pluronic® block copolymers for overcoming drug resistance in cancer

Advanced Drug Delivery Reviews 54 (2002) 759–779www.elsevier.com/ locate/drugdeliv

P luronic block copolymers for overcoming drug resistance incancer

a , a b*Alexander V. Kabanov , Elena V. Batrakova , Valery Yu. AlakhovaDepartment of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025Nebraska Medical Center,

Omaha, NE 68198,USAbSupratek Pharma Inc., 531 Blvd. des Prairies, Build. 18, Laval, Quebec H7B 1B7, Canada

Received 28 January 2002; accepted 21 May 2002

Abstract

Pluronic block copolymers have been used extensively in a variety of pharmaceutical formulations including delivery oflow 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-resistantcancer (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 ofdrug efflux transporters, abolishing drug sequestration in acidic vesicles as well as inhibiting the glutathione/glutathioneS-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 chemosensitizationof 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 clinicalevaluations of these systems are in progress. 2002 Elsevier Science B.V. All rights reserved.

Keywords: Anthracyclines; Cancer; MDR; P-glycoprotein; Multidrug resistant protein; MRP; Block copolymer

Contents

1 . Introduction ............................................................................................................................................................................ 7602 . Mechanisms of resistance to antineoplastic agents ..................................................................................................................... 761

2 .1. Drug efflux proteins ......................................................................................................................................................... 7612 .2. Drug sequestration in cytoplasmic vesicles......................................................................................................................... 7622 .3. Metabolic detoxification systems....................................................................................................................................... 7622 .4. Inhibition or prevention of drug-induced apoptosis ............................................................................................................. 763

3 . Structure and solution behavior of Pluronic block copolymers .................................................................................................. 7634 . Pluronic block copolymers sensitize drug-resistant cancers....................................................................................................... 764

5 . Several drug resistance mechanisms are affected by Pluronic ........................................................................................ 7655 .1. Inhibition of Pgp drug efflux system.................................................................................................................................. 765

*Corresponding author. Tel.:11-402-559-9364; fax:11-402-559-9543.E-mail address: [email protected](A.V. Kabanov).

0169-409X/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0169-409X( 02 )00047-9

mailto:[email protected]

760 A.V. Kabanov et al. / Advanced Drug Delivery Reviews 54 (2002) 759–779

5 .2. Effects on other drug transporters ...................................................................................................................................... 7665 .3. Effects on drug sequestration within cytoplasmic vesicles ................................................................................................... 7675 .4. Effect on GSH/GST system ............................................................................................................................................. 767

6 . ATP depletion induced by Pluronic in MDR cells.................................................................................................................... 7677 . Membrane fluidization and inhibition of Pgp ATPase activity by Pluronic ........................................................................ 770

8 . Interplay of ATP depletion and membrane fluidization as a possible mechanism for Pluronic activity in MDR cells ..................... 7709 . Dose dependence of Pluronic effects in MDR cells: unimers vs. micelles .................................................................................. 771

1 0. Relationship between structure of Pluronic and its activity in MDR cells ................................................................................. 7721 1. Effects of Pluronic on cytotoxicity of various drugs in resistant cells....................................................................................... 773

1 2. Clinical trials of doxorubicin–Pluronic formulation (SP1049C) .............................................................................................. 7741 3. Conclusion............................................................................................................................................................................ 774Acknowledgements...................................................................................................................................................................... 775References .................................................................................................................................................................................. 775

1 . Introduction The glutathione/glutathione S-transferase detoxifica-tion system is frequently activated in MDR cells

Chemotherapy remains the primary treatment op- contributing to drug resistance [11,12]. MRP acts intion for cancer. Unfortunately, the efficacy of chemo- concert with this system, providing for the efflux oftherapy treatment in many types of cancers is glutathione conjugates of xenobiotics from the cellsseverely limited by drug resistance [1,2]. As a result [11]. Another impediment to treatment, which isof inherent drug resistance, the response rate follow- present in MDR cells, involves the sequestration ofing treatment remains very low for many malignan- drugs within cytoplasmic vesicles, followed by theircies, including acute leukemias, malignant extrusion out of the cell [13]. Drug sequestration inmelanomas, metastatic forms of breast, prostatic and MDR cells is achieved through the maintenance ofother cancers [1–3]. Additionally, prior treatment abnormally elevated pH gradients across organelle

1with anticancer agents is an adverse prognostic membranes—by the activity of H -ATPase, an ATP-factor, presumably as a result of acquired drug dependent pump [14].resistance, resulting in low long-term survival rates While a number of experimental and clinicalfor patients with relapsed and refractory tumors approaches have been studied to overcome MDR,[3,4]. Intensive laboratory and clinical studies aimed including the use of MDR chemosensitizers, theat overcoming drug resistance in cancer have, so far, appearance of several distinct transporters in resistantproduced only limited success with some novel cells may limit the success of those agents, whichtherapeutic regimens and antineoplastic agents [5,6]. target a single drug efflux pump. Furthermore, theStill, in many cases, no remedy has been found to combination of several independent mechanisms ofovercome these drug resistance mechanisms to im- drug resistance might complicate chemotherapy andprove clinical outcome in resistant cancers [7,8]. reinforces the need for development of novel drugs

One problem in treating drug resistance in cancer, and drug formulations effective against drug resistantwhich makes it a formidable task to tackle, is that cancers.there are many mechanisms through which the One novel approach using polymers to overcomeresistance is exhibited. In some cases several mecha- MDR, reported by Kopecek’s group, consists ofnisms act simultaneously and/or in concert, which conjugating drug to a soluble polymer carriermay further complicate therapy. For example, tumors [15,16]. A conjugate of doxorubicin with copolymerwith the multiple drug resistance (MDR) phenotype ofN-(2-hydroxypropyl)methacrylamide (pHPMA)have been widely recognized as one of the most has been shown to be effective against both MDRdifficult types to treat. MDR cells overexpress efflux and non-MDR cancers [17]. Furthermore, in contrasttransporters belonging to a superfamily of ATP to the effects of free drug, chronic exposure tobinding cassette (ABC) proteins, such as P-glycopro- pHPMA–doxorubicin conjugates did not inducetein (Pgp) and multidrug resistance-associated pro- MDR in cancer cells [18,19]. The reason for theteins (MRP) that pump drugs out of a cell [9,10]. differential behavior of the pHPMA–doxorubicin

A.V. Kabanov et al. / Advanced Drug Delivery Reviews 54 (2002) 759–779 761

appears to be due to differences in the mechanism of induces such a significant decrease in ATP levels,its transport into the cell compared to that of the free observed selectively in MDR-expressing cells, as

drug. As a result of the conjugation to the polymer Pluronic block copolymers do. Therefore, the find-chain, doxorubicin is rendered inaccessible to the ing of energy-depleting effects of Pluronic block

