Distribution of Droplets and Immune Responses After Aerosol and Intra-Nasal Delivery of Influenza Virus to the Respiratory Tract of Pigs.

Recent evidence indicates that local immune responses and tissue resident memory T cells (TRM) are critical for protection against respiratory infections but there is little information on the contributions of upper and lower respiratory tract (URT and LRT) immunity. To provide a rational basis for designing methods for optimal delivery of vaccines to the respiratory tract in a large animal model, we investigated the distribution of droplets generated by a mucosal atomization device (MAD) and two vibrating mesh nebulizers (VMNs) and the immune responses induced by delivery of influenza virus by MAD in pigs. We showed that droplets containing the drug albuterol, a radiolabel (99mTc-DTPA), or a model influenza virus vaccine (S-FLU) have similar aerosol characteristics. 99mTc-DTPA scintigraphy showed that VMNs deliver droplets with uniform distribution throughout the lungs as well as the URT. Surprisingly MAD administration (1ml/nostril) also delivered a high proportion of the dose to the lungs, albeit concentrated in a small area. After MAD administration of influenza virus, antigen specific T cells were found at high frequency in nasal turbinates, trachea, broncho-alveolar lavage, lungs, tracheobronchial nodes, and blood. Anti-influenza antibodies were detected in serum, BAL and nasal swabs. We conclude that the pig is useful for investigating optimal targeting of vaccines to the respiratory tract.

Nasal Administration and Plasma Pharmacokinetics of Parathyroid Hormone Peptide PTH 1-34 for the Treatment of Osteoporosis.

Nasal delivery of large peptides such as parathyroid 1-34 (PTH 1-34) can benefit from a permeation enhancer to promote absorption across the nasal mucosa into the bloodstream. Previously, we have published an encouraging bioavailability (78%), relative to subcutaneous injection in a small animal preclinical model, for a liquid nasal spray formulation containing the permeation enhancer polyethylene glycol (15)-hydroxystearate (Solutol® HS15). We report here the plasma pharmacokinetics of PTH 1-34 in healthy human volunteers receiving the liquid nasal spray formulation containing Solutol® HS15. For comparison, data for a commercially manufactured teriparatide formulation delivered via subcutaneous injection pen are also presented. Tc-99m-DTPA gamma scintigraphy monitored the deposition of the nasal spray in the nasal cavity and clearance via the inferior meatus and nasopharynx. The 50% clearance time was 17.8 min (minimum 10.9, maximum 74.3 min). For PTH 1-34, mean plasma Cmax of 5 pg/mL and 253 pg/mL were obtained for the nasal spray and subcutaneous injection respectively; relative bioavailability of the nasal spray was ≤1%. Subsequently, we investigated the pharmacokinetics of the liquid nasal spray formulation as well as a dry powder nasal formulation also containing Solutol® HS15 in a crossover study in an established ovine model. In this preclinical model, the relative bioavailability of liquid and powder nasal formulations was 1.4% and 1.0% respectively. The absolute bioavailability of subcutaneously administered PTH 1-34 (mean 77%, range 55-108%) in sheep was in agreement with published human data for teriparatide (up to 95%). These findings have important implications in the search for alternative routes of administration of peptides for the treatment of osteoporosis, and in terms of improving translation from animal models to humans.

Riluzole: real-world evidence supports significant extension of median survival times in patients with amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is the commonest form of motor neuron disease and is a fatal, degenerative, multisystem disorder affecting upper and/or lower motor neurons in the motor cortex, brain stem, and spinal cord. ALS is characterized by progressive atrophy of associated bulbar, limb, thoracic, and abdominal muscles and supporting cells manifesting in a range of muscular symptoms such as weakness and wasting and eventual paralysis; the majority of patients will die from respiratory failure within 2-5 years of onset. Riluzole, a synthetic benzothiazole drug with glutamine antagonist activity, is indicated for the treatment of patients with ALS and is the only drug that has been shown to slow the course of the disease and extend survival in ALS patients. The original analyses, and subsequent meta-analyses, of data obtained from randomized controlled trials (RCTs) suggest that riluzole typically extends survival by 2-3 months and increases the chance of an additional year of survival by ~9%. However, published real-world evidence (RWE) from 10 clinical ALS databases indicates that riluzole therapy may afford much greater extension of survival, and improvements in median survival times of more than 19 months have been reported in the overall ALS patient population. This article will review the available data from RCTs and RWE on riluzole therapy.

Intranasal H5N1 vaccines, adjuvanted with chitosan derivatives, protect ferrets against highly pathogenic influenza intranasal and intratracheal challenge.

