Development of small molecule PET probes for imaging of the mTOR pathway
AuthorWichmann, Christian Werner
AffiliationSchool of Chemistry
Document TypePhD thesis
Access StatusOpen Access
© 2018 Dr Christian Werner Wichmann
HER2 positive breast cancer accounts for about one third of all breast cancers and often results in poor prognosis. The development of resistant patient populations has led to the clinical evaluation of rationally designed combination therapies such as mTOR inhibitor / Herceptin therapy. The BOLERO-1/3 phase III clinical trials indicated a correlation between mTOR activity and treatment response. The aim of this work is to develop a mTOR specific PET ligand by creating a fluorine-18 labelled mTOR inhibitor derivative. Two classes of mTOR inhibitors were synthesized and radiolabelled. The cytotoxicity of new derivatives was determined using SRB assays and compared to reference compounds. In vitro cell uptake of radiolabelled derivatives was investigated using internalization assays and the specificity of observed uptake was confirmed in competition studies. The cellular localization of promising compounds was explored using confocal microscopy. Radiolabelled compounds were evaluated in vivo in murine xenografts of human breast cancer cell lines. The biodistribution was determined using dynamic PET/MR images over 2 hours. Synthesis of class 1 compounds was achieved via a convergent synthesis with a typical yield of 28% over the longest linear sequence (4 steps). Synthesis of class 2 reference compound and radiolabeling precursor was achieved in 38% and 12% yield over 3 and 6 linear steps, respectively. Synthesis of fluorine-19 derivatives of the class 1 and 2 PET ligands was achieved in 7% and 5% yield over 5 and 7 linear steps, respectively. Fluorine-18 labelling of class 1 PET ligand was achieved via copper-catalysed Click Chemistry in 40% RCY after 90 minutes. Radiolabelling of class 2 PET ligand proceeded in 1.5% RCY after 70 minutes. Typical molar activities of class 1 and 2 PET ligands were 27.6 and 22.1 GBq/μmol, respectively, and the radiochemical purity was > 99% in both cases. The logP of class 1 PET ligand was 0.9 at pH 7.4 and no metabolic breakdown was detected in mouse, rat and human S9 liver fractions over 3 hours. Cytotoxicity assays showed an increase in the IC50 values of class 1 PET compared to the reference compound, whereas radiolabeling of class 2 molecules preserved their potency. Internalization studies with [18F]MPC-1-1 showed 2.5- and 1.7-fold increased uptake in HCC-1419 and BT-474 compared to the negative control. In competition assays, the cell uptake could be reduced by 75% and 65%, respectively. Internalization assays with [18F]MPC-2-1 showed limited correlation of cell uptake with RAD001 sensitivity. Confocal microscopy using a fluorescently labelled class 1 derivative showed uptake in the cytoplasm of BT-474 cells. The biodistribution in mice was dominated by intestinal and bladder uptake, 15% and 25% ID/ccm at 90 minutes p.i., respectively. Tumour uptake in BT-474 xenografts increased over time whereas muscle tissue showed clearance of the PET ligand. The mean tumour-to-muscle ratios at 90 minutes p.i. were 1.7 ± 0.2 for RAD001 sensitive and 0.9 ± 0.2 for insensitive xenografts. The mean uptake in BT-474 tumours at 90 minutes p.i. was 2.8% ± 0.3% ID/ccm compared to 1.8% ± 0.2% ID/ccm in MDA-MB-231 and 1.7% ± 0.4% ID/ccm in MDA-MB- 468 xenografts. In conclusion, [18F]MPC-1-1 showed potential as a PET ligand capable of discriminating between RAD001 sensitive and insensitive breast cancer cell lines. Its cellular localization in the cytoplasm was confirmed and uptake was shown to be specific in competition assays. Further studies investigating the uptake threshold which correlates to positive response to mTOR inhibitor treatment are warranted.
KeywordsPET; small molecule; fluorine-18; breast cancer; HER2; mTOR; RAD001; in vitro; in vivo
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