GPER-Targeted, Tc-Labeled, Nonsteroidal Ligands Demonstrate

Published OnlineFirst July 16, 2014; DOI: 10.1158/1541-7786.MCR-14-0289

Molecular Cancer Oncogenes and Tumor Suppressors Research

GPER-Targeted, 99mTc-Labeled, Nonsteroidal Ligands Demonstrate Selective Tumor Imaging and In Vivo Estrogen Binding

Tapan K. Nayak1,2, Chinnasamy Ramesh3, Helen J. Hathaway1,4, Jeffrey P. Norenberg2,4, Jeffrey B. Arterburn3,4, and Eric R. Prossnitz1,4

Abstract Our understanding of estrogen (17b-estradiol, E2) receptor biology has evolved in recent years with the discovery and characterization of a 7-transmembrane-spanning G protein–coupled estrogen receptor (GPER/GPR30) and the development of GPER-selective functional chemical probes. GPER is highly expressed in certain breast, endometrial, and ovarian cancers, establishing the importance of noninvasive methods to evaluate GPER expression in vivo.Here, we developed 99mTc-labeled GPER ligands to demonstrate the in vivo status of GPER as an estrogen receptor (ER) and for GPER visualization in whole animals. A series of 99mTc(I)-labeled nonsteroidal tetrahydro-3H-cyclopenta[c]quinolone derivatives was synthesized utilizing pyridin-2-yl hydrazine and picolylamine chelates. Radioligand receptor binding studies revealed binding affinities in the 10 to 30 nmol/L range. Cell signaling assays previously demonstrated that derivatives retaining a ketone functionality displayed agonist properties, whereas those lacking such a hydrogen bond acceptor were antagonists. In vivo biodistribution and imaging studies performed on mice bearing human endometrial and breast cancer cell xenografts yielded significant tumor uptake (0.4–1.1%ID/g). Blocking studies revealed specific uptake in multiple organs (adrenals, uterus, and mammary tissue), as well as tumor uptake with similar levels of competition by E2 and G-1, a GPER-selective agonist. In conclusion, we synthesized and evaluated a series of first-generation 99mTc-labeled GPER-specific radioligands, demonstrating GPER as an estrogen- binding receptor for the first time in vivo using competitive binding principles, and establishing the utility of such ligands as tumor imaging agents. These results warrant further investigation into the role of GPER in estrogen- mediated carcinogenesis and as a target for diagnostic/therapeutic/image-guided drug delivery.

Implications: These studies provide a molecular basis to evaluate GPER expression and function as an ER through in vivo imaging. Mol Cancer Res; 12(11); 1635–43. 2014 AACR.

Introduction in premenopausal women compared with postmenopausal Estrogens mediate profound effects throughout the body women or age-matched men is widely attributed to the presence of 17b-estradiol (E2), the predominant and most and regulate physiologic and pathologic processes in both – women and men. The lower prevalence of many diseases potent endogenous estrogen (1 3). E2, although common- ly recognized as the female sex hormone, also has critical roles in additional normal physiologic processes within the nervous, immune, vascular, muscular, skeletal, and endo- 1Department of Cell Biology and Physiology, School of Medicine, Univer- – sity of New Mexico Health Science Center, Albuquerque, New Mexico. crine systems (2, 4 8). In addition, E2 signaling plays an 2College of Pharmacy, University of New Mexico Health Science Center, important role in various pathologic conditions and dis- Albuquerque, New Mexico. 3Department of Chemistry and Biochemistry, orders, including cancer, cardiovascular diseases, hyperten- New Mexico State University, Las Cruces, New Mexico. 4University of New Mexico Cancer Center, University of New Mexico Health Science Center, sion, osteoporosis, cognitive and behavioral alterations, Albuquerque, New Mexico. neurodegenerative diseases, as well as metabolic and im- – Note: Supplementary data for this article are available at Molecular Cancer mune disorders (2, 4 8). However, elucidating the exact Research Online (http://mcr.aacrjournals.org/). role(s) of E2 in these processes is often complicated by the Current address for T.K. Nayak: F Hoffmann-La Roche AG, Grenzacher- existence of several types of E2 receptors (ERs) and mul- strasse 124, 4070 Basel, Switzerland. tiple modes of cellular signaling mechanisms that span time Corresponding Author: Eric R. Prossnitz, Department of Cell Biology and frames from seconds to hours, or even days (6, 9). The Physiology, University of New Mexico, Albuquerque, NM 87131. Phone: actions of E2 have traditionally been ascribed to one of the 505-272-5647; Fax: 505-272-1421; E-mail: [email protected] two closely related classical nuclear hormone receptors, doi: 10.1158/1541-7786.MCR-14-0289 ERa and ERb, which are best characterized for regulating 2014 American Association for Cancer Research. gene expression (10, 11), and their membrane-localized

