Drug Transport Research Articles

Characterization of two melanoma cell lines resistant to BRAF/MEK inhibitors (vemurafenib and cobimetinib)

BackgroundBRAF (v-raf murine sarcoma viral oncogene homolog B1)/MEK (mitogen-activated protein kinase kinase) inhibitors are used for melanoma treatment. Unfortunately, patients treated with this combined therapy develop resistance to treatment quite quickly, but the mechanisms underlying this phenomenon are not yet fully understood. Here, we report and characterize two melanoma cell lines (WM9 and Hs294T) resistant to BRAF (vemurafenib) and MEK (cobimetinib) inhibitors.MethodsCell viability was assessed via the XTT test. The level of selected proteins as well as activation of signaling pathways were evaluated using Western blotting. The expression of the chosen genes was assessed by RT-PCR. The distribution of cell cycle phases was analyzed by flow cytometry, and confocal microscopy was used to take photos of spheroids. The composition of cytokines secreted by cells was determined using a human cytokine array.ResultsThe resistant cells had increased survival and activation of ERK kinase in the presence of BRAF/MEK inhibitors. The IC50 values for these cells were over 1000 times higher than for controls. Resistant cells also exhibited elevated activation of AKT, p38, and JNK signaling pathways with increased expression of EGFR, ErbB2, MET, and PDGFRβ receptors as well as reduced expression of ErbB3 receptor. Furthermore, these cells demonstrated increased expression of genes encoding proteins involved in drug transport and metabolism. Resistant cells also exhibited features of epithelial-mesenchymal transition and cancer stem cells as well as reduced proliferation rate and elevated cytokine secretion.ConclusionsIn summary, this work describes BRAF/MEK-inhibitor-resistant melanoma cells, allowing for better understanding the underlying mechanisms of resistance. The results may thus contribute to the development of new, more effective therapeutic strategies.

FRAP analysis of peptide diffusion in extracellular matrix mimetic hydrogels as an in vitro model for subcutaneous injection

Subcutaneous (SC) injection is a common route of administration for drug compounds with poor oral bioavailability. However, bioavailability is often variable and incomplete, and there is as yet no standard accepted medium for simulation of the human SC environment. In this work we evaluate a FRAP based method for quantitative determination of local self-diffusion coefficients within extracellular matrix (ECM) mimetic hydrogels, potentially useful as in vitro models for drug transport in the ECM after SC injection. Gels were made consisting of either agarose, cross-linked collagen (COL) and hyaluronic acid (HA) or cross-linked HA. The diffusivities of uncharged FITC-dextran (FD4), the highly charged poly-lysine (PLK20) and poly-glutamic acid (PLE20) as well as the GLP-1 analogue exenatide were determined within the gels using FRAP. The diffusion coefficients in uncharged agarose gels were in the range of free diffusion in PBS. The diffusivity of cationic PLK20 in gels containing anionic HA was substantially decreased due to strong electrostatic interactions. Peptide aggregation could be observed as immobile fractions in experiments with exenatide. We conclude that the FRAP method provides useful information of peptides’ interactions and transport properties in hydrogel networks, giving insight into the mechanisms affecting absorption of drug compounds after subcutaneous injection.

Heterogeneous Organohydrogel Toward Automated and Interference-Free Gradient Feeding of Drugs in Cell Screening.

Cell-based microarrays are widely used in the fields of drug discovery and toxicology. Precise gradient generation and automated drug feeding are essential for high-throughput screening of live cells in tiny droplets. However, most existing technologies either require sophisticated robotic equipment or cause mechanical/physiological interference with cells. Here, a heterogeneous organohydrogel is presented for automated gradient drug feeding, while ensuring minimal interference with cells. The heterogeneous organohydrogel comprises three crucial components. The bottom surface can automatically generate gradients functioning as a gradient generator, the organohydrogel bulk allows unidirectional transport of drugs without backflow, and the top surface with hydrophilic arrays can firmly anchor the cell-based droplet array to evaluate the concentration-dependent bioeffects of drugs accurately. Such a unique structure enables universal screening of different cell types and drugs dissolved in different solvents, requiring neither additional accessories nor arduous drug functionalization. The heterogeneous organohydrogel with unprecedented automation and non-interference possesses the enormous potential to be a next-generation platform for drug screening.

