Drug Transport Studies Research Articles

Transdermal Iontophoretic Delivery of Propofol: A General Anaesthetic in the Form of its Phosphate Salt

The main objective of this study was to investigate the feasibility of delivery of propofol phosphate (PP), a prodrug of propofol, via transdermal route using iontophoresis in combination with chemical permeation enhancers (CPEs). PP, a prodrug, was synthesized and its structure was characterized. In vitro passive and iontophoretic drug transport studies were carried out using Franz diffusion cell across freshly excised hairless rat skin at different concentrations of PP in combination with CPE. Among all the CPEs screened, 0.1% sodium dodecyl sulfate (SDS) increased the passive transdermal flux to 13.43 ± 0.73 μg/(cm(2) h) from 8.52 ± 0.82 μg/(cm(2) h) (control). Cathodal iontophoresis in combination with 0.1% SDS synergistically enhanced the flux [249.24 ± 6.12 μg/(cm(2) h)] of PP. The Pharmacokinetic studies were performed in rat model to assess the feasibility of transdermal delivery of PP. The amount of propofol present in plasma samples in control group (passive) was below the detectable levels at all the time points during the study. The plasma concentration-time profile of iontophoresis group of rats was fit to a noncompartmental model and the pharmacokinetic parameters were calculated. These studies suggest the plausibility of achieving therapeutically relevant levels of propofol when delivered via transdermal route by combining iontophoresis with CPE.

Alternative functional in vitro models of human intestinal epithelia

Physiologically relevant sources of absorptive intestinal epithelial cells are crucial for human drug transport studies. Human adenocarcinoma-derived intestinal cell lines, such as Caco-2, offer conveniences of easy culture maintenance and scalability, but do not fully recapitulate in vivo intestinal phenotypes. Additional sources of renewable physiologically relevant human intestinal cells would provide a much needed tool for drug discovery and intestinal physiology. We compared two alternative sources of human intestinal cells, commercially available primary human intestinal epithelial cells (hInEpCs) and induced pluripotent stem cell (iPSC)-derived intestinal cells to Caco-2, for use in in vitro transwell monolayer intestinal transport assays. To achieve this for iPSC-derived cells, intestinal organogenesis was adapted to transwell differentiation. Intestinal cells were assessed by marker expression through immunocytochemical and mRNA expression analyses, monolayer integrity through Transepithelial Electrical Resistance (TEER) measurements and molecule permeability, and functionality by taking advantage the well-characterized intestinal transport mechanisms. In most cases, marker expression for primary hInEpCs and iPSC-derived cells appeared to be as good as or better than Caco-2. Furthermore, transwell monolayers exhibited high TEER with low permeability. Primary hInEpCs showed molecule efflux indicative of P-glycoprotein (Pgp) transport. Primary hInEpCs and iPSC-derived cells also showed neonatal Fc receptor-dependent binding of immunoglobulin G variants. Primary hInEpCs and iPSC-derived intestinal cells exhibit expected marker expression and demonstrate basic functional monolayer formation, similar to or better than Caco-2. These cells could offer an alternative source of human intestinal cells for understanding normal intestinal epithelial physiology and drug transport.

Establishment of MDCKII Cell Monolayer with Metabolic Activity by CYP3A4 Transduced with Recombinant Adenovirus

This study aims to establish an in vitro system that can assess intestinal first-pass metabolism of CYP3A4 substrate drugs using adenoviral transduction. Madin-Darby canine kidney II (MDCKII) cells were used as a model of intestinal epithelial cells. Recombinant adenovirus expressing green fluorescent protein (AdGFP) and CYP3A4 (AdCYP3A4) was used as vectors. On day 2 after seeding MDCKII cells onto a semipermeable membrane, cells were infected with each adenovirus vector at various MOIs (multiplicities of infection) ranging from 0 to 200. On day 5, cell monolayers were used for drug transport study. The expression of GFP in monolayers of MDCKII cells transduced with AdGFP increased MOI-dependently and adenoviral infection showed no effect on the membrane permeability of drugs. The metabolite formation rate of midazolam, a CYP3A4 substrate, in the permeation process of a monolayer linearly increased with an increase in the MOI of AdCYP3A4. When the period that vectors and cells were located adjacent to each other was prolonged, the rate increased 2-fold compared with that calculated from a result with a monolayer obtained from a shorter period of adjacency. This study indicates that monolayers of MDCKII cells transduced with AdCYP3A4 have the potential to enable estimation of the first-pass metabolism by CYP3A4 in the intestinal absorption process.

