As CD13 is selectively expressed in angiogenesis, it can serve as a target for molecular imaging tracers to noninvasively visualize angiogenic processes in vivo. The CD13-targeting moiety NGR was synthesized and cyclized by native chemical ligation (NCL) instead of disulfide bridging, leading to a cyclic peptide backbone: cyclo(Cys-Asn-Gly-Arg-Gly) (coNGR). Beside this new monomeric coNGR, a tetrameric NGR peptide co(NGR)4 was designed and synthesized. After radiolabeling, their in vitro and in vivo characteristics were determined. Both coNGR-based imaging agents displayed considerably higher standardized uptake values (SUVs) at infarcted areas compared to the previously reported disulfide-cyclized cNGR imaging agent. Uptake patterns of 111In-coNGR and 111In-co(NGR)4 coincided with CD13 immunohistochemistry on excised hearts. Blood stability tests indicated better stability for both novel imaging agents after 50 min blood incubation compared to the disulfide-cyclized cNGR imaging agent. In mice, both coNGR peptides cleared rapidly from the blood mainly via the kidneys. In addition, co(NGR)4 showed a significantly higher specific uptake in infarcted myocardium compared to coNGR and thus is a promising sensitive imaging agent for detection of angiogenesis in infarcted myocardium.
Angiogenesis is an endogenous healing process which serves to restore tissue blood supply in response to ischemic injury [
Frequently employed imaging agents for noninvasive nuclear imaging of cardiovascular angiogenesis in animal models are based either on the
The CD13-targeting NGR motif has been explored at our institute as a molecular angiogenesis imaging agent for fluorescence microscopy [
Whereas the ring structure of our previously developed cyclic NGR peptide cyclic(NAc-Cys-Asn-Gly-Arg-Cys-Gly-Gly-Lys) was cyclized using a disulfide bond (henceforth called “cNGR”), we now cyclized the ring structure via a peptide bond by native chemical ligation (NCL, Figure
Reaction mechanism of cyclization of CNGRG-MpaL via NCL.
Structural formulas of DTPA-conjugated coNGR (a), co(NGR)4 (b), and cNGR (c).
Boc-Cys(MeBzl)-OH, Boc-Asn(Xan)-OH, and Boc-Arg(Tos)-OH were purchased from Bachem (Bubendorf, Switzerland). Boc-Gly-OH was obtained from Peptide Institute, Inc. (Osaka, Japan).
Linear CNGRG-MpaL thioester peptide was synthesized on methylbenzhydrylamine- (MBHA-) polystyrene resin (ChemPep, Wellinton, FL, USA; 0.2–0.4 mmol scale) as described previously [
For cyclization, the peptide was dissolved in 50 mM (NH4)2CO3 pH 7.8 (Sigma Aldrich, Steinheim, Germany) at a maximum concentration of 1 mg/mL. The reaction was performed at 37°C and was followed over time by UPLC-MS analysis. Generally, cyclization was complete after 1 h. After cyclization, coNGR was purified as described above.
To synthesize a tetrameric cyclic NGR peptide, a scaffold peptide with 4 NGR-coupling sites was necessary. Therefore, a lysine wedge was synthesized on MBHA resin (0.2 mmol scale). First, Boc-Lys(Fmoc)-OH (Bachem; Fmoc = 9-fluorenylmethoxycarbonyl) was coupled to the solid support, followed by two coupling cycles of Boc-Lys(Boc)-OH (Bachem). After chain assembly of the
Maleimide-DTPA was prepared via the method described by Dirksen et al. [
Analytical mass data of linear CNGRG-MpaL, coNGR, Lys(Thz)-(Lys)2-(SMCC)4, DTPA-coNGR, and DTPA-co(NGR)4 are given in Table
Analytical mass data of coNGR-based tracer constructs. Masses are given in Da. From top to bottom, mass of linear peptide (thioester), cyclic peptide after NCL, and the lysine wedge with four SMCC linkers are represented. Last two rows represent mass of DTPA-coNGR and DTPA-co(NGR)4, respectively.
