-
3618
Therapeutics, Targets, and Chemical Biology
Cancer
Research
Selective Visualization of Cyclooxygenase-2 in Inflammationand
Cancer by Targeted Fluorescent Imaging Agents
Md. Jashim Uddin1, Brenda C. Crews1, Anna L. Blobaum1, Philip J.
Kingsley1, D. Lee Gorden2, J. Oliver McIntyre2,Lynn M. Matrisian2,
Kotha Subbaramaiah4, Andrew J. Dannenberg4, David W. Piston3, and
Lawrence J. Marnett1
Abstract
Authors'
AResearch,Vanderbilt2Departme3DepartmeAstronomTennesseeCornell
Uni
Note: SupResearch O
Corresponical Center615-343-73
doi: 10.115
©2010 Am
Cancer R
Down
Effective diagnosis of inflammation and cancer by molecular
imaging is challenging because of interferencefrom nonselective
accumulation of the contrast agents in normal tissues. Here, we
report a series of novelfluorescence imaging agents that
efficiently target cyclooxygenase-2 (COX-2), which is normally
absent fromcells, but is found at high levels in inflammatory
lesions and in many premalignant and malignant tumors.After either
i.p. or i.v. injection, these reagents become highly enriched in
inflamed or tumor tissue comparedwith normal tissue and this
accumulation provides sufficient signal for in vivo fluorescence
imaging. Further,we show that only the intact parent compound is
found in the region of interest. COX-2–specific delivery
wasunambiguously confirmed using animals bearing targeted deletions
of COX-2 and by blocking the COX-2active site with high-affinity
inhibitors in both in vitro and in vivo models. Because of their
high specificity,contrast, and detectability, these fluorocoxibs
are ideal candidates for detection of inflammatory lesionsor
early-stage COX-2–expressing human cancers, such as those in the
esophagus, oropharynx, and colon.Cancer Res; 70(9); 3618–27. ©2010
AACR.
Introduction
Molecular imaging presents exciting opportunities for
theselective detection of specific cell populations, such as
thosebearing markers of disease (1, 2). Cyclooxygenase-2 (COX-2)is
an attractive target for molecular imaging because it isexpressed
in only a few normal tissues and is greatly upre-gulated in
inflamed tissues as well as many premalignantand malignant tumors
(3, 4). COX-2 is an importantcontributor to the etiology of
inflammation and canceras illustrated by the efficacy of
COX-2–selective inhibitorsas anti-inflammatory agents, cancer
preventive agents, andadjuvant cancer therapeutic agents (5). The
importance ofCOX-2 in tumor progression has been thoroughly
documen-ted in the esophagus and colon where COX-2 is detected
inpremalignant lesions and its levels seem to increase duringtumor
progression (6–8). The importance of COX-2 insurvival and response
to therapy has been elegantly shown
ffiliations: 1A.B. Hancock, Jr. Memorial Laboratory for
CancerDepartments of Biochemistry, Chemistry, and
Pharmacology,Institute of Chemical Biology, Center in Molecular
Toxicology;nt of Cancer Biology, Vanderbilt-Ingram Cancer Center;
andnts of Molecular Physiology and Biophysics and Physics andy,
Vanderbilt University School of Medicine, Nashville,; and
4Department of Medicine, Weill Medical College ofversity, New York,
New York
plementary data for this article are available at Cancernline
(http://cancerres.aacrjournals.org/).
ding Author: Lawrence J. Marnett, Vanderbilt University Med-,
23rd Avenue and Pierce, Nashville TN, 37232-0146. Phone:29; Fax:
615-343-7534; E-mail: [email protected].
8/0008-5472.CAN-09-2664
erican Association for Cancer Research.
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by Edelman and colleagues (9) who reported that non–smallcell
lung cancer patients expressing high levels of COX-2 intheir tumors
have reduced survival compared with patientsexpressing low levels
of COX-2. Patients with high tumor ex-pression of COX-2 benefit
from the combination of carbopla-tin and gemcitabine plus the COX-2
inhibitor, celecoxib,whereas patients with low expression exhibit a
poorerresponse to carboplatin/gemcitabine/celecoxib than
tocarboplatin/gemcitabine alone (9).Positron emission tomography or
single-photon emission
computed tomography imaging agents (18F-, 11C-, or 123I-labeled
COX-2 inhibitors) have been described for nuclearimaging (10–17).
