Influence of human skin specimens consisting of different skin layers on the result of in vitro permeation experiments

The literature exhibits high variation in results from drug permeation experiments across human skin. Our purpose was to investigate the influence of human skin specimens, consisting of different skin layers and resulting from different skin preparation techniques, on the in vitro permeation of a model drug, i.e. flufenamic acid (FFA). FFA permeation across human (1) trypsin-isolated stratum corneum, (2) heat-separated epidermis and (3) dermis, (4) dermatomized skin and (5) full-thickness skin (FTS) from either a hydrophilic or lipophilic donor was investigated in Franz-type diffusion cells. Cumulative permeated drug amounts were plotted versus time, and a fit to Fick's 2nd law of diffusion was performed. Since performing skin diffusion experiments in the laboratory is time consuming and expensive, especially when using FTS, we also investigated the possibility of calculating the resistances of composite skin layers from the diffusion resistances of the individual skin layers. Due to short lag time, practical handling and economic preparation, heat-separated epidermis appears to be superior in human skin in vitro permeation experiments compared to separated stratumcorneum sheets, dermatomized skin and FTS. Furthermore, we found a good correlation between calculated and experimental resistances which underlines that calculation of the total diffusion resistance of composed skin preparations from resistances of individual skin layers is legitimate and useful. Considering our findings, improved interpretation of literature data and more consistent results for future permeation experiments are possible.     

Influence of cyclodextrin complexation on the in vitro permeation and skin metabolism of dexamethasone

The influence of complexation of a model drug, dexamethasone acetate (DMA), with beta-cyclodextrin (beta-CyD) and hydroxypropyl-beta-cyclodextrin (HP-beta-CyD) on the in vitro permeation through hairless mouse skin and on skin metabolism have been investigated. Complexation with CyDs increased the amount of DMA permeated in the order of 2.0 and 3.0 times for beta-CyD and HP-beta-CyD, respectively. The partition coefficient, between stratum corneum and buffer (K(SC/buffer)), for DMA decreased when the drug was an inclusion complex, being greatest for DMA/HP-beta-CyD complex. Complexation protected the drug against skin metabolism. The increase of skin permeation and stability of the model drug in the skin suggest that the complexation with beta-CyD and HP-beta-CyD is a rational way to improve the physical-chemical properties of drugs for use in transdermal delivery systems.     

Evaluation of the permeation of peat substances through human skin in vitro

Peat and various peat extracts have been successfully applied for a variety of clinical indications. Quite apart from the physico-thermal effects, new studies point towards the so-called "chemical effects" of peat containing substances. These effects include a stimulatory response of the spontaneous contractile activity (SCA) of smooth muscle (SM) tissue. The effects are, however, dependent on the possible permeability of pharmacologically active substances as naturally occurring ingredients of peat. Since peat is a mixture of various products it is necessary to examine the various peat types based upon their biological activity on SM tissue. In order to unequivocally prove the pharmacological activity of cutaneous peat treatment, in vitro permeation measurements of these actives across excised human skin can be used.HPLC analysis revealed that aqueous peat extracts contain up to 18 fractions of water-soluble compounds of fulvic and ulmic acids. These compounds have been found to have a stimulatory response on the contractile activity of SM tissue. In vitro diffusion studies showed that the permeability of these substances across human full thickness skin (thickness: 200 um(-1)) is highly selective and the resulting stimulatory activity is dependent on the permeated fraction. Especially, the HPLC fractions 7-11 and 14 are able to permeate human skin. Fractions 7-11 show a moderate stimulatory effect of SCA on SM for more than 90 min whereas fraction 14 shows the strongest stimulatory effect which was, however, suppressed after 87 min. These results show that the cutaneous therapy with peat treatment results in transcutaneaous permeation of biologically active fulvic and ulmic acid derivatives explaining the additional "chemical" effect of peat treatment in clinical practice.     

