Characteristics of Shed Snake Skin Permeability to Indomethacin and Fatty Alcohols

To investigate the utilities of a shed snake skin as a model membrane for preclinical studies of transdermal drug delivery, the flux of indomethacin was determined under various conditions by using a diffusion cell. The flux of fatty alcohols was determined and compared with that in human skin reported in references. The esterase activity of shed snake skin was also determined. It was found that the flux of indomethacin decreased with an increase of pH and the amount of ethanol in a vehicle. The flux of indomethacin increased by the addition of Azone, N-methyl?2?pyrroridone and N,N-dimethyl-m-toluamide in the cream. The flux of fatty alcohols in shed snake skin was greater than that reported in human skin, and shed snake skin had similar esterase activity to human skin.     

Evaluation of Simultaneous Permeation and Metabolism of Methyl Nicotinate in Human,Snake, and Shed Snake Skin

The transdermal permeation and metabolic characteristics of methyl nicotinate (MN) in stratum corneum and split-thickness human skin and three species of shed snake and snake skin (Elaphae obsoleta, Naja kaouthia, and Python molurus bivittatus) were evaluated. In vitro skin transport using excised skin and hydrolysis experiments using skin homogenate were carried out. The flux of MN, a metabolite, nicotinic acid (NA), and the total (MN+NA), as well as kinetic parameters (V_max and K_m) for hydrolysis of MN were determined and compared among various skin types. The total flux from MN-saturated solution through human skin was not significantly different from that through snake and shed snake skin of Elaphae obsoleta, Naja kaouthia but was significantly higher than that through snake and shed snake skin of Naja kaouthia (p < 0.05). A great difference in skin esterase activity was observed between human and snake in both snake skin and shed snake skin of all species. In all skins except the stratum corneum of human skin, NA flux increased with an increase in MN donor concentration and reached a plateau, suggesting that metabolic saturation was taking place in the skin. NA flux at the plateau and MN donor concentrations at which the NA flux reached a plateau also varied by species. These findings indicated that the discrepancy in transdermal profiles of MN among skins tested was predominantly due to the difference in the esterase activity in the skin.     

A Method to Predict the Percutaneous Permeability of Various Compounds: Shed Snake Skin as a Model Membrane

Penetration of various compounds through shed snake skin was measured in vitro to examine the effect of lipophilicity and molecular size of a compound on permeability through this model membrane. The permeabilities were found to be controlled by the lipophilicity and the molecular size of the permeant. The smaller and the more lipophilic the compound, the greater the permeability. Equations have been developed to predict the permeability from the molecular weight and the distribution coefficient of a compound. Further, the lipophilicity of shed snake skin is similar to that of human skin and the response of shed snake skin to the molecular size of a permeant is more similar to human skin than to hairless mouse skin. Considering the similarities between shed snake skin and human stratum coraeum in terms of structure, composition, and permeability characteristics, the same considerations may apply to permeability through human stratum corneum.     

Use of Shed Snake Skin as a Model Membrane for in Vitro Percutaneous Penetration Studies: Comparison with Human Skin

The potential usefulness of shed snake skin as a model membrane for transdermal research was examined. There are similarities between shed snake skin and human stratum corneum in terms of structure, composition, lipid content, water permeability, etc. The permeability of various compounds and the contribution of several functional groups to the permeability were also found to be similar between shed snake skin and human skin. Moreover, the permeability of compounds through shed snake skin was increased by Azone, one of the most extensively studied transdermal penetration enhancers. Considering the similarities between shed snake skin and human skin, ease of storage and handling, and low cost, shed snake skin may offer a good model membrane for transdermal research.     

Percutaneous Permeation of Basic Compounds Through Shed Snake Skin as a Model Membrane

Abstract— Relationships between the in-vitro permeability of basic compounds through shed snake skin as a suitable model membrane for human stratum corneum and their physicochemical properties were investigated. Compounds with low pK_a values were selected to compare the permeabilities of non-ionized forms of the compounds. Steady-state penetration was achieved immediately without a lag time for all compounds. Flux rate and permeability coefficient were calculated from the steady-state penetration data and relationships between these parameters and the physicochemical properties were investigated. The results showed that permeability may be controlled by the lipophilicity and the molecular size of the compounds. Equations were developed to predict the permeability from the molecular weights and the partition coefficients of basic compounds.     

