Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.6.3.44 (P-glycoprotein)
 13,344 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new combination of fluorescent dyes (rhodamine 123 and hydroethidine) was used to internally label hybridoma fusion partners. Murine hybridoma 520C9 (recognizing human c-erbB-2) was labeled with hydroethidine. Murine hybridoma 3G8 (recognizing human Fc gamma receptor III) was labeled with rhodamine 123, and verapamil was used to block rhodamine efflux via P-glycoprotein. Viability assays showed little cytotoxicity from these dyes at the concentrations used. The labeled cells were fused with polyethylene glycol, sorted for dual fluorescence on an Epics V cell sorter, and cloned. Hybrid hybridomas producing bispecific antibodies were selected for ability to promote lysis of SK-Br-3 breast cancer cells by human mononuclear cells. Several positive clones were obtained and shown to have a double content of DNA. Bispecific antibody produced by subclone 2B1 was purified by anion exchange chromatography and shown to bind both tumor cells and Fc gamma R III bearing cells. Using two parameter flow cytometric analysis, we were able to measure a 'bridging' effect of this bispecific antibody, which caused formation of complexes between PMNs and SK-Br-3 cells. Either parental antibody could compete with bispecific antibody to block such complexing. This fusion method provides several advantages over other techniques presently used (speed, convenience, low toxicity and automatic exclusion of dead cells) and can be applied to produce other hybrid hybridomas.
 ...
PMID:Selection of hybrid hybridomas by flow cytometry using a new combination of fluorescent vital stains. 171 68

Our human T-cell leukemia line, CEM/VM-1, selected for resistance to VM-26 (teniposide), is cross-resistant to several drugs that interact with topoisomerase II, including VP-16 (etoposide), 4'-(9-acridinylamino)methanesulphon-m-anisidide, daunorubicin, and mitoxantrone. However, in contrast to cell lines exhibiting multidrug resistance (MDR) associated with overexpression of P-glycoprotein, this line is not cross-resistant to the Vinca alkaloids, is not impaired in drug accumulation, and does not overexpress the mdrl gene (Cancer Res., 47: 1297, 5455, 1987). More recently we found that nuclear extracts of these cells exhibit decreased topoisomerase II catalytic and cleavage activity, compared to the drug-sensitive line (Biochemistry, 1988). These results suggest that an alteration in topoisomerase II or a modulator of this enzyme may be responsible for this altered topoisomerase II-form of multidrug resistance (at-MDR). In the present work, we studied the somatic cell genetics of at-MDR. We produced hybrid cell lines by polyethylene glycol-mediated fusion of the CEM/VM-1 line with a hypoxanthine-guanine phosphoribosyl transferase-deficient, ouabain-resistant CEM line (CEM.AG1.OU1.5) that exhibits VM-26 sensitivity. Ten of the hybrid lines that grew in selective medium were randomly chosen for expansion and four were analyzed for both DNA content by flow cytometry and VM-26 sensitivity in a 72-h growth inhibition assay. The hybrid lines all contained approximately 2x DNA compared to unfused controls, indicating that the fusions were successful. The IC50 for VM-26 in 3 of the 4 lines was the same as that of the sensitive controls, ranging from 4.7 to 7.4 x 10(-8) M, and another was 76 x 10(-8) M. These data indicate that drug sensitivity was reconstituted by the hybridization procedure. By comparison, the VM-26 IC50 values in the CEM/VM-1 cells and CEM/VM-1 x CEM/VM-1 control "fusions" were 360 and 750 x 10(-8) M, respectively. To determine whether a topoisomerase II-mediated function was reconstituted in the hybrids, we measured drug-stimulated DNA cleavage ("cleavable complex formation"). Using 32P-labeled pBR322 DNA as substrate with nuclear extracts from drug sensitive cells, 100 microM VM-26 maximally stimulated DNA cleavage by approximately 11-fold compared to no-drug controls.(ABSTRACT TRUNCATED AT 400 WORDS)
 ...
PMID:Genetic characterization of the multidrug-resistant phenotype of VM-26-resistant human leukemic cells. 253 2

