Targeting caveolae to pump bispecific antibody to TGF-ß into diseased lungs enables ultra-low dose therapeutic efficacy.

The long-sought-after "magic bullet" in systemic therapy remains unrealized for disease targets existing inside most tissues, theoretically because vascular endothelium impedes passive tissue entry and full target engagement. We engineered the first "dual precision" bispecific antibody with one arm pair to precisely bind to lung endothelium and drive active delivery and the other to precisely block TGF-ß effector function inside lung tissue. Targeting caveolae for transendothelial pumping proved essential for delivering most of the injected intravenous dose precisely into lungs within one hour and for enhancing therapeutic potency by >1000-fold in a rat pneumonitis model. Ultra-low doses (µg/kg) inhibited inflammatory cell infiltration, edema, lung tissue damage, disease biomarker expression and TGF-ß signaling. The prodigious benefit of active vs passive transvascular delivery of a precision therapeutic unveils a new promising drug design, delivery and therapy paradigm ripe for expansion and clinical testing.

Propylene Glycol Caprylate-Based Nanoemulsion Formulation of Plumbagin: Development and Characterization of Anticancer Activity.

Plumbagin, a bioactive naphthoquinone, has demonstrated potent antitumor potential. However, plumbagin is a sparingly water-soluble compound; therefore, clinical translation requires and will be facilitated by the development of a new pharmaceutical formulation. We have generated an oil-in-water nanoemulsion formulation of plumbagin using a low-energy spontaneous emulsification process with propylene glycol caprylate (Capryol 90) as an oil phase and Labrasol/Kolliphor RH40 as surfactant and cosurfactant excipients. Formulation studies using Capryol 90/Labrasol/Kolliphor RH40 components, based on pseudoternary diagram and analysis of particle size distribution and polydispersity determined by dynamic light scattering (DLS), identified an optimized composition of excipients for nanoparticle formulation. The nanoemulsion loaded with plumbagin as an active pharmaceutical ingredient had an average hydrodynamic diameter of 30.9 nm with narrow polydispersity. The nanoemulsion exhibited long-term stability, as well as good retention of particle size in simulated physiological environments. Furthermore, plumbagin-loaded nanoemulsion showed an augmented cytotoxicity against prostate cancer cells PTEN-P2 in comparison to free drug. In conclusion, we generated a formulation of plumbagin with high loading drug capacity, robust stability, and scalable production. Novel Capryol 90-based nanoemulsion formulation of plumbagin demonstrated antiproliferative activity against prostate cancer cells, warranting thus further pharmaceutical development.

Plumbagin-Serum Albumin Interaction: Spectral, Electrochemical, Structure-Binding Analysis, Antiproliferative and Cell Signaling Aspects with Implications for Anticancer Therapy.

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) is a small molecule with potent anticancer activity. Like other 1,4-naphthoquinones, it exhibits electrophilic reactivity towards biological nucleophiles. We demonstrate that plumbagin and structurally related 1,4-naphthoquinones with at least one unsubstituted quinoid carbon (C2 or C3) bind to albumin, an ubiquitously present nucleophile, with minimum recovery of free drug. Extraction recovery of plumbagin from albumin in solution showed one-phase exponential decline with a half-live of 9.3 min at 10 µmol/L. In the presence of albumin, plumbagin exhibited instant changes in UV/Vis absorption bands. Electrochemical analysis using cyclic voltammetry showed a decrease in redox peak currents over time until electro-inactivity, thus suggesting the formation of a supramolecular adduct inaccessible for electron transfer. The adduct inhibited cell growth and caused cell-cycle arrest of prostate cancer cells, in part by decreasing levels of the cell-cycle regulator RBBP. The conjugate displayed similar cellular effects to those described for plumbagin, such as decreased levels of androgen receptor and protein kinase C epsilon. The effect of plumbagin-albumin on cancer cells was species-specific, suggesting a receptor-mediated mechanism. Furthermore, it was blocked by cathepsin inhibitor pepstatin A, indicating that lysosomal degradation is involved in the processing of plumbagin-albumin adduct. The spontaneously formed adduct of plumbagin with serum albumin is likely to mediate the biological activities of plumbagin in vivo and to fundamentally influence its pharmacodynamics.

