Presence Of P-glycoprotein Research Articles

Polyethylene glycol-phospholipid functionalized single-walled carbon nanotubes for enhanced siRNA systemic delivery

Small interfering RNAs (siRNA) technology has emerged as a promising therapeutic tool for human health conditions like cancer due to its ability to regulate gene silencing. Despite FDA-approved, their delivery remains localized and limiting their systemic use. This study used single-walled carbon nanotubes (SWNTs) functionalized with polyethylene glycolated (PEGylated) phospholipids (PL-PEG) derivatives for systemic siRNA delivery. We developed an siRNA systemic delivery vehicle (SWNT-siRNA) by conjugating SWNT functionalized with PL-PEG containing either amine (PA) or maleimide (MA). The functionalized SWNT with a lower molecular weight of PA produced the SWNT-siRNA conjugate system with the highest stability and high siRNA loading quantity. The system delivered siRNA to a panel of tumour cell lines of different organs (i.e. HeLa, H1299 and MCF-7) and a non-cancerous human embryonic kidney 293 cells (HEK293T) with high biocompatibility and low toxicity. The cellular uptake of SWNT-siRNA conjugates by epithelial cells was found to be energy dependent. Importantly, the presence of P-glycoprotein, a marker for drug resistance, did not inhibit SWNT-mediated siRNA delivery. Mouse xenograft model further confirmed the potential of SWNT-siRNA conjugates with a significant gene knock-down without signs of acute toxicity. These findings pave the way for potential gene therapy applications using SWNTs as delivery vehicles.

Safety considerations with the current treatments for peripheral T-cell lymphoma

ABSTRACT Introduction Peripheral T-Cell Lymphomas (PTCL) constitute a heterogeneous group of aggressive T – and natural killer (NK)-cell disorders and are associated with a poor prognosis. Frontline treatments often consist of anthracycline-based combination chemotherapy with the exception of NK-T cell lymphomas, where such combinations are ineffective due to the presence of P-glycoprotein which leads to multidrug resistance. Infectious and immune mediated side effects might be more pronounced in or unique to T-cell lymphomas due to the selection of agents which target multiple T-cell subtypes and also an immunocompromised state induced by the lymphomas themselves. Areas covered This review provides a comprehensive overview of safety considerations of treatment regimens used for peripheral T-cell lymphomas. We cover regimens used in both frontline and relapsed settings including combination chemotherapy, single agent chemotherapies and immunotherapies. Expert opinion Treatment of T-cell lymphomas often requires sequencing of several therapies due to lower efficacy of available treatment regimens in curing the disease compared to that seen in B-cell non-Hodgkin lymphomas. In addition, certain complications are more common in T-cell lymphomas due to their unique immunobiology. An understanding of these salient aspects is important for all providers who treat patients with this challenging disease group.

Glabratephrin reverses doxorubicin resistance in triple negative breast cancer by inhibiting P-glycoprotein

Triple-negative breast cancer is one of the most aggressive breast cancer. The first therapeutic option is chemotherapy, often based on anthracycline as doxorubicin. However, chemotherapy efficacy is limited in by the presence of P-glycoprotein (Pgp), a membrane transporter protein that effluxes doxorubicin, reducing its cellular accumulation and toxicity. Inhibiting Pgp activity with effective and non-toxic products is still an open challenge. In this work, we demonstrated that the natural product Glabratephrin (Glab), a prenylated flavonoid from Tephrosia purpurea with a unique chemical structure, increased doxorubicin accumulation and cytotoxicity in triple negative breast cancer cells with high levels of Pgp, characterized by both acquired or intrinsic resistance to doxorubicin. Glab also reduced the growth of Pgp-expressing tumors, without adding significant extra-toxicities to doxorubicin treatment. Interestingly, Glab did not change the expression of Pgp, but it reduced the affinity for Pgp and the efflux of doxorubicin, as suggested by the increased Km and the reduced Vmax. In silico molecular docking predicted that Glab binds two residues (phenylalanine 322, glutamine 721) localized in the transmembrane domains of Pgp, facing the extracellular environment. Moreover, site-directed mutagenesis identified glycine 185 as a critical residue mediating the reduced catalytic efficacy of Pgp elicited by Glab. We propose Glab as an effective and safe compound able to reverse doxorubicin resistance mediated by Pgp in triple negative breast cancers, opening the way to a new combinatorial approach that may improve chemotherapy efficacy in the most refractory and aggressive breast cancer.

