Excised Guinea Pig Dorsal Skin Research Articles

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 Delivery of Retinoic Acid to Skin by Cationic Liposomes

We studied the delivery of retinoic acid to skin by using cationic liposomes consisting of double-chained cationic surfactant, phosphatidylcholine (PC) and retinoic acid in excised guinea pig dorsal skin. Egg yolk PC liposomes contaning retinoic acid at a molar ratio of 4 : 1 increased the delivery of retinoic acid about two-fold, compared with its addition as an isopropyl myristate solution. Cationic liposomes containing 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) further enhanced the incorporation dependent on the DOTAP content. Liposomes consisting of DOTAP, egg yolk PC, and retinoic acid at a molar ratio of 2 : 2 : 1 induced a 3.7-fold increase in the skin incorporation compared with the egg yolk PC liposomes without DOTAP. Significant difference was not observed when either dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC) was used instead of egg yolk PC as well as when dimethyldipalmitylammonium was used instead of DOTAP. These results suggest the potential of the use of the cationic liposomes for the intradermal delivery of lipophilic drugs like retinoic acid.

Enhanced Skin Permeation of Salicylate by Ion-Pair Formation in Non-aqueous Vehicle and Further Enhancement by Ethanol and l-Menthol

Enhancement of skin permeability of salicylate from non-aqueous vehicle by ion-pair formation with either alkylamines or benzylamine as model cationic ions was examined in excised guinea pig dorsal skin. Solubility of salicylate in isopropyl myristate (IPM) was increased by the addition of either alkylamines or benzylamine as counter ions. The increase was more significant in the presence of amines with longer alkyl chains. Flux of salicylate increased in the presence of these amines due to the increase in the solubility. Maximum flux was observed in the presence of n-hexylamine, which induced an 11-fold increase due to 137-fold increase in solubility. Flux and permeability coefficients of salicylate in the presence of n-butylamine, n-hexylamine, iso-octylamine and benzylamine as counter ions in IPM were larger than those of the non-ionic form of salicylic acid. Flux of 3-methylsalicylate (3-CH3 substituent) and that of 5-hydroxysalicylate (5-OH substituent) were smaller than that of salicylate in the presence of n-hexylamine. After partition to the skin surface, the ion-pair is suggested to dissociate and permeate separately according to the study using lidocaine as the counter ion. Flux of salicylate increased in the presence of benzylamine as the counter ion by the addition of 15% ethanol and 15% ethanol plus 1% l-menthol due to further improvement in the solubility as well as an increase in the permeability coefficient.

Permeability of Ionized Salicylate Derivatives through Guinea Pig Dorsal Skin

pH-dependency of skin permeability to salicylic acid was examined in excised guinea pig dorsal skin. Permeation followed the pH-partition theory at acidic pH. However, above pH 5.0 the observed permeability coefficients were larger than the estimated values obtained from the ratio of the undissociated forms. These findings are quite different from those obtained using the same drug and a silicone rubber membrane, in which permeability coefficients were consistent with the pH-partition theory. The findings suggested that permeation of salicylate as anions occurred at a neutral skin pH. The permeability coefficient of the ionized form was estimated to be about 1.6% of the nonionized form. We also examined the skin permeability of salicylate and its five 5-substituents and two 3-substituents at pH 7.4. We investigated the relationship between their permeability coefficients and the physico-chemical properties of the substituents. Multi regression analysis on the permeability coefficients showed a parabolic relationship between the values of the hydrophobic parameter (pi) and the logarithms of the permeability coefficients. These findings suggested that the ionic permeation pathway of salicylate derivatives is controlled by hydrophobic as well as hydrophilic properties.

Enhanced skin permeation of cationic drug ketotifen through excised guinea pig dorsal skin by surfactants with different electric charges.

