Water soluble polymers in tumor targeted delivery

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

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Fig. 1. Structures of polymer–anticancer drug conjugates in clinical use (A) and in clinical trials (B).

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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.

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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

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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

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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

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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.

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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)

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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

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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

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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.