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Antitumor Activity and Pharmacodynamic Biomarkers of aNovel and Orally Available Small-Molecule Antagonist ofInhibitor of Apoptosis Proteins

Hiroyuki Sumi1, Masato Yabuki1, Kenichi Iwai1, Megumi Morimoto1, Ryosuke Hibino1, Masakazu Inazuka1,Kentaro Hashimoto1, Yohei Kosugi2, Kazunobu Aoyama2, Shunsuke Yamamoto2, Mie Yoshimatsu3,Hideki Yamasaki4, Ryuichi Tozawa5, Tomoyasu Ishikawa1, and Sei Yoshida6

AbstractInhibitor of apoptosis proteins (IAP), which are key regulators of apoptosis, are inhibited by second

mitochondria-derived activator of caspase (SMAC). Small-molecule IAP antagonists have recently been

reported as novel therapeutic treatments for cancer. In this study, we showed that the octahydro-

pyrrolo[1,2-a]pyrazine derivative, T-3256336, is a novel and orally available small-molecule IAP antagonist.

T-3256336 selectively binds to and antagonizes protein interactions involving cellular IAP-1 (cIAP-1), cIAP-2,

and X-linked IAP (XIAP). T-3256336 induced the rapid proteasomal degradation of cIAP-1 and activated TNF-

a–dependent extrinsic apoptosis signaling in cultured cells. In a MDA-MB-231-Luc breast cancer xenograft

model, T-3256336 induced cIAP-1 degradation, TNF-a production, and caspase activation in tumors, which

resulted in strong antitumor activities. T-3256336 induced increases in the plasma levels of TNF-a and

fragmented cytokeratin-18, which correlated with the antitumor potency in MDA-MB-231-Luc xenograft

models. This study provided further insights into biomarkers of IAP antagonists. Furthermore, our data

provided evidence that T-3256336 is a promising new anticancer drug worthy of further evaluation and

development. Mol Cancer Ther; 12(2); 230–40. �2012 AACR.

IntroductionThe targeting of critical apoptosis inhibitors is an attrac-

tivecancer therapeutic strategy (1–3). Inhibitor ofapoptosisproteins (IAP) are a class of proteins that contain tandemduplications of a unique motif known as the baculoviralIAP repeat (BIR) motif, which was originally identified inbaculoviruses (insect viruses; v-IAPs; ref. 4). Inmammals, 8IAPs [X-linked IAP (XIAP), cellular IAP-1 (cIAP-1), cIAP-2,IAP-like protein-2 (ILP2), NAIAP, melanoma IAP(MLIAP), survivin, and BRUCE] have been identified(5). Among these IAP proteins, cIAP-1 and cIAP-2 play acritical role in the regulation of TNF receptor–mediatedapoptosis, andXIAP is a central regulator of both the deathreceptor- andmitochondria-mediated apoptosis pathways

(6–8). The third BIR domain (BIR3) of XIAP selectivelybinds to and inhibits the initiator caspase-9 (9),whereas thesecond BIR (BIR2) domain binds to and inhibits effectorcaspase-3/caspase-7 (10–12). Consistent with their role inthe inhibition of apoptosis, XIAP and cIAP-1 are highlyexpressed in cancers ofdiverse tumor types (13–16) andareconsidered new cancer therapeutic targets (17, 18).

The second mitochondria-derived activator of caspase(SMAC)/direct IAP-binding protein with low pI (DIA-BLO) is anendogenousantagonist of IAPs (19, 20). Thepro-apoptotic functionofSMACisdependentona conserved4-residue IAP interaction motif (Ala-Val-Pro-Ile) that isfound at the amino terminus of the mature protein (21,22). Recent independent studies have shown that IAPantagonists induce the rapid degradation of cIAP-1, whichleads toNF-kBactivation, and theproductionandsecretionof TNF-a, and the TNF-a–dependent apoptosis (23–26).Several IAP antagonists, includingAT-406, LCL-161,GDC-0152, TL-32711, and HGS-1029, which mimic the interac-tions of the SMAC amino-terminal peptide with IAP pro-teins, have been developed and are currently being eval-uated in clinical settings (27–30). The identification andestablishment of biomarkers that report apoptotic celldeath in tumors or in surrogate tissues, such as blood, isoneof thekey issues in thedevelopmentof IAPantagonists.

