of tumor cell hypoxia may be associated with a moreaggressive biologic behavior (1,2) has initiated studies suchas those by Reynolds et al. (3). Using a tumorigenic cell linecarrying a recoverable, chromosomal-based X-phage shuttlevector, exposure to hypoxia resulted in an elevated mutationfrequency and a mutation pattern similar to that seen inhypoxic tumors (3). Hypoxia has been shown to upregulategene products (4), which in turn have been associated withpoor clinical prognosis. Clinically, tumor hypoxia has beenassociated with decreased local tumor control after radiationor surgery for cervical cancers (1), metastasis of high-gradeextremity sarcomas (5), and local recurrence of head andneck cancers (2). In these studies, the presence of hypoxiahas been determined using the Eppendorf needle electrodesystem. This system is somewhat limited in its generalclinical application, primarily because of tissue access andthe need for a very skilled operator to obtain and interpretthe resulting P02 histograms. Two other clinically relevanttechniques for assaying the presence of hypoxia are thecomet assay (6) and the binding of 2-nitroimidazole drugsunder hypoxic conditions (7—12).Assessment of the numberand location of adducts formed by 2-nitroimidazole bindinghas been accomplished using immunohistochemistry (13)and nuclear medicine techniques (11). The primary advantage afforded by nuclear medicine techniques is theirnoninvasive nature. Noninvasive imaging of tumor hypoxiais currently being studied using either nuclear scintigraphyofiodine-labeled 2-nitroimidazoles, such as [131I]IAZA(14),technetium-labeled molecules (15), or PET imaging of18F-labeled drugs such as fluoromisonidazole (FMISO) (11).The advantages of PET imaging over conventional scintigraphy techniques include the use of nanomolar doses of drug(lower dose of radioactivity) and better resolution. However,the short half-lives of the radionuclides used may limit thetumor-to-normal tissue true contrast in resulting images, as aresult of insufficient clearance of nonspecifically bounddrug.

EF5 has been studied extensively, using immunohistochemical detection of the reduced adducts in tissues. Monoclonal antibodies conjugated with fluorescent dyes havebeen reported and extensively characterized (8, 13, 16). Correlation of EF5 binding to several independent endpoints,including radiation response (7) and response to tirapaza

The noninvasive assessment of tumor hypoxia in vivo is underactive investigationbecause hypoxia has been shown to be animportantprognosticfactorfortherapyresistance.Variousnuclearmedicine imaging modalities are being used, including PETimagingof 18F-containingcompounds.In this study,we reportthedevelopment of 18F-IabeledEF1 for noninvasive imaging ofhypoxia.EF1 is a 3-monofluoroanalog of the well-characterizedhypoxia marker EF5, 2(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide,which has been used to detecthypoxiain tumorandnontumorsystemsusingimmunohistochemical methods.Methods: We have studied 2 rat tumor types: thehypoxic Morris 7777 (07) hepatoma and the oxic 9LF gliomatumor, each grown in subcutaneous sites. PET studies wereperformed using a pharmacological dose of nonradioactivecarrierin additionto [18F]EF1to optimizeandassessdrugbiodistribution. After PET imaging of the tumor-bearing rats,tissueswere obtainedfor ‘y-countingof the 18Fin varioustissuesand immunohistochemicaldetectionof intracellulardrug adductsintumors.Inonepairoftumors,Eppendortneedleelectrodestudies were performed.Results: [‘8F]EF1was excreteddominantly through the urinarytract. The tumor-to-muscle(TIM) ratioof [18F]EF1in the Q7 tumors was 2.7 and 2.4 based on PETstudies and 2.1, 2.5, and 3.0 basedon ‘y-countingof the tissues(n = 3). In contrast, the T/M ratio of [18F]EF1in the 9LF gliomatumor was 0.8 and 0.5 based on PET studies and 1.0, 1.2, and1.4 based on v-counting ofthe tissues (n = 3). lmmunohistochemical analysisof drug adductsfor the two tumor types agreedwiththe radioactivityanalysis.IntheQ7 tumor,substantialheterogeneousbindingwasobservedthroughoutthe tumor,whereasinthe 9LFtumor minimalbindingwas found.Conclusion: [18F]EF1is an excellent radiotracer for noninvasive imaging of tumorhypoxia.

Key Words: hypoxia;tumor;PET;EFI ; EF3; imaging;18F;rat;hepatoma;glioma

J NuclMed2000;41:327—336

n recent years, understanding of the clinical and biologicsignificance of the tumor microenvironment has increased.The role of hypoxia in determining clinical outcome hasbeen of particular interest. The observation that the presence

ReceivedJan.4, 1999;revisionacceptedJun.21, 1999.For correspondence or reprints contact: Sydney M. Evans, VMD, University

ofPennsylvaniaSchoolofMedicine,DMsionofOncologyResearch,195JohnMorgan Bldg., Philadelphia, PA19104.

DETECTION OF TUMOR H@oxIA WITH [‘8FIEF1 •Evans et al. 327

Noninvasive Detection of Tumor HypoxiaUsing the 2-Nitroimidazole [18F]EF1Sydney M. Evans, Alexander V. Kachur, Chyng-Yann Shiue, Roland Hustinx, W. Timothy Jenkins, Grace G. Shive,Joel S. Karp, Abass Alavi, Edith M. Lord, William R. Dolbier, Jr., and Cameron J. Koch

Schools ofMedicine and VeterinaryMedicine, University ofPennsylvania, Philadelphia, Pennsylvania; Department of Chemistry,University ofFlorida, Gainesville, Florida; and the Cancer Center@University ofRochestei Rochester@New York

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mine and comet assay (17) have been made. In this study, wedevelop and present the PET imaging of [‘@F]EFl.

