Inter- and intra-individual variability in human skin barrier function: a large scale retrospective study.

In vitro transepidermal tritiated water flux measurements are frequently used to evaluate skin barrier integrity for quality control purposes. However, research in this area to date has been largely based upon small-scale studies, each involving relatively few skin permeation measurements. In order to enhance our understanding in this area, we have conducted a much larger scale retrospective statistical analysis of tritiated water kp values. These values reflected the permeability of 2400 skin samples that were derived from 112 female volunteers over a 4 year period. It was found that the population of tritiated water kp values constituted a positively skewed, non-Normal distribution. Mean kp was 2.04 x 10(-3)cm/h while the 95th percentile was 4.50 x 10(-3)cm/h. Both values are higher than those reported in previous smaller studies. Hence, our study indicates that previously suggested upper limits for tritiated water flux are too low and that they be revised upwards to a value of 4.5 x 10(-3)cm/h. Analysis was also performed on smaller data subsets allowing inter-individual and intra-individual comparisons. For intra-individual kp variability, site-related differences yielded a non-Normal, positively skewed pattern in most individuals. Inter-individual variability was Normally-distributed and showed scatter that was much smaller in magnitude.

Factors influencing hydrocortisone permeation into human hair follicles: use of the skin sandwich system.

The aim of the present study was to use the in vitro human skin sandwich system in order to quantify the influence of formulation variables on intrafollicular hydrocortisone permeation. The investigated variables were the pH and the viscosity of the topical formulation as well as the presence of chemical enhancers (carvone, menthone, oleic acid and sodium lauryl sulphate). Furthermore, skin sandwich hydration was also varied in order to determine if the method itself can be run using only partially hydrated skin tissues. It was determined that the follicular contribution to hydrocortisone flux decreased marginally with increasing alkalinity in the pH range 3-8.8. Intrafollicular penetration was markedly reduced when HPMC gels were used instead of an aqueous solution. Pretreating the skin with chemical enhancers also reduced the follicular contribution to flux, probably due to permeabilisation of the continuous stratum corneum. Furthermore, it was not possible to satisfactorily modify the skin sandwich method so that it could be deployed using less hydrated skin.

In-vitro permeation of drugs into porcine hair follicles: is it quantitatively equivalent to permeation into human hair follicles?

It is already well-established that the general permeability properties of porcine skin are close to those of human skin. However, very little is known with respect to drug absorption into hair follicles and the similarities if any between the two types of tissue. The aim of this study was to use the skin sandwich system to quantify follicular drug absorption into porcine hair follicles. To our knowledge, this is the first time that the skin sandwich has been extended to porcine tissue. For this purpose, seven different drugs -- estradiol, corticosterone, hydrocortisone, aldosterone, cimetidine, deoxyadenosine and adenosine -- exhibiting a wide range of log octanol-water partition coefficients (log K(o/w)), but comparable molecular weights, were chosen as candidate solutes. The results showed a parabolic profile with maximal follicular contribution occurring at intermediate log K(o/w) values. Linear regression analysis indicated that the follicular contributions in porcine skin correlated well with previously published follicular contributions in human skin (r(2) = 0.87). The novelty of this research is that we show that porcine tissue is a good surrogate for modelling human skin permeability within the specific context of quantifying drug absorption into hair follicles.

Evidence that drug flux across synthetic membranes is described by normally distributed permeability coefficients.

Over recent decades, the use of in vitro diffusion cell studies to assess skin permeability has evolved into a major research tool, providing key insights into the relationships between skin, drug and formulation. Sometimes, such studies involve synthetic membranes as this approach can yield useful inferences with respect to drug-skin partitioning and diffusion phenomena. Yet despite the popularity of such studies, it is still not at all known whether typical solute transport across synthetic barriers results in a normal distribution of permeability coefficients or alternatively some type of skewed distribution. The present study aims to shed light on this issue. To this end, five compounds (testosterone, oestradiol, corticosterone, aldosterone and adenosine) exhibiting a broad range of octanol-water partition coefficient values were selected as test penetrants. The protocol involved taking multiple replicate measurements of each drug's passive steady state flux through poly(dimethylsiloxane) membrane. Each penetrant's resultant permeability coefficient database was subjected to a Kolmogorov-Smirnov (KS) test for normality. It was found that the permeability coefficients of all five drugs were distributed in a Gaussian-normal fashion. The theoretical significance and practical impact of these findings are discussed.

