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Coman D.,Yale University | Kiefer G.E.,Macrocyclics | Rothman D.L.,Yale University | Sherry A.D.,University of Texas Southwestern Medical Center | And 2 more authors.
NMR in Biomedicine | Year: 2011

Responsive contrast agents (RCAs) composed of lanthanide(III) ion (Ln 3+) complexes with a variety of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (DOTA 4-) derivatives have shown great potential as molecular imaging agents for MR. A variety of LnDOTA-tetraamide complexes have been demonstrated as RCAs for molecular imaging using chemical exchange saturation transfer (CEST). The CEST method detects proton exchange between bulk water and any exchangeable sites on the ligand itself or an inner sphere of bound water that is shifted by a paramagnetic Ln 3+ ion bound in the core of the macrocycle. It has also been shown that molecular imaging is possible when the RCA itself is observed (i.e. not its effect on bulk water) using a method called biosensor imaging of redundant deviation in shifts (BIRDS). The BIRDS method utilizes redundant information stored in the nonexchangeable proton resonances emanating from the paramagnetic RCA for ambient factors such as temperature and/or pH. Thus, CEST and BIRDS rely on exchangeable and nonexchangeable protons, respectively, for biosensing. We posited that it would be feasible to combine these two biosensing features into the same RCA (i.e. dual CEST and BIRDS properties). A complex between europium(III) ion (Eu 3+) and DOTA-tetraglycinate [DOTA-(gly)] was used to demonstrate that its CEST characteristics are preserved, while its BIRDS properties are also detectable. The in vitro temperature sensitivity of EuDOTA-(gly) was used to show that qualitative MR contrast with CEST can be calibrated using quantitative MR mapping with BIRDS, thereby enabling quantitative molecular imaging at high spatial resolution. © 2011 John Wiley & Sons, Ltd.

Ferreira C.L.,MDS Nordion | Lamsa E.,MDS Nordion | Woods M.,MDS Nordion | Duan Y.,Ottawa Heart Institute | And 6 more authors.
Bioconjugate Chemistry | Year: 2010

Ga radioisotopes, including the generator-produced positron-emitting isotope 68Ga (t 1/2 = 68 min), are of increasing interest for the development of new radiopharmaceuticals. Bifunctional chelates (BFCs) that can be efficiently radiolabeled with Ga to yield complexes with good in vivo stability are needed. To this end, we undertook a systematic comparison of four BFCs containing different chelating moieties: two novel BFCs, p-NO 2-Bn-OxO (l-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid) and p-NO 2-Bn-PCTA (3,6,9,15-tetraazabicyclo [9.3.1]pentadeca-l(1.5),11, 13-triene-3,6,9-triacetic acid), and two more commonly used BFCs, p-NO 2-Bn-DOTA (1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid) and p-NO 2-Bn-NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid). Each BFC was compared with respect to radiolabeling conditions, radiochemical yield, stability, and in vivo clearance properties. p-NO 2-Bn-PCTA, p-NO 2-Bn-OxO, and p-NO 2-Bn-NOTA were all more efficiently radiolabeled with Ga compared to p-NO 2-Bn-DOTA. p-NO 2-Bn-DOTA required longer reaction time, higher concentrations of BFC, or heating to obtain equivalent radiochemical yields. Better stability was observed for p-NO 2-Bn-NOTA and p-NO 2-Bn-PCTA compared to p-NO 2-Bn-DOTA and p-NO 2-Bn-OxO, especially with respect to transmetalation to transferrin. Ga-radiolabled p-NO 2-Bn-OxO was found to be kinetically labile and therefore unstable in vivo. Ga-radiolabeled p-NO 2-Bn-NOTA and p-NO 2-Bn-PCTA were relatively inert, while Ga-radiolabeled p-NO 2-BnDOTA had intermediate stability, losing >20% of Ga in less than one hour when incubated with apo-transferrin. Similar stability differences were seen when incubating at. pH 2. In vivo PET imaging and biodistribution studies in mice showed that 68Ga- radiolabeled p-NO 2-Bn-PCTA, p-NO 2-Bn-NOTA, and p-NO 2-Bn-DOTA all cleared through the kidneys. While there was no statistical difference in the biodistribution results of 68Ga- radiolabeled p-NO 2-Bn-PCTA and p-NO 2-Bn-DOTA, 68Ga-radiolabeled p-NO 2-Bn-NOTA cleared more rapidly from blood and muscle tissue but retained at up to 5 times higher activity in the kidneys. © 2010 American Chemical Society.

