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It is striking that all marketed SARS-CoV-2 vaccines are developed for intramuscular administration designed to produce humoral and cell mediated immune responses, preventing viremia and the COVID-19 syndrome. They have a high degree of efficacy in humans (70-95%) depending on the type of vaccine. However, little protection is provided against viral replication and shedding in the upper airways due to the lack of a local sIgA immune response, indicating a risk of transmission of virus from vaccinated individuals. A range of novel nasal COVID-19 vaccines are in development and preclinical results in non-human primates have shown a promising prevention of replication and shedding of virus due to the induction of mucosal immune response (sIgA) in upper and lower respiratory tracts as well as robust systemic and humoral immune responses. Whether these results will translate to humans remains to be clarified. An IM prime followed by an IN booster vaccination would likely result in a better well-rounded immune response, including prevention (or strong reduction) in viral replication in the upper and lower respiratory tracts.

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&reg, HS15). We report here the plasma pharmacokinetics of PTH 1-34 in healthy human volunteers receiving the liquid nasal spray formulation containing Solutol&reg, 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 &le, 1%. Subsequently, we investigated the pharmacokinetics of the liquid nasal spray formulation as well as a dry powder nasal formulation also containing Solutol&reg, 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&ndash, 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.

The ability to deliver therapeutically relevant amounts of drugs directly from the nasal cavity to the central nervous system to treat neurological diseases is dependent on the availability of efficient drug delivery systems. Increased delivery and/or therapeutic effect has been shown for drugs encapsulated in nanoparticles; however, the factors governing the transport of the drugs and/or the nanoparticles from the nasal cavity to the brain are not clear. The present study evaluates the potential transport of nanoparticles across the olfactory epithelium in relation to nanoparticle characteristics. Model systems, 20, 100, and 200 nm fluorescent carboxylated polystyrene (PS) nanoparticles that were nonmodified or surface modified with polysorbate 80 (P80-PS) or chitosan (C-PS), were assessed for transport across excised porcine olfactory epithelium mounted in a vertical Franz diffusion cell. Assessment of the nanoparticle content in the donor chamber of the diffusion cell, accompanied by fluorescence microscopy of dismounted tissues, revealed a loss of nanoparticle content from the donor suspension and their association with the excised tissue, depending on the surface properties and particle size. Chitosan surface modification of PS nanoparticles resulted in the highest tissue association among the tested systems, with the associated nanoparticles primarily located in the mucus, whereas the polysorbate 80-modified nanoparticles showed some penetration into the epithelial cell layer. Assessment of the bioelectrical properties, metabolic activity, and histology of the excised olfactory epithelium showed that C-PS nanoparticles applied in pH 6.0 buffer produced a damaging effect on the epithelial cell layer in a size-dependent manner, with fine 20 nm sized nanoparticles causing substantial tissue damage relative to that with the 100 and 200 nm counterparts. Although histology showed that the olfactory tissue was affected by the application of citrate buffer that was augmented by addition of chitosan in solution, this was not reflected in the bioelectrical parameters and the metabolic activity of the tissue. Regarding transport across the excised olfactory tissue, none of the nanoparticle systems tested, irrespective of particle size or surface modification, was transported across the epithelium to appear in measurable amounts in the receiver chamber.

Osteoporosis treatment with PTH 1-34 injections significantly reduces the incidence of bone fracture. Potential further reductions in fracture rate should be observed through nasal spray delivery to address the poor compliance associated with patient dislike of repeated PTH 1-34 subcutaneous injections. In vitro human osteoblast-like Saos-2 cell intracellular cAMP levels were used to define PTH 1-34 nasal spray formulation bioactivity. The chemically synthesised PTH 1-34 had an EC50 of 0.76nM. Absorption enhancers polyethylene glycol (15)-hydroxystearate (Solutol® HS15), poloxamer 407, chitosan or sodium hyaluronate did not diminish the bioactivity of PTH 1-34 within an in vitro cell culture model (p >0.05). We also demonstrated the effectiveness of the transmucosal absorption enhancer Solutol® HS15 in a nasal spray formulation using a preclinical pharmacokinetic model. In Sprague-Dawley rats without the absorption enhancer the uptake of PTH 1-34 into the blood via intranasal delivery produced a Cmax of 2.1±0.5ng/ml compared to 13.7±1.6ng/ml with Solutol® HS15 enhancer (p=0.016) and a Cmax14.8±8ng/ml in subcutaneous injections. Together these data illustrate that the nasal spray formulation bioactivity in vitro is not affected by the nasal spray absorption enhancers investigated, and the Solutol® HS15 nasal spray formulation had an equivalent pharmacokinetic profile to subcutaneous injection in the rat model. The Solutol® HS15 formulation therefore demonstrated potential as a PTH 1-34 nasal spray formulation for the treatment of osteoporosis.

