DRM02, a novel phosphodiesterase-4 inhibitor with cutaneous anti-inflammatory activity.

Chronic inflammatory skin disorders are frequently associated with impaired skin barrier function. Selective phosphodiesterase-4 (PDE4) inhibition constitutes an effective therapeutic strategy for the treatment of inflammatory skin diseases. We now report the pharmacological anti-inflammatory profile of DRM02, a novel pyrazolylbenzothiazole derivative with selective in vitro inhibitory activity toward PDE4 isoforms A, B and D. DRM02 treatment of cultured primary human and mouse epidermal keratinocytes interfered with pro-inflammatory cytokine production elicited by interleukin-1α and tumor necrosis factor-α. Similarly, DRM02 inhibited the production of pro-inflammatory cytokines by human peripheral blood mononuclear cells ex vivo and cultured THP-1 monocyte-like cells, with IC50 values of 0.6-14 µM. These anti-inflammatory properties of DRM02 were associated with dose-dependent repression of nuclear factor-κB (NF-κB) transcriptional activity. In skin inflammation in vivo mouse models, topically applied DRM02 inhibited the acute response to phorbol ester and induced Th2-type contact hypersensitivity reactivity. Further, DRM02 also decreased cutaneous clinical changes and expression of Th17 immune pathway cytokines in a mouse model of psoriasis evoked by repeated topical imiquimod application. Thus, the overall pharmacological profiling of the PDE4 inhibitor DRM02 has revealed its potential as a topical therapy for inflammatory skin disorders and restoration of skin homeostasis.

Inhibition of Sebum Production with the Acetyl Coenzyme A Carboxylase Inhibitor Olumacostat Glasaretil.

Olumacostat glasaretil (OG) is a small molecule inhibitor of acetyl coenzyme A (CoA) carboxylase (ACC), the enzyme that controls the first rate-limiting step in fatty acid biosynthesis. Inhibition of ACC activity in the sebaceous glands is designed to substantially affect sebum production, because over 80% of human sebum components contain fatty acids. OG inhibits de novo lipid synthesis in primary and transformed human sebocytes. TrueMass Sebum Panel analyses showed a reduction in saturated and monounsaturated fatty acyl chains across lipid species, including di- and triacylglycerols, phospholipids, cholesteryl esters, and wax esters in OG-treated sebocytes. There was no shift to shorter acyl chain lengths observed, suggesting that the fatty acid chain elongation process was not affected. OG is a pro-drug of the ACC inhibitor 5-(tetradecyloxy)-2-furoic acid and was designed to enhance delivery in vivo. Topical application of OG but not 5-(tetradecyloxy)-2-furoic acid significantly reduced hamster ear sebaceous gland size, indicating that this pro-drug approach was critical to obtain the desired activity in vivo. High-performance liquid chromatography analyses of hamster ear extracts showed that OG treatment increased ACC levels and the ratio of acetyl-CoA to free CoA in these animals, indicating increased fatty acid oxidation. These changes are consistent with ACC inhibition. Matrix-assisted laser desorption/ionization imaging showed that OG applied onto Yorkshire pig ears accumulated in sebaceous glands relative to the surrounding dermis. Sebaceous gland ACC represents an attractive therapeutic target given its central role in formation of sebum, a key factor in acne pathogenesis.

Influence of interleukin-1alpha on androgen receptor expression and cytokine secretion by cultured human dermal papilla cells.

