Advanced Drug Delivery Reviews 54 (2002) 759779

www.elsevier.com / locate / drugdeliv

Pluronic block copolymers for overcoming drug resistance in

cancer
Alexander V. Kabanov a , *, Elena V. Batrakova a , Valery Yu. Alakhov b
a

Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center,
Omaha, NE 68198, USA
b
Supratek Pharma Inc., 531 Blvd. des Prairies, Build. 18, Laval, Quebec H7 B 1 B7, 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 of
low molecular mass drugs and polypeptides. This review describes novel applications of Pluronic block copolymers in the
treatment of drug-resistant tumors. It has been discovered that Pluronic block copolymers interact with multidrug-resistant
cancer (MDR) tumors resulting in drastic sensitization of these tumors with respect to various anticancer agents, particularly,
anthracycline antibiotics. Furthermore, Pluronic affects several distinct drug resistance mechanisms including inhibition of
drug efflux transporters, abolishing drug sequestration in acidic vesicles as well as inhibiting the glutathione / glutathione
S-transferase detoxification system. All these mechanisms of drug resistance are energy-dependent and therefore ATP
depletion induced by Pluronic block copolymers in MDR cells is considered as one potential reason for chemosensitization
of these cells. Following validation using in vitro and in vivo models, a formulation containing doxorubicin and Pluronic
mixture (L61 and F127), SP1049C, has been evaluated in phase I clinical trials. Further mechanistic studies and clinical
evaluations of these systems are in progress.
2002 Elsevier Science B.V. All rights reserved.
Keywords: Anthracyclines; Cancer; MDR; P-glycoprotein; Multidrug resistant protein; MRP; Block copolymer

Contents
1. Introduction ............................................................................................................................................................................
2. Mechanisms of resistance to antineoplastic agents .....................................................................................................................
2.1. Drug efflux proteins .........................................................................................................................................................
2.2. Drug sequestration in cytoplasmic vesicles.........................................................................................................................
2.3. Metabolic detoxification systems .......................................................................................................................................
2.4. Inhibition or prevention of drug-induced apoptosis .............................................................................................................
3. Structure and solution behavior of Pluronic block copolymers ..................................................................................................
4. Pluronic block copolymers sensitize drug-resistant cancers.......................................................................................................
5. Several drug resistance mechanisms are affected by Pluronic ........................................................................................
5.1. Inhibition of Pgp drug efflux system ..................................................................................................................................
*Corresponding author. Tel.: 11-402-559-9364; fax: 11-402-559-9543.
E-mail address: akabanov@unmc.edu (A.V. Kabanov).
0169-409X / 02 / $ see front matter 2002 Elsevier Science B.V. All rights reserved.
PII: S0169-409X( 02 )00047-9

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5.2. Effects on other drug transporters ......................................................................................................................................

5.3. Effects on drug sequestration within cytoplasmic vesicles ...................................................................................................
5.4. Effect on GSH / GST system .............................................................................................................................................
6. ATP depletion induced by Pluronic in MDR cells....................................................................................................................
7. Membrane fluidization and inhibition of Pgp ATPase activity by Pluronic ........................................................................
8. Interplay of ATP depletion and membrane fluidization as a possible mechanism for Pluronic activity in MDR cells .....................
9. Dose dependence of Pluronic effects in MDR cells: unimers vs. micelles ..................................................................................
10. Relationship between structure of Pluronic and its activity in MDR cells .................................................................................
11. Effects of Pluronic on cytotoxicity of various drugs in resistant cells.......................................................................................
12. Clinical trials of doxorubicinPluronic formulation (SP1049C) ..............................................................................................
13. Conclusion............................................................................................................................................................................
Acknowledgements ......................................................................................................................................................................
References ..................................................................................................................................................................................

1. Introduction
Chemotherapy remains the primary treatment option for cancer. Unfortunately, the efficacy of chemotherapy treatment in many types of cancers is
severely limited by drug resistance [1,2]. As a result
of inherent drug resistance, the response rate following treatment remains very low for many malignancies, including acute leukemias, malignant
melanomas, metastatic forms of breast, prostatic and
other cancers [13]. Additionally, prior treatment
with anticancer agents is an adverse prognostic
factor, presumably as a result of acquired drug
resistance, resulting in low long-term survival rates
for patients with relapsed and refractory tumors
[3,4]. Intensive laboratory and clinical studies aimed
at overcoming drug resistance in cancer have, so far,
produced only limited success with some novel
therapeutic regimens and antineoplastic agents [5,6].
Still, in many cases, no remedy has been found to
overcome these drug resistance mechanisms to improve clinical outcome in resistant cancers [7,8].
One problem in treating drug resistance in cancer,
which makes it a formidable task to tackle, is that
there are many mechanisms through which the
resistance is exhibited. In some cases several mechanisms act simultaneously and / or in concert, which
may further complicate therapy. For example, tumors
with the multiple drug resistance (MDR) phenotype
have been widely recognized as one of the most
difficult types to treat. MDR cells overexpress efflux
transporters belonging to a superfamily of ATP
binding cassette (ABC) proteins, such as P-glycoprotein (Pgp) and multidrug resistance-associated proteins (MRP) that pump drugs out of a cell [9,10].

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The glutathione / glutathione S-transferase detoxification system is frequently activated in MDR cells
contributing to drug resistance [11,12]. MRP acts in
concert with this system, providing for the efflux of
glutathione conjugates of xenobiotics from the cells
[11]. Another impediment to treatment, which is
present in MDR cells, involves the sequestration of
drugs within cytoplasmic vesicles, followed by their
extrusion out of the cell [13]. Drug sequestration in
MDR cells is achieved through the maintenance of
abnormally elevated pH gradients across organelle
membranesby the activity of H 1 -ATPase, an ATPdependent pump [14].
While a number of experimental and clinical
approaches have been studied to overcome MDR,
including the use of MDR chemosensitizers, the
appearance of several distinct transporters in resistant
cells may limit the success of those agents, which
target a single drug efflux pump. Furthermore, the
combination of several independent mechanisms of
drug resistance might complicate chemotherapy and
reinforces the need for development of novel drugs
and drug formulations effective against drug resistant
cancers.
One novel approach using polymers to overcome
MDR, reported by Kopeceks group, consists of
conjugating drug to a soluble polymer carrier
[15,16]. A conjugate of doxorubicin with copolymer
of N-(2-hydroxypropyl)methacrylamide (pHPMA)
has been shown to be effective against both MDR
and non-MDR cancers [17]. Furthermore, in contrast
to the effects of free drug, chronic exposure to
pHPMAdoxorubicin conjugates did not induce
MDR in cancer cells [18,19]. The reason for the
differential behavior of the pHPMAdoxorubicin

