Fe(III) and Cu(II) Complexes of Chlorogenic Acid: Spectroscopic, Thermal, Anti-/Pro-Oxidant, and Cytotoxic Studies.

Complexes of chlorogenic acid (5-CQA) with copper(II) and iron(III) were synthesized in a solid state and examined by means of FT-IR, thermogravimetric, and elemental analyses. The molar stoichiometric ratios of metal:ligand for the solid forms of the complexes were established as Cu(II):L = 1:2 and Fe(III):L = 2:3 (L: 5-CQA), with the possible coordination through the carboxylate group and the hydroxyl group from the catechol moiety. In an aqueous solution at pH = 7.4, the composition of the complexes was Cu(II):L = 1:1, and Fe(III):L = 1:1 and 1:2. The Cu(II) and Fe(III) complexes with 5-CQA showed lower antioxidant properties, as estimated by the spectrophotometric methods with DPPH•, ABTS•+, and HO• radicals, than the ligand alone, whereas in the lipid peroxidation inhibition assay, the metal complexes revealed a higher antioxidant activity than 5-CQA. Cu(II) 5-CQA showed the highest pro-oxidant activity in the Trolox oxidation assays compared to the other studied compounds. The lipophilic parameters of the compounds were estimated using the HPLC method. 5-CQA and its complexes with Fe(III) and Cu(II) were not toxic to HaCaT cells in a tested concentration range of 0.15-1000 nM after a 24 h incubation time.

Preclinical Evaluation of 225Ac-Labeled Single-Domain Antibody for the Treatment of HER2pos Cancer.

Human epidermal growth factor receptor type 2 (HER2) is overexpressed in various cancers; thus, HER2-targeting single-domain antibodies (sdAb) could offer a useful platform for radioimmunotherapy. In this study, we optimized the labeling of an anti-HER2-sdAb with the α-particle-emitter 225Ac through a DOTA-derivative. The formed radioconjugate was tested for binding affinity, specificity and internalization properties, whereas cytotoxicity was evaluated by clonogenic and DNA double-strand-breaks assays. Biodistribution studies were performed in mice bearing subcutaneous HER2pos tumors to estimate absorbed doses delivered to organs and tissues. Therapeutic efficacy and potential toxicity were assessed in HER2pos intraperitoneal ovarian cancer model and in healthy C57Bl/6 mice. [225Ac]Ac-DOTA-2Rs15d exhibited specific cell uptake and cell-killing capacity in HER2pos cells (EC50 = 3.9 ± 1.1 kBq/mL). Uptake in HER2pos lesions peaked at 3 hours (9.64 ± 1.69% IA/g), with very low accumulation in other organs (<1% IA/g) except for kidneys (11.69 ± 1.10% IA/g). α-camera imaging presented homogeneous uptake of radioactivity in tumors, although heterogeneous in kidneys, with a higher signal density in cortex versus medulla. In mice with HER2pos disseminated tumors, repeated administration of [225Ac]Ac-DOTA-2Rs15d significantly prolonged survival (143 days) compared to control groups (56 and 61 days) and to the group treated with HER2-targeting mAb trastuzumab (100 days). Histopathologic evaluation revealed signs of kidney toxicity after repeated administration of [225Ac]Ac-DOTA-2Rs15d. [225Ac]Ac-DOTA-2Rs15d efficiently targeted HER2pos cells and was effective in treatment of intraperitoneal disseminated tumors, both alone and as an add-on combination with trastuzumab, albeit with substantial signs of inflammation in kidneys. This study warrants further development of [225Ac]Ac-DOTA-2Rs15d.

The Road to Personalized Myeloma Medicine: Patient-specific Single-domain Antibodies for Anti-idiotypic Radionuclide Therapy.

To this day, multiple myeloma remains an incurable cancer. For many patients, recurrence is unavoidably a result of lacking treatment options in the minimal residual disease stage. This is due to residual and treatment-resistant myeloma cells that can cause disease relapse. However, patient-specific membrane-expressed paraproteins could hold the key to target these residual cells responsible for disease recurrence. Here, we describe the therapeutic potential of radiolabeled, anti-idiotypic camelid single-domain antibody fragments (sdAbs) as tumor-restrictive vehicles against a membrane-bound paraprotein in the syngeneic mouse 5T33 myeloma model and analogously assess the feasibility of sdAb-based personalized medicine for patients with multiple myeloma. Llamas were immunized using extracts containing paraprotein from either murine or human sera, and selective sdAbs were retrieved using competitive phage display selections of immune libraries. An anti-5T33 idiotype sdAb was selected for targeted radionuclide therapy with the ß--particle emitter 177Lu and the α-particle emitter 225Ac. sdAb-based radionuclide therapy in syngeneic mice with a low 5T33 myeloma lesion load significantly delayed tumor progression. In five of seven patients with newly diagnosed myeloma, membrane expression of the paraprotein was confirmed. Starting from serum-isolated paraprotein, for two of three selected patients anti-idiotype sdAbs were successfully generated.