Purpose CriticalSorb™, with the principal component Solutol® HS15, is a novel mucosal drug delivery system demonstrated to improve the bioavailability of selected biotherapeutics. The intention of this study is to elucidate mechanism(s) responsible for the enhancement of trans-mucosal absorption of biological drugs by Solutol® HS15. Methods Micelle size and CMC of Solutol® HS15 were determined in biologically relevant media. Polarised airway Calu-3 cell layers were used to measure the permeability of a panel of biological drugs, and to assess changes in TEER, tight junction and F-actin morphology. The rate of cell endocytosis was measured in vitro in the presence of Solutol® HS15 using a membrane probe, FM 2–10. Results This work initially confirms surfactant-like behaviour of Solutol® HS15 in aqueous media, while subsequent experiments demonstrate that the effect of Solutol® HS15 on epithelial tight junctions is different from a ‘classical’ tight junction opening agent and illustrate the effect of Solutol® HS15 on the cell membrane (endocytosis rate) and F-actin cytoskeleton. Conclusion Solutol® HS15 is the principle component of CriticalSorb™ that has shown an enhancement in permeability of medium sized biological drugs across epithelia. This study suggests that its mechanism of action arises primarily from effects on the cell membrane and consequent impacts on the cell cytoskeleton in terms of actin organisation and tight junction opening.

Objective The aim of this study was to determine the antimicrobial activity of different chitosans (CS) against typical colonizing pathogens of the urinary tract and to assess their efficacy against bacterial adhesion and the subsequent biofilm formation on urinary catheters. Methods The antimicrobial activity of high and low molecular weight CS (50 and 150 kDa) at pH 5.0 and 6.0 was tested against Klebsiella pneumoniae and Escherichia coli clinical isolates by time–kill studies. The anti-adhesion assays on Foley urinary catheters were performed in Artificial Urine Medium (AUM) with the addition of each CS (AUM-CS) at the same pH values. Finally, the efficacy over time of chitosan treatments on bacterial adhesion on urinary catheters was determined. Results A viability reduction of K. pneumoniae and E. coli isolates, regardless of pH value, was evidenced in time–kill studies, in particular in the presence of CS 50 kDa. As regards the anti-adhesion efficacy on urinary catheters, high and low molecular weight CS evidenced a higher efficacy to reduce bacterial adhesion at pH 5.0. A low number of viable K. pneumoniae and E. coli cells were recovered from catheters after CS treatments, highlighting a promising efficacy over time. Conclusion Our data show the potential of chitosans to reduce or prevent not only the adhesion of well-known human uropathogens on urinary catheters but also the re-growth ability of the uropathogens.

The production of microparticles using a supercritical carbon dioxide based PGSS technique (CriticalMix™) has been exploited to develop blended systems targeted at pulmonary delivery. Hence, PEG based polymers of different molecular weights (1000-6000 Da) were blended in situ with fatty acids (stearic, palmitic or myristic acid) or with commercially available PEG-stearates. The effect of the different thermodynamic properties of the polymers was evaluated by characterising the microparticles produced in terms of their melting temperature by conventional DSC and in the presence of high pressure CO(2) using a high pressure variable volume view cell. The microparticles produced were also assessed by SEM and particle size distribution. It is well known that as the molecular weight of the PEG chains increases, so does the viscosity of the melt and this leads to an increase in the particle size. In the paper we show that blending with myristic acid provides optimal control of particle size when the blend is sprayed from scCO(2) leading to high yields in the optimal aerodynamic size range of 2-5 μm for the deep lung delivery. The highest yield and smallest particles (~5 μm) were produced with a blend of PEG 3000 and myristic acid (1:1) whereas the batches containing palmitic acid and stearic acid showed lower yields and larger particle sizes.

To exert its activity a drug must reach its pharmacological site(s) of action(s) within the body. One of the current approaches to achieve site specific delivery utilises the use of a carrier. This review focuses on the physicochemical and biological properties of polymeric particulate carriers in the nanometre size range surface modified by poly(ethylene oxide) (PEO). Such systems are able to bypass the normal physiological defence processes occurring after the intravenous injection of particulates and, depending on the particle size and PEO layer properties, remain for a prolonged period of time in the systemic circulation, or have a degree of selectivity for sites of deposition within the body.

