Nasal delivery of chitosan-DNA plasmid expressing epitopes of respiratory syncytial virus (RSV) induces protective CTL responses in BALB/c mice.

Respiratory syncytial virus (RSV), an important pathogen of the lower respiratory tract, is responsible for severe illness both in new born and young children and in elderly people. Due to complications associated with the use of the early developed vaccines, there is still a need for an effective vaccine against RSV. Most pathogens enter the body via mucosal surfaces and therefore vaccine delivery via routes such as the nasal, may well prove to be superior in inducing protective immune responses against respiratory viruses, since both local and systemic immunity can be induced by nasal immunisation. Previously we have shown that intradermal immunisation of a plasmid DNA encoding the CTL epitope from the M2 protein of RSV induced protective CTL responses. In the present study, the mucosal delivery of plasmid DNA formulated with chitosan has been investigated. Chitosan is a polysachharide consisting of copolymers of N-acetylglucosamine and glucosamine that is derived from chitin, a material found in the shells of crustacea. Intranasal immunisation with plasmid DNA formulated with chitosan induced peptide- and virus-specific CTL responses in BALB/c mice that were comparable to those induced via intradermal immunisation. Following RSV challenge of chitosan/DNA immunised mice, a significant reduction (P<0.001) in the virus load was observed in the lungs of immunised mice compared to that in the control group. These results indicate the potential of immunisation with chitosan-formulated epitope-based vaccines via the intranasal route.

Nasal delivery of insulin using novel chitosan based formulations: a comparative study in two animal models between simple chitosan formulations and chitosan nanoparticles.

PURPOSE: To investigate whether the widely accepted advantages as sociated with the use of chitosan as a nasal drug delivery system might be further improved by application of chitosan formulated a nanoparticles. METHODS: Insulin-chitosan nanoparticles were prepared by the ionotropic gelation of chitosan glutamate and tripolyphosphate pentasodium and by simple complexation of insulin and chitosan. The nasal absorption of insulin after administration in chitosan nanoparticle formulations and in chitosan solution and powder formulations wa evaluated in anaesthetised rats and/or in conscious sheep. RESULTS: Insulin-chitosan nanoparticle formulations produced a pharmacological response in the two animal models, although in both cases the response in terms of lowering the blood glucose levels was less (to 52.9 or 59.7% of basal level in the rat, 72.6% in the sheep than that of the nasal insulin chitosan solution formulation (40.1% in the rat, 53.0% in the sheep). The insulin-chitosan solution formulation was found to be significantly more effective than the complex and nanoparticle formulations. The hypoglycaemic response of the rat to the administration of post-loaded insulin-chitosan nanopar ticles and insulin-loaded chitosan nanoparticles was comparable. As shown in the sheep model, the most effective chitosan formulation for nasal insulin absorption was a chitosan powder delivery system with a bioavailability of 17.0% as compared to 1.3% and 3.6% for the chitosan nanoparticles and chitosan solution formulations, respectively. CONCLUSION: It was shown conclusively that chitosan nanoparticles did not improve the absorption enhancing effect of chitosan in solution or powder form and that chitosan powder was the most effective for mulation for nasal delivery of insulin in the sheep model.

Intranasal delivery of morphine.

Morphine administered nasally to humans as a simple solution is only absorbed to a limited degree, with a bioavailability of the order of 10% compared with intravenous administration. This article describes the development of novel nasal morphine formulations based on chitosan, which, in the sheep model, provide a highly increased absorption with a 5- to 6-fold increase in bioavailability over simple morphine solutions. The chitosan-morphine nasal formulations have been tested in healthy volunteers in comparison with a slow i.v. infusion (over 30 min) of morphine. The results show that the nasal formulation was rapidly absorbed with a T(max) of 15 min or less and a bioavailability of nearly 60%. The shape of the plasma profile for nasal delivery of the chitosan-morphine formulation was similar to the one obtained for the slow i.v. administration of morphine. Furthermore, the metabolite profile obtained after the nasal administration of the chitosan-morphine nasal formulation was essentially identical to the one obtained for morphine administered by the intravenous route. The levels of both morphine-6-glucuronide and morphine-3-glucuronide were only about 25% of that found after oral administration of morphine. It is concluded that a properly designed nasal morphine formulation (such as one with chitosan) can result in a non-injectable opioid product capable of offering patients rapid and efficient pain relief.

Chitosan as a novel nasal delivery system for vaccines.

