Inorganica Chimica Acta 363 (2010) 1316–1319

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Inorganica Chimica Acta

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An automated method for regular productions of copper-64 for PETradiopharmaceuticals

Paul Burke, Oksana Golovko, John C. Clark, Franklin I. Aigbirhio *

Wolfson Brain Imaging Centre, Department of Clinical Neurosciences, University of Cambridge, Box 65 Addenbrooke’s Hospital, Cambridge CB2 0QQ, United Kingdom

a r t i c l e i n f o

Article history:Received 30 June 2009Received in revised form 21 January 2010Accepted 23 January 2010Available online 4 February 2010

Dedicated to Jonathan R. Dilworth.

Keywords:Copper-64PETRadiopharmaceuticalsBis(thiosemicarbazones)

0020-1693/$ - see front matter � 2010 Elsevier B.V. Adoi:10.1016/j.ica.2010.01.038

* Corresponding author. Tel.: +44 (0)1223 331823;E-mail address: fia20@wbic.cam.ac.uk (F.I. Aigbirh

a b s t r a c t

To facilitate the development of PET radiopharmaceuticals labelled with the positron-emitting radioiso-tope copper-64 (t1/2 = 12.7 h) we have developed a fully automated method for its regular productions.Using the 64Ni(p,n)64Cu nuclear reaction applied on a 16.5 MeV PETtrace cyclotron the radioisotope isgenerated in good yields (up to 2 GBq at end-of-synthesis) within 4 h irradiations on nickel-64 (99.6%enrichment) plated onto a gold disk. Based on ion exchange chromatography an automated methodhas been devised for efficient extraction of the copper-64 in good radionuclide and chemical purity, withICP-OES analysis determining the concentration of the copper to be 0.14–1.5 ppm. The specific radioac-tivities of the copper-64 at end-of-synthesis were calculated to be 9.62–77 GB/lmol. The copper-64radioisotope obtained from this method was then applied to the radiosynthesis of the hypoxia markers,64Cu-ATSM and 64Cu-ATSE, which were obtained in good radiochemical yields of >95%.

� 2010 Elsevier B.V. All rights reserved.

1. Introduction

The molecular imaging technique of positron emission tomog-raphy (PET) has become a major technology for biomedical re-search, with tremendous potential for understanding normalfunction in man as well as abnormal and diseased states [1]. How-ever a major restriction for its progression arises from the posi-tron-emitting radiopharmaceuticals central to the technique. Inparticular, a lack of suitable radiopharmaceuticals for a wide rangeof biological targets and methods to prepare them simply and reli-ably at reduced cost. Due to the short half-lives of the most com-monly used radioisotopes, carbon-11 (t1/2 = 20.4 min) andfluorine-18 (t1/2 = 110 min) the preparations of their PET radio-pharmaceuticals, starting from very high radioactivities generatedfrom on-site cyclotrons, is highly demanding; requiring precisemanipulations, expensive automated apparatus and highly skilledpersonnel. Thus, there are requirements for PET probes whichcan be prepared more simply and reliable, with minimal automa-tion and can be used at sites without cyclotron.

These requirements have created an interest in developing andapplying the radioisotopes of copper [2,3], in particular copper-64[4], which has distinct advantages; its half-life of 12.7 h allows it tobe produced centrally and distributed widely, the radiosynthesis ofthe probes are generally simple, robust and amenable for automa-tion, while its low-energy b+ (0.655 MeV) enable high-resolution

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images to be derived, a particular advantage for small animal imag-ing. These features outweigh its disadvantages of a low b+ abun-dance (19.3%), which combined with its longer half-life can resultin poor dosimetry for clinical use, and the requirement of solid tar-gets for production.

This growing interest has driven the need for regular access tothe radioisotope, in particular to be produced from biomedicalcyclotrons which are present in major PET centres. For regular highradioactivity productions it is essential that the method of produc-tion is amenable to full automation to minimise radiation exposureto operators and it has the required reliability to underpin a PETimaging programme. For our radiotracer development programmea key need is copper-64 to be produced at high specific radioactiv-ities and with good radionuclidic and chemical purity to deriveradiopharmaceuticals that can be used for accurate delineation oflow population binding sites in vivo e.g. small animal imaging.