Vehicle effects on in vitro skin permeation of thiocolchicoside

Thiocolchicoside, a semi-synthetic derivative of colchicoside, is used in topical formulations for its anti-inflammatory and muscle-relaxant properties. The objective of this study was to evaluate the effect of a (propylene glycol diperlagonate) DPPG and (propylene glycol) PG mixture present in an innovative foam formulation (Miotens) on the flux of thiocolchicoside through excised human skin. Furthermore, the in vitro permeation behaviour of this new formulation (Miotens foam) was compared to another commercial product (Muscoril ointment) and to a control gel formulation (thiogel), both enhancer free. The best permeation profile was obtained from the foam formulation (Miotens) which was able to increase the thiocolchicoside flux about three fold compared to control formulation (thiogel) and about two fold compared to the commercial formulation Muscoril ointment.     

Influence of membrane-solvent-solute interactions on solute permeation in skin

The relative importance of solubility parameters and other solvent properties on membrane diffusion processes has not been fully elucidated in the literature. Previously, we have studied the effect of different vehicles on the permeation of caffeine, benzoic acid (BA) and salicylic acid (SA) through silicone membranes. The present paper investigates diffusion of the selected permeants from different saturated solutions through human epidermis.

The permeation of caffeine was strongly affected by the vehicle chosen and the maximum enhancement observed for the permeation of caffeine was 288-fold. A maximum of 12-fold enhancement in the flux was observed for the permeation of SA and a maximum of 10-fold enhancement was observed for the permeation of BA. The diffusion profiles obtained for SA in the different solvents were very similar when compared with those obtained for BA but the permeation rates were higher for BA than for SA. This similarity results from the similar chemical structure and lipophilicity.     

透皮促进剂对白花前胡甲素体外经皮渗透的影响

目的:考察透皮促进剂对白花前胡甲素(dl-praeruptorin A,Pd-Ia)体外经皮渗透的影响。方法:采用改进的Franz扩散池,以大鼠离体皮肤为渗透屏障,用高效液相色谱法对Pd-Ia进行含量测定,考察月桂氮酮(Azone)及1%Azone与不同浓度丙二醇(PG)混合物对Pd-Ia透皮吸收的影响。结果:使用Azone对Pd-Ia有促透作用,1%Azone效果较好,平均渗透速率达到4.064μg.cm-2.h-1;1%Azone与15%PG合用促透效果最好,平均渗透速率达到4.889μg.cm-2.h-1,且与单用1%Azone有显著性差异(P<0.05)。结论:1%Azone与15%PG合用时,含0.5%Pd-Ia溶液体外渗透具有最大促透效果,体现出协同作用。     

Influence of different penetration enhancers on in vitro skin permeation and in vivo photoprotective effect of flavonoids

In the present study the permeation of two flavonoids (naringenin and hesperetin) through excised human skin mounted in Franz diffusion cells and its possible optimization following skin pretreatment with two penetration enhancers (d-limonene and lecithin) were determined. Hesperetin and naringenin were able to permeate through excised human skin; moreover, skin pretreatment with d-limonene and lecithin increased, to different degrees, their cutaneous permeation. On the basis of findings obtained in these in vitro experiments, we designed a schedule for a series of in vivo experiments, in which the protective effect of topically applied naringenin and hesperetin against UV-B-induced skin damage was assessed monitoring the extent of erythema in human volunteers by means of reflectance spectrophotometry. Hesperetin and naringenin from formulations containing the flavonoid alone were completely ineffective in decreasing UV-B-induced erythema. Furthermore, both d-limonene and lecithin have enhanced, to a significant extent, the photoprotective activity of naringenin and hesperetin. Taken together, these data demonstrate that hesperetin and naringenin may be successfully employed as topical photoprotective agents. However their topical activity needs to be optimized by using suitable penetration enhancers.     

