Advanced Drug Delivery Reviews 62 (2010) 150–166
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Advanced Drug Delivery Reviews j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a d d r
Structural and chemical aspects of HPMA copolymers as drug carriers☆ Karel Ulbrich ⁎, Vladimír Šubr Institute of Macromolecular Chemistry v.v.i., Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic
a r t i c l e
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Article history: Received 18 September 2009 Accepted 21 October 2009 Available online 18 November 2009 Keywords: Drug delivery systems N-(2-hydroxypropyl)methacrylamide Polymer drug conjugates Synthesis of HPMA copolymers Drug targeting Anticancer drugs
a b s t r a c t Synthetic strategies and chemical and structural aspects of the synthesis of HPMA copolymer conjugates with various drugs and other biologically active molecules are described and discussed in this chapter. The discussion is held from the viewpoint of design and structure of the polymer backbone and biodegradable spacer between a polymer and drug, structure and methods of attachment of the employed drugs to the carrier and structure and methods of conjugation with targeting moieties. Physicochemical properties of the water-soluble polymer–drug conjugates and polymer micelles including mechanisms of drug release are also discussed. Detailed description of biological behavior of the polymer–drug conjugates as well as application of the copolymers for surface modification and targeting of gene delivery vectors are not included, they are presented and discussed in separate chapters of this issue. © 2009 Elsevier B.V. All rights reserved.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . Polymer backbone . . . . . . . . . . . . . . . . . 2.1. Nondegradable linear copolymers . . . . . . . 2.2. Biodegradable branched and graft copolymers . 2.3. Micelle-forming copolymers . . . . . . . . . 3. Spacers . . . . . . . . . . . . . . . . . . . . . . 3.1. Enzymatically degradable spacers . . . . . . . 3.2. Spacers susceptible to chemical hydrolysis . . 3.3. Other biodegradable spacers . . . . . . . . . 4. Polymer-conjugated biologically active molecules. . . 4.1. Cancerostatics and other low-molecular-weight 4.2. Proteins and enzymes . . . . . . . . . . . . 4.3. Imaging agents and diagnostics . . . . . . . . 5. Targeting moieties . . . . . . . . . . . . . . . . . 5.1. Antibodies and their fragments . . . . . . . . 5.2. Peptides and oligopeptides . . . . . . . . . . 5.3. Saccharides . . . . . . . . . . . . . . . . . 5.4. Other targeting moieties . . . . . . . . . . . 6. Outlook and concluding remarks . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “HPMA Copolymers: 30 Years of Advances”. ⁎ Corresponding author. Tel.: + 420 296 809 231; fax: + 420 296 809 410. E-mail address:
[email protected] (K. Ulbrich). 0169-409X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2009.10.007
Since the first synthesis of the N-(2-hydroxypropyl)methacrylamide (HPMA) monomer and its homopolymer, poly(HPMA) [1], application of poly(HPMA) (DUXON) as blood plasma expander [2–4] and synthesis of first reactive HPMA copolymers [5,6] in the early seventies, water-soluble HPMA copolymers have developed into a group of most frequently studied hydrophilic polymers utilizable for synthesis of polymer drug
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conjugates. Even though a majority of the conjugates were proposed for treatment of malignant diseases, numerous examples of application of HPMA copolymer–drug conjugates for treatment of other diseases (such as antibiotics, immunosuppressives, vaccines) were also demonstrated. The structure of most water-soluble HPMA copolymer-based therapeutics follows the model suggested by Ringsdorf [7] and accomplished by Kopeček, illustrating an ideal composition of the water-soluble polymer– drug conjugate. In this relatively simple model composed of four components, the polymer–drug conjugate consisted of water-soluble biocompatible copolymer chain in which some comonomer units contained drug and targeting moieties bound to the polymer backbone via biodegradable oligopeptide spacers tailored as substrates for selected intracellular peptidases. Pioneering work at this first stage of development of HPMA copolymer–drug conjugates was focused on methods of synthesis of nondegradable HPMA polymers and copolymers [1,8], optimization of structure of enzymatically degradable oligopeptide spacers using a simple drug model (4-nitroanilide) [9–13] and synthesis and degradation of enzymatically degradable branched high-molecularweight (HMW) polymer carriers [12–15]. Studies of synthesis and properties of actively targeted HPMA copolymer carriers started in the early eighties [16–19] and at the same time first polymer conjugates with biologically active molecules (drugs) were synthesized and tested [20–22]. First HPMA copolymer conjugates with doxorubicin (PK1 and PK2) were clinically tested for their toxicity and anticancer activity in early nineties. Moreover, HPMA copolymers have been also used for formation, coating, modification and targeting of gene delivery vectors (polyplexes, adenoviruses) [23–27]. In this chapter we are dealing with chemical, structural and synthetic aspects of development of HPMA copolymer conjugates designed for delivery of drugs and other biologically active substances and, in some cases, their physicochemical characteristics are also discussed. The discussion is held from the viewpoint of design and structure of the polymer carrier and biodegradable spacer as well as of the structure of the employed drugs and targeting moieties. Biological behavior of the polymer– drug conjugates as well as application of HPMA copolymers for surface modification and targeting of viral and non-viral gene delivery vectors are presented and discussed in other chapters of this issue. 2. Polymer backbone Development of polymer–drug conjugates efficient in vivo requires a properly designed polymer carrier enabling specific delivery of the drug to its target, in case of malignant diseases to tumors or, more specifically, into tumor cells. Two concepts of polymer drug targeting are used. Active targeting is based on specific interactions of the targeting moiety and cell membrane receptors, and it can be used generally in all cases when the active molecule — targeting moiety and its cell membrane-associated receptor are known. Passive targeting is directed to solid tumors; it utilizes high molecular weight of the polymer carrier and its accumulation in solid tumors due to the enhanced permeability and retention (EPR) effect [28]. It was shown, that, in accordance with the behavior of other polymers, the efficiency of tumor accumulation of HPMA copolymers is molecular weight dependent [29]. It is clear that proper design, length and structure of the polymer carrier significantly influence the body distribution of the conjugate and thus its biological activity. 2.1. Nondegradable linear copolymers The simplest structure of a HPMA copolymer–drug conjugate consists of the nondegradable linear HPMA copolymer chain bearing the drug attached with its amide bond through spacers degradable or nondegradable in the living body. In some cases, the spacers terminate also in a low-molecular-weight (LMW) targeting moiety attached to the same copolymer chain. Three methods of synthesis of
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targeted polymer conjugates have been developed: (a) synthesis of a HPMA copolymer bearing reactive groups (polymer precursor) enabling attachment of other components of the system with covalent bonds, e.g. by aminolysis [30,31]; (b) copolymerization of HPMA with comonomers containing drug or targeting moiety in their structure [30,32] and (c) combination of both methods, i.e. copolymerization of HPMA with comonomers containing bioactive molecule and reactive groups. Polymerization is followed by aminolytic attachment of a second active moiety [33]. For their syntheses, see Fig. 1. An original synthesis of the polymer precursors, HPMA copolymers bearing reactive 4-nitrophenyl ester groups (–ONp), is based on radical precipitation copolymerization of HPMA with respective comonomers in acetone initiated with 2,2′-azobis-(isobutyronitrile) [6,8]. By changing the copolymer composition, polymer precursors with varying amounts of reactive groups randomly distributed along polymer chains have been synthesized and employed in most syntheses of polymer–drug conjugates [34,35]. Molecular weights of the copolymers are limited to a range from 20 000 to 35 000 due to chain-transfer reactions and steric hindrance in precipitation of growing polymer radicals [8]. Polydispersity of the copolymers is rather low (∼ 1.5) but their molecular weight cannot be easily controlled, e.g. by changing composition of the polymerization mixture or temperature. Indeed, homogenous copolymerizations carried out in organic solvents (DMSO, DMF) afford higher molecular weights and slightly better control of the reaction. Unfortunately, polydispersity is higher and the content of reactive groups in reactive copolymers does not correspond to the content of spacers due to hydrolysis during polymerization so that the structure of such copolymers is not well defined. HPMA copolymers with carbonyl thiazolidine-2-thione (–TT) reactive groups (Fig. 2) have been developed [36] with the aim of improving properties of polymer precursors. These copolymers can be prepared by solution polymerization, their molecular weight can be easily controlled and specific reactivity of –TT groups makes it possible to perform aminolytic conjugation reactions both in organic and aqueous solutions. The –TT copolymers namely show low rates of hydrolysis and high rates of aminolysis in aqueous solutions and in certain cases also exhibit selective reactivity. The –TT group-containing copolymers were successfully employed in the synthesis of antibodytargeted and protein conjugates [36] or in coating viral gene delivery vectors [26] in aqueous solutions. All the reactive polymer precursors mentioned above are multivalent HPMA copolymers bearing 10–15 reactive groups randomly distributed along the polymer chain. Synthesis of some conjugates (graft copolymers, antibody-targeted star conjugates, micellar systems, etc.) required development of HPMA homopolymer and HPMA copolymers with only one reactive group at the end of polymer chain (semitelechelic polymers). In principle, two methods of synthesis of semitelechelic polymers were employed: radical polymerization or copolymerization of HPMA carried out in the presence of chain-transfer agents [25,37–40], mainly thiol group-containing acids, or radical copolymerization using special bifunctional azo-initiators containing –TT [41,42] or 2-pyridyldisulfanyl [43] groups. Modification of the terminal –TT group was employed in the synthesis of other semitelechelic polymers terminating, e.g., with maleimido [44] or 2-pyridyldisulfanyl [42] groups. Such semitelechelic HPMA copolymers were used for coating of viral or nanoparticulate delivery vectors, for conjugation with proteins and glycoproteins [44] or for the synthesis of branched and hyperbranched high-molecular-weight (HMW) polymer–Dox conjugates [42]. Each of the methods shows some advantages and disadvantages. Molecular weights of polymers prepared by chain-transfer polymerization are from 3 to 15 kDa; the presence of a polymer without reactive end group requires further purification while the functionality of polymers prepared using specific initiators is often higher than one per chain. Recent data show that “living” radical polymerization could improve the situation in synthesis of hydrophilic monofunctional polymers significantly.
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Fig. 1. Scheme of synthesis of HPMA copolymer precursors and targeted linear polymer–drug conjugates. (A) Synthesis of reactive HPMA copolymer with –ONp groups; (B) synthesis of a copolymer with a drug and reactive –ONp groups and its use for synthesis of targeted conjugate; (C) synthesis of the conjugate by copolymerization.
Low polydispersity of polymer drug conjugates is one of the most important requirements demanded from conjugates designed for in vivo applications. Unfortunately, the abovementioned HPMA homopolymers and copolymers exhibited polydispersity higher than 1.5, rather close to 2. This was why attempts at controlled polymerization of HPMA have appeared in recent years. First attempts at atom transfer radical polymerization (ATRP) carried out in the bulk or solution led to low conversions when common ATRP ligands were employed [45]. The HPMA polymer with molecular weights Mn 15–97 kDa and polydispersity below 1.1 were prepared by reversible addