Functional Evaluation of Anchored Skin Equivalent Cultured in Vitro: Percutaneous Absorption Studies and Lipid Analysis

Pharmaceutical Research -     

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 The samples withdrawn from the receptor cell were analyzed for osthol content by high-performance liquid chromatography (HPLC).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.     

氮酮促进复方替硝唑凝胶经皮渗透作用研究

目的 :研究不同浓度的氮酮对复方替硝唑凝胶透皮吸收的作用 ,并筛选出最佳浓度。方法 :采用垂直式Franz扩散池 ,以不同浓度的氮酮作为促渗剂 ,应用反相高效液相色谱法测定处方中替硝唑、氯霉素、水杨酸3组分的含量并计算其单位面积累积透皮量。结果与结论 :不同浓度的氮酮对复方替硝唑凝胶中3组分的渗透性均有促进作用 ,其中浓度为2 %的氮酮透皮促渗作用最显著。     

Low Dose Lipid Formulations: Effects on Gastric Emptying and Biliary Secretion

Purpose Food stimulates changes to gastrointestinal secretion and motility patterns, however, the effect of smaller quantities of lipid, such as that contained in a lipid-based drug formulation, has not been detailed. This study aimed to examine the effects of small quantities of lipid on gastric emptying and biliary secretion. Methods The influence of oral administration of three lipid-based formulations and a negative control formulation on gastric emptying and biliary secretion was evaluated in 16 healthy male subjects using gamma scintigraphy, ultrasonography and duodenal aspiration. Results Low quantities (2 g) of long chain lipid stimulated gall bladder contraction and elevated intestinal bile salt, phospholipid and cholesterol levels. Changes in gastric emptying were also evident, although these did not reach statistical significance. Administration of a similar quantity of medium chain lipid, however, had little effect on gastric emptying and gallbladder contraction and did not stimulate appreciable increases in intestinal concentrations of biliary-derived lipids. Conclusions The quantities of long chain lipid that might be administered in a pharmaceutical formulation stimulate gallbladder contraction and elevate intestinal levels of bile salt and phospholipid. This effect is a likely contributor to the ability of lipid based formulations to enhance the absorption of poorly water-soluble drugs.     

Drug Solubilization Behavior During in Vitro Digestion of Simple Triglyceride Lipid Solution Formulations

PURPOSE: The purpose of this study was to characterize the solubilization and precipitation characteristics of a range of poorly water-soluble drugs during digestion of either long-chain or medium-chain triglyceride (TG) lipid formulations. METHODS: TG solution formulations of five selected drugs (griseofulvin, diazepam, danazol, cinnarizine, and halofantrine) were digested in ritro and drug distribution/solubilization behavior in the resulting digests assessed. RESULTS: For the less lipophilic drugs, the mass of drug dissolved in either medium or long-chain TG was low and the drugs partitioned rapidly into the aqueous digestion phase. For the higher log P drugs, drug transfer to the aqueous phase was limited by accumulation in undigested long-chain TG. In contrast, medium-chain TG was digested completely producing a dispersed aqueous phase that was capable, at least in the case of the high log P drugs, of supporting supersaturated drug concentrations. CONCLUSIONS: The solubilization behavior of lipophilic drugs on digestion of simple TG lipid formulations is a function of the lipophilicity of the drug (which dictates the drug dose and the partitioning behavior), the nature of the colloidal phases produced on digestion of the different formulation lipids, and the kinetics of drug transfer between the digesting formulation and the colloidal phases produced.     

干扰素α脂质体凝胶剂的透皮吸收作用研究

目的探讨基因重组人干扰素α-2b脂质体凝胶剂经家兔皮肤给药后的吸收作用。方法给家兔皮肤分别涂抹干扰素α-2b脂质体凝胶（试验组）和干扰素α-2b凝胶（对照组），用双抗体夹心酶联免疫吸附法（ELISA法）检测干扰素含量，测定给药后不同时间内血浆中干扰素含量及24h后皮肤层中的干扰素含量。结果与对照组比较，试验组干扰素在家兔皮肤层中有较多的滞留量（P〈0．01）；两组家兔不同时间血浆中干扰素的含量均极微少（P〉0．05）。结论脂质体作为干扰素皮肤局部给药的载体，能够提高干扰素在皮肤中的含量，但并不增加进入血液循环的干扰素量。     

