Download 1

Journal of Controlled Release 74 (2001) 147–158
www.elsevier.com / locate / jconrel
Water soluble polymers in tumor targeted delivery
ˇ a,b , *, P. Kopeckova
ˇ
´ a , T. Minko a , Z.-R. Lu a , C.M. Peterson c
J. Kopecek
a
Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, 30 South 2000 East, Room 301, Salt Lake City,
UT 84112, USA
b
Department of Bioengineering, University of Utah, 30 South 2000 East, Room 301, Salt Lake City, UT 84112, USA
c
Department of Obstetrics and Gynecology, University of Utah, 30 South 2000 East, Room 301, Salt Lake City, UT 84112, USA
Abstract
The rationales for the use of water soluble polymers for anticancer drug delivery include: the potential to overcome some
forms of multidrug resistance, preferential accumulation in solid tumors due to enhanced permeability and retention (EPR)
effect, biorecognizability, and targetability. The utility of a novel paradigm for the treatment of ovarian carcinoma in an
experimental animal model, which combines chemotherapy and photodynamic therapy with polymer-bound anticancer drugs
is explained. Research and clinical applications as well as directions for the future development of macromolecular
therapeutics are discussed.  2001 Elsevier Science B.V. All rights reserved.
Keywords: HPMA copolymers; PK1; Ovarian carcinoma; Antibody fragments; Long-circulating macromolecular therapeutics; Combination
therapy; Photodynamic therapy
1. Introduction
The concept of macromolecular carriers of (anticancer) drugs has evolved continuously over the last
century. In 1906, Ehrlich coined the phrase ‘magic
bullet’, and recognized the importance of biorecognition for successful drug delivery [1]. DeDuve discovered the lysosomotropism of macromolecules and the
high enzymatic activity localized in the lysosomal
compartment [2]. The conjugation of drugs to synthetic and natural macromolecules was initiated
nearly 50 years ago. Jatzkewitz used a dipeptide
spacer to attach a drug (mescaline) to polyvinylpyrrolidone in the early 1950s [3]. Ushakov’s group
*Corresponding author. Tel.: 11-801-581-4532; fax: 11-801581-3674.
ˇ
E-mail address: [email protected] (J. Kopecek).
synthesized numerous water soluble polymer–drug
conjugates in the 1960s and 1970s [4]. Mathe´ et al.
pioneered conjugation of drugs to immunoglobulins,
setting the stage for targeted delivery [5]. Finally,
Ringsdorf presented a clear concept of the use of
polymers as targetable drug carriers in 1975 [6].
The field of macromolecular therapeutics has
developed considerably during the last 20 years
(reviewed in Refs. [7,8]). Numerous conjugates are
used clinically or are being evaluated in clinical trials
(Fig. 1). SMANCS, a poly(styrene-co-maleic acid)
conjugated neocarzinostatin is used clinically in
Japan [9]. Poly(ethylene glycol) (PEG)-bound camptothecin [10], poly( L-glutamic acid)-bound paclitaxel
[11],
and
N-(2-hydroxypropyl)methacrylamide
(HPMA) copolymer conjugates with doxorubicin
[12,13], doxorubicin and N-acylgalactosamine as a
targeting moiety [14], and camptothecin [15] are
0168-3659 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved.
PII: S0168-3659( 01 )00330-3
148
ˇ et al. / Journal of Controlled Release 74 (2001) 147 – 158
J. Kopecek
Fig. 1. Structures of polymer–anticancer drug conjugates in clinical use (A) and in clinical trials (B).
ˇ et al. / Journal of Controlled Release 74 (2001) 147 – 158
J. Kopecek
presently in clinical trials. Synthetic macromolecules
are also used in protein and lysosomal constructs
approved by the FDA. PEG modified adenosine
deaminase [16] is indicated for patients with severe
combined immunodefficiency disease. PEG modified
L-asparaginase [17] is indicated for patients with
acute lymphoblastic leukemia who require L-asparaginase in their treatment regimen, but are hypersensitive to the native forms of L-asparaginase. PEG
modified liposomes containing doxorubicin are indicated for patients with AIDS-related advanced
Kaposi’s sarcoma [18].
Macromolecular therapeutics has now reached a
stage of maturity which will foster further development in two distinct pathways. First, there is the
potential to synthesize more conjugates using numerous low molecular weight (e.g. poorly soluble) drugs
and to evaluate them on various cancer models. This
is a feasible, immediately applicable option which is
being pursued. The other pathway, looking further in
the future for direct clinical translation, is to initiate
detailed studies on the differences in the mechanism
of anticancer action of free and polymer-bound drugs
at the cellular and subcellular levels. The identification of new intracellular molecular targets, specific
for macromolecular conjugates, will permit the design of second generation of macromolecular therapeutics. In this overview we have summarized our
recent efforts in the design, synthesis, and evaluation
of HPMA copolymer–anticancer drug conjugates.
Additional details may be found in (ref. [19] and
accompanying short communications in this volume
[20–25].
2. Rationale and hypotheses
A major rationale for the use of water-soluble
polymers as carriers of anticancer drugs is based on
the mechanism of cell entry [7,8]. Consequently,
targetable polymer–drug conjugates should be
biorecognizable at two levels [26–28]: the plasma
membrane to increase the recognition and internalization by a subset of target cells and intracellularly
by lysosomal enzymes to release the drug from the
carrier. The latter is a prerequisite for transport of the
drug into the cytoplasm and nucleus resulting in
biological activity.
