Introduction

Alpha-emitting radionuclides are of increasing interest in targeted cancer therapy. The range of alpha particles in tissue is only a few cell diameters (less than 100 μm), offering the possibility of extremely local irradiation of malignant cells while leaving neighboring cells intact, in particular bone marrow and healthy tissue. Alpha-emitters have greater biological effectiveness due to their high linear energy transfer (LET around 100 keV/μm) compared to both conventional external beam radiation and beta particles. 223Ra is one of the most promising alpha-emitting radionuclides for bone-targeting therapy [1]. A number of recent scientific and clinical investigations are devoted to 223Ra as a treatment of bone metastases occurring when cancer cells (prostate, breast and others) spread from their original site to skeletal surfaces [2–8]. As calcium mimic and thus a natural bone-seeking agent 223Ra accumulates at areas of increased bone turnover without special carriers. Bone mineral hydroxyapatite, which forms 50 % of the bone matrix, is its target. A novel 223Ra-based radiopharmaceutical, named Alpharadin (developed by Algeta ASA, Oslo, Norway), a solution of 223Ra dichloride (223RaCl2) to be administrated intravenously is of particular benefit in the treatment of prostate cancer bone metastases demonstrating not only symptom relief but improvement on overall survival. Alpharadin, marketed as Xofigo, was approved for treatment of patients with castration-resistant prostate cancer by the U.S. Food and Drug Administration (FDA) on 15 May 2013 [9, 10].

223Ra injected intravenously retains in skeletal metastases, realizing an in vivo generator of short-lived α-emitting daughters. A low injected activity is required for therapy because of high energy deposition of 223Ra (28.2 MeV of total energy with 95.3 % of the decay energy emitted as alpha-particles). The 223Ra decay chain is as follows:

The most promising approach for routine production of 223Ra in radiochemical laboratories and hospitals is a radioisotope generator method because of the device simplicity, reliability of operating with highly radioactive materials and convenience to be transported. A long-term operating 227Ac/227Th-based generator is usually used for 223Ra production. It represents an extraction chromatographic column containing P,P′-di(2-ethylhexyl)methanediphosphonic acid on silica (Dipex-2 or AC resin) [11]. The common procedure includes separation of 223Ra from parent radionuclides 227Ac/227Th by elution of 223Ra from the generator column with 1 M HCl or HNO3. For enhanced separation of 223Ra, two-coupled columns of Dipex-2 are used. The next step is sorption of 223Ra on the cation-exchange column (AG-50W × 12 resin) and its subsequent stripping with 8 M HNO3. The obtained solution is evaporated and 223Ra residue is dissolved with sodium chloride/sodium citrate solution [1, 11]. The alternative tandem generator system for production and on-line isolation of 223Ra were developed in our previous works. The system consists of two columns: the anion-exchange generator column with loaded 227Ac/227Th-source from which 223Ra is separated with an aqueous-alcoholic solution and the cation-exchange column intended to prepare 223Ra solution in the complexing form [12, 13].

The goal of the present work was to produce 223Ra in a form suitable for direct biomedical application using the developed generator system. The cation-exchange behavior of radium in saline solutions was investigated and the optimal elution conditions were found for 223Ra isolation in the form of 223Ra-EDTA complexes in isotonic NaCl solutions at pH close to the normal pH of blood. A scheme for on-line production of 223Ra as a radiopharmaceutical for direct intravenous administration to treat bone metastases as well as for targeted radioimmunotherapy was suggested.

Experimental

Reagents and equipment

HNO3, CH3OH, NaCl, disodium salt of ethylenediaminetetraacetic acid Na2EDTA, NH4OH («Acros Organics») were of analytical grade. Stock NaCl and Na2EDTA solutions were prepared by dissolution of a weighed reagent portion in double-distilled water or 0.9 % NaCl (for Na2EDTA). Dowex-1 × 8 strong base anion-exchanger and Dowex-50 × 8 strong acid cation-exchanger of 100–200 mesh («Serva») were placed into glass columns (0.3–0.5 × 5–10) cm and used as generator and for elution experiments.