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variants. Recent studies have revealed the contribution of tive 99mTc-labeled agents for demonstrating the status of a novel G protein-coupled estrogen receptor GPER (pre- GPER as an ER in vitro and in vivo. In vivo biodistribution viously GPR30), which belongs to the family of seven- and competition binding studies with E2 and G-1 were transmembrane G protein–coupled receptors, to many of performed in mice bearing ERa/b-negative and GPER- the rapid cellular and biologic responses to E2 (6, 12–14). expressing type II human endometrial carcinoma Hec50 GPER is expressed in numerous tissues and the scope of tumors and ERa/b- and GPER-positive human breast research into its many functions has increased dramatically adenocarcinoma MCF7/HER2-18 tumors. Our results over the last decade (15–23); nevertheless, isolated reports demonstrate not only that GPER functions in vivo as have failed to observe GPER-mediated estrogenic responses an E2-binding receptor, but also that GPER-selective in the uterus or the mammary gland or GPER-dependent 99mTc-labeled ligands can be used to visualize breast and estrogen binding in cell-based systems (24–26). endometrial tumors in vivo. GPER protein is (over) expressed in approximately 50% of all breast cancers and correlates with clinical and pathologic biomarkers of poor outcome, such as tumor size and the Materials and Methods presence of metastases, regardless of ER status (27). Addi- Chemical synthesis of nonsteroidal GPER-specific tional studies have found that GPER protein is overexpressed ligands in ovarian cancer where it is associated with lower survival Synthetic derivatives of the GPER-targeting tetrahydro- rates (28, 29). Similarly, GPER is overexpressed in tumors 3H-cyclopenta[c]quinoline scaffold possessing different che- where E2 and progesterone receptors are downregulated and lating heterocyclic aminocarboxylate ligands with demon- in high-risk endometrial cancer patients with lower survival strated capacity for the formation of neutral tricarbonylrhe- rates (30, 31). GPER is also widely expressed in cancer cell nium(I) and tricarbonyltechnetium(I) complexes at the C8 lines isolated from diverse organs as well as primary tumors of position were prepared as previously described (54). Ligand the thyroid, lung, prostate, pancreas, and testicular germ compounds containing a pyridin-2-yl-hydrazinylethanoic cells, in addition to the breast, endometrium, and ovaries acid group (1,3,4), or pyridin-2-yl-methylaminoethanoic (6, 12, 32–34). acid (2), were prepared as the tert-butyl esters with nitrogen Importantly, in patients with breast cancer treated only groups protected as tert-butoxycarbonyl derivatives, and with tamoxifen, GPER protein expression increased and deprotected with trifluoroacetic acid in dichloromethane at survival was markedly reduced in patients with initial GPER- ambient temperature before labeling. The nonradioactive positive tumors, suggesting that patients with breast cancer tricarbonylrhenium(I) complexes (5-Re - 8-Re) were pre- who have high GPER protein expression should not be pared as previously described (54). treated exclusively with tamoxifen (35). Cellular effects of tamoxifen via GPER were further demonstrated through Radiosynthesis of 99mTc(I)-labeled nonsteroidal tamoxifen-mediated stimulation of tumor cell proliferation GPER-specific ligands and migration (36–38). Thus, although anti-estrogens such The organometallic aqua ion labeling agent [99mTc a þ as tamoxifen, fulvestrant, and raloxifene function as ER (CO)3(H2O)3] was prepared by adding 3.7 GBq of freshly – 99m antagonists, they act as GPER agonists (39 42), stimulating eluted Na- TcO4 to the Isolink kit (Tyco healthcare, proliferation and other cellular activities via the GPER- Mallinckrodt) as previously described (55). The alkaline 99m þ mediated transactivation of EGFR (43). [ Tc(CO)3(H2O)3] mixture was then neutralized to Several radiopharmaceuticals have been developed pH 7 with acetic acid. The synthetic chelates 1-4 were for the noninvasive imaging and assessment of ER status dissolved in ethanol and 10 mg of each derivative was added – a 99m þ (44 48). The most successful E2 radiopharmaceutical 16 - to the prepared [ Tc(CO)3(H2O)3] mixture. The reac- [18F]fluoroestradiol-17b (FES) is under clinical investigation mixture was stirred for 2 hours at room temperature. tion and to date has produced promising results in PET Alternatively, the mixture was heated to 80C for 30 imaging of ER-expressing tumors, in particular for the evalua- minutes as an alternative rapid radiosynthesis approach. tion of responsiveness of breast tumors to anti-estrogen drugs Inorganic impurities from the Isolink kit, aqua ions of such as tamoxifen (47, 49, 50). However, FES binds to all 99mTc (if any) and excess ligand were separated using solid subtypes of the classical ERs and furthermore does not phase extraction (SPE). SPE was performed using C-18 differentiate between classical ERs and GPER. Therefore, SepPak Plus cartridges (Waters). The impurities and excess to identify potentially aggressive forms of cancer, as well as for ligand were eluted with 4 0.5 mL fractions of the weak risk stratification of patients about to undergo endocrine solvent (50% ethanol in water). Elution of the final non- therapy, the assessment of GPER status is likely to be vital. steroidal GPER-specific 99mTc-labeled product was per- Therefore, with the ultimate goal of noninvasive assess- formed with 4 0.5 mL fractions of the strong solvent ment of GPER expression in patients, we developed a (100% ethanol). High-performance liquid chromatography series of nonsteroidal 99mTc-labeled agents based on the (HPLC) was performed to assess radiochemical purity and previously reported GPER-selective agonist G-1 and specific activity. To assess radiochemical purity and specific GPER antagonists G15 and G36 (51–53). Using com- activity, 10 mL of the final product was diluted in 200 mLof petitive binding and radiotracer principles, the primary HPLC grade ethanol (JT Baker), and 10 mL of the diluted objectives of this study were to characterize GPER-selec- sample was injected on a reverse-phase C-18 column

