Pharmacokinetic Pharmacodynamic Analysis of Vismodegib in ...clincancerres.aacrjournals.org/content/clincanres/17/14/4682.full.pdf · pathway inhibitor (HPI) vismodegib (GDC-0449)

Cancer Therapy: Preclinical

Pharmacokinetic–Pharmacodynamic Analysis of Vismodegib in PreclinicalModels of Mutational and Ligand-Dependent Hedgehog Pathway Activation

Harvey Wong2, Bruno Alicke1, Kristina A. West1, Patricia Pacheco1, Hank La2, Tom Januario3, Robert L. Yauch3,Frederic J. de Sauvage4, and Stephen E. Gould1

AbstractPurpose: Vismodegib (GDC-0449) is a potent and selective inhibitor of the Hedgehog (Hh) pathway

that shows antitumor activity in preclinical models driven bymutational or ligand-dependent activation of

the Hh pathway.We wished to characterize the pharmacokinetic–pharmacodynamic (PK/PD) relationship

of vismodegib in both model systems to guide optimal dose and schedule for vismodegib in the clinic.

Experimental Design: Preclinical efficacy and PK/PD studies were carried out with vismodegib in a

Ptchþ/� allograft model of medulloblastoma exhibiting mutational activation of the Hh pathway and

patient-derived colorectal cancer (CRC) xenograft models exhibiting ligand-dependent pathway activation.

Inhibition of the hedgehog pathway was related to vismodegib levels in plasma and to antitumor efficacy

using an integrated population-based PK/PD model.

Results: Oral dosing of vismodegib caused tumor regressions in the Ptchþ/� allograft model of

medulloblastoma at doses �25 mg/kg and tumor growth inhibition at doses up to 92 mg/kg dosed

twice daily in two ligand-dependent CRC models, D5123, and 1040830. Analysis of Hh pathway activity

and PK/PD modeling reveals that vismodegib inhibits Gli1 with a similar IC50 in both the medullo-

blastoma and D5123 models (0.165 mmol/L �11.5% and 0.267 mmol/L �4.83%, respectively). Pathway

modulation was linked to efficacy using an integrated PK/PD model revealing a steep relationship

where > 50% of the activity of vismodegib is associated with >80% repression of the Hh pathway.

Conclusions: These results suggest that even small reductions in vismodegib exposure can lead to

large changes in antitumor activity and will help guide proper dose selection for vismodegib in the clinic.

Clin Cancer Res; 17(14); 4682–92. ’2011 AACR.

Introduction

The hedgehog (Hh) pathway has been implicated in anumber of cancers including basal cell carcinoma (BCC)(1, 2), medulloblastoma (3, 4), colorectal, pancreatic (5),prostate (6), and lymphoma (7). Hh pathway activationcan occur through mutational activation in the case of BCCand medulloblastoma where aberrant proliferation is dri-ven by ligand-independent cell autonomous Hh signaling.These cancers driven by mutational activation of the Hhpathway have been referred to as type-1 cancers whereas thelarger set of cancers, driven by paracrine Hh signalingbetween tumor cells and the surrounding stroma, havebeen referred to as ligand-dependent or type-3 cancers (8).

In the latter cases Hh ligands (Sonic, Indian, and Desert)bind to a twelve-pass transmembrane receptor calledpatched (PTCH) which, in the absence of ligand, inhibitsa seven-pass transmembrane protein named smoothened(SMO). In the presence of Hh ligand the repressive actionof PTCH on SMO is relieved and SMO initiates signaltransduction through theGLI family of transcription factorsleading to induction of downstream targets which includeGLI1 and PTCH forming both positive and negative feed-back loops, respectively. Additional transcriptional targetsinclude the cell-cycle regulator cyclin D2 (9, 10), the anti-apoptotic protein BCL-2 (11, 12), and the proangiogenicfactors VEGF and angiopoietin (13). In the case of BCCapproximately 90% of sporadic cases have been associatedwith inactivatingmutations of the negative regulator PTCH,which leads to constitutive activation of the pathway (14)and up to 10% contain activating mutations in the positiveregulator SMO(15, 16). Sporadic cases ofmedulloblastomahave also been associated with inactivating mutations inPTCH albeit at a lower frequency of approximately 30% ofcases (17). Taken together these findings have spurredinterest in the development of inhibitors of this pathwayas potential antitumor therapeutics the most advanced ofwhich is vismodegib (GDC-0449), currently in late-phaseclinical development (8, 18).

Authors' Affiliations: Departments of Translational Oncology1, DrugMetabolism and Pharmacokinetics2, Molecular Diagnostics3, and Mole-cular Biology4 Genentech Inc, 1 DNAWay, South San Francisco, California

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

Corresponding Author: Stephen E. Gould, Genentech, Inc., 1 DNA Way,South San Francisco, CA 94080. Phone: 650-676-0319; Fax: 650-225-1411; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-0975

’2011 American Association for Cancer Research.

