Preparation of [61Cu]pyruvaldehyde-bis (N4 ...

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Radiochim. Acta 94, 113–117 (2006) / DOI 10.1524/ract.2006.94.2.113 © by Oldenbourg Wissenschaftsverlag, München

Preparation of [61Cu]pyruvaldehyde-bis (N4-methylthiosemicarbazone) complex as a possible PET radiopharmaceutical By A. R. Jalilian1 , ∗, P. Rowshanfarzad1 and M. Sabet2 1 2

Cyclotron and Nuclear Medicine Department, Nuclear Research Center for Agriculture and Medicine, Atomic Energy Organization of Iran, Karaj, P.O. Box: 31485-498, Iran SSDL and Health Physics Department, Nuclear Research Center for Agriculture and Medicine, Atomic Energy Organization of Iran, Karaj, P.O. Box: 31485-498, Iran

(Received March 22, 2005; accepted in revised form August 18, 2005)

Copper-61 / PTSM / Radiopharmaceutical / Cyclotron / PET Summary. Copper-61 (T1/2 = 3.33 h) produced via the nat Zn( p, x)61 Cu nuclear reaction using natural zinc target, was separated from the irradiated target material by a two-step method developed in our laboratory and was used for the preparation of [61 Cu]-pyruvaldehydebis(N 4 -methylthiosemicarbazone) ([61 Cu]-PTSM) using an in house-made PTSM ligand. An electroplated natural zinc layer on a gold-coated copper backing (zinc and gold layer thicknesses were 80 and 50 µm respectively) was irradiated with 22 MeV protons (22–12 MeV on the target). 61 Cu was separated by a two-step chromatography method using a cation and an anion exchange column which gave satisfactory results. After 180 µA irradiation of the target for 3.2 hours, about 6.006 Ci of 61 Cu2+ was obtained with a radiochemical separation yield of more than 95% and a radionuclidic purity of more than 99% (60 Cu as impurity). Colorimetric methods showed that traces of chemical impurities in the product were below the accepted limits. The solution of [61 Cu]-PTSM was prepared with a radiochemical yield of higher than 80%, radiochemical purity of better than 98% and specific activity of about 246 Ci/mmol.

1. Introduction Copper offers a unique selection of radioisotopes (60 Cu, 61 Cu, 62 Cu, 64 Cu, and 67 Cu) with half-lives ranging from 9.8 minutes to 61.9 hours, suitable for imaging and/or radiotherapy [1]. The most commonly used copper radioisotopes 62 Cu and 64 Cu, provide very good physical properties for therapeutic and/or diagnostic purposes. Copper-62 is usually provided by 62 Zn/62 Cu generator, but has a short half life of 8 minutes [2–4]. Copper-64 is used for diagnostic imaging as well as therapy [5–7]. Copper-61 is a positron emitter (T1/2 = 3.33 h, β + : 62%, E.C.: 38%). Few production methods of copper-61 have been reported for radiolabeling of biomolecules and other applications [8–11]. *Author for correspondence (E-mail: [email protected]).

Interestingly, it has been shown that the tomographic images obtained using 61 Cu were superior to those using 64 Cu, based on the larger abundance of positrons emitted by 61 Cu compared with 64 Cu (62% vs. 18%) [9]. As an example of copper-labelled radiopharmaceutical, Cu-PTSM has been used as a PET perfusion tracer for tumor blood flow and cerebral/myocardial perfusion [12–14]. Based on the interesting properties of copper-61 and the possibility of its production via nat Zn( p, x)61 Cu, we were interested in the production and purification of this radionuclide and its ultimate use in radiolabeling of PTSM as a possible PET tracer.

2. Experimental 2.1 Materials Production of 61 Cu was performed at the NRCAM1 30 MeV cyclotron (Cyclone-30, IBA). Natural zinc chloride with a high purity of more than 98% was provided commercially (Merck chemical company, Darmstadt, Germany). Other chemicals were purchased from Aldrich Chemical Company (Milwaukee, USA). All exchange resins were provided commercially (Bio-Rad Laboratories, Canada). 1 H-NMR spectrum was obtained on a Bruker FT-80 (80 MHz) instrument with tetramethylsilane as the internal standard. Infrared spectrum was taken on a Perkin-Elmer 781 (KBr disks). Mass spectrum was recorded using a Finnigan Mat TSQ-70 spectrometer. Radio thin layer chromatography (RTLC) was performed on polymer-backed silica gel (F 1500/LS 254, 20 × 20 cm, TLC Ready Foil, Schleicher & Schuell® , Germany). Ethyl acetate and normal saline used for labeling were of high purity. Radio-chromatography was performed by counting 5 mm-slices of polymer-backed silica gel paper using a Canberra™ high purity germanium (HPGe) detector (model GC1020-7500SL). Radionuclidic purity was checked by the same detector. All calculations and RTLC counting were based on 283 keV peak. 1

