Pharmacokinetics of hedgehog pathway inhibitor vismodegib (GDC-0449) in patients with locally advanced or metastatic solid tumors: the role of alpha-1-acid glycoprotein binding.

Abstract

In a phase I trial for patients with refractory solid tumors, hedgehog pathway inhibitor vismodegib (GDC-0449) showed little decline in plasma concentrations over 7 days after a single oral dose and nonlinearity with respect to dose and time after single and multiple dosing. We studied the role of GDC-0449 binding to plasma protein alpha-1-acid glycoprotein (AAG) to better understand these unusual pharmacokinetics.
Sixty-eight patients received GDC-0449 at 150 (n = 41), 270 (n = 23), or 540 (n = 4) mg/d, with pharmacokinetic (PK) sampling at multiple time points. Total and unbound (dialyzed) GDC-0449 plasma concentrations were assessed by liquid chromatography/tandem mass spectrometry, binding kinetics by surface plasmon resonance-based microsensor, and AAG levels by ELISA.
A linear relationship between total GDC-0449 and AAG plasma concentrations was observed across dose groups (R(2) = 0.73). In several patients, GDC-0449 levels varied with fluctuations in AAG levels over time. Steady-state, unbound GDC-0449 levels were less than 1% of total, independent of dose or total plasma concentration. In vitro, GDC-0449 binds AAG strongly and reversibly (K(D) = 13 μmol/L) and human serum albumin less strongly (K(D) = 120 μmol/L). Simulations from a derived mechanistic PK model suggest that GDC-0449 pharmacokinetics are mediated by AAG binding, solubility-limited absorption, and slow metabolic elimination.
GDC-0449 levels strongly correlated with AAG levels, showing parallel fluctuations of AAG and total drug over time and consistently low, unbound drug levels, different from previously reported AAG-binding drugs. This PK profile is due to high-affinity, reversible binding to AAG and binding to albumin, in addition to solubility-limited absorption and slow metabolic elimination properties.

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2011;17:2512-2520. Published OnlineFirst February 7, 2011.Clin Cancer Res


Richard A. Graham, Bert L. Lum, Sravanthi Cheeti, et al.
Tumors: the Role of Alpha-1-Acid Glycoprotein Binding
(GDC-0449) in Patients with Locally Advanced or Metastatic Solid
Pharmacokinetics of Hedgehog Pathway Inhibitor Vismodegib



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Cancer Therapy: Clinical
See commentary p. 2071
Pharmacokinetics of Hedgehog Pathway Inhibitor Vismodegib
(GDC-0449) in Patients with Locally Advanced or Metastatic Solid
Tumors: the Role of Alpha-1-Acid Glycoprotein Binding
Richard A. Graham1, Bert L. Lum1, Sravanthi Cheeti1, Jin Yan Jin1, Karin Jorga1, Daniel D. Von Hoff2,
Charles M. Rudin3, Josina C. Reddy1, Jennifer A. Low1, and Patricia M. LoRusso4
Abstract
Purpose: In a phase I trial for patients with refractory solid tumors, hedgehog pathway inhibitor
vismodegib (GDC-0449) showed little decline in plasma concentrations over 7 days after a single oral dose
and nonlinearity with respect to dose and time after single and multiple dosing. We studied the role of
GDC-0449 binding to plasma protein alpha-1-acid glycoprotein (AAG) to better understand these unusual
pharmacokinetics.
Experimental Design: Sixty-eight patients received GDC-0449 at 150 (n ¼ 41), 270 (n ¼ 23), or 540
(n ¼ 4) mg/d, with pharmacokinetic (PK) sampling at multiple time points. Total and unbound (dialyzed)
GDC-0449 plasma concentrations were assessed by liquid chromatography/tandem mass spectrometry,
binding kinetics by surface plasmon resonance–based microsensor, and AAG levels by ELISA.
Results: A linear relationship between total GDC-0449 and AAG plasma concentrations was observed
across dose groups (R2¼ 0.73). In several patients, GDC-0449 levels varied with fluctuations in AAG levels
over time. Steady-state, unbound GDC-0449 levels were less than 1% of total, independent of dose or
total plasma concentration. In vitro, GDC-0449 binds AAG strongly and reversibly (KD¼ 13 mmol/L) and
humanserumalbuminlessstrongly(KD¼120mmol/L).SimulationsfromaderivedmechanisticPKmodel
suggest that GDC-0449 pharmacokinetics are mediated by AAG binding, solubility-limited absorption,
and slow metabolic elimination.
Conclusions: GDC-0449 levels strongly correlated with AAG levels, showing parallel fluctuations of
AAG and total drug over time and consistently low, unbound drug levels, different from previously
reported AAG-binding drugs. This PK profile is due to high-affinity, reversible binding to AAG and binding
to albumin, in addition to solubility-limited absorption and slow metabolic elimination properties. Clin
Cancer Res; 17(8); 2512–20. ?2011 AACR.
