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

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Journal of Controlled Release 82 (2002) 189–212 www.elsevier.com / locate / jconrel Review Pluronic block copolymers as novel polymer therapeutics for drug and gene delivery a, a b * Alexander V. Kabanov , Elena V. Batrakova ,Valery Yu. Alakhov a Department of Pharmaceutical Sciences, College of Pharmacy, 986025 Nebraska Medical Center, Omaha, NE 68198-6025, USA b Supratek Pharma Inc., 531 Blvd. des Prairies, Build. 18, Laval, Quebec, Canada H7B 1B7 Received 5 September 2001; accepted 7 January 2002 Abstract 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. 2002 Elsevier Science B.V. All rights reserved. Keywords: Block copolymers; Blood–brain barrier; Gene therapy; Cancer chemotherapy; Drug delivery 1. Introduction site within the body, where it can perform its biological role. In addition, delivery systems can Development of efficient drug delivery systems address and correct problems related to the physical has attracted tremendous attention during the last two characteristic of a drug, including solubility and decades. The principal reason for the incredible stability. Consequently, the technologies currently growth of drug delivery technology is the realization under development for drug delivery and drug that the best prospect for achieving substantial targeting systems will have a tremendous impact on improvements over current therapies will occur the improvement of novel drug therapies. through improved delivery of both existing drugs and A major subset of existing drug delivery systems, yet undiscovered drugs. This necessity arises pri- those based on synthetic polymers, have attracted marily due to the enormous barriers that a drug significant attention, as they appear particularly molecule must overcome before it reaches its target promising [1]. This has led to the emergence of a new field called ‘polymer therapeutics’. Generally, polymer therapeutics refers to any polymer that is *Corresponding author. Tel.: 11-402-559-9364; fax: 11-402- used as a component of a drug product for the 559-9543. purpose of eliciting or modifying drug action. This E-mail addresses: [email protected] (A.V. Kabanov), [email protected] (V.Y. Alakhov). includes polymers, which are inherently biologically 0168-3659 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0168-3659(02)00009-3

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Page 1: Pluronic® block copolymers as novel polymer therapeutics for drug and gene delivery

Journal of Controlled Release 82 (2002) 189–212www.elsevier.com/ locate/ jconrel

ReviewPluronic block copolymers as novel polymer therapeutics for

drug and gene deliverya , a b*Alexander V. Kabanov , Elena V. Batrakova , Valery Yu. Alakhov

aDepartment of Pharmaceutical Sciences, College of Pharmacy, 986025Nebraska Medical Center, Omaha, NE 68198-6025,USAbSupratek Pharma Inc., 531 Blvd. des Prairies, Build. 18, Laval, Quebec, Canada H7B 1B7

Received 5 September 2001; accepted 7 January 2002

Abstract

Pluronic block copolymers are found to be an efficient drug delivery system with multiple effects. The incorporation ofdrugs 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 ofthese cells with respect to various anticancer agents. Furthermore, the single molecular chains of copolymer, unimers, inhibitdrug 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 invarious drug delivery and gene delivery systems are considered. 2002 Elsevier Science B.V. All rights reserved.

Keywords: Block copolymers; Blood–brain barrier; Gene therapy; Cancer chemotherapy; Drug delivery

1. Introduction site within the body, where it can perform itsbiological role. In addition, delivery systems can

Development of efficient drug delivery systems address and correct problems related to the physicalhas attracted tremendous attention during the last two characteristic of a drug, including solubility anddecades. The principal reason for the incredible stability. Consequently, the technologies currentlygrowth of drug delivery technology is the realization under development for drug delivery and drugthat the best prospect for achieving substantial targeting systems will have a tremendous impact onimprovements over current therapies will occur the improvement of novel drug therapies.through improved delivery of both existing drugs and A major subset of existing drug delivery systems,yet undiscovered drugs. This necessity arises pri- those based on synthetic polymers, have attractedmarily due to the enormous barriers that a drug significant attention, as they appear particularlymolecule must overcome before it reaches its target promising [1]. This has led to the emergence of a

new field called ‘polymer therapeutics’. Generally,polymer therapeutics refers to any polymer that is

*Corresponding author. Tel.:11-402-559-9364; fax:11-402-used as a component of a drug product for the559-9543.purpose of eliciting or modifying drug action. ThisE-mail addresses: [email protected] (A.V. Kabanov),

[email protected] (V.Y. Alakhov). includes polymers, which are inherently biologically

0168-3659/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0168-3659( 02 )00009-3

Page 2: Pluronic® block copolymers as novel polymer therapeutics for drug and gene delivery

190 A.V. Kabanov et al. / Journal of Controlled Release 82 (2002) 189–212

active, polymer–drug conjugates, polymeric mi- copolymers in aqueous media. These solutions existcelles, nanoparticles and polymer-coated liposomes. in the form of either a molecular dispersion or asA growing number of polymer therapeutics are micelles of the block copolymers. The core–shellapproved by the regulatory authorities in North architecture of the micelles is essential for theirAmerica, Europe and Asia for clinical use in treat- utility in drug delivery. The core is a water-incom-ment of cancer, infectious and genetic diseases. patible compartment that is segregated from theThere are several fundamental properties of polymers aqueous exterior by the hydrophilic chains of theuseful in solving drug delivery problems. First, shell, thereby forming, within the core, a ‘cargo’ forpolymers are large molecules that can be designed to the incorporation of various therapeutic or diagnosticbe intrinsically multifunctional and thus can be reagents. As a result, polymeric micelles can be usedcombined either covalently or non-covalently with as efficient carriers for compounds, which alonedrugs to overcome multiple problems such as solu- exhibit poor solubility, undesired pharmacokineticsbility, stability, permeability, etc. Second, polymers and low stability in a physiological environment.can be combined with various targeting vectors to Furthermore, specifically in relation to drug and genedirect drugs to specific sites in the body. Third, delivery, there exists a distinct set of properties of

polymers are ideal for design of environmental Pluronic block copolymers, which are potentially ofstimulus-responsive materials allowing for controlled enormous importance for pharmaceutical applica-and sustained release of the drug at the site of the tions. These properties bestow the ability of

action. Finally, polymers can have biological activity Pluronic block copolymers to enhance drug per-of their own and are capable of interacting with and formance by acting as biological response-modifyingmodifying the activity of various endogenous drug agents, which act directly upon the target cells. One

transport systems within the body, thus affecting such example is the ability of Pluronic blockdrug delivery. copolymers to enhance the effectiveness of chemo-

One important and promising example of novel therapeutic micelle formulation. Furthermore, thesepolymer therapeutics that benefit from each of the activities may prove very useful in enhancing bothabove properties of the polymers is polymeric mi- oral and brain bioavailability of pharmaceuticalcelles formed by amphiphilic block copolymers. agents, which are currently only poorly transportedPolymeric micelles have been evaluated in multiple across the small intestine or the blood–brain barrier

pharmaceutical applications as drug and gene deliv- (BBB). Finally, very recently, Pluronic block co-ery systems, as well as in diagnostic imaging as polymers were found exciting opportunities for thecarriers for various contrasting agents [2–11]. This development of novel gene therapies and vaccinationpaper presents an updated overview of the use of strategies.

Pluronic block copolymers in drug and gene deliv- The following sections discuss: (i) the structureery. This focus is justified because the magnitude of and self-assembly of Pluronic block copolymers;

experimental and theoretical data available with the (ii) physicochemical principles of micellar drugPluronic block copolymers allows for a systematic formulations; (iii) pharmacokinetic consideration of

and in depth consideration of the use of these Pluronic formulations; (iv) Pluronic formulationscompounds as polymer therapeutics. The use of for the treatment of drug-resistant tumors; (v)

Pluronic block copolymers in experimental medi- Pluronic effects on both brain and oral bioavail-cine and pharmaceutical sciences has a long history. ability; and (vi) Pluronic formulations for gene

A number of excellent reviews have been published therapy.that contain detailed discussions of many aspects of

Pluronic -based formulations, particularly, thoseusing gels, w/o and o/w emulsions, nanoparticles 2. Pluronic block copolymers: structure and

coated by the block copolymer and solid polymer solution behaviorblends [4,12–20]. These systems are formed either in

the condensed state or with a high concentration of 2.1. Pluronic structure and synthesislipophilic components. The current review focuses

on the relatively dilute isotropic solutions of block Pluronic block copolymers (also known under

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A.V. Kabanov et al. / Journal of Controlled Release 82 (2002) 189–212 191

altered. The structure formula of Pluronic blockcopolymers is shown in Fig. 1. Table 1 presents a list

of selected Pluronic copolymers available fromBASF Corp. Copolymers with variousx andy valuesare characterized by distinct hydrophilic–lipophilicbalance (HLB).

