Stability of ⁴⁷Sc-complexes with acyclic polyamino-polycarboxylate ligands

Abstract

The aim of this study was to evaluate acyclic ligands which can be applied for labeling proteins such as monoclonal antibodies and their fragments with scandium radionuclides. Recently, scandium isotopes (⁴⁷Sc, ⁴⁴Sc) are more available and their properties are convenient for radiotherapy or PET imaging. They can be used together as “matched pair” in theranostic approach. Because proteins denaturize at temperature above 42 °C, ligands which efficiently form complexes at room temperature, are necessary for labelling such biomolecules. For complexation of scandium radionuclides open chain ligands DTPA, HBED, BAPTA, EGTA, TTHA and deferoxamine have been chosen. We found that the ligands studied (except HBED) form strong complexes within 10 min and that the radiolabelling yield varies between 96 and 99 %. The complexes were stable in isotonic NaCl, but stability of ⁴⁶Sc-TTHA, ⁴⁶Sc-BAPTA and ⁴⁶Sc-HBED in PBS buffer was low, due to formation by Sc³⁺stronger complexes with phosphates than with the studied ligands. From the radiolabelling studies with n.c.a. ⁴⁷Sc we can conclude that the most stable complexes are formed by the 8-dentate DTPA and EGTA ligands.

Introduction

Radionuclides emitting low and medium energy beta-particles and having several days half-life are attractive candidates for radioimmunotherapy. The most promising radioisotope in this category is ¹⁷⁷Lu, which has favourable decay characteristics, as e.g. half-life of 6.71 days.

This radionuclide can be produced in nuclear reactors by n,γ reaction and is commercially available at high levels of specific activity and chemical purity.

Having large cross-section of 2090 b [1], ¹⁷⁷Lu can be directly produced with a relatively high specific activity by neutron activation of ¹⁷⁶Lu (2.6 % of natural abundance). Nevertheless, some amount of stable ¹⁷⁶Lu cannot be avoided, which may cause some problems concerning receptor saturation with biomolecules labelled with stable isotope, especially when the number of receptors is limited. For this purpose an alternative production route via neutron capture, starting with enriched ¹⁷⁶Yb targets, has been demonstrated [2–4]. In this method, the isotopically enriched ¹⁷⁶Yb target undergoes the (n,γ) reaction to produce ¹⁷⁷Yb, which subsequently decays by β⁻-emission (T _1/2 = 1.9 h) to ¹⁷⁷Lu. However, this indirect production route requires difficult radiochemical separation of ¹⁷⁷Lu from the irradiated Yb₂O₃ target. Taking into account that ytterbium and lutetium are neighbouring trivalent lanthanides and that the Yb/Lu mass ratio in the irradiated target can be as high as several thousand, separation is a very difficult task [2, 5, 6].

The usefulness of other radionuclides, which may enhance the therapeutic effect of radiopharmaceuticals, should be also investigated. As a good alternative to application of carrier-free ¹⁷⁷Lu Lehenberger et al. [7] proposed application of reactor produced ¹⁶¹Tb. The properties of this radionuclide are similar to those of ¹⁷⁷Lu, but separation from the target is considerably easier.

Application of ⁴⁷Sc, as an alternative radionuclide to ¹⁷⁷Lu, was proposed in earlier works [8–11]. ⁴⁷Sc is a low energy β⁻-emitter with a 3.35 days half-life, and shows a primary γ-ray at 159 keV, which is suitable for imaging. It is important to mention that other scandium radionuclides, ⁴³Sc and ⁴⁴Sc, are also promising β⁺-emitters, suitable for PET technique. Therefore, therapeutic ⁴⁷Sc together with diagnostic ⁴⁴Sc or ⁴³Sc can be used as “matched pair” in teranostic approach. Essential decay characteristics and production parameters for n.c.a. ⁴⁷Sc, ¹⁷⁷Lu and ¹⁶¹Tb are presented in Table 1.

Table 1.

Production parameters of n.c.a. ¹⁷⁷Lu, ¹⁶¹Tb and ⁴⁷Sc

Radionuclide	T1/2 (days)	Nuclear reactions	Eβmax (MeV)	Main photons, keV (%)	Cross-section (barn) (Vertes 2011)	Cost of enriched target ($/mg)	Separation from the target
177Lu	6.647	176Yb(n,γ)177Yb \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \underrightarrow {\beta {\kern 1pt} - } $$\end{document} 177Lu	0.14	54 (33.5)	3	13	Very difficult
				208.4 (60.9)
				229 (37.2)
161Tb	6.906	160Gd(n,γ)161Gd \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \underrightarrow {\beta {\kern 1pt} - } $$\end{document} 161Tb	0.15	25.7 (21)	1.5	5	Difficult
				48.9 (16)
				74.6 (9.8)
47Sc	3.341	47Ti(n,p)47Sc	0.16	159.4 (68)	0.15	10	Easy

