Ben Forbes

Hyaluronan: Pharmaceutical Characterization and Drug Delivery

Hyaluronic acid (HA), is a polyanionic polysaccharide that consists of N-acetyl-D-glucosamine and β-glucoronic acid. It is most frequently referred to as hyaluronan because it exists in vivo as a polyanion and not in the protonated acid form. HA is distributed widely in vertebrates and presents as a component of the cell coat of many strains of bacteria. Initially the main functions of HA were believed to be mechanical as it has a protective, structure stabilizing and shock-absorbing role in the body. However, more recently the role of HA in the mediation of physiological functions via interaction with binding proteins and cell surface receptors including morphogenesis, regeneration, wound healing, and tumor invasion, as well as in the dynamic regulation of such interactions on cell signaling and behavior has been documented. The unique viscoelastic nature of hyaluronan along with its biocompatibility and nonimmunogenicity has led to its use in a number of cosmetic, medical, and pharmaceutical applications. More recently, HA has been investigated as a drug delivery agent for ophthalmic, nasal, pulmonary, parenteral, and dermal routes. The purpose of our review is to describe the physical, chemical, and biological properties of native HA together with how it can be produced and assayed along with a detailed analysis of its medical and pharmaceutical applications.

Human airway epithelial cell lines for in vitro drug transport and metabolism studies

The pharmaceutical industry relies on appropriate in vitro models for the evaluation of drug absorption and metabolism. Despite increasing interest in drug delivery via the lung, there is currently no widely accepted cell culture model of the airway epithelium. This review considers the airway epithelium, the culture of airway epithelial cells and the need for cell lines which can model the airway epithelium. Three of the most promising human bronchial cell lines, 16HBE14o-, Calu-3 and BEAS-2B, are reviewed, with emphasis on their recent application for the study of drug transport, drug metabolism and gene delivery. Current limitations and future directions for the development of these cell lines as models of the airway epithelium are discussed.

The human bronchial epithelial cell line 16HBE14o- as a model system of the airways for studying drug transport.

The 16HBE14o- cell line, which forms polarised cell layers in vitro, provides a promising opportunity to develop a convenient epithelial cell culture model in which respiratory drug transport can be evaluated in vitro. This study investigated the effect of cell seeding density, collagen substratum and time in culture on the development of barrier properties in this cell line, after which the permeability of the 16HBE14o- cell layers to a series of solutes was studied. Seeding cells at a density of 2.5 x 10(5) cells per cm(2) on a monofibrillar Vitrogen-100 collagen substratum, followed by culture at an air-liquid interface for 6 days resulted in cell layers with a transepithelial electrical resistance (TER) of 247+/-47 omegacm(2) and an apparent permeability coefficient of 2.5 x 10(-6)cms(-1) for mannitol. The permeability of the 16HBE14o- cells to hydrophilic molecules (logP<1.9) was of an order of magnitude greater than that of typical alveolar cell cultures, possibly reflecting barrier properties more representative of the airways. More lipophilic drugs showed higher permeabilities indicating a sigmoidal relationship between permeability and lipophilicity similar to that observed for solute transport across primary cultured epithelial cell layers. These results indicate that under appropriate culture conditions, 16HBE14o- cell layers provide a discriminatory barrier to solute transport.

In vivo evaluation of novel hyaluronan/chitosan microparticulate delivery systems for the nasal delivery of gentamicin in rabbits

Biodegradable microparticles containing gentamicin were prepared using chitosan hydroglutamate (CH), hyaluronic acid (HA) and a combination of both polymers by a solvent evaporation method. These formulations were administered nasally via an insufflator. Gentamicin was also administered nasally into rabbits as a solution and powder (a physical mixture of gentamicin and lactose), intravenously (IV) and intramuscularly (IM). The resultant serum levels of gentamicin were determined by Fluorescence Polarisation Immunoassay (FPIA). The bioavailability of gentamicin was poor when administered as a nasal solution (1.1%) and dry powder (2.1%) when compared with IV. However, the microparticulate systems composed of CH and HA/CH considerably enhanced the bioavailability of gentamicin (31.4 and 42.9%, respectively,) with HA microparticles inducing a less significant enhancement (23.3%). Previous in vitro dissolution and frog palate studies indicated that these microparticulate formulations were all mucoadhesive and demonstrated prolonged drug release. Such findings were translated into an increase in the bioavailability of gentamicin when compared with a simple nasal solution in vivo. When HA and CH were combined in the HA/CH formulation, the polymers appeared to improve the absorption of incorporated gentamicin synergistically in comparison to the individual polymers, suggesting a promising nasal delivery system.

