Yucatan Micropig Skin Research Articles

Influence of Skin Thickness on the in Vitro Permeabilities of Drugs through Sprague-Dawley Rat or Yucatan Micropig Skin

The purpose of this study was to clarify the influence of skin thickness on the in vitro permeabilities of 3 model drugs with different physicochemical properties (nicorandil (NR), isosorbide dinitrate (ISDN) and flurbiprofen (FP)) through Sprague-Dawley rat (rat) or Yucatan micropig (YMP) skin. Intact, dermis-split, stratum corneum-stripped or stratum corneum-stripped and dermis-split rat or YMP skin (rat skin thickness: approximately 0.4, 0.9 or 1.2 mm; YMP skin thickness: approximately 0.4, 0.9, 1.8 or 2.8 mm) were set in Franz-type diffusion cells to determine the permeation rate, lag time and resistance ratio of the viable epidermis and dermis against whole skin (R(ved)/R(tot)) of the drugs. The YMP skin permeabilities of the drugs decreased with an increase in the skin thickness, and significant differences were observed in the permeation rates and lag times between intact and dermis-split (0.4 mm) YMP skins. The decreases in the permeabilities of the drugs through the YMP skin were larger than those through the rat skin. The influence of resistances of ISDN and FP through the dermis-split rat or YMP skin was greater at 0.9 mm skin thickness than 0.4 mm skin thickness. The R(ved)/R(tot) values for the YMP skins were relatively large for lipophilic drugs (ISDN and FP), and these ratios increased with an increase in the dermis thickness. These results suggest that in vitro skin permeation studies must be done using dermis-split (0.4 mm) skin with the thinnest dermis for predicting in vivo human percutaneous absorption rate.

Distribution of Polyphenols and a Surfactant Component in Skin during Aerosol OT Microemulsion-Enhanced Intradermal Delivery

As for most other polyphenols, intradermal delivery of curcumin and resveratrol is limited; however, it was significantly improved by a microemulsion using Aerosol OT (Aerosol OT microemulsion) and Tween 80 (Tween 80 microemulsion) as surfactants. Aerosol OT microemulsion was more effective and the incorporation ratio of these polyphenols into skin by Aerosol OT microemulsion was five-fold or ten-fold that by Tween 80 microemulsion. To clarify the mechanism of the enhancement we examined the distribution of these polyphenols and the surfactant component, Aerosol OT, using excised guinea pig skin and Yucatan micropig (YMP) skin. During permeation, polyphenols distributed deep in the skin. In particular, a small molecule, resveratrol, was mainly present in the dermis in YMP skin. Aerosol OT also distributed deep in the skin. These findings suggest the possible involvement of the interaction of surfactant molecules with skin components in the enhanced delivery process of polyphenols. The distribution ratio between the dermis and epidermis of the polyphenols, including quercetin, in the presence of Aerosol OT microemulsion decreased with the increase of molecular weight in YMP skin, suggesting the possibility that distribution to the dermis is regulated by the molecular size.

Prediction of Blood Concentration of Nicotine or Lidocaine after Application of Their Dermal Tapes in Human from the in Vitro Permeation through Yucatan Micropig Skin

Prediction of Blood Concentration of Nicotine or Lidocaine after Application of Their Dermal Tapes in Human from the <i>in Vitro</i> Permeation through Yucatan Micropig Skin

