Investigations of N-linked macrocycles for 111in and 90Y labeling of proteins

Wu, Chao‐Liang; Virzi, F.; Hnatowich, Donald J.

doi:10.1016/0883-2897(92)90012-n

Cited by 12 publications

(12 citation statements)

References 6 publications

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“…It is known to form very stable metal complexes. [73][74][75] The high electron density of heavy metals enables high resolution imaging of the block polymer in transmission electron microscopy (TEM) studies, as discussed below. Indium was used to form a complex with the DOTA-functionalized polymer.…”

Section: Resultsmentioning

confidence: 99%

Facile synthesis of thiol-functionalized amphiphilic polylactide–methacrylic diblock copolymers

2014

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Biodegradable amphiphilic diblock copolymers based on an aliphatic ester block and various hydrophilic methacrylic monomers were synthesized using a novel hydroxyl-functionalized trithiocarbonate-based chain transfer agent. One protocol involved the one-pot simultaneous ring-opening polymerization (ROP) of the biodegradable monomer (3S)-cis-3,6-dimethyl-1,4-dioxane-2,5-dione (L-lactide, LA) and reversible addition-fragmentation chain transfer (RAFT) polymerization of 2-(dimethylamino)ethyl methacrylate (DMA) or oligo(ethylene glycol) methacrylate (OEGMA) monomer, with 4dimethylaminopyridine being used as the ROP catalyst and 2,2 0-azobis(isobutyronitrile) as the initiator for the RAFT polymerization. Alternatively, a two-step protocol involving the initial polymerization of LA followed by the polymerization of DMA, glycerol monomethacrylate or 2-(methacryloyloxy)ethyl phosphorylcholine using 4,4 0-azobis(4-cyanovaleric acid) as a RAFT initiator was also explored. Using a solvent switch processing step, these amphiphilic diblock copolymers self-assemble in dilute aqueous solution. Their self-assembly provides various copolymer morphologies depending on the block compositions, as judged by transmission electron microscopy and dynamic light scattering. Two novel disulfide-functionalized PLA-branched block copolymers were also synthesized using simultaneous ROP of LA and RAFT copolymerization of OEGMA or DMA with a disulfide-based dimethacrylate. The disulfide bonds were reductively cleaved using tributyl phosphine to generate reactive thiol groups. Thiol-ene chemistry was utilized for further derivatization with thiol-based biologically important molecules and heavy metals for tissue engineering or bioimaging applications, respectively.

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Section: Resultsmentioning

confidence: 99%

Facile synthesis of thiol-functionalized amphiphilic polylactide–methacrylic diblock copolymers

2014

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“…Covalent attachment of DOTA to amines by acylation with the isobutyl formate mixed anhydride of the chelating agent has been employed to synthesize a variety of monofunctionalized DOTA amides. Wu et al (48) conjugated DOTA to biotin in this manner and studied the serum stabilities of mIn-labeled and 90Y-labeled DOTAbiotin-avidin conjugates. Sherry et al (49) used the same method to synthesize the Gd(III) complex of the monopropylamide of DOTA, a model for magnetic resonance imaging contrast agents consisting of monoconjugate d DOTA macromolecules.…”

Section: Resultsmentioning

confidence: 99%

“…Wu et al (48) conjugated DOTA to biotin in this manner and studied the serum stabilities of ll1In-labeled and goY-labeled DOTAbiotin-avidin conjugates. Sherry et al (49) used the same method to synthesize the Gd(II1) complex of the monopropylamide of DOTA, a model for magnetic resonance imaging contrast agents consisting of monoconjugated DOTA macromolecules.…”

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confidence: 99%

A Facile, Water-Soluble Method for Modification of Proteins with DOTA. Use of Elevated Temperature and Optimized pH To Achieve High Specific Activity and High Chelate Stability in Radiolabeled Immunoconjugates

Lewis¹,

Raubitschek²,

Shively³

1994

Bioconjugate Chem.