Pleiotropic activities of nitric oxide-releasing doxorubicin on P-glycoprotein/ABCB1

Doxorubicin is one of the first-line chemotherapeutic drugs for osteosarcoma, but the rate of success is below 60% of patients. The main cause of this low success is the presence of P-glycoprotein (P-gp/ABCB1) that effluxes the drug, limiting the intracellular accumulation and toxicity of Doxorubicin. P-gp also inhibits immunogenic cell death promoted by Doxorubicin. Nitric oxide-releasing Doxorubicin is a synthetic anthracycline effective against P-gp-positive osteosarcoma cells. It is not known how it impacts on P-gp expression and immunogenic cell death induction. To address this point, we treated human Doxorubicin-sensitive osteosarcoma U-2OS cells and their resistant variants with increasing amount of P-gp, with Dox and Nitric oxide-releasing Doxorubicin. While Doxorubicin was cytotoxic only in U-2OS cells, Nitric oxide-releasing Doxorubicin maintained its cytotoxic properties in all the resistant variants. Nitric oxide-releasing Doxorubicin elicited a strong nitrosative stress in whole cell extracts, endoplasmic reticulum and plasma membrane. P-gp was nitrated in all these compartments. The nitration caused protein ubiquitination and lower catalytic efficacy. The removal of P-gp from cell surface upon Nitric oxide-releasing Doxorubicin treatment disrupted its interaction with calreticulin, an immunogenic cell death-inducer that is inhibited by P-gp. Drug resistant cells treated with Nitric oxide-releasing Doxorubicin exposed calreticulin, were phagocytized by dendritic cells and expanded anti-tumor CD8+ T-lymphocytes. The efficacy of Nitric oxide-releasing Doxorubicin was validated in Dox-resistant osteosarcoma xenografts and was higher in immune-competent humanized mice than in immune-deficient mice, confirming that part of Nitric oxide-releasing Doxorubicin efficacy relies on the restoration of immunogenic cell death. Nitric oxide-releasing Doxorubicin was a pleiotropic anthracycline reducing activity and expression of P-gp, and restoring immunogenic cell death. It can be an innovative drug against P-gp-expressing/ Doxorubicin-resistant osteosarcomas.

Discovery of a Tin-Piperonal-Entecavir Schiff Base Compound That Overcomes Multidrug Resistance by Inhibiting P-Glycoprotein.

The presence of P-glycoprotein in the human intestine represents a significant barrier to effective drug therapy. These proteins form a multidrug-resistant barrier to most drugs, especially those administered orally. Thus, strategies are needed to prepare molecules to combat these resistant proteins and enable an increase in drug efficacy. We developed a novel tin-Schiff base complex using an ultrasonic bath, a new technique in small molecule synthesis. New bond formation was confirmed using ultraviolet and Fourier transform spectroscopies. A computational study was carried out using Absorption, Distribution, Metabolism, Excretion and Toxicity software. The novel tin-entecavir (ETV)-piperonal Schiff base acts as a potent P-glycoprotein inhibitor, which overcomes the multidrug resistance to all drugs that are substrates for P-glycoprotein. Further study showed that the novel tin complex is less toxic than the parent compound at the same dose. The development of this tin-piperonal-ETV Schiff base complex is a major breakthrough for overcoming multidrug resistance barriers and can be applied to other drug molecules.

Intranasal administration of carbamazepine-loaded carboxymethyl chitosan nanoparticles for drug delivery to the brain

Epilepsy is considered as a common and diverse set of chronic neurological disorders and its symptoms can be controlled by antiepileptic drugs (AEDs). The presence of p-glycoprotein and multi-drug resistance transporters in the blood-brain barrier could prevent the entry of AEDs into the brain, causing drug resistant epilepsy. To overcome this problem, we propose using carboxymethyl chitosan nanoparticles as a carrier to deliver carbamazepine (CBZ) intra-nasally with the purpose to bypass the blood-brain barrier thus to enhance the brain drug concentration and the treatment efficacy. Results so far indicate that the developed CBZ-NPs have small particle size (218.76 ± 2.41 nm) with high drug loading (around 35%) and high entrapment efficiency (around 80%). The in vitro release profiles of CBZ from the NPs are in accordance with the Korsmeyer-peppas model. The in vivo results show that both encapsulation of CBZ in nanoparticles and the nasal route determined the enhancement of the drug bioavailability and brain targeting characteristics.