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

appears to be due to differences in the mechanism of

its transport into the cell compared to that of the free
drug. As a result of the conjugation to the polymer
chain, doxorubicin is rendered inaccessible to the
Pgp efflux pump in MDR. The resulting conjugate
most likely enters the cells via endocytosis, a Pgpindependent pathway, while the free drug is transported across the membrane through diffusion, a
pathway that in MDR cells is affected by Pgp [20].
A recent study by Minko et al. [21] suggested that
HPMA-bound doxorubicin induced additional caspase-dependent apoptosis signaling pathways, which
led to more pronounced apoptosis when compared to
the free drug. Therefore, the polymerdrug conjugate
not only enhanced drug delivery in MDR cells but
also acted as a biological response modifier that
potentiates the drug cytotoxic effect in a cell.
Another approach that has recently attracted increasing attention uses poly(ethylene oxide)poly(propylene oxide) block copolymers (Pluronic ) in
formulations to treat drug-resistant cancers [2224].
Following validation using in vitro and in vivo
models, a formulation that contains doxorubicin and
Pluronic mixture (L61 and F127),1 SP1049C, has
been evaluated in clinical trials [25].
Experimental studies have demonstrated that
Pluronic block copolymers sensitize MDR cells,
resulting in an increase in the cytotoxic activity of
anthracyclines and other cytotoxic drugs by 2 to 3
orders of magnitude [23,24]. Remarkably Pluronic
affects several distinct drug resistance mechanisms
including inhibiting of drug efflux transporters, abolishing drug sequestration and inhibiting the glutathione (GSH) / glutathione S-transferase (GST) detoxification system. Furthermore, recent studies demonstrated that Pluronic block copolymers induce a
dramatic reduction in ATP levels selectively in MDR
cells, while non-MDR cells are not responsive to this
block copolymer in this manner. It has long been
suggested that a broadly successful strategy for
killing drug-resistant cancer cells could be based on
selective energy depletion in these cells, since many
mechanisms of drug resistance are energy-dependent
[26]. However, no other agent was known so far that

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induces such a significant decrease in ATP levels,

observed selectively in MDR-expressing cells, as
Pluronic block copolymers do. Therefore, the finding of energy-depleting effects of Pluronic block
copolymers, in combination with their very high
sensitization effects and ability to inhibit multiple
mechanisms of drug resistance in MDR cells, is of
considerable theoretical and practical significance.
This review describes the effects of Pluronic block
copolymers in drug-resistant cancer and discusses the
latest knowledge regarding fundamental mechanisms
of their action as well as clinical aspects related to
the use of this technology in cancer chemotherapy.

2. Mechanisms of resistance to antineoplastic

agents
Various mechanisms of drug resistance have been
described for all major classes of antineoplastic
agents (Table 1). Historically, investigators examined alterations in the targets of drug action as the
most likely mechanisms involved in drug resistance
[27]. The well-known examples include doxorubicin,
which targets topoisomerase II [28] or methotrexate,
which inhibits dihydrofolate reductase [29,30]. However, more recently, it became evident that distinct
cellular mechanisms, which often occur simultaneously in the same cell, may take part in resistance
of cancer cells to a broad range of drugs. One group
of such mechanisms involves reduction of effective
concentration of the drug prior to its interaction with
the target [1]. This is achieved as a result of one or a
combination of the following processes: (i) drug
efflux out of the cells; (ii) drug sequestration in
vesicles inside the cell; and (iii) drug elimination by
detoxification systems. In addition to the mechanisms reducing drug concentration in the cell, there
appears to be an important group of mechanisms that
decrease drug-induced damage to the cell after drug
interaction with the target. The mechanisms for
reduction of drug concentration and mechanisms for
reduced damage are considered below.

2.1. Drug efflux proteins

1

Here and below we omit the term Pluronic, using the letter and
numeric code to define the corresponding block copolymers, for
example, L61 for Pluronic L61.

Resistance to anticancer drugs is often mediated

by the overexpression of membrane pumps belong-

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

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Table 1
Drug resistance mechanisms described for antineoplastic agents (based on Refs. [111,2636,43,44,50,114120])a
Drug classes and examples

Mechanism of
cytotoxicity

Molecules implicated in
resistance mechanism

Intercalators
Alkylators

Doxorubicin, daunomycin
Cyclophosphamide
Cisplatin

Topo II inhibitor, SO
DNA alkylation
DNA alkylation

BCNU
Methotrexate

DNA alkylation
Folic acid antagonist

5-Fluorouracil
Vinblastine,
vincristine
Etoposide
Paclitaxel

Uracil analog
Tubulin
polymerization inhibitors
Topo II inhibitor
Inhibitor of microtubule assembly

Pgp; MRP; GST; Topo II

GSH; ALDH; AGT (?)b
GSH; MT; DNA repair
enzymes; cMOAT (?)
AGT
Amplification of DHFR; MRP (?);
decreased RFC expression
Amplification of TS
Pgp; MRP; tubulin
mutation
MRP; GSH; Pgp; Topo I
Pgp, altered a / b tubulin

Antimetabolites

Vinca alkaloids
Epidophylotoxins
Taxanes

a
AGT, O 6 -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

to extrude many xenobiotics out of tumor cells
[9,10]. Examples of ATP-dependent efflux pumps,
which are overexpressed in resistant tumors, include
P-glycoprotein (Pgp) [31], multidrug resistance-associated protein (MRP or MRP1), canalicular multispecific organic anion transporter cMOAT (MRP2)
and several other MRP-like proteins [32,33]. Pgp has
a broad specificity to a range of structurally diverse
drugs including anthracyclines (doxorubicin,
daunorubicin),
epipodophyllotoxins
(etoposide,
teniposide), vinca alkaloids (vinblastine, vincristine),
paclitaxel, and other compounds [34]. MRP acts as a
transporter of organic anions, particularly, of glutathione conjugates, and apparently works in concert
with the glutathione detoxification system [35]. As a
result, the action of MRP as a drug transporter
depends on intracellular levels of glutathione [11,36].

2.2. Drug sequestration in cytoplasmic vesicles

Many studies on MDR cells show ATP-dependent
sequestration of the drugs inside cytoplasmic vesicles
followed by extrusion of the drug from the cell
[3740]. Retention of drug molecules in the vesicles
can decrease the amount of drug that actually reaches
the site of action and thus, might contribute to drug
resistance [13]. This phenomenon appears to be
associated with abnormally elevated pH in the

organelles in MDR cells and is characteristic of the

weak base drugs (e.g. doxorubicin) that are positively
charged at acidic pH [13,27]. A major protein
responsible for generating pH gradients across organelle membranes is a vacuolar H 1 -ATPase [41].
Inhibitors of the H 1 -ATPase abolish sequestration of
the drug in the vesicles and reverse drug resistance in
MDR cells [14,39]. Furthermore, involvement of the
vesicles in drug extrusion from the cells has been
proposed [14,37]. These studies demonstrated the
relationship between vesicular sequestration and drug
resistance conferred by the overexpression of MRP
in MDR cells [14,37,42].

