Revolutionizing Psoriasis Topical Treatment: Enhanced Efficacy Through Ceramide/Phospholipid Composite Cerosomes Co-Delivery of Cyclosporine and Dithranol: In-Vitro, Ex-Vivo, and in-Vivo Studies.

Purpose: Improving the treatment of psoriasis is a serious challenge today. Psoriasis is an immune-mediated skin condition affecting 125 million people worldwide. It is commonly treated with cyclosporine-A (CsA) and dithranol (DTH). CsA suppresses the activation of T-cells, immune cells involved in forming psoriatic lesions. Meanwhile, DTH is a potent anti-inflammatory and anti-proliferative drug that effectively reduces the severity of psoriasis symptoms such as redness, scaling, and skin thickness. CsA and DTH belong to BCS class II with limited oral bioavailability. We aim to develop a drug delivery system for topical co-delivery of CsA and DTH, exploring its therapeutic potential. Methods: Firstly, we developed a niosomal drug delivery system based on ceramide IIIB to form Cerosomes. Cerosomes were prepared from a mixture of Ceramide, hyaluronic acid, and edge activator using a thin-film hydration technique. To co-deliver CsA and DTH topically for the treatment of psoriasis. These two hydrophobic drugs encapsulated into our synthesized positively charged particle cerosomes. Results:  Cerosomes had an average particle size of (222.36 nm± 0.36), polydispersity index of (0.415±0.04), Entrapment Efficiency of (96.91%± 0.56), and zeta potential of (29.36±0.38mV) for selected formula. In vitro, In silico, in vivo, permeation, and histopathology experiments have shown that cerosomes enhanced the skin penetration of both hydrophobic drugs by 66.7% compared to the CsA/DTH solution. Imiquimod (IMQ) induced psoriatic mice model was topically treated with our CsA/DTH cerosomes. We found that our formulation enhances the skin penetration of both drugs and reduces psoriasis area and severity index (PASI score) by 2.73 times and 42.85%, respectively, compared to the CsA/DTH solution. Moreover, it reduces the levels of proinflammatory cytokines, TNF-α, IL-10, and IL-6 compared to CsA/DTH solution administration. Conclusion: The Cerosomes nano-vesicle-containing CsA/DTH represents a more promising topical treatment for psoriasis, giving new hope to individuals with psoriasis, compared to commercial and other conventional alternatives.

Induction of IL-1ß and antimicrobial peptides as a potential mechanism for topical dithranol.

Topical dithranol is effective in autoimmune conditions like alopecia areata, inducing hair regrowth in a high percentage of cases. Exact mechanisms of dithranol in alopecia areata, with seemingly healthy epidermis besides altered hair follicles, are not well understood. To better understand dithranol's mechanisms on healthy skin, we analysed its effect on normal murine as well as xenografted human skin. We found a strong increase in mRNA expression of anti-microbial peptides (AMPs) (eg Lcn2, Defb1, Defb3, S100a8, S100a9), keratinocyte differentiation markers (eg Serpinb3a, Flg, Krt16, Lce3e) and inflammatory cytokines (eg Il1b and Il17) in healthy murine skin. This effect was paralleled by inflammation and disturbed skin barrier, as well as an injury response resulting in epidermal hyperproliferation, as observed in murine and xenografted adult human skin. This contact response and disturbed barrier induced by dithranol might lead via a vicious loop between AMPs such as S100a8/a9 (that led to skin swelling itself after topical application) and cytokines such as IL-1ß to an immune suppressive environment in the skin. A better understanding of the skin's physiologic response to dithranol may open up new avenues for the establishment of novel therapeutics (including AMP-related/interfering molecules) for certain skin conditions, such as alopecia areata.

Chemical Modulators of Fibrinogen Production and Their Impact on Venous Thrombosis.