All drugs and precursors described in this study arederivativesof N-propyl-2-(2-nitroimidazol-l[H]-yl)-acetamide with 1 or more hydrogen atoms substituted byfluorine in the propyl group. The substitution occurs at thesecond and third carbon atoms (last 2 carbons of thepropylamine sidechain). The name of each compound comprises the letter E (originally chosen to indicate that the sidechains were similar to etanidazole), followed by the letter F(for fluorine), followed by 2 numbers separated by a comma(signifying the number of fluorine atoms on carbons 2 and 3,respectively). In cases in which the fluorine atoms arelocated only at the terminal carbon or in which there are noisomers, only a single number will be used (i.e., EF5 ratherthan EF2,3; EF3 rather than EFO,3; EF1 rather than EFO,1).Some precursors of the final drug are designated with a final“Br,―indicating a bromine on carbon 3. For example,N-(3-bromo-propyl)-2-(2-nitroimidazol-1[H]-yl)-acetamideis named EFOBr. The common synthesis procedure has beento make halogen-substituted propylamine and then conjugate it to 2-nitroimidazole acetate as previously describedfor EF5 (16).

1\vo unique aspects of nomnvasive hypoxic imaging with[‘8FJEF1using PET are described. First, a new rationaleregarding the importance and significance ofdrug half-life ispresented. We suggest that it is not necessary to require rapidclearance ofradioactive drug, as long asthe drug biodistribution is uniform. Second, we present immunohistochemicalstudies ofEFi + EF3 drug binding to compare with the PETimages. Both aspects are accomplished by administering the[‘8F]EF1with unlabeled carrier (EF1) plus EF3 at wholebody drug levels of (30 + 100) @imol/L,respectively. Use ofcarrierdrugin largeexcessoverradioactivedrugallowstheindependent assessment of drug biodistribution by conventional means (imaging, high-performance liquid chromatography [HPLC] determination) and, in addition, allows forimmunohistochemical detection of intracellular EF1IEF3adducts in the tumors using specific antibodies for EF3. The

FIGURE 1. Chemicalformulasof EFI,EF3,EF5, IAZA,FMISO,andBMS-181321.

correspondence of these analysis methods is presented in 2rat tumor types, 1 of which is hypoxic (Moths 7777hepatoma, Q7) and 1 of which is oxic (9LF glioma, 9LF).

MATERIALS AND METhODS

DrugsPreparation of EF1 (Fig. 1) from 3-fluoropropylamine and

2-(2-nitroimidazol-l[H]-yl)-acetic acid, and [‘tF]EFlfrom EFOBrand 2-(2-nitroimidazol-l[H]-yl)-acetic acid have been previouslyreported (18). Briefly, EF1 was prepared by conjugation of3-fluoropropylaminewith 2-(2-nitroimidazole-l[H]-yl)-acetic acidusing the method of mixed anhydride with isobutylchloroformate(16). EFOBr was synthesized by an analogous method using3-bromopropylamine. [18F]EF1 was prepared from EFOBr andpotassiumkryptofix[2.2.2] @Fin dimethylsulfoxide(DMSO)at120°C.A typicalreactionmixturecontained 1 mLDMSO, 10 pmolEFOBr (2.9 mg), 7 iimol of [KKrflF, and 1.5 pmol potassiumkryptofix carbonate ([KKrf]2CO3).The chemical yield of EF1 wasup to 2%ofthe initialEFOBr.Purificationwas achievedby dilutingthe DMSO mixture with water and passing it through an anionexchange column. This simple method achieved up to 95%radiochemical purity, although clinical usewould clearly require anadditionalpurificationby HPLC.The final specific activity of theEFI used for injection was 5.6 X 10' MBq/mol. This low specificactivity was the result of the addition of nonradioactive fluoride (7mmol/L) during [‘8F]EFIsynthesis and nonradioactive EF1 ascarrier for injection. EF3 (Fig. I) was synthesized by Dr. MikeTracy (SRI International, Palo Alto, CA).

Tumor ModelsQ7 cells were obtained from the American Type Culture

Collection (Rockville, MD). They were maintained in exponentialgrowthat 37°Cby transfersat 3.5-dintervals.GrowthmediumwasmodifiedEagle'smediumsupplementedwith 15%fetalcalf serumand standard penicillin and streptomycin. 9LF cells were obtainedfrom Dr. Alan Franko (Cross Cancer Institute, Edmonton, Albe@ta,Canada). These cells have morphologic characteristics of 9Lglioma tumors but, when grown in vivo in our laboratory, wereradiation sensitive and bound minimal EF5 (Koch and Evans,unpublished data, 1995).They were maintained as described for Q7cells, except that bovine calf serum was used as a media supplement.

