Assessment of cyclooxygense-2 expression with 99mTc-labeled celebrex.

Abstract

Cyclooxygenase-2 (COX-2) plays an important role in angiogenesis and cancer progression. Since many tumor cells exhibit COX-2 expression, functional imaging of COX-2 expression using celebrex (CBX, a COX-2 inhibitor) may provide not only a non-invasive, reproducible, quantifiable alternative to biopsies, but it also greatly complements pharmacokinetic studies by correlating clinical responses with biological effects. Moreover, molecular endpoints of anti-COX-2 therapy could also be assessed effectively. This study aimed at measuring uptake of Tc-EC-CBX in COX-2 expression in tumor-bearing animal models. In vitro Western blot analysis and cellular uptake assays were used to examine the feasibility of using Tc-EC-CBX to measure COX-2 activity. Tissue distribution studies of Tc-EC-CBX were evaluated in tumor-bearing rodents at 0.5-4 h. Dosimetric absorption was then estimated. Planar scintigraphy was performed in mice, rats and rabbits bearing tumors. In vitro cellular uptake indicated that cells with higher COX-2 expression (A549 and 13762) had higher uptake of Tc-EC-CBX than lower COX-2 expression (H226). In vivo biodistribution of Tc-EC-CBX in tumor-bearing rodents showed increased tumor:tissue ratios as a function of time. In vitro and biodistribution studies demonstrated the possibility of using Tc-EC-CBX to assess COX-2 expression. Planar images confirmed that the tumors could be visualized with Tc-EC-CBX from 0.5 to 4 h in tumor-bearing animal models. We conclude that Tc-EC-CBX may be useful to assess tumor COX-2 expression. This may be useful in the future for selecting patients for treatment with anti-COX-2 agents.

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Preclinical report255
Assessment of cyclooxygense-2 expression with
99mTc-labeled celebrex
David J. Yanga, Jerry Bryanta, Joe Y. Changb, Richard Mendeza,
Chang-Sok Oha, Dong-Fang Yua, Megumi Itoa, Ali Azhdariniaa,
Sahar Kohanima, E. Edmund Kima, Edward Lincand Donald A. Podoloffa
Cyclooxygenase-2 (COX-2) plays an important role in
angiogenesis and cancer progression. Since many tumor
cells exhibit COX-2 expression, functional imaging of COX-
2 expression using celebrex (CBX, a COX-2 inhibitor) may
provide not only a non-invasive, reproducible, quantifiable
alternative to biopsies, but it also greatly complements
pharmacokinetic studies by correlating clinical responses
with biological effects. Moreover, molecular endpoints of
anti-COX-2 therapy could also be assessed effectively. This
study aimed at measuring uptake of99mTc-EC–CBX in
COX-2 expression in tumor-bearing animal models. In vitro
Western blot analysis and cellular uptake assays were used
to examine the feasibility of using99mTc-EC–CBX to
measure COX-2 activity. Tissue distribution studies of
99mTc-EC–CBX were evaluated in tumor-bearing rodents at
0.5–4h. Dosimetric absorption was then estimated. Planar
scintigraphy was performed in mice, rats and rabbits
bearing tumors. In vitro cellular uptake indicated that cells
with higher COX-2 expression (A549 and 13762) had
higher uptake of99mTc-EC–CBX than lower COX-2
expression (H226). In vivo biodistribution of99mTc-EC–CBX
in tumor-bearing rodents showed increased tumor:tissue
ratios as a function of time. In vitro and biodistribution
studies demonstrated the possibility of using99mTc-EC–
CBX to assess COX-2 expression. Planar images
confirmed that the tumors could be visualized with
99mTc-EC–CBX from 0.5 to 4h in tumor-bearing animal
models. We conclude that99mTc-EC–CBX may be useful to
assess tumor COX-2 expression. This may be useful in the
future for selecting patients for treatment with anti-COX-2
agents. Anti-Cancer Drugs 15:255–263? c 2004 Lippincott
Williams & Wilkins.
Anti-Cancer Drugs 2004, 15:255–263
Keywords: COX-2 imaging,99mTc-EC–celebrex
Divisions ofaDiagnostic Imaging,bRadiation Oncology andcCancer Medicine,
University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.
