The effects of serine palmitoyltransferase inhibitor, ISP-I, on UV-induced barrier disruption in the stratum corneum

We examined the effects of ISP-I (myriocin, thermozymocidin) - a potent inhibitor of serine palmitoyltransferase (SPT) which is involved in the ceramide synthetic pathway-on skin barrier function in post-UVB-irradiated hairless mouse skin. Disruption of the skin barrier function after UVB irradiation as represented by the increase in transepidermal water loss (TEWL) was significantly suppressed with ISP-I treatment. In the ISP-I-treated skin, the peak of cell proliferation was observed 24 h earlier than in vehicle-treated skin. In addition, the number of apoptotic cells in ISP-I-treated skin showed a sharp decrease at 48 and 72 h post-irradiation. The number of stratum corneum cell layers was increased in ISP-I-treated skin at 72 h after UVB irradiation; at this time, TEWL in ISP-I-treated skin was lower than that in the vehicle-treated skin. We suggest ISP-I treatment altered cell proliferation and apoptosis after UVB exposure by modulating ceramide synthesis in epidermal cells, resulting in an increase of stratum corneum layers which lessened the effects of irradiation-induced barrier disruption.     

Promoting mechanism of menthol derivative, 1-O-ethyl-3-buthylcyclohexanol, on the percutaneous absorption of ketoprofen

Menthol derivatives were synthesized and evaluated for their promoting activity on the percutaneous absorption of ketoprofen and skin irritation in vivo, choosing O-ethylmenthol (MET) as the mother compound. The compound having a C-3 positionned n-butyl group (1-O-ethyl-3-n-buthylcyclohexanol, OEBC) indicated the most promoting activity and caused relatively little skin irritation. In order to understand enhancement mechanism of OEBC an in vitro permeation study of ketoprofen was performed. The time course of the cumulative amounts of drug permeated through the rat skin exhibited a linear relation after an initial lag time. This was analyzed in membrane diffusion model and the diffusion and partition parameters of ketoprofen were estimated. Both parameters were remarkably enhanced when a hydrogel containing a small quantity of OEBC (0.5%) was applied. Furthermore, to clarify the site of action of OEBC, we also investigated in vitro permeation study of ketoprofen employing different skins of state, reversed skin and stratum corneum stripped skin. When OEBC was added to the hydrogels which were applied to the reversed and stripped skins, almost no changes of the flux were observed compared with the control (without OEBC). These results suggested that the site of action of OEBC was stratum corneum. Morphological changes of the stratum corneum surface were microscopically observed with 0-2% OEBC. The spaces between the stratum corneum cells treated with 0.5-2% OEBC became extended and the shape of each cell became clear. This may suggest that the site of action of OEBC was the intercellular of stratum corneum. Furthermore, an electron spin resonance study was performed to investigate the effect of OEBC on the intercellular lipid bilayer fluidity of the stratum corneum and the rotational correlation times were calculated. 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) were used as the spin label. In use of OEBC, the fluidity of TEMPO labeled the stratum corneum lipid increased as the addition of OEBC. The results suggested that OEBC promote the penetration of drugs by enhancing fluidity of the local lipid bilayers around TEMPO.     

黄芩苷渗透裸鼠皮肤的动力学研究

目的 :研究黄芩苷渗透裸鼠皮肤的动力学。方法 :以裸鼠皮肤为实验屏障 ,测定黄芩苷渗透裸鼠皮肤的速率常数。结果 :黄芩苷渗透裸鼠皮肤的动力学方程为Q=76 07T1/2—32 11;黄芩苷渗透去除角质层皮肤的动力学方程为Q=134 23T1/2+29 07。去除贴片后皮肤内仍有黄芩苷渗出。结论 :所制备黄芩苷贴片应属于骨架型。黄芩苷渗透皮肤的主要阻力来自于角质层 ,去除角质层的皮肤对黄芩苷有一定的贮库效应     

