Post on 26-Apr-2023
ORIGINAL ARTICLE
The impact of bevacizumab on temozolomide concentrationsin intracranial U87 gliomas
Rachel Grossman • Michelle A. Rudek •
Harry Brastianos • Patti Zadnik • Henry Brem •
Betty Tyler • Jaishri O. Blakeley
Received: 7 February 2012 / Accepted: 2 April 2012 / Published online: 27 May 2012
� Springer-Verlag 2012
Abstract
Purpose An important question in the sequencing of anti-
cancer therapies in patients with glioblastoma (GBM) is
whether concurrent anti-angiogenesis therapies improve or
impair brain concentrations of concomitantly administered
cytotoxic therapies. The purpose of this study is to assess
the intratumoral disposition of temozolomide (TMZ) via
microdialysis before and after bevacizumab in an intra-
cranial GBM xenograft model.
Methods Microdialysis probes were placed within tumor
and contralateral brain in athymic rats bearing U87 intra-
cerebral gliomas. TMZ (50 mg/kg oral) was administered
10 days thereafter. Extracellular fluid (ECF) was collected
for 6 h. BEV was administered (10 mg/kg IV), and TMZ
was re-dosed (50 mg/kg oral) 36 h thereafter with addi-
tional ECF collection. All ECF samples were assessed for
TMZ concentration with liquid chromatography–tandem
mass spectrometry.
Results Tumor TMZ mean area under the concentration–
time curve (AUC0–?) was 3.35 lg h/mL pre-BEV. Post-
BEV, tumor mean TMZ AUC0–? was 3.98 lg h/mL. In
non-tumor brain, mean TMZ AUC0–? pre-BEV was
3.22 lg h/mL and post-BEV was 3.34 lg h/mL.
Conclusions There were no statistically significant
changes in TMZ pharmacokinetics before or after BEV in
the athymic rat U87 intracranial glioma model. BEV and
TMZ are being investigated as a combination therapy in
several ongoing studies for patients with glioma. These
data reassuringly suggest that BEV does not significantly
change the ECF tumor concentrations of TMZ in either
tumor-bearing or normal brain when dosed 36 h prior to
TMZ.
Keywords Microdialysis � Brain tumor � Angiogenesis �Temozolomide � Bevacizumab � U87 glioma
Introduction
Glioblastoma (GBM) was considered a chemoresistant
tumor until the alkylating therapy temozolomide (TMZ)
was added to radiation therapy resulting in an improved
median overall survival (OS) from 10 % with radiotherapy
alone to 26 % with chemoradiation at 2 years in patients
Rachel Grossman and Michelle A. Rudek have contributed equally to
the work performed and the generation of the manuscript.
R. Grossman
Department of Neurosurgery, Tel-Aviv Medical Center,
Tel Aviv, Israel
M. A. Rudek
The Sidney Kimmel Comprehensive Cancer Center, Johns
Hopkins University School of Medicine, Baltimore, MD, USA
H. Brastianos
Department of Medicine, McMaster University,
Hamilton, ON, Canada
P. Zadnik � B. Tyler
Department of Neurosurgery, Johns Hopkins University
School of Medicine, Baltimore, MD, USA
H. Brem
Department of Neurosurgery, Oncology and Biomedical
Engineering, Johns Hopkins University School of Medicine,
Baltimore, MD, USA
J. O. Blakeley (&)
Department of Neurology, Neurosurgery and Oncology, Johns
Hopkins University School of Medicine, CRB II, Suite 1M16,
1550 Orleans Street, Baltimore, MD 21231, USA
e-mail: jblakel3@jhmi.edu
123
Cancer Chemother Pharmacol (2012) 70:129–139
DOI 10.1007/s00280-012-1867-1
with newly diagnosed glioblastoma (GBM) [1]. The plat-
form of TMZ in combination with radiotherapy has been
further enhanced by the addition of some standard (i.e.,
carmustine wafers) and several experimental anti-cancer
drugs, achieving up to 37 % OS at 2 years in some patients
[2, 3]. Despite these gains, the enthusiasm for the devel-
opment of effective drug therapies for GBM has been
tempered by the repeated failure to achieve durable clinical
responses.
One factor contributing to the poor clinical performance
of many drugs against brain cancer is limited access to
tumor across the blood brain barrier (BBB) [4, 5]. The
BBB is a physical and physiologic barrier that restricts
entry of exogenous compounds, including many anti-can-
cer therapies, to the brain [4, 6]. TMZ is a small (MW
194.15) alkylating drug known to cross the BBB with an
average AUCextracellular fluid (ECF)/plasma of 18 ± 4 % in
patients [7] and 19.3 ± 9.6 % in rat glioma models [8].
