Drug Uptake Mechanisms Research Articles

Abstract B017: Understanding the impact of transporter heterogeneity on the efficacy of PARP inhibitors

Abstract B017: Understanding the impact of transporter heterogeneity on the efficacy of PARP inhibitors

Enhancing pre-clinical research with simplified intestinal cell line models

Two-dimensional culture remains widely employed to determine the bioavailability of orally delivered drugs. To gain more knowledge about drug uptake mechanisms and risk assessment for the patient after oral drug admission, intestinal in vitro models demonstrating a closer similarity to the in vivo situation are needed. In particular, Caco-2 cell-based Transwell® models show advantages as they are reproducible, cost-efficient, and standardized. However, cellular complexity is impaired and cell function is strongly modified as important transporters in the apical membrane are missing. To overcome these limitations, primary organoid-based human small intestinal tissue models were developed recently but the application of these cultures in pre-clinical research still represents an enormous challenge, as culture setup is complex as well as time- and cost-intensive. To overcome these hurdles, we demonstrate the establishment of primary organoid-derived intestinal cell lines by immortalization. Besides exhibiting cellular diversity of the organoid, these immortalized cell lines enable a standardized and more cost-efficient culture. Further, our cell line-based Transwell®-like models display an organ-specific epithelial barrier integrity, ultrastructural features and representative transport functions. Altogether, our novel model systems are cost-efficient with close similarity to the in vivo situation, therefore favoring their use in bioavailability studies in the context of pre-clinical screenings.

Multimodal imaging of a liver-on-a-chip model using labelled and label-free optical microscopy techniques.

A liver-on-a-chip model is an advanced complex in vitro model (CIVM) that incorporates different cell types and extracellular matrix to mimic the microenvironment of the human liver in a laboratory setting. Given the heterogenous and complex nature of liver-on-a-chip models, brightfield and fluorescence-based imaging techniques are widely utilized for assessing the changes occurring in these models with different treatment and environmental conditions. However, the utilization of optical microscopy techniques for structural and functional evaluation of the liver CIVMs have been limited by the reduced light penetration depth and lack of 3D information obtained using these imaging techniques. In this study, the potential of both labelled as well as label-free multimodal optical imaging techniques for visualization and characterization of the cellular and sub-cellular features of a liver-on-a-chip model was investigated. (1) Cellular uptake and distribution of Alexa 488 (A488)-labelled non-targeted and targeted antisense oligonucleotides (ASO and ASO-GalNAc) in the liver-on-a-chip model was determined using multiphoton microscopy. (2) Hyperspectral stimulated Raman scattering (SRS) microscopy of the C-H region was used to determine the heterogeneity of chemical composition of circular and cuboidal hepatocytes in the liver-on-a-chip model in a label-free manner. Additionally, the spatial overlap between the intracellular localization of ASO and lipid droplets was explored using simultaneous hyperspectral SRS and fluorescence microscopy. (3) The capability of light sheet fluorescence microscopy (LSFM) for full-depth 3D visualization of sub-cellular distribution of A488-ASO and cellular phenotypes in the liver-on-a-chip model was demonstrated. In summary, multimodal optical microscopy is a promising platform that can be utilized for visualization and quantification of 3D cellular organization, drug distribution and functional changes occurring in liver-on-a-chip models, and can provide valuable insights into liver biology and drug uptake mechanisms by enabling better characterization of these liver models.

Membranes and Transport Mechanisms

Cellular life depends on the movement of membrane ions. For cells to continue functioning as they go through their life cycle, there must be a tremendous amount of exchange. Cell membranes are active structures rather than merely passive barriers separating cells from their surroundings. Cell membranes are known to be selectively permeable, allowing some chemicals to pass through while preventing others from doing so. Therefore, all cellular exchange activities can have active participation from cell membranes. Through the membrane interface, elements needed for cellular existence, communication among cells, and interaction between cells and their environment are taken in. Additionally, cells have the ability to direct and influence input and export processes, as well as moderate the subsequent signal response. Effective drug transportation across cellular membranes is essential for all treatment procedures. The majority of medications used in cancer therapy require carrier proteins for their transmembrane transport, even though hydrophobic small molecules can enter the cell membrane through straightforward diffusion. As a result, many scientists concentrate on creating novel strategies to improve drug delivery and uptake into desired cells. A live cell's plasma membrane serves as its outer wall. Although it acts as a barrier, it also permits transport between the cell and its surroundings.
 While the hydrophilic head groups are in contact with the surrounding water, the bilayer's hydrophobic interior, which is made up of fatty acid chains, renders the membrane impermeable to water-soluble compounds. To reduce the accessibility of hydrophobic fatty acid chains, phospholipids can spontaneously form stable bilayers in an aqueous environment due to their amphipathic nature. As lipids and proteins positioned in the plasma membrane can rotate or diffuse laterally and are distributed asymmetrically in the two leaflets, membranes are extremely dynamic structures. In addition to glycolipids, the outer leaflet of the plasma membrane mostly consists of the phospholipids phosphatidylcholine and sphingomyelin. Phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol are the three main substances that make up the inner leaflet.
 Additionally, both leaflets contain cholesterol, which affects the fluidity of the membrane. Lipid rafts are particular domains abundant in sphingolipids and cholesterol. The plasma membrane's basic role as a selectively permeable wall dividing the inside from the outside of the cell is determined by the structure of the phospholipid bilayer. Membrane proteins are essential for guaranteeing the selective translocation of molecules across the membrane as well as the regulation of cellular connections. They can function as energy converters, ion channels, pumps, receptors, or enzymes. The maintenance of the osmotic pressure and cellular pH, as well as the regulation of drug uptake mechanisms, account for the regulation of fundamental cellular activities.

Proliposome: A Novel Solution for the Stability of Liposomes - A Review

Proliposome are phospholipid-based vesicular drug delivery systems that have specialized applications in drug delivery via various routes of administration. They are widely researched as potential carriers for improving bioavailability through oral administration of drugs with low bioavailability and are composed of the drug, phospholipid, and a water-soluble carrier or porous powder. The technology is based on the inherent ability of hydrated membrane lipids to form liposomal vesicles when exposed to water. It is specifically designed for molecular dispersion and delivery of hydrophilic and hydrophobic drugs to achieve good bioavailability, local, systemic effects, and site-specific drug delivery strategies. By utilizing phospholipids and various carriers, these vesicular systems play critical roles in improving bioavailability and solubility. An in-depth understanding of the physical and biological behavior of Proliposome is required for designing an effective delivery system that will increase drug availability at the anticipated site. Proliposome provide an opportunity to improve drug therapeutic action by modifying drug uptake mechanisms. The Proliposome approach was developed as a simple, repeatable, and dependable manufacturing technique for large-scale production of liposome dispersions. The present review discusses the use of Proliposome to improve the oral bioavailability of poorly water-soluble drugs, components for the formulation, effects of excipients, techniques for preparation, evaluation of Proliposome, and other key issues related to various routes of administration. Proliposome formulation is an alternative, novel solution for liposome stability.

Fabrication of HKUST-1/ZnO/SA nanocomposite for Doxycycline and Naproxen adsorption from contaminated water

Doxycycline and Naproxen are among the most widely used drugs in the therapy of CoVID 19 disease found in surface water. Water scarcity in recent years has led to research to treat polluted water. One of the easy and low-cost methods for treatment is adsorption. The utilize of Metal-Organic Frameworks (MOFs) to evacuate pharmaceutical contaminants from water sources has been considered by researchers in the last decade. In this research, HKUST-1/ZnO/SA composite with high adsorption capacity, chemical and water stability, recovery, and reuse properties has been synthesized and investigated. By adding 10 wt% of ZnO and 50 wt% of sodium alginate to HKUST-1, at 25 °C and pH = 7, the specific surface area is reduced by 60%. The parameters of drugs concentration C0 =(5,80) mg/L, time=(15,240) min, and pH= (2,12) were investigated, and the results showed that the HKUST-1/ZnO/SA is stable in water for 14 days and it can be used in 10 cycles with 80% removal efficiency. The maximum Adsorption loading of doxycycline and Naproxen upon HKUST-1/ZnO/SA is 97.58 and 80.04 mg/g, respectively. Based on the correlation coefficient (R2), the pseudo-second-order and the Langmuir isotherm models were selected for drug adsorption. The proposed mechanism of drug uptake is by MOFs, hydrogen bonding, electrostatic bonding, and acid-base interaction.

