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1.  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
2.  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
3.  Comparative gallium-68 labeling of TRAP-, NOTA-, and DOTA-peptides: practical consequences for the future of gallium-68-PET 
EJNMMI Research  2012;2:28.
Currently, 68Ga-labeled 1,4,7,10-tetraazacyclododecane-tetraacetic acid (DOTA)-peptides are the most widely used class of 68Ga radiotracers for PET, although DOTA is not optimal for 68Ga complexation. More recently, 1,4,7-triazacyclononane-triacetic acid (NOTA) and particularly triazacyclononane-phosphinate (TRAP) chelators have been shown to possess superior 68Ga binding ability. Here, we report on the efficiency, reproducibility, and achievable specific activity for fully automated 68Ga labeling of DOTA-, NOTA-, and TRAP-peptide conjugates.
Compared to NOTA- and DOTA-peptides, achievable specific activity (AS) for TRAP-peptide is approximately 10 and 20 times higher, respectively. AS values in the range of 5,000 GBq/μmol were routinely obtained using 1 GBq of 68Ga, equivalent to 0.11 μg of cold mass for a 185-MBq patient dose of a 3-kDa conjugate. The TRAP-peptide could be 68Ga-labeled with excellent reproducibility and > 95% radiochemical yield for precursor amounts as low as 1 nmol.
High 68Ga labeling efficiency of TRAP-peptides could facilitate realization of kit labeling procedures. The good reproducibility of the automated synthesis is of relevance for GMP production, and the possibility to provide very high specific activities offers a high degree of safety in first clinical trials, due to reduction of cold mass content in tracer formulations.
PMCID: PMC3538506  PMID: 22682112
macrocyclic ligands; gallium-68; positron-emission tomography; peptides; bioconjugates; radiolabeling
4.  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
5.  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
6.  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
7.  Synthesis and radiolabeling of chelator-RNA aptamer bioconjugates with copper-64 for targeted molecular imaging 
Bioorganic & medicinal chemistry  2011;19(13):4080-4090.
Ribonucleic acid (RNA) aptamers with high affinity and specificity for cancer-specific cell-surface antigens are promising reagents for targeted molecular imaging of cancer using positron emission tomography (PET). For this application, aptamers must be conjugated to chelators capable of coordinating PET-radionuclides (e.g. copper-64, 64Cu) to enable radiolabeling for in vivo imaging of tumors. This study investigates the choice of chelator and radiolabeling parameters such as pH and temperature for the development of 64Cu-labeled RNA-based targeted agents for PET imaging. The characterization and optimization of labeling conditions are described for four chelator-aptamer complexes. Three commercially available bifunctional macrocyclic chelators (1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid mono N-hydroxysuccinimide [DOTA-NHS]; S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid [p-SCN-Bn-NOTA]; and p-SCN-Bn-3,6,9,15-tetraazabicyclo [9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid [p-SCN-Bn-PCTA]), as well as the polyamino-macrocyclic diAmSar (3,6,10,13,16,19-hexaazabicyclo[6.6.6] icosane-1,8-diamine) were conjugated to A10–3.2, a RNA aptamer which has been shown to bind specifically to a prostate cancer-specific cell-surface antigen (PSMA). Although a commercial bifunctional version of diAmSar was not available, RNA conjugation with this chelator was achieved in a two-step reaction by the addition of a disuccinimidyl suberate linker. Radiolabeling parameters (e.g. pH, temperature, and time) for each chelator-RNA conjugate were assessed in order to optimize specific activity and RNA stability. Furthermore, the radiolabeled chelator-coupled RNA aptamers were evaluated for binding specificity to their target antigen. In summary, key parameters were established for optimal radiolabeling of RNA aptamers for eventual PET imaging with 64Cu.
