Evaluation of Topical Antifungals Using a New Predictive Animal Model for Efficacy against Severe Tinea Unguium: A Comparison of Efinaconazole and Luliconazole.

Development of new topical drugs requires an animal onychomycosis model that can predict the drug efficacy against moderate to severe human onychomycosis because the severity of onychomycosis varies and affects the drug efficacy. This study established a non-immunosuppressive guinea pig tinea unguium model under 8-week infection condition in addition to a previously reported model under 4-week infection condition. In the tinea unguium model, most fungi were tightly present in the arthrospore form, like in human onychomycosis. The topical formulations of efinaconazole and luliconazole, two azole class anti-onychomycosis drugs, were evaluated for their efficacy in these models. In the untreated group, the nail fungal burden in the 8-week model was higher than that in the 4-week model and the stronger infection intensity affected the efficacy of the drugs, suggesting that the 8-week model was more severe. The 90% efficacy rate (42%) of luliconazole in the 8-week model was significantly lowered than that (83%) in the 4-week model, and its 99% efficacy rates were 0% in both models. Conversely, the 90% and 99% efficacy rates of efinaconazole (92% and 50% in the 4-week model, and 75% and 25% in the 8-week model, respectively) were not significantly different between the two infection durations. In addition, efinaconazole was more effective than luliconazole in reducing the nail fungal burden. Considering the relevance of clinical reports of the effectiveness of efinaconazole on severe onychomycosis, the new severe tinea unguium model would predict drug efficacy against moderate to severe onychomycosis.

A novel method for predicting the efficacy of topical drugs on onychomycosis: A comparison of efinaconazole and luliconazole.

To be effective against onychomycosis, topically applied drugs have to reach the infection site at an effective concentration to exert antifungal activity against the parasitic form of dermatophytes. We established a novel in vitro method for predicting drug efficacy at the infection site and verified the method by comparing the efficacy of two azole class topical anti-onychomycosis drugs. To predict drug efficacy in the nail plate, a human nail permeability test was conducted and the activities of the free-drugs in the upper, middle, and lowest layers of the nail plate were determined by measuring the growth inhibitory zone. Efinaconazole permeated the nail more efficiently than luliconazole, and the amount of efinaconazole in the middle and lowest layers was higher compared with that of luliconazole. Efinaconazole demonstrated antifungal activities at the concentrations in all of the nail layers, whereas luliconazole was only active at the concentrations in the upper and middle layers. The results could be explained by differences in their affinity for keratin and nail permeability. The established method enables the evaluation of nail permeability and anti-arthrospore activity of free-drugs in the nail plate to predict drug efficacy. This method will be useful for new topical drug development.

In Vitro Combination Effect of Topical and Oral Anti-Onychomycosis Drugs on Trichophyton rubrum and Trichophyton interdigitale.

To evaluate the combination effects of anti-onychomycosis drugs, the minimum inhibitory concentrations of topical (efinaconazole, luliconazole, and tavaborole) and oral (itraconazole and terbinafine) drugs for Trichophyton rubrum and Trichophyton interdigitale (8 each, with a total of 16 strains) were determined using the microdilution checkerboard technique based on the Clinical and Laboratory Standard Institute guidelines. No antagonism was observed between the topical and oral drugs against all the tested strains. Efinaconazole with terbinafine exerted a synergistic effect on 43.8% of the strains tested (7/16 strains) and efinaconazole with itraconazole on 12.5% (2/16 strains). Conversely, luliconazole showed no synergistic effect with terbinafine but was synergistically effective with itraconazole against 31.3% of the strains (5/16 strains). Tavaborole showed no synergistic effect with terbinafine and was synergistically effective with itraconazole against 18.8% of the strains (3/16 strains). The results suggest that a combination of topical and oral drugs could be a potential clinical option for onychomycosis treatment, and overall, the efinaconazole and oral drug combination would be the most advantageous among the tested combinations.

Contrasting volcano spacing along SW Japan arc caused by difference in age of subducting lithosphere.