Fatty acid ester surfactants, e.g., Cremophor EL and Solutol HS 15, that modulate multi-drug resistance (MDR) have been described; however, the drug potential of these preparations is unclear because of the molecular heterogeneity of these and other commercial surfactants. In previous experiments, an active but still polydisperse preparation, designated CRL 1337, was synthesized by reacting purified oleic acid with a 20-fold molar excess of ethylene oxide. We have subjected this preparation to chromatographic separation, and infrared analysis of the active fractions revealed a significant component of diester structures (fatty acid-PEG-fatty acid). A new generation of diester compounds has now been synthesized. Preparations comprised of 99% diesters were significantly more potent than monoester preparations for MDR modification activity in vitro. As previously determined for ethylene oxide-derived preparations similar to CRL 1337, the nature of the fatty acid domains proved to be important for activity, as was the relative length of the polyethylene glycol domain (which determines the hydrophile-lipophile balance). The ester linkage appeared unimportant since homologous diethers and diamides had activity similar to that of diesters. Stearic acid diester was 1.5- to 7-fold more potent than CRL 1337 when tested in cell proliferation inhibition assays. In light of these structural restrictions on drug potentiation, and since these surfactants are active at relatively low concentrations (below 1 microgram/ml), investigations of their mechanism of action were initiated by exploring specific interactions with P-glycoprotein. Both active and inactive diesters inhibited azidopine labeling of P-glycoprotein, suggesting that fatty acid-PEG diesters can interfere with P-glycoprotein substrate binding. Other attributes of these preparations must contribute to their ability to reverse MDR.
 ...
PMID:Reversal of multi-drug resistance in vitro by fatty acid-PEG-fatty acid diesters. 854

We evaluated the effect of water-soluble vitamin E (d-alpha-tocopheryl polyethylene glycol 1000 succinate [TPGS]; Liqui-E) on the oral pharmacokinetics of the cyclosporine, a poorly available (approximately 30%) drug, in healthy volunteers. Ten healthy subjects were given two doses of oral cyclosporine (10mg/kg) separated by a 7-day washout period. Oral TPGS (2.6 IU/kg) was administered concomitantly with one of the cyclosporine doses in a randomized order. A significant increase was observed in area under the blood concentration-time curve (AUC;mean +/ SD) with concomitant TPGS administration (3908 +/- 2601 versus 6296 +/- 5102 ng x hr/ml). Significant decreases were observed in apparent oral clearance (0.24 +/- 0.14 versus 0.15 +/- 0.08 L/hr/kg) and apparent oral steady-state volume of distribution (1.57 +/- 0.95 versus 1.07 +/- 0.73 L/kg). No significant changes were observed in the ratios of metabolites to parent drug AUC values. The comparable relative decreases in apparent oral clearance (38%) and apparent oral steady-state volume of distribution (30%) with TPGS are most likely explained by enhanced absorption, decreased counter transport back into the intestine by P-glycoprotein, or some unknown mechanism by which cyclosporine is protected from metabolism in the gut, thereby increasing bioavailability.
 ...
PMID:The effect of water-soluble vitamin E on cyclosporine pharmacokinetics in healthy volunteers. 865 92

Fatty acid ester surfactants Cremophor EL and Solutol HS 15 were described earlier as modulators of multidrug resistance mediated by MDR1 P-glycoprotein (Pgp). We have shown that the most active components of these polydisperse surfactants are fatty acid-polyethylene glycol-fatty acid diesters (FA-PEG-FA). A new generation of Pgp-surfactant inhibitors of defined structure was therefore synthesized. In the present study we show that these compounds are also able to inhibit up-regulation of MDR1 gene expression caused by cytarabine (ARA-C) and doxorubicin in human tumor cell lines H9 and KB 3-1, which express minimal levels of MDR1 mRNA. The surfactant inhibitors, however, had no effect on the induction of MDR1 gene expression by protein kinase C agonists. Using a set of FA-PEG-FA diesters with various fatty acids and different lengths of the PEG domain, we demonstrated that the activity of diester preparations as inhibitors of drug-induced MDR1 activation was in proportion to their activity as inhibitors of Pgp function. Oleic and stearic acid diesters with PEG 900 (20 ethylene oxide units) were the most potent. The poloxamer analogs of these diesters demonstrated similar effects. In contrast, the well-known, structurally unrelated inhibitors of Pgp activity, verapamil, cyclosporin A and PSC 833, had no inhibitory effect on drug-induced MDR1 activation. The ability of FA-PEG-FA diesters to inhibit both Pgp function and drug-induced MDR1 activation suggests that these chemomodulators may be uniquely useful for the prophylaxis of Pgp-mediated multidrug resistance in drug-treated tumors.
 ...
PMID:Inhibition of cytarabine-induced MDR1 (P-glycoprotein) gene activation in human tumor cells by fatty acid-polyethylene glycol-fatty acid diesters, novel inhibitors of P-glycoprotein function. 890 Apr 36