PMCID: PMC4083585  PMID: 21658962
8.  Synthesis of Novel Neutrophil-Specific Imaging Agents for Positron Emission Tomography (PET) Imaging 
A neutrophil specific peptide, cinnamoyl-F(D)LF(D)LFK (cFLFLFK), was conjugated consecutively with a polyethylene glycol moiety (3.4 K) and 2,2′,2″,2‴-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) to form cFLFLFK-PEG-DOTA. After 64Cu labeling, Positron Emission Tomography (PET) imaging was successfully able to detect mouse lung inflammation.
PMCID: PMC2612577  PMID: 17959381
Neutrophil antagonist; Peptide tracer; Inflammation imaging; 64Cu
9.  Pharmacokinetics and Biodistribution of a Human Monoclonal Antibody to Oxidized LDL in Cynomolgus Monkey Using PET Imaging 
PLoS ONE  2012;7(9):e45116.
Oxidized low-density lipoprotein (LDL) plays an essential role in the pathogenesis of atherosclerosis. The purpose of this study was to characterize the pharmacokinetics (PK) of a human recombinant IgG1 antibody to oxidized LDL (anti-oxLDL) in cynomolgus monkey. The tissue biodistribution of anti-oxLDL was also investigated using positron emission tomography (PET) imaging.
Anti-oxLDL was conjugated with the N-hydroxysuccinimide ester of DOTA (1,4,7,10-tetraazacyclododecane 1,4,7,10-tetraacetic acid) and radiolabeled by chelation of radioactive copper-64 (64Cu) for detection by PET. Anti-oxLDL was administered as a single intravenous (IV) dose of 10 mg/kg (as a mixture of radiolabeled and non-labeled material) to two male and two female cynomolgus monkeys. Serum samples were collected over 29 days. Two ELISA methods were used to measure serum concentrations of anti-oxLDL; Assay A was a ligand binding assay that measured free anti-oxLDL (unbound and partially bound forms) and Assay B measured total anti-oxLDL. The biodistribution was observed over a 48-hour period following dose administration using PET imaging.
Anti-oxLDL serum concentration-time profiles showed a biphasic elimination pattern that could be best described by a two-compartment elimination model. The serum concentrations obtained using the two ELISA methods were comparable. Clearance values ranged from 8 to 17 ml/day/kg, while beta half-life ranged from 8 to12 days. The initial volume of distribution and volume of distribution at steady state were approximately 55 mL/kg and 150 mL/kg, respectively. PET imaging showed distribution predominantly to the blood pool, visible as the heart and great vessels in the trunk and limbs, plus diffuse signals in the liver, kidney, spleen, and bone marrow.
The clearance of anti-oxLDL is slightly higher than typical IgG1 antibodies in cynomolgus monkeys. The biodistribution pattern appears to be consistent with an antibody that has no large, rapid antigen sink outside the blood space.
PMCID: PMC3444451  PMID: 23028793
10.  In vivo evaluation of 64Cu-labeled Magnetic Nanoparticles as a Dual-Modality PET/MR Imaging Agent 
Bioconjugate chemistry  2010;21(4):715-722.
A novel nanoparticle-based dual-modality positron emission tomograph/magnetic resonance imaging (PET/MRI) contrast agent was developed. The probe consisted of a superparamagnetic iron oxide (SPIO) core coated with PEGylated phospholipids. The chelator 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to PEG termini to allow labeling with positron-emitting 64Cu. Radiolabeling with 64Cu at high yield and high purity was readily achieved. The 64Cu-SPIO probes produced strong MR and PET signals, and were stable in mouse serum for 24 hr at 37 °C. Biodistribution and in vivo PET/CT imaging studies of the probes showed a circulation half-life of 143 min, and high initial blood retention with moderate liver uptake, making them an attractive contrast agent for disease studies.
PMCID: PMC2865436  PMID: 20353170
11.  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
12.  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
13.  PET imaging of CXCR4 using copper-64 labeled peptide antagonist 
Theranostics  2011;1:251-262.