Described in this article is a fully automated method for theproduction of copper-64 for regular high yield production thatachieves these specifications.

2. Experimental

2.1. Material

Concentrated hydrochloric acid 33–36%, nitric acid 67–69%,(Romil-UpA�, Ultra Purity), 99.6% nickel-64 (Isoflex�), gold disk(99.99%, 20 mm diameter � 2 mm thickness, Goodfellows) AG�

1-X8 ion exchange resin, chloride form, 200–400 mesh (Bio-Rad),

P. Burke et al. / Inorganica Chimica Acta 363 (2010) 1316–1319 1317

trifluoroacetic acid, acetonitrile HPLC grade (Fisher Scientific),deionised 18-megohm water (Simplicity 185, Millipore) glasswareand other tools were pre-treated with 0.1 N nitric acid for a mini-mum of 2 h. Precursors for 64Cu-ASTM and 64Cu-ATSE were kindlysupplied by Prof. Jon R. Dilworth and Dr. Paul S. Donnelly of OxfordUniversity.

2.2. Plating solution

The nickel-64 powder (25–50 mg) was dissolved in concen-trated nitric acid (0.3 ml) and heated at 90 �C until evaporated todryness. The residue was then dissolved in concentrated HCl(1 ml) then heated (90 �C) to dryness. This procedure was repeateda further two times then the NiCl2 residue was dissolved in water(10 ml) producing a solution at pH 7.

2.3. Preparation of nickel target

A gold disk was cleaned by immersion in concentrated nitricacid, heated at 50 �C for 30 min, followed by rinsing in water(10 ml) until the rinse water is pH 7. The disk was oven dried,placed in a transport shuttle, which was then inserted into theelectro-plating cell (20 ml volume, Peek) opposite a platinum an-ode. The cell was then heated to 45 �C and connected to a volt-age/current regulated 0–5 V DC supply with the voltage set at4.5 V; the target disk forming the cathode. The plating solution(10 ml) was added to the cell and with continuous stirring 5 M HCl(0.1 ml) was added which resulted in an increase in the current to10 mA. The supply output current was adjusted to 5 mA at whichlevel the voltage fell to 3.8 V. During this plating process, over a12–24 h period depending on the amount of nickel, the current/voltage relationship was maintained by adding, drop-wise,amounts of 5 M HCl (0.1–0.2 ml). On completion the disk was re-moved, washed with water, oven dried and the plating weightdetermined.

Selector v

Pump

12 3

45

6V1

EtchCell

1 2 3

Cu64

Air ac

P1

GM1

9M 9M

Etchant Nickel rinse Co

HCL HCL HCL

Ion Exchange Column

Lead Pots

1ml vial copper fraction

GM tube in contact with tube monitors radiation in ectchant

Fig. 1. Schematic of the copper-64 processing rig installed in the production hot cell. Sdriven table for the collection vials. Also outlined are their connections to the main tran

2.4. Irradiation of the nickel-64 target

Copper-64 was produced on a GE PETtrace� cyclotron by the64Ni(p,n)64Cu nuclear reaction [5] with a incident proton beam en-ergy of 16.5 MeV, beam current of 25–30 lA and a run time of240 min. The target face was presented normal to the cyclotronbeam at 90�, forced against a gas seal and indirectly cooled onthe rear surface by forced contact with a water-cooled ramprobe.

2.5. Separation of copper from nickel and cobalt

Using a pneumatic process the irradiated Ni-64 target shuttle,was transferred from the cyclotron targetry system through a plas-tic piping conduit, to the copper-64 process module (Fig. 1) in-stalled in a lead-shielded ‘‘hot cell”. The shuttle was positionedsuch that the target is presented horizontally to the cell with itsplated area bounded by a Kalrez� sealing O-ring held in the cellbody. With semi-automated control of the processing system theprocedure to extract the copper-64 was then performed:

Etching: Using peristaltic pump (P1), 9 M HCl (1 ml) was circu-lated from Vial 1 through the etching cell (via V2), and back to Vial1. During this procedure the back face of the target disk was heatedto 90 �C, resulting in the digestion of the nickel/copper-64 plate bythe acid flow. Completion of the digestion process, over a period of10–20 min depending on the target weight, was determined by theincreased, then stable radioactivity level in Vial 1. This was assayedby a radioactivity detector placed next to Vial 1. The nickel/coppersolution was drawn back from etching cell and recirculation loopinto Vial 1 by reversal of P1 and the etching cell rinsed using0.5 ml 9 M HCL from Vial 2. The rinse was drawn back into Vial 1and the solution allowed to cool.