Permeability Profiles of M-Alkoxysubstituted Pyrrolidinoethylesters of Phenylcarbamic Acid Across Caco-2 Monolayers and Human Skin

Purpose. The purpose of the present research was to study 10 m-alkoxysubstituted pyrrolidinoethylesters of phenylcarbamic acid—potential local anesthetics. The relationships between the structure of the molecule, its physicochemical parameters (log D_oct, log k, R_M, solubility) were correlated to the permeability data obtained from permeation experiments in Caco-2 monolayers and excised human skin in vitro. Methods. The extent and mechanism(s) of permeability of the series were studied through a Caco-2 monolayer in the apical-to-basolateral (a-b) and basolateral-to-apical (b-a) directions. The MTT test was performed to determine cellular damage. In vitro transdermal permeability data were obtained from permeation experiments on excised human skin by using side-by-side chambers. Passive diffusion and iontophoretically enhanced permeability were measured. Results. In Caco-2 monolayers, similar results in the shape of the permeability curves were obtained for the two directions. In the b-a direction, the values of P_app were 2-6 times greater than in the a-b direction. A plot of drug permeability vs. the number of carbons in the alkoxychain plateaued first, after which the permeability decreased by the increasing lipophilicity of the drug. If the log D_oct of the ester was 3.4 and the MW > 385 Da, no measurable Caco-2 permeability was found. Cell damage was also higher by the more lipophilic compounds. In excised human skin, the relationship between the passive diffusion of the drugs and the number of carbons in the alkoxychain was parabolic (r ² = 0.95). Introducing low-level electrical current (iontophoresis), transdermal permeability of the more hydrophilic phenylcarbamic acid esters increased clearly. Conclusions. Lipophilicity and solubility of a compound have crucial roles in the permeation process. A very high lipophilicity has, however, a negative influence on the permeability, both intestinally and transdermally. Iontophoresis significantly increases the diffusion of small and less lipophilic compounds.     

高效液相色谱法同时测定人血浆中单硝酸异山梨酯、阿司匹林和水杨酸

目的:建立同时测定人血浆中单硝酸异山梨酯、阿司匹林及其代谢物水杨酸浓度的高效液相色谱法。方法:以茶碱为内标,血浆经酸化后,乙酸乙酯提取,高效液相色谱质谱检测器(MSD)和二极管阵列检测器(DAD)串联在线测定血浆中单硝酸异山梨酯、阿司匹林和水杨酸的浓度;分析柱:Zorbax DB-C18(5μm,2.1 mmx150mm),保护柱:Zorbax DB-C8(5μm,2.1mm×12.5 mm),柱温30℃;流动相为A:3 mmol·L-1乙酸铵溶液(含冰乙酸0.022％),流速为0.17 mL·min-1,B:甲醇-乙腈(85:15),流速为0.03 mL·min-1。单硝酸异山梨酯和阿司匹林用高效液相-电喷雾质谱(HPLC/ESI-MS)选择离子检测,水杨酸用DAD检测。结果:血浆中单硝酸异山梨酯、阿司匹林和水杨酸线性范围分别为0.00625-1.2 μg·mL-1,0.025-1.2μg·mL-1,0.0625-12μg·mL-1;相对回收率分别为101.3％-103.0％,99.8％-119.7％,98.4％-104.0％;提取回收率均高于80％;日内、日间精密度均小于8％。结论:方法简便、快速、重现性好,可用于临床血药浓度监测和复方制剂的药代动力学和生物利用度研究。     

Role of Appendages in Skin Resistance and lontophoretic Peptide Flux: Human Versus Snake Skin

