Pediatric Safety of Polysorbates in Drug Formulations.

Polysorbates 20 and 80 are the most frequently used excipients in biotherapeutics, the safety data for which have been well documented in adults. The polysorbate content in therapeutic formulations that are administered to children, however, has been less clearly regulated or defined with regard to safety. In pediatric patients, excessive amounts of polysorbate in biotherapeutics have been linked to hypersensitivity and other toxicity-related effects. To determine safe levels of polysorbates for young patients, we have developed the progressive pediatric safety factor (PPSF), an age- and weight-based tool that estimates the amount of parenterally administered polysorbates 20 and 80 in formulations that will avoid excipient-related adverse events. Compared with existing modalities for calculating maximum acceptable doses of excipients for initial clinical trials in pediatrics, the PPSF is far more conservative, thus constituting an added margin of safety for excipient exposure in the most sensitive subpopulations-i.e., neonates and infants. Further, the PPSF may be applied to any relevant excipient, aiding pharmaceutical developers and regulatory authorities in conservatively estimating the safety assessment of a biotherapeutic's formulation, based on excipient levels.

Pathogenic Autoreactive T and B Cells Cross-React with Mimotopes Expressed by a Common Human Gut Commensal to Trigger Autoimmunity.

Given the immense antigenic load present in the microbiome, we hypothesized that microbiota mimotopes can be a persistent trigger in human autoimmunity via cross-reactivity. Using antiphospholipid syndrome (APS) as a model, we demonstrate cross-reactivity between non-orthologous mimotopes expressed by a common human gut commensal, Roseburia intestinalis (R. int), and T and B cell autoepitopes in the APS autoantigen ß2-glycoprotein I (ß2GPI). Autoantigen-reactive CD4+ memory T cell clones and an APS-derived, pathogenic monoclonal antibody cross-reacted with R. int mimotopes. Core-sequence-dependent anti-R. int mimotope IgG titers were significantly elevated in APS patients and correlated with anti-ß2GPI IgG autoantibodies. R. int immunization of mice induced ß2GPI-specific lymphocytes and autoantibodies. Oral gavage of susceptible mice with R. int induced anti-human ß2GPI autoantibodies and autoimmune pathologies. Together, these data support a role for non-orthologous commensal-host cross-reactivity in the development and persistence of autoimmunity in APS, which may apply more broadly to human autoimmune disease.

Commensal orthologs of the human autoantigen Ro60 as triggers of autoimmunity in lupus.

The earliest autoantibodies in lupus are directed against the RNA binding autoantigen Ro60, but the triggers against this evolutionarily conserved antigen remain elusive. We identified Ro60 orthologs in a subset of human skin, oral, and gut commensal bacterial species and confirmed the presence of these orthologs in patients with lupus and healthy controls. Thus, we hypothesized that commensal Ro60 orthologs may trigger autoimmunity via cross-reactivity in genetically susceptible individuals. Sera from human anti-Ro60-positive lupus patients immunoprecipitated commensal Ro60 ribonucleoproteins. Human Ro60 autoantigen-specific CD4 memory T cell clones from lupus patients were activated by skin and mucosal Ro60-containing bacteria, supporting T cell cross-reactivity in humans. Further, germ-free mice spontaneously initiated anti-human Ro60 T and B cell responses and developed glomerular immune complex deposits after monocolonization with a Ro60 ortholog-containing gut commensal, linking anti-Ro60 commensal responses in vivo with the production of human Ro60 autoantibodies and signs of autoimmunity. Together, these data support that colonization with autoantigen ortholog-producing commensal species may initiate and sustain chronic autoimmunity in genetically predisposed individuals. The concept of commensal ortholog cross-reactivity may apply more broadly to autoimmune diseases and lead to novel treatment approaches aimed at defined commensal species.

Cutting Edge: Nanogel-Based Delivery of an Inhibitor of CaMK4 to CD4+ T Cells Suppresses Experimental Autoimmune Encephalomyelitis and Lupus-like Disease in Mice.