The thermodynamic behaviour of selected polymeric components for preparation of controlled release microparticles using supercritical carbon dioxide (scCO 2 ) processing was investigated. The polymeric materials selected were egg lecithin (a model for the lung surfactant phospholipid), poly(ethyleneglycol) (PEG) of different molecular weights, fatty acids (C18, C16, and C14), and physical blends of PEGs and fatty acids. In addition a range of PEG-stearates was also assessed. Analysis of thermodynamic behaviour was performed by differential scanning calorimetry (DSC) and by assessment of their interaction with scCO 2 in a high-pressure variable volume view cell. The key criterion was to demonstrate a strong interaction with scCO 2 and to show liquefaction of the polymeric material at acceptable processing temperatures and pressures. Positive results should then indicate the suitability of these materials for processing by the Particle from Gas Saturated Solutions (PGSS) technique using scCO 2 to create microparticles for pulmonary administration. It was found that the materials tested interacted with scCO 2 and showed a sufficient lowering of their melting temperature ( T m ) to make them suitable for use in the PGSS microparticle production rig. Fatty acids of low T m were shown to act as a plasticising agent and to lower the T m of PEG further during interaction with scCO 2.

Systemic delivery of proteins via the nasal route has to date been limited by their poor absorption across the nasal mucosa, and the less than optimal tolerability of known permeation enhancers. We have recently developed a highly effective nasal delivery system (CriticalSorb™) based on Solutol HS15. Extensive toxicology studies have shown CriticalSorb™ to be very well tolerated, non-toxic and non-irritant. Cell culture and ex vivo-isolated tissue studies have shown it to promote transport of molecules mainly via transcellular but also to some extent, via paracellular routes. Pharmacokinetic/pharmacodynamic studies in rats, rabbits, non-human primates and recently in man have demonstrated significantly enhanced systemic delivery of nasally administered proteins including insulin (~6 kDa) and human growth hormone (~22 kDa), and pharmacodynamics similar to those after subcutaneous injection. CriticalSorb™ therefore opens up the possibility of developing nasal spray formulations for macromolecules such as proteins.

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 [14C]-mannitol, [14C]-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 [14C]-mannitol, FD-4 [14C]-antipyrine across ileal and colonic mucosae, accompanied by a decrease in TEER. In Caco-2 monolayers, it also increased the Papp of [14C]-mannitol FD-4 and [14C]-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. R10809 SFI Cluster IDDN SB. 13/6/2013

This review sets out to describe and discuss the synthetic approaches and the fields of application of PEGylated chitosan copolymers especially for medical use. The PEGylation of chitosan and chitosan derivatives is able to add new physicochemical properties to the cationic polysaccharide polymers, thereby overcoming some limitations, especially regarding their solubility and their use in drug and gene delivery (DNA and siRNA). All methods of derivatization have been considered and described together with the different methods of characterization of the copolymers. The capacities of PEGylated chitosan to reduce chitosan toxicity, to enhance membrane permeation and to form thermosensitive hydrogels have also been discussed.

The instability of human growth hormone (hGH) to temperature and interfaces makes its formulation into injectable, sustained-release drug delivery systems challenging. A novel method of encapsulating hGH in polymeric microparticles has been developed using supercritical CO(2) (scCO(2)) technology, but there is limited understanding of the stability of hGH within this system. The aim of this study was to evaluate the stability of hGH in scCO(2) processing. The integrity of the protein was assessed following exposure to scCO(2) using a range of different analytical techniques. Mass spectrometry showed that no peptide cleavage occurred as a result of processing or exposure to scCO(2). Size-exclusion chromatography detected formation of aggregates at high temperatures, but not as a result of the encapsulation process. Reverse-phase chromatography demonstrated that the production of deamidation products occurred as a function of temperature, but only at temperatures higher than those used for the encapsulation process. Circular dichroism and infrared spectroscopy demonstrated that the use of scCO(2) was not detrimental to the secondary molecular structure of hGH. Together, these results show that the structural integrity of hGH is unaffected by scCO(2) processing and that hGH can be successfully encapsulated in polymer microparticles using this technique.

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.

