A "click chemistry" approach to the efficient synthesis of multiple imaging probes derived from a single precursor.

Different imaging modalities can provide complementary information on biological processes at the cellular or molecular level in vitro and in vivo. However, specific molecular probes suitable for a comparison of different imaging modalities are often not readily accessible because their preparation is usually accomplished by individually developed and optimized syntheses. Herein, we present a general, modular synthetic approach that provides access to multiple probes derived from a single precursor by application of the same, efficient functionalization strategy, the Cu(I)-catalyzed cycloaddition of terminal alkynes and azides (click chemistry). To demonstrate the viability and efficiency of this approach, folic acid (FA) was selected as a targeting vector because the preparation of FA-based imaging probes used for SPECT, PET, MRI, and NIRF by reported synthetic strategies is usually difficult to achieve and often results in low overall yields. We prepared a versatile γ-azido-FA precursor as well as a set of alkyne functionalized probes and precursors including ligand systems suitable for the chelation of various (radio)metals, an NIR dye and (18)F- and (19)F-derivatives, which enabled the parallel development of new FA-imaging probes. The Cu(I)-mediated coupling of the alkynes with the γ-azido-FA precursor was accomplished in high yields and with minimal use of protective groups. The various probes were fully characterized spectroscopically as well as in vitro and in vivo. In vitro, all new FA-derivatives exhibited high affinity toward the folic acid receptor (FR) and/or were specifically internalized into FR-overexpressing KB cells. In vivo experiments with nude mice showed that all probes (except the MRI probes which have not been tested yet) accumulated specifically in FR-positive organs and human KB-cell xenografts. However, in vivo imaging revealed significant differences between the various FA-derivatives with respect to unspecific, off-target localization. In general, the comparison of different probes proved the superiority of the more hydrophilic, radiometal-based imaging agents, a result which will guide future efforts for the development of FA-based imaging probes and therapeutic agents. In addition, the strategy presented herein should be readily applicable to other molecules of interest for imaging and therapeutic purposes and thus represents a valuable alternative to other synthetic approaches.

A putative biomarker signature for clinically effective AKT inhibition: correlation of in vitro, in vivo and clinical data identifies the importance of modulation of the mTORC1 pathway.

Our identification of dysregulation of the AKT pathway in ovarian cancer as a platinum resistance specific event led to a comprehensive analysis of in vitro, in vivo and clinical behaviour of the AKT inhibitor GSK2141795. Proteomic biomarker signatures correlating with effects of GSK2141795 were developed using in vitro and in vivo models, well characterised for related molecular, phenotypic and imaging endpoints. Signatures were validated in temporally paired biopsies from patients treated with GSK2141795 in a clinical study. GSK2141795 caused growth-arrest as single agent in vitro, enhanced cisplatin-induced apoptosis in vitro and reduced tumour volume in combination with platinum in vivo. GSK2141795 treatment in vitro and in vivo resulted in ~50-90% decrease in phospho-PRAS40 and 20-80% decrease in fluoro-deoxyglucose (FDG) uptake. Proteomic analysis of GSK2141795 in vitro and in vivo identified a signature of pathway inhibition including changes in AKT and p38 phosphorylation and total Bim, IGF1R, AR and YB1 levels. In patient biopsies, prior to treatment with GSK2141795 in a phase 1 clinical trial, this signature was predictive of post-treatment changes in the response marker CA125. Development of this signature represents an opportunity to demonstrate the clinical importance of AKT inhibition for re-sensitisation of platinum resistant ovarian cancer to platinum.

Synthesis, radiolabeling, in vitro and in vivo evaluation of [18F]-FPECMO as a positron emission tomography radioligand for imaging the metabotropic glutamate receptor subtype 5.

INTRODUCTION: [18F]-(E)-3-((6-Fluoropyridin-2-yl)ethynyl)cyclohex-2-enone O-methyl oxime ([18F]-FPECMO) is a novel derivative of [11C]-ABP688. [18F]-FPECMO was characterized as a PET imaging agent for the metabotropic glutamate receptor subtype 5 (mGluR5). METHODS: [18F]-FPECMO was synthesized in a one-step reaction sequence by reacting [(18)F]-KF-K(222) complex with (E)-3-((6-bromopyridin-2-yl)ethynyl)cyclohex-2-enone O-methyl oxime in dry DMSO. The in vitro affinity of FPECMO was determined by displacement assays using rat whole brain homogenates (without cerebellum) and the mGluR5-specific radioligand [(3)H]-M-MPEP. Further in vitro characterization involved metabolite studies, lipophilicity determination and autoradiographical analyses of brain slices. In vivo evaluation was performed by postmortem biodistribution studies and PET experiments using Sprague-Dawley rats. RESULTS: The radiochemical yield after semipreparative HPLC was 35+/-7% and specific activity was >240 GBq/micromol. [(18)F]-FPECMO exhibited optimal lipophilicity (logD=2.1) and high metabolic stability in vitro. Displacement studies revealed a K(i) value of 3.6+/-0.7 nM for FPECMO. Biodistribution studies and ex vivo autoradiography showed highest radioactivity accumulation in mGluR5-rich brain regions such as the striatum and hippocampus. Co-injection of [18F]-FPECMO and ABP688 (1 mg/kg body weight), an mGluR5 antagonist, showed 40% specific binding in the striatum, hippocampus and cortex, regions known to contain high densities of the mGluR5. PET imaging, however, did not allow the visualization of mGluR5-rich brain regions in the rat brain due to a fast washout of [18F]-FPECMO from mGluR5-expressing tissues and rapid defluorination. CONCLUSIONS: [18F]-FPECMO showed significant potential for the detection of mGluR5 in vitro; however, its in vivo characteristics are not optimal for a clear-cut visualization of the mGluR5 in rats.