fluorinated cox-2 inhibitors as agents in pet imaging of...

Research ArticleSee perspective on p. 1523

Fluorinated COX-2 Inhibitors as Agents in PET Imaging ofInflammation and Cancer

Md. Jashim Uddin1, Brenda C. Crews1, Kebreab Ghebreselasie1, Imran Huda1, Philip J. Kingsley1,Mohammad Sib Ansari2, Mohammed N. Tantawy2, Jeffery Reese3, and Lawrence J. Marnett1

AbstractCOX-2 is a major contributor to the inflammatory response and cancer progression so it is an important

target for prevention and therapy. COX-2 is absent or expressed at low levels in most epithelial cells but is

found at high levels in inflammatory lesions, and many premalignant and malignant tumors. Thus, it is an

attractive target for molecular imaging.We report a series of novel fluorinated imaging agents, derived from

indomethacin or celecoxib that selectively inhibit COX-2. The most promising lead, compound 7, was a

fluorinated derivative of celecoxib. Kinetic analysis revealed that this fluorinated compound is a slow, tight-

binding inhibitor of COX-2 and exhibits minimal inhibitory activity against COX-1. Efficient incorpo-

ration of 18F into compound 7 by radiochemical synthesis and intravenous injection provided sufficient

signal for in vivo positron emission tomography (PET) imaging. Selective uptake of 18F-7 was observed in

inflamed rat paws compared with the noninflamed contralateral paws and uptake was blocked by

pretreatment with the COX-2 inhibitor, celecoxib. Uptake of 18F-7 was not observed when inflammation

was induced in COX-2–null mice. In nudemice bearing both a COX-2–expressing human tumor xenograft

(1483) and a COX-2–negative xenograft (HCT116), 18F-7 selectively accumulated in the COX-2–expressing

tumor. Accumulation was blocked by pretreatment of the animals with celecoxib. The in vitro and in vivo

properties of compound 7 suggest it will be a useful probe for early detection of cancer and for evaluation of

the COX-2 status of premalignant and malignant tumors. Cancer Prev Res; 4(10); 1536–45. �2011 AACR.

Introduction

Molecular imaging promises to revolutionize the diag-nosis and treatment of cancer. Detection of early-stagecancer or of chronically inflamed tissue is one of themajor clinical challenges to which the development ofradiotracer- or fluorescent-based imaging techniques hasbeen directed (1–4). Efforts have been made to maximizethe delivery of ligands into the tumor to achieve a highsignal-to-noise ratio (5–7). COX-2 is an inducible enzymethat is expressed at elevated levels at sites of inflammationand malignant transformation, distinct from most normaltissues (8). This fact, combined with an extensive founda-

tion of COX-2–selective inhibitors, makes this enzyme anideal target for imaging inflammation and cancer.

The COX active site is located at the terminus of a longchannel that runs from the protein surface to the interior ofthe protein. The initial portion of the channel has a largevolume, or lobby, which narrows at a constriction com-prised of Arg-120, Tyr-355, and Glu-524 (Fig. 1). Theconstriction constitutes a gate that must open and closefor substrates and inhibitors to pass into or out of the COXactive site, which is located above it. All COX inhibitorsbind in the active site above the constriction (Fig. 1A and B)and the constriction site residues play an important role inbinding carboxylic acid–containing inhibitors by a com-bination of ion pairing and hydrogen bonding (9). Indo-methacin is a nonselective inhibitor of COX-1 and COX-2,but it can be converted in a single step to a wide variety ofamide or ester derivatives that selectively inhibit COX-2(10). Celecoxib is a COX-2–selective inhibitor, whichinserts its sulfonamide group into a side pocket of theactive site that is only accessible in COX-2. Extensivestructure–activity studies suggest that bulky substituentson the sulfonamide or sulfone are not tolerated (11).

We recently reported that COX-2–targeted fluorescentimaging agents can be selectively delivered into inflamma-tory tissues and COX-2–expressing tumors in vivo (12) andthat COX-2–targeted single-photon emission computedtomography (SPECT) imaging agents are selectively taken

Authors' Affiliations: 1A.B. Hancock, Jr., Memorial Laboratory for CancerResearch, Department of Biochemistry, Chemistry and Pharmacology,Vanderbilt Institute of Chemical Biology, Center for Molecular Toxicologyand Vanderbilt-Ingram Cancer Center; and 2Department of Radiology andRadiological Sciences, and Vanderbilt University Institute of ImagingSciences; 3Division of Neonatology, Department of Pediatrics, VanderbiltUniversity Medical Center, Nashville, Tennessee

Note: The supplementary data for this article are available at CancerPrevention Research Online (http://cancerprevres.aacrjournals.org).

Corresponding Author: Lawrence J. Marnett, 23rd Ave at Pierce, 850Robinson Research Building, Vanderbilt University School of Medicine,Nashville TN 37232-1046. Phone: 615-343-7329; Fax: 615-343-7534;E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-11-0120

�2011 American Association for Cancer Research.

CancerPreventionResearch

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Published OnlineFirst September 7, 2011; DOI: 10.1158/1940-6207.CAPR-11-0120

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up by inflamed tissue (13). Attempts have been made todevelop COX-2–targeted positron emission tomography(PET) imaging agents and although compounds havebeen reported that are taken up into COX-2–expressingcells, no agents have been reported that are effective in vivo(14–16). Multiple factors may contribute to the lack ofsuccess including poor COX-2 inhibitory activity or rapiddefluorination of 18F-based agents (17). As an enormousamount of medicinal chemistry has been conducted tocreate COX-2–specific small molecule inhibitors, thereare numerous classes of potential building blocks thatare available for the preparation of molecular imaging

agents for COX-2 (11, 18, 19). To enhance the possibilityfor developing an efficient COX-2–targeted PET imagingagent, we have synthesized a series of fluorinated com-pounds derived from the indomethacin or celecoxib scaf-fold. Here, we report their synthesis, and biochemical andcellular evaluation as inhibitors of COX-2. We also reportthe radiochemical synthesis of the 18F-derivative of themost promising compound and its uptake into inflamma-tory lesions and tumors in a COX-2–dependent fashion.This compound represents the first PET imaging agentvalidated for the detection of COX-2 in inflammationand cancer.