2.3. Metabolic detoxification systems

Several cellular thiols (GSH, thioredoxin, metallothioneins) are believed to contribute to resistance
to many antineoplastic drugs (Table 1). The GSH /
GST-related metabolic pathway is, probably, the best
studied [4345]. Conjugation of electrophilic molecules with the reduced glutathione produces species
that are usually less toxic and more hydrophilic than
the original compounds and can be partially metabolized and excreted [12,46]. In cells with acquired
resistance to antineoplastic agents, both GSH content
and GST activity are frequently elevated, which
apparently results in protection of the cells from
these agents [47].

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

2.4. Inhibition or prevention of drug-induced

apoptosis
DNA damage and apoptosis are the main causes of
drug-induced cytotoxicity [48,49]. Enhanced DNA
repair by O 6 -alkylguanine-DNA alkyltransferase has
been known as a common mechanism of resistance
to drugs [50,51]. More recently, alteration in deathinducing signaling pathways, resulting in inhibition
or prevention of apoptosis, has been suggested as
another mechanism of chemoresistance [52,53].
Overexpression of anti-apoptotic genes (e.g. Bcl-2),
as well as mutation or reduced expression of proapoptotic genes (e.g. p53), has been shown to render
cancer cells resistant to drug effects [52,53]. Contribution of Pgp to protection of tumors from apoptosis
has recently been discussed in view of observations
that inhibition of Pgp by chemosensitizing agents can
restore the normal apoptotic cascade in cells with
defective signaling pathways [5457].
Overall, there are multiple, complex and, possibly,
interrelated mechanisms of drug resistance. These
mechanisms are often found simultaneously, which
can further complicate the chemotherapy of tumors.
3. Structure and solution behavior of Pluronic
block copolymers
Pluronic block copolymers (also known under
their non-proprietary name poloxamers) consist of

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hydrophilic ethylene oxide (EO) and hydrophobic

propylene oxide (PO) blocks arranged in a basic
ABA structure: EO n / 2 PO m EO n / 2 . The structure
formula of Pluronic block copolymers is shown in
Fig. 1. Copolymers with various numbers of hydrophilic EO (n) and hydrophobic PO (m) units are
characterized by a distinct hydrophiliclipophilic
balance (HLB). Pluronic block copolymers are
synthesized using step-wise anionic polymerization
by sequential addition of PO and EO monomers in
the presence of an alkaline catalyst, such as sodium
or potassium hydroxide [58]. The reaction is initiated
by polymerization of the PO block followed by the
growth of EO chains at both ends of the PO block. It
usually produces polymers with a relatively low
polydispersity index (Mn /Mw ).

The solubility of Pluronic block copolymers in

water depends on their structure, namely, the lengths
of the hydrophobic and hydrophilic blocks, as well
as the temperature. Elevation of the temperature
promotes dehydration of alkylene oxide units of the
block copolymer decreasing the solubilityfirstly, of
the PO block and secondly, of the EO block. At the
body temperature, 37 8C, PO chains are water-insoluble, while EO chains are still well-hydrated and
water-soluble. Molecular dispersions form at the
block copolymer concentrations below the critical
micelle concentration (CMC). Above the CMC the
individual block copolymer molecules, termed unimers, self-assemble into micelles through a process
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 PO
block.

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micellization is the hydrophobic interactions of the

PO blocks. As a result, the PO blocks self-assemble
into the inner core of the micelles covered by the
hydrophilic corona from EO blocks. Pluronic micelles are commonly pictured as spheres composed
of a PO core and an EO corona. This portrayal is
correct for most block copolymers, which have an
EO content above 30%, especially in relatively dilute
solutions at body temperature. However, additional
micelle morphologies, including lamella and rods,
can also form in Pluronic systems [59]. When
spherical micelles are formed, depending on the
Pluronic type, the micelles commonly have an
average hydrodynamic diameter ranging from about
20 to about 80 nm [59].
The coreshell architecture of Pluronic micelles
is essential for their utility in drug delivery applications [60]. The core formed by the PO chains is a
water-incompatible compartment that is segregated
from the aqueous exterior by the hydrophilic chains
of the EO corona, thereby forming, within the core, a
cargo hold for the incorporation of various therapeutic reagents. The process of transfer of waterinsoluble 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
for compounds, which alone exhibit poor solubility,
undesired pharmacokinetics and low stability in a
physiological environment. The hydrophilic shell
contributes greatly to the pharmaceutical behavior of
Pluronic formulations by maintaining the micelles
in a dispersed state, as well as by decreasing
undesirable drug interactions with cells and proteins
through steric-stabilization effects.
CMC is of paramount significance to drug delivery
using Pluronic block copolymers [60,61]. Firstly,
the CMC determines the stability of micelles against
possible dilution of the solution in body fluids
[60,62]. Secondly, the CMC determines the maximal
achievable concentration of Pluronic unimers, to
which cells will be exposed, thereby defining the
biological effects, which Pluronic itself, will exert
on these cells [63]. Typically, Pluronic block
copolymers, which are used for drug delivery, at
body temperature (37 8C) have a CMC ranging from
1 mM to 1 mM (ca. 5310 23 to 1 wt.%). The CMC
depends on the lengths of the PO and EO blocks
[64,65]. An increase in the length of the PO block

Fig. 2. Effects of molecular composition of Pluronic block

copolymer on CMC. CMCs were determined at 37 8C using the
pyrene solubilization technique and plotted as a function of the
length of the PO block, NPO . From Ref. [63] (with permission).

elevates the net hydrophobicity of the Pluronic

molecule and favors the formation of the micelle
core, which results in a CMC decrease [59,66,67].
Conversely, an increase in the lengths of the EO
blocks decreases the hydrophobicity and destabilizes
the micelle, resulting in the CMC increase [64,65].
Fig. 2 presents the dependence of the log CMC of
Pluronic block copolymers on the length of the PO
block (the effect of EO block length is usually less
drastic than the effect of the PO block length).
4. Pluronic block copolymers sensitize drugresistant cancers
The use of Pluronic block copolymers to treat
drug-resistant cancers is a rapidly developing area of
drug delivery for cancer chemotherapy. Studies by
Alakhov et al. demonstrated that Pluronic block
copolymers sensitize resistant cancer cell lines, resulting in an increase in the cytotoxic activity of the
drug by 2 to 3 orders of magnitude [23,24,63,68]. By
addition of P85 or L61, the cytotoxic effects of

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

Fig. 3. Cytotoxicity of free daunorubicin (1, 2) and daunorubicin

with 1 wt.% P85 (3, 4) with respect to resistant SKVLB (1, 4) and
sensitive SKOV3 (2, 3) cells. After exposure for 120 min to the
drug or drug Pluronic , cells were cultured in drug-free media for
4 days and cytotoxicity was determined using a tetrazolium-based
assay. Based on Ref. [23] (with permission).