ClinicalCancer

Research

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Vismodegib is a potent, selective Hedgehog pathwayinhibitor (HPI) that targets SMO with an EC50 of 2.8nmol/L on a human palatal mesenchymal cell line(HEPM) stably expressing a GLI-responsive luciferasereporter gene (19–21). Vismodegib shows favorablepharmacokinetics in preclinical species characterized bymoderate clearance in monkeys and low to very lowclearance in mouse, rat, and dog (20). Based on the datapresented to date, the pharmacokinetics of vismodegib isconsistent with it being a very low clearance compound inhumans (22). Vismodegib has shown dramatic antitumoractivity in clinical trials treating autonomous-driven, ortype-1 cancers, including metastatic or locally advancedbasal cell carcinoma (23) and metastatic medulloblas-toma (24).HPI’s have been shown to be active in preclinical tumor

models dependent on mutational activation (25, 26) andparacrine ligand-dependent Hh-signaling (5). Here weexplore the pharmacokinetic–pharmacodynamic (PK/PD) relationship of vismodegib in models of both muta-tional (Ptchþ/� medulloblastoma allograft) and ligand-dependent (patient-derived colorectal xenograft) Hh path-way activation and provide an integrated PK/PD-efficacymodel that describes the PD-efficacy relationship. Surpris-ingly, despite targeting the Hh pathway in different tumorcompartments and the Hh pathway being driven by eithermutation or ligand expression, we report very similarrelationships among the two tumor types. In both caseswe find that >50% of the antitumor activity is associatedwith pathway inhibition of >80%. In addition, we findthat there is a steep relationship between antitumor

response and pathway modulation suggesting a switch-likebehavior. Collectively, these findings have importantimplications for the development of HPI’s in the clinicand suggest that inhibitors must be used at or abovetheir IC95 to ensure maximal activity and that dosingeven marginally below the IC95 can lead to dramaticreduction in antitumor efficacy leading to subtherapeuticactivity.

Materials and Methods

Subcutaneous xenograft and allograft models in micePatient-derived colorectal cancer xenograft tumor mod-

els (D5123 and 1040830) were established from directimplantation of surgical material obtained from the Coop-erative Human Tissue Network funded by the NationalCancer Institute into female CD-1 nu/nu mice 6 to 8 weeksof age (Charles River Laboratories) and propagated in vivoas described (5). Mice were housed andmaintained accord-ing to the animal use guidelines of Genentech, Inc, con-forming to California State legal and ethical practices.Medulloblastoma allografts were established from directtransplantation of spontaneous central nervous system(CNS) tumors that formed in Ptchþ/� mice (27) via directtransplantation into female CD-1 nu/nu mice. Allografttumors were serially transplanted via dissection of thetumor, mechanical disaggregation, and subcutaneousinoculation of 5 to 10million cells. Calu6 xenograft tumorswere established through inoculation of 4� 106 viable cells(ATCC) subcutaneously in the hind flank of female CD-1nu/numice 6 to 8weeks of age (Charles River Laboratories)using a 1 cc syringe fitted with a 22-gauge needle as a 50:50suspension in growth factor depleted Matrigel (No.354230, BD Biosciences). Tumor-bearing mice were dis-tributed into tumor volume-matched cohorts when thetumors reached between 200 and 350 mm3. The vismo-degib-resistant medulloblastoma allograft, sg274, wasdeveloped by intermittent suboptimal dosing of aPtchþ/�, p53�/� medulloblastoma allograft as describedpreviously (21). Vismodegib was formulated as a suspen-sion in 0.5% methyl-cellulose, 0.2% tween-80 (MCT), andwas administered orally. Tumor volumes were determinedusing digital calipers (Fred V. Fowler Company, Inc.) usingthe formula (L � W � W)/2. Tumor growth inhibition(%TGI) was calculated as the percentage of the area underthe fitted curve (AUC) for the respective dose group per dayin relation to the vehicle, such that %TGI ¼ 100 � 1–(AUCtreatment/day)/(AUCvehicle/day). Curve fitting wasapplied to Log2 transformed individual tumor volume datausing a linear mixed-effects model using the R packagenlme, version 3.1–97 in R v2.12.0.

Pharmacokinetic studiesFemale CD-1 nude mice (weighing 25–28 g) (Charles

River Laboratories) were administered oral doses of 5, 15,50, and 100 mg/kg (free base equivalent) of vismodegibhydrochloride salt in 0.5% methylcellulose/0.2%Tween 80 (MCT). Blood samples (�1 mL) were collected

Translational Relevance

The current work defines the pharmacokinetic–phar-macodynamic (PK/PD) relationship of the Hedgehogpathway inhibitor (HPI) vismodegib (GDC-0449) inpreclinical tumor models driven by either mutationalactivation or ligand-dependent activation of the Hedge-hog pathway. Cancers that contain mutational activa-tion of the Hh pathway, such as basal cell carcinomaand medulloblastoma, display a cell-autonomousdependency on Hh-signaling whereas cancers such ascolorectal and pancreatic adenocarcinoma depend onHh ligand-dependent paracrine signaling betweentumor and the surrounding stroma. Surprisingly,despite these differences, we find similar PK/PD rela-tionships amongst these two types of models. Using anintegrated population-based PK/PD model we definehere the interaction between Hh pathway suppressionand antitumor response. We find a steep PD response–effect relationship that operates like an on–off switchwhere even a small reduction in pathway suppressioncan lead to dramatic reductions in efficacy. These obser-vations are pertinent to all HPI being tested in the clinicand will help guide proper dose and schedule to achievemaximal clinical benefit.