Treatment of autoimmune diseases is still largely based on the use of systemically acting immunosuppressive drugs, which invariably cause severe side effects. Calcium/calmodulin-dependent protein kinase IV is involved in the suppression of IL-2 and the production of IL-17. Its pharmacologic or genetic inhibition limits autoimmune disease in mice. In this study, we demonstrate that KN93, a small-molecule inhibitor of calcium/calmodulin-dependent protein kinase IV, targeted to CD4(+) T cells via a nanolipogel delivery system, markedly reduced experimental autoimmune encephalomyelitis and was 10-fold more potent than the free systemically delivered drug in the lupus mouse models. The targeted delivery of KN93 did not deplete T cells but effectively blocked Th17 cell differentiation and expansion as measured in the spinal cords and kidneys of mice developing experimental autoimmune encephalomyelitis or lupus, respectively. These results highlight the promise of cell-targeted inhibition of molecules involved in the pathogenesis of autoimmunity as a means of advancing the treatment of autoimmune diseases.

Modelling of a targeted nanotherapeutic 'stroma' to deliver the cytokine LIF, or XAV939, a potent inhibitor of Wnt-ß-catenin signalling, for use in human fetal dopaminergic grafts in Parkinson's disease.

The endogenous reparative capacity of the adult human brain is low, and chronic neurodegenerative disorders of the central nervous system represent one of the greatest areas of unmet clinical need in the developing world. Novel therapeutic strategies to treat them include: (i) growth factor delivery to boost endogenous repair and (ii) replacement cell therapy, including replacing dopaminergic neurons to treat Parkinson's disease (PD). However, these approaches are restricted not only by rapid degradation of growth factors, but also by the limited availability of cells for transplant and the poor survival of implanted cells that lack the necessary stromal support. We therefore hypothesised that provision of a transient artificial stroma for paracrine delivery of pro-survival factors could overcome both of these issues. Using leukaemia inhibitory factor (LIF) - a proneural, reparative cytokine - formulated as target-specific poly(lactic-co-glycolic acid) (PLGA) nano-particles (LIF-nano-stroma), we discovered that attachment of LIF-nano-stroma to freshly isolated fetal dopaminergic cells improved their survival fourfold: furthermore, in vivo, the number of surviving human fetal dopaminergic cells tended to be higher at 3 months after grafting into the striatum of nude rats, compared with controls treated with empty nanoparticles. In addition, we also analysed the effect of a novel nano-stroma incorporating XAV939 (XAV), a potent inhibitor of the developmentally important Wnt-ß-catenin signalling pathway, to investigate whether it could also promote the survival and differentiation of human fetal dopaminergic precursors; we found that the numbers of both tyrosine-hydroxylase-positive neurons (a marker of dopaminergic neurons) and total neurons were increased. This is the first demonstration that LIF-nano-stroma and XAV-nano-stroma each have pro-survival effects on human dopaminergic neurons, with potential value for target-specific modulation of neurogenic fate in cell-based therapies for PD.

Neurotrophin receptor TrkB promotes lung adenocarcinoma metastasis.

Lung cancer is notorious for its ability to metastasize, but the pathways regulating lung cancer metastasis are largely unknown. An in vitro system designed to discover factors critical for lung cancer cell migration identified brain-derived neurotrophic factor, which stimulates cell migration through activation of tropomyosin-related kinase B (TrkB; also called NTRK2). Knockdown of TrkB in human lung cancer cell lines significantly decreased their migratory and metastatic ability in vitro and in vivo. In an autochthonous lung adenocarcinoma model driven by activated oncogenic Kras and p53 loss, TrkB deficiency significantly reduced metastasis. Hypoxia-inducible factor-1 directly regulated TrkB expression, and, in turn, TrkB activated Akt signaling in metastatic lung cancer cells. Finally, TrkB expression was correlated with metastasis in patient samples, and TrkB was detected more often in tumors that did not have Kras or epidermal growth factor receptor mutations. These studies demonstrate that TrkB is an important therapeutic target in metastatic lung adenocarcinoma.

Multi-compartmental oral delivery systems for nucleic acid therapy in the gastrointestinal tract.

