The heat shock protein 90 inhibitor, AT13387,displays a long duration of action in vitro andin vivo in non-small cell lung cancerBrent Graham,1 Jayne Curry, Tomoko Smyth, Lynsey Fazal, Ruth Feltell,2 Isobel Harada,3 Joe Coyle,Brian Williams,7 Matthias Reule,4 Hayley Angove,5 David M. Cross,6 John Lyons, Nicola G. Wallis andNeil T. Thompson

Astex Pharmaceuticals Ltd., Cambridge, UK

(Received July 19, 2011 ⁄ Revised November 20, 2011 ⁄ Accepted December 1, 2011 ⁄ Accepted manuscript online December 19, 2011 ⁄ Article first published online February 9, 2012)

A ubiquitously expressed chaperone, heat shock protein 90(HSP90) is of considerable interest as an oncology target becausetumor cells and oncogenic proteins are acutely dependent onits activity. AT13387 (2,4-dihydroxy-5-isopropyl-phenyl)-[5-(4-methyl-piperazin-1-ylmethyl)-1,3-dihydro-isoindol-2-yl] methanone,L-lactic acid salt) a novel, high-affinity HSP90 inhibitor, which iscurrently being clinically tested, has shown activity against awide array of tumor cell lines, including lung cancer cell lines.This inhibitor has induced the degradation of specific HSP90client proteins for up to 7 days in tumor cell lines in vitro. Theprimary driver of cell growth (mutant epidermal growth factorreceptors) was particularly sensitive to HSP90 inhibition. Thelong duration of client protein knockdown and suppression ofphospho-signaling seen in vitro after treatment with AT13387was also apparent in vivo, with client proteins and phospho-sig-naling suppressed for up to 72 h in xenograft tumors aftertreatment with a single dose of AT13387. Pharmacokinetic anal-yses indicated that while AT13387 was rapidly cleared fromblood, its retention in tumor xenografts was markedlyextended, and it was efficacious in a range of xenograft mod-els. AT13387’s long duration of action enabled, in particular, itsefficacious once weekly administration in human lung carcinomaxenografts. The use of longer-acting HSP90 inhibitors, such asAT13387, on less frequent dosing regimens has the potential tomaintain antitumor efficacy as well as minimize systemic expo-sure and unwanted effects on normal tissues. (Cancer Sci 2012;103: 522–527)

T he super-chaperone system is involved in the folding andmaturation of newly synthesized proteins.(1,2) HSP90 in

particular aids in the folding and maturation of a distinct sub-set of proteins, which includes kinases, cell surface receptorsand transcription factors.(3,4) The N-terminal domain ATPaseactivity of HSP90 is essential for this function.(5) Inhibition ofthis domain induces remodeling of the HSP90 chaperone com-plex, resulting in the recruitment of ubiquitin ligases, polyubiq-uitination and subsequent proteasomal degradation of HSP90client proteins.(6,7) Through this mechanism, the inhibition of asingle target enzyme can have a wide effect on the stabilityand, hence, the function of a large set of client proteins. Asmany oncogenic proteins are HSP90 clients, HSP90 inhibitionhas been found to have broad antitumor effects.(8–10) In con-trast to more recent targeted therapies, where the appearanceof new driver mutations or resistance mutations result in a lossof efficacy, client protein mutation increases dependence onHSP90 chaperoning activity as these mutations tend to renderthe proteins less stable.(11–13) Previous studies have also dem-onstrated that the constitutively activated mutant forms ofEGFR are particularly dependent on HSP90 both in vitro and

in vivo,(14–16) indicating an HSP90 inhibitor may be particu-larly efficacious in mutant EGFR tumors.After HSP90 inhibition, it has been observed that client pro-

tein depletion is transient and that levels return to normal upondrug withdrawal.(17,18) Extending the duration of target inhibi-tion in a tumor would be expected to extend the antitumor effect.Inhibitor accumulation in tumors, coupled with rapid clearancefrom blood and normal tissue, has been observed for multipleclasses of HSP90 inhibitors and may be due in part to the kinet-ics of the inhibitor binding to tumor-derived HSP90 com-plexes.(19–23) The potential advantage of compounds binding toHSP90 for a longer duration has been illustrated previously bycomparing the monomeric ansamycin inhibitor 17-AAG with adimeric variant that exhibited slow off-rate kinetics.(18,24)

