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Reversal of multidrug resistance by the inhibition of ATP-binding cassette
pumps employing “Generally Recognized As Safe” (GRAS) nanopharmaceu-
ticals: A review
To appear in:
Advanced Drug Delivery Reviews
Accepted date: 10 September 2013
Please cite this article as: Alejandro Sosnik, Reversal of multidrug resistance by the
inhibition of ATP-binding cassette pumps employing “Generally Recognized As Safe”
(GRAS) nanopharmaceuticals: A review, Advanced Drug Delivery Reviews (2013), doi:
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ADDR Theme Edition on “Nanotechnology and drug resistance”
(Edited by Tamara Minko)
Reversal of multidrug resistance by the inhibition of ATP-binding
cassette pumps employing “Generally Recognized As Safe”
(GRAS) nanopharmaceuticals: A review
1The Group of Biomaterials and Nanotechnology for Improved Medicines
(BIONIMED), Department of Pharmaceutical Technology, Faculty of Pharmacy and
Biochemistry, University of Buenos Aires, Argentina
2National Science Research Council (CONICET), Argentina
3Department of Materials Science and Engineering, Technion-Israel Institute of
Technology, Technion City, Haifa 32000, Israel
*Contact email: email@example.com
NBD – Nucleotide-binding domain
List of abbreviations
AUC – Area-Under-the-Curve
BCS – Biopharmaceutic Classification System
BCRP – Breast cancer resistance protein
CD – Cyclodextrins
CFR – Code of Federal Regulations
CMC – Critical Micellar Concentration
DDS – Drug Delivery Systems
Dox – Doxorubicin
FDC – Fixed Dose Combinations
US-FDA – U.S. Food and Drug Administration
GRAS – Generally Recognized As Safe
HBV – Hepatitis B virus
HIV – Human Immunodeficiency Virus
HLB – Hydrophilic-Lipophilic Balance
IV – Intravenous
MDR – Multidrug-resistant
MRP – Multidrug resistance-associated protein
NRTIs – Nucleoside reverse transcriptase inhibitors
PAMAM – Polyamidoamines
P-gp – P-glycoprotein
PCL – Poly(epsilon-caprolactone)
PEG – Poly(ethylene glycol)
PEO-PPO – Poly(ethylene oxide)-poly(propylene oxide) block copolymer
PI – Protease inhibitor
PLA – Poly(lactic acid)
R123 – Rhodamine 123
TB – Tuberculosis
US-FDA – US Food and Drug Administration
function of the different ABCs, with emphasis on those pumps related to drug
Pumps of the ATP-binding cassette superfamily (ABCs) regulate the access of
drugs to the intracellular space. In this context, the overexpression of ABCs is a
well-known mechanism of multidrug resistance (MDR) in cancer and infectious
diseases (e.g., viral hepatitis and the human immunodeficiency virus) and is
associated with therapeutic failure. Since their discovery, ABCs have emerged as
attractive therapeutic targets and the search of compounds that inhibit their genetic
expression and/or their functional activity has gained growing interest. Different
generations of pharmacological ABC inhibitors have been explored over the last
four decades to address resistance in cancer, though clinical results have been
somehow disappointing. “Generally Recognized As Safe” (GRAS) is a U.S. Food
and Drug Administration designation for substances that are accepted as safe for
addition in food. Far from being “inert”, some amphiphilic excipients used in the
production of pharmaceutical products have been shown to inhibit the activity of
ABCs in MDR tumors, emerging as a clinically translatable approach to overcome
resistance. The present article initially overviews the classification, structure and
resistance. Then, the different attempts to capitalize on the activity of GRAS
nanopharmaceuticals as ABC inhibitors are discussed.
ATP-binding casette superfamily pumps (ABCs); multidrug resistance (MDR);
cancer; infectious diseases; “Generally Recognized As Safe” (GRAS)
pharmaceutical excipients; nanopharmaceuticals; polymeric amphiphiles.
4.3.2. Poly(ethylene oxide)-polyester block copolymers
4.4. Thiolated polymers
Reversal of multidrug resistance by nanopharmaceuticals
2. Pumps of the ATP-binding cassette superfamily-Classification, structure and
3. Pharmacological inhibition
4. Inhibition by “Generally Recognized As Safe” (GRAS) nano-pharmaceuticals
4.1. Natural polymers
4.2. Poly(ethylene glycol) and is derivatives
4.3. Amphiphilic copolymers
4.3.1. Poly(ethylene oxide)-poly(propylene oxide) block copolymers
4.5. Miscellaneous polymers
5. Clinical status and perspectives
P-glycoprotein (P-gp, ABCB1, MDR1) was the first ever identified ABC [33,34],
Pumps of the ATP-binding cassette superfamily, ABCs , are the largest group of
transmembrane proteins. According to their sequence and homology, they are
classified into seven subfamilies named from A to G . ABCs are involved in the
active efflux of a broad variety of endogenous molecules (e.g., lipids, proteins and
products of the metabolism) and xenobiotics (e.g. drugs) out of the cell against a
concentration gradient . The energy source is the hydrolysis of ATP.
There are 49 human ABCs [4-8] distributed ubiquitously or tissue-specifically in
liver, kidney, lung, pancreas, stomach , intestine , central nervous system
[10,11], and different anatomical barriers [9,10,12-16]. Since ABCs regulate the
access of drugs to the intracellular space , their overexpression is a
mechanism of multidrug resistance (MDR) in cancer [2,18-20], hepatitis B [21,22]
and the human immunodeficiency virus (HIV) [23-25]. ABCs also preclude the
intestinal absorption of drugs administered by the oral route, reducing their
bioavailability [26-29]. In addition, they might alter the renal excretion of drugs and
their metabolites [30-32].
followed by the multidrug resistance-associated protein-1 (MRP-1, ABCC1) [35,36]
and the breast cancer resistance protein (BCRP, ABCG2) [37,38].
It is estimated that at least 50% of the compounds listed in the Sigma catalogue
are substrates of P-gp or MRP (ABCC) . Drug/ABC interactions are quite
specific and they need to be individually assessed. In addition, drugs might be
substrates of more than one pump. For example, antiretrovirals of the family of
nucleoside reverse transcriptase inhibitors (NRTIs) are known substrates of BCRP
ABC inhibitors in a new intended use. Conversely, second (e.g., valspodar )
. However, the intracellular accumulation and brain distribution of abacavir, an
NRTI, is also reduced by P-gp . This phenomenon makes the screening very
laborious and predictions very difficult.
Since their discovery, ABCs have emerged as attractive therapeutic targets . In
this context, the search of inhibitors of the genetic expression and/or the functional
activity of ABCs as pharmacotherapy adjuvants in cancer  and HIV  has
gained interest in the scientific community. In addition, patient pharmacogenetic
polymorphism is being studied to correlate underexpression or overexpression of
specific ABCs to altered pharmacological outcomes ; polymorphism is
established when there are changes in the nucleotide sequence of a specific gene
in the general population with a frequency greater than 1%.
