Year : 2011 | Volume
: 10 | Issue : 1 | Page : 73--89
Abstracts of Poster Presentations (Chemistry)
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Gallium-68 Labelling of NOTA-peptide Conjugates for Imaging Infection - Especially Tuberculosis
T. Ebenhan 1,3 , N. Chadwick 2 , T. Govender 2 , H.G. Kruger 3 , M. Sathekge 4 , J.R. Zeevaart 5
1 Radiochemistry, Nuclear Energy Corporation of South Africa, Pretoria, 2 School of Pharmacy and Pharmacology, UKZN Westville Campus, Durban, 3 School of Chemistry, UKZN Westville Campus, Durban, 4 Nuclear Medicine Department, Steve Biko Academic Hospital, Pretoria, 5 School of Pharmacy, North West University, Potchefstroom, South Africa
Objective: Anti-tuberculosis active compounds are the key to effective control and early diagnosis of the infection in South Africa. Known anti-TB active peptides, conjugated with 1,4,7-triazacyclononanetriacetic acid(NOTA)and radiolabelled with Gallium-68 are to be evaluated via pre-clinical trials on small laboratory animals and on primates for prospective PET-imaging of tuberculosis. 
Materials and Methods: Peptides were synthesised on 0.10 mmol scale using a CEM microwave peptide synthesizer by standard Fmoc/tert-butyl (tBu)-solid phase synthesis. Following cleavage from the resin, the peptides were purified using semi-preparative HPLC running a methanol:water gradient on a C-18 column. Sample purity was determined using LC-MS. The NOTA group was conjugated with peptides using solid-phase synthesis.  Cytotoxicity was tested using MTS assay in Vero cells. Prior to radiolabeling 110 nmol gallium trichloride was added to 20 nmol NOTA-peptide for 5 min at room temperature to allow for chelating followed by LC-MS to determine the ratio of bound to unbound Gallium. Radiolabeling of a NOTA-peptide with Gallium-68 was performed in adaption to DOTATOC labeling.  DOTATATE and c(RGDyK)SCN-Bz-NOTA (Kits)  were used as reference peptides for radiolabeling. Quantitative and qualitative analysis was performed on ITLS-SG using 0.1 M citric acidas eluent. Radiochemical purity was determined by scanning chromatograms on a TLC scanner using a gamma radiation detector followed by ROI analysis.
Results: The NOTA-conjugated peptides are non-toxic, considering the nanomolar concentration administered to patients; however cytotoxicity for free gallium (III) chloride is being determined. At pH 7 >99% Gallium is chelated into NOTA-peptides. Radiolabeling of a NOTA-peptide shows sensitivity for both, pH and temperature. Optimally, a yield range of 77-85% for 10 min heating on 85 degrees Celsius at pH 6 was determined. Gallium-68-DOTATATE shows a yield range of 77-83% (radiochemical purity >97%) whereas labeling yield range c(RGDyK)SCN-Bz-NOTA is 67-80% (radiochemical purity 72-88%).
Conclusion: Radiolabeling of both, reference peptides and NOTA-peptide was carried out successfully. Thus, the Gallium-68-labeled NOTA-peptide may significantly contribute to improved early diagnosis and treatment of TB. Unlike Gallium-68-DOTATATE, all peptides, conjugated with NOTA, have still to be investigated towards higher radiochemical purities.
1. M. Sathekge, Nucl Med Commun 2008; 29:663-5.
2. B. Guérin et al. Org Lett 2010; 12:280-3.
3. W.A. Breeman et al. Eur J Nucl Med Mol Imaging 2005; 32:478-85.
4. J.M. Jeong et al. J Nucl Med 2008; 49:830-6.
Ga-68 Labelling of RGD Peptides for Imaging Angiogenesis
P. Knetsch 1 , M. Petrik 1 , C. Rangger 1 , A. Helbok 1 , M. Fani 3 , B. Pichler 2 , E. von Guggenberg 1 , H.-J. Pietzsch 4 , I. Virgolini 1 , C. Decristoforo 1 , R. Haubner 1
1 Department for Nuclear Medicine, Medical University Innsbruck, Austria, 2 Department of Preclinical Imaging, Universtiy of Tübingen, Germany, 3 Department of Nuclear Medicine, University of Freiburg, Germany, and 4 Research Center Rossendorf, Germany
Ga-68 attracts increasing interest in molecular imaging with PET, based on its favourable properties and generator availability, especially for radiolabelling of peptides. An interesting molecular target involved in the angiogenic process is the αvβ3 integrin. It has been demonstrated in preclinical as well as in clinical studies that radiolabeled RGD-peptides such as [ 18F]Galacto-RGD allow non-invasive monitoring of αvβ3 expression using PET. The preparation of [18 F]Galacto-RGD is time consuming and requires purification steps in the synthesis. Here we report on the development of a 68 Ga-labeled RGD-peptide selected from a small series of different bifunctional chelators. Peptides were synthesised using standard SPPS protocols. After cyclisation in solution and selective deprotection of the amino function of the lysine the chelating moieties were conjugated via in situ activation. The chelating systems include 1,4,7,10-tetraazacyclododecane-1,4,7,10-acetic acid (DOTA), a 1,4,7-triaaza-10-oxocyclododecane-1,4,7-acetic acid derivative (B505), 1,4,7-triaazacyclononane-4,7-acetic acid-1-2-glutaric acid (NODAGA), and a tris (2-mercaptoethyl) amine derivative (NS3). Labelling was carried out using the fractionated elution method in sodium acetate or phosphate buffer, respectively. In vitro evaluation included determination of the partition coefficient, protein binding properties, metabolic stability, binding affinity, and cell uptake characteristics. In vivo evaluation was carried out using nude mice bearing alpha(v)beta3-positive and alpha(v)beta3-negative tumors. For all tracer biodistribution data were collected. For the most promising also small animal PET imaging was carried out. All peptides could be labelled with Ga-68 in good radiochemical yields. Labelling of NODAGA-RGD could be achieved even at room temperature. Whereas labelling of NS3-RGD has to be followed by Seppak separation to obtain the product in high radiochemical purity. The compounds showed comparable partition coefficients, binding affinity for the alpha(v)beta3 integrin as well as receptor specific uptake. However, great differences were found in the protein binding properties. Out of the four peptides tested only NODAGA-RGD showed low protein binding. This is also reflected in the biodistribution data. Lowest activity concentration in blood and best tumor/background ratios were found for NODAGA-RGD. Subsequent small animal imaging showed best imaging properties for NODAGA-RGD, which seems to be comparable with F-18-Galacto-RGD. In this series NODAGA-RGD revealed most promising properties for molecular imaging applications. Easy radiolabelling at room temperature, low amount of protein bound activity and the resulting lower activity concentration found in blood compared to the other compounds makes it to an interesting alternative to F-18-Galacto-RGD for imaging alpha(v)beta3 expression combining easy accessibility with high stability and good imaging properties with considerable advantages over other bifunctional chelators.
Imaging of Protein Synthesis: Comparison of 68 Ga-DOTA-Puromycin, [3H]tyrosine and 2-fluoro-[3H]tyrosine
Sebastian Eigner, R. Denis, Beckford Vera, Marco Fellner, Natalia S. Loktionova, Markus Piel, Frantisek Melichar, Frank Rösch, Tobias L. Ross, Ondrej Lebeda, Katerina Eigner Henke
Nuclear Physics Institute, Academy of Science of the Czech Republic, Institute for Nuclear Chemistry, Johannes Gutenberg-University Mainz, Germany
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-tRNA [1,2] (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 anti-tumor-therapy, to enhance long time survival of tumor patients and minimize therapy caused side effects. Multiple attempts to reach this goal have been made through the last decades, using mostly radiolabeled amino acids, with limited or unsatisfactory success.  The aim of this study was to estimate the possibility of determining protein synthesis ratios with 68 Ga-DOTA-Puromycin ( 68 Ga-DOTA-Pur) [3H]tyrosine and 2-fluoro-[3H]tyrosine and to estimate the possibility of different pathways due to the fluorination of tyrosine.
Materials and Methods: DOTA-Puromycin was synthesized using a puromycin-tethered CPG support by the usual protocol for automated DNA and RNA synthesis following our design. 68 Ga was obtained from 68 Ge/ 68 Ga-generator as described previously by Zhernosekov et al. in 2007.  The purified eluate was used for labeling of DOTA-Puromycin at 95°C for 20 minutes. [3H]tyrosine and 2-fluoro-[3H]tyrosine have been purchased with the highest purity available 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 PC-3 prostate carcinoma cells (ATCC, USA). 68 Ga-DOTA-Pur was additionally used for μPET imaging of Walker carcinomas and AT-1 tumors in rats. Dynamic scans were performed for 45 minutes after IV application (tail vein) of 20-25 MBq of 68 Ga-DOTA-Pur.
Results: No significant differences in the behavior of [3H]tyrosine and 2-fluoro-[3H]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 is 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 uptake of 68 Ga-DOTA-Pur is directly connected with the process of protein synthesis and shows high tumor uptake during μPET imaging, neither [3H]tyrosine nor 2-fluoro-[3H]tyrosine, can be considered usefully for determination of protein synthesis.
Acknowledgement: This work has been supported by the Ministry of Education, Youth and Sports of the Czech Republic and The Grant Agency of the Czech Republic.
1. Nathans D, Neidle A, Structural requirements for puromycin inhibition of protein synthesis. Nature 1963;197:1076-7.
2. Nathans D, Puromycin Inhibition of Protein Synthesis: Incorporation of Puromycin into Peptide Chains. Proceedings of the National Academy of Sciences of the United States of America 1964;51:585-92.
3. Haubner R, PET radiopharmaceuticals in radiation treatment planning - synthesis and biological characteristics. Radiotherapy and oncology : Journal of the European Society for Therapeutic Radiology and Oncology 2010;96:280-7.
4. Zhernosekov KP, Filosofov DV, Baum RP, Aschoff P, Bihl H, Razbash AA, et al. Processing of generator-produced 68 Ga for medical application. Journal of nuclear medicine : official publication, Society of Nuclear Medicine: 2007;48:1741-8.