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GPER-Targeted 99mTc-Labeled Imaging Agents

(JT Baker) using HPLC grade ethanol and HPLC grade pellet was removed. After a 1-week recovery period, biodis- water as previously described (55). Stability, transchelation, tribution and imaging studies were performed. All protocols and partition coefficient studies were performed as previ- were approved by the Institutional Animal Care and Use ously described (55). Committee of the University of New Mexico Health Sciences Center (Albuquerque, NM). Cell culture and receptor binding studies ERa/b-negative/GPER-positive human endometrial carci- Histologic staining of tumors noma Hec50 cells (42, 56) and ERa/b-positive/GPER-posi- Sections (5 mm) from paraffin-embedded approximately tive human breast adenocarcinoma MCF7/HER2-18 cells 10-week Hec50 cell tumors were prepared for IHC using (57) were cultured in DMEM medium supplemented with an affinity purified polyclonal antibody raised against the FBS (10%), 100 U/mL penicillin, and 100 mg/mL strepto- carboxy-terminus of GPER as previously described (31). mycin. Cells were grown as a monolayer at 37C, in a humi- Briefly, sections were deparaffinized in CitriSolv (Fisher), fi di ed atmosphere of 5% CO2 and 95% air. Competition followed by rehydration in increasing H2O:ethanol solu- binding studies of the corresponding Re-labeled agents were tions. Antigen retrieval was carried out by microwaving slides performed using an 125I-labeled GPER-specificradioligand in 0.01 mol/L sodium citrate buffer (pH 6.0) for 25 minutes, (1-{2-[4-(6-bromo-benzo[1,3]dioxol-5-yl)-3a,4,5,9b-tetrahy- followed by incubation in fresh 2% H2O2 for 10 minutes. dro-3H-cyclopenta[c]quinolin-8-yl]-ethyl}-3-(3-iodo(125)- Permeabilization and blocking were carried out by incubat- phenyl)-urea (8 in ref. 58)) as previously described (58). ing the slides for 30 minutes in 200 mL 0.1% Triton X-100 in PBS containing 3% BSA in a humid chamber. Slides were Animal and tumor models then incubated with the GPER carboxy-terminal antibody GPER-targeted 99mTc-labeled agents were evaluated in diluted to a final protein concentration of 2 mg/mL in 3% mice bearing Hec50 or MCF7/HER2-18 tumors. Hec50 cell normal goat serum for 1 hour. Following washes, bound tumors were generated by injecting 3 to 4 million Hec50 cells antibody was detected using goat anti-rabbit IgG conjugated subcutaneously in 8-week-old female ovariectomized athy- to horseradish peroxidase (diluted 1:250 in 3% normal goat mic, Crl:Nu/Nu-nuBR "athymic nude" mice (Harlan Inc.). serum, 45 minutes), which was detected with 30,3-diami- MCF7/HER2-18 tumor models were similarly generated by nobenzidine tetrahydrochloride (Sigma). injecting 4 to 6 million cells subcutaneously. One day before injection of cells, a 60-day release E2 (1.7 mg) pellet (Inno- Biodistribution and SPECT/CT imaging studies vative Research of America) was implanted subcutaneously. Conscious tumor-bearing mice were injected in the tail After 8 to 10 weeks, palpable tumors were observed and the vein with selected GPER-targeted 99mTc-labeled agents. To