Different generations of pharmacological ABC inhibitors have been explored over
the last four decades to overcome MDR in cancer [44-47]. Several of them, such
as verapamil, cyclosporine A and quinidine  belong to a first generation of ABC
blockers. The main common characteristic is that they are drugs already approved
by the regulatory agencies for other uses and they were clinically evaluated as
and third generation inhibitors (e.g., elacridar , tariquidar [51, laniquidar ,
zosuquidar ) were specifically designed for this application and do not display
other pharmacodynamic effects. In general, clinical results were disappointing due
to insufficient therapeutic benefit, unacceptable systemic toxicity, lack of validated,
reproducible and sensitive methods to measure ABC concentrations in different
cells, tissues and organs, and the co-existence of other pumps that distort the
“Generally Recognized As Safe” (GRAS) is a U.S. Food and Drug Administration
(US-FDA) designation for substances that are accepted as safe for addition in food
(under the conditions of use) by qualified experts . GRAS compounds are listed
in the Code of Federal Regulations Title 21 (21 CFR) parts 182 and 184.
Pharmaceutical excipients are conceived as inert constituents incorporated into a
pharmaceutical product to improve the performance of a drug (e.g.,
physicochemical stability, dissolution rate, bioavailability), to make the
manufacturing process more efficient and to prolong the product shelf life [57,58].
For example, a broad spectrum of lipidic and polymeric drug carriers [59,60] have
been used to overcome the poor aqueous solubility of approximately 50% of the
drugs on the market [61-63]. The application of amphiphilic polymeric excipients
that self-assemble into nanoscopic micelles has emerged as one of the most
appealing and popular technology platforms [64,65]. However, far from being
“inert”, some of these amphiphiles have been shown to inhibit the activity of ABCs
in MDR tumors . Over the last two decades, Kabanov and colleagues have
investigated the relationship between the structure of different poly(ethylene
oxide) (PEO-PPO-PEO) block
copolymers (poloxamer, Pluronic®) and the inhibition of the functional activity of
ABCs [67,68]. Their investigations revealed that the inhibitory activity relies not
only on the direct interaction of the copolymer with the transporter and the plasma
cell membrane but also from the hindrance of several cellular pathways [67,68].
This comprehensive work led to the development of an innovative formulation of
the antitumoral drug doxorubicin, namely SP-1049C (Supratek Pharma Inc.,
Canada), that incorporates a combination of poloxamers as inhibitors of P-gp
cellular membrane [1-3]. Forty nine genes that encode for the corresponding ABC
[69,70]. This product showed increased effectiveness in preclinical and clinical
phases and it was granted Orphan Drug status for the treatment of gastric cancer
by the U.S.-FDA in 2008 . These investigations constitute a solid basis for the
implementation of new therapeutic approaches to overcome ABC-mediated
resistance in other types of cancer and diseases.
First, the present article succinctly describes the classification, structure and
function of the different ABCs. Those pumps directly associated with resistance to
drugs are emphasized. Then, the attempts to capitalize on the activity of GRAS
nanopharmaceuticals as ABC inhibitors are overviewed. Finally, the clinical impact
and the perspectives of the field are discussed.
2. Pumps of the ATP-binding cassette superfamily: Classification, structure
ABCs are integral membrane proteins that play a fundamental role in the active
translocation of amino acids, proteins, lipids, lipopolysaccharides, inorganic ions,
peptides, sugars, metal ions, metabolism products and drugs across the plasma
have been identified in humans and classified into seven subfamilies from ABCA to
ABCG according to genomic organization, order of domains and sequence
conservation [72,73]. A summary of the different ABCs is presented in Table 1.
ABCs present one cytosolic and one membrane domain. The cytosolic nucleotide-
binding domain (NBD) is highly conserved and members of the same subfamily
share approximately 30% to 40% of the residues . NBDs comprise three
sequence elements, the Walker A and Walker B motifs separated by approximately
proposed for P-gp . For a comprehensive description of the main ABCs,
120 aminoacids and the C motif or ABC signature. The former two are common to
different nucleotide-binding proteins, while the signature is characteristic of each
ABC . All these elements are fundamental for ATP binding and hydrolysis and
for the link between this process and molecular transport in the so-called
'conformational coupling' . The membrane domain is formed by four to eight
transmembrane -helices that give place to putative pathways and it would confer
specificity for the passage of a broad variety of substrates across the membrane
. The term 'ATP-binding cassette' was coined owing to this modular structure
. Some ABCs can co-transport different substrates at the same time, some
times synergistically. For example, the transport of vincristine and etoposide by
MRP-1 demands the presence of reduced glutathione [75,76]. The same was
observed for the transport of fluorescent dyes Hoechst 33342 and rhodamine 123
by P-gp . These results would indicate the presence of two binding sites in the
pump with differential affinity for the different substrates. Thus, efficient
translocation would only take place once both sites are occupied by their
respective substrates . Moreover, the presence of a third binding site was
readers are referred to the review by Schinkel and Jonker .
The study of drug/ABC interactions is usually addressed employing different in
vitro, ex vivo and in vivo models and it often results in limited reproducibility and
comparability among laboratories [80-82]. To standardize the study and make it
more reproducible and robust, some research groups focused on the development
of non-living nanotechnology platforms (e.g., liposomes) that take advantage of the
Juliano and Ling in 1976 . The term MDR was coined based on its ability to
latest biochemical tools available in the market . This is another complementary
area of development associated with the employment of ABC as therapeutic
targets. In advance, a brief description of the different ABC subfamilies is
introduced, with emphasis on those that are intimately associated with drug
resistance. More detailed information on each one can be found in Table 1 and the
ABCAs are mainly involved in the trafficking of lipids in different cells and organs.
For example, ABCA1 is involved in the efflux of phospholipids and cholesterol from
peripheral cells, while ABCA4 is specific of N-retinylidene-
phosphatidylethanolamine. ABCA3 and ABCA6 have been related to poor
response to chemotherapy, the former identified in childhood acute myeloid
The most extensively investigated transporter of this family is P-gp discovered by
confer resistance to several drugs at the same time. P-gp mainly localizes in the
apical membrane of epithelia, thus translocating substrates in the basolateral-to-
apical direction. The presence of P-gp in the intestinal epithelium leads to reduced
oral biovailability of a broad variety of antitumorals and protease inhibitor (PI)
antiretrovirals [31,84]. For example, to increase the bioavailability of PIs (e.g.,
amprenavir, atazanavir, darunavir, indinavir, lopinavir, saquinavir, tipranavir) in the
combined pharmacotherapy of HIV, these drugs are co-administered with a low
(e.g., intestine, liver). Thus, MRP-2 reduces oral absorption and enhances
dose of ritonavir as boosting agent [85,86]. The expression of P-gp in the human
intestine gradually grows from the proximal to the distal small intestine and the
colon, the latter segment displaying the highest concentration of this pump .
