Mechanism of skin penetration-enhancing effect by laurocapram.

In order to clarify the mechanism of action of laurocapram (Azone) on the skin permeation of drugs, the following experiments were done. First, the effect of Azone on the skin components was compared with that of other penetration enhancers. Azone markedly fluidized liposomal lipids (as a model lipid system) compared with other enhancers. Ethanol extracted large amounts of the stratum corneum lipids, whereas Azone did not. These results suggest that the effect of Azone on the lipids in the stratum corneum is not the same as that of ethanol. In addition, ethanol increased the amount of free sulfhydryl (SH) group of keratin in the stratum corneum, whereas Azone did not directly affect the stratum corneum protein. Azone increased water content in the stratum corneum, as measured by skin conductance. This effect might be a reason for the action of Azone. For further understanding, the enhancing effects of Azone on the skin permeation of several model compounds (alcohols, sugars, and inorganic ions) were compared with the effects of pretreatment with distilled water, which was thought to increase water-holding capacity, and pretreatment with ethanol, which was thought to affect the lipids and protein in the skin barrier (i.e., stratum corneum). Pretreatment with water or ethanol enhanced skin permeation of hydrophilic compounds, whereas they decreased that of octanol, a hydrophobic compound. The tendency of Azone to increase or decrease the skin permeation rate of most compounds was similar to that of pretreatment with water or ethanol. However, the effect of Azone on the skin permeation of inorganic ions was relatively low, whereas that of pretreatment with water or ethanol was high.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of molecular weight on the dermatopharmacokinetics and systemic disposition of drugs after intracutaneous injection

The aim of this study was to investigate the effect of molecular weight on the topical migration and systemic absorption of drugs, sodium salicylate (SA), calcein sodium (CAL) and FITC-dextrans (FD-4, FD-10, FD-20 and FD-40), after intracutaneous (i.c.) injection. The apparent diffusion coefficients in the skin (D(s)) of SA, CAL and FD-4 after i.c. injection were almost the same and slightly less than 1x10(-4)cm(2)/min. The D(s) value drastically decreased with molecular weight more than 10kDa. The plasma concentration-time curve after i.c. injection was predicted by the convolution method. The ratio of AUC (observed/predicted) was approximately unity for SA, CAL and FD-4 although the systemic absorption of only FD-4 was delayed. In contrast, the AUC ratios of FD-10, FD-20 and FD-40 were about 0.5, 0.2 and 0.06, respectively, and obviously decreased with the molecular weight. The elimination of FD-10, FD-20 and FD-40 from the skin was slow and the drugs tended to remain in the muscle. These results indicated the importance of diffusion in the skin and permeability through the blood vessels for local migration and systemic absorption of drugs after penetration into viable skin.     

Utility of MTT assay in three-dimensional cultured human skin model as an alternative for draize skin irritation test: approach using diffusion law of irritant in skin and toxicokinetics-toxicodynamics correlation

Purpose. A cytotoxicity assay using a three-dimensional cultured human skin model, Living Skin Equivalent-high (LSE-high) was evaluated as an alternative to the Draize skin irritation tests using animals. A relation between the cytotoxicity and calculated concentration of an irritant in skin was also evaluated. Methods. Colorimetric thiazoyl blue (MTT) conversion assay and a surfactant, cetylpyridinium chloride (CPC), were selected as a cytotoxicity assay and a model irritant. The fraction of dead cell number in the MTT assay or the Draize irritation score (in vitro and in vivo irritation data, respectively) was treated as a function of CPC concentration in the viable skin of LSE-high and guinea pig. Separately, in vitro permeations of CPC through the LSE-high or excised guinea pig skin were determined to calculate the average concentration of CPC in the viable skin using the Fickian diffusion theory. The obtained relations between the irritation scores and CPC concentration were evaluated by the Emax model (Hill equation). Results. CPC concentration showing 50% irritation (IC ₅₀) was similar for the MTT assay (18.9%) and Draize test (12.3%), and a good relationship (r = 0.981) was observed between the fraction of dead cell number and the Draize score. In contrast, IC ₅₀, 1.32%, for the MTT assay in LSE-high was much lower than that using guinea pig skin. We then corrected the results for the MTT assay using a ratio of IC ₅₀ in guinea pig skin against LSE-high, resulting in a good relation between both MTT results in guinea pig skin and LSE-high. Conclusion. The present results suggest that the MTT assay using LSE-high may be utilized as an alternative for the Draize test in animals for evaluating skin irritation.     

