Evaluation of Nanotargeted 111In-Cyclic RGDfK-Liposome in a Human Melanoma Xenotransplantation Model.

Nanotargeted liposomes may be modified with targeting peptide on the surface of a prepared liposome to endow specificity and elevate targeting efficiency. The aim of this study was to develop a radioactive targeted nanoparticle, the 111In-cyclic RGDfK-liposome, and its advantage of recognizing the αVß3 integrin was examined. The cyclic RGDfK modified liposomes were demonstrated the ability to bind the αVß3 integrin expressed on the surface of human melanoma cell in vitro and in vivo. The effects of the cyclic RGDfK-liposome on the functioning of phagocytes was also examined, showing no considerable negative effects on the engulfment of bacteria and the generation of reactive oxygen species. Based upon these findings, the cyclic RGDfK- liposome is said to be a promising agent for tumor imaging.

Truncated Escherichia coli thioredoxin induces proliferation of human blood mononuclear cells and production of reactive oxygen species as well as proinflammatory cytokines.

UNLABELLED: Thioredoxin (Trx) is a redox protein characterized by a Trx fold. A naturally occurring truncated human Trx, Trx 80, which lacks the C-terminal strand-helix of the Trx fold, stimulates proliferation of peripheral blood mononuclear cells (PBMCs). It has not been clear how Trx80 gains this function. This study investigates whether a peptide with substantial sequence difference from Trx80, but retaining an abridged Trx fold can elicit PBMC proliferation. We genetically truncated a carboxy-terminal ß-α motif of Escherichia coli Trx to produce a peptide, Trx83, which shares low sequence identity with human Trx80. Addition of reduced-form Trx83 to resting human PBMCs promoted cell proliferation, while oxidized-form Trx83 lacked the function. By contrast, oxidized-form Trx80 exhibited a high activity in promoting PBMC proliferation, indicating the importance of sequence context of an abridged thioredoxin in influencing PBMC proliferation. Trx83 increases cellular reactive oxygen species and proinflammatory cytokines TNF-α and IL-1ß, suggesting that Trx83 modulates inflammatory pathways. This notion is supported by the observation that cystine or cysteine abolishes the Trx83 induced PBMC proliferation. The PBMC stimulatory activity of Trx83 may have potential for pharmacological developments. SIGNIFICANCE OF THE STUDY: Elicitation of primary proliferative responses of PBMCs by a protein is generally difficult. We show that Escherichia coli Trx83 with a truncated Trx fold induces PBMC proliferation, but only in the disulfide-reduced form. In contrast, oxidized-form human Trx80 is a potent stimulator. These results demonstrate that the sequence context of an abridged Trx fold is influential in inducing PBMC proliferation. The stimulatory effect of Trx83 is associated with an increase of inflammatory response. The possibility of eliciting PBMC proliferation and switching this activity on/off by redox control provides a perspective for developing Trx83 as a PBMC stimulatory agent. Copyright © 2016 John Wiley & Sons, Ltd.

Single dose acute toxicity testing for N,N-bis(2-mercaptoethyl)-N',N' diethylethylenediamine in beagles.

N,N-Bis(2-mercaptoethyl)-N',N'-diethylenediamine (BMEDA) is used in the preparation of the (188)Re-BMEDA-liposome as a chelator to deliver rhenium 188 into liposomes. Although the safety of the (188)Re-BMEDA-liposome had been established, the use of BMEDA in preparing the liposome is of interest; however, an assessment of its safety is warranted. In this present work, we report on the acute toxicity study of BMEDA in beagles to identify doses causing no adverse effect and doses causing life-threatening toxicity. In a single dose 14-day systemic toxicity study conducted in beagles, BMEDA was without compound-related adverse effects at doses of up to 2mg/kg in a series of clinical observations and clinical pathology examinations. The results of these studies could aid in choosing doses for repeat-dose studies and in the selection of starting doses for Phase 1 human studies.

Pharmacokinetics of BMEDA after intravenous administration in beagle dogs.

The pharmacokinetics of N,N-bis(2-mercapatoethly)-N',N'-diethylenediamine (BMEDA), a molecule that can form a chelate with rhenium-188 (188Re) to produce the 188Re-BMEDA-liposomes, was studied. In this work, beagles received a single injection of BMEDA, at doses of 1, 2, or 5 mg/kg; the concentration of BMEDA in the beagles' plasma was then analyzed and determined by liquid chromatography-mass spectrometry/mass spectrometry. Based on the pharmacokinetic parameters of BMEDA, we found that male and female animals shared similar patterns indicating that the pharmacokinetics of BMEDA is independent of gender differences. In addition, the pharmacokinetics of BMEDA was seen to be non-linear because the increase of mean AUC0-t and AUC0-∞ values tend to be greater than dose proportional while the mean Vss and CL values of BMEDA appeared to be dose dependent. The information on the pharmacokinetics of BMEDA generated from this study will serve as a basis to design appropriate pharmacology and toxicology studies for future human use.

Leukocyte nicotinamide adenine dinucleotide phosphate-reduced oxidase is required for isocyanate-induced lung inflammation.

