Cancer therapy by antibody-targeted Cerenkov light and metabolism-selective photosensitization.

Photodynamic therapy (PDT) is an effective cancer treatment option, but it suffers from penetration limit of light, making it available only for superficial and endoscopically accessible cancers. Recently, there have been reports that Cerenkov luminescence originated from radioisotopes can be utilized as an excitation source for PDT without external light illumination. Here, cancer-selective agents, i.e., (1) clinically available 5-aminolevulinic acid (5-ALA), which promotes cancer metabolism-specific accumulation of protoporphyrin IX (PpIX), and (2) 64Cu-DOTA-trastuzumab, which has HER2-expressing cancer selective uptake, are separately applied as a photosensitizer and an in situ radiator, respectively, to potentiate tumor-specific Cerenkov luminescence energy transfer (CLET) from 64Cu to PpIX for high-precision PDT of cancer. It is shown that the combinational administration and tumor colocalization of 5-ALA and 64Cu-DOTA-trastuzumab exert significant in vitro cytotoxicity (cell viability <9%) as well as in vivo antitumor effects (tumor volume ratio of 0.50 on 14 days post-injection) on HER2-expressing breast and gastric cancer models. This study proves that high-precision treatment regimen using dual-targeted CLET-based PDT is feasible for HER2-expressing cancers. Furthermore, the results offer great potential for clinical translation to the dual-targeted CLET-based PDT because the treatment regimen uses components, 5-ALA and 64Cu-DOTA-trastuzumab, which are already in clinical uses.

Novel Chelating Agents for Zirconium-89-Positron Emission Tomography (PET) Imaging: Synthesis, DFT Calculation, Radiolabeling, and In Vitro and In Vivo Complex Stability.

We report the synthesis and evaluation of novel chelating agents for zirconium-89 (89Zr) with positron emission tomography (PET) imaging applications. New chelating agents NODHA, NOTHA, and NODHA-PY were constructed on 1,4,7-triazacyclononane (TACN) and possess hydroxamic acid or a pyridine ring as an acyclic binding moiety. The new chelating agents were theoretically studied for complexation with Zr(IV). Structures of Zr(IV)-NODHA, Zr(IV)-NOTHA, and Zr(IV)-NODHA-PY were predicted using density functional methods. NODHA was found to form stronger bonds with Zr(IV) when compared to NOTHA and NODHA-PY. The new chelating agents were evaluated for radiolabeling efficiency in binding 89Zr. The corresponding [89Zr]Zr-labeled chelators were evaluated for complex stability in human serum. All new chelating agents rapidly bound to 89Zr in excellent radiolabeling efficiency at room temperature. Among the new [89Zr]Zr-labeled chelators evaluated, [89Zr]Zr-NODHA showed the highest stability in human serum without losing 89Zr, and [89Zr]Zr-NODHA-PY released a considerable amount of 89Zr in human serum. [89Zr]Zr-NODHA, [89Zr]Zr-NODHA-PY, and [89Zr]Zr-DFO were comparatively evaluated for in vivo complex stability by performing biodistribution studies using normal mice. [89Zr]Zr-DFO had the lowest bone uptake at all time points, while [89Zr]Zr-NODHA-PY showed poor stability in mice as evidenced by high bone accumulation at the 24 h time point. [89Zr]Zr-NODHA exhibited better renal clearance but higher bone uptake than [89Zr]Zr-DFO.

Pyridine-containing octadentate ligand NE3TA-PY for formation of neutral complex with 177Lu(III) and 90Y(III) for radiopharmaceutical applications: Synthesis, DFT calculation, radiolabeling, and in vitro complex stability.