In this study, we investigated the therapeutic potentialof the novel and orally available IAP antagonist, T-3256336. We showed that T-3256336 effectively inhibited

Authors' Affiliations: 1Oncology Drug Discovery Unit, PharmaceuticalResearch Division,2Drug Metabolism and Pharmacokinetics ResearchLaboratories, 3BioMolecular Research Laboratories, 4Drug SafetyResearch Laboratories, 5Metabolic Disease Drug Discovery, and 6Inflam-mation Drug Discovery Unit, Takeda Pharmaceutical Company Ltd., Japan

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

Corresponding Authors: Hiroyuki Sumi, Takeda Pharmaceutical Compa-ny Ltd., 26-1 Muraoka Higashi 2-chome Fujisawa Kanagawa 251-8555,Japan. Phone: 81-466-32-2645; Fax: 81-466-29-4412; E-mail:[email protected]; and Sei Yoshida. Phone: 81-466-32-2642;E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-12-0699

�2012 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 12(2) February 2013230

on June 29, 2020. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst December 12, 2012; DOI: 10.1158/1535-7163.MCT-12-0699

http://mct.aacrjournals.org/

tumor growth and caused tumor regression without sig-nificant body weight loss. In the pharmacodynamic stud-ies, we showed that the circulating levels of TNF-a, themarker of cell death, correlated with tumor growth inhi-bition (TGI).Ourdata provided evidence that T-3256336 isa promising new anticancer drug that is worthy of furtherevaluation and development. Our data also showed thepotential of circulating TNF-a and cytokeratin-18 as bio-markers to predict the clinical efficacy of IAP antagonist.

Materials and MethodsChemical synthesisThe (7R)-ethoxy-octahydro-pyrrolo[1,2-a]pyrazinederi-

vative, T-3256336, was synthesized and purified accord-ing to methods described in Supplementary Informa-tion and the patents filed previously (31).

Cell lines, proteins, peptides, and reagentsMDA-MB-231, MDA-MB-468, BT-474 SK-OV-3, and T-

47D, HL-60, and NCI-H1703 cancer cell line and MRC5normal lung fibroblasts were obtained from theAmericanType Culture Collection. The culture medium that wasrecommended by the suppliers was used for the cultiva-tion of each cell line.MDA-MB-231 cells stably expressingluciferase (MDA-MB-231-Luc) were established atTakeda Pharmaceutical Company, Ltd. (TPC) by trans-fecting a firefly luciferase expression vector (PromegaCorporation) into MDA-MB-231 cells. Commerciallyobtained cells were not authenticated by the authors. Anantibody against cIAP-1 (AF8181), cIAP-2 (AF8171), andhuman TNF-a (MAB210) was purchased from R&D Sys-tems, Inc. Anti-XIAP (610762) was purchased from BDBiosciences. Anti-Livin antibody (88C570) was fromIMGENEX Corporation. Anti-glyceraldehydes-3-phos-phate dehydrogenase (GAPDH; MAB374) antibody wasfrom Millipore. Anti-IkBa (#4814), anti-phospho-IkBa(Ser32) (#2859), anti-NF-kB p65 (#3034), anti-phospho-NF-kB p65 (Ser536) (#3033), anti-c-Jun N-terminal kinase(JNK) (#9258), anti-phospho-JNK (Thr183/Tyr185)(#9251), anti-phospho-p38 (Thr180/Tyr182) (#9211),anti-p38 (9217), anti-caspase-8 (#9746), and anti-cas-pase-3 (#9665) were purchased from Cell Signaling Tech-nology, Inc. MG-132 (#474791), pan-caspase inhibitor z-BAD-fmk (#219007), and caspase-8 inhibitor z-IETD-fmk(#218759) were purchased from Calbiochem. The recom-binant human XIAP (residues 124–357, XIAP_BIR2-BIR3)and caspase-9 were prepared at Takeda California, andthe 6-His–tagged recombinant BIR3 domains of humancIAP-1 (residues 250–350, cIAP-1_BIR3) and cIAP-2 (resi-dues 238–349, cIAP-2_BIR3) were prepared at TPC. Therecombinant BIR3 domain of human XIAP (residues 252–356) fused to an N-terminal His-tag (XIAP_BIR3) waspurchased from R&D Systems, Inc., and the SMAC-N7peptide (AVPIAQK)waspurchased fromMerckKgaA.C-terminal-biotinylated SMAC-N7 peptide [AVPIAQ-K(biotin)-NH2] (biotinyl-SMAC) was synthesized at Pep-tide Institute Inc. The cryptate-conjugated mouse mono-

clonal antibody anti-6-Histidine (Anti-6HIS Cryptate),high-grade XL665-conjugated streptavidin (SA-XL), andhomogeneous time-resolvedfluorescence resonance ener-gy transfer (HTRF) detection buffer were purchased fromSceti Medical Labo K.K. Recombinant human caspase-3and caspase-7were purchased fromWako Pure ChemicalIndustries, Ltd.

