Silvia S. Jurisson

Radiometals for Combined Imaging and Therapy

Cathy S. Cutler,*,† Heather M. Hennkens,† Nebiat Sisay,†,‡ Sandrine Huclier-Markai, and Silvia S. Jurisson‡ †University of Missouri Research Reactor Center, Columbia, Missouri 65211, United States ‡Department of Chemistry, University of Missouri, Columbia, Missouri 65211, United States Laboratoire Subatech, UMR 6457, Ecole des Mines de Nantes/Universite de Nantes/CNRS-IN2P3, 4 Rue A. Kastler, BP 20722, F-44307 Nantes Cedex 3, France

In Vivo Evaluation and Small-Animal PET/CT of a Prostate Cancer Mouse Model Using 64Cu Bombesin Analogs: Side-by-Side Comparison of the CB-TE2A and DOTA Chelation Systems

The BB2 receptor subtype, of the bombesin family of receptors, has been shown to be highly overexpressed in a variety of human tumors, including prostate cancer. Bombesin (BBN), a 14-amino acid peptide, has been shown to target the BB2 receptor with high affinity. 64Cu (half-life = 12.7 h, β+: 18%, Eβ+max = 653 keV; β−: 37%, Eβ−max = 578 keV) is a radioisotope that has clinical potential for application in both diagnostic imaging and radionuclide therapy. Recently, new chelation systems such as 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A) have been reported to significantly stabilize the 64Cu radiometal in vivo. The increased stability of the 64Cu-CB-TE2A chelate complex has been shown to significantly reduce nontarget retention compared with tetraazamacrocycles such as 1,4,7,10-tetraazacyclodoadecane-N,N′,N″,N‴-tetraacetic acid (DOTA). The aim of this study was to determine whether the CB-TE2A chelation system could significantly improve the in vivo stability of 64Cu bombesin analogs. The study directly compares 64Cu bombesin analogs using the CB-TE2A and DOTA chelation systems in a prostate cancer xenograft SCID (severely compromised immunodeficient) mouse model. Methods: The CB-TE2A-8-AOC-BBN(7–14)NH2 and DOTA-8-AOC-BBN(7–14)NH2 conjugates were synthesized and radiolabeled with 64Cu. The receptor-binding affinity and internalization profile of each metallated conjugate was evaluated using PC-3 cells. Pharmacokinetic and small-animal PET/CT studies were performed using female SCID mice bearing PC-3 xenografts. Results: In vivo BB2 receptor targeting was confirmed by tumor uptake values of 6.95 ± 2.27 and 4.95 ± 0.91 %ID/g (percentage injected dose per gram) at the 15-min time point for the 64Cu-CB-TE2A and 64Cu-DOTA radioconjugates, respectively. At the 24-h time point, liver uptake was substantially reduced for the 64Cu-CB-TE2A radioconjugate (0.21 ± 0.06 %ID/g) compared with the 64Cu-DOTA radioconjugate (7.80 ± 1.51 %ID/g). The 64Cu-CB-TE2A-8-AOC-BBN(7–14)NH2 radioconjugate demonstrated significant clearance, 98.60 ± 0.28 %ID, from the mouse at 24 h after injection. In contrast, only 67.84 ± 5.43 %ID of the 64Cu activity was excreted using the 64Cu-DOTA-8-AOC-BBN(7–14)NH2 radioconjugate because of nontarget retention. Conclusion: The pharmacokinetic and small-animal PET/CT studies demonstrate significantly improved nontarget tissue clearance for the 64Cu-CB-TE2A8-AOC-BBN(7–14)NH2. This is attributed to the improved in vivo stability of the 64Cu-CB-TE2A chelate complex as compared with the 64Cu-DOTA chelate complex.

Radioactive gold nanoparticles in cancer therapy: therapeutic efficacy studies of GA-198AuNP nanoconstruct in prostate tumor–bearing mice

