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1.  Lead-203-Labeled Alpha-Melanocyte Stimulating Hormone Peptide as an Imaging Probe for Melanoma Detection 
Peptide-targeted alpha therapy with 7.4 MBq of 212Pb-[1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-ReO-[Cys3,4,10, d-Phe7, Arg11]α-MSH3-13 {212Pb-DOTA-Re(Arg11)CCMSH} cured 45% of B16/F1 murine melanoma-bearing C57 mice in a 120-day study, highlighting its melanoma treatment potential. However, there is a need to develop an imaging surrogate for patient-specific dosimetry and to monitor the tumor response to 212Pb-DOTA-Re(Arg11)CCMSH therapy. The purpose of this study was to evaluate the potential of 203Pb-DOTA-Re(Arg11)CCMSH as a matched-pair SPECT imaging agent for 212Pb-DOTA-Re(Arg11)CCMSH.
DOTA-Re(Arg11)CCMSH was labeled with 203Pb in 0.5 M NH4OAc buffer at pH 5.4. The internalization and efflux of 203Pb-DOTA-Re(Arg11)CCMSH were determined in B16/F1 melanoma cells. The pharmacokinetics of 203Pb-DOTA-Re(Arg11)CCMSH was examined in B16/F1 melanoma-bearing C57 mice. A micro-SPECT/CT imaging study was performed with 203Pb-DOTA-Re(Arg11)CCMSH in a B16/F1 melanoma-bearing C57 mouse at 2 h post-injection.
Lead-203-DOTA-Re(Arg11)CCMSH was easily prepared in NH4OAc buffer and completely separated from the excess non-radiolabeled peptide by RP-HPLC. Lead-203-DOTA-Re(Arg11)CCMSH displayed fast internalization and extended retention in B16/F1 cells. Approximately 73% of 203Pb-DOTA-Re(Arg11)CCMSH activity internalized after a 20-min incubation at 25°C. After incubating the cells in culture media for 20 min, 78% of internalized activity remained in the cells. Lead-203-DOTA-Re(Arg11)CCMSH exhibited similar biodistribution pattern with 212Pb-DOTA-Re(Arg11)CCMSH in B16/F1 melanoma-bearing mice. Lead-203-DOTA-Re(Arg11)CCMSH exhibited the peak tumor uptake of 12.00±3.20 %ID/g at 1 h post-injection. The tumor uptake gradually decreased to 3.43±1.12 %ID/g at 48 h post-injection. Lead-203-DOTA-Re(Arg11)CCMSH exhibited a peak tumor to kidney uptake ratio of 1.53 at 2 h post-injection. The absorbed doses to the tumor and kidneys were 4.32 and 4.35 Gy/37 MBq, respectively. Whole-body clearance of 203Pb-DOTA-Re(Arg11)CCMSH was fast, with approximately 89% of the injected activity cleared through urinary system by 2 h post-injection. Lead-203 showed 1.6 mm SPECT imaging resolution, which was comparable to 99mTc. Melanoma lesions were visualized through SPECT/CT images of 203Pb-DOTA-Re(Arg11)CCMSH at 2 h post-injection.
Lead-203-DOTA-Re(Arg11)CCMSH exhibited favorable pharmacokinetic and tumor imaging properties, highlighting its potential as a matched-pair SPECT imaging agent for 212Pb-DOTA-Re(Arg11)CCMSH melanoma treatment.
PMCID: PMC3610567  PMID: 18413404
2.  A comparison of 111In- or 64Cu-DOTA-trastuzumab Fab fragments for imaging subcutaneous HER2-positive tumor xenografts in athymic mice using microSPECT/CT or microPET/CT 
EJNMMI Research  2011;1:15.
Our objective was to compare 111In- or 64Cu-DOTA-trastuzumab Fab fragments for imaging small or large s.c. tumor xenografts in athymic mice that display a wide range of human epidermal growth factor receptor-2 (HER2) expression using microSPECT/CT or microPET/CT.
Trastuzumab Fab were labeled with 111In or 64Cu by conjugation to 1,4,7,10-tetraazacyclododecane N, N', N'', N'''-tetraacetic acid (DOTA). The purity of 111In- and 64Cu-DOTA-trastuzumab Fab was measured by SDS-PAGE and HPLC. HER2 binding affinity was determined in saturation radioligand binding assays using SKBR-3 cells (1.3 × 106 HER2/cell). MicroSPECT/CT and microPET/CT were performed in athymic mice bearing s.c. BT-20 and MDA-MB-231 xenografts with low (0.5 to 1.6 × 105 receptors/cell), MDA-MB-361 tumors with intermediate (5.1 × 105 receptors/cell) or SKOV-3 xenografts with high HER2 expression (1.2 × 106 receptors/cell) at 24 h p.i. of 70 MBq (10 μg) of 111In-DOTA-trastuzumab Fab or 22 MBq (10 μg) of 64Cu-DOTA-trastuzumab Fab or irrelevant 111In- or 64Cu-DOTA-rituximab Fab. Tumor and normal tissue uptake were quantified in biodistribution studies.
111In- and 64Cu-DOTA-trastuzumab were > 98% radiochemically pure and bound HER2 with high affinity (Kd = 20.4 ± 2.5 nM and 40.8 ± 3.5 nM, respectively). MDA-MB-361 and SKOV-3 tumors were most clearly imaged using 111In- and 64Cu-DOTA-trastuzumab Fab. Significantly higher tumor/blood (T/B) ratios were found for 111In-DOTA-trastuzumab Fab than 111In-DOTA-rituximab Fab for BT-20, MDA-MB-231 and MDA-MB-361 xenografts, and there was a direct association between T/B ratios and HER2 expression. In contrast, tumor uptake of 64Cu-DOTA-trastuzumab Fab was significantly higher than 64Cu-DOTA-rituximab Fab in MDA-MB-361 tumors but no direct association with HER2 expression was found. Both 111In- and 64Cu-DOTA-trastuzumab Fab imaged small (5 to 10 mm) or larger (10 to 15 mm) MDA-MB-361 tumors. Higher blood, liver, and spleen radioactivity were observed for 64Cu-DOTA-trastuzumab Fab than 111In-DOTA-trastuzumab Fab.
We conclude that 111In-DOTA-trastuzumab Fab was more specific than 64Cu-DOTA-trastuzumab Fab for imaging HER2-positive tumors, especially those with low receptor density. This was due to higher levels of circulating radioactivity for 64Cu-DOTA-trastuzumab Fab which disrupted the relationship between HER2 density and T/B ratios. Use of alternative chelators that more stably bind 64Cu may improve the association between T/B ratios and HER2 density for 64Cu-labeled trastuzumab Fab.
PMCID: PMC3250982  PMID: 22214307
indium-111; copper-64; HER2; MicroSPECT; MicroPET; DOTA; trastuzumab Fab; breast cancer; ovarian cancer
3.  Evaluation of 111In-Labeled Cyclic RGD Peptides: Tetrameric Not Tetravalent 
Bioconjugate chemistry  2010;21(5):969-978.