Table 1. Time-dependent uptake of 99mTc-G derivatives

1-h PI 1-h PI (Block)a 3-h PI Organ 5-99mTc 6-99mTc 7-99mTc 5-99mTc 6-99mTc 7-99mTc 5-99mTc 6-99mTc 7-99mTc Heart 1.7 0.2 1.8 0.1 1.2 0.1 1.5 0.3 1.9 0.1 1.5 0.2 1.9 0.5 1.1 0.1 1.3 0.2 Blood 0.5 0.2 1.5 0.2 3.2 0.7 1.4 0.5 1.6 0.2 4.4 0.6 1.4 0.1 1.2 0.1 1.7 0.2 Lungs 6.2 0.4 1.5 0.3 2.1 0.6 4.6 0.2 2.6 0.8 2.3 0.3 4.6 0.9 2.1 0.1 1.6 0.3 Liver 13.0 1.9 15.4 1.5 21.5 1.3 12.5 1.4 16.0 0.9 23.2 0.7 13.4 0.6 16.9 3.0 27.9 0.9 Gall bladder 10.9 1.4 5.9 2.1 3.7 2.6 7.8 3.4 8.7 2.7 3.6 0.7 15.0 1.5 2.0 0.4 3.4 1.5 Spleen 1.0 0.2 1.1 0.3 3.1 1.0 2.5 1.0 1.2 0.1 1.4 0.3 2.2 0.7 1.6 0.4 1.0 0.2 Large intestine 0.8 0.3 1.9 0.8 2.6 1.4 2.5 1.3 1.6 0.3 1.4 0.7 1.5 0.3 1.3 0.3 2.3 0.7 Small intestine 0.8 0.4 1.0 0.2 2.4 1.3 1.5 0.4 1.2 0.2 1.7 0.3 0.5 0.1 3.1 1.4 1.1 0.2 Stomach 1.6 0.4 1.6 0.1 2.0 0.4 1.8 0.7 2.0 0.2 0.8 0.3 1.1 0.3 0.8 0.1 0.8 0.2 Kidneys 2.7 0.3 1.8 0.1 2.4 0.6 2.5 0.6 1.2 0.2 3.2 0.4 1.6 0.5 1.8 0.3 2.6 0.3 Adrenal Gland 1.4 0.2 1.0 0.1 1.0 0.2 0.7 0.1 0.4 0.1 0.5 0.0 1.9 0.2 1.3 0.5 1.1 0.1 Bone 0.2 0.0 0.3 0.0 0.3 0.1 0.2 0.1 0.3 0.0 0.3 0.0 0.6 0.2 0.2 0.0 0.1 0.0 Muscle 0.1 0.0 0.1 0.0 0.1 0.0 0.1 0.0 0.1 0.0 0.1 0.0 0.2 0.0 0.2 0.1 0.2 0.0 Uterus 0.9 0.0 0.6 0.0 0.4 0.0 0.6 0.1 0.4 0.0 0.3 0.0 0.8 0.0 0.7 0.1 0.4 0.0 Mammary Gland 0.7 0.1 0.5 0.1 0.5 0.0 0.2 0.0 0.3 0.0 0.2 0.0 1.0 0.1 0.5 0.1 0.4 0.0 Pituitary 0.6 0.0 0.5 0.0 0.9 0.0 0.2 0.1 0.1 0.0 0.3 0.1 1.0 0.1 0.1 0.0 0.6 0.1 Brain 0.2 0.0 0.1 0.0 0.1 0.0 0.1 0.0 0.1 0.0 0.1 0.0 0.1 0.0 0.1 0.0 0.1 0.0 Urinary bladder 0.4 0.1 0.1 0.0 0.2 0.0 0.2 0.0 0.1 0.0 0.3 0.1 0.2 0.0 0.4 0.0 0.4 0.2 Hec50 tumor 0.8 0.1 0.5 0.0 0.5 0.1 0.3 0.0 0.4 0.0 0.3 0.0 1.0 0.1 0.7 0.1 0.4 0.0

aReceptor blocking studies were performed by coinjecting 5 mg G-1 with the radiotracer.

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determine receptor specificity, 5 mg GPER-selective agonist G-1 or 5 mg receptor-nonselective ligand 17b-E2 was coinjected with the radiotracer. At the desired time points, the 100 animals were sacrificed by carbon dioxide inhalation. 80 Tumor, blood, and selected normal organs were harvested, weighed, and the radioactivity measured in a Wallace 60 Wizard 1480 gamma counter (PerkinElmer). The percent- 5-Re age of the injected dose per gram of tissue (% ID/g) was 40 6-Re calculated by comparison with standards representing 10% 7-Re of the injected dose per animal. High-resolution in vivo 20 8-Re SPECT/CT imaging studies were performed using a multi- Specific binding (%) pinhole NanoSPECT/CT small animal imager (Bioscan 0 Inc) with whole body and focused imaging studies carried –11 –10 –6–7–8–9 – fl out on anesthetized mice (1.5% 1.7% iso urane) on a Concentration (log mol/L) temperature-controlled bed (36–38C). Images were coregistered and analyzed using InVivoScope software pro- gram (Bioscan Inc). Figure 2. Competition binding curves from radioligand receptor binding studies performed with an iodinated GPER-targeted G-1 derivative as the radiotracer and the nonradioactive Re-labeled derivatives (5-Re - 8-Re) as competitor using GPER-expressing (ERa- and ERb-negative) human endometrial carcinoma Hec50 cells. n 3.

