Synthesis and Evaluation of the First 68Ga-Labeled C-Terminal Hydroxamate-Derived Gastrin-Releasing Peptide Receptor-Targeted Tracers for Cancer Imaging with Positron Emission Tomography.

Gastrin-releasing peptide receptor (GRPR), overexpressed in many solid tumors, is a promising imaging marker and therapeutic target. Most reported GRPR-targeted radioligands contain a C-terminal amide. Based on the reported potent antagonist D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH, we synthesized C-terminal hydroxamate-derived [68Ga]Ga-LW02075 ([68Ga]Ga-DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH) and [68Ga]Ga-LW02050 ([68Ga]Ga-DOTA-Pip-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHOH), and compared them with the closely related and clinically validated [68Ga]Ga-SB3 ([68Ga]Ga-DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt). Binding affinities (Ki) of Ga-SB3, Ga-LW02075, and Ga-LW02050 were 1.20 ± 0.31, 1.39 ± 0.54, and 8.53 ± 1.52 nM, respectively. Both Ga-LW02075 and Ga-LW02050 were confirmed to be GRPR antagonists by calcium release assay. Imaging studies showed that PC-3 prostate cancer tumor xenografts were clearly visualized at 1 h post injection by [68Ga]Ga-SB3 and [68Ga]Ga-LW02050 in PET images, but not by [68Ga]Ga-LW02075. Ex vivo biodistribution studies conducted at 1 h post injection showed that the tumor uptake of [68Ga]Ga-LW02050 was comparable to that of [68Ga]Ga-SB3 (5.38 ± 1.00 vs. 6.98 ± 1.36 %ID/g), followed by [68Ga]Ga-LW02075 (3.97 ± 1.71 %ID/g). [68Ga]Ga-SB3 had the highest pancreas uptake (37.3 ± 6.90 %ID/g) followed by [68Ga]Ga-LW02075 (17.8 ± 5.24 %ID/g), while the pancreas uptake of [68Ga]Ga-LW02050 was only 0.53 ± 0.11 %ID/g. Our data suggest that [68Ga]Ga-LW02050 is a promising PET tracer for detecting GRPR-expressing cancer lesions.

Imidazolium-methylene-trifluoroborate: A novel radioprosthetic group validated with preclinical 18 F-Positron Emission Tomography imaging of Prostate Specific Membrane Antigen in mice.

Organotrifluoroborates have gained acceptance as radioprosthetic groups for radiofluorination. Of these, the zwitterionic prosthetic group "AMBF3 " with a quaternary dimethylammonium ion dominates the trifluoroborate space. Herein, we report on imidazolium-methylene trifluoroborate (ImMBF3 ) as an alternative radioprosthetic group and report on its properties in the context of a PSMA-targeting EUK ligand that was previously been conjugated to AMBF3 . The ImMBF3 is readily synthesized from imidazole and conjugated via CuAAC "click" chemistry to give a structure similar to PSMA-617. 18 F-labeling proceeded in one step per our previous reports and imaged in LNCaP-xenograft bearing mice. The [18 F]-PSMA-617-ImMBF3 tracer proved to be less polar (LogP7.4 = -2.95 ± 0.03) while showing a significantly lower solvolytic rate (t1/2 = 8100 min) and slightly higher molar activity (Am) at 174 ± 38 GBq/µmol. Tumor uptake was measured at 13.7 ± 4.8%ID/g and a tumor:muscle ratio of 74.2 ± 35.0, tumor:blood ratio of 21.4 ± 7.0, tumor:kidney ratio of 0.29 ± 0.14, and tumor:bone ratio of 23.5 ± 9.5. In comparison with previously reported PSMA-targeting EUK-AMBF3 conjugates, we have altered the LogP7.4 value, tuned the solvolytic half-life of the prosthetic, and increased radiochemical conversion while achieving similar tumor uptake, contrast ratios, and molar activities compared with AMBF3 bioconjugates.

Synthesis and Evaluation of 99mTc-Labeled PSMA-Targeted Tracers Based on the Lys-Urea-Aad Pharmacophore for Detecting Prostate Cancer with Single Photon Emission Computed Tomography.

