Young-Seung Kim

Improving Tumor Uptake and Pharmacokinetics of 64Cu-Labeled Cyclic RGD Peptide Dimers with Gly3 and PEG4 Linkers

Radiolabeled cyclic RGD (Arg-Gly-Asp) peptides represent a new class of radiotracers with potential for early tumor detection and noninvasive monitoring of tumor metastasis and therapeutic response in cancer patients. This article describes the synthesis of two cyclic RGD peptide dimer conjugates, DOTA-PEG(4)-E[PEG(4)-c(RGDfK)](2) (DOTA-3PEG(4)-dimer: DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; PEG(4) = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) and DOTA-G(3)-E[G(3)-c(RGDfK)](2) (DOTA-3G(3)-dimer: G(3) = Gly-Gly-Gly). Integrin alpha(v)beta(3) binding affinities of cyclic RGD peptides were determined by competitive displacement of (125)I-echistatin bound to U87MG human glioma cells and follow the order of DOTA-E{E[c(RGDfK)](2)}(2) (DOTA-tetramer: IC(50) = 10 +/- 2 nM) > DOTA-3G(3)-dimer (IC(50) = 62 +/- 6 nM) approximately DOTA-3PEG(4)-dimer (IC(50) = 74 +/- 3 nM) > DOTA-E[c(RGDfK)](2) (DOTA-dimer: IC(50) = 102 +/- 5 nM). The addition of PEG(4) and G(3) linkers between two cyclic RGD motifs in DOTA-3G(3)-dimer and DOTA-3PEG(4)-dimer makes it possible for them to achieve the simultaneous integrin alpha(v)beta(3) binding in a bivalent fashion. Both (64)Cu(DOTA-3PEG(4)-dimer) and (64)Cu(DOTA-3G(3)-dimer) were prepared in high yield with specific activity being >50 Ci/mmol. Biodistribution and imaging studies were performed in athymic nude mice bearing U87MG human glioma xenografts. The results from those studies show that PEG(4) and G(3) linkers are particularly useful for improving tumor uptake and clearance kinetics of (64)Cu radiotracers from the nontumor organs, such as kidneys, liver, and lungs. There is a linear relationship between the tumor size and %ID tumor uptake, suggesting that (64)Cu(DOTA-3PEG(4)-dimer) and (64)Cu(DOTA-3PEG(4)-dimer) might be useful for noninvasive monitoring of tumor growth or shrinkage during antiangiogenic therapy. MicroPET imaging data clearly demonstrate the utility of (64)Cu(DOTA-3G(3)-dimer) as a new PET radiotracer for imaging integrin alpha(v)beta(3)-positive tumors.

Effects of Targeting Moiety, Linker, Bifunctional Chelator, and Molecular Charge on Biological Properties of 64Cu-Labeled Triphenylphosphonium Cations

In this report, we present the synthesis and evaluation of six new 64Cu-labeled triphenylphosphonium (TPP) cations. Biodistribution studies were performed using the athymic nude mice bearing U87MG human glioma xenografts to explore the impact of TPP moieties, linkers, bifunctional chelators (BFCs), and molecular charge on biological properties of 64Cu radiotracers. On the basis of the results from this study, it is concluded that (1) mTPP (tris(4-methoxyphenyl)phosphonium) is a better mitochondrion-targeting molecule than TPP and 3mTPP (tris(2,4,6-trimethoxyphenyl)phosphonium); (2) DO3A (1,4,7,10-tetraazacyclododecane-4,7,10-triacetic acid) and DO2A (1,4,7,10-tetraazacyclododecane-4,7-diacetic acid) are suitable BFCs for the 64Cu-labeling of TPP cations; (3) NOTA-Bn ( S-2-(4-thioureidobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid) has a significant adverse effect on the radiotracer tumor uptake and tumor-to-background ratios; and (4) monoanionic BFCs should be avoided to ensure that 64Cu chelate has a neutral or negative charge. Considering the tumor uptake and tumor/liver ratios, 64Cu(DO2A-xy-TPP)+ is the best candidate for more extensive evaluations in different tumor-bearing animal models.