H CHELATE±M(CO)3 H Statistical analysis O N All numerical data were expressed as the mean of the values H SEM. GraphPad Prism version 5 was used for statistical O Br analysis and analysis of competitive binding data, which was fi P M = Re, 99mTc performed using a single t model. A value 0.05 was considered statistically significant. H H H H N O N O N O N O O Results N OH N M OC CO Radiochemistry 5-M 1 CO The 99mTc- and Re-labeled derivatives of 1-4 (Fig. 1) were prepared using the tricarbonyl approach. The [99mTc H þ H O (CO)3(H2O)3] intermediate was prepared with a radio- O N O N O chemical purity of >95% and mixed with the corresponding O N OH N M chelating ligands and stirred at room temperature for 2 OC CO hours. Radiolabeling yields were high with more than 95% 2 6-M CO 99m þ of the [ Tc(CO)3(H2O)3] incorporated into the derivatives 1, 2 (Supplementary Fig. S1A and S1B, respective- H H H H O 99m 99m 99m N O N ly), and 3 to generate 5- Tc, 6- Tc and 7- Tc, and N N O more than 85% incorporation into derivative 4 (Supple- N OH N M mentary Fig. S1C) to produce 8-99mTc. On the basis of the OC CO elution profile of the 99mTc generator and the transient 3 7-M CO equilibrium between the parent and the daughter radionu- fi fi H H clide, the calculated speci c activity of the nal products H H N O N O N ranged from 22.8 to 41.6 TBq/mmol with radiochemical N purities exceeding 95%. Typical radiochemical yields after OH N O N M purification ranged from 60% to 75%. Although the rate of CO þ OC radiolabeling using the [99mTc(CO) (H O) ] intermedi- 4 8-M CO 3 2 3 ate was increased with heating at 80C for 30 minutes, which allows access to radioligands with increased specific Figure 1. Chemical structures of GPER-targeted derivatives 1-4 and the activity, HPLC analysis revealed the presence of trace corresponding metal (M) chelates 5-8. Synthetic derivatives possessing degradation products and the radiochemical purity was less heterocyclic aminocarboxylate chelate ligands: pyridin-2-yl- than 70% under these conditions. The higher radiolabeling hydrazinylethanoic acid (1,3,4); and pyridin-2-yl-methylaminoethanoic acid (2). Neutral complexes (5-Re - 8-Re) and (5-99mTc - 8-99mTc) yields and increased purity obtained using the room tem- þ 99m þ isolated from labeling with Re (CO)3 and Tc (CO)3 in aqueous perature procedure were advantageous for the subsequent ethanol, respectively. in vivo studies.

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Figure 3. Receptor-mediated AB uptake of 6-99mTc (A), 7-99mTc (B), 2.0 5.0 and 5-99mTc (C) in ovariectomized 1 h PI 4.0 athymic (NCr) nu/nu female mice 1.5 1 h PI G-1 Block 3.0 1 h PI bearing human endometrial Hec50 1 h PI G-1 Block tumors 1 hour postinjection in the 1.0 1.0 % ID/g absence (open bars) or presence % ID/g (solid bars) of 5 mg coinjected 0.5 0.5 GPER-selective agent G-1. D, GPER-selectivity and receptor- 0.0 0.0 99m mediated uptake of 5- Tc in Blood Bone Blood Bone Tumor MuscleAdrenalUterus Tumor MuscleAdrenalUterus ovariectomized athymic (NCr) Mammary Mammary Organ Organ nu/nu female mice bearing human CD endometrial Hec50 tumors at 1, 2, 1.5 and 3 hours PI determined by 2.0 coinjecting 5 mg E2 or GPER- 1.5 1 h PI Uterus selective agent G-1 (indicated in 1 h PI G-1 Block 1.0 Uterus (G-1) parentheses). All uptake values are 1.0 Uterus (E2) expressed as % ID/g. Data % ID/g represent the mean value SEM % ID/g 0.5 Tumor 0.5 from at least three determinations. Tumor (G-1) , P < 0.05 compared with uptake in Tumor (E2) 0.0 0.0 the absence of the indicated 123 Blood Bone coinjected competitor. MuscleAdrenalUterus Tumor Mammary Time (hours after injection) Organ