Expression of CXCR4 in cancer has been found to correlate with poor prognosis and resistance to chemotherapy. In this study we developed a derivative of the CXCR4 peptide antagonist, T140-2D, that can be labeled easily with the PET isotope copper-64, and thereby enable in vivo visualization of CXCR4 in tumors. T140 was conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono (N-hydroxysuccinimide ester) (DOTA-NHS) to give T140-2D, which contains a DOTA molecule on each of the two lysine residues. 64Cu-T140-2D was evaluated in vitro by migration and binding experiments, and in vivo by microPET imaging and biodistribution, in mice bearing CXCR4-positive and CXCR4-negative tumor xenografts. T140-2D was labeled with copper-64 to give 64Cu-T140-2D in a high radiochemical yield of 86 ± 3% (not decay-corrected) and a specific activity of 0.28 - 0.30 mCi/μg (10.36 – 11.1 MBq/μg). 64Cu-T140-2D had antagonistic and binding characteristics to CXCR4 that were similar to those of T140. In vivo, 64Cu-T140-2D tended to bind to red blood cells and had to be used in a low specific activity form. In this new form 64Cu-T140-2D enabled specific imaging of CXCR4-positive, but not CXCR4-negative tumors. Undesirably, however, 64Cu-T140-2D also displayed high accumulation in the liver and kidneys. In conclusion, 64Cu-T140-2D was easily labeled and, in its low activity form, enabled imaging of CXCR4 in tumors. It had high uptake, however, in metabolic organs. Further research with imaging tracers targeting CXCR4 is required.
PMCID: PMC3085282  PMID: 21544263
T140 peptide; CXCR4 imaging; PET; copper-64
14.  PET imaging of CXCR4 using copper-64 labeled peptide antagonist 
Theranostics  2011;1:251-262.
Expression of CXCR4 in cancer has been found to correlate with poor prognosis and resistance to chemotherapy. In this study we developed a derivative of the CXCR4 peptide antagonist, T140-2D, that can be labeled easily with the PET isotope copper-64, and thereby enable in vivo visualization of CXCR4 in tumors. T140 was conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono (N-hydroxysuccinimide ester) (DOTA-NHS) to give T140-2D, which contains a DOTA molecule on each of the two lysine residues. 64Cu-T140-2D was evaluated in vitro by migration and binding experiments, and in vivo by microPET imaging and biodistribution, in mice bearing CXCR4-positive and CXCR4-negative tumor xenografts. T140-2D was labeled with copper-64 to give 64Cu-T140-2D in a high radiochemical yield of 86 ± 3% (not decay-corrected) and a specific activity of 0.28 - 0.30 mCi/µg (10.36 - 11.1 MBq/µg). 64Cu-T140-2D had antagonistic and binding characteristics to CXCR4 that were similar to those of T140. In vivo, 64Cu-T140-2D tended to bind to red blood cells and had to be used in a low specific activity form. In this new form 64Cu-T140-2D enabled specific imaging of CXCR4-positive, but not CXCR4-negative tumors. Undesirably, however, 64Cu-T140-2D also displayed high accumulation in the liver and kidneys. In conclusion, 64Cu-T140-2D was easily labeled and, in its low activity form, enabled imaging of CXCR4 in tumors. It had high uptake, however, in metabolic organs. Further research with imaging tracers targeting CXCR4 is required.
PMCID: PMC3085282  PMID: 21544263
T140 peptide; CXCR4 imaging; PET; copper-64
15.  Non-Invasive Imaging of Cysteine Cathepsin Activity in Solid Tumors Using a 64Cu-Labeled Activity-Based Probe 
PLoS ONE  2011;6(11):e28029.
