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Purpose: The integrin αvβ6 is overexpressed in a variety of aggressive cancers and serves as a prognosis marker. This study describes the conjugation, radiolabeling, and in vitro and in vivo evaluation of four chelators to determine the best candidate for (64)Cu radiolabeling of A20FMDV2, an αvβ6 targeting peptide., Procedures: Four chelators were conjugated onto PEG28-A20FMDV2 (1): 11-carboxymethyl-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4-methanephosphonic acid (CB-TE1A1P), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and 4,4'-((3,6,10,13,16,19-hexazazbicyclo[6.6.6]ico-sane-1,8-diylbis(aza-nediyl))bis(methylene)dibenzoic acid (BaBaSar). All peptides were radiolabeled with (64)Cu in ammonium acetate buffer at pH 6 and formulated to pH 7.2 in PBS for use. The radiotracers were evaluated using in vitro cell binding and internalization assays and serum stability assays. In vivo studies conducted include blocking, biodistribution, and small animal PET imaging. Autoradiography and histology were also conducted., Results: All radiotracers were radiolabeled in good radiochemical purity (>95 %) under mild conditions (37-50 °C for 15 min) with high specific activity (0.58-0.60 Ci/μmol). All radiotracers demonstrated αvβ6-directed cell binding (>46 %) with similar internalization levels (>23 %). The radiotracers (64)Cu-CB-TE1A1P-1 and (64)Cu-BaBaSar-1 showed improved specificity for the αvβ6 positive tumor in vivo over (64)Cu-DOTA-1 and (64)Cu-NOTA-1 (+/- tumor uptake ratios-3.82 +/- 0.44, 3.82 ± 0.41, 2.58 ± 0.58, and 1.29 ± 0.14, respectively). Of the four radiotracers, (64)Cu-NOTA-1 exhibited the highest liver uptake (10.83 ± 0.1 % ID/g at 4 h)., Conclusions: We have successfully conjugated, radiolabeled, and assessed the four chelates CB-TE1A1P, DOTA, NOTA, and BaBaSar both in vitro and in vivo. However, the data suggests no clear "best candidate" for the (64)Cu-radiolabeling of A20FMDV2, but instead a trade-off between the different properties (e.g., stability, selectivity, pharmacokinetics, etc.) with no obvious effects of the individual chelators.

The Glucagon-like peptide 1 receptor (GLP-1R) has become an important target for imaging due to its elevated expression profile in pancreatic islets, insulinoma, and the cardiovascular system. Because native GLP-1 is degraded rapidly by dipeptidyl peptidase-IV (DPP-IV), several studies have conjugated different chelators to a more stable analog of GLP-1 (such as exendin-4) as PET or SPECT imaging agents with various advantages and disadvantages. Based on the recently developed Sarcophagin chelator, here, we describe the construction of GLP-1R targeted PET probes containing monomeric and dimeric exendin-4 subunit. The in vitro binding affinity of BarMalSar-exendin-4 and Mal2Sar-(exendin-4)2 was evaluated in INS-1 cells, which over-express GLP-1R. Mal2Sar-(exendin-4)2 demonstrated around 3 times higher binding affinity compared with BaMalSar-exendin-4. After (64)Cu labeling, microPET imaging of (64)Cu-BaMalSar-exendin-4 and (64)Cu-Mal2Sar-(exendin-4)2 were performed on subcutaneous INS-1 tumors, which were clearly visualized with both probes. The tumor uptake of (64)Cu-Mal2Sar-(exendin-4)2 was significantly higher than that of (64)Cu-BaMaSarl-exendin-4, which could be caused by polyvalency effect. The receptor specificity of these probes was confirmed by effective blocking of the uptake in both tumor and normal positive organs with 20-fold excess of unlabeled exendin-4. In conclusion, sarcophagine cage conjugated exendin-4 demonstrated persistent and specific uptake in INS-1 insulinoma model. Dimerization of exendin-4 could successfully lead to increased tumor uptake in vivo. Both (64)Cu-BaMalSar-exendin-4 and (64)Cu-Mal2Sar-(exendin-4)2 hold a great potential for GLP-1R targeted imaging.

18F positron emission tomography (PET) has a number of attributes that make it clinically attractive, including nearly 100% positron efficiency, very high specific radioactivity, and a short half-life of ≈ 110 minutes. However, the short half-life of 18F and the poor nucleophilicity of fluoride introduce challenges for the incorporation of 18F into complex molecules. Recently, the tetrazine-trans-cyclooctene ligation was introduced as a novel 18F labeling method that proceeds with fast reaction rates without catalysis. Herein we report an efficient method for 18F labeling of free cysteines of peptides and proteins based on sequential ligation with a bifunctional tetrazinyl-maleimide and an 18F-labeled trans-cyclooctene. The newly developed method was tested for site-specific labeling of both c(RGDyC) peptide and vascular endothelial growth factor (VEGF)-SH protein. Starting with 4 mCi of 18F-trans-cyclooctene and only 10 μg of tetrazine-RGD (80-100 μM) or 15 μg of tetrazine-VEGF (6.0 μM), 18F-labeled RGD peptide and VEGF protein could be obtained within 5 minutes in 95% yield and 75% yield, respectively. The obtained tracers were then evaluated in mice. In conclusion, a highly efficient method has been developed for site-specific 18F labeling of cysteine-containing peptides and proteins. The special characteristics of the tetrazine-trans-cyclooctene ligation provide unprecedented opportunities to synthesize 18F-labeled probes with high specific activity for PET applications.