The aim of the present work is to investigate if conditions can be devised where PEGylation of chitosan would reduce its toxicity toward the nasal mucosa while maintaining its ability to open the cellular tight junctions and, consequently, produce an enhancement of macromolecular permeability. A series of mPEG-g-chitosan copolymers with varying levels of mPEG substitution, mPEG molecular weight, and chitosan molecular weight were synthesized by grafting carboxylic acid-terminated mPEGs (Mw 1.9 and 5.0 × 10(3) g mol(-1)) to chitosans (Mw 28.9 and 82.0 × 10(3) g mol(-1)) using a NHS/EDC coupling system. The synthesized mPEG-g-chitosans were fully characterized using a number of techniques, including FT-IR, (1)H NMR, and SEC-MALLS and their physicochemical properties were analyzed by TGA and DSC. Thereafter, the conjugates were tested for their cytotoxicity and tight junction modulating property in a relevant cell model, a mucus producing Calu-3 monolayer. mPEG-g-chitosan conjugates exhibited reduced toxicity toward cells, as compared to unmodified chitosan counterparts. Furthermore, the conjugates demonstrated a dramatic effect on cell monolayer transepithelial electrical resistance (TEER) and enhancement of permeability of model macromolecules. TEER and permeability-enhancing effects, as measurable indicators of tight junction modulation, were found to be pH-dependent and were notably more pronounced than those exhibited by unmodified chitosans. This work therefore demonstrates that conditions can be contrived where PEGylation improves the toxicity profile of chitosan, while preserving its effect on epithelial tight junctions in the nose.

Proteins constitute an increasing proportion of the drugs in development. The barriers to their entry into the blood stream and rapid clearance means that they often have to be injected several times a day, affecting patient compliance. This paper reviews the major technologies enabling the development of injectable sustained-release products and formulation strategies to maintain protein integrity and modify release rates. Whilst many injectable sustained-release products are on the market, these are all delivering small molecular weight drugs and peptides. This is due to the manufacturing processes that denature and degrade the proteins upon encapsulation and release into the body. Formulation strategies are discussed and a number of new technologies reviewed that are able to overcome the issues with conventional manufacturing processes. The reliance of many processes on organic solvents has prevented their application to the development of injectable sustained release protein products. The development of entirely solvent free and aqueous methods of manufacture of these products has meant that numerous sustained-release protein products are close to reaching the market.

Small molecular weight drugs, peptides, and nanoparticles have previously been shown to localize in the central nervous system after intraneural administration. A basic understanding of direct nose-to-brain drug delivery, particularly for nanoparticles with different physicochemical characteristics, remains unclear. In this study, fluorescence microscopy and stereology were used to track intranasally administered chitosan-coated polystyrene (C-PS) or polysorbate-coated polystyrene (P80-PS) nanoparticles (100 nm or 200 nm in diameter) in olfactory and respiratory nasal epithelia and olfactory bulbs in mice. Chitosan coating caused particles to adhere to the extracellular mucus which could provide useful modality for paracellular drug transport. Nanoparticle transport was exclusively transcellular. None of the nanoparticle formulations showed preference for uptake into olfactory axons over other nasal epithelial cells. Both 100 nm PS and 100 nm P80-PS were observed in olfactory epithelial cells but were absent from the olfactory bulbs; therefore, it is speculated that an optimal nanoparticle diameter for axonal transport is

Biodegradable polymeric nanoparticles of different architectures based on polyethylene glycol-co-poly(ε-caprolactone) block copolymers have been loaded with noscapine (NOS) to study their effect on its anticancer activity. It was intended to use solubility of NOS in an acidic environment and ability of the nanoparticles to passively target drugs into cancer tissue to modify the NOS pharmacokinetic properties and reduce the requirement for frequent injections.Linear and star-shaped copolymers were synthetized and used to formulate NOS loaded nanoparticles. Cytotoxicity was performed using a sulforhodamine B method on MCF-7 cells, while biocompatibility was determined on rats followed by hematological and histopathological investigations.Formulae with the smallest particle sizes and adequate entrapment efficiency revealed that NOS loaded nanoparticles showed higher extent of release at pH 4.5. Colloidal stability suggested that nanoparticles would be stable in blood when injected into the systemic circulation. Loaded nanoparticles had IC50 values lower than free drug. Hematological and histopathological studies showed no difference between treated and control groups. Pharmacokinetic analysis revealed that formulation P1 had a prolonged half-life and better bioavailability compared to drug solution.Formulation of NOS into biodegradable polymeric nanoparticles has increased its efficacy and residence on cancer cells while passively avoiding normal body tissues. Graphical Abstract ᅟ.

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.

