Carolyn J. Anderson

Efficient production of high specific activity 64Cu using a biomedical cyclotron.

Copper-64 (T 1/2 = 12.7 h) is an intermediate-lived positron-emitting radionuclide that is a useful radiotracer for positron emission tomography (PET) as well as a promising radiotherapy agent for the treatment for cancer. Currently, copper-64 suitable for biomedical studies is produced in the fast neutron flux trap (irradiation of zinc with fast neutrons) at the Missouri University Research Reactor. Access to the fast neutron flux trap is only possible on a weekly basis, making the availability of this tracer very limited. In order to significantly increase the availability of this intermediate-lived radiotracer, we have investigated and developed a method for the efficient production of high specific activity Cu-64 using a small biomedical cyclotron. It has been suggested that it may be possible to produce Cu-64 on a small biomedical cyclotron utilizing the 64Ni(p,n)64Cu nuclear reaction. We have irradiated both natural nickel and enriched (95% and 98%) Ni-64 plated on gold disks. Nickel has been electroplated successfully at thicknesses of approximately 20-300 mm and bombarded with proton currents of 15-45 microA. A special water-cooled target had been designed to facilitate the irradiations on a biomedical cyclotron up to 60 microA. We have shown that it is possible to separate Cu-64 from Ni-64 and other reaction byproducts rapidly and efficiently by using ion exchange chromatography. Production runs using 19-55 mg of 95% enriched Ni-64 have yielded 150-600 mCi of Cu-64 (2.3-5.0 mCi/microAh) with specific activities of 94-310 mci/microgram Cu. The cyclotron produced Cu-64 had been used to radiolabel PTSM [pyruvaldehyde bis-(N4-methylthiosemicarbazone), used to quantify myocardial, cerebral, renal, and tumor blood flow], MAb 1A3 [monoclonal antibody MAb to colon cancer], and octreotide. A recycling technique for the costly Ni-64 target material has been developed. This technique allows the nickel eluted off the column to be recovered and reused in the electroplating of new targets with an overall efficiency of greater than 90%.

Copper Chelation Chemistry and its Role in Copper Radiopharmaceuticals

Molecular imaging is an important scientific discipline that plays a major role in clinical medicine and pharmaceutical development. While several imaging modalities including X-ray computed tomography (CT) and magnetic resonance imaging (MRI) generate high-resolution anatomical images, positron emission tomography (PET) and single photon emission computed tomography (SPECT) offer insight into the physiological processes that occur within a living organism. Of these two nuclear medicine imaging techniques, PET has advantages with respect to sensitivity and resolution, and this has led to the production and development of many positron emitting radionuclides that include non-traditional radionuclides of the transition metals. Copper-64 (t(1/2) = 12.7 h, beta(+): 17.4%, E(beta+max) = 656 keV; beta(-): 39%, E(beta-max) = 573 keV) has emerged as an important positron emitting radionuclide that has the potential for use in diagnostic imaging and radiotherapy. However, (64)Cu must be delivered to the living system as a stable complex that is attached to a biological targeting molecule for effective imaging and therapy. Therefore, significant research has been devoted to the development of ligands that can stably chelate (64)Cu. This review discusses the necessary characteristics of an effective (64)Cu chelator, while highlighting the development and evaluation of (64)Cu-complexes attached to biologically-targeted ligands.

Metal complexes as diagnostic tools

Abstract This review summarizes some of the developments of metal complexes and metal-complex-bioconjugates for the diagnosis of disease states that have occurred over the past 10 years. The diagnostic imaging modalities discussed are gamma scintigraphy, positron emission tomography (PET) and magnetic resonance imaging (MRI). Metal complexes are utilized in all three imaging modalities to image a broad array of diseases, including heart disease, brain disorders and cancer. There are a wide variety of different radiometals that have been utilized in the synthesis of coordination compounds for gamma scintigraphy and PET, and these will be discussed individually. The field of metal-complex-bioconjugate chemistry is covered extensively, describing radiometals labeled to biomolecules such as receptor ligands for diseases such as neurological disorders and cancer. A section is devoted to the development of coordination compounds for MRI enhancement agents, and specifically details the agents that have been evaluated in vivo, both in animal models and in humans. Overall, the goal of this review is to demonstrate the significant progress made in the field of coordination chemistry as it applies to the development of diagnostic imaging agents.

