The Plant Defensin Ppdef1 Is a Novel Topical Treatment for Onychomycosis.

Onychomycosis, or fungal nail infection, causes not only pain and discomfort but can also have psychological and social consequences for the patient. Treatment of onychomycosis is complicated by the location of the infection under the nail plate, meaning that antifungal molecules must either penetrate the nail or be applied systemically. Currently, available treatments are limited by their poor nail penetration for topical products or their potential toxicity for systemic products. Plant defensins with potent antifungal activity have the potential to be safe and effective treatments for fungal infections in humans. The cystine-stabilized structure of plant defensins makes them stable to the extremes of pH and temperature as well as digestion by proteases. Here, we describe a novel plant defensin, Ppdef1, as a peptide for the treatment of fungal nail infections. Ppdef1 has potent, fungicidal activity against a range of human fungal pathogens, including Candida spp., Cryptococcus spp., dermatophytes, and non-dermatophytic moulds. In particular, Ppdef1 has excellent activity against dermatophytes that infect skin and nails, including the major etiological agent of onychomycosis Trichophyton rubrum. Ppdef1 also penetrates human nails rapidly and efficiently, making it an excellent candidate for a novel topical treatment of onychomycosis.

Skin permeation and penetration of mometasone furoate in the presence of emollients: An ex vivo evaluation of clinical application protocols.

Background: Topical corticosteroids (TCS) and emollients are developed independently by the pharmaceutical industry but are often used together in practice. There is potential for the TCS and emollient formulations to interact on the skin surface affecting TCS absorption into the skin. Clinical guidelines acknowledge this issue but lack an evidence base and differ in their recommendations. There is a current clinical need to establish whether the application protocol employed for TCS and emollient products can impact delivery of TCS to the skin. Objectives: To investigate whether the sequence of application of a TCS and emollient and the time between their application can affect TCS skin absorption. Methods: The delivery of mometasone furoate (MF) to ex vivo human skin was evaluated following the application of Elocon cream either 5 or 30 min, before and after three different emollients. Mechanistic explanation of the changes in drug absorption was provided by modelling the skin permeation data and Raman microscopy of mixed Elocon cream and emollient formulations. Results: A circa fivefold difference in MF absorption was observed depending on the emollient and application protocol. Applying Elocon cream at short intervals in relation to Hydromol intensive significantly increased MF absorption regardless of the application protocol. In contrast, applying Elocon cream after Diprobase cream or ointment significantly reduced MF absorption relative to Elocon cream alone or when Elocon cream was applied before these emollients. The changes in drug absorption observed were attributed to the presence of emollients altering Elocon cream formulation performance through different mechanisms, including introduction of penetration enhancing excipients and inducing drug crystallization in the mixed TCS emollient layer on the skin surface. Conclusions: Emollients can affect MF absorption in different ways depending on the emollient and sequence of administration. Using a 30 min gap between product applications may not be sufficient to mitigate emollient effects on TCS absorption.

Accelerating topical formulation development for inflammatory dermatoses; an ex vivo human skin culture model consistent with clinical therapeutics.

Although animal models have been extensively used to evaluate human topical therapeutics, they exhibit marked physiological differences to human skin. Our objective was to develop a human ex vivo skin culture model to explore the pathophysiology of inflammatory dermatoses and for preclinical testing of potential therapeutic treatments. Ex vivo skin barrier integrity and metabolic activity was retained for 5 days and stimulation of T-helper cells (Th1), which produce proinflammatory cytokines, provided inflammatory responses similar to those reported from in vivo biopsy. Tissue responses to established therapies of pimecrolimus (Elidel) and clobetasol propionate (Dermovate) were evaluated using the human ex vivo skin culture, assessing pharmacodynamic changes in gene expression alongside the pharmacokinetics of drug penetration with both products showing time dependent efficacies. The translational utility of the human ex vivo skin culture model of inflammatory dermatoses was demonstrated through comparison with an in vivo clinical study, with similar reductions in inflammatory gene expression recorded for both drug treatments. Thus, this model can reduce, replace or refine animal testing and also mitigate the risk of failure in costly and time-consuming clinical trials associated with novel topical therapeutic development.

