Preclinical Pharmacokinetic and Pharmacodynamic Evaluation
of Metronomic and Conventional Temozolomide Dosing
Qingyu Zhou, Ping Guo, Xiaomin Wang, Silpa Nuthalapati, and James M. Gallo
Department of Pharmaceutical Sciences, School of Pharmacy, Temple University, Philadelphia, Pennsylvania
Received December 11, 2006; accepted January 25, 2007
Metronomic dosed (MD) chemotherapy as opposed to conven-
tional dosed (CD) chemotherapy is considered an alternate
strategy to target angiogenesis and limit host toxicity. Although
this approach is promising, there has not been any attempt to
define optimal metronomic dosing regimens by integrating
pharmacokinetic (PK) with pharmacodynamic (PD) measure-
ments. The aim of this study was to compare the pharmacoki-
netics and pharmacodynamics of temozolomide [TMZ, 8-car-
MD and CD regimens. In vivo studies were carried out in
xenografted athymic rats treated with either 18 mg/kg/day TMZ
for 5 days or 3.23 mg/kg/day TMZ for 28 days. PK studies were
performed on the first and last days of dosing. PD measure-
ments consisted of gene and protein expression of various
angiogenic markers, tumor size, tumor pH, and interstitial fluid
pressure (IFP). The results demonstrated that the PK parame-
ters (total clearance, volume of distribution, and tumor/plasma
accumulation) were quite similar for MD and CD groups, consis-
tent with the linear PK properties of TMZ. Both TMZ treatment
schedules caused a significant decrease in IFP and tumor size
compared with vehicle control treatment, demonstrating compa-
rable effectiveness of MD and CD regimens. Using real-time poly-
merase chain reaction and Western blot analyses, some differ-
ences were noted in expression levels of vascular endothelial
MD regimen may be superior to the CD regimen by preventing
tumors from progressing to a proangiogenic state. In conclusion,
several PK/PD factors contributing to the antitumor activity of the
MD TMZ therapy have been identified and form a foundation for
further investigations of low-dose TMZ regimens.
Gliomas represent the most common and aggressive type of
primary brain tumors, accounting for 42% of all primary
nervous system tumors and 77% of all malignant primary
central nervous system tumors (Ashby and Ryken, 2006).
Despite advances in the multidisciplinary approach and di-
agnostic imaging techniques, the overall prognosis for pa-
tients with high-grade malignant gliomas still remains
bleak, with a median survival measured in months and an
overall 5-year survival rate of ?2% (Surawicz et al., 1998). In
the past, the chemotherapeutic approach to malignant brain
tumors relied mainly on the use of nitrosoureas, which
showed only modest antitumor activity (Nieder et al., 2000).
More recently, 8-carbamoyl-3-methylidazo(5,1-d)-1,2,3,5-ter-
razin-4(3H)-one (TMZ, temozolomide) has emerged as a well-
tolerated oral alkylating agent that is able to cross the blood-
brain barrier with demonstrated encouraging activity in
primary and recurrent gliomas (Newlands et al., 1992; Deh-
dashti et al., 2006).
TMZ is rapidly and well absorbed after oral administration
(Stevens et al., 1987; Newlands et al., 1992) and undergoes
spontaneous hydrolysis at physiological pH to form its active
(MTIC), which further degrades to 5(4)-aminomidazole-4(5)-
carboxamide and a highly reactive methyl-diazonium cation
(Stevens et al., 1987; Denny et al., 1994). MTIC exerts its
cytotoxic effect primarily through methylation of genomic
DNA at the O6position of guanine. The formation of O6-
methylguanine (O6MeG) subsequently induces futile cycling
of a mismatch repair pathway, leading to inhibition of DNA
replication and cell-cycle arrest at the G2-M phase transition
(Denny et al., 1994; Roos et al., 2007). The O6MeG lesion can
This work was supported by National Institutes of Health Grants CA72937
and CA85577 (to J.M.G.).
Article, publication date, and citation information can be found at
ABBREVIATIONS: TMZ, temozolomide, 8-carbamoyl-3-methylidazo(5,1-d)-1,2,3,5-terrazin-4(3H)-one; MTIC, 3-methyl-(triazen-1-yl)imidazole-4-
carboxamide; O6MeG, O6-methylguanine; HUVEC, human umbilical vein endothelial cell; PK, pharmacokinetic; PD, pharmacodynamic; CD,
conventional dosed; MD, metronomic dosed; PAC, paclitaxel; Ang, angiopoietin; VEGF, vascular endothelial growth factor; HIF-1?; hypoxia-
inducible factor-1?; TSP-1, thrombospondin-1; FBS, fetal bovine serum; LC, liquid chromatography; MS/MS, mass spectrometry/mass spec-
trometry; IFP, interstitial fluid pressure; IF, interstitial fluid; PCR, polymerase chain reaction.
