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Efinaconazole is a triazole developed as a 10% solution for topical treatment of onychomycosis, a common fungal nail infection. Efinaconazole solution and topical formulation vehicle administered dermally to mice (13weeks), rats (6months) and minipigs (9months) produced transient erythema, minimal to modest hyperkeratosis, and mild microscopic skin inflammation. The liver was the target organ of systemic toxicity; reversible, minimal to moderate vacuolated changes were noted in the rat dermal study at 15 and 50mg/kg/day. No systemic toxicity was observed in mice and minipigs, at approximate high dermal doses of 930 and 170mg/kg/day, respectively. Daily subcutaneous injection of propylene glycol vehicle or efinaconazole to rats for 6months produced severe local inflammation and systemic spread, evidenced by peritoneal adhesions, spinal cord necrosis and urinary tract disease. Mortalities occurred in all groups but were increased at the high dose (30 or 40mg/kg/day), suggesting that vehicle effects were exacerbated by efinaconazole. Efinaconazole was not carcinogenic in a 2-year mouse dermal study and was not genotoxic. Exposure-based safety margins at the NOAEL were 70-698 relative to onychomycosis patients. In conclusion, efinaconazole demonstrated low/moderate toxicity, consistent with other azole antifungals, and high safety margins for topical onychomycosis therapy.

Onychomycosis is a common fungal nail disease that is difficult to treat topically due to the deep location of the infection under the densely keratinized nail plate. Keratin affinity of topical drugs is an important physicochemical property impacting therapeutic efficacy. To be effective, topical drugs must penetrate the nail bed and retain their antifungal activity within the nail matrix, both of which are adversely affected by keratin binding. We investigated these properties for efinaconazole, a new topical antifungal for onychomycosis, compared with those of the existing topical drugs ciclopirox and amorolfine. The efinaconazole free-drug concentration in keratin suspensions was 14.3%, significantly higher than the concentrations of ciclopirox and amorolfine, which were 0.7% and 1.9%, respectively ( P < 0.001). Efinaconazole was released from keratin at a higher proportion than in the reference drugs, with about half of the remaining keratin-bound efinaconazole removed after washing. In single-dose in vitro studies, efinaconazole penetrated full-thickness human nails into the receptor phase and also inhibited the growth of Trichophyton rubrum under the nail. In the presence of keratin, efinaconazole exhibited fungicidal activity against Trichophyton mentagrophytes comparable to that of amorolfine and superior to that of ciclopirox. In a guinea pig onychomycosis model with T. mentagrophytes infection, an efinaconazole solution significantly decreased nail fungal burden compared to that of ciclopirox and amorolfine lacquers ( P < 0.01). These results suggest that the high nail permeability of efinaconazole and its potent fungicidal activity in the presence of keratin are related to its low keratin affinity, which may contribute to its efficacy in onychomycosis.

The mechanism of action of efinaconazole, a new triazole antifungal, was investigated with Trichophyton mentagrophytes and Candida albicans . Efinaconazole dose-dependently decreased ergosterol production and accumulated 4,4-dimethylsterols and 4α-methylsterols at concentrations below its MICs. Efinaconazole induced morphological and ultrastructural changes in T. mentagrophytes hyphae that became more prominent with increasing drug concentrations. In conclusion, the primary mechanism of action of efinaconazole is blockage of ergosterol biosynthesis, presumably through sterol 14α-demethylase inhibition, leading to secondary degenerative changes.

Arsenic is a human toxin and carcinogen commonly found as a contaminant in drinking water. Arsenite (As(III)) is the most toxic inorganic form, but recent evidence indicates that the metabolite monomethylarsonous acid (MMA(III)) is even more toxic. We have used a chemical genomics approach to identify the genes that modulate the cellular toxicity of MMA(III) and As(III) in the yeast Saccharomyces cerevisiae. Functional profiling using homozygous deletion mutants provided evidence of the requirement of highly conserved biological processes in the response against both arsenicals including tubulin folding, DNA double-strand break repair, and chromatin modification. At the equitoxic doses of 150 microM MMA(III) and 300 microM As(III), genes related to glutathione metabolism were essential only for resistance to the former, suggesting a higher potency of MMA(III) to disrupt glutathione metabolism than As(III). Treatments with MMA(III) induced a significant increase in glutathione levels in the wild-type strain, which correlated to the requirement of genes from the sulfur and methionine metabolic pathways and was consistent with the induction of oxidative stress. Based on the relative sensitivity of deletion strains deficient in GSH metabolism and tubulin folding processes, oxidative stress appeared to be the primary mechanism of MMA(III) toxicity whereas secondary to tubulin disruption in the case of As(III). Many of the identified yeast genes have orthologs in humans that could potentially modulate arsenic toxicity in a similar manner as their yeast counterparts.