Biological functions of exosomes in the liver in health and disease.

Biological functions of exosomes in the liver in health and disease.

Intercellular Transfer of P-Glycoprotein from the Drug Resistant Human Bladder Cancer Cell Line BIU-87 Does Not Require Cell-to-Cell Contact

The efflux activity of transmembrane P-glycoprotein prevents various therapeutic drugs from reaching lethal concentrations in cancer cells, resulting in multidrug resistance. We investigated whether drug resistant bladder cancer cells could transfer functional P-glycoprotein to sensitive parental cells. Drug sensitive BIU-87 bladder cancer cells were co-cultured for 48 hours with BIU-87/ADM, a doxorubicin resistant derivative of the same cell line, in a Transwell® system that prevented cell-to-cell contact. The presence of P-glycoprotein in recipient cell membranes was established using fluorescein isothiocyanate, laser scanning confocal microscopy and Western blot. P-glycoprotein mRNA levels were compared between cell types. Rhodamine 123 efflux assay was done to confirm that P-glycoprotein was biologically active. The amount of P-glycoprotein protein in BIU-87 cells co-cultured with BIU-87/ADM was significantly higher than in BIU-87 cells (0.44 vs 0.25) and BIU-87/H33342 cells (0.44 vs 0.26, each p <0.001), indicating P-glycoprotein transfer. P-glycoprotein mRNA expression was significantly higher in BIU-87/ADM cells than in co-cultured BIU-87 cells (1.28 vs 0.30), BIU-87/H33342 (0.28) and BIU-87 cells (0.25, each p <0.001), ruling out a genetic mechanism. After 30 minutes of efflux, rhodamine 123 fluorescence intensity was significantly lower in BIU-87/ADM cells (5.55 vs 51.45, p = 0.004) and co-cultured BIU-87 cells than in BIU-87 cells (14.22 vs 51.45, p <0.001), indicating that P-glycoprotein was functional. Bladder cancer cells can acquire functional P-glycoprotein through a nongenetic mechanism that does not require direct cell contact. This mechanism is consistent with a microparticle mediated process.

Study on intestinal absorption and pharmacokinetic characterization of diester diterpenoid alkaloids in precipitation derived from Fuzi–Gancao herb-pair decoction for its potential interaction mechanism investigation

Ethnopharmacological relevanceAconitum carmichaelii Debx. (Fuzi in Chinese) has been widely clinically used to treat heart failure and rheumatism. Whereas its serious toxicity, Radix et Rhizoma Glycyrrhizae (Gancao in Chinese) was combined with it as traditional Chinese medicine (TCM) herb-pair for toxicity reduction and pharmacological effect improvement. Though some previous viewpoints about that has been reported, the underlying interaction mechanism of two herbs remain unknown and definitely worthy of investigating. Aim of studyIn present study, we focus on Fuzi–Gancao herb-pair precipitation (FGP), considering it related to the compatibility mechanism of Fuzi–Gancao herb-pair. The intestinal absorption and pharmacokinetic characters of 3 diester diterpenoid alkaloids in the precipitation were investigated. Materials and methodsBoth everted gut sac model and in situ single-pass intestinal perfusion model were used to investigate rat small intestinal permeability and transport mechanism of aconitine, hypaconitine and mesaconitine. Moreover, by means of determination of the plasma concentration, the pharmacokinetic characters of 3 alkaloid compounds in rats have been developed. ResultsIn everted gut sac permeability experiment, the permeability of hypaconitine appeared best in ileum. Furthermore, their uptakes were increased in the presence of P-glycoprotein (P-gp) inhibitors. In situ single-pass intestinal perfusion uptake experiment, results revealed that the transport mechanism may fit the active transport mechanism. And 3 alkaloids in FPG could be absorbed well in rats, fitting 2-compartment model with 1st order absorption and lag time. ConclusionsOur results in present study indicated that 3 diester diterpenoid alkaloids in FGP could be dissolved out in gastrointestinal tract firstly and then absorbed in blood after oral administration, which could result in prolonging their mean residence time and adding their absorbed doses, avoiding dose dumping. The current study has significant enlightenments for further investigation on the interaction mechanisms of other acid-base herb-pairs as well as Fuzi–Gancao herb-pair.