Mono- and Bivalent Ga-68-Labeled Folates
Johanna Seemann 1 , Cristina Müller 2 , Tobias L. Ross 1
1 Institute of Nuclear Chemistry, Johannes Gutenberg-University, Fritz-Strassmann-Weg 2, 55128 Mainz, Germany and 2 Paul Scherrer Institute, 5232 Villigen, Switzerland
Aim of the Study: The folate receptor presents an ideal target for receptor-based tumor imaging and targeting as it is overexpressed on a variety of malignant human cancer cell lines. A diversity of folic acid derivatives tested as radiopharmaceuticals and imaging agents showed already the success of this principle. Monovalent radiofolates labeled with Ga-67/68 were already synthesized using non-cyclic chelating agents and recently also the cyclic chelator DOTA. In contrast to DOTA, the cyclic bifunctional chelator (BFC) DO2A allows a stable complexation of Ga-67/68 and additionally the coupling to more than one targeting moiety and thus to take advantage of multivalency effects.
The aim of this project is the development of new multivalent Ga-68-radiofolates for PET-imaging using DO2A as chelating moiety. Comparing hexyl- and PEG-spacers in mono- and bifunctional Ga-68-radiofolates will give information about effects on affinity, pharmacokinetics and stability in vitro and in vivo. Moreover, these comparative studies will show the influence of multivalency effects on the folate receptor system. To our best knowledge, these are the first studies regarding multivalent radiofolates.
Materials and Methods: Glutamic acid was coupled with an azidopropyl-spacer in the γ-position. Reaction with protected pteroic acid yielded the corresponding azido-functionalized folic acid derivative. The synthesized BFC was derivatized with one or two hexyl- or PEG-spacers. A terminal alkyne group was introduced to enable subsequent click-reactions with the azido-folate. Preliminary labeling with Ga-68 was performed in 0.25M HEPES buffer at 95°C for 10 minutes.
Results: The folic acid moiety and four different mono- and bivalent chelating building blocks were synthesized. The conditions for the click-reaction were tested on a model derivative before applying it to the azido-folate molecule. HPLC-monitoring showed complete conversion within 4h for the model derivative whereas the folate-compound needed 12h to yield the desired triazole structure. First radiolabeling tests resulted in promising labeling yields after 10 minutes.
Summary: Folic acid is an ideal targeting-vector addressing the folate receptor on malignant cells and inflammatory tissues. Mono- and bivalent Ga-68-DO2A-folates are ready to confirm this via in vivo studies using μ-PET imaging. The high efficiency and selectivity of the click-reaction was proved by a model system and successfully transferred to the accordant folic acid derivative. Optimization of the Ga-68-labeling and first in vitro and in vivo evaluations using KB cells and μPET imaging are in progress.
Synthesis of Gallium-68-NODAPA-NCS-Glibenclamide for ß-cell Imaging
Melanie Zimny, V. Nagel, F. Rösch
Institute of Nuclear Chemistry, Johannes Gutenberg University, Mainz, Germany
Aim of the Study: More than 246 million people worldwide suffer from diabetes mellitus which is a metabolic disease. This large number of patients can be divided into two major groups: Type 1 and 2 diabetes. In type 1 diabetes an autoimmune attack causes the loss of most of the insulin-producing β-cells of the islets of Langerhans in the pancreas. This results in an absolute deficiency of insulin and the patient has to be treated with insulin. At the moment a lot of effort is put into the field of transplantation of β-cells, but till now a suitable method for monitoring or to control the success of the transplantation is lacking. An interesting target to access the β-cell mass might be the SUR1 (sulfonylurea receptor 1) which is almost exclusively expressed on β-cells. Several hypoglycemic drugs have a high binding affinity to this receptor and are therefore used in the treatment of type 2 diabetes (e.g. nateglinides, repaglinides and glibenclamide). On this basis various 18F-labelled radiotracers have been developed but due to their high lipophilicity, a pronounced liver uptake precluded a visualization of the pancreatic β-cells. The incorporation of the positron-emitter 68Ga via the NODAPA-NCS chelator helps to improve the hydophilicity and is assumed to provide improved pharmacokinetics enabling quantification of the pancreatic β-cells mass in vivo by positron-emission-tomography (PET).
Materials and Methods: The modificatedglibenclamide was synthesized starting from 5-chlorhydroxybenzoic acid. At first, the carboxyl group was protected by esterification. The linker tert-butyl-3-bromopropyl-carbamate was attached. The ester was cleaved by using a mixture of NaOH and ethylenedioxine. The free carboxyl function was converted into an activated ester in situ by use of ethyl chloroformate and subsequently 4-(2-aminoethyl)-benzsulfonamide was coupled. Via a Cu(I)-catalysed reaction N-cyclohexylformamide was attached. The Boc-protecting group of the linker was removed using TFA. The obtained glibenclamide derivative was coupled to NODAPA-NCS by thiourea formation.
Results: 5-Chlorhydroxybenzoic acid was converted into the corresponding ester by using a mixture of sulphuric acid and methanol in 88% yield. Under basic conditions the tert-butyl-3-bromopropylcarbamate was attached at the phenolic hydroxyl group and the ester was cleaved in good yields. Ethyl chloroformate was added to produce an activated intermediate which was coupled with 4-(2-amino-ethyl)benzoesulfonamide in 84% yield. Cyclohexylisocyanate was used in a copper(I)-catalysed reaction to obtain the glibenclamide derivative (43%). For deprotection of the linker group the sulfonylurea was stirred in TFA (almost quantitative). The glibenclamide derivative was coupled under basic conditions to NODAPA-NCS to give the corresponding thiourea.
Summary: We successfully synthesized a new NOTA-derivative for SUR 1 targeting. After purification and deprotection of the NODAPA-NCS-glibenclamide, this tracer with improved hydrophilicity will be labelled with 68 Ga and used for in vivo PET imaging of the pancreas.
Acknowledgement: This work is supported by a fellowship of the Dr. Georg Scheuing-Foundation.
Synthesis of NODAPA-glucosamine Derivatives for Imaging Arthrosis using the PET-nuclide 68 Ga
Verena Nagel, Patrick Riss, Frank Rösch
Institute of Nuclear Chemistry, Johannes Gutenberg University Mainz, Fritz-Strassmann Weg 2, 55128 Mainz, Germany
Aim of the Study: Arthrosis is a prevalent joint disease lot of people over 65 years are suffering from. The loss of cartilage protecting the bone surface leads to pain and stiffness. As the small biomolecule glucosamine is part of the cartilage and synovial liquid, it is used in the treatment of arthrosis and is therefore a potential targeting vector in molecular imaging of arthrosis.  Already developed 99mTc-labeled glucosamine derivatives showed promising uptake in the cartilage  at patients with manifested arthrosis. 68 Ga, with the half-life of 68 minutes and its availability by the 68 Ge/ 68 Ga-generator, is a favoured PET-nuclide. 1,4,7-triazacyclononan-1,4,7-triacetic acid (NOTA) is an established chelator for 68 Ga and needs a free functionality for attaching targeting vectors. The isothiocyanate group is a convenient moiety which allows mild coupling reactions with biomolecules carrying a free amino function that forms rapidly thioureas as products. Two versatile p-isothiocyanatephenyl derivatives of NOTA have been developed. In order to be able to image arthrosis applying the PET technique, glucosamine was used as targeting vector.
Materials and Methods: Triazacyclononan (TACN) in MeCN and K 2 CO 3 as base was coupled with 2-(p-nitrophenyl)-2-bromo acetic acid tert-butylester over night at RT. Mono- and dialkylated products were isolated by column chromatography. Alkylation with tert-butyl-bromo acetate resulted in the NODAPA-(tert-bu) 3 products. Reduction of the nitro moiety was facilitated by catalytic hydrogenation of NODAPA-(tert-bu) 3 -(NO 2 )n in MeOH/H 2 O 9:1 with Pd/C (5%) within 2h. NODAPA-(tert-bu) 3 -(NH 2 )n was dissolved in a mixture of CHCl 3 / NaHCO 3 -solution and cooled to 0°C. After adding thiophosgene dropwise, stirring 1h at RT yielded the pure NODAPA-(tert-bu) 3 -(NCS)n. Glucosamine hydrochloride in a MeOH/NaHCO 3 -solution was stirred for 30 min at RT, followed by the addition of NODAPA-(tert-bu) 3 -(NCS)n. After 1h, the solvent was evaporated and the residue re-dissolved in MeOH, filtered and concentrated. The hydroxyl groups of glucosamine were protected as acetoxy functionalities by using acetic anhydride to enable subsequent deprotection of the tert-butyl esters.
Results: TACN was converted with 2-(p-nitrophenyl)-2-bromo acetic acid-tert-butylester resulting in the mono (58%) and divalent (13%) product. Reaction with tert-butyl bromo acetate gave NODAPA-(tert-bu) 3 in good yields. Reduction of the nitro moiety was successfully performed using 5% Pd/C. The amino compounds were isolated in high yields (80% NODAPA-(tert-bu) 3 -NH2 and 71% NODAPA-(tert-bu) 3 -(NH2)2). Without further purification, the reaction with thiophosgene at pH 9 in CHCl 3 /H 2 O yielded NODAPA-(tert-bu) 3 -(NCS)n in high yields (79% NODAPA-(tert-bu) 3 -NCS and 74% NODAPA-(tert-bu) 3 -(NCS) 2 ). The targeting vector glucosamine hydrochloride was coupled to NODAPA-(tert-bu) 3 -(NCS)n under basic conditions. Both thiourea products were isolated in excellent yields (90% NODAPA-(tert-bu) 3 -glucosamine and 81% NODAPA-(tert-bu) 3 -(glucosamine) 2 ). Acetylation of the hydroxyl functions gave the fully protected products in quantitative yields.
Summary: Two new complex NOTA derivatives, mono- and divalent NODAPA-(tert-bu) 3 -(NCS)n have been synthesized. The targeting vector glucosamine to image joints affected by arthrosis was coupled to both isothiocyanate derivatives successfully. Conversion with acetic anhydride yielded the completely protected molecules. After subsequent deprotection, the new NOTA precursors are available for 68 Ga-labeling.