PK/PD Analysis of Vismodegib

www.aacrjournals.org Clin Cancer Res; 17(14) July 15, 2011 4683




up to 24 hours postdose via cardiac puncture (terminalcollection) into tubes containing potassium ethylenedia-minetetraacetic acid (K2EDTA) anticoagulant. Immediatelyon collection, the blood was mixed with K2EDTA andstored on ice. Within 30 minutes, blood samples werecentrifuged at approximately 1000 to 1500 � g for 5minutes at 4�C, and plasma was harvested. The plasmasamples were stored at �80�C until analysis. Concentra-tions of vismodegib were determined by LC/MS/MS asdescribed previously (20).

Pharmacodynamic studies in miceTumor-bearing mice were treated with vismodegib in

0.5% methylcellulose/0.2% Tween 80 (MCT) either as asingle dose (medulloblastoma model), or as a series of 5consecutive doses given on a twice-daily schedule [D5123CRC, Calu6 non–small cell lung carcinoma (NSCLC)models]. Tumors were harvested at various times followingthe last dose and total RNA extracted for qPCR using theQiagen RNeasy Mini Kit and RNA samples treated withAmbion rDNAse according to the manufacturers protocol(Ambion). qPCR was carried out on an Applied Biosystems7500 theromocycler (Apllied Biosystems), using 100 ngRNA/reaction. Relative gene expression was calculatedusing the following equation: 2-deltaCt*1000. Data arepresented either as relative expression or as fold changefrom untreated tumors using RPL19 as a loading control, asindicated. Primers and probes sequences were; Gli1, F 50-GCA GTG GGT AAC ATG AGT GTC T-30, R 50-AGG CACTAG AGT TGA GGA ATT GT-30, P 50-CTC TCC AGG CAGAGA CCC CAG C-30, RPL19 F 50-AGA AGG TGA CCT GGATGA GA-30, R 50-TGA TAC ATA TGG CGG TCA ATC T-30,P 50-CTT CTC AGG AGA TAC CGG GAA TCC AAG-30.Blood samples were collected and processed at varioustimepoints as described above.

PK/PD modelingMean vismodegib plasma concentration-time profiles

determined from the pharmacokinetic studies were usedto estimate oral-pharmacokinetic parameters, AUC (areaunder the concentration-time profile from time zero to thelast measurable timepoint), Cmax (the maximum observedconcentrations), tmax (the time at which Cmax wasobserved), and t1/2 (half-life), by noncompartmental ana-lysis (28). For purposes of modeling and simulation,plasma concentration-time data were fitted to a one-com-partment model with oral absorption. Because the phar-macokinetics of vismodegib appeared nonlinear over thedose range tested, elimination was described by theMichaelis–Menten equation. Mean estimates of the absorp-tion rate constant (ka), the maximum elimination rate(Vmax), the substrate concentration resulting in 50% ofVmax (Km; Michaelis–Menten constant) and the apparentvolume of distribution (V/F) were obtained by simulta-neously fitting plasma concentration-time data from alldose groups using SAAM II (SAAM Institute, University ofWashington, Seattle, WA). These estimates were used to

simulate vismodegib concentrations in the modeling oftumor efficacy studies.

Gli1 mRNA inhibition studies in medulloblastomaallografts and D5123 xenografts

The PK/PD relationship of vismodegib plasma concen-trations to Gli1 mRNA inhibition was characterized usingan indirect response model (29) where vismodegib con-centrations inhibit the formation of Gli1 mRNA asdescribed by the following equation:

dðGli1Þdt

¼ kin 1� C

IC50 þ C

� �� koutðGli1Þ ðAÞ

Gli1 (PD units) is defined as the % normalized Gli1mRNA, t (hr) is the time, kin (PD units/hr) is the formationrate of Gli1 mRNA, C (mmol/L) is the concentration ofvismodegib, IC50 (mmol/L) is the vismodegib concentra-tion where there is 50% inhibition of Gli1 mRNA, kout(hr�1) is the rate constant describing the loss of Gli1mRNA. At steady state, kin ¼ kout (Gli1); therefore koutwas replaced by kin/(Gli1)initial where (Gli1) initial is thecontrol (predose) value of Gli1 mRNA. Plasma and tumorGli1 mRNA were fit simultaneously using SAAM II. Phar-macodynamic parameters are presented as the estimatefollowed by the %SE (percent standard error of theestimate ¼ (standard error of the estimate/parameter esti-mate) � 100) in parentheses. Free IC50 and IC95 for bothmedulloblastoma allografts and CRC xenografts were cal-culated using amean vismodegib unbound fraction of 1.34� 0.17% determined ex vivo by equilibrium dialysis fromplasma collected from these pharmacodynamic studies.