Methods for in vitro percutaneous absorption studies II. Animal models for human skin

The percutaneous absorption of compounds through the skin of selected animal species was compared to that occurring with human skin, using in vitro diffusion cell techniques. The permeability of back skin of the following animals was examined: Hormel miniature pigs, Osborne-Mendel rats, NIH hairless mice, and Swiss mice. Benzoic acid, acetylsalicylic acid, and urea, dissolved in a petrolatum vehicle, were applied and permeability constants were determined. With the faster penetrating compounds, benzoic acid and acetylsalicylic acid, mouse skin and hairless mouse skin were similar and were the most permeable. Little difference was observed in acetylsalicylic acid absorption of human, pig, and rat skin. Pig skin and hairless mouse skin were the best animal models for the slower penetrating urea. The thicknesses of the stratum corneum, epidermis, and whole skin were determined from microtome sections prepared from frozen skin. In addition, the density of hair follicles in each type of skin was determined. In general terms, thickness of the stratum corneum was found to be important: the thickest stratum corneum was that of pig skin and the thinnest was that of the mouse. Often however, differences in permeability and stratum corneum thickness did not correlate on a 1:1 basis, which can be indicative of differences in structure of the different types of skin. The animal model of choice is dependent on the compound. For benzoic acid and acetylsalicylic acid, the pig and rat are good models for human skin. In the case of slow absorbing compounds, such as urea, diffusion through appendages in the skin may make undesirable the use of skin of densely haired animals.     

Deposition of viprostol (a synthetic PGE2 vasodilator) in the skin following topical administration to laboratory animals

1. Topical application of 14C-viprostol, a synthetic prostaglandin E2 analogue, to laboratory animals resulted in a significant depot of radioactivity in the skin at the application site in all species studied: mouse, rat, guinea pig, rabbit and monkey, with longer residence times in the larger species. 2. The location of the 14C-label in the skin in mice and monkeys was determined by microscopic autoradiography. Evaluation of the autoradiograms show rapid penetration of the drug into the skin via the hair follicles. 3. In mouse distribution of radioactivity was evident in the stratum corneum and down the hair shafts by 30 min. after dosing. By 2 h radioactivity was also observed throughout the viable epidermis; in the dermis only the hair shafts contained significant radioactivity. At 72 h after dose removal, radioactivity was evident only in the hair follicles and hair shaft. 4. In monkey the residence time of radioactivity in the skin was significantly longer than in mouse, but the general distribution pattern was similar in both species. 5. The presence of viprostol in the hair follicles and epidermal layer after topical administration is consistent with its extensive skin metabolism previously reported.     

花椒毒素在人体皮肤及角质层中的渗透动力学探讨

目的探讨花椒毒素在人体完整皮肤及其角质层中的渗透动力学特性。方法采用人体离体皮肤及从完整皮肤中分离出的角质层，在Franz-cell扩散池中进行不同浓度的花椒毒素乙醇溶液(0.1, 0.5, 2.5和5.0 mg·mL^-1)的渗透实验，均采用1.4%的人血清溶液作为接收液，用HPLC法测定各接收液的药物量和皮肤中的贮存药物量。结果花椒毒素乙醇溶液单位面积透过完整人皮肤速率随着浓度增加而增加，2.5 mg·mL^-1以上的浓度对其速率几乎无影响。在角质层中也存在同样的现象，但其单位面积透皮速率最大值提前了约6 h。而且随着给药浓度的增加，24 h后完整皮肤和角质层的药物贮存量也相应增加，但达到一定浓度后存在饱和现象。且花椒毒素乙醇溶液在完整皮肤中的透皮时滞(0.82 h)明显大于在角质层中的透皮时滞(0.47 h)。结论该结果对开发花椒毒素外用制剂时浓度的选择提供了依据，并对其外用药物后照射长波段紫外光(UVA)的时间提供了参考。     

Does Epidermal Turnover Reduce Percutaneous Penetration?

