Sensitivities of gel entrapped hepatocytes in hollow fibers to hepatotoxic drug

The aim of this study was to determine the feasibility of detecting hepatotoxicity using gel entrapped hepatocytes in simple hollow fibers. Four typical hepatotoxic drugs were tested for hepatotoxicity in gel entrapped hepatocyte as opposed to hepatocyte monolayer, a hepatocyte system extensively used for hepatotoxicity studies in vitro. Hepatotoxicity or cell damage was assessed by the methyl tetrazolium (MTT) assay, liver-specific functions and the intracellular glutathione (GSH) content. After exposure to acetaminophen, significant cell damage of gel entrapped hepatocytes was detected at 48 h while hepatocyte monolayer was not so sensitive except for albumin synthesis and this difference between two hepatocyte systems was similar on hepatotoxic response to antituberculosis drugs including rifampicin and isoniazid. At low concentrations of either rifampicin or isoniazid, time-dependent hepatotoxicity was only evidenced in gel entrapped hepatocytes after treatment and no cell damage occurred in hepatocyte monolayer at an incubation time as long as 96 h. Interestingly, hepatotoxicities of acetaminophen, isoniazid and rifampicin are all reportedly relevant to drug metabolisms of cytochrome P450. For sodium salicylate whose hepatotoxicity is unassociated to P450 activities, more significant reductions on cell viability and albumin synthesis at 5 mM than those at 1 mM apparently illustrated the concentration-dependent hepatotoxicities of gel entrapped hepatocytes as well as hepatocyte monolayer. It is highly suggested that gel entrapped hepatocyte are more sensitive in evaluation of hepatotoxicities than hepatocyte monolayer if this hepatotoxicity is related to drug metabolism. Thus, gel entrapment culture of hepatocytes with simple hollow fibers could be recommended for hepatotoxicity studies in vitro.     

Isoniazid-induced hepatotoxicity in rat hepatocytes of gel entrapment culture

Gel entrapment culture of rat hepatocytes in hollow fibers were evaluated as a potential in vitro model for studies on isoniazid-induced hepatotoxicity. After exposure to isoniazid (0.11 mM and 1.1 mM) for 24-96 h, gel entrapped hepatocytes were more severely damaged than hepatocyte monolayers according to the assays on methyl thiazolyl tetrazolium (MTT) reduction, intracellular glutathione (GSH) content, reactive oxygen species (ROS) levels, and albumin secretion. Furthermore, CYP 2E1 activity detected by 4-nitrocatechol (4-NC) formation maintained at least 7 days in gel entrapped hepatocytes but decreased to an undetectable level within 2 days in hepatocyte monolayer. And the addition of CYP 2E1 inhibitor, diethyl-dithiocarbamate (DDC), significantly reduced isoniazid-induced GSH depletion in gel entrapped hepatocytes. In addition, the protective effects of N-acetylcysteine (NAC), GSH, liquorice extract and glycyrrhizic acid (GA), a purified compound from liquorice extract, against isoniazid hepatotoxicity were clearly observed in gel entrapped hepatocytes at 72 h incubation. Overall, gel entrapped hepatocytes were more susceptible to isoniazid-induced hepatotoxicity than hepatocyte monolayers by a possible mechanism that higher CYP 2E1 activity in gel entrapped hepatocytes could enhance isoniazid toxicity. This indicates that gel entrapped hepatocytes in hollow fibers could be a more effective model than hepatocyte monolayer for hepatotoxicity research in vitro.     

Species-specific toxicity of troglitazone on rats and human by gel entrapped hepatocytes

Troglitazone, despite passing preclinical trials on animals, was shortly withdrawn from market due to its severe hepatotoxicity in clinic. As rat hepatocyte monolayer consistently showed sensitive troglitazone toxicity as human hepatocyte monolayer in contrast to the species-specific toxicity in vivo, this paper utilized both hepatocytes in three-dimensional culture of gel entrapment to reflect the species difference on hepatotoxicity. Rat hepatocytes in gel entrapment did not show obvious cellular damage even under a long-term exposure for 21 days while gel entrapped human hepatocytes significantly displayed oxidative stress, steatosis, mitochondrial damage and cell death at a short exposure for 4 days. As a result, the detected species-specific toxicity of troglitazone between gel entrapped rat and human hepatocytes consisted well with the situation in vivo but was in a sharp contrast to the performance of two hepatocytes by monolayer culture. Such contradictory toxicity of rat hepatocytes between monolayer and gel entrapment culture could be explained by the fact that troglitazone was cleared more rapidly in gel entrapment than in monolayer culture. Similarly, the differential clearance of troglitazone in rat and human might also explain its species-specific toxicity. Therefore, gel entrapment of hepatocytes might serve as a platform for evaluation of drug toxicity at early stage of drug development by reducing costs, increasing the likelihood of clinical success and limiting human exposure to unsafe drugs.     