Gene and RNA interference therapies have significant potential for alleviating countless diseases, including many associated with the gastro-intestinal (GI) tract. Unfortunately, oral delivery of genes and small interfering RNA (siRNA) is very challenging due to the extracellular and intracellular barriers. In this review, we discuss the utilization of multi-compartmental delivery systems for oral administration of nucleic acid therapies. Some of the illustrative examples of multi-compartmental systems include solid nanoparticles-in-microsphere, solid nanoparticles-in-emulsion, and liquid nanoparticles-in-emulsion. Using type B gelatin nanoparticles encapsulated in poly(ε-caprolactone) microspheres, we have prepared nanoparticles-in-microsphere oral system (NiMOS) for gene and siRNA delivery for the treatment of inflammatory bowel disease (IBD). The results of these studies show that the multi-compartmental formulations can overcome many of the barriers for effective oral gene and siRNA delivery.

Characterization of the cell of origin for small cell lung cancer.

Small cell lung carcinoma (SCLC) is a neuroendocrine subtype of lung cancer that affects more than 200,000 people worldwide every year with a very high mortality rate. Here, we used a mouse genetics approach to characterize the cell of origin for SCLC; in this mouse model, tumors are initiated by the deletion of the Rb and p53 tumor suppressor genes in the lung epithelium of adult mice. We found that mouse SCLCs often arise in the lung epithelium, where neuroendocrine cells are located, and that the majority of early lesions were composed of proliferating neuroendocrine cells. In addition, mice in which Rb and p53 are deleted in a variety of non-neuroendocrine lung epithelial cells did not develop SCLC. These data indicate that SCLC likely arises from neuroendocrine cells in the lung.

Oral TNF-α gene silencing using a polymeric microsphere-based delivery system for the treatment of inflammatory bowel disease.

The purpose of this study was to evaluate down-regulation of tumor necrosis factor (TNF)-α by oral RNA interference therapy. Control (scrambled sequence) or TNF-α specific small interfering RNA (siRNA) was encapsulated in type B gelatin nanoparticles and further entrapped in poly(epsilon-caprolactone) (PCL) microspheres to form a nanoparticles-in-microsphere oral system (NiMOS). Upon confirmation of the dextran sulfate sodium (DSS)-induced acute colitis model, mice were divided into several treatment groups receiving no treatment, blank NiMOS, NiMOS with scramble siRNA, or NiMOS with TNF-α silencing siRNA by oral administration. Successful gene silencing led to decreased colonic levels of TNF-α, suppressed expression of other pro-inflammatory cytokines (e.g., interleukin (IL)-1ß, interferon (IFN)-γ) and chemokines (MCP-1), an increase in body weight, and reduced tissue myeloperoxidase activity. Results of this study established the clinical potential of a NiMOS-based oral TNF-α gene silencing system for the treatment of inflammatory bowel disease as demonstrated in an acute colitis model.

Dual TNF-α/Cyclin D1 Gene Silencing With an Oral Polymeric Microparticle System as a Novel Strategy for the Treatment of Inflammatory Bowel Disease.

OBJECTIVES: RNA silencing utilizing short interfering RNA (siRNA) offers a new and exciting means to overcome the limitations of current treatment options of many diseases. However, delivery of these molecules still poses a great challenge to date. METHODS: In the present study, a multicompartmental biodegradable polymer-based nanoparticles-in-microsphere oral system (NiMOS) using gelatin nanoparticles encapsulating a combination of siRNA duplexes specifically targeted against tumor necrosis factor-α (TNF-α) and cyclin D1 (Ccnd1) was employed to study its effects on a dextran sulfate sodium (DSS)-induced acute colitis mouse model mimicking inflammatory bowel disease (IBD). DSS colitis-bearing animals were divided into several control and treatment groups and received either no treatment, blank NiMOS, NiMOS-encapsulating inactive (scrambled), active TNF-α silencing, CyD1 silencing siRNA, or a combination of both active siRNAs by repeated oral administration of three NiMOS doses. RESULTS: Successful gene silencing with the aid of dual siRNA treatment led to decreased colonic levels of TNF-α or CyD1, suppressed expression of certain pro-inflammatory cytokines (interleukin-1α and -ß, interferon-γ), an increase in body weight, and reduced tissue myeloperoxidase activity, while the silencing effect of CyD1 siRNA or the dual treatment was more potent than that of TNF-α siRNA alone. CONCLUSION: Results of this study demonstrate the therapeutic potential of a NiMOS-based oral combined TNF-α and CyD1 gene silencing system for the treatment of IBD as shown in an acute colitis model.