Percutaneous Penetration of Acyclovir Through Excised Hairless Mouse and Rat Skin: Effect of Vehicle and Percutaneous Penetration Enhancer

The effects of vehicle and percutaneous penetration enhancer on the penetration of acyclovir through excised hairless mouse and rat skin were investigated. Four solvents, propylene glycol (PG), ethanol (ET), isopropanol (IPA), and isopropyl myristate (IPM), were employed as vehicles, in combination with four enhancers, l-farnesylazacycloheptan-2-one (7FU), l-geranylazacycloheptan-2-one (7GU), l-geranylazacyclopentan-2-one (5GU), and l-dodecylazacycloheptan-2-one (Azone). Acyclovir was suspended in vehicles to avoid the effect of the thermodynamic activity of acyclovir in the vehicle. The penetration of acyclovir through hairless mouse skin from IPA was enhanced by 7GU, whereas that from IPM was not affected. All combinations of vehicle and penetration enhancer were examined using rat skin. No effect of the enhancers was observed in the IPM vehicle. The estimated solubility parameters of vehicles and enhancers indicated that the polarities of IPM and the enhancers are similar, which prevents effective penetration of the enhancers from IPM. However, the penetration of acyclovir from the other vehicles was increased by the enhancers. The combination of hydrophilic vehicle and hydrophobic enhancer resulted in a large enhancing effect. The disappearance of the enhancers from the vehicle correlated with their enhancing activity, but other factors also seemed to affect the penetration enhancement of acyclovir.     

氮酮对鬼臼毒素固体脂质纳米粒透皮性的影响

目的 考察氮酮对鬼臼毒素固体脂质纳米粒透皮性的影响。方法 以大鼠腹部皮肤为屏障，考察氮酮浓度（体积比）对鬼臼毒素固体脂质纳米粒表现透皮系数的影响。结果 当氯酮浓度分别为0，1％，2％，3％。4％，5％，6％时，鬼臼毒素脂质纳誊粒的表观透皮系数（x10^5）分别为24．82，53．05，55．14，43．92，20．57，25．86，23，30。结论 在有效浓度范围内，氮酮可增加鬼臼毒索固体脂质纳米粒的透皮吸收，含不同浓度氮酮的鬼臼毒素固体脂质纳米粒的透皮能力有差异。     

An Acute and Coincident Increase in FABP Expression and Lymphatic Lipid and Drug Transport Occurs During Intestinal Infusion of Lipid-Based Drug Formulations to Rats

Purpose To determine a) whether administration of lipid-based formulations can acutely up-regulate the intestinal expression of I-FABP and L-FABP and b) whether this occurs coincidentally with an increase in intestinal lymphatic lipid and drug transport.Methods The expression of I-FABP and L-FABP mRNA (using q-PCR) and protein (using immunohistochemistry and Western blotting) in enterocytes was compared with data describing transport of lipid and drug into intestinal lymph following infusion of a set of lipid-based formulations.Results Administration of relatively small amounts of oleic acid (5–20 mg/h) over a 5 h period to rats acutely up-regulated the expression, and altered the intracellular distribution of, I-FABP and L-FABP in the enterocytes of the small intestinal epithelia. The increase in expression of I-FABP and L-FABP correlated well with previous data describing the transport of lipid and drug into intestinal lymph following infusion of the same formulations.Conclusion The expression and intracellular distribution of I-FABP and L-FABP are acutely influenced by lipid infusion over a time period relevant to feeding or the administration of pharmaceutical lipidic formulations, and these changes occur coincidentally with increased drug transport into the lymphatics.     

