Bioconjugate Chem. 2008, 19, 201–210
Linker Effects on Biological Properties of of a Cyclic RGDfK Dimer
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In-Labeled DTPA Conjugates
Bing Jia,†,‡ Zhaofei Liu,† Jiyun Shi,†,‡ Zilin Yu,† Zhi Yang,§ Huiyun Zhao,† Zhengjie He,4 Shuang Liu,*,4 and Fan Wang*,† Medical Isotopes Research Center, Peking University, Beijing 100083, Department of Isotopes, China Institute of Atomic Energy, Beijing 102413, Department of Nuclear Medicine, Peking University Oncology School, Beijing 100036, China, and School of Health Sciences, Purdue University, Indiana 47907. Received August 8, 2007; Revised Manuscript Received October 7, 2007
In this report, we present in Vitro and in ViVo evaluation of three 111In-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 Rvβ3 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 125I-c(RGDyK) in binding to integrin Rvβ3, suggesting that E or Cys residue has little effect on the integrin Rvβ3 affinity of E[c(RGDfK)]2. It was also found that the 111In-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 111In-labeling under mild conditions (e.g., room temperature). Biodistribution studies were performed using BALB/c nude mice bearing U87MG human glioma xenografts. 111In-DTPABn-SU016, 111In-DTPA-Bn-E-SU016, and 111In-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. 111InDTPA-Bn-E-SU016 had a significantly lower (p < 0.01) kidney uptake than 111In-DTPA-Bn-SU016 and 111 In-DTPA-Bn-Cys-SU016. The liver uptake of 111In-DTPA-Bn-SU016 was 1.69 ( 0.18% ID/g at 24 h p.i., while the liver uptake values of 111In-DTPA-Bn-E-SU016 and 111In-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 111In radiotracers evaluated in this study, 111In-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 (98% before being used for radiolabeling and animal studies. The ESI-MS data were completely consistent with the proposed formula for DTPA-Bn-SU016, DTPA-BnE-SU016, and DTPA-Bn-Cys-SU016. Integrin rvβ3 Binding Affinity. The integrin Rvβ3 binding affinities of DTPA-Bn-SU016, DTPA-Bn-E-SU016, and DTPABn-Cys-SU016 were determined using the integrin Rvβ3-positive U87MG human glioma cells. For comparison purposes, c(RGDyK) and SU016 were also evaluated in the same assay. Figure 2 shows the displacement curves of c(RGDyK), SU016,
Jia et al.
Figure 2. Inhibition of 125I-c(RGDyK) binding to Rvβ3 integrin on U87MG human glioma cells by c(RGDyK) (b), SU016 (9), DTPABn-SU016 (O), 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-CysSU016, respectively.
DTPA-Bn-SU016, DTPA-Bn-E-SU016, and DTPA-Bn-CysSU016 against the binding of 125I-c(RGDyK) to integrin Rvβ3 on U87MG glioma cells. The 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-ESU016, and 6.9 ( 0.9 nM for DTPA-Bn-Cys-SU016. Radiochemistry. 111In-DTPA-Bn-SU016, 111In-DTPA-BnE-SU016, and 111In-DTPA-Bn-Cys-SU016 were prepared by reacting 111InCl3 with the DTPA conjugate in NH4OAc buffer (200 mM, pH ) 5.5). Radiolabeling was almost instantaneous at room temperature, particularly at higher concentrations of the DTPA conjugate. Their radiochemical purity (RCP) was >99% after Sep-Pak purification. The specific activity was in the range 100–150 mCi/µmol, which is consistent with that obtained for the 111In-labeled DTPA conjugate of a cyclic RGDfK monomer (40). The specific activity obtained at room temperature for all three 111In radiotracers in this study was 3–5 times better than that of the 111In-labeled DOTA conjugates (∼30 mCi/µmol