JPET Fast Forward. Published on August 18, 2005 as DOI:10.1124/jpet.105.091066
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BP-1107: A Novel, Synthetic Thiazolidinedione That Inhibits Epidermal Hyperplasia in Psoriatic Skin – SCID Mouse Transplants Following Topical Application
Narasimharao Bhagavathula, Kamalakar C. Nerusu, Mahendranath Reddy, Charles N. Ellis, Amar Chittiboyina, Mitchell Avery, Harrihar A. Pershadsingh, Theodore W. Kurtz and James Varani
(NB, KN, MR, JV) Department of Pathology, University of Michigan Medical School, Ann Arbor, MI (CE) Department of Dermatology, University of Michigan Medical School, and Dermatology Service; Veterans Affairs Medical Center, Ann Arbor, MI (AC, MA) Department of Medicinal Chemistry, University of Mississippi, Oxford, MS (HP) Departments of Family Medicine, Kern Medical Center & University of California, Irvine, CA; and Bethesda Pharmaceuticals, Inc., Bakersfield, CA (TK) Department of Laboratory Medicine, University of California, San Francisco, CA
1 Copyright 2005 by the American Society for Pharmacology and Experimental Therapeutics.
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Short title: BP-1107 and psoriasis
Send Communications to: James Varani, Ph.D. Department of Pathology The University of Michigan 1301 Catherine Road/Box 0602 Ann Arbor, MI 48109 Tele: 734 615-0298 Fax: 734 763-6476 Email:
[email protected] Number of text pages Number of tables Number of figures Number of references Number of words in abstract Number of words in Introduction Number of words in Discussion
Abbreviations: PPAR-γ KGM KBM DMEM-FBS MMP MMP-1 MMP-2 MMP-9 SDS-PAGE MAP kinase ERK1/2
: 31 :0 :8 : 41 : 146 : 445 : 1018
Peroxisome proliferator-activated receptor-γ Keratinocyte growth medium Keratinocyte basal medium Dulbecco’s modified minimal essential medium Matrix metalloproteinase Interstitial collagenase 72-kD gelatinase A 92-kD gelatinase B Sodium dodecylsulfate – polyacrylamide gel electrophoresis Mitogen activated protein kinase Extracellular factor-regulated kinase – 1/2
Recommended section assignment: Cellular and Molecular
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ABSTRACT
Recent studies have demonstrated that orally administered thiazolidinedione ligands of the peroxisome proliferator-activated receptor-γ (PPAR-γ) can ameliorate clinical features of psoriasis in humans. Thiazolidinediones also inhibit the proliferation of psoriatic keratinocytes in monolayer and organ culture, and at least one of these agents (troglitazone) inhibits epidermal hyperplasia of human psoriatic skin transplanted to severe combined immunodeficient (scid) mice. In the present study we show that a novel, synthetic, thiazoladinedione derivative (BP1107) is capable of inhibiting psoriatic hyperplasia in the scid mouse transplant model after topical application. Like other thiazolidinediones, BP-1107 inhibits proliferation of rapidly growing keratinocytes in monolayer culture, but compared to these agents, the effective dose of BP-1107 needed to suppress keratinocyte proliferation is much lower. Concentrations of BP1107 that effectively inhibit keratinocyte function have no detrimental effect on dermal fibroblasts. These data suggest that effective topical anti-psoriatic therapy may be provided with this agent.
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INTRODUCTION
Psoriasis is an inflammatory skin disease characterized by excessive keratinocyte proliferation, leading to a significant thickening of the epidermis, expansion of epidermal rete pegs into papillary dermal space and continuous shedding of the thickened epidermis. The etiology of the disease is complex and not well-understood. T-cells are almost certainly involved in the initiation of psoriatic lesions. Activated T cells in the region of the dermal-epidermal junction are thought to drive the hyperplastic proliferative response through elaboration of Th1 cytokines including tumor necrosis factor-α, interferon-γ and various interleukins (IL-2, IL-6 and IL-8) (Nickoloff, 1991; Valdimarsson, et al., 1995; Austin, et al., 1999). Although the immune system is likely to be responsible for initiating the disease, the subsequent hyper-proliferative response in the keratinocytes appears to be a direct consequence of pro-proliferative intraepidermal events.
