Development of a neopentyl 211At-labeled activated ester providing in vivo stable 211At-labeled antibodies for targeted alpha therapy.

In this study we developed a neopentyl 211At-labeled activated ester that incorporates a triazole spacer and applied it to the synthesis of an 211At-labeled cetuximab. The activated ester was synthesized via the nucleophilic 211At-astatination of a neopentyl sulfonate carrying two long alkyl chains that serve as a lipid tag, which was followed by the hydrolysis of an acetal. Additionally, we developed a novel Resin-Assisted Purification and Deprotection (RAPD) protocol involving a solid-phase extraction of the protected 211At-labeled compound from the mixture of the labeling reaction, hydrolysis of the acetal on the resin, and finally an elution of the 211At-labeled activator from the resin. This method allows the synthesis of an 211At-labeled activated ester with high purity through a simplified procedure that circumvents the need for HPLC purification. Using this 211At-labeled activated ester, we efficiently synthesized 211At-labeled cetuximab in 27±1% radiochemical yield with 95% radiochemical purity. This 211At-activated ester demonstrated high reactivity, and enabled the completion of the reaction with the antibody within 10 min. In comparative biodistribution studies between 211At-labeled cetuximab and the corresponding 125I-labeled cetuximab in normal mice, both the thyroid and stomach showed radioactivity levels that were less than 1.0% of the injected dose.

In vivo stable 211At-labeled prostate-specific membrane antigen-targeted tracer using a neopentyl glycol structure.

BACKGROUND: Prostate cancer is a common cancer among men worldwide that has a very poor prognosis, especially when it progresses to metastatic castration-resistant prostate cancer (mCRPC). Therefore, novel therapeutic agents for mCRPC are urgently required. Because prostate-specific membrane antigen (PSMA) is overexpressed in mCRPC, targeted alpha therapy (TAT) for PSMA is a promising treatment for mCRPC. Astatine-211 (211At) is a versatile α-emitting radionuclide that can be produced using a cyclotron. Therefore, 211At-labeled PSMA compounds could be useful for TAT; however, 211At-labeled compounds are unstable against deastatination in vivo. In this study, to develop in vivo stable 211At-labeled PSMA derivatives, we designed and synthesized 211At-labeled PSMA derivatives using a neopentyl glycol (NpG) structure that can stably retain 211At in vivo. We also evaluated their biodistribution in normal and tumor-bearing mice. RESULTS: We designed and synthesized 211At-labeled PSMA derivatives containing two glutamic acid (Glu) linkers between the NpG structure and asymmetric urea (NpG-L-PSMA ((L-Glu)2 linker used) and NpG-D-PSMA ((D-Glu)2 linker used)). First, we evaluated the characteristics of 125I-labeled NpG derivatives because 125I was readily available. [125I]I-NpG-L-PSMA and [125I]I-NpG-D-PSMA showed low accumulation in the stomach and thyroid, indicating their high in vivo stability against deiodination. [125I]I-NpG-L-PSMA was excreted in urine as hydrophilic radiometabolites in addition to the intact form. Meanwhile, [125I]I-NpG-D-PSMA was excreted in urine in an intact form. In both cases, no radioactivity was observed in the free iodine fraction. [125I]I-NpG-D-PSMA showed higher tumor accumulation than [125I]I-NpG-L-PSMA. We then developed 211At-labeled PSMA using the NpG-D-PSMA structure. [211At]At-NpG-D-PSMA showed low accumulation in the stomach and thyroid in normal mice, indicating its high stability against deastatination in vivo. Moreover, [211At]At-NpG-D-PSMA showed high accumulation in tumor similar to that of [125I]I-NpG-D-PSMA. CONCLUSIONS: [211At]At-NpG-D-PSMA showed high in vivo stability against deastatination and high tumor accumulation. [211At]At-NpG-D-PSMA should be considered as a potential new TAT for mCRPC.

Approaches to Reducing Normal Tissue Radiation from Radiolabeled Antibodies.