These have all been based on the diaryl-heterocycle structural
class analogous to celecoxib androfecoxib. Although selective
uptake into macrophages ortumor cells expressing COX-2 has been
shown in vitro forsome compounds, such selectivity has not been
rigorouslyshown in vivo and significant nonspecific binding has
beenobserved (18). Thus, despite recognition of the potential
ofCOX-2–targeted imaging agents, in vivo proof-of-conceptfor this
strategy is lacking.Fluorescent COX-2 inhibitors are attractive
candidates as
targeted imaging agents. Such compounds have the advan-tage that
each molecule bears the fluorescent tag and thecompounds are
nonradioactive and stable. Thus, they canbe used conveniently for
cellular imaging, animal imaging,and clinical imaging of tissues in
which topical or endolumi-nal illumination is possible (e.g.,
esophagus, colon, and upperairway through endoscopy, colonoscopy,
and bronchoscopy,respectively). Prior work from our laboratory
showed thatfluorescent COX-2 inhibitors can be useful
biochemicalprobes of protein binding but these earlier compounds
were
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COX-2 Imaging
neither potent inhibitors of COX-2 nor did they possess
ap-propriate fluorescence properties to be useful for cellular orin
vivo imaging (19). Thus, we initiated a program to designand
synthesize a series of fluorescent COX-2 inhibitors thatcould be
used for these applications. The design strategy forcandidate
development was based on our prior discovery thatamide derivatives
of the nonselective COX inhibitor, indo-methacin, are selective
COX-2 inhibitors. Many compoundswere synthesized and screened for
COX-2 inhibition in vitroand in intact cells. Then, the most
promising compoundswere evaluated as imaging agents in intact cells
and in animalmodels of inflammation and cancer. We describe herein
theoptimized candidates, their selective uptake by COX-2–expressing
cells and tumors, and genetic and pharmacologicvalidation that
their in vivo target is COX-2.
Materials and Methods
Synthesis and characterization of all compounds isdescribed in
Supplementary Data.Inhibition assay using purified COX-1 and
COX-2.
Cyclooxygenase activity of ovine COX-1 or human COX-2was assayed
by a method that quantifies the conversion of[1-14C]arachidonic
acid to [1-14C]prostaglandin products. Re-action mixtures of 200 μL
consisted of hematin-reconstitutedprotein in 100 mmol/L Tris-HCl
(pH 8.0), 500 μmol/L phenol,and [1-14C]arachidonic acid (50 μmol/L,
approximately 55–57 mCi/mmol, Perkin-Elmer). For the time-dependent
inhi-bition assay, hematin-reconstituted COX-1 (44 nmol/L) orCOX-2
(66 nmol/L) was preincubated at 25°C for 17 minutesand 37°C for 3
minutes with varying inhibitor concentrationsin DMSO followed by
the addition of [1-14C]arachidonic acid(50 μmol/L) for 30 seconds
at 37°C. Reactions were termi-nated by solvent extraction in
Et2O/CH3OH/1 mol/L citrate(pH 4.0; 30:4:1). The phases were
separated by centrifugationat 2,000 g for 2 minutes and the organic
phase was spottedon a TLC plate (EMD Kieselgel 60, VWR). The plate
wasdeveloped in EtOAc/CH2Cl2/glacial AcOH (75:25:1) at
4°C.Radiolabeled products were quantified with a
radioactivityscanner (Bioscan, Inc.). The percentage of total
productsobserved at different inhibitor concentrations was
dividedby the percentage of products observed for protein
samplespreincubated for the same time with DMSO.Cell culture and in
vitro intact cell metabolism assay.
HCT116, ATCC CCL-247 human colorectal carcinoma cells,passage 8
to 18, Mycoplasma negative by a PCR detec-tion method (Sigma
VenorGem) were grown in DMEM(Invitrogen/Life Technologies) + 10%
fetal bovine serum(FBS; Atlas) to 70% confluence. RAW264.7, ATCC
TIB-71murine macrophage-like cells, passage number 8 to 15,
Myco-plasma negative by a PCR detection method were grownin DMEM +
10% heat-inactivated FBS to 40% confluence(six-well plates,
Sarstedt) and activated for 7 hours in 2 mLserum-free DMEM with 200
ng/mL lipopolysaccharide (LPS;Calbiochem) and 10 μ/mL IFN γ
(Calbiochem). Human 1483head and neck squamous cell carcinoma
(HNSCC) cells (20),derived, characterized, and provided by Dr.