Unlabelled: Because islet transplantation has become a promising treatment option for patients with type 1 diabetes, a noninvasive imaging method is greatly needed to monitor these islets over time. Here, we developed an (18)F-labeled exendin-4 in high specific activity for islet imaging by targeting the glucagonlike peptide-1 receptor (GLP-1R)., Methods: Tetrazine ligation was used to radiolabel exendin-4 with (18)F. The receptor binding of (19/18)F-tetrazine trans-cyclooctene (TTCO)-Cys(40)-exendin-4 was evaluated in vitro with INS-1 cell and in vivo on INS-1 tumor (GLP-1R positive) and islet transplantation models., Results: (18)F-TTCO-Cys(40)-exendin-4 was obtained in high specific activity and could specifically bind to GLP-1R in vitro and in vivo. Unlike the radiometal-labeled exendin-4, (18)F-TTCO-Cys(40)-exendin-4 has much lower kidney uptake. (18)F-TTCO-Cys(40)-exendin-4 demonstrated its great potential for transplanted islet imaging: the liver uptake value derived from small-animal PET images correlated well with the transplanted β-cell mass determined by immunostaining. Autoradiography showed that the localizations of radioactive signal indeed corresponded to the distribution of islet grafts in the liver of islet-transplanted mice., Conclusion: (18)F-TTCO-Cys(40)-exendin-4 demonstrated specific binding to GLP-1R. This PET probe provides a method to noninvasively image intraportally transplanted islets.

Early evaluation of cancer response to a therapeutic regimen can help increase the effectiveness of treatment schemes and, by enabling early termination of ineffective treatments, minimize toxicity, and reduce expenses. Biomarkers that provide early indication of tumor therapy response are urgently needed. Solid tumors require blood vessels for growth, and new anti-angiogenic agents can act by preventing the development of a suitable blood supply to sustain tumor growth. The purpose of this study is to develop a class of novel molecular imaging probes that will predict tumor early response to an anti-angiogenic regimen with the humanized vascular endothelial growth factor antibody bevacizumab. Using a bevacizumab-sensitive LS174T colorectal cancer model and a 12-mer bacteriophage (phage) display peptide library, a bevacizumab-responsive peptide (BRP) was identified after six rounds of biopanning and tested in vitro and in vivo. This 12-mer peptide was metabolically stable and had low toxicity to both endothelial cells and tumor cells. Near-infrared dye IRDye800-labeled BRP phage showed strong binding to bevacizumab-treated tumors, but not to untreated control LS174T tumors. In addition, both IRDye800- and (18)F-labeled BRP peptide had significantly higher uptake in tumors treated with bevacizumab than in controls treated with phosphate-buffered saline. Ex vivo histopathology confirmed the specificity of the BRP peptide to bevacizumab-treated tumor vasculature. In summary, a novel 12-mer peptide BRP selected using phage display techniques allowed non-invasive visualization of early responses to anti-angiogenic treatment. Suitably labeled BRP peptide may be potentially useful pre-clinically and clinically for monitoring treatment response.

Purpose: A reliable and routine process to introduce a new F-labeled dimeric RGD-peptide tracer ([F]FPP(RGD)) for noninvasive imaging of αβ expression in tumors needed to be developed so the tracer could be evaluated for the first time in man. Clinical-grade [F]FPP(RGD) was screened in mouse prior to our first pilot study in human. Procedures: [F]FPP(RGD) was synthesized by coupling 4-nitrophenyl-2-[F]fluoropropionate ([F]NPE) with the dimeric RGD-peptide (PEG-c(RGDyK)). Imaging studies with [F]FPP(RGD) in normal mice and a healthy human volunteer were carried out using small animal and clinical PET scanners, respectively. Results: Through optimization of each radiosynthetic step, [F]FPP(RGD) was obtained with RCYs of 16.9 ± 2.7% ( n = 8, EOB) and specific radioactivity of 114 ± 72 GBq/μmol (3.08 ± 1.95 Ci/μmol; n = 8, EOB) after 170 min of radiosynthesis. In our mouse studies, high radioactivity uptake was only observed in the kidneys and bladder with the clinical-grade tracer. Favorable [F]FPP(RGD) biodistribution in human studies, with low background signal in the head, neck, and thorax, showed the potential applications of this RGD-peptide tracer for detecting and monitoring tumor growth and metastasis. Conclusions: A reliable, routine, and automated radiosynthesis of clinical-grade [F]FPP(RGD) was established. PET imaging in a healthy human volunteer illustrates that [F]FPP(RGD) possesses desirable pharmacokinetic properties for clinical noninvasive imaging of αβ expression. Further imaging studies using [F]FPP(RGD) in patient volunteers are now under active investigation. [ABSTRACT FROM AUTHOR]