Pluronic block copolymers are synthesized bysequential addition of PO and EO monomers in thepresence of an alkaline catalyst, such as sodium orpotassium hydroxide [12]. The reaction is initiated

by polymerization of the PO block followed by theFig. 1. Pluronic block copolymers available from BASF (Wyan-dotte, MI), contain two hydrophilic EO blocks and a hydrophobic growth of EO chains at both ends of the PO block.PO block. Anionic polymerization usually produces polymers

with a relatively low polydispersity index (M /M ).n w

their non-proprietary name ‘poloxamers’ consist of Further chromatographic fractionation was employedethylene oxide (EO) and propylene oxide (PO) in procedures for the manufacture of highly purifiedblocks arranged in a basic A–B–A structure: EO – block copolymers [21,22]. This reduces the presencex

PO –EO . This arrangement results in an am- of admixtures, particularly, of the PO homopolymery x

phiphilic copolymer, in which the number of hydro- and block copolymers with lower content of EO thanphilic EO (x) and hydrophobic PO (y) units can be expected.

Table 1Physicochemical characteristics of Pluronic block copolymers

a c dCopolymer MW Average no. of Average no. of HLB Cloud point in CMC (M)b bEO units (x) PO units (y) 1% aqueous

csolution (8C)23L35 1900 21.59 16.38 19 73 5.331023L43 1850 12.61 22.33 12 42 2.231023L44 2200 20.00 22.76 16 65 3.631024L61 2000 4.55 31.03 3 24 1.131024L62 2500 11.36 34.48 7 32 4.031024L64 2900 26.36 30.00 15 58 4.831024F68 8400 152.73 28.97 29 .100 4.831025L81 2750 6.25 42.67 2 20 2.331025P84 4200 38.18 43.45 14 74 7.131025P85 4600 52.27 39.66 16 85 6.531025F87 7700 122.50 39.83 24 .100 9.131024F88 11 400 207.27 39.31 28 .100 2.531025L92 3650 16.59 50.34 6 26 8.831025F98 13 000 236.36 44.83 28 .100 7.731026L101 3800 8.64 58.97 1 15 2.131026P103 4950 33.75 59.74 9 86 6.131026P104 5900 53.64 61.03 13 81 3.431026P105 6500 73.86 56.03 15 91 6.231025F108 14 600 265.45 50.34 27 .100 2.231026L121 4400 10.00 68.28 1 14 1.031026P123 5750 39.20 69.40 8 90 4.431026F127 12 600 200.45 65.17 22 .100 2.8310

a The average molecular weights provided by the manufacturer (BASF, Wyandotte, MI).b The average numbers of EO and PO units were calculated using the average molecular weights.c HLB values of the copolymers; the cloud points were determined by the manufacturer.d CMC values were determined previously using pyrene probe [38].

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2.2. Micellization and solubilization will vary 3–10 times depending on the method ofmeasurement employed.

A defining property of amphiphilic block copoly- Some of the methods used for determination ofmers is the ability of individual block copolymer CMC in Pluronic aqueous dispersions include

molecules, termed ‘unimers’, to self-assemble into surface tension measurements [24–27], chromatog-micelles in aqueous solutions. The ‘unimers’ form raphy [28], light scattering [29–33], small anglemolecular solutions in water at block copolymer neutron scattering (SANS) [34], small angle X-rayconcentrations below that of the critical micelle scattering (SAXS) [35], differential scanningconcentration (CMC). At concentrations of the block calorimetry [35,36], viscosimetry [35], acoustic mea-copolymer above the CMC, unimer molecules aggre- surements [32,35], and utilization of fluorescentgate and form micelles, a process called ‘micelliza- probes [25,37,38].

tion’. The micelles can be spherical, rod-like, or Typically, the Pluronic copolymers, which arelamellar depending on the length of the EO and PO used for drug delivery, at body temperature (378C)blocks, concentration of the block copolymers, and have a CMC ranging from 1mM to 1 mM (ca.

23the temperature [23]. All these types of micelles 5310 to 1% wt.) (Table 1). There is comprehen-have hydrophobic core of the corresponding shape sive work available which relates the CMC to the

formed by PO chains and hydrophilic shell formed structure of Pluronic block copolymers [27,37]. Theby EO chains. The number of block copolymer CMC of the block copolymers is strongly dependentmolecules forming one micelle is called the ‘aggre- on the lengths of the blocks. An increase in thegation number’. The aggregation numbers for a length of the hydrophobic PO block elevates the net

spherical micelle usually vary from several to over hydrophobicity of the Pluronic molecule and favors100. The PO core can serve as a ‘pool’ for the the segregation of the PO chains into the micelleincorporation of various hydrophobic compounds core [23,39,40]. As a result, an increase in the lengthinto the micelles. As a result of the incorporation of the PO block results in a CMC decrease [37].into the PO core water-insoluble compounds are Conversely, an increase in the lengths of the EOtransferred into the micellar solution, a process blocks elevates the probability of contacts of the POcalled ‘solubilization’. units with the EO units within the core of the

micelles. This effect, decreases the hydrophobicity ofthe core and results in destabilization of the micelle.

2.3. Critical micelle concentration (CMC) Therefore, the CMC increases as the hydrophilic EOblock length is increased [27,37].

The CMC is most simply defined as the con- CMC is of paramount significance to drug deliverycentration at which micelles are formed. At con- using block copolymers [25,41]. First, the CMCcentrations below the CMC the block copolymer in determines the stability of micelles against possiblesolution is in the form of unimers. When the block dilution of the drug delivery system in body fluidscopolymer is at a concentration above CMC there [7,25]. Second, the CMC determines the maximal

exist a dynamic equilibrium exchange between mi- achievable concentration of Pluronic unimers, tocelles and unimers. As the concentration of block which cells will be exposed, thereby defining thecopolymer increases above the CMC, the number of biological response modifying effects, which

micelles elevates, while the concentration of the Pluronic itself, will exert on these cells [42]. Bothunimers in equilibrium with the micelles usually issues are addressed in greater detail in the followingremains constant (equal to CMC). Although the sections.CMC is defined as a single concentration point the

micellization and micelle structure transitions can 2.4. Structure of Pluronic micellesoccur in a relatively broad range of concentration in

the vicinity of the CMC and may also extend above Pluronic micelles are commonly pictured asthe CMC. For Pluronic block copolymers, it is spheres with PO core and EO corona. Table 2

generally considered acceptable that the CMC value presents some molecular characteristics of Pluronic

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A.V. Kabanov et al. / Journal of Controlled Release 82 (2002) 189–212 193

Table 2Molecular characteristics of the P85 micelles

a bMicelle parameter Experiment Theory3Partial specific volume,v (cm /g) 0.84 N/a

o 2 27Translation diffusion coefficient,D (cm /s) 3.7310 N/azo 213Sedimentation coefficient,S (s) 5.9310 N/a

Molecular mass,M (kDa) 256674 243.8Aggregation number,N 57616 53a

Micelle radius,R (nm) 7.360.3 6.46Hydrophobic core radius,r (nm) 3.760.3 3.63Hydrophilic corona thickness,R2r (nm) 3.660.6 2.83

3Mean volume per EO unit,v (nm ) 0.49 N/aEO3 3Core volume, 4/3pr (nm ) 210 200

3 3Micelle volume, 4/3pR (nm ) 1630 1130a Characteristics of the micelles at 378C as reported by Kabanov et al. [25]. The micelle volume and the core volume were calculated by

us based on the data for the hydrodynamic radius of the micelle and the core radius reported in this reference.b Theoretical values calculated by Nagarajan [23]. The molecular mass of the micelle, micelle volume and core volume were calculated,

by us, based on the data reported in this reference.

micelles using P85 as an example. Generally, these size range [16]. Furthermore, particles, which arecharacteristics are strongly dependent on the com- smaller than 100 nm can also be accommodated inposition of the block copolymer. Literature con- endocytic vesicles allowing entry into target cells viataining experimental studies as well as the theoretical endocytosis [51]. Following systemic administration

considerations related to Pluronic micelles is avail- in the body particles ranging from ca. 70 to 200 nmable [24,39,40,43–46]. These works relate the mi- demonstrate the most prolonged circulation timescelle aggregation number and micelle size to the [4,49]. However, deviation from the preferred rangelengths of the EO and PO chains. In general, for a of the sizes of the particles used for systemicfixed PO block, the block copolymers with a higher administration is usually accompanied by the de-EO block content are less aggregated and have a crease in the blood circulation times. For example,smaller core size. When spherical micelles are on one hand, the use of particles with diameters

formed, depending on the Pluronic type the mi- more than 200 nm is not recommended because suchcelles commonly have an average hydrodynamic particles are frequently sequestered by the spleen asdiameter ranging from about 20 to about 80 nm, and a result of mechanical filtration, followed by eventu-aggregation numbers ranging from 10 to several al removal by the cells of the phagocyte systemdozens [23]. [4,48]. On the other hand, small particles with

