Radiotracers for in situ infection imaging: Experimental considerations for in vitro microbial uptake of gallium-68-labeled siderophores.

In vitro screening of gallium-68(68Ga)-siderophores in pathogens relevant to infections is valuable for determining species specificity, their effect on cell viability, and potential clinical applications. As the recognition and internalization of siderophores relies on the presence of receptor- and/or siderophore-binding proteins, the level of uptake can vary between species. Here, we report in vitro uptake validation in Escherichia coli with its native siderophore, enterobactin (ENT) ([68Ga]Ga-ENT), considering different experimental factors. Compared with other reporting methods of uptake, '% Added dose/109 CFU/mL (% AD/109 CFU/mL),' considering the total viable count, showed a better comparison among microbial species. Later, in vitro screening with [68Ga]Ga-desferrioxamine B (DFO-B) showed high uptake by Staphylococcus aureus and S. epidermidis; moderate uptake by Pseudomonas aeruginosa; poor uptake by E. coli, Candida albicans, and Aspergillus fumigatus; and no uptake by Enterococcus faecalis and C. glabrata. Except for S. epidermidis, [68Ga]Ga-DFO-B did not reduce the cell viability.

Does gait influence biomechanics in a distal femoral osteotomy? An early post operative fracture after DFO above a Tomofix® plate in a multiple sclerosis and low-density bone affected patient: choose a longer plate-a case report.

BACKGROUND: Distal femur osteotomies are a well known and valuable treatment option to manage valgus malalignment with unicompartmental arthritis. Early postoperative complications are well known, and risk factors, such as pulmonary diseases, smoke, high dependent functional status, and body mass index, have been studied, but no study is available about osteotomies when gait is abnormal because of neurodegenerative conditions or when mineral density is below the normal rate. CASE PRESENTATION: We report the case of a 44 year-old female Mediterranean patient who underwent a biplanar distal femur opening wedge osteotomy surgery following a lateral meniscus total removal, which led to the subsequent development of lateral compartment osteoarthritis and pain, despite general comorbidities, such as multiple sclerosis. Additionally, 2 months later a supracondylar femur fracture above the previously applied Tomofix® plate was reported. Fracture was treated by applying a LCP condylar 16 hole (336 mm) plate, a structural fibular graft, and strut fibular graft on the opposite side. CONCLUSION: The overall aim of this case report is to provide a lesson to surgeons who want to perform a realignment surgery of the lower limb in patients with abnormal gait. Not only mechanical axes are to be considered, but also bone density, patient's gait, and load force distribution along the bone stock. Emerging literature on three-dimensional cutting guides fails to account for these factors, thus promoting a standardized approach to surgery across all patients. The present case highlights a patient with low bone density and abnormal force distribution resulting from a pathologic neurodegenerative gait. In such cases, treatment decisions must carefully consider the biomechanical vulnerabilities of the native bone and the distribution of vector forces. These conditions must lead the choice toward a longer plate if an osteotomy is indicated, because surgery is more likely to fail.

Mechanisms of exacerbation of Th2-mediated eosinophilic allergic asthma induced by plastic pollution derivatives (PPD): A molecular toxicological study involving lung cell ferroptosis and metabolomics.

Polystyrene microplastics (PS-MP) and dibutyl phthalate (DBP) are plastic pollution derivatives (PPDs) commonly found in the natural environment. To investigate the effects of PPD exposure on the risk of allergic asthma, we established a PPD exposure group in a mouse model. The dose administered for PS-MP was 0.1 mg/d and for DBP was 30 mg/kg/d, with a 5-week oral administration period. The pathological changes of airway tissue and the increase of oxidative stress and inflammatory response confirmed that PPD aggravated eosinophilic allergic asthma in mice. The mitochondrial morphological changes and metabolomics of mice confirmed that ferrotosis and oxidative stress played key roles in this process. Treatment with 100 mg/Kg deferoxamine (DFO) provided significant relief, and metabolomic analysis of lung tissue supported the molecular toxicological. Our findings suggest that the increased levels of reactive oxygen species (ROS) in the lungs lead to Th2-mediated eosinophilic inflammation, characterized by elevated IL-4, IL-5, and eosinophils, and reduced INF-γ levels. This inflammatory response is mediated by the NFκB pathway and exacerbates type I hypersensitivity through increased IL-4 production. In this study, the molecular mechanism by which PPD aggravates asthma in mice was elucidated, which helps to improve the understanding of the health effects of PPD and lays a theoretical foundation for addressing the health risks posed by PPD.

