Differential permeation of propranolol enantiomers across human skin in vitro from formulations containing an enantioselective excipient.

This work tested the hypothesis that a stereospecific topical formulation could be used to engineer differential permeation rates for each enantiomer of an applied racemate across human skin in vitro. Racemic and enantiomerically pure R or S propranolol HCI were formulated with cellulose tris(3,5-dimethyl phenyl carbamate) (CDMPC) and applied to excised human skin using side-by-side Franz-type diffusion cells. When the pure enantiomers were used, there was a marked difference between the penetration rates of R and S propranolol (flux ratio: 2.06; P = 0.04). When racemic propranolol was used, the difference was reduced, although still statistically significant (flux ratio: 1.2; P = 0.08), particularly in view of the differential activities of the two enantiomers. Control experiments, in which no CDMPC was present, produced equal permeation rates. The results can be rationalised in terms of differential adsorption onto CDMPC within the vehicle, whereby S-propranolol is preferentially bound relative to R-propranolol. This causes an imbalance in the apparent donor phase concentrations that (in accordance with Fickian diffusion laws and thermodynamic activity) gives rise to differences in permeation rates. The diminished differential observed when the racemate was used, rather than individual enantiomers, is less easily rationalised. In this work, it was the permeation of the eutomer (S-propranolol) that was retarded, although the general principle of stereoselectively retarded skin permeation has been established.     

Cell model of inflammation

Chemical and physical stimuli trigger a cutaneous response by first inducing the main epidermal cells, keratinocytes, to produce specific mediators that are responsible for the initiation of skin inflammation. Activation modulates cell communication, namely leucocyte recruitment and blood-to-skin extravasation through the selective barrier of the vascular ECs (endothelial cells). In the present study, we describe an in vitro model which takes into account the various steps of human skin inflammation, from keratinocyte activation to the adhesion of leucocytes to dermal capillary ECs. Human adult keratinocytes were subjected to stress by exposure to UV irradiation or neuropeptides, then the conditioned culture medium was used to mimic the natural micro-environmental conditions for dermal ECs. A relevant in vitro model must include appropriate cells from the skin. This is shown in the present study by the selective reaction of dermal ECs compared with EC lines from distinct origins, in terms of leucocyte recruitment, sensitivity to stress and nature of the stress-induced secreted mediators. This simplified model is suitable for the screening of anti-inflammatory molecules whose activity requires the presence of various skin cells.     

Liquid chromatography assay for 5-aminolevulinic acid: application to in vitro assessment of skin penetration via Dermaportation

The purpose of the present study was to develop a reverse-phase high performance liquid chromatographic (HPLC) assay for quantifying 5-aminolevulinic acid (ALA). The assay was applied to study the skin permeation of ALA and the influence of a novel skin penetration enhancement technology. Separation was achieved utilizing a Phenomenex Jupiter C(18) column following fluorescence derivatization with fluorescamine. The assay was linear (r(2)>0.99) with a minimum limit of quantitation of 400 ng/mL. The inter- and intraday variation was 1.6 and 0.9% at the lower end of the linear range and 1.5 and 1.9% at the upper end, respectively. The HPLC assay and fluorescence derivatization procedure is sensitive, simple, rapid, accurate and reproducible and offers advantages with regard to stability of ALA in comparison to other fluorescence derivatization methods. Results from the preliminary skin permeation study demonstrated substantial skin penetration of ALA only when applied with Dermaportation as a skin penetration enhancement device.     

Dual polarization interferometry size and density characterisation of DNA immobilisation and hybridisation

Investigation of nucleic acid interactions was performed using dual polarization interferometry, a novel approach to elucidating molecular interactions. This paper presents a preliminary study of adsorption of single stranded DNA onto functionalised silicon oxynitride, compared with covalent linkage, and avidin-biotin immobilisation. The effect of probe concentration on hybridisation efficiency was also examined. We found that increasing the electrolyte concentration resulted in a decrease of adsorbed DNA and that capture of a biotinylated duplex DNA on an adsorbed avidin layer resulted in four times fewer molecules per cm(2) than for duplex DNA covalently bound via an amine end terminal. The rate of thickness increase of a biotin probe layer on an adsorbed avidin capture layer increased 10-fold when the probe concentration was increased from 0.1 microM to 1 microM. The close grafting density of the higher concentration probe meant that the immobilised probes were unavailable for hybridisation.     

