A single immunization with a dry powder anthrax vaccine protects rabbits against lethal aerosol challenge.

Here we confirm that intranasal (IN) dry powder anthrax vaccine formulations are able to protect rabbits against aerosol challenge 9 weeks after a single immunization. The optimum dose of rPA in our dry powder anthrax vaccine formulation in rabbits was experimentally determined to be 150microg and therefore was chosen as the target dose for all subsequent experiments. Rabbits received a single dose of either 150microg rPA, 150microg rPA+150microg of a conjugated 10-mer peptide representing the Bacillus anthracis capsule (conj), or 150microg of conj alone. All dry powder formulations contained MPL and chitosan (ChiSys). Significant anti-rPA titers and anthrax lethal toxin neutralizing antibody (TNA) levels were seen with both rPA containing vaccines, although rPA-specific IgG and TNA levels were reduced in rabbits immunized with rPA plus conj. Nine weeks after immunization, rabbits were exposed to a mean aerosol challenge dose of 278 LD50 of Ames spores. Groups immunized with rPA or with rPA+conj had significant increases in survivor proportions compared to the negative control group by Logrank test (p=0.0001 and 0.003, respectively), and survival was not statistically different for the rPA and rPA+conj immunized groups (p=0.63). These data demonstrate that a single immunization with our dry powder anthrax vaccine can protect against a lethal aerosol spore challenge 9 weeks later.

An intranasal vaccine targeting both the Bacillus anthracis toxin and bacterium provides protection against aerosol spore challenge in rabbits.

An intranasal vaccine targeting the Bacillus anthracis toxin and vegetative bacterium was tested for the ability to protect immunized rabbits against aerosol B. anthracis spore exposure. Rabbits were vaccinated intranasally with PA-based vaccines formulated as dry powders with or without chitosan (ChiSys, Archimedes Development Limited), a compound that exhibits muco-adhesive properties, or as a liquid. Formulations also contained MPL adjuvant and PA. Some vaccines contained PA conjugated to a 10-mer peptide of the poly-d-glutamic acid capsule of B. anthracis. Rabbits were immunized on days 0 and 28 and aerosol challenged with an average 250LD50 Ames spores on day 85. Serum antibody was measured before and after challenge. Significant anti-PA serum IgG levels were obtained, particularly with use of ChiSys based formulations. PA-Conj induced significant anti-capsule responses, although a formulation containing free capsule peptide did not. All immunized rabbits survived the challenge, but differences in morbidity, as evidenced by anorexia, between vaccine groups were observed. Only rabbits immunized with PA+PA-Conj appeared normal throughout the post-challenge observation period (14 days), while all that received PA with the free capsule peptide appeared ill at times as evidenced by a failure to eat normally. One negative control rabbit received a lower inhaled spore dose (183LD50) and survived the challenge, although it was anorexic post-challenge. It also had a high level of anti-LF antibodies in its convalescent serum (5400 U/ml), indicating an extensive infection. In contrast, 75% of the immunized rabbits had no LF-specific antibody in their post-challenge sera, and the rest had low levels (< or = 138 U/ml), indicating that infections resulting in toxin production were avoided or greatly reduced. Thus, intranasal immunization with a chitosan-based powder vaccine combining PA and capsule epitopes provided superior protection against B. anthracis infection compared to a single antigen (PA) vaccine, as evidenced by a reduction in morbidity and prevention of death.

The effect of blood sampling site and physicochemical characteristics of drugs on bioavailability after nasal administration in the sheep model.

PURPOSE: Investigate the effect of blood sampling site and physicochemical characteristics of drugs on the pharmacokinetic (PK) parameters obtained after intravenous and nasal administration in sheep and compare results with computer simulations. METHODS: Three drugs, insulin, morphine, and nicotine, were administered nasally and by intravenous (IV) injection to sheep, and serial blood samples collected concurrently from the carotid artery (insulin, morphine) or cephalic vein (nicotine) and jugular vein. Plasma drug concentrations were measured, and pharmacokinetic and statistical analyses performed, to evaluate sampling site differences. RESULTS: After nasal insulin, bioavailabilities calculated from the two blood sampling site data were comparable. In contrast, apparent bioavailabilities following nasal morphine or nicotine were significantly higher when sampling was from the jugular vein. These results were supported by computer simulations. These observations are attributed to the greater effects of noninstantaneous mixing of drugs for jugular vein sampling following nasal dosing, compared to the other sampling sites, which is significant for drugs that are rapidly and well absorbed and that have a high volume of distribution (Vd). CONCLUSION: The results clearly show that the characteristics of the drug and the blood sampling site can have a significant effect on the pharmacokinetic results obtained after nasal administration in sheep.

