Enhanced in Vitro Skin Permeation of Cationic Drugs

The lipophilicity of cationic drugs can be increased by forming ion pairs with the carboxylate anion of fatty acids. Transport of cations across an isopropyl myristate (IPM) membrane was facilitated in the presence of oleic acid and lauric acid, providing an appropriate pH gradient existed. Enhancement of in vitro skin permeation of various drugs, in the presence of fatty acids, was shown to be more dramatic with the slow-permeating neutral caffeine and anionic salicylate. Since both molecules are unable to form ion pairs it is probable that the fatty acids are capable of exerting a disruptive influence on the skin. The cationic drugs appeared to traverse excised human skin more rapidly than predicted by the model membrane data. This may be due to ion pairing with free fatty acids or other anionic groups within the skin. Consequently, the enhancing ability of fatty acids was less marked for neutral or anionic permeants.     

In Vitro Human Skin Barrier Modulation by Fatty Acids: Skin Permeation and Thermal Analysis Studies

Purpose. This study aims to elucidate the skin permeation enhancement and the skin perturbation effects of a number of fatty acids, i.e. straight-chain saturated (SFA), monounsaturated (MUFA) and polyunsaturated acids (PUFA). Methods. The skin permeation enhancement effects were studied using human stratum corneum (SC) and p-aminobenzoic acid (PABA) as a model permeant. The fatty acids in propylene glycol (FA/PG) were applied according to a pre-treatment/co-treatment protocol. The perturbation effects were studied using differential thermal analysis (DTA) on SC after pretreatment with FA/PG. Results. SFA with 6 to 12 carbons exhibit a parabolic correlation between enhancement effect and chain-length, with a maximum at nonanoic-decanoic acids (with 9 and 10 carbons). Nonanoic and decanoic acids exert barely noticeable effects on the thermal behaviour of SC, suggesting that they easily mix with the skin lipids. All cis-6-, 9-, 11- or 13-octadecenoic acids (MUFA) enhance the permeation of PABA to the same extent. DTA revealed that the cis-9- and 13-isomers form a separate domain containing mostly the pure fatty acids within the SC lipids and suppress the lipid transitions at 70°/80°C. PUFA—linoleic (LA), -linolenic (ALA) and arachidonic acids—enhance PABA permeation stronger than MUFA but additional double bonds do not further increase the degree of enhancement. LA and ALA form separate domains but do not completely suppress the SC lipid transitions at 70°/ 80°C. Increase in the enthalpy changes of 70°/80° transitions linearly correlates to the decrease in the permeability coefficients, suggesting that an increased perturbation of the skin lipids not necessarily has to yield an increased PABA permeation. Conclusions. The enhancement effects of fatty acids on the PABA penetration through SC are structure-dependent, associated with the existence of a balance between the permeability of pure fatty acids across SC and the interaction of the acids to skin lipids.     

Enhanced Permeation of Methotrexate In Vitro by Ion Pair Formation With L-Arginine

Ion paired solutions of methotrexate in L-arginine/water/propylene glycol systems were evaluated for their potential to enhance the permeation of methotrexate across rabbit nasal mucosa in vitro. The partition coefficient of methotrexate in the methotrexate: L-arginine ion paired systems was observed to be 24 times greater than that of the methotrexate system without L-arginine. The ion pair formation between methotrexate and L-arginine was confirmed by a decrease in the conductivity of the systems in the presence of propylene glycol, a dielectric constant reducing agent. The permeation of methotrexate across the rabbit nasal mucosa from the ion paired systems was observed to be significantly greater (p < 0.05) as compared to control systems of methotrexate solution in water and a sodium salt. Furthermore, a threefold increase in the flux of methotrexate was observed when propylene glycol was added to the ion paired systems. These results suggest that methotrexate: L-arginine ion paired systems have potential in improving the permeation of methotrexate across rabbit nasal mucosa and may form the basis for further development of an intranasal therapeutic system of methotrexate. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3633–3639, 2009     

