PET Imaging of Matrix Metalloproteinase Activity in Abdominal Aortic Aneurysm; Toward Clinical Translation

Introduction: Abdominal aortic aneurysm (AAA) is responsible for ~10,000 deaths per year in the United States, mainly due to rupture. Hence, there is a pressing need for better evaluation of AAA rupture risk. Matrix metalloproteinases (MMPs) play a key role in AAA progression and rupture, and MMP imaging could potentially improve AAA risk stratification and reduce related mortality and morbidity. Clinical translation of MMP imaging requires access to robust radiotracers and may be facilitated with PET imaging. Here, we describe the synthesis and evaluation of ⁶⁴Cu-RYM2, the first in class PET MMP radiotracer for AAA imaging.

Methods: RYM2 functionalized with a NOTA-chelator was synthesized, starting from 4-methylpentanoyl chloride, in 20 synthetic steps. Structural characterization and purity analysis were performed using nuclear magnetic resonance (NMR) and liquid chromatography mass spectrometry (LCMS). Radiolabeling was achieved by conjugation of ⁶⁴Cu, as copper acetate, to the NOTA chelator using a single step radiolabeling procedure. The radiolabeling efficiencies were measured using radio high performance liquid chromatography (HPLC). The binding affinities (K_i) of RYM2 and Cu-RYM2 towards different activated recombinant human MMPs were measured using a competitive inhibition assay. ⁶⁴Cu-RYM2 stability and blood partition were determined by incubation in blood in vitro. Binding of ⁶⁴Cu-RYM2 to human AAA and normal aorta was evaluated by autoradiography. The biodistribution and blood kinetics of ⁶⁴Cu-RYM2 were evaluated in mice by gamma counting. In vivo binding to AAA was studied by microPET/CT using Apoe^-/- mice infused with Angiotensin II (Ang II) for 4 weeks and correlated with ex vivo measured MMP activity and Cd68 gene expression. Toxicology evaluation was performed in wild type mice using murine equivalent of 100 times human dose.

Results: NMR and LCMS characterization confirmed the structure, and HPLC analysis confirmed ³ 95% purity of ⁶⁴Cu-RYM2. RYM2 and Cu-RYM2 showed high affinity binding to several activated recombinant human MMPs, with K_i as low as 9 nM for MMP-12. ⁶⁴Cu-RYM2 showed minimal degradation after 2 h incubation in human and mouse blood in vitro, with 71 –74 % remaining in the free fraction. In vivo, 84.4 ± 8.1 % of the radiotracer remained in the intact form in mouse blood at 1 h post-injection (p.i.) (n = 3). There was significantly higher specific binding to human AAA (n = 9) compared to normal aortic tissue (n = 5, P < 0.01). In vivo evaluation of ⁶⁴Cu-RYM2 biodistribution and blood kinetics showed rapid, primarily renal blood clearance and low blood activity (0.7 ± 0.1 % injected dose/g) at 1 h p.i.. ⁶⁴Cu-RYM2 uptake in AAA was readily detectable on microPET/CT images in Ang II-infused Apoe^-/- mice (Figure). Quantification of the suprarenal abdominal aorta PET signal showed 2.2- and 3.1-fold higher radiotracer uptake in animals with AAA (n = 10, SUV_max = 1.82 ± 0.50) compared to Ang II-infused mice with low remodeling (n = 13, SUV_max = 0.83 ± 0.16) and control animals (n = 6, SUV_max = 0.59 ± 0.04, P < .01 and P < .0001, respectively). Radiotracer uptake in vivo correlated with ex vivo measures of MMP activity and Cd68 gene expression. The specificity of radiotracer uptake in vivo in murine aneurysm was confirmed by a 63% reduction in the PET signal in blocking studies (Figure). Preliminary toxicology evaluation showed no adverse reaction in mice.

Conclusions: A novel MMP-targeted PET radiotracer, ⁶⁴Cu-RYM2, was developed and validated in preclinical studies. These along with binding to human tissues and toxicology data suggest that ⁶⁴Cu-RYM2 is a promising candidate for first in human imaging studies of MMP activity in AAA.

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