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Pluronic block copolymer composition. A tumor

panel included intraperitoneal murine leukemias
(P388, P388-Dox), subcutaneous murine myelomas
(Sp2 / 0, Sp2 / 0-Dnr), intravenous and subcutaneous
Lewis lung carcinoma (3LL-M27), subcutaneous
human breast carcinomas (MCF-7, MCF-7 /ADR),
and human head and neck carcinoma (KBv) [69]. By
the National Cancer Institute criteria for tumor
inhibition and increased lifespan, Pluronic -formulated doxorubicin has met the efficiency criteria in all
models (9 of 9), while doxorubicin alone was
effective only in selected tumors (2 of 9) [69]. The
results demonstrated that the tumors were more
responsive in the Pluronic copolymer treatment
group than in the control groups that received only
doxorubicin. Together these studies indicate improved treatment of drug-resistant cancers with
Pluronic block copolymers.

5. Several drug resistance mechanisms are

affected by Pluronic

5.1. Inhibition of Pgp drug efflux system

doxorubicin in the resistant lines significantly surpassed those observed in the sensitive lines. For example, Fig. 3 illustrates typical effects of P85 on the
cytotoxicity of the anthracycline drug, daunorubicin,
with respect to resistant and sensitive tumors. In this
experiment, human ovarian carcinoma cells (SKVLB
and SKOV3) were exposed for 120 min to free
daunorubicin or daunorubicin with P85. Cytotoxicity
studies in the sensitive cell line, SKOV3, showed
little difference between the control group and
groups exposed to P85. However, in the MDR cell
line, SKVLB, there was a significant enhancement in
cytotoxicity to daunorubicin in the presence of P85.
Effects of the block copolymers in the resistant cells
are expressed in the form of a resistance reversion
index (RRI), i.e. ratio of IC 50 of the drug in the
assay buffer and Pluronic solution (IC 50,0 / IC 50 ). In
the particular case presented in Fig. 3, the resistance
reversion index was ca. 700 [23].
Effects of Pluronic block copolymers in drugresistant tumors have also been reported in vivo
[22,69]. In these studies, mice bearing drug-sensitive
and drug-resistant tumors were treated with doxorubicin alone or doxorubicin formulated with

One major reason for the enhanced cytotoxicity

observed with Pluronic in drug-resistant cancer
cells appears to be related to the affects of the
copolymer on the Pgp drug efflux transport system.
Similar affects have been observed with other
nonionic surfactants such as Cremophor EL, Solutol
HS15, Triton X-100, Nonidet P-40 or polyoxyethylated derivatives of fatty acids that all exhibit
an ability to enhance the cytotoxicity of antineoplastic agents in MDR cells [7078]. There is overwhelming evidence to support inhibition of Pgp by
Pluronic block copolymers. This is supported by
the observation that the intracellular accumulation of
doxorubicin in resistant cancer cells expressing Pgp
can be greatly enhanced by treatment with Pluronic
[24,63]. No alteration in drug uptake in the presence
of Pluronic was observed with non-Pgp-expressing
cancer cells, i.e. the copolymers affect specifically
the Pgp-controlled transport route in MDR cells
[24,63]. This conclusion has been reinforced by the
recent studies by Evers et al. [79] and Batrakova et
al. [80] demonstrating that Pluronic block copolymers (L61, P85) profoundly increase accumulation

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and permeability of various Pgp-dependent drugs in

mdr1-transfected cells, which overexpress Pgp. At
the same time, these copolymers have little on no
effect on drug transport in non-transfected cells,
which display low endogenous Pgp expression.
Therefore, enhanced transport of Pgp substrates,
induced by exposure of the cells to Pluronic ,
correlates with the level of Pgp expression in the
cells, suggesting that the effects are exerted through
a Pgp-dependent route. Furthermore, the block copolymer has no or little affect on the accumulation of
non-Pgp-dependent compounds in both resistant and
sensitive cells [8082]. Therefore, the increased
absorption of the Pgp substrates in Pgp-expressing
cells is attributable to the effects of the block
copolymer on the Pgp efflux system, rather than to
non-specific alterations in membrane permeability of
the substrates. Finally, since the efflux of Pgp-dependent drugs was decreased in the presence of
Pluronic block copolymers, the mechanism for the
increased drug accumulation observed with these
block copolymers appears to be related to inhibition
of the drug efflux system [24,82].

5.2. Effects on other drug transporters

There is increasing evidence suggesting that the
effects of Pluronic block copolymers might stem
beyond inhibition of the Pgp efflux pump alone.
Studies by Miller et al. [83], using the human
pancreatic adenocarcinoma cell line, Panc-1, that
expresses the MRP1 efflux pump, suggested that P85
inhibits efflux and increases cellular accumulation of
the MRP-dependent probe, fluorescein, in these cells.
Similar to the case of Pgp inhibition discussed
above, the effects of Pluronic in Panc-1 cells
appeared to be specific with respect to fluorescein
transport system(s), since no accumulation increases
were observed for an organic cation, rhodamine 123,
a substrate of Pgp that is not expressed in these cells.
However, more recently it was concluded that, in
addition to MRP1, some other organic anion transporters, including MRP2, might also be present in
Panc-1 (D.W. Miller, personal communication). Thus,
Pluronic could possibly inhibit these transporters as
well, resulting in enhancement of fluorescein accumulation in Panc-1 cells. Therefore, although the
affect of Pluronic on some organic anion transport

system, distinct from Pgp, was evident based on this

study, the support of inhibition of a specific MRP
transport system(s) remained inconclusive.
To address Pluronic effects on MRP, a recent
study by Evers et al. [79] has examined drug
transport in canine kidney cells, MDCK II that had
been transfected to stably express either MRP1 or
MRP2. The results of this study suggested that L61
partially inhibited MRP2-mediated transport of vinblastine in the MRP2-expressing cell line. However,
the potency of this block copolymer, with respect to
inhibition of MRP2, in these cells appeared to be
lower than its potency with respect to Pgp in mdr1transfected cells that express Pgp. Furthermore, these
authors concluded that L61 had little if any influence
on MRP1- and MRP2-mediated transport of another
MRP-substrate, dinitrophenol S-glutathione [79]. The
dinitrophenol S-glutathione experiments, however,
are difficult to interpret because the total lengths of
exposure of the cells to the block copolymer in this
study were relatively short (30 min or less). This
might be insufficient in view of the previous report
that inhibition of the drug efflux system in MRPexpressing cells requires an exposure of at least 30 to
60 min to the block copolymer prior to adding the
substrate to the cells [83].
These studies of the effects of Pluronic on MRP
are complicated by the possible interference of
effects on both Pgp and MRP, commonly co-expressed in cells, and by the lack of selective MRP
substrates that would allow the exclusion of nonMRP drug transport mechanisms that can be affected
by the block copolymer. To address this problem we
have recently examined effects of P85 on transport
of several substrates in cells expressing MRP (manuscript in preparation). In addition to canine kidney
cells, MDCKIIMDR1 and MDCKIIMDR2, the
same as in the paper by Evers et al. [79], this study
used human lung carcinoma epithelial cell line,
COR-L23 / R, selected with doxorubicin, which overexpresses MRP1 but has little if any Pgp. Using
several substrates, which are either selective to
organic anion transporters, such as fluorescein and
29,79-bis-(2-carboxyethyl)-5-carboxyfluorescein acetoxymethylester, or are substrates of both Pgp and
MRP, such as vinblastine and doxorubicin, this study
suggests that P85 is a potent inhibitor of both MRP1
and MRP2 in these cells. In all cases, pre-exposure
of the cells to P85 was necessary to fully exhibit the

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

effects of this block copolymer on the MRP transport

systems, which may explain the discrepancy of the
results with the prior study by Evers et al. [79].
Thus, although the evaluation of the effects of
Pluronic on MRP is far from complete, there is
substantial evidence to suggest that these block
copolymers affect multiple drug transport systems in
MDR cells.

