New technologies for drug delivery across the blood brain barrier.

The blood-brain barrier (BBB) efficiently restricts penetration of therapeutic agents to the brain from the periphery. Therefore, discovery of new modalities allowing for effective delivery of drugs and biomacromolecules to the central nervous system (CNS) is of great need and importance for treatment of neurodegenerative disorders. This manuscript focuses on three relatively new strategies. The first strategy involves inhibition of the drug efflux transporters expressed in BBB by Pluronic block copolymers, which allows for the increased transport of the substrates of these transporters to the brain. The second strategy involves the design of nanoparticles conjugated with specific ligands that can target receptors in the brain microvasculature and carry the drugs to the brain through the receptor mediated transcytosis. The third strategy involves artificial hydrophobization of peptides and proteins that facilitates the delivery of these peptides and proteins across BBB. This review discusses the current state, advantages and limitations of each of the three technologies and outlines their future prospects.

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.

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.

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.

Pluronic P85 increases permeability of a broad spectrum of drugs in polarized BBMEC and Caco-2 cell monolayers.

PURPOSE: Previous studies demonstrated that inhibition of P glycoprotein (P-gp) by Pluronic P85 (P85) block copolymer increases apical (AP) to basolateral (BL) transport of rhodamine 123 (R123) in the polarized monolayers of bovine brain microvessel endothelial cells (BBMEC) and Caco-2 cells. The present work examines the effects of P85 on the transport of fluorescein (Flu), doxorubicin (Dox), etoposide (Et), taxol (Tax), 3'-azido-3'-deoxythymidine (AZT), valproic acid (VPA) and loperamide (Lo) using BBMEC and Caco-2 monolayers as in vitro models of the blood brain barrier and intestinal epithelium respectively. METHODS: Drug permeability studies were performed on the confluent BBMEC and Caco-2 cell monolayers mounted in Side-Bi-Side diffusion cells. RESULTS: Exposure of the cells to P85 significantly enhanced AP to BL permeability coefficients of Flu, Tax, Dox and AZT in both cell models. Further, P85 enhanced AP to BL transport of Et, VPA and Lo in Caco-2 monolayers. No changes in the permeability coefficients of the paracellular marker mannitol were observed in the presence of the copolymer. CONCLUSIONS: P85 increases AP to BL permeability in BBMEC and Caco-2 monolayers with respect to a broad panel of structurally diverse compounds, that were previously shown to be affected by P-gp and/ or multidrug resistance associated protein (MRP) efflux systems. Broad specificity of the block copolymer effects with respect to drugs and efflux systems appears to be a valuable property in view of developing pharmaceutical formulations to increase drug accumulation in selected organs and overcome both acquired and intrinsic drug resistance that limits the effectiveness of many chemotherapeutic agents.

Inhibition of multidrug resistance-associated protein (MRP) functional activity with pluronic block copolymers.

PURPOSE: Using monolayers of human pancreatic adenocarcinoma cells (Panc-1) that express multidrug resistance-associated protein (MRP), the present work investigates the effects of Pluronic block copolymers on the functional activity of MRP. METHODS: The studies examined the accumulation and efflux of the MRP selective probe fluorescein (FLU) in Panc-1 cell monolayers with and without Pluronic P85 (P85), Pluronic L81 (L81) and Pluronic F108 (F108). RESULTS: Treatment of Panc-1 cells with P85 resulted in concentration-dependent increases in FLU accumulation and elimination of FLU sequestration in vesicular compartments in these cells. The effects of P85 were selective for FLU in the Panc-1 cell monolayers. Inhibition of MRP-mediated transport was dependent on the composition of Pluronic block copolymer: the more hydrophobic copolymer had the greater effect on FLU uptake in Panc-1 monolayers (L81 > P85 > F108). CONCLUSIONS: This paper demonstrates for the first time that Pluronic block copolymers inhibit multidrug resistance-associated protein (MRP). The similarities in the effects of Pluronic block copolymers on MRP and P-glycoprotein drug efflux systems suggest that a single unifying mechanism may explain the inhibition observed.

Effects of pluronic P85 unimers and micelles on drug permeability in polarized BBMEC and Caco-2 cells.

PURPOSE: Using polarized bovine brain microvessel endothelial cells (BBMEC) monolayers as in vitro model of the blood brain barrier and Caco-2 monolayers as a model of the intestinal epithelium, the present work investigates the effects of Pluronic P85 block copolymer (P85) on the transport of the P-gycoprotein (P-gp)- dependent probe, rhodamine 123 (R123). METHODS: The permeability and cell efflux studies are performed with the confluent cell monolayers using Side-Bi-Side diffusion cells. RESULTS: At concentrations below the critical micelle concentration, P85 inhibits P-gp efflux systems of the BBMEC and Caco-2 cell monolayers resulting in an increase in the apical to basolateral permeability of R123. In contrast, at high concentrations of P85 the drug incorporates into the micelles, enters the cells and is then recycled back out to the apical side resulting in decrease in R123 transport across the cell monolayers. Apical to basolateral permeability of micelle-incorporated R123 in BBMEC monolayers was increased by prior conjugation of P85 with insulin, suggesting that modified micelles undergo receptor-mediated transcytosis. CONCLUSIONS: Pluronic block copolymers can increase membrane transport and transcellular permeability in brain microvessel endothelial cells and intestinal epithelium cells. This suggests that these block copolymers may be useful in designing formulations to increase brain and oral absorption of select drugs.

