Increased Pgp Expression Research Articles

Non‐Nutritive Sweeteners Disturb P‐Glycoprotein Function and Liver Detoxification

Over the course of 9 months, nearly all pregnant women are exposed to toxins, potentially including environmental chemicals, prescription drugs, over-the-counter medicine, social substances, illicit drugs, etc. In this critical window of development, slight fetal exposures to toxic molecules can be teratogenic, leading to severe neurological and growth defects. In our previously published mouse study, we emphasized that pregnancy consumption of Non-Nutritive Sweeteners (NNS) increased fetal toxicity, including lower liver detoxification enzymes and increased intermediary metabolites. About 35% of adults in the United States acknowledge consuming NNS, seeking to limit sugars consumption and their adverse health effects. While increasingly prevalent in food products, three in four adults cannot identify NNS and may therefore be involuntary exposed. Although recognized as safe for the general population, NNS's effects on a specific population or period of human development are incompletely understood. According to various studies, NNS alter gut hormonal secretion, glucose absorption, appetite, kidney function, in vitro insulin secretion, and adipogenesis. Most NNS circulate in the body and are found in blood, urine, amniotic fluid, and breast milk and bind sweet receptors in target organs. Despite an apparent maternofetal transmission, little is known about NNS consequences during pregnancy on fetal toxicity. To protect the fetus, the placenta and both mother's and fetus' livers are essential cleansing organs. Mainly, transporters such as the P-Glycoprotein (Pgp) are critical to flush foreign metabolites out of the cells and back to mother's blood circulation. In rats, Abou-Donia et al. previously demonstrated that the intestinal Pgp and various Cytochromes P450 expression varies according to sucralose consumption.Therefore we hypothesize that NNS directly impact Pgp expression and activity in the liver, increasing fetal toxicity. Using human hepatic cells, we demonstrated that in vitro incubation of a combined sucralose and Acesulfame-K mixture significantly altered detoxification actors' expression, including aldehyde dehydrogenases and alcohol dehydrogenases, the aspartate aminotransferase (AST) and efflux transporters such as the Pgp. Individually, both sucralose and Acesulfame-K slightly decrease Pgp expression at low concentrations. On the contrary, elevated concentrations of both NNS increased Pgp expression in hepatic cells, suggesting an elevated detoxification response and often associated with multi-drug resistant cells. Finally, in both in vitro and cell-free assays, NNS inhibited Pgp-mediated drug transport. Altogether, these preliminary data suggest that NNS disturb drug metabolism by changing Pgp expression and function in vitro, and potentially participate in pregnancy complication and fetal development defects.

P-glycoprotein Expression Is Upregulated in a Pre-Clinical Model of Traumatic Brain Injury.

Athletes participating in contact sports are at risk for sustaining repeat mild traumatic brain injury (rmTBI). Unfortunately, no pharmacological treatment to lessen the pathophysiology of brain injury has received U.S. Food and Drug Administration (FDA) approval. One hurdle to overcome for potential candidate agents to reach effective therapeutic concentrations in the brain is the blood–brain barrier (BBB). Adenosine triphosphate (ATP)-binding cassette (ABC) transporters, such as P-glycoprotein (Pgp), line the luminal membrane of the brain capillary endothelium facing the vascular space. Although these transporters serve to protect the central nervous system (CNS) from damage by effluxing neurotoxicants before they can reach the brain, they may also limit the accumulation of therapeutic drugs in the brain parenchyma. Thus, increased Pgp expression following brain injury may result in reduced brain availability of therapeutic agents.We therefore questioned if repeat concussive injury increases Pgp expression in the brain. To answer this question, we used a rodent model of repeat mild closed head injury (rmCHI) and examined the messenger RNA (mRN) and protein expression of both isoforms of rodent Pgp (Abcb1a and Abcb1b). Compared with sham-operated controls (n = 5), the mRNA levels of both Abcb1a and Abcb1b were found to be increased in the hippocampus at day 1 (n = 5) and at day 5 (n = 5) post-injury. Using a validated antibody, we show increased immunolabeling for Pgp in the dorsal cortex at day 5 and in the hippocampus at day 1 (n = 5) and at day 5 (n = 5) post-injury compared with sham controls (n = 6). Taken together, these results suggest that increased expression of Pgp after rmCHI may reduce the brain accumulation of therapeutic drugs that are Pgp substrates. It is plausible that including a Pgp inhibitor with a candidate therapeutic agent may be an effective approach to treat the pathophysiology of rmCHI.