The pH of Na2EDTA solutions was adjusted with NH4OH or HNO3 and measured with pH-meter (HANNA Instruments).

Alpha-activities of the radionuclides were measured using an a-Analyst Spectrometer from Canberra. Gamma-activities were measured with a NaI(Tl) crystal. Gamma-spectrometric determination of radionuclides was carried out with a high-purity Ge detector from Canberra and resolution of 2 keV at 1,332 keV. The spectra were analysed using the Genie-2000 Spectra Analysis Software. The radiochemical purity of short-lived radionuclides was also controlled by measuring the half-lives. 227Ac was identified through the measurement of γ-lines of the 227Th daughter radionuclide after its accumulation for several days.

Generator system and procedures

223Ra was regularly produced using the 227Ac-based generator described earlier [14]. Briefly, 227Ac was loaded on the column filled with 0.5 g of Dowex-1 × 8 anion-exchanger utilized as generator of 223Ra since 2002. Aqueous methanol-nitric acid solutions (0.70–0.75 M HNO3—80 % CH3OH) were used as eluents for separation of 223Ra from 227Ac/227Th. No breakthrough of the parent radionuclides 227Ac and 227Th in the eluate was observed (Fig. 1). The decontamination factor of 223Ra from 227Ac/227Th was ≥106.

Fig. 1
figure 1

α-Spectra of 227Ac (aliquot) loaded on generator column 13.11.2002 (a) and 223Ra in 0.7 M HNO3—80 % CH3OH solution after elution from generator column 02.12.2013 (b)

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The solution obtained from the generator containing 223Ra in 0.70–0.75 M HNO3—80 % CH3OH was passed through the column with 0.05 g of Dowex-50 × 8 cation-exchanger which completely retains radium. Investigation of 223Ra elution from the cation-exchange column was carried out with saline solutions (0.9 % NaCl) containing Na2EDTA in the range of concentration from 0.001 to 0.1 M, at pH = 4.5–8. A special preliminary procedure was developed to precondition the column before elution: after sorption of 223Ra from HNO3 aqueous-alcoholic solution the column was subsequently washed with 2 mL of double-distilled water and 4 mL of 0.9 % NaCl. After each sorption-elution cycle the cation-exchange column was washed with 2 mL of 2 M HNO3, large amounts of double-distilled water (until pH = 6), and then saturated with 0.70–0.75 M HNO3—80 % CH3OH solution. Solutions were passed through the column at a flow rate 0.2–0.3 ml/min in all experiments.

Results and discussion

In previous work we studied the cation-exchange behavior of 223Ra as EDTA and DTPA complexes in aqueous solutions. It was shown that 223Ra could be eluted from the cation-exchange column at pH ≥ 9 [12, 13]. The present studies on 223Ra isolation were carried out using EDTA in normal saline solutions (0.9 % or 0.15 M NaCl), which are usually used for intravenous injection. It is known that radium can form chloride complexes, so the presence of NaCl can influence radium sorption/elution. The cation-exchange behavior of radium was studied in saline solutions in dependence on NaCl concentration. The experimental data have shown that 223Ra is strongly retained by Dowex-50 × 8 cation-exchanger when [NaCl] ≤ 0.2 M, the distribution coefficients are more than n 103 g/mL. Thus, isotonic NaCl solution does not decrease the 223Ra retention on the cation-exchanger, however, in the case of elution with the complexing agent, differences between aqueous and saline eluent solutions were observed. As presented in Fig. 2. when Na2EDTA-NaCl solutions are used, radium starts to elute already at pH = 6.2 and its almost complete elution is achieved at pH ≥ 7.2. On the contrary, quantitative elution with Na2EDTA in the absence of NaCl is observed only in the alkaline range (pH = 9–10).

