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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2008-0050
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The Journal of Clinical Endocrinology & Metabolism Vol. 93, No. 6 2358-2365
Copyright © 2008 by The Endocrine Society

[123I]Iodometomidate for Molecular Imaging of Adrenocortical Cytochrome P450 Family 11B Enzymes

Stefanie Hahner1, Andrea Stuermer1, Michael Kreissl, Christoph Reiners, Martin Fassnacht, Heribert Haenscheid, Felix Beuschlein, Martina Zink, Katharina Lang, Bruno Allolio1 and Andreas Schirbel1

Endocrinology and Diabetes Unit (S.H., A.St., M.F., M.Z., K.L., B.A.), Department of Medicine I, and Department of Nuclear Medicine (M.K., C.R., H.H., A.Sc.), University of Wuerzburg, D-97080 Wuerzburg, Germany; and Medical Clinic (F.B.), University Hospital Innenstadt, Ludwig Maximilians University, D-80336 Munich, Germany

Address all correspondence and requests for reprints to: Prof. Dr. Bruno Allolio, M.D., Endocrinology and Diabetes Unit, Department of Medicine, University of Wuerzburg, Josef-Schneider-Strasse 2, D-97080 Wuerzburg, Germany. E-mail: allolio_b{at}medizin.uni-wuerzburg.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Due to advances in conventional imaging, adrenal tumors are detected with increasing frequency. However, conventional imaging provides only limited information on the origin of these lesions, which represent a wide range of different pathological entities. New specific imaging methods would therefore be of great clinical value. We, therefore, studied the potential of iodometomidate (IMTO) as tracer for molecular imaging of cytochrome P450 family 11B (Cyp11B) enzymes.

Methods: Inhibition of Cyp11B1 and Cyp11B2 by IMTO, etomidate, metomidate, and fluoroetomidate was investigated in NCI-h295 cells and in Y1 cells stably expressing hsCyp11B1 or hsCyp11B2. Pharmacokinetics and biodistribution after iv injection of [123/125I]IMTO were analyzed in mice in biodistribution experiments and by small-animal single-photon emission computed tomography (SPECT). Furthermore, four patients with known adrenal tumors (two metastatic adrenal adenocarcinomas, one bilateral adrenocortical adenoma, and one melanoma metastasis) were investigated with [123I]iodometomidate-SPECT.

Results: In cell culture experiments, all compounds potently inhibited both Cyp11B1 and Cyp11B2. Adrenals showed high and specific uptake of [123/125I]IMTO and were excellently visualized in mice. In patients, adrenocortical tissue showed high and specific tracer uptake in both primary tumor and metastases with short investigation time and low radiation exposure, whereas the non-adrenocortical tumor did not exhibit any tracer uptake.

Conclusion: We have successfully completed the development of an in vivo detection system of adrenal Cyp11B enzymes by [123I]IMTO scintigraphy in both experimental animals and humans. Our findings suggest that [123I]IMTO is a highly specific radiotracer for imaging of adrenocortical tissue. Due to the general availability of SPECT technology, we anticipate that [123I]IMTO scintigraphy may become a widely used tool to characterize adrenal lesions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Adrenal masses, often detected incidentally, belong to the most prevalent human tumors (1, 2, 3). These tumors comprise a variety of different entities and, accordingly, require highly variable therapies ranging from immediate surgery to observational follow-up (4). Although both computerized tomography (CT) and magnetic resonance imaging (MRI) contribute significantly to the characterization of adrenal masses (5, 6), they often fail to differentiate lesions with low fat content. A tumor biopsy may be needed to definitely characterize the origin of the tumor, but this invasive procedure has been associated with a variety of adverse events, in particular with dissemination of tumor cells in adrenocortical cancer (7). Thus, noninvasive tools to characterize the tissue specificity of an adrenal lesion are of considerable interest.

The presently available norcholesterol scintigraphy, with [131I]iodomethyl norcholesterol (NP59) and [75Se]selenomethyl norcholesterol (Scintadren), is able to differentiate adrenal adenomas from other adrenal masses (8). However, the approach is time consuming and leads to a considerable patient radiation dose. Moreover, this technique is limited by poor spatial resolution and low specificity, because adrenal cancers may show highly variable uptake. Because of these limitations, the role of norcholesterol scintigraphy in the evaluation of adrenal tumors remains a matter of debate (2).

