29. Pietrzyk U, Herholz K, Fink K, et al. An interactive technique for three-dimensionalimage registration: validation for PET, SPECT, MRI and CT brain studies. J NucI Med1994;35:2011—2018.

30. Podreka I, Asenbaum 5, BrUcke T, et al. lntraindividual reproducibility of HMPAObrain uptake [Abstract]. J Cereb Blood Flow Metab 199 1;1l(suppl l):S776.

3 1. Bonte FJ, Stokely EM, Devous MD, Homan RW. Single-photon tomographicdetermination of regional cerebral blood flow in epilepsy. Am J Neuroradiol 1983;3:544—546.

32. Duncan R, Patterson J, Hadley DM, Wyper DJ, McGeorge AP, Bone I. Technetium99m HMPAO single photon emission CT in temporal lobe epilepsy. Acta NeurolScand 1990;8l:287—293.

33. Marks D, Chou A, Katz A, HotTer P, Spencer S. SPECT in patients with CNSheterotopias [Abstract]. Epilepsia 1990;3 1:669.

34. Henkes H, Hosten N, Cordes M, Neumann K, Hansen ML. Increased rCBF in gray

matter heterotopias detected by SPECT using 9@―Tchexamethyl-propylenamine

oxime. Neuroradiology 1991:310—312.35. Leiderman DB, Balish M, Sato 5, Kufta C, Reeves P. Theodore WH. Comparison of

PETmeasurementsofcerebral bloodflow andglucose metabolismforthe localizationofhuman epileptic foci. Epilepsy Res l992;l3:153—l58.

36. Elger CE, Lehnertz K. Ictogenesis and chaos. In: Wolf P, ed. Epileptic seizures and

syndromes. London: John Libbey and Company Ltd.; 1994:541—546.

37. Lehnertz K, Elger CE. Spatio-temporal dynamics of the primary epileptogenic area in

temporal lobe epilepsy characterized by neuronal complexity loss. Electroencephalogr

Clin Neurophvsiol 1995;95: 108 —117.

Key Words: PET; adrenal cortex; steroid synthesis; etomidate;11j3-hydroxylase

J NuciMed199839982-989

S a result of the increased use of CT, MRI and ultrasound,

incidentally found massesin the locationof the adrenalglandshave emerged as a clinical problem. Patients undergoing CT forreasons other than suspected adrenal disease manifest adrenallesions with a frequency of approximately 1% (1—3). Thepotential extent of this problem is illustrated by the fact thatadrenal lesions are found at autopsy in 1.9%—8.7% (4,5). Inmost cases (7O%—94%), these incidentally found tumors,termed “incidentalomas,―consist of benign cortical nonhypersecretory adenomas (6). If an adrenal lesion has been radiologicallydetectedinapatient,abiochemicalhormonalscreeningisperformed to rule out both adrenal medullary and corticalhypersecretory tumors. If hypersecretion is established biochemically, these patients are normally considered candidatesfor surgery. For patients without evidence of adrenal hypersecretion, the differentiation ofthe common adenomas from otherbenign lesions such as cysts and lipomas or from malignantlesions,i.e., adrenalcortical carcinomaor extra-adrenalmetastasis,mustbe established.In view of the low specificityof CT,MRI and other imaging methods for such differentiation,patients with nonhypersecretory tumors that are >3 cm indiameter are often referred for surgery to rule out a primaryadrenal cortical carcinoma.

Iodine-123-iodomethylnorcholesterol(NP-59) imaging (7—9)has a high specificity and accuracy in defining adrenal corticalmasses. A limitation of the method is the long waiting period(4—7days) from injection to imaging, making it cumbersomeand costly. Both hypersecretory and nonhypersecretory adenomas (and some hypersecretory adrenal cortical carcinomas)accumulate NP-59, whereas nonfunctioning primary and secondary malignant lesions, as well as other space-occupyinglesions, demonstrate decreased, distorted or absent uptake.