Ion exchange chromatography: The nickel/copper-64 solution(1.5 ml) was transferred with the use of the peristaltic pump (P1)from Vial 1 and loaded onto the ion exchange column (AG1-X8,5 � 13 mm, EX1). The solution was then allowed to elute (at an

alve

Nitrogen inlet

4

GM2

AG1-8X

Ni & Co

tuated table

2M 0.1M

balt eluatant Copper eluant

HCL

10ml vial

Shielded GM tube measuring activity in drips for ion exchange column

hown are the etching cell, the reagent vials, ion exchange column and the linearlysfer selection valve.

Fig. 2. A typical radiochromatogram obtained from radioactivity monitoring (GM2) of the eluate from the ion exchange column during elution with 2 M HCl, followed by0.1 M HCl. Shown are the times during which fractions F1 and F2 are collected.

1318 P. Burke et al. / Inorganica Chimica Acta 363 (2010) 1316–1319

average of 0.04 ml/min) through the column over a period of35 min and the eluate which contained nickel was collected in avials installed in a remotely controlled linear carrier carousel. Thiswas followed by a further elution of the column with 9 M HCl(2.0 ml) from Vial 3 to remove residual nickel, with the copper-64 still retained on the column. The column was then eluted with2 M HCl (0.5 ml) contained in Vial 4 and with radioactivity moni-toring of the eluate (Fig. 2) the initial radioactive fraction was col-lected. This fraction (F1) was later shown by ICP-OES analysis andby monitoring radioactivity decay times to contain a mixture ofnickel, cobalt-55 and copper-64. Finally the column was elutedwith 0.1 M HCl (0.3 ml) from Vial 5. With radioactivity monitoringof the eluate the main copper-64 fraction (F2) was then collected inapproximately 0.1–0.2 ml. The final radiochemical product was0.1–2 GBq (n = 30) end-of-synthesis.

2.6. Recovery of enriched nickel-64

The combined nickel fraction in 9 M HCL (2–4 ml) was loadedonto an ion exchange column (AG1-X8, 5 � 13 mm) and the eluatecollected. An additional 2 ml of 9 M HCL was then passed throughthe column and the eluate collected. The combined eluate containsthe required enriched nickel. The solution was evaporated to dry-ness with a combination of heating and nitrogen flow to leave aresidue, which was then dissolved in water (2 ml). This solutionwas evaporated to dryness to leave a residue and then dissolvedin water (10 ml) to produce a solution with pH 7.

2.7. Analysis of Cu-64 productions by ICP-OES

With a combination of heating and a nitrogen flow the 64CuCl2

samples (0.1 ml) were evaporated to dryness and re-dissolved in0.1 M HNO3 (1 ml). The samples were then analysed for Cu, Ni,Co and Zn content on a Varian Vista Pro ICP-OES at the Universityof Cambridge Department of Earth Sciences with blank samples of0.1 M HNO3 utilised as control samples.

2.8. Radiosynthesis of copper-64 complexes

64Cu(CH3CO2)2 was prepared by neutralising a solution of64CuCl2 in 0.1 M HCl (20 ll, 20–50 MBq) with 0.1 M Sodium acetate(pH 5.5, 2.0 ml). To 64Cu(CH3CO2)2 (100 ll) were added water(400 ll) and the ligand in dimethyl sulfoxide (50 ll, 1 mg/1 ml).This was then stirred at RT for 10 min then subjected to analysisby radio-HPLC.

