Pain, disability, and quality of life in participants after concurrent onabotulinumtoxinA treatment of upper and lower limb spasticity: Observational results from the ASPIRE study.

INTRODUCTION: Upper and lower limb spasticity is commonly associated with central nervous system disorders including stroke, traumatic brain injury, multiple sclerosis, cerebral palsy, and spinal cord injury, but little is known about the concurrent treatment of upper and lower limb spasticity with botulinum toxins. OBJECTIVE: To evaluate onabotulinumtoxinA (onabotA) utilization and to determine if concurrent onabotA treatment of the upper and lower limbs has supported improvements in participants with spasticity. DESIGN: Sub-analysis of a 2-year, international, prospective, observational registry (ASPIRE, NCT01930786). SETTING: International clinic sites (54). PARTICIPANTS: Adult spasticity participants across etiologies, who received ≥1 concurrent onabotA treatment of the upper and lower limbs during the study. INTERVENTION: Participants were treated with onabotA at the clinician's discretion. OUTCOMES: Baseline characteristics and outcomes of disability (Disability Assessment Scale [DAS]), pain (Numeric Pain Rating Scale [NPRS]), participant satisfaction, physician satisfaction, and quality of life (QoL; Spasticity Impact Assessment [SIA]) were evaluated. Adverse events were monitored throughout the study. RESULTS: Of 744 participants enrolled, 730 received ≥1 dose of onabotA; 275 participants received treatment with onabotA in both upper and lower limbs during ≥1 session; 39.3% of participants were naïve to onabotA for spasticity. The mean (SD) total dose per treatment session ranged from 421.2 (195.3) to 499.6 (188.6) U. The most common baseline upper limb presentation was clenched fist (n = 194, 70.5%); lower limb was equinovarus foot (n = 219, 66.9%). High physician and participant satisfaction and improvements in pain, disability and QoL were reported after most treatments. Nine participants (3.3%) reported nine treatment-related adverse events; two participants (0.7%) reported three serious treatment-related severe adverse events. No new safety signals were identified. CONCLUSION: More than a third of enrolled participants received at least one concurrent onabotA treatment of the upper and lower limbs, with reduced pain, disability, and improved QoL after treatment, consistent with the established safety profile of onabotA for the treatment of spasticity.

Safety and Real-World Dosing of OnabotulinumtoxinA for the Treatment of Adult Spasticity: Post Hoc Analysis of the Adult Spasticity International Registry Study.

OBJECTIVE: The aim of the study is to evaluate the safety of onabotulinumtoxinA treatment for spasticity across dose ranges in real-world practice. DESIGN: Adult Spasticity International Registry was a multicenter, prospective, observational study (NCT01930786) of onabotulinumtoxinA treatment for adult spasticity over 2 yrs. Adverse events, serious adverse events, treatment-related adverse events, and serious treatment-related adverse events were sorted into five categories (≤200, 201-400, 401-600, 601-800, ≥801 U) based on cumulative dose per session. RESULTS: In 3103 treatment sessions ( T ), 730 patients received ≥1 dose of onabotulinumtoxinA. Dose categories included the following: ≤200 U ( n = 312, T = 811), 201-400 U ( n = 446, T = 1366), 401-600 U ( n = 244, T = 716), 601-800 U ( n = 69, T = 149), and ≥801 U ( n = 29, T = 61). Of these patients, 261 reported 827 adverse events, 94 reported 195 serious adverse events, 20 reported 23 treatment-related adverse events, and 2 patients treated with 201-400 U onabotulinumtoxinA reported 3 serious treatment-related adverse events. Treatment-related adverse events reported included ≤200 U (8/811, 0.9%), 201-400 U (7/1366, 0.5%), 401-600 U (6/716, 0.8%), 601-800 U (1/149, 0.7%), and ≥801 U (1/61, 1.6%). CONCLUSIONS: In this post hoc analysis, most treatment sessions were performed with 201-400 U onabotulinumtoxinA. Patients treated with 201-400 U onabotulinumtoxinA had an adverse event profile consistent with onabotulinumtoxinA package inserts globally (e.g., United States, European Union, United Kingdom, Canada). No new safety signals were identified.

Characterization of a Nanovaccine Platform Based on an α1,2-Mannobiose Derivative Shows Species-non-specific Targeting to Human, Bovine, Mouse, and Teleost Fish Dendritic Cells.

