CriticalSorb™ promotes permeation of flux markers across isolated rat intestinal mucosae and Caco-2 monolayers.

PURPOSE: CriticalSorb™ is a novel absorption enhancer based on Solutol(®) HS15, one that has been found to enhance the nasal transport. It is in clinical trials for nasal delivery of human growth hormone. The hypothesis was that permeating enhancement effects of the Solutol(®)HS15 component would translate to the intestine. METHODS: Rat colonic mucosae were mounted in Ussing chambers and Papp values of [(14)C]-mannitol, [(14)C]-antipyrine, FITC-dextran 4000 (FD-4), and TEER values were calculated in the presence of CriticalSorb™. Tissues were fixed for H & E staining. Caco-2 monolayers were grown on Transwells™ for similar experiments. RESULTS: CriticalSorb™(0.01% v/v) significantly increased the Papp of [(14)C]-mannitol, FD-4 [(14)C]-antipyrine across ileal and colonic mucosae, accompanied by a decrease in TEER. In Caco-2 monolayers, it also increased the Papp of [(14)C]-mannitol FD-4 and [(14)C]-antipyrine over 120 min. In both monolayers and tissues, it acted as a moderately effective P-glycoprotein inhibitor. There was no evidence of cytotoxicity in Caco-2 at concentrations of 0.01% for up to 24 h and histology of tissues showed intact epithelia at 120 min. CONCLUSIONS: Solutol(®) HS15 is the key component in CriticalSorb™ that enables non-cytotoxic in vitro intestinal permeation and its mechanism of action is a combination of increased paracellular and transcellular flux.

Single shot tetanus vaccine manufactured by a supercritical fluid encapsulation technology.

Single shot vaccines of tetanus toxoid (TT) were manufactured using the NanoMix process - a low temperature solvent free encapsulation technology using supercritical fluids. The formulations were injected into mice, and compared to multiple injections of a commercially available alum adsorbed TT vaccine. After 5 months the antibody titres were found to be similar for both the alum adsorbed and microparticle formulations, demonstrating for the first time the potential of formulating antigens in PLA microparticles using the supercritical fluid (NanoMix) technique to produce single shot vaccines. The results are likely to be due to the maintenance of toxoid bioactivity and some degree of sustained release of the encapsulated antigens, resulting in repeated stimulation of antigen presenting cells eliminating the need for multiple immunisations. This demonstrates the potential of this supercritical fluid processing technique to reduce the need for booster doses in a vaccine regimen.

Sustained release hGH microsphere formulation produced by a novel supercritical fluid technology: in vivo studies.

Novel sustained release formulations of hGH prepared by supercritical fluid processing of PLGA/PLA (the CriticalMix process) were produced in the form of microparticles for subcutaneous injection. The basis of the process is that PLGA/PLA polymers liquefy when exposed to supercritical CO(2), thereby allowing the hGH to be mixed efficiently into the polymers at an ambient temperature and in the absence of solvents. The CO(2) was removed from the mixture by depressurisation through a nozzle, resulting in the production of microparticles containing the hGH, which were collected in a cyclone. The best microparticle formulations showed an initial in vitro burst of around 35% and a sustained release over 14 days. When tested in the rat model, which displays a faster clearance rate of hGH than other animal models, two formulations showed prolonged release over 2-3 days with sustained plasma levels at 1-5 ng/ml whereas the soluble hGH formulation was cleared within 24h. Two selected sustained release formulations were tested in cynomolgus monkeys and compared to a single injection of soluble hGH. The burst release from the sustained release formulations was similar in magnitude to a daily dose of hGH and serum hGH levels were maintained for a seven day period. It is probable from the data that the sustained release would have continued for up to 14 days if sampling had been continued. The IGF-1 results showed there was no significant difference between the levels obtained for once daily injection of soluble hGH and the two sustained release formulations.

Nasal administration of an angiotensin antagonist in the rat model: effect of bioadhesive formulations on the distribution of drugs to the systemic and central nervous systems.