Stability, transchelation, and partition coefﬁcient studies of tricarbonylrhenium(I)-labeled derivatives 5-Re, 6-Re, All of the 99mTc-labeled derivatives demonstrated good 7-Re, and 8-Re, were 16.3, 11.3, 12.5, and 29.2 nmol/L, stability (more than 95%) in mouse plasma and PBS buffer respectively (see Table 3 for summary). after incubation at 37C for 24 hours. Overall, less than 10% transchelation was observed upon incubation with a solution Biodistribution in tumor-bearing animals of 1 mmol/L cysteine solution or 1 mmol/L histidine at Biodistribution studies were performed in ovariectomized 37C for 24 hours. The radioligands 5-99mTc, 6-99mTc, athymic (NCr) nu/nu female mice bearing Hec50 and 99m 99m P 7- Tc, and 8- Tc exhibited log (o/w) values of 4.6 MCF7/HER2-18 cell tumor xenografts by injecting 1.5 0.3, 5.0 0.1, 4.9 0.1, and 5.5 0.1 (mean SEM from MBq 99mTc-labeled derivatives in the tail vein of conscious four determinations), respectively. mice. The 13% to 16% and 15% to 18% ID/g liver uptake of 5-99mTc and 6-99mTc, respectively, were up to 1.5–2-fold Radioligand receptor binding studies lower than the 21% to 28% ID/g liver uptake of 7-99mTc Competition binding studies on the corresponding stable (Table 1), despite the similar log P values of 6-99mTc and isotope Re-labeled derivatives were performed on ERa/ 7-99mTc, suggesting a correlation between the presence of b-negative and GPER-expressing human endometrial car- the ethanone moiety in the linker with decreased liver cinoma Hec50 cells using an 125I-labeled GPER-speciﬁc uptake. Three hours postinjection (PI), the 1.05% ID/g 99m – derivative as the radiotracer (Fig. 2). The mean IC50 values tumor uptake for 5- Tc was more than 1.5 2-fold more

Table 2. Selective uptake of 5-99mTc in selected organs of ovariectomized female athymic (NCr) nu/nu mice bearing ERa/b-positive and GPER-positive human breast adenocarcinoma MCF-7/HER2-18 tumors

Organ 3-h PI (% ID/g) 3-h PI (G-1 block)a (% ID/g) 3-h PI (E2 block)a (% ID/g) Blood 1.43 0.10 2.14 0.59 1.97 0.37 Bone 0.50 0.11 0.44 0.17 0.52 0.25 Muscle 0.18 0.02 0.16 0.01 0.18 0.01 Adrenal gland 2.20 0.22 1.11 0.10 1.29 0.24 Uterus 0.80 0.04 0.45 0.07 0.56 0.01 Mammary gland 1.07 0.12 0.56 0.08 0.67 0.15 MCF-7/HER2-18 tumor 0.70 0.03 0.41 0.05 0.49 0.02

aReceptor blocking studies were performed by coinjecting 5 mg G-1 or E2 as indicted with the 5-99mTc.

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Table 3. Comparison of selected chemical and biologic characteristics of 99mTc-labeled GPER-targeted agents

GPER-targeted Tumor/blood Tumor uptake Liver uptake a agent LogP(o/w) IC50 (nmol/L) (3-h PI) (% ID/g, 3-h PI) (% ID/g, 3-h PI) 5-99mTc 4.6 0.3 16 (8.6–31) 0.76 1.05 0.09 13.38 0.60 6-99mTc 5.0 0.1 11 (6.4–20) 0.59 0.69 0.09 16.91 2.97 7-99mTc 4.9 0.1 13 (6.9–23) 0.24 0.40 0.03 27.95 0.91 8-99mTc 5.5 0.1 29 (18–48) ND ND ND

Abbreviation: ND, not determined. aDetermined by competition binding with the respective Re complex. Values represent best-ﬁt values using a one-site competition analysis with 95% conﬁdence intervals provided in parentheses.