Dendritic cells serve as the main immune cells that trigger the immune response. We developed a simple and cost-effective nanovaccine platform based on the α1',2-mannobiose derivative for dendritic cell targeting. In previous work, we have formulated the α1,2-mannobiose-based nanovaccine platform with plasmid DNA and tested it in cattle against BoHV-1 infection. There, we have shown that the dendritic cell targeting using this nanovaccine platform in vivo can boost the immunogenicity, resulting in a long-lasting immunity. In this work, we aim to characterize the α1',2-mannobiose derivative, which is key in the nanovaccine platform. This DC-targeting strategy takes advantage of the specific receptor known as DC-SIGN and exploits its capacity to bind α1,2-mannobiose that is present at terminal ends of oligosaccharides in certain viruses, bacteria, and other pathogens. The oxidative conjugation of α1',2-mannobiose to NH2-PEG2kDa-DSPE allowed us to preserve the chemical structure of the non-reducing mannose of the disaccharide and the OH groups and the stereochemistry of all carbons of the reducing mannose involved in the binding to DC-SIGN. Here, we show specific targeting to DC-SIGN of decorated micelles incubated with the Raji/DC-SIGN cell line and uptake of targeted liposomes that took place in human, bovine, mouse, and teleost fish DCs in vitro, by flow cytometry. Specific targeting was found in all cultures, demonstrating a species-non-specific avidity for this ligand, which opens up the possibility of using this nanoplatform to develop new vaccines for various species, including humans.

siRNA delivery: from basics to therapeutic applications.

The chance to selectively intervene and stop the development of any gene-dependent disease in different organs and pathologies makes siRNA an ideal therapeutic agent. However, serious issues should be addressed before the real therapeutic use of siRNA. The poor pharmacokinetic properties of siRNA, its short half-life, its low in vivo stability, its fast elimination by kidney filtration and its low transfection efficiency complicate the use of siRNA as a therapeutic molecule. In this review, we will describe the latest and most advanced approaches and strategies undertaken to address these limitations and improve siRNA delivery and its gene silencing efficacy as well as the prospects for its therapeutic applications.

Highly sensitive microscale in vivo sensor enabled by electrophoretic assembly of nanoparticles for multiple biomarker detection.

This paper describes a microscale in vivo sensor platform device for the simultaneous detection of multiple biomarkers. We designed the polymer-based biosensors incorporating multiple active isolated areas, as small as 70 µm × 70 µm, for antigen detection. The fabrication approach involved conventional micro- and nano-fabrication processes followed by site-specific electrophoretic directed assembly of antibody-functionalized nanoparticles. To ensure precise and large-scale manufacturing of these biosensors, we developed a semi-automated system for the attachment of the 250-µm biosensor to a 300-µm catheter probe. Our fabrication and post-processing procedures should enable large-scale production of such biosensor devices at lower manufacturing cost. The principle of detection with these biosensors involved a simple fluorescence-based enzyme-linked immunosorbent assay. These biosensors exhibit high selectivity (ability to selectively detect multiple biomarkers of different diseases), specificity (ability to target generic to specific disease biomarkers), rapid antigen uptake, and low detection limits (for carcinoembryonic antigen, 31.25 pg mL(-1); for nucleosomes, 62.5 pg mL(-1)), laying the foundation for potential early detection of various diseases.

Size-selective template-assisted electrophoretic assembly of nanoparticles for biosensing applications.

The precise, size-selective assembly of nanoparticles gives rise to many applications where the assembly of nano building blocks with different biological or chemical functionalizations is necessary. We introduce a simple, fast, reproducible-directed assembly technique that enables a complete sorting of nanoparticles with single-particle resolution. Nanoparticles are size-selectively assembled into prefabricated via arrays using a sequential template-directed electrophoretic assembly method. Polystyrene latex (PSL) nanoparticles with diameters ranging from 200 to 50 nm are selectively assembled into vias comparable to nanoparticle diameter. We investigate the effects of particle size and via size on the sorting efficiency. We show that complete sorting can be achieved when the size of the vias is close to the diameter of the nanoparticles and the size distribution of the chosen nanoparticles does not overlap. The results also show that it is necessary to keep the electric field on during the insertion and removal of the template. To elucidate the versatility and nil effects that the electrophoresis assembly technique has on the assembled nanoparticle characteristics, we have assembled cancer-specific monoclonal antibody-2C5-coated nanoparticles and have also shown that they can successfully measure low concentrations of the nucleosome (NS) antigen.