Preparation and Biological Evaluation of Copper-64–Labeled Tyr3-Octreotate Using a Cross-Bridged Macrocyclic Chelator

Purpose: Somatostatin receptors (SSTr) are expressed on many neuroendocrine tumors, and several radiotracers have been developed for imaging these types of tumors. For this reason, peptide analogues of somatostatin have been well characterized. Copper-64 (t1/2 = 12.7 hours), a positron emitter suitable for positron emission tomography (PET) imaging, was shown recently to have improved in vivo clearance properties when chelated by the cross-bridged tetraazamacrocycle 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo(6.6.2)hexadecane (CB-TE2A) compared with 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA). Experimental Design: CB-TE2A and TETA were conjugated to the somatostatin analogue tyrosine-3-octreotate (Y3-TATE) for evaluation of CB-TE2A as a bifunctional chelator of 64Cu. The in vitro affinity of each compound for SSTr was determined using a homologous competitive binding assay. In vivo characteristics of both radiolabeled compounds were examined in biodistribution and microPET studies of AR42J tumor-bearing rats. Results: Cu-CB-TE2A-Y3-TATE (Kd = 1.7 nmol/L) and Cu-TETA-Y3-TATE (Kd = 0.7 nmol/L) showed similar affinities for AR42J derived SSTr. In biodistribution studies, nonspecific uptake in blood and liver was lower for 64Cu-CB-TE2A-Y3-TATE. Differences increased with time such that, at 4 hours, blood uptake was 4.3-fold higher and liver uptake was 2.4-fold higher for 64Cu-TETA-Y3-TATE than for 64Cu-CB-TE2A-Y3-TATE. In addition, 4.4-times greater tumor uptake was detected with 64Cu-CB-TE2A-Y3-TATE than with 64Cu-TETA-Y3-TATE at 4 hours postinjection. MicroPET imaging yielded similar results. Conclusions: CB-TE2A appears to be a superior in vivo bifunctional chelator of 64Cu for use in molecular imaging by PET or targeted radiotherapy due to both improved nontarget organ clearance and higher target organ uptake of 64Cu-CB-TE2A-Y3-TATE compared with 64Cu-TETA-Y3-TATE.

Comparative studies of Cu-64-ATSM and C-11-acetate in an acute myocardial infarction model: ex vivo imaging of hypoxia in rats.

Copper labeled diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) is a promising agent for the imaging of hypoxic tissues. In the present study 64Cu(t1/2 = 12.8 h) labeled Cu-ATSM was used in combination with 11C (t1/2 = 20.3 min) labeled acetate as a regional perfusion marker to visualize hypoxic rat heart tissue in an acute left anterior descending (LAD) coronary artery occluded rat model using an ex vivo tissue slice imaging technique. 64Cu-ATSM was injected intravenously c.a. 10 min after occlusion and rats were sacrificed by cervical dislocation 10 min after injection. Carbon-11-acetate was injected 1 min before sacrifice to obtain a measure of blood flow. The heart was dissected, frozen, and cut into 1-mm thick slices with a gauged slicer, and 11C images were obtained with an electronic autoradiography instrument. After decay of 11C, 64Cu images were obtained in the same manner. In ischemic regions, where there was low 11C accumulation, 64Cu showed high accumulation when compared with normal regions. In rats with a large occlusion, the center of the ischemia did not show any accumulation of either 11C or 64Cu, indicating no blood supply. Cu-ATSM appears to be useful for the detection of hypoxia with contrast being observed at short times (10 min) postinjection.

Receptor-binding, biodistribution, and metabolism studies of 64Cu-DOTA-cetuximab, a PET-imaging agent for epidermal growth-factor receptor-positive tumors

The epidermal growth-factor receptor (EGFR) and its ligands have been recognized as critical factors in the pathophysiology of tumorigenesis. Overexpression of the EGFR plays a significant role in the tumor progression of a wide variety of solid human cancers. Therefore, the EGFR represents an attractive target for the design of novel diagnostic and therapeutic agents for cancer. Cetuximab (C225, Erbitux) was the first monoclonal antibody targeted against the ligand-binding site of EGFR approved by the Food and Drug Administration for the treatment of patients with EGFR-expressing, metastatic colorectal carcinoma, although clinical trials showed variability in the response to this treatment. The aim of this study involved using cetuximab to design a positron emission tomography (PET) agent to image the overexpression of EGFR in tumors. Cetuximab was conjugated with the chelator, DOTA, for radiolabeling with the positron-emitter, 64Cu (T(1/2) = 12.7 hours). 64Cu-DOTA-cetuximab showed high binding affinity to EGFR-positive A431 cells (K(D) of 0.28 nM). Both biodistribution and microPET imaging studies with 64Cu-DOTA-cetuximab demonstrated greater uptake at 24 hours postinjection in EGFR-positive A431 tumors (18.49% +/- 6.50% injected dose per gram [ID/g]), compared to EGFR-negative MDA-MB-435 tumors (2.60% +/- 0.35% ID/g). A431 tumor uptake at 24 hours was blocked with unlabeled cetuximab (10.69% +/- 2.72% ID/g), suggesting that the tumor uptake was receptor mediated. Metabolism experiments in vivo showed that 64Cu-DOTA-cetuximab was relatively stable in the blood of tumor-bearing mice; however, there was significant metabolism in the liver and tumors. 64Cu-DOTA-cetuximab is a potential agent for imaging EGFR-positive tumors in humans.

Radiolabeling of TETA- and CB-TE2A-conjugated peptides with copper-64.