Effect of Ethanol Pretreatment on Skin Permeation of Drugs

It has been demonstrated that ethanol (EtOH) can enhance skin permeation of drugs when simultaneously applied with drugs. However, only a few studies have reported on the pretreatment effect of EtOH on skin permeations. In this study, the pretreatment effects of EtOH on skin permeation of drugs were investigated by measuring changes in skin permeation and electrical skin resistance. Permeabilities of deuterium oxide (D2O), isosorbide mononitrate (ISMN), isosorbide dinitrate (ISDN), calcein sodium (CA-Na), and fluorescein isothiocyanate-dextran 4 kDa (FD-4, 3.3-4.4 kDa) were evaluated through Yucatan micropig skin pretreated with different concentrations of EtOH solution. From the results, almost constant skin permeabilities of D2O and ISDN were observed independent of EtOH concentration. Skin permeabilities of ISMN, CA, and FD-4 increased with low concentrations of EtOH, but decreased with high concentrations of EtOH. At 99.5% EtOH pretreatment, skin permeabilities of hydrophilic compounds (ISMN, CA, and FD-4) decreased to non-detectable levels. In addition, low molecular ion transports were almost constant at any EtOH concentration. Since molecular (ion) sizes of ISMN, CA, and FD-4 are larger than Na+, Cl-, and D2O, permeation pathway sizes for hydrophilic compounds in the skin barrier may be remarkably decreased by pretreatment with high concentrations of EtOH. However, the permeability coefficient of ISDN was not influenced by any EtOH concentration, since ISDN is a lipophilic, low-molecular compound that permeated through the lipophilic stratum corneum pathway. The present results show useful information for repeatedly and topically applied formulations containing EtOH, and also contribute to the effective use of alcohol formulations.

Variation Assessment for in Vitro Permeabilities through Yucatan Micropig Skin

The purpose of this study was to evaluate the variations in the in vitro Yucatan micropig (YMP) skin permeabilities of drugs and to clarify whether YMP skin can be used to predict human skin permeability. In vitro permeation studies of the three model drugs, nicorandil, isosorbide dinitrate and flurbiprofen, through YMP skin were performed using Franz-type diffusion cells. The permeation rates of the three model drugs were determined, and their variations were evaluated. The inter-individual variations in YMP skin permeability for the three model drugs were smaller than that in human skin permeability, and the permeation rates of the three model drugs through the YMP skin were approximately half that through human skin. In addition, the intra-individual variations in YMP skin permeability for nicorandil and flurbiprofen were much smaller than the inter-individual variations in YMP skin. The inter- and intra-regional variations in YMP skin permeability were very small. The markedly smaller variation in the permeability through YMP skin as compared with that through human skin indicated that in vitro permeation studies using YMP skin would be particularly useful for evaluating differences in the skin permeability of the three model drugs as well as for predicting human skin permeability.

Efficient Topical Delivery of Chlorogenic Acid by an Oil-in-Water Microemulsion to Protect Skin against UV-Induced Damage

We examined the intradermal delivery of a hydrophilic polyphenol chlorogenic acid by in vitro study using excised guinea pig dorsal skin and Yucatan micropig skin. Skin accumulation as well as the solubility of chlorogenic acid in aqueous vehicles was much greater than for other polyphenols such as quercetin and genistein. However, since enhancement of skin delivery seemed to be necessary to exhibit its protective effects against oxidative damage of skin, we examined the effects of microemulsions as vehicles. Using microemulsions consisting of 150 mM NaCl solution, isopropyl myristate, polyoxyethylene sorbitan monooleate (Tween 80) and ethanol, skin accumulation as well as solubility of chlorogenic acid further increased. Enhancement effect of an oil-in-water (o/w-type) microemulsion was greater than that of a water-in-oil (w/o-type) microemulsion possibly due to the greater increase in solubility. This finding was quite different from previous findings on relatively hydrophobic polyphenols such as quercetin and genistein. Pretreatment of guinea pig dorsal skin with chlorogenic acid containing microemulsion gel prevented erythema formation induced by UV irradiation. These findings indicate the potential use of hydrophilic chlorogenic acid with o/w-type microemulsion as a vehicle to protect skin against UV-induced oxidative damage.