Several structurally distinct HSP90 inhibitors are progressingthrough clinical development with early indications of clinicalresponses in published phase I and II data.(25–27) While thereare many similarities in the pharmacology of these differentagents,(28,29) one prominent variable is the duration of thepharmacodynamic effect reported.(20,30,31)

We have used fragment-based drug discovery to identify thehigh-affinity, long-acting HSP90 inhibitor, AT13387, which iscurrently being evaluated in clinical trials. In the studiesdescribed here, we have used this inhibitor to demonstrate thatmaximizing the duration of effect in the target tissue enabledsustained client protein depletion and extended tumor growthinhibition with less frequent dosing of AT13387. This wasespecially manifested for the non-small lung cancer cell lineNCI-H1975, in which mutant EGFR is a particularly sensitiveHSP90 client. The use of longer-acting HSP90 inhibitors intumors could lead to less frequent dosing regimens, limit expo-sure of non-target tissues to active drugs and reduce potentialsafety concerns in the clinic.

Materials and Methods

Materials. AT13387 was synthesized at Astex Pharmaceuti-cals (Cambridge, UK) and stored as a lyophilized powder(Table 1). Synthesis of AT13387 is as described by Woodheadet al.(32) All other reagents were purchased from Sigma (Poole,UK) unless otherwise stated.

Protein production. The ATPase domain of HSP90a wasexpressed as a His6-tagged fusion and purified using Ni-NTA

1To whom correspondence should be addressed. E-mail: Brent.Graham@astx.com2Present address: Horizon Discovery Services Ltd., Cambridge, UK.3Present address: Glaxo Smithkline, Stevenage, UK.4Present address: Merck, KGaA Germany, Merck Serono Research, Darmstadt,Germany.5Present address: Domainex, Cambridge, UK.6Present address: Pfizer Global Research and Development, Sandwich, UK.7In memory of Brian Williams, who sadly died during the course of this work.

Cancer Sci | March 2012 | vol. 103 | no. 3 | 522–527 doi: 10.1111/j.1349-7006.2011.02191.x© 2011 Japanese Cancer Association and Astex Pharmaceuticals Ltd.

metal-affinity chromatography and Superdex 75 gel-filtrationchromatography. Proteins were concentrated in 20 mM Tris(pH 7.4) containing 150 mM NaCl and 1.0 mM DTT.

HSP90 competition isothermal calorimetry. Kd values forAT13387 and 17-AAG binding to HSP90 were determinedwith a competition Isothermal Calorimetry (ITC) format. ITCexperiments were performed on a MicroCal VP-ITC (North-hampton, MA, USA) at 25°C in a buffer comprising 25 mMTris, 100 mM NaCl, 1 mM MgCl2 and 1 mM Tris(2-carboxy-ethyl)phosphine at pH 7.4 in order to maintain the higher affin-ity, reduced form of 17-AAG.(32,33)

Cell culture and reagents. The human cell lines A375, 22RV1,BT474, DU145, LNCaP, MCF-7, MDA-MB-468, MV4-11,NCI-H1650, NCI-H1975, NCI-H1993, PANC1, PC-3, SKBr3,U266, and SK-MEL-28 cells were obtained from the AmericanType Culture Collection (Teddington, UK). A549, HCT-116,HL60, HT-29, K562, MCF-7, MDA-MB-231, MES-SA, MES-SA/DX5, PNT2, SW620 and T47D cells were obtained from theEuropean Collection of Cell Cultures (Porton Down, UK).RPMI-8226 and TFK1 cells were obtained from DeutscheSammlung von Mikroorganismen und Zellkulturen (Braunsch-wieg, Germany). Cells were grown in their recommended cul-ture medium, supplemented with 10–20% fetal bovine serumand maintained at 37°C in an atmosphere of 5% CO2.

Proliferation assays. Cells were seeded into 96-well platesbefore the addition of compound in 0.1% (v/v) DMSO. GI50were determined using a 10-point dose response curve for

three cell doubling times. After compound incubation 10%(v/v), Alamar blue (Biosource International, Camarillo, CA,USA) was added, and cells were incubated for a further 4 h.Fluorescence was read at kex = 535 nm and kem = 590 nm.

