/2 ¼ 3.4 hours) than the other HSP90 inhibitors(t1/2 ¼ 7.1–19.1 hours), thereby demonstrating its lower retinaldistribution profile (Fig. 3A and Supplementary Table S3).Furthermore, the drug exposure ratio between tumor and retinaof each compound (T/R ratio) was calculated to assess whichcompound was distributed more in tumor than in retina; TAS-116 showed the highest T/R ratio (T/R ¼ 5.7) from among thetested HSP90 inhibitors (T/R ¼ 0.7–2.3), indicating its uniquetissue distribution profile (Fig. 3E).

TAS-116 significantly suppresses tumor growth in anorthotopic lung tumormodel expressing EGFR (L858R/T790M)

TAS-116 demonstrated antitumor activity without ocular tox-icity in a subcutaneous xenograft model. However, it is possiblethat the favorable tissue distribution profile of TAS-116 is onlyobserved under such subcutaneous conditions. To rule out thispossibility, we evaluated the antitumor activity of TAS-116 byusing anorthotopic human lung cancermodel inmice. Luciferase-transduced NCI-H1975 cells harboring EGFR (L858R/T790M;ref. 37) were directly transplanted into the lungs of mice, andTAS-116 was administered six weeks after transplantation. Tumorvolume was assessed by means of bioluminescence imaging(Fig. 4). Although the intensity of luminescence was increasedin the vehicle-treated group, TAS-116 administration resulted insignificantly decreased luminescence. These results suggested thatthe tumor growth in themouse lung was suppressed by treatmentwith TAS-116.

DiscussionHSP90 inhibitors simultaneously affectmultiple cancer-related

proteins, and are therefore potentially useful for treating targetedtherapy–resistant tumors that have arisen frommutations withinthe target itself or from activation of alternative signaling path-ways. For example, NVP-AUY922 is active against ALK-TKI– orEGFR-TKI–resistant non–small cell lung carcinoma (8, 38). How-ever, becauseHSP90 also plays a role in normal cells, it is essentialto balance efficacy and toxicity to maximize the antitumor poten-tial of HSP90 inhibitors. Here, we characterize a novel HSP90inhibitor that showed superior druggability with respect to targetselectivity and its pharmacologic, pharmacokinetic, and safetyprofiles.

TAS-116 represents one of the most selective HSP90a and binhibitors developed to date; it shows greater specific binding toHSP90a and b than to the highly homologous HSP90 familymembers GRP94 and TRAP1. The relative importance of the fourHSP90 paralogs with respect to cancer treatment remains poorlyunderstood. Although inhibition or downregulation of GRP94inhibits growth and induces apoptosis in human multiple mye-loma and HER2-expressing cell lines (39, 40), the role of GRP94in liver tumorigenesis remains controversial, with recent studiesindicating the potential for GRP94 to act as both a tumor sup-pressor and an oncogene (41, 42). Inhibition of TRAP1 by amitochondria-directed variant of 17-AAG leads to specific apo-ptosis in cancer cells (43); however, TRAP1 may also play, in acontext-dependent manner, a tumor suppressor or an oncogenerole (44). Our present finding that the highly selective HSP90a

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Figure 2.Antitumor activity of TAS-116 inhuman tumor xenograft mousemodels. Antitumor activity of TAS-116in NCI-N87 (A) and MV-4-11 (D)xenograft models. Athymic nude micebearing the indicated tumors(n ¼ 6 mice per group) were orallyadministered TAS-116 at 3.6, 7.1, or14.0mg/kg/day. T/C, tumor volumeofTAS-116-treated mice versus vehicle-treated mice. Data are presented asmean� SD. �� , P < 0.01 versus control[either vehicle-treated (NCI-N87) oruntreated (MV-4-11)]. B, body weightchange of mice corresponding to A.Effects of TAS-116 treatment onHSP90 client protein levels in anNCI-N87 (C) or MV-4-11 (E) mousexenograft model. Mice bearingNCI-N87 tumors were orallyadministered TAS-116 at 14.0 mg/kg/day for 3 days. Mice bearing MV-4-11tumors were treated with a single oraldose of 14.0 mg/kg of TAS-116. Micewere euthanized at 12 hours after thefinal dose, and tumorswere harvested.Protein extracts (20 mg) fromtumors were subjected to Westernblot analysis.






