European Journal of Pharmacology 711 (2013) 19–26

Contents lists available at SciVerse ScienceDirect

European Journal of Pharmacology

0014-29http://d

n CorrVillageIndia. Te

E-m

journal homepage: www.elsevier.com/locate/ejphar

Molecular and cellular pharmacology

RBx10080307, a dual EGFR/IGF-1R inhibitor for anticancer therapy

Ruchi Tandon a,n, V. Senthil a, D. Nithya a, Venu Pamidiboina a, Ankur Kumar a, Sumit Malik a,Tridib Chaira c, Manish Diwan a, Praful Gupta b, R. Venkataramanan b, Renu Malik a,Biswajit Das b, Sunanda G. Dastidar a, Ian Cliffe b, Abhijit Ray a, Pradip Kumar Bhatnagar a,b,c

a Department of Pharmacology, Gurgaon, Haryana, Indiab Department of Medicinal Chemistry, Gurgaon, Haryana, Indiac Department of Metabolism and Pharmacokinetics, New Drug Discovery Research, Ranbaxy Laboratories Limited, Plot No. 20, Sector-18, Gurgaon 122001,Haryana, India

a r t i c l e i n f o

Article history:Received 9 January 2013Received in revised form5 April 2013Accepted 13 April 2013Available online 29 April 2013

Keywords:IGF-1REGFRSignaling pathwayCancerResistanceMutant cell line

99/$ - see front matter & 2013 Elsevier B.V. Ax.doi.org/10.1016/j.ejphar.2013.04.016

espondence to: Daiichi Sankyo Life Science RSarhaul, Sector-18, Udyog Vihar Industrial Arel.: +91 124 284 8737; fax: +91 124 239 7546.ail address: ruchi.tandon.fg@dsin.co.in (R. Tan

a b s t r a c t

Pharmacological intervention of epidermal growth factor receptor (EGFR) family members by antibodiesor small molecule inhibitors has been one of the most successful approaches for anticancer therapy.However this therapy has its own limitations due to the development of resistance, over a period of time.One of the possible causes of the development of resistance to the therapy with EGFR inhibitors could bethe simultaneous activation of parallel pathways. Both EGFR and insulin like growth factor-1 receptor(IGF-1R) pathways are reported to act reciprocal to each other and converge into the mitogen activatedprotein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways. Inhibiting one pathway alonemay therefore not be sufficient and could be a cause of development of resistance. The other cause couldbe mutations of EGFR which would be less sensitive to the inhibitors. We, therefore, suggest that co-targeting IGF-1R and EGFR kinases by dual inhibitors can lead to improved efficacy and address theproblems of resistance.

In the present manuscript, we report the identification of a novel, small molecule dual EGFR/IGF-1Rinhibitor, RBx10080307 which displayed in vitro activity at the molecular level and oral efficacy in mousexenograft model. The compound also showed in vitro activity in an EGFR mutant cell line and may thushave the potential to show activity in resistant conditions. Additional efficacy studies are needed in EGFRresistant mouse cancer model and if found efficacious, this can be a major advantage over standaloneerlotinib and other existing therapies.

& 2013 Elsevier B.V. All rights reserved.

1. Introduction

Clinical studies have shown that epidermal growth factorreceptor (EGFR) is over-expressed in a variety of cancer typesincluding breast cancer, non-small cell lung cancers (NSCLCs) andB-cell acute lymphoblastic leukemia (B-ALL) (Baselga, 2000). Smallmolecule inhibitors of EGFR family members like gefitinib anderlotinib have been developed and are currently used in the clinic.However, most tumors develop resistance to this therapeutics overa median period of 6–12 months (Yamasaki et al., 2007). Rigorousresearch has been carried out by various groups in a variety ofclinical and pre-clinical studies to suggest the mechanisms under-lying the development of resistance of these drugs despite aninitial positive response. The most common resistance mechan-isms deciphered so far are development of secondary EGFR

ll rights reserved.

esearch Centre in India (RCI)a, Gurgaon 122015, Haryana,

don).

mutations and activation of alternate signaling mediators likeIGF-1R, c-Met, ras or src.

In our previous study we have shown some preliminaryfindings by following a systems biology approach supported withconfirmatory in vitro data (Tandon et al., 2011) that dual targetingof EGFR and IGF-1R may be a more effective approach compared tosingle targeting of any of these two receptors. In addition, thereare reports which suggest that IGF-1R can interact with EGFR toaugment the malignant behavior of tumors (Wilsbacher et al.,2008; Werner et al., 1991; Baserga et al., 2003; Baserga, 1995;Yu and Rohan, 2000; LeRoith et al., 1995; Arteaga, 1992; Sachdevet al., 2003; Dunn et al., 1998; Chernicky et al., 2000) andoveractivation of IGF-1R could be an important contributorof EGFR resistance (Adams et al., 2004; Gilmore et al., 2002).Co-expression of members of epidermal growth factor receptor(EGFR) family and insulin like growth factor-1 (IGF-1R) has alsobeen linked to clinical outcomes in several solid tumors. Wetherefore suggest that dual inhibition of EGFR and IGF-1R couldbe a viable approach to achieve a more effective treatmentresponse and to overcome resistance.

Table 1

Erlotinib

AEW-541Erlotinib AEW-541

RBx10080307RBx10080307In vitro profile of RBx10080307

RBx No. Cell free IC50 (nM) Cell proliferation (IC50 nM)

IGF-1R EGFR A431 A549 HT29 Hela MIA PaCa-2 HFF H1975

RBx10080307 277 54 317 131 97 209 266 410,000 1140Erlotinib 410,000 45 1080 410,000 2275 NA 410,000 410,000 410,000AEW-541 291 410,000 899 615 9540 1200 NA 755 410,000

IGF-1R assay was performed using fluorescence based assay using a fluorescent quencher (IQ reagent. EGF-R kinase assay was performed using an ELISA based method using Poly-Glu-Tyr as a substrate and antibody. AEW 541 anderlotinib were used as standard IGF-1R and EGFR inhibitors. For cell proliferation assay, 2�103 cells/well in growth media were treated with 1 ml of test drug and incubated for 48 h. Cell numbers were quantitated by MTT assay andpercentage inhibition of the cells in the presence of compound, as compared to DMSO control was calculated and used in Graph Pad Prism 4 software to calculate the IC50 values.