5.3. Effects on drug sequestration within

cytoplasmic vesicles
In MDR cells, drug can be sequestrated within
cytoplasmic vesicles and then extruded from the cell
before the drug can exert its intended action on the
cell, which is another potential impediment to the
treatment of resistant tumors [13,3740]. Drug sequestration in MDR cells is achieved through the
maintenance of abnormally elevated pH gradients
across organelle membranes through the activity of
H 1 -ATPase, an ATP-dependent pump [14]. Recent
studies by Venne et al. [24] examined effects of
Pluronic block copolymers on intracellular localization of doxorubicin in the MDR cancer cell line,
MCF-7 /ADR. In these cells, free doxorubicin is
sequestered in cytoplasmic vesicles, which might
further diminish the amount of the drug available for
interaction with the nucleus [13]. Following incubation of these cells with doxorubicin and Pluronic ,
drug was released from the vesicles and accumulated, primarily, in the nucleus [24]. Furthermore, we
have observed vesicular sequestration of fluorescein
in Panc-1 cells overexpressing MRP [83]. This
sequestration was also abolished and fluorescein was
released in the cytoplasm by treating the cells with
Pluronic .

5.4. Effect on GSH /GST system

The activity of MRP drug efflux transporter with
respect to many substrates is closely tied to the
GSH / GST detoxification system in MDR cells [13].
Therefore, studies of the effects of Pluronic block
copolymers on GSH / GST system have begun. For
example, significant decreases in both intracellular
levels of GSH and GST activity were observed in
MDCK cells expressing MRP, following exposure of
these cells to P85 (manuscript in preparation). GST
activity, as assessed using a colorimetric substrate

767

1-chloro-2,4-dinitrobenzene, was significantly inhibited immediately following 2-h exposure of the

MDCKII, MDCKIIMRP1 and MDCKIIMRP2
cells to P85. The levels of GSH did not decrease
during 2-h exposure of these cells to P85. However,
the GSH pools in these cells were significantly
depleted when the cells were first exposed to
Pluronic and then incubated in Pluronic -free
media during 22 h. Inhibition of the GST / GSH
detoxification system should result in the decrease of
glutathione conjugation of select substrates, such as
doxorubicin, and additionally decrease the extent of
elimination of these substrates from the cells through
the MRP-mediated drug efflux pathway. The complex and interrelated mechanisms of drug elimination
through MRP and GST / GSH detoxification systems
result in different responses of these systems to
Pluronic when different substrates are used. Firstly,
the organic anion substrates of MRP that do not
require glutathione conjugation, such as fluorescein
[84,85], usually respond to Pluronic in the same
way as the Pgp substrates do, resulting in decreased
efflux and increased substrate accumulation in the
cells [83]. Secondly, the compounds that become
MRP substrates only after glutathione conjugation,
such as doxorubicin [86,87], can exhibit a complex
dependence on GSH levels, as well as MRP and
GST activities. Finally, compounds that are co-transported with GSH without glutathione conjugation,
through the MRP route, such as vinblastine [35,88],
can depend on GSH levels as well as MRP activity
but not exhibit it. The effects of Pluronic block
copolymers on the efflux and accumulation of such
compounds in the resistant cells can be amplified
through affects on multiple components of the
detoxification system present in these cells, providing that these component(s) are critical (rate
limiting) for the elimination of the substrate. Overall,
these studies suggest that Pluronic block copolymers affect several important mechanisms of drug
resistance in cancer cells.
6. ATP depletion induced by Pluronic in MDR
cells
Various drug resistance mechanisms, including
drug transport and detoxification systems, require
consumption of energy to sustain their function in

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

MDR cells. Therefore, mechanistic studies have

focused on the effects of Pluronic block copolymers on metabolism and energy conservation in
drug-resistant cells [89,90]. Slepnev et al. [91] were
first to demonstrate that intracellular levels of ATP
were depleted following a 2-h exposure of Jurkat
T-cell lymphoma cells to the Pluronic block copolymer, P85. The following key observation, in the
context of the drug resistance phenomena, was the
study by Batrakova et al. [89,90] that compared the
effects of the P85 on ATP levels in several cell types
that either exhibit MDR phenotype, or do not exhibit
MDR phenotype. In this study, exposure of MDR
and non-MDR cells to different doses of P85 resulted
in a transient energy depletion, which was reversed
following removal of the block copolymer. The
MDR cells were much more responsive to P85,
exhibiting a profound decrease in ATP levels at
substantially lower concentrations of the block copolymer, compared to the non-MDR cells. The
effective concentrations of P85 that induced a 50%
decrease in ATP levels in the cells (EC 50 ), as
determined from the doseresponse curves, are
presented in Table 2. This table also presents the
relative responsiveness of the MDR cells compared
to the non-MDR counterparts. These data suggest

that the responsiveness of the cells to P85 correlated

with appearance of MDR in these cells, rather than
to the amount of ATP available in the cells prior to
treatment. Based on the results of this study, the
presence of the MDR phenotype is one factor that
renders cellular metabolism responsive to treatment
with Pluronics .
The mitochondria are responsible for carrying out
much of the metabolic activities of the cell, and
might be a potential site of action for Pluronic
block copolymers. It has long been known that
nonionic polymeric detergents, such as Tween 80
and Pluronic , can decrease oxidative metabolism of
tissues, cells and isolated mitochondria [9294].
Rapoport et al. [94] has shown that two Pluronic
copolymers, P85 and P105, reduce the activity of the
electron transport chains in mitochondria, as assessed
by the rates of bioreduction of lipophilic spin-probes
in HL-60 cancer cells. A recent confocal microscopy
study using P85 that was labeled with a fluorescent
tag to examine its transport and localization inside
cells suggested that within a 15 to 60-min interval,
the block copolymer molecules spread throughout
the cell, where they may interact with various
intracellular organelles, including mitochondria [95].
There could be multiple reasons for the inhibitory