In Vitro Skin Permeation of Osthol from Hydro-Alcohofic Gel Formulations

Aim To evaluate the in vitro percutaneous absorption behavior of osthol from a series of hydro-alcoholic gel formulations containing three penetration enhancers through excised human skin (stratum cormeum and epidermis,SCE). Methods Excised human skin was mounted in Franz-type diffusion cells. The samples withdrawn from the receptor cell were analyzed for osthol content by high-performance liquid chromatography (HPLC). Results The enhancers azone, menthol and chenopodium increased the osthol percutaneous steady-state fluxes 3.12, 2.00 and 1.25 times those of the enhancer-free formulations (controls), separately. Conclusions The main enhancement mechanism of the skin penetration enhancers azone, menthol and chenopodium is to destroy the barrier fimction of stratum corneum, reducing the resistance of drug transport through the skin and increasing the diffusion coefficients of osthol.     

蛇床子素凝胶药物经皮吸收的体外研究

目的体外测定含有渗透促进剂的蛇床子素凝胶经人体皮肤的吸收.方法以离体人皮肤为渗透模型,应用Franz扩散池进行实验.样品以高效液相法测定蛇床子素的含量.结果与对照组相比,渗透促进剂Azone、薄荷醇、土荆芥油可以使得蛇床子素的稳态流量分别提高3.12、2.00、1.25倍.结论三种渗透促进剂的作用机理为破坏了皮肤角质层的屏障作用,降低了药物的扩散阻力,因而提高了蛇床子素的扩散系数.     

An in Vitro Study of Diamorphine Permeation Through Premature Human Neonatal Skin

The permeation kinetics of diamorphine through human premature neonatal cadaver skin over a range of gestational ages between 24 and 36 weeks was investigated using small diffusion cells. A strong inverse correlation was noted between the apparent permeability coefficient and the gestational age of the skin (P < 0.01; n = 26). The calculated apparent permeability coefficients decreased with gestational age from 6.0 × 10 ^–2 cm · hr^–1 at 24 weeks' gestation to 5.2 × 10^–6 cm · hr^–1 at 36 weeks' gestation. The amount of diamorphine remaining bound within the skin at the end of the in vitro experiments did not change significantly with gestational age of the skin. Diamorphine was subject to degradation over the course of the in vitro experiments to produce significant amounts of 6-mono-acetylmorphine and evidence is presented to suggest that this was due to residual skin esterase activity. It is calculated that the steady-state flux rate of diamorphine through neonatal skin observed in these experiments would be sufficient to obtain a therapeutic plasma concentration of morphine assuming a 2-cm² area for application and a delivery rate of 15 µg hr ^–1 kg^–1. However, the prolonged half-life of morphine in the premature neonate would result in a delay of some hours before the attainment of this level.     

In Vitro Absorption of Triethanolamine Through Human Skin

The human skin penetration of triethanolamine (TEA) was measured using in vitro diffusion cell techniques. [¹⁴C]TEA was applied to viable skin in an oil-in-water emulsion containing TEA stearate as an emulsifying agent to simulate cosmetic exposure. The percent of the applied dose of TEA absorbed into the receptor fluid was similar with both 1% and 5% TEA formulations. Absorption of TEA was reduced by lowering the pH of the formulation, presumably due to the increased ionization of TEA. Absorption of TEA into the receptor fluid (1% formulation, pH 7.0) was 0.43% of the applied dose in a 24 h study. Substantial amounts of TEA remained in the skin at the end of the study (9.4% of dose), but only minimal amounts diffused into the receptor fluid when the collection time was extended to 72 h in separate studies. The amount of TEA remaining in skin at the end of the 24 h studies should not be included in estimates of systemic absorption.     

An Examination of Excised Skin Tissues Used for In Vitro Membrane Permeation Studies

The morphology and layer thicknesses of excised human and hairless mouse skin have been examined. Excised tissues were prepared either by heating at 60°C or by incubation in an ethylenediamine-tetracetic acid (EDTA) solution. Either process yielded spearated sheets of stratum corneum plus attached viable epidermis (SCE), the thicknesses of which were determined microscopically. These measurements indicated that initial skin separation occurred at the dermal/epidermal junction for both separation processes. The two techniques produced SCE sections showing consistent differences in thickness of the attached viable epidermis layer. This effect depended upon the presence or absence of epidermal invaginations. In the former case, EDTA-separated tissue gave narrower viable epidermis of more uniform thickness than that seen with heat-separated tissue. In the latter case, both techniques produced SCE having viable epidermis layers of similar thickness.     