Purpose. 1. The assessment of the role of hair follicles and sweat glands in skin resistance and percutaneous iontophoretic flux of 9-desglycinamide, 8-arginine vasopressin (DGAVP) by comparing two skin species: human stratum corneum which contained hair follicles, sweat and sebaceous glands, and shed snake skin which lacked all appendages. 2. The effect of l-dodecylazacycloheptan-2-one (dodecyl-Azone, a lipid perturbing agent) on the iontophoretic DGAVP flux. Methods. Iontophoresis in vitro was performed in a transport cell (0.79 cm² area available for percutaneous transport) by 8-hours application of a pulsed constant current of 100 Hz, 50% duty cycle and 0.26 mA.cm^–2 current density delivered by a pair of Ag/AgCl electrodes, of which the anode was facing the anatomical surface of the skin samples. Results. The initial resistances of human stratum corneum and shed snake skin samples were of the same order of magnitude (20–24 k.cm²) and both skin species showed a comparable resistance-decrease profile during 8-hours iontophoresis, indicating that the resistances were mainly determined by the stratum corneum and not greatly influenced by the appendageal structures. The initial resistances of the skin samples pretreated with dodecyl-azone were less than 50% of the values of untreated samples. Because dodecyl-azone is known to perturb the ordering of the intercellular lipids, the effect of azone on the resistance confirms that the resistance mainly resides within the intercellular lipids of the stratum corneum. No correlation was found between the iontophoretic DGAVP-flux and the conductance of human skin. For shed snake skin, however, a good correlation was found, indicating that the iontophoretic permeability of human skin in vitro for a peptide such as DGAVP is, unlike shed snake skin, not related to its overall permeability to ions. While the initial resistances of both human and snake skin were in the same order of magnitude and showed the same declining profile during iontophoresis, the steady state iontophoretic DGAVP flux across human stratum corneum was approximately 140 times larger than through shed snake skin. These findings suggest that small ions follow pathways common to both skin types, presumably the intercellular route, while the peptide on the other hand is transported differently: across snake skin presumably along intercellular pathways only, but across human stratum corneum along additional pathways (most likely of appendageal origin) as well. This interpretation is supported by the observations made of the effects of dodecyl-azone on DGAVP-iontophoresis. Pretreatment with dodecyl-azone did not significantly change steady state fluxes and lag times of DGAVP-iontophoresis across human stratum corneum, but resulted in a significant 3-fold lag time decrease and a 3-fold flux increase of DGAVP-iontophoresis across snake skin. Conclusions. The results of these in vitro studies emphasize the importance of the appendageal pathway for iontophoretic peptide transport across human stratum corneum.     

Percutaneous Permeability to Paraqut: In Vitro Experiments with Human Skin

Abstract

Percutaneous permeability to paraquat through intact and mechanically damaged human skin was measured in vitro from diluted solutions (1 mg/ ml). In intact human skin under occlusion, a permeability to paraquat of 3 × 10^?5 cdhr was measured at steady state. The binding of paraquat to skin was determined and found to be negligible. The measured permeabilities were used in a pharmacokinetic model to predict paraquat levels in blood and lungs. The model predicts that for intact human skin and diluted paraquat solutions systemic toxicity is unlikely. However, the risk increased significantly when damaged skin or concentration paraquat solutions are involved.     

Effectiveness and Mode of Action of Isopropyl Myristate as a Permeation Enhancer for Naproxen through Shed Snake Skin

The effectiveness and mode of action of isopropyl myristate (IPM) as an enhancer for the permeation of naproxen through shed snake skin have been investigated. The highest naproxen permeability was afforded by IPM (36.2 × 10^?4 cm h^?1), followed by menthol (25.0 × 10^?4 cm h^?1), oleic acid (11.1 × 10^?4 cm h^?1), azone (7.3 × 10^?4 cm h^?1)_and control (1.4 × 10^?4 cm h^?1). Whereas the permeability of un-ionized naproxen (47.4 × 10^?5 cm h^?1) was much greater than that of ionized naproxen (1.11 × 10^?5 cm h^?1), IPM-treatment of the intact skin increased the flux of ionized naproxen significantly more (50?fold) than that of un-ionized naproxen (15?fold). The large effect of pH on the permeation of naproxen through the intact stratum corneum became insignificant after extraction of lipids from the skin. Similar permeation of naproxen through intact and delipidized skin after IPM treatment indicated that the lipid barrier of the skin was largely impaired by IPM. Direct application of IPM to skin yielded a 2.6?fold higher naproxen permeability than the application of IPM as a gel. A greater amount of naproxen was absorbed from 1% test gel (pH 5) containing IPM than from 10% commercial gel (pH 7) containing no IPM. These results show that use of IPM can significantly improve the bioavailability of naproxen in topical preparations.     

Absorption of Salicylic Acid from the Perfused Human Rectum

Abstract: The absorption of salicylic acid from solutions, in which it is highly ionized, was investigated by means of a new in vivo perfusion technique in human subjects. The absorption rate shows proportionality to the concentration (15.6 mM-250 mM), while the rate decreases with increasing pH (5.9-8.9). This is in agreement with the theory of passive diffusion of the unionized form of the drug. From a 47 mM sodium salicylate solution, buffered to pH 7.9 with phosphate, 7.5 cm of rectum absorbs about 100 mg per hour. When a glycine buffer is used instead of the phosphate buffer, the absorption rate increases about 2.5 times.     