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics
JPET 321:265–275, 2007
Vol. 321, No. 1
Printed in U.S.A.
at ASPET Journals on December 30, 2015
be repaired by cellular O6-methylguanine-DNA methyltrans-
ferase, which transfers the methyl group to a cysteine resi-
due (Trivedi et al., 2005).
The standard recommended dose of TMZ as a single agent
or in combination is 150 to 200 mg/m2daily for 5 consecutive
days repeated every 4 weeks (Brandes et al., 2002). Other
administration schedules, including compressed and ex-
tended dosing protocols, evaluated with the intention of
maximizing O6-methylguanine-DNA methyltransferase de-
pletion and hence potentiating the cytotoxicity, have demon-
strated limited improvement of clinical efficacy with poten-
tially increased hematological toxicity (Danson et al., 2003;
Tosoni et al., 2006). Thus, there is a need to reappraise the
dose and schedule of TMZ to further improve its effectiveness
while reducing its toxicity.
Metronomic chemotherapy, which refers to the frequent
administration of chemotherapeutics at doses significantly
below the maximum tolerated dose without prolonged drug-
free breaks, has been evaluated as an alternative strategy to
achieve long-term therapeutic control (Gasparini, 2001; Ker-
bel and Kamen, 2004). The primary mechanism for metro-
nomic chemotherapy is thought to be inhibition of tumor
angiogenesis by direct killing of endothelial cells in the tumor
neovasculature (Miller et al., 2001) and suppressing the mo-
bilization and levels of bone marrow-derived circulating en-
dothelial progenitor cells (Bertolini et al., 2003). As such,
metronomic chemotherapy may possess several advantages
over conventional chemotherapy, including delaying the on-
set of acquired drug resistance and reducing host toxicity.
Two milestone studies by Browder et al. (2000) and by Kle-
ment et al. (2000) followed by several subsequent studies
have illustrated the capacity of different cytotoxic agents to
preferably target the endothelial cell compartment of tumors
when lower doses were given more frequently (Shaked et al.,
2005; Klink et al., 2006).
Several studies have been carried out to assess the anti-
angiogenic potential of the protracted low-dose TMZ treat-
ment. An early preclinical study demonstrated that angio-
genesis was inhibited by 5 ?M TMZ in both chorioallantoic
membrane and human umbilical vein endothelial cell
(HUVEC)-based Matrigel assays (Kurzen et al., 2003). A
recent clinical study showed that after receiving continuous
low-dose TMZ plus rofecoxib treatment, patients harboring
highly angiogenic glioblastoma multiforme had a signifi-
cantly longer time to progression compared with patients
with low angiogenic glioblastoma multiforme (Tuettenberg et
al., 2005), which suggested an antiangiogenic activity for
continuous low-dose TMZ plus rofecoxib. A more recent pre-
clinical study demonstrated that metronomic treatment with
TMZ exerted both antiangiogenic and antitumor effects in a
TMZ-resistant C6/LacZ rat glioma model (Kim et al., 2006).
Although previous studies showed that TMZ might possess
potential antiangiogenic properties when given metronomi-
cally, further investigation is warranted to understand
PK-PD relationships that can guide the selection of optimal
metronomic dosing regimens of TMZ. Therefore, the aim of
the present study was to characterize and contrast the phar-
macokinetics and pharmacodynamics of TMZ in nude rats
with human glioma xenografts receiving either CD or MD
TMZ treatment, and in so doing, provide a foundation for the
design of optimal MD TMZ treatments.
Materials and Methods
Materials. TMZ was generously provided by Schering Plough
Research Institute (Kenilworth, NJ). Paclitaxel (PAC), protease in-
hibitor cocktail, and a mouse monoclonal anti-angiopoietin (Ang) 1
antibody were purchased from Sigma-Aldrich (St. Louis, MO). Rab-
bit polyclonal antivascular endothelial growth factor (VEGF), anti-
hypoxia-inducible factor-1? (HIF-1?), anti-Tie-2 antibodies, and a
goat polyclonal anti-Ang-2 antibody were purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). A mouse monoclonal anti-
thrombospondin-1 (TSP-1) antibody was from NeoMarkers (Fre-
mont, CA). Matrigel was from BD Biosciences (Bedford, MA).
