Percutaneous penetration of squaric acid and its esters in hairless mouse and human skin in vitro

Summary Squaric acid diethylester and squaric acid dibutylester have been used in contact sensitization therapy of alopecia areata. This study investigated the application of these esters or squaric acid alone to hairless mouse and human skin in vitro to determine squaric acid flux from the various preparations. Measurable amounts of squaric acid were delivered through skin by squaric acid itself, but flux was lower than for that delivered by the two esters. These results support the proposal by Noster that the esters combine with a protein to form an antigen while squaric acid can not and that this explains why the esters are active in contact sensitization and the acid is not. We suggest that the results of previous studies showing that the diethyl ester of squaric acid was a less effective sensitizer than the dibutyl ester may have been due to decomposition of the ethyl ester to squaric acid.     

The percutaneous penetration of nandrolone decanoate.

A study has been carried out on the penetration of nandrolone decanoate through human cadaver skin in vitro using radioactive tracer material. The radiochemical purity of the material desorbed from the dermal skin surface is very much less than that of the material absorbed into the epidermal surface. These observations are a consequence of the low level of overall penetration of the skin by the steroid. There is also a possibility that a small percentage of the steroid is being metabolized by micro-organisms present in the skin samples.     

Factors affecting the attachment of Treponema pallidum to mammalian cells in vitro.

Attachment of Treponema pallidum (Nichols) to mammalian cells is probably the first step in the pathogenesis of syphilis. It may also be important for the multiplication of T pallidum in vitro. When factors affecting the attachment of T pallidum to mammalian cells in vitro were studied significantly greater numbers of treponemes were found to attach to baby rabbit genital organ (BRGO) cells than to five other mammalian cell lines. When attached to BRGO cells T pallidum survived longer in vitro than unattached treponemes. Eagle's minimal essential medium was superior to three other culture media in increasing attachment and maintaining the survival of treponemes. Dithiothreitol (0.25-1.0 mmol/l) had no effect on the attachment of T pallidum to BRGO cells. Anaerobic conditions were superior to microaerophilic conditions, and the latter were superior to aerobic conditions for the attachment and survival of T pallidum to BRGO cells. Within the range of concentrations tested the number of treponemes attached to the BRGO cells was directly dependent on the concentrations of viable treponemes in the inoculum. Greater numbers of treponemes attached to actively metabolising BRGO cells than to quiescent or slowly growing cells.     

In vitro kinetics of 8-methoxypsoralen penetration into human lymphoid cells.

Extracorporeal photochemotherapy (ECPC) requires ex vivo UVA irradiation of blood lymphocytes during the time of the theoretical peak 8-methoxsalen (8-MOP) concentration. The aims of this study were to determine the mechanism of cellular uptake of 8-MOP, its possible saturation and the time needed to reach maximal concentration (Tmax) in lymphoid cells. 8-MOP was measured by liquid chromatography in the supernatant of lymphoid cell suspensions incubated with a known amount of 8-MOP. The kinetics of cellular uptake were determined and showed that equilibrium had already been reached after 2 min and remained constant for at least 60 min. The uptake was independent of temperature (4, 25 and 37 degrees C) and was proportional to the 8-MOP concentration in the supernatant. This indicated that 8-MOP penetrated into lymphoid cells by passive diffusion, rather than by active transport or facilitated diffusion, and was thus a non-saturable process. In addition, intracellular metabolism was negligible. These findings demonstrated that the plasma and lymphocytic Tmax were reached simultaneously and statistical analysis showed them to be significantly correlated, thereby validating the standard ECPC protocol for drug ingestion and lymphocyte irradiation.     