Figure 1. COX-2 crystal structure.A single subunit of the COX-2homodimer is shown. A, themembrane-binding domain (teal)is connected to the C-terminal endof the epidermal growth factordomain (pink) and comprises 4a-helices. One of the helices (theD-helix) connects the membrane-binding domain to the catalyticdomain (blue). NSAIDs andCOXIBs fill the active site locatedabove the constriction comprisedof Arg-120, Tyr-355, and Glu-524(red stick structures), whichseparates the active site from themembrane-binding domain.B, view down from top of activesite. The active site of COX-2 witha bound molecule of SC558, aCOX-2 selective inhibitor andcelecoxib analog. The SO2NH2

pharmacophore of SC558 insertsinto the COX-2 side-pocket,where one sulfonamide oxygenatom forms a H-bond withArg-513, and the nitrogen atomforms a H-bond with the carbonyloxygen of Phe-518. The CF3moiety of SC558 projects towardthe constriction site residues ofCOX-2 active site.

Catalyticdomain(blue)

A

B

Membrane-binding domain

(teal) COX-2active site

EGFdomain(pink)

THR-521 PHE-

381PHE-518 ARG-

513ASN-195

ASN-581

ILE-568

ALA-516

TYR-355 ARG-

120ILE-124

GLU-524

PHE-529

ILE-377

VAL-582

PET Imaging of COX-2

www.aacrjournals.org Cancer Prev Res; 4(10) October 2011 1537




Materials and Methods

ChemistrySyntheses and characterizations of all compounds are

described in Supplementary Data.

Inhibition assay using purified COX-1 and COX-2COX activity of ovine COX-1 or human COX-2 was

assayed by TLC (20). Briefly, reaction mixtures of 200 mLconsisted of hematin-reconstituted protein in 100 mmol/LTris-HCl (pH 8.0), 500 mmol/L phenol, and [1-14C]-arachidonic acid (50 mmol/L, �55–57 mCi/mmol; PerkinElmer). For the time-dependent inhibition assays, hematin-reconstituted COX-1 (44 nmol/L) or COX-2 (66 nmol/L)was preincubated at 25�C for 17 minutes and 37�C for 3minutes with varying inhibitor concentrations in dimethylsulfoxide (DMSO) followed by the addition of[1-14C]arachidonic acid (50 mmol/L) for 30 seconds at37�C. Reactions were terminated by solvent extraction inEt2O/CH3OH/1 mol/L citrate, pH 4.0 (30:4:1). The phaseswere separated by centrifugation at 2,000� g for 2 minutesand the organic phase was spotted on a TLC plate (EMDKieselgel 60; VWR). The plate was developed in EtOAc/CH2Cl2/glacial AcOH (75:25:1) at 4�C. Radiolabeled pros-tanoid products were quantitated with a radioactivity scan-ner (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 intact cell metabolism assayCOX-2–negative human colorectal carcinoma (HCT116)

cells, ATCC CCL-247 human colorectal carcinoma cells,passage 8 to 18, Mycoplasma negative by a PCR detectionmethod (5/17/10 and 12/08/10 testing; Sigma Venor-Gem), were grown in Dulbecco’s modified Eagles medium(DMEM; Invitrogen/Life Technologies) þ 10% FBS (Atlas)to 70% confluence. RAW264.7, ATCC TIB-71 murine mac-rophage-like cells, passage 5 to 15, Mycoplasma negative bya PCR detection method (5/17/10 and 12/08/10 testing),were grown in DMEM (Invitrogen/Gibco) þ 10% heat-inactivated FBS (Atlas) in 6-well plates (Sarstedt) to 40%confluence and activated for 7 hours in serum-free DMEMwith 200 ng/mL lipopolysaccharide (LPS; Calbiochem)and 10 U/mL IFN-g (Calbiochem). COX-2–expressing hu-man head and neck squamous cell carcinoma (1483HNSCC) cells (gift of Dr. A.J. Dannenberg, Cornell MedicalCollege), passage 9 to 19, Mycoplasma negative by a PCRdetection method (5/17/10 and 12/08/10 testing), weregrown in DMEM/F12þ 10% FBS in 6-well plates until 60%confluence. The ability of compounds to inhibit COX-2 inintact cells was assayed (21). Serum-free medium (2 mL)was added, and the cells were treated with inhibitor dis-solved in DMSO (0–5 mmol/L, final concentration) for 30minutes at 37�C followed by the addition of [1-14C]-arachidonic acid (10 mmol/L, �55 mCi/mmol; PerkinElmer) for 20 minutes at 37�C. Reactions were terminatedby solvent extraction in Et2O/CH3OH/1 mol/L citrate, pH

4.0 (30:4:1), and the organic phase was spotted on a 20 �20 cm TLC plate (EMD Kieselgel 60; VWR). The plate wasdeveloped in EtOAc/CH2Cl2/glacial AcOH (75:25:1), andradiolabeled prostaglandins were quantified with a radio-activity scanner (Bioscan, Inc.). The percentage of totalproducts observed at different inhibitor concentrationswas divided by the percentage of products observed forcells preincubated with DMSO.

