Fluorine-18 labelling of a novel series of chimeric, mdm2 oncogene targeting, peptide-pna oligomers using [18F]FPyME

Complete text of publication follows: Peptide nucleic acids (PNAs) form a unique class of synthetic macromolecules, originally designed as ligands for the recognition of double stranded DNA, where the deoxyribose phosphate backbone of original DNA is replaced by a pseudo-peptide N-(2-aminoethyl)glycyl backbone, while retaining the nucleobases of DNA. PNAs have already showed promising therapeutic potential as antisense and anti-gene agents and are inspiring the development of a variety of research and diagnostic assays, including their use as imaging tools. Within our intensive programs of development of oligonucleotide-based probes for PET-imaging, a novel series of chimeric peptide-PNA oligomers has been designed as complementary antisense probes targeting a specific 15-base sequence located at the intron-exon junction of the pre-mRNA of the murine double minute (mdm2) oncogene. This gene codes for a p53 interacting protein that represses p53 transcriptional activity, and appears to be over expressed in several tumor types including soft tissue sarcomas and osteosarcomas as well as breast tumors. For in vivo 3D-imaging purposes, all oligomers include a cysteine thus providing a sulfhydryl function permitting prosthetic conjugation with maleimide-based reagents such as AlexaFluor680R (AF680) for optical fluorescence imaging and [18F]FPyME (1-[3-(2-[18F]fluoropyridin-3-yloxy)propyl]pyrrole-2, 5-dione), a prosthetic reagent labeled with the positron-emitter fluorine-18 for PET imaging, which latter work is presented herein. Methods: [18F]FPyME was prepared using a three-step radiochemical pathway already reported and includes an HPLC-purification (semi-preparative SiO2 ZorbaxR Rx-SIL, Hewlett Packard). [18F]FPyME was conjugated with the peptide-PNA oligomers (PNA3132, PNA3133, and PNA3135, 0.25-0.30 micro-moles) in 1/9 (v:v) mixture (1 mL) of DMSO and 0.1 M aq. PBS (pH 8) at room temperature for 15 min. The [18F]FPyME-conjugated products (c-[18F]PNA3132, c-[18F]PNA3133 and c-[18F]PNA3135, respectively) were purified using semi-preparative RP-HPLC (μBondapak columnR (Waters), gradient elution: water (+0.1 % TFA) / MeCN from 95/5 to 50/50 in 20 min, flow rate: 6 mL/min) and finally retaken in aq. 0.9% NaCl (3 mL) after HPLC-solvent evaporation under reduced pressure. Results: 2.5-3.7 GBq batches of HPLC-purified [18F]FPyME could be obtained in 110 min starting from 20-25 GBq of [18F]fluoride (RCY: 25-30%). Conjugation of [18F]FPyME with PNA3132, PNA3133, and PNA3135 provided the target conjugated PNAs (1.1-1.8 GBq batches) in 45 min (formulation included) and with yields ranging from 60% to 65% (HPLC-isolated, decay-corrected). As demonstrated by TLC and HPLC, the c- [18F]PNAs were shown to be≥ 95% radiochemically pure. Conclusions: Three selected chimeric peptide-PNA oligomers have been successfully labeled with fluorine-18 using [18F]FPyME and our Zymate XP robotic system. Whole-body bio-distributions and pharmacokinetics of c- [18F]PNA3132, c-[18F]PNA3133 and c-[18F]PNA3135 are currently evaluated in nude mice bearing SJSA1 tumours (human osteosarcoma)