Potential pitfalls in skin permeation experiments: Influence of experimental factors and subsequent data evaluation

There is a growing demand for skin penetration and permeation data considering toxicological and potential drug delivery aspects for an increasing number of substances. Although there are official guidelines available, results from different skin diffusion studies are often inconsistent and sometimes even controversial. The aim of our study is to address and to investigate the influence of experimental parameters as well as mathematical problems for subsequent evaluation of the permeation raw data. To create a reliable database diffusion experiments across human stratum corneum were performed under highly standardized conditions. The experimental data were evaluated using linear and non-linear regression analysis to determine the influence on the permeability coefficient and the lag-time. Additionally, the influence of two critical experimental parameters, temperature and unstirred water layers, on the permeability was investigated in silico. Based on our results we suggest that the influence of temperature on the permeability coefficient is small compared to the effect of other experimental parameters. Thickness of unstirred water layers has a tremendous effect on the permeation and may lead to underestimation of the permeability by more than 90%. Non-linear regression analysis seems to be superior compared to linear algorithms hence is advisable for evaluation of the experimental data. Our findings may help to optimize the experimental set-up and to reduce total costs for future skin diffusion testing. With regards to EU’s REACH-Initiative this will also help to create more and reliable data on safety issues of industrial materials.     

Effect of chemical enhancers and iontophoresis on thiocolchicoside permeation across rabbit and human skin in vitro

The aim of this work was to study the permeation of thiocolchicoside across the skin in vitro. The effect of the chemical enhancer lauric acid and the physical technique of iontophoresis was investigated. Permeation experiments were performed in vitro using rabbit ear skin as barrier. The effect of lauric acid at different concentrations (2% and 4%) and of the vehicle (water, ethanol, or ethanol/water) was investigated. The primary effect of lauric acid was on the partitioning parameter, whereas the diffusive parameter did not change significantly. When human epidermis was used, the permeation parameters were generally lower, although not significantly different from rabbit ear skin. The data obtained with full-thickness human skin indicate that, despite the hydrophilic nature of thiocolchicoside, the resistance to drug transport is not limited to the stratum corneum, but that the underlying dermal tissue can also contribute. Iontophoresis enhanced the flux of thiocolchicoside compared with the passive control. The mechanism by which iontophoresis enhanced thiocolchicoside transport across the skin was electroosmosis. The permeation of thiocolchicoside across the skin can be enhanced using chemical or physical penetration enhancers.     

Transdermal permeation of WIN 55,212-2 and CP 55,940 in human skin in vitro

Synthetic cannabinoids have a promising future as treatments for nausea, appetite modulation, pain, and many neurological disorders. Transdermal delivery is a convenient and desirable dosage form for these drugs and health conditions. The aim of the present study was to investigate the in vitro transdermal permeation of two synthetic cannabinoids, WIN 55,212-2 and CP 55,940. Transdermal flux, drug content in the skin, and lag times were measured in split-thickness human abdominal skin in flow-through diffusion cells with receiver solutions of 4% bovine serum albumin (BSA) or 0.5% Brij 98. Differential thermal analysis (DSC) was performed in order to determine heats of fusion, melting points, and relative thermodynamic activities. The in vitro diffusion studies in 0.5% Brij 98 indicated that WIN 55,212-2 diffuses across human skin faster than CP 55,940. The WIN 55,212-2 skin disposition concentration levels were also significantly higher than that of CP 55,940. Correspondingly, CP 55,940 was significantly metabolized in the skin. WIN 55,212-2 flux and skin disposition were significantly lower into 4% BSA than into 0.5% Brij 98 receiver solutions. There was no significant difference in the flux, lag time, and drug content in the skin of CP 55,940 in 4% BSA versus 0.5% Brij 98 receiver solutions. The DSC studies showed that CP 55,940 had a significantly lower melting point, smaller heat of fusion, and corresponding higher calculated thermodynamic activity than the more crystalline WIN 55,212-2 mesylate salt. The permeation results indicated that WIN 55,212-2 mesylate, CP 55,940, and other potent synthetic cannabinoids with these physicochemical properties could be ideal candidates for the development of a transdermal therapeutic system.     

