Cross-coupling of [11C]methyllithium for 11C-labelled PET tracer synthesis

The cross-coupling of aryl bromides with [11C]CH3Li for the labelling of a variety of tracers for positron emission tomography (PET) is presented. The radiolabelled products were obtained in excellent yields, at rt and after short reaction times (3-5 min) compatible with the half-life of 11C (20.4 min). The automation of the protocol on a synthesis module is investigated, representing an important step towards a fast method for the synthesis of 11C-labelled compounds for PET imaging

Development and evaluation of PET tracers for imaging ÿØ-glucuronidase activity in cancer and inflammation

Een belangrijke uitdaging voor het vinden van een effectieve behandeling van kanker of bepaalde ontstekingsziekten is het uitroeien van de ziekte zonder schade aan te brengen aan gezonde weefsels van de patient. Het gebrek aan effectiviteit van behandelingen is vaak te wijten aan de fysiologische overeenkomsten tussen gezonde cellen en aangedane cellen, waardoor de selectieve therapeutische werking van cytotoxische geneesmiddelen in uitsluitend de zieke cellen niet mogelijk is. Met dit probleem in gedachte werd een nieuw concept ontwikkeld, genaamd prod rug therapie, waarbij het geneesmiddel wordt gemodificeerd tot een minder reactieve of een minder cytotoxische prodrug. De prod rug kan weer worden omgezet in het actieve geneesmiddel door een enzym dat aileen in het beoogde weefsel (zieke cellen) in hoge concentraties voorkomt. Het enzym ~-glucuronidase zou een aantrekkelijk enzym kunnen zijn voor deze prodrug benadering. Het is bekend dat in bepaalde ziekten -bijvoorbeeld verschillende soorten kanker, bacteriele en virale infecties, reumato'ide artritis en neurologische aandoeningen -hoge concentraties ~-glucuronidase worden gevonden in de ruimte tussen de cellen in aangedane weefsels. In gezonde weefsels daarentegen wordt het enzym uitsluitend gevonden binnenin de cellen in de Iysosomen. Selectieve prod rugs voor ~-glucuronidase zijn meestal glucuronide verbindingen van het geneesmiddel, die relatief niet-toxisch zijn vanwege het water-oplosbare karakter van glucuronide groep. Dit voorkomt dat prod rugs de cel kunnen binnendringen en daardoor dat de prodrugs worden geactiveerd door ~-glucuronidase in de Iysosomen in gezonde cellen. In het zieke weefsel daarentegen is het enzym aanwezig buiten de cellen, waardoor de prodrug kan worden omgezet in het toxische oorspronkelijke geneesmiddel dat ter plekke zijn therapeutische effect kan bewerkstelligen. p-Glucuronidase is a lysosomal enzyme that might be explored in the treatment of several diseases. Especially in those where p-glucuronidase is upregulated in inflammatory lesions (either due to inflammation or infection) or necrotic lesions of large solid tumors. In these pathological conditions, p-glucuronidase-mediated prodrug therapy could be applied. Over the past decades, preclinical studies have shown exciting results, especially for the treatment of cancer, where the treatment with cytostatics is hindered by serious side-effects which can be either by the lack of selectivity or acquired resistance. Glucuronide-conjugates are substrates for p-glucuronidase, thus they can be used as prod rugs for p-glucuronidase-based prod rug therapy. These prodrug therapy strategies are based on selective activation of the prodrug by the higher p-glucuronidase activity in tumor/inflammatory lesions, called Prodrug monotherapy. The efficacy of glucuronide prodrugs can be further improved by combined use of the prodrugs with a tumor-specific antibody (called Antibody-directed enzyme prodrug therapy, ADEPT) or by transfection of the tumor cells with a gene encoding the desired exogenous enzyme (called Gene-directed enzyme prodrug therapy, GDEPT). Despite the appealing concept of prod rug therapy and the encouraging preclinical results, each of these different strategies has its disadvantages and therefore, the quest for a clinical applicable strategy is continuing. For such specific strategies, knowledge of the expression levels and the distribution of the prodrugconverting enzyme is vital. Therefore non-invasive imaging techniques like positron emission tomography (PET) can be attractive tools to evaluate new enzyme-prod rug combinations and to optimize treatment strategies. Thus, the aim of this thesis was to develop PET tracers for monitoring extracellular p-glucuronidase activity in different disease models, with the intention to use this non-invasive method to support the optimization of p-glucuronidase-mediated prod rug therapy.

Synthesis and Evaluation of [F-18]-FEAnGA as a PET Tracer for beta-Glucuronidase Activity

To increase the therapeutic index of chemotherapeutic drugs, prodrugs have been investigated as anticancer agents, as they may present fewer eytotoxic side effects than conventional cytotoxic drugs, while therapeutic efficacy is maintained or even increased. Extracellular beta-glucuronidase (beta-GUS) in the tumors has been investigated as a target enzyme for prodrug therapy, as it can convert nontoxic prodrugs into cytostatic drugs. To optimize beta-GUS-based prodrug therapies, PET imaging could be a useful tool by providing information regarding the localization and quantification of beta-GUS. Here, we describe our first PET tracer for extracellular beta-GUS, [F-18]-FEAnGA, which consists of a 2-[F-18]fluorocthylamine ([F-18]-FEA) group bound to a glucuronic acid via a self-immolative nitrophenyl spacer. [F-18]-FEAnGA was synthesized by alkylation of its imidazole carbamate precursor with [F-18]-FEA, followed by deprotection of the sugar moiety with NaOH in 10-20% overall radiochemical yield. [F-18]-FEAnGA is about 10-fold more hydrophilic than the cleavage product [F-18]-FEA, and it is stable in PBS and rat plasma for at least 3 h. In the presence of either Escherichia coli beta-GUS or bovine liver beta-GUS, in vitro cleavage of [F-18]-FEAnGA with complete release of [F-18]-FEA was observed within 30 mm. C6 glioma cells incubated with the tracer and Escherichia coli beta-GUS or bovine liver beta-GUS showed a 4- and 1.5-fold higher uptake of radioactivity, respectively, as compared to control C6 cells without beta-GUS. Incubation of CT26 murine colon adenocarcinoma cells or the genetically engineered CT26m beta GUS cells, which expressed membrane-anchored GUS on the outer cell membrane, with the tracer, resulted in a 3-fold higher uptake into GUS-expressing cells as compared to control cells. In a preliminary microPET study in mice bearing both CT26 and CT26m beta GUS tumors, [F-18]-FEAnGA exhibited a 2-fold higher retention of radioactivity in the tumor expressing beta-GUS than in the control tumor. [F-18]-FEA did not show any difference in tracer uptake between tumors. These results suggest that [F-18]-FEAnGA may be a suitable PET tracer for evaluation of beta-GUS activity, since it is specifically cleaved by beta-GUS and the released [F-18]-FEA remains attached to targeted cells