Radiolabeled antibodies are powerful tools for both imaging and therapy in the field of nuclear medicine. Radiolabeling methods that do not release radionuclides from parent antibodies are essential for radiolabeling antibodies, and practical radiolabeling protocols that provide high in vivo stability have been established for many radionuclides, with a few exceptions. However, several limitations remain, including undesirable side effects on the biodistribution profiles of antibodies. This review summarizes the numerous efforts made to tackle this problem and the recent advances, mainly in preclinical studies. These include pretargeting approaches, engineered antibody fragments and constructs, the secondary injection of clearing agents, and the insertion of metabolizable linkages. Finally, we discuss the potential of these approaches and their prospects for further clinical application.

Reduction of the hepatic radioactivity levels of [111In]In-DOTA-labeled antibodies via cleavage of a linkage metabolized in lysosomes.

INTRODUCTION: Radiolabeled antibodies are promising tools for cancer diagnosis using nuclear medicine. A DOTA-chelating system is useful for preparing immuno-positron emission tomography and immuno-single-photon emission computed tomography probes with various radiometals. Radiolabeled antibodies are generally metabolized in the reticuloendothelial system, producing radiometabolites after proteolysis in hepatic lysosomes. Because of the bulkiness and extremely high hydrophilicity of DOTA, radiometabolites containing a radiometal-DOTA complex typically exhibit high and persistent localization in hepatic lysosomes. Radioactivity in the liver impairs the accurate diagnosis of cancer surrounding the liver and liver metastasis, and a high tumor/liver ratio is desirable. In this study, we reduced the hepatic radioactivity of radiometal-labeled antibodies containing a DOTA-chelating system. A cleavable linkage was inserted to liberate the radiometabolite, which exhibited a short residence time in hepatocytes. METHODS: Using indium-111 (111In)-labeled antibodies, we prepared 111In-labeled galactosyl-neoglycoalbumins (NGAs) because they are useful for evaluating the residence time of radiometabolites in the liver. An 111In-labeled NGA with a cleavable linkage ([111In]In-DO3AiBu-Bn-FGK-NGA) was administered to normal mice, and biodistribution studies and metabolic analyses of urinary and fecal samples were performed with comparison to an 111In-labeled NGA prepared by a conventional method ([111In]In-DOTA-Bn-SCN-NGA). Then, 111In-labeled antibodies ([111In]In-DO3AiBu-Bn-FGK-IgG and [111In]In-DOTA-Bn-SCN-IgG) were prepared using a procedure similar to that for 111In-labeled NGAs. In vitro plasma stability and biodistribution were investigated for both 111In-labeled antibodies in U87MG tumor-bearing mice. RESULTS: Through the liberation of radiometabolites including [111In]In-DO3AiBu-Bn-F, [111In]In-DO3AiBu-Bn-FGK-NGA was cleared more rapidly from the liver than [111In]In-DOTA-Bn-SCN-NGA (4.07 ± 1.54%ID VS 71.68 ± 3.03%ID at 6 h postinjection). [111In]In-DO3AiBu-Bn-FGK-IgG exhibited lower tumor accumulation (8.83 ± 1.48%ID/g) but a significantly higher tumor/liver ratio (2.21 ± 0.53) than [111In]In-DOTA-Bn-SCN-IgG (11.65 ± 2.17%ID/g in the tumor and a tumor/liver ratio of 0.85 ± 0.18) at 72 h after injection. CONCLUSION: A molecular design that reduces the high and persistent hepatic radioactivity of radiolabeled antibodies by liberating radiometabolites with a short hepatic residence time in lysosomes would be applicable for radiometal-labeled antibodies using a DOTA-chelating system.

Neopentyl glycol-based radiohalogen-labeled amino acid derivatives for cancer radiotheranostics.