Fig. 2
figure 2

Elution of 223Ra with 1 mL of 0.05 M Na2EDTA versus pH: aqueous solutions (open symbols), saline 0.15 M NaCl solutions (solid symbols); Dowex-50 × 8, 0.05 g

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The data on 223Ra elution with saline Na2EDTA solutions as a function of [Na2EDTA] are presented in Fig. 3. It can be seen that more than 90 % of radium is eluted with 1 ml of Na2EDTA when its concentration is ≥0.05 M. For the further studies the 0.05 M Na2EDTA-0.15 M NaCl solution was chosen as eluent.

Fig. 3
figure 3

Elution of 223Ra versus Na2EDTA concentration; 1 mL of 0.15 M NaCl; pH ≈ 7.4; Dowex-50 × 8, 0.05 g

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It is known that biologic systems are characterized by definite pH values, e.g. the normal pH of blood is 7.4. To determine optimal conditions for 223Ra isolation in the form suitable for biomedical trials we studied elution of 223Ra-EDTA complexes in the range of pH closed to 7–8. The elution curves of 223Ra-EDTA with 0.05 M Na2EDTA-0.15 M NaCl solution at pH values from 6.99 to 8.04 are presented in Fig. 4.

Fig. 4
figure 4

Elution curves of 223Ra with 0.05 M Na2EDTA-0.15 M NaCl at pH value: 6.99 (1), 7.21 (2), 7.42 (3), 7.63 (4), 7.75 (5), 8.04 (6); Dowex-50 × 8, 0.05 g

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As it is shown at pH = 7.4–8.0 more than 90 % of 223Ra is eluted from the column with 1 mL of the 0.05 M Na2EDTA-0.15 M NaCl solution. The higher pH of the eluent the lower volume is needed for almost complete 223Ra elution (0.5 mL is enough at pH = 7.6–8.0). Separate experiments were performed to verify the stability of the complexes of Ra with EDTA. The eluates containing the 223Ra-EDTA complexes were repeatedly passed through the same cation-exchange column (preconditioned appropriately). The obtained elution curves were identical to the curves presented in Fig. 4. It allows to conclude, that sufficiently stable 223Ra-EDTA complexes are formed in the 0.05 M Na2EDTA–0.15 M NaCl at the investigated pH range.

Based on the obtained data a simple and effective method for 223Ra production from an anion-exchange generator column and subsequent isolation of 223Ra-EDTA complexes in normal saline solution using a cation-exchange column was developed. The scheme of the method is presented in Fig. 5.

Fig. 5
figure 5

The scheme of 223Ra production using anion-exchange generator column and subsequent isolation of 223Ra-EDTA in saline solutions using cation-exchange column

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The final saline 223Ra-EDTA solution does not contain any other radionuclides except 223Ra and its daughters. A γ-spectrum of the 223Ra eluate in 0.05 M Na2EDTA-0.15 M NaCl solution at pH = 7.4 from Dowex-50 × 8 cation-exchange column is shown in Fig. 6.

Fig. 6
figure 6

γ-Spectrum of 223Ra in 0.05 M Na2EDTA-0.15 M NaCl solution at pH = 7.4 after elution from Dowex-50 × 8

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Conclusions

The cation-exchange behavior of 223Ra in Na2EDTA-NaCl solutions was studied concerning dependence on composition of the solution, concentration of complexing agent and pH value. The optimal conditions for 223Ra elution in the form of complexes with Na2EDTA in 0.9 % NaCl solutions were determined. A novel scheme of 223Ra-EDTA preparation using a tandem generator system for targeted alpha-radiotherapy was developed. The scheme is adaptable to automation for routine clinical processes, excluding the need of evaporation of the high acid radioactive solutions, reducing hazards to technical staff. The produced radium is directly applicable as a 223Ra-based bone-seeking radiopharmaceutical. Alternatively, it may be used for the preparation of 223Ra complexes with biomolecules for targeted alpha therapy of various organs.