Crystal Structure, Spectroscopic Characterization, Antioxidant and Cytotoxic Activity of New Mg(II) and Mn(II)/Na(I) Complexes of Isoferulic Acid.

The Mg(II) and heterometallic Mn(II)/Na(I) complexes of isoferulic acid (3-hydroxy-4-methoxycinnamic acid, IFA) were synthesized and characterized by infrared spectroscopy FT-IR, FT-Raman, electronic absorption spectroscopy UV/VIS, and single-crystal X-ray diffraction. The reaction of MgCl2 with isoferulic acid in the aqueous solutions of NaOH resulted in synthesis of the complex salt of the general formula of [Mg(H2O)6]⋅(C10H9O4)2⋅6H2O. The crystal structure of this compound consists of discrete octahedral [Mg(H2O)6]2+ cations, isoferulic acid anions and solvent water molecules. The hydrated metal cations are arranged among the organic layers. The multiple hydrogen-bonding interactions established between the coordinated and lattice water molecules and the functional groups of the ligand stabilize the 3D architecture of the crystal. The use of MnCl2 instead of MgCl2 led to the formation of the Mn(II)/Na(I) complex of the general formula [Mn3Na2(C10H7O4)8(H2O)8]. The compound is a 3D coordination polymer composed of centrosymmetric pentanuclear subunits. The antioxidant activity of these compounds was evaluated by assays based on different antioxidant mechanisms of action, i.e., with •OH, DPPH• and ABTS•+ radicals as well as CUPRAC (cupric ions reducing power) and lipid peroxidation inhibition assays. The pro-oxidant property of compounds was measured as the rate of oxidation of Trolox. The Mg(II) and Mn(II)/Na(I) complexes with isoferulic acid showed higher antioxidant activity than ligand alone in DPPH (IFA, IC50 = 365.27 µM, Mg(II) IFA IC50 = 153.50 µM, Mn(II)/Na(I) IFA IC50 = 149.00 µM) and CUPRAC assays (IFA 40.92 µM of Trolox, Mg(II) IFA 87.93 µM and Mn(II)/Na(I) IFA 105.85 µM of Trolox; for compounds' concentration 10 µM). Mg(II) IFA is a better scavenger of •OH than IFA and Mn(II)/Na(I) IFA complex. There was no distinct difference in ABTS•+ and lipid peroxidation assays between isoferulic acid and its Mg(II) complex, while Mn(II)/Na(I) complex showed lower activity than these compounds. The tested complexes displayed only slight antiproliferative activity tested in HaCaT human immortalized keratinocyte cell line within the solubility range. The Mn(II)/Na(I) IFA (16 µM in medium) caused an 87% (±5%) decrease in cell viability, the Mg salt caused a comparable, i.e., 87% (±4%) viability decrease in a concentration of 45 µM, while IFA caused this level of cell activity attenuation (87% ± 5%) at the concentration of 1582 µM (significant at α = 0.05).

Production and Supply of α-Particle-Emitting Radionuclides for Targeted α-Therapy.

Encouraging results from targeted α-therapy have received significant attention from academia and industry. However, the limited availability of suitable radionuclides has hampered widespread translation and application. In the present review, we discuss the most promising candidates for clinical application and the state of the art of their production and supply. In this review, along with 2 forthcoming reviews on chelation and clinical application of α-emitting radionuclides, The Journal of Nuclear Medicine will provide a comprehensive assessment of the field.

Radiochemical separation of 224Ra from 232U and 228Th sources for 224Ra/212Pb/212Bi generator.

The application of diagnostic and therapeutic radionuclides in nuclear medicine has grown significantly and has translated into the increased interest in radionuclide generators and their development. 224Ra and its shorter-lived daughters, 212Pb and 212Bi, are very interesting radionuclides from Targeted Alpha Therapy point of view for treatment of small cancers or metastatic forms. The purpose of the present work was to develop a simple generator for rapid elution of carrier-free 224Ra from 232U or 228Th sources by radiochemical separation based on extraction chromatography with the utilization of a home-made material. The bis(2-ethylhexyl) hydrogen phosphate (HDEHP) extractant was immobilized on polytetrafluroethylene (PTFE) grains and its ability to selectively adsorb 232U and 228Th, with simultaneous high elution recovery of 224Ra, was checked over few years. The 224Ra was quantitatively eluted with small volume (3-5 mL) of 0.1 M HNO3 with low breakthrough (<0.005%) and was used for further milking of 212Bi and 212Pb from DOWEX 50WX12 by 0.75 M and 2.0 M HCl, respectively. The elaborated here methods allowed high recovery of 224Ra, 212Pb and 212Bi radionuclides and their application in radiolabeling of various biomolecules.