Recent developments in lipid-based pharmaceutical nanocarriers.

Within the broad spectrum of nanoparticulate carriers, polymeric and lipid-core micelles, liposomes, solid nanoparticles and many others have demonstrated great biological properties which make them excellent pharmaceutical delivery systems. In particular, micelles and liposomes have been shown to have good longevity in the blood that allows their accumulation in pathological areas with a compromised vasculature; can possess specific targeting to disease sites when various targeting ligands are attached to the surface of the nanocarriers or to surface-attached cell-penetrating molecules (like TAT peptide) to enhance intracellular penetration; possess stimulus-sensitivity allowing for drug release from the carriers under certain pathological conditions; and show contrast properties with carrier loading of various contrast materials that allow for direct carrier visualization in vivo. The engineering of "multifunctional pharmaceutical nanocarriers" based on the combination of several useful properties in the same system can significantly enhance the efficacy of many therapeutic and diagnostic protocols. This review considers the current status and next future directions in the emerging area of nanomedicine with particular attention to two lipid-based nanoparticulate systems: liposomes and micelles.

Delivery of siRNA into breast cancer cells via phage fusion protein-targeted liposomes.

Efficacy of siRNAs as potential anticancer therapeutics can be increased by their targeted delivery into cancer cells via tumor-specific ligands. Phage display offers a unique approach to identify highly specific and selective ligands that can deliver nanocarriers to the site of disease. In this study, we proved a novel approach for intracellular delivery of siRNAs into breast cancer cells through their encapsulation into liposomes targeted to the tumor cells with preselected intact phage proteins. The targeted siRNA liposomes were obtained by a fusion of two parental liposomes containing spontaneously inserted siRNA and fusion phage proteins. The presence of pVIII coat protein fused to a MCF-7 cell-targeting peptide DMPGTVLP in the liposomes was confirmed by Western blotting. The novel phage-targeted siRNA-nanopharmaceuticals demonstrate significant down-regulation of PRDM14 gene expression and PRDM14 protein synthesis in the target MCF-7 cells. This approach offers the potential for development of new anticancer siRNA-based targeted nanomedicines. FROM THE CLINICAL EDITOR: In this study, the authors report a novel approach for targeted intracellular delivery of siRNAs into breast cancer cells through encapsulation into liposomes targeted to the tumor cells with preselected intact phage proteins.

Effective stabilization and delivery of siRNA: reversible siRNA-phospholipid conjugate in nanosized mixed polymeric micelles.

siRNA is a powerful tool to control cellular processes at the post-transcriptional level. However, its therapeutic potential is limited because of low stability in biological fluids and the lack of simple and efficient delivery systems. Chemical modification of siRNA could be used to increase its intracellular delivery, but may affect its specific activity. To overcome these obstacles, we suggest a simple and effective system capable of stabilization, delivery, and subsequent release of free active siRNA within cells. With this in mind, we reversibly modified the double-stranded GFP-siRNA with a phosphothioethanol (PE) portion via the reducible disulfide bond and incorporated the resulting siRNA-S-S-PE conjugate in nanosized PEG-PE micelles. In the mixed siRNA-S-S-PE/PEG-PE micelles obtained, siRNA was well-protected against degradation by nucleases for at least 24 h, and was released easily from these nanoparticles in free form in the presence of glutathione (GSH) at a concentration mimicking the intracellular levels. In GFP-C166 endothelial cells, mixed GFP-siRNA-S-S-PE/PEG-PE micelles down-regulate the GFP production 50-fold more effectively than free siRNA. In addition, siRNA-containing micelles showed none of the cytotoxic side effects typical for siRNA delivery systems that are based on electrostatic association of siRNA with cationic carriers. Thus, a reversible siRNA-phospholipid conjugate formulated into mixed micelles with PEG-PE can be an effective, nontoxic system for stabilization and delivery of siRNA.

Targeting glioma cells in vitro with ascorbate-conjugated pharmaceutical nanocarriers.