Pg-p is also expressed in liver, kidney and it is maximal in the blood-brain barrier,
where it limits the passage of drugs into the central nervous system [87,88].
This family is formed by six pumps, MRP-1 being the most important exponent.
ABCCs display a unique amino-proximal membrane-spanning domain represented
by an extension of approximately 200 aminoacids. This structure distinguishes it
from other ABCs . MRP-1 is a co-transporter that effluxes amphipathic anions
and non-anionic hydrophobic drugs conjugated or complexed with glutathione,
glucuronic acid or sulfate. MRP-1 was primarily identified in the context of cellular
resistance to doxorubicin, though more thorough studies indicated that it confers
resistance to other anthracyclines (e.g., daunorubicin), Vinca alkaloids,
epipodophyllotoxins and methotrexate [35,90,91]. MRP-2 is another MDR-mediator
that, as opposed to MRP-1, localizes in the apical membrane of polarized cells
hepatobiliary clearance of drugs [32,92]. MRP-2 is expressed in liver, kidney and
the intestinal epithelium; the highest concentration is found in the duodenum and it
decreases towards the colon . The role of this ABC in intestine is still unclear.
Substrates are usually anions, hydrophobic molecules conjugated with anions 
and uncharged amphipathic molecules . Also, the transport of cationic or
uncharged molecules could be stimulated by reduced glutathione through the
formation of labile complexes or by co-transport (see above) . MRP-3 has been
much less investigated than the previous counterparts. It displays 58% similarity
with MRP-1 and it is found in the basolateral membrane of polarized cells,
transporting organic anions. The rest of ABCCs remain almost unexplored and
their localization and most relevant substrates are still under screening.
This subfamily is formed by four half-transporters that dimerise to form functional
homo or heterodimers [73,85]. ABCD1 was the first half-transporter to be
discovered and it would be involved in the translocation of acyl-CoA esters across
There exists one single member in this subfamily, ABCE1, entailed to transport
organic anions . Since it lacks the transmembrane domain, its role as pump is
As previously described for ABCE1, it lacks transmembrane domain and its
performance in drug efflux is unclear .
The human ABCG subfamily is formed by five half-transporters that display an N-
terminal NBD followed by a C-terminal membrane domain. Half-transporters
undergo dimerisation to form the active pump [72,95]. ABCG2 (BCRP) is one of
the pumps associated with MDR. Some of its substrates are anthracyclines,
camptothecins (e.g., topotecan, SN-38), methothrexate and other antitumorals .
In addition, it is also involved in the efflux of antibiotics such as erythromycin,
tetracycline and ofloxacin  and different inhibitors of the HIV reverse
Chemotherapy remains the most effective approach to treat disseminated and
transcriptase [7,40]. Complementary data indicated that the ABCG2/ABCG2
homodimer is the minimum functional unit and it is independent of additional
proteins [41,97]. On the other hand, studies have also demonstrated the existence
of ABCG2 sequence variability in drug-selected cell lines . For example,
mutations in arginine of position 482 would lead to changes in the function and
interaction with substrates . ABCG2 is expressed in the apical membrane of
cells of tissues with excretory function. Among them, the canalicular membrane of
hepatocytes, the luminal membrane of villous epithelial cells in the small and large
intestine, ducts and lobules of the breast, cortical and proximal tube of kidney, the
apical membrane of trophoblasts that give place to a rate-limiting barrier in the
placenta [99,100], and apical membranes of capillary vessels in the blood-brain
barrier . BCRP would also play a role as a novel biomarker of cancer stem
cells . For a more comprehensive reading on BCRP, readers are
recommended the review article by Robey et al. . Finally, ABCG5 and ABCG8
are also half-transporters that heterodimerise to transport sitosterol .
3. Pharmacological inhibition of ABCs
hematological tumors. However, drug resistance accounts for about 90% of the
therapeutic failures . In fact, 50% of the cancers express detectable levels of
P-gp . Evidence on the relationship between MRP-1 and BCRP expression and
MDR is more limited. There is solid evidence showing that these ABCs mediate
resistance in non-small cell lung cancer and leukemia, respectively, resulting in
poor clinical response [38,108,104,105]. As mentioned above, the initial discovery
might result in reduced drug clearance and severe drug toxicity .
of P-gp and more recently of MRP-1 and BCRP motivated the search of reversal
agents that would underregulate their genetic expression or block their functional
activity [7,18,106-108]. Regretfully, the clinical impact of exploiting ABCs as
therapeutic targets has been less encouraging, leaving researchers and clinicians
with a 'bittersweet feeling'.
The first generation of inhibitors was formed by drugs displaying pharmacological
activity per se (e.g., verapamil [47,109,110], cyclosporin A [47,111]) and that were
approved to treat other medical conditions (Table 2). The main constraint with
these compounds was that the required dose to inhibit ABCs in vivo was too high
and it resulted in serious adverse effects. These frustrating results motivated the
search for more selective inhibitors that would be deprived of any
pharmacodynamic activity and that would not display strong interactions with
metabolizing pathways or alter the pharmacokinetics of other drugs. A series of
selective blockers have been clinically assessed with limited success (Table 2). On
the other hand, it is pertinent to stress that attaining this goal is not simple because
the uncontrolled inhibition of an ABC without the appropriate dose adjustment
One additional challenge stems from the fact that the different subfamilies of ABCB
and ABCC also play fundamental roles in the immune system [18,113-115]. In this
context, the clinical assessment of novel ABC blockers needs to be carefully
planned to prevent the emergence of undesired secondary effects due to the poor
intrinsic antitumoral response of the host. Other researchers have proposed other
strategies to circumvent MDR: the design of novel anticancer drugs that are not
substrates of the specific ABC or the use of molecules that exploit the
remained sole until the early 1990s, when different research groups reported that
overexpression of the pump . However, the design of new drugs and their
clinical trial is a long and expensive endeavor and it appears as even more
challenging and hard to accomplish, especially in poverty-related diseases hitting
mainly developing nations. Under the current circumstances, the application of
inherently 'inert' pharmaceutical excipients comes out as an exceptional tool to
overcome drug resistance and improve the treatment of disease.
4. Inhibition of ABCs by “Generally Recognized As Safe” (GRAS)
The first report on the use of a macromolecular or polymeric pharmaceutical
excipient as ABC blocker most likely appeared in the literature in 1972, when
Rhiem and Biedlerm observed that polysorbate 80 (Tween® 80) enhanced the
response of MDR Chinese hamster cells to daunorubicin and actinomycin D .