Ga(III) Complexes of NOTA-Bisphosphonate Ligands - New Probes for Bone Targeting
Jan Holub, Vojtěch Kubíček, Petr Hermann
Chearles University in Prague, Department of Inorganic Chemistry; Hlavova 2030, 128 40 Prague 2, Czech Republic
Bisphosphonates are known to show high affinity to bones and other calcified tissues. In radiodiagnostic methods, a lot of attention is aimed at bone targeting compounds due to diagnosis of bone metastases. Recently, we have studied 68 Ga complexes with DOTA-like ligands bearing bisphosphonate in a side chain.  The complexes should serve as a PEt alternative to well established SPECT diagnosis of bone metastases with 99mTc-bisphosphonates. The complexes show excellent uptake in metastatic tissue. However, the cavity of DOTA-like ligands is not the best choice for Ga(III) ion due to high denticity (8 donor groups) and large diameter. Thus, complexation of Ga(III) ion with DOTA-like ligands is limited to a narrow pH range. Among macrocyclic ligands, NOTA and its analogues fit best the size and nature of Ga(III) ion and, thus, Ga-NOTA complex shows high thermodynamic stability and kinetic inertness. In order to improve coordination properties of our chelators, we have studied NOTA analogues that are modified with distant bisphosphonate. The bisphosphonate group is attached through an amide or phosphinate pendant arm. The ligands were synthesized in large amounts by multistep procedures starting from 1,4,7-triazacyclononane. Complexation of Ga(III) and Fe(III) ions was studied by means of NMR and UV-VIS spectroscopy. The complexes were tested using agueous suspension of hydroxyapatite as a model for bone tissue. Similar to other bisphosphonates, the complexes show good binding. This makes them good candidates for oncomming studies as potential imaging probes for diagnosis of bone tissue and metastases.
1. M. Fellner, R. P. Baum, V. Kubíček, P. Hermann, I. Lukeš, V. Prasat, F. Rösch; Eur. J. Nucl. Med. Mol. Imaging 2010, 37, 834
A New High Efficient NaCl Based Cationic 68 Ge/ 68 Ga Generator Eluate Purification
D. Müller, I. Klette, R.P. Baum
Zentralklinik Bad Berka, Robert Koch Allee 9, 99437 Bad Berka, Germany
Aim of the Study: The aim of our investigation was to develop efficient 68-Ga labelling procedures for a routinely available application in the clinical praxis. The purification procedure for the 68-Ge/68-Ga eluate should reduce the handling with concentrated HCl, should yield the labelled final product in a high yield with a high purity and the use of acetone or other organic solvents should be avoided.
Materials and Methods: All reagents were purchased from commercial sources and used as received. The mentioned cartridges are commercially available. For all experiments a 68-Ga generator from Obninsk (Eckert & Ziegler Europe) and IGG100 68-Ga generator (Eckert & Ziegler Europe) were used. Two generators were connected and the older generator was eluted through the new one with 10 mL of 0.1 M HCl (Merck, Germany). The 68-Ga of the generator eluate was collected on a SCX cartridge and directly eluted with a hydrochloric solution of sodium chloride into the reaction vial with 40 μg of DOTATOC, 3 mL water and 0.5 ml ammonium acetate buffer.
Results: After heating the solution for seven minutes at 90°C the reaction is finished. The concentration of unbounded 68-Ga is lower than 5%. The radiochemical purity of the labelled DOTATOC is higher than 95%. The reaction mixture contains no toxic substances or substances of concern so that no subsequent purification is not necessary. After a steril filtration the radiochemical yield is about 82% (n.d.c.) and 17% higher than the cationic labelling based on an elution of the activity with the help of a acetonic hydrochloric acid as described by Zhernosekov et al. (J Nucl Med 2007;48:1741-1748).
Summary: We could develop a new high efficient NaCl based cationic 68 Ge/ 68 Ga generator eluate purification.With this procedure the radiochemical yield for the labelling of DOTATOC with 68-Ga is about 82%. A subsequent purification step is not necessary.
The Combined Cationic-anionic Purification of the 68 Ge/ 68 Ga Generator Eluate for the Labelling of Fragile Peptides
D. Müller, I. Klette, R.P. Baum
Zentralklinik Bad Berka, Robert Koch Allee 9, 99437 Bad Berka, Germany
Aim of the Study: In the case of larger and fragile peptides or proteins like DOTA conjugated Affibody the cationic concentration of the generator eluate as described by Zhernosekov et al. (J Nucl Med 2007;48:1741-1748) does not work. A higher purity of the 68 Ga is necessary for a successful labelling. The basic idea for this procedure was the combination of the cationic followed by a subsequent anionic purification method. The purification procedure for the 68-Ge/68-Ga eluate should reduce the volume of the used concentrated HCl, the volume of 68-Ga activity and should deliver the labelled final product in a good yield with a high purity and the use of acetone or other organic solvents should be avoided.
Materials and Methods: The 68-Ga generators were eluted with a total of 10 mL 0.1 M HCl and the 68-Ga was collected on the SCX cartridge. The activity was eluted with 1.0 mL 5.5 M HCl and directly colleced on the SAX anionic exchanger cartridge. After drying of the cartridge with a steam of helium (or air) for 1 min, 60% of the 68Ga were eluted with 0.4 - 1.0 mL water into the reaction vial with 0.4 mL 1.5 M HEPES buffer and 100 μg of DOTA-Affibody (DOTA-ZHer2:342-pep2) with a subsequent heating to 80°C for 5 minutes.
Results: This procedure leads to a final product with a radiochemical purity greater than 95% without further purification steps. The described purification delivers 68-Ga in a high chemical and radiochemical purity.
Summary: We could develop a purification procedure for 68-Ga which removes efficient foreign ions. This method allow the labelling of fragile peptides with a higher molecular mass than DOTATOC like DOTA-Affibody (DOTA-ZHer2:342-pep2) or proteins.
Quality and Validation of Silica-based Resin Column of 68 Ge/ 68 Ga Generator for Use in Medicinal Product
Wilberforce Oware, K.K. Solanki, N. Bird and C. Solanki
Radiopharmacy, Department of Nuclear Medicine, Cambridge University Hospital NHS Foundation Trust. Cambridge, UK
Introduction: None of the commercially available gallium generators in UK has a product licence however under EU guide to GMP Annex 15 all facilities, equipment and process which may affect quality of medicinal product should be validated before use. Secondly, there are concerns about maintenance of sterility with prolonged use of these generators. Thirdly presence of metallic content and acidity during radiolabelling of peptide results in poor yield and so far the 68 Ge/ 68 Ga generators in the market mainly contain a modified SnO 2 or TiO 2 glass column which could potentially breakthrough. More significantly it is not easy to detect these in clinical environment. A new generator from ITG is a silica-based resin column with attached organic molecule for efficient 68 Ga using 0.05M acid elution is a novel approach. We present the first UK validation of silica-based resin column 68 Ge/ 68 Ga generator.
Aim: This study aims to undertake comprehensive validation programme to comply with EU GMP Annex 15. Another is to evaluate and examine the advantages and limitations of this generator.
Materials and Methods: A validation master plan was established which included optimal conditions for aseptic preparation and radiation protection. Assessment included establishment and standardization of sterile suprapure solvent (0.05M HCl), bio-burden, elution speed, column yield, fractionation and build-up. Eluate ( 68 GaCl 3 ) was analysis for radionuclide, radiochemical purity, pH, 68 Ge breakthrough, trace metal impurities, LAL and sterility. A radiation risk assessment was also performed.
Results: Elution profile: The optimal elution solvent was 0.05M HCl. The optimal volume for elution was 4.0mL (yield=81%±2.1 n=20). First 1.5mL using fractionation elution yield 85%±1.1, n=20 of total radioactivity. Quality Control Parameters: Radionuclide purity (99.97%%±0.02 n=20, Radiochemical purity (95.90%%±0.8 n=20), pH (4.3%±0.1, n=20) Sterility: (0 cfu, n=4), LAL (<175EU n=4). Microbiologically TSA 90mm plates exposure during elution were also within limits. Radiation Protection: A 50mm lead to house the generator had a surface dose <1.2uSv/hr. However during elution, the surface dose rate increased to 20uSv/hr. Dose rate on surface of 20 mm lead shielded vial containing 250 MBq was ~ 0.8mSv/hr. The time in contact during elution was <1 min corresponding to a skin dose of <15uSv.
Conclusion: The systematic validation was useful. Novel non-metallic column 68 Ge/ 68 Ga generator system has been validated to EU GMP Annex 15. However ongoing process, pharmaceutical, microbiology assessment continues. It displays suitable characteristic for peptide and other radiopharmaceuticals preparations to widen clinical use of 68 Ga for PET imaging.
Method for Post-elution Concentration and Acidity Reduction of Eluate Obtained from SnO 2 Based 68 Ge/ 68 Ga Generator
Dariusz Pawlak, Wioletta Wojdowska, Renata Mikolajczak
Institute of Atomic Energy, Radioisotope Centre POLATOM; Otwock-Swierk, Poland
Aim of the Study: Several approaches to concentrate and purify the eulates of 68 Ge/ 68 Ga generators were proposed in literature, utilizing cation exchange, anion exchange and eluate fractionation. Recently E. Blois et al. described the features of SnO 2 based 68 Ge/ 68 Ga generator and presented modifications to the anion exchange based concentration of 68 Ga eluate. In order to reduce the eluate acidity these authors used 5 M NaCl and ethanol for washing the anion exchange column before eluting 68 Ga with small volume of water. In our work we propose to add the cation exchange column before the anion exchanger to obtain similar effect in terms of eluate concentration and improve purification from 68 Ge. Three different strong cation exchangers were tested for their potential for 68 Ga absorption and recovery followed by evaluation of anion exchange using 3 anionites alredy described in literature. Finally, the process of 68 Ga eluate concentration and purifcation was carried out in the tandem of columns using elected cation and anion exchangers.
Materials and Methods: The SnO 2 based 68 Ge/ 68 Ga generator (iThembaLABS, SA) was eluted with 7 ml of 0.6M HCl (223-317 MBq) at a flow rate of 2 ml/min. The eluate was directly passed through a cartridge containing strong cation-exchange resin. Following cationites were tested: AG 50W-X12, Sepra SCX 50 μm and Strata X-C 33 μm, each in quantity of 100 mg. 68 Ga was then desorbed with 2 ml of 4.5 M HCl. The yield of adsorption and desorption was measured taking into account the radioactivity of 68 Ga in eluate, in waste and that retained on columns. The 68 Ge eluates obtained in 4.5 M HCl were loaded on anion-exchange columns (AG 1X8, Oasis Wax 30 μm, Strata X-AW in quantitites 50 mg, 30 mg and 30 mg, respectively). The anionite column was then rinsed with 2 ml of 5 M NaCl followed by 2 ml of EtOH. Subsequently, 68 Ga was desorbed from the column with 2 ml of water or 0.005 M HCl. The yields of adsorption and desporption from the anionites were measured. Each experiment was repeated 5 times. In the next step the selected cation exchange column and anion exchange column were connected in series and the processs of concentration/purification was carried on line. The 68Ge content in the product was measured after 1 day of cooling using a γ-spectrometer equipped with a coaxial HPGe detector.