R.Tandon

etal./

EuropeanJournal

ofPharm

acology711

(2013)19

–2620

R. Tandon et al. / European Journal of Pharmacology 711 (2013) 19–26 21

In the present manuscript, we report the identificationof a novel dual EGFR/IGF-1R inhibitor, RBx10080307 (N4-(3-cyclo-propyl-1H-pyrazol-5-yl)-5-fluoro-N2-[4-(piperazin-1-yl) phenyl]pyrimidine-2,4-diamine) (Table 1), with in vivo efficacy in a mousexenograft model. We have also shown that RBx10080307 is42 times more efficacious compared to erlotinib, an EGFR inhibitorin the cell proliferation assays using EGFR mutant cell line, H1975,which supports our concept that a dual EGFR/IGF-1R inhibitorwould be capable of overcoming EGFR resistance. Our findingsprovide a base to further evaluate the efficacy of EGFR/IGF-1R dualinhibitors with an objective to get a drug with an improvedefficacy and ability to overcome clinical resistance of EGFRinhibitors.

2. Materials and methods

2.1. Reagents and cell culture

2.1.1. ReagentsRecombinant IGF-1R was purchased from Upstate, USA, and

polyGlu-Tyr, Sodium ortho vanadate, 3,3′,5,5 Tetramethylbenzi-dine (TMB) and mammalian cell lysis/extraction reagent, Cell lytic-M from Sigma. IGF-1R peptide (Rhodamine-KKKSPGEYVNIEFG)was custom synthesized from Sigma. Protease inhibitor cocktailtablets were purchased from Roche, USA, and enhanced chemilu-minescence (ECL) reagent from Millipore, USA. AEW 541 andRBx10080307 were synthesized in house with 497% purity.Erlotinib HCl purity499% was procured commercially fromAuspure Biotechnology Co., Ltd., China. JC-1 (5,5′,6,6′-tetrachloro-1,1′3,3′-tetra ethyl benzimidazo carbocyanine iodide dye) andCCCP were purchased from Molecular Probes, USA.

2.1.2. AntibodiesAntibodies were purchased from commercial sources as indi-

cated: mouse monoclonal anti-phosphotyrosine, clone PT-66 per-oxidase conjugate antibody, rabbit anti-phospho-IR/IGF-1R(pTyr1158/1162/1163) and monoclonal anti-β-actin peroxidasefrom Sigma, USA, rabbit polyclonal anti-EGFR from Santacruzbiotechnology, UK, and rabbit anti-phospho-EGFR(Tyr1068), rabbitanti-phospho Akt (Ser473), anti-Erk1/2 pTpY185/ 187, cyclin-D1,rabbit anti-IGF-1R β from Cell signaling technology, UK.

2.1.3. Cell lines and cell culture conditionsAll the cell lines in the study were purchased from the

American Type Culture Collection (ATCC, USA). Culture media,fetal calf serum and antibiotics (streptomycin and penicillin) werepurchased from GIBCO laboratories, USA. A431 (human epider-moid cancer), A549 (human lung carcinoma), HT29 (human coloncarcinoma) were maintained in DMEM and H1975 (human lungcarcinoma with EGFR mutations) was maintained in RPMI mediasupplemented with 10% fetal bovine serum. The culture mediumfor all the cell lines also contained L-glutamine supplemented with10% fetal bovine serum, 100 units/ml penicillin G and 100 μg/mlstreptomycin (1% penicillin/streptomycin), and cells were allowedto propagate at 37 1C in a humidified 5% CO2 incubator. Pre-wet,sterile hollow fibers made of PVDF with pore size of 500 kDa wereprocured from Spectrum Medical, USA, and CCK-8 kit fromDojindo Labs, Japan.

2.2. Animals

Balb/c Nude mice, age 4–12 weeks of both sexes were obtainedfrom the Animal Handling and Breeding Facility, Ranbaxy Labora-tories Ltd. For Hollow fiber studies, 8–12 week old mice weretaken while for Xenograft studies younger animals of 4–6 weeks

age were used. For pharmacokinetic study male Swiss mice(2572 g) were obtained from the Animal Handling and BreedingFacility, Ranbaxy Laboratories Ltd. Animals were acclimated forthree days before initiation of the study. The animals were housedin standard cages and maintained at a temperature of 2472 1Cwith controlled illumination to provide a light and dark cycle of12 h with access to food and water ad libitum. All animal experi-ments were performed with Institutional animal ethics committeeapproval.

2.3. Experimental procedures

2.3.1. Kinase assays for EGFREGFR kinase assays were performed using an ELISA based

method. Poly-Glu-Tyr was used as tyrosine kinase substrate.Ninety microliters of tyrosine kinase buffer, containing 50 mMHEPES, 20 mM MgCl2, 100 mM MnCl2 200 mM Na3VO4 and 100 mMATP, was added to the poly-Glu-Tyr coated 96 well ELISA platefollowed by the addition of 1 ml test compound, dissolved indimethyl-sulfoxide (DMSO). Purified EGFR enzyme (25 ng) wasadded to each well and reaction incubated at 25 1C for 30 min.Assay plates were washed with phosphate buffer saline (PBS) and100 ml anti-phospho tyrosine–peroxidase conjugated antibody wasadded to each well. Plate was incubated at 25 1C for an additional30 min and washed with PBS. 100 ml of tetra methyl benzidine(TMB) solution from Sigma was added to each well followed by theaddition of 2.5 N H2SO4 to stop the reaction. A readout was takenin ELISA reader at 450 nm. Percent enzyme inhibition was calcu-lated compared to the DMSO vehicle control and IC50 values werecalculated using the Graph pad prism version 4.

