A rapid in vitro assay for evaluating the effects of acetylcholinesterase inhibitors and reactivators in the rat basolateral amygdala.

We established a novel brain slice assay to test the ability of acetylcholinesterase (AChE) reactivators to prevent ACh-induced M1 muscarinic acetylcholine receptor (mAChR) dependent hyperexcitability observed after exposure to the organophosphate (OP)-based AChE inhibitor and sarin surrogate 4-nitrophenyl isopropyl methylphosphonate (NIMP). Whole-cell patch clamp recordings were used to evaluate the response of pyramidal neurons in the rat basolateral amygdala (BLA) to brief (1 min) bath application of ACh (100 µM), either in control conditions, or after exposure to NIMP ± an AChE reactivator. Bath application of ACh produced atropine- and pirenzepine-sensitive inward currents in voltage clamped BLA pyramidal neurons, and increased the frequency of spontaneous EPSCs, suggesting robust activation of M1 mAChRs. Responses to ACh were increased ~3-5 fold in slices that had been preincubated in NIMP, and these effects were reversed in a concentration dependent manner by exposure to a commercially available AChE reactivator. The current work outlines a simple assay that can be used to evaluate the efficacy of both known and novel AChE reactivators in an area of the limbic system that likely contributes to seizures after acute exposure to OP-based AChE inhibitors.

Topical doxycycline monohydrate hydrogel 1% targeting proteases/PAR2 pathway is a novel therapeutic for atopic dermatitis.

Atopic Dermatitis (AD) is characterized by skin barrier disruption and an aberrant immune response. Doxycycline is tetracycline antibiotics broadly used systemically to treat inflammatory dermatologic conditions. Several studies have shown doxycycline has anti-inflammatory and pro-healing properties, mainly by blocking tissue proteolytic activity. It is our hypothesis that daily application of a novel doxycycline topical formulation in AD subjects will reduce severity of the disease, by blocking cutaneous proteases activity and restoring skin barrier function and inflammation. To test this hypothesis, we performed a proof of concept, open-label clinical study. Subjects enrolled in the study (n = 15) applied NanoDOX® Hydrogel 1% daily for 4 weeks on a chosen eczematous area. Investigational drug was well tolerated, and no local or systemic adverse events due to investigational drug were reported. Notably, a significant clinical improvement was observed based on a modified Eczema Area & Severity Index (EASI) score of the treated area from start of treatment to 14 and 28 days post-treatment (P < .001). A significant improvement of pruritus was also observed (P = .02). This proof of concept clinical trial is first to explore the impact of a non-systemic doxycycline treatment on AD patients. Our results provide evidence to investigate novel AD treatment strategies targeting cutaneous proteases activity.

Quantitative Assessment of Pulmonary Targeting of Inhaled Corticosteroids Using Ex Vivo Receptor Binding Studies.

The goal of locally acting inhaled corticosteroids is to achieve distinct pulmonary effects with reduced systemic side effects. The present work using an ex vivo receptor binding model in rats was interested in assessing pulmonary targeting for several commercially available corticosteroids by monitoring receptor occupancies in the lung and systemic organs (liver, kidney, spleen, and brain) after intravenous (IV) injection or intratracheal (IT) instillation of a dry powder administration at a dose of 100 µg/kg. Pulmonary targeting, defined as the difference in cumulative receptor occupancies (AUCE) between the lung and kidney after pulmonary delivery, differed across the investigated corticosteroids (ΔAUCE range, 33 ± 46 to 143 ± 52% *h) with the highest degree found for corticosteroids with high systemic clearance and pronounced lipophilicity (presumably allowing a long pulmonary residence time). Additionally, this study demonstrated differences in the receptor occupancies across systemic organs. Using kidney receptor occupancies as the comparator, liver receptor occupancies were reduced (ΔAUCE range: - 157 ± 43 to 178 ± 42% *h) after IV and IT administration for corticosteroids with high intrinsic clearance, while they were increased for corticosteroid prodrugs due to hepatic activation. Spleen receptor occupancies were increased after IT (ΔAUCE range: 33 ± 35 to 135 ± 28% *h), but not after IV administration. This was especially true for slowly dissolving drugs. Reduced brain uptake was also observed for ciclesonide (CIC) and des-ciclesonide (desCIC), two compounds previously not investigated. In summary, ex vivo receptor binding studies represent a powerful tool to assess the fate of ICSs.

Mucosal vaccine efficacy against intrarectal SHIV is independent of anti-Env antibody response.