Plumbagin-Loaded Nanoemulsion Drug Delivery Formulation and Evaluation of Antiproliferative Effect on Prostate Cancer Cells.

BACKGROUND: Plumbagin, a medicinal plant-derived 5-hydroxy-2-methyl-1,4-naphthoquinone, is an emerging drug with a variety of pharmacological effects, including potent anticancer activity. We have previously shown that plumbagin improves the efficacy of androgen deprivation therapy (ADT) in prostate cancer and it is now being evaluated in phase I clinical trial. However, the development of formulation of plumbagin as a compound with sparing solubility in water is challenging. METHODS: We have formulated plumbagin-loaded nanoemulsion using pneumatically controlled high pressure homogenization of oleic acid dispersions with polyoxyethylene (20) sorbitan monooleate as surfactant. Nanoemulsion formulations were characterized for particle size distribution by dynamic light scattering (DLS). The kinetics of in vitro drug release was determined by equilibrium dialysis. Anticancer activity toward prostate cancer cells PTEN-P2 was assessed by MTS (Owen's reagent) assay. RESULTS: Particle size distribution of nanoemulsions is tunable and depends on the surfactant concentration. Nanoemulsion formulations of plumbagin with 1-3.5% (w/w) of surfactant showed robust stability of size distribution over time. Plumbagin-loaded nanoemulsion with average hydrodynamic diameter of 135 nm showed exponential release of plumbagin with a half-life of 6.1 h in simulated gastric fluid, 7.0 h in simulated intestinal fluid, and displayed enhanced antiproliferative effect toward prostate cancer cells PTEN-P2 compared to free plumbagin. CONCLUSION: High drug-loading capacity, retention of nanoparticle size, kinetics of release under simulated physiological conditions, and increased antiproliferative activity indicate that oleic-acid based nanoemulsion formulation is a suitable delivery system of plumbagin.

Differential gene expression induced by anti-cancer agent plumbagin is mediated by androgen receptor in prostate cancer cells.

Treatment of mice harboring PTEN-P2 tumors in the prostate or on prostate tissue in vivo with 5-hydroxy-2-methyl-1,4-naphthoquinone, also known as plumbagin, results in tumor regression in castrated mice, but not in intact mice. This suggested that dihydrotestosterone (DHT) production in the testes may prevent cell death due to plumbagin treatment, but the underlying mechanism is not understood. We performed RNA-seq analysis on cells treated with combinations of plumbagin and DHT, and analyzed differential gene expression, to gain insight into the interactions between androgen and plumbgin. DHT and plumbagin synergize to alter the expression of many genes that are not differentially regulated by either single agent when used alone. These experiments revealed that, for many genes, increases in mRNAs caused by DHT are sharply down-regulated by plumbagin, and that many transcripts change in response to plumbagin in a DHT-dependent manner. This suggests that androgen receptor mediates some of the effects of plumbagin on gene expression.

Plumbagin improves the efficacy of androgen deprivation therapy in prostate cancer: A pre-clinical study.

BACKGROUND: Plumbagin is a candidate drug for the treatment of prostate cancer. Previous observations indicated that it may improve the efficacy of androgen deprivation therapy (ADT). This study evaluates the effectiveness of treatment with combinations of plumbagin and alternative strategies for ADT in mouse models of prostate cancer to support its clinical use. METHODS: Plumbagin was administered per oral in a new sesame oil formulation. Standard toxicology studies were performed in rats. For tumor growth studies, mouse prostate cancer cell spheroids were placed on top of grafted prostate tissue in a dorsal chamber and allowed to form tumors. Mice were separated in various treatment groups and tumor size was measured over time by intra-vital microscopy. Survival studies were done in mice after injection of prostate cancer cells in the prostate of male animals. Androgen receptor (AR) levels were analyzed by Western blot from prostate cancer cells treated with plumbagin. RESULTS: Plumbagin caused a decrease in AR levels in vitro. In mice, plumbagin at 1 mg/kg in sesame oil displayed low toxicity and caused a 50% tumor regression when combined with castration. The combination of plumbagin with various forms of chemical ADT including treatment with a GnRH receptor agonist, a GnRH receptor antagonist, or CYP17A1 inhibitors, outperformed ADT alone, increasing mouse survival compared to the standard regimen of castration alone. In contrast, the combination of plumbagin with AR antagonists, such as bicalutamide and enzalutamide, showed no improvement over AR antagonists alone. Thus, plumbagin is effective in combination with drugs that prevent the synthesis of testosterone or its conversion to dihydrotestosterone, but not with drugs that bind to AR. CONCLUSION: Plumbagin significantly improves the effect of ADT drugs currently used in the clinic, with few side effects in mice.