149
The successful design of new macromolecular
therapeutics is based on a multidisciplinary approach
to hypothesis formulation and problem solving. Early
work on the design of HPMA copolymer conjugates
has been published [26–28]. This overview addresses questions, which are critical to more sophisticated developmental issues in macromolecular therapeutics. The following hypotheses have been tested
on human ovarian carcinomas with HPMA copolymer–anticancer drug conjugates: (1) polymer–anticancer drug conjugates (macromolecular therapeutics), being internalized in membrane limited
organelles, activate different signaling pathways and
are more effective than free drugs in the treatment of
sensitive and multidrug resistant cancers; (2) longcirculating (high-molecular-weight) macromolecular
therapeutics will preferentially accumulate in tumors
with concomitant increase of therapeutic efficacy;
(3) novel targeted macromolecular therapeutics containing polymerizable antibody fragments accumulate in the target tissue to a higher extent than current
targeted systems; (4) combination chemotherapy and
photodynamic therapy provides treatment results
superior to individual therapies; and (5) evaluation
of signaling pathways involved in the mechanism of
action of macromolecular therapeutics will permit
the design and tailor-made synthesis of highly potent
second-generation conjugates.
2.1. Multidrug resistance and gene expression
Resistance of malignant tumors to chemotherapeutic agents remains one of the major causes of
failure in cancer therapy. The ultimate success of the
treatment of ovarian carcinomas will to a large extent
depend on the successful elimination of multidrug
resistant (MDR) cells [29]. A membrane glycoprotein, termed P-glycoprotein, has been shown to be
responsible for cross-resistance to a broad range of
structurally and functionally distinct cytotoxic
agents. This glycoprotein, encoded in humans by the
mdr1 gene, functions as an energy-dependent efflux
pump to remove cytotoxic agents from the resistant
cells. The elucidation of the function of P-glycoprotein [30], other ATP-driven efflux pumps [31] as
well as other mechanisms of multidrug resistance
[32] have had a major impact on the understanding
of multidrug resistance in human tumors. This
150
ˇ et al. / Journal of Controlled Release 74 (2001) 147 – 158
J. Kopecek
understanding permits the rational design of novel
polymeric anticancer drugs which are effective in the
treatment of multidrug resistant cells. The exclusion
of the polymer–drug conjugate from the cytoplasm
of the cell, through intracellular trafficking in membrane limited organelles, renders the efflux pumps
ineffective. Furthermore, subcellular trafficking
along the endocytic pathway from the plasma membrane to the perinuclear region changes the gradient
of distribution of drugs inside cells [33,34]. The
concentration gradient of free drugs is directed from
the plasma membrane to the perinuclear region, in
contrast to polymer-bound drugs which have a
gradient in exactly the opposite direction. The drug,
released from the polymeric carrier in the lysosomal
compartment, enters the cytoplasm in the perinuclear
region. Consequently, the probability of its interaction with nuclear DNA and / or topoisomerase II is
higher than the probability of its recognition by the
P-glycoprotein efflux pump [33].
We hypothesized that macromolecular therapeutics, being internalized in membrane-limited organelles, could activate different signaling pathways
and possess different anticancer effects than free
drug. As a result, macromolecular therapeutics might
be more protected from intracellular detoxification
mechanisms than free drugs. Our recent in vitro
study [35] was devoted to verifying this hypothesis.
We used free and HPMA copolymer bound doxorubicin (DOX) and A2780 sensitive and A2780 /AD
DOX resistant human ovarian carcinoma cells. While
free DOX up-regulated genes encoding ATP-driven
efflux pumps (MDR1, MRP), HPMA copolymer–
DOX conjugate overcame existing pumps and downregulated the MRP gene. Free DOX also activated
cell metabolism and expression of the genes responsible for detoxification and DNA repair. HPMA
copolymer–DOX conjugate down-regulated HSP-70,
GST-p, BUDP, Topo-IIa,b and TK1 genes. Additionally, apoptosis, lipid peroxidation and DNA
damage were significantly higher after exposure to
HPMA copolymer-DOX conjugate, as reflected by
simultaneous activation of p53, c-fos (in A2780
cells) or c-jun (in A2780 /AD cells) signaling pathways and inhibition of the bcl-2 gene.
The anticancer activity of free DOX and HPMA
copolymer–DOX conjugate (PK1) was studied on
solid tumor mice models of DOX sensitive (A2780)
and DOX resistant (A2780 /AD) human ovarian
carcinoma [36]. Free DOX was effective only in
DOX sensitive tumors decreasing the tumor size
about three times, while HPMA copolymer–DOX
conjugate decreased the tumor size 28 and 18 times,
respectively when compared to controls. The HPMA
copolymer–DOX conjugate effectively killed both
tumor types inducing apoptosis and necrosis through
the activation of p53, Apaf-1, Caspase-9, c-fos, c-jun
pathways and the down-regulation of the bcl-2 gene.
An excellent correlation was found between in vitro
and in vivo data on gene expression after exposure to
free and HPMA copolymer-bound DOX.
In summary, HPMA copolymer–DOX conjugate
overcame drug efflux pumps, more significantly
induced apoptosis and lipid peroxidation, and inhibited DNA repair, replication, and biosynthesis
when compared to free DOX. These differences
demonstrate the need to define and quantify the
molecular targets of drug action such as oncogenes,
tumor suppressor genes, drug-resistance-mediating
transporters, cell cycle proteins, apoptotic pathways,
DNA repair enzymes, components of the cytoarchitecture, and drug metabolizing enzymes [37,38].
The mechanism of action of HPMA copolymerbound DOX toward human ovarian carcinomas is
shown in Fig. 2.