It is widely believed that protection against acquisition of HIV or SIV infection requires anti-envelope (anti-Env) antibodies, and that cellular immunity may affect viral loads but not acquisition, except in special cases. Here we provide evidence to the contrary. Mucosal immunization may enhance HIV vaccine efficacy by eliciting protective responses at portals of exposure. Accordingly, we vaccinated macaques mucosally with HIV/SIV peptides, modified vaccinia Ankara-SIV (MVA-SIV), and HIV-gp120-CD4 fusion protein plus adjuvants, which consistently reduced infection risk against heterologous intrarectal SHIVSF162P4 challenge, both high dose and repeated low dose. Surprisingly, vaccinated animals exhibited no anti-gp120 humoral responses above background and Gag- and Env-specific T cells were induced but failed to correlate with viral acquisition. Instead, vaccine-induced gut microbiome alteration and myeloid cell accumulation in colorectal mucosa correlated with protection. Ex vivo stimulation of the myeloid cell-enriched population with SHIV led to enhanced production of trained immunity markers TNF-α and IL-6, as well as viral coreceptor agonist MIP1α, which correlated with reduced viral Gag expression and in vivo viral acquisition. Overall, our results suggest mechanisms involving trained innate mucosal immunity together with antigen-specific T cells, and also indicate that vaccines can have critical effects on the gut microbiome, which in turn can affect resistance to infection. Strategies to elicit similar responses may be considered for vaccine designs to achieve optimal protective efficacy.

Intranasal delivery of a bivalent norovirus vaccine formulated in an in situ gelling dry powder.

The global health community is beginning to understand the burden of norovirus-associated disease, which has a significant impact in both developed and developing countries. Norovirus virus like particle (VLP)-based vaccines are currently under development and have been shown to elicit systemic and mucosal immune responses when delivered intranasally. In the present study, we describe the use of a dry powder formulation (GelVac™) with an in situ gelling polysaccharide (GelSite™) extracted from Aloe vera for nasal delivery of a bivalent vaccine formulation containing both GI and GII.4 norovirus VLPs. Dose-ranging studies were performed to identify the optimal antigen dosages based on systemic and mucosal immune responses in guinea pigs and determine any antigenic interference. A dose-dependent increase in systemic and mucosal immunogenicity against each of the VLPs were observed as well as a boosting effect for each VLP after the second dosing. A total antigen dose of ≥50 µg of each GI and GII.4 VLPs was determined to be the maximally immunogenic dose in guinea pigs. The immunogenicity results of this bivalent formulation, taken together with previous work on monovalent GelVac™ norovirus vaccine formulation, provides a basis for future development of this norovirus VLP vaccine.

Zika Virus: Medical Countermeasure Development Challenges.

INTRODUCTION: Reports of high rates of primary microcephaly and Guillain-Barré syndrome associated with Zika virus infection in French Polynesia and Brazil have raised concerns that the virus circulating in these regions is a rapidly developing neuropathic, teratogenic, emerging infectious public health threat. There are no licensed medical countermeasures (vaccines, therapies or preventive drugs) available for Zika virus infection and disease. The Pan American Health Organization (PAHO) predicts that Zika virus will continue to spread and eventually reach all countries and territories in the Americas with endemic Aedes mosquitoes. This paper reviews the status of the Zika virus outbreak, including medical countermeasure options, with a focus on how the epidemiology, insect vectors, neuropathology, virology and immunology inform options and strategies available for medical countermeasure development and deployment. METHODS: Multiple information sources were employed to support the review. These included publically available literature, patents, official communications, English and Lusophone lay press. Online surveys were distributed to physicians in the US, Mexico and Argentina and responses analyzed. Computational epitope analysis as well as infectious disease outbreak modeling and forecasting were implemented. Field observations in Brazil were compiled and interviews conducted with public health officials.

Preclinical dose-ranging studies of a novel dry powder norovirus vaccine formulation.

Norovirus is the primary cause of viral gastroenteritis in humans with multiple genotypes currently circulating worldwide. The development of a successful norovirus vaccine is contingent on its ability to induce both systemic and mucosal antibody responses against a wide range of norovirus genotypes. Norovirus virus-like particles (VLPs) are known to elicit systemic and mucosal immune responses when delivered intranasally. Incorporation of these VLPs into an intranasal powder vaccine offers the advantage of simplicity and induction of neutralizing systemic and mucosal antibodies. Nasal immunization, which provides the advantage of ease of administration and a mucosal delivery mechanism, faces the real issue of limited nasal residence time due to mucociliary clearance. Herein, we describe a novel dry powder (GelVac™) formulation of GI or GII.4 norovirus VLPs, two dominant circulating genotypes, to identify the optimal antigen dosages based on systemic and mucosal immune responses in guinea pigs. Systemic and mucosal immunogenicity of each of the VLPs was observed in a dose-dependent manner. In addition, a boosting effect was observed after the second dosing of each VLP antigen. With the GelVac™ formulation, a total antigen dose of ≥ 15 µg was determined to be the maximally immunogenic dose for both GI and GII.4 norovirus VLPs based on evaluation for 56 days. Taken together, these results indicate that norovirus VLPs could be used as potential vaccine candidates without using an immunostimulatory adjuvant and provide a basis for the development of a GelVac™ bivalent GI/GII.4 norovirus VLP vaccine.