Prostate-specific membrane antigen (PSMA) is a well-validated prostate cancer marker but reported PSMA-targeted tracers derived from the Lys-urea-Glu pharmacophore including the clinically validated [99mTc]Tc-EDDA/HYNIC-iPSMA have high off-target uptake in kidneys, spleen, and salivary glands. In this study, we synthesized and evaluated three novel 99mTc-labeled PSMA-targeted tracers and investigated if the tracers derived from the Lys-urea-Aad pharmacophore could have minimized uptake in off-target organs/tissues. In vitro competition binding assays showed that compared with HYNIC-iPSMA, the three novel ligands had slightly weaker PSMA binding affinity (average Ki = 3.11 vs. 8.96-11.6 nM). Imaging and ex vivo biodistribution studies in LNCaP tumor-bearing mice showed that [99mTc]Tc-EDDA/HYNIC-iPSMA and the three novel tracers successfully visualized LNCaP tumor xenografts in SPECT images and were excreted mainly via the renal pathway. The average tumor uptake at 1 h post-injection varied from 5.40 to 18.8%ID/g, and the tracers derived from the Lys-urea-Aad pharmacophore had much lower uptake in the spleen and salivary glands. Compared with the clinical tracer [99mTc]Tc-EDDA/HYNIC-iPSMA, the Lys-urea-Aad-derived [99mTc]Tc-EDDA-KL01127 had lower background uptake and superior tumor-to-background contrast ratios and is therefore promising for clinical translation to detect prostate cancer lesions with SPECT.

68Ga-Labeled [Thz14]Bombesin(7-14) Analogs: Promising GRPR-Targeting Agonist PET Tracers with Low Pancreas Uptake.

With overexpression in various cancers, the gastrin-releasing peptide receptor (GRPR) is a promising target for cancer imaging and therapy. However, the high pancreas uptake of reported GRPR-targeting radioligands limits their clinical application. Our goal was to develop 68Ga-labeled agonist tracers for detecting GRPR-expressing tumors with positron emission tomography (PET), and compare them with the clinically validated agonist PET tracer, [68Ga]Ga-AMBA. Ga-TacBOMB2, TacBOMB3, and TacBOMB4, derived from [Thz14]Bombesin(7-14), were confirmed to be GRPR agonists by a calcium mobilization study, and their binding affinities (Ki(GRPR)) were determined to be 7.62 ± 0.19, 6.02 ± 0.59, and 590 ± 36.5 nM, respectively, via in vitro competition binding assays. [68Ga]Ga-TacBOMB2, [68Ga]Ga-TacBOMB3, and [68Ga]Ga-AMBA clearly visualized PC-3 tumor xenografts in a PET imaging study. [68Ga]Ga-TacBOMB2 showed comparable tumor uptake but superior tumor-to-background contrast ratios when compared to [68Ga]Ga-AMBA. Moreover, [68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3 showed a much lower rate of uptake in the pancreas (1.30 ± 0.14 and 2.41 ± 0.72%ID/g, respectively) than [68Ga]Ga-AMBA (62.4 ± 4.26%ID/g). In conclusion, replacing Met14 in the GRPR-targeting sequence with Thz14 retains high GRPR-binding affinity and agonist properties. With good tumor uptake and tumor-to-background uptake ratios, [68Ga]Ga-TacBOMB2 is promising for detecting GRPR-expressing tumors. The much lower pancreas uptake of [68Ga]Ga-TacBOMB2 and [68Ga]Ga-TacBOMB3 suggests that [Thz14]Bombesin(7-14) is a promising targeting vector for the design of GRPR-targeting radiopharmaceuticals, especially for radioligand therapy application.

Synthesis and Preclinical Evaluation of Three Novel 68Ga-Labeled Bispecific PSMA/FAP-Targeting Tracers for Prostate Cancer Imaging.

Tumor heterogeneity limits the efficacy and reliability of monospecific radiopharmaceuticals in prostate cancer diagnosis and therapy. To overcome this limitation and improve lesion detection sensitivity, we developed and evaluated three bispecific radiotracers that can target both prostate-specific membrane antigen (PSMA) and fibroblast activation protein (FAP), which are the two key proteins overexpressed in prostate cancer. Three FAP-targeting ligands with various linker lengths were synthesized through multistep organic synthesis, and then connected to the PSMA-targeting motif. IC50(PSMA) and IC50(FAP) values of Ga-complexed bispecific ligands, Ga-AV01017, Ga-AV01030, and Ga-AV01038 were 25.2-71.6 and 1.25-2.74 nM, respectively. The uptake values in PSMA-expressing LNCaP tumor xenografts were 4.38 ± 0.55, 5.17 ± 0.51, and 4.25 ± 0.86 %ID/g for [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, and [68Ga]Ga-AV01038, respectively, which were lower than the monospecific PSMA-targeting tracer [68Ga]Ga-HTK03041 (23.1 ± 6.11 %ID/g). The uptake values in FAP-expressing HEK293T:hFAP tumor xenografts were 2.99 ± 0.37, 3.69 ± 0.81, 3.64 ± 0.83 %ID/g for [68Ga]Ga-AV01017, [68Ga]Ga-AV01030, and [68Ga]Ga-AV01038, respectively, which were also lower than the monospecific FAP-targeting tracer, [68Ga]Ga-FAPI-04 (12.5 ± 2.00 %ID/g). We observed that the bispecific tracers had prolonged blood retention, in which tracers with a longer linker tend to have a higher blood uptake and lower tumor uptake. Further investigations are needed to optimize the linker selection to generate promising bispecific PSMA/FAP-targeting tracers for prostate cancer imaging.