Purpose. After its removal from the skin surface, chemical remaining within the skin can become systemically available. The fraction of chemical in the skin that eventually enters the body depends on the relative rates of percutaneous transport and epidermal turnover (i.e., stratum corneum desquamation). Indeed, some investigators have claimed that desquamation is an efficient mechanism for eliminating dermally absorbed chemical from the skin. Methods. The fate of chemical within the skin following chemical contact was examined using a mathematical model representing turnover of and absorption into the stratum corneum and viable epidermis. The effects of turnover rate, exposure duration, penetrant lipophilicity, and lag time for chemical diffusion were explored. Results. These calculations show that significant amounts of chemical can be removed from skin by desquamation if epidermal turnover is fast relative to chemical diffusion through the stratum corneum. However, except for highly lipophilic and/or high molecular weight (>350 Da) chemicals, the normal epidermal turnover rate is not fast enough and most of the chemical in the skin at the end of an exposure will enter the body. Conclusions. Epidermal turnover can significantly reduce subsequent chemical absorption into the systemic circulation only for highly lipophilic or high molecular weight chemicals.     

Percutaneous absorption of corticosteroids: age, site, and skin-sectioning influences on rates of permeation of hairless mouse skin by hydrocortisone

As part of a long-range plan to decipher the mechanism of mass transfer of corticosteroids across skin, the permeation of [3H]hydrocortisone through hairless mouse skin was characterized by in vitro diffusion cell techniques. Age and anatomical site-related behaviors were explored mainly with whole skin but also with skin stripped of its horny layer. Permeability of the mouse skin was low shortly after birth and increased during the singular normal hair cycle exhibited by the SKH-hr-1 mouse strain. The return to a hairless state over a period of 20-35 d was accompanied by decreased permeability of hydrocortisone. A permeability coefficient of approximately 2 X 10(-4) cm/h was found for the mature mouse. For whole skin, there was no difference in the permeabilities of dorsal and abdominal skin sections, but for stratum corneum-free membranes and dermal membranes, the abdominal site appeared to be more permeable, which is consistent with its thinner dimensions. Totally stripped skin and isolated dermis are approximately 500 and approximately 1000 times more permeable than intact skin, respectively; the unaltered stratum corneum of the hairless mouse is thus shown to be the major barrier to the mass transfer of hydrocortisone.     

Deposition of viprostol (a synthetic PGE2 vasodilator) in the skin following topical administration to laboratory animals

1. Topical application of ¹⁴C-viprostol, a synthetic prostaglandin E₂ analogue, to laboratory animals resulted in a significant depot of radioactivity in the skin at the application site in all species studied: mouse, rat, guinea pig, rabbit and monkey, with longer residence times in the larger species.

2. The location of the ¹⁴C-label in the skin in mice and monkeys was determined by microscopic autoradiography. Evaluation of the autoradiograms show rapid penetration of the drug into the skin via the hair follicles.

3. In mouse distribution of radioactivity was evident in the stratum corneum and down the hair shafts by 30?min. after dosing. By 2?h radioactivity was also observed throughout the viable epidermis; in the dermis only the hair shafts contained significant radioactivity. At 72?h after dose removal, radioactivity was evident only in the hair follicles and hair shaft.

4. In monkey the residence time of radioactivity in the skin was significantly longer than in mouse, but the general distribution pattern was similar in both species.

5. The presence of viprostol in the hair follicles and epidermal layer after topical administration is consistent with its extensive skin metabolism previously reported.     

The effects of an alpha hydroxy acid (glycolic acid) on hairless guinea pig skin permeability.

