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In our previous study, mesenchymal–epithelial transition factor (c-Met)-binding peptides (cMBP) had been readily radiolabeled with radioactive iodide for glioma imaging because of five histidine amino acids. However, iodinated cMBP showed relatively unfavorable in vivo kinetics. For this reason, we tried to design dual peptide ligands that would be advantageous in recognizing both c-Met receptor and integrin αvβ3. A cMBP-click-cRGDyk (cyclic Arg-Gly-Asp-Tyr-Lys) heterodimer was synthesized from mini polyethylene glycol-conjugated cMBP-3 glycine (GGG)-a single name of amino acids (SC) (Ser-Cys) and cRGDyk through a click (1 + 3 cycloaddition), and then labeled with iodine 125 (I-125) via histidine in the cMBP and tyrosine in the cRGDyk. The receptor-binding characteristics and tumor-targeting efficacy of cMBP-click-cRGDyk were tested in vitro and in vivo. A cMBP-click-cRGDyk had comparable integrin αvβ3-binding affinity with cRGDyk. The results of the biodistribution of 125I-cMBP-click-cRGDyk at 4 h showed higher tumor-to-blood, tumor-to-liver, and tumor-to-muscle ratios: 10.07, 6.76, and 11.12, compared to 2.34, 1.99, and 5.18 of 125I-cMBP-GGG-SC, respectively. U87MG tumor xenografts could be visualized by single photon emission computed tomography (SPECT)/CT using 125I-cMBP-click-cRGDyk and also image contrast and overall quality were improved compared to 125I-cMBP-GGG-SC. As the results of in vivo inhibition using free cRGDyk or cMBP-GGG-SC indicated, the tumoral uptake of 125I-cMBP-click-cRGDyk decreased. This finding means that 125I-cMBP-click-cRGDyk was specifically uptaken by integrin αvβ3 and the c-Met receptor. Although imaging quality was improved, additional experiments are needed to acquire significant image-quality improvement. (Cancer Sci 2011; 102: 1516–1521)
Many types of receptors are uniquely expressed or markedly overexpressed in tumors, and have been used as potential targets for cancer diagnosis and therapy. The ability to measure and identify specific receptor expression is crucial for the accurate diagnosis, staging, restaging, and classification of tumors, and for monitoring patient response to therapy.(1,2) Many research groups are therefore interested in peptide radiopharmaceuticals. Compared to high molecular weight polymers, small peptides are structurally well defined and are generally cleared from circulation much faster, which might lead to a higher target-to-background ratio.
Multivalent ligands can be homomultivalent, with multiple copies of the same ligand, or heteromultivalent, with different types of ligands targeting different types of receptors. Multivalent ligands consist of multiple binding moieties (pharmacophores) that are bound together via chemical linkers. Multivalent binding can lead to increased functional affinity and binding specificity.(3–5) A wide spectrum of binding moieties has been studied, including small peptide fragments, truncated versions of antibodies, and carbohydrate analogs.(5–7)
Integrin αvβ3 is highly expressed in invasive tumors, such as late-stage glioblastomas, breast and prostate tumors, malignant melanomas, and ovarian carcinomas, as well as in newborn blood vessels.(2,8) The expression level of integrin αvβ3 is an important factor in determining malignant invasiveness and metastatic potential in both preclinical animal models and cancer patients.(9) Over the past several years, many researchers have successfully developed series RGD peptide radiotracers with favorable in vivo kinetics for tumor integrin αvβ3 imaging.(10,11) Mesenchymal–epithelial transition factor (c-Met) is a receptor tyrosine kinase known to stimulate invasive cancer cell growth and increase metastatic potential; it is also expressed and mutates in a variety of solid tumors.(12–14) Mesenchymal–epithelial transition factor is overly expressed in human glioblastomas, with expression levels correlating to glioma malignancy grade and vascularity. The activated endothelial cells around c-Met-positive tumor tissues express high levels of integrin during tumor angiogenesis, invasion, and metastasis. We previously reported that iodine 125 (I-125)-radiolabeled, c-Met-binding peptides (cMBP) bound specifically to U87MG cells and in vivo tumors.(14) A glioma tumor (U87MG) expresses both c-Met and integrin αvβ3, and we hypothesize that a peptide ligand recognizing both receptors would be advantageous over a single receptor-binding probe. Chen et al. previously studied this concept.(7)
Currently, the copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition of azides and alkynes, commonly called “click chemistry”, plays a crucial role in a wide range of biomedical applications.