Pgp efflux pump in MDR. The resulting conjugate copolymers, in combination with their very highmost likely enters the cells via endocytosis, a Pgp- sensitization effects and ability to inhibit multipleindependent pathway, while the free drug is trans- mechanisms of drug resistance in MDR cells, is ofported across the membrane through diffusion, a considerable theoretical and practical significance.

pathway that in MDR cells is affected by Pgp [20]. This review describes the effects of Pluronic blockA recent study by Minko et al. [21] suggested that copolymers in drug-resistant cancer and discusses theHPMA-bound doxorubicin induced additional cas- latest knowledge regarding fundamental mechanismspase-dependent apoptosis signaling pathways, which of their action as well as clinical aspects related toled to more pronounced apoptosis when compared to the use of this technology in cancer chemotherapy.the free drug. Therefore, the polymer–drug conjugatenot only enhanced drug delivery in MDR cells butalso acted as a biological response modifier that 2 . Mechanisms of resistance to antineoplasticpotentiates the drug cytotoxic effect in a cell. agents

Another approach that has recently attracted in-creasing attention uses poly(ethylene oxide)–poly- Various mechanisms of drug resistance have been

(propylene oxide) block copolymers (Pluronic ) in described for all major classes of antineoplasticformulations to treat drug-resistant cancers [22–24]. agents (Table 1). Historically, investigators ex-Following validation using in vitro and in vivo amined alterations in the targets of drug action as themodels, a formulation that contains doxorubicin and most likely mechanisms involved in drug resistance

1Pluronic mixture (L61 and F127), SP1049C, has [27]. The well-known examples include doxorubicin,been evaluated in clinical trials [25]. which targets topoisomerase II [28] or methotrexate,

Experimental studies have demonstrated that which inhibits dihydrofolate reductase [29,30]. How-Pluronic block copolymers sensitize MDR cells, ever, more recently, it became evident that distinct

resulting in an increase in the cytotoxic activity of cellular mechanisms, which often occur simultan-anthracyclines and other cytotoxic drugs by 2 to 3 eously in the same cell, may take part in resistance

orders of magnitude [23,24]. Remarkably Pluronic of cancer cells to a broad range of drugs. One groupaffects several distinct drug resistance mechanisms of such mechanisms involvesreduction of effectiveincluding inhibiting of drug efflux transporters, abol- concentration of the drug prior to its interaction withishing drug sequestration and inhibiting the gluta- the target [1]. This is achieved as a result of one or athione (GSH)/glutathione S-transferase (GST) de- combination of the following processes: (i) drugtoxification system. Furthermore, recent studies dem- efflux out of the cells; (ii) drug sequestration in

onstrated that Pluronic block copolymers induce a vesicles inside the cell; and (iii) drug elimination bydramatic reduction in ATP levels selectively in MDR detoxification systems. In addition to the mecha-cells, while non-MDR cells are not responsive to this nisms reducing drug concentration in the cell, thereblock copolymer in this manner. It has long been appears to be an important group of mechanisms thatsuggested that a broadly successful strategy for decrease drug-induced damage to the cell after drugkilling drug-resistant cancer cells could be based on interaction with the target. The mechanisms forselective energy depletion in these cells, since many reduction of drug concentration and mechanisms formechanisms of drug resistance are energy-dependent reduced damage are considered below.[26]. However, no other agent was known so far that

2 .1. Drug efflux proteins1Here and below we omit the term ‘Pluronic’, using the letter and

Resistance to anticancer drugs is often mediatednumeric code to define the corresponding block copolymers, forexample, L61 for Pluronic L61. by the overexpression of membrane pumps belong-


Table 1aDrug resistance mechanisms described for antineoplastic agents (based on Refs. [1–11,26–36,43,44,50,114–120])

Drug classes and examples Mechanism of Molecules implicated incytotoxicity resistance mechanism

Intercalators Doxorubicin, daunomycin Topo II inhibitor, SO Pgp; MRP; GST; Topo IIbAlkylators Cyclophosphamide DNA alkylation GSH; ALDH; AGT (?)

Cisplatin DNA alkylation GSH; MT; DNA repairenzymes; cMOAT (?)

BCNU DNA alkylation AGTAntimetabolites Methotrexate Folic acid antagonist Amplification of DHFR; MRP (?);

decreased RFC expression5-Fluorouracil Uracil analog Amplification of TS

Vinca alkaloids Vinblastine, Tubulin Pgp; MRP; tubulinvincristine polymerization inhibitors mutation

Epidophylotoxins Etoposide Topo II inhibitor MRP; GSH; Pgp; Topo ITaxanes Paclitaxel Inhibitor of microtubule assembly Pgp, altereda /b tubulin

a 6AGT, O -alkylguanine-DNA alkyltransferase; ALDH, aldehyde dehydrogenase; cMOAT, multispecific organic anion transporter(MRP2); DHFR, dihydrofolate reductase; GSH, glutathione; MT, metallothionein; Pgp, P-glycoprotein; RFC, reduced folate carrier (RFC);SO, superoxides and free radicals; TS, thymidylate synthase; Topo II, topoisomerase II.

b Question mark means that a specific mechanism is suspected but not yet completely proven.

ing to the ABC protein superfamily, which are able organelles in MDR cells and is characteristic of theto extrude many xenobiotics out of tumor cells weak base drugs (e.g. doxorubicin) that are positively[9,10]. Examples of ATP-dependent efflux pumps, charged at acidic pH [13,27]. A major proteinwhich are overexpressed in resistant tumors, include responsible for generating pH gradients across or-

1P-glycoprotein (Pgp) [31], multidrug resistance-asso- ganelle membranes is a vacuolar H -ATPase [41].1ciated protein (MRP or MRP1), canalicular multis- Inhibitors of the H -ATPase abolish sequestration of

pecific organic anion transporter cMOAT (MRP2) the drug in the vesicles and reverse drug resistance inand several other MRP-like proteins [32,33]. Pgp has MDR cells [14,39]. Furthermore, involvement of thea broad specificity to a range of structurally diverse vesicles in drug extrusion from the cells has beendrugs including anthracyclines (doxorubicin, proposed [14,37]. These studies demonstrated thedaunorubicin), epipodophyllotoxins (etoposide, relationship between vesicular sequestration and drugteniposide), vinca alkaloids (vinblastine, vincristine), resistance conferred by the overexpression of MRPpaclitaxel, and other compounds [34]. MRP acts as a in MDR cells [14,37,42].transporter of organic anions, particularly, of gluta-thione conjugates, and apparently works in concert 2 .3. Metabolic detoxification systemswith the glutathione detoxification system [35]. As aresult, the action of MRP as a drug transporter Several cellular thiols (GSH, thioredoxin, metal-depends on intracellular levels of glutathione [11,36]. lothioneins) are believed to contribute to resistance

to many antineoplastic drugs (Table 1). The GSH/2 .2. Drug sequestration in cytoplasmic vesicles GST-related metabolic pathway is, probably, the best

studied [43–45]. Conjugation of electrophilic mole-Many studies on MDR cells show ATP-dependent cules with the reduced glutathione produces species

sequestration of the drugs inside cytoplasmic vesicles that are usually less toxic and more hydrophilic thanfollowed by extrusion of the drug from the cell the original compounds and can be partially metabo-[37–40]. Retention of drug molecules in the vesicles lized and excreted [12,46]. In cells with acquiredcan decrease the amount of drug that actually reaches resistance to antineoplastic agents, both GSH contentthe site of action and thus, might contribute to drug and GST activity are frequently elevated, whichresistance [13]. This phenomenon appears to be apparently results in protection of the cells fromassociated with abnormally elevated pH in the these agents [47].