Peter Sacks (NewYork University School of Dentistry, New York, NY),
were
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grown at passage 8 to 18, Mycoplasma negative by a PCRdetection
method, in DMEM/F12 + 10% FBS + Antibiotic/Antimycotic in six-well
plates to 60% confluence. Serum-freemedium (2 mL) was added and the
cells were treated with in-hibitor dissolved in DMSO (0–5 μmol/L,
final concentration)for 30 minutes at 37°C followed by the addition
of [1-14C]-arachidonic acid (10 μmol/L, ∼55 mCi/mmol) for 20
minutesat 37°C. Reactions were terminated by solvent extraction
inEt2O/CH3OH/1 mol/L citrate (pH 4.0; 30:4:1) and the organicphase
was spotted on a 20 × 20 cm TLC plate (EMD Kieselgel60, VWR). The
plate was developed in EtOAc/CH2Cl2/glacialAcOH (75:25:1) and
radiolabeled products were quantified witha radioactivity scanner
(Bioscan, Inc.). The percentage of totalproducts observed at
different inhibitor concentrations wasdivided by the percentage of
products observed for cellspreincubated with DMSO.Fluorescence
microscopy of RAW264.7 cells or 1483
HNSCC cells. RAW264.7 cells were plated on 35-mm MatTekdishes
(MatTek Corp.) such that the cells were 40% confluentand human 1483
HNSCC cells were 60% confluent on the dayof the experiment. The
RAW264.7 cells were activated for6 hours in serum-free DMEM with
200 ng/mL LPS and10 μ/mL IFN γ. Both cell lines were incubated in
2.0 mLHBSS/Tyrode's buffer with 200 nmol/L compound 1, 2, or 3for
30 minutes at 37°C. To block the COX-2 active site, thecells were
preincubated with 10 μmol/L indomethacin or5 μmol/L celecoxib for
20 minutes before the addition ofcompound 1 or 2. The cells were
then washed brieflythrice and incubated in HBSS/Tyrode's buffer for
30 minutesat 37°C. Following the required washout period, the
cellswere imaged in 2.0 mL fresh HBSS/Tyrode's buffer on aZeiss
Axiovert 25 Microscope with the propidium iodidefilter (0.5–1.0
second exposure, gain of 2). All treatmentswere performed in
duplicate dishes in at least three separateexperiments.Confocal
microscopy of 1483 cells treated with
compound 2/mitotrackerGR. 1483 HNSCC were plated inMatTek dishes
(MatTek Corp.) and grown to 60% to 70% con-fluence for 48 hours.
DMSO or compound 2 (100 nmol/L) wasadded to each dish containing
2.0mLHBSS/Tyrode's buffer for30 minutes at 37°C. After four quick
HBSS washes, cells wereincubated for 30 minutes in 2.0 mL
HBSS/Tyrode's buffer andimaged with a Zeiss LSM510 confocal
microscope using a 63 ×1.4 NA plan-Apochromat objective lens. To
visualize cellularmitochondria, 100 nmol/L Mitotracker GR was added
for15 minutes at 37°C followed by three quick washes before
im-aging. Four hundred eighty-eight nanometer excitation wereused
to image Mitotracker GR through a 500- to 530-nm band-pass filter
and compound 2 was imaged using 532 nm excita-tion and collection
through a 565- to 615-nm bandpass filter.The pinhole was set to 1
Airy unit and images were collectedthroughout the focus of the
cells. To assure a full sampling ofthe perinuclear region, analysis
was performed on the opticalsections through the middle of the
nucleus.In vivo imaging of COX-2 in inflammation. Carrageenan
(50 μL 1% in sterile saline) was injected in the rear
leftfootpad of female C57BL/6 mice, followed by compound1 or 2 (1
mg/kg, i.p.) at 24 hours postcarrageenan. Animals
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Uddin et al.
3620
were imaged 3 hours later in a Xenogen IVIS 200 (DsRed fil-ter,
1.5 cm depth, 1 s). For comparison, animals also weredosed with
compound 3, which does not inhibit COX-2. To testfurther the
molecular target for compound 2, parallel experi-ments were
performed using COX-2 (−/−) mice. Experimentsalso were performed in
which compound 1 was administeredto the same animals by repetitive
i.p. injection on days 1, 3, 5,and 7 to monitor the time course of
compound uptake follow-ing carrageenan induction of
inflammation.Establishment of xenografts in nude mice. Female
nude
mice, NU-Fox1nu, were purchased at 6 to 7 weeks of agefrom
Charles River Laboratories. Human 1483 HNSCC cellsand HCT116
colorectal carcinoma cells were trypsinizedand resuspended in cold
PBS containing 30% Matrigel suchthat 1 × 106 cells in 100 μL were
injected s.c. on the left flank.The HCT116 or 1483 xenografts
required only 2 to 3 weeks ofgrowth.In vivo imaging of nude mice
with xenografts. Female
nude mice bearing medium-sized HCT116 or 1483 xenografttumors on
the left flank were dosed by i.p. injection with2 mg/kg compound 2
or by retro-orbital injection with1 mg/kg compound 2. The animals
were lightly anesthetizedwith 2% isoflurane for fluorescence
imaging in the XenogenIVIS 200 with the DSRed filter at 1.5-cm
depth and 1-secondexposure (f2). For the COX-2 active site–blocking
experi-ments, nude mice bearing 1483 xenografts were predosedby
i.p. injection with 2 mg/kg indomethacin at 24 hoursand 1 hour