HUVECs and the HUVEC media kit, EGM-2 BulletKit, were from
Cambrex Bio Science (Walkersville, MD). Dulbecco’s modified Ea-
gle’s medium was from Mediatech (Herndon, VA). Fetal bovine se-
rum (FBS) and TRIzol reagent were purchased from Invitrogen
(Carlsbad, CA). A protein assay kit was obtained from Bio-Rad
PerkinElmer Life and Analytical Sciences (Boston, MA). All other
chemicals and solvents were obtained from commercial sources.
Drug analyses were conducted by liquid chromatography (LC)/
mass spectrometry/mass spectrometry (MS/MS)(API 4000 triple
quadrupole system; Applied Biosystems, Foster City, CA). Microdialy-
sis apparatus including microdialysis probes (CMA/20) with a 20-kDa
molecular mass cutoff polycarbonate dialysis membrane, a microdialy-
sis pump (CMA/102), and a refrigerated fraction collector (CMA/170)
were purchased from CMA Microdialysis (North Chelmsford, MA).
Male athymic nude rats (rnu/rnu) were purchased from Taconic
Farms (Germantown, NY) and used for xenografting at the age of 6
to 7 weeks. The care and use of animals were approved by the
Institutional Animal Care and Use Committee in accordance with
National Institutes of Health guidelines.
Cell Culture and in Vitro Cytotoxicity Assay. HUVECs were
cultured in an EGM-2 BulletKit composed of endothelial cell basal
medium-2 supplemented with ascorbic acid, FBS, hydrocortisone,
fibroblast growth factor, VEGF, human epidermal growth factor,
long R insulin-like growth factor-1, gentamicin sulfate, and heparin
as described by the manufacturer. A human SF188 glioma cell line
that was transfected with the mouse full-length VEGF164cDNA as
reported previously (Ma et al., 1998) and thereby overexpressing
VEGF (V?) were grown in Dulbecco’s modified Eagle’s medium
supplemented with 10% standard FBS. Cells were maintained in a
humidified atmosphere of 5% CO2in air at 37°C.
For the in vitro cytotoxicity study, 24- and 144-h exposures to TMZ
and PAC in HUVECs and SF188V? cells were compared. Cells were
seeded in 96-well plates and allowed to attach overnight and then
were treated with TMZ and PAC at various concentrations for 24 h
(2 ? 103cells/well in 100 ?l of medium) or for 144 h (500 cells/well in
100 ?l of medium). During the 144-h treatment period, culture media
were changed every 24 h along with fresh drug solutions. At the end
of the treatment, cells were fixed with trichloroacetic acid and
stained with sulforhodamine B. Optical densities were measured at
570 nm with a SpectraMax M2 microplate reader equipped with
SoftMax Pro software (Molecular Devices, Sunnyvale, CA). The
growth of treated cells was expressed as a percentage of control
cultures (vehicle alone). The concentration of drugs that decreased
the number of viable cells by 50% (i.e., IC50) compared with un-
treated cells was calculated by nonlinear fitting of the experimental
data obtained from three independent experiments performed in
Xenografts and in Vivo Study Protocol. SF188V? tumor cells
at logarithmic growth in vitro were harvested and washed twice with
phosphate-buffered saline. Tumor cells (5 ? 106) suspended in 0.3 ml
of Matrigel were inoculated s.c. in the dorsal neck region of the
athymic rats (6- to 7-week-old male rats) through a 23-gauge needle.
Tumor growth was monitored twice a week. Two perpendicular di-
ameters (a and b) were measured with a vernier caliper (Fisher
Scientific Co., Newark, DE), and tumor volumes (V) were calculated
Zhou et al.
at ASPET Journals on December 30, 2015
mulation in tumor cells or whether endothelial cells alone
serve as sufficient targets. Although we have not yet identi-
fied a breakthrough MD for TMZ, the current study indicates
that TMZ activity as a MD agent requires drug accumulation
in tumor cells.
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Address correspondence to: Dr. James M. Gallo, Department of Pharma-
ceutical Sciences, School of Pharmacy, Temple University, 3307 N. Broad St.,
Philadelphia, PA 19140. E-mail: email@example.com
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