Evaluation of PET/CT imaging with [89Zr]Zr-DFO-girentuximab: a phase 1 clinical study in Japanese patients with renal cell carcinoma (Zirdac-JP).

BACKGROUND: PET/CT imaging with Zirconium-89 labeled [89Zr]Zr-DFO-girentuximab, which targets tumor antigen CAIX, may aid in the differentiation and characterization of clear cell renal cell carcinomas (RCC) and other renal and extrarenal lesions, and has been studied in European and American cohorts. We report results from a phase I study that evaluated the safety profile, biodistribution, and dosimetry of [89Zr]Zr-DFO-girentuximab in Japanese patients with suspected RCC. METHODS: Eligible adult patients received 37 MBq (± 10%; 10 mg mass dose) of intravenous [89Zr]Zr-DFO-girentuximab. Safety and tolerability profile was assessed based on adverse events, concomitant medications, physical examination, vital signs, hematology, serum chemistry, urinalysis, human anti-chimeric antibody measurement, and 12-lead electrocardiograms at predefined intervals. Biodistribution and normal organ and tumor dosimetry were evaluated with PET/CT images acquired at 0.5, 4, 24, 72 h and Day 5 ± 2 d after administration. RESULTS: [89Zr]Zr-DFO-girentuximab was administered in six patients as per protocol. No treatment-emergent adverse events were reported. Dosimetry analysis showed that radioactivity was widely distributed in the body, and that the absorbed dose in healthy organs was highest in the liver (mean ± standard deviation) (1.365 ± 0.245 mGy/MBq), kidney (1.126 ± 0.190 mGy/MBq), heart wall (1.096 ± 0.232 mGy/MBq), and spleen (1.072 ± 0.466 mGy/MBq). The mean effective dose, adjusted by the radioactive dose administered, was 0.470 mSv/MBq. The radiation dose was highly accumulated in the targeted tumor, while any abnormal accumulation in other organs was not reported. CONCLUSIONS: This study demonstrates that [89Zr]Zr-DFO-girentuximab administered to Japanese patients with suspected RCC has a favorable safety profile and is well tolerated and has a similar dosimetry profile to previously studied populations.

Improving Routine 89Zr-Immuno-PET Applications: Mild Iron Removal Can Favor the Use of Fe-DFO-N-suc-TFP Ester Over p-NCS-Bz-DFO.

A key aspect for the applicability of 89Zr-radioimmunoconjugates is inert modification and radiolabeling. The two commercially available bifunctional variants of the siderophore desferrioxamine (DFO), Fe-DFO-N-suc-TFP-ester and p-NCS-Bz-DFO, are most often used for clinical 89Zr-immuno-PET. The use of Fe-DFO-N-suc-TFP-ester is advantageous with regard to higher radiolysis stability and more facile assessment of radiochemical purity as well as chelator-to-mAb ratio. However, not all mAbs withstand the Fe-removal step at relatively low pH (4-4.5) using EDTA, which is needed after conjugation to allow 89Zr labeling. In this study, it was investigated whether hydroxybenzyl ethylenediamine (HBED) or the clinically approved deferiprone (DFP) can serve as an alternative for EDTA to establish a pH-independent mild method for Fe-removal and thereby broaden the applicability of Fe-DFO-N-suc-TFP-ester. Carrier-added [59Fe]Fe-DFO-N-suc-TFP-ester was used for mAb modification to enable direct tracking of the Fe-removal efficiency under various conditions. Whereas incomplete Fe-removal with HBED was observed at pH 5 or higher, Fe-removal with DFP was possible at a broad pH range (4-9). This provides a mild, pH-independent method for Fe-removal, improving the applicability and attractiveness of Fe-DFO-N-suc-TFP-ester for 89Zr-mAb preparation.

Good practices for 89Zr radiopharmaceutical production and quality control.

