Hoof Membranes Research Articles

Influence of penetration enhancers and molecular weight in antifungals permeation through bovine hoof membranes and prediction of efficacy in human nails

This work aimed to evaluate the effect of different substances on the permeation of geraniol through bovine hoof membranes. Different penetration enhancers were able to increase the permeability up to 25 times compared to control. It was demonstrated that acetilcysteine in association with ascorbic acid increased the permeation, even in acid formulations. In addition, some antifungal drugs were incorporated into a gel formulation of HPMC containing acetylcysteine 5% and ascorbic acid 0.2% and then the permeation coefficient through bovine hoof membranes was evaluated. The relationship between permeability and molecular weight was established for fluconazole, miconazole, terbinafine, butenafine, geraniol and nerol. Geraniol and nerol, the antifungals with lower molecular weight, had the better permeability results. Permeability coefficients for nail plates were estimated and geraniol demonstrated similar or even better efficacy index values against T. rubrum, T. menthagrophytes and M. canis compared with terbinafine and miconazole.

Effect of pressure sensitive adhesive and vehicles on permeation of terbinafine across porcine hoof membrane

The purpose of this study was to investigate characteristics of transungual drug delivery and the feasibility of developing a drug-in-adhesive formulation of terbinafine. The permeation of terbinafine from a PSA matrix across porcine hoof membrane was determined using a plate containing poloxamer gel. The permeation rate of terbinafine across hairless mouse skin was evaluated using a flow-through diffusion cell system. The permeation of terbinafine across the hoof membranes was the highest from the silicone adhesive matrix, followed by PIB, and most of the acrylic adhesives, SIS, and SBS. The rank order of permeation rate across mice skin was different from the rank order across porcine hooves. The amount of terbinafine permeated across the porcine hoof membranes poorly correlated with the amount of terbinafine remaining inside the hooves after 20 days, however, the ratio between rate of terbinafine partitioning into the hoof membrane and its rate of diffusion across the membrane was relatively constant within the same type of PSA. For influence of various vehicles in enhancing permeation of terbinafine across the hoof membrane, all vehicles except Labrasol(®) showed tendency to improve permeation rate. However, the enhancement ratio of a given vehicle differed from one adhesive to another with a moderate correlation between them. The infrared spectrum of the hoof treated with NMP, PPG 400 or PEG 200 indicated that the conformation of keratin changed from a non-helical to a helical structure.

Antimycotic activity and penetration through bovine hoof membranes of an innovative terbinafine nail solution (P-3058)

Antimycotic activity and penetration through bovine hoof membranes of an innovative terbinafine nail solution (P-3058)

Thermogelling hydrogels of cyclodextrin/poloxamer polypseudorotaxanes as aqueous-based nail lacquers: Application to the delivery of triamcinolone acetonide and ciclopirox olamine

This work investigated the use of in situ gelling hydrogels based on polypseudorotaxanes of Pluronic F-127 and partially methylated β-cyclodextrin as aqueous nail lacquers. N-acetylcysteine and urea were incorporated as penetration enhancers. The formulations were tested for their ability to deliver ciclopirox and triamcinolone across human nail plate and bovine hoof. Simple aqueous solutions of the drugs with N-acetylcysteine provided measurable fluxes across hoof membranes but became quickly depleted of drug. Further, these solutions would have a short residence time upon nail application. Addition of Pluronic F-127 facilitated drug solubilization and provided the formulations with in situ gelling properties but drug entrapment into the micelles slowed down the delivery process. This was solved by addition of methylated β-cyclodextrin; the formulations retained the thermogelling properties, drug solubilization was further increased, and drug delivery was accelerated. The polymer chains compete with the drugs for the cyclodextrin cavity forming polypseudorotaxanes, which facilitated drug release. The permeability of both drugs was higher across bovine hoof than human nail. The new polypseudorotaxanes formulation delivered more ciclopirox across human nail than a marketed organic lacquer which supports the growing hypothesis that aqueous-based nail lacquers represent a superior formulation strategy in nail topical delivery.

