[A Strategic Partnership for Medicine Creation and Access to Medicine for Neglected Tropical Diseases].

Neglected tropical diseases (NTDs) are communicable diseases that are uncommon in developed countries but epidemic in developing countries of the tropical and subtropical regions around the world. One of the important contributions expected of pharmaceutical companies is the development and provision of drugs effective against NTDs. Firstly, Eisai has distributed at price zero to endemic countries worldwide diethylcarbamazine (DEC) tablets for patients suffering with lymphatic filariasis (LF). Eisai's efforts toward improving global health have resulted in a rich portfolio of assets addressing six infectious diseases: malaria, tuberculosis, Chagas disease, LF, leishmaniasis, and mycetoma. As the most advanced project, Eisai has developed E1224, which is available in both intravenous and oral formulations, and delivers ravuconazole, the active form of fosravuconazole, with a long plasma half-life. The first clinical trials of E1224 for Chagas disease have already been completed, led by the Drugs for Neglected Diseases initiative (DNDi). As a result, parasite clearance was observed with E1224 during the treatment phase, but parasite regrowth was observed after the end of drug administration. On the other hand, a clinical trial for eumycetoma in collaboration with DNDi is ongoing supported by the Global Health Innovative Technology (GHIT) Fund. In this manner, Eisai will continue its Medicine Creation research projects in collaboration with various Product Development Partnerships (PDPs) and academia.

Development of E1224 by leveraging a strategic partnership for the medicines creation against neglected tropical diseases.

Neglected tropical diseases (NTDs) are communicable diseases that are uncommon in developed countries but epidemic in developing countries in tropical and subtropical regions of the world. One of the important contributions expected of pharmaceutical companies is the development and provision of drugs effective against NTDs. Eisai's efforts toward improving global health have resulted in a rich portfolio of assets addressing six infectious diseases: malaria, tuberculosis, Chagas disease, lymphatic filariasis, leishmaniasis, and mycetoma. As the most advanced project, Eisai has developed E1224 (fosravuconazole l-lysine ethanolate), which is available in both intravenous and oral formulations, and provides ravuconazole, an active form of fosravuconazole, with a long plasma half-life. The first clinical trials of E1224, for Chagas disease, have already been completed, led by the Drugs for Neglected Diseases initiative (DNDi). As a result, parasite clearance was observed with E1224 during the treatment phase, but parasite regrowth was observed after the end of drug administration, suggesting that the mechanism of action of E1224 on Trypanosoma cruzi is static rather than parasiticidal. On the other hand, a clinical trial for eumycetoma in collaboration with DNDi is ongoing supported by the Global Health Innovative Technology Fund, and is examining the efficacy of weekly treatment with E1224 versus the current standard of care, daily treatment with itraconazole. In this manner, Eisai will continue its drug-discovery research projects in collaboration with various PDPs and academia supported by funding agencies.

E1210, a new broad-spectrum antifungal, suppresses Candida albicans hyphal growth through inhibition of glycosylphosphatidylinositol biosynthesis.

Continued research toward the development of new antifungals that act via inhibition of glycosylphosphatidylinositol (GPI) biosynthesis led to the design of E1210. In this study, we assessed the selectivity of the inhibitory activity of E1210 against Candida albicans GWT1 (Orf19.6884) protein, Aspergillus fumigatus GWT1 (AFUA_1G14870) protein, and human PIG-W protein, which can catalyze the inositol acylation of GPI early in the GPI biosynthesis pathway, and then we assessed the effects of E1210 on key C. albicans virulence factors. E1210 inhibited the inositol acylation activity of C. albicans Gwt1p and A. fumigatus Gwt1p with 50% inhibitory concentrations (IC(50)s) of 0.3 to 0.6 µM but had no inhibitory activity against human Pig-Wp even at concentrations as high as 100 µM. To confirm the inhibition of fungal GPI biosynthesis, expression of ALS1 protein, a GPI-anchored protein, on the surfaces of C. albicans cells treated with E1210 was studied and shown to be significantly lower than that on untreated cells. However, the ALS1 protein levels in the crude extract and the RHO1 protein levels on the cell surface were found to be almost the same. Furthermore, E1210 inhibited germ tube formation, adherence to polystyrene surfaces, and biofilm formation of C. albicans at concentrations above its MIC. These results suggested that E1210 selectively inhibited inositol acylation of fungus-specific GPI which would be catalyzed by Gwt1p, leading to the inhibition of GPI-anchored protein maturation, and also that E1210 suppressed the expression of some important virulence factors of C. albicans, through its GPI biosynthesis inhibition.