NO2

NA/\,N H@,CH2@

\_J EFI

NO2

NN ,NH@,,,CH2

EF3

NO@

N@,N ,NH@,,@CF2

EF5

:@z; @@r20. @0

H

N'@@'N@K@=J CH2F

Fluoromisenidazole IAZA BMS-181321

328 THE Jou@N@i. OF NUCLEARMEDICINE •Vol. 41 •No. 2 •February 2000

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All animal studies conformed to the regulations of the University of Pennsylvania InstitutionalAnimal Care and Use Committee.For normal tissue biodistribution studies, 4—6-wk-oldBALB/cnu/nu female mice were used. Male Buffalo rats (Harlan SpragueDawley, Indianapolis, IN) and male Fischer rats (Charles River,Wilmington, MA) were used for Q7 and 9LF studies, respectively.Tumors were produced by injecting 1 million tumor cells subcutaneously. The 9L tumor model takes 10—14d to grow to a I-cctumor, and the Q7 tumor takes 14—20d to grow to the same size.

In VivoStudiesTwo typesof in vivo experimentswereperformed:

I . Nontumor-bearing mice: biodistribution (normal tissues andblood) of [‘tF]EFI

2. Thmor-bearing rat studies (6 animals total):

a. PET imaging (n = 4)b. Biodistributionof[‘tFJEFlintumor,normaltissues,and

blood (n = 6)C. Immunohistochemical detection of EF1IEF3 in tumor

(n 4)d. Eppendorf needle electrode studies (n 2)

In 4 rats (3 9LF-bearing rats and 1 Q7-bearing rat), the bladderwas exteriorized. This allowed for the bladder to be expressedpreceding each image. This technique avoided the problem of highbladder activity dominating the camera's available response range.ForPETimagingstudies(totaltime, —2.5h), rodentswere initiallyanesthetized with intraperitoneal ketamine (130 mg/kg) and xylazinc (1.3 mgfkg), and anesthesia was maintained using additionalsmaller intraperitoneal injections of anesthetic, as needed. Drugwas injected in a lateral tail or femoral vein; the total volumeinjected (in milliliters of physiological saline) was 1% of theanimal's weight (in grams). The injection volume containedapproximately 1.9 MBq [‘tflEFl,3 mmol/L carrier EF1, and 7mmol/L EF3. Assuming an even biodistribution, the whole-bodyconcentration of EFI + EF3 was therefore 130 @imolIL.In onestudy of 2 rats, only 0.56 MBq [‘tF]EFIcould be synthesized, andthe resulting PET studies were not interpretable because of poorcounting statistics. After PET imaging studies, tumor, normaltissues, urine, and blood were removed, and the mass andradioactivity of a portion of each sample were determined. Tumorand liver were frozen for immunohistochemical analysis of EF3biodistribution. EF3 was used as the dominant carrier, because itwas expected that its biodistribution would be more similar to EFI(because of increased polarity) than to EF5. Mice were injectedwith a proportionately smaller volume of drug mixture through thetail vein.

PETImagingandinterpretationThe HEAD Penn-PET scanner (University of Pennsylvania,

Philadelphia, PA) has a large axial extent, without septa, to achievehigh sensitivity with 3-dimensional imaging techniques. Detailsregarding the scanner have been previously reported (19). Immediately before image reconstruction, the data were corrected forscatter and randoms. Image counts were collected for a 5-mminterval at approximately 10, 30, 60, and 120 mm after druginjection.

Regions of interest (ROIs) were manually drawn over thenormal tissues and tumors. Similar regions were placed at the samelevel for each image. The activity within these ROIs was thenmeasured and the values, in counts per second per voxel, were

plotted against time. The kidneys could not be evaluated for I ratbecause high bowel (stomach) activity precluded reliably distinguishing the right kidney (data not shown). As noted earlier, ROIscouldnotbe reliablydrawnin 1 pairof ratsbecauseof poorcounting statistics, and only the tissue -y-countsare included fromthese2 animals.

BiodIstributlon StudiesAt various times after drug injection, tissues, blood, and urine

were collected. Each sample was weighed. The radioactivity ofeach sample was measured in a sodium iodide well counter(Gamma Counter 5000 Series; Packard Instrument Company,Downers Grove, IL), and the activity was expressed as a percentageinjected dose per gram of sample. This value was calculated bycomparison of the tissue counts to a known percentage of theinjected activity, measured at the same time.

Tissue Preparation for Antibody StainingandAnalysisof Sections

Tumor sections were cut at 14-jim thickness using a MicromHM 505N cryostat and collected onto poly-L-lysine--coated mmcroscope slides. The sections were fixed for 1h in ice-cold Dulbecco'sphosphate-buffered saline (1X PBS) containing freshly dissolvedparaformaldehyde(4%,pH 7.1—7.4,P-6148;SigmaChemicalCo.,St. Louis, MO). The rinsing and blocking of tissue sections wasidentical to that described previously for EF5 (13). Monoclonalantibodies (designated ELK5-A8) made against EF3 tissue adductswere similar to those previously described for EF5 (16). Theseantibodies were conjugated with the green-excited, orangeemitting fluorescent dye, Cy3 (Amersham, Arlington Heights, IL).

EF3 binding was assessed by imaging the tissue sections at theappropriate wavelengths for Cy3. Slides were imaged using aPhotometrics “Quantix―cooled CCD camera (Photometrics, Thcson, AZ), run by a Macintosh 9600 computer (Apple Computers,Inc., Cupertino, CA) and IPlab software (Scanalytics, Fairfax, VA).Sections were automatically scanned at constant camera exposureusing a Ludl electronic stage, run by the same computer andsoftware. Preceding micro@ope use, the brightness of the fluorescent bulb was calibrated so that the absolute brightness of thefluorescence could be determined. Cy3 dye with absorbency of1.25 at 549 nm was loaded into a hemocytometer, and a fluorescenceimage was recordedafter focusing the microscopeon theruled grid of the hemocytometer. Image fields of 1.05 X 0.7 mmwere obtained for a lOX objective.

