Evaluation of agonist and antagonist radioligands for somatostatin receptor imaging of breast cancer using positron emission tomography

Background: The somatostatin receptor subtype 2 (sstr2) is expressed on a majority of luminal breast cancers, however SPECT and scintigraphy imaging with agonistic sstr2 probes has been sub-optimal. High affinity antagonists can access more binding sites on the cell surface, resulting in higher tumor uptake and improved sensitivity. We compared the tumor uptake and biodistribution of the antagonist 68Ga-NODAGA-JR11 with two agonists 68Ga-DOTA-Tyr3-octreotide (68Ga-DOTATOC) and 68Ga-DOTA-Tyr3-octreotate (68Ga-DOTATATE), in the human, sstr2-positive, luminal breast cancer model: ZR-75-1. Results: Peptides were assayed for binding affinity using a filtration-based competitive assay to sstr2. natGa-DOTATOC and natGa-DOTATATE had excellent affinity (inhibition constant Ki: 0.9 ± 0.1 nM and 1.4 ± 0.3 nM respectively) compared to natGa-NODAGA-JR11 (25.9 ± 0.2 nM). The number of binding sites on ZR-75-1 cells was determined in vitro by saturation assays. Agonist 67/natGa-DOTATOC bound to 6.64 ± 0.39 × 104 sites/cells, which was 1.5-fold higher than 67/natGa-NODAGA-JR11 and 2.3-fold higher than 67/natGa-DOTATATE. All three 68Ga-labeled peptides were obtained in good decay-corrected radiochemical yield (61-68%) and were purified by high performance liquid chromatography to ensure high specific activity (137 – 281 MBq/nmol at the end of synthesis). NOD scid gamma mice bearing ZR-75-1 tumors were injected intravenously with the labeled peptides and used for PET/CT imaging and biodistribution at 1 h post-injection. We found that 68Ga-DOTATOC had the highest tumor uptake (18.4 ± 2.9%ID/g), followed by 68Ga-DOTATATE (15.2 ± 2.2%ID/g) and 68Ga-NODAGA-JR11 (12.2 ± 0.8%ID/g). Tumor-to-blood and tumor-to-muscle ratios were also higher for the agonists (>40 and >150 respectively), compared to the antagonist (15.6 ± 2.2 and 45.2 ± 11.6 respectively). Conclusions: The antagonist 68Ga-NODAGA-JR11 had the lowest tumor uptake and contrast compared to agonists 68Ga-DOTATOC and 68Ga-DOTATATE in ZR-75-1 xenografts. The main contributing factor to this result could be the use of an endogenously expressing cell line, which may differ from previously published transfected models in the number of low-affinity, antagonist-specific binding sites. The relative merit of agonists versus antagonists for sstr2 breast cancer imaging warrants further investigation, first in preclinical models with other sstr2-positive breast cancer xenografts, and ultimately in luminal breast cancer patients.Medicine, Faculty ofNon UBCRadiology, Department ofReviewedFacult

Synthesis and Evaluation of ⁶⁸Ga-Labeled (2<i>S</i>,4<i>S</i>)-4-Fluoropyrrolidine-2-Carbonitrile and (4<i>R</i>)-Thiazolidine-4-Carbonitrile Derivatives as Novel Fibroblast Activation Protein-Targeted PET Tracers for Cancer Imaging

Fibroblast activation protein α (FAP-α) is a cell-surface protein overexpressed on cancer-associated fibroblasts that constitute a substantial component of tumor stroma and drive tumorigenesis. FAP is minimally expressed by most healthy tissues, including normal fibroblasts. This makes it a promising pan-cancer diagnostic and therapeutic target. In the present study, we synthesized two novel tracers, [68Ga]Ga-SB03045 and [68Ga]Ga-SB03058, bearing a (2S,4S)-4-fluoropyrrolidine-2-carbonitrile or a (4R)-thiazolidine-4-carbonitrile pharmacophore, respectively. [68Ga]Ga-SB03045 and [68Ga]Ga-SB03058 were evaluated for their FAP-targeting capabilities using substrate-based in vitro binding assays, and in PET/CT imaging and ex vivo biodistribution studies in an HEK293T:hFAP tumor xenograft mouse model. The IC50 values of natGa-SB03045 (1.59 ± 0.45 nM) and natGa-SB03058 (0.68 ± 0.09 nM) were found to be lower than those of the clinically validated natGa-FAPI-04 (4.11 ± 1.42 nM). Contrary to the results obtained in the FAP-binding assay, [68Ga]Ga-SB03058 demonstrated a ~1.5 fold lower tumor uptake than that of [68Ga]Ga-FAPI-04 (7.93 ± 1.33 vs. 11.90 ± 2.17 %ID/g), whereas [68Ga]Ga-SB03045 (11.8 ± 2.35 %ID/g) exhibited a tumor uptake comparable to that of [68Ga]Ga-FAPI-04. Thus, our data suggest that the (2S,4S)-4-fluoropyrrolidine-2-carbonitrile scaffold holds potential as a promising pharmacophore for the design of FAP-targeted radioligands for cancer diagnosis and therapy

