Tamila J Stott Reynolds1, Rebecca Schehr2, Dijie Liu3, Jingli Xu4, Yubin Miao5, Timothy J Hoffman6, Tammy L Rold7, Michael R Lewis3, Charles J Smith8. 1. Research Division, Harry S. Truman Memorial Veterans' Hospital, Columbia, Missouri, United States, 65201; Department of Veterinary Pathobiology, Comparative Medicine Program, University of Missouri College of Veterinary Medicine, Columbia, MO, United States, 65211. Electronic address: stottreynoldst@missouri.edu. 2. Veterinary Research Scholars Program, University of Missouri College of Veterinary Medicine, Columbia, MO, United States, 65211. 3. Research Division, Harry S. Truman Memorial Veterans' Hospital, Columbia, Missouri, United States, 65201; Department of Radiology, University of Missouri School of Medicine, Columbia, MO, United States, 65211. 4. College of Pharmacy, University of New Mexico, Albuquerque, NM, United States, 87131. 5. College of Pharmacy, University of New Mexico, Albuquerque, NM, United States, 87131; Cancer Research and Treatment Center, University of New Mexico, Albuquerque, NM, United States, 87131; Department of Dermatology, University of New Mexico, Albuquerque, NM, United States, 87131. 6. Research Division, Harry S. Truman Memorial Veterans' Hospital, Columbia, Missouri, United States, 65201; Department of Internal Medicine, University of Missouri School of Medicine, Columbia, MO, United States, 65211; Department of Chemistry, University of Missouri, Columbia, MO, United States, 65211. 7. Research Division, Harry S. Truman Memorial Veterans' Hospital, Columbia, Missouri, United States, 65201; Department of Internal Medicine, University of Missouri School of Medicine, Columbia, MO, United States, 65211. 8. Research Division, Harry S. Truman Memorial Veterans' Hospital, Columbia, Missouri, United States, 65201; Department of Radiology, University of Missouri School of Medicine, Columbia, MO, United States, 65211; University of Missouri Research Reactor Center, University of Missouri, Columbia, MO, United States, 65211. Electronic address: smithcj@health.missouri.edu.
Abstract
INTRODUCTION: Here we present the metallation, characterization, in vivo and in vitro evaluations of dual-targeting, peptide-based radiopharmaceuticals with utility for imaging and potentially treating prostate tumors by virtue of their ability to target the αVβ3 integrin or the gastrin releasing peptide receptor (GRPr). METHODS: [RGD-Glu-6Ahx-RM2] (RGD: Arg-Gly-Asp; Glu: glutamic acid; 6-Ahx: 6-amino hexanoic acid; RM2: (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2)) was conjugated to a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) bifunctional chelator (BFCA) purified via reversed-phase high-performance liquid chromatography (RP-HPLC), characterized by electrospray ionization-mass spectrometry (ESI-MS), and radiolabeled with (111)In or (177)Lu. Natural-metallated compounds were assessed for binding affinity for the αVβ3 integrin or GRPr in human glioblastoma U87-MG and prostate PC-3 cell lines and stability prior to in vivo evaluation in normal CF-1 mice and SCID mice xenografted with PC-3 cells. RESULTS: Competitive displacement binding assays with PC-3 and U87-MG cells revealed high to moderate binding affinity for the GRPr or the αVβ3 integrin (IC50 range of 5.39±1.37 nM to 9.26±0.00 nM in PC-3 cells, and a range of 255±47 nM to 321±85 nM in U87-MG cells). Biodistribution studies indicated high tumor uptake in PC-3 tumor-bearing mice (average of 7.40±0.53% ID/g at 1h post-intravenous injection) and prolonged retention of tracer (mean of 4.41±0.91% ID/g at 24h post-intravenous injection). Blocking assays corroborated the specificity of radioconjugates for each target. Micro-single photon emission computed tomography (microSPECT) confirmed favorable radiouptake profiles in xenografted mice at 20h post-injection. CONCLUSIONS: [RGD-Glu-[(111)In-DO3A]-6-Ahx-RM2] and [RGD-Glu-[(177)Lu- DO3A]-6-Ahx-RM2] show favorable pharmacokinetic and radiouptake profiles, meriting continued evaluation for molecular imaging in murine U87-MG/PC-3 xenograft models and radiotherapy studies with (177)Lu and (90)Y conjugates. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: These heterovalent, peptide-targeting ligands perform comparably with many mono- and multivalent conjugates with the potential benefit of increased sensitivity for detecting cancer cells exhibiting differential expression of target receptors. Published by Elsevier Inc.
INTRODUCTION: Here we present the metallation, characterization, in vivo and in vitro evaluations of dual-targeting, peptide-based radiopharmaceuticals with utility for imaging and potentially treating prostate tumors by virtue of their ability to target the αVβ3 integrin or the gastrin releasing peptide receptor (GRPr). METHODS: [RGD-Glu-6Ahx-RM2] (RGD: Arg-Gly-Asp; Glu: glutamic acid; 6-Ahx: 6-amino hexanoic acid; RM2: (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2)) was conjugated to a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) bifunctional chelator (BFCA) purified via reversed-phase high-performance liquid chromatography (RP-HPLC), characterized by electrospray ionization-mass spectrometry (ESI-MS), and radiolabeled with (111)In or (177)Lu. Natural-metallated compounds were assessed for binding affinity for the αVβ3 integrin or GRPr in humanglioblastoma U87-MG and prostate PC-3 cell lines and stability prior to in vivo evaluation in normal CF-1 mice and SCIDmice xenografted with PC-3 cells. RESULTS: Competitive displacement binding assays with PC-3 and U87-MG cells revealed high to moderate binding affinity for the GRPr or the αVβ3 integrin (IC50 range of 5.39±1.37 nM to 9.26±0.00 nM in PC-3 cells, and a range of 255±47 nM to 321±85 nM in U87-MG cells). Biodistribution studies indicated high tumor uptake in PC-3 tumor-bearing mice (average of 7.40±0.53% ID/g at 1h post-intravenous injection) and prolonged retention of tracer (mean of 4.41±0.91% ID/g at 24h post-intravenous injection). Blocking assays corroborated the specificity of radioconjugates for each target. Micro-single photon emission computed tomography (microSPECT) confirmed favorable radiouptake profiles in xenografted mice at 20h post-injection. CONCLUSIONS: [RGD-Glu-[(111)In-DO3A]-6-Ahx-RM2] and [RGD-Glu-[(177)Lu- DO3A]-6-Ahx-RM2] show favorable pharmacokinetic and radiouptake profiles, meriting continued evaluation for molecular imaging in murine U87-MG/PC-3 xenograft models and radiotherapy studies with (177)Lu and (90)Y conjugates. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: These heterovalent, peptide-targeting ligands perform comparably with many mono- and multivalent conjugates with the potential benefit of increased sensitivity for detecting cancer cells exhibiting differential expression of target receptors. Published by Elsevier Inc.