Advanced drug delivery to the lymphatic system: lipid-based nanoformulations

The delivery of drugs and bioactive compounds via the lymphatic system is complex and dependent on the physiological uniqueness of the system. The lymphatic route plays an important role in transporting extracellular fluid to maintain homeostasis and in transferring immune cells to injury sites, and is able to avoid first-pass metabolism, thus acting as a bypass route for compounds with lower bioavailability, ie, those undergoing more hepatic metabolism. The lymphatic route also provides an option for the delivery of therapeutic molecules, such as drugs to treat cancer and human immunodeficiency virus, which can travel through the lymphatic system. Lymphatic imaging is useful in evaluating disease states and treatment plans for progressive diseases of the lymph system. Novel lipid-based nanoformulations, such as solid lipid nanoparticles and nanostructured lipid carriers, have unique characteristics that make them promising candidates for lymphatic delivery. These formulations are superior to colloidal carrier systems because they have controlled release properties and provide better chemical stability for drug molecules. However, multiple factors regulate the lymphatic delivery of drugs. Prior to lymphatic uptake, lipid-based nanoformulations are required to undergo interstitial hindrance that modulates drug delivery. Therefore, uptake and distribution of lipid-based nanoformulations by the lymphatic system depends on factors such as particle size, surface charge, molecular weight, and hydrophobicity. Types of lipid and concentration of the emulsifier are also important factors affecting drug delivery via the lymphatic system. All of these factors can cause changes in intermolecular interactions between the lipid nanoparticle matrix and the incorporated drug, which in turn affects uptake of drug into the lymphatic system. Two lipid-based nanoformulations, ie, solid lipid nanoparticles and nanostructured lipid carriers, have been administered via multiple routes (subcutaneous, pulmonary, and intestinal) for targeting of the lymphatic system. This paper provides a detailed review of novel lipid-based nanoformulations and their lymphatic delivery via different routes, as well as the in vivo and in vitro models used to study drug transport in the lymphatic system. Physicochemical properties that influence lymphatic delivery as well as the advantages of lipid-based nanoformulations for lymphatic delivery are also discussed.

Use of in vitro release models in the design of sustained and localized drug delivery systems for subcutaneous and intra-articular administration

Predictive in vitro release methods for parenteral depots should be developed with emphasis on the intended administration site as well as the formulation principles and the properties of drug administered. For subcutaneous/intra-articular administration, drug release from the depot and subsequent tissue transport away from the injection site involve diffusive and/or convective processes. The present review gives an account of drug transport in the synovial joint and the subcutaneous (or connective) tissue and how to implement such drug transport processes in the release model, e.g. the use of dialysis membrane for simulating the synovium and hydrogels for mimicking the subcutaneous tissue. This review summarizes the in vitro release studies performed using the rotating dialysis cell model including drug release assessment for the design of lipophilic drug/prodrug solutions. The initial exploration of the UV imaging technology for studying drug transport and release processes for depots intended for subcutaneous injection is commented on.

Precision-cut Intestinal Slices as In Vitro Tool for Studies on Drug Metabolism

The role of the intestine in drug metabolism has long been underestimated as a consequence of the technical difficulty to discern the role of the intestine from that of the liver in in vivo experiments and of the lack of in vitro models that are sufficiently viable and fully representing the physiology and anatomy of the intestine. Recently the precision-cut slice model, which is widely used for liver and kidney, was also adapted for the small and large intestine. In this review the application of precision-cut intestinal slices (PCIS) for research in drug metabolism and transport is discussed. PCIS can be prepared from animal and human tissues from all regions of the intestine allowing investigation of species differences and regional gradients of activities of metabolizing enzymes. They are viable for 8-24 h of incubation and show high activity of drug metabolizing enzymes, representative for the in vivo activity. They have been successfully used to study drug-drug interactions such as induction, inhibition and regulation of drug metabolizing enzymes, transporters and nuclear factors. Moreover they appear to be a suitable model for studies on cold preservation of donor organs for transplantation, and allow exploring inter-organ interactions by co-incubation with precision-cut slices of other organs. Their application as model for drug-induced intestinal toxicity is still in its infancy but appears to be promising. PCIS, prepared from human and animal tissues, represent a powerful translational model for drug metabolism, transport and toxicity studies and as such contributes to the reduction and replacement of animal experiments.