We investigated the protective efficacy of two intranasal chitosan (CSN and TM-CSN) adjuvanted H5N1 Influenza vaccines against highly pathogenic avian Influenza (HPAI) intratracheal and intranasal challenge in a ferret model. Six groups of 6 ferrets were intranasally vaccinated twice, 21 days apart, with either placebo, antigen alone, CSN adjuvanted antigen, or TM-CSN adjuvanted antigen. Homologous and intra-subtypic antibody cross-reacting responses were assessed. Ferrets were inoculated intratracheally (all treatments) or intranasally (CSN adjuvanted and placebo treatments only) with clade 1 HPAI A/Vietnam/1194/2004 (H5N1) virus 28 days after the second vaccination and subsequently monitored for morbidity and mortality outcomes. Clinical signs were assessed and nasal as well as throat swabs were taken daily for virology. Samples of lung tissue, nasal turbinates, brain, and olfactory bulb were analysed for the presence of virus and examined for histolopathological findings. In contrast to animals vaccinated with antigen alone, the CSN and TM-CSN adjuvanted vaccines induced high levels of antibodies, protected ferrets from death, reduced viral replication and abrogated disease after intratracheal challenge, and in the case of CSN after intranasal challenge. In particular, the TM-CSN adjuvanted vaccine was highly effective at eliciting protective immunity from intratracheal challenge; serologically, protective titres were demonstrable after one vaccination. The 2-dose schedule with TM-CSN vaccine also induced cross-reactive antibodies to clade 2.1 and 2.2 H5N1 viruses. Furthermore ferrets immunised with TM-CSN had no detectable virus in the respiratory tract or brain, whereas there were signs of virus in the throat and lungs, albeit at significantly reduced levels, in CSN vaccinated animals. This study demonstrated for the first time that CSN and in particular TM-CSN adjuvanted intranasal vaccines have the potential to protect against significant mortality and morbidity arising from infection with HPAI H5N1 virus.

Chitosan: a promising safe and immune-enhancing adjuvant for intranasal vaccines.

The nasal route is attractive for the delivery of vaccines in that it not only offers an easy to use, non-invasive, needle-free alternative to more conventional parenteral injection, but it also creates an opportunity to elicit both systemic and (crucially) mucosal immune responses which may increase the capability of controlling pathogens at the site of entry. Immune responses to "naked" antigens are often modest and it is widely accepted that incorporation of an adjuvant is a prerequisite for the achievement of clinically effective nasal vaccines. Many existing adjuvants are sub-optimal or unsuitable because of local toxicity or poor enhancement of immunogenicity. Chitosan, particularly chitosan salts, have now been used in several preclinical and clinical studies with good tolerability, excellent immune stimulation and positive clinical results across a number of infections. Particularly significant evidence supporting chitosan as an adjuvant for nasal vaccination comes from clinical investigations on a norovirus vaccine; this demonstrated the ability of chitosan (ChiSys®), when combined with monophosphoryl lipid, to evoke robust immunological responses and confer protective immunity following (enteral) norovirus challenge. This article summarizes the totality of the meaningful information (including key unpublished data) supporting the development of chitosan-adjuvanted vaccines.

A study of Chitosan and c-di-GMP as mucosal adjuvants for intranasal influenza H5N1 vaccine.

BACKGROUND: Highly pathogenic avian influenza A/H5N1 virus remains a potential pandemic threat, and it is essential to continue vaccine development against this subtype. A local mucosal immune response in the upper respiratory tract may stop influenza transmission. It is therefore important to develop effective intranasal pandemic influenza vaccines that induce mucosal immunity at the site of viral entry. OBJECTIVES: We evaluated the humoral and cellular immune responses of two promising mucosal adjuvants (Chitosan and c-di-GMP) for intranasal influenza H5N1 vaccine in a murine model. Furthermore, we evaluated the concept of co-adjuvanting an experimental adjuvant (c-di-GMP) with chitosan. METHODS: BALB/c mice were intranasally immunised with two doses of subunit NIBRG-14 (H5N1) vaccine (7·5, 1·5 or 0·3 µg haemagglutinin (HA) adjuvanted with chitosan (CSN), c-di-GMP or both adjuvants. RESULTS: All adjuvant formulations improved the serum and local antibody responses, with the highest responses observed in the 7·5 µg HA CSN and c-di-GMP-adjuvanted groups. The c-di-GMP provided dose sparing with protective single radial haemolysis (SRH), and haemagglutination inhibition (HI) antibody responses found in the 0·3 µg HA group. CSN elicited a Th2 response, whereas c-di-GMP induced higher frequencies of virus-specific CD4+T cells producing one or more Th1 cytokines (IFN-γ+, IL-2+, TNF-α+). A combination of the two adjuvants demonstrated effectiveness at 7·5 µg HA and triggered a more balanced Th cytokine profile. CONCLUSION: These data show that combining adjuvants can modulate the Th response and in combination with ongoing studies of adjuvanted intranasal vaccines will dictate the way forward for optimal mucosal influenza vaccines.

