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High purity production and potential applications of copper-60 and copper-61
Nuclear Medicine & Biology, Vol. 26, pp. 351–358, 1999 Copyright © 1999 Elsevier Science Inc. All rights reserved.
ISSN 0969-8051/99/$–see front matter PII S0969-8051(98)00113-9
High Purity Production and Potential Applications of Copper-60 and Copper-61 Deborah W. McCarthy,1 Laura A. Bass,1 P. Duffy Cutler,1 Ruth E. Shefer,2 Robert E. Klinkowstein,2 Pilar Herrero,1 Jason S. Lewis,1 Cathy S. Cutler,1 Carolyn J. Anderson1 and Michael J. Welch1 1
MALLINCKRODT INSTITUTE OF RADIOLOGY, WASHINGTON UNIVERSITY SCHOOL OF MEDICINE, DIVISION OF RADIOLOGICAL SCIENCES, ST. LOUIS, MISSOURI, USA; AND 2NEWTON SCIENTIFIC, INC., CAMBRIDGE, MASSACHUSETTS, USA
ABSTRACT. Previously we described the high yield production of 64Cu using a target system designed specifically for low energy, biomedical cyclotrons. In this study, the use of this target system for the production of 60Cu and 61Cu is described and the utility of these isotopes in the labeling of biomolecules for tumor and hypoxia imaging is demonstrated. 60Cu and 61Cu were produced by the 60Ni(p,n)60Cu, 61 Ni(p,n)61Cu, and 60Ni(d,n)61Cu nuclear reactions. The nickel target (>99% enriched or natural nickel) was plated onto a gold disk as described previously (54 –225 mm thickness) and irradiated (14.7 MeV proton beam and 8.1 MeV deuteron beam). The copper isotopes were separated from the nickel via ion exchange chromatography and the radioisotopic purity was assessed by gamma spectroscopy. Yields of up to 865 mCi of 60Cu have been achieved using enriched 60Ni. 61Cu has been produced with a maximum yield of 144 mCi using enriched 61Ni and 72 mCi using enriched 60Ni. Specific activities (using enriched material) ranged from 80 to 300 mCi/mg Cu for 60Cu and from 20 to 81 mCi/mg Cu for 61Cu. Bombardments of natural Ni targets were performed using both protons and deuterons. Yields and radioisotopic impurities were determined and compared with that for enriched materials. 60Cu was used to radiolabel diacetyl-bis(N4methylthiosemicarbazone), ATSM. 60Cu-ATSM was injected into rats that had an occluded left anterior descending coronary artery. Uptake of 60Cu-ATSM in the hypoxic region of the heart was visualized clearly using autoradiography. In addition, 60Cu-ATSM was injected into dogs and excellent images of the heart and heart walls were obtained using positron emission tomography (PET). 61Cu was labeled to 1,4,8,11tetraazacyclotetradecane-N,N’,N”,N”’-tetraacetic acid-octreotide (TETA-octreotide) and the PET images of tumor-bearing rats were obtained up to 2 h postinjection. After decay of the 61Cu, the same rat was injected with 64Cu-TETA-octreotide and the images were compared. The tumor images obtained using 61Cu were found to be superior to those using 64Cu as predicted based on the larger abundance of positrons emitted by 61 Cu vs. 64Cu. NUCL MED BIOL 26;4:351–358, 1999. © 1999 Elsevier Science Inc. All rights reserved. KEY WORDS. Cu-60, Cu-61, Cu-64, Production, PET
INTRODUCTION 62
67
64
Cu, Cu, and Cu are copper radionuclides already being used for several nuclear medicine applications. 62Cu (t1/2 5 9.76 min) is generator produced and has been used to label agents to quantify both blood flow and hypoxia (5– 8, 19). A commercial version of the generator is now being investigated (9). The short half-life (9 h) of the 62Zn parent limits the potential usefulness of this nuclide. 67 Cu is a longer-lived isotope (t1/2 5 61.9 h), which is produced in high yield using the high energy proton beam at Brookhaven National Laboratories (BLIP) Facility and Los Alamos National Laboratory. The need for these high energy physics facilities allows only limited availability of this radionuclide. 64Cu has an intermediate half-life of 12.7 h and has applications both for imaging and therapy. Previously we reported the high yield production of 64Cu using a target system designed specifically for low energy, biomedical cyclotrons (13). Address correspondence to: Michael J. Welch, Ph.D., Mallinckrodt Institute of Radiology, Washington University School of Medicine, 510 S. Kingshighway Blvd., St. Louis, MO 63110, USA; e-mail: welch@mirlink. wustl.edu. Received 15 August 1998. Accepted 20 November 1998.
We are interested in developing positron emission tomography (PET) radiopharmaceuticals labeled with other positron-emitting Cu radionuclides (see Table 1). 60Cu has a half-life of 23.7 min and decays 93% of the time by positron emission. 61Cu has a longer half-life (3.32 h) and 60% of its emissions are by positron decay. We are particularly interested in preparing agents for imaging hypoxic tissue in tumor, brain, and heart, as well as developing techniques for labeling peptides and proteins. In this paper, we demonstrate the utility of these isotopes in the imaging of tumors and hypoxic tissues at times ranging from tens of minutes to a few hours. If a single target and production system can be used to produce these nuclides as well as 64Cu, copper nuclides would be available for a wide variety of biological studies. PET studies using compounds labeled with Cu-60 may be possible, depending on the type of quantitative information necessary. Cu-60 emits prompt gamma rays in cascade with each positron in the 1–2-MeV range that are virtually unaffected by the typical lead and lead/tungsten shielding in the PET gantry. These gammas or their secondary radiations contribute substantial noise to the data by means of random coincidences. The thin tungsten septa in commercially available PET scanners are normally effective at controlling the randoms fraction by limiting the solid angle seen by each detector. With this shielding, virtually invisible to the high-
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TABLE 1. Positron-Emitting Cu Radionuclides Nuclide
Half-life
Decay mode
Major gamma (keV)
Emissions (%)
60
23.7 min
93% b1 7% EC
61
3.32 h
62
0.16 h
80 52 15 12 10.7 0.15
64
12.7 h
60% b1 40% EC 98% b1 2% EC 19% b1, 43% EC, 38% b2
1333 1760 850 284 656 875 1345
0.47
Cu Cu Cu Cu
b1 3.92 3.00 2.00 1.22
max
MeV, 6% MeV, 18% MeV, 69% MeV
2.91 MeV 0.656 MeV
Data from: Table of Isotopes (9a).
energy gammas, any activity inside and within a few centimeters of the axial field-of-view will flood all detectors in the tomograph with single photon events. This action has the dual effect of adding noise via increased random coincidences, and reducing good signal via increased deadtime. Among the casualties of the higher-energy gammas is the deadtime correction. The calculation of an accurate correction factor for the data depends on an expected ratio of single events to coincident events. Phantom studies have been carried out to determine the magnitude of the effect of the prompt gamma emissions.
to 64,61,60Cu and the percent complexation monitored by radio-thin layer chromatography (radio-TLC) (Bioscan, Washington, DC). The samples were spotted on silica plates and the plates developed using 1:1 MeOH:10% NH4OAc. Cu acetate remained at the origin whereas complexed copper in the form of Cu(TETA)2- migrated with Rf 5 0.42. The minimum TETA concentration for which 100% labeling occurred was assumed to be equal to the concentration of Cu(II) present.
Radiochemical Synthesis of MATERIALS AND METHODS
Nickel Targets Preparations High purity reagents used in the electroplating and separation studies were the same as those described previously for the production of 64Cu (13). Isotopically enriched 60Ni (99.59%) and 61Ni (99.44%) were purchased from Trace Sciences International (Richmond Hills, Ontario, Canada). Natural nickel (99.99% purity) was purchased from Aldrich Chemical Company (Milwaukee, WI). The electroplating procedure was an adaptation of the methodology reported by Piel and co-workers (15). Briefly, appropriate quantities of nickel metal were dissolved in 6 N HNO3 and evaporated to dryness. The residue was treated with H2SO4, diluted with deionized water, and evaporated to almost dryness. The residue was cooled and dissolved with deionized water. The pH was adjusted to 9 with concentrated ammonium hydroxide and ammonium sulfate electrolyte was added. The solution was quantitatively transferred to an electrolytic cell in which the anode was a graphite rod and the cathode was a gold disk (0.06250 thick 3 0.750 diameter). The cells were typically operated at 2.4 –2.6 V and at currents between 10 and 25 mA. Electroplating was accomplished in 12–24 h. The diameter of the Ni targets were 0.5 cm unless otherwise indicated.