Compound | Monoisotopic mass | Molecular weight | Measured |
---|---|---|---|
CNGRG-MpaL | 705.31 | 705.81 | 705.32 |
coNGR | 487.20 | 487.54 | 487.22 |
Lys(Thz)-(Lys)2-(SMCC)4 | 1520.77 | 1521.84 | 1520.83 |
DTPA-coNGR | 1170.50 | 1171.25 | 1170.49 |
DTPA-co(NGR)4 | 4140.86 | 4143.69 | 4140.76 |
coNGR and co(NGR)4 were radiolabeled with 111InCl3 (Mallinckrodt, Petten, The Netherlands) analogous to what has been described previously [
111In-cNGR, 111In-coNGR, or 111In-co(NGR)4 was added to 1 × 10 mL human blood in heparin (BD Biosciences, Vianen, The Netherlands). Blood stability was tested at 0, 10, 30, and 50 min. For each time point imaging agents were separated from blood cells and proteins by adding 0.5 mL MeCN (VWR International BV, Breda, The Netherlands) to 0.5 mL blood sample followed by centrifugation (2,000 ×g, 3 min). Supernatant samples were analyzed by HPLC using the above-mentioned method.
The
Freshly prepared
In 10–12-week-old male Swiss mice, we induced MI by ligation of the left anterior descending coronary artery (LAD) as described before [
Mice were anesthetized with isoflurane (induction 2.5%; maintenance 1.5%), a catheter was placed in a tail vein, and animals were positioned in the SPECT camera (MILabs, Utrecht, The Netherlands). Prior to image acquisition, a bolus injection of
Acquired list mode data was reconstructed using MILabs reconstruction software (version 2.51) employing the POS-EM algorithm (6 iterations and 16 subsets, reconstructed at a voxel size of 0.4 mm).
To allow quantification of imaging agent uptake, in vivo isotope-specific conversion factors (CF) were determined for the 0.6 mm collimator in a representative phantom with a known activity. Using the previously described method [
PMOD 3.7 cardiac tool PCARD (PMOD technologies, Zürich, Switzerland) was used to segment the heart in the 17-segment model. Uptake per segment was subsequently expressed as a mean standardized uptake value (
After imaging, the vital organs were harvested and kept for gamma-counting (Wallac Wizard, Turku, Finland). Acquired data were expressed as percentage injected dose per gram tissue (% ID/g).
Hearts were dissected and fixated in HEPES-buffered formaldehyde containing 150 mM saline for 24 h at 4°C. Hereafter, hearts were placed in 70% ethanol for maximal one month before embedding in paraffin. Paraffin-embedded hearts were cut at 4
All data were expressed as mean ± SEM. To test for significant differences we performed an unpaired student’s
In this study, an NGR peptide cyclized via native chemical ligation (NCL) and its tetravalent analog were designed and synthesized. The feasibility of these two new NGR peptide-based ligands for radionuclide imaging of CD13 expression in a mouse MI model with SPECT was explored. The peptides were radiolabeled with 111In and used for dual-isotope SPECT with
All observed masses of the constructs fell within the range of theoretical monoisotopic and average masses. Structural formulas of final products are given in Figures
Blood stability of 111In-labeled cyclic NGR-based SPECT tracers coNGR, co(NGR)4, and cNGR [
Tracer | Intact product (%) |
---|---|
coNGR | 20.22 |
co(NGR)4 | 25.31 |
cNGR | 7.55 |
Octanol-water partition coefficients indicated that coNGR was more hydrophilic than co(NGR)4 (Table
Tracer |
|
|
|
---|---|---|---|
coNGR |
|
|
|
co(NGR)4 |
|
|
|
cNGR |
|
|
|
The 17-segment model was used to determine standardized uptake values (SUVs) of the tracers [
Overview of the SUVs for coNGR and co(NGR)4 in MI animals. Infarcted areas (significantly decreased
Polar perfusion maps from each mouse in the MI group displaying the uptake pattern of
Representative in vivo fusion images of
Representative in vivo fusion images of
To examine whether multimerization indeed resulted in higher target uptake, SUVs of co(NGR)4 and coNGR in infarcted and noninfarcted areas were compared for each tracer separately. Uptake of co(NGR)4 in segments that were affected by MI was significantly higher (0.57 ± 0.03) than in segments that were not affected by MI (0.43 ± 0.02), whereas uptake of coNGR in MI-affected segments (0.83 ± 0.05) was not significantly higher than in unaffected segments (0.76 ± 0.05). This suggests that co(NGR)4 has a more specific uptake in infarcted areas than coNGR.