Integrated PK/PD-efficacy modelTo understand the relationship between vismodegib

plasma concentrations, Gli1 mRNA inhibition and tumorgrowth inhibition, an integrated PK/PD-efficacy model wasconstructed by adding an antitumor efficacy component tothe PK/PD model used to characterize the relationshipbetween vismodegib concentrations and Gli1 mRNA inhi-bition described above (Supplementary Fig. S1). Briefly,the PK/PDmodel describing Gli1mRNA inhibition (Eq. A)was used to simulate Gli1 mRNA inhibition for all xeno-graft/allograft dose groups. The following equationsdescribe the model used in the fitting process to relateGli1 mRNA inhibition to tumor volume:

dðTVÞdt

¼ kngðTVÞ �KðTVÞ ðBÞ

where

K ¼ Kmax � ð%IÞnKð%IÞn50 þ ð%IÞn ðCÞ

TV (mm3) is defined as the tumor volume, t (day) is time,kng (day

�1) is the net growth rate constant, K (day�1) is the

Wong et al.

Clin Cancer Res; 17(14) July 15, 2011 Clinical Cancer Research4684




rate constant describing the antitumor tumor effect ofvismodegib, Kmax (day�1) is the maximum value of K, nis the Hill coefficient, and K (%I)50 is defined as the %Iwhere K is 50% of Kmax. %I is defined as the percentinhibition of Gli1 mRNA and was calculated as follows:

%I ¼ ðGli1Þinitial � ðGli1ÞðGli1Þinitial

� �� 100% ðDÞ

Concentrations of vismodegib in mice were simulatedbased on the pharmacokinetic parameters obtained fromthe pharmacokinetic studies. The S-ADAPT program, anaugmented version of ADAPT II with population analysiscapabilities (30, 31) was used to fit individual tumorvolumes from all dose levels simultaneously. Intersubjectvariability was assumed to be log-normally distributedand fitted using an exponential-variance model. Residualvariability error was modeled using a proportional errormodel. In cases where intersubject variance was small andcould not be estimated reliably, the intersubject variancewas fixed to 0.00001. Population parameters estimatesare presented as the estimate followed by the %SE inparentheses.The described integrated PK/PD-efficacy model was fit

simultaneously to tumor volumes from three medulloblas-toma allograft studies. For study 1, medulloblastoma allo-graft mice were given vehicle and daily doses of 0.3 to 75mg/kg vismodegib; for study 2, allograft mice were givenvehicle and daily doses of 10 or 75mg/kg vismodegib (datanot shown); and for study 3, allograft mice were givenvehicle and daily doses of 1 to 175mg/kg vismodegib (datanot shown). Of these medulloblastoma allograft studies,study 1 best captures the dose response relationship basedon the doses selected (Fig. 1A and B) and serves as therepresentative study. This integrated PK/PD-efficacy modelwas also used to fit tumor volumes from the D5123 studywhere xenograft mice were given twice daily doses of 46 to92 mg/kg vismodegib (Fig. 1C).

Results

Vismodegib is highly efficacious in medulloblastomaallograft tumorsWe first characterized the dose response of vismodegib in

the subcutaneous murine Ptchþ/� medulloblastoma-allo-graft model. Tumor growth in this model is driven bymutational inactivation of the tumor suppressor Ptch,and therefore directly tests the dependency of tumorgrowth on autocrine Hh pathway activation (27). Micewere implanted subcutaneously with serially passaged allo-graft tumor fragments originally derived from a sponta-neous medulloblastoma tumor that formed in a Ptchþ/�

mouse. Daily oral dosing with 0.3 to 75 mg/kg of vismo-degib for 14 days leads to dose responsive antitumoractivity with tumor regressions occurring at or above 25mg/kg (Fig. 1A, B). Tumor regressions occur rapidly withthe bulk of the activity observed within a week of initiatingtreatment. Antitumor activity reached a plateau above 25

mg/kg with little increase in activity seen with higher dosessuggesting the effect was saturated (Fig. 1B). The pharma-cokinetics study of vismodegib showed a dose-dependentincrease in exposure (AUC) at doses above 15 mg/kg, andtherefore the saturation of efficacy was not due to dose-limiting exposure (Fig. 1E; Table 1).

Vismodegib causes growth delay in patient-derivedcolorectal xenografts

Unlike inmedulloblastomawhere Hh pathway activity isdriven by mutational activation of the Hh pathway, path-way activity is driven by paracrine ligand signaling incolorectal, pancreatic, and ovarian cancers (5). In thisparacrine model of Hh-dependent tumorigenesis, ligandproduced by epithelial-derived tumors signals to the sur-rounding stromal cells which respond by providing directgrowth signals and or by promoting a favorable environ-ment for tumor growth. Early passage patient-derived col-orectal xenografts were implanted subcutaneously into theflanks of nude mice and treated twice daily with 23 to 92mg/kg vismodegib for 18 to 21 days. Two independentmodels (D5123 and 1040830) revealed that vismodegib atdoses > 46 mg/kg twice daily caused tumor growth delay(52 and 69% TGI, respectively, Fig. 1C, D).