UNLABELLED Biocompatibility studies and cancer therapeutic applications of nanoparticulate beta-emitting gold-198 (198Au; beta(max) = 0.96 MeV; half-life of 2.7 days) are described. Gum arabic glycoprotein (GA)-functionalized gold nanoparticles (AuNPs) possess optimum sizes (12-18 nm core diameter and 85 nm hydrodynamic diameter) to target individual tumor cells and penetrate through tumor vasculature and pores. We report the results of detailed in vivo therapeutic investigations demonstrating the high tumor affinity of GA-198AuNPs in severely compromised immunodeficient (SCID) mice bearing human prostate tumor xenografts. Intratumoral administration of a single dose of beta-emitting GA-198AuNPs (70 Gy) resulted in clinically significant tumor regression and effective control in the growth of prostate tumors over 30 days. Three weeks after administration of GA-198AuNPs, tumor volumes for the treated animals were 82% smaller as compared with tumor volume of control group. The treatment group showed only transitory weight loss in sharp contrast to the tumor-bearing control group, which underwent substantial weight loss. Pharmacokinetic studies have provided unequivocal evidence for the optimum retention of therapeutic payload of GA-198AuNPs within the tumor site throughout the treatment regimen with minimal or no leakage of radioactivity to various nontarget organs. The measurements of white and red blood cells, platelets, and lymphocytes within the treatment group resembled those of the normal SCID mice, thus providing further evidence on the therapeutic efficacy and concomitant in vivo tolerance and nontoxic features of GA-198AuNPs. FROM THE CLINICAL EDITOR In this study, the biocompatibility and cancer therapeutic applications of glycoprotein (GA) functionalized gold nanoparticles containing b-emitting Au-198 are described in SCID mice bearing human prostate tumor xenografts. The findings of significant therapeutic efficacy, good in vivo tolerance and non-toxic features make these particles ideal candidates for future human applications.

Development of an in vitro model for assessing the in vivo stability of lanthanide chelates

An in vitro model was developed to evaluate the in vivo stability of lanthanide polyaminocarboxylate complexes. The ligand-to-metal ratios for the chelates EDTA, CDTA, DTPA, MA-DTPA (monoamide-DTPA) and DOTA with the lanthanides lanthanum, samarium, and lutetium were optimized to achieve > or = 98% complexation yield for the resultant radiolanthanide complexes. The exchange of the radiolanthanides from their EDTA, CDTA, DTPA, MA-DTPA and DOTA complexes with Ca(2+) was determined by in vitro adsorption and in vitro column studies using hydroxyapatite (HA), an in vitro bone model. In vitro serum stability of these radiolanthanide complexes was used as an additional indicator of in vivo stability, although the mechanism of instability in serum will be different than with bone. The in vitro studies were consistent with the expected findings that the smallest lanthanide (Lu) formed the most stable complexes. In vivo studies were done to validate the in vitro model. Biodistribution studies in normal CF-1 mice showed that in vivo stability of the complex (i.e., the more lanthanide remaining in complex form) could be assessed by a combination of the urinary, bone and liver uptake. For example, biodistribution studies demonstrate that high urinary excretion correlated with complex stability, while high liver plus bone uptake correlated with complex instability. The urinary excretion of the EDTA complexes decreased from (177)Lu to (140)La indicating a loss in stability in the direction of (140)La, consistent with the in vitro studies. The more stable a lanthanide complex is, the lower its exchange with HA in vitro will be, and the lower its combined bone plus liver uptake and higher its urinary excretion will be in vivo. This investigation indicates that the in vivo stability can be determined by a screening method that measures the degree of exchange from the lanthanide chelate with hydroxyapatite (HA) and its serum stability.

Modification of the Structure of a Metallopeptide: Synthesis and Biological Evaluation of 111In-Labeled DOTA-Conjugated Rhenium-Cyclized α-MSH Analogues