In recent studies, we (Ellis, et al., 2000; Bhagavathula, et al., 2004) and other investigators (Komuves, et al., 1998; Kubota, et al., 1998; Mueller, et al., 1998; Rivier, et al., 1998; Sarraf, et al., 1998; Rivier, et al., 2000; Mao-Qiang, et al., 2004) have shown that agonists of the peroxisome proliferator-activated receptor – γ (PPAR-γ) including thiazolidinediones such as troglitazone, rosiglitazone and pioglitazone reduce epithelial cell proliferation and induce differentiation.
At least one of the thiazolidinediones (i.e., troglitazone) normalizes the
histological appearance of human psoriatic skin in organ culture and in the human skin – severe combined immunodeficient (scid) mouse model (Ellis, et al., 2000). Both troglitazone and pioglitazone have been shown to reduce the clinical and histological presentation of psoriasis
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following systemic treatment (Pershadsingh, et al., 1998; Ellis, et al., 2000; Robertshaw and Friedmann, 2005; Shafiq, et al., 2005). Unfortunately, these agents cause serious side effects (fluid retention, weight gain) in a subset of patients. Although the first clinically approved thiazolidinedione ligand of PPAR-γ (troglitazone) was withdrawn from the market because of hepatotoxicity, this does not appear to be a class effect because liver toxicity has not been an issue with other PPAR-γ activators such as pioglitazone or rosiglitazone (Lebovitz, 2002).
Recently, we synthesized a series of thiazolidinedione PPAR-γ agonists. One of these agents (referred to as BP-1107) was shown to have high affinity for PPAR-γ (EC50 of 26 pM as compared to 64 nM for rosiglitazone) (Bhagavathula, et al., 2004). Given the high affinity of BP-1107 for PPAR-γ, we conducted a series of experiments to assess the ability of this agent to modulate keratinocyte proliferation in vitro and epidermal thickness in the human skin – scid mouse transplant model. Our results show that BP-1107 is a potent keratinocyte growth inhibitor in vitro and effectively reduces epidermal hyperplasia of human psoriatic skin transplanted to scid mice. Of significance, BP-1107 is effective when delivered topically.
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METHODS
BP-1107. BP-1107, a novel, high-affinity PPAR-γ ligand, was obtained from the Department of Medicinal Chemistry, University of Mississippi (Dr. Avery) and Bethesda Pharmaceuticals, Inc. (Bakersfield, CA). Structurally, BP-1107 is an adamantyl-coupled thiazolidinedione (Figure 1{2-[4-(2,4-dioxo-thiazolidin-5-ylmethyl)-phenoxy]-ethyl}-methyl-amide).
BP1107
was
designated as BP107 in International Patent Publication Number WO 2005/009437.
Other reagents. Commercial reagents utilized in this study included antibodies to phosphoERK1/2, total-ERK1/2, phospho-c-jun and total-c-jun, all obtained from Cell Signaling Technologies, Inc. (Beverly, MA). A rabbit polyclonal antibody to matrix metalloproteinase-1 (MMP-1) was obtained from Chemicon International, Inc. (Temecula, CA), and an antibody to β-tubulin was obtained from Santacruz Biotech (Santa Cruz, CA). Epidermal growth factor (EGF) was from R&D Systems (Minneapolis, MN).
Human tissue. Six mm punch biopsies of psoriatic lesional skin (four biopsies per volunteer) were obtained from two individuals with active psoriasis on the trunk and/or hip. Neither of the tissue donors was on therapy at the time of biopsy and neither had been on systemic therapy for a period of at least six months. Six mm punch biopsies of skin were also obtained from four nonpsoriatic volunteers (four biopsies per volunteer). The use of human skin in this study was approved by the University of Michigan Institutional Review Board, and biopsies were obtained after receiving written informed consent from the donors.
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Transplantation procedure. Scid mice (CB-17 strain; Taconic Farms Inc., Germantown, NY) were used as tissue recipients. One 6-mm punch biopsy was transplanted onto the dorsal surface of a recipient mouse as described previously (Zeigler, et al., 2001). Briefly, mice were anesthetized and skin from the dorsal region shaved. Mouse skin was surgically removed to size and replaced with the human tissue. The human tissue was secured to the back of the mouse with absorbable sutures (4-0 Dexon"S", Davis-Geck, Manati, Puerto Rico). The transplants were then bandaged with Xeroform petrolatum dressing (Kendall Company; Mansfield, MA) for 3-4 days. The animals were maintained in a pathogen-free environment throughout the preparation and treatment phases. Treatment was initiated one to two weeks post-transplantation, depending on how rapidly the tissue healed. The human skin – scid mouse transplant model has been used previously to study the pathophysiology of psoriasis (Nickoloff, et al., 1995; Wrone-Smith and Nickoloff, 1996; Gilhar, et al., 1997) and to assess potential anti-psoriatic therapies (Ellis, et al., 2000; Zeigler, et al., 2001; Bhagavathula, et al., 2005).
