Polymer conjugates: nanosized

medicines for treating cancer

Mara J. Vicent
1,2
and Ruth Duncan
1
1
Centre for Polymer Therapeutics, Welsh School of Pharmacy, Cardiff University, King Edward VII Avenue, Cardiff, UK, CF10 3XF
2
Centro de Investigacio n Pr ncipe Felipe-FVIB, Av. Autopista del Saler 16, E-46013 Valencia, Spain
Interdisciplinary research at the interface of polymer
chemistry and the biomedical sciences has produced the
rst polymer-based nanomedicines for the diagnosis
and treatment of cancer. These water-soluble hybrid
constructs, designed for intravenous administration, fall
into two main categories: polymerprotein conjugates
or polymerdrug conjugates. Polymer conjugation to
proteins reduces immunogenicity, prolongs plasma
half-life and enhances protein stability. Polymerdrug
conjugation promotes tumor targeting through the
enhanced permeability and retention (EPR) effect and,
at the cellular level following endocytic capture, allows
lysosomotropic drug delivery. The successful clinical
application of polymerprotein conjugates (PEGylated
enzymes and cytokines) and promising results arising
from clinical trials with polymer-bound chemotherapy
(e.g. doxorubicin, paclitaxel, camptothecins) has
provided a rm foundation for more sophisticated
second-generation constructs that deliver the newly
emerging target-directed anticancer agents (e.g. modu-
lators of the cell cycle, signal transduction inhibitors and
antiangiogenic drugs) in addition to polymerdrug
combinations (e.g. endocrine- and chemo-therapy).
Introduction
Cancer is a major cause of mortality affecting one in three
individuals in the UK and killing one in four. Worldwide
incidence of cancer continues to increase, largely owing to
the aging population. Two distinct approaches are being
taken in the search for improved anticancer treatments.
The rst involves the use of genomics and proteomics
research to assist in the identication of unique targets [1]
that will aid the synthesis of the perfect t drug molecule
with exquisite therapeutic activity and no side effects.
Despite some successes, such as the use of the tyrosine
kinase inhibitor Glivec
w
(Gleevec
w
in the US) (http://www.
gleevec.com), as a treatment for chronic myeloid leukemia
(CML) [2] and gastrointestinal cancer, this concept has
proved difcult to realize in practice. Improvements in the
survival of patients with common metastatic tumors, such
as breast, prostate, lung and gastrointestinal cancers,
have been slow to materialize. Poorly predictive preclini-
cal models, lack of tumor target specicity, lack of effective
cellular and intracellular delivery, and, most importantly,
development of drug resistance are blamed for the
slow progress.
A second, and complementary, approach is the design
of systems for drug targeting that are better able to
guide drugs precisely to the tumor cells, at increased
concentrations and away from sites of dose-limiting
toxicity. First-generation technologies include liposomes,
for example DaunoXomee (http://www.gilead.com/wt/
tert_uk/daunoxome_uk) and Doxil
w
/Caelyx
w
(http://www.
doxil.com) [3,4], antibodydrug conjugates, for example
Mylotarg
w
(http://www.pharmacist.com/pdf/mylotarg.pdf)
[5], and also several polymer conjugates carrying either
low-molecular-weight drugs or proteins [610]. There is
increasing optimism that nanotechnology applied to
medicine will bring signicant advances in the diagnosis
and treatment of cancer [11] and these hybrid multi-
component polymer-containing systems can be viewed as
the rst-generation of nanomedicines to be used for
cancer treatment.
Synthetic and natural polymers have an established
role as biomedical materials, including their use to
fabricate prostheses and soft contact lenses [12]. They
are also used as the pharmaceutical excipients in drug
formulations [13]. During the past decade, the importance
of polymeric drug-delivery systems in oncology has grown
exponentially with the advent of biodegradable polymeric
implants. In this form, they are used as a subcutaneous
depot that slowly releases luteinising hormone-releasing
hormone (LHRH) analogues, for example Zoladex
w
(http://
www.zoladex.net) and Lupron Depot
w
(http://www.lupron.
com) for treatment of prostate and other hormone-
dependent cancers [14], and, when implanted post-
surgery, they provide local delivery of chemotherapy for
the treatment of brain cancer, for example Gliadel
w
Wafer
(http://www.gliadel.com) [15].