Context: The development of an improved, efficacious human GH (hGH) product administered by a noninjectable route of delivery such as the nasal route is highly desirable. We have developed a novel nasal hGH product (CP024) that showed excellent nasal absorption in animal models; however, the translation of these results into the clinical setting is essential because past attempts to develop such formulations by other groups have been unable to induce IGF-1 in man. Objective: The objective of the study was to assess the pharmacokinetics, pharmacodynamics, and tolerability of CP024 compared with a sc hGH injection. Design: This was a single-center, nonrandomized placebo-controlled, open-label, five-way crossover study in eight healthy volunteers. Setting: The study was carried out at a contract research organization, Quotient Bioresearch. Volunteers: Eight healthy male volunteers, given an iv infusion of octreotide to suppress the endogenous GH secretion during the study period, participated in the study. No volunteers were withdrawn due to side effects. Main Outcome Measures: Measurement of hGH and IGF-1 levels and tolerability of the drug product was performed. Results: No serious adverse events were reported and no subjects withdrawn from study due to the treatment. After the nasal administration of CP024, 3-fold higher hGH blood levels were obtained as compared with hGH nasal control. The relative bioavailability was about 3%. CP024 (given twice daily) induced a significant increase in IGF-1 levels up to 19 hours after administration, with no significant difference to those obtained after the sc injection of hGH. Conclusions: The study indicates that CP024 is a promising candidate for an efficacious nasal product for the treatment of GH deficiency due to induction of IGF-1 similar to that after a sc injection, despite the lower plasma hGH concentration obtained. A dose-response study is needed to evaluate the optimal nasal dose.

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.

We previously reported that intranasal immunization with a non-toxic mutant cross-reacting material (CRM)197 of diphtheria toxin, formulated with chitosan, generated protective neutralizing antibodies in mice and guinea pigs. Furthermore, we demonstrated that intranasal delivery of a powder formulation of the CRM197-based vaccine was well tolerated and significantly boosted antibody responses in adult volunteers. Here we report that intranasal booster immunization with CRM197 alone or with chitosan induced systemic T cell responses. We addressed for the first time the induction of T cell subtypes following intranasal vaccination in humans. Intranasal vaccination with CRM197, like parenteral immunization with a conventional diphtheria toxoid vaccine, enhanced antigen-specific IFN-gamma production. However, formulation of the nasal diphtheria vaccine with chitosan significantly augmented Th2-type responses, which correlated with protective levels of toxin-neutralizing antibodies in intranasally boosted individuals. The results suggest that vaccines capable of inducing strong Th2-type responses, such as CRM197 formulated with chitosan, have potential for the development of a protective mucosal vaccine against diphtheria in humans. Furthermore, our findings demonstrate that mucosal subunit vaccines with appropriate delivery systems have considerable potential for booster immunization of adults.

An understanding of the deposition and clearance of nasal drug formulations from the human nasal cavity is important in order to optimally exploit the nasal route for delivery of drugs intended for local or systemic applications. Three radionuclide imaging methods may be used to assess nasal drug delivery: (i) gamma scintigraphy; (ii) single photon emission computed tomography (SPECT); and (iii) positron emission tomography (PET). Gamma scintigraphy is used to image the nasal cavity in two dimensions, while SPECT and PET are 3-dimensional imaging methods. By quantifying the deposition and clearance of drug formulations, radionuclide imaging studies allow a comparison to be made between the performance of new nasal drug delivery systems and those already marketed. Imaging data have been used to demonstrate the bioadhesive properties of novel formulations in the nasal cavity and to show that little or no drug usually penetrates directly to the lungs when delivered intranasally. These data may have an important regulatory role by clarifying whether small in vitro differences between products are likely to be clinically significant. Gamma scintigraphy is likely to remain the most widely used radionuclide imaging method, at least for the time being, although a larger future role is likely for SPECT and PET.

The blood–brain barrier that segregates the brain interstitial fluid from the circulating blood provides an efficient barrier for the diffusion of most, especially polar, drugs from the blood to receptors in the central nervous system (CNS). Hence limitations are evident in the treatment of CNS diseases, such as Parkinson's and Alzheimer's diseases, especially exploiting neuropeptides and similar polar and large molecular weight drugs. In recent years interest has been expressed in the use of the nasal route for delivery of drugs to the brain, exploiting the olfactory pathway. A wealth of studies has reported proof of nose-to-brain delivery of a range of different drugs in animal models, such as the rat. Studies in man have mostly compared the pharmacological effects (e.g. brain functions) of nasally applied drugs with parenterally applied drugs and have shown a distinct indication of direct nose-to-brain transport. Recent studies in volunteers involving cerebrospinal fluid sampling, blood sampling and pharmacokinetic analysis after nasal, and in some instances parenteral administration of different drugs, have in my opinion confirmed the likely existence of a direct pathway from nose to brain.