Unusual Reduction of the In Vitro Skin Permeation of [3H]dexetimide by Atropine

Pharmaceutical Research -     

Transdermal Permeation of Alniditan by Iontophoresis: In Vitro Optimization and Human Pharmacokinetic Data

Purpose. The aim of this paper was to assess the feasibility of electrically enhanced transdermal delivery of alniditan, a novel 5 HT_1D agonist for the treatment of migraine. Methods. An in vitro study was first performed to optimize the different parameters affecting iontophoresis efficiency. The mechanism of alniditan permeation by iontophoresis was investigated. Finally, a phase I clinical trial was performed to assess systemic delivery of alniditan by iontophoresis. Results. i) In vitro: The optimal conditions were found with a buffer like ethanolamine at a pH of 9.5, with Ag/AgCl electrodes and a direct current application. Alniditan permeation was enhanced when increasing the current density, the duration of current application and the drug concentration. Iontophoresis slightly increased drug quantities in stratum corneum compared to passive diffusion but it strongly increased alniditan quantities in viable skin, ii) The objective to deliver in vivo 0.5 mg of alniditan within less than 1 h was reached but an erythema was detected at the anode. Conclusions. This study demonstrates the feasibility of iontophoretic delivery system for antimigraine compounds.     

A Mechanistic Bayesian Inferential Workflow for Estimation of In Vivo Skin Permeation from In Vitro Measurements

Computational models can play an integral role in the chemical risk assessment of dermatological products. However, a limitation on the ability of mathematical models to extrapolate from in vitro measurements to in human predictions arises from context-dependence: modeling assumptions made in one setting may not carry over to another scenario. Mechanistic models of dermal absorption relate the skin penetration kinetics of permeants to their partitioning and diffusion across elementary sub-compartments of the skin. This endows them with a flexibility through which specific model components can be adjusted to better reflect dermal absorption in contexts that differ from the in vitro setting, while keeping fixed any context-invariant parameters that remain unchanged in the two scenarios. This paper presents a workflow for predicting in vivo dermal absorption by integrating a mechanistic model of skin penetration with in vitro permeation test (IVPT) measurements. A Bayesian approach is adopted to infer a joint posterior distribution of context-invariant model parameters. By populating the model with samples of context-invariant parameters from this distribution and adjusting context-dependent parameters to suit the in vivo setting, simulations of the model yield estimates of the likely range of in vivo dermal absorption given the IVPT data. This workflow is applied to five compounds previously tested in vivo. In each case, the range of in vivo predictions encompassed the range observed experimentally. These studies demonstrate that the proposed workflow enables the derivation of mechanistically derived upper bounds on dermal absorption for the purposes of chemical risk assessment.     

In Vitro Skin Permeation and Bioassay of Chlorhexidine Phosphanilate,a New Antimicrobial Agent

An in vitro technique was developed to study the permeation and antimicrobial activity of graded concentrations of a new antibacterial agent, chlorhexidine phosphanilate (CHP), in cream formulations using Franz diffusion cells. Formulations containing from 0.2 to 2% CHP were quantitatively applied to intact excised skin and to skin from which the stratum corneum and partial epidermis had been enzymatically removed. Receptor fluids from diffusion cells were sampled over time and assayed by HPLC methods for chlorhexidine and phosphanilic acid; 24- and 48-hr samples of the diffusate from studies with damaged skin were also bioassayed using clinical isolates of appropriate microbial species. Through intact skin almost no permeation of CHP was observed over 48 hr. The failure of CHP to penetrate intact human skin suggests that normal stratum corneum is the rate-limiting barrier to penetration by this antimicrobial agent. In damaged skin lacking stratum corneum barrier, the release of CHP from the formulation becomes the rate-determining step. Coincident with penetrating damaged skin, CHP dissociates, and the molar ratio of the chlorhexidine and phosphanilate moieties in the diffusate changes to favor phosphanilic acid. The extent of changes in the permeation rates of both moieties of CHP was directly related to the CHP concentration in cream. Both CHP moieties were found to reach equilibrium in the dermis within 24 hr after application. It was also observed that CHP creams down to 0.2% concentration yielded diffusates with activity exceeding the minimum inhibitory concentration of all test microorganisms within 24 hr.     