MethOdS With the purpose of developing a PET imaging agent fortumors of the adrenal cortex, we developed syntheses for@ 1C-etomidate and its methyl analog,@ 1C-metomidate. (R)-[O-ethyl-1-1 1C]Etomidate and (R)-[O-methyl-1 1C]metomidate were prepared

by reaction of the appropriate respective@ 1C-Iabeledalkyl iodideand the tetrabutylammoniumsatt of the carboxylic acid derivative.The specificity of binding to the adrenalcortex was tested throughthe use of frozen section autoradiographyof different tissues of therat, pig and human. Inhibitionof tracer binding was evaluatedwithetomidate, ketoconazoleand metyrapone,well-known inhibitors ofenzymes for steroid synthesis. Tracer binding to different humantumor samples was compared to immunohistochemical stainingwith antibodies for the steroid synthesis enzymes P450 11/3(11f3-hydroxylase),P450scc (cholesterolside-chaincleavageenzyme),P450 C21 (21-hydroxylase) and P450 17a (17ca-hydroxylase).ThreePET investigations, one with@ 1C-etomidate and two with@metomidate,were performed in rhesus monkey sections, includingthe adrenals,liverand kidneys.Time-activitycurves were generatedfrom measured tracer uptake in these organs. Results: In frozensectionautoradiographyof varioustissues,high bindingwas seeninthe adrenal cortex from all species, as well as in the tumors ofadrenal cortical origin. The level of liver binding was about 50% ofthat in the adrenals, whereas that of all other organs was <10% ofthe adrenalbinding.The adrenalbinding was blocked by etomidateand ketoconazoleat low doses but not by metyrapone.The bindingin the adrenal tumor samples correlated with immunostaining forP450 11g. PET studies in the monkey demonstrated high uptake inthe adrenalswith excellentvisualization.The uptake increasedwithtime without indicationof washout.Slightlyloweruptakewas seeninthe liver as compared to the adrenals, and in the late images, noorgansotherthanadrenalsandliverwereseen.Conclusion:Theseinvestigations indicate that@ 1C-etomidate and@ 1C-metomidatehavethe potentialto be usefulspecific agentsfor the visualizationofthe normal adrenal cortex and to provide positive identificationofadrenalcorticaltumors.

Received Jun. 18, 1997; revision accepted Sep. 27, 1997.For correspondence or reprints contact: Mats BergstrOm, PhD, Uppsa@ University

PET Centre, University Hospital, S-751 85 Uppsaia, Sweden.

982 THEJOURNALOFNUCLEARMEDICINE•Vol. 39 â€No. 6 . June 1998

In Vitro and In Vivo Primate Evaluation of Carbon11-EtomidateandCarbon-11-MetomidateasPotentialTracers for PET Imaging of the Adrenal Cortexand Its TumorsMats Bergstrom, Thomas A. Bonasera, Li Lu, Elisabeth Bergstrom, Cam Backlin, Claes Juhlin and Bengt LAngstromSubftmtomole Biorecognition Project, Japan Sciences Technology Corporation and Uppsala University PET Centre, andDepartment ofSurgery, University Hospital, Uppsala, Sweden

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Using NP-59 scintigraphy in 229 patients, Gross et al. (10)demonstrated 100% specificity and 70% sensitivity in charactenzing clinically silent adrenal masses found by CT. In theirreport, however, only 15% of the NP-59 avid lesions (adrenaladenomas) were confirmed by surgery. Boland et al. (11) usedPET with ‘8F-fluorodeoxyglucose to show an excellent discrimination between benign adrenal lesions with no uptake andadrenal metastases characterized by high uptake. The development of adrenal cortical imaging agents based on enzymeinhibitors was reported by Beierwaltes et al. (12,13). The ‘‘Cradiolabelingofananalogofmetyrapone,apotentandselectiveinhibitor of 1lf3-hydroxylase activity, has been reported (14),but these researchers concluded from results of rat biodistribution studies, that this tracer was not a viable candidate for the invivo PET imaging of 1lf3-hydroxylase activit@' in humans.“Faintadrenal images―were reported when 12 1-metyraponewas used in one patient with bilateral hyperplasia (15). Thus,successful human studies of enzyme inhibitor-based adrenalcortical imaging have not been reported.

Tomoreefficientlydiscriminateadrenalcorticalmassesfromnonadrenal masses presenting as incidentalomas on CT and tovisualize and lateralize small hyperfunctioning adrenal adenomas that are not clearly depicted by CT, we hypothesized thatPET might be potentially valuable, with its vast potential forusing radiolabeled substances.