Amonafide: a potential role in treating acute myeloid leukemia

Introduction: Amonafide is a novel topoisomerase II (Topo II) inhibitor and DNA intercalator that induces apoptotic signaling by blocking the binding of Topo II to DNA. Amonafide retains cytotoxic activity even in the presence of P-glycoprotein (Pgp)-mediated multi-drug resistance (MDR), a major contributor to clinical treatment failure.Areas covered: In vitro, Pgp-mediated transport (efflux) of amonafide from myeloblasts obtained from patients with secondary acute myeloid leukemia (sAML) was significantly less than efflux of daunorubicin. Amonafide has shown efficacy in patients with sAML, as well as in patients with poor prognostic characteristics such as older age and unfavorable cytogenetics, all associated with MDR. Improved antileukemic activity is observed when amonafide is given together with cytarabine, rather than as monotherapy, with a complete remission rate of ∼ 40% in a recent Phase II trial in sAML. The efficacy of amonafide was maintained among poor-risk subsets of patients, including older patients and patients who had previous myelodysplastic syndrome or previous leukemogenic therapy. The safety profile was acceptable and manageable.Expert opinion: Amonafide plus cytarabine may have clinical utility in patients with sAML and in other poor-risk subgroups of acute myeloid leukemia (AML). Ongoing trials will help define the role for amonafide in the treatment of poor-risk AML.

Studies on Function of P-Glycoprotein in Photodynamic Therapy of Endometriosis

The aim of the present study was to examine whether the effects of endometriosis-targeted photodynamic therapy (PDT), dependent on 5-aminolevulinic acid (ALA), rely on the presence of P-glycoprotein (P-gp), which is regarded as constituting one of the causes of multidrug resistance phenomenon. The significance of the undertaken studies reflects the complete absence of reports related to the modulation of P-gp activity and efficacy of PDT in patients with endometriosis. Tissue samples of normal endometria were obtained from eight women after hysterectomy who were diagnosed with cervical intra-epithelial neoplasia. Fragments of ovarian endometriosis were obtained from 15 women. Epithelial cells were isolated from the material and in in vitro conditions were preincubated with P-gp blocker-verapamil-before ALA-PDT. The cytotoxicity was evaluated using the XTT test, allowing us to estimate cell growth inhibition. Statistical analysis of the results involved the nonparametric Wilcoxon paired rank test and the Mann-Whitney U-test using the Statistica v5 software (p < 0.05). In parallel, P-gp presence in the analyzed material was evaluated using immunohistochemistry. In normal endometrial epithelium, verapamil was shown to intensify phototoxic effects at 2 and 4 mmol/L ALA (p < 0.05). In endometriotic epithelium, such intensification was noted in all examined concentrations of ALA (p < 0.001). Moreover, the ectopic epithelial cells were more sensitive than eutopic epithelial cells to PDT upon ALA alone, as well as after preincubation with verapamil. Immunohistohemical analysis allowed us to demonstrate the absence of glycoprotein P in normal endometrium. In endometriosis, P-gp was localised in both the epithelium and the stroma of the examined material. Phototoxic effects could be amplified in epithelial cells of endometriotic foci by appropriate action of verapamil and 5-aminolevulinic acid.

Effect of the presence of P-glycoprotein (MDR1) on the ability of AML patients to achieve complete remission: Results of a meta-analysis of the literature.

