Valery Alakhov

Pluronic® block copolymers as novel polymer therapeutics for drug and gene delivery

Pluronic block copolymers are found to be an efficient drug delivery system with multiple effects. The incorporation of drugs into the core of the micelles formed by Pluronic results in increased solubility, metabolic stability and circulation time for the drug. The interactions of the Pluronic unimers with multidrug-resistant cancer cells result in sensitization of these cells with respect to various anticancer agents. Furthermore, the single molecular chains of copolymer, unimers, inhibit drug efflux transporters in both the blood-brain barrier and in the small intestine, which provides for the enhanced transport of select drugs to the brain and increases oral bioavailability. These and other applications of Pluronic block copolymers in various drug delivery and gene delivery systems are considered.

Pluronic® block copolymers for overcoming drug resistance in cancer

Pluronic block copolymers have been used extensively in a variety of pharmaceutical formulations including delivery of low molecular mass drugs and polypeptides. This review describes novel applications of Pluronic block copolymers in the treatment of drug-resistant tumors. It has been discovered that Pluronic block copolymers interact with multidrug-resistant cancer (MDR) tumors resulting in drastic sensitization of these tumors with respect to various anticancer agents, particularly, anthracycline antibiotics. Furthermore, Pluronic affects several distinct drug resistance mechanisms including inhibition of drug efflux transporters, abolishing drug sequestration in acidic vesicles as well as inhibiting the glutathione/glutathione S-transferase detoxification system. All these mechanisms of drug resistance are energy-dependent and therefore ATP depletion induced by Pluronic block copolymers in MDR cells is considered as one potential reason for chemosensitization of these cells. Following validation using in vitro and in vivo models, a formulation containing doxorubicin and Pluronic mixture (L61 and F127), SP1049C, has been evaluated in phase I clinical trials. Further mechanistic studies and clinical evaluations of these systems are in progress.

Evaluation of polyether-polyethyleneimine graft copolymers as gene transfer agents

Cationic copolymers consisting of polycations linked to non-ionic polymers are evaluated as non-viral gene delivery systems. These copolymers are known to produce soluble complexes with DNA, but only a few studies have characterized the transfection activity of these complexes. This work reports the synthesis and characterization of a series of cationic copolymers obtained by grafting the polyethyleneimine (PEI) with non-ionic polyethers, poly (ethylene oxide) (PEO) or Pluronic 123 (P123). The PEO–PEI conjugates differ in the molecular mass of PEI (2 kDa and 25 kDa) and the degree of modification of PEI with PEO. All of these conjugates form complexes upon mixing with plasmids, which are stable in aqueous dispersion for several days. The sizes of the particles formed in these systems vary from 70 to 200 nm depending on the composition of the complex. However, transfection activity of these systems is much lower than that of PEI (25 kDa) or Superfect as assessed in in vitro transfection experiments utilizing a luciferase reporter expression in Cos-7 cells as a model system. In contrast, conjugate of P123 with PEI (2 kDa) mixed with free P123 (9:1(wt)) forms small and stable complexes with DNA (110 nm) that exhibit high transfection activity in vitro. Furthermore, gene expression is observed in spleen, heart, lungs and liver 24 h after i.v. injection of this complex in mice. Compared to 1,2-bis(oleoyloxy)-(trimethylammonio) propane:cholesterol (DOTAP:Chol) and PEI (25 kDa) transfection systems, the P123-PEI system reveals a more uniform distribution of gene expression between these organs, allowing a significant improvement of gene expression in liver.

PLURONIC BLOCK COPOLYMERS: NOVEL FUNCTIONAL MOLECULES FOR GENE THERAPY

Pluronic block copolymers are recognized pharmaceutical excipients listed in the US and British Pharmacopoeia. They have been used extensively in a variety of pharmaceutical formulations including delivery of low molecular mass drugs and polypeptides. This review describes novel applications of Pluronic block copolymers in gene therapy. In particular, these molecules can modify the biological response during gene therapy in the skeletal muscle, resulting in an enhancement of the transgene expression as well as an enhancement of the therapeutic effect of the transgene. Furthermore, Pluronic block copolymers are versatile molecules that can be used as structural elements of the polycation-based gene delivery systems (polyplexes). Based on these studies, the use of block copolymers in gene delivery is a promising area of research, in which new and important developments are expected.

