Target Tissue Uptake Research Articles

Rapid precision targeting of nanoparticles to lung via caveolae pumping system in endothelium.

Modern medicine seeks precision targeting, imaging and therapy to maximize efficacy and avoid toxicities. Nanoparticles (NPs) have tremendous yet unmet clinical potential to carry and deliver imaging and therapeutic agents systemically with tissue precision. But their size contributes to rapid scavenging by the reticuloendothelial system and poor penetration of key endothelial cell (EC) barriers, limiting target tissue uptake, safety and efficacy. Here we discover the ability of the EC caveolae pumping system to outpace scavenging and deliver NPs rapidly and specifically into the lungs. Gold and dendritic NPs are conjugated to antibodies targeting caveolae of the lung microvascular endothelium. SPECT-CT imaging and biodistribution analyses reveal that rat lungs extract most of the intravenous dose within minutes to achieve precision lung imaging and targeting with high lung concentrations exceeding peak blood levels. These results reveal how much ECs can both limit and promote tissue penetration of NPs and the power and size-dependent limitations of the caveolae pumping system. This study provides a new retargeting paradigm for NPs to avoid reticuloendothelial system uptake and achieve rapid precision nanodelivery for future diagnostic and therapeutic applications.

Non-monotonic dose-response of di-(2-ethylhexyl) phthalate isolated from Penicillium citrinum XT6 on adipogenesis and expression of PPARγ and GLUT4 in 3T3-L1 adipocytes.

Adipogenesis is the fat cell formation process regulated by peroxisome proliferator-activated receptors (PPARγ). The insulin-responsive glucose transporter 4 (GLUT4) has a major role in glucose uptake and metabolism in insulin target tissues (i.e.,adipose and muscle cells). The interplay between PPARγ and GLUT4 is essential for proper glucose homeostasis. This study aimed to isolate, elucidate, and investigate the effect of an isolated compound from Penicillium citrinum XT6 on adipogenesis, PPARγ, and GLUT4 expression in 3T3-L1 adipocytes. The isolated compound was determined by analyzing spectroscopic data (LC-MS, FT-IR, Spectrophotometry UV-Vis, and NMR). The adipogenesis activity of the isolated compound in 3T3-L1 cells was determined by the Oil Red O staining method. RT-PCR was used to analyze the gene expression of PPARγ and GLUT4. Di-(2-ethylhexyl)-phthalate (DEHP) was the isolated compound from P.citrinum XT6. The results revealed adipogenesis stimulation and inhibition, as well as PPARγ and GLUT4 expressions. DEHP showed a non-monotonic dose-response (NMDR) effect on adipogenesis and PPARγ and GLUT4 expression. It is the first study that reveals DEHP's NMDR effects on lipid and glucose metabolism in adipocytes.

Hepatic expression of GAA results in enhanced enzyme bioavailability in mice and non-human primates

Pompe disease (PD) is a severe neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). PD is currently treated with enzyme replacement therapy (ERT) with intravenous infusions of recombinant human GAA (rhGAA). Although the introduction of ERT represents a breakthrough in the management of PD, the approach suffers from several shortcomings. Here, we developed a mouse model of PD to compare the efficacy of hepatic gene transfer with adeno-associated virus (AAV) vectors expressing secretable GAA with long-term ERT. Liver expression of GAA results in enhanced pharmacokinetics and uptake of the enzyme in peripheral tissues compared to ERT. Combination of gene transfer with pharmacological chaperones boosts GAA bioavailability, resulting in improved rescue of the PD phenotype. Scale-up of hepatic gene transfer to non-human primates also successfully results in enzyme secretion in blood and uptake in key target tissues, supporting the ongoing clinical translation of the approach.

Fluorescent Dye Labeling Changes the Biodistribution of Tumor-Targeted Nanoparticles.