TMZ given in standard dosing achieves better intratumoral
concentrations than many cytotoxic therapies [4, 7]; how-
ever, there is preclinical evidence that there is incremental
improvement in TMZ’s efficacy against glioma cells with
higher intratumoral concentrations [9, 10]. These data
suggest that interventions that either increase or decrease
intratumoral TMZ concentrations may correspondingly
improve or impair the efficacy of TMZ against GBM.
Tumor-driven angiogenesis results in irregular and leaky
endothelium in GBM [11]. This process contributes to the
abnormal BBB common in GBM and is driven largely by
vascular endothelial growth factor (VEGF) signaling [12].
Intensive investigation of anti-angiogenesis agents in GBM
is ongoing, and several such agents have been shown to
normalize the BBB in preclinical models and in patients
[11, 13]. Treatment with bevacizumab (BEV), a mono-
clonal antibody against VEGF, has resulted in durable
objective radiographic response rates and improved clinical
function in some patients with recurrent GBM, leading to
accelerated approval by the Food and Drug Administration
(FDA) for this indication [14, 15]. BEV is currently under
investigation for patients with newly diagnosed GBM
in combination with standard TMZ and radiotherapy
(NCT00884741; NCT00943826). Combining anti-angio-
genesis agents with cytotoxic therapies such as TMZ may
be an effective strategy for patients with GBM [14–16].
However, to date there has been no demonstrated improve-
ment in OS with this approach, and it is unknown whether
normalization of the BBB via anti-angiogenesis agents
enhances [17–19] or restricts [18, 20, 21] delivery of con-
current chemotherapies to glioma cells. Restriction of TMZ
to glioma cells may be an unintended adverse effect of BEV
combination therapy that could negatively impact overall
therapeutic efficacy. To assess the impact of BEV on intra-
tumoral TMZ concentrations, we used microdialysis to
measure the brain extracellular fluid (ECF) concentration of
TMZ before and after BEV in vivo in an intracranial U87
glioma model. Such data are critical for enhancing our
understanding of potential therapeutic interactions that may
influence clinical dosing schedules.
Materials and methods
Materials
TMZ (Schering Corporation, a subsidiary of Merck & Co.,
Inc. NJ, USA) and BEV (lot # 705413, Genentech, Inc. CA,
USA) were purchased from the Johns Hopkins pharmacy.
CMA12 microdialysis brain probes (membrane length
2 mm, shaft diameter 0.6 mm, shaft length 14 mm, mem-
brane diameter 0.5 mm, polyarylethersulfone membrane),
CMA 120 bowl system for freely moving animals, CMA
402 Syringe Pump, microsyringes 10-mL glass with piston
stroke 60 mm were commercially purchased from CMA
(CMA Microdialysis Inc. MA, USA). The study was
approved by the Johns Hopkins Animal Care and Use
Committee, and all procedures were conducted with com-
pliance with their regulations.
Cell culture
The U87 human glioma cell line (provided by Dr. John
Laterra, Johns Hopkins University, Baltimore, MD) [22]
was grown in MEM with Earle Salts and L-glutamine
(MEM 1*Mediatech, Inc.) supplemented with 10 % fetal
bovine serum (Gemini Bioproducts, Inc.), 2 mMol/L
sodium pyruvate (Mediatech, Inc.), 0.1 mmol/L MEM-
non-essential amino acids (Mediatech, Inc.), and penicil-
lin–streptomycin (Mediatech, Inc.). The cells were grown
at 37 �C in a humidified incubator with 5 % CO2.
Implantation of tumor cells and microdialysis probes
Five male, athymic nude rats, 200–300 g each (Harlan
Bioproducts, Madison, WI), were anesthetized with
0.6–0.8 mL of pharmaceutical grade ketamine and xyal-
zine, IP. The dosing for ketamine was 15 mg, and the
dosing for xylazine was 1.5 mg. Anesthetized animals were
secured in a stereotactic apparatus. The scalp was cleansed,
and a midline incision was made to expose the skull. Two
holes were drilled by an automatic drill in the skull 2 mm
lateral (right and left) and posterior to the Bregma. Two
guide cannulas with dummy catheters were placed to a
depth of 4 mm. The guide cannula was secured to the skull
with dental cement (Geristore Syringeable value kit A2,
Denmat, Santa-Maria, CA,). A 25-lL Hamilton syringe
with a 26-gauge needle attached to the stereotactic frame
130 Cancer Chemother Pharmacol (2012) 70:129–139
123
was used to inject 1 million U87 cells at a depth of 5 mm
from the skull via guide cannula on the left striatum over
3–4 min. The needle was then withdrawn, and the skin was
closed with sutures. After the surgery, the animals were
given buprenorphine, 1 mg/kg SC, for analgesia. They
were returned to their individual cages and received a
regular rat diet and water ad libitum. The animals were
monitored daily for weight loss or new neurological deficit.