The effect of bioadhesive formulations on the direct transport of an angiotensin antagonist drug ((14)C-GR138950) from the nasal cavity to the central nervous system was evaluated in a rat model. Three different bioadhesive polymer formulations (3% pectin LM-5, 1.0% pectin LM-12 and 0.5% chitosan G210) containing the drug were administered nasally to rats by inserting a dosing cannula 7mm into the nasal cavity after which the plasma and brain tissue levels were measured. It was found that the polymer formulations provided significantly higher plasma levels and significantly lower brain tissue levels of drug than a control, in the form of a simple drug solution. Changing the depth of insertion of the cannula from 7 to 15mm, in order to reach the olfactory region in the nasal cavity significantly decreased plasma levels and significantly increased brain tissue levels of drug for the two formulations studied (1.0% pectin LM-12 and a simple drug solution). There was no significant difference between the drug availability for the bioadhesive formulation and the control in the brain when the longer cannula was used for administration. It is suggested that the conventional rat model is not suitable for evaluation of the effects of bioadhesive formulations in nose-to-brain delivery.

Distribution and clearance of bioadhesive formulations from the olfactory region in man: effect of polymer type and nasal delivery device.

There is an increasing need to identify novel approaches by which to improve the efficiency of drug transport from the nasal cavity (olfactory region) to the CNS, especially for treatment of central nervous system disorders. It is suggested, that one approach is the combination of active targeting of a bioadhesive formulation, that will retain the drug at the absorption site, potentially in combination with, an absorption enhancer. Two low methylated pectins, LM-5 and LM-12 were selected for evaluation as drug delivery systems, due to their ability to gel in the nasal cavity and their bioadhesive characteristics, together with chitosan G210, which acts both as a bioadhesive material and as an efficient absorption enhancer. It was found that all of the bioadhesive formulations were able to reach the olfactory region in the nasal cavity of human volunteers when delivered using a simple nasal drop device. Furthermore, the formulations displayed a significantly increased residence time on the epithelial surface. This was in contrast to a non-bioadhesive control delivered with the same device. In contrast, a pectin formulation administered with a nasal spray system did not show an increase in residence time in the olfactory region. It was further shown that the reproducibility of olfactory delivery of a polymer formulation was significantly better intra-subject than inter-subject.

Evaluation of bioadhesive polymers as delivery systems for nose to brain delivery: in vitro characterisation studies.

There is an increasing need for nasal drug delivery systems that could improve the efficiency of the direct nose to brain pathway especially for drugs for treatment of central nervous system disorders. Novel approaches that are able to combine active targeting of a formulation to the olfactory region with controlled release bioadhesive characteristics, for maintaining the drug on the absorption site are suggested. If necessary an absorption enhancer could be incorporated. Low methylated pectins have been shown to gel and be retained in the nasal cavity after deposition. Chitosan is known to be bioadhesive and also to work as an absorption enhancer. Consequently, two types of pectins, LM-5 and LM-12, together with chitosan G210, were selected for characterisation in terms of molecular weight, gelling ability and viscosity. Furthermore, studies on the in vitro release of model drugs from candidate formulations and the transport of drugs across MDCK1 cell monolayers in the presence of pectin and chitosan were also performed. Bioadhesive formulations providing controlled release with increased or decreased epithelial transport were developed. Due to their promising characteristics 3% LM-5, 1% LM-12 pectin and 1% chitosan G210 formulations were selected for further biological evaluation in animal models.

Differences in the adsorption behaviour of poly(ethylene oxide) copolymers onto model polystyrene nanoparticles assessed by isothermal titration microcalorimetry correspond to the biological differences.