Precision-cut Intestinal Slices as In Vitro Tool for Studies on Drug Metabolism

The role of the intestine in drug metabolism has long been underestimated as a consequence of the technical difficulty to discern the role of the intestine from that of the liver in in vivo experiments and of the lack of in vitro models that are sufficiently viable and fully representing the physiology and anatomy of the intestine. Recently the precision-cut slice model, which is widely used for liver and kidney, was also adapted for the small and large intestine. In this review the application of precision-cut intestinal slices (PCIS) for research in drug metabolism and transport is discussed. PCIS can be prepared from animal and human tissues from all regions of the intestine allowing investigation of species differences and regional gradients of activities of metabolizing enzymes. They are viable for 8-24 h of incubation and show high activity of drug metabolizing enzymes, representative for the in vivo activity. They have been successfully used to study drug-drug interactions such as induction, inhibition and regulation of drug metabolizing enzymes, transporters and nuclear factors. Moreover they appear to be a suitable model for studies on cold preservation of donor organs for transplantation, and allow exploring inter-organ interactions by co-incubation with precision-cut slices of other organs. Their application as model for drug-induced intestinal toxicity is still in its infancy but appears to be promising. PCIS, prepared from human and animal tissues, represent a powerful translational model for drug metabolism, transport and toxicity studies and as such contributes to the reduction and replacement of animal experiments.

A novel matrix for the short-term storage of cells: utility in drug metabolism and drug transporter studies with rat, dog and human hepatocytes

1. The SureTran™ matrix is a novel method facilitating short-term maintenance of fresh primary hepatocyte cellular function and offers the potential use of primary cells “as fresh” for several days post isolation. In the study presented, the maintenance of several key phase I and II drug metabolizing enzyme and drug transporter activities is demonstrated with rat and dog hepatocytes preserved for up to 7 days after cell isolation.2. Intrinsic clearance values were determined for 60 new chemical entities using rat hepatocytes freshly isolated at AstraZeneca and rat hepatocytes prepared at the facilities of Abcellute Ltd (SureTran™ purveyors), stored and incubated 24 hours after isolation. A very good correspondence in the intrinsic clearance values underlines the utility of the cell maintenance matrix.3. For human hepatocytes many of the enzyme activities assayed were well maintained for 7 days of storage but some declined to below 50% of initial values between day 4 and 7 of storage. Human OATP1B1 activity was only determined with one batch and declined to 51% of the initial test value by day 4 and further down to 35% by day 7.

Renal Transporters in Drug Development

The kidney plays a vital role in the body's defense against potentially toxic xenobiotics and metabolic waste products through elimination pathways. In particular, secretory transporters in the proximal tubule are major determinants of the disposition of xenobiotics, including many prescription drugs. In the past decade, considerable progress has been made in understanding the impact of renal transporters on the disposition of many clinically used drugs. In addition, renal transporters have been implicated as sites for numerous clinically important drug-drug interactions. This review begins with a description of renal drug handling and presents relevant equations for the calculation of renal clearance, including filtration and secretory clearance. In addition, data on the localization, expression, substrates, and inhibitors of renal drug transporters are tabulated. The recent US Food and Drug Administration drug-drug interaction draft guidance as it pertains to the study of renal drug transporters is presented. Renal drug elimination in special populations and transporter splicing variants are also described.

Zebrafish as a Model to Study Drug Transporters at the Blood–Brain Barrier

The blood–brain barrier (BBB) restricts the delivery of therapeutics into the central nervous system (CNS). This limitation is due largely to localization of drug transport mechanisms to the brain endothelial cells (BECs). Although mammalian and in vitro models are currently used to study drug transport, zebrafish (Danio rerio) are advantageous for dissecting complex biological problems such as the BBB. This report focuses on zebrafish as a suitable model system for studying the BBB and drug transport. Clinical Pharmacology & Therapeutics (2012); 92 5, 567–570. doi:10.1038/clpt.2012.168