Rhenium-cyclized CCMSH analogues are novel melanoma-targeting metallopeptides with high tumor uptake, long tumor retention, and low background in normal tissues, which make these metallopeptides an ideal structural motif for designing novel melanoma-targeting agents. ReCCMSH has been derivatized with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelate so that it can be labeled with a wide variety of radionuclides for imaging and therapeutic applications. This study involved optimization of the in vivo biological properties of DOTA-ReCCMSH (S), through modification of the structure of the metallopeptide. Several DOTA-ReCCMSH analogues, Ac-Lys(DOTA)-ReCCMSH (4) DOTA-ReCCMSH(Arg(11)) (6), DOTA-ReCCMSH-OH (8), and DOTA-ReCCMSH-Asp-OH (10), were synthesized using solid phase peptide synthesis followed by rhenium cyclization. The IC(50) values of the metallopeptides were determined through competitive binding assays against (125)I-(Tyr(2))-NDP. Radiolabeling of the DOTA-rhenium-cyclized peptides with (111)In was carried out in NH(4)OAc (0.1 M; pH 5.5)-buffered solution for 30 min at 70 degrees C. The stability of the radiolabeled complexes was evaluated in 0.01 M, pH 7.4, phosphate-buffered saline/0.1% bovine serum albumin solution. After separation of the radiolabeled peptide from the unlabeled peptide by reverse phase high-performance liquid chromatography, the biodistribution of the radiolabeled complex was performed in C57 mice bearing B16/F1 murine melanoma tumors. All radiolabeled complexes showed fast blood clearance (2 h postinjection (pi): (111)In-S, 0.07 +/- 0.03% ID/g; (111)In-4, 0.09 +/- 0.06% ID/g; (111)In-6, 0.21 +/- 0.08% ID/g; (111)In-8, 0.11 +/- 0.10% ID/g; and (111)In-10, 0.05 +/- 0.03% ID/g), and their clearance was predominantly through the urine (4 h pi: 93.5 +/- 1.7, 87.8 +/- 6.5, 89.8 +/- 4.2, 93.3 +/- 1.1, and 93.8 +/- 1.8 (% ID) for (111)In-labeled S, 4, 6, 8, and 10, respectively). Tumor uptake values of 9.45 +/- 0.90, 6.01 +/- 2.36, 17.41 +/- 5.61, 9.27 +/- 0.68, and 7.32 +/- 2.09 (% ID/g) for (111)In-labeled S, 4, 6, 8, and 10, respectively, were observed at 4 h pi. The kidney uptake was 9.27 +/- 2.65% ID/g for (111)In-S, 19.02 +/- 2.63% ID/g for (111)In-4, 7.37 +/- 1.13% ID/g for (111)In-6, 8.70 +/- 0.88% ID/g for (111)In-8, and 8.13 +/- 1.47% ID/g for (111)In-10 at 4 h pi. Complex 6 showed high melanoma uptake and lower kidney uptake than the corresponding Lys(11) analogues, supporting 6 for further investigations as a potential therapeutic radiopharmaceutical.

Optimization, biological evaluation and microPET imaging of copper-64-labeled bombesin agonists, [64Cu-NO2A-(X)-BBN(7–14)NH2], in a prostate tumor xenografted mouse model☆

UNLABELLED Gastrin-releasing peptide receptors (GRPr) are a member of the bombesin (BBN) receptor family. GRPr are expressed in high numbers on specific human cancers, including human prostate cancer. Therefore, copper-64 ((64)Cu) radiolabeled BBN(7-14)NH(2) conjugates could have potential for diagnosis of human prostate cancer via positron-emission tomography (PET). The aim of this study was to produce [(64)Cu-NO2A-(X)-BBN(7-14)NH(2)] conjugates for prostate cancer imaging, where X=pharmacokinetic modifier (beta-alanine, 5-aminovaleric acid, 6-aminohexanoic acid, 8-aminooctanoic acid, 9-aminonanoic acid or para-aminobenzoic acid) and NO2A=1,4,7-triazacyclononane-1,4-diacetic acid [a derivative of NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid)]. METHODS [(X)-BBN(7-14)NH(2)] Conjugates were synthesized by solid-phase peptide synthesis (SPPS), after which NOTA was added via manual conjugation. The new peptide conjugates were radiolabeled with (64)Cu radionuclide. The receptor-binding affinity was determined in human prostate PC-3 cells, and tumor-targeting efficacy was determined in PC-3 tumor-bearing severely combined immunodeficient (SCID) mice. Whole-body maximum intensity microPET/CT images of PC-3 tumor-bearing SCID mice were obtained 18 h postinjection (pi). RESULTS Competitive binding assays in PC-3 cells indicated high receptor-binding affinity for the [NO2A-(X)-BBN(7-14)NH(2)] and [(nat)Cu-NO2A-(X)-BBN(7-14)NH(2)] conjugates. In vivo biodistribution studies of the [(64)Cu-NO2A-(X)-BBN(7-14)NH(2)] conjugates at 1, 4 and 24 h pi showed very high uptake of the tracer in GRPr-positive tissue with little accumulation and retention in nontarget tissues. High-quality, high-contrast microPET images were obtained, with xenografted tumors being clearly visible at 18 h pi. CONCLUSIONS NO2A chelator sufficiently stabilizes copper(II) radiometal under in vivo conditions, producing conjugates with very high uptake and retention in targeted GRPr. Preclinical evaluation of these new peptide conjugates in tumor-bearing mice provides some impetus for clinical evaluation in human patients.