Targeted radionuclide therapy is a developing therapeutic modality for cancer and employs a cytotoxic radionuclide bound to a chelating agent and a bioactive molecule with high binding affinity for a specific biomarker in tumors. An optimal chelator is one of the critical components to control therapeutic efficacy and toxicity of targeted radionuclide therapy. We designed a new octadentate ligand NE3TA-PY (7-[2-[(carboxymethyl)(2-pyridylmethyl)amino]ethyl]-1,4,7-triazacyclononane-1,4-diacetic acid) for ß-particle-emitting 177Lu and 90Y with targeted radionuclide therapy applications. The pyridine-containing polyaminocarboxylate ligand was proposed to form a neutral complex with Lu(III) and Y(III). The new chelator NE3TA-PY was synthesized and experimentally and theorectically studied for complexation with 177Lu(III) and 90Y(III). DFT-optimized structures of Y(III)-NE3TA-PY and Lu(III)-NE3TA-PY complexes were predicted. NE3TA-PY displayed excellent radiolabeling efficiency with both 177Lu and 90Y. The new chelator (NE3TA-PY) bound to 177Lu was more stable in human serum and better tolerated when challenged by EDTA than 90Y-labeled NE3TA-PY. Our findings suggest that the new chelator (NE3TA-PY) produced excellent Lu-177 radiolabeling and in vitro complex stability profiles.

New Bifunctional Chelator 3p-C-NEPA for Potential Applications in Lu(III) and Y(III) Radionuclide Therapy and Imaging.

We have developed structurally unique bifunctional chelators in the NETA, NE3TA, and DEPA series for potential radiopharmaceutical applications. As part of our continued research efforts to generate efficient bifunctional chelators for targeted radionuclide therapy and imaging of various diseases, we designed a scorpion-like chelator that is proposed to completely saturate the coordination spheres of Y(III) and Lu(III). We herein report the synthesis and evaluation of a new chelator (3p-C-NEPA) with 10 donor groups for complexation with ß-emitting radionuclides 90Y(III), 86Y(III), and 177Lu(III). The chelator was synthesized and evaluated for radiolabeling kinetics with the readily available radioisotopes 90Y and 177Lu, and the corresponding 90Y or 177Lu-radiolabeled complexes were evaluated for in vitro stability in human serum and in vivo complex stability in mice. The new chelator rapidly bound 90Y or 177Lu and formed a stable complex with the radionuclides. The new chelator 3p-C-NEPA radiolabeled with either 90Y or 177Lu remains stable in human serum without dissociation for 10 days. 177Lu-labeled 3p-C-NEPA produced a favorable in vivo biodistribution profile in normal mice.

Heterochiral Assembly of Amphiphilic Peptides Inside the Mitochondria for Supramolecular Cancer Therapeutics.

Self-assembly of peptides containing both l- and d-isomers often results in nanostructures with enhanced properties compared to their enantiomeric analogues, such as faster kinetics of formation, higher mechanical strength, and enzymatic stability. However, occurrence and consequences of the heterochiral assembly in the cellular microenvironment are unknown. In this study, we monitored heterochiral assembly of amphiphilic peptides inside the cell, specifically mitochondria of cancer cells, resulting in nanostructures with refined morphological and biological properties owing to the superior interaction between the backbones of opposite chirality. We have designed a mitochondria penetrating tripeptide containing a diphenyl alanine building unit, named as Mito-FF due to their mitochondria targeting ability. The short peptide amphiphile, Mito-FF co-assembled with its mirror pair, Mito-ff, induced superfibrils of around 100 nm in diameter and 0.5-1 µm in length, while enantiomers formed only narrow fibers of 10 nm in diameter. The co-administration of Mito-FF and Mito-ff in the cell induced drastic mitochondrial disruption both in vitro and in vivo. The experimental and theoretical analyses revealed that pyrene capping played a major role in inducing superfibril morphology upon the co-assembly of racemic peptides. This work shows the impact of chirality control over the peptide self-assembly inside the biological system, thus showing a potent strategy for fabricating promising peptide biomaterials by considering chirality as a design modality.

Synthesis and Feasibility Evaluation of a new Trastuzumab Conjugate Integrated with Paclitaxel and 89Zr for Theranostic Application Against HER2-Expressing Breast Cancers.