Recently, high-affinity binding of metomidate (MTO) to adrenal steroidogenic enzymes has led to its use as a radiotracer for adrenal steroidogenic tissue (9, 10, 11, 12, 13). Accordingly, [11C]MTO has been introduced as a tracer for positron emission tomography (PET), differentiating adrenocortical from nonadrenocortical tissue with high specificity (14). However, due to the short half-life of 11C (20 min) [11C]MTO-PET is restricted to PET centers with an on-site cyclotron. Moreover, the short half-life also limits its use to the early uptake of the tracer potentially missing the optimal target to background ratio. Therefore, longer-lived radionuclides and a better general availability of radiotracers for adrenocortical imaging are prerequisites for their successful use.

We have recently shown that iodometomidate (IMTO) binds to adrenal membranes with high affinity in vitro (15). Therefore, we hypothesized that the use of [123I]IMTO for single-photon emission CT (SPECT) and planar scintigraphy may provide a valuable alternative to PET imaging.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Synthesis of IMTO and fluoroetomidate (FETO)

Etomidate (ETO) and MTO were purchased from Janssen Pharmaceuticals (Janssen-Cilag, Neuss, Germany). Synthesis of IMTO was performed as previously described (16). For synthesis of FETO, MTO [(R)-(+)-methyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylate] (3.0 g, 13.0 mmol) was hydrolyzed for 10 min in 30 ml refluxing 10% NaOH. The solution was buffered to a pH of 4.0–4.2 and extracted three times with dichloromethane. The combined organic phases were dried over Na2SO4, filtered, and evaporated to dryness. The product was purified by recrystallization from water and dried in vacuum yielding 2.25 g (80.0%) white crystals. The resulting (R)-(+)-1-(1-phenylethyl)-1H-imidazole-5-carboxylic acid (541 mg, 2.5 mmol) was dissolved in a mixture of 1,2-dichloroethane (10 ml), sulfuric acid (1.4 ml), and 2-fluoroethanol (4.0 g, 62.5 mmol). The mixture was stirred at 80 C overnight, poured on ice, neutralized with sodium bicarbonate, and extracted three times with dichloromethane. The combined organic phases were dried over Na2SO4, filtered, and evaporated to dryness. The residue was purified by flash chromatography on silica gel (ethyl acetate/hexanes 50/50) yielding FETO (423 mg, 64.4%) as a brown oil.

Radiosynthesis of [123/125I]IMTO

Labeling was performed in a sealed conical vial containing 30 µg of the stannylated precursor in 30 µl ethanol and Na123/125I in 0.02 N NaOH. To initiate the reaction, 6 µl 1 N HCl and 10 µl chloramine-T (1.5 mg/ml) were added. The reaction was allowed to proceed for 3 min at room temperature and quenched by the addition of 7 µl 1 N NaOH. Purification of [123/125I]IMTO was performed by HPLC (Nucleosil 100-7 250 x 4.6 mm; CS Chromatographie Service, Langerwehe, Germany), with eluent CH3OH/H2O/diethylamine (60/40/0.2) at a flow rate of 1.5 ml/min. Capacity factor of [123/125I]IMTO k' = 6.8. The [123/125I]IMTO-containing HPLC fraction was evaporated to dryness at room temperature under reduced pressure. For iv injection, the residue was redissolved in a suitable volume of 0.9% saline and passed through a sterile 0.22-µm Millipore filter into a sterile vial.

Plasmid constructs and transfection

To induce expression of human cytochrome P450 family 11B1 (Cyp11B1) and Cyp11B2 enzymes in Y1 cells, the full-length cDNAs for the proteins were subcloned into the multicloning site of pcDNA3.1(zeo) (Invitrogen, Eggenstein, Germany). The cDNA fragments were isolated by PCR and digested by EcoRI. The individual fragments were ligated into the linearized vectors digested by EcoRI.