The term polymer therapeutics [8] (Box 1) has been
adopted to encompass several different families of
polymer-based drugs, polymeric pro-drugs and polymer-
based delivery systems; this term includes the water-
soluble polymer conjugates of the drugs and proteins
described here. Unlike polymeric implants designed for
sustained or local drug release, these water soluble,
therapeutic polymer-based conjugates are designed for
parenteral administration, often in the treatment of
disseminated metastatic disease.
Both polymerdrug and polymerprotein conjugates
must be carefully designed for their individual use, taking
Corresponding author: Vicent, M.J. (mjvicent@cipf.es).
Available online 22 November 2005
Review TRENDS in Biotechnology Vol.24 No.1 January 2006
www.sciencedirect.com 0167-7799/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2005.11.006
into account the nature of the individual drug payload
(protein, low-molecular-mass drug) and the location of each
molecular pharmacological target. Both families of polymer
conjugates are tailor-made using a basic tripartite structure
(Table 1) and they contain a water-soluble polymer, a linker
and the bioactive agent. It has become clear that the
molecular mass and physico-chemical properties of the
polymer are frequently the most important drivers gover-
ning biodistribution, elimination and metabolism of the
conjugate as a whole; therefore, the choice of a suitable
polymer is vital. In all cases, the water-soluble polymeric
carrier must be non-toxic, non-immunogenic and suitable
for repeated administration. Poly(ethyleneglycol) (PEG)
[6,7], N-(2-hydroxypropylmethacrylamide) (HPMA)
copolymers [9,10], and poly(glutamic acid) (PGA) [16,17]
are the most widely tested in a clinical setting. Of these
polymers, only PGA is biodegradable; consequently, the
molecular masses of PEGand HPMAcopolymers have been
limitedto !40 kDa to ensure eventual renal elimination. In
the case of non-degradable polymers, the polymerdrug or
protein linker is an important design feature (Table 1).
Although polymerprotein and polymerdrug conjugates
possess many similarities, the biological rationales behind
their respective designs have been very different.
Polymerprotein conjugates
During the past 15 years, an increasing number of
polymerprotein conjugates have entered into routine
clinical use in oncology (Table 2).
SMANCS: a polymerprotein conjugate for localized
administration
In 1990, SMANCS (Zinostatin stimalamer
w
, http://www.
yamanouchi.com) was the rst polymerprotein conjugate
to be brought to market. It was designed by Maeda and
colleagues [18,19] with the aim of creating a conjugate
suitable for local administration, using the femoral artery
to access the hepatic artery, in patients suffering from
primary liver cancer (hepatocellular carcinoma) [18]. The
conjugate contains two polymer chains of styrene-co-
maleic anhydride (SMA) covalently bound to the anti-
tumor protein neocarzinostatin (NCS). This increases
lipid-solubility and enables the administration of
SMANCS in the phase-contrast agent Lipiodol, which
increases plasma half-life, allows tumor visualization and
improves the degree of tumor targeting. A remarkable
tumor to blood ratio of O2500 was recorded using
SMANCS [20]. Furthermore, a large number of hepatoma
Box 1. Denition of polymer therapeutics
The umbrella term polymer therapeutics was coined to describe
polymers used as either polymeric drugs or as components of
polymerdrug conjugates, polymerprotein conjugates, polymeric
micelles (to which a drug is covalently bound) and multi-component
polyplexes being developed as non-viral vectors [8]. In each case, a
specic water-soluble polymer is tailored to be the bioactive agent,
or to act as an inert functional part of a multi-faceted construct. The
aim is to improve drug, protein or gene delivery, and there is
considerable hope that such nanosized medicines, designed using
advanced polymer chemistry and precision engineering at a
molecular level, together with an appreciation of the patho-
physiology of normal and diseased tissue will help to realize the
full therapeutic potential of the post-genomics era. From the
industrial standpoint, these nanosized medicines (5100 nm) are
more like new chemical entities rather than conventional drug
delivery systems or formulations, which simply entrap, solubilize or
control drug release without resorting to chemical conjugation.
