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Preparation and biological evaluation of 64Cu-CB-TE2A-sst2-ANT, a somatostatin antagonist for PET imaging of somatostatin receptor-positive tumors.

ABSTRACT: Recently, the somatostatin receptor subtype 2 (SSTR2) selective antagonist sst2-ANT was determined to have a high affinity for SSTR2. Additionally, 111In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-sst2-ANT showed high uptake in an SSTR2-transfected, tumor-bearing mouse model and suggested that radiolabeled SSTR2 antagonists may be superior to agonists for imaging SSTR2-positive tumors. This report describes the synthesis and evaluation of 64Cu-CB-4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-sst2-ANT (64Cu-CB-TE2A-sst2-ANT) as a PET radiopharmaceutical for the in vivo imaging of SSTR2-positive tumors.Receptor-binding studies were performed to determine the dissociation constant of the radiopharmaceutical 64Cu-CB-TE2A-sst2-ANT using AR42J rat pancreatic tumor cell membranes. The internalization of 64Cu-CB-TE2A-sst2-ANT was compared with that of the 64Cu-labeled agonist 64Cu-CB-TE2A-tyrosine3-octreotate (64Cu-CB-TE2A-Y3-TATE) in AR42J cells. Both radiopharmaceuticals were also compared in vivo through biodistribution studies using healthy rats bearing AR42J tumors, and small-animal PET/CT of 64Cu-CB-TE2A-sst2-ANT was performed.The dissociation constant value for the radiopharmaceutical was determined to be 26 +/- 2.4 nM, and the maximum number of binding sites was 23,000 fmol/mg. 64Cu-CB-TE2A-sst2-ANT showed significantly less internalization than did 64Cu-CB-TE2A-Y3-TATE at time points from 15 min to 4 h. Biodistribution studies revealed that the clearance of 64Cu-CB-TE2A-sst2-ANT from the blood was rapid, whereas the clearance of 64Cu-CB-TE2A-sst2-ANT from the liver and kidneys was more modest at all time points. Tumor-to-blood and tumor-to-muscle ratios were determined to be better for 64Cu-CB-TE2A-sst2-ANT than those for 64Cu-CB-TE2A-Y3-TATE at the later time points, although liver and kidney uptake was significantly higher. Small-animal imaging using 64Cu-CB-TE2A-sst2-ANT revealed excellent tumor-to-background contrast at 4 h after injection, and standardized uptake values remained high even after 24 h.The PET radiopharmaceutical 64Cu-CB-TE2A-sst2-ANT is an attractive agent, worthy of future study as a PET radiopharmaceutical for the imaging of somatostatin receptor-positive tumors.

SUBMITTER: Wadas TJ

PROVIDER: S-EPMC2794832 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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Preparation and biological evaluation of 64Cu-CB-TE2A-sst2-ANT, a somatostatin antagonist for PET imaging of somatostatin receptor-positive tumors.

Wadas Thaddeus J TJ Eiblmaier Martin M Zheleznyak Alexander A Sherman Christopher D CD Ferdani Riccardo R Liang Kexian K Achilefu Samuel S Anderson Carolyn J CJ

Journal of nuclear medicine : official publication, Society of Nuclear Medicine 20081016 11

<h4>Unlabelled</h4>Recently, the somatostatin receptor subtype 2 (SSTR2) selective antagonist sst2-ANT was determined to have a high affinity for SSTR2. Additionally, 111In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-sst2-ANT showed high uptake in an SSTR2-transfected, tumor-bearing mouse model and suggested that radiolabeled SSTR2 antagonists may be superior to agonists for imaging SSTR2-positive tumors. This report describes the synthesis and evaluation of 64Cu-CB-4,11-bis(carboxy ...[more]

