Pharmacokinetic Role of P-Glycoprotein in Oral Bioavailability and Intestinal Secretion of Grepafloxacin in Vivo

P-glycoprotein (P-gp; also known as MDR1 or ABCB1) is an ATP-driven multidrug transporter that extrudes various hydrophobic toxic compounds to the extracellular space. P-gp consists of two transmembrane domains (TMDs) that form the substrate translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. At least two P-gp states are required for transport. In the inward-facing (pre-drug transport) conformation, the two NBDs are separated, and the two TMDs are open to the intracellular side; in the outward-facing (post-drug transport) conformation, the NBDs are dimerized, and the TMDs are slightly open to the extracellular side. ATP binding and hydrolysis cause conformational changes between the inward-facing and the outward-facing conformations, and these changes help translocate substrates across the membrane. However, how ATP hydrolysis is coupled to these conformational changes remains unclear. In this study, we used a new FRET sensor that detects conformational changes in P-gp to investigate the role of ATP binding and hydrolysis during the conformational changes of human P-gp in living HEK293 cells. We show that ATP binding causes the conformational change to the outward-facing state and that ATP hydrolysis and subsequent release of γ-phosphate from both NBDs allow the outward-facing state to return to the original inward-facing state. The findings of our study underscore the utility of using FRET analysis in living cells to elucidate the function of membrane proteins such as multidrug transporters.

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Potential Role of Dual NF-κB and Oxidative Stress Pathway Inhibitor, OT-304 as a Novel Drug Resistance-Reversing Agent.

Blood ◽

10.1182/blood.v110.11.4211.4211 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4211-4211

Author(s):

Shaker A. Mousa ◽

Ghanshyam Patil ◽

Abdelhadi Rebbaa

Keyword(s):

Oxidative Stress ◽

Drug Resistance ◽

Tumor Cells ◽

Genetic Alterations ◽

Potential Candidate ◽

Drug Resistant ◽

P Glycoprotein ◽

And Oxidative Stress

Abstract The development of resistance to chemotherapy represents an adaptive biological response by tumor cells that leads to treatment failure and patient relapse. During the course of their evolution (intrinsic resistance) or in response to chemotherapy (acquired resistance), tumor cells may undergo genetic alterations to possess a drug resistant phenotype. Dysregulation of membrane transport proteins and cellular enzymes, as well as altered susceptibility to commit to apoptosis are among the mechanisms that contribute to the genesis of acquired drug resistance. Recently, the development of approaches to prevent and/or to reverse this phenomenon has attracted special interest and a number of drug candidates have been identified. Despite strong effects observed for these candidates in vitro, however, most of them fail in vivo. In the present study, we have identified a novel small molecule inhibitor of dual NF-κB and oxidative stress pathways, OT-304, as a potential candidate to reverse drug resistance. Initial investigations indicate that this compound effectively inhibits proliferation of doxorubicin-sensitive and doxorubicin-resistant cells to the same extent, suggesting that it is capable of bypassing the development of drug resistance. Additional experiments reveal that OT-304 enhances cancer cell sensitivity to doxorubicin and to etoposide, particularly in cells characterized by the over-expression of the drug transporter P-glycoprotein. These findings suggest that either the expression/and or the function of P-glycoprotein could be affected by OT-304. In vivo studies using tumor xenografts in nude mice showed that OT-304 is also capable of preventing the growth of drug resistant cancer cells. This later finding further confirms the role of OT-304 as a drug resistance-reversing agent and warrants further pre-clinical and clinical investigation to determine its efficacy in treating aggressive tumors.

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A novel in vivo regulatory role of P-glycoprotein in alloimmunity

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2010.03.040 ◽

2010 ◽

Vol 394 (3) ◽

pp. 646-652 ◽

Cited By ~ 5

Author(s):

Atsushi Izawa ◽

Tobias Schatton ◽

Natasha Y. Frank ◽

Takuya Ueno ◽

Kazuhiro Yamaura ◽

...

Keyword(s):

Regulatory Role ◽

P Glycoprotein

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Cellular Accumulation and Pharmacodynamic Evaluation of the Intracellular Activity of CEM-101, a Novel Fluoroketolide, against Staphylococcus aureus, Listeria monocytogenes, and Legionella pneumophila in Human THP-1 Macrophages

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00203-09 ◽

2009 ◽

Vol 53 (9) ◽

pp. 3734-3743 ◽

Cited By ~ 39

Author(s):

Sandrine Lemaire ◽

Françoise Van Bambeke ◽

Paul M. Tulkens

Keyword(s):