Attempts have been made to correlate estimates of molecular weight for a group of cationic polysaccharides known as chitosans between the highly popular technique of size-exclusion chromatography coupled to multi-angle laser light scattering, "SEC-MALLS", and the less convenient but more established technique of sedimentation equilibrium in the analytical ultracentrifuge. Four pharmaceutical grade chitosans of various molecular weights and degrees of acetylation (4-30%) were chosen. Better correlation than previous was achieved, although some batch variability was observed. Despite the broad spectrum in degree of acetylation, a log s degrees(20,w) versus log Mw scaling plot appeared to fit a straight line with power-law exponent b=0.25 +/- 0.04, i.e. between the limits of rod (0.15) and coil (0.4-0.5), although this may be the average of a lower b value at low Mw and higher b at high Mw. With regard to viscosity, a logeta versus logMw scaling plot appeared to also fit a straight line with power-law exponent a=0.96 +/- 0.10, again between the coil (0.5-0.7) and rod (1.8) limits.

This paper discusses the problems associated with nasal drug delivery and how it is possible, sometimes by means of quite simple concepts, to improve transport across the nasal membrane. In this way it is feasible to deliver efficiently challenging drugs such as small polar molecules, peptides and proteins and even the large proteins and polysaccharides used in vaccines or DNA plasmids exploited for DNA vaccines. The transport of drugs from the nasal cavity directly to the brain is also described and examples of studies in man, where this has been shown to be feasible, are discussed. Recent results from Phase I/II studies in man with a novel nasal chitosan vaccine delivery system are also described. Finally, the author's thoughts about the future for nasal drug delivery are also depicted.

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.

This paper describes the basic concepts for the transmucosal delivery of drugs, and in particular the use of the nasal route for delivery of challenging drugs such as polar low-molecular-weight drugs and peptides and proteins. Strategies for the exploitation of absorption enhancers for the improvement of nasal delivery are discussed, including consideration of mechanisms of action and the correlation between toxic effect and absorption enhancement. Selected enhancer systems, such as cyclodextrins, phospholipids, bioadhesive powder systems and chitosan, are discussed in detail. Examples of the use of these enhancers in preclinical and clinical studies are given. Methods for assessing irritancy and damage to the nasal membrane from the use of absorption enhancers are also described. Finally, the mucosal use of absorption enhancers (chitosan) for the improved nasal delivery of vaccines is reported with reference to recent phase I/II clinical studies.

Self-consistent field (SCF) modelling studies can be used to predict the properties of poly(lactic acid):poly(ethyleneoxide) (PLA:PEG) nanoparticles using the theory developed by Scheutjens and Fleer. Good agreement in the results between experimental and modelled data has been observed previously for a series of PLA:PEG nanoparticles with a constant 5 kDa PEG block and a variable PLA block. In the present work, a comparison is performed between PLA:PEG nanoparticles having 5 and 10 kDa PEG blocks with varying PLA blocks (2-4 kDa). The calculated volume fraction profiles show that the PLA core region of the PLA:PEG 5 kDa series is larger than for the PLA:PEG 10 kDa series, whereas the PEG corona region of the PLA:PEG 10 kDa series is larger than that of the PLA:PEG 5 kDa series due to the presence of the longer PEG chain. The results indicate that the aggregation numbers of the 5 kDa series are larger than for the 10 kDa series due to steric effects associated with the PEG layer, even though the particle radii for the 10 kDa series are larger than for the 5 kDa series. The surface area available on the PLA core for each PEG chain is greater for the 10 kDa PEG series indicating that the 5 kDa PEG series has a denser PEG layer. (C) 2002 Elsevier Science B.V. All rights reserved.