This report presents the synthesis and evaluation of 111In(DOTA-6G-RGD4) (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid; 6G-RGD4 = E{G3-E[G3-c(RGDfK)]2}2 and G3 = Gly-Gly-Gly), 111In(DOTA-RGD4) (RGD4 = E{E[c(RGDfK)]2}2) and 111In(DOTA-3G-RGD2) (3G-RGD2 = G3-E[G3-c(RGDfK)]2) as new radiotracers for imaging integrin αvβ3–positive tumors. The IC50 values of DOTA-6G-RGD4, DOTA-RGD4 and DOTA-3G-RGD2 were determined to be 0.4 ± 0.1, 1.5 ± 0.2 and 1.3 ± 0.2 nM against 125I-c(RGDyK) bound to integrin αvβ3–positive U87MG human glioma cells. 111In(DOTA-6G-RGD4), 111In(DOTA-RGD4) and 111In(DOTA-3G-RGD2) were prepared by reacting 111InCl3 with the respective DOTA conjugate in NH4OAc buffer (100 mM, pH = 5.5). Radiolabeling could be completed by heating the reaction mixture at 100 °C for 15 – 20 min. The specific activity was ~1850 MBq/μmol for 111In(DOTA-3G-RGD2) and ~1480 MBq/μmol for 111In(DOTA-6G-RGD4). The athymic nude mice bearing U87MG human glioma xenografts were used to evaluate tumor uptake and excretion kinetics of 111In(DOTA-6G-RGD4), 111In(DOTA-RGD4) and 111In(DOTA-3G-RGD2). The results from both the integrin αvβ3 binding assay and biodistribution studies suggest that the tetrameric cyclic RGD peptides, such as RGD4 and 6G-RGD4, are most likely bivalent in binding to the integrin αvβ3. Both 111In(DOTA-6G-RGD4) and 111In(DOTA-RGD4) had significantly higher tumor uptake than 111In(DOTA-3G-RGD2) at 24 – 72 h post-injection due to the extra RGD motifs in RGD4 and 6G-RGD4. 111In(DOTA-3G-RGD2) had very little metabolism while 111In(DOTA-6G-RGD4) had a 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 90Y and 177Lu analogs M(DOTA-6G-RGD4) (M = 90Y and 177Lu) might be useful as therapeutic radiotracers for treatment of integrin αvβ3-positive solid tumors.
PMCID: PMC2874107  PMID: 20387808
integrin αvβ3; 111In-labeled cyclic RGD peptides; tumor imaging
4.  Evaluation of 64Cu Labeled GX1: A Phage Display Peptide Probe for PET Imaging of Tumor Vasculature 
Molecular imaging using positron emission tomography (PET) radiotracers targeted to tumor vasculature offers a noninvasive method for early detection of tumor angiogenesis and efficient monitoring of response to anti-tumor vasculature therapy. The previous in vitro results demonstrated that the GX1 peptide, identified by phage display technology, is a tumor vasculature endothelium-specific ligand. In this study, we evaluated a 64Cu-labeled GX1 peptide as a potential radiotracer for microPET imaging of tumor vasculature in a U87MG tumor xenografted mouse model.
Macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane-N, N′, N″, N‴-tetraacetic acid (DOTA)-conjugated GX1 peptide was synthesized and radiolabeled with 64Cu (t1/2=12.7 h) in ammonium acetate buffer. The 64Cu-labeled GX1 peptide was then subjected to in vitro tumor cell uptake study, small animal PET and direct tissue sampling biodistribution studies in a U87MG tumor xenografted mouse model.
The in vitro experiment demonstrated that 64Cu-DOTA-GX1 is stable in PBS with more than 91% of 64Cu-DOTA-GX1 peptide remaining intact after 24 h of incubation. Cellular uptake and retention studies revealed 64Cu-DOTA-GX1 binds to U87MG glioma cells and has good tumor cell retention. For small animal PET imaging studies, the U87MG tumors were all clearly visible with high contrast to contralateral background at all measured time points after injection of 64Cu-DOTA-GX1 while high accumulation in liver and kidneys were also observed at early time points. The U87MG tumor uptake was determined to be the highest (7.97±0.75%ID/g) at 24 h pi. The blocking experiment was achieved by co-injection of 64Cu-DOTA-GX1 with non-radiolabeled GX1 peptide (20 mg/kg) at 24 h pi, suggesting 64Cu-DOTA-GX1 is a target-specific tracer. Furthermore, the biodistribution results were consistent with the quantification of microPET imaging, demonstrating the highest ratio (16.09±1.21) of tumor/muscle uptake of 64Cu-DOTA-GX1 at 24 h pi for non-blocking group and significant decreased ratio (6.57±0.58) for blocking group. Finally, metabolic studies suggested that 64Cu-DOTA-GX1 is stable in mouse blood and urine in vivo at early time point while the metal transchelation may also occur in mouse liver and kidneys.
Our studies demonstrate that 64Cu-DOTA-GX1 is a promising radiotracer for imaging tumor vasculature.
PMCID: PMC3629956  PMID: 21360213
64Cu-Labeled GX1 peptide; PET imaging; Tumor vasculature; Phage display
5.  Development of Novel Radiogallium-Labeled Bone Imaging Agents Using Oligo-Aspartic Acid Peptides as Carriers 
PLoS ONE  2013;8(12):e84335.
68Ga (T1/2 = 68 min, a generator-produced nuclide) has great potential as a radionuclide for clinical positron emission tomography (PET). Because poly-glutamic and poly-aspartic acids have high affinity for hydroxyapatite, to develop new bone targeting 68Ga-labeled bone imaging agents for PET, we used 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as a chelating site and conjugated aspartic acid peptides of varying lengths. Subsequently, we compared Ga complexes, Ga-DOTA-(Asp)n (n = 2, 5, 8, 11, or 14) with easy-to-handle 67Ga, with the previously described 67Ga-DOTA complex conjugated bisphosphonate, 67Ga-DOTA-Bn-SCN-HBP. After synthesizing DOTA-(Asp)n by a Fmoc-based solid-phase method, complexes were formed with 67Ga, resulting in 67Ga-DOTA-(Asp)n with a radiochemical purity of over 95% after HPLC purification. In hydroxyapatite binding assays, the binding rate of 67Ga-DOTA-(Asp)n increased with the increase in the length of the conjugated aspartate peptide. Moreover, in biodistribution experiments, 67Ga-DOTA-(Asp)8, 67Ga-DOTA-(Asp)11, and 67Ga-DOTA-(Asp)14 showed high accumulation in bone (10.5±1.5, 15.1±2.6, and 12.8±1.7% ID/g, respectively) but were barely observed in other tissues at 60 min after injection. Although bone accumulation of 67Ga-DOTA-(Asp)n was lower than that of 67Ga-DOTA-Bn-SCN-HBP, blood clearance of 67Ga-DOTA-(Asp)n was more rapid. Accordingly, the bone/blood ratios of 67Ga-DOTA-(Asp)11 and 67Ga-DOTA-(Asp)14 were comparable with those of 67Ga-DOTA-Bn-SCN-HBP. In conclusion, these data provide useful insights into the drug design of 68Ga-PET tracers for the diagnosis of bone disorders, such as bone metastases.
PMCID: PMC3877283  PMID: 24391942
6.  In vivo targeting of HER2-positive tumor using 2-helix affibody molecules 
Amino acids  2011;43(1):405-413.
Molecular imaging of human epidermal growth factor receptor type 2 (HER2) expression has drawn significant attention because of the unique role of the HER2 gene in diagnosis, therapy and prognosis of human breast cancer. In our previous research, a novel cyclic 2-helix small protein, MUT-DS, was discovered as an anti-HER2 Affibody analog with high affinity through rational protein design and engineering. MUT-DS was then evaluated for positron emission tomography (PET) of HER2-positive tumor by labeling with two radionuclides, 68Ga and 18F, with relatively short half-life (t1/2 < 2 h). In order to fully study the in vivo behavior of 2-helix small protein and demonstrate that it could be a robust platform for labeling with a variety of radionuclides for different applications, in this study, MUT-DS was further radiolabeled with 64Cu or 111In and evaluated for in vivo targeting of HER2-positive tumor in mice. Design 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid (DOTA) conjugated MUT-DS (DOTA–MUT-DS) was chemically synthesized using solid phase peptide synthesizer and I2 oxidation. DOTA–MUTDS was then radiolabeled with 64Cu or 111In to prepare the HER2 imaging probe (64Cu/111In-DOTA–MUT-DS). Both biodistribution and microPET imaging of the probe were evaluated in nude mice bearing subcutaneous HER2-positive SKOV3 tumors. DOTA–MUT-DS could be successfully synthesized and radiolabeled with 64Cu or 111In. Biodistribution study showed that tumor uptake value of 64Cu or 111In-labeled DOTA–MUT-DS was 4.66 ± 0.38 or 2.17 ± 0.15%ID/g, respectively, in nude mice bearing SKOV3 xenografts (n = 3) at 1 h post-injection (p.i.). Tumor-to-blood and tumor-to-muscle ratios for 64Cu-DOTA-MUT-DS were attained to be 3.05 and 3.48 at 1 h p.i., respectively, while for 111In-DOTA–MUT-DS, they were 2.04 and 3.19, respectively. Co-injection of the cold Affibody molecule ZHER2:342 with 64Cu-DOTA-MUT-DS specifically reduced the SKOV3 tumor uptake of the probe by 48%. 111In-DOTA–MUT-DS displayed lower liver uptake at all the time points investigated and higher tumor to blood ratios at 4 and 20 h p.i., when compared with 64Cu-DOTA–MUT-DS. This study demonstrates that the 2-helix protein based probes, 64Cu/111In DOTA–MUT-DS, are promising molecular probes for imaging HER2-positive tumor. Two-helix small protein scaffold holds great promise as a novel and robust platform for imaging and therapy applications.