Correspondence to D. J. Yang, Department of Nuclear Medicine, University of
Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX
77030, USA.
Tel: +1 713 794-1053; fax: +1 713 794-5456;
e-mail: dyang@di.mdacc.tmc.edu
Received 8 November 2003 Accepted 24 November 2003
Introduction
Epidemiological studies have shown that non-steroidal
anti-inflammatory drugs (NSAIDs) such as aspirin
significantly reduce the risk of colorectal, esophageal,
gastric, lung and breast cancers. NSAIDs inhibit pros-
taglandinendoperoxide
(COX)] activity. This enzyme introduces two molecules
of O2into arachidonic acid to form prostaglandin (PG)
G2, which is then reduced to PGH2. PGH2is converted
to PGE2, PGF2a, PGI2and thromboxanes by separate
enzymes [1–3]. PGs are critical mediators of physiologic
processes and inflammation. They are produced by two
different isoforms of the COX enzyme, i.e. COX-1 and
COX-2. In particular, COX-2 was demonstrated to be
crucial for PG synthesis in inflammation, and its
concentrations are elevated in the epithelial cells within
human colorectal, esophageal, head and neck, and lung
cancers. Although COX-1 is considered a housekeeping
gene in most tissues, COX-1 is constitutively expressed
in most tissues, with highest levels found in the stomach,
platelets, renal tubules and liver. COX-1 produces PGs
synthase[cyclooxygenase
necessary for normal physiologic functions, e.g. maintain-
ing the integrity of the gastrointestinal tract and platelet
function. The COX-1 gene may also be important in
temperature regulation and in mediating the pyresis
that occurs in the absence of infection [4,5]. In contrast,
COX-2 is not expressed in normal tissues; however,
it is greatly induced during inflammation or tumorigen-
esis [6].
Specific COX-2 inhibitor such as celecoxib [celebrex
(CBX)] spare COX-1 inhibition associated with NSAIDs
which induced peptic ulcer disease, reduced platelet
function and renal tubule toxicity. Celecoxib, a specific
COX-2 inhibitor, was approved for osteoarthritis or
rheumatoid arthritis based on its potent anti-inflamma-
tory activity and favorable toxicity profile with a reduced
incidence of peptic ulcer [7]. Inhibition of COX-2 by
celecoxib delays tumor growth and metastasis in xeno-
graft tumor models as well as suppresses basic fibroblast
growth factor 2-induced neovascularization of the rodent
cornea. In addition, celecoxib, given daily in the diet,
0959-4973 ? c 2004 Lippincott Williams & Wilkins
DOI: 10.1097/01.cad.0000119732.04668.6d
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Page 2

significantly inhibited the induction of rat mammary
tumors by 7,12-dimethylbenz[a]anthracene [7]. These
results indicate that celecoxib has significant antineo-
plastic activity. Non-invasive molecular imaging of COX-2
expression in vivo would be useful to assess clinical
endpoints in validating the COX-2 response with anti-
COX-2-based clinical studies.
Due to favorable physical characteristics as well as
extremely low cost,99mTc has been preferred for labeling
radiopharmaceuticals. Several compounds have been
labeled with
using
chelates[8,9].Bis-aminoethanethiol
ligands, also called diaminodithiol compounds, are known
to form very stable Tc(V)O complexes on the basis of
efficient bindingofthe
twothiolsulfurandtwo
99mTc-L,L-ethylenedicysteine (99mTc-EC) is a recent
and successful example of N2S2 chelates [10,11].
EC can be labeled with99mTc easily and efficiently with
high radiochemical purity and stability and is excreted
through the kidneys by active tubular transport [12,13].
A series of
imaging in oncology have been previously reported
[14–21]. EC–agent conjugates
structural alteration. In this paper, tumor uptake of
99mTc-EC–celebrex in various cell lines, and biodistribu-
tion and planar imaging in tumor-bearing animal models
are presented.
99mTc nitrogen and
tetradentate
sulfur
oxotechnetium
amine
groupto
nitrogenatoms.