BACKGROUND: During the previous two decades, PET imaging of biopharmaceuticals radiolabeled with zirconium-89 has become a consistent tool in preclinical and clinical drug development and patient selection, primarily due to its advantageous physical properties that allow straightforward radiolabeling of antibodies (89Zr-immuno-PET). The extended half-life of 78.4 h permits flexibility with respect to the logistics of tracer production, transportation, and imaging and allows imaging at later points in time. Additionally, its relatively low positron energy contributes to high-sensitivity, high-resolution PET imaging. Considering the growing interest in radiolabeling antibodies, antibody derivatives, and other compound classes with 89Zr in both clinical and pre-clinical settings, there is an urgent need to acquire valuable recommendations and guidelines towards standardization of labeling procedures. MAIN BODY: This review provides an overview of the key aspects of 89Zr-radiochemistry and radiopharmaceuticals. Production of 89Zr, conjugation with the mostly used chelators and radiolabeling strategies, and quality control of the radiolabeled products are described in detail, together with discussions about alternative options and critical steps, as well as recommendations for troubleshooting. Moreover, some historical background on 89Zr-immuno-PET, coordination chemistry of 89Zr, and future perspectives are provided. This review aims to serve as a quick-start guide for scientists new to the field of 89Zr-immuno-PET and to suggest approaches for harmonization and standardization of current procedures. CONCLUSION: The favorable PET imaging characteristics of 89Zr, its excellent availability due to relatively simple production and purification processes, and the development of suitable bifunctional chelators have led to the widespread use of 89Zr. The combination of antibodies and 89Zr, known as 89Zr-immuno-PET, has become a cornerstone in drug development and patient selection in recent years. Despite the advanced state of 89Zr-immuno-PET, new developments in chelator conjugation and radiolabeling procedures, application in novel compound classes, and improved PET scanner technology and quantification methods continue to reshape its landscape towards improving clinical outcomes.

MALDI-mass spectrometry imaging as a new technique for detecting non-heme iron in peripheral tissues via caudal vein injection of deferoxamine.

As one of the most common iron-chelating agents, deferoxamine (DFO) rapidly chelates iron in the body. Moreover, it does not compete for the iron characteristic of hemoglobin in the blood cells, which is common in the clinical treatment of iron poisoning. Iron is a trace element necessary to maintain organism normal life activities. Iron deficiency can lead to anemia, whereas iron overload can cause elevated levels of cellular oxidative stress and cell damage. As a consequence, detection of the iron content in tissues and blood is of great significance. The traditional techniques for detecting the iron content include inductively coupled plasma-mass spectrometry and atomic absorption spectrometry, which cannot be used for imaging purposes. Laser ablation-ICP-MS and synchrotron radiation micro-X-ray fluorescence can map the concentration and distribution of iron in tissues. However, these methods can only be used to measure the total iron levels in blood or tissues. In recent years, due to the deepening understanding of iron metabolism, diseases related to iron overload have attracted increasing attention. Therefore, we took advantage of the properties of DFO in terms of chelating iron and investigated different sampling times following DFO injection in the tail vein of mice. We used mass spectrometry imaging (MSI) technology to detect the DFO and ferrioxamine content in the blood and different tissues to indirectly characterize the non-heme iron content.

Preparation of a Zirconium-89 Labeled Clickable DOTA Complex and Its Antibody Conjugate.

Desferrioxamine B (DFO) is the clinical standard chelator for preparing zirconium-89 labeled antibodies. In the current study, the stabilities of a zirconium-89 labeled panitumumab (PAN; Vectibix®) with three different chelators (DFO, DFO*, and DOTA) were compared. PAN is an anti-HER1/EGFR monoclonal antibody approved by the FDA for the treatment of HER1-expressing colorectal cancers and was used as the model antibody for this study. DFO/DFO* conjugates of PAN were directly radiolabeled with zirconium-89 at room temperature to produce [89Zr]Zr-DFO/DFO*-PAN conjugates following a well-established procedure. A zirconium-89 labeled DOTA-PAN conjugate was prepared by an indirect radiolabeling method. A cyclooctyne-linked DOTA chelator (BCN-DOTA-GA) was first radiolabeled with zirconium-89 at 90 °C under a two-step basic pH adjustment method followed by conjugation with PAN-tetrazene at 37 °C to produce a labeled conjugate, BCN-[89Zr]Zr-DOTA-GA-PAN. High reproducibility of the radiolabeling was observed via this two-step basic pH adjustment. The overall radiochemical yield was 40-50% (n = 12, decay uncorrected) with a radiochemical purity of >95% in 2 h synthesis time. All three conjugates were stable in whole human serum for up to 7 days at 37 °C. The kinetic inertness of the conjugates was assessed against the EDTA challenge. BCN-[89Zr]Zr-DOTA-GA-PAN exhibited excellent inertness followed by [89Zr]Zr-DFO*-PAN. [89Zr]Zr-DFO-PAN displayed the lowest level of inertness.

H Vessel Formation as a Marker for Enhanced Bone Healing in Irradiated Distraction Osteogenesis.