61/64
Cu-TETA-Octreotide
Octreotide was purchased from DePaul Pharmaceutical (Bridgeton, MO). TETA z 4HCl z 4H2O was purchased from Aldrich. TETA-octreotide was prepared and radiolabeled with copper as described previously (1). For the labeling, 7–15 mCi of 64Cu or 61 Cu were labeled to 3 mg of TETA-octreotide in 0.1 M ammonium acetate buffer, pH 5.5 by a 45-min room temperature incubation. Samples were purified by a C-18 SepPakt to remove uncomplexed 64/61 Cu-acetate. Radiochemical purity was assessed by radio-TLC using reversed-phase C-18 plates developed in 70:30 methanol:5% NH4OAc. Cu-TETA-octreotide migrates with Rf 5 0.5. Radiochemical purity of the labeled complexes was typically above 97%. Approximately 300 mCi (0.2 mg) of the Cu-TETA-octreotide radioconjugates were injected into the rats.
Isotope Production and Analysis Targets were irradiated in the previously described holder (13) using approximately 14.7 MeV protons and 8.1 MeV deuterons on a Cyclotron Corporation CS-15 cyclotron at Washington University. Isotope yield measurements were determined using either a calibrated Ge detector (Canberra model 1510, Meriden, CT) or a radioisotope dose calibrator (Capintec CRC-10, Pittsburgh, PA) in combination with the Ge detector. The Ge detector was used to detect radionuclidic impurities. Specific activities were determined as described previously for 64Cu by titration of 64Cu-acetate with the macrocycle TETA (13). Briefly, aliquots of TETA were added
FIG. 1. Structure of Cu-diacetyl-bis(N 4-methylthiosemicarbazone) (Cu-ATSM).
Production and Application of Cu-60 and Cu-61
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TABLE 2. Yields of Copper Isotopes Using 991% Enriched Materials
Isotope
Nuclear Reaction
Target thickness (microns)
Maximum EOB yield (mCi)
Typical EOB yield (mCi)
60
60
225
865
450
61
61
61
60
118 122 215
144 72 921
100 60 200
Cu
Cu Cu 64 Cu
Ni(p,n)60Cu
Ni(p,n)61Cu Ni(d,n)61Cu 64 Ni(p,n)64Cu
Yield (mCi/mA z h)
Major isotopic impurities 0.05% 61Cu 0.025% 57Co 0.04% 58Co 0.04% 58Co 0.012% 55Co
58 7.6 2.44 8.0
EOB 5 end of bombardment.
TABLE 3. Natural Ni Bombardment: 14.7 MeV Protons
Target thickness (microns)
EOBsat yield Cu-60 (mCi/mA)
116
10.42 (EOB yield 5 0.875 mCi/mA)
116b
7.86 (EOB yield 5 6.5 mCi/mA)
a
a b
Radionuclidic impurities per 10 mCi Cu-60
% Radionuclidic impurities (relative to Cu-60) EOB
EOB
45 min post-EOB
Co-55 5 0.65
Co-55 5 0.065 mCi
Co-55 5 0.235 mCi
Cu-61 5 0.60 Co-55 5 1.0
Cu-61 5 0.060 mCi Co-55 5 100 mCi
Cu-61 5 0.192 mCi Co-55 5 361.8 mCi
Co-57 5 5.3 E-3 Co-58 5 5.2 E-4 Ni-57 5 6.7 E-3 Cu-64 5 0.38 Cu-61 5 1.12
Co-57 5 0.53 mCi Co-58 5 0.052 mCi Ni-57 5 0.67 mCi Cu-64 5 38 mCi Cu-61 5 112 mCi
Co-57 5 1.975 mCi Co-58 5 0.1937 mCi Ni-57 5 2.462 mCi Cu-64 5 135.92 mCi Cu-61 5 356.9 mCi
Sample bombarded at 4 mA for 3 min, 0.6 cm diameter target. Sample bombarded at 20 mA (avg current) for 1 h.
TABLE 4. Radionuclidic Impurities per 10 mCi
60
Cu Produced Using 991% Enriched
60
Ni
Radionuclidic impurities per 10 mCi Cu-60
EOBsat yield Cu-60 (mCi/mA) 45
EOB
45 min post-EOB
100 min post-EOB
Co-55 5 3.6 E-5 mCi Co-57 5 0.036 mCi Cu-61 5 5 mCi
Co-55 5 0.13 mCi Co-57 5 2.24 mCi Cu-61 5 15.93 mCi
Co-55 5 0.627 mCi Co-57 5 11.16 mCi Cu-61 5 65.6 mCi
EOB 5 end of bombardment. Conditions included using a 125-mm thick target, 14.7 MeV proton beam, ;15 mA avg. current, 44-min bombardment.
TABLE 5.
60
Cu production (60Ni(p,n)60Cu) Using 99.59 Enriched
60
Ni EOBsat yield
Target thickness (microns) 68.1 54.36 129.33 115.02 125.32
Production parameters (EOB activity, irradiation time, average beam current) 152 178 613 309 494
mCi, mCi, mCi, mCi, mCi,
44 72 49 20 44
min, min, min, min, min,
12.68 mA 8.5 mA 15.3 mA 14.4 mA 14.86 mA
EOB 5 end of bombardment prior to column separation. Production parameters 5 14.7 MeV protons.
(mCi) Before column separation
(mCi/mA) Before column separation
(mCi) After separation
(mCi/mA) After separation
210 203 805 697 683
16.56 23.9 52.6 48.4 45.93
201 256 458 688 456
15.7 30.1 30.0 47.8 30.67
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TABLE 6. Natural Ni Bombardment: 8.1 MeV Deuterons Target thickness (microns)
EOB yield Cu-61 (mCi/mA z h)
% Radionuclidic impurities (relative to Cu-61) EOB
0.91 0.29
Co-56 5 0.07 Co-56 5 0.08 Co-58 5 0.05 Ni-65 5 6.6
117a 130b
Radionuclidic impurities per 10mCi Cu-61 EOB Co-56 5 7 mCi Co-56 5 8.0 mCi Co-58 5 5.0 mCi Ni-65 5 660 mCi
EOB 5 end of bombardment. a Sample bombarded at 4 mA for 3 min, 0.6-cm diameter target. b Sample bombarded at 10 mA for 2 h. Sample was analyzed 3 h after EOB: short half-lived isotopes not analyzed for.
Preparation of
60
Cu-ATSM
H2ATSM was synthesized as described by Petering and co-workers (14). All chemicals were purchased from Aldrich, unless stated otherwise. Briefly, 4-methyl-3-thiosemicarbazide (97%) was dissolved in 5% acetic acid (99.99%) at 50 – 60°C with stirring. 2,3-Butanedione (97%) was taken up in water (distilled, deionized, .18 MV resistivity) and added to the 4-methyl-3-thiosemicarbazide solution dropwise. The hot solution was filtered through a course fritted-glass filter and a precipitate was collected. This solid was washed with H2O and then ethanol and dried at 75°C overnight. The solid was purified by suspension in hot 80% acetic acid under reflux. The mixture was again filtered hot and the undissolved material collected and dried overnight at 75°C. 60Cu was used to radiolabel ATSM using methods identical to Fujibayashi and co-workers (4).
Phantom Studies To evaluate the quantitative accuracy of imaging Cu-60 with PET, a phantom study was performed where the measured activity was compared with the true activity. A cylinder was filled with a dilute solution of 60Cu and scanned over several half-lives using a Siemens ECAT Exact PET scanner (12). For comparison, a similar experiment was carried out utilizing fluorine-18.
Imaging Studies All animal studies were performed in compliance with guidelines set forth by the Washington University Animal Studies Committee. Autoradiographic images of tissue slices were obtained utilizing an InstantImagert electronic autoradiography system (Packard Instrument Company (Meriden, CT). Images of rats were obtained utilizing the Siemens 953 B PET scanner at Washington University. PET images of the canine were obtained using PET Electronics SP3000E scanner at Washington University. Radiopharmaceuticals used for all imaging studies were prepared using isotopes produced from enriched target materials.
Tissue-Tek embedding medium (Miles Inc., Elkhart, IN), and 1-mm slices cut. The sections were placed on a grid and loaded onto the Packard InstantImager to obtain the 60Cu-ATSM images.
Rat Tumor Imaging Studies Lewis rats were implanted with somatostatin receptor-positive rat pancreatic tumors (CA20948) in the nape of the neck (10). The original tumor cells were purchased from the Tumor Bank at Biomeasure, Inc. (Hopkinton, MA). At the time of study, the tumor was approximately 1,340 mm3 and weighed 1.3 g. For the PET imaging study, the tumor-bearing female rat (170 g) was anesthetized and 253 mCi of 61Cu-TETA-octreotide were injected via intracardiac puncture. Scanning commenced immediately and PET images were collected for 1 h post injection. After the 61Cu had been allowed to decay (22 h later), 353 mCi of 64Cu-TETAOctreotide were injected via intracardiac puncture. Scanning began immediately and images of the entire animal were collected for 1 h.
