Molecular size as the main determinant of solute maximum flux across the skin

One of the most important determinants of dermatological and systemic penetration after topical application is the delivery or flux of solutes into or through the skin. The maximum dose of solute able to be delivered over a given period of time and area of application is defined by its maximum flux (J(max), mol per cm(2) per h) from a given vehicle. In this work, J(max) values from aqueous solution across human skin were acquired or estimated from experimental data and correlated with solute physicochemical properties. Whereas epidermal permeability coefficients (k(p)) are optimally correlated to solute octanol-water partition coefficient (K-ow) and molecular weight (MW) was found to be the dominant determinant of J(max) for this literature data set: log J(max)=-3.90-0.0190MW (n=87, r(2)=0.847, p

The effect of region of application on absorption of ethanol and hexanol through canine skin

The effect of region of application on the percutaneous penetration of solutes with differing lipophilicity was investigated in canine skin. Skin from the thorax, neck, back, groin, and axilla regions was harvested from Greyhound dogs and placed in Franz-type diffusion cells. Radiolabelled (C-14) ethanol (Log P 0.19) or hexanol (Log P 1.94) was applied to each skin section for a total of 5 h. The permeability coefficient (k(P), cm h(-1)) and residue of alcohol remaining in the skin were significantly (P = 0.001) higher for hexanol compared to ethanol. In contrast, ethanol had a far greater maximum flux (J(max), mol (cm(2))(-1) h(-1)) than hexanol (P = 0.001). A comparison of regional differences shows the k(P) and Jmax for ethanol in the groin was significantly lower (P = 0.035) than the back. The k(P) and Jmax for hexanol were significantly higher (P = 0.001) in the axilla than the other four skin sites. An understanding of factors influencing percutaneous drug movement is important when formulating topical preparations for the dog. (C) 2003 Elsevier Ltd. All rights reserved.

Multiphoton high-resolution 3D imaging of Langerhans cells and keratinocytes in the mouse skin model adopted for epidermal powdered immunization

Langerhans cells (LCs) can be targeted with DNA-coated gold micro-projectiles ("Gene Gun") to induce potent cellular and humoral immune responses. It is likely that the relative volumetric distribution of LCs and keratinocytes within the epidermis impacts on the efficacy of Gene Gun immunization protocols. This study quantified the three-dimensional (3D) distribution of LCs and keratinocytes in the mouse skin model with a near-infrared multiphoton laser-scanning microscope (NIR-MPLSM). Stratum corneum (SC) and viable epidermal thickness measured with MPLSM was found in close agreement with conventional histology. LCs were located in the vertical plane at a mean depth of 14.9 mum, less than 3 mum above the dermo-epidermal boundary and with a normal histogram distribution. This likely corresponds to the fact that LCs reside in the suprabasal layer (stratum germinativum). The nuclear volume of keratinocytes was found to be approximately 1.4 times larger than that of resident LCs (88.6 mum3). Importantly, the ratio of LCs to keratinocytes in mouse ear skin (1:15) is more than three times higher than that reported for human breast skin (1:53). Accordingly, cross-presentation may be more significant in clinical Gene Gun applications than in pre-clinical mouse studies. These interspecies differences should be considered in pre-clinical trials using mouse models.

Transdermal drug delivery: Basic principles for the veterinarian

The use of topical pharmaceutical formulations is increasingly popular in veterinary medicine. A potential concern is that not all formulations are registered for the intended species, yet current knowledge strongly suggests that simple extrapolation of transdermal drug pharmacokinetics and pharmacodynamics between species, including humans, cannot be done. In this review, an overview is provided of the underlying basic principles determining the movement of topically applied molecules into and through the skin. Various factors that may affect transdermal drug penetration between species, between individuals of a particular species and regional differences in an individual are also discussed. A good understanding of the basic principles of transdermal drug delivery is critical to avoid adverse effects or lack of efficacy when applying topical formulations in veterinary medicine. (c) 2005 Elsevier Ltd. All rights reserved.