Gel entrapment culture of rat hepatocytes for investigation of tetracycline-induced toxicity

This paper aimed to explore three-dimensionally cultured hepatocytes for testing drug-induced nonalcoholic steatohepatitis. Gel entrapped rat hepatocytes were applied for investigation of the tetracycline-induced steatohepatitis, while hepatocyte monolayer was set as a control. The toxic responses of hepatocytes were systematically evaluated by measuring cell viability, liver-specific function, lipid accumulation, oxidative stress, adenosine triphosphate content and mitochondrial membrane potential. The results suggested that gel entrapped hepatocytes showed cell death after 96 h of tetracycline treatment at 25 μM which is equivalent to toxic serum concentration in rats, while hepatocyte monolayer showed cell death at a high dose of 200 μM. The concentration-dependent accumulation of lipid as well as mitochondrial damage were regarded as two early events for tetracycline hepatotoxicity in gel entrapment culture due to their detectability ahead of subsequent increase of oxidative stress and a final cell death. Furthermore, the potent protection of fenofibrate and fructose-1,6-diphosphate were evidenced in only gel entrapment culture with higher expressions on the genes related to β-oxidation than hepatocyte monolayer, suggesting the mediation of lipid metabolism and mitochondrial damage in tetracycline toxicity. Overall, gel entrapped hepatocytes in three-dimension reflected more of the tetracycline toxicity in vivo than hepatocyte monolayer and thus was suggested as a more relevant system for evaluating steatogenic drugs.     

Prediction of cytochrome 450 mediated drug-drug interactions by three-dimensional cultured hepatocytes

Cytochrome P450 (CYP) inhibition and induction are the key mechanisms in drug-drug interactions which should be avoided in clinic for the uncertain influence on the efficacy and safety of drug co-administration. The CYPmediated drug-drug interactions urgently need to be predicted by in vitro models before animal and clinical trials, while the primary hepatocytes may represent the most appropriate experimental system by now. However, traditional twodimensional (2D) culture of hepatocyte monolayer, regardless of the good facilitation and repeatability, rapidly loses its CYP-inducibility during short-term culture due to the deviated microenvironments from in vivo. Also, 2D culture did not reproduce the CYP-inhibition mediated hepatotoxicity in drug co-administration. Recently, three-dimensional (3D) cultured hepatocytes have been recognized as the promising models for predicting in vivo drug-drug interactions. The 3D cultures such as sandwich and gel entrapped hepatocytes showed the comparable response to CYP inhibitors and inducers as in vivo and well reflected the inhibitor/inducer mediated hepatotoxicity. In this regard, this review, for the first time, compares the effect of 2D and 3D hepatocyte cultures on reflecting CYP-mediated drug-drug interactions in vivo. And the advantage of 3D cultured hepatocytes on predicting in vivo CYP-induction/inhibition will be particularly discussed.     

Re-evaluation of tacrine hepatotoxicity using gel entrapped hepatocytes

Controversial results about the involvement of CYP 1A2 and oxidative stress in tacrine-induced hepatotoxicity have been described by the different research groups. We suggested that different expression levels of CYP 1A2 in cell lines and primary hepatocytes in vitro may be the cause of the controversial results above. Therefore, this paper re-evaluated the toxicity of tacrine by using gel entrapment culture of rat hepatocytes. The toxic effect of tacrine on hepatocytes was assayed by the reduction of methyl thiazolyl tetrazolium (MTT) and intracellular glutathione (GSH), as well as by albumin synthesis. It was found that the detectable hepatotoxic dose of tacrine is much lower in hepatocytes entrapped in gel than in those in monolayer cultures. The fact that fluvoxamine, a potent cytochrome P450 (CYP) 1A2 inhibitor, reduced tacrine toxicity and the expression of the CYP 1A2 gene was maintained in gel entrapped hepatocytes, but not in hepatocyte monolayers, could illustrate a close association between CYP 1A2 expression levels and tacrine toxicity. Glycyrrhetinic acid (GA), a free radical scavenger, protected gel entrapped hepatocytes from tacrine toxicity, but was ineffective in hepatocyte monolayers. Hence, gel entrapped hepatocytes could reflect higher tacrine hepatotoxicity in vivo than hepatocyte monolayers.     

