Targeted tumor radiotherapy uses radionuclides conjugated to chemically-guided agents such as labelled monoclonal antibodies or isotopically-labelled polypeptide. These agents require radioisotopes with high specific activity, high LET particle emissions, photon emissions for monitoring therapy with imaging and follow-up as well as adsorbed dose distribution and half-lives long enough to allow the preparation and distribution of radiopharmaceuticals [1] - [6] . High energy beta emitting radionuclides are preferentially used in these agents to kill tumoral cells [4] . Radioactive lanthanides such as ¹⁴⁹Pm, ¹⁶¹Tb, ¹⁶⁶Ho and ¹⁷⁷Lu, have a great potential in radiotherapy because they are beta or Auger-electron emitters with just enough gammas to enable imaging, with half- lives long enough to allow preparation and distribution of the radiopharmaceuticals, and can be prepared at high specific activities (carrier-free) (see Table 1).

Chemically, lanthanides (Ln³⁺) have the ability to replace Ca²⁺ in biological systems (e.g. enzymes, proteins, cells, cytoplasm). Additionally, they could cause the inhibition of collagenase or lymphocyte activation, the sta-

bilization of collagen fibrils, the stimulus-mediated cell secretion, neutrophil chemotaxis and aggregation, etc. [1] - [5] [7] . Lanthanides display strong oxyphilicity and form high thermodynamic stability complexes, especially those derived from poly (aminocarboxilic) acids, which enables them to remain intact while diffusing into extracellular spaces with rapid clearance through kidneys [8] . On the other hand, all lanthanides have similar chemical properties regarding labeling, therefore, radiolanthanides that have been used for therapy, such as ¹⁵³Sm and ¹⁷⁷Lu, might easily be replaced with other radiolanthanides in accordance with their application. For example, high energy beta emitters such as ¹⁴⁹Pm or ¹⁶⁶Ho are efficient for metastases while the low-energy electron emitters as ¹⁶¹Tb and ¹⁷⁷Lu might be suitable for micrometastases therapy [4] [9] .

A large number of clinical studies focused on the use of ¹⁷⁷Lu for therapeutic treatment have been reported [5] [10] - [13] : ¹⁷⁷Lu-DOTATOC, ¹⁷⁷Lu-DOTATATE,¹⁷⁷Lu-DOTA-HSAM and ¹⁷⁷Lu-DOTA-Octreotate have been used as receptor-positive tumors [10] [11] [14] [15] , ¹⁷⁷Lu-octreotate in advanced low-grade neuroendocrine tumors [15] [16] , ¹⁷⁷Lu-RM2 for prostate cancer [17] , ¹⁷⁷Lu-antitenascin monoclonal antibody (mAb) 81C6 as a targeted radiotherapeutic in patients with brain tumors [18] , [¹⁷⁷Lu]Lu-AMBA and ¹⁷⁷Lu DOTA-chelated bombesin (BN) as targeted radiotherapy of tumors expressing gastrin releasing peptide receptors [19] [20] or ¹⁷⁷Lu- monoclonal antibody (mAb) L8A4 for the treatment of residual tumor margins remaining after surgical debulking of brain tumors [21] [22] .

¹⁶¹Tb is a low-energy β⁻ emitter similar to ¹⁷⁷Lu that has been less used and studied than ¹⁷⁷Lu. However possesses a high potential for treating small tumor cell clusters or even targeting single cells, due to its ability to emit a significant amount of conversion and Auger electrons, which provides much higher local dose density due to their shorter range in tissue (0.5 - 30 μm). For example, ¹⁶¹Tb-DTPA-octreotide has been reported as a somatostatin analogue for intraoperative scanning [2] [11] .

The high beta energy emitter ¹⁶⁶Ho has being used with: ¹⁶⁶Ho-DOTMP, ¹⁶⁶Ho-EDTMP as bone agent in the treatment of multiple myeloma (for bone marrow ablation) [5] [12] [13] , ¹⁶⁶Ho macroaggregates such as ¹⁶⁶Ho- FHMA or ¹⁶⁶Ho-poly(L-lactic acid) in radiation synovectomy or hepatic tumors [2] [23] [24] , ¹⁶⁶Ho and immature DCs to treat irreversible melanoma or as an adjuvant therapy after surgery [25] , [¹⁶⁶Dy]Dy/¹⁶⁶Ho-(EDTMP), from in vivo ¹⁶⁶Dy/¹⁶⁶Ho generator system, for myeloablative radiotherapy and subsequent stem cell transplantation [26] or self-expandable covered metallic stent incorporated with ¹⁶⁶Ho for delivering intraluminal brachytherapy as well as for internal bile drainage in malignant biliary stricture [27] .