Microemulsion and Microporation Effects on the Genistein Permeation Across Dermatomed Human Skin

This study reports the microemulsion (ME) effects on the permeation of genistein across normal (intact) and microporated human skin. The genistein formulation was optimized to know the stable ME region in the pseudo-ternary phase diagrams and to maximize the skin permeation and retention of genistein. The phase diagrams were constructed with different oil phases, surfactants, and their combinations. The influence of formulation factors on the permeation through intact and microporated human skin was determined. Based on its wide ME region, as well as permeation enhancement effects, oleic acid was used as an oil phase with various surfactants and co-surfactants to further maximize the ME region and skin permeation. The water content in the formulation played an important role in the ME stability, droplet size, and flux of genistein. For example, the ME with 20% water exhibited 4- and 9-fold higher flux as compared to the ME base (no water) and aqueous suspension, respectively. Likewise, this formulation had demonstrated 2- and 4-fold higher skin retention as compared to the ME base (no water) and aqueous suspension, respectively. The skin microporation did not significantly increase the skin permeation of genistein from ME formulations. The ME composition, water content, and to a lesser extent the ME particle size played a role in improving the skin permeation and retention of genistein.     

Role of volatile oil pretreatment and skin cholesterol on permeation of ion-paired diclofenac sodium

This study was designed to investigate the influence of volatile oil pretreated skin on in vitro permeation from films containing ionized and dodecylamine ion-paired diclofenac sodium (DS). The involvement of skin cholesterol was investigated to determine its possible role in enhancing the permeation of ion-paired DS. Cardamom oil produced the maximum (10 x) in vitro permeation enhancement for ion-paired DS. The carrageenan induced rat paw oedema reduction (up to 12 hr) by cardamom oil was comparable to that of diclofenac injection (s c). Leaching of cholesterol from excised skin in addition to increased partition coefficient following volatile oil skin pretreatment appears to be responsible for in vitro permeation enhancement of DS. Whereas, a mild barrier perturbation effect due to altered cholesterol levels following pretreatment with volatile oils appears to increase the permeation of ion-paired DS across viable skin, thereby producing significant reduction of carrageenan induced paw oedema.     

Elucidation of the percutaneous absorption of chromium compounds by functional proteomics

Chromium compounds are known to be associated with cytotoxicity and carcinogenicity when applied via a skin route. The aim of this study was to evaluate the skin permeability and toxicological profiles of four chromium species. Chromium permeation across the skin, as determined by an in vitro Franz cell, decreased in the order of sodium chromate>potassium chromate>potassium dichromate>chromium nitrate. The uptake of chromium species within the skin generally showed a contrary trend to the results of permeation, although differences among the various compounds were not large. Levels of in vivo skin deposition of the four compounds showed no statistically significant differences. Potassium chromate produced the greatest disruption of the skin structure as determined by HE staining, followed in order by sodium chromate, potassium dichromate, and chromium nitrate. This indicates that hexavalent chromium elicited greater toxicity to the skin compared to trivalent chromium. A similar result was observed for the viability of skin fibroblasts. To improve our understanding of the molecular mechanisms leading to functional changes in proteins, proteomic tools, including 2‐DE and MS techniques combined with sequence database correlations, were applied to identify target proteins altered by pathologic states. Eight protein spots, corresponding to cutaneous enzymes involved in energy metabolism and chaperon proteins, which were identified and discussed in this study, were associated with skin cytotoxicity, immunity, and carcinogenesis. In addition, functional proteomics of skin tissues may provide a promising tool for developing therapeutic strategies and can serve as the basis for further research.     

Fluidity enhancement: a critical factor for performance of liposomal transdermal drug delivery system

Liposomes are well known lipid carriers for drug delivery of bioactive molecules encapsulated inside their membrane. Liposomes as skin drug delivery systems were initially promoted primarily for localized effects with minimal systemic delivery. Subsequently, a novel vesicular system, transferosomes was reported for transdermal delivery with efficiency similar to subcutaneous injection. The multiple bilayered organizations of lipids applied in these vesicles structure are somewhat similar to complex nature of stratum corneal intercellular lipids domains. The incorporation of novel agents into these lipid vesicles results in the loss of entrapped markers but it is similar to fluidization of stratum corneum lipids on treatment with a penetration enhancer. This approach generated the utility of penetration enhancers/fluidizing agents in lipids vesicular systems for skin delivery. For the transdermal and topical applications of liposomes, fluidity of bilayer lipid membrane is rate limiting which governs the permeation. This article critically reviews the relevance of using different types of vesicles as a model for skin in permeation enhancement studies. This study has also been designed to encompass all enhancement measurements and analytical tools for characterization of permeability in liposomal vesicular system.     

