In vitro antifungal activity of luliconazole against nondermatophytic moulds.

In vitro antifungal activity of luliconazole against nondermatophytic moulds causing superficial infections was compared with that of five classes of 12 topical and systemic drugs. The minimum inhibitory concentration (MIC) of the drugs against the genera of Neoscytalidium, Fusarium, Aspergillus, Scedosporium, and Alternaria was measured via modified microdilution method. In results, the nondermatophytic moulds were found to be less susceptible to drugs to which Neoscytalidium spp. and Fusarium spp. were typically drug resistant. However, luliconazole was effective against all the genera tested, including afore-mentioned two species, and had the lowest MICs among the drugs tested.

Guinea pig seborrheic dermatitis model of Malassezia restricta and the utility of luliconazole.

Seborrheic dermatitis (SD) is a multifactorial disease in which Malassezia restricta has been proposed as the predominant pathogenic factor. However, experimental evidence supporting this hypothesis is limited. A guinea pig SD model using a clinical isolate of M. restricta was used to elucidate the pathogenicity of M. restricta. Also, the efficacy of 1% luliconazole (LLCZ) cream, a topical imidazole derivative, against M. restricta was compared with that of a 2% ketoconazole (KCZ) cream in the same guinea pig model. Dorsal skin hairs of guinea pig were clipped and treated with M. restricta by single or repeated inoculations without occlusion. Skin manifestations were examined macroscopically and histologically. A quantitative polymerase chain reaction (PCR) assay was also performed for mycological evaluation. An inflammatory response mimicking SD occurred after repeated as well as single inoculation but not in abraded skin. The inflammation score attained its maximum on day 11 and persisted until day 52. The yeast form of the fungal elements was distributed on the surface of stratum corneum and around the follicular orifices, and an epidermal and dermal histological reaction was observed. Application of 1% LLCZ or 2% KCZ cream significantly improved the skin manifestations and decreased the quantity of M. restricta rDNA in the skin lesions. The efficacy of topical antifungal drugs suggested that M. restricta is a pathogenic factor contributing to SD.

[In vitro Antifungal Activity of Luliconazole against Trichophyton spp].

The minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) of luliconazole against Trichophyton rubrum (14 strains) and Trichophyton mentagrophytes (14 strains), which are the most common cause of tinea, were compared with those of 6 topical antifungal drugs of lanoconazole, bifonazole, efinaconazole, liranaftate, naftifine and terbinafine.　Luliconazole showed the most potent antifungal activity (MIC90 =0.00098 µg/ml and MFC90 =0.0078 µg/ml) among the compounds tested against the two species. Efinaconazole and bifonazole, the drug of azole-class, showed a large MFC/MIC ratio. On the other hand, these ratios of luliconazole and lanoconazole were as small as those of liranaftate, naftifine and terbinafine which are thought to possess fungicidal mechanism. These results suggest that luliconazole possesses fungicidal activity against both species of Trichophyton.　In this study, we found that luliconazole had the most potent antifungal activity among the major topical antimycotics used in Japan and the US. Luliconazole would be the best-in-class drug for dermatophytosis in clinics.

[Affinity of Luliconazole to Keratin Prepared from Healthy Human Nailand Porcine Hoof].

Luliconazole (LLCZ), an imidazole derivative with a broad spectrum of potent antifungal activity especially for T. rubrum and T. mentagrophytes, is under development as a new drug for treatment of tinea unguium.　It is well known that curative effect of an antifungal agent in dermatophytosis is affected by the pharmacokinetics of an agent at the infection loci as well as its antifungal activity, but there is no report about the affinity of LLCZ to nail keratin. We studied LLCZ affinity to keratin powder prepared from healthy human nail and porcine hoof. The LLCZ adsorbed to keratin preparations was washed with phosphate buffer, and its concentration in the buffer supernatant was measured by HPLC. Antifungal titer of the supernatant was also biologically confirmed by disk diffusion assay. Adsorption rate of LLCZ was 80% or more, and LLCZ was gradually liberated into washing buffer. Cumulative liberation rate in 10 times repeated washing against initially adsorbed drug amount was 47.4% for keratin from human nail and was either 52.5% or 50.8% (depending on the LLCZ concentration) for keratin from porcine hoof. The supernatant showed antifungal potential to T. rubrum. These results indicate that LLCZ applied to the nail surface is fully adsorbed to nail keratin and gradually liberated from it. The nail keratin could function as drug reservoir to supply biologically active LLCZ to the nail tissue region of infection loci. The LLCZ delivered to the loci would exert its antifungal potential on tinea unguium. This study also suggests the versatility of porcine hoof powder as an alternative to human nail keratin preparation for non-clinical study.

