Natalia Loktionova

A 44 Ti/ 44 Sc radionuclide generator for potential application of 44 Sc-based PET-radiopharmaceuticals

Abstract 44Ti/44Sc radionuclide generators are of interest for molecular imaging. The 3.97 h half-life of 44Sc and its high positron branching of 94% may stimulate the application of 44Sc labelled PET radiopharmaceuticals. However, both 44Ti production and 44Ti/44Sc generator design represent challenges for basic radiochemistry. About 5 mCi (185 MBq) of 44Ti were obtained via the Sc(p, 2n) nuclear reaction. 44Ti was separated from 1.5 g of massive scandium targets in multi-step procedures, including exchange chromatography on cation exchange resin (AG 50W-X8, 200−400 mesh, H+-form). In order to design a robust 44Ti/44Sc generator concept, distribution coefficients of Ti(IV) and Sc(III) on both AG 1-X8 (200−400 mesh, Cl--form) and AG 50W-X8 (200−400 mesh, H+-form) resins eluted with HCl and HCl/H2C2O4 solution of various concentrations were investigated systematically. Optimal conditions for efficient separations of both radionuclides have been determined for AG 1-X8 resin and mixtures of 0.07 M HCl and 0.005 M H2C2O4. A 5 mCi generator was prepared on an AG 1-X8 column (H=150 mm, D=3 mm, V0=0.55 mL). The system achieved elution of 180 MBq 44Sc in 20 mL of eluate solution. The breakthrough of 44Ti is 90 Bq. This corresponds to an excellent separation factor of 2×106. In the context of long-term stability of 44Ti/44Sc generators, a “reverse” type of washing steps after each elution using 0.07 M HCl/0.005 M H2C2O4 mixtures appeared to be essential.

Post-elution processing of 44Ti/44Sc generator-derived 44Sc for clinical application

The (44)Ti/(44)Sc (T(1/2)(44)Ti=60a) generator provides cyclotron-independent access to positron-emitting (44)Sc (T(1/2)=3.97d) for PET imaging. This work aims to post-elution processing of initial (44)Sc generator eluates in order to reduce its volume, HCl concentration and remove the oxalate anions. The on-line adsorption of (44)Sc on cationic resin AG 50W-X8 (200-400 mesh, H(+)-form) is achieved with >98% efficacy. Subsequently, the purified (44)Sc is desorbed by using 3ml of 0.25M ammonium acetate (pH=4.0). The post-processing takes 10min. The overall yield of the post-processing reached 90%, which is referred to the (44)Sc obtained from the (44)Ti/(44)Sc generator. In addition to the chemical purification, the content of (44)Ti breakthrough was further reduced by one order of magnitude. The 185MBq generator finally provides 150MBq of (44)Sc containing <10Bq of (44)Ti ready for labeling.

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

The positron-emitting radionuclide (44)Sc 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 (44)Ti/(44)Sc generator in Mainz, where long-lived (44)Ti decays to no-carrier-added (nca) (44)Sc. The (44)Sc 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 (44)Sc for labeling of DOTA-conjugated peptides. DOTA-D-Phe(1)-Tyr(3)-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 (44)Sc 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. (44)Sc-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 (Fe(3+), Ca(2+), Cu(2+), Mg(2+)), as well as other ligand competitors, like EDTA and DTPA, even after almost 25 h incubation at 37°C. Present study shows that nca (44)Sc forms stable complexes with the macrocyclic ligand DOTA and that (44)Sc-DOTATOC and analog targeting vectors may be synthesized for further preclinical and clinical investigations.

Improved column-based radiochemical processing of the generator produced 68Ga

An improved chemical strategy for processing of the generator produced (68)Ga was developed based on processing of the original (68)Ge/(68)Ga generator eluate on a micro-column. Direct pre-concentration and purification of the eluted (68)Ga is performed on a cation-exchange resin in hydrochloric acid/acetone media. A supplementary step based on a second micro-column filled with a second resin allows direct re-adsorption of (68)Ga eluted from the cation exchanger. (68)Ga is finally striped from the second resin with a small volume of pure water. For this purpose a strong anion exchanger and a novel extraction chromatographic resin based on tetraalkyldiglycolamides are characterized. The strategy allows online pre-concentration and purification of (68)Ga from the original generator eluate. The supplementary column allows transferring (68)Ga with high radionuclide and chemical quality in the aqueous solution with small volume and low acidity useful for direct radiolabeling reactions.