Trastuzumab Modified Barium Ferrite Magnetic Nanoparticles Labeled with Radium-223: A New Potential Radiobioconjugate for Alpha Radioimmunotherapy.

Barium ferrite nanoparticles (BaFeNPs) were investigated as vehicles for 223Ra radionuclide in targeted α-therapy. BaFe nanoparticles were labeled using a hydrothermal Ba2+ cations replacement by 223Ra with yield reaching 61.3 ± 1.8%. Radiolabeled nanoparticles were functionalized with 3-phosphonopropionic acid (CEPA) linker followed by covalent conjugation to trastuzumab (Herceptin®). Thermogravimetric analysis and radiometric method with the use of [131I]-labeled trastuzumab revealed that on average 19-21 molecules of trastuzumab are attached to the surface of one BaFe-CEPA nanoparticle. The hydrodynamic diameter of BaFe-CEPA-trastuzumab conjugate is 99.9 ± 3.0 nm in water and increases to 218.3 ± 3.7 nm in PBS buffer, and the zeta potential varies from +27.2 ± 0.7 mV in water to -8.8 ± 0.7 in PBS buffer. The [223Ra]BaFe-CEPA-trastuzumab radiobioconjugate almost quantitatively retained 223Ra (>98%) and about 96% of 211Bi and 94% of 211Pb over 30 days. The obtained radiobioconjugate exhibited high affinity, cell internalization and cytotoxicity towards the human ovarian adenocarcinoma SKOV-3 cells overexpressing HER2 receptor. Confocal studies indicated that [223Ra]BaFe-CEPA-trastuzumab was located in peri-nuclear space. High cytotoxicity of the [223Ra]BaFe-CEPA-trastuzumab bioconjugate was confirmed by radiotoxicity studies on SKOV-3 cell monolayers and 3D-spheroids. In addition, the magnetic properties of the radiobioconjugate should allow for its use in guide drug delivery driven by magnetic field gradient.

Design and Evaluation of 223Ra-Labeled and Anti-PSMA Targeted NaA Nanozeolites for Prostate Cancer Therapy-Part I.

Prostate cancer is the second most frequent malignancy in men worldwide. Unfortunately, current therapies often lead to the onset of metastatic castration-resistant prostate cancer (mCRPC), causing significant mortality. Therefore, there is an urgent need for new and targeted therapies that are advantageous over the current ones. Recently, the PSMA-targeted radioligand therapy of mCRPC has shown very promising results. In line with this, we described the synthesis of a new radioimmunoconjugate, 223RaA-silane-PEG-D2B, for targeted mCRPC therapy. The new compound consists of a NaA zeolite nanocarrier loaded with the α-particle emitting Ra-223 radionuclide, functionalized with the anti-PSMA D2B antibody. Physicochemical properties of the synthesized compound were characterized by standard methods (HR-SEM, TEM, XRD, FTIR, EDS, NTA, DLS, BET, TGA). The targeting selectivity, the extent of internalization, and cytotoxicity were determined in LNCaP C4-2 (PSMA+) and DU-145 (PSMA-) cells. Our results supported the 223RaA-silane-PEG-D2B synthesis and revealed that the final product had a diameter ca. 120 nm and specific activity 0.65 MBq/1mg. The product was characterized by a high yield of stability (>95% up to 12 days). The conjugation reaction resulted in approximately 50 antibodies/nanoparticle. The obtained radioimmunoconjugate bound specifically and internalized into PSMA-expressing LNCaP C4-2 cells, but not into PSMA-negative DU-145 cells. 223RaA-silane-PEG-D2B demonstrated also potent cytotoxicity in LNCaP C4-2 cells. These promising results require further in vivo evaluation of 223RaA-silane-PEG-D2B with regard to its toxicity and therapeutic efficacy.

Trastuzumab Conjugated Superparamagnetic Iron Oxide Nanoparticles Labeled with 225Ac as a Perspective Tool for Combined α-Radioimmunotherapy and Magnetic Hyperthermia of HER2-Positive Breast Cancer.