obtained at 100 °C) (33). It should be noted that the radiolabeling conditions used in this study are not optimized. The specific activity of 111In-DTPA-Bn-SU016, 111 In-DTPA-Bn-E-SU016, and 111In-DTPA-Bn-Cys-SU016 could be further improved at elevated temperatures. All three 111In radiotracers were analyzed using the same HPLC method (method 2). Their HPLC retention times were 18.2, 11.3, and 11.7 min, respectively. Their log P values were determined in an equal volume mixture of n-octanol and 25 mM phosphate buffer (pH ) 7.4), and were calculated to be -2.7 ( 0.1 (n ) 3), -3.4 ( 0.1 (n ) 3), and -3.3 ( 0.2 (n ) 3), respectively. Both HPLC retention time and log P values suggest that incorporation of E and Cys amino acid linkers increases hydrophilicity of the 111In-labeled cyclic RGDfK dimer. Solution Stability. High solution stability is a requirement for both diagnostic and therapeutic radiotracers. In this study, we studied solution stability of 111In-DTPA-Bn-SU016, 111InDTPA-Bn-E-SU016, and 111In-DTPA-Bn-Cys-SU016 both in saline and in the presence of EDTA challenge. Figure 3 shows their solution stability data in saline (a) and 6 mM EDTA solution (b). It was clear that all three 111In radiotracers remained stable for >24 h in saline and in aqueous solution of 6 mM EDTA. Biodistribution Characteristics. Biodistribution properties of 111In-DTPA-Bn-SU016, 111In-DTPA-Bn-E-SU016, and 111InDTPA-Bn-Cys-SU016 were evaluated using the BALB/c nude mice bearing the U87MG human glioma xenografts. Selected biodistribution data are listed in Tables 1, 2, and 3. Figure 4
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In-Labeled DTPA Conjugates of RGDfK Dimer
Bioconjugate Chem., Vol. 19, No. 1, 2008 205 Table 3. Selected Biodistribution Data of 111In-DTPA-Bn-Cys-SU016 in BALB/c Nude Mice Bearing U87MG Human Glioma Xenograftsa organ
0.5 h
1h
4h
24 h
tumor blood heart liver spleen kidney muscle gut stomach bone
2.72 ( 0.28 0.83 ( 0.16 1.19 ( 0.16 3.08 ( 0.60 2.08 ( 0.19 14.78 ( 2.31 0.57 ( 0.04 7.09 ( 0.86 4.12 ( 0.25 1.01 ( 0.26
1.78 ( 0.09 0.24 ( 0.06 0.72 ( 0.18 2.41 ( 0.15 1.74 ( 0.62 12.20 ( 1.76 0.37 ( 0.05 5.03 ( 0.81 2.77 ( 0.44 0.54 ( 0.12
1.53 ( 0.64 0.06 ( 0.01 0.46 ( 0.03 2.32 ( 0.38 1.58 ( 0.11 10.70 ( 1.19 0.23 ( 0.03 3.48 ( 0.44 1.61 ( 0.16 0.53 ( 0.23
0.98 ( 0.29 0.03 ( 0.01 0.29 ( 0.02 0.79 ( 0.15 1.08 ( 0.24 4.96 ( 0.62 0.14 ( 0.02 3.41 ( 0.82 1.10 ( 0.07 0.35 ( 0.03
a The organ uptake is expressed %ID/g. Each data point represents an average of biodistribution data in four animals.
Figure 3. Solution stability data of 111In-DTPA-Bn-SU016, 111InDTPA-Bn-E-SU016, and 111In-DTPA-Bn-Cys-SU016 in saline (a) and 6 mM EDTA solution (b). Table 1. Selected Biodistribution Data of 111In-DTPA-Bn-SU016 in BALB/c Nude Mice Bearing U87MG Human Glioma Xenograftsa organ
0.5 h
1h
4h
24 h
tumor blood heart liver spleen kidney muscle gut stomach bone
2.53 ( 0.63 0.69 ( 0.02 1.07 ( 0.09 2.48 ( 0.22 2.04 ( 0.13 9.07 ( 1.29 0.49 ( 0.10 6.46 ( 0.25 3.88 ( 0.39 0.93 ( 0.07
2.21 ( 0.65 0.22 ( 0.04 0.95 ( 0.40 2.49 ( 0.38 2.56 ( 1.11 7.34 ( 0.70 0.35 ( 0.04 5.08 ( 0.92 2.86 ( 0.15 0.76 ( 0.14
1.86 ( 0.34 0.09 ( 0.06 0.49 ( 0.05 2.86 ( 0.53 1.83 ( 0.37 7.05 ( 1.49 0.24 ( 0.06 3.18 ( 0.19 1.78 ( 0.15 0.52 ( 0.23
1.69 ( 0.18 0.05 ( 0.02 0.43 ( 0.09 1.93 ( 0.28 1.94 ( 0.41 6.14 ( 1.34 0.21 ( 0.04 4.16 ( 0.77 1.55 ( 0.24 0.51 ( 0.11
a The organ uptake is expressed as %ID/g. Each data point represents an average of biodistribution data in four animals.