The papain family of cysteine cathepsins are actively involved in multiple stages of tumorigenesis. Because elevated cathepsin activity can be found in many types of human cancers, they are promising biomarkers that can be used to target radiological contrast agents for tumor detection. However, currently there are no radiological imaging agents available for these important molecular targets. We report here the development of positron emission tomography (PET) radionuclide-labeled probes that target the cysteine cathepsins by formation of an enzyme activity-dependent bond with the active site cysteine. These probes contain an acyloxymethyl ketone (AOMK) functional group that irreversibly labels the active site cysteine of papain family proteases attached to a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) tag for labeling with 64Cu for PET imaging studies. We performed biodistribution and microPET imaging studies in nude mice bearing subcutaneous tumors expressing various levels of cysteine cathepsin activity and found that the extent of probe uptake by tumors correlated with overall protease activity as measured by biochemical methods. Furthermore, probe signals could be reduced by pre-treatment with a general cathepsin inhibitor. We also found that inclusion of a Cy5 tag on the probe increased tumor uptake relative to probes lacking this fluorogenic dye. Overall, these results demonstrate that small molecule activity-based probes carrying radio-tracers can be used to image protease activity in living subjects.
PMCID: PMC3221694  PMID: 22132198
16.  Positron Emission Tomography Imaging of CD105 Expression with a 64Cu-Labeled Monoclonal Antibody: NOTA Is Superior to DOTA 
PLoS ONE  2011;6(12):e28005.
Optimizing the in vivo stability of positron emission tomography (PET) tracers is of critical importance to cancer diagnosis. In the case of 64Cu-labeled monoclonal antibodies (mAb), in vivo behavior and biodistribution is critically dependent on the performance of the bifunctional chelator used to conjugate the mAb to the radiolabel. This study compared the in vivo characteristics of 64Cu-labeled TRC105 (a chimeric mAb that binds to both human and murine CD105), through two commonly used chelators: 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Flow cytometry analysis confirmed that chelator conjugation of TRC105 did not affect its CD105 binding affinity or specificity. PET imaging and biodistribution studies in 4T1 murine breast tumor-bearing mice revealed that 64Cu-NOTA-TRC105 exhibited better stability than 64Cu-DOTA-TRC105 in vivo, which resulted in significantly lower liver uptake without compromising the tumor targeting efficiency. In conclusion, this study confirmed that NOTA is a superior chelator to DOTA for PET imaging with 64Cu-labeled TRC105.
PMCID: PMC3235104  PMID: 22174762
17.  Peptoid-based PET imaging of vascular endothelial growth factor receptor (VEGFR) expression 
Non-invasive detection of vascular endothelial growth factor receptor 2 (VEGFR2) by positron emission tomography (PET) would allow the evaluation of tumor vascular activity in vivo. Recently, a dimeric peptoid, GU40C4, was reported as a highly potent antagonist of VEGFR2 activation inhibiting angiogenesis and tumor growth in vivo. The purpose of this work was to evaluate the potential of this peptoid for PET imaging of VEGFR2 expression. To label GU40C4 and a control peptoid with a positron emitter, 64Cu (t1/2 = 12.7 h; β+: 0.653 MeV, 17.4%), a cysteine was introduced to the C-terminus of the peptoids and then conjugated to a bifunctional chelator (DOTA: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) through the maleimide-thiol coupling chemistry. The in vitro binding assay showed a negligible effect of the DOTA conjugation on the VEGFR2 binding affinity of GU40C4. Both peptoid conjugates were efficiently labeled with 64Cu in high radiochemical yields (> 90%); the specific activity was in the range of 10 – 80 GBq/μmol. PET imaging evaluation using a prostate cancer xenograft (PC3) mouse model showed that 64Cu-DOTA-GU40C4 had a prominent and steady accumulation in the VEGFR2 positive PC3 tumors (2.25 ± 0.24, 2.15 ± 0.34, and 1.90 ± 0.18 %ID/g at 1, 4, and 20 h p.i., respectively; n = 3), which is significantly higher than the control peptoid conjugate (0.3 – 0.5 %ID/g; p < 0.001 at 1, 4, and 20 h p.i.). Interestingly, the mouse salivary glands were also clearly visualized by 64Cu-DOTA-GU40C4 (3.17 ± 0.25, 3.00 ± 0.36, and 1.83 ± 0.21 %ID/g at 1, 4, and 20 h p.i., respectively; n = 3) rather than its control peptoid conjugate. VEGFR2 expression in the salivary glands was shown by polymerase chain reaction (PCR) assay. Our results demonstrate that 64Cu-DOTA-GU40C4 can be used to image the expression of VEGFR2 in vivo.