In vitro activity of E1210, a novel antifungal, against clinically important yeasts and molds.

E1210 is a new antifungal compound with a novel mechanism of action and broad spectrum of antifungal activity. We investigated the in vitro antifungal activities of E1210 compared to those of fluconazole, itraconazole, voriconazole, amphotericin B, and micafungin against clinical fungal isolates. E1210 showed potent activities against most Candida spp. (MIC(90) of ≤0.008 to 0.06 µg/ml), except for Candida krusei (MICs of 2 to >32 µg/ml). E1210 showed equally potent activities against fluconazole-resistant and fluconazole-susceptible Candida strains. E1210 also had potent activities against various filamentous fungi, including Aspergillus fumigatus (MIC(90) of 0.13 µg/ml). E1210 was also active against Fusarium solani and some black molds. Of note, E1210 showed the greatest activities against Pseudallescheria boydii (MICs of 0.03 to 0.13 µg/ml), Scedosporium prolificans (MIC of 0.03 µg/ml), and Paecilomyces lilacinus (MICs of 0.06 µg/ml) among the compounds tested. The antifungal action of E1210 was fungistatic, but E1210 showed no trailing growth of Candida albicans, which has often been observed with fluconazole. In a cytotoxicity assay using human HK-2 cells, E1210 showed toxicity as low as that of fluconazole. Based on these results, E1210 is likely to be a promising antifungal agent for the treatment of invasive fungal infections.

Efficacy of oral E1210, a new broad-spectrum antifungal with a novel mechanism of action, in murine models of candidiasis, aspergillosis, and fusariosis.

E1210 is a first-in-class, broad-spectrum antifungal with a novel mechanism of action-inhibition of fungal glycosylphosphatidylinositol biosynthesis. In this study, the efficacies of E1210 and reference antifungals were evaluated in murine models of oropharyngeal and disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. Oral E1210 demonstrated dose-dependent efficacy in infections caused by Candida species, Aspergillus spp., and Fusarium solani. In the treatment of oropharyngeal candidiasis, E1210 and fluconazole each caused a significantly greater reduction in the number of oral CFU than the control treatment (P < 0.05). In the disseminated candidiasis model, mice treated with E1210, fluconazole, caspofungin, or liposomal amphotericin B showed significantly higher survival rates than the control mice (P < 0.05). E1210 was also highly effective in treating disseminated candidiasis caused by azole-resistant Candida albicans or Candida tropicalis. A 24-h delay in treatment onset minimally affected the efficacy outcome of E1210 in the treatment of disseminated candidiasis. In the Aspergillus flavus pulmonary aspergillosis model, mice treated with E1210, voriconazole, or caspofungin showed significantly higher survival rates than the control mice (P < 0.05). E1210 was also effective in the treatment of Aspergillus fumigatus pulmonary aspergillosis. In contrast to many antifungals, E1210 was also effective against disseminated fusariosis caused by F. solani. In conclusion, E1210 demonstrated consistent efficacy in murine models of oropharyngeal and disseminated candidiasis, pulmonary aspergillosis, and disseminated fusariosis. These data suggest that further studies to determine E1210's potential for the treatment of disseminated fungal infections are indicated.

In vitro activity of a novel broad-spectrum antifungal, E1210, tested against Candida spp. as determined by CLSI broth microdilution method.

The in vitro activity of the novel antifungal agent E1210 and four comparators (caspofungin, fluconazole, posaconazole, and voriconazole) was determined against 90 clinical isolates of Candida using Clinical and Laboratory Standards Institute methods. The collection was composed of 21 Candida albicans, 20 C. glabrata, 25 C. parapsilosis, and 24 C. tropicals, and also included 21 fluconazole-resistant and 15 caspofungin-resistant strains. E1210 was highly active against all the species tested and was more potent than all comparators. The MIC(90) results (µg/mL) for E1210, caspofungin, fluconazole, posaconazole, and voriconazole, respectively, were as follows by species: C. albicans (0.06, 4, ≥64, 0.5, 0.5), C. glabrata (0.06, 2, 32, 1, 1), C. parapsilosis (0.06, 4, 16, 0.12, 0.25), and C. tropicalis (0.06, 4, ≥64, 0.5, 2). E1210 was also the most active agent against fluconazole-resistant strains of C. albicans (MIC range, 0.015-0.12 µg/mL), C. glabrata (0.06 µg/mL), C. parapsilosis (MIC range, 0.06-0.05 µg/mL), and C. tropicalis (MIC range, 0.008-0.06 µg/mL), and was the most potent agent tested against caspofungin-resistant strains of C. albicans (MIC range, 0.008-0.12 µg/mL), C. glabrata (MIC range, 0.03-0.06 µg/mL), and C. tropicalis (MIC range, 0.015-0.06 µg/mL).