2.2. Long-circulating macromolecular therapeutics
Another important design feature of macromolecular therapeutics is the relationship between physicochemical characteristics and efficacy. To guarantee
biocompatibility, the molecular weight distribution
of synthetic polymer carriers must be below the renal
threshold [39]. However, such macromolecules (molecular weight less than approximately 50 kDa for
HPMA copolymers) may be rapidly lost from the
circulation. To obtain a long circulating half-life and
retain renal elimination properties, the molecular
weight of water soluble drug carriers may be increased by introducing lysosomally degradable crosslinks (bridges) connecting short polymer chains
[40]. Such long-circulating (high-molecular weight)
polymer conjugates have the potential to accumulate
efficiently in tumor tissue [41,42] due to the leaky
tumor vasculature and the enhanced permeability and
retention (EPR) effect [43,44]. We have designed a
ˇ et al. / Journal of Controlled Release 74 (2001) 147 – 158
J. Kopecek
151
Fig. 2. Mechanism of action HPMA copolymer-bound doxorubicin toward human ovarian carcinoma. Adapted from Ref. [36].
new reproducible way of synthesis of long-circulating HPMA copolymer-drug conjugates by crosslinking copolymerization (Fig. 3). Copolymerization
of HPMA, a polymerizable derivative of DOX
(N-methacryloylglycylphenylalanylleucylglycyl doxorubicin), and a newly designed crosslinking
agent, N2,N5-bis(N-methacryloylglycylphenylalanylleucylglycyl)ornithine resulted in a high-molecular
weight, branched, water soluble HPMA copolymer
containing lysosomally degradable oligopeptide sequences in the crosslinks as well as in side-chains
terminating in DOX [45]. Four conjugates with Mw
of 22, 160, 895, 1230 kDa were prepared and the
influence of their molecular weight on the biodistribution and efficacy to treat human ovarian carcinoma
xenografts in mice was studied [42]. The half-life of
conjugates in the blood was up to five times longer
and the elimination rate from the tumor was up to 25
times slower as the Mw of conjugates increased from
22 to 1230 kDa. The treatment with HPMA copolymer-bound DOX possessing a Mw higher than
160 kDa inhibited the tumor growth more efficiently
than that of 22 kDa or free DOX (P,0.02). The
administration of long-circulating HPMA copolymerDOX conjugates resulted in molecular weight dependent enhanced tumor accumulation with a concomitant increase in therapeutic efficacy [42].
2.3. New targeting strategies
Targetability of polymer–drug conjugates is a
significant improvement over free drug administration. We have previously demonstrated the advantageous properties of HPMA copolymer–anticancer
drug–antibody conjugates [34,46–48]. Synthetic
methods were developed [47] for the binding of
antibodies via amino groups, oxidized saccharide
152
ˇ et al. / Journal of Controlled Release 74 (2001) 147 – 158
J. Kopecek
Fig. 3. Structure and therapeutic efficacy of long-circulating HPMA copolymer–doxorubicin conjugates. Growth inhibition of s.c. human
ovarian OVCAR-3 carcinoma xenografts in nu / nu mice after i.v.administration of 2.2 mg / kg DOX equivalent dose is shown for conjugates
with different molecular weights [42,45].
moieties close to hinge regions, and antibody fragments.
2.3.1. Polymerizable antibody fragments
Recently, using new macromonomers–polymerizable antibody fragments, we have developed a new
technology for the synthesis of targetable macromolecular therapeutics [20,49]. Polymerizable antibody Fab’ fragments of OV-TL 16 antibody (IgG1),
which recognize the OA-3 antigen expressed on the
majority of human ovarian carcinomas, were first
synthesized. These polymerizable Fab’ fragments
then can readily copolymerize with HPMA to
produce Fab’ containing HPMA copolymers. The
polymerizable Fab’ fragments and their HPMA
copolymers maintain the bioactivity of the Fab’
fragment and can effectively bind to OVCAR-3
human ovarian carcinoma cells. A targetable polymeric meso-chlorin e 6 mono(N-2-aminoethylamide)
(Mce 6 ) conjugate, poly(HPMA-co-MA-Fab’-coMA-GFLG-Mce 6 ), has been synthesized by copolymerization of HPMA, MA-Fab’ and MA-GFLGMce 6 in the presence of a water-soluble azo-initiator
(MA is methacryloyl, one letter abbreviations for
amino acids have been used). The Fab’ targeted
Mce 6 containing copolymer (IC 50 52.6 mM) possessed a much higher in vitro cytotoxicity than the
non-targeted Mce 6 containing HPMA copolymer
(IC 50 5230 mM) against OVCAR-3 human ovarian
carcinoma cells. The cytotoxicity of the targeted
copolymer was even higher than the free Mce 6
(IC 50 57.9 mM). The confocal microscopic investigation of the internalization of different drug forms
confirmed the rapid internalization and accumulation
of the Fab’ targeted drug containing copolymer in
the lysosomes of OVCAR-3 cells via receptor-mediated endocytosis. The internalization of the nontargeted drug containing copolymer was considerably
slower and below the detection sensitivity at the
experimental conditions used. The therapeutic efficacy of the conjugates correlated well with their
biorecognition [20].
The concept of using polymerizable Fab’ fragments as macromonomers provides a new paradigm
for the synthesis of targeted polymeric drug delivery
systems, and may have unique applications in other
areas, such as immunoassays, biosensor technology
and affinity chromatography.
ˇ et al. / Journal of Controlled Release 74 (2001) 147 – 158
J. Kopecek
2.3.2. Synthetic receptor-binding epitopes
It now appears that the use of small synthetic
peptides as receptor binding epitopes may be an
advantageous strategy in targetable conjugate design.