体外三维肝细胞模型在药物肝毒性评价中的应用

肝毒性是药物治疗过程中常见的副作用,也是导致新药临床试验失败以及药物撤市的主要原因。建立可以有效识别药物肝毒性的体外模型对于新药研发至关重要。传统的二维（2D）模型在培养过程中会逐渐丧失肝脏特异性功能,无法准确评价药物肝毒性。新兴的三维（3D）培养技术可模拟体内细胞微环境,有利于肝细胞的体外组装,从而使3D肝细胞模型表现出与体内肝细胞相似的表型以及药物毒性反应,在肝毒性评价方面具有很大潜力。     

Synergistic drug-cytokine induction of hepatocellular death as an in vitro approach for the study of inflammation-associated idiosyncratic drug hepatotoxicity

Idiosyncratic drug hepatotoxicity represents a major problem in drug development due to inadequacy of current preclinical screening assays, but recently established rodent models utilizing bacterial LPS co-administration to induce an inflammatory background have successfully reproduced idiosyncratic hepatotoxicity signatures for certain drugs. However, the low-throughput nature of these models renders them problematic for employment as preclinical screening assays. Here, we present an analogous, but high-throughput, in vitro approach in which drugs are administered to a variety of cell types (primary human and rat hepatocytes and the human HepG2 cell line) across a landscape of inflammatory contexts containing LPS and cytokines TNF, IFNγ, IL-1α, and IL-6. Using this assay, we observed drug-cytokine hepatotoxicity synergies for multiple idiosyncratic hepatotoxicants (ranitidine, trovafloxacin, nefazodone, nimesulide, clarithromycin, and telithromycin) but not for their corresponding non-toxic control compounds (famotidine, levofloxacin, buspirone, and aspirin). A larger compendium of drug-cytokine mix hepatotoxicity data demonstrated that hepatotoxicity synergies were largely potentiated by TNF, IL-1α, and LPS within the context of multi-cytokine mixes. Then, we screened 90 drugs for cytokine synergy in human hepatocytes and found that a significantly larger fraction of the idiosyncratic hepatotoxicants (19%) synergized with a single cytokine mix than did the non-hepatotoxic drugs (3%). Finally, we used an information theoretic approach to ascertain especially informative subsets of cytokine treatments for most highly effective construction of regression models for drug- and cytokine mix-induced hepatotoxicities across these cell systems. Our results suggest that this drug-cytokine co-treatment approach could provide a useful preclinical tool for investigating inflammation-associated idiosyncratic drug hepatotoxicity.     

Establishment of a methodology for investigating protectants against ethanol-induced hepatotoxicity

Ethanol-induced liver injury has been extensively reported in clinic, but still lacks an efficient in vitro platform for investigating its hepatotoxicity and protectants. This study aimed to establish a methodology on the culture conditions regarding the sealability against evaporation of ethanol, culture medium and 2D/3D culture of hepatocytes. Based on the experimental findings, it was indicated that the ethanol evaporation from culture plates was a severe problem reducing its toxicity in hepatocyte. According to the detected ethanol toxic response marked by reduced cell viability, 3D cultured hepatocytes in gel entrapment were suggested to be better than 2D hepatocyte in monolayer, but the cultures in either William’s Medium E or DMEM exhibited comparable sensitivity to ethanol toxicity. Subsequently, 3D cultured hepatocytes with Parafilm sealing were systematically illustrated to well reflect the ethanol-induced lipid accumulation, reactive oxygen species/malondialdehyde generation, glutathione depletion and cytochrome 2E1 induction. Finally, such hepatocyte models were proposed as a platform for screening of herbal component against ethanol hepatotoxicity. Nano-silibinin, for the first time, found to perform significant protection against ethanol-induced hepatotoxicity while silibinin in normal particles could not inhibit such toxicity. This protection of nano-silibinin might relate to its improved bioavailability compared to normal insoluble silibinin and could act as an anti-oxidative and anti-steatosis agent against ethanol-induced hepatotoxicity.     