Decorporation of systemically distributed americium by a novel orally administered diethylenetriaminepentaacetic acid (DTPA) formulation in beagle dogs.

Novel decorporation agents are being developed to protect against radiological accidents and terrorists attacks. Radioactive americium is a significant component of nuclear fallout. Removal of large radioactive materials, such as 241Am, from exposed persons is a subject of significant interest due to the hazards they pose. The objective of this study was to evaluate the dose-related efficacy of daily doses of NanoDTPA™ Capsules for decorporating Am administered intravenously as a soluble citrate complex to male and female beagle dogs. In addition, the efficacy of the NanoDTPA™ Capsules for decorporating 241Am was directly compared to intravenously administered saline and DTPA. Animals received a single IV administration of 241Am(III)-citrate on Day 0. One day after radionuclide administration, one of four different doses of NanoDTPA™ Capsules [1, 2, or 6 capsules d(-1) (30 mg, 60 mg, or 180 mg DTPA) or 2 capsules BID], IV Zn-DTPA (5 mg kg(-1) pentetate zinc trisodium) as a positive control, or IV saline as a placebo were administered. NanoDTPA™ Capsules, IV Zn-DTPA, or IV saline was administered on study days 1-14. Animals were euthanized on day 21. A full necropsy was conducted, and liver, spleen, kidneys, lungs and trachea, tracheobronchial lymph nodes (TBLN), muscle samples (right and left quadriceps), gastrointestinal (GI) tract (stomach plus esophagus, upper and lower intestine), gonads, two femurs, lumbar vertebrae (L1-L4), and all other soft tissue remains were collected. Urinary and fecal excretion profiles were increased approximately 10-fold compared to those for untreated animals. Tissue contents were decreased compared to untreated controls. In particular, liver content was decreased by approximately eightfold compared to untreated animals. The results from this study further demonstrate that oral NanoDTPA™ Capsules are equally efficient compared to IV Zn-DTPA in decorporation of actinides.

Mechanical and biocompatible characterization of a cross-linked collagen-hyaluronic acid wound dressing.

Collagen scaffolds have been widely employed as a dermal equivalent to induce fibroblast infiltrations and dermal regeneration in the treatment of chronic wounds and diabetic foot ulcers. Cross-linking methods have been developed to address the disadvantages of the rapid degradation associated with collagen-based scaffolds. To eliminate the potential drawbacks associated with glutaraldehyde cross-linking, methods using a water soluble carbodiimide have been developed. In the present study, the glycosaminoglycan (GAG) hyaluronic acid (HA), was covalently attached to an equine tendon derived collagen scaffold using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) to create ntSPONGE The HA was shown to be homogeneously distributed throughout the collagen matrix. In vitro analyses of the scaffold indicated that the cross-linking enhanced the biological stability by decreasing the enzymatic degradation and increasing the thermal denaturation temperature. The material was shown to support the attachment and proliferation of mouse L929 fibroblast cells. In addition, the cross-linking decreased the resorption rate of the collagen as measured in an intramuscular implant model in rabbits. The material was also shown to be biocompatible in a variety of in vitro and in vivo assays. These results indicate that this cross-linked collagen-HA scaffold, ntSPONGE has the potential for use in chronic wound healing.

Osteoconductivity and osteoinductivity of NanoFUSE(®) DBM.

Bone graft substitutes have become an essential component in a number of orthopedic applications. Autologous bone has long been the gold standard for bone void fillers. However, the limited supply and morbidity associated with using autologous graft material has led to the development of many different bone graft substitutes. Allogeneic demineralized bone matrix (DBM) has been used extensively to supplement autograft bone because of its inherent osteoconductive and osteoinductive properties. Synthetic and natural bone graft substitutes that do not contain growth factors are considered to be osteoconductive only. Bioactive glass has been shown to facilitate graft containment at the operative site as well as activate cellular osteogenesis. In the present study, we present the results of a comprehensive in vitro and in vivo characterization of a combination of allogeneic human bone and bioactive glass bone void filler, NanoFUSE(®) DBM. NanoFUSE(®) DBM is shown to be biocompatible in a number of different assays and has been cleared by the FDA for use in bone filling indications. Data are presented showing the ability of the material to support cell attachment and proliferation on the material thereby demonstrating the osteoconductive nature of the material. NanoFUSE(®) DBM was also shown to be osteoinductive in the mouse thigh muscle model. These data demonstrate that the DBM and bioactive glass combination, NanoFUSE(®) DBM, could be an effective bone graft substitute.