The production method of highly active ⁴⁷Sc in a nuclear reactor was described by Mausner and coworkers [8, 12]. Enriched ⁴⁷TiO₂ target was irradiated with high energy neutrons (E_n > 1 MeV) to produce ⁴⁷Sc in the ⁴⁷Ti(n,p)⁴⁷Sc reaction. Various methods of ⁴⁷Sc separation from metallic Ti and TiO₂ targets, based on TBP extraction or cation and anion exchange processes, have been reported [12, 13]. Recently, to avoid the slow dissolution of the target in hot concentrated H₂SO₄, a new, simple and fast method based on irradiation of the Li⁴⁷₂TiF₆ or ⁴⁷TiO₂ target, dissolution in HF solution and ion exchange isolation of ⁴⁷Sc has been investigated [11].

As shown in Table 1, the advantage of ⁴⁷Sc production contrary to that of ¹⁷⁷Lu and ¹⁶¹Tb, is relatively easy isolation of the radionuclide from the target, but the disadvantage is smaller cross-section of the nuclear reaction.

To date, only few reports concerning labelling studies with Sc radionuclides were published [14–17] Authors of these papers concluded, that for labelling peptides, such as octreotide, the macrocyclic ligand 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), is the most suitable. The stability of the DOTA complexes results from their kinetical inertness, therefore efficient labelling of DOTA-conjugates requires elevated (90 °C) temperature. Due to its rather long half-life, ⁴⁷Sc can be also considered for preparation of alternative therapeutic agents with longer pharmacokinetics, such as ⁴⁷Sc-labelled proteins, as e.g. monoclonal antibodies, its fragments and nanobodies. Unfortunately, because proteins denaturize at temperature above 42 °C, ligands other than DOTA, which form complexes at room temperature, are necessary for labelling such biomolecules. The acyclic chelators are not as kinetically stable as the macrocyclic chelators (DOTA, NOTA, TETA etc.), but formation of their complexes at room temperature is much faster. In this paper we report formation and stability studies on ⁴⁷Sc complexes with various acyclic polydentate ligands, which exhibit faster than DOTA kinetics of complex formation.

Experimental

Materials

The analytical grade reagents were used for radiochemical investigations. Water was obtained from a Millipore water purification system. Ammonium acetate and hydrofluoric acid (40 %) were purchased from Sigma-Aldrich. All resins (Chelex^® 100, Dowex^® 1X8, Dowex^® 50WX4) were purchased in 100–200 mesh size from Dow Chemical.

The analytical grade TiO₂ was obtained from Sigma-Aldrich. Enriched ⁴⁷TiO₂ was supplied by Isoflex, San Francisco, USA. The isotopic composition was: ⁴⁶Ti-4.0, ⁴⁷Ti-65.8, ⁴⁸Ti-26.8, ⁴⁹Ti-1.8, ⁵⁰Ti-1.6 %.

We have chosen the following acyclic ligands for the studies: 8-dentate diethylenetriaminepentaacetic acid (DTPA), 6-dentate N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid (HBED), 6-dentate 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), 8-dentate ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 10-dentate triethylenetetraamine-N,N′,N′′,N′′′-hexaacetic acid (TTHA) and deferoxamine (DFO). For comparison, cyclic 8-dentate ligand, the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) has been chosen.

Radionuclides

For reasons of availability we used in preliminary experiments instead of ⁴⁷Sc, the longer lived ⁴⁶Sc radionuclide. The former is produced by fast neutron irradiation of the ⁴⁷Ti target, while ⁴⁶Sc can be produced in a simple way by direct thermal neutron irradiation of natural scandium. The ⁴⁶Sc radionuclide was obtained by neutron irradiation of the Sc₂O₃ target at a neutron flux of 1.5 × 10¹⁴ n cm⁻² s⁻¹ for 200 h in the Maria research reactor in Świerk (Poland). The specific activity of the radionuclide was 3.8 GBq/mg. The target was then dissolved in 0.1 M HCl.