Experimental model of transthoracic, vascular-targeted, photodynamically induced myocardial infarction.

We describe a novel model of myocardial infarction (MI) in rats induced by percutaneous transthoracic low-energy laser-targeted photodynamic irradiation. The procedure does not require thoracotomy and represents a minimally invasive alternative to existing surgical models. Target cardiac area to be photodynamically irradiated was triangulated from the thoracic X-ray scans. The acute phase of MI was histopathologically characterized by the presence of extensive vascular occlusion, hemorrhage, loss of transversal striations, neutrophilic infiltration, and necrotic changes of cardiomyocytes. Consequently, damaged myocardium was replaced with fibrovascular and granulation tissue. The fibrotic scar in the infarcted area was detected by computer tomography imaging. Cardiac troponin I (cTnI), a specific marker of myocardial injury, was significantly elevated at 6 h (41 ± 6 ng/ml, n = 4, P < 0.05 vs. baseline) and returned to baseline after 72 h. Triphenyltetrazolium chloride staining revealed transmural anterolateral infarcts targeting 25 ± 3% of the left ventricle at day 1 with a decrease to 20 ± 3% at day 40 (n = 6 for each group, P < 0.01 vs. day 1). Electrocardiography (ECG) showed significant ST-segment elevation in the acute phase with subsequent development of a pathological Q wave and premature ventricular contractions in the chronic phase of MI. Vectorcardiogram analysis of spatiotemporal electrical signal transduction revealed changes in inscription direction, QRS loop morphology, and redistribution in quadrant areas. The photodynamically induced MI in n = 51 rats was associated with 12% total mortality. Histological findings, ECG abnormalities, and elevated cTnI levels confirmed the photosensitizer-dependent induction of MI after laser irradiation. This novel rodent model of MI might provide a platform to evaluate new diagnostic or therapeutic interventions.

The combination of plumbagin with androgen withdrawal causes profound regression of prostate tumors in vivo.

BACKGROUND: Hormonal ablation is the standard treatment for disseminated androgen-dependent prostate cancer. Although tumor growth is controlled at first, the tumor invariably recurs in the form of castration-resistant prostate cancer. This study assessed the efficacy of a new therapeutic strategy that combines plumbagin, a naturally occurring naphthoquinone, with androgen ablation. METHODS: Viewing microscopy chambers were placed in the dorsal skinfold of mice. Syngeneic prostate tissue was grafted within the chambers and allowed to vascularize. H2B-GFP/PTEN-P2 prostate cancer cells were co-implanted on top of the grafted prostate tissue. Androgen ablation was achieved using surgical castration. Intact and castrated mice were administered plumbagin or sham treatment. Tumor growth, mitosis and apoptosis were monitored in real-time using fluorescent Intra-Vital Microscopy. The mechanism of action of plumbagin was explored using human and mouse prostate cancer cells. RESULTS: Whereas both plumbagin and castration alone impeded tumor growth, only the combination of plumbagin and castration caused profound tumor regression in vivo, mostly due to increased apoptosis of the tumor cells. The cytotoxicity of plumbagin was not affected by androgens in vitro, suggesting that microenvironmental factors not present in culture play a crucial role in the combination effect. Plumbagin-induced cell death was mediated, at least in part, by activation of ERK and was due to generation of reactive oxygen species, because it was abolished by the anti-oxidant N-acetyl-L-cysteine. CONCLUSION: Androgen deprivation in combination with plumbagin may provide a significant improvement over androgen deprivation alone and deserves further evaluation.

Laser-targeted photosensitizer-induced lung injury: noninvasive rat model of pulmonary infarction.