Cell Surface Transporters and Novel Drug Developments.

Despite the numerous scientific and technological advances made within the last decade the attrition rates for new drug discovery remain as high as 95% for anticancer drugs. Recent drug development has been in part guided by Lipinski’s Rule of 5 (Ro5) even though many approved drugs do not comply to these rules. With Covid-19 vaccine development strategy dramatically accelerating drug development perhaps it is timely to question the generic drug development process itself to find a more efficient, cost effective, and successful approach. It is widely believed that drugs permeate cells via two methods: phospholipid bilayer diffusion and carrier mediated transporters. However, emerging evidence suggests that carrier mediated transport may be the primary mechanism of drug uptake and not diffusion as long believed. Computational biology increasingly assists drug design to achieve desirable absorption, distribution, metabolism, elimination and toxicity (ADMET) properties. Perfecting drug entry into target cells as a prerequisite to intracellular drug action is a logical and compelling route and is expected to reduce drug attrition rates, particularly gaining favour amongst chronic lifelong therapeutics. Novel drug development is rapidly expanding from the utilisation of beyond the rule of five (bRo5) to pulsatile drug delivery systems and fragment based drug design. Utilising transporters as drug targets and advocating bRo5 molecules may be the solution to increasing drug specificity, reducing dosage and toxicity and thus revolutionising drug development. This review explores the development of cell surface transporter exploitation in drug development and the relationship with improved therapeutic index.

Distribution profiles of diphenhydramine and lidocaine in scalp, axillary, and pubic hairs measured by micro-segmental hair analysis: good indicator for discrimination between administration and external contamination of the drugs

PurposeDrug distribution in scalp hair can provide historical information about drug use, such as the date and frequency of drug ingestion. We previously developed micro-segmental hair analysis, which visualizes drug distribution at 0.4-mm intervals in individual hairs. The present study examines whether the distribution profiles of drugs can be markers for the administration or external contamination of the drugs using scalp, axillary, and pubic hairs.MethodsA single dose of anti-itch ointment containing diphenhydramine (DP) and lidocaine (LD) was topically applied to the axillary or pubic areas of two volunteers; DP was also orally administered; and LD was intra-gingivally injected. Scalp, axillary, and pubic hairs were assessed using our micro-segmental analysis.ResultsThe localization of DP and LD differed within individual scalp hair strands, implying DP and LD were predominantly incorporated into scalp hair via the bloodstream and via sweat/sebum, respectively, showing double-peak profiles. However, DP and LD were distributed along the shafts of axillary and pubic hairs without appearance of the double-peak profiles when the ointment had been applied to the axillary and pubic areas. The distributions of DP and LD in scalp hairs did not significantly differ according to administration routes, such as oral administration, gingival injection, and topical application.ConclusionsMicro-segmental analysis revealed differences in the distribution profiles of drugs in hairs, and distinguished hairs with and without external contamination. These findings will be useful for understanding of the mechanism of drug uptake into hair and for estimating the circumstances for a drug use.

Carrier-Mediated Drug Uptake in Fungal Pathogens.