Introduction
Aberrant hedgehog pathway activation has been impli-
cated in a number of cancers (1–9). Vismodegib, GDC-
0449; 2-chloro-N-[4-chloro-3-(pyridin-2-yl)phenyl]-4-
[methylsulfonyl]benzamide, is a potent hedgehog
signaling pathway inhibitor in development for treatment
of various cancers (10–13). GDC-0449 binds to and inhi-
bits smoothened (SMO), a 7-transmembrane hedgehog
pathway signaling protein. Activity of GDC-0449 was first
shown in vivo in preclinical models of medulloblastoma
(11), colon, and pancreatic tumors (9). In a phase I study
for patients with advanced malignancies, GDC-0449 was
well-tolerated, with pharmacodynamic (PD) evidence of
hedgehog pathway inhibition and tumor regressions in
patients with basal cell carcinoma and medulloblastoma
(10, 11, 13).
Preclinical pharmacokinetic (PK) properties of GDC-
0449 were favorable, with low in vivo clearance and good
oral bioavailability across animal species (14). In vitro
studies in human hepatocytes suggested that GDC-0449
was very metabolically stable; nearly 100% of the com-
pound remained intact following coincubations (14). At
physiologic pH, GDC-0449 exhibits limited solubility in
vitro (0.99 mg/mL, at pH 0.1, compared with 0.0001 mg/
mL, at pH 6.5–7.4).
In a phase I study, an atypical PK profile was observed,
with little decline in GDC-0449 plasma concentrations
during a 7-day observation period following a single oral
dose (10, 13). After continuous daily dosing, steady-state
plasma concentrations were achieved earlier than expected
(within 7–14 days); plasma concentrations did not
increase with increasing dose levels, suggesting nonlinear
pharmacokinetics with regard to dose and time.
Authors' Affiliations:1Genentech Inc., South San Francisco, California;
2Translational Genomics Research Institute (TGen) and TGen Clinical
Research Institute, Scottsdale, Arizona;3Johns Hopkins University, Balti-
more, Maryland; and the4Karmanos Cancer Institute, Detroit, Michigan
Corresponding Author: Richard A. Graham, Clinical Pharmacology,
Genentech, 1 DNA Way, South San Francisco, CA 94080. Phone: 650-
225-5791; Fax: 650-742-5167. E-mail: graham.richard@gene.com
doi: 10.1158/1078-0432.CCR-10-2736
?2011 American Association for Cancer Research.
Clinical
Cancer
Research
Clin Cancer Res; 17(8) April 15, 2011
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Like many drugs, GDC-0449 binds to human serum
albumin (HSA) but GDC-0449 also binds to alpha-1-acid
glycoprotein (AAG) with high affinity. AAG is an acute-
phase reactant protein and carrier of basic and neutrally
charged lipophilic drugs (15–18). Binding to AAG results
in clinically pertinent alterations in pharmacokinetics and/
or pharmacodynamics for many classes of pharmacologic
agents, including anticancer drugs (18) such as docetaxel
(19), erlotinib (20), gefitinib (21), imatinib (22), and
UCN-01 (23, 24). Previous in vitro experiments had shown
that GDC-0449 is highly bound (>95%) to human plasma
proteins at clinically relevant concentrations (14). In vitro
equilibrium dialysis experiments with GDC-0449 concen-
trationsof5,25,and75mmol/LandAAGconcentrationsof
0.5, 1, and 5 mg/mL showed that binding of GDC-0449 to
AAG was saturable within a clinically relevant concentra-
tion range for GDC-0449 and physiologically relevant
range for AAG. Specifically, binding was saturated by
GDC-0449 at the low and medium concentrations of
AAG when drug concentration was greater than 5 mmol/L.
Using surface plasmon resonance (SPR) methodology, we
found that the binding dissociation constant for AAG (KD
AAG ¼ 13 mmol/L), was lower than KDHSA (120 mmol/L),
suggesting stronger binding to AAG than to HSA (18).
Given this in vitro protein-binding data, we conducted
a preliminary analysis of AAG and HSA concentrations
in 40 patients on a phase I study who received GDC-0449
at 150, 270, or 540 mg/d. A single plasma sample from
each patient was analyzed for AAG, HSA, and GDC-0449
21 days after initiation of daily dosing; a full AAG, HSA,
and AAG PK profile was determined for 3 of these patients.
Exploratoryanalyses indicated
between clinical GDC-0449 plasma and AAG (but not
a strongcorrelation
HSA) concentrations, as well as parallel fluctuations in
plasma GDC-0449 and AAG concentrations over time
(18).