Pulmonary infarction is a life-threatening lung injury that requires rapid and accurate diagnosis for proper treatment. Targetable and reproducible small-animal models that would allow experimental development and preclinical evaluation of diagnostic methods for detecting pulmonary infarction are critically missing. The authors report here a novel procedure to selectively induce pulmonary infarction by photodestructive laser-light irradiation in a targeted location within a specific lung compartment after administration of a photosensitizer. Histopathological analysis of the illuminated lung tissue revealed massive hemorrhage and vascular occlusion after acute injury localized to the site of irradiation. Collapse of alveolar structure, neutrophil influx, and necrosis were subsequently observed. Computed tomography (CT) scans showed evidence of abnormal density and airspace consolidation in the irradiated area of the lung, but not elsewhere in the lung compartment. Perfusion imaging using 99mTc-labeled macroaggregated albumin by single-photon emission computed tomography revealed diminished scintigraphic signal in the opaque area of infarcted lung tissue. The histological changes, CT findings, and perfusion characteristics of pulmonary infarction are mimicked using laser-irradiated, photosensitizer-mediated photodestruction to selectively induce chronic lung injury in a localized area. This small-animal model can be easily and readily used for targeted induction of pulmonary infarction in a designated area of lung compartment and offers the potential for use in evaluating novel diagnostic and therapeutic methods.

Overcoming in vivo barriers to targeted nanodelivery.

Nanoparticles have been investigated as promising nanocarriers for delivery of imaging and therapeutic agents for several decades, but have met with limited success. Although enormous progress in the fields of nanotechnology and nanoscience has been achieved, basic discoveries have not yet translated into effective targeted therapies. Nanoparticles can potentially improve the pharmacokinetics and pharmacodynamics of drugs; however, the complexity of in vivo systems imposes multiple barriers that severely inhibit efficiency and have to be overcome to fully exploit the theoretical potential of nanoparticles. Here, we address two major challenges to effective systemic nanodelivery. Both limited penetration across the vascular endothelium and uptake by the reticuloendothelial system (RES) substantially impede effectiveness of nanoparticle delivery into tissues. Although the design of nanoparticles with extended circulation half-life is essential, it is not sufficient for effective penetration of nanoparticles across the formidable barrier formed by the vascular endothelium. Current nanodelivery systems rely on passive transvascular exchange and tissue accumulation. They require high dosages to create large concentration gradients that drive nanoparticles passively across the blood-tissue interface. However, passive accumulation has resulted in only a fractional dosage of nanoparticles penetrating into target tissue. This inevitably diminishes therapeutic efficacy and aggravates potential side effects. Although there are multiple ways to augment passive delivery, active delivery of targeted nanoparticles across the vascular endothelium could significantly increase the therapeutic index and decrease side effects of nanoparticle-based drug delivery systems. Use of active transendothelial transport pathways, such as caveolae, may provide an effective solution to both target and deliver nanoparticles.

Iodine-125 radiolabeling of silver nanoparticles for in vivo SPECT imaging.

Silver nanoparticles are increasingly finding applications in medicine; however, little is known about their in vivo tissue distribution. Here, we have developed a rapid method for radiolabeling of silver nanoparticles with iodine-125 in order to track in vivo tissue uptake of silver nanoparticles after systemic administration by biodistribution analysis and single-photon emission computerized tomography (SPECT) imaging. Poly(N-vinyl-2 -pyrrolidone)-capped silver nanoparticles with an average size of 12 nm were labeled by chemisorption of iodine-125 with a > 80% yield of radiolabeling efficiency. Radiolabeled silver nanoparticles were intravenously injected in Balb/c mice, and the in vivo distribution pattern of these nanoparticles was evaluated by noninvasive whole-body SPECT imaging, which revealed uptake of the nanoparticles in the liver and spleen. Biodistribution analysis confirmed predominant accumulation of the silver nanoparticles in the spleen (41.5%ID/g) and liver (24.5%ID/g) at 24 h. Extensive uptake in the tissues of the reticuloendothelial system suggests that further investigation of silver nanoparticle interaction with hepatic and splenic tissues at the cellular level is critical for evaluation of the in vivo effects and potential toxicity of silver nanoparticles. This method enables rapid iodine-125 radiolabeling of silver nanoparticles with a specific activity sufficient for in vivo imaging and biodistribution analysis.