2.3.2. Kinase assay for IGF-1RIGF-1R Kinase assay was performed using fluorescence based

assay. Briefly, 8 ml buffer A (20 mM HEPES, 1 mM DTT, 0.05%TritonX-100, pH 7.4) and 4 μM ATP were added to a 384 well platefollowed by the addition of 1 μl of test drug/DMSO. Sixteenmicroliters of buffer B (20 mM HEPES, 5 mM MgCl2, 2 mM MnCl2,pH 7.4) containing 5 mM substrate peptide (Rhodamine-KKKSPGEYVNIEFG, sigma) and 25–50 ng of purified enzyme(US Biologicals) was added to each well. Reaction was incubatedfor 90 min at 25 1C. This was followed by adding 50 ml of 0.25� IQreagent (Pierce Technology) to each well. Fluorescence intensitywas determined at 544/590 nm and enzyme activity was calcu-lated compared to no drug vehicle control. IC50 was calculatedusing the graph pad prism software version 4.

2.3.3. Cell proliferation assaysCell proliferation assays were carried out in A431, A549, HT29,

Mia-Paca-2, HeLa and H1975 in a 96 well plate format using3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra sodium bromide(MTT) method (van de Loosdrecht et al., 1994). Cells were seededin 96 well plates with a cell density of 2500 cells in each well. Cellswere treated with test drug, dissolved in DMSO (0.5% final conc.)for 48 h. Cells were then treated with the MTT reagent for 4 hfollowed by the addition of DMSO to lyse the cells and solubilizethe formazan crystals. The samples were read using an ELISA platereader at a wavelength of 570 nm. The amount of color producedwas directly proportional to the number of viable cells. Inhibitionof cell growth, in the presence of test compounds, with respect tocontrol wells was calculated as percentage inhibition and used tocalculate the IC50 values using Graph pad prism version 4.

2.3.4. Combination assaysEGFR Kinase inhibitor, erlotinib and EGFR and IGF-1R, dual

inhibitor, RBx10080307 were used in combination to determine

R. Tandon et al. / European Journal of Pharmacology 711 (2013) 19–2622

the inhibition of cell growth in H1975 cells after 48 h of drugtreatment. IC50 of erlotinib was determined in the presence of300 nM and 1 mM concentrations of RBx10080307. The effect ofthe combination was compared with the results of individualcompounds and combination index (CI) (Tang et al., 1998) calcu-lated using the following formula:

CI ¼D1=Dx1þ D2=Dx2þ αD1D2=Dx1Dx2

where (D)1 and (D)2 are the doses of drugs 1 and 2, which arerequired to produce x% effect in combination. (Dx)1 and (Dx)2 arethe doses of drugs 1 and 2 required to produce x% effectindividually, α¼0 for mutually exclusive drugs (CI∼1 denotesadditive effect, CIo0.8 indicates synergism and CI41.2 indicatesantagonism).

2.3.5. Determination of phosphorylationCells were treated with drugs at different concentrations in

growth media containing 10% bovine serum for 48 h followed bylysis of cells using CelLytic buffer (Sigma, USA) with 1 mM sodiumortho vanadate and protease inhibitors cocktail (Roche). Sampleswere analyzed for the levels of phosphorylated and total signalingproteins by western blot using BioRad electrophoresis and transfersystems and enhanced chemiluminescence (ECL) detection. Den-sitometry analysis was also done to obtain a quantitative assess-ment of effect of inhibitors on the levels of protein expression ofsignaling mediators of the EGFR/IGF-1R pathway by measuring theband densities in western blot experiments using NIH ImageJsoftware. All the band intensities were normalized from thebackground value and percent expression calculated with respectto control (Test/control�100¼% expression). Percent inhibitionwas calculated from the above expression levels (100−%expression¼% inhibition). Graph pad prism 4 (Non-linear regres-sion fit−Log (inhibitor) vs response) was used to calculate the IC50

values.

2.3.6. Analysis of mitochondrial membrane potentialA431 cells were plated in 96 well plate at a density of 50,000

cells/well in 200 μl complete medium and then treated with thedrugs next day for 24 h. CCCP (carbonyl cyanide 3-chlorophenyl-hydrazone) was used as a positive control and treated for 3 h. Cellswere then treated with JC-1; Molecular probes) at a final conc. of2 μM/well for 45 min. Final DMSO conc. in each well was 0.75%.The cells were then washed with PBS to remove unbound dye,trypsinized and then transferred to a 96 well black wall clearbottom plates (Costar). Mitochondrial membrane potential forsingle cells was calculated by taking the readout in flex stationat two wavelengths 590 nm (red) and 525 nm (green). The ratio ofthe fluorescence reading at 525/590 gives a direct measure of thecells undergoing apoptosis.

2.3.7. Metabolic stability in liver microsomesThe test compound (0.5 mM) was incubated in a reaction

mixture consisting of liver microsomes and NADPH regeneratingsystem. Aliquots were withdrawn at 3 min intervals until 30 minand were analyzed for parent compound by LC–MS/MS. The loss ofparent compound was expressed as percentage of test compoundremaining with time and the rate of decay was estimated bymono-exponential decay kinetics. The rate of decay was normal-ized to microsomal protein expressed as ml/min/g liver.

2.3.8. Mouse pharmacokineticsMouse pharmacokinetic study was carried out at three doses

4 mg/kg intravenous, 20 mg/kg oral or 100 mg/kg oral. Animalswere fasted for 12 h before dosing and 2 h after dosing. Water wasallowed throughout the study period. Intravenous formulation was

prepared as 1.25 mg/ml solution in N-saline. Oral formulation of5 mg/ml concentration was prepared in normal saline and12.5 mg/ml concentration was prepared in 0.25% w/v methylcellulose for 20 mg/kg and 100 mg/kg dose, respectively. Oraladministration was done using an 18 G stainless steel intubationcannula and intravenous administration as a bolus through tailvein. Sparse plasma samples, from ∼300 ml bleeds, were obtainedunder ether anesthesia in a tube containing sodium citrate asanticoagulant at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h post-dose(intravenous administered rats) or at 0.25, 0.5, 1, 2, 4, 8, 12 and24 h post-dose (oral administered rats). The plasma samples wereanalyzed in the Perkin-Elmer HPLC system (200 Series) interfacedto API-4000 mass spectrometer (Applied Biosystems, USA) con-trolled by Analyst 1.4.1 software. The plasma concentration–timedata analysis was performed using NCA module of WinNonlinProfessional software (Version 4.1).