The influence of drug partition coefficient on follicular penetration: in vitro human skin studies.

The aim of this study was to employ the novel skin sandwich system in order to quantify the influence of the octanol-water partition coefficient on follicular drug absorption in human skin. To this end, seven different drugs - estradiol, corticosterone, hydrocortisone, aldosterone, cimetidine, deoxyadenosine and adenosine - exhibiting a wide range of log octanol-water partition coefficients (logK(o/w)) but relatively similar molecular weights were selected as candidate solutes. Application of the skin sandwich technique yielded an interesting relationship between % follicular contribution and logK(o/w). The follicular contribution to total flux was small (4 and 2%) for the two most lipophilic drugs but varied between 34 and 60% for the remaining drugs of intermediate and low logK(o/w) values. Lipophilicity seems to be an important modulator of drug absorption into follicular orifices only above a critical logK(o/w) threshold. Below this critical logK(o/w) value, lipophilicity does not apparently influence the follicular contribution in an obvious way and the process is probably governed by other molecular properties. Identification of these other active properties would require performing this kind of a study on a much larger set of candidate drugs.

Towards a correlation between drug properties and in vitro transdermal flux variability.

Over recent years, there has been growing evidence that the permeability coefficient variability describing any specific transdermal drug delivery system is not always normally distributed. However, since different researchers have used different test compounds, methodologies and skin types, it has been difficult to identify any general correlation between drug properties and flux variability. The aim of the present study was to investigate whether there was a relationship between these two variables. To this end, six different compounds (sucrose, adenosine, aldosterone, corticosterone, oestradiol and testosterone) exhibiting a range of partition coefficients but relatively similar molecular weights were screened by taking multiple replicate measurements of their permeation profiles as they penetrated across porcine skin in vitro. It was found that for relatively hydrophilic solutes (log P(o/w)< or = approximately 2.5), physicochemical properties that facilitated slow transdermal flux were associated with more positively skewed permeability coefficient distributions while rapid flux was associated with more symmetric distributions. However, no correlation could be found between molecular properties and the extent of statistical fit to either the normal or log-normal distribution.

Transfollicular drug delivery--is it a reality?

Once regarded as merely evolutionary remnants, the hair follicles and sebaceous glands are increasingly recognised as potentially significant elements in the percutaneous drug delivery paradigm. Interest in pilosebaceous units has been directed towards their use as depots for localised therapy, particularly for the treatment of follicle-related disorders such as acne or the alopecias. Furthermore, considerable attention has also been focused on exploiting the follicles as transport shunts for systemic drug delivery. This paper reviews various key facets of this field including; relevant aspects of pilosebaceous anatomy and physiology, the design and efficacy of follicle-targeting formulations and the emergence of quantitative modeling systems. Several novel developments in this area promise to greatly expand our understanding of this field in the near future.

Assessment of drug permeability distributions in two different model skins.

Past in vitro studies with human skin have indicated that drug permeability coefficient (Kp) distributions do not always follow a Gaussian-normal pattern. This has major statistical implications, exemplified by the fact that use of t-tests to evaluate significance is limited to normally distributed populations. Percutaneous absorption research often involves using animal or synthetic skins to simulate less readily available human skin. However, negligible work has been performed on assessing the permeability variabilities of these model membranes. This paper aims to fill this gap. To this end, four studies were undertaken representing two different drugs (caffeine and testosterone) with each drug penetrating through two different model skins (silicone membrane and pig skin). It was determined that in the silicone membrane studies, both compounds' Kp distributions could be fitted to a normal pattern. In contrast, in the pig skin studies, there were notable differences between each drug. While the testosterone Kp values could be fitted to a normal distribution, this was not possible with the caffeine Kp data, which could be fitted to a log-normal distribution. There is some evidence from the literature as well as physicochemical considerations that these outcomes may reflect general trends that are dependent upon both membrane and penetrant properties.

Mucosal drug delivery: membranes, methodologies, and applications.