Candida, Aspergillus, and Cryptococcus species are the most frequent cause of severe human fungal infections. Clinically relevant antifungal drugs are scarce, and their effectiveness are hampered by the ability of fungal cells to develop drug resistance mechanisms. Drug effectiveness and drug resistance in human pathogens is very often affected by their “transportome”. Many studies have covered a panoply of drug resistance mechanisms that depend on drug efflux pumps belonging to the ATP-Binding Cassette and Major Facilitator Superfamily. However, the study of drug uptake mechanisms has been, to some extent, overlooked in pathogenic fungi. This review focuses on discussing current knowledge on drug uptake systems in fungal pathogens, highlighting the need for further studies on this topic of great importance. The following subjects are covered: (i) drugs imported by known transporter(s) in pathogenic fungi; and (ii) drugs imported by known transporter(s) in the model yeast Saccharomyces cerevisiae or in human parasites, aimed at the identification of their homologs in pathogenic fungi. Besides its contribution to increase the understanding of drug-pathogen interactions, the practical implications of identifying drug importers in human pathogens are discussed, particularly focusing on drug development strategies.

Antibiotic-in-Cyclodextrin-in-Liposomes: Formulation Development and Interactions with Model Bacterial Membranes.

Gram-negative bacteria possess numerous defenses against antibiotics, due to the intrinsic permeability barrier of their outer membrane (OM), explaining the recalcitrance of some common and life-threatening infections. We report the formulation of a new drug, PPA148, which shows promising activity against all Gram-negative bacteria included in the ESKAPEE pathogens. PPA148 was solubilized by inclusion complexation with cyclodextrin followed by encapsulation in liposomes. The complex and liposomal formulation presented increased activity against E. coli compared to the pure drug when assessed with the Kirby Bauer assay. The novel formulation containing 1 μg PPA148 reached similar efficacy levels equivalent to those of 30 μg of pure rifampicin. A range of biophysical techniques was used to explore the mechanism of drug uptake. Langmuir trough (LT) and neutron reflectivity (NR) techniques were employed to monitor the interactions between the drug and the formulation with model membranes. We found evidence for liposome fusion with the model Gram-negative outer membrane and for cyclodextrins acting as inner membrane (IM) permeation enhancers without presenting intrinsic antimicrobial activity. An antibiotic-in-cyclodextrin-in-liposomes (ACL) formulation was developed, which targets both the bacterial OM and IM, and offers promise as a means to breach the Gram-negative cell envelope.

Measurement of three-dimensional distributions of drugs in nails using liquid chromatography/tandem mass spectrometry after micro-segmentation to elucidate drug uptake routes

Ingested drugs can be taken up into nails and hairs from the bloodstream and these drugs can be stably retained over several months or more. Free edges of nails can often be used as a specimen to prove drug intake. However, the mechanism of drug uptake into the nails is unclear. Although it is presumed that there are horizontal uptake routes at the nail root and vertical uptake routes at the nail bed against the direction of growth, three-dimensional distribution analysis is required to verify this phenomenon. Herein, we first developed a method to measure the three-dimensional distribution of drugs in the nails using the combination of micro-segmentation of nails (0.2 × 1.5 × 0.06 mm size) and highly sensitive quantification of drugs by LC-MS/MS. Carbinoxamine was administered as a model compound to a subject in a single dose, and then the free edges of big toenails were collected every several weeks over a year. The three-dimensional distribution of carbinoxamine in each free edge was visualized and arranged along the collection period. Carbinoxamine was localized in the lower layer during the early collection period (up to 78 days after drug ingestion) and in the upper and middle layers later (134 days or later). The changes in the drug distribution in the nails along the collection period implied that two-way drug uptake takes place in the whole nail through both the nail bed and the nail root simultaneously. The developed analytical method could be useful to elucidate the mechanism of drug uptake into the nails.

Multivalency in Drug Delivery-When Is It Too Much of a Good Thing?

Multivalency plays a large role in many biological and synthetic systems. The past 20 years of research have seen an explosion in the study of multivalent drug delivery systems based on scaffolds such as dendrimers, polymers, and other nanoparticles. The results from these studies suggest that when it comes to the number of ligands, sometimes, to quote Shakespeare, "too much of a good thing" is an apt description. Recent theoretical studies on multivalency indicate that the field may have had a misplaced emphasis on maximizing binding strength where in fact it is the selectivity of multivalent drug delivery systems that is the key to success. This Topical Review will summarize these theoretical developments. We will then illustrate how these developments can be used to rationalize the immunoresponses and drug uptake mechanisms for multivalent systems and show the path forward toward the design of better multivalent drug delivery systems.