It has been proven and confirmed in numerous repeated tests, that the use of a combination of several therapeutic methods gives much better treatment results than in the case of separate therapies. Particularly promising is the combination of ionizing radiation and magnetic hyperthermia in one drug. To achieve this objective, magnetite nanoparticles have been modified in their core with α emitter 225Ac, in an amount affecting only slightly their magnetic properties. By 3-phosphonopropionic acid (CEPA) linker nanoparticles were conjugated covalently with trastuzumab (Herceptin®), a monoclonal antibody that recognizes ovarian and breast cancer cells overexpressing the HER2 receptors. The synthesized bioconjugates were characterized by transmission electron microscopy (TEM), Dynamic Light Scattering (DLS) measurement, thermogravimetric analysis (TGA) and application of 131I-labeled trastuzumab for quantification of the bound biomolecule. The obtained results show that one 225Ac@Fe3O4-CEPA-trastuzumab bioconjugate contains an average of 8-11 molecules of trastuzumab. The labeled nanoparticles almost quantitatively retain 225Ac (>98%) in phosphate-buffered saline (PBS) and physiological salt, and more than 90% of 221Fr and 213Bi over 10 days. In human serum after 10 days, the fraction of 225Ac released from 225Ac@Fe3O4 was still less than 2%, but the retention of 221Fr and 213Bi decreased to 70%. The synthesized 225Ac@Fe3O4-CEPA-trastuzumab bioconjugates have shown a high cytotoxic effect toward SKOV-3 ovarian cancer cells expressing HER2 receptor in-vitro. The in-vivo studies indicate that this bioconjugate exhibits properties suitable for the treatment of cancer cells by intratumoral or post-resection injection. The intravenous injection of the 225Ac@Fe3O4-CEPA-trastuzumab radiobioconjugate is excluded due to its high accumulation in the liver, lungs and spleen. Additionally, the high value of a specific absorption rate (SAR) allows its use in a new very perspective combination of α radionuclide therapy with magnetic hyperthermia.

Trastuzumab-Modified Gold Nanoparticles Labeled with 211At as a Prospective Tool for Local Treatment of HER2-Positive Breast Cancer.

Highly localized radiotherapy with radionuclides is a commonly used treatment modality for patients with unresectable solid tumors. Herein, we propose a novel α-nanobrachytherapy approach for selective therapy of human epidermal growth factor receptor 2 (HER2)-positive breast cancer. This uses local intratumoral injection of 5-nm-diameter gold nanoparticles (AuNPs) labeled with an α-emitter (211At), modified with polyethylene glycol (PEG) chains and attached to HER2-specific monoclonal antibody (trastuzumab). The size, shape, morphology, and zeta potential of the 5 nm synthesized AuNPs were characterized by TEM (Transmission Electron Microscopy) and DLS (Dynamic Light Scattering) techniques. The gold nanoparticle surface was modified by PEG and subsequently used for antibody immobilization. Utilizing the high affinity of gold for heavy halogens, the bioconjugate was labelled with 211At obtained by α irradiation of the bismuth target. The labeling yield of 211At was greater than 99%. 211At bioconjugates were stable in human serum. Additionally, in vitro biological studies indicated that 211At-AuNP-PEG-trastuzumab exhibited higher affinity and cytotoxicity towards the HER2-overexpressing human ovarian SKOV-3 cell line than unmodified nanoparticles. Confocal and dark field microscopy studies revealed that 211At-AuNP-PEG-trastuzumab was effectively internalized and deposited near the nucleus. These findings show promising potential for the 211At-AuNP-PEG-trastuzumab radiobioconjugate as a perspective therapeutic agent in the treatment of unresectable solid cancers expressing HER2 receptors.

d-Amino Acid Peptide Residualizing Agents for Protein Radioiodination: Effect of Aspartate for Glutamate Substitution.

The residualizing prosthetic agent Nε-(3-[*I]iodobenzoyl)-Lys5-Nα-maleimido-Gly¹-d-GEEEK ([*I]IB-Mal-d-GEEEK) showed promise for the radioiodination of monoclonal antibodies (mAbs) that bind to internalizing molecular targets. Although enhanced tumor uptake was achieved in these studies, elevated kidney accumulation also was observed, particularly with low-molecular-weight, single-domain antibody fragments (sdAbs). Here, we developed an analogous agent (IB-Mal-d-GDDDK), in which glutamate residues (E) were replaced with aspartates (D) to determine whether this modification could decrease renal uptake. [125I]IB-Mal-d-GDDDK and [131I]IB-Mal-d-GEEEK were synthesized with similar radiochemical yields (60⁻80%) and coupled to the anti-HER2 sdAb 5F7 at 50⁻60% efficiency. Paired-label internalization assays in vitro indicated similar levels of intracellular activity residualization in HER2-expressing BT474M1 cells for [125I]IB-Mal-d-GDDDK-5F7 and [131I]IB-Mal-d-GEEEK-5F7. A paired-label biodistribution comparison of the two labeled conjugates was performed in mice with HER2-expressing SKOV-3 xenografts, and the results of this study indicated that renal uptake at 1 h was 127.5 ± 18.7% ID/g and 271.4 ± 66.6% ID/g for [125I]IB-Mal-d-GDDDK-5F7 and [131I]IB-Mal-d-GEEEK-5F7, respectively. The tumor uptake of the two radioconjugates was not significantly different. These results demonstrate that substitution of E with D in the IB-Mal-d-GEEEK construct reduced kidney accumulation of the sdAb. However, renal activity levels need to be reduced further if d-amino acid derived prosthetic agents are to be of practical value for labeling low molecular weight biomolecules such as sdAbs.