Relationship of MDR1 gene polymorphism and P-glycoprotein expression in Chinese refractory lupus nephritis

To evaluate the association of multidrug resistance 1 (MDR1) polymorphism and the expression of P-glycoprotein (Pgp) in Chinese refractory lupus nephritis (LN) patients. Polymerase chain reaction-direct sequencing was used to analyze MDR1 polymorphism. The genotype distribution of MDR1 polymorphism in 132 SLE (systemic lupus erythematosus) patients was evaluated. ELISA was used to measure the expression of Pgp. Relationship among Pgp expression, MDR1 polymorphism, SLEDAI (SLE disease activity index), and kidney pathological score was analyzed by using One-way ANOVA and Pearson linear correlation. The frequency distribution of the MDR1 gene was consistent with the Hardy-Weinberg equilibrium. Compared with CT and CC, patients with T/T homozygote in MDR1 C3435T had significantly increased Pgp expression in the refractory group (p < 0.05). Additionally, SLEDAI score was positively correlated with Pgp expression (r = 0.481, p < 0.05). Also, Pgp expression was positively correlated with renal pathological activity index (r = 0.76, p < 0.05). MDR1 C3435T polymorphism is significantly associated with Pgp expression in patients with refractory LN. Pgp expression is closely related to SLEDAI and renal pathological score. Thus, Pgp may be useful in evaluation of the prognosis of patients with refractory LN.

Intracellular Doppler Spectroscopy detects altered drug response in SKOV3 tumor spheroids with silenced or inhibited P-glycoprotein

Intracellular Doppler spectroscopy is a form of low-coherence digital holography based upon Doppler detection of scattered light that measures drug response/resistance in tumor spheroids, xenografts, and clinical biopsies. Multidrug resistance (MDR) is one of the main causes of ineffective cancer treatment. One MDR mechanism is mediated by the MDR1 gene that encodes the drug efflux pump P-glycoprotein (Pgp). Overexpression of Pgp in some cancers is associated with poor chemotherapeutic response. This paper uses intracellular Doppler spectroscopy to detect Pgp-mediated changes to drug response in 3D tissues grown from an ovarian cancer cell line (SKOV3). The SKOV3 cell line was incrementally exposed to cisplatin to create a cell line with increased Pgp expression (SKOV3cis). Subsequently, MDR1 in a subset of these cells was silenced in SKOV3cis using shRNA to create a doxycycline inducible, Pgp-silenced cell line (SKOV3cis-sh). A specific Pgp inhibitor, zosuquidar, was used to study the effects of Pgp inhibition on the Doppler spectra. Increased drug sensitivity was observed with Pgp silencing or inhibition as determined by drug IC50s of paclitaxel-response of silenced Pgp and doxorubicin-response of inhibited Pgp, respectively. These results indicate that intracellular Doppler spectroscopy can detect changes in drug response due to silencing or inhibition of a single protein associated with drug resistance with important consequences for personalized medicine.

Tumor stressors induce two mechanisms of intracellular P-glycoprotein–mediated resistance that are overcome by lysosomal-targeted thiosemicarbazones