Toward 18 F-Labeled Theranostics: A Single Agent that Can Be Labeled with 18 F, 64 Cu, or 177 Lu.

We report a single-molecule radiotracer that can be labeled independently with 18 F-fluoride or radiometals (64 Cu, 177 Lu) in a single step. A prostate-specific membrane antigen (PSMA)-targeting ligand, armed with both an organotrifluoroborate and a metal-chelator (DOTA), was designed to optionally afford 18 F-, 64 Cu- or 177 Lu-labeled products that were injected into mice bearing prostate cancer (LNCaP) xenografts. PET/CT images and ex vivo biodistribution data show high, specific tumor uptake irrespective of which radionuclide is used, thereby demonstrating a new approach to combining, in a single molecule, 18 F-labeling capabilities for PET imaging with radiometalation for potential imaging and therapeutic applications.

[nat/44Sc(pypa)]-: Thermodynamic Stability, Radiolabeling, and Biodistribution of a Prostate-Specific-Membrane-Antigen-Targeting Conjugate.

44Sc is an attractive positron-emitting radionuclide for PET imaging; herein, a new complex of the Sc3+ ion with nonmacrocyclic chelator H4pypa was synthesized and characterized with high-resolution electrospray-ionization mass spectrometry (HR-ESI-MS), as well as different nuclear magnetic resonance (NMR) spectroscopic techniques (1H, 13C, 1H-13C HSQC, 1H-13C HMBC, COSY, and NOESY). In aqueous solution (pH = 7), [Sc(pypa)]- presented two isomeric forms, the structures of which were predicted using density functional theory (DFT) calculation with a small energy difference of 22.4 kJ/mol, explaining their coexistence. [Sc(pypa)]- was found to have superior thermodynamic stability (pM = 27.1) compared to [Sc(AAZTA)]- (24.7) and [Sc(DOTA)]- (23.9). In radiolabeling, [44Sc][Sc(pypa)]- formed efficiently at RT in 15 min over a range of pH (2-5.5), resulting in a complex that is highly stable (>99%) in mouse serum over at least six half-lives of scandium-44. Similar labeling efficiency was observed with the PSMA (prostate-specific membrane antigen)-targeting H4pypa-C7-PSMA617 at pH = 5.5 (RT, 15 min), confirming negligible disturbance from the bifunctionalization on scandium-44 scavenging. Moreover, the kinetic inertness of the radiocomplex was proved in vivo. Surprisingly, the molar activity was found to have profound influence on the pharmacokinetics of the radiotracers where lower molar activity drastically reduced the background accumulations, particularly, kidney, and thus, yielded a much higher tumor-to-background contrast.

Comparison of biological properties of [177 Lu]Lu-ProBOMB1 and [177 Lu]Lu-NeoBOMB1 for GRPR targeting.

The gastrin-releasing peptide receptor (GRPR) is overexpressed in prostate cancer and other solid malignancies. Following up on our work on [68 Ga]Ga-ProBOMB1 that had better imaging characteristics than [68 Ga]Ga-NeoBOMB1, we investigated the effects of substituting 68 Ga for 177 Lu to determine if the resulting radiopharmaceuticals could be used with a therapeutic aim. We radiolabeled the bombesin antagonist ProBOMB1 (DOTA-pABzA-DIG-D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-ψ-Pro-NH2 ) with lutetium-177 and compared it with [177 Lu]Lu-NeoBOMB1 (obtained in 54.2 ± 16.5% isolated radiochemical yield with >96% radiochemical purity and 440.8 ± 165.1 GBq/µmol molar activity) for GRPR targeting. Lu-NeoBOMB1 had better binding affinity for GRPR than Lu-ProBOMB1 (Ki values: 2.26 ± 0.24 and 30.2 ± 3.23nM). [177 Lu]Lu-ProBOMB1 was obtained in 53.7 ± 5.4% decay-corrected radiochemical yield with 444.2 ± 193.2 GBq/µmol molar activity and >95% radiochemical purity. In PC-3 prostate cancer xenograft mice, tumor uptake of [177 Lu]Lu-ProBOMB1 was 3.38 ± 1.00, 1.32 ± 0.24, and 0.31 ± 0.04%ID/g at 1, 4, and 24 hours pi. However, the uptake in tumor was lower than [177 Lu]Lu-NeoBOMB1 at all time points. [177 Lu]Lu-ProBOMB1 was inferior to [177 Lu]Lu-NeoBOMB1, which had better therapeutic index for the organs receiving the highest doses.

Evaluation of Met-Val-Lys as a Renal Brush Border Enzyme-Cleavable Linker to Reduce Kidney Uptake of 68Ga-Labeled DOTA-Conjugated Peptides and Peptidomimetics.

