Novel molecular imaging ligands targeting matrix metalloproteinases 2 and 9 for imaging of unstable atherosclerotic plaques

Abstract

Molecular imaging of matrix metalloproteinases (MMPs) may allow detection of atherosclerotic lesions vulnerable to rupture. In this study, we develop a novel radiolabelled compound that can target gelatinase MMP subtypes (MMP2/9) with high selectivity and inhibitory potency. Inhibitory potencies of several halogenated analogues of MMP subtype-selective inhibitors (N-benzenesulfonyliminodiacetyl monohydroxamates and N-halophenoxy-benzenesulfonyl iminodiacetyl monohydroxamates) were in the nanomolar range for MMP2/9. The analogue with highest inhibitory potency and selectivity was radiolabelled with [123I], resulting in moderate radiochemical yield, and high radiochemical purity. Biodistribution studies in mice, revealed stabilization in blood 1 hour after intravenous bolus injection. Intravenous infusion of the radioligand and subsequent autoradiography of excised aortas showed tracer uptake in atheroprone mice. Distribution of the radioligand showed co-localization with MMP2/9 immunohistochemical staining. In conclusion, we have developed a novel selective radiolabeled MMP2/9 inhibitor, suitable for single photon emission computed tomography (SPECT) imaging that effectively targets atherosclerotic lesions in mice.

Fig 1. Hydroxamate target compounds.

Schematic depicting halogenated forms of two sulfonamide hydroxamate based compounds examined (4a-e and 10a-d).

Fig 2. Synthesis of hydroxamate target compound 4a-e from 1a-e.

(i): HN(CH₂CO₂tbu)₂,TEA, DCM, rt, o/n, yield 39–75%; (ii): HCOOH, rt, o/n, yield 30–93%; (iii): ECF, NMM, THF, NH₂OH x HCl, MeOH, 0°C, 2 h, yield 63–97%.

Fig 3

Synthesis of 10 a-d. (i): HN(CH₂COOH, THF/H₂O, rt, o/n, yield 3–35%; (ii): HN(CH₂CO₂tbu)₂, TEA, DCM, rt, o/n, 78–99%; (iii): HCOOH, rt, o/n, yield 75–94%; (iv): ECF, NMM, THF, NH₂OH x HCl, MeOH, 0°C, 2 h, yield 52–91%.

Fig 4. Radiosynthesis of [¹²³I]10c from 10d by nucleophilic radioiodination.

(i): Na[¹²³I]I, ethanol, citric acid, 2,5 dihydroxybenzoic acid, CuSO₄, SnSO_4, 130°C, 40 min, yield 51%.

Fig 5. Biodistribution of [¹²³I]10c following intravenous infusion in healthy mice (n = 3–4 /group).

Injected dose (ID) of radioactivity was measured in fat (A), muscle (B), liver (C), kidney (D), spleen (E), blood (F), heart (G) and lung (H) at varying time-points after injection. Values shown are mean ± SEM.

Fig 6. Binding of [¹²³I]10c to atherosclerotic lesions containing MMP2 and MMP9.

A) Storage phosphor image depicting binding of [¹²³I]10c in a cross-section of the aortic arch of an ApoE-/- mouse. The region with highest apparent radioactivity has been outlined. B) Hematoxylin and eosin staining of the respective aortic arch cross-section shown in (A). The respective outlined region in (A) coincides with an atherosclerotic lesion at the base of a branching artery, as outlined in (B). C) Enlargement of the outlined region in (B). D) Cross-section of the atherosclerotic plaque in a consecutive section, displaying MMP2 (brown) distribution mainly in the smooth muscle cell-rich medial area of the aorta, and nuclei (blue). E) Cross-section of the atherosclerotic lesion in a consecutive section, depicting MMP9 (brown) distribution mainly in foam cell macrophages of the atherosclerotic plaque, and nuclei (blue). F) Respective section in (E) stained with anti-alpha-smooth muscle actin depicting smooth muscle rich layer (purple) in the aorta vessel wall, adjacent to the plaque core where apparent foam cells are present.