RESUMO
Radioimmunoconjugates (RICs) composed of tumor-targeting monoclonal antibodies and radionuclides have been developed for diagnostic and therapeutic application. A new radiolabeling method using microfluidic devices is expected to facilitate simpler and more rapid synthesis of RICs. In the microfluidic method, microfluidic chips can promote the reaction between reactants by mixing them efficiently, and pumping systems enable automated synthesis. In this study, we synthesized RICs by the pre-labeling method, in which the radiometal is coordinated to the chelator and then the radiolabeled chelator is incorporated into the antibodies, using microfluidic devices for the first time. As a result of examining the reaction parameters including the material of mixing units, reaction temperature, and flow rate, RICs with radiochemical purity (RCP) exceeding 90% were obtained. These high-purity RICs were successfully synthesized without any purification simply by pumping three solutions of a chelating agent, radiometal, and antibody into microfluidic devices. Under the same conditions, the RCP of RICs labeled by conventional methods was below 50%. These findings indicate the utility of microfluidic devices for automatic and rapid synthesis of high-quality RICs.
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In the field of radiopharmaceutical development targeting cancer, an albumin binder (ALB) is commonly used to improve accumulation of radioligands in tumors because it has high binding affinity for albumin and extends the circulation time of radioligands. The further development of ALB-containing radioligands is also expected to regulate their pharmacokinetics. In this study, we newly designed and synthesized [111In]In-PNT-DA1 derivatives, prostate-specific membrane antigen (PSMA)-targeting radioligands including a functional linker (d-glutamic acid or 4-(aminomethyl)benzoic acid), and evaluated the relationships among the structure, albumin-binding affinity, and pharmacokinetics. These derivatives showed a different binding affinity for albumin by the introduction of a linker. Biodistribution studies revealed that the introduction of a linker affects the pharmacokinetics of each derivative. The biodistribution studies also suggested that moderate albumin-binding affinity enhances the tumor/kidney ratio of the derivative. SPECT imaging using [111In]In-PNT-DA3 with the highest tumor/kidney ratio among [111In]In-PNT-DA1 derivatives led to clear visualization of a PSMA-positive LNCaP tumor. The results suggest that the appropriate introduction of linker entities may be necessary to improve the pharmacokinetics of PSMA-targeting radioligands.
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Cathepsin B (CTSB) is a lysosomal protease that is overexpressed in tumor cells. Radioimmunoconjugates (RICs) composed of CTSB-recognizing chelating agents are expected to increase the molecular weights of their radiometabolites by forming conjugates with CTSB in cells, resulting in their improved retention in tumor cells. We designed a novel CTSB-recognizing trifunctional chelating agent, azide-[111In]In-DOTA-CTSB-substrate ([111In]In-ADCS), to synthesize a RIC, trastuzumab-[111In]In-ADCS ([111In]In-TADCS), and evaluated its utility to improve tumor retention of the RIC. [111In]In-ADCS and [111In]In-TADCS were synthesized with satisfactory yield and purity. [111In]In-ADCS was markedly stable in murine plasma until 96 h postincubation. [111In]In-ADCS showed binding to CTSB in vitro, and the conjugation was blocked by the addition of CTSB inhibitor. In the internalization assay, [111In]In-TADCS exhibited high-level retention in SK-OV-3 cells, indicating the in vitro utility of the CTSB-recognizing unit. In the biodistribution assay, [111In]In-TADCS showed high-level tumor accumulation, but the retention was hardly improved. In the first attempt to combine a CTSB-recognizing unit and RIC, these findings show the fundamental properties of the CTSB-recognizing trifunctional chelating agent to improve tumor retention of RICs.
Assuntos
Catepsina B , Quelantes , Imunoconjugados , Catepsina B/metabolismo , Quelantes/química , Quelantes/síntese química , Animais , Camundongos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Distribuição Tecidual , Linhagem Celular Tumoral , Humanos , Radioisótopos de Índio/química , Técnicas de Química Sintética , Trastuzumab/químicaRESUMO
INTRODUCTION: The pretargeting approach consists of in vivo ligation between pre-injected antibodies and low-molecular-weight radiolabeled effectors. The advantage of the pretargeting approach is to improve a tumor-to-background ratio, but the disadvantage is to compromise tumor accumulation. In this study, we applied albumin binder (ALB) to the pretargeting approach to overcome low tumor accumulation. METHODS: We synthesized two novel trifunctional effectors containing an ALB moiety, a chelator, and a different tetrazine and two corresponding effectors without an ALB moiety. Albumin-binding assays and stability assays were performed using 111In-labeled effectors. Measurements of reaction rate constant were conducted using 111In-labeled effectors and anti-HER2 antibody trastuzumab modified by trans-cyclooctene, which drives the click reaction with tetrazine. Biodistribution studies using HER2-expressing tumor-bearing mice were performed with or without the pretargeting approach. RESULTS: In albumin-binding assays, ALB-containing effectors exhibited a marked binding to albumin. Two ALB-containing effectors showed the difference in the reactivity and the slight difference in the stability. In biodistribution studies without the pretargeting approach, two ALB-containing effectors showed different pharmacokinetics in blood retention. With the pretargeting approach, the tumor accumulation was improved by the introduction of ALB and the highest tumor accumulation was observed in using the ALB-containing effector with higher blood retention. CONCLUSION: These results suggest that the application of ALB to the pretargeting approach is effective to improve tumor accumulation, and the structure of tetrazine influences the utility of ALB-containing effectors.