Fluorescent dye labeling is a common strategy to analyze the fate of administered nanoparticles in living organisms. However, to which extent the labeling processes can alter the original nanoparticle biodistribution has been so far neglected. In this work, two widely used fluorescent dye molecules, namely, ATTO488 (ATTO) and Sulfo-Cy5 (S-Cy5), have been covalently attached to a well-characterized CXCR4-targeted self-assembling protein nanoparticle (known as T22-GFP-H6). The biodistribution of labeled T22-GFP-H6-ATTO and T22-GFP-H6-S-Cy5 nanoparticles has been then compared to that of the non-labeled nanoparticle in different CXCR4+ tumor mouse models. We observed that while parental T22-GFP-H6 nanoparticles accumulated mostly and specifically in CXCR4+ tumor cells, labeled T22-GFP-H6-ATTO and T22-GFP-H6-S-Cy5 nanoparticles showed a dramatic change in the biodistribution pattern, accumulating in non-target organs such as liver or kidney while reducing tumor targeting capacity. Therefore, the use of such labeling molecules should be avoided in target and non-target tissue uptake studies during the design and development of targeted nanoscale drug delivery systems, since their effect over the fate of the nanomaterial can lead to considerable miss-interpretations of the actual nanoparticle biodistribution.

Acacetin enhances glucose uptake through insulin-independent GLUT4 translocation in L6 myotubes

BackgroundLowering blood glucose levels by increasing glucose uptake in insulin target tissues, such as skeletal muscle and adipose tissue, is one strategy to discover and develop antidiabetic drugs from natural products used as traditional medicines. PurposeOur goal was to reveal the mechanism and activity of acacetin (5,7-dihydroxy-4′-methoxyflavone), one of the major compounds in Agastache rugose, in stimulating glucose uptake in muscle cells. MethodsTo determine whether acacetin promotes GLUT4-dependent glucose uptake in cultured L6 skeletal muscle cells, we performed a [14C] 2-deoxy-D-glucose (2-DG) uptake assay after treating differentiated L6-GLUT4myc cells with acacetin. ResultsAcacetin dose-dependently increased 2-DG uptake by enhancing GLUT4 translocation to the plasma membrane. Our results revealed that acacetin activated the CaMKII-AMPK pathway by increasing intracellular calcium concentrations. We also found that aPKCλ/ζ phosphorylation and intracellular reactive oxygen species (ROS) production were involved in acacetin-induced GLUT4 translocation. Moreover, acacetin-activated AMPK inhibited intracellular lipid accumulation and increased 2-DG uptake in HepG2 cells. ConclusionTaken together, these results suggest that acacetin might be useful as an antidiabetic functional ingredient. Subsequent experiments using disease model animals are needed to verify our results.

Инсулиномиметически активные макромолекулярные металлокомплексы ванадия на основе поли-N-винилпирролидона. Часть 1. Синтез макромолекулярного металлокомплекса ванадия и оценка его острой пероральной токсичности

Diabetes mellitus (DM) is a severe chronic disease affecting virtually all kinds of metabolic processes in the human body. As incidence and prevalence of DM are increasing worldwide, WHO estimates the number of people living with diabetes to be as high as half a billion and still rising. Long-term health complications of DM, such as diabetic nephropathy, retinopathy, neuropathy, and cardiovascular pathology, although preven-table in most cases, are known to be severe, potentially life-threatening and often irreversible, which makes the search for new effective antidiabetic drugs a research priority and one of the most pressing issues in health care today. To date, the insulin-mimetic properties of vanadium compounds have been the subject of quite a significant number of studies. In vivo experiments have shown that vanadium, acting in an insulin-like fashion, takes part in the regulation of glucose and lipid metabolism. In particular, vanadium stimulates glucose uptake and metabolism in insulin target tissues, increases the intensity of glycogen and lipid biosynthesis, and inhibits that of gluconeogenesis, glycogenolysis, and lipolysis. Organic vanadium complexes are known to be less toxic than its inorganic salts, which are characterized by a number of potentially serious adverse effects, mainly but not exclusively on the central nervous system and the kidneys; in addition to lower toxicity, organic vanadium compounds have been found to have higher bioavailability when to compared to those of inorganic nature. Such observations clearly hold promise for the development of an entirely novel therapeutic class of antidiabetic drugs. The aims of this study are to obtain new polymeric vanadyl(VO2+) derivatives based on poly-N-vinylpyrrolidone (PVP) that would exert hypoglycemic effect in vivo, and to explore the possibilities of using such compounds or formulations derived thereof for type 2 DM prophylaxis and/or treatment. This work gives a method of obtaining and isolation of an oxovanadium(IV)-polymer metallocomplex, and describes the structural features of said compound, confirmed experimentally. The results of the acute oral toxicity study revealed that the newly synthesized compound is of low toxicity, having a median lethal dose (LD50) that is at a level well above those typical of inorganic vanadium salts.