A variety of different types of nasal vaccine systems has been described to include cholera toxin, microspheres, nanoparticles, liposomes, attenuated virus and cells and outer membrane proteins (proteosomes). The present review describes our work on the use of the cationic polysaccharide, chitosan as a delivery system for nasally administered vaccines. Several animal studies have been carried out on influenza, pertussis and diphtheria vaccines with good results. After nasal administration of the chitosan-antigen nasal vaccines it was generally found that the nasal formulation induced significant serum IgG responses similar to and secretory IgA levels superior to what was induced by a parenteral administration of the vaccine. Animals vaccinated via the nasal route with the various chitosan-antigen vaccines were also found to be protected against the appropriate challenge. So far the nasal chitosan vaccine delivery system has been tested for vaccination against influenza in human subjects. The results of the study showed that the nasal chitosan influenza vaccine was both effective and protective according to the CPMP requirements. The mechanism of action of the chitosan nasal vaccine delivery system is also discussed.

Polymeric lamellar substrate particles for intranasal vaccination.

In recent years, several strategies have been under investigation to achieve safe and effective immunisation, in terms of new antigens, adjuvants and routes of vaccination. The latter include mucosal sites such as oral, rectal, vaginal and nasal. Biodegradable microparticles produced from polymers such as poly(D,L-lactide) (PLA) and poly(D,L-lactide-co-glycolide) (PLGA) containing encapsulated vaccine antigens have been extensively studied for immunisation. These microparticles allow controlled release of vaccines with the aim to develop as single dose vaccines. However there are concerns regarding the integrity and immunogenicity of the antigen during the encapsulation process when the antigen is exposed to organic solvents, high shear stresses and the exposure of antigen to low pH which is caused by polymer degradation. Polymeric lamellar substrate particles (PLSP) produced by simple precipitation of PLA, form a novel polymeric system for the adsorption of antigens. This procedure avoids pH changes, exposure to organic solvents and hence allows the integrity of the antigen to be retained. The aim of this article is to discuss the factors affecting the characteristics of PLSP and adsorption of antigens onto PLSP and consider their potential as adjuvants for the nasal delivery of protein, peptide or viral vaccines.

Bioadhesive starch microspheres and absorption enhancing agents act synergistically to enhance the nasal absorption of polypeptides.

This paper investigates the effect of starch microspheres on the absorption enhancing efficiency of various enhancer systems in formulations with insulin after application in the nasal cavity of sheep. The enhancers studied were lysophosphatidylcholine, glycodeoxycholate and sodium taurodihydroxyfusidate, a bile salt derivative. The enhancers were selected on the basis of their perceived or proven mechanism of action and worked predominantly by interacting with the lipid membrane. The bioadhesive starch microspheres were shown to increase synergistically the effect of the absorption enhancers on the transport of the insulin across the nasal membrane. Dependent on the potency of the enhancer system the increment in absorption enhancement was shown to be from 1.4 times to 5 times that obtained for the absorption enhancer in solution.

Carbohydrate biopolymers enhance antibody responses to mucosally delivered vaccine antigens.

We have evaluated the ability of two carbohydrate biopolymers, chitosan and gellan, to enhance antibody responses to subunit influenza virus vaccines delivered to the respiratory tracts of mice. Groups of mice were vaccinated three times intranasally (i.n.) with 10 microg of purified influenza B/Panama virus surface antigens (PSAs), which consist of hemagglutinin (HA) and neuraminidase (NA), either alone or admixed with chitosan or gellan solutions. Separate groups were vaccinated subcutaneously (s.c.) with PSAs adsorbed to Alhydrogel or chitosan or gellan alone i.n. Serum antibody responses were determined by enzyme-linked immunosorbent assay (ELISA) for influenza virus-specific immunoglobulin G (IgG) and by HA inhibition (HAI) and NA inhibition (NAI) assays. The local respiratory immune response was measured by assaying for influenza virus-specific IgA antibody in nasal secretions and by enumerating nasal and pulmonary lymphocytes secreting IgA, IgG, and IgM anti-influenza virus-specific antibodies by enzyme-linked immunospotting (ELISPOT). When administered alone i.n., B/Panama PSA was poorly immunogenic. Parenteral immunization with B/Panama PSA with Alhydrogel elicited high titers of anti-B/Panama antibodies in serum but a very poor respiratory anti-B/Panama IgA response. In contrast, i.n. immunization with PSA plus chitosan stimulated very strong local and systemic anti-B/Panama responses. Gellan also enhanced the local and serum antibody responses to i.n. PSA but not to the same extent as chitosan. The ability of chitosan to augment the immunogenicity of influenza vaccines given i.n. was confirmed using PSA prepared from an influenza A virus (A/Texas H1N1).