In vitro permeation of levothyroxine across the skin

The aim of this work was to investigate the in vitro transdermal permeation characteristics of sodium levothyroxine, in view of its topical application. Permeation experiments were performed in vitro, using rabbit ear skin as barrier. At the end of the experiments levothyroxine retained in the skin was extracted and quantified by HPLC. The formulations tested were solutions and a commercial cream. The use of dimethyl beta-cyclodextrin as solubilizing agent increased to a significant extent levothyroxine solubility, but reduced its skin accumulation. Skin stripping before drug application produced a considerable increase in the amount retained and levothyroxine was found also in the receptor compartment. The application of the commercial cream in occlusive conditions increased to a significant extent drug retention in the skin. In conclusion, levothyroxine skin administration is promising in view of a localized effect, because it was retained in the skin. On the contrary, transdermal administration in view of systemic effect does not represent a concrete possibility.     

6种促进剂对西替利嗪体外经皮渗透的影响

目的 :研究 6种不同的促进剂对西替利嗪体外经皮渗透的促进作用。方法 :用Valia Chien水平扩散池 ,选择了丙二醇、月桂氮芯卓 酮 (azone)、柠烯 (dipentene)、水杨酸甲酯、含 10 %薄荷脑的丙二醇、含 10 %樟脑的丙二醇作为促进剂 ,采用离体SD大鼠腹部皮肤用促渗剂预处理的方式 ,建立以去氯羟嗪为内标的反相离子对高效液相色谱法 ,测定接收液中西替利嗪的含量。结果 :除丙二醇和水杨酸外其余几种促进剂对西替利嗪体外经皮渗透都有显著的促进作用 (P <0 .0 1)。以含 10 %薄荷脑的丙二醇的促渗效果最好。结论 :月桂氮芯卓 酮、柠烯、薄荷脑、樟脑可以作为促渗剂用于西替利嗪经皮吸收制剂     

促渗剂对氟比洛芬体外经皮渗透的影响

目的研究不同的促渗剂对氟比洛芬体外经皮渗透的促渗作用。方法采用TK-6A型透皮扩散仪,用人皮进行体外经皮渗透实验,考察不同的促渗剂[二甲基亚砜、月桂醇、丙二醇、月桂氮酮(氮酮)、尿素、油酸]及其组合对氟比洛芬体外透皮吸收的促渗作用,以HPLC法测定各时间点接受室中药物浓度,求算透皮吸收的有关参数,比较各促渗剂的促渗作用。结果15%二甲基亚砜、3%氮酮、1%尿素可使氟比洛芬经皮渗透速率分别提高1.8,1.5,1.1倍,促渗剂联用取得的促渗效果更佳,5%油酸 20%丙二醇 1%尿素可使该药物的经皮渗透速率提高6倍。结论单用促渗剂对氟比洛芬经皮渗透促渗效果有限,促渗剂联合使用可以显著提高氟比洛芬经皮渗透速率。     

Reversible effects of permeation enhancers on human skin.

This study outlines a systematic approach for investigating a desired characteristic of chemicals used to facilitate the permeation of drugs across the skin that is, the reversibility of the permeation enhancement effect. This implies that the vital skin barrier function is restored and not permanently impaired after the application of these enhancers. The reversible effects of two terpene enhancers, (R)-(-)-carvone and eucarvone, on excised human skin were evaluated by in vitro permeation and extraction studies on normal (untreated) and enhancer-pretreated epidermis, respectively. For the permeation studies on normal epidermis, the donor solutions were the model drug, haloperidol (HP, 3mg/ml), in propylene glycol (PG) with or without 5% (w/v) enhancer and for the extraction studies using epidermis pretreated with enhancer, a solution of HP (3mg/ml) in PG was used. The solubilities of the enhancers in 0.03% lactic acid (receptor solution) and of HP in PG (donor solution) were determined to demonstrate that the sink and saturated conditions were maintained in the respective compartments of the flow-through cells throughout the in vitro experiments. (R)-(-)-Carvone cleared out of the skin faster than eucarvone. This could be due to the 4-fold higher skin permeability of (R)-(-)-carvone compared to that of eucarvone. The amount of HP deposited in the epidermis was much lower in the eucarvone-pretreated epidermis than that pretreated with (R)-(-)-carvone. The permeation profile of HP across the enhancer-pretreated skin was 4-fold greater than in the vehicle alone (control), but similar to that across untreated skin with enhancer present in the donor solution, indicating that permeation across the enhancer-pretreated skin did not change. The enhancing effects of both terpenes on the skin were found to be reversible and the permeability of the skin was left intact after the passage of the drug in the vehicle with these enhancers.     