Effects of vehicle and region of application on absorption of hydrocortisone through canine skin

Objective-To determine the effects of various vehicles on the penetration and retention of hydrocortisone applied to canine skin. Sample Population-20 canine skin samples obtained from the thorax, neck, and groin regions of 5 Greyhounds. Procedure-Skin was harvested from dogs after euthanasia and stored at -20 degrees C until required. The skin was then defrosted and placed into diffusion cells, which were maintained at approximately 32 degrees C by a water bath. Saturated solutions of hydrocortisone that contained trace amounts of radiolabelled [C-14]-hydrocortisone in each vehicle (ie, PBS solution [PBSS] alone, 50% ethanol [EtOH] in PBSS [wt/wt], and 50% propylene glycol in PBSS [wt/wt]) were applied to the outer (stratum corneum) surface of each skin sample, and aliquots of receptor fluid were collected for 24 hours and analyzed for hydrocortisone. Results-The maximum flux of hydrocortisone was significantly higher for all sites when dissolved in a vehicle containing 50% EtOH, compared with PBSS alone or 50% propylene glycol, with differences more prominent in skin from the neck region. In contrast, higher residues of hydrocortisone were found remaining within the skin when PBSS alone was used as a vehicle, particularly in skin from the thorax and neck. Conclusions and Clinical Relevance-Penetration of topically applied hydrocortisone is enhanced when EtOH is used in vehicle formulation. Significant regional differences (ie, among the thorax, neck, and groin areas) are also found in the transdermal penetration and skin retention of hydrocortisone. Variability in clinical response to hydrocortisone can be expected in relation to formulation design and site of application.

Optimization of a transplant model to assess skin reconstitution from stem cell-enriched primary human keratinocyte populations

Given that an important functional attribute of stem cells in vivo is their ability to sustain tissue regeneration, we set out to establish a simple and easy technique to assess this property from candidate populations of human keratinocyte stem cells in an in vivo setting. Keratinocytes were inoculated into devitalized rat tracheas and transplanted subcutaneously into SCID mice, and the epithelial lining regenerated characterized to establish the validity of this heterotypic model. Furthermore, the rate and quality of epidermal tissue reconstitution obtained from freshly isolated unfractionated vs. keratinocyte stem cell-enriched populations was tested as a function of (a) cell numbers inoculated; and (b) the inclusion of irradiated support keratinocytes and dermal cells. Rapid and sustained epidermal tissue regeneration from small numbers of freshly isolated human keratinocyte stem cells validates the utilization of this simple and reliable model system to assay for enrichment of epidermal tissue-reconstituting cells.

An in vivo comparative study of sonic, desert and Indian hedgehog reveals that hedgehog pathway activity regulates epidermal stem cell homeostasis

Despite the well-characterised role of sonic hedgehog (Shh) in promoting interfollicular basal cell proliferation and hair follicle downgrowth, the role of hedgehog signalling during epidermal stem cell fate remains largely uncharacterised. In order to determine whether the three vertebrate hedgehog molecules play a role in regulating epidermal renewal we overexpressed sonic (Shh), desert (Dhh) and Indian (Ihh) hedgehog in the basal cells of mouse skin under the control of the human keratin 14 promoter. We observed no overt epidermal morphogenesis phenotype in response to Ihh overexpression, however Dhh overexpression resulted in a range of embryonic and adult skin manifestations indistinguishable from Shh overexpression. Two distinct novel phenotypes were observed amongst Shh and Dhh transgenics, one exhibiting epidermal progenitor cell hyperplasia with the other displaying a complete loss of epidermal tissue renewal indicating deregulation of stem cell activity. These data suggest that correct temporal regulation of hedgehog activity is a key factor in ensuring epidermal stem cell maintenance. In addition, we observed Shh and Dhh transgenic skin from both phenotypes developed lesions reminiscent of human basal cell carcinoma (BCC), indicating that BCCs can be generated despite the loss of much of the proliferative (basal) compartment. These data suggest the intriguing possibility that BCC can arise outside the stem cell population. Thus the elucidation of Shh (and Dhh) target gene activation in the skin will likely identify those genes responsible for increasing the proliferative potential of epidermal basal cells and the mechanisms involved in regulating epidermal stem cell fate.