In vivo measurement of the surface energy of human fingernail plates

The surface energy of the human nail plate is expected to influence the adhesion of microorganisms (and subsequent colonization and infections) as well as that of medicines (and subsequent drug permeation) and of cosmetics. The aim of the study was therefore to measure the surface energy of nail plates in vivo. The surface energy of healthy human fingernails (untreated, hydrated and abraded) and of hoof membranes (often used as a model for the nail plate) was estimated from contact angle measurements of liquids (water, formamide, diiodomethane and glycerol) on the nail plate and subsequent computation using the Lifshitz-van der Waals/acid-base (LW-AB) approach. The surface energy of untreated fingernail plates was found to be 34 mJ m(-2) . Most of this total energy was from the apolar Lifshitz-van der Waals component. When the polar component of the surface energy was analysed, the electron donor component was considerably larger than the electron acceptor one. Hydrating the nail plate had no significant influence on the surface energy. In contrast, abrasion caused a small, but statistically significant increase in the apolar surface energy component. The surface energy of bovine hoof membrane was similar to that of the fingernail plate. We conclude that the human fingernail plate is a low-energy surface and that bovine hoof membranes may be used as a substitute for the nail plate in certain experiments.

In vitro Antimycotic Activity and Nail Permeation Models of a Piroctone Olamine (Octopirox) Containing Transungual Water Soluble Technology

Several in vitro studies with a new medical device (Myfungar) containing 0.5% of piroctone olamine (CAS 68890-66-4, octopirox) in a hydroxypropyl chitosan hydroalcoholic solution were performed. The chemical name of the active ingredient is 1-hydroxy-4-methyl-6 (2,4,4-trimethylpentyl)-2(1H)-pyridone; combination with 2-amino-ethanol (1:1). The antimycotic activity was determined in the most common fungi responsible of nail infections such as Candida parapsilosis, Scopulariopsis brevicaulis or Trichophyton rubrum. The minimum inhibitory concentration (MIC), found by means of the broth dilution susceptibility method, ranged between 0.0003% and 0.006% for all pathogens considered. The in vitro permeation study was performed by using bovine hoof membranes inserted in a modified Gummer vertical permeation cell. The experiment showed, at 30 h, a retention of the product in the nail membranes by 11.1% of the applied dose. No piroctone permeation through bovine hoof membranes could be detected by HPLC due to the limit of quantitation of this method. On the other hand, permeation of nail membranes has been demonstrated by a biological assay: the study of in vitro permeation through bovine hoof membranes, performed by biological assay, showed dose-dependent inhibition rings of T. rubrum growth by the tested device, placed either on disks for antibiogram or on nail fragments. The placebo did not show any inhibition. In vitro experimental infection by T. rubrum showed a preventive activity of the tested device on fungal growth as well as a curative activity, as the pathogen was eradicated by the tested solution in previously infected nails.

Validation of bovine hoof slices as a model for infected human toenails: in vitro ciclopirox transungual permeation

Topical therapy has recently been proposed for treating onychomycosis and other nail disturbances. However, the clinical outcome may be limited by the difficulty of active ingredients effectively penetrating the nail plate. Bovine hoof membranes have been widely used to predict in vitro efficacy of drug products in nail diseases. Many studies have compared bovine hooves with human healthy nails, considering the difference between healthy and unhealthy nails to be negligible. To validate bovine hoof slices as a model for human unhealthy nails by investigating the transungual permeation/retention of ciclopirox (CPX) through bovine hoof slices and excised infected human toenails after application of a new film-forming formulation (P-3051). To investigate the ability of CPX to achieve fungicidal concentrations in and through infected toenails. A new experimental technique based on a permeation unit allowed analysis by high-performance liquid chromatography of the amounts of CPX permeating through and retained in the membranes. The efficacy index was evaluated as follows: amount of permeated CPX/Trichophyton rubrum minimum inhibiting concentration. Extrapolated CPX flux through bovine hoof slices was about 14-fold higher than through infected human toenails, the difference being mainly due to the fourfold higher thickness of the toenails. In toenails, the CPX efficacy index for T. rubrum was positive (>1·0) soon after P-3051 application. This study confirms the validity of bovine hoof slices as a model for infected human nails, and suggests a substantial equivalence between the two models. Following P-3051 application, CPX reaches fungicidal concentrations in and through human infected toenails.

In-vitro permeability of the human nail and of a keratin membrane from bovine hooves: prediction of the penetration rate of antimycotics through the nail plate and their efficacy.