In the setting of bone defects, the injured vasculature and loss of hemodynamic inflow leads to hematoma formation and low oxygen tension which stimulates vascular expansion through the HIf-1α pathway. Most importantly, this pathway upregulates sprouting of type H vessels (CD31hiEmcnhi vessels). H vessels engage in direct interaction with perivascular osteoprogenitor cells (OPCs), osteoblasts, and preosteoclasts of bone formation and remodeling. This angiogenic-osteogenic coupling leads to synchronous propagation of vascular and bony tissue for regenerative healing. A growing body of literature demonstrates that H vessels constitute a large portion of bone's innate capacity for osteogenic healing. We believe that CD31hiEmcnhi vessels play a role in bone healing during distraction osteogenesis (DO). DO is a procedure that utilizes traction forces to facilitate induction of endogenous bone formation and regeneration of surrounding soft tissues such as skin, muscle, tendon, and neurovascular structures. While the H vessel response to mechanical injury is adequate to facilitate healing in normal healthy tissue, it remains inadequate to overcome the devastation of radiation. We posit that the destruction of CD31hiEmcnhi vessels plays a role in precluding DO's effectiveness in irradiated bone defect healing. We aim, therefore, to recapitulate the normal pathway of bony healing by utilizing the regenerative capacity of H vessels. We hypothesize that using localized application of deferoxamine (DFO) will enhance the H vessel-mediated vasculogenic response to radiation damage and ultimately enable osteogenic healing during DO. This discovery could potentially be exploited by developing translational therapeutics to hopefully accelerate bone formation and shorten the DO consolidation period, thereby potentially expanding DO's utilization in irradiated bone healing. Sprague-Dawley rats were divided into three groups: DO, radiation with DO (xDO), and radiation with DO and DFO implantation (xDODFO). Experimental groups received 35 Gy of radiation. All groups underwent DO. The treatment group received injections into the osteotomy site, every other day, beginning on postoperative day (POD) 4 of DFO. Animals were sacrificed on POD 40. For immunohistochemical analysis, mandibles were dissected and fixed in 4% paraformaldehyde for 48 hours, decalcified in Cal-Ex II for 2 days, dehydrated through graded ethanol of increasing concentration, and then embedded in paraffin. Samples were cut into 7-µm thick longitudinally oriented sections including the metaphysis and diaphysis. CD31 and Emcn double immunofluorescent staining were performed to evaluate the extent of CD31hiEmcnhi vessel formation. Bone sections were then stained with conjugated antibodies overnight at 4°C. Nuclei were stained with Hoechst. Slides were also double stained with Osterix and CD31 to study the quantity of H vessel-mediated recruitment of OPCs to accelerate bone healing. Images were acquired with a Nikon Ti2 widefield microscope and analyzed in NIS- Elements Advanced Research 5.41.02 software. The abundance of type H vessels is represented by the area fraction of CD31 + Emcn+ vessel area inside the regenerate sample. OPC concomitant proliferation into the distraction gap is represented by the area fraction of Osterix+ cell area inside of the regenerate sample. There were 6× more type H vessels in DO groups than in xDO groups. Localized DFO significantly increased the abundance of type H vessels of irradiated DO animals compared to xDO by 15× ( p = 0.00133531). Moreover, the DO and xDODFO groups with higher abundance of type H vessels also demonstrated better angiogenesis and osteogenesis outcomes. Interestingly, xDODFO groups doubled the quantity of H vessel formation compared to DO, indicating a supraphysiologic response ( p = 0.044655055). Furthermore, H vessel-mediated recruitment of OPCs mimicked the described H vessel formation trend in our study groups. Irradiated DO groups contained 3× less OPCs compared to DO controls. DFO treatment to xDO animals remediated irradiation damage by containing 12× Osterix+ cells. Finally, DFO treatment of irradiated animals quadrupled osteoprogenitor recruitment into the distraction gap compared to DO controls. In this study, we developed a novel approach to visualize CD31hiEmcnhi in paraffin sections to study DO regeneration. Normal DO demonstrated a significant upregulation of H vessel formation and associated angiogenic-osteogenic coupling. Radiation severely decreased H vessel formation along with an associated significant diminution of new bone formation and nonunion. DFO administration, however, resulted in vascular replenishment and the restoration of high quantities of CD31hiEmcnhi and OPCs, recapitulating the normal process of bony regeneration and repair. DFO treatment remediated new bone formation and bony union in irradiated fields associated with increased H vessel angiogenic-osteogenic coupling. While further studies are required to optimize this approach, the results of this study are incredibly promising for the long-awaited translation of localized DFO into the clinical arena.

Biosynthesis of novel desferrioxamine derivatives requires unprecedented crosstalk between separate NRPS-independent siderophore pathways.

