SPECT Imaging of Lysyl Oxidase-like 2 in a Model of Idiopathic Pulmonary Fibrosis.

Noninvasive imaging of idiopathic pulmonary fibrosis (IPF) remains a challenge. The aim of this study was to develop an antibody-based radiotracer targeting Lysyl Oxidase-like 2 (LOXL2), an enzyme involved in the fibrogenesis process, for SPECT/CT imaging of pulmonary fibrosis. The bifunctional chelator DOTAGA-PEG4-NH2 was chemoenzymatically conjugated to the murine antibody AB0023 using microbial transglutaminase, resulting in a degree of labeling (number of chelators per antibody) of 2.3. Biolayer interferometry confirmed that the binding affinity of DOTAGA-AB0023 to LOXL2 was preserved with a dissociation constant of 2.45 ± 0.04 nM. DOTAGA-AB0023 was then labeled with 111In and in vivo experiments were carried out in a mice model of progressive pulmonary fibrosis induced by intratracheal administration of bleomycin. [111In]In-DOTAGA-AB0023 was injected in three groups of mice (control, fibrotic, and treated with nintedanib). SPECT/CT images were recorded over 4 days p.i. and an ex vivo biodistribution study was performed by gamma counting. A significant accumulation of the tracer in the lungs of the fibrotic mice was observed at D18 post-bleomycin. Interestingly, the tracer uptake was found selectively upregulated in fibrotic lesions observed on CT scans. Images of mice that received the antifibrotic drug nintedanib from D8 up to D18 showed a decrease in [111In]In-DOTAGA-AB0023 lung uptake associated with a decrease in pulmonary fibrosis measured by CT scan. In conclusion, we report the first radioimmunotracer targeting the protein LOXL2 for nuclear imaging of IPF. The tracer showed promising results in a preclinical model of bleomycin-induced pulmonary fibrosis, with high lung uptake in fibrotic areas, and accounted for the antifibrotic activity of nintedanib.

Tripodal scaffolds with three appended imidazole thiones for Cu(I) chelation and protection from Cu-mediated oxidative stress.

Imidazole thiones appear as interesting building blocks for Cu(I) chelation and protection against Cu-mediated oxidative stress. Therefore, a series of tripodal molecules derived from nitrilotriacetic acid appended with three imidazole thiones belonging either to histamine-like or histidine-like moieties were synthesized. These tripods demonstrate intermediate affinity between that previously measured for tripodal analogues bearing three thiol moieties such as cysteine and those grafted with three thioethers, like methionines, consistently with the thione group in the imidazole thione moiety existing as a tautomer between a thiol and a thione. The two non-alkylated tripods derived from thioimidazole, TH and TH* demonstrated three orders of magnitude larger affinity for Cu(I) (logKpH 7.4 = 14.3) than their analogues derived from N,N'-dialkylated thioimidazole TMe and TEt (logKpH 7.4 = 11-11.6). Their efficiency to inhibit Cu-mediated oxidative stress is demonstrated by several assays involving ascorbate consumption or biomolecule damages and correlates with their ability to chelate Cu(I), related to their conditional complexation constants at pH 7.4. The two non-alkylated tripods derived from thioimidazole, TH and TH* are significantly more powerful in reducing Cu-mediated oxidative stress than their analogues derived from N,N'-dialkylated thioimidazole TMe and TEt.

Systemic Delivery of Tumor-Targeted Bax-Derived Membrane-Active Peptides for the Treatment of Melanoma Tumors in a Humanized SCID Mouse Model.

Melanoma is a highly metastatic and deadly form of cancer. Invasive melanoma cells overexpress integrin αvß3, which is a well-known target for Arg-Gly-Asp-based (RGD) peptides. We developed a sophisticated method to synthetize milligram amounts of a targeted vector that allows the RGD-mediated targeting, internalization, and release of a mitochondria-disruptive peptide derived from the pro-apoptotic Bax protein. We found that 2.5 µM Bax[109-127] was sufficient to destabilize the mitochondria in ten different tumor cell lines, even in the presence of the anti-apoptotic Bcl2 protein, which is often involved in tumor resistance. This pore-forming peptide displayed antitumor activity when it was covalently linked by a disulfide bridge to the tetrameric RAFT-c[RGD]4-platform and after intravenous injection in a human melanoma tumor model established in humanized immuno-competent mice. In addition to its direct toxic effect, treatment with this combination induced the release of the immuno-stimulating factor monocyte chimoattractant protein 1 (MCP1) in the blood and a decrease in the level of the pro-angiogenic factor FGF2. Our novel multifunctional, apoptosis-inducing agent could be further customized and assayed for potential use in tumor-targeted therapy.