The preparation and in  vitro evaluation of a theranostic conjugate composed of trastuzumab, paclitaxel (PTX), and deferoxamine (DFO)-chelated 89Zr have been reported. These comounds have potential applications against HER2 receptor positive breast cancers. We conjugated DFO and PTX to trastuzumab by exploiting simple conjugation chemistry. The conjugate (DFO-trastuzumab-PTX) showed excellent radiolabeling efficiency with 89Zr and the labeled conjugate had high in vitro stability in human serum. Furthermore, DFO-trastuzumab-PTX displayed comparable cytotoxicity with PTX and 89Zr-DFO-trastuzumab-PTX exhibited HER2 receptor-mediated binding on HER2-positive MDA-MB-231 breast cancer cells. The results of our in vitro study indicate high potential of 89Zr-DFO-trastuzumab-PTX to be utilized in the theranostic application against HER2-postive breast cancers.

Design, Synthesis, and Biological Evaluation of Polyaminocarboxylate Ligand-Based Theranostic Conjugates for Antibody-Targeted Cancer Therapy and Near-Infrared Optical Imaging.

We report the design, synthesis, and evaluation of polyaminocarboxylate ligand-based antibody conjugates for potential application in targeted cancer therapy and near-infrared (NIR) fluorescence imaging. We synthesized a new polyaminocarboxylate chelate (CAB-NE3TA) as a potential anticancer agent. CAB-NE3TA displayed potent inhibitory activities against various cancer cell lines. We then designed a multifunctional theranostic platform (CAB-NE3TA-PAN-IR800) constructed on an epidermal growth factor receptor (EGFR)-targeted antibody (panitumumab, PAN) labeled with a NIR fluorescent dye. We also built the first atomistic model of the EGFR-PAN complex and loaded it with the cytotoxic CAB-NE3TA and the NIR dye. The therapeutic (CAB-NE3TA-PAN) and theranostic (CAB-NE3TA-PAN-IR800) conjugates were evaluated using an EGFR-positive A431 (human skin cancer) cell xenograft mouse model. Biodistribution studies using NIR fluorescence imaging demonstrated that the CAB-NE3TA-PAN labeled with the IR800 dye selectively targeted the A431 tumors in mice and resulted in prolonged retention in the tumor tissue and displayed excellent clearance in blood and normal organs. The therapeutic conjugate was capable of significantly inhibiting tumor growth, leading to nearly complete disappearance of tumors in the mice. The results of our pilot in vivo studies support further evaluation of the novel ligand-based therapeutic and theranostic conjugates for targeted iron chelation cancer therapy and imaging applications.

Theranostic Polyaminocarboxylate-Cyanine-Transferrin Conjugate for Anticancer Therapy and Near-Infrared Optical Imaging.

Iron chelation therapy has been recognized as a promising antitumor therapeutic strategy. Herein we report a novel theranostic agent for targeted iron chelation therapy and near-infrared (NIR) optical imaging of cancers. The theranostic agent was prepared by incorporation of a polyaminocarboxylate-based cytotoxic chelating agent (N-NE3TA; 7-[2-[(carboxymethyl)amino]ethyl]-1,4,7-triazacyclononane-1,4-diacetic acid) and a NIR fluorescent cyanine dye (Cy5.5) onto a tumor-targeting transferrin (Tf). The N-NE3TA-Tf conjugate (without Cy5.5) was characterized and evaluated for antiproliferative activity in HeLa, HT29, and PC3 cancer cells, which have elevated expression levels of the transferrin receptor (TfR). The N-NE3TA-Tf conjugate displayed significant inhibitory activity against all three cancer cell lines. The NIR dye Cy5.5 was then incorporated into N-NE3TA-Tf, and the resulting cytotoxic and fluorescent transferrin conjugate N-NE3TA-Tf-Cy5.5 was shown by microscopy to enter TfR-overexpressing cancer cells. This theranostic conjugate has potential application for dual use in targeted iron chelation cancer therapy and NIR fluorescence imaging.

Size-engineered biocompatible polymeric nanophotosensitizer for locoregional photodynamic therapy of cancer.