As a biochemical target, we selected the cortisol- andaldosterone-synthesizingenzymes,whichshouldbefoundexclusively in lesions originating from the adrenal cortex. As thepotentially best tracer substance, we chose etomidate, which hasbeen shown to be the most potent available inhibitor of11f3-hydroxylase (16—19),a key enzyme in the biosynthesis ofcortisol and aldosterone. Etomidate, first reported in the 1960sand l970s and manufactured by Janssen Pharmaceutica, is animidazole derivative with short-acting hypnotic properties whenit is administered intravenously and has, thus, been used as ananesthetic in humans (20). Incubation studies with humanadrenocortical tissue have shown that etomidate predominantlyblocks the conversions of 1l-deoxycortisol to cortisol (IC50 =0. 15 pM) and of 1l-deoxycorticosterone to corticosterone (IC50= 0.03 pM) (16, 1 7) by inhibition of the cytochrome P450-

linked 1l@-hydroxylase enzyme system. Etomidate also inhibits l7a-hydroxylase (IC50 = 6—8 pAl) and, much less potently,the production of androstendione from l7a-hydroxyprogesterone (IC50 = 380 @.tM).Etomidate has no inhibitory effect on2l-hydroxylase.

During the development of the labeling methods for ‘‘C-etomidate, we also investigated the methylated form, 1metomidate. Both ofthese compounds were tested in vitro usingfrozen section autoradiography and in vivo in biodistributionstudies in the rhesus monkey to explore their potential as tracersfor the adrenal cortex and its tumors.

MATERIALSAND METhODS

ChemistrySynthesis of Carbon-i i-Labeled Ethyl Iodide. Carbon-i 1-

CO2 was produced on a Scanditronix MC-l7 cyclotron. Aftercryogenic focusing, 1‘C-CO2was transferred in a stream ofnitrogen to a glass reaction vessel that had been charged withMeMgBr (36 @mol,0.3 ml of a 0.12 M solution diluted 1:24 inEt20-tetrahydrofuran; Aldrich) 30 sec after the cryogenic ‘‘C-CO2 trap was removed from its liquid nitrogen bath. After theamount of radioactivity that passed through the reaction vesselandtrappedin an Ascante tubehadpeaked(-@-2.5mm), LiA1H4(0.2 mmol, 0.2 ml of a 1 M solution in tetrahydrofuran; Aldrich)

was added. The nitrogen flow was increased to 200 mi/mm, andthe vessel was heated to 130°Cover 3 mm. The nitrogen flowwas reduced to 10 mi/mm. A second reaction vessel connectedin series to the first was charged with HI (0.5 ml, 57%; Fluka),and the same amount ofHI was added to the first reaction vesselover 30 sec. After an additional 30 sec, the second reactionvessel was heated to 130°Cover 3 mm, and the volatilecomponents were distilled through a Sicapent drying columninto the substrate vial over 7 mm.

Preparation of the Precursor. (R)-1-(l-Phenylethyl)-lH-imidazole-5-carboxylic acid (R28 141, 22.8 mg, 105 @mol;CilagJanssen Pharmaceutica) was weighed into a 5-ml glass vial.Water (2 ml) and methylene chloride (2 ml) were added,followed by tetra-n-butylammonium hydroxide (60 pi of a 1.5M aqueous solution, 90 p@mol;Aldrich). The vial was equippedwith a magnetic stirring bar and capped, and the solution wasstirred for 18 hr at room temperature.The organic phasewasremoved, passed through a MgSO4 plug and filtered into aflask. Solvent was removed in vacuo and the residue wasdissolved in 2.2 ml DMF(N,N-dimethylformamide)(Aldrich).Each of eight oven-dried 0.8-ml glass high-performance liquidchromatography (HPLC) vials was charged with 250 ml of theresulting solution. The vials were crimp-capped and storedbelow 0°Cbefore use.

Synthesis of [O-Ethyl-i-Carbon-li]Etomidate. Carbon-i 1-ethyl iodide was distilled into the precursor solution in theHPLC vial described above. The vial was heated in a 130°Caluminum block for 7 mm. The vial contents were purified byHPLC (Beckman Ultrasphere ODS, 5 mm, 10 X 250 mm, 45%ethanol/55% water, 5 mllmin, 254-nm mass detection, radioactivity detection), and [0-ethyl-i-' ‘C]etomidate (ETO) wascollected from —9.9—i1.8 mm. For in vitro experiments, thetracer was diluted directly from the HPLC eluent. For in vivoPET experiments, the mobile phase was removed in vacuo at120°C,and the residue was dissolved in 10% ethanol/90%phosphatebuffer(pH 7.4). The productwasanalyzedbyanalytical HPLC for the concentration of etomidate and forradiochemicalpurity [BeckmanUltrasphereODS, 5 mm, 4.6 X250 mm, 65% acetonitrile:methanol (1 :1)135% water, 1 ml/min,254-nm mass detection, radioactivity detection, tR (etomidatestandard) = 7.7 mm].

