Pluronic P85 enhances the delivery of digoxin to the brain: in vitro and in vivo studies.
Drug delivery across the blood-brain barrier is limited by several mechanisms. One important mechanism is drug efflux, mediated by several transport proteins, including P-glycoprotein. The goal of this work was to examine the effect of a novel drug delivery system, Pluronic block copolymer P85, on P-glycoprotein-mediated efflux from the brain using in vitro and in vivo methods. The hypothesis was that specific Pluronic copolymer systems enhance drug delivery to the central nervous system through the inhibition of P-glycoprotein. The effect of P85 on the cellular accumulation and transport of digoxin, a model P-glycoprotein substrate, was examined in porcine kidney epithelial cells (LLC-PK1) transfected with the human MDR1 gene. The effect of P85 on the directional flux across an in vitro BBB was also characterized. In vivo brain distribution studies were accomplished using wild-type and P-glycoprotein knockout mice. Pluronic increased the cellular accumulation of digoxin 3-fold in LLC-PK1 cells and 5-fold in the LLC-PK1-MDR1-transfected cells. Similar effects were observed for a prototypical P-glycoprotein substrate rhodamine-123. P85 treatment decreased the basolateral-to-apical and increased the apical-to-basolateral digoxin flux across LLC-PK1-MDR1 cell monolayers, and analogous results were observed with the in vitro BBB monolayers. The coadministration of 1% P85 with radiolabeled digoxin in wild-type mice increased the brain penetration of digoxin 3-fold and the digoxin level in the P85-treated wild-type mice was similar to that observed in the P-glycoprotein-deficient animals. These data indicate that Pluronic P85 can enhance the delivery of digoxin to the brain through the inhibition of the P-glycoprotein-mediated efflux mechanism. (+info)
Mechanism of pluronic effect on P-glycoprotein efflux system in blood-brain barrier: contributions of energy depletion and membrane fluidization.
Pluronic block copolymer, P85, inhibits the P-glycoprotein (Pgp) drug efflux system and increases the permeability of a broad spectrum of drugs in the blood-brain barrier (BBB). This study examines the mechanisms by which P85 inhibits Pgp using bovine brain microvessel endothelial cells (BBMEC) as an in vitro model of the BBB. The hypothesis was that simultaneous alterations in intracellular ATP levels and membrane fluidization in BBMEC monolayers by P85 results in inhibition of the drug efflux system. The methods included the use of 1) standard Pgp substrate rhodamine 123 to assay the Pgp efflux system in BBMEC, 2) luciferin/luciferase assay for ATP intracellular levels, and 3) 1,6-diphenyl-1,3,5-hexatriene for membrane microviscosity. Using 3H-labeled P85 and fluorescein-labeled P85 for confocal microscopy, this study suggests that P85 accumulates in the cells and intracellular organelles such as the mitochondria where it can interfere with metabolic processes. Following exposure of BBMEC to P85, the ATP levels were depleted, and microviscosity of the cell membranes was decreased. Furthermore, P85 treatment decreased Pgp ATPase activity in membranes expressing human Pgp. A combination of experiments examining the kinetics, concentration dependence, and directionality of P85 effects on Pgp-mediated efflux in BBMEC monolayers suggests that both energy depletion (decreasing ATP pool available for Pgp) and membrane fluidization (inhibiting Pgp ATPase activity) are critical factors contributing to the activity of the block copolymer in the BBB. (+info)
Mechanism of sensitization of MDR cancer cells by Pluronic block copolymers: Selective energy depletion.