PET Dog Study A 22.73-kg male mongrel dog was fasted 24 h prior to the procedure. The dog was anesthetized, placed in the SP3000E PET scanner, and administered 19.1 mCi of 11CO. PET scanning commenced immediately and images were collected for 5 min. Twenty minutes after the 11CO administration, 20 mCi of H15 2 O were injected intravenously and PET data were again collected for 5 min. Finally, 10 min after the H15 2 O injection, 1 mCi of 60 Cu-ATSM was injected intravenously and PET data collected for 10 min. TABLE 7. Production of 61Cu Using 99.59% Enriched60Ni and 99.44% Enriched 61Ni(d,n)61Cu and 61Ni(p,n)61Cu Nuclear Reactions Nuclear reaction 60
Rat Heart Model Studies 60
Cu-ATSM (structure shown in Fig. 1) was used to visualize hypoxia in an acute left anterior descending (LAD) coronary artery occluded heart model by ex vivo tissue slicing as described previously (4). Briefly, a male Wistar rat (360 g) was anesthetized, intubated, and ventilated with room air. Blood flow was occluded to the LAD coronary artery. Thirteen minutes postocclusion, 270 mCi 60CuATSM were injected through the femoral vein. The animal was sacrificed 10 min postinjection, the heart removed, frozen in
Ni(d,n)61Cu Ni(d,n)61Cu 60 Ni(d,n)61Cu 61 Ni(p,n)61Cu 61 Ni(p,n)61Cu 61 Ni(p,n)61Cu 61 Ni(p,n)61Cu 61 Ni(p,n)61Cu 60
Target thickness (microns)
EOB yield (mCi)
EOB yield (mCi/mA z h)
120a 122a 105 117 118 57.8 52.07 116
56 72 45 112.4 144 60.5 98.2 168
1.11 2.44 1.62 11.24 7.62 3.02 3.07 9.6
EOB 5 end of bombardment. 8.1 MeV deuteron beam, 14.7 MeV proton beam. a 0.6 cm diameter target.
Production and Application of Cu-60 and Cu-61
TABLE 8. Cost Comparison for Making 20 mCi Nuclear reaction 61
Yield (mCi/mA z h) 61
Ni(p,n) Cu Ni(d,n)61Cu 60 Ni(d,n)61Cu
7.62 2.44 0.91
60
355
61
Target material
Cu Cost of recycling lossa
# Cyclotron hours
Cyclotron cost (@ $245/hr)
Total cost
$165 $40 Negligible
0.13 0.82 2.20
$32 $201 $539
$197 $241 $539
61
Ni Ni nat Ni 60
Conditions included 14.7 MeV, 20-mA proton beam, 8.1 MeV, 10 mA deuteron beam. a Assuming 85% recovery of the enriched 61Ni. For the 60Ni, because of the relatively low cost, the material is not recovered.
RESULTS AND DISCUSSION 64
61
60
Cu, Cu, and Cu were produced in high yield using the target system described previously (13) . The maximim production yields of the copper radionuclides are shown in Table 2. 61Cu was produced by two nuclear reactions 61Ni(p,n)61Cu and 60Ni (d,n)61Cu. Although higher yields of 61Cu are produced using the (p,n) reaction, the target material for this reaction is approximately 25 times the cost of the target material for the d,n reaction ($55/mg Ni-61 vs $2/mg for Ni-60). None of the proton irradiation targets used in this work are “thick targets.” Of the targets described in Table 2, the ;220-mm thick targets degrade the 14.7-MeV proton beam by 5.2 MeV, whereas the ;120-mm thick target degrades the beam by approx. 2.5 MeV. The cross-sections of the 60Ni/61Ni(p,n) reactions do not decrease dramatically until 6 –7 MeV (15), so higher yields could be obtained at greater target expense with thicker targets (up to 350 mm). The ;120-mm thick target used for the deuteron irradiation is a “thick target.” The yields obtained are somewhat variable presumably due to minor alterations in the beam alignment, which affects the 0.5-cm and 0.6-cm diameter target used. Typical specific activities of these cyclotron-produced Cu isotopes as determined by TETA titrations are: 64Cu 5 40 –250 mCi/mg Cu, 61Cu 5 20 – 81 mCi/mg Cu, 60Cu 5 80 –300 mCi/mg Cu. Several authors have reported the production of 60Cu on biomedical cyclotrons using natural Ni. Martin (11) irradiated natural Ni (thick target) with 1 mA of 11-MeV protons for 23 min and reported the following end of bombardment (EOB) yields: 0.01 mCi 55 Co, 0.01 mCi 57Co, 5.0 mCi 60Cu, 0.07 mCi 61Cu, 3.8 mCi 62Cu, and 0.02 mCi 64Cu. Martin’s EOBsat (end of saturation bombardment) yield of Copper-60 was 10 mCi/mA (thick target yield). Similar yields can be obtained using a 14.7-MeV proton beam. Our EOBsat yields using a 14.7-MeV proton beam with natural nickel ;120 mm thick) for the 60Ni(p,n)60Cu reaction were 7.86 mCi /mA and 10.42 mCi/mA (see Table 3). The yields obtained were in agreement with those predicted by Piel et al. (15). Martin (11) described an electrochemical dissolution of the target material and separation of the Ni and Co from the copper by solvent extraction followed by anion exchange separation. Martin’s separation method removed 99% of the cobalt contaminants and TABLE 9. Cost Comparison for Making 50 mCi Nuclear reaction 61
Yield (mCi/mA z h) 61
Ni(p,n) Cu Ni(d,n)61Cu 60 Ni(d,n)61Cu 60
7.62 2.44 0.91
Target material 61
Ni Ni nat Ni 60
61
they were not considered in his dosimetry calculations with Cu-60PTSM. Using 99.59% enriched material for the 60Ni(p,n)60Cu reaction, we produced much less Co radioisotopic impurities than production from natural Ni, even with the higher energy protons used (see Table 4). This allowed for our separation procedure (anion exchange chromatography) to be less complicated. In addition, the 60 Cu yield using enriched material was four times higher than using nat Ni, which is important in certain circumstances in which minimal preparation time is required. Table 5 shows the 60Cu yields using enriched nickel for the 60Ni(p,n)60Cu nuclear reaction. In some cases, the EOBsat yields of the separated material were less than that of the whole target due to loss of 60Cu during the separation procedure. Production of 61Ni using natural Ni has also been reported (17). Thick natural nickel foils were bombarded with 6.0 MeV deuterons. A yield of 10 MBq/mA z h (0.27 mCi/mA z h) was reported for 61Cu and the only radioisotopic impurity reported was 62Cu. We observed low levels of radioisotopic impurities but with higher yields at '8.1 MeV deuterons (see Table 6). Zweit et al. (20) reported cross-section data for 60Ni(d,n)61Cu and 61Ni(d,2n)61Cu; however, values were given only for energies of 7.2–18.9 MeV. Szelecse´nyi et al. (16) irradiated enriched 61Nielectroplated targets with protons and measured the cross-sections of the reactions. Energy range for the production of the 61Cu was Ep 5 12–9 MeV, corresponding to a target thickness of 0.119 g/cm2 (134 mm). The theoretical yield for producing 61Cu with protons using 100% 61Ni is 17.5 mCi /mA z h. Our 61Cu yields from the proton bombardment of 112–118-mm thick 61Ni targets were in very good agreement with Szelecse´nyi (16) (Table 7). We conducted a cost comparison (using our production parameters) for the production of 61Cu using either 60Ni, 61Ni, or natNi as the target material. For producing 20 mCi of 61Cu, the cost of production using 60Ni or 61Ni was similar ($241 vs. $197 respectively). The advantage of using 60Ni (60Ni(d,n)61Cu) as the target material is its low cost. Process time is reduced if it is not necessary to recycle the enriched material. Production of 61Cu by deuteron irradiation of natNi was considerably more expensive ($539) due to the longer bombardment time required (Table 8). If larger quantities of 61Cu are required, i.e., 50 mCi, proton bombardment of 61Ni
Cu Cost of recycling lossa
# Cyclotron hours
Cyclotron cost (@ $245/hr)
Total cost
$165 $40 Negligible
0.33 2.05 5.5
$80 $502 $1346
$245 $542 $1346
Conditions included 14.7 MeV, 20 mA proton beam, 8.1 MeV, 10 mA deuteron beam. a Assuming 85% recovery of the enriched 61Ni. For the 60Ni, because of the relatively low cost, the material is not recovered.