RadiochemistryThe procedure for the radiosynthesis of the compound 7

is described in Supplementary Data.

In vivo PET imaging of COX-2 in inflammationAnimal studies were conducted in accordance with the

standards of humane animal care described in the NIHGuide for the Care and Use of Laboratory Animals, usingprotocols approved by the Vanderbilt University institu-tional animal care anduse committee. Animalswerehousedin an Association for the Assessment and Accreditation ofLaboratory Animal Care (AAALAC)-approved animal carefacility. Targeted disruption of the Ptgs2 genewas previouslyaccomplished by insertion of a PGK-neo cassette in place ofa 1.8-kb EcoRV genomic fragment spanning exon 1 andflanking sequences (22). Genotype was determined by tailPCR using the primers: sense, 50-ACCTCTGCGATGCT-CTTCC; antisense, 50-CACCATAGAATCCAGTCCGG; neo,50-ATCGCCTTCTTGACGAGTTC. Because of infertility andmultiple reproductive deficits, Ptgs2 deficiency was reestab-lished in albino CD1 strain outbred mice (Charles RiverLaboratory) through 10-generation outcross matings tocreate Ptgs2 deletion in a strain with vigorous reproductivecharacteristics. Heterozygous Ptgs2 females (20–25 g, 48–60 days old) were mated with homozygous null malesto produce sufficient Ptgs2-null offspring.

Carrageenan (50 mL, 1% in sterile saline) was injectedsubcutaneously in the rear right footpad of wild-type CD-1or COX-2–null mice (20–25 g) under anesthesia, followedby the administration of radiotracer 18F-7 [200 mL intraper-itoneally (i.p.), 300–400 mCi, �3.5 mCi/mL] at 24 hoursafter carrageenan administration, when inflammation wasmaximal. At 2 hours after radiotracer injection, the micewere anesthetized with 2% isoflurane, positioned on amouse bed, and placed in the microPET Focus 220(Siemens). They were imaged for 30 minutes with the rearright footpad being the center field of view. Computedtomography (CT) images of the rear right footpad werethen produced by the microCAT II (Siemens) at X-ray beamintensity of 500 mA and tube voltage of 80 kV. The PETimages were reconstructed using an OSEM3D/MAP algo-rithm into transaxial slices (128�128�95)with voxel sizesof 0.095 cm� 0.095 cm� 0.08 cm. The PET and CT imageswere coregistered to each other on the basis of bed position.Volumetric regions of interest (ROI) were drawn aroundthe left and right hind limbs of the coregistered images,using the medical imaging analysis tool AMIDE (23). Themaximum concentration within the ROI of the right hindlimb was compared with that of the left hind limb.

Uddin et al.

Cancer Prev Res; 4(10) October 2011 Cancer Prevention Research1538




Carrageenan (100 mL, 1% in sterile saline) was injectedsubcutaneously in the rear right footpad of male SpragueDawley rats (300–350 g; Charles River Laboratory) underanesthesia, followed by the administration of radiotracer18F-7 (200–400 mL, tail vein, �1 mCi, �3.5 mCi/mL) at 2hours after carrageenan administration. The rats wereallowed to wake, placed in their cages, and fed ad libitum.They were anesthetized with 2% isoflurane, positioned ona rat bed, and placed in the microPET Focus 220 (Siemens)at 2 hours postinjection. They were imaged for 30 minuteswith the rear right footpad being the center field of view. CTimages of the rear right footpad were then produced by themicroCAT II (Siemens) at X-ray beam intensity of 27 mAand tube voltage of 80 kV. The PET images were recon-structed using an OSEM3D/MAP algorithm into transaxialslices (128 � 128 � 95) with voxel sizes of 0.095 cm �0.095 cm� 0.08 cm. The PET and CT images were followedby coregistration of CT images with each other. VolumetricROIs were drawn around the left and right hind limbs ofthe coregistered images, using the medical imaging analysistool AMIDE (23). The maximum concentration within theROI of the right hind limb was compared with that of theleft hind limb.

Establishment of xenografts in nude miceFemale nude mice, NU-Fox1nu, were purchased at 6 to

7 weeks of age from Charles River Labs. Human 1483HNSCC cells or HCT116 colorectal carcinoma cells weretrypsinized and resuspended in cold PBS containing 30%Matrigel such that 1 � 106 cells in 100 mL were injectedsubcutaneously on the left or right flank. The HCT116 or1483 xenografts required 2 to 3 weeks of growth.

In vivo PET imaging of nude mice with xenograftsFemale nudemice bearing medium-sized 1483 xenograft

tumors on the left hip or HCT116 xenograft tumors on theright hip were dosed with approximately 350 mCi of 18F-7via retro-orbital injection. Two hours later, the mice wereanesthetized with 2% isoflurane, positioned on a bed andplaced in themicroPETFocus220 (Siemens) and imaged for30 minutes. CT images of the hips were then produced bythe microCAT II (Siemens, Knoxville, TN) at X-ray beamintensity of 500 mA and tube voltage of 80 kVp. The PETimages were reconstructed using an OSEM3D/MAP algo-rithm into transaxial slices (128�128�95)with voxel sizesof 0.095 cm� 0.095 cm� 0.08 cm. The PET and CT imageswere coregistered to each other on the basis of bed position.