6-Ascorbate-PEG-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (6-ascorbate-PEG-PE) was synthesized according to a two-step procedure: (1) activation of ascorbic acid with bromine, and (2) synthesis of 6-ascorbate-PEG-PE by reacting 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (poly(ethylene glycol))-2000] with an excess of 6-Br-ascorbic acid. The 6-ascorbate-PEG-PE was recovered by precipitation in diethyl ether and purified by gel permeation chromatography. The analysis of the product by 1H NMR and UV-vis spectroscopy confirmed the identity of the conjugate. Liposomes and PEG-PE-based lipid-core micelles were prepared by thin film hydration technique incorporating 6-ascorbate-PEG-PE as targeting moiety. The targeting properties of the ascorbate-decorated nanosystems were tested by fluorescence-activated cell sorting (FACS) analysis and fluorescent microscopy on a panel of tumor cell lines preliminary selected for their ability to express the SVCT2 ascorbate transporter. Cell lines had been selected on the basis of the immunological properties assessed by FACS, which showed that two glioma cell lines, C6 and F98, and fibroblasts NIH/3T3 express plasma membrane-associated SVCT2 transporter for reduced ascorbic acid. Ascorbate-decorated pharmaceutical nanocarriers were endowed with selective targeting properties toward the SVCT2 transporter expressed in glioma cell models. This study shows that SVCT2 transporter for ascorbic acid expressed both in peculiar epithelial cells of the choroid plexus responsible for the filtering of vitamin C into the central nervous system (CNS) and, in some brain tumor cell lines, can be conceivably exploited as a potential target for delivery of drug-loaded pharmaceutical nanocarriers to the brain.

PEG-PE micelles loaded with paclitaxel and surface-modified by a PBR-ligand: synergistic anticancer effect.

Selective ligands to the peripheral benzodiazepine receptor (PBR) may induce apoptosis and cell cycle arrest. An overexpression of PBR in certain cancers allowed us to consider the use of highly selective ligands to PBR for receptor-mediated drug targeting to tumors. With this in mind, we prepared PBR-targeted nanoparticulate drug delivery systems (PEG-PE micelles) loaded with the anticancer drug paclitaxel (PCL) to test possible synergistic anticancer effects. PEG2k-PE-based polymeric micelles with and without PCL were prepared in HBS, pH 7.5, and conjugated with a PBR-ligand (CB86) in 0.45% of DMSO. The cytotoxic effect of such micelles against the LN 18 human glioblastoma cell line was studied in cell culture. The micelles maintained their size and size distribution and remained intact without drug release after the PBR-ligand conjugation. The PCL-loaded PBR-targeted micelles showed a significantly enhanced toxicity against human glioblastoma LN 18 cancer cells in vitro. Thus, PBR-targeted nanopreparations may potentially serve as a new nanomedicine for targeted cancer therapy.

Peripheral benzodiazepine receptor ligand-PLGA polymer conjugates potentially useful as delivery systems of apoptotic agents.

Poly(d,l-lactic-co-glycolic acid) (PLGA) polymers having different average molecular weights were chemically conjugated to two imidazopyridinacetamides (1 and 2), chosen as model Peripheral Benzodiazepine Receptor (PBR) ligands, via an ester or amide linkage. It is in order to evaluate these conjugates as delivery systems of PBR ligands endowed with apoptosis inducing activity. Various coupling reaction conditions were tested to optimize the conjugation process. After purification by extensive dialysis procedures, the macromolecular conjugates were characterized by FT-IR, UV, (1)H NMR spectroscopy, DSC and the average molecular weights of synthesized conjugates were determined by GPC. PBR ligand released from these conjugates occurred in human serum and in 0.1 N HCl solution at a faster rate than that observed in phosphate buffer, pH 7.4. Moreover, the macromolecular conjugates displayed high affinity and selectivity for PBR. Cytotoxicity studies demonstrated that PBR ligand-PLGA polymer conjugates induce survival inhibition in rat C6 glioma cell line. Fluorescence microscopy studies evidenced the cellular uptake of FITC-conjugated probes 10 and 11 and moreover, the mitochondrial morphology modification induced by compounds 1 and 4a. Therefore, this study demonstrates that this PBR ligand-PLGA combination may provide a new mitochondrial targeted approach useful for improved cancer chemotherapy.