On the basis of these preliminary protein-binding
results, and the important role of AAG binding on the
PK profile of a number of other drugs, the role of AAG
binding on the clinical PK profile of GDC-0449 was inves-
tigated; results are presented herein. In addition, a mechan-
istic PK model was derived to further assess the role of AAG
binding.
Methods
Study design
The phase I trial was an open-label multicenter trial
evaluating escalating doses of GDC-0449 administered
orally once daily. Descriptions of study design, patient
eligibility, and assessments are provided in the accompa-
nying article (13). Human investigations were conducted
after approval by a local Human Investigations Committee
inaccordancewithassurancesapprovedbytheDepartment
of Health and Human Services. All patients provided writ-
ten informed consent according to federal and institutional
guidelines before study procedures began.
Trialenrollment occurredin2 stages.Stage1 consistedof
dose escalations to estimate a maximum tolerated dose.
Stage 2 consisted of 3 cohorts: (i) an expanded cohort, at
the proposed phase II dose of 150 mg, foradditional safety,
and PK and PD data, (ii) an additional cohort of patients
with locally advanced or metastatic basal cell carcinoma at
150 and 270 mg dose levels, and (iii) a cohort to evaluate
pharmacokinetics of a new 150 mg phase II drug formula-
tion (smaller particles with faster in vitro dissolution than
the phase I formulation). Comparisons of GDC-0449
pharmacokinetics between each of these cohorts are
beyond the scope of this article but are included in the
accompanying article (13).
Treatment and biosampling
Serial blood samples were obtained as specified in the
following text to determine plasma concentrations of AAG
and of total and unbound GDC-0449:
Stage 1: Three increasing dose levels of GDC-0449 were
used: 150, 270, and 540 mg. On day 1, each patient
received GDC-0449 in capsule form at the assigned dose,
followed by a 7-day (washout) observation period.
Planned blood sampling included 5 samples within 24
hours after the firstdose, and additional sampleson days 2,
3,4,and8.Continuous daily dosingwasinitiated onday 8;
samples were obtained predose on days 15, 22, 29, 36, and
every 28 days thereafter during treatment.
Stage 2: Patients received 150 or 270 mg GDC-0449 daily.
Samples were collected predose on days 8, 15, 22, 29, 36,
and every 28 days thereafter during treatment. For patients
receiving the phase II formulation, blood samples were
additionally collected at 2, 8, and 24 hours following first
dose.
Translational Relevance
Vismodegib(GDC-0449),asmall-moleculehedgehog
pathway inhibitor, has shown encouraging antitumor
activity in advanced basal cell carcinoma and medullo-
blastoma. In the first phase 1 study, GDC-0449 showed
an unusual pharmacokinetic (PK) profile with an unex-
plained elimination half-life of more than 7 days and
accumulation that unexpectedly plateaued within the
first 14 days. This article is the first to describe some
of the phenomena contributing to the GDC-0449 PK
profile: high-affinity binding to alpha-1-acid glycopro-
tein (AAG) with tight correlation to plasma AAG levels
over time and consistently low, unbound drug levels.
This is unique compared with previously reported AAG-
binding drugs, possibly due to unprecedented satura-
tion of plasma AAG. These phenomena, in combination
with binding to serum albumin, solubility-limited gas-
trointestinal absorption, and slow metabolic elimina-
tion, are captured in a biologically based GDC-0449 PK
model, presented here for the first time. The model will
be used to simulate alternate dose regimens and will be
validated through ongoing clinical trials of GDC-0449.
GDC-0449 Pharmacokinetics and AAG
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HSA concentrations were determined at the same time
points, except for days 2, 3, and 4 for stage 1 patients and 2,
8, and 24 hours after the first dose for stage 2 patients
receiving the phase II formulation.
GDC-0449 was administered on an empty stomach;
patients consumed only water for 1 hour pre- and postdose
and took their dose more than 1 hour prior to the first meal
of the day, around the same time each day.
Bioanalysis of GDC-0449 in plasma
GDC-0449 plasma concentrations were determined
by Tandem Labs, using a validated solid-phase extrac-
tion liquid chromatography/tandem mass spectrometry
(LC/MS-MS) method (25). Human plasma (K2EDTA)
samples containing GDC-0449 were analyzed in 200 mL
aliquots. GDC-0449 concentrations were calculated
using a 1/x2quadratic regression over a concentration
range of 5.00 to 5,000 ng/mL (0.012–11.9 mmol/L) with
GDC-0449-d5 as an internal standard. The API 3000 was
operated in the selected reaction monitoring (SRM)
mode under optimized conditions for detection of
GDC-0449 and GDC-0449-d5 positive ions formed by
electrospray ionization.