Tissue analysisTo evaluate the potential for defluorination, we analyzed

the footpads (inflamed and control) of rats injected withunlabeled compound 7. We injected 100 mL 1% carrageen-an into the right hind paw of male Sprague Dawley rats(body weight, 300–350 g) and waited 3 hours for inflam-mation to develop. Rats (n¼ 3)were dosedwith compound7 (1 mg/kg, i.p.). At 2 hours postdosing of compound 7,the rats were euthanized with isoflurane overdose and foot-pads were collected, weighed, and snap-frozen at�80�C for

analysis. The tissue was thawed and digested with Protein-ase K (Sigma) overnight. An aliquot of the digestion solu-tion was extracted with 1.2 volumes of EtOAc/n-Hexane(2:1). The organic layer was removed, dried, and reconsti-tuted inMeOHandwater. The sampleswere analyzed on anLC-ESI-MS system (Thermo) and 7 and its defluorinatedmetabolite were detected via selected reaction monitoring.The unknown samples were quantitated against a 5-pointstandard curve, which was subjected to the Proteinase Kdigestion and liquid–liquid extraction. The compoundswere separated in reverse-phase mode on a C18 column(5 � 0.2 cm; Phenomenex), using gradient elution. Mobilephase component A was water and B was acetonitrile, eachwith 0.1% acetic acid. The gradient used was 50% B to 80%B over 4 minutes, followed by a brief hold and return toinitial conditions. The flow rate was 0.35 mL/min.

Results

Inhibition of purified COX-1 and COX-2A series of fluorine-containing indomethacin and cele-

coxib derivatives was synthesized and evaluated for inhibi-tion of COX-2 in vitro and in intact cells. The syntheses oftarget molecules and their complete chemical characteriza-tion are described in Supplementary Data. The IC50 valuesfor inhibition of purified human COX-2 or ovine COX-1 bytest compounds were determined by a standard assay (20).Table 1 summarizes the data from this assay. Indomethacinfluoroalkylamide 1 displayed selective COX-2 inhibition(IC50 ¼ 0.14) with no inhibition of COX-1 up to 4 mmol/Linhibitor. Incorporation of functionalities, like an arylgroup, in the alkyl chain led to derivative 2, which revealeda significant increase in selectivity andpotency againstCOX-2 (2, COX-2 IC50 ¼ 0.09 mmol/L, COX-1 IC50 > 4 mmol/L).Chain length extension of 2 to higher alkyldiamide analo-gues 3–6 retained the selective inhibition of COX-2 withslightly decreased potency (3, COX-2 IC50 ¼ 0.26 mmol/L,COX-1 IC50 > 4 mmol/L). Although compounds 1–6 wereeffective COX-2 inhibitors in intact cells, their p-chloroben-zoyl groupwasnot stable to the conditionsof radiochemicalsynthesis, so we abandoned this series and focused oncelecoxib derivatives. Replacement of the CF3 group ofcelecoxib by a CH2F substituent gave compound 7, whichshowed potent and selective inhibition of COX-2 with anIC50 of 0.16 mmol/L. However, when the SO2NH2 group of7 was replaced with a SO2Me group, the COX-2 inhibitorypotencywas decreased (8, IC50¼ 0.60mmol/L). Compound9, which contains a fluoropropyl group at the C-3 positionof celecoxib showed COX-2 inhibitory activity (IC50¼ 1.95mmol/L) with a significant loss of potency. A further drop ofpotency was observed when the SO2NH2 pharmacophoreof compound 9 was replaced by the SO2Me group incompound 10 (IC50 ¼ 3.4 mmol/L).

Inhibition of COX-2 in cell cultureThe ability of promising compounds to inhibit COX-2

in intact cells was assayed in the RAW264.7 murine mac-rophage-like cell line (21). RAW264.7 cells were incubated






Table 1. Biochemical properties of fluorinated indomethacin and celecoxib derivatives

Purified enzyme IC50

(mmol/L)RAW264.7 cellsa IC50

(mmol/L), COX-2Compound no. Structure COX-1 COX-2

1

NH

NCH3

H3CO

O

Cl

O

F

>4 0.14 0.68

2

NH

NCH3

H3CO

O

Cl

O

F

>4 0.09 0.01

3

NH

NCH3

H3CO

O

Cl

O

NH

O

F

>4 0.26 0.16

4 NH

NCH3

H3CO

O

Cl

O

NH

S

OO

F

>4 0.10 0.04

5NH

NCH3

H3CO

O

Cl

OHN

O

F

>4 0.16 0.24

6

NH

NCH3

H3CO

O

Cl

OHN

S

F

O O

>4 0.13 0.11

7N

N

H3C

H2NO2S

F

>4 0.16 0.08

8

NN

H3C

H3CO2S

F

>4 0.60 N.T.

9N

N

H3C

H2NO2S

F>4 1.95 N.T.

(Continued on the following page)

Uddin et al.





with LPS (200 ng/mL) and IFN-g (10 U/mL) for 7 hours toinduce COX-2 expression and then were treated withvehicle or the test compounds at several concentrations(0–5 mmol/L) for 30 minutes followed by the addition of10 mmol/L [1-14C]-arachidonic acid (�55 mCi/mmol).IC50 values for inhibition of RAW264.7 cell prostaglandinproduction by compounds 1–7 were determined. Com-pounds 2, 4, and 7 showed the highest potency with IC50

values in the low nanomolar range (Table 1).