One of the main advantages is the possibility of
attaching numerous biorecognizable moieties to one
macromolecule. Resulting polyvalent interactions
may collectively be much stronger than the corresponding monovalent interactions [50]. The
biorecognition of HPMA copolymers containing
side-chains terminated in N-acylated galactosamine
by the asialoglycoprotein receptor is dependent on
the amount of bound ligand [51]. The similar advantage of multivalent interactions (cooperative binding)
was observed in the inhibition of virus mediated
agglutination of erythrocytes by polyacrylamides
with pendant sialoside groups [52], lectin recognition
of HPMA copolymers with pendant fucosylamine
residues [53], and mouse lymphoma binding of short
peptides fused via a semi-rigid hinge region with the
coiled-coil assembly domains into a multivalent
binding molecule [54].
The Epstein–Barr virus (EBV) envelope glycoprotein mediates virus attachment to the EBV/ C3dg
receptor on human B lymphocytes [55] and specific
binding to some receptors on human T cell lymphomas [56]. Two regions of amino acid similarity
have been found in the gp350 and C3d coding
sequences and it was suggested that they may
represent CD21 (CR2) binding sites of gp350 / 220.
Multimeric forms of the N-terminal gp350 / 220
peptide, composed from nine amino acid residues
(EDPGFFNVE), conjugated to albumin efficiently
blocked recombinant gp350 / 220 and C3dg binding
to B cells [55].
Attachment of the nonapeptide (NP) via a tetrapeptide (GFLG) side-chain to HPMA copolymers
resulted
in
a
tridecapeptide
epitope
(GFLGEDPGFFNVE) and in a modified biorecognition by B and T cells [57]. These conjugates
interacted with Raji B-cells and CCRF-HSB-2 Tcells in a multipoint attachment mode, indicating that
a cooperative effect was effective. A conjugate
containing four tridecapeptide epitopes per HPMA
copolymer chain possessed a binding activity (constant) that was one order of magnitude higher than
the binding activity of a conjugate containing one
epitope per macromolecule [57]. These data (Fig. 4)
153
strongly suggest the possibility of manipulating the
structure of epitopes with the aim to produce targetable drug delivery systems suitable for the treatment
of lymphomas.
Detailed studies are needed to find the optimal
epitope-receptor
combination
to
maximize
biorecognition and targeting to immunocompetent
cells. To this end, we established a model for
biorecognition studies based on an epitope presentation scaffold — a coiled-coil stem loop (CCSL)
peptide self-assembled on a solid substrate [58,59].
The CCSL peptide self-assembly represents a feasible model of exposing epitopes for biorecognition
studies.
2.4. Combination chemotherapy and photodynamic
therapy
Photodynamic therapy (PDT) uses the combination of light and certain absorbing molecules, called
photosensitizers, which in the presence of oxygen,
lead to rapid cell destruction [60,61]. The generation
of singlet oxygen is ultimately responsible for the
majority of such phototoxic effects, although other
reactions, e.g. the formation of radicals, do indeed
occur. A reactive excited state of molecular oxygen,
singlet oxygen, lies only 90 kJ mol 21 above the
triplet ground state, enabling photosensitizers to
efficiently catalyze its formation [62]. Because the
lifetime of singlet oxygen is short (|10 26 s) its site
of action is largely determined by its location.
Polymer bound photosensitizers will end up in the
lysosomal compartment of target cells where they
will be inactive without light. A double targeting
effect can be achieved by biorecognition and tumor
accumulation followed by the subsequent localized
application of light [63].
A novel treatment approach using combination
chemotherapy and photodynamic therapy (PDT)
delivered as HPMA copolymer bound anticancer
agents has been developed [64]. On two cancer
models, Neuro 2A neuroblastoma [65] and human
ovarian carcinoma heterotransplanted in the nude
mouse [66] we have shown that combination therapy
with HPMA copolymer-anticancer drug (DOX and
Mce 6 ) conjugates showed tumor cures which could
not be obtained with either chemotherapy or PDT
154
ˇ et al. / Journal of Controlled Release 74 (2001) 147 – 158
J. Kopecek
Fig. 4. Langmuir binding isotherms of HPMA copolymer–NP conjugates containing different amounts of NP per polymer chain to Raji B
cells. Various concentrations of conjugates were added to cells in Dulbecco’s phosphate buffered saline (containing 30 mM NaN 3 and 0.5%
bovine serum albumin) at 08C and incubated for 4 h [57].
alone. Cooperativity of the action of both drugs
contributed to the observed effect [67].
Recently, we performed a series of studies to
optimize the efficacy of chemotherapy and photodynamic therapy in the treatment of experimental
ovarian carcinoma. An association of hydrophobic
side-chains terminated with the drug may result in a
decreased quantum yield of singlet oxygen formation
(P(GFLG)-Mce 6 ) or a modified rate of DOX release
(P(GFLG)-DOX). Consequently, we optimized the
structure of the conjugates based on their solution
properties [68,69]. A biodistribution study of
P(GFLG)-Mce 6 and P(GFLG)-DOX revealed the
optimum time lag between the administration of both
macromolecular therapeutics and irradiation of the
tumor [70]. Based on these data, we hypothesized
that combination therapies of s.c. human ovarian
carcinoma OVCAR-3 xenografts in nude mice using
multiple doses / irradiation of P(GFLG)-Mce 6 and
P(GFLG)-DOX utilizing low doses could be effective without sacrificing the therapeutic efficacy (Fig.
5). Indeed, 10 out of 12 tumors exhibited complete
responses in the group of mice receiving multiple
PDT plus multiple chemotherapy [71]. The results
demonstrated that: (a) HPMA copolymer-bound
drugs exhibited selective tumor accumulation contrary to free drugs; (b) PDT using an HPMA
copolymer-Mce 6 conjugate with multiple light irradiations was a better therapy than that with single
light irradiation; and finally, (c) combination chemotherapy and photodynamic therapy with HPMA
copolymer–DOX and HPMA copolymer–Mce 6
conjugates was the most effective regimen [66,71].