Using excised guinea pig dorsal skin, we examined the effects of three surfactants, anionic sodium dodecylsulfate (SDS), cationic n-dodecyltrimethylammonium bromide and non-ionic n-dodecyl-beta-D-maltoside, all of which commonly have an n-dodecyl group, on in vitro skin permeation of the cationic drug ketotifen. All these three surfactants increased the skin permeation of ketotifen. Among the surfactants tested, anionic SDS had the largest enhancement effects, and significantly increased the permeation at concentrations over 1 mM. The enhancement effect of the same anionic surfactant on the permeation of anionic salicylate was smaller and similar to that of cationic n-dodecyltrimethylammonium. The enhancement effects of SDS on ketotifen permeation were more marked than those of the cationic surfactant but differed from previous findings of their effects on other drugs permeation. Analysis of the retention of ketotifen in the skin suggested that SDS-induced increase in the transfer of hydrophilic ketotifen to the skin is the main reason for the marked increase in skin permeation.

Enhancement effects of double-chained cationic surfactants of n-dimethyldialkylammoniums on permeability of salicylate through guinea pig dorsal skin.

We examined the enhancement effects of the double-chained cationic surfactants of n-dimethyldialkylammoniums (CH(3))(2)N(+)(C(n)H(2n+1))(2) on the permeation of anionic salicylate through excised guinea pig dorsal skin at pH 7.4. Among them, n-dimethyldidecylammonium (2C10), which seemed to form micelles, had dose-dependent enhancement effects at concentrations of more than 0.1 mM, and about a ninety-fold increase in the permeability coefficient of salicylate was observed at 2 mM. The enhancement effect of 2C10 was larger than those of single-chained cationic surfactants of n-alkyltrimethylammoniums. n-Dimethyldilaurylammonium (2C12), which seemed to form bilayer vesicles, induced about a twenty five-fold increase in the permeability coefficient. The enhancement effects of n-dimethyldialkylammoniums decreased with the increase in their alkyl chain lengths. In contrast, only slight stimulation by these cationic surfactants was observed for silicon rubber membrane permeation of salicylate. Analysis of the retention of the salicylate in the skin suggested that the double-chained cationic surfactants-induced increase in the transfer of salicylate to the skin is the main reason for the marked stimulation of the skin permeation.

Analysis of electron spin resonance spectra of alkyl spin labels in excised guinea pig dorsal skin, its stratum corneum, delipidized skin and stratum corneum model lipid liposomes.

The electron spin resonance (ESR) spectra of alkyl spin labels were observed in the excised guinea pig dorsal skin, its stratum corneum, delipidized skin and stratum corneum model lipid liposomes. The spectrum of 5-doxylstearic acid (5-NS) in the stratum corneum and order parameter obtained from the spectrum, indicated that the spin label was present in highly ordered lipid lamella. On the other hand, the spectrum of methyl ester of 5-NS (5-NMS) and its apparent rotational correlation time calculated from the spectrum, showed only a weakly immobilized component in the stratum corneum as well as in the whole excised skin. The ester spin label seemed to be scarcely present in the rigid lipid lamella, but mainly in the relatively fluid environment. On the other hand, cationic alkyl spin labels showed quite different spectra depending on their alkyl chain lengths. Long-chain 4-(N,N-dimethyl-N,-pentadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl (CAT-15) seemed to be present in the protein region of the stratum corneum as we recently reported, whereas hydrophilic quaternary ammonium spin label 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-1-oxyl (CAT-1) seemed to be present in the bulk water of the skin, even in delipidized skin. These findings indicated that the different interaction and different localization of the alkyl spin labels depended on their electronic charge as well as their alkyl chain lengths.

Effects of n-alkyltrimethylammonium on skin permeation of benzoic acid through excised guinea pig dorsal skin.

Effects of cetyltrimethylammonium and two other n-alkyltrimethylammoniums on the permeation of benzoic acid through excised guinea pig dorsal skin were examined. Cetyltrimethylammonium markedly increased both the flux and permeability coefficient in the concentration range of 0.5 and 5 mm. However, 50mM cetyltrimethylammonium decreased them. The presence of 2 mm cetyltrimethylammonium, which induced a maximal enhancement effect, also increased the fluxes of 4-methyl, 4-ethyl and 4-n-propyl substituents of benzoic acid, but the enhancement effects were less. Analysis of the free energy of transfer of the methylene group of the substituents from the aqueous phase to skin suggested that cetyltrimethylammonium made the skin relatively more hydrophilic. Electron spin resonance spectra analysis by a long-chain quaternary alkylammonium analog, 4-(N,N-dimethyl-N-pentadecyl)ammonium-2,2,6,6-tetramethylpiperidine-1-ox yl iodide (CAT-15), showed that the spin label was present in a nearly solid state in both excised skin and its stratum corneum. This finding suggested the high affinity of the long-chain alkylammoniums to proteins in the stratum corneum and the involvement of the interaction between the cationic surfactants and the proteins in their improvement of the hydrophilic property of the skin, as well as their marked enhancement effects on the skin permeation of relatively hydrophilic drugs.

