Byeong Deog Park

Inhibition of atopic dermatitis-like skin lesions by topical application of a novel ceramide derivative, K6PC-9p, in NC/Nga mice.

Abstract:  Atopic dermatitis (AD) is a chronic inflammatory skin disease that commonly begins in childhood. K6PC‐9p (N‐(Ethyl dihydrogenphosphate)‐2‐hexyl‐3‐oxo‐decanamide) is a synthetic ceramide derivative of PC‐9S (N‐Ethanol‐2‐mirystyl‐3‐oxo‐staramide), which was known to be effective in atopic patients. In this study, we examined the effect of topical application of K6PC‐9p on skin inflammation and AD‐like skin lesions in mouse models. K6PC‐9p dose‐dependently inhibited phorbol ester‐induced increase in ear thickness in BALB/c mice. Moreover, topical application of K6PC‐9p suppressed dust mite extract‐induced AD‐like skin lesions in NC/Nga mice. Histopathological analysis revealed that both ear swelling and leucocyte infiltration were suppressed by K6PC‐9p treatment. K6PC‐9p also suppressed IL‐4 and TNF‐α expression in the ears and mast cell infiltration into the ears in NC/Nga mice. Further study demonstrated that K6PC‐9p inhibited ConA‐induced IL‐4 secretion and LPS‐induced macrophage activation. Taken together, our results showed that topical application of K6PC‐9p exerts beneficial effects in animal model of skin inflammation and AD, suggesting that K6PC‐9p might be a promising topical agent for the treatment of inflammatory skin diseases.

K6PC-5, a Direct Activator of Sphingosine Kinase 1, Promotes Epidermal Differentiation Through Intracellular Ca2+ Signaling

Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, regulates multiple cellular responses such as Ca(2+) signaling, growth, survival, and differentiation. Because sphingosine kinase (SphK) is the enzyme directly responsible for production of S1P, many factors have been identified that regulate its activity and subsequent S1P levels. Here we synthesized a previously unidentified SphK activator, K6PC-5, and have studied its effects on intracellular Ca(2+) signaling in HaCaT cells and epidermal differentiation in murine skin. K6PC-5, a hydrophobic compound chemically named N-(1,3-dihydroxyisopropyl)-2-hexyl-3-oxo-decanamide, activated SphK (obtained from C57BL/6 murine blood and F9-12 cell lysates) in a dose-dependent manner. K6PC-5 induced both intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations in HaCaT cells and Ca(2+) mobilization in hairless mouse epidermis. Both dimethylsphingosine (DMS) and dihydroxysphingosine (DHS), SphK inhibitors, and transfection of SphK1-siRNA blocked K6PC-5-induced increases in [Ca(2+)](i). The K6PC-5-induced [Ca(2+)](i) oscillations were dependent on thapsigargin-sensitive Ca(2+) stores and Ca(2+) entry, but independent of the classical phospholipase C-mediated pathway. In addition, K6PC-5 enhanced the expression of involucrin and filaggrin, specific differentiation-associated marker proteins in HaCaT cells, whereas transfection of SphK1-siRNA blocked the increase of involucrin. Topical K6PC-5 also enhanced the expression of involucrin, loricrin, filaggrin, and keratin 5 in intact murine epidermis. Finally, topical K6PC-5 inhibited epidermal hyperplasia by exerting antiproliferative effects on keratinocytes in murine epidermis. These results suggest that K6PC-5 acts to regulate both differentiation and proliferation of keratinocytes via [Ca(2+)](i) responses through S1P production. Thus, regulation of S1P levels may represent a novel approach for treatment of skin disorders characterized by abnormal differentiation and proliferation, such as atopic dermatitis and psoriasis.

K6PC-5, a sphingosine kinase activator, induces anti-aging effects in intrinsically aged skin through intracellular Ca2+ signaling.

