Development of new copper-64 labeled rhodamine: a potential PET myocardial perfusion imaging agent.

BACKGROUND: Myocardial perfusion imaging (MPI) is one of the most commonly performed investigations in nuclear medicine procedures. Due to the longer half-life of the emerging positron emitter copper-64 and its availability from low energy cyclotron, together with its well-known coordination chemistry, we have synthesized 64Cu-labeled NOTA- and 64Cu-NOTAM-rhodamine conjugates as potential cardiac imaging agents using PET. RESULTS: 64Cu-NOTA- and 64Cu-NOTAM-rhodamine conjugates were synthesized using a traightforward and one-step simple reaction. Radiochemical yields were greater than 97% (decay corrected), with a total synthesis time of less than 25 min. Radiochemical purities were always greater than 98% as assessed by TLC and HPLC. These synthetic approaches hold considerable promise as a simple method for 64Cu-rhodamine conjugates synthesis, with high radiochemical yield and purity. Biodistribution studies in normal Fischer rats at 60 min post-injection, demonstrated significant heart uptake and a good biodistribution profile for both the radioconjugates. However, the 64Cu-NOTAM-rhodamine conjugate has shown more heart uptake (~ 10% ID/g) over the 64Cu-NOTA-rhodamine conjugate (5.6% ID/g). CONCLUSIONS: These results demonstrate that these radioconjugates may be useful probes for the PET evaluation of MPI.

Identification of Activation Isotopes in a CS-30 Cyclotron Vault.

A CS-30 cyclotron has been in operation at King Faisal Specialist Hospital and Research Center (KFSHRC) since 1982. The CS-30 cyclotron has been used to produce medical radioisotopes for positron emission tomography (PET) and single-photon emission computed tomography (SPECT). Some of the nuclear reactions of radionuclide production are associated with the intense release of a wide range of fast neutrons. In this work, we investigated the radionuclides produced from neutron interactions with the cyclotron facility walls. Activation isotopes were determined by performing gamma ray spectrometry utilizing a high-purity germanium (HPGe) detector. The major radionuclides found were 152Eu, 154Eu, 134Cs, 65Zn and 60Co. Activation isotope accumulation had increased the dose rate inside the facility. The surface dose rates were measured at all of the surrounding walls. The maximum surface dose rate was found to be 1.2 µSv/h, which is much lower than the permissible occupational exposure of 15 µSv/h based daily 5 work hours.

IAEA contribution to the development of 64Cu radiopharmaceuticals for theranostic applications.

Copper-64 is a very attractive radioisotope with unique nuclear properties that allow using it as both a diagnostic and therapeutic agent, thus providing an almost ideal example of a theranostic radionuclide. A characteristic of Cu-64 stems from the intrinsic biological nature of copper ions that play a fundamental role in a large number of cellular processes. Cu-64 is a radionuclide that reflects the natural biochemical pathways of Cu-64 ions, therefore, can be exploited for the detection and therapy of certain malignancies and metabolic diseases. Beside these applications of Cu-64 ions, this radionuclide can be also used for radiolabelling bifunctional chelators carrying a variety of pharmacophores for targeting different biological substrates. These include peptide-based substrates and immunoconjugates as well as small-molecule bioactive moieties. Fueled by the growing interest of Member States (MS) belonging to the International Atomic Energy Agency (IAEA) community, a dedicated Coordinated Research Project (CRP) was initiated in 2016, which recruited thirteen participating MS from four continents. Research activities and collaborations between the participating countries allowed for collection of an impressive series of results, particularly on the production, preclinical evaluation and, in a few cases, clinical evaluation of various 64Cu-radiopharmaceuticals that may have potential impact on future development of the field. Since this CRP was finalized at the beginning of 2020, this short review summarizes outcomes, outputs and results of this project with the purpose to propagate to other MS and to the whole scientific community, some of the most recent achievements on this novel class of theranostic 64Cu-pharmaceuticals.

Development of the Tumor-Specific Antigen-Derived Synthetic Peptides as Potential Candidates for Targeting Breast and Other Possible Human Carcinomas.