A Dual-Modality Linker Enables Site-Specific Conjugation of Antibody Fragments for 18F-Immuno-PET and Fluorescence Imaging.

Antibody-based dual-modality (PET/fluorescence) imaging enables both presurgery antigen-specific immuno-PET for noninvasive whole-body evaluation and intraoperative fluorescence for visualization of superficial tissue layers for image-guided surgery. Methods: We developed a universal dual-modality linker (DML) that facilitates site-specific conjugation to a cysteine residue-bearing antibody fragment, introduction of a commercially available fluorescent dye (via an amine-reactive prosthetic group), and rapid and efficient radiolabeling via click chemistry with 18F-labeled trans-cyclooctene (18F-TCO). To generate a dual-modality antibody fragment-based imaging agent, the DML was labeled with the far-red dye sulfonate cyanine 5 (sCy5), site-specifically conjugated to the C-terminal cysteine of the anti-prostate stem cell antigen (PSCA) cys-diabody A2, and subsequently radiolabeled by click chemistry with 18F-TCO. The new imaging probe was evaluated in a human PSCA-positive prostate cancer xenograft model by sequential immuno-PET and optical imaging. Uptake in target tissues was confirmed by ex vivo biodistribution. Results: We successfully synthesized a DML for conjugation of a fluorescent dye and 18F. The anti-PSCA cys-diabody A2 was site-specifically conjugated with either DML or sCy5 and radiolabeled via click chemistry with 18F-TCO. Immuno-PET imaging confirmed in vivo antigen-specific targeting of prostate cancer xenografts as early as 1 h after injection. Rapid renal clearance of the 50-kDa antibody fragment enables same-day imaging. Optical imaging showed antigen-specific fluorescent signal in PSCA-positive xenografts and high contrast to surrounding tissue and PSCA-negative xenografts. Conclusion: The DML enables site-specific conjugation away from the antigen-binding site of antibody fragments, with a controlled linker-to-protein ratio, and combines signaling moieties for 2 imaging systems into 1 molecule. Dual-modality imaging could provide both noninvasive whole-body imaging with organ-level biodistribution and fluorescence image-guided identification of tumor margins during surgery.

99mTc(I) carbonyl-radiolabeled lipid based drug carriers for temozolomide delivery and bioevaluation by in vitro and in vivo

Abstract In preclinical research radiolabeled nanoparticles have been attracting interest as a new class of imaging probes. Assuming good stability of solid lipid nanoparticles (SLNs) under physiological conditions, radiolabeled SLNs can be used for imaging and measuring uptake in target tissue. Present study was performed to evaluate biological behavior of temozolomide (TMZ) loaded solid lipid nanoparticles (SLN-TMZ) in vivo and in vitro. Lipid nanoparticles were prepared by emulsification and low-temperature solidification method. ζ potential, morphology and particle size of nanoparticles were determined. Biological behavior of 99mTc(CO)3 + radiolabeled SLN-TMZ were investigated in vitro on U87/Daoy cell lines and in vivo on female Wistar Albino rats. Obtained results of in vitro incorporation, in vivo biodistribution and gamma imaging studies on radiolabeled SLN-TMZ show that the radiolabeled solid lipid nanoparticles could have potential as a drug delivery system for TMZ.

Genetic Heterogeneity of SLC22 Family of Transporters in Drug Disposition.