Results: Our investigations showed that the AG 50W-12 resin had the highest capacity for selective purification of 68 Ga (the yields for 68 Ga adsprption and desorption were over 98% and 94%, respectively). Among tested anion exchngers, the Oasis WAX 30 μm showed the highest adsprption and desorption yields of over 98% and 92%, respectively, when 0.005 M HCl was used as eluent. Elution with water resulted in a few percent lower yields. When these two selected cation and anion exchange columns were connected in series, the overall yield of the process was 82.66±3.46%, time corrected. The loss of 68 Ga in waste solutions was very low, not more than 1% at each step and the radioactivity retained on columns was around 5% for cation exchange and around 8% for anion exchange column. The content of 68 Ge in the product solution was below 2x10-3% (<5 kBq), which was around 8 times lower than the initial content of 68 Ge in the generator eluate. One should note that at the time of experiments the generator was already exploited for over 12 months.
Summary: The resin AG 50W-12 had the highest capacity for selective purification of 68 Ga. Similar results were obtained by de Blois et al. using AG 50W-8 resin, these authors confirmed also the suitability of Oasis WAX 30. The advantage of the approach presented in our work is the on-line reduction of eluate acidity and at the same time the reduction in the level of radionuclidic impurities, mainly 68 Ge. The method is very efficient and easy to adopt in the laboratory set up.
Purification of 68 Ga from 68 Ge/ 68 Ga-Radionuclide Generator Combining Two Different Methods
N.S. Loktionova 1 , A. Belozub 2 , K.P. Zhernosekov 3 , D.V. Filosofov 2 , F. Rösch 1
1 Institute of Nuclear Chemistry, Johannes Gutenberg-University Mainz, Germany; 2 Joint Institute of Nuclear Research, DLNP, 141980 Dubna, Russian Federation; 3 Chair of Radiochemistry, Technical University of Munich, Germany; 55122 Mainz, Fritz-Strassmann-Weg 2, Germany
Aim of the Study: The 68 Ge/ 68 Ga radionuclide generator provides an excellent source of the positron emitting 68 Ga for the routine synthesis and application of 68 Ga-labeled compounds using PET. Recently, we have introduced a post-processing approach absorbing 68 Ga online from generator eluates on a small cation exchange resin with a purification step using a 0.15 M HCl/80% acetone mixture (N1) and a quantitative desorption of 68 Ga from the resin by 0.4 ml of a 0.05 M HCl/98.5% acetone mixture (N2). The overall content of acetone in the purified 68 Ga fraction is small and non-toxic.  However, it may be reasonable to reduce it further and improve final radioactivity concentrations and purity of 68 Ga.
Materials and Methods: In the first step, 68 Ga was transferred onto the cation exchange column (AG 50W-X8, 200-400 mesh) from the generator with 7 ml of 0.1 M HCl. This concentrates 68 Ga from the generator eluate and removes major impurities. In the next step, the column was eluted with 1 ml of N1 solution to wash the column from residual impurities, c.f. Fe, Mg, Ge. The 68 Ga fraction is online transferred to a small column equipped with an anion exchange resin (AG 1-X8, 200-400 mesh, 50 mg) or a chromatographic resin (TODGA, 50-100 μm particle size, 100 mg) using 4 M or 5 M HCl, respectively. In a final desorption step, highly pure 68 Ga is eluted by 100-300 μl water from AG 1-X8 or TODGA resin, respectively.
Results: All 68 Ga eluted from the cation exchange cartridge is almost quantitatively trapped on both anion exchange resins (about 97% for AG 1-X8 and 99% for TODGA). The elution profile of 68 Ga from the anion exchanger and TODGA in initial generator eluate showed already highest efficacies in the first 100 μl water fraction. Altogether, about 87±5% of 68 Ga were successively desorbed in only 300 μl of water from AG 1-X8. [1-3] In comparison, about 96% of 68Ga are recovered in 1000 μl of water from TODGA. Only 10±5 Bq of 68 Ge were detected in the final 300-1000 μl water fractions.
Summary: The complete process takes only about 5 min. The high purification factor concerning 68 Ge and other metallic impurities is still preserved due to the initial cation exchange resin. With only 10±5 Bq of 68 Ge detected in the final 300 μl water fractions, the overall breakthrough of 68 Ge is about 10-6%. The pH of the final product it highly acidic, but it can be modified by using 300 μl 0.1-0.5 M NaOH instead of 300 μl water as eluent. Using the combination of two post-processing methods, provides 68 Ga in very high purity, higher radioactivity concentration and avoids the use of acetone.
Acknowledgement: This project was supported by the EC via the COST action D38.
1. K. P. Zhernosekov et al. (2007), J. Nucl. Med. 48, 1741-1748
2. Hofmann M. et al. (2001),Eur. J. Nucl. Med. 28, 1751-1757
3. Meyer G.-J. et al. (2004), Eur. J. Nucl. Med. 31, 1097-1104.
Separation of 68 Ga and 68 Ge on TLC Plate
N.S. Loktionova, F. Rösch
Institute of Nuclear Chemistry, 55122 Mainz, Fritz-Strassmann-Weg 2, Germany
Aim of the Study: The breakthrough of 68 Ge from a 68 Ge/ 68 Ge generator is a critical parameter in the context of the clinical use of 68 Ga pharmaceuticals. Due to the lack of sufficient characteristic radiation, 68 Ge is "invisible" within an excess of 68 Ga activity. Consequently, 68 Ga eluate (or product) solutions usually need to be stored waiting for the complete decay of the excess of initially present 68 Ga (which is the case not before one day after the elution) for subsequent spectroscopy of 68 Ge according to its 68 Ge-68Ga equilibrium. The aim of this study is to separate 68 Ga from 68 Ge on a TLC plate to get an instant possibility to determine the 68 Ge breakthrough from a 68 Ge/ 68 Ge generator within one hour post elution already.
Materials and Methods: A two years old 68 Ge/ 68 Ga generator was used with a yield of 68 Ga of about 100 MBq and a breakthrough of 68 Ge of about 10 kBq. The generator was eluted with 5 ml of 0.1 M HCl. 2 μl aliquots were taken for TLC. Silica gel 60 TLC from MERCK was used for separation of 68 Ga and 68 Ge. A mixture of 5% NaCl, methanol and 25% ammonia in ratio 3:1:1 was identified as most effective mobile phase. TLC measurements were performed at 0, 1, 2, 3, 4, 5, 10, 15, 20, 25 and 30 hours after preparation of the TLC chromatograms. Radio-analytical determination was carried out via an Instant Imager from Packard Canberra.
Results: In the mixture 5% NaCl : methanol : 25% ammonia=3:1:1 on Silica gel, 68 Ga and 68 Ge show different Rf values of has Rf=0 and Rf=0.3-0.6, respectively. The same TLC is measured 1 and 10 hours after the TLC development. While the activity of the 68 Ga spot is decreasing according to its half-life, the 68 Ge spot shows increasing count rate, again according to the 67.7 min half-life of 68 Ga. The determination of the count rate 67.7 min after the development of the TLC thus represents half of the total activity of 68 Ge present. Using a calibration between count rate and absolute activity, the breakthrough of 68 Ge can be quantified instantaneously, i.e. after about 1 hour already.
Summary: 68 Ga and 68 Ge were effectively separated on TLC Silica gel 60, using a mixture 5% NaCl : methanol : 25% ammoniac=3:1:1 as mobile phase. From the subsequent counting of the activities or by cutting the TLC plate on two parts it is possible to quantify the breakthrough of 68 Ge almost directly after elution during few minutes only without using gamma-detectors.
Method for Concentration and Chemical Purification of Eluates from Ge-68/Ga-68 Generator-based Tin Oxide
Dana Niculae, Ioana Patrascu, Catalin Tuta, Ioan Ursu
National Institute for Physics and Nuclear Engineering Horia Hulubei. Reactorului 30, Magurele 077125, Ilfov, Romania
Aim: The aims of the study were to characterize the performance of a Ge-68/Ga-68 generator (tin oxide column, elution with 0.6 - 1.0 M HCl) and its long therm efficient utilization for Ga-68 radiopharmaceuticals preparation and to select the optimal method for pre-concentration and purification of the Ge-68/Ga-68 generator eluate, in order to increase the specific radioactivity (SRA) of labelled conjugate.
Materials and Methods: We have tested 4 different methods for pre-concentration and purification of the eluate: fractionated elution, anionic exchange resin purification, cationic exchange resin purification, cationic and anionic columns purification. The radiochemical and chemical purities were evaluated by RP HPLC on C18 column with UV and radiodetection. The high SRA Ga-68 was coupled with DOTA-VIP and the influence of metallic impurities was tested by measuring the radiolabelling yield of different Ga-68 containing fractions of the purified eluates.
Results: The fractionated elution concentrates about 93% of Ga-68 activity in 1.0 ml. By using the anionic exchange resin columns cartridge we were able to concentrate up to 78% of Ga-68 activity from the eluate in 0.5 ml and additional purification from the most of chemical impurities and Ge-68 impurities was achieved.This resulted in a high yield of DOTA-VIP labelling.
Summary: The generator-produced positron-emitting isotope 68 Ga (t1/2= 68 min) is of increasing interest for the development of new radiopharmaceuticals, taking the advantage of in-house preparation of Ga-68 without necessity of cyclotron, with future possibility of freeze-dried kits.The purification and concentration of eluates is needed to avoid some of the backdrawns: the chemical form of 68 Ga after elution, the high volume of elution, the contamination with other cations coming from the column or eluent impurities, breakthrough of 68 Ge. These methods gave effective purification and concentration of 68 Ga eluates, independent of the volume of the eluate and allow high radiolabelling yield of DOTA-VIP.