Developmental Expression of Drug Transporters and Conjugating Enzymes Involved in Enterohepatic Recycling: Implication for Pediatric Drug Dosing.

Around 50% of the drugs used in children have never been tested for safety and efficacy in this vulnerable population. Immature drug elimination pathways can lead to drug toxicity when pediatric doses are determined using empirical methods such as body-surface area or body-weight-normalized adult dosing. In the absence of clinical data, physiologically-based pharmacokinetic (PBPK) modeling has emerged as a useful tool to predict drug pharmacokinetics in children. These models utilize developmental physiological data, including age-dependent differences in the abundance of drug-metabolizing enzymes and transporters (DMET), to mechanistically extrapolate adult pharmacokinetic data to children. The reported abundance data of hepatic DMET proteins in subcellular fractions isolated from frozen tissue are prone to high technical variability. Therefore, we carried out the proteomics-based quantification of hepatic drug transporters and conjugating enzymes in 50 pediatric and 8 adult human hepatocyte samples. Out of the 34 studied proteins, 28 showed a significant increase or decrease with age. While MRP6, OAT7, and SULT1E1 were highest in < 1-year-old samples, the abundance of P-gp and UGT1A4 was negligible in < 1-year-old samples and increased significantly after 1 year of age. Incorporation of the age-dependent abundance data in PBPK models can help improve pediatric dose prediction, leading to safer drug pharmacotherapy in children.

Human enteroid monolayers as a potential alternative for Ussing chamber and Caco-2 monolayers to study passive permeability and drug efflux.

After oral administration, the intestine is the first site of drug absorption, making it a key determinant of the bioavailability of a drug, and hence drug efficacy and safety. Existing non-clinical models of the intestinal barrier in vitro often fail to mimic the barrier and absorption of the human intestine. We explore if human enteroid monolayers are a suitable tool for intestinal absorption studies compared to primary tissue (Ussing chamber) and Caco-2 cells. Bidirectional drug transport was determined in enteroid monolayers, fresh tissue (Ussing chamber methodology) and Caco-2 cells. Apparent permeability (Papp) and efflux ratios for enalaprilat (paracellular), propranolol (transcellular), talinolol (P-glycoprotein (P-gp)) and rosuvastatin (Breast cancer resistance protein (BCRP)) were determined and compared between all three methodologies and across intestinal regions. Bulk RNA sequencing was performed to compare gene expression between enteroid monolayers and primary tissue. All three models showed functional efflux transport by P-gp and BCRP with higher basolateral to apical (B-to-A) transport compared to apical-to-basolateral (A-to-B). B-to-A Papp values were similar for talinolol and rosuvastatin in tissue and enteroids. Paracellular transport of enalaprilat was lower and transcellular transport of propranolol was higher in enteroids compared to tissue. Enteroids appeared show more region- specific gene expression compared to tissue. Fresh tissue and enteroid monolayers both show active efflux by P-gp and BCRP in jejunum and ileum. Hence, the use of enteroid monolayers represents a promising and versatile experimental platform to complement current in vitro models.

