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Synthesis and Evaluation of Radiolabelled Galactose Derivatives for In Vivo Visualization of LacZ Reporter Gene Expression using Positron Emission tomography
Abstract/OtherAbstract :
Non-invasive in vivo imaging of reporter gene expression is useful for many medical and biological research applications. The combination of an appropriate reporter gene and radiolabelled reporter probe offers the potential for in vivo imaging and follow-up of therapeutic transgenes and reporter gene expressing cells. The aim of this study was to develop 18F- and 11C-labelled reporter probes, based on derivatives of galactose, for in vivo visualization of LacZ reporter gene expression using positron emission tomography (PET).<br/>The LacZ gene encodes for bacterial beta-galactosidase, an enzyme that hydrolyses beta-galactosidic bonds such as in its natural substrate lactose. Three classes of radiolabelled galactose-derivatives were synthesized, all of which carry the radioisotope in the aglycon part and have the beta-galactosyl configuration in order to have affinity for the enzyme.<br/>As a first approach, 18F- and 11C-labelled phenyl beta-D-galactopyranosides were synthesized in good radiochemical yield and purity. Since these tracers have an ‘acetal ether’ linker (galactosidic bond) between galactose and the aglycon, they release radiolabelled phenols upon enzymatic hydrolysis. Although both tracers were good substrates of the enzyme in vitro, the accumulation in LacZ expressing 293T cells was relatively low. Since no mammalian active transporters are known for this kind of galactose-derivatives, passage through lipophilic membranes is assumed to occur via passive diffusion. Therefore, the low cell uptake can be partly ascribed to the hydrophilicity of the tracers (-1< log P <0), hampering efficient cell entry. RP-HPLC analysis of the cell lysate and supernatant of LacZ cells after incubation with [18F]fluoroethoxyphenyl beta-D-galactopyranoside revealed that once inside the cell, the tracer was efficiently and specifically hydrolysed by the enzyme into the corresponding radiolabelled phenol. However, a large fraction of the radiolabelled aglycon was also present in the supernatant of the LacZ expressing cells. This can be due to efflux of the phenolic metabolite out of the cells or can be caused by the presence of beta-galactosidase in the extracellular fluid (vide infra). In vivo evaluation of the 18F-labelled galactosyl ether in a mouse inoculated with a LacZ expressing tumour using µPET imaging, revealed low uptake in the tumour, presumably due to poor tumourcell membrane penetration.<br/>In a second attempt to develop tracers with a better retention in LacZ expressing cells, we synthesized 18F- and 11C-labelled beta-galactosyl esters. In these probes the ‘acetal ether’ is replaced by an ‘acetal ester’ linker. Upon hydrolysis, radiolabelled benzoic acids are released that are hypothesized to have a better retention in beta-gal expressing cells due to their lower pKA value compared to the phenolic aglycons of the radiolabelled galactosyl ethers. The synthesis and radiosynthesis were somewhat more complicated due to the presence of the labile acetal ester function, resulting in lower radiochemical yields. Despite the presence of the acetal ester, both probes were good substrates of the enzyme in vitro. In contrast to the galactosyl ethers, these tracers were metabolized in vivo into the corresponding more polar benzoic acids, resulting in a more efficient clearance from blood into the urine. Incubation experiments with LacZ gene-transduced 293T cells showed no difference in cellular uptake and retention compared to control 293T cells and analysis of the cell lysates and supernatant fractions using RP-HPLC revealed a high amount of radiolabelled benzoic acid in the supernatant of the LacZ expressing cells. This was found to be predominantly due to the presence of beta-galactosidase in the extracellular fluid, possibly caused by cell lysis or cellular secretion of the enzyme.<br/>Nevertheless, even in the case of extracellular hydrolysis one would expect a steeper radiotracer accumulation curve for the LacZ cells compared to the control cells, indicating that diffusion of the hydrophilic intact tracers (-1< log P <0) through the cell membrane and not their hydrolysis to the corresponding benzoic acid derivative (or phenol as with the galactosyl ethers of class I) is rate limiting.<br/>As a last approach, we developed 11C-labelled phenylic and naphthylic beta-galactosyl triazoles. The precursors and reference compounds were synthesized using a Cu(I)-catalyzed ‘click’ cycloaddition reaction between acetylated beta-galactosyl azide and the corresponding terminal alkynes. In vitro incubation experiments with beta-gal in the presence of the chromogenic substrate ONPG, showed that the triazolic compound is not a substrate of the enzyme, but acts as an inhibitor. Of all the tracers discussed in this thesis, the naphthylic triazole was the most lipophilic (log P: 1.4). Nevertheless, just like with the more polar tracers, biodistribution studies showed no brain uptake. This may be explained in terms of the high polar surface area of the compounds. All studied tracers have four polar sugar-hydroxyl functions, resulting in strong hydrogen bond formation and poor passive diffusion across lipophilic membranes. Both triazolic tracers were very stable in vivo, what was expected in view of the rigid triazole linkage. Cell uptake experiments showed a higher cell uptake for the more lipophilic naphthylic triazole compared to the phenylic tracer, however for both tracers there was no significant difference in specific retention between LacZ and control HEK cells, which is probably due to the fact that the affinity of this series of triazoles for the beta-gal enzyme is too low.<br/>In conclusion we can state that three classes of radiolabelled galactose-derivatives were synthesized and evaluated as reporter probes for imaging LacZ reporter gene expression in vivo. Development of efficient radiolabelled LacZ probes is not so evident, since several factors have to be taken into account and have to be optimized in order to have a good in vivo target to background signal. The major problem encountered was the compatibility of the polar sugar-containing tracers with the efficient transport across lipophilic membranes. As was shown with the lipophilic naphthylic triazole of class III, increasing the log P value by modification of the aglycon has a positive effect on cell uptake. Since the tracers of class I and II were good substrates of the enzyme, synthesis of more lipophilic derivatives of these compounds may be a promising future perspective in those studies were no transporters are present. On the other hand, when a LacZ vector could be constructed that also expresses the lactose permease gene (LacY), which is responsible for transport of lactose and other beta-galactopyranosides into bacteria, the problem of the hampered cell diffusion and uptake could be solved.
Authors :
Celen, Sofie
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Bormans, Guy
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Publisher :  K.U.Leuven     Type :  Electronic thesis or dissertation, Elektronische thesis of dissertatie     Format :  1294797 bytes, application/pdf    
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