Synthesis and Evaluation of Novel 68Ga-Labeled [D-Phe6,Leu13ψThz14]bombesin(6-14) Analogs for Cancer Imaging with Positron Emission Tomography

Gastrin-releasing peptide receptor (GRPR) is overexpressed in various cancers and is a promising target for cancer diagnosis and therapy. However, the high pancreas uptake and/or metabolic instability observed for most reported GRPR-targeted radioligands might limit their clinical applications. Our group recently reported a GRPR-targeted antagonist tracer, [⁶⁸Ga]Ga-TacsBOMB2 ([⁶⁸Ga]Ga-DOTA-Pip-D-Phe⁶-Gln⁷-Trp⁸-Ala⁹-Val¹⁰-Gly¹¹-His¹²-Leu¹³ψThz¹⁴-NH₂), which showed a minimal pancreas uptake in a preclinical mouse model. In this study, we synthesized four derivatives with unnatural amino acid substitutions (Tle¹⁰-derived Ga-LW01158, NMe-His¹²-derived Ga-LW01160, α-Me-Trp⁸- and Tle¹⁰-derived Ga-LW01186, and Tle¹⁰- and N-Me-Gly¹¹-derived Ga-LW02002) and evaluated their potential for detecting GRPR-expressing tumors with positron emission tomography (PET). The binding affinities (Ki(GRPR)) of Ga-LW01158, Ga-LW01160, Ga-LW01186, and Ga-LW02002 were 5.11 ± 0.47, 187 ± 17.8, 6.94 ± 0.95, and 11.0 ± 0.39 nM, respectively. [⁶⁸Ga]Ga-LW01158, [⁶⁸Ga]Ga-LW01186, and [⁶⁸Ga]Ga-LW02002 enabled clear visualization of subcutaneously implanted human prostate cancer PC-3 tumor xenografts in mice in PET images. Ex vivo biodistribution studies showed that [⁶⁸Ga]Ga-LW01158 had the highest tumor uptake (11.2 ± 0.65 %ID/g) and good tumor-to-background uptake ratios at 1 h post-injection. Comparable in vivo stabilities were observed for [⁶⁸Ga]Ga-LW01158, [⁶⁸Ga]Ga-LW01186, and [⁶⁸Ga]Ga-LW02002 (76.5–80.7% remaining intact in mouse plasma at 15 min post-injection). In summary, the Tle¹⁰ substitution, either alone or combined with α-Me-Trp⁸ or NMe-Gly¹¹ substitution, in Ga-TacsBOMB2 generates derivatives that retained good GRPR binding affinity and in vivo stability. With good tumor uptake and tumor-to-background imaging contrast, [⁶⁸Ga]Ga-LW01158 is promising for detecting GRPR-expressing lesions with PET.Medicine, Faculty ofRadiology, Department ofReviewedFacultyResearche

Unnatural amino acid substitutions to improve in vivo stability and tumor uptake of 68Ga-labeled GRPR-targeted TacBOMB2 derivatives for cancer imaging with positron emission tomography

Abstract Background Overexpressed in various solid tumors, gastrin-releasing peptide receptor (GRPR) is a promising cancer imaging marker and therapeutic target. Although antagonists are preferable for the development of GRPR-targeted radiopharmaceuticals due to potentially fewer side effects, internalization of agonists may lead to longer tumor retention and better treatment efficacy. In this study, we systematically investigated unnatural amino acid substitutions to improve in vivo stability and tumor uptake of a previously reported GRPR-targeted agonist tracer, [68Ga]Ga-TacBOMB2 (68Ga-DOTA-Pip-D-Phe6-Gln7-Trp8-Ala9-Val10-Gly11-His12-Leu13-Thz14-NH2). Results Unnatural amino acid substitutions were conducted for Gln7, Trp8, Ala9, Val10, Gly11 and His12, either alone or in combination. Out of 25 unnatural amino acid substitutions, tert-Leu10 (Tle10) and NMe-His12 substitutions were identified to be preferable modifications especially in combination. Compared with the previously reported [68Ga]Ga-TacBOMB2, the Tle10 and NMe-His12 derived [68Ga]Ga-LW01110 showed retained agonist characteristics and improved GRPR binding affinity (Ki = 7.62 vs 1.39 nM), in vivo stability (12.7 vs 89.0% intact tracer in mouse plasma at 15 min post-injection) and tumor uptake (5.95 vs 16.6 %ID/g at 1 h post-injection). Conclusions Unnatural amino acid substitution is an effective strategy to improve in vivo stability and tumor uptake of peptide-based radiopharmaceuticals. With excellent tumor uptake and tumor-to-background contrast, [68Ga]Ga-LW01110 is promising for detecting GRPR-expressing cancer lesions with PET. Since agonists can lead to internalization upon binding to receptors and foreseeable long tumor retention, our optimized GRPR-targeted sequence, [Tle10,NMe-His12,Thz14]Bombesin(7–14), is a promising template for use for the design of GRPR-targeted radiotherapeutic agents