⁴⁷Sc was produced by fast neutrons in the ⁴⁷Ti(n,p)⁴⁷Sc reaction on enriched (66 %) ⁴⁷Ti target. The 2 mg samples of ⁴⁷TiO₂ target were irradiated in the fast neutron channel of nuclear reactor in Świerk (Poland) for 143 h at fast neutron flux (>1 MeV) of 3 × 10¹³ n cm⁻² s⁻¹, and thermal neutron flux of 2.5 10¹⁴ n cm⁻² s⁻¹. The irradiated ⁴⁷TiO₂ was then dissolved in 1 ml of concentrated hydrofluoric acid (2–4 h with gentle heating at 80 °C) and the solution was diluted to 20 ml with 1 M HF. The solution was next passed through anion exchange bed (Dowex^® 1X8). ⁴⁷Sc was quantitatively eluted using the 0.04 M HNO₃ + 0.06 M HF solution. For additional purification the eluent was evaporated to dryness and the residue was dissolved in 0.1 M HNO₃. Afterwards ⁴⁷Sc was adsorbed on cation exchange column Dowex^® 50WX4 and finally eluted in two 1 ml fractions using 0.25 M ammonium acetate.

Syntheses of radiolabelled complexes

The experimental conditions for labelling, such as metal-to-ligand molar ratio and time of reaction were optimized to achieve high complexation yield. The ⁴⁶Sc complexes were synthesized by mixing 164 nmol of carrier added ⁴⁶Sc in chloride form with aqueous solutions of chelators in various molar ligand-to-metal ratios (1/1, 2/1, 5/1, 10/1). In the case of n.c.a. ⁴⁷Sc 1 MBq was used and the ligand amount varied from 2 to 15 nmol. The complexes were prepared at room temperature at pH = 6.0 (20 mM acetate buffer). The radiolabelling yield was determined by thin layer chromatography (TLC) using silica gel plates (Polygram, Macherey–Nagel). The NH₃/H₂O (1/25) mixture was used as the mobile phase. The distribution of activity on paper strips was measured by cutting the paper into 1-cm pieces and counting in the NaI (Tl) well counter. The ⁴⁶Sc-complexes moved with the solvent front (R _f = 1), while free ⁴⁶Sc remained at the starting point.

Stability of complexes

Stability of the ^46,47Sc acyclic complexes in isotonic NaCl solution and PBS buffer was assessed by adding 20 μl of radiocomplex solution to 500 μl of 0.9 % NaCl and 0.01 M PBS buffer. The solutions were incubated at 37 °C and kinetics of the Sc complex dissociation was measured by taking aliquots of the NaCl and PBS solution at different time points and measuring the liberated ⁴⁶Sc by ITLC analysis.

Results and discussion

Radiolabelling and kinetics

As already mentioned, the cyclic polyamino-polycarboxylate ligands like DOTA, due to formation of kinetically inert complexes, are excellent ligands for binding M³⁺ to biomolecules. DOTA labelled with ⁹⁰Y, ¹⁷⁷Lu, ²¹³Bi and ⁶⁸Ga is widely used in peptide radiopharmaceuticals such as octreotide, bombesin, substance P etc. However, complex formation with macrocyclic DOTA derivatives generally requires, in contrast to open-chain analogues, heating at elevated temperatures (>90 °C). Because the aim of our studies was elaboration of a method suitable for labelling proteins with ⁴⁷Sc, we studied at first the kinetics of formation of Sc-DOTA complexes at room temperature. Figure 1 presents kinetics of scandium complexation by DOTA at 25 and 70 °C.

Fig. 1.

Labelling yield of the DOTA ligand with ⁴⁶Sc at room and elevated temperature

As shown in Fig. 1 the kinetics of complexation at room temperature is slow, while increase of temperature to 70 °C drastically increases kinetics of labelling. Therefore, macrocyclic ligands, like DOTA, are not suitable for labelling of thermal non-resistant molecules, like proteins. In the present work, we examined selected acyclic polyamino-polycarboxylate ligands, which form complexes with scandium cations more rapidly than does DOTA. The ligands demonstrating high affinity for 3+ metal cations such as Fe³⁺, Ga³⁺ and lanthanides were selected for our studies. Structures of the ligands are presented in Fig. 2.

Fig. 2.

Structures of the ligands

Kinetics of ⁴⁶Sc complexation by open chain ligands was studied, in contrary to the DOTA, only at room temperature (Fig. 3). Comparison of Fig. 3. with the Fig. 1 shows much faster labelling at room temperature, of the open chain ligands than of the cyclic DOTA. In the case of DTPA the labelling yield after 10 min reached more than 99 % of the equilibrium value, while in the case of DOTA achievements of about 90 % value required more than 20 h. However, as shows in Fig. 3, the open chain ligand HBED seems, not to attain within 30 h equilibrium value.

Fig. 3.

Labelling yield of DTPA and HBED ligands with ⁴⁶Sc at room temperature

The labelling yield of acyclic ligands with ⁴⁶Sc was studied by us at different metal-to-ligand molar ratios, see Table 2. Our studies showed that at pH = 6.0 more than 96 % of ⁴⁶Sc was complexed with just 1:1 ligand-to-metal molar ratio.