The enhancing effect of a triethanolamine-ethanol-isopropyl myristate mixed system on the skin permeation of acidic drugs

The effect of a TEI enhancer mixed system consisting of triethanolamine (T), ethanol (E) and isopropyl myristate (IPM) on the skin permeation of acidic, basic and neutral drugs were evaluated in vitro using excised hairless rat skin. The binary enhancer system consisting of IPM and ethanol (El) produced marked improvement on the penetration of all the drugs tested. When T was added to the EI system, a greater enhancing effect was found only on acidic drugs with a carboxyl group, compared with the flux in the EI system. On addition of another amine to the EI system, instead of T, mefenamic acid (MA), which exhibited the highest enhancing effect of the model drugs, showed an approximately 14-180 times greater flux than when delivered by the EI system. On simultaneous application of isosorbide dinitrate (ISDN) with MA in the TEI system, the flux of MA increased on increasing the T concentration in the TEI system, while, the flux of ISDN, a neutral drug, was unaffected by the T concentration. Application of MA in the EI system after pretreatment of the TEI system showed that the residual amount of T in the skin plays an important role in the skin permeation of MA. Furthermore, at a fixed concentration of MA, the flux of MA increased on increasing the T concentration in the TEI system, while the flux of E remained unchanged. Finally, the infrared spectrum of MA with amine in the E solution indicated that the carboxyl group of MA was ionized. These results demonstrated that the formation of an ion pair between MA and T, but not the effect of T on the skin, may be responsible for the enhanced skin permeation of MA using the TEI system.     

Electric field analysis on the improved skin concentration of benzoate by electroporation

The objective in the present study was to understand the relationship between the increased skin concentration of benzoate as a model drug after topical application of its sodium salt and the electric field intensity produced in the skin barrier, the stratum corneum, by electroporation. A piece of excised abdominal hairless rat skin was set in a Franz type diffusion cell, and 0.5% sodium benzoate and physiological saline were applied to the stratum corneum and dermis sides, respectively. Two needle electrodes made of Ag were connected to an electrical power source, which produced exponentially decaying pulses. The electrodes were placed on the skin surface with a distance of 0.5 cm between both electrodes. After the 4 h passive permeation experiment, an electrical pulse was applied to the rat skin at 300 V every minute for 10 min. The skin was then removed from the diffusion cell, and the amounts of benzoate in different positions of the skin specimen were measured. Field intensity generated in the stratum corneum by electroporation was determined by a finite element method using a computer program. The amounts of benzoate at different sites in the skin were almost proportional to the mean field intensity in the corresponding stratum corneum. These results suggested that the enhancing effect of electroporation can be evaluated by the field intensity more directly than the application voltage.     

Effect of poly-L-arginine on the nasal absorption of FITC-dextran of different molecular weights and recombinant human granulocyte colony-stimulating factor (rhG-CSF) in rats

The effect of poly-L-arginine (poly-L-Arg) on the in vivo nasal absorption of FITC-dextrans with a mean molecular weight ranging from 4.3 to 167 kDa and recombinant human granulocyte colony-stimulating factor (rhG-CSF) in rats were studied. When FITC-dextrans were co-administered intranasally with 1.0 w/v% poly-L-Args of different molecular weight (MW, ca. 45.5 and 92 kDa, poly-L-Arg (50) and poly-L-Arg (100)), the bioavailability (F(infinity)) increased markedly compared with that after administration of FITC-dextran alone. However, the F(infinity) decreased exponentially with the increasing molecular weight of FITC-dextrans. There was no significant difference between the enhanced nasal absorption of FITC-dextrans achieved by the co-administration of poly-L-Arg (50) and poly-L-Arg (100). Moreover, the relationship between the F(infinity) and the molecular weight of FITC-dextrans indicated that the molecular weight of protein drugs, which exhibited efficient absorption with poly-L-Arg, was about 20 kDa, when the lower limit of bioavailability for developing a potent transnasal delivery system was assumed to be about 10%. Indeed, the nasal absorption of rhG-CSF, which has a molecular weight of 18.8 kDa, was also increased after co-administration of 1.0 w/v% poly-L-Arg (50) and the F(infinity) was about 11%. It seems likely that poly-L-Arg can be used to provide adequate nasal absorption of various protein drugs which have a molecular weight of about 20 kDa, thereby allowing the successful development of a variety of transnasal drug delivery systems.     