In vivo and in vitro characterization of novel microparticulates based on hyaluronan and chitosan hydroglutamate

This study examined the application of previously characterized microparticles composed of hyaluronan (HA) and chitosan hydroglutamate (CH) as well as novel microparticles consisting of both polymers (HA/CH) to improve the nasal delivery of a model drug. The rabbit bioavailabilities of gentamicin incorporated in HA, CH, and HA/CH microparticles were increased 23-, 31-, and 42-fold, respectively, compared with the control intranasal solution of gentamicin, indicating that all test microparticles were retained for longer periods on the nasal mucosa of the rabbits as supported by previous in vitro dissolution as well as frog palate mucoadhesion studies, thereby improving drug absorption. The higher bioavailabilities of CH-based formulations (CH and HA/CH) suggest the penetration-enhancing effects of CH may also be partially responsible for the improvement. A model was developed, based on a glass impinger device, to deliver dry powder formulations reproducibly onto the surface of cultured cell monolayers. In vitro permeability and fluorescence microscopy studies on the tight junctions of the 16HBE14o- cell lines further confirmed the ability of CH-based formulations to enhance penetration. Furthermore, the in vitro absorption profile from cell culture studies was consistent with those determined from in vivo studies. The complementary effect from the mucoadhesive nature of HA coupled with the penetration-enhancing effects of CH makes the novel HA/CH formulation a promising nasal delivery system.

Nail Swelling as a Pre-formulation Screen for the Selection and Optimisation of Ungual Penetration Enhancers

IntroductionTargeting drug treatment to fungal infections that reside within or below the nail plate is problematic due to the highly restrictive barrier of the human nail. To optimise topical formulations for ungual drug delivery, inclusion of an effective penetration enhancer (PE) is imperative. At present, in vitro nail permeation studies can take weeks or months in order to obtain any meaningful data because the lack of a simple in vitro model to identify and develop nail PEs makes the selection and optimisation of novel PEs an empirical and inefficient process. The aim of this study was to compare three methods for pre-formulation screening of putative ungual PEs and then to select the most suitable technique for screening candidates that may enhance the permeation of therapeutic agents through the human nail.MethodsThree screening techniques were evaluated; nail swelling (weight increase of human nail clippings), horse hoof swelling (weight increase of horse hoof clippings) and nail penetration of a radiolabelled permeability probe. Four test PEs were evaluated using each screening method and nail swelling was identified as a simple, rapid, economic, relevant and reliable technique. This screen was then used to evaluate 20 potential PEs. Thioglycolic acid (TA), hydrogen peroxide (H2O2) and urea H2O2 produced the greatest nail weight increases; 71.0 ± 4.6%, 69.2 ± 6.6%, and 69.0 ± 9.9 respectively. To confirm the relationship between human nail swelling and altered ungual barrier function, a permeation study was performed in human nails using caffeine as a model penetrant.Results and DiscussionHuman nails pre-treated with TA in vitro had a 3.8-fold increase in caffeine flux compared to the control (TA-free solution). This study illustrated the potential to use human nail clipping swelling as a surrogate marker of PE activity for topical ungual drug delivery.

The permeability of large molecular weight solutes following particle delivery to air-interfaced cells that model the respiratory mucosa.