The size of the micelles is particularly important diameters of less than 5–10 nm are rapidly removedfrom the drug delivery standpoint. The size variation through extravasation and renal clearance [47].in nanoscale range strongly affects the blood circula-tion times and the bioavailability of the particles 2.5. Temperature dependence of micelle formationwithin the body [4,16,47–50]. For many pharma-ceutical applications using nanoscale particles the Micelle formation, in aqueous solutions of

preferred size range is from ca. 10 to 100 nm. The Pluronic , is critically dependent upon temperature.micelles of Pluronic block copolymers are clearly This is a result of the successive dehydration of PO

within this preferred size range. For example, par- and EO chains upon the increase in temperatureticles ranging from ca. 10 to 70 nm offer effective [24,28,36,39,40,44,52]. Below room temperature,

distribution in certain tissues [4,16]. Also, some both types of blocks within a Pluronic molecule arepharmaceutical applications requiring faster drainage hydrated and are relatively soluble in water. Whenfrom the sites of injection following subcutaneous the temperature increases, the PO block dehydratesinjection can benefit from using the particles of this and becomes insoluble, resulting in the formation of

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micelles. The temperature at which micelles are drug delivery systems containing both micelles andformed is referred to as the ‘critical micelle tempera- unimers.

ture’ or CMT. For most Pluronic copolymers, theCMT values range from ca. 25 to 408C, i.e. below ornear body temperature. 3. Micellar drug formulation

Consideration of the temperature dependence ofmicelle formation in drug delivery is important in 3.1. Methods of incorporation of drugs in blockseveral aspects. In particular, one must be aware thatcopolymer micellesmicelles can disintegrate and release solubilized drugif they are stored below room temperature. Although The simplest ways of preparing drug-containingthe micelle disintegration is reversible and micelles micelles is to solubilize solid drug or inject a smallcan reassemble when the temperature is increased, volume of a drug solution, in a water-miscible

the solubilized drug can precipitate and may be organic solvent, into micellar solutions of Pluronicdifficult to re-solubilize as a consequence of kinetics in water [54,55]. Depending on the type of drugfactors. Furthermore, recent studies utilize the tem- used, activity of the drug and route of administration,

perature dependence of Pluronic micelle formation the desired concentration of the drug in a pharma-to design various temperature responsive drug deliv- ceutical formulation may vary. Quite often, forery systems, such as, for example, thermotropic gels example, a relatively high concentration of drug in

from Pluronic -poly(acrylic acid) conjugates and formulation (usually, several mg/ml) is needed forothers [53]. injection. In these cases, techniques that facilitate

formation of equilibrium dispersions can be used.For example, drug and block copolymer are first

2.6. Biological significance of micelles and dissolved in a common organic solvent and then theunimers phases are reversed by slow addition of the aqueous

component [9]. The residual organic solvent is Pluronic micelles and Pluronic unimers play removed by dialysis or evaporation. Another tech-

very important roles in a drug formulation. Micelles nique involves evaporation of the solvent from aare used as carriers for various drugs [2–9]. The common organic solution of the drug and blockincorporation of drugs into the core of the micelle copolymer, usually obtaining a dry film from theresults in increased solubility, metabolic stability and component mixture, which is then re-dispersed in thecirculation time for the drug. By attaching a peptide aqueous media.or other biospecific molecule that can promote site-specific drug delivery to surface of the EO corona, a 3.2. Solubilization capacity of the micellesmicelle can be targeted to a specific site in the body

[54,55]. Pluronic unimers also have an important Quite often scientists developing micellar drugbiological role in drug delivery. It has been discov- formulations face limitations to the solubilization

ered that Pluronic unimers exhibit biological re- capacity of the micelles with respect to certainsponse modifying activities in certain drug formula- lipophilic drugs. For example, an estimate of thetions. For example, the interactions of the unimers ‘cargo space’ in P85 micelles suggests that thewith multidrug-resistant cancer cells result in sensiti- volume of the core is less than 13% of the totalzation of these cells with respect to various anti- volume of the micelle (Table 2). Obviously, the

cancer agents [56,57]. Further, Pluronic unimers actual space available for incorporation of the drughave been shown to inhibit drug efflux transporters in the core is even lower than the above number,in both the blood–brain barrier and in the small which further limits the solubilization capacity.intestine [41,58]. This can provide for enhanced Significant variation between the solubilizationtransport of select drugs to the brain and enhanced capacity of micelles formed by the same blockoral bioavailability. The following chapters consider copolymer with respect to various solutes is ex-

pharmaceutical and biochemical aspects of Pluronic plained by differences in the structure of this solute

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A.V. Kabanov et al. / Journal of Controlled Release 82 (2002) 189–212 195

[23]. Many studies have indicated that the most micelles in the formulation administered to theimportant factor related to the drug solubilization patient, as well as of the amount of the drug, whichcapacity is the compatibility between the solubilizate can be released upon dilution of this formulation inand the core-forming block [23,39,59–61]. For ex- the body fluids.ample, Nagarajan demonstrated that the amount of Several methods have been proposed for determin-the incorporated solubilizate increases as the molecu- ing the partition coefficients of solutes in the blocklar volume of the solubilizate decreases [23]. Further, copolymer systems. Fluorescence technique is onethe solubilization capacity is greater when the core convenient method that allows determination of theblock–solubilizate interactions are favorable and the partition coefficients of solutes in block copolymersolubilizate-water surface tension is lower [23,59]. micelles [25,63]. In the case of non-fluorescentAs a result, for example, aromatic hydrocarbons are solutes that contain chromophor groups, a method

incorporated in Pluronic micelles to a greater extent for partition coefficient determination is based onthan aliphatic hydrocarbons [23,61]. Overall, com- measuring the electronic spectra of these solutes. Anpared to conventional low molecular weight surfac- alternative method for determination of the partition

tants, Pluronic block copolymers have much higher coefficients involves measuring the rates of diffusionsolubilization capacities and are more selective to- of the solute from the block copolymer solutionswards aromatic and heterocyclic compounds than through a semi-permeable membrane [64,65]. Atowards aliphatic molecules [23,61]. Since many recent work compared the fluorescence- and dif-drug molecules contain aromatic and heterocyclic fusion-based techniques and revealed that the parti-

groups, Pluronic micelles appear to be particularly tion coefficient values obtained by these two meth-suited for the preparation of pharmaceutical formula- ods are in very good agreement with each other [65].tions of such drugs.

3.4. Interaction of biopolymers with Pluronicmicelles3.3. Solute partitioning

Pluronic unimers can bind to hydrophobic sitesIn every micellar system containing solubilizate,on biopolymer molecules, even at concentrationsthere is always a dynamic exchange between thebelow the CMC when micelles are not present.solute molecules incorporated in the micelle andHydrophobic effect should obviously play a majorthose dissolved in the external solution, a processrole as a driving force for binding of biopolymers tocalled ‘partitioning’. If the rate of transfer of the Pluronic aggregates or unimers. Madden et al. [66]molecules from micelles to solution equals the rate studied the effect of Pluronic on protein conforma-of the transfer of these molecules from the solutiontion, using P105 and a peripheral membrane protein,to micelles, i.e. equilibrium is reached, then thecytochrome c, as examples. Using electron para-partitioning is characterized by a thermodynamicmagnetic resonance measurements on a cytochromeconstant, called the ‘partition coefficient’, P [62]. Byc modified with a paramagnetic label, this studydefinition, the partition coefficient stands for thesuggested that the protein did indeed interact withratio: the Pluronic micelles, inducing changes in therotational correlation time of the protein label. The[S ]m

]]P 5 protein did not appear to insert deeply in the[S ]w hydrophobic core of the Pluronic micelle, i.e.where (S ) and (S ) are the solute concentrations in assumed peripheral localization, and the size of them w

the micelle microphase and in the aqueous phase, micelle did not change.respectively. Furthermore, the interactions of proteins with