In vivo treatment protocol. Normal human skin and psoriatic lesional plaque skin transplanted onto scid mice were treated topically with BP-1107. Briefly, 100 µl of solution – either DMSO alone or BP-1107 (100 µM) in DMSO – was applied daily for 21 days. At the end of the treatment period, animals were sacrificed. The transplanted skin with a small amount of surrounding mouse skin was removed and fixed in 10% buffered formalin. After staining with hematoxylin and eosin, skin sections were examined by light microscopy. Tissue sections were visualized by light microscopy at 200X magnification. Epidermal thickness measurements were made at four or more sites on three or four different histological sections from each transplant
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(two control and two BP-1107-treated biopsies per subject) and averaged. From these values, mean epidermal thickness was determined.
Human epidermal keratinocytes and dermal fibroblasts in monolayer culture. Normal human epidermal keratinocytes were isolated as described previously (Varani, et al., 1994). Primary and early passage cells were maintained in Keratinocyte Growth Medium (KGM) (Cambrex, Inc.). KGM is a modification of MCDB-153 medium containing a low level (0.15 mM) of extracellular Ca2+ and further supplemented with a mixture of growth factors including 0.1 ng per ml EGF, 0.5 µg per ml insulin, and 2% bovine pituitary extract. Fibroblasts were obtained from the same tissue and grown in monolayer culture using Dulbecco's modified minimal essential medium supplemented with nonessential amino acids and 10% fetal bovine serum (DMEM-FBS). Both keratinocytes and fibroblasts were maintained at 37°C in an atmosphere of 95% air and 5% CO2. Cells were subcultured by exposure to trypsin/ethylenediamine tetraacetic acid (EDTA) and used at passage 2-3.
Proliferation assays. For dose-response studies, keratinocytes and fibroblasts were seeded at 5 X 104 cells per well in their respective growth media (24-well plate) and allowed to attach overnight. Cells were treated with different concentrations of BP-1107.
Proliferation was
measured on day-3 by releasing the cells with trypsin/EDTA and enumerating them using a particle counter (Coulter Electronics, Hialeah, FL). For time-course studies, 5 X 104 cells were seeded per well in a 24-well plate, allowed to attach overnight, and treated with 1 µM BP-1107. Cell counts were made on days 1 through 3. KGM was used for keratinocyte proliferation studies and KBM supplemented with 1.4 mM Ca2+ was used for fibroblast proliferation assays. KBM consists of the same basal medium as KGM but is not supplemented with growth factors.
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Cytotoxicity assays. Keratinocytes were plated at 5 X 104 cells per well in KGM and incubated overnight to allow the cells to attach. Next day, cells were exposed to control conditions or to different concentrations of BP-1107 for four hours. At the end of the incubation period, the cells were harvested, counted and replated in growth medium. Eighteen hours later, the percentage of cells that had reattached and spread was determined. The ability of cells to reattach and spread after treatment was used as a measure of cell viability (Varani, et al., 1985).
Motility assay. Motility was assessed using the “Scratch Wound” assay. The cells were seeded into a 24-well dish at 1x105 cells per well and incubated in KGM to near confluency. At that point, a “scratch-wound” approximately 100 µm in width was made through the monolayer. The wounded monolayers were incubated in KGM with different concentrations of BP-1107 for two days. At the end of the incubation period, the cultures were photographed under phase-contrast microscopy at 200X magnification. The distance the cells migrated into the wounded area was measured using a micrometer.
MMP assays. Keratinocyte- or fibroblast-conditioned medium was assayed for MMP activity. SDS-PAGE substrate embedded enzymography (zymography) was used to identify enzymes with gelatinase activity. Assays were carried out as described in a previous report (Gibbs, et al., 1999). Briefly, denatured but non-reduced culture fluid samples were resolved in 10% SDSPAGE gels prepared with incorporation of gelatin (1 mg per ml) prior to casting. After electrophoresis, gels were washed twice for 15 minutes in 50 mM Tris buffer containing 1 mM Ca2+, 0.5 mM Zn2+ and 2.5% Triton X-100. The gels were then incubated overnight in Tris buffer with 1% Triton X-100 and stained the following day with Brilliant Blue 250-R. Following
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destaining, zones of enzyme activity were detected as regions of negative staining against the dark background. Gelatin zymography is used for detection of MMP-2 (72-kD gelatinase A) and MMP-9 (92-kD gelatinase B).