6586 Background: The expression of P-gp is highly prevalent in acute myeloid leukemia (AML). Current treatment options, including anthracyclines and anthracenediones, are substrates for P-gp. A meta-analysis was carried out to evaluate the influence of P-gp on treatment outcome in AML. Methods: Studies investigating P-gp (function and/or protein and/or mRNA) in AML and reporting the relevant outcomes (in particular, complete remission [CR]) were considered for this meta-analysis. Only patients diagnosed with AML, and who either proceeded to treatment with anthracycline drugs or mitoxantrone, or who had previously been treated with anthracyclines or mitoxantrone, were eligible to be included in the meta-analysis. The primary analysis population for the meta-analysis was the evaluable population (EP), that is, all patients from included studies whose P-gp status was known. Studies for inclusion in the meta-analysis were identified in a thorough literature search, including the PubMed database, using a pre-specified search algorithm. The incidence of P-gp was tabulated by publication and overall. The odds ratio (OD) for CR (positive versus negative P-gp status) was tabulated by publication with 95% confidence interval (CI). Results: 74 studies were included in the analysis. Individual study odds ratios were calculated from 53 studies which provided 4,069 patients evaluable for both CR and P-gp status. The median CR rate across all studies was 65%; CR rates were 78% in Pgp- and 47% in Pgp+ patients. Of the 53 publications, 47 gave OR < 1, including 30 with statistical significance (p < 0.05). Of the 6 publications which gave OR > 1, none reached statistical significance. The overall pooled odds ratio for CR (positive versus negative P-gp status) was 0.302 (95% CI, 0.260-0.350). Conclusions: CR is much less likely to occur in patients with P-gp expression. These patients should be considered for treatment options that are unaffected by P-gp. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Antisoma Antisoma Antisoma

Methods for estimation of brain active site concentration of drugs and other chemicals

Event Abstract Back to Event Methods for estimation of brain active site concentration of drugs and other chemicals Margareta Hammarlund-Udenaes1* 1 Uppsala University, Division of Pharmacokinetics and Drug Therapy, Department of Pharmaceutical Biosciences, Sweden Brain active site concentrations are defined as the unbound, pharmacologically active concentrations at the site of action. The site of action may be located extracellularly in the brain interstitial fluid (ISF) or at some intracellular target within the parenchymal cells. Unbound blood concentrations are not representative of the brain ISF concentrations when the drug is exposed to active efflux or influx transport at the blood-brain barrier (BBB). The brain ISF concentrations may neither be representative of the intracellular concentrations depending on type of transport to the intracellular target. Methods intended for estimation of brain active site concentrations have to directly measure or estimate the unbound concentration. Recent development has made it possible to measure and/or estimate brain ISF concentrations, and also to estimate average intracellular concentrations. When discussing methods, it is important to distinguish between methods for estimation of rate of transport across the BBB and methods for estimation of the extent of equilibration between brain and blood [1]. Examples of "rate methods" are in situ brain perfusion, brain efflux index and cell culture models that measure the permeability surface area product or uptake clearance into the brain. Only the "extent methods" can be used to estimate active site concentrations. The extent of equilibration can be determined for total drug (Kp) or unbound drug (Kp,uu). Kp,uu is by definition the ratio of unbound concentrations in brain ISF and blood. Total drug ratios can be determined with i.e. logBB methods and with PET. A simple method with which to determine Kp is to administer an i.v. infusion to steady state [1, 2]. A suggestion is to administer an infusion for 4 hours to rats and thereafter take brain and blood samples (Fig. 1). In order to obtain Kp,uu, the infusion should be combined with plasma protein binding determination and brain slice measurement. This method increases the possibility of obtaining good estimations of unbound target concentrations already in the drug discovery setting. Unbound ratios can be measured directly with microdialysis for drugs that do not stick to tubings and probes. The unspecific binding in brain can be determined with Vu,brain or fu,brain measurements, where Vu,brain is the unbound volume of distribution in brain (Fig. 1), and fu,brain is the unbound fraction in brain. Vu,brain is equal to 1/fu,brain under certain circumstances. CSF has for long been valued as a substitute for unbound brain concentrations but with a lack of comparison. This has mainly been due to the lack of methods to measure brain ISF concentrations. The unbound concentration ratio between CSF and blood (Kp,uu,CSF) was recently evaluated as a substitute for Kp,uu [3]. There was a rather good correlation (Fig. 2 A). However, the CSF ratio over-predicted the brain Kp,uu ratio at low values, and underpredicted the ratio at high values. At low values, this can be explained as caused by the presence of P-glycoprotein (P-gp) at the BBB but a different function of P-gp at the bloodcerebrospinal fluid barrier (BCSFB). At high values, it is speculated that active uptake transporters are present in the BBB but not in the BCSFB. Human and rat data had the same rank-order, but human Kp,uu,CSF values were on average 3-fold higher than those in rats (Fig. 2 B). Intracellular exposure can be addressed with a combination of the brain slice and the brain homogenate methods [4]. Development is ongoing to further sub-divide estimations into cellular components. In conclusion, methods for estimation of brain active site concentrations need to measure or estimate the unbound moiety. There is a rather limited availability of methods, but recent development has made it possible to increase the knowledge about the relationship between brain active site concentrations and unbound concentrations in blood. Pic 1 Pic 2