High kidney uptake is a common feature of peptide-based radiopharmaceuticals, leading to reduced detection sensitivity for lesions adjacent to kidneys and lower maximum tolerated therapeutic dose. In this study, we evaluated if the Met-Val-Lys (MVK) linker could be used to lower kidney uptake of 68Ga-labeled DOTA-conjugated peptides and peptidomimetics. A model compound, [68Ga]Ga-DOTA-AmBz-MVK(Ac)-OH (AmBz: aminomethylbenzoyl), and its derivative, [68Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH, coupled with the PSMA (prostate-specific membrane antigen)-targeting motif of the previously reported HTK01166 were synthesized and evaluated to determine if they could be recognized and cleaved by the renal brush border enzymes. Additionally, positron emission tomography (PET) imaging, ex vivo biodistribution and in vivo stability studies were conducted in mice to evaluate their pharmacokinetics. [68Ga]Ga-DOTA-AmBz-MVK(Ac)-OH was effectively cleaved specifically by neutral endopeptidase (NEP) of renal brush border enzymes at the Met-Val amide bond, and the radio-metabolite [68Ga]Ga-DOTA-AmBz-Met-OH was rapidly excreted via the renal pathway with minimal kidney retention. [68Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH retained its PSMA-targeting capability and was also cleaved by NEP, although less effectively when compared to [68Ga]Ga-DOTA-AmBz-MVK(Ac)-OH. The kidney uptake of [68Ga]Ga-DOTA-AmBz-MVK(HTK01166)-OH was 30% less compared to that of [68Ga]Ga-HTK01166. Our data demonstrated that derivatives of [68Ga]Ga-DOTA-AmBz-MVK-OH can be cleaved specifically by NEP, and therefore, MVK can be a promising cleavable linker for use to reduce kidney uptake of radiolabeled DOTA-conjugated peptides and peptidomimetics.

Functionally Versatile and Highly Stable Chelator for 111In and 177Lu: Proof-of-Principle Prostate-Specific Membrane Antigen Targeting.

Here, we present the synthesis and characterization of a new potentially nonadentate chelator H4pypa and its bifunctional analogue tBu4pypa-C7-NHS conjugated to prostate-specific membrane antigen (PSMA)-targeting peptidomimetic (Glu-urea-Lys). H4pypa is very functionally versatile and biologically stable. Compared to the conventional chelators (e.g., DOTA, DTPA), H4pypa has outstanding affinities for both 111In (EC, t1/2 ≈ 2.8 days) and 177Lu (ß-,γ, t1/2 ≈ 6.64 days). Its radiolabeled complexes were achieved at >98% radiochemical yield, RT within 10 min, at a ligand concentration as low as 10-6 M, with excellent stability in human serum over at least 5-7 days (<1% transchelation). The thermodynamic stabilities of the [M(pypa)]- complexes (M3+ = In3+, Lu3+, La3+) were dependent on the ionic radii, where the smaller In3+ has the highest pM value (30.5), followed by Lu3+ (22.6) and La3+ (19.9). All pM values are remarkably higher than those with DOTA, DTPA, H4octapa, H4octox, and H4neunpa. Moreover, the facile and versatile bifunctionalization enabled by the p-OH group in the central pyridyl bridge of the pypa scaffold (compound 14) allows incorporation of a variety of linkers for bioconjugation through easy nucleophilic substitution. In this work, an alkyl linker was selected to couple H4pypa to a PSMA-targeting pharmacophore, proving that the bioconjugation sacrifices neither the tumor-targeting nor the chelation properties. The biodistribution profiles of 111In- and 177Lu-labeled tracers are different, but promising, with the 177Lu analogue particularly outstanding.

[68Ga]Ga/[177Lu]Lu-BL01, a Novel Theranostic Pair for Targeting C-X-C Chemokine Receptor 4.