Conditions of equilibrium partitioning are rarely block copolymers are dependent on the hydropho-realized in the body. However, partitioning coeffi- bicity and ionic state of the protein globule. Indeed,cient can be useful, as first estimation, to determine Svensson et al. [67] recently demonstrated this in athe amount of the drug, which is incorporated in the study of partitioning of various peptides and proteins

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in aqueous two-phase system containing solutions of unable to cross the cell membrane. These studiesPluronic as one phase and dextran as another phase. suggested that attachment of protein molecules to the

The biopolymers used in this study included: (i) micelles could have beneficial effects on the deliveryrelatively hydrophilic proteins (lysozyme, bovine of biologically active molecules to critical sites in theserum albumin and cytochromec); (ii) membrane- body or within a cell.bound hydrophobic peptide and protein (gramicidin One strategy developed for incorporation of hy-

D and bacteriorhodopsin), and (iii) a recombinant B drophilic proteins into Pluronic micelles involvesdomain of staphylococcal protein A fused with covalent attachment of the protein to the blocktryptophan-rich peptides of various lengths. Two copolymer molecule [56]. The sites of attachment ofblock copolymers, F68 and P105, were examined at the protein are the free terminal hydroxyl groups in

two temperatures, 5 and 308C, which were below the Pluronic molecule (Fig. 1). The conjugatesand above the CMT of the copolymers. Generally, obtained are then mixed with the unmodified blockpartitioning of the hydrophobic proteins to the copolymer at concentrations above the CMC, which

Pluronic phase increased as the temperature was results in the formation of mixed micelles, withincreased and the micelles were formed, compared to protein molecules being attached to the EO corona.the lower temperature when only the unimers werepresent. This behavior was consistent with the dehy-dration of the PO chains upon increase in tempera- 4. Pharmacokinetics of block copolymer micelleture and the contribution of the hydrophobic effect to formulationsthe partitioning. With hydrophilic proteins the tem-perature only marginally affected the partitioning. 4.1. Pharmacokinetics and biodistribution of

Overall, binding of the proteins with Pluronic Pluronicmicelles is a complex function of hydrophobicity andionization state of the protein globule as well as the There are few reports on the subject of phar-block copolymer structure and environmental param- macokinetics and tissue distribution of block co-eters (pH, ionic strength, temperature). When a polymers in the body. One such study was carriedprotein is too hydrophilic to be incorporated into the out in our laboratory. This study examined the

3block copolymer micelle, it can be ‘hydrophobized’ pharmacokinetics and biodistribution of H-labeledas a result of point modification with hydrophobic P85 after a single i.v.-bolus administration in mice. It

groups to improve binding. For example, various was found that Pluronic remained in the circulationhydrophilic proteins (enzymes, antibodies, toxins) for a substantial period of time following a singlewere transferred into membrane forms by covalent injection. Thus, at the sampling time of 19 h, ca.modification with fatty acid moieties [68,69]. 17% of the administered dose of the block co-

polymer was still found in the blood. In addition,3.5. Modification of Pluronic micelles with relatively high concentrations of the block copolymer

peptides and proteins (ca. 0.001–0.01% wt.) were delivered to the organs,where it remained accumulated for the entire length

To allow for a drug delivery of Pluronic micelles of the study.to specific sites in the body or specific receptors on A separate study compared the biodistribution of

cells, the micelle corona was modified with peptide various Pluronic copolymers having various ratiosor protein molecules [41,54,55]. Furthermore, Slep- of the lengths of the EO and PO segments [73]. Thisnev [70] has demonstrated that peroxidase conju- study determined the areas under the curves (AUC)gated with P85 and then incorporated into the P85 for blood, liver, and spleen, as well as the tissuemicelles can be taken up into cells and subsequently distribution coefficients (P ) for both liverorgan / blood

released from endosomic compartments into the and spleen. As is seen in Table 3, the tissuecytoplasm. Since then, Topchieva et al. [71,72] used distribution coefficients increase in the following

a similar technique of modification to enable in- Pluronic order: F68,F108,P85,L61. This sug-tracellular delivery of proteins that were otherwise gests that the retention of the block copolymer in the

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Table 3Pharmacokinetic and tissue distribution parameters of Pluronic block copolymers after i.v. administration in mice

Tissue F68 F108 P85 L61

AUC P AUC P AUC P AUC Porg / blood org / blood org / blood org / blood

Blood 0.111 – 0.026 – 2.180 – 0.044 –Liver 0.225 2.03 0.079 3.03 7.365 3.38 0.215 4.93Spleen 0.066 0.59 0.032 1.23 4.240 1.94 0.118 2.68

Based on the data reported by Kabanov and Alakhov [73].P is the tissue distribution coefficient defined as the ratio of the AUC inorg / blood

organ to AUC in blood.

organs increases as the length of the hydrophobic PO The CMC values observed for Pluronic block23block increases, or as the HLB decreases. The copolymers are in the range from ca. 5310 to 1%

described pharmacokinetics and biodistribution wt. In terms of micelle stability, this range can bestudies show that the block copolymer concentrations characterized as ‘from moderately stable to relativelyin the plasma remain quite high for several hours unstable micelles’.following administration [73]. These concentrations In addition, block copolymer micelles may exhibitare in the same range as the established CMCs for highkinetic stability due to long lasting relaxation

the studied Pluronics , suggesting that micelles processes that result in a slow dissociation of themight be present in the circulation. micelles after dilution to concentrations below the

CMC. Micelles that are formed by block copolymers4.2. Micelle stability and drug release containing a hydrophobic block with a glass transi-

tion temperature exceeding 378C are particularlyThe stability of a block copolymer micelle as a stable in this respect [74,75]. The molecular motion

drug carrier can be considered in terms of the of the chains in the core of such micelles is ‘frozen’equilibrium behavior (‘thermodynamic stability’) and and, thus, the block copolymer molecules are strong-the dynamic behavior (‘kinetic stability’) [7]. The ly physically attached to each other [9]. The drugCMC and the partition coefficient are the major release rates in such systems are also low due tothermodynamic constants determining the stability of slow diffusion of the solute through the core. Blockthe micellar carrier and the drug release in equilib- copolymers of this type offer high blood circulationrium conditions (Fig. 2). The dilution of the mi- times combined with slow release of the free drug incelles, for example, in the body fluids, results in a the body.decrease in the portion of the micelle-incorporated Direct characterization of the intermoleculardrug. Furthermore, if the system is diluted below the stability of the block copolymer formulation, par-CMC, the micelles are completely disintegrated and ticularly, in the context of the determining of

the drug is completely released in the external media. whether the Pluronic micelles formed in vitro arealso preserved in vivo, is technically difficult, if notimpossible. This is because the physicochemicaltechniques normally employed for micelle characteri-zation, such as dynamic light scattering, fluorescentprobing and ultracentrifugation, are severely limitedby the presence of cells, proteins and other macro-molecular components in whole blood. These bloodcomponents have characteristics that hinder theobservation of micelles in whole blood. On the otherhand, techniques involving the isolation of theFig. 2. Mechanisms of drug release from the micelle: (A) dis-micelles from blood components, such as chromatog-integration of the micelles below CMC; (B) release of the drug as

a result of partitioning. raphy, cannot be used because of the dynamic

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characteristic of the block copolymers to disassemble effective release of the drug from the carrier withininto single chains upon dilution. the critical site of action. Once this optimum is

Nevertheless, some attempts to address the stabili- achieved, the therapeutic index is maximal.ty of Pluronic micelles in the presence of selected The potential strength of the block copolymer

blood components, such as serum proteins have been approach in therapeutics is that the critical physico-reported. Due to dynamic and equilibrium character chemical parameters can be adjusted in a very broadof micellar system, the concentration of the block range within a single homologous block copolymercopolymers in the blood is very important in view of set in order to maximize the therapeutics index forthe micelle stability problem. The pharmacokinetics given drug delivery situation. This possibility can be

study described above [73] suggested that the con- illustrated using Pluronic block copolymers. Forcentrations of the block copolymer in the plasma these copolymers a relationship of partition coeffi-could be sufficient to form micelles. Another po- cient of a hydrophobic probe, pyrene, and CMC hastential problem is that the micelles may disintegrate been evaluated [38]. The experimental curve under-as a result of interactions with blood components, lying this relationship is presented in Fig. 3. As issuch as serum proteins [25]. To address this issue, seen in this figure, the CMC andP-values are varied