A patient-derived amyotrophic lateral sclerosis blood-brain barrier model for focused ultrasound-mediated anti-TDP-43 antibody delivery

BackgroundAmyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disorder with minimally effective treatment options. An important hurdle in ALS drug development is the non-invasive therapeutic access to the motor cortex currently limited by the presence of the blood-brain barrier (BBB). Focused ultrasound and microbubble (FUS+ MB) treatment is an emerging technology that was successfully used in ALS patients to temporarily open the cortical BBB. However, FUS+ MB-mediated drug delivery across ALS patients’ BBB has not yet been reported. Similarly, the effects of FUS+ MB on human ALS BBB cells remain unexplored.MethodsHere we established the first FUS+ MB-compatible, fully-human ALS patient-cell-derived BBB model based on induced brain endothelial-like cells (iBECs) to study anti-TDP-43 antibody delivery and FUS+ MB bioeffects in vitro.ResultsGenerated ALS iBECs recapitulated disease-specific hallmarks of BBB pathology, including reduced BBB integrity and permeability, and TDP-43 proteinopathy. The results also identified differences between sporadic ALS and familial (C9orf72 expansion carrying) ALS iBECs reflecting patient heterogeneity associated with disease subgroups. Studies in these models revealed successful ALS iBEC monolayer opening in vitro with no adverse cellular effects of FUS+ MB as reflected by lactate dehydrogenase (LDH) release viability assay and the lack of visible monolayer damage or morphology change in FUS+ MB treated cells. This was accompanied by the molecular bioeffects of FUS+ MB in ALS iBECs including changes in expression of tight and adherens junction markers, and drug transporter and inflammatory mediators, with sporadic and C9orf72 ALS iBECs generating transient specific responses. Additionally, we demonstrated an effective increase in the delivery of anti-TDP-43 antibody with FUS+ MB in C9orf72 (2.7-fold) and sporadic (1.9-fold) ALS iBECs providing the first proof-of-concept evidence that FUS+ MB can be used to enhance the permeability of large molecule therapeutics across the BBB in a human ALS in vitro model.ConclusionsTogether, this study describes the first characterisation of cellular and molecular responses of ALS iBECs to FUS+ MB and provides a fully-human platform for FUS+ MB-mediated drug delivery screening on an ALS BBB in vitro model.

Precision Medicine in Childhood Cancer: The Influence of Genetic Polymorphisms on Vincristine-Induced Peripheral Neuropathy.

Cancer is the leading cause of disease-related death among children. Vincristine (VCR), a key component of childhood cancer treatment protocols, is associated with the risk of peripheral neuropathy (PN), a condition that may be reversible upon drug discontinuation but can also leave lasting sequelae. Single nucleotide polymorphism (SNP) in genes involved in VCR pharmacokinetics and pharmacodynamics have been investigated in relation to an increased risk of PN. However, the results of these studies have been inconsistent. A retrospective cohort study was conducted to investigate the potential association of drug transporter genes from the ATP-binding cassette (ABC) family and the centrosomal protein 72 (CEP72) gene with the development of PN in 88 Caucasian children diagnosed with cancer and treated with VCR. Genotyping was performed using real-time PCR techniques for the following SNPs: ABCB1 rs1128503, ABCC1 rs246240, ABCC2 rs717620, and CEP72 rs924607. The results indicated that age at diagnosis (OR = 1.33; 95% CI = 1.07-1.75) and the ABCC1 rs246240 G allele (OR = 12.48; 95% CI = 2.26-100.42) were associated with vincristine-induced peripheral neuropathy (VIPN). No association was found between this toxicity and CEP72 rs924607. Our study provides insights that may contribute to optimizing childhood cancer therapy in the future by predicting the risk of VIPN.

Functionalization of a porous copper(ii) metal-organic framework and its capacity for loading and delivery of ibuprofen.