Chemotherapeutic efficiency of drugs in vitro: Comparison of doxorubicin exposure in 3D and 2D culture matrices

The interest in the use of 3D matrices for in vitro analysis, with a view to increasing the relevance of in vitro studies and reducing the dependence on in vivo studies, has been growing in recent years. Cells grown in a 3D in vitro matrix environment have been reported to exhibit significantly different properties to those in a conventional 2D culture environment. However, comparison of 2D and 3D cell culture models have recently been noted to result in differing responses of cytotoxic assays, without any associated change in viability. The effect was attributed to differing conversion rates and effective concentrations of the resazurin assay in 2D and 3D environments, rather than differences in cellular metabolism. In this study, the efficacy of a chemotherapeutic agent, doxorubicin, is monitored and compared in conventional 2D and 3D collagen gel exposures of immortalized human cervical cells. Viability was monitored with the aid of the Alamar Blue assay and drug internalisation was verified using confocal microscopy. Drug uptake and retention within the collagen matrix was monitored by absorption spectroscopy. The viability studies showed apparent differences between the 2D and 3D culture systems, the differences attributed in part to the physical transition from 2D to a 3D environment causing alterations to dye resazurin uptake and conversion rates. The use of 3D culture matrices has widely been interpreted to result in “reduced” toxicity or cellular “resistance” to the chemotherapeutic agent. The results of this study show that the reduced efficiency of the drug to cells grown in the 3D environment can be accounted for by a sequential reduction of the effective concentration of the test compound and assay. This is due to absorption within the collagen gel inducing a higher uptake of both drug and assay thereby influencing the toxic impact of the drug and conversion rate of resazurin, and. The increased effective surface area of the cell exposed to the drug and assay in the 3D environment. The effect was noted to be higher after shorter exposure periods and should be accounted for in in vitro 2D and 3D culture environment comparisons.     

改性中空纤维-离心超滤-HPLC法测定吲哚美辛凝胶含量

目的：研究中空纤维的改性处理方法以消除超滤膜对吲哚美辛的吸附问题，建立准确、简单的样品前处理方法以去除凝胶剂中的高分子辅料，进而对主药含量进行测定。方法：采用3种表面活性剂对PVDF-HF进行改性处理后，取1 g凝胶样品经25 mL甲醇溶解后，转移至表面活性剂改性处理的中空纤维离心装置（HFCF-UF）中，4 000 r·min^-1离心10 min，腔内滤液经稀释后直接进HPLC分析。结果：采用2%吐温-80处理的聚偏氟乙烯中空纤维膜对样品进行超滤处理后，对吲哚美辛消除了膜对药物的非特异性吸附（NSB）且有效去除了高分子凝胶辅料的干扰。吲哚美辛在5.05~30.3 μg·mL^-1范围内线性关系良好，平均回收率为99.6%，RSD小于1.5%。结论：所建立的方法简便、快速，定量准确，可用于吲哚美辛凝胶的含量测定。     

微型中空纤维生物反应器肝细胞培养技术及其在药物肝毒性研究中的应用

传统的细胞培养方法中,肝细胞在体外不能实现高活性高密度的生长,因而无法模拟体内的三维立体生长环境,从而制约了药物肝毒性的体外评价;利用微型中空纤维凝胶包埋技术可以在体外固定肝细胞而模拟体内的立体环境。本文综述了微型中空纤维凝胶包埋技术的发展及其在体外药物肝毒性方面的应用。     

Rifampicin exacerbates isoniazid-induced toxicity in human but not in rat hepatocytes in tissue-like cultures

BACKGROUND AND PURPOSE: Rifampicin has been extensively reported to exacerbate the hepatotoxicity of isoniazid in patients with tuberculosis. However, this was controversially claimed by previous reports using rat models. This study evaluated the effect of rifampicin on isoniazid-induced hepatocyte toxicity by using human and rat hepatocytes in tissue-like culture.EXPERIMENTAL APPROACH: Hepatocytes in tissue-like gel entrapment were used to examine isoniazid toxicity, as shown by cell viability, intracellular glutathione content and albumin secretion. For demonstration of the differential effects of rifampicin on human and rat hepatocytes, induction by rifampicin of cytochrome P450 (CYP) 2E1, a major enzyme associated with isoniazid hepatotoxicity, was detected by 4-nitrocatechol formation and RT-PCR analysis.KEY RESULTS: Rifampicin (12 microM) enhanced isoniazid-induced toxicity in human hepatocytes but not in rat hepatocytes. Enhanced CYP 2E1 enzymic activity and mRNA expression were similarly detected in human hepatocytes but not in rat hepatocytes. Both rat and human hepatocytes in gel entrapment were more sensitive to isoniazid treatment compared with the corresponding hepatocytes in a monolayer culture.CONCLUSIONS AND IMPLICATIONS: The difference in induction of CYP 2E1 by rifampicin between rat and human hepatocytes accounted for the difference in exacerbation of isoniazid hepatocyte toxicity by rifampicin, with more significant toxicity in gel entrapment than in monolayer cultures. Thus, human hepatocytes in tissue-like cultures (gel entrapment) could be an effective model for hepatotoxicity research in vitro, closer to the in vivo situation.     