It is remarkable that regardless of the fact that the overexpression of P-gp in this
cell type was discovered four years later (1976), there was already indirect
evidence that its inhibition enhanced the efficacy of antitumorals . This report
non-cytotoxic concentrations of the non-ionic surfactants polyethoxylated castor oil
(Cremophor® EL) and polysorbate 80 (Tween® 80) modulated the resistance of
different MDR cell lines of leukaemia, Ehrlich ascites tumor, human squamous lung
cancer, human myeloma, ovarian cancer and colon carcinoma that overexpress P-
gp to daunorobucin [117-121]. This phenomenon was already ascribed to the direct
binding of the surfactant to P-pg. Cremophor® EL was also shown to improve the
activity of cyclosporin A in acute myeloid leukemia cells . In 1996, Kabanov et
The present section comprehensively addresses the state-of-the-art in this field by
al. described the hypersensitization of human ovarian carcinoma cells that
overexpress P-gp (SKVLB) to daunorubicin by exposure to a different type of
excipient, a non-ionic PEO-PPO-PEO block copolymer, namely Pluronic® P85
. During the same year, a similar study reported on the response of MDR
Chinese hamster ovarian cells (CH(R)C5) and human breast carcinoma cells
(MCF-7/ADR) treated with Pluronic® L61 and doxorubicin . In both studies,
the cytotoxicity of the antitumoral drug was increased by up to 700 times. Since
then, a profuse research has been conducted to increase the release of drugs to
the intracellular space of tumoral cells and their passage across barriers such as
the intestinal epithelium and the blood-brain barrier by means of the inhibition of
ABCs with GRAS nanopharmaceuticals. One of the most striking features of these
products is a more straightforward bench-to-bedside translation when compared to
pharmacologically active inhibitors (Table 2), owing to the fact that they have been
already accepted as safe and they are deprived of major toxic effects. Being aware
of their great therapeutic potential, scientists began to investigate this approach
also in infectious diseases [22,43,125].
discussing the most relevant contributions done in the context of ABC inhibition for
each group of GRAS nanopharmaceuticals.
4.1. Natural polymers
The inhibition of ABCs to improve the oral bioavailability of drugs and to overcome
MDR in cancer using anionic polysaccharides such as xanthan gum, gellan gum,
alginates, dextran and fucoidan has been explored by Carreno-Gomez and
Duncan and the intellectual property comprehensively protected (Table 3) .
studies to explore P-gp/PEG interactions and the passage of different molecules in
Even though the claims of this patent application covered a broad spectrum of
transporters and substrates, the examples provided were focused on two
antitumorals, vinblastin and doxorubicin .
4.2. Poly(ethylene glycol) and its derivatives
Poly(ethylene glycol)s (PEGs) are highly biocompatible polyethers that have been
investigated in many biomedical applications [127,128]. PEGs are soluble in
aqueous and organic solvents and they display limited cyto and biotoxicity and
immunogenicity. PEG is not cleaved in vivo, though it is eliminated by renal
filtration in a molecular weight-dependent manner. Mono and bifunctional PEGs
have been used to modify nanoparticles and biologically active molecules in a
process known as PEGylation . A few works reported on the ability of PEG (of
molecular weight between 400 and 20,000 g/mol) to increase the accumulation of
digoxin , rhodamine 123 , paclitaxel and doxorubicin [132,133],
prednisolone, methyl-prednisolone and quinidine . These results were used as
the basis for the synthesis of a paclitaxel-PEG conjugate that by-passed the efflux
by intestinal P-gp and increased the bioavailability of the drug . Automated
cell monolayers were also described .
As previously described Cremophor EL and polysorbates (e.g., Tween® 80), two
PEG containing surfactants, were the first pharmaceutical excipients assessed as
ABC blockers [116-122,137-141]. Following this rationale, other amphiphilic
conjugates of PEG with small molecules such as D--tocopheryl succinate 1000
have been evaluated with positive results ; this specific derivative displaying
discovery of this inhibitory activity. It would not be surprising to realize that the
optimal molecular weight for the inhibitory activity (Figure 1) . Results were
further supported with in vivo experiments . A conjugate of PEG with the
lipophilic vitamin D, PEG-cholecalciferol polyethylene glycol succinate, showed
similar efficacy . PEG stearate (Myrj®) and alkylates (Brij®) also showed this
behaviour, increasing the transport of epirubicin across CaCo2 cell monolayers
. Other approved pharmaceutical excipients that present certain structural
analogy with PEG (e.g. caprylocaproyl macrogol glycerides, Labrasol®; glyceryl
showed a similar beneficial effect [146,147,148]. It is
noteworthy that most of these studies focused on P-gp, the first pump linked to
MDR, and did not explored interactions with additional ABCs. At this point, it would
be important to stress that the use of these excipients is common in
pharmaceutical development, for example to increase the aqueous solubility and
physicochemical stability of different drugs, independently of their activity as ABC
blocker. Thus, their approval by regulatory agencies took place probably before the
incorporation of these excipients to different formulations that were assessed in
clinical trials has altered their pharmacokinetics. If this were the case, many GRAS
nanopharmaceuticals considered pharmacologically 'inert' at a previous time and
approved for use in pharmaceutical products, should be clinically revaluated. In this
framework, it would be interesting to retrospectively investigate the performance of
the same drugs without these excipients to determine whether they are safe or not
for use in new products.
FDA for the treatment of esophageal cancer [69,70].
4.3. Amphiphilic copolymers
4.3.1. Poly(ethylene oxide)-poly(propylene oxide) block copolymers. Amphiphilic
poly(ethylene oxide)-poly(propylene oxide) block copolymers (PEO-PPOs) with
different block arrangements and architectures are among the most investigated
biomaterials for the production of polymeric micelles [64,65,149] and thermo-
responsive physical and chemically crosslinked gels [150-159]. There exist two
groups of PEO-PPOs commercially available in a broad spectrum of molecular
weights and hydrophilic-lipophilic balance (HLB), the linear poloxamers (see
above, Pluronic®) and the branched poloxamines (Tetronic®) . These
copolymers have showed good cell [154-157,160] and biocompatibility for
administration by different routes [161-163]. In addition, several poloxamers (e.g.,
Pluronic® F127, F108, F68, F87, L44) are included as excipients in medicines and
medical devices approved by the regulatory agencies. Thus, they are commercially
available in pharmaceutical quality [164-166]. Pluronic® L61 is an interesting
example where the approval of the GRAS nanopharmaceutical came as part of an
innotative doxorubicin formulation that was granted orphan drug status by the U.S.-
Poloxamines show several structural differences with respect to poloxamers. On
one hand, the presence of a central ethylenediamine residue makes them
responsive not only to the temperature (as in the case of poloxamers) but also to
the pH of the medium [167-169]. In addition, these tertiary amine moieties could be
chemically modified [156,170,171]. On the other, poloxamines display a greater
number of terminal blocks than poloxamers. As with any amphiphile, PEO-PPO
polymeric micelles are formed above the critical micellar concentration (CMC) in an
in these early stages, findings suggested that molecules interact directly with the
entropy-driven process associated with the release of water hydration molecules
from the hydrophobic PPO segments . Conversely, below the CMC,
copolymer molecules form unimolecular aggregates known as unimers. Depending
on structural properties (e.g., molecular weight, HLB, architecture) and
environmental conditions (e.g., temperature, pH, ionic strength, etc.), concentrated
solutions of these copolymers could undergo a reverse gelation upon heating.