Designed auto-assembly of nanostreptabodies for rapid tissue-specific targeting in vivo.

Molecular medicine can benefit greatly from antibodies that deliver therapeutic and imaging agents to select organs and diseased tissues. Yet the development of complex and defined composite nanostructures remains a challenge that requires both designed stoichiometric assembly and superior in vivo testing ability. Here, we generate nanostructures called nanostreptabodies by controlled sequential assembly of biotin-engineered antibody fragments on a streptavidin scaffold with a defined capacity for additional biotinylated payloads such as other antibodies to create bispecific antibodies as well as organic and non-organic moieties. When injected intravenously, these novel and stable nanostructures exhibit exquisite targeting with tissue-specific imaging and delivery, including rapid transendothelial transport that enhances tissue penetration. This "tinkertoy construction" strategy provides a very flexible and efficient way to link targeting vectors with reporter and/or effector agents, thereby providing virtually endless combinations potentially useful for multipurpose molecular and functional imaging in vivo as well as therapies.

Immunotargeting and cloning of two CD34 variants exhibiting restricted expression in adult rat endothelia in vivo.

Mapping protein expression of endothelial cells (EC) in vivo is fundamental to understanding cellular function and may yield new tissue-selective targets. We have developed a monoclonal antibody, MAb J120, to a protein expressed primarily in rat lung and heart endothelium. The antigen was identified as CD34, a marker of hematopoietic stem cells and global marker of endothelial cells in human and mouse tissues. PCR-based cloning identified two CD34 variant proteins, full length and truncated, both of which are expressed on luminal endothelial cell plasma membranes (P) isolated from lung. Truncated CD34 predominated in heart P, and neither variant was detected in P from kidney or liver. CD34 in lung was readily accessible to (125)I-J120 inoculated intravenously, and immunohistochemistry showed strong CD34 expression in lung EC. Few microvessels stained in heart and kidney, and no CD34 was detected in vessels of other organs or in lymphatics. We present herein the first complete sequence of a rat CD34 variant and show for the first time that the encoded truncated variant is endogenously expressed on EC in vivo. We also demonstrate that CD34 expression in rat EC, unlike mouse and human, is restricted in its distribution enabling quite specific lung targeting in vivo.

Ubiquitous yet distinct expression of podocalyxin on vascular surfaces in normal and tumor tissues in the rat.

BACKGROUND/AIMS: The vasculature has become an important target in the development of therapies for increasing numbers of human diseases, yet there are few reliable markers available to identify the endothelium in rodent models. We have characterized the expression, subcellular localization and accessibility of the rat pan-endothelial marker podocalyxin (podxl) using a newly developed monoclonal antibody (mAb), G278. METHODS: podxl expression and accessibility to binding by G278 were determined in the rat by a variety of experimental approaches. RESULTS: mAb G278 reliably immunostained blood vessels of all types and of every size in fresh-frozen, fixed-frozen and paraffin-embedded sections of all tissues, but did not stain lymphatic vessels. Western blotting, in vivo imaging and biodistribution analyses demonstrated that the highest levels of endothelial podxl were found in the lung and heart. We also determined that podxl is not enriched in caveolae and that its expression can be modulated in the tumor microenvironment. CONCLUSION: Our study shows that podxl is a better identifier of rat endothelia than are some of the more commonly used markers and that mAb G278 is a robust antibody for use not only in identifying rat blood vessels but also as a tool to elucidate podxl function.

Live dynamic imaging of caveolae pumping targeted antibody rapidly and specifically across endothelium in the lung.

How effectively and quickly endothelial caveolae can transcytose in vivo is unknown, yet critical for understanding their function and potential clinical utility. Here we use quantitative proteomics to identify aminopeptidase P (APP) concentrated in caveolae of lung endothelium. Electron microscopy confirms this and shows that APP antibody targets nanoparticles to caveolae. Dynamic intravital fluorescence microscopy reveals that targeted caveolae operate effectively as pumps, moving antibody within seconds from blood across endothelium into lung tissue, even against a concentration gradient. This active transcytosis requires normal caveolin-1 expression. Whole body gamma-scintigraphic imaging shows rapid, specific delivery into lung well beyond that achieved by standard vascular targeting. This caveolar trafficking in vivo may underscore a key physiological mechanism for selective transvascular exchange and may provide an enhanced delivery system for imaging agents, drugs, gene-therapy vectors and nanomedicines. 'In vivo proteomic imaging' as described here integrates organellar proteomics with multiple imaging techniques to identify an accessible target space that includes the transvascular pumping space of the caveola.