99mTc-EC–agent conjugates for functional
provide minimal
Materials and methods
Chemicals and analysis
Mass spectral analyses were conducted at the University
of Texas Health Science Center (Houston, TX). NMR
spectra were recorded on a Bruker 500 Spectrometer. The
mass data were obtained by fast atom bombardment on a
Kratos MS 50 instrument. N-hydroxysulfosuccinimide
(Sulfo-NHS) and 1-ethyl-3-(3-dimethylaminopropyl) car-
bodiimide–HCl (EDC) were purchased from Pierce
(Rockford, IL). All other chemicals were purchased from
Aldrich (Milwaukee, WI). Celebrex capsules (Pfizer, New
York, NY) were obtained from M. D. Anderson Hospital
Pharmacy. [18F]FDG was purchased from PET NET
(Houston, TX). Silica gel-coated thin-layer chromatogra-
phy (TLC) plates were purchased from Whatman
(Clifton, NJ).
Synthesis of EC
EC was prepared in a two-step synthesis according to the
previously described methods [10,11]. Briefly, cysteine–
HCl (41.52g) was dissolved in water (106ml). To this,
formaldehyde was added (26.1ml) and the reaction
mixture was stirred overnight at room temperature.
Pyridine (26.6ml) was then added and the precipitate
formed. The crystals were separated and washed with
ethanol (54ml) for 25min at room temperature, then
filtered with a Buchner funnel. The crystals were
triturated with petroleum ether (150ml), again filtered
and then lyophilized for 3 days. The precursor,
thiazolidine-4-carboxylic acid (m.p. 1951C, reported
196–1971C), was used for synthesis of EC. The precursor
(22g) was dissolved in liquid ammonia (200ml) and
refluxed. Sodium metal was added until a persistent blue
color appeared. Ammonium chloride was added to the
blue solution and then the solvents were evaporated
to dryness. The residue was dissolved in water (200ml)
and the pH was adjusted to 2. A precipitate formed, and
was filtered and washed with water (500ml). The
solid (EC) was dried in a calcium chloride dessicator
and weighed 10.7g (48.2% yield, m.p. 2471C, reported
251–2531C). The structure was confirmed by1H-NMR
andfast-atombombardment
(FAB-MS).
L-
mass spectroscopy
Synthesis of ethylamino COX-2 (EA–CBX)
N-4-(5-p-tolyl-3-trifluoromethyl-pyrazol-1-yl)benzenesul-
fonylamide (CBX) was extracted from celebrex capsules.
Briefly, whole capsule contents were suspended in
chloroform. The mixture was filtered and the chloroform
layer was evaporated to dryness, yielded crude CBX
product. CBX (114.4mg, 0.3mmol) was dissolved in
chloroform (2ml). Tothis
(0.3mmol in chloroform 0.5ml) and ethyl isocyanatoace-
tate 33.7ml (0.3mmol in chloroform 0.5ml) were
added. The reaction was stirred at room temperature
for 6h. The solvent was evaporated in vacuo. The product
was isolated from a silica gel-packed column using
chloroform/methanol as an eluant. The yield of the ester
form of CBX (compound I) was 135mg (88.1%). The
synthetic scheme is shown in Figure 1. NMR spectra data
of compound I is shown in Figure 2. Compound I
(102mg, 0.2mmol) was then dissolved in 2ml of
methanol and ethylene diamine (72.9ml) was added.
The reaction was stirred at room temperature for
24h. The product was isolated from silica gel-packed
column using chloroform:methanol as an eluant. The
desired EA–CBX (compound II) was isolated (91mg,
86.7% yield). NMR spectra data of compound II is shown
Figure 3.
solution,DBU 44.9ml
Synthesis of EC–ethylamino CBX conjugate (EC–CBX)
To dissolve EC, NaOH (1 N, 0.6ml) was added to a
stirred solution of EC (42.3 ma, 0.15mmol) in water
(3ml). To this colorless solution, Sulfo–NHS (65.1mg,
0.3mmol) and EDC (57.5mg, 0.3mmol) were added. EA-
CBX (78.6mg, 0.15mmol) was then added. The mixture
was stirred at room temperature for 24h. The mixture
was dialyzed for 48h using a Spectra/POR molecular
porous membrane with molecule cut–off at 500 (Spec-
trum Medical Industries, Houston, TX). After dialysis,
the product was dried under lyophilization. The product
weighed 87.5mg (yield 75%). NMR spectra data of EC–
CBX is shown in Figure 4.