Equilibrium dialysis
Equilibrium dialysis was conducted by QPS, using a
validated method, with a Single-Use Plate Rapid Equili-
brium Dialysis device equipped with dialysis membranes
of molecular weight cutoff of approximately 8,000
(Thermo Scientific). The dialysis plate was placed on an
orbitalshakeratapproximately500rpmandincubatedina
5% CO2humid incubator (Napco 5400) at 37?C. The
dialysis of plasma samples (0.3 mL) was conducted against
0.133 mol/L isotonic potassium phosphate buffer (0.5mL)
at pH 7.4 for 6 hours. Protein-binding samples in mixed
matrix(plasma/buffer¼1:1)wereshippedtoTandemLabs
for analysis, as described later.
Bioanalysis of GDC-0449 in plasma/buffer
Tandem Labs conducted analyses of human plasma
(K2EDTA): PBS (50:50 v/v) samples containing GDC-
0449 in 100 mL aliquots by a validated solid-phase extrac-
tion procedure followed by LC/MS-MS. GDC-0449 con-
centrations were calculated using a 1/x2linear regression
over a concentration range of 0.100 to 100 ng/mL
(0.00024–0.24 mmol/L), using GDC-0449-d5 as an inter-
nal standard. The API 5000 was operated in the SRM mode
under optimized conditions for detection of GDC-0449
and GDC-0449-d5 positive ions formed by electrospray
ionization.
AAG and HSA analytic methods
Concentrations of AAG in human K2EDTA plasma were
determined using a commercially available kit (Dade Behr-
ing Marburg, Germany) modified for assessment by 96-
well ELISA. HSA concentrations were determined as part of
each patient’s standard chemistry panel.
In vitro protein-binding interaction methods
The binding affinities and kinetic profiles of GDC-0449
with AAG and albumin were assessed using SPR-based
optical biosensors (Biacore; GE Healthcare) by methods
similar to those described by Rich and colleagues (26) and
Frostell-Karlsson and colleagues (27).
PK and statistical analyses
Individual patient GDC-0449 PK parameter values for
total and unbound plasma concentration–time data were
derived using noncompartmental methods (WinNonlin
version 5.2.1; Pharsight Corp.).
Summary statistics were tabulated for calculated and
observed PK parameters. A linear mixed-effects model
was used to examine relationships between total plasma
drug concentration and protein (AAG and albumin) con-
centration.
A mechanism-based conceptual PK simulation approach
was used to explore multiple hypotheses for the observed
total and unbound GDC-0449 concentrations, as well as
the relationship to levels of AAG. Multiple mathematical
model structures were tested incorporating one or both of
the principal factors, AAG binding and solubility-limited
absorption, in various mathematical permutations. Para-
meter values were selected on the basis of in vitro or in
vivo measurements and to best represent phase I PK ob-
servations. Simulations were conducted using Berkeley
Madonna software (version 8.3.11). Results were evaluated
on the basis of visual inspection of observed versus pre-
dicted PK profiles.
Results
Pharmacokinetics of GDC-0449 after single or
multiple doses
PK data for total and unbound GDC-0449 in plasma
following single-dose administration are summarized in
Table 1. Concentration–time profiles at 150, 270, or
540 mg showed that maximum total or unbound plasma
concentrations (Cmax) were achieved by day 2, with little
decline in concentrations over the ensuing 6-day washout
period (Table 1, Fig. 1A and B). Cmaxincreased with dose
escalation from 150 to 270 mg. At 540 mg, the mean total
and unbound plasma Cmaxwas similar to that observed at
270 mg as shown in Table 1.
For patients in stage 1 who received a single dose of
GDC-0449 on day 1 and continuous daily dosing begin-
ning on day 8, the observed time to reach steady-state
plasma concentrations ranged from 7 to 14 days (corre-
sponding to study days 14–21). Steady-state GDC-0449
plasma concentrations were calculated as an average of
plasma concentrations from study day 21 (stage 2) or
study day 28 (stage 1) onward. Similar levels (total and
unbound) were observed across all dosing cohorts, includ-
ing the 150 mg phase II capsule formulation (Fig. 1E
and F).
On average, steady-state, unbound GDC-0449 concen-
trations in plasma were less than 1% of total GDC-0449
Graham et al.
Clin Cancer Res; 17(8) April 15, 2011 Clinical Cancer Research
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Page 5

concentrations, and similar levels were detected regardless
ofdoseortotalplasmaconcentration(rangingfrom5.46to
56.0 mmol/L; Fig. 2).