Eppendorf Needle Electrode MeasurementsThe Eppendorf needle electrode was calibrated by alternately

bubbling N2and air through the reference chamber and producing acalibration set before and after in vivo tumor measurements. Asmall portion of the skin overlying the tumor was removed toprovide access for needle electrode insertion, and a scalpel bladewas used to nick the capsular surface of the tumor. The electrodewas advanced (typically 2—3mm) through this opening until stablereadings were observed. The needle progressed in 0.4-mm steps byadvancing 0.7 mm followed by 0.3 mm withdrawal before the nextmeasurement. P02 values were normalized to pre- and postmeasurementcalibrations.

RESULTS

Structures of drugs used in this study, as well as FMISO,IAZA, and BMS-18l32l, are shown in Figure 1.

DETECTION OF TUMOR Hypoxi@ wim [‘8F]EF1 •Evans et al. 329

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The biodistribution of [‘8FIEFIwith carrier EF1IEF3 wasdetermined in 5 mice. Tissues were removed and counted ina ‘y-counterat 1 (n = 1), 5 (n = 2), and 90 (n = 2) mm afterinjection. At 1 mm, the radioactivity was in substantialdisequilibrium, with higher specific activities in tissues withlarge fractional blood volumes (blood, liver, heart, musclekidney, lung; data not shown). By 5 mm, most nonexcretorytissuecontainedsimilardrugconcentrations(Fig. 2A). Anexception was brain, with about 10-fold reduced specificactivity compared with other organs and tissues. [‘8F]EF1was excreted primarily through the urinary tract, as cvidencedby the tremendousincreasein urinecountsby 90mm; at 5 mm, the fractional activity per gram was negligible, whereas by 90 mm it had increased to 0.6 (Fig. 2B). Toa much lesser extent there was excretion through thegastrointestinal tract, as evidenced by increasing counts inthe cecum. However, at 90 mm, the fractional activity pergram was 20-fold higher in urine than in cecum. In all othertissues except bone (tibia), the radioactivity counts decreasedsubstantiallyovertime.Boneactivitylikelyreflectscontamination of the injectate by [‘8F]F.

Transverseandsagittalimagesatthelevelof thetumorin2 rats, 120 and 135 mm after [‘8F]EF1injection, arepresented in Figure 3. Figures 3A and B show the oxic 9LFtumor and Figures 3C and D show the hypoxic Q7 tumor. InFigures 3A and C, the 9LF tumor could not be discriminatedfrom other tissue along the right body wall proximal to thekidney, despite presenting Figure 3C at maximal sensitivity.Asaresultof thishighcontrast,activitycouldbeseenintheskeletal system (midline activity: spine).

At 120 mm after injection, the Q7 tumor was easily seen

in the sagittal image on the rat's left body wall, adjacent tothe region of the lung, diaphragm, and liver (Fig. 3C). Highlevelsof activitycouldalsobeseenin theupperquadrantofthe abdomen. These represented activity in the gastrointestinal tract (stomach) and kidney. In the transverse sectionthrough the midportion of the Q7 tumor (Fig. 3D), the onlysubstantial activity was seen in the tumor. The only manipulation to the Q7 image was to collimate out bladder activitybefore analysis. This was necessary because of the very highurine activity (corrected in later experiments by exteriorizing the bladder; thus, the portion of the body presented in thesagittal image for the Q7 was shorter than that for the 9L).Similarfindingswereobtainedin a secondQ7-bearingrat,but the tumor was located in the subcutaneous tissuesadjacent to the abdominal organs (data not shown). Differencesin bowelretentionbetweenthe9LF andQ7-bearingrat were possibly attributable to differences in reagentpreparation purity resulting in variations in route of excretion (e.g., almost entirely renal versus partially through thegastrointestinal tract) but may also have been caused byanimal-to-animal variations, relative food intake, etc.

At the completion of the PET studies, tissues wereremoved from 2 Q7-bearing rats and 2 9LF-bearing rats andanalyzed for radioactivity using -y-counting (Fig. 4). -y-countstudies(Fig.4A) supportthedatapresentedin Figure2 formice (e.g., high renal activity as a result of drug excretion).Irrespective of tumor type, activities in muscle, heart,spleen, and brain were similar and relatively low. Higherlevelswerepresentin theboneandQ7 tumor.Figure4Bpresentsthetumor-to-muscle(TIM) ratioof -y-countsfor 6rats(Q7 tumors:2.1,2.5,3.0;9LF tumors:I.0,1.2,1.4).

A III B

1

Brain

@om@h

Brain

Spleen

Lung

Muscle

Heart

Stomach

Kidney

Intestine

Liver

Cecum

TibiaS

BloodS

Urine

FIGURE2. Relativeretention(fractionofinjectedactivity/g)ofradioactivityafter injection of [18F]EFI into mice, with tissuescollected at 5 (A) and 90 (B) mm. Withexception of brain, uptake was nearlyequivalentfor all but excretorytissues by 5mm (urine counts were not different frombackground).By 90 mm,braintissuewas inequilibrium with blood and other low-binding tissues,and most radioactivitywas nowfound in urine. Bone retention was likelycausedby contaminatingfluoride ion.