before dosing with compound 2 (2 mg/kg, i.p.).Pharmacokinetics of
candidate compounds. Female
nude mice with medium-sized 1483 HNSCC xenografttumors on the
left flank were injected i.p. with 2 mg/kg com-pound 2. At 0, 0.5,
3, 12, and 24 hours, the mice (n = 4 for eachtime point, duplicate
experiments) were anesthetized withisoflurane. Blood samples were
taken by cardiac punctureinto a heparinized syringe into a 1.5-mL
heparinized tubeon ice, followed by removal of the liver, kidney,
contralateralleg muscle, and xenograft tumor. All organs/tissues
wererinsed briefly in ice-cold PBS, blotted dry, weighed, andsnap
frozen in liquid nitrogen. The blood samples werecentrifuged at 4°C
at 6,000 rpm for 5 minutes and theplasma was transferred to clean
tubes and frozen at −80°C.Compound 2 was extracted by homogenizing
the tissuein 100 mmol/L Tris (pH 7.0) buffer and mixing an ali-quot
of the homogenate with 1.2× volume of acetonitrile.The acetonitrile
was removed and the samples were dried,reconstituted, and analyzed
through reversed-phase high-performance liquid chromatography
(HPLC)-UV using aPhenomenex 10 × 0.2 cm C18 or a Phenomenex 7.5 ×
0.2 cmSynergi Hydro-RP column held at 40°C. The samples
werequantified against a standard curve prepared by addingcompound
2 to tissue homogenates of undosed animalsfollowed by the workup
described. Cochromatography wasperformed with multiple columns and
elution conditions asdescribed in the Supplemental Data.In vivo
imaging of Min mice. C57BL/6 APC-Min mice
maintained on a high-fat (11%) diet for 18 weeks developed20 to
30 intestinal polyps per mouse. Before imaging, Minmice were
anesthetized (2% inhaled isoflurane) for retro-
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orbital injection of compound 2 at 1 mg/kg. At 2 hours
post-injection, the mice were euthanized and the intestines
wereresected, washed with PBS, and fixed in 10% formalin beforeex
vivo imaging by fluorescence dissecting microscopy (ZeissM2Bio; n =
5).
Results
COX-2 is a potentially ideal target for molecular imagingbecause
its active site (and the active site of COX-1) is burieddeep inside
each subunit of the homodimeric protein (21–23).Access to the
active site is controlled by a constrictionthat separates it from a
large opening in the membrane-binding domain that we have termed
the lobby (Supple-mentary Fig. S1). All substrates or inhibitors
bind in the lobbyand then diffuse through the constriction into the
active site(24). The constriction is composed of Tyr-355, Glu-524,
andArg-120 and serves as the binding site for the carboxylic
acidgroup of substrates and certain inhibitors (25). We have
re-ported that neutral derivatives (esters and amides) of
certaincarboxylic acid inhibitors (e.g., indomethacin) bind to
COX-2but not to COX-1 (26). A three-dimensional structure ofCOX-2
complexed to such a conjugate has not been solvedbut structures of
related complexes suggest the indomethacinunit binds in the active
site with the tethered amide, breech-ing the constriction and
projecting into the lobby (22, 27).These structural and functional
analyses provide the designprinciples for the construction of
COX-2–targeted imagingagents.Synthesis of candidate compounds and
cellular imaging.
Three carboxylic acid cores—i.e., indomethacin, a
celecoxibcarboxylic acid derivative, and an indolyl carboxamide
ana-logue of indomethacin—were tethered through a series
ofalkylenediamines, piperazines, polyethylene glycol, or
pheny-lenediamines to a diverse range of fluorophores. The
fluo-rophores attached included dansyl, dabsyl,
coumarin,fluorescein, rhodamine, alexa-fluor, nile blue, cy5, cy7,
nearIR, and IR dyes as well as lanthanide chelators. Nearly200
compounds were synthesized and each conjugate wastested for its
ability to selectively inhibit COX-2 in assaysusing purified
proteins in vitro. Promising molecules weretested for their ability
to inhibit COX-2 in LPS-treatedRAW264.7 macrophages. Preliminary
experiments indicatedthat indomethacin conjugates bound most
tightly and selec-tively to COX-2; therefore, most of the compounds
synthe-sized were derived from this core.Indomethacin conjugates to
dansyl, dabsyl, coumarin,
fluorescein, and rhodamine-derived fluorophores
exhibitedpromising COX-2 inhibition and selectivity both in
vitroand in intact cells. The carboxy-X-rhodamine (6-ROX-
and5-ROX)–based conjugates, 1 and 2, displayed the best ba-lance of
cellular activity and optical properties (λex = 581nm, λemit = 603
nm) and were used for all subsequent experi-ments (Table 1). A
detailed kinetic analysis indicated that1 and 2 require lengthy
preincubations with COX-2 toachieve maximal inhibition but once
bound, they dissociatevery slowly (Supplementary Fig. S2). Thus,
they are slow,tight-binding inhibitors with very low rates of
association
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COX-2 Imaging
ww
Table 1. Biochemical properties of carboxy-X-rhodamine
derivatives
Compound no.