Effects of removal of stratum corneum, delipidization and addition of enhancers, ethanol and l-menthol, on skin permeation of benzoic acid and its 4-n-alkyl substituents in excised guinea pig dorsal skin.

Skin penetration of benzoic acid and its 4-alkyl substituents (methyl, ethyl, n-propyl and n-butyl) through excised guinea pig dorsal skin was examined, and effects of removal of stratum corneum, delipidization and addition of the penetration enhancers, ethanol and l-menthol plus ethanol, were observed. Permeability coefficients, which increased with the increase in their alkyl chain lengths, depended on the ratio of undissociated form of the derivatives. Removal of stratum corneum by tape stripping and delipidization by a chloroform-methanol mixture, whose effects on the permeation were similar, increased the permeability coefficients of the derivatives, especially those of relatively hydrophilic derivatives. Addition of 1% l-menthol plus 15% ethanol increased the permeability coefficient of benzoic acid, but decreased those of ethyl-, n-propyl- and n-butyl-substituents, and differences in the permeability coefficients among these acids almost disappeared. A similar though weaker tendency was observed for the effects of 15% ethanol itself. Analysis of transfer free energy of the methylene group from vehicle to skin revealed that tape stripping and delipidization induced the reduction of lipophilic barrier property, although it still remained after these treatments. The analysis also showed that the addition of the enhancers made the skin relatively more hydrophilic compared to the vehicle, which induced an increase in permeability coefficient of benzoic acid and decreases in those of its lipophilic substituents.

Permeability of benzoic acid derivatives in excised guinea pig dorsal skin and effects of l-menthol

Penetration through excised guinea pig dorsal skin was examined for nine mono-substituents of benzoic acid derivatives. Permeability coefficients of the derivatives correlated well with their n-octanol/water partition coefficients. Since the regression coefficient was similar to the value obtained in human skin, it is suggested that the lipid lamellae of guinea pig skin resembled that of human skin in lipophilicity. Addition of penetration enhancer, 1% l-menthol in 15% ethanol markedly increased the flux and permeability coefficients of relatively hydrophilic derivatives and decreased the dependency of the permeability coefficients on the partition coefficients. Electron spin resonance analysis using 5-doxylstearic acid revealed the presence of a strongly immobilized component of the spin label in the skin and its disappearance in the presence of 1% l-menthol in 15% ethanol. These results suggest that the rigid lamellar structure of the stratum corneum was disrupted by l-menthol with ethanol, and caused the enhancement of penetration of relatively hydrophilic benzoic acid derivatives.

Effect of phosphatidylglycerol on the in vitro percutaneous drug penetration through the dorsal skin of guinea pigs, and analysis of the molecular mechanism, using (ATR-FTIR) spectroscopy

Standard in vito skin percutaneous penetration methods using excised guinea pig dorsal skin were employed, to characterize the penetration of a water-insoluble drug: tenoxicam (TEX), and a water-soluble drug: diclofenac sodium salt (DFS), enhanced by phosphatidylglycerol (PG); and an attenuated total reflectance fourier transform infrared (ATR-FTIR) spectroscopy was used to analyze the molecular mechanism of the drug penetration route. The C-H bond stretching absorbance frequency shift in the stratum corneum (SC) induced a higher and a broader absorbance, and the shift was dependent on the PG concentrations. The percutaneous penetration of TEX was dependent on the PG concentration (up to 6%). The enhancing mechanism of PG to TEX may not only increase the diffusion coefficient (D) and the partition coefficient (K) in the percutaneous TEX penetration but also increase fluidity of the route (intercellular lipid domain) for TEX, while that of PG to DFS, excepting 1%, PG system, may be increasing the D value in the percutaneous DFS penetration only. The percutaneous penetration of DFS was not dependent on the PG concentrations. Furthermore, the percutaneous penetration of TEX was proportional to the C-H bond stretching absorbance frequency shifts. In contrast, the percutaneous penetration of DFS was not proportional to the C-H bond stretching absorbance frequency shifts. Furthermore, the accumulation of TEX in skin was proportional to the C-H bond stretching absorbance frequency shifts. The striking parallel between the enhancement of the percutaneous penetration of TEX and the measured SC lipid fluidity shifts caused by PG, suggests that the transdermal water-insoluble drug penetration may be ultimately related to the SC lipid structure. Overall, these results suggest that PG mainly affects the intercellular lipid pathway (lipid-rich domains).