Use of Pre-Clinical In Vitro and In Vivo Pharmacokinetics for the Selection of a Potent Hepatitis C Virus Protease Inhibitor, Boceprevir, for Clinical Development

Boceprevir is a novel hepatitis C virus (HCV) protease inhibitor undergoing clinical investigation for use in the treatment of human HCV infection. Preclinical in vivo pharmacokinetic studies have been performed in rat, dog, and/or monkey after single administration by oral and intravenous routes for about one thousand compounds prior to the selection of boceprevir as the development candidate. In vitro pharmacokinetic assessments of metabolic stability, cell permeability, and plasma protein binding have been performed. The compound has physicochemical properties (molecular weight of 519.7 and moderate lipophilicity and H-bonding functionalities) that are borderline for good pharmacokinetic behavior. Absorption in animals ranges from 12 to 37%, indicating incomplete absorption. Based on in vitro permeability studies in Caco-2 cells, boceprevir appears to be an efflux substrate displaying a saturable efflux profile at high dosing concentrations and a reversible efflux profile in the presence of P-glycoprotein (P-gp) inhibitors. Boceprevir displayed a rather high liver/plasma average ratio of 30 in rats, indicating good uptake by the target tissue. The rat liver/plasma ratio of boceprevir appeared to be higher than those for compounds that are under clinical evaluation. Based on the preclinical pharmacokinetic data, boceprevir appears to have limited absorption, but reasonable liver distribution which is a primary factor for selecting boceprevir as a development candidate. Recent clinical proof-of-concept study confirmed that boceprevir is efficacious in reducing the viral load in patients. Keywords: HCV protease inhibitor, In vivo pharmacokinetics, In vitro pharmacokinetics

P-Glycoprotein is not present in mitochondrial membranes

Recent reports have indicated the presence of P-glycoprotein in crude mitochondrial membrane fractions, leading to the assumption that P-glycoprotein is present in mitochondrial membranes, and may be involved in transport across these membranes. To determine the validity of this claim, two cell lines overexpressing endogenous P-glycoprotein were investigated. Using various centrifugation steps, mitochondria were purified from these cells and analyzed by Western blot reaction with the anti-P-glycoprotein antibody C219 and organelle-specific antibodies. While P-glycoprotein is present in crude mitochondrial fractions, these fractions are contaminated with plasma membranes. Further purification of the mitochondria to remove plasma membranes revealed that P-glycoprotein is not expressed in mitochondria of the KB-V1 (vinblastine-resistant KB-3-1 cells) or MCF-7 ADR (adriamycin-resistant MCF-7 cells) cell lines. To further substantiate these findings, we used confocal microscopy and the anti-P-glycoprotein antibody 17F9. This demonstrated that in intact cells, P-glycoprotein is not present in mitochondria and is primarily localized to the plasma membrane. These findings are consistent with the role of P-glycoprotein in conferring multidrug resistance by decreasing cellular drug accumulation. Therefore, contrary to previous speculation, P-glycoprotein does not confer cellular protection by residing in mitochondrial membranes.

Quantitative Immunoassay To Measure Plasma and Intracellular Atazanavir Levels: Analysis of Drug Accumulation in Cultured T Cells