Human Cyp11B1 and Cyp11B2 enzymes were expressed in Y1 cells using liposome/lipid-mediated DNA transfection. Purified plasmid DNA was mixed with Lipofectamine (Invitrogen) transfection reagents according to the manufacturer’s protocol. To generate a stable Y1-Cyp11B1 and Y1-Cyp11B2 cell line, Y1 cells were transfected with the pcDNA3.1(zeo)-Cyp11B1 and pcDNA3.1(zeo)-Cyp11B2 vector, respectively. Transfected cells were selected with 1000 µg/ml zeocin (Invitrogen). To screen colonies, Western blotting and real-time PCR were used to determine the level of Cyp11B1 and Cyp11B2 expression. Colonies with the highest Cyp11B expression were further tested for their ability to synthesize cortisol or aldosterone from deoxycortisol (RSS) and 11-deoxycorticosterone (DOC), respectively. Experimental protocols were standardized regarding substrate concentrations and incubation periods.

Evaluation of specificity for CYB11B1 and CYP11B2 inhibition

To evaluate Cyp11B1 and Cyp11B2 inhibition by ETO, MTO, IMTO, and FETO, Y1-Cyp11B1 and Y1-Cyp11B2 cells were subcultured on six-well plates (0.5 x 106 cells per well) in 2 ml culture medium. The enzyme reaction was started after 24 h by the addition of 1 ml culture medium containing either RSS or DOC as substrate and the corresponding inhibitor. RSS and DOC were dissolved in ethanol to a final test concentration of 1 µM. For determination of IC50 values, the inhibitors were added to the culture medium at concentrations between 0.6 nM and 60 µM and incubated for 48 h. Y1-Cyp11B1 and Y1-Cyp11B2 cells, which were treated in the same way but without inhibitors, served as controls. As additional controls, untransfected Y1 cells were also incubated with RSS and DOC, respectively. Both RSS and DOC were obtained from Sigma (Deisenhofen, Germany).

Human tissue, isolation of RNA from solid tissues, and cDNA synthesis

Tissue preparation, RNA isolation, and cDNA synthesis were performed as described previously (17). Total RNA from human lung, kidney, and testis were purchased from BD Clontech (Heidelberg, Germany).

For details on cell culture, steroid determination, real-time PCR, and immunohistochemistry, see supplemental information (published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org).

Animal experiments

Male CD-1 mice were injected iv with 1 µCi (37 kBq) [125I]IMTO. At predefined time points (15 min, 30 min, 2 h, and 4 h), mice were killed (n = 6 per time point). Blood was collected, and heart, lung, liver, intestine, stomach, spleen, kidneys, adrenals, testes, and brain were excised and weighed. Radioactivity was measured using a {gamma}-counter. Results were expressed as a percentage of the injected dose per gram of organ weight.

Animal experiments were approved by and performed in compliance with the guidelines of the local animal care authorities (Az. 621-2531.01-48/03).

For SPECT imaging, mice (n = 10) were anesthetized with 2,2,2-tribromoethanol (Avertin) (0.5 mg/g body weight sc). The animals were placed on an animal holder and warmed to maintain body temperature during anesthesia. We injected into the tail vein 40 MBq [123I]IMTO dissolved in 200 µl 0.9% saline and, for visualization of the kidneys, 37 MBq [99mTc]2,3-dimercaptosuccinic acid ([99mTc]DMSA) dissolved in 200 µl 0.9% saline. Scintigraphy was performed with a single-head SPECT system (Ecam Signature; Siemens, Erlangen, Germany) with a multi-pinhole collimator. Data were acquired for 10–20 min with subsequent iterative reconstruction into three-dimensional datasets (HiSPECT, Scivis; Göttingen, Germany).

The toxicology and mutagenicity studies of non-radioactive IMTO were performed by BSL Bioservice (Planegg, Germany) and followed internationally accepted guidelines and recommendations. Five male and five female mice were treated in a single exposition with 2.0 µg/kg body weight of IMTO by iv injection. This dosing regime ensured a dosage per animal that is 100-fold the expected dosage for clinical use. A careful examination was made once a day. At the end of the observation period of 14 d, the animals were killed and necroscopy was carried out to record gross pathological changes. To investigate the potential of IMTO for its ability to induce gene mutations, plate incorporation and preincubation tests were performed with different Salmonella typhimurium strains. IMTO was tested in two independent experiments at several concentrations. Each assay was conducted with and without metabolic activation. The concentrations, including controls, were tested in triplicate. The highest dose group (100 µg/plate) corresponds to the 100-fold clinical dose.

