Entry inhibition of HSV-1 and -2 protects mice from viral lethal challenge.

The present study focused on inhibition of HSV-1 and -2 replication and pathogenesis in vitro and in vivo, through the selective targeting of the envelope glycoprotein D. Firstly, a human monoclonal antibody (Hu-mAb#33) was identified that could neutralise both HSV-1 and -2 at nM concentrations, including clinical isolates from patients affected by different clinical manifestations and featuring different susceptibility to acyclovir in vitro. Secondly, the potency of inhibition of both infection by cell-free viruses and cell-to-cell virus transmission was also assessed. Finally, mice receiving a single systemic injection of Hu-mAb#33 were protected from death and severe clinical manifestations following both ocular and vaginal HSV-1 and -2 lethal challenge. These results pave the way for further studies reassessing the importance of HSV entry as a novel target for therapeutic intervention and inhibition of cell-to-cell virus transmission.

Topical formulations containing finasteride. Part II: determination of finasteride penetration into hair follicles using the differential stripping technique.

The differential stripping technique consists of a tape-stripping phase followed by a cyanoacrylate biopsy. This technique not only allows the quantification of drug retained in the stratum corneum (SC) and in the hair follicles but also differentiates transepidermal from transfollicular penetration. Our study aimed at both validating the differential stripping procedure on hairless rat skin and assessing the role of the hair follicle in the cutaneous penetration of finasteride (FNS) after application of two experimental formulations for 6 or 24 h: P-08-016, a hydroxypropyl chitosan (HPCH)-based formulation and P-10-008, an anhydrous formulation devoid of HPCH. Microscopic and histological evaluation showed that after 15 tape strips both the SC and the viable epidermis were completely removed. A subsequent cyanoacrylate skin surface biopsy led to the removal of the infundibula content. The largest amounts of FNS were found in the epidermis and in the appendages after application of P-08-016, regardless of the time from application. In contrast, smaller and statistically significant amounts of FNS were recovered with P-10-008 6 h after application, compared with that at 24 h. In conclusion, the differential stripping technique allowed determination of the amount of FNS localized in different skin districts, focusing particularly on the follicular contribution.

Topical formulations containing finasteride. Part I: in vitro permeation/penetration study and in vivo pharmacokinetics in hairless rat.

In hair follicle (Hf) cells, the type-2 5-α-reductase enzyme, implicated in androgenetic alopecia, is selectively inhibited by finasteride (FNS). Because an effective topical formulation to deliver FNS to Hf is currently unavailable, this investigation aimed at evaluating in vitro FNS skin permeation and retention through and into hairless rat and human abdominal skin. Four hydroxypropyl chitosan (HPCH)-based formulations (P-08-012, P-08-016, P-08-063, and P-08-064) and one anhydrous formulation without HPCH (P-10-008) were tested. The pharmacokinetics in plasma and skin after application of P-08-016 or P-10-008 on dorsal rat skin with single and repeated doses was investigated. P-08-016 performed the best in driving FNS to the reticular dermis without producing a high transdermal flux. Neither the in vivo single nor the repeated dose experiments produced plasma levels of FNS and no differences were found between formulations concerning skin retention. No increase in the amount of drug retained in the skin was obtained with the repeated dose experiment. In conclusion, the HPCH-based formulation P-08-016 might represent an alternative to systemic therapy for its ability to promote a cutaneous depot of FNS in the region of hair bulbs, minimizing systemic absorption even after repeated treatments.

Potential of Ergosterol synthesis inhibitors to cause resistance or cross-resistance in Trichophyton rubrum.

Superficial mycoses caused by Trichophyton rubrum are among the most common infections worldwide. T. rubrum infections are difficult to treat and are often associated with recurrences after interruption of the antifungal therapy. Nevertheless, reports on T. rubrum resistance to commonly used antifungal drugs are rare. In this study, we compared the in vitro resistance frequencies and development of resistance to terbinafine, itraconazole, amorolfine, and ciclopirox in T. rubrum. Results demonstrated that naturally occurring mutants were isolated at a frequency of 10(-7) for itraconazole and 10(-9) for terbinafine and amorolfine. To mimic conditions of body sites in which low drug levels are reached during therapy, T. rubrum was propagated for 10 transfers in media containing subinhibitory drug concentrations. Resistance to itraconazole, terbinafine, and amorolfine emerged at a higher frequency than was seen with spontaneous mutation. Itraconazole-resistant mutants also showed decreased susceptibility to amorolfine as well as to terbinafine, and amorolfine-resistant mutants were also less susceptible to terbinafine. No mutant resistant to ciclopirox was isolated, suggesting no propensity of T. rubrum to develop resistance to this drug. How different drug mechanisms of action can influence the onset of resistance is discussed.