Microdialysis methodology and ECF sample collection
Based on prior serial MRI assessments of 4 rats with
intracranial U87 tumors, we determined that the mean
tumor diameter was 2.8 ± 0.93 mm at day 10 (Fig. 1).
This is an optimal size as it ensures the probe is surrounded
by tumor cells, but that the tumor size is not going to result
in imminent herniation. Hence, on day 11 after tumor
inoculation, the dummy catheters were replaced with 2 mm
CMA12 microdialysis catheters, bilaterally (left = tumor
side, right = normal brain). The inlet tubing was attached
to a CMA microsyringe and pump, and lactated ringers
solution was perfused through the catheter tip at a rate of
1 lL/min (Fig. 2). After a 60 min period of equilibration,
TMZ was administered to rats as a single oral dose at
50 mg/kg dissolved in water. Brain ECF dialysate collec-
tions were collected at baseline and every 60 min for 6 h.
In vivo dialysate recovery experiments were performed at
the end of the 6 h dialysate collection for bilateral probes.
At the conclusion of the initial collection period, the
microdialysis catheters were removed from the cannula and
placed in lactated ringer solution. The dummy catheters
were replaced. A single dose of bevacizumab (10 mg/kg)
was injected via tail vein. The rats were returned to their
cages with ad libitum diet, water, and activity and monitored
for comfort.
Thirty-six hours after the BEV was dosed, rats were
transiently re-anesthetized with ketamine and xyalzine, the
microdialysis catheters were put in place of the dummy
catheters, and the rats were replaced into the collection
cages. The catheters were again perfused with a lactated
ringer solution of 1 lL/min and allowed to equilibrate over
60 min. TMZ was again given as described above, now
36 h after BEV. Dialysate collection was continued every
60 min for 6 h. In vivo dialysate recovery experiments
Fig. 1 T1w gadolinium MRI of
a rat brain with catheters in U87
tumor (left) and contralateral
normal brain (right) 10 days
after U87 cell inoculation and
dummy probe placement
Cancer Chemother Pharmacol (2012) 70:129–139 131
123
were done again at the conclusion of collection. All ECF
samples were assessed for drug concentrations using liquid
chromatography–tandem mass spectrometry (LC/MS/MS)
over the concentration range of 0.02–5 lg/mL [8]. The
inter-assay precision were all \15 %, and the accuracy
expressed as the percentage error was within the range of
±15 % for microdialysate.
In vivo assessment of probe recovery
In vivo dialysate recovery experiments were done at the
end of each collection period to allow estimation of in vivo
recovery and assess the integrity of the microdialysis sys-
tem. At the end of ECF collection, the probes were per-
fused at a rate of 1 lL/min with lactated ringer solution
containing TMZ to determine the in vivo probe recovery
using the retrodialysis method described elsewhere [8].
Microdialysate samples were collected at 10-min intervals
for 40 min, and the percentage relative recovery was cal-
culated as follows [23, 24]:
Invivo recovery ¼ Cperfusate � Cdialysate
Cperfusate
where Cperfusate is the drug concentration (2 lg/mL) in the
perfusate and Cdialysate is the drug concentration in the
microdialysate, which was the average concentration for 2–
4 samples at each collection time, for each probe. In the
case of 1 animal where samples were not obtained, the
average in vivo recovery for the other time points for that
animal was used for further calculations. The recovery was
utilized to estimate the estimated ECF concentration
according to the equation: [23, 24].
Estimated ECF concentration
¼ measured microdialysis sample
invivo recovery
All animals were euthanized by perfusion, and their
brains harvested, sectioned, and stained with H/E to
demonstrate peri-probe tumor histology (Fig. 3). In 2
animals, MRI of brain was also done with the probe in
place to confirm position relative to the tumor (Fig. 1).
Pharmacokinetic analysis
Individual pharmacokinetic parameters for both raw and
estimated ECF before and after administration of BEV
were calculated by standard non-compartmental analysis
using the WinNonlin version 5.3 (Pharsight Corporation,
Mountain View, CA). The maximum plasma concentration
(Cmax) and the time of Cmax after oral administration (Tmax)
were obtained by visual inspection of the concentration–
time curve. The area under the plasma concentration–time
curve (AUC) was calculated using the log-linear trapezoi-
dal rule to the end of sample collection (AUC0–5.5h) and
extrapolated to infinity (AUC0–?) by dividing the last
quantifiable concentration by the terminal disposition rate
constant (kz), which was determined from the slope of the
terminal phase of the concentration–time profile. The half-
life (T1/2) was determined by dividing 0.693 by kz. If the
percent AUC extrapolated was greater than 50 %, then
only the AUC0–5.5h was reported.