The oral absolute bioavailability was calculated from theDNAUC (Dose Normalized AUC) of oral and intravenous exposuresusing the following formula:

%F ¼ DNAUCpo

DNAUCiv� 100

2.3.9. In vivo activity screening in hollow fiber modelHollow fiber (HF) studies were performed as a pre-screen

(Hollingshead et al., 1995), before undertaking longer xenograftstudies. Since RBx10080307 had shown efficacy in multiple celllines in the cell proliferation assays, hollow fiber studies werecarried out to select a cell line which is most sensitive to thismolecule for further in vivo xenograft studies. RBx10080307 wastested simultaneously in the same animal, against 3 tumor celllines, selected on the basis of in vitro cellular data.

Tumor cell lines were individually packed aseptically in hollowfibers and heat sealed to make small fragments of about 2 cmlength. Different colored hollow fibers were used for differenttumor cell lines. Under anesthesia, 3 hollow fiber fragments wereimplanted in mice subcutaneously with the help of trocar needles.Animals were grouped as control, test and reference standard.RBx10080307 was tested at a dose of 100 mg/kg/day given in3 divided doses daily for 7 days and erlotinib at 100 mg/kg/day asa single dose once daily for 7 days as reference standard. Controlanimals received vehicle. Both compounds were administered asfreshly prepared 0.25% methyl cellulose suspension by oral route.

On the 7th day, post-2 h dosing, terminal blood samples(∼500 μl) were collected for plasma concentration measurements.Animals were euthanized to retrieve the implanted HF fragmentsfrom all the groups. The excised HF fragments were cleaned toremove any adherent tissues and incubated in accutase solutionfor 20 min. The cells were retrieved in 200 μl complete media bygently flushing individual fragment. The viability of the retrievedcells was determined using WST-8 containing CCK-8 kit basedcolorimetric readout as per manufacturer's instructions.

The tumor cell growth inhibition was calculated as follows:

Percent growth inhibition¼Absorbance units of control−Absorbance units of treated

Absorbance units of control� 100

2.3.10. Efficacy study in HT29 xenograft modelApproximately 30 mg of HT29 tumor fragments was implanted

subcutaneously on the back of nude mice using a trocar needleand observed for tumor induction. Animals showing tumor growthof 100–200 mm3 were randomized and recruited in the study tostart the treatment. Experimental mice were monitored at leastonce a week for body weight measurements and up to two timesin a week for tumor growth using a digital Vernier calipers (Make:

R. Tandon et al. / European Journal of Pharmacology 711 (2013) 19–26 23

CD-6”CS, Mitutoyo, Japan). Test group animals received RBx10080307 at 100 mg/kg, BD and positive control group receivederlotinib at 100 mg/kg, OD for 21 days by oral route. Animals ofcontrol group received vehicle, i.e., 0.25% methyl cellulose at 5 ml/kg, OD for 21 days. Tumor volumes and tumor growth inhibitionwere calculated as follows (Higgins et al., 2004):

Tumor volume¼ ðL�W2Þ=2 ðUnit : mm3Þwhere L¼ length, W¼width

Percent tumor growth inhibition¼ ð1−T=CÞ � 100

where T is the change in tumor volume of Test group from day 0 today n; C is the corresponding change in the Control group.

Details of animals in each experimental group are given below.

Groupno.

Treatment

Dose Groupsize (n)

Group I

Vehicle control(0.25% methylcellulose solution)

–

6–8

Group II

RBx10080307 100 mg/kg bodyweight/day; BD,qd�21

6–8

Group III

Erlotinib 100 mg/kg bodyweight/day, OD,qd�21

6–8

2.3.11. Statistical analysisGrowth in tumor volume for experimental animals of different

treatment groups was represented as Mean7S.E.M. and analyzedfor statistical significance by unpaired t-test with respect tocontrol group. P-valueo0.05 was considered as statisticallysignificant.

Table 2Erlotinib and RBx10080307 combinations synergistically decrease cell numbers inH1975 cell line.

Individual IC50s in nM Erlotinib dose–response in the presence ofRBx10080307 (concentration in nM)

Erlotinib(DX1)

RBx10080307(DX2)

Erlotinib IC50(D1)

RBx10080307 conc.(D2)

CI atIC50

40140 1140 22 1000 0.873785 300 0.36

Cells were grown for 48 h in DMEM with 10% FBS in the presence of dose–responseof either erlotinib alone or in combination with RBx100080307 Live cells werequantified with MTT reagent. Combination indices (CI) were calculated using theformula as given in the experimental procedures section. Combination indexo in-dicates synergism, combination index¼1 index indicates additivity and combina-tion index41 indicates antagonism. Data are average of 2 independentexperiments with triplicate samples.

3. Results

3.1. In vitro activity of RBx10080307

IC50 of RBx10080307 for EGFR inhibition was found to be54 nM which was comparable to erlotinib in our test system(Table 1). IGF-1R IC50 of RBx10080307 was found to be 277 nMwhich was comparable to AEW-541 (Table 1).

RBx10080307 was tested against a panel of cancer cell lines andfound efficacious in A431, A549, HT29, MiaPaCa-2 and HeLa cell lineswith IC50 values in the range of 97–317 nM. All the three moleculeswere also tested in a non-cancer, human fibroblast cell line HFFwhich does not over-express the target receptors. RBx10080307 anderlotinib did not show any inhibition up to 10 μM, while for AEW-541IC50 was 755 nM in this cell line (Table 1).