BACKGROUND: L-type amino acid transporter 1 (LAT1) is overexpressed in various cancers; therefore, radiohalogen-labeled amino acid derivatives targeting LAT1 have emerged as promising candidates for cancer radiotheranostics. However, 211At-labeled amino acid derivatives exhibit instability against deastatination in vivo, making it challenging to use 211At for radiotherapy. In this study, radiohalogen-labeled amino acid derivatives with high dehalogenation stability were developed. RESULTS: We designed and synthesized new radiohalogen-labeled amino acid derivatives ([211At]At-NpGT, [125I]I-NpGT, and [18F]F-NpGT) in which L-tyrosine was introduced into the neopentyl glycol (NpG) structure. The radiolabeled amino acid derivatives were recognized as substrates of LAT1 in the in vitro studies using C6 glioma cells. In a biodistribution study using C6 glioma-bearing mice, these agents exhibited high stability against in vivo dehalogenation and similar biodistributions. The similarity of [211At]At-NpGT and [18F]F-NpGT indicated that these pairs of radiolabeled compounds would be helpful in radiotheranostics. Moreover, [211At]At-NpGT exhibited a dose-dependent inhibitory effect on the growth of C6 glioma-bearing mice. CONCLUSIONS: [211At]At-NpGT exhibited a dose-dependent inhibitory effect on the tumor growth of glioma-bearing mice, and its biodistribution was similar to that of other radiohalogen-labeled amino acid derivatives. These findings suggest that radiotheranostics using [18F]F-NpGT and [123/131I]I-NpGT for diagnostic applications and [211At]At-NpGT and [131I]I-NpGT for therapeutic applications are promising.

Enhanced Therapeutic Effect of Liposomal Doxorubicin via Bio-Orthogonal Chemical Reactions in Tumors.

Liposomes are highly biocompatible drug carriers in drug delivery systems (DDSs). Preferential accumulation of liposomes and acceleration of drug release at target tumor sites are essential for effective cancer therapy using liposomal formulations; however, conventional liposomes are unsuitable for on-demand drug release. We have previously reported that drug release can be accelerated via a bio-orthogonal inverse electron demand Diels-Alder (IEDDA) reaction between amphiphilic tetrazine (Tz)-containing liposomes and norbornene (NB) derivatives in vitro. In this study, we prepared HSTz-liposomes composed of hydrogenated soybean phosphatidylcholine (HSPC) and Tz compound (2-hexadecyl-N-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)octadecanamide) with particle sizes of 60-80 nm and ζ-potentials of -5 to 0 mV. Similar to our previous report, the addition of 5-norbornene-2-carboxylic acid (NBCOOH) to HSTz-liposomes accelerated drug release from the liposomes in vitro. In the biodistribution study using colon26 tumor-bearing mice, the radiolabeled HSTz-liposomes were accumulated and retained in the tumor at 6-48 h post-injection, whereas the radioactivity in the blood almost disappeared at 48 h. Therefore, the timing of the injection of NBCOOH was selected to be 48 h after the injection of the HSTz-liposome to avoid the IEDDA reaction in the bloodstream. We investigated the in vivo drug release by evaluating the intratumoral localization of doxorubicin (DOX) encapsulated in HSTz-liposomes labeled with fluorescent lipids. In the tumors treated with HSTz-liposomes and NBCOOH, DOX was more widely dispersed in the tumor compared with fluorescent lipid, suggesting that the release of encapsulated drugs (DOX) from HSTz-liposomes was enhanced in the tumor tissue via the bio-orthogonal IEDDA reaction. Furthermore, the combination of DOX-encapsulated HSTz-liposomes with NBCOOH significantly suppressed tumor growth compared to conventional DOX-encapsulated liposomes. In conclusion, the bio-orthogonal IEDDA reactions in the liposomal membrane enabled the acceleration of drug release from HSTz-liposomes in vivo, suggesting a promising strategy for effective cancer therapy.

Inverse electron-demand diels-alder reactions of tetrazine and norbornene at the air-water interface.