In recent years, extensive research into novel forms of drug delivery has suggested that mucosal approaches offer a promising therapeutic alternative, especially for systemically acting drugs. Transmucosal drug delivery offers many benefits, including noninvasive administration, convenience, rapid onset, as well as elimination of hepatic first-pass metabolism. The investigated absorptive surfaces consist of the nasal, buccal, ocular, vaginal, and rectal mucosae. Among these, the nasal and buccal routes have proved the most promising to date. The bioavailability achieved mainly depends upon the pathophysiological state of the mucosa and the properties of both the drug and delivery systems. Various agents can increase the efficacy of transmucosal drug delivery. These include cyclodextrins, bile salts, surfactants, fusidic acid derivatives, microspheres, liposomes, and bioadhesive agents. The mechanisms of action, effectiveness, and toxicity profiles of these enhancers have been investigated extensively in both animal and human models.

Enhanced iontophoretic delivery of buspirone hydrochloride across human skin using chemical enhancers.

Buspirone hydrochloride (BH) is a structurally and pharmacologically unique anxiolytic that is used to treat a variety of different anxiety conditions. The marketed product is named BuSpar. The in vitro iontophoretic delivery of BH through human skin was investigated in order to evaluate the feasibility of delivering a therapeutic dose of BH by this route. We also examined the influence of co-formulations of chemical enhancers (Azone, oleic acid, menthone, cineole, and terpineol) on BH permeation, both without iontophoresis and with iontophoresis-to look for possible synergistic effects. By applying iontophoresis at 0.5 mA/cm(2), it was possible to achieve a BH steady state flux of approximately 350 microg/cm(2)h, which would be therapeutically effective if clinically duplicated. Importantly, 24 h of iontophoresis at 0.5 mA/cm(2) did not affect skin morphology and after the current was switched off, the skin's permeability to BH rapidly reverted to its pre-iontophoretic level. Without iontophoreis, BH transdermal flux was significantly enhanced by the application of 2.5% (v/v) concentrations of Azone, oleic acid, or menthone but not cineole or terpineol. Furthermore, this paper identified a synergistic transport enhancement effect developing when very low current (0.025 mA/cm(2)) iontophoresis was applied in conjunction with Azone treatment.

Iontophoretic transdermal delivery of buspirone hydrochloride in hairless mouse skin.

The transdermal delivery of buspirone hydrochloride across hairless mouse skin and the combined effect of iontophoresis and terpene enhancers were evaluated in vitro using Franz diffusion cells. Iontophoretic delivery was optimized by evaluating the effect of drug concentration, current density, and pH of the vehicle solution. Increasing the current density from 0.05 to 0.1 mA/cm2 resulted in doubling of the iontophoretic flux of buspirone hydrochloride, while increasing drug concentration from 1% to 2% had no effect on flux. Using phosphate buffer to adjust the pH of the drug solution decreased the buspirone hydrochloride iontophoretic flux relative to water solutions. Incorporating buspirone hydrochloride into ethanol:water (50:50 vol/vol) based gel formulations using carboxymethylcellulose and hydroxypropylmethylcellulose had no effect on iontophoretic delivery. Incorporation of three terpene enhancers (menthol, cineole, and terpineol) into the gel resulted in a synergistic effect when combined with iontophoresis. Menthol was the most active enhancer, and when combined with iontophoresis it was possible to deliver 10 mg/cm2/day of buspirone hydrochloride.

Preparation and characterization of in situ ionic cross-linked pectin films: unique biodegradable polymers.

The study aimed to investigate the swelling and degradation of calcium pectinate (CaP) films that were cross-linked by the innovative approach of adding aqueous calcium chloride (CaCl2) to pre-formed pectin films in situ. The films, cast from low methoxy pectin, were dried and cross-linked by immersion in a selected CaCl2 solution for a selected period. It was found that CaCl2 concentration, immersion time, and temperature affected the films' dissolution and swelling behaviors in simulated intestinal fluid. With lower CaCl2 concentration, more time was needed to form a proper film. Heat accelerated the cross-linking reaction, probably by elevating the cross-linked solution flux into the matrix. Depending upon cross-linking conditions, similar calcium contents in the CaP films resulted in different swelling and degradation behaviors. The degree of pectin esterification (DE) affected the films' degradation rate. The role of pectin molecular weight and DE on the films' mechanical properties was determined by stress/strain analysis.

Effect of microneedle treatment on the skin permeation of a nanoencapsulated dye.