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2.2 Selection of the production parameters

2.5 Quality control of the product

In this research, 64 Zn( p, α)61 Cu was selected as the best nuclear reaction for the production of 61 Cu, but natural zinc was used as the target material, due to the small amount of radioactive copper impurities produced, and also for costeffectiveness. Other impurities such as gallium and zinc radioisotopes were also produced as a result of proton bombardment of natural zinc, but they could be easily removed in the radiochemical separation process. The optimum proton beam energy was calculated and types of possible impurities were predicted by ALICE nuclear code [15]. It must be noted that the main nuclear reaction for the production of 61 Cu as a result of nat Zn bombardment, was actually limited to 64 Zn( p, α)61 Cu, due to the isotopic abundance of 64 Zn in natural zinc (48.6%).

2.5.1 Control of radionuclidic purity

2.3 Targetry Target thickness was determined using SRIM nuclear code [16]. The target had to decrease the energy of the incident proton beam (22 MeV) to 12 MeV according to the results of ALICE nuclear code. According to the necessary considerations, an 80 micron natural zinc layer was electrodeposited on a gold coated (50 microns) copper backing. The target was cooled by a flow of 18 ◦ C distilled water with a rate of 50 lit/min through the backing grooves. For the target preparation process, nat ZnCl2 was dissolved in 0.05 M HCl to prepare a zinc cation-containing solution. The mass of zinc ions in the cell had to be twice that of the electrodeposited layer. Hydrazine hydrate (2 ml, 80%) was added as a reducing agent. The electrodeposition was performed at pH = 2.5–3, with a cell volume of 480 ml and a current density of 35 mA/cm2 and platinum was used as the anode material to give an 80 micron zinc layer on the copper backing after 50 minutes.

2.4 Radiochemical separation After target irradiation, chemical separation was carried out in no-carrier-added form. The method used in this research was a two step ion exchange chromatography method. In the first step, gallium was separated from zinc and copper. For this purpose, the irradiated target was dissolved by 10 M HCl (15 ml, H2 O2 added) and the solution was passed through a cation exchange resin (Dowex 50 W×8, H+ form, 200–400 mesh, h: 10 cm, I.D.: 1 cm) which had been preconditioned by passing 25 ml of 9 M HCl. The column was then washed by 25 ml of 9 M HCl with a rate of 1 ml/min to remove copper and zinc ion contents and gallium remained on the column [17]. In the second step, copper was separated from zinc ions. The solution obtained from the cation exchange column was heated almost to dryness and the remainder was dissolved in 6 M HCl. This solution was loaded on an anion exchange chromatography AG 1 × 8 column (100–200 mesh, Cl− form, 25 cm high, 1.5 cm I.D.) preconditioned with 25 ml of distilled water and 100 ml of 6 M HCl. The loading rate was 2 ml/min. Copper (61 Cu) was washed out of the column by passing 50 ml of 2 M HCl with a rate of 2 ml/min. After the quality control process, the resulting [61 Cu]CuCl2 solution was ready for the labeling step [18].

The gamma spectroscopy of the final sample was carried out by a HPGe detector coupled with a Canberra™ multichannel analyzer. The peaks were observed and the area under curve was counted for 1000 seconds. 2.5.2 Chemical purity control The formation of colored dithizone-zinc complex was measured using visible spectroscopic assay to determine Zn cation concentrations according [19] using dithizone organic reagent (0.002% in CCl4 ). Briefly, the presence of pinkish color of zinc-dithizone complex was checked for the test samples, 1, 5, 10 ppm standards and finally a blank solution (1 ml each). The color of the test tube had been less than that of the standard. The amount of gold cation was checked in the final solution using color formation of acidic rhodamine B reagent reacting with gold dilutions based on a previously reported colorimetric method [20].