The barrier integrity of hairless guinea pig skin after treatment with an alpha hydroxy acid was assessed through in vivo topical application of an oil-in-water emulsion containing 5 or 10% glycolic acid at pH 3.0. The control was a commercial moisturizing lotion, pH 7.8. A dosing regimen for the glycolic acid formulations that was tolerated by the hairless guinea pigs and significantly decreased stratum corneum turnover time was determined using the dansyl chloride staining technique. Once-daily dosing of hairless guinea pig skin for 3 weeks with the glycolic acid formulations resulted in approximately a 36-39% decrease in stratum corneum turnover time compared with the control lotion. After this treatment, hairless guinea pigs were sacrificed for the in vitro measurement of the percutaneous absorption of [14C]hydroquinone and [14C]musk xylol. No significant differences in the 24-hour absorption of either test compound were found for skin treated with the control lotion or the glycolic acid formulations. There were also no significant differences found in the absorption of [3H]water through skin from the different treatment groups. Although no increase in skin penetration occurred after treatment with the glycolic acid formulations, histology revealed approximately a twofold increase in epidermal thickness. Also the number of nucleated cell layers nearly doubled in skin treated with 5% and 10% glycolic acid compared with the control lotion and untreated skin. These studies demonstrate that substantial changes in the structure of hairless guinea pig epidermis can occur without significant effect on skin permeability of two model compounds.     

Correlation between stratum corneum/water-partition coefficient and amounts of flufenamic acid penetrated into the stratum corneum

The stratum corneum of various donors differs in particular in the composition of the lipoidal phase. Considering the drug amounts penetrating into the stratum corneum a simple methodology to correlate these differences in the stratum corneum composition with the drug amounts detectable within the stratum corneum is desirable. Penetration experiments investigating several incubation times were carried out with three different skin flaps using the Saarbruecken penetration model and the lipophilic model drug flufenamic acid. The drug amounts within the stratum corneum were obtained with the tape-stripping technique, while the drug amounts present in the deeper skin layers were achieved by cryosectioning. The stratum corneum/water-partition coefficient was determined with the same three skin flaps to characterize the lipoidal stratum corneum phase in general, and the differences were attributed to the different amounts of ceramides and sterols. In addition, for the lipophilic drug flufenamic acid, a direct linear correlation was found between the stratum corneum/water-partition coefficients and the drug amounts penetrated into the stratum corneum for all investigated time intervals (correlation coefficients of r(30 min) = 0.998, r(60 min) = 0.998 and r(180 min) = 0.987). In contrast to the stratum corneum/water-partition coefficients, the determination of a corresponding relationship for the stratum corneum and the deeper skin layers failed due to the reason that steady-state conditions could not be achieved for the deeper skin layers during the investigated time intervals. In summary, the stratum corneum/water-partition coefficients offer the possibility to predict drug amounts within the stratum corneum of different donor skin flaps without a time consuming determination of the lipid composition of the stratum corneum.     

Fate of fluazifop butyl in rat and human skin in vitro

Enzyme mediated hydrolysis of fluazifop butyl has been measured with rat and human skin post-mitochondrial fractions. Rat skin had a ten times greater capacity to metabolise fluazifop butyl than human skin, but the enzyme affinities were similar. The post-mitochondrial fraction metabolism was compared to that seen during absorption in a flow through diffusion cell with viable skin. Limited hydrolysis of absorbed fluazifop butyl was seen in rat skin, but increased two fold if the stratum corneum was removed. The stratum corneum was found to retain fluazifop butyl. When the skin was pre-incubated with the esterase inhibitor bis (p-nitrophenol) phosphate (BNPP), reduced metabolism was seen. No metabolism of fluazifop butyl was seen in human skin during absorption. Retention of the compound by the stratum corneum is postulated to restrict the accessibility of the compound to the enzyme site, thus influencing the observed metabolism during the absorption process.     

Mechanism of urea effect on percutaneous absorption of clonidine

The urea effect on skin permeation of clonidine was investigated to reduce a log time and to increase a permeability. ICR mouse skin and human skin were used and were assumed to be a two-layer membrane consisted of stratum corneum and viable epidermis. The urea acted as a skin denaturant and humectant in the whole epidermis. Also it enhanced the skin permeability of clonidine about 3.5 times. On the other hand, it enhanced the skin permeability by acting as a humectant in the viable epidermis. But the urea effect on the whole epidermis was shown to be greater than that on the viable epidermis. Therefore, it was found that the effect of urea was greater on the stratum corneum than the viable epidermis. Variation of enhancing effect according to the concentration of urea was not found in the range of 1% to 20%.     