Enzyme–inhibitor binding kineticsThe high potency of compounds such as celecoxib and

indomethacin derives from the fact that they are slow, tight-binding inhibitors. They establish a rapid equilibriumwith aloosely bound enzyme–inhibitor complex, then slowly con-vert to a much more tightly bound complex. Their rate ofdissociation from the tightly bound complex is very slow.Figure 2A shows the time- and concentration-dependentinhibition of mCOX-2 by compound 7. The inhibition ofCOX-2 proceeded rapidly, and the inhibition plateau forcompound 7 at high concentrations approached 20% activ-ity remaining. Figure 2B displays a plot of the single expo-

nential rate constants for inhibition as a function ofconcentration, which allows the determination of the equi-librium constant for initial association (KI ¼ k�1/k1). Com-pound 7 has a KI value of 7.3 � 1.6 mmol/L, as comparedwith indomethacin and celecoxib, which have KI values of13.2 � 2.3 mmol/L and 4.0 mmol/L, respectively. The inac-tivation rate (k2) of compound 7 was slower (compound 7,k2¼ 0.035 s�1) than that of indomethacin (k2¼ 0.061 s�1).The dissociation rate (k�2) of compound 7 was very slow(k�2¼0.0003s�1; equationsAandB,where,E¼ enzyme, I¼inhibitor, EI ¼ loosely bound enzyme-inhibitor complexEI* ¼ tightly bond enzyme-inhibitor complex. Thus, onceassociatedwithCOX-2, compound 7 remains tightly bound.

Eþ I Ðk1

k�1½EI� Ð

k2

k�2EI* ðAÞ

kobs ¼ ððk2 � ½I�=ðKI þ ½I�ÞÞþk�2 ðBÞ

In vivo PET imaging of carrageenan-inducedinflammation

The rat footpad model is well documented for the role ofCOX-2–derived prostaglandins as a major driving force for

Table 1. Biochemical properties of fluorinated indomethacin and celecoxib derivatives (Cont'd )

Purified enzyme IC50

(mmol/L)RAW264.7 cellsa IC50

(mmol/L), COX-2Compound no. Structure COX-1 COX-2

10N

N

H3C

H3CO2S

F

>4 3.40 N.T.

11N

N

H3C

H2NO2S

OH

>4 1.3 N.T.

Indomethacin

OH

NCH3

H3CO

O

Cl

O

0.05 0.75 0.01

CelecoxibN

N

H3C

H2NO2S

CF3

>4 0.03 N.T.

NOTE: The IC50s for inhibition of purified ovine COX-1, human COX-2, or COX-2 in LPS activated macrophage-like RAW264.7 cells.aAssays were conducted as described inMaterials andMethods. Compounds that are poor inhibitors of COX-2 in the purified enzymeassay were not tested (N.T.) in intact cells.






the acute edema that results after carrageenan injection intothe paw (24). One of the significant advantages of thisanimal model of inflammation is the ability to image theinflamed footpad in comparison with the noninflamedcontralateral footpad, which does not express COX-2.In vivo biodistribution of 18F-7was studied, in the inflamedand contralateral footpad of male Sprague Dawley rats(body weight, 300–350 g). We injected 100 mL of 1%carrageenan in the rear right footpad and waited 2 hoursfor inflammation to develop. 18F-7, dissolved in a formu-lation solvent consisting of ethanol (20%) and sterile saline(80%; 300 mL,�1mCi), was then injected (tail vein) underanesthesia. At 1.5 hours postinjection, the rats were anes-thetized with 2% isoflurane and placed in themicroPET/CTfor data acquisition. 18F-7 targeted the swollen footpadselectively over the contralateral footpad (Fig. 3A). The PETimages were analyzed using AMIDE (23) and the imageswere corrected for injected dose and body weight. Three-dimensional ROIs were drawn around the left and righthind limbs separately. The ROIs included the entire limbfrom toes to ankle. Uptake in the right hind limb ROI wasgreater than that of the left hind limb. The mechanism ofdelivery was confirmed by blocking the COX-2 active sitein the inflamed footpad with celecoxib. At 2 hours aftercarrageenan administration, we administered celecoxib

(10 mg/kg, 25 mL, tail vein) within 2 minutes of the dosingwith 18F-7 (�800 mCi, 300 mL, tail vein) to block the COX-2active site. Reduced uptake in the inflamed footpad wasapparent (Fig. 3B).

Inflamed Noninflamed

Inflamed

B

A

Noninflamed

Figure 3. In vivo labeling of COX-2–expression in inflammation bycompound 18F-7. A, male Sprague Dawley rats with carrageenan-inducedinflammation in the rear right footpad were dosed with 18F-7 (300 mL,�1 mCi, tail vein). At 1.5 hours postinjection, the rat was anesthetized andimaged in the microPET Focus 220 for 30 minutes. The PET image showsthat the radiotracer targeted the swollen footpad (inflamed) selectively overthe contralateral footpad (control). B, rats with carrageenan-inducedinflammation were predosed with celecoxib (10 mg/kg, 25 mL, tail vein)immediately prior to 18F-7 injection (300 mL,�1 mCi, tail vein). At 1.5 hourspostinjection, the rat was anesthetized and imaged in the microPET Focus220 for 30 minutes. The PET image shows minimal radiotracer uptake inthe inflamed footpad compared with the control footpad.

0.3Inflamed

Noninflamed

0.2

0.1

0.085 90 95 100 105 110

Time (min)

Upta

ke o

f 18F

-7 (

SU

V)

Figure 4. Time course of in vivo uptake of 18F-7 in inflamed versusnoninflamed footpad of a typical Sprague Dawley rat. Standardized uptakevalues (SUV) at the ROI were determined by the Medical Imaging AnalysisTool AMIDE (22). The error bars are the variance generated automaticallyby the software.