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FIG. 2. Activity concentration values as measured by a cylindrical phantom. F-18 activity (left) is accurate over the 0 –1 mCi/mL measured here, and is within 5% out to 5 mCi/mL (not shown). A decaying phantom filled with Cu-60 activity in ionic solution is shown on the right. The deadtime correction restores data loss up to about 0.8 mCi/mL. Above this level, minor modifications to the deadtime model will be required to account for the effects of Cu-60’s high-energy gammas. (61Ni(p,n)61Cu) is preferable (cost 5 $245 versus $542 for 60 Ni(d,n)61Cu and $1,346 for deuteron bombardment of natNi) (Table 9). The radionuclide, 60Cu, decays by the emission of high energy positrons and high energy gamma rays. For this reason, an experiment was performed in which the measured activity was compared with the true activity using a cylindrical phantom. It was determined that the 60Cu activity could be quantified to a concentration of 0.8 mCi/mL. In human studies using 60Cu-ATSM, we anticipate an activity concentration averaging just 0.18 mCi/mL, and potentially ranging as high as 0.9 mCi/mL. Thus, except perhaps at the high end of this range, the effects shown in Figure 2 will be small to negligible. With minor adjustments to the deadtime calculation, calibrated for Cu-60 imaging, we will be able to obtain accurate parameter estimates over a much wider activity range if necessary. The data obtained for 60Cu were compared with that from 18F in Figure 2. With the standard deadtime correction provided with the PET system, a linear response was obtained for F-18, as expected, but not for Cu-60. The accuracy diverged at a concentration of 0.8 mCi/mL. This corresponds to approximately 4 mCi total activity in the field of view of the tomograph. In a PET study of myocardial perfusion, a relatively small bolus of Cu-60 activity, perhaps 10 mCi total, would be sufficient to exceed the linear range of the tomograph. In other studies, in which the activity can be infused over time, or in which the measurement of an input function is not needed, then a much larger dose could be used while still obtaining accurate results. This approximates to a uniform distribution of approximately 50 mCi in a human. It is highly unlikely that any radiopharmaceutical would require such a high concentration, so linear data can be obtained utilizing appropriate amounts of this nuclide. As anticipated, the 60Cu data are affected by the high energy gammas. 60 Cu has been evaluated in two imaging studies. Images were obtained ex vivo following administration of 60Cu-ATSM into a rat in which the LAD had been tied off (Fig. 3). The images obtained with 60Cu were similar to images obtained previously with 64Cu (4). In addition, 60Cu-ATSM, a hypoxic tissue marker, was injected into a dog under normoxic conditions. 60Cu-ATSM is selectively
trapped in hypoxic tissues but is rapidly washed out of normoxic cells (4). A high quality image of a dog heart was obtained showing some retention of 60Cu-ATSM in the walls of the dog heart (Fig. 4). The image showed a reconstructed midventricular PET image of the heart from a normal dog obtained 30 – 640 s after administration of 60 Cu-ATSM. The image to the left was not corrected for blood pool, whereas the image on the right was corrected for tracer remaining in the blood pool. Even though Cu-ATSM washes out from normoxic tissue (;20% retention), high quality images are obtained in vivo. The image was obtained using 1 mCi of tracer. Our preliminary dose estimates allow ;13 mCi of 60Cu-ATSM to be administered to a human. This method demonstrates the viability of using 60Cu-labeled radiopharmaceuticals for PET imaging. Studies on the uptake of Cu-ATSM in ischemic heart are in progress (18). A potential application of 61Cu is for labeling proteins and peptides for which maximum uptake occurs from 2 to 6 h post administration. An agent that falls into this category is 61CuTETA-octreotide. Comparison images with 64Cu-TETA-octreotide are shown in Figure 5. These images were obtained using 0.2 mg of 61/64 Cu-TETA-octreotide. The small amount of injected dose was chosen because it was significantly lower than the amount previously used to block the uptake of 64Cu-TETA-octreotide (250 mg) (2). This small amount is unlikely to cause any decrease in the uptake of Cu-64-TETA-octreotide. Higher quality images were obtained with 61Cu than with 64Cu, due to the higher positron yield (60% vs. 19%) and the shorter half-life. Because high quality human images (3) can be obtained 4 h postadministration of 64 Cu-TETA-octreotide, higher quality images could be obtained (at the same radiation dose) using 61Cu-TETA-octreotide.
CONCLUSION The cyclotron target described previously for the production of Cu can be used to produce 60Cu and 61Cu in high yield and at high specific activitiy. For certain biological applications, the shorter half-life of 60Cu and 61Cu may be preferable to the 12.7-h 64
Production and Application of Cu-60 and Cu-61
357
FIG. 3. Ex vivo imaging of a left anterior descending (LAD) coronary artery occluded heart model. At the top are photographic images of the heart slices. At the bottom are images obtained on the Packard InstantImager of the distribution of Cu-60-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-60-ATSM) in hypoxic tissue. half-life of 64Cu. Despite the high b1 energy and prompt gamma emission of 60Cu, high quality images are obtained both autoradiographically (using the Packard InstantImager) and in PET scans of 60 Cu-ATSM. For studies in which maximum contrast is obtained in less than 1 h, 60Cu will be preferred. If optimal uptake occurs in 1– 4 h, 61Cu will bepreferred.
This work was supported by research grants from the Department of Energy DE-FG02-87ER60512 (M.J.W.) and NIH CA64475 (C.J.A.). We thank Bill Margenau, Dave Ficke and Todd Perkins for assistance in isotope production, and Lynne A. Jones, Terry Sharpe and Lennis Lich for their contribution to the animal imaging studies.
FIG. 4. Reconstructed midventricular positron emission tomography (PET) images of the heart from a normal canine obtained 30 – 640 s after administration of Cu-60-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-60-ATSM). The raw image is shown to the left, and the image to the right represents the raw image after being corrected for tracer remaining in the blood pool.
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FIG. 5. Images of tumor-bearing Lewis rats injected with Cu-1,4,8,11-tetraazacyclotetradecane-N,N*,N(,N***-tetraacetic acid (TETA)-octreotide (octreotide is a somatostatin analog, used in imaging tumors that contain somatostatin receptors). The bottom images were obtained after 353 mCi of 61Cu -TETA-octreotide were injected (;3.6 E, 6 total counts per slice). Twenty-two hours later, 253 mCi of 64Cu-TETA-octreotide was injected into the same rat (;7.8 E, 5 total counts per slice) and positron emission tomography (PET) images were obtained again (top images).
References 1. Anderson C. J., Jones L. J., Bass L. A., Sherman E. L., McCarthy D. W., Cutler P. D., Lanahan M. V., Cristel M. E., Lewis J. S. and Schwarz S. W. (1998) Radiotherapy, toxicity and dosimetry of copper-64labeled-TETA-octreotide in tumor-bearing rats. J. Nucl. Med. in press. 2. Anderson C. J., Pajeau T. S., Edwards W. B., Sherman E. L. C., Rogers B. E. and Welch M. J. (1995) In vitro and in vivo evaluation of copper-64-octreotide conjugates. J. Nucl. Med. 36, 2315–2325. 3. Dehdashti F., Anderson C. J., Trask D. D., Bass L. A., Schwarz S. W., Cutler P. D., McCarthy D. W. and Lanahan M. V. (1997) Initial results with PET imaging using Cu-64-labeled TETA-octreotide in patients with carcinoid tumor. J. Nucl. Med 38, 103. 4. Fujibayashi Y., Cutler C. S., Anderson C. J., McCarthy D. W., Jones L. A., Sharp T., Yonekura Y. and Welch M. J. (1998) Comparative studies of Cu-64-ATSM and C-11 Acetate in an acute myocardial infarction model: Ex vivo imaging of hypoxia in rats. Nucl. Med. Biol. in press. 5. Fujibayashi Y., Taniuchi H., Yonekura Y., Ohtani H., Konishi J. and Yokoyama A. (1997) Copper-62-ATSM: A new hypoxia imaging agent with high membrane permeability and low redox potential. J. Nucl. Med. 38, 1155–1160. 6. Fujibayashi Y., Wada K., Matsumoto K., Yonekura Y., Konishi J. and Yokoyama A. (1991) Retention mechanism of Cu-62-PTSM, a generator-produced positron emitting Cu-62 labeled blood flow tracer. J. Nucl. Med. 32, 974. 7. Green M. A. and Goldenberg D. M. (1987) A potential copper radiopharmaceutical for imaging the heart and brain: Copper-labeled pyruvaldehyde bis(N-methylthiosemicarbazone). Nucl. Med. Biol. 14, 59 – 61. 8. Green M. A., Welch M. J., Mathias C. J., Bergmann S. R., Perlmutter J. S., Raichle M. E., Rubio F. F. and Janik M. (1990) Copper-62-PTSM: Production and evaluation of a generator produced multi-purpose PET perfusion agent. J. Nucl. Med. 31, 815. 9. Lacy J. L., Haynes N. G., Nayak N., Martin C. S., Dai D. and Green M. A. (1998) Performance of a Zn-62/Cu-62 generator in clinical trials of the PET perfusion agent Cu-62-PTSM. J. Nucl. Med. 39, 142. 9a.Lederer C. M. and Shirley V. S. (1978) Table of Isotopes, 7th edn. MacMillan, New York. 10. Longnecker D. S., Lilja H. S., French J., Kuhlmann E. and Noll W.