Skin permeation of parabens in excised guinea pig dorsal skin, its modification by penetration enhancers and their relationship with n-octanol/water partition coefficients.

Skin penetration of methyl, ethyl, propyl and butyl parabens through excised guinea pig dorsal skin was examined, and effects of the penetration enhancers, l-menthol plus ethanol itself and N-dodecyl-2-pyrrolidone, were observed. Permeability of coefficients of the parabens correlated with n-octanol/water partition coefficients. Addition of 1% l-menthol in 15% ethanol about sixteen times increased the permeability coefficient of methyl paraben, whereas this enhancer decreased that of butyl paraben to about one fifth of the control value. A similar, though weaker, tendency was observed for the effects of 15% ethanol itself. 0.025% suspension of N-dodecyl-2-pyrrolidone increased the permeability coefficient of methyl paraben about seven times, whereas it did not change that of butyl paraben significantly. Therefore, dependency of the permeability coefficients of the parabens on n-octanol/water partition coefficients almost disappeared in the presence of this compound. A spin label study with stratum corneum lipid liposomes revealed that increase of fluidity of the lipid bilayer by these penetration enhancers corresponded with their enhancement effects on skin penetration of methyl paraben. Perturbation of stratum corneum lipid lamella thus seems to be related with their enhancement of the absorption of hydrophilic paraben.

Effect of phosphatidylcholine on the percutaneous penetration of drugs through the dorsal skin of guinea pigs in vitro; and analysis of the molecular mechanism, using attenuated total reflectance-fourier transform infrared (ATR-FTIR) spectroscopy

Phospholipids are effective penetration enhancers for the transdermal delivery of certain co-applied drugs. Standard in vitro skin percutaneous penetration methods using excised guinea pig dorsal skin were employed to characterize the comparison with penetration of a water-insoluble drug, prednisolone (PR), and a water-soluble drug, diclofenac sodium salt (DFS) enhanced by phosphatidylcholine (PC) from soybean. An ATR-FTIR spectroscopy was used to analyze the molecular mechanism of enhancement of the drug penetration, and to measure the lipid fluidity in the stratum corneum (SC) of guinea pig skin caused by PC. The C-H bond stretching absorbance frequency shifts in the SC of guinea pigs induced a higher and a broader absorbance shifts, and a shift was dependent on the PC concentrations. In addition, the percutaneous penetration of PR was dependent on the PC concentration (up to 10% concentration). On the other hand, the percutaneous penetration of DFS was not dependent on the PC concentration. Further, the percutaneous penetration of PR was proportional to the C-H bond stretching absorbance frequency shifts. In contrast, the percutaneous penetration of DFS was not proportional to the C-H bond stretching absorbance frequency shifts. Furthermore, the accumulation of PR in skin was inversely proportional to the C-H bond stretching absorbance frequency shifts (without a plot of the control). The accumulation of DFS in skin was inversely proportional to the C-H bond stretching absorbance frequency shifts (without a plot of the control). The striking parallelism between the enhancement of the percutaneous penetration of PR and the measured SC lipid fluidity shifts caused by PC suggests that the transdermal water-insoluble drug penetration may be ultimately related to the SC lipid structure. On the other hand, the non-parallelism between the enhancement of the percutaneous penetration of DFS and the measured SC lipid fluidity shifts measurement caused by PC suggests that the mechanism of the transdermal water-soluble drug penetration may not be intimately related to the SC lipid structure.