Multidrug resistance (MDR) is a major obstacle in cancer treatment due to the ability of tumor cells to efflux chemotherapeutics via drug transporters (e.g. P-glycoprotein (Pgp; ABCB1)). Although the mechanism of Pgp-mediated drug efflux is known at the plasma membrane, the functional role of intracellular Pgp is unclear. Moreover, there has been intense focus on the tumor micro-environment as a target for cancer treatment. This investigation aimed to dissect the effects of tumor micro-environmental stress on subcellular Pgp expression, localization, and its role in MDR. These studies demonstrated that tumor micro-environment stressors (i.e. nutrient starvation, low glucose levels, reactive oxygen species, and hypoxia) induce Pgp-mediated drug resistance. This occurred by two mechanisms, where stressors induced 1) rapid Pgp internalization and redistribution via intracellular trafficking (within 1 h) and 2) hypoxia-inducible factor-1α expression after longer incubations (4-24 h), which up-regulated Pgp and was accompanied by lysosomal biogenesis. These two mechanisms increased lysosomal Pgp and facilitated lysosomal accumulation of the Pgp substrate, doxorubicin, resulting in resistance. This was consistent with lysosomal Pgp being capable of transporting substrates into lysosomes. Hence, tumor micro-environmental stressors result in: 1) Pgp redistribution to lysosomes; 2) increased Pgp expression; 3) lysosomal biogenesis; and 4) potentiation of Pgp substrate transport into lysosomes. In contrast to doxorubicin, when stress stimuli increased lysosomal accumulation of the cytotoxic Pgp substrate, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), this resulted in the agent overcoming resistance. Overall, this investigation describes a novel approach to overcoming resistance in the stressful tumor micro-environment.

Bronchiolitis obliterans syndrome is associated with increased p-glycoprotein expression and loss of glucocorticoid receptor from steroid-resistant proinflammatory CD8+ T cells.

Immunosuppressive therapy fails to suppress the production of proinflammatory cytokines, particularly by CD8+ T cells, in stable lung transplant recipients and those undergoing chronic rejection, suggesting that some patients may become relatively resistant to immunosuppressants such as glucocorticoids (GC). We have shown loss of GC receptor (GCR) from the CD8+ cells, and we hypothesized that the drug membrane efflux pump, p-glycoprotein-1 (Pgp), may also be involved in lymphocyte steroid resistance following lung transplant. Pgp/GCR expression and interferon (IFN)-γ/tumour necrosis factor (TNF)-α proinflammatory cytokine production was measured in blood lymphocytes from 15 stable lung transplant patients, 10 patients with bronchiolitis obliterans syndrome (BOS) and 10 healthy aged-matched controls (±prednisolone±Pgp inhibitor, cyclosporin A±GCR activator, Compound A) using flow cytometry. Both Pgp+ and Pgp- lymphocyte subsets from all subjects produced IFN-γ/TNF-α proinflammatory cytokines. Pgp expression was increased in CD8+ Pgp+ T cells and correlated with IFN-γ/TNF-α expression and BOS grade. Reduced GCR was observed in CD8+ Pgp- T, natural killer (NK)T-like and NK cells from stable patients compared with controls, and reduced further in CD8+ Pgp- T cells in BOS. The addition of 2·5ng/ml cyclosporin A and 1µM prednisolone inhibit IFN-γ/TNF-α production significantly by CD8+ Pgp+ T cells from BOS patients. The addition of 10µM Compound A and 1 µM prednisolone inhibit IFN-γ/TNF-α production significantly by CD8+ Pgp- T cells from BOS patients. BOS is associated with increased Pgp expression and loss of GCR from steroid-resistant proinflammatory CD8+ T cells. Treatments that inhibit Pgp and up-regulate GCR in CD8+ T cells may improve graft survival.

Knockout of P-glycoprotein does not alter antiepileptic drug efficacy in the intrahippocampal kainate model of mesial temporal lobe epilepsy in mice

