Hiroaki Todo

Prediction of skin permeation by chemical compounds using the artificial membrane, Strat-M™.

PURPOSE The usefulness of the synthetic membrane, Strat-M™ as an alternative to human and animal skins was evaluated by estimating the skin permeabilities of chemical compounds. METHOD Thirteen chemical compounds with molecular weights (M.W.) of 152-289 and lipophilicities (log Ko/w) of -0.9 to 3.5 were selected. Strat-M™, excised human skin, or hairless rat skin was set in a Franz-type diffusion cell and a saturated solution of each chemical compound was applied to determine membrane permeation profiles. The obtained permeability coefficients (log P) were compared among these membranes. RESULTS AND DISCUSSION Elevations were observed in log P for Strat-M™ with an increase in the log Ko/w of the applied compounds, and similar results were observed with the human and hairless rat skins. A correlation was obtained in log P values between Strat-M™ and human or hairless rat skin. Furthermore, the diffusion and partition parameters of chemicals in Strat-M™ were similar to those in the excised human and rat skins. These results suggest that Strat-M™ could be used as an alternative to animal or human skin in permeation studies.

Transdermal drug delivery by in-skin electroporation using a microneedle array.

The aim of the present work was to develop a minimally invasive system for the delivery of macromolecular drugs to the deep skin tissues, so-called in-skin electroporation (IN-SKIN EP), using a microneedle (MN) electrode array. Fluorescein isothiocyanate (FITC)-dextran (FD-4: average molecular weight, 4.3 kDa) was used as the model macromolecular drug. MNs were arranged to puncture the skin barrier, the stratum corneum, and electrodes were used for EP so that a high electric field could be applied to skin tissues to promote viable skin delivery. In vitro skin permeation experiments showed that IN-SKIN EP had a much higher skin penetration-enhancing effect for FD-4 than MN alone or ON-SKIN EP (conventional EP treatment), and that higher permeation was achieved by applying a higher voltage and longer pulse width of EP. In addition, no marked skin irritation was observed by IN-SKIN EP, which was determined by the LDH leaching test. These results suggest that IN-SKIN EP can be more effectively utilized as a potential skin delivery system of macromolecular drugs than MN alone and conventional ON-SKIN EP.

Mathematical Model to Predict Skin Concentration of Drugs: Toward Utilization of Silicone Membrane to Predict Skin Concentration of Drugs as an Animal Testing Alternative

PurposeTo calculate the skin concentration of active ingredients in cosmetics and topical pharmaceuticals using silicone membrane permeation.MethodsA series of parabens were used as model ingredients. Skin concentration of parabens was calculated using silicone membrane permeability. Their partition coefficient from formulations to the silicone membrane was determined by the membrane permeation profiles, and used to calculate their silicone membrane concentration, under an assumption that the membrane is one homogenous diffusion layer. The same procedure was applied for hairless rat skin.ResultsThe calculated concentration of parabens in silicone membrane was very close to their observed values. However, the skin concentration calculated by skin permeability was not similar to the observed concentration. Re-calculation was performed under the assumption that the skin consists of two diffusion layers. This modification using permeation data through full-thickness and stripped skin enabled precise prediction of the skin concentration of parabens. In addition, the partition coefficient to the silicone membrane was useful to estimate their skin concentration.ConclusionsIngredient concentration in skin can be precisely predicted using diffusion equations and partition coefficients through permeation experiments using a silicone membrane. The calculated in-skin concentration is useful for formulation studies of cosmetics and topical pharmaceuticals.

Effect of liquid crystals with cyclodextrin on the bioavailability of a poorly water-soluble compound, diosgenin, after its oral administration to rats

Diosgenin, found in wild yam (Dioscorea villosa), has been shown to ameliorate diabetes and hyperlipidemia, increase cell proliferation in a human 3D skin model, and inhibits melanin production in B16 melanoma cells. It is also an active element in cosmeceutical and dietary supplements. Although the bioavailability of diosgenin is low due to its poor solubility and intestinal permeability, it was subsequently improved using a β-cyclodextrin (β-CD) inclusion complex. Recently liquid crystals (LCs) were shown to enhance the bioavailability of poorly water-soluble drugs. The purpose in the present study was to prepare diosgenin LCs and investigate the interaction between LC and β-CD in order to improve its bioavailability of diosgenin. Crystallinity and particle diameters of LCs in water were determined by small angle X-ray scattering (SAXS) and Zetasizer. Pharmacokinetic parameters were calculated using the plasma content of diosgenin after its oral administration to Wistar rats. Regarding the formation of glyceryl monooleate (GMO) and phytantriol (PHY) LC, SAXS patterns showed the hexagonal and cubic phases, respectively. Bioavailability was significantly enhanced after oral administration of LCs prepared by GMO than after diosgenin alone. The bioavailability was further improved with the combination of LC and β-CD than LC and water.