2.6 Preparation of pyruvaldehyde-bis(N4 -methylthiosemicarbazone) H2 PTSM was prepared according the reported method for the production of thiosemicarbazones starting N 4 methylthiosemicarbazide and pyruvaldehyde which was consistent with previous report [21] (60%) m.p. 241–243 ◦ C. 1 H NMR (D6 -DMSO) δ (ppm) 11.74 (s, 1H, NH−N2 ), 10.33 (s, 1H, NH−N2 ), 9.43 (m, 2H, NH−N4 ), 7.68 (s, 1H, H−C=N), 3.04 (s, 3H, CH3 −N4 ), 2.98 (s, 3H, CH3 −N4 ), 2.18 (s, 3H, CH3 −C=N). IR (KBr) λmax 3208, 3132 (N−H), 1429 (C=N), 1111 (C=S). Mass (electrospray) 246.1 (14%) M+ , calculated; 246.

2.7 Preparation of [61 Cu]pyruvaldehyde-bis(N4 -methylthiosemicarbazone) s Preparation of [61 Cu]pyruvaldehyde-bis(N 4 -methylthiosemicarbazone) was accomplished according to the former works with slight modifications [22]. Briefly [61 Cu]CuOAc was reacted with a mixture of H2 PTSM followed by C18 Sep-Pak column purification. The tracer was finally washed by ethanol fractions and formulated by the addition of normal saline followed by passing the solution through a 0.22 µm filter. The radiochemical purity was checked by RTLC.

2.8 Stability of [61 Cu]PTSM complex in the final product Stability tests were based on previous studies performed for radiolabeled copper complexes [23]. A sample of [61 Cu]PTSM (5 mCi) was kept at room temperature for 5 hours while checked by RTLC every half an hour. A micropipet sample (5 µl) was taken from the shaking mixture and the ratio of free radiocopper to [61 Cu]PTSM was checked by radio thin layer chromatography (eluent: dry ethyl acetate).

Preparation of [61 Cu]pyruvaldehyde-bis (N 4 -methylthiosemicarbazone) complex as a possible PET radiopharmaceutical

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3. Results and discussion Various nuclear reactions have been suggested for the production of 61 Cu. Different research studies have been reported using 61 Ni( p, n)61 Cu [24, 25], 60 Ni(d, n)61 Cu [24], 63 Cu( p, t)61 Cu and 65 Cu( p, t2n)61 Cu [26], 59 Co(α, 2n)61 Cu [9, 27] reactions for the production of 61 Cu, but we were interested in 64 Zn( p, α)61 Cu reaction, due to its higher production yield and radionuclidic and chemical purity compared with other methods. However, the idea of replacing 64 Zn with natural zinc arose in order to reduce costs and to provide easily available targets. The possibility of using natural zinc targets was investigated by running ALICE nuclear code for reactions of all zinc isotopes with protons in the energy range of 3–30 MeV (30 MeV is the maximum energy of accelerated protons in our cyclotron). Investigation of the results showed that natural zinc can be used as the target material between 22–12 MeV proton beam energy with high yield and low level of isotopic impurities (Fig. 2). SRIM code was used for target thickness determination. The target had to be thick enough to reduce the incident proton beam energy from 22 MeV to 12 MeV. In the IBACyclone 30 systems, the target is placed at an angle of 6 degrees toward the proton beam, in order to achieve higher production yield with lower target thickness (0.1 times the calculated thickness) and better cooling (Fig. 3). According to the results of SRIM code and the characteristics of the system, an 80-micron zinc layer was electrodeposited on a gold layered copper backing. The 50 micron gold layer

Fig. 1. Radioactivity of eluted ethanol fractions from C18 . 70 nat

Cross Section (mb)

60

60-69

Zn(p,x )

Cu Cu-69 (3 min )

50

Cu-67 (61.9 h) Cu-66 (5.1 min )

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Cu-65 (stable ) Cu-64 (12.7 h)

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Cu-63 (stable ) Cu-62 (9.74 min ) Cu-61 (3.33 h)

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Cu-60 (23 min )

10 0 3

6

9

12

15

18

21

24

27

30

Proton Energy (MeV)

Fig. 2. Results of ALICE code for nat Zn( p, x)60−69 Cu reactions.