Challenges of Ge-68 Separation from Ga-68 eluates
M. Kosinski, C. Wastiel, S. Baechler, F. Bochud
Institut de Radiophysique, Rue du Grand-Pré 1 1007 Lausanne, Switzerland
Aim: The goal of this study was to determine the Ge-68 radionuclidic impurity level of Ga-68 eluates, which requires a rapid (less than an hour) but adequate (high efficiency of separation) method appropriate to any commercial Ga-68 generators.
Materials and Methods: The separation of the radioelements has been performed by two different methods, one using a cation exchange column Dowex 50WX8 and the other using an extraction chromatography UTEVA Resin, in both cases with and without pre-filtration through a 0.22 μm membrane filter (Millipore). In the case of Dowex 50WX8, the eluate solution (HCl 0.1M) has been set directly through the column whereas in the case of UTEVA, it has been previously acidified by HCl 4M. The Ge-68 has been eluted with 10 ml HCl 0.1M for Dowex and 10 ml HCl 4M for UTEVA. The activity of Ga-68 has been measured directly in a dose calibrator while the Ge-68 was indirectly deduced 24 h afterwards using the Ga-68 activity value. Generators from Eckert Ziegler (IGG 100) and Obninsk were used for this study.
Results: Depending on the origin of the eluate, some surprising results were obtained. In the case of the Obninsk generator, the chemical form of Ge-68 seems to be ionic and homogeneous: 99.6% of Ge-68 is eluted from the Dowex and UTEVA column without and with pre-filtration. The retention on the membrane filter was negligible (<0.3%). In the case of Eckert Ziegler generator, the non filtered eluate was 57% retained on the Dowex column (HCl 0.1M used for elution) and 48% on the UTEVA column (HCl 4M used for elution). The pre-filtration showed a retention of 31% with a solution HCl 0.1M and 14% with a solution HCl 4M.
Summary: We found that the separation of Ge-68 from Ga-68 depends on the type of generator. It is easily feasible with Obninsk generators but not possible for generators with different chemical forms of eluates (complexes, particular forms, etc.). In remediation, an aggressive chemical treatment can be used to obtain a homogeneous ionic form but this method cannot be performed routinely in a nuclear medicine department.
68 Ga and 67 Ga Purification Studies: Preliminary Studies
R.F. Costa, M.F. Barboza, J.A. Osso Jr
Instituto de Pesquisas Energéticas e Nucleares (IPEN/CNEN-SP), Av. Prof. Lineu Prestes, 2242 - 05508-000 - Sao Paulo-SP, Brazil
Aim: The aim of this work is to present the results of two different methods developed for handmade purification of 68 Ga and 67 Ga for future radiolabeling with biomolecules.
Materials and Methods: A commercial generator based on a TiO 2 phase adsorbing 68 Ge was obtained from Cyclotron Co. (Obninsk, Russian Federation). The 67 Ga, obtained from MDS Nordion® , was used as surrogate for 68 Ga. The amount of chemical impurities present in 68 Ga and 67 Ga was evaluated by ICP-OES before and after the purification. Two purification methods were employed. The first one used the cation exchange resin AG50W-X8 (H+, 200-400 mesh and >400 mesh, BioRad, USA). A solution of 68 Ga in 0.1 mol.L-1 HCl was loaded in the column and 68 Ga was further eluted with a solution of acetone/12 mol.L-1 HCl (97:0.4)%. Two geometries of column were tested containing 5mL and 0.5mL of resin. The second method of purification was the conventional solvent extraction using diisopropyl ether with added TiCl 3 . A solution containing 67 Ga in 7 mol.L-1 HCl was extracted with diisopropyl ether and 67 Ga was extracted with H 2 O. The purification studies shifted towards the preparation of extraction chromatography column based in the absorption of diisopropyl ether in XAD-16 (20-60 mesh, Amberlite, USA), which was tested with both radionuclides.
Results: The results of the purification performed with 5mL of resin were not satisfactory and after changing for a smaller column, the yield of elution was 5% higher. With this effort, the best results of elution yield were achieved with AG50W-X8 (H+, >400 mesh) resin (87.1±0.8)%. The levels of Ge and Ti were decreased. However the levels of contaminants such as Zn and Fe were still high. The recovery yield for the solvent extraction was lower (72.52±0.1)% and the levels of contaminants decreased, but this method was time consuming and not useful for the short half life of 68 Ga. Therefore, the same methodology was transferred to an extraction chromatography column and the recovery yield was increased (78.0±3.8)%. The level of all contaminants was decreased for 68 Ga, except for Fe. Nevertheless, the levels of Zn, although decreased, were still high in all experiments.
Summary: The extraction chromatography column based in the absorption of diisopropyl ether in XAD-16 is the most promising purification method. However, efforts must be done to decrease the levels of Zn and Fe in the 68 Ga eluate.
Preparation High-purified 68 Ga Solutions via Combination of Cation and Anion Exchange Processes
Anton A. Larenkov, A.B. Bruskin, G.E. Kodina
Burnasyan Federal Medical Biophysical Center, Moscow, Russia
68 Ga radionuclide obtained from 68 Ge/ 68 Ga generator is one of the most promising for the synthesis of radiopharmaceuticals (RP) for positron emission tomography because of its nuclear and chemical properties. Chemical form of 68 Ga in the eluate of the 68 Ge/ 68 Ga generator, theoretically means, that it can be directly used as universal sourse of 68 Ga for RPs synthesis, provided the availability of a suitable chelating agent. However, the presence of competing impurity of metallic cations in the eluate prevents the formation of complexes 68 Ga 3+ . The presence of impurities (Cd 2+ ; Co 2+ ; Cu 2+ ; In 3+ ; Fe 2+ ; Fe 3+ ; Lu 3+ ; Ni 2+ ; Zn 2+) with concentration of about 1 μM in the stock solution is frequently unacceptable for preparation of 68 Ga-RPs with high quality. Breakthrough of the long-lived parent 68 Ge through the column with the sorbent is about 10 -3 % of total activity 68 Ge in the generator at the time of elution. Furthermore large volume of eluate (5÷10 ml) and its acidity requires concentration of eluate activity for peptide targeting in nanomolar amounts. Thus, purification and concentration of the 68 Ga generator eluate before the actual reaction of the bioconjugates targeting is necessary.
Aim of the Study: The aim of the study was to design the method of purification and concentration of 68 Ga solutions. This method will allowed to obtain concentrated 68 Ga-solutions with high chemical and radiochemical purity without organic solvents. These solutions can be used for the synthesis of radiopharmaceuticals with high specific (molar) activity and radiochemical purity (RCP) in the most convenient form for clinical use.
Materials and Methods: This goal has been reached with the combination of the cation and anion exchange in mixed media. On the first stage 68 Ga from the 68 Ge/ 68 Ga generator (Cyclotron Ltd., Obninsk) eluate quantitatively sorbed on strong cation exchange resin (e.g. Dowex 50W×8). Part of the non-isotopic carrier, contained in the eluate, and parent 68 Ge radionuclide passes through the resin. The greater part of the non-isotopic carriers sorbed on the cation exchange resin, can be quantitatively eluted with the mixture of hydrochloric acid and acetone with the specific molar ratio at which 68 Ga is not eluted from the resin. After washing, 68 Ga was completely and almost selectively eluted from the resin with a mixture of hydrochloric acid and acetone with the molar ratio different from previous one. In the subsequent interaction of the obtained eluate with strong anion exchange resin (e.g. Dowex 1×8) quantitative sorption of 68 Ga takes place. Anion exchange resin was washed with ethanol for fast and complete removing of acetone and hydrochloric acid traces and then dried with air or inert gas for complete removing of ethanol traces. After that 68 Ga was quantitatively eluted from the anion exchange resin with 0.01 - 0.1 M hydrochloric acid.
Results: Purification and concentration technique was tested with model solutions (solutions of metal chlorides in 0,1 M HCl), as well as with 68 Ge/ 68 Ga generator eluates. Both 68 Ge/ 68 Ga generator original eluate and purified solution were subjected to quantitative analysis (ICP-MS). Results are given in the table below:
The cleaning process takes not more15 minutes. Purification process yield without decay correction is 82%, with decay correction - 98%. The minimum volume of final solution was 200-250 μl after the purification and concentration procedure. Production of purified and concentrated solution of 68 Ga chloride complexes in 0,01 - 0,1 M hydrochloric acid significantly simplifies the process of synthesis of the RPs, as it becomes possible to use freeze-dried forms of precursor, without any subsequent purification such as solid phase extraction and others. 1 ml concentrated and purified solution 68Ga (293 MBq) obtained by the presented method was added to the freeze-dried composition consisting 10 mg sodium acetate and 20 μg of the DOTA-TATE peptide. The vial was thermostated at 95°C for 10 min. The labeling reaction yield (i.e. RCP) was more than 99% (radio-TLC and HPLC data).
Summary: Experimental data show, that the method of purification and concentration of the 68 Ge/ 68 Ga generator eluate using cation and anion exchange resins is very effective and promising for synthesis of the 68 Ga-RPs in everyday medical practice. Designed method is allows obtaining of concentrated 68 Ga-solutions with high chemical and radiochemical purity in 0,01 - 0,1 M hydrochloric acid medium without organic solvents.
Optimisation of the Conditions of 68 Ga-AMBA Radiolabelling with Sodium Acetate Buffer
V. Nataf, J. Rose, A. Prignon, F. Montravers, J.N. Talbot
Animal PET Platform LIMP, Tenon Hospital, 4 rue de la Chine 75020 Paris, France
Aim of the Study: The animal PET platform (LIMP) at Tenon Hospital performed 68 Ga-DOTA-peptide radiolabelling using an Eckert-Ziegler 68 Ge/ 68 Ga generator and a fully-automated, PC-controlled, radiopharmaceutical synthesis device (SynChrom R&D, Raytest). The radiolabelling method of 68 Ga-AMBA (DO3A-CH 2 CO-G-4-aminobenzoyl-Q-W-A-V-G-H-L-M-NH2), a bombesin (BN)-like peptide, described by A. Nunn and al. from Bracco Research, uses a sodium acetate solution to obtain a pH between 3 and 3.5. This value is an optimal pH to radiolabel DOTA-peptide with 68 Ga. But these conditions don't allow reproducible radiochemical yield (28% to 100% of radiochemical purity) due to variations in the pH values in the reactor. The aim of this study is to optimise the pH value with sodium acetate buffer.