We have developed an enzyme immunoassay to measure atazanavir (ATV) levels in plasma and cells. Anti-ATV polyclonal antibodies were raised in rabbits by using a synthetic ATV derivative coupled to bovine serum albumin as the immunogen, and the enzyme tracer was prepared by chemically coupling the ATV derivative with acetylcholinesterase. These reagents were used to develop a sensitive competitive enzyme immunoassay performed in microtitration plates, and the lowest limit of quantification was 150 pg/ml, which is about 10 times more sensitive than previously published techniques. The plasma assay was performed, after a simple methanol extraction, with a minimum of 30 microl of plasma. This assay showed good precision and efficiency, since the rates of recovery from human plasma and cell extracts spiked with ATV ranged form 93 to 113%, with coefficients of variation of less than 10%. ATV concentrations were measured in peripheral blood mononuclear cells incubated with various ATV concentrations and in CEM cells in the absence or presence of antiretroviral drugs and drug transporter inhibitors. The results indicated a dose-dependent uptake (intracellular concentration/extracellular concentration ratio range, 0.04 to 19). A significant increase in the accumulation of ATV was noticed in the presence of P-glycoprotein and MRP1 inhibitors (dipyridamole, inter alia). Interestingly, efavirenz significantly increased the baseline accumulation of ATV, whereas nevirapine induced a marked reduction. This new enzyme immunoassay for measuring plasma and intracellular ATV levels was fully validated and provides an inexpensive and useful tool for routine therapeutic drug monitoring. Moreover, in vitro results suggested the implication of drug transporters and interactions with other antiviral drugs that should be further explored in human immunodeficiency virus-infected patients.

Evaluation of the immortalized human brain capillary endothelial cell line BB19 as a human cell culture model for the blood–brain barrier

In vitro models of the blood–brain barrier (BBB) play a major role in the study of BBB permeability of drug candidates. However, most established in vitro models use cells of non-human origin, which is not optimal for the prediction of brain permeability in humans. The aim of this study was to assess the human brain capillary endothelial cell line BB19 for its usefulness as an in vitro model of the human BBB. Restrictive tight junctions are a prerequisite for drug transport studies. Sucrose permeability of BB19 cells on different filters was compared to porcine brain capillary endothelial cells (BCEC). Tightness of BB19 cell monolayers still needs further optimization. Hardly any discrimination between Sucrose and Propranolol ( P app = 1.30 × 10 −5 vs. 2.18 × 10 −5 cm/s) was seen. Cells showed an improvement towards a more primary BCEC morphology with C6 conditioned medium, dexamethasone, and 1,25-dihydroxyvitamin D 3. The presence of P-glycoprotein (P-gp), MRP4 and BCRP, ABC-transporters located in the BBB, has been shown on mRNA level, by immunostaining, and western blot. MRP1, MRP2, MRP5, OAT3, and OAT4 were also detected by RT-PCR. Functional properties of the BBB were shown with uptake of propranolol, morphine, and sucrose. Uptake studies with daunomycin and the P-gp inhibitor verapamil showed functional activity of P-gp. We conclude that BB19 cells might be feasible as a human in vitro model of the BBB for drug uptake studies. However, for the assessment of transport studies, further improvements of this model are necessary.

Enhanced oral paclitaxel absorption with vitamin E-TPGS: Effect on solubility and permeability in vitro, in situ and in vivo

Solubility and permeability being important determinants of oral drug absorption, this study was aimed to investigate the effect of d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) on the solubility and intestinal permeability of paclitaxel in vitro, in situ and in vivo, in order to estimate the absorption enhancement ability of TPGS. Aqueous solubility of paclitaxel is significantly enhanced by TPGS, where a linear increase was demonstrated above a TPGS concentration of 0.1 mg/ml. Paclitaxel demonstrated asymmetric transport across rat ileum with significantly greater (26-fold) basolateral-to-apical (B–A) permeability than that in apical-to-basolateral (A–B) direction. Presence of P-glycoprotein (P-gp) inhibitor, verapamil (200 μM), diminished asymmetric transport of paclitaxel suggesting the role of P-gp-mediated efflux. TPGS showed a concentration-dependent increase in A–B permeability and decreased B–A permeability. The maximum efflux inhibition activity was found at a minimum TPGS concentration of 0.1 mg/ml, however, further increase in TPGS concentration resulted in decreased A–B permeability with no change in B–A permeability. Thus, the maximum paclitaxel permeability attained with 0.1 mg/ml TPGS was attributed to the interplay between TPGS concentration dependent P-gp inhibition activity and miceller formation. In situ permeability studies in rats also demonstrated the role of efflux in limiting permeability of paclitaxel and inhibitory efficiency of TPGS. The plasma concentration of [ 14C]paclitaxel following oral administration (25 mg/kg) was significantly increased by coadministration of TPGS at a dose of 50 mg/kg in rats. Bioavailability is enhanced about 4.2- and 6.3-fold when [ 14C]paclitaxel was administrated with verapamil (25 mg/kg) and TPGS, respectively, as compared to [ 14C]paclitaxel administered alone. The effect of verapamil on oral bioavailability of [ 14C]paclitaxel was limited relative to the TPGS, consistent with the in vitro solubility and permeability enhancement ability of TPGS. In conclusion, the current data suggests that the coadministration of TPGS may improve the bioavailability of BCS class II–IV drugs with low solubility and/or less permeable as a result of significant P-gp-mediated efflux.