A construct encoding a single chain variable fragment of the anti-P-glycoprotein monoclonal antibody C219 was made by combining the coding sequences for the heavy and light chain variable domains with a sequence encoding the flexible linker (GGGGS)3, an OmpA signal sequence, a c-myc identification tag, and a five-histidine purification tag. The construct was expressed in Escherichia coli and purified from the periplasmic fraction using a nickel chelate column and ion exchange chromatography. Three-step Western blot analysis showed that the construct retains binding affinity for P-glycoprotein. Crystals of 1.0 x 0.2 x 0.2 mm were grown in 100 mM citrate, pH 4.5, 21% polyethylene glycol 6000 in the presence of low concentrations of subtilisin, resulting in proteolytic removal of the linker and purification tags. The structure was solved to a resolution of 2.4 A with an R factor of 20.6, an Rfree of 28.5, and good stereochemistry. This result could lead to a clinically useful product based on antibody C219 for the diagnosis of P-glycoprotein-mediated multidrug resistance. The molecule will also be useful in biophysical studies of functional domains of P-glycoprotein, as well as studies of the intact molecule.
 ...
PMID:A single chain Fv fragment of P-glycoprotein-specific monoclonal antibody C219. Design, expression, and crystal structure at 2.4 A resolution. 936 49

Antisense oligonucleotides (ON) are regarded as potential therapeutic agents for controlling gene expression at the mRNA level. The strength of the interaction with the target sequence is one critical factor for the therapeutic efficiency of an ON. Herein, the results of studies on antisense 15mer and 20mer ONs against mdr1b-mRNA are described. The mdr1b is a member of the group that encodes the P-glycoprotein (Pgp), responsible for the phenomenon of multidrug resistance. The effects of backbone modification (DNA, phosphorothioate (PTO)), terminal modifications (hexadecyl, cholesteryl, tocopherol, polyethylenglycol, 2'-O-methyl-modified RNA) and base sequence misalignments (1 to 3 bases) on interaction kinetics and binding strength were investigated. The interaction of an immobilized sense strand with the dissolved antisense ON was monitored with a label-free optical transducer based on thin film interference (RIfS). Association kinetics were detected at a low density of immobilized ON. Thermodynamics were investigated by homogeneous phase titration of sense and antisense ON and subsequent quantification of equilibrium concentrations of unbound ON at a transducer highly loaded with sense ON. Association rate constants varied from 3.1 (+/- 0.2) x 10(4) M-1 s-1 (poly(ethylene glycol)-modified DNA strand) to 4.3 (+/- 0.1) x 10(4) M-1 s-1 (hexadecyl-modified strand). Binding constants varied from 1.9 (+/- 0.1) x 10(8) M-1 (cholesteryl modification) to 5 (+/- 0.4) x 10(7) M-1 (tocopherol modification). Phosphorothioate ON showed a reduction in binding strength of more than 1 order of magnitude. The data presented give valuable information for the efficiency of modified antisense oligonucleotides.
 ...
PMID:Interaction of chemically modified antisense oligonucleotides with sense DNA: a label-free interaction study with reflectometric interference spectroscopy. 1042 71

Graft-copolymers, containing poly(ethylene glycol) (PEG) and polyethyleneimine (PEI) chains have been proposed as carriers for delivery of phosphorothioate oligonucleotides (SODNs). Complexes of such copolymers with SODN self-assemble into particles having a core of neutralized PEI and SODN and a corona of PEG. Transferrin molecules are attached to the PEG corona using avidin/biotin construct. For this purpose, biotin moieties are covalently linked to the free ends of the PEG chains in the PEG-g-PEI copolymer. SODNs are reacted with mixtures of biotinylated and biotin-free PEG-g-PEI copolymers of various compositions to adjust the number of the biotin moieties in the complex. Resulting complexes have small size (ca. 40 nm) and do not aggregate in aqueous solutions for at least several days. To attach transferrin, they are supplemented first with avidin and then with biotin-transferrin conjugate. This increases the effective diameter of the particles to ca. 75-103 nm, depending on the composition of the complex. Cellular accumulation and fluorescence microscopy studies characterize the effects of these modifications on interaction of fluorescently labeled SODNs with KBv cell monolayers. The data suggest significant enhancement of SODN association with cells resulting from modification of the complex with transferrin. SODN complimentary to the site 546-565 of human mdr 1-mRNA was used to inhibit expression of the drug efflux transporter, P-glycoprotein (P-gp), in multiple drug resistant (MDR) cancer cells (KBv, MCF-7 ADR). Accumulation of a P-gp specific probe, rhodamine 123, in the cell monolayers is used to characterize the effects on P-gp efflux system following the treatment of the cells with antisense SODN or its complexes. This study suggests that antisense SODN incorporated in the complexes retain the ability to inhibit P-gp efflux system, while complexes of the randomized control SODN are inactive. Therefore, the antisense SODN is released from the complex and interacts with its intracellular target upon interaction of the complexes with the cells. Furthermore, modification of the complexes with transferrin leads to a significant increase of the effects of the antisense SODN on the P-gp efflux system in the cells. Overall, this study suggests that polyion complex micelles with protein-modified corona are promising tools for the delivery of antisense SODN.
 ...
PMID:Polyion complex micelles with protein-modified corona for receptor-mediated delivery of oligonucleotides into cells. 1050 53

