Evaluation of the radiolabeled boronic acid-based FAP inhibitor MIP-1232 for atherosclerotic plaque imaging

Abstract

Research towards the non-invasive imaging of atherosclerotic plaques is of high clinical priority as early recognition of vulnerable plaques may reduce the incidence of cardiovascular events. The fibroblast activation protein alpha (FAP) was recently proposed as inflammation-induced protease involved in the process of plaque vulnerability. In this study, FAP mRNA and protein levels were investigated by quantitative polymerase chain reaction and immunohistochemistry, respectively, in human endarterectomized carotid plaques. A published boronic-acid based FAP inhibitor, MIP-1232, was synthetized and radiolabeled with iodine-125. The potential of this radiotracer to image plaques was evaluated by in vitro autoradiography with human carotid plaques. Specificity was assessed with a xenograft with high and one with low FAP level, grown in mice. Target expression analyses revealed a moderately higher protein level in atherosclerotic plaques than normal arteries correlating with plaque vulnerability. No difference in expression was determined on mRNA level. The radiotracer was successfully produced and accumulated strongly in the FAP-positive SK-Mel-187 melanoma xenograft in vitro while accumulation was negligible in an NCI-H69 xenograft with low FAP levels. Binding of the tracer to endarterectomized tissue was similar in plaques and normal arteries, hampering its use for atherosclerosis imaging.

Figure 1

Relative mRNA expression levels of FAP (A) and SMA (B) in normal arteries (n = 2), stable plaques (n = 11) and vulnerable plaques (n = 9). For both proteins no significant difference was detected between stable and vulnerable plaques. (C) Comparison of the relative mRNA expression levels of FAP and SMA. mRNA expression was quantified by qPCR, shown are averages of three independent analyses. Lines indicate mean values. The square bracket indicates an outlier that was excluded from statistical analyses.

Figure 2

Hematoxylin/eosin (HE; A–C) and immunohistochemical (A–D) staining for FAP and SMA of representative 2 μm paraffin-embedded sections of a normal artery (A), a stable plaque (B) and vulnerable plaques (C,D). Boxed higher-magnification images show a small blood vessel (normal artery A1), regions in the fibrous cap (stable B1, B2 and vulnerable plaque C1) and FAP-positive macrophages (C2, arrows). (D) High magnification images show FAP-positive giant cells (D1, arrowheads) and macrophages (D1, D2, arrows) in a vulnerable plaque. The endarterectomized plaques are composed of tunica intima and part of the media. Lu: lumen. Scale bar, low magnification 2000 μm; A1, B1, B2, C1, 200 μm; C2, D1, D2, 50 μm.

Scheme 1

Synthesis of reference compound 4 and corresponding precursor 6.

Scheme 2

Radioiodination scheme of precursor 6 to [¹²⁵I]MIP-1232.

Figure 3

(A) In vitro autoradiogram of representative sections of human carotid plaques under baseline ([¹²⁵I]MIP-1232) and blockade condition ([¹²⁵I]MIP-1232 with excess unlabeled MIP-1232). Hematoxylin/eosin (HE) staining below represents plaque morphology. Scale bar 3 mm. (B) Quantified total and specific binding of [¹²⁵I]MIP-1232 to normal arteries (n = 5), stable plaques (n = 16) and vulnerable plaques (n = 15) determined by autoradiography and corrected for tissue size. No significant intergroup differences were determined. Lines indicate mean values, diamonds indicate the specimens shown in A. (C) In vitro autoradiography with xenografts under baseline and blockade conditions. IHC staining for FAP of the SK-Mel-187 and the NCI-H69 xenograft (20 µm cryosections). Scale bar 3 mm for autoradiography; 50 µm for IHC images. Color scales for minimal to maximal binding.