A new ex vivo skin model for mechanistic understanding of putative anti-inflammatory topical therapeutics.

Several in vitro models have been designed as test systems for inflammatory skin conditions, commonly using cell-culture or reconstructed human epidermis approaches. However, these systems poorly recapitulate the physiology and, importantly, the metabolism and biochemical activity of skin in vivo, whereas ex vivo skin culture models can retain these features of the tissue. Our objective was to develop a human ex vivo skin culture model to explore the pathophysiology of inflammatory dermatoses and for preclinical testing of potential therapeutic treatments. Following exogenous stimulation, tissue integrity and ability to induce inflammatory gene expression was retained, and stimulant concentrations and duration was optimised to mimic published data from inflammatory clinical biopsies of dermatitis and psoriasis patients. The validity and utility of the model was demonstrated when challenged with 5 drugs including a corticosteroid and vitamin D3 analogue, where inflammatory biomarkers were regulated in a manner consistent with the drugs' reported in vivo mechanisms of action. This model retains important inflammatory gene signals observed in human inflammatory dermatoses for preclinical evaluation of novel therapeutics.

Human skin explant model for the investigation of topical therapeutics.

The development of in vitro and ex vivo models to mimic human illness is important not only for scientific understanding and investigating therapeutic approaches but also to mitigate animal testing and bridge the inter-species translational gap. While in vitro models can facilitate high-throughput and cost-efficient evaluation of novel therapeutics, more complex ex vivo systems can better predict both desirable and adverse in vivo effects. Here we describe an ex vivo cultured human skin explant model in which we have characterized pathological tissue integrity, barrier function and metabolic stability over time. Our findings suggest that human skin can be successfully cultured for pharmacodynamic use up to and beyond 9 days without any adverse physiological consequence.

Determination of the Permeation and Penetration of Flurbiprofen into Cadaveric Human Pharynx Tissue.

OBJECTIVE: Flurbiprofen 8.75 mg spray and lozenge have a rapid onset of action for sore throat relief, suggesting local action, although tissue penetration and the mechanism of local relief have not been determined. This investigation aimed to quantify the permeation and penetration of flurbiprofen, applied as local pharmaceutical forms, into full-thickness cadaveric human mucosal pharynx tissue, representing the clinical scenario as far as possible. METHODS: A validated high-performance liquid chromatography method quantified the permeation and penetration of flurbiprofen (spray and lozenge formulations) into human cadaveric pharynx tissue using a micro Franz cell model mimicking physiological and anatomical conditions. Full-thickness mucosal pharynx tissue, consisting of oral epithelium, basement membrane, and lamina propria, was utilized to imitate the in vivo setting. Flurbiprofen was analyzed on the surface of the pharynx tissue, within the pharynx tissue and in receiver fluid, over 60 mins. RESULTS: Flurbiprofen was detected in receiver fluid from 10 mins following spray application and was quantifiable from 20 mins. Flurbiprofen from lozenge was detected from 10 mins and was above the limit of quantitation in receiver fluid from 40 mins. Flurbiprofen recovered from the surface of the pharynx tissue was 24.45% and 8.48% of applied dose for spray and lozenge, respectively. Flurbiprofen recovered within pharynx tissue was 46.50% and 54.65% of applied dose for spray and lozenge, respectively. For flurbiprofen lozenge, recovery within pharynx tissue was 6-fold higher relative to recovery from the pharynx tissue surface. CONCLUSION: Flurbiprofen from spray and lozenge formulations penetrated human cadaveric pharynx tissue, indicating that flurbiprofen can reach all layers of the pharynx mucosal tissue, including the underlying lamina propria, which contains blood vessels and nerve fibers that contribute to pain during sore throat. This suggests that flurbiprofen may have a local mechanism of action for sore throat, although this has yet to be determined.

Diffusion through the ex vivo vitreal body - Bovine, porcine, and ovine models are poor surrogates for the human vitreous.

The human vitreous humour is a complex gel structure whose composition and physical properties can vary considerably from person to person and also change with age. To date, the viscoelastic properties of the human vitreous gel has not been thoroughly investigated and despite many years of intensive research, an ideal vitreous substitute remains a challenge. Understanding the physical structure and properties of the vitreous is of fundamental and therapeutic interest, providing a clear insight into diffusion and transport of administered ophthalmic drug molecules into the vitreous. A number of mammalian surrogates, mainly bovine, porcine and ovine vitreous humours have been used in the literature as a means of studying ophthalmic drug transport and diffusion. In this study, the mechanical, physical and rheological properties of ovine, porcine, and bovine surrogates were investigated and compared to human vitreous. In addition, a bespoke Franz cell construct was used to compare the diffusion of a model drug (fluorescein) through vitreous samples. Despite the similarity in rheological properties between bovine, porcine and human vitreous samples, diffusion of fluorescein through the different vitreous samples revealed great differences in values of steady-state flux and diffusion coefficient. In addition, a first-generation vitreous mimic, composed of 4.5 mg/mL hyaluronic acid with complex viscosity of 0.3 ±â€¯0.01 Pa has been evaluated and was demonstrated to be a better mimic of the human vitreous than the mammalian samples investigated.

Sebomic identification of sex- and ethnicity-specific variations in residual skin surface components (RSSC) for bio-monitoring or forensic applications.

BACKGROUND: "Residual skin surface components" (RSSC) is the collective term used for the superficial layer of sebum, residue of sweat, small quantities of intercellular lipids and components of natural moisturising factor present on the skin surface. Potential applications of RSSC include use as a sampling matrix for identifying biomarkers of disease, environmental exposure monitoring, and forensics (retrospective identification of exposure to toxic chemicals). However, it is essential to first define the composition of "normal" RSSC. Therefore, the aim of the current study was to characterise RSSC to determine commonalities and differences in RSSC composition in relation to sex and ethnicity. METHODS: Samples of RSSC were acquired from volunteers using a previously validated method and analysed by high-pressure liquid chromatography-atmospheric pressure chemical ionisation-mass spectrometry (HPLC-APCI-MS). The resulting data underwent sebomic analysis. RESULTS: The composition and abundance of RSSC components varied according to sex and ethnicity. The normalised abundance of free fatty acids, wax esters, diglycerides and triglycerides was significantly higher in males than females. Ethnicity-specific differences were observed in free fatty acids and a diglyceride. CONCLUSIONS: The HPLC-APCI-MS method developed in this study was successfully used to analyse the normal composition of RSSC. Compositional differences in the RSSC can be attributed to sex and ethnicity and may reflect underlying factors such as diet, hormonal levels and enzyme expression.

RNA Aptamer Delivery through Intact Human Skin.

It is generally recognized that only relatively small molecular weight (typically < ∼ 500 Da) drugs can effectively permeate through intact stratum corneum. Here, we challenge this orthodoxy using a 62-nucleotide (molecular weight = 20,395 Da) RNA-based aptamer, highly specific to the human IL-23 cytokine, with picomolar activity. Results demonstrate penetration of the aptamer into freshly excised human skin using two different fluorescent labels. A dual hybridization assay quantified aptamer from the epidermis and dermis, giving levels far exceeding the cellular half maximal inhibitory concentration values (>100,000-fold), and aptamer integrity was confirmed using an oligonucleotide precipitation assay. A T helper 17 response was stimulated in freshly excised human skin resulting in significantly upregulated IL-17f, and IL-22; topical application of the IL-23 aptamer decreased both IL-17f and IL-22 by approximately 45% but did not result in significant changes to IL-23 mRNA levels, confirming that the aptamer did not globally suppress mRNA levels. This study demonstrates that very-large-molecular-weight RNA aptamers can permeate across the intact human skin barrier to therapeutically relevant levels into both the epidermis and dermis and that the skin-penetrating aptamer retains its biologically active conformational structure capable of binding to endogenous IL-23.

Polymeric gels for intravaginal drug delivery.

Intravaginal drug delivery can elicit a local effect, or deliver drugs systemically without hepatic first pass metabolism. There are a number of emerging areas in intravaginal drug delivery, but the vagina is a challenging route of administration, due to the clearance mechanisms present which result in poor retention of dosage forms, and the potential for irritation and other adverse reactions. Gel formulations are desirable due to the ease of application, spreading and that they cause little to no discomfort to the patient. However, these dosage forms, in particular, are poorly retained and traditional gels typically have little control over drug release rates. This has led to a large number of studies on improving the retention of vaginal gels and modulating the controlled release of drugs from the gel matrix. This review outlines the anatomy and physiology of the vagina, focussing on areas relevant to drug delivery. Medical applications of vaginally administered medicines is then discussed, followed by an overview of polymeric gels in intravaginal drug delivery. The sensorial properties of intravaginal gels, and how these relate to user compliance are also summarised. Finally, some important barriers to marketing approval are described.