99mTc-Labeled Cyclic RGD Peptides for Noninvasive Monitoring of Tumor Integrin αvβ3 Expression

This report describes the biologic evaluations of [99mTc(HYNIC-3P-RGD2)(tricine)(TPPTS)] (99mTc-3P-RGD2: 6-hydrazinonicotinyl; 3P-RGD2 = PEG4-E[PEG4-c(RGDfK)]2; PEG4 = 15-amino-4,7,10,13-tetraoxapentadecanoic acid; and TPPTS = trisodium triphenylpho-sphine-3,3′,3“-trisulfonate), [99mTc(HYNIC-3G-RGD2)(tricine)(TPPTS)] (99mTc-3G-RGD2: 3G-RGD2 = G3-E[G3-c(RGDfK)]2 and G3 = Gly-Gly-Gly), and 99mTcO(MAG2−3G-RGD2) (MAG2 = mercaptoacetylglycylglycyl) as radiotracers for noninvasive imaging of tumor integrin αvβ3 expression in five xenografted tumor-bearing models. Biodistribution and imaging studies were performed in athymic nude mice bearing U87MG, MDA-MB-435, A549, HT29, or PC-3 tumor xenografts. Immunochemistry was performed using the cultured primary tumor cells and xenografted tumor tissues. It was found that the radiotracer tumor uptake followed the trend U87MG > MDA-MB-435 ≈ HT29 ≈ A549 > PC-3. The total integrin β3 expression levels followed the general trend: U87MG > MDA-MB-435 ≈ A549~HT29 > PC-3. There is a linear relationship between the radiotracer injected dose per gram tumor uptake and the total integrin β3 expression levels. On the basis of these, it was concluded that radiotracer tumor uptake is contributed by integrin αVβ3 expressed on tumor cells and activated endothelial cells of the tumor neovasculature. 99mTc-3P-RGD2 has the capability to monitor integrin αvβ3 expression in a noninvasive fashion.

Evaluation of 111In-Labeled Cyclic RGD Peptides: Tetrameric Not Tetravalent

This report presents the synthesis and evaluation of (111)In(DOTA-6G-RGD(4)) (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid; 6G-RGD(4) = E{G(3)-E[G(3)-c(RGDfK)](2)}(2) and G(3) = Gly-Gly-Gly), (111)In(DOTA-RGD(4)) (RGD(4) = E{E[c(RGDfK)](2)}(2)) and (111)In(DOTA-3G-RGD(2)) (3G-RGD(2) = G(3)-E[G(3)-c(RGDfK)](2)) as new radiotracers for imaging integrin alpha(v)beta(3)-positive tumors. The IC(50) values of DOTA-6G-RGD(4), DOTA-RGD(4), and DOTA-3G-RGD(2) were determined to be 0.4 +/- 0.1, 1.4 +/- 0.1 and 1.1 +/- 0.1 nM against (125)I-c(RGDyK) bound to integrin alpha(v)beta(3)-positive U87MG human glioma cells. (111)In(DOTA-6G-RGD(4)), (111)In(DOTA-RGD(4)), and (111)In(DOTA-3G-RGD(2)) were prepared by reacting (111)InCl(3) with the respective DOTA conjugate in NH(4)OAc buffer (100 mM, pH = 5.5). Radiolabeling could be completed by heating the reaction mixture at 100 degrees C for 15-20 min. The specific activity was approximately 1850 MBq/micromol for (111)In(DOTA-3G-RGD(2)) and approximately 1480 MBq/micromol for (111)In(DOTA-6G-RGD(4)). The athymic nude mice bearing U87MG human glioma xenografts were used to evaluate tumor uptake and excretion kinetics of (111)In(DOTA-6G-RGD(4)), (111)In(DOTA-RGD(4)), and (111)In(DOTA-3G-RGD(2)). The results from both the integrin alpha(v)beta(3) binding assay and biodistribution studies suggest that the tetrameric cyclic RGD peptides, such as RGD(4) and 6G-RGD(4), are most likely bivalent in binding to the integrin alpha(v)beta(3). Both (111)In(DOTA-6G-RGD(4)) and (111)In(DOTA-RGD(4)) had significantly higher tumor uptake than (111)In(DOTA-3G-RGD(2)) at 24-72 h postinjection due to the extra RGD motifs in RGD(4) and 6G-RGD(4). (111)In(DOTA-3G-RGD(2)) had very little metabolism, while (111)In(DOTA-6G-RGD(4)) had significant metabolism during its excretion via both renal and hepatobiliary routes over the 2 h period, probably due to its much larger size. The combination of high tumor uptake with long tumor retention suggests that their corresponding (90)Y and (177)Lu analogues M(DOTA-6G-RGD(4)) (M = (90)Y and (177)Lu) might be useful as therapeutic radiotracers for treatment of integrin alpha(v)beta(3)-positive solid tumors.