Optimization of the HepaRG cell model for drug metabolism and toxicity studies

The HepaRG cell line is the first human cell line able to differentiate in vitro into mature hepatocyte-like cells. Our main objective within the framework of the EEC-LIINTOP project was to optimize the use of this cell line for drug metabolism and toxicity studies, especially after repeat treatments. The main results showed that differentiated HepaRG cells: (i) retained their drug metabolism capacity (major CYPs, phase 2 enzymes, transporters and nuclear receptors) and responsiveness to prototypical inducers at relatively stable levels for several weeks at confluence. The levels of several functions, including some CYPs such as CYP3A4, were dependent on the addition of dimethyl sulfoxide in the culture medium; (ii) sustained the different types of chemical-induced hepatotoxicity, including steatosis, phospholipidosis and cholestasis, after acute and/or repeat treatment with reference drugs. In particular, drug-induced vesicular steatosis was demonstrated in vitro for the first time. In conclusion, our results from the LIINTOP project, together with other studies reported concomitantly or more recently in the literature, support the conclusion that the metabolically competent human HepaRG cells represent a surrogate to primary human hepatocytes for investigating drug metabolism parameters and both acute and chronic effects of xenobiotics in human liver.     

Human hepatocytes as a tool for studying toxicity and drug metabolism

Drugs are usually biotransformed into new chemical species that may have either toxic or therapeutic effects. Drug metabolism studies are routinely performed in laboratory animals but, due to metabolic interspecies differences when compared to man, they are not accurate enough to anticipate the metabolic profile of a drug in humans. Human hepatocytes in primary culture provide the closest in vitro model to human liver and the only model that can produce a metabolic profile of a given drug that is very similar to that found in vivo. However their availability is limited due to the restricted access to suitable tissue samples. The scarcity of human liver has led to optimising the cryopreservation of adult hepatocytes for long-term storage and regular supply. Human hepatocytes in primary culture express typical hepatic functions and express drug metabolising enzymes. Moreover, qualitative and quantitative similarities between in vitro and in vivo metabolism of drugs were observed. Different strategies have been envisaged to prolong cell survival and delay the spontaneous decay of the differentiated phenotype during culture. Thus, hepatocytes represent the most appropriate model for the evaluation of integrated drug metabolism, toxicity/metabolism correlations, mechanisms of hepatotoxicity, and the interactions (inhibition and induction) of xenobiotics and drug-metabolising enzymes. However, in view of limitations of primary hepatocytes, efforts are made to develop alternative cellular models (i.e. metabolic competent CYP-engineered cells stably expressing individual CYPs and transient expression of CYPs by transduction of hepatoma cells with recombinant adenoviruses). In summary, several cellular tools are available to address key issues at the earliest stages of drug development for a better candidate selection and hepatotoxicity risk assessment.     

Rat primary hepatocytes show enhanced performance and sensitivity to acetaminophen during three-dimensional culture on a polystyrene scaffold designed for routine use

The in vitro evaluation of hepatotoxicity is an essential stage in the research and development of new pharmaceuticals as the liver is one of the most commonly impacted organs during preclinical toxicity studies. Fresh primary hepatocytes in monolayer culture are the most commonly used in vitro model of the liver but often exhibit limited viability and/or reduction or loss of important liver-specific functions. These limitations could potentially be overcome using three-dimensional (3D) culture systems, but their experimental nature and limited use in liver toxicity screening and drug metabolism has impaired their uptake into commercial screening programs. In this study we use a commercially available polystyrene scaffold developed for routine 3D cell culture to maintain primary rat hepatocytes for use in metabolism and toxicity studies over 72?h. We show that primary hepatocytes retain their natural cuboidal morphology with significantly higher viability (>74%) than cells grown in monolayer culture (maximum of 57%). Hepatocytes in the 3D scaffolds exhibit differential expression of genes associated with phase I, II, and III drug metabolism under basal conditions compared with monolayer culture and can be induced to stably express significantly higher levels of the cytochrome-P450 enzymes 1A2, 2B1, and 3A2 over 48?h. In toxicity studies the hepatocytes in the 3D scaffolds also show increased sensitivity to the model toxicant acetaminophen. These improvements over monolayer culture and the availability of this new easy to use 3D scaffold system could facilitate the uptake of 3D technologies into routine drug screening programs.     