While the generation of micelles and gels demand minimum concentrations of
approximately 1% and 15-20%, respectively, the ABC inhibitory activity of these
copolymers has been exclusively ascribed to the unimers . This implies that
relatively low copolymer concentrations (as low as 0.01%), usually below both the
cytotoxic levels and the accepted administration limits, might be enough to
increase the intracellular accumulation of an ABC substrate.
As in many of the applications of these biomaterials, most of the research
assessing the inhibition of ABCs has been devoted to poloxamers. The potential of
these copolymers to revert MDR in cancer was first demonstrated in vitro by the
group of Kabanov employing drugs that are substrates of P-gp [123,124]. Already
outer side of the cell membrane, facilitating the influx of the free drug . Then,
these studies were extended to other copolymers and pumps associated with MDR
in different in vitro and in vivo models [174-183]. For example, Batrakova et al.
compared the activity of epirubicin and doxorubicin administered alone or with
Pluronic® L61, P85 and F108 on murine leukaemia P388 and daunorubicin-
sensitive Sp2/0 and daunorubicin-resistant Sp2/0 myeloma cells grown
subcutaneously in mice . The co-administration of hydrophobic copolymers
paclitaxel, zidovudine, valproic acid and loperamide were assessed . Findings
L61 and P85 prolonged the mice lifespan. The strong relationship between activity
and copolymer concentration and hydrophobicity estimated by the EO/PO molar
ratio was suggested in that work for the first time . The passage of drugs to
the central nervous system is controlled by the presence of the blood-brain barrier,
an anatomical barrier that displays a unique combination of ABCs . Depending
on the concentration, Pluronic® P85 could increase drug absorption in microvessel
endothelial cells by two different pathways . Below the CMC by P-gp
inhibition, while above it by the uptake of drug loaded micelles. An additional
contribution of this work was that it discussed for the first time the potential specific
interaction between PEO-PPOs and P-gp . The question of whether micelles
were indispensable or not to attain the inhibition began to be revealed also in early
works of this group that assessed the transport of rhodamine 123 across confluent
Caco2 cell monolayers (an in vitro model of the intestinal epithelium) and brain
microvessel endothelial cells monolayers (an in vitro model of the blood-brain
barrier) in the absence and the presence of different concentrations of Pluronic®
P85 [175,176]. Different molecules such as fluorescein, doxorubicin, etoposide,
with rhodamine 123 showed that unimers increased the accumulation to a
significantly greater extent than the micelles, pointing out them as the key player in
the inhibition process (Figure 2) . In contrast, above the CMC, the dye was
incorporated into the micelles that then enter the cell and are effluxed to the apical
side . Similar results were observed with the accumulation of fluorescein in
human pancreatic adenocarcinoma cells that express MRP . At the same
time, it should be stressed that the detrimental effect of micelles depends on their
Another aspect of interest was the investigation of the various inhibition pathways
ability to sequester the substrate. In addition, the apical-to-basolateral transport of
fluorescein, doxorubicin, paclitaxel and zidovudine increased with Pluronic® P85 in
both cell models, while for etoposide, valproic acid and loperamide this
phenomenon was observed only in Caco2 cell monolayers . It is convenient to
remark at this point that regardless of the fact that these excipients block a broad
spectrum of ABC pumps, their qualitative and quantitative activity could differ
among cell types and sources. Moreover, these pumps show at least two binding
sites that selectively bind specific substrates. One inhibitor could effectively block
the transport of some substrates but not of others because it binds only one of the
sites. In this context, the specific interactions need to be thoroughly assessed and
predictions are difficult. At the same time, there are compositional features that
govern the inhibition. In general, poloxamers of intermediate-length PPO blocks
(30-60 PO units) and relatively high hydrophobicity (or low HLB value) display the
best inhibitory activity (Figure 3) . However, these features also depend on
the copolymer architecture, as it will be discussed below for the branched PEO-
PPO derivatives, poloxamines.
in cell and animal models [68,184]. The direct interaction of these copolymers with
phospholipid bilayers has been modelled by Firestone et al. [185,186]. These
studies highlighted the number of poly(propylene oxide) repeating units (NPO) as
the parameter that governs the association with the bilayer. PEO-PPO-PEOs with
very short hydrophobic blocks and radius of gyration (7 Å) smaller than that of the
hydrophobic tails of the lipid bilayer (~20 Å) probably intercalate in the cell
membrane, both PEO terminal blocks being exposed at the outer membrane
decrease oxygen consumption . This effect is transient and ATP levels
surface (Figure 4A). Conversely, copolymers that contain longer PPO blocks
(radius of gyration of ~15 Å) would span the bilayer (Figure 4B). Thus, fluidization
and decrease of the membrane microviscosity that alters the conformation of the
pump is one of the mechanisms behind the inhibition process [187,188].
Interestingly, the interaction of the copolymers with normal and tumor cells led to
an increase and a decrease of the cell microviscosity, respectively . At the
same time, different studies suggested that exposure to these copolymers reduces
drastically the intracellular ATP levels due to interferences in the electron transport
chain of mitochondria, this phenomenon being exclusive of MDR cells [188-192].
This relationship between ATP-depletion and activity was demonstrated in different
cells expressing P-gp and MRPs employing Pluronic® P85 as the inhibitor .
Highly responsive cells showed ATP depletion and at least 100-fold increase in
doxorubicin cytotoxicity with copolymer concentrations below 0.01%; the greater
the depletion, the greater the sensitization. More recently, a work confirmed that
poloxamer molecules rapidly cross the membrane of MDR cells and co-localize
with mitochondria, where they block the respiratory chain at different stages and
recover once the copolymer is removed from the medium. Other mechanisms
behind the inhibition are (i) inhibition of a detoxification pathway, namely
glutathione/glutathione-S-transferase, that is most relevant in human breast cancer
cells , (ii) direct interaction with the pump, (iii) release of cytochrome c and
increase of intracellular reactive oxygen species and (iv) decreased sequestration
of drugs within cytoplasmatic vesicles . Poloxamers would also generate ion
channels that facilitate the transport across the cell membrane of neutral drugs and
downregulation of mdr1 and abcg2 genes, while the expression of mrp1 remained
ions, the nature of the substrate conditioning the structure of the channel .