Thrombosis is a leading cause of morbidity and mortality. Fibrinogen, the soluble substrate for fibrin-based clotting, has a central role in haemostasis and thrombosis and its plasma concentration correlates with cardiovascular disease event risk and a prothrombotic state in experimental models. We aimed to identify chemical entities capable of changing fibrinogen production and test their impact on experimental thrombosis. A total of 1,280 bioactive compounds were screened for their ability to alter fibrinogen production by hepatocyte-derived cancer cells and a selected panel was tested in zebrafish larvae. Anthralin and all-trans retinoic acid (RA) were identified as fibrinogen-lowering and fibrinogen-increasing moieties, respectively. In zebrafish larvae, anthralin prolonged laser-induced venous- occlusion times and reduced thrombocyte accumulation at injury sites. RA had opposite effects. Treatment with RA, a nuclear receptor ligand, increased fibrinogen mRNA levels. Using an antisense morpholino oligonucleotide to deplete zebrafish fibrinogen, we correlated a shortening of laser-induced venous thrombosis times with RA treatment and fibrinogen protein levels. Anthralin had little effect on fibrinogen mRNA in zebrafish larvae, despite leading to lower detectable fibrinogen. Therefore, we made a proteomic scan of anthralin-treated cells and larvae. A reduced representation of proteins linked to the canonical secretory pathway was detected, suggesting that anthralin affects protein secretion. In summary, we found that chemical modulation of fibrinogen levels correlates with measured effects on experimental venous thrombosis and could be investigated as a therapeutic avenue for thrombosis prevention.

The African natural product knipholone anthrone and its analogue anthralin (dithranol) enhance HIV-1 latency reversal.

A sterilizing or functional cure for HIV is currently precluded by resting CD4+ T cells that harbor latent but replication-competent provirus. The "shock-and-kill" pharmacological ap-proach aims to reactivate provirus expression in the presence of antiretroviral therapy and target virus-expressing cells for elimination. However, no latency reversal agent (LRA) to date effectively clears viral reservoirs in humans, suggesting a need for new LRAs and LRA combinations. Here, we screened 216 compounds from the pan-African Natural Product Library and identified knipholone anthrone (KA) and its basic building block anthralin (dithranol) as novel LRAs that reverse viral latency at low micromolar concentrations in multiple cell lines. Neither agent's activity depends on protein kinase C; nor do they inhibit class I/II histone deacetylases. However, they are differentially modulated by oxidative stress and metal ions and induce distinct patterns of global gene expression from established LRAs. When applied in combination, both KA and anthralin synergize with LRAs representing multiple functional classes. Finally, KA induces both HIV RNA and protein in primary cells from HIV-infected donors. Taken together, we describe two novel LRAs that enhance the activities of multiple "shock-and-kill" agents, which in turn may inform ongoing LRA combination therapy efforts.

Dithranol: An Insight into its Novel Delivery Cargos for Psoriasis Management.

OBJECTIVE: Dithranol (DTH) is a well-known moiety that has long been used promisingly to impede and treat skin disorders, particularly psoriasis. Nowadays, a rekindled interest in the use of DTH for this disorder has been observed. Side effects associated with conventional topical formulations of this moiety have aroused the interest of the scientific community in investigating novel cargos of DTH for psoriasis management. RESULTS: Previous research has evidenced the anti-inflammatory and anti-proliferating potential of DTH. Numerous studies have indicated that DTH inhibits polymorphonuclear (PMN) leucocyte, modulates epidermal cell receptors and promotes anti-psoriatic action. However, some deterrent factors like poor solubility, stability, toxicity, staining and skin irritation hamper its use as a potential therapeutic agent. With the adoption of novel drug delivery technologies, the above mentioned inherent limitations of DTH have been compensated to reestablish this drug moiety. CONCLUSION: This article reviews novel drug delivery aspects, safety concerns, clinical evidence, current status, and future opportunities of DTH in the management of psoriasis. Further, it will update researchers on this promising drug moiety, which is free from systemic adverse responses in comparison to other therapeutic molecules like steroids, for psoriasis treatment.

Dithranol targets keratinocytes, their crosstalk with neutrophils and inhibits the IL-36 inflammatory loop in psoriasis.