Materials and Methods: The 68 Ge/ 68 Ga generator is eluted without pre-purification, with 5 mL of 0.1M HCl. 1.9 mL of 68 Ga(Cl)3 are collected in the reactor. But this collected volume is not the same for each elution due to variations in Argon gas pressure in the module and the rhythm of elutions performed with the generator. These variations induce modifications of pH values in the reactor that the fluctuations in radiolabelling yield.
To compare the reproducibility of the 68 Ga radiolabelling yield with sodium acetate buffer to the reproducibility with sodium acetate solution, we prepared AMBA samples in the reactor according to two different methods: for method 1, the sample contained 100 μL of AMBA solution, 92 μL of sodium acetate solution 2M and 908 μL of Traceselect® water. For method 2, the sample contained 100 μL of AMBA solution and 1000 μL of sodium acetate buffer 1.5 M, pH 4. After elution, the sample in the reactor was heated for 10 minutes at 95°C. At the end of the heating step, the sample was analysed by HPLC with a UV detector and a radioactivity detector sequentially to assess radiochemical purity. The pH was measured with pH paper strips. Each method was performed 4 times.
Results: pH values were between 2 and 3.5 with method 1 while method 2 provided pH values between 3 and 3.5. The radiolabelling yields of 68 Ga-AMBA obtained with method 1 and method 2 were 61.5±34% and 90.5±4% respectively. Method 2 using sodium acetate buffer is more reproducible than method 1.
Conclusion: these results show that sodium acetate buffer can lessen the impact of HCl volume variations with stabilisation of the pH in the reactor around 3 to 3.5. This method allows us to perform radiolabelling with a good reproducibility while avoiding the volume changes.
Determination of Optimum pH for Gallium-68 DOTA-TATE Labelling
Fakhrurazi Bin Ahmad Fadzil, Suharzelim Abu Bakar, Mohd Borhanuddin Md Hassan
Nuclear Medicine Department, Putrajaya Hospital, 62150 Wilayah Persekutuan Putrajaya, Malaysia
Objectives: Gallium-68 tracers possess favorable nuclear properties for nuclear medicine applications; mainly for diagnostic purpose. It is a known PET radionuclide that can easily be obtained with milking of the generator and there are several types of generator are commercially available depending on their column type. The pH plays an important role in Gallium-68 labelling, as too acidic pH creates more proton (H+) that will compete with gallium-68 for labelling. Meanwhile, if too alkaline, Gallium Hydroxide complexes will form. The aim of this study is to determine the optimum pH for Gallium-68 DOTA-TATE labelling with the self-prepared chemicals.
Materials and Methods: In our centre, we are using a SnO 2 -based 68 Ge/ 68 Ga generator. The peptide that we used for labelling is DOTA-TATE, a pharmaceutical grade peptide from ABX lab. Gallium-68 was eluted with 10ml 0.6M HCl, as recommended by the manufacturer. A fractionation method was applied in order to concentrate and purify gallium-68 eluate. 2 ml fraction containing Ga-68 is transferred into the reactor and it is mix with a vial containing 150uL of DOTA-TATE in a 280mg/ml HEPES buffer with different range of volume; 1.0 ml to 2.0 ml of the buffer. The pH of each mixture is determined and the radiochemical purity of Gallium-68 DOTA-TATE preparation will be compared.
Results: The radiochemical purity of each Gallium-68 DOTA-TATE preparation varies when the volume of the 280 mg/ml HEPES buffer was varied. It shows that at pH more than 3 and at pH less than 2.5, the RCP of Gallium-68 DOTA-TATE preparation is less than 90%. The highest RCP (>95%) was obtained with a mixture of 1.3 ml 280 mg/ml HEPES buffer with the 2 ml fraction of Gallium-68 eluate in 0.6 M HCl. The pH for this mixture was 2.85.
Summary: The pH plays an important role in Gallium-68 labelling as too low or high pH will result in low labelling yield. The optimum pH for gallium-68 DOTATATE labelling is between 2.5 to 3.0. However, the highest RCP was obtained at a pH of 2.85. Due to its narrow range of pH (2.5-3.0) the chemicals have to be well prepared.
Improving Radiochemical Purity and Quality Control of 68 Ga-DOTATATE
R. Hesselmann, A. Johayem, U. Özdemir, M. Dragic, A. Blainc, L. Mu, R. Schibli
Center of Radiopharmaceutical Science, University Hospital Zürich, Nuclear Medicine, Rämistrasse 100, 8091 Zürich, Switzerland
Aim: To improve the radiochemical purity of 68 Ga-DOTATATE manufactured with the standard process of a Modular-Lab Pharm Tracer (Eckert&Ziegler), we tested the addition of three different radical scavengers and compared three HPLC methods for the quality control.
Synthesis Method : The commercially available 68 Ge/ 68 Ga generator (Eckert & Ziegler, Berlin, Germany) and commercially available synthetic cassette (C4-Ga68-PP) were used for the labeling. Briefly, around 820 MBq 68 Ga 3+ was generated and reacted with DOTATATE in sodium-acetate buffer (pH=4) at 95°C for 400 sec. The influence of different anti-oxidants such as ethanol, ascorbic acid and sodium thiosulfate on the radiochemical purity was investigated with the published and our new established analytical HPLC method.
Analytical Methods: Analytical HPLC system used was a Merck Hitachi Lachrom Elite system. UV chromatograms were recorded at 220 nm. Eluents for all methods were: 0.1% TFA in water (solvent A), 0.1% TFA in acetonitrile (solvent B). Method 1: ACE column, 3 μm, 150 × 4 mm, grad: 0-2 min, 18% B; 2-11 min, 18-60% B; 11-14 min, 60% B; 14-15 min 60-18% B. The flow rate was 0.6 ml/min. Method 2: with the same type of column, isocratic 24% B in 12 min. flowrate: 0.6 ml/min. Method 3. ACE column, 3 μm C18, 50 mm × 4.6 mm, grad 17-25% B in 10 min. The flow rate was 2 ml/min.
Results: When the starting amount radioactivity was around 200 MBq, the radiochemical purity was more than 95% with all the analytical methods, while when the starting amount of radioactivity over 800 MBq, although method 1 gave more than 95% radiochemical purity, more than 10% side peaks were obtained with method 2. The radiochemical impurities could be well separated from the 68 Ga-DOTATATE peak. The percentage of these radiochemical impurities was dependent on the starting radioactivity of 68 Ga and could up to more than 15%. Unfortunately, the 68 Ga-DOTATATE peak shows a strong tailing with the method 2, which may hide other radioimpurities eluting directly after the product peak. The peak shape and the resolution of the radioimpurities could be further improved by using a shorter ACE column at a higher flow rate and the adapted gradient. Based on our experience with other similar indicated that the radioimpurities might be oxidised 68 Ga-DOTATATE. It is planned to verify the chemical nature of the impurities by LC-MS. The influence of Na 2 S 2 O 3 addition on the radiochemical purity of 68 Ga-DOTATATE is still under our investigation. Addition of the radical scavengers ascorbic acid or ethanol could dramatically improve the radiochemical purity of 68 Ga-DOTATATE to always more than 95%,
Summary: A suitable analytical method was established for 68 Ga-DOTATATE quality control. Addition of ascorbic acid or ethanol could dramatically improve the radiochemical purity of 68 Ga-DOTATATE.
(Ga-68)DOTA TATE0 Labeling Using An Automated System with Disposable Cassettes
Matthew J. Combs, Damion Stimson, David MacFarlane, Alex Yordanov
Bioscan, Inc., Royal Prince Alfred Hospital, Sidney, NSW and Royal Brisbane and Womens Hospital, Brisbane, NWS, Australia, 4590 MacArthur Blvd. NW, Washington DC 20007, USA
Aim: Radiolabeled peptides are becoming of increased interest to the nuclear medicine community due to their ability to specifically bind to receptors expressed in a variety of tumors. Peptide labeling with radiometals is much easier and overcomes the challenges of labeling with F-18. Radiometal generators such as Ga-68 are also readily available and more practical in some situations than F-18. There is a need for a flexible system that automatically elutes the generator or introduces the starting material, labels the radiopeptides and delivers the final product using sterile, disposable cassettes.
The objective of this work was to develop an automated radiometal peptide labeling unit that is flexible and uses disposable cassettes. The ReFORM-Plus (Bioscan) chemistry module was used to meet this objective. The ReFORM-Plus was coupled to a Ge-68/Ga-68 generator (Eckert & Ziegler) and peristaltic pump. Software specific to peptide radiolabeling was developed and implemented. The software is flexible allowing for user selection of peptide/metal. Sterile, disposable cassettes were also developed for the system. An automated kit test is performed by the synthesizer to ensure the integrity of the disposable cassette and alert the user to any problems. The software also provides a peptide-specific synthesis report. Pump control and selection of eluent/wash allows for automated post-synthesis cleaning and storage of the system.
Materials and Methods: For this work, a Ga-68 DOTATATE method was implemented on the system using the method of Decristoforo et al. (2007)* with purification using a Sep-Pak® Light C18 cartridge (Waters), followed by sterile filtration via a Millex® -GV (Millipore) filter. The final product was tested for radiochemical purity by both HPLC and TLC, pH and radionuclidic purity. Product samples were also tested for sterility and apyrogenicity.
Results: Initial (Ga-68)DOTATATE yields gave 40% non-corrected radiochemical yield in 20 minutes (320 MBq product from 800 MBq generator elution) including purification, formulation and sterile filtration. Product passed all quality control specifications. Subsequent improvements in the methods have resulted in yield of 50%. The system is under validation for clinical use and other radiopeptides and radiometals are under investigation to be implemented on the system.
Summary: The system meets our needs of automatically labeling peptides with radiometals and provides flexibility for future projects past Ga-68 DOTATATE. The disposable cassettes allow for regulatory compliance and convenience and are flexible to adapt to many labeling methods. Further enhancements to the system are ongoing.