Tc-99m-N-MPO : Novel cationic Tc-99m radiotracer for myocardial perfusion imaging

AbstractBackground. Technetium 99m-N-MPO ([Tc-99m-N(mpo)(PNP5)]+) is a cationic Tc-99m nitrido complex. The objective of this study is to evaluate its potential as a new radiotracer for myocardial perfusion imaging. Methods and Results. Biodistribution studies were performed in Sprague-Dawley rats and guinea pigs to compare the myocardial uptake and excretion kinetics of Tc-99m-N-MPO from noncardiac organs, such as the liver and lungs, with those of the known cationic Tc-99m radiotracers: Tc-99m-N-DBODC5 and Tc-99m-sestamibi. Planar imaging was performed in Sprague-Dawley rats to evaluate the utility of Tc-99m-N-MPO as a myocardial perfusion imaging agent. Metabolism studies were carried out by use of both Sprague-Dawley rats and guinea pigs. In general, the heart uptake of Tc-99m-N-MPO was between that of Tc-99msestamibi and Tc-99m-N-DBODC5 over the 2-hour study period. However, the heart-liver ratio of Tc-99m-N-MPO (12.75±3.34) at 30 minutes after injection was more than twice that of Tc-99m-N-DBODC5 (6.01±1.45) and approximately 4 times higher than that of Tc-99msestamibi (2.90±0.22). The heart uptake and heart-liver ratio of Tc-99m-N-MPO and Tc-99m-sestamibi in guinea pigs were significantly lower than those obtained in Sprague-Dawley rats. The metabolism studies demonstrated no detectable Tc-99m-N-MPO metabolites in the urine and feces samples of the Sprague-Dawley rats at 120 minutes after injection. In guinea pigs no Tc-99m-N-MPO metabolites were detected in the urine at 120 minutes, but only approximately 60% of Tc-99m-N-MPO remained intact in the feces samples. In contrast, there was no intact Tc-99m-sestamibi detected in urine samples, and less than 15% of Tc-99m-sestamibi remained intact in the feces samples. Planar imaging studies indicated that clinically useful images of the heart may be obtained as early as 15 minutes after injection of Tc-99m-N-MPO. Conclusion. The combination of favorable organ biodistribution and myocardial uptake with rapid liver clearance makes Tc-99m-N-MPO a very promising myocardial perfusion radiotracer worthy of further evaluation in various preclinical animal models.

The IDO1 selective inhibitor epacadostat enhances dendritic cell immunogenicity and lytic ability of tumor antigen-specific T cells.