Fig. 3. Results of SRIM code for determination of target thickness.

was used to prevent the interference of the backing copper with the product during radiochemical separation. [61 Cu]CuCl2 was prepared by 22 MeV proton bombardment of the nat Zn target. The target was bombarded with a current intensity of 180 µA for 3.2 hours (500 µAh). The chemical separation process was based on a no-carrieradded method. The resulting activity of 61 Cu was 6.006 Ci at the end of bombardment (E.O.B.) and the production yield was 12.01 mCi/µAh. Radiochemical separation was performed by a two-step ion exchange chromatography method with a yield of more than 95%. Quality control of the product was performed in two steps. Radionuclidic control showed the presence of 67.41 (4.23%), 282.96 (12.2%), 373 (2.15%), 511 (122.9%), 656 (10.77%), 1186 (3.75%) keV gamma energies, all originating from 61 Cu and showed a radionuclidic purity higher than 99% (E.O.S.). The rest of activity was attributed to 60 Cu (0.23%). In order to check the chemical purity, concentration of zinc (from target material) and gold (from target support) were determined using visible colorimetric assays. The colorimetric assays demonstrated that the zinc concentration was below the internationally accepted levels, i.e. 5 ppm [28] while gold concentration confirmed to be less than 0.9 ppm. Because of the engagement of several polar functional groups in its structure, labeling of PTSM with copper cation greatly affects its chromatographic properties and the final complex is highly lipophilic. Thus the labeled and unlabeled PTSM can easily be separated using solid phase C18 SepPak column. In TLC studies, the more polar un-complexed PTSM and free copper fractions, correlate to smaller Rf s (Rf = 0.1–0.2), while the PTSM complex migrates at the higher Rf (Rf = 0.8). In all radiolabeling runs (n = 9), the integral ratio of the two peaks were constant (98 : 2), showing the high radiochemical purity and consistency of the labeling method. In order to obtain the best labeling reaction conditions, the complex formation was studied for temperature. Heating the reaction mixture to 50 ◦ C did not change the yield, while some degradation products were obtained via TLC and RTLC. The final radiolabeled complex diluted in normal saline was then passed through a 0.22 micron (Millipore) filter (filtration was used to sterilize the product). Due to its thermal instability, [61 Cu]PTSM preparation could totally be

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degraded and left detectable amounts of free copper after autoclaving. The chemical stability of [61 Cu]PTSM was high enough to perform further studies. Radio thin layer chromatography was performed to control the radiochemical purity of the product, using a mixture of dry ethyl acetate as the mobile phase for both pre-column and post column fractions. The radiochromatogram showed a major and distinct radio peak at the Rf of 0.90, using an in-house made radiochromatogram scanner coupled with a HPGe detector. The step motor was installed to count 0.4 cm-piece each 30 second through the slot of a shielded chamber. Uncomplexed 61 Cu eluted at Rf = 0.0. The radiochemical yields (more than 98% in each case, n = 9) were determined by comparison of uncomplexed 61 Cu and the major radio peak at Rf = 0.80. RTLC of the final product showed no change in stability and the patterns for trace [61 Cu]CuOAc and [61 Cu]PTSM were not changed during 5 hours.

4. Conclusion The method used in this research for the production and chemical separation of 61 Cu was quite simple and cost effective, while none of the previous studies given in the literature have reported such a high production yield with low level of contaminants. The 60 Ni(d, n)61 Cu reaction has resulted in a yield of 2.44 mCi/µAh [6, 24], while 59 Co(α, 2n)61 Cu has yielded 6.16 mCi/µAh [27]. Although 61 Ni( p, n)61 Cu has been reported to give a maximum yield of 15.5 mCi/µAh, the 60 Cu and 64 Cu contamination levels were 14.59% and 0.086% respectively [25] which is not satisfactory. A recent report on a similar nuclear reaction and beam energy as our study has given a yield of 9.9 mCi/µAh in the proton energy range of 19–10 MeV [10], but use of higher energy has led to an increase in thick target yield in our study. Total labeling and formulation of [61 Cu]PTSM took about 10 minutes, with a yield of 98%. A significant specific activity (≈ 246 Ci/mmol) was formed via insertion of [61 Cu]copper cations. No unlabelled and/or labeled byproducts were observed upon RTLC analysis of the final preparations after SPE purification. The radio-labeled complex was stable in aqueous solutions for at least 5 hours and no significant amount of other radioactive species were detected by RTLC, 12 hours after labeling. Trace amounts of [61 Cu]copper acetate (≈ 2%) were detected by RTLC which showed that radiochemical purity of the [61 Cu]PTSM was higher than 98%. [61 Cu]PTSM is a PET radiotracer with an intermediate half life, and the high chemical stability of this radiopharmaceutical makes it a very suitable candidate for diagnostic applications.

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