Binding activities using HTRF technologyFive microliters of IAP proteins (40 nmol/L of XIAP_-

BIR3 and 8 nmol/L of cIAP-1/-2_BIR3) and 5 mL ofincreasing concentrations of compounds were added tothewells containing assay buffer [25mmol/L (4-(2-hydro-xyethyl)-1-piperazineethanesulfonic acid (HEPES), 100mmol/L NaCl, 0.1% bovine serum albumin, 0.1% TritonX-100, pH 7.5]. After shaking, 5 mL of biotinyl-SMAC (20nmol/L of XIAP_BIR3, 80 nmol/L of cIAP-1_BIR3, and120nmol/L of cIAP-2_BIR3dissolved in assay buffer)wasadded to the well, which was followed by adding 5 mL ofthe mixture of Anti-6HIS cryptate and SA-XL, which wasdiluted 100 times with HTRF detection buffer. After anovernight incubation at room temperature in the dark,HTRF measurements were conducted with a EnVisionmulti-label reader (PerkinElmer Inc.). Fluorescence at 615nm (F615 nm) is the total europium signal, and fluores-cence at 665 nm (F665 nm) is the FRET signal. The ratio[(F665 nm/F615 nm) � 10,000] was calculated, and IC50

values were determined using the ratio with nonlinearregression curve fitting with Prism (Version 5.01, Graph-Pad Software, Inc.).

Cell-free functional assayVarious concentrations of T-3256336 or SMAC-N7,

XIAP_BIR2þ3 (40 nmol/L for caspase-3, 4 mmol/L forcaspase-7, or 300 nmol/L for caspase-9), and 1 unit ofcaspase-3/caspase-7/caspse-9 was added to wells in 384-well plates (Corning Incorporated) at a final volume of 30mL in assay buffer [20 mmol/L HEPES, 0.1% CHAPS, 1mmol/L EDTA, 10% sucrose, and 10mmol/L dithiothrei-tol (DTT), pH 7.5]. After incubating at room temperaturefor 5 minutes, 10 mL of 40 or 160 mmol/L Ac-DEVD-AMCsolution (Enzo Life Sciences, Inc.) for caspase-3 and cas-pase-7, 50 mmol/L of Ac-LEHD-AMC for caspase-9 wereadded to the wells, respectively. Following incubation for30 minutes at room temperature with shaking, fluores-cence at 380 nm excitation and 460 nm emission wave-lengths was measured using a multimode microplatereader Spectra Max M5e (Molecular Devices, Inc.). Activ-ity was expressed as EC50, whichwas the concentration atwhich half-maximum recoverywas achieved,with Prism.

Cell viability assay and measurement of caspaseactivity

Cells were seeded at 3 � 103 cells per well in 96-wellplates (Sumitomo Bakelite Co., Ltd.) and cultured over-night. On the following day, test compoundswere dilutedin growth medium to the desired final concentration andthen added to the cells. After 24 hours of incubation,

Preclinical Study of T-3256336, a Selective IAP Antagonist

www.aacrjournals.org Mol Cancer Ther; 12(2) February 2013 231




caspase activities were measured with Caspase-Glo-3/Caspase-Glo-7, Caspase-Glo-8, or Caspase-Glo-9 Assays(Promega Corporation). After 3 days of incubation, cellviability was measured with a CellTiter-Glo LuminescentCell Viability Assay (Promega Corporation). GI50 valueswere determined with the ratio by nonlinear regressioncurve fitting with Prism.

SDS-PAGE and Western blottingCells were lysed with 100 mL of SDS sample buffer

(BioRad Laboratories, Inc.) and heated at 95�C for 5minutes. Each cell lysate was subjected to SDS-PAGE andtransferred onto Sequi-Blot PVDF Membranes (BioRadLaboratories, Inc.). The membranes were blocked withStartingBlock T20 (PBS) Blocking Buffer (Thermo FisherScientific, Inc.) and probed overnight with an antibodydiluted 500- to 2,000-fold with Can Get Signal Immunor-eaction Enhancer Solution I (Toyobo Co., Ltd.). The mem-brane was washed with PBS containing 0.1% Tween-20(Wako Pure Chemical Industries, Ltd.) and incubatedwith horseradish peroxidase-labeled secondary antibody(GEHealthcare) thatwasdiluted 20,000-foldwithCanGetSignal Immunoreaction Enhancer Solution II (ToyoboCo.,Ltd.) for 2 hours at room temperature. Themembranewaswashed in the samemanner as above, andproteins labeledwith the antibody became chemically luminescent withSuperSignal West Femto Maximum Sensitivity Substrate(ThermoFisher Scientific, Inc.) andwere detectedwith theluminoimage analyzer LAS-1000 (Fujifilm Corporation).

Quantitative real-time PCRCells were seeded at 5 � 105 cells per well in 6-well

plates and cultured overnight. On the following day, testcompounds were diluted in growth medium to thedesired final concentration and then added to the cells.The isolation of total RNAwas conductedwith anRNeasyMini Kit (Qiagen Inc.). About 1 ng of total RNA from eachsample was reverse transcribed with the SuperScriptVILO cDNA Synthesis Kit (Life Technologies). For thequantitative analysis of the levels of mRNA expression, areal-time PCR assay was conducted (TaqMan with the7900HT Fast Real-Time PCR System; Life Technologies)using TaqMan probes.