Pluronic micelles, were examined in vitro in the by over two orders of magnitude, when the blockpresence of bovine serum albumin using an ultracen- copolymer structure is changed. The copolymers attrifugation technique. This study suggested that the upper left corner of this graph have the lowestmicelles of P85 are preserved in solutions of bovine CMC and highestP, characterized by the highestserum albumin at concentrations of the protein as stability and strongest retention of the solute. Inhigh 3% wt., which is comparable to the protein contrast, the copolymers at the lower left corner,concentration in plasma [25]. having the highest CMC and lowestP, are readily

disintegrating and releasing drug upon dilution. Byvarying the molecular parameters of the copolymers

4.3. Effects of Pluronic structure on micelles one can adjust the block copolymer-based formula-stability and drug release tion to achieve the desired characteristics of micelle

stability and drug release.Core-forming chains in Pluronic micelles disso-

ciate and release drug in vivo relatively fast. Drugformulations based on these block copolymers pro-vide for relatively small increases in drug circulationtimes compared to the circulation times of free drug[25]. Drug extravasation in the cases of such formu-lations can be high due to the rapid release of thedrug from the micelles. Usually, the bioavailabilityof the drug in this delivery system is the same oreven higher than the bioavailability of the free drugs.It is important to recognize that an increase in thecirculation time through stabilization of the drug–carrier complex will not necessarily improve thetherapeutic index of the drug. Indeed, too strong anattachment of the drug to the micelle carrier de-creases drug release, which may result in lowerconcentrations of the drug in the body. There mustbe some optimum ratio between: (i) effective in- Fig. 3. Relationship between the partitioning coefficients of

corporation of the drug in the carrier, that increases pyrene and CMC in Pluronic block copolymer systems (re-stability and circulation time of the drug, and (ii) produced from Ref. [38] with permission).

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4.4. Effects of Pluronic on biodistribution of a various solid tumors, compared to the free drugdrug [57,76].

At present, little is known about the effects of Pluronic block copolymers on pharmacokinetics 5. Pluronic formulations for treatment of

and biodistribution of drugs in the body. For exam- drug-resistant tumorsple, Alakhov et al. [57] examined the phar-macokinetics and tissue distribution of doxorubicin 5.1. Sensitization of drug-resistant cancer by

formulated with the mixed micelle of Pluronic L61 Pluronic block copolymersand F127 (termed ‘SP1049C’, Supratek Pharma Inc.,Montreal, Canada) in normal and tumor-bearing Tumors with the multidrug-resistant (MDR) phe-animals. Table 4 presents the AUC data obtained in notype are among some of the most difficult types tothis study for plasma, and various tissues. By treat. MDR cells overexpress efflux proteins belong-comparing the conventional doxorubicin formulation ing to a superfamily of ATP binding cassette (ABC),and SP1049C formulation, the authors concluded such as P-glycoprotein (Pgp) and multidrug resis-that the block copolymer has little affect on the tance-associated proteins (MRP) that pump drugs outpharmacokinetic profiles of the drug in liver, kidney, of a cell [77,78]. Furthermore, several other proteinsheart and lung of both normal and tumor-bearing (e.g. glutathione-S-transferase, metallothionein,

6mice. At the same time, the brain AUCs were thioredoxin, topoisomerase I, II, O -alkylguanine-increased 2.9- and 1.7-times in normal and tumor- DNA alkyltransferase, etc.) are believed to contributebearing mice, respectively. The increase in the brain to the resistant phenotype as well [77]. The combina-accumulation of doxorubicin may be related to tion of several independent mechanisms of druginhibition of the drug efflux systems, such as P- resistance complicates chemotherapy and reinforcesglycoprotein (Pgp), expressed in the blood–brain the need for the development of novel drugs and

barrier, by Pluronic . A substantial, 1.7-times in- drug formulations effective against drug-resistantcrease in the accumulation of doxorubicin in solid cancers. Alakhov et al. [56,79,80] demonstrated that

tumor was also observed with SP1049C formulation Pluronic block copolymers sensitize resistant cells,compared to the conventional formulation [57]. The resulting in an increase in the cytotoxic activity ofincrease in the drug delivery to the tumor is im- the drug by two to three orders of magnitude. By

portant in view of the fact that Pluronic -based addition of P85 or L61, the cytotoxic effects ofcompositions of doxorubicin have been shown to be doxorubicin in the resistant lines significantly surpas-substantially more effective in vivo in treating sed those observed in the sensitive lines. Similar

Table 4The plasma pharmacokinetic parameters of free doxorubicin and doxorubicin in SP1049C formulation in normal and tumor-bearing mice

Animal / formulation AUC (mg3h per g)

Plasma Brain Heart Kidney Liver Lung Tumor

Normal miceDoxorubicin 7.1 9.0 111.5 271.5 147.1 307.5 –Doxorubicin /SP1049C 14.6 26.0 139.8 312.1 192.3 282.2 –

Tumor-bearing miceDoxorubicin 7.1 5.6 156.2 263.7 154.2 207.5 30.1Doxorubicin /SP1049C 8.5 9.2 177.2 270.9 173.5 268.2 50.8

Based on the data reported by Alakhov et al. [57].

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effects of Pluronic block copolymers have also efflux system, rather than to nonspecific alterationsbeen reported in vivo [57,76]. In these studies, mice in membrane permeability of the substrates.bearing drug-sensitive and drug-resistant tumors Recently, however, evidence has begun to mount

were treated with doxorubicin alone and doxorubicin suggesting that the effects of Pluronic block co-in Pluronic compositions. The tumor panel included polymers might stem beyond solely inhibition of the

i.p. murine leukemias (P388, P388-Dox), s.c. murine Pgp efflux pump. Studies by Miller et al. [83], usingmyelomas (Sp2/0, Sp2/0-Dnr), i.v. and s.c. Lewis the human pancreatic adenocarcinoma cell line,lung carcinoma (3LL-M27), s.c. human breast car- Panc-1, that expresses the MRP efflux pump, sug-cinomas (MCF-7, MCF-7ADR), and human head gested that P85 inhibits efflux and increases cellularand neck carcinoma (KBv) [57]. Using the National accumulation of the MRP-dependent probe, fluores-Cancer Institute criteria for tumor inhibition and cein, in these cells. A recent study by Evers et al.

increased lifespan, Pluronic -formulated doxorubicin [81] reported that L61 partially inhibited MRP2-has met the efficiency criteria in all models (nine of mediated transport of vinblastine in transfectednine), while doxorubicin alone was effective only in canine kidney cells that stably express MRP2. How-selected tumors (two of nine) [57]. Together these ever, the potency of this inhibitor with respect tostudies indicate improved treatment of drug-resistant MRP2 in these cells was substantially lower than its

cancers with Pluronic block copolymers. potency with respect to the Pgp in the mdr1-trans-fected cells that express Pgp. Thus, the evaluation of

5.2. Effects of Pluronic on drug resistance the effects of Pluronic block copolymers on MRP ismechanisms far from completion.

Another impediment to treatment, which is presentEnhanced cytotoxicity observed with Pluronic in MDR cells, involves the sequestration of drugs

block copolymers in drug-resistant cancer cells ap- within cytoplasmic vesicles, followed by extrusion ofpears to be related to the effects of the copolymer on the drug from the cell [84–88]. Drug sequestration inthe Pgp drug efflux transport system. Evidence to MDR cells is achieved through the maintenance of

support inhibition of Pgp by Pluronic block co- abnormally elevated pH gradients across organelle1polymers is the observation that defects in the membranes—by the activity of H -ATPase, an ATP-

intracellular accumulation of doxorubicin in resistant dependent pump [89]. Recent studies by Venne et al.cancer cells expressing Pgp can be overcome by [80] examined effects of Pluronic block copolymers

treatment with Pluronic [42,80]. No alteration in on intracellular localization of doxorubicin in thedrug uptake in the presence of Pluronic was MDR cancer cell line, MCF-7/ADR. In these cells,

observed with non-Pgp expressing parental cancer free doxorubicin is sequestered in cytoplasmic vesi-cells, providing additional support for the specific cles, which might further diminish the amount ofeffects of the copolymer on Pgp transport system in drug available for interaction with the nucleus [88].MDR cells. This conclusion has been reinforced by Following incubation of the cells with doxorubicin

recent studies by Evers et al. [81] and Batrakova et and Pluronic , the drug was released from theal. [58] demonstrating that Pluronic block copoly- vesicles and accumulated, primarily, in the nucleus

mers (L61, P85) have pronounced effects, increasing [80]. Overall, these studies suggest that Pluronicaccumulation and permeability of various Pgp-de- copolymers affect several important mechanisms ofpendent drugs in mdr1-transfected cells that over- drug resistance in cancer cells (Fig. 4).express Pgp. An additional support for the Pgp-

mediated mechanism of Pluronic in Pgp expressing 5.3. Energy depletion phenomenoncells is that the block copolymer has no or littleeffect on the accumulation of non-Pgp dependent Various drug resistance mechanisms, includingcompounds in both resistant and parental cells drug transport and detoxification systems, require[58,61,82]. Therefore, the increased absorption of the consumption of energy to sustain their function inPgp substrates in Pgp expressing cells is attributable MDR cells. Hence, mechanistic studies have focused

to the effects of the block copolymer on the Pgp on the effects of Pluronic block copolymers on