Despite the introduction of biologics, topical dithranol (anthralin) has remained one of the most effective anti-psoriatic agents. Serial biopsies from human psoriatic lesions and both the c-Jun/JunB and imiquimod psoriasis mouse model allowed us to study the therapeutic mechanism of this drug. Top differentially expressed genes in the early response to dithranol belonged to keratinocyte and epidermal differentiation pathways and IL-1 family members (i.e. IL36RN) but not elements of the IL-17/IL-23 axis. In human psoriatic response to dithranol, rapid decrease in expression of keratinocyte differentiation regulators (e.g. involucrin, SERPINB7 and SERPINB13), antimicrobial peptides (e.g. ß-defensins like DEFB4A, DEFB4B, DEFB103A, S100 proteins like S100A7, S100A12), chemotactic factors for neutrophils (e.g. CXCL5, CXCL8) and neutrophilic infiltration was followed with much delay by reduction in T cell infiltration. Targeting keratinocytes rather than immune cells may be an alternative approach in particular for topical anti-psoriatic treatment, an area with high need for new drugs.

Potent inhibitors of equine steroid isomerase EcaGST A3-3.

Equine glutathione transferase A3-3 (EcaGST A3-3) belongs to the superfamily of detoxication enzymes found in all higher organisms. However, it is also the most efficient steroid double-bond isomerase known in mammals. Equus ferus caballus shares the steroidogenic pathway with Homo sapiens, which makes the horse a suitable animal model for investigations of human steroidogenesis. Inhibition of the enzyme has potential for treatment of steroid-hormone-dependent disorders. Screening of a library of FDA-approved drugs identified 16 out of 1040 compounds, which at 10 µM concentration afforded at least 50% inhibition of EcaGST A3-3. The most potent inhibitors, anthralin, sennoside A, tannic acid, and ethacrynic acid, were characterized by IC50 values in the submicromolar range when assayed with the natural substrate Δ5-androstene-3,17-dione.

Anthralin modulates the expression pattern of cytokeratins and antimicrobial peptides by psoriatic keratinocytes.

BACKGROUND: Psoriasis is an inflammatory skin disease with aberrant keratinocyte proliferation, presumably as a result of immune cell activation. Th17 cytokines like IL-17A and IL-22 are critically implicated in epidermal thickening, altered keratinocyte differentiation and production of innate factors such as antimicrobial peptides. Psoriasis treatment options include modern targeted therapies using anti-cytokine antibodies and traditional non-targeted treatments like anthralin (dithranol). While the mode of action of anti-cytokine antibodies is defined, the effects of topical anthralin on psoriatic skin are not fully understood. OBJECTIVE: This study aims to unravel the direct effects of anthralin on keratinocyte proliferation, differentiation and production of psoriasis-associated factors. METHODS: We tested the effects of anthralin on cell proliferation, cytokeratin expression and changes in the expression of antimicrobial peptides using primary keratinocytes and 3D psoriasis tissue models with and without stimulation of the psoriasis-promoting cytokines IL-17A and IL-22. Moreover, we compared the findings derived from monolayer and multilayer cultures to data derived from lesional skin of patients with psoriasis before and under treatment with anthralin. RESULTS: Our study shows that anthralin directly induces cell apoptosis in vitro in monolayer cultures but not in 3D psoriasis tissue models treated with IL-17A and IL-22. Yet, keratinocyte proliferation as determined by Ki-67 staining is impaired by anthralin in vivo. In lesional skin but not in 3D psoriasis tissue models anthralin rapidly normalizes cytokeratin (CK)16 expression. Furthermore, anthralin directly inhibits DEFB4 expression in vitro and in vivo, while other antimicrobial peptides and cytokines studied like IL-6 and IL-8 are regulated differently in vitro and in vivo. CONCLUSIONS: Our results show that anthralin directly regulates DEFB4A expression. However, its beneficial effects on psoriasis cannot be explained by direct effects on keratinocyte differentiation or cytokine expression.

Serum Fatty Acid-Binding Protein 4 is Increased in Patients with Psoriasis.