In vivo efficacy studyMice were housed and maintained within the facility

at TPC in accordance with the Takeda ExperimentalAnimal Care and Use Committee–approved protocol.Athymic nude mice (BALB/cAJcl-nu/nu) of approxi-mately 5 weeks of age were obtained from CLEA Japan,Inc. For the subcutaneous-implanted tumor xenograftmodels, nude mice were injected with 5 � 106 cells permouse. When tumor volumes reached approximately 200mm3, mice were randomly assigned to treatment groups.The compounds were orally administered to the mice in0.5% methylcellulose (Shin-Etsu Chemical Co., Ltd.).Tumor growth and body weights were measured twiceweekly, and average tumor volumes were calculated, as

estimated from the formula [(L � W2)/2] throughout thestudy.

In vivo pharmacokinetics/pharmacodynamics studyNude mice were injected with 5 � 106 MDA-MB-231-

Luc cells.When the tumor volumes reached approximate-ly 200 mm3, mice were randomly assigned to control ortreatment groups. Tumors and plasma were collected atmultiple time points after the oral administration of T-3256336. The concentrations of T-3256336 in the plasmaand tumors were determined by liquid chromatography/tandem mass spectrometry. Homogenization wasconductedwith a Physcotron (NS-310E)with radioimmu-noprecipitation assay buffer containing a phosphataseinhibitor cocktail and a protease inhibitor cocktail (Sig-ma-Aldrich Co.). The protein concentration in the tumorlysate was determined by bicinchoninic acid (BCA) pro-tein assay kit (Thermo Fisher Scientific, Inc.). cIAP-1protein levels were determined as described above. Plas-ma human TNF-a levels were determined with a humanTNF-a Quantikine ELISA Kit (R&D Systems, Inc.). Thelevels of caspase-cleaved (M30) and total (M65) cytoker-atin-18 in plasma were determined with an ELISA kit(Peviva AB).

Measurement of caspase activity in tumorsCaspase-3/caspase-7 activities in tumors were mea-

sured with a Caspase-Glo assay kit (Promega Corpora-tion). Cytosolic extracts from MDA-MB-231-Luc xeno-grafts were prepared by homogenization in extractionbuffer (25 mmol/L HEPES, pH 7.5; 5 mmol/L MgCl2; 1mmol/L EDTA) and subsequently centrifuged (5 min-utes, 10,000 rpm, 4�C). The protein concentration of thesupernatant was adjusted to 1 mg/mL with extractionbuffer, and an equal volume of reagents and 10 mg/mLcytosolic protein was mixed and incubated at room tem-perature for 30minutes. The luminescence of each samplewas measured in a luminometer.

Histopathological examination of xenograft athymicnude mice

To determine the effect of T-3256336 on xenograft mod-el, necropsy was conducted on the day after 2-week oraladministration of T-3256336. The animals were eutha-nized and were macroscopically examined the externalsurface of the carcass, the thoracic and abdominal cavities,organs, and tissues. Tumor tissues, liver, spleen, skin, andintestines fromthe control, 30 and100mg/kggroupswerefixed in 10 vol% neutral-buffered formalin, embedded inparaffin, sectioned, stained with hematoxylin and eosin(H&E), and examined histopathologically.

ResultsT-3256336 bound to the BIR3 domains of cIAP-1,cIAP-2, and XIAP

We discovered the octahydro-pyrrolo[1,2-a]pyrazinederivative, T-3256336, was a novel small-molecule IAPantagonist (Fig. 1A). The binding affinities of T-3256336 to

Sumi et al.

Mol Cancer Ther; 12(2) February 2013 Molecular Cancer Therapeutics232




the BIR3 domains of human XIAP, cIAP-1, and cIAP-2were tested in an HTRF-binding assay (Fig. 1B). T-3256336 showed high affinities for cIAP-1 and cIAP-2with IC50 values of 1.3 and 2.2 nmol/L, respectively. TheIC50 value of T-3256336 for XIAP was 200 nmol/L. Theintermediate of T-3256336 showed very weak activityagainst XIAP, cIAP-1, and cIAP-2 (IC50 > 30 mmol/L;Supplementary Information). We assessed T-3256336for its ability to inhibit the function of XIAP in a cell-free functional assay. Recombinant human XIAP pro-

tein (XIAP_BIR2-BIR3) inhibited the activity of caspase-3 dose dependently and achieved 80% inhibition at 40nmol/L (Fig. 1C). In these conditions, T-3256336 dosedependently antagonized XIAP and promoted the activ-ities of caspase-3 with an EC50 value of 1.3 mmol/L (Fig.1D). In addition, T-3256336 promoted the activities ofcaspase-7 and caspase-9 dose dependently (Supplemen-tary Fig. S1B). These data showed that T-3256336 canbind to cIAP-1, cIAP-2, and XIAP and can functionallyinhibit XIAP.