Molecular dynamics simulation of water permeation through the alpha-hemolysin channel

The alpha-hemolysin (AHL) nanochannel is a non-selective channel that allows for uncontrolled transport of small molecules across membranes leading to cell death. Although it is a bacterial toxin, it has promising applications, ranging from drug delivery systems to nano-sensing devices. This study focuses on the transport of water molecules through an AHL nanochannel using molecular dynamics (MD) simulations. Our results show that AHL can quickly transport water across membranes. The first-passage time approach was used to estimate the diffusion coefficient and the mean exit time. To study the energetics of transport, the potential of mean force (PMF) of a water molecule along the AHL nanochannel was calculated. The results show that the energy barriers of water permeation across a nanopore are always positive along the channel and the values are close to thermal energy (k_BT). These findings suggest that the observed quick permeation of water is due to small energy barriers and a hydrophobic inner channel surface resulting in smaller friction. We speculate that these physical mechanisms are important in how AHL causes cell death.     

Design and development of ethosomal transdermal drug delivery system of valsartan with preclinical assessment in Wistar albino rats

Abstract

Valsartan (VLT) is a highly selective and orally active antihypertensive drug. However, its oral administration is associated with drawbacks like low bioavailability. The objective of this study was to design and develop a transdermal delivery system for VLT using ethosomal carriers to investigate their enhanced transdermal delivery potential. VLT ethosomes were prepared by cold method. VLT ethosomes were characterized by scanning electron microscopy. The prepared ethanolic liposomes were characterized to be spherical having low polydispersity of nano-size range with good entrapment efficiency. ETC5 ethosomal suspension with 4% of phospholipon 90H and 40% of ethanol was found to have highest entrapment efficiency, i.e. 80.230?±?0.8748%. The permeation study of ethosomes was evaluated by ex vivo diffusion study through rat abdominal skin using Franz’s diffusion cells and ETC5 ethosomal suspension was found to have highest permeation with flux of 92.819?±?1.539?µg/cm²/h, when compared to the permeation profiles of drug solutions either in water or in a water–ethanol mixture. Transdermal application of ethosomal VLT on Wistar rats showed better and prolonged antihypertensive activity in comparison to orally administered VLT suspension by virtue of transdermal permeation through Wistar rat skin. Histopathological study of skin applied with ETC5 showed intercellular permeation across skin by dissolving intercellular lipids in epidermis without causing any rigorous changes in the skin cellular structure. In conclusion, ethosomes enabled the transdermal permeation of VLT, which amply proves its superiority over oral administration for antihypertensive treatment.     

The in vitro use of the hair follicle closure technique to study the follicular and percutaneous permeation of topically applied drugs

Recent studies on follicular permeation emphasise the importance of hair follicles as diffusion pathways, but only a limited amount of data are available about the follicular permeation of topically applied drugs. This study examines the use of a hair follicle closure technique in vitro, to determine the participation of hair follicles in transdermal drug penetration. Various substances, with different lipophilicities, were tested: caffeine, diclofenac, flufenamic acid, ibuprofen, paracetamol, salicylic acid and testosterone. Diffusion experiments were conducted with porcine skin, the most common replacement material for human skin, in Franz-type diffusion cells over 28 hours. Different experimental settings allowed the differentiation between interfollicular and follicular permeation after topical application of the test compounds. A comparison of the apparent permeability coefficients of the drugs demonstrates that the percutaneous permeations of caffeine and flufenamic acid were significantly higher along the hair follicles. In the cases of paracetamol and testosterone, the follicular pathway appears to be of importance, while no difference was found between interfollicular and follicular permeation for diclofenac, ibuprofen and salicylic acid. Thus, the hair follicle closure technique represents an adequate in vitro method for gaining information about follicular or percutaneous permeation, and can replace in vivo testing in animals or humans.     

The use of reconstructed human epidermis for skin absorption testing: Results of the validation study

A formal validation study was performed, in order to investigate whether the commercially-available reconstructed human epidermis (RHE) models, EPISKIN, EpiDerm and SkinEthic, are suitable for in vitro skin absorption testing. The skin types currently recommended in the OECD Test Guideline 428, namely, ex vivo human epidermis and pig skin, were used as references. Based on the promising outcome of the prevalidation study, the panel of test substances was enlarged to nine substances, covering a wider spectrum of physicochemical properties. The substances were tested under both infinite-dose and finite-dose conditions, in ten laboratories, under strictly controlled conditions. The data were subjected to independent statistical analyses. Intra-laboratory and inter-laboratory variability contributed almost equally to the total variability, which was in the same range as that in preceding studies. In general, permeation of the RHE models exceeded that of human epidermis and pig skin (the SkinEthic RHE was found to be the most permeable), yet the ranking of substance permeation through the three tested RHE models and the pig skin reflected the permeation through human epidermis. In addition, both infinite-dose and finite-dose experiments are feasible with RHE models. The RHE models did not show the expected significantly better reproducibility, as compared to excised skin, despite a tendency toward lower variability of the data. Importantly, however, the permeation data showed a sufficient correlation between all the preparations examined. Thus, the RHE models, EPISKIN, EpiDerm and SkinEthic, are appropriate alternatives to human and pig skin, for the in vitro assessment of the permeation and penetration of substances when applied as aqueous solutions.     