According to this work, only unimers and dimers would be involved in the transport
of drugs, while ions would be transported upon the formation of aggregates. It is
also worth stressing that studies conducted in membranes overexpressing P-gp,
MRP-1 and MRP-2 indicated that the effect of poloxamers on P-gp ATPase is
stronger than that on MRP-1 and MRP-2 ATPases . Another mechanism
would be related to changes in the equilibrium between proapoptotic and
antiapoptotic activity in MDR cells. Free doxorubicin activates a proapoptotic and
an antiapoptotic signal at the same time, while doxorubicin/Pluronic® P85 mixtures
increase the proapoptotic activity at the expense of the antiapoptotic one (Figure
5) . These observations were supported with data on the downregulation of
the intracellular concentrations of antiapoptotic genes BCL2 and BCLXL and the
overexpression of proapoptotic ones BAX, P53, APAF1, caspase 9 and caspase 3
in MDR cells . A recent work by Cuestas et al. investigated for the first time
the effect of branched PEO-PPOs on the expression of mRNA encoding for the
main ABCs in a human hepatoma cell line . Findings indicated the
unaltered (Figure 6) . Another noteworthy phenomenon is related to the ability
of these copolymers to prevent the development of P-gp-mediated MDR in vitro
and in vivo over the exposure to antitumoral drugs .
Cancer has undoubtedly led innovation in the nanopharmaceutical field for more
than three decades and several products have reached the market . The
profuse research carried out by Kabanov at the interface of nanotechnology and
cancer MDR employing pharmaceutical excipients as ABC inhibitors has turned,
their performance with verapamil and several poloxamers of proven inhibitory
over the years, into a very robust platform. This groundbreaking work probably
reached a pinnacle with the approval of SP1049C, an injectable combination of
doxorubicin with Pluronic® F127 and L61, for the therapy of chemosensitive and
chemoresistant cancers where the copolymers are used because they disrupt the
functionality of mitochondria [69-71]. Based on the same technology called
BioModTM, other products for the treatment of MDR cancers are in the company
pipeline . For example, SP1015C is a novel formulation of cabazitaxel, a
semi-synthetic derivative of a natural taxoid developed by Sanofi-Aventis and
approved by the U.S.-FDA for the treatment of hormone-refractory prostate cancer
in 2010. The mechanism of action is the inhibition of the microtubule assembly.
SP1012C follows the same rationale though the antitumoral is docetaxel.
Other groups followed the steps of this pioneering work and began to explore other
aspects of the inhibition. For example, to elucidate the incidence of the molecular
architecture of PEO-PPOs on the inhibition of P-gp, the group of Sosnik assessed
for the first time the ability of a broad spectrum of pristine and N-methylated
poloxamines to inhibit this pump in Caco2 cell monolayers ; they contrasted
activity (e.g., Pluronic® P85). A main difference of poloxamines is that the PPO
content is fragmented into four shorter segments bound to a central
ethylenediamine moiety, while poloxamers present one single central PPO block.
Results indicated that some poloxamines of high to intermediate hydrophobicity
such as Tetronic® 304, 904, 1301 and 901 enhanced the relative accumulation of
doxorubicin with respect to verapamil (Figure 7) . Poloxamines did not
straightforwardly comply with the structural features previously stated for
The use of these ABC inhibitors to overcome resistance in infectious diseases
poloxamers to display an optimal inhibitory activity . Considering poloxamines
as two PEO-PPO-PEO triblocks covalently linked in the center of the molecule by
the ethylenediamine residue, the number of 'effective' PO units in the central PPO
block of each virtual PEO-PPO-PEO triblock would be the total NPO divided by 2.
Thus, in terms of the effect on the dimensions of the core, two PO units in
poloxamine would probably be equivalent to one PO unit in poloxamer. In this
work, an updated diagram of the inhibitory activity of poloxamers and poloxamines
was introduced and three main areas of low, medium and high inhibitory activity
were defined . Another aspect was the evaluation of the activity of positively-
charged poloxamines. N-methylation increased the inhibitory activity of Tetronic®
1107 that surpassed that of verapamil. In contrast, more hydrophobic derivatives
were more cytotoxic, undermining their potential application as ABC inhibitors. A
challenge that would need to be overcome is that poloxamines are not approved
yet for pharmaceutical use. This reduces the chances of bench-to-bedside
translation. At the same time, they open new possibilities to expand the application
of these copolymers as ABC blockers.
such as HIV and viral hepatitis [198,203-206] has attracted the attention of a few
researchers, especially because they would represent relatively economic and
scalable formulation upgrades, more compatible with innovation in less profitable
markets [207,208]. However, the pace of progress in these diseases has been
notoriously slower than in cancer.
The impact that ABCs have in HIV can be illustrated for indinavir, a PI approved for
the treatment of HIV-1 in 1996 that is substrate of P-gp in the intestine . This
bioavailabilty of antiretrovirals in the brain and the cerebrospinal fluid . Thus,
drug was initially classified into Class IV of the Biopharmaceutic Classification
System (BCS) due to its apparently low ability to permeate the intestinal epithelium
[61,209]. At a later time, the drug was reclassified into Class II because it
intrinsically crosses this barrier, though the active efflux mechanism removes it in
the basolateral-to-apical direction . As previously suggested, most of the
antiretrovirals are substrates of at least one ABC pump and the interaction
drug/pump needs to be specifically assessed because predictions based on
previous data are complex [211,212]. For example, saquinavir is not only a
substrate of P-gp but also of MRP-1 and MRP-2 in the kidney . A similar
phenomenon is observed for abacavir that is a substrate of at least BCRP and P-
gp . In this framework, the incorporation of GRAS nanopharmaceuticals that
inhibit ABCs to antiretroviral formulations with the aim of increasing their
bioavailability when administered by different administration routes is an appealing
strategy to improve the therapeutic outcome.
The central nervous system is one of the most challenging HIV reservoirs owing to
the presence of a broad variety of ABCs in the blood-brain barrier that reduce the
there is a special interest to improve this aspect of the pharmacotherapy .
Spitzenberger et al. investigated the biodistribution of a combination of zidovudine,
lamivudine and nelfinavir co-administered with Pluronic® P85 in a murine model of
HIV-1 encephalitis . First, studies conducted in vitro indicated that the pure
copolymer in concentrations ranging between 0.001% and 0.01% decreased the
replication of the HIV in infected monocyte-derived macropages by 55% and 44%,
respectively, at day 12. In addition, exposure of infected cells to P85
also increased the accumulation in wild type cells, suggesting the inhibition of
(0.01%)/nelfinavir (0.01-0.001 mol/L) showed greater inhibition than free
nelfinavir. When an antiretroviral cocktail was administered to HIV-infected mice,
brain exposure increased and infection levels decreased owing to the inhibition of
P-gp by While 39.9% and 68.5% of the infected
macrophages was HIV-positive at days 7 and 14, the administration of P85
(0.2%)/antiretroviral cocktail reduced these levels to 9.4% and 7%, respectively.