Biomolecular assemblies combining two orthogonal copper-mediated ligations in a one-pot reaction.

The access to multifunctional biomolecular compounds involves multistep reactions usually with a complicated protection scheme and lengthy separation processes. The development of a strategy combining several orthogonal ligations is highly desirable. Herein, we introduce a new method that involves two orthogonal copper-mediated ligations of azide with alkyne, and amine with thioacid. We established compatible conditions to carry out molecular assemblies of three different chemical components in a single one-pot reaction. The effectiveness of the method was demonstrated in the synthesis of biomolecular compounds that are known to target tumor tissue. The simple reaction conditions suggest that this strategy of combining several orthogonal ligations could have wide potential for the chemical synthesis of complex macromolecules.

Redox-driven host-guest interactions allow the controlled release of captured cells on RGD-functionalized surfaces.

A quartz crystal microbalance technique with dissipation monitoring and a complementary optical microscopy technique were used for monitoring the capture and release of specific cells on a surface displaying a bifunctional molecular device, composed of a molecular scaffold endowed with the cell recognition property of an RGD ligand and a ß-CD/Fc redox-switchable system.

PET imaging and biodistribution analysis of the effects of succinylated gelatin combined with L-lysine on renal uptake and retention of 64Cu-cyclam-RAFT-c(-RGDfK-)4 in vivo.

(64)Cu-cyclam-RAFT-c(-RGDfK-)4, an αVß3 integrin-targeting tetrameric cyclic RGD peptide probe, is a potential theranostic compound for positron emission tomography (PET) of tumor angiogenesis and for internal radiotherapy owing to the multiple decay modes of (64)Cu. Since kidneys are dose-limiting organs in internal radiotherapy, we aimed to reduce the renal accumulation of (64)Cu-cyclam-RAFT-c(-RGDfK-)4 by co-injection with Gelofusine (GF), a succinylated gelatin solution, and/or L-lysine (Lys), and to explore, for the first time, the related mechanisms using the noninvasive and quantitative PET imaging technology. Biodistribution assays, dynamic and static PET scans, and metabolism studies with radio-thin-layer chromatography (radio-TLC) were performed in healthy or αVß3-positive tumor-bearing mice. In the results, co-injection with GF markedly reduced the renal uptake and slightly increased the tumor uptake of (64)Cu-cyclam-RAFT-c(-RGDfK-)4. L-Lysine alone had no effect on the probe biodistribution, but the combined use of Lys and GF tended to enhance the effect of GF. Dynamic PET and metabolite analysis by radio-TLC highly revealed that GF blocks the renal reabsorption of (64)Cu-cyclam-RAFT-c(-RGDfK-)4, but does not interfere with its metabolism and excretion. In conclusion, administration of GF and Lys is a useful strategy for kidney protection in (64)Cu-cyclam-RAFT-c(-RGDfK-)4-based internal radiotherapy.

Micro-positron emission tomography/contrast-enhanced computed tomography imaging of orthotopic pancreatic tumor-bearing mice using the αvß3 integrin tracer 64Cu-labeled cyclam-RAFT-c(-RGDfK-)4.

The purpose of this study was to develop a clinically relevant orthotopic xenotransplantation model of pancreatic cancer and to perform a preclinical evaluation of a new positron emission tomography (PET) imaging probe, 64Cu-labeled cyclam-RAFT-c(-RGDfK-)4 peptide (64Cu-RAFT-RGD), using this model. Varying degrees of αvß3 integrin expression in several human pancreatic cancer cell lines were examined by flow cytometry and Western blotting. The cell line BxPC-3, which is stably transfected with a red fluorescence protein (RFP), was used for surgical orthotopic implantation. Orthotopic xenograft was established in the pancreas of recipient nude mice. An in vivo probe biodistribution and receptor blocking study, preclinical PET imaging coregistered with contrast-enhanced computed tomography (CECT) comparing 64Cu-RAFT-RGD and ¹8F-fluoro-2-deoxy-d-glucose (¹8F-FDG) accumulation in tumor, postimaging autoradiography, and histologic and immunohistochemical examinations were done. Biodistribution evaluation with a blocking study confirmed that efficient binding of probe to tumor is highly αvß3 integrin specific. 64Cu-RAFT-RGD PET combined with CECT provided for precise and easy detection of cancer lesions. Autoradiography, histologic, and immunohistochemical examinations confirmed the accumulation of 64Cu-RAFT-RGD in tumor versus nontumor tissues. In comparative PET studies, 64Cu-RAFT-RGD accumulation provided better tumor contrast to background than ¹8F-FDG. Our results suggest that 64Cu-RAFT-RGD PET imaging is potentially applicable for the diagnosis of αvß3 integrin-expressing pancreatic tumors.