A porous copper(ii) metal-organic framework (MOF) of 4,4',4''-tri-tert-butyl-2,2':6',2''-terpyridine(N3ttb) and 5-nitroisophthalic acid (npd) formulated as [Cu(npd)(N3ttb)]·(DMF)(H2O) 1 (DMF = dimethylformamide) was synthesized and characterized by elemental analyses, spectroscopic techniques, single crystal X-ray crystallography, and scanning electron microscopy. Single crystal X-ray crystallographic analysis of the copper(ii) metal-organic framework reveals a monoclinic crystal system with space group P21/c. The copper(ii) ion is in a five-coordinate geometry consisting of three meridional nitrogen atoms of 4,4',4''-tri-tert-butyl-2,2':6',2''-terpyridine and two oxygen atoms of 5-nitroisophthalic acid to form a square pyramidal structure. The compound was functionalized with ethylenediamine (ED) to form [Cu(npd)(N3ttb)]-ED 2 that was characterized by FT-IR, PXRD, SEM-EDX and BET and the drug loading capacity was investigated and compared with that of as-synthesized MOFs. The amount of ibuprofen loaded was 916.44 mg g-1 (15.27%) & 1530.20 mg g-1 (25.50%) over 1 and 2, respectively. The results indicate that the functionalized MOFs 2 have a higher loading capacity for ibuprofen than 1 by 613.76 mg g-1 (10.23%), which could be ascribed to the acid-base interactions in the functionalized molecules. The results show that [Cu(npd)(N3ttb)]-ED 2 is a better drug transporter than [Cu(npd)(N3ttb)]·(DMF)(H2O) 1 due to the presence of an amine functional group that interacts with the acid group on the ibuprofen through non-covalent bonds interactions.

A Model for Reversible Electroporation to Deliver Drugs into Diseased Tissues.

Drug delivery through electroporation could be highly beneficial for the treatment of different types of diseased tissues within the human body. In this work, a mathematical model of reversible tissue electroporation is presented for injecting drug into the diseased cells. The model emphasizes the tissue boundary where the drug is injected as a point source. In addition, the effect of drug loss at tissue boundaries through extracellular space is studied elaborately. Multiple pulses are applied to deliver a sufficient amount of drug into the targeted cells. The set of differential equations that model the physical circumstances are solved numerically. This model obtains a mass transfer coefficient (MTC), in terms of pore fraction coefficient and drug permeability that controls the drug transport from extracellular to intracellular space. The drug penetration throughout the tissue is captured for the application of different pulses. The boundary effects on drug concentration are highlighted in this study. The advocated model is able to perform homogeneous drug transport into the cells so that the affected tissue is treated completely. This model can be applied to optimize clinical experiments by avoiding the lengthy and costly in vivo and in vitro experiments.

Age-Specific ADME Gene Expression in Infant Intestinal Enteroids.

In childhood, developmental changes and environmental interactions highly affect orally dosed drug disposition across the age range. To optimize dosing regimens and ensure safe use of drugs in pediatric patients, understanding this age-dependent biology is necessary. In this proof-of-concept study, we aimed to culture age-specific enteroids from infant tissue which represent its original donor material, specifically for drug transport and metabolism. Enteroid lines from fresh infant tissues (n = 8, age range: 0.3-45 postnatal weeks) and adult tissues (n = 3) were established and expanded to 3D self-organizing enteroids. The gene expression of drug transporters P-gp (ABCB1), BCRP (ABCG2), MRP2 (ABCC2), and PEPT1 (SLC15A1) and drug metabolizing enzymes CYP3A4, CYP2C18, and UGT1A1 was determined with RT-qPCR in fresh tissue and its derivative differentiated enteroids. Expression levels of P-gp, BCRP, MRP2, and CYP3A4 were similar between tissues and enteroids. PEPT1 and CYP2C18 expression was lower in enteroids compared to that in the tissue. The expression of UGT1A1 in the tissue was lower than that in enteroids. The gene expression did not change with the enteroid passage number for all genes studied. Similar maturational patterns in tissues and enteroids were visually observed for P-gp, PEPT1, MRP2, CYP3A4, CYP2C18, and VIL1. In this explorative study, interpatient variability was high, likely due to the diverse patient characteristics of the sampled population (e.g., disease, age, and treatment). To summarize, maturational patterns of clinically relevant ADME genes in tissue were maintained in enteroids. These findings are an important step toward the potential use of pediatric enteroids in pediatric drug development, which in the future may lead to improved pediatric safety predictions during drug development. We reason that such an approach can contribute to a potential age-specific platform to study and predict drug exposure and intestinal safety in pediatrics.