Dodecyl N,N-Dimethylamino Acetate and Azone Enhance Drug Penetration Across Human,Snake, and Rabbit Skin

The effectiveness of the penetration enhancers, dodecyl N, N-dimethylamino acetate (DDAA) and Azone, on pretreated human epidermis for the permeation of model drugs, indomethacin, 5-fluorouracil, and propranolol-HCl, was studied in in vitro diffusion cells. Snakeskin (Elaphe obsoleta) and rabbit pinna skin were compared as possible models for human skin. The drug concentrations were analyzed by HPLC. With all skins and all model drugs, DDAA increased drug permeability at least as well as Azone, and in most cases it was a more effective permeation enhancer. The relative permeation improvements in human skin, snakeskin, and rabbit skin were 10- to 20-, 5- to 50-, and 20- to 120-fold, respectively. Tritiated water served as an indicator of skin condition. Its penetration in the skin samples was independent of the drugs used, and both penetration enhancers significantly increased the flux of tritiated water through all skins. Thus, DDAA and Azone significantly increased the permeation of lipophilic and hydrophilic model compounds. Rabbit pinna skin was a poor model for human skin in vitro, while snakeskin was much closer to human skin in terms of transdermal permeability. In most cases drug permeability decreased in the order rabbit human > or < snake.     

高效液相色谱法测定复方水杨酸散中水杨酸的含量

目的 建立测定复方水杨酸散中水杨酸含量的高效液相色谱 (HPLC)法. 方法采用Hypersil C18色谱柱(250 mm×4.6 mm,5 μm),以甲醇-水(用稀磷酸调节pH 3.0)(45:55)为流动相,检测波长303 nm.结果 水杨酸在5.08～50.80 μg·mL-1 浓度范围内与色谱峰面积线性关系良好,r=0.999 5,高、中、低 3种浓度的回收率分别为99.22%,100.32%,99.99%,RSD分别为0.19%,1.01%,0.68%.结论 该方法操作简便,准确度和精密度高,耐用性好,能用于复方水杨酸散中水杨酸含量的测定.     

Are Water Permeability Measurements Sufficient to Characterize in Vitro Cultured Human Skin Surrogates?

Abstract

We have found previously that human neonatal foreskin keratinocyte air/liquid (A/L) cultures developed normal-appearing epidermis, and that water flux was only two to three times higher than that found with intact human skin. To understand further the barrier properties of these A/L cultures, we have analyzed the composition and the gross conformational structures of the cultured stratum corneum (SC) lipids, and compared them with those of human SC. Electron microscopic examination demonstrated that cultured SC has high intracellular lipid content, but that it lacks both the basic unit repetition (e.g., broad/narrow/broad/broad/narrow/ broad pattern of electronlucent bands) normally found in human SC intercellular lipids and the covalently bound lipid envelope. These results indicate that the cultures are hyperproliferative, with characteristics similar to those found in SC obtained from patients with atopic dermatitis or psoriasis, or from animals whose diets lack essential fatty acids. In addition, the high free sterol content and the altered fatty acid/ceramide composition of these cultures argue that the compromised barrier function is linked to hyperproliferation and lipid synthesis, or vice versa. Infrared spectroscopic analysis of the SC lipid thermal transitions confirm that there are major conformational differences between the lipids of cultured and human SC, which clearly have an impact on permeability. The hyperproliferative state of growth and the profound differences between cultured and human SC in their lipid structural, compositional, and conformational properties together attest that water permeability alone is not a sufficiently sensitive marker of keratinocyte terminal differentiation for in vitro culture systems. It is not completely obvious how these differences in the cultured SC may influence the utility of this surrogate in studies involving skin biochemistry, pharmacology, permeability, metabolism, and toxicology. Nevertheless, it is clear that one needs to characterize the end product fully before one can judiciously interpret any results obtained from this or other similar skin surrogates.     