Bisphenol A affects human endometrial endothelial cell angiogenic activity in vitro

The widespread Bisphenol A (BPA) is classified as an endocrine-disrupting chemical (EDC) with estrogenic properties. Human endometrial endothelial cells (HEECs) play a key role in the endometrial angiogenesis that is under the control of estradiol. The hypothesis was that BPA may affect endometrial angiogenesis by disturbing some functional properties of the HEEC.To study this, primary HEECs were exposed to environmentally relevant doses of BPA. The HEECs were co-cultured with primary endometrial stromal cells to create conditions as similar to the in vivo situation as possible. The effects of BPA were evaluated by proliferation and viability assays, tube-formation assays, quantitative PCRs, Western blots and ELISAs.BPA slightly increased HEEC tube formation and VEGF-D protein expression compared with vehicle, without affecting HEEC viability or proliferation.Bisphenol A thus caused changes in HEEC activities in vitro, and may therefore have disturbing effects on endometrial angiogenesis.     

Enhancing effects of ion-pair complexes on skin permeation of cromolyn in vitro

Lipophilic ion-pair complexes of 3-dl-alpha-tocopherylcarbonyl-1-n-alkyl-pyridinium-cromolyn (TAP-CG) were designed to enhance the percutaneous absorption of cromolyn (CG), and the effect of n-alkyl chainlength of the ion-pair complexes on the CG permeation through hairless mouse skin was evaluated in vitro. The permeation rates of CG were examined in isopropyl myristate (IPM) suspension using static Keshany-Chien type diffusion cells at 32 degrees C. The permeation parameters, steady-state flux, diffusion coefficient, partition coefficient between skin and IPM, and permeability coefficient were determined. Steady-state fluxes of CG increased linearly with the increasing n-alkyl chain-length of TAP-CG, and 3-dl-alpha-tocopherylcarbonyl-1-n-hexyl-pyridinium-cromolyn (THP-CG) produced the highest CG flux (0.62 +/- 0.11 nmol.cm-2.h-1), which was 14-fold greater than that of CG.Na in IPM suspension and more than 480-fold greater than that of CG.Na in aqueous solution due to increasing lipophilicity. In the case of TAP-CG with longer n-alkyl chainlength than THP-CG, however, the steady-state fluxes of CG decreased due to the high molecular weight and/or the high lipophilicity of the ion-pair complexes. It is suggested that lipophilic ion-pair complexes, especially THP-CG, are effective in absorption of cromoglicate through the skin. The results would be useful for studies on the role of each counterion in the lipophilic ion-pair complexes.     

薄荷脑促进氯霉素经皮渗透作用研究

本文对薄荷脑促进氯霉素的经皮渗透作用作了初步观察，２％薄荷脑在氯霉素醇溶液或在４：３：３溶液中，对１％氯霉素均有显著的促进经皮渗透作用，该作用在４ｈ即有显著性差异，随时间增加透皮量也进一步增加。研究表明２％月桂氮酮（氮酮）在上述两种溶剂中也有明显的促进氯霉素的经皮渗透作用，且随时间增加而增加。１％月桂氮酮和ｌ％薄荷脑同时应用，比两者在２％浓度单独应用时的助渗作用更强。     