The human epidermal growth factor receptor 2 (HER2) represents one of the most studied tumor-associated antigens for cancer immunotherapy. The receptors for HER2 are overexpressed in various human cancers, such as breast and ovarian cancer. The relatively low expression of this antigen on normal tissues makes it a clinically useful molecular target for tumor imaging and targeted therapy. HER2 overexpression is correlated with aggressive tumor behavior and poor clinical outcomes. Thus, HER2 has become an important prognostic and predictive factor, as well as a potential molecular target. Due to the heterogeneity of breast cancer and possible discordance in HER2 status between primary tumors and distant metastases, assessment of HER2 expression by noninvasive imaging is important. Molecular imaging of HER2 expression may provide essential prognostic and predictive information concerning disseminated cancer and aid in the selection of an optimal therapy. Another tumor-specific antigen is MUC1, which is silent on normal tissues, but overexpressed in almost all human epithelial cell cancers, including >90% of human breast, ovarian, pancreatic, colorectal, lung, prostate, and gastric cancers and is a promising tumor antigen with diagnostic as well as the therapeutic potential of cancer. Radiolabeled small peptide ligands are attractive as probes for molecular imaging, as they reach and bind the target receptor efficiently and clear from blood and non-target organs faster than bulky antibodies. In this study, HER2 and MUC1-based peptides were synthesized and preclinically evaluated in an effort to develop peptide-based SPECT radiopharmaceuticals derived from tumor-associated antigens for the detection of breast cancer. Our findings demonstrate that the tumor antigen peptides radiolabeled efficiently with 99mTc and showed high metabolic stability in human plasma in vitro. The data from breast tumor cell binding confirmed the high affinity (in low nanomolar range) towards respective breast cancer cell lines. In healthy mice, 99mTc-labeled peptides displayed favorable pharmacokinetics, with high excretion by the renal system. In tumor xenografts nude mice models, good uptake by the SKBR3, MCF7, and T47D tumors were found, with good tumor-to-blood and tumor to muscle ratios. Additionally, tumor lesions can be seen in γ-camera imaging. Our data suggest that based on its ability to detect HER2- and MUC1-positive breast cancer cells in vivo, 99mTc-HER2 and 99mTc-MUC1-targeted peptides may be promising tumor imaging probes and warrant further investigation.

A convenient and efficient total solid-phase synthesis of DOTA-functionalized tumor-targeting peptides for PET imaging of cancer.

INTRODUCTION: An efficient and cost-effective synthesis of the metal chelating agents that couple to tumor-targeting peptides is required to enhance the process of preclinical research toward the clinical translation of molecular imaging agents. DOTA is one of the most widely used macrocyclic ligands for the development of new metal-based imaging and therapeutic agents owing to its ability to form stable and inert complexes under physiological conditions. Although solid-phase synthesis compatible DOTA-tris-(t-Bu ester) is a commercial product, it is expensive and contain chemical impurities. There is a need to explore new and cost-effective methods for the preparation of metal chelating agents, i.e., DOTA, directly on solid support to facilitate rapid, cost-effective, and high purity preparation of DOTA-linked peptides for imaging and therapy. In the present study, we describe a facile synthetic strategy of DOTA preparation and its linkage to peptides directly on solid-phase support. METHODS: Bombesin (BN) peptides were functionalized with DOTA chelator prepared from cyclen precursor on solid-phase and from commercial DOTA-tris and radiolabeled with 68Ga. In vitro BN/GRP receptor binding affinities of the corresponding radiolabeled peptides were determined by saturation binding assays on human breast MDA-MB-231, MCF7, T47D, and PC3 prostate cancer cells. Pharmacokinetics were studied in Balb/c mice and in vivo tumor targeting in MDA-MB-231 tumor-bearing nude mice. RESULTS: DOTA was prepared successfully from cyclen on solid-phase support, linked specifically to BN peptides and resultant DOTA-coupled peptides were radiolabeled efficiently with 68Ga. The binding affinities of all the six BN peptides were comparable and in the low nanomolar range. All 68Ga-labeled peptides showed high metabolic stability in plasma. These radiopeptides exhibited rapid pharmacokinetics in Balb/c mice with excretion mainly through the urinary system. In nude mice, MDA-MB-231 tumor uptake profiles were slightly different; the BN peptide with Ahx spacer and linked to DOTA through cyclen exhibited higher tumor uptake (2.32% ID/g at 1 h post-injection) than other radiolabeled BN peptides investigated in this study. The same leading BN peptide also displayed favorable pharmacokinetic profile in Balb/c mice. The PET images clearly visualized the MDA-MB-231 tumor. CONCLUSIONS: DOTA prepared from cyclen on solid-phase support showed comparable potency and efficiency to DOTA-tris in both in vitro and in vivo evaluation. The synthetic methodology described here allows versatile, site-specific introduction of DOTA into peptides to facilitate the development of DOTA-linked molecular imaging and therapy agents for clinical translation.