Fibroblast-conditioned medium was assayed for MMP-1 (interstitial collagenase) by western blotting as described previously (Bhagavathula, et al., 2004). Briefly, samples were separated in 10% SDS-PAGE under denaturing and reducing conditions and transferred to nitrocellulose membranes. After blocking with a 5% nonfat milk solution in Tris-buffered saline (TBS) at 4oC overnight, membranes were incubated for one hour at room temperature with a rabbit polyclonal anti-human MMP-1 antibody, diluted 1:6000 in 0.5% nonfat milk/0.1% TweenTBS (TTBS). Thereafter the membranes were washed with TTBS and bound antibody detected using the Phototope-HRP Western blot detection kit (Cell Signaling Technologies, Inc., Beverly, MA).
Type I procollagen assay. Fibroblast-conditioned medium was assayed for type I procollagen by enzyme-linked immunosorbant assay (ELISA) (Pan Vera Corp., Madison, WI) as described previously (Varani, et al., 2000). The procollagen assay uses an antibody to the C-terminal propeptide region that is part of the collagen molecule as it is synthesized and secreted (before being proteolytically cleaved). As such, this assay is a measure of newly synthesized collagen.
Preparation of cell lysates and immunoblot analysis of signaling intermediates. Cells were lysed in 1X cell lysis buffer from Cell Signaling Technologies. Lysis was performed at 4oC by scraping the cells into lysis buffer and sonicating the samples. Cell lysates were incubated on ice for 30 minutes and then cleared by microcentrifugation at 16000 g for 15 minutes. The
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supernatant fluids were collected and protein concentration was estimated using the BioRad DC protein assay kit (BioRad, Hercules, CA). Cell extracts containing equivalent amounts of protein (40 µg of total protein per lane) were electrophoresed in 10% SDS-polyacrylamide gels. Western blotting for signaling intermediates was carried out as described above for MMP-1.
Statistical analysis. Measurements were expressed as means and standard errors or means and standard deviations. Statistical analyses were carried out using the Student t-test where two groups were compared or by ANOVA followed by paired group comparisons where there were multiple groups. P < 0.05 was considered statistically significant.
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RESULTS
Effects of topical treatment of human skin – scid mouse transplants with BP-1107. In the first series of experiments, psoriatic lesional skin from two individuals was transplanted to scid mice (four animals per subject). After allowing the skin to heal, two animals from each group were treated topically with BP-1107 (100 µl of a 100 µM solution dispensed over the treatment area with a micropipette on 21 consecutive days) while the other two received vehicle alone. At the end of the treatment period, skin sites were photographed. Grossly, the human tissue treated with BP-1107 became visibly thinned (as compared to skin from animals treated with vehicle alone). In places, the epidermis appeared to be completely eroded. In other areas, the treated tissue shrank such that the area covered by the transplant decreased. The upper panels of Figure 2 demonstrate the gross features of control and BP-1107-treated psoriatic skin transplants.
The middle panels of Figure 2 demonstrate histological features of skin from vehicletreated and BP-1107-treated mice. The vehicle-treated skin had the typical features of psoriatic plaque skin including thickened epidermis with associated rete ridges and pegs, along with inflammatory foci and with focal loss of the granular layer (Panel C). In contrast, the epidermis from animals treated with BP-1107 was much thinner than skin from the untreated animals. There was no evidence of rete pegs or ridges (Panel D). The lower portion of Figure 2 provides quantitative information based on epidermal thickness measurements. Overall, the epidermal thickness of skin from BP-1107-treated animals was thinned, as compared to skin from untreated mice.