Influence of surface geometry on the culture of human cell lines: A comparative study using flat, round-bottom and v-shaped 96 well plates.

In vitro cell based models have been invaluable tools for studying cell behaviour and for investigating drug disposition, toxicity and potential adverse effects of administered drugs. Within this drug discovery pipeline, the ability to assess and prioritise candidate compounds as soon as possible offers a distinct advantage. However, the ability to apply this approach to a cell culture study is limited by the need to provide an accurate, in vitro-like, microenvironment in conjunction with a low cost and high-throughput screening (HTS) methodology. Although the geometry and/or alignment of cells has been reported to have a profound influence on cell growth and differentiation, only a handful of studies have directly compared the growth of a single cell line on different shaped multiwell plates the most commonly used substrate for HTS, in vitro, studies. Herein, the impact of various surface geometries (flat, round and v-shaped 96 well plates), as well as fixed volume growth media and fixed growth surface area have been investigated on the characteristics of three commonly used human cell lines in biopharmaceutical research and development, namely ARPE-19 (retinal epithelial), A549 (alveolar epithelial) and Malme-3M (dermal fibroblastic) cells. The effect of the surface curvature on cells was characterised using a combination of a metabolic activity assay (CellTiter AQ/MTS), LDH release profiles (CytoTox ONE) and absolute cell counts (Guava ViaCount), respectively. In addition, cell differentiation and expression of specific marker proteins were determined using flow cytometry. These in vitro results confirmed that surface topography had a significant effect (p < 0.05) on cell activity and morphology. However, although specific marker proteins were expressed on day 1 and 5 of the experiment, no significant differences were seen between the different plate geometries (p < 0.05) at the later time point. Accordingly, these results highlight the impact of substrate geometry on the culture of a cell line and the influence it has on the cells' correct growth and differentiation characteristics. As such, these results provide important implications in many aspects of cell biology the development of a HTS, in vitro, cell based systems to further investigate different aspects of toxicity testing and drug delivery.

Application of sebomics for the analysis of residual skin surface components to detect potential biomarkers of type-1 diabetes mellitus.

Metabolic imbalance in chronic diseases such as type-1 diabetes may lead to detectable perturbations in the molecular composition of residual skin surface components (RSSC). This study compared the accumulation rate and the composition of RSSC in type-1 diabetic patients with those in matched controls in order to identify potential biomarkers of the disease. Samples of RSSC were collected from the foreheads of type-1 diabetic (n = 55) and non-diabetic (n = 58) volunteers. Samples were subsequently analysed to identify individual components (sebomic analysis). There was no significant difference in the rate of accumulation of RSSC between type-1 diabetics and controls. In terms of molecular composition, 171 RSSC components were common to both groups, 27 were more common in non-diabetics and 18 were more common in type-1 diabetic patients. Statistically significant (P < 0.05) differences between diabetic and non-diabetic volunteers were observed in the recovered amounts of one diacylglyceride (m/z 594), six triacylglycerides (m/z 726-860) and six free fatty acids (m/z 271-345). These findings indicate that sebomic analysis can identify differences in the molecular composition of RSSC components between type-1 diabetic and non-diabetic individuals. Further work is required to determine the practical utility and identity of these potential biomarkers.

Using Salt Counterions to Modify ß2-Agonist Behavior in Vivo.