The number of radiopharmaceuticals containing copper radionuclides for diagnostic imaging and targeted radiotherapy has grown considerably over the past few decades. This expansion has created the need for protocols allowing for the efficient chelation of 64Cu to peptide-chelator conjugates. Step 1A of this protocol describes a 64Cu-radiolabeling procedure for 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA)-conjugated peptides. This reaction is facile and requires the incubation of 64CuCl2 in 0.1 M ammonium acetate buffer with the TETA-peptide for 30 min at room temperature (20–23 °C). Step 1B of this protocol describes the radiolabeling procedure for 4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (CB-TE2A)-conjugated peptides. The CB-TE2A-peptide can be labeled with 64Cu in 0.1 M ammonium acetate buffer in 2 h at 95 °C. In both cases, the conjugates can be radiolabeled with 64Cu at greater than 95% purity and with specific activities of 37–111 MBq μg−1 (1–3 mCi μg−1). Both protocols are straightforward and can be completed within 3 h.

The Growing Impact of Bioorthogonal Click Chemistry on the Development of Radiopharmaceuticals

Click chemistry has become a ubiquitous chemical tool with applications in nearly all areas of modern chemistry, including drug discovery, bioconjugation, and nanoscience. Radiochemistry is no exception, as the canonical Cu(I)-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, inverse electron demand Diels-Alder reaction, and other types of bioorthogonal click ligations have had a significant impact on the synthesis and development of radiopharmaceuticals. This review will focus on recent applications of click chemistry ligations in the preparation of imaging agents for SPECT and PET, including small molecules, peptides, and proteins labeled with radionuclides such as 18F, 64Cu, 111In, and 99mTc.

64Cu-Labeled Inhibitors of Prostate-Specific Membrane Antigen for PET Imaging of Prostate Cancer

Prostate-specific membrane antigen (PSMA) is a well-recognized target for identification and therapy of a variety of cancers. Here we report five 64Cu-labeled inhibitors of PSMA, [64Cu]3–7, which are based on the lysine–glutamate urea scaffold and utilize a variety of macrocyclic chelators, namely NOTA(3), PCTA(4), Oxo-DO3A(5), CB-TE2A(6), and DOTA(7), in an effort to determine which provides the most suitable pharmacokinetics for in vivo PET imaging. [64Cu]3–7 were prepared in high radiochemical yield (60–90%) and purity (>95%). Positron emission tomography (PET) imaging studies of [64Cu]3–7 revealed specific accumulation in PSMA-expressing xenografts (PSMA+ PC3 PIP) relative to isogenic control tumor (PSMA– PC3 flu) and background tissue. The favorable kinetics and high image contrast provided by CB-TE2A chelated [64Cu]6 suggest it as the most promising among the candidates tested. That could be due to the higher stability of [64Cu]CB-TE2A as compared with [64Cu]NOTA, [64Cu]PCTA, [64Cu]Oxo-DO3A, and [64Cu]DOTA chelates in vivo.

Toxicity and dosimetry of 177Lu‐DOTA‐Y3‐octreotate in a rat model

Radiolabeled somatostatin analogs have demonstrated effectiveness for targeted radiotherapy of somatostatin receptor‐positive tumors in both tumor‐bearing rodent models and humans. A radionuclide of interest for cancer therapy is reactor‐produced 177Lu (t1/2 = 6.64 d; β− [100%]). The high therapeutic efficacy of the somatostatin analog 177Lu‐DOTA‐Tyr3‐octreotate (DOTA‐Y3‐TATE, where DOTA is 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid) was previously demonstrated in a tumor‐bearing rat model (Erion et al., J. Nucl. Med. 1999;40:223P; de Jong et al., Int. J. Cancer, 2001; 92:628–633). In the current study, the toxicity and dosimetry of 177Lu‐DOTA‐Y3‐TATE were determined in both normal and tumor‐bearing rats. Doses of 177Lu‐DOTA‐Y3‐TATE ranging from 0 to 123 mCi/kg were administered to rats and complete blood counts (CBCs) and blood chemistries were analyzed out to 6 weeks. No overt signs of toxicity were observed with 177Lu‐DOTA‐Y3‐TATE (i.e., lethargy, weight loss, scruffy coat or diarrhea) at any of the dose levels. Blood chemistries and CBCs were normal except for the white blood cell counts, which showed a dose‐dependent decrease. The maximum tolerated dose was not reached at 123 mCi/kg. The biodistribution of 177Lu‐DOTA‐Y3‐TATE was determined in CA20948 rat pancreatic tumor‐bearing rats, and the data were used to estimate human absorbed doses to normal tissues. The dose‐limiting organ was determined to be the pancreas, followed by the adrenal glands. The absorbed dose to the rat CA20948 tumor was estimated to be 336 rad/mCi (91 mGy/MBq). These data demonstrate that 177Lu‐DOTA‐Y3‐TATE is an effective targeted radiotherapy agent at levels that show minimal toxicity in this rat model.