Relationship between AAG and GDC-0449
pharmacokinetics
A linear mixed model with subjects as a random effect
wasusedtostudytherelationshipbetweentotalGDC-0449
plasma concentration and AAG concentration. All GDC-
0449 and AAG plasma samples collected after day 21 for
each patient were included; data were pooled across
cohorts and dose groups for the analysis. A strong relation-
ship was observed (R2¼ 0.73). The population estimate
describing the relationship between GDC-0449 and AAG
(the fixed-effect part of the model) was as follows:
GDC-0449totalconcentration
¼ ð0:482 ? AAGplasmaconcentrationÞþ4:658
where the value þ4.658 was the population intercept
and 0.482 was the population slope of the regression line
(Fig. 3). There was no significant correlation between HSA
levels and GDC-0449 total plasma concentration (shown
in Fig. 4 for representative patients).
In individual patients, GDC-0449 concentrations varied
with AAG concentrations over the study period. Concen-
tration–time plots for representative patients are shown in
Figure 4. Patient A received 150 mg daily for up to 200 days
and showed fairly consistent plasma GDC-0449 and AAG
concentrations after initial dosing. Over a similar time
period, patients B and C received 270 mg GDC-0449 daily;
plasma concentrations of GDC-0449 fluctuated over time
in parallel with AAG concentrations, whereas HSA levels
remained stable. On a molar basis, AAG concentrations
remained higher than total GDC-0449 concentrations in
each of these patients, consistent with overall results
(Fig. 3).
Development of a mechanistic PK model
A mechanism-based conceptual PK model was devel-
oped to provide a quantitative description of the observed
GDC-0449 concentration data. Figure 5A illustrates the
factors considered in model development that are most
likely relevant for GDC-0449 PK and pharmacologic inter-
actions at the target tumor site: GDC-0449 absorption,
distribution, elimination, and binding to AAG. In this
model, plasma drug exists in 3 possible forms: unbound
(Du), drug–AAG complex (D-AAG), and drug–HSA com-
plex (D-HSA; Fig. 5A). Kinetics of protein binding are
described using a rapid equilibrium binding equation with
the dissociation constants designated as KDAAG and KD
HSA. The final model assumed that drug was absorbed
from the gastrointestinal (GI) tract into vasculature at a
constant zero-order rate(k0) due tolimited solubility inthe
GI tract. In addition, drug can be absorbed only during a
limited period of GI transit time (T0), which limits its
bioavailability, especially athigher doses andafter multiple
doses. Drug elimination from plasma is incorporated into
the model as a first-order elimination of the unbound drug
with rate constant (kel).
Simulations suggested that solubility-limited absorption
could primarily explain the observed nonlinearity with
respect to dose and time whereas a protein-binding com-
ponent is necessary to explain the tight correlation between
total GDC-0449 and AAG, as well as the low, unbound
fraction. Incorporation of solubility-limited absorption,
slow elimination, and protein-binding components into
a single model provides an explanation for the observed PK
characteristics of GDC-0449.
Figure 5B–D show concentration profiles from PK simu-
lations for single-dose GDC-0449 with a 7-day washout,
followed by continuous daily dosing. Total GDC-0449
concentrations (Fig. 5B), unbound GDC-0449 concentra-
tions (Fig. 5C), and the correlation of the AAG concentra-
tion with the total GDC-0449 concentration (Fig. 5D)
are shown. The model prediction represented most of
the key phase 1 observations robustly, including dose-
proportional exposure increase from 150 to 270 mg after
single dose but no further increase at 540 mg, similar
steady-state total GDC-0449 concentrations across all
doses, and consistently low, unbound fraction. The ob-
served linear relationship between total GDC-0449 plasma
concentrations and AAG was also predicted by the simula-
tion (Fig. 5D).
Discussion
Ina phase I clinical trial, GDC-0449 was shown to have a
unique PK profile characterized by (i) little decline in
Table 1. Single-dose unbound and total GDC-0449 PK parameters from patients enrolled in stage 1
Stage 1 patients
by dose cohort
Mean ? SD
Unbound GDC-0449Total GDC-0449
Tmax, dCmax, mmol/LAUClast, mmol/L hTmax, dCmax, mmol/LAUClast, mmol/L h
150 mg (n ¼ 7)
270 mg (n ¼ 9)
540 mg (n ¼ 4)
2.43 ? 2.22
1.61 ? 1.16
0.834 ? 0.871
0.0093 ? 0.0121
0.0324 ? 0.0247
0.0292 ? 0.0289
0.577 ? 0.769
2.41 ? 1.37
2.43 ? 1.45
2.43 ? 2.22
2.11 ? 0.928
2.04 ? 1.34
3.58 ? 1.34
6.34 ? 3.40
6.81 ? 2.69
322 ? 185
839 ? 458
1,010 ? 446
GDC-0449 Pharmacokinetics and AAG
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Page 6

plasma concentrations over a 7-day washout period fol-
lowing a single dose and (ii) nonlinearity with regard to
dose and time after continuous daily dosing (10, 13). To
explain these observations, we considered in vitro and
preliminary clinical protein-binding data for GDC-0449
and the established importance of in vivo AAG binding to a
number of drugs.