PK/PD relationship of vismodegib inmedulloblastoma allograft tumors

We next wished to explore the PK/PD relationship in themedulloblastoma allograft model using Gli1 mRNA levelsas a direct readout of Hh pathway activity. We dosed micebearing subcutaneous Ptchþ/� allograft tumors with 0, 1,10, and 50 mg/kg vismodegib and assayed pathway mod-ulation in tumors via qPCR from 2 to 32 hours following asingle dose to capture the dynamics of vismodegib activity.A fully efficacious dose of 50 mg/kg of vismodegib (119%TGI) suppressed Gli1 for greater than 12 hours with Gli1levels increasing from 16 to 24 hours (Fig. 2A). This isin contrast to the partially efficacious dose of 10 mg/kg(85% TGI) which failed to inhibit Gli1 to the same extentand which was associated with increasing levels ofGli1 by 8hours (Fig. 2A). Little, if any, Gli1 inhibition was observedin response to 1 mg/kg of vismodegib, consistent with thelack of antitumor activity observed at this dose (Fig. 1A;14% TGI). Plasma was also collected and analyzed forvismodegib levels (Fig. 2D). Maximal repression of thepathway was associated with 3.2–13 mmol/L vismodegibduring the first 12 hours whereas levels below 0.1 mmol/Lfailed to inhibit pathway activity relative to the vehicletreatment (Fig. 2A and D).

PK/PD relationship of vismodegib in ligand-drivenxenograft tumors

As with the mutational driven tumors we wished toexplore the PK/PD relationship in ligand-dependenttumors using Gli1 mRNA levels as the readout of Hhpathway activity. However, unlike in the medulloblastomamodel where Hh pathway activation occurs within tumorcells, Gli1 activity was monitored in the tumor stroma to






detect paracrine Hh signaling between the xenograftedtumor and infiltrating stroma using PCR primers andprobes that specifically detect murine Gli1 (5). Two sepa-rate models were chosen to characterize the PK/PD rela-tionship, a model that responds to vismodegib treatmentby exhibiting growth delay (D5123) and second model(Calu6) that is insensitive to vismodegib treatment.Tumor-bearing mice were dosed orally with 11.5 to 92mg/kg vismodegib bid for a total of 5 doses to achievesteady state levels in plasma and to mimic the dosingparadigm used in efficacy studies. In both cases, doses of46 mg/kg or higher of vismodegib were required to causereduction in stromal Gli1 expression over vehicle treatedanimals 12 hours following dosing (Fig. 2B, C). Nochanges in human GLI1 were detected within the tumorsin either model showing that vismodegib was inhibitingparacrine Hh-signaling in these tumors [data not shown,

(5)]. Significant inhibition of Gli1 at 12 hours was asso-ciated with 7.2 to 10.1 mmol/L of vismodegib in plasma(Fig. 2E, F).

Single-dose pharmacokinetic assessment ofvismodegib in mice

To fully characterize the PK parameters of vismodegibfollowing oral administration in mice we administered asingle dose of vismodegib at 5, 15, 50, and 100 mg/kg inMCT and analyzed vismodegib levels in plasma for morethan 24 hours. Estimated oral pharmacokinetic parametersare presented in Table 1. Exposure (AUC) increased withincreasing dose over the dose range tested. However, thisincrease in AUC was greater than dose proportional con-sistent with nonlinear elimination. Half-life also increasedwith increasing dose, consistent with nonlinear elimina-tion. For modeling and simulation purposes, the following

Figure 1. In vivo efficacy ofvismodegib. Vismodegib causestumor regression in Ptchþ/�

allograft medulloblastoma tumors.A, groups of 5 mice bearingsubcutaneous Ptchþ/� allografttumors were treated with vehicleor 0.3–75 mg/kg vismodegib oncedaily. The population mean foreach dose group is plotted using aspline fit. B, dose response plot ofthe tumor growth inhibition with95%confidence intervals showingsigmoidal dose response andsaturated effect. Doses at orabove 25 mg/kg cause tumorregressions in this model and thiseffect is saturated at doses at andabove 50 mg/kg. Dashed linerepresents tumor stasis. C and D,twice daily dosing of vismodegibcauses growth delay in patient-derived xenograft modelsexhibiting a paracrine requirementfor hedgehog pathway activity.Two patient-derived xenografts(C, D5123, D, 1040830) weretreated with 0–92 mg/kgvismodegib (free-baseequivalents) and tumorsmeasured twice weekly for18–21 days. The population meanfor each dose group is plottedusing a spline fit. Maximal tumorgrowth inhibition of 52% and 69%was observed in the D5123 and1040830 models, respectively. Noincrease in efficacy was notedabove 69 mg/kg vismodegib in theD5123 model, higher doses werenot tested in the 1040830 model.E, pharmacokinetic profiles ofvismodegib following single oraldoses to CD-1 nude mice.

Wong et al.





PK parameters were estimated by fitting an oral one-com-partment model with nonlinear elimination to vismodegibconcentration-time profiles from all doses simultaneously:ka ¼ 4.04 hr�1, Vmax ¼ 1.94 mmol/hr/kg hr�1, Km ¼ 4.22mmol/L, and V/F ¼ 4.43 L/kg (Fig. 1E and Table 1). Theseestimated parameters were used to simulate vismodegibplasma concentrations when fitting tumor-volume datafrom efficacy studies.