盐酸尼卡地平凝胶家兔在体透皮吸收研究

进行了盐酸尼卡地平凝胶的家兔在体透皮吸收研究，评价月桂氮Chuo酮对其透皮吸收的促进作用。通过测定家兔皮肤给药后不同时间的血药浓度值，得到了血药－时间曲线，经拟合获得了透皮吸收的主要药动学参数，结果表明加入月桂氮Chuo酮后尼卡地平的峰浓（Cmax)，血药浓度曲线下面积（AUC0－24h)和平均滞留时间（MRT）三个参数均显著增加（P＜0．0001），而达峰时间（Tmax)和时滞（Tlag)两个参数无显著性改变（P＞0．05）。     

The Influence of an Electric Field on Ion and Water Accessibility to Stratum Corneum Lipid Lamellae

Purpose. To study ion transport through stratum corneum (SC) lipid lamellae under passive and iontophoretic conditions. Methods. Iodide ion transport was measured by fluorescence quenching. Since the process involves diffusion of an iodide ion to the fluorophore located within the SC lamellae, the accessibility of iodide ions was measured. Moreover, the use of anthroyloxy fatty acid probes, provided information as a function of depth within the lamellae. Results. Fluorescence quenching by iodide ions increased with iontophoretic current density, suggesting increased ion accessibility within the SC lamellae. In addition, at constant current, quenching decreased as the fluorophore was located deeper within the lamellae. This gradient in ion accessibility suggests that more iodide is found near the head-group than near the core of the SC lipid lamellae. Results obtained in the absence of iodide also show increased water accessibility during iontophoresis. Conclusions. These results show that in the presence of an applied electric field the SC lipid lamellae interior becomes more accessible to water and ions. These results imply that during iontophoresis, ion and water transport through human skin is associated, at least in part, with the SC lipid lamellae.     

Comparative Analysis of Percutaneous Absorption Enhancement by d-Limonene and Oleic Acid Based on a Skin Diffusion Model

Percutaneous absorption-enhancing effects of d-limonene and oleic acid were investigated using three model drugs with different lipophilicities in in vitro diffusion experiments with guinea pig skin. Pretreatment of the skin with d-limonene resulted in a large penetration enhancement for the lipophilic butylparaben (BP) and amphiphilic 6-mercaptopurine (6-MP) but had little effect on the hydrophilic mannitol (MT). Oleic acid caused a large effect only on 6-MP penetration. The penetration profiles were analyzed with a two-layer skin diffusion model consisting of stratum corneum with polar and nonpolar routes and viable epidermis plus dermis. Through curve-fitting, six parameters corresponding to drug diffusivity and partitioning in these three regions of the skin were obtained, and the mechanisms of enhancers were assessed in comparison with those of l-geranylazacycloheptan-2-one (GACH) reported previously. Increased penetration was caused mainly by modification of the barrier property of the nonpolar route in the stratum corneum in all cases. In the nonpolar route, d-limonene increased mainly drug diffusivity, while GACH enhanced predominately drug partitioning. On the other hand, oleic acid moderately increased both parameters.     

Determination of Amiodarone Hydrochloride in Pharmaceutical Formulations by Derivative UV Spectrophotometry and High-Performance Liquid Chromatography (HPLC)

Assay procedures based on derivative ultraviolet spectrophotometry and high-performance liquid chromatography (HPLC) have been developed for the specific determination of amiodarone hydrochloride in pharmaceutical dosage forms. The use of first- and second-order derivative spectrophotometry was found to have suppressed the background absorption from the excipients with comparable accuracy and precision to the reversed-phased HPLC reference method. A conventional UV absorption method ( = 242 nm) is subject to possible interference by formulation excipients.     

羟乙桂胺离子导入和涂抹兔体透皮吸收的对比

采用离子导入和直接经皮给药方法，以高效液相色谱法研究了羟乙桂胺乳剂（1．5g／40cm2）对10只家兔的透皮吸收及药物动力学。结果表明，离子导入方法组平均值（n=5）Ka为1．57h-1，T1／2α为1．15h和AUC为16．05μg·h·ml-1。离子导人组与直接外敷组Cmax、Tmax和AUC均有显著性差异（P＜0．01），表明离子导入方法组较涂抹给药的透皮吸收性能好。     

Spectral Reflectance Measurement of Evaporating Chemical Films: Initial Results and Application to Skin Permeation