PMCID: PMC4172459  PMID: 21984380
Affibody; HER2; PET; Molecular imaging; 111In; 64Cu
7.  Improving Tumor Uptake and Pharmacokinetics of 64Cu-Labeled Cyclic RGD Peptide Dimers with Gly3 and PEG4 Linkers 
Bioconjugate chemistry  2009;20(4):750-759.
Radiolabeled cyclic RGD (Arg-Gly-Asp) peptides represent a new class of radiotracers with potential for the early tumor detection and non-invasive monitoring of tumor metastasis and therapeutic response in cancer patients. This report describes the synthesis of two cyclic RGD peptide dimer conjugates, DOTA-PEG4-E[PEG4-c(RGDfK)]2 (DOTA-3PEG4-dimer: DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid; PEG4 = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) and DOTA-G3-E[G3-c(RGDfK)]2 (DOTA-3G3-dimer: G3 = Gly-Gly-Gly). Integrin αvβ3 binding affinities of cyclic RGD peptides were determined by competitive displacement of 125I-echistatin bound to U87MG human glioma cells, and follow the order of DOTA-E{E[c(RGDfK)]2}2 (DOTA-tetramer: IC50 = 10 ± 2 nM) > DOTA-3G3-dimer (IC50 = 62 ± 6 nM) ~ DOTA-3PEG4-dimer (IC50 = 74 ± 3 nM) > DOTA-E[c(RGDfK)]2 (DOTA-dimer: IC50 = 102 ± 5 nM). The addition of PEG4 and G3 linkers between two cyclic RGD motifs in DOTA-3G3-dimer and DOTA-3PEG4-dimer makes it possible for them to achieve the simultaneous integrin αvβ3 binding in a bivalent fashion. Both 64Cu(DOTA-3PEG4-dimer) and 64Cu(DOTA-3G3-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 PEG4 and G3 linkers are particularly useful for improving tumor uptake and clearance kinetics of 64Cu radiotracers from the non-tumor organs, such as kidneys, liver and lungs. There is a linear relationship between the tumor size and %ID tumor uptake, suggesting that 64Cu(DOTA-3PEG4-dimer) and 64Cu(DOTA-3PEG4-dimer) might be useful for noninvasive monitoring of tumor growth or shrinkage during anti-angiogenic therapy. MicroPET imaging data clearly demonstrate the utility of 64Cu(DOTA-3G3-dimer) as a new PET radiotracer for imaging integrin αvβ3-positive tumors.
PMCID: PMC2676896  PMID: 19320477
integrin αvβ3; 64Cu-labeled cyclic RGD peptides; PET tumor imaging
8.  Evaluation of 111In-Labeled Cyclic RGD Peptides: Effects of Peptide and Linker Multiplicity on Their Tumor Uptake, Excretion Kinetics and Metabolic Stability 
Theranostics  2011;1:322-340.
Purpose: The purpose of this study was to demonstrate the valence of cyclic RGD peptides, P-RGD (PEG4-c(RGDfK): PEG4 = 15-amino-4,710,13-tetraoxapentadecanoic acid), P-RGD2 (PEG4-E[c(RGDfK)]2, 2P-RGD4 (E{PEG4-E[c(RGDfK)]2}2, 2P4G-RGD4 (E{PEG4-E[G3-c(RGDfK)]2}2: G3 = Gly-Gly-Gly) and 6P-RGD4 (E{PEG4-E[PEG4-c(RGDfK)]2}2) in binding to integrin αvβ3, and to assess the impact of peptide and linker multiplicity on biodistribution properties, excretion kinetics and metabolic stability of their corresponding 111In radiotracers.
Methods: Five new RGD peptide conjugates (DOTA-P-RGD (DOTA =1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid), DOTA-P-RGD2, DOTA-2P-RGD4, DOTA-2P4G-RGD4, DOTA-6P-RGD4), and their 111In complexes were prepared. The integrin αvβ3 binding affinity of cyclic RGD conjugates were determined by a competitive displacement assay against 125I-c(RGDyK) bound to U87MG human glioma cells. Biodistribution, planar imaging and metabolism studies were performed in athymic nude mice bearing U87MG human glioma xenografts.
Results: The integrin αvβ3 binding affinity of RGD conjugates follows the order of: DOTA-6P-RGD4 (IC50 = 0.3 ± 0.1 nM) ~ DOTA-2P4G-RGD4 (IC50 = 0.2 ± 0.1 nM) ~ DOTA-2P-RGD4 (IC50 = 0.5 ± 0.1 nM) > DOTA-3P-RGD2 (DOTA-PEG4-E[PEG4-c(RGDfK)]2: IC50 = 1.5 ± 0.2 nM) > DOTA-P-RGD2 (IC50 = 5.0 ± 1.0 nM) >> DOTA-P-RGD (IC50 = 44.3 ± 3.5 nM) ~ c(RGDfK) (IC50 = 49.9 ± 5.5 nM) >> DOTA-6P-RGK4 (IC50 = 437 ± 35 nM). The fact that DOTA-6P-RGK4 had much lower integrin αvβ3 binding affinity than DOTA-6P-RGD4 suggests that the binding of DOTA-6P-RGD4 to integrin αvβ3 is RGD-specific. This conclusion is consistent with the lower tumor uptake for 111In(DOTA-6P-RGK4) than that for 111In(DOTA-6P-RGD4). It was also found that the G3 and PEG4 linkers between RGD motifs have a significant impact on the integrin αvβ3-targeting capability, biodistribution characteristics, excretion kinetics and metabolic stability of 111In-labeled cyclic RGD peptides.
Conclusion: On the basis of their integrin αvβ3 binding affinity and tumor uptake of their corresponding 111In radiotracers, it was conclude that 2P-RGD4, 2P4G-RGD4 and 6P-RGD4 are most likely bivalent in binding to integrin αvβ3, and extra RGD motifs might contribute to the long tumor retention times of 111In(DOTA-2P-RGD4), 111In(DOTA-2P4G-RGD4) and 111In(DOTA-6P-RGD4) than that of 111In(DOTA-3P-RGD3) at 72 h p.i. Among the 111In-labeled cyclic RGD tetramers evaluated in the glioma model, 111In(DOTA-2P4G-RGD4) has very high tumor uptake with the best tumor/kidney and tumor/liver ratios, suggesting that 90Y(DOTA-2P4G-RGD4) and 177Lu(DOTA-2P4G-RGD4) might have the potential for targeted radiotherapy of integrin αvβ3-positive tumors.
PMCID: PMC3157017  PMID: 21850213
integrin αvβ3; 111In-labeled cyclic RGD peptides; tumor imaging
9.  Effects of the Amino Acid Linkers on the Melanoma-Targeting and Pharmacokinetic Properties of Indium-111-labeled Lactam Bridge-Cyclized α-MSH Peptides 
The purpose of this study was to examine the profound effects of the amino acid linkers on the melanoma targeting and pharmacokinetic properties of novel 111In-labeled lactam bridge-cyclized DOTA-[X]-CycMSHhex {1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid-[X]-c[Asp-His-dPhe-Arg-Trp-Lys]-CONH2, X=GlyGlyNle, GlyGluNle or NleGlyGlu} peptides.