256
Anti-Cancer Drugs
2004, Vol 15 No 3
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Page 3

Radiolabeling of EC–CBX with99mTc
Radiosynthesis of
adding required amount of [99mTc]pertechnetate into a
home–made kit containing the lyophilized residue of EC–
CBX (5mg), SnCl2 (100mg), Na2HPO4 (13.5mg),
ascorbic acid (0.5mg), glutamic acid (2mg) and EC
(0.5mg). Final pH of the preparation was 7.4. Radio-
chemical purity was determined by TLC (ITLC SG;
Gelman Sciences, Ann Arbor, MI) eluted with, respec-
tively, ammonium acetate (1M in water):methanol (4:1).
From radio–TLC (Bioscan, Washington, DC) analysis, the
radiochemical purity was >95%.
99mTc–EC–CBX was achieved by
In vitro cellular uptake studies
To evaluate if the uptake of99mTc-EC–CBX correlates to
COX-2 expression, in vitro cellular uptake assay was
conducted using high, intermediate and low COX-2-
expressing cancer cell lines 13762 (breast), A549 (lung)
and H226 (lung), respectively. H226 was known to have
lower COX-2 expression than A549 [22,23]. However,
little was known about 13762 rat-driven breast tumor
cells, thus Western blot analysis was performed on 13762
and A549 (positive control) cancer cells. Cancer cells
were treated with celebrex (10mg) and then lysed with
buffer containing 20% SDS in dimethyl formamide/H2O,
pH 4.7, at 371C. The supernatants were cleared by
centrifugation and the protein concentrations were
measured by the Lowry method. Equal amounts of
protein were subjected to immunoprecipitation in the
presence of mouse anti-human COX-2 antibody (Santa
Cruz Biotechnology, San Diego, CA) for 2h at 41C,
followed by incubation with immobilized Protein G Plus/
Protein A–agarose beads (Oncogene Research Products,
Cambridge, MA) overnight at 41C. For Western immuno-
blotting, the immunoprecipitates or equal amounts of
crude extract were boiled in Laemmli SDS sample buffer,
resolved by SDS–PAGE, transferred to nitrocellulose and
probed with mouse antiphosphorylation primary antibody
(Upstate Biotechnology, Lake Placid, NY) at 41C over-
night. After the blots were incubated for another 1h at
Fig. 1
S
O
H2N
N
CF3
O
DBU/CHCl3
+
EtO
NCO
O
EDC/S-NHS
+
S
O
N
CF3
O
EtO
O
H
N
O
H
N
H2N
NH2
S
O
N
CF3
O
N
H
O
H
N
O
H
N
H2N
MeOH
H2O
NH HN
HSSH
OO
HO
OH
NHHN
HSSH
OO
HO
S
O
N
CF3
O
H
N
O
N
H
O
N
H
N
H
(I)
(II)
(III)
N
N
N
N
Synthetic scheme of EC–CBX.
Assessment of COX-2 expression with99mTc-labeled celebrex Yang et al.
257
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Page 4

room temperature with horseradish peroxidase-labeled
anti-mouse secondary antibody (Amersham Life Science,
Arlington Heights, IL), signals were detected by the
enhanced chemiluminescence assay (Amersham Life
Science) according to the manufacturer’s instructions.
Three cancer cell lines (13762, A549 and H226) with
different COX-2 expression and activities were used to
study the effect of cellular uptake after CBX induction at
various doses. All cell lines were maintained in culture
with Dulbecco’s modified Eagle medium supplemented
with 10% fetal calf serum, glutamine and antibiotics, in a
humidified atmosphere with 5% CO2and 95% air. The
cells were treated with celebrex (0.25–5mg/well) for
30min, followed by adding 4mCi (0.148 MBq) of99mTc-
EC–CBX (0.1mg/well) to each well. Cells were incu-
bated with radiotracers at 371C at 0.5–4h. After
incubation, cells were washed with ice-cold phosphate-
buffered saline (PBS) twice and trypsinized with 0.5ml
of trypsin solution. Then cells were collected and the
radioactivity was measured by a g-counter. Data are
expressed in mean±SD percent uptake ratio of three
measurements.