Large intestine-targeted, nanoparticle-releasing oral vaccine to control genitorectal viral infection.

Both rectal and vaginal mucosal surfaces serve as transmission routes for pathogenic microorganisms. Vaccination through large intestinal mucosa, previously proven protective for both of these mucosal sites in animal studies, can be achieved successfully by direct intracolorectal (i.c.r.) administration, but this route is clinically impractical. Oral vaccine delivery seems preferable but runs the risk of the vaccine's destruction in the upper gastrointestinal tract. Therefore, we designed a large intestine-targeted oral delivery with pH-dependent microparticles containing vaccine nanoparticles, which induced colorectal immunity in mice comparably to colorectal vaccination and protected against rectal and vaginal viral challenge. Conversely, vaccine targeted to the small intestine induced only small intestinal immunity and provided no rectal or vaginal protection, demonstrating functional compartmentalization within the gut mucosal immune system. Therefore, using this oral vaccine delivery system to target the large intestine, but not the small intestine, may represent a feasible new strategy for immune protection of rectal and vaginal mucosa.

Aerosolized gentamicin reduces the burden of tuberculosis in a murine model.

Tuberculosis (TB) is a major infectious disease problem: 1.7 million people annually die due to TB. Emergence of drug-resistant Mycobacterium tuberculosis and the lack of new antibiotics have exacerbated the situation. There is an urgent need to develop or repurpose drugs against TB. We evaluated inhaled gentamicin as direct respiratory system-targeted therapy in a murine model of TB. Aerosolized-gentamicin-treated mice showed significantly reduced lung M. tuberculosis loads and fewer granulomas relative to untreated controls. These results suggest that direct delivery of antibiotics to the respiratory system may provide therapeutic benefit to conventional treatment regimes for treatment of pulmonary TB.

Evaluation of inhaled cidofovir as postexposure prophylactic in an aerosol rabbitpox model.

Smallpox is considered a biological threat based upon the possibility of deliberate reintroduction into the population, creating an urgent need for effective antivirals. The antiviral drug cidofovir (Cr) has shown to be effective against poxviruses, although route-specific nephrotoxicity has hampered its development for emergency post-exposure prophylaxis (PEP). In this study, we use a micronized dry powder formulation of pharmaceutical-grade Cr (NanoFOVIRTM; Nf) to treat rabbits exposed to aerosolized rabbitpox virus (RPXV) to further evaluate the effectiveness of direct drug delivery to the lung. Naïve rabbits were infected with RPXV by aerosol; three subsets received aerosolized Nf at 0.5, 1.0 or 1.75mg/kg daily for 3days post-exposure, positive and negative control groups received intravenous (IV) Cr treatments and no treatment, respectively. Nf groups showed an antiviral-dose associated survival of 50% (0.5mg/kg), 80% (1.0mg/kg) and 100% (1.75mg/kg). All animals (100%) from the IV-Cr treatment group and none (0%) from the untreated controls survived. Nf (1.75) protected rabbits from RPX at approximately 10% of the equivalent IV-Cr dose. A dose-related effect was observed in clinical development of RPX disease in Nf groups. Significant reduction of RPX-induced pathological changes was observed in Nf (1.75) and IV-Cr groups. Results suggest that Nf may be a viable antiviral for emergency post-exposure prophylaxis and should be evaluated in other models of poxviral disease.

Brain permeability of inhaled corticosteroids.

The aim of this study was to evaluate if the permeability of inhaled corticosteroids entering the brain is reduced and if P-glycoprotein (P-gp) transporters are involved. Currently employed inhaled corticosteroids were given intravenously and intratracheally to rats at a dose of 100 microg kg-1. An ex-vivo receptor binding assay was used to monitor over 12 h the glucocorticoid receptor occupancy in the brain and a systemic reference organ (kidney). The involvement of P-gp in the brain permeability of triamcinolone acetonide was assessed in wild-type mice and mdr1a(-/-) knockout mice (mice lacking the gene for expressing P-gp). After both forms of administration, the average brain receptor occupancies were 20-56% of those of the reference organ, with the more lipophilic drugs showing a more pronounced receptor occupation. While the receptor occupancies in the liver of wild-type and mdr1a(-/-) mice were similar after administration of triamcinolone acetonide, brain receptor occupancies in mdr1a(-/-) mice were significantly greater (mdr1a(-/-): 47.6%, 40.2-55.0%, n=14; 2; wild-type: 11.5+/-33.0%, n=14; 3). Penetration into the brain for inhaled corticosteroids (especially those of lower lipophilicity) is reduced. Experiments in mdr1a(-/-) mice confirmed the involvement of P-gp transporters. Further studies are needed to assess whether potential drug interactions at the transporter level are of pharmacological significance.