Intranasal delivery of recombinant human growth hormone (somatropin) in sheep using chitosan-based powder formulations.

The effectiveness of chitosan in promoting the intranasal bioavailability of recombinant human growth hormone (hGH) has been evaluated. hGH was formulated with chitosan to produce a powder blend (Formulation A) and granules (Formulation B) for intranasal administration. The in vivo pharmacokinetic performance of the formulations was evaluated in a group of six sheep in a randomised crossover study. A subcutaneous injection of hGH solution was administered as a control. The intranasal and subcutaneous doses of hGH were 0.3 and 0.03 mg/kg, respectively. The intranasal formulations appeared to be well tolerated. Mean bioavailabilities of hGH from Formulations A and B were 14 and 15%, respectively relative to subcutaneous injection. It is concluded that chitosan-based intranasal powder formulations may provide a practical means for non-injectable delivery of hGH and, potentially, other therapeutic protein molecules.

Effect of chitosan on the intranasal absorption of salmon calcitonin in sheep.

The effects of a chitosan-based delivery system on the pharmacokinetics of intranasally administered salmon calcitonin (sCT) were investigated in a sheep model. In particular, the feasibility of producing a formulation with a comparable or improved bioavailability and/or less variability than the currently marketed nasal product (Miacalcin nasal spray, Novartis Pharmaceuticals) was assessed. A comparator (control) formulation comprising sCT solution was also tested. Sheep (n=6) were dosed intranasally according to a randomized crossover design. The intranasal sCT dose was 1100 IU (equivalent to approximately 17 IU kg-1). After completion of the nasal dosing legs, five of the sheep received 300 IU sCT (equivalent to approximately 5 IU kg-1) by subcutaneous injection to estimate relative bioavailability. After intranasal or subcutaneous dosing, serial blood samples were taken and plasma separated by centrifugation before measuring sCT concentrations by ELISA. Pharmacokinetic (non-compartmental) and statistical (analysis of variance or non-parametric alternative) analyses were performed. No systemic or local adverse effects were observed following intranasal or subcutaneous administration of sCT. The mean relative bioavailability of sCT from the chitosan solution was improved twofold compared with Miacalcin nasal spray and threefold compared with sCT control solution. Inter-animal variability in sCT absorption appeared to be lower with use of the chitosan-based solution compared with the control solution or commercial product. Based on the reported sheep data, a chitosan delivery system could offer the potential to significantly improve the intranasal absorption of sCT and reduce the variability in absorption. In the clinical setting, this may allow relatively lower doses of the drug to be given intranasally and/or lead to improvements in the efficacy or quality of intranasal therapy.

Mathematical modeling and optimization of drug delivery from intratumorally injected microspheres.

PURPOSE: Paclitaxel is a highly promising phase-sensitive antitumor drug that could conceivably be improved by extended lower dosing as opposed to intermittent higher dosing. Although intratumoral delivery of paclitaxel to the whole tumor at different loads and rates has already been achieved, determining an optimal release mode of paclitaxel for tumor eradication remains difficult. This study set out to rationally design such an optimal microsphere release mode based on mathematical modeling. EXPERIMENTAL DESIGN: A computational reaction-diffusion framework was used to model drug release from intratumorally injected microspheres, drug transport and binding in tumor interstitum, and drug clearance by microvasculature and intracellular uptake and binding. RESULTS: Numerical simulations suggest that interstitial drug concentration is characterized by a fast spatially inhomogeneous rise phase, during which interstitial and intracellular binding sites are saturated, followed by a slow spatially homogeneous phase that is governed by the rate of drug release from microspheres. For zero-order drug release, the slow phase corresponds to a plateau drug concentration that is proportional to the ratio of the rate of blood clearance of drug to the rate of drug release from microspheres. Consequently, increasing the duration of intratumoral drug release extends the duration of cell exposure to the drug but lowers the plateau drug concentration. This tradeoff implies that intratumoral drug release can be designed to optimize tumor cell kill. Synthesizing our modeling predictions with published dose-response data, we propose an optimal protocol for the delivery of paclitaxel-loaded microspheres to small solid tumors.