Figure 1.
PPAR β/δ selective antagonists used in this study.
Chemical structures and in vitro pharmacodynamic data shown are taken from the references listed. The structure of the PPAR β/δ selective agonist GW501516 used in this study is given for comparison.
Figure 2.
Low systemic absorption of topically applied PPAR β/δ antagonists.
A. Peak blood concentrations of PPAR β/δ agonist GW501516, and antagonists GSK0660 and compound 3 h, respectively, at 1 h after topical application to skin. Left: Amount of drugs detected in systemic circulation, expressed as fraction of total amount applied, was calculated as detailed in methods. Right: Drug concentration expressed as molar concentration. B. GSK0660 concentration in blood (left) and total amount of circulating drug as fraction of amount applied (right, calculated as described in Methods) at the indicated time points after drug application. The horizontal dashed line represents the reported IC50 for GSK0660 acting on PPAR β/δ reported previously (300 nmol/L). Data shown represent average ± s.d. of n = 3 mice per data point.
Figure 3.
Half – life of GSK0660 after topical application to skin.
42 mg of GSK0660 ointment was applied to dorsal skin of C57Bl/6j wild type mice. Mice were sacrificed at the time after drug application indicated in the figure and drug concentration determined by mass spectrometry, as detailed in Methods. Data shown represent average ± s.d. of n = 3 mice per data point.
Figure 4.
Absence of inflammatory changes induced by PPAR β/δ antagonists in skin after topical application.
(a) C57Bl/6j wild type mice were treated with ointments containing GSK0660 or compound 3 h applied twice daily to shaved dorsal skin for one week. Mice were sacrificed 1 h after the last ointment application and skin tissue processed for H&E based histology and mass spectrometry, as described in Methods. Data shown represent average ± s.d. of n = 3 mice per data point (left) treated with GSK (blue columns) or compound 3 h (red). Representative histology sections of all treated mice are shown on right. The inset in the middle panel shows a section of GSK0660-treated epidermis showing apoptotic looking cells (marked by red arrow head). Horizontal bar represents 5 µm. (b) Representative H&E sections of C57Bl/6j wild type mice treated for one week with either GSK0660 (top) or GSK3787 (bottom). Red arrow-heads denoting apoptotic looking cells.
Figure 5.
Prevention of epidermal hyperplasia by transdermal application of selective PPAR β/δ antagonists.
Both the PPAR β/δ agonist GW501516 (GW) and the antagonists GSK0660 (GSK) or compound 3 h were applied topically to the skin, as described in the text. Left: representative H&E-stained paraffin-sections of dorsal skin from PPAR β/δ transgenic mice after treatment with ointments containing the indicated drugs for twenty days, as detailed in Methods. Horizontal bar represents 5 µm. Right: quantification of H&E-based epidermal thickness observed in n = 4 mice per group, performed as detailed in Methods. * p<0.05 in a two-sided independent t-test.
Figure 6.
Reversal of psoriasis-like skin disease in PPAR β/δ mice by PPAR β/δ antagonists.
Skin disease was induced by systemic oral administration of the PPAR β/δ agonist GW501516. (Control mice received standard chow). Subsequently, GW501516 dose was lowered to allow maintenance of phenotype as described in the text and mice were treated twice daily with either vehicle only (GW501516 group) or antagonist-containing ointments, as indicated. A. Reversal of epidermal hyperplasia, performed as described in the legend for figure 5. Horizontal bar represents 5 µm. B. Reduction of T cell infiltration. Skin samples of the treated skin regions were processed and stained for FACS analysis as described in Methods. Scatter plots shown on left show representative data, column diagrams on right show quantification of FACS data in n = 4 mice per group. The scatter plot shown at the bottom indicates the lympocyte gate used for quantification of CD4/CD8 cells, as previously described [21]. C. Quantification of target genes previously been shown to be induced in PPAR β/δ transgenic mice by qPCR, as detailed in Methods. * p<0.05 in a two-sided independent t-test.
Figure 7.
Control of PPAR β/δ – mediated skin disease using reduced-frequency application of ointment containing an irreversible PPAR β/δ antagonist.
Skin disease in PPAR β/δ transgenic mice was induced by i.p. injection of the agonist GW501516. Additionally, mice were shaved on their abdomen and were treated with vehicle-ointment or ointment containing either GSK0660 (twice daily) or GSK3787 at the indicated frequencies. Red arrow denotes apoptotic cells noted in the GW-only treatment group. A. Top: Representative H&E stains from 3 different mice in each treatment group. Horizontal bar represents 5 µm. Bottom: Quantification of epidermal thickness (p<0.01 in all treatment groups vs. GW-only). B. Quantification of dermal infiltrate. Data shown represent average ± s.d. of five mice per group.
Figure 8.
Effect of GSK3787 on PPAR β/δ -mediated skin disease applied either twice daily or three times per week (once per day).
A. Left: representative H&E stains from 3 different mice in each treatment group. Horizontal bar represents 5 µm. Right: Quantification of epidermal thickness and dermal nuclei, respectively. * p<0.05 vs. GW501516 only. B. Q-PCR based quantification of the PPAR β/δ target genes LCE3f, IL1β, and HBEGF, as in figure 6c. Data shown represent n = 5 mice per treatment group.