Iron is essential to many biological processes but its poor solubility in aerobic environments restricts its bioavailability. To overcome this limitation, bacteria have evolved a variety of strategies, including the production and secretion of iron-chelating siderophores. Here, we describe the discovery of four series of siderophores from Streptomyces ambofaciens ATCC23877, three of which are unprecedented. MS/MS-based molecular networking revealed that one of these series corresponds to acylated desferrioxamines (acyl-DFOs) recently identified from S. coelicolor. The remaining sets include tetra- and penta-hydroxamate acyl-DFO derivatives, all of which incorporate a previously undescribed building block. Stable isotope labeling and gene deletion experiments provide evidence that biosynthesis of the acyl-DFO congeners requires unprecedented crosstalk between two separate non-ribosomal peptide synthetase (NRPS)-independent siderophore (NIS) pathways in the producing organism. Although the biological role(s) of these new derivatives remain to be elucidated, they may confer advantages in terms of metal chelation in the competitive soil environment due to the additional bidentate hydroxamic functional groups. The metabolites may also find application in various fields including biotechnology, bioremediation, and immuno-PET imaging.IMPORTANCEIron-chelating siderophores play important roles for their bacterial producers in the environment, but they have also found application in human medicine both in iron chelation therapy to prevent iron overload and in diagnostic imaging, as well as in biotechnology, including as agents for biocontrol of pathogens and bioremediation. In this study, we report the discovery of three novel series of related siderophores, whose biosynthesis depends on the interplay between two NRPS-independent (NIS) pathways in the producing organism S. ambofaciens-the first example to our knowledge of such functional cross-talk. We further reveal that two of these series correspond to acyl-desferrioxamines which incorporate four or five hydroxamate units. Although the biological importance of these novel derivatives is unknown, the increased chelating capacity of these metabolites may find utility in diagnostic imaging (for instance, 89Zr-based immuno-PET imaging) and other applications of metal chelators.

Mechanism of damage of HIF-1 signaling in chronic diabetic foot ulcers and its related therapeutic perspectives.

Diabetic foot ulcer (DFU) is a chronic complication of diabetes. Wound healing in patients with DFU is generally very slow, with a high recurrence rate even after the ulcer healed. The DFU remains a major clinical challenge due to a lack of understanding of its pathogenesis. Given the significant impact of DFU on patient health and medical costs, enhancing our understanding of pathophysiological alterations and wound healing in DFU is critical. A growing body of research has shown that impaired activation of the HIF-1 pathway in diabetics, which weakens HIF-1 mediated responses to hypoxia and leads to down-regulation of its downstream target genes, leading to incurable diabetic foot ulcers. By analyzing and summarizing the literature in recent years, this review summarizes the mechanism of HIF-1 signaling pathway damage in the development of DFU, analyzes and compares the application of PHD inhibitors, VHL inhibitors, biomaterials and stem cell therapy in chronic wounds of diabetes, and proposes a new treatment scheme mediated by participation in the HIF-1 signaling pathway, which provides new ideas for the treatment of DFU.

Chelating Effect of Siderophore Desferrioxamine-B on Uranyl Biomineralization Mediated by Shewanella putrefaciens.

In contaminated water and soil, little is known about the role and mechanism of the biometabolic molecule siderophore desferrioxamine-B (DFO) in the biogeochemical cycle of uranium due to complicated coordination and reaction networks. Here, a joint experimental and quantum chemical investigation is carried out to probe the biomineralization of uranyl (UO22+, referred to as U(VI) hereafter) induced by Shewanella putrefaciens (abbreviated as S. putrefaciens) in the presence of DFO and Fe3+ ion. The results show that the production of mineralized solids {hydrogen-uranium mica [H2(UO2)2(PO4)2·8H2O]} via S. putrefaciens binding with UO22+ is inhibited by DFO, which can both chelate preferentially UO22+ to form a U(VI)-DFO complex in solution and seize it from U(VI)-biominerals upon solvation. However, with Fe3+ ion introduced, the strong specificity of DFO binding with Fe3+ causes re-emergence of biomineralization of UO22+ {bassetite [Fe(UO2)2(PO4)2·8(H2O)]} by S. putrefaciens, owing to competitive complexation between Fe3+ and UO22+ for DFO. As DFO possesses three hydroxamic functional groups, it forms hexadentate coordination with Fe3+ and UO22+ ions via these functional groups. The stability of the Fe3+-DFO complex is much higher than that of U(VI)-DFO, resulting in some DFO-released UO22+ to be remobilized by S. putrefaciens. Our finding not only adds to the understanding of the fate of toxic U(VI)-containing substances in the environment and biogeochemical cycles in the future but also suggests the promising potential of utilizing functionalized DFO ligands for uranium processing.