3.2. RBx10080307 inhibits the proliferation of the EGFR mutant cellline resistant to erlotinib

An EGFR double mutation (T790M) has been identified inNSCLC patients who initially responded to gefitinib or erlotinibbut later developed drug resistance (Gow et al., 2005). To assessthe potential of RBx10080307 and erlotinib to inhibit the activityof a cell line harboring the T790M EGFR mutation, cell prolifera-tion assays were performed using the bronchoalveolar cell lineH1975 which harbors both an activating (L858R) and resistanceassociated mutation (T790M). Cell proliferation assays were alsoperformed in A431 cells, which express WT EGFR in parallel, for

comparison. H1975 cells were found to be resistant to EGFRinhibitor erlotinib as compared to the cells harboring WT receptor.IC50 of erlotinib in H1975 cell line was found to be 410 μM (actualvalue 40.1 μM). IGF-1R inhibitor, AEW-541, alone was also notactive in the mutant cell line and its IC50 was found to be 410 μM(actual value 27.2 μM). RBx10080307 on the other hand was foundto be 410 times more active than erlotinib and its IC50 was foundto be 1.1 μM in H1975 cells. This data suggests that RBx10080307 ismore potent than erlotinib or AEW-541 in the H1975 cell pro-liferation assay (Table 1).

3.3. Combination of erlotinib and RBx10080307 synergisticallyblocks cell proliferation and increases cell death in H1975 Cells

In our earlier manuscript (Tandon et al., 2011) we have shown thatthe combined use of erlotinib and AEW-541 synergistically decreasescell numbers in A431 cell line. Since erlotinib has been very effective inthe initial treatment periods in cancer patients, but shows resistanceafter a few weeks of treatment, we performed in vitro experiments inEGFR resistant cell line, H1975, to evaluate whether a combination ofRBx10080307 can improve the efficacy of erlotinib in this cell line. Forthe combination study, cell proliferation assays were conducted inwhich cells were treated with compounds for 48 h. Dose–responsecurves were generated for erlotinib and RBx10080307 alone and forerlotinib in the presence of 300 nM or 1 mM concentration ofRBx10080307 (sub-optimal concentration of RBx10080307 in H1975cells). Combination indices (CI) were calculated as shown in Table 3.We have shown that combination of 1000 nM conc. of RBx10080307reduced the IC50 of erlotinib from 40.14 μM to 22 nM and when acombination of 300 nM RBx10080307 was used, IC50 of erlotinib,reduced to 3.8 mM. In both the cases the combination index (CI) wascalculated using the formula given above and was found to be o1,which is suggestive of synergistic inhibition (Table 2). Our studiessuggest that a combination of RBx10080307 increases the sensitivityof H1975 cells towards erlotinib. It is possible that inhibition of IGF-1Ralong with EGFR by RBx10080307 increases the sensitivity of erlotinibin the resistant cell line and therefore provides an experimentalevidence for our rationale for a dual EGFR/IGF-1R inhibitor.

3.4. RBx10080307 decreases the levels of phosphorylated targetreceptors and downstream mediators of the signaling pathways

Western blot studies suggest that erlotinib and AEW-541decreased the phosphorylation levels of their respective targets,EGFR and IGF-1R. RBx10080307 on the other hand decreased thelevels of both pEGFR and pIGF-1R in a dose dependent manner.Since Erk 1 and 2 are the common downstream mediators of theEGFR and IGF-1R pathways, it was hypothesized that

A431 A549 HeLa HT29 MiaPacaIGF-1R

EGFR

β Actin

Fig. 1. Effect of RBx10080307 on the protein expression levels of phosphorylated receptors and downstream mediators of the signaling pathway. (a) Levels of total andphosphorylated IGF-1R, Erk1/2, after 48 h treatment of A431 cells with different concentrations of RBx10080307, erlotinib and AEW-541. β actin was used as an internalcontrol and blots are representative of 2 independent experiments. (b) Levels of IGF-1R and EGFR proteins in different cancer cell lines.

Table 3Effect of dual IGF-1R/EGFR inhibitor on the expression levels of phosphorylatedreceptors and downstream mediators of the signaling pathway.

Drug Inhibition of phosphorylation (IC50, nM)

pIGF-1R pEGFR pErk1/2

RBx10080307 1041 700 209AEW541 o100 ND 10,000Erlotinib ND 1883 7988

Densitometric representation of percent inhibition of protein expression levels ofsignaling mediators of the EGFR/IGF-1R pathway by measuring the decrease in theband densities in Western blot experiments after treatment with RBx10080307.

00.20.40.60.8

11.21.41.6

Vehicle(DMSO) 1 3 10CCCP

RBx10080307 Conc (in uM)

525n

M/5

90nM

ratio

Fig. 2. Effect of RBx10080307 on apoptosis in A431 as determined by mitochon-drial membrance potential assay using JC-1 dye. 525/590 nM ratio as a measure ofdecreased mitochondrial membrane potential after treatment of A431 cells withRBx10080307 for 24 h.

Table 4Metabolic clearance of RBx10080307 using human and mouse liver microsomes.

RBx No. Intrinsic clearance (ml/min/g liver)

Human Mice

RBx10080307 0.6 1.6Erlotinib 1 4.4

Compound (0.5 mM) was incubated with liver microsomes (0.5 mg/ml) at 37 1C inphosphate buffer (pH 7.4) with an NADPH regenerating system present. At the

R. Tandon et al. / European Journal of Pharmacology 711 (2013) 19–2624

RBx10080307 being a dual inhibitor would significantly diminishthe levels of the activated/phosphorylated form of this protein,more than erlotinib or AEW-541 alone. Changes in the phosphor-ylation of Erk1/2 by all the three drugs were therefore investigatedby western blot to prove this concept. It was observed thattreatment with erlotinib decreased the levels of phosphor Erk1/2but IGF-1R inhibitor (AEW-541) alone did not show any decreasein the levels of this protein. Interestingly, RBx10080307 was foundto be more potent than erlotinib in decreasing the levels ofphospho Erk1/2 (Fig. 1 and Table 3).

indicated time points samples were withdrawn, reaction quenched, and the parentcompound quantitated by LC–MS/MS; data are reported as intrinsic clearancecalculated from the slope of decay.