Nanotechnology solutions for mucosal immunization.

The current prevalence of infectious diseases in many developing regions of the world is a serious burden, impacting both the general health as well as economic growth of these communities. Additionally, treatment with conventional medication becomes increasingly challenging due to emergence of new and drug resistant strains jeopardizing the progress made in recent years towards control and elimination of certain types of infectious diseases. Thus, from a public health perspective, prevention such as through immunization by vaccination, which has proven to be most effective, might be the best alternative to prevent and combat infectious diseases in these regions. To achieve this, development of wide-scale immunization programs become necessary including vaccines that can easily and widely be distributed, stored and administered. Mucosal vaccines offer great potential since they can be administered via oral or intranasal delivery route which does not require trained personnel, avoids the use of needles and improves overall patient compliance and acceptance. However, it necessitates the implementation of specific immunization strategies to improve their efficacy. Application of nanotechnology to design and create particle mediated delivery systems that can efficiently encapsulate vaccine components for protection of the sensitive payload, target the mucosal immune system and incorporate mucosal adjuvants maximizing immune response is key strategy to improve the effectiveness of mucosal vaccines.

Delivery strategies to enhance mucosal vaccination.

BACKGROUND: Vaccines capable of eliciting cellular and humoral immune responses in tandem could provide prophylactic and therapeutic responses against infectious diseases and cancer. These responses can be induced systemically and at mucosal surfaces by activating the mucosal immune system, but rarely successfully due to challenges associated with mucosal delivery. OBJECTIVE: To investigate delivery strategies to improve the effectiveness of mucosal vaccines. METHODS: Different challenges are associated with different types of vaccines. We consider administration routes, schedules, carrier systems and adjuvants that can be used to overcome these challenges to enhance mucosal vaccination. RESULTS/CONCLUSIONS: The use of particle-mediated delivery systems is an effective strategy to enhance mucosal vaccination by protecting immunogenic material during delivery, providing targeted delivery systems, and allowing incorporation of adjuvant material.

Fabrication, functionalization, and application of electrospun biopolymer nanofibers.

The use of novel nanostructured materials has attracted considerable interest in the food industry for their utilization as highly functional ingredients, high-performance packaging materials, processing aids, and food quality and safety sensors. Most previous application interest has focused on the development of nanoparticles. However, more recently, the ability to produce non-woven mats composed of nanofibers that can be used in food applications is beginning to be investigated. Electrospinning is a novel fabrication technique that can be used to produce fibers with diameters below 100 nm from (bio-) polymer solutions. These nanofibers have been shown to possess unique properties that distinguish them from non-woven fibers produced by other methods, e.g., melt-blowing. This is because first the process involved results in a high orientation of polymers within the fibers that leads to mechanically superior properties, e.g., increased tensile strengths. Second, during the spinning of the fibers from polymer solutions, the solvent is rapidly evaporated allowing the production of fibers composed of polymer blends that would typically phase separate if spun with other processes. Third, the small dimensions of the fibers lead to very high specific surface areas. Because of this the fiber properties may be greatly influenced by surface properties giving rise to fiber functionalities not found in fibers of larger sizes. For food applications, the fibers may find uses as ingredients if they are composed solely of edible polymers and GRAS ingredients, (e.g., fibers could contain functional ingredients such as nutraceuticals, antioxidants, antimicrobials, and flavors), as active packaging materials or as processing aids (e.g., catalytic reactors, membranes, filters (Lala et al., 2007), and sensors (Manesh et al., 2007; Ren et al., 2006; Sawicka et al., 2005). This review is therefore intended to introduce interested food and agricultural scientists to the concept of nano-fiber manufacturing with a particular emphasis on the use of biopolymers. We will review typical fabrication set-ups, discuss the influence of process conditions on nanofiber properties, and then review previous studies that describe the production of biopolymer-based nanofibers. Finally we briefly discuss emerging methods to further functionalize fibers and discuss potential applications in the area of food science and technology.