Polystyrene microplastics mitigate lead-induced neurotoxicity by reducing heavy metal uptake in zebrafish larvae.

The combined pollution of lead (Pb) and polystyrene microplastics (PS-MPs) is common in aquatic environments. However, the combined neurotoxicity of these two pollutants is still poorly understood. In this study, zebrafish (Danio rerio) larvae were used to assess the combined neurotoxicity and mechanism of Pb and PS-MPs at environmentally relevant concentrations. The results showed that Pb (10 µg/L) induced abnormal behavior including significantly reduced movement distance, maximum acceleration, and average velocity (P < 0.05) along with altered expression of neurodevelopment-related genes (gap43 and α1-tubulin) (P < 0.05). PS-MPs (25 µg/L, 250 µg/L; diameter at 25 µm) co-exposure not only significantly reduced the concentration of Pb in the exposed solution (P < 0.01), but also decreased the uptake of Pb by downregulating the divalent metal transporter 1 gene (dmt1) (P < 0.01), thereby alleviating Pb-induced neurotoxicity. However, to demonstrate that PS-MPs alleviate the neurotoxicity of Pb by reducing Pb uptake, upregulation of dmt1 by addition of deferoxamine (DFO, an efficient iron chelator, 100 µM) significantly increased the Pb uptake and exacerbated neurotoxicity in zebrafish. In summary, our results demonstrated that PS-MPs alleviate Pb neurotoxicity by downregulating the mRNA level of dmt1 and decreasing the Pb uptake. This study provides a new insight into the combined neurotoxicity and underlying mechanisms of PS-MPs and Pb on zebrafish.

Developing Iron Nanochelating Agents: Preliminary Investigation of Effectiveness and Safety for Central Nervous System Applications.

Excessive iron levels are believed to contribute to the development of neurodegenerative disorders by promoting oxidative stress and harmful protein clustering. Novel chelation treatments that can effectively remove excess iron while minimizing negative effects on the nervous system are being explored. This study focuses on the creation and evaluation of innovative nanobubble (NB) formulations, shelled with various polymers such as glycol-chitosan (GC) and glycol-chitosan conjugated with deferoxamine (DFO), to enhance their ability to bind iron. Various methods were used to evaluate their physical and chemical properties, chelation capacity in diverse iron solutions and impact on reactive oxygen species (ROS). Notably, the GC-DFO NBs demonstrated the ability to decrease amyloid-ß protein misfolding caused by iron. To assess potential toxicity, in vitro cytotoxicity testing was conducted using organotypic brain cultures from the substantia nigra, revealing no adverse effects at appropriate concentrations. Additionally, the impact of NBs on spontaneous electrical signaling in hippocampal neurons was examined. Our findings suggest a novel nanochelation approach utilizing DFO-conjugated NBs for the removal of excess iron in cerebral regions, potentially preventing neurotoxic effects.

Straightforward Synthesis of DFO* - An Octadentate Chelator for Zirconium-89.

DFO* is an octadentate chelator able to form highly stable chelates with Zirconium-89 (89 Zr) for nuclear medicinal applications in Positron Emission Tomography (PET).[1,2] The synthesis of DFO* and its scale-up remains challenging by reported synthetic protocols. For this reason, we set out to develop a de novo synthesis of a hydroxamate-containing building block suitable for the coupling to the commercially available DFO (desferrioxamine B, mesylate salt) yielding, after deprotection, the desired chelator DFO* in a more efficient procedure. Highlights of the new synthesis of DFO* reported herein are less synthetic steps and the isolation of the desired product DFO* by using solid phase extraction (SPE), thus avoiding tedious HPLC purification. DFO* is obtained in excellent purity (92-98 %) and an overall yield of approximately 29 %. In addition, the isolated trifluoroacetic acid (TFA)-salt of DFO* displays an improved solubility in organic solvents (DMSO, DMF, methanol), which will facilitate its use for the preparation of structurally diverse derivatives suitable for bioconjugation chemistry and the development of 89 Zr-labeled radiotracers.

Evaluation of Chelator-to-Antibody Ratio on Development of 89Zr-iPET Tracer for Imaging of PD-L1 Expression on Tumor.

