On the stability of radiolabelled esters: A Carboxylesterase assay

Autoren:Nics, Lukas (Medizinische Universität Wien); Häusler, Daniela (Medizinische Universität Wien); Mien, Leonhard-Key (Medizinische Universität Wien); Wadsak, Wolfgang (Medizinische Universität Wien); Ettlinger, Dagmar (Medizinische Universität Wien); El-Samahi, H. (Universität Wien); Wagner, Karl-Heinz; Dudczak, Robert (Medizinische Universität Wien); Kletter, Kurt (Medizinische Universität Wien); Mitterhauser, Markus (Medizinische Universität Wien)
Abstrakt:Aim: Amongst functional groups for radiolabelling, ester functions play an important role. Esters are accessible via direct radiolabelling procedures on the corresponding acid functions or indirectly by suitable leaving groups. Biologically, they are cleaved by ubiquitous carboxyl esterase enzymes. These enzymes belong to the most prominent biocatalysts in Phase I metabolism, yielding in the unlabelled acidic core and the corresponding radioalcohol. Hence, our aim was to access the stability of a series of radiolabelled esters (-methyl, -ethyl, -fluoroethyl) to give an estimate and to compare the influence of those different substituents on ester stabilities. Materials and methods: Experiments were performed in triplicate (n=3), using different amounts of the particular unlabelled substrates, knowing that the radiolabelled structures exhibit comparable chemical behavior. Experiments were conducted with β-CIT, FE@CIT, FP-CIT, MTO, ETO, FETO, FMZ, FFMZ, CFN, FE@SUPPY and FE@SUPPY:2 under physiological conditions (porcine carboxyl esterase 80IU, 36°C, phosphate-buffered saline ph7.4). The incubation reactions were stopped by adding acetonitrile/methanol (10:1) at 0, 60, 120, 180, 240, 360 minutes, respectively. After centrifugation (10000rpm, 3min) of the reaction mixtures, the obtained supernatant was subjected to HPLC analysis: Agilent 1100 system with a diode array UV detector at a range from 235 to 255nm; stationary phase was a 5µm particle size RP-18 column, in each case. The mobile phase was a mixture of different amounts of buffered hydrophilic solution and acetonitrile, optimized for the respective substrates. Results: The in vitro assays showed Michaelis-Menten constants (Km) and limiting velocities (Vmax) as follows: Substrate Km[µM] Vmax[µM/min] β-CIT 175,1 0,101 FE@CIT 182,6 0,142 FP-CIT 521,4 0,131 MTO 162,0 1,470 ETO 168,6 1,351 FETO 115,1 1,543 FMZ 39,8 0,208 FFMZ 54,4 0,245 CFN 2133,0 0,041 FE@SUPPY 20,1 0,038 FE@SUPPY:2 13,1 0,015 Conclusion: Differences in Michaelis-Menten constants and limiting velocities represent differences in steric and electronic properties of the whole structures. Within comparable structured groups, stability differences between methyl-, ethyl- and fluoroethyl substituents are negligible. Therefore, with respect to carboxylesterase stability [18F]-fluorethyesters are a useful alternative.
Bibliographische Notiz:L.Nics1,2, D.Haeusler1,3, L.-K.Mien1,3,W.Wadsak1,4, D.Ettlinger1, H.El-Samahi3, K.H.Wagner2, R.Dudczak1, K.Kletter1, M.Mitterhauser1,3,5 1 Department of Nuclear Medicine, Medical University of Vienna, Austria 2 Department of Nutritional Sciences, University of Vienna, Austria 3 Department of Pharmaceutic Technology and Biopharmaceutics, University of Vienna, Austria 4 Department of Inorganic Chemistry, University of Vienna, Austria 5 Hospital Pharmacy of the General Hospital of Vienna, Austria