Specific interaction between GroEL and denatured protein measured by compression-free force spectroscopy

We investigated the interaction between GroEL and a denatured protein from a mechanical point of view using an atomic force microscope. Pepsin was bound to an atomic force microscope probe and used at a neutral pH as an example of denatured proteins. To measure a specific and delicate interaction force, we obtained force curves without pressing the probe onto GroEL molecules spread on a mica surface. Approximately 40 pN of tensile force was observed for approximately 10 nm while pepsin was pulled away from the chaperonin after a brief contact. This length of force duration corresponding to the circumference of GroEL's interior cavity was shortened by the addition of ATP. The relation between the observed mechanical parameters and the chaperonin's refolding function is discussed.     

Effect of size, surface charge, and hydrophobicity of poly(amidoamine) dendrimers on their skin penetration

The barrier functions of the stratum corneum and the epidermal layers present a tremendous challenge in achieving effective transdermal delivery of drug molecules. Although a few reports have shown that poly(amidoamine) (PAMAM) dendrimers are effective skin-penetration enhancers, little is known regarding the fundamental mechanisms behind the dendrimer-skin interactions. In this Article, we have performed a systematic study to better elucidate how dendrimers interact with skin layers depending on their size and surface groups. Franz diffusion cells and confocal microscopy were employed to observe dendrimer interactions with full-thickness porcine skin samples. We have found that smaller PAMAM dendrimers (generation 2 (G2)) penetrate the skin layers more efficiently than the larger ones (G4). We have also found that G2 PAMAM dendrimers that are surface-modified by either acetylation or carboxylation exhibit increased skin permeation and likely diffuse through an extracellular pathway. In contrast, amine-terminated dendrimers show enhanced cell internalization and skin retention but reduced skin permeation. In addition, conjugation of oleic acid to G2 dendrimers increases their 1-octanol/PBS partition coefficient, resulting in increased skin absorption and retention. Here we report that size, surface charge, and hydrophobicity directly dictate the permeation route and efficiency of dendrimer translocation across the skin layers, providing a design guideline for engineering PAMAM dendrimers as a potential transdermal delivery vector.     

Structure of the skin barrier and its modulation by vesicular formulations

The natural function of the skin is to protect the body from unwanted influences from the environment. The main barrier of the skin is located in the outermost layer of the skin, the stratum corneum. Since the lipids regions in the stratum corneum form the only continuous structure, substances applied onto the skin always have to pass these regions. For this reason the organization in the lipid domains is considered to be very important for the skin barrier function. Due to the exceptional stratum corneum lipid composition, with long chain ceramides, free fatty acids and cholesterol as main lipid classes, the lipid phase behavior is different from that of other biological membranes. In stratum corneum crystalline phases are predominantly present, but most probably a subpopulation of lipids forms a liquid phase. Both the crystalline nature and the presence of a 13 nm lamellar phase are considered to be crucial for the skin barrier function. Since it is impossible to selectively extract individual lipid classes from the stratum corneum, the lipid organization has been studied in vitro using isolated lipid mixtures. These studies revealed that mixtures prepared with isolated stratum corneum lipids mimic to a high extent stratum corneum lipid phase behavior. This indicates that proteins do not play an important role in the stratum corneum lipid phase behavior. Furthermore, it was noticed that mixtures prepared only with ceramides and cholesterol already form the 13 nm lamellar phase. In the presence of free fatty acids the lattice density of the structure increases. In stratum corneum the ceramide fraction consists of various ceramide subclasses and the formation of the 13 nm lamellar phase is also affected by the ceramide composition. Particularly the presence of ceramide 1 is crucial. Based on these findings a molecular model has recently been proposed for the organization of the 13 nm lamellar phase, referred to as "the sandwich model", in which crystalline and liquid domains coexist. The major problem for topical drug delivery is the low diffusion rate of drugs across the stratum corneum. Therefore, several methods have been assessed to increase the permeation rate of drugs temporarily and locally. One of the approaches is the application of drugs in formulations containing vesicles. In order to unravel the mechanisms involved in increasing the drug transport across the skin, information on the effect of vesicles on drug permeation rate, the permeation pathway and perturbations of the skin ultrastructure is of importance. In the second part of this paper the possible interactions between vesicles and skin are described, focusing on differences between the effects of gel-state vesicles, liquid-state vesicles and elastic vesicles.     