An important aspect of modern medicine is its orientation to achieve more personalized pharmacological treatments. In this context, transporters involved in drug disposition have gained well-justified attention. Owing to its broad spectrum of substrate specificity, including endogenous compounds and xenobiotics, and its strategical expression in organs accounting for drug disposition, such as intestine, liver and kidney, the SLC22 family of transporters plays an important role in physiology, pharmacology and toxicology. Among these carriers are plasma membrane transporters for organic cations (OCTs) and anions (OATs) with a marked overlap in substrate specificity. These two major clades of SLC22 proteins share a similar membrane topology but differ in their degree of genetic variability. Members of the OCT subfamily are highly polymorphic, whereas OATs have a lower number of genetic variants. Regarding drug disposition, changes in the activity of these variants affect intestinal absorption and target tissue uptake, but more frequently they modify plasma levels due to enhanced or reduced clearance by the liver and secretion by the kidney. The consequences of these changes in transport-associated function markedly affect the effectiveness and toxicity of the treatment in patients carrying the mutation. In solid tumors, changes in the expression of these transporters and the existence of genetic variants substantially determine the response to anticancer drugs. Moreover, chemoresistance usually evolves in response to pharmacological and radiological treatment. Future personalized medicine will require monitoring these changes in a dynamic way to adapt the treatment to the weaknesses shown by each tumor at each stage in each patient.

Effects of Tumor Burden on Reference Tissue Standardized Uptake for PET Imaging: Modification of PERCIST Criteria.

Purpose To examine the effect metabolic burden (tumor and/or cardiac myocyte uptake) has on fluorine 18 fluorodeoxyglucose (FDG) distribution in organs and tissues of interest. Materials and Methods Positron emission tomographic (PET)/computed tomographic (CT) scans at the Ann Arbor Veterans Affairs hospital from January to July 2015 were reviewed. A total of 107 scans (50 patients; mean age, 64.3 years ± 13.2 [standard deviation]) had metabolic tissue burden assessed by using total lesion glycolysis (TLG) obtained from autosegmentation of the tumor and/or cardiac tissue. Standardized uptake value (SUV) and subsequent normalized SUV uptake in target organs and tissues were compared with 436 FDG PET/CT scans previously reported in 229 patients as a function of TLG to describe the effect(s) that metabolic burden has on reference tissue (blood pool, liver, and brain) FDG uptake. Subsequent regression by using linear mixed-effects models was used. If the slope of the regression was significantly (P < .05) different than zero, then an effect from TLG was present. Results There was a negative inverse relationship (P < .0001) between FDG uptake within reference tissues (blood pool, liver, and brain) and TLG in comparison to the study population at similar blood glucose levels. This TLG effect was no longer statistically significant (P > .05) when FDG uptake was normalized to a reference tissue (eg, blood pool or liver). Conclusion Metabolic tissue burden can have a significant effect on SUV measurements for PET imaging. This effect can be mitigated by normalizing FDG uptake to a reference tissue. © RSNA, 2018.

Uptake and Utilization of Selenium from Selenoprotein P

Selenoprotein P (SELENOP) is a serum glycoprotein that is required for proper selenium distribution in mammals, particularly in supplying selenium to the brain and testes. As the sole mechanism for providing essential selenium to developing spermatozoa, SELENOP metabolism is central to male fertility in all mammals. In addition, this process is important for proper brain function, especially under conditions of limited dietary selenium. Several specific and nonspecific mechanisms for SELENOP uptake in target tissues have been described, but the utilization of SELENOP as a source of selenium for intracellular selenoprotein production has not been systematically characterized. In this report, we examine the process of SELENOP uptake using a robust selenium uptake assay that measures selenium utilization in cells fed 75Se-SELENOP. Using a series of inhibitors and modulators we have identified specific regulators of the process and found that SELENOP must be in an oxidized state for uptake. This assay also demonstrates that SELENOP uptake is not highly sequence specific as thezebrafish protein is recognized and processed by mammalian cells.

Epigallocatechin-3-gallate (EGCG) activates AMPK through the inhibition of glutamate dehydrogenase in muscle and pancreatic ß-cells: A potential beneficial effect in the pre-diabetic state?