C-X-C chemokine receptor type 4 (CXCR4) is overexpressed in hematological and solid malignancies. LY2510924 is a potent peptide antagonist of CXCR4. A derivative of LY2510924, BL01, was evaluated for theranostic applications targeting CXCR4. Methods: BL01 was synthesized by solid phase approach. A Lys(ivDde) residue was added at the C-terminus of LY2510924 (cyclo[Phe-Tyr-Lys(iPr)-d-Arg-2-Nal-Gly-d-Glu]-Lys(iPr)-NH2). A DOTA chelator was conjugated to the side chain of the deprotected exogenous Lys residue. The binding affinity of Ga/Lu-BL01 was determined by competitive radioligand binding assays. BL01 was radiolabeled with 68GaCl3 or 177LuCl3. Biodistribution studies were performed in mice bearing Daudi Burkitt's lymphoma tumor xenografts at selected time points. PET imaging studies were performed with [68Ga]Ga-BL01, with blocking experiments performed with preinjection of LY2510924. The stability of [68Ga]Ga/[177Lu]Lu-BL01 was assessed in mouse plasma. Results: Ga-BL01 and Lu-BL01 have nanomolar affinity for CXCR4. [68Ga]Ga-BL01 was obtained in 58 ± 5% decay-corrected radiochemical yields and >99% radiochemical purity with a molar activity of 40 ± 11 GBq/µmol, while [177Lu]Lu-BL01 was obtained in 65 ± 6% decay-corrected radiochemical yields and >99% radiochemical purity with a molar activity of 120 ± 21 GBq/µmol. [68Ga]Ga-BL01 and [177Lu]Lu-BL01 were excreted primarily through the renal pathway. Daudi xenografts were clearly delineated in PET images with good contrast. On the basis of biodistribution data, tumor uptake of [68Ga]Ga-BL01 was 10.2 ± 2.56% injected dose per gram (%ID/g) at 1 h postinjection (p.i.). Spleen (12.6 ± 2.36 %ID/g) and lungs (13.2 ± 2.98 %ID/g), organs that express CXCR4, had high uptake as well. Preinjection of LY2510924 reduced average uptake of [68Ga]Ga-BL01 in tumors by 88%, demonstrating target specificity. The uptake of [68Ga]Ga-BL01 in tumor increased to 15.3 ± 1.86 %ID/g at 2 h p.i., with improved contrast. [177Lu]Lu-BL01 has similar pharmacokinetics as [68Ga]Ga-BL01 at 1 h p.i. The highest uptake was observed in tumor (14.0 ± 1.11 %ID/g), followed by the lungs (13.0 ± 1.27 %ID/g) and spleen (11.6 ± 1.78 %ID/g). The tumor uptake increased to 16.2 ± 2.69 %ID/g at 4 h p.i., before declining slightly to 10.1 ± 1.41 %ID/g at 24 h p.i. Both compounds were stable in vivo, as no metabolites were observed at 5 min p.i. Conclusions: [68Ga]Ga-BL01 and [177Lu]Lu-BL01 are a promising theranostic pair for imaging and endoradiotherapy of CXCR4-expressing malignancies.

Effects of Linker Modification on Tumor-to-Kidney Contrast of 68Ga-Labeled PSMA-Targeted Imaging Probes.

68Ga-PSMA-11 is currently the most popular prostate-specific membrane antigen (PSMA) radioligand used in the clinic to detect prostate cancer and metastases. However, the high uptake of 68Ga-PSMA-11 in kidneys can create halo-artifacts resulting in lower detection sensitivity for lesions adjacent to the kidneys. In this study, we developed two 68Ga-labeled PSMA-targeted tracers, 68Ga-HTK01166 and 68Ga-HTK01167, based on 68Ga-PSMA-617 with the goal of improving tumor-to-kidney ratio compared to 68Ga-PSMA-11. The 2-naphthylalanine (2-Nal) in PSMA-617 was replaced with 2-indanylglycine (Igl) or 3,3-diphenylalanine (Dip) to synthesize HTK01166 and HTK01167, respectively. Binding affinities ( Ki) of Ga-PSMA-11, Ga-PSMA-617, Ga-HTK01166, and Ga-HTK01167 to PSMA were 3.13 ± 0.40, 1.23 ± 0.08, 5.74 ± 2.48, and 25.7 ± 9.84 nM, respectively, as determined by in vitro competition binding assays. 68Ga labeling was performed in HEPES buffer with microwave heating, and 68Ga-labeled PSMA-11, PSMA-617, HTK01166, and HTK01167 were obtained in 46-69% average decay-corrected radiochemical yield with >99% radiochemical purity and 62.9-152 GBq/µmol average specific activity. PET imaging and biodistribution studies were performed in mice bearing PSMA-expressing LNCap prostate cancer xenografts. All tracers enabled clear visualization of tumors in PET images with excellent tumor-to-background contrast. The uptake values (%ID/g) for tumor and kidneys at 1 h postinjection were 8.91 ± 0.86 and 204 ± 70.6 for 68Ga-PSMA-11, 16.7 ± 2.30 and 29.2 ± 5.14 for 68Ga-PSMA-617, 14.1 ± 4.40 and 147 ± 59.6 for 68Ga-HTK01166, and 7.79 ± 1.65 and 4.30 ± 1.80 for 68Ga-HTK01167. The tumor-to-kidney ratios for 68Ga-labeled PSMA-11, PSMA-617, HTK01166, and HTK01167 were 0.05 ± 0.02, 0.63 ± 0.10, 0.10 ± 0.02, and 1.98 ± 0.63, respectively. Compared with 68Ga-PSMA-617, 68Ga-HTK01166 showed comparable tumor uptake and almost 5-fold higher kidney uptake, whereas 68Ga-HTK01167 exhibited lower tumor and kidney uptake. Compared with 68Ga-PSMA-11, 68Ga-HTK01167 had similar tumor uptake and tumor-to-blood contrast ratio (23.8 ± 6.71 vs 20.4 ± 4.98) but higher tumor-to-background contrast ratios for other background organs especially for kidneys. Our data indicate that substitution of 2-Nal in PSMA-617 with other lipophilic amino acid can modulate PSMA binding affinity and their pharmacokinetics in vivo.

