Radiolabeling of DOTATOC with the long-lived positron emitter 44Sc

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Abstract

The positron-emitting radionuclide 44Sc with a half-life of 3.97 h and a β+ branching of 94.3% is of potential interest for clinical PET. As so far it is available from a 44Ti/44Sc generator in Mainz, where long-lived 44Ti decays to no-carrier-added (nca) 44Sc. The 44Sc is a trivalent metal cation and should be suitable for complexation with many well established bifunctional chelators conjugated to peptides or other molecular targeting vectors. Thus, the aim of this work was to investigate the potential of 44Sc for labeling of DOTA-conjugated peptides. DOTA-D-Phe1-Tyr3-octreotide (DOTATOC) was used as a model molecule to study and optimize labeling procedure. Reaction parameters such as buffer conditions, concentration of peptide, pH range, reaction temperature and time were optimized. Addition of 21 nmol of DOTATOC to 44Sc in ammonium acetate buffer pH 4.0 provided labeling yields >98% within 25 min of heating in an oil-bath at 95 °C. This time can be reduced to 3 min only by applying microwave supported heating. 44Sc-DOTATOC was found to be stable in 0.9% NaCl, PBS pH 7.4, fetal calf and human serums, and also in the presence of competing metal cations (Fe3+, Ca2+, Cu2+, Mg2+), as well as other ligand competitors, like EDTA and DTPA, even after almost 25 h incubation at 37 °C. Present study shows that nca 44Sc forms stable complexes with the macrocyclic ligand DOTA and that 44Sc-DOTATOC and analog targeting vectors may be synthesized for further preclinical and clinical investigations.

Highlights

► Labeling of somatostatin analouges with positron emitter 44Sc was tested. ► DOTATOC was labeled with 44Sc isotope. ► Optimal conditions for 44Sc-DOTATOC synthesis were found. ► Stability of 44Sc-DOTATOC was investigated in details in different solutions.

Introduction

Positron emission tomography has become a powerful and widely used imaging technology in the last decade. While the basic principles of PET are similar to those of SPECT, PET systems are generally more sensitive than SPECT, have better spatial resolution and provide the possibility of more accurate attenuation correction (Lodge et al., 2005, Rahmim and Zaidi, 2008). In fact, recent PET imaging is dominated by the use of short-lived radionuclides such as 18F, 11C, 13N and 15O produced in on-site accelerators and mostly applied in the same medical centers. However, PET could expand beyond the large medical centers and become a truly routine clinical tool in the case additional sources of long-lived positron-emitting radionuclides are available. One possibility is to supply hospitals with “non-standard/unconventional” PET radionuclides directly produced at cyclotrons, e.g. 76Br (16.1 h), 124I (4.2 d), 89Zr (78.4 h), 64Cu (12.7 h) or 86Y(14.7 h) (Haddad et al., 2008, Qaim, 2008, Hao et al., 2010).

Another source of positron-emitting radionuclides is a radionuclide-generator-system, similar to the 99Mo/99mTc generator commonly used in SPECT. For PET, such a generator should consist of a long-lived parent radionuclide absorbed on a column from which a shorter-lived, positron-emitting decay product is eluted by passing a suitable solvent through the column. The interest on application of metallic radionuclides, especially generator-based, for PET diagnosis is increasing during the recent years. An excellent example of this trend is a 68Ge/68Ga generator which provides high (85%) β+ branching 68Ga (T1/2=67.7 min) from long-lived 68Ge (T1/2=270.8 d) (cf. e.g. Roesch and Riss, 2010, for review). This generator is turning into an important source of new 68Ga-labeled radiopharmaceuticals for routine use in clinical PET/CT. However, PET imaging with 68Ga may be restricted by its rather short physical half-life, especially when proteins with long biological half-lives, like monoclonal antibodies or their fragments, are used. Therefore, intense studies focused on development of generators providing radionuclides with longer physical half-life, such as 72As (T1/2=26 h) from the 72Se/72As generator, or 44Sc (T1/2=3.97 h) from the 44Ti/44Sc generator (Rösch and Knapp, 2003). Especially, the latter radionuclide with its convenient physicochemical properties can find an application in PET. 44Sc has almost 4-times longer half-life and higher β+ branching than commonly used 68Ga. Its decay product nuclide, 44Ca, is stable and non-toxic. 44Sc can be obtained from a 44Ti/44Sc generator. 44Ti is obtained in reaction 45Sc(p, 2n)44Ti and decays by electron capture into 44Sc. The 44Ti, with its long half-life of 59.2±0.6 years, could provide a cyclotron-independent source of 44Sc for several decades (Ahmad et al., 1998, Alenitzky et al., 2005, Filosofov et al., 2010). Several approaches have been investigated in the past to develop the chemistry of 44Ti/44Sc generators (Greene and Hillman, 1967, Mirza and Aziz, 1969, Seidl and Lieser, 1973, Schumann et al., 2007). Recently, Filosofov et al. (2010) described a 185 MBq (5 mCi) generator system with the relevant radiochemical parameters, such as >97% elution efficacy for 44Sc and very low breakthrough of <5×10−5% of 44Ti. This generator was further studied in the terms of post-processing of the eluate in order to provide high radiochemical purity and radionuclide concentration batches of 44Sc in an aqueous system applicable to subsequent labeling reactions (Pruszyński et al., 2010).