A mucosal vaccine against diphtheria: formulation of cross reacting material (CRM(197)) of diphtheria toxin with chitosan enhances local and systemic antibody and Th2 responses following nasal delivery.

The development of new generation vaccines against diphtheria is dependent on the identification of antigens and routes of immunization that are capable of stimulating immune responses similar to, or greater than, those obtained with the parenterally-delivered toxoid vaccine, while reducing the adverse effects that have been associated with the traditional vaccine. In this study, we examined the cellular and humoral immune responses in mice generated after both parenteral and mucosal immunizations with cross-reacting material (CRM(197)) of diphtheria toxin. We found that both native and mildly formaldehyde-treated CRM(197) and conventional diphtheria toxoid (DT) induced mixed Th1/Th2 responses and similar levels of anti-DT serum IgG following parenteral immunization. In contrast, CRM(197) preparations were poorly immunogenic when administered intranasally in solution. However, formulation of the antigens with chitosan significantly enhanced their immunogenicity, inducing high levels of antigen-specific IgG, secretory IgA, toxin-neutralizing antibodies and T cell responses, predominately of Th2 subtype. Furthermore, intranasal immunization with CRM(197) and chitosan induced protective antibodies against the toxin in a guinea pig passive challenge model. We also found that priming parenterally with DT in alum and boosting intranasally with CRM(197) was a very effective method of immunization in mice, capable of inducing high levels of anti-DT IgG and neutralizing antibodies in the serum and secretory IgA in the respiratory tract. Our findings suggest that boosting intranasally with CRM(197) antigen may be very effective in adolescents or adults who have previously been parenterally immunized with a conventional diphtheria toxoid vaccine.

Potential of polymeric lamellar substrate particles (PLSP) as adjuvants for vaccines.

In recent years microspheres or microparticles produced from biodegradable polymers such as poly(D,L-lactide) (PLA) and poly(D, L-lactide-co-glycolide) (PLGA) containing encapsulated vaccine antigens have been investigated for administration via parenteral, oral, and intranasal routes. These microparticles allow the controlled release of vaccines with an aim to reduce the number of doses for primary immunisation or to develop single dose vaccines. The polymer materials have been widely regarded as being of minimal toxicity. Evaluation of candidate systems in animal studies have shown antibody levels and cell responses similar to or greater than those observed with adjuvants such as alum. However, there are concerns regarding the integrity and immunogenicity of the antigen during the encapsulation process when the antigen is exposed to organic solvents, high shear stresses and the exposure of antigen to low pH which is caused by polymer degradation. An alternative approach would be to adsorb antigens to the surface of biodegradable polymer particles. Polymeric lamellar substrate particles (PLSP), produced by a simple precipitation of PLA, are suitable for this purpose. The adsorption of antigens onto these particles is a simple procedure. It avoids pH changes due to bulk polymer degradation and the use of solvents and therefore will be less damaging to the vaccine. Moreover, such systems will be much easier to scale up for a clinical study and eventual manufacture. The aim of this article is to discuss the preparation and physical characteristics of PLSP, antigen adsorption, in vivo efficacy of PLSP antigen systems and to consider the potential of PLSP as controlled release adjuvants for protein, peptide or viral vaccines.

Stimulation of mucosal and systemic antibody responses against Bordetella pertussis filamentous haemagglutinin and recombinant pertussis toxin after nasal administration with chitosan in mice.

Mice were intranasally immunised with a mixture of Bordetella pertussis filamentous haemagglutinin (FHA) and recombinant pertussis toxin, PT-9K/129G (rPT) in combination with chitosan. For both antigens, this formulation induced systemic responses as measured by serum IgG and also mucosal responses as measured by secretory IgA in lung lavage and nasal washes. Immunosorbant assays were used to measure these responses. Both the systemic and mucosal responses were considerably higher than those produced when a mixture of rPT and FHA was administered nasally without chitosan. In comparison, intraperitoneally administered rPT/FHA adsorbed to Alhydrogel elicited only a systemic response, and nasal chitosan solution produced neither systemic nor mucosal response. This study clearly demonstrated that chitosan potentiated the serum and mucosal immune responses to nasally administered FHA and rPT in mice. Hence, this nasal chitosan delivery system has potential as a new non-injectable vaccine for the prophylaxis of whooping cough.