Table 2
Effects of P85 on ATP levels in MDR and non-MDR cells a
Cells

MDR1
phenotype

Initial ATP levels

nmol / mg protein b

EC 50 , %

Relative responsiveness
to P85 c

MCF-7
MCF-7 /ADR
KB
KBv
C2C12
HUVEC
Caco-2
BBMEC
LLC-PK1
LLC-MDR1

No
Yes
No
Yes
No
No
Yes
Yes
No
No

3061.5
300620
160.01
460.1
1561.4
4064.9
5.560.4
1.660.04
6166.9
7961.7

2.25
0.009
0.675
0.036
4.5
0.0675
0.00067
0.018
0.45
0.0045

250 d

19 e

6670 f
250 f

100 g

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

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

activity of these compounds in mitochondria. The

likely components contributing to the anti-metabolic
effects of nonionic detergents include their ability to
serve as K 1 ionophores [9698] and their ability to
uncouple oxidative phosphorylation [99,100]. It is
also possible that these compounds directly inhibit
the NADH dehydrogenase complex by interacting
with the hydrophobic sites of this complex that are
located in the mitochondrial membrane [93,99].
However, the reasons for a remarkable selectivity
of Pluronic block copolymers with respect to MDR
cells are not completely understood. One hypothesis
is that the high rates of energy consumption by the
drug efflux pumps, combined with Pluronic -induced inhibition of respiration, determine the responsiveness of the resistant cells to the block copolymer.
Under conditions in which the respiration necessary
for ATP synthesis is inhibited, the high rates of
ATPase activity by the drug efflux pumps (and
possibly, some other energy-dependent mechanisms)
could result in a rapid exhaustion of the intracellular
ATP pools in the resistant cells. Alternatively, cells,
which do not exhibit these resistance mechanisms,
would appear to be less responsive to inhibition of
respiration and would not exhibit energy depletion,
at least to the extent observed in the resistant cells.
Such a hypothesis is in line with the earlier observation that resistant cells have an increased glucose
utilization rate compared to sensitive cells [101,102].
Furthermore, the toxicity of the inhibitor of glycolysis, 2-deoxyglucose was found to be consistently
higher in MDR cells than in the non-MDR lines
[102]. According to Batrakova et al. [90] the extent
of the selectivity of P85 with respect to MDR cells is
the same or even greater than that of 2-deoxyglucose. Furthermore, P85 was significantly more selective than the inhibitors of respiration, rotenone and
sodium azide.
Most recently, evidence has begun to mount
suggesting that there might be an alternative explanation for the high selectivity of the block copolymers
with respect to MDR cells. A study, from our
laboratory, suggested that P85 significantly inhibits
ATPase activity of Pgp in isolated cell membranes
[90]. Since Pgp is one of the major ATPases
overexpressed in MDR cells, and the fact that
Pluronic inhibits this activity, it makes the high
energy consumption in MDR cells a less likely cause

769

for the block copolymer selectivity in these cells. An

alternative hypothesis would be that, for some
reason, the metabolic processes in MDR cells are
more sensitive to inhibition with Pluronic than the
metabolic processes in non-MDR cells. This could
result in the more pronounced ATP depletion observed following exposure of MDR cells to the block
copolymer. Furthermore, it has to be emphasized that
in addition to overexpression of Pgp some other
factors implicated in the appearance of the MDR
phenotype, such as other efflux proteins, high levels
of H 1 -ATPase and activated GSH / GST detoxification system, could also impose high energy requirements in MDR cells and collectively contribute to
the elevated responsiveness of these cells to
Pluronic . The high responsiveness to P85 of the
cell lines, which have relatively low levels of Pgp
expression, such as HUVEC (Table 2) may be one
indication of the possible involvement of alternative
mechanisms in the energy depletion [90].
The energy depletion phenomenon is obviously
extremely important in order to understand the
reasons for the elevated anticancer activity of
Pluronic -based formulations in drug-resistant
tumors. Transient energy depletion, as a result of
exposure of the cells to Pluronic in the absence of
the chemotherapeutic agent, does not induce a cytotoxic effect in either MDR or sensitive cells [89,90].
However, if the chemotherapeutic agent, doxorubicin, is present, then the exposure of MDR cells to the
combination of both drug and block copolymer
results in a pronounced potentiation of the cytotoxic
activity, i.e. the sensitization effect [89,90]. The
apparent relationship between energy depletion and
enhanced cytotoxicity in cancer cells is supported by
the literature. For example, metabolic modulators
alone, such as 2-deoxyglucose, iodo-acetic acid,
fluoro-acetic acid, oligomycine, azide, antimycin and
rotenone, which can inhibit energy conservation at
various levels (i.e. glucose uptake, glycolysis, citric
acid cycle, oxidative phosphorylation), have been
shown to induce apoptotic cell death in cancer cells
[103]. Furthermore, a three-drug combination, PMA
((N-phosphonacetyl)-aspartate,
6-methylmercaptopurine, 6-aminonicotinamide), which contains inhibitors of pyrimidine and purine pools, can potentiate drug activity and enhance drug-induced apoptosis in cancer tumors [104].

770

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

7. Membrane fluidization and inhibition of Pgp

ATPase activity by Pluronic
Pluronic block copolymers are known to induce
drastic changes in the microviscosity of cell membranes, as assessed using the hydrophobic membrane
probe, 1,6-diphenyl-1,3,5-hexatriene (DPH) [105].
These changes can be attributed to the alterations in
the structure of the lipid bilayers as a result of
adsorption of the block copolymer molecules on the
membranes. In particular, recent studies using DPH
as a probe, have demonstrated that exposure of cells
to Pluronic block copolymers, such as P85, results
in fluidization of the cellular membranes of both
normal and cancerous cells expressing high levels of
Pgp [95]. Membrane fluidization by various agents,
including nonionic surfactants such as Tween 20,
Nonidet P-40 and Triton X-100, is known to contribute to inhibition of Pgp efflux function [106].
Furthermore, it is believed that inhibition of Pgp
ATPase activity, presumably, through nonspecific
changes in lipid and protein conformation and
mobility, has a major contribution to the inhibition of
Pgp efflux function [106]. A recent study evaluated
the effects of Pluronic block copolymer, P85, on
the Pgp ATPase activity using membranes containing
human Pgp [95]. In this work the membranes were
exposed to P85 and then the ATPase activity of Pgp
was assayed by determining the liberated inorganic
phosphate [107]. The study also evaluated a Pgp
substrate, verapamil, to determine whether binding of
a specific substrate with Pgp could modulate the
effects of P85 on Pgp ATPase activity. P85 at
concentrations as low as 0.001% induced dramatic
decreases in Pgp ATPase activity compared to the
copolymer-free control. Verapamil alone in the absence of the block copolymer induced a significant
increase in Pgp ATPase activity. This effect is
believed to be due to the binding of verapamil in the
active center of Pgp [108,109]. However, the inhibitory effect of P85 was observed in the presence of
verapamil at all block copolymer concentrations
examined (0.001% to 1%). Overall, this study suggested that P85 is a potent inhibitor of the Pgp
ATPase activity. A similar study was recently conducted using membranes isolated from MRP1 overexpressing lung carcinoma cell line, COR-L23 / R
(paper in preparation). P85 inhibited the ATPase

activity in these cells as well, providing initial

evidence that Pluronic block copolymers might
inhibit MRP ATPase.

