Markus Jahn

Processing of Generator-Produced 68Ga for Medical Application

The 68Ge/68Ga generator provides an excellent source of positron-emitting 68Ga. However, newly available “ionic” 68Ge/68Ga radionuclide generators are not necessarily optimized for the synthesis of 68Ga-labeled radiopharmaceuticals. The eluates have rather large volumes, a high concentration of H+ (pH of 1), a breakthrough of 68Ge, increasing with time or frequency of use, and impurities such as stable Zn(II) generated by the decay of 68Ga, Ti(IV) as a constituent of the column material, and Fe(III) as a general impurity. Methods: We have developed an efficient route for the processing of generator-derived 68Ga eluates, including the labeling and purification of biomolecules. Preconcentration and purification of the initial generator eluate are performed using a miniaturized column with organic cation-exchanger resin and hydrochloric acid/acetone eluent. The purified fraction was used for the labeling of nanomolar amounts of octreotide derivatives either in pure aqueous solution or in buffers. Results: Using the generator post-eluate processing system, >97% of the initially eluated 68Ga activity was obtained within 4 min as a 0.4-mL volume of a hydrochloric acid/acetone fraction. The initial amount of 68Ge(IV) was decreased by a factor of 104, whereas initial amounts of Zn(II), Ti(IV), and Fe(III) were reduced by factors of 105, 102, and 10, respectively. The processed 68Ga fraction was directly transferred to solutions containing labeling precursors—for example, DOTA-dPhe1-Tyr3-octreotide (DOTATOC) (DOTA = 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid). Labeling yields of >95% were achieved within 10 min. Overall yields reached 70% at 20 min after generator elution relative to the eluted 68Ga activity, not corrected for decay. Specific activities of 68Ga-DOTATOC were 50 MBq/nmol using a standard protocol, reaching 450 MBq/nmol under optimized conditions. Conclusion: Processing on a cation-exchanger in hydrochloric acid/acetone media represents an efficient strategy for the concentration and purification of generator-derived 68Ga(III) eluates. The developed scheme guarantees high yields and safe preparation of injectable 68Ga-labeled radiopharmaceuticals for routine application and is easy to automate. Thus, it is being successfully used in clinical environments and might contribute to a new direction for clinical PET, which could benefit significantly from the easy and safe availability of the radionuclide generator-derived metallic positron-emitter 68Ga.

Radioactive Labeling of Defined HPMA-Based Polymeric Structures Using [18F]FETos for In Vivo Imaging by Positron Emission Tomography

During the last decades polymer-based nanomedicine has turned out to be a promising tool in modern pharmaceutics. The following article describes the synthesis of well-defined random and block copolymers by RAFT polymerization with potential medical application. The polymers have been labeled with the positron-emitting nuclide fluorine-18. The polymeric structures are based on the biocompatible N-(2-hydroxypropyl)-methacrylamide (HPMA). To achieve these structures, functional reactive ester polymers with a molecular weight within the range of 25,000-110,000 g/mol were aminolyzed by 2-hydroxypropylamine and tyramine (3%) to form (18)F-labelable HPMA-polymer precursors. The labeling procedure of the phenolic tyramine moieties via the secondary labeling synthon 2-[(18)F]fluoroethyl-1-tosylate ([(18)F]FETos) provided radiochemical fluoroalkylation yields of ∼80% for block copolymers and >50% for random polymer architectures within a synthesis time of 10 min and a reaction temperature of 120 °C. Total synthesis time including synthon synthesis, (18)F-labeling, and final purification via size exclusion chromatography took less than 90 min and yielded stable (18)F-labeled HPMA structures in isotonic buffer solution. Any decomposition could be detected within 2 h. To determine the in vivo fate of (18)F-labeled HPMA polymers, preliminary small animal positron emission tomography (PET) experiments were performed in healthy rats, demonstrating the renal clearance of low molecular weight polymers. Furthermore, low metabolism rates could be detected in urine as well as in the blood. Thus, we expect this new strategy for radioactive labeling of polymers as a promising approach for in vivo PET studies.

72/74As-labeling of HPMA based polymers for long-term in vivo PET imaging.

In the context of molecular imaging, various polymers based on the clinically approved N-(2-hydroxypropyl)-methacrylamide (HPMA) have been radio-labeled using longer-living positron emitters 72As t1/2=26 h or 74As t1/2=17.8 d. This approach may lead to non-invasive determination of the long-term in vivo fate of polymers by PET (positron emission tomography). Presumably, the radio label itself will not strongly influence the polymer structure due to the fact that the used nuclide binds to already existing thiol moieties within the polymer structure. Thus, the use of additional charges or bulky groups can be avoided.

Iron oxide/hydroxide nanoparticles with negatively charged shells show increased uptake in Caco-2 cells.