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Figure 3.Comparative pharmacokinetic and retinal toxicity analysis of TAS-116 and NVP-AUY922. A, biodistribution of TAS-116 or NVP-AUY922 in athymic nude rats withestablished NCI-H1975 xenografts after oral or intravenous administration of TAS-116 at 12.0 mg/kg or NVP-AUY922 at 20.0 mg/kg, respectively. Data arepresented as mean � SD (n ¼ 3). po, oral administration; iv, intravenous administration. B, effects of TAS-116 or NVP-AUY922 on retinal morphology andphotoreceptor cell death in SD rats. TAS-116 was administered orally every day for two weeks. NVP-AUY922 was administered intravenously three times a week fortwo weeks. Eye sections were subjected to HE staining and TUNEL. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; po qd, daily oraladministration; w, week. Scale bars, 50 mm. Effect on HSP90 clients (C) and antitumor activity of TAS-116 (D) in an NCI-H1975 rat xenograft model. Forpharmacodynamic analysis, rats with established NCI-H1975 xenografts were treated with an oral dose of 12.0 mg/kg of TAS-116. Tumors (n ¼ 3 per group) wereharvested at 8 hours after administration. The levels of theHSP90 clientswere evaluated bymeans ofWestern blotting. To evaluate the antitumor activity, NCI-H1975tumor–bearing rats (n ¼ 5 rats per group) were orally treated with TAS-116 at 12.0 mg/kg daily for two weeks. �� , P < 0.01 versus untreated control. E, comparisonof the drug exposure ratio between tumor and retina (AUC0–24, tumor/AUC0–24, retina) of HSP90 inhibitors in an NCI-H1975 rat xenograft model. HSP90 inhibitorswere administered in tumor-bearing rats with the routes and doses described in Supplementary Table S3 and then subjected to pharmacokinetic analysis.

Ohkubo et al.





and b inhibitor TAS-116 has strong antitumor activity againstseveral xenografted tumors indicates that inhibition of theHSP90a and b members of the HSP90 family is sufficient toexert antitumor activity.

The impact of paralog selectivity on the therapeutic indexand toxic effects of HSP90 inhibitors is yet to be sufficientlyexplored (3). Recently, it was discovered that GRP94, a criticalchaperone for the Wnt coreceptor LRP6, has a functional role ingut homeostasis via regulation of the canonical Wnt signalingpathway (29). Conditional deletion of grp94 results in rapidweight loss, diarrhea, and hematochezia in mice that are charac-terized by a gut-intrinsic defect in the proliferation of intestinalcrypts, as well as compromised nuclear b-catenin translocation,loss of crypt–villus structure, and impaired barrier function (29).It however remains unclear whether pharmacologic inhibition ofGRP94 would mimic such effect. Gastrointestinal toxicity is amajor clinical adverse event associated with HSP90 inhibitors(17–19, 21, 45) that is considered an on-target effect of HSP90inhibition. However, because TAS-116 did not inhibit the inter-action between GRP94 and LRP6 in the present study, it isconceivable that a selective inhibitor such as TAS-116 may not

cause GRP94-related gastrointestinal toxicity. This howeverremains to be validated.

One approach to balancing the efficacy and toxicity ofantitumor agents is optimization of the dosing schedule. Inthis regard, oral rather than intravenous administration ispreferable as it provides greater dose and schedule flexibility,and TAS-116 has been shown to have high oral availability inseveral species without the need for special formulation orprodrug development.

Sustained tumoral exposure with minimal exposure of otherorgans is another approach to achieve a maximal therapeuticwindow for antitumor agents. TAS-116 addresses the issues thathave hampered the clinical development of HSP90 inhibitorsbecause it shows a favorable tissue distribution profile. One ofthe most notable HSP90-related adverse events universallyobserved but to differing degrees in the clinic is visual distur-bance (18–21, 45–48). The observed symptoms include nightblindness, photopsia, blurred vision, and visual impairment,which are likely associated with retinal dysfunction (20, 21).Two independent reports of rodent models suggest that HSP90plays a critical role in retinal function and that sustained HSP90

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Figure 4.Effects of TAS-116 in a lung cancer mouse model based on orthotopically xenografted luciferase-transduced NCI-H1975 cells. A, representative photographs ofphoton emissions from individual mice with an orthotopic lung tumor with or without TAS-116 administration. Color gradation scale indicates the number oflive tumor cells (purple, low bioluminescence; red, high bioluminescence). V, ventral image; D, dorsal image; ROI, region of interest. B, emitted bioluminescence(photons/s) at the indicated time points (n ¼ 8 mice per group). Data are presented as mean � SD. �� , P < 0.01 versus vehicle-treated control.