Luliconazole Retention in Stratum Corneum and Prevention of Fungal Infection in a Guinea Pig Tinea Pedis Model.

OBJECTIVE: To compare drug concentrations in the stratum corneum following daily application of luliconazole and terbinafine cream in a guinea pig tinea pedis model. METHODS: Luliconazole 1% cream or terbinafine 1% cream were topically applied once daily to hind limbs of guinea pigs for 14 days. Drug concentration in stratum corneum of plantar skin was measured by HPLC-UV on days 1, 3, 7, 10, and 14. Separately, creams were applied daily for 5 days to the hind limbs of guinea pigs and skin drug release determined. In addition, drug retention in the stratum corneum was assessed by infecting guinea pigs with Trichophyton mentagrophytes, 14 and 21 days after a single application of luliconazole or terbinafine creams. RESULTS: Luliconazole stratum corneum concentrations were higher than those of terbinafine throughout the study. Concentrations of luliconazole and terbinafine were 71.6µg/g and 36.6µg/g, respectively, after a single application (P<.05), reaching steady state after 10 days. Cumulative release of luliconazole from the stratum corneum was 4.5 times greater than with terbinafine. Unlike terbinafine, no fungal invasion of the stratum corneum was seen 14 days post-treatment with luliconazole. CONCLUSIONS: Drug concentrations of luliconazole in the stratum corneum and subsequent release are greater than those achieved with terbinafine and may contribute to clinical efficacy. Luliconazole may also provide greater protection against disease recurrence.

Antifungal susceptibility testing method for dermatophytes -determination of MIC and MFC values.

In vitro antifungal susceptibility of pathogenic fungi is important information for physicians when selecting an appropriate antifungal drug, deciding the route of drug administration, surveying resistant strains, and so on. Although both the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) are well known endpoints of antifungal susceptibility, the MIC is by far the more highly referred in clinical laboratories. In fact, while methods for determining the MIC have been standardized in Japan and the West to ensure accuracy and reproducibility of the results, by contrast, scant attention has been paid to standardizing methods for determining the MFC. The same preference for MIC topical antifungal drugs for dermatophytes are concerned. In 1999, the Japanese Society of Medical Mycology published a new, standardized method of testing the MIC for dermatophytes and this has since been widely adopted. Nonetheless, the fact remains that the MFC is still determined using methods derived from antifungal pharmacology. Recently in Japan, however, the MFC of topical antifungal drugs has begun attracting more attention due to the development of new antifungal agents with fungicidal activity. These new developments call for improving our understanding of both the MIC and MFC as endpoints of antifungal susceptibility, and for standardizing methods for determining the MFC.The present paper has two objectives : first, to overview the MIC and MFC for topical drugs as endpoints of antifungal susceptibility; and second, to describe a novel test based on the standardized broth microdilution method combined with the trans-well system and neutral red, which we recently developed in our laboratory for directly measuring the MFC.

Spontaneous intraocular hemorrhage in rats during postnatal ocular development.