Synthesis of [0-Methyl-Carbon-i i]Metomidate. Carbon-i 1-methyl iodide (prepared from [‘‘C]C02,LiA1H4 and HI) wasdistilled into the precursor vial, proceeding as described abovefor ETO. [0-Methyl-' ‘C]metomidate (MTO) was collectedfrom the preparative HPLC column from —6.5—7.8mm. For invivo experiments, the product was formulated directly bydiluting a l-ml aliquot of HPLC eluent with 8 ml of phosphatebuffer. The product was analyzed by analytical HPLC for theconcentration of metomidate and for radiochemical purity[Beckman Ultrasphere ODS, 5 mm, 4.6 X 250 mm, 60%acetonitrile:water(50:7)140% 50 mM ammonium formate (jH3.5), 1 ml/min, 254-nm mass detection, radioactivitydetection,tR (metomidate standard) = 5.2—5.3 mm].

Frozen-Sec@on AutoradiographyVarious tissues from rats and pigs (adrenal gland, liver, lung,

heart, kidney, bladder, colon, rectum, prostate, pancreas and brain),complemented with human tissues (normal adrenal gland, adrenaltumors, pancreatic cancer, uterine leiomyoma and synovia fromrheumatoid arthritis), were rapidly frozen and stored at —70°C.Frozen sections with a thickness of 25 @mwere cut with a cryostatmicrotome, adhered to gelatinized glass slides, well-dried at +4°Cand stored at —20°Cawaiting the experiments. For the bindingstudies, the glass-mounted tissues were preincubated for 10 mm in

PET IMAGINGOFTHEADRENALCORTEX•Bergstrom et al. 983

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@...,.

FIGURE1. Frozen-sectionautoradkgraphyperformedby incubatingtissuesliceswith 11C-etomidateat a 30 nM concentration for 30 mm,washingand exposingon phosphorimagingplate.ParallelsampIes were coincubated with 10 pM etomklate to indicatenonspecfficbinding.

Tris-HC1 buffer (pH 7.4). Carbon-l i-etomidate or ‘‘C-metomidatewas addedat a concentration of 30 nM, and incubation proceededfor 30 mm at room temperature. Parallel sections were incubatedwith radioactivity plus 10 p.M of etomidate for blocking specificbinding. Thereafter, the sections were washed three times for 1 mmon each occasion in buffer and then once in purified water andfinally dried for 10 mm at +37°C.A reference incubation standardwas preparedby placing 20 @tlofthe incubationsolutionon a smallfilter paper attached to a glass slide. The mounted sections wereplaced on a storage phosphor screen (Phosphorlmager; MolecularDynamics) for an exposure of 40 mm (21,22). The plate was readby a laser, and a quantitative image of the distribution of radioactivity was viewed on a computer screen. For quantification ofbinding, regions of interest (ROIs) were outlined on the sections,the incubation standard, the blocked sections and a backgroundarea of the screen. After subtraction of the background, specificbinding in a tissue section was calculated as total binding minus

uptake in a blocked section of the corresponding tissue, and thefraction of specific binding was calculated as the ratio of specificbinding to total binding. The specific binding was further correctedfor variations in specific radioactivity by dividing by the totalmeasured radioactivity in the incubation standard.

In separate experiments, the influence of incubation time wasinvestigated by incubation of pig adrenal and pig liver sections foriO, 20, 30, 40 and 50 mm. The influence of washing time wasinvestigated by incubating for 30 mm and washing three times for1 mm each time, and thereafter, the sections were left in the lastwashing bath for another 10, 20, 30 or 40 mm. The inhibition of‘‘C-metomidate binding to pig adrenal and liver sections was

examined either by adding nonradioactive etomidate at concentrations up to 10 pM together with the radioactivity or by addingetomidate at concentrations up to 3.3 @M60 mm before theradioactivitywasadded.Similarblockingstudiesat similar concentrations were performed with ketoconazole and metyrapone.