2 .4. Inhibition or prevention of drug-induced hydrophilic ethylene oxide (EO) and hydrophobicpropylene oxide (PO) blocks arranged in a basicapoptosisA–B–A structure: EO –PO –EO . The structuren / 2 m n / 2

formula of Pluronic block copolymers is shown inDNA damage and apoptosis are the main causes ofFig. 1. Copolymers with various numbers of hydro-drug-induced cytotoxicity [48,49]. Enhanced DNA

6 philic EO (n) and hydrophobic PO (m) units arerepair by O -alkylguanine-DNA alkyltransferase hascharacterized by a distinct hydrophilic–lipophilicbeen known as a common mechanism of resistance

balance (HLB). Pluronic block copolymers areto drugs [50,51]. More recently, alteration in death-synthesized using step-wise anionic polymerizationinducing signaling pathways, resulting in inhibitionby sequential addition of PO and EO monomers inor prevention of apoptosis, has been suggested asthe presence of an alkaline catalyst, such as sodiumanother mechanism of chemoresistance [52,53].or potassium hydroxide [58]. The reaction is initiatedOverexpression of anti-apoptotic genes (e.g. Bcl-2),by polymerization of the PO block followed by theas well as mutation or reduced expression of pro-growth of EO chains at both ends of the PO block. Itapoptotic genes (e.g. p53), has been shown to renderusually produces polymers with a relatively lowcancer cells resistant to drug effects [52,53]. Contri-polydispersity index (M /M ).bution of Pgp to protection of tumors from apoptosis n w

has recently been discussed in view of observations The solubility of Pluronic block copolymers inthat inhibition of Pgp by chemosensitizing agents can water depends on their structure, namely, the lengthsrestore the normal apoptotic cascade in cells with of the hydrophobic and hydrophilic blocks, as welldefective signaling pathways [54–57]. as the temperature. Elevation of the temperature

Overall, there are multiple, complex and, possibly, promotes dehydration of alkylene oxide units of theinterrelated mechanisms of drug resistance. These block copolymer decreasing the solubility—firstly, ofmechanisms are often found simultaneously, which the PO block and secondly, of the EO block. At thecan further complicate the chemotherapy of tumors. body temperature, 378C, PO chains are water-insolu-

ble, while EO chains are still well-hydrated andwater-soluble. Molecular dispersions form at the

3 . Structure and solution behavior of Pluronic block copolymer concentrations below the criticalblock copolymers micelle concentration (CMC). Above the CMC the

individual block copolymer molecules, termed ‘un-Pluronic block copolymers (also known under imers’, self-assemble into micelles through a process

their non-proprietary name ‘poloxamers’) consist of called ‘micellization’. The driving force for the

Fig. 1. Pluronic block copolymers available from BASF (Wyandotte, MI), contain two hydrophilic EO blocks and a hydrophobic POblock.


micellization is the hydrophobic interactions of thePO blocks. As a result, the PO blocks self-assembleinto the inner core of the micelles covered by the

hydrophilic corona from EO blocks. Pluronic mi-celles are commonly pictured as spheres composedof a PO core and an EO corona. This portrayal iscorrect for most block copolymers, which have anEO content above 30%, especially in relatively dilutesolutions at body temperature. However, additionalmicelle morphologies, including lamella and rods,

can also form in Pluronic systems [59]. Whenspherical micelles are formed, depending on the

Pluronic type, the micelles commonly have anaverage hydrodynamic diameter ranging from about20 to about 80 nm [59].

The core–shell architecture of Pluronic micellesis essential for their utility in drug delivery applica-tions [60]. The core formed by the PO chains is awater-incompatible compartment that is segregatedfrom the aqueous exterior by the hydrophilic chains

Fig. 2. Effects of molecular composition of Pluronic blockof the EO corona, thereby forming, within the core, acopolymer on CMC. CMCs were determined at 378C using the‘cargo hold’ for the incorporation of various thera-pyrene solubilization technique and plotted as a function of thepeutic reagents. The process of transfer of water-length of the PO block,N . From Ref. [63] (with permission).POinsoluble compounds into the PO core of the micellar

solution is referred to as ‘solubilization’. As a result,polymeric micelles can be used as efficient carriers elevates the net hydrophobicity of the Pluronic

for compounds, which alone exhibit poor solubility, molecule and favors the formation of the micelleundesired pharmacokinetics and low stability in a core, which results in a CMC decrease [59,66,67].physiological environment. The hydrophilic shell Conversely, an increase in the lengths of the EOcontributes greatly to the pharmaceutical behavior of blocks decreases the hydrophobicity and destabilizes

Pluronic formulations by maintaining the micelles the micelle, resulting in the CMC increase [64,65].in a dispersed state, as well as by decreasing Fig. 2 presents the dependence of the log CMC of

undesirable drug interactions with cells and proteins Pluronic block copolymers on the length of the POthrough steric-stabilization effects. block (the effect of EO block length is usually less

CMC is of paramount significance to drug delivery drastic than the effect of the PO block length).using Pluronic block copolymers [60,61]. Firstly,

the CMC determines the stability of micelles againstpossible dilution of the solution in body fluids 4 . Pluronic block copolymers sensitize drug-

[60,62]. Secondly, the CMC determines the maximal resistant cancersachievable concentration of Pluronic unimers, to

which cells will be exposed, thereby defining the The use of Pluronic block copolymers to treatbiological effects, which Pluronic itself, will exert drug-resistant cancers is a rapidly developing area of

on these cells [63]. Typically, Pluronic block drug delivery for cancer chemotherapy. Studies bycopolymers, which are used for drug delivery, at Alakhov et al. demonstrated that Pluronic block

body temperature (378C) have a CMC ranging from copolymers sensitize resistant cancer cell lines, re-231 mM to 1 mM (ca. 5310 to 1 wt.%). The CMC sulting in an increase in the cytotoxic activity of the

depends on the lengths of the PO and EO blocks drug by 2 to 3 orders of magnitude [23,24,63,68]. By[64,65]. An increase in the length of the PO block addition of P85 or L61, the cytotoxic effects of