Psoriasis is associated with metabolic syndrome and cardiovascular disease. Fatty acid-binding proteins (FABP) have been recognized as predictors of these systemic disorders. The aim of this study was to assess correlations between levels of serum heart and adipocyte fatty acid-binding proteins (FABP3, FABP4) and disease severity, indicators of inflammation or metabolic disturbances, and topical treatment in psoriatic patients. Thirty-seven patients with relapse of plaque-type psoriasis and 16 healthy volunteers were recruited. Blood samples were collected before and after 14 days of therapy. Serum FABP concentrations were examined by enzyme-linked immunosorbent assay for correlation with Psoriasis Area and Severity Index (PASI), body mass index (BMI), inflammatory or metabolic parameters, and treatment used. The median FABP4 serum levels were significantly increased (p = 0.038) in psoriatic patients, while FABP3 levels did not differ (p = 0.47) compared to the controls. No significant correlations were noted between the proteins and PASI, C-reactive protein (CRP), BMI, or levels of glucose or lipids. FABP3 significantly correlated with white blood count (p = 0.03) and aspartate aminotransferase (p = 0.04). After topical treatment, there was no significant change in serum FABP3 [11.5 (4.9-30.3) vs. 12.9 (3.5-30.3) ng/ml] (p = 0.96), whereas FABP4 was decreased [27,286 (20,344-32,257) vs. 23,034 (18,320-29,874) pg/ml] (p = 0.12), losing its basal significance. FABP4 may be a marker of psoriasis, and FABP3 may be associated with inflammation or liver disorders in psoriatic patients. FABP do not appear to be useful for determining disease severity or the effectiveness of antipsoriatic treatment.

Antibacterial activities of the methanol extracts, fractions and compounds from Harungana madagascariensis Lam. ex Poir. (Hypericaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Harungana madagascariensis Lam. ex Poir. (Hypericaceae) is used in folk medicine to treat a variety of human ailments, mainly antibacterial, antifungal, antiviral and viral infections. In the present study, the methanol extract from the leaves (HML) and bark (HMB) of this plant as well as fractions (HMBa-c), sub-fractions (HMBa1-5) and compounds isolated from HMBa and HMBb namely betulinic acid (1), madagascin (2), ferruginin A (3) and Kaempferol-3-O-ß-d-glucopyranoside (4) were tested for their antimicrobial activities against a panel of 28 g-negative bacteria including multidrug resistant (MDR) phenotypes. MATERIALS AND METHODS: The broth microdilution method was used to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the above samples; column chromatography was used for the fractionation and purification of the bark extract whilst the chemical structures of compounds were determined using spectroscopic techniques. RESULTS: Crude extract HMB together with fraction HMBa and sub-fraction HMBa3 were active on the 28 tested bacterial strains. HML as well as fractions HMBb, HMBc and sub-fractions HMBa1, HMBa2, HMBa4 and HMBa5 were selectively active. MIC values below or equal to 1024µg/mL were recorded with these samples on 92.9% (for HML and HMBa 4), 82.1% (for HMBb), 78.6% (for HMBa2), 50.0% (for HMBa5) and 42.9% (for HMBc) tested bacteria. For crude material, the lowest MIC value below 8µg/mL was obtained with HMB against Escherichia coli ATCC10536 and W3110 strains, and with sub-fraction HMBa3 against Klebsiella pneumoniae K2 strains. MIC values below 10µg/mL were recorded with compound 3 against E. coli ATCC10536, Enterobacter aerogenes ATCC13048 and EA294, Pseudomonas aeruginosa PA01, K. pneumoniae K2 and Kp55 and Enterobacter cloacae BM67. CONCLUSIONS: Harungana madagascariensis is a potential source of antimicrobial drugs to fight against MDR bacteria. The anthranol 3 is the main antibacterial constituents of the bark of the plant. HMB and compound 3 deserve further investigations to develop natural drug to combat Gram-negative bacteria and otherwise MDR phenotypes.

Dithranol treatment of plaque-type psoriasis increases serum TNF-like weak inducer of apoptosis (TWEAK).

PURPOSE: TNF-like weak inducer of apoptosis (TWEAK) mediates not only apoptosis, but also inflammation, cell growth and angiogenesis. The role of TWEAK in psoriasis remains unknown. The aim of the study was to assess serum levels of TWEAK in psoriatic patients before and after topical treatment with dithranol in relation to the clinical activity of the disease. MATERIAL AND METHODS: Serum samples were collected from 40 patients with plaque type psoriasis before and after topical treatment with dithranol. The concentrations of serum TWEAK were measured by ELISA and next compared with 16 healthy controls. The data were analyzed with respect to Psoriasis Area and Severity Index (PASI). RESULTS: Baseline serum TWEAK concentrations of psoriatic patients (685±166pg/ml) were significantly greater compared to healthy controls (565±110pg/ml). Topical treatment resulted in further increase in serum TWEAK (749±179pg/ml; p<0.01). In case of patients with initial serum TWEAK concentrations above the median, PASI after topical treatment was lower compared to the individuals with initial TWEAK below the median. CONCLUSION: According to the study, serum Tweak was increased in psoriasis patients compared with controls. Moreover, dithranol topical treatment caused further increase in serum TWEAK. Also, a higher effectiveness of topical treatment was observed in case of patients with higher initial TWEAK concentrations. The results suggest a potential role of TWEAK in psoriasis therapy.

α-Glucosidase Inhibitory Prenylated Anthranols from Harungana madagascariensis.

Four new prenylated anthranols, harunganols C-F (1-4), along with kenganthranol A (5), harunganin (6), and ferruginin A (7), were identified from the leaves of Harungana madagascariensis. The structures of compounds 2, 5, and 7 were confirmed by single-crystal X-ray diffraction analysis. Compound 1 is a unique symmetrical anthranol dimer connected via a CH2 group. Compound 4 possesses a unique C-10 hemiketal group. All anthranols were evaluated for their α-glucosidase inhibitory activities. They displayed a higher potency compared to acarbose except for 3 and 4. In particular, harunganol C (1) showed an IC50 value of 1.2 µM.

Synthesis and biological evaluation of new C-10 substituted dithranol pleiotropic hybrids.

Selective alkylation of the antipsoriatic drug dithranol (DTR) at C-10 with tert-butyl bromoacetate, followed by acid-mediated deprotection, produced the corresponding carboxylic acid 4 which was coupled with selectively protected polyamines (PAs), such as putrescine (PUT), spermidine (SPD) and spermine (SPM), dopamine and aliphatic amines and substituted benzylamines producing a series of DTR-PA hybrids, after acid-mediated deprotection, as well as simple amides. The compounds were tested as antioxidants and inhibitors of lipoxygenase (LOX). The amides 4,4'-dimethoxybenzhydrylamide 13 (86% and 95%), 2,4-dimethoxybenzylamide 12 (87% and 81%) and dodecylamide 9 (98% and 74%), and the hybrid DTR-SPM (7) (93% and 87%), showed the highest antioxidant activity in the DPPH and AAPH assays, whereas the most potent inhibitors of LOX were amide 13 (IC50=7 µM), the benzylamide 10 (IC50=7.9 µM) and the butylamide 8 (IC50=10 µM). Molecular binding studies showed that binding of these derivatives into the hydrophobic domain blocks approach of substrate to the active site, inhibiting soybean LOX. Amide 13 presented the highest anti-inflammatory activity (79.7%). The DTR moiety was absolutely necessary for securing high anti-inflammatory potency. Ethyl ester 3 (IC50=0.357 µM) and the amides 9 (IC50=0.022 µM) and 13 (IC50=0.56 µM) exhibited higher antiproliferative activity than DTR (IC50=0.945 µM) on HaCaT keratinocytes whereas amide 13 generally presented better cytocompatibility. Amide 13 is a very promising lead compound for further development as an anti-inflammatory and antiproliferative agent.

Em08red, a dual functional antiproliferative emodin analogue, is a downregulator of ErbB2 expression and inducer of intracellular oxidative stress.