Conversely, a P85-free antiretroviral combination reduced the viral infection to
35.5% at day 14, being even less effective than pure P85 0.2% solution that
reduced it to 13.4%. Similar studies assessed the effect of Pluronic® P85, F88 and
F127 on the accumulation of nelfinavir and saquinavir Madin-Darby canine
[204,205]. The inhibitory activity depended on the copolymer molecular features
and the intracellular drug accumulation declined above the CMC due to drug
sequestration. Pluronic® P85 inhibited both the basal and the nelfinavir-induced P-
gp activity and increased the accumulation of the drug. Conversely, Pluronic®
F127 and F88 did not. Results with saquinavir followed a similar trend, though it
additional pumps such as MRPs .
Efavirenz is another first-line antiretroviral that is a substrate of BCRP in the
intestine [43,82] and the blood-brain barrier of rat . Due to its poor aqueous
solubility, efavirenz shows limited oral bioavailability and inter- and intra-individual
variability. Different single and mixed poloxamer and poloxamine polymeric
micelles were investigated to encapsulate the drug and improve its oral
To enhance the micellization tendency, other polyethers where the PPO block is
pharmacokinetics [169,216-218]. Even if the focus of these comprehensive work
was not the assessment of the inhibition of BCRP, systems containing Tetronic®
904, a copolymer that inhibits both BCRP and P-gp [202,206], further increased the
oral bioavailability of the drug in rats . These findings constituted indirect
evidence of the poloxamine-mediated inhibition of BCRP. Studies that compared
the bioavailability of efavirenz in the brain after the IV and the intranasal
administration followed a similar trend .
Other diseases that offer limited therapeutic alternatives, especially in resistant
cases are viral hepatitis . Aiming to explore the potential of PEO-PPOs to
overcome resistance in viral hepatitis, Cuestas et al. extended the study of the
multiple-inhibitory activity of poloxamines on P-gp, BCRP and MRP1 to two human
liver carcinoma cell lines, Huh7 and HepG2 . Copolymers of intermediate to
high hydrophobicity (e.g., Tetronic® 304, 904 and 1301) inhibited P-gp and BCRP
but not MRP-1 in both cell lines (Table 4) . The activity was associated with
both copolymer concentration and hydrophobicity. In contrast, a more hydrophilic
counterpart, Tetronic® 1107, did not show any inhibitory effect.
replaced by the more hydrophobic poly(butylene oxide), poly(styrene oxide) and
poly(glycidyl oxide) were developed (Table 3) [220,221]. Very recently, Barbosa
and collaborators assessed the ability of two poly(styrene oxide)-poly(ethylene
oxide) triblock copolymers to inhibit P-gp efflux in a MDR cell line (NCI/ADR-RES)
. Copolymer unimers inhibited the functional activity of P-gp to a similar extent
that Pluronic® P85 and increased the accumulation of doxorubicin and its
cytotoxicity in MDR cells, but not in sensitive cells.
to poloxamers that decreased the activity of the enzyme . The modification of
4.3.2. Poly(ethylene oxide)-polyester block copolymers. Poly(ether)s such as PEG,
poloxamers and poloxamines are bioeliminable by renal filtration in a molecular
weight dependent manner, though they are not biodegradable. Partially
biodegradable amphiphiles that combine hydrophilic blocks of PEG with
hydrophobic ones of poly(lactic acid) and/or poly(epsilon-caprolactone) and display
diverse molecular architectures have been synthesized [223-230]. Based on the
previous experience with PEO-PPOs, the ABC inhibitory activity of non-ionic
surfactants of poly(ethylene oxide)-b-poly(epsilon-caprolactone) has been
investigated (Table 3) [231-233]. For example, Zastre et al. increased the transport
of rhodamine 123 across Caco2 cell monolayers by co-formulating the model drug
with a low molecular weight methoxypolyethylene glycol-b-poly(epsilon-
caprolactone) diblock . The results were comparable to those with verapamil.
In a later study, the same researchers evaluated how the perturbation of the
cellular membrane affected the activity of P-gp ATPase . The diblock
decreased the fluidity of the membrane of Caco2 cells, as previously shown for
poloxamers. This fluidity drop increased the ATPase activity by 3-fold, as opposed
Pluronic® P105 with poly(epsilon-caprolactone) blocks has been recently reported
. In vitro cytotoxicity assays showed the sensitization of resistant SKOV-
3/PTX tumor cells with the paclitaxel/copolymer formulation. In addition, the IV
administration reduced tumor volumes with respect to the free drug owing to the
inhibition of ATPase of P-gP, the decrease of the membrane microviscosity and the
loss of mitochondrial membrane potential and subsequent decrease of ATP levels
(Figure 9) . A question that emerges from this work is why the activity of the
serves as both drug carrier and ABC inhibitor, they recently reported on liposomes
modified poloxamer was not compared to that of the pristine one. In this context,
results suggested that the inhibitory activity is exclusively conferred by the
poloxamer component that is active per se  and that the modification with PCL
only contributed to increase the encapsulation extent of paclitaxel. In a similar way,
Pluronic® L61 and L92, were modified with poly(acrylic acid) to produce microgels
that enhanced the transport of doxorubicin across Caco2 cell monolayers by the
combination of two mechanisms: the intrinsic inhibition of P-gp by the poloxamer
constituent and the increase of the passive absorption .
4.4. Thiolated copolymers
Thiolated derivatives of chitosan, a biocompatible muco-adhesive polysaccharide
with diverse biomedical applications [235,236], and other approved polymeric
pharmaceutical excipients (e.g., polycarbophil, poly(acrylic acid), poly(allylamine)s,
hydroxyethylcellulose) have been primarily developed by the group of Bernkop-
Schnurch to improve the interaction of the drug delivery system with the intestinal
mucosa [237-246]. At the same time, their ability to block P-gp and MRPs was
explored with positive results (Table 3) [237-246]. In a more complex system that
of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and a maleimide-functionalized
lipid that were surface-modified with chitosan-thioglycolic acid and its protected
version with 6-mercaptonicotinamide . These nanocarriers improved the
transport of fluorescein isothiocyanate-dextran and rhodamine 123 across rat
intestinal mucosa . Regardless of these promising results and the fact that
polymeric platforms such as poly(acrylic acid) have been approved for oral and
topical administration routes, these thiolated derivatives have not been approved
improved when dendrimers are added to the medium . In any event, the
as pharmaceuticals. This constitutes a hard nut to crack towards their clinical
implementation, especially if parenteral administration is envisioned.