PMID: 18927338

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Project description:Copper-64 is an attractive radionuclide for PET imaging and is frequently used in clinical applications. The aim of this study was to perform a side-by-side comparison of the in vitro and in vivo performance of 64Cu-NODAGA-JR11 (NODAGA = 1,4,7-triazacyclononane,1-glutaric acid,4,7-acetic acid, JR11 = p-Cl-Phe-cyclo(D-Cys-Aph(Hor)-D-Aph(cbm)-Lys-Thr-Cys)D-Tyr-NH2), a somatostatin receptor 2 antagonist, with the clinically used sst2 agonist 64Cu-DOTA-TATE ((TATE = D-Phe-cyclo(Cys-Tyr-D-Trp-Lys-Thr-Cys)Thr). In vitro studies demonstrated Kd values of 5.7±0.95 nM (Bmax = 4.1±0.18 nM) for the antagonist 64/natCu-NODAGA-JR11 and 20.1±4.4. nM (Bmax = 0.48±0.18 nM) for the agonist 64/natCu-DOTA-TATE. Cell uptake studies showed the expected differences between agonists and antagonists. Whereas 64Cu-DOTA-TATE (the agonist) showed very effective internalization in the cell culture assay (with 50% internalized at 4 hours post-peptide addition under the given experimental conditions), 64Cu-NODAGA-JR11 (the antagonist) showed little internalization but strong receptor-mediated uptake at the cell membrane. Biodistribution studies of 64Cu-NODAGA-JR11 showed rapid blood clearance and tumor uptake with increasing tumor-to-relevant organ ratios within the first 4 hours and in some cases, 24 hours, respectively. The tumor washout was slow or non-existent in the first 4 hours, whereas the kidney washout was very efficient, leading to high and increasing tumor-to-kidney ratios over time. Specificity of tumor uptake was proven by co-injection of high excess of non-radiolabeled peptide, which led to >80% tumor blocking. 64Cu-DOTA-TATE showed less favorable pharmacokinetics, with the exception of lower kidney uptake. Blood clearance was distinctly slower and persistent higher blood values were found at 24 hours. Uptake in the liver and lung was relatively high and also persistent. The tumor uptake was specific and similar to that of 64Cu-NODAGA-JR11 at 1 h, but release from the tumor was very fast, particularly between 4 and 24 hours. Tumor-to-normal organ ratios were distinctly lower after 1 hour. This is indicative of insufficient in vivo stability. PET studies of 64Cu-NODAGA-JR11 reflected the biodistribution data with nicely delineated tumor and low background. 64Cu-NODAGA-JR11 shows promising pharmacokinetic properties for further translation into the clinic.

Project description:UnlabelledMelanoma is a malignant tumor derived from epidermal melanocytes, and it is known for its aggressiveness, therapeutic resistance, and predisposition for late metastasis. Very late antigen-4 (VLA-4; also called integrin α4β1) is a transmembrane noncovalent heterodimer overexpressed in melanoma tumors that plays an important role in tumor growth, angiogenesis, and metastasis by promoting adhesion and migration of cancer cells. In this study, we evaluated 2 conjugates of a high-affinity VLA-4 peptidomimetic ligand, LLP2A, for PET/CT imaging in a subcutaneous and metastatic melanoma tumor.MethodsLLP2A was conjugated to 1,4,8,11-tetraazacyclotetradecane-1-(methane phosphonic acid)-8-(methane carboxylic acid) (CB-TE1A1P) and 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentanedioic acid (NODAGA) chelators for (68)Ga and (64)Cu labeling. The conjugates were synthesized by solid-phase peptide synthesis, purified by reversed-phase high-performance liquid chromatography, and verified by liquid chromatography mass spectrometry. Saturation and competitive binding assays with B16F10 melanoma cells determined the affinity of the compounds for VLA-4. The biodistributions of the LLP2A conjugates were evaluated in murine B16F10 subcutaneous tumor-bearing C57BL/6 mice. Melanoma metastasis was induced by intracardiac injection of B16F10 cells. PET/CT imaging was performed at 2, 4, and 24 h after injection for the (64)Cu tracers and 1 h after injection for the (68)Ga tracer.Results(64)Cu-labeled CB-TE1A1P-PEG4-LLP2A and NODAGA-PEG4-LLP2A showed high affinity to VLA-4, with a comparable dissociation constant (0.28 vs. 0.23 nM) and receptor concentration (296 vs. 243 fmol/mg). The tumor uptake at 2 h after injection was comparable for the 2 probes, but (64)Cu-CB-TE1A1P-PEG4-LLP2A trended toward higher uptake than (64)Cu-NODAGA-PEG4-LLP2A (16.9 ± 2.2 vs. 13.4 ± 1.7 percentage injected dose per gram, P = 0.07). Tumor-to-muscle and tumor-to-blood ratios from biodistribution and PET/CT images were significantly higher for (64)Cu-CB-TE1A1P-PEG4-LLP2A than (64)Cu-NODAGA-PEG4-LLP2A (all P values < 0.05). PET/CT imaging of metastatic melanoma with (68)Ga-NODAGA-PEG4-LLP2A and (64)Cu-NODAGA-PEG4-LLP2A showed high uptake of the probes at the site of metastasis, correlating with the bioluminescence imaging of the tumor.ConclusionThese data demonstrate that (64)Cu-labeled CB-TE1A1P/NODAGA LLP2A conjugates and (68)Ga-labeled NODAGA-LLP2A are excellent imaging agents for melanoma and potentially other VLA-4-positive tumors. (64)Cu-CB-TE1A1P-PEG4-LLP2A had the most optimal tumor-to-nontarget tissue ratios for translation into humans as a PET imaging agent for melanoma.