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Structure
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Purified enzyme IC50(μmol/L)
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Cell IC50(μmol/L)
COX-1
COX-2
1
>25 0.83 0.36
2
>25 0.7 0.31
3
>25 >4 ND
4
>25 >4 ND
5
>25 >4 ND
6
0.92 0.21 NT
7
>25 0.14 NT
8
0.05 0.75 0.01
NOTE: Assays were conducted as described in Materials and
Methods. IC50s for inhibition of ovine COX-1 or human
COX-2.Compounds also were tested in intact RAW264.7 macrophages
(Cell IC50).Abbreviations: ND, no inhibition detected up to 5
μmol/L; NT, not tested.
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and dissociation. Compounds 1 and 2 were less potentthan
celecoxib or rofecoxib as inhibitors of COX-2 (Table 1).A negative
control molecule (3) was synthesized thatcontained 6-ROX bound to
indomethacin through a shorterethylenediamine tether, which
eliminated COX-2 inhibition(Table 1).The human head and neck cancer
cell line, 1483, which
expresses high levels of COX-2 (28), exhibited strong
labelingwith compounds 1 or 2 (Fig. 1A). Preincubation of the
cellswith the COX-2–selective inhibitor, celecoxib, prevented
thelabeling of 1483 cells by either compound (Fig. 1B). In all
ofthese in vitro experiments, the labeling seemed to be
intracel-lular, so confocal microscopy was performed to verify the
lo-calization. Incubation of compound 2 with 1483 cells led tothe
perinuclear labeling of membraneous structures that ap-peared to be
endoplasmic reticulum or Golgi (Fig. 1C). Theperinuclear labeling
correlated well to multiple previous re-ports of the intracellular
localization of COX-2 (29–32). Incu-bation of the same cells with
Mitotracker showed that themitochondria did not colocalize with
compound 2 (Fig. 1C).The mouse macrophage–like cell line, RAW
264.7, does not
express COX-2 and exhibited very weak labeling with 1 or 2(e.g.,
Supplementary Fig. S3A), whereas LPS-pretreated cellslabeled more
strongly (Supplementary Fig. S3B). The labelingof the
COX-2–expressing RAW cells by 1 or 2 was preventedby pretreatment
of the cells with indomethacin (Supplemen-tary Fig. S3C) or
celecoxib, which block the COX-2 active site.Importantly, no
labeling was observed when either control orLPS-pretreated RAW
cells were incubated with compound 3,which does not inhibit COX-2
(Supplementary Fig. S3D). Theextent of compound 2 uptake increased
at 4 hours with theappearance of COX-2 protein. A further increase
in uptakewas not observed at 7 hours although there was higherCOX-2
protein as detected by Western blotting (Supplemen-tary Fig. S4).
Comparison of the amount of compound 2taken up at 7 hours to the
amount of COX-2 estimated byWestern blotting in the LPS-treated
cells suggested a stoichi-ometry of binding of 0.90 (Supplementary
Data).Imaging carrageenan–induced inflammation. Com-
pounds 1 and 2 seemed promising based on these in vitroimaging
experiments, so their potential for in vivo imagingwas evaluated
using carrageenan-induced inflammation inthe mouse footpad, human
tumor xenografts in nude mice,and spontaneous tumors arising in
mouse models. Themouse footpad model is well documented for the
role ofCOX-2–derived prostaglandins as a major driving force forthe
acute edema that results 24 hours after carrageenan in-jection into
the paw (33). One of the significant advantages ofthis animal model
of inflammation is the ability to image theinflamed footpad
compared with the noninflamed contra-lateral footpad, which does
not express COX-2. We injectedfemale C57BL/6 mice with 50 μL 1%
carrageenan in the rearleft footpad, followed by compound 1 or 2 (1
mg/kg, i.p.)at 24 hours postcarrageenan. Animals were imaged 3
hourslater in a Xenogen IVIS 200 (DsRed filter, 1.5 cm depth, 1
s).Both compounds 1 and 2 targeted the swollen footpad withan
average 4.5-fold increase in fluorescence over that of
thecontralateral, uninjected footpad (Fig. 2). For comparison,
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Figure 1. Labeling of COX-2–expressing cells by compound 2.
Theexperimental protocols are described inMaterials andMethods. A,
1483HNSCCcells treated with 200 nmol/L compound 2 for 30 min. B,
1483 HNSCCcells pretreated with 5 μmol/L celecoxib for 20 min
before compound 2treatment. C, confocal microscopy of 1483 HNSCC
cells treated with bothmitotrackerGR (blue; mitochondria) and
compound 2 (red; perinuclear).