Urine

O.ftOl 41.4)1 0.1 O.(N)I 41.01 0.1

FRACTIONINJECTEDDOSEPERGRAM

330 THE JOURNALOF NUCLEARMEDICINE •Vol. 41 •No. 2 •February 2000

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0

FIGURE 3. Transverse and sagittal images in 2 tumor-bearingrats120—135 mmafter [18F]EFIinjection.(A) Sagittalimageof rat bearing 9LF tumor adjacent to rightthorax (arrow). (C) Transverse image ofsame animal at plane of tumor.Despite

__@—@=-i adjustment of camera sensitivity and con

trast, tumor cannot be visualized adjacentto body wall on either image. (B) Sagittalimage of rat bearing 07 tumor (arrow). (D)Transverseimageof sameanimalat planeof tumor (arrow). Note that tumor uptake ismuch higherthan in any other tissue inplane.k = kidney;s = stomach.

D

0

PET images (e.g., Fig. 3) were analyzed to obtaintime—activitycurves for various tissues (Fig. 5). In all 4 ratsstudied, renal activity decreased over time (data not shown).There was no difference between the activity within themuscles, which were ipsilateral versus contralateral to thetumor, and these data were pooled. For Q7 tumors, the T/Mactivity ratios (Fig. 5A) initially decreased, then rose to aplateau. For 9L tumors, the T/M was constant with time (Fig.SA). For the Q7 tumors, T/M ratios of 2.4 and 2.7 wereachieved; in contrast, the T/M ratio for the 9LF tumors neverexceeded 1. Similar conclusions arose from measuringabsolute activity in each tumor versus time, normalized tothe earliest time point (Fig. SB). In the Q7 tumors, tumoractivity decreased slightly over the first 50 mm, thenincreased at 125 mm, followed by a plateau. By contrast, inthe 9LF tumors, there was a steady decrease in activity overtime.

In 2 rats, 1 bearing a 9LF and 1 bearing a Q7 tumor,Eppendorf needle electrode studies were performed. In theQ7 tumor, the median P°2was 2.25 mm Hg and in the 9LF,8.2 mm Hg. These data corresponded to the observation of a2.5 (Q7) and 1.0 (9LF) T/M ratio using y-counts.

After PET imaging, a portion of the tumor was frozen forsectioning and immunohistochemical staining with ELK5-A8antibodies (specific against EF3 adducts; Lord and Koch,unpublished data, 1994) to corroborate the presence of

hypoxia in the PET imaged tumors (Fig. 6). A typical I X0.7 mm region of one Q7 tumor is presented in the lowerpanel (Fig. 6B). Diffuse EF1/F3 binding was seen throughout the section. Regions of highest drug binding wereadjacent to areas of cell death and necrosis. Nonbindingregionsrepresentingviable,aerobiccellswerealsoseen.Incontrast, there was a relative absence of binding in a 9LFtumor (Fig. 6A). Despite a camera exposure almost identicalto that in Figure 6B (6 versus 6.5 s) only a few scatteredregions with limited EFI/EF3 binding could be identified.Regions of necrosis were not identified in the 9LF tumor.

DISCUSSION

In recent years, there has been increasing interest in thediagnosis of hypoxia as a prognostic marker of disease. Thisinterest has been intense in the field of oncology, in whichhypoxia, based on Eppendorfelectrode data, has been shownto predict tumor recurrence and metastasis (5). There arenow preclinical and early clinical data supporting the use ofantibody recognition of nitroimidazole adduct formation intissues as a measure of hypoxia (13,20), and human clinicaltrials are underway (21). However, I limitation of this

technique is the invasiveness of the assay (e.g., the requirement to obtain a tissue sample). In nononcologic diseases,such as myocardial infarction (22) or stroke, an invasive

DEmcrIoN OF TUMOR HYPOXIA WITH [‘8FJEF1•Evans et al. 331

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A

Blood

Liver

o.@1

FRACTION INJECTED DOSE PER GRAM

B

z

assay would be contraindicated or impossible. Thus, severalgroups have been developing noninvasive assays to detectthe distribution of hypoxic markers using nuclear medicinetechniques. Examples include SPECT scanning of['311}IAZA(23), nuclear medicine imaging of [@Tc]-labeled HL91(24), and PET scans of [‘8F]FMISO(25,26). Thus far,several scientific and technical problems have been overcome, resulting in limited success. For the ‘311-basedtechniques, whole-body radiation dose and deiodination (27) areproblematic, but the available clinical data using 123!areencouraging (23,28). @mTc@HL9lhas been studied as both atumor (24) and myocardial imaging agent (29). It is unclearfrom the data presented whether this drug represents a bloodflow agent or truly images hypoxia. Ballinger et al. (30)

Q7 TUMORS 9LF TUMORS

FIGURE4. (A)-y-countsof tissuesfromtumor-bearingrats. High renal activity wasthe result of drug excretion. In 4 rats,irrespectiveof tumortype, heart,muscle,spleen, and brain levels were similar andrelativelylow.Higherlevelswerepresentinliver,kidney,and Q7 tumor. (B)T/M ratiofor3 Q7tumors(blackbars)and3 9LFtumors(whitebars).