Pharmacoresistance to antiepileptic drugs (AEDs) is a major challenge in epilepsy therapy, affecting at least 30% of patients. Thus, there is considerable interest in the mechanisms responsible for such pharmacoresistance, with particular attention on the specific cellular and molecular factors that lead to reduced drug sensitivity. Current hypotheses of refractory epilepsy include the multidrug transporter hypothesis, which posits that increased expression or function of drug efflux transporters, such as P-glycoprotein (Pgp), in brain capillaries reduces the local concentration of AEDs in epileptic brain regions to subtherapeutic levels. In the present study, this hypothesis was addressed by evaluating the efficacy of six AEDs in wildtype and Pgp deficient Mdr1a/b−/− mice in the intrahippocampal kainate model of mesial temporal lobe epilepsy. In this model, frequent focal electrographic seizures develop after an initial kainate-induced status epilepticus. These seizures are resistant to major AEDs, but the mechanisms of this resistance are unknown. In the present experiments, the focal nonconvulsive seizures were resistant to carbamazepine and phenytoin, whereas high doses of valproate and levetiracetam exerted moderate and phenobarbital and diazepam marked anti-seizure effects. All AEDs suppressed generalized convulsive seizures. No significant differences between wildtype and Pgp-deficient mice were observed in anti-seizure drug efficacies. Also, the individual responder and nonresponder rates in each experiment did not differ between mouse genotypes. This does not argue against the multidrug transporter hypothesis in general, but indicates that Pgp is not involved in the mechanisms explaining that focal electrographic seizures are resistant to some AEDs in the intrahippocampal mouse model of partial epilepsy. This was substantiated by the finding that epileptic wildtype mice do not exhibit increased Pgp expression in this model.

Drug-Induced Trafficking of P-Glycoprotein in Human Brain Capillary Endothelial Cells as Demonstrated by Exposure to Mitomycin C

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality.

Marked Differences in the Effect of Antiepileptic and Cytostatic Drugs on the Functionality of P-Glycoprotein in Human and Rat Brain Capillary Endothelial Cell Lines

The expression of P-glycoprotein (Pgp) is increased in brain capillary endothelial cells (BCECs) of patients with pharmacoresistant epilepsy. This may restrict the penetration of antiepileptic drugs (AEDs) into the brain. However, the mechanisms underlying increased Pgp expression in epilepsy patients are not known. One possibility is that AEDs induce the expression and functionality of Pgp in BCECs. Several older AEDs that induce human cytochrome P450 enzymes also induce Pgp in hepatocytes and enterocytes, but whether this extends to Pgp at the human BBB and to newer AEDs is not known. This prompted us to study the effects of various old and new AEDs on Pgp functionality in the human BCEC line, hCMEC/D3, using the rhodamine 123 (Rho123) efflux assay. For comparison, experiments were performed in two rat BCEC lines, RBE4 and GPNT, and primary cultures of rat and pig BCECs. Furthermore, known Pgp inducers, such as dexamethasone and several cytostatic drugs, were included in our experiments. Under control conditions, GPNT cells exhibited the highest and RBE4 the lowest Pgp expression and Rho123 efflux, while intermediate values were determined in hCMEC/D3. Known Pgp inducers increased Rho123 efflux in all cell lines, but marked inter-cell line differences in effect size were observed. Of the various AEDs examined, only carbamazepine (100 μM) moderately increased Pgp functionality in hCMEC/D3, while valproate (300 μM) inhibited Pgp. These data do not indicate that treatment with AEDs causes a clinically relevant induction in Pgp functionality in BCECs that form the BBB.

In-vitro evaluation of chronic alcohol effects on expression of drug-metabolizing and drug-transporting proteins

In alcoholics without alcoholic liver disease, boosted drug elimination has been reported. However, mechanistic explanations for this phenomenon remain uncertain. In particular, data on the potential role of drug transporters are sparse. Using a well-established in-vitro model for induction of human drug-metabolizing and drug-transporting proteins, we evaluated the potency of ethanol and the major fermentation side-product isopentanol to alter expression and function of these proteins by quantitative real-time polymerase chain reaction, Western blotting and flow cytometry. P-glycoprotein (Pgp)-inhibiting properties of ethanol and isopentanol were investigated via calcein extrusion assay. Ethanol and isopentanol significantly changed expression levels of drug-metabolizing and drug-transporting proteins that normalized within 2 weeks upon withdrawal. Cytochrome P-450 2C19 and Pgp were most strongly induced. Ethanol-induced Pgp at the messenger RNA (mRNA) (twofold to eightfold) and protein level (twofold), but not at the functional level. Both compounds did not inhibit Pgp. Ethanol is demonstrated to increase mRNA and protein expression of human drug transporters such as Pgp in vitro. Withdrawal of ethanol exposure causes return to non-induced conditions within weeks. Functional consequences of increased Pgp expression in alcoholics need to be evaluated by clinical trials applying selective Pgp substrates such as digoxin.

