Liposome-skin interactions and their effects on the skin permeation of drugs

The aim of the study was to evaluate the interaction of phospholipid liposomes with skin and stratum corneum lipid liposomes (SCLLs). The influence of phospholipid liposomes on the skin permeability of model drugs was also studied. The transdermal flux of the drugs applied in various phospholipid containing formulations through human epidermis was studied in diffusion chambers. Liposomes in water solutions did not enhance the skin permeability of the drugs, but when ethanol (32% w/v) was present in the donor with EPC (egg yolk lecithin), permeabilities of some model drugs were substantially increased. Confocal microscopy studies revealed that EPC do not penetrate into the skin from water solutions, while from ethanol solutions, EPC penetrates deeply into the stratum corneum. Also, resonance energy transfer between different liposome compositions and the release of calcein from SCLLs showed that interactions between phospholipid liposomes and SCLLs increased with increasing ethanol concentration in the liposome solutions.     

The effect of preoperative intra-aortic balloon pump support in high risk patients requiring myocardial revascularization

Differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM) have been used to determine the influence of beta-cyclodextrin (beta-CyD), hydroxypropyl-beta-CyD (HP-beta-CyD) and gamma-CyD on the structural properties of the stratum corneum from the hairless mouse. Some modest changes in the stratum corneum lipid transition temperature were induced by HP-beta-CyD and blue shifts were observed in the FTIR spectra of the C-H asymmetric and symmetric stretching of the lipids from the stratum corneum. Results from TEM studies indicated that HP-beta-CyD caused removal and possible disorganization of the lipid matrix that envelopes the corneocytes of the stratum corneum, whereas no effect was seen after treatment of the samples with beta-CyD and gamma-CyD. These results suggest that HP-beta-CyD can increase the permeability of the stratum corneum possibly as a result of extraction of lipids, and might thus enhance drug permeation through the skin.     

Thermally induced molecular disorder in human stratum corneum lipids compared with a model phospholipid system; FT-Raman spectroscopy

The molecular basis of lipid packing in human stratum corneum and a model phospholipid system has been studied as a function of temperature using Fourier Transform (FT) Raman spectroscopy. Thermally induced molecular rearrangements of the model lipid system, dipalmitoylphosphatidyl choline (DPPC), and stratum corneum were investigated using FT Raman spectroscopy coupled to a heating chamber. Spectra were recorded for a range of sample temperatures and the results for the two systems were compared, producing previously unreported information of the thermal behaviour for the different systems. Discrete thermal events were recorded for both systems by plotting band separation of the lipid v(CH2) symmetric and asymmetric stretching modes against temperature. The main thermal events observed for DPPC included a 'pre-melting' between 37 and 39 degrees C, the main transition observed between 41 and 42 degrees C, a 'post-transition' between 42 and 43 degrees C and three minor transitions at 58-60, 65-70 and 75-80 degrees C. No evidence was found for the pre-transition of DPPC, previously observed at 34-35 degrees C. The main transitions for human stratum corneum were observed at 35-45, 55, 72 and 83 degrees C, measured from lipid CH2 stretching and bending vibrations. The keratin thermal transition at about 100 degrees C exerted little effect on the lipid bands and no characterisable structural changes were reflected in the keratotic bands.     

A new HPLC-based method for the quantitative analysis of inner stratum corneum lipids with special reference to the free fatty acid fraction