The SW Japan arc built by subduction of the Philippine Sea (PHS) plate exhibits uneven distribution of volcanoes: thirteen Quaternary composite volcanoes form in the western half of this arc, Kyushu Island, while only two in the eastern half, Chugoku district. Reconstruction of the PHS plate back to 14 Ma, together with examinations based on thermal structure models constrained by high-density heat flow data and a petrological model for dehydration reactions suggest that fluids are discharged actively at depths of 90-100 km in the hydrous layer at the top of the old (> 50 Ma), hence, cold lithosphere sinking beneath Kyushu Island. In contrast, the young (15-25 Ma) oceanic crust downgoing beneath Chugoku district releases fluids largely at shallower depths, i.e. beneath the non-volcanic forearc, to cause characteristic tectonic tremors and low-frequency earthquakes (LFEs) and be the source of specific brine springs. Much larger amounts of fluids supplied to the magma source region in the western SW Japan arc could build more densely-distributed volcanoes.

Giant rhyolite lava dome formation after 7.3 ka supereruption at Kikai caldera, SW Japan.

Kikai submarine caldera to the south of the Kyushu Island, SW Japan, collapsed at 7.3 ka during the latest supereruption (>500 km3 of magma) in the Japanese Archipelago. Multi functional research surveys of the T/S Fukae Maru in this caldera, including multi-beam echosounder mapping, remotely operated vehicle observation, multi-channel seismic reflection survey, and rock sampling by dredging and diving, provided lines of evidence for creation of a giant rhyolite lava dome (~32 km3) after the caldera collapse. This dome is still active as water column anomalies accompanied by bubbling from its surface are observed. Chemical characteristics of dome-forming rhyolites akin to those of presently active small volcanic cones are different from those of supereruption. The voluminous post-caldera activity is thus not caused simply by squeezing the remnant of syn-caldera magma but may tap a magma system that has evolved both chemically and physically since the 7.3-ka supereruption.

Fungicidal Activity in the Presence of Keratin as an Important Factor Contributing to In Vivo Efficacy: A Comparison of Efinaconazole, Tavaborole, and Ciclopirox.

Use of oral antifungals in the treatment of onychomycosis is commonplace; but their use can be limited by safety and patient concerns. Due to their broader safety margins, topical antifungals (efinaconazole, tavaborole, and ciclopirox) are a useful option in the treatment of mild-to-moderate onychomycosis in the USA, but their antifungal activity has yet to be directly compared. This study aims to identify important factors contributing to in vivo efficacies of the three topical antifungals. Minimum inhibitory concentrations (MICs) were determined by Clinical and Laboratory Standards Institute (CLSI) M38-A2 broth microdilution. The MIC90 values of efinaconazole, tavaborole, and ciclopirox for T. rubrum were 0.0078, 8.0, and 0.50 µg/mL, respectively. The MIC90 values for T. mentagrophytes were 0.016, 8.0, and 0.50 µg/mL, respectively. Efinaconazole showed potent fungicidal activity in keratin-containing medium, whereas tavaborole was fungistatic, and ciclopirox not active. In the guinea pig model of onychomycosis, the therapeutic efficacy of efinaconazole was superior to those of tavaborole and ciclopirox. This study suggests that not only fungistatic activity (MIC), but also fungicidal activity in the presence of keratin, is an important factor contributing to the in vivo efficacy of topical antifungal drugs against onychomycosis.

Efficacy Coefficients Determined Using Nail Permeability and Antifungal Activity in Keratin-Containing Media Are Useful for Predicting Clinical Efficacies of Topical Drugs for Onychomycosis.

Onychomycosis is difficult to treat topically due to the deep location of the infection under the densely keratinized nail plate. In order to obtain an in vitro index that is relevant to the clinical efficacy of topical anti-onychomycosis drugs, we profiled five topical drugs: amorolfine, ciclopirox, efinaconazole, luliconazole, and terbinafine, for their nail permeabilities, keratin affinities, and anti-dermatophytic activities in the presence of keratin. Efinaconazole and ciclopirox permeated full-thickness human nails more deeply than luliconazole. Amorolfine and terbinafine did not show any detectable permeation. The free-drug concentration of efinaconazole in a 5% human nail keratin suspension was 24.9%, which was significantly higher than those of the other drugs (1.1-3.9%). Additionally, efinaconazole was released from human nail keratin at a greater proportion than the other drugs. The MICs of the five drugs for Trichophyton rubrum were determined at various concentrations of keratin (0-20%) in RPMI 1640 medium. The MICs of ciclopirox were not affected by keratin, whereas those of efinaconazole were slightly increased and those of luliconazole and terbinafine were markedly increased in the presence of 20% keratin. Efficacy coefficients were calculated using the nail permeation flux and MIC in media without or with keratin. Efinaconazole showed the highest efficacy coefficient, which was determined using MIC in media with keratin. The order of efficacy coefficients determined using MIC in keratin-containing media rather than keratin-free media was consistent with that of complete cure rates in previously reported clinical trials. The present study revealed that efficacy coefficients determined using MIC in keratin-containing media are useful for predicting the clinical efficacies of topical drugs. In order to be more effective, topical drugs have to possess higher efficacy coefficients.