Evaluation of an Anti-HER2 Nanobody Labeled with 225Ac for Targeted α-Particle Therapy of Cancer.

Human epidermal growth factor receptor type 2 (HER2) is overexpressed in numerous carcinomas. Nanobodies (Nbs) are the smallest antibody-derived fragments with beneficial characteristics for molecular imaging and radionuclide therapy. Therefore, HER2-targeting nanobodies could offer a valuable platform for radioimmunotherapy, especially when labeled with α-particle emitters, which provide highly lethal and localized radiation to targeted cells with minimal exposure to surrounding healthy tissues. In this study, the anti-HER2 2Rs15d-nanobody was conjugated with 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid ( p-SCN-Bn-DOTA) and radiolabeled with an α-emitter 225Ac with a high yield (>90%) and a radiochemical purity above 95%. The 225Ac-DOTA-Nb binding affinity was 4.12 ± 0.47 nM with an immunoreactive fraction above 80%. Binding to low HER2-expressing MDA-MB-231 cells was negligible, whereas HER2-overexpressing SKOV-3 cells could be blocked with an excess of unlabeled nanobody, confirming the specificity of binding. Noncompeting binding to HER2 was observed in the presence of an excess of trastuzumab. The cell-associated fraction of 225Ac-DOTA-Nb was 34.72 ± 16.66% over 24 h. In vitro, the radioconjugate was toxic in an HER2-mediated and dose-dependent manner, resulting in IC50 values of 10.2 and 322.1 kBq/mL for 225Ac-DOTA-Nb and the 225Ac-DOTA control, respectively, on SKOV-3 cells, and 282.2 kBq/mL for 225Ac-DOTA-Nb on MDA-MB-231 cells. Ex vivo biodistribution studies, performed in mice bearing subcutaneous HER2-overexpressing and low HER2-expressing tumors, showed a fast uptake in SKOV-3 tumors compared to MDA-MB-231 (4.01 ± 1.58% ID/g vs 0.49 ± 0.20% ID/g after 2 h), resulting also in high tumor-to-normal tissue ratios. In addition, coinjection of 225Ac-DOTA-Nb with Gelofusine reduced kidney retention by 70%. This study shows that 225Ac-DOTA-Nb is a promising new radioconjugate for targeted α-particle therapy and supports its further development.

Functionalized TiO2 nanoparticles labelled with 225Ac for targeted alpha radionuclide therapy.

The 225Ac radioisotope exhibits very attractive nuclear properties for application in radionuclide therapy. Unfortunately, the major challenge for radioconjugates labelled with 225Ac is that traditional chelating moieties are unable to sequester the radioactive daughters in the bioconjugate which is critical to minimize toxicity to healthy, non-targeted tissues. In the present work, we propose to apply TiO2 nanoparticles (NPs) as carrier for 225Ac and its decay products. The surface of TiO2 nanoparticles with 25 nm diameter was modified with Substance P (5-11), a peptide fragment which targets NK1 receptors on the glioma cells, through the silan-PEG-NHS linker. Nanoparticles functionalized with Substance P (5-11) were synthesized with high yield in a two-step procedure, and the products were characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS) and thermogravimetric analysis (TGA). The obtained results show that one TiO2-bioconjugate nanoparticle contains in average 80 peptide molecules on its surface. The synthesized TiO2-PEG-SP(5-11) conjugates were labelled with 225Ac by ion-exchange reaction on hydroxyl (OH) functional groups on the TiO2 surface. The labelled bioconjugates almost quantitatively retain 225Ac in phosphate-buffered saline (PBS), physiological salt and cerebrospinal fluid (CSF) for up to 10 days. The leaching of 221Fr, a first decay daughter of 225Ac, in an amount of 30% was observed only in CSF after 10 days. The synthesized 225Ac-TiO2-PEG-SP(5-11) has shown high cytotoxic effect in vitro in T98G glioma cells; therefore, it is a promising new radioconjugate for targeted radionuclide therapy of brain tumours.

131I-labeled Anti-HER2 Camelid sdAb as a Theranostic Tool in Cancer Treatment.

Purpose: Camelid single-domain antibody-fragments (sdAb) have beneficial pharmacokinetic properties, and those targeted to HER2 can be used for imaging of HER2-overexpressing cancer. Labeled with a therapeutic radionuclide, they may be used for HER2-targeted therapy. Here, we describe the generation of a 131I-labeled sdAb as a theranostic drug to treat HER2-overexpressing cancer.Experimental Design: Anti-HER2 sdAb 2Rs15d was labeled with 131I using [131I]SGMIB and evaluated in vitro Biodistribution was evaluated in two HER2+ murine xenograft models by micro-SPECT/CT imaging and at necropsy, and under challenge with trastuzumab and pertuzumab. The therapeutic potential of [131I]SGMIB-2Rs15d was investigated in two HER2+ tumor mouse models. A single-dose toxicity study was performed in mice using unlabeled [127I]SGMIB-sdAb at 1.4 mg/kg. The structure of the 2Rs15d-HER2 complex was determined by X-ray crystallography.Results: [131I]SGMIB-2Rs15d bound specifically to HER2+ cells (Kd = 4.74 ± 0.39 nmol/L). High and specific tumor uptake was observed in both BT474/M1 and SKOV-3 tumor xenografted mice and surpassed kidney levels by 3 hours. Extremely low uptake values were observed in other normal tissues at all time points. The crystal structure revealed that 2Rs15d recognizes HER2 Domain 1, consistent with the lack of competition with trastuzumab and pertuzumab observed in vivo [131I]SGMIB-2Rs15d alone, or in combination with trastuzumab, extended median survival significantly. No toxicity was observed after injecting [127I]SGMIB-2Rs15d.Conclusions: These findings demonstrate the theranostic potential of [131I]SGMIB-2Rs15d. An initial scan using low radioactive [*I]SGMIB-2Rs15d allows patient selection and dosimetry calculations for subsequent therapeutic [131I]SGMIB-2Rs15d and could thereby impact therapy outcome on HER2+ breast cancer patients. Clin Cancer Res; 23(21); 6616-28. ©2017 AACR.

211At labeled substance P (5-11) as potential radiopharmaceutical for glioma treatment.

INTRODUCTION: The purposes of the present work were to label substance P (5-11) with 211At using a rhodium(III) complex with a bifunctional ligand-2-(1,5,9,13-tetrathiacyclohexadecan-3-yloxy)acetic acid ([16aneS4]-COOH) and to assess the in vitro stability and toxicity of the obtained radiobioconjugate. METHODS: Two approaches were evaluated to obtain 131I/211At-Rh[16aneS4]-SP5-11 radiobioconjugates, based on 2-step and 1-step syntheses. In the first method 131I/211At-Rh[16aneS4]-COOH complexes were obtained that required further coupling to a biomolecule. In the second approach, the bioconjugate [16aneS4]-SP5-11 was synthesized and further labeled with 131I and 211At through the utilization of a Rh(III) metal cation bridge. The synthesized compounds were analyzed by HPLC, TLC and paper electrophoresis. RESULTS: The 131I/211At-Rh[16aneS4]-COOH complexes were obtained in high yield and possessed good stability in PBS and CSF. Preliminary studies on coupling of 131I-Rh[16aneS4]-COOH to substance P (5-11) in 2-step synthesis showed that this procedure was too long with respect to 211At half-life, prompting us to improve it by finally using a 1-step synthesis. This strategy not only shortened the labeling time, but also increased final yield of 131I/211At-Rh[16aneS4]-SP5-11 radiobioconjugates. The stability of both compounds in PBS and CSF was high. Toxicity studies with the 211At-Rh[16aneS4]-SP5-11 demonstrated that radiobioconjugate significantly reduced T98G cell viability in a dose dependent manner reaching 20% of survival at the highest radioactivity 1200kBq/mL. CONCLUSIONS: The radiobioconjugate 211At-Rh[16aneS4]-SP5-11 revealed its potential in killing glioma T98G cells during in vitro studies; therefore further animal studies to are required to determine its in vivo stability and treatment potential in normal and xenografted mice.

N-Succinimidyl 3-((4-(4-[(18)F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([(18)F]SFBTMGMB): a residualizing label for (18)F-labeling of internalizing biomolecules.

Residualizing labeling methods for internalizing peptides and proteins are designed to trap the radionuclide inside the cell after intracellular degradation of the biomolecule. The goal of this work was to develop a residualizing label for the (18)F-labeling of internalizing biomolecules based on a template used successfully for radioiodination. N-Succinimidyl 3-((4-(4-[(18)F]fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(bis-Boc-guanidinomethyl)benzoate ([(18)F]SFBTMGMB-Boc2) was synthesized by a click reaction of an azide precursor and [(18)F]fluorohexyne in 8.5 ± 2.8% average decay-corrected radiochemical yield (n = 15). An anti-HER2 nanobody 5F7 was labeled with (18)F using [(18)F]SFBTMGMB ([(18)F]RL-I), obtained by the deprotection of [(18)F]SFBTMGMB-Boc2, in 31.2 ± 6.7% (n = 5) conjugation efficiency. The labeled nanobody had a radiochemical purity of >95%, bound to HER2-expressing BT474M1 breast cancer cells with an affinity of 4.7 ± 0.9 nM, and had an immunoreactive fraction of 62-80%. In summary, a novel residualizing prosthetic agent for labeling biomolecules with (18)F has been developed. An anti-HER2 nanobody was labeled using this prosthetic group with retention of affinity and immunoreactivity to HER2.