In contrast to the partition coefficient octanol/water the molecular size of penetrating drugs has a noticeable influence on the permeability of the human nail plate and a keratin membrane from bovine hooves. The relationship between permeability and molecular weight is founded on well-established theories. The correlation between the permeability of the nail plate and that of the hoof membrane allows a prediction of the nail permeability after determination of the drug penetration through the hoof membrane. The maximum flux of ten antimycotics (amorolfine, bifonazole, ciclopirox, clotrimazole, econazole, griseofulvin, ketoconazole, naftifine, nystatin and tolnaftate) through the nail plate was predicted on the basis of their penetration rates through the hoof membrane and their water solubilities. An efficacy coefficient against onychomycoses was calculated from the maximum flux and the minimum inhibitory concentration. Accordingly, amorolfine, ciclopirox, econazole and naftifine are expected to be especially effective against dermatophytes, whereas in the case of an infection with yeasts only, amorolfine and ciclopirox are promising.

In-vitro permeability of the human nail and of a keratin membrane from bovine hooves: influence of the partition coefficient octanol/water and the water solubility of drugs on their permeability and maximum flux.

Penetration of homologous nicotinic acid esters through the human nail and a keratin membrane from bovine hooves was investigated by modified Franz diffusion cells in-vitro to study the transport mechanism. The partition coefficient octanol/water PCOct/W of the esters was over the range 7 to > 51,000. The permeability coefficient P of the nail plate as well as the hoof membrane did not increase with increasing partition coefficient or lipophilicity of the penetrating substance. This indicates that both barriers behave like hydrophilic gel membranes rather than lipophilic partition membranes as in the case of the stratum corneum. Penetration studies with the model compounds paracetamol and phenacetin showed that the maximum flux was first a function of the drug solubility in water or in the swollen keratin matrix. Dissociation hindered the diffusion of benzoic acid and pyridine through the hoof membrane. Since keratin, a protein with an isoelectric point of about 5, is also charged, this reduction can be attributed to an exclusion of the dissociating substance due to the Donnan equilibrium. Nevertheless, the simultaneous enhancement of the water solubility makes a distinct increase of the maximum flux possible. In order to screen drugs for potential topical application to the nail plate, attention has to be paid mainly to the water solubility of the compound. The bovine hoof membrane may serve as an appropriate model for the nail.

Teratohyla sornozai Cisneros-Heredia, Yánez-Muñoz y Ortega-Andrade es un sinónimo junior de Rulyrana orejuela Duellman y Burrowes (Amphibia, Anura, Centrolenidae)

Teratohyla sornozai fue recientemente descrita [1] sobre la base de especímenes colectados en tres localidades en las provincias de Esmeraldas, Imbabura y Pichincha, noroccidente del Ecuador. En la descripción original [1], T sornozai se diferenció de Rulyrana orejuela [2] por la forma del hocico (proyectado de perfil en T sornozai, truncado de perfil en R. orejuela), la extensión de la membrana en manos y pies (mayor en T. sornozai), la presencia de melanóforos en las manos y pies (mayor en R. orejuela), el color del iris (gris oscuro con un anillo circumpupilar amarillo en R. orejuela, vs. dorado con reticulaciones oscuras en T. sornozai), y el tamaño corporal (mayor en R. orejuela). Sin embargo, la adquisición de mayor material de R. orejuela y la re-evaluación de los especímenes asignados a T. sornozai permitieron determinar que ambos nombres corresponden a una misma especie. Las diferencias observadas en los especímenes asignados a Teratohyla sornozai se deben a variación intraespecífica (por ejemplo, el color del iris) o a cambios ontogénicos (los juveniles de R. orejuela presentan menos melanóforos en las patas, una mayor extensión en las membranas manuales y pediales). La variación en la forma del hocico parece corresponder a un patrón común observado en varias especies de Centrolenidae, donde los juveniles tienen el hocico proyectado en vista lateral o de perfil. Basados en esta nueva evidencia, colocamos a Teratohyla sornozai Cisneros-Heredia, Yánez-Muñoz y Ortega-Andrade como un sinónimo junior de Rulyrana orejuela Duellman y Burrowes (Figuras 1-2). Con este cambio, el número de localidades conocidas para Rulyrana orejuela en Ecuador se incrementa a cuatro: Mashpi, Saguangal [3], Río Naranjal y Canadé [1].