Patient imaging

Patients were pretreated with 600 mg sodium perchlorate orally to prevent [123I]iodide uptake into the thyroid gland. Subsequently, 185 MBq [123I]IMTO was administered iv. Series of planar whole-body images (dorsal and ventral views) were acquired for patients between 5 min and 28 h after injection with a dual-head SPECT camera (Ecam duet; Siemens). The whole-body scans of patient 1 were performed with a standard activity of [123I]iodine in the field of view and were used to deduce time activity curves and to determine residence times in tissue with specific uptake. Activity not taken up in accumulating tissue was assumed to be distributed uniformly throughout the remaining body and excreted by the renal system with reasonable residence times in kidney and bladder. Patients 2–4 were also imaged using a dual-head SPECT-CT camera (Symbia T2; Siemens); in addition to a SPECT dataset, a non-contrast-enhanced low-dose CT was performed to enable anatomical allocation and correction for photon attenuation.

Statistical analysis

Significance of differences was evaluated by ANOVA using the statistical software program StatView 4.51. A P value of <0.05 was considered statistically significant with post hoc analysis carried out by Fisher protected least significant difference test. All results are expressed as means ± SD.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inhibition of steroidogenic enzymes by IMTO in vitro

To detect steroidogenic tissue in vivo, high-affinity binding of IMTO to steroidogenic enzymes is required. We, therefore, as-sessed the effect of IMTO in comparison with MTO, ETO, and FETO on steroidogenesis in human adrenocortical cancer cells (NCI-h295 cells). All compounds potently blocked adrenal steroid secretion in a dose-dependent manner with evidence of inhibition of both 11β-hydroxylase (Cyp11B1) and aldosterone synthase (Cyp11B2). At higher concentrations, also side chain cleavage enzyme (Cyp11A1) was inhibited. Accordingly, at lower doses (0.6–60 nM), accumulation of steroids upstream of the enzyme block was demonstrated with a shift toward increased androgen production (17-OH-progesterone, dehydroepiandrosterone, and androstenedione). At higher doses (>6 µM), inhibition of Cyp11A1 completely shuts down adrenocortical steroidogenesis in NCI-h295 cells (Fig. 1AGo). A comparison of the different compounds revealed similar potency concerning inhibition of steroidogenesis. In particular, iodination of MTO was not associated with a reduction of inhibitory activity.


Figure 1
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  		FIG. 1. A, Steroid secretion in NCI-h295 cells after incubation with ETO, MTO, IMTO or FETO (0.6 nM-60 µM). NCI-h295 cells were incubated for 48 h with the respective agents. Each experiment was performed in triplicate. At the end of the incubation period, steroid hormones [cortisol, 17-OH-progesterone (17-OH-P), dehydroepiandrosterone (DHEA), androstenedione, and aldosterone] were determined in the cell supernatant by commercially available RIAs. B, Expression of Cyp11B protein in Y1 cells stably expressing either human Cyp11B1 or Cyp11B2 in comparison with untransfected Y1 cells and human NCI-h295 adrenocortical cancer cells. No Cyp11B expression could be detected in untransfected Y1 cells, whereas high expression levels are detected in NCI-h295 and in Y1-Cyp11B1 and Y1-Cyp11B2 cells. C, Determination of IC50 values for Cyp11B1 and Cyp11B2 activity in stably transfected Y1-Cyp11B1 and Y1-Cyp11B2 cells. For determination of IC50 values, cells were incubated for 48 h with ETO, MTO, IMTO, or FETO (0.6 nM to 60 µM) together with the corresponding substrate RSS and DOC (1 µM), respectively. Results were calculated from three individual experiments, each performed in duplicate.

 
To further characterize selective binding to the closely related human Cyp11B1 and Cyp11B2 enzymes, we used adrenocortical Y1 cells stably transfected with either human Cyp11B1 or Cyp11B2 (Fig. 1BGo).

Cyp11B1 (11β-hydroxylase) and Cyp11B2 (aldosterone synthase) catalyze the final steps in adrenal steroid biosynthesis. Cyp11B1 converts RSS to cortisol, and Cyp11B2 catalyzes the conversion of DOC to aldosterone.