The inner stratum corneum is likely to represent the location of the intact skin barrier, unperturbed by degradation processes. In our studies of the physical skin barrier a new high-performance liquid chromatography (HPLC)-based method was developed for the quantitative analysis of lipids of the inner stratum corneum. All main lipid classes were separated and quantitated by HPLC/light scattering detection (LSD) and the free fatty acid fraction was further analysed by gas-liquid chromatography (GLC). Mass spectrometry (MS) was used for peak identification and flame ionization detection (FID) for quantitation. Special attention was paid to the free fatty acid fraction since unsaturated free fatty acids may exert a key function in the regulation of the skin barrier properties by shifting the physical equilibrium of the multilamellar lipid bilayer system towards a noncrystalline state. Our results indicated that the endogenous free fatty acid fraction of the stratum corneum barrier lipids in essence exclusively consisted of saturated long-chain free fatty acids. This fraction was characterized as a very stable population (low interindividual peak variation) dominated by saturated lignoceric acid (C24:0, 39 molar%) and hexacosanoic acid (C26:0, 23 molar%). In addition, trace amounts of very long-chain (C32-C36) saturated and monounsaturated free fatty acids were detected in human forearm inner stratum corneum. Our analysis method gives highly accurate and precise quantitative information on the relative composition of all major lipid species present in the skin barrier. Such data will eventually permit skin barrier model systems to be created which will allow a more detailed analysis of the physical nature of the human skin barrier.     

Permeation characteristics of 8-methoxypsoralen through human skin; relevance to clinical treatment

The permeation characteristics through human skin of 8-methoxypsoralen (8-MOP) and its physical attributes were investigated. The log octanol/water partition coefficient and saturated aqueous solubility of 8-MOP at 32 degrees C were 1-98 and 55.8 micrograms mL-1 respectively, 8-MOP showed Fickian diffusion, with its flux being linearly related to the concentration of drug in the donor solution. The permeability coefficient of 8-MOP through human skin from different concentrations of aqueous solutions and a 2.6 micrograms mL-1 bath lotion (as used in clinics) were statistically identical with mean values of 1.76 +/- 0.12 x 10(-2) and 1.70 +/- 0.32 x 10(-2) cm h-1 respectively (P > or = 0.05). An ethanol/water (1:1 w/v) receptor solution did not improve the clearance of 8-MOP from the dermis when compared with an aqueous vehicle. Complete removal of the stratum corneum by tape stripping from full-thickness membranes produced a threefold increase in the flux of 8-MOP thus suggesting that the main barrier to 8-MOP permeation resides in the stratum corneum although the aqueous epidermal and dermal tissue provide a significant resistance to transdermal drug permeation. The equilibrium uptake of 8-MOP into psoriatic plaques and the 8-MOP aqueous plaque partition coefficient were found to be more than twofold greater than for normal stratum corneum. The absorption of 8-MOP from the total applied topical dose (396 mg) was assessed as approximately 0.25% and only 2.5% of an oral dose, a significant reduction in the possible toxic hazard. The peak concentration of 8-MOP permeating through the skin was observed at about 35 min after limited exposure for 15 min. Our results suggest that following a 15 min bath in the drug solution, there may be a need for an interval of about 20 min before patients are irradiated to ensure the optimization of photosensitizer with UVA irradiation (PUVA) therapy. Alternatively, UV irradiation could be applied at a lower flux over a longer time.     

Transdermal delivery of narcotic analgesics: comparative metabolism and permeability of human cadaver skin and hairless mouse skin

The permeation of hairless mouse skin and human cadaver skin by narcotic analgesics was investigated to determine the interspecies variation. Permeability coefficients of morphine, fentanyl, and sufentanil across full-thickness hairless mouse skin were 1 order of magnitude higher than those found for human epidermis. The permeability coefficient of morphine for stripped hairless mouse skin was 500-fold higher than that for intact skin, showing the stratum corneum to be the principal barrier to its penetration. The permeability coefficient of fentanyl for stripped hairless mouse skin was also raised, but stripping caused an inappreciable increase in the permeation rate of sufentanil. The thick dermis of excised mouse skin obviously offered a significant resistance to the permeation of these lipophilic compounds. In comparison, the permeability coefficients of fentanyl and sufentanil through stripped cadaver epidermis (n > or = 25) were 67 and 37 higher than for intact human epidermis, respectively. The skin metabolism of the narcotics was investigated. No significant metabolic degradation of morphine, fentnayl, and sufentanil was observed in either fresh human cadaver skin or hairless mouse skin homogenates in the presence of NADPH cofactor, suggesting a low monooxygenase enzyme presence in skin. Moreover, no measurable glucuronidation of morphine took place in human skin or hairless mouse skin. Both processes proceeded rapidly in liver homogenates (mouse) under identical circumstances. It thus appears that these drugs pass through in intact form.     