studied the @‘@‘Tc-labeledcompound BMS181321 in vitro.Their studies suggest that this compound accumulates inboth aerobic and hypoxic cells, with the latter being higherand time-dependent. For PET studies, clinical data haveemphasized [18F]FMISO. This agent has been studied inseveral anatomic sites in humans, including gliomas (25),lung cancer (31), and nasopharyngeal carcinoma (26).Because picomolar drug concentrations were administered,drug biodistribution was difficult to assess. In addition,becauseof low tumor-to-normaltissueratios,it wasnecessary to perform kinetic analyses to assess what has beentermed “fractionalhypoxic volumes― (31). FDG has alsobeen studied from the perspective of imaging tumor metabolism, especially glucose. Minn et al. (32) have performed

332 THE Joui@.r@iOF NUCLEARMEDICINE•Vol. 41 •No. 2 •February 2000

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was used to avoid the problem ofextremely small concentrations of radioactive drug being sequestered or metabolizedbefore whole-body biodistribution occurred. Both EF1 andEF3 were added as components of the nonradioactivecarrier. We have specific antibodies to EF5 and EF3, but notEF1 . We chose EF3 rather than EF5 because the former isthe more polar (and thus more similar to EF1). The resultingbiodistribution data (Figs. 2 and 4) support the work ofLaughlin et al. (33) using EF5 in mice (e.g., the drug distributeswell to all organs and is excreted primarily through the urinarytract). One exception is that EFI, unlike EF5 (33), does notdistribute optimally into the central nervous system, probably because it is much less lipophilic than EF5 (octanol:water partition coefficient, 1:5 versus 7:1, respectively). Ithas been argued that a polar drug is required to avoidneurotoxicity at high drug concentrations (34,35). In studiesof EF5 in rodents (7, 13), larger mammals (Koch and Evans,unpublished data, 1997), and in ongoing human studies,neurotoxicity with EF5 has not been found. Thus, development of an EF5-analog-based noninvasive assay of hypoxiais unlikely to be limited by neurotoxicity. However, ourbiodistribution data suggest that EF1 is too polar for optimalbrain tissue access.

One of the most controversial aspects of the interpretationof nuclear medicine studies has been understanding exactlywhich physiological processes are being tracked. For cxample, FDG was originally developed to identify neoplasticcells based on their metabolic changes (e.g., atypical glucosemetabolism) (36). Continuing studies on this agent haveshown that its incorporation into cells is influenced by cellcycle (37), cell proliferative status (38), and hypoxia (32).Hypoxia would be expected only for cells showing a Pasteureffect (39). Similar questions regarding mechanism ofactivity have arisen with respect to labeled hypoxia imagingagents, because there has been no way to localize the agentsindependently and/or correlate their presence to an independent laboratory endpoint. Therefore, we set out to correlate 3techniques to determine the biodistribution of injected drug:‘y-countingand PET imaging for ‘tF,HPLC for parent drug,and immunohistochemistry for EF1IEF3 adducts. In theQ7-bearing rat shown in Figure 3A, for example, substantialbinding is seen in PET images of the tumor at 135 mm,especially compared with adjacent muscle. Analysis of theseimages result in a T/M ratio of 2.5 (Fig. SB). ‘y-countsfromthese tissues demonstrate aT/M ratio of3.0 (Fig. 4). Immunohistochemical analysis of the tumor tissue shows moderate bindingof EF1/EF3 in hypoxic regions adjacent to regions of necrosisand distant from vessels (Fig. 6B). Conversely, in the 9LFtumor, the T/M ratio based on ‘y-countswas 1.4 and thatbased on time—activitycurves was 0.8. In the immunohistochemical images, only a few small regions of EFI/EF3binding were seen. These data, especially in light of previouswork on EF5 (7,8,13), are highly supportive of the conceptthat late PET images of [@F]EF1 represent tumor hypoxia.Early uptake in the time course (5 miii) for the Q7 tumorslikely represents perfusion-related effects.

A

.@

0 50 100 150TIMEAFTEREF1 INJECTION(mm)

B 1.6@

@ 1.2.

@ 0.8

@ 0.4.

z

0.

0 50 100 150

TIMEAFTEREF1 INJECTION(mm)

FIGURE5. (A) For Q7 tumors,T/M activityratiosinitiallydecreased,then rose to plateau (D,U). For 9L tumors,T/M wasconstantwithtime(O,•).ForQ7tumors,T/Mratiosof2.4and2.7 were achieved; in contrast, T/M ratio for 9LF tumors neverexceeded1. (B) Absoluteactivityin each tumorversustime,normalized to earliest time point. In Q7 tumors (0$), tumoractivitydecreasedslightlyoverfirst 50 mm,then increasedat 125mm followed by plateau. By contrast, in 9LF tumors (0,1), therewassteadydecreaseinactivityovertime.

studiessuggestingthatFDG bindingincreasesin thepresence of hypoxia.

Herein we report biodistribution studies of [‘tF}EFlinnormal mice. Mice were chosen for this aspect of the studybecause of their small and consistent size and ease ofhandling tissues. Tumor and normal tissue studies wereperformed in 9LF-bearing Fischer rats and Q7-bearingBuffalo rats. These 2 tumor lines (and associated rat strains)werechosenbecausesubstantialdataareavailableontumorbiology and physiology in these tumors, e.g., that 9LFtumors are predictably oxic and Q7 tumors are moderatelyhypoxic when the tumors are of moderate size (1—2cmdiameter, as used herein). Biodistribution of 2-nitroimidazole drugshas not been reportedto be affectedby rodentstrain.