Efinaconazole Topical Solution, 10%: Factors Contributing to Onychomycosis Success.

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.

Cause and risk of catastrophic eruptions in the Japanese Archipelago.

The Japanese Archipelago is characterized by active volcanism with variable eruption styles. The magnitude (M)-frequency relationships of catastrophic caldera-forming eruptions (M ≥ 7) are statistically different from those of smaller eruptions (M ≤ 5.7), suggesting that different mechanisms control these eruptions. We also find that volcanoes prone to catastrophic eruptions are located in regions of low crustal strain rate (<0.5 × 10(8)/y) and propose, as one possible mechanism, that the viscous silicic melts that cause such eruptions can be readily segregated from the partially molten lower crust and form a large magma reservoir in such a tectonic regime. Finally we show that there is a ∼1% probability of a catastrophic eruption in the next 100 years based on the eruption records for the last 120 ky. More than 110 million people live in an area at risk of being covered by tephra >20 cm thick, which would severely disrupt every day life, from such an eruption on Kyushu Island, SW Japan.

In vitro and in vivo assessment of dermatophyte acquired resistance to efinaconazole, a novel triazole antifungal.

Efinaconazole is a novel triazole antifungal drug for the topical treatment of onychomycosis, a nail infection caused mainly by dermatophytes. We assessed the potential of efinaconazole to induce resistance in dermatophytes by continuous exposure of Trichophyton rubrum strains to efinaconazole in vitro (12 passages) and in a guinea pig onychomycosis model (8 weeks). There was no evidence of efinaconazole resistance development in the tested strains under the experimental conditions used.

The low keratin affinity of efinaconazole contributes to its nail penetration and fungicidal activity in topical onychomycosis treatment.

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.

Transungual delivery of efinaconazole: its deposition in the nail of onychomycosis patients and in vitro fungicidal activity in human nails.

BACKGROUND: Effective transungual delivery of topical antifungal agents in onychomycosis has been hampered by poor nail permeation. To be effective they must have antifungal efficacy, and effectively permeate through the dense keratinized nail plate to the site of infection in the nail bed and nail matrix. The therapeutic efficacy of efinaconazole topical solution, 10% has been established in two phase 3 clinical trials in distal lateral subungual onychomycosis. OBJECTIVE: To investigate the transungual delivery of efinaconazole in onychomycosis patients and its fungicidal activity in the toenail. METHODS: Concentrations of efinaconazole were determined as part of a multi-center, open label study in forty onychomycosis patients following repeated application of efinaconazole topical solution, 5% and 10% to the toenails over 28 days, with a 2-week follow-up. Fungicidal activity against T. rubrum in the ventral layer of human nails was determined using an in vitro human nail infection model (ChubTur®). RESULTS: Efinaconazole concentrations in the nail were four orders of magnitude higher than MIC values of efinaconazole against dermatophytes. Further, nail drug concentrations were not influenced by the presence of disease or nail thickness, and maintained at high antifungal levels post-treatment. Efinaconazole was effective in reducing fungal viability, suggesting that sufficient amounts of efinaconazole were being delivered into the ventral layer of the nail plate.
CONCLUSIONS: Effective transungual delivery of efinaconazole was demonstrated. The high efinaconazole concentrations in patient toenails and fungicidal activity in vitro potentially contribute to the clinical efficacy reported in phase 3 studies.

Mechanism of action of efinaconazole, a novel triazole antifungal agent.

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.

Comparison of in vitro antifungal activities of efinaconazole and currently available antifungal agents against a variety of pathogenic fungi associated with onychomycosis.

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 (MIC90) 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 (MIC50) 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.

Introducing site-specific glycosylation using protein engineering techniques reduces the immunogenicity of ß-lactoglobulin.