The Dual Cyclooxygenase/5-Lipoxygenase Inhibitor Licofelone Attenuates P-Glycoprotein-Mediated Drug Resistance in the Injured Spinal Cord

There are currently no proven effective treatments that can improve recovery of function in spinal cord injury (SCI) patients. Many therapeutic compounds have shown promise in pre-clinical studies, but clinical trials have been largely unsuccessful. P-glycoprotein (Pgp, Abcb1b) is a drug efflux transporter of the blood-spinal cord barrier that limits spinal cord penetration of blood-borne xenobiotics. Pathological Pgp upregulation in diseases such as cancer causes heightened resistance to a broad variety of therapeutic drugs. Importantly, several drugs that have been evaluated for the treatment of SCI, such as riluzole, are known substrates of Pgp. We therefore examined whether Pgp-mediated pharmacoresistance diminishes delivery of riluzole to the injured spinal cord. Following moderate contusion injury at T10 in male Sprague-Dawley rats, we observed a progressive, spatial spread of increased Pgp expression from 3 days to 10 months post-SCI. Spinal cord uptake of i.p.-delivered riluzole was significantly reduced following SCI in wild type but not Abcb1a-knockout rats, highlighting a critical role for Pgp in mediating drug resistance following SCI. Because inflammation can drive Pgp upregulation, we evaluated the ability of the new generation dual anti-inflammatory drug licofelone to promote spinal cord delivery of riluzole following SCI. We found that licofelone both reduced Pgp expression and enhanced riluzole bioavailability within the lesion site at 72 h post-SCI. This work highlights Pgp-mediated drug resistance as an important obstacle to therapeutic drug delivery for SCI, and suggests licofelone as a novel combinatorial treatment strategy to enhance therapeutic drug delivery to the injured spinal cord.

P‐glycoprotein imaging in temporal lobe epilepsy: In vivo PET experiments with the Pgp substrate [11C]‐verapamil

Overexpression of the multidrug efflux transporter P-glycoprotein (Pgp) at the blood-brain barrier (BBB) is thought to be involved in pharmacoresistance in epilepsy by extruding antiepileptic drugs (AEDs) from their target site. To explore this hypothesis, positron emission tomography (PET) scans were performed with the Pgp substrate-verapamil (VPM) in animal models before and after status epilepticus (SE) and in patients with temporal lobe epilepsy (TLE) and healthy controls. In addition to baseline scans, a second VPM-PET scan was performed after administration of the Pgp inhibitor tariquidar (TQD), showing that VPM uptake at baseline and its increase after Pgp inhibition are reduced in animals following SE compared to baseline, and in refractory TLE relative to healthy controls. In animal models, brain regions with increased Pgp expression (cerebellum, thalamus, and hippocampus) showed reduced influx rate constants from blood to brain, K(1), of the radiolabeled Pgp substrate relative to control animals. In human studies, preliminary findings are lower K(1) values in refractory compared to seizure-free patients and attenuated increase of K(1) for temporal lobe regions in patients with TLE compared to healthy controls. In summary, there is lower brain uptake of the Pgp substrate VPM in Pgp-rich areas of animals 2 days following SE, as well as lower increase in VPM brain uptake after TQD in patients with refractory TLE compared to healthy controls, supporting the hypothesis of increased cerebral Pgp function following prolonged seizures and as a mechanism contributing to drug resistance in refractory epilepsy. The observation of reduced VPM uptake in refractory compared to seizure-free patients with TLE is consistent with multiple mechanisms affecting Pgp function, including uncontrolled seizures.