Glucose homeostasis is determined by insulin secretion from the ß-cells in pancreatic islets and by glucose uptake in skeletal muscle and other insulin target tissues. While glutamate dehydrogenase (GDH) senses mitochondrial energy supply and regulates insulin secretion, its role in the muscle has not been elucidated. Here we investigated the possible interplay between GDH and the cytosolic energy sensing enzyme 5′-AMP kinase (AMPK), in both isolated islets and myotubes from mice and humans. The green tea polyphenol epigallocatechin-3-gallate (EGCG) was used to inhibit GDH. Insulin secretion was reduced by EGCG upon glucose stimulation and blocked in response to glutamine combined with the allosteric GDH activator BCH (2-aminobicyclo-[2,2,1] heptane-2-carboxylic acid). Insulin secretion was similarly decreased in islets of mice with ß-cell-targeted deletion of GDH (ßGlud1−/−). EGCG did not further reduce insulin secretion in the mutant islets, validating its specificity. In human islets, EGCG attenuated both basal and nutrient-stimulated insulin secretion. Glutamine/BCH-induced lowering of AMPK phosphorylation did not operate in ßGlud1−/− islets and was similarly prevented by EGCG in control islets, while high glucose systematically inactivated AMPK. In mouse C2C12 myotubes, like in islets, the inhibition of AMPK following GDH activation with glutamine/BCH was reversed by EGCG. Stimulation of GDH in primary human myotubes caused lowering of insulin-induced 2-deoxy-glucose uptake, partially counteracted by EGCG. Thus, mitochondrial energy provision through anaplerotic input via GDH influences the activity of the cytosolic energy sensor AMPK. EGCG may be useful in obesity by resensitizing insulin-resistant muscle while blunting hypersecretion of insulin in hypermetabolic states.

Enhanced cellular uptake and tumor penetration of nanoparticles by imprinting the "hidden" part of membrane receptors for targeted drug delivery.

The widely existing transmembrane helices can serve as a novel type of binding site for recognizing corresponding membrane receptors. Through imprinting the transmembrane domain of certain receptors, here we report the construction of polymeric nanoparticles which can achieve enhanced cellular uptake and permeability in target tissues for tumor-targeted drug delivery.

Effect of anti-laronidase antibodies on efficacy and safety of laronidase enzyme replacement therapy for MPS I: A comprehensive meta-analysis of pooled data from multiple studies.

Enzyme replacement therapy (ERT) with laronidase has an important role in the treatment of patients with mucopolysaccharidosis type I (MPS I). Laronidase is safe and has demonstrated effectiveness in terms of stabilizing or improving conventional clinical and laboratory markers of the disease. However, like most ERTs, laronidase produces an anti-drug IgG antibody response in more than 90% of patients during the first few months of treatment. Preclinical data from the MPS I canine model suggest that anti-drug antibodies (ADA) impair enzyme uptake in target tissues. In patients, the effects on tissue glycosaminoglycan (GAG) clearance are difficult to assess directly but data from clinical studies have suggested an association between ADA and both a reduced pharmacodynamic response and hypersensitivity reactions. This comprehensive meta-analysis of pooled data from patients in three clinical studies of laronidase (including one study with an extension) was undertaken to provide a more robust assessment of the relationship between the ADA response to laronidase, clinical and laboratory markers of MPS I, and hypersensitivity reactions. The meta-analysis demonstrated an inverse relationship between the ADA response and the percent reduction in urinary GAG (uGAG) levels. However, no relationships between the ADA response and changes in percent predicted forced vital capacity and six-minute walk test were seen. The study also re-assayed stored serum samples from the original trials with a novel method to determine the inhibitory effect of ADA. Patients with higher ADA exposure over time were found to have higher inhibition of enzyme uptake into cells. High ADA exposure can result in a commensurate level of enzyme uptake inhibition that decreases the pharmacodynamic effect of the exogenously administered therapeutic enzyme, but with no clear effect on clinical efficacy.

Enhancement of glucose uptake in mouse skeletal muscle cells and adipocytes by P2Y6 receptor agonists.