Synergistic effects of iontophoresis and jet injector pretreatment on the in-vitro skin permeation of diclofenac and angiotensin II

A non-needle syringe (jet injector) was utilized to increase skin permeation of drugs by iontophoresis. Briefly, physiological saline was initially flushed by the injector to make a pore in the stratum corneum of excised hairless rat skin, and the iontophoretic skin permeation of two model compounds, sodium diclofenac and angiotensin II, was followed using a 2-chamber diffusion cell. Constant voltage and constant current iontophoresis treatments were evaluated. Pretreatment using the jet injector alone resulted in about 13- and 22-fold increases in the steady-state flux of diclofenac and angiotensin II, respectively, through the skin, compared with non-treated controls. Jet injector pretreatment with constant voltage iontophoresis further enhanced skin permeation of diclofenac and angiotensin II, and the enhancement was also greater than that by constant voltage iontophoresis alone. Thus, a synergistic effect was observed. The ratio of enhancement was greater compared with the control. Jet injector pretreatment with constant current iontophoresis, however, did not always yield higher skin permeation of the drugs than injector pretreatment alone, although the lag time was shortened. The difference in the enhancement between the constant voltage- and constant current iontophoresis can be explained by the electric current through the excised skin. Constant current iontophoresis after a short period of constant voltage iontophoresis with multiple jet injector pretreatments may be the best way to increase drug permeability while preventing severe skin damage.     

Improved nasal absorption of drugs using poly-L-arginine: effects of concentration and molecular weight of poly-L-arginine on the nasal absorption of fluorescein isothiocyanate-dextran in rats.

The effects of the concentration and molecular weight of poly-L-arginine (poly-L-Arg) on the in vivo nasal absorption of fluorescein isothiocyanate-labeled dextran (MW, 4 kDa, FD-4) in rats were studied. When poly-L-Arg with a range of different molecular weights (MW, 8.9, 45.5 and 92.0 kDa) was applied intranasally at various concentrations, the bioavailability (F(0-9 h)) of FD-4 increased with the increasing concentration of poly-L-Arg. The enhanced absorption was also dependent on the molar concentration, in that the poly-L-Arg with a higher molecular weight increased F(0-9 h) at a lower molar concentration. In addition, for each applied concentration, the poly-L-Arg exhibited a molecular weight-dependence as far as the enhancement of FD-4 absorption was concerned. On the other hand, the maximum absorption rate (MAR) of FD-4, calculated by means of a deconvolution method, tended to reach a maximum plateau level at a lower applied concentration for the poly-L-Arg with the highest molecular weight, but this plateau level was almost the same for poly-L-Arg with molecular weights of 45.5 and 92.0 kDa. Moreover, the simulated absorption profiles of FD-4 indicate that the degree of enhancement (the level of MAR and the subsequent reduction in the absorption rate) was dependent on the molecular weight of poly-L-Arg, while the effect of poly-L-Arg was maintained for a longer period, depending on the applied concentration, although the MAR was relatively similar. These results indicate that the molecular weight of poly-L-Arg appears to affect both the enhancing efficiency (absorption rate) and the time-frame of this enhancing effect, whereas the concentrations of each poly-L-Arg system applied only have an effect on the time-frame. These effects may also be associated with the charge density of a poly-L-Arg molecule.     

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

Purpose  

To calculate the skin concentration of active ingredients in cosmetics and topical pharmaceuticals using silicone membrane permeation.     

In Vitro Release Profile of Mitomycin C from Albumin Microspheres: Extrapolation from Macrospheres to Microspheres

To analyze the in vitro release profiles of mitomycin C from albumin microspheres prepared by chemical denaturation in a multiparticulate system, a method to calculate the total cumulative amount of mitomycin C released from a batch of microspheres was developed. Mitomycin C-loaded albumin macrospheres (diameter in mm range) were prepared, and the in vitro release kinetics of mitomycin C from individual macrospheres were determined. Then the relationship between the kinetic parameters and the physical parameters (e.g., diameter, weight) was investigated under the assumption that macrospheres and microspheres behave identically. Further, the size distribution of microspheres was measured, and the total cumulative amount of mitomycin C released from albumin microspheres was calculated. The release profiles of mitomycin C from individual macrospheres fitted first-order release kinetics better than spherical matrix kinetics. The calculated initial mitomycin C contents and first-order release rate constants for individual macrospheres were correlated with the weight and reciprocal of surface area of the macrospheres, respectively. The observed in vitro release profile for the microspheres agreed with the calculated values. These results suggest that this method is valid for calculating drug release from albumin microspheres.