Therefore, Y1-HsCyp11B1 and Y1-HsCyp11B2 cells were incubated with the enzyme substrate RSS or DOC, respectively, and the corresponding Cyp11B inhibitor. In this experimental model, ETO, MTO, and their derivatives IMTO and FETO demonstrated potent inhibition of both Cyp11B1 and Cyp11B2 with IC50 values in the nanomolar range. IC50 values for inhibition of Cyp11B1 were as follows (nmol/liter): ETO, 0.99 ± 0.62; MTO, 4.60 ± 2.39; IMTO, 1.83 ± 1.83; and FETO, 2.94 ± 1.42.

IC50 values for inhibition of Cyp11B2 were as follows (nmol/liter): ETO, 4.80 ± 0.21; MTO, 16.7 ± 3.28; IMTO, 6.90 ± 1.37; and FETO, 20.2 ± 9.67 (Fig. 1CGo). However, Cyp11B1 was more potently blocked than Cyp11B2. In comparison with MTO, IMTO rather showed increased inhibitory activity with a significantly lower IC50 value for Cyp11B2 (P < 0.05) (Fig. 1CGo).

In vivo studies in experimental animals

For optimal imaging results in vivo high specific binding and low background activity are required. We therefore studied the biodistribution of [125I]IMTO in CD-1 mice (Fig. 2AGo). We observed fast and specific uptake in adrenal tissue with maximal target to nontarget ratios 15 min after iv injection followed by a rapid clearance within 4 h. Uptake in the adrenal glands was an order of magnitude higher than in other tissues, suggesting excellent imaging potential of IMTO [target to nontarget ratios 15 min after iv injection: adrenal gland (ag)/blood 11.8 ± 6.6; ag/heart 34.4 ± 11.9; ag/lung 12.8 ± 6.0; ag/liver 15.2 ± 7.3; ag/intestine 40.0 ± 12.8; ag/stomach 56.6 ± 25.3; ag/spleen 57.4 ± 20.9; ag/kidney 16.1 ± 6.9; ag/testis 30.9 ± 12.4; ag/brain 90.6 ± 31.1].


Figure 2
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  		FIG. 2. A, Biodistribution and pharmacokinetics of [125I]IMTO in male CD-1 mice. Mice were injected with 1 µCi (37 kBq) [125I]IMTO iv. Organ dosimetry was determined after defined time intervals between 15 min, 30 min, 2 h, and 4 h (n = 6 per time point). Results are expressed as percentage of injected dose per gram organ. Maximal tracer uptake in the adrenals is obtained 15 min after injection with rapid clearance during the following 4 h. B, Small-animal SPECT with [123I]IMTO in mice. Mice were injected with 40 MBq [123I]IMTO into the tail vein alone (left) or in combination with the kidney tracer [99mTc]DMSA. Data were acquired for 10–20 min with subsequent iterative reconstruction into three-dimensional datasets. For visualization of the kidneys, 37 MBq [99mTc]DMSA was coadministered (right). Image fusion of [123I]IMTO- and [99mTc]DMSA-SPECT clearly visualized the two adrenal glands at the cranial pole of the kidneys (arrows) shortly after tracer injection with only little background activity.

 
We next performed in vivo imaging studies with [123I]IMTO in male CD-1 mice using small-animal SPECT. Two distinct regions with high uptake were visible in the upper abdomen shortly after injection of [123I]IMTO with low background activity, consistent with the biodistribution data. To prove that the uptake was located in the adrenal glands, in addition to [123I]IMTO, we coadministered [99mTc]DMSA, which is predominantly taken up into renal tissue. The two adrenal glands clearly could be visualized at the cranial pole of the corresponding kidneys (Fig 2BGo and supplemental movie 2C, published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org).

These findings further indicated that [123I]IMTO is a highly suitable radiotracer for imaging of CYP11B expressing tissue in vivo. No relevant uptake of [123I]IMTO in testicular tissue of male CD-1 mice was detectable.

In toxicity testing studies, IMTO (2 µg/kg body weight) caused no compound-related mortality, either in female or in male animals within 14 d after the dose. All animals showed normal food intake and weight gain. At necroscopy, no evidence of gross pathology was found. Thus, IMTO exhibits no acute toxicity.

Furthermore, tests for mutagenicity revealed no biologically relevant increases in revertant colony numbers of bacterial strains after treatment with IMTO over a wide range of concentrations (1–100 µg/plate). In addition, IMTO did not cause gene mutations by base-pair changes or frameshifts (data not shown). Therefore, IMTO is considered to be nonmutagenic.