2.9. Radio-HPLC

Analysis was performed on a system comprising an Agilent1100 series HPLC system consisting of a binary pump and a vari-able wavelength detector (Agilent Technologies, Waldbronn, Ger-many). This was coupled with a Flow-count radio-HPLC unit witha sodium iodide/PMT high energy gamma detector (Bioscan) tomonitor the effluent radioactivity. Chromatographic method con-sisted of Primesphere C18-HC, 250 � 4.60 mm 5 micron column(Phenomenex) that was eluted at 1 ml/min for 30 min with a sol-vent gradient of water/acetonitrile/trifluoroacetic acid; initially95/5/0.1 (v/v), then 65/35/0.1 at 20 min and finally 95/5/0.1 at25 min. UV monitoring was at 254 nm. Data was acquired with aLaura Lite 3 radio chromatography software (Lablogic SystemsLTD, Sheffield, Great Britain).

3. Results and discussion

Over recent years there have been several publications describ-ing methods for the production of copper-64 [6–9] from biomedi-cal cyclotrons, with key variations centred on obtaining aneffective separation of the copper-64 from the nickel startingmaterial and other impurities e.g. cobalt-55. However thesemethods are generally not appropriate for regular automated pro-ductions of this isotope; often requiring precise manual manipula-tion in various aspects of the production, in particular the strippingof the target material and the subsequent chemical separation. Indesigning our process our aim was to refine these various stagesto produce a method with distinct advantages for routine produc-tion, most importantly being amenable for complete automation toreduce any radiation exposure to the operator, while achievinggood extraction of the copper-64 radioisotope.

One refinement was the nickel plating solution we used differedfrom previous methods [7] in that an all nickel chloride solution isused. The advantage this bestows is that at the end of the overallprocess the nickel-64, which must be recovered for re-use due tohigh cost, is already in the chloride form, enabling a more rapidand simpler preparation of a new plating solution. Due to the highincident proton beam energy of the PETtrace cyclotron, of approx-imately 16.5 MeV, to achieve high radiochemical yields of copper-64 within a short irradiation time, we produced targets with anickel plate thickness of 33–110 lm (mean 58.55 lm). This en-abled us to obtain yields of 0.1–2 GBq at end-of-synthesis(n = 30) with irradiation of 4 h at beam currents of 25–30 lA, withthe radiochemical yield dependent on the target loading (15–

N NN

S

N

S NHRRHN

Me Me

64Cu R = Me, ATSMEt, ATSE

Fig. 3. Copper-64 bis(thiosemicarbazones).

P. Burke et al. / Inorganica Chimica Acta 363 (2010) 1316–1319 1319

50 mg of nickel-64, mean of 26 mg). Our ‘‘standard” productionsbased on 26 mg of nickel, produces approximately 1 GBq of radio-chemical yield (RCY) at end-of-synthesis with target yields of18 Mq MBq/lA*h. These are generally lower radiochemical yieldsat end-of-synthesis than reported by other groups, e.g. McCarthyet al. [7] RCY of 5.55–25.05 GBq (85.1–185 MBq/lA*h) and Obataet al. [8] RCY of 2.22–24.3 GBq (22.2–111 MBq/lA*h). However di-rect comparisons are difficult due to different irradiation condi-tions been used.