100

A

B

75

50

25

% C

OX

-2 a

ctiv

ity r

em

ain

ing

k ob

s (

S–

1)

0

30

20

10

00 5,000 10,000 15,000 20,000 25,000

Compound 7 (nmol/L)

0 1 2 3

Time (min)

4 5

250 nmol/L

500 nmol/L

1,000 nmol/L

4,000 nmol/L

Figure 2.Kinetics of the time-dependent inhibition of COX-2 by compound7. The purified mCOX-2 enzyme was reconstituted with heme andpreincubated with the inhibitor at 37�C for various times (0, 0.5, 1, 3, 5, 10,15, 30, 45, 60, and 120 minutes) prior to the addition of the substrate (50mmol/L). A, time-dependent inhibition of COX-2 by compound 7 at theindicated concentrations. B, secondary plot of kobs versus inhibitorconcentration used to generate values for KI, k2, and k-2, using equations Aand B (see text).

Uddin et al.





The time course of uptake of 18F-7 was evaluated bysignal integration at different times. Figure 4 compares theuptake of 18F-7 in the carrageenan-injected footpad overthe noninflamed footpad obtained from measurements ofindividual footpad signals at various time points between1.5 and 2 hours.To quantitatively compare uptake, we euthanized the

animals at 2 hours postinjection of 18F-7, removed the hindfeet, and measured the radioactivity of the individualfootpads in a well gamma counter. There was a 1.53-fold� 0.5 (n ¼ 5) increase in the inflamed compared with thenoninflamed footpad. In contrast, no increase of radio-tracer in the inflamed footpad compared with the nonin-flamed footpad was detected in celecoxib-pretreatedanimals (0.9-fold � 0.14, n ¼ 5). Celecoxib dosing resultsin a highly significant decrease in 18F-7 uptake at the site ofinflammation (P ¼ 0.0006).COX-2–null mice on a CD-1 genetic background were

used to validate further the COX-2 specificity of our target-ing probe. We injected 50 mL of 1% carrageenan s.c. in therear right footpad of wild-type or COX-2–null mice andwaited 24 hours for maximal inflammation to develop.18F-7, dissolved as described, was injected (200 mL, i.p.,�350 mCi). At 2.5 hours postinjection of radiotracer, micewere anesthetized with 2% isoflurane and placed in themicroPET/CT Focus 220 for imaging of rear legs (acquisi-tion time, 30 minutes). At 3 hours postinjection, weeuthanized the mice, removed their feet, and counted themin a well gamma counter. Software analysis using AMIDE(23) determined no statistical difference in 18F-7 uptake inthe inflamed foot versus the contralateral control foot inthe COX-2–null mouse. Well counting of the COX-2–nullfeet, normalized to Bq/g, showed a 1.08 � 0.09 ratio ofradioactivity in the inflamed foot compared with the non-inflamed foot (n ¼ 5). In contrast, the wild-type mouseinflamed foot to noninflamed foot uptake ratio was 1.48�0.04, significantly different from the COX-2–null mouseratio (P ¼ 0.0002). The absence of COX-2 in the site of theinflammation reduces the 18F-7 uptake by greater than80%.The potential for defluorination was evaluated by

administration of unlabeled 7 followed by extractionand analysis of both 7 and its oxidatively defluorinatedmetabolite, 11, by LC-MS. Intact compound 7 was iden-tified in the inflamed footpad (486.3 pmol/g tissue). Alow level of the defluorinated metabolite, 11, wasdetected in the inflamed footpad (37.9 pmol/g tissue).The calculated extent of defluorination was 8.8 � 5.0%(n ¼ 3).

In vivo PET imaging of COX-2–expressing tumorsThe results in the footpad inflammation model showed

that the presence of COX-2 protein is associated withselective uptake of a COX-2–targeted PET imaging agent.We next evaluated the ability of these compounds to targetCOX-2 in human tumor xenografts. Female nude micewere injected in the left hip with 1483 HNSCC cells andin the right hip with HCT116 cells. The tumor xenografts

were allowed to grow to approximately 750 to 1,000 mm3.Animals were dosed by retro-orbital injection with 18F-7(�350 mCi) and then lightly anesthetized with 2%isoflurane in preparation for imaging. At 3 hours postin-jection, the animals were placed in the microPET/CT Focus220 for imaging (acquisition time ¼ 30 minutes). A sig-nificant uptake of 18F-7 was documented in the COX-2expressing 1483 tumor (Fig. 5A, sagittal view). Minimaluptake was documented in the COX-2–null HCT116tumor (Fig. 5B, sagittal view). Complete biodistributionis presented in the Supplementary Figure S2. After imaging,the mice were sacrificed by isoflurane overdose. The 1483HNSCC and HCT116 tumors were removed and weighed,and radioactivity associated with each tumor was countedwith a well gamma counter. After normalization of uptake(Bq/g), the 1483 tumor showed approximately a 3-foldhigher uptake of the radiotracer compared with theHCT116 tumor (Fig. 6A; n ¼ 3, P ¼ 0.0004,). In a typicalexperiment, the uptake of the radiotracer in the 1483tumors was 1.5% to 2% of the injected dose/g. When wepredosed the mice bearing 1483 xenografts with celecoxib(20 mg/kg, i.p.) within 2 minutes of the 18F-7 dosing toblock the COX-2 active site, the radioactivity (Bq/g) in the1483 tumor was comparable to that in leg muscle bywell gamma counting (1.01-fold� 0.16, n¼ 4). The vehiclepredosed mice averaged 2.94-fold � 0.36 uptake inthe 1483 tumors compared with leg muscle (Fig. 6B; Sup-plementary Fig. S3; n ¼ 5, P ¼ 0.00001 for celecoxib vs.vehicle). These results suggest that the difference in uptakeof radiotracer in 1483 and HCT116 xenografts is theirdifferential in COX-2 expression and that blockingthe COX-2 active site in 1483 xenografts precludes 18F-7binding.