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not induce permeabilization of the cellular membranesince no leakage of intracellular ATP in the externalmedia was observed [98]. Therefore, ATP depletionwas likely to be a result of inhibition of cellularmetabolism rather than due to a loss of ATP in theenvironment. The following groundbreaking ob-servation, in the context of the drug resistancephenomena, was the study that compared the effectsof the P85 on ATP levels in several cell types thateither express Pgp and MRP, or do not express Pgpand MRP [90,91]. Exposure of both resistant andsensitive cells to P85 resulted in energy depletion,which was reversed when the block copolymer wasremoved. However, the resistant cells were muchmore responsive to P85, exhibiting profound de-creases in ATP levels at substantially lower con-Fig. 4. Multiple mechanisms of Pluronic block copolymercentrations of the block copolymer compared to theeffects in MDR cells.sensitive cells. The effective concentrations of P85that induced a 50% decrease in ATP levels in the

metabolism and energy conservation in drug-resistant cells (EC ), as determined from the dose–response50

cells [90,91]. The basis for such studies was the curves are presented in Table 5. This table alsoearlier reports that Pluronic block copolymers can presents the relative responsiveness of the Pgp and

affect mitochondria function in non-MDR cells. MRP overexpressing cells compared to the non-PgpThere could be multiple reasons for the inhibitory and non-MRP overexpressing cells. These data sug-activity of these compounds in mitochondria. The gest that the responsiveness of the cells to P85likely components contributing to the anti-metabolic correlated with expression of Pgp and MRP. Basedeffects of nonionic detergents include their ability to on the results of this study, the appearance of the

1serve as K ionophores [92,93], and uncouple MDR phenotype is one factor that renders cellularoxidative phosphorylation [94,95]. It is also possible metabolism responsive to treatment with Pluronics

that these detergents directly inhibit the NADH (Fig. 4).dehydrogenase complex by interacting with the

hydrophobic sites of this complex in the mitochon- 5.4. Membrane fluidization by Pluronic blockdria membrane [95,96]. Rapoport et al. [97], using copolymerslipophilic spin-probes, has directly shown that two

Pluronic copolymers, P85 and P105, reduce the Pluronic block copolymers are known to induceactivity of the electron transport chains in mito- drastic changes in the microviscosity of cell mem-chondria as assessed by the rates of bioreduction of branes as assessed using a hydrophobic membranethese probes in HL-60 cancer cells. These findings probe, 1,6-diphenyl-1,3,5-hexatriene (DPH)

indicate that Pluronic block copolymers could be [99,100]. These changes can be attributed to thetransported inside the cells and reach mitochondria. alterations in the structure of the lipid bilayers as aThis, in fact, was directly shown for selected result of adsorption of the block copolymer mole-

Pluronics (e.g. P85) that were labeled with fluores- cules on the membranes. Interestingly, it appears thatcent tags to examine their transport and localization Pluronic block copolymers have different effects

inside cells [41,55]. with respect to the membranes of some normal andSlepnev et al. [98] were first to demonstrate that cancer cells. Melik-Nubarov et al. [99] reported that

following a 2-h exposure of Jurkat T-cell lymphoma the treatment with the same doses of P85 or L61cells to a Pluronic block copolymer, P85, intracel- increased microviscosity (‘fluidized’) of the mem-

lular levels of ATP were depleted. This treatment did branes of cancerous cells, while, in contrast, de-

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Table 5Effects of P85 on ATP levels in Pgp and MRP overexpressing cells and cells that do not overexpress Pgp or MRP

Cells Pgp or MRP Initial ATP levels EC (%) Relative responsiveness50a boverexpression (nmol /mg protein) to P85

MCF-7 No 3061.5 2.25 –cMCF-7/ADR Pgp, MRP 300620 0.009 250

KB No 160.01 0.675 –dKBv Pgp 460.1 0.036 19

C2C12 No 1561.4 4.5 –HUVEC No 4064.9 0.0675 –

eCaco-2 Pgp, MRP 5.560.4 0.00067 6670eBBMEC Pgp, MRP 1.660.04 0.018 250

LLC-PK1 No 6166.9 0.45 –fLLC-MDR1 Pgp 7961.7 0.0045 100

Based on the data presented by Batrakova et al. [90]. In this study, cells were exposed to P85 for 120 min prior to determining theintracellular ATP levels.

a Mean6S.E.M. (n54).b Calculated as the ratio of EC of non-Pgp non-MRP cells to EC of corresponding Pgp expressing cells.50 50c Compared to MCF-7 cells.d Compared to KB cells.e Compared to C2C12 cells.f Compared to LLC-PK1 cells.

creased the microviscosity (‘solidified’) of mem- crucial for the proper functioning of Pgp. It wasbranes of the normal blood cells. Membrane fluidiza- suggested that binding of ATP in one domain causestion by various agents including nonionic surfactants, a conformational change in the Pgp molecule neces-such as Tween 20, Nonidet P-40 and Triton X-100, sary for the hydrolysis of ATP and translocation ofis known to contribute to inhibition of Pgp efflux the substrate [103]. Therefore, the structural per-function [101]. MDR modulation by membrane turbations in the lipid membranes induced by thefluidizers occurs by abolishment of Pgp ATPase block copolymer may decrease the affinity of ATP toactivity that results in the loss of Pgp-mediated drug its binding site and interfere with the ATPaseefflux [101]. Furthermore recent studies demonstra- activity. This means that higher concentrations ofted that P85 inhibits Pgp ATPase activity and intracellular ATP would be required for normalinhibition of this activity is observed with the same functioning of Pgp, i.e. drug efflux system woulddoses of the block copolymer as those that inhibit become more vulnerable to decreases in intracellularPgp efflux in Pgp expressing cells [91,100]. There- ATP. It is possible that different drug transporters

fore, it is likely that these Pluronic block co- have different energy requirements in MDR cellspolymers have a ‘double-punch’ effect in Pgp ex- and/or different sensitivities to changes in membrane

pressing cells: through ATP depletion and membrane microviscosity. As a result, the potency of Pluronicfluidization, which both have a combined result of block copolymers with respect to modulation ofpotent inhibition of Pgp [91,100]. Both factors are these drug transport mechanisms may also vary.critical for exhibition of the effect of P85 on Pgp

efflux system in Pgp expressing cells. The interrela- 5.5. Dose dependence of Pluronic effects intionship between the membrane fluidization and MDR cells

energy depletion components of Pluronic affect canbe better understood in view of the current picture of As is the case with any chemosensitizer, the

Pgp structure describing Pgp as a two-domain pro- effects of Pluronic block copolymers in MDR cellstein with ATP-binding sites in each domain [102]. exhibit significant dose-dependent behavior. How-Proper interaction of these two ATP-binding sites is ever, due to the ability of the block copolymers to

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self-assemble into micelles and to solubilize hydro- of Pluronic unimers in sensitization of the MDRphobic drugs, the dose dependencies observed with cells increased with the increase in the copolymerthese compounds are drastically different compared hydrophobicity. As follows from the discussion

to the dose dependencies of many other Pgp above it appears that the effects of Pluronic on Pgpmodulators. Batrakova et al. [42] have studied the are at least in part induced by alterations in the

dose–response to various Pluronic block copoly- structure of the cell membranes by the block co-mers in MDR cells. These studies demonstrated that: polymers. Such interactions with the membranes(i) increases in accumulation of the Pgp probe, involve incorporation of the hydrophobic PO chainsrhodamine 123 and (ii) potentiation of doxorubicin of the block copolymer in the lipid bilayers [106]. In

activity by Pluronic in MDR cancer cells occur at contrast, hydrophilic EO chains do not interact withblock copolymer concentrations below the CMC. lipid membranes and are used to prevent binding ofThis means that both effects are due to the block other polymers with the membranes [107,108]. Atcopolymer single chains, i.e. the unimers. As a the same time, it is possible that hydrophobic blockresult, the accumulation and cytotoxicity of drugs in copolymers are better transported inside the cellsMDR cells increase with increasing concentrations of and/or are more potent inhibitors of metabolic

