The impact of bevacizumab on temozolomide concentrations in intracranial U87 gliomas

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: [email protected]

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


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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


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


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


123

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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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The impact of bevacizumab on temozolomide concentrations in intracranial U87 gliomas

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