Enhanced skin delivery of polyphenols by microemulsion and prevention against UV irradiation-induced erythema formation

Polyphenols have been applied for topical purposes such as photoprotection against UV-induced skin damage. However, intradermal delivery of most polyphenols is inefficient. To improve the efficiency, we tried to clarify the usefulness of microemulsion using excised guinea pig dorsal skin and Yucatan micropig skin. We used two types of microemulsions consisting of isopropyl myristate (IPM), 150 mM NaCl solution, Tween 80 and ethanol. Weight ratio of IPM, 150 mM NaCl solution, Tween 80 and ethanol was 8:25:20:47 in microemulsion A, and 33:7:30:30 in microemulsion D. By using these microemulsions solubility and skin delivery of polyphenols such as genistein and chlorogenic acid were markedly improved. For example, solubility of genistein in 150 mM NaCl was only 0.059 mM, it increased to 80 mM in o/w-type microemulsion A, and further increased to 140 mM in w/o-type microemulsion D. Skin delivery of genistein also increased 36 times using microemulsion A and further increased about 60 times using microemulsion D at saturated concentration. On the other hand, enhancement effect of microemulsion A was larger than that of microemulsion D for skin accumulation of hydrophilic chlorogenic acid. Genistein and chlorogenic acid retained in the skin significantly inhibited lipid peroxidation dose-dependently in vitro. When genistein was applied with microemulsion D at saturated concentration (140 mM), lipid peroxidation tested by ammonium iron (II) sulfate and sodium citrate decreased to less than 10%. We furthermore revealed that pretreatment of guinea pig dorsal skin with microemulsion gel which contains either genistein or chlorogenic acid prevented UV-irradiation-induced erythema formation. These findings revealed the potential use of microemulsion for the delivery of these polyphenols to protect skin against UV-irradiation-induced damage.

Enhanced Skin Delivery of Genistein and Other Two Isoflavones by Microemulsion and Prevention against UV Irradiation-Induced Erythema Formation

To improve the efficiency of the intradermal delivery of genistein and other two isoflavones (daidzein and biochanin A), we tried to clarify the usefulness of microemulsion by in vitro study on excised guinea pig dorsal skin and Yucatan micropig skin. Using microemulsion consisting of isopropyl myristate (IPM), 150 mM NaCl solution, Tween 80 and ethanol as a vehicle, the solubility of all the isoflavones markedly increased and significant amounts of isoflavones were delivered to the skin. The effect of water-in-oil (w/o)-type microemulsion D was larger than that of oil-in-water (o/w)-type microemulsion A. Among three isoflavones tested, the increase of genistein was most marked on both solubility and skin accumulation. Genistein retained in the skin significantly inhibited lipid peroxidation in vitro dose-dependently. Furthermore, pretreatment of guinea pig dorsal skin with genistein containing gel-like microemulsion D prevented UV irradiation-induced erythema formation. These findings indicate the potential use of w/o-type microemulsion for the delivery of genistein to protect skin against UV-induced oxidative damage.

Enhanced skin delivery of quercetin by microemulsion

Abstract Objectives For topical application of quercetin it is necessary to improve the low efficiency of its intradermal delivery as well as its low solubility in aqueous and organic vesicles. The aim of this study was to determine the usefulness of a microemulsion for that purpose. Methods A microemulsion consisting of isopropyl myristate, 150 mm NaCl solution, Tween 80 and ethanol was prepared. The skin delivery of quercetin by microemulsion using excised guinea-pig and Yucatan micropig skin in Franz diffusion cells was examined. Lipid peroxidation in skin was also tested using iron(II) and citrate. Key findings Using a w/o microemulsion as a vehicle, intradermal delivery of quercetin was significantly increased, as was its solubility. Quercetin penetrated deep into the skin, but no transfer was observed into the receptor compartment. It was confirmed that quercetin retained in the skin dose-dependently inhibited lipid peroxidation. Conclusions The findings indicate the potential use of microemulsions for the skin delivery of quercetin, where it exerts antioxidative effects.

Enhanced skin delivery of quercetin by microemulsion

For topical application of quercetin it is necessary to improve the low efficiency of its intradermal delivery as well as its low solubility in aqueous and organic vesicles. The aim of this study was to determine the usefulness of a microemulsion for that purpose. A microemulsion consisting of isopropyl myristate, 150 mM NaCl solution, Tween 80 and ethanol was prepared. The skin delivery of quercetin by microemulsion using excised guinea-pig and Yucatan micropig skin in Franz diffusion cells was examined. Lipid peroxidation in skin was also tested using iron(II) and citrate. Using a w/o microemulsion as a vehicle, intradermal delivery of quercetin was significantly increased, as was its solubility. Quercetin penetrated deep into the skin, but no transfer was observed into the receptor compartment. It was confirmed that quercetin retained in the skin dose-dependently inhibited lipid peroxidation. The findings indicate the potential use of microemulsions for the skin delivery of quercetin, where it exerts antioxidative effects.