Different localizations of drugs simultaneously administered in a strand of hair by micro‐segmental analysis

Segmental hair analysis is used to estimate the time of drug intake at monthly precision in drug-related crimes. Previously, we advanced this analytical method to specify the day of drug intake by cutting a strand of hair into 0.4-mm segments, which correspond to daily hair growth. Herein, we investigated the distributions of 7 compounds in a strand of hair using micro-segmental analysis. Several strands of hair were collected 33.1-229.4 days after subjects were administered 4 pharmaceutical products that contained 10 drugs in single doses within 32 hours. The administered drugs and resulting metabolites were extracted from 0.4-mm hair segments and quantified using liquid chromatography-tandem mass spectrometry. Acidic and neutral compounds were detected at low amounts in any of the hair segments analyzed. Epinastine, fexofenadine, dihydrocodeine, chlorpheniramine, and the chlorpheniramine metabolite, desmethylchlorpheniramine each was localized to 2 regions within a strand of hair. By contrast, methylephedrine and its metabolite, ephedrine, each was localized to only a region. Among 20 individual strands of hair associated with different subjects and head regions, few differences in the shapes of drug concentration-hair segment curves for each compound were detected. Our data indicated that 2 mechanisms for drug uptake into hair can operate depending on drug properties and that co-administered drugs can be localized to different regions in a strand of hair. Micro-segmental analysis may aid in the identification of the day of drug intake and help to elucidate the mechanisms of drug uptake into hair.

Targeted photodynamic therapy as potential treatment modality for the eradication of colon cancer and colon cancer stem cells.

Colorectal cancer is commonly treated by tumour resection, as chemotherapy and radiation have proven to be less effective, especially if the tumour has metastasized. Resistance to therapies occurs in almost all patients with colorectal cancer, especially in those with metastatic tumours. Cancer stem cells have the ability to self-renew, and their slow rate of cycling enhances resistance to treatment and increases the likelihood of tumour recurrence. Most metastatic tumours are unable to be surgically removed, thus creating a need for treatment modalities that target cancers directly and destroy cancer stem cells. Photodynamic therapy involves a photosensitizer that when exposed to a light source of a particular wavelength becomes excited and produces a form of oxygen that kills cancer cells. Photodynamic therapy is currently being investigated as a treatment modality for colorectal cancer, and new studies are exploring enhancing photodynamic therapy efficacy with the aid of drug carriers and immune conjugates. These modifications could prove effective in targeting cancer stem cells that are thought to be resistant to photodynamic therapy. In order for photodynamic therapy to be an effective treatment in colorectal cancer, it requires treatment of both primary tumours and the metastatic secondary disease that is caused by colon cancer stem cells. This review focuses on current photodynamic therapy treatments available for colorectal cancer and highlights proposed actively targeted photosynthetic drug uptake mechanisms specifically mediated towards colon cancer stem cells, as well as identify the gaps in research which need to be investigated in order to develop a combinative targeted photodynamic therapy regime that can effectively control colorectal cancer primary and metastatic tumour growth by eliminating colon cancer stem cells.

Computational Modelling of Three-phase Stent-based Delivery

Background and ObjectiveTreatment of arterial lesions using drug-eluting stent is now a gold standard method, however, the mechanisms of drug uptake and its retention is not well understood. In most of the computational studies, only the binding of drug to specific receptor is considered; but it is well established that when the drug binds to the specific receptor, there is also occurrence of non-specific binding caused by the trapping of drug in the extracellular matrix. When non-specific binding is not subtracted from total binding to give receptor-mediated binding, a description of receptor/ligand requires that effects of non-specific binding be considered.