BACKGROUND Sphingosine-1-phosphate (S1P), a bioactive sphingolipid metabolite, regulates multiple cellular responses such as Ca(2+) signaling, growth, survival, and differentiation. Because sphingosine kinase (SK) is the enzyme directly responsible for the production of S1P, many factors have been identified that regulate its activity and subsequent S1P levels. To date, there are no reports to demonstrate a chemically induced, direct activation of SK. OBJECTIVE Here we have studied the effects of K6PC-5 as a newly synthesized SK activator on fibroblast proliferation in both human fibroblasts and aged mouse skin. To demonstrate that K6PC-5 has S1P-mediated action mechanism in fibroblasts, we have measured SK-dependent intracellular Ca(2+) signaling. METHODS Fibroblasts were cultured primarily from human foreskin and were used to study the effect of K6PC-5 and S1P on intracellular Ca(2+) signaling and fibroblast proliferation. Changes in intracellular Ca(2+) were detected by fluorescence with fura-2/AM. To study skin anti-aging effects of K6PC-5, we used intrinsically aged hairless mice (56 weeks old). RESULTS K6PC-5 promoted fibroblast proliferation and procollagen production in human fibroblasts significantly. K6PC-5 induced intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations in human fibroblasts. Both dimethylsphingosine and dihydroxysphingosine, SK inhibitors, and the transfection of SK1-siRNA blocked the K6PC-5-induced increases in [Ca(2+)](i), an effect independent of the classical PLC/IP(3)-mediated pathway. The K6PC-5-induced [Ca(2+)](i) oscillations were dependent on thapsigargin-sensitive Ca(2+) stores and Ca(2+) entry. Topical application of K6PC-5 for 2 weeks to intrinsically aged hairless mice enhanced fibroblast proliferation, collagen production, and eventually increased dermal thickness (10%). K6PC-5 also promoted specific epidermal differentiation marker proteins, including involucrin, loricrin, filaggrin, and keratin 5, without any alterations on epidermal barrier function. CONCLUSION These results suggest that K6PC-5 acts to regulate fibroblast proliferation through intracellular S1P production, and can further promote keratinocyte differentiation. We anticipate that the regulation of S1P levels may represent a novel approach for the treatment of skin disorders, including skin aging.

Novel synthetic ceramide derivatives increase intracellular calcium levels and promote epidermal keratinocyte differentiation

Ceramide is an important constituent of stratum corneum lipids, which act as both physical barriers and signal modulators. We synthesized several ceramide derivatives and investigated their effects on keratinocyte differentiation. RT-PCR and Western blotting showed that the novel synthetic ceramide derivatives K6PC-4 (N-ethanol-2-hexyl-3-hydroxy-decanamide), K6PC-5, (N-ethanol-3-oxo-2-tetradecyl/hexadecyl-octadecanamide/eicosanamide)and K6PC-9 [N-(1,3-dihydroxypropyl)-2-hexyl-3-oxo-decanamide] markedly increased keratin 1 and involucrin expression in normal human epidermal keratinocytes cultured in vitro. These ceramide derivatives elicited a rapid transient increase in intracellular calcium levels, which were measured using laser scanning confocal microscopy. In addition, K6PC-4, K6PC-5, and K6PC-9 stimulated the phosphorylation of p42/44 extracellular signal-regulated kinase and c-Jun N-terminal kinase. In a reconstituted epidermis model, K6PC-4, K6PC-5, and K6PC-9 significantly increased keratin 1 expression in the suprabasal layer. These results indicate that these novel synthetic ceramide derivatives have the potential to promote keratinocyte differentiation, suggesting that the lipid molecules are applicable for treating skin diseases involving abnormal keratinocyte differentiation.

Effects of a multilamellar emulsion on glucocorticoid-induced epidermal atrophy and barrier impairment