Solitary splenic metastasis of an adenocarcinoma of the ovaries

BACKGROUND: Topical application of inhibitors of HMGCoA reductase, the rate-limiting enzyme of cholesterol synthesis, has been shown to induce impairment of barrier function. OBJECTIVE: Assessing whether oral administration of statins used for reducing blood levels of cholesterol induces functional changes in stratum corneum barrier. MATERIALS AND METHODS: 69 subjects of both sexes under-going treatment for hypercholesterolemia (mean age 48 +/- 11 years) entered the study; 43 had been treated with simvastatin and 11 with pravastatin for 6 months; 15 only on dietary regimen served as controls. Efficiency of stratum corneum water barrier was evaluated by transepidermal water loss (TEWL) measurement using an evaporimeter; water-holding capacity of the stratum corneum was assessed by the sorption-desorption test measured by capacitance. Statistical analysis was performed using ANOVA. RESULTS: No differences were found between the groups (simvastatin, pravastatin, diet) concerning both basal TEWL and the dynamic of water binding in the stratum corneum. CONCLUSIONS: Prolonged treatment with cholesterol-lowering drugs based on inhibition of HMGCoA reductase does not alter the permeability barrier of the skin.     

X-ray diffraction analysis of isolated skin lipids: reconstitution of intercellular lipid domains

Low- and wide-angle X-ray diffraction were used to determine the structural organization of lipids isolated from the stratum corneum extracellular matrix that forms the major water permeability barrier in mammalian epidermis. Hydrated pig skin ceramides gave a single low-angle reflection of about 62 angstroms and a wide-angle-reflection at 4.15 angstroms. The addition of either cholesterol or fatty acid, the other major lipid components of the skin stratum corneum extracellular matrix, modified this diffraction pattern, depending on the lipid mole ratios. In the absence of water, lipid mixtures exhibited lipid phase separation, as shown by low- and wide-angle reflections typical of a separate cholesterol phase. However, a hydrated 2:1:1 mole ratio of ceramide:cholesterol:palmitic acid (similar to that found in stratum corneum) produced a diffraction pattern with a single sharp wide-angle reflection at 4.10 angstroms and low-angle reflections which indexed as the first eight orders of a single repeat period of 130 angstroms. The repeat period and intensity distribution of the low-angle data were similar to those found in intact stratum corneum [White et al. (1988) Biochemistry 27, 3725-3732; Bouwstra et al. (1994) Biochim. Biophys. Acta 1212, 183-192]. Higher concentrations of cholesterol or palmitic acid resulted in lipid phase separations. The 130 angstrom repeat period decreased only about 3 angstroms as water was removed by incubation in low-relative humidity atmospheres. The 130 angstrom repeat period depended on the presence of a particular ceramide, N-(omega-acyloxy)-acylsphingosine, which is found only in the epidermis. In contrast, 2:1:1 mixtures of brain ceramide:cholesterol:palmitic acid gave reflections of 56 and 34 angstroms. These results indicate that a structure with dimensions similar to those of the lamellar repeating unit found in skin stratum corneum does not depend on the presence of protein but does depend on the presence of specific skin ceramides and appropriate concentrations of cholesterol and fatty acid.     