Dermal papilla cells (DPC) control the growth character of the hair follicle through their elaboration of mitogenic factors and extracellular matrix components. Further, the dermal papilla is a primary site of androgen action in the hair follicle. Interleukin-1alpha (IL-1alpha) is prominent in skin wounding and inflammatory responses although regarded as a negative hair growth regulator. We studied the effect of IL-1alpha and the potent androgen 5alpha-dihydrotestosterone (DHT) on the expression of the androgen receptor (AR) and various factors secreted by cultured human temporal scalp DPC. IL-1alpha triggered cellular changes consistent with nuclear factor-kappaB pathway activation as well as reduced AR mRNA and protein expression levels for DHT-stimulated DPC. This cytokine also increased DPC supernatant keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) concentrations. IL-1alpha did not influence DPC supernatant levels of transforming growth factor-beta1, a negative hair growth regulator. The stimulatory effect of IL-1alpha on DPC VEGF, GM-CSF, KGF, and IL-8 expression was also evident at the mRNA level for these cytokines. IL-1alpha also increased mRNA transcript levels of protease-nexin-1, a secreted serine protease inhibitor expressed in the dermal papilla of anagen-stage hair follicles. Although DHT did not affect supernatant cytokine concentrations, the androgen altered mRNA transcript levels of several factors for DPC co-stimulated with IL-1alpha. In consideration of its in vitro activity profile, IL-1alpha may be an important modifier of dermal papilla activity as well as potentially influence androgen-regulated gene expression in DPC.

Ultraviolet B light stimulates interleukin-20 expression by human epithelial keratinocytes.

The proinflammatory cytokine interleukin-20 (IL-20) may exert the majority of its activity in the skin. We examined the effect of various treatments including several forms of phototherapy on IL-20 expression using cultured normal human epithelial keratinocytes (NHEK). Broadband UVB light, recombinant (r) IL-1 and rIL-8 increased, while hydrocortisone reduced, NHEK supernatant IL-20 levels. Elevation of NHEK IL-20 mRNA and maximal supernatant IL-20 levels occurred with a UVB light dose (40 mJ cm(-2)) that reduced cell viability by approximately 50%. While this UVB light dose also elevated supernatant IL-1 alpha and IL-8 levels, antibody neutralization studies indicated that neither of these cytokines was directly responsible for this increase in IL-20 expression. However, the elevation in IL-20 levels was fully inhibited by the p38 mitogen-activated protein kinase (MAPK) inhibitor SB-203580, suggesting involvement of this stress signaling pathway in this UVB light response. Photodynamic therapy (PDT) with the photosensitizer lemuteporfin, UVA light, cisplatin, lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha) or recombinant interferon-gamma (rIFN-gamma) either had little effect or decreased NHEK supernatant IL-20 levels. Reduced IL-20 levels paralleled the cytotoxic actions of PDT, UVA light or cisplatin and the antiproliferative effect of rIFN-gamma. Neither rIL-20 supplementation nor anti-IL-20 antibody treatments affected cell viability indicating that soluble IL-20 did not affect the short-term survival of UVB light-irradiated NHEK. Stimulation of IL-20 expression in keratinocytes by UVB light suggests that this cytokine might participate in skin responses to this ever-present environmental factor and potentially has a role in UV light-associated dermatoses.

Bcl-2 and Bcl-xL overexpression inhibits cytochrome c release, activation of multiple caspases, and virus release following coxsackievirus B3 infection.

Coxsackievirus B3, a cytopathic virus in the family Picornaviridae, induces degenerative changes in host cell morphology. Here we demonstrate cytochrome c release and caspases-2, -3, -6, -7, -8, and -9 processing. Enforced Bcl-2 and Bcl-xL expression markedly reduced release of cytochrome c, presentation of the mitochondrial epitope 7A6, and depressed caspase activation following infection. In comparison, cell death using TRAIL ligand caused caspase-8 processing prior to cytochrome c release and executioner caspases and cell death was only partially rescued by Bcl-2 and Bcl-xL overexpression. Disruption of the mitochondrial inner membrane potential following CVB3 infection was not inhibited by zVAD.fmk treatment. Bcl-2 or Bcl-xL overexpression or zVAD.fmk treatment delayed the loss of host cell viability and decreased progeny virus release following infection. Our data suggest that mitochondrial release of cytochrome c may be an important early event in caspase activation in CVB3 infection, and, as such, may contribute to the loss of host-cell viability and progeny virus release.

