The in vitro antifungal susceptibilities of six clinical Trichophyton rubrum isolates obtained sequentially from a single onychomycosis patient who failed oral terbinafine therapy (250 mg/day for 24 weeks) were determined by broth microdilution and macrodilution methodologies. Strain relatedness was examined by random amplified polymorphic DNA (RAPD) analyses. Data obtained from both broth micro-and macrodilution assays were in agreement and revealed that the six clinical isolates had greatly reduced susceptibilities to terbinafine. The MICs of terbinafine for these strains were >4 g/ml, whereas they were <0.0002 g/ml for the susceptible reference strains. Consistent with these findings, the minimum fungicidal concentrations (MFCs) of terbinafine for all six strains were >128 g/ml, whereas they were 0.0002 g/ml for the reference strain. The MIC of terbinafine for the baseline strain (cultured at the initial screening visit and before therapy was started) was already 4,000-fold higher than normal, suggesting that this is a case of primary resistance to terbinafine. The results obtained by the broth macrodilution procedure revealed that the terbinafine MICs and MFCs for sequential isolates apparently increased during the course of therapy. RAPD analyses did not reveal any differences between the isolates. The terbinafine-resistant isolates exhibited normal susceptibilities to clinically available antimycotics including itraconazole, fluconazole, and griseofulvin. However, these isolates were fully cross resistant to several other known squalene epoxidase inhibitors, including naftifine, butenafine, tolnaftate, and tolciclate, suggesting a target-specific mechanism of resistance. This is the first confirmed report of terbinafine resistance in dermatophytes.

A new class of synthetic antifungal agents, the allylamines , has been developed by modification of naftifine , a topical antimycotic. SF 86-327, the most effective of these compounds so far, is highly active in vitro against a wide range of fungi and exceeds clinical standards in the oral and topical treatment of guinea pig dermatophytoses. SF 86-327 is a powerful specific inhibitor of fungal squalene epoxidase, a key enzyme in sterol biosynthesis.

Host factors involved in viral replication are potentially attractive antiviral targets that are complementary to specific inhibitors of viral enzymes, since resistant mutations against the latter are likely to emerge during long-term treatment. It has been reported recently that cyclosporine, which binds to a family of cellular proteins, cyclophilins, inhibits hepatitis C virus (HCV) replication in vitro. Here, the activities of various cyclosporine derivatives were evaluated in the HCV replicon system. There was a strong correlation between the anti-HCV activity and cyclophilin-binding affinity of these compounds. Of these, NIM811 has been selected as a therapeutic candidate for HCV infection, since it binds to cyclophilins with higher affinity than cyclosporine but is devoid of the significant immunosuppressive activity associated with cyclosporine. NIM811 induced a concentration-dependent reduction of HCV RNA in the replicon cells with a 50% inhibitory concentration of 0.66 M at 48 h. Furthermore, a greater than three-log 10 viral RNA reduction was achieved after treating the cells with as little as 1 M of NIM811 for 9 days. In addition, the combination of NIM811 with alpha interferon significantly enhanced anti-HCV activities without causing any increase of cytotoxicity. Taken together, these promising in vitro data warrant clinical investigation of NIM811, an inhibitor of novel mechanism, for the treatment of hepatitis C.

There has only been one clinically confirmed case of terbinafine resistance in dermatophytes, where six sequential Trichophyton rubrum isolates from the same patient were found to be resistant to terbinafine and cross-resistant to other squalene epoxidase (SE) inhibitors. Microsomal SE activity from these resistant isolates was insensitive to terbinafine, suggesting a target-based mechanism of resistance (B. Favre, M. Ghannoum, and N. S. Ryder, Med. Mycol. 42:525-529, 2004). In this study, we have characterized at the molecular level the cause of the resistant phenotype of these clinical isolates. Cloning and sequencing of the SE gene and cDNA from T. rubrum revealed the presence of an intron in the gene and an open reading frame encoding a protein of 489 residues, with an equivalent similarity (57%) to both yeast and mammalian SEs. The nucleotide sequences of SE from two terbinafine-susceptible strains were identical whereas those of terbinafine-resistant strains, serially isolated from the same patient, each contained the same single missense introducing the amino acid substitution L393F. Introduction of the corresponding substitution in the Candida albicans SE gene (L398F) and expression of this gene in Saccharomyces cerevisiae conferred a resistant phenotype to the transformants when compared to those expressing the wild-type sequence. Terbinafine resistance in these T. rubrum clinical isolates appears to be due to a single amino acid substitution in SE.Dermatophytosis is a common infection of the keratinized tissues skin, hair, and nails caused by dermatophytes. Among the three known genera of dermatophytes, Epidermophyton, Microsporum, and Trichophyton, Trichophyton species, especially T. mentagrophytes, T. tonsurans, and T. rubrum, are the most common pathogens, with T. rubrum being the most prevalent isolated organism. T. rubrum is particularly involved in tinea pedis and tinea unguium (onychomychosis). While most superficial infections can be effectively cured with different topical agents with various mechanisms of action, tinea capitis and onychomychosis require the use of oral drugs such as fluconazole, itraconazole, griseofulvin, and terbinafine to be sucessfully treated. Nail infections require extended periods of therapy with at least 3 months of daily or intermittent dosing regimens.Despite the high incidence of dermatophytosis and the difficult and long-term treatment of some of these infections, with associated uneven patient compliance, antifungal resistance in dermatophytes appears to be rare. This is in contrast with candidiasis and aspergillosis, where numerous isolates resistant to various antifungals have been identified and then characterized (1,18,24,27,29,30). Systematic susceptibility testing of clinical isolates from patients with onychomychosis who failed on therapy with terbinafine did not reveal any correlation between the MIC of terbinafine against these isolates and clinical failure (20). Nevertheless, in one case terbinafine-resistant T. rubrum was identified (20). All isolates...

SF 86-327 is a new antimycotic agent of the ailylamine type. Its primary action appears to be the inhibition of ergosterol biosynthesis at the point of squalene epoxidation, as was previously found with the related compound naftifine. Biosynthesis was measured by incorporation of [14C]acetate into sterols in cells of Candida albicans, Candida parapsilosis, Torulopsis glabrata, and the dermatophyte Trichophyton mentagrophytes. There was a positive correlation between the SF 86-327 concentrations needed for inhibition of growth and of sterol synthesis in these four fungi. The greater antifungal efficacy of SF 86-327 in comparison with naftifine was also reflected in the relative activities of the two compounds as sterol synthesis inhibitors. Inhibition was maximal at neutral pH. A similar degree of inhibition was found in cell-free extracts when [14C]mevalonate was used as substrate. In all cases, inhibition of sterol synthesis was accompanied by a parallel accumulation of labeled squalene. SF 86-327 and naftifine had no significant effect on initial enzymes of the ergosterol pathway, measured by incorporation of [14C]acetyl coenzyme A, or on steps distal to squalene epoxidation, measured by conversion of labeled squalene 2,3-epoxide or lanosterol. Both allylamines were highly selective for fungal, as opposed to mammalian, sterol biosynthesis. SF 86-327 caused slight inhibition of squalene epoxidation in a rat liver cell-free system, but at concentrations three to four orders of magnitude greater than those required for inhibition of the fungal pathway.