Epacadostat is a novel inhibitor of indoleamine-2,3-dioxygenase-1 (IDO1) that suppresses systemic tryptophan catabolism and is currently being evaluated in ongoing clinical trials. We investigated the effects of epacadostat on (a) human dendritic cells (DCs) with respect to maturation and ability to activate human tumor antigen-specific cytotoxic T-cell (CTL) lines, and subsequent T-cell lysis of tumor cells, (b) human regulatory T cells (Tregs), and (c) human peripheral blood mononuclear cells (PBMCs) in vitro. Simultaneous treatment with epacadostat and IFN-γ plus lipopolysaccharide (LPS) did not change the phenotype of matured human DCs, and as expected decreased the tryptophan breakdown and kynurenine production. Peptide-specific T-cell lines stimulated with DCs pulsed with peptide produced significantly more IFN-γ, TNFα, GM-CSF and IL-8 if the DCs were treated with epacadostat. These T cells also displayed higher levels of tumor cell lysis on a per cell basis. Epacadostat also significantly decreased Treg proliferation induced by IDO production from IFN-γ plus LPS matured human DCs, although the Treg phenotype did not change. Multicolor flow cytometry was performed on human PBMCs treated with epacadostat; analysis of 123 discrete immune cell subsets revealed no changes in major immune cell types, an increase in activated CD83+ conventional DCs, and a decrease in immature activated Tim3+ NK cells. These studies show for the first time several effects of epacadostat on human DCs, and subsequent effects on CTL and Tregs, and provide a rationale as to how epacadostat could potentially increase the efficacy of immunotherapeutics, including cancer vaccines.

64Cu-labeled 2-(diphenylphosphoryl)ethyldiphenylphosphonium cations as highly selective tumor imaging agents: effects of linkers and chelates on radiotracer biodistribution characteristics.

Radiolabeled organic cations, such as triphenylphosphonium (TPP), represents a new class of radiotracers for imaging cancers and the transport function of multidrug resistance P-glycoproteins (particularly MDR1 Pgp) by single photon emission computed tomography (SPECT) or positron emission tomography (PET). This report presents the synthesis and biological evaluation of (64)Cu-labeled 2-(diphenylphosphoryl)ethyldiphenylphosphonium (TPEP) cations as novel PET radiotracers for tumor imaging. Biodistribution studies were performed using the athymic nude mice bearing subcutaneous U87MG human glioma xenografts to explore the impact of linkers, bifunctional chelators (BFCs), and chelates on biodistribution characteristics of the (64)Cu-labeled TPEP cations. Metabolism studies were carried out using normal athymic nude mice to determine the metabolic stability of four (64)Cu radiotracers. It was found that most (64)Cu radiotracers described in this study have significant advantages over (99m)Tc-Sestamibi for their high tumor/heart and tumor/muscle ratios. Both BFCs and linkers have significant impact on biological properties of (64)Cu-labeled TPEP cations. For example, (64)Cu(DO3A-xy-TPEP) has much lower liver uptake and better tumor/liver ratios than (64)Cu(DO3A-xy-TPP), suggesting that TPEP is a better mitochondrion-targeting molecule than TPP. Replacing DO3A with DO2A results in (64)Cu(DO2A-xy-TPEP) (+), which has a lower tumor uptake than (64)Cu(DO3A-xy-TPEP). Substitution of DO3A with NOTA-Bn leads to a significant decrease in tumor uptake for (64)Cu(NOTA-Bn-xy-TPEP). The use of DOTA-Bn to replace DO3A has little impact on the tumor uptake, but the tumor/liver ratio of (64)Cu(DOTA-Bn-xy-TPEP) (-) is not as good as that of (64)Cu(DO3A-xy-TPEP), probably due to the aromatic benzene ring in DOTA-Bn. Addition of an extra acetamido group in (64)Cu(DOTA-xy-TPEP) results in a lower liver uptake, but tumor/liver ratios of (64)Cu(DOTA-xy-TPEP) and (64)Cu(DO3A-xy-TPEP) are comparable due to a faster tumor washout of (64)Cu(DOTA-xy-TPEP). Substitution of xylene with the PEG 2 linker also leads to a significant reduction in both tumor and liver uptake. MicroPET imaging studies on (64)Cu(DO3A-xy-TPEP) in athymic nude mice bearing U87MG glioma xenografts showed that the tumor was clearly visualized as early as 1 h postinjection with very high T/B contrast. There was very little metabolite (<2%) detectable in the urine and feces samples for (64)Cu(DO3A-xy-TPEP), (64)Cu(DOTA-Bn-xy-TPEP)(-), and (64)Cu(NOTA-Bn-xy-TPEP). Considering both tumor uptake and T/B ratios (particularly tumor/heart, tumor/liver, and tumor/muscle), it was concluded that (64)Cu(DO3A-xy-TPEP) is a promising PET radiotracer for imaging the MDR-negative tumors.