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In addition to transplanting and treating psoriatic plaque skin, skin from four nonpsoriatic donors was also transplanted to scid mice and treated topically with BP-1107. With non-psoriatic skin samples, BP-1107 was used at two concentrations (25 and 100 µM). Consistent with past reports (Zeigler, et al., 2001; Bhagavathula, et al., 2005), skin from nonpsoriatic tissue donors became hyperplastic upon transplantation. When the non-psoriatic skin transplants were treated topically with a 100 µM concentration, findings were similar to those with psoriatic skin, i.e., shrinkage of the tissue and areas of epidermal erosion were observed. These gross changes were not observed in animals treated with the lower concentration. Histological findings including thinning of the epidermis with loss of rete pegs and ridges were observed with both drug concentrations (Figure 3).
Effects of BP-1107 on keratinocyte function in monolayer culture.
Human epidermal
keratinocytes were treated with BP-1107 in monolayer culture and effects on proliferation, motility and elaboration of MMP-9 assessed. As shown in Figure 4, keratinocyte proliferation was inhibited by BP-1107 in a dose- and time-dependent manner. Significant inhibition of growth was achieved at a concentration of 0.5 µM (ED50 = 0.65 µM). Growth inhibition did not appear to reflect cytotoxicity since there was no evidence of cell death in the 4-hour cytotoxicity assay with drug concentrations between 0.1 and 2 µM. Additionally, studies were carried out in which keratinocytes were treated with concentrations of BP-1107 between 0.1 and 2 µM for a two-day period. At the end of the treatment period, the cells were washed. Half were reincubated in medium with the same concentrations of BP-1107 and the other half were incubated in control growth medium. After two additional days of incubation, cells were harvested and counted. As shown in Figure 5, growth inhibition with BP-1107 was fully reversible. That is,
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cells treated with BP-1107 for two days and then incubated in drug-free medium grew as rapidly between days 2 and 4 as cells not exposed to drug initially.
In contrast to these results, exposure of cells to higher concentrations of BP-1107 (10-20 µM) was cytotoxic. Specifically, the number of cells recovered from wells at the end of the 3day incubation period was fewer than the number plated (indicative of cell killing) and this was verified by cell death in the 4-hour cytotoxicity assay (not shown).
Keratinocyte motility was assessed next. As shown in Figure 6, cell migration was inhibited in the presence of BP-1107. The dose-response for inhibition of motility (ED50 = 0.55 µM) was slightly lower than that for proliferation.
Supernatant fluids were collected from untreated and BP-1107-treated keratinocytes and assayed for MMP-2 and MMP-9 by gelatin zymography. Results from this study indicated no significant effect on either enzyme (not shown).
Effects of BP-1107 on intracellular signaling events that underlie proliferation, motility and MMP production in keratinocytes. Previous studies have demonstrated the importance of mitogen-activated protein (MAP) kinase signaling (in particular, signaling through the ERK1/2 pathway) to EGF-induced biological events in keratinocytes (Zeigler, et al., 1999). To determine if the inhibitory effects of BP-1107 on keratinocyte function could be related to interference with MAP kinase signaling, we assessed the effects of the synthetic PPAR-γ agonist on ERK1/2 phosphorylation in EGF-stimulated keratinocytes.
For this experiment, keratinocytes were
treated with 10 ng/ml EGF in the absence or presence of BP-1107. At various times later, 14
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ERK1/2 phosphorylation was assessed. Inhibition was seen as early as 5 minutes after EGF stimulation, was maximal at 15 minutes and gradually returned to baseline by 120 minutes (Figure 7). In addition to the rapid/transient reduction in the phosphorylation of both ERK1 and ERK2, there was also a longer sustained reduction. This was observed by assessing phosphoERK1/2 levels in control and BP-1107 – treated keratinocytes after one and two days of treatment. On both days, levels of phospho-ERK1/2 were lower in treated than control cells (Figure 7). In contrast to effects on phospho-ERK1/2, there was no measurable effect of BP1107 treatment on total ERK1/2 levels in either the short-term (5-120 minutes) or long-term (one and two day) studies.
In a final set of experiments, we examined the ability of BP-1107 to reduce c-jun phosphorylation in keratinocytes. In contrast to what was observed with ERK1/2, treatment with BP-1107 produced essentially no change in the amount of phosphorylated c-jun seen in keratinocytes over a 6-hour time period following EGF stimulation (not shown). Likewise, there was no change in total c-jun expression. The lack of effect on c-jun is of interest since there was no change in MMP-9 expression under the same conditions. Previous studies have shown a role of c-jun in MMP-9 induction (Fisher, et al., 1998).