Glucose uptake by peripheral tissues such as skeletal muscles and adipocytes is important in the maintenance of glucose homeostasis. We previously demonstrated that P2Y6 receptor (P2Y6R) agonists protect pancreatic islet cells from apoptosis and stimulate glucose-dependent insulin release. Here, we investigated the effects of P2Y6R activation on glucose uptake in insulin target tissues. An agonist of the P2Y6R, P1-(5′-uridine)-P3-(5′-N4-methoxycytidine)-triphosphate (MRS2957), significantly increased the uptake of [3H]2-deoxyglucose in mouse C2C12 myotubes and 3T3-L1 adipocytes, and this stimulation was significantly decreased by a selective P2Y6R antagonist N,N″-1,4-butanediyl-bis[N′-(3-isothiocyanatophenyl)thiourea] (MRS2578). Pre-incubation with Compound C (an inhibitor of 5′-AMP-activated protein kinase, AMPK), or AMPK siRNA abolished the stimulatory effect of MRS2957 on glucose uptake. Also, MRS2957 (60 min incubation) increased recruitment of the facilitated glucose transporter-4 (GLUT4) to the cell membrane, which was blocked by MRS2578. Treatment of C2C12 myotubes with MRS2957 induced significant phosphorylation of AMPK, which increase GLUT4 expression through histone deacetylase (HDAC)5 signaling. Glucose uptake in primary mouse adipocytes from wild-type mice was stimulated upon P2Y6R activation by either MRS2957 or native agonist UDP, and the P2Y6R effect was antagonized by MRS2578. However, in adipocytes from P2Y6R-knockout mice P2Y6R agonists had no effect on glucose uptake, and there was no change in the glucose uptake by insulin. Our results indicate that the P2Y6R promotes glucose metabolism in peripheral tissues, which may be mediated through AMPK signaling.

Pre-Targeted Radioimmunotherapy Employing a Recombinant Bispecific Antibody Using a Murine Xenograft Model of Human Leukemia