Assessment of the retinal pigment epithelial functions‐ modelling approach

AbstractPurpose Our purpose is to construct mathematical models of functions of retinal pigment epithelium (RPE). Here we show the development and results of two models: 1) a compartmental model of Ca2+ dynamics 2) finite element model of epithelial transport and trans‐epithelial resistance.Methods A model of Ca2+ dynamics of APRE‐19 cells based on an experimental data and literature was constructed. Ca+2 dynamics of APRE‐19 cells were recorded using fluorescent microscope after mechanical stimulation. Various Ca+2 functional conditions were tested. The computational model constructed (Matlab SimBiology ) comprises over 40 cell function parameters and twelve variables ranging from stretch‐sensitive ion channels to ryanodine receptor dynamics and sarco/endoplasmic reticulum ATPases. Further, a model of RPE epithelia barrier was developed based on finite element modelling (FEM) of the epithelia and trans‐epithelial resistance simulating the spatial distribution of epithelial properties originating of the tight junction distribution on epithelia.Results The Ca+2 kinetics model of RPE was able to reproduce the Ca+2 dynamics of APRE cells with high accuracy with above 0.97 cross‐correlation. Further, including stretch‐sensitive ion channels explained the Ca+2 dynamics of the cells close to the mechanically stimulated cell. The epithelial model showed that epithelial inhomogeneity may play a crucial role in epithelial tightness and even very small cellular changes produce large variation on the measures of the epithelial properties.Conclusion The models constructed provide new insight of the functions of the RPE with applications from drug transport studies to assessment of the functionality of stem cell derived RPE.

Long-term stability, biocompatibility and oral delivery potential of risperidone-loaded solid lipid nanoparticles

A solid lipid nanoparticles (SLN) formulation to improve the oral delivery of risperidone (RISP), a poorly water-soluble drug, was designed and tested. Initially, lipid-RISP solubility was screened to select the best lipid for SLN preparation. Compritol(®)-based formulations were chosen and their long-term stability was assessed over two years of storage (at 25 °C and 4 °C) by means of particle size, polydispersity index (PI), zeta potential (ZP) and encapsulation efficiency (EE) measurements. SLN shape was observed by transmission electron microscopy (TEM) at the beginning and end of the study. The oxidative potential (OP) of the SLN was measured and their biocompatibility with Caco-2 cells was evaluated using the (4,5-dimethylthiazol-2-yl)2,5-dyphenyl-tetrazolium bromide (MTT) assay. In vitro drug release and transport studies were performed to predict the in vivo release profile and to evaluate the drug delivery potential of the SLN formulations, respectively. The RISP-loaded SLN systems were stable and had high EE and similar shape to the placebo formulations before and after storage. Classical Fickian diffusion was identified as the release mechanism for RISP from the SLN formulation. Biocompatibility and dose-dependent RISP transport across Caco-2 cells were observed for the prepared SLN formulations. The viability of SLN as formulations for oral delivery of poorly water-soluble drugs such as RISP was illustrated.

Iontophoresis Across the Proximal Nail Fold to Target Drugs to the Nail Matrix

The main objective of the present study was to investigate the plausibility of iontophoretic delivery of drugs to the nail matrix via proximal nail fold. The in vitro drug transport studies were performed in Franz diffusion cells across folded epidermis, which is used as a model for the proximal nail fold. The amount of drug transported into the receiver compartment following iontophoresis for 3 h at 0.5 mA/cm(2) was 150-fold higher than the control (0.008 ± 0.002 μg/cm(2)). The amount of drug present in the skin after iontophoresis (0.45 ± 0.12 μg/mg) was approximately fivefold higher as compared with that of the control (0.08 ± 0.01 μg/mg). Iontophoresis of terbinafine across the proximal nail fold was assessed using excised cadaver toe model as well. A custom-designed foam-pad-type patch system was used for iontophoresis in cadaver toes. The amount of the drug delivered into the nail matrix following iontophoresis for 3 h was significantly higher than the minimum inhibition concentration of terbinafine. However, on the contrary, passive delivery for about 24 h did not result in any detectable drug levels in the nail matrix. Iontophoresis across the proximal nail fold could be developed as a potential method to target drugs to nail matrix.

Development of a Model for Functional Studies of ABCG2 (Breast Cancer Resistance Protein) Efflux Employing a Standard BeWo Clone (B24)

Human choriocarcinoma-derived BeWo cells express high levels of breast cancer resistance protein (BCRP/ABCG2) with no functional P-glycoprotein (P-gp) (ABCB1) activity, making them a potential model to study bidirectional ABCG2-mediated drug transport. However, the original BeWo clone (B24) available to researchers does not form confluent monolayers with tight junctions required by the model. Our aim was to adapt culture conditions to attempt to generate confluent BeWo monolayers for drug transport studies using the standard B24 clone. BeWo cells (B24; American Type Culture collection [ATCC]) were cultured in six-well plates or polycarbonate millicell inserts in a number of media formulations, growth supplements, and basement membrane substitutes. Cells were examined for confluence by microscopy, and transepithelial electrical resistance (TEER) was measured daily; monolayer permeability was assessed when TEER had stabilized. Optimal growth rates were achieved in culture conditions consisting of Medium 199 (M199) supplemented with epidermal growth factor (EGF; 20 ng/mL), vitamin supplements, and 10% fetal calf serum (FCS) with collagen coating. A TEER of 170 Ω in 0.6 cm(2) inserts was achieved 2 weeks after seeding under optimal conditions. The cell-impermeable diffusion marker 5(6) carboxy-2,7dichlorodihydrofluorescein (C-DCDHF) had a permeability coefficient of 3.5×10(-6) cm/s, indicative of minimal paracellular permeability. ABCG2 expression, as determined by immunoblotting, remained unaffected by confluency. In conclusion, we describe culture conditions for the B24 BeWo clone that facilitate the formation of monolayers with tighter junctions and reduced paracellular transport compared to previously published models. These growth conditions provide a good model of ABCG2-mediated drug transport in a human placental cell line.