Preparation and In Vitro and In Vivo Characterization of the Tumor-specific Antigen-derived Peptide as a Potential Candidate for Targeting Human Epidermal Growth Factor Receptor 2-positive Breast Carcinomas.

BACKGROUND/AIM: The human epidermal growth factor receptor (HER2) is considered as one of the most well-characterized tumor-associated antigens for cancer therapy and plays an important role in the growth and progression of breast cancer. Overexpression of HER2 in various cancers and the availability of its extracellular region makes it a clinically useful target for the development of tumor-antigen specific agents. In this study, we have prepared a HER2-targeted hybrid peptide as a single-photon emission computed tomography (SPECT) imaging probe and evaluated its tumor targeting potential in subcutaneous HER2-positive breast cancer xenograft models. MATERIALS AND METHODS: The HER2-targeted hybrid peptide was prepared by solid-phase peptide synthesis and radiolabeled with 99mTc by the ligand exchange method. In vitro tumor cell binding properties of 99mTc-HER2 were evaluated in HER2-positive (SKBR3) and ER-positive (MCF7 and T47D) breast cancer cell lines. In vivo tumor targeting characteristics were investigated in both SKBR3 (HER2-positive) and MDA-MB-231 (HER2-negative) xenografted animal models. RESULTS: A high labeling efficiency of greater than 95% was achieved when HER2 peptide was radiolabeled with 99mTc by the standard ligand exchange method. 99mTc-HER2 displayed a high binding affinity (Kd=49.95±14.11 nM) to HER2-positive SKBR3 cell line whereas in the case of the ER-positive cell lines (MCF-7 and T47D), the binding affinity was found to be 2-3-fold lower than SKBR3. In vivo tumor uptake in nude mice with SKBR3 tumor xenografts was 2.81±0.79% ID/g as early as 60 min p.i. The uptake in SKBR3 tumors was always higher than the uptake in the blood and muscle, with good tumor-to-blood and tumor-to-muscle ratios. In contrast, low accumulation in ER-positive tumors (MCF7 and T47D) was observed compared to HER2-positive SKBR3 tumor mice. A low to moderate (less than 5% ID/g) accumulation and retention of 99mTc-HER2 was found in most of the major organs excluding the kidneys in both healthy and tumor-bearing mice. CONCLUSION: In view of its ability to detect HER2-positive breast cancer cells in vivo, 99mTc-HER2-targeted peptide may be a promising tumor imaging probe and warrants further investigation.

Quantitative imaging characteristics of zirconium-89 on Gemini Time-Of-Flight PET/CT.

PURPOSE: Interest in PET imaging using zirconium-89 (Zr) (t1/2=78.41 h)-labeled tracers for the tracking and quantification of monoclonal antibodies (mAbs) is growing, mainly because of its well-matched physical half-life with the biological half-life of intact mAbs. This study aims to evaluate the imaging characteristics of Zr-PET in comparison with those obtained using fluorine-18 fluorodeoxyglucose (F-FDG) PET (gold standard tracer in PET imaging) using a Time-Of-Flight (TOF) PET/computed tomography (CT) scanner. MATERIALS AND METHODS: The system's spatial resolution, sensitivity, scatter fraction (SF), image uniformity, and image quality were measured on a Gemini TOF PET/CT scanner according to the NEMA NU2-2001 protocols. The NEMA 2001 kit was used to carry out these measurements. Timing and energy resolutions were measured using Na and F-FDG point sources only. RESULTS: Spatial resolution in transverse and axial planes measured at 10 mm off access were 4.7 and 4.6 mm for Zr and F-FDG, respectively. At 100 mm, radial, tangential, and axial spatial resolution values were 5.2, 5.1, and 5.2 mm for Zr and 5.1, 4.9, and 5.2 mm for F-FDG, respectively. Sensitivity measured at the center of the field of view was 14.6 and 4.16 cps/kBq for Zr and F-FDG, respectively. SF was 32.6% for Zr in comparison with 31.8% for F-FDG. Image contrast for Zr-PET images was 36.9 and 29.7% for F-FDG for the smallest (10 mm)-sized sphere, and it was 70.6 and 72.8% for Zr and F-FDG, respectively, for the largest (37 mm)-sized sphere. Background variation was 10.3% for Zr and 6.8% for F-FDG for the smallest-sized sphere and 3.4 and 3.8% for Zr and F-FDG, respectively, for the largest-sized sphere. CONCLUSION: In this study, we measured imaging characteristics of Zr on a Gemini TOF PET/CT scanner. Our results show that Zr has lower spatial resolution and noise-equivalent count rate with increased SF and background variation; however, it offered superior sensitivity and improved image contrast in comparison with F-FDG. Zr is an ideal radiotracer for immuno-PET imaging because of its physical half-life, which is well matched with mAbs, in addition to its affinity to be trapped inside the target cell after internalization of the mAbs.

Novel synthesis and initial preclinical evaluation of (18)F-[FDG] labeled rhodamine: a potential PET myocardial perfusion imaging agent.

Myocardial perfusion imaging is one of the most commonly performed investigations in nuclear medicine studies. Due to the clinical importance of [(18)F]-fluoro-2-deoxy-D-glucose ([(18)F]-FDG) and its availability in almost every PET center, a new radiofluorinated [(18)F]-FDG-rhodamine conjugate was synthesized using [(18)F]-FDG as a prosthetic group. In a convenient and simple one-step radiosynthesis, [(18)F]-FDG-rhodamine conjugate was prepared in quantitative radiochemical yields, with total synthesis time of nearly 20 min and radiochemical purity of greater than 98%, without the need for HPLC purification, which make these approaches amenable for automation. Biodistribution studies in normal rats at 60 min post-injection demonstrated a high uptake in the heart (>11% ID/g) and favorable pharmacokinetics. Additionally, [(18)F]-FDG-rhodamine showed an extraction value of 27.63%±5.12% in rat hearts. These results demonstrate that [(18)F]-FDG-rhodamine conjugate may be useful as an imaging agent for the positron emission tomography evaluation of myocardial perfusion.

Development and pre-clinical evaluation of new 68Ga-NOTA-folate conjugates for PET imaging of folate receptor-positive tumors.

In an attempt to develop new folate radiotracers with favorable biochemical properties for detecting folate receptor-positive cancers, we synthesized 68Ga-NOTA- and 68Ga-NOTAM-folate conjugates using a straightforward and a one-step simple reaction. Radiochemical yields were greater than 95% (decay-corrected) with total synthesis time of less than 20 min. Radiochemical purities were always greater than 98% without high-performance liquid chromatography (HPLC) purification. These synthetic approaches hold considerable promise as a rapid and simple method for 68Ga-folate conjugate preparation with high radiochemical yield in a short synthesis time. In vitro tests on the KB cell line showed that significant amounts of the radioconjugates were associated with cell fractions. Biodistribution studies in nude mice bearing human KB xenografts, demonstrated a significant tumor uptake and favorable biodistribution profile for 68Ga-NOTA-folate over the 68Ga-NOTAM-folate conjugate. The uptake in the tumors was blocked by excess injection of folic acid, suggesting a receptor-mediated process. These results demonstrate that the 68Ga-NOTA-folate conjugate may be useful as a molecular probe for detection and staging of folate receptor-positive cancers, such as ovarian cancer and their metastasis, as well as monitoring tumor response to treatment.