Stability and in vivo behavior of Rh[16aneS4-diol]211 at complex: a potential precursor for astatine radiopharmaceuticals.

INTRODUCTION: The heavy halogen (211)At is of great interest for targeted radiotherapy because it decays by the emission of short-range, high-energy α-particles. However, many astatine compounds that have been synthesized are unstable in vivo, providing motivation for seeking other (211)At labeling strategies. One relatively unexplored approach is to utilize prosthetic groups based on astatinated rhodium (III) complex stabilized with a tetrathioether macrocyclic ligand - Rh[16aneS(4)-diol](211)At. The purpose of the current study was to evaluate the in vitro and in vivo stability of this complex in comparison to its iodine analog - Rh[16aneS(4)-diol](131)I. METHODS: Rh[16aneS(4)-diol](211)At and Rh[16aneS(4)-diol](131)I complexes were synthesized and purified by HPLC. The stability of both complexes was evaluated in vitro by incubation in phosphate-buffered saline (PBS) and human serum at different temperatures. The in vivo behavior of the two radiohalogenated complexes was assessed by a paired-label biodistribution study in normal Balb/c mice. RESULTS: Both complexes were synthesized in high yield and purity. Almost no degradation was observed for Rh[16aneS(4)-diol](131)I in PBS over a 72 h incubation. The astatinated analog exhibited good stability in PBS over 14 h. A slow decline in the percentage of intact complex was observed for both tracers in human serum. In the biodistribution study, retention of (211)At in most tissues was higher than that of (131)I at all time points, especially in spleen and lungs. Renal clearance of Rh[16aneS(4)-diol](211)At and Rh[16aneS(4)-diol](131)I predominated, with 84.1 ± 2.3% and 94.6 ± 0.9% of injected dose excreted via the urine at 4 h. CONCLUSIONS: The Rh[16aneS(4)-diol](211)At complex might be useful for constructing prosthetic groups for the astatination of biomolecules and further studies are planned to evaluate this possibility.

D-Amino acid peptide residualizing agents bearing N-hydroxysuccinimido- and maleimido-functional groups and their application for trastuzumab radioiodination.

INTRODUCTION: Proteins that undergo receptor-mediated endocytosis are subject to lysosomal degradation, requiring radioiodination methods that minimize loss of radioactivity from tumor cells after this process occurs. To accomplish this, we developed the residualizing radioiodination agent N(ϵ)-(3-[(*)I]iodobenzoyl)-Lys(5)-N(α)-maleimido-Gly(1)-D-GEEEK (Mal-D-GEEEK-[(*)I]IB), which enhanced tumor uptake but also increased kidney activity and necessitates generation of sulfhydryl moieties on the protein. The purpose of the current study was to synthesize and evaluate a new D-amino acid based agent that might avoid these potential problems. METHODS: N(α)-(3-iodobenzoyl)-(5-succinimidyloxycarbonyl)-D-EEEG (NHS-IB-D-EEEG), which contains 3 D-glutamates to provide negative charge and a N-hydroxysuccinimide function to permit conjugation to unmodified proteins, and the corresponding tin precursor were produced by solid phase peptide synthesis and subsequent conjugation with appropriate reagents. Radioiodination of the anti-HER2 antibody trastuzumab using NHS-IB-D-EEEG and Mal-D-GEEEK-IB was compared. Paired-label internalization assays on BT474 breast carcinoma cells and biodistribution studies in athymic mice bearing BT474M1 xenografts were performed to evaluate the two radioiodinated D-peptide trastuzumab conjugates. RESULTS: NHS-[(131)I]IB-D-EEEG was produced in 53.8%±13.4% and conjugated to trastuzumab in 39.5%±7.6% yield. Paired-label internalization assays with trastuzumab-NHS-[(131)I]IB-D-EEEG and trastuzumab-Mal-D-GEEEK-[(125)I]IB demonstrated similar intracellular trapping for both conjugates at 1h ((131)I, 84.4%±6.1%; (125)I, 88.6%±5.2%) through 24h ((131)I, 60.7%±6.8%; (125)I, 64.9%±6.9%). In the biodistribution experiment, tumor uptake peaked at 48 h (trastuzumab-NHS-[(131)I]IB-D-EEEG, 29.8%±3.6%ID/g; trastuzumab-Mal-D-GEEEK-[(125)I]IB, 45.3%±5.3%ID/g) and was significantly higher for (125)I at all time points. In general, normal tissue levels were lower for trastuzumab-NHS-[(131)I]IB-D-EEEG, with the differences being greatest in kidneys ((131)I, 2.2%±0.4%ID/g; (125)I, 16.9%±2.8%ID/g at 144 h). CONCLUSION: NHS-[(131)I]IB-D-EEEG warrants further evaluation as a residualizing radioiodination agent for labeling internalizing antibodies/fragments, particularly for applications where excessive renal accumulation could be problematic.