Western blot samples and analysis. Cells were incubatedovernight at 37°C, followed by treatment with AT13387 or17-AAG for the indicated time. Floating and adherent cellswere collected and treated as described previously.(34) Sampleswere resolved by SDS-PAGE and immunoblotted with anti-bodies specific for Akt, pAkt, androgen receptor, cleavedPARP, c-Met, EGFR, phospho-EGFR, Erk, phospho-Erk, S6,phospho-S6, Stat3, phospho-Stat3 (Cell Signaling Technolo-gies, Cambridge, UK), B-Raf, CDK4, HER2 Raf-1 (SantaCruz Antibodies, Santa Cruz, CA, USA), actin (Abcam, Cam-bridge, UK), HSP70 (StressGen Biotechnologies, Victoria, BC,Canada), or GAPDH (Chemicon International, Temecula, CA,USA). This was followed by infrared dye labeled anti-rabbit oranti-mouse antibodies (Licor Bioscience, Lincoln, NE, USA).Blots were scanned to detect infrared fluorescence on theOdyssey Infrared Imaging System (Licor Bioscience).Activation of signaling pathways was accomplished by

serum-starving BT474 or U266 cells for 18 h, followed by 15min stimulation with either insulin-like growth factor-1 or IL-6, before cell lysates were harvested.

Xenograft models. Male athymic BALB/c mice (nu/nu) wereobtained from Harlan UK (Bicester, UK) and were given foodand water ad libitum. The care and treatment of experimentalanimals were in accordance with the United Kingdom Coordi-nating Committee for Cancer Research guidelines and with Uni-ted Kingdom Animals (Scientific Procedures) Act 1986.(35,36)

Mice were injected subcutaneously with tumor cells,NCI-H1975 at 2 9 106 cells in 100 lL serum-free medium. Ani-mals were randomized into groups of seven or eight, and treat-ment was started when tumors were approximately 100 mm3 inmean diameter. AT13387 was dissolved in 17.5% (w/v) hy-droxypropyl-b-cyclodextrin and administered intraperitoneallyat various dose levels in a dose volume of 10 mL/kg. Tumor bur-den was estimated from caliper measurements. Tumor growthdelay was analyzed with the log–rank test on the survival curves(Graphpad Software, GraphPad Prism v3.02, La Jolla, CA,USA). Tolerability was estimated by monitoring body weightloss and survival over the course of the study.

Pharmacokinetic analysis. Pharmacokinetic parameters weredetermined after intraperitoneal (80 mg/kg) administration ofAT13387 to athymic BALB/c mice bearing subcutaneous NCI-H1975 xenografts. Blood and tumor samples were collected atindicated times following dosing. Blood was drawn from theheart into a heparinized syringe. Tumors were dissected andall samples were stored at �20°C until analysis. Samples wereprepared for analysis as described previously.(34) Quantificationwas by comparison with a standard calibration line. The limitof detection for AT13387 was 150 nM in blood and 120 nMin tumor.

Analysis of tumor sample pharmacodynamic markers. Analysisof tumor sample pharmacodynamic markers was carried out asdescribed previously using the indicated antibodies.(17,34)

Results

Binding of AT13387 to HSP90 in vitro. AT13387 is a novelsmall molecule inhibitor of HSP90 that was discovered with afragment-based drug discovery approach that has beendescribed in detail elsewhere.(32)

X-ray crystallographic analysis of AT13387 in complex withthe N-terminal domain of HSP90 showed that it bound withinthe N-terminal ATPase catalytic site and overlapped with thebinding site for ATP.(32) Direct binding of AT13387 to HSP90was investigated with isothermal calorimetry. The Kd for

Table 1. Anti-proliferative effect of AT13387 on a panel of human

tumor cell lines

Origin Cell lineAT13387

GI50 (nM)

Normal prostate PNT2 480

Colon carcinoma HCT116 48

HT-29 78

SW620 210

Lung carcinoma A549 22

NCI-H1975 27

NCI-H1993 63

NCI-H1650 13

Breast carcinoma MCF-7 53

MDA-MB-231 260

MDA-MB-468 25

SK-BR3 63

T47D 29

BT474 13

Multiple myeloma U266 58

RPMI 8226 70

Pancreatic cells PANC1 55

Hepatoma Huh-7 22

Prostate carcinoma DU145 94

PC3 120

LNCaP 77

22Rv1 46

Uterine sarcoma MES-SA 53

MES-SA/Dx5 42

Ovarian SKOV3 44

Leukemia HL60 22

K562 47

MV4-11 13

Melanoma A375 18

SkMel 28 44

Cholangiocarcinoma TFK-1 19

ATT13387, (2,4-dihydroxy-5-isopropyl-phenyl)-[5-(4-methyl-piperazin-1-

ylmethyl)-1,3-dihydro-isoindol-2-yl] methanone, L-lactic acid salt.