There is a paucity of data describing the impact of salt counterions on the biological performance of inhaled medicines in vivo. The aim of this study was to determine if the coadministration of salt counterions influenced the tissue permeability and airway smooth muscle relaxation potential of salbutamol, formoterol, and salmeterol. The results demonstrated that only salbutamol, when formulated with an excess of the 1-hydroxy-2-naphthoate (1H2NA) counterion, exhibited a superior bronchodilator effect (p < 0.05) compared to salbutamol base. The counterions aspartate, maleate, fumarate, and 1H2NA had no effect on the ability of formoterol or salmeterol to reduce airway resistance in vivo. Studies using guinea pig tracheal sections showed that the salbutamol:1H2NA combination resulted in a significantly faster (p < 0.05) rate of tissue transport compared to salbutamol base. Furthermore, when the relaxant activity of salbutamol was assessed in vitro using electrically stimulated, superfused preparations of guinea pig trachea, the inhibition of contraction by salbutamol in the presence of 1H2NA was greater than with salbutamol base (a total inhibition of 94.13%, p < 0.05). The reason for the modification of salbutamol's behavior upon administration with 1H2NA was assigned to ion-pair formation, which was identified using infrared spectroscopy. Ion-pair formation is known to modify a drug's physicochemical properties, and the data from this study suggested that the choice of counterion in inhaled pharmaceutical salts should be considered carefully as it has the potential to alter drug action in vivo.

In Vitro Cell Models for Ophthalmic Drug Development Applications.

Tissue engineering is a rapidly expanding field that aims to establish feasible techniques to fabricate biologically equivalent replacements for diseased and damaged tissues/organs. Emerging from this prospect is the development of in vitro representations of organs for drug toxicity assessment. Due to the ever-increasing interest in ocular drug delivery as a route for administration as well as the rise of new ophthalmic therapeutics, there is a demand for physiologically accurate in vitro models of the eye to assess drug delivery and safety of new ocular medicines. This review summarizes current existing ocular models and highlights the important factors and limitations that need to be considered during their use.

A Novel Vehicle for Enhanced Drug Delivery Across the Human Nail for the Treatment of Onychomycosis.

The aim of this study was to use in vitro nail models to investigate the potential of a novel base formulation (Recura) containing either fluconazole or miconazole for the treatment of onychomycosis in comparison to two commercial comparators (Jublia and a Penlac generic). Initially, a modified Franz cell was used, where sections of human nail served as the barrier through which drug penetrated into an agar-filled chamber infected with dermatophytes. A second study was performed using a novel infected nail model where dermatophytes grew into human nail and adenosine triphosphate levels were used as biological marker for antimicrobial activity. The novel enhancing system Recura increased the permeation of both existing drugs through human nail sections mounted in a modified Franz cell. Furthermore, the infected nail model also confirmed that the system also enhanced the permeation through infected nail resulting in a decrease in adenosine triphosphate levels superior (P ≤ 0.05) to Penlac generic and equivalent (P > 0.05) to the commercial comparator Jublia. This study demonstrated that with the use of a novel permeation-enhancing formulation base, Recura enhances delivery of miconazole and fluconazole when applied ungually such that the efficacy was equivalent or superior to commercial comparators. Such a topically applied system has the possibility of overcoming the systemic side effects of antifungals when taken orally.

New insights into eutectic cream skin penetration enhancement.

The manner in which the eutectic cream EMLA enhances the percutaneous penetration of lidocaine and prilocaine into human skin is still not fully understood. The purpose of this study was to investigate if the modification of drug aggregation played a role in the way EMLA facilitates delivery. Light scattering analysis of lidocaine alone in water gave a critical aggregation concentration (CAC) of 572 µM and a mean aggregate size of 58.8 nm. The analysis of prilocaine in identical conditions gave a CAC of 1177 µM and a mean aggregate size of 105.7 ± 24.8 nm. When the two drugs were mixed at their eutectic 1:1 ratio in water the CAC reduced to 165.8 µM and the aggregate size was 43.82 nm. This lidocaine-prilocaine interaction in water was further modified upon addition of polyoxyethylene hydrogenated castor oil, the surfactant in the EMLA aqueous phase, to produce aggregates of <20 nm. The physical characterisation data suggested that it was the EMLA cream's surfactant that modified the drug molecular interactions in the aqueous continuous phase and caused a 6 fold higher drug penetration through human epidermal tissue compared to the oil formulations tested in this study.

Effect of ethnicity, gender and age on the amount and composition of residual skin surface components derived from sebum, sweat and epidermal lipids.