¹⁴⁹Pm, a moderate beta energy emitter, has been the least exploited of these radiolanthanides. Just two studies have been reported: ¹⁴⁹Pm-DO3A-amide-βAla-BBN concerning in vivo tracking of the therapeutic dose [28] and biodistribution studies conducted with ¹⁴⁹Pm and ¹⁷⁷Lu-DOTA-conjugated vitronectin receptor (α_vβ₃) antagonist-RGD [29] .

Carrier-free radiolanthanides of high specific activity can be produced in nuclear reactors via neutron irradiation of massive lanthanide targets (>1 mg), as described in the reaction 1, followed by a radiochemical separation of the daughter radionuclide (, <1 mg) from the macro-amounts of the parent target () [11] [30] - [37] .

Considering that the development of potential radiotherapy agents greatly depends on a consistent and reasonably priced supply of high specific activity radiolanthanides (carrier-free), the objective of this work was to develop a device production in order to obtain the carrier free radiolanthanides: ¹⁴⁹Pm, ¹⁶¹Tb, ¹⁶⁶Ho and ¹⁷⁷Lu. The process, previously developed by our group, is based on radiochemical separation of these radiolanthanides from irradiated natural targets, using an extraction chromatographic resin and for this specific purpose a device was designed and built.

The separation conditions of the Nd/Pm, Gd/Tb, Dy/Ho and Yb/Lu pairs, shown in Table 2, were selected from the K_d values of Nd, Pm, Gd, Tb, Dy, Ho, Yb and Lu as a function of HNO₃ concentration in Ln SPS Eichrom resin (See Figure 1(a)) [38] [39] and the separation factors (a) of the Nd/Pm, Gd/Tb, Dy/Ho and Yb/Lu (see Figure 1(b)) pairs calculated from their K_d values.

The methodology of radiolanthanide separation is divided into six stages, as outlined in Figure 2: 1) Irradiation of lanthanide nitrates in the nuclear reactor to form the parent/daughter pairs (¹⁴⁹Nd/¹⁴⁹Pm, ¹⁶¹Gd/¹⁶¹Tb, ¹⁶⁶Dy/¹⁶⁶Ho and ¹⁷⁷Yb/¹⁷⁷Lu) (See Table 3), 2) Dissolution of the radioactive salts containing the parent/ daughter pair in 0.15 mol/L HNO₃ and adsorption into the chromatographic column loaded with Ln SPS Eichrom resin, 3) Desorption of parent by elution with HNO₃, 4) Desorption of daughter by elution with HNO₃, 5) Precipitation of lanthanide hydroxides Ln(OH)₃ by addition of NaOH to daughter’s eluate, and 6) Re-dissolution of lanthanide hydroxides Ln(OH)₃ with 0.1 mol/L HCl.

(a) (b)

Figure 1. (a) Effect of HNO₃ concentration on the distribution coefficients of Nd, Pm, Gd, Tb, Gd, Ho, Yb and Lu in Ln SPS Eichrom resin; (b) Separation factors (a) of the Nd/Pm, Gd/Tb, Dy/Ho and Yb/Lu pairs in Ln SPS Eichrom resin as a function of [HNO₃] [37] [38] .

Figure 2. Separation protocol by produce ¹⁴⁹Pm, ¹⁶¹Tb, ¹⁶⁶Ho and ¹⁷⁷Lu.

Radiolanthanides are produced by irradiation of 50 mg ¹⁴⁸Nd(NO₃)₃, ¹⁶⁰Gd(NO₃)₃, ¹⁶⁴Dy(NO₃)₃ and ¹⁷⁶Yb(NO₃)₃ in the TRIGA MARK III Reactor of the National Institute of the Nuclear Research (ININ) in Mexico, to a neutron fluence rate of 1 × 10¹³ n cm⁻²∙s⁻¹, under the conditions marked in Table 3.

The parent lanthanide of the parent/daughter pairs: Nd/Pm, Gd/Tb, Dy/Ho and Yb/Lu is eluted at first during the chromatographic separation process. This solution can be re-crystallized as nitrate and the enriched target recover for a new irradiation. The ¹⁴⁹Pm, ¹⁶¹Tb, ¹⁶⁶Ho and ¹⁷⁷Lu solutions obtained at high concentrations of nitric acid (3 to 8 M) are transformed to lanthanide chloride salts by adding NaOH, until reaching a pH value of 7 to form lanthanide hydroxides Ln(OH)₃, which were re-dissolved in 4 mL of 0.1 N HCl to get lanthanide chlorides solutions (¹⁴⁹PmCl₃, ¹⁶¹TbCl₃, ¹⁶⁶HoCl₃ and ¹⁷⁷LuCl₃) carrier-free with a radionuclide purity higher than 99.9%.