8. Interplay of ATP depletion and membrane

fluidization as a possible mechanism for
Pluronic activity in MDR cells
As evidenced in the previous section, there could
be multiple reasons for inhibition of the Pgp efflux
system by P85 in MDR cells. These reasons first
include dramatic energy depletion induced by the
block copolymer, which might abolish the ATPdependent drug efflux. Another reason includes the
inhibition of the Pgp ATPase activity, which is
essential for the functioning of this transporter.
Therefore, it is likely that these Pluronic block
copolymers have a double-punch effect in MDR
cells: through ATP depletion and membrane fluidization, which both have a combined result of potent
inhibition of Pgp (Fig. 4). The most compelling
evidence to support the idea that the energy depletion
contributes to the sensitization of the MDR cells by
Pluronic was obtained in experiments using an
energy supplementation system [90,95]. These
studies were based on the observation that treatment
of cells with dodecylamine in combination with P85
allows transport of ATP into the cells from the
extracellular media [91]. The use of this system with
MDR cells allowed restoration of intracellular ATP
levels, which abolished inhibition of the Pgp efflux
system by P85 and substantially reduced the cytotoxic effect of doxorubicin formulated with P85 in these
cells [90,95]. Since the block copolymer in this
experiment was still present and, presumably, bound
with the cell membranes, the ATP supplementation
study indicates that membrane fluidization alone may
not be sufficient for inhibition of the Pgp efflux
system in MDR cells. On the other hand, the directionality studies and experiment involving P85 removal from MDR cells suggest that energy depletion
alone in the absence of interaction of the block
copolymer with the Pgp-containing membranes
might be insufficient to inhibit the efflux system.
Therefore, both factors are critical for exhibition of
the effect of P85 on the Pgp efflux system in MDR
cells.

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

771

Fig. 4. Schematic presenting multiple effects of Pluronic block copolymers displayed in MDR cell. These effects include (a) decrease in
membrane 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 not
sufficiently studied at present.

The interrelationship between the membrane

fluidization and energy depletion components of
Pluronic action can be better understood in view of
the current picture of Pgp structure describing Pgp as
a two-domain protein with ATP-binding sites in each
domain [110]. Proper interaction of these two ATPbinding sites is crucial for the proper functioning of
Pgp. It was suggested that binding of ATP in one
domain causes a conformational change in the Pgp
molecule necessary for the hydrolysis of ATP and
translocation of the substrate [111]. Therefore, the
structural perturbations in the lipid membranes induced by Pluronic may decrease the affinity of ATP
to its binding site and interfere with the ATPase

activity. This means that higher concentrations of

intracellular ATP would be required for normal
functioning of Pgp, i.e. the drug efflux system would
become more vulnerable to decreases in intracellular
ATP. Future studies of the kinetics of the Pgp
function in the presence of Pluronic are needed to
confirm this hypothesis.
9. Dose dependence of Pluronic effects in
MDR cells: unimers vs. micelles
Due to the ability of Pluronic block copolymers
to self-assemble into micelles and to solubilize

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

hydrophobic drugs, the dose dependencies observed

with these agents in MDR cells are drastically
different compared to the dose dependencies of many
other MDR chemosensitizers. The studies by Batrakova et al. [63] have demonstrated that both
inhibition of the Pgp efflux system by Pluronic and
potentiation of doxorubicin activity by Pluronic in
MDR cancer cells, occur at block copolymer concentrations below the CMC. This means that both
effects are due to Pluronic unimers. As a result, the
accumulation and cytotoxicity of drugs in MDR cells
increase with increasing concentrations of Pluronic
until the CMC is reached and unimer concentration
levels off. This behavior is illustrated in Fig. 5 using
the effects of the block copolymers on doxorubicin
cytotoxic activity in MDR cells as an example. At
concentrations above the CMC, drug accumulation
and cytotoxicity in MDR cells tend to first level off
and then decrease [63]. Decreases in drug absorption
in cells at block copolymer concentrations above the
CMC are similar to those reported previously for
other nonionic detergent MDR modulators, such as

Fig. 5. Effects of Pluronic block copolymers on cytotoxicity of

doxorubicin with respect to MDR cells: curve 1, MCF7 /ADR
cells were treated with doxorubicinL61 compositions; curve 2,
KBv cells were treated with doxorubicinP85 compositions
containing varying concentrations of the block copolymer. Resistance reversion indexes (ratio of IC 50 of doxorubicin in the assay
buffer and Pluronic solution) are plotted as functions of the
concentration of the block copolymers. The vertical arrows from
left to right show the CMC of P85 and L61. From Ref. [63] (with
permission).

Cremophor EL [112,113]. The inhibition of cell

transport in the presence of micelles is attributed to
incorporation of the drug into the micelles, resulting
in a decreased free drug available for diffusion
through the cell membrane into the cells.
10. Relationship between structure of Pluronic
and its activity in MDR cells
Batrakova et al. [63] reported that both the inhibition of the Pgp efflux system as well as the hypersensitization effects of Pluronic in MDR cells
depend on the molecular composition of the block
copolymer. This study examined a panel of over 20
block copolymers having different lengths of hydrophilic (EO) and hydrophobic (PO) segments using
MDR cancer cell line, KBv. From the standpoint of
their affect on Pgp these copolymers were subdivided into three groups: group 1, hydrophilic
copolymers with HLB varying from 20 to 29 having
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 from
30 to 60 PO repeating units, which are the most
efficacious 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 copolymers
of group 2. To evaluate how efficacious the block
copolymers are at inhibiting Pgp, a doseresponse
curve for rhodamine 123 accumulation was obtained
for each block copolymer. Next, the maximal accumulation levels observed with the most effective
doses of each Pluronic were plotted as a function of
the length of the hydrophobic PO block (NPO ). This
yielded a bell-shaped dependency of the net efficacy
of Pluronic copolymers in inducing rhodamine 123
accumulation in MDR cells (Fig. 6). Furthermore,
the resistance reversion indexes determined in the
doxorubicin cytotoxicity study in MDR cells followed exactly the same pattern as the drug accumulation data shown in Fig. 6 [63].
In order to understand the reasons for the observed
structurefunctional relationships in Pluronic block
copolymers, we have examined intracellular transport of the copolymers as well as their affects on
ATP depletion and Pgp ATPase activity (paper in