Simultaneous transdermal extraction of glucose and lactate from human subjects by reverse iontophoresis

This study investigated the possibility of simultaneously extracting glucose and lactate from human subjects, at the same skin location, using transdermal reverse iontophoresis. Transdermal monitoring using iontophoresis is made possible by the skin's permeability to small molecules and the nanoporous and microporous nature of the structure of skin. The study was intended to provide information which could be used to develop a full, biosensor-based, monitoring system for multiple parameters from transdermal extraction. As a precursor to the human study, in vitro reverse iontophoresis experiments were performed in an artificial skin system to establish the optimum current waveforms to be applied during iontophoresis. In the human study, a bipolar DC current waveform (with reversal of the electrode current direction every 15 minutes) was applied to ten healthy volunteers via skin electrodes and utilized for simultaneous glucose and lactate transdermal extraction at an applied current density of 300 microA/cm2. Glucose and lactate were successfully extracted through each subject's skin into the conducting gel that formed part of each iontophoresis electrode. The results suggest that it will be possible to noninvasively and simultaneously monitor glucose and lactate levels in patients using this approach and this could have future applications in diagnostic monitoring for a variety of medical conditions.     

The effect of lipids on transepidermal water permeation in snakes

Transepidermal water permeation was measured for the shed skin of 8 species of snakes under conditions where the skin formed an interface between liquid water and dry air. Extraction with chloroform: methanol removed lipids amounting to 4.9-8.7% of the dry weight of skin and increased transepidermal water permeation 35- to 175-fold. Initial extraction of snake skin with hexane removed only 25-35% of the total extractable lipid but increased water permeation 3- to 10-fold. For totally delipidized skin, water-to-air permeation was much faster than air-to-air permeation, and was also faster than evaporation from a free water surface.     

Liquid Crystalline Systems for Transdermal Delivery of Celecoxib: In Vitro Drug Release and Skin Permeation Studies

Liquid crystalline systems of monoolein/water could be a promising approach for the delivery of celecoxib (CXB) to the skin because these systems can sustain drug release, improve drug penetration into the skin layers and minimize side effects. This study evaluated the potential of these systems for the delivery of CXB into the skin based on in vitro drug release and skin permeation studies. The amount of CXB that permeated into and/or was retained in the skin was assayed using an HPLC method. Polarizing light microscopy studies showed that liquid crystalline systems of monoolein/water were formed in the presence of CXB, without any changes in the mesophases. The liquid crystalline systems decreased drug release when compared to control solution. Drug release was independent of the initial water content of the systems and CXB was released from cubic phase systems, irrespective of the initial water content. The systems released the CXB following zero-order release kinetics. In vitro drug permeation studies showed that cubic phase systems allowed drug permeation and retention in the skin layers. Cubic phase systems of monoolein/water may be promising vehicles for the delivery of CXB in/through the skin because it improved CXB skin permeation compared with the control solution.KEY WORDS: celecoxib, drug delivery systems, liquid crystalline system, monoolein, skin permeation     

Development of an aerosol surface inoculation method for bacillus spores

A method was developed to deposit Bacillus subtilis spores via aerosolization onto various surface materials for biological agent decontamination and detection studies. This new method uses an apparatus coupled with a metered dose inhaler to reproducibly deposit spores onto various surfaces. A metered dose inhaler was loaded with Bacillus subtilis spores, a surrogate for Bacillus anthracis. Five different material surfaces (aluminum, galvanized steel, wood, carpet, and painted wallboard paper) were tested using this spore deposition method. This aerosolization method deposited spores at a concentration of more than 10(7) CFU per coupon (18-mm diameter) with less than a 50% coefficient of variation, showing that the aerosolization method developed in this study can deposit reproducible numbers of spores onto various surface coupons. Scanning electron microscopy was used to probe the spore deposition patterns on test coupons. The deposition patterns observed following aerosol impaction were compared to those of liquid inoculation. A physical difference in the spore deposition patterns was observed to result from the two different methods. The spore deposition method developed in this study will help prepare spore coupons via aerosolization fast and reproducibly for bench top decontamination and detection studies.