Effect of topically applied sphingomyelin-based liposomes on the ceramide level in a three-dimensional cultured human skin model

Sphingomyelin-based liposomes were prepared and applied to the stratum corneum side or basal layer side of a three-dimensional (3D) cultured human skin model, and the increase in the type II ceramide (ceramide II) content of the cultured skin model was evaluated. The sphingomyelin-based liposomes were prepared by a high-pressure emulsification method, and the obtained liposomes were characterized; the particle diameter and zeta potential of the liposomes were 155.3 nm and −11.4 mV, respectively. Their spherical shape and lamella structure were observed by transmission electron microscopy. The sphingomyelin-based liposomes or saline were applied to the cultured skin model, and ceramide II was extracted from the skin model. The extracted ceramide II was separated by high-performance thin-layer chromatography and quantified by a densitometer. The amount of ceramide II in the cultured skin model was significantly increased by the application of the sphingomyelin-based liposomes, compared with the nonapplication group. Thus, sphingomyelin-based liposomes are useful for enriching the ceramide level in 3D cultured skin models.

Molecular mechanisms of action of different concentrations of ethanol in water on ordered structures of intercellular lipids and soft keratin in the stratum corneum.

Ethanol (EtOH) is one of the bases in topically applied medicines that promote the skin permeation of drugs. Although the effects of EtOH have been attributed to structural modifications in the stratum corneum, the underlying mechanisms, especially the influence of different concentrations of EtOH, have not been examined extensively. Structural modifications in the stratum corneum of hairless mouse due to the application of EtOH/water mixture were herein investigated at the molecular level using synchrotron X-ray diffraction. The results revealed that all EtOH concentrations examined greatly modified the short lamellar structures containing the aqueous layer in intercellular lipids and the structure of keratin fibrils in corneocytes, which can take up hydrophilic compounds. However, the long lamellar and the hydrocarbon-chain packing structures were unaffected by EtOH. Changes to the short lamellar structures were not proportional to the concentration of EtOH. However, the keratin fibril structures changed gradually with increasing EtOH concentration. The X-ray diffraction experiments enabled the effects of different EtOH concentrations on the morphology of the stratum corneum to be assessed by using a number of experimental samples to avoid variations due to individual differences. The results indicated that alterations to the short lamellar structures appeared to be related to the skin permeability of drugs with the application of EtOH/water mixture, and monotonous structural changes in the keratin fibrils with an increase in EtOH concentration may contribute to this permeation as supplement. These results will be useful for the development of new drug formulations containing EtOH.

Effects of soybean peptide and collagen peptide on collagen synthesis in normal human dermal fibroblasts.

The collagen present in the dermis of the skin is a fibrous protein that fills the gaps between cells and helps maintain tissue flexibility. Effectively increasing the collagen present in the skin is an important goal for cosmetic research. Recent research has shown that soybean peptide (SP) has anti-fatigue activity, antioxidant activity, and the ability to increase type I collagen, while collagen peptide (CP) has the ability to enhance corneal moisture content and viscoelasticity, as well as to increase levels of hyaluronic acid synthesizing enzymes in human skin. Little documented research, however, has been conducted on collagen formation in relation to these peptides. Therefore, this research applied SP and CP with molecular weights primarily around 500 and preparations containing both SP and CP to normal human dermal fibroblasts together with magnesium ascorbyl phosphate (VC-PMg), and used real-time PCR to determine the gene expression of type I collagen (COL1A1), which contributes to collagen synthesis, and Smad7, which contribute to collagen breakdown. In addition, enzyme linked immuno sorbent assay (ELISA) was used to measure collagen content in the media. COL1A1 gene expression at 24 h after sample addition showed higher tendency in all samples and increased with time at 4, 8 and 24 h after addition. Smad7 gene expression was not substantially different at 4 h after addition. matrix metalloproteinase-1 gene expression was higher following SP addition, but was lower after the addition of CP and SP+CP. Medium collagen content was higher in all samples and increased with time at 8 h after addition. Collagen levels were higher when SP and CP were added together.