Studies in humans

Because IMTO binds specifically to both Cyp11B enzymes, significant expression of Cyp11B in adrenocortical lesions is a prerequisite for the clinical use of IMTO as a radiotracer in humans. We therefore investigated expression of Cyp11B mRNA in different human tissues. Cyp11B1 and Cyp11B2 expression was detectable in all investigated adrenal tissues as assessed by real-time PCR. Expression of Cyp11B1 and Cyp11B2 mRNA was high in all hormone-producing tumors and in most of the inactive adenomas. In adrenal adenocarcinomas (ACCs), Cyp11B1 and Cyp11B2 mRNAs were detectable in all tumors (Fig 3AGo), whereas no expression was detectable in samples derived from human liver, kidney, or testis, which served as negative controls. Furthermore, using an antibody directed against human Cyp11B, expression of Cyp11B protein was detected immunohistochemically in all investigated normal adrenals (n = 5) and adrenocortical adenomas (n = 15). Cyp11B immunoreactivity was not detectable in one of four adrenocortical cancers (Fig. 3BGo) and in none of the six non-ACCs.


Figure 3
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  		FIG. 3. A, Quantitative PCR analysis of relative levels of Cyp11B1 and Cyp11B2 mRNA expression in human adrenal tissues. Expression was analyzed in 11 normal adrenal glands (NAG), four hormonally inactive adenomas (EIA), five cortisol-producing adenomas (CPA), five aldosterone-producing adenomas (APA), and 14 ACC. Every tumor sample (500 ng cDNA) was analyzed in duplicate. No Cyp11B1 or Cyp11B2 mRNA expression was detected in human liver, kidney, or testis (data not shown). B, Immunohistochemical detection of Cyp11B protein in normal adrenal glands (NAG), aldosterone-producing adenoma (APA), Cushing adenoma (CPA), and adrenal ACC. Expression was analyzed in five normal, five Conn adenomas, five Cushing adenomas and four ACCs. All benign tumors exhibited moderate to strong Cyp11B protein expression. Three of four ACCs had clear Cyp11B expression at variable expression levels; one tumor showed negative Cyp11B staining (lower panel). C, Imaging of adrenocortical tissue in patients by [123I]IMTO scintigraphy. Patient 1, Depiction of bilateral adrenal tumors 24 h after tracer administration. At this time point, tracer accumulation was solely detectable in the adrenal tissue. Overlay with CT images allowed attribution of tracer uptake to the tumors. Patient 2, SPECT-CT images of the abdomen 4 h after administration of 185 MBq [123I]IMTO showing the unaffected adrenal gland at the right side. No tracer uptake was detectable in the adrenal mass of the left adrenal gland, indicating a lesion of non-adrenocortical origin, which was later confirmed by histological examination as a malignant melanoma. Patient 3, SPECT-CT images of the pelvis 4 h after of 185 MBq [123I]IMTO showing significant tracer uptake in bone lesions, indicating metastases of adrenocortical cancer. Patient 4, upper panel, whole-body scan 5 h after administration of 185 MBq [123I]IMTO showing the large primary tumor on the right side and multiple metastatic lesions in lung, liver, right mammary gland, gluteal muscle, sc tissue, intraabdominal soft tissue, and the abdominal wall. Patient 4, lower panel, SPECT-CT images of the tumor region with coronal and sagittal CT projections with and without image fusion demonstrating high tracer uptake in the primary tumor.

 
After approval by the ethical committee of the University of Wuerzburg (Permit No. 100/05), the German Federal Institute for Drugs and Medical Devices (BfArM) (Permit No. 4031230), the German Federal Office for Radiation Protection (BfS) (Permit No. Z5-22461/2-2006-024) and written informed consent, four patients were investigated by [123I]IMTO-SPECT. Patient 1 was a 49-yr-old woman diagnosed with bilateral adrenal masses. Both tumors were 3.5 cm in diameter. Hounsfield units (HU) in unenhanced CT were more than 10 HU on the right side, suggesting a nonadenoma lesion, and less than 10 HU on the left side, indicating an adrenal adenoma. Urinary catecholamine levels were marginally elevated, whereas cortisol and aldosterone excretion was normal. Patient 2 was a 67-yr-old woman with a diagnosis of breast cancer 12 yr ago. In a CT scan that was performed for restaging due to a newly recognized nodular lesion of the contralateral mammary gland, a mass of 10 cm diameter in the region of the left adrenal gland was detected. Imaging features were suspicious for a malignant lesion. Hormonal workup did not reveal any significant endocrine activity. Patient 3 was a 50-yr-old woman who first presented with paresthesia of the right part of her face, exophthalmus, and headache. Further clinical workup revealed an intracranial tumor infiltrating the orbita and the base of the skull. Histological examination of the resected lesion revealed an epithelial malignancy. Subsequent staging by CT revealed a right-sided 10-cm adrenal tumor and multiple bone lesions, suggesting metastatic adrenocortical cancer. Hormonal workup did not reveal any endocrine activity of the tumor. Patient 4 was a 64-yr-old woman suffering from progressive metastatic adrenal adenocarcinoma with excessive glucocorticoid and androgen production. Local resection of the primary tumor had not been performed due to the advanced tumor stage.