Coadministration of anticancer drugs and multidrug resistance modulators directed against P-glycoprotein over-expressed in tumors also results in nonspecific blockade of this drug efflux pump in excretory tissues such as the liver and kidneys. These interactions often result in impaired renal and biliary clearance for anticancer agents such as doxorubicin (DOX). In the present investigation, we characterized the excretory processes associated with liposomal DOX administration to elucidate how liposome encapsulation may bypass adverse pharmacokinetic interactions between DOX and (3'-keto-Bmt1)-(Val2)-cyclosporin (Valspodar). Renal and biliary clearance properties of liposome-encapsulated DOX were compared with those for nonencapsulated DOX in the presence and absence of Valspodar using an instrumented rat model with implanted jugular vein and bile duct catheters for continuous sampling. Two types of liposomal DOX formulations were used, a drug-permeable egg phosphatidyl choline/cholesterol system and a sterically stabilized polyethylene glycol/1,2 distearoyl-sn-glycero-3-phosphocholine/cholesterol system to establish the relative roles of liposome-encapsulated and released drug on the pharmacokinetic and excretion alterations induced by Valspodar. DOX and its primary metabolites were quantitated using high-performance liquid chromatography. When Valspodar was coadministered with nonencapsulated DOX, 3.5- and 37.5-fold reductions in renal clearance (CLr) and biliary clearance (CLb), respectively, were observed, which resulted in increased plasma DOX concentrations and total exposure. However, Valspodar-induced alterations in CLr and CLb were less profound with egg phosphatidyl choline/cholesterol DOX (1.7- and 2.0-fold reductions, respectively) and negligible with the long-circulating polyethylene glycol-containing liposomal formulation. These results indicate that liposomes may circumvent Valspodar-induced DOX pharmacokinetic changes by reducing the rate of drug excretion in liver and kidney tissue to a level that is within the renal and biliary excretion capacity in the presence of P-glycoprotein blockade.
 ...
PMID:Doxorubicin encapsulated in sterically stabilized liposomes exhibits renal and biliary clearance properties that are independent of valspodar (PSC 833) under conditions that significantly inhibit nonencapsulated drug excretion. 1053 63

Poly(ethylene glycol), or PEG, conjugation to proteins and peptides is a growing technology used to enhance efficacy of therapeutics. This investigation assesses pharmacodynamic and pharmacokinetic characteristics of PEG-conjugated [D-Pen2,D-Pen5]-enkephalin (DPDPE), a met-enkephalin analog, in rodent (in vivo, in situ) and bovine (in vitro) systems. PEG-DPDPE showed increased analgesia (i.v.) compared with nonconjugated form (p < 0.01), despite a 172-fold lower binding affinity for the delta-opioid receptor. [125I]PEG-DPDPE had a 36-fold greater hydrophilicity (p < 0.01) and 12% increase in the unbound plasma protein fraction (p < 0.01), compared with [(125)I]DPDPE. [125I]PEG-DPDPE had a 2.5-fold increase in elimination half-life (p < 0.01), 2.7-fold decrease in volume of distribution (p < 0.01), and a 7-fold decrease in plasma clearance rate (p < 0.01) to [125I]DPDPE. Time course distribution showed significant concentration differences (p < 0.01) in plasma, whole blood, liver, gallbladder, gastrointestinal (GI) content, GI tract, kidneys, spleen, urine, and brain (brain, p < 0.05), between the conjugated and nonconjugated forms. Increased brain uptake of [(125)I]PEG-DPDPE corresponded to analgesia data. [125I]PEG-DPDPE in brain was shown to be 58.9% intact, with 41.1% existing as [125I]DPDPE (metabolite), whereas [125I]DPDPE was 25.7% intact in the brain (at 30 min). In vitro P-glycoprotein affinity was shown for [125I]DPDPE (p < 0.01) but not shown for [125I]PEG-DPDPE. In vitro saturable uptake, with 100 microM DPDPE, was shown for [125I]PEG-DPDPE (p < 0.05). In this study, PEG-conjugated DPDPE seems to act as a prodrug, enhancing peripheral pharmacokinetics, while undergoing hydrolysis in the brain and allowing nonconjugated DPDPE to act at the receptor.
 ...
PMID:Pharmacodynamic and pharmacokinetic characterization of poly(ethylene glycol) conjugation to met-enkephalin analog [D-Pen2, D-Pen5]-enkephalin (DPDPE). 1145 51


1 2 3 4 5 6 7 8 9 10 Next >>