Enhancing Treatment Efficacy of 177Lu-PSMA-617 with the Conjugation of an Albumin-Binding Motif: Preclinical Dosimetry and Endoradiotherapy Studies.

We designed and evaluated a novel albumin-binder-conjugated 177Lu-PSMA-617 derivative, 177Lu-HTK01169, with an extended blood retention time to maximize the radiation dose delivered to prostate tumors expressing prostate-specific membrane antigen (PSMA). PSMA-617 and HTK01169 that contained N-[4-( p-iodophenyl)butanoyl]-Glu as an albumin-binding motif were synthesized using the solid-phase approach. Binding affinity to PSMA was determined by in vitro competition-binding assay. 177Lu labeling was performed in acetate buffer (pH 4.5) at 90 °C for 15 min. SPECT/CT imaging, biodistribution, and endoradiotherapy studies were conducted in mice bearing PSMA-expressing LNCaP tumor xenografts. Radiation dosimetry was calculated using OLINDA software. Lu-PSMA-617 and Lu-HTK01169-bound PSMA with high affinity ( Ki values = 0.24 and 0.04 nM, respectively). SPECT imaging and biodistribution studies showed that 177Lu-PSMA-617 and 177Lu-HTK01169 were excreted mainly via the renal pathway. With fast blood clearance (0.68%ID/g at 1 h postinjection), the tumor uptake of 177Lu-PSMA-617 peaked at 1 h postinjection (15.1%ID/g) and gradually decreased to 7.91%ID/g at 120 h postinjection. With extended blood retention (16.6 and 2.10%ID/g at 1 and 24 h, respectively), the tumor uptake of 177Lu-HTK01169 peaked at 24 h postinjection (55.9%ID/g) and remained at the same level by the end of the study (120 h). Based on dosimetry calculations, 177Lu-HTK01169 delivered an 8.3-fold higher radiation dose than 177Lu-PSMA-617 to LNCaP tumor xenografts. For the endoradiotherapy study, the mice treated with 177Lu-PSMA-617 (18.5 MBq) all reached humane end point (tumor volume >1000 mm3) by Day 73 with a median survival of 58 days. Mice treated with 18.5, 9.3, 4.6, or 2.3 MBq of 177Lu-HTK01169 had a median survival of >120, 103, 61, and 28 days, respectively. With greatly enhanced tumor uptake and treatment efficacy compared to 177Lu-PSMA-617 in preclinical studies, 177Lu-HTK01169 warrants further investigation for endoradiotherapy of prostate cancer.

Synthesis and evaluation of an 18F-labeled boramino acid analog of aminosuberic acid for PET imaging of the antiporter system xC.

In this study, we synthesized 18F-ASu-BF3, a close boramino acid analog of 5-[18F]fluoro-aminosuberic acid (18F-ASu), via 18F-19F isotope exchange reaction and evaluated its potential for imaging with positron emission tomography (PET). 18F-ASu-BF3 was stable in mouse plasma and taken up into PC3 prostate cancer cells via the system xC- amino acid transporter. The continuous use of isoflurane for anesthesia during dynamic imaging acquisition slowed down the excretion of 18F-ASu-BF3 and enabled visualization of PC3 tumor xenografts in mice. In contrast, no tumor visualization was observed from static images of 18F-BF3-ASu due to its rapid renal excretion mediated in part by the organic anion transporter. Our data indicate that the pharmacokinetics of amino acids could be altered after being converted into their boramino acid analogs. Therefore, care should be taken when using the boramino acid strategy to design and prepare 18F-labeled tracers for imaging amino acid transporters/receptors with PET.

Synthesis and evaluation of an 18 F-labeled trifluoroborate derivative of 2-nitroimidazole for imaging tumor hypoxia with positron emission tomography.