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Figure 1. Chemical structure and binding affinity to the BIR3 domain of XIAP, cIAP-1, and cIAP-2. A, chemical structure of T-3256336. B, inhibition curves of T-3256336 on binding to the BIR3 domain of XIAP, cIAP-1, or cIAP-2 as determinedwith anHTRFmethod. C, XIAP_BIR2þ3 protein inhibited caspase-3 activitydose dependently. D, recovery effect of T-3256336 on caspase-3 activity that was inhibited by XIAP_BIR2þ3. Caspase-3 was incubated with variousconcentrations of T-3256336 and XIAP_BIR2þ3 for 5 minutes at room temperature. Ac-DEVD-AMC was then added and incubated for 30 minutes.Fluorescence detection of substrate cleavage was assessed.






T-3256336 induced the proteasomal degradation ofcIAP-1, the activation of NF-kB, and the extrinsicapoptosis in MDA-MB-231 breast cancer cells

It has been reported that IAP antagonists induce thedegradation of cIAP-1, which leads activation of NF-kBthrough the stabilization of NIK and the recruitment ofRIP1, resulting in TNF-a production and kills sensitivetumor cells through an extrinsic apoptosis in a subset ofsensitive tumor cells apoptotic signaling pathway (23–26).We therefore tested the activity of our novel small-mol-ecule compound, T-3256336, with respect to cIAP-1 deg-radation, NF-kB activation, and caspase activation inMDA-MB-231 breast cancer cells. T-3256336 efficientlyinduced cIAP-1/-2 degradation (IC50 < 5 nmol/L), where-as it did not affect other IAPs (Fig. 2A). The proteasomeinhibitor,MG-132, prevented theT-3256336–induceddeg-radation of cIAP-1 protein, which was consistent with theobservations for other IAP antagonists (SupplementaryFig. S2A). The rapid degradation of cIAP-1was associatedwith the increased phosphorylation of IkBa and NF-kBp65, which was indicative of NF-kB activation (Supple-mentary Fig. S2B). TNF-amRNA levels were time depen-dently induced by treatment with T-3256336 (Fig. 2B).TNF-a secretion into culture medium was induced bytreatment with T-3256336 (Supplementary Fig. S3A). Thecleavage of caspase-8 and caspase-3,which is indicative oftheir activations, was also observed (Supplementary Fig.S2B). T-3256336 activated the executioner caspase-3/cas-pase-7 and the initiator caspase-8 but not caspase-9 with 4

hours of treatment (Fig. 2C). T-3256336 inhibited theproliferation of MDA-MB-231 breast cancer cell with amean GI50 value of 1.8 nmol/L, whereas proliferation ofthe normal human lung fibroblast MRC5 cells was notinhibited (Fig. 2D). T-3256336 also inhibited the growth ofMDA-MB-468,NCI-H1703, andSK-OV-3 cells but not thatof T-47D and BT-474 cells (Supplementary Fig. S4). Theprecise mechanisms underlying the different sensitivityremain to be elucidated. Human TNF-neutralizing anti-body, Z-Bad-FMK, which is a pan-caspase inhibitor, andZ-IETD-FMK, which is a selective caspase-8 inhibitor,markedly inhibited the activity of T-3256336 (Supplemen-tary Fig. S3B). These data indicated that T-3256336 func-tions as a potent and selective antagonist of IAPs in cells.

Pharmacokinetic profile of orally administeredT-3256336 in nude mice

To investigate the pharmacokinetic properties of T-3256336, T-3256336 was orally administered to mice withxenograft tumors. The concentration of T-3256336 wasmeasured during the time range of 0 to 72 hours in plasmaand xenograft tumors. The area under the curve (AUC)values of the concentration of T-3256336 were 0.29, 1.85,3.53, and 8.03 mg/mL in plasma and 4.02, 11.20, 23.05, and63.09 mg h/mg in tumors when administered at doses of10, 30, 50, and 100 mg/kg, respectively (Fig. 3, Supple-mentary Table S1). The AUC values in the tumors were 6-to 14-fold higher than those in plasma. These data clearly

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Figure 2. Biochemical activity of T-3256336 in MDA-MB-231 cells. A,T-3256336 causes a rapid loss ofcIAP-1. MDA-MB-231 cells weretreated with T-3256336 at theindicated concentration for 8hours, and cell lysates wereexamined by Western blotting withantibodies against cIAP-1, cIAP-2,XIAP, and Livin. B, T-3256336stimulates TNF-a mRNAexpression. A quantitative real-time PCR analysis of TNF-amRNAexpression levels was done on theRNA samples derived from MDA-MB231 treated with T-3256336(0.5 mmol/L) for the indicated timeperiods. All valueswere normalizedto a GAPDH internal control.C, T-3256336 treatment activatedcaspase-8 and caspase-3/caspase-7. MDA-MB-231 cellswere treated with T-3256336(0.5 mmol/L) for the indicated timeperiods, and caspase activity wasdetermined. D, T-3256336 inhibitsthe cellular proliferation of MDA-MB231 cells but not in MRC-5cells. MDA-MB-231 and MRC-5cells were treated with theindicated concentrations ofT-3256336 for 3 days, and cellviability was determined.