Introduction: Acute Myeloid Leukemia (AML) has a high rate of relapse despite hematopoietic cell transplantation (HCT). Pre-targeted radioimmunotherapy (PRIT) prior to HCT offers a promising approach to reduce relapse for high-risk patients. PRIT studies with antibody-streptavidin (Ab-SAV) conjugates have shown promise, but may be limited by SAV immunogenicity. We have developed a recombinant anti-human CD45 bispecific Ab with picomolar affinity to radiolabeled DOTA chelates for targeting AML xenografts in a preclinical model.Methods: We engineered a bispecific Ab recognizing CD45 and DOTA-metal chelates. The light chain sequence from anti-human CD45 Ab (BC8) was cloned into a pFUSE vector along with a single-chain variable region developed to bind to DOTA chelates of multiple radionuclides. Ab was co-transfected into HEK 293 F cells with a pFUSE vector containing heavy chain sequence from Rituximab. Cultures were grown to extinction, and Ab purified via Protein A ion-exchange chromatography. Ab was characterized by SDS-PAGE, HPLC, and flow cytometry to determine specificity and integrity of material. Ab was tested for in vivo targeting in a xenograft model of human AML. Athymic mice bearing flank HEL tumors (30 to 100 µg) were injected with 280 µg bispecific Ab or 300 µg Ab-SAV. Twenty two hours later mice were treated with or without clearing agent (5 µg DOTAY-dextran for bispecific Ab and 50 µg NAGB for Ab-SAV groups). Two hours later mice were given Yttrium-90 (90Y)-DOTA-biotin. Blood was taken at time points over 22 hours post-injection for pharmacokinetic study; xenografts and normal tissues were taken 4, 24, and 48 hours after DOTA-biotin injection for gamma counts.Results: Bispecific Ab targeting CD45 bound to human AML cells in vitro with an increase in mean fluorescence intensity >2 logs compared to control. SDS-PAGE and HPLC demonstrated a construct of expected size. Blood clearance of 90Y-DOTA-biotin was assessed following injection of bispecific Ab with or without clearing agent and compared to mice that received BC8-SAV as the first-step reagent. Even without clearing agent, mice that had received bispecific Ab showed rapid blood clearance of 90Y-DOTA-biotin similar to that of Ab-SAV-injected mice that had received clearing agent, with 8.15 ± 0.56 % ID/g in the blood 5 minutes post-DOTA-biotin injection falling to 1.02 ± 0.62% ID/g within 4 hours and 0.40 ± 0.16 % ID/g at 20 hours. Mice that received BC8-SAV followed by clearing agent had 7.32 ± 2.0 % ID/g in the blood 5 minutes post-injection, falling to 1.13 ± 0.77 % within 4 hours and 0.73 ± 0.46 % at 20 hours. Mice receiving bispecific Ab followed by DOTAY-dextran clearing agent and 90Y-DOTA-biotin showed a rapid blood clearance with a radioactivity concentration of 8.07 ± 3.0% ID/g at 5 minutes and falling to 0.14 ± 0.03 % within 4 hours post-injection. In vivo targeting of the bispecific Ab was similar to Ab-SAV groups; within 4 hours 3.62 ± 0.91 % ID/g was detected in the tumor compared to 0.12 ± 0.05 % in the blood for mice receiving bispecific Ab, while Ab-SAV mice had a tumor value of 3.85 ± 2.5 % and blood was 1.17 ± 0.63 %. Uptake in non-target tissues was low at 4 hours post-injection compared to tumor, with liver and kidney values of 0.05 ± 0.014 % ID/g and 0.518 ± 0.09 % ID/g, respectively. Tumor retention remained steady at 24 and 48 hours with values of 4.13 ± 1.0% ID/g and 4.14 ± 2.47% ID/g respectively. Groups that received bispecific Ab as the first-step agent followed by clearing agent and then 90Y-DOTA-biotin showed slightly higher uptake in normal organs early on, but displayed higher uptake in target tissue. Tumor values at 4, 24, and 48 hours post-injection were 4.65 ± 2.4, 5.89 ± 4.2, and 3.95 ± 2.3 % ID/g, respectively.Conclusion: Recombinant bispecific Ab targeting both human CD45 and radiometal-labeled DOTA is promising as a novel therapeutic approach for AML prior to HCT. Bispecific Ab targets human AML cells in vitro and in vivo with favorable biodistribution showing specific uptake in tumors while normal organs displayed minimal uptake. Blood clearance of the bispecific Ab is rapid and comparable to that of Ab-SAV, even without the addition of a clearing agent. Future studies will assess therapeutic potential of this bispecific Ab PRIT approach in a murine model of AML.Funding Source Acknowledgment: Research reported in this publication was supported by the National Cancer Institute under award numbers R01CA109663, R01CA136639, and R01CA138720. DisclosuresNo relevant conflicts of interest to declare.

The effect of chelator type on in vitro receptor binding and stability in 177Lu-labeled cetuximab and panitumumab.

Monoclonal antibodies are used in the therapy of various diseases. Thanks to their high specific uptake in target tissues, these antibodies can be utilized in targeted radioimmunotherapy as carriers of radioisotopes to tumors. However, important characteristics of antibodies such as target binding and stability in the organism may be affected by various structural parameters. This study has focused on the potential influence of selected chelators on radiochemical quality and in vitro receptor binding capacity in two modified monoclonal antibodies-cetuximab and panitumumab, both ligands of the epidermal growth factor receptor (EGFR). These two antibodies were each coupled with three macrocyclic chelators (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 1,4,7-triazacyclononane-1,4,7-triacetic acid, and 3,6,9,15-tetraazabicyclo[9.3.1]-pentadeca-1(15),11,13-triene-4-(S)-(4-isothiocyanatobenzyl)-3,6,9-triacetic acid) and labeled with lutetium-177. The stability of the preparations was checked, and the cell binding to EGFR-expressing cell lines was examined. The used method led to very stable radiolabeled preparations. The results showed that binding to the target cells was not affected by the type of chelator. All three chelators may be useful for the labeling of cetuximab and panitumumab with lutetium-177 in future preclinical or clinical studies. Our study revealed previously unpublished fact that the type of chelator selected does not affect binding of EGFR-targeted antibodies labeled with lutetium-177.