Role of AcrAB-TolC and Its Components in Influx-Efflux Dynamics of QAC Drugs in Escherichia coli Revealed Using SHG Spectroscopy.

Multidrug efflux pumps, especially those belonging to the class of resistance-nodulation-division (RND), are the key contributors to the rapidly growing multidrug resistance in Gram-negative bacteria. Understanding the role of efflux pumps in real-time drug transport dynamics across the complex dual-cell membrane envelope of Gram-negative bacteria is thus crucial for developing efficient antibiotics against them. Here, we employ second harmonic generation-based nonlinear spectroscopy to study the role of the tripartite efflux pump and its individual components. We systematically investigate the effect of periplasmic adaptor protein AcrA, inner membrane transporter protein AcrB, and outer membrane channel TolC on the overall drug transport in live Acr-type Escherichia coli and its mutant strain cells. Our results reveal that when one of its components is missing, the tripartite AcrAB-TolC efflux pump machinery in Escherichia coli can effectively function as a bipartite system, a fact that has never been demonstrated in live Gram-negative bacteria.

The effect of pregnancy-related hormones on hepatic transporters: studies with premenopausal human hepatocytes.

Pregnancy results in significant changes in drug pharmacokinetics (PK). While previous studies have elucidated the impact of pregnancy-related hormones (PRH) on mRNA or protein expression and activity of major hepatic metabolizing enzymes, their effect on hepatic drug transporters remains largely unexplored. Therefore, we investigated the effect of a cocktail of PRH on the mRNA expression and activity of hepatic transporters. Plated human hepatocytes (PHH) from 3 premenopausal donors were incubated, in triplicate, for 72h, with vehicle (DMSO < 0.01%), rifampin (10μM; positive control) or a cocktail of PRH consisting of estrone, estradiol, estriol, estetrol, progesterone, cortisol, testosterone, oxytocin, and placental growth hormone. The PRH concentrations replicated 0.1×, 1×, or 10× of the plasma concentrations of these hormones observed during each of the three trimesters of pregnancy. After treatment, mRNA expression (quantified by qPCR) of hepatic influx and efflux transporters as well as the activity of influx transporters was quantified (uptake of a selective substrate ± corresponding transporter inhibitor). The data were expressed relative to that in the control (vehicle) group. Significance was evaluated by ANOVA (followed by Dunn's multiple comparisons) or unpaired t-test when the within-lot data were analyzed, or repeated measures ANOVA (followed by Dunn's multiple comparisons) or paired t-test when data from all 3 lots were analyzed (p < 0.05). In general, a) PRH cocktails significantly induced transporter mRNA expression in the following order OAT2 ≈ NTCP ≈ OCT1 > OATP2B1 and repressed mRNA expression in the following order OATP1B3 > OATP1B1; b) these changes translated into significant induction of OAT2 (T1-T3) and NTCP (T2-T3, in only two lots) activity at the 1× PRH concentration. Compared with the influx transporters, the induction of mRNA expression of efflux transporters was modest, with mRNA expression of MRP2 and BSEP being induced the most. Once these data are verified through in vivo probe drug PK studies in pregnancy, they can be populated into physiologically based pharmacokinetic (PBPK) models to predict, for all trimesters of pregnancy, transporter-mediated clearance of any drug that is a substrate of the affected transporters.

Blood-Brain Barrier Permeability is Affected by Changes in Tight Junction Protein Expression at High-Altitude Hypoxic Conditions-this may have Implications for Brain Drug Transport.