1483 TumorA B HCT116 Tumor

Head

Figure 5. In vivo PET imaging of COX-2–expressing or COX-2 negativetumors by 18F-7. Tumor-bearing female nude mice were dosed by retro-orbital injection with compound 18F-7 (100 mL, �350 mCi, retro-orbital)under anesthesia. At 3 hours postinjection, the animals were imaged in themicroPET/CT instrument (30-minute acquisition); A, female nude mousemicroPET/CT image with COX-2 expressing HNSCC 1483 tumor on theleft hip (dotted circle), and B, COX-2 negative HCT116 tumor on the righthip (dotted circle).






Discussion

These studies show the feasibility of specific in vivotargeting of COX-2 in inflammatory lesions and tumors,using fluorinated organic compounds. Compound 18F-7displays a high degree of selectivity of uptake by inflam-matory tissue and tumors in live animals relative to

surrounding normal tissue as determined by PET imaging.Uptake of the compound requires the expression of COX-2at the target site and declines as the COX-2 active site isblocked or does not occur in the absence of COX-2 expres-sion. Compound 7 is a slow, tight-binding inhibitor ofCOX-2, thus leading to a detectable enrichment in the siteof interest.

To achieve this success, a number of fluorinated deriva-tives of indomethacin or celecoxib were evaluated as COX-2–targeted PET imaging agents. A significant percentageshowed inhibitory activity against purified COX-2 andCOX-2 in intact cells. Among the compounds that emergedfrom this development pathway, only compound 7 exhib-ited sufficient stability in radiochemistry or metabolicstability to survive long enough to distribute to inflamma-tory lesions or xenograft tumors.

The extent of uptake in inflamed tissue or tumorsdepends upon several factors such as the permeability ofCOX-2–expressing cells to the radiotracer, kinetics of bind-ing and release from the COX-2 active site, vascularizationof the tissue, and possible expulsion of the fluorinatedtracer by transporters. The specificity for COX-2 bindingof compound 7 was illustrated by multiple observations:(a) uptake into inflamed over noninflamed tissues;(b) absence of uptake by inflamed tissues in COX-2–nullanimals; (c) blockage of uptake into inflamed tissue or intoCOX-2–expressing tumors by celecoxib predosing of theanimals; and (d) uptake into COX-2–expressing xenografttumors over COX-2–negative xenograft tumors. Thesein vivo studies provide support for the conclusion thatbinding to COX-2 is the major determinant of uptake intoinflamed or tumor tissues. Thus, compound 7 represents afeasible PET imaging reagent for detection of tissues con-taining high levels of COX-2 in clinical settings. It shouldfind utility in the detection of premalignant lesions andthe stratification of patients for the presence of COX-2in their tumors. The latter is an important determinantof the responsiveness of tumors to adjuvant celecoxibtherapy (25).

Disclosure of Potential Conflicts of Interests

No potential conflicts of interest were disclosed.

Acknowledgments

Radiochemical synthesis and imaging was conducted in the Center forSmall Animal Imaging at the Vanderbilt University Institute for ImagingSciences. The authors thank Dr. Carol Rouzer for critical reading of thismanuscript.

Grant Support

This work was supported by grants from the NIH (CA89450, CA128323, andCA136465).

Received March 23, 2011; revised September 2, 2011; acceptedSeptember 2, 2011; published online October 4, 2011.

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Figure 6. In vivo uptake statistics in tumors. A, female nude mice bearing1483 HNSCC and HCT116 tumors were dosed with compound 18F-7(�350 mCi, retro-orbital). The animals were imaged at 3 hourspostinjection in the microPET/CT Focus 220 instrument. After imaging,the animals were sacrificed by isoflurane overdose. The 1483 HNSCCand HCT116 tumors and leg muscles were removed and weighed, andradioactivity associated with each tissue was counted with a wellgamma counter. The plot shows the increased radiotracer (Bq/g) inCOX-2–expressing 1483 tumors versus COX-2–negative HCT116tumors (n ¼ 3, P ¼ 0.0004). B, nude mice bearing 1483 xenograft tumorswere predosed with vehicle or with celecoxib (20 mg/kg, i.p.)immediately prior to 18F-7 dosing (�350 mCi, retro-orbital) and imaged at3 hours postinjection. After imaging, the animals were sacrificed byisoflurane overdose, and tissues were removed and counted as in 6A.The plot shows the fold increase of radiotracer in the 1483/vehicle tumorto muscle ratio compare with the 1483/celecoxib tumor to muscle ratio(n ¼ 4, P ¼ 0.0004). (* ¼ statistically significant).

Uddin et al.





References1. Baker M. Whole-animal imaging: the whole picture. Nature 2010;463:

977–80.2. Giepmans BNG, Adams SR, Ellisman MH, Tsien RY. The fluorescent

toolbox for assessing protein location and function. Science 2006;312:217–24.

3. Neri D, Carnemolla B, Nissim A, Leprini A, Querz�e G, Balza E, et al.Targeting by affinity-matured recombinent antibody fragments of anangiogenesis-associated fibronectin isoform. Nat Biotechnol 1997;15:1271–5.

4. Becker A, Riefke B, Ebert B, Sukowski U, Rinneberg H, Semmler W,et al. Macromolecular contrast agents for optical imaging of tumors:comparison of indotricarbocyanine-labeled human serum albuminand transferin. Photochem Photobiol 2000;72:234–41.

5. Ntziachristos V, Tung C-H, Bremer C, Weissleder R. Fluorescencemolecular tomography resolves protesase activity in vivo. Nat Med2002;8;757–60.