Although coNGR and co(NGR)4 were not directly compared with cNGR in an in vivo study, both coNGR-based ligands showed a higher uptake than cNGR in the infarcted area. However, only co(NGR)4 indicated a more target-specific uptake which makes co(NGR)4 a more optimal agent than cNGR or coNGR for imaging of angiogenesis after MI.
One hour postinjection (p.i.), urinary excretion in MI mice was 72.5 ± 5.2 for coNGR and 54.9 ± 9.1 for co(NGR)4 (% ID/g ± SEM,
Biodistribution of coNGR and co(NGR)4 in MI mice and co(NGR)4 in sham-operated mice. Substantial kidney uptake of coNGR and co(NGR)4 was observed. coNGR had significantly higher uptake than co(NGR)4 in blood, lungs, and intestines whereas uptake in other organs was similar.
Unlike with cyclic RGD, cyclic NGR multimerization did not result in higher target uptake as coNGR exceeded co(NGR)4 uptake. Changing the currently used short SMCC spacer, which only allows statistical rebinding, for a longer and less rigid spacer that might enable binding to multiple CD13 receptors simultaneously, could improve the avidity and thereby the affinity of the tracer. For example, a flexible PEG spacer with a length that could bridge the width of the CD13 receptor of 131 Å [
To validate uptake patterns of both coNGR-based tracers, cardiac CD13 expression was evaluated through immunohistological staining. A low level of CD13 expression was observed within the myocardium of sham-operated control animals (Figures
Representative pictures of CD13 expression in myocardium of sham and MI operated animals. Low baseline expression of CD13 was observed on photomicrographs (magnification 40x) of the myocardium of sham-operated animals (a). Dramatically increased CD13 expression was observed on photomicrographs (magnification 40x) of infarcted myocardium of MI operated animals (b). On photomicrographs (magnification 40x) of noninfarcted myocardium, a marginally higher expression of CD13 was noticed (c). Photomicrographs (magnification 200x) in (d), (e), and (f) represent a magnification of the region indicated in (a), (b), and (c), respectively. Note that expression of CD13 is not restricted to blood vessels but that endothelium of blood vessels is positive in the infarcted heart as opposed to the sham (arrows).
The enhanced uptake of coNGR and co(NGR)4 in the healthy myocardium of infarcted hearts points towards an overall angiogenic response of the heart in MI animals. Additionally, for co(NGR)4 the highest level of uptake was found in and around the infarcted areas of MI animals which also correlated to the histological findings as the highest level of CD13 expression was found in and around the infarcted areas. The same trend was observed for coNGR. It is highly likely that the uptake in the infarcted area as well as in the healthy myocardium is specific and related to increased CD13 expression.
Two CD13-targeting SPECT tracers for angiogenesis, coNGR and co(NGR)4, were designed and synthesized. However, target uptake of cyclic NGR-based imaging agents does not seem to increase with increasing valency. Instead, the key to enhance cyclic NGR-based imaging agent uptake was to stabilize the ring structure through NCL. Additional studies with different linkers conjugated to the lysine scaffold are warranted to investigate a possible increased avidity effect with coNGR-based imaging agents.
The authors declare that there are no conflicts of interest regarding the publication of this article.
This study was performed within the framework of the Center for Translational Molecular Medicine (CTMM), Project EMINENCE (Grant 01C-204 to Geert Hendrikx, Tilman M. Hackeng, Mark J. Post, and Ingrid Dijkgraaf), the Weijerhorst Foundation (Matthias Bauwens; Felix M. Mottaghy), and The Netherlands Organisation for Scientific Research (NWO; VIDI 723.013.009 to Ingrid Dijkgraaf). CTMM, Weijerhorst Foundation, and NWO are cordially acknowledged for their financial support.