Pluronic block copolymer composition. A tumorpanel included intraperitoneal murine leukemias(P388, P388-Dox), subcutaneous murine myelomas(Sp2/0, Sp2/0-Dnr), intravenous and subcutaneousLewis lung carcinoma (3LL-M27), subcutaneoushuman breast carcinomas (MCF-7, MCF-7/ADR),and human head and neck carcinoma (KBv) [69]. Bythe National Cancer Institute criteria for tumor

inhibition and increased lifespan, Pluronic -formu-lated doxorubicin has met the efficiency criteria in allmodels (9 of 9), while doxorubicin alone waseffective only in selected tumors (2 of 9) [69]. Theresults demonstrated that the tumors were more

responsive in the Pluronic copolymer treatmentgroup than in the control groups that received onlydoxorubicin. Together these studies indicate im-proved treatment of drug-resistant cancers with

Fig. 3. Cytotoxicity of free daunorubicin (1, 2) and daunorubicin Pluronic block copolymers.with 1 wt.% P85 (3, 4) with respect to resistant SKVLB (1, 4) andsensitive SKOV3 (2, 3) cells. After exposure for 120 min to the

drug or drug Pluronic , cells were cultured in drug-free media for5 . Several drug resistance mechanisms are4 days and cytotoxicity was determined using a tetrazolium-based

affected by Pluronicassay. Based on Ref. [23] (with permission).

5 .1. Inhibition of Pgp drug efflux systemdoxorubicin in the resistant lines significantly surpas-sed those observed in the sensitive lines. For exam- One major reason for the enhanced cytotoxicity

ple, Fig. 3 illustrates typical effects of P85 on the observed with Pluronic in drug-resistant cancercytotoxicity of the anthracycline drug, daunorubicin, cells appears to be related to the affects of thewith respect to resistant and sensitive tumors. In this copolymer on the Pgp drug efflux transport system.experiment, human ovarian carcinoma cells (SKVLB Similar affects have been observed with otherand SKOV3) were exposed for 120 min to free nonionic surfactants such as Cremophor EL, Solutoldaunorubicin or daunorubicin with P85. Cytotoxicity HS15, Triton X-100, Nonidet P-40 or polyox-studies in the sensitive cell line, SKOV3, showed yethylated derivatives of fatty acids that all exhibitlittle difference between the control group and an ability to enhance the cytotoxicity of antineoplas-groups exposed to P85. However, in the MDR cell tic agents in MDR cells [70–78]. There is over-line, SKVLB, there was a significant enhancement in whelming evidence to support inhibition of Pgp by

cytotoxicity to daunorubicin in the presence of P85. Pluronic block copolymers. This is supported byEffects of the block copolymers in the resistant cells the observation that the intracellular accumulation ofare expressed in the form of a ‘resistance reversion doxorubicin in resistant cancer cells expressing Pgp

index’ (RRI), i.e. ratio of IC of the drug in the can be greatly enhanced by treatment with Pluronic50

assay buffer and Pluronic solution (IC / IC ). In [24,63]. No alteration in drug uptake in the presence50,0 50the particular case presented in Fig. 3, the resistance of Pluronic was observed with non-Pgp-expressing

reversion index was ca. 700 [23]. cancer cells, i.e. the copolymers affect specificallyEffects of Pluronic block copolymers in drug- the Pgp-controlled transport route in MDR cells

resistant tumors have also been reported in vivo [24,63]. This conclusion has been reinforced by the[22,69]. In these studies, mice bearing drug-sensitive recent studies by Evers et al. [79] and Batrakova et

and drug-resistant tumors were treated with doxoru- al. [80] demonstrating that Pluronic block copoly-bicin alone or doxorubicin formulated with mers (L61, P85) profoundly increase accumulation


and permeability of various Pgp-dependent drugs in system, distinct from Pgp, was evident based on thismdr1-transfected cells, which overexpress Pgp. At study, the support of inhibition of a specific MRPthe same time, these copolymers have little on no transport system(s) remained inconclusive.

effect on drug transport in non-transfected cells, To address Pluronic effects on MRP, a recentwhich display low endogenous Pgp expression. study by Evers et al. [79] has examined drugTherefore, enhanced transport of Pgp substrates, transport in canine kidney cells, MDCK II that had

induced by exposure of the cells to Pluronic , been transfected to stably express either MRP1 orcorrelates with the level of Pgp expression in the MRP2. The results of this study suggested that L61cells, suggesting that the effects are exerted through partially inhibited MRP2-mediated transport of vin-a Pgp-dependent route. Furthermore, the block co- blastine in the MRP2-expressing cell line. However,polymer has no or little affect on the accumulation of the potency of this block copolymer, with respect tonon-Pgp-dependent compounds in both resistant and inhibition of MRP2, in these cells appeared to besensitive cells [80–82]. Therefore, the increased lower than its potency with respect to Pgp in mdr1-absorption of the Pgp substrates in Pgp-expressing transfected cells that express Pgp. Furthermore, thesecells is attributable to the effects of the block authors concluded that L61 had little if any influencecopolymer on the Pgp efflux system, rather than to on MRP1- and MRP2-mediated transport of anothernon-specific alterations in membrane permeability of MRP-substrate, dinitrophenolS-glutathione [79]. Thethe substrates. Finally, since the efflux of Pgp-depen- dinitrophenolS-glutathione experiments, however,dent drugs was decreased in the presence of are difficult to interpret because the total lengths of

Pluronic block copolymers, the mechanism for the exposure of the cells to the block copolymer in thisincreased drug accumulation observed with these study were relatively short (30 min or less). Thisblock copolymers appears to be related to inhibition might be insufficient in view of the previous reportof the drug efflux system [24,82]. that inhibition of the drug efflux system in MRP-

expressing cells requires an exposure of at least 30 to60 min to the block copolymerprior to adding the

5 .2. Effects on other drug transporters substrate to the cells [83].These studies of the effects of Pluronic on MRP

There is increasing evidence suggesting that the are complicated by the possible interference ofeffects of Pluronic block copolymers might stem effects on both Pgp and MRP, commonly co-ex-

beyond inhibition of the Pgp efflux pump alone. pressed in cells, and by the lack of selective MRPStudies by Miller et al. [83], using the human substrates that would allow the exclusion of non-pancreatic adenocarcinoma cell line, Panc-1, that MRP drug transport mechanisms that can be affectedexpresses the MRP1 efflux pump, suggested that P85by the block copolymer. To address this problem weinhibits efflux and increases cellular accumulation of have recently examined effects of P85 on transportthe MRP-dependent probe, fluorescein, in these cells.of several substrates in cells expressing MRP (manu-Similar to the case of Pgp inhibition discussed script in preparation). In addition to canine kidney

above, the effects of Pluronic in Panc-1 cells cells, MDCKII–MDR1 and MDCKII–MDR2, theappeared to be specific with respect to fluorescein same as in the paper by Evers et al. [79], this studytransport system(s), since no accumulation increasesused human lung carcinoma epithelial cell line,were observed for an organic cation, rhodamine 123, COR-L23/R, selected with doxorubicin, which over-a substrate of Pgp that is not expressed in these cells.expresses MRP1 but has little if any Pgp. UsingHowever, more recently it was concluded that, in several substrates, which are either selective toaddition to MRP1, some other organic anion trans- organic anion transporters, such as fluorescein andporters, including MRP2, might also be present in 29,79-bis-(2-carboxyethyl)-5-carboxyfluorescein acet-Panc-1 (D.W. Miller, personal communication). Thus, oxymethylester, or are substrates of both Pgp and

