Synthesis and biological evaluation of a novel decadentate ligand DEPA.
ABSTRACT: An efficient and short synthetic route to a novel decadentate ligand 7-[2-(bis-carboxymethyl-amino)-ethyl]-4,10-bis-carboxymethyl-1,4,7,10-tetraaza-cyclododec-1-yl-acetic acid (DEPA) with both macrocyclic and acyclic binding moieties is reported. A reproducible and scalable synthetic method to a precursor molecule of DEPA, 1,4,7-tris(tert-butoxycarbonylmethyl)tetraazacyclododecane was developed. DEPA was evaluated as a chelator of (177)Lu, (212)Bi, and (213)Bi for potential use in an antibody-targeted cancer therapy, radioimmunotherapy (RIT) using Arsenazo III based spectroscopic complexation kinetics, in vitro serum stability, and in vivo biodistribution studies.
Project description:A new bifunctional ligand C-DEPA was designed and synthesized as a component for antibody-targeted radiation therapy (radioimmunotherapy, RIT) of cancer. C-DEPA was conjugated to a tumor targeting antibody, trastuzumab, and the corresponding C-DEPA-trastuzumab conjugate was evaluated for radiolabeling kinetics with (205/6)Bi. C-DEPA-trastuzumab conjugate rapidly bound (205/6)Bi, and (205/6)Bi-C-DEPA-trastuzumab conjugate was stable in human serum for 72 h. The in vitro radiolabeling kinetics and serum stability data suggest that C-DEPA is a potential chelate for preclinical RIT applications using (212)Bi and (213)Bi.
Project description:Zevalin® is an antibody-drug conjugate radiolabeled with a cytotoxic radioisotope ((90)Y) that was approved for radioimmunotherapy (RIT) of B-cell non-Hodgkin's lymphoma. A bifunctional ligand that displays favorable complexation kinetics and in vivo stability is required for effective RIT. New bifunctional ligands 3p-C-DE4TA and 3p-C-NE3TA for potential use in RIT were efficiently prepared by the synthetic route based on regiospecific ring opening of aziridinium ions with prealkylated triaza- or tetraaza-backboned macrocycles. The new bifunctional ligands 3p-C-DE4TA and 3p-C-NE3TA along with the known bimodal ligands 3p-C-NETA and 3p-C-DEPA were comparatively evaluated for potential use in targeted radiotherapy using ?-emitting radionuclides (90)Y and (177)Lu. The bifunctional ligands were evaluated for radiolabeling kinetics with (90)Y and (177)Lu, and the corresponding (90)Y or (177)Lu-radiolabeled complexes were studied for in vitro stability in human serum and in vivo biodistribution in mice. The results of the comparative complexation kinetic and stability studies indicate that size of macrocyclic cavity, ligand denticity, and bimodality of donor groups have a substantial impact on complexation of the bifunctional ligands with the radiolanthanides. The new promising bifunctional chelates in the DE4TA and NE3TA series were rapid in binding (90)Y and (177)Lu, and the corresponding (90)Y- and (177)Lu-radiolabeled complexes remained inert in human serum or in mice. The in vitro and in vivo data show that 3p-C-DE4TA and 3p-C-NE3TA are promising bifunctional ligands for targeted radiotherapy applications of (90)Y and (177)Lu.
Project description:A new bifunctional ligand 3p-C-DEPA was synthesized and evaluated for use in targeted ?-radioimmunotherapy. 3p-C-DEPA was efficiently prepared via regiospecific ring opening of an aziridinium ion and conjugated with trastuzumab. The 3p-C-DEPA-trastuzumab conjugate was extremely rapid in binding (205/6)Bi, and the corresponding (205/6)Bi-3p-C-DEPA-trastuzumab complex was stable in human serum. Biodistribution studies were performed to evaluate in vivo stability and tumor targeting of (205/6)Bi-3p-C-DEPA-trastuzumab conjugate in tumor bearing athymic mice. (205/6)Bi-3p-C-DEPA-trastuzumab conjugate displayed excellent in vivo stability and targeting as evidenced by low organ uptake and high tumor uptake. The results of the in vitro and in vivo studies indicate that 3p-C-DEPA is a promising chelator for radioimmunotherapy of (212)Bi and (213)Bi.
Project description:Alkylation of the hydrobromide salts of 1,4,7-tris(methoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane and 1,4,7-tris(ethoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane with appropriate ?-bromoacetamides, followed by hydrolysis, provides convenient access to 10-(2-alkylamino-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid derivatives that contain acid-sensitive functional groups. The utility of the method is demonstrated by improved syntheses of two known DOTA monoamides bearing acid-sensitive ?-tritylthio alkyl chains in much higher yields based on cyclen as the starting material.
