Peptide optimization and conjugation strategies in the development of molecularly targeted magnetic resonance imaging contrast agents.

Peptides are highly selective, high-affinity ligands for a diverse array of disease targets, but suitably derivatizing them for application as diagnostic or therapeutic agents often presents a significant challenge. Covalent modification with metal chelates frequently results in decreased binding affinity, so a variety of strategies must be explored to find suitable locations for modification and facile peptide conjugation chemistries that maintain or enhance binding affinity. In this chapter, we present a paradigm for systematically optimizing peptide binding and determining the favorable sites and methods for peptide conjugation. This strategy is illustrated by two case studies of peptide-based targeted gadolinium contrast agents: EP-2104R for diagnosis of thrombosis and EP-3533 for diagnosis of cardiac perfusion and fibrosis. Two different architectures for the peptide-metal complex conjugation were designed: EP-2104R contains a total of four gadolinium (Gd) chelates linked at the N- and C-termini, whereas EP-3533 is derivatized with three Gd chelates, two on the N-terminus and one on a lysine side chain. Detailed protocols are provided for two Gd chelate conjugation methods.

EP-2104R: a fibrin-specific gadolinium-Based MRI contrast agent for detection of thrombus.

Thrombus (blood clot) is implicated in a number of life threatening diseases, e.g., heart attack, stroke, pulmonary embolism. EP-2104R is an MRI contrast agent designed to detect thrombus by binding to the protein fibrin, present in all thrombi. EP-2104R comprises an 11 amino acid peptide derivatized with 2 GdDOTA-like moieties at both the C- and N-terminus of the peptide (4 Gd in total). EP-2104R was synthesized by a mixture of solid phase and solution techniques. The La(III) analogue was characterized by and 1D and 2D NMR spectroscopy and was found to have the expected structure. EP-2104R was found to be significantly more inert to Gd(III) loss than commercial contrast agents. At the most extreme conditions tested (pH 3, 60 degrees C, 96 hrs), less than 10% of Gd was removed from EP-2104R by a challenge with a DTPA based ligand, while the commercial contrast agents equilibrated within minutes to hours. EP-2104R binds equally to two sites on human fibrin (Kd = 1.7 +/- 0.5 microM) and has a similar affinity to mouse, rat, rabbit, pig, and dog fibrin. EP-2104R has excellent specificity for fibrin over fibrinogen (over 100-fold) and for fibrin over serum albumin (over 1000-fold). The relaxivity of EP-2104R bound to fibrin at 37 degrees C and 1.4 T was 71.4 mM(-1) s(-1) per molecule of EP-2104R (17.4 per Gd), about 25 times higher than that of GdDOTA measured under the same conditions. Strong fibrin binding, fibrin selectivity, and high molecular relaxivity enable EP-2104R to detect blood clots in vivo.