Table 2. Selected Biodistribution Data of 111In-DTPA-Bn-E-SU016 in BALB/c Nude Mice Bearing U87MG Human Glioma Xenograftsa organ
0.5 h
1h
4h
24 h
4.0 h/block
tumor blood heart liver spleen kidney muscle gut stomach bone
2.45 ( 0.43 0.71 ( 0.10 1.06 ( 0.14 2.94 ( 0.38 2.13 ( 0.26 6.04 ( 1.40 0.52 ( 0.06 5.36 ( 1.98 3.92 ( 0.28 0.97 ( 0.20
2.33 ( 0.55 0.21 ( 0.07 0.72 ( 0.10 3.12 ( 0.38 2.01 ( 0.43 5.29 ( 0.51 0.37 ( 0.06 5.36 ( 0.49 3.00 ( 0.30 0.77 ( 0.23
1.86 ( 0.15 0.06 ( 0.01 0.47 ( 0.05 2.15 ( 0.09 1.60 ( 0.22 3.80 ( 0.33 0.22 ( 0.03 4.31 ( 0.52 1.64 ( 0.11 0.40 ( 0.08
0.89 ( 0.17 0.03 ( 0.01 0.20 ( 0.04 0.55 ( 0.11 0.79 ( 0.25 1.37 ( 0.16 0.12 ( 0.02 1.73 ( 0.22 0.70 ( 0.06 0.34 ( 0.04
0.25 ( 0.06 0.04 ( 0.01 0.06 ( 0.00 0.29 ( 0.04 0.18 ( 0.03 2.58 ( 0.42 0.04 ( 0.00 0.40 ( 0.09 0.22 ( 0.07 0.08 ( 0.02
a The organ uptake is expressed %ID/g. Each data point represents an average of biodistribution data in four animals.
shows the comparison of excretion kinetics and T/B ratios among 111In-DTPA-Bn-SU016, 111In-DTPA-Bn-E-SU016, and 111 In-DTPA-Bn-Cys-SU016. Since they share the same cyclic RGDfK dimer (SU016) and 111In-DTPA chelate, the differences
in their biodistribution and excretion characteristics is caused by the linker (E and Cys). In general, all three 111In radiotracers displayed a rapid blood clearance predominantly via the renal route. Their blood clearance curves were almost identical (Figure 4), and their bone uptake was relatively low (Tables 1,, and 3). The tumor uptake of 111In-DTPA-Bn-SU016, 111In-DTPA-Bn-E-SU016, and 111InDTPA-Bn-Cys-SU016 was comparable between 0.5 and 4 h p.i. within the experimental error. At 24 h p.i., however, 111InDTPA-Bn-SU016 had the tumor uptake (1.69 ( 0.18% ID/g) that was significantly higher (p < 0.05) than that of 111In-DTPABn-E-SU016 (0.89 ( 0.17% ID/g) and 111In-DTPA-Bn-CysSU016 (0.98 ( 0.29% ID/g). The kidney uptake of 111In-DTPABn-E-SU016 was much lower (p < 0.001) than that of 111 In-DTPA-Bn-SU016 and 111In-DTPA-Bn-Cys-SU016 over the 24 h study period. The liver uptake of 111In-DTPA-BnSU016, 111In-DTPA-Bn-E-SU016, and 111In-DTPA-Bn-CysSU016 was comparable between 0.5 and 1 h p.i. (Figure 4), but it was significantly higher (p < 0.001) for 111In-DTPA-BnSU016 than that for 111In-DTPA-Bn-E-SU016 and 111In-DTPABn-Cys-SU016 at 24 h p.i. For example, the liver uptake of 111 In-DTPA-Bn-SU016 was 1.69 ( 0.18% ID/g at 24 h p.i., and the liver uptake of 111In-DTPA-Bn-E-SU016 and 111InDTPA-Bn-Cys-SU016 was only 0.55 ( 0.11% ID/g and 0.79 ( 0.15% ID/g at 24 h p.i., respectively. Among the three 111In radiotracers evaluated in this animal model, 111In-DTPA-BnE-SU016 has the lowest uptake in liver and kidneys, and the best tumor/liver and tumor/kidney ratios (Figure 4) at 24 h p.i. The blocking experiment was used to demonstrate tumor specificity of the radiotracer. In this experiment, E[c(RGDfK)]2 (15 mg/kg or ∼300 µg/mouse) was used as the blocking agent. Such a high dose was used to make sure that the integrin Rvβ3 is almost completely blocked. Figure 5 illustrates biodistribution data of 111In-DTPA-Bn-E-SU016 at 4 h p.i. in the presence of E[c(RGDfK)]2. Apparently, pre-injection of excess E[c(RGDfK)]2 significantly reduced the tumor uptake of 111In-DTPABn-E-SU016, suggesting that its tumor uptake is indeed integrin Rvβ3-mediated. However, its uptake in liver, spleen, stomach, gut, and kidneys was also reduced significantly, most likely due to the blockage of integrin Rvβ3 in these organs. Metabolism. Metabolism studies on 111In-DTPA-Bn-SU016, 111 In-DTPA-Bn-E-SU016, and 111In-DTPA-Bn-Cys-SU016 were performed using normal BALB/c nude mice. Urine samples were analyzed to determine whether they are able to retain their chemical integrity at 1 h p.i. We also tried to analyze feces samples at 1 h p.i., but the radioactivity level was too low to be detected. Figure 6 shows HPLC chromatograms of 111InDTPA-Bn-SU016, 111In-DTPA-Bn-E-SU016, and 111In-DTPABn-Cys-SU016 in the urine at 1 h p.i. It is quite clear that there is little metabolite (