Skin atrophy is one of the most frequent side‐effects of the topical glucocorticoid. Skin barrier impairment has also been reported as a steroid‐induced side effect. Although there have been various studies on preventing or minimizing this atrophogenic effect, little has been reported about preventing barrier impairment. This study was performed to determine the effects of a multilamellar emulsion (MLE) that had a well‐ordered lamellar structure on the steroid‐induced barrier impairment and epidermal atrophy. To confirm these effects of MLE, 0.05% clobetasol‐17‐propionate (CP) and 0.05% clobetasol‐17‐propionate in MLE (MLE/CP) were topically applied to both flanks of hairless mice for 9 days. The topically applied CP induced a significant impairment of the epidermal permeability barrier, and MLE/CP also did not have a preventive effect on this change. However, skinfold thickness studies and histological studies showed that MLE/CP significantly reduced the steroid‐induced atrophy. The topical application of MLE/CP was also shown to have a preventive effect on the steroid‐induced increase of the stratum corneum (SC) surface pH. In addition, the electron microscopic findings showed relatively well‐conserved lamellar bilayers in the skin treated with MLE, as compared to CP only. The results showed that the topical application of MLE immediately after CP treatment prevented the glucocorticoid‐induced transepidermal water loss values increase. Light microscopy measurements showed that the skin treated with MLE immediately after CP treatment for 1 week had a slightly lower decline of skin thickness than did the CP‐treated skin. These results suggest that MLE should be effective for preventing glucocorticoid‐induced epidermal atrophy and for repairing the barrier impairment.

K6PC-5, a novel sphingosine kinase activator, improves long-term ultraviolet light-exposed aged murine skin.

Abstract:  Sphingosine‐1‐phosphate (S1P), which is formed by phosphorylation of sphingosine through a process catalysed by sphingosine kinase (SK), is a multifunctional mediator of a variety of cellular responses including proliferation, differentiation, motility, and survival. K6PC‐5, which was recently synthesized as a novel SK activator, is expected to increase S1P levels. Indeed studies have already demonstrated that K6PC‐5 exhibits anti‐aging effects on intrinsic aged murine skin by increasing fibroblasts, collagen synthesis, dermal thickness, and epidermal differentiation. However, photoaging and intrinsic aging have highly different clinical and histopathological properties. In this study, we developed a photoaged murine model by exposing mice that were 56 weeks old to ultraviolet (UV)B and UVA radiation for 8 weeks. We then investigated whether K6PC‐5, as an SK activator, had anti‐aging effects on photoaged murine skin in addition to its effects on intrinsic aged murine skin and determined the mechanism. K6PC‐5 increased dermal collagen density in photoaged skin through increases in fibroblasts and collagen production. Photoaged murine skin treated with K6PC‐5 showed an increase in stratum corneum (SC) integrity with increased corneodesmosome density and an improvement in barrier recovery rate. Matrix metalloproteinase 13 remained unchanged. These results indicate that topical application of K6PC‐5 improves photoaged skin by improving skin barrier and increasing fibroblast count and function. In conclusion, K6PC‐5, as an S1P activator, improves long‐term UV‐exposed aged skin as well as intrinsic aged skin.

Effectiveness of Topical Chia Seed Oil on Pruritus of End-stage Renal Disease (ESRD) Patients and Healthy Volunteers.

BACKGROUND Several studies have been performed to evaluate the efficacy of dietary n-3 fatty acid for patients with renal dysfunction. While about 40% to 80% of patients with end-stage renal disease (ESRD) complain about pruritus and xerosis, there are few reports on the effects of topical n-3 fatty acid on these symptoms. OBJECTIVE In order to investigate the possible beneficial effects of topical n-3 fatty acid, oils extracted from chia (Salvia hispanica) seed were formulated into topical products, the effects of which were measured. METHODS Five healthy volunteers having xerotic pruritus symptoms and 5 patients with pruritus caused by either ESRD or diabetes were involved in this study. A topical formulation containing 4% chia seed oils were applied for an 8-week duration. Subjective itching symptoms were assessed on a 6-point scale, as were other skin functions, namely transepidermal water loss and skin capacitance. RESULTS After the 8 weeks of application, significant improvements in skin hydration, lichen simplex chronicus, and prurigo nodularis were observed in all patients. A similar improvement was also observed among healthy volunteers with xerotic pruritus. Improvement of epidermal permeability barrier function and skin hydration, represented by trans-epidermal water loss and skin capacitance, respectively, were also observed. No adverse effects were observed in all the tested patients and volunteers. CONCLUSION Chia seed oil can be used as an adjuvant moisturizing agent for pruritic skin, including that of ESRD patients.

Anti-ageing effects of a new synthetic sphingolipid (K6EAA-L12) on aged murine skin.