A Scaled-up Production Method for [ 68 Ga]DOTA-TATE, using Eluates from a SnO 2 -based 68 Ge/ 68 Ga generator
Daniel D. Rossouw, Wouter A.P. Breeman
iThemba LABS, P.O.Box 722, Somerset West, South Africa
Aim of the Study: A simple, practical method to prepare multi-dose quantities of 68 Ga-labelled DOTA-TATE, using un-purified 68 Ga eluates from a SnO 2 -based generator, is reported. In order to achieve optimal elution efficiencies with this generator, 0.6 M HCl is used as eluant, as opposed to the 0.1 M HCl required for other types of generators.
Our aims were to adapt existing labelling recipes in order to accommodate the more acidic eluate, to scale up labelling reactions without compromising radiochemical yields and to improve existing solid phase C18 purification techniques in order to optimise recovery yields and to bring about an injection-ready post-purified product.
Materials and Methods: A SnO 2 -based 1850 MBq 68 Ga generator (double-loaded with 3700 MBq 68 Ge) was prepared in house at iThemba LABS, South Africa. The first 50 drop 0.6 M HCl eluate fraction (4.0 - 4.3 ml) was used directly for labelling. The pH was adjusted with 2.5 M sodium acetate, using eluate:NaOAc v/v ratios of 80:23 or 80:29. DOTA-TATE (BioSynthema, St Louis, MO, USA; 30 μg or 50 μg) was added and the mixture was heated at 90 - 95°C for 10 or 20 min. It was subsequently purified on a Sep-Pak C18 cartridge (500 mg). Free radiogallium and traces of 68 Ge were washed out with saline solution, and [ 68 Ga]DOTA-TATE was eluted with ethanol or an ethanol/saline mixture (1:1, v/v).
Results: The eluate fraction used for labelling comprised 90 - 95% of the total eluted activity. Neutralization with 2.5 M NaOAc afforded a suitable pH-range of 3.6 - 4.2 for labelling, resulting in reaction volumes of up to 5.6 ml. Under these conditions, a DOTA-TATE concentration of 3.7 - 4.0 μM (30 μg) led to very inconsistent labelling efficiencies, while a concentration range of 6.2 - 6.8 μM (50 μg) resulted in consistently high labelling efficiencies. There was no significant reduction in the labelling efficiency when employing a shorter reaction time (10 min). Purification on C18 resulted in decay-corrected recovery yields in excess of 80%. A 50:50 mixture of ethanol and saline proved to be a more efficient de-sorption eluant than 100% ethanol, giving close to 90% decay-corrected yields and resulting in less residual activity on the C18. Radiochemical purities were in excess of 98%. Under optimized conditions, up to 900 MBq purified [ 68 Ga]DOTA-TATE could be prepared, using a 2-3 month old generator, while a 15 month old generator delivered up to 300 MBq purified product.
Summary and Conclusions: The methodology reported here can be used to prepare several patient doses of [ 68 Ga]DOTA-TATE, utilizing up to 95% of the total eluted activity. There is no need for a pre-elution step as recommended in several papers, thereby simplifying the elution procedure. It allows for a radiochemically pure injection-ready product. It also has the potential of being converted into a semi- or fully automated procedure, thereby causing a further reduction in synthesis and processing time.
Development of 68 Ga Generator at ANSTO
Van So Le, Michael Izard, Paul Pellegrini, Myint Zaw
ANSTO LifeSciences, Australian Nuclear Science and Technology Organisation; Locked Bag 2001 Kirawee DC, NSW, 2232, Australia
A 68 Ge/ 68 Ga generator combined with automated 68 Ga eluate purification unit was developed to produce 68 Ga solution suitable for labelling peptide ligands for PET radiopharmaceutical applications. The sorbent of a Ti-Zr ceramic structure  was used as a generator column packing material. 68 Ga eluate of around 5 mL volume in 0.1 M HCl solution was purified on a small cation exchanger column with an aqueous alcohol solution mixture of hydrochloric acid, ascorbic acids and halide salts. An alkali solution was used for elution of 68 Ga from the ion exchange resin column to obtain a purified 68 Ga solution which is conditioned with acidic solutions to obtain a final 68 Ga product of pH=3-4 in 0.75 mL 0.5 M NaCl or 0.5 M sodium acetate solution. The organic solvent free 68 Ga solution product of acidity suitable for coordination chemistry based labelling of the peptide ligands was successfully used for preparation of DOTATATE and DOTATOC PET radiopharmaceuticals.
The process of 68 Ga elution from the generator followed by 68 Ga eluate purification was performed using a low-cost automation bench-top system.  This system is designed based on the timing sequence of seven processing steps without feedback control. The variable flow rate of eluents used for elution/purification in this system also ensure the optimisation of operating times with respect to different adsorption/ desorption kinetics of 68 Ga ion species, which is controlled by the sorbent and ion exchange resin used in the generator and purification columns.
1. Van So Le, Sorbent material, Patent, PCT/AU2011/000245
2. Van So Le, 68 Gallium purification, Patent, PCT/AU2011/000244
Production of 68 Ge from Natural Zink by Cyclotron
Thomas Ebenhan 1 , Z. Szucs 2 , C. Cimpeanu 3 , D. Dudu 3 , L. Craciun 3 , A. Luca 3 , M. Sahagia 3 , J.R. Zeevaart 4
1 Radiochemistry, Nuclear Energy Corporation of South Africa; 2 Institute of Nuclear Research of H.A.S., Hungary; 3 Horia Hulubei National Institute of Physics and Nuclear Engineering, Romania; 4 School of Pharmacy, North West University, Potchefstroom, South Africa
Objective: The production of 68 Ge by cyclotron is well known using the 69 Ga(p,2n) 68 Ge nuclear reaction.  The cross section of this reaction is enough high for the routine production of 68 Ge. However there is another promising nuclear reaction which was somehow forgotten. It is the natZn(ρ,xn) 68 Ge, reaction which was studied 42 years ago in detail.  Its cross section data indicate that the reaction increase up to 800 mbarn, which is higher than the above mentioned proton induced nuclear reaction which goes up to approximately 550 mbarn. Zink as a target material and the associated radiochemical knowledge relating Zink target processing are also well known for the 67 Ga production. The remaining task is to produce 68 Ge from natZn and separation of the produced 68 Ge from the irradiated target. An easy and elegant solution will be described in this paper.
Materials and Methods: The Zn target was prepared from a 250 μm thick, 99.99+% pure Zn foil. The α-beam irradiation was carried out in the U120 cyclotron, in Horia Hulubei Institute of Nuclear Physics, Bucharest. The parameters of the irradiation were as follows: beam current was 5 μA, the energy was 27 MeV, the irradiation time was 163 h. After the irradiation the target was kept for 4 weeks to "cool" and decaying of short lived radioisotopes, produced by the side nuclear reactions. For the chemical separation the freeze dry distillation was chosen. The Zn target was put on frozen hydrochloric acid into a two-finger glass chamber and then gently heated. During the dissolution the temperature of the reaction mixture was regulated by cooling with liquid Nitrogen. The dissolved material was frozen back and evacuated. In the reduced atmosphere the second finger of the glass chamber was kept cold, while the glass-finger containing the mixture was kept at room temperature. During the night the 68 Ge transferred to the cold finger in form of GeCl4 together with the hydrochloric acid, while the target material and contaminating 65Zn and 58Co radioisotopes remained in the reaction-finger.
Results: The irradiation of Zn was carried out according to the routine process using the same equipment and knowhow from the 67Zn(p,n) 67 Ga production. The irradiated target contained 15% of 65Zn and 0,2% of 58Co radioisotopes even after the cooling time due to the Cu and Fe impurities. A 78% chemical yield of the separation was achieved. The radiochemical purity was 100% by γ-spectrum evaluation. The 68 Ge was tracked by its daughter, 68 Ga.
2. Karpeles J. Radiochimica Acta, 1969;12:115 In German
Screening of Different 68 Ga-Schiff-Base Amines in vitro Test for Myocardial Imaging
Melanie Zimny, M. Fellner, F. Rösch
Institute of Nuclear Chemistry, Johannes Gutenberg-University, Mainz, Johannes Gutenberg-Universität Mainz, Institut für Kernchemie, Fritz Strassmann Weg 2, 55128 Mainz, Germany
Aim of the Study: Coronary artery disease (CAD) develops over years with almost no symptoms and causes most of the sudden deaths worldwide. Routinely SPECT-tracers like 99mTc-tetrofosmin and 99mTc-sestamibi are used to gain information about heart perfusion and myocardial cell damage. The development of a 68 Ga-labelled myocardial-tracer for cardiac PET-applications is of great interest. 68 Ga-[bis(4,6-dimethoxysalicylaldimino)-N,N'-bis(3-aminopropyl)ethylenediamine]+ showed a good uptake and retention in myocardial cells.  Therefore, Hexadentatebis(salicylaldimines) might be ideal candidates to access myocardial perfusion. New monocationicbis(salicylaldimine) derivatives were synthesized and compared by in vitro experiments.
Materials and Methods: 10 different aldehydes were coupled with a tetra-amino backbone. The obtained Schiff base amines were labelled with 68 Ga at 80°C in HEPES buffer (yield ≥ 70%) and purified by solid phase extraction. The lipophilicity and the uptake of the tracers in HL-1 rat heart cells were determined. The ionophorvalinomycin was added to investigate the influence of the cell membrane and mitochondrial potential.
Results: 10 Schiff-base derivatives were successfully synthesized and labelled in high yields (>70%) with 68 Ga. The lipophilicity of the 68 Ga-Schiff-base-complexes was in the range of 1.3 - 2.7. Valinomycin slightly decreased the uptake of the 68 Ga-tracers. Two compounds showed an extremely high cell-uptake (45% and 40% respectively).
Summary: New 68 Ga-Schiff base derivatives were synthesized and evaluated. The 68 Ga-tracers showed varying uptake with or without the ionophorvalinomycin. In vivo evaluation using μ-PET imaging will reveal their qualification for myocardial imaging.
Acknowledgement: This work is supported by a fellowship of the Dr. Georg Scheuing-Foundation.
1. Tsang et al., (1993), J Nucl Med, 34, 1127-1131
DOTA-TYR3-Octreotate Labelled with LU-177 and I-131 - Preclinical Comparative Evaluation
Dana Niculae, Valeria Lungu, Mihai Radu, Ioana Patrascu
National Institute for Physics and Nuclear Engineering Horia Hulubei; Reactorului 30, Magurele 077125, Ilfov, Romania
Aim: Our goal was to study, select and optimizing the radiolabelling parameters of DOTA-Tyr3-Octreotate with 131I and 177Lu, for obtaining DOTA-131I-Tyr3-Octreotate, 177Lu-DOTA-Tyr3-Octreotate and 177Lu-DOTA-131I-Tyr3-Octreotate and to determine their binding affinity to the specific membrane surface receptors.