Spatially periodic orbits in coupled-map lattices

We believe that it is unlikely that intact liposomes can cross the stratum corneum and act as carriers of drugs through the skin. However, it is probable that liposomes have the potential to modify the cutaneous biodistribution of applied chemicals (e.g., drugs, toxins, humectants) and thus modify their activity. Liposomal lipids can also penetrate the skin barrier. The effect of this is likely to be varied and lipid dependent, but this phenomenon could hold the most potential for the application of liposomes to topical therapy.     

Systemic vascular resistance in intradialytic hypotension determined by means of impedance cardiography

Topical corticosteroids (TCS) are among the most frequently used topical therapeutics. Recently, it has been shown that TCS not only has antiproliferative actions, but also inhibits the differentiation of the epidermis and finally perturbates stratum corneum (s.c.) barrier function. It is well established that epidermal barrier function resides within the intercellular lipids of the SC. However, to date, little is known about the effects of TCS on the structure and composition of s.c. lipids. We therefore used hairless mouse skin to study the sequential changes of the s.c. permeability barrier and their intercellular lipids by ruthenium tetroxide staining and high-performance thin-layer chromatography (HPTLC) during topical use of corticosteroids. The results demonstrated a progressive increase in transepidermal water loss accompanied by a diminution in the SC intercellular lipid lamellae, which showed a normal structure of individual lamella. Analysis of lipid composition by HPTLC after a 6-week application of TCS also showed an obvious decrease in all the main components of s.c. lipids, which are known to constitute the permeability barrier of the skin. In light of these results, our work provides direct morphological evidence that TCS deteriorates the permeability barrier of epidermis when applied to normal skin.     

Ultrasonically enhanced transdermal drug delivery. Experimental approaches to elucidate the mechanism

The effect of therapeutic range ultrasound on skin permeability was studied in vitro. Permeating molecule ionization state, pH, ultrasound duration, reversibility of the enhancement phenomenon, and skin structural alterations were evaluated. It was found that ultrasound affects the permeability of both ionized and unionized molecules. No irreversible structural alterations due to the ultrasound exposure were detected in the stratum corneum. Ultrasound enhancing mechanism was discussed.     

Testosterone skin permeation enhancement by menthol through formation of eutectic with drug and interaction with skin lipids

The aim of this investigation was to elucidate the mechanism of skin permeation enhancement of the lipophilic drug, testosterone, by menthol. Menthol was found to form eutectic mixtures with testosterone, cholesteryl oleate, and ceramides. DSC measurements showed that menthol drastically lowers the Tm of testosterone, from 153.7 to 39.9 degrees C. The decrease in Tm resulted in an increase in the solubility of testosterone in an aqueous ethanol vehicle by 2.8-fold, which caused a corresponding 2.8-fold increase in the flux of testosterone. A further increase in skin flux, to eight times the base line, could be attributed to the effect of menthol on the skin barrier properties. This assumption is supported by DSC results showing that menthol decreases the Tm of cholesteryl oleate and ceramides and modifies the thermogram profile of isolated stratum corneum. The results of this investigation indicate that menthol affects skin permeation by a dual mechanism: by forming a eutectic with the penetrating compound, thereby increasing its solubility, and by altering the barrier properties of the stratum corneum. Moreover, this study indicates that both types of interactions must be taken into consideration when using chemical enhancers and that decreasing the melting temperature of the permeant through formation of a eutectic could be one approach for increasing solubility and permeation rates.     

In vitro percutaneous absorption enhancement of a lipophilic drug tamoxifen by terpenes

Tamoxifen is a highly lipophilic drug that is widely used in breast malignancies and also as a prophylactic therapy in women at high risk for the development of this disease. Recently, the terpenes have been reported to show an enhancement effect on percutaneous drug absorption. The effect of terpenes (e.g. carvone, 1,8-cineole, menthol, and thymol) was studied on the in vitro percutaneous absorption of tamoxifen through porcine epidermis. The above terpenes (5% w/v) in combination with 50% ethanol significantly (P < 0.01) increased the permeability coefficient of tamoxifen in comparison to the control (50% ethanol). The solubility of tamoxifen was determined in the control and enhancer solutions to correct the permeability enhancement by way of fractional solubility adjustment. Binding of tamoxifen to powdered stratum corneum from control and enhancer solutions was also determined. Binding studies reveal that the enhancement in the permeability coefficient of tamoxifen by menthol and thymol is due at least in part, to improvement in the partitioning of the drug to the stratum corneum. In conclusion, terpenes in combination with ethanol can be used to enhance the percutaneous absorption of the highly lipophilic drug tamoxifen.     