44Sc-DOTA-BN[2-14]NH2 in comparison to 68Ga-DOTA-BN[2-14]NH2 in pre-clinical investigation. Is 44Sc a potential radionuclide for PET?

AIM In the present study we demonstrate the in vitro and in vivo comparison of the (44)Sc and (68)Ga labeled DOTA-BN[2-14]NH(2). (44)Sc is a positron emitter with a half life of 3.92 h. Hence it could be used for PET imaging with ligands requiring longer observation time than in the case of (68)Ga. METHODS The binding affinity of (nat)Sc-DOTA-BN[2-14]NH(2) and (nat)Ga-DOTA-BN[2-14]NH(2) to GRP receptors was studied in competition to [(125)I-Tyr(4)]-Bombesin in the human prostate cancer cell line PC-3. A preliminary biodistribution in normal rats was performed, while first microPET images were assessed in male Copenhagen rats bearing the androgen-independent Dunning R-3327-AT-1 prostate cancer tumor. RESULTS The affinity to GRP receptors in the PC-3 cell line was higher for (nat)Ga-DOTA-BN[2-14]NH(2) (IC(50)(nM)=0.85 ± 0.06) than that of (nat)Sc-DOTA-BN[2-14]NH(2) (IC(50) (nM)=6.49 ± 0.13). The internalization rate of (68)Ga labeled DOTA-BN[2-14]NH(2) was slower than that of (44)Sc, but their final internalization percents were comparable. (68)Ga-DOTA-BN[2-14]NH(2) was externalized faster than (44)Sc-DOTA-BN[2-14]NH(2). The biodistribution of (44)Sc-DOTA-BN[2-14]NH(2) and (68)Ga-DOTA-BN[2-14]NH(2) in normal rats revealed a higher uptake in target organs and tissues of the first one while both excreted mainly through urinary tract. In microPET images both tracers were accumulated in the tumor with similar uptake patterns. CONCLUSIONS Despite the differences in the receptor affinity both the (68)Ga- and the (44)Sc-labeled DOTA-BN[2-14]NH(2) tracers showed comparable distribution and similar time constants of uptake and elimination. Moreover no differences in tumor accumulation (neither in the overall uptake nor in the dynamics) were observed from the microPet imaging. From that perspective the use of either (44)Sc or (68)Ga for detecting tumors with GRP receptors is equivalent.

Comparison of different phosphorus-containing ligands complexing 68Ga for PET-imaging of bone metabolism

Abstract 99mTc-phosphonate structures are well established tracers for bone tumour imaging. Our objective was to investigate different 68Ga-labelled phosphonate ligands concerning labelling kinetics, binding to hydroxyapatite and bone imaging using μ-PET. Seven macrocyclic phosphorus-containing ligands and EDTMP were labelled in nanomolar scale with n.c.a. 68Ga in Na-HEPES buffer at pH∼4. Except for DOTP, all ligands were labelled with >92% yield. Binding of the 68Ga-ligand complexes on hydroxyapatite was analysed to evaluate the effect of the number of the phosphorus acid groups on adsorption parameters. Adsorption of 68Ga-EDTMP and 68Ga-DOTP was >83%. For the 68Ga-NOTA-phosphonates an increasing binding with increasing number of phosphonate groups was observed but was still lower than 68Ga-DOTP and 68Ga-EDTMP. μ-PET studies in vivo were performed with 68Ga-EDTMP and 68Ga-DOTP with Wistar rats. While 68Ga-EDTMP-PET showed uptake on bone structures, an excess amount of the ligand (>1.5 mg EDTMP/kg body weight) had to be used, otherwise the 68Ga3+ is released from the complex and forms gallium hydroxide or it is transchelated to 68Ga-transferrin. As a result, the main focus of further phosphonate structures has to be on complex formation in high radiochemical yields with macrocyclic ligands with phosphonate groups that are not required for complexing 68Ga.