Materials and Methods: Radioiodinated DOTA-TATE was synthesized using the Chloramine-T (Cl-T) method. The radiolabelling method of DOTA-TATE with 177Lu was optimized. For both radiolabelling procedures the optimal values for beta-emitters to peptide molar ratios, pH, temperature and incubation time were established, taking into account the radioactive and biological therapeutic doses. 177LuCl 3 in 0.05N HCl, 285 GBq/mg 177Lu (Polatom) and 1660 GBq/mg 177Lu (Nordion) respectively Na131I, 18 500 MBq/mL, carrier free, were used in the experiments. The stability of DOTA-TATE labelled with different radionuclides was performed by incubation in 0.9% NaCl and human serum. The competitive binding and the saturation binding assays were performed using rat brain cortex membrane.
Results: 177Lu-DOTA-TATE and DOTA-131I-Tyr3-TATE with high specific activity and radiochemical purity higher than 95% was obtained. The 131I-DOTA-TATE stability in 0.9% NaCl is higher than that of 177Lu-DOTA-TATE, while in human serum 177Lu-DOTA-TATE proved to be more stable than 131I-DOTA-TATE.The in vivo studies were performed using normal as well as hepatoma HRS1 tumor bearing rats, which overexpress somatostatin receptors. The results show a high and specific uptake of the luthetium radiolabelled somatostatin, starting at 24 h post injection, up to 1 68 h, followed by renal elimination. The in vitro stability show that DOTA is a stable chelator for 177Lu while Tyr3 has high affinity for the electrophylic iodine. The IC50 and Kd values of DOTA-131I-Tyr3-TATE show a high binding affinity to somatostatin receptors.
Summary: The in vitro receptor binding assays of DOTA-131I-Tyr3-TATE and 177Lu-DOTA-Tyr3-TATE using rat brain cortex membrane shows a comparable affinities in nanomolar range and places these products in the family of radiopeptides with high binding affinity for somatostatin expressive receptors. The biodistribution data shows that the 177Lu-DOTA-Tyr3-TATE is more stable and shows better uptake compared to DOTA-131I-Tyr3-TATE, the competitive localization index of 177Lu-DOTA-Tyr3-TATE being 3 times higher.The biodistribution confirms the in vivo stability of the 177Lu-DOTA-TATE and its selectivity for HRS1 tumors
Synthesis and Quality Control of 177Lu-BPAMD
D. Müller, I. Klette, R.P. Baum
Zentralklinik Bad Berka, Robert Koch Allee 9, 99437 Bad Berka, Germany
Aim of the Study: For a clinical treatement of 177-Lu-BPAMD an useful and applicable labelling and quality control procedure is needed. Therefore, close to the clinical application the development of an applicable synthesis and quality control procedure necessary. The synthesis should deliver the 177Lu labelled BPAMD in a high yield and the qualtity control should detect the amount of the formed 177-Lu-hydroxid.
Materials and Methods: The labelling procedure for the synthesis of Lu-BPAMD was performed under GMP conditions using a laminar flow box. To prevent radiolysis 2,5 dihydroxy benzoic acid was used and the reaction was finished after 30 min. For the quality control a HPLC method, combined with different ITLC methodes were used and the radiochemical purity was higher than 95%.
Results: By the developing of this synthesis and quality control procedure the first treatment of patients with 177-Lu- BPAMD could start.
Summary: We could develop a reproducible and applicable labelling procedure for the synthesis of 177-Lu- BPAMD. The reaction delivers the product with a radiochemical purity higher than 95%. An applicable and acceptable quality control procedure for a routinely used clinical application was developed.
Title of Presentation: A New Synthesis Pathway and Quality Control of 68-Ga-BPAMD for Clinical Application
D. Müller, I. Klette, R.P. Baum
Zentralklinik Bad Berka, Robert Koch Allee 9, 99437 Bad Berka, Germany
Aim of the Study: In the case of the bisphosphonate monoamide analogue of DOTA (BPAMD) the new NaCl based labelling procedure (Mueller et al.; Poster:"A new high efficient NaCl based cationic 68 Ge/ 68 Ga generator eluate purification") leads to an increase of formation of 68-Ga hydroxide. The cationic labelling procedure by Zhernosekov et al. (J Nucl Med 2007;48:1741-1748) leads to a non physiologic final product with a pH lower than 2. The neutralisation of the final product with sodium hydrogencarbonate (8,4%) is not possible. During the addition of the sodium hydrogencarbonate solution an unknown compound is detectable in the HPLC. Furthermore the final product contains non reacted and free 68 Ga3+ in different concentrations, so that a purification step is neccessary. A reproducible labelling method for a clinical application is needed here.
Materials and Methods: The modified combined cationic/anionic method (Mueller et al., J Labelled Compd Radiopharm 2009;52:477) is necessary for this labelling reaction. The 68 Ga generators were eluted with a total of 10 mL 0.1 M HCl and the 68-Ga was collected on the SCX cartridge and then eluted with 5.5 M HCl directly through the SAX cartridge. The 68 Ga was then eluted with water/ammonium acetate solution into the reaction vial with an aqueous ammonium acetate solution and 20 μg of BPAMD. After heating the solution to 100°C for 12 minutes the 68-Ga3+ was completely bonded and after sterile filtration the radiochemical yield is about 55% (n.d.c.).
Results: In contrast to the cationic labelling procedure (Zhernosekov et al.) after heating the solution to 100°C for 12 minutes the 68-Ga3+ was completely bonded. The radiochemical yield is about 55% (n.d.c.) after sterile filtration. This procedure leads to a final product with a radiochemical purity greater than 95% without further purification steps. The described procedure delivers 68-Ga labelled BPAMD in a high radiochemical purity and the pH of the final solution is about 4.
Summary: We could develop a reproducible and applicable labelling procedure for the synthesis of 68-Ga- BPAMD. The reaction delivers the product with a radiochemical purity higher than 95%. A subsequent purification is not necessary and the pH of the reaction solution is about 4.
Stability Investigations of [177Lu]BPAMD - A Potential Agent for Bone Pain Palliation
Marco Fellner, Vojtěch Kubíček, Petr Hermann, Frank Rösch
Institute of Nuclear Chemistry, Johannes Gutenberg University Mainz, Fritz-Strassmann-Weg 2, D-55128 Mainz, Germany
Purpose: Bone metastases are a serious aggravation for patients suffering from cancer. To live as normal as possible, palliative pain treatment for these patients as well as the prolonging of their life expectancy is of great importance. Therefore treatment of bone lesions with therapeutic radionuclides such as 177Lu together with highly potent bone seeking ligands comes into focus of medicine. As the DOTA-based bisphosphonate BPAMD was already labelled and investigated with 68 Ga for PET-imaging, this ligand is also suitable for lanthanides such as 177Lu.
Materials and Methods: The ligand BPAMD was labelled with 177Lu and investigated concerning stability depending on activity per volume as well as the usage of radical scavengers such as ethanol and gentisic acid. Activity concentratoins were investigated in the range of 0.50 - 15.71 GBq/mL up to 72 h. In addition also the impact of gentisic acid addition prior to the complex formation and after finishing the reaction was analysed.
Results: BPAMD shows high radiochemical yields in complexation with 177Lu. However the final complex exhibits very low stability at high activity concentrations (>4 GBq/mL). For lower activity concentrations (<1 GBq/mL) and the addition of ethanol or gentisic acid yields stable [177Lu]BPAMD. However, gentisic acid is superior to ethanol in terms of complex stability, especially adding the scavenger prior to the complex formation. Applying this procedure [177Lu]BPAMD was stabilized to more than 95% intact complex after 24 h.
Summary: [177Lu]BPAMD is of great interest for radionuclide therapy on bone metastases. The stable final product can be applied in many clinics, as the complexation as well as the stabilization is very convenient.
The Diversity of [ 68 Ga]Ga-based Imaging Agents
Uppsala Applied Science Lab, GE Healthcare, Division of Biomedical Radiation Sciences, Department of Radiology, Oncology, and Radiation Science, Uppsala University, Sweden
Introduction: Positron Emission Tomography (PET) field and, in particular utilization of 68 Ga radiometal is getting momentum. The introduction of new radiopharmaceuticals and their accessibility are important factors determining the expansion of clinical nuclear medicine for early disease detection and personalized medicine with higher therapeutic efficiency. Further, the availability of the technology for GMP compliant automated tracer production can facilitate the introduction of new radiopharmaceuticals due to the ability to conduct standardized and harmonized multi-center studies for regulatory approval.
Materials and Methods: The labelling chemistry was conducted using pre-concentrated and purified 68 Ga at room or elevated tempreture using conventional or microvawe heating mode. The synthesis was automated. In vitro cell and tissue cryosection assays as well as in vivo and ex vivo animal studies were perfomed for the biological evaluation of the tracers.
Results: A number of 68 Ga-based radiotracers were developed for targeted imaging of receptors, pre-targeted imaging using small effector or hapten molecules as well as non-targeted imaging of pulmonary perfusion and ventilation. The labelling was direct or chelator mediated. Macrocyclicchelators such as DOTA and NOTA coupled to the ligand molecules provided fast and stable complexation with 68 Ga respectively at elevated and room tempreture. The binding specificity of the imaging agents for targeted and pre-targeted imaging was confirmed using in vitro, ex vivo, and in vivo assays. The performance assessment of the radiotracers for non-targeted imaging was conducted in piglet models using PET scanner. The impact of peptide mass on the tumor uptake of [ 68 Ga]Ga-DOTA-TOC was assessed in a patient study for the assistance to personalized cancer therapy. The labelling synthesis was automated for GMP compliant production.
Summary: 68 Ga was used for the labelling of a broad range of molecules (small organic molecules, peptides, proteins, oligonucleotides) as well as particles. It has the potential to facilitate development of clinically practical PET and may worldwide promote PET technique for earlier better diagnostics and further for individualized medicine. 68 Ga may become PET analogue to legendary generator produced 99mTc with added value of higher sensitivity, resolution, quantitation and personalized medicine.