Copper is an essential micronutrient for all biological systems. Multiple proteins require one or more atoms of copper for proper structure and function, but excess of copper is toxic. To prevent the consequences of copper deficiency and overload, living organisms have evolved molecular mechanisms that regulate its uptake, intracellular traffic, storage, and efflux. Underlying some of the cellular responses to variations in copper levels are changes in the expression of genes encoding molecular components of copper metabolism. In recent years, genome-scale expression analysis in several eukaryotic models has allowed the identification of copper-responsive genes involved in copper homeostasis. Characterization of the transcriptional changes in response to varying copper levels include both genes directly involved in copper homeostasis and genes involved in different cellular process that, even though they are not directly connected to copper metabolism, change their expression during the cellular adaptation to copper availability. Evaluation of these gene expression patterns could aid in the identification of biologically relevant markers to monitor copper status in humans.

To provide an adequate therapeutic effect against onychomycosis, it has been suggested that topical drugs should have two properties: drug permeability through the nail plate and into the nail bed, and retention of their antifungal activity in the disease-affected areas. Only recently has the importance of other delivery routes (such as subungual) been discussed. Efinaconazole has been shown to have a more potent antifungal activity in vitro than the most commonly used onychomycosis treatments. The low keratin affinity of efinaconazole contributes to its effective delivery through the nail plate and retention of its antifungal activity. Its unique low surface tension formulation provides good wetting properties affording drug delivery both through and under the nail. High antifungal drug concentrations have been demonstrated in the nail of onychomycosis patients, and effectiveness of efinaconazole topical solution, 10% confirmed in two large well-controlled multicenter Phase 3 clinical studies in patients with mild-to-moderate disease.

Onychomycosis is a common fungal nail infection in adults that is difficult to treat. The in vitro antifungal activity of efinaconazole, a novel triazole antifungal, was evaluated in recent clinical isolates of Trichophyton rubrum , Trichophyton mentagrophytes , and Candida albicans , common causative onychomycosis pathogens. In a comprehensive survey of 1,493 isolates, efinaconazole MICs against T. rubrum and T. mentagrophytes ranged from ≤0.002 to 0.06 μg/ml, with 90% of isolates inhibited (MIC 90 ) at 0.008 and 0.015 μg/ml, respectively. Efinaconazole MICs against 105 C. albicans isolates ranged from ≤0.0005 to >0.25 μg/ml, with 50% of isolates inhibited (MIC 50 ) by 0.001 and 0.004 μg/ml at 24 and 48 h, respectively. Efinaconazole potency against these organisms was similar to or greater than those of antifungal drugs currently used in onychomycosis, including amorolfine, ciclopirox, itraconazole, and terbinafine. In 13 T. rubrum toenail isolates from onychomycosis patients who were treated daily with topical efinaconazole for 48 weeks, there were no apparent increases in susceptibility, suggesting low potential for dermatophytes to develop resistance to efinaconazole. The activity of efinaconazole was further evaluated in another 8 dermatophyte, 15 nondermatophyte, and 10 yeast species (a total of 109 isolates from research repositories). Efinaconazole was active against Trichophyton , Microsporum , Epidermophyton , Acremonium , Fusarium , Paecilomyces , Pseudallescheria , Scopulariopsis , Aspergillus , Cryptococcus , Trichosporon , and Candida and compared favorably to other antifungal drugs. In conclusion, efinaconazole is a potent antifungal with a broad spectrum of activity that may have clinical applications in onychomycosis and other mycoses.

Background In humans, inorganic arsenic (iAs) is metabolized to methylated arsenical species in a multistep process mainly mediated by arsenic (+3 oxidation state) methyltransferase (AS3MT). Among these metabolites is monomethylarsonous acid (MMAIII), the most toxic arsenic species. A recent study in As3mt-knockout mice suggests that unidentified methyltransferases could be involved in alternative iAs methylation pathways. We found that yeast deletion mutants lacking MTQ2 were highly resistant to iAs exposure. The human ortholog of the yeast MTQ2 is N-6 adenine-specific DNA methyltransferase 1 (N6AMT1), encoding a putative methyltransferase. Objective We investigated the potential role of N6AMT1 in arsenic-induced toxicity. Methods We measured and compared the cytotoxicity induced by arsenicals and their metabolic profiles using inductively coupled plasma–mass spectrometry in UROtsa human urothelial cells with enhanced N6AMT1 expression and UROtsa vector control cells treated with different concentrations of either iAsIII or MMAIII. Results N6AMT1 was able to convert MMAIII to the less toxic dimethylarsonic acid (DMA) when overexpressed in UROtsa cells. The enhanced expression of N6AMT1 in UROtsa cells decreased cytotoxicity of both iAsIII and MMAIII. Moreover, N6AMT1 is expressed in many human tissues at variable levels, although at levels lower than those of AS3MT, supporting a potential participation in arsenic metabolism in vivo. Conclusions Considering that MMAIII is the most toxic arsenical, our data suggest that N6AMT1 has a significant role in determining susceptibility to arsenic toxicity and carcinogenicity because of its specific activity in methylating MMAIII to DMA and other unknown mechanisms.