Current approaches in use of water-insoluble photosensitizers for photodynamic therapy (PDT) of cancer often demand a nano-delivery system. Here, we report a photosensitizer-loaded biocompatible nano-delivery formulation (PPaN-20) whose size was engineered to ca. 20nm to offer improved cell/tissue penetration and efficient generation of cytotoxic singlet oxygen. PPaN-20 was fabricated through the physical assembly of all biocompatible constituents: pyropheophorbide-a (PPa, water-insoluble photosensitizer), polycaprolactone (PCL, hydrophobic/biodegradable polymer), and Pluronic F-68 (clinically approved polymeric surfactant). Repeated microemulsification/evaporation method resulted in a fine colloidal dispersion of PPaN-20 in water, where the particulate PCL matrix containing well-dispersed PPa molecules inside was stabilized by the Pluronic corona. Compared to a control sample of large-sized nanoparticles (PPaN-200) prepared by a conventional solvent displacement method, PPaN-20 revealed optimal singlet oxygen generation and efficient cellular uptake by virtue of the suitably engineered size and constitution, leading to high in vitro phototoxicity against cancer cells. Upon administration to tumor-bearing mice by peritumoral route, PPaN-20 showed efficient tumor accumulation by the enhanced cell/tissue penetration evidenced by in vivo near-infrared fluorescence imaging. The in vivo PDT treatment with peritumorally administrated PPaN-20 showed significantly enhanced suppression of tumor growth compared to the control group, demonstrating great potential as a biocompatible photosensitizing agent for locoregional PDT treatment of cancer.

Development of highly efficient nanocarrier-mediated delivery approaches for cancer therapy.

Nanocarriers (NCs) are a group of nano-sized vehicles devised to deliver drugs to targeted malignant tissues or organs that provide remarkably improved targeting efficiency and therapeutic efficacy for cancer therapy. A variety of NCs have been developed to accommodate appropriate loading and release of drugs with a wide spectrum of chemical and physical characteristics. In addition, physicochemical modifications to the surface or interior of NCs allow for modulation of pharmacokinetic features reflecting clinical demands. However, cancer-related mortality is still high and drug-mediated cancer treatment remains a challenging research field despite the remarkable advances in targeting efficiency and therapeutic efficacy resulting from NCs. In this review, we focus on typical approaches and recent trends in NC-mediated drug delivery systems and their potential for targeted cancer therapy.

Transferrin conjugates of triazacyclononane-based bifunctional NE3TA chelates for PET imaging: Synthesis, Cu-64 radiolabeling, and in vitro and in vivo evaluation.

Three different polyaminocarboxylate-based bifunctional NE3TA (7-[2-[carboxymethyl)amino]ethyl]-1,4,7-triazacyclononane-1,4-diacetic acid) chelating agents were synthesized for potential use in copper 64-PET imaging applications. The bifunctional chelates were comparatively evaluated using transferrin (Tf) as a model targeting vector that binds to the transferrin receptor overexpressed in many different cancer cells. The transferrin conjugates of the NE3TA-based bifunctional chelates were evaluated for radiolabeling with (64)Cu. In vitro stability and cellular uptake of (64)Cu-radiolabeled conjugates were evaluated in human serum and prostate (PC-3) cancer cells, respectively. Among the three NE3TA-Tf conjugates tested, N-NE3TA-Tf was identified as the best conjugate for radiolabeling with (64)Cu. N-NE3TA-Tf rapidly bound to (64)Cu (>98% radiolabeling efficiency, 1min, RT), and (64)Cu-N-NE3TA-Tf remained stable in human serum for 2days and demonstrated high uptake in PC-3 cancer cells. (64)Cu-N-NE3TA-Tf was shown to have rapid blood clearance and increasing tumor uptake in PC-3 tumor bearing mice over a 24h period. This bifunctional chelate presents highly efficient chelation chemistry with (64)Cu under mild condition that can be applied for radiolabeling of various tumor-specific biomolecules with (64)Cu for potential use in PET imaging applications.

Promising bifunctional chelators for copper 64-PET imaging: practical (64)Cu radiolabeling and high in vitro and in vivo complex stability.