Effects of BP-1107 on fibroblast function in monolayer culture. In addition to assessing BP1107 on keratinocyte function, human dermal fibroblasts isolated from the same skin samples as the keratinocytes were also examined for response to BP-1107. Fibroblast proliferation and procollagen synthesis proved to be resistant to the effects of this agent. When treated with a concentration of 1 µM, there was no significant effect on fibroblast proliferation over the 3-day observation period (Figure 8; upper panel).
Likewise, there was no inhibition of type I
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procollagen elaboration under the same conditions (Figure 8; middle panel). On the other hand, fibroblast production of MMP-1 (interstitial collagenase), which is the major collagen-degrading enzyme in human skin (Brennan, et al., 2003), was reduced in the presence of BP-1107 (Figure 8; lower panel).
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DISCUSSION
Thiazolidinediones such as troglitazone, rosiglitazone and pioglitazone were originally developed for use in the treatment of type II diabetes, but it was observed early-on that when individuals with psoriatic plaques were treated with troglitazone, there was an improvement in their psoriasis (Pershadsingh, et al., 1998; Ellis, et al., 2000). Subsequently, it was demonstrated in controlled studies that these synthetic PPAR-γ agonists were potent suppressors of epithelial proliferation and inducers of epithelial differentiation (Komuves, et al., 1998; Kubota, et al., 1998; Mueller, et al., 1998; Rivier, et al., 1998; Sarraf, et al., 1998; Ellis, et al., 2000; Rivier, et al., 2000; Mao-Qiang, et al., 2004). Our own studies confirmed the growth-suppressing effects of troglitazone, rosiglitazone and other synthetic thiazolidinediones on human epidermal keratinocytes (Ellis, et al., 2000; Bhagavathula, et al., 2004; Venkatraman, et al., 2004). Motility, another commonly-observed feature in psoriatic keratinocytes, was also substantially down-regulated following treatment with rosiglitazone (Bhagavathula, et al., 2004).
In a significant subset of patients, systemic administration of thiazolidinediones is associated with several disconcerting side effects including fluid retention, edema, and weight gain. While systemic delivery may be required for treatment of conditions such as type II diabetes, psoriasis is, at least in some individuals, amenable to topical therapy. If agents with PPAR-γ agonist activity could be delivered topically, many of the unwanted effects associated with systemic delivery might be averted. In the present study we demonstrate that topical treatment of human psoriatic skin transplants on scid mice with a novel, adamantyl-coupled thiazolidinedione (BP-1107) significantly reduces the hyperplasia and normalizes differentiation in the transplanted skin.
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Our previous studies have demonstrated that normal human skin becomes hyperplastic as a consequence of transplantation to scid mice (Zeigler, et al., 2001). Therapeutic approaches that interfere with the immune basis of psoriasis (i.e., cyclosporine and anti-CD11a) inhibited psoriatic hyperplasia but not the hyperplasia developing in normal skin upon transplantation. In contrast, agents that interfere with the biochemical events that are directly involved in keratinocyte growth control (i.e., antibody to amphiregulin and corticosteroids) suppressed hyperplasia in both normal and psoriatic skin (Zeigler, et al., 2001; Bhagavathula, et al., 2005). Not surprisingly, given what is known about PPAR-γ receptors in epidermal growth control, BP1107 was found to suppress the reactive hyperplasia in non-psoriatic skin transplants as well as the hyperplasia in the psoriatic skin transplants.
Likewise, inhibition of human epidermal
keratinocyte proliferation in monolayer was also demonstrated with BP-1107, as was downregulation of keratinocyte motility. In these regards, the effects of BP-1107 were similar to those we have reported previously with troglitazone and rosiglitazone (Ellis, et al., 2000; Bhagavathula, et al., 2004), the differences being that BP-1107 functions at significantly lower concentrations than the other thiazolidinediones, and works in vivo following topical application.
In addition to assessing effects on keratinocyte function, BP-1107 was also examined for effects on dermal fibroblasts. No detrimental effects on fibroblast proliferation or type I procollagen elaboration, were seen at concentrations that effectively interfered with epidermal proliferation.
Unexpectedly, MMP-1 (interstitial collagenase) production was significantly
suppressed by BP-1107. The mechanism of MMP-1 – suppression or its possible significance are not yet known. Given these actions of topical BP-1107 in the epidermis and dermis, one
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might expect anti-psoriatic activity without the dermal atrophy associated with corticosteroid use. On the other hand, since BP-1107 appears to function at the keratinocyte level to suppress epidermal hyperplasia, there is the possibility for erosion of the reactive epidermis at the edge of the psoriatic lesion. Likewise, the possibility exists that the normal wound-healing response would be disturbed. Additional studies will be needed to determine if these consequences materialize. In any event, topical use of the agent is unlikely to produce a generalized effect on normal epidermal function or on wound healing.