Project description:A dinuclear Nd(III) macrocyclic complex of 1 (1,4-bis[1-(4,7,10-tris(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane]-p-xylene) and mononuclear complexes of 1,4,7-tris-1,4,7,10-tetraazacyclododecane, 2, and 1,4,7-tris[(N-N-diethyl)carbamoylmethyl]-1,4,7,10-tetraazacyclododecane, 3, are prepared. Complexes of 1 and 2 give rise to a PARACEST (paramagnetic chemical exchange saturation transfer) peak from exchangeable amide protons that resonate approximately 12 ppm downfield from the bulk water proton resonance. The dinuclear Nd(III) complex is promising as a PARACEST contrast agent for MRI applications, because it has an optimal pH of 7.5 and the rate constant for amide proton exchange (2700 s(-1)) is nearly as large as it can be within slow exchange conditions with bulk water. Dinuclear Ln(2)(1) complexes (Ln(III) = Nd(III), Eu(III)) bind tightly to anionic ligands including carbonate, diethyl phosphate, and DNA. The CEST amide peak of Nd(2)(1) is enhanced by certain DNA sequences that contain hairpin loops, but decreases in the presence of diethyl phosphate or carbonate. Direct excitation luminescence studies of Eu(2)(1) show that double-stranded and hairpin-loop DNA sequences displace one water ligand on each Eu(III) center. DNA displaces carbonate ion despite the low dissociation constant for the Eu(2)(1) carbonate complex (K(d) = 15 microM). Enhancement of the CEST effect of a lanthanide complex by binding to DNA is a promising step toward the preparation of PARACEST agents containing DNA scaffolds.
Project description:Radionuclide molecular imaging of human epidermal growth factor receptor 3 (HER3) expression using affibody molecules could be used for patient stratification for HER3-targeted cancer therapeutics. We hypothesized that the properties of HER3-targeting affibody molecules might be improved through modification of the radiometal-chelator complex. Macrocyclic chelators NOTA (1,4,7-triazacyclononane-N,N',N''-triacetic acid), NODAGA (1-(1,3-carboxypropyl)-4,7-carboxymethyl-1,4,7-triazacyclononane), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), and DOTAGA (1,4,7,10-tetraazacyclododececane,1-(glutaric acid)-4,7,10-triacetic acid) were conjugated to the C-terminus of anti-HER3 affibody molecule Z08698 and conjugates were labeled with indium-111. All conjugates bound specifically and with picomolar affinity to HER3 in vitro. In mice bearing HER3-expressing xenografts, no significant difference in tumor uptake between the conjugates was observed. Presence of the negatively charged 111In-DOTAGA-complex resulted in the lowest hepatic uptake and the highest tumor-to-liver ratio. In conclusion, the choice of chelator influences the biodistribution of indium-111 labeled anti-HER3 affibody molecules. Hepatic uptake of anti-HER3 affibody molecules could be reduced by the increase of negative charge of the radiometal-chelator complex on the C-terminus without significantly influencing the tumor uptake.
Project description:PURPOSE:The integrin ?v?6 is overexpressed in a variety of aggressive cancers and serves as a prognosis marker. This study describes the conjugation, radiolabeling, and in vitro and in vivo evaluation of four chelators to determine the best candidate for (64)Cu radiolabeling of A20FMDV2, an ?v?6 targeting peptide. PROCEDURES:Four chelators were conjugated onto PEG28-A20FMDV2 (1): 11-carboxymethyl-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4-methanephosphonic acid (CB-TE1A1P), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and 4,4'-((3,6,10,13,16,19-hexazazbicyclo[6.6.6]ico-sane-1,8-diylbis(aza-nediyl))bis(methylene)dibenzoic acid (BaBaSar). All peptides were radiolabeled with (64)Cu in ammonium acetate buffer at pH 6 and formulated to pH 7.2 in PBS for use. The radiotracers were evaluated using in vitro cell binding and internalization assays and serum stability assays. In vivo studies conducted include blocking, biodistribution, and small animal PET imaging. Autoradiography and histology were also conducted. RESULTS:All radiotracers were radiolabeled in good radiochemical purity (>95 %) under mild conditions (37-50 °C for 15 min) with high specific activity (0.58-0.60 Ci/?mol). All radiotracers demonstrated ?v?6-directed cell binding (>46 %) with similar internalization levels (>23 %). The radiotracers (64)Cu-CB-TE1A1P-1 and (64)Cu-BaBaSar-1 showed improved specificity for the ?v?6 positive tumor in vivo over (64)Cu-DOTA-1 and (64)Cu-NOTA-1 (+/- tumor uptake ratios-3.82?+/-?0.44, 3.82?±?0.41, 2.58?±?0.58, and 1.29?±?0.14, respectively). Of the four radiotracers, (64)Cu-NOTA-1 exhibited the highest liver uptake (10.83?±?0.1 % ID/g at 4 h). CONCLUSIONS:We have successfully conjugated, radiolabeled, and assessed the four chelates CB-TE1A1P, DOTA, NOTA, and BaBaSar both in vitro and in vivo. However, the data suggests no clear "best candidate" for the (64)Cu-radiolabeling of A20FMDV2, but instead a trade-off between the different properties (e.g., stability, selectivity, pharmacokinetics, etc.) with no obvious effects of the individual chelators.