Sumi et al.





showed that T-3256336 was orally absorbed and efficient-ly distributed to tumor tissues.

T-3256336 induced rapid degradation of cIAP-1TNF-a–dependent apoptosis in tumor tissuesTo investigate the in vivo activities, T-3256336wasorally

administered to mice bearing xenografts of MDA-MB-231-Luc cells at doses of 10, 30, 50, and 100 mg/kg. Asingle administration of T-3256336 at 10mg/kgmarkedlydecreased the levels of cIAP-1 protein in tumors within 30minutes and the effect lasted for about 24 hours. The cIAP-1 protein degradationwas inducedmore rapidlywith 100mg/kg (Fig. 4A, Supplementary Fig. S5). Human TNF-alevels in plasma were dose dependently increased, andthe maximum level was observed 6 hours after adminis-tration (Fig. 4B). Robust activations of caspase-3/caspase-7 in tumors were observed with 30 mg/kg or more, andthe effects were dose dependently prolonged (Fig. 4C). Inaddition, we analyzed the levels of caspase-cleaved cyto-keratin-18 (M30) and total cytokeratin 18 (M65). BothM30and M65 levels in plasma were dose dependentlyincreased with 30 mg/kg or more (Fig. 4D and E). These

data showed that orally administered T-3256336 exertedits effects in xenograft tumors and that the effect could bemonitored with serum biomarkers.

In vivo efficacy of T-3256336 in xenograft micemodels

To evaluate the therapeutic potential, T-3256336 wasadministered to mice bearing MDA-MB-231-Luc xeno-graft tumors once a day for 14 days, and the effects ontumor growth, aswell as on bodyweight, were examined.Treatment with T-3256336 at 10 mg/kg completely inhib-ited tumor growth during the treatment (T/C < 5%).Treatment with T-3256336 at 30, 50, and 100 mg/kgreduced the tumor volume from around 200 to 84, 56,and 39 mm3, respectively, at the end of the treatment,whichwas a reduction of 79%, 86%, and 90%, respectively(Fig. 5A, Supplementary Table S2). Importantly, no sig-nificant body weight loss was observed in mice when 10,30, or 50 mg/kg of T-3256336 was administered (Fig. 5B).In HL-60 xenograft model, T-3256336 dose dependentlyinhibited tumor growth, and after the 2-week treatment,dose-dependent increases of necrotic area and apoptoticcells in tumor were observed (Supplementary Fig. S6Aand S6B). In a histopathologic analysis, atrophy of hairfollicles and hyperkeratosis in the skin and slight increaseof granulopoiesis in the spleen were noted at 100 mg/kg.No histopathologic abnormalities in the liver and intes-tines were detected up to 100mg/kg (Supplementary Fig.S7). Taken together, our data showed that T-3256336 canexert antitumor effects without severe adverse effects.

Correlation between pharmacodynamic biomarkersand antitumor effects in the MDA-MB-231-Lucxenograft model

To analyze the correlations between pharmacodynam-ics and antitumor efficacy, we calculated the area underthe effect (AUE) of pharmacodynamic parameters. Thetotal pharmacodynamic responses of cIAP-1 degradation[AUEcIAP-1 degradation(0–24 h)] in the tumor were saturatedaround 10 mg/kg (Fig. 6A). The AUE of TNF-a secretion[AUETNF-a(0–24 h)], M30 [AUEM30(0–48 h)], and M65[AUEM65(0–48 h)] in plasma increased dose dependentlywith 10 to 50 mg/kg of T-3256336. However, they weresimilar at around 50mg/kg (Fig. 6B,D, andE). In contrast,the AUE of caspase activation [AUEcaspase activation(0–48 h)]in tumors increased dose dependently up to 100 mg/kg(Fig. 6C). TGI was positively correlated with the AUE ofTNF-a, M30, and M65 levels in plasma in MDA-MB-231-Luc xenograftmodels (Supplementary Fig. S8). Thesedatasuggested that pharmacodynamic parameter levels, suchas TNF-a, M30, and M65 in plasma, could predict theeffects of the compound in the clinic.

DiscussionTargeting of the IAP family is a widely accepted cancer

therapeutic strategy for the induction of tumor-selectivecell death (23–26). In this study,wedeveloped a novel and

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orally available small-molecule IAP antagonist, T-3256336, that binds specifically to cIAP-1, cIAP-2, andXIAP. We showed that T-3256336 induced the rapiddegradation of cIAP-1, activation of NF-kB, the produc-tion and secretion of TNF-a, and TNF-a–dependentapoptosis. In addition, we showed that the oral admin-istration of T-3256336 significantly induced rapid cIAP-1 degradation and apoptosis in tumor tissues. Consis-tent with its potent activity in apoptosis induction inxenograft tumor tissues, T-3256336 was highly effectivein the inhibition of tumor growth xenograft micemodel.