Colon targeting of fluticasone propionate inclusion complex: a novel approach in inflammatory bowel disease

Purpose of the present research was to present fluticasone propionate, a glucocorticoid, as a novel formulation exhibiting improved aqueous solubility, and targeting the drug directly to colon for the treatment of inflammatory bowel disease. Inclusion complex of the drug with hydroxypropyl betacyclodextrin were prepared by solvent evaporation and subsequently the granules of the inclusion complex were coated with Eudragit S100, in order to achieve colon targeting. Inclusion complex was characterized by FTIR, DSC, XRD and 1H-NMR studies. In vitro drug release from coated granules and the drug transport across excised rat colon using modified Ussing chamber were also attempted. The drug was found to be present in amorphous form, when included in HPβCD cavities. Furthermore, intrinsic dissolution of the drug was found to increase by ~18 times. Coated granules exhibited no drug release in 0.01 N HCl as dissolution medium, indicating gastro-resistance, while 92 % of the drug was released in 120 min, in phosphate buffer (pH 7.4) as dissolution medium. The drug transport studies with rat colon led to more drug transport and concentration in target tissue, when presented as inclusion complex. The formulation releases the drug with improved aqueous solubility in colonic region, and thus concentrating the drug at the target tissue itself.

Application of biopharmaceutics classification system (BCS) in drug transport studies across human respiratory epithelial cell monolayers

Transport studies of model drugs were conducted across the human nasal epithelial (HNE) and normal human bronchial epithelial (NHBE) cell monolayers cultured by air–liquid interface method. Physicochemical properties (e.g., molecular weight, calculated partition coefficient, dose number) of model drugs were quoted from literatures and apparent permeability coefficients (P app) across the HNE and NHBE cell monolayers were directly measured. A linear relationship was observed between the P app values of model drugs in the HNE and NHBE cell monolayers. As the molecular weight of model drugs increased, the P app showed a decreasing pattern while the increase of partition coefficients resulted in the increment of P app. These results indicated that the transport of model drugs across both cell monolayers followed mainly the passive diffusion mechanism, although substrates mediated by drug transporters showed a deviating pattern. It was also interesting to note that almost all model drugs could be grouped into the same biopharmaceutics classification system as that classified by the human intestinal permeability when the P app was plotted as a function of dose number (D 0) of each drug.

Verbascoside, Isoverbascoside, and Their Derivatives Recovered from Olive Mill Wastewater as Possible Food Antioxidants

Olive oil processing industries generate substantial quantities of phenolic-rich byproducts, which could be valuable natural sources of antioxidants. This work is focused on the recovery and structural characterization of antioxidant compounds from olive mill wastewater (OMWW), a polluting byproduct of the olive oil production process. Phenolics were extracted from the waste material using a membrane technology coupled to low-pressure gel filtration chromatography on a Sephadex LH-20. The LH-20 fraction was, in turn, characterized for its phenolic composition by HPLC-DAD-MS/MS analyses. Verbascoside, isoverbascoside, β-hydroxyverbascoside, β-hydroxyisoverbascoside, and various oxidized phenolics were identified. Uptake of verbascoside, purified from the LH-20 fraction, by HT-29 cells, an established model system for studying drug transport properties, was also assayed. Finally, the antioxidant activities of the LH-20 fraction and verbascoside were characterized by two different techniques. Individual verbascoside was more active as a scavenger of reactive oxygen species and as a chemopreventive agent protecting low-density lipoproteins from oxidative damage than the LH-20 fraction.

Efflux protein expression in human stem cell-derived retinal pigment epithelial cells.