3.5. Dual inhibition of IGF-1R and EGFR decreases the mitochondrialmembrane potential and increases apoptosis

To further characterize the effect of RBx10080307 on apop-tosis, mitochondrial membrane potential assay was performedin A431 cells. As mitochondria appear to be critically involved intriggering the apoptotic cell death, we explored whether thecombination of the two inhibitors or the dual inhibitor couldalter mitochondrial membrane potential (Ψ). Analysis of Ψ byusing JC-1 dye showed an increase in the ratio of fluorescence at525 nm/590 nM in cells undergoing apoptosis. Significant dropin mitochondrial membrane potential was observed withRBx10080307 (Fig. 2).

3.6. Microsomal stability of RBx10080307

Experiments were carried out to determine the intrinsicclearance (CLint) of the compound using human and mouse livermicrosomes, RBx10080307 was found to be stable in both humanand liver microsomes (Table 4).

3.7. Preclinical pharmacokinetics

To evaluate the bioavailability of RBx10080307, before under-taking in vivo studies, a PK study was performed in female Swissmice. RBx10080307 showed high plasma clearance (CLp42� liver

R. Tandon et al. / European Journal of Pharmacology 711 (2013) 19–26 25

plasma flow), and was well distributed (Vd∼35 times total bodywater). Absolute oral bioavailability from 20 mg/kg solution was47%. Plasma concentration time profile after intravenous admin-istration at 5.0 mg/kg appeared bi-exponential with a terminalhalf-life of 2.9 h. Oral administration of RBx10080307 at 100 mg/kg oral dose shows dose proportional increase in exposure from20 mg/kg oral dose (Table 5). The oral absolute bioavailability wascalculated from 20 mg/kg, p.o. and 5 mg/kg, i.v. plasma exposuresand is presented in Fig. 3.

3.8. In vivo activity screening in hollow fiber model

RBx10080307 was well tolerated at 100 mg/kg/day dose givenin 3 divided doses by all the animals. There was no change evidentin body weight profile between Control and Test groups. Cellgrowth inhibition of 50% or more was considered as the criterionfor an ‘active’ compound. RBx10080307 showed low to moderateactivity against 2 out of 3 cell lines tested, i.e., HT29 and A431(Table 6). Among these, HT29 was found to be most sensitive whileno activity was observed against A549 cell line. Based on this data,HT-29 was selected as the target tumor type for the xenograftstudy. Erlotinib showed inhibition of cell growth in all the 3 celllines tested.

Table 5In vivo PK of RBx10080307 in mice.

Compound RBx 10080307a Erlotinibb

Route p.o. p.o. i.v. p.o. p.o.Dose, mg/kg 20 100 5 20 100Cmax, ng/ml 264.4 869.9 – 9100 24,000Tmax, h 2 0.5 – 0.5–1 0.5–1AUClast, ng h/ml 560.2 2825.1 295.0 33,500 196,000DNAUClast, ng h kg/ml/mg 28.0 28.3 59.0 1675 1960T1/2, h 2.9CLp, ml/min/kg – – 281.2 – –

Vdss , l/kg – – 25.1 – –

F, % 47 – – – –

All values mentioned as mean of 3 animals.a Female Swiss mice were used for the study. Oral formulation was made in

0.25% w/v methylcellulose and intravenous formulation in 0.9% w/v normal saline.Following dosing, sparse blood samples were collected at different time points till24 h post-dose; plasma samples were obtained from blood samples after centrifu-gation and analyzed by an LC–MS/MS method.

b Literature value. Female nu/nu athymic mice were used for the study.Compound was formulated as a fine suspension with sodium carboxymethylcellu-lose and Tween 80 in water for injection. Following dosing, sparse blood sampleswere collected.

1

10

100

1000

0 1 2 3 4 5 6 7 8Time post-dose (h)

Con

cent

ratio

n (n

g/m

L)

5 mg/kg iv

20 mg/kg po

Fig. 3. Pharmacokinetic profile of RBx10080307 in Swiss albino mice. Mice werefasted 4–6 h pre-dose and 2 h post-dose; water was provided ad libitum. Plasmasamples were analyzed for RBx10080307 by LC–MS/MS. Normal saline was used asvehicle. Data is presented as Mean7SEM from n¼3 mice for each time point.

The mean plasma concentrations of 4717121 nM observed inthe RBx10080307 treated mice post-2 h last dose were marginallyhigher than the desired cellular IC50 values of 317 nM. Therefore,in the subsequent efficacy study in xenograft model, dose ofRBx10080307 was increased to 200 mg/kg daily to enhance theexposure levels, which was given in two divided doses.

3.9. Efficacy study in HT29 xenograft model

RBx10080307 at 100 mg/kg, twice daily dose when tested byoral route, showed significant tumor growth inhibition of 62% inmice by day 41 (Po0.05) which was comparable to erlotinib arm(100 mg/kg, once daily; Fig. 4). The body weight profile was similarfor the test and control group animals.

4. Discussion

Epidermal growth factor receptors (EGFR) are the transmembranereceptors which play an important role in controlling normal cellgrowth, apoptosis and other cellular functions. EGFR inhibitors areused in clinics to treat a variety of cancers where there is up-regulation of EGFR, including breast, pancreatic, and non-small-celllung cancer, However, resistance often develops in the clinic follow-ing longer term treatment with these inhibitors, compromising theirclinical utility. In preclinical models, interactions between IGF-1Rand EGFR signaling have been shown to contribute to the develop-ment of resistance to anti-EGFR therapies. Preclinical data available

Table 6Cell growth inhibition activity of RBx10080307 in hollow fiber model againstdifferent tumor cell lines.