Three novel DOTA-GGNle-CycMSHhex, DOTA-GENle-CycMSHhex and DOTA-NleGE-CycMSHhex peptides were designed and synthesized. The melanocortin-1 (MC1) receptor binding affinities of the peptides were determined in B16/F1 melanoma cells. The melanoma targeting and pharmacokinetic properties of 111In-DOTA-GGNle-CycMSHhex and 111In-DOTA-GENle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice.
DOTA-GGNle-CycMSHhex and DOTA-GENle-CycMSHhex displayed 2.1 and 11.5 nM MC1 receptor binding affinities, whereas DOTA-NleGE-CycMSHhex showed 873.4 nM MC1 receptor binding affinity. The introduction of the -GlyGly- linker maintained high melanoma uptake while decreased the renal and liver uptakes of 111In-DOTA-GlyGlyNle-CycMSHhex. The tumor uptake values of 111In-DOTA-GGNle-CycMSHhex were 19.05 ± 5.04 and 18.6 ± 3.56 % injected dose/gram (%ID/g) at 2 and 4 h post-injection. 111In-DOTA-GGNle-CycMSHhex exhibited 28, 32 and 42% less renal uptake values than 111In-DOTA-Nle-CycMSHhex we reported previously, and 61, 65 and 68% less liver uptake values than 111In-DOTA-Nle-CycMSHhex at 2, 4 and 24 h post-injection, respectively.
The amino acid linkers exhibited the profound effects on the melanoma targeting and pharmacokinetic properties of the 111In-labeled lactam bridge-cyclized α-MSH peptides. Introduction of the -GlyGly- linker maintained high melanoma uptake while reducing the renal and liver uptakes of 111In-DOTA-GlyGlyNle-CycMSHhex, highlighting its potential as an effective imaging probe for melanoma detection, as well as a therapeutic peptide for melanoma treatment when labeled with a therapeutic radionuclide.
PMCID: PMC3065533  PMID: 21421725
Alpha-melanocyte stimulating hormone; Radiolabeled cyclic peptide; Melanoma imaging
10.  Assessment of blood flow with 68Ga-DOTA PET in experimental inflammation: a validation study using 15O-water 
Increased blood flow and vascular permeability are key events in inflammation. Based on the fact that Gadolinium-1,4,7,10-tetraazacyclododecane-N,N‘,N‘‘,N‘‘‘-tetraacetic acid (Gd-DOTA) is commonly used in magnetic resonance (MR) imaging of blood flow (perfusion), we evaluated the feasibility of its Gallium-68 labeled DOTA analog (68Ga-DOTA) for positron emission tomography (PET) imaging of blood flow in experimental inflammation. Adult, male Sprague-Dawley rats with turpentine oil induced sterile skin/muscle inflammation were anesthetized with isoflurane, and imaged under rest and adenosine-induced hyperemia by means of dynamic 2-min Oxygen-15 labeled water (H2 15O) and 30-min 68Ga-DOTA PET. For the quantification of PET data, regions of interest (ROIs) were defined in the focus of inflammation, healthy muscle, myocardium and heart left ventricle. Radioactivity concentration in the ROIs versus time after injection was determined for both tracers and blood flow was calculated using image-derived input. According to the H2 15O PET, blood flow was 0.69 ± 0.15 ml/min/g for inflammation and 0.15 ± 0.03 ml/min/g for muscle during rest. The blood flow remained unchanged during adenosine-induced hyperemia 0.67 ± 0.11 and 0.12 ± 0.03 ml/min/g for inflammation and muscle, respectively, indicating that adenosine has little effect on blood flow in peripheral tissues in rats. High focal uptake of 68Ga-DOTA was seen at the site of inflammation throughout the 30-min PET imaging. According to the 68Ga-DOTA PET, blood flow measured as the blood-to-tissue transport rate (K1) was 0.60 ± 0.07 ml/min/g for inflammation and 0.14 ± 0.06 ml/min/g for muscle during rest and 0.63 ± 0.08 ml/min/g for inflammation and 0.09 ± 0.04 ml/min/g for muscle during adenosine-induced hyperemia. The H2 15O-based blood flow and 68Ga-DOTA-based K1 values correlated well (r = 0.94, P < 0.0001). These results show that 68Ga-DOTA PET imaging is useful for the quantification of increased blood flow induced by inflammation.
PMCID: PMC4171843  PMID: 25250206
Blood flow; DOTA; Gallium-68; inflammation; Oxygen-15 water; PET; rat
11.  Evaluation of a 64Cu-Labeled Cystine-Knot Peptide Based on Agouti-Related Protein for PET of Tumors Expressing αvβ3 Integrin 
Recently, a truncated form of the agouti-related protein (AgRP), a 4-kDa cystine-knot peptide of human origin, was used as a scaffold to engineer mutants that bound to αvβ3 integrin with high affinity and specificity. In this study, we evaluated the potential of engineered integrin-binding AgRP peptides for use as cancer imaging agents in living subjects.
Engineered AgRP peptides were prepared by solid-phase peptide synthesis and were folded in vitro and purified by reversed-phase high-performance liquid chromatography. Competition assays were used to measure the relative binding affinities of engineered AgRP peptides for integrin receptors expressed on the surface of U87MG glioblastoma cells. The highest-affinity mutant, AgRP clone 7C, was site-specifically conjugated with 1,4,7,10-tetraazacyclododecane-N,N′,N′′N′′′-tetraacetic acid (DOTA). The resulting bioconjugate, DOTA-AgRP-7C, was radiolabeled with 64Cu for biodistribution analysis and small-animal PET studies in mice bearing U87MG tumor xenografts. In addition to serum stability, the in vivo metabolic stability of 64Cu-DOTA-AgRP-7C was assessed after injection and probe recovery from mouse kidney, liver, tumor, and urine.
AgRP-7C and DOTAAgRP-7C bound with high affinity to integrin receptors expressed on U87MG cells (half maximal inhibitory concentration values, 20 ± 4 and 14 ± 2 nM, respectively). DOTA-AgRP-7C was labeled with 64Cu with high radiochemical purity (>99%). In biodistribution and small-animal PET studies, 64Cu-DOTA-AgRP-7C displayed rapid blood clearance, good tumor uptake and retention (2.70 ± 0.93 percentage injected dose per gram [%ID/g] and 2.37 ± 1.04 %ID/g at 2 and 24 h, respectively), and high tumor-to-background tissue ratios. The integrin-binding specificity of 64Cu-DOTA-AgRP-7C was confirmed in vitro and in vivobyshowing thata large molar excessofthe unlabeled peptidomimetic c(RGDyK) could block probe binding and tumor uptake. Serum stability and in vivo metabolite assays demonstrated that engineered AgRP peptides are sufficiently stable for in vivo molecular imaging applications.
A radiolabeled version of the engineered AgRP peptide 7C showed promise as a PET agent for tumors that express the αvβ3 integrin. Collectively, these results validate AgRP-based cystine-knot peptides for use in vivo as molecular imaging agents and provide support for the general use of AgRP as a scaffold to develop targeting peptides, and hence diagnostics, against other tumor receptors.
PMCID: PMC4143171  PMID: 20124048
cystine-knot; agouti-related protein (AgRP); αvβ3 integrin; PET; 64Cu
12.  Metastatic Melanoma Imaging with an 111In-labeled Lactam Bridge-cyclized Alpha-Melanocyte Stimulating Hormone Peptide 
Nuclear medicine and biology  2009;36(3):267-276.
The purpose of this study was to examine whether a novel lactam bridge-cyclized 111In-labeled 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid-Gly-Glu-c[Lys-Nle-Glu-His-dPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp] {DOTA-GlyGlu-CycMSH} could be an effective imaging probe for metastatic melanoma detection.