Synthesis and evaluation of novel antifungal agents-quinoline and pyridine amide derivatives.

Quinoline amide, azaindole amide and pyridine amides were synthesized and tested for in vitro antifungal activity against fungi. These synthesized amides have potent antifungal activity against Candida albicans and Aspergillus fumigatus. Our results suggest that hetero ring amides may be potent antifungal agents that operate by inhibiting the function of Gwt1 protein in the GPI biosynthetic pathway.

Medicinal genetics approach towards identifying the molecular target of a novel inhibitor of fungal cell wall assembly.

Glycosylphosphatidylinositol (GPI)-anchored cell wall mannoproteins are required for the adhesion of pathogenic fungi, such as Candida albicans, to human epithelium. Small molecular inhibitors of the cell surface presentation of GPI-anchored mannoproteins would be promising candidate drugs to block the establishment of fungal infections. Here, we describe a medicinal genetics approach to identifying the gene encoding a novel target protein that is required for the localization of GPI-anchored cell wall mannoproteins. By means of a yeast cell-based screening procedure, we discovered a compound, 1-[4-butylbenzyl]isoquinoline (BIQ), that inhibits cell wall localization of GPI-anchored mannoproteins in Saccharomyces cerevisiae. Treatment of C. albicans cells with this compound resulted in reduced adherence to a rat intestine epithelial cell monolayer. A previously uncharacterized gene YJL091c, named GWT1, was cloned as a dosage-dependent suppressor of the BIQ-induced phenotypes. GWT1 knock-out cells showed similar phenotypes to BIQ-treated wild-type cells in terms of cell wall structure and transcriptional profiles. Two different mutants resistant to BIQ each contained a single missense mutation in the coding region of the GWT1 gene. These results all suggest that the GWT1 gene product is the primary target of the compound.

GWT1 gene is required for inositol acylation of glycosylphosphatidylinositol anchors in yeast.

Glycosylphosphatidylinositol (GPI) is a conserved post-translational modification to anchor cell surface proteins to plasma membrane in all eukaryotes. In yeast, GPI mediates cross-linking of cell wall mannoproteins to beta1,6-glucan. We reported previously that the GWT1 gene product is a target of the novel anti-fungal compound, 1-[4-butylbenzyl]isoquinoline, that inhibits cell wall localization of GPI-anchored mannoproteins in Saccharomyces cerevisiae (Tsukahara, K., Hata, K., Sagane, K., Watanabe, N., Kuromitsu, J., Kai, J., Tsuchiya, M., Ohba, F., Jigami, Y., Yoshimatsu, K., and Nagasu, T. (2003) Mol. Microbiol. 48, 1029-1042). In the present study, to analyze the function of the Gwt1 protein, we isolated temperature-sensitive gwt1 mutants. The gwt1 cells were normal in transport of invertase and carboxypeptidase Y but were delayed in transport of GPI-anchored protein, Gas1p, and were defective in its maturation from the endoplasmic reticulum to the Golgi. The incorporation of inositol into GPI-anchored proteins was reduced in gwt1 mutant, indicating involvement of GWT1 in GPI biosynthesis. We analyzed the early steps of GPI biosynthesis in vitro by using membranes prepared from gwt1 and Deltagwt1 cells. The synthetic activity of GlcN-(acyl)PI from GlcN-PI was defective in these cells, whereas Deltagwt1 cells harboring GWT1 gene restored the activity, indicating that GWT1 is required for acylation of inositol during the GPI synthetic pathway. We further cloned GWT1 homologues in other yeasts, Cryptococcus neoformans and Schizosaccharomyces pombe, and confirmed that the specificity of acyl-CoA in inositol acylation, as reported in studies of endogenous membranes (Franzot, S. P., and Doering, T. L. (1999) Biochem. J. 340, 25-32), is due to the properties of Gwt1p itself and not to other membrane components.