64Cu-Labeled Lissamine Rhodamine B: A Promising PET Radiotracer Targeting Tumor Mitochondria

Enhanced mitochondrial potential in carcinoma cells is an important characteristic of cancer. It is of great current interest to develop a radiotracer that is sensitive to mitochondrial potential changes at the early stage of tumor growth. In this report, we present the synthesis and evaluation of (64)Cu-labeled Lissamine rhodamine B (LRB), (64)Cu(DOTA-LRB) (DOTA-LRB = 2-(6-(diethylamino)-3-(diethyliminio)-3H-xanthen-9-yl)-5-(N-(2-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclo-dodecan-1-yl)acetamido)ethyl)sulfamoyl)benzenesulfonate) as a new radiotracer for imaging tumors in athymic nude mice bearing U87MG human glioma xenografts by positron emission tomography (PET). We also explored its localization mechanism using Cu(DOTA-LRB) as the fluorescent probe in both the U87MG human glioma cell line and the cultured primary U87MG glioma cells. It was found that (64)Cu(DOTA-LRB) had the highest tumor uptake (6.54 ± 1.50, 6.91 ± 1.26, 5.68 ± 1.13, 7.58 ± 1.96, and 5.14 ± 1.50%ID/g at 0.5, 1, 2, 4, and 24 h postinjection, respectively) among many (64)Cu-labeled organic cations evaluated in the same animal model. The cellular staining study indicated that Cu(DOTA-LRB) was able to localize in mitochondria of U87MG glioma cells due to the enhanced negative mitochondrial potential. This statement is completely supported by the results from decoupling experiment with carbonylcyanide-m-chlorophenylhydrazone (CCCP). MicroPET data showed that the U87MG glioma tumors were clearly visualized as early as 30 min postinjection with (64)Cu(DOTA-LRB). (64)Cu(DOTA-LRB) remained stable during renal excretion, but underwent extensive degradation during hepatobiliary excretion. On the basis of the results from this study, it was concluded that (64)Cu(DOTA-LRB) represents a new class of promising PET radiotracers for noninvasive imaging of the MDR-negative tumors.

Evaluation of 64Cu(DO3A-xy-TPEP) as A Potential PET Radiotracer for Monitoring Tumor Multidrug Resistance