The adsorption behaviour of a tetrafunctional copolymer of poly (ethylene oxide)-poly (propylene oxide) ethylene diamine (commercially available as Poloxamine 908) and a diblock copolymer of poly (lactic acid)-poly (ethylene oxide) (PLA/PEG 2:5) onto a model colloidal drug carrier (156 nm sized polystyrene latex) is described. The adsorption isotherm, hydrodynamic thickness of the adsorbed layers and enthalpy of the adsorption were assessed. The close similarity in the conformation of the poly (ethylene oxide) (PEO) chains (molecular weight 5,000 Da) in the adsorbed layers of these two copolymers was demonstrated by combining the adsorption data with the adsorbed layer thickness data. In contrast, the results from isothermal titration microcalorimetry indicated a distinct difference in the interaction of the copolymers with the polystyrene colloid surface. Poloxamine 908 adsorption to polystyrene nanoparticles is dominated by an endothermic heat effect, whereas, PLA/PEG 2:5 adsorption is entirely an exothermic process. This difference in adsorption behaviour could provide an explanation for differences in the biodistribution of Poloxamine 908 and PLA/PEG 2:5 coated polystyrene nanoparticles observed in previous studies. A comparison with the interaction enthalpy for several other PEO-containing copolymers onto the same polystyrene colloid was made. The results demonstrate the importance of the nature of the anchoring moiety on the interaction of the adsorbing copolymer with the colloid surface. An endothermic contribution is found when an adsorbing molecule contains a poly (propylene oxide) (PPO) moiety (e.g. Poloxamine 908), whilst the adsorption is exothermic (i.e. enthalpy driven) for PEO copolymers with polylactide (PLA/PEG 2:5) or alkyl moieties.

Use of the ninhydrin assay to measure the release of chitosan from oral solid dosage forms.

This study evaluated and optimised the ninhydrin assay as a tool for measuring the in vitro release and dissolution of chitosan from solid dosage forms. The precision and accuracy of the assay for the type of chitosan used in the study were examined by measuring the inter- and intra-sample variation and found to be within acceptable limits. The assay was applied practically to construct a pH/solubility profile for chitosan and subsequently to measure the release and dissolution of chitosan from dosage forms in the presence and absence of a model drug, sodium salicylate. Assay performance was found to be satisfactory over a wide range of physiologically relevant pH values. It is concluded that the ninhydrin assay is an essential aid in the design and testing of solid dosage forms with different chitosan-drug release profiles.

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 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.

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.

Transport of nanoparticles across the rat nasal mucosa.

The transport of 125I-radiolabelled latex nanoparticles across the nasal mucosa of rats was studied using a range of particle sizes and surface coatings. Translocation of the particles into the blood stream was examined by means of monitoring the radiolabel associated with the particles. Particles were detected in the blood after 5 minutes. The number of particles in the blood peaked at 60 minutes, and then remained constant for a further 2 hours. The smallest particles (20 nm) showed greater uptake than the largest particles investigated (1000 nm). The total maximum uptake seen for the smallest particles was in the order of 3.25% of administered dose. 100 nm particles coated with chitosan showed an increase in both the extent and rate of uptake, with the concentration in the blood peaking at 15 minutes rather than at 60 minutes. It is suggested that transport of the particles across the nasal membrane is due mainly to a transcellular transport mechanisms by the nasal associated lymphoid tissue (NALT), especially the M-cell like cells. However, some paracellular transport cannot totally be ruled out for the smallest particles, especially if coated with chitosan.

The effect of surface coverage and conformation of poly(ethylene oxide) (PEO) chains of poloxamer 407 on the biological fate of model colloidal drug carriers.