Status of therapies in development for the treatment of age-related macular degeneration.

Age-related macular degeneration (AMD) is among the leading causes of visual impairment in the elderly. The FDA has approved only two treatments, laser photocoagulation and Visudyne photodynamic therapy (PDT), approved for the wet form of AMD, a progressive condition characterized by the presence of choroidal neovascularization (CNV). Current pharmaceutical activities aimed at the treatment of wet-type AMD are largely focused on the development of anti-angiogenic drugs that would inhibit further CNV formation or even reduce existing CNV. However, other lines of attack for the treatment of wet AMD include anti-inflammatory agents as well as new PDT agents. This review will summarize ongoing activities for these different approaches.

Selective action of the photosensitizer QLT0074 on activated human T lymphocytes.

A new photosensitizer, presently designated QLT0074, may have the potential for the treatment of immune and nonimmune conditions with photodynamic therapy (PDT). The activity of QLT0074 was tested against human peripheral blood T cells and Jurkat T lymphoma cells. At low nanomolar concentrations of QLT0074 in combination with blue light, apoptosis was rapidly induced in Jurkat and blood T cells in vitro as indicated by the expression of the apoptosis-associated mitochondrial 7A6 marker and Annexin-V labeling. Further studies performed with Jurkat T cells showed that PDT-induced apoptosis with QLT0074 was associated with caspase-3 activation and the cleavage of the caspase substrate poly(adenosine diphosphate-ribose)polymerase. Flow cytometry studies revealed that blood T cells with high expression of the interleukin-2 receptor (CD25) took up greater amounts of QLT0074 and were eliminated to a greater extent with PDT than T cells with low levels of this activation marker. This selective action of PDT was confirmed by similar reductions in the percentage of T cells that expressed other activation-related markers, including very late activation antigen-4 (CD49d), human leukocyte antigen DR (HLA-DR), intercellular adhesion molecule-1 (CD54) and Fas (CD95). For activated T cells treated with a specific dose of QLT0074 and light 24 h earlier, CD25 expression density was significantly less, whereas CD54, CD95 and HLA-DR levels were similar to those for control cells treated with light alone. This work shows that PDT with QLT0074 exerts selective, dose-related effects on T cells in vitro.

Rostaporfin (Miravant Medical Technologies).

Pharmacia Corp, under license from Miravant Medical Technologies (formerly PDT Inc), is developing rostaporfin (SnET2, Purlytin), a light-activated cytotoxic drug developed as part of Miravant's PhotoPoint photodynamic therapy (PDT) program, for the potential treatment of wet age-related macular degeneration (AMD) [180314]. In January 2002, results of phase III trials indicated that rostaporfin had not met the primary efficacy endpoint for the wet form of AMD. At this time, a full review of the data was to be undertaken, and decisions about future development of the drug were to be made after additional analyses had been completed [435577]. The original licensing agreements included the development of rostaporfin for several ophthalmology, oncology and urology indications [289078], and for dermatological applications including certain skin cancers [267521]. However, in August 1998, Miravant reported that it no longer intended to pursue cutaneous metastatic breast cancer (CMBC), in order to focus on AMD [439372], [439384]. Also in 1998, studies in basal cell carcinoma and AIDS-related Kaposi's sarcoma were discontinued because of business considerations [439372]. Rostaporfin is activated by red light with a wavelength of 664 nm. It is injected into the patient, where it distributes and selectively binds to plasma lipoproteins, which are produced in high concentrations by hyperproliferating cells such as cancer cells. After 24 h, the targeted cells are stimulated by red light to activate the compound. This triggers the formation of toxic free radical species that destroy the cells without affecting the surrounding normal tissue [85236]. In January 2002, Credit Suisse First Boston estimated sales for Pharmacia of 40 million US dollars in 2003 and 80 million US dollars in 2004 [436118], while in the same month, Argus Research predicted peak annual sales for Pharmacia of less than 250 million US dollars[436279].