Pharmacological activity of natural lipids on a skin barrier disruption model.

The lipids of the stratum corneum are considered responsible for the most important functions of the skin, such as the transepidermal water loss, as well as the transdermal penetration of the chemical substances. Topical application of lipids similar to the physiological stratum corneum (SC) on barrier disrupted skin, could enhance the recovery rate of the skin barrier. A mixture of natural lipids or liposomes with the same lipid composition, were applied and their pharmacological action was investigated. The tests were done in vivo, on the back of hairless mice. Comparative results were obtained and showed that the liposomes had a higher turnover of the skin barrier in contrast to that of the mechanical mixture of lipids.     

Feasibility of Measuring the Bioavailability of Topical Betamethasone Dipropionate in Commercial Formulations Using Drug Content in Skin and a Skin Blanching Bioassay

An in vivo technique has been developed which simultaneously compares a skin blanching bioassay with drug content in human stratum corneum following topical application of four 0.05% beta-methasone dipropionate formulations. Bioavailability of drug from commercial cream and ointment formulations was assessed by quantification of drug content in tape-stripped stratum corneum and skin blanching in the treated skin site under occluded conditions. Tape-stripping removed stratum corneum to a varying degree between individuals but was consistent (35%) within an individual with all formulations, day to day. A correlation (r = 0.9935) between the amount of drug in the treated stratum corneum normalized for surface area and the corresponding skin blanching score was observed with four 0.05% betamethasone dipropionate formulations. Increasing the amount of drug in the tape-stripped stratum corneum correlated with an increased skin blanching score. Ointment formulations delivered more drug to the skin and produced greater blanching scores than the cream formulations. Topical corticosteroid content in the treated skin site can therefore be quantified and correlates well with the resulting pharmacodynamic activity.     

Enhanced transdermal delivery of triprolidine from the ethylene-vinyl acetate matrix.

Triprolidine-containing matrix was fabricated with ethylene-vinyl acetate (EVA) copolymer to control the release of the drug. The permeation rate of triprolidine in the stripped skin was greatly larger than that in the whole skin. Thus it showed that the stratum corneum acts as a barrier of skin permeation. The effect of penetration enhancer and stripping of skin on the permeation of triprolidine through the excised mouse skin was studied. Penetrating enhancers showed increased flux probably due to the enhancing effect on the skin barrier, the stratum corneum. Among enhancers used such as glycols, fatty acids and non-ionic surfactants, polyoxyethylene-2-oleyl ether showed the best enhancement. The permeability of triprolidine was markedly increased with stripping of the mouse skin to remove the stratum corneum that acts as a barrier of skin permeation. For the controlling transdermal delivery of triprolidine, the application of EVA membrane containing permeation enhancer could be useful in the development of transdermal drug delivery system.     

Changes in electrophysiological properties of rat skin with age

The age-related changes in the electrical and physiological properties of the skin were examined in rats at the ages of 5, 10, 21, 90, and 180 d. The resistance of the stratum corneum, the resistance of the viable skin (epidermis and dermis), and the capacitance of the stratum corneum were analyzed from skin impedance data using an equivalent circuit. With development and aging, the resistance of the stratum corneum and the viable skin increased, whereas the capacitance of the stratum corneum decreased. Physiological characteristics such as the thickness of skin strata and the content of lipid and water in the stratum corneum were also measured. The lipid content in the stratum corneum was constant at all ages. The water content in the stratum corneum decreased, and the thickness of skin strata increased with age. Comparison between electrical data and physiological properties suggested that the increase in the resistance of the stratum corneum with aging is primarily caused by the decrease in the water content and that the capacitance of the stratum corneum and the resistance of the viable skin depend on age-related increases in the thickness of skin strata. In conclusion, the age dependency of cutaneous electrical properties may affect the permeation profile of drugs through the skin, and impedance analysis can be used to estimate age-related changes in transdermal drug delivery.     