Through the clinical use of polymerprotein conjugates, and clinical
development of polymeranticancer drug conjugates (Table 2),
polymer therapeutics is already becoming well established as a
new class of therapy for cancer treatment.
Table 1. Chemical characteristics of polymer anticancer conjugates
Compound Polymer characteristics Linker Drug (loading) Cleavage conditions
Polymer M mass (g/mol)
Polymerprotein conjugates
SMANCS Styren-maleic anhydride
(SMA) copolymer
15000 Amine Neocarzinostatin Non-biodegradable
Oncaspar
w
m-PEG 5000 Amide L-asparaginase Non-biodegradable
Neulastae m-PEG 20000 Amide G-CSF Non-biodegradable
PEG-Asys
w
Branched m-PEG 40000 Amide IFNa-2a Non-biodegradable
PEG-introne m-PEG 12000 Carbamate IFNa2b b-lactamase or basic
hydrolysis
Polymerdrug conjugates
XYOTAXe Poly-L-glutamic acid
(PGA)
40000 Ester Paclitaxel (37wt%) PGA degraded by
cathepsin B and ester
linker by esterases or acid
hydrolysis
PK1 HPMA copolymer 30000 Amide Doxorubicin (8.5 wt%) Thiol protease cathepsin B
PK2 HPMA copolymer 25000 Amide Doxorubicin (7.5 wt%)
and galactosamine
(1.52.5 mol%)
Thiol protease cathepsin B
AP5280;
AP5346
HPMA copolymer 25000 Malonate;
Malonate-DACH
Pt (w7 wt%) Hydrolysis
CT-2106 Poly-L-glutamic acid
(PGA)
40000 Ester Camptothecin
(3335 wt%)
PGA degraded by
cathepsin B and ester
linker by esterases or
acid hydrolysis
PROTHECANe PEG 40000 Ester Camptothecin
(1.7 wt%)
Esterases or acid
hydrolysis
Mw: Molecular weight; SMANCS: poly(styrene-co-maleic anhydride)-neocarzinostatin; G-CSF: granulocyte colony-stimulating factor; HPMA: N-(2-hydroxypropyl)
methacrylamide; m-PEG: monomethoxy poly(ethylene glycol); PEG: poly(ethylene glycol); DACH: diaminocyclohexane; IFN-a: interferon-a.
Review TRENDS in Biotechnology Vol.24 No.1 January 2006 40
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patients treated with SMANCS had tumor shrinkage
(95%) and decreased afetoprotein levels (86%) [18].
PEG-ylated proteins: a step towards more convenient
and safer protein therapeutics
Although the number of peptide-, protein- and antibody-
based drugs entering clinical use is growing rapidly, the
limitations of these often include a short plasma half-life,
poor stability and, in the case of proteins, immunogeni-
city. Through their pioneering research in 1970s, Davis,
Abuchowski and colleagues developed the concept of
polyethylene glycol (PEG)protein conjugation (PEGyla-
tion) [21], and the continuing entry of PEGylated
proteins into routine clinical use has been responsible
for a paradigm shift in the acceptance of polymer-based
macromolecular medicines. This technique is used to
increase protein solubility and stability, and reduce
protein immunogenicity; moreover, polymer conjugation
prolongs plasma half-life through prevention of renal
elimination and avoidance of receptor-mediated protein
uptake by cells of the reticuloendothelial system (RES).
Consequently, polymer-conjugated therapeutics require
less frequent dosing, which is a great benet to the
patient [6,7,22].
For optimized synthesis of a polymerprotein conjugate,
a semi-telechelic polymer (one with a single reactive group
at one terminal end) is required to avoid protein cross-
linking during conjugation; furthermore, the linker used
must be chosen carefully to ensure that the linking
chemistry will not generate toxicity or immunogenicity
(Table 1). Appropriate stability characteristics are also
required, and these are dependent on the protein being
bound [6,7]. The synthetic approach requires reproducible,
site-specic protein modication. Linear and branched
PEGs with molecular masses of 5 000 40 000 g/mol have
been used to create protein conjugates, and the PEG
molecular mass chosen has a great impact on their
pharmacokinetics [6,7]. In some cases, multiple PEGs
have been attached per protein molecule, but today a 1 to
1 polymer to protein ratio is often preferred [23].