Attachment of OV-TL16 monoclonal antibodies to
both HPMA copolymer conjugates further increased
the efficacy of the therapy. Combination treatment
ˇ et al. / Journal of Controlled Release 74 (2001) 147 – 158
J. Kopecek
155
Fig. 5. Multiple combination chemotherapy and photodynamic therapy of s.c. human ovarian OVCAR-3 carcinoma xenografts in nu / nu
mice. Solutions of HPMA copolymer conjugates with DOX and Mce 6 were administered i.v on day 0 for single therapy and on days 0, 5,
and 10 for multiple therapy. In PDT, the tumor was irradiated with laser light (110 J / cm 2 , 650 nm) at 12 and 18 h, respectively. The control
group received saline buffer. Adapted from Ref. [71].
with targeted HPMA copolymer conjugates with
DOX and Mce 6 at drug equivalent doses of 2.2
mg / kg DOX and 1.5 mg / kg Mce 6 produced longterm survivors [21].
3. Conclusions and future prospects
Macromolecular therapeutics utilizing biocompatible HPMA copolymer–anticancer drug conjugates
provide the unique advantages of subcellular trafficking and localization, as well as enhanced accumulation in solid tumors. Multiple avenues for targeting
and alternative activities on various cellular signaling
pathways are available in macromolecular delivered
agents compared to free agents. The clinical applications of macromolecular therapeutics, and particularly the novel treatment approach of combination
chemotherapy and photodynamic therapy are demonstrated by multiple preclinical studies. Reduced nonspecific toxicity has been a constant advantage in all
preclinical studies. Clinical trials are in development.
Research efforts continue to define specific cellular
and subcellular targets which will result in the
optimal design of second generation macromolecular
drugs. The macromolecular anticancer therapeutics
will have widespread applications in oncology.
Acknowledgements
We thank our students and coworkers for their
support and participation. Their names are shown in
the references. The research was supported by NIH
grants CA51578 and CA88047 from the National
Cancer Institute.
References
[1] P. Ehrlich, Studies in Immunity, Plenum Press, New York,
1906.
[2] C. De Duve, T. De Barsy, B. Poole, A. Trouet, P. Tulkens, F.
van Hoof, Lysosomotropic agents, Biochem. Pharmacol. 23
(1974) 2495–2531.
[3] H. Jatzkewitz, Peptamin (glycyl-L-leucyl-mescaline) bound
to blood plasma expander (polyvinylpyrrolidone) as a new
depot form of a biologically active primary amine (mescaline), Z. Naturforsch. 10b (1955) 27–31.
[4] E.F. Panarin, S.N. Ushakov, Synthesis of polymer salts and
amidopenicillines (in Russian), Khim. Pharm. Zhur. 2 (1968)
28–31.
´ T.B. Loc, J. Bernard, Effect sur la leucemie
´
[5] G. Mathe,
L1210
´
de la Souris d’une combinaison par diazotation d’A methop´
terine
et de g-globulines de hamsters porteurs de cette
´
´´
´
leucemie
par heterogreffe,
Compte-rendus del’Academie
des
Sciences 3 (1958) 1626–1628.
[6] H. Ringsdorf, Structure and properties of pharmacologically
156
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
ˇ et al. / Journal of Controlled Release 74 (2001) 147 – 158
J. Kopecek
active polymers, J. Polym. Sci., Polym. Symp. 51 (1975)
135–153.
ˇ
D. Putnam, J. Kopecek,
Polymers with anticancer activity,
Adv. Polym. Sci. 122 (1995) 55–123.
ˇ
ˇ
´ T. Minko, Z.-R. Lu, HPMA
J. Kopecek,
P. Kopeckova,
copolymer–anticancer drug conjugates: design, activity, and
mechanism of action, Eur. J. Pharm. Biopharm. 50 (2000)
61–81.
K. Tsuchia, T. Uchida, M. Kobayashi, H. Maeda, T. Konno,
H. Yamanaka, Tumor-targeted chemotherapy with SMANCS
in lipiodol for renal cell carcinoma: longer survival with
larger size tumors, Urology 55 (2000) 495–500.
C.D. Conover, R.B. Greenwald, A. Pendri, C.W. Gilbert,
K.L. Shum, Camptothecin delivery systems: enhanced efficacy and tumor accumulation of camptothecin to polyethylene
glycol via a glycine linker, Cancer Chemother. Pharmacol.
42 (1998) 407–414.
C. Li, J.E. Price, L. Milas, N.R. Hunter, S. Ke, D.F. Yu, C.
Charnsangavej, S. Wallace, Antitumor activity of poly( Lglutamic acid)–paclitaxel on syngeneic and xenografted
tumors, Clin. Cancer Res. 54 (1999) 891–897.
P.A. Vasey, C. Twelves, S.B. Kaye, P. Wilson, R. Morrison,
R. Duncan, A. Thomson, L. Murray, T.E. Hilditch, T.
Murray, S. Burtles, E. Frigerio, D. Fraier, J. Cassidy, Phase I
clinical and pharmacokinetic study of PK1 (HPMA copolymer–doxorubicin): First member of a new class of
chemotherapeutic agents–drug-polymer conjugates, Clin.
Cancer Res. 5 (1999) 83–94.
A.H. Thompson, P.A. Vasey, L.S. Murray, J. Cassidy, D.
Fraier, E. Frigerio, C. Twelves, Population pharmacokinetics
in phase I drug development: A phase I study of PK1 in
patients with solid tumors, Br. J. Cancer 81 (1999) 99–107.
P.J. Julyan, L.W. Seymour, D.R. Ferry, S. Daryani, C.M.
Boivin, J. Doran, M. David, D. Anderson, C. Christodolou,
A.M. Young, S. Hesselwood, D.J. Kerr, Preliminary clinical
study of the distribution of HPMA copolymers bearing
doxorubicin and galactosamine, J. Control. Release 57
(1999) 281–290.