A new class of drug carriers: micelles of poly(oxyethylene)-poly(oxypropylene) block copolymers as microcontainers for drug targeting from blood in brain☆

Abstract A new concept of design of drug delivery systems based on using self-assembling supramacromolecular complexes is formulated. Microcontainers for drug targeting were prepared using polymeric surfactant poly(oxyethylene)-poly(oxypropylene) block copolymer (pluronic). Molecules of a drug are solubilized in a pluronic micelle being incorporated into its inner hydrophobic core, formed by poly(oxypropylene) chain blocks. The outer hydrophilic shell of such micelles is formed by nontoxic and nonimmunogenic poly(oxyethylene) blocks. Solubilization of low molecular weight compounds (fluorescein isotbiocyanate (FITC), haloperidol etc.) in pluronic micelles was studied using fluorescence and ultracentrifugation. The dimensions of the aggregates formed in the solutions of various pluronics (P85, F64, L68, L101) and its mixtures were determined using quasielastic light-scattering technique. In a majority of cases the diameter of pluronic micelles (including those containing solubilized compounds) was in the range of 12–36 nm. For targeting of such microcontainers to a certain cell the pluronic molecules were conjugated with antibodies against a target-specific antigen or with protein ligands selectively interacting with target cell receptors. The obtained conjugates were then incorporated into the drug-containing micelles by simple mixing of the corresponding components. It was found that solubilization of FITC in pluronic micelles considerably influences its distribution in animal (mouse) tissues resulting, in particular, in the drastic increase of FITC fluorescence in lung. Conjugation of FITC-containing micelles with insulin vector results in increase of FITC penetration in all tissues including the brain. The specific targeting of the solubilized FITC in brain was observed in the case when the pluronic conjugate with antibodies to the antigen of brain glial cells ( α 2 -glycoprotein) was incorporated into micelles. Under these conditions the considerable increase of FITC fluorescence in the brain and decrease of its fluorescence in the lungs has been registered. Possibility of using micellar microcontainers for targeting of solubilized neuroleptics (haloperidol) in brain was studied. Incorporation of antibodies to α 2 glycoprotein into haloperidol-containing micelles results in a drastic increase of drug effect. This result indicates that vector-containing pluronic micelles provide an effective transport of solubilized neuroleptics across blood-brain barrier.

Block copolymer-based formulation of doxorubicin. From cell screen to clinical trials

Abstract A new doxorubicin formulation (SP1049C) has been developed using a combination of two polyethylene oxide polypropylene oxide block copolymers, in particular Pluronic L61 and Pluronic F127. The analysis of cytotoxic activity of this product on the cell screen panel has shown that SP1049C is highly effective against multidrug resistant cells that are normally not susceptible to doxorubicin and most other cytotoxic drugs. Further mechanistic studies have revealed that SP1049C has higher activity than doxorubicin due to: (i) increase in the drug uptake; (ii) inhibition of the energy-dependent drug efflux; and (iii) changes in intracellular drug trafficking. The experiments on in vivo tumour models have confirmed high efficacy of SP1049C against drug-resistant tumours, as well as suggested that this product has considerably broader efficacy than doxorubicin. The analysis of pharmacokinetics and biodistribution of SP1049C has shown that it accumulates in tumour tissue more effectively than doxorubicin, while distribution of the formulation in normal tissues is similar to that of doxorubicin. The toxicity studies of the copolymer composition used in SP1049C and of the product itself have demonstrated that the carrier has high safety margin, while toxicity of SP1049C is similar to that of doxorubicin suggesting that no additional adverse effects should be expected in clinical trials of SP1049C.

A combination of poloxamers increases gene expression of plasmid DNA in skeletal muscle.