Graham et al. Cancer Sci | March 2012 | vol. 103 | no. 3 | 523© 2011 Japanese Cancer Association and Astex Pharmaceuticals Ltd.

AT13387 binding was 0.7 nM. This compares to a Kd of 6.7nM for the binding of the ansamycin 17-AAG to the same site;the mean stoichiometry of binding for AT13387 was 1.03.The inhibition of a number of isolated kinases by AT13387

was also investigated including CDK1, CDK2, CDK4, FGFR3,PKB-b, JAK2, VEGFR2, PDGFR-b and Aurora B. None ofthe tested kinases were significantly inhibited at concentrationsbelow 30 lM (data not shown).

Antitumor activity of AT13387. AT13387 was a potent inhibi-tor of the proliferation and survival of many different cell linesfrom a variety of different tumor types in vitro. Across a panelof 30 tumor cell lines, AT13387 potently inhibited cell proli-feration with GI50 (n = 2) values in the range 13–260 nM(Table 1). The similarity in the GI50 values for AT13387 inthe MES-SA cell line and the P-glycoprotein–expressing vari-ant, MES-SA/Dx5, (53 vs 42 nM) indicated that it is not asubstrate for the P-glycoprotein transporter. AT13387 alsoinhibited proliferation of the non-tumorigenic human prostateepithelial cell line PNT2 with a GI50 (n = 2) value of 480 nM.Among the cell lines most sensitive to AT13387 were sev-

eral known to be dependent on receptor tyrosine kinases, suchas EGFR, HER2, c-Met, and FLT3. These included non-smallcell lung cancer cell lines with mutant EGFR (NCI-H1975;GI50, 27 nM) or amplified c-Met (NCI-H1993; GI50, 63 nM).Other sensitive cell lines included a cholangiocarcinoma line(TFK1; GI50, 19 nM) and IL-6–responsive tumor cell lines,notably the multiple myeloma lines RPMI-8226 (GI50, 70 nM)and U266 (GI50, 58 nM). We are unaware of any other reportsdemonstrating HSP90 inhibitor sensitivity in a cholangiocarci-noma cell line. It is noteworthy that IL-6 has been shown tocontribute to tumorigenesis and cell survival in cholangiocarci-nomas.(37–39) The benefit of current chemotherapeutic optionsfor cholangiocarcinomas is controversial, with some reportsclaiming a survival benefit while others report no impact ondisease or survival benefit.(40–43) The sensitivity of IL-6 recep-tor-signaling and TFK1 cells to AT13387 suggests that cho-langiocarcinomas are attractive candidates for furtherinvestigation in preclinical and clinical studies.

Cell-based mechanism of action studies. The molecular finger-print of HSP90 inhibition is characterized by an increase inHSP72 and corresponding depletion in HSP90 client proteins(e.g. CDK4, Raf-1 and Akt).(44,45) The time- and concentra-tion-dependence of these effects for AT13387 were demon-strated in the non-small cell lung cancer cell line NCI-H1975,which harbors two activating mutations (L858R and T790M)in EGFR (Fig. 1A,B). At concentrations of 30 nM and above,AT13387 induced depletion of a range of HSP90 client pro-teins in NCI-H1975 cells within 6 h of exposure. In furtherstudies, client proteins believed to be the primary drivers ofcell growth were investigated. In addition to loss of mutantEGFR in NCI-H1975 cells, AT13387 also caused depletion ofBRAF in the melanoma cell line A375, the androgen receptorin the prostate cancer cell line 22Rv1, Her2 in the breast carci-noma cell line BT474 and c-Met in the lung carcinoma lineNCI-H1993 (Fig. 1C). Inhibition of signaling via growth andsurvival pathways (e.g. Erk1/2 and PI3K/Akt) is an importantconsequence of HSP90 inhibition, and suppression of phos-phorylation in these signaling pathways is often the earliestevent observed.(28) We have found IL-6–dependent cells to bevery sensitive to AT13387, which correlates with the inhibitionof the downstream signaling pathway. IL-6–induced phospho-signaling through both the Stat3 and MAPK pathways wasablated in the presence of AT13387 in the multiple myelomacell line U266 and breast carcinoma cell line BT474 (Fig. 1D).