The present study has 2 aims. First, the method of spectral reflectance was used to measure evaporation rates of thin (～25-300 μm) films of neat liquid volatile organic chemicals exposed to a well-regulated wind speed u. Gas-phase evaporation mass transfer coefficient (k_evap) measurements of 10 chemicals, 9 of which were measured at similar u, are predicted (slope of log-log data = 1.01; intercept = 0.08; R² = 0.996) by a previously proposed mass transfer correlation. For one chemical, isoamyl alcohol, the dependence of k_evap on u^0.52 was measured, in support of the predicted exponent value of ½. Second, measured k_evap of nicotine was used as an input in analytical models based on diffusion theory to estimate the absorbed fraction (F_abs) of a small dose (5 μL/cm²) applied to human epidermis in vitro. The measured F_abs was 0.062 ± 0.023. Model-estimated values are 0.066 and 0.115. Spectral reflectance is a precise method of measuring k_evap of liquid chemicals, and the data are well described by a simple gas-phase mass transfer coefficient. For nicotine under the single exposure condition measured herein, F_abs is well-predicted from a theoretical model that requires knowledge of k_evap, maximal dermal flux, and membrane lag time.     

Physicochemical Aspects of Percutaneous Penetration and Its Enhancement

The classic diffusion model-based interpretation of percutaneous absorption is compared to a simple kinetic analysis. The physicochemical significance and the major deductions of the two approaches are shown to be in general agreement. In particular, the effect of penetrant oil/water partition coefficient on transdermal flux is consistently predicted by the two models. Diffusional and kinetic assessments of skin penetration enhancement are then shown to reveal similar dependencies upon penetrant physical chemistry. It is demonstrated that the requirements for successful promotion of a lipophilic drug's transdermal flux are quite different from those necessary for a hydrophilic penetrant. Finally, in light of published transport data and our increased comprehension of the stratum corneum barrier function, the evidence for (and significance of) different absorption paths across the stratum corneum is considered. In addition, the impact of penetrant size on transport is addressed. It is argued that currently held beliefs concerning (i) a putative polar route through the stratum corneum and (ii) the dependence of flux on molecular weight warrant considerable further attention before their unequivocal acceptance is appropriate.     

Liposomal Formulations of Cyclosporin A: Influence of Lipid Type and Dose on Pharmacokinetics

Purpose. Liposomal formulations of Cyclosporin A (CyA)³ have been described in more than 30 publications to substitute Cremophor EL (CrEL), a triricinoleate ester of ethoxylated glycerol, as drug carrier. However, conflicting reports did not allow to draw consistent conclusions about the influence of liposomes on CyA pharmacokinetics (PK) and pharmacodynamics. Methods. A series of liposomal CyA-formulations with varying liposome composition and lipid dose but constant CyA dose was compared in rats. Data were analysed with a PK-model taking into account the varying volume of distribution with the varying lipid concentration in blood. Results. Surface properties and lipid type of liposomes are not important PK predictors of liposomal CyA, at least for small dosages of liposomes. Rather, the absolute lipid amount and the lipophilicity of cyclosporins are critical factors influencing the PK of liposomal CyA. The higher the concentration of lipid in blood and the greater the lipophilicity of cyclosporin is, the higher are the concentrations of CyA in blood. Conclusions. These relations may explain the inconsistent literature results. Together with earlier observations from our group the above findings indicate, that CyA is not caged in the liposomal membranes. Reports in literature, which claim lower clearance and a lower volume of distribution of CyA in obese rats compared to lean rats, support our assumption about the involved mechanisms. A semi-quantitative model of CyA distribution is presented, which points to the variable free fraction of CyA in plasma as the crucial factor for all previously reported phenomena in liposomal CyA formulations.     

Evaluation of a Noninvasive Method for Monitoring Percutaneous Absorption of Lidocaine in Vivo

The pharmacodynamic measurement of in vivo skin penetration of lidocaine was explored with an instrument used in dentistry to determine tooth pulp vitality. The instrument delivers a low-current, pulsatile electrical waveform of increasing intensity with time. The readings, which are reproducible, are in arbitrary units on a scale of 0–80. Testing of naive sites showed variation as a function of location, even over relatively small distances. The response at a marked site over a 12-hr period generally was consistent in five subjects. Following intradermal administration of 1 or 2% lidocaine hydrochloride injection in one subject, the instrument reached its maximum value within 2 min. This was followed by a sustained plateau and then a gradual falloff of the effect. Topical formulations containing 5% lidocaine base and corresponding blank formulations were applied under occlusion within Hilltop chambers to intact skin on the forearms of human volunteers for 3 hr. While the response to a 40% propylene glycol formulation was not significantly different from the corresponding control, a cream exhibited slow development of profound anesthesia that lasted for several hours following chamber removal.