The radiolanthanide separation device, called DISER, was designed and built from separation methodology described previously, for routinely production of carrier-free ¹⁴⁹Pm,¹⁶¹Tb, ¹⁶⁶Ho and ¹⁷⁷Lu with a radionuclide purity of more than 99.9%. The DISER was built at the National Institute of Nuclear Research (ININ) in Mexico by groups from the Radioactive Materials Research Laboratory (LIMR) and the Prototype Fabrication Department. The device subsequently described is the result of several arrangements and modifications.

The DISER was placed in a hot cell consisted of 1) Labware support stands, 2) Chromatographic columns support stand, 3) Reagent Access System and 4) Opening system of the irradiation container (see Figure 3). A description of each component and system is presented below.

Labware support stand. The DISER has three acrylic support stands, two are held at the top of the cell to place beakers used in the separation process (A1). Both support stands are rotatory in order to facilitate the handling and arrangement of labware during the process. The third is placed on the left wall of the cell and is constituted by eight semi-hollow cylindrical bases where the chromatographic columns are stocked (A2) (see Figure 3).

Chromatographic columns support carousel. The support carousel is the DISER’s core and is there where the separation processes of the parent/daughter pairs (¹⁴⁹Nd/¹⁴⁹Pm, ¹⁶¹Gd/¹⁶¹Tb, ¹⁶⁶Dy/¹⁶⁶Ho and ¹⁷⁷Yb/¹⁷⁷Lu) is performed. The carousel is constructed all in acrylic and its stainless steel shaft is screwed onto the cell (see Figure 3). The support carousel consists of: 1) a rotating disk which can hold beakers to receive the eluates from the separation process (B1) and 2) two supports fixed on the carousel’s shaft, one holds 2 chromatographic columns for the separation process and dissolution of lanthanide hydroxides (B2), and the other one in shape of funnel to add the NaOH to the daughter’s eluates (B3). This neutralization reaction is air-cooled. The columns base is also gyratory and can be fitted to varing heights.

The glass chromatographic columns (80 × 12 mm) with a coarse fritted disc are used in the separation process―packaged with 2 g of Eichrom’s Ln SPS resin (50 - 100 μm)―and the dissolution of lanthanide hydroxides, filled with glass wool and Whatman No.1 filter paper.

Reagent Access System is located outside the hot cell, at the top and consists of a glass separatory funnel with cap and stopcork holded to an acrilic support (C). The funnel tip is connected to a glass pipette located directly on the chromatographic column. This system allows to introduce to the cell the HNO₃ solutions requiered for the separation and dissolution process, easily and safely without openning the hot cell.

Opening system of the irradiation container. The radioactive lanthanide nitrate salts contained in quartz ampoules are uncapped in the opening system. The latter is constituted by an acrylic support in the form of inverted “L” (D); the ampoule is held into the large base (D1) which can slide and in the cutter ampoule base (D2) the tip of the quartz ampoule is inserted into the hole opener and by applying a torque, the top is cracked.

Before separation process

The DISER is perfectly cleaned; the chromatographic columns (Wheaton 80 × 12 mm) are packed with with Ln SPS Eichrom resin (B2) and with glass wool and Whatman No. 1 filter paper (B3). All solutions are prepared: HNO₃ solutions and 4 mol/L HCl. The chromatographic columns are placed into the Labware support stand (A2), the beakers properly numbered and the final product vial into the carousel (B1) and the NaOH pearls and the 0.15 mol/L HNO₃ solution into support stands (A1).

Separation Process

a) The ampoule containing the radioactive nitrate salt is introduced into the DISER and placed in the support (D2) of the opening system (D). The ampoule is then opened, as shown in Figure 4.

b) Radioactive nitrate salt [ⁿLn(NO₃)₃/*⁽ⁿ⁺¹⁾Ln(NO₃)₃] is then dissolved by 0.15 mol/L HNO₃ and this solution is added to the separation column (B2) using a Pasteur pipette.

c) 0.15 mol/L HNO₃ is added to the reservoir of the Reagent Access System (C), which is then opened to introduce this solution to the separation column (B2). Eluates are recovered in the beaker No. 1.

d) The separation column (B2) is aligned to the position of the beaker No. 2 and the corresponding solution of HNO₃ used to desorb the parent radiolanthanide (see Figure 2) is loaded to the Reagent Access System (C). This solution is added to the chromatographic separation column (B2) and the eluate, contained the parent radiolanthanide, is recovered in the beaker No. 2.

e) Separation column (B2) is rotated to the position of the beaker No. 3, the Reagent Access System (C) is loaded with the nitric acid solution to desorb the daughter radiolanthanide (Figure 2) and the eluate contained the daughter radiolanthanide, is recovered in the beaker No. 3.

f) Sodium hydroxide pearls are added slowly to the beaker No. 3 to until complete dissolution of NaOH.

g) The recovery column (B3) is turned to the position of the beaker No. 4, and the content of the beaker No. 3 is added to the column. Liquid phase is recovered in the beaker No. 4 and the solid phase constituted by the daughter radiolanthanide hydroxide remains into the column.

h) The recovery column (B3) is rotated to align to the final product vial and the Reagent Access System (C) is loaded with 0.1 M HCl. This solution is then added to the recovery column (B3) and after 10 minutes of contact, in order to completely dissolve the lanthanide hydroxides, the column is open and the liquid phase, contained the lanthanides chlorides is finally recovered in the final product vial.