984 THEJouiu'@.i.OFNUCLEARMEDICINE•Vol. 39 . No. 6 . June 1998

Tracer dosecull 1-etomidate

Blocking 10 uM

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RGURE4. Semilogarithmicplotof inhibitionof11C-metomidatebindingtop@adrenalcortex exertedby etomidate(0 and •)and ketoconazo@(i@andA)at increasingdoses.Theinhibitorwasaddedalther60mmbefore(•andA) or slmuftaneousIy with tracer (0 and L@4.

The immunohistochemical staining was performed on 6-smacetone-fixed cryosections. As primary antisera, rabbit polyclonalantibodiesgeneratedagainstthe following enzymeswere used:bovine cytochrome P450 11 @3-hydroxylase(P450 11j3, diluted1:50; Oxygene Dallas, Dallas, TX) (24), porcine l7a-hydroxylase(P450 i7a, diluted 1:70; Oxygene Dallas) (25) and bovine sidechain cleavage enzyme (P450 5cc, diluted 1:30; Oxygene Dallas)(26). In addition, human antisera containing autoantibodies tocytochrome P450 2 i-hydroxylase were used (P450 C2 1, diluted1:150) (27). The sections were then incubated with biotinylatedgoat antirabbit IgG (BA1000, diluted 1:200; Vector Laboratories,Burlinghame, CA) or peroxidase-labeled rabbit antihuman IgG(P2 14, diluted 1:200; DAKO, Glostrup, Denmark), followed byincubation of avidin—biotin—horseradishperoxidase complex(PK4000; Vector). As negative control normal rabbit IgG (ZOi 13;

FIGURE2. Rslath,especificbindingof 11C-etomldatetofrozensecticns fromrat, @gand human @ssues.NOrmalizedto rat adrenalcortex binding(100%).Bars,s.d.

Adrenal Tissues and ImmunhistochemistryOne biopsy from a normal adrenal gland was obtained during

nephrectomy and biopsies from 13 adrenocortical tumors duringadrenal surgery. The adrenal tumors consisted of six adenomas(1—4cm in diameter) and seven adrenal cortical carcinomas (7—15cm in diameter). Of the adenomas, two were associated withprimary hyperaldosteronism (Conn's syndrome), two were associated with hypersecretion of cortisol and two were associated withnonfunctioning status. Of the carcinomas, one was associated withhypersecretion of estradiol, one was associated with hypersecretionof estradiol, cortisol and androgens and the remaining five wereassociated with no biochemical evidence of hormone excess. Allpatients with carcinomas demonstrated a pathological urine steroidprofile with increased urinary excretion of mainly 3f3-hydroxy-5-ene steroids and tetrahydro-l 1-deoxycortisol (23).

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FIGURE5. Relathiebindingof 11C-metomklateto 13 operative samples ofadrenaltumors correlatedwith the degreeof immunostaining(scale,0 = nostainingto 3 = strongstaining)for 11@3-hydrox@@4ase.0 = cancers;•=adenomas.

FIGUREa Time course of bindingof 11C-etomklate(0 and L@)and 11C-metomidate(•and A) to p@adrenalslices. Lower lines(t@and A) indicateamountof nonspecfficbinding.

PET Ir@ii@om@oOFTHEADRENALCORTEX•Bergstrom et al. 985

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FiGURE6. PE@images obtained afterintravenousinjection of 11C-metomidateinrhesusmonkeys.Leftimages(AandC)show the uptake pattern 0.5 mm afterinjection, and nght images (B and D)correspondingsections 20 mm after in_on. Upper images (A and B) showaxial sections,and lower images(C andD)show parasagittalsec@onsthroughthenght kidneyand adrenal.i@zrowindicatesadrenalcortex.

DAKO) and normal human serum obtained from a blood donorwere used in the same concentrations as the primary antisera.Staining was visualized by 3-amino-9-ethylcarbazole (KEBO Laboratories,SpAnga,Sweden)and counterstainedwith Mayer's hematoxylin.All antiserawere dilutedin phosphate-bufferedsalinecontaining 1% bovine serum albumin (Sigma Chemical Co., St.Louis, MO), and all incubations were performed at room temperature.

Immunoreactivity, reported as semiquantitative staining intensity, was classified as follows: 0 = none, 1 = weak, 2 moderateand 3 = strong.

