Development and Evaluation of Selective Matrix Metalloproteinase-13 PET Radiotracers for Imaging Atherosclerosis

Abstract

Atherosclerosis is a chronic inflammatory disease characterized by the buildup of lipid-rich plaques within the coronary arteries and a leading cause of death worldwide. Given that plaque rupture occurs due to molecular changes in plaque composition and is considered the primary cause of heart attack and stroke, molecular imaging by positron emission tomography (PET) provides the opportunity to assess disease severity and may assist in the prevention of severe cardiovascular events and associated fatal outcomes. Matrix metalloproteinases (MMPs) represent a class of zinc-chelating enzymes with diverse functions in extracellular matrix (ECM) remodeling during normal physiological processes and inflammatory diseases. As MMPs possess distinct substrate specificity and differential roles in disease progression, selective targeting of individual MMPs should be prioritized for diagnosis and therapy. Upregulated MMP-13 activity has been extensively implicated with atherosclerotic plaque destabilization due to uncontrolled ECM degradation and formation of thin-capped and collagen-poor plaques that are most susceptible to rupture. As such, MMP-13 represents a compelling molecular biomarker of unstable atherosclerosis for selective PET radiotracer development. This thesis outlines the synthesis, optimization, and evaluation of MMP-13 selective PET radiotracers for imaging atherosclerosis. Chapter I introduces atherosclerosis, provides an overview of MMPs as therapeutics and diagnostics, and describes available strategies for MMP-13 selective radiotracer development. Chapter II outlines a head-to-head comparison of MMP-13 selective and broad-spectrum MMP imaging with existing fluorine-18 labeled PET radiotracers. In this study, the first in vivo evaluation of [18F]FMBP and [18F]BR-351, as MMP-13 selective and non-selective MMP-targeted radiotracers based on the pyrimidine-dicarboxamide and non-peptidic aryl sulfonamide inhibitor classes, was performed in a mouse model of atherosclerosis. Determination of radiotracer pharmacokinetics, target specificity, and sensitivity to atherosclerotic tissue validated the feasibility of using an MMP-13 selective PET radiotracer to detect ECM remodeling in atherosclerotic plaques and demonstrated several advantages to this strategic imaging approach. Chapter III describes the development and evaluation of novel MMP-13 selective PET radiotracers based on the most potent and selective quinazoline-2-carboxamide inhibitor class for imaging atherosclerosis. Structure-activity relationship (SAR) studies were performed with a particular focus on modifications that would facilitate late-stage radiolabeling with carbon-11 or fluorine-18 and pharmacokinetic characterization in atherosclerotic mice. The first biological evaluation of three candidate radiotracers [11C]5b, [11C]5f, and [18F]5j was conducted and elucidated the structural determinants required to obtain in vivo functional activity, MMP-13 specificity/selectivity, and desirable pharmacokinetics for vascular imaging. This study further highlighted the superiority of the quinazoline-2-carboxamide scaffold for selective MMP-13 PET radiotracer development and identified [18F]5j as a promising lead for ex vivo atherosclerotic plaque imaging. Chapter IV focuses on the synthesis and design of highly functionalized second-generation MMP-13 selective PET radiotracers based on the quinazoline-2-carboxamide scaffold with greater contrast for non-invasive atherosclerotic plaque imaging. Structural modifications focused on restoring critical binding interactions, incorporating a new site for fluorine-18 or carbon-11 radiolabeling, and reducing lipophilicity. SAR uncovered the optimized inhibitor 29f as a promising lead radiotracer candidate with remarkably enhanced MMP-13 potency and off-target selectivity. In vivo evaluations of [11C]29f in atherosclerotic mice demonstrated its favorable pharmacokinetic properties and assessed its potential utility for atherosclerosis.