We propose to develop a novel, modular arrangement of synthetic peptides and radiolabeling prosthetic groups to develop imaging probes that target cytokine / growth factor receptor expression in CNS disease states. The synthetic peptides, referred to as RTD probes are single- or double-chain constructs. In the simplest application, they are intended to target homodimeric receptors. Potentially, heterodimeric receptors can be targeted with modification of the basic RTD probe design. Further, members of this research team have previously been involved in the development of a dual radioisotopic labeling prosthetic group - a benzophenone that harbors fluorine and iodine atoms which can be exchanged for F-18 and/or 1-124/125 isotopes. Conjugating the RTD peptide with the benzophenone will yield a suitably radiolabeled positron emission tomography (PET) imaging probe. Since both the receptor targeting domain and the PET labeling group are modular, this imparts a great deal of flexibility to this technology. We present proof-of-concept data on imaging FGF-receptors in a CNS demyelination disease model and propose to use similar methods and validation strategies to generate imaging probes for other cytokine receptors, namely VEGF- and PDGF- receptors. The project will consist of recursive rounds of in vitro screening using several biochemical and cell biochemistry assays to assess receptor binding parameters. RTD peptides passing these screens will be tested in in vivo biodistribution studies and in animal models using ex vivo autoradiography. Finally, successful probes will be utilized for PET imaging. The rat experimental autoimmune encephalitis (EAE) model for human multiple sclerosis (MS) was chosen for the in vivo validation studies. The combination of focal inflammation at the site of EAE/MS lesions and blood brain barrier (BBB) disruption at the lesions unmask cytokine receptors on parenchymal brain cells that are normally hidden from circulation and that have been upregulated as a result of the disease process. With our microMRI and microPET facilities, we can monitor the lesions and determine if an RTD probe is effective when the MRI and PET images are co- registered and the same lesions are identified by both imaging modalities. Clinically, MS is adequately imaged by MRI alone, but this passive water content-based modality cannot provide direct information on active biological processes, such as the upregulation of cytokine receptors in the inflammatory and recovery phase of a demyelinating lesion... but an RTD-PET probe can. The primary goal is not just to develop PET probes for MS per se, but to develop methods and protocols for creating, screening, and validating probes that can image cytokine receptors in any demyelinating disease, or potentially any CNS disease where there is an overexpression of cytokine receptor. ? ? ?