Monkey StudiesThree rhesus monkeys were selected for PET investigations. The

monkeys were anesthetized with diprivan and tracrium and prepared for artificial ventilation. The monkeys were placed supine onthe bed ofthe PET camera (GE 4096) (28) and positioned to allowthe 10-cm field of view to image the abdomen including theadrenals. A transmission scan was performed using an external68Gesource.Carbon-i 1 etomidate (one study) or@ ‘C-metomidate(two studies) (95—135MBq) was injected intravenously andscanning was initiated with a dynamic imaging sequence in which14 frames were acquired over 45 mm. Quantitative images werereconstructed with attenuation and scatter correction. In the imagesobtained,ROIs weremanuallyoutlinedto representadrenals,liver,kidney and pancreas. Well-perfused organs, such as the kidneysand the pancreas, were identified in early images. For each regionand each study, time-activity data were generated, expressed as thestandardized uptake value (SUV) by dividing the regional tracerconcentrationby the ratio of total administeredradioactivitytobody weight. Time-activity data were plotted to allow a compari

sonofbehavior ofthe two tracersin the selectedorgans.To furtherdemonstrate the selective tracer uptake in the adrenals as comparedto liver, the ratios of adrenal to liver were formed and plottedversus time.

RESULTS

Rad@Syntheses ofETO and MTO were prepared by reaction of the

appropriate respective ‘‘C-labeledalkyl iodide and the tetrabutylammonium salt of the carboxylic acid derivative. Up to 1.6GBq of ETO with a specific radioactivity of up to 6.4GBq4i.mol was prepared in less than 60 mm from the end ofcyclotron bombardment, whereas up to 10 GBq of MTO with aspecific radioactivity of up to 120 GBq/p.mol was prepared inunder40mm.Autoradiography

The images from frozen-section autoradiography performedwith ‘‘C-etomidatedemonstrated high binding in adrenals fromall speciesincludingman,aswell as in theConn'sadenoma(Fig. 1). Relatively high binding was also noted in the livers,whereas other organs had low binding. Quantitatively, thebinding in the adrenals from rats, pigs and humans differed bylessthan 10% (Fig. 2). Liver binding was 40%—50%of adrenalbinding, kidney binding was 6%—l6% and binding of all otherorgans was <10%.

When binding studies were performed with ‘‘C-metomidatein different tissues, the highest uptake was noted in the adrenalsfrom the rat and pig (‘‘C-metomidatewas not studied in humanadrenals), followed by livers from these two species. Liverbinding was about 26% of the respective adrenal binding.

986 THEJOURNALOFNUCLEARMEDICINE•Vol. 39 •No. 6 •June 1998

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A B

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FIGURE7. Axialimages of the monkeyabdomenat the levslsofthe nghtadrenal‘@t and C) and left adrenal (B and D).

Upper images @Aand B) were obtainedwith the injection of 11C-etomldate,andlower images (C and D) were obtainedwith 11C-metomidate.

Binding to rat pancreas was 4%, and binding to rat and pigkidney was about 1%. All other tissues, including humanpancreascancers,uterine leiomyoma and inflammatory synovia, had binding of < 1% of that seen in the adrenals.

There was a good correlation between the binding of@ ‘C-etomidate and ‘‘C-metomidate (R2 = 0.95), but in general, thebinding to the adrenals was higher for ‘‘C-metomidateand thebindingto otherorganswaslowerfor thistracer.

Incubation with ‘‘C-etomidateor ‘‘C-metomidate for different lengths of time resulted in a nearly linear increase in thebound fraction (Fig. 3). With the longest incubation time (50mm), a tendency for equilibration was seen. After a 30-mmincubation time, the nonspecific binding was 13% for ‘‘C-etomidate and 2% for ‘‘C-metomidate. A 30-mm incubation,followed by washing for different time periods, indicated noloss of radioactivity for either of the tracers.