Treatment Number of HF fragments showing 450% inhibition out oftotal number of HF fragments

HT29 A431 A549

Erlotinib 10/16 9/16 2/4RBx10080307 6/15 3/15 0/3

Nude mice were implanted with coded hollow fiber fragments (n¼3/mice/site)carrying different tumor cell lines. Animals were treated with either RBx10080307(100 mg/kg/day in 3 divided doses, p.o., �7 d), erlotinib (100 mg/kg/day, p.o.,�7 d) or Vehicle (0.25% methyl cellulose) alone. At the end of study, cell growthinhibition was measured in fiber fragments excised from mice. Data has beenpooled from two separate studies.

Fig. 4. HT29 xenografts study in nude mice to evaluate tumor growth profile.RBx10080307 showed significant tumor growth inhibition of 62% in mice by day 41(Po0.05). In the erlotinib arm also, animals showed a similar trend. The bodyweight of animals did not show any change between different treatment groupsand control group.

R. Tandon et al. / European Journal of Pharmacology 711 (2013) 19–2626

from co-targeting EGFR and IGF-1R suggests the superior efficacy ofsuch a combination, compared to treatment with the respectivemonotherapies in various assay models (Fidanze et al., 2010;Hubbard et al., 2009; Tandon et al., 2011; Wang et al., 2010;Wilsbacher et al., 2008). Clinical data from combination studiesusing small molecules or antibodies or both, targeting EGF and IGF-1 receptors (Buck et al., 2008; Goetsch et al., 2005; please refer towww.clinicaltrials.gov for current status of these combinations),further provides a scientific basis to pursue a single moleculetargeting both the receptors. A single molecule with dual targetapproach may also provide a simpler clinical design and avoid thechances of any risks of drug–drug interactions.

Although there are a few reports available in literature forEGFR–IGF–1R combination using a single molecule (Fidanze et al.,2010; Hubbard et al., 2009; Tandon et al., 2011; Wang et al., 2010;Wilsbacher et al., 2008) they all lack in vivo efficacy due to eitherpoor PK profile, physicochemical properties or selectivity of thecompounds tested.

In the present manuscript we are reporting for the first time,in vivo efficacy data of a single molecule dual inhibitor of EGFR andIGF-1R. The outcome of our in vitro and in vivo studies usingRBx10080307 provides the proof of concept for a dual EGFR/IGF-1R kinase inhibitor. Detailed in vitro profiling of RBx10080307suggested that this molecule possesses acceptable in vitro enzymeand cell based potencies. It also had good oral bioavailability andwas therefore tested for in vivo efficacy. The AUC levels ofRBx10080307 at 20 and 100 mg/kg, p.o. dose were about 60–70fold lower compared to erlotinib. Despite such low levels ofcompound in circulation, the xenograft study with RBx10080307showed significant tumor growth inhibition that was comparableto erlotinib. It is possible that dual inhibitor compounds with PKproperties similar to erlotinib may show a better in vivo efficacy.Since, RBx10080307 has also been found to be effective in theEGFR mutant cell line, H1975, it would further be interesting to seeif differentiation in EGFR resistant tumors can be achieved in theH1975 xenograft studies using this compound. Our findingsprovide a base for further evaluation of this concept. Since noneof the existing therapies show efficacy in a resistant state, effec-tiveness of an EGFR/IGF-1R dual inhibitor in this situation wouldbe a major differentiation factor and could be the basis of futurestrategy for the development of next generation inhibitors forcancer therapy.

Acknowledgments

We thank Mr. Pradeep Sharma and Mr. Biju Benjamin fortechnical help provided in the DMPK studies.

References

Adams, T.E., McKem, N.M., Ward, C.W., 2004. Signaling by the type-1 insulin likegrowth factor receptor: interplay with the epidermal growth factor receptor.Growth Factors 22, 89–95.

Arteaga, C.L., 1992. Interference of the IGF system as a strategy to inhibit breastcancer growth. Breast Cancer Res. Treat. 22, 101–106.

Baselga, J., 2000. The EGF receptor family as targets for breast cancer therapy.Breast Cancer Res. 2 (Suppl. 1), S13.

Baserga, R., 1995. The insulin-like growth factor 1 receptor: a key to tumor growth?Cancer Res. 55, 249–252.

Baserga, R., Peruzzi, F., Reiss, K., 2003. IGF-1 receptor in cancer biology. Int. J. Cancer107, 873–877.

Buck, E, Eyzaguirre, A, Rosenfeld-Franklin, M, Thomson, S, Mulvihill, M., Barr, S.,Brown, E., O'Connor, M., Yao, Y., Pachter, J., Miglarese, M., Epstein, D., Iwata, K.K.,Haley, J.D., Gibson, N.W., Ji, Q.S., 2008. Feedback mechanisms promote co-operativity for small molecule inhibitors of epidermal and insulin-like growthfactor receptors. Cancer Res. 68, 8322–8332.

Chernicky, C.L, Yi, L., Tan, H., Gan, S.U., Ilan, J., 2000. Treatment of human breastcancer cells with antisense RNA to the type I insulin-like growth factor receptorinhibits cell growth, suppresses tumorigenesis, alters the metastatic potentialand prolongs survival in vivo. Cancer Gene Ther. 7, 384–395.

Dunn, S.E., Ehrlich, M., Sharp, N.J, Reiss, K., Solomon, G., Hawkins, R., Baserga, R.,Barrett, J.C., 1998. A dominant negative mutant of the insulin-like growthfactor-1 receptor inhibits the adhesion, invasion and metastasis of breastcancer. Cancer Res. 58, 3353–3361.

Fidanze, S.D., Erickson, S.A., Wang, G.T., Mantei, R., Clark, R.F., Sorensen, B.K.,Bamaung, N.Y., Kovar, P., Johnson, E.F., Swinger, K.K., Stewart, K.D., Zhang, Q.,Tucker, L.A., Pappano, W.N., Wilsbacher, J.L., Wang, J., Sheppard, G.S., Bell, R.L.,Davidsen, S.K., Hubbard, R.D., 2010. Imidazo[2,1-b]thiazoles: multi-targetedinhibitors of both the insulin-like growth factor receptor and members of theepidermal growth factor family of receptor kinases. Bioorg. Med. Chem. Lett. 20,2452–2455.