2-Nitroimidazole-based hypoxia imaging tracers such as 18 F-FMISO are normally imaged at late time points (several hours post-injection) due to their slow clearance from background tissues. Here, we investigated if a hydrophilic zwitterion-based ammoniomethyl-trifluoroborate derivative of 2-nitroimidazole, 18 F-AmBF3 -Bu-2NI, could have the potential to image tumor hypoxia at earlier time points. AmBF3 -Bu-2NI was prepared in 4 steps. 18 F labeling was conducted via 18 F-19 F isotope exchange reaction, and 18 F-AmBF3 -Bu-2NI was obtained in 14.8 ± 0.4% (n = 3) decay-corrected radiochemical yield with 24.5 ± 5.2 GBq/µmol specific activity and >99% radiochemical purity. Imaging and biodistribution studies in HT-29 tumor-bearing mice showed that 18 F-AmBF3 -Bu-2NI cleared quickly from blood and was excreted via the hepatobiliary and renal pathways. However, the tumor was not visualized in PET images until 3 hours post-injection due to low tumor uptake (0.54 ± 0.13 and 0.19 ± 0.04%ID/g at 1 and 3 hours post-injection, respectively). The low tumor uptake is likely due to the highly hydrophilic motif of ammoniomethyl-trifluoroborate that prevents free diffusion of 18 F-AmBF3 -Bu-2NI across the cell membrane. Our results suggest that highly hydrophilic 18 F-labeled ammoniomethyl-trifluoroborate derivatives might not be suitable for imaging intracellular targets including nitroreductase, a common tumor hypoxia imaging target.

Radiolabeled R954 Derivatives for Imaging Bradykinin B1 Receptor Expression with Positron Emission Tomography.

Peptide receptors have emerged as promising targets for diagnosis and therapy. The aberrant overexpression of these receptors in different cancer subtypes allows for the adoption of new treatment strategies that complement conventional chemotherapies. Bradykinin B1 receptor (B1R) is a G protein-coupled receptor that is overexpressed in many cancers, with limited expression in healthy tissues. Previously, we developed 68Ga- and 18F-labeled derivatives of B1R antagonist peptides B9858 and B9958, and successfully targeted B1R-expressing tumor xenografts in vivo. R954 (Ac-Orn-Arg-Oic-Pro-Gly-αMePhe-Ser-d-2-Nal-Ile), a potent B1R antagonist, is reportedly more stable than B9858 against peptidase degradation. We evaluated two radiolabeled derivatives of R954 (68Ga-HTK01083 and 18F-HTK01146) for B1R PET imaging. Peptides were synthesized via solid phase strategy. Nonradioactive standards were obtain by reacting GaCl3 with DOTA-dPEG2-R954 and by clicking N-propargyl-N,N-dimethylammoniomethyl-trifluoroborate with azidoacetyl-dPEG2-R954. Binding affinity for B1R was determined by an in vitro competition binding assay. 68Ga-HTK01083 was obtained by incubating DOTA-dPEG2-R954 with 68GaCl3 under acidic conditions, while 18F-HTK01146 was prepared via an 18F-19F isotope exchange reaction. Biodistribution and imaging studies were conducted at 1 h postinjection (p.i.) in mice inoculated with B1R-expressing (B1R+) and B1R-nonexpressing (B1R-) cells. HTK01083 and HTK01146 bound B1R with good affinity (Ki = 30.5 and 24.8 nM, respectively). 68Ga/18F-labeled R954 were obtained on average in ≥10% decay-corrected radiochemical yield with >99% radiochemical purity and ≥52 GBq/µmol specific activity. For both tracers, clearance was predominantly renal with minimal involvement of the hepatobiliary system. For PET images, B1R+ tumors, kidneys, and bladder were visible. At 1 h p.i., uptake in B1R+ tumor was comparable between 68Ga-HTK01083 (8.46 ± 1.44%ID/g) and 18F-HTK01146 (9.25 ± 0.69%ID/g). B1R+ tumor-to-blood and B1R+ tumor-to-muscle ratios were 6.32 ± 1.44 and 20.7 ± 3.58 for 68Ga-HTK01083, and 7.24 ± 2.56 and 19.5 ± 4.29 for 18F-HTK01146. Our results indicate R954 is a good lead sequence for optimization of B1R tracers for cancer imaging.

Synthesis and evaluation of 18F-labeled CJ-042794 for imaging prostanoid EP4 receptor expression in cancer with positron emission tomography.

The potent and selective prostanoid EP4 receptor antagonist CJ-042794 was radiolabeled with 18F, and evaluated for imaging EP4 receptor expression in cancer with positron emission tomography (PET). The fluorination precursor, arylboronic acid pinacol ester 4, was prepared in 4 steps with 42% overall yield. 18F-CJ-042794 was synthesized via a copper-mediated 18F-fluorination reaction followed by base hydrolysis, and was obtained in 1.5±1.1% (n=2) decay-corrected radiochemical yield. PET/CT imaging and biodistribution studies in mice showed that 18F-CJ-042794 was excreted through both renal and hepatobiliary pathways with significant retention in blood. The EP4-receptor-expressing LNCaP prostate cancer xenografts were clearly visualized in PET images with 1.12±0.08%ID/g (n=5) uptake value and moderate tumour-to-muscle contrast ratio (2.73±0.22) at 1h post-injection. However, the tumour uptake was nonspecific as it could not be blocked by co-injection of cold standard, precluding the application of 18F-CJ-042794 for PET imaging of EP4 receptor expression in cancer.