The most remarkable characteristic of GDC-0449 phar-
macokinetics that we observed in this study was the strong
correlation between GDC-0449 and AAG plasma concen-
trations. We further investigated the role of AAG binding
on the pharmacokinetics of GDC-0449, including deriva-
tion of a semimechanistic PK model and measurement of
AAG and unbound GDC-0449 levels in all phase I patient
samples. The results from this report suggest that the PK
profile of GDC-0449 is largely dictated by 3 factors: solu-
bility-limited absorption, associated with dose and time
nonlinearity;aslowrateofsystemicelimination,associated
with the long half-life; and binding to AAG (the focus of
this report), which resulted in the surprisingly tight corre-
lation between in vivo levels of GDC-0449 and AAG. The
binding of AAG strongly impacted the pharmacokinetics of
GDC-0449, as evidenced by intrapatient parallel fluctua-
tion in AAG and GDC-0449 levels.
Binding of AAG impacts pharmacokinetics and/or phar-
macodynamics of a number of drugs across a broad spec-
trum ofclasses. Intheoncology setting (28,29), AAGbinds
to chemotherapeutic agents such as docetaxel (19) and
150270 540Ph II-150
Dose (mg)
0.5
0.4
0.3
0.2
0.1
0
Unbound GDC-0449 Css (µmol/L)
Average steady-state concentrations
of unbound GDC-0449
Total GDC-0449 Css (µmol/L)
150 270540 Ph II-150
Dose (mg)
60
50
40
30
20
10
0
Average steady-state concentrations
of total GDC-0449
0.3
0.2
0.1
0.0
Unbound GDC-0449 plasma
concentration (µmol/L)
0 7 14 21 28
Time (d)
35 42 49 56 63
150 mg (n = 40)
270 mg (n = 23)
540 mg (n = 4)
Multiple dose unbound (all subjects)Multiple dose unbound (all subjects)
40
30
20
10
0
Total GDC-0449 plasma
concentration (µmol/L)
0 7 14 21 28
Time (d)
35 42 49 56 63
150 mg (n = 40)
270 mg (n = 23)
540 mg (n = 4)
0.06
0.04
0.02
0.00
Unbound GDC-0449 plasma
concentration (µmol/L)
0 1 2 3 4 5 6 7
Time (d)
150 mg (n = 7)
270 mg (n = 9)
540 mg (n = 4)
Single dose unbound (stage 1 subjects)
15
10
5
0
Total GDC-0449 plasma
concentration (µmol/L)
0 1 2 3 4 5 6 7
Time (d)
150 mg (n = 7)
270 mg (n = 9)
540 mg (n = 4)
Single dose total (stage 1 subjects)
AB
CD
EF
Figure 1. Pharmacokinetics of
GDC-0449 after single- and
multiple-dose administration.
Plasma concentrations of total (A)
and unbound (B) GDC-0449 over
time are shown after a single dose
and after multiple daily doses
(C, total; D, unbound). (For C and
D, PK samples from a patient who
discontinued from the study early
were not collected after the
initiation of multiple dosing.)
Average steady-state
concentrations of total (E) and
unbound (F) GDC-0449 in all
subjects in the 150, 270, 540, and
150 mg phase II formulation
cohorts are also presented. In E
and F, The line in the middle of the
box represents the median, the
top and bottom box limits
represent the 25th and 75th
percentiles, and the top and
bottom bars represent 1.5 times
the interquartile range.
Graham et al.
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Page 7

several small-molecule tyrosine and protein kinase inhibi-
tors, including erlotinib (20), gefitinib (21), imatinib (22),
and UCN-01 (23, 24). Plasma concentrations of AAG are
normally around 0.28 to 0.92 g/L but increase during
inflammatory or stress reactions (28, 29). Levels of AAG
in patients with malignant tumors are elevated up to 5-fold
(30). In addition, a mixture of 2 or 3 genetic variants of
AAG exists in most individuals. Although many drugs have
similar binding constants for AAG, other drugs (such as
imatinib) have shown differences in binding to genetic
variants (30, 31). These properties of AAG largely explain
the alterations or interpatient differences in pharmacoki-
netics and/or pharmacodynamics for a variety of drugs
(19–24).