AT13387 inhibition of HSP90 results in prolonged client proteindepletion. Following the observations that AT13387 treatmentdepleted client proteins and ablated phospho-signaling, weinvestigated the duration of these biochemical effects in vitro.Melanoma (A375), breast carcinoma (BT474), and lung carci-noma cell lines (NCI-H1993 and NCI-H1975) were treatedwith 1 lM of AT13387 to ensure complete inhibition ofHSP90. The duration of this inhibition was then studied atvarious times after the inhibitor had been removed (Fig. 2A–D). At doses above the IC50 value, the results demonstratedthat the extent and duration of client protein depletion wasgenerally dependent on the length of exposure to compound

(A) (B)

(C)

(D)

Fig. 1. Effects of AT13387 treatment on HSP90 client protein levels. (A) NCI-H1975 cells treated with the indicated doses of AT13387 for 18 hbefore lysates were harvested and equivalent amounts of protein from each lysate were resolved by SDS-PAGE and immunoblotting with theindicated antibodies. Control samples are vehicle only treated cells. (B) NCI-H1975 cells treated with the indicated doses of AT13387 for 1, 3, 6,12, 24 or 48 h before lysates were harvested and immunoblotted with anti-EGFR antibody. (C) 22Rv1, NCI-H1975, BT474, A375 and NCI-H1993cells were untreated or treated for 18 h with 1 lM AT13387 before they were harvested for immunoblot analyses. (D) Serum-starved U266 andBT474 cells were treated with 1 lM AT13387 for 18 h before they were induced with IL-6 or IGF-1 and harvested for immunoblot analyses.AT13387, (2,4-dihydroxy-5-isopropyl-phenyl)-[5-(4-methyl-piperazin-1-ylmethyl)-1,3-dihydro-isoindol-2-yl] methanone, L-lactic acid salt; CTL,control; EGFR, epidermal growth factor receptor; HSP, heat shock protein; pAkt, phospho-Akt.

524 doi: 10.1111/j.1349-7006.2011.02191.x© 2011 Japanese Cancer Association and Astex Pharmaceuticals Ltd.

and that 24-h exposure maximally suppressed levels of clientproteins and phospho-signaling in these cells (Fig. 1A,B).After removal of AT13387 from the medium, A375 melanomacells showed significant suppression of all tested HSP90 clientproteins for at least 48 h (Fig. 2A). Additionally, signalingthrough the MAPK (phospho-Erk1/2) and PI3K (phospho-S6)pathways was suppressed for at least 48 h in AT13387-treatedA375 cells. Suppression by AT13387 of the primary drivers ofgrowth in BT474 (HER2), NCI-H1993 (c-Met) and NCI-H1975 (EGFR) cell lines was found to last in excess of 7 daysafter removal of the AT13387 (Fig. 2B–D). Mutant EGFRfound in NCI-H1975 cells was particularly sensitive to deple-tion following AT13387 treatment, with this prolonged clientdepletion requiring only a 7-h exposure to AT13387 (Fig. 2D).Suppression of phospho-signaling was also maintained overthis extended period. An expected consequence of this longduration of action is reduced survival of these cells. Indeed,the corresponding level of cleaved PARP in cells treated withAT13387 correlates with a significant decrease in PI3K signal-ing.

Pharmacodynamic profile of AT13387. Given the sensitivity ofEGFR to AT13387 in vitro, we wanted to determine the dura-tion of response to AT13387 elicited in vivo. The extended

duration of action of HSP90 inhibition following a single max-imum dose of AT13387 (80 mg/kg) was investigated in vivo inmice bearing NCI-H1975 xenograft tumors. Pharmacodynamicanalyses revealed that the HSP90 client protein EGFR wassuppressed for up to 72 h following treatment with AT13387.Indicative of the strength of growth and survival signals in thetumors, Phospho-S6 and pAkt were also suppressed for up to72 h (Fig. 3). Similar results for Phospho-S6 and pAkt wereobtained from studies using mice bearing A375 xenografts(data not shown).