In vitro and in vivo evaluation of cubosomes containing 5-fluorouracil for liver targeting

The objective of this study was to prepare cubosomal nanoparticles containing a hydrophilic anticancer drug 5-fluorouracil (5-FU) for liver targeting. Cubosomal dispersions were prepared by disrupting a cubic gel phase of monoolein and water in the presence of Poloxamer 407 as a stabilizer. Cubosomes loaded with 5-FU were characterized in vitro and in vivo. In vitro, 5-FU-loaded cubosomes entrapped 31.21% drug and revealed nanometer-sized particles with a narrow particle size distribution. In vitro 5-FU release from cubosomes exhibited a phase of rapid release of about half of the entrapped drug during the first hour, followed by a relatively slower drug release as compared to 5-FU solution. In vivo biodistribution experiments indicated that the cubosomal formulation significantly (P<0.05) increased 5-FU liver concentration, a value approximately 5-fold greater than that observed with a 5-FU solution. However, serum serological results and histopathological findings revealed greater hepatocellular damage in rats treated with cubosomal formulation. These results demonstrate the successful development of cubosomal nanoparticles containing 5-FU for liver targeting. However, further studies are required to evaluate hepatotoxicity and in vivo antitumor activity of lower doses of 5-FU cubosomal formulation in treatment of liver cancer.Key words: 5-Fluorouracil, Hydrophilic drug, Cubosomes, Liver targeting, Hepatotoxicity     

Characterization of primary mouse hepatocyte spheroids as a model system to support investigations of drug-induced liver injury

Primary mouse hepatocytes isolated from genetically defined and/or diverse lines and disease models are a valuable resource for studying the impact of genetic and environmental factors on drug response and disease. However, standard monolayer cultures result in a rapid decline in mouse hepatocyte viability and functionality. Therefore, we evaluated 3D spheroid methodology for long-term culture of primary mouse hepatocytes, initially to support investigations of drug-induced liver injury (DILI). Primary hepatocytes isolated from male and female C57BL/6J mice were used to generate spheroids by spontaneous self-aggregation in ultra-low attachment plates. Spheroids with well-defined perimeters were observed within 5 days after seeding and retained morphology, ATP, and albumin levels for an additional 2 weeks in culture. Global microarray profiling and quantitative targeted proteomics assessing 10 important drug metabolizing enzymes and transporters demonstrated maintenance of mRNA and protein levels in spheroids over time. Activities for 5 major P450 enzymes were also stable and comparable to activities previously reported for human hepatocyte spheroids. Time- and concentration-dependent decreases in ATP and albumin were observed in response to the DILI-causing drugs acetaminophen, fialuridine, AMG-009, and tolvaptan. Collectively, our results demonstrate successful long-term culture of mouse hepatocytes as spheroids and their utility to support investigations of DILI.     

Improved stability and release control of levodopa and metaraminol using ion-exchange fibers and transdermal iontophoresis

Achievement of controlled drug delivery and stability of drugs during storage is a problem also in transdermal drug delivery. The objective of this study was to determine, whether an easily oxidized drug, levodopa, could be stabilized during storage using pH-adjustment and ion-exchange fibers. Controlled transdermal delivery of the zwitterionic levodopa was attempted by iontophoresis and ion-exchange fiber. Ion-exchange kinetics and transdermal permeation of a cationic (presumably more stable) model drug, metaraminol, were compared to the corresponding data of levodopa. Levodopa was rapidly oxidized in the presence of water, especially at basic pH-values. At acidic pH-values the stability was improved significantly. Ion-exchange group and the pH had a clear effect on the release of both the levodopa and metaraminol from the ion-exchange fiber. The adsorption/release kinetics of metaraminol were more easily controllable than the corresponding rate and extent of levodopa adsorption/release. Iontophoretic enhancement of drug permeation across the skin was clearly more significant with the positively charged metaraminol than with the zwitterionic levodopa. Ion-exchange fibers provide a promising alternative to control drug delivery and to store drugs that are degraded easily.