In this study, we evaluated the potential of (64)Cu(DO3A-xy-TPEP) (DO3A-xy-TPEP = (2-(diphenylphosphoryl)ethyl)diphenyl(4-((4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)methyl)benzyl)phosphonium) as a PET (positron emission tomography) radiotracer for noninvasive monitoring of multidrug resistance (MDR) transport function in several xenografted tumor models (MDR-negative: U87MG; MDR-positive: MDA-MB-435, MDA-MB-231, KB-3-1, and KB-v-1). It was found that (64)Cu(DO3A-xy-TPEP) has a high initial tumor uptake (5.27 +/- 1.2%ID/g at 5 min p.i.) and shows a steady uptake increase between 30 and 120 min p.i. (2.09 +/- 0.53 and 3.35 +/- 1.27%ID/g at 30 and 120 min p.i., respectively) in the MDR-negative U87MG glioma tumors. (64)Cu(DO3A-xy-TPEP) has a greater uptake difference between U87MG glioma and MDR-positive tumors (MDA-MB-231: 1.57 +/- 0.04, 1.00 +/- 0.17, and 0.93 +/- 0.15; MDA-MB-435: 1.15 +/- 0.19, 1.12 +/- 0.20, and 0.81 +/- 0.11; KB-3-1: 1.45 +/- 0.31, 1.43 +/- 0.16, and 1.08 +/- 0.19; and KB-v-1: 1.63 +/- 0.47, 1.81 +/- 0.31, and 1.14 +/- 0.22%ID/g at 30, 60, and 120 min p.i., respectively) than (99m)Tc-Sestamibi. Regardless of the source of MDR, the overall net effect is the rapid efflux of (64)Cu(DO3A-xy-TPEP) from tumor cells, which leads to a significant reduction of its tumor uptake. It was concluded that (64)Cu(DO3A-xy-TPEP) is more efficient than (99m)Tc-Sestamibi as the substrate for MDR P-glycoproteins (MDR Pgps) and multidrug resistance-associated proteins (MRPs), and might be a more efficient radiotracer for noninvasive monitoring of the tumor MDR transport function. (64)Cu(DO3A-xy-TPEP) and (99m)Tc-Sestamibi share almost identical subcellular distribution patterns in U87MG glioma tumors. Thus, it is reasonable to believe that (64)Cu(DO3A-xy-TPEP), like (99m)Tc-Sestamibi, is able to localize in mitochondria due to the increased plasma and mitochondrial transmembrane potentials in tumor cells.

In Vitro and In Vivo Analysis of Indocyanine Green-Labeled Panitumumab for Optical Imaging—A Cautionary Tale

Indocyanine green (IC-Green), the only FDA approved near-infrared (NIR) fluorophore for clinical use, is attractive to researchers for the development of targeted optical imaging agents by modification of its structure and conjugation to monoclonal antibodies (mAbs) or their fragments. IC-Green derivative, ICG-sulfo-OSu (ICG-sOSu), is frequently used for antibody conjugation. However, ICG-sOSu is amphiphilic and readily facilitates aggregation of mAbs that is not easily separable from the desired immunoconjugates. Complications originating from this behavior are frequently overlooked by researchers. This study examined detailed chemical and biological characteristics of an ICG-sOSu-labeled mAb, panitumumab, and provided a clinically applicable strategy to deliver a pure conjugation product. Size-exclusion high-performance liquid chromatography (SE-HPLC) analysis of conjugation reactions, performed at molar reaction ratios of ICG-sOSu: mAb of 5, 10, or 20, resulted in isolable desired ICG-sOSu-panitumumab conjugation product in 72%, 53%, and 19% yields, respectively, with the remainder consisting of high molecular weight aggregates (>150 kDa) 14%, 30%, and 51%, respectively. The HPLC-purified ICG-sOSu-panitumumab products were analyzed by native and SDS polyacrylamide gel electrophoresis (PAGE) followed by optical imaging. Results indicated that the interaction between ICG-sOSu and panitumumab was due to both covalent and noncovalent binding of the ICG-sOSu to the protein. Noncovalently bound dye in the ICG-sOSu-panitumumab conjugate products was removed by extraction with ethyl acetate to further purify the HPLC-isolated conjugation products. With conserved immunoreactivity, excellent target-specific uptake of the doubly purified bioconjugates was observed with minimal liver retention in athymic nude mice bearing HER1-expressing tumor xenografts. In summary, the preparation of well-defined bioconjugate products labeled with commercial ICG-sOSu dye is not a simple process and control of the conjugation reaction ratio and conditions is crucial. Furthermore, absolute purification and characterization of the products is necessitated prior to in vivo optical imaging. Use of validated and characterized dye conjugate products should facilitate the development of clinically viable and reproducible IC-Green derivative and other NIR dye mAb conjugates for optical imaging applications.