Pharmacokinetic profile of AT13387. To determine whetherthe distribution of AT13387 to the xenograft tumor may inpart account for its extended duration of action, we investi-gated both the blood and tumor pharmacokinetic profiles in thesame mice used to establish the pharmacodynamic profiles.After intraperitoneal administration of 80 mg/kg AT13387 toNCI-H1975 xenograft-bearing mice, AT13387 was rapidlycleared from the blood and could not be detected beyond 24 h.In contrast, high levels of AT13387 were measured in thetumors, and the compound was detectable for at least 240 hafter dosing (Fig. 4). In summary, this study demonstrated that,in mice, AT13387 was cleared rapidly from blood and distrib-uted readily to tumors. Tumor retention of AT13387 was

(A) (B) (C) (D)

Fig. 2. AT13387 has a long duration of inhibition in vitro. (A) A375, (B) BT474 and (C) NCI-H1993 cells were treated with 1 lM of AT13387 for24 h, washed with PBS and re-incubated with fresh culture medium. (D) NCI-H1975 cells were treated with 1 lM AT13387 for 7 h, washed withPBS and re-incubated with fresh culture medium. Samples were taken at the indicated times after PBS wash for immunoblot analyses. AT13387,(2,4-dihydroxy-5-isopropyl-phenyl)-[5-(4-methyl-piperazin-1-ylmethyl)-1,3-dihydro-isoindol-2-yl] methanone, L-lactic acid salt; EGFR, epidermalgrowth factor receptor; HSP, heat shock protein; pAkt, phospho-Akt.

Fig. 3. AT13387 in vivo duration of action. Athymic mice bearingNCI-H1975 xenografts were given intraperitoneal injections of 80 mg/kg AT13387. Control animals were untreated. Samples were taken atthe indicated times for immunoblot analyses. AT13387, (2,4-dihydroxy-5-isopropyl-phenyl)-[5-(4-methyl-piperazin-1-ylmethyl)-1,3-dihydro-iso-indol-2-yl] methanone, L-lactic acid salt; CTL, control; EGFR, epidermalgrowth factor receptor; HSP, heat shock protein; pAkt, phospho-Akt.

Fig. 4. Pharmacokinetic analyses of AT13387. Mean exposure param-eters for AT13387 in male athymic Balb/c mice bearing NCI-H1975tumors after intraperitoneal administration of 80 mg/kg AT13387. (●)AT13387 concentrations in tumors; (■) AT13387 concentrations inblood. The dashed line represents the limit of detection. AT13387,(2,4-dihydroxy-5-isopropyl-phenyl)-[5-(4-methyl-piperazin-1-ylmethyl)-1,3-dihydro-isoindol-2-yl] methanone, L-lactic acid salt.

Graham et al. Cancer Sci | March 2012 | vol. 103 | no. 3 | 525© 2011 Japanese Cancer Association and Astex Pharmaceuticals Ltd.

markedly extended, which may contribute to extended targetinhibition in this target tissue. The extended tumor retention ofAT13387 may also mean that less frequent dosing is requiredin order to maintain efficacy in xenograft models.

Antitumor efficacy of AT13387 in vivo. The dose and scheduledependence of the antitumor effects of AT13387 were investi-gated in NCI-H1975 xenografts in athymic mice (Fig. 5).When given on an intermittent basis, AT13387 could be toler-ated at doses of up to 70 mg/kg twice weekly or 90 mg/kgonce weekly. Bodyweight loss in mice did not exceed 20%before recovering in all cases except one, and loss was highestfollowing the second dose. Tumor growth inhibition was simi-lar in NCI-H1975 for both dosing regimens. The maintenanceof antitumor effects with such a prolonged off-treatment periodis consistent with the extended pharmacodynamic action ofAT13387 observed for mutant EGFR and other biomarkersin vitro and in vivo and the extended retention of AT13387in tumors.

Discussion

In the course of characterizing AT13387, we observed it tohave a long duration of action in tumor cells. In several celllines, 24-h treatment in vitro with AT13387 suppressed someclient proteins for longer than 7 days. One of the more sensi-tive cell lines tested was the NCI-H1975 lung carcinoma cellline, which suppressed mutant EGFR after only being exposedto AT13387 for 7 h. By 7 days post-exposure, the viability ofthe remaining cells was very low and the effect could be con-sidered irreversible. In all lines tested to date, the duration ofaction of AT13387 persisted longer than has been reported fora range of other small molecule HSP90 inhibitors. For exam-ple, SNX-2112 was reported to suppress HER2 for only 24–48h in BT474 cells in vitro.(18) Prolonged pharmacodynamiceffects were also noted for AT13387 in vivo when client pro-teins and phospho-signaling were monitored in NCI-H1975xenograft tumors in mice following a single dose. In thesexenograft tumors, the suppression of specific sensitive clientproteins lasted up to 72 h. HSP90 client proteins weresuppressed for 24–48 h with NVP-AUY922,(30) for only 24 hfollowing treatment with SNX-5422 (the pro-drug of SNX-2112),(20) and <24 h following treatment with BIIB021.(31) Anoverall review of the data for different HSP90 inhibitors inclinical development suggests a correlation between thepotency of the inhibitors and their duration of action in tumors.The estimated Kd of AT13387 for HSP90 was 0.7 nM, makingit the most potent inhibitor at the N-terminal ATP site reportedto date compared with other inhibitors (e.g. SNX-2112, 30