PEGylation of a therapeutic protein can lead to a reduction
in bioactivity and even the possibility of inactivation. To
avoid a randomly modied protein surface, altered overall
protein charge and reduced substrate or receptor binding
afnity, site-specic PEGylation of proteins following
mutagenesis can be performed (Table 1) [24]. Moreover, a
degradable PEGprotein linkage can be used to maximize
the protein bioactivity [25].
PEGenzyme conjugates
In 1994, PEG-L-asparaginase (Oncaspar
w
) (http://www.
enzon.com) was the rst anti-tumor PEGylated protein to
be approvedfor clinical use [26,27]. This conjugate contains
multiple PEG chains (molecular mass w5 000 g/mol)
linked to the enzyme, and it is used as a treatment for
acute lymphoblastic leukemia(ALL) adisease that has an
essential requirement for the amino acid L-asparagine. The
native enzyme induces hypersensitivity reactions and has
a relatively short plasma half-life (830 h), which necessi-
tates dailyadministrationfor 4weeks, whereas the PEGL-
asparaginase conjugate has a plasma half-life of w14 days
and can, therefore, be administered by a 1 h infusion every
2 weeks. Furthermore, Oncaspar
w
is used in combination
with chemotherapy to treat those children with ALL who
are hypersensitive to the native enzyme [8] due to the
reduced immunogenicity of the conjugate compared with
that of the native protein [28].
Currently, other PEGylated enzymes are undergoing
clinical development. PEGrecombinant arginine deimi-
nase (rhArg) is being developed as a treatment for
Table 2. Polymerprotein and polymerdrug conjugates
Compound Name Status Comment
Polymerprotein conjugates
SMANCS Zinostatin Stimalmer
w
Market Hepatocellular carcinoma
PEGL-asparaginase Oncaspar
w
Market Acute lymphoblastic leukamia
PEGGCSF Neulastae Market Prevention of neutropenia associated with
cancer and AIDS chemotherapy
PEGinterferon a 2a PEG-Asys
w
Market Hepatitis B and C
Phase I/II Melanoma, chronic myelogenous leukemia
and renal cell carcinoma
PEGinterferon a 2b PEG-Introne Market Hepatitis C
Phase I/II Melanoma, multiple myeloma and renal cell
carcinoma
PEGarginine deiminase ADI-PEG20 Phase I Hepatocellular carcinoma
PEGglutaminase combined with a
glutamine antimetabolite
6-diazo-5-oxo-L-norleucine (DON)
PEG-PGA and DON Phase I/II Various
Polymerdrug conjugates
Polyglutamatepaclitaxel CT-2103; XYOTAXe Phase II/III Various, particularly non small cell lung
cancer; ovarian cancer
Polyglutamatecamptothecin CT-2106 Phase I Various
HPMA copolymerdoxorubicin PK1; FCE28068 Phase II Various, particularly lung and breast cancer
HPMA copolymerdoxorubicin-galacto-
samine
PK2; FCE28069 Phase I/II Particularly hepatocellular carcinoma
HPMA copolymercarboplatin platinate AP5280 Phase I/II Various
HPMA copolymerDACHplatinate AP5346 Phase I/II Various
PEGcamptothecin PROTHECANe Phase II Various
SMANCS: poly(styrene-co-maleic anhydride)-neocarzinostatin; G-CSF: granulocyte colony-stimulating factor; HPMA: N-(2-hydroxypropyl)methacrylamide; PEG:
poly(ethylene glycol); DACH: diaminocyclohexane.
Review TRENDS in Biotechnology Vol.24 No.1 January 2006 41
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hepatocellular carcinoma as a single agent, which depletes
arginine, and also in combination with 5-uorouracil
(5-FU) [29]. In a phase I/II study involving 35 patients,
rhArg was administered by weekly intramuscular injec-
tion: one patient showed tumor shrinkage, two had stable
disease, and all patients had arginine levels !2 mM[30]. A
combination of PEGylatedglutaminase (PEGglut) and
the glutamine antimetabolite 6-diazo-5-oxo-L-norleucine
(DON), is also being clinically evaluated, based on the
hypothesis that DON will be more effective when
glutamine levels are depleted. In a phase I study [31],
patients were treated with 120 IU m
K2
of PEGglut in
combination with an increasing dose of DON. Both agents
were administered twice a week, with DON administered
4 h after the PEGglut conjugate. To date, ve patients
have stable disease and one patient, with colorectal
cancer, has a decline in levels of the carcinoembryonic
antigen marker.