89Zr-iPET has been widely used for preclinical and clinical immunotherapy studies to predict patient stratification or evaluate therapeutic efficacy. In this study, we prepared and evaluated 89Zr-DFO-anti-PD-L1-mAb tracers with varying chelator-to-antibody ratios (CARs), including 89Zr-DFO-anti-PD-L1-mAb_3X (tracer_3X), 89Zr-DFO-anti-PD-L1-mAb_10X (tracer_10X), and 89Zr-DFO-anti-PD-L1-mAb_20X (tracer_20X). The DFO-anti-PD-L1-mAb conjugates with varying CARs were prepared using a random conjugation method and then subjected to quality control. The conjugates were radiolabeled with 89Zr and evaluated in a PD-L1-expressing CT26 tumor-bearing mouse model. Next, iPET imaging, biodistribution, pharmacokinetics, and ex vivo pathological and immunohistochemical examinations were conducted. LC-MS analysis revealed that DFO-anti-PD-L1-mAb conjugates were prepared with CARs ranging from 0.4 to 2.0. Radiochemical purity for all tracer groups was >99% after purification. The specific activity levels of tracer_3X, tracer_10X, and tracer_20X were 2.2 ± 0.6, 8.2 ± 0.6, and 10.5 ± 1.6 µCi/µg, respectively. 89Zr-iPET imaging showed evident tumor uptake in all tracer groups and reached the maximum uptake value at 24 h postinjection (p.i.). Biodistribution data at 168 h p.i. revealed that the tumor-to-liver, tumor-to-muscle, and tumor-to-blood uptake ratios for tracer_3X, tracer_10X, and tracer_20X were 0.46 ± 0.14, 0.58 ± 0.33, and 1.54 ± 0.51; 4.7 ± 1.3, 7.1 ± 3.9, and 14.7 ± 1.1; and 13.1 ± 5.8, 19.4 ± 13.8, and 41.3 ± 10.6, respectively. Significant differences were observed between tracer_3X and tracer_20X in the aforementioned uptake ratios at 168 h p.i. The mean residence time and elimination half-life for tracer_3X, tracer_10X, and tracer_20X were 25.4 ± 4.9, 24.2 ± 6.1, and 25.8 ± 3.3 h and 11.8 ± 0.5, 11.1 ± 0.7, and 11.7 ± 0.6 h, respectively. No statistical differences were found between-tracer in the aforementioned pharmacokinetic parameters. In conclusion, 89Zr-DFO-anti-PD-L1-mAb tracers with a CAR of 1.4-2.0 may be better at imaging PD-L1 expression in tumors than are traditional low-CAR 89Zr-iPET tracers.

Mesenchymal Stem Cell-conditioned Medium Protecting Renal Tubular Epithelial Cells by Inhibiting Hypoxia-inducible Factor-1α and Nuclear Receptor Coactivator-1.

BACKGROUND: Acute kidney injury (AKI) is characterized by inflammatory infiltration and damage and death of renal tubular epithelial cells (RTECs), in which hypoxia plays an important role. Deferoxamine (DFO) is a well-accepted chemical hypoxia-mimetic agent. Mesenchymal stem cell-conditioned medium (MSC-CM) can reduce local inflammation and repair tissue. In this study, we explored the effect and molecular mechanism of MSC-CM-mediated protection of RTECs under DFO-induced hypoxia. METHODS: Rat renal proximal tubule NRK-52E cells were treated with different concentrations of DFO for 24 hours, followed by evaluation of RTEC injury, using a Cell Counting Kit-8 (CCK-8) to detect cell viability and western blotting to evaluate the expression of transforming growth factor- beta 1 (TGF-ß1), α-smooth muscle actin (α-SMA), and hypoxia-inducible factor-1 alpha (HIF-1α) in NRK-52E cells. Then, three groups of NRK-52E cells were used in experiments, including normal control (NC), 25 µM DFO, and 25 µM DFO + MSC-CM. MSC-CM was obtained from the human umbilical cord. MSC-CM was used to culture cells for 12 hours before DFO treatment, then fresh MSC-CM and 25 µM DFO were added, and cells were cultured for another 24 hours before analysis. RESULTS: Western blotting and cellular immunofluorescence staining showed culture of NRK-52E cells in 25 µM DFO for 24 hours induced HIF-1α and nuclear receptor coactivator-1 (NCoA-1), simulating hypoxia. MSC-CM could inhibit the DFO-induced up-regulation of α-SMA, TGF-ß1, HIF-1α and NCoA-1. CONCLUSION: Our results suggest that MSC-CM has a protective effect on RTECs by down-regulating HIF-1α and NCoA-1, which may be the harmful factors in renal injury.

Iron Chelator Deferoxamine Alleviates Progression of Diabetic Nephropathy by Relieving Inflammation and Fibrosis in Rats.