The findings presented here may help advance understanding of how thiazolidinediones function in psoriasis. The pathophysiology of psoriasis is complex and multi-faceted. A T-cell – mediated immune response is involved in the initiation of psoriatic lesions, but a variety of studies suggest that intra-epidermal events directly mediate hyperplastic epidermal growth. In particular, hyperplastic epidermal proliferation in psoriatic lesional skin is thought to reflect autocrine or paracrine stimulation through the EGF receptor. Ligands for the EGF receptor, including transforming growth factor-α, amphiregulin and heparin-binding EGF are elevated in psoriatic lesional skin and/or in psoriatic keratinocytes relative to control skin/cells (Gottlieb, et al., 1988; Elder, et al., 1989; Cook, et al., 1992; Piepkorn, et al., 1998; Piepkorn, et al., 2003). Amphiregulin may be particularly important, since transgenic mice over-expressing the amphiregulin gene develop a psoriasiform phenotype (Cook, et al., 1997; Cook, et al., 2004). Furthermore, our own recent studies have shown that interfering with human amphiregulin in the human skin – scid mouse model suppresses hyperplasia in the transplanted skin (Bhagavathula, et al., 2005). While thiazolidinediones do not appear to interfere with the initial ligand-receptor – mediated events (Bhagavathula, et al., 2004), by interfering with down-stream signaling events
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(i.e., ERK1,2 activation), these agents prevent the cellular responses to EGF receptor activation that control proliferation.
While the findings presented here strongly suggest that a direct effect of the thiazolidinediones on keratinocyte function contributes to their anti-psoriatic activity, this does not rule out additional effects mediated through immune modulation. A number of PPAR-γ agonists, including members of the thiazoladinedine family, have been shown to be beneficial in immune/inflammatory conditions (Augstein, et al., 2003; Culver, et al., 2004; Schaefer, et al., 2005; Hasegawa, et al., 2005). Efficacy is presumed to reflect modulation of the Th1/Th2 cytokine balance. Obviously, anti-proliferative activity for keratinocytes and modulation of immune function are not mutually exclusive. Additional studies will be necessary to completely delineate the relative importance of these activities to the overall anti-psoriatic activity of the thiazolidinedione compounds.
In summary, the findings presented here demonstrate that a novel, high-affinity PPARγ ligand, consisting of an adamantyl-coupled thiazolidinedione, reduces epidermal hyperplasia of human skin transplanted to scid mice.
Inhibition is observed following topical treatment,
suggesting that effective anti-psoriatic therapy may be provided without the inherent consequences observed with systemic use of this class of agents. Given these findings, it is reasonable to consider further development of BP-1107 as a topical agent for use in psoriasis and perhaps in other conditions in which epidermal hyperplasia constitutes a major part of the pathology.
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Footnotes:
This study was supported in part by grants R41 AR44767 and R41 AR 50330 from the National Institutes of Health, Bethesda, MD.
This is to indicate that two of the authors (Harrihar A Pershadsingh and Theodore W. Kurtz) have equity in Bethesda Pharmaceuticals, Inc., and could have financial gain if the drug under study proves to be effective. Within the past three years, Dr. Ellis has served as an unpaid consultant to Bethesda Pharmaceuticals, Inc.
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FIGURE LEGENDS
Figure 1. Structure of BP-1107
Figure 2. Effect of BP-1107 on human psoriatic skin transplanted to scid mice.
Upper
panels: Gross appearance of human psoriatic skin 35 days after transplantation to a scid mouse and topical treatment for 21 days with 100 µl/day of vehicle alone (A) or 100 µM BP-1107 (B). Middle Panels: Histological appearance of human psoriatic skin 35 days after transplantation to scid mice and topical treatment for 21 days with 100 µl/day of vehicle alone (C) or 100 µM BP1107 (D). Quantitative information is presented in Panel E. Values shown represent mean epidermal thickness + standard errors based on at least four measurements in each of three sections from two different transplants per subject and two subjects. Statistical significance was determined using the Student t-test. Differences were significant at the p