PMCID: PMC3477717  PMID: 23133797
VEGFR2; peptoid; PET; 64Cu; prostate cancer; tumor angiogenesis
18.  A novel Indium-111-labeled gonadotropin-releasing hormone peptide for human prostate cancer imaging 
The purpose of this study was to evaluate the tumor targeting and imaging properties of a novel 111In-labeled gonadotropin-releasing hormone (GnRH) peptide {1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-Ahx-(d-Lys6-GnRH1)} for human prostate cancer. The biodistribution and tumor imaging properties of 111In-DOTA-Ahx-(d-Lys6-GnRH1) were determined in DU145 human prostate cancer-xenografted nude mice. 111In-DOTA-Ahx-(d-Lys6-GnRH1) exhibited rapid tumor uptake (1.27 ± 0.40 %ID/g at 0.5 h post-injection) coupled with fast whole-body clearance through the urinary system. The DU145 human prostate cancer-xenografted tumor lesions were clearly visualized by single photon emission computed tomography (SPECT)/CT at 0.5 h post-injection of 111In-DOTA-Ahx-(d-Lys6-GnRH1). The successful imaging of DU145 human prostate cancer-xenografted tumor lesions using 111In-DOTA-Ahx-(d-Lys6-GnRH1) highlighted its potential as a novel imaging probe for human prostate cancer imaging.
PMCID: PMC3159814  PMID: 21821417
Gonadotropin-releasing hormone receptor; receptor-targeting peptide; prostate cancer; imaging
19.  Pilot study of 68Ga-DOTA-F(ab′)2-trastuzumab in patients with breast cancer 
Nuclear Medicine Communications  2013;34(12):1157-1165.
68Ga-1,4,7,10-Tetraazacyclododecane-N,N′,N′′,N′′′-tetraacetic acid (DOTA)-F(ab′)2-trastuzumab [68Ga-DOTA-F(ab′)2-trastuzumab] has been developed at our institution as a positron imaging reagent for assessing human epidermal growth factor receptor 2 (HER2) expression status by in-vivo imaging. Initial studies on animals demonstrated promising results in the monitoring of treatment response to heat shock protein 90-targeted drugs that inhibit the client protein HER2. We report here our initial clinical experience in the assessment of the toxicity, pharmacokinetics, biodistribution, and dosimetry profile of 68Ga-DOTA-F(ab′)2-trastuzumab with PET/computed tomography using a mean of 236 MBq/5 mg administered intravenously.
Materials and methods
A group of 16 women with breast cancer were enrolled in this study. The one patient who did not receive 68Ga-DOTA-F(ab′)2-trastuzumab was excluded from analysis. Both HER2-negative (n=7) and HER2-positive (n=8) cases were studied. Among the latter, seven had undergone trastuzumab treatment previously and one had not.
It was determined that 68Ga-DOTA-F(ab′)2-trastuzumab was well tolerated, with a T½ of ∼3.6±0.9 h; the critical organ was the kidney, with a mean dose of 0.383 cGy/37 MBq; and tumor targeting was seen in 4/8 patients with HER2-positive disease.
The reagent is safe, and assessments through additional studies in a better-defined group of patients, using larger administered masses of antibodies, with a better immunoreactive fraction are needed.
PMCID: PMC3815146  PMID: 24100444
breast cancer; F(ab′)2-trastuzumab; 68Ga; Herceptin; PET/CT
20.  Dynamic PET and Optical Imaging and Compartment Modeling using a Dual-labeled Cyclic RGD Peptide Probe 
Theranostics  2012;2(8):746-756.
Purpose: The aim of this study is to determine if dynamic optical imaging could provide comparable kinetic parameters to that of dynamic PET imaging by a near-infrared dye/64Cu dual-labeled cyclic RGD peptide.