A solid-in-oil nanodispersion for transcutaneous protein delivery

Transcutaneous delivery attracts much attention but remains a challenging strategy for hydrophilic macromolecular drug administration. In the present study, we demonstrated that a solid-in-oil (S/O) nanodispersion, an oil-based nanodispersion of hydrophilic drugs, effectively enhanced the permeation of proteins into the skin. All of the different model proteins, FITC-labeled insulin (MW ca. 6 kDa), enhanced green fluorescent protein (EGFP, MW ca. 27 kDa) and horseradish peroxidase (HRP, MW ca. 40 kDa), permeated through the stratum corneum of Yucatan micropig skin in vitro by forming a S/O nanodispersion. The penetrated EGFP and HRP exhibited green fluorescence and catalytic activity, respectively, suggesting that these proteins can permeate into the skin in a functional form. The results indicated the potential utility of the S/O nanodispersion as a novel vehicle for transcutaneous protein delivery.

Local Anesthetic Cream Prepared from Lidocaine-Tetracaine Eutectic Mixture

Local anesthetic creams for the clinical treatment of conditions such as postherpetic neuralgia were prepared as an in-house formulation from the eutectic mixture of lidocaine-tetracaine (LT cream) using two eutectic mixtures of local anesthetic (EMLA) type bases. The LT formulation was compared with a lidocaine-prilocaine (LP cream) eutectic mixture formulated using the same base as EMLA. The chemical stability of lidocaine was examined in advance and was found to be stable for more than 3 months either in LT cream or in LP cream. The release rate of lidocaine from the formulated creams was examined using a cellulose ester membrane. The release rate of lidocaine from LT cream was similar to that from LP cream. The release rate of tetracaine was slightly slower than that of lidocaine in LT cream reflecting the larger molecular size of tetracaine. The penetration rate was examined in vitro using a Yucatan micropig skin. The penetration rate of lidocaine was similar between LT and LP creams. Infiltration anesthesia action examined in guinea pigs indicated that the difference between the two creams was statistically insignificant. The present study suggests the equivalence of the LT and LP creams as a local anesthetic and the potential of LT cream for clinical use either in the easy formulation or in the low-cost formulation.

A Novel Solid-in-oil Nanosuspension for Transdermal Delivery of Diclofenac Sodium

We formulated a solid-in-oil nanosuspension (SONS) as a novel transdermal delivery carrier for diclofenac sodium (DFNa). The basic transdermal characteristics of the SONS were evaluated using a Yucatan micropig (YMP) skin model. DFNa-sucrose erucate (i.e. surfactant) complexes were prepared via the formation of a water-in-oil emulsion. The complexes were suspended in isopropyl myristate (IPM) to form a SONS. The basic transdermal characteristics of the SONS were examined using full-thickness YMP dorsal skin in a Franz-type diffusion cell. DFNa powder suspended in IPM without complex formation was used as a control. The effect of the weight ratio of surfactant to DFNa on DFNa penetration of the skin was evaluated. DFNa was successfully dispersed into IPM as a nanosized suspension via complex formation with sucrose erucate. The resultant SONS increased the permeability flux of DFNa across the YMP skin by up to 3.8-fold compared with the control. The size of the SONS depended on the weight ratio of the surfactant to DFNa. The optimal weight ratio for the highest DFNa permeation was 8.8, at which point the mean diameter of the SONS was 14.4 nm. The SONS formulation can enhance the percutaneous absorption of DFNa.