Effect of dipolar aprotic permeability enhancers on the basal stratum corneum

The effect of dimethyl sulfoxide, dimethyl formamide, and dimethyl acetamide on the basal stratum corneum of excised nude mouse skin was investigated. All of these dipolar aprotic solvents caused a swelling of the basal stratum corneum cells and a disruption of the normal keratin pattern. This behavior suggests that dipolar aprotic solvents might alter the barrier properties of the basal stratum corneum cells. To test this hypothesis, the distribution of topically applied, electron-dense divalent metal ions (Hg2+ and Ni2+) was studied in excised nude mouse skin which had been perturbed by the application of dipolar aprotic solvents, and in controls which had not been so treated. In control skin membranes, Hg2+ and Ni2+ were located almost exclusively in the intercellular space of the stratum corneum. However, with the application of a dipolar aprotic solvent, Hg2+ and Ni2+ were found in the intercellular spaces and inside the basal stratum corneum cells, where they appeared to be primarily associated with the cytoplasmic filaments. Sulfide precipitation allowed for the localization of Hg2+ and Ni2+, and subsequent chemical identification by energy-dispersive X-ray microanalysis. The spatial resolution of X-ray microanalysis studies was approximately 0.5-0.75 micron. The spatial alteration in mercury and nickel precipitate distribution, which occurs when the skin is pretreated with a dipolar aprotic solvent, is consistent with the hypothesis that the pathway of Hg2+ and Ni2+ diffusion through the basal stratum corneum has also been modified.     

Effects of isopropanol-isopropyl myristate binary enhancers on in vitro transport of estradiol in human epidermis: a mechanistic evaluation

The purpose of this study was to mechanistically investigate effects of isopropanol (IPA)-isopropyl myristate (IPM) binary enhancers on transport of a model drug, estradiol (E2) in human epidermis (stratum corneum + viable epidermis) in vitro. The study was focused on use of the same IPA-IPM compositions on both sides of the skin ("symmetric" configuration) with saturated E2 (maximum thermodynamic activity). For E2 transport in all IPA-IPM compositions tested, stratum corneum still was the rate-limiting layer of human epidermis. The relative contributions to E2 flux enhancement were separated into the changes in solubility and diffusivity of E2 in stratum corneum. As a major factor, E2 solubility in stratum corneum was enhanced by 35 times with increasing IPA from neat IPM to neat IPA. E2 diffusivity in stratum corneum also played a significant role, which increased by 8 times from neat IPM to 50% IPA. Stratum corneum swelled more in IPM-rich region, decreased with increasing IPA, and even deswelled in neat IPA. IPA uptake correlated well to E2 solubility in stratum corneum; both linearly increased with increasing IPA. IPM uptake appeared to correlate to E2 diffusivity in stratum corneum; both maximized around 50% IPA.     

Irritant contact dermatitis: effect of age

Knowledge on age-relationship to irritant contact dermatitis (ICD) remains of interest. We searched for articles and textbooks on age-relationship to ICD and evaluated relevant data. Irritant response may be enhanced in children and decline with increasing age. In general, older skin reacts more slowly and with less intensity to irritants when compared with young skin. Such age-related changes may depend: (i) on differences in percutaneous penetration in old and young skin, and/or on (ii) differences in the microcirculatory efficiency, which serves as the route by which inflammatory cells make their way to the site of inflammation. Additionally, stratum corneum turnover time increases with age which means that an irritant remains longer on the skin; a compromised cutaneous blood vessel network with ageing may lead to a decreased inflammatory response, decreased absorption and decreased clearance. In conclusion, age-related differences of ICD are present despite some conflicting data. Investigations elucidating this interesting subject may benefit in prevention and intervention strategies.