than 0.40% ID/g tumor uptake for 7-99mTc and 0.69% where similar levels of blocking were observed with coin- ID/g for 6-99mTc. Overall, all three derivatives exhibited jection of either G-1 or E2 (Table 2), suggesting that binding comparable receptor binding affinity and specificity, yet sites for 5-99mTc were equally accessible to both G-1 and E2. showed different uptake values for target and nontarget organs. At 24 hours PI, washout of the tracer was observed Imaging studies in tumor and nontarget organs except the liver (Supplemen- Imaging studies were carried out after intravenous injec- tary Table S1). tion of 19 MBq 5-99mTc via the tail vein of Hec50 endo- All of the evaluated 99mTc-labeled derivatives demonstrat- metrial tumor-bearing mice. Whole body SPECT/CT (60 s/ ed receptor specificity when GPER-blocking studies were projection) imaging studies, carried out under 1.5% to 1.7% performed by coinjecting 5 mg G-1 with the corresponding isoflurane using a temperature-controlled bed (36–38C), radiotracer (Fig. 3). GPER-mediated uptake was observed in revealed high liver, gall bladder, and intestine uptake. To the adrenals, uterus, mammary tissue, and the Hec50 and better visualize the tumor, a limited field of view SPECT MCF7/HER2-18 tumors (Fig. 3; Table 2). Of the 99mTc- image (200 s/projection) was obtained from live mice. The labeled derivatives evaluated, 7-99mTc exhibited the lowest GPER-expressing tumor, which shows intense intracellular target/blood ratio, whereas 5-99mTc exhibited the highest expression of GPER (Fig. 4A), was clearly visualized at 3 target/blood ratio (Table 3). However, all of the 99mTc- hours (Fig. 4B). Image quantification revealed no significant labeled derivatives evaluated were rapidly metabolized as the washout of the tracer from the tumor for up to 6 hours PI collected urine showed the presence of radiometabolites. At (1.10 0.27% ID at 3 hours PI vs. 1.23 0.16% ID at 6 1 hour PI, approximately 30% of 5-99mTc was found in the hours PI). More than 70% ID was in the liver, gall bladder, urine. The remaining radioactive species were hydrophilic and the intestines at 3 and 6 hours PI. radiometabolites (Supplementary Fig. S2A). At 2 hours PI, 5-99mTc was completely metabolized (Supplementary Fig. S2B). At 4 hours PI, most of the radiometabolites were Discussion excreted and the only radioactive species circulating in the Roles for the membrane-bound GPER in resistance to blood on HPLC analysis of the plasma sample was the endocrine therapy and aggressive forms of breast, ovarian, injected radiotracer (Supplementary Fig. S2C). Similarly, and endometrial carcinoma have been reported (6, 29, for 6-99mTc, at 2 hours PI most of the radioactive species 31, 35, 59, 60). The GPER-selective ligands G-1, G15, were hydrophilic radiometabolites. The excretion of the and G36 have been extensively used (with >180 publica- three radiotracers ranged from 8% to 14% ID at 3 hours PI. tions) to elucidate the roles and functions of this receptor in A detailed in vivo study of GPER ligand specificity was normal physiology and pathology (6, 37, 39, 52). Although performed using 5-99mTc in which receptor binding was consensus supports the involvement of GPER in many blocked by coinjecting 5 mg of either G-1 or E2. The estrogenic responses, a small number of conflicting reports 5-99mTc agent exhibited specific binding to target organs have suggested that GPER may not function as an ER in vitro at 1, 2, and 3 hours PI, suggesting limited nonspecific uptake or in vivo (24, 26, 61). To further address the functions of of a radiometabolite in the target organs (Fig. 3D). Further- GPER in vivo, we developed radiolabeled GPER-specific more, at 3 hours PI, the level of inhibition of 5-99mTc uptake agents and performed in vivo competition binding studies was similar when either G-1 or E2 was coinjected, demon- using radiotracer principles to demonstrate the expression strating 5-99mTc binding specificity (with respect to both E2 profile of GPER in various tissues and in different ERa/b- and G-1) to GPER in target organs such as mammary tissue and/or GPER-expressing tumor xenograft cancer models. and the uterus. This GPER selectivity was further demon- We also evaluated whether these radiolabeled GPER-specific strated in ERa/b-and GPER-positive human breast adeno- agents can be used for noninvasive GPER visualization carcinoma MCF7/HER2-18 tumor-bearing mice (Table 2) of tumors.

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GPER-Targeted 99mTc-Labeled Imaging Agents

Figure 4. A, IHC staining of GPER in human endometrial Hec50 tumor xenograft. Scale bar 10 mm. B, reconstructed coregistered transverse SPECT/CT image slice focusing on the ﬂank tumor at 3 hours PI with 5-99mTc. The tumor is designated with an arrow. For the SPECT image acquisition, a focused scan of 200 s/projection was used; the CT image was acquired with 180 projections (1.5 pitch and an energy of 45 kVp). The energy window for SPECT acquisition for 99mTc was set at 140 14 keV.