In the competition experiments, the specific binding ofâ€â€˜C-metomidate to pig adrenal sections was inhibited in a

dose-dependentmannerbyetomidatewithanIC50of 0.4 pM ifthe blocking agent was added simultaneously with the tracerand with an IC50 of 0. 13 pM if it was added 60 mm before (Fig.4). In studiesofthe ‘‘C-etomidateand ‘‘C-metomidatebindingto liver slices, the specific binding was much lower than in theadrenals. This binding was inhibited at lower etomidate concentrations, 0.09 and 0.07 j.@M,respectively. Ketoconazoleinhibited the ‘‘C-metomidate binding to adrenal sections withan IC50 of9.5 @Mwhen it was given simultaneously with tracerand with an IC50 of 2.3 @..tMwhen it was given 60 mm before.In the liver sections, the inhibition constants were 6. 1 and 2.1,.LAI,respectively. When attempts were made to inhibit ‘‘C-metomidate binding with metyrapone, no effect was seen up to10pM.

Immunohistochemistry revealed significant differences be

tween the different tissue samples studied. No correlation couldbe found between the degree of ‘‘C-metomidatebinding and theimmunohistochemicalstaining for P450 i7a. Compared toimmunohistochemistry for P450 scc and P450 C21, the ‘‘C-metomidate binding was, on average, higher in those sectionswith intense staining, but there was a wide range from close tozero to the highest values. There was a clear overlap inI ‘C-metomidate binding between sections with weak staining

and those with strong staining.With respect to staining for P450 1if3, there was a clear

correlation to ‘‘C-metomidatebinding with high binding inthose sections showing strong staining and low binding in thosewith weak staining (Fig. 5). There was no overlap in bindingbetween those with strong staining compared to the others.There was also a clear separation ofadenomas and cancers, withthe adenomas all having strong staining and high binding ofI ‘C-metomidate (mean ± s.d., 1 .40 ± 0.76) and the cancers

showingfaintor weakstainingandlow binding(0.17 ±0.15).

MonkeyStudiesIn the PET images acquired immediately after injection, high

uptake was noted in the kidney cortex and pancreas (Fig. 6).After only a few minutes, the adrenals exhibited the highestuptake of those tissues in the field of view of the PET camera.At the end ofthe study, only the adrenals and the liver could beseen, with significantly higher uptake in the adrenals. Visually,no clear difference was noted between@ ‘C-metomjdateandâ€â€˜C-etomidate (Fig. 7). The time course of uptake in the kidney

and pancreas was characterized by very high initial values(SUV -‘.-15), followed by a marked washout (SUV @‘2—3)(Fig.8). The adrenal uptake increased rapidly within the first fewminutes, followed by a slower increase to SUV levels of 15—20.The liver showed a tendency to reach a plateau (ETO) or slow

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0 10 20

TIme (mln)

0 10 20 30

Time (mm)

etomidate, has similar inhibitor@jproperties. Each inhibitorcould be readily synthesized in ‘C-labeledform in amountssuitable for in vivo and in vitro studies, although both theradiochemical yields and specific radioactivities were significantly higher for MTO than ETO. The lower yields and specificactivities obtained in the synthesis of ETO can be directlyattributed to the nonoptimized synthesis of [‘‘C]ethyliodide.The method provided a mixture of ‘‘Ciodides (methyl, ethyland isopropyl) (29), and the presence ofethyl iodide mass couldlikely be causedby a combination of the high reactivity ofmethyl magnesium bromide with atmospheric carbon dioxideand hydrolysis ofresidual diethyl ether in the reaction medium.

Frozen-section autoradiography performed with ETO andMTO demonstrated that the binding ofthese tracers was high innormal adrenal cortex tissues without any major interspeciesdifferences.The binding was basically irreversible in the timespan available for ‘‘C-labeledcompounds. As an indication thatthe binding observed is likely to be related to binding tolif3-hydroxylase, this binding is blocked by etomidate andketoconazoleat dosescorrelating well with 1ij3-hydroxylaseinhibition constants (i6—i9). Finally, there was a good conelation betweenthebinding of' ‘C-metomidateandstainingwithan antibody to 11f3-hydroxylase. However, one factor thatremains to be explained is the lack of competition by metyrapone, a well-known inhibitor of 1ij3-hydroxylase. The reasonfor this discrepancy may be that ETO and MTO bind preferentially to one isoenzymewhile metyraponebinds to anotherorthat the compoundsbind to different siteson the enzyme.

The monkey studies demonstrate that ‘‘C-metomidate and1‘C-metomidate are also suitable in vivo tracers. Although theadrenal cortex in the monkey is smaller than the resolutionelementofthe PETcamera,theadrenalswereclearlydelineatedand had higher uptake than did any other structure in theabdomen.This points to the possibility of visualizing theadrenals and lesions derived from them in vivo in humans, andindeed,we havealreadyshownthis in a pilot study(BergstromM, unpublished data, 1997).