nM; NVP-AUY922, 1.7 nM; BIIB021, 1.7 nM; 17-AAG, 6.7nM).(20,21,31) The high potency and extended action ofAT13387 correlated with a long tumor retention in xenograftmodels.Previous reports have suggested that high-affinity binding,

specifically to tumor-derived HSP90 protein complexes, might

account for tumor-specific distribution of HSP90 inhibitors.(23)

However, another study suggested there was no difference inthe affinity of 17-AAG to HSP90 alone or to HSP90 in amultiprotein complex.(46) That study found that high-affinitybinding is related to a slow dissociation rate for certain inhib-itors and suggested that the observed tumor-specific accumu-lation of these compounds may simply be related to increasedlevels of HSP90 found in tumor cells and the law of massaction.We explored a number of intermittent dosing schedules in

NCI-H1975 xenografts in mice to investigate the relevance tothe antitumor effect of the prolonged pharmacodynamic actionassociated with AT13387. Based on the observation that thetested client proteins were suppressed for up to 72 h in vivo,we explored twice weekly dosing and found it to be effectivein a broad range of different tumor types. We also foundAT13387 to be efficacious with once weekly dosing in a lungcarcinoma xenograft model. Published data on other agentssuggests that only compounds that have pharmacodynamicactions lasting for 48 h or more are able to maintain efficacywhen dosed once a week.(30)

One advantage of exploiting the long tumor retention andextended pharmacodynamic effect of HSP90 inhibitors is lessfrequent dosing, such that systemic exposure to the agent isreduced. This is particularly apparent for AT13387, wherethese effects are combined with rapid clearance from the bloodin mice. Notable toxicities reported preclinically and clinicallyfor HSP90 inhibitors include liver and gastrointestinal toxic-ity.(17,27) Thus, the therapeutic index of HSP90 inhibitors inthe clinic might be improved by exploiting longer actingagents such as AT13387.

Acknowledgments

We thank Dr Matthew Squires and Dr Jon Lewis for their invaluablediscussions.

Disclosure Statement

Brent Graham, Jayne Curry, Tomoko Smyth, Lynsey Fazal, Joe Coyle,John Lyons, Nicola G. Wallis and Neil T. Thompson are employees ofAstex Pharmaceuticals.

(A) (B)

Fig. 5. AT13387 is efficacious in multiple xenograft models on a once a week dosing schedule. (A) Athymic mice bearing NCI-H1975 xenograftswere treated for 16 days with intraperitoneal injection of (♦) 17.5% cyclodextrin or AT13387: (■) 70 mg/kg on days 1, 4, 8, 12 and 16; (▲) 55mg/kg on days 1, 4, 8, 12 and 16; or (♢) 90 mg/kg on days 1, 8 and 16. Eight mice were included in each group. Results shown indicate meantumor volume (mm3) with error bars representing ± SE. (B) Dosing schedules and body weight changes corresponding to (A). AT13387, (2,4-di-hydroxy-5-isopropyl-phenyl)-[5-(4-methyl-piperazin-1-ylmethyl)-1,3-dihydro-isoindol-2-yl] methanone, L-lactic acid salt.

526 doi: 10.1111/j.1349-7006.2011.02191.x© 2011 Japanese Cancer Association and Astex Pharmaceuticals Ltd.

Abbreviations

17-AAG 17-N-allylamino-17-demethoxygeldanamycinATT13387 (2,4-dihydroxy-5-isopropyl-phenyl)-[5-(4-methyl-

piperazin-1-ylmethyl)-1,3-dihydro-isoindol-2-yl]methanone, L-lactic acid salt

EGFR epidermal growth factor receptorHSP heat shock proteinpAkt phospho-Akt

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Graham et al. Cancer Sci | March 2012 | vol. 103 | no. 3 | 527© 2011 Japanese Cancer Association and Astex Pharmaceuticals Ltd.