The reported half-lives of most AAG-binding drugs (e.g.,
more than 20 tyrosine kinase inhibitors in a recent report;
ref. 32) are much shorter than the reported half-life of AAG
(?5 days; ref. 33), suggesting that factors other than AAG
binding also determine their PK characteristics. UCN-01 is
an investigational anticancer drug with a binding affinity
for AAG that is stronger by approximately 4 orders of
magnitude (reported KAof 8.0 ? 108L/mol, which corre-
sponds to a KDof 1.25 nmol/L; ref. 24) than GDC-0449
and aslowerdissociation rate(18). This isassociated witha
very prolonged elimination phase after intravenous (IV)
administration; UCN-01 total plasma concentrations
approximate plasma concentrations of AAG (23). The
pharmacokinetics of UCN-01 seem to essentially reflect
the strong binding to AAG, with drug taking on the kinetics
of the protein. In comparison, the elimination half-life of
GDC-0449 after IV administration is also long (34); how-
ever, for GDC-0449, this is not strictly a result of AAG
binding, as indicated by the complete reversibility of GDC-
0449 binding to AAG and a much lower binding affinity
compared with UCN-01.
In addition to binding kinetics, we considered whether
nonlinear binding to AAG was an important contributing
factor to the PK profile of GDC-0449. For example, ima-
tinib–AAG binding results in a nonlinear relationship
between total and unbound imatinib plasma concentra-
tions (35) and levels of unbound imatinib correlate with
PD responses to treatment. Similarly to GDC-0449, ima-
tinib binds strongly to AAG (KAof 1.7 ? 106L/mol, which
corresponds to a KDof 0.6 mmol/L) and less strongly to
HSA (KAof 3.0 ? 104L/mol, which corresponds to a KDof
33mmol/L; ref.32).However, forimatinib,high AAGlevels
result in low available plasma concentrations of unbound
drug and lower clearance, associated with hematologic
toxicity and imatinib resistance in leukemia (36, 37).
Unlike imatinib, the concentration of unbound GDC-
0449 in plasma remained low and a relatively constant
proportion of total steady-state plasma concentration
across the observed range (?5.46–56.0 mmol/L); our data
and PK modeling strongly suggest that this can be
explained only by the binding of GDC-0449 to both
AAG and HSA. Once AAG binding becomes saturated by
GDC-0449, the unbound GDC-0449 fraction remains low
due to binding to HSA, which serves as a high-capacity
drug-binding protein relativeto AAGdue to itshigh levelin
plasma. Therefore, saturation of overall protein binding is
not achieved by GDC-0449 at clinically relevant drug
concentrations. Notably, the relative difference in binding
affinity of imitinib for AAG and HSA is 2 orders of magni-
tude but only 1 order of magnitude for GDC-0449, which
could explain why there is a nonlinear relationship
between total and unbound imatinib plasma concentra-
tions but not GDC-0449 plasma concentrations.
Ahypothesistoexplaintheuniqueinterpatientandintra-
patient (over time) correlations between AAG and GDC-
0449 levels is that due to the poor solubility of GDC-0449,
the intestine acts as a continuous source of drug to the
systemic circulation with a zero-order input rate whereas
AAG in the systemic circulation acts as a sink for GDC-
0449. In the event of an increase or decrease in plasma
AAG, the absorption rate may transiently increase or
4
3
2
1
0
Percentage unbound
0 10 20 30 40 50 60
Total GDC-0449 plasma concentration (µmol/L)
150 mg (n = 7)
270 mg (n = 9)
540 mg (n = 4)
Figure 2. The percentage of unbound GDC-0449 is low and consistent
across the range of GDC-0449 concentrations achieved within the study.
R2 = 0.73
60
50
40
30
20
10
0
Total GDC-0449 concentration (µmol/L)
0 20 40
AAG (µmol/L)
60 80 100
150 mg
270 mg
540 mg
Figure 3. Total GDC-0449 and AAG plasma concentrations are
highly correlated. The scatter plot includes all data from each
patient after day 21 of daily dosing and is reflective of steady-state
conditions.
GDC-0449 Pharmacokinetics and AAG
www.aacrjournals.orgClin Cancer Res; 17(8) April 15, 2011
2517
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Page 8

GDC-0449
AAG
HSA
50
40
30
20
10
0
800
600
400
200
0
GDC-0449 or AAG alasma
concentration (µmol/L)
HSA concentration (µmol/L)HSA concentration (µmol/L) HSA concentration (µmol/L)
Patient C
0 100 200
Time (d)
300 400
50
40
30
20
10
0
800
600
400
200
0
GDC-0449 or AAG plasma
concentration (µmol/L)
Patient B
0 100 200
Time (d)
300 400
GDC-0449
AAG
HSA
50
40
30
20
10
0
800
600
400
200
0
GDC-0449 or AAG plasma
concentration (µmol/L)
Patient A
Time (d)
0 50 100 150 200 250
GDC-0449
AAG
HSA
ABC
Figure 4. Concentration-time profiles in three representative patients (A), (B), and (C). Left y-axis reflects plasma concentration of total GDC-0449 and AAG,
whereas right y-axis reflects serum concentration of HSA.