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.

Development of a novel nasal nicotine formulation comprising an optimal pulsatile and sustained plasma nicotine profile for smoking cessation.

A novel nasal formulation, in the form of a nicotine-Amberlite resin complex powder has been developed that provided an optimal combined pulsatile and sustained plasma nicotine profile for smoking cessation. The adsorption isotherms of nicotine hydrogen tartrate salt on two types of Amberlite resins (IRP69 and IR120) were evaluated and the subsequent in vitro release properties of nicotine from the nicotine-Amberlite complex powders were tested using a Franz diffusion cell. Amberlite IRP69 and Amberlite IR120 are similar cationic exchange materials with the same ion-exchange capacity but due to a smaller particle size range (10-150 microm) Amberlite IRP69 had a better flow property and a better adsorptive capacity than Amberlite IR120. The material is used as an excipient in marketed pharmaceutical formulations. The highly water soluble salt, nicotine hydrogen tartrate, displayed good adsorption onto both types of Amberlite resin. The maximum adsorption of nicotine onto Amberlite IRP69 was 1.071 mg drug per mg resin. The cumulative release of drug from nicotine hydrogen tartrate-Amberlite complex powders showed that the higher the drug loading, the faster was the rate of release of the drug. Based on these results, various nicotine hydrogen tartrate-Amberlite IRP69 powder formulations containing different ratios of free to bound drug (50% to 100% bound) and a control solution were prepared and evaluated in a sheep model by nasal administration. The nicotine plasma profiles demonstrated that an initial rapid peak plasma level of nicotine followed by a sustained elevated level could be achieved by adjusting the ratio of free to bound nicotine in the Amberlite powder formulation. The curves obtained from some of the formulations were comparable to those predicted from a computer-generated pharmacokinetic model.

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.

Clearance characteristics of chitosan based formulations in the sheep nasal cavity.

This paper describes the clearance characteristics of two bioadhesive nasal delivery systems in the form of chitosan microspheres and chitosan solution, from the nasal cavity of conscious sheep. The pattern of deposition and clearance of the nasal dosage forms were evaluated using a radioactive tracer and the non-invasive technique of gamma scintigraphy. The clearance of chitosan microsphere and solution formulations was compared with that of a control solution. The data show that the control was cleared rapidly from the sheep nasal cavity with a half-time of clearance (time taken for 50% clearance; t(50%)) of about 15 min. The bioadhesive chitosan delivery systems were cleared at a slower rate, with half-times of clearance of 43 min and 115 min, for solution and microsphere formulations respectively. From the results reported in this study it can be concluded that the chitosan delivery systems investigated had significantly reduced rates of clearance from the sheep nasal cavity, as compared to the control. Consequently, chitosan delivery systems have the ability to increase the residence time of drug formulations in the nasal cavity thereby providing the potential for improved systemic medication. The nasal clearance rates recorded in the sheep model mimic very closely the clearance rates found in a previous study using human subjects. It can also be concluded that the sheep can be considered a suitable model for in vivo nasal clearance studies of novel bioadhesive drug delivery systems.

Gastrointestinal transit of dosage forms in the pig.

The gastrointestinal transit of liquid, pellet and tablet formulations was measured under fasted conditions in the domestic pig (n = 4) using the technique of gamma scintigraphy. The mean times for 50% gastric emptying for liquid and pellet systems were 1.4 and 2.2 h, respectively; tablets emptied between 1.5 and 6.0 h. Total transit times were in the order of 50 h. These data conform well to published values for the transit of liquid and solid food materials in the pig. The times are much shorter than those previously published for the transit of solid dosage forms in the pig. We conclude that the domestic pig would be a good model to study the gastrointestinal transit of pharmaceutical formulations and the absorption of drug compounds.

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).

Intranasal insulin delivery and therapy.