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COX-2 Imaging
animals also were dosed with compound 3, which doesnot inhibit
COX-2. In Fig. 2A, the left mouse was dosed withcompound 3 and the
right mouse with compound 1. Com-pound 3 yielded minimal
fluorescence in the inflamed pawcompared with the contralateral
paw, whereas compound 1yielded a strong signal in the inflamed paw.
To test furtherthe molecular target of compounds 1 or 2, parallel
expe-riments were performed using mice bearing targeted dele-tions
in COX-2. Figure 2B depicts the fold difference in thecompound
2–derived fluorescence signal in the 24-hourcarrageenan-injected
footpad over the control footpad forwild-type versus COX-2 (−/−)
mice. The COX-2 null miceconsistently showed approximately a 40%
increase in signalin the swollen footpad apparently due to
nonspecific binding.This contrasts with a 400% to 600% increase in
the swollenfootpad in wild-type mice. Finally, experiments were
con-ducted to evaluate the uptake of compound 1 during the
res-olution of inflammation. Following carrageenan
injection,compound 1 was administered i.p. 1, 3, 5, and 7 days
laterand the animals were imaged. Uptake of compound 1 wasmaximal
at 24 hours but declined thereafter (Fig. 2C). At-tempts to
estimate active COX-2 protein by the quantificationof
prostaglandins in paw extracts were unsuccessful becauseof poor
recovery.Imaging COX-2–expressing tumors. The results in the
footpad inflammation model show that COX-2–targetedfluorescent
conjugates are taken up in inflamed paws ofCOX-2–expressing mice
but not in COX-2 null animals. Wenext evaluated the ability of
these compounds to targetCOX-2 in human tumor xenografts. Female
nude mice wereinjected in the left flank with HCT-116 or 1483 cells
and thexenografts were allowed to grow to approximately 750 to1,000
mm3. Animals were dosed by retro-orbital injectionwith compound 2
(1 mg/kg) then lightly anesthetized with2% isoflurane in
preparation for imaging. No fluorescencewas observed during the
first 60 minutes postinjection, butsignal was reproducibly detected
in the COX-2–expressing1483 tumors starting at 3 to 5 hours and
persisted as longas 26 hours postinjection. At 3.5 hours
postinjection, theHCT116 tumor, which does not express COX-2
(34),showed minimal fluorescence (Fig. 3A) whereas the 1483tumor
exhibited bright fluorescence (Fig. 3B). In anothercontrol
experiment, nude mice bearing 1483 xenograftswere treated with the
fluorophore alone, 5-ROX (2 mg/kg, i.p.), which is neither an
inhibitor of COX-2 norCOX-1. No signal from 5-ROX alone accumulated
in the tu-mors at any time point. This result showed that the
fluoro-phore moiety was not responsible for the tumor uptake
ofcompound 2, supporting the conclusion that the difference
inlabeling of 1483 and HCT116 xenografts is due their differen-tial
in COX-2 expression.Nude mice with 1483 xenografts were pretreated
with
either DMSO or indomethacin in DMSO (2 mg/kg, i.p.)
beforecompound 2 dosing (2 mg/kg, i.p.). At 3 hours
postinjection,the DMSO-pretreated mice showed strong fluorescence
intheir tumors (Fig. 3C) compared with weak signals in thetumors of
the indomethacin-pretreated mice (Fig. 3D). Inthe mouse xenograft
model, indomethacin was able to block
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Figure 2. In vivo labeling of COX-2–expression in inflammation
bycompound 1, 2, or 3. A, C57BL/6 mouse with
carrageenan-inducedinflammation in the left foot pad. The left
mouse was dosed with thenegative control molecule 3 (1 mg/kg, i.p.)
and the right mouse wasdosed with compound 1 (1 mg/kg, i.p.). Both
mice were imaged at3 h postinjection. B, fold increase of
fluorescence in inflamed versuscontralateral paw of wild-type (WT)
and COX-2 (−/−) mice at 3 hpostinjection of compound 2 (1 mg/kg,
i.p.; n = 6). RFU, relativefluorescence unit. C, carrageenan was
injected in the rear left footpads offemale C57BL/6 mice, followed
by dosing compound 1 (1 mg/kg i.p.)24 h later. Animals were
reinjected with compound 1 at 3, 5, and7 d postcarrageenan (n = 9).
Mice were imaged at 3 h aftercompound injection. The plot shows the
fold increase of fluorescence inswollen versus contralateral foot
(n = 6).