Intramuscular administration of plasmid DNA is a promising strategy to express therapeutic genes, however, it is limited by a relatively low level of gene expression. We report here that a non-ionic carrier, SP1017, composed of two amphiphilic block copolymers, pluronics L61 and F127, also known as poloxamers, significantly increases intramuscular expression of plasmid DNA. Two reporter genes, luciferase and β-galactosidase, and one therapeutic gene, erythropoietin, were injected intramuscularly with and without SP1017 into C57Bl/6 and Balb/C mice and Sprague–Dawley rats. SP1017 increased gene expression by about 10-fold and maintained higher gene expression compared with naked DNA. Comparison of SP1017 with polyvinyl pyrrolidone (PVP) showed that SP1017 exhibited a significantly higher efficacy and its optimal dose was 500-fold lower. Experiments with β-galactosidase using X-gal staining suggested that SP1017 considerably increased plasmid DNA diffusion through the tissue. SP1017 also improved expression of the erythropoietin gene leading to an increase in its systemic level and hematocrits. Previous toxicity studies have suggested that SP1017 has over a 1000-fold safety margin. Poloxamers used in SP1017 are listed in the US Pharmacopeia as inactive excipients and are widely used in a variety of clinical applications. We believe that the described system constitutes a simple and efficient gene transfer method to achieve local or systemic production of therapeutic proteins.

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.

Effect of Pluronic P85 on ATPase activity of drug efflux transporters

No HeadingPurpose.Pluronic block copolymers are potent sensitizers of multidrug resistant (MDR) cancer cells. The sensitization effect by Pluronics is a result of two processes acting in concert: i) intracellular ATP depletion, and ii) inhibition of ATPase activity of drug efflux proteins. This work characterizes effects of Pluronic P85 on ATPase activities of Pgp, MRP1, and MRP2 drug efflux transport proteins and interaction of these proteins with their substrates, vinblastine, and leucotriene C4.Methods.Using membranes overexpressing Pgp, MRP1, and MRP2, the current study evaluates effects of Pluronic P85 (P85) on the kinetic parameters (Vmax, Km, Vmax/Km) of ATP hydrolysis by these ATPases.Results.The decreases in the maximal reaction rates (Vmax) and increases in apparent Michaelis constants (Km) for these transporters in the presence of various concentrations of P85 were observed. The mechanism of these effects may involve i) conformational changes of the transporter due to membrane fluidization and/or ii) nonspecific steric hindrance of the drug-binding sites by P85 chains embedded into cellular membranes. The extent of these alterations was increased in the row MRP1 < MRP2 << Pgp.Conclusions.These data suggest that there are unifying pathways for the inhibition of Pgp and MRPs by the block copolymer. However, the effect of P85 on Pgp ATPase activity is considerably greater compared with the effects on MRP1 and MRP2 ATPases. This may be a reason for greater inhibitory effects of Pluronic in Pgp-compared with MRP-overexpressing cells.

Sensitization of cells overexpressing multidrug-resistant proteins by pluronic P85.

AbstractPurpose. This study evaluated the chemosensitizing effects of Pluronic P85 (P85) on cells expressing multidrug resistance-associated proteins, MRP1 and MRP2. Methods. Cell models included MRP1- and MRP2-transfected MDCKII cells as well as doxorubicin-selected COR-L23/R cells overexpressing MRP1. Effects of P85 on cellular accumulation and cytotoxicity of vinblastine and doxorubicin were determined. Mechanistic studies characterized the effects of P85 on ATP and reduced glutathione (GSH) intracellular levels as well as MRP ATPase and glutathione-S-transferase (GST) activities in these cells. Results. Considerable increases of vinblastine and doxorubicin accumulation in the cells overexpressing MRP1 and MRP2 in the presence of P85 were observed, although no statistically significant changes in drug accumulation in the parental cells were found. P85 treatment caused an inhibition of MRP ATPase activity. Furthermore, P85 induced ATP depletion in these cells similar to that previously reported for Pgp-overexpressing cells. In addition, reduction of GSH intracellular levels and decrease of GST activity were observed following P85 treatment. Finally, significant enhancement of cytotoxicity of vinblastine and doxorubicin by P85 in MRP-overexpressing cells was demonstrated. Conclusions. This study suggests that P85 can sensitize cells overexpressing MRP1 and MRP2, which could be useful for chemotherapy of cancers that display these resistant mechanisms.