V.R. Caiolfa, M. Zamai, A. Fiorino, E. Frigerio, C. Pellizoni,
R. d’Argy, A. Ghiglieri, M.G. Castelli, M. Farao, E. Pesenti,
M. Gigli, F. Angelucci, A. Suarato, Polymer-bound camptothecin: initial biodistribution and antitumor activity studies,
J. Control. Release 65 (2000) 105–119.
M.S. Hershfield, Biochemistry and immunology of poly(ethylene glycol)-modified adenosine deaminase (PEGADA), in: J.M. Harris, S. Zalipsky (Eds.), Poly(Ethylene
Glycol) Chemistry and Biological Applications, ACS Symposium Series 680, American Chemical Society, Washington,
DC, 1997, pp. 145–154.
L.M. Holle, Pegaspargase: an alternative?, Ann. Pharmacother. 31 (1997) 616–624.
for the International SL-DOX Study Group, F.-D. Goebel, D.
Goldstein, M. Goos, H. Jablonowski, J.S. Stewart, Efficacy
and safety of Stealth liposomal doxorubicin in AIDS-related
Kaposi’s sarcoma, Br. J. Cancer 73 (1996) 989–994.
ˇ
´ J. Kopecek,
ˇ
T. Minko, P. Kopeckova,
The role of caspases in
apoptosis induced be free and HPMA copolymer-bound
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
[35]
doxorubicin in human ovarian carcinoma cell, J. Control.
Release 71 (2001) 227–237.
ˇ
´ J. Kopecek,
ˇ
Z.-R. Lu, J.-G. Shiah, P. Kopeckova,
Preparation
and biological evaluation of polymerizable antibody Fab’
fragment targeted polymeric drug delivery system, J. Control. Release 74 (2001) 263–268.
ˇ
´ C.M. Peterson, R.C.
J.-G. Shiah, Y. Sun, P. Kopeckova,
ˇ
Straight, J. Kopecek,
Combination chemotherapy and photodynamic
therapy
of
targetable
N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin / mesochlorin
e 6 –OV-TL16 antibody immunoconjugates, J. Control. Release 74 (2001) 249–253.
ˇ
´ J. Kopecek,
ˇ
M. Tijerina, P. Kopeckova,
The effects of
subcellular
localization
of
N-(2-hydroxypropyl)methacrylamide copolymer-Mce 6 conjugates in a
human ovarian carcinoma, J. Control. Release 74 (2001)
269–273.
ˇ
´ T. Minko, S.E. Tabibi, J.
Y. Kasuya, Z.-R. Lu, P. Kopeckova,
ˇ
Kopecek,
Synthesis of HPMA copolymer–aminopropylgeldanamycin conjugates, J. Control. Release 74 (2001) 203–
211.
´
ˇ
´ J. Kopecek,
ˇ
S. Wroblewski,
M. Berenson, P. Kopeckova,
Potential of lectin-N-(2-hydroxypropyl)methacrylamide copolymer–drug conjugates for the treatment of pre-cancerous
conditions, J. Control. Release 74 (2001) 283–293.
L. Varticovski, Z.-R. Lu, I. de Aos, T. Kagawa, K. Mitchell,
ˇ
R. Christensen, J. Kopecek,
A water-soluble HPMA copolymer–wortmannin conjugate retains phosphoinositide 3kinase inhibitory activity in vitro and in vivo, J. Control.
Release 74 (2001) 275–281.
ˇ
J. Kopecek,
Biodegradation of polymers for biomedical use,
in: H. Benoit, P. Rempp (Eds.), IUPAC Macromolecules,
Pergamon Press, Oxford, 1982, pp. 305–320.
ˇ
J. Kopecek,
Controlled biodegradability of polymers — a
key to drug delivery systems, Biomaterials 5 (1984) 19–25.
ˇ
´ J. Strohalm, K. Ulbrich, B.
J. Kopecek,
P. Rejmanova,
ˇ´
´ V. Chytry,
´ J.B. Lloyd, R. Duncan, Synthetic polyRıhova,
meric drugs, US Patent 5,037,883 (1991).
A. Persidis, Cancer Multidrug Resistance, Nat. Biotechnol.
17 (1999) 94–95.
U.A. German, P-glycoprotein: A mediator of multidrug
resistance in tumour cells, Eur. J. Cancer 32A (1996) 927–
944.
D.W. Loe, R.G. Decley, S.P.C. Cole, Biology of the multidrug resistance associated protein, MRP, Eur. J. Cancer 32A
(1996) 945–957.
K.D. Tew, Gluthatione-associated enzymes in anticancer
drug resistance, Cancer Res. 54 (1994) 4313–4320.
ˇ
´ C. Gentry, J. Kopecek,
ˇ
V. Omelyanenko, P. Kopeckova,
Targetable HPMA copolymer–adriamycin conjugates. Recognition, internalization, and subcellular fate, J. Control.
Release 53 (1998) 25–37.
ˇ
´ J. Kopecek,
ˇ
V. Omelyanenko, C. Gentry, P. Kopeckova,
HPMA copolymer–anticancer drug-OV-TL16 antibody
conjugates. 2. Processing in epithelial ovarian carcinoma
cells in vitro, Int. J. Cancer 75 (1998) 600–608.
ˇ
´ J. Kopecek,
ˇ
T. Minko, P. Kopeckova,
Comparison of the
anticancer effect of free and HPMA copolymer-bound
ˇ et al. / Journal of Controlled Release 74 (2001) 147 – 158
J. Kopecek
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
[44]
[45]
[46]
[47]
[48]
adriamycin in human ovarian carcinoma cells, Pharm. Res.