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92 ± 6% (n = 8) of the COX-2–expressing tumor uptake ofcompound
2.We next investigated whether the COX-2 inhibitory activity
of our imaging probes correlated with their in vivo efficacyin
targeting COX-2–expressing tumors. Nude mice bearing1483 xenografts
were dosed (2 mg/kg, i.p.) with compound 4(no COX inhibition at 3
μmol/L), compound 5 (30% COX-2inhibition at 3 μmol/L), and compound
2 (90% COX-2 inhi-bition at 3 μmol/L; Table 1). At 3.5 hours
postinjection, fluo-rescence from the tumor region was directly
proportionalto the compound potency as a COX-2 inhibitor
(Supplemen-tary Fig. S5).Experiments were conducted to determine
the identity of
the fluorescent material(s) detected in vivo and to monitorthe
time course of its distribution and tissue uptake
followinginjection of compound 2 into nude mice bearing 1483
humantumor xenografts. Extracts of plasma, liver, kidney, tumor,and
adjacent muscle were quantitatively analyzed by HPLCat different
times after i.p. administration of the compound.A single
fluorescent compound was detected in all the ex-tracts, which
coeluted with a standard of compound 2 inmultiple HPLC systems.
This compound displayed an identi-cal mass spectrum to the
unmetabolized parent molecule,compound 2 (Fig. 4A). The time
courses of uptake and dis-tribution of compound 2 in plasma and
various tissues aredisplayed in Fig. 4B. Compound 2 was rapidly
distributed fol-lowing i.p. administration and reached nearly
maximal levelsin plasma, liver, and kidney 30 minutes after
injection. Com-pound 2 levels declined substantially over the
course of 12 to24 hours to a small fraction of its initial levels
in all three ofthese compartments. In contrast, the time course for
uptakeof compound 2 into the 1483 tumors lagged substantially
andrequired ∼3 hours to reach near maximal levels. The levels
ofcompound 2 remained relatively high in the tumor so by24 hours,
the tumor levels were as high as the levels in liveror kidney. This
indicates both slow uptake and release ofcompound 2 into and out of
the tumor.APCmin mice bear the same mutation (Apc−) that is
caus-
ative for familial adenomatous polyposis in human beingsand
these mice primarily develop small intestinal tumorsthat express
COX-2 (35, 36). Crossing APCmin mice withCOX-2 (−/−) mice reduces
intestinal tumor development by85% and treatment of APCmin mice
with COX-2 inhibitorsalso reduces tumorigenesis (37, 38). APCmin
mice (ages 18–20 wk, fed a high-fat diet) were injected
retro-orbitally withcompound 2 (1 mg/kg), and after 2 hours,
animals were sac-rificed and their intestines were removed. The
tissue waswashed thoroughly with PBS, opened longitudinally,
andimaged. Figure 5A shows the low background fluorescenceof a
section of small intestine without polyps. A single polyp(Fig. 5B)
and a five-polyp cluster (Fig. 5C) displayed highfluorescence, with
greatly increased detection compared withbright-field
visualization. The signal enrichment of com-pound 2 in the polyps
was estimated to be >50:1. COX-2 ex-pression in the polyps seems
to be required for this selectiveuptake although other factors
beside the level of COX-2protein may contribute to the relative
enrichment over sur-rounding normal tissue.
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Discussion
These studies show the feasibility of specific in vivo
target-ing of COX-2 in inflammatory lesions and tumors usingorganic
fluorophores tethered to indomethacin through anamide linkage.
Compounds 1 and 2 display a very high degreeof selectivity of
uptake by inflammatory tissue and tumors inlive animals relative to
surrounding normal tissue or muscleas determined by either imaging
or mass spectrometry. Thisselectivity seems greater than that
reported in previous liter-ature reports of fluorescent tumor
imaging in which the ratioof tumor fluourescence was compared with
muscle fluores-cence (39). Uptake of our compounds requires the
expression
Figure 3. In vivo labeling of COX-2–expressing xenografts
bycompound 2. A, nude mice with HCT116 xenograft (COX-2 negative)or
1483 xenograft (B; COX-2 positive) were dosed (retro-orbital) with1
mg/kg compound 2 and imaged at 3.5 h postinjection. C, nude
micewith 1483 xenografts were predosed with DMSO before injection
ofcompound 2 (2 mg/kg, i.p.), or predosed with indomethacin (D; 2
mg/kg,i.p.) 24 and 1 h before compound 2 and imaged at 3 h
postinjection(Xenogen IVIS, DsRed filter, 1 s, f2, 1.5 cm depth).
The emission observedaround the peritoneal cavity in C and D is due
to residual compound2 at the site of injection.
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-
COX-2 Imaging
of COX-2 at the target site and declines as the level of
COX-2decreases. Although uptake into inflamed or tumor tissueseems
to be slower than expected from simple distributionin the body, the
kinetics of compound release seem to beextremely slow, thus leading
to a detectable buildup of thelabel. Similar results are observed
by both imaging com-pounds 1 or 2 (Figs. 2 and 3) or by direct
quantitative analysisof compound 2 (Supplementary Fig. S4; Fig.
