Linker effects on biological properties of 111In-labeled DTPA conjugates of a cyclic RGDfK dimer

Abstract

In this report, we present in vitro and in vivo evaluation of three 111 In-labeled DTPA conjugates of a cyclic RGDfK dimer: DTPA-Bn-SU016 (SU016 = E[c(RGDfK)] 2; DTPA-Bn = 2-( p-isothioureidobenzyl)diethylenetriaminepentaacetic acid), DTPA-Bn-E-SU016 ( E = glutamic acid) and DTPA-Bn-Cys-SU016 (Cys = cysteic acid). The integrin alpha vbeta 3 binding affinities of SU016, DTPA-Bn-SU016, DTPA-Bn-E-SU016, and DTPA-Bn-Cys-SU016 were determined to be 5.0 +/- 0.7 nM, 7.9 +/- 0.6 nM, 5.8 +/- 0.6 nM, and 6.9 +/- 0.9 nM, respectively, against 125 I-c(RGDyK) in binding to integrin alpha vbeta3, suggesting that E or Cys residue has little effect on the integrin alpha vbeta3 affinity of E[c(RGDfK)] 2. It was also found that the 111 In-labeling efficiency of DTPA-Bn-SU016 and DTPA-Bn-E-SU016 is 3-5 times better than that of DOTA analogues due to fast chelation kinetics and high-yield 111 In-labeling under mild conditions (e.g., room temperature). Biodistribution studies were performed using BALB/c nude mice bearing U87MG human glioma xenografts. 111 In-DTPA-Bn-SU016, 111 In-DTPA-Bn-E-SU016, and 111 In-DTPA-Bn-Cys-SU016 all displayed rapid blood clearance. Their tumor uptake was comparable between 0.5 and 4 h postinjection (p.i.) within experimental error. 111 In-DTPA-Bn-E-SU016 had a significantly lower ( p < 0.01) kidney uptake than 111 In-DTPA-Bn-SU016 and 111 In-DTPA-Bn-Cys-SU016. The liver uptake of 111 In-DTPA-Bn-SU016 was 1.69 +/- 0.18% ID/g at 24 h p.i., while the liver uptake values of 111 In-DTPA-Bn-E-SU016 and 111 In-DTPA-Bn-Cys-SU016 were 0.55 +/- 0.11% ID/g and 0.79 +/- 0.15% ID/g at 24 h p.i., respectively. Among the three 111 In radiotracers evaluated in this study, 111 In-DTPA-Bn-E-SU016 has the lowest liver and kidney uptake and the best tumor/liver and tumor/kidney ratios. Results from metabolism studies indicated that there is little metabolism (<10%) for three 111 In radiotracers at 1 h p.i. Imaging data showed that tumors can be clearly visualized at 4 h p.i. with good contrast in the tumor-bearing mice administered with 111 In-DTPA-Bn-E-SU016. It is concluded that using a glutamic acid linker can significantly improve excretion kinetics of the 111 In-labeled E[c(RGDfK)] 2 from liver and kidneys.

Figure 1

Schematic presentation of ¹¹¹In-labeled cyclic RGDfK dimer DTPA conjugates: ¹¹¹In(DTPA-Bn-PKM-SU016) (PKM = direct bond, glutamic acid (E) and cysteic acid (Cys); SU016 = E[c(EGDfK)]₂).

Figure 2

Inhibition of ¹²⁵I-c(RGDyK) binding to α_vβ₃ integrin on U87MG human glioma cells by c(RGDyK) (•), SU016 (■), DTPA-Bn-SU016 (○), DTPA-Bn-E-SU016 (△), and DTPA-Bn-Cys-SU016 (×). IC₅₀ values were calculated to be 31.9 ± 4.0 nM for c(RGDyK), 5.0 ± 0.7 nM for SU016, 7.9 ± 0.6 nM for DTPA-Bn-SU016, 5.8 ± 0.6 nM for DTPA-Bn-E-SU016 and 6.9 ± 0.9 nM for DTPA-Bn-Cys-SU016, respectively.

Figure 3

Solution stability data of ¹¹¹In-DTPA-Bn-SU016, ¹¹¹In-DTPA-Bn-E-SU016, and ¹¹¹In-DTPA-Bn-Cys-SU016 in saline (a) and 6 mM EDTA solution (b).

Figure 4

Direct comparison of the excretion kinetics and tumor-to-background ratios between ¹¹¹In-DTPA-Bn-SU016, ¹¹¹In-DTPA-Bn-E-SU016, and ¹¹¹In-DTPA-Bn-Cys-SU016 in selected organs of the BALB/c nude mice bearing the U87MG human glioma xenografts.

Figure 5

Biodistribution data of ¹¹¹In-DTPA-Bn-E-SU016 at 4 h p.i. with/without the presence of excess E[c(EGDfK)]₂ in BALB/c nude mice bearing the U87MG human glioma xenografts.

Figure 6

Radio-HPLC chromatograms of ¹¹¹In-DTPA-Bn-SU016 (top), ¹¹¹In-DTPA-Bn-E-SU016 (middle), and ¹¹¹In-DTPA-Bn-Cys-SU016) (bottom) in the kit matrix and urine at 1 h p.i. Each normal BALB/c mouse was administered with 200 μCi of radiotracer. Two mice were used for each radiotracer.

Figure 7

A representative scintigraphic image of the tumor-bearing mouse administered with ~200 μCi of ¹¹¹In-DTPA-Bn-E-SU016. The arrow indicates presence of tumor.