Melanoma Imaging Using ¹⁸F‑Labeled α‑Melanocyte-Stimulating Hormone Derivatives with Positron Emission Tomography

Melanocortin 1 receptor (MC1R) is specifically expressed in the majority of melanomas, a leading cause of death related to skin cancers. Accurate staging and early detection is crucial in managing melanoma. Based on the α-melanocyte-stimulating hormone (αMSH) sequence, MC1R-targeted peptides have been studied for melanoma imaging, predominately for use with single-photon emission computed tomography, with few attempts made for positron emission tomography (PET). ¹⁸F is a commonly used PET isotope due to readily available cyclotron production, pure positron emission, and a favorable half-life (109.8 min). In this study, we aim to design and evaluate αMSH derivatives that enable radiolabeling with ¹⁸F for PET imaging of melanoma. We synthesized three imaging probes based on the structure of Nle⁴-cyclo­[Asp⁵-His-d-Phe⁷-Arg-Trp-Lys¹⁰]-NH₂ (Nle-CycMSH_hex), with a Pip linker (CCZ01064), an Acp linker (CCZ01070), or an Aoc linker (CCZ01071). ¹⁸F labeling was enabled by an ammoniomethyl-trifluoroborate (AmBF₃) moiety. <i>In vitro</i> competition binding assays showed subnanomolar inhibition constant (<i>K</i>_i) values for all three peptides. The ¹⁸F radiolabeling was performed via a one-step ¹⁸F–¹⁹F isotope exchange reaction that resulted in high radiochemical purity (>95%) and good molar activity (specific activity) ranging from 40.7 to 66.6 MBq/nmol. All three ¹⁸F-labeled peptides produced excellent tumor visualization with PET imaging in C57BL/6J mice bearing B16-F10 tumors. The tumor uptake was 7.80 ± 1.77, 5.27 ± 2.38, and 5.46 ± 2.64% injected dose per gram of tissue (%ID/g) for [¹⁸F]­CCZ01064, [¹⁸F]­CCZ01070, and [¹⁸F]­CCZ01071 at 1 h post-injection (p.i.), respectively. Minimal background activity was observed except for kidneys at 4.99 ± 0.20, 4.42 ± 0.54, and 13.55 ± 2.84%ID/g, respectively. The best candidate [¹⁸F]­CCZ01064 was further evaluated at 2 h p.i., which showed increased tumor uptake at 11.96 ± 2.31%ID/g and further reduced normal tissue uptake. Moreover, a blocking study was performed for CCZ01064 at 1 h p.i., where tumor uptake was significantly reduced to 1.97 ± 0.60%ID/g, suggesting the tumor uptake was receptor mediated. In conclusion, [¹⁸F]­CCZ01064 showed high tumor uptake, low normal tissue uptake, and fast clearance and is therefore a suitable and promising candidate for PET imaging of melanoma

p-NO2-Bn-H(4)neunpa and H(4)neunpa-Trastuzumab:Bifunctional Chelator for Radiometalpharmaceuticals and In-111 Immuno-Single Photon Emission Computed Tomography Imaging

Potentially nonadentate (N5O4) bifunctional chelator p-SCN-Bn-H(4)neunpa and its immunoconjugate H(4)neunpa-trastuzumab for In-111 radiolabeling are synthesized. The ability of p-SCN-Bn H(4)neunpa and H(4)neunpa-trastuzumab to quantitatively radiolabel (InCl3)-In-111 at an ambient temperature within 15 or 30 min, respectively, is presented. Thermodynamic stability determination with In3+, Bi3+, and La3+ resulted in high conditional stability constant (pM) values. In vitro human serum stability assays have demonstrated both In-111 complexes to have high stability over 5 days. Mouse biodistribution of [In-111][In(p-NO2 Bn neunpa)](-), compared to that of [In-111][In(p-NH2-Bn-CHX-A"-diethylenetriamine pentaacetic acid (DTPA))](2-), at 1, 4, and 24 h shows fast clearance of both complexes from the mice within 24 h. In a second mouse biodistribution study, the immunoconjugates In-neunpa trastuzumab and In-111-CHX-A"-DTPA-trastuzumab demonstrate a similar distribution profile but with slightly lower tumor uptake of In-111-neunpa-trastuzumab compared to that of In-111-CHX-A"-DTPA-trastuzumab. These results were also confirmed by immuno-single photon emission computed tomography (immuno-SPECT) imaging in vivo. These initial investigations reveal the acyclic bifunctional chelator p-SCN-Bn-H(4)neunpa to be a promising chelator for In-111 (and other radiometals) with high in vitro stability and also show H(4)neunpa trastuzumab to be an excellent In-111 chelator with promising biodistribution in mice