A molecular insight into the electro-transfer of small molecules through electropores driven by electric fields

The transport of chemical compounds across the plasma membrane into the cell is relevant for several biological and medical applications. One of the most efficient techniques to enhance this uptake is reversible electroporation. Nevertheless, the detailed molecular mechanism of transport of chemical species (dyes, drugs, genetic materials, …) following the application of electric pulses is not yet fully elucidated. In the past decade, molecular dynamics (MD) simulations have been conducted to model the effect of pulsed electric fields on membranes, describing several aspects of this phenomenon. Here, we first present a comprehensive review of the results obtained so far modeling the electroporation of lipid membranes, then we extend these findings to study the electrotransfer across lipid bilayers subject to microsecond pulsed electric fields of Tat11, a small hydrophilic charged peptide, and of siRNA. We use in particular a MD simulation protocol that allows to characterize the transport of charged species through stable pores. Unexpectedly, our results show that for an electroporated bilayer subject to transmembrane voltages in the order of 500mV, i.e. consistent with experimental conditions, both Tat11 and siRNA can translocate through nanoelectropores within tens of ns. We discuss these results in comparison to experiments in order to rationalize the mechanism of drug uptake by cells. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

Atorvastatin as a potential anti-malarial drug: in vitro synergy in combinational therapy with quinine against Plasmodium falciparum

BackgroundQuinine (QN) remains the first line anti-malarial drug for the treatment of complicated malaria in Europe and Africa. The emergence of QN resistance has been documented. QN resistance is not yet a significant problem, but there is an urgent need to discover partners for use in combination with QN. The aim of the study was to assess the in vitro potentiating effects of atorvastatin (AVA), a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, in combination with QN against Plasmodium falciparum and to evaluate whether the effects of AVA could be associated with gene copy number or mutations in genes involved in QN resistance, such as pfcrt, pfmdr1, pfmrp and pfnhe.MethodsThe susceptibilities to combination of AVA with QN were assessed against 21 parasite strains using the in vitro isotopic microtest. Genotypes and gene copy number were assessed for pfcrt, pfmdr1, pfmdr2, pfmrp genes. In addition, the number of DNNND, DDNHNDNHNN repeats in pfnhe-1 ms4760 and the ms4760 profile were determined for each strains of P. falciparum.ResultsAVA demonstrated synergistic effects in combination with QN against 21 P. falciparum strains. The QN IC50 was reduced by 5% (0% to 15%; 95%CI: 1%-8%), 10% (3% to 23%; 95%CI: 7%-14%) and 22% (14% to 40%; 95%CI: 19%-25%) in presence of AVA at concentrations of 0.1, 0.5 and 1.0 μM, respectively. These reductions were all significant (p < 0.009). The reduction in the QN IC50 in presence of AVA was not significantly correlated with the QN IC50 (r = 0.22, P = 0.3288) or the AVA IC50 (r = 0.03, P = 0.8946). The synergistic effect of AVA in combination with QN was not significantly associated with polymorphisms in the pfcrt, pfmdr1, pfmrp, and pfnhe-1 genes that could be involved in QN resistance. The synergistic effect of AVA on QN responses was not significantly associated with pfmdr1 copy number (P = 0.0428).ConclusionThe synergistic effect of AVA in combination with QN was found to be unrelated to mutations occurring in transport protein genes involved in QN drug resistance. The different mechanisms of drug uptake and/or mode of action for AVA compared to the other anti-malarial drugs, as well as the AVA-mediated synergy of the anti-malarial effect of QN, suggests that AVA will be a good candidate for combinatorial malaria treatment. All of these observations support calls for both an in vivo evaluation with pharmacokinetic component and clinical trials of AVA as an anti-malarial therapy.