Pluronic could possibly inhibit these transporters as MRP, such as vinblastine and doxorubicin, this studywell, resulting in enhancement of fluorescein ac- suggests that P85 is a potent inhibitor of both MRP1cumulation in Panc-1 cells. Therefore, although the and MRP2 in these cells. In all cases, pre-exposure

affect of Pluronic on some organic anion transport of the cells to P85 was necessary to fully exhibit the


effects of this block copolymer on the MRP transport 1-chloro-2,4-dinitrobenzene, was significantly in-systems, which may explain the discrepancy of the hibited immediately following 2-h exposure of theresults with the prior study by Evers et al. [79]. MDCKII, MDCKII–MRP1 and MDCKII–MRP2Thus, although the evaluation of the effects of cells to P85. The levels of GSH did not decrease

Pluronic on MRP is far from complete, there is during 2-h exposure of these cells to P85. However,substantial evidence to suggest that these block the GSH pools in these cells were significantlycopolymers affect multiple drug transport systems in depleted when the cells were first exposed to

MDR cells. Pluronic and then incubated in Pluronic -freemedia during 22 h. Inhibition of the GST/GSH

5 .3. Effects on drug sequestration within detoxification system should result in the decrease ofcytoplasmic vesicles glutathione conjugation of select substrates, such as

doxorubicin, and additionally decrease the extent ofIn MDR cells, drug can be sequestrated within elimination of these substrates from the cells through

cytoplasmic vesicles and then extruded from the cell the MRP-mediated drug efflux pathway. The com-before the drug can exert its intended action on the plex and interrelated mechanisms of drug eliminationcell, which is another potential impediment to the through MRP and GST/GSH detoxification systemstreatment of resistant tumors [13,37–40]. Drug se- result in different responses of these systems to

questration in MDR cells is achieved through the Pluronic when different substrates are used. Firstly,maintenance of abnormally elevated pH gradients the organic anion substrates of MRP that do notacross organelle membranes through the activity of require glutathione conjugation, such as fluorescein

1 H -ATPase, an ATP-dependent pump [14]. Recent [84,85], usually respond to Pluronic in the samestudies by Venne et al. [24] examined effects of way as the Pgp substrates do, resulting in decreased

Pluronic block copolymers on intracellular localiza- efflux and increased substrate accumulation in thetion of doxorubicin in the MDR cancer cell line, cells [83]. Secondly, the compounds that becomeMCF-7/ADR. In these cells, free doxorubicin is MRP substrates only after glutathione conjugation,sequestered in cytoplasmic vesicles, which might such as doxorubicin [86,87], can exhibit a complexfurther diminish the amount of the drug available for dependence on GSH levels, as well as MRP andinteraction with the nucleus [13]. Following incuba- GST activities. Finally, compounds that are co-trans-

tion of these cells with doxorubicin and Pluronic , ported with GSH without glutathione conjugation,drug was released from the vesicles and accumu- through the MRP route, such as vinblastine [35,88],lated, primarily, in the nucleus [24]. Furthermore, we can depend on GSH levels as well as MRP activity

have observed vesicular sequestration of fluorescein but not exhibit it. The effects of Pluronic blockin Panc-1 cells overexpressing MRP [83]. This copolymers on the efflux and accumulation of suchsequestration was also abolished and fluorescein was compounds in the resistant cells can be amplifiedreleased in the cytoplasm by treating the cells with through affects on multiple components of the

Pluronic . detoxification system present in these cells, pro-viding that these component(s) are critical (rate

5 .4. Effect on GSH /GST system limiting) for the elimination of the substrate. Overall,these studies suggest that Pluronic block copoly-

The activity of MRP drug efflux transporter with mers affect several important mechanisms of drugrespect to many substrates is closely tied to the resistance in cancer cells.GSH/GST detoxification system in MDR cells [13].

Therefore, studies of the effects of Pluronic blockcopolymers on GSH/GST system have begun. For 6 . ATP depletion induced by Pluronic in MDR

example, significant decreases in both intracellular cellslevels of GSH and GST activity were observed inMDCK cells expressing MRP, following exposure of Various drug resistance mechanisms, includingthese cells to P85 (manuscript in preparation). GST drug transport and detoxification systems, requireactivity, as assessed using a colorimetric substrate consumption of energy to sustain their function in


MDR cells. Therefore, mechanistic studies have that the responsiveness of the cells to P85 correlated with appearance of MDR in these cells, rather thanfocused on the effects of Pluronic block copoly-

to the amount of ATP available in the cells prior tomers on metabolism and energy conservation intreatment. Based on the results of this study, thedrug-resistant cells [89,90]. Slepnev et al. [91] werepresence of the MDR phenotype is one factor thatfirst to demonstrate that intracellular levels of ATPrenders cellular metabolism responsive to treatmentwere depleted following a 2-h exposure of Jurkat

with Pluronics .T-cell lymphoma cells to the Pluronic block co-The mitochondria are responsible for carrying outpolymer, P85. The following key observation, in the

much of the metabolic activities of the cell, andcontext of the drug resistance phenomena, was themight be a potential site of action for Pluronicstudy by Batrakova et al. [89,90] that compared the

block copolymers. It has long been known thateffects of the P85 on ATP levels in several cell typesnonionic polymeric detergents, such as Tween 80that either exhibit MDR phenotype, or do not exhibit

and Pluronic , can decrease oxidative metabolism ofMDR phenotype. In this study, exposure of MDRtissues, cells and isolated mitochondria [92–94].and non-MDR cells to different doses of P85 resulted

in a transient energy depletion, which was reversed Rapoport et al. [94] has shown that two Pluronicfollowing removal of the block copolymer. The copolymers, P85 and P105, reduce the activity of theMDR cells were much more responsive to P85, electron transport chains in mitochondria, as assessedexhibiting a profound decrease in ATP levels at by the rates of bioreduction of lipophilic spin-probessubstantially lower concentrations of the block co- in HL-60 cancer cells. A recent confocal microscopypolymer, compared to the non-MDR cells. The study using P85 that was labeled with a fluorescenteffective concentrations of P85 that induced a 50% tag to examine its transport and localization insidedecrease in ATP levels in the cells (EC ), as cells suggested that within a 15 to 60-min interval,50

determined from the dose–response curves, are the block copolymer molecules spread throughoutpresented in Table 2. This table also presents the the cell, where they may interact with variousrelative responsiveness of the MDR cells compared intracellular organelles, including mitochondria [95].to the non-MDR counterparts. These data suggest There could be multiple reasons for the inhibitory

Table 2aEffects of P85 on ATP levels in MDR and non-MDR cells

Cells MDR1 Initial ATP levels EC , % Relative responsiveness50b cphenotype nmol /mg protein to P85

MCF-7 No 3061.5 2.25 –dMCF-7/ADR Yes 300620 0.009 250

KB No 160.01 0.675 –eKBv Yes 460.1 0.036 19

C2C12 No 1561.4 4.5 –HUVEC No 4064.9 0.0675 –

fCaco-2 Yes 5.560.4 0.00067 6670fBBMEC Yes 1.660.04 0.018 250

LLC-PK1 No 6166.9 0.45 –gLLC-MDR1 No 7961.7 0.0045 100

a Based on Ref. [90] as well as data in preparation (MDCK cells). In this study cells were exposed to P85 for 120 min prior todetermining the intracellular ATP levels.