To reduce the immunogenicity of ß-lactoglobulin (BLG), we prepared wild-type bovine BLG variant A (wt) and three site-specifically glycosylated BLGs (D28N, D137N/A139S, and P153A), and expressed them in the methylotrophic yeast Pichia pastoris by fusion of the cDNA to the sequence coding for the α-factor signal peptide from Saccharomyces cerevisiae. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the glycosylated BLGs were conjugated with a ~4 kDa high-mannose chain. Each glycosylated BLG retained ∼80% of the retinol-binding activity of BLG. Structural analyses by intrinsic fluorescence, CD spectra, and ELISA with monoclonal antibodies indicated that the surface structure was slightly changed by using protein engineering techniques, but that the site-specifically glycosylated BLGs were covered by high-mannose chains without substantial disruption of wt conformation. Antibody responses to the glycosylated BLGs tended to be weaker in BALB/c, C57BL/6, and C3H/He mice. We conclude that site-specific glycosylation is an effective method to reduce the immunogenicity of BLG, and that masking of epitopes by high-mannose chains is effective to reduce immunogenicity.

[Characteristic evaluation and clinical usefulness of dose area product meter at radiography].

Philips DigitalDiagnost, a digital radiographic system mounting flat panel detector (FPD), can display dose area data (DiDi dose) calculated by examination parameters. We evaluated its fundamental characteristics and compared the values of DiDi dose andactual measured data obtained by the area dose product meter (PD-4100L). Tendency of varied values of mAs, X-ray tube values and exposure area from both the area dose product meter and the DiDi dose were coincided. Further, in clinical images of chest PA 100 cases, chest lateral 50 cases and abdomen stand 25 cases, the determination coefficient was overly high as R(2)=0.99. Based on these results, it is clear that the DiDi dose can be treated the same as the area dose product meter. Under increasing of patient X-ray exposure dose is a concern in digital general radiography, this research indicates that maximum values of histogram obtained by DiDi dose contributes dose awareness for radiographer.

The structure of iron in Earth's inner core.

Earth's solid inner core is mainly composed of iron (Fe). Because the relevant ultrahigh pressure and temperature conditions are difficult to produce experimentally, the preferred crystal structure of Fe at the inner core remains uncertain. Static compression experiments showed that the hexagonal close-packed (hcp) structure of Fe is stable up to 377 gigapascals and 5700 kelvin, corresponding to inner core conditions. The observed weak temperature dependence of the c/a axial ratio suggests that hcp Fe is elastically anisotropic at core temperatures. Preferred orientation of the hcp phase may explain previously observed inner core seismic anisotropy.

F2928-1 and -2, new antifungal antibiotics from Cladobotryum sp.

Two new antifungal antibiotics, F2928-1 (1) and -2 (2), were isolated from the culture broth of Cladobotryum sp. These compounds were purified by solvent extraction, silica gel column chromatography and preparative HPLC, consecutively. The structures of these compounds were assigned as a decalin compound on the basis of various spectral analyses. These compounds showed antimicrobial activity against fungi including clinically important fungus, Aspergillus fumigatus.

Therapeutic efficacy of topically applied KP-103 against experimental tinea unguium in guinea pigs in comparison with amorolfine and terbinafine.

The therapeutic efficacy of KP-103, a novel topical triazole, in a guinea pig tinea unguium model was investigated. Experimental tinea unguium and tinea pedis were produced by inoculation of Trichophyton mentagrophytes SM-110 between the toes of the hind paw of guinea pigs. One percent solution (0.1 ml) of KP-103, amorolfine, or terbinafine was topically applied to the nails and whole sole of an infected foot once daily for 30 consecutive days, and terbinafine was also orally administered at a daily dose of 40 mg/kg of body weight for 30 consecutive days, starting on day 60 postinfection. The fungal burdens of nails and plantar skin were assessed using a new method, which makes it possible to recover infecting fungi by removing a carryover of the drug remaining in the treated tissues into the culture medium. Topically applied KP-103 inhibited the development of nail collapse, significantly reduced the fungal burden of the nails, and sterilized the infected plantar skin. On the other hand, topical amorolfine and topical or oral terbinafine were ineffective for tinea unguium, although these drugs eradicated or reduced the fungal burden of plantar skin. The in vitro activities of amorolfine and terbinafine against T. mentagrophytes SM-110 were 8- and 32-fold, respectively, decreased by the addition of 5% keratin to Sabouraud dextrose broth medium. In contrast, the activity of KP-103 was not affected by keratin because its keratin affinity is lower than those of the reference drugs, suggesting that KP-103 largely exists in the nails as an active form that was not bound to keratin and diffuses in the nail without being trapped by keratin. The effectiveness of KP-103 against tinea unguium is probably due to its favorable pharmacokinetic properties in the nails together with its potent antifungal activity.