This reaction can be carried out in high yields under mild conditions, and the 1,2,3-triazole formed has a similar polarity and size with an amide bond. Due to these favorable aspects, the use of click chemistry to conjugate two (bio)molecular components has recently been reported.(12,15,16)
In this study, we hypothesized that the introduction of cRGDyk using click reaction methodology would help reduce radioactive accumulation in major organs by enhancing elimination, while increasing tumoral uptake. We developed a 1,2,3-triazole-associated, cMBP-click-cRGDyk peptide heterodimer that recognizes c-Met receptors through the cMBP motif and integrin αvβ3 through the RGD motif. After radiolabeling this synthesized heterodimer, we conducted studies targeting integrin and c-Met receptors both in vitro and in vivo.
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Since U87MG tumor cells express both c-Met and integrin αvβ3, we explored whether a dual c-Met- and integrin αvβ3-targeting approach would allow us to develop improved imaging probes over 125I-cMBP-GGG-SC that only recognizes one receptor type. In a previous study, cMBP was conjugated with two types of linkers, such as GGG and 8-aminooctanoic acid (AOC). The tissue distribution of three different peptides, 125I-cMBP, 125I-cMBP-GGG-SC, and 125I-cMBP-AOC-C, were determined in U87MG-xenografted mice.(14)
As determined by a biodistribution study, 125I-cMBP-GGG-SC exhibited the highest T/B at 4 h. However, static pin-hole images of 125I-cMBP-GGG-SC showed a relatively low tumor uptake and high body background activity at 1 and 4 h, and even higher pancreatic and renal activities at all time points. Therefore, a modification of cMBP-GGG-SC through heterodimerization of two ligands, one targeting c-Met and the other targeting integrin, was needed to improve the targetability for an in vivo cancer model.
Peptide modifications to be used as imaging agents can be achieved by various methods. In order to improve binding affinity and receptor selectivity and to avoid the fast degradation of peptides in vivo, several researchers introduced cyclization methods.(17) Heteromultimerization and homomultimerization of ligands with multitargeting properties have also been developed by many researchers.(2–6) These approaches have successfully improved tumor-targeting efficacy and pharmacokinetics compared to single analog methodologies. We therefore synthesized radiolabeled 125I-cMBP-click-cRGDyk (Fig. 1). The linking groups in small peptide receptor-targeted radiopharmaceutical designs have largely been viewed as merely convenient ways to adjust blood retention and overall clearance of radiopharmaceuticals, without making substantial alterations to the targeting vector. In this study, two different peptides were connected, forming a 1,2,3-trizole ring between an alkyne and an azide terminal.
Our data from the receptor-binding assays demonstrated that cMBP-click-cRGDyk is similar to cMBP-GGG-SC for c-Met binding and to cRGDyk for integrin αvβ3 binding (Fig. 2). However, the binding affinity of cMBP-click-cRGDyk for both receptors was not improved. As shown in Table 1, 125I-cMBP-click-cRGDyk tends to have a faster washout than 125I-cMBP-GGG-SC, but logP values of the two radiolabeled compounds were similar (−2.40 ± 0.05 and −2.75 ± 0.05, respectively). In another previous heterodimer study, 18F-FB-BBN-RGD tended to have a slower washout than 18F-FB-BBN, which might be the result of enhanced, effective binding due to dual targeting.(7) In comparing our results, although 125I-cMBP-click-cRGDyk and 125I-cMBP-GGG-SC showed similar binding affinity and hydrophilicity, 125I-cMBP-click-cRGDyk tended to have a faster clearance rate in vivo than 125I-cMBP-GGG-SC.