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


activity of these compounds in mitochondria. The for the block copolymer selectivity in these cells. Anlikely components contributing to the anti-metabolic alternative hypothesis would be that, for someeffects of nonionic detergents include their ability to reason, the metabolic processes in MDR cells are

1 serve as K ionophores [96–98] and their ability to more sensitive to inhibition with Pluronic than theuncouple oxidative phosphorylation [99,100]. It is metabolic processes in non-MDR cells. This couldalso possible that these compounds directly inhibit result in the more pronounced ATP depletion ob-the NADH dehydrogenase complex by interacting served following exposure of MDR cells to the blockwith the hydrophobic sites of this complex that are copolymer. Furthermore, it has to be emphasized thatlocated in the mitochondrial membrane [93,99]. in addition to overexpression of Pgp some other

However, the reasons for a remarkable selectivity factors implicated in the appearance of the MDRof Pluronic block copolymers with respect to MDR phenotype, such as other efflux proteins, high levels

1cells are not completely understood. One hypothesis of H -ATPase and activated GSH/GST detoxifica-is that the high rates of energy consumption by the tion system, could also impose high energy require-

drug efflux pumps, combined with Pluronic -in- ments in MDR cells and collectively contribute toduced inhibition of respiration, determine the respon- the elevated responsiveness of these cells to

siveness of the resistant cells to the block copolymer. Pluronic . The high responsiveness to P85 of theUnder conditions in which the respiration necessary cell lines, which have relatively low levels of Pgpfor ATP synthesis is inhibited, the high rates of expression, such as HUVEC (Table 2) may be oneATPase activity by the drug efflux pumps (and indication of the possible involvement of alternativepossibly, some other energy-dependent mechanisms) mechanisms in the energy depletion [90].could result in a rapid exhaustion of the intracellular The energy depletion phenomenon is obviouslyATP pools in the resistant cells. Alternatively, cells, extremely important in order to understand thewhich do not exhibit these resistance mechanisms, reasons for the elevated anticancer activity of

would appear to be less responsive to inhibition of Pluronic -based formulations in drug-resistantrespiration and would not exhibit energy depletion, tumors. Transient energy depletion, as a result of

at least to the extent observed in the resistant cells. exposure of the cells to Pluronic in the absence ofSuch a hypothesis is in line with the earlier observa- the chemotherapeutic agent, does not induce a cyto-tion that resistant cells have an increased glucose toxic effect in either MDR or sensitive cells [89,90].utilization rate compared to sensitive cells [101,102]. However, if the chemotherapeutic agent, doxorubi-Furthermore, the toxicity of the inhibitor of gly- cin, is present, then the exposure of MDR cells to thecolysis, 2-deoxyglucose was found to be consistently combination of both drug and block copolymerhigher in MDR cells than in the non-MDR lines results in a pronounced potentiation of the cytotoxic[102]. According to Batrakova et al. [90] the extent activity, i.e. the sensitization effect [89,90]. Theof the selectivity of P85 with respect to MDR cells is apparent relationship between energy depletion andthe same or even greater than that of 2-deoxyglu- enhanced cytotoxicity in cancer cells is supported bycose. Furthermore, P85 was significantly more selec- the literature. For example, metabolic modulatorstive than the inhibitors of respiration, rotenone and alone, such as 2-deoxyglucose, iodo-acetic acid,sodium azide. fluoro-acetic acid, oligomycine, azide, antimycin and

Most recently, evidence has begun to mount rotenone, which can inhibit energy conservation atsuggesting that there might be an alternative explana- various levels (i.e. glucose uptake, glycolysis, citriction for the high selectivity of the block copolymers acid cycle, oxidative phosphorylation), have beenwith respect to MDR cells. A study, from our shown to induce apoptotic cell death in cancer cellslaboratory, suggested that P85 significantly inhibits [103]. Furthermore, a three-drug combination, PMAATPase activity of Pgp in isolated cell membranes ((N-phosphonacetyl)-aspartate, 6-methylmercap-[90]. Since Pgp is one of the major ATPases topurine, 6-aminonicotinamide), which contains in-overexpressed in MDR cells, and the fact that hibitors of pyrimidine and purine pools, can poten-Pluronic inhibits this activity, it makes the high tiate drug activity and enhance drug-induced apop-energy consumption in MDR cells a less likely cause tosis in cancer tumors [104].


7 . Membrane fluidization and inhibition of Pgp activity in these cells as well, providing initial ATPase activity by Pluronic evidence that Pluronic block copolymers might

inhibit MRP ATPase.Pluronic block copolymers are known to induce

drastic changes in the microviscosity of cell mem-branes, as assessed using the hydrophobic membrane8 . Interplay of ATP depletion and membraneprobe, 1,6-diphenyl-1,3,5-hexatriene (DPH) [105]. fluidization as a possible mechanism for

These changes can be attributed to the alterations inPluronic activity in MDR cellsthe structure of the lipid bilayers as a result ofadsorption of the block copolymer molecules on the As evidenced in the previous section, there couldmembranes. In particular, recent studies using DPH be multiple reasons for inhibition of the Pgp effluxas a probe, have demonstrated that exposure of cells system by P85 in MDR cells. These reasons first

to Pluronic block copolymers, such as P85, results include dramatic energy depletion induced by thein fluidization of the cellular membranes of both block copolymer, which might abolish the ATP-normal and cancerous cells expressing high levels of dependent drug efflux. Another reason includes thePgp [95]. Membrane fluidization by various agents, inhibition of the Pgp ATPase activity, which isincluding nonionic surfactants such as Tween 20, essential for the functioning of this transporter.