The nasal cavity has for more than three decades been widely explored as a potential alternative route to oral or parenteral administration for systemically active drugs. The nasal route has shown remarkable advantages that include a rapid and high systemic availability, avoidance of first pass metabolism by the liver, and the possibility of targeting drugs directly from the nasal cavity to the brain [1, 2]. Considerable knowhow and data have been accumulated over the years from investigational work carried out by various excellent research groups in academia and industry. New nasal delivery carriers and emerging technologies have been used for product design and efficient clinical translation to nasal medicines. The nasal route for systemic administration is attractive in many therapeutic areas where a rapid onset of action is required, e.g., pain, erectile dysfunction, frigidity, migraine, seizures, insomnia, panic attacks, Parkinson rigidity, hot flushes, emesis, Alzheimer or MS attacks, and cardiovascular events. An increasing number of small molecules are being marketed as nasal products such as Zomig® (zolmitriptan), Imitrex® (sumatriptan), and Stadol NS (butorphanol tartrate) for migraine treatment, Aerodiol® (estradiol hemihydrate) for menopausal syndrome treatment, PecFent® and Instanyl® (fentanyl) for severe pain treatment, and Nicorette® (nicotine) for smoking cessation [3]. With this growing number of applications, the US market of intranasal drug products is expected to reach US$5.2 billion by 2017 [4]. It is noteworthy that most of the marketed products are based on molecules sufficiently lipophilic to enable therapeutic levels of the drug to reach the systemic circulation, thus requiring no nasal absorption enhancers. Despite the obvious advantages of intranasal drug delivery, the nasal cavity presents a number of limitations for drug absorption, including low intrinsic permeability for some drugs, such as hydrophilic molecules, peptides, proteins, and nucleotides, rapid mucociliary clearance, and enzymatic degradation [1, 2]. In order to achieve efficient and safe intranasal drug products, a number of strategies for overcoming nasal delivery barriers can be applied. In the design of a nasal product, three main cooperative entities should be taken in consideration: the drug, the delivery carrier, and the administration device. The factors to be considered in the design and development of an efficient nasal product, related to these three components, are schematically presented as a three-lobe fleur-de-lys in Fig. 1. Several important morphological and physiological constraints on nasal drug delivery including limited volume of administration requiring high drug potency or mucosal enzymatic drug degradation should be kept in mind when formulating new nasal products. Also, the key properties required from drug candidates for development of successful intranasal products would be potency, lipophilicity, and water solubility. The nasal delivery of hydrophilic or high molecular weight drugs would be in need of a nasal absorption promoter in order for the drug to be transported across the nasal membrane in sufficient quantity for therapeutic use. Innovative strategy approaches to design efficient nasal delivery systems for specific drugs are currently in various stages of research and development. These include new nasal enhanced delivery technologies, design of carriers that impede drug degradation by mucosal enzymes, modulation E. Touitou (*) Institute for Drug Research, School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem 91120, Israel e-mail: touitou@cc.huji.ac.il

The use of the nasal route for the delivery of challenging drugs has created much interest in recent years in the pharmaceutical industry. Consequently, drug delivery companies are actively pursuing the development of novel nasal drug-delivery systems and the exploitation of these for administration of conventional generic drugs and peptides, both in-house and with partners in the pharmaceutical industry. This review sets out to discuss some new developments and strategies in nasal drug delivery. An exiting discovery that drugs can be transported directly from nose to brain via the olfactory pathway is discussed and examples of proof-of-concept in man are given.

A series of poly(lactic acid)-poly(ethylene oxide) (PLA-PEG) AB block copolymers have been synthesized and used to produce aqueous dispersions of micellar-like nanoparticles. The hydrodynamic radius of the PLA core plus the stabilizing 5 kDa PEG layer, as determined by photon correlation spectroscopy, increased with the molecular weight of the PLA block. PLA-PEG particles have possible application as drug carriers for targeted drug delivery. In addition to particle size, the layer thickness and mobility of the hydrated PEG chains will be important in determining the particle's ability to avoid uptake by the defense system of the body, the phagocytic cells of the reticuloendothelial system. Viscoelastic measurements were used to investigate interparticle interactions in concentrated dispersions of the PLA-PEG nanoparticles. This approach has not previously been used to study how the interactions in dispersions of micellar-like assemblies depend on the copolymer geometry. When the volume fraction of the dispersions, score, was increased, the storage modulus, G', became greater than the loss modulus, G", indicating steric interaction between the PEG chains. At higher Φ c o r e values, compression of the grafted polymer chains was observed. The critical volume fraction at which overlap of the PEG chains commences, Φ c o r e crit (i.e., G' = G"), was used in conjunction with the overall hydrodynamic radius to determine the grafted PEG chain layer thickness. Micellar-like nanoparticles assembled from a PLA-PEG copolymer with a 5 kDa PEG block and a relatively low molecular weight 3 kDa PLA chain had an appreciable steric layer thickness of 6.3 nm. Hence, interparticle interactions in the concentrated dispersion were "soft" in nature as a result of the long-range interactions, as shown by the weak scaling dependence of the storage modulus on the volume fraction. The layer became more extended as the molecular weight of the PLA block was increased from 3 to 6 kDa, and as a consequence the PEG chains became less compressible. By using this method to characterize the PEG layer, it should be possible to rationalize the in vivo performance of the PLA-PEG nanoparticles and therefore aid the design of particulate drug carriers.

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.