Hydrosoluble medicated nail lacquers: in vitro drug permeation and corresponding antimycotic activity

Two nail lacquers, containing ciclopirox (CPX) or amorolfine (MRF), based on water-insoluble polymers are currently considered mainstays of topical treatment of onychomycosis. The present study aimed at evaluating the antimycotic activity of a new water-soluble nail lacquer containing CPX (CPX/sol), easily removable by washing with water and applicable to periungual skin. To compare transungual permeation of CPX with that of MRF in the same hydroxypropyl chitosan-based nail lacquer (MRF/sol) and with a nonwater-soluble reference (Loceryl); Galderma International, La Défense, France), and to evaluate the antimycotic activity of CPX/sol and Loceryl against the most common fungal strains that cause onychomycosis. Methods In vitro drug permeation experiments with CPX/sol, MRF/sol and Loceryl were carried out through bovine hoof slices. Experimental permeates from CPX/sol and Loceryl underwent in vitro susceptibility testing against clinical isolates of dermatophytes, moulds and yeast. Results MRF transungual flux from MRF/sol lacquer was significantly higher when compared with Loceryl. CPX was able to permeate hoof membranes more easily compared with MRF. CPX and MRF concentrations in the subungual fluids collected after application of CPX/sol or Loceryl were sufficient to inhibit fungal growth, with the exception of Candida parapsilosis. Smaller amounts of fluid containing CPX were required for complete inhibition of fungal growth. Efficacy index values were significantly higher for CPX/sol. Conclusions Application of the CPX/sol nail lacquer allows rapid nail penetration of CPX, providing CPX levels sufficient to inhibit fungal growth for a prolonged period of time (30 h) after application of lacquer dose. CPX/sol nail lacquer appeared superior to the market reference Loceryl in terms of both vehicle (hydroxypropyl chitosan) and active ingredient (CPX) as witnessed by its higher efficacy on all nail pathogens.

An investigation into keratinolytic enzymes to enhance ungual drug delivery

The topical therapy of nail diseases is limited by the low permeability of drugs through the nail plate. To increase drug penetration, the integrity of the nail plate must be compromised to a certain extent. We hypothesised that keratinolytic enzymes might decrease the barrier properties of the nail plate by hydrolysing the nail keratins, and thereby enhance ungual drug permeation. To determine enzyme action on nail plates, nail clippings were incubated at 35 °C, in the presence of keratinase at optimal pH for 48 h, after which the nail plates were examined using scanning electron microscopy. It was found that the enzyme acted on the intercellular matrix which holds nail cells together, such that corneocytes on the dorsal surface separated from one another and ‘lifted off’ the nail plate. In addition, the surface of the corneocytes was corroded. Permeation studies using modified Franz diffusion cells and bovine hoof membranes as a model for the nail plate showed that the enzyme enhanced drug permeation through the hoof membrane. The permeability and partition coefficients, and the drug flux were found to be significantly increased in the presence of the enzyme. We can conclude that the enzyme, via its hydrolytic action on nail plate proteins, could increase ungual drug delivery.

Permeation of ciclopirox across porcine hoof membrane: effect of pressure sensitive adhesives and vehicles.

The purpose of this study was to investigate the effects of various pressure sensitive adhesives (PSA) on the penetration of ciclopirox across porcine hoof membrane. In addition, the influence of various vehicles on the permeation of ciclopirox from a PSA matrix across porcine hoof membrane was evaluated using a plastic penetration chamber containing poloxamer gel at 32 degrees C. Ciclopirox in poloxamer gel was extracted, derivatized and analyzed by HPLC. Ciclopirox showed highest permeability from an acrylic adhesive with a hydroxyl functional group, and this was followed by styrene-isoprene-styrene, styrene-butadiene-styrene, silicone and polyisobutylene adhesives in order. In addition, N-methyl-2-pyrrolidone (NMP), polypropylene glycol 400 (PPG 400), dimethylsulfoxide (DMSO), Labrasol, mercaptoethanol and Transcutol showed excellent permeation enhancing effects in a styrene-isoprene-styrene adhesive. A porcine hoof membrane treated with NMP changed from the non-helical phase to the helical phase, and another treated with DMSO showed a S=O peak shift to lower wavelength.