Cyclosporin D

Improved closed-loop recycling counter-current chromatography (CLR CCC) with a two-phase solvent system composed of n-hexane-acetonitrile (1:1, v/v) was developed for separation, purification and preparation of cyclosporin D from the crude extract of fungus Hypoxylon Spp. (sj18). 28 mg cyclosporin D was successfully purified from 300 mg crude extract sample. The purity was 95.2% after five cycles, determined by HPLC. The structure of cyclosporin D was identified and assigned by 1H NMR, 13C NMR and mass spectrometric analyses. In addition, in the study, we show an interesting phenomenon that cyclosporin D can be prepared by the conventional CCC in n-hexane-ethyl acetate-methanol-water solvent system (2.5:1:2.5:1, v/v/v/v), and can also be prepared by the improved closed-loop recycling CCC in n-hexane-acetonitrile solvent system (1:1, v/v), but the efficiency of preparation varies greatly.

Background & aims: Cyclosporin A specifically suppresses hepatitis C virus (HCV) replication in vitro at clinically achievable concentrations. In this study, we investigated the mechanisms of action of cyclosporin A against HCV replication. Methods: The in vitro effects of cyclosporin A on HCV replication were analyzed using an HCV replicon system that expresses chimeric luciferase reporter protein. Results: The significant effects of cyclosporin A on expression of an HCV replicon and the absence of such effects of FK506, which shares mechanisms of action with cyclosporin A, suggested the involvement of intracellular ligands of cyclosporin A, the cyclophilins. Transient and stable knockdown of the expression of cytoplasmic cyclophilins A, B, and C by short hairpin RNA-expressing vectors suppressed HCV replication significantly. A cyclosporin analogue, cyclosporin D, which lacks immunosuppressive activity but exhibits cyclophilin binding, induced a similar suppression of HCV replication. Furthermore, cyclosporin A treatment of Huh7 cells induced an unfolded protein response exemplified by expression of cellular BiP/GRP78. Treatment of cells with thapsigargin and mercaptoethanol, which induce the unfolded protein responses, suppressed HCV replication, suggesting that the cyclosporin-induced unfolded protein responses might contribute to the suppression of HCV protein processing and replication. Conclusions: The anti-HCV activity of cyclosporin A is mediated through a specific blockade of cyclophilins, and these molecules may constitute novel targets for anti-HCV therapeutics.

Psoriasis vulgaris is a chronic inflammatory disorder of the skin, in which activation of keratinocytes and crosstalk between keratinocytes and T cells or dendritic cells are considered to be involved in the pathogenesis of psoriasis vulgaris. Cyclosporin (Cy) A, an immunomodulator, has been used for the treatment of psoriasis vulgaris, but the mechanism of its action on keratinocytes has not been well elucidated as its function on T cells is well known. Previous study indicated that the expression of the unfolded protein response (UPR) markers, GRP78/Bip and HRD1 were poorly expressed in psoriasis vulgaris. To investigate if the UPR in keratinocytes is involved in the pathogenesis of psoriasis vulgaris we assessed immunocytochemistry of normal human skin and psoriatic lesions, quantitative PCR of keratinocyte cell line (HaCaT) treated with TGFβ. Moreover, to elucidate how CyA effects on the UPR in keratinocytes, we set out quantitative PCR and western blotting, HaCaT and squamous cell carcinoma cell lines (HSC-1) treated with CyA and CyA analog, cyclosporin D. Furthermore, the siRNA-mediated knockdown effect of cyclophilin (Cyp) A, Cyp B and Cyp C on HaCaT cells were also examined. As a result, the UPR was downregulated in keratinocytes from psoriatic lesions, characterized by immunocytochemical staining of GRP78/Bip, CHOP/GADD153, HRD1 and C/EBPβ. TGFβ induced UPR markers in HaCaT cells. CyA treatment and siRNA-mediated knockdown of Cyp B induced the UPR in HaCaT cells or HSC-1 cells. Altogether, we demonstrate that in psoriasis vulgaris CyA or reduction in Cyp B by RNA interference might induce the UPR in keratinocytes.