Poloxamer 407 was adsorbed onto the surface of model colloidal drug carriers, polystyrene nanoparticles of 40, 70 and 137 nm in diameter, and the effect of the degree of surface coverage and the conformation of the poly(ethylene oxide) (PEO) chains on biological fate was studied. The relationship between the physicochemical and the biological properties of the nanoparticle systems was also investigated. The adsorbed layer of poloxamer 407 was characterised in terms of percentage surface coverage, thickness of the adsorbed layer and average surface area per PEO chain. Computer modelling of the adsorbed layer was performed (applying the self-consistent field technique), to obtain the structural information of the PEO chains in the layer. The in vitro interaction of the nanoparticles with different degrees of poloxamer 407 surface coverage with serum components and the in vivo biodistribution in the rat model were assessed. The results demonstrated that an increase in the surface coverage with poloxamer 407 resulted in an increased volume fraction of the PEO in the adsorbed layer, further extension of the PEO chains from the surface and closer packing of the chains at the surface. With regard to the interaction with the serum components, an increased surface coverage resulted in a reduction of the amount of serum proteins adsorbed, and, importantly, affected the type of proteins adsorbed. High molecular weight proteins were not adsorbed onto the nanoparticles with a surface coverage above approx. 25%. Following the intravenous administration to rats, even the nanoparticles with the lowest degree of surface coverage (approx. 5%) showed improved circulation profiles relative to the uncoated nanoparticles. The effect was more pronounced for the 40 nm nanoparticles. A further increase in the surface coverage to approx. 25% resulted in a significant increase in circulation time, as compared to uncoated and 5% coated systems, for all sizes of nanoparticles. Importantly, it was found that a long in vivo blood circulation time could be achieved for nanoparticles with a relatively low degree of surface coverage with PEO chains.

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.

Drug-polyionic block copolymer interactions for micelle formation: physicochemical characterisation.

While covalent attachment of small drug molecules to AB copolymers for the formation of polymeric micelles for drug delivery has been investigated, few studies have focused on non-covalent interactions. The aim of this study was therefore to explore the potential of non-covalent interactions between an AB copolymer, Poly(aspartic acid)-poly(ethylene glycol) (Pasp-PEG), with anionic pendant groups and diminazene aceturate, a small molecular weight cationic drug. Micelles were prepared by mixing solutions of Pasp-PEG and diminazene in 25 mM Tris-HCl buffer. At all Pasp-PEG concentrations studied, the micelles appeared to be water soluble with a unimodal size distribution and ranged in size from approximately 22 to 60 nm. The polyionic micelles also displayed similar and small absolute zeta potential values at various drug:monomer molar ratios which confirmed stabilisation by the PEG corona. The scattering intensity was maximal and remained unchanged, while particle size increased slightly at pH range from 3.4 to 7.2. At this pH range both the polymer and drug would be ionised and ionic interactions possible to drive micellar formation. An increase in size and scattering intensity with addition of NaCl to the micelles was attributed to dehydration of the PEG corona which may have led to aggregation of the micelles. The absence of micellar dissociation upon addition of salt was attributed to the dominance of hydrogen bonding between Pasp and diminazene aceturate, as assessed by isothermal titration microcalorimetry. Morphological evaluation of these constructs showed them to be discrete and fairly uniform in size and shape. This study was therefore successful in confirming the potential of non-covalent interactions using an AB copolymer to form polyionic micelles for drug delivery.

Development of systems for targeting the regional lymph nodes for diagnostic imaging: in vivo behaviour of colloidal PEG-coated magnetite nanospheres in the rat following interstitial administration.

PURPOSE: Nanoparticles can be utilised for targeting drugs to the regional lymph nodes or as diagnostic agents. The surface modification of magnetite nanospheres with poly(ethylene glycol) (PEG) has been assessed by in vitro characterisation and in vivo studies following subcutaneous administration to the rat. METHODS: Magnetite nanospheres were prepared with a grafted PEG layer using various PEG lengths from 350 to 1,000 Da. Thermogravimetric analysis was utilised to measure the adsorbed amount of PEG. Colloid stability was confirmed by measurement of the particle size and electrophoretic mobility. The kinetics of injection site drainage and lymph node retention were determined 2 hours after subcutaneous administration, for nanospheres coated with PEG lengths of 350, 550. 750, and 1,000 Da. For the 750 PEG coated nanospheres, the kinetics of distribution was determined over a 48-hour time course. RESULTS: The distribution of the nanospheres was modified and the lymph node localisation enhanced by altering the surface coverage of PEG on the magnetic surface. CONCLUSIONS: PEG-coated magnetite nanospheres with different surface characteristics can be utilised to target a diagnostic agent to regional lymph nodes.