80
60
40
20
0
Total GDC-0449 (µmol/L)
AAG (µmol/L)
0 10 20 30 40 50 60 70 80
150 mg
270 mg
540 mg
Simulation of correlation between
GDC-0449 and AAG
150 mg
270 mg
540 mg
0.06
0.04
0.02
0.00
Unbound GDC-0449 (µmol/L)
0 7 14
Time (d)
21 28 35
Simulation of unbound GDC-0449
150 mg
270 mg
540 mg
35
28
21
14
7
0
Total GDC-0449 (µmol/L)
Time (d)
0 7 14 21 28 35
Simulation of total GDC-0449
k0
kel
(kidney/liver)
KDAAG
KDHSA
Du
HSA
Du
Du
AAG
D-HSA
D-AAG
GLI
Hh
PTCH1
SMO
Tumor site
BCD
A
Figure 5. Mechanistic PK model and associated simulations. A, schematic depicting the physiologic processes that likely dictate GDC-0449
pharmacokinetics. The model assumes that the unbound drug and the drug–HSA binding complex are both available for binding to AAG in order to reproduce
the strong correlation between GDC-0449 and AAG plasma concentrations. Unbound drug elimination from plasma is incorporated into the model as a first-
orderrateconstant(kel)viametabolismand/or parent excretion. Unbound drugcanbind toandinhibit SMOactivity,leading toblockade of hedgehog signaling
at the target tumor site. The final model assumed that drug was absorbed from the GI tract into vasculature at a constant zero-order rate (k0) due to limited
solubility in the GI tract. In addition, drug can be absorbed only during a limited period of GI transit time (T0), which limits its bioavailability, especially at higher
doses and after multiple doses. Model simulations for total GDC-0449 (B), unbound GDC-0449 (C), and the correlation between GDC-0449 and AAG
concentrations (D) are shown.
Graham et al.
Clin Cancer Res; 17(8) April 15, 2011 Clinical Cancer Research
2518
Published OnlineFirst February 7, 2011; DOI:10.1158/1078-0432.CCR-10-2736

Page 9

decrease, respectively. Additional experiments and mathe-
maticalmodeling are needed to further test this hypothesis.
To our knowledge, this is the first report in which plasma
AAG levels explain most (>70%) of the observed PK varia-
bility. The ability of AAG to influence the PK profile of a
particular drug depends on a balance of several factors,
including level of affinity, binding kinetics, binding capa-
city, relative affinity to HSA compared with AAG, and other
factors such as intrinsic hepatic clearance and oral absorp-
tion. Taken together, our study results, PK model simula-
tions, and comparisons with other AAG-binding drugs
suggest that the pharmacokinetics of GDC-0449 are dic-
tated by its solubility-limited absorption following oral
administration, slow rate of metabolic elimination, and
interaction with AAG. Although the current PK model has
been predictive of the key PK properties of the molecule,
additional enhancement and validation of this model
would permit the incorporation of elements that fully
describe other PK properties, such as AAG and unbound
GDC-0449 fluctuations, as they relate to total GDC-0449
over time. Ongoing studies of GDC-0449, including the
assessment of absolute bioavailability (by IV and oral
administration), alternative dose schedules, binding to
different AAG genetic variants, and mechanistic and popu-
lation PK modeling and simulations, will further define
factors that contribute to the unique PK characteristics of
GDC-0449.
Disclosure of Potential Conflicts of Interest
D. D. Von Hoff received a grant to his institution for the phase I trial of
GDC-0449; P.M. LoRusso has research funding from Genentech, is on
Genentech’s speakers bureau (compensated) and has participated in Gen-
entech Advisory Boards (compensated); CM Rudin has been a paid con-
sultant to Genentech (not on projects related to Hedgehog inhibitors). R.A.
Graham, B.L. Lum, S. Cheeti, J.Y. Jin, K. Jorga, J.C. Reddy, and J.A. Low are
employees of Genentech, A Member of the Roche Group, and shareholders
of Roche.
Acknowledgments
The authors thank Vikram Malhi for clinical pharmacokinetic data
analysis, Dr. Mark Dresser for his thoughtful review and input, Drs. Harvey
Wong, Anthony Giannetti, and Alan Deng for bioanalytic contributions,
Dr. Ilsung Chang for biostatistics input, and Lisa Beckel and Hilary Nelson
for their work in clinical operations. We also thank Dr. Leslie Benet for his
insightful input and discussion. Dr. Abie Craiu at Genentech, Inc., provided
assistance with preparation of the manuscript.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
ReceivedOctober12,2010;revisedDecember29,2010;acceptedJanuary
10, 2011; published OnlineFirst February 7, 2011.
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