Indirect response PK/PD model characterizing thevismodegib-Gli1 relationshipTo more fully characterize the PK/PD relationship of

vismodegib in plasma to Gli1 mRNA levels in both themedulloblastoma allograft and D5123 CRC xenograft

models we fitted an indirect response model to the vismo-degib plasma concentration and Gli1 mRNA data (29). Inthis model, the concentration of vismodegib in plasma (C)influences the rate of Gli1 mRNA production (kin) accord-ing to Eq. A such that steady stateGli1 levels are determinedby a balance of production and degradation (kout). We usedan indirect response model as vismodegib binds to andinhibits SMO, which then allows accumulation of a cleavedform of GLI3, a repressor of Hh target gene transcription,including Gli1 (32). Using this approach we find thatvismodegib inhibits Gli1 with a similar IC50 in both themedulloblastoma allograft model as well as the CRC xeno-graft model D5123 (0.165 and 0.267 mmol/L, respectively;Table 2, Fig. 3) despite the different cellular compartmentstargeted (tumor vs. stroma for medulloblastoma and

Table 1. Oral pharmacokinetics of vismodegib in CD-1 nude mice

Dose AUC Cmax tmax t1/2(mmol/L � hr) (mmol/L) (hr) (hr)

5 mg/kg 10.7 2.47 1 1.7015 mg/kg 33.3 8.08 0.5 2.8850 mg/kg 155 37.1 1 3.90100 mg/kg 413 34.7 1 25.3

Figure 2. Hedgehog pathway modulation in Ptchþ/� medulloblastoma allograft tumors following a single dose of vismodegib (A). Animals (n ¼ 3) weregiven a single dose of 1, 10, and 50 mg/kg vismodegib and tumors harvested for analysis from 2–32 hours following drug administration. Hedgehogpathway activity was assayed through quantitative PCR of murine Gli1 mRNA. Data are plotted relative to vehicle treated tumors following normalization toGAPDH. D, levels of vismodegib were assayed in the plasma at each time point and dose level. Maximal repression of the pathway was associatedwith 3.2–13 mmol/L vismodegib during the first 12 hours whereas levels below 0.1 mmol/L failed to significantly alter pathway activity. Hedgehog pathwaymodulation in stroma from a patient-derived colorectal tumor (B, D5123) or a NSCLC xenograft (C, Calu6) following five doses of vismodegib dosed twice daily.Animals (n ¼ 3) were given a single dose of 11.5–92 mg/kg vismodegib and tumors harvested for analysis at either 1, 6, 12, 18, or 24 hours followingthe final dose of vismodegib. Hedgehog pathway activity was assayed through quantitative PCR of murine Gli1 using species-specific primers and probes.Data are plotted relative to vehicle treated tumors following normalization to murine Rpl19. E and F, levels of vismodegib were assayed in the plasma at eachtime point and dose level in the D5123 (E) and Calu6 models (F).






D5123, respectively) and the different mechanisms ofpathway activation (loss of PTCH function vs Hh-ligandexpression).

Integrated population pharmacokinetic/pharmacodynamic–efficacy model

We next wished to understand the relationship betweenvismodegib plasma concentrations, Gli1mRNA inhibition,and tumor-growth inhibition to determine the degree ofpathway suppression associated with tumor-growth inhi-bition in both tumor models. The PK/PD model used tocharacterize the relationship between vismodegib concen-trations and Gli1 mRNA inhibition described above wascombined with an indirect response populationmodel andthis integrated PK/PD model was fit to the individualtumor volume data from the efficacy studies (see Materialand Methods, Supplementary Fig. S1). The integratedmodel adequately characterizes the tumor volume datafrom the efficacy studies as evident in plots of predictedvs. observed tumor volume presented in Figs. 4A and B.Estimated pharmacodynamic parameters are presented inTable 3. The estimated pharmacodynamic parametersalong with the inter- and intra-individual variability wereestimated with good precision with % standard error of allestimates being �30%. The integrated PK/PD-efficacymodel predicts that in both mutation- and ligand-driventumors 50% of the maximum antitumor effect is achievedat, or above, approximately 82% of pathway inhibition(Table 3, Fig. 4C). In addition, the relationship betweenGli1 inhibition and tumor growth is characterized by asteepHill slope (n) observed for both themedulloblastomaand D5123 models (8.7 and 8.9, respectively; Table 3,Fig. 4C). Such steep Hill slopes are indicative of on–offswitches whereby narrow changes in pathway modulationmay have disproportionately large impacts on physiologicoutcome and have been described for several pathways thatcontrol binary cell fate decisions including Hh (33, 34).

The net growth rate constant of the medulloblastoma-allograft model was approximately 2-fold higher than forthe D5123 xenograft model (kng ¼ 0.154 and 0.0810day�1, respectively). The maximum value of the rate con-stant describing the antitumor effect of vismodegib (Kmax)differed between the two models with Kmax being approxi-mately 11-fold greater in the medulloblastoma-allograftmodel compared with the D5123 CRC model (0.589 vs.0.0524 day�1, respectively; Table 3, Fig. 4D). These differ-ences in Kmax are apparent from the maximal tumor growthinhibition noted in the efficacy studies with a maximal

52% tumor growth inhibition (TGI) observed in theD5123CRC xenograft model and tumor regression (125% TGI)being observed in the medulloblastoma-allograft model(Fig. 1A, C).