Polymercytokine conjugates
PEGgranulocyte colony stimulating factor (PEGGCSF)
(Neulastae/PEG-lgrastim) (http://www.amgen.com) is
currently used to prevent severe cancer chemotherapy-
induced neutropenia [3234]. Administration of one
dose of PEGGCSF (100 mg kg
K1
), by subcutaneous
injection on day 2 of each chemotherapy cycle, gives the
equivalent neutrophil support when compared with GCSF
(5 mg kg
K1
day
K1
) given by daily injection throughout the
chemotherapy cycle [32]. The half-life of PEGGCSF is
longer (1580 h) compared with that of the native protein
(1.37.2 h). Although some allergic reactions have been
seen following administration of GCSF, none was observed
in clinical trials using the PEG conjugate, and the major
side effect observed with both free and conjugated GCSF is
bone pain (%26% patients) [3234].
PEGinterferon-a conjugates PEG-Introne (http://
www.schering-plough.com) and PEGASYS
w
(http://www.
roche.com) are being investigated as anti-tumor agents
in their own right [35]. Interferon-a (IFN-a) is active in
melanoma and renal cell carcinoma (RCC) patients, but
the side-effects associated with IFN-a include mild-to-
moderate nausea, anorexia, fatigue and depression. The
half-life of IFN-a (2.3 h) necessitates administration three
times per week. Both of these PEGIFN-a conjugates have
been marketed for the treatment of hepatitis C (http://
www.pegasys.com and http://www.pegintron.com) and
their usefulness as anticancer agents is currently being
investigated. In a phase I/II study, patients with advanced
solid tumors (primarily RCC) were given once-weekly
PEGylated IFNa-2b at doses from 0.75 to 7.5 mg kg
K1
for 12 weeks; the maximum tolerated dose (MTD) for
PEGylated IFNa-2b at 12 weeks was 6.0 mg kg
K1
week
K1
.
The conjugate was active and well-tolerated in patients
with metastatic solid tumors, and in a group of 44
previously untreated RCC patients: the objective response
rate was 14% [35]. Recently, a phase II clinical trial has
investigated the combination of PEGylated IFNa-2b,
GM-CSF and thalidomide as a treatment for RCC. The
rationale behind this choice is the desire to achieve
synergistic enhancement of their respective apoptotic,
anti-angiogenic and immunomodulatory activities. Of the
four patients evaluated so far, one patient has stable
disease [36].
At this point, the important contribution that PEG-
based medicines have already made to the use of new
macromolecular drugs in the clinic is clear. Based on these
observations, it is expected that this eld will expand at an
exponential rate.
Polymerdrug conjugates
Parallel to the emergence of polymerprotein conjugates,
and from the combination of the realization by De Duve
that the endocytic pathway might be useful for
lysosomotropic drug delivery [37] and the vision of
Ringsdorf for the idealized polymer chemistry for drug
conjugation [38], the concept of targetable polymerdrug
conjugates was developed (Table 1 and Figure 1). Drug
conjugation to a water-soluble polymer platform restricts
cellular uptake to the endocytic pathway and, hence,
allows tumor-specic targeting of low-molecular-mass
chemotherapeutic agents in addition to limiting the
access of the conjugate to the normal sites of toxicity.
Use of a water-soluble polymer as a platform also helps
to solubilize hydrophobic drugs (e.g. doxorubicin and
paclitaxel), making a more convenient formulation to
administer intravenously. Examples of polymerdrug
conjugates that have entered the clinical development
stage are listed in Table 2 and Figure 1. The proposed
mechanism of action of HPMA copolymerdoxorubicin is
shown schematically in Figure 2 [10].