Methods: The integrin αvβ3 binding RGD peptide was conjugated with a macrocyclic chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for copper labeling and PET imaging and a near-infrared dye ZW-1 for optical imaging. The in vitro biological activity of RGD-C(DOTA)-ZW-1 was characterized by cell staining and receptor binding assay. Sixty-min dynamic PET and optical imaging were acquired on a MDA-MB-435 tumor model. Singular value decomposition (SVD) method was applied to compute the dynamic optical signal from the two-dimensional optical projection images. Compartment models were used to quantitatively analyze and compare the dynamic optical and PET data.
Results: The dual-labeled probe 64Cu-RGD-C(DOTA)-ZW-1 showed integrin specific binding in vitro and in vivo. The binding potential (Bp) derived from dynamic optical imaging (1.762 ± 0.020) is comparable to that from dynamic PET (1.752 ± 0.026).
Conclusion: The signal un-mixing process using SVD improved the accuracy of kinetic modeling of 2D dynamic optical data. Our results demonstrate that 2D dynamic optical imaging with SVD analysis could achieve comparable quantitative results as dynamic PET imaging in preclinical xenograft models.
PMCID: PMC3425122  PMID: 22916074
dual modality imaging; dynamic imaging; kinetic modeling; Singular value decomposition (SVD); integrin αvβ3; RGD peptide.
21.  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
22.  Rapid intracerebroventricular delivery of Cu-DOTA-etanercept after peripheral administration demonstrated by PET imaging 
BMC Research Notes  2009;2:28.
The cytokines interleukin-1 and tumor necrosis factor (TNF), and the cytokine blocker interleukin-1 receptor antagonist, all have been demonstrated to enter the cerebrospinal fluid (CSF) following peripheral administration. Recent reports of rapid clinical improvement in patients with Alzheimer's disease and related forms of dementia following perispinal administration of etanercept, a TNF antagonist, suggest that etanercept also has the ability to reach the brain CSF. To investigate, etanercept was labeled with a positron emitter to enable visualization of its intracranial distribution following peripheral administration by PET in an animal model.
Radiolabeling of etanercept with the PET emitter 64Cu was performed by DOTA (1,4,7,10-tetraazadodecane-N,N',N",N"'-tetraacetic acid) conjugation of etanercept, followed by column purification and 64Cu labeling. MicroPET imaging revealed accumulation of 64Cu-DOTA-etanercept within the lateral and third cerebral ventricles within minutes of peripheral perispinal administration in a normal rat anesthesized with isoflurane anesthesia, with concentration within the choroid plexus and into the CSF.
Synthesis of 64Cu-DOTA-etanercept enabled visualization of its intracranial distribution by microPET imaging. MicroPET imaging documented rapid accumulation of 64Cu-DOTA-etanercept within the choroid plexus and the cerebrospinal fluid within the cerebral ventricles of a living rat after peripheral administration. Further study of the effects of etanercept and TNF at the level of the choroid plexus may yield valuable insights into the pathogenesis of Alzheimer's disease.
PMCID: PMC2651903  PMID: 19284700
23.  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
24.  Early Tumor Response to Hsp90 Therapy Using HER2 PET: Comparison with 18F-FDG PET 
We compared 68Ga-DOTA-F(ab′)2-herceptin (DOTA is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid [HER2 PET]) and 18F-FDG PET for imaging of tumor response to the heat shock protein 90 (Hsp90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17AAG).
Mice bearing BT474 breast tumor xenografts were scanned with 18F-FDG PET and HER2 PET before and after 17AAG treatment and then biweekly for up to 3 wk.
Within 24 h after treatment, a significant decrease in HER2 was measured by HER2 PET, whereas 18F-FDG PET uptake, a measure of glycolysis, was unchanged. Marked growth inhibition occurred in treated tumors but became evident only by 11 d after treatment. Thus, Her2 downregulation occurs independently of changes in glycolysis after 17AAG therapy, and Her2 reduction more accurately predicts subsequent tumor growth inhibition.
HER2 PET is an earlier predictor of tumor response to 17AAG therapy than 18F-FDG PET.
PMCID: PMC3193602  PMID: 16644749
25.  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

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