The key challenge in the production of copper-64 is the separa-tion of the radioisotope from the much larger mass of enrichednickel, which needs to be recovered for further re-use, and fromthe other metal and radionuclide products e.g. cobalt-55, whichcan be generated by the 58Ni(p, a)55Co process, due to presenceof low amounts of nickel-58 in the enriched nickel-64. Previousmethods [6–9] also based on the use of ion exchange chromatogra-phy vary in the elution of the various concentration of HCl or HCl-ethanol mixtures and water for selectivity elution of the metalions. However a common aspect are the large volumes used forthese elution’s (8–25 ml) with the final copper-64 product ob-tained in volumes of 10–15 ml of water or 0.1 M HCl. In contrastwe are able to perform this elution and separation with signifi-cantly smaller volumes of solutions (1–2 ml). The target is posi-tioned in the process system (Fig. 1), which is effectively a closedloop, through which the small volume of acid (1.5 ml) can be re-circulated to effectively dissolve and remove the nickel/copper-64 material. The small liquid volumes are enabled by the use of amuch smaller ion exchange resin bed volume of 0.2 ml for the sep-aration process. The short resin column is capped with a Teflon fritthat ensure the resin remains wet between elution stages, and alsogoverns the elution flow rate through the column. On loading theentire radioactive product is retained in the first 1–2 mm of the re-sin with four resin bed volumes of 9 M HCl been sufficient to re-move the main bulk of the nickel from the column. With thesmaller resin bed the final copper-64 fraction is eluted in only100–200 ll volume of 0.1 M HCl. This saves on the need for a sig-nificant amount of evaporation procedures to concentrate or re-move excess HCl solution, enabling shorter processing times aswell as been more amenable to full automation. The overall pro-cess from end-of-bombardment to final copper-64 product is lessthan 90 min, which would enable it to be applied to other copperPET radioisotopes of interest which have shorter half-life’s e.g. cop-per-61 (t1/2 = 3.32 h, b+ 1.22 MeV, 60%).

Our analysis of the copper-64 fractions by ICP-OES (n = 7) con-firmed the chemical purity obtained by this method comparedfavourable to those achieved by other groups in particular in lownickel content, with no detectable amounts of cobalt (Table 1).With careful control of the quality of the reagents and materials,including the ion exchange resin, we were able to minimise theamount of stable copper they introduce into the copper-64 prod-uct, with the major source of stable copper considered to arisefrom the cyclotron targetry, which contain copper pipes for the he-lium cooling system. The resulting amounts of stable copper in thecopper-64 product solutions samples were determined to be in thelow ppm levels (0.14–1.5 ppm). As a result our specific radioactiv-ities based on the total amounts of stable copper determined by

Table 1The total amount of stable metal impurities present in a typical copper-64 solutionbatch produced by our method compared to published values.

Cu (lg) Ni (lg) Fe (lg) Co (lg) Zn (lg)

WBIC preparation 3.1 1.18 3.52 n/d 0.96Avila-Rodriguez et al. [9] 3.1 0.88 1.19 0.01McCarthy et al. [7] 0.60–

2.9712.4 11.4 0.17 1.5

n/d = Not detected.

ICP-OES (n = 7) varied between 9.62 and 77 GB/lmol (RCY 0.5–2 GBq). Again, these values are lower than those obtained by othergroups (55–185 GB/lmol [8] and 222–734 GBq/lmol [7]) howeverthese were obtained through productions of higher activities.Based on our stable copper content comparable specific radioactiv-ities could be obtained with higher activity productions. Howeverit must be noted that the effective specific radioactivity for a cop-per radiotracer maybe less due to a chelate binding to other metalimpurities in addition to the copper e.g. the nickel and zinc. Basedon the average amount of impurities in our copper-64 samples asdetermined by ICP-OES this would reduce the specific radioactivi-ties from 0.15–1.2 GBq/lg to 0.089–0.71 GBq/lg.

Additional characterisation of the radioisotope product was itssuccessful application to the radiosynthesis of the copper-64bis(thiosemicarbazones), 64Cu-ATSM and 64Cu-ATSE (Fig. 3), inhigh radiochemical yields of >95% (as determined by radio-HPLC).These PET radiopharmaceuticals are of significant interest for thedelineation of hypoxia [10] by PET in cancer and various neurolog-ical pathologies.

We have attained our key objectives of developing a process forproducing copper-64 that is automated throughout the whole pro-cedure from transfer of the nickel plated gold disk from the cyclo-tron vault to obtaining the purified copper-64 fraction, requiringno manual manipulations and therefore no radioactive dose tothe operators. Importantly good yields of the radioisotope couldbe produced with the requisite good radiochemical and chemicalpurities. This method is now being regularly applied for our andcollaborators programmes in developing and applying copper-64PET radiopharmaceuticals [11–14].

Acknowledgements

The authors would like to thank their colleagues in the WBICChemistry group and Mr Jason Day of Dept of Earth Sciences forthe ICP-OES analysis.

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