The separation efficiency and the radionuclide purities are then determined to the final product with a coaxial gamma detector HPGe (Canberra 7229P) connected to a PC-multichannel analyzer (ACCUSSPECT-A, Canberra). Chemical purity should be determined by ICP.

Radiochemical separations of the ¹⁴⁹Pm, ¹⁶¹Tb, ¹⁶⁶Ho and ¹⁷⁷Lu from the macro-amounts of the neodinium, gadolinium, dysprosium and ytterbium targets, performed in the DISER by the methodology proposed, allow to separate: 1) Gadolinium and Terbium with an efficiency of 100%, recovering the ¹⁶¹Tb with a radionuclide purity of 100%; 2) Neodymium and Promethium with an efficiency of 98.4% and a ¹⁴⁹Pm radionuclide purity of 99.9%; 3) Dysprosium and Holmium with an efficiency of 100%, obtaining 100% pure ¹⁶⁶Ho; and 4) Ytterbium and Lutetium with an efficiency of 89.7%_, recovering the ¹⁷⁷Lu at a 99.9% radionuclide purity if the first 6 mL of the ¹⁷⁷Lu eluate are removed.

The recovery efficiencies of the lanthanides chlorides after precipitation of the hydroxides and the dissolution with 0.1 mol/L HCl are: 98.5%, 96.5%, 96.9% and 99.6% for ¹⁴⁹Pm, ¹⁶¹Tb, ¹⁶⁶Ho and ¹⁷⁷Lu respectively. The remotion of

HNO₃ is commonly performed by evaporation to dryness; however, this process consumes time, requires the installation of a heating equipment inside the hot cell and produces highly corrosive vapors (NO_x) by the decomposition of nitric acid. To avoid these drawbacks, the eluates of Ln(NO₃)₃ (Ln = ¹⁴⁹Pm, ¹⁶¹Tb, ¹⁶⁶Ho and ¹⁷⁷Lu) were transformed to lanthanides hydroxides [Ln(OH)₃], and dissolved in 0.1 M HCl [40] :

The sodium nitrate salts are removed in the aqueous phase and only the hydroxide precipitates are redissolved. In the separation protocol proposed, the pH values to precipitate the lanthanide nitrate solutions was fixed at 7, considering that the pH values reported by the literature, at which lanthanides precipitate are: Nd (6.7), Pm (6.6), Gd(6.4), Tb (6.3), Dy (6.1), Ho (6.1), Yb (5.6) and Lu (5.5), values lower than 7 [40] .

A radiolanthanide separation device (DISER) was designed and built to standardize the separation process of the four pairs: ¹⁴⁹Nd/¹⁴⁹Pm, ¹⁶¹Gd/¹⁶¹Tb, ¹⁶⁶Dy/¹⁶⁶Ho, ¹⁷⁷Yb/¹⁷⁷Lu in order to obtain carrier-free ¹⁴⁹Pm, ¹⁶¹Tb, ¹⁶⁶Ho or ¹⁷⁷Lu. The separation process of pairs reported here consists to six stages: 1) Target Irradiation; 2) Dissolution of the irradiated target to be loaded into the chromatographic column; 3) Desorption of parent; 4) Desorption of daughter; 5) Formation of lanthanide hydroxides; and 6) Re-dissolution of lanthanide hydroxides with HCl. The adequate execution of this process, for a period of 20 - 25 min, is able to produce carrier-free ¹⁴⁹Pm, ¹⁶¹Tb, ¹⁶⁶Ho or ¹⁷⁷Lu and with a radionuclide purity of more than 99.9%. It is important to remark that for an optimal performance is important to use the following separation conditions: for Neodymium and Promethium use 0.18 and 1.5 M HNO₃, for Gadolinium and Terbium use 0.8 and 3 M HNO₃, for Dysprosium and Holmium use 1.5 M HNO₃ and for Ytterbium and Lutetium use 3.4 and 8 M HNO₃ respectively.

The authors indebted to the technical staff of reactor Triga Mark III (Mexico): Maximiano Hernández, Wenceslao Nava, Margarito Alva, Braulio Ortega, Edgar Herrera and Roberto Raya; and Angel Jesus Juárez Neri by drawing DISER.

This work was supported by the “Consejo Nacional de Ciencia y Tecnología” grant number: CONACYT- SALUD-2004-C01-001.