Ciclopirox vs amorolfine: in vitro penetration into and permeation through human healthy nails of commercial nail lacquers.

One of the pre-requisite for a successful topical antifungal drug indicated for onychomycosis is its bioavailability into the nail unit for achieving fungal eradication and clinical benefit. The aim of this study was to compare in vitro permeation/penetration through and into human nails of amorolfine (MRF) from a 5% anhydrous commercial formulation (Loceryl®) and ciclopirox (CPX) from the 8% aqueous formulation in hydroxypropyl chitosan (HPCH) technology (Onytec®). The ability of the active ingredient to reach efficacious concentrations to inhibit nail pathogens was also evaluated. The amounts of drug permeated and retained in human healthy nails were determined using a suitably modified diffusion apparatus. HPLC analysis of the samples was performed. The HPCH-based CPX formulation demonstrated an efficient penetration into and permeation through the nail plates. Conversely, Loceryl® produced an amount of MRF permeated through and penetrated into the human toenails significantly lower than CPX. The evaluation of the efficacy index showed a higher potential efficacy of Onytec® with respect to Loceryl® on nail pathogens. The present work not only reinforced the previous results on different experimental substrates, but pointed out the superiority of HPCH-based Onytec® formulation containing CPX with respect to Loceryl® commercial product with MRF, both in terms of higher permeation through and penetration into the human nail, and for the efficacy towards the most common ungual pathogens.

In vitro activity of nifuratel on vaginal bacteria: could it be a good candidate for the treatment of bacterial vaginosis?

Bacterial vaginosis is characterized by a shift of the physiological flora to a diverse spectrum of bacteria, where Gardnerella vaginalis and Atopobium vaginae are the most important markers. In this study, the antimicrobial activity of nifuratel against G. vaginalis, A. vaginae, and lactobacilli was compared with that of the two currently used antibiotics metronidazole and clindamycin. Results suggest that nifuratel has a better spectrum of activity, being highly active against G. vaginalis and A. vaginae without affecting lactobacilli.

Ciclopirox: recent nonclinical and clinical data relevant to its use as a topical antimycotic agent.

Ciclopirox is a topical antimycotic agent belonging to the chemical class of hydroxypyridones and not related to azoles or any other class of antifungal agents. Its antimicrobial profile includes nearly all of the clinically relevant dermatophytes, yeasts and moulds, and is therefore broader than that of most other antimycotics. It is also active against certain frequently azole-resistant Candida species and against some bacteria. The mechanism of action of ciclopirox is different from that of other topical antifungal drugs, which generally act through ergosterol inhibition. The high affinity of ciclopirox for trivalent metal cations, resulting in inhibition of the metal-dependent enzymes that are responsible for the degradation of peroxides within the fungal cell, appears to be the major determinant of its antimicrobial activity. This unique and multilevel mechanism of action provides a very low potential for the development of resistance in pathogenic fungi, with cases of resistance rarely reported. Ciclopirox also displays mild anti-inflammatory effects in biochemical and pharmacological models; effects also shown in small clinical studies. Scavenging of reactive oxygen species released from inflammatory cells is a likely contributor to these anti-inflammatory effects. Ciclopirox, and its olamine salt, is available in multiple topical formulations, suitable for administration onto the skin and nails and into the vagina. The pharmaceutical forms most widely investigated are 1% ciclopirox olamine cream and 8% ciclopirox acid nail lacquer, but lotion, spray, shampoo, pessary, solution, gel and douche formulations have also been used. Ciclopirox penetrates into the deep layers of the skin, mucosal membranes and nail keratin, reaching concentrations exceeding the minimal fungicidal concentrations for most medically important fungi. A large number of clinical trials were and are still being performed with ciclopirox, starting in the early 1980s. Ciclopirox was first developed for fungal skin infections and vaginal candidiasis, and is currently well established in these indications. More recently, the drug has been clinically investigated in seborrhoeic dermatitis and onychomycosis, showing good efficacy and excellent tolerability. Emphasis in this review is given to a ciclopirox medicated nail lacquer, which is based on an original technology and has superior properties in terms of its affinity to keratin and nail permeation. It has been found to have superior efficacy and safety to another commercially available formulation in the treatment of onychomycosis. The safety features of ciclopirox are well known. The topical drug is devoid of systemic adverse reactions. Mild local reactions characterized by a burning sensation of the skin, irritation, redness, pain or pruritus, generally in less than 5% of treated patients, can be observed following skin and vaginal application. With nail application, the most common adverse event is the appearance of mild erythema in 5% of the treated population. As a general conclusion, although less effective than some oral antimycotic agents in various indications, ciclopirox compares very well in terms of the benefit/risk ratio due to its excellent tolerability and complete absence of serious adverse effects.