Changes to blood-brain barrier structure and function may affect the delivery of drugs into the brain. It is worthwhile to exploring more study on how the blood-brain barrier changes in structure and function and how that affects drug transport in high-altitude hypoxic environment. The DIA high-throughput sequencing technique indicate that the rats blood-brain barrier has been identified to have 7252 proteins overall and 8 tight junction proteins, among which Claudin-7 was a plateau-specific tight junction protein under high-altitude hypoxia, and based on the interaction network study, 2421 proteins are found to interact with one another, with ZO-1 being the primary target. The results of the projected gene function analysis demonstrated that changes in tight junction proteins are related to the control of TRP channels by inflammatory mediators, the wnt signaling pathway, the ABC transporter system, and drug metabolism-CYP450 enzyme regulation. Additionally, the electron microscopy, the Evans blue combination with confocal laser scanning microscopy, and the Western Blot and RT-qPCR revealed that high-altitude hypoxic environment induces blood-brain barrier tight junctions to open, blood-brain barrier permeability increases, ZO-1, Occludin, Claudin-5 protein and mRNA expression decreased. Our research implies that structural and functional alterations in the blood-brain barrier induced by high altitude hypoxia may impact drug transport inside the central nervous system, and that drug transporters and drug-metabolizing enzymes may be key players in this process.

Aquasomes: A Promising Delivery Systems for Poorly Soluble Bioactives

Aquasomes are considered an excellent and efficient carrier system for the transport of drugs or biochemically active long chain macromolecules such as proteins and peptides, various hormones, antigens, enzymes, and genes in the recently burgeoning field of nanobiotechnology research. These are three-layer self-collecting structures composed of an oligomeric film covering a strong stage nanocrystalline centre to which biochemically dynamic particles adhere to, independent of changes in the environment and which self-assembles through non-covalent or ionic connections. Aquasomes are circular particles, 60–300 nm in size, that are used to deliver antigens and medications. The structural stability is provided by the solid core, while the active drug molecules are stabilized and protected from dehydration by the polyhydroxy oligomer covering. The most common way to distribute aquasome formulations is parenterally, however recent research indicates that there may be additional, oral, ways as well. A combination of targeted molecular shielding and prolonged release mechanism is used by aquasome to deliver their bioactive chemicals. The present paper offers an overview of the aquasome as a potentially useful medication delivery technique. It covers every facet of aquasome, such as their structure, the processes for preparing them, ways to characterize them, and medical uses as a drug delivery system.

Drug Metabolism and Transport Capacity of Endothelial Cells, Pericytes, and Astrocytes: Implications for CNS Drug Disposition.

Therapeutically targeting the brain requires interactions with endothelial cells, pericytes, and astrocytes at the blood brain barrier (BBB). We evaluated regional and cell-type specific drug metabolism and transport mechanisms using rhesus macaques and in vitro treatment of primary human cells. Here, we report heterogenous distribution of representative drugs, tenofovir (TFV), emtricitabine (FTC), and their active metabolites, which cerebrospinal fluid measures could not reflect. We found that all BBB cell types possessed functional drug metabolizing enzymes and transporters that promoted TFV and FTC uptake and pharmacologic activation. Pericytes and astrocytes emerged as pharmacologically dynamic cells that rivaled hepatocytes and were uniquely susceptible to modulation by disease and treatment. Together, our findings demonstrate the importance of considering the BBB as a unique pharmacologic entity, rather than viewing it as an extension of the liver, as each cell type possesses distinct drug metabolism and transport capacities that contribute to differential brain drug disposition.

ATP-binding cassette transporter inhibitor potency and substrate drug affinity are critical determinants of successful drug delivery enhancement to the brain