Influence of Admixture with Urea Ointment and White Petrolatum on the Penetration of Corticosteroids into Yucatan Micropig Skin

Admixtures of two primary corticosteroid ointments, Almeta® and Myser® ointments, were made with urea ointment and white petrolatum. The release rate of corticosteroids from the admixed ointments with urea ointment and white petrolatum to a buffer solution were significantly lower than those expected from a dilution ratio 0.5 for both Almeta® and Myser® ointments. Also, for the admixtures with urea ointment, the amount of corticosteroids penetrating into the model skin was larger than that expected from the dilution ratio, especially for Myser® ointment, while the amounts of corticosteroids penetrating into the skin from the admixtures with white petrolatum were smaller than those expected from the dilution ratio, for both primary ointments.An electron probe micro analyzer was used to observe the actual distribution profile of corticosteroid molecules in a cross section of the skin. In the case of the Myser®-urea ointment admixture, the distribution profile of difulprednate was quite homogeneous, probably due to the softening effect of urea on the skin and the affinity of difulprednate with skin tissue, though the urea ointment did not have much effect on the distribution profile of alclometasone released from the Almeta® ointment. On the other hand, difulprednate was concentrated at the surface for the admixture with white petrolatum.

Partial ablation of porcine stratum corneum by argon-fluoride excimer laser to enhance transdermal drug permeability

To enhance skin permeability to medicine, the argon-fluoride excimer laser (ArF laser) was used to partially ablate the stratum corneum. Skin permeability to dextran (20 kDa) was studied in the Yucatan micro-pig skin in vitro. The cumulative amount of dextran permeating across the full-thickness skin was not detected for 30 h in the unirradiated skin; we obtained up to 90.5 microg/cm2 in laser-irradiated skin. In the case where the total laser energy was kept constant at 7.1 J/cm2, permeability was mainly influenced by laser fluence rather than laser pulses. Many granular structures of about 2 microm were found on the stratum corneum surface of ablated skin. Size and density of these structures were changed according to irradiation conditions. Skin permeability may be estimated by these structural changes. Our partial stratum corneum ablation by ArF laser could be suitable for transdermal drug delivery.

Enhancement Effect of p‐Menthane‐3,8‐diol on In Vitro Permeation of Antipyrine and Indomethacin Through Yucatan Micropig Skin

The enhancing effect of p‐menthane‐3,8‐diol (MDO) on skin permeation of antipyrine (ANP) and indomethacin (IM) through Yucatan micropig skin in vitro was compared with l‐menthol. p‐menthane‐3,8‐diol is a metabolite of l‐menthol and has little odor. It is easy to combine the vehicle because of lower lipophilicity than l‐menthol. All formulations contained 40% (v/v) ethanol. The permeation of ANP increased with MDO about three times that without enhancer by increasing ANP concentration in the skin. However, the MDO effect was about a quarter that of l‐menthol. The permeation of IM with MDO was about 15 times that with no enhancer and it was almost the same as that with l‐menthol. The lag time of permeation was not significantly changed by MDO, which was not so in the case of l‐menthol. Skin concentration of IM increased about 11 times and six times with MDO and l‐menthol, respectively. MDO and l‐menthol partitioned to the skin relatively high concentrations, 5.9 and 2.5 mg/cm3, respectively. The solubility of IM in the skin was improved by MDO, and consequently, the permeation of IM was enhanced.

Comparison of skin permeation enhancement by 3- l-menthoxypropane-1,2-diol and l-menthol: the permeation of indomethacin and antipyrine through Yucatan micropig skin and changes in infrared spectra and X-ray diffraction patterns of stratum corneum

3- l-Menthoxypropane-1,2-diol (MPD) is a derivative of l-menthol, which has an enhancement effect on drug permeation through skin. In this study, the effect of MPD on drug permeation through skin was compared with that of l-menthol. MPD or l-menthol at final concentrations of 3% in 40% ethanol was added to the drugs indomethacin or antipyrine and each mix then applied to Yucatan micropig skin in vitro. Drug concentrations in the skin were higher in the presence of either MPD or l-menthol, however, only l-menthol shortened the lag time of permeation. MPD enhanced the skin permeation of the drugs only by increasing the skin concentration of the drugs. In contrast, l-menthol enhanced the skin permeation of the drugs by increasing both the skin concentration and the diffusion rate in skin. The infrared (IR) spectra and X-ray diffraction patterns of stratum corneum after treatment with MPD did not differ from those of intact stratum corneum. A change in the IR spectra of stratum corneum after treatment with l-menthol was observed at the CH band, and the peaks representative of the lipid structure in the X-ray diffraction patterns decreased in intensity. These results suggest that l-menthol, but not MPD, disrupts the intercellular lipid structure of stratum corneum. Thus, MPD is expected to be a moderate skin permeation enhancer.