To study spontaneous intraocular hemorrhage in rats during postnatal ocular development and to elucidate the underlying mechanism, postnatal ocular development in the albino Wistar Hannover (WH) and Sprague-Dawley (SpD) and pigmented Long-Evans (LE) strains was analyzed. Pups (n = 2 to 5) from each strain were euthanized daily on postnatal days (PND) 0 through 21 and their eyes examined macroscopically and histologically; similar analyses were performed in 26 to 39 additional WH pups daily from PND 7 to 14. At necropsy, ring-shaped red regions and red spots were present in the eyes of WH and SpD rats. These lesions were attributed histologically to hemorrhage of the tunica vasculosa lentis or of the retina, choroid, and hyaloid artery, respectively. Similar intraocular hemorrhages occurred in LE rats, although the macroscopic alterations found in WH and SpD rats were not present in this strain. Among the 3 strains evaluated, the incidence of the intraocular hemorrhage was highest in WH rats. We here showed that intraocular hemorrhage occurs spontaneously during normal ocular development in rats regardless of the strain; however, the region, degree, and incidence of intraocular hemorrhage differ among strains. Hemorrhage in the tunica vasculosa lentis and hyaloid artery may result from the leakage of erythrocytes from the temporary vasculature of these tissues during regression. The mechanisms underlying hemorrhage in the retina and choroid remain unclear. To our knowledge, this report is the first to describe the spontaneous intraocular hemorrhage that occurs during postnatal ocular development in rats.

Ligand-independent androgen receptor antagonism caused by the newly developed pesticide pyrifluquinazon (PFQ).

Androgen receptor (AR) is an essential component to activate AR dependent gene transcriptions. Despite wide acceptance of pharmacological controls on transcriptional pathway depending on competitive inhibitions of ligand binding, only a few examples, AR antagonism via ligand-independent mechanisms, have been recognized. Pyrifluquinazon(PFQ), a newly developed pesticide, induced representative AR antagonism against rats in in vivo and in vitro. Intriguingly, this AR antagonism was not based on inhibition of ligand binding. Instead, the evidence suggested that the AR antagonism was induced as a consequence of decline of cellular AR protein level. This study demonstrated that AR N-terminal region could be an essential element for a ligand-independent mechanism underling the AR antagonism by PFQ. Our findings should provide a novel insight into the regulation of AR-mediated transcription.

Short-term therapy with luliconazole, a novel topical antifungal imidazole, in guinea pig models of tinea corporis and tinea pedis.

Luliconazole is a novel topical antifungal imidazole with broad-spectrum and potent antifungal activity. The drug is under clinical development in the United States for management of dermatophytosis with a short-term treatment regimen. The present study was undertaken to investigate the clinical benefit of short-term therapy with luliconazole cream in guinea pig models of tinea corporis and tinea pedis induced with Trichophyton mentagrophytes. The dose-dependent therapeutic efficacy of topical luliconazole cream (0.02 to 1%), measured by macroscopic improvement of skin lesions and by fungal eradication as determined by a culture assay, was demonstrated using a tinea corporis model. The improvement in skin lesions seen with luliconazole cream was observed even at a concentration of 0.02%, and its efficacy at 0.1% was equal to that of 1% bifonazole cream. The efficacy of short-term therapy with 1% luliconazole cream, which is used for clinical management, was investigated using the tinea corporis model (4- and 8-day treatment regimens) and the tinea pedis model (7- and 14-day treatment regimens). The 1% luliconazole cream completely eradicated the fungus in half or less of the treatment time required for 1% terbinafine cream and 1% bifonazole cream, as determined by a culture assay for both models. These results clearly indicate that 1% luliconazole cream is sufficiently potent for short-term treatment for dermatophytosis compared to existing drugs. Luliconazole is expected to be useful in the clinical management of dermatophytosis.

[Animal model for superficial mycosis].

Tinea corporis and the tinea pedis model in guinea pig with Trichophyton mentagrophytes are well established models of dermatophytoses. We attempted to provide animal infection models for T. tonsurans, endemic in Japan, and Malassezia restricta, an important pathogenic factor in seborrhoeic dermatitis, by utilizing the tinea corporis model. An inoculum of the organisms was applied to the back skin of male guinea pigs. T. tonsurans infected animals showed follicular inflammation mimicking those seen in humans. Interestingly, anthropophilic T. tonsurans showed a high infection rate in animal skin. Meanwhile, a single application of M. restricta, as well as consecutive applications to the surface of the skin without any pretreatment, succeeded in producing scales mimicking seborrhoeic dermatitis, but application of the pathogens after the tape stripping of the stratum corneum failed to induce infection. These models using guinea pigs were considered to be useful for studying the pathogenesis of, and evaluating therapies for, T. tonsurans infection and seborrhoeic dermatitis.