HIF-1 activation induces doxorubicin resistance in MCF7 3-D spheroids via P-glycoprotein expression: a potential model of the chemo-resistance of invasive micropapillary carcinoma of the breast

BackgroundInvasive micropapillary carcinoma (IMPC) of the breast is a distinct and aggressive variant of luminal type B breast cancer that does not respond to neoadjuvant chemotherapy. It is characterized by small pseudopapillary clusters of cancer cells with inverted cell polarity. To investigate whether hypoxia-inducible factor-1 (HIF-1) activation may be related to the drug resistance described in this tumor, we used MCF7 cancer cells cultured as 3-D spheroids, which morphologically simulate IMPC cell clusters.MethodsHIF-1 activation was measured by EMSA and ELISA in MCF7 3-D spheroids and MCF7 monolayers. Binding of HIF-1α to MDR-1 gene promoter and modulation of P-glycoprotein (Pgp) expression was evaluated by ChIP assay and FACS analysis, respectively. Intracellular doxorubicin retention was measured by spectrofluorimetric assay and drug cytotoxicity by annexin V-FITC measurement and caspase activity assay.ResultsIn MCF7 3-D spheroids HIF-1 was activated and recruited to participate to the transcriptional activity of MDR-1 gene, coding for Pgp. In addition, Pgp expression on the surface of cells obtained from 3-D spheroids was increased. MCF7 3-D spheroids accumulate less doxorubicin and are less sensitive to its cytotoxic effects than MCF7 cells cultured as monolayer. Finally, HIF-1α inhibition either by incubating cells with 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (a widely used HIF-1α inhibitor) or by transfecting cells with specific siRNA for HIF-1α significantly decreased the expression of Pgp on the surface of cells and increased the intracellular doxorubicin accumulation in MCF7 3-D spheroids.ConclusionsMCF7 breast cancer cells cultured as 3-D spheroids are resistant to doxorubicin and this resistance is associated with an increased Pgp expression in the plasma membrane via activation of HIF-1. The same mechanism may be suggested for IMPC drug resistance.

Do ATP-Binding Cassette Transporters Cause Pharmacoresistance in Epilepsy? Problems and Approaches in Determining which Antiepileptic Drugs are Affected

Resistance to multiple antiepileptic drugs (AEDs) is a common problem in epilepsy, affecting at least 30% of patients. One prominent hypothesis to explain this resistance suggests an inadequate penetration or excess efflux of AEDs across the blood - brain barrier (BBB) as a result of overexpressed efflux transporters such as P-glycoprotein (Pgp), the encoded product of the multidrug resistance- 1 (MDR1, ABCB1) gene. Pgp and MDR1 are markedly increased in epileptogenic brain tissue of patients with AED-resistant partial epilepsy and following seizures in rodent models of partial epilepsy. In rodent models, AED-resistant rats exhibit higher Pgp levels than responsive animals; increased Pgp expression is associated with lower brain levels of AEDs; and, most importantly, co-administration of Pgp inhibitors reverses AED resistance. Thus, it is reasonable to conclude that Pgp plays a significant role in mediating resistance to AEDs in rodent models of epilepsy - however, whether this phenomenon extends to at least some human refractory epilepsy remains unclear, particularly because it is still a matter of debate which AEDs, if any, are transported by human Pgp. The difficulty in determining which AEDs are substrates of human Pgp is mainly a consequence of the fact that AEDs are highly permeable compounds, which are not easily identified as Pgp substrates in in vitro models of the BBB, such as monolayer (Transwell(®)) efflux assays. By using a modified assay (concentration equilibrium transport assay; CETA), which minimizes the influence of high transcellular permeability, two groups have recently demonstrated that several major AEDs are transported by human Pgp. Importantly, it was demonstrated in these studies that Pgp-mediated transport highly depends on the AED concentration and may not be identified if concentrations below or above the therapeutic range are used. In addition to the efflux transporters, seizure-induced alterations in BBB integrity and activity of drug metabolizing enzymes (CYPs) affect the brain uptake of AEDs. For translating these findings to the clinical arena, in vivo imaging studies using positron emission tomography (PET) with (11)C-labelled AEDs in epileptic patients are under way.