Permeabilities of alkyl p-aminobenzoates through living skin equivalent and cadaver skin

The in vitro permeabilities of alkyl p-aminobenzoates through living skin equivalent (LSE) and cadaver skin were compared. Methyl, ethyl, and butyl p-aminobenzoates were used as model compounds. The permeabilities of these compounds through LSE and cadaver skin from an aqueous drug suspension were determined with a flow-through diffusion cell. The permeability coefficients of these esters in LSE were an order of magnitude higher than in cadaver skin. This was primarily because of low resistances offered by the outermost layer (i.e., stratum corneum) of LSE. In the case of cadaver skin, the permeability coefficient increased as the carbon chain length increased, whereas no appreciable change in the permeability coefficients of these esters in LSE was observed. These results clearly suggest that the LSE membrane offered very little resistance as opposed to cadaver skin. Therefore, the LSE membrane may not quantitatively represent a good human skin model for evaluating skin permeation of a drug from topical or transdermal formulations.     

Functional characterisation of tachykinin receptors mediating ion transport in porcine jejunum

Reconstructed human skin was prepared from human keratinoblasts. After 1 week of cultivation at the air-liquid interface a stratified layer developed, similar to native human epidermis. Liposomes with an average diameter of 50 nm, made of phosphatidylcholine (PC), phosphatidylserine (PS) and human stratum corneum lipids (hSCL) were applied on top of this culture system. The rate of penetration through the reconstructed human epidermis was 1.38, 0.55 and 0.013 ng lipidh-1cm-2 for PC, hSCL and PS liposomes, respectively. Electron microscopy and confocal laser scanning microscopy showed that PS and hSCL liposomes aggregated at the skin surface, while PC liposomes remained homogeneously dispersed. Fluorescence measurements demonstrated that vesicles, made of native human stratum corneum lipids rapidly mixed with PS liposomes, weakly with hSCL liposomes and did not mix with PC liposomes.     

Stratum corneum lipids: the effect of ageing and the seasons

Stratum corneum lipids play a predominant role in maintaining the water barrier of the skin. In order to understand the biological variation in the levels and composition of ceramides, ceramide 1 subtypes, cholesterol and fatty acids, stratum corneum lipids collected from tape strippings from three body sites (face, hand, leg) of female Caucasians of different age groups were analysed. In addition, we studied the influence of seasonal variation on the lipid composition of stratum corneum from the same body sites. The main lipid species were quantified using high-performance thin-layer chromatography and individual fatty acids using gas chromatography. Our findings demonstrated significantly decreased levels of all major lipid species, in particular ceramides, with increasing age. Similarly, the stratum corneum lipid levels of all the body sites examined were dramatically depleted in winter compared with spring and summer. The relative levels of ceramide 1 linoleate were also depleted in winter and in aged skin whereas ceramide 1 oleate levels increased. The other fatty acid levels remained fairly constant with both season and age, apart from lignoceric and heptadecanoic acid which showed a decrease in winter compared with summer. The decrease in the mass levels of intercellular lipids and the altered ratios of fatty acids esterified to ceramide 1, are likely to contribute to the increased susceptibility of aged skin to perturbation of barrier function and xerosis, particularly during the winter months.     

HLA-DP-associated susceptibility to the optico-spinal form of multiple sclerosis in the Japanese

Ceramides are the major lipid components of the stratum corneum, the major permeability barrier of the skin. Here we report a chemical synthesis of ceramide analogs covalently bonded on the silica particles, that can be used to predict the skin permeability of chemicals via HPLC methods.     