The surface chemistry of the inverse electron-demand Diels-Alder (IEDDA) reaction at the air-water interface is elucidated. Tetrazine (C18-Tz) and norbornene derivatives (C16-NCA) were used as the reactants. Langmuir monolayers of C18-Tz, C16-NCA, and their binary mixtures were prepared on aqueous substrates. The surface properties were analyzed using the surface pressure (π)-molecular area (A) and surface potential (ΔV)-A isotherms, as well as fluorescence microscopy to monitor the progress of the reaction. First, to provide comparison data to evaluate the reaction on the surface, the two components were mixed in stock solutions of organic solvents for the IEDDA reaction. The Langmuir monolayer spread from the reaction solution was characterized as a function of the reaction time. In the subsequent experiments, the Langmuir monolayers were deposited onto the surface of the substrate solutions by spreading from separate stock solutions of C18-Tz and C16-NCA. The variation of the surface behavior of the monolayers with the molecular area, surface composition of the two components, compression speed of the monolayers, and the temperature was studied. We discuss the effects of the air phase in the reaction field on the reaction efficiency by comparing the results obtained from the two methods.

Inverse Electron Demand Diels-Alder Reactions in the Liposomal Membrane Accelerates Release of the Encapsulated Drugs.

Bio-orthogonal inverse electron demand Diels-Alder (IEDDA) reactions between liposomes containing a tetrazine-based (Tz) compound and 2-norbornene (2-NB) could be a novel trigger for accelerating drug release from the liposomes via temporary membrane destabilization, as shown in our previous report. Herein, we evaluated the in vitro drug release using NB derivatives with carboxyl groups [5-norbornene-2-carboxylic acid (NBCOOH) and 5-norbornene-2,3-dicarboxylic acid (NB(COOH)2)] to investigate the effects of substituents at the NB backbone on the drug release rate. First, POTz-liposome composed of a Tz compound (2-hexadecyl-N-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)octadecanamide) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) were prepared. The mass spectrometry analysis revealed the binding of NB derivatives to the Tz compound via the IEDDA reaction after the POTz-liposome reacted with the NB derivatives. Indium-111-labeled diethylenetriaminepentaacetic acid (111In-DTPA) was encapsulated inside the liposomes, and the drug release rate was quantified by measuring radioactivity. At 24 h after incubation with 2-NB, NBCOOH, and NB(COOH)2, the release rates of 111In-DTPA from POTz-liposome were 21.0, 80.8, and 23.3%, respectively, which were significantly higher than those of POTz-liposome that was not treated with NB derivatives (4.2%), indicating the involvement of the IEDDA reaction for prompting drug release. Additionally, a thermodynamic evaluation using Langmuir monolayers was conducted to explore the mechanism of the accelerated drug release. An increase in membrane fluidity and a reduction in intermolecular repulsion between POPC and the Tz compound were observed after the reaction with NB derivatives, especially for NBCOOH. Thus, the IEDDA reaction in the liposomal membrane could be a potent trigger for accelerating the release of encapsulated drugs by regulating membrane fluidity and intermolecular repulsion in the liposomal membrane.

Synthesis of an amphiphilic tetrazine derivative and its application as a liposomal component to accelerate release of encapsulated drugs.

Tetrazine irreversibly reacts with dienophiles, and its derivatives find wide applications in the fields of biochemistry and biophysics. We have synthesized an amphiphilic tetrazine derivative (2-hexadecyl-N-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazine-3-yl)pyridin-3-yl)octadecanamide; 1), which has a hydrophilic tetrazine structure and hydrophobic alkyl chains. Liposomes composed of compound 1 and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) (PTz-liposome) were prepared. In search of a new drug delivery system (DDS), we investigated the viability of inverse electron-demand Diels-Alder, a reaction between tetrazine and 2-norbornene, on the surface of the liposomes to change membrane fluidity and promote spatial and temporal controlled release of the encapsulated drugs. Compound 1 was synthesized with a yield of 71%. MS analysis after incubation of 2-norbornene with PTz-liposome revealed the binding of 2-norbornene to tetrazine. Indium-111-labeled diethylenetriaminepentaacetic acid (111In-DTPA) was encapsulated inside PTz-liposome to evaluate the leakage of free 111In-DTPA from the liposomes quantitatively. After 24 h of adding 2-norbornene, the release percentage for PTz-liposome was significantly higher than that for the control liposome (without tetrazine structure). Furthermore, the membrane fluidity of the PTz-liposome was increased by adding 2-norbornene. These results suggested that the combination of dienophile and liposome containing a newly synthesized tetrazine derivative can be used as a controlled release DDS carrier.