Arsenic, a human carcinogen that is associated with an increased risk of bladder cancer, is commonly found in drinking water. An important mechanism by which arsenic is thought to be carcinogenic is through the induction of epigenetic changes that lead to aberrant gene expression. Previously, we reported that the SAS2 gene is required for optimal growth of yeast in the presence of arsenite (As(III)). Yeast Sas2p is orthologous to human MYST1, a histone 4 lysine 16 (H4K16) acetyltransferase. Here, we show that H4K16 acetylation is necessary for the resistance of yeast to As(III) through the modulation of chromatin state. We further explored the role of MYST1 and H4K16 acetylation in arsenic toxicity and carcinogenesis in human bladder epithelial cells. The expression of MYST1 was knocked down in UROtsa cells, a model of bladder epithelium that has been used to study arsenic-induced carcinogenesis. Silencing of MYST1 reduced acetylation of H4K16 and induced sensitivity to As(III) and to its more toxic metabolite monomethylarsonous acid (MMA(III)) at doses relevant to high environmental human exposures. In addition, both As(III) and MMA(III) treatments decreased global H4K16 acetylation levels in a dose- and time-dependent manner. This indicates that acetylated H4K16 is required for resistance to arsenic and that a reduction in its levels as a consequence of arsenic exposure may contribute to toxicity in UROtsa cells. Based on these findings, we propose a novel role for the MYST1 gene in human sensitivity to arsenic.

Benzene is an established human hematotoxicant and leukemogen but its mechanism of action is unclear. To investigate the role of single-nucleotide polymorphisms (SNPs) on benzene-induced hematotoxicity, we analyzed 1395 SNPs in 411 genes using an Illumina GoldenGate assay in 250 benzene-exposed workers and 140 unexposed controls. Highly significant findings clustered in five genes (BLM, TP53, RAD51, WDR79 and WRN) that play a critical role in DNA repair and genomic maintenance, and these regions were then further investigated with tagSNPs. One or more SNPs in each gene were associated with highly significant 10-20% reductions (P values ranged from 0.0011 to 0.0002) in the white blood cell (WBC) count among benzene-exposed workers but not controls, with evidence for gene-environment interactions for SNPs in BLM, WRN and RAD51. Further, among workers exposed to benzene, the genotype-associated risk of having a WBC count4000 cells/microl increased when using individuals with progressively higher WBC counts as the comparison group, with some odds ratios8-fold. In vitro functional studies revealed that deletion of SGS1 in yeast, equivalent to lacking BLM and WRN function in humans, caused reduced cellular growth in the presence of the toxic benzene metabolite hydroquinone, and knockdown of WRN using specific short hairpin RNA increased susceptibility of human TK6 cells to hydroquinone toxicity. Our findings suggest that SNPs involved in DNA repair and genomic maintenance, with particular clustering in the homologous DNA recombination pathway, play an important role in benzene-induced hematotoxicity.

Iron and copper are essential nutrients for life as they are required for the function of many proteins but can be toxic if present in excess. Accumulation of these metals in the human body as a consequence of overload disorders and/or high environmental exposures has detrimental effects on health. The budding yeast Saccharomyces cerevisiae is an accepted cellular model for iron and copper metabolism in humans primarily because of the high degree of conservation between pathways and proteins involved. Here we report a systematic screen using yeast deletion mutants to identify genes involved in the toxic response to growth-inhibitory concentrations of iron and copper sulfate. We aimed to understand the cellular responses to toxic concentrations of these two metals by analyzing the different subnetworks and biological processes significantly enriched with these genes. Our results indicate the presence of two different detoxification pathways for iron and copper that converge toward the vacuole. The product of several of the identified genes in these pathways form molecular complexes that are conserved in mammals and include the retromer, endosomal sorting complex required for transport (ESCRT) and AP-3 complexes, suggesting that the mechanisms involved can be extrapolated to humans. Our data also suggest a disruption in ion homeostasis and, in particular, of iron after copper exposure. Moreover, the identification of treatment-specific genes associated with biological processes such as DNA double-strand break repair for iron and tryptophan biosynthesis for copper suggests differences in the mechanisms by which these two metals are toxic at high concentrations.