Statistical analysis
The PK parameters were summarized using descriptive
statistics. Differences in the PK parameters between
Fig. 2 Drawing of microdialysis experimental procedure with cath-
eters in the tumor (A) and contralateral normal brain (B). Inlet tubing
carries perfusion fluid and outlet tubing carries dialysate. Rats were
free-moving in individual cages throughout collection. The insert
(C) shows the microdialysis probe structure allowing diffusion of
low-molecular-weight agents across the semipermeable membrane
Fig. 3 A sample of the H/E sections created from rats bearing U87
gliomas after euthanization. The tract of the microdialysis probe is
seen in the center of the tumor on the left
132 Cancer Chemother Pharmacol (2012) 70:129–139
123
Table 1 Summary of temozolomide pharmacokinetics in brain extracellular fluid in tumor (section A, top) and in contralateral normal brain
(section B, bottom) before (left) and after (right) bevacizumab
Pre-bevacizumab Post-bevacizumab
Recovery
(%)
Tmax
(h)
Cmax
(lg/mL)
AUC0–?
(lg h/mL)
T1/2
(h)
Recovery
(%)
Tmax
(h)
Cmax
(lg/mL)
AUC0–?
(lg h/mL)
T1/2
(h)
A.
Tumor ECF—raw data
1 a 2.5 0.25 0.7 0.8 62 1.5 0.1 N.R.b N.R.b
2 61 2.5 0.36 1.3 1.6 78 0.5 1.0 N.R.b N.R.b
3 91 1.5 0.13 0.66 1.6 60 1.5 0.3 1.0 1.6
4 58 0.5 1.07 4.1 3.7 81 1.5 1.3 3.9 1.1
5 32 1.5 0.52 1.8 1.5 56 1.5 1.1 3.1 1.2
Summaryc (% C.V.) 60 ± 24
(40)
1.5
(0.5, 2.5)
0.5 ± 0.4
(79)
1.1 ± 0.6
(52)
1.8 ± 1.1
(59)
68 ± 11
(16)
1.5
(0.5, 1.5)
0.6 ± 0.6
(101)
2.7 ± 1.5
(56)
1.3 ± 0.3
(20)
Tumor ECF—corrected for recovery
1 2.5 0.4 1.2 0.8 1.5 0.2 N.R.b N.R.b
2 2.5 0.6 2.1 1.6 0.5 0.1 N.R.b N.R.b
3 1.5 0.1 0.6 1.6 1.5 0.4 1.7 1.6
4 0.5 1.9 7.1 3.7 1.5 1.6 4.8 1.1
5 1.5 1.6 5.7 1.5 1.5 1.9 5.5 1.2
Summaryc (% C.V.) 1.5
(0.5, 2.5)
0.9 ± 0.8
(83)
3.4 ± 2.9
(87)
1.8 ± 1.1
(59)
1.5
(0.5, 1.5)
0.9 ± 0.9
(99)
4.0 ± 2.0
(51)
1.3 ± 0.3
(20)
B.
Non-tumor brain ECF—raw data
1 77 1.5 0.5 1.6 1.3 62 1.5 0.2 0.8 2.2
2 5 1.5 0.2 0.8 2.0 77 1.5 0.1 0.4 3.1
3 90 1.5 0.1 0.5 2.6 52 1.5 0.3 1.2 1.5
4 49 1.5 0.7 3.0 1.7 54 1.5 1.1 3.5 1.0
5 2 1.5 0.4 1.3 1.8 49 1.5 1.1 3.0 1.3
Summaryc (% C.V.) 59 ± 26
(44)
1.5
(1.5, 1.5)
0.4 ± 0.3
(70)
1.4 ± 1.0
(66)
1.9 ± 0.5
(27)
59 ± 11
(19)
1.5
(1.5, 1.5)
0.6 ± 0.5
(90)
1.8 ± 1.4
(78)
1.8 ± 0.8
(46)
Non-tumor brain ECF—corrected for recovery
1 1.5 0.6 2.1 1.3 1.5 0.3 1.2 2.2
2 1.5 0.3 1.4 2.0 1.5 0.1 0.6 3.1
3 1.5 0.1 0.5 2.6 1.5 0.6 2.3 1.5
4 1.5 1.5 6.0 1.7 1.5 2.1 6.5 1.0
5 1.5 1.6 6.0 1.8 1.5 2.2 6.1 1.3
Summaryc (% C.V.) 1.5
(1.5, 1.5)
0.8 ± 0.7
(83)
3.2 ± 2.6
(81)
1.9 ± 0.5
(27)
1.5
(1.5, 1.5)
1.1 ± 1.0
(95)
3.3 ± 2.8
(83)
1.8 ± 0.8
(46)
AUC0–? area under the concentration–time curve extrapolated to infinity, Cmax maximum concentration, % C.V. percent coefficient of variation,
Min minimum, Max maximum, N.R. not reported, SD standard deviation, Tmax time to maximum concentrationa No retrodialysis samples were collected due to catheter failure. 60 % is the average recovery over all conditionsb Not reported due to inability to calculate the terminal disposition rate constant (kz) coupled with the last quantifiable time point being before
5.5 hc Median (Min, Max) is reported for Tmax. Mean ± SD (% C.V.) are reported for all other parameters
Cancer Chemother Pharmacol (2012) 70:129–139 133
123
pre- and post-bevacizumab were evaluated statistically by
use of a Wilcoxon matched pairs signed-rank test. The
statistical analysis was done using JMP Statistical Dis-
covery Software version 3.2.6 (SAS Institute, Cary, NC).