In vitro percutaneous penetration: evaluation of the utility of hairless mouse skin

The permeability barrier of hairless mouse skin has been determined in vitro after exposure of the epidermal surface to volumes of acetone typically used in human in vivo skin penetration studies. It has been shown that the transport of tritiated water (when applied for limited 5-h periods) across hairless mouse skin is not affected by acetone treatments of approximately 15 microliters/cm2. Submersion of the membranes between aqueous donor and receptor phases for periods greater than 24 h, however, leads to significant and catastrophic barrier impairment. The acetone dose in the experiments reported is greater than that employed in vivo when the solvent is used to deposit a penetrant on human skin. We suggest, therefore, that acetone-mediated facilitation of percutaneous absorption in humans is unlikely. A further conclusion of this work is that in vitro solvent-deposition penetration experiments using hairless mouse skin should provide reliable transport information for at least 48 h postadministration. Although hairless mouse skin is more permeable than its human counterpart, in vitro measurements using the murine barrier should, therefore, provide useful and relevant guidelines for risk assessment calculations and bioavailability determinations.     

Lead in human scalp hair: some factors affecting its variability.

The accumulation of lead in human scalp hair was compared in male and female children and adults from various locations in the United Sates, Japan, Yugoslavia, Iran, and South Africa. The most significant variables which influenced the concentration of lead in hair were ingestion of lead-containing substances, exposure to lead of environmental origin, place of residence, site from which the hair specimen was sampled relative to its distance from the scalp, and age. The least significant variables were sex and nutritional deficiencies.     

Factors affecting the attachment of Treponema pallidum to mammalian cells in vitro

Attachment of Treponema pallidum (Nichols) to mammalian cells is probably the first step in the pathogenesis of syphilis. It may also be important for the multiplication of T pallidum in vitro. When factors affecting the attachment of T pallidum to mammalian cells in vitro were studied significantly greater numbers of treponemes were found to attach to baby rabbit genital organ (BRGO) cells than to five other mammalian cell lines. When attached to BRGO cells T pallidum survived longer in vitro than unattached treponemes. Eagle's minimal essential medium was superior to three other culture media in increasing attachment and maintaining the survival of treponemes. Dithiothreitol (0.25-1.0 mmol/l) had no effect on the attachment of T pallidum to BRGO cells. Anaerobic conditions were superior to microaerophilic conditions, and the latter were superior to aerobic conditions for the attachment and survival of T pallidum to BRGO cells. Within the range of concentrations tested the number of treponemes attached to the BRGO cells was directly dependent on the concentrations of viable treponemes in the inoculum. Greater numbers of treponemes attached to actively metabolising BRGO cells than to quiescent or slowly growing cells.     

In vitro regeneration of the barrier to water penetration in human epidermis

A method is presented for simultaneous in vitro growth and measurement of permeability of human skin. During culture of specimens, the parakeratotic layer formed is shown to possess considerable barrier function. Maximum barrier capacity of this layer in vitro is attained at 14–17 days’ incubation. When the stratum corneum is removed prior to incubation, the maximum barrier capacity is reached in 3–4 days. The difference in behaviour between intact and stripped skin is reflected in the pattern of DNA synthesis of the basal cells, which show an enhanced proliferation after stripping. Barrier regeneration of stripped skin is seen closely to resemble the parallel situation in vivo.     

Sodium lauryl sulphate penetration in an in vitro model using human skin

Because of their ability to impair the skin barrier function, detergents constitute a major risk factor for the development of irritant contact dermatitis. Sodium lauryl sulphate (SLS) is a commonly used detergent for experimental studies within the area of irritant contact dermatitis. In the present study, penetration of S³⁵-labelled SLS was studied in an in vitro model using human cadaver skin. The investigations showed that SLS is capable of permeating the skin barrier when applied under occlusion. SLS could be detected in the dermis and the amount of SLS found here was shown to depend on the dose of SLS applied on the skin. Penetration of SLS continued after removal of the SLS applied as a patch test on the skin surface. Considerable inter-individual variation in the penetration of SLS was demonstrated between different donors.     