Analysis of hair follicle penetration of lidocaine and fluorescein isothiocyanate-dextran 4 kDa using hair follicle-plugging method

Abstract Objective: Skin appendages including hair follicles (hfs) and the stratum corneum (sc) are beginning to be recognized as important permeation pathways for the skin permeation of drugs, but their detailed role is not yet clear. To investigate the contribution of hfs to drug permeation, we conducted skin permeation tests by controlling the hf contribution with a hf-plugging method. Method: Lidocaine (LC) and fluorescein isothiocyanate-dextran 4 kDa (FD-4) were selected as model drugs and pig ear skin was used as model skin. Results: Skin permeabilities of ionized LC and FD-4 decreased with hf-plugging, whereas no change was observed for the skin permeation of unionized LC. A fairly good correlation was found for ionized LC and FD-4 between skin permeability and the number of hfs plugged. Permeation parameters of model drugs for both skin pathways were calculated utilizing Ficks second law of diffusion. Consequently, the sc pathway could highly contribute to the permeation of unionized LC, since unionized LC shows markedly high partition to the sc. In contrast, the hf pathway could contribute to the permeation of ionized LC and FD-4, since these had high distributions to the hf pathway in spite of its very small surface area relative to whole skin surface area. Conclusion: The hf pathway must be important for the skin permeation of ionized compounds and hydrophilic high molecular compounds. hf-plugging is also a useful method for assessing the skin permeability of compounds through the hf pathway.

Mathematical Model to Predict Skin Concentration after Topical Application of Drugs

Skin permeation experiments have been broadly done since 1970s to 1980s as an evaluation method for transdermal drug delivery systems. In topically applied drug and cosmetic formulations, skin concentration of chemical compounds is more important than their skin permeations, because primary target site of the chemical compounds is skin surface or skin tissues. Furthermore, the direct pharmacological reaction of a metabolically stable drug that binds with specific receptors of known expression levels in an organ can be determined by Hill’s equation. Nevertheless, little investigation was carried out on the test method of skin concentration after topically application of chemical compounds. Recently we investigated an estimating method of skin concentration of the chemical compounds from their skin permeation profiles. In the study, we took care of “3Rs” issues for animal experiments. We have proposed an equation which was capable to estimate animal skin concentration from permeation profile through the artificial membrane (silicone membrane) and animal skin. This new approach may allow the skin concentration of a drug to be predicted using Fick’s second law of diffusion. The silicone membrane was found to be useful as an alternative membrane to animal skin for predicting skin concentration of chemical compounds, because an extremely excellent extrapolation to animal skin concentration was attained by calculation using the silicone membrane permeation data. In this chapter, we aimed to establish an accurate and convenient method for predicting the concentration profiles of drugs in the skin based on the skin permeation parameters of topically active drugs derived from steady-state skin permeation experiments.

Estimation of skin concentrations of topically applied lidocaine at each depth profile

Skin concentrations of topically administered compounds need to be considered in order to evaluate their efficacies and toxicities. This study investigated the relationship between the skin permeation and concentrations of compounds, and also predicted the skin concentrations of these compounds using their permeation parameters. Full-thickness skin or stripped skin from pig ears was set on a vertical-type diffusion cell, and lidocaine (LID) solution was applied to the stratum corneum (SC) in order to determine in vitro skin permeability. Permeation parameters were obtained based on Ficks second law of diffusion. LID concentrations at each depth of the SC were measured using tape-stripping. Concentration-depth profiles were obtained from viable epidermis and dermis (VED) by analyzing horizontal sections. The corresponding skin concentration at each depth was calculated based on Ficks law using permeation parameters and then compared with the observed value. The steady state LID concentrations decreased linearly as the site became deeper in SC or VED. The calculated concentration-depth profiles of the SC and VED were almost identical to the observed profiles. The compound concentration at each depth could be easily predicted in the skin using diffusion equations and skin permeation data. Thus, this method was considered to be useful for promoting the efficient preparation of topically applied drugs and cosmetics.