In this case, the ideal polymerdrug linker is stable
during transport to the tumor, but able to release drug at
an optimum rate on arrival; because many of the drugs
being transported exert their effects through an intra-
cellular pharmacological receptor, it is essential that drug
release actually occurs. Peptidyl linkages, which are
stable in plasma but have been optimized for cleavage
following endocytic capture by lysosomal thiol-dependant
proteases, have been used to synthesize the clinically
tested HPMA copolymerdrug conjugates [9,10]. PGA
paclitaxel (XYOTAXe) (http://www.cticseattle.com) has a
high paclitaxel content (w37 wt%) and, in this case, the
drug is attached by an ester bond. The conjugate, with its
high drug-payload, is stable when traveling to the site of
action, but intracellular degradation of the polymer
backbone, by lysosomal thiol-dependant proteases [39],
releases diglutamyl paclitaxel, which subsequently
liberates the drug. Linkers based on the cis-aconityl, acetal
or hydrazone moiety undergo pH-dependent hydrolysis
following internalization of the conjugate into the
acidic endosomal and lysosomal compartments. An HPMA
copolymerhydrazonedoxorubicin conjugate has recently
shown signicantly improved anti-tumor activity against
lymphoma in vivo [40]. Adequate drug carrying capacity, in
relation to the potency of the agent being carried, is
essential, as is the ability to target the tumor by an
active (receptorligand) or a passive (pathophysiological)
mechanism: both of these are important design features.
The hyperpermeability of tumor vasculature is one of
the keyfactors governingthe successful targetingof atumor
by polymer-based cancer therapies. After intravenous
administration, the leakiness of the angiogenic tumor
Review TRENDS in Biotechnology Vol.24 No.1 January 2006 42
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vasculature allows selective extravasation of the conjugate
in tumor tissue. Additionally, tumor tissue frequently lacks
an effective lymphatic drainage, which subsequently
promotes polymer retention. The combination of these
factors leads to an accumulation of the conjugate in tumor
tissue a passive targeting phenomenon named by Maeda
as the enhanced permeability and retention (EPR) effect
[41] (Figure 2a). EPR-mediated tumor targeting is
HN
HO
O
HN
NH
O
O
O
NH
O
O
O
NH
OH
O
O O
OH
OH O
HO
O
HO
HN
NH
O
O
O
NH
O
O
R
x
y
z
Ligand(R) Target cell
Galactosamine hepatocytes
MSH melanoma
RGD endothelial
EBV peptide lymphocytes
OV-TL ovarian carcinoma
Anti-Thy-1.2 mAb leukemia and lymphoma
HN
HO
O
HN
NH
O
O
O
NH
O
O
O
NH
OH
O
O O
OH
OH O
HO
O
HO
95
5
PK1 AP5280
HN
HO
O
HN
NH
O
O
O
NH
O
O
95
5
O
Pt
N
+
Na
-
O
O
O
NH
3
NH
3
CT-2106
H
N
O
H
N
O
n m
O
O
-
Na
+
O
O
O
N
N
O
O
H
N
O
H
N
O
n m
O
O
-
Na
+ O
XYOTAX
TM
NH
O
O
HO
O
O
O
O
O
OH
O
O
O
O
O
H
N
O
p
O
HN
O
O
OH
O
O
O
O
O
O
O
O
O
O
OH
(a)
(b)
Figure 1. Polymerdrug conjugates being evaluated as anticancer agents. (a) Conjugates designed for passive targeting through the EPR effect: N-(2-hydroxypropylmetha-
crylamide (HPMA) copolymerdoxorubicin (PK1; FCE28068); HPMA copolymerplatinate (AP5280); poly-L-glutamic acidcamptothecin conjugate (CT-2106); Poly-L-glutamic
acidpaclitaxel conjugate (XYOTAXe; CT-2103). (b) A conjugate designed for tumor targeting using receptor-mediated endocytosis: HPMA copolymerdoxorubicin
galactosamine (PK2; FCE28069) designed to promote liver targeting through the asialoglycoprotein receptor. Other ligands explored for targeting are also shown.
Review TRENDS in Biotechnology Vol.24 No.1 January 2006 43
www.sciencedirect.com
ultimatelydrivenbythe circulatingplasmaconcentrationof
the polymer conjugate [42].