PET Imaging of Carbonic Anhydrase IX Expression of HT-29 Tumor Xenograft Mice with (68)Ga-Labeled Benzenesulfonamides.

Carbonic anhydrase IX (CA-IX) is a HIF-1-inducible enzyme that is overexpressed in many cancer subtypes to promote survival and invasion in hypoxic niches. Pharmacologic inhibition of CA-IX is achievable through sulfonamide-based inhibitors and has been shown to reduce primary growth of cancers and distant metastasis in preclinical models. We explored a multivalent approach for targeting CA-IX in vivo, noninvasively, with positron emission tomography. Three (68)Ga-polyaminocarboxylate chelator complex-conjugated tracers containing one, two, or three 4-(2-aminoethyl)benzenesulfonamide moieties were synthesized and evaluated for protein binding and imaging properties. Binding affinity to CA-I, -II, -IX, and -XII were determined using a stopped-flow CA catalyzed CO2 hydration assay. Biodistribution and PET/CT imaging were performed using immunocompromised mice bearing CA-IX expressing HT-29 colorectal tumors. Compounds demonstrated good binding affinity to CA-IX (Ki: 7.7-25.4 nM). (68)Ga-labeled sulfonamides were obtained in 64-91% decay-corrected average radiochemical yields with 50-536 GBq/µmol specific activity and >97% average radiochemical purity. All three tracers allowed for the visualization of tumor xenografts at 1 h postinjection, with the monomer displaying the highest contrast. Tumor uptake of the monomer was blockable in the presence of acetazolamide, confirming target specificity. The monomer was excreted predominantly through the kidneys, while the dimer and trimer were cleared by both renal and hepatobiliary pathways. According to biodistribution analysis, tumor uptake (%ID/g) of the monomeric, dimeric, and trimeric tracers were 0.81 ± 0.15, 1.93 ± 0.26, and 2.30 ± 0.53 at 1 h postinjection. This corresponded to tumor-to-muscle ratios of 5.02 ± 0.22, 4.07 ± 0.87, and 4.18 ± 0.84, respectively. Our data suggest that (68)Ga-polyaminocarboxylate chelator-conjugated sulfonamides can be used to noninvasively image CA-IX. These CA-IX targeting PET tracers may be used to identify patients who can benefit from treatments targeting this protein or serve as surrogate imaging agents for tumor hypoxia.

Design, synthesis and evaluation of (18)F-labeled bradykinin B1 receptor-targeting small molecules for PET imaging.

Two fluorine-18 ((18)F) labeled bradykinin B1 receptor (B1R)-targeting small molecules, (18)F-Z02035 and (18)F-Z02165, were synthesized and evaluated for imaging with positron emission tomography (PET). Z02035 and Z02165 were derived from potent antagonists, and showed high binding affinity (0.93±0.44 and 2.80±0.50nM, respectively) to B1R. (18)F-Z02035 and (18)F-Z02165 were prepared by coupling 2-[(18)F]fluoroethyl tosylate with their respective precursors, and were obtained in 10±5 (n=4) and 22±14% (n=3), respectively, decay-corrected radiochemical yield with >99% radiochemical purity. (18)F-Z02035 and (18)F-Z02165 exhibited moderate lipophilicity (LogD7.4=1.10 and 0.59, respectively), and were stable in mouse plasma. PET imaging and biodistribution studies in mice showed that both tracers enabled visualization of the B1R-positive HEK293T::hB1R tumor xenografts with better contrast than control B1R-negative HEK293T tumors. Our data indicate that small molecule antagonists can be used as pharmacophores for the design of B1R-targeting PET tracers.

Synthesis and evaluation of (18)F-labeled 4-nitrobenzyl derivatives for imaging tumor hypoxia with positron emission tomography: Comparison of 2-[(18)F]fluoroethyl carbonate and 2-[(18)F]fluoroethyl carbamate.

Two 4-nitrobenzyl derivatives, 2-fluoroethyl 4-nitrobenzyl carbonate 1 and 4-nitrobenzyl N-2-fluoroethyl carbamate 2, were radiolabeled with (18)F and evaluated for imaging tumor hypoxia with positron emission tomography. Although good tumor uptake was observed for [(18)F]1 and [(18)F]2 (>2.5%ID/g at 3-h post-injection), the tracers cleared slowly from nontarget tissues (>1.5%ID/g) and exhibited extensive defluorination in vivo (>4.0%ID/g for bone). Therefore, [(18)F]1 and [(18)F]2 are not suitable for imaging tumor hypoxia due to suboptimal tumor-to-background contrasts.