The tumoral uptake decreased with time compared to 125I-cMBP-GGG-SC (6.53 ± 2.29, 6.85 ± 1.89, and 7.05 ± 1.24 at 30 min, 2 and 4 h). Moreover, high kidney uptake appeared in three time points compared to 125I-cMBP-GGG-SC (27.54 ± 5.11, 9.54 ± 1.91, and 6.18 ± 1.92 at 30 min, 2 and 4 h), slowly decreasing from 40.67 ± 4.67 to 15.83 ± 1.56. As shown Figure 4(c,d), the T/B, T/L, and T/M ratios of 125I-cMBP-click-cRGDyk were higher than those of 125I-cMBP-GGG-SC. While the initial tumor accumulation of 125I-cMBP-click-cRGDyk was less than 125I-cMBP-GGG-SC, the T/B, T/L, and T/M ratios increased with time due to the relatively low activities in major organs, such as the heart, lung, blood, liver, intestines, muscles, and spleen. Iodine-125-cMBP-click-cRGDyk demonstrated a quicker clearance rate than 125I-cMBP-GGG-SC (blood activity: 4.22 ± 0.75, 2.77 ± 0.38 and 2.39 ± 0.50 at 30 min, 2 and 4 h) and lower background activity.
The imaging quality of 125I-cMBP-click-cRGDyk was evaluated in a U87MG tumor xenografted model. The quality was also improved by 125I-cMBP-click-cRGDyk compared to 125I-cMBP-GGG-SC in the liver, heart, pancreas, and kidneys at 1 and 4 h (Fig. 5). However, thyroid uptake was observed in the 4 h post-injection image (Fig. 5d), but this might be due to the deiodination of 125I-cMBP-click-cRGDyk. In a previous study, the labeling stability of 125I-cMBP-GGG-SC was stable, and cMBP-GGG-SC did not internalize into the U87MG cells; thyroid uptake did not appear in the in vivo study. However, as labeled radioiodine on the tyrosine ring of RGD peptides was found to be unstable in vivo,(18,19)125I-cMBP-click-cRGDyk was thought to be internalized like 125I-cMBP-AOC-C and then deiodinated. Taking this into consideration, a direct comparison between two compounds with radioiodine activity might be difficult to achieve, since there is the possibility of underestimating cMBP-click-cRGDyk binding with integrin due to deiodination from the heterodimer compound, compared to cMBP-GGG-SC for c-Met only. When labeling efficiency and the stability of 125I-cRGDyk was estimated, the in vitro labeling stability of 125I-cRGDyk quickly decreases with time. Consequently, 125I-cMBP-click-cRGDyk might demonstrate faster clearance than 125I-cMBP-GGG-SC.
Moreover, although the tumor volumes used in this study were smaller than those of 125I-cMBP-GGG-SC, they were well visualized. For the identification of integrin receptor targetability, free cRGDyk (48 nmol) and cMBP-GGG-SC were co-injected, and images were acquired (Fig. 5e). These inhibition results suggest that 125I-cMBP-click-cRGDyk was specifically taken up by integrin αvβ3 and the c-Met receptor.
In conclusion, we successfully developed a heterodimeric peptide that binds to both the c-Met receptor and integrin αvβ3 using click reaction methodology. Dual integrin and/or c-Met receptor recognition showed slightly improved tumor-targeting efficacy and imaging quality compared to 125I-cMBP-GGG-SC single analogs, despite high renal clearance activity and decreasing tumoral uptake over time. However, additional experiments are necessary to improve clinical pharmacokinetics, such as decreased kidney uptake and prolonged tumor uptake.