Rationale: Myocardial infarction (MI) is one of the leading causes of death worldwide and inflammation is central to the tissue response and patient outcomes. The 18kDa translocator protein (TSPO) has been utilized in positron emission tomography (PET) as an inflammatory biomarker. The aims of this study were to: 1) screen novel, fluorinated, TSPO radiotracers for susceptibility to the rs6971 genetic polymorphism using in vitro competition binding assays in human brain and heart, 2) assess whether the in vivo characteristics of our lead radiotracer, 18 F-LW223, are suitable for clinical translation and 3) validate whether 18 F-LW223 can detect macrophage driven inflammation in a rat myocardial infarction model. Methods: Fifty-one human brain and twenty-nine human heart tissue samples were screened for the rs6971 polymorphism. Competition binding assays were conducted with 3 H-PK11195 and the following ligands: PK11195, PBR28 and our novel compounds (AB5186 and LW223). Naive rats and mice were used for in vivo PET kinetic studies, radiometabolite studies and dosimetry experiments. Rats underwent permanent coronary artery ligation and were scanned using PET/CT with invasive input function at 7 days following MI. For quantification of PET signal in the hypoperfused myocardium, K 1 was used as a surrogate marker of perfusion to correct the binding potential for impaired radiotracer transfer from plasma to tissue (BP TC). Results: LW223 binding to TSPO was not susceptible to the rs6971 genetic polymorphism in human brain and heart samples. In rodents, 18 F-LW223 displayed a specific uptake consistent with TSPO expression, a slow metabolism in blood (62% of parent at 120 min), a high plasma free fraction of 38.5% and a suitable dosimetry profile Brain Tissue for Binding Assays Heart Tissue for Binding Assays

Pluripotent stem cell-derived differentiated endothelial cells offer high potential in regenerative medicine in the cardiovascular system. With the aim of translating the use of a human stem cell-derived endothelial cell product (hESC-ECP) for treatment of critical limb ischemia (CLI) in man, we report a good manufacturing practice (GMP)-compatible protocol and detailed cell tracking and efficacy data in multiple preclinical models. The clinical-grade cell line RC11 was used to generate hESC-ECP, which was identified as mostly endothelial (60% CD31/CD144), with the remainder of the subset expressing various pericyte/mesenchymal stem cell markers. Cell tracking using MRI, PET, and qPCR in a murine model of limb ischemia demonstrated that hESC-ECP was detectable up to day 7 following injection. Efficacy in several murine models of limb ischemia (immunocompromised/immunocompetent mice and mice with either type I/II diabetes mellitus) demonstrated significantly increased blood perfusion and capillary density. Overall, we demonstrate a GMP-compatible hESC-ECP that improved ischemic limb perfusion and increased local angiogenesis without engraftment, paving the way for translation of this therapy.

Myo-inositol hexakisphosphate (IP6) is a natural product known to inhibit vascular calcification (VC), but with limited potency and low plasma exposure following bolus administration. Here we report the design of a series of inositol phosphate analogs as crystallization inhibitors, among which 4,6-di-O-(methoxy-diethyleneglycol)-myo-inositol-1,2,3,5-tetrakis (phosphate), (OEG 2) 2-IP4, displays increased in vitro activity, as well as more favorable pharmacokinetic and safety profiles than IP6 after subcutaneous injection. (OEG 2) 2-IP4 potently stabilizes calciprotein particle (CPP) growth, consistently demonstrates low micromolar activity in different in vitro models of VC (i.e., human serum, primary cell cultures, and tissue explants), and largely abolishes the development of VC in rodent models, while not causing toxicity related to serum calcium chelation. The data suggest a mechanism of action independent of the etiology of VC, whereby (OEG 2) 2-IP4 disrupts the nucleation and growth of pathological calcification.

Assessing the retention of cell therapies following implantation is vital and often achieved by labelling cells with 2′-[18F]-fluoro-2′-deoxy-D-glucose (18F-FDG). However, this approach is limited by local retention of cell-effluxed radiotracer. Here, in a preclinical model of critical limb ischemia, we assessed a novel method of cell tracking using 3′-deoxy-3′-L-[18F]-fluorothymidine (18F-FLT); a clinically available radiotracer which we hypothesise will result in minimal local radiotracer reuptake and allow a more accurate estimation of cell retention. Human endothelial cells (HUVECs) were incubated with 18F-FDG or 18F-FLT and cell characteristics were evaluated. Dynamic positron emission tomography (PET) images were acquired post-injection of free 18F-FDG/18F-FLT or 18F-FDG/18F-FLT-labelled HUVECs, following the surgical induction of mouse hind-limb ischemia. In vitro, radiotracer incorporation and efflux was similar with no effect on cell viability, function or proliferation under optimised conditions (5 MBq/mL, 60 min). Injection of free radiotracer demonstrated a faster clearance of 18F-FLT from the injection site vs. 18F-FDG (p ≤ 0.001), indicating local cellular uptake. Using 18F-FLT-labelling, estimation of HUVEC retention within the engraftment site 4 hr post-administration was 24.5 ± 3.2%. PET cell tracking using 18F-FLT labelling is an improved approach vs. 18F-FDG as it is not susceptible to local host cell reuptake, resulting in a more accurate estimation of cell retention.