Peripheral inflammatory hyperalgesia modulates morphine delivery to the brain: a role for P‐glycoprotein

P-glycoprotein (Pgp, ABCB1) is a critical efflux transporter at the blood-brain barrier (BBB) where its luminal location and substrate promiscuity limit the brain distribution of numerous therapeutics. Moreover, Pgp is known to confer multi-drug resistance in cancer chemotherapy and brain diseases, such as epilepsy, and is highly regulated by inflammatory mediators. The involvement of inflammatory processes in neuropathological states has led us to investigate the effects of peripheral inflammatory hyperalgesia on transport properties at the BBB. In the present study, we examined the effects of lambda-carrageenan-induced inflammatory pain (CIP) on brain endothelium regulation of Pgp. Western blot analysis of enriched brain microvessel fractions showed increased Pgp expression 3 h post-CIP. In situ brain perfusion studies paralleled these findings with decreased brain uptake of the Pgp substrate and opiate analgesic, [(3)H] morphine. Cyclosporin A-mediated inhibition of Pgp enhanced the uptake of morphine in lambda-carrageenan and control animals. This indicates that the CIP induced decrease in morphine transport was the result of an increase in Pgp activity at the BBB. Furthermore, antinociception studies showed decreased morphine analgesia following CIP. The observation that CIP modulates Pgp at the BBB in vivo is critical to understanding BBB regulation during inflammatory disease states.

Nematodes as Models for the Study of the Regulation of Activity of Pglycoproteins in Multidrug Resistance (MDR)

Alterations in drug transport are a major cause of chemotherapy failures due to nonspecific resistance mechanisms. These mechanisms involve several ABC transporter proteins among which the P-glycoproteins have been the most extensively studied in vertebrates. Increased Pgp expression and/or activity in organisms leads to resistance to many chemically unrelated compounds and therapeutic agents. Such resistance has been observed in cancer and in other diseases due to infectious and parasitic pathogens. Most of the available information on these transporters has been obtained from genetic analyses, although useful data for understanding the cellular mechanisms of such resistance remains limited. As resistance affects several types of organism comparative information on the function and activity of these cellular pumps in various biological environments would be welcome. As such, nematodes represent a possible model. Recent data have been obtained for efflux function and modulation by different molecules in these invertebrates. The observed data are summarized here and the future of these studies is discussed. Keywords: ABC transporters, P-glycoprotein, multidrug resistance, nematodes, efflux, pathogens, drugs, lipids

Neuronal expression of the drug efflux transporter P-glycoprotein in the rat hippocampus after limbic seizures

In the brain, the efflux transporter P-glycoprotein (Pgp) is predominantly located on the luminal membrane of endothelial cells lining brain microvessels and forming the blood–brain barrier. Many lipophilic drugs, including antiepileptic drugs, are potential substrates for Pgp. Overexpression of Pgp in endothelial cells of the blood–brain barrier has been determined in patients with drug resistant forms of epilepsy such as temporal lobe epilepsy and rodent models of temporal lobe epilepsy and suggested to lead to reduced penetration of antiepileptic drugs into the brain. Expression of Pgp after seizures has also been described in astrocytes, whereas it is not clear whether neurons can express Pgp. In the present study, Pgp expression was studied by immunohistochemistry in rats 24 h after a status epilepticus induced by either pilocarpine or kainate, widely used models of temporal lobe epilepsy. Unexpectedly, in addition to endothelial Pgp staining, intense Pgp staining was found in neurons in the CA3c/CA4 sectors and hilus of the hippocampus formation, but not in other brain regions examined. The neuronal Pgp staining was confirmed by two different Pgp antibodies. Double immunolabeling and confocal microscopy showed that Pgp was colocalized with the neuronal marker neuronal nuclear antigen, but not with the glial marker glial fibrillary acidic protein. No neuronal Pgp staining was seen in control rats. The expression of Pgp in neurons after limbic seizures was substantiated by determining Pgp encoding genes ( mdr1a, mdr1b) in neurons by real time quantitative RT-PCR. Increased Pgp expression in hippocampal neurons is likely to affect the action of drugs with intraneuronal targets and, in view of recent evidence from other cell types, could be associated with prevention of apoptosis which is involved in neuronal damage developing after seizures such as produced by pilocarpine.