The binding-inhibitory activities of T-3256336 againstcIAP-1-BIR3 and cIAP-2-BIR3 were stronger than thebinding-inhibitory activity against XIAP-BIR3. ThesecIAP-dominant profiles of T-3256336 were confirmed bya co-crystal structural analysis of T-3256336 with bothXIAP and cIAP-1, which indicated a higher binding affin-ity of the T-3256336 against cIAP-1 (manuscript in prep-aration). A cell-free functional assay with recombinantXIAP containing BIR2-BIR3 showed that T-3256336 hadXIAP inhibition potency, which has been reported to beimportant for the sufficient induction of apoptosis byIAP antagonists. In the pharmacokinetic analysis, dose-

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Figure 4. T-3256336 showspharmacodynamic activity inMDA-MB-231-Luc xenograft models.MDA-MB-231-Luc tumor–bearingmice were orally administeredT-3256336 at 10, 30, 50, or100 mg/kg. Mice were sacrificed atthe indicated time after the singletreatment of T-3256336, and tumorand plasma pharmacodynamicsparameters were measured. A,tumor cIAP-1 protein levels weredetermined by Western blotting.Plasma human TNF-a levels (B)were determined by ELISA. C,tumor caspase-3/caspase-7activity was determined after theindicated time periods. PlasmaM30 (D) and M65 (E) levels weredetermined by ELISA. The data arepresented as means and SDs from3 mice in each group (N ¼ 3).

Sumi et al.





dependent pharmacokinetics and a relatively high tumorconcentration were observed. The concentration of T-3256336 in the tumors at 30, 50, and 100 mg/kg dosingwas sufficient for the functional inhibition of XIAP. There-fore, these data suggested that T-3256336 has the potentialto show strong efficacy as an orally available IAP antag-onist in mouse models.Our in vivo data clearly showed that a single dose of T-

3256336 was effective in inducing rapid cIAP-1 proteindegradation, TNF-a secretion, caspase activation, andapoptosis in the MDA-MB-231-Luc tumor tissues. cIAP-1 protein levels were decreased within 30 minutes after asingle administration of T-3256336 at 10, 30, 50, and 100mg/kg, and the effect lasted for at least 24 hours. Fur-thermore, T-3256336 induced TNF-a secretion (maximum

concentration at 6 hours post-dose) and caspase activation(maximum activity at 12 hours post-dose). The time dif-ference in the maximum concentration between cIAP-1protein degradation and TNF-a secretion/caspase acti-vation was a reasonable response based on the mechan-isms of action of IAP antagonists. We found thatAUEcIAP-1 degradation(0–24 h) and AUETNF-a(0–24 h) increaseddose dependently in the dose range of 10 to 50 mg/kg inthe MDA-MB-231-Luc xenograft model. However, therewas no difference between cIAP-1 protein degradationandTNF-a secretion levels above 50mg/kg, and, thus, theEmax for the pharmacodynamic response could be satu-rated at a dose around 30 to 50 mg/kg. Both cIAP-1degradation and TNF-a secretionwere expected to reflectthe cIAP-1 inhibition potency. Therefore, the concentra-tion of T-3256336 in tumor tissues could be adequate fortriggering the induction of TNF-a–dependent apoptosisin MDA-MB-231-Luc xenograft tumors based on thestrong cIAP-1 inhibition potency of T-3256336. However,we found that AUEcaspase activation(0–48 h) increased dosedependently. Caspase activation at the peak time wasalmost the same as those for doses at 50 and 100 mg/kgof T-3256336. However, caspase activation lasted longerfor doses at 100 mg/kg of T-3256336 than doses of50 mg/kg of T-3256336, reflecting the prolonged concen-tration of T-3256336 in tumors at 100 mg/kg. Therefore,XIAP inhibition potency could contribute to the pro-longed caspase activation inMDA-MB-231-Luc xenograftmodels. As a reflection of this prolonged caspase activa-tion, T-3256336 showed strong TGI potencywith a once-a-week administration regimen of 100 mg/kg inMDA-MB-231-Luc xenograft models (data not shown). These datasuggested that both cIAP-1 inhibition andXIAP inhibitionare necessary for efficient apoptosis induction in TNF-a–dependent tumors. Consistent with this profile of T-3256336, our data indicated that T-3256336 exhibited avery strong in vivo antitumor activity at nontoxic doseschedules. However, an optimal human dose and sched-ule need to be determined in clinical studies.