Project description:Self-assembly of a range of carboxylic acids (benzoic acid, dinicotinic acid, nicotinic acid, and isophthalic acid) with the europium complex of 5-nitro-?,?'-bis(DO3Ayl)-m-xylene (where DO3A is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid) has been explored to establish the thermodynamics of binding in a range of solvent systems and in a range of aqueous buffer solutions. In this system, profound effects are observed as a consequence of competition by the hydroxide ion, which outcompetes even dinicotinate at high pH. In the case of isophthalate, which binds most strongly, and dinicotinate, both enthalpic and entropic contributions to binding have been identified. The europium complex with 5-nitro-?,?'-bis(DO3Ayl)-m-xylene is found to have a solution structure significantly different from the related europium complex of 5-amino-?,?'-bis(DO3Ayl)-m-xylene. It is found that phosphate binds strongly to the europium complex of the nitro derivate but not to the europium complex of amino derivative. Lactate, citrate, and pyruvate also bind strongly to 5-nitro-?,?'-bis(Eu?DO3Ayl)-m-xylene, and it is concluded that the solution structure of this binuclear lanthanide complex is significantly different from that of the amino-substituted complex.
Project description:An efficient synthetic route to a bifunctional chelating agent C-NE3TA-NCS for antibody-targeted radioimmunotherapy (RIT) applications was developed. Various synthetic methods centered on the key reaction steps including bimolecular cyclization, ring opening reactions of aziridine and aziridinium cations, and reductive aminiation were explored to optimize the preparation of a tetraaza-based chelate TANPA and C-NE3TA analogues. Heptadentate C-NE3TA-NCS was conjugated to a tumor targeting antibody and compared to hexadentate C-NOTA-NCS for radiolabeling reaction kinetics with lanthanides for RIT. C-NE3TA-antibody conjugate displayed significantly enhanced complexation kinetics with 90Y as compared to C-NOTA-antibody conjugate. The synthetic methods for TANPA and C-NE3TA-NCS reported herein have broad applications for preparation of bifunctioanl macrocyclic chelating agents.
Project description:Alpha particles possess an exquisite degree of cytotoxicity when employed for targeted ?-particle therapy (TAT) or radioimmunotherapy (RIT). (212)Pb, which acts as an in vivo generator of the ?-emitting nuclide (212)Bi has shown great promise in pre-clinical studies when used to label the HER2 binding antibody, trastuzumab. Currently, the first RIT clinical trial employing (212)Pb radiolabeled trastuzumab is in progress. This report provides detailed current protocol operations and steps that were generated for use in the clinical trial as well as the relevant pre-clinical experimentation, and describes in detail the labeling of proteins or peptides with (212)Pb as provided via a (224)Ra based generator system.(212)Pb was eluted from the (224)Ra/(212)Pb generator using hydrochloric acid (2M). The generator eluate was evaporated and digested with nitric acid (8M) followed by extraction of the (212)Pb with dilute nitric acid (0.1M). The dilute nitric acid solution of (212)Pb was used to label the immunoconjugate Trastuzumab-TCMC (2-(4-isothiocyanatobenzyl-1,4,7,10-tetraaza-1,4,7,10,tetra-(2-carbamonylmethyl)-cyclododecane) at pH5.5.Elution of (212)Pb from the generator was efficient yielding>90% of available (212)Pb. Trastuzumab-TCMC was efficiently labeled with a radiochemical yield of 94% ± 4% (n=7) by ITLC and an isolated yield of 73% ± 3% (n=7).The results show the feasibility of generating radioimmunoconjugates and peptide conjugates for use as in vivo ? generator systems in the clinic. The technology holds promise in applications involving the treatment of minimal disease such as micrometastases and residual tumor after surgical debulking, hematological cancers, infections, and compartmental cancers, such as ovarian cancer.