BACKGROUND/PURPOSE: The superficial layer on the skin surface, known as the acid mantle, comprises a mixture of sebum, sweat, corneocyte debris and constituents of natural moisturizing factor. Thus, the phrase 'residual skin surface components' (RSSC) is an appropriate term for the mixture of substances recovered from the skin surface. There is no general agreement about the effects of ethnicity, gender and age on RSSC. The aim of this human volunteer study was to evaluate RSSC in relation to ethnicity, gender and age. A suitable acquisition medium for RSSC collection was identified and samples of RSSC were subsequently analysed using gas chromatography-mass spectrometry (GC-MS) and gravimetry. METHODS: A total of 315 volunteers participated in the study from a range of self-declared ethnic backgrounds. Six acquisition media were compared to determine the most suitable media for RSSC collection. The effect of age, gender and ethnicity on RSSC collection was evaluated by gravimetric analysis while GC-MS was used to determine the composition of RSSC. RESULTS: Of the six candidate materials assessed, cigarette paper provided the most practical and reproducible sample acquisition medium. There was no significant difference in the amount of RSSC collected when based on gender and ethnicity and no significant correlation between RSSC recovery and age. Up to 49 compounds were detected from human RSSC when analysed by GC-MS. CONCLUSIONS: The results of the present study suggest that RSSC can be effectively collected using cigarette paper and analysed by GC-MS. Ethnicity, gender and age had no significant impact on the quantity of RSSC recovered from the skin surface.

Topical corticosteroid delivery into human skin using hydrofluoroalkane metered dose aerosol sprays.

Drug loaded hydrofluoroalkane (HFA) sprays can generate effective pharmaceutical formulations, but a deeper understanding of the manner in which these dynamic systems drive the process of in situ semi-solid dosage form assembly is required. The aim of this study was to investigate the effect of the matrix assembly and composition on drug localisation in human skin. Comparing the characteristics of sprays constituting HFA 134a, ethanol (EtOH), poly(vinyl pyrrolidone) K90, isopropyl myristate (IPM), and poly(ethylene glycol) (PEG) demonstrated that the addition of non-volatile solvents acted to delay EtOH evaporation, control the degree of drug saturation (DS) and enhance the corticosteroid delivery from HFA spray formulations. In a dose matched skin penetration study the HFA sprays containing only EtOH as a co-solvent delivered 2.1 µg BMV (DS 13.5) into the tissue, adding IPM to the EtOH HFA delivered 4.03 µg BMV (DS 11.2), whist adding PEG to the EtOH HFA delivered 6.1 µg BMV (DS 0.3). Compared to commercial cream (delivering 0.91 µg BMV) the EtOH/PEG HFA spray deposited over 6 times (p<0.05) more drug into the skin. Post spray deposition characterisation of the semi-solid suggested that the superior performance of the EtOH/PEG HFA spray was a consequence of retarding EtOH evaporation and presenting the drug in an EtOH rich PEG residual phase, which promoted BMV passage through the SC and into epidermis.

In silico prediction of aqueous solubility using simple QSPR models: the importance of phenol and phenol-like moieties.

Recently the authors published a robust QSPR model of aqueous solubility which exploited the computationally derived molecular descriptor topographical polar surface area (TPSA) alongside experimentally determined melting point and logP. This model (the "TPSA model") is able to accurately predict to within ± one log unit the aqueous solubility of 87% of the compounds in a chemically diverse data set of 1265 molecules. This is comparable to results achieved for established models of aqueous solubility e.g. ESOL (79%) and the General Solubility Equation (81%). Hierarchical clustering of this data set according to chemical similarity shows that a significant number of molecules with phenolic and/or phenol-like moieties are poorly predicted by these equations. Modification of the TPSA model to additionally incorporate a descriptor pertaining to a simple count of phenol and phenol-like moieties improves the predictive ability within ± one log unit to 89% for the full data set (1265 compounds -8.48 < logS < 1.58) and 82% for a reduced data set (1160 compounds 6.00 < logS < 0.00) which excludes compounds at the sparsely populated extremities of the data range. This improvement can be rationalized as the additional descriptor in the model acting as a correction factor which acknowledges the effect of phenolic substituents on the electronic characteristics of aromatic molecules i.e. the generally positive contribution to aqueous solubility made by phenolic moieties.