16 (1999) 986–996.
ˇ
´ J. Kopecek,
ˇ
T. Minko, P. Kopeckova,
Efficacy of the
chemotherapeutic action of HPMA copolymer-bound doxorubicin in a solid tumor model of ovarian carcinoma, Int. J.
Cancer 86 (2000) 108–117.
J.N. Weinstein, T.G. Meyers, P.M. O’Connor, S.H. Friend,
A.J. Fornace Jr., K.W. Kohn, T. Fojo, S.E. Bates, L.V.
Rubinstein, N.L. Anderson, J.K. Buolamwini, W.W. van
Osdol, A.P. Monks, D.A. Scudiero, E.A. Sausville, D.W.
Zaharevitzm, B. Bunow, V.N. Viswanadhan, G.S. Johnson,
R.E. Wittes, K.D. Paul, An information-intensive approach to
the molecular pharmacology of cancer, Science 275 (1997)
343–349.
M.I. Rivera, S.F. Stinson, D.T. Vistica, J.L. Jorden, S.
Kenney, E.A. Sausville, Selective toxicity of the tricyclic
thiophene NSC 652287 in renal carcinoma cell lines, Biochem. Pharmacol. 57 (1999) 1283–1295.
ˇ
J. Kopecek,
Soluble polymers in medicine, in: D.F. Williams
(Ed.), Systemic Aspects of Biocompatibility, Vol. 2, CRC
Press, Boca Raton, FL, 1981, pp. 159–180.
ˇ
´
´ P. Rejmanova,
´ J. Strohalm, B.
J. Kopecek,
I. Cıfkova,
Obereigner, K. Ulbrich, Polymers containing enzymatically
degradable bonds. 4. Preliminary experiments in vivo, Makromol. Chem. 182 (1981) 2941–2949.
E. Marecos, R. Weissleder, A. Bogdanov Jr., Antibodymediated versus nontargeted delivery in a human small cell
lung carcinoma model, Bioconjugate Chem. 9 (1998) 184–
191.
ˇ´
ˇ
´ Y. Sun, C.M.
J.-G. Shiah, M. Dvorak,
P. Kopeckova,
ˇ
Peterson, J. Kopecek,
Biodistribution and antitumor efficacy
of long-circulating N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin conjugates in nude mice, Eur. J. Cancer
37 (2001) 131–139.
Y. Matsumura, H. Maeda, A new concept for macromolecular therapeutics in cancer chemotherapy: Mechanism of
tumoritropic accumulation of proteins and the antitumor
agent SMANCS, Cancer Res. 6 (1986) 6387–6392.
Y. Noguchi, J. Wu, R. Duncan, J. Strohalm, K. Ulbrich, T.
Akaike, H. Maeda, Early phase tumor accumulation of
macromolecules: A great difference in clearance rate between tumor and normal tissues, Jpn. J. Cancer Res. 89
(1998) 307–314.
ˇ´
ˇ
´ J. Kopecek,
ˇ
M. Dvorak,
P. Kopeckova,
High-molecular
weight HPMA copolymer–adriamycin conjugates, J. Control. Release 60 (1999) 321–332.
ˇ´
´ P. Kopeckova,
ˇ
´ J. Strohalm, P. Rossmann, V.
B. Rıhova,
ˇ
Vetvicka, J. Kopecek,
Antibody directed affinity therapy
applied to the immune system: In vivo effectiveness and
limited toxicity of daunomycin conjugates to HPMA copolymers and targeting antibody, Clin. Immunol. Immunopathol. 46 (1988) 100–114.
ˇ
´ C. Gentry, J.-G. Shiah, J.
V. Omelyanenko, P. Kopeckova,
ˇ
Kopecek,
HPMA Copolymer–anticancer drug–OV-TL16
antibody conjugates. 1. Influence of the method of synthesis
on the binding affinity to OVCAR-3 ovarian carcinoma cells
in vitro, J. Drug Targeting 3 (1996) 357–373.
ˇ
´ T. Minko, J. Kopecek,
ˇ
K. Kunath, P. Kopeckova,
HPMA
[49]
[50]
[51]
[52]
[53]
[54]
[55]
[56]
[57]
[58]
[59]
[60]
[61]
[62]
157
copolymer–anticancer drug–OV-TL16 antibody conjugates.
3. The effect of free and polymer-bound adriamycin on the
expression of some genes in the OVCAR-3 human ovarian
carcinoma cell line, Eur. J. Pharm. Biopharm. 49 (2000)
11–15.
ˇ
´ J. Kopecek,
ˇ
Z.-R. Lu, P. Kopeckova,
Polymerizable Fab’
antibody fragments for targeting of anticancer drugs, Nat.
Biotechnol. 17 (1999) 1101–1104.
M. Mammen, S.-K. Choi, G.M. Whitesides, Polyvalent
interactions in biological systems: Implications for design
and use of multivalent ligands and inhibitors, Angew. Chem.
Int. Ed. 37 (1998) 2754–2794.
R. Duncan, L.C.W. Seymour, L. Scarlett, J.B. Lloyd, P.
´
ˇ
Rejmanova,
J. Kopecek,
Fate of N-(2-hydroxypropyl)methacrylamide copolymers with pendent galactosamine residues after intravenous administration to rats,
Biochim. Biophys. Acta 880 (1986) 62–71.
G.B. Sigal, M. Mammen, G. Dahman, G.M. Whitesides,
Polyacrylamides bearing pendant sialoside groups strongly
inhibit agglutination of erythrocytes by influenza virus: The
strong inhibition reflects enhanced binding through cooperative polyvalent interactions, J. Am. Chem. Soc. 118 (1996)
3789–3800.
