Journal of Nuclear Medicine, published on August 18, 2011 as doi:10.2967/jnumed.111.088906 Targeting Somatostatin Receptors: Preclinical Evaluation of Novel 18F-Fluoroethyltriazole-Tyr3-Octreotate Analogs for PET Julius Leyton1, Lisa Iddon2, Meg Perumal1, Bard Indrevoll3, Matthias Glaser2, Edward Robins2, Andrew J.T. George4, Alan Cuthbertson3, Sajinder K. Luthra2, and Eric O. Aboagye1 1Comprehensive Cancer Imaging Center at Imperial College, Faculty of Medicine, Imperial College London, London, United Kingdom; 2MDx Discovery (part of GE Healthcare) at Hammersmith Imanet Ltd., Hammersmith Hospital, London, United Kingdom; 3GE Healthcare AS, Oslo, Norway; and 4Section of Immunobiology, Faculty of Medicine, Imperial College London, London, United Kingdom Key Words: somatostatin receptor; octreotide; 18F-fluoroethyl- The incidence and prevalence of gastroenteropancreatic triazole-Tyr3-octreotate; positron emission tomography; and neuroendocrine tumors has been increasing over the past 3 neuroendocrine decades. Because of high densities of somatostatin receptors J Nucl Med 2011; 52:1–8 (sstr)—mainly sstr-2—on the cell surface of these tumors, 111In- DOI: 10.2967/jnumed.111.088906 diethylenetriaminepentaacetic acid-octreotide scintigraphy has become an important part of clinical management. 18F-radio- labeled analogs with suitable pharmacokinetics would permit PET with more rapid clinical protocols. Methods: We compared the affinity in vitro and tissue pharmacokinetics by PET of 5 structurally related 19F/18F-fluoroethyltriazole-Tyr3-octreotate The incidence and prevalence of gastroenteropancreatic (FET-TOCA) analogs: FET-G-polyethylene glycol (PEG)-TOCA, neuroendocrine tumors (GEP-NETs) has increased signifi- FETE-PEG-TOCA, FET-G-TOCA, FETE-TOCA, and FET-bAG- cantly over the past 3 decades (1). The most common site TOCA to the recently described 18F-aluminum fluoride NOTA- of primary GEP-NETs is the gastrointestinal tract (60%). octreotide (18F-AIF-NOTA-OC) and the clinical radiotracer Within the gastrointestinal tract, the single most common 68 19 3 Ga-DOTATATE. Results: All F-fluoroethyltriazole-Tyr - site is the small intestine (34%), followed by the rectum octreotate compounds retained high agonist binding affinity to sstr-2 in vitro (half-maximal effective concentration, 4–19 nM vs. (23%), colon (19%), stomach (8%), pancreas (8%), and appen- somatostatin at 5.6 nM). Dynamic PET showed that incorpora- dix (7%). There is no standard management of GEP-NETs, tion of PEG linkers, exemplified by 18F-FET-G-PEG-TOCA and although the recent PROMID trial suggested a response ben- 18F-FETE-PEG-TOCA, reduced uptake in high sstr-2–express- efit in the use of sandostatin LAR Depot (octreotide acetate 18 ing AR42J pancreatic cancer xenografts. F-FET-bAG-TOCA for injection suspension; Novartis Pharmaceuticals) in pa- showed the lowest nonspecific uptake in the liver. Tumor tients with metastatic midgut tumors (2). Targeted therapies, uptake increased in the order 68Ga-DOTATATE , 18F-AIF- NOTA # 18F-FET-bAG-TOCA , 18F-FET-G-TOCA. The uptake including everolimus and sunitinib, are being investigated of 18F-FET-bAG-TOCA was specific: a radiolabeled scrambled (3). Treatment of pancreatic neuroendocrine patients with peptide, 18F-FET-bAG-[W-c-(CTFTYC)K], did not show tumor everolimus, for example, has shown prolongation of progres- uptake; there was lower uptake of 18F-FET-bAG-TOCA in sion-free survival (4). Other treatment options include radio- AR42J xenografts when mice were pretreated with 10 mg of labeled somatostatin analogs and surgical debulking; unlabeled octreotide per kilogram; and there was low uptake however, no improvements in survival have been reported, of 18F-FET-bAG-TOCA in low sstr-2–expressing HCT116 xeno- grafts. Conclusion: We have developed novel fluoroethyltria- and validated biomarkers for response are lacking. zole-Tyr3-octreotate radioligands that combine high specific Currently somatostatin-receptor (sstr) imaging is the binding with rapid target localization and rapid pharmacoki- method of choice for the staging of GEP-NETs. Somato- netics for high-contrast PET. 18F-FET-bAG-TOCA and 18F- statin [H-Ala1-Gly2-c(Cys3-Lys4-Asn5-Phe6-Phe7-Trp8-Lys9- FET-G-TOCA are candidates for future clinical evaluation. Thr10-Phe11-Thr12-Ser13-Cys14)-OH] is a small regulatory peptide that has an inhibitory effect on the production of several exocrine hormones (prolactin, insulin, glucagon, Received Feb. 3, 2011; revision accepted May 10, 2011. and thyroid-stimulating hormone) in the gastrointestinal For correspondence or reprints contact: Eric O. Aboagye, Molecular tract and antiproliferative effects. sstrs are G-protein– Therapy Group, Faculty of Medicine, Imperial College London, Room 240 MRC Cyclotron Building, Hammersmith Hospital, Du Cane Rd., London, W12 coupled receptors expressed on cell membranes, which on ONN, United Kingdom ligand binding generate a transmembrane signal. Five re- E-mail: [email protected] COPYRIGHT ª 2011 by the Society of Nuclear Medicine, Inc. ceptor subtypes (sstr-1–sstr-5) have been identified to date PET WITH 18F-OCTREOTATE • Leyton et al. 1 jnm088906-pm n 8/17/11 Copyright 2011 by Society of Nuclear Medicine. (5). GEP-NETs frequently express a high density of sstr, in 18F radiolabeling of peptides under mild reaction condition, particular sstr-2, that can be exploited for imaging, from with good synthetic yields (25,26). The Tyr3-octreotate core identifying a small primary lesion after biochemical syn- was selected over octreotide because of its superior sstr-2 drome diagnosis and selecting patients for radiotherapy to specificity, higher affinity, and tumor uptake properties exploring the extent of metastatic disease (6–8). Somato- (8,12). Rapid tumor accumulation, together with rapid statin analogs have been developed because somatostatin clearance of labeled peptides from normal tissues including itself has a short plasma half-life (;3 min). The current the liver and muscle, is a prerequisite for use of short-lived somatostatin receptor–based clinical standard for functional positron-emitting radioisotopes such as 18F. Recognizing imaging of GEP-NETs is scintigraphy with 111In-diethyl- that incorporation of polyethylene glycol (PEG) moiety enetriaminepentaacetic acid (DTPA)-octreotide (half-life and length or type of linker used to connect the peptide of 111In, 2.8 d; photon energy, 254 keV) (9); clinical imag- core to the 18F-containing prosthetic group can change peptide ing is then performed at 1–2 d after injection of the radio- disposition, we performed a comparative study to evaluate pharmaceutical to ensure sufficient target-to-background tissue pharmacokinetics and imaging properties of 5 struc- contrast. Octreotide [(D)-Phe1-c(Cys2-Phe3-(D)-Trp4-Lys5- turally related 18F-fluoroethyltriazole-Tyr3-octreotate (FET- Thr6-Cys7)Thr-ol8] binds with high affinity to sstr-2 and TOCA) analogs—FET-G-PEG-TOCA, FETE-PEG-TOCA, to a lesser extent sstr-5 (10). Substitution of Phe3 by Tyr3, FET-G-TOCA, FETE-TOCA, and FET-bAG-TOCA (Fig. 1)— ½Fig: 1 as well as C-terminal Thr(ol)8 by Thr8, gives Tyr3-octreo- and compared these to a scrambled labeled peptide, the recently tate [(D)-Phe1-c(Cys2-Tyr3-(D)-Trp4-Lys5-Thr6-Cys7)Thr8], described 18F-aluminum fluoride NOTA-octreotide (18F-AIF- with improved selectivity and affinity for sstr-2, compared NOTA-OC (19)) and the clinically used radiotracer 68Ga- with octreotide (8,11,12). DOTATATE. Assessment of the pharmacokinetics and imaging PET offers better image contrast than scintigraphy, and properties in vivo should provide structure–activity relation- indeed octreotide analogs using radiometals such as 68Ga ship data for selection of the optimal radiotracer. 1 (1.1-h half-life, 90% b , 1.9-MeV Eb1 [positron energy]) 64 1 MATERIALS AND METHODS and Cu (12.7-h half-life, 19% b , 0.6-MeV Eb1; also 2 emission of 30% b particles) have been developed for Radiochemicals and Log D Measurements peptide-based imaging by PET (13–15). A good correlation Five structurally related 19F/18F fluoroethyltriazole-Tyr3-octreo- between the uptake of 68Ga-labeled peptides and sstr-2 tate analogs (Supplemental Fig. 1; supplemental materials are immunohistochemistry has been demonstrated in patients, available online only at http://jnm.snmjournals.org) and a related supporting the use of these radioligands for imaging of sstr- scrambled peptide were prepared. All commercially available 2(16). Peptides labeled with the 18F radioisotope, having chemicals were of analytic grade and used without further purifi- 19 the optimal combination of half-life, photon flux, and pho- cation. Stable F was used for assays in which radioactivity was 1 not needed. The synthesis of the radiotracers by click radiochem- ton energy (1.8-h half-life, 97% b , 0.6-MeV E 1), should b istry (Supplemental Fig. 1) is reported in detail elsewhere (27). be superior to the metal-based tracers and be devoid of Labeled products were eluted with ethanol in 100-mL fractions nonspecific uptake resulting from transchelation (17). Con- and made up to a 10% ethanol/phosphate-buffered saline solution 18 sequently, F-labeled octreotide analogs have previously (pH 7.4). 18F-AIF-NOTA was synthesized as previously described been developed (18–20). Tyr3-octreotate has been radiola- (19). 68Ga-DOTATATE was purchased from Covidien (U.K.) Com- beled with both 68Ga and 18F radioisotopes for PET of mercial Ltd. The lipophilicity (Log D) of the radiotracers has been tumors (21,22). In this article, we explored the relatively reported (27). new copper-catalyzed azide-alkyne cycloaddition reaction In Vitro Binding Assay (click reaction) to design several octreotate analogs. Marik Tyr3-octreotate analogs bind mainly to sstr-2. The affinity of ˚ and Sutcliffe (23), and Glaser and Arstad (24) were the first stable 19F-fluoroethyltriazole-Tyr3-octreotate analogs for somato- 18 to apply click chemistry to F radiolabeling of various statin receptor subtype sstr-2, versus sstr-4 as control low-affinity- building blocks or peptides. Since then, the click reaction receptor subtype, was determined using a fluorometric imaging has been used for rapid chemoselective and regiospecific plate reader assay. The assay involved measuring the 19F-fluoro- FIGURE 1.