Assuntos
Quelantes , Animais , Camundongos , Quelantes/química , Quelantes/síntese química , Distribuição Tecidual , Linhagem Celular Tumoral , Humanos , Técnicas de Química Sintética , Feminino , Albuminas/química , Receptor ErbB-2/metabolismo , Trastuzumab/química , Trastuzumab/farmacocinéticaRESUMO
Chelator-containing radioimmunoconjugates (RICs) composed of monoclonal antibodies, chelators, and radiometals exhibit broad potential for cancer diagnosis or therapy. In this study, we developed novel trifunctional chelating agents that enhance the tumor retention of RICs, MDPEI2, and MDPEI4, which contain the metal chelator DOTA, a maleimide moiety, and diethylenetriamine (PEI2) or tetraethylenepentamine (PEI4), respectively, as a poly(ethylenimine) (PEI) scaffold for the addition of positive charges to the radiometabolites of RICs to reduce their release from tumor cells. Trastuzumab radiolabeled by [111In]In-MDPEI2 ([111In]In-TMDPEI2) or [111In]In-MDPEI4 ([111In]In-TMDPEI4) showed high immunoreactivity and lower rates of exportations of their radiometabolites from tumor cells than RICs without PEI scaffolds. The tumor uptake of [111In]In-TMDPEI2 and [111In]In-TMDPEI4 was enhanced compared with RICs without PEI scaffolds, and [111In]In-TMDPEI2 exhibited the highest tumor/blood ratio. These results indicate the utility of MDPEI2 to synthesize RICs with favorable tumor-targeting properties in vivo by controlling the radioactivity distribution in tumor cells.
Assuntos
Imunoconjugados , Neoplasias , Humanos , Quelantes , Anticorpos Monoclonais , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológico , Trastuzumab , Linhagem Celular TumoralRESUMO
Prostate-specific membrane antigen (PSMA) is a promising target for metastatic castration-resistant prostate cancer. We previously reported the effectiveness of PSMA-DA1 as a PSMA-targeting radiotheranostic agent containing an albumin binder moiety. To further enhance tumor uptake, we newly designed PSMA-NAT-DA1 (PNT-DA1) by the introduction of a lipophilic linker into PSMA-DA1. The PSMA affinity of [111In]In-PNT-DA1 was increased (Kd = 8.20 nM) compared with that of [111In]In-PSMA-DA1 (Kd = 89.4 nM). [111In]In-PNT-DA1 showed markedly high tumor accumulation (131.6% injected dose/g at 48 h post-injection), and [111In]In-PNT-DA1 enabled the visualization of the tumor clearly at 24 h post-injection with SPECT/CT imaging. The administration of [225Ac]Ac-PNT-DA1 (2.5 kBq) led to shrinkage of the tumor without marked toxicity, and the antitumor effects of [225Ac]Ac-PNT-DA1 were superior to those of [225Ac]Ac-PSMA-DA1 and [225Ac]Ac-PSMA-617, which is the current gold standard for PSMA-targeting 225Ac-endoradiotherapy. These results suggest that the combination of [111In]In-PNT-DA1 and [225Ac]Ac-PNT-DA1 comprises a promising method of PSMA-targeting radiotheranostics.
Assuntos
Glutamato Carboxipeptidase II , Neoplasias da Próstata , Humanos , Masculino , Albuminas , Antígenos de Superfície , Linhagem Celular Tumoral , Índio/química , Neoplasias da Próstata/patologia , Compostos Radiofarmacêuticos/uso terapêutico , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único , Radioisótopos de Índio/química , Radioisótopos de Índio/uso terapêuticoRESUMO
We have reported that the chelator-based clickable radiotheranostic platform, ADIBO-DOTADG-ALB (ADA), has favorable properties as a radiotheranostic platform for low-molecular-weight ligands. In this study, we evaluated the applicability of ADA to moderate-molecular-weight ligands to expand the utility of the ADA platform. As a moderate-molecular-weight ligand, we selected exendin-4, a peptide-based agonist to glucagon-like peptide-1 receptor (GLP-1R). An exendin-4-incorporated ADA derivative, exendin-4-Cys40-triazole-DOTADG-ALB (EtDA), was radiolabeled with 111In by the conjugation of exendin-4-Cys40 azide to [111In]In-ADA. The click ligation of exendin-4-Cys40 azide to [111In]In-ADA was quantitatively completed in 10 min under ambient conditions. In the in vitro cell-binding assay and albumin-binding assay, [111In]In-EtDA showed strong binding to both a GLP-1R-expressing cell and albumin. In the biodistribution assay, [111In]In-EtDA showed markedly protracted tumor uptake, which was significantly decreased by the coinjection of exendin-4-Cys40. The single photon emission computed tomography (SPECT) image of [111In]In-EtDA visualized the tumor clearly. These results indicated the utility of [111In]In-EtDA as a radiotheranostic agent, suggesting the applicability of the ADA platform to moderate-molecular-weight ligands.