11. 12. 13.
14. 15.
16.
17. 18.
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(1979) Transplantation of azaserine-induced carcinomas of pancreas in rats. Cancer Lett. 7, 197–202. Martin C. C. (1992) The kinetics of copper pyruvaldehyde bis (N4-methylthiosemicarbazone): A blood flow tracer for positron emission tomography. PhD Dissertation, University of Wisconsin-Madison, Madison. Martin C. C., Christian B. T., Satter M. R., Nickerson L. D. H. and Nickles R. J. (1995) Quantitative PET with positron emitters that emit prompt gamma rays. IEEE Trans. Med. Imaging 14, 681– 687. McCarthy D. W., Shefer R. E., Klinkowstein R. E., Bass L. A., Margenau W. H., Cutler C. S., Anderson C. J. and Welch M. J. (1997) Efficient production of high specific activity 64Cu using a biomedical cyclotron. Nucl. Med. Biol. 24, 35– 43. Petering H. G., Buskirk H. H. and Underwood G. E. (1964) The anti-tumor activity of 2-keto-3-ethoxybutyraldehyde bis(thiosemicarbazone) and related compounds. Cancer Res. 24, 367–372. Piel H., Qaim S. M. and Sto¨cklin G. (1992) Excitation functions of (p,xn)-reactions on natNi and highly enriched 62Ni: Possibility of production of medically important radioisotope 62Cu at a small cyclotron. Radiochim. Acta 57, 1–5. Szelecse´nyi F., Blessing G. and Qaim S. M. (1993) Excitation functions of proton induced nuclear reactions on enriched 61Ni and 64Ni: Possibility of production of no-carrier-added 61Cu and 64Cu at a small cyclotron. Appl. Radiat. Isot. 44, 575–580. Tolmachev V., Lundqvist H. and Einarsson L. (1998) Production of 61 Cu from a natural nickel target. Appl. Radiat. Isot. 49, 79 – 81. Welch M. J., Lewis J. S., McCarthy D. W., Sharp T., Herrero P. and Fujibayashi Y. (1998) Evaluation of copper-60-dicaetyl-bis(N9-methylthiosemicarbazone)(Cu-ATSM), a hypoxia imaging agent, in canine models of ischemia. Eur. J. Nucl. Med. 25, 978. Zweit J., Goodall R., Cox M., Babich J. W., Potter G. A., Sharma H. L. and Ott R. J. (1992) Development of high performance zinc-62/ copper-62 radionuclide generator for positron emission tomography. Eur. J. Nucl. Med. 19, 418 – 425. Zweit J., Smith A. M., Downey S. and Sharma H. L. (1991) Excitation functions for deuteron induced reactions in natural nickel: Production of no-carrier-added 64Cu from enriched 64Ni targets for positron emission tomography. Appl. Radiat. Isot. 42, 193–197.
ISSN 0969-8051/99/$–see front matter PII S0969-8051(98)00113-9
High Purity Production and Potential Applications of Copper-60 and Copper-61 Deborah W. McCarthy,1 Laura A. Bass,1 P. Duffy Cutler,1 Ruth E. Shefer,2 Robert E. Klinkowstein,2 Pilar Herrero,1 Jason S. Lewis,1 Cathy S. Cutler,1 Carolyn J. Anderson1 and Michael J. Welch1 1
MALLINCKRODT INSTITUTE OF RADIOLOGY, WASHINGTON UNIVERSITY SCHOOL OF MEDICINE, DIVISION OF RADIOLOGICAL SCIENCES, ST. LOUIS, MISSOURI, USA; AND 2NEWTON SCIENTIFIC, INC., CAMBRIDGE, MASSACHUSETTS, USA
ABSTRACT. Previously we described the high yield production of 64Cu using a target system designed specifically for low energy, biomedical cyclotrons. In this study, the use of this target system for the production of 60Cu and 61Cu is described and the utility of these isotopes in the labeling of biomolecules for tumor and hypoxia imaging is demonstrated. 60Cu and 61Cu were produced by the 60Ni(p,n)60Cu, 61 Ni(p,n)61Cu, and 60Ni(d,n)61Cu nuclear reactions. The nickel target (>99% enriched or natural nickel) was plated onto a gold disk as described previously (54 –225 mm thickness) and irradiated (14.7 MeV proton beam and 8.1 MeV deuteron beam). The copper isotopes were separated from the nickel via ion exchange chromatography and the radioisotopic purity was assessed by gamma spectroscopy. Yields of up to 865 mCi of 60Cu have been achieved using enriched 60Ni. 61Cu has been produced with a maximum yield of 144 mCi using enriched 61Ni and 72 mCi using enriched 60Ni. Specific activities (using enriched material) ranged from 80 to 300 mCi/mg Cu for 60Cu and from 20 to 81 mCi/mg Cu for 61Cu. Bombardments of natural Ni targets were performed using both protons and deuterons. Yields and radioisotopic impurities were determined and compared with that for enriched materials. 60Cu was used to radiolabel diacetyl-bis(N4methylthiosemicarbazone), ATSM. 60Cu-ATSM was injected into rats that had an occluded left anterior descending coronary artery. Uptake of 60Cu-ATSM in the hypoxic region of the heart was visualized clearly using autoradiography. In addition, 60Cu-ATSM was injected into dogs and excellent images of the heart and heart walls were obtained using positron emission tomography (PET). 61Cu was labeled to 1,4,8,11tetraazacyclotetradecane-N,N’,N”,N”’-tetraacetic acid-octreotide (TETA-octreotide) and the PET images of tumor-bearing rats were obtained up to 2 h postinjection. After decay of the 61Cu, the same rat was injected with 64Cu-TETA-octreotide and the images were compared. The tumor images obtained using 61Cu were found to be superior to those using 64Cu as predicted based on the larger abundance of positrons emitted by 61 Cu vs. 64Cu. NUCL MED BIOL 26;4:351–358, 1999. © 1999 Elsevier Science Inc. All rights reserved. KEY WORDS. Cu-60, Cu-61, Cu-64, Production, PET
INTRODUCTION 62
67
64
Cu, Cu, and Cu are copper radionuclides already being used for several nuclear medicine applications. 62Cu (t1/2 5 9.76 min) is generator produced and has been used to label agents to quantify both blood flow and hypoxia (5– 8, 19). A commercial version of the generator is now being investigated (9). The short half-life (9 h) of the 62Zn parent limits the potential usefulness of this nuclide. 67 Cu is a longer-lived isotope (t1/2 5 61.9 h), which is produced in high yield using the high energy proton beam at Brookhaven National Laboratories (BLIP) Facility and Los Alamos National Laboratory. The need for these high energy physics facilities allows only limited availability of this radionuclide. 64Cu has an intermediate half-life of 12.7 h and has applications both for imaging and therapy. Previously we reported the high yield production of 64Cu using a target system designed specifically for low energy, biomedical cyclotrons (13). Address correspondence to: Michael J. Welch, Ph.D., Mallinckrodt Institute of Radiology, Washington University School of Medicine, 510 S. Kingshighway Blvd., St. Louis, MO 63110, USA; e-mail: welch@mirlink. wustl.edu. Received 15 August 1998. Accepted 20 November 1998.
We are interested in developing positron emission tomography (PET) radiopharmaceuticals labeled with other positron-emitting Cu radionuclides (see Table 1). 60Cu has a half-life of 23.7 min and decays 93% of the time by positron emission. 61Cu has a longer half-life (3.32 h) and 60% of its emissions are by positron decay. We are particularly interested in preparing agents for imaging hypoxic tissue in tumor, brain, and heart, as well as developing techniques for labeling peptides and proteins. In this paper, we demonstrate the utility of these isotopes in the imaging of tumors and hypoxic tissues at times ranging from tens of minutes to a few hours. If a single target and production system can be used to produce these nuclides as well as 64Cu, copper nuclides would be available for a wide variety of biological studies. PET studies using compounds labeled with Cu-60 may be possible, depending on the type of quantitative information necessary. Cu-60 emits prompt gamma rays in cascade with each positron in the 1–2-MeV range that are virtually unaffected by the typical lead and lead/tungsten shielding in the PET gantry. These gammas or their secondary radiations contribute substantial noise to the data by means of random coincidences. The thin tungsten septa in commercially available PET scanners are normally effective at controlling the randoms fraction by limiting the solid angle seen by each detector. With this shielding, virtually invisible to the high-
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TABLE 1. Positron-Emitting Cu Radionuclides Nuclide
Half-life
Decay mode
Major gamma (keV)
Emissions (%)
60
23.7 min
93% b1 7% EC
61
3.32 h
62
0.16 h
80 52 15 12 10.7 0.15
64
12.7 h
60% b1 40% EC 98% b1 2% EC 19% b1, 43% EC, 38% b2
1333 1760 850 284 656 875 1345
0.47
Cu Cu Cu Cu
b1 3.92 3.00 2.00 1.22
max
MeV, 6% MeV, 18% MeV, 69% MeV
2.91 MeV 0.656 MeV
Data from: Table of Isotopes (9a).
energy gammas, any activity inside and within a few centimeters of the axial field-of-view will flood all detectors in the tomograph with single photon events. This action has the dual effect of adding noise via increased random coincidences, and reducing good signal via increased deadtime. Among the casualties of the higher-energy gammas is the deadtime correction. The calculation of an accurate correction factor for the data depends on an expected ratio of single events to coincident events. Phantom studies have been carried out to determine the magnitude of the effect of the prompt gamma emissions.
to 64,61,60Cu and the percent complexation monitored by radio-thin layer chromatography (radio-TLC) (Bioscan, Washington, DC). The samples were spotted on silica plates and the plates developed using 1:1 MeOH:10% NH4OAc. Cu acetate remained at the origin whereas complexed copper in the form of Cu(TETA)2- migrated with Rf 5 0.42. The minimum TETA concentration for which 100% labeling occurred was assumed to be equal to the concentration of Cu(II) present.
Radiochemical Synthesis of MATERIALS AND METHODS
Nickel Targets Preparations High purity reagents used in the electroplating and separation studies were the same as those described previously for the production of 64Cu (13). Isotopically enriched 60Ni (99.59%) and 61Ni (99.44%) were purchased from Trace Sciences International (Richmond Hills, Ontario, Canada). Natural nickel (99.99% purity) was purchased from Aldrich Chemical Company (Milwaukee, WI). The electroplating procedure was an adaptation of the methodology reported by Piel and co-workers (15). Briefly, appropriate quantities of nickel metal were dissolved in 6 N HNO3 and evaporated to dryness. The residue was treated with H2SO4, diluted with deionized water, and evaporated to almost dryness. The residue was cooled and dissolved with deionized water. The pH was adjusted to 9 with concentrated ammonium hydroxide and ammonium sulfate electrolyte was added. The solution was quantitatively transferred to an electrolytic cell in which the anode was a graphite rod and the cathode was a gold disk (0.06250 thick 3 0.750 diameter). The cells were typically operated at 2.4 –2.6 V and at currents between 10 and 25 mA. Electroplating was accomplished in 12–24 h. The diameter of the Ni targets were 0.5 cm unless otherwise indicated.
61/64
Cu-TETA-Octreotide
Octreotide was purchased from DePaul Pharmaceutical (Bridgeton, MO). TETA z 4HCl z 4H2O was purchased from Aldrich. TETA-octreotide was prepared and radiolabeled with copper as described previously (1). For the labeling, 7–15 mCi of 64Cu or 61 Cu were labeled to 3 mg of TETA-octreotide in 0.1 M ammonium acetate buffer, pH 5.5 by a 45-min room temperature incubation. Samples were purified by a C-18 SepPakt to remove uncomplexed 64/61 Cu-acetate. Radiochemical purity was assessed by radio-TLC using reversed-phase C-18 plates developed in 70:30 methanol:5% NH4OAc. Cu-TETA-octreotide migrates with Rf 5 0.5. Radiochemical purity of the labeled complexes was typically above 97%. Approximately 300 mCi (0.2 mg) of the Cu-TETA-octreotide radioconjugates were injected into the rats.
Isotope Production and Analysis Targets were irradiated in the previously described holder (13) using approximately 14.7 MeV protons and 8.1 MeV deuterons on a Cyclotron Corporation CS-15 cyclotron at Washington University. Isotope yield measurements were determined using either a calibrated Ge detector (Canberra model 1510, Meriden, CT) or a radioisotope dose calibrator (Capintec CRC-10, Pittsburgh, PA) in combination with the Ge detector. The Ge detector was used to detect radionuclidic impurities. Specific activities were determined as described previously for 64Cu by titration of 64Cu-acetate with the macrocycle TETA (13). Briefly, aliquots of TETA were added
FIG. 1. Structure of Cu-diacetyl-bis(N 4-methylthiosemicarbazone) (Cu-ATSM).
Production and Application of Cu-60 and Cu-61
353
TABLE 2. Yields of Copper Isotopes Using 991% Enriched Materials
Isotope
Nuclear Reaction
Target thickness (microns)
Maximum EOB yield (mCi)
Typical EOB yield (mCi)
60
60
225
865
450
61
61
61
60
118 122 215
144 72 921
100 60 200
Cu
Cu Cu 64 Cu
Ni(p,n)60Cu
Ni(p,n)61Cu Ni(d,n)61Cu 64 Ni(p,n)64Cu
Yield (mCi/mA z h)
Major isotopic impurities 0.05% 61Cu 0.025% 57Co 0.04% 58Co 0.04% 58Co 0.012% 55Co
58 7.6 2.44 8.0
EOB 5 end of bombardment.
TABLE 3. Natural Ni Bombardment: 14.7 MeV Protons
Target thickness (microns)
EOBsat yield Cu-60 (mCi/mA)
116
10.42 (EOB yield 5 0.875 mCi/mA)
116b
7.86 (EOB yield 5 6.5 mCi/mA)
a
a b
Radionuclidic impurities per 10 mCi Cu-60
% Radionuclidic impurities (relative to Cu-60) EOB
EOB
45 min post-EOB
Co-55 5 0.65
Co-55 5 0.065 mCi
Co-55 5 0.235 mCi
Cu-61 5 0.60 Co-55 5 1.0
Cu-61 5 0.060 mCi Co-55 5 100 mCi
Cu-61 5 0.192 mCi Co-55 5 361.8 mCi
Co-57 5 5.3 E-3 Co-58 5 5.2 E-4 Ni-57 5 6.7 E-3 Cu-64 5 0.38 Cu-61 5 1.12
Co-57 5 0.53 mCi Co-58 5 0.052 mCi Ni-57 5 0.67 mCi Cu-64 5 38 mCi Cu-61 5 112 mCi
Co-57 5 1.975 mCi Co-58 5 0.1937 mCi Ni-57 5 2.462 mCi Cu-64 5 135.92 mCi Cu-61 5 356.9 mCi
Sample bombarded at 4 mA for 3 min, 0.6 cm diameter target. Sample bombarded at 20 mA (avg current) for 1 h.
TABLE 4. Radionuclidic Impurities per 10 mCi
60
Cu Produced Using 991% Enriched
60
Ni
Radionuclidic impurities per 10 mCi Cu-60
EOBsat yield Cu-60 (mCi/mA) 45
EOB
45 min post-EOB
100 min post-EOB
Co-55 5 3.6 E-5 mCi Co-57 5 0.036 mCi Cu-61 5 5 mCi
Co-55 5 0.13 mCi Co-57 5 2.24 mCi Cu-61 5 15.93 mCi
Co-55 5 0.627 mCi Co-57 5 11.16 mCi Cu-61 5 65.6 mCi
EOB 5 end of bombardment. Conditions included using a 125-mm thick target, 14.7 MeV proton beam, ;15 mA avg. current, 44-min bombardment.
TABLE 5.
60
Cu production (60Ni(p,n)60Cu) Using 99.59 Enriched
60
Ni EOBsat yield
Target thickness (microns) 68.1 54.36 129.33 115.02 125.32
Production parameters (EOB activity, irradiation time, average beam current) 152 178 613 309 494
mCi, mCi, mCi, mCi, mCi,
44 72 49 20 44
min, min, min, min, min,
12.68 mA 8.5 mA 15.3 mA 14.4 mA 14.86 mA
EOB 5 end of bombardment prior to column separation. Production parameters 5 14.7 MeV protons.
(mCi) Before column separation
(mCi/mA) Before column separation
(mCi) After separation
(mCi/mA) After separation
210 203 805 697 683
16.56 23.9 52.6 48.4 45.93
201 256 458 688 456
15.7 30.1 30.0 47.8 30.67
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TABLE 6. Natural Ni Bombardment: 8.1 MeV Deuterons Target thickness (microns)
EOB yield Cu-61 (mCi/mA z h)
% Radionuclidic impurities (relative to Cu-61) EOB
0.91 0.29
Co-56 5 0.07 Co-56 5 0.08 Co-58 5 0.05 Ni-65 5 6.6
117a 130b
Radionuclidic impurities per 10mCi Cu-61 EOB Co-56 5 7 mCi Co-56 5 8.0 mCi Co-58 5 5.0 mCi Ni-65 5 660 mCi
EOB 5 end of bombardment. a Sample bombarded at 4 mA for 3 min, 0.6-cm diameter target. b Sample bombarded at 10 mA for 2 h. Sample was analyzed 3 h after EOB: short half-lived isotopes not analyzed for.
Preparation of
60
Cu-ATSM
H2ATSM was synthesized as described by Petering and co-workers (14). All chemicals were purchased from Aldrich, unless stated otherwise. Briefly, 4-methyl-3-thiosemicarbazide (97%) was dissolved in 5% acetic acid (99.99%) at 50 – 60°C with stirring. 2,3-Butanedione (97%) was taken up in water (distilled, deionized, .18 MV resistivity) and added to the 4-methyl-3-thiosemicarbazide solution dropwise. The hot solution was filtered through a course fritted-glass filter and a precipitate was collected. This solid was washed with H2O and then ethanol and dried at 75°C overnight. The solid was purified by suspension in hot 80% acetic acid under reflux. The mixture was again filtered hot and the undissolved material collected and dried overnight at 75°C. 60Cu was used to radiolabel ATSM using methods identical to Fujibayashi and co-workers (4).
Phantom Studies To evaluate the quantitative accuracy of imaging Cu-60 with PET, a phantom study was performed where the measured activity was compared with the true activity. A cylinder was filled with a dilute solution of 60Cu and scanned over several half-lives using a Siemens ECAT Exact PET scanner (12). For comparison, a similar experiment was carried out utilizing fluorine-18.
Imaging Studies All animal studies were performed in compliance with guidelines set forth by the Washington University Animal Studies Committee. Autoradiographic images of tissue slices were obtained utilizing an InstantImagert electronic autoradiography system (Packard Instrument Company (Meriden, CT). Images of rats were obtained utilizing the Siemens 953 B PET scanner at Washington University. PET images of the canine were obtained using PET Electronics SP3000E scanner at Washington University. Radiopharmaceuticals used for all imaging studies were prepared using isotopes produced from enriched target materials.
Tissue-Tek embedding medium (Miles Inc., Elkhart, IN), and 1-mm slices cut. The sections were placed on a grid and loaded onto the Packard InstantImager to obtain the 60Cu-ATSM images.
Rat Tumor Imaging Studies Lewis rats were implanted with somatostatin receptor-positive rat pancreatic tumors (CA20948) in the nape of the neck (10). The original tumor cells were purchased from the Tumor Bank at Biomeasure, Inc. (Hopkinton, MA). At the time of study, the tumor was approximately 1,340 mm3 and weighed 1.3 g. For the PET imaging study, the tumor-bearing female rat (170 g) was anesthetized and 253 mCi of 61Cu-TETA-octreotide were injected via intracardiac puncture. Scanning commenced immediately and PET images were collected for 1 h post injection. After the 61Cu had been allowed to decay (22 h later), 353 mCi of 64Cu-TETAOctreotide were injected via intracardiac puncture. Scanning began immediately and images of the entire animal were collected for 1 h.
PET Dog Study A 22.73-kg male mongrel dog was fasted 24 h prior to the procedure. The dog was anesthetized, placed in the SP3000E PET scanner, and administered 19.1 mCi of 11CO. PET scanning commenced immediately and images were collected for 5 min. Twenty minutes after the 11CO administration, 20 mCi of H15 2 O were injected intravenously and PET data were again collected for 5 min. Finally, 10 min after the H15 2 O injection, 1 mCi of 60 Cu-ATSM was injected intravenously and PET data collected for 10 min. TABLE 7. Production of 61Cu Using 99.59% Enriched60Ni and 99.44% Enriched 61Ni(d,n)61Cu and 61Ni(p,n)61Cu Nuclear Reactions Nuclear reaction 60
Rat Heart Model Studies 60
Cu-ATSM (structure shown in Fig. 1) was used to visualize hypoxia in an acute left anterior descending (LAD) coronary artery occluded heart model by ex vivo tissue slicing as described previously (4). Briefly, a male Wistar rat (360 g) was anesthetized, intubated, and ventilated with room air. Blood flow was occluded to the LAD coronary artery. Thirteen minutes postocclusion, 270 mCi 60CuATSM were injected through the femoral vein. The animal was sacrificed 10 min postinjection, the heart removed, frozen in
Ni(d,n)61Cu Ni(d,n)61Cu 60 Ni(d,n)61Cu 61 Ni(p,n)61Cu 61 Ni(p,n)61Cu 61 Ni(p,n)61Cu 61 Ni(p,n)61Cu 61 Ni(p,n)61Cu 60
Target thickness (microns)
EOB yield (mCi)
EOB yield (mCi/mA z h)
120a 122a 105 117 118 57.8 52.07 116
56 72 45 112.4 144 60.5 98.2 168
1.11 2.44 1.62 11.24 7.62 3.02 3.07 9.6
EOB 5 end of bombardment. 8.1 MeV deuteron beam, 14.7 MeV proton beam. a 0.6 cm diameter target.
Production and Application of Cu-60 and Cu-61
TABLE 8. Cost Comparison for Making 20 mCi Nuclear reaction 61
Yield (mCi/mA z h) 61
Ni(p,n) Cu Ni(d,n)61Cu 60 Ni(d,n)61Cu
7.62 2.44 0.91
60
355
61
Target material
Cu Cost of recycling lossa
# Cyclotron hours
Cyclotron cost (@ $245/hr)
Total cost
$165 $40 Negligible
0.13 0.82 2.20
$32 $201 $539
$197 $241 $539
61
Ni Ni nat Ni 60
Conditions included 14.7 MeV, 20-mA proton beam, 8.1 MeV, 10 mA deuteron beam. a Assuming 85% recovery of the enriched 61Ni. For the 60Ni, because of the relatively low cost, the material is not recovered.
RESULTS AND DISCUSSION 64
61
60
Cu, Cu, and Cu were produced in high yield using the target system described previously (13) . The maximim production yields of the copper radionuclides are shown in Table 2. 61Cu was produced by two nuclear reactions 61Ni(p,n)61Cu and 60Ni (d,n)61Cu. Although higher yields of 61Cu are produced using the (p,n) reaction, the target material for this reaction is approximately 25 times the cost of the target material for the d,n reaction ($55/mg Ni-61 vs $2/mg for Ni-60). None of the proton irradiation targets used in this work are “thick targets.” Of the targets described in Table 2, the ;220-mm thick targets degrade the 14.7-MeV proton beam by 5.2 MeV, whereas the ;120-mm thick target degrades the beam by approx. 2.5 MeV. The cross-sections of the 60Ni/61Ni(p,n) reactions do not decrease dramatically until 6 –7 MeV (15), so higher yields could be obtained at greater target expense with thicker targets (up to 350 mm). The ;120-mm thick target used for the deuteron irradiation is a “thick target.” The yields obtained are somewhat variable presumably due to minor alterations in the beam alignment, which affects the 0.5-cm and 0.6-cm diameter target used. Typical specific activities of these cyclotron-produced Cu isotopes as determined by TETA titrations are: 64Cu 5 40 –250 mCi/mg Cu, 61Cu 5 20 – 81 mCi/mg Cu, 60Cu 5 80 –300 mCi/mg Cu. Several authors have reported the production of 60Cu on biomedical cyclotrons using natural Ni. Martin (11) irradiated natural Ni (thick target) with 1 mA of 11-MeV protons for 23 min and reported the following end of bombardment (EOB) yields: 0.01 mCi 55 Co, 0.01 mCi 57Co, 5.0 mCi 60Cu, 0.07 mCi 61Cu, 3.8 mCi 62Cu, and 0.02 mCi 64Cu. Martin’s EOBsat (end of saturation bombardment) yield of Copper-60 was 10 mCi/mA (thick target yield). Similar yields can be obtained using a 14.7-MeV proton beam. Our EOBsat yields using a 14.7-MeV proton beam with natural nickel ;120 mm thick) for the 60Ni(p,n)60Cu reaction were 7.86 mCi /mA and 10.42 mCi/mA (see Table 3). The yields obtained were in agreement with those predicted by Piel et al. (15). Martin (11) described an electrochemical dissolution of the target material and separation of the Ni and Co from the copper by solvent extraction followed by anion exchange separation. Martin’s separation method removed 99% of the cobalt contaminants and TABLE 9. Cost Comparison for Making 50 mCi Nuclear reaction 61
Yield (mCi/mA z h) 61
Ni(p,n) Cu Ni(d,n)61Cu 60 Ni(d,n)61Cu 60
7.62 2.44 0.91
Target material 61
Ni Ni nat Ni 60
61
they were not considered in his dosimetry calculations with Cu-60PTSM. Using 99.59% enriched material for the 60Ni(p,n)60Cu reaction, we produced much less Co radioisotopic impurities than production from natural Ni, even with the higher energy protons used (see Table 4). This allowed for our separation procedure (anion exchange chromatography) to be less complicated. In addition, the 60 Cu yield using enriched material was four times higher than using nat Ni, which is important in certain circumstances in which minimal preparation time is required. Table 5 shows the 60Cu yields using enriched nickel for the 60Ni(p,n)60Cu nuclear reaction. In some cases, the EOBsat yields of the separated material were less than that of the whole target due to loss of 60Cu during the separation procedure. Production of 61Ni using natural Ni has also been reported (17). Thick natural nickel foils were bombarded with 6.0 MeV deuterons. A yield of 10 MBq/mA z h (0.27 mCi/mA z h) was reported for 61Cu and the only radioisotopic impurity reported was 62Cu. We observed low levels of radioisotopic impurities but with higher yields at '8.1 MeV deuterons (see Table 6). Zweit et al. (20) reported cross-section data for 60Ni(d,n)61Cu and 61Ni(d,2n)61Cu; however, values were given only for energies of 7.2–18.9 MeV. Szelecse´nyi et al. (16) irradiated enriched 61Nielectroplated targets with protons and measured the cross-sections of the reactions. Energy range for the production of the 61Cu was Ep 5 12–9 MeV, corresponding to a target thickness of 0.119 g/cm2 (134 mm). The theoretical yield for producing 61Cu with protons using 100% 61Ni is 17.5 mCi /mA z h. Our 61Cu yields from the proton bombardment of 112–118-mm thick 61Ni targets were in very good agreement with Szelecse´nyi (16) (Table 7). We conducted a cost comparison (using our production parameters) for the production of 61Cu using either 60Ni, 61Ni, or natNi as the target material. For producing 20 mCi of 61Cu, the cost of production using 60Ni or 61Ni was similar ($241 vs. $197 respectively). The advantage of using 60Ni (60Ni(d,n)61Cu) as the target material is its low cost. Process time is reduced if it is not necessary to recycle the enriched material. Production of 61Cu by deuteron irradiation of natNi was considerably more expensive ($539) due to the longer bombardment time required (Table 8). If larger quantities of 61Cu are required, i.e., 50 mCi, proton bombardment of 61Ni
Cu Cost of recycling lossa
# Cyclotron hours
Cyclotron cost (@ $245/hr)
Total cost
$165 $40 Negligible
0.33 2.05 5.5
$80 $502 $1346
$245 $542 $1346
Conditions included 14.7 MeV, 20 mA proton beam, 8.1 MeV, 10 mA deuteron beam. a Assuming 85% recovery of the enriched 61Ni. For the 60Ni, because of the relatively low cost, the material is not recovered.