In all patients, both adrenals and adrenocortical tumor tissue were first detected within the first 60 min after injection of [123I]IMTO with best delineation of the adrenals or lesions 4–6 h after injection. At 24 h after injection, specific uptake was detected exclusively in adrenocortical tissue. In patient 1, both tumors exhibited high tracer uptake, suggesting an adrenocortical origin of the lesions (Fig. 3CGo). The right-sided tumor was resected, and subsequent histopathology confirmed an adrenocortical adenoma. Because the left tumor showed all features of an adenoma, in particular low HU indicating high fat content, it was not removed and showed no evidence of growth during follow-up. In patient 2, the right adrenal was clearly visualized (Fig. 3CGo). However, no tracer uptake was detected in the large adrenal mass on the left side. In a subsequent biopsy, the lesion was diagnosed as a malignant melanoma. Patient 3 showed significant tracer uptake in the adrenal tumor as well as in the contralateral unaffected adrenal. Furthermore, bone lesions that had been described as suspicious for metastasis exhibited high tracer uptake. Three additional vertebral lesions that had not been described by CT showed significant tracer uptake. Patient 4 exhibited high tracer uptake in both the primary tumor and the distant metastases (Fig. 3CGo and supplemental movie 3D, published as supplemental data on The Endocrine Society’s Journals Online web site at http://jcem.endojournals.org). The contralateral adrenal gland, which was suppressed by the excessive glucocorticoid production, could still be visualized despite low tracer uptake. With the exception of some small pulmonary metastases, [123I]IMTO visualized all metastases that had previously been detected by CT.

The whole-body effective dose ranged from 1.9–3.2 mSv in the respective patients, which is approximately 1/10 of the effective radiation dose of [131I]norcholesterol scintigraphy (20–30 mSv).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have established [123I]IMTO as a highly specific radiotracer for adrenal imaging. The half-life of 123I (13.2 h) and the use of SPECT imaging indicate that [123I]IMTO is suitable for widespread use as a radiotracer for adrenal tissue both in experimental animals and in humans.

Our in vitro studies demonstrate that IMTO binds specifically to Cyp11B enzymes, which are expressed in high amounts exclusively in tissues of adrenocortical origin. Thus, high uptake of [123I]IMTO in adrenal tumors noninvasively defines these lesions as steroidogenic tissue. The binding of both IMTO and FETO to Cyp11B enzymes resembles the effect of ETO and MTO, suggesting that iodination and fluorination do not significantly alter the pharmacodynamic properties of these agents. Accordingly, toxicity and mutagenicity testing revealed no untoward effects. Because IMTO also binds to aldosterone synthase (Cyp11B2), [123I]IMTO may also be useful for imaging of Conn adenomas, which are characterized by high expression of Cyp11B2 (18).

Although both ETO and MTO have been shown to bind to intracerebral GABAA receptors (19, 20), we observed only weak tracer uptake in the brain, indicating that binding of IMTO to GABAA receptors plays no significant role in IMTO imaging. Furthermore, the very low tracer uptake in the testes, indicates that binding to side chain cleavage enzyme (CYP11A1), is not sufficient to lead to relevant tracer uptake in the testes.