Abstract: Labeling biomolecules with 18F is usually done through coupling with prosthetic groups, which generally requires several time-consuming radiosynthetic steps resulting in low labeling yield. Recently, the tetrazine-trans-cyclooctene ligation has been introduced as a method of bioconjugation that proceeds with fast reaction rates without need for catalysis. Herein, we report the development of an extremely fast and efficient method for generating 18F labeled probes based on the tetrazine-trans-cyclooctene ligation. Starting with only 30μg (78μM) of a tetrazine-RGD conjugate and 2mCi (5μM) of 18F-trans-cyclooctene, the 18F labeled RGD peptide could be obtained in more than 90% yield within five minutes. The 18F labeled RGD peptide demonstrated prominent tumor uptake in vivo. The receptor specificity was confirmed by blocking experiments. These results successfully demonstrate that the tetrazine-trans-cyclooctene ligation serves as an efficient labeling method for PET probe construction. [Copyright &y& Elsevier]

peptide heterodimer comprises two different receptor-targeting peptide ligands. Molecular imaging probes based on dual-receptor targeting peptide heterodimers exhibit improved tumor targeting efficacy for multi-receptor expressing tumors compared with their parent single-receptor targeting peptide monomers. Previously we have developed bombesin (BBN)-RGD (Arg-Gly-Asp) peptide heterodimers, in which BBN and RGD are covalently connected with an asymmetric glutamate linker (J Med Chem 52:425-432, 2009). Although F-labeled heterodimers showed significantly better microPET imaging quality than F-labeled RGD and BBN monomers in a PC-3 xenograft model which co-expresses gastrin-releasing peptide receptor (GRPR) and integrin αvβ3, tedious heterodimer synthesis due to the asymmetric nature of glutamate linker restricts their clinical applications. In this study, we report the use of a symmetric linker AEADP [AEADP = 3,3′-(2-aminoethylazanediyl)dipropanoic acid] for the synthesis of BBN-RGD peptide heterodimer. The F-labeled heterodimer (F-FB-AEADP-BBN-RGD) showed comparable microPET imaging results with glutamate linked BBN-RGD heterodimers, indicating that the replacement of glutamate linker with AEADP linker did not affect the biological activities of BBN-RGD heterodimer. The heterodimer synthesis is rather easy and straightforward. Because tumors often co-express multiple receptors, the use of a symmetric linker provides a general method of fast assembly of various peptide heterodimers for imaging multi-receptor expressing tumors. [ABSTRACT FROM AUTHOR]

Purpose: In vivo imaging of α(v)β(3) has important diagnostic and therapeutic applications. (18)F-Galacto-arginine-glycine-aspartic acid (RGD) has been developed for positron emission tomography (PET) imaging of integrin α(v)β(3) expression and is now being tested on humans. Dimerization and multimerization of cyclic RGD peptides have been reported to improve the integrin α(v)β(3)-binding affinity due to the polyvalency effect. Here, we compared a number of new dimeric RGD peptide tracers with the clinically used (18)F-galacto-RGD.Procedures: RGD monomers and dimers were coupled with galacto or PEG(3) linkers, and labeled with (18)F using 4-nitrophenyl 2-(18)F-fluoropropionate ((18)F-NFP) or N-succinimidyl 4-(18)F-fluorobenzoate as a prosthetic group. The newly developed tracers were evaluated by cell-based receptor-binding assay, biodistribution, and small-animal PET studies in a subcutaneous U87MG glioblastoma xenograft model.Results: Starting with (18)F-F(-), the total reaction time for (18)F-FP-SRGD2 and (18)F-FP-PRGD2 is about 120 min. The decay-corrected radiochemical yields for (18)F-FP-SRGD2 and (18)F-FP-PRGD2 are 52 ± 9% and 80 ± 7% calculated from (18)F-NFP. Noninvasive small-animal PET and direct tissue sampling experiments demonstrated that the dimeric RGD peptides had significantly higher tumor uptake as compared to (18)F-galacto-RGD.Conclusion: Dimeric RGD peptide tracers with relatively high tumor integrin-specific accumulation and favorable in vivo kinetics may have the potential to be translated into clinic for integrin α(v)β(3) imaging. [ABSTRACT FROM AUTHOR]