Effects of pluronic block copolymers on drug absorption in Caco-2 cell monolayers.

PURPOSE: The present work characterizes the effects of Pluronic copolymers on the transport of a P-gp-dependent probe, rhodamine 123 (R123) in Caco-2 cell monolayers. METHODS: The accumulation and efflux studies were performed on the confluent Caco-2 monolayers using fluorescent probes with and without Pluronic copolymers. RESULTS: At concentrations below the critical micelle concentration single chains ("unimers") of Pluronic P85 enhanced the accumulation and inhibited the efflux of R123 in Caco-2 monolayers. The transport of the P-gp-independent probe, rhodamine 110 was not altered under these conditions. In contrast the micelles increased R123 accumulation to a much lower extent when compared to the unimers and enhanced R123 efflux in Caco-2 monolayers. CONCLUSIONS: Pluronic P85 unimers increase accumulation of a P-gp-dependent drug in Caco-2 monolayers through inhibition of the P-gp efflux system. The mechanism of the micelle effect is not known, however, it is very similar to the micelle effects in BBMEC. This has been previously shown to involve vesicular transport of the micelle-incorporated drug. The study suggests that Pluronic copolymers can be useful in increasing oral absorption of select drugs.

Interactions of pluronic block copolymers with brain microvessel endothelial cells: evidence of two potential pathways for drug absorption.

Pluronic block copolymers have been previously reported to increase the delivery of agents to the brain [Kabanov et al. (1992) J. Controlled Release 22, 141-158]. In the present study, primary cultured bovine brain microvessel endothelial cells (BBMEC) were used as an in vitro model of the blood-brain barrier to examine the membrane interactions of Pluronic P85 (P85) and potential mechanisms for drug absorption. At concentrations below the critical micelle concentration (cmc), P85 enhanced the accumulation of the fluorescent probe rhodamine 123 (R123) in BBMEC through inhibition of P-glycoprotein (P-gp)-mediated drug efflux. The effects of P85 on the cellular accumulation of R123 were also observed in KBv cells (P-gp positive) but not in human umbilical vein endothelial cells (P-gp negative). In contrast to the effects with P85 below the cmc, the enhanced absorption of R123 observed with Pluronic micelles was transient and not dependent on P-gp. A transient increase in R123 accumulation was observed in both P-gp positive cells (brain microvessel endothelial cells and KBv) and P-gp negative cells (human umbilical vein endothelial cells). Therefore, it appears that P85 affects the absorption of drugs in brain microvessel endothelial cells through (1) inhibition of the P-gp-mediated drug efflux at low concentrations of the copolymer and (2) increased vesicular transport at higher concentrations of the copolymer. Furthermore, both interactions of P85 with the brain endothelial cells appear to be energy-dependent as demonstrated by the inhibitory effects of the metabolic inhibitor 2-deoxyglucose.

Anthracycline antibiotics non-covalently incorporated into the block copolymer micelles: in vivo evaluation of anti-cancer activity.

The chemosensitising effects of poly(ethylene oxide)-poly(propylene oxide)-poly-(ethylene oxide) (PEO-PPO-PEO) block copolymers (Pluronic) in multidrug-resistant cancer cells has been described recently (Alakhov VY, Moskaleva EY, Batrakova EV, Kabanov AV 1996, Biocon. Chem., 7, 209). This paper presents initial studies on in vivo evaluation of Pluronic copolymers in the treatment of cancer. The anti-tumour activity of epirubicin (EPI) and doxorubicin (DOX), solubilised in micelles of Pluronic L61, P85 and F108, was investigated using murine leukaemia P388 and daunorubicin-sensitive Sp2/0 and -resistant Sp2/0(DNR) myeloma cells grown subcutaneously (s.c.). The study revealed that the lifespan of the animals and inhibition of tumour growth were considerably increased in mice treated with drug/copolymer compositions compared with animals treated with the free drugs. The anti-tumour activity of the drug/copolymer compositions depends on the concentration of the copolymer and its hydrophobicity, as determined by the ratio of the lengths of hydrophilic PEO and hydrophobic PPO segments. The data suggest that higher activity is associated with more hydrophobic copolymers. In particular, a significant increase in lifespan (T/C> 150%) and tumour growth inhibition (> 90%) was observed in animals with Sp2/0 tumours with EPI/P85 and DOX/L61 compositions. The effective doses of these compositions caused inhibition of Sp2/0 tumour growth and complete disappearance of tumour in 33-50% of animals. Future studies will focus on the evaluation of the activity of Pluronic-based compositions against human drug-resistant tumours.