Patched1 functions as a gatekeeper by promoting cell cycle progression

Mutations in the Hedgehog receptor, Patched 1 (Ptch1), have been linked to both familial and sporadic forms of basal cell carcinoma (BCC), leading to the hypothesis that loss of Ptch1 function is sufficient for tumor progression. By combining conditional knockout technology with the inducible activity of the Keratin6 promoter, we provide in vivo evidence that loss of Ptch1 function from the basal cell population of mouse skin is sufficient to induce rapid skin tumor formation, reminiscent of human BCC. Elimination of Ptch1 does not promote the nuclear translocation of beta-catenin and does not induce ectopic activation or expression of Notch pathway constituents. In the absence of Ptch1, however, a large proportion of basal cells exhibit nuclear accumulation of the cell cycle regulators cyclin D1 and B1. Collectively, our data suggest that Ptch1 likely functions as a tumor suppressor by inhibiting G(1)-S phase and G(2)-M phase cell cycle progression, and the rapid onset of tumor progression clearly indicates Ptch1 functions as a gatekeeper. In addition, we note the high frequency and rapid onset of tumors in this mouse model makes it an ideal system for testing therapeutic strategies, such as Patched pathway inhibitors.

A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse

The c-fms gene encodes the receptor for macrophage colony-stimulating factor (CSF-1). The gene is expressed selectively in the macrophage and trophoblast cell lineages. Previous studies have indicated that sequences in intron 2 control transcript elongation in tissue-specific and regulated expression of c-fms. In humans, an alternative promoter was implicated in expression of the gene in trophoblasts. We show that in mice, c-fms transcripts in trophoblasts initiate from multiple points within the 2-kilobase (kb) region flanking the first coding exon. A reporter gene construct containing 3.5 kb of 5' flanking sequence and the down-stream intron 2 directed expression of enhanced green fluorescent protein (EGFP) to both trophoblasts and macrophages. EGFP was detected in trophoblasts from the earliest stage of implantation examined at embryonic day 7.5. During embryonic development, EGFP highlighted the large numbers of c-fms-positive macrophages, including those that originate from the yolk sac. In adult mice, EGFP location Was consistent with known F4/80-positive macrophage populations, including Langerhans cells of the skin, and permitted convenient sorting of isolated tissue macrophages from disaggregated tissue. Expression of EGFP in transgenic mice was dependent on intron 2 as no lines with detectable EGFP expression were obtained where either all of intron 2 or a conserved enhancer element FIRE (the Fms intronic regulatory element) was removed. We have therefore defined the elements required to generate myeloid- and trophoblast-specific transgenes as well as a model system for the study of mononuclear phagocyte development and function. (C) 2003 by The American Society of Hematology.

Regulation of MAPK activation, AP-1 transcription factor expression and keratinocyte differentiation in wounded fetal skin

Fetal epithelium retains the ability to re-epithelialize a wound in organotypic culture in a manner not dependent on the presence of underlying dermal substrata. This capacity is lost late in the third trimester of gestation or after embryonic day 17 (E-17) in the rat such that embryonic day 19 (E-19) wounds do not re-epithelialize. Moreover, wounds created in E-17 fetuses in utero heal in a regenerative, scar-free fashion. To investigate the molecular events regulating re-epithelialization in fetal skin, the wound-induced expression profile and tissue localization of activator protein 1 (AP-1) transcription factors c-Fos and c-Jun was characterised in E-17 and E-19 skin using organotypic fetal cultures. The involvement of mitogen-activated protein kinase (MAPK) signaling in mediating wound-induced transcription factor expression and wound re-epithelialization was assessed, with the effect of wounding on the expression of keratinocyte differentiation markers determined. Our results show that expression of AP-1 transcription factors was induced immediately by wounding and localized predominantly to the epidermis in E-17 and E-19 skin. c-fos and c-jun induction was transient in E-17 skin with MAPK-dependent c-fos expression necessary for the re-epithelialization of an excisional wound in organotypic culture. In E-19 skin, AP-11 expression persisted beyond 12 h post-wounding, and marked upregulation of the keratinocyte differentiation markers keratin 10 and loricrin was observed. No such changes in the expression of keratin 10 or loricrin occurred in E-17 skin. These findings indicate that re-epithelialization in fetal skin is regulated by wound-induced AP-1 transcription factor expression via MAPK and the differentiation status of keratinocytes.