It is hoped that the use of receptor-targeting ligands
will lead to improved tumor targeting by polymerdrug
conjugates by building on the EPR effect [43]. Although
many HPMA copolymer conjugates have been designed
to contain saccharides [44], antibodies [45,46], proteins
and peptides [47,48] as targeting ligands, only one
conjugate has progressed into clinical trial, so far:
HPMA copolymerdoxorubicingalactosamine (PK2,
FCE28069) (Figure 1b). In this case, the overhanging
galactosamine moieties localize to the asialoglycoprotein
receptor in hepatocytes. Hepatocellular carcinoma cells,
and the conjugate, have been used to assess the potential
of this as a possible treatment for liver cancer [44].
During the past decade, eleven polymerdrug con-
jugates have entered phase I/II clinical trials as anti-
cancer therapeutics (Table 2); the reduced toxicity of the
bound drug and anti-tumor activity in chemotherapy-
refractory patients have clearly achieved proof-of-
concept status [49]. Most of these conjugates have
used HPMA copolymer as the water-soluble polymer
platform. Most recently, two HPMA copolymer
platinates, AP5280 and AP5346, have shown promising
results in phase I trials (with MTD of 4500 mg Pt m
K2
and 1280 mg Pt m
K2
, respectively), and are currently
entering phase II [50,51]. In December 2004, an
investigational new drug application was led with
the Food and Drug Administration (FDA) for the
DACHplatinate conjugate, AP5346.
TRENDS in Biotechnology
(a) Whole organism level
Kidney
(b) Cellular level
H
+
H
+
H
+
enz
(ii) Increased tumor targeting
due to the EPR effect
Tumor
(iii) Effects on endothelial cells
and vascular permeability
(i) Decreased exposure of normal
tissues due to retention in the
bloodstream
Administration
of conjugate
(iv) Immunostimulation
(v) Increased renal
clearance
of conjugated drug
Uptake by fluid-phase
pinocytosis
Uptake by receptor-mediated
pinocytosis
Exocytosis
and receptor
recycling
Recycling
Endosome
Lysosome
Drug release
trigerred by
pH or lysosomal
enzymes
H
+
Bypasses
P-gp and MRP
MDR1; MRP
Exocytosis of non
degradable polymer
Figure 2. The mechanism of action of polymeranticancer drug conjugates at (a) the whole organism level, and (b) the cellular level, focusing on the intracellular
lysosomotropic delivery.
Review TRENDS in Biotechnology Vol.24 No.1 January 2006 44
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HPMA copolymerGlyPheLeuGlydoxorubicin (or
epirubicin), containing human immunoglobulin (HuIg),
was synthesized for the treatment of patients on a case-by-
case basis, as required. So far, these have also been tested
clinically in six patients [52,53]. The HuIg used was either
prepared from autologous IgG removed from sera by
precipitation with 40% ammonium sulfate or was a
commercially available allogenic human g-globulin.
These studies monitored the progression of the disease
in addition to a large number of biochemical and
immunological parameters. Although it is difcult to
assess the data obtained, objectively, in the context of
good clinical practice (GCP) guidelines, it is interesting to
note that, in some patients, anti-tumor effects were seen.
The conjugate did not seem to induce anti-Ig antibodies,
and increased levels of CD16
C
56 and CD4
C
cells in
peripheral blood along with the activation of NK and LAK
cells supported the suggestion that HPMA copolymer
doxorubicin conjugates can be immunostimulatory.
The PGApaclitaxel conjugate XYOTAXe is the most
advanced anticancer drug conjugate in clinical trials. It
has shown anti-tumor activity against several different
cancers. Recently reported phase III trials show that
XYOTAXe offers a less toxic and more convenient
treatment option for poor performance status (PS2) non-
small cell lung cancer (NSCLC) patients. As a single
agent, or combined with other drugs or radiotherapy,
XYOTAXe can prolong patient survival, for example in
PS2 patients with NSCLC [54], and when administered as
a rst-line therapy in combination with carboplatin to
advanced stage III/IV primary ovarian cancer patients
with a 98% response rate [55]. XYOTAXe has also been
reported as a novel radiation sensitizer: when given in
combination with radiation therapy, it elicited a major
tumor response in 81% of patients with esophaegeal or
gastric cancer [56]. Lessons have also been learnt from
many of the early clinical studies, for example the failure
of HPMA conjugates of paclitaxel and camptothecin in
phase I clinical trials. Such negative outcomes underline
the importance of designing a polymerdrug linker that is
stable in transit to the tumor [57,58].