Transport of Nucleoside Analogs Across the Plasma Membrane: A Clue to Understanding Drug-Induced Cytotoxicity

Nucleoside analogs are widely used in the treatment of cancer and viral-induced diseases. Efficacy of treatments relies upon a variety of events, including transport across tissue and target barriers, which determine drug pharmacokinetics and target cell bioavailability. To exert their action, nucleosides have to be chemically modified, thus compromising cellular uptake by those routes which are responsible for the uptake of natural nucleosides and nucleobases. In this review we will focus on established knowledge and recent advances in the understanding of nucleoside- and nucleobase-derived drug uptake mechanisms. Basically, these drug uptake processes involve the gene families SLC22, SLC28 and SLC29. These gene families encode Organic Anion Transporter (OAT)/Organic Cation Transporter (OCT), Concentrative Nucleoside Transporter (CNT) and Equilibrative Nucleoside Transporter (ENT) proteins, respectively. The pharmacological profiles of these plasma membrane carriers as well as their basic physiological and regulatory properties, including their tissue and subcellular distribution will be reviewed. This knowledge is crucial for the understanding of nucleoside- and nucleobase-derived drug bioavailability and therapeutic action. Moreover, changes in both transporter expression and/or transporter function (for instance as a consequence of gene variability) might also modulate response to treatment, thereby anticipating a putative diagnostic and predictive added value to the analysis of transporter expression and their corresponding genetic variants.

Sequestration of amitriptyline by liposomes

We study the uptake of amitriptyline, which is a common cause of overdose-related fatalities, in aqueous solutions by 1 , 2 -dimyristoyl- sn-glycero-3-phosphocholine (DMPC) liposomes and liposomes composed of a mixture of DMPC and 1 , 2 -dioleoyl- sn-glycero-3-[phospho- rac(1-glycerol)] (DOPG) lipids. The effect of drug concentration, liposomal charge, pH, salt, and protein presence on the drug uptake is investigated using two different methodologies, a precipitation and a centrifugation method. Furthermore, the time scale of the drug uptake is studied through qualitative observations at high pH and through conductivity measurements at neutral pH and found to be < 5 s . The results of the quantitative studies show that the fractional drug uptake decreases with increasing drug concentration, and for a given concentration it increases with the pH and decreases in the presence of salt. We find that a larger amount of drug is sequestered by negatively charged liposomes (those containing DOPG) than liposomes with no net charge (DMPC). We speculate that the mechanism of drug uptake is due to both electrostatic interactions as well as hydrophobic effects. The fractional uptake by DMPC:DOPG in a 70:30 ratio is as high as 95% in water and about 90% in physiological buffer. The fractional uptake is also measured in presence of 2% (w/w) bovine serum albumin (BSA), which is approximately the protein concentration in the intercellular fluid. In presence of protein the fractional uptakes by 70:30 DMPC:DOPG liposomes and 50:50 DMPC:DOPG liposomes are 82 and 90%, respectively, at 125 μM drug amitriptyline. In the absence of liposomes, 67% of the drug is taken up by the protein in a 2% (w/w) BSA, 125 μM amitriptyline solution. Thus, addition of 50:50 DMPC:DOPG liposomes reduces the free drug concentration by a factor of about 3.5, making them attractive candidates for drug detoxification.

The applicability of rat and human liver slices to the study of mechanisms of hepatic drug uptake

In the present study we investigated the applicability of the liver slice model to study mechanisms of drug uptake. Four model compounds were investigated that enter hepatocytes via entirely different membrane transport mechanisms. Rhodamine B (RB), which enters hepatocytes by passive diffusion, was homogeneously distributed throughout the rat liver slice (250 μm thickness) within 5 min, indicating that the penetration rate into the slice and the diffusion rate into the cells are rapid. In contrast, lucigenin (LU), which is taken up by hepatocytes through adsorptive endocytosis, was detected in the inner cell layers after 15 min. Digoxin uptake into the slice showed a temperature-dependent component and was stereoselectively inhibited by quinine, which is compatible with the involvement of a carrier-mediated uptake mechanism. The neo-glycoalbumin Lactose 27-Human Serum Albumin (Lact 27-HSA) and the negatively charged Succinylated-Human Serum Albumin (Suc-HSA) entered the slices and were taken up temperature-dependently into hepatocytes and endothelial cells, respectively. The liver slice preparation is a valuable tool to investigate the mechanisms of cellular uptake of drugs. Moreover, the precision-cut liver slices offer the unique possibility to study both hepatocyte and endothelial cell function in human and rat liver.