Enhanced In Vitro Skin Deposition Properties of Retinyl Palmitate through Its Stabilization by Pectin

The purpose of this study was to examine the effect of stabilization of retinyl palmitate (RP) on its skin permeation and distribution profiles. Skin permeation and distribution study were performed using Franz diffusion cells along with rat dorsal skin, and the effect of drug concentration and the addition of pectin on skin deposition profiles of RP was observed. The skin distribution of RP increased in a concentration dependent manner and the formulations containing 0.5 and 1 mg of pectin demonstrated significantly increased RP distributions in the epidermis. Furthermore, it was found that skin distribution of RP could be further improved by combined use of pectin and ascorbyl palmitate (AP), due largely to their anti-oxidative effect. These results clearly demonstrate that the skin deposition properties of RP can be improved by stabilizing RP with pectin. Therefore, it is strongly suggested that pectin could be used in the pharmaceutical and cosmetic formulations as an efficient stabilizing agent and as skin penetration modulator.     

Effects of Pulsed Electromagnetic Fields on Rat Osteoblasts

目的:研究脉冲电磁场(PEMFs)对大鼠成骨细胞的作用.方法:取新生SD大鼠头盖骨,通过骨组织块培养法分离、培养成骨细胞.取传代第3代细胞分为5组,1-70组、2-70组、3-70组、4-70组、对照组,其中前4组为作用组,接受频率为70 Hz,峰值磁场分别为1、2、3、4mT的PEMFs作用,对照组不接受PEMFs作用.按照分组剂量进行离体PEMFs作用(0.5 h/d,连续作用4 d),作用结束后,对各组分别进行细胞碱性磷酸酶(ALP)活性,核结合因子α1 (cbfα1)表达,体外成骨能力指标一骨结节形成能力检测.结果:经PEMFs作用后,与对照组相比,成骨细胞ALP活性降低,cbfα1表达增多,骨结节数量显著增多.结论:PEMFs作用成骨细胞可促进成骨细胞功能分化,增强成骨细胞的细胞外基质矿化.     

博莱霉素脂质体凝胶的制备和体外透皮性比较

目的：研制博莱霉素（BLM）脂质体凝胶,并对其皮肤靶向性进行体外评价。方法：采用逆相蒸发-冻融法制备BLM脂质体,再用卡渡姆940为基质制成BLM脂质体凝胶;以离心法测定BLM脂质体的包封率;以体外透皮渗透释药法,比较BLM脂质体凝胶与BLM普通凝胶的透过作用。结果：BLM脂质体平均粒径为（885．20±12．08）nm,平均包封率为（66．80±1．38）％。在24h内,BLM脂质体凝胶累积透过量（Q）及稳态透皮速率（J）与BLM普通脂质体相比,均明显提高,而在皮肤中的滞留药量也显著提高（P〈0．05）。结论：BLM脂质体凝胶在体外可显著增加BLM的透皮吸收,增加皮肤中的滞留量,值得进一步研究。     

Dermal Stability and In Vitro Skin Permeation of Collagen Pentapeptides (KTTKS and palmitoyl-KTTKS)