In vivo evaluation of 99mTc/188Re-labeled linear alpha-melanocyte stimulating hormone analogs for specific melanoma targeting

Radiolabeled alpha-melanocyte stimulating hormone (alpha-MSH) analogs were examined in melanoma-bearing mice to determine the effects of peptide length, structure, and radiometal chelation chemistry on tumor targeting and in vivo biodistribution. The linear alpha-MSH analogs [Nle4,D-Phe7]alpha-MSH (NDPMSH) and [D-Phe7]alpha-MSH(5-10) (DPMSH) were radiolabeled with 99mTc and 188Re via the addition of tetrafluorophenyl mercapto-acetylglycylglycyl-gamma-aminobutyrate (MAG2) or tetrapeptide Ac-Cys-Gly-Cys-Gly (CGCG) chelation moieties. 125I-Tyr2-NDPMSH was obtained by direct iodination of the Tyr2 residue. Tumor uptake of 99mTc-labeled CGCG- and MAG2-NDPMSH analogs at 30 min postinjection were 6.52 +/- 1.11 %ID/g and 4.17 +/- 1.34 %ID/g, respectively, resulting in a significantly higher tumor-to-blood uptake ratio than that of 125I-NDPMSH or a shorter alpha-MSH analog, 99mTc-CGCG-DPMSH. The combination of radiolabeling efficacy and in vivo tumor uptake highlights the potential of 99mTc-CGCG-NDPMSH as a melanoma imaging agent.

Pm-149 DOTA bombesin analogs for potential radiotherapy: in vivo comparison with Sm-153 and Lu-177 labeled DO3A-amide-βAla-BBN(7–14)NH2

Promethium-149 (149Pm) is one of only three radiolanthanides that can be prepared in no carrier added concentrations. This high specific activity radiolanthanide is thus suitable for targeting limited numbers of specific receptors found on many tumor cells. Promethium-149 is a moderate energy beta(-) emitter (1.07 MeV (95.9%)) with a half-life of 2.21 days. Pm-149 also emits a low abundance of an imageable gamma ray (286 keV (3%)) that may allow in vivo tracking of the therapeutic dose. The 149Pm and Sm complexes with the DO3A-amide chelator with zero and three carbon spacers to the bombesin peptide analog BBN(7-14)NH(2) were synthesized and characterized. The Sm complexes were synthesized for macroscopic characterization purposes (ESI-MS, in vitro cell binding) since no stable isotopes of Pm are known. The biological properties of the 149Pm, 153Sm and 177Lu-DO3A-amide-betaAla-BBN complexes were compared in normal mouse biodistribution studies.

Pertechnetate immobilization with amorphous iron sulfide

Abstract The reduction of pertechnetate (TcO4−) with freshly prepared amorphous iron sulfide was investigated. The amorphous iron sulfide (FeS) was shown to have an elemental composition of FeS0.97 for all of the size fractions and a point of zero charge of pHpzc=7.4. Solubility studies of FeS in various buffers indicated that in the pH range 6.1–9.0, the concentrations of dissociated Fe2+ and S2− were negligible. The reductive immobilization of TcO4− with FeS was shown to be accelerated by increasing ionic strength and strongly pH dependent. At pH values below the pHpzc, the positively charged FeS surface reacted much faster with TcO4− and had higher immobilization yields relative to the negatively charged FeS surface at pH values above pHpzc. The TcO4−−FeS reaction is consistent with a surface mediated reaction through ligand exchange. The TcO4−−FeS reductive immobilization reaction product was characterized by X-ray absorption near edge spectroscopy (XANES), extended X-ray absorption fine structure (EXAFS), Fourier transform infrared spectroscopy (FT-IR), and energy dispersive X-ray spectroscopy (EDS) and found to be predominantly TcO2. Studies on the reductive capacity of the FeS and the long term stability of the TcO4−−FeS reaction product under both anaerobic and aerobic environments shows the potential utility of the in situ gaseous (hydrogen sulfide gas) immobilization technology in solidification of TcO4− by creating a FeS permeable reaction barrier in the vadose zone.

OPT3 Is a Component of the Iron-Signaling Network between Leaves and Roots and Misregulation of OPT3 Leads to an Over-Accumulation of Cadmium in Seeds

SUMMARY Long-distance communication between leaves and roots are key to properly regulate the uptake of trace metals from the soil. The molecular basis of this shoot-to-root signaling is currently unknown. In this manuscript, we describe the role of OPT3 in the shoot-to-root signaling of the iron status in Arabidopsis. We also show that reduced expression of OPT3 induces an over-accumulation of the toxic metal cadmium, but not other metals, in seeds.