Delivery of Therapeutic siRNA to the Lung Endothelium via Novel Lipoplex Formulation DACC

Posttranscriptional gene silencing by RNA interference can be therapeutically exploited to inhibit pathophysiological gene expression. However, in contrast to the established effectiveness of RNAi in vitro, safe and effective delivery of siRNAs to specific organs and cell types in vivo remains the major hurdle. Here, we report the development and in vivo characterization of a novel siRNA delivery system (DACC lipoplex) suitable for modulating target gene expression specifically in the lung vasculature. Systemic administration of DACC in mice delivered siRNA cargo functionally to the lung pulmonary endothelium. A single dose of DACC lipoplexes administered by bolus injection or by infusion was sufficient to specifically silence genes expressed in pulmonary endothelial cells such as CD31, Tie-2, VE-cadherin, or BMP-R2. When tested in a mouse model for lung cancer, repeated treatment with DACC/siRNA(CD31) reduced formation of lung metastases and increased life span in a mouse model of experimental lung metastasis.

Biodegradable Guar Gum Nanoparticles as Carrier for Tamoxifen Citrate in Treatment of Breast Cancer

We prepared, characterized and studied the biodistribution of tamoxifen citrate (TMX) loaded cross-linked guar gum (GG) nanoparticles (NPs). NPs were prepared via a single step emulsion process and particle size evaluated. The extent of tissue distribution and retention following oral administration of TMX loaded GG NPs and TMX tablet in female albino mice was analyzed over a period of 48 hours. Till 48 hours, the particles remained detectable in both mammary and ovary tissue (estrogen receptors). Uptake and retention of TMX from NPs and tablet in mammary gland and ovary tissue changed with time. Results showed that the uptake and retention of NPs was more in the mammary gland between 24 - 48 hours (11.2% at 24 h; 4.65% at 48 h). As mammary gland is the target organ in breast cancer therapy, it may be concluded that the cross-linked GG NPs are capable of releasing the drug at the target and minimize the uptake and retention in non target tissue, the ovary (7.98% at 24 h; 1.9% at 48 h). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) with time were measured. No abnormal changes in the liver enzymes were observed. GG NPs under study can be used as a drug carrier system for treating cancer.

Mechanisms Regulating GLUT4 Transcription in Skeletal Muscle Cells Are Highly Conserved across Vertebrates

The glucose transporter 4 (GLUT4) plays a key role in glucose uptake in insulin target tissues. This transporter has been extensively studied in many species in terms of its function, expression and cellular traffic and complex mechanisms are involved in its regulation at many different levels. However, studies investigating the transcription of the GLUT4 gene and its regulation are scarce. In this study, we have identified the GLUT4 gene in a teleost fish, the Fugu (Takifugu rubripes), and have cloned and characterized a functional promoter of this gene for the first time in a non-mammalian vertebrate. In silico analysis of the Fugu GLUT4 promoter identified potential binding sites for transcription factors such as SP1, C/EBP, MEF2, KLF, SREBP-1c and GC-boxes, as well as a CpG island, but failed to identify a TATA box. In vitro analysis revealed three transcription start sites, with the main residing 307 bp upstream of the ATG codon. Deletion analysis determined that the core promoter was located between nucleotides -132/+94. By transfecting a variety of 5´deletion constructs into L6 muscle cells we have determined that Fugu GLUT4 promoter transcription is regulated by insulin, PG-J2, a PPARγ agonist, and electrical pulse stimulation. Furthermore, our results suggest the implication of motifs such as PPARγ/RXR and HIF-1α in the regulation of Fugu GLUT4 promoter activity by PPARγ and contractile activity, respectively. These data suggest that the characteristics and regulation of the GLUT4 promoter have been remarkably conserved during the evolution from fish to mammals, further evidencing the important role of GLUT4 in metabolic regulation in vertebrates.