111In-DOTA-GlyGlu-CycMSH was prepared and purified by reverse phase high performance liquid chromatography (RP-HPLC). The internalization and efflux of 111In-DOTA-GlyGlu-CycMSH were examined in B16/F10 melanoma cells. The biodistribution of 111In-DOTA-GlyGlu-CycMSH was determined in B16/F10 pulmonary metastatic melanoma-bearing and normal C57 mice. Pulmonary metastatic melanoma imaging was performed by small animal SPECT/CT (Nano-SPECT/CT®) using 111In-DOTA-GlyGlu-CycMSH as an imaging probe and compared with 18F-FDG PET imaging.
111In-DOTA-GlyGlu-CycMSH was readily prepared with greater than 95% radiolabeling yield. 111In-DOTA-GlyGlu-CycMSH displayed rapid internalization and extended efflux in B16/F10 cells. 111In-DOTA-GlyGlu-CycMSH exhibited significantly (p<0.05) higher uptakes (2.00±0.74 %ID/g at 2 h post-injection and 1.83±0.12 %ID/g at 4 h post-injection) in metastatic melanoma-bearing lung than that in normal lung (0.08±0.08 %ID/g and 0.05±0.05 %ID/g at 2 and 4 h post-injection, respectively). The activity accumulation in normal organs were low (<0.5 %ID/g) except for the kidneys 2 and 4 h post-injection. B16/F10 pulmonary melanoma metastases were clearly visualized with 111In-DOTA-GlyGlu-CycMSH 2 h post-injection rather than with 18F-FDG 1 h post-injection.
111In-DOTA-GlyGlu-CycMSH exhibited favorable metastatic melanoma targeting and imaging properties, highlighting its potential as an effective imaging probe for metastatic melanoma detection.
PMCID: PMC2685149  PMID: 19324272
Metastatic melanoma imaging; radiolabeled lactam bridge-cyclized peptide; alpha-melanocyte stimulating hormone
13.  64Cu-Labeled Alpha-Melanocyte-Stimulating Hormone Analog for MicroPET Imaging of Melanocortin 1 Receptor Expression 
Bioconjugate chemistry  2007;18(3):765-772.
The alpha-melanocyte-stimulating hormone (α-MSH) receptor (melanocortin type 1 receptor, or MC1R) plays an important role in the development and growth of melanoma cells. It was found that MC1R was overexpressed on most murine and human melanoma, making it a promising molecular target for melanoma imaging and therapy. Radiolabeled α-MSH peptide and its analogs that can specifically bind with MC1R have been extensively explored for developing novel agents for melanoma detection and radionuclide therapy. The goal of this study was to evaluate a 64Cu-labeled α-MSH analog, Ac-Nle-Asp-His-d-Phe-Arg-Trp-Gly-Lys(DOTA)-NH2 (DOTA–NAPamide), as a potential molecular probe for microPET imaging of melanoma and MC1R expression in melanoma xenografted mouse models. 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated NAPamide was synthesized and radiolabeled with 64Cu (t1/2=12 h) in NH4OAc (0.1 M; pH 5.5) buffered solution for 60 min at 50 °C. Cell culture studies reveal rapid and high uptake and internalization of 64Cu–DOTA–NAPamide in B16F10 cells. Over 90% of receptor-bound tracer is internalized at 3 h incubation. A cellular retention study demonstrates that the receptor-bound 64Cu–DOTA–NAPamide is slowly released from the B16F10 cells into the medium; 66% of the radioactivity is still associated with the cells even after 3 h incubation. The biodistribution of 64Cu–DOTA–NAPamide was then investigated in C57BL/6 mice bearing subcutaneous murine B16F10 melanoma tumors with high capacity of MC1R and Fox Chase Scid mice bearing human A375M melanoma with a relatively low number of MC1R receptors. Tumor uptake values of 64Cu–DOTA–NAPamide are found to be 4.63 ± 0.45% and 2.49 ± 0.31% ID/g in B16F10 and A375M xenografted melanoma at 2 h postinjection (pi), respectively. The B16F10 tumor uptake at 2 h pi is further inhibited to 2.29 ± 0.24% ID/g, while A375M tumor uptake at 2 h pi remains 2.20 ± 0.41% ID/g with a coinjection of excess α-MSH peptide. MicroPET imaging of 64Cu–DOTA–NAPamide in B16F10 tumor mice clearly shows good tumor localization. However, low A375M tumor uptake and poor tumor to normal tissue contrast were observed. This study demonstrates that 64Cu–DOTA–NAPamide is a promising molecular probe for α-MSH receptor positive melanoma PET imaging as well as MC1R expression imaging in living mice.
PMCID: PMC4143155  PMID: 17348700
14.  Functional Imaging of HER2-Positive Metastatic Breast Cancer Using 64Cu-DOTA-Trastuzumab Positron Emission Tomography 
Women with human epidermal growth factor receptor 2 (HER2)-positive breast cancer are candidates for treatment with the anti-HER2 antibody trastuzumab. Assessment of HER2 status in recurrent disease is usually made by core needle biopsy of a single lesion which may not be representative of the larger tumor mass or other sites of disease. Our long-range goal is to develop positron emission tomography (PET) of radiolabeled trastuzumab for systemically assessing tumor HER2 expression and identifying appropriate use of anti-HER2 therapies. The purpose of this study was to evaluate PET-CT of 64Cu-DOTA-trastuzumab for detecting and measuring tumor uptake of trastuzumab in patients with HER2-positive metastatic breast cancer.
Eight women with biopsy-confirmed HER2-positive metastatic breast cancer and no anti-HER2 therapy for ≥ 4 mo underwent complete staging, including 18F-fluorodeoxyglucose (FDG)/PET-CT. For 6 of the 8 patients, 64Cu-DOTA-trastuzumab injection (364-512 MBq, 5 mg trastuzumab) was preceded by trastuzumab infusion (45 mg). PET-CT (PET scan duration 1 h) was performed 21-25 (“Day 1”) and 47-49 (“Day 2”) h after 64Cu-DOTA-trastuzumab injection. Scan fields of view were chosen based on 18F-FDG/PET-CT. Lesions visualized relative to adjacent tissue on PET were considered PET-positive; analysis was limited to lesions identifiable on CT. Radiolabel uptake in prominent lesions was measured as maximum single-voxel standardized uptake value (SUVmax).
Liver uptake of 64Cu was reduced approximately 75% with the 45 mg trastuzumab pre-dose, without significant effect on tumor uptake. The study included 89 CT-positive lesions; detection sensitivity was 77, 89 and 93% for Day 1, Day 2 and 18F-FDG, respectively. On average, tumor uptake was similar for 64Cu-DOTA-trastuzumab and 18F-FDG [SUVmax (mean, range): Day 1 (8.1, 3.0-22.5, n=48); Day 2 (8.9, 0.9-28.9, n=38); 18F-FDG (9.7, 3.3-25.4, n=56)], but the extent of same-lesion uptake was not correlated between the 2 radiotracers. No toxicities were observed, and estimated radiation dose from 64Cu-DOTA-trastuzumab was similar to 18F-FDG.
64Cu-DOTA-trastuzumab visualizes HER2-positive metastatic breast cancer with high sensitivity, and is effective in surveying disseminated disease. A 45 mg trastuzumab pre-dose provides a 64Cu-DOTA-trastuzumab biodistribution favorable for tumor imaging. 64Cu-DOTA-trastuzumab/PET-CT warrants further evaluation for assessing tumor HER2 expression and measuring delivery of trastuzumab-based therapy.
PMCID: PMC4084518  PMID: 24337604
64Cu-labeled trastuzumab; HER2; breast cancer
15.  Synthesis and Evaluation of Novel Gonadotropin-Releasing Hormone Receptor-Targeting Peptides 
Bioconjugate chemistry  2011;22(8):1682-1689.