Expression of ErbB2 protein is inversely correlated with the prognosis in cancer patients. Consequently, strategies targeting ErbB2 remain an attractive option in treating several types of malignancies, including oral cancer. In addition, many studies have shown that emodin and emodin derivatives are able to inhibit growth of ErbB2-overexpressing tumor cells. In this study, a series of computer modeling-generated emodin analogues were synthesized and tested for their antiproliferative activity against oral cancer cell lines overexpressing ErbB2. Among these analogues, em08red (1,8-dihydroxy-9(10H)-anthracenone) demonstrated potent antiproliferative activity against all three tested ErbB2-overexpressing cell lines, ie, FaDu, HSC3, and OECM1. Treatment with em08red significantly downregulated activation of ErbB2 as well as the ErbB2 protein expression level in the tested cell lines and induced G2 arrest. Antiapoptosis protein (Bcl-xl and Bcl-2) expression levels were also downregulated, and active caspase-3 and caspase-9 was detected in cells after treatment with em08red. Moreover, treatment with em08red stimulated production of cytotoxic reactive oxygen species in treated cells, and this could be partially reversed by pretreatment with N-acetylcysteine. Overall, we demonstrated inhibition of ErbB2 function and induction of reactive oxygen species in tumor cells by em08red, which prevented proliferation of tumor cells and induced apoptotic cell death.

Anthralin/dithranol in dermatology.

Anthralin (1,8-dihydroxy-9anthrone, dithranol) was first synthesized as a derivative of chrysarobin, prepared from the araroba tree in Brazil over a century ago. Drawbacks to the use of anthralin include irritation and discoloration of the skin. This property of the molecule prompted workers to investigate details of its pharmacology, mode of action, and indications. The major point of this article is to highlight and revisit these aspects for pertinent future use. Therefore, it is worthwhile to consider that the drug is relatively innocuous, yet effective, and is devoid of any systemic side effects in contrast to a wide variety of systemic and topical therapies available for psoriasis and other dermatoses.

An investigation of the effects of dithranol-induced apoptosis in a human keratinocyte cell line.

OBJECTIVES: Dithranol, one of the most successful topical agents for the treatment of psoriasis, has been shown to exert its therapeutic effect by inducing keratinocyte apoptosis. To gain further insights into dithranol-induced apoptotic events in vitro, a detailed investigation of its time- and dose-dependent effects has been performed through the evaluation of selected apoptotic markers, using a human keratinocyte cell line (HaCaT) as a model. METHODS: The time- and dose-dependent effects of dithranol on a human keratinocyte cell line (HaCaT) were investigated through the evaluation of a series of apoptotic markers; morphological changes (electron microscopy), phosphatidylserine externalisation (flow cytometry), and caspase-3/7 activation. KEY FINDINGS: The dithranol-induced apoptotic cascade was found to follow a well-defined dose and time-course, with the concentration and the period of exposure to the drug acting as the two major factors influencing the events and nature of cell death. The earliest apoptotic event detected was caspase activation (after 6 h), followed by the occurrence of phosphatidylserine externalisation (after 9 h) and subsequently the morphological characteristics associated with early and late stage apoptosis/necrosis (after 12 h). CONCLUSIONS: This study has elucidated the dose- and time-response effects of dithranol-induced apoptosis in human keratinocytes in vitro.

Dithranol downregulates expression of Id1 mRNA in human keratinocytes in vitro.

The precise causes of psoriasis, a chronic skin disorder characterized by hyperproliferation of keratinocytes and incomplete keratinization, are unclear. It is known that expression of helix-loop-helix transcription factor Id1, which functions as an inhibitor of differentiation, is upregulated in psoriatic skin. We investigated the effect of the antipsoriatic drug dithranol on mRNA and protein expression levels of Id1 in the HaCaT keratinocyte cell line. Cultured HaCaT cells were treated with 0-0.5 µg/mL dithranol for 30 min. After 2 and 4 h, total cellular RNA and total proteins were isolated from HaCaT cells, and quantitative real-time reverse transcriptase (RT-PCR) and Western blot were used to determine the mRNA and protein levels of Id1, respectively. Changes in normalized Id1 mRNA levels were observed only after 4 h of dithranol treatment. There was reduced expression of Id1 mRNA transcripts in the HaCaT cells treated with 0.1 µg/mL dithranol, but the reduction was not significant. The expression of Id1 mRNA was significantly downregulated (almost 50%) when 0.25 or 0.5 µg/mL dithranol was applied to the HaCaT cells. However, the normalized Id1 protein levels were not significantly affected. The molecular mechanisms underlying this finding should be investigated further to help determine the therapeutic action of this drug.