In vivo biodistribution and dosimetry of99mTc-EC–CBX
in tumor-bearing rats
The animal experiments were approved by the University
of Texas M. D. Anderson Institutional Animal Care and
Use Committee. Female Fischer 344 rats (150±25g)
(Harlan Sprague-Dawley, Indianapolis, IN) were inocu-
lated s.c. with 0.1ml of mammary tumor cells from the
13762 tumor cell line suspension (106cells/rat, a tumor
cell line specific to Fischer rats) into the hind legs using
25-gauge needles. Studies were performed 14–17 days
after implantation when tumors reached approximately
Fig. 2
Observed (p.p.m.)
7.86 (2H, d, J=8.6Hz)
7.33 (2H, d, J=8.6Hz)
7.03 (2H, d, J=8.2Hz)
6.98 (2H, d, J=8.2Hz)
6.62 (1H, s)
4.41 (2H, s)
4.01 (2H, q, J=7.1Hz)
2.22 (3H, s)
1.11 (3H, t, J=7.1Hz)
g
h
a
I
S
N
CF3
O
O
O
O
N
H
O
N
H
a
b
b
c
c
d
e
e
f
f
g
h
i
N
d
b
c
e
f
1H-NMR spectra data of ester analog of celebrex (compound I).
Fig. 3
Observed (p.p.m.)
7.83 (2H, d, J=8.6Hz)
7.27 (2H, d, J=8.6Hz)
7.03_7.09 (4H, m)
6.68 (1H, s)
3.56 (2H, br)
3.38 (2H, br)
2.94 (2H, br)
2.26 (3H, s)
f
e
c,b
d
g
h
I
a
S
N
CF3
O
O
H
N
O
N
H
O
N
H
H2N
a
b
b
c
c
d
e
e
f
f
g
h
i
N
1H-NMR spectra data of ethylenediamine analog of celebrex
(compound II).
Fig. 4
Observed (p.p.m.)
7.80 (2H, d, J=8.6Hz)
7.33 (2H, d, J=8.6Hz)
7.01_7.11 (4H, m)
6.90 (1H, s)
4.15 (2H, s)
3.55_3.65 (2H, m)
3.43_3.53 (4H, m)
2.99 (2H, d, J=7.0Hz)
2.94 (2H, d, J=9.0Hz)
2.41 (4H, d, J=3.0Hz)
2.16 (3H, s)
f
e
c,b
d
g
k, n
h, i
j
o
l, m
a
NHHN
HSSH
OO
HO
k
lm
n
o
S
O
N
CF3
O
H
N
O
N
H
O
N
H
N
H
a
b
b
c
c
d
e
e
f
f
g
h
i
j
N
1H-NMR spectra data of EC–CBX.
258
Anti-Cancer Drugs
2004, Vol 15 No 3
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Page 5

1cm in diameter. Rats were anesthetized with ketamine
(10–15mg/rat, i.p.) before each procedure. Each animal
was injected i.v. with 370–550kBq of99mTc-EC–CBX or
99mTc-EC (n=3/time point). The injected mass of
99mTc-EC–CBX was 100mg per rat. At 0.5, 2 and 4h
following administration of the radiotracers, the rats were
sacrificed and the selected tissues were excised, weighed
and counted for radioactivity by using a g-counter
(Packard Instruments, Downers Grove, IL). The biodis-
tribution of tracer in each sample was calculated as
percentage of the injected dose per gram of tissue wet
weight (%ID/g). Tumor/non-tumor tissue count density
ratios were calculated from the corresponding %ID/g
values. Student’s t-test was used to assess the signifi-
cance of differences between groups.
Dosimetric calculations were performed using in-house
curve-fitting software. Time–activity curves were gener-
ated for each organ. Analytic integration of the curves was
used to determine the area under the curve (AUC) which
was then divided by injected dose to yield the residence
time of each organ. Residence times were then used to
calculate target organ absorbed radiation doses based on
the MIRD methodology for the normal adult male using
the MIRDose 3.1 soft ware package [24].