Staphylococcus Aureus ◽

Listeria Monocytogenes ◽

Legionella Pneumophila ◽

Critical Role ◽

Intracellular Accumulation ◽

Cellular Accumulation ◽

Intracellular Activity ◽

P Glycoprotein

ABSTRACT CEM-101 is a novel fluoroketolide with lower MICs than those of telithromycin and macrolides. Our aim was to assess the cellular accumulation and intracellular activity of CEM-101 using models developed for analyzing the pharmacokinetics and pharmacological properties of antibiotics against phagocytized bacteria. We used THP-1 macrophages and Staphylococcus aureus (ATCC 25923 [methicillin (meticillin) sensitive]), Listeria monocytogenes (strain EGD), and Legionella pneumophila (ATCC 33153). CEM-101 reached cellular-to-extracellular-concentration ratios of about 350 within 24 h (versus approximately 20, 30, and 160 for telithromycin, clarithromycin, and azithromycin, respectively). This intracellular accumulation was suppressed by incubation at a pH of ≤6 and by monensin (proton ionophore) and was unaffected by verapamil (P-glycoprotein inhibitor; twofold accumulation increase for azithromycin) or gemfibrozil. While keeping with the general properties of the macrolide antibiotics in terms of maximal efficacy (E max; approximately 1-log10-CFU decrease compared to the postphagocytosis inoculum after a 24-h incubation), CEM-101 showed significantly greater potency against phagocytized S. aureus than telithromycin, clarithromycin, and azithromycin (for which the 50% effective concentration [EC50] and static concentrations were about 3-, 6-, and 15-fold lower, respectively). CEM-101 was also about 50-fold and 100-fold more potent than azithromycin against phagocytized L. monocytogenes and L. pneumophila, respectively. These differences in EC50s and static concentrations between drugs were minimized when data were expressed as multiples of the MIC, demonstrating the critical role of intrinsic drug activity (MIC) in eliciting the antibacterial intracellular effects, whereas accumulation per se was unimportant. CEM-101 should show enhanced in vivo potency if used at doses similar to those of the comparators tested here.

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Vagal control of fluid transport, transmural potential difference, and motility in the ferret jejunum

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1985.249.6.g651 ◽

1985 ◽

Vol 249 (6) ◽

pp. G651-G654 ◽

Cited By ~ 2

Author(s):

B. Greenwood ◽

N. W. Read

Keyword(s):

Fluid Transport ◽

Vagal Stimulation ◽

Contractile Activity ◽

Intestinal Transport ◽

Intestinal Secretion ◽

Potential Difference ◽

Intraluminal Pressure ◽

Jejunal Loop

The role of the vagus nerve in the control of intestinal transport was investigated in the ferret jejunum in vivo. Fluid transport was measured in an isolated 10-cm segment of jejunum by means of a single-pass perfusion technique with radioactive markers introduced into the perfusion fluid and the bloodstream of the animal. Transmural potential difference (PD) and intraluminal pressure in the perfused jejunal loop were also monitored. Vagal stimulation (20 Hz, 20 V, and 0.5 ms for 1 min) resulted in jejunal fluid movement in the direction of secretion, a rise in transmural PD, and an increase in jejunal contractile activity. Similar changes were induced by close intra-arterial injection of acetylcholine (20 micrograms X kg-1). The contractile response to vagal stimulation was abolished by atropine. Moreover, atropine did not block the changes in fluid transport and transmural PD that were induced by vagal stimulation, although the transmural PD response was reduced. The results suggest that vagal stimulation induces intestinal secretion accompanied by a rise in transmural PD; the events are mediated at least in part by a noncholinergic transmitter as yet undetermined.

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Assessment of Pharmacokinetic Drug–Drug Interactions in Humans: In Vivo Probe Substrates for Drug Metabolism and Drug Transport Revisited

The Annual Review of Pharmacology and Toxicology ◽

10.1146/annurev-pharmtox-010818-021909 ◽

2019 ◽

Vol 59 (1) ◽

pp. 507-536 ◽

Cited By ~ 16

Author(s):

Uwe Fuhr ◽

Chih-hsuan Hsin ◽

Xia Li ◽

Wafaâ Jabrane ◽

Fritz Sörgel

Keyword(s):

Drug Transport ◽

Quantitative Prediction ◽

Pharmacokinetic Parameters ◽

Glycoprotein P ◽

Therapeutic Drugs ◽

P Glycoprotein ◽

Clinical Drug ◽

Pharmacokinetic Drug

Pharmacokinetic parameters of selective probe substrates are used to quantify the activity of an individual pharmacokinetic process (PKP) and the effect of perpetrator drugs thereon in clinical drug–drug interaction (DDI) studies. For instance, oral caffeine is used to quantify hepatic CYP1A2 activity, and oral dagibatran etexilate for intestinal P-glycoprotein (P-gp) activity. However, no probe substrate depends exclusively on the PKP it is meant to quantify. Lack of selectivity for a given enzyme/transporter and expression of the respective enzyme/transporter at several sites in the human body are the main challenges. Thus, a detailed understanding of the role of individual PKPs for the pharmacokinetics of any probe substrate is essential to allocate the effect of a perpetrator drug to a specific PKP; this is a prerequisite for reliably informed pharmacokinetic models that will allow for the quantitative prediction of perpetrator effects on therapeutic drugs, also in respective patient populations not included in DDI studies.

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