inhibitionmight adversely affect visual function (34, 49). There-fore, ocular effects may be minimized by reducing retinal expo-sure. In the present study, TAS-116 was distributed less in retinathan in plasma in rats; consequently, TAS-116 did not produceany detectable photoreceptor injury. It has been proposed thatthe retina/plasma drug-exposure ratio and retinal eliminationprofile might be useful indicators of the ocular toxic potential ofHSP90 inhibitors (33). Indeed, our results are consistent withprevious results showing that ganetespib does not cause thesame degree of visual symptoms and is eliminated from retinamore rapidly than other HSP90 inhibitors (33). However, thereis the possibility that the lower retinal distribution of someHSP90 inhibitors is merely correlated with lower tissue pene-tration of the compounds. If so, such compounds weakenthe risk of ocular toxicities; however, this is at the cost of lessantitumor potential. Importantly, in addition to the lowerretinal distribution, TAS-116 showed a much higher distribu-tion in tumor and antitumor activity in an NCI-H1975 subcu-taneous xenograft rat model. To assess the risk-to-benefitratio of HSP90 inhibitors, we calculated the tumor/retina (T/R) drug-exposure ratio for several HSP90 inhibitors in thisxenograft setting. Except for TAS-116, all the tested compoundsshowed comparable drug exposure between tumor and retina(T/R ¼ 0.9–2.3). However, TAS-116 demonstrated a higher T/Rratio (T/R¼ 5.7), indicating that it has a unique, favorable tissuedistribution profile. Though the molecular mechanisms bywhich TAS-116 achieves this higher T/R exposure ratio remainto be elucidated, we expect TAS-116 to show ahigher therapeuticwindow in the clinic than the other HSP90 inhibitors withrespect to ocular toxicity. The drug T/R exposure ratio may alsobe useful in future preclinical screenings of candidate HSP90inhibitor compounds that show an improved therapeutic index.

In conclusion, TAS-116 is an orally available HSP90 inhibitorthat selectively binds to HSP90a and b. TAS-116 showed anti-tumor activity without detectable ocular toxicities in a rodentmodel, probably due to its favorable tissue distribution. Like-wise, TAS-116 did not inhibit CYP450 within the effective

concentration range, indicating a low drug–drug interactionpotential that might allow combination administration withother drugs. These tractable yet specific properties of TAS-116support the selection of this compound as a candidate forclinical evaluation as an antitumor agent characterized by max-imal antitumor activity and minimal HSP90-related toxicitiessuch as ocular toxicity.

Disclosure of Potential Conflicts of InterestS.Ohkubo, C. Yoshimura, andM. Kitade have ownership interest in a patent,

WO2011004610 A1. No potential conflicts of interest were disclosed by theother authors.

Authors' ContributionsConception and design: S. Ohkubo, Y. Kodama, C. YoshimuraDevelopment of methodology: Y. Kodama, H. Muraoka, H. Hitotsumachi,A. Gomori, K. Takahashi, Y. ShibataAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S.Ohkubo, Y. Kodama,H.Muraoka,H.Hitotsumachi,C. Yoshimura, A. Hashimoto, K. Ito, A. Gomori, K. Takahashi, Y. Shibata,A. KanohAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Ohkubo, Y. Kodama, H. Muraoka, H. Hitotsuma-chi, C. Yoshimura, A. Hashimoto, K. Ito, A. Gomori, K. Takahashi, Y. ShibataWriting, review, and/or revision of the manuscript: S. Ohkubo, Y. Kodama,C. YoshimuraStudy supervision: S. Ohkubo, K. YonekuraOther (chemical design and synthesis): M. Kitade

AcknowledgmentsThe authors thankDrs. Teruhiro Utsugi and FumioMorita for their insightful

discussions, and Takamasa Suzuki for his technical assistance.

Grant SupportAll research work was funded by Taiho Pharmaceutical Co., Ltd.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 13, 2014; revised October 13, 2014; accepted November 9,2014; published OnlineFirst November 21, 2014.

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Published OnlineFirst November 21, 2014.Mol Cancer Ther Shuichi Ohkubo, Yasuo Kodama, Hiromi Muraoka, et al. Minimal Ocular Toxicity in Preclinical Models

, Demonstrates Potent Antitumor Activity andβ and α90TAS-116, a Highly Selective Inhibitor of Heat Shock Protein

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