The purpose of this study was to develop novel radiolabeled gonadotropin-releasing hormone (GnRH) receptor-targeting peptides for breast cancer imaging. Three novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated GnRH peptides were designed and synthesized. The radiometal chelator DOTA was conjugated to the epsilon or alpha amino group of D-lysine, or the epsilon amino group of L-lysine via an Ahx {aminohexanoic acid} linker to generate DOTA-Ahx-(D-Lys6-GnRH1), DOTA-Ahx-(D-Lys6-GnRH2) and DOTA-Ahx-(L-Lys6-GnRH3), respectively. The conjugation of the DOTA to the epsilon amino group of D-lysine (rather than alpha amino group of D-lysine nor epsilon amino group of L-lysine) maintained the nanomolar GnRH receptor binding affinity. The IC50 values of DOTA-Ahx-(D-Lys6-GnRH1), DOTA-Ahx-(D-Lys6-GnRH2) and DOTA-Ahx-(L-Lys6-GnRH3) were 36.07 nM, 10.6 mM and 4.3 mM, respectively. Since only DOTA-Ahx-(D-Lys6-GnRH1) displayed nanomolar receptor binding affinity, the specific GnRH receptor binding of 111In-DOTA-Ahx-(D-Lys6-GnRH1) was determined in human GnRH receptor membrane preparations. Furthermore, the biodistribution and tumor imaging properties of 111In-DOTA-Ahx-(D-Lys6-GnRH1) were examined in MDA-MB-231 human breast cancer-xenografted nude mice. 111In-DOTA-Ahx-(D-Lys6-GnRH1) exhibited specific GnRH receptor binding and rapid tumor uptake (1.76 ± 0.58 %ID/g at 0.5 h post-injection) coupled with fast whole-body clearance through the urinary system. The MDA-MB-231 human breast cancer-xenografted tumor lesions were clearly visualized by single photon emission computed tomography (SPECT)/CT at 1 h post-injection of 111In-DOTA-Ahx-(D-Lys6-GnRH1). The profound impact of DOTA position on the binding affinity of the GnRH peptide provided a new insight into the design of novel radiolabeled GnRH peptides. The successful imaging of MDA-MB-231 human breast cancer-xenografted tumor lesions using 111In-DOTA-Ahx-(D-Lys6-GnRH1) suggested its potential as a novel imaging probe for human breast cancer imaging.
PMCID: PMC3157568  PMID: 21749045
Gonadotropin-releasing hormone receptor; receptor-targeting peptide
16.  111In-labeled Lactam Bridge-cyclized Alpha-Melanocyte Stimulating Hormone Peptide Analogues for Melanoma Imaging 
Bioconjugate chemistry  2008;19(2):539-547.
The purpose of this study was to examine the influence of the lactam bridge cyclization on melanoma targeting and biodistribution properties of the radiolabeled conjugates. Two novel lactam bridge-cyclized α-MSH peptide analogues, DOTA-CycMSH (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]) and DOTA-GlyGlu-CycMSH (DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]), were synthesized and radiolabeled with 111In. The internalization and efflux of 111In-labeled CycMSH peptides were examined in B16/F1 melanoma cells. The melanoma targeting properties, pharmacokinetics and SPECT/CT imaging of 111In-labeled CycMSH peptides were determined in B16/F1 melanoma-bearing C57 mice. Both 111In-DOTA-CycMSH and 111In-DOTA-GlyGlu-CycMSH exhibited fast internalization and extended retention in B16/F1 cells. The tumor uptake values of 111In-DOTA-CycMSH and 111In-DOTA-GlyGlu-CycMSH were 9.53±1.41 %injected dose/gram (%ID/g) and 10.40±1.40 %ID/g at 2 h post-injection, respectively. Flank melanoma tumors were clearly visualized with 111In-DOTA-CycMSH and 111In-DOTA-GlyGlu-CycMSH by SPECT/CT images at 2 h post-injection. Whole-body clearance of the peptides was fast, with greater than 90% of the radioactivities cleared through urinary system by 2 h post-injection. There was low radioactivity (<0.8 %ID/g) accumulated in blood and normal organs except kidneys at all time points investigated. Introduction of a negatively-charged linker (-Gly-Glu-) into the peptide sequence decreased the renal uptake by 44% without affecting the tumor uptake at 4 h post-injection. High receptor-mediated melanoma uptakes coupled with fast whole-body clearance in B16/F1 melanoma-bearing C57 mice demonstrated the feasibility of using 111In-labeled lactam bridge-cyclized α-MSH peptide analogues as a novel class of imaging probes for receptor-targeting melanoma imaging.
PMCID: PMC3617549  PMID: 18197608
Melanoma imaging; lactam bridge cyclization; alpha-MSH; melanocortin-1
17.  Correlating EGFR Expression with Receptor-Binding Properties and Internalization of 64Cu-DOTA-Cetuximab in 5 Cervical Cancer Cell Lines 
The anti–epidermal growth factor receptor (anti-EGFR) antibody cetuximab is clinically approved for the treatment of EGFR-expressing metastatic colorectal cancer and advanced head and neck cancer. Overexpression of EGFR has also been found in more than 70% of carcinomas of the cervix. The overall goal of this study was to determine whether 64Cu-1,4,7,10-tetraazacyclododecane-N,N′, N′′N′′′-tetraacetic acid (DOTA)-cetuximab has potential as an agent for measuring EGFR concentration by PET imaging in cervical cancer tumors.
Cetuximab was conjugated to the bifunctional chelator DOTA and labeled with 64Cu. EGFR messenger RNA (mRNA) expression was correlated with EGFR densities on the cell surface of 5 different cervical cancer cell lines and with receptor function, measured by internalization of 64Cu-DOTA-cetuximab. Imaging in tumor-bearing mice with small-animal PET was performed using the highest-expressing cervical cancer cell line.
The affinity of 64Cu-DOTA-cetuximab binding for the EGFR was similar in 4 EGFR-positive lines, varying from 0.1 to 0.7 nM. The mRNA expression corresponded well with EGFR densities and levels of internalization, with responses decreasing in the order of CaSki > ME-180 > DoTc2 4510 > HeLa > C-33A. Biodistribution and small-animal PET studies with 64Cu-DOTA-cetuximab in CaSki tumor-bearing nude mice showed relatively high tumor uptake at 24 h after injection (13.2 ± 1.2 percentage of injected activity per gram), although there was also significant retention of activity in the blood and liver accumulation.
64Cu-DOTA-cetuximab was successfully used to detect and quantify EGFR expression in cervical cancer tumors, and small-animal PET/CT of EGFR-expressing CaSki tumors suggests potential for PET/CT of EGFR-positive tumors.
PMCID: PMC4277815  PMID: 18703609
64Cu; cervical cancer; cetuximab
18.  64Cu-Labeled Lissamine Rhodamine B: A Promising PET Radiotracer Targeting Tumor Mitochondria 
Molecular pharmaceutics  2011;8(4):1198-1208.
The 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 the mitochondrial potential changes at the early stage of tumor growth. In this report, we present the synthesis and evaluation of 64Cu-labeled Lissamine Rhodamine B (LRB), 64Cu(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 U87MG human glioma cell line and the cultured primary U87MG glioma cells. It was found that 64Cu(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 post-injection, respectively) among many 64Cu-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 post-injection with 64Cu(DOTA-LRB). 64Cu(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 64Cu(DOTA-LRB) represents a new class of promising PET radiotracers for noninvasive imaging of the MDR-negative tumors.
PMCID: PMC3148415  PMID: 21545131
64Cu PET radiotracers; mitochondrial potential and tumor imaging
19.  Radiolabeled Affibody-Albumin Bioconjugates for HER2 Positive Cancer Targeting 
Bioconjugate chemistry  2011;22(3):413-421.