Imaging Bradykinin B1 Receptor with ⁶⁸Ga-Labeled [des-Arg¹⁰]Kallidin Derivatives: Effect of the Linker on Biodistribution and Tumor Uptake

Bradykinin B1 receptor (B1R) that is overexpressed in cancers but minimally expressed in normal healthy tissues represents an attractive biomarker for the development of cancer imaging agents. The goal of this study was to evaluate the effect of different linkers on the pharmacokinetics and tumor uptake of a B1R-targeting radio-peptide sequence, ⁶⁸Ga-DOTA-linker-Lys-Arg-Pro-Hyp-Gly-Cha-Ser-Pro-Leu. Four peptides, SH01078, P03034, P04115, and P04168, with 6-aminohexanoic acid, 9-amino-4,7-dioxanonanoic acid, Gly-Gly, and 4-amino-(1-carboxymethyl)­piperidine, respectively, as the linker were synthesized and evaluated. In vitro competition binding assays showed that the <i>K</i>_i values of SH01078, P03034, P04115, and P04168 were 27.8 ± 4.9, 16.0 ± 1.9, 11.4 ± 2.5, and 3.6 ± 0.2 nM, respectively. Imaging and biodistribution studies were performed in mice bearing both B1R-positive HEK293T::hB1R and B1R-negative HEK293T tumors. All tracers showed mainly renal excretion with excellent tumor visualization and minimal background activity except for kidneys and bladder. The average uptake of ⁶⁸Ga-labeled SH01078, P03034, and P04115 in HEK293T::hB1R tumor was similar (1.96–2.17%ID/g) at 1 h postinjection. ⁶⁸Ga-P04168 generated higher HEK293T::hB1R tumor uptake (4.15 ± 1.13%ID/g) and lower background activity, leading to a >2-fold improvement in HEK293T::hB1R tumor-to-background (HEK293T tumor, blood, muscle, and liver) contrasts over those of ⁶⁸Ga-labeled SH01078, P03034, and P04115. Our results indicate that the choice of linker affects binding affinity, pharmacokinetics, and tumor targeting. The use of the cationic 4-amino-(1-carboxymethyl)­piperidine linker improved tumor visualization, and the resulting ⁶⁸Ga-P04168 might be promising for clinical application for imaging B1R-expressing tumors with positron emission tomography

<i>p</i>‑NO₂–Bn–H₄neunpa and H₄neunpa–Trastuzumab: Bifunctional Chelator for Radiometalpharmaceuticals and ¹¹¹In Immuno-Single Photon Emission Computed Tomography Imaging

Potentially nonadentate (N₅O₄) bifunctional chelator <i>p</i>-SCN–Bn–H₄neunpa and its immunoconjugate H₄neunpa–trastuzumab for ¹¹¹In radiolabeling are synthesized. The ability of <i>p</i>-SCN–Bn–H₄neunpa and H₄neunpa–trastuzumab to quantitatively radiolabel ¹¹¹InCl₃ at an ambient temperature within 15 or 30 min, respectively, is presented. Thermodynamic stability determination with In³⁺, Bi³⁺, and La³⁺ resulted in high conditional stability constant (<i>p</i>M) values. In vitro human serum stability assays have demonstrated both ¹¹¹In complexes to have high stability over 5 days. Mouse biodistribution of [¹¹¹In]­[In­(<i>p</i>-NO₂–Bn–neunpa)]⁻, compared to that of [¹¹¹In]­[In­(<i>p</i>-NH₂–Bn–CHX-A″–diethylenetriamine pentaacetic acid (DTPA))]^2–, at 1, 4, and 24 h shows fast clearance of both complexes from the mice within 24 h. In a second mouse biodistribution study, the immunoconjugates ¹¹¹In-neunpa–trastuzumab and ¹¹¹In–CHX-A″–DTPA–trastuzumab demonstrate a similar distribution profile but with slightly lower tumor uptake of ¹¹¹In-neunpa–trastuzumab compared to that of ¹¹¹In–CHX-A″–DTPA–trastuzumab. These results were also confirmed by immuno-single photon emission computed tomography (immuno-SPECT) imaging in vivo. These initial investigations reveal the acyclic bifunctional chelator <i>p</i>-SCN–Bn–H₄neunpa to be a promising chelator for ¹¹¹In (and other radiometals) with high in vitro stability and also show H₄neunpa–trastuzumab to be an excellent ¹¹¹In chelator with promising biodistribution in mice