Direct production of 44Sc consists in bombarding enriched with 44Ca target with proton beam (Haddad et al., 2008, Kamel et al., 2011). The production of this radionuclide by ARRONAX group is considered as a “great interest at short term” together with other PET radionuclides like 64Cu, 68Ga, 82Rb or 124I, and even before other positron emitters such as 86Y, 89Zr or 52Fe (8.3 h) (Haddad et al., 2008).

The 44Sc with its physicochemical properties of a trivalent rare earth metal appears to be appropriate candidate in PET/CT diagnosis. It may be used to synthesize radiopharmaceuticals based on bifunctional chelators (DOTA, DTPA, NOTA, etc) established to coordinate currently used trivalent radionuclides in diagnosis and therapy, such as 68Ga and 111In or 90Y and 177Lu, as well as non-radioactive Gd(III). As a relatively longer-lived β+ emitter, it could be used for more accurate planning and dosimetric calculations in endoradiotherapy based on the radionuclides mentioned above, but also for direct matching β emitting 47Sc radiopharmaceuticals (Mausner et al., 1995). Thus, 44Sc/47Sc can join the other matched-pairs of β+/β radionuclides, which would permit coordinated dosimetric PET imaging and therapy.

Macrocyclic chelators are well known to form stable complexes with metal cations and are of great interest for radiopharmaceutical design. DOTA-conjugated peptides, such as octreotide and octreotate derivatives or substance P, as well as some small proteins, e.g. affibodies or nanobodies, are readily labeled with metal radionuclides e.g. 68Ga, 90Y, 177Lu or 213Bi (Breeman et al., 2003, Cordier et al., 2010, Tolmachev et al., 2010, de Blois et al., 2011).

The aim of this work was to determine optimal conditions for radiolabeling DOTA-conjugated octreotides using DOTATOC as a model molecule. Parameters that influence reaction kinetics, like incubation time and temperature, amount of chelate-peptide and pH of the reaction were investigated. Influence of microwave supported heating on time and completeness of complexation reaction was compared with the conventional heating method in an oil-bath. Stability of the formed conjugate was checked in 0.9% NaCl, phosphate buffered-saline (PBS, pH 7.4), fetal calf and human serums, and in the presence of different metal cations as well as other competing chelators, like EDTA and DTPA.

Section snippets

Chemicals and reagents

DOTA-D-Phe1-Tyr3-octreotide (DOTATOC) was obtained as GMP-grade from piChem R&D (Graz, Austria) and an aqueous stock solution of 1 μg/μL was prepared. All chemicals were analytical or pure reagent grade and used as received unless otherwise specified. Deionized Milli-Q water (18.2  cm; Millipore) was used in all reactions. Fetal Calf Serum (FCS) and Human Serum Albumin (HSA) were purchased from Sigma-Aldrich (USA).

44Sc and the 44Ti/44Sc radionuclide generator

44Sc was available from 44Ti/44Sc generator system developed in Mainz with 44Ti

Results and discussion

Macrocyclic chelators are widely used in radiopharmacy for metallic radionuclides complexation, because they offer higher stability compare to acyclic ones, e.g. thermodynamic stability constant (log K) of Sc(III) with DOTA is 27.0, whereas for Sc(III)-DTPA it is 20.99 (Majkowska-Pilip and Bilewicz, 2011, Masuda et al., 1991). However, the complex formation with macrocyclic DOTA derivatives generally requires heating at elevated temperatures in contrast to open-chain analogs. Therefore,

Conclusions

Synthesis of DOTATOC with the new generator-derived PET radionuclide 44Sc was investigated in details. Incorporation of 44Sc into DOTATOC was almost quantitative (>98%) at pH 4.0 after 25 min heating in an oil-bath at 95 °C. This time can be significantly reduced to 3 min only when microwave heating is adopted for synthesis. We also performed optimization studies with DOTA-D-Phe1-Tyr3-octreotate (DOTATATE, abx, Germany) and obtained the same results. Therefore, only experimental data for 44

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (DFG Ro 985/18) and the European Commission Grant (FP 6, ToK, POL-RAD-PHARM, MTKD-CT-2004-509224). European Union COST Actions BM0607 and COST D38 are acknowledged for their support.

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