Affibody molecules have received significant attention in the fields of molecular imaging and drug development. However, Affibody scaffolds display an extremely high renal uptake, especially when modified with chelators and then labeled with radiometals. This unfavorable property may impact their use as radiotherapeutic agents in general and as imaging probes for the detection of tumors adjacent to kidneys in particular. Herein, we present a simple and generalizable strategy for reducing the renal uptake of Affibody molecules while maintaining their tumor uptake. Human serum albumin (HSA) was consecutively modified by 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono-N-hydroxysuccinimide ester (DOTA-NHS ester) and the bifunctional crosslinker sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Sulfo-SMCC). The HER2 Affibody analog, Ac-Cys-ZHER2:342, was covalently conjugated with HSA, and the resulting bioconjugate DOTA-HSA-ZHER2:342 was further radiolabeled with 64Cu and 111In and evaluated in vitro and in vivo. Radiolabeled DOTA-HSA-ZHER2:342 conjugates displayed a significant and specific cell uptake into SKOV3 cell cultures. Positron emission tomography (PET) investigations using 64Cu-DOTA-HSA-ZHER2:342 were performed in SKOV3 tumor-bearing nude mice. High tumor uptake values (> 14% ID/g at 24 h and 48 h) and high liver accumulations but low kidney accumulations were observed. Biodistribution studies and single-photon emission computed tomography (SPECT) investigations using 111In-DOTA-HSA-ZHER2:342 validated these results. At 24 h post injection, the bio distribution data revealed high tumor (16.26% ID/g) and liver uptake (14.11% ID/g) but relatively low kidney uptake (6.06% ID/g). Blocking studies with co-injected, non-labeled Ac-Cys-ZHER2:342 confirmed the in vivo specificity of HER2. Radiolabeled DOTA-HSA-ZHER2:342 Affibody conjugates are promising SPECT and PET-type probes for the imaging of HER2 positive cancer. More importantly, DOTA-HSA-ZHER2:342 is suitable for labeling with therapeutic radionuclides (e.g. 90Y or 177Lu) for treatment studies. The approach of using HSA to optimize the pharmacokinetics and biodistribution profile of Affibodies, may be extended to the design of many other targeting molecules.
PMCID: PMC3059402  PMID: 21299201
20.  Effect of DOTA Position on Melanoma Targeting and Pharmacokinetic Properties of 111In-labeled Lactam Bridge-Cyclized Alpha-Melanocyte Stimulating Hormone Peptide 
Bioconjugate chemistry  2009;20(11):2162-2168.
The purpose of this study was to examine the effect of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) position on melanoma targeting and pharmacokinetics of radiolabeled lactam bridge-cyclized alpha-melanocyte stimulating hormone (α-MSH) peptide.
A novel lactam bridge-cyclized α-MSH peptide, Ac-GluGlu-CycMSH[DOTA] {Ac-Glu-Glu-c[Lys(DOTA)-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp]}, was synthesized using standard 9-fluorenylmethyloxycarbonyl (Fmoc) chemistry. DOTA was directly attached to the alpha amino group of Lys in the cyclic ring while the N-terminus of the peptide was acetylated to generate Ac-GluGlu-CycMSH[DOTA]. The MC1 receptor binding affinity of Ac-GluGlu-CycMSH[DOTA] was determined in B16/F1 melanoma cells. Melanoma targeting and pharmacokinetic properties of Ac-GluGlu-CycMSH[DOTA]-111In were determined in B16/F1 melanoma-bearing C57 mice and compared to that of 111In-DOTA-Gly-Glu-c[Lys-Nle-Glu-His-DPhe-Arg-Trp-Gly-Arg-Pro-Val-Asp] (111In-DOTA-GlyGlu-CycMSH, DOTA was coupled to the N-terminus of the peptide).
Ac-GluGlu-CycMSH[DOTA] displayed 0.6 nM MC1 receptor binding affinity in B16/F1 cells. Ac-GluGlu-CycMSH[DOTA]-111In was readily prepared with greater than 95% radiolabeling yield. Ac-GluGlu-CycMSH[DOTA]-111In exhibited high tumor uptake (11.42 ± 2.20% ID/g 2 h post-injection) and prolonged tumor retention (9.42 ± 2.41% ID/g 4 h post-injection) in B16/F1 melanoma-bearing C57 mice. The uptake values for non-target organs were generally low (<1.3% ID/g) except for the kidneys 2, 4 and 24 h post-injection.
DOTA position exhibited profound effect on melanoma targeting and pharmacokinetic properties of Ac-GluGlu-CycMSH[DOTA]-111In, providing a new insight into the design of lactam bridge-cyclized peptide for melanoma imaging and therapy.
PMCID: PMC2786309  PMID: 19817405
Melanoma detection; lactam bridge cyclization; alpha-melanocyte stimulating hormone peptide; melanocortin-1 receptor
21.  68Ga-DOTA-Siglec-9 PET/CT imaging of peri-implant tissue responses and staphylococcal infections 
EJNMMI Research  2014;4:45.
Staphylococcus epidermidis (S. epidermidis) has emerged as one of the leading pathogens of biomaterial-related infections. Vascular adhesion protein-1 (VAP-1) is an inflammation-inducible endothelial molecule controlling extravasation of leukocytes. Sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) is a leukocyte ligand of VAP-1. We hypothesized that 68Ga-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-conjugated Siglec-9 motif containing peptide (68Ga-DOTA-Siglec-9) could detect inflammatory response due to S. epidermidis peri-implant infection by positron emission tomography (PET).
Thirty Sprague-Dawley rats were randomized into three groups. A sterile catheter was implanted into the medullary canal of the left tibia. In groups 1 and 2, the implantation was followed by peri-implant injection of S. epidermidis or Staphylococcus aureus (S. aureus) with adjunct injections of aqueous sodium morrhuate. In group 3, sterile saline was injected instead of bacteria and no aqueous sodium morrhuate was used. At 2 weeks after operation, 68Ga-DOTA-Siglec-9 PET coupled with computed tomography (CT) was performed with the measurement of the standardized uptake value (SUV). The presence of the implant-related infection was verified by microbiological analysis, imaging with fluorescence microscope, and histology. The in vivo PET results were verified by ex vivo measurements by gamma counter.
In group 3, the tibias with implanted sterile catheters showed an increased local uptake of 68Ga-DOTA-Siglec-9 compared with the intact contralateral bones (SUVratio +29.5%). 68Ga-DOTA-Siglec-9 PET detected inflammation induced by S. epidermidis and S. aureus catheter-related bone infections (SUVratio +58.1% and +41.7%, respectively). The tracer uptake was significantly higher in the S. epidermidis group than in group 3 without bacterial inoculation, but the difference between S. epidermidis and S. aureus groups was not statistically significant. The difference between the S. aureus group and group 3 was neither statistically significant.
PET/CT imaging with novel 68Ga-DOTA-Siglec-9 tracer was able to detect inflammatory tissue response induced by catheter implantation and staphylococcal infections.