Cytokeratins are expressed inmost epithelial cells, and,in many carcinomas, fragmented or complexed cytoker-atins have been detected in the circulation of patientswithepithelial malignancies. Thus, they have been evaluatedas tumor biomarkers (32–35). The M65 assay detects full-length and caspase-cleaved cytokeratin-18 and thus hasbeen proposed as a biomarker of caspase-dependent and-independent cell death (36). TheM30 assay detects only acytokeratin-18 neoepitope that is generated followingcaspase cleavage and that is considered a specific assayfor epithelial apoptosis (37–39). Several reports haverecently suggested that the circulating form of cytoker-atin-18 is predictive of tumor response to drug treatmentand may have prognostic significance (40–42). In thisstudy, both the circulating levels of M30 and M65 inplasma increaseddosedependently, peaking 9hours aftera single dose of T-3256336. Recently, several reports haverevealed that the loss of cIAP proteins can modulateprogrammed necrosis, necroptosis, as well as apoptosis

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Figure 5. Single-agent efficacy of T-3256336 in MDA-MB-231-Lucxenograft models. MDA-MB-231-Luc tumor–bearing mice were treatedwith T-3256336 daily at 10, 30, 50, or 100mg/kg/day for 14 days. A tumorgrowth curve (A) is shown, and body weight changes (B) are shown.The data represent the means and SDs from 5 mice in each group.Statistical significance was determined by Shirley–Williams test.�, P < 0.05; ��, P < 0.01 versus vehicle treatment. q.d., once daily dosing.






(43, 44). Therefore, not onlyM30 but alsoM65 levels couldbe increased dose dependently as a response to T-3256336administration. In addition, both AUEM30(0–48 h) andAUEM65(0–48 h) were positively correlated with the TGI inMDA-MB-231-Luc xenograft models. These data sug-gested that the measurement of circulating M30 andM65 levels in patient samples could be a promisingmethod for determining the efficiency of IAP antagonistsin the clinic.

In this study, we showed that cIAP-1 expression levelsand caspase activation in tumor tissues and circulatingTNF-a, M30, andM65 levels were useful for detecting the

in vivo efficiency of IAP antagonists at an early stage oftreatment as these pharmacodynamic parameters corre-lated with TGI. This study provided further insights intothe biomarkers of IAP antagonists. Circulating TNF-a,M30, and M65 levels may be potential biomarkers fordetecting cell death and clinical efficiency as invasivebiomarkers in the clinic. Furthermore, our data providedevidence that T-3256336 is a promising new anticancerdrug worthy of further evaluation and development.

Disclosure of Potential Conflicts of InterestAll authors are employees of Takeda Pharmaceutical Company, Ltd.

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Figure 6. Total pharmacodynamicresponses of thepharmacodynamic parameterAUEs in MDA-MB-231-Lucxenograft models. The AUEs ofcIAP-1 degradation (A) and humanTNF-a (B) up to 24 hours after asingle dose of T-3256336.Caspase activation (C) andM30 (D)and M65 (E) levels up to 48 hoursafter a single dose of T-3256336were calculated by multiplying thecumulative mean values by time (1hour equals 1 unit).

Sumi et al.





Authors' ContributionsConception and design: H. Sumi, M. Yabuki, K. Iwai, R. Hibino, T.IshikawaDevelopment of methodology: H. Sumi, R. Hibino, K. Hashimoto, M.YoshimatsuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H. Sumi, M. Yabuki, K. Iwai, M. Morimoto, R.Hibino, Y.Kosugi, K.Aoyama, S. Yamamoto,M. Yoshimatsu,H.YamasakiAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): H. Sumi, M. Yabuki, M. Morimoto, R.Hibino, Y.Kosugi, K.Aoyama, S. Yamamoto,M. Yoshimatsu,H.YamasakiWriting, review, and/or revision of themanuscript:H. Sumi,M. Inazuka,K. HashimotoAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M. YoshimatsuStudy supervision: H. Sumi, R. Tozawa, T. Ishikawa, S. YoshidaDesign and synthesis of IAP antagonist, T-3256336: K. Hashimoto

AcknowledgmentsThe authors thank Akihiro Ohashi, Takeo Arita, Akito Nakamura,

Yuichi Hikichi, and Osamu Nakanishi for helping with insightful discus-sions, suggestions, and reagents and the entire IAP teammembers for theirsupport and many contributions to this project.

Grant SupportAll works were funded by Takeda Pharmaceutical Company, Ltd. All

authors have not received any grant.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received July 6, 2012; revised October 30, 2012; accepted November 29,2012; published OnlineFirst December 12, 2012.

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2013;12:230-240. Published OnlineFirst December 12, 2012.Mol Cancer Ther Hiroyuki Sumi, Masato Yabuki, Kenichi Iwai, et al. Apoptosis Proteinsand Orally Available Small-Molecule Antagonist of Inhibitor of Antitumor Activity and Pharmacodynamic Biomarkers of a Novel

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