Pluronic until the CMC is reached and unimer processes than the hydrophilic copolymers. Further-concentration levels off. In contrast, above the CMC, more, in the energy depletion studies, exposure ofblock copolymer added to the system is consumed in the MDR cells to a hydrophilic copolymer, F108, didthe formation of the micelles. Under these con- not induce such a drastic decrease in intracellularditions, the drug accumulation and cytotoxicity in ATP levels as those observed following exposure ofMDR cells, first, level off and then decrease [42]. the cells to the more hydrophobic, P85 and L61 [91].Therefore, the CMC provides the ‘cut-off point’ for Therefore, combined differences in membrane inter-the maximal drug accumulation in MDR cells. The actions, cellular transport and energy depletion ac-decreases in drug absorption in the cells above the tivity may contribute to the observed dependence ofCMC are very similar to those reported previously the potency of block copolymers on their structure infor other nonionic detergents that are MDR MDR cells.modulators, such as Cremophor EL [104,105]. The To evaluate how efficacious the block copolymersinhibition of cell transport in the presence of micelles are in inhibiting Pgp, the absorption of rhodamineis attributed to incorporation of the drug into the 123 was examined in Pgp expressing cells [42]. Amicelles, resulting in a decrease in the amount of the dose–response curve for rhodamine 123 accumula-free probe available for diffusion through the cell tion was obtained for each block copolymer asmembrane into cells. discussed in the previous section. Next, the maximal

accumulation levels (‘R123 enhancement factors’)5.6. Effects of composition of Pluronic block observed with the most effective doses of each

copolymers Pluronic were plotted as a function of the length ofthe hydrophobic PO block (y). This yielded a bell-

Both the inhibition of the Pgp efflux system as shaped dependency of the net efficacy of Pluronicwell as the hypersensitization effects of Pluronic in copolymers in inducing rhodamine 123 accumulation

MDR cells depend on the molecular composition of in cells (Fig. 5). As is seen in the figure, the mostthe block copolymer. For example, Batrakova et al. efficacious block copolymers are those with inter-[42] evaluated a panel of block copolymers, differing mediate lengths of the hydrophobic block rangingin the lengths of EO and PO chains in the resistant from 30 to 60 PO repeating units, while the blockKBv cell line. First, the studies of rhodamine 123 copolymers with shorter or longer PO blocks are lessaccumulation in the cells suggested that more hydro- efficient. Furthermore, the resistance reversion index-phobic block copolymers (having lower HLB) are es determined in the doxorubicin cytotoxicity studymore active than more hydrophilic block copolymers in KBv cells followed exactly the same pattern as the(having higher HLB). Second, the cytotoxicity drug accumulation data shown in Fig. 5 [42]. Thisstudies using doxorubicin suggested that the potency behavior was explained by the interplay of both

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6. Pluronic effects on brain and oralbioavailability of drugs

6.1. Effects of Pluronic on brain accumulation ofdrugs

There is an increasing interest in the literature tothe role of Pgp and MRP in the tissue distribution oftherapeutic agents based on the reports that thesetransporters are expressed in ‘normal’ cells in thebody, including brain microvessel endothelial cells(BMEC), intestinal epithelial cells, and hepatocytes[109–116]. For example, immunohistochemical andfunctional studies indicate that Pgp is expressed onthe luminal (apical) plasma membrane of BMEC thatform the BBB [106,109]. Less is known regardingthe role of the MRP drug efflux transport system inthe BBB. There are several isoforms of MRP thatappear to be expressed in the BBB, including MRP1,MRP4, MRP5, and MRP6 [110]. In contrast to Pgp,which transports lipophilic and cationic compounds,MRP transports anionic compounds and/or glucuro-Fig. 5. Optimization of Pluronic block copolymer compositionnide and glutathione conjugates [111,112]. Severalin MDR cells. The entire set of hydrophilic copolymers with HLBstudies indicate the presence of outwardly directedvarying from 20 to 29 had no or little effect on drug transport and

is not presented in this figure. Only the copolymers with HLB less efflux transport systems for organic anions in thethan 20 exhibiting varying degrees of activity in MDR cells are BBB [113–115].presented (reproduced from Ref. [42] with permission). Batrakova et al. [114] evaluated the effects of

Pluronic block copolymers on the permeability of abroad panel of drugs using bovine BMEC mono-layers as an in vitro model of BBB. Consequently,

hydrophobic-hydrophilic properties of Pluronic the panel of compounds used in this study includedmolecules and their self-assembly behavior in aque- known substrates for Pgp such as rhodamine 123,ous solutions. The copolymers with long PO seg- doxorubicin, digoxin, ritonavir, taxol and vinblastine.ments and short EO segments are the most potent. In addition, this panel included substrates of organicHowever, they also have much lower solubility and anion transporters: fluorescein, ziduvidin and metho-CMC (Table 1), which limits the effective con- trexate, as well as compounds with less studiedcentration of the unimers in the solution. On the specificity for the efflux pumps, such as loperamide,other hand, the unimers with short PO segments have valproic acid and L-DOPA. Table 6 presents thehigh CMC and their concentration in solution can be apparent permeability coefficients (P ) of the com-app

very high. However, the potency of these unimers is pounds in bovine BMEC monolayers in the apicallow. As a result, the optimal net efficacy is observed (AP) to basolateral (BL) direction. As is seen from

with Pluronic copolymers, which have intermediate the data, P85 increased AP to BL permeability inlengths of PO chains, and relatively short EO bovine BMEC monolayers with respect to a broadsegments, such as P85 and L61. The unimers of panel of structurally diverse compounds. The en-these copolymers are sufficiently potent and rela- hancement effects ranged from 1.3 times, in the casetively high concentration of unimers can be reached of methotrexate, to almost 20 times, in the case ofin solution. vinblastine.

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Table 6Effects of P85 on the permeability of various solutes in bovine BMEC monolayers in AP to BL direction

6Solute Brain to blood P 310 (cm/s) Effectapp

drug transporter Assay buffer P85

Mannitol None 5.760.4 5.760.4 n.s.Loperamide Unknown 25.262.8 24.561.1 n.s.Valproic acid Unknown 25.861.4 22.860.3 n.s.

aMethotrexate MRP 8.260.8 11.160.4 1.3*Fluorescein MRP 16.560.6 24.460.2 1.5*Rhodamine 123 Pgp 2.860.2 4.560.3 1.6*Ziduvidin MRP 16.561.8 31.169.1 2.0*Doxorubicin Pgp, MRP 13.260.2 31.567.3 2.4*

bDigoxin Pgp 3.860.4 15.561.0 4.1*aRitonavir Pgp 0.960.1 6.560.6 7.7*

Taxol Pgp 1.560.1 16.562.1 11.2*aVinblastine Pgp, MRP 0.660.1 12.262.0 19.0*

The data used in this table were reported by Batrakova et al. [114] unless is stated different.*Statistically significant effects of the copolymer on the permeability of the drug.

a Data in preparation, not included in Ref. [114].b Calculated using data reported by Batrakova et al. [58].

To date, there are two studies that indicate demonstrated that delivery to the CNS of a prototypi-Pluronic can enhance the brain penetration of cal Pgp substrate could be significantly enhanced by

selected drugs in vivo. In the first study, Kabanov et co-administration of Pluronic (Fig. 6, pathway A).al. [54,55] demonstrated increased delivery of the These studies provide a foundation for future de-neuroleptic drug, haloperidol, to the brain in mice velopment of a therapeutic method for enhancing the

with the use of insulin or antibody conjugated brain penetration of drugs using Pluronic blockPluronic micelles. In addition, haloperidol delivery copolymer technology.

to the brain was also increased in the presence ofP85 [54,55]. In the second study, Batrakova et al. 6.2. Targeting of Pluronic micelles using protein

[58] examined the brain accumulation of digoxin in ligandswild-type mice, mdr1a knockout mice and wild-typemice treated with P85. The Pgp-dependent drug, Attachment of a specific ligand to the drug carrierdigoxin, demonstrated greater permeability enhance- enables binding of the carrier species with the cellment than doxorubicin in the in vitro panel study surface, entrapment of these species into endosomes(Table 6). As expected, the brain accumulation of and the trafficking of these species inside the cell.digoxin was significantly higher in the mdr 1a/b For example, the corona forming chains in the block(2 /2) knockout mice, due to the absence of Pgp copolymer micelles can be modified to incorporateefflux transport of digoxin at the BBB [58]. The ligand moieties and redirect the micelles alongkinetic profiles of radiolabeled digoxin in the plasma endocytic pathways. Receptor-mediated transport

and the brain of wild-type mice suggested that P85 using Pluronic micelles as drug carriers has beenprolonged the residence time and concentrations of reported by Kabanov et al. [55]. This study revealed

Pluronic in the brain [58]. The relative contribution that the P85 micelles, containing a fluorescent dye,of Pgp inhibition to this central nervous system are taken up into cells via an endocytosis mecha-(CNS) delivery enhancement was evaluated by com- nism. The absorption was drastically increased whenparing P85-treated wild-type female FBV mice with a protein molecule, enterotoxin B, capable of bindingnontreated Pgp-deficient mice. These experiments with a cell receptor, was covalently linked to the

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cellular processing could be altered to favor trans-cytosis as the major pathway and re-cycling to thelumenal side as the minor transport pathway, whichresults in enhancement in the flux of the drug acrossin the BBB.