Pharmacokinetic/Pharmacodynamic relationship ofvismodegib in a resistant model of medulloblastoma

To further explore the prediction that small decreases inGli1 inhibition can lead to disproportionately large effectson efficacy we characterized the PK/PD relationship ofvismodegib in a model (sg274) of medulloblastoma thatcontains a SMOmutant (D477G) that is largely resistant tovismodegib (21). Treatment of established allografts of thesg274model with 100mg/kg bid of vismodegib led to 52%tumor growth inhibition whereas a considerably lowerdose of vismodegib (25 mg/kg) was required to regressSMOWT sensitive tumors (Fig. 1A). In the same study asecond HPI, Hh-Antag, at 100 mg/kg bid led to tumorregression in the sg274 model. Tumor-growth inhibitionwas associated with 76% inhibition of the Hh pathway inresponse to vismodegib and 99% inhibition in response toHh-Antag (Fig. 4E, F). These observations are consistentwith our predictions based on the integrated PK/PDmodelthat small decreases in the inhibition of Gli1 can result inlarge losses of antitumor activity.

Discussion

The development of rationally targeted agents for thetreatment of cancer requires a full understanding of thepharmacokinetic–pharmacodynamic relationships of theinvestigational agent (35, 36). This information can pro-vide useful pharmacokinetic exposure targets as well aspharmacodynamic endpoints that can be used as gates forfurther clinical development. Vismodegib is a potent andselective HPI currently in clinical development. We, andothers have previously shown that two different modes ofHh-signaling can drive tumorigenesis: (i) ligand-indepen-dent mutational activation of the Hh pathway in the case ofBCC and medulloblastoma, and (ii) paracrine ligand-dependent signaling observed in colorectal, pancreatic,and ovarian cancer (5, 6, 26). We have used preclinicalmodels of both modes of Hh pathway activation to char-acterize the relationship of vismodegib in plasma to path-way inhibition, and pathway inhibition to efficacy with theaim of identifying any differences between Hh ligand-independent and Hh ligand-dependent cancers and toestablish exposure targets for vismodegib in clinical trials.

Table 2. Pharmacodynamic parameter estimates from Gli1 mRNA inhibition studies

Parameter Medulloblastoma (%SE) D5123 Model (%SE)

kin (PD units/hr) 47.2 (4.0) 12.2 (5.73)IC50 (mmol/L) 0.165 (11.5) 0.267 (4.83)Free IC50 (nmol/L) 2.21 3.58Free IC95 (nmol/L) 42.0 68.1

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We have found that vismodegib is a potent inhibitor ofmutation-driven tumorigenesis leading to tumor regressionin an allograft model of medulloblastoma driven by loss offunction of PTCHwith an approximate ED50 of 6mg/kg. Incontrast, vismodegib at doses up to 92 mg/kg twice dailycaused moderate tumor growth delay in two patient-derived CRC xenograft models that exhibit paracrine Hh-signaling. These more moderate effects on tumor-growthinhibition seen in the CRCmodels reported here are typicalof the level of antitumor activity seen in additional para-crine models such as patient-derived and cell-line xeno-grafts treated with HPIs including signal-blockingantibodies (5). These differences in responsiveness mayreflect the nature of pathway activation between these totypes of tumor. Medulloblastoma allografts are driven byconstitutive activation of the Hh pathway owing to theirloss of the tumor suppressor PTCH and are likely depen-

dent on this activity for growth and survival. On thecontrary, the patient-derived xenografts, which exhibitparacrine Hh-signaling between the tumor cells and sur-rounding stroma do not have a direct requirement forpathway activity. In this case the Hh pathway is not theoncogenic driver but Hh-signaling to the stroma leadsindirectly to increased tumor growth presumably due toinduction of signals that directly or indirectly promotetumor growth.

Despite the differences noted in the antitumor effectsbetween the two types of models we found a very similarrelationship between the IC50 for vismodegib in plasmaand pathway suppression as measured by Gli1 mRNA intumor or stroma in the case of medulloblastoma allografts(0.165 mmol/L) and CRC patient-derived xenografts (0.267mmol/L), respectively. These observations are consistentwith vismodegib acting at the level of SMO which lies

Figure 3. Modeling of the PK/PDrelationship of vismodegib plasmaconcentrations to Gli1 mRNAinhibition in the D5123 CRCxenograft (A) andmedulloblastoma allografts (B).Traces represent the predictedvalues from the PKPD model andopen symbols represent observedvalues �SE. Data in A weremodeled assuming 5 doses on abid schedule followed by PKPDsampling whereas a single dosewas modeled in B to match theexperimental conditions.






Figure 4. Integrated populationPK/PD-efficacy model of both theD5123 xenograft andmedulloblastoma allograftmodels. A and B, plots ofpredicted tumor volumes derivedfrom the integrated populationPK/PD-efficacy model versus theobserved values for the D5123 (A)and medulloblastoma models (B)showing that model nicelycharacterizes tumor volume datafor both models. C, relativemaximal inhibition-responsecurves for D5123 (dashed) andmedulloblastoma (solid) modelsshowing a steep relationshipcharacterized by Hill slopes of 8.9and 8.7, respectively. D, the samedata in (C) are plotted as absolutevalues showing the approximately11-fold higher antitumor rateconstant (K) seen in themedulloblastoma model versusthe D5123 model. E, Treatment ofestablished sg274 allografts witheither 100 mg/kg bid ofvismodegib (open triangles) or 100mg/kg Hh-Antag bid (filled circles)led to 52% tumor growth inhibitionor tumor regression, respectively.F, qRT-PCR from tumors treatedin (E) 6 hours following a final doseof 100 mg/kg of either vismodegibor Hh-Antag revealing 76%inhibition of the Hh pathway inresponse to vismodegib and99% inhibition in response toHh-Antag. Error bars representþ/� SEM.