The Effect of Cell Culture Conditions on Saquinavir Transport Through, and Interactions with, MDCKII Cells Overexpressing hMDR1

MDCK cells are cultured using wide‐ranging conditions and can produce variable results. To develop a standard protocol for studying saquinavir transport using MDCKII cells, stably transfected MDCKII cells overexpressing human Pgp (MDCKII‐PGP) and MDCKII wild‐type cells (MDCKII/wt) were used to evaluate the combined effects of seeding density (6.9 × 105 or 5 × 104 cells/cm2), substratum (polycarbonate ± collagen coating) and saquinavir presence on monolayer integrity, Pgp expression, and saquinavir transport. The saquinavir efflux ratio (ratio of BL → AP/AP → BL permeability) for MDCKII‐PGP cells (6.9 × 105 cells/cm2) was 57 with variable mannitol permeabilities. Consistent mannitol permeabilities and higher saquinavir efflux ratios were obtained with 5 × 104 cells/cm2 on polycarbonate (78) or collagen‐coated polycarbonate (126). The MDCKII/wt saquinavir efflux ratio was 9. Saquinavir presence increased paracellular permeability for all treatments relative to cells seeded onto collagen‐coated membranes. Collagen coating caused increased Pgp expression and saquinavir efflux ratios correlated (r2 = 0.96) with Pgp expression levels [MDCKII‐PGP (on collagen‐coated polycarbonate) > MDCKII‐PGP (on polycarbonate) > MDCKII/wt (on collagen‐coated polycarbonate)]. These results directly and quantitatively link interrelated differences in cell culture conditions to changes in monolayer integrity, transporter expression, and active transport; and emphasize the critical application of controls in cell culture models. © 2003 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 92:1957–1967, 2003

Multidrug resistance factor - glycoprotein P in rheumatoid arthritis

Система комплексной поддержки и сопровождения научного журнала Elpub позволяет запустить двуязычный сайт журнала со встроенной системой электронной редакции в соответствии с лучшими издательскими практиками, а так же предусматривает техническую и методическую поддержку со стороны специалистов НЭИКОН.

Characterization and application of a vinblastine-selected CACO-2 cell line for evaluation of p-glycoprotein.

The role of the adenosine triphosphate-binding cassette (ABC) superfamily of membrane transporters is well documented in tumor cell multidrug resistance. More recently, growing evidence of their influence on oral bioavailability, drug excretion rates, and drug-drug interaction potential at the intestinal level has stimulated much investigation. Our laboratory is interested in evaluating the apical (AP) ABC transporter P-glycoprotein (Pgp [mdr-1]) for its role in xenobiotic efflux at the intestinal level. We propagated Caco-2 cells in the presence of vinblastine (a cytotoxic, Pgp substrate) to promote transporter expression though selection. That is, the cell population expressing Pgp, or with the capacity to up-regulate Pgp expression, survived and expanded in the presence of vinblastine. We have used this selected cell line (Caco-2 VinB) to develop a fluorescent-based assay to study the chemical modulators of Pgp activity. Using the Caco-2 VinB cells, we have successfully demonstrated the differential potency of previously characterized Pgp inhibitors. In addition, we conducted a morphological evaluation of the two cell lines using transmission, scanning, and confocal microscopy. Both cell strains differentiated into highly functional, polarized columnar epithelium, although the vinblastine-selected cell line had lost the phenotypic diversity observed in native Caco-2 populations. Increased Pgp expression was noted in Caco-2 VinB cells compared with the native cell line on Western blot analysis, which was localized to the AP surface using confocal microscopy and functionally demonstrated using transport assays. We believe that the Caco2 VinB cell line is a versatile tool for application in pharmaceutical drug development.