NMR studies on a series of poly(lactic acid)−poly(ethylene oxide) (PLA−PEG) diblock copolymers have been carried out in d6-acetone and in D2O. Nanoparticles of the PLA−PEG copolymers were obtained using a modified interfacial polymer deposition−solvent evaporation technique (Fessi, H.; Devissaguet, J. P.; Puisieux, F.; Thies, C. French Patent 2 608 988, 1986). In D2O, the PLA block forms a central hydrophobic core, while the PEG block forms an hydrophilic corona layer. In D2O, the hydrophobic core of the nanoparticle is generally not seen, while the PEG corona is observed. Only the PLA methyl protons at the interface between the two regions are observed, and these are seen as a double doublet structure. For nanoparticles with a low molecular weight PLA block length, an additional methyl multiplet signal is seen suggesting that PLA methyl groups are in more than one chemical environment. This is not seen for nanoparticles with a high molecular weight PLA block length indicating more uniform structure in th...

Purpose. To investigate whether the widely accepted advantages associated with the use of chitosan as a nasal drug delivery system, might be further improved by application of chitosan formulated as nanoparticles.

The purpose of this study was to develop and evaluate a delivery system comprising a thermosensitive gel for the sustained release of steroidal hormones in fish, over an extended period of time after a single intramuscular (i.m.) injection and for the improved reproductive performance in fish. Controlled delivery systems based on thermosensitive gels are easy to prepare, low cost and high versatility dosage forms, which have been shown to be effective in several animal species for sustained release of hormones. In this work, a thermosensitive gel system based on poloxamer 407 in water:ethanol medium, able to work as a prolonged release carrier for 17β-estradiol (E2), has been developed. Such a system was able to solubilize the lipophilic E2 and to gel at the required water temperature for fish rearing (20 °C). Moreover, the system exhibited the best injection condition at temperatures below 15 °C when the system behaved as a low viscosity Newtonian liquid. The thermosensitive gel system was tested in vivo in the fish model, Carassius auratus, and the results compared with a single i.m. injection of E2 dissolved in corn-oil and other relevant control systems not containing E2. The results were particularly interesting, since fish injected with the E2 thermosensitive gel formulation, showed significantly higher levels of the circulating hormone than corn oil-E2 treated animals at 72 and 96 h after injection. In addition, the thermogel system was able to sustain the plasma level of E2 for about 11 days. The increased plasma levels of E2 were also accompanied by maintained higher values of plasma vitellogenin (VTG), thus suggesting that the thermosensitive polymer based delivery system could prevent rapid hepatic clearance of E2, resulting in prolonged stimulation of estrogen receptor-mediated pathways in goldfish.

There is an obvious need for efficient and safe nasal absorption enhancers for the development of therapeutically efficacious nasal products for small hydrophilic drugs, peptides, proteins, nucleic acids and polysaccharides, which do not easily cross mucosal membranes, including the nasal. Recent years have seen the development of a range of nasal absorption enhancer systems such as CriticalSorb (based on Solutol HS15) (Critical Pharmaceuticals Ltd), Chisys based on chitosan (Archimedes Pharma Ltd) and Intravail based on alkylsaccharides (Aegis Therapeutics Inc.), that is presently being tested in clinical trials for a range of drugs. So far, none of these absorption enhancers have been used in a marketed nasal product. The present review discusses the evaluation of chitosan and chitosan derivatives as nasal absorption enhancers, for a range of drugs and in a range of formulations such as solutions, gels and nanoparticles and finds that chitosan and its derivatives are able to efficiently improve the nasal bioavailability. The revirtew also questions whether chitosan nanoparticles for systemic drug delivery provide any real improvement over simpler chitosan formulations. Furthermore, the review also evaluates the use of chitosan formulations for the improvement of transport of drugs directly from the nasal cavity to the brain, based on its mucoadhesive characteristics and its ability to open tight junctions in the olfactory and respiratory epithelia. It is found that the use of chitosan nanoparticles greatly increases the transport of drugs from nose to brain over and above the effect of simpler chitosan formulations.

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.

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.

The effect of the molecular weight, reaction temperature and polymer concentration were investigated. The PEG coverage could be controlled by the selection of the polymer; the molecular coverage increased with decrease in polymer molecular weight. A method for synthesising PEG-coated magnetite particles for use in diagnostic imaging is described. Magnetite particles in aqueous conditions were reacted with Si(OR) 2 R′R′′ (trimethoxysilane-PEG, R=Me; R′=Ome; R′′=PEG). These PEG-coated magnetite particles showed greatly enhanced colloid stability properties with respect to the uncoated particles.

Poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles of 150-nm mean size were produced by an interfacial deposition method. The polar model drug Rose Bengal was successfully loaded into the nanoparticles during production and the surface of these particles was subsequently modified with poloxamer 407 and poloxamine 908 in order to create a steric stabilising layer of PEG on the surface. Drug loading was low (

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.

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 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.