Positron emission tomography imaging of tumor angiogenesis and monitoring of antiangiogenic efficacy using the novel tetrameric peptide probe 64Cu-cyclam-RAFT-c(-RGDfK-)4.

64Cu-cyclam-RAFT-c(-RGDfK-)4 is a novel multimeric positron emission tomography (PET) probe for αVß3 integrin imaging. Its uptake and αVß3 expression in tumors showed a linear correlation. Since αVß3 integrin is strongly expressed on activated endothelial cells during angiogenesis, we aimed to determine whether 64Cu-cyclam-RAFT-c(-RGDfK-)4 PET can be used to image tumor angiogenesis and monitor the antiangiogenic effect of a novel multi-targeted tyrosine kinase inhibitor, TSU-68. Athymic nude mice bearing human hepatocellular carcinoma HuH-7 xenografts, which expressed negligible αVß3 levels on the tumor cells, received intraperitoneal injections of TSU-68 or the vehicle for 14 days. Antiangiogenic effects were determined at the end of therapy in terms of 64Cu-cyclam-RAFT-c(-RGDfK-)4 uptake evaluated using PET, biodistribution assay, and autoradiography, and they were compared with microvessel density (MVD) determined by CD31 immunostaining. 64Cu-cyclam-RAFT-c(-RGDfK-)4 PET enabled clear tumor visualization by targeting the vasculature, and the biodistribution assay indicated high tumor-to-blood and tumor-to-muscle ratios of 31.6 ± 6.3 and 6.7 ± 1.1, respectively, 3 h after probe injection. TSU-68 significantly slowed tumor growth and reduced MVD; these findings were consistent with a significant reduction in the tumor 64Cu-cyclam-RAFT-c(-RGDfK-)4 uptake. Moreover, a linear correlation was observed between tumor MVD and the corresponding standardized uptake value (SUV) (r = 0.829, P = 0.011 for SUV(mean); r = 0.776, P = 0.024 for SUV(max)) determined by quantitative PET. Autoradiography and immunostaining showed that the distribution of intratumoral radioactivity and tumor vasculature corresponded. We concluded that 64Cu-cyclam-RAFT-c(-RGDfK-)4 PET can be used for in vivo angiogenesis imaging and monitoring of tumor response to antiangiogenic therapy.

Reduction of renal uptake of 111In-DOTA-labeled and A700-labeled RAFT-RGD during integrin αvß3 targeting using single photon emission computed tomography and optical imaging.

Integrin α(v)ß(3) expression is upregulated during tumor growth and invasion in newly formed endothelial cells in tumor neovasculature and in some tumor cells. A tetrameric RGD-based peptide, regioselectively addressable functionalized template-(cyclo-[RGDfK])4 (RAFT-RGD), specifically targets integrin α(v)ß(3) in vitro and in vivo. When labeled with indium-111, the RAFT-RGD is partially reabsorbed and trapped in the kidneys, limiting its use for further internal targeted radiotherapy and imaging investigations. We studied the effect of Gelofusine on RAFT-RGD renal retention in tumor-bearing mice. Mice were imaged using single photon emission computed tomography and optical imaging 1 and 24 h following tracer injection. Distribution of RAFT-RGD was further investigated by tissue removal and direct counting of the tracer. Kidney sections were analyzed by confocal microscopy. Gelofusine significantly induced a >50% reduction of the renal reabsorption of (111)In-DOTA-RAFT-RGD and A700-RAFT-RGD, without affecting tumor uptake. Injection of Gelofusine significantly reduced the renal retention of labeled RAFT-RGD, while increasing the tumor over healthy tissue ratio. These results will lead to the development of future therapeutic approaches.