The absorption of commonly used ferrous iron salts from intestinal segments at neutral to slightly alkaline pH is low, mainly because soluble ferrous iron is easily oxidized to poorly soluble ferric iron and because ferrous iron, but not ferric iron, is carried by the divalent metal transporter DMT-1. Moreover, ferrous iron frequently causes gastrointestinal side effects. Iron hydroxide nanoparticles with neutral and hydrophilic carbohydrate shells are alternatively used to ferrous salts. In these formulations gastrointestinal side effects are rare because hundreds of ferric iron atoms are safely packed in nanoscaled cores surrounded by the solubilizing shell; nevertheless, iron bioavailability is even worse compared to ferrous salts. In this study the cell uptake of iron hydroxide and iron oxide nanoparticles (FeONP) with negatively charged shells of different chemical types and sizes was compared to the uptake of those with neutral hydrophilic shells, ferrous sulfate and ferric chloride. The nanoparticle uptake was measured in Caco-2 cells with the iron detecting ferrozine method and visualized by transmission electron microscopy. The toxicity was evaluated using the MTT assay. For nanoparticles with a negatively charged shell the iron uptake was about 40 times higher compared to those with neutral hydrophilic carbohydrate shell or ferric chloride and in the same range as ferrous sulfate. However, in contrast to ferrous sulfate, nanoparticles with negatively charged shells showed no toxicity. Two different uptake mechanisms were proposed: diffusion for hydroxide nanoparticles with neutral hydrophilic shell and adsorptive endocytosis for nanoparticles with negatively charged shells. It needs to be determined whether iron hydroxide nanoparticles with negatively charged shells also show improved bioavailability in iron-deficient patients compared to iron hydroxide nanoparticles with a neutral hydrophilic shell, which exist in the market today.

Separation and purification of no-carrier-added arsenic from bulk amounts of germanium for use in radiopharmaceutical labelling

Abstract Radioarsenic labelled radiopharmaceuticals could add special features to molecular imaging with positron emission tomography (PET). For example the long physical half-lives of 72As (T1/2=26 h) and 74As (T1/2=17.8 d) in conjunction with their high positron branching rates of 88% and 29%, respectively, allow the investigation of slow physiological or metabolical processes, like the enrichment and biodistribution of monoclonal antibodies in tumour tissue or the characterization of stem cell trafficking. A method for separation and purification of no-carrier-added (nca) arsenic from irradiated metallic germanium targets based on distillation and anion exchange is developed. It finally converts the arsenic into an *As(III) synthon in PBS buffer and pH 7 suitable for labelling of proteins via As-S bond formations. The method delivers radioarsenic in high purity with separation factors of 106 from germanium and an overall yield from target to labelling synthon of >40%. In a proof-of-principle experiment, the monoclonal antibody Bevacizumab, directed against the human VEGF receptor, was labelled with a radiochemical yield >90% within 1 h at room temperature with nca 72/74/77As.

Hemin-coupled iron(III)-hydroxide nanoparticles show increased uptake in Caco-2 cells

Objectives  The absorption of commonly used ferrous iron salts from intestinal segments at neutral to slightly alkaline pH is low, mainly because soluble ferrous iron is easily oxidized to poorly soluble ferric iron and ferrous iron but not ferric iron is carried by the divalent metal transporter DMT‐1. Moreover, ferrous iron frequently causes gastrointestinal side effects. In iron(III)‐hydroxide nanoparticles hundreds of ferric iron atoms are safely packed in nanoscaled cores surrounded by a solubilising carbohydrate shell, yet bioavailability from such particles is insufficient when compared with ferrous salts. To increase their intestinal uptake iron(III)‐hydroxide nanoparticles were coupled in this study with the protoporphyrin hemin, which undergoes carrier‐mediated uptake in the intestine.

Visualisation of a somatostatin receptor-expressing tumour with 67Ga-DOTATOC SPECT.

In comparison to In-DTPAOC (Octreoscan), galliumlabelled DOTATOC shows better binding affinity to human somatostatin receptor subtype 2 and improved pharmacology in vivo [1, 2]. Especially if Ga-DOTATOC and PET/ CT are applied, somatostatin receptor-expressing tumour tissue is excellently visualised. However, SPECT is still a more widely available imaging method. Here we present the first visualisation of a human somatostatin receptorexpressing tumour with Ga-DOTATOC SPECT/CT. A 65-year-old man with known mesenteric lymph node metastases of a surgically removed carcinoid in the small bowel received 180 MBq of In-DTPAOC and, 1 week later, 230 MBq of Ga-DOTATOC (50 MBq/μg specific activity). All metastases detected with In-DTPAOC (a) could be visualised with Ga-DOTATOC as well. Scans of Ga-DOTATOC (SPECT/CT) were performed less than 4 h post injection to generate excellent images of the mesenteric manifestations (b), with a higher tumour to background ratio compared to that on In-DTPAOC images. The presence of only faint renal Ga-DOTATOC uptake constitutes a further favourable characteristic of this radiolabelled peptide. a