氢定乳膏的制备及大鼠体外经皮渗透性研究

目的制备氢定乳膏并考察其在离体大鼠皮肤中的渗透行为。方法以乳剂型基质制备氢定乳膏,采用改良Franz扩散池进行体外经皮渗透实验,采用HPLC法测定药物在接受介质中的含量。结果氢定乳膏中氢化可的松和醋酸氯己定的经皮渗透系数(Ps)和滞后时间(Tlag)分别为1.4μg.cm-.2h-1、0.5 h;3.0μg.cm-.2h-1、1.0 h。结论氢定乳膏对离体大鼠皮肤的渗透行为符合零级动力学过程,其透过皮肤的药量少。     

Piperazinylalkyl prodrugs of naproxen improve in vitro skin permeation.

Novel morpholinyl (4a) and piperazinylalkyl (4b-e) esters were synthesized and evaluated in vitro for their properties as bioreversible topically administered dermal prodrugs of naproxen. These ionizable prodrugs exhibited various aqueous solubilities and lipophilicities, depending on the pH of medium. As indicated by octanol-buffer partition coefficients (logP(app)) at pH 7.4, all of the prodrugs were significantly more lipophilic (logP(app)=0.7-3.9) than naproxen (logP(app)=0.3). Furthermore, the most aqueous of the soluble prodrugs (4b-d) were only 2-3-fold less soluble in an aqueous buffer of pH 7.4 ( approximately 30-50 mM) than was naproxen ( approximately 100 mM). At a pH of 5.0, prodrugs showed a generally higher aqueous solubility and similar logP(app) values, compared to naproxen. The chemical and enzymatic hydrolysis of prodrugs at 37 degrees C was investigated in aqueous buffer solutions (pH 5.0 and 7.4) and in 80% human serum (pH 7.4), respectively. The prodrugs showed moderate chemical stability (t(1/2)=15-150 days at pH 5.0), and they were hydrolyzed enzymatically to naproxen, with half-lives ranging from 0.4 to 77 min. In permeation studies using post-mortem human skin in vitro, the flux of naproxen was 6.5 and 1.6 nmol/cm(2). h in a saturated aqueous buffer vehicle of pH 7.4 and 5.0, respectively. Among the prodrugs, two piperazinyl derivatives (4c and 4d) resulted in a 9- and 4-fold enhancement of permeation, respectively, when compared to naproxen itself at pH 7.4. 4c also resulted in a significantly (4-fold) better permeation than naproxen at pH 5.0. In conclusion, piperazinyl esters improved skin permeation of naproxen and are promising prodrugs of naproxen for topical drug delivery.     

Heparin penetration into and permeation through human skin from aqueous and liposomal formulations in vitro

The transport of unfractionated (UH) and low molecular weight Heparin (LMWH) in human skin was investigated in vitro using heat separated epidermal membrane and dermis and the effect of liposomal formulations with Phospholipon(R) 80 (PL80) and Sphingomyelin (SM) was assessed. The distribution of Heparin within skin tissue was studied by the tape stripping method. Heparin concentrations were measured with a biological assay. Transepidermal water loss was determined to characterize barrier properties of skin. No consistent permeation of Heparin through epidermal membrane was detected. Penetration into the epidermal membrane was for LMWH significantly greater than for UH. Accumulation of UH was largely restricted to the outermost layers of the stratum corneum while LMWH penetrated into deeper epidermal layers. UH penetration into epidermis was detected for the PL80 liposomal formulation only. The extent of LMWH penetration was independent of the formulation, LMWH, however, showed a trend to accumulate in deeper epidermal layers for the PL80 compared to the aqueous formulation. Thus, molecular weight and liposomal formulations influenced the penetration pattern of Heparin in the epidermis. It can not be concluded whether the concentration of LMWH achieved at the blood capillaries is sufficient to exert a pharmacological effect. UH permeated readily through dermis irrespectively of formulation and its accumulation in the dermis was significantly enhanced and its lag time of permeation increased in the presence of SM liposomes.