Ultrastructural study of the skin after facial chemical peels and the effect of moisturization on wound healing

Ultrastructural changes occurring in the skin at early times after chemical peels as well as effects on the wound healing with moisturization after these peels have been examined. This study evaluated the changes seen in the skin 3 days and 5 days after 35% trichloroacetic acid peels, and the effect of moisturization on this healing was evaluated. Biopsies at 3 days showed an outermost layer of necrotic stratum corneum and stratum granulosum and an underlying layer of new stratum corneum. There were increased cytoplasmic vacuoles in the keratinocytes of the stratum spinosum, stratum granulosum, and stratum basale layers. There was extensive intercellular spacing between the basal keratinocytes. At 5 days the necrotic layer of stratum corneum and stratum granulosum was gone. The lower epidermis at 5 days showed less intercellular spacing, and there was less vacuolization within keratinocytes. In seven of eight patients treated with moisturization after the peel (p = 0.0325), the ultrastructural changes at 5 days were consistent with a more advanced state of healing compared with those that were treated dry. Ultrastructural morphology at this time showed less intercellular spacing and fewer cytoplasmic vacuoles, indicative of an advanced state of wound repair. These moisturized skin specimens had returned to an almost normal state of structure compared with the skin that had been treated dry.     

Entropy-driven binding/partition of amino acids/dipeptides to stratum corneum lipid vesicles

This study employed large unilamillar vesicles composed of purchased stratum corneum lipids to investigate the binding/partition of amino acids/dipeptides to stratum corneum lipid vesicles. The partition coefficients of amino acids/dipeptides between the stratum corneum lipid vesicles and the acetate buffer were determined by HPLC. In addition, the binding/partition enthalpy of amino acids/dipeptides with the stratum corneum lipid vesicles was derived by directly measuring the binding/partition heat with isothermal titration calorimetry. According to the binding/petition Gibbs free energy and the binding/partition enthalpy, all the binding/partition of amino acids/dipeptides with the stratum corneum lipid vesicles is endothermic, implying an entropy-driven binding/partition. Also, the equilibrium binding/partition results demonstrate that the partition coefficients of amino acids/dipeptides do not correlate with the transdermal permeability. This finding suggests that either the interaction between the penetrants and the lipid bilayer between corneocytes may not be a determining step or that the paracellular path is not a dominant route of transdermal penetration.     

Difference in the enhancing effects of ultrasound on the skin permeation of polar and non-polar drugs

The effect of ultrasound (150 kHz, 111 mW/cm2) on the permeability of isosorbide dinitrate (ISDN) and antipyrine (ANP) through excised hairless rat skin was evaluated using an Arrhenius plot. The permeability coefficients of ISDN across skin (at various temperatures) in the presence and absence of ultrasound were virtually isolinear on the Arrhenius plot. It has been suggested that the temporal increase in the ISDN flux, which was observed when ultrasound was applied in our previous study, was only a result of the thermal effect of ultrasound, i.e., an increase in the temperature of the donor solution. On the other hand, ultrasound influenced the Arrhenius plot of ANP, suggesting that the enhancement effect for ANP permeation could be not explained only by the thermal effect of ultrasound. In addition, the effective diffusion (D) and partition coefficients (K) of ISDN and ANP were estimated using their skin permeation profiles across the ultrasonic pretreated skin. The coefficients of ISDN with ultrasonic pretreatment were comparable to those without pretreatment. On the other hand, the D value of ANP with ultrasonic pretreatment was increased about 4 times by ultrasonic pretreatment, in spite of an insignificant change in the K value. These results suggest that the ultrasound used in the present study increased the effective diffusivity across the aqueous region in the stratum corneum to enhance the skin permeation of the polar compound, ANP.