The rapid and specific uptake of IMTO into adrenal tissue in mice suggested excellent imaging properties also in humans, a prediction confirmed by our first investigations in humans. The effective dose in humans was only 1/10 compared with norcholesterol scintigraphy (21). Best visualization of tumor manifestations in humans was observed 4–6 h after [123I]IMTO, making IMTO imaging much less time consuming and more convenient than imaging with [131I]iodomethylnorcholesterol. On the other hand, very low background activity was found after this time and even after 24 h, at a time when [11C]MTO PET imaging is no longer feasible due to the short half-life of 11C (20 min).

The potential clinical use of [123I]IMTO is highlighted by our patients. In patient 1, CT suggested a nonadenomatous adrenal lesion with a risk of malignancy. However, high [123I]IMTO uptake by this lesion strongly suggested an adrenocortical tumor, which was later confirmed by histopathology. In patient 2, although the missing uptake of [123I]IMTO strongly indicated a nonadrenocortical lesion, a positive [123I]IMTO scan of the adrenal lesion would have identified this lesion as adrenocortical tumor, thereby helping to avoid a bioptic diagnosis. Because we could demonstrate high expression of Cyp11B also in hormonally inactive adrenocortical tumors, [123I]IMTO-SPECT will be particularly helpful in characterizing hormonally silent adrenal lesions, excluding metastatic disease by a significant [123I]IMTO uptake. Cyp11B expression is significant in most ACCs suggesting that [123I]IMTO-SPECT might be highly suitable to detect metastatic lesions as has also been reported for [11C]MTO (22). This is demonstrated in our patients three and four, showing that both bone metastases (patient 3) and soft tissue metastases (patient 4) can be detected.

PET imaging has a higher spatial resolution than SPECT, and more studies are needed to define the size of adrenal or metastatic lesions detectable by [123I]IMTO-SPECT. On the other hand, the use of radioiodine and the very high expression of Cyp11B enzymes in some cases of ACC also may open up the avenue for the treatment of patients with [131I]IMTO. This potential is highlighted in our fourth patient. This woman suffered from ACC with florid Cushing’s syndrome that failed to respond to mitotane and different cytotoxic regimens. The high tumor uptake of [123I]IMTO in this patient suggests that [131I]IMTO treatment may be potentially useful for palliative therapy of ACC because effective treatment options in advanced disease are still missing (23). Radiosensitivity of ACC has been a matter of debate but has recently been confirmed, because radiation treatment of the tumor bed in patients with ACC leads to significant reduction of local recurrences (24). [131I]Iodomethylnorcholesterol (NP59) has been used to differentiate malignant from benign adrenal lesions, because most malignant tumors exhibit no uptake of NP59 (25). In contrast, similar to [11C]MTO-PET, [123I]IMTO-SPECT is unlikely to differentiate benign from malignant adrenocortical lesions. However, uptake of [131I]iodomethylnorcholesterol into ACC has been studied in only a small number of cases and in some cases of ACC. [131I]Iodomethylnorcholesterol uptake has also been observed, reducing its specificity (26).

In short, [123I]IMTO-SPECT seems to be highly useful for molecular imaging of Cyp11B expression in adrenocortical tissue. The high and specific tracer uptake and its pharmacokinetics and low radiation dose suggest that [123I]IMTO-SPECT has the potential to become a valuable and widely available tool to characterize adrenal lesions in both experimental animals and humans. However, more studies are needed to extend our proof-of-concept investigations and to fully define the diagnostic potential of [123I]IMTO-SPECT.


   Acknowledgments
 
We thank Dr. Patrick Adam, Department of Pathology, University of Wuerzburg, for performing the immunohistochemistry studies.


   Footnotes
 
This work was supported by the Wilhelm-Sander-Stiftung (Project Grants 2003.175.1 and 2003.175.2 to A.Sc., B.A., and S.H.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online April 8, 2008

1 S.H. and A.St. as well as B.A. and A.Sc. contributed equally to this work. Back

Abbreviations: ACC, Adrenocortical carcinoma; CT, computed tomography; Cyp11B1, cytochrome P450 family 11B1; DMSA, 2,3-dimercaptosuccinic acid; DOC, 11-deoxycorticosterone; ETO, etomidate; FETO, fluoroetomidate; HU, Hounsfield units; IMTO, iodometomidate; MRI, magnetic resonance imaging; MTO, metomidate; PET, positron emission tomorgaphy; RSS, deoxycortisol; SPECT, single-photon emission computed tomography.

Received January 8, 2008.

Accepted March 28, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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