151

125

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We have developed a method for attachment of the macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane N,N',N",N"'-tetraacetic acid (DOTA) to proteins by activation of a single carboxyl group with N-hydroxysulfosuccinimide (sulfo-NHS). The sulfo-NHS active ester of DOTA was prepared in a single step using 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), and DOTA conjugates of cytochrome c and the anti-carcinoembryonic antigen chimeric monoclonal antibody cT84.66 were prepared by adding the DOTA active ester reaction mixture to the proteins at pH 8.5-9.0. Mass spectrometry of the cytochrome c conjugates showed that as the molar ratio of DOTA active ester to protein in the reaction mixture was increased from 10:1 to 100:1, the average number of chelators attached to the protein molecule increased from 2.64 to 8.79. When DOTA active ester reacted with the antibody at a molar ratio of 100:1, the conjugate averaged 3.8 chelates per antibody. Immunoreactivity of the antibody conjugate radiolabeled with 111In(III) and 90Y(III) remained quantitative. Variation of the DOTA:sulfo-NHS:EDC activation stoichiometry from 2:2:1 to 10:10:1 revealed that the kinetic stability of the radioconjugates increased as the molar ratio of carbodiimide, relative to DOTA and sulfo-NHS, was decreased. Radiolabeling of the protein conjugates with 111In(III) and 90Y(III) proved to be sensitive to pH, buffer, and temperature effects. The optimum pH for the labeling reaction was different for each protein and may be related to the isoelectric point of the protein. Radiometal incorporation at high specific activity was accomplished in acetate and Tris buffers, but the presence of citrate inhibited the labeling reaction. Increasing the temperature of the radiolabeling reaction from 25 to 43 degrees C greatly increased both the efficiency of radiometal incorporation and the kinetic stability of the radioconjugates. Stability studies of the conjugates in human serum and in the presence of a 5000- to 250,000-fold excess of diethylenetriaminepentaacetic acid (DTPA) demonstrated that the radiolabeled proteins are kinetically inert under physiological conditions. In serum, the 111In(III)-labeled antibody showed a rate of radiometal loss of approximately 0.08% per day. In the presence of excess DTPA, both conjugates lost 111In(III) at a rate of about 0.3% per day. No loss of 90Y(III) from the conjugates was observed in serum, but in excess DTPA, both 90Y(III) labeled proteins showed a rate of radiometal loss of approximately 0.2% per day. Therefore, kinetic analysis of metal loss from a radiolabeled immunoconjugate in the presence of a vast excess of DTPA may provide a better indication of the in vivo stability of that immunoconjugate than serum stability studies.

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“…The purpose of this study was to develop a new 111 Inlabelling agent suitable for estimating protein pharmacokinetics. 1,4,7,10-Tetraazacyclododecane-N,N« ,N« « ,N« « « -tetraacetic acid (DOTA), which produced a highly stable and hydrophilic 111 In chelate (Wu et al 1992), was selected as the chelating site, and the monoreactive DOTA derivative with a tetra¯uorophenyl group as the protein binding site (mDOTA) was designed and synthesized to avoid crosslinking of proteins. The plasma stability, radioactivity retention in the catabolic site and radiochemical yields of 111 In-DOTA-proteins were investigated using human serum albumin (HSA), galactosyl-neoglycoalbumin (NGA) and cytochrome c (cyt c), respectively, as model proteins.…”

Section: Introductionmentioning

confidence: 99%

Synthesis and evaluation of a monoreactive DOTA derivative for indium-111-based residualizing label to estimate protein pharmacokinetics

Mukai

Namba

Arano

et al. 2002

Journal of Pharmacy and Pharmacology

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The purpose of this study was to develop an indium-111 (111In)-based residualizing label for estimating the pharmacokinetics of proteins. 1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), which produced a highly stable and hydrophilic 111In chelate, was selected as the chelating site, and the monoreactive DOTA derivative with a tetrafluorophenyl group as the protein binding site (mDOTA) was designed to avoid cross-linkings of proteins. mDOTA was synthesized with an overall yield of 11%. The stability in murine plasma, the radioactivity retention in the catabolic sites of proteins and the radiochemical yields of 111In-labelled proteins via mDOTA were investigated using human serum albumin (HSA), galactosyl-neoglycoalbumin (NGA) and cytochrome c (cyt c) as model proteins. 111In-labelled HSA via mDOTA was highly stable for 5 days after incubation in murine plasma. Long retention of radioactivity in the catabolic sites was observed after injection of 111In-DOTA-NGA in mice, due to the slow elimination of the radiometabolite from the lysosome. At a chelator concentration of 42.2 microM, 111In-DOTA-cyt c was produced with over 91% radiochemical yield. On the other hand, 111In-DOTA-lysine and 111In-DOTA were obtained with high radiochemical yields at lower chelator concentrations. These findings indicated that mDOTA would be an appropriate 111In-labelling agent for estimating protein pharmacokinetics. These findings also suggested that the introduction of a protein binding site at a position distal from the unmodified DOTA structure would be preferable to preparing 111In-DOTA-labelled proteins with higher specific activity.

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Investigations of N-linked macrocycles for 111in and 90Y labeling of proteins

Cited by 12 publications

References 6 publications

Facile synthesis of thiol-functionalized amphiphilic polylactide–methacrylic diblock copolymers

Facile synthesis of thiol-functionalized amphiphilic polylactide–methacrylic diblock copolymers

A Facile, Water-Soluble Method for Modification of Proteins with DOTA. Use of Elevated Temperature and Optimized pH To Achieve High Specific Activity and High Chelate Stability in Radiolabeled Immunoconjugates

Synthesis and evaluation of a monoreactive DOTA derivative for indium-111-based residualizing label to estimate protein pharmacokinetics

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