Table 3. Pharmacodynamic parameter estimates from the integrated population PK/PD-efficacy model

Parameter Medulloblastoma D5123 colorectal

Populationmean (%SE)

Interindividualvariance (%SE)

Populationmean (%SE)

Interindividualvariance (%SE)

kng (day�1) 0.154 (4.4) 0.095 (21.4) 0.0810 (3.0) 0.0374 (22.3)Kmax (day

�1) 0.589 (4.3) 0.046 (30.3) 0.0524 (5.1) 0.0867 (23.8)K(Gli1)50 (% Gli1 mRNA inhibition) 81.5 (0.3) fixed 95.6 (2.2) 0.0171 (23.8)n 8.68 (0.4) fixed 8.89 (9.4) 0.250 (25.6)Initial tumor volume (mm3) 290 (3.1) 0.078 (18.0) 249 (4.6) 0.110 (20.7)Residual variability (s) 0.254 (3.6) 0.158 (9.2)

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downstream of the signaling defect present in the medul-loblastoma model (loss of PTCH), inhibition of whichshould lead to similar suppression of the downstreamtranscriptional target Gli1. This also suggests that the dif-ference in potency of vismodegib observed in the twosystems is not due to differences in pathway dynamicsbut rather varying dependencies on the Hh pathway itself.Equal pathway inhibition leads to regression in oneinstance and tumor-growth delay in the other.Using an integrated PK/PD-efficacy model we were able

to characterize the relationship between Gli1 inhibitionand efficacy in both mutation-driven and paracrine-drivenmodels. Of great interest was the observed steep relation-ship between Gli1 inhibition and the rate constant describ-ing the measure of antitumor effect (K). The Hill slopeobserved for both the D5123 and medulloblastoma modelwere �9 suggesting that the pathway is acting as an on–offswitch in both systems. During embryonic developmentthe Hh pathway plays a fundamental role in specifying avariety of cell-fate choices that require precise temporal andspatial control of pathway activity. Hh pathway activity isregulated by both positive (GLI1) and negative (PTCH)feedback loops that help to restrict activity to cells in anarrow range of Hh ligand expression. Pathways character-ized by the presence of both positive and negative feedbackloops have been reported to exhibit switch-like behaviorconsistent with what we have observed (33). Based on theshapes and K(%I)50 of the response effect curves, it is clearthat a high degree of inhibition of the pathway is needed toachieve maximal antitumor response, regardless of themechanism of pathway activation. A similar requirementfor near maximal repression of pathway signaling has beenrecently reported for the B-RAF inhibitor PLX4032 inmelanoma patients where >80% of pERK inhibition wasassociated with tumor regression (37). In addition, anearlier report described a steep Hill slope for the responseeffect curve of a second B-RAF inhibitor, GDC-0879, inxenograft models (38) consistent with empirical modelingof the MEK-ERK pathway that suggested a switch-likebehavior (39). The steep slope of the response effect curveseen with vismodegib also predicts that even small

decreases in drug levels, and hence pathway suppression,can have disproportionately large effects on efficacybecause the switch operates over a narrow range of Gli1inhibition. This hypothesis is supported by the large dif-ference in efficacy observed with Hh-Antag (completetumor regression, 125% tumor growth inhibition) andvismodegib (52% tumor growth inhibition) in the sg274resistance model despite the more modest differencesnoted in pathway suppression between the two com-pounds (99% and 76%, respectively). This requirementfor near maximal pathway suppression has importantimplications for the clinical development of HPI’s as wellas other agents that target pathways characterized by asimilar steep response effect curve. To achieve maximalclinical benefit one must achieve near complete pathwayinhibition and therefore the target needs to be nonessentialfor normal tissues to enable a favorable therapeutic index.Based on the data in this study and to account for potentialspecies differences in protein binding, we prospectively seta target plasma concentration for visomodegib in ourclinical trials to meet, or exceed, the free IC95 for GLI1inhibition (42 to 68 nmol/L, Table 2) to ensure maximalclinical benefit. Recent reports suggest that at the recom-mended dose and frequency of visomedegib (150 mg oncedaily) patients exceed these targets for unbound drug(109 nmol/L �0.058 SEM) (40).

Disclosure of Potential Conflicts of Interest

All of the authors are employees of Genentech, Inc.

Acknowledgments

The authors wish to thank Cornelis Hop, Lori Friedman, and JenniferLow for their insightful comments and careful review of the manuscript.Vismodegib was discovered by Genentech and was jointly validated througha series of preclinical studies carried out under a collaborative agreementbetween Genentech, Inc. and Curis, Inc.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 14, 2011; accepted May 11, 2011; published OnlineFirstMay 24, 2011.

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2011;17:4682-4692. Published OnlineFirst May 24, 2011.Clin Cancer Res Harvey Wong, Bruno Alicke, Kristina A. West, et al. Hedgehog Pathway Activationin Preclinical Models of Mutational and Ligand-Dependent

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