BackgroundPharmacotherapy for brain diseases is severely compromised by the blood-brain barrier (BBB). ABCB1 and ABCG2 are drug transporters that restrict drug entry into the brain and their inhibition can be used as a strategy to boost drug delivery and pharmacotherapy for brain diseases.MethodsWe employed elacridar and tariquidar in mice to explore the conditions for effective inhibition at the BBB. Abcg2;Abcb1a/b knockout (KO), Abcb1a/b KO, Abcg2 KO and wild-type (WT) mice received a 3 h i.p. infusion of a cocktail of 8 typical substrate drugs in combination with elacridar or tariquidar at a range of doses. Abcg2;Abcb1a/b KO mice were used as the reference for complete inhibition, while single KO mice were used to assess the potency to inhibit the remaining transporter. Brain and plasma drug levels were measured by LC-MS/MS.ResultsComplete inhibition of ABCB1 at the BBB is achieved when the elacridar plasma level reaches 1200 nM, whereas tariquidar requires at least 4000 nM. Inhibition of ABCG2 is more difficult. Elacridar inhibits ABCG2-mediated efflux of weak but not strong ABCG2 substrates. Strikingly, tariquidar does not enhance the brain uptake of any ABCG2-subtrate drug. Similarly, elacridar, but not tariquidar, was able to inhibit its own brain efflux in ABCG2-proficient mice. The plasma protein binding of elacridar and tariquidar was very high but similar in mouse and human plasma, facilitating the translation of mouse data to humans.ConclusionsThis work shows that elacridar is an effective pharmacokinetic-enhancer for the brain delivery of ABCB1 and weaker ABCG2 substrate drugs when a plasma concentration of 1200 nM is exceeded.

Dysregulation of Human Hepatic Drug Transporters by Proinflammatory Cytokines.

Proinflammatory cytokines, elevated during inflammation caused by infection and/or autoimmune disorders, result in reduced clearance of drugs eliminated primarily by cytochrome P450 enzymes (CYPs). However, the effect of cytokines on hepatic drug transporter expression or activity has not been well-studied. Here, using plated human hepatocytes (PHHs; n = 3 lots), we investigated the effect of interleukin (IL)-6, IL-1β, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ), on the mRNA expression and activity of hepatic drug transporters. PHHs were incubated for 72 hours at their pathophysiologically relevant plasma concentrations, both individually (0.01, 0.1, 1, 10 ng/ml) or as a cocktail (i.e., when each was combined at 0.1 or 1 ng/ml). Following cytokine cocktail exposure (1 ng/ml), significant downregulation of mRNA expression of organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, sodium/taurocholate cotransporting polypeptide (NTCP), breast cancer resistance protein (BCRP), P-glycoprotein (P-gp), multidrug and toxin extrusion protein 1, multidrug resistance proteins (MRP) 2, 3, and 4 was observed. While the mRNA expression of organic anion transporter (OAT) 2 and organic cation transporter (OCT) 1 was downregulated in two lots, it was upregulated in one lot. In agreement (mostly), the 1 ng/ml cytokine cocktail reduced OATP1B1/3, OATP2B1, OAT2, OCT1, and NTCP activity by 75%, 44%, 82%, 47%, and 80%, respectively. Interestingly, upregulation of OAT2 and OCT1 mRNA in one donor did not translate into the same directional change in activity. Although significant interlot variability was observed, in general, the above effects, using individual cytokines, could be attributed to IL-1β, TNF-α, and IFN-γ. SIGNIFICANCE STATEMENT: To date, this is the first comprehensive study to investigate the effect of four major proinflammatory cytokines, both individually and as a cocktail, on the mRNA expression and activity of human hepatic drug transporters. The data obtained can be used in the future to predict transporter-mediated drug clearance changes during inflammation through physiologically based pharmacokinetic modeling and simulation.

Proof of Concept of an All-in-One System for Measuring Hepatic Influx, Egress, and Metabolic Clearance Based on the Extended Clearance Concept.