The a priori level of significance was P \ 0.05.
Results
Pharmacokinetic of TMZ in brain ECF
Similar maximal and total exposure (Cmax and AUC0–?),
Tmax, and T1/2 values were found when TMZ was admin-
istered alone and with BEV (raw data: Cmax P = 0.32,
AUC0–? P = 0.75, Tmax P = 0.75, T1/2 P = 1.00; cor-
rected data: Cmax P = 0.38, AUC0–? P = 1.00, Tmax
P = 0.75, T1/2 P = 1.00) (Table 1; Fig. 4). The mean
corrected TMZ ECF Cmax on the tumor side was
0.93 ± 0.77 lg/mL (mean ± SD), which occurred at a
median time of 1.50 h. The area under the concentration
curve (AUC0–?) was 3.35 ± 2.90 lg h/mL. After the
administration of BEV, the mean corrected Cmax of ECF
concentration of TMZ on the tumor side was 0.85 ±
0.85 lg/mL, which occurred at a median time of 1.50 h, and
AUC0–? was 3.98 ± 2.02 lg h/mL. This represented a 0.9-
fold decrease in the Cmax and a 1.2-fold increase in TMZ
mean AUC0–? after BEV administration. The half-life was
slightly decreased after BEV administration (1.84 ± 1.08 h
pre vs. 1.30 ± 0.27 h post).
On the contralateral side (non-tumor-bearing brain),
before BEV administration, corrected Cmax of ECF of TMZ
was 0.82 ± 0.68 lg/mL (mean ± SD), which occurred at
a median time of 1.50 h, and AUC0–? was 3.22 ±
2.62 lg h/mL. After the administration of BEV, the cor-
rected Cmax of ECF concentration of TMZ was 1.06 ±
1.01 lg/mL, which occurred at a median time of 1.50 h,
and the AUC0–? was 3.34 ± 2.78 lg h/mL. This repre-
sented a 1.3-fold increase in the Cmax of ECF concentration
of TMZ, and no change in mean AUC0–? of ECF con-
centration of TMZ after BEV administration (Fig. 4). The
half-life was slightly prolonged after BEV administration
(1.86 ± 0.50 h pre vs. 1.82 ± 0.83 h post). There was no
statistical difference in parameters on the contralateral side
(non-tumor bearing brain) (P [ 0.05).
Discussion
Cytotoxic and anti-angiogenic therapies will theoretically
complement each other to decrease tumor cell prolifera-
tion, reduce tumor associated inflammation, and induce
cancer cell death. Based on this principle, there are several
ongoing clinical studies assessing the combination thera-
pies of TMZ and BEV in patients with malignant gliomas.
However, there is not yet any evidence that BEV improves
OS in patients with GBM or that concurrent cytotoxic
therapy with BEV has significant clinical benefit over BEV
monotherapy [14, 15]. Moreover, it has been suggested that
agents such as BEV may inadvertently decrease the intra-
tumoral concentration of TMZ that has been proven to
prolong OS when given with radiation therapy to patients
with newly diagnosed GBM [17, 21]. We address the
critical clinical question of the influence of BEV on TMZ
intratumoral PK via direct sampling from intracranial U87
tumors with microdialysis catheters in the presence and
absence of BEV.