In vitro antifungal activities of luliconazole, a new topical imidazole.

Luliconazole is a topical antifungal drug newly developed in Japan. The present study compares the in vitro antifungal activity of luliconazole against clinically important dermatomycotic fungi with that of other representative antifungal drugs. The reference drugs chosen were five classes of nine topical agents, i.e., allylamine (terbinafine), thiocarbamate (liranaftate), benzylamine (butenafine), morpholine (amorolfine), and azole (ketoconazole, clotrimazole, neticonazole, miconazole and bifonazole). The minimum inhibitory concentrations (MIC) of luliconazole and the reference drugs against Trichophyton spp. (T. rubrum, T. mentagrophytes and T. tonsurans) and Candida albicans were measured by the standardized broth microdilution method. Luliconazole demonstrated greater potency against Trichophyton spp. (MIC range:

[A case of bacterial meningitis due to Streptococcus bovis in an infant with normal cerebrospinal fluid findings at the first CSF examination].

Streptococcus bovis very occasionally causes rarely sepsis, endocarditis, and meningitis in newborns and the elderly. We report the case of infant meningitis caused by S. bovis despite normal cerebrospinal fluid (CSF) findings at the first CSF examination. A 77-day-old boy with 21-trisomy and patent foramen ovale and seen for a high fever underwent blood examination and lumbar puncture due to toxic appearance despite a lack of meningeal signs, and was admitted. His CSF findings were normal and he was given intravenous ceftriaxone against potential bacteremia. He had systemic seizures with continuous fever for 2 days after admission and a second CSF examination. Gram-positive coccus grew from his CSF at the first examination, and CSF cells from the second lumbar puncture increased to 4060/tL (86% neutrophils), so vancomycin was added against potential enterococcal meningitis. S. bovis was finally grown from the first CSF, ceftriaxone discontinued, and intravenous ampicillin added. He recovered after 20 days of antibiotic administration. S. bovis becomes a potential pathogen for meningitis in infants, and must be considered as a cause of meningitis despite its very rarity. CSF findings at the first lumbar puncture may be normal for meningitis in newborns and infants at the first CSF examination, so we must be very careful in the diagnosis of bacterial meningitis even with normal CSF findings, and considered antibiotic treatment against potential bacterial meningitis.

Genotypes of macrolide-resistant pneumococci from children in southwestern Japan: raised incidence of strains that have both erm(B) and mef(A) with serotype 6B clones.

MICs of penicillin G, erythromycin, clarithromycin, clindamycin, azithromycin, and telithromycin were tested for 189 clinical isolates collected during 2002 to 2005 from children in southwestern Japan. Serotyping and polymerase chain reaction for presence of erm(B) and mef(A) were performed. All strains with erm(B) + mef(A) were analyzed by pulsed-field gel electrophoresis (PFGE) and compared to 3 global clones: Spain(23F)-1; Spain(9V)-3 and its variant -14; a South Korean strain same as Taiwan (19F)-14 clone and 5 strains with erm(B) + mef(A) from other countries. Of the 173 macrolide-resistant (erythromycin MIC > or =0.5 microg/mL) strains, 104 (60.1%) had erm(B), 47 (27.2%) had mef(A), and 22 (12.7%) had erm(B) + mef(A). Strains expressing erm(B) or both erm(B) and mef(A) had high macrolide MIC(90)s (>64 microg/mL), except telithromycin (MIC(90), 0.25 microg/mL). Of the 22 erm(B) + mef(A) strains, 10 had 4 distinct PFGE patterns and were mainly serotype 6B clones, which differed from those described in previous reports; 5 other strains had unique profiles.

[Dipylidium caninum infection in an infant].

Dipylidium caninum, the dog tapeworm, is a common intestinal cestode of domestic dogs and cats, but few cases have been reported of human infection by this parasite in Japan. We repot a case of D. caninum infection in a 17 month-old girl, who sometimes had symptoms of abdominal pain, diarrhea, and dysphoria at night. Her mother noted the appearance of small white worms in her stool, and she was seen by a local pediatrician. Despite antiparasitic therapy wiht pyrantel pamoate, the problem persisted and was eventually referred for further workup to Kurume University Hospital. The diagnosis was made by microscopic examination of the excreted proglottids, which contained characteristic egg capsules. She was successfully treated with a singledose of praziquantel and four adult parasites were recovered. The longest intact worm was 32cm. Her family had household pets (a dog and a cat). The pets were seen by the local veterinary and both were evidenced D. caninum. Humans, primarily children, become infected when they accidentally ingest fleas. Parents usually find proglottids as multiple white objects, often described as cucumber, melon, or pumpkin seeds, in stool, diapers, or on the perineum. Most general practitioners and pediatricians may treat children with enterobiasis (pinworm) infection, and in case the treatment fails, other parasite infection should be considered such as this worm. A history of dog or cat pets, fleas, and flea bites may be important clues to diagnosis. Pets found to be infected should also be treated.

[In vitro activities of antifungal drugs against clinical isolates of Trichophyton tonsurans].

To determine drug susceptibility of Trichophyton tonsurans endemic in Japan, in vitro MICs of antifungal drugs against a total of 10 clinical isolates of T. tonsurans collected from dermatophytosis patients were measured by the agar dilution method and the broth microdilution method. The agar dilution method was not appropriate as the growth of T. tonsurans on the agar medium was too slow to determine drug activity, while the broth microdilution method was thought to be an appropriate method for this study. The MIC90 values determined by the broth microdilution method for terbinafine, itraconazole, miconazole and ketoconazole were 0.013, 0.1, 0.8 and 0.4 microg/ml, respectively. Meanwhile, the MIC90 values of lanoconazole and luliconazole, known to be antifungal drugs potent against dermatomycosis, were 0.00078 and 0.00039 microg/ ml, respectively. The drug susceptibility of these T. tonsurans isolates to the aforementioned antifungal drugs was found to be on a similar level with that of T. mentagrophytes and T. rubrum, major causative agents of dermatomycosis. The results also demonstrated the strong antifungal activity of lanoconazole and luliconazole against T. tonsurans.

In vitro antifungal activity of luliconazole against clinical isolates from patients with dermatomycoses.

The in vitro antifungal activity of luliconazole, a novel topical imidazole, against pathogenic fungi implicated in dermatomycoses was studied. A total of 91 clinical isolates, consisting of 59 Trichophyton rubrum isolates, 26 T. mentagrophytes isolates, 1 Epidermophyton floccosum isolate, and 5 Candida albicans isolates were tested by the broth microdilution method, employing lanoconazole, terbinafine, and bifonazole as reference drugs. The minimum inhibitory concentrations (MICs) of luliconazole against T. rubrum and T. mentagrophytes were in the range of 0.00012-0.004 microg/ml and 0.00024-0.002 microg/ml, respectively. The MIC90 of luliconazole for these two species of dermatophytes was the same, at 0.001 microg/ml, and these values were 4 times, 30 times, and more than 1000 times lower than those of lanoconazole, terbinafine, and bifonazole, respectively. Similarly, the 1 isolate of E. floccosum tested was inhibited by luliconazole with an MIC of 0.001 microg/ml. Luliconazole also proved to be very potent against C. albicans (MIC range, 0.031-0.25 microg/ml), nearly on par, in terms of efficacy, with lanoconazole (0.063-0.25 microg/ml) and more potent than terbinafine (2->64 microg/ml) and bifonazole (0.5-4 microg/ml). These results showed that luliconazole was very potent in vitro against pathogenic fungi isolated from patients with dermatomycoses, and these findings emphasized the utility of luliconazole for the topical management of this condition.