Collagen pentapeptide (Lys-Thr-Thr-Lys-Ser, KTTKS) and its palmitoylated derivative (pal-KTTKS) have received a great deal of attention as cosmeceutical ingredients for their anti-wrinkle effects. The objective of this study was to evaluate stability and permeability of KTTKS and pal-KTTKS in hairless mouse skin. In this study, a liquid chromatography-tandem mass spectrometric method was developed for the quantification of pal-KTTKS, and used for stability and permeability studies. Stability studies were performed using skin extracts and homogenates. Both KTTKS and pal-KTTKS were rapidly degraded, but pal-KTTKS was more stable than KTTKS. When protease inhibitors were added, the stability of both compounds (KTTKS and pal-KTTKS) improved significantly. In the skin permeation study, neither KTTKS nor pal-KTTKS was detected in the receptor solution, which indicates that neither compound could permeate through the full-thickness hairless mouse skin in the experimental conditions of this study. While KTTKS was not detected in any of the skin layers (the stratum corneum, epidermis, and dermis), pal-KTTKS was observed in all skin layers: 4.2 ± 0.7 μg/cm² in the stratum corneum, 2.8 ± 0.5 μg/cm² in the epidermis, and 0.3 ± 0.1 μg/cm² in the dermis. In conclusion, this study indicated that pal-KTTKS had greater stability and permeability than that of un-modified KTTKS, and may be a useful anti-wrinkle and anti-aging cosmeceutical agent.     

Physicochemical Evaluation,in Vitro Human Skin Diffusion,and Concurrent Biotransformation of 3-O-Alkyl Carbonate Prodrugs of Naltrexone

PURPOSE: The purpose of this study was to evaluate the physicochemical properties and in vitro human skin diffusion of the 3-O-alkyl carbonate prodrugs of naltrexone (NTX). METHODS: Melting points and heats of fusion (deltaHf) were determined using differential scanning calorimetry. In vitro human skin permeation rates of NTX and its prodrugs were measured using a flowthrough diffusion cell system. Drug disposition in the skin was quantified at the end of the diffusion experiment. The solubilities of the drugs were determined in mineral oil and isotonic buffer. Partitioning of the prodrugs from vehicle to skin was determined using isolated sheets of human stratum corneum (SC). RESULTS: All the prodrugs hydrolyzed to NTX on passing through the skin, and the methyl NTX-3-O-carbonate (ME-NTX) provided the highest NTX flux, apparent permeability coefficient (Kp), and calculated relative thermodynamic activity from the melting point and deltaHf. The ME-NTX SC/vehicle partition coefficient was the highest of the prodrug series, although similar to the NTX SC/vehicle partition coefficient value. The shortest chain prodrugs underwent the highest extent of bioconversion to NTX upon passing through the skin. CONCLUSIONS: Within this 3-O-alkyl carbonate prodrug series, the shortest chain prodrug was the most skin-permeable compound with the highest partition coefficient and a significant extent of bioconversion.     

Enhanced Anti-Inflammatory Effects of Cu,Zn-Superoxide Dismutase Delivered by Genetically Modified Skin Fibroblasts In Vitro and In Vivo

Purpose. The purpose of this work was to evaluate the anti-inflammatory effects of secretable human Cu, Zn-superoxide dismutase (hSOD) delivered by genetically modified skin fibroblasts in vitro and in vivo. Methods. Rat skin fibroblasts were transfected with pRc/CMV-ILSOD including secretable SOD-coding cDNA. The effects of host and transformants on oxidative stress in vitromodels using the xanthine/xanthine oxidase (X/XO) system were examined to study the paracrine SOD action. The anti-inflammatory effects by transplantation of host and transformants were evaluated in an acute inflammation model, carrageenin-induced paw edema, in rats. Results. The transformants (ILSOD cells) secreted SOD protein into the extracellular space, and the extracellular SOD activity in ILSOD cells cultures was significantly increased compared with that in host cell cultures. ILSOD cells diminished the cytotoxic activity by X/XO in a paracrine fashion. These protective effects of ILSOD cells against X/XO-induced cytotoxicity correlated well with the decrease in lipid peroxidation in the damaged cells. The in vivo study showed that transplantation of ILSOD cell suspensions into the hind paw in rats inhibited carrageenin-induced paw edema for at least 7 days, and the degree and the durability of these inhibitory effects were dependent on the number of ILSOD cells transplanted. These inhibitory effects of ILSOD cell suspensions were reduced by co-administration of antiserum for hSOD. Furthermore, the healing of paw edema caused by carrageenin was markedly enhanced by transplantation of ILSOD cells into the edemics hind paw. Conclusions. The findings suggested that genetically modified skin fibroblasts are a suitable delivery system for obtaining an efficient and continuous supply of SOD to the target site, and this strategy may be a useful drug delivery system for therapeutic proteins.