The results of investigations reported herein are unique,because the carrier drug (EF1/EF3) was used to a finalwhole-body concentration of 130 pmol/L. This technique

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FIGURE6. Immunohistochemicalanaly515of EF3 binding in Q7 (B) versus 9L (A)tumor tissue; each panel illustrates typical1.0 x 0.7 mm area. DiffuseEF1/F3bindingisseenthroughoutQ7section.Regionsofhighestdrug bindingwere seen adjacenttoareasof necrosis.Nonbindingregionsrepresentingviable,aerobiccellsarealsoseen.Note relative absence of binding in 9LFtumor (A). Despite camera exposure almost identical to that in B (6 versus 6.5 s),only scattered regions of limited EF1/EF3bindingcanbe identified.Forbothimages,pixel intensityhas been increasedby factorof 3 to improve visibility of binding in 9LFtumor.No alterationsin backgroundor contrast were made.

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Wiebe and Stypinski (40) provided an excellent review ofthe importanceof the pharmacokineticcharacterizationofradiopharmaceuticals, with emphasis on those that are beingdeveloped for imaging hypoxia. They have characterized theideal hypoxia pharmaceutical in the following way: “[it]must concentrate efficiently and selectively in target cells,while keeping the radiation dose to the patient withinacceptable levels. The radiopharmaceutical must be clearedquickly from the blood to minimize radioactivity to producea clear image of the (hypoxic) tissue in the patient. Perhapsthe most important requirement is that it must have a meanresidence time long enough to allow it to diffuse into thehypoxic areas, which are regions of diminished bloodsupply.―In furtherdiscussion,theynotethat“ideally,100%of the dose should be eliminated renally― and that “toaccurately interpret the results of an imaging study, theintracellular uptake must be detailed along with overallbiodistribution― (40). Some of these desirable properties

may be incompatible. Rapid renal clearance is typicallyassociated with highly polar compounds, and such compounds are known to have quite heterogeneous whole-bodydistributions, with particular emphasis on exclusion from thecentral nervous system and other neural tissues. Even withdominant renal clearance, drug half-lives will increasesubstantially in humans compared with the smaller animalsused in preclinical studies. This can be problematic whenradioactive isotopes are used, since the half-lives of theisotopes are fixed. Thus, it has been suggested that noninvasive hypoxia assays using PET will always suffer from thevery short half-lives of the available isotopes (14). Notmentioned in this and other reviews (14,40) is the potentialproblem of drug availability in vivo. Many nitroimidazolesare known to fragment to other molecules, which may ormay not have the desired labeling characteristics. Thisproblem is further exacerbated by the chemical stability ofthe isotope—drug linkage, with halogens other than fluorine

334 THEJOURNALOFNUCLEARMEDICINE•Vol. 41 •No. 2 •February2000

.d @$‘

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5. Brizel DM, Scully SP, Harrelson JM, et al. Tumor oxygenation predicts for thelikelihood of distant metastases in human soft tissue sarcoma. Cancer Res.1996:56:941—943.

6. Olive PL, DurandRE,LeRicheJ, JacksonSM. Gel electrophoresisof individualcells to quantify hypoxic fraction in human breast cancers. Cancer Res.1993:53:733—736.

7. EvansSM, JenkinsWT, JoinerB, Lord EM, Koch Ci. 2-Nitroimidazole(EF5)binding predicts radiation resistance in individual 9L s.c. tumors. Cancer Res.1996:56:405—411.

8. Koch Ci, Evans SM, Lord EM. Oxygen dependence of cellular uptake of EF5[2-(2-nitro-l H-imidazol-1-yl)-N-(2.2,3.3.3-pentatluoropropyl)acetamidej: analysis of drug adducts by fluorescent antibodies vs bound radioactivity. Br J Cancer.1995:72:869—874.

9. HodgkissRi, Jones0, LongA, et al Flowcytometricevaluationof hypoxiccellsinsolidexperimentaltumoursusingfluo.rescenceimmunodetection.BriCancer 1991:63:119-125.

10. Raleigh JA, Miller GO, Franko AJ, Koch Ci, Fuciarelli AF, Kelley DA.

Fluorescence immunohistochemical detection of hypoxic cells in spheroids andWmours. BrJ Cancer 1987:56:395—400.

II. Rasey iS, Grunbaum Z, Magee 5, et al. Characterization of radiolabeledfluoromisonidazole as a probe for hypoxic cells. Radial Res. 1987:111:292—304.

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13. Evans SM, Joiner B, Jenkins WT, Laughlin KM. Lord EM, Koch Ci. Identiflcation of hypoxia in cells and tissues of epigastric 9L rat glioma using EF5[2-(2-nitro- 1H-imidazol- I -yl)-N-(2.2,3,3,3-pentafluoropropyl) acetamidel. Br ICancer 1995:72:875—882.

14. ChapmanJD, EngethardtEL, StobbeCC, SchneiderRF,HanksGE. Measuringhypoxia and predicting tumor radioresistance with nuclear medicine assays.Radiother OncoL 1998:46:229—237.