Table 2.

Labelling of the ligands with carrier added ⁴⁶Sc

Sc:ligand molar ratio (Sc = 1.6×10−7 M)	Labelling yield (%)
	EGTA	TTHA	DTPA	DFO	BAPTA
1:1	99.3	98.0	99.1	96.2	98.2
1:2	100	100	100	100	99.0
1:10	100	100	100	100	100

In vitro stability studies

For radionuclide therapy applications, radionuclides must remain associated with the targeting chelate-protein to avoid the toxicity released in their dissociation. As shown in Fig. 4, all synthesized ⁴⁶Sc-complexes exhibited high stability in isotonic NaCl solution. After 120 h incubation in the NaCl solution more than 96 % of ⁴⁶Sc remained in complexed form. In 0.1 M PBS buffer ⁴⁶Sc-DTPA and ⁴⁶Sc-EGTA complexes were stable over the whole course of the experiment. Stability of ⁴⁶Sc-TTHA and ⁴⁶Sc-BABTA in PBS solution was very low due to replacement of ligands by phosphates in the first coordination sphere. In the case of ⁴⁶Sc-HBED slow decomposition of the complex was observed.

Fig. 4.

Stability of ⁴⁶Sc-BABTA, ⁴⁶Sc-TTHA, ⁴⁶Sc-EGTA, ⁴⁶Sc-DTPA and ⁴⁶Sc-HBED complexes in 0.9 % NaCl and 0.1 M PBS buffer

Labelling of the ligands with n.c.a. ⁴⁷Sc

The obtained results on complex formation and stability of ⁴⁶Sc complexes in PBS buffer shows that only Sc-DTPA and Sc-EGTA can be used as precursors for scandium radiopharmaceuticals. Therefore, we have chosen the two ligands for further studies with n.c.a. ⁴⁷Sc. Various concentrations of the ligands were investigated in order to evaluate their usability for binding ⁴⁷Sc to biomolecules. Radiolabelling was performed with quantities of the ligands from 2 to 15 nmol (Table 3). For comparison, results of labelling the commonly used macrocyclic ligand DOTA [8] is presented in Table 4.

Table 3.

Labelling of the open chain ligands with n.c.a. ⁴⁷Sc

Amount of ligand (nmol)	Labelling yield (%)
	DTPA	EGTA
2	38.81	97.20
5	94.89	98.80
10	95.40	99.40
15	100.0	100.0

Table 4.

Labelling of the macrocyclic ligand with n.c.a. ⁴⁷Sc

Amount of DOTA (nmol)	Labelling efficiency (%)
22.5	79.4
36.0	96.2
58.5	97.3

From comparison of the Table 3 with the Table 4 one can conclude that both ligands, DTPA and EGTA, form complexes with n.c.a. ⁴⁷Sc, in lower amount of ligand than those formed by DOTA. Particularly in the case of EGTA, only 2 nmol of the ligand is sufficient to achieve labelling higher than 97 %. The reason is that EGTA and DTPA as a 8-dentate ligand, forms strong complexes by binding via four carboxylic oxygen atoms, two ether oxygen atoms and two nitrogen atoms (EGTA) [18] or five carboxylic and tree nitrogen (DTPA). As was reported by Thakur et al. [19], contrary to our results Am³⁺, Cm³⁺ and Eu³⁺ form stronger complexes with DTPA than EGTA. Formation of stronger complexes by Sc³⁺ with EGTA than DTPA is probably related with stronger interaction of smaller and harder Sc³⁺ with ether than with carboxylic oxygen atoms.

Conclusion

In this paper formation and in vitro stability of a series of polyamino-polycarboxylate ligands labelled with ⁴⁶Sc and ⁴⁷Sc has been studied. We found that acyclic ligands (except HBED) form complexes much faster than the cyclic DOTA. The radiolabelling yield of acyclic ligands was very high and for the Sc:L molar ratio of 1:1 varied after 10 min from 96 to 99 %. The obtained complexes were stable in isotonic NaCl solution. However, stability of ⁴⁶Sc-TTHA, ⁴⁶Sc-BABTA and ⁴⁶Sc-HBED in PBS buffer was low, due to formation by Sc³⁺stronger complexes with phosphates than with TTHA, BABTA and HBED. We have shown that when using n.c.a. ⁴⁷Sc only 2 nmol of the EGTA is sufficient to obtain labelling yield greater than 97 %. Therefore, Sc-EGTA is a promising moiety for coupling ⁴⁷Sc to proteins. However, biological studies on animal models are needed for evaluation the in vivo stability of radiometal-labelled chelates.