Nonidet P-40 and Triton X-100, is known to contrib- Therefore, it is likely that these Pluronic blockute to inhibition of Pgp efflux function [106]. copolymers have a ‘double-punch’ effect in MDRFurthermore, it is believed that inhibition of Pgp cells: through ATP depletion and membrane fluidiza-ATPase activity, presumably, through nonspecific tion, which both have a combined result of potentchanges in lipid and protein conformation and inhibition of Pgp (Fig. 4). The most compellingmobility, has a major contribution to the inhibition of evidence to support the idea that the energy depletionPgp efflux function [106]. A recent study evaluated contributes to the sensitization of the MDR cells by

the effects of Pluronic block copolymer, P85, on Pluronic was obtained in experiments using anthe Pgp ATPase activity using membranes containing energy supplementation system [90,95]. Thesehuman Pgp [95]. In this work the membranes were studies were based on the observation that treatmentexposed to P85 and then the ATPase activity of Pgp of cells with dodecylamine in combination with P85was assayed by determining the liberated inorganic allows transport of ATP into the cells from thephosphate [107]. The study also evaluated a Pgp extracellular media [91]. The use of this system withsubstrate, verapamil, to determine whether binding of MDR cells allowed restoration of intracellular ATPa specific substrate with Pgp could modulate the levels, which abolished inhibition of the Pgp effluxeffects of P85 on Pgp ATPase activity. P85 at system by P85 and substantially reduced the cytotox-concentrations as low as 0.001% induced dramatic ic effect of doxorubicin formulated with P85 in thesedecreases in Pgp ATPase activity compared to the cells [90,95]. Since the block copolymer in thiscopolymer-free control. Verapamil alone in the ab- experiment was still present and, presumably, boundsence of the block copolymer induced a significant with the cell membranes, the ATP supplementationincrease in Pgp ATPase activity. This effect is study indicates that membrane fluidization alonemaybelieved to be due to the binding of verapamil in the not be sufficient for inhibition of the Pgp effluxactive center of Pgp [108,109]. However, the inhib- system in MDR cells. On the other hand, the direc-itory effect of P85 was observed in the presence of tionality studies and experiment involving P85 re-verapamil at all block copolymer concentrations moval from MDR cells suggest that energy depletionexamined (0.001% to 1%). Overall, this study sug- alone in the absence of interaction of the blockgested that P85 is a potent inhibitor of the Pgp copolymer with the Pgp-containing membranesATPase activity. A similar study was recently con- might be insufficient to inhibit the efflux system.ducted using membranes isolated from MRP1 over- Therefore, both factors are critical for exhibition ofexpressing lung carcinoma cell line, COR-L23/R the effect of P85 on the Pgp efflux system in MDR(paper in preparation). P85 inhibited the ATPase cells.


Fig. 4. Schematic presenting multiple effects of Pluronic block copolymers displayed in MDR cell. These effects include (a) decrease inmembrane viscosity (‘fluidization’); (b) ATP depletion; (c, d) inhibition of drug efflux transport systems; (e) reduction in GSH/GST

detoxification activity; and (f) drug release from acidic vesicles in the cell. Effects of Pluronic block copolymers on apoptosis (g) are notsufficiently studied at present.

The interrelationship between the membrane activity. This means that higher concentrations offluidization and energy depletion components of intracellular ATP would be required for normal

Pluronic action can be better understood in view of functioning of Pgp, i.e. the drug efflux system wouldthe current picture of Pgp structure describing Pgp as become more vulnerable to decreases in intracellulara two-domain protein with ATP-binding sites in each ATP. Future studies of the kinetics of the Pgp

domain [110]. Proper interaction of these two ATP- function in the presence of Pluronic are needed tobinding sites is crucial for the proper functioning of confirm this hypothesis.Pgp. It was suggested that binding of ATP in onedomain causes a conformational change in the Pgp

molecule necessary for the hydrolysis of ATP and 9 . Dose dependence of Pluronic effects intranslocation of the substrate [111]. Therefore, the MDR cells: unimers vs. micellesstructural perturbations in the lipid membranes in-

duced by Pluronic may decrease the affinity of ATP Due to the ability of Pluronic block copolymersto its binding site and interfere with the ATPase to self-assemble into micelles and to solubilize


hydrophobic drugs, the dose dependencies observed Cremophor EL [112,113]. The inhibition of cellwith these agents in MDR cells are drastically transport in the presence of micelles is attributed todifferent compared to the dose dependencies of many incorporation of the drug into the micelles, resultingother MDR chemosensitizers. The studies by Bat- in a decreased free drug available for diffusionrakova et al. [63] have demonstrated that both through the cell membrane into the cells.

inhibition of the Pgp efflux system by Pluronic andpotentiation of doxorubicin activity by Pluronic in

MDR cancer cells, occur at block copolymer con- 1 0. Relationship between structure of Pluroniccentrations below the CMC. This means that both and its activity in MDR cells

effects are due to Pluronic unimers. As a result, theaccumulation and cytotoxicity of drugs in MDR cells Batrakova et al. [63] reported that both the inhibi-

increase with increasing concentrations of Pluronic tion of the Pgp efflux system as well as the hyper-until the CMC is reached and unimer concentration sensitization effects of Pluronic in MDR cells

levels off. This behavior is illustrated in Fig. 5 using depend on the molecular composition of the blockthe effects of the block copolymers on doxorubicin copolymer. This study examined a panel of over 20cytotoxic activity in MDR cells as an example. At block copolymers having different lengths of hydro-concentrations above the CMC, drug accumulation philic (EO) and hydrophobic (PO) segments usingand cytotoxicity in MDR cells tend to first level off MDR cancer cell line, KBv. From the standpoint ofand then decrease [63]. Decreases in drug absorption their affect on Pgp these copolymers were sub-in cells at block copolymer concentrations above the divided into three groups: group 1, hydrophilicCMC are similar to those reported previously for copolymers with HLB varying from 20 to 29 havingother nonionic detergent MDR modulators, such as no or little affect on Pgp (e.g. F68, F88, F108,

F127); group 2, hydrophobic copolymers (HLB,19)with intermediate length of PO block ranging from30 to 60 PO repeating units, which are the mostefficacious in inhibition of Pgp (e.g. P85, P81, L61);and group 3, hydrophobic copolymers (HLB,19)with shorter or longer PO blocks (e.g. L35, L44,L121), which are less efficient than the copolymersof group 2. To evaluate how efficacious the blockcopolymers are at inhibiting Pgp, a dose–responsecurve for rhodamine 123 accumulation was obtainedfor each block copolymer. Next, the maximal ac-cumulation levels observed with the most effective

doses of each Pluronic were plotted as a function ofthe length of the hydrophobic PO block (N ). ThisPO

yielded a bell-shaped dependency of the net efficacyof Pluronic copolymers in inducing rhodamine 123

accumulation in MDR cells (Fig. 6). Furthermore,the resistance reversion indexes determined in theFig. 5. Effects of Pluronic block copolymers on cytotoxicity ofdoxorubicin cytotoxicity study in MDR cells fol-doxorubicin with respect to MDR cells: curve 1, MCF7/ADRlowed exactly the same pattern as the drug accumu-cells were treated with doxorubicin–L61 compositions; curve 2,

KBv cells were treated with doxorubicin–P85 compositions lation data shown in Fig. 6 [63].containing varying concentrations of the block copolymer. Resist- In order to understand the reasons for the observed

ance reversion indexes (ratio of IC of doxorubicin in the assay50 structure–functional relationships in Pluronic blockbuffer and Pluronic solution) are plotted as functions of thecopolymers, we have examined intracellular trans-concentration of the block copolymers. The vertical arrows fromport of the copolymers as well as their affects onleft to right show the CMC of P85 and L61. From Ref. [63] (with

permission). ATP depletion and Pgp ATPase activity (paper in


groups 1 and 3 were much less efficacious. Thisstudy provided evidence that the block copolymersthat are most efficacious in inhibiting Pgp (group 2)are also the most active in energy depletion and viseversa.