Certain disorders that manifest themselves as lesions in theadrenal cortex may have steroid-synthesizing properties similarto or quite different from those of the surrounding tissue.Benign adenoma, for example, has been shown to have increased levels of 11j3-hydroxylase (30,3i). Other lesions foundin the adrenal cortex, such as adrenal cancer, may have varyinglevels of this and other steroid biosynthetic enzymes, as thelimited number of tumors investigated implies. Lesions ofnonadrenal origin, such as metastases originating from lung,breast and colorectal cancers, which frequently metastasize tothe adrenalcortex,areexpectedto not havethe mechanismsofadrenal steroid biosynthesis and, therefore, to not containenzymes such as 1l@3-hydroxylase. The method presented whenapplied in vivo in humans, should, therefore, allow a gooddiscrimination betweenlesionsof adrenalcortical origin andmetastasesfrom other tumor sites or pheochromocytomas andcouldpotentially influencetherapeuticandsurgicaldecisionsinpatientcare.

CONCLUSiONIn vitro studies using ‘‘C-etomidate and ‘‘C-metomidate

have indicated high uptake in the adrenal cortex of threespecies, including man, and very low uptake in most otherorgans. In vivo imaging of the adrenalswas possible in themonkey with good visualization. It is thus likely that either ofthese two tracers will allow in vivo imaging of the adrenalcortex and its tumors to be usedfor diagnosticor differentialdiagnostic purposes. With a shorter synthesis time and higher

A

(I)

B

>

Cl)

20

15

10'

Adrenals

Liver

Kidn.y

5.

20'

15'

10

5

Adrenals

Liver

Kidney

40 50

FiGURE& 1ime-ac@vftycurvesofadrenals,lNerandkidney,expressedinSW, in PET studies in monkeys performed with 11C-etomidate(A) and11C-metomidate(B).

decrease (MTO) after a maximum at about 10 mm afterinjection. The fmal liver SUV level achieved was about 10 forboth tracers.

The adrenal-to-liver ratio showed a minimum at about 5 mmafter injection, followed by almost linear increases with time toreach ratios of 1.3 for ‘‘C-metomidate and 1.8 for@etomidate.

DISCUSSIONWhile searching the literature for suitable agents that could

be used for the identification of adrenal cortex tumors, it waslogical to focus on one specific property of the adrenal cortex,namely, the biosynthesis of the steroids cortisol and aldosterone. This synthesis proceeded from cholesterol through severalhydroxylation and other cytochrome P450 enzymes. We assumed that tracers with specific binding to any of theseenzymes should have a high specificity for binding to theadrenal cortex and lesions originating from there. Etomidate, anethyl carboxylic acid ester, has been reported to be the mostpotent inhibitor of the adrenal steroid synthesis system known(i9) and, hence, was a logical choice for the attempt to find asuitable PET tracer. Metomidate, the methyl ester derivative of

988 THEJOURNALOFNUCLEARMEDICINE•Vol. 39 •No. 6 •June 1998

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radiochemical yields and specific radioactivities, ‘1C-metomidate is more suitable for future clinical studies.

ACKNOWLEDGMENTSThis work was supported by grants from the Swedish Cancer

Society, Uppsala Lions Foundation, Ake Wibergs Foundation andthe SwedishNaturalSciencesResearchCouncil(GrantK3463toBengt LAngstrom) and by postdoctoral fellowships from the Swedish Cancer Fund (to Thomas A. Bonasera), National ScienceFoundation(Grant INT-9505394 to Thomas A. Bonasera)andFulbright Commissions of the United States and Sweden (toThomas A. Bonasera). Etomidate, metomidate and R28l4l werekindly supplied by Janssen Research Foundation (Beerse, Belgium).

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PET IMAGINGOFTHEADRENALCORTEX•Bergstrom et al. 989

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1998;39:982-989.J Nucl Med.   Mats Bergström, Thomas A. Bonasera, Li Lu, Elisabeth Bergström, Carin Backlin, Claes Juhlin and Bengt Långström  Its TumorsCarbon-11-Metomidate as Potential Tracers for PET Imaging of the Adrenal Cortex and In Vitro and In Vivo Primate Evaluation of Carbon-11-Etomidate and

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