Project description:Radiolabeled sst 2 and sst 3 antagonists are better candidates for tumor targeting than agonists with comparable binding characteristics (Ginj, M.; Zhang, H.; Waser, B.; Cescato, R.; Wild, D.; Erchegyi, J.; Rivier, J.; Mäcke, H. R.; Reubi, J. C. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 16436-16441.). Because most of the neuroendocrine tumors express sst 2, we used the known antagonists acetyl- pNO 2Phe (2)- c[ dCys (3)-Tyr (7)- dTrp (8)-Lys (9)-Thr (10)-Cys (14)]- dTyr (15)-NH 2 ( 1) (Bass, R. T.; Buckwalter, B. L.; Patel, B. P.; Pausch, M. H.; Price, L. A.; Strnad, J.; Hadcock, J. R. Mol. Pharmacol. 1996, 50, 709-715. Bass, R. T.; Buckwalter, B. L.; Patel, B. P.; Pausch, M. H.; Price, L. A.; Strnad, J.; Hadcock, J. R. Mol. Pharmacol. 1997, 51, 170; Erratum.) and H-Cpa (2)- c[ dCys (3)-Tyr (7)- dTrp (8)-Lys (9)-Thr (10)-Cys (14)]-2Nal (15)-NH 2 ( 7) (Hocart, S. J.; Jain, R.; Murphy, W. A.; Taylor, J. E.; Coy, D. H. J. Med. Chem. 1999, 42, 1863-1871.) as leads for analogues with increased sst 2 binding affinity and selectivity. Among the 32 analogues reported here, DOTA- pNO 2Phe (2)- c[ dCys (3)-Tyr (7)- dAph (8)(Cbm)-Lys (9)-Thr (10)-Cys (14)- dTyr (15)-NH 2 ( 3) and DOTA-Cpa (2)- c[ dCys (3)-Aph (7)(Hor)- dAph (8)(Cbm)-Lys (9)-Thr (10)-Cys (14)]- dTyr (15)-NH 2 ( 31) had the highest sst 2 binding affinity and selectivity. All of the analogues tested kept their sst 2 antagonistic properties (i.e., did not affect calcium release in vitro and competitively antagonized the agonistic effect of [Tyr (3)]octreotide). Moreover, in an immunofluorescence-based internalization assay, the new analogues prevented sst 2 internalization induced by the sst 2 agonist [Tyr (3)]octreotide without being active by themselves. In conclusion, several analogues (in particular 3, 31, and 32) have outstanding sst 2 binding and functional antagonistic properties and, because of their DOTA moiety, are excellent candidates for in vivo targeting of sst 2-expressing cancers.

Project description:Mesenteric branch arteries isolated from cannabinoid type 1 receptor knockout (CB(1)(-/-)) mice, their wild-type littermates (CB(1)(+/+) mice), and C57BL/J wild-type mice were studied to test the hypothesis that murine arteries undergo high sensitivity Ca(2+)-induced relaxation that is CB(1) receptor dependent. Confocal microscope analysis of mesenteric branch arteries from wild-type mice showed the presence of Ca(2+) receptor-positive periadventitial nerves. Arterial segments of C57 control mice mounted on wire myographs contracted in response to 5 micromol/L norepinephrine and responded to the cumulative addition of extracellular Ca(2+) with a concentration-dependent relaxation that reached a maximum of 72.0 +/- 6.3% of the prerelaxation tone and had an EC(50) for Ca(2+) of 2.90 +/- 0.54 mmol/L. The relaxation was antagonized by precontraction in buffer containing 100 mmol/L K(+) and by pretreatment with 10 mmol/L tetraethylammonium. Arteries from CB(1)(-/-) and CB(1)(+/+) mice also relaxed in response to extracellular Ca(2+) with no differences being detected between the knockout and their littermate controls. SR141716A, a selective CB(1) antagonist, caused concentration-dependent inhibition of Ca(2+)-induced relaxation in both the knockout and wild-type strains (60% inhibition at 1 micromol/L). O-1918, a cannabidiol analog, had a similar blocking effect in arteries of both wild-type and CB(1)(-/-) mice at 10 micromol/L. In contrast, 1 micromol/L SR144538, a cannabinoid type 2 receptor antagonist, or 50 micromol/L 18alpha-glycyrrhetinic acid, a gap junction blocker, were without effect. SR141716A (1 to 30 micromol/L) was also assessed for nonspecific actions on whole-cell K(+) currents in isolated vascular smooth muscle cells. SR141716A inhibited macroscopic K(+) currents at concentrations higher than those required to inhibit Ca(2+)-induced relaxation, and appeared to have little effect on currents through large conductance Ca(2+)-activated K(+) channels. These data indicate that arteries of the mouse relax in response to cumulative addition of extracellular Ca(2+) in a hyperpolarization-dependent manner and rule out a role for CB(1) or CB(2) receptors in this effect. The possible role of a nonclassical cannabinoid receptor is discussed.

Dataset Information

Preparation and biological evaluation of 64Cu-CB-TE2A-sst2-ANT, a somatostatin antagonist for PET imaging of somatostatin receptor-positive tumors.

Publications

Preparation and biological evaluation of 64Cu-CB-TE2A-sst2-ANT, a somatostatin antagonist for PET imaging of somatostatin receptor-positive tumors.

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