6. Blum G, Mullins SR, Keren K, Fonovic M, Jedeszko C, Rice MJ, et al.Dynamic imaging of protease activity with fluorescently quenchedactivity-based probes. Nat Chem Biol 2005;1:203–9.

7. Achilefu S, Bloch S,MarkiewiczMA, Zhong T, Ye Y, DorshowRB, et al.Synergistic effects of light-emitting probes and peptides for targetingand monitoring integrin expression. Proc Natl Acad Sci U S A2005;102:7976–81.

8. Taketo MM. Cyclooxygenase-2 inhibitors in tumorigenesis (Part II). JNatl Can Inst 1998;90:1529–36.

9. Kurumbail RG, Stevens AM, Gierse JK, McDonald JJ, Stegeman RA,Pak JY, et al. Structural basis for selective inhibition of cyclooxygen-ase-2 by anti-inflammatory agents. Nature 1996;384:644–8.

10. Kalgutkar AS, Marnett AB, Crews BC, Remmel RP, Marnett LJ. Esterand amide derivatives of the nonsteroidal antiinflammatory drug,indomethacin, as selective cyclooxygenase-2 inhibitors. J Med Chem2000;43:2860–70.

11. Penning TD, Talley JJ, Bertenshaw SR, Carter JS, Collins PW, DocterS, et al. Synthesis and biological evaluation of the 1,5-diarylpyrazoleclass of cyclooxygenase-2 inhibitors: identification of 4-[5-(4-methyl-phenyl)-3- (trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (SC-58635, celecoxib). J Med Chem 1997;40:1347–75.

12. Uddin MJ, Crews BC, Blobaum AL, Kingsley PJ, Gorden DL, McIntyreJO, et al. Selective visualization of cyclooxygenase-2 in inflammationandcancerwith targeted imagingagents.CancerRes2010;70:3618–27.

13. Uddin MJ, Crews BC, Ghebreselasie K, Marnett L J. [123I]-Celecoxibanalogs as SPECT tracer of cyclooxygenase-2 (COX-2) in inflamma-tion. ACS Med Chem Lett 2011;2:160–4.

14. Prabhakaran J, Underwood MD, Parsey RV, Arango V, Majo VJ,Simpson NR, et al. Synthesis and in vivo evaluation of [18F]-4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamideas a PET imaging probe for COX-2 expression. Bioorg Med Chem2007;15:1802–7.

15. Vries EFJD, Waarde AV, Buursma AR, Vaalburg W. Synthesis and invivo evaluation of 18F-desbromo-DuP-697 as a PET tracer for cyclo-oxygenase-2 expression. J Nucl Med 2003;44:1700–6.

16. McCarthy TJ, Sheriff AU, Graneto MJ, Talley JJ, Welch MJ. Radio-synthesis, in vitro validation, and in vivo evaluation of 18F-labeledCOX-1 and COX-2 inhibitors. J Nucl Med 2002;43:117–24.

17. Kharasch ED, Thummel KE. Identification of cytochrome P450 2E1 asthe predominant enzyme catalyzing human liver microsomal defluor-ination of sevoflurane, isoflurane, and methoxyflurane. Anesthesiol1993;79:795–807.

18. Hart FD, Boardman PL. Indomethacin: a new non-steroid anti-inflam-matory agent. Br Med J 1963;5363:965–70.

19. Prasit P, Wang Z, Brideau C, Chan CC, Charleson S, CromlishW, et al.The discovery of rofecoxib, [MK 966, VIOXX�, 4-(40-methylsulfonyl-phenyl)-3-phenyl-2(5H)-furanone], an orally active cyclooxygenase-2inhibitor. Bioorg Med Chem Lett 1999;9:1773–8.

20. Kalgutkar AS, Crews BC, Rowlinson SW, Marnett AB, Kozak KR,Remmel RP, et al. Biochemically based design of cyclooxygenase-2(COX-2) inhibitors: facile conversion of nonsteroidal anti-inflammatorydrugs to potent and highly selective COX-2 inhibitors. Proc Natl AcadSci U S A 2000;97:925–30.

21. Kalgutkar AS, Kozak KR, Crews BC, Hochgesang GP, Marnett LJ.Covalent modification of cyclooxygenase-2 (COX-2) by 2-acetoxy-phenyl alkyl sulfides, a new class of selective COX-2 inactivators.J Med Chem 1998;41:4800–18.

22. Dinchuk JE, Car BD, Focht RJ, Johnston JJ, Jaffee BD, CovingtonMB, et al. Renal abnormalities and an altered inflammatory responsein mice lacking cyclooxygenase II. Nature 1995;378:406–9.

23. Loening AM, Gambhir SS. AMIDE: a free software tool for multi-modality medical image analysis. Mol Imaging 2003;2:131–37.

24. Winter CA, Risley EA, Nuss GW. Carrageenin-induced edema in hindpaw of the rat as an assay for antiinflammatory drugs. Proc Soc ExpBiol Med 1962;111:544–7.

25. Edelman MJ, Watson D, Wang X, Morrison C, Kratzke RA, Jewell S,et al. Eicosanoid modulation in advanced lung cancer: cycloxygen-ase-2 expression is a positive predictive factor, for celecoxib þchemotherapy–Cancer and Leukemia Group B trial 30203. J ClinOncol 2008;26,848–55.






2011;4:1536-1545. Published OnlineFirst September 7, 2011.Cancer Prev Res Md. Jashim Uddin, Brenda C. Crews, Kebreab Ghebreselasie, et al. Inflammation and CancerFluorinated COX-2 Inhibitors as Agents in PET Imaging of

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