Microdialysis is a technique that allows direct mea-
surement of compounds in the ECF. The catheters are FDA
approved for use in humans. They have been predomi-
nantly used in the setting of brain trauma and ischemia
[25]. More recently, microdialysis catheters have been used
to assess the delivery of cytotoxic chemotherapy to brain
Fig. 4 Concentrations of TMZ in brain ECF obtained by microdi-
alysis in the tumor a or non-tumor brain b. The open symbols
represent the pre-bevacizumab concentrations, while closed symbols
are post-bevacizumab. Symbols, mean; bars, SD
134 Cancer Chemother Pharmacol (2012) 70:129–139
123
tumors in both preclinical and clinical studies [4, 7, 8, 17–
20, 26, 27]. This technique allows sampling of amenable
drugs in the ECF surrounding tumor cells. Drug concen-
trations in the ECF compartment are thought to best rep-
resent bioavailable drug. Furthermore, the catheters can
stay in place for several days allowing each animal to serve
as its own control for both tumor and normal brain as well
as for before and after treatment comparisons.
Although microdialysis is an informative technique, it
has several limitations that have to be considered. Insertion
of the microdialysis probe into tissue transiently disrupts
the normal BBB. This is maximal immediately after
insertion and is largely resolved 3–4 days after insertion
[28]. We administered TMZ and performed the microdi-
alysis studies on day 11 after insertion of the guide cann-
ulas and tumor cells in order to both minimize the effect of
the catheter placement on BBB disruption and ensure that
the catheter membrane was centered in the tumor core.
Another limitation of microdialysis is that the drug con-
centrations in dialysate are estimates of the true ECF
concentrations [29]. To ensure as accurate as possible an
estimation of ECF, we used a low flow rate (1 lL/min) and
a drug shown to have excellent in vitro recovery [8]. In
addition, we estimated the percent recovery via in vivo
retrodialysis at the end of each study, for each catheter, to
estimate TMZ recovery within the tissue environment [30].
Our retrodialysis recovery rates ranged between 58.5 ±
11.4 and 67.7 ± 10.7 % compared to rates of 87 ± 5.5 %
seen in in vitro studies [7]. These rates suggest that the
catheters were functional and with adequate recovery even
at the end of an extended collection period.
The data from this study showed that there was no sta-
tistically significant difference in the intratumoral AUC of
TMZ with or without BEV in U87 xenograft tissue versus
normal brain (Fig. 4). One possible explanation for this is
that BEV, a humanized monoclonal antibody to VEGF,
does not influence VEGF in a rat model. However, avail-
able data show that BEV does demonstrate therapeutic
efficacy in orthotropic U87 models in athymic rats [31–33]
as it was shown to cause a dose-dependent decrease in the
relative cerebral blood volume (rCBV) as well as a
decrease in the rate of tumor growth [31, 32]. BEV was
also shown to influence brain tumor perfusion as assessed
by ferumoxytol MRI in rats with U87 gliomas [31, 32, 34].
Hence, there is evidence for a biological effect of BEV in
the U87 rat model.
Another possible explanation for the lack of observed
difference between pre- and post-BEV TMZ concentrations
is that U87 tumors are not angiogenic and therefore would
not be expected to respond to BEV. However, the available
literature shows that U87 tumors have an angiogenic phe-
notype [35, 36]. Specifically, U87 cells (in vitro) and
tumors (in vivo) have been characterized as expressing
vimentin and demonstrating angiogenic properties such as
neovascularization in addition to malignant features such
as hypercellularity, pleomorphism, nuclear atypia, and
inflammation similar to human GBM [35]. Although U87
tumor do not generally show necrosis or invasion, they do
demonstrate high vessel density, abnormally large and
irregular blood vessels and a high density of cells staining
for VEGF [36]. These properties make U87 a favorable
model to assess the effects of anti-angiogenesis therapies
on intratumoral PK. Finally, a well-recognized property of
U87 tumors is rapid and predictable growth. This is an
important quality for intracranial microdialysis studies
allowing for optimal catheter placement within tumor core
and sequential dosing of medications over time without
animal morbidity due to large tumors.
A limitation of this study is that serial MRIs could not
be done with the cannulas in place to demonstrate the
variance in the degree of vascularization or the change in
vascularization in response to BEV. In addition, rats were
euthanized at the conclusion of the study, and tissue was
harvested to confirm the spatial relationship of catheter to
tumor (Fig. 3), but additional testing of vascular markers
was not available. An additional limitation of this study is
that we do not have plasma levels to assess systemic TMZ
exposure. However, TMZ plasma disposition has been well
characterized in prior TMZ PK studies, and the comparison
of tumor versus normal brain within animals across treat-
ment conditions serves as an internal control. Overall, our
TMZ PK values are similar to prior reports of brain TMZ
concentrations both within human non-enhancing brain [7]
and other preclinical glioma studies [17, 18, 37] (Table 2).