6.3. Enhancement of oral bioavailability of drugsby Pluronic block copolymers

There is an increasing body of evidence thatoutwardly directed drug efflux systems hinder oralbioavailability of selected drugs. Intestinal epithelialcells are known to express a functionally active Pgpas well as various isoforms of MRP, includingMRP2, MRP3 and, to a lesser extent, MRP1 andMRP5 [116]. In this respect, it is believed that byinhibiting the function of these drug efflux systems itFig. 6. Proposed mechanism of drug transport in brain microves-

is possible to increase efficiency of delivery of thesesel endothelial cells with Pluronic block copolymers: (A) inhibi-drugs through the oral route. The potential effective-tion of Pgp results in increased flux of drug from blood to brain,

(B) solubilization of drugs in micelles decelerates drug transport ness of inhibition of the Pgp efflux system inacross BBB, micelles undergo fluid phase endocytosis, and (C) enhancing oral bioavailability can be found in theconjugation of micelles with insulin vector enhances drug trans-

recent studies by Nerukar et al. [104,105] examiningport through adsorptive endocytosis (reproduced from Ref. [41]the effects of nonionic detergents on peptide per-with permission).meability in monolayers of the human colon epi-thelium cell line, Caco-2. In these studies, the

surface of a micelle. Excess of the free enterotoxin B permeability of Caco-2 monolayers to selected pep-effectively inhibited the uptake of the micelles with tides was significantly enhanced by nonionic surfac-the enterotoxin B modified corona, which suggested tants, Polysorbate 80 and Cremophor EL. Conse-specific interactions of the micelles with the cells quently, in vitro studies examined the effects of

through a receptor. Pluronic block copolymers on drug absorption andA similar approach was recently used to evaluate permeability in Caco-2 monolayers [41,82]. The

the effects of the conjugation of specific ligands with results obtained using rhodamine 123 were veryPluronic micelles on drug processing and per- similar to those observed during studies of drug

meability in BMEC [41]. To target receptor-me- transport in BMEC monolayers. Specifically, thediated endocytosis in the BMEC, P85 micelles were unimers of Pluronic block copolymers inhibited the

conjugated to insulin. In the presence of insulin Pgp efflux system in Caco-2 monolayers, resulting invector the transport of the probe was significantly a significant enhancement of absorption and per-increased compared to the transport of the probe in meability of the probe. Table 7 presents the apparentthe insulin-free micelles or in the micelles with the permeability coefficients (P ) of various com-app

same concentration of free insulin (Fig. 6, pathway B pounds in Caco-2 monolayers in the AP to BLand C). Furthermore, the competitive inhibition of direction [114]. As is seen from the data, P85transport of insulin-vectorized micelles with an increases permeability in the monolayers with re-excess of the free insulin was demonstrated. This spect to a broad panel of drug and probe molecules,study suggested that conjugation of insulin to suggesting that it can be useful for increasing oral

Pluronic micelles resulted in a shift from a vesicu- bioavailability of these compounds. This approachlar transport of the micelle, through simple endo- has been further validated in an in vivo study incytosis, to a receptor-mediated endocytosis. Further- which C57 Bl /6 mice were given p.o. rhodaminemore, by modifying the micelles with a ligand, the 123. When the compound was formulated with a

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A.V. Kabanov et al. / Journal of Controlled Release 82 (2002) 189–212 207

Table 7 [120]. For example, a micellar formulation based onEffects of P85 on the permeability of various solutes in Caco-2 the mixture of the block copolymers, L61 and F127monolayers in AP to BL direction

(termed ‘SP1017’, Supratek Pharma Inc., Montreal,6Solute Drug P 310 (cm/s) Effectapp Canada), has been identified that increases gene

transporter Assay buffer P85 expression by 5- to 20-fold compared to naked DNA.Unlike cationic DNA carriers, such as polycations orMannitol None 0.260.004 0.260.004 n.s.cationic lipids, SP1017 does not condense DNA.Valproic acid Unknown 10.460.5 14.360.5 1.4*

Loperamide Unknown 1.660.1 3.360.2 2.1* Furthermore, this formulation does not improve theDoxorubicin Pgp, MRP 14.561.2 35.260.7 2.4* in vitro transfection of either the myoblast or the

aMethotrexate MRP 3.960.2 10.160.3 2.6* myofiber developmental stages of the murine muscleEtoposide Pgp, MRP 4.060.6 10.660.8 2.7*

cell line, C2C12, when compared to plasmid DNARhodamine 123 Pgp 0.760.1 2.360.5 3.3*alone. However, during intramuscular administration,Fluorescein MRP 3.860.4 13.960.5 3.7*

aVinblastine Pgp, MRP 3.160.4 13.360.3 4.3* SP1017 was shown to promote expression of bothaRitonavir Pgp 3.360.3 14.460.4 4.4* reporter and therapeutic genes [120]. Importantly,

Ziduvidin MRP 2.960.1 14.560.4 5.0* maximal stimulation of gene expression with thisTaxol Pgp 0.3660.01 4.760.4 13.1*

formulation was observed at a relatively low con-The data used in this table were reported by Batrakova et al. centration of the block copolymer (0.01%), pro-

[114] unless is stated different.viding for at least a 500-fold safety margin in*Statistically significant effects of the copolymer on the per-animals. Recent data demonstrated that plasmidmeability of the drug.

a Data in preparation, not included in Ref. [114]. DNAs driven by either CMV- or NFkB-responsiveelements were considerably more responsive to theSP1017 effect than plasmid DNAs, which were

lipophilic Pluronic block copolymer, its oral ab- under the control of either SV-40- or an AP-1-sorption rate increased by more than threefold response element cassette [121]. The promoter de-(manuscript in preparation). Furthermore, recent pendence is unusual and it suggests that SP1017 mayreports have successfully demonstrated increased act as a synthetic biological response modifier,oral uptake of two drugs, amikacin and tobramicin, probably by affecting mechanisms of the transcrip-which could be Pgp substrates, following oral ad- tional control of the transgene expression. Comparedministration to mice in the presence of poloxamer to poly(vinyl pyrrolidone), another non-ionic carrierCRL-1605 [117,118]. Taken together these studies that is under development for non-viral gene therapy

provide substantial evidence that Pluronic block [122,123], SP1017 appears to be more efficient andcopolymers can be useful in increasing oral absorp- requires less DNA to produce the same amount oftion of select drugs by inhibiting drug efflux systems transgene [120]. These studies demonstrated that

in intestinal epithelial cells. Pluronic block copolymers are promising agents fornon-viral gene therapy. It provides for a simple andefficient gene transfer method, which is potentiallyapplicable in multiple gene therapy protocols involv-

7. Pluronic formulations for gene therapy ing intramuscular injection of plasmid DNA in orderto synthesize therapeutic proteins or to vaccinate by

It is well known that plasmid DNA injected into expression of a particular gene product.skeletal muscle can generate gene expression [119]. Overall, Pluronic block copolymers are among

However, in many cases, the relatively low level and the most potent drug targeting systems with a broadrelatively short duration of gene expression achieved spectrum of biological response modifying activity,limit the applicability of naked DNA as a therapeutic and an ability to self-assemble into micelles, in-

agent. Therefore, a recent discovery that Pluronic corporate drug molecules and transport them withinblock copolymers increase levels and duration of the body. These properties warrant further inves-

expression of a transgene by a plasmid DNA in tigation regarding the use of Pluronic formulationsskeletal muscle in mice is of considerable interest as novel promising drug delivery systems.

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