Gilmore, A.P., Valentijn, A.J., Wang, P., 2002. Activation of BAD by therapeuticinhibition of epidermal growth factor receptor and transactivation by insulin-like growth factor. J. Biol. Chem. 277, 27643–27650.

Goetsch, L., Gonzalez, A., Leger, O., Beck, A., Pauwels, P.J., Haeuw, J.F., Corvaia, N.,2005. A recombinant humanized anti-insulin-like growth factor receptor type Iantibody (h7C10) enhances the antitumor activity of vinorelbine and anti-epidermal growth factor receptor therapy against human cancer xenografts. Int.J. Cancer 113, 316–328.

Gow, C.H., Shih, J.Y., Chang, Y.L., Yu, C.J., 2005. Acquired gefitinib-resistant mutationof EGFR in a chemonaive lung adenocarcinoma harboring gefitinib-sensitivemutation L858R. PLoS Med. 2, e269.

Higgins, B., Kolinsky, K., Smith, M., Beck, G., Rashed, M., Adames, V., Linn, M.,Wheeldon, E., Gand, L., Birnboeck, H., Hoffmann, G., 2004. Antitumor activity oferlotinib (OSI-774, Tarceva) alone or in combination in human non-small celllung cancer tumor xenograft models. Anti-Cancer Drugs 15, 503–512.

Hollingshead, M.G., Alley, M.C., Camalier, R.F., Abbott, B.J., Mayo, J.G., Malspeis, L.,Grever, M.R., 1995. In vivo cultivation of tumor cells in hollow fibers. Life Sci. 57,131–141.

Hubbard, R.D., Bamaung, N.Y., Fidanze, S.D., Erickson, S.A., Palazzo, F., Wilsbacher, J.L., Zhang, Q., Tucker, L.A., Hu, X., Kovar, P., Osterling, D.J., Johnson, E.F., Bouska,J., Wang, J., Davidsen, S.K., Bell, R.L., Sheppard, G.S., 2009. Development ofmultitargeted inhibitors of both the insulin-like growth factor receptor (IGF-IR)and members of the epidermal growth factor family of receptor tyrosinekinases. Bioorg. Med. Chem. Lett. 19, 1718–1721.

LeRoith, D., Baserga, R., Helman, L., Roberts Jr., C.T., 1995. Insulin-like growth factorsand cancer. Ann. Intern. Med. 122, 54–59.

Sachdev, D., Li, S.L., Hartell, J.S., Fujita-Yamaguchi, Y., Miller, J.S., Yee, D., 2003.A chimeric humanized single-chain antibody against the type I insulin likegrowth factor (IGF) receptor renders breast cancer cells refractory to themitogenic effects of IGF-I. Cancer Res. 63, 627–635.

Tandon, R., Kapoor, S., Vali, S., Senthil, V., Nithya, D., Venkataramanan, R., Sharma,A., Talwadkar, A., Ray, A., Bhatnagar, P.K., Dastidar, S.G., 2011. Dual epidermalgrowth factor receptor (EGFR)/insulin-like growth factor-1 receptor (IGF-1R)inhibitor: a novel approach for overcoming resistance in anticancer treatment.Eur. J. Pharmacol. 667, 56–65.

Tang, W.X., Cheng, P.Y., Luo, Y.P., Wang, R.X., 1998. Interaction between cisplatin,5-fluorocil and vincristine on human hepatoma cell line (7721). World J.Gastroenterol. 4, 418–420.

van de Loosdrecht, A.A., Beelen, R.H., Ossenkoppele, G.J., Broekhoven, M.G.,Langenhuijsen, M.M., 1994. A tetrazolium-based colorimetric MTT assay toquantitate human monocyte mediated cytotoxicity against leukemic cells fromcell lines and patients with acute myeloid leukemia. J. Immunol. Methods 174,311–320.

Wang, G.T., Mantei, R.A., Hubbard, R.D., Wilsbacher, J.L., Zhang, Q., Tucker, L., Hu, X.,Kovar, P., Johnson, E.F., Osterling, D.J., Bouska, J., Wang, J., Davidsen, S.K, Bell, R.L., Sheppard, G.S., 2010. Substituted 4-amino-1H-pyrazolo[3,4-d]pyrimidines asmulti-targeted inhibitors of insulin-like growth factor-1 receptor (IGF1R) andmembers of ErbB-family receptor kinases. Bioorg. Med. Chem. Lett. 19,6067–6071.

Werner, H., Woloschak, M., Stannard, B., Shen-Orr, Z., Roberts Jr., C.T., LeRoith, D.H., 1991.The insulin-like growth factor-1 receptor: molecular biology, heterogeneity andregulation. In: LeRoith, D. (Ed.), Insulin-like Growth Factors: Molecular and CellularAspects. CRC Press, Boca Raton, FL, pp. 17–47.

Wilsbacher, J.L., Zhang, Q., Tucker, L.A., Hubbard, R.D., Sheppard, G.S., Bamaung, N.Y., Fidanze, S.D., Wang, G.T., Hu, X., Davidsen, S.K., Bell, R.L., Wang, J., 2008.Insulin-like growth factor-I receptor and erbb kinase inhibitor combinationsblock proliferation and induce apoptosis through cyclin D1 reduction and Baxactivation. J. Biol. Chem. 283, 23721–23730.

Yamasaki, F., Johansen, M.J., Zhang, D., Krishnamurthy, S., Felix, E., Bartholomeusz,C., Aguilar, R.J., Kurisu, K., Mills, G.B., Hortobagyi, G.N., Ueno, N.T., 2007.Acquired resistance to erlotinib in A-431 epidermoid cancer cells requiresdown-regulation of MMAC1/PTEN and up-regulation of phosphorylated Akt.Cancer Res. 67, 5779–5788.

Yu, H., Rohan, T., 2000. Role of insulin-like growth factor family in cancerdevelopment and progression. J. Natl. Cancer Inst. 92, 1472–1489.