Positron emission tomography (PET) using copper-64 is a sensitive and non-invasive imaging technique for diagnosis and staging of cancer. A bifunctional chelator that can present rapid radiolabeling kinetics and high complex stability with (64)Cu is a critical component for targeted PET imaging. Bifunctional chelates 3p-C-NE3TA, 3p-C-NOTA, and 3p-C-DE4TA were evaluated for complexation kinetics and stability with (64)Cu in vitro and in vivo. Hexadentate 3p-C-NOTA and heptadentate 3p-C-NE3TA possess a smaller TACN-based macrocyclic backbone, while nonadentate 3p-C-DE4TA is constructed on a larger CYCLEN-based ring. The frequently explored chelates of (64)Cu, octadentate C-DOTA and hexadentate C-NOTA were also comparatively evaluated. Radiolabeling kinetics of bifunctional chelators with (64)Cu was assessed under mild conditions. All bifunctional chelates instantly bound to (64)Cu in excellent radiolabeling efficiency at room temperature. C-DOTA was less efficient in binding (64)Cu than all other chelates. All (64)Cu-radiolabeled bifunctional chelates remained stable in human serum without any loss of (64)Cu for 2 days. When challenged by an excess amount of EDTA, (64)Cu complexes of C-NOTA, 3p-C-NE3TA and 3p-C-NOTA were shown to be more stable than (64)Cu-C-DOTA and (64)Cu-3p-C-DE4TA. (64)Cu complexes of the new chelates 3p-C-NE3TA and 3p-C-NOTA displayed comparable in vitro and in vivo complex stability to (64)Cu-C-NOTA. In vivo biodistribution result indicates that the (64)Cu-radiolabeled complexes of 3p-C-NOTA and 3p-C-NE3TA possess excellent in vivo complex stability, while (64)Cu-3p-C-DE4TA was dissociated as evidenced by high renal and liver retention in mice. The results of in vitro and in vivo studies suggest that the bifunctional chelates 3p-C-NE3TA and 3p-C-NOTA offer excellent chelation chemistry with (64)Cu for potential PET imaging applications.

Multifunctional Photonics Nanoparticles for Crossing the Blood-Brain Barrier and Effecting Optically Trackable Brain Theranostics.

Theranostic photonic nanoparticles (TPNs) that cross the blood-brain barrier (BBB) and efficiently deliver a therapeutic agent to treat brain diseases, simultaneously providing optical tracking of drug delivery and release, are introduced. These TPNs are constructed by physical encapsulation of visible and/or near-infrared photonic molecules, in an ultrasmall micellar structure (<15 nm). Phytochemical curcumin is employed as a therapeutic as well as visible-emitting photonic component. In vitro BBB model studies and animal imaging, as well as ex vivo examination, reveal that these TPNs are capable of transmigration across the BBB and subsequent accumulation near the orthotopic xenograft of glioblastoma multiforme (GBM) that is the most common and aggressive brain tumor whose vasculature retains permeability-resistant properties. The intracranial delivery and release of curcumin can be visualized by imaging fluorescence produced by energy transfer from curcumin as the donor to the near-infrared emitting dye, coloaded in TPN, where curcumin induced apoptosis of glioma cells. At an extremely low dose of TPN, a significant therapeutic outcome against GBM is demonstrated noninvasively by bioluminescence monitoring of time-lapse proliferation of luciferase-expressing U-87 MG human GBM in the brain. This approach of TPN can be generally applied to a broad range of brain diseases.

Synthesis and comparative biological evaluation of bifunctional ligands for radiotherapy applications of (90)Y and (177)Lu.

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 ß-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.

Novel (64)Cu-radiolabeled bile acid conjugates for targeted PET imaging.

A promising bifunctional chelate (N-NE3TA) was conjugated to bile acids, cholic acid (CA), deoxycholic acid (DCA), and chenodeoxycholic acid (CDCA) as tumor targeting vectors. Bile acid conjugates of N-NE3TA (CA-N-NE3TA, DCA-N-NE3TA, and CDCA-N-NE3TA) were comparatively evaluated for complexation with (64)Cu, an imaging probe for positron emission tomography (PET). N-NE3TA-bile acid conjugates were evaluated for radiolabeling kinetics with (64)Cu, and the corresponding (64)Cu-radiolabeled conjugates were screened for complex stability in human serum and EDTA solution. The NE3TA-bile acid conjugates instantly bound to (64)Cu with excellent radiolabeling efficiency at room temperature. All NE3TA-bile acid conjugates radiolabeled with (64)Cu remained inert in human serum for 2days without releasing a considerable amount of the radioactivity. The (64)Cu-radiolabeled complexes were further challenged by EDTA in a 100-fold molar excess. Bile acid-N-NE3TA conjugates radiolabeled with (64)Cu were quite stable with a minimal transfer of (64)Cu to EDTA at 4h time point. The in vitro data indicate that the bile acid-N-NE3TA conjugates deserve further biological evaluation for (64)Cu-based targeted PET imaging applications.

Application of aziridinium ring opening for preparation of optically active diamine and triamine analogues: Highly efficient synthesis and evaluation of DTPA-based MRI contrast enhancement agents.

Ring opening of aziridinium ions with nitrogen nucleophiles was applied to the highly efficient synthesis of optically active vicinal diamines and diethylene triamine pentaacetic acid (DTPA) analogues as potential magnetic resonance imaging (MRI) contrast enhancement agents. The synthetic method features a column-free isolation of the regiospecific and stereospecific nucleophilic substitution products of enantiomerically enriched aziridinium ions in excellent yield.

Synthesis and evaluation of a new bifunctional NETA chelate for molecular targeted radiotherapy using(90)Y or(177)Lu.

INTRODUCTION: Therapeutic potential of ß-emitting cytotoxic radionuclides (90)Y and (177)Lu has been demonstrated in numerous preclinical and clinical trials. A bifunctional chelate that can effectively complex with the radioisotopes is a critical component for molecular targeted radiotherapy (90)Y and (177)Lu. A new bifunctional chelate 5p-C-NETA with a relatively long alkyl spacer between the chelating backbone and the functional unit for conjugation to a tumor targeting moiety was synthesized. 5p-C-NETA was conjugated to a model targeting moiety, a cyclic Arg-Gly-Asp-D-Tyr-Lys (RGDyK) peptide binding integrin αvß3 protein overexpressed on various cancers. 5p-C-NETA was conjugated to c(RGDyK) peptide and evaluated for potential use in molecular targeted radiotherapy of (90)Y and (177)Lu. METHODS: 5p-C-NETA conjugated with c(RGDyK) was evaluated in vitro for radiolabeling, serum stability, binding affinity, and the result of the in vitro studies of 5p-C-NETA-c(RGDyK) was compared to that of 3p-C-NETA-c(RGDyK). (177)Lu-5p-C-NETA-c(RGDyK) was further evaluated for in vivo biodistribution using gliobastoma bearing mice. RESULT: The new chelate rapidly and tightly bound to a cytotoxic radioisotope for cancer therapy, (90)Y or (177)Lu with excellent radiolabeling efficiency and maximum specific activity under mild condition (>99%, RT, <1 min). (90)Y- and (177)Lu-radiolabeled complexes of the new chelator remained stable in human serum without any loss of the radiolanthanide for 14 days. Introduction of the tumor targeting RGD moiety to the new chelator made little impact on complexation kinetics and stability with (90)Y or (177)Lu. (177)Lu-radiolabeled 5p-C-NETA-c(RGDyK) conjugate was shown to target tumors in mice and produced a favorable in vivo stability profile. CONCLUSION: The results of in vitro and in vivo evaluation suggest that 5p-C-NETA is an effective bifunctional chelate of (90)Y and (177)Lu that can be applied for generation of versatile molecular targeted radiopharmaceuticals.

Novel hexadentate and pentadentate chelators for 64Cu-based targeted PET imaging.

A series of new hexadentate and pentadentate chelators were designed and synthesized as chelators of (64)Cu. The new pentadentate and hexadentate chelators contain different types of donor groups and are expected to form neutral complexes with Cu(II). The new chelators were evaluated for complex kinetics and stability with (64)Cu. The new chelators instantly bound to (64)Cu with high labeling efficiency and maximum specific activity. All (64)Cu-radiolabeled complexes in human serum remained intact for 2 days. The (64)Cu-radiolabeled complexes were further challenged by EDTA in a 100-fold molar excess. Among the (64)Cu-radiolabeled complexes evaluated, (64)Cu-complex of the new chelator E was well tolerated with a minimal transfer of (64)Cu to EDTA. (64)Cu-radiolabeled complex of the new chelator E was further evaluated for biodistribution studies using mice and displayed rapid blood clearance and low organ uptake. (64)Cu-chelator E produced a favorable in vitro and in vivo complex stability profiles comparable to (64)Cu complex of the known hexadentate NOTA chelator. The in vitro and in vivo data highlight strong potential of the new chelator E for targeted PET imaging application.