N-Succinimidyl guanidinomethyl iodobenzoate protein radiohalogenation agents: influence of isomeric substitution on radiolabeling and target cell residualization.

INTRODUCTION: N-succinimidyl 4-guanidinomethyl-3-[(*)I]iodobenzoate ([(*)I]SGMIB) has shown promise for the radioiodination of monoclonal antibodies (mAbs) and other proteins that undergo extensive internalization after receptor binding, enhancing tumor targeting compared to direct electrophilic radioiodination. However, radiochemical yields for [(131)I]SGMIB synthesis are low, which we hypothesize is due to steric hindrance from the Boc-protected guanidinomethyl group ortho to the tin moiety. To overcome this, we developed the isomeric compound, N-succinimidyl 3-guanidinomethyl-5-[(131)I]iodobenzoate (iso-[(131)I]SGMIB) wherein this bulky group was moved from ortho to meta position. METHODS: Boc2-iso-SGMIB standard and its tin precursor, N-succinimidyl 3-((1,2-bis(tert-butoxycarbonyl)guanidino)methyl)-5-(trimethylstannyl)benzoate (Boc2-iso-SGMTB), were synthesized using two disparate routes, and iso-[*I]SGMIB synthesized from the tin precursor. Two HER2-targeted vectors - trastuzumab (Tras) and a nanobody 5F7 (Nb) - were labeled using iso-[(*)I]SGMIB and [(*)I]SGMIB. Paired-label internalization assays in vitro with both proteins, and biodistribution in vivo with trastuzumab, labeled using the two isomeric prosthetic agents were performed. RESULTS: When the reactions were performed under identical conditions, radioiodination yields for the synthesis of Boc2-iso-[(131)I]SGMIB were significantly higher than those for Boc2-[(131)I]SGMIB (70.7±2.0% vs 56.5±5.5%). With both Nb and trastuzumab, conjugation efficiency also was higher with iso-[(131)I]SGMIB than with [(131)I]SGMIB (Nb, 33.1±7.1% vs 28.9±13.0%; Tras, 45.1±4.5% vs 34.8±10.3%); however, the differences were not statistically significant. Internalization assays performed on BT474 cells with 5F7 Nb indicated similar residualizing capacity over 6h; however, at 24h, radioactivity retained intracellularly for iso-[(131)I]SGMIB-Nb was lower than for [(125)I]SGMIB-Nb (46.4±1.3% vs 56.5±2.5%); similar results were obtained using Tras. Likewise, a paired-label biodistribution of Tras labeled using iso-[(125)I]SGMIB and [(131)I]SGMIB indicated an up to 22% tumor uptake advantage at later time points for [(131)I]SGMIB-Tras. CONCLUSION: Given the higher labeling efficiency obtained with iso-SGMIB, this residualizing agent might be of value for use with shorter half-life radiohalogens.

Radiolabeling and in vitro evaluation of (67)Ga-NOTA-modular nanotransporter--a potential Auger electron emitting EGFR-targeted radiotherapeutic.

INTRODUCTION: Modular nanotransporters (MNTs) are vehicles designed to transport drugs from the cell surface via receptor-mediated endocytosis and endosomal escape to nucleus. Hence their conjugation to Auger electron emitters, can cause severe cell killing, by nuclear localization. Herein we evaluate the use of MNT as a platform for targeted radiotherapy with (67)Ga. METHODS: EGF was the targeting ligand on the MNT, and NOTA was selected for its radiolabeling with (67)Ga. In the radiolabeling study we dealt with the precipitation of MNT (pI 5.7) at the labeling pH (4.5-5.5) of (67)Ga. Cellular and nuclei uptake of (67)Ga-NOTA-MNT by the A431 cell line was determined. Its specific cytotoxicity was compared to that of (67)Ga-EDTA, (67)Ga-NOTA-BSA and (67)Ga-NOTA-hEGF, in A431 and U87MGWTT, cell lines, by clonogenic assay. Dosimetry studies were also performed. RESULTS: (67)Ga-NOTA-MNT was produced with 90% yield and specific activity of 25.6mCi/mg. The in vitro kinetics revealed an increased uptake over 24h. 55% of the internalized radioactivity was detected in the nuclei at 1h. The cytotoxicity of (67)Ga-NOTA-MNT on A431 cell line was 17 and 385-fold higher when compared to non-specific (67)Ga-NOTA-BSA and (67)Ga-EDTA. While its cytotoxic potency was 13 and 72-fold higher when compared to (67)Ga-NOTA-hEGF in the A431 and the U87MGWTT cell lines, respectively, validating its nuclear localization. The absorbed dose, for 63% cell killing, was 8Gy, confirming the high specific index of (67)Ga. CONCLUSION: These results demonstrate the feasibility of using MNT as a platform for single cell kill targeted radiotherapy by Auger electron emitters.