Conclusions and future opportunities
With the rst polymerprotein anti-tumor drugs on the
market, and an increasing number of polymerdrug
conjugates in clinical testing with a clear possibility of
reaching the market within the next year, polymer
anticancer conjugates are becoming an established
addition to the anticancer armory. However, there are
still many challenges to address and opportunities to
develop this technology further.
Novel polymeric carriers
The development of better polymeric carriers is ongoing.
There is a need to develop high-molecular-mass, biode-
gradable polymeric carriers that can better exploit EPR-
mediated tumor targeting. PEGpolyacetals that show
pH-dependent degradation [59] and dextrins [60] that are
degraded by amylase might be two practical options for
such carriers. There is an urgent need to move away from
heterogeneous, random-coiled, polymeric carriers towards
better dened polymer structures. Dendrimers and
dendronized polymers combine the features of a mono-
disperse nanoscale geometry with high end-group density
at their surface, and are attractive options for immobili-
zation of anticancer drugs [61], as imaging agents [62]
and/or as targeting moieties [63,64]. Although dendrimer
imaging agents have been evaluated clinically, there is
still a need to establish the safety and chemical
characteristics of many of the dendrimer structures
currently being developed. Other interesting polymer
architectures under evaluation include: hyperbranched
polymers [65], block copolymers [66], stars [67] and hybrid
glyco- [68] and peptide-derivatives.
From the therapeutic viewpoint mentioned at the
outset, there is a need to improve the existing therapeutic
strategies. For example, a potential mechanism of
resistance affecting polymer conjugates that require
endocytic uptake and lysosomal enzyme activation could
be reduced internalization or a decrease in the levels of an
activating enzyme; therefore, polymer-based strategies for
extracellular drug delivery are also being developed.
Polymer-directed enzyme pro-drug therapy (PDEPT) is a
two-step approach that uses a polymer-bound enzyme to
activate a polymerdrug conjugate, present in the tumor
interstitium, where the linker is a substrate for the
activating enzyme. In vivo proof-of-concept has been
reported using this strategy [69,70].
Polymer conjugates containing drugs directed against
novel anticancer targets [7176] are also emerging. The
rst polymeric anti-angiogenic conjugate, HPMA copoly-
merTNP-470, has been recently described [77]. Prelimi-
nary attempts have been made to develop therapies that
target the apoptotic signaling cascade at the molecular
level: promising in vivo results have been seen for a
targeted pro-apoptotic anticancer drug delivery system
that has been developed for the treatment of ovarian
cancer [78]. The combination of chemotherapy and
photodynamic therapy using immunoconjugates is also
being explored [79].
Finally, it is important to mention that use of polymer
drug conjugates in combination therapy is also seen as an
important opportunity to enhance tumor response rates
[54,55]. The polymeric carrier provides an ideal platform
for the delivery of a cocktail of drugs simultaneously. We
have recently reported the rst endocrinechemotherapy
combination in the form of the model compound HPMA
copolymeraminoglutethimidedoxorubicin [80]. It was
discovered that conjugates containing both drugs showed
markedly enhanced cytotoxicity compared with HPMA
copolymerdoxorubicin, a conjugate that has already
shown clinical activity in breast cancer patients [49],
whereas mixtures of polymer conjugates containing only
aminoglutethimide or only doxorubicin did not show a
synergistic benet. These observations underline the
possibility of designing polymerdrug combinations for
improved treatment of breast and prostate cancer in
the future.
Realization of the full therapeutic potential of these
novel nanomedicines has only been possible through
multidisciplinary research involving biologists, chemists,
clinicians and industry. It is important to note that
Review TRENDS in Biotechnology Vol.24 No.1 January 2006 45
www.sciencedirect.com
polymerprotein conjugates are also being used to
successfully treat diseases other than cancer (e.g. PEG
interferon-a can be used to cure hepatitis C). It is clear
that the application of polymer therapeutics for the
treatment of cancer is just the beginning.
Acknowledgements
M.J.V. would like to thank the European Commission for a Marie Curie
Fellowship (HPMF-CT-200201555).
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