One important consideration for the planned use of con-
comitant TMZ and BEV dosing is the intrinsic sensitivity of
the tumor cells to TMZ and whether that may be influenced
by concomitant BEV. U87 cells are sensitive to TMZ with
an IC50 of 472 ± 14 lM [38, 39] and show susceptibility to
TMZ as measured by tumor response [40, 41] as well as
prolonged survival [42]. There are also data to suggest that
the positive anti-tumor effects of TMZ require adequate
drug concentration [40]. Although assessment of tumor
response to TMZ and BEV, alone or in combination, was
beyond the scope of this PK study, the PK data presented
here suggest that there is no interruption of TMZ access to
tumor with BEV and therefore, no clear mechanism by
which BEV would adversely influence the therapeutic effect
of TMZ. In fact, it is possible that BEV could enhance the
effect of TMZ on gliomas cells. Although there are no
published data about the effect of BEV and TMZ in U87
models, in the Hs683 intracranial orthotropic glioma model,
concurrent BEV and TMZ resulted in improved survival
times versus either drug alone [38].
It is expected that normal brain would not have a sub-
stantial change in TMZ with or without BEV, as was seen
Cancer Chemother Pharmacol (2012) 70:129–139 135
123
Ta
ble
2T
emo
zolo
mid
e(T
MZ
)p
har
mac
ok
inet
ics
inp
recl
inic
alg
lio
ma
mo
del
s(b
rain
and
per
iph
ery
)an
do
ne
clin
ical
stu
dy
,w
ith
and
wit
ho
ut
exp
erim
enta
lan
ti-a
ng
iog
enes
isag
ents
TM
Zm
on
oth
erap
yS
ub
ject
sS
amp
lin
g
loca
tio
n
TM
Zd
osi
ng
AU
C0–?
(lg
h/m
L)
Tm
ax
(h)
Cm
ax
(lg
/mL
)
Po
rtn
ow
etal
.[7
]P
atie
nts
Bra
in1
50
mg
/m2
PO
2.7
±1
.02
.0±
0.8
0.6
0±
0.3
Zh
ou
etal
.[3
7]
Rat
sB
rain
20
mg
/kg
IV5
.1±
4.3
0.3
6±
0.0
94
.0±
2.6
Zh
ou
etal
.[8
]R
ats
Per
iph
ery
18
mg
/kg
/day
95
day
3.2
3m
g/k
g/d
ay9
28
day
31
±1
5(d
ay5
)
4.6
±2
.6(d
ay2
8)
0.7
7±
0.2
2
0.4
5±
0.3
4
11
±5
.9
2.0
±0
.58
TM
Z?
anti
-an
gio
gen
esis
ther
apy
(all
inra
ts)
An
ti-a
ng
iog
enic
agen
tS
amp
lin
g
loca
tio
n
TM
Zd
osi
ng
AU
C0–?
(lg
h/m
L)
Tm
ax
(h)
Cm
ax
(lg
/mL
)
Dev
inen
iet
al.
[20
].T
NP
-47
03
0m
g/
kg
95
do
ses
Per
iph
ery
40
mg
/kg
IAC
on
tro
l:9
1±
32
TN
P-4
70
:6
9±
30
N.R
.N
.R.
Ma
etal
.[1
7]
TN
P-4
70
30
mg
/
kg
94
–6
do
ses
Bra
inC
on
tin
uo
us
IAin
fusi
on
(40
lg
/mL
)to
reac
h
stea
dy
stat
e
Co
ntr
ol:
8.6
±2
.3
TN
P-4
70
:4
.2±
0.5
N.R
.N
.R.
Per
iph
ery
Co
ntr
ol:
17
±4
.3
TN
P-4
70
:1
2±
1.8
N.R
.N
.R.
Ma
etal
.[1
8]
Su
nit
inib
25
mg
/kg
IPB
rain
Co
nti
nu
ou
sIA
infu
sio
n
(20
lg
/mL
)to
reac
h
stea
dy
stat
e
Co
ntr
ol:
2.8
±1
.2
TN
P-4
70
:5
.3±
2.6
N.R
.N
.R.
Per
iph
ery
Co
ntr
ol:
17
±3
.7
TN
P-4
70
:1
3±
3.9
N.R
.N
.R.