Abstract:  Recently, we reported on the anti‐ageing effects of K6PC‐5. This compound induced keratinocyte differentiation and fibroblast proliferation by increasing sphingosine‐1 phosphate synthesis. We performed this study to confirm the anti‐ageing effects of new synthetic products (the K6EAA series) derived from K6PC‐5 through an amino group induction. Cellular responses such as differentiation, proliferation and calcium mobilization were investigated using cultured human keratinocytes and fibroblasts. Also, we measured the expressions of collagen mRNA and protein using real time RT‐PCR and ELISA, respectively. The K6EAA‐L12 product, selected by in vitro screening, was evaluated for anti‐ageing effects on intrinsically and extrinsically (photo) aged models of hairless mice. In the intrinsically aged murine skin, K6EAA‐L12 showed anti‐ageing effects by activating collagen synthesis, eventually causing dermal thickening. Also, in the photo‐aged skin, the dermal collagen density and dermal thickness were increased. In photo‐aged murine skin, K6EAA‐L12 increased stratum corneum integrity by increasing corneodesmosome density and improved the barrier recovery rate. However, there were no changes in the expressions of epidermal differentiation maker proteins. In conclusion, topical K6EAA‐L12, a new synthetic K6PC‐5 derivative, improves intrinsically and extrinsically (photo) aged skin by increasing the collagen density and improving the skin barrier function.

Inhibition of skin inflammation and atopic dermatitis by topical application of a novel ceramide derivative, K112PC-5, in mice

PC-9S (N-Ethanol-2-mirystyl-3-oxo-stearamide) is a synthetic ceramide and has been known to be effective in atopic and psoriatic patients. K112PC-5 (2-Acetyl-N-(1,3-dihydroxyisopropyl)-tetradecanamide) is a novel ceramide derivative of PC-9S. In the present study, we examined the effect of K112PC-5 on macrophage and T lymphocyte function in primary macrophages and splenocytes, respectively, as well as the effect of topical application of K112PC-5 on skin inflammation and atopic dermatitis (AD) in mouse models. K112PC-5 inhibited lipopolysaccharide-induced nitrite generation in mouse peritoneal macrophages in a dose-dependent manner. However, K112PC-5 did not affect concanavalin A-induced proliferation, interleukin (IL)-2 secretion and IL-4 secretion in mouse splenocytes. In addition, K112PC-5 significantly suppressed the increase in phorbol ester-induced ear thickness in BALB/c mice. Further study demonstrated that topical application of K112PC-5 also inhibited AD induced by extracts of dust mites, Dermatophagoides pteronyssinus and Dermatophagoides farinae, in NC/Nga mice. Taken together, these results showed that K112PC-5 exerted an anti-inflammatory effect both in vitro and in vivo and proved to be beneficial in an animal model of AD. Our results suggest that K112PC-5 might be beneficial as a topical agent for the treatment of AD.

Preventive Effects of Multi-Lamellar Emulsion on Low Potency Topical Steroid Induced Local Adverse Effect

Background Topical steroid treatment induces diverse local Wand systemic adverse effects. Several approaches have been tried to reduce the steroid-induced adverse effects. Simultaneous application of physiological lipid mixture is also suggested. Objective Novel vehicles for topical glucocorticoids formulation were evaluated for the efficacy of reducing side-effects and the drug delivery properties of desonide, a low potency topical steroid. Methods Transcutaneous permeation and skin residual amount of desonide were measured using Franz diffusion cells. The in vivo anti-inflammatory activity was evaluated using murine model. Results Topical steroids formulation containing desonide, in either cream or lotion form, were prepared using multi-lamellar emulsion (MLE), and conventional desonide formulations were employed for comparison. MLE formulations did not affect the anti-inflammatory activity of the desonide in phobol ester-induced skin inflammation model, compared with conventional formulations. While the penetrated amounts of desonide were similar for all the tested formulations at 24 hours after application, the increased lag time was observed for the MLE formulations. Interestingly, residual amount of desonide in epidermis was significantly higher in lotion type MLE formulation. Steroid-induced adverse effects, including permeability barrier function impairment, were partially prevented by MLE formulation. Conclusion Topical desonide formulation using MLE as a vehicle showed a better drug delivery with increased epidermal retention. MLE also partially prevented the steroid-induced side effects, such as skin barrier impairment.