RESUMO
225Ac-based radiotheranostics targeting prostate-specific membrane antigen (PSMA) has induced impressive responses in patients with metastatic castration-resistant prostate cancer. To enhance the therapeutic effects of radioligands labeled with 225Ac (half-life: 10 days), a radioligand that shows longer tumor retention would be useful. Here, we designed and synthesized a straight-chain PSMA-targeting radioligand, PSMA-DA1, which includes an (iodophenyl)butyric acid derivative as an albumin binder (ALB). We performed preclinical evaluations of PSMA-DA1 as a tool for PSMA-targeting radiotheranostics using 111In, 90Y, and 225Ac. [111In]In-PSMA-DA1 demonstrated significantly greater tumor uptake and retention than a corresponding non-ALB-conjugated compound. In mice, single-photon emission computed tomography performed with [111In]In-PSMA-DA1 produced clear tumor images, and the administration of [90Y]Y-PSMA-DA1 or [225Ac]Ac-PSMA-DA1 inhibited tumor growth. [225Ac]Ac-PSMA-DA1 had antitumor effects in mice at a lower radioactivity level than [225Ac]Ac-PSMA-617, which has been reported to be clinically useful. These results indicate that PSMA-DA1 may be a useful PSMA-targeting radiotheranostic agent.
Assuntos
Antineoplásicos/uso terapêutico , Glutamato Carboxipeptidase II/metabolismo , Glicoproteínas de Membrana/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Compostos Radiofarmacêuticos/uso terapêutico , Actínio/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Ciclamos/síntese química , Ciclamos/metabolismo , Ciclamos/farmacocinética , Ciclamos/uso terapêutico , Humanos , Ligantes , Masculino , Camundongos Endogâmicos ICR , Camundongos SCID , Medicina de Precisão/métodos , Neoplasias de Próstata Resistentes à Castração/diagnóstico por imagem , Neoplasias de Próstata Resistentes à Castração/metabolismo , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único , Ureia/análogos & derivados , Ureia/metabolismo , Ureia/farmacocinética , Ureia/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Radiotheranostics has attracted attention as a powerful strategy for treating cancer patients with precision medicine. We designed and synthesized a novel DOTA-based trifunctional agent, ADIBO-DOTADG-ALB (ADA), which allowed compounds with targeting ligands, radiometals, and an albumin binder to be readily prepared. ADA exhibited promising properties as a theranostic platform.
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INTRODUCTION: Carbonic anhydrase-IX (CA-IX) is markedly overexpressed in many types of solid tumors promoting tumorigenicity and tumor growth. We synthesized novel 68Ga-labeled imidazothiadiazole sulfonamide (IS) derivatives ([68Ga]Ga-DO3A-IS1 and [68Ga]Ga-DO2A-IS2), and evaluated their utility as positron emission tomography (PET) probes targeting CA-IX. METHODS: [67/68Ga]Ga-DO3A-IS1 and [67/68Ga]Ga-DO2A-IS2 were synthesized from corresponding precursors by ligand substitution reaction in acetate buffer. Cell binding assays were performed using HT-29 cells, which highly express CA-IX, and MDA-MB-231 cells, which show lower-level expression of CA-IX, and a biodistribution assay with model mice bearing the HT-29 or MDA-MB-231 tumor was performed. [68Ga]Ga-DO3A-IS1 was further evaluated by PET/CT. RESULTS: To evaluate their fundamental properties, [67Ga]Ga-DO3A-IS1 and [67Ga]Ga-DO2A-IS2 were synthesized by conjugation with 67Ga, which has a much longer decay half-life and can be utilized more easily than 68Ga. [67/68Ga]Ga-DO3A-IS1 and [67/68Ga]Ga-DO2A-IS2 were prepared from corresponding precursors with preferable yield and purity. [67Ga]Ga-DO3A-IS1 and [67Ga]Ga-DO2A-IS2 showed significantly greater binding to HT-29 cells than MDA-MB-231 cells in vitro and the binding of [67Ga]Ga-DO2A-IS2 to HT-29 cells was much greater than that of [67Ga]Ga-DO3A-IS1, suggesting multivalent interactions. [67Ga]Ga-DO3A-IS1 and [67Ga]Ga-DO2A-IS2 showed significant selectivity for the HT-29 tumor in vivo, while tumor uptake of [67Ga]Ga-DO3A-IS1 was greater than that of [67Ga]Ga-DO2A-IS2. PET/CT of [68Ga]Ga-DO3A-IS1 showed selectivity for the HT-29 tumor, although [68Ga]Ga-DO3A-IS1 could not be used to visualize the HT-29 tumor clearly because of its strong background signals. CONCLUSION: These results indicate that 68Ga-labeled IS derivatives may be useful 68Ga-PET probes targeting CA-IX with further structural modifications.