The transepithelial transport rates of compounds after deposition as aerosolised particles onto respiratory cell layers and allowing dissolution in the cell surface secretions has not been reported in a comprehensive manner to date. Here, the twin-stage impinger (TSI) was used to deposit potentially respirable particles (aerodynamically <6.4 microm) of varying molecular weight dextrans labelled with fluorescein isothiocyanate (FITC-dex) onto Calu-3 cells, a model of the bronchial epithelium. The TSI functioned as a particle size segregator, with >96% of the deposited particles being geometrically <6.4 microm (as measured by microscopy) and the particles being deposited discretely with a uniform distribution. Cell layers tolerated particle deposition at an air flow of 60 L/min. A small reduction in transepithelial electrical resistance (TER) of <10% occurred initially, but the original TER was recovered within 10 min and there was no significant effect on apparent permeability (P(app)) of FITC-dex 4 over 4 h. Interleukin 8 (IL-8) secretion in the apical and basolateral directions over 24 h was not increased by exposure to the TSI and particle deposition. The rate of FITC-dex 4 (4 kDa) transport across the cell layer after deposition and dissolution of the particles in the cell surface secretions was approximately 20-fold higher (P<0.05) than if applied as a solution. The volume of cell surface secretions was estimated by tracer dilution (3.44+/-1.90 microl, mean+/-SEM) and this value was used to calculate the P(app) of compound once deposited as a particle. The Papp value was found to be similar to that obtained when the compound was applied in solution (P<0.05). Thus, the increased transport rate was attributable to the differences in donor chamber solute concentration rather than any change in the permeability of the cell layer itself. Following particle deposition, transport of FITC-dex with molecular weights between 4 and 70 kDa correlated well (r(2)=0.918) with reported in vivo canine pulmonary clearance after intratracheal instillation of dextrans of similar molecular weight. The use of the TSI and the Calu-3 cell line for the assessment of compound dissolution and transport rates after particle deposition may allow more realistic analyses to be made with respect to the in vivo situation.

Adaptive resistance to benzalkonium chloride, amikacin and tobramycin: the effect on susceptibility to other antimicrobials.

Aims: To produce strains of antimicrobial‐resistant Pseudomonas aeruginosa via adaptation to benzalkonium chloride, amikacin and tobramycin and to then examine the incidence, or otherwise, of cross‐resistance between antibiotics and between antibiotics and benzalkonium chloride.

Sparing methylation of β-cyclodextrin mitigates cytotoxicity and permeability induction in respiratory epithelial cell layers in vitro

Cyclodextrins (CDs) are promising solubility enhancers for inhaled drug delivery. However, they have dose-dependent effects on the respiratory epithelium, which may have advantages for permeability enhancement but also gives rise to safety concerns. In this study, the methyl thiazol tetrazolium (MTT) assay was used to compare a new sparingly methylated beta-CD, Kleptose Crysmebeta (Crysmeb) with the more established CD derivatives hydroxypropyl-gamma-cyclodextrin (HPgammaCD), randomly methylated beta-cyclodextrin (Rameb) and hydroxypropyl-beta-cyclodextrin (HPbetaCD). The betaCD derivatives affected cell metabolism in A549 cells in a concentration dependent manner with LD(50) of 56, 31 and 11 mM obtained for HPbetaCD, Crysmeb and Rameb, respectively. Calu-3 cells were less susceptible to betaCD with an LD(50) of 25 mM being obtained for Rameb only. Permeability increases in Calu-3 cell layers were observed with betaCD derivatives and a concentration dependency shown. The mechanism of permeability enhancement and its reversibility was investigated. Rameb produced an irreversible loss of cell layer barrier function at > or = 25 mM, but perturbations of epithelial integrity were moderate and reversible in the case of HPbetaCD and Crysmeb (25-50 mM). Given its high solubilisation capacity, the low toxicity and transient absorption promoting properties, this study identifies Crysmeb as a promising adjuvant in formulations for inhalation.

The relationships and susceptibilities of some industrial, laboratory and clinical isolates of Pseudomonas aeruginosa to some antibiotics and biocides.

Aims: To provide evidence to support or refute the hypothesis that cross‐resistance between antibiotics and biocides can occur.