4).To achieve this success, ~200 compounds were evaluated
as candidate COX-2–targeted imaging agents. Although a sig-
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nificant percentage showed COX-2 inhibitory activity
againstpurified protein, only a fraction of these compounds
inhib-ited COX-2 activity in intact cells, and of those, most
didnot possess fluorescence properties suitable for in vivo
imag-ing. Among the compounds that emerged from our develop-ment
pathway, only compounds 1 and 2 exhibited sufficientmetabolic
stability to survive long enough to distribute toinflammatory
lesions or xenograft tumors. The low overallsuccess rate (∼1%)
likely underscores why COX-2–targetedimaging agents have proven
difficult to develop.
Figure 4. Analysis of fluorescentmaterial in xenografts and
severalmouse tissues. A, representativeHPLC-UV
chromatogram(detection, 581 nm) of1483 tumor extract (4
hpostadministration) revealed asingle major fluorescentcompound
that coeluted withcompound 2 (15.3 min). Thepeak eluting at 13.3
minintegrates for
-
Uddin et al.
3626
The specificity for COX-2 binding of these compounds
wasillustrated by multiple observations: (a) only cultured
cellsthat express COX-2 took up fluorocoxibs and uptake was
in-hibited by the COX inhibitors indomethacin and celecoxib.
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The intracellular localization of the probes matches that
ofCOX-2 protein and the stoichiometry of uptake was ∼0.9molecule of
beacon per subunit; (b) uptake into inflamedover noninflamed tissue
was blocked by indomethacinpretreatment of the animals and was not
observed inCOX-2 (−/−) animals. No uptake was observed with aclose
structural analogue of compound 1 that does not in-hibit COX-2; (c)
uptake into COX-2–expressing tumors wasblocked by indomethacin
pretreatment of the animals anda correlation was found between the
amount of light emis-sion from the tumor and the COX-2 inhibitory
potencyof the beacon. The nontargeted fluorophores 5-ROX and6-ROX
did not accumulate in COX-2–expressing xenografts;and (d) >95%
of the fluorescent material present in the tu-mors is the
unmetabolized parent compound. Thus, in vitroand in vivo studies
provide strong support for the conclusionthat binding to COX-2 is
the major determinant of uptake intoinflamed, premalignant, or
malignant tissue. Although thestoichiometry of compound 2 binding
to COX-2 protein wasestimated to be 0.9 in activated RAW cells,
such high stoichi-ometry cannot be assumed in all situations. The
extent ofuptake in cells, inflamed tissue, or tumors will depend
uponseveral factors such as the permeability of
COX-2–expressingcells to the probe, kinetics of binding and release
from theCOX-2 active site, vascularization of the tissue, and
possibleexpulsion of the probes by transporters. Further studies
willbe needed to explore in greater detail the quantitative
aspectsof the use of these compounds in vivo.Compounds 1 and 2
represent the first feasible reagents
for clinical detection of tissues containing high levels ofCOX-2
in settings amenable to fluorescent excitation andanalysis by
surface measurement or endoscopy (e.g., skin,esophagus, intestine,
and bladder). Although such fluoroco-xibs will not be useful for
applications in internal organs thatare not accessible for optical
imaging, their developmentprovides rigorous proof of concept for
the feasibility ofmolecular targeting of COX-2 in inflammatory
lesions,premalignant lesions, and tumors.
Disclosure of Potential Conflicts of Interest
L.J. Marnett: sponsored research and consulting, XL Tech Group.
The otherauthors disclosed no potential conflicts of interest.
Acknowledgments
We thank S.K. Dey for the COX-2 null animals and Melissa Turman
for theassistance with molecular graphics.
Grant Support
Research and Center grants from the NIH (CA86283, CA89450,
CA105296,CA68485, CA60867, CA126588, CA111469, and GM72048), the
Medical Free-Electron Laser Program of the US Department of
Defense, XL TechGroup,and New York Crohn's Foundation.
The costs of publication of this article were defrayed in part
by the paymentof page charges. This article must therefore be
hereby marked advertisement inaccordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Received 07/16/2009; revised 02/20/2010; accepted 02/23/2010;
publishedonline 04/29/2010.
Figure 5. In vivo labeling of COX-2–expression in intestinal
polyps bycompound 2. C57BL/6J-Min/+ mice bearing small intestinal
polypswere euthanized at 2 h after retro-orbital injection of
compound 2(1 mg/kg) and small intestines were washed, opened, and
examined bydissecting fluorescence microscopy. A, section of small
intestine withno polyp, 90-millisecond exposure. B, single polyp,
90-millisecondexposure. C, polyp cluster, 90-millisecond
exposure.
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COX-2 Imaging
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