Ferreira C.L.,Nordion | Yapp D.T.T.,BC Cancer Agency | Mandel D.,Nordion | Gill R.K.,BC Cancer Agency | And 5 more authors.
Bioconjugate Chemistry | Year: 2012

In this study, a bifunctional version of the chelate PCTA was compared to the analogous NOTA derivative for peptide conjugation, 68Ga radiolabeling, and small peptide imaging. Both p-SCN-Bn-PCTA and p-SCN-Bn-NOTA were conjugated to cyclo-RGDyK. The resulting conjugates, PCTA-RGD and NOTA-RGD, retained their affinity for the peptide target, the αvβ 3 receptor. Both PCTA-RGD and NOTA-RGD could be radiolabeled with 68Ga in >95% radiochemical yield (RCY) at room temperature within 5 min. For PCTA-RGD, higher effective specific activities, up to 55 MBq/nmol, could be achieved in 95% RCY with gentle heating at 40 °C. The 68Ga-radiolabeled conjugates were >90% stable in serum and in the presence of excess apo-transferrin over 4 h; 68Ga-PCTA-RGD did have slightly lower stability than 68Ga-NOTA-RGD, 93 ± 2% compared to 98 ± 1%, at the 4 h time point. Finally, the tumor and nontarget organ uptake and clearance of 68Ga-radiolabeled PCTA-RGD and NOTA-RGD was compared in mice bearing HT-29 colorectal tumor xenografts. Activity cleared quickly from the blood and muscle tissue with >90% and >70% of the initial activity cleared within the first 40 min, respectively. The majority of activity was observed in the kidney, liver, and tumor tissue. The observed tumor uptake was specific with up to 75% of the tumor uptake blocked when the mice were preinjected with 160 nmol (100 μg) of unlabeled peptide. Uptake observed in the blocked tumors was not significantly different than the background activity observed in muscle tissue. The only significant difference between the two 68Ga-radiolabeled bioconjugates in vivo was the kidney uptake. 68Ga-radiolabeled PCTA-RGD had significantly lower (p < 0.05) kidney uptake (1.1 ± 0.5%) at 2 h postinjection compared to 68Ga-radiolabeled NOTA-RGD (2.7 ± 1.3%). Overall, 68Ga-radiolabeled PCTA-RGD and NOTA-RGD performed similarly, but the lower kidney uptake for 68Ga-radiolabeled PCTA-RGD may be advantageous in some imaging applications. © 2012 American Chemical Society.

Ait-Mohand S.,Université de Sherbrooke | Fournier P.,Université de Sherbrooke | Dumulon-Perreault V.,Université de Sherbrooke | Kiefer G.E.,Macrocyclics | And 4 more authors.
Bioconjugate Chemistry | Year: 2011

Several bifunctional chelates (BFCs) were investigated as carriers of 64Cu for PET imaging. The most widely used chelator for 64Cu labeling of BFCs is DOTA (1,4,7,10-tetraazacyclododecane-N, N′,N″,N″-tretraacetic acid), even though this complex exhibits only moderate in vivo stability. In this study, we prepared a series of alternative chelator-peptide conjugates labeled with 64Cu, measured in vitro receptor binding affinities in human breast cancer T47D cells expressing the gastrin-releasing peptide receptor (GRPR) and compared their in vivo stability in mice. DOTA-, NOTA-(1,4,7-triazacyclononane-1,4,7-triacetic acid), PCTA-(3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9- triacetic acid), and Oxo-DO3A-(1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid) peptide conjugates were prepared using H 2N-Aoc-[d-Tyr 6,βAla 11,Thi 13,Nle 14] bombesin(6-14) (BBN) as a peptide template. The BBN moiety was selected since it binds with high affinity to the GRPR, which is overexpressed on human breast cancer cells. A convenient synthetic approach for the attachment of aniline-BFC to peptides on solid support is also presented. To facilitate the attachment of the aniline-PCTA and aniline-Oxo-DO3A to the peptide via an amide bond, a succinyl spacer was introduced at the N-terminus of BBN. The partially protected aniline-BFC (p-H 2N-Bn-PCTA(Ot-Bu) 3 or p-H 2N-Bn-DO3A(Ot-Bu) 3) was then coupled to the resulting N-terminal carboxylic acid preactivated with DEPBT/ClHOBt on resin. After cleavage and purification, the peptide-conjugates were labeled with 64Cu using [ 64Cu]Cu(OAc) 2 in 0.1 M ammonium acetate buffer at 100 °C for 15 min. Labeling efficacy was >90% for all peptides; Oxo-DO3A-BBN was incubated an additional 150 min at 100 °C to achieve this high yield. Specific activities varied from 76 to 101 TBq/mmol. Competition assays on T47D cells showed that all BFC-BBN complexes retained high affinity for the GRPR. All BFC-BBN 64Cu-conjugates were stable for over 20 h when incubated at 37 °C in mouse plasma samples. However, in vivo, only 37% of the 64Cu/Oxo-DO3A complex remained intact after 20 h while the 64Cu/DOTA-BBN complex was completely demetalated. In contrast, both 64Cu/NOTA- and 64Cu/PCTA-BBN conjugates remained stable during the 20 h time period. Our results indicate that it is possible to successfully conjugate aniline-BFC with peptide on solid support. Our data also show that 64Cu-labeled NOTA- and PCTA-BBN peptide conjugates are promising radiotracers for PET imaging of many human cancers overexpressing the GRP receptor. © 2011 American Chemical Society.

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