Membrane transport of camptothecin: facilitation by human P-glycoprotein (ABCB1) and multidrug resistance protein 2 (ABCC2)

BackgroundThe purpose of the present study was to continue the investigation of the membrane transport mechanisms of 20-(S)-camptothecin (CPT) in order to understand the possible role of membrane transporters on its oral bioavailability and disposition.MethodsThe intestinal transport kinetics of CPT were characterized using Caco-2 cells, MDCKII wild-type cells and MDCKII cells transfected with human P-glycoprotein (PGP) (ABCB1) or human multidrug resistance protein 2 (MRP2) (ABCC2). The effects of drug concentration, inhibitors and temperature on CPT directional permeability were determined.ResultsThe absorptive (apical to basolateral) and secretory (basolateral to apical) permeabilities of CPT were found to be saturable. Reduced secretory CPT permeabilities with decreasing temperatures suggests the involvement of an active, transporter-mediated secretory pathway. In the presence of etoposide, the CPT secretory permeability decreased 25.6%. However, inhibition was greater in the presence of PGP and of the breast cancer resistant protein inhibitor, GF120918 (52.5%). The involvement of additional secretory transporters was suggested since the basolateral to apical permeability of CPT was not further reduced in the presence of increasing concentrations of GF120918. To investigate the involvement of specific apically-located secretory membrane transporters, CPT transport studies were conducted using MDCKII/PGP cells and MDCKII/MRP2 cells. CPT carrier-mediated permeability was approximately twofold greater in MDCKII/PGP cells and MDCKII/MRP2 cells than in MDCKII/wild-type cells, while the apparent Km values were comparable in all three cell lines. The efflux ratio of CPT in MDCKII/PGP in the presence of 0.2 μM GF120918 was not completely reversed (3.36 to 1.49). However, the decrease in the efflux ratio of CPT in MDCKII/MRP2 cells (2.31 to 1.03) suggests that CPT efflux was completely inhibited by MK571, a potent inhibitor of the Multidrug Resistance Protein transporter family.ConclusionsThe current results provide evidence that PGP and MRP2 mediate the secretory transport of CPT in vitro. However, the involvement of other transporters cannot be ruled out based on these studies. Since these transporters are expressed in the intestine, liver and kidney variations in their expression levels and/or regulation may be responsible for the erratic oral absorption and biliary excretion of CPT observed in human subjects.

Uptake and efflux of quinacrine, a candidate for the treatment of prion diseases, at the blood-brain barrier.

1. A clinical trial of quinacrine in patients with Creutzfeldt-Jakob disease is now in progress. The permeability of drugs through the blood-brain barrier (BBB) is a determinant of their therapeutic efficacy for prion diseases. The mechanism of quinacrine transport across the BBB was investigated using mouse brain endothelial cells (MBEC4). 2. The permeability of quinacrine through MBEC4 cells was lower than that of sodium fluorescein, a BBB-impermeable marker. The basolateral-to-apical transport of quinacrine was greater than its apical-to-basolateral transport. In the presence of P-glycoprotein (P-gp) inhibitor, cyclosporine or verapamil, the apical-to-basolateral transport of quinacrine increased. The uptake of quinacrine by MBEC4 cells was enhanced in the presence of cyclosporine or verapamil. 3. Quinacrine uptake was highly concentrative, this event being carried out by a saturable and carrier-mediated system with an apparent Km of 52.1 microM. Quinacrine uptake was insensitive to Na+-depletion and changes in the membrane potential and sensitive to changes in pH. This uptake was decreased by tetraethylammonium and cimetidine, a substrate and an inhibitor of organic cation transporters, respectively. 4. These findings suggest that quinacrine transport at the BBB is mediated by the efflux system (P-gp) and the influx system (organic cation transporter-like machinery).