OBJECTIVES: The aim of the study was to investigate the effect of microneedle (MN) pretreatment on the transdermal delivery of a model drug (Rhodamine B, Rh B) encapsulated in polylactic-co-glycolic acid (PLGA) nanoparticles (NPs) focusing on the MN characteristics and application variables. METHODS: Gantrez MNs were fabricated using laser-engineered silicone micro-mould templates. PLGA NPs were prepared using a modified emulsion-diffusion-evaporation method and characterised in vitro. Permeation of encapsulated Rh B through MN-treated full thickness porcine skin was performed using Franz diffusion cells with appropriate controls. KEY FINDINGS: In-vitro skin permeation of the nanoencapsulated Rh B (6.19 ± 0.77 µg/cm²/h) was significantly higher (P < 0.05) compared with the free solution (1.66 ± 0.53 µg/cm²/h). Mechanistic insights were supportive of preferential and rapid deposition of NPs in the MN-created microconduits, resulting in accelerated dye permeation. Variables such as MN array configuration and application mode were shown to affect transdermal delivery of the nanoencapsulated dye. CONCLUSIONS: This dual MN/NP-mediated approach offers potential for both the dermal and transdermal delivery of therapeutic agents with poor passive diffusion characteristics.

Methods for quantifying intrafollicular drug delivery: a critical appraisal.

IMPORTANCE OF THE FIELD: In recent years there has been increasing awareness that the hair follicles and their associated pilosebaceous structures may act as significant permeation pathways and/or reservoirs for topically applied drugs. This has implications in terms of dermatological therapy for acne, hirsutism, alopecias or certain skin cancers as well as systemic drug delivery. As the processes modulating follicular drug penetration are poorly understood at present, there is an emergent need for methodologies that can quantify follicular drug penetration and deposition. So far, a review article specifically dedicated to these methodological aspects has not yet been written. AREAS COVERED IN THIS REVIEW: This paper reviews the available quantitative follicular methodologies that have been developed over the years, describing the advantages and disadvantages of each approach. This review covers comparative techniques that are based on measuring drug flux through 'follicle-free' and 'follicle-containing' integuments, the skin sandwich, differential stripping and optical imaging-based technologies. Techniques for measuring drug-sebum interactions are also discussed. WHAT THE READER WILL GAIN: The reader will develop an understanding of the complexities involved in quantifying drug delivery through follicles and pilosebaceous units. The Expert opinion section will give the reader insights into how more broad-ranging future research could allow identification of the most useful methods for quantifying follicular drug transport. TAKE HOME MESSAGE: This is still a poorly understood field. It clearly warrants much larger scale studies than have been performed so far involving multiple techniques and multiple drugs.

A two-layer diffusive model for describing the variability of transdermal drug permeation.

There is mounting evidence that the permeability coefficients (k(p)) that describe any given transdermal drug permeation process generally follow some form of positively skewed, non-symmetrical distribution rather than a simple normal distribution. Yet a suitable theoretical treatment of this area has not been undertaken to date. In this paper, we describe a two-layer model that can explain five drugs'k(p) variabilities as measured in two previously published papers. The model shows why rapidly permeating drugs would tend to exhibit more symmetrical k(p) distributions while progressively more slowly permeating drugs would tend to exhibit progressively more positively skewed k(p) distributions. Future research should take this effect into account when comparing the flux variabilities of hydrophilic and lipophilic drugs.

Effects of microneedle length, density, insertion time and multiple applications on human skin barrier function: assessments by transepidermal water loss.

Microneedle (MN) arrays have attracted considerable attention in recent years due to their ability to facilitate effective transdermal drug delivery. Despite appreciable research, there is still debate about how different MN dimensions or application modes influence permeabilization. This study aimed to investigate this issue by taking transepidermal water-loss measurements of dermatomed human skin samples following the insertion of solid polymeric MNs. Insertions caused an initial sharp drop in barrier function followed by a slower incomplete recovery - a paradigm consistent with MN-generation of microchannels that subsequently contract due to skin elasticity. While 600 µm-long MNs were more skin-perturbing than 400 µm MNs, insertion of 1000 µm-long MNs caused a smaller initial drop in integrity followed by a degree of long term permeabilization. This is explainable by the longest needles compacting the tissue, which then decompresses over subsequent hours. Multiple insertions had a similar effect as increasing MN length. There was some evidence that increasing MN density suppressed the partial barrier recovery caused by tissue contraction. Leaving MNs embedded in skin seemed to reduce the initial post-insertion drop in barrier function. Our results suggest that this in vitro TEWL approach can be used to rapidly screen MN-effects on skin.

Transepidermal water loss for probing full-thickness skin barrier function: correlation with tritiated water flux, sensitivity to punctures and diverse surfactant exposures.