4.5. Miscellaneous polymers
Different pharmaceutical excipients and drug carriers displaying a variety of
structural and compositional features have been also sporadically explored as ABC
blockers or means to circumvent resistance, though these investigations have not
been comprehensive enough to take them to a higher level. Dendrimers are
hyperbranched polymers with a well-defined, three-dimensional architecture and a
relatively low molecular weight polydispersity and a high and tunable functionality.
D´Emanuele et al. conjugated propranolol and terfenadine to modified
polyamidoamine dendrimers and improved the transport across Caco2 cell
monolayers [248,249]. However, the mechanism for the improved passage was not
related to the inhibition of P-gp; the permeability to the drug did not increase when
cells were exposed to the free dendrimer. These findings indicated that the drug-
polymer conjugate, a new chemical entity, was not a substrate of the pump and
evaded its efflux transport. Other groups showed that the accumulation could be
inconsistent evidence of the potential of these nanocarriers in ABC-mediated
resistance and the current regulatory status make their clinical translation unlikely.
Cyclodextrins (CDs) are macrocyclic oligosaccharides that display a hydrophobic
cavity and a hydrophilic surface . The formation of drug/CD inclusion or non-
inclusion complexes enables their aqueous solubilization and physicochemical
stabilization. Due to their good biocompatibility and approval by the U.S.-FDA, the
and P-gp in mouse mastocytoma P-815 cells . CD depleted cholesterol,
European Medicines Agency, CDs are among the most extensively used
pharmaceutical excipients. At the same time, it should be stressed that CDs are
not inert and that some derivatives (e.g. -CD) can interact with the lipid rafts of the
cell membrane and to extract cholesterol, leading to cytotoxic effects and to
alterations of different cellular pathways . For example, cholesterol levels
modulate the activity of BCRP  and ABCA1 and ABCG1 [253,254] and
changes could affect the functional activity of the pump. In this context, this
interaction could be capitalized to improve the therapeutic outcomes of those drugs
that are substrates of cholesterol-dependent pumps. Only a few works explored
this effect. Several decades ago, Okada and co-workers described the synthesis of
a new chemically modified -CD, namely 6-O--maltosyl--cyclodextrin that
conserves the capacity to form complexes with cholesterol and presents negligible
cytotoxic effects on erythrocytes and Caco2 cells [255,256]. More recently, this
research group resumed the investigations and explored the effect of cholesterol
depletion with 6-O--maltosyl--cyclodextrin and restoration with 6-O--maltosyl--
cyclodextrin/cholesterol complex on the expression of the proteins ABCA1, ABCG1
resulting in a decrease of the expression of ABCA1 and ABCG1. Then, incubation
of cells with a cholesterol/CD complex restored the levels of expression of both
pumps. Conversely, no changes were observed in the accumulation of rhodamine
123, indicating that it does not have any effect on P-gp. These results were in
agreement with previous studies conducted in P-gp-bearing liposomes lacking
added cholesterol . It should be stressed that P-gp can migrate within the
Clinical status and future perspectives
membrane from zones of low to those of high cholesterol concentration. Thus,
these changes in cholesterol levels could modulate the activity of the pump, as
shown by Arima et al. . Other authors also suggested that changes in
cholesterol levels might lead to the modification of the intracellular trafficking of
membrane proteins and the concentration of functional P-gp molecules in the
membrane. For example, cholesterol saturation accelerated the internalization of
P-gp molecules, while cholesterol depletion did not have any significant effect
. In any event, the experience gained with CDs is still far from being robust
enough to address preclinical studies. Moreover, owing to this ability to sequester
cholesterol, -CDs are not feasible for administration by the IV route. In this
context, their incorporation into pharmaceutical products as ABC inhibitors appears
at least as controversial, if not unfeasible. On the other hand, other less risky
administration routes (e.g. oral) could found in these approved excipients a Trojan
horse to overcome ABC-mediated drug resistance or decreased bioavailability.
Studies in this direction are still missing.
ABC-mediated drug resistance has become a great hurdle towards the attainment
of therapeutic success in many different types of cancer. More recently, these
mechanisms were also unveiled in infectious diseases that claim millions of lives
every year. After the discovery of P-gp, several transmembrane proteins involved
in MDR were proposed as valuable cellular targets to improve the treatment of
cancer [261-268]. In this context, the co-administration of antitumoral drugs
advantageous feature of these components is that, since they do not display any
together with inhibitors of specific transporters was envisioned as a breakthrough
in the struggle of clinicians with multidrug resistance . However, the initial
prospects were probably overestimated. The use of pharmacologically active ABC
inhibitors such as verapamil and cyclosporin A was rapidly turned down due to
severe adverse effects. Moreover, the effect of the inhibition on the accumulation
of drugs and the imbalance of the immune response in cancer (that is also
governed by these pumps) has not been comprehensively investigated. The
search for specific blockers gained impulse over the last years and novel agents
began to be designed and clinically trialed [269-271]. At the same time, imagining a
scenario where these molecules are completely deprived of any secondary
pharmacological effect probably appears as an utopy, posing also complex
questions regarding the versatility of these pharmacological blockers in different
types of cancer and in combination with different antitumoral drugs.
The present article overviewed the different investigations aiming to improve the
accumulation and transport of ABC substrates by means of the inhibtion of the
pump employing 'inert' GRAS nanopharmaceutical excipients. The most
inherent pharmacological activity, less potentially adverse effects could be
expected. The analysis of each study referred was careful and concious in terms of
the goals, the methodologies and the findings and only studies where the
improvement stemmed from the inhibition of the ABC were considered.
The application of 'inert' pharmaceutical excipients emerged first as an interesting
academic niche that after many years of research was ascertained in preclinical
and clinical studies with the approval of an innovative medicine for the treatment of
esophageal cancer. This is the most convincing evidence that one could expect for
a development of this kind. On the other hand, while it supports the value of the
technology, it does not pave the way to the indiscriminated use of any GRAS
nanopharmaceutical in MDR. In other words, each therapeutic application should
be specifically evaluated. At the same time, this is only a promising beginning and
the potential of this strategy could be explored and capitalized in other diseases
such as HIV, where the development of novel drugs or treatment adjuvants is
jeopardized by the fact that most of the patients live in developing nations and the
market profitability is more restricted. Still, questions such as whether the inhibition
of ABCs could make treatments independent of pharmacogenetic polymorphism or
not, remain unanswered. Furthermore, even if 'inert', the uncontrolled inhibition of
ABCs without adverse effects ascribed to the excipient could represent a
disadvantage due to drug accumulation and possible toxicity. In this framework, the
investigation of these fundamental issues is still pending and should be addressed
before we will be able to appreciate the potential of this therapeutic strategy.
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