反相高效液相色谱法测定复方水杨酸酊中水杨酸和苯甲酸含量

目的建立测定复方水杨酸酊中水杨酸和苯甲酸含量的反相高效液相色谱法。方法采用PhenomenexLunaC18柱（150mm×4．6mm,5μm）,乙腈-四氢呋喃-冰醋酸-水（20：5：5：70）为流动相,流速为1．0mL／min,检测波长为231nm,柱温为30℃。结果水杨酸的质量浓度线性范围为10．3～51．6μg／mL（r=0．9996）,平均回收率为100．00％,RSD为1．46％（n=9）;苯甲酸的质量浓度线性范围为20．0-100．0μg／mL（r=0．9999）,平均回收率为101．41％,RSD为1．36％（n=9）。结论该方法简便、准确,专属性好,灵敏度高,可同时测定复方水杨酸酊中水杨酸和苯甲酸的含量。     

HPLC法测定水杨酸软膏中主药及有关物质的含量

目的:建立以高效液相色谱法测定水杨酸软膏中主药及有关物质含量的方法。方法:色谱柱为Kromasil C18,流动相为甲醇-水(冰醋酸调节pH至2.5)=50∶50,检测波长为270nm,流速为1.0mL·min-1,进样量为20μL。结果:主峰与有关物质可完全分离,水杨酸检测浓度的线性范围为10.014～100.14μg.mL-1(r=0.9999),平均回收率为99.35%,RSD=0.99%,最低检测限为5ng·mL-1,有关物质标示量均小于1.01%。结论:本方法操作简便、结果准确、专属性强,可用于该制剂的含量测定。     

紫外分光光度法测定甲醛水杨酸涂剂中水杨酸含量

目的研究甲醛水杨酸涂剂中水杨酸的含量测定方法。方法采用紫外分光光度法,在296．4nm波长处直接测定。结果水杨酸质量浓度在11．25～33．75μg／mL范围内与吸光度线性关系良好,r=0．9999（n=5）;平均回收率为99．80％,RSD为0．35％（n=6）。结论紫外分光光度法准确、简单、快捷,适用于医院制剂的快速分析。     

高效液相色谱法测定水杨酸醇溶液中水杨酸含量

目的采用高效液相色谱（HPLC）法测定水杨酸醇溶液中水杨酸的含量。方法以Eclipse XDB—C18柱（250mm×4．6mm,5μm）为色谱柱,以甲醇-0．4％磷酸溶液（58：42）为流动相,检测波长为298nm,流速为1．0mL／min,柱温为25℃。结果水杨酸进样量在0．16-0．80μg范围内与峰面积积分值呈良好的线性关系,r=1．0000（12=6）,平均回收率为99．51％,RSD为0．2％（n=6）。结论HPLC法简单、结果准确、灵敏度高,可用于水杨酸醇溶液中水杨酸的含量测定。     

不同基质及促渗剂对氢溴酸高乌甲素凝胶经皮渗透的影响

［摘要］目的 研究不同凝胶基质及促渗剂对氢溴酸高乌甲素经皮吸收的影响. 方法 分别用羟丙基甲基纤维素和卡波姆934作为凝胶基质制备氢溴酸高乌甲素凝胶,并加入月桂氮卓酮或油酸作为经皮吸收促进剂,采用TK－12A型透皮扩散池测定并比较其吸收速率. 结果 与羟丙基甲基纤维素凝胶比较,以卡波姆934作为凝胶基质的氢溴酸高乌甲素其经皮渗透效果较好. 加入3%月桂氮卓酮+乙醇作为复合促渗剂能显著促进氢溴酸高乌甲素卡波姆凝胶中药物的吸收,3%油酸对药物的经皮渗透影响不大. 结论 氢溴酸高乌甲素在以3%月桂氮卓酮和乙醇为复合促渗剂,卡波姆为基质的凝胶剂中具有较好的经皮渗透效果.     

中空膜液相微萃取-HPLC法用于人血浆中水杨酸的测定

建立灵敏、准确地测定人血浆中水杨酸浓度的HPLC法。样品经过酸化处理后,以甲苯-正辛醇(4:6,V/V)为萃取溶剂、NaOH水溶液(pH=10)为接收相进行中空纤维膜微萃取。在该条件下,血浆中水杨酸浓度在2~250 ng.mL-1范围内,线性关系良好(r=0.9994);检测限为0.7ng.mL-1(S/N=3);精密度(RSD)<8%(n=5);回收率为87.6%~102.0%。该方法灵敏、有效、所需有机溶剂少、富集效率高,适于血浆中水杨酸浓度的测定。