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FIG. 2. Activity concentration values as measured by a cylindrical phantom. F-18 activity (left) is accurate over the 0 –1 mCi/mL measured here, and is within 5% out to 5 mCi/mL (not shown). A decaying phantom filled with Cu-60 activity in ionic solution is shown on the right. The deadtime correction restores data loss up to about 0.8 mCi/mL. Above this level, minor modifications to the deadtime model will be required to account for the effects of Cu-60’s high-energy gammas. (61Ni(p,n)61Cu) is preferable (cost 5 $245 versus $542 for 60 Ni(d,n)61Cu and $1,346 for deuteron bombardment of natNi) (Table 9). The radionuclide, 60Cu, decays by the emission of high energy positrons and high energy gamma rays. For this reason, an experiment was performed in which the measured activity was compared with the true activity using a cylindrical phantom. It was determined that the 60Cu activity could be quantified to a concentration of 0.8 mCi/mL. In human studies using 60Cu-ATSM, we anticipate an activity concentration averaging just 0.18 mCi/mL, and potentially ranging as high as 0.9 mCi/mL. Thus, except perhaps at the high end of this range, the effects shown in Figure 2 will be small to negligible. With minor adjustments to the deadtime calculation, calibrated for Cu-60 imaging, we will be able to obtain accurate parameter estimates over a much wider activity range if necessary. The data obtained for 60Cu were compared with that from 18F in Figure 2. With the standard deadtime correction provided with the PET system, a linear response was obtained for F-18, as expected, but not for Cu-60. The accuracy diverged at a concentration of 0.8 mCi/mL. This corresponds to approximately 4 mCi total activity in the field of view of the tomograph. In a PET study of myocardial perfusion, a relatively small bolus of Cu-60 activity, perhaps 10 mCi total, would be sufficient to exceed the linear range of the tomograph. In other studies, in which the activity can be infused over time, or in which the measurement of an input function is not needed, then a much larger dose could be used while still obtaining accurate results. This approximates to a uniform distribution of approximately 50 mCi in a human. It is highly unlikely that any radiopharmaceutical would require such a high concentration, so linear data can be obtained utilizing appropriate amounts of this nuclide. As anticipated, the 60Cu data are affected by the high energy gammas. 60 Cu has been evaluated in two imaging studies. Images were obtained ex vivo following administration of 60Cu-ATSM into a rat in which the LAD had been tied off (Fig. 3). The images obtained with 60Cu were similar to images obtained previously with 64Cu (4). In addition, 60Cu-ATSM, a hypoxic tissue marker, was injected into a dog under normoxic conditions. 60Cu-ATSM is selectively
trapped in hypoxic tissues but is rapidly washed out of normoxic cells (4). A high quality image of a dog heart was obtained showing some retention of 60Cu-ATSM in the walls of the dog heart (Fig. 4). The image showed a reconstructed midventricular PET image of the heart from a normal dog obtained 30 – 640 s after administration of 60 Cu-ATSM. The image to the left was not corrected for blood pool, whereas the image on the right was corrected for tracer remaining in the blood pool. Even though Cu-ATSM washes out from normoxic tissue (;20% retention), high quality images are obtained in vivo. The image was obtained using 1 mCi of tracer. Our preliminary dose estimates allow ;13 mCi of 60Cu-ATSM to be administered to a human. This method demonstrates the viability of using 60Cu-labeled radiopharmaceuticals for PET imaging. Studies on the uptake of Cu-ATSM in ischemic heart are in progress (18). A potential application of 61Cu is for labeling proteins and peptides for which maximum uptake occurs from 2 to 6 h post administration. An agent that falls into this category is 61CuTETA-octreotide. Comparison images with 64Cu-TETA-octreotide are shown in Figure 5. These images were obtained using 0.2 mg of 61/64 Cu-TETA-octreotide. The small amount of injected dose was chosen because it was significantly lower than the amount previously used to block the uptake of 64Cu-TETA-octreotide (250 mg) (2). This small amount is unlikely to cause any decrease in the uptake of Cu-64-TETA-octreotide. Higher quality images were obtained with 61Cu than with 64Cu, due to the higher positron yield (60% vs. 19%) and the shorter half-life. Because high quality human images (3) can be obtained 4 h postadministration of 64 Cu-TETA-octreotide, higher quality images could be obtained (at the same radiation dose) using 61Cu-TETA-octreotide.
CONCLUSION The cyclotron target described previously for the production of Cu can be used to produce 60Cu and 61Cu in high yield and at high specific activitiy. For certain biological applications, the shorter half-life of 60Cu and 61Cu may be preferable to the 12.7-h 64
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FIG. 3. Ex vivo imaging of a left anterior descending (LAD) coronary artery occluded heart model. At the top are photographic images of the heart slices. At the bottom are images obtained on the Packard InstantImager of the distribution of Cu-60-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-60-ATSM) in hypoxic tissue. half-life of 64Cu. Despite the high b1 energy and prompt gamma emission of 60Cu, high quality images are obtained both autoradiographically (using the Packard InstantImager) and in PET scans of 60 Cu-ATSM. For studies in which maximum contrast is obtained in less than 1 h, 60Cu will be preferred. If optimal uptake occurs in 1– 4 h, 61Cu will bepreferred.
This work was supported by research grants from the Department of Energy DE-FG02-87ER60512 (M.J.W.) and NIH CA64475 (C.J.A.). We thank Bill Margenau, Dave Ficke and Todd Perkins for assistance in isotope production, and Lynne A. Jones, Terry Sharpe and Lennis Lich for their contribution to the animal imaging studies.
FIG. 4. Reconstructed midventricular positron emission tomography (PET) images of the heart from a normal canine obtained 30 – 640 s after administration of Cu-60-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-60-ATSM). The raw image is shown to the left, and the image to the right represents the raw image after being corrected for tracer remaining in the blood pool.
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FIG. 5. Images of tumor-bearing Lewis rats injected with Cu-1,4,8,11-tetraazacyclotetradecane-N,N*,N(,N***-tetraacetic acid (TETA)-octreotide (octreotide is a somatostatin analog, used in imaging tumors that contain somatostatin receptors). The bottom images were obtained after 353 mCi of 61Cu -TETA-octreotide were injected (;3.6 E, 6 total counts per slice). Twenty-two hours later, 253 mCi of 64Cu-TETA-octreotide was injected into the same rat (;7.8 E, 5 total counts per slice) and positron emission tomography (PET) images were obtained again (top images).
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