Hypersensitization of multidrug resistant human ovarian carcinoma cells by pluronic P85 block copolymer.

The chemosensitizing effect of Pluronic P85 block copolymer were studied using two human ovarian carcinoma sublines: the glycoprotein P (P-gp) multidrug resistant (MDR) SKVLB cells and non-MDR SKOV3 cells. The dramatic increase (up to 700 times) in the daunorubicin cytotoxic activity was observed in the presence of 0.01% (22 microM) to 1% (2.2 mM) copolymer in the case of SKVLB cells. By contrast, the copolymer induced a less than 3-fold increase in the drug activity in SKOV3 cells. As a result, the MDR subline demonstrated much higher response ("hypersensitivity") to the daunorubicin/ Pluronic compared to that of the non-MDR cells. The copolymer increased the cytotoxic effects of other MDR type drugs (doxorubicin, epirubicin, vinblastine, and mitomycin C) by a factor of 20-1000 and non-MDR type drugs (methotrexate and cisplatin) by a factor of 2-5.5. The daunorubicin influx in the cytoplasm and nuclei of SKVLB cells was also increased in the presence of the copolymer, while in SKOV3 cells, it remained practically unchanged. However, the hypersensitization of the MDR cells by the copolymer could not be merely explained by the P-gp modulation. Therefore, the possible role of the copolymer in inhibition of non-P-gp drug resistance is hypothesized, which may also explain the sensitization of MDR cells with respect to non-MDR type drugs as well as sensitization of parental cells. The concentration dependence of the IC50 in MDR cells indicates that just the copolymer unimers are responsible for the hypersensitization effect. The results obtained suggest that Pluronic P85 can be used as a delivery system to enhance the activity of antineoplastic agents against MDR tumors.

Pluronic micelles as a tool for low-molecular compound vector delivery into a cell: effect of Staphylococcus aureus enterotoxin B on cell loading with micelle incorporated fluorescent dye.

Micelles of pluronic P85 (poly(oxyethylene)-poly(oxypropylene) block copolymer) are used as microcontainers for in vitro delivery of fluorescein into Jurkat and MDCK cells. In order to target the fluorescein containing micelles into the cell, Staphylococcus aureus enterotoxin B (SEB) is covalently conjugated with a pluronic molecule and the conjugate is incorporated into the micelle content. The incorporation of SEB capable of receptor-mediated endocytosis results in a drastic enhancement of the efficiency of cell loading with the fluorescent dye. This effect is not observed under the conditions (4 degrees C) when endocytosis is abolished.

Micelles of poly(oxyethylene)-poly(oxypropylene) block copolymer (pluronic) as a tool for low-molecular compound delivery into a cell: phosphorylation of intracellular proteins with micelle incorporated [gamma-32P]ATP.

Pluronic P85 (poly(oxyethylene)-poly(oxypropylene) block copolymer) was used for in vitro delivery of [gamma-32P]ATP into intact Jurkat cells. Negatively charged ATP molecules are not able to penetrate cell plasma membrane. Hence, exogenous [gamma-32P]ATP added to a cell culture does not participate in phosphorylation of intracellular proteins. The addition to cells of [gamma-32P]ATP solubilized in positively charged (containing dodecylamine) pluronic micelles results in considerable increase of protein phosphorylation. In this case the treatment of intact cells with alkaline phosphatase (resulting in dephosphorylation of external proteins) causes no essential decrease of [32P]-incorporation in cell proteins. That gives an evidence of delivery of solubilized ATP into a cell. Under the experimental conditions used, pluronic micelles neither influence the viability of cells nor permeabilize cell plasma membrane.

Increasing cytostatic effects of ricin A chain and Staphylococcus aureus enterotoxin A through in vitro hydrophobization with fatty acid residues.

In order to impart an ability for receptor-independent transmembrane transfer to water-soluble proteins, it has been suggested that they be hydrophobized by lipid groups (fatty acids, etc.). To this end, systems of reversed micelles of surfactants in organic solvents were used as reaction media for protein modification. It was shown that after introduction of a hydrophobic anchor (stearic acid residue) the toxic effect of ricin A-chain (in the absence of B-chain) on intact cells became very close to that of the native toxin. As a result of stearic acid acylation, the activity of Staphylococcal enterotoxin A increased by nearly 1.5-2 orders. The observed phenomena can be explained by receptor-independent intracellular translocation of the hydrophobized toxins.

The neuroleptic activity of haloperidol increases after its solubilization in surfactant micelles. Micelles as microcontainers for drug targeting.

It has been suggested to use surfactant micelles as microcontainers for increasing the efficiency of neuroleptic targeting from blood flow into the brain. The neuroleptic action of haloperidol, intraperitoneally injected into mice in micellar solution of non-ionic block copolymer surfactant (pluronic P-85) in water, increased several-fold if compared with that observed for haloperidol aqueous solution. Incorporation of brain-specific antibodies into haloperidol-containing micelles resulted in additional drastic increase (more than by 2 orders of magnitude) in the drug effect.