Diabetic nephropathy (DN) is one of the most devastating diabetic microvascular complications. It has previously been observed that iron metabolism levels are abnormal in diabetic patients. However, the mechanism by which iron metabolism levels affect DN is poorly understood. This study was designed to evaluate the role of iron-chelator deferoxamine (DFO) in the improvement of DN. Here, we established a DN rat model induced by diets high in carbohydrates and fat and streptozotocin (STZ) injection. Our data demonstrated that DFO treatment for three weeks greatly attenuated renal dysfunction as evidenced by decreased levels of urinary albumin, blood urea nitrogen, and serum creatinine, which were elevated in DN rats. Histopathological observations showed that DFO treatment improved the renal structures of DN rats and preserved podocyte integrity by preventing the decrease of transcripts of nephrin and podocin. In addition, DFO treatment reduced the overexpression of fibronectin 1, collagen I, IL-1ß, NF-κB, and MCP-1 in DN rats, as well as inflammatory cell infiltrates and collagenous fibrosis. Taken together, our findings unveiled that iron chelation via DFO injection had a protective impact on DN by alleviating inflammation and fibrosis, and that it could be a potential therapeutic strategy for DN.

Distal femoral osteotomy for the valgus knee: indications, complications, clinical and radiological outcome.

INTRODUCTION: The aim of this study was to describe the indications and technical aspects of medial closing and lateral opening distal femoral osteotomy (MCDFO and LODFO) for patients with a valgus knee and to report clinical and radiological outcomes and complications. METHODS: Over 6 years, 28 DFOs (22 MCDFO, 6 LODFO) were performed in 22 Patients. In this cohort study, we retrospectively analyzed clinical and radiological outcome measures as well as complications. RESULTS: The median (range) age was 47 (17-63) years, height 1.68 (1.56-1.98) m, body mass 80 (49-105) kg, and body mass index (BMI) 27.4 (18.6-37.0) kg/m2. The clinical follow-up was 21 (7-81) months, the need for total or unicompartmental knee arthroplasty (TKA/UKA) and hardware removal was followed up for 59 (7-108) months postoperatively. Preoperatively, hip-knee-ankle angle (HKA, negative values denote varus) was 7.0 (2.0-13.0)°, mechanical lateral distal femoral angle (mLDFA) was 83.7 (79.9-88.2)°, and mechanical proximal tibial angle (MPTA) was 89.0 (86.6-94.5)°. Postoperatively, HKA was -1.3 (-9.0-1.2)° and mLDFA was 90.8 (87.3-97.3)°. The incidence of minor and major complications was 25% and 14%, the incidence of delayed and nonunion was 18% and 4%, respectively. At the last follow-up, 18% of the patients had pain at rest, 25% during activities of daily living, and 39% during physical activity, and 71% were satisfied with the outcome. 7% of the cases received a TKA/UKA, 71% received a hardware removal. CONCLUSION: DFO is a reasonable treatment for lateral osteoarthritis in younger patients to avoid disease progression and the need for an UKA/TKA. However, there is a long rehabilitation time, a considerable risk for complications, and a high need for hardware removal. While many patients experienced symptoms at the long-term follow-up, most were satisfied with the outcome. Appropriate patient information is essential. Level of evidence Level IV, Case Series. Trial registration number NCT04382118, clinicaltrials.gov, May 11, 2020.

3D hypoxia-mimicking and anti-synechia hydrogel enabling promoted neovascularization for renal injury repair and regeneration.

In-situ renal tissue engineering is promising yet challenging for renal injury repair and regeneration due to the highly vascularized structure of renal tissue and complex high-oxidative stress and ischemic microenvironment. Herein, a novel biocompatible 3D porous hydrogel (DFO-gel) with sustained release capacity of hypoxia mimicking micromolecule drug deferoxamine (DFO) was developed for in-situ renal injury repair. In vitro and in vivo experimental results demonstrated that the developed DFO-gels can exert the synchronous benefit of scavenging excess reactive oxygen species (ROS) regulating inflammatory microenvironment and promoting angiogenesis for effective renal injury repair by up-regulating hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF). The in-situ neogenesis of neonatal glomerular- and tubular-like structures in the implanted areas in the partially nephrectomized rats also suggested the potential for promoting renal injury repair and regeneration. This multifunctional hydrogel can not only exhibit the sustained release and promoted bio-uptake capacity for DFO, but also improve the renal injured microenvironment by alleviating oxidative and inflammatory stress, accelerating neovascularization, and promoting efficient anti-synechia. We believe this work offers a promising strategy for renal injury repair and regeneration.