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

Fig. 6. Efficacy of Pluronic block copolymer composition in

MDR KBv cells. Figure presents data for groups 2 and 3 block
copolymers only. Rhodamine 123 (R123) enhancement factors are
defined here as the ratios of rhodamine 123 accumulation in the
cells in the presence of the block copolymer to rhodamine 123
accumulation in the assay buffer (reproduced from Ref. [63] with
permission).

preparation). These studies used Pluronic block

copolymers that are representative of each of the
above three groups.
Firstly, the transport of fluorescent-labeled copolymers in the bovine brain microvessel endothelial
cells (BBMEC) was examined by confocal microscopy. These experiments suggested that hydrophilic
copolymers of group 1 did not transport within the
cells. Hydrophobic copolymers of group 3 were
accumulated in the intracellular endosomal compartments but did not release in the cytoplasm. Finally,
the hydrophobic copolymers of group 2 were effectively transported within the cells. This provided
initial evidence that the ability of Pluronic block
copolymers to inhibit the activity of the Pgp efflux
system can be correlated with their ability to be
transported within the cell.
Secondly, the study of ATP levels in MDR cells
suggested that only the copolymers of group 2
effectively depleted ATP, while the copolymers of

773

groups 1 and 3 were much less efficacious. This

study provided evidence that the block copolymers
that are most efficacious in inhibiting Pgp (group 2)
are also the most active in energy depletion and vise
versa.
Thirdly, the affects of block copolymers on the
Pgp ATPase activity were evaluated using membranes containing human Pgp. The copolymers of
group 2 induced dramatic decreases in Pgp ATPase
activity compared to the copolymer-free control as
described Section 7. Conversely, the copolymers of
group 3 had much less affect on the ATPase activity,
while the copolymers of group 1 practically did not
change the ATPase activity. These experiments
provided further support for the fact that the ability
of Pluronic block copolymers to inhibit the activity
of the Pgp efflux system can be correlated with their
ability to inhibit Pgp ATPase activity.
Overall, combined differences in membrane interactions, cellular transport and energy depletion activity may contribute to the observed dependence, in
MDR cells, of the potency of the block copolymers
on their structure.
11. Effects of Pluronic on cytotoxicity of
various drugs in resistant cells
One question arising in relation to the studies
described in the manuscript is whether Pluronic
copolymers can potentiate activity in drug-resistant
tumors of a broader range of antineoplastic agents
beyond anthracyclines, that are currently the best
characterized? One study examined the effects of
P85 on the cytotoxicity of a panel of antineoplastic
agents including anthracyclines (daunorubicin), vinca
alkaloids (vinblastine), alkylating agents (mitomycin
C, cisplatin), and antimetabolites (methotrexate) [23].
The studies were conducted using the human ovarian
carcinoma cell line SKVLB, which has been selected
for vinblastine-resistance and has the MDR phenotype. As is seen in Table 3, the IC 50 s of the MDRtype drugs, daunorubicin, vinblastine, and mitomycin
C, were increased 80 to 1090 times in the presence
of 2.2 mM Pluronic P85. Less significant changes
(only 2 to 6 times) were observed in the IC 50 s of
cisplatin and methotrexate in SKVLB cells, which
are not resistant to these drugs. This result indicates

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

Table 3
Effects 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 50,0

ng / ml

Drug / Pluronic IC 50
ng / ml

RRI (IC 50,0 / IC 50 )

Daunorubicin
Vinblastine
Mitomycin C
Methotrexate
Cisplatin

5000
1200
800
500
400

7
1.1
10
90
200

700
1090
80
6
2

that the sensitization of resistant cells by Pluronic

block copolymers is a general phenomenon characteristic for a broad range of structurally diverse
drugs.
12. Clinical trials of doxorubicinPluronic
formulation (SP1049C)
Pluronic block copolymer, L61, was selected for
further preclinical development based on the in vitro
and in vivo efficacy evaluation studies of anthracyclines [22,24]. The final block copolymer system
used in the clinical studies contains 0.25% L61 and
2% F127 formulated in isotonic buffered saline and
is called SP1049C (Supratek Pharma, Montreal,
PQ, Canada) [69]. This system contains mixed
micelles of L61 and F127 with an effective diameter
of ca. 22 to 27 nm, which do not aggregate in the
presence of the serum proteins. Prior to administration, doxorubicin is mixed with this system, resulting
in the spontaneous incorporation of the drug into
micelles. The formulation is safe following systemic
administration based on acute and subacute toxicity
studies in animals [69]. An open labeled, phase I
dose escalation and pharmacokinetics clinical trial of
SP1049C has been recently completed under the
sponsorship of the UK Cancer Research Campaign in
two sites; Christie Hospital in Manchester (UK) and
Queen Elisabeth Hospital, Birmingham (UK) [25].
The primary goal of this trial was to determine the
maximal tolerable dose (MTD), toxicity and the
pharmacokinetics profile of SP1049C after intravenous administration. A total of 26 patients entered the
trial. The dose limiting toxicity of myelosuppression
was observed at MTD 90 mg / m 2 . Other toxicities
observed were reversible: alopecia, nausea, and
lethargy. It was established that a dose level of 70

mg / m 2 is suitable for evaluation in phase II / III

trials. Plasma pharmacokinetics showed that
SP1049C exhibited slightly longer half-life compared
to conventional doxorubicin (50 h vs. 30 h). One
patient with relapsed previously-treated esophageal
sarcoma showed temporarily a 50% reduction in
tumor size, which progressed rapidly on completion
of treatment. Furthermore, antitumor activity was
seen in relapsed previously-treated soft tissue sarcoma. Phase II clinical trials are scheduled to begin
in early 2002.

13. Conclusion
In conclusion, although the studies described in
this paper are relatively recent, the results obtained
already suggest that Pluronic block copolymers are
promising agents for utilization in treatment of drugresistant tumors. The combination of several independent mechanisms of drug resistance might
complicate chemotherapy and reinforces the need for
development of novel drugs and drug formulations
effective against drug resistant cancers. It has long
been suggested that a broadly successful strategy for
killing drug-resistant cancer cells could be based on
selective energy depletion in these cells, since many
mechanisms of drug resistance are energy-dependent
[26]. Therefore, the recognition of energy-depleting
effects of Pluronic block copolymers, in combination with their very high sensitization effects and
their ability to inhibit multiple mechanisms of drug
resistance in MDR cells, is of considerable theoretical and practical significance. Both the mechanistic
studies and clinical evaluations of these systems are
in progress and we expect fascinating new developments in this area from these on-going studies in the
near future.

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

Acknowledgements
AVK acknowledges support from the National
Cancer Institute (CA89225) and the National Institute for Neurological Disorders and Stroke
(NS36229). We would like to thank Drs. William F.
Elmquist and Donald W. Miller (both Omaha, NE,
USA) and Brian Leyland-Jones (Montreal, Canada)
for stimulating discussions and valuable advice. The
authors have been co-founders, shareholders and / or
employees of Supratek Pharma Inc. (Montreal, PQ,
Canada).

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