Preparation of griseofulvin for topical application using N-methyl-2-pyrrolidone.

We attempted to prepare a new griseofulvin formulation for topical application using N-methyl-2-pyrrolidone (NMP). Griseofulvin dissolves poorly in both water and oil, but dissolves in NMP to a concentration of about 100 mg/ml. A soybean oil-water emulsion with soybean lecithin and NMP as emulsifier and co-solvent, respectively, was prepared using a Microfluidizer, a high-pressure homogenizer. The size of the droplets in emulsion was about 200 nm, and the emulsion was stable for over 3 months. The skin permeation of griseofulvin through Yucatan micropig skin was studied in vitro using vertical type cells under donor phase open conditions. The permeation of griseofulvin from the NMP-water mixture (0-40%) into the skin tended to increase with increasing NMP concentration, although this finding was not statistically significant. Permeation from emulsion (oil phase, 20%; NMP 10-40%) was significantly higher than that from the water-NMP mixture. Permeation from the oil-NMP mixture was highest among the formulations investigated, and permeation from emulsion under donor phase closed conditions was significantly lower than that under open conditions. We believe that the evaporation of water from the emulsion after application to the skin was an important factor in skin permeation enhancement. When the emulsion containing 3% l-menthol was applied, a sufficient skin concentration (47 microg/cm3 in dermis) was obtained.

In vitro release of tranilast from oily gels and penetration of the drug into Yucatan micropig skin.

For the transdermal delivery of tranilast (TL), a drug used for the treatment of skin diseases such as keloids and hypertrophic scars, its oily gels were prepared; its in vitro release and penetration into Yucatan micropig skin were evaluated. In the gels that consisted of hydrogenated soybean phospholipids (HSL) and octyl isononanoate (IOIN), a fatty-acid ester, the release of TL from the gels was proportional to the drug content, and the extent of TL released up to 6 h from them was approximately 70% of the amount of applied TL. On the other hand, with the gels consisting of HSL and isocetyl isostearate (ICIS), the release of TL from the gels was about half of that from IOIN gels, even at the same drug concentration. When oily gels were used, the TL skin concentration was rapidly increased compared with the level obtained with suspensions. With 0.1% IOIN gel, a high concentration of TL (ca. 160 microg/g) in the dermis was obtained and continued until at least 48 h. These results suggest that oily gels may be useful for the topical application of TL.

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3-l-menthoxypropan-1, 2-diol (MPD), which is a derivative of l-menthol, and l-menthol enhance the penetration of indomethacin (IM) through hairless rat (HR) skin. It is well known that degree of enhancement differs with skin type, so we studied the enhancement of MPD and l-menthol on the penetration of IM through Yucatan micropig (YMP) skin. Ethanol (40%) containing 0.5% IM and 3% enhancer was applied to the donor phase of a modified Franz-type cell. The permeation rate of IM through YMP skin without enhancer was 1.7 μg/cm2/hr, which was about 1/3 that through HR skin. In the presence of MPD or l-menthol the rate of IM permeation through YMP skin were 2.5 and 5.6 μg/cm2/hr, respectively, much lower than through HR skin, 90 and 144 μg/cm2/hr. IM concentration in the skin without enhancer was 240 μg/g, with MPD, 380 μg/g and with l-menthol, 490 μg/g. MPD and l-menthol concentrations in the skin were 2.9 and 2.2 mg/g, respectively. It was considered that each enhancer was partitioned in the skin so that the partition coefficient of IM in the skin increased, though the increasing factor was smaller than that in the case of HR skin. The rate of IM permeation through epidermal membrane and stripped YMP skin were also determined.