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16.LordEM, HarwellL, KochCi. Detectionof hypoxiccellsby monoclonalantibody-recognizing 2-nitroinsidazole adducts. CancerRes. l993;53:5271—5276.

17. Brown JM, Giaccia AJ. The unique physiology of solid tumors: opportunities (andproblems) for cancer therapy. Cancer Res. 1998:58:1408—1416.

18. KachurA, Evans SM, Shive C-Y, etal. Synthesis ofnew hypoxia markers EFI and[‘5F]-EF1.App! Radiat iso:. 1999:51:643—650.

19. KarpiS, FreifelderR, GeaganMJ. et al. Three-dimensionalimagingcharacteristics ofthe HEAD PENN-PET scanner. JNucl Med. 1997:38:636-643.

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21. Raleigh IA. Calkins-Adams DP, Rinker LH. et al. Hypoxia and VEGF expressionin human squamous cell carcinomas using pimonidazole as a hypoxia marker.Cancer Res. 1998:58:3765—3768.

22. Bialik S. Greenen DL, Sasson IE, et al. Myocyte apoptosis during acutemyocardial infarction in the mouse localizes to hypoxic regions but occursindependently of p53. J C!in Invest. 1997:100:1363—1372.

23. Parliament MB. Chapman JD, Urtasun RC, et al. Non-invasive assessmentofhuman tumour hypoxia with l231-iodoazomycin arabinoside: preliminary reportof a clinical study. Br J Cancer 1992:65:90-95.

24. Cook Gi, Houston S. Barrington SF. Fogelman I. Technetium-99m-labeled HL91to identify tumor hypoxia: correlation with fluorine-18-FDG. I Nuci Med.1998:39:99—103.

25. Valk PE, Mathis CA, Prados MD, Gilbert IC, Budinger TF. Hypoxia in humangliomas: demonstration by PETwith fluorine-18-fluoromisonidazole. JNucI Med.1992:33:2133—2137.

26. Yeh SH, Uu RS, Wu LC. et al. Fluorine-l8 fluoromisonidazole tumour to muscleretention ratio for the detection of hypoxia in nasopharyngeal carcinoma. Eur INuciMed. 1996:23:1378-1383.

27. Cherif A, Wallace S. Yang Di, et al. Development of new markers for hypoxiccells: [‘3tljiodonrisonidazoleand [‘311]iodoerythronitroimidazole.J Drug Target.1996:4:31—39.

28. Groshar D, McEwan AJ, Parliament MB, et xi. Imaging tumor hypoxia and tumorperfusion. INuci Med. 1993:34:885—888.

29. Okada an, Johnson G Ill, Nguyen KN, Edwards B, Archer CM, Kelly JD.@“Tc-HL91.Effects oflow flow and hypoxia on a new ischemia-avid myocardial

imaging agent. Circulation. 1997:95:1892—1899.30. Baffinger JR. Kee 1W, Rauth AM. In vitro and in vivo evaluation of a

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being particularly susceptible to metabolism, exchange, orhepaticelimination.The pharmacokineticanalysesof thedrugs used in nuclear medicine assays often have createduncertainties because of the low drug concentrations usedand because assays are limited to detection of the associatedradioactive atom. This problem is paramount with technetium, because stable isotopes are not available.

In light of the preceding discussion, we believe that ourapproach to noninvasive assays of hypoxia differs in 3primary ways from previous studies:

1. In vivo drug stability of EF1 and its analogs isexceptionally high. This extends, as with other fluorinated compounds, to the stability of the bound isotope.

2. Use of high carrier drug concentrations allows assessment of the biodistribution of drug and its metabolitesby conventional (immunohistochemical) and HPLCbasedassays,in additiontoradioactiveassays.

3. High drug concentrations and reduced polarity willresult in a longer drug half-life than isotope half-life.Thus, we suggest emphasizing a uniform drug biodistribution. Imaging thus entails differentiating metabolisminduced increases on a constant background of nonmetabolized drug, possibly complicated by perfusionartifacts at early times (14).

The results provided herein demonstrate that EF1 providesmany ofthe advantageous characteristics ofa PET molecule withfew of the problematic ones. We clearly demonstrate that PETimaging with [‘tF]EFlis able to differentiate hypoxic versusaerobic tumors in rodents. The high T/M ratio seen in thehypoxic Q7 tumors allows an easy delineation of the lesionwithout complex image or kinetic analyses. Our ongoingwork is directed at further developing drugs in the EF1—EF5series for PET imaging. This study will emphasize increasing lipophilicity through multiple fluorinated sites, such as[‘8F]EF3and [‘tF]EFS,while maintaining pharmacologicconcentrations (up to 100 jimol/L) of nonradioactive drug.

ACKNOWLEDGMENTS

This work was supported in part by grants from theEducation and Research Foundation, Society of NuclearMedicine; Department of Radiation Oncology, University ofPennsylvania (Gillies McKenna, chairman); and grantsCA28332 and CA74071.

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2000;41:327-336.J Nucl Med.   Karp, Abass Alavi, Edith M. Lord, William R. Dolbier, Jr. and Cameron J. KochSydney M. Evans, Alexander V. Kachur, Chyng-Yann Shiue, Roland Hustinx, W. Timothy Jenkins, Grace G. Shive, Joel S. 

F]EF118Noninvasive Detection of Tumor Hypoxia Using the 2-Nitroimidazole [

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