Skin barrier function is a key parameter to consider when performing in vitro percutaneous absorption studies. Whilst tritiated water flux measurements were often used to assess skin integrity, recent decades have witnessed the emergence of the more rapid and user-friendly transepidermal water loss (TEWL) approach. Yet to date, the nature of the correlation between TEWL and skin barrier function in vitro has still not been comprehensively established. In this study, a novel TEWL device, operating on a cold-induced vapour sink principle, was used to probe the barrier function of full-thickness porcine skin. The method was sufficiently sensitive to show the influence of punctures on barrier function although the observed non-linear pattern suggested tissue swelling processes and/or capillary action could be occurring. The results of various surfactant application experiments strongly suggested that TEWL was indeed largely predictive of skin sample integrity. A key finding was that basal TEWL was linearly correlated with basal tritiated water flux (r(2)=0.80, n=63). Thus, a dedicated TEWL method can be used as a good alternative to water flux measurements for assessing full-thickness skin barrier function.

Drug release kinetics from tablet matrices based upon ethylcellulose ether-derivatives: a comparison between different formulations.

The present study involved the preparation of ibuprofen-containing controlled release tablets formulated from either the established granular product, Ethocel Standard Premium, or the novel finely-milled product, Ethocel Standard FP Premium. The tablets were prepared by either direct compression or wet granulation. The aim was to explore the influence of different parameters on the kinetics and mechanisms of ibuprofen release from the tablets. These parameters were; polymer particle size, polymer molecular weight, drug : polymer ratio, preparation methodology and partial replacement of lactose with the coexcipient-hydroxypropyl methylcellulose (HPMC). The derived drug release data were analyzed with reference to various established mathematical models while the f2-metric technique was used in order to determine profile equivalency. It was found that drug release was mostly modulated by several interactive factors apparently exhibiting crosstalk. Nevertheless, it was possible to identify some simple rules. Incorporation of Ethocel FP polymers and application of the wet granulation technique facilitated greater efficiency in controlling ibuprofen release behavior from the matrices. Furthermore, drug release profiles could be modulated by partial substitution of the primary excipient with HPMC. Polymer concentrations and particle sizes, rather than viscosity grade, were found to be decisive factors in controlling drug release rates.

Transdermal and buccal delivery of methylxanthines through human tissue in vitro.

We examined the in vitro permeation of central nervous stimulants - caffeine, theophylline, and theobromine across human skin with the aid of six chemical enhancers. It was found that oleic acid was the most potent enhancer for all three methylxanthines. Further optimization studies with different solvents showed that caffeine transport could be enhanced to give flux values up to 585 microg/cm2.hr-1. Theobromine and theophylline delivery rates proved insufficient. An additional study involving a buccal tissue equivalent showed that this membrane was more permeable than skin for all model actives tested and would offer an alternate way of delivery.

Specific lipoplex-mediated antisense against Bcl-2 in breast cancer cells: a comparison between different formulations.

G3139 is an antisense oligonucleotide (ODN) that can down-regulate bcl-2, thus potentially acting as a potent anticancer drug. However, effective therapy requires efficient ODN delivery, which may be achieved by employing G3139 lipoplexes. Yet, lipofection is a complex, multifactorial process that is still poorly understood. In order to shed more light on this issue, we prepared 18 different G3139 lipoplex formulations and compared them in terms of their capability to transfect MCF-7 breast cancer cells. Each formulation was composed of a cationic lipid and sometimes a helper lipid. The cationic lipid was either DOTAP (N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride), DC-CHOL (3ss[N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol), or CCS (ceramide carbomoyl spermine). The helper lipid was either DOPC, DOPE, or cholesterol. Each lipid combination existed in two different structural forms--either large unilamellar vesicles (approximately 100 nm LUV) or unsized heterolamellar vesicles (UHV). Cell proliferation assays were used to evaluate the cytotoxicity of G3139 lipoplexes, control cationic lipid assemblies, and free G3139. Western blots were used to confirm the specific activity of G3139 as an anti-bcl-2 antisense agent. We determined that treatment of MCF-7 cells with G3139:CCS lipoplexes (UHV-derived) produced a maximal 50-fold improvement in antisense efficacy compared to treatment with free G3139. The other G3139 lipoplexes were not superior to free G3139. Thus, successful lipofection requires precise optimization of lipoplex lipid composition, structure, and concentration.