ˇ
´ J. Kopecek,
ˇ
R.C. Rathi, P. Kopeckova,
Biorecognition of
sugar containing N-(2-hydroxypropyl)methacrylamide copolymers by immobilized lectin, Macromol. Chem. Phys.
198 (1997) 1165–1180.
A.V. Tersikh, J.-M. Le Doussal, R. Crameri, I. Fisch, J.-P.
Mach, A.V. Kajava, ‘Peptabody’: A new type of high avidity
binding protein, Proc. Natl. Acad. Sci. USA 94 (1997)
1663–1668.
G.R. Nemerow, R.A. Houghten, M.D. Moore, N.R. Cooper,
Identification of an epitope in the major envelope protein of
Epstein–Barr virus that mediates viral binding to the B
lymphocyte EBV receptor (CR2), Cell 56 (1989) 369–377.
D. Watry, J.A. Hedrick, S. Siervo, G. Rhodes, J.J. Lamberti,
J.D. Lambris, C.D. Tsoukas, Infection of human thymocytes
by Eppstein–Barr virus, J. Exp. Med. 173 (1991) 971–980.
ˇ
´ R.K. Prakash, C.D. Ebert, J.
V. Omelyanenko, P. Kopeckova,
ˇ
Kopecek,
Biorecognition of HPMA copolymer–adriamycin
conjugates by lymphocytes mediated by synthetic receptor
binding epitopes, Pharm. Res. 16 (1999) 1010–1019.
ˇ
A. Tang, C. Wang, R. Stewart, J. Kopecek,
Self-assembled
peptides exposing epitopes recognizable by human lymphoma cells, Bioconjugate Chem. 11 (2000) 363–371.
ˇ
A. Tang, C. Wang, R. Stewart, J. Kopecek,
The coiled-coil in
the design of protein-based constructs: Hybrid hydrogels and
epitope displays, J. Control. Release 72 (2001) 57–70.
ˇ´
´ K. Ulbrich, J. Strohalm, J.
N.L. Krinick, B. Rıhova,
ˇ
Kopecek,
Targetable photoactivatable drugs. 2. Synthesis of
N-(2-hydroxypropyl)methacrylamide
copolymer–anti-Thy
1.2 antibodies–chlorin e 6 conjugates and a preliminary study
of their photodynamic effect on mouse splenocytes in vitro,
Makromol. Chem. 191 (1990) 839–856.
B.W. Henderson, T.J. Dougherty, How does photodynamic
therapy work?, Photochem. Photobiol. 55 (1992) 145–157.
J.D. Spikes, Photosensitization, in: K.C. Smith (Ed.), The
158
[63]
[64]
[65]
[66]
[67]
ˇ et al. / Journal of Controlled Release 74 (2001) 147 – 158
J. Kopecek
Science of Photobiology, 2nd Edition, Plenum Press, New
York, 1989, pp. 79–110.
ˇ´
ˇ
´ N.L. Krinick, Targetable photoacJ. Kopecek,
B. Rıhova,
tivatable drugs, J. Control. Release 16 (1991) 137–143.
ˇ
J. Kopecek,
N.L. Krinick, Drug delivery system for the
simultaneous delivery of drugs activatable by enzymes and
light, US Patent 5,258,453 (1993).
N.L. Krinick, Y. Sun, D. Joyner, J.D. Spikes, R.C. Straight, J.
ˇ
Kopecek,
A polymeric drug delivery system for the simultaneous delivery of drugs activatable by enzymes and / or light,
J. Biomat. Sci., Polym. Ed. 5 (1994) 303–324.
C.M. Peterson, J.M. Lu, Y. Sun, C.A. Peterson, J.-G. Shiah,
ˇ
R.C. Straight, J. Kopecek,
Combination chemotherapy and
photodynamic therapy with N-(2-hydroxypropyl)meth-acrylamide copolymer-bound anticancer drugs inhibit human
ovarian carcinoma heterotransplanted in nude mice, Cancer
Res. 56 (1996) 3980–3985.
J.M. Lu, C.M. Peterson, J.G. Shiah, Z.-W. Gu, C.A. Peterson,
ˇ
R.C. Straight, J. Kopecek,
Cooperativity between free and
N-(2-hydroxypropyl)methacrylamide copolymer bound ad-
[68]
[69]
[70]
[71]
riamycin and mesochlorin e 6 monoethylene diamine induced
photodynamic therapy in human epithelial ovarian carcinoma
in vivo, Int. J. Oncol. 15 (1999) 5–16.
ˇ ˇ ´ J.D. Spikes, J. Kopecek,
ˇ
J.-G. Shiah, C. Konak,
Solution and
photoproperties of N-(2-hydroxypropyl)methacrylamide copolymer–meso-chlorin e 6 conjugates, J. Phys. Chem. B 101
(1997) 6803–6809.
ˇ ˇ ´ J.D. Spikes, J. Kopecek,
ˇ
J.-G. Shiah, C. Konak,
Influence of
pH on aggregation and photoproperties of N-(2-hydroxypropyl)methacrylamide copolymer–meso-chlorin e 6 conjugates, Drug Delivery 5 (1998) 119–126.
ˇ
J.-G. Shiah, Y. Sun, C.M. Peterson, J. Kopecek,
Biodistribution of free and N-(2-hydroxypropyl)methacrylamide copolymer-bound mesochlorin e 6 and adriamycin in nude mice
bearing human ovarian carcinoma OVCAR-3 xenografts, J.
Control. Release 61 (1999) 145–157.
J.-G. Shiah, Y. Sun, C.M. Peterson, R.C. Straight, J.
ˇ
Kopecek,
Antitumor activity of HPMA copolymer-meso
chlorin e 6 and adriamycin conjugates in combination treatments, Clin. Cancer Res. 6 (2000) 1008–1015.