Preclinical evaluation of NETA-based bifunctional ligand for radioimmunotherapy applications using 212Bi and 213Bi: radiolabeling, serum stability, and biodistribution and tumor uptake studies.

INTRODUCTION: Despite the great potential of targeted α-radioimmunotherapy (RIT) as demonstrated by pre-clinical and clinical trials, limited progress has been made on the improvement of chelation chemistry for (212)Bi and (213)Bi. A new bifunctional ligand 3p-C-NETA was evaluated for targeted α RIT using (212)Bi and (213)Bi. METHODS: Radiolabeling of 3p-C-NETA with (205/6)Bi, a surrogate of (212)Bi and (213)Bi, was evaluated at pH5.5 and room temperature. In vitro stability of the (205/6)Bi-3p-C-NETA-trastuzumab conjugate was evaluated using human serum (pH7, 37 °C). Immunoreactivity and specific activity of the (205/6)Bi-3p-C-NETA-trastuzumab conjugate were measured. An in vivo biodistribution study was performed to evaluate the in vivo stability and tumor targeting properties of the (205/6)Bi-3p-C-NETA-trastuzumab conjugate in athymic mice bearing subcutaneous LS174T tumor xenografts. RESULT: The 3p-C-NETA-trastuzumab conjugate was extremely rapid in complexing with (205/6)Bi, and the corresponding (205/6)Bi-3p-C-NETA-trastuzumab was stable in human serum. (205/6)Bi-3p-C-NETA-trastuzumab was prepared with a high specific activity and retained immunoreactivity. (205/6)Bi-3p-C-NETA-trastuzumab conjugate displayed excellent in vivo stability and targeting as evidenced by low normal organ and high tumor uptake. CONCLUSION: The results of the in vitro and in vivo studies indicate that 3p-C-NETA is a promising chelator for RIT applications using (212)Bi and (213)Bi. Further detailed in vivo evaluations of 3p-C-NETA for targeted α RIT are warranted.

Synthesis and preclinical evaluation of bifunctional ligands for improved chelation chemistry of 90Y and 177Lu for targeted radioimmunotherapy.

We report a practical and high-yield synthesis of a bimodal bifunctional ligand 3p-C-NETA-NCS containing the isothiocyanate group for conjugation to a tumor targeting antibody. 3p-C-NETA-NCS was conjugated to a tumor-targeting antibody, trastuzumab, and the corresponding 3p-C-NETA-trastuzumab conjugate was evaluated and compared to trastuzumab conjugates of the known bifunctional ligands C-DOTA, C-DTPA, and 3p-C-DEPA for radiolabeling kinetics with (90)Y and (177)Lu. 3p-C-NETA-trastuzumab conjugate exhibited extremely rapid complexation kinetics with (90)Y and (177)Lu. (90)Y-3p-C-NETA-trastuzumab and (177)Lu-3p-C-NETA-trastuzumab conjugates were stable in human serum for 2 weeks. A pilot biodistribution study was conducted to evaluate in vivo stability and tumor targeting of (177)Lu-radiolabeled trastuzumab conjugate using nude mice bearing ZR-75-1 human breast cancer. (177)Lu-3p-C-NETA-trastuzumab conjugate displayed low radioactivity level at blood (1.6%), low organ uptake (<2.2%), and high tumor-to-blood ratio (6.4) at 120 h. 3p-C-NETA possesses favorable in vitro and in vivo profiles and is an excellent bifunctional chelator that can be used for targeted RIT applications using (90)Y and (177)Lu and has the potential to replace DOTA and DTPA analogues in current clinical use.