Biodistribution and pharmacokinetics of 125I-labeled monoclonal antibody M75 specific for carbonic anhydrase IX, an intrinsic marker of hypoxia, in nude mice xenografted with human colorectal carcinoma.

Carbonic anhydrase IX (CA IX) is frequently expressed in human carcinomas and absent from the corresponding normal tissues. Strong induction by tumor hypoxia predisposes CA IX to serve as a target for cancer diagnostics and therapy. Here we evaluated targeting properties and pharmacokinetics of CA IX-specific monoclonal antibody (MAb) M75. Binding parameters of (125)I-labeled M75, including equilibrium dissociation constant, hypoxia-related binding to various cell lines and internalization, were analyzed in vitro. Biodistribution of (125)I-M75 in nude mice bearing HT-29 human colorectal carcinoma xenografts with hypoxic pattern of CA IX expression was studied by measurements of radioactivity in dissected tissues and macroautoradiography of tissue sections. Pharmacokinetics of intravenously administered (125)I-M75 was described using a 2-compartment model. Blood clearance showed a distribution phase t(1/2)(alpha) = 3.4 hr and an elimination phase t(1/2)(beta) = 55.3 hr postinjection. Despite predominant CA IX localization in less accessible perinecrotic regions, (125)I-M75 exhibited specific accumulation in xenograft, with a mean uptake of 15.3 +/- 3.6% of injected dose per gram of tumor tissue at 48 hr postadministration. Specificity of M75 localization was confirmed by low tumor uptake of control antibody. Altogether, our data demonstrate that M75 MAb is a promising tool for selective immunotargeting of hypoxic human tumors that express CA IX.

Lowered oxygen tension induces expression of the hypoxia marker MN/carbonic anhydrase IX in the absence of hypoxia-inducible factor 1 alpha stabilization: a role for phosphatidylinositol 3'-kinase.

Transcription of the gene coding for the tumor-associated antigen MN/carbonic anhydrase IX (CAIX) is regulated by hypoxia-inducible factor 1 (HIF-1). Previous studies identified CAIX expression in areas adjacent to hypoxic regions in solid tumors and suggested supplementary/alternative modes of regulation. To better understand the mechanisms activating CAIX expression, we characterized the cell density-dependent induction of CAIX in HeLa cells. This process is anchorage and serum independent and is not mediated by a soluble factor, decreased pH, or lowered glucose concentration. Stabilization of HIF-1 alpha was not observed in dense cultures. In contrast to sparse cell culture conditions, phosphatidylinositol 3'-kinase (PI3K) activity was significantly increased in dense HeLa cultures. The PI3K inhibitors LY294002 and wortmannin inhibited CAIX expression in dense cultures in a dose-dependent manner, specifically targeting the CA9 promoter (-173/+31 region) that was transactivated by constitutively active p110 PI3K subunit. The mechanism controlling CAIX expression in dense cultures is, however, dependent on lowered O(2) tension because stirring abrogates induction of CAIX expression. Hypoxia- and cell density-induced CAIX expressions were mediated by two seemingly independent mechanisms, as documented by the additive effect of increased cell density and treatment with the hypoxia-mimic CoCl(2) on levels of CAIX expression. The minimal cell density-dependent region within the CA9 promoter consists of the juxtaposed protected region 1 and hypoxia-response elements. However cell density-dependent CAIX expression was abrogated in the HIF-1 alpha-deficient Kal3.5 cells, suggesting an important role of HIF-1 in the corresponding mechanism. Thus, induction of CAIX in high-density cultures requires separate but interdependent pathways of PI3K activation and a minimal level of HIF-1 alpha. These interdependent pathways function at a lowered O(2) concentration that is, however, above that necessary for HIF-1 alpha stabilization.