This paper, for the first time, demonstrates that exposure of cells to the poly(ethylene oxide)-poly(propylene oxide) block copolymer, Pluronic P85, results in a substantial decrease in ATP levels selectively in MDR cells. Cells expressing high levels of functional P-glycoprotein (MCF-7/ADR, KBv; LLC-MDR1; Caco-2, bovine brain microvessel endothelial cells [BBMECs]) are highly responsive to Pluronic treatment, while cells with low levels of P-glycoprotein expression (MCF-7, KB, LLC-PK1, human umbilical vein endothelial cells [HUVECs] C2C12 myoblasts) are much less responsive to such treatment. Cytotoxicity studies suggest that Pluronic acts as a chemosensitizer and potentiates cytotoxic effects of doxorubicin in MDR cells. The ability of Pluronic to inhibit P-glycoprotein and sensitize MDR cells appears to be a result of ATP depletion. Because many mechanisms of drug resistance are energy dependent, a successful strategy for treating MDR cancer could be based on selective energy depletion in MDR cells. Therefore, the finding of the energy-depleting effects of Pluronic P85, in combination with its sensitization effects is of considerable theoretical and practical significance. (+info)
Inducing neutrophil recruitment in the liver of ICAM-1-deficient mice using polyethyleneimine grafted with Pluronic P123 as an organ-specific carrier for transgenic ICAM-1.
Coordinated expression of cell adhesion molecules and chemokines on the surface of vascular endothelium is responsible for the homing of immune effector cells to targeted sites. One way to attract non-activated immune cells to targeted organs is to use transgenically expressed adhesion molecules responsible for leukocyte recruitment. We have previously shown that polyethyleneimine (PEI) grafted with non-ionic amphiphilic Pluronic P123 block copolymer (P123PEI) modifies biodistribution of plasmid DNA toward the liver. In the present study, a P123PEI-formulated plasmid carrying the gene encoding for the murine ICAM-1 molecule was injected i.v. into transgenic ICAM-1-deficient mice. The RT-PCR analysis of ICAM-1 mRNA expression showed that P123PEI induced a dose-dependent expression of ICAM-1 in the liver. Furthermore, this expression of ICAM-1 induced neutrophil invasion in the liver, while no such invasion was observed in mice injected with formulated control plasmid or naked DNA. These results suggest that P123PEI allows functional transgene expression in the liver following i.v. injection and that ICAM-1 could be used to enhance immune response locally by attracting immune effector cells. (+info)
Peptide-based drug development is a rapidly growing field within pharmaceutical research. Nevertheless, peptides have found limited clinical use due to several physiological and pathological factors. Pluronic block copolymers represent a growing technology with the potential to enhance efficacy of peptide therapeutics. This investigation assesses Pluronic P85 (P85) and its potential to enhance opioid peptide analgesia. Two opioid peptides, [D-Pen(2),D-Pen(5)]-enkephalin (DPDPE) and biphalin, were examined as to the benefits of P85 coadministration, above (1.0%) and below (0.01%) the critical micelle concentration, with morphine as a nonpeptide control. P85 was examined in vitro to assess blood-brain barrier uptake in association with P-glycoprotein effect, DPDPE and morphine being P-glycoprotein substrates. P85 coadministration with DPDPE and biphalin showed increased (p < 0.01) analgesia with both 0.01 and 1.0% P85. Morphine showed increased (p < 0.01) analgesia with 0.01% P85 only. This increase in analgesia is due to both an increase in peak effect, as well as a prolongation of effect. P85 increased cellular uptake of (125)I-DPDPE and [(3)H]morphine at 0.01% (p < 0.01) and 1.0% (p < 0.01 and p < 0.05, respectively). Cyclosporin-A coadministration with (125)I-DPDPE and [(3)H]morphine increased cellular uptake (p < 0.01 and p < 0.05, respectively). (125)I-DPDPE and [(3)H]morphine coadministered with 0.01% P85 and cyclosporin-A increased cellular uptake compared with control (p < 0.01) and compared with cyclosporin-A coadministration without P85 (p < 0.01 and p < 0.05, respectively). This indicates that, in addition to P-gp inhibition, 0.01% P85 increased (125)I-DPDPE and [(3)H]morphine uptake. In our examination, we determined that P85 enhanced the analgesic profile of biphalin, DPDPE, and morphine, both above and below the critical micelle concentration. (+info)
Optimal structure requirements for pluronic block copolymers in modifying P-glycoprotein drug efflux transporter activity in bovine brain microvessel endothelial cells.