Retinal pigment epithelial (RPE) cells in the back of the eye nourish photoreceptor cells and form a selective barrier that influences drug transport from the blood to the photoreceptor cells. At the molecular level, ATP-dependent efflux transporters have a major role in drug delivery in human RPE. In this study, we assessed the relative expression of several ATP-dependent efflux transporter genes (MRP1, -2, -3, -4, -5, -6, p-gp, and BCRP), the protein expression and localization of MRP1, MRP4, and MRP5, and the functionality of MRP1 efflux pumps at different maturation stages of undifferentiated human embryonic stem cells (hESC) and RPE derived from the hESC (hESC-RPE). Our findings revealed that the gene expression of ATP-dependent efflux transporters MRP1, -3, -4, -5, and p-gp fluctuated during hESC-RPE maturation from undifferentiated hESC to fusiform, epithelioid, and finally to cobblestone hESC-RPE. Epithelioid hESC-RPE had the highest expression of MRP1, -3, -4, and P-gp, whereas the most mature cobblestone hESC-RPE had the highest expression of MRP5 and MRP6. These findings indicate that a similar efflux protein profile is shared between hESC-RPE and the human RPE cell line, ARPE-19, and suggest that hESC-RPE cells are suitable in vitro RPE models for drug transport studies. Embryonic stem cell model might provide a novel tool to study retinal cell differentiation, mechanisms of RPE -derived diseases, drug testing and targeted drug therapy.

Hierarchy of evidence in interpretation of clinical significance of drug interactions

Since drug interactions may result in serious adverse effects or failure of therapy, it is of huge importance that health professionals base their decisions about drug prescription, dispensing and administration on reliable research evidence, taking into account the hierarchy of data sources for evaluation. CLINICAL SIGNIFICANCE OF POTENTIAL INTERACTIONS--INFORMATION SOURCES: The sources of data regarding drug interactions are numerous, beginning with various drug reference books. However, they are far from uniformity in the way of choosing and presenting putative clinically relevant interactions. CLINICAL SIGNIFICANCE OF POTENTIAL INTERACTIONS--INTERPRETATION OF INFORMATION: The difficulties in interpretation of drug interactions are illustrated through the analysis of a published example involving assessment made by two different groups of health professionals. SYSTEMATIC EVALUATION OF DRUG--DRUG INTERACTION: The potential for interactions is mainly investigated before marketing a drug. Generally, the in vitro, followed by in vivo studies are to be performed. The major metabolic pathways involved in the metabolism of a new molecular entity, as well as the potential of induction of human enzymes involved in drug metabolism are to be examined. In the field of interaction research it is possible to make use of the population pharmacokinetic studies as well as of the pharmacodynamic assessment, and also the post-registration monitoring of the reported adverse reactions and other literature data. In vitro and in vivo drug metabolism and transport studies should be conducted to elucidate the mechanisms and potential for drug-drug interactions. The assessment of their clinical significance should be based on well-defined and validated exposure-response data.

Targeting p53-Null Neuroblastomas through RLIP76

The search for p53-independent mechanism of cancer cell killing is highly relevant to pediatric neuroblastomas, where successful therapy is limited by its transformation into p53-mutant and a highly drug-resistant neoplasm. Our studies on the drug-resistant p53-mutant as compared with drug-resistant p53 wild-type neuroblastoma revealed a novel mechanism for resistance to apoptosis: a direct role of p53 in regulating the cellular concentration of proapoptotic alkenals by functioning as a specific and saturable allosteric inhibitor of the alkenal-glutathione conjugate transporter, RLIP76. The RLIP76-p53 complex was showed by both immunoprecipitation analyses of purified proteins and immunofluorescence analysis. Drug transport studies revealed that p53 inhibited both basal and PKCα-stimulated transport of glutathione conjugates of 4HNE (GSHNE) and doxorubicin. Drug resistance was significantly greater for p53-mutant as compared with p53 wild-type neuroblastoma cell lines, but both were susceptible to depletion of RLIP76 by antisense alone. In addition, inhibition of RLIP76 significantly enhanced the cytotoxicity of cisplatin. Taken together, these studies provide powerful evidence for a novel mechanism for drug and apoptosis resistance in p53-mutant neuroblastoma, based on a model of regulation of p53-induced apoptosis by RLIP76, where p53 is a saturable and specific allosteric inhibitor of RLIP76, and p53 loss results in overexpression of RLIP76; thus, in the absence of p53, the drug and glutathione-conjugate transport activities of RLIP76 are enhanced. Most importantly, our findings strongly indicate RLIP76 as a novel target for therapy of drug-resistant and p53-mutant neuroblastoma.