68Ge content quality control of 68Ge/68Ga-generator eluates and 68Ga radiopharmaceuticals – A protocol for determining the 68Ge content using thin-layer chromatography

(68)Ge breakthrough from a (68)Ge/(68)Ga-generator appears to be one of the most critical parameters for the routine clinical application of this generator and (68)Ga-radiopharmaceuticals. We report a TLC-based (thin-layer chromatography) protocol which allows the (68)Ge breakthrough of a generator to be determined within 1 h post-initial elution. The protocol can also be adapted to allow the (68)Ge content of a (68)Ga-radiopharmaceutical preparation to be determined prior to in vivo application.

Measurement of Protein Synthesis: In Vitro Comparison of 68 Ga-DOTA-Puromycin, [ 3 H]Tyrosine, and 2-Fluoro-[ 3 H]tyrosine

AIM Puromycin has played an important role in our understanding of the eukaryotic ribosome and protein synthesis. It has been known for more than 40 years that this antibiotic is a universal protein synthesis inhibitor that acts as a structural analog of an aminoacyl-transfer RNA (aa-tRNA) in eukaryotic ribosomes. Due to the role of enzymes and their synthesis in situations of need (DNA damage, e.g., after chemo- or radiation therapy), determination of protein synthesis is important for control of antitumor therapy, to enhance long-term survival of tumor patients, and to minimize side-effects of therapy. Multiple attempts to reach this goal have been made through the last decades, mostly using radiolabeled amino acids, with limited or unsatisfactory success. The aim of this study is to estimate the possibility of determining protein synthesis ratios by using (68)Ga-DOTA-puromycin ((68)Ga-DOTA-Pur), [(3)H]tyrosine, and 2-fluoro-[(3)H]tyrosine and to estimate the possibility of different pathways due to the fluorination of tyrosine. METHODS DOTA-puromycin was synthesized using a puromycin-tethered controlled-pore glass (CPG) support by the usual protocol for automated DNA and RNA synthesis following our design. (68)Ga was obtained from a (68)Ge/(68)Ga generator as described previously by Zhernosekov et al. (J Nucl Med 48:1741-1748, 2007). The purified eluate was used for labeling of DOTA-puromycin at 95°C for 20 min. [(3)H]Tyrosine and 2-fluoro-[(3)H]tyrosine of the highest purity available were purchased from Moravek (Bera, USA) or Amersham Biosciences (Hammersmith, UK). In vitro uptake and protein incorporation as well as in vitro inhibition experiments using cycloheximide to inhibit protein synthesis were carried out for all three substances in DU145 prostate carcinoma cells (ATCC, USA). (68)Ga-DOTA-Pur was additionally used for μPET imaging of Walker carcinomas and AT1 tumors in rats. Dynamic scans were performed for 45 min after IV application (tail vein) of 20-25 MBq (68)Ga-DOTA-Pur. RESULTS No significant differences in the behavior of [(3)H]tyrosine and 2-fluoro-[(3)H]tyrosine were observed. Uptake of both tyrosine derivatives was decreased by inhibition of protein synthesis, but only to a level of 45-55% of initial uptake, indicating no direct link between tyrosine uptake and protein synthesis. In contrast, (68)Ga-DOTA-Pur uptake was directly linked to ribosomal activity and, therefore, to protein synthesis. (68)Ga-DOTA-Pur μPET imaging in rats revealed high tumor-to-background ratios and clearly defined regions of interest in the investigated tumors. SUMMARY Whereas the metabolic pathway of (68)Ga-DOTA-Pur is directly connected with the process of protein synthesis and shows high tumor uptake during μPET imaging, neither [(3)H]tyrosine nor 2-fluoro-[(3)H]tyrosine can be considered useful for determination of protein synthesis.