Effect of anthralin on cell viability in human prostate adenocarcinoma.

The study revealed the key role of serine protease hepsin activity in transition of in situ prostate adenocarcinoma into the metastasizing form. Inhibition of hepsin activity suppresses the invasive growth of the tumor. Hepsin is an convenient target for pharmacological agents, so the study of its inhibitory mechanisms is a promising avenue in drug development. Assay of proteolytic activity in various tumor cell lines in vitro showed that this activity in prostate adenocarcinoma cells significantly surpasses proteolytic activity in other examined tumor cell lines. Selective cytotoxic action of anthralin, an inhibitor of hepsin activity, on human adenocarcinoma cells was demonstrated in comparison with other tumor cell lines.

Effects of the antipsoriatic drug dithranol on E2A and caspase-9 gene expression in vitro.

Although the precise causes of psoriasis remain to be elucidated, psoriasis has been known as a disorder in which factors in the immune system, enzymes and other biochemical substances that regulate skin cell division are functionally imbalanced, thereby resulting in rapid proliferation of keratinocytes and incomplete keratinization. The expression of candidate genes such as E2A and caspase-9, which have been recognized to play a critical role in cellular proliferation/differentiation and apoptosis, is of great interest. They may be therapeutically targeted by the antipsoriatic drug, dithranol. We examined the molecular effects of dithranol on the mRNA and protein expression levels of E2A and caspase-9 in the HaCaT keratinocyte cell line. The HaCaT cells were treated with 0-0.5 µg/mL dithranol for 30 min. After dithranol was washed out, the HaCaT cells were cultured for 2 h, and their total cellular RNA and proteins were isolated. Quantitative real-time reverse transcriptase-polymerase chain reaction and Western blot were performed to determine the mRNA and protein levels of these two genes. We found that dithranol treatment in the range of 0.25-0.5 µg/mL slightly upregulated the mRNA expression of E2A and caspase-9 approximately 1.5- and 1.2-fold, respectively. However, undetectable change and minor downregulation of the protein expression levels were observed for E2A and caspase-9, respectively. Consequently, these genes appear not to be viable therapeutic targets for dithranol.

Therapeutic and cytotoxic effects of the novel antipsoriasis codrug, naproxyl-dithranol, on HaCaT cells.

A novel topical codrug, naproxyl-dithranol (Nap-DTH), in which dithranol and naproxen are linked via an ester in a 1:1 ratio to form a single chemical entity, was synthesized. The antiproliferative, anti-inflammatory and toxic effects of Nap-DTH were assessed, at the cellular level, using various in vitro methods. Cultured HaCaT keratinocytes were treated with Nap-DTH, and the cellular effects were compared with those of the parent compounds, individually and as a 1:1 mixture of naproxen:dithranol to mimic 1:1 in situ liberation from Nap-DTH. The results demonstrate that Nap-DTH did not modify proliferation and only exhibited slight toxic effects after 24 h at concentrations >21 µM. At a lower concentration (3.4 µM), Nap-DTH did not alter cell proliferation or inflammation, which suggests that the codrug is therapeutically inert. Relating to this, the 1:1 mixture of naproxen:dithranol exhibited the lowest toxic effect and the highest antiproliferative effect on HaCaT keratinocytes compared to dithranol at the same concentration. Moreover, the 1:1 mixture exhibited a reduced inflammatory effect compared to dithranol alone, as reflected by the upregulation of cyclooxygenase-2 by 45% and 136%, respectively. In spite of the 1:1 mixture showing a greater downregulation of Ki-67 and a 2-fold reduction of proliferating cell nuclear antigen (both cellular markers of proliferation) than dithranol, dithranol showed a much greater induction of cleaved caspase-3 protein expression (upregulated by 287%, compared to 85% for the 1:1 mixture). This suggests that when dithranol was administered with naproxen, inhibition of cell growth plays a more important role in the antiproliferation effects than the induction of apoptotic cell death. These results confirm that the codrug would lead to a better therapeutic profile and fewer adverse effects compared to its parent compounds.