On the basis of our previous studies, we had demonstrated as the poorer outcome for patients with GPER-positive the need for an uncharged neutral agent for intracellular endometrial cancers (31), noninvasive assessment of GPER targeting of GPER (62, 63); therefore, we synthesized a series expression in tumors is also likely to become a critical factor of uncharged neutral GPER-selective agents based on organ- in the diagnosis and selection of potential treatment options ometallic rhenium(I)/technetium(I) tricarbonyl chemistry in endometrial cancers. In addition to its importance in (54). Radioligand receptor-binding studies revealed binding breast and endometrial cancers, GPER expression has also affinities in the 10 to 30 nmol/L range, demonstrating been associated with poor survival, or recognized as a minimal to moderate loss of affinity relative to the parent diagnostic biomarker, in ovarian (29), pancreatic (34), lung molecules G-1 and G15. Cellular signaling assays previously (65), testicular germ cell (66), and other cancers. demonstrated that derivatives retaining a hydrogen bond In conclusion, we have synthesized and evaluated a series acceptor ketone functionality in the linker displayed agonist of first generation 99mTc-labeled GPER-specific radioima- properties whereas those lacking this moiety were antago- ging agents and demonstrated for the first time the status of nists (54). In vivo biodistribution studies revealed further GPER as an E2-binding receptor in vivo, using competitive differences associated with the structures of the linkages; the radioligand binding principles with both the endogenous radiolabeled conjugate 7-99mTc with a flexible hydrophobic hormone E2 as well as a synthetic GPER-selective ligand G- ethane linkage displayed greater liver uptake and lower 1. Although we chose 99mTc as the radionuclide for the tumor uptake than radiolabeled derivatives with an ethanone development GPER-targeted agents due to the widespread linkage (5-99mTc and 6-99mTc). availability of this isotope for clinical radiopharmacy, with In vivo competition studies with excess G-1 and E2 in further structural optimization, 18F- and 11C-labeled GPER- ERa/b-negative and GPER-positive human endometrial targeted agents could be developed with potentially carcinoma Hec50 tumors as well as in ERa/b- and improved biodistribution and imaging characteristics. Tak- GPER-positive human breast adenocarcinoma MCF7/ en together, these studies advance investigations into the HER2-18 tumors demonstrated GPER-specific uptake in roles of GPER in E2-mediated carcinogenesis (67) as well the mammary gland, uterus, and tumor, thus demonstrating as its clinical application as both a diagnostic and therapeutic for the first time the status of GPER as an E2-binding target in breast, endometrial, and other cancers. receptor in vivo. Although liver and intestine uptake values were high, selective tumor targeting was achieved. SPECT/ Disclosure of Potential Conflicts of Interest CT imaging further demonstrated tumor localization and No potential conflicts of interest were disclosed. retention of the 99Tc-labeled G derivatives for up to 6 hours postinjection. GPER expression patterns within primary and metastatic breast cancers could play an important diagnostic Authors' Contributions Conception and design: T.K. Nayak, H.J. Hathaway, J.P. Norenberg, J.B. Arter- role because GPER is expressed in approximately 50% of burn, E.R. Prossnitz breast cancers, regardless of ER status. GPER (over)expres- Development of methodology: T.K. Nayak, C. Ramesh, J.P. Norenberg, sion is also correlated with tumor size, Her2 status, and the E.R. Prossnitz Acquisition of data (provided animals, acquired and managed patients, provided presence of metastases (27) as well as hormone therapy facilities, etc.): T.K. Nayak, C. Ramesh, J.P. Norenberg resistance (35, 60) and recurrence in triple-negative breast Analysis and interpretation of data (e.g., statistical analysis, biostatistics, compu- tational analysis): T.K. Nayak, H.J. Hathaway, J.P. Norenberg, J.B. Arterburn, cancer (64). In addition, with the recent report that ER- E.R. Prossnitz negative Hec50 endometrial tumors respond to E2-stimu- Writing, review, and/or revision of the manuscript: T.K. Nayak, H.J. Hathaway, lation in vivo with enhanced tumor growth via GPER, J.P. Norenberg, J.B. Arterburn, E.R. Prossnitz Administrative, technical, or material support (i.e., reporting or organizing data, as evidenced by the inhibition of E2-stimulated tumor constructing databases): C. Ramesh, J.P. Norenberg, E.R. Prossnitz growth by the GPER-selective antagonist G36 (42) as well Study supervision: J.P. Norenberg, J.B. Arterburn, E.R. Prossnitz

www.aacrjournals.org Mol Cancer Res; 12(11) November 2014 1641

Nayak et al.

Grant Support Sciences Center, the UNM College of Pharmacy, and the UNM Cancer Center (NIH This work was supported by the NIH (grants R01 CA127731, to J.B. Arterburn P30 CA118100). The costs of publication of this article were defrayed in part by the payment of page and E.R. Prossnitz; CA118743, to E.R. Prossnitz; CA116662, to E.R. Prossnitz; advertisement CA163890, to E.R. Prossnitz; and P20-GM103451, to J.B. Arterburn), the New charges. This article must therefore be hereby marked in accordance with Mexico Cowboys for Cancer Research Foundation (to J.B. Arterburn and E.R. 18 U.S.C. Section 1734 solely to indicate this fact. Prossnitz), the Oxnard Foundation (to E.R. Prossnitz), and the Stranahan Foundation (to E.R. Prossnitz). SPECT/CT images were acquired in the Keck-UNM Small Received May 20, 2014; revised June 16, 2014; accepted June 19, 2014; Animal Imaging Shared Resource supported by the University of New Mexico Health published OnlineFirst July 16, 2014.

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GPER-Targeted, 99mTc-Labeled, Nonsteroidal Ligands Demonstrate Selective Tumor Imaging and In Vivo Estrogen Binding

Tapan K. Nayak, Chinnasamy Ramesh, Helen J. Hathaway, et al.

Mol Cancer Res 2014;12:1635-1643. Published OnlineFirst July 16, 2014.

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