Scintigraphic imaging studies
Scintigraphic imaging studies was performed in rats and
rabbits animal models. For rats, female Fischer 344 rats
(150±25g) (Harlan Sprague-Dawley, Indianapolis, IN)
were inoculated s.c. with 0.1ml of mammary tumor cells
from the 13762 tumor cell line suspension (106cells/rat, a
tumor cell line specific to Fischer rats) into the hind legs.
For rabbits, New Zealand white rabbits were inoculated
with VX-2 tumor mass (rabbit-driven squamaceous
mammary tumor). For mice, athymic nude mice were
inoculated s.c. with 0.1ml of tumor cells from the A549
and H226 tumor cell line suspension (2? 106cells/
mouse) into the hind legs. Studies performed 12–17 days
after implantation when tumors reached approximately
1cm in diameter. The animals were administered with
99mTc-EC–CBX or99mTc-EC (0.3 mCi/rat; 1 mCi/rabbit,
0.1 mCi/mouse, n=3, i.v.) and the scintigraphic images,
using a g camera equipped with a low-energy, parallel-
hole collimator, were obtained at 0.5–4h after i.v.
injection of the radiotracers. To assess locoregional
radioactivity, a small hand-held g camera (eZ-SCOPE;
Anzai Medical, Tokyo, Japan) was also used with a low-
energy parallel-hole collimator (high sensitive type). The
field of view for eZ-SCOPE is 32mm?32mm with 16?
16 pixels, of which the intrinsic spatial resolution is 2mm
and sensitivity is 770000c.p.s./MBq. To demonstrate
whether inducible COX-2 expression could be imaged by
99mTc-EC–CBX, a group of nude mice bearing human
lung tumors (A549 and H226) was pretreated with CBX
(1mg, i.p.) and the images were acquired at 2h after i.v.
injection of99mTc-EC–CBX. Computer-outlined regions
of interest (ROI) (counts per pixel) of the tumor lesion
site and symmetric normal muscle site were used to
determine tumor:background count density ratios. The
ratios were used to compare dynamic tumor uptake pre-
and post-treatment of CBX.
Acute toxicity studies
Swiss-Webster mice (17-20g, male and female, 12 each)
were purchased from Harlan Sprague-Dawley (Indiana-
polis, IN) and housed at all times in animal rooms in the
M. D. Anderson Cancer Center with controlled tempera-
ture (21–231C), humidity (50–55%) and light period (on
at 0600h, off at 1800h). Following 2 control days, mice
were administered an i.v. injection (tail vein) of saline and
EC–CBX (20, 40 and 100mg/kg) (n=3/dosage), and
body weight was measured daily. Control groups were
administered saline only. EC–CBX (5mg) was dissolved
in 1ml of water. The final concentration used for animal
studies was 5mg/ml. The injection volume was 0.08–
0.4ml per mouse.
Results
In vitro cellular uptake studies
Western blot analysis indicated that 13762 and A549
cancer cells showed positive COX-2 expression (Fig. 5).
There was a significant increase in uptake after pretreat-
ing cells with CBX. At higher dose (5mg CBX/well), the
uptake was decreased. The findings suggest COX-2
enzyme might be induced by pretreatment with CBX
(Figs 6–8). In addition, higher COX-2 expression cell
lines (A549 and 13762) had higher uptake than lower
COX-2 expression cell line (H226) [22].
Biodistribution and dosimetric estimates
Biodistribution of99mTc-EC–CBX in tumor-bearing rats
showed increased tumor:tissue count density ratios as a
function of time compared to99mTc-EC (Tables 1 and 2).
Based upon preclinical biodistribution studies, dosimetry
was estimated from MIRDose (Table 3). If the subject
does not involve other radiation exposure, whole-body,
liver and effective dose equivalent for the proposed single
Fig. 5
Western immunoblotting assays indicated that A549 and 13762 cells
showed positive COX-2 expression.
Assessment of COX-2 expression with99mTc-labeled celebrex Yang et al.
259
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