Thirdly, the affects of block copolymers on thePgp ATPase activity were evaluated using mem-branes containing human Pgp. The copolymers ofgroup 2 induced dramatic decreases in Pgp ATPaseactivity compared to the copolymer-free control asdescribed Section 7. Conversely, the copolymers ofgroup 3 had much less affect on the ATPase activity,while the copolymers of group 1 practically did notchange the ATPase activity. These experimentsprovided further support for the fact that the ability

of Pluronic block copolymers to inhibit the activityof the Pgp efflux system can be correlated with theirability to inhibit Pgp ATPase activity.

Overall, combined differences in membrane inter-actions, cellular transport and energy depletion ac-tivity may contribute to the observed dependence, inFig. 6. Efficacy of Pluronic block copolymer composition inMDR cells, of the potency of the block copolymersMDR KBv cells. Figure presents data for groups 2 and 3 blockon their structure.copolymers only. Rhodamine 123 (R123) enhancement factors are

defined here as the ratios of rhodamine 123 accumulation in thecells in the presence of the block copolymer to rhodamine 123

accumulation in the assay buffer (reproduced from Ref. [63] with 1 1. Effects of Pluronic on cytotoxicity ofpermission).

various drugs in resistant cells

preparation). These studies used Pluronic block One question arising in relation to the studiescopolymers that are representative of each of the described in the manuscript is whether Pluronicabove three groups. copolymers can potentiate activity in drug-resistant

Firstly, the transport of fluorescent-labeled co- tumors of a broader range of antineoplastic agentspolymers in the bovine brain microvessel endothelial beyond anthracyclines, that are currently the bestcells (BBMEC) was examined by confocal micro- characterized? One study examined the effects ofscopy. These experiments suggested that hydrophilic P85 on the cytotoxicity of a panel of antineoplasticcopolymers of group 1 did not transport within the agents including anthracyclines (daunorubicin), vincacells. Hydrophobic copolymers of group 3 were alkaloids (vinblastine), alkylating agents (mitomycinaccumulated in the intracellular endosomal compart- C, cisplatin), and antimetabolites (methotrexate) [23].ments but did not release in the cytoplasm. Finally, The studies were conducted using the human ovarianthe hydrophobic copolymers of group 2 were effec- carcinoma cell line SKVLB, which has been selectedtively transported within the cells. This provided for vinblastine-resistance and has the MDR pheno-

initial evidence that the ability of Pluronic block type. As is seen in Table 3, the IC s of the MDR-50

copolymers to inhibit the activity of the Pgp efflux type drugs, daunorubicin, vinblastine, and mitomycinsystem can be correlated with their ability to be C, were increased 80 to 1090 times in the presencetransported within the cell. of 2.2 mM Pluronic P85. Less significant changes

Secondly, the study of ATP levels in MDR cells (only 2 to 6 times) were observed in the IC s of50

suggested that only the copolymers of group 2 cisplatin and methotrexate in SKVLB cells, whicheffectively depleted ATP, while the copolymers of are not resistant to these drugs. This result indicates


Table 3Effects of Pluronic P85 (2 mM) on the cytotoxicity of various drugs in resistant SKVLB cell line. Based on Ref. [23]

Antineoplastic agent Free drug IC Drug/Pluronic IC RRI (IC / IC )50,0 50 50,0 50

ng/ml ng/ml

Daunorubicin 5000 7 700Vinblastine 1200 1.1 1090Mitomycin C 800 10 80Methotrexate 500 90 6Cisplatin 400 200 2

2that the sensitization of resistant cells by Pluronic mg/m is suitable for evaluation in phase II / IIIblock copolymers is a general phenomenon charac- trials. Plasma pharmacokinetics showed thatteristic for a broad range of structurally diverse SP1049C exhibited slightly longer half-life compareddrugs. to conventional doxorubicin (50 h vs. 30 h). One

patient with relapsed previously-treated esophagealsarcoma showed temporarily a 50% reduction in

1 2. Clinical trials of doxorubicin–Pluronic tumor size, which progressed rapidly on completionformulation (SP1049C) of treatment. Furthermore, antitumor activity was

seen in relapsed previously-treated soft tissue sar-Pluronic block copolymer, L61, was selected for coma. Phase II clinical trials are scheduled to begin

further preclinical development based on the in vitro in early 2002.and in vivo efficacy evaluation studies of anthra-cyclines [22,24]. The final block copolymer systemused in the clinical studies contains 0.25% L61 and 1 3. Conclusion2% F127 formulated in isotonic buffered saline andis called ‘SP1049C’ (Supratek Pharma, Montreal, In conclusion, although the studies described inPQ, Canada) [69]. This system contains mixed this paper are relatively recent, the results obtained

micelles of L61 and F127 with an effective diameter already suggest that Pluronic block copolymers areof ca. 22 to 27 nm, which do not aggregate in the promising agents for utilization in treatment of drug-presence of the serum proteins. Prior to administra- resistant tumors. The combination of several in-tion, doxorubicin is mixed with this system, resulting dependent mechanisms of drug resistance mightin the spontaneous incorporation of the drug into complicate chemotherapy and reinforces the need formicelles. The formulation is safe following systemic development of novel drugs and drug formulationsadministration based on acute and subacute toxicity effective against drug resistant cancers. It has longstudies in animals [69]. An open labeled, phase I been suggested that a broadly successful strategy fordose escalation and pharmacokinetics clinical trial of killing drug-resistant cancer cells could be based onSP1049C has been recently completed under the selective energy depletion in these cells, since manysponsorship of the UK Cancer Research Campaign in mechanisms of drug resistance are energy-dependenttwo sites; Christie Hospital in Manchester (UK) and [26]. Therefore, the recognition of energy-depleting

Queen Elisabeth Hospital, Birmingham (UK) [25]. effects of Pluronic block copolymers, in combina-The primary goal of this trial was to determine the tion with their very high sensitization effects andmaximal tolerable dose (MTD), toxicity and the their ability to inhibit multiple mechanisms of drugpharmacokinetics profile of SP1049C after intraven- resistance in MDR cells, is of considerable theoret-ous administration. A total of 26 patients entered the ical and practical significance. Both the mechanistictrial. The dose limiting toxicity of myelosuppression studies and clinical evaluations of these systems are

2was observed at MTD 90 mg/m . Other toxicities in progress and we expect fascinating new develop-observed were reversible: alopecia, nausea, and ments in this area from these on-going studies in thelethargy. It was established that a dose level of 70 near future.


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pluronic® block copolymers for overcoming drug resistance in cancer

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