Zh
ou
etal
.[2
6]
Su
nit
inib
10
mg
/kg
/
day
91
4d
ay
40
mg
/kg
91
4d
ay
Per
iph
ery
20
mg
/kg
PO
Co
ntr
ol:
20
±6
.7
Su
nit
inib
10
mg
:2
9±
8.6
Su
nit
inib
40
mg
:2
4±
8.3
0.6
9±
0.1
8
0.7
8±
0.1
0
0.8
3±
0.0
0
11
±3
.6
16
±4
.4
13
±2
.7
PO
ora
l,IV
intr
aven
ou
s,IA
intr
a-ar
teri
al,
N.R
.n
ot
rep
ort
ed
136 Cancer Chemother Pharmacol (2012) 70:129–139
123
in this study. However, the lack of difference in TMZ PK
between U87 tissue and normal brain was unexpected. It is
possible that due to the relatively high penetration of TMZ
across the BBB at baseline (Table 2), the small sample size
was not adequate to reflect the difference in TMZ con-
centrations between tumor and normal brain. In humans,
disruption of the BBB as evidenced by contrast enhance-
ment has been associated with higher levels of metho-
trexate than in non-contrast enhancing regions of brain
[27]. The only human data about TMZ disposition in brain
are from regions of relative BBB integrity [7]. In preclin-
ical models, combining TMZ and angiogenesis inhibitors
has led to inconsistent TMZ concentration results depen-
dent on the tumor model, tumor location, anti-angiogenesis
agent, and vehicle used (Table 2) [8, 17, 18, 20, 37]. In a
U87 orthotropic model, Zhou and Gallo showed that TMZ
concentrations were higher in tumor versus normal brain
[19]. However, this study assessed drug concentrations in
tumor homogenate at steady state and hence is not directly
comparable to the ECF concentrations reported here.
Devineni et al. showed reduction in TMZ in a subcutaneous
(SC) C6 tumor model with TNP-470 (a synthetic analog of
fumagillin shown to inhibit angiogenesis) [20]. Ma et al.
found similar results in both SC and intracranial models
using the human-derived cells engineered to express high
levels of VEGF (SF188V?) treated with TNP-470 [17].
However, in that same tumor model, the pan-kinase
inhibitor sunitinib resulted in decreased TMZ concentra-
tions in the peripheral tumors but increased TMZ concen-
trations in intracranial tumors [18]. Further investigation of
the effect of sunitinib on intratumoral concentration of
TMZ was undertaken in an intracranial U87 model and
again showed a dose-dependent effect of sunitinib such that
low dose enhanced TMZ concentrations intracranially, but
high dose reduced TMZ concentrations [19]. The cumu-
lative view of this data is that the impact of anti-angio-
genesis agents on cytotoxic drug concentrations in the
brain is dependent on the agents in question as well as the
tumor response to anti-angiogenesis therapy. In this study,
we chose to evaluate BEV as it is the only anti-angiogen-
esis agent with FDA approval for use in recurrent GBM.
TMZ is currently used to treat patients with newly
diagnosed and recurrent GBM. Of note, TMZ showed
mixed activity as a single agent in patients with both
recurrent and newly diagnosed GBM in early clinical
investigation and was initially approved for use in patients
with recurrent anaplastic astrocytoma [43, 44]. TMZ
given concurrently with radiation therapy has shown
significantly improved OS in patients with GBM [1]. It is
possible that TMZ and radiation therapy are synergistic.
An alternative hypothesis is that there is improved access
of TMZ to tumor across a BBB disrupted by radiation
therapy [45].
There are currently two large phase III trials investigating
the combination of BEV, TMZ, and radiation therapy
in patients with newly diagnosed GBM (NCT00884741;
NCT00943826). The results from the present study show
that there are no statistically significant changes in TMZ
concentrations before and after BEV in a U87 glioma
xenograft model. These results are encouraging and suggest
that with a dosing scheme similar to what is being used
clinically, TMZ is not restricted from brain tissue with
concomitant BEV. Additional studies with agents that have
lower brain penetration than TMZ will provide additional
data about the role of BEV in enhancing or restricting access
of drugs that have low baseline penetration across the BBB.
Preclinical assessment of intracranial PK in experimental
models may prove a significant clinical predictor of effective
clinical dosing strategies assisting in the rationale develop-
ment of clinical paradigms with combination therapies.
Acknowledgments We would like to thank Dr. John Laterra for
donation of the U87 cell line and Ming Zhao, Aleksander Mnat-
sakanyan, Ping He, Bachu Lal, and Jinyuan Zhou for their technical
support and advice. This work was supported in part by grants from
American Physicians Fellowship (APF) for Medicine in Israel
(Rachel Grossman), by the AANS/CNS Section on Tumors/Brain-
LAB International Fellowship (Rachel Grossman), the Research
Scholar Grant #116293-RSG-08-119-01-CCE from the American
Cancer Society (Betty Tyler), and by the Analytical Pharmacology
Core of the Sidney Kimmel Comprehensive Cancer Center at Johns
Hopkins (NIH grants P30 CA006973 and UL1 RR025005), the
Shared Instrument Grant (1S10RR026824-01). Grant Number UL1
RR 025005 from the National Center for Research Resources
(NCRR), a component of the National Institutes of Health (NIH) and
NIH Roadmap for Medical Research, and its contents are solely the
responsibility of the authors and do not necessarily represent the
official view of NCRR or NIH.
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