Intranasal insulin delivery has been widely investigated as an alternative to subcutaneous injection for the treatment of diabetes. The pharmacokinetic profile of intranasal insulin is similar to that obtained by intravenous injection and, in contrast to subcutaneous insulin delivery, bears close resemblance to the 'pulsatile' pattern of endogenous insulin secretion during meal-times. The literature suggests that intranasal insulin therapy has considerable potential for controlling post-prandial hyperglycaemia in the treatment of both IDDM and NIDDM. However, effective insulin absorption via the nasal route is unlikely without employing the help of absorption enhancers or promoters which are able to modulate nasal epithelial permeability to insulin and/or prolong the residence time of the drug formulation in the nasal cavity. This article discusses the structure and function of the nasal cavity, the barriers which prevent nasal insulin absorption and through the use of absorption enhancers or promoters methods by which these barriers may be overcome.

Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery.

Chitosan is a polysaccharide that demonstrates much potential as a gene delivery system. The ability of a commercially available chitosan and depolymerized chitosan oligomers to condense plasmid was determined using TEM and microtitration calorimetry, while the diameter and stability of the resultant complexes were measured using laser light scattering. Selected complexes were physically stable to challenge with both serum and salt solutions. Parameters such as chitosan molecular weight, plasmid concentration and charge ratio influenced such stability. The effect of including a pH-sensitive endosomolytic peptide on the physicochemical properties of the complex was determined. The presence of a pH-sensitive endosomolytic peptide enhanced the levels of reporter gene expression in Cos-1 cells 4-fold. A selected complex containing a lytic peptide was administered in the upper small intestine and colon of rabbits, and reporter gene expression was measured in defined intestinal tissues. Reporter gene expression was enhanced in defined intestinal tissues, although levels of expression remained low. The combination of strong complex stability and low in vivo expression levels suggest that uptake and/or decomplexation, but not endosomal release, may be the critical rate-limiting steps in the uptake process.

Is the pig a good animal model for studying the human ileal brake?

This study was designed to investigate the existence of an ileal brake mechanism in the pig model. The test substances used (oleic acid, deoxycholic acid, taurocholic acid) had all been previously shown to affect the ileal brake mechanism in other species including man. The substances were infused directly into the terminal ileum of surgically modified pigs, 45 min after the pigs had ingested a meal containing a drug marker. The marker used was sulfasalazine, which is cleaved to form a metabolite, sulfapyridine, when it reaches the colon. The subsequent HPLC analysis of collected blood samples allowed the appearance of sulfapyridine in the plasma and hence the arrival of sulfasalazine in the colon to be determined. Any differences in transit between control and test could be evaluated from a profile of plasma concentrations and corresponding values of AUC. The findings from this study show that the various substances did not affect transit of a test meal in the pig and suggest that it is not possible to use this pig model to make predictions about the human ileal brake.

Provocation and amplification of the transvalvular pressure gradient in rheumatic tricuspid stenosis.

A low cardiac output and high compliance of the systemic venous system may mask a resting tricuspid diastolic gradient in patients with significant rheumatic tricuspid stenosis. Thirty-three patients (mean age 28 +/- 10 years) with rheumatic tricuspid stenosis evidenced by 2-dimensional echocardiography (doming and restricted motion of all 3 tricuspid valve leaflets) were studied to expose occult and to amplify borderline and basal tricuspid valve gradients. At cardiac catheterization, the right atrium and right ventricular pressures were recorded simultaneously in the basal state, after intravenous infusion of 200, 400, 500, 700 or 1,000 ml of normal saline until a mean right atrial pressure of 12 mm Hg was achieved, and after 0.6 mg of intravenous atropine. Eleven patients (33%) had a mean tricuspid diastolic gradient of greater than 2 mm Hg at rest (group 1). After 483 +/- 240 ml of saline infusion, the mean tricuspid diastolic gradient increased from 5 +/- 2 to 9 +/- 3 mm Hg (p less than 0.001), secondary to a marked rise in right atrial pressure from 8 +/- 3 to 12 +/- 2 mm Hg (p less than 0.001). Concomitantly, there was no increase in right ventricular end-diastolic pressure, although the heart rate increased from 76 +/- 13 to 79 +/- 12 beats/min (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)