BACKGROUND: Isocyanates are low-molecular-weight compounds noted for inducing occupational and environmental asthma. Isocyanate-induced lung disease, an oxidant stress-dependent pulmonary inflammation, is the leading cause of occupational asthma. OBJECTIVES: To address the role of leukocyte-produced oxidants in airway inflammation induced by toluene diisocyanate (TDI), and to elucidate the role of leukocyte nicotinamide adenine dinucleotide phosphate-reduced (NADPH) oxidase in pathogenesis by TDI. METHODS: Wild-type mice and NADPH oxidase-deficient mice (neutrophil cytosolic factor 1 mutant, Ncf1(-/-)) were intranasally injected, challenged with inhalatory TDI, and then investigated for lung inflammation. RESULTS: Cell infiltration in lung tissue and leukocytes in bronchoalveolar lavage, airway reactivity to a methacholine challenge, and TDI-induced inflammatory cytokine expression and nuclear factor activation in the lung tissue were all markedly lower in Ncf1(-/-) mice. Wild-type mice treated with blocking antibodies against CD4 and IL-17 showed markedly lower TDI-induced airway hyperresponsiveness. CONCLUSION: Leukocyte NADPH oxidase is an essential regulator in TDI-induced airway inflammation through redox modification of immune responses.

The PEGylated liposomal doxorubicin improves the delivery and therapeutic efficiency of 188Re-Liposome by modulating phagocytosis in C26 murine colon carcinoma tumor model.

Liposome in delivering radionuclide for cancer therapy has been expansively studied; however, liposome itself can be deliberately entrapped and destroyed by the reticuloendothelial system, causing an insufficiency of the drug delivery, which in turn would restrict the effectiveness of the drug. In this study, mice with subcutaneous implantation of C26 murine colon cancer received an experimental treatment regimen in which mice took delivery of PEGylated liposomal doxorubicin (LipoDox) first, after a three-day interval, of Rhenium-188 encapsulated into PEGylated liposome ((188)Re-Liposome) subsequently and by which suppressed the functioning of reticuloendothelial system for the short term. The data showed that based upon the biodistribution assay and the evaluation of the therapeutic efficacy, (188)Re-Liposome was more sufficiently delivered to tumor sites in mice with this treatment regimen than mice without the regimen, and that cancer mortalities in mice with the treatment regimen were much lower than the mortalities in mice without the regimen. Taken together, a new strategy proposed in this study significantly improved both the (188)Re-Liposome delivery and the effectiveness of (188)Re-Liposome, suggesting that the strategy can be an ideal treatment for cancer.

Thapsigargin and flavin adenine dinucleotide ex vivo treatment rescues trafficking-defective gp91phox in chronic granulomatous disease leukocytes.

Mutations in leukocyte NADPH oxidase genes lead to defective respiratory burst in leukocytes and cause chronic granulomatous diseases (CGD) in humans. The most common form of CGD is caused by mutations in the membrane-bound oxidase component gp91phox, which is encoded by the CYBB gene on the X chromosome. We previously reported on a patient with a CYBB mutation (H338Y) that prevents the intracellular trafficking and expression of gp91phox on leukocytes. The capacity of the leukocytes to produce reactive oxygen species (ROS) was rescued by treatment with thapsigargin and flavin adenine dinucleotide (FAD). The increase in ROS production was not due to the increase in cytoplasmic calcium induced by thapsigargin because the treatment of calcium ionophore did not have the same effect. Protein and cellular analyses on leukocytes and cells transfected with GFP-tagged gp91phox mutant showed that treated cells expressed more Endo H-resistant gp91phox protein on the cell surface and are more effective in killing bacteria. Thapsigargin- and FAD-treated CGD leukocytes had enhanced activity in protecting mice from Staphylococcus-induced peritoneal abscess formation in a mouse model of CGD. These results indicate that thapsigargin-FAD ex vivo treatment is effective in rescuing the ROS-producing activity of leukocytes in selected CGD patients.

Ligand binding of leukocyte integrin very late antigen-4 involves exposure of sulfhydryl groups and is subject to redox modulation.

Activation of leukocyte integrins is important for selective recruitment of cells from the circulation to tissues. Our previous studies showed that the binding between the integrin very late antigen-4 (VLA-4) and vascular cell adhesion molecule-1 (VCAM-1) is modulated by reactive oxygen species. In this study, we investigated the molecular nature of redox modulation on the activation states of VLA-4 on human leukocytes. We found that ligand binding of VLA-4 induced exposure of sulfhydryl groups on the alpha4 peptide. Low concentrations (5-10 microM) of exogenous hydrogen peroxide in the presence or absence of added glutathione enhanced the ligand binding ability of VLA-4 to VCAM-1 and cell rolling on VCAM-1, while higher concentrations (> or = 100 microM) of hydrogen peroxide inhibited the binding. Exogenous hydrogen peroxide and glutathione induced molecular modification of S-glutathionylation on the alpha4 peptide. The redox regulation of the VLA-4 binding activity required outside-in signaling and cytoskeleton rearrangement. Our results indicate that ligand binding of VLA-4 involves redox modulations which may play a pivotal role in regulating the activation states of VLA-4 in inflammatory tissues and hence direct leukocyte trafficking.