PMCID: PMC4265888  PMID: 25520903
68Ga-DOTA-Siglec-9; Implant infection; Osteomyelitis; PET; S. aureus; S. epidermidis; VAP-1
22.  High-throughput flow injection analysis of labeled peptides in cellular samples – ICP-MS analysis versus fluorescence based detection 
A high throughput method based on flow injection analysis was developed and validated for the quantification of the peptide Bβ15-42 in cellular samples comparing different labeling strategies and detection methods. The used labels were 1,4,7,10-tetraazacyclododecane-N, N′, N′′, N′′′-tetraaceticacid (In-DOTA) and 2-(4-isothiocyanatobenzyl) - 1,4,7,10-tetraazacyclododecane-N, N′, N′′, N′′′-tetraacetic acid (In-DOTA-Bn) for elemental labeling. 6-Hydroxy-9-(2-carboxyphenyl)- (3H)-xanthen-3-on (fluorescein) was employed as fluorescence label. The explored peptide (mass = 3 kD) is a novel candidate drug, which shows an anti-inflammatory effect after an event of myocardial infarction. The analysed samples were fractioned cell compartments of human umbilical cord vein endothelial cells (HUVEC) maintained via lysis with Triton X buffer. In order to enhance sensitivity and selectivity of peptide quantification via flow injection the peptide was labeled prior to incubation using elemental and fluorescence labels. Quantification of the elemental and fluorescence labeled peptide was performed via flow injection analysis combined with inductive coupled plasma sector field mass spectrometry (FIA-ICP-SFMS) or fluorescence detection (FIA-FLD), respectively. The employed quantification strategies were external calibration in the case of fluorescence detection and external calibration with and without internal standardization and on-line IDMS in the case of ICP-MS detection
The limit of detection (LOD) for FIA-ICP-MS was 9 pM In-DOTA-Bβ15-42 (0.05 fmol absolute) whereas FIA-FLD showed a LOD of 100 pM (3 fmol absolute) for the fluorescein labeled peptide. Short term precision of FIA-ICP-MS was superior for all ICP-MS based quantification strategies compared to FIA-FLD (FIA-ICP-SFMS: 0.3-3.3%; FIA-FLD: 6.5%). Concerning long term precision FIA-ICP-SFMS with on-line IDMS and internal standardization showed the best results (3.1 and 4.6%, respectively) whereas the external calibration of both applied methodological approaches was only in the range of 10 %.
The concentrations in the Triton X soluble fraction relative to the applied amount of Indium in the cell culture were in the range of 0.75-1.8% for In-DOTA or 0.30-0.79% for the 2-(4-isothiocyanatobenzyl) - 1,4,7,10-tetraazacyclododecane-N, N′, N′′, N′′′-tetraacetic acid (In-DOTA-Bn) labeled peptide Bβ15-42. In the Triton X insoluble fraction the relative concentrations of Indium were 0.03-0.18% for the In-DOTA labeled peptide and 0.03-0.13% for Bβ15-42-In-DOTA-Bn.
PMCID: PMC3378036  PMID: 22723737
ICP-MS; quantification; peptide; labeling; fluorescence
23.  Imaging carotid body chemodectomas with 68Ga-DOTA-NOC PET-CT 
The British Journal of Radiology  2012;85(1016):1140-1145.
The purpose of the present study was to evaluate the role of 68-gallium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-1-Nal3-octreotide (68Ga-DOTA-NOC) positron emission tomography CT (PET-CT) in carotid body chemodectomas (CBCs).
: 5 patients with known CBCs (conventional imaging and angiography) were evaluated retrospectively (3 males, 2 females; mean age 36.6 years). All underwent PET-CT after injection of 148 MBq (4 mCi) of 68Ga-DOTA-NOC.
: At baseline there were four bilateral CBCs and one unilateral CBC with an additional glomus tympanicum and bilateral glomus jugulare tumour in these five patients. All these lesions showed intense uptake on 68Ga-DOTA-NOC PET-CT. Apart from these known lesions, additional tumours were detected in three patients; glomus jugulare in one, aortic body tumour and multiple abdominal paragangliomas in another and unsuspected vertebral metastasis in the last. Overall it led to a change in management in 3 of the 5 patients (60%).
: 68Ga-DOTA-NOC PET-CT is a promising non-invasive imaging modality in the detection and characterisation of CBCs.
PMCID: PMC3587100  PMID: 22096221
24.  Reduction of the Ring Size of Radiolabeled Lactam Bridge-Cyclized Alpha-MSH Peptide Resulting in Enhanced Melanoma Uptake 
The purpose of this study was to examine the profound effect of the ring size of the radiolabeled lactam bridge-cyclized α-melanocyte stimulating hormone (α-MSH) peptide on its melanoma targeting properties.
A novel cyclic α-MSH peptide, DOTA-Nle-CycMSHhex {1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-CONH2}, was synthesized and radiolabeled with 111In. The melanocortin-1 (MC1) receptor binding affinity of DOTA-Nle-CycMSHhex was determined in B16/F1 melanoma cells. The internalization and efflux of 111In-DOTA-Nle-CycMSHhex were examined in B16/F1 cells. The melanoma targeting properties and single photon emission computed tomography (SPECT)/CT imaging of 111In-DOTA-Nle-CycMSHhex were determined in B16/F1 melanoma-bearing C57 mice.
DOTA-Nle-CycMSHhex displayed 1.77 nM receptor binding affinity. 111In-DOTA-Nle-CycMSHhex exhibited rapid internalization and extended retention in B16/F1 cells. The tumor uptake of 111In-DOTA-Nle-CycMSHhex was 24.94 ± 4.58 and 10.53 ± 1.11% injected dose/gram (%ID/g) at 0.5 and 24 h post-injection, respectively. Greater than 82% of the injected radioactivity was cleared through the urinary system by 2 h post-injection. The tumor/kidney uptake ratios reached 2.04 and 1.70 at 2 and 4 h post-injection, respectively. Flank melanoma tumors were clearly visualized by SPECT/CT using 111In-DOTA-Nle-CycMSHhex as an imaging probe at 2 and 24 h post-injection. The radioactivity accumulation in normal organs was low except for the kidneys at 2, 4 and 24 h post-injection.
The reduction of the peptide ring size dramatically increased the melanoma uptake and decreased the renal uptake of 111In-DOTA-Nle-CycMSHhex, providing a new insight into the design of novel radiolabeled lactam bridge-cyclized α-MSH peptide for melanoma imaging and treatment.
PMCID: PMC2998183  PMID: 20150256
Melanoma imaging; radiolabeled cyclic peptide; alpha-melanocyte stimulating hormone; small animal imaging
25.  Characterization and Evaluation of 64Cu-Labeled A20FMDV2 Conjugates for Imaging the Integrin αvβ6 
The integrin αvβ6 is overexpressed in a variety of aggressive cancers and serves as a prognosis marker. This study describes the conjugation, radiolabeling, and in vitro and in vivo evaluation of four chelators to determine the best candidate for 64Cu radiolabeling of A20FMDV2, an αvβ6 targeting peptide.
Four chelators were conjugated onto PEG28-A20FMDV2 (1): 11-carboxymethyl-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4-methanephosphonic acid (CB-TE1A1P), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and 4,4′-((3,6,10,13,16,19-hexazazbicyclo[6.6.6]ico-sane-1,8-diylbis(aza-nediyl)) bis(methylene)dibenzoic acid (BaBaSar). All peptides were radiolabeled with 64Cu in ammonium acetate buffer at pH 6 and formulated to pH 7.2 in PBS for use. The radiotracers were evaluated using in vitro cell binding and internalization assays and serum stability assays. In vivo studies conducted include blocking, biodistribution, and small animal PET imaging. Autoradiography and histology were also conducted.
All radiotracers were radiolabeled in good radiochemical purity (>95 %) under mild conditions (37–50 °C for 15 min) with high specific activity (0.58–0.60 Ci/µmol). All radiotracers demonstrated αvβ6-directed cell binding (>46 %) with similar internalization levels (>23 %). The radiotracers 64Cu-CB-TE1A1P-1 and 64Cu-BaBaSar-1 showed improved specificity for the αvβ6 positive tumor in vivo over 64Cu-DOTA-1 and 64Cu-NOTA-1 (+/− tumor uptake ratios—3.82 +/−0.44, 3.82±0.41, 2.58±0.58, and 1.29±0.14, respectively). Of the four radiotracers, 64Cu-NOTA-1 exhibited the highest liver uptake (10.83±0.1 % ID/g at 4 h).
We have successfully conjugated, radiolabeled, and assessed the four chelates CB-TE1A1P, DOTA, NOTA, and BaBaSar both in vitro and in vivo. However, the data suggests no clear “best candidate” for the 64Cu-radiolabeling of A20FMDV2, but instead a trade-off between the different properties (e.g., stability, selectivity, pharmacokinetics, etc.) with no obvious effects of the individual chelators.
PMCID: PMC4277820  PMID: 24448825
Integrin αvβ6; Copper-64; PET imaging

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