Hepatic clearance (CLH ) prediction is a critical parameter to estimate human dose. However, CLH underpredictions are common, especially for slowly metabolized drugs, and may be attributable to drug properties that pose challenges for conventional in vitro absorption, distribution, metabolism, and elimination (ADME) assays, resulting in nonvalid data, which prevents in vitro to in vivo extrapolation and CLH predictions. Other processes, including hepatocyte and biliary distribution via transporters, can also play significant roles in CLH Recent advances in understanding the interplay of metabolism and drug transport for clearance processes have aided in developing the extended clearance model. In this study, we demonstrate proof of concept of a novel two-step assay enabling the measurement of multiple kinetic parameters from a single experiment in plated human primary hepatocytes with and without transporter and cytochrome P450 inhibitors-the hepatocyte uptake and loss assay (HUpLA). HUpLA accurately predicted the CLH of eight of the nine drugs (within twofold of the observed CLH ). Distribution clearances were within threefold of observed literature values in standard uptake and efflux assays. In comparison, the conventional suspension hepatocyte stability assay poorly predicted the CLH The CLH of only two drugs was predicted within twofold of the observed CLH Therefore, HUpLA is advantageous by enabling the measurement of enzymatic and transport processes concurrently within the same system, alleviating the need for applying scaling factors independently. The use of primary human hepatocytes enables physiologically relevant exploration of transporter-enzyme interplay. Most importantly, HUpLA shows promise as a sensitive measure for low-turnover drugs. Further evaluation across different drug characteristics is needed to demonstrate method robustness. SIGNIFICANCE STATEMENT: The hepatocyte uptake and loss assay involves measuring four commonly derived in vitro hepatic clearance endpoints. Since endpoints are generated within a single test system, it blunts experimental error originating from assays otherwise conducted independently. A key advantage is the concept of removing drug-containing media following intracellular drug loading, enabling the measurement of drug reappearance rate in media as well as the measurement of loss of total drug in the test system unencumbered by background quantities of drug in media otherwise present in a conventional assay.

Recent Updates on Interaction Studies and Drug Delivery of Antimalarials with Serum Albumin Proteins.

This review focuses on recent trends in the binding study of various antimalarial agents with serum albumins in detail. Serum albumin has a significant role in the transport of drugs and endogenous ligands. The nature and magnitude of serum albumin and drug interactions have a tremendous impact on the pharmacological behavior and toxicity of that drug. Binding of drug to serum albumin not only controls its free and active concentration, but also provides a reservoir for a long duration of action. This ultimately affects drug absorption, distribution, metabolism, and excretion. Such interaction determines the actual drug efficacy as the drug action can be correlated with the amount of unbound drug. With the advancement in spectroscopic techniques and simulation studies, binding studies play an increasingly important role in biophysical and biomedical science, especially in the field of drug delivery and development. This review assesses the insight we have gained so far to improve drug delivery and discovery of antimalarials on the basis of a plethora of drug-serum protein interaction studies done so far.

Three-dimensional culture of human proximal tubular epithelial cells for an in vitro evaluation of drug-induced kidney injury

Drug-induced kidney injury (DIKI) is the major cause of acute kidney injury (AKI). Renal proximal tubular epithelial cells (RPTECs) are the primary target sites of DIKI and express transporters involved in renal drug disposition. In the present study, we focused on three-dimensionally cultured human RPTECs (3D-RPTECs) with elevated expression of drug transporters to investigate their utility in DIKI evaluation. Intracellular ATP levels in 3D-RPTECs are reduced by tenofovir and cisplatin that are substrates of an organic anion transporter 1 and an organic cation transporter 2, respectively. In addition, 3D-RPTECs were exposed to 17 and 15 drugs that are positive and negative to RPTEC toxicity, respectively, for up to 28 d. The 20 % decreasing concentration of drugs for ATP amount (EC20) was obtained, and the ratio of EC20 values and clinical maximum concentration (Cmax) ≤100 were used as cut-off value to evaluate potential of DIKI. The sensitivities of 3D-RPTECs were 82.4 % and 88.2 % after 7 d and 28 d of drug exposure, respectively, and the specificities were 100 % and 93.3 %, respectively. The predictive performance of 3D-RPTECs was higher than that of two-dimensional cultured RPTECs and the kidney cell line HK-2. In conclusion, 3D-RPTECs are useful for in vitro evaluation of RPTEC injury by measuring intracellular ATP levels.