Pluronic block copolymer P85 was shown to inhibit the P-glycoprotein (Pgp) drug efflux system and to increase the permeability of a broad spectrum of drugs in the blood-brain barrier (BBB). However, there is an entire series of Pluronics varying in lengths of propylene oxide and ethylene oxide and overall lipophilicity. This study identifies those structural characteristics of Pluronics required for maximal impact on drug efflux transporter activity in bovine brain microvessel endothelial cells (BBMECs). Using a wide range of block copolymers, differing in hydrophilic-lipophilic balance (HLB), this study shows that lipophilic Pluronics with intermediate length of propylene oxide block (from 30 to 60 units) and HLB <20 are the most effective at inhibiting Pgp efflux in BBMECs. The methods used included 1) cellular accumulation studies with the Pgp substrate rhodamine 123 in BBMECs to assess Pgp activity; 2) luciferin/luciferase ATP assay to evaluate changes in cellular ATP; 3) 1,6-diphenyl-1,3,5-hexatriene membrane microviscosity studies to determine alterations in membrane fluidity; and 4) Pgp ATPase assays using human Pgp-expressing membranes. Pluronics with intermediate lipophilic properties showed the strongest fluidization effect on the cell membranes along with the most efficient reduction of intracellular ATP synthesis in BBMEC monolayers. The relationship between the structure of Pluronic block copolymers and their biological response-modifying effects in BBMECs are useful for determining formulations with maximal efficacy for increasing BBB permeability. (+info)
Phagocytic activation of human neutrophils by the detergent component of fluosol.
Fluosol (Alpha Therapeutic Corporation, Los Angeles, CA) an emulsion of perfluorocarbons with a high oxygen-carrying capacity, was approved as an adjunct to alleviate myocardial ischemia during coronary angioplasty. This drug also significantly enhances myocardial salvage presumably related to an action on the neutrophil. The mechanism by which fluosol and its individual components, including the detergent Pluronic F-68, affected neutrophil function was examined. During the incubation of neutrophils with fluosol, a rapid stimulation of superoxide anion production and degranulation which progressively increased over a 30-minute period was detected. Neutrophils incubated with only Pluronic F-68 produced similar amounts of superoxide anion. Cytochalasin B, an inhibitor of phagocytosis, significantly inhibited this superoxide anion generation. As shown previously, neutrophils incubated with fluosol for 30 minutes and then subsequently stimulated manifested a reduction in lysozyme release as compared with untreated cells. Results of an electron microscopic examination confirmed the cellular uptake of the fluosol within phagocytic vacuoles. Neutrophil viability determined by trypan blue was unaffected after fluosol treatment. These observations show that the fluosol emulsion, primarily through micelles formed by the detergent Pluronic F-68, activates human neutrophils by serving as a phagocytic stimulus, which produces a cell refractory to subsequent stimulation. (+info)
Catalysis by alpha-chymotrypsin entrapped into surface-modified polymeric nanogranules in organic solvent.
Physicochemical characteristics of previously suggested surface-modified polymeric nanogranules (SMPN) and catalytic and stability properties of alpha-chymotrypsin entrapped into such nanogranules in a nonpolar solvent were investigated in more details. SMPN were obtained by polymerization of an acrylamide/N,N'-methylene-bisacrylamide mixture in a mixed reversed micellar system composed of Aerosol OT [sodium di(2-ethylhexyl)sulfosuccinate] and the polymeric surfactant Pluronic F-108 modified with polymerizable groups, followed by the chromatographic removal of the auxiliary surfactant, Aerosol OT. An optimal solvent system was found providing the required orientation of the polymeric surfactant in starting mixed micelles, i.e. with polar fragments immersed into the micellar interior and apolar fragments protruding into organic solvent. The hydrodynamic diameter of SMPN in benzene solution was estimated by means of quasi-elastic light scattering to be 84 +/- 1 nm. Catalytic and stability properties of alpha-chymotrypsin entrapped into SMPN strongly depended on conditions of preparation of SMPN. The optimal concentration of acrylamide monomers in the micellar interior and hydration degree of starting reversed micelles were found to be 20% by mass and wo = 15, respectively. alpha-Chymotrypsin-containing SMPN were used as a catalyst in the synthesis of N-acetyl-L-tyrosine ethyl ester from N-acetyl-L-tyrosine and ethanol, performed in a membrane reactor. (+info)