Dipyridamole potentiates the growth-inhibitory action of methotrexate and 5-fluorouracil in human keratinocytes in vitro

Human keratinocytes transport extracellular thymidine across the plasma membrane and incorporate it into DNA. Data presented here show that dipyridamole, a well-known inhibitor of facilitated diffusion of nucleosides, blocks the transport of thymidine into human keratinocytes in vitro. Dipyridamole (1.0 microM) inhibited the transport of 3H-thymidine (0.2 microM) into intracellular material by 75% and its subsequent salvage and incorporation into DNA by 48%. Dipyridamole (1 microM) did not affect the growth of keratinocytes in vitro but did potentiate the growth inhibition caused by methotrexate (MTX) or 5-fluorouracil (5-FU). The growth of keratinocytes exposed to 0.1 microM MTX for 8 d was inhibited by 32%. However, in combination with a noninhibitory concentration of dipyridamole (1 microM), this concentration of MTX (0.01 microM) inhibited the growth of keratinocytes by 93%. Thymidine in culture medium reversed the cytotoxicity of MTX. However, in the presence of dipyridamole, thymidine in the culture medium did not reverse the action of MTX. The synergistic interaction between MTX and dipyridamole was also observed with 5-FU and dipyridamole. 5-FU (0.5 microM) inhibited cell growth by 30% but in combination with dipyridamole (1 microM), inhibited cell growth by 86%. These data are consistent with the theory that inhibiting thymidine salvage by blocking transport of extracellular thymidine potentiates the growth inhibitory action of inhibitors of de novo pyrimidine biosynthesis in human keratinocytes. Combination chemotherapy, such as methotrexate plus dipyridamole, might be efficacious in the treatment of hyperproliferative diseases of the epidermis.     

Effect of sodium lauryl sulfate (SLS) on in vitro percutaneous penetration of water, hydrocortisone and nickel

The dose- and time-related effect of sodium lauryl sulfate (SLS) on in vitro percutaneous penetration was studied using 3 radiolabeled tracer compounds with different physicochemical properties: tritiated water, hydrocortisone and nickel. Human cadaver abdominal skin from caucasian women was used as membrane in static in vitro penetration cells. Simultaneous application of SLS together with 1 of the tracer compounds showed, after 48 h, a significant dose-effect relationship between SLS concentration (0.25%, 2% and 10%) and penetration of tritiated water or nickel ( p < 0.001, Spearman), whereas SLS had no significant effect on penetration of hydrocortisone. When 4% SLS was applied as pretreatment, a significant time-effect relationship, after 48 h, was found between pretreatment time (0.5, 2 and 8 h) and penetration of tritiated water. A similar relationship was not found for penetration of nickel or hydrocortisone. Pretreatment of the skin with SLS for 2 h using 3 concentrations (0.25%, 4% and 10%) showed, after 48 h, a significant dose-effect relationship between SLS treatment and penetration of tritiated water or nickel ( p < 0.001, Spearman). Pretreatment had no effect on penetration of hydrocortisone. Pretreatment simulates a cleaning-washing situation. The present in vitro skin penetration model, using human cadaver skin, described the dose-effect and time-effect relationships for SLS on the penetration profiles of 3 different compounds. The model may be extended to other compounds with suspected irritant/damaging effect on the skin barrier. It should be kept in mind that the model uses a dead skin membrane without the barrier repair mechanisms of live skin.     

Factors affecting the binding of lectins to normal human skin

Factors affecting the binding of a wide range of lectins to normal human skin were examined in order to evaluate current discrepancies in the literature. The profile of specific binding characteristic for each lectin was found to be variously influenced by the source of conjugate, tissue-processing method, the effectiveness of saccharide inhibitors, and by individual and minor body site variations. Most significantly, the use of routine histological processing not only greatly reduced binding intensity overall but also altered the binding pattern.     

Human percutaneous penetration of hydrocortisone: The vulva

Summary Percutaneous penetration of ¹⁴C hydrocortisone through normal vulvar skin (labia majora) was measured in six subjects and compared with that of the forearm. Of the topically applied hydrocortisone 7.7% penetrated vulvar skin whereas 1.3% penetrated forearm skin. This regional variation of pecutaneous penetration may have toxicologic and therapeutic significance.