Antifungal activity, experimental infections and nail permeation of an innovative ciclopirox nail lacquer based on a water-soluble biopolymer.

P-3051 is an innovative 8% ciclopirox nail lacquer, based on hydroxypropyl chitosan (HPCH) as a film-forming agent. The authors' aim was to investigate P-3051's in vitro antifungal activity, as well as its in vitro and in vivo nail permeation. The dilution susceptibility tests performed for Trichophyton rubrum (T. rubrum) and Candida parapsilosis (C. parapsilosis) showed that the minimum inhibitory concentrations (MICs) of P-3051, as percent of ciclopirox, was for both fungi < or = 0.0015% (equivalent to a concentration of 15.6 mg/ ml). In the biological assay of in vitro nail permeation and fungal inhibition, the authors observed that P-3051 permeated well through bovine hoof membranes and produced dose-dependent inhibitory effects on dermatophyte, yeast and mold strains. Moreover, the inhibition effects were higher than those obtained by equal amounts of the ciclopirox reference nail lacquer. P-3051 and the reference showed the same protective activity in experimental infections with strains of dermatophytes isolated from clinical samples. The amount of ciclopirox remained in cut fingernails washed six hours after in vivo application of P-3051 ranged between 18 and 35% of the applied dose. After in vitro application to cut human nails, 40-50% of the applied ciclopirox penetrated during the first six hours, independent of nails being infected or uninfected, intact or filed. In both experiments, the concentration of ciclopirox is largely higher (three to four orders of magnitude) than the MICs for nail pathogens.

Computerized image analysis of nails affected by fungal infection: evaluation using digital photographs and manually defined areas.

Despite the relevant increase in clinical trials on the efficacy of various systemic and/or topical antifungal agents in onychomycosis therapy, the evaluation of the results is largely subjective. The aim of this study was to set up and ensure an objective, reproducible and reliable method to measure nail plate involvement in onychomycosis. In order to validate a specifically designed software for the computerized image analysis of affected areas of the nail, standardized clinical pictures of onychomycosis were prepared by six different clinicians using a sample of 11 affected nails. Diseased areas and total nail plates were measured both on the clinical pictures and on their drawings traced by the different clinicians on transparent tapes adhering to sample nails. The computerized procedure was undertaken by a trained operator who was not a dermatologist. The variation coefficients of measurements on clinical pictures (automatically detected) and on drawings were compared. In addition, the agreement between automatic evaluation and drawing was evaluated by means of Bland-Altman analysis. To consider the effect of possible variations linked to different operators using the computerized method, 11 clinical pictures (one for each clinical case considered) were selected and submitted to computerized image analysis by six different trained operators. The computerized detection of affected nail areas showed a coefficient of variation (vc=8.5%) lower than that observed on drawings (vc=14.7%). The two methods showed appreciable agreement, as demonstrated by Bland-Altman plot. The coefficient of variation of image analysis conducted by six different operators was very low for the total area calculation (vc=0.9%) and acceptable for pathological area detection (vc=4.8%). Based on the results obtained, we conclude that automatic evaluation is a reliable and helpful method for the measurement of the clinical involvement of the nail plate in onychomycosis and for the evaluation of therapies, since it can increase the objectivity and reproducibility of data. However, in a minority of difficult cases, expert dermatological evaluation is needed.

Microbiological and pharmaco-toxicological profile of nifuratel and its favourable risk/benefit ratio for the treatment of vulvo-vaginal infections. A review.

Nifuratel (CAS 4936-47-4) is a furane-derivative provided with a strong trichomonicidal activity equivalent to that of metronidazole (CAS 443-48-1); it has a broad antibacterial spectrum of action, including both Gram-negative and Gram-positive organisms. It is active against Chlamydia trachomatis and Mycoplasma spp. and has also some degree of activity against Candida spp. and mycetes. Its broad spectrum of action, confirmed by in vitro and in vivo studies, covers virtually all the micro-organisms responsible for the infections of the genito-urinary tract. Nifuratel has a very safe toxicological profile. It is practically non-toxic in acute tests in mice and rats and is also well tolerated after repeated oral and intravaginal administrations. Nifuratel is devoid of teratogenic effects. The comparison among past and recent clinical studies confirms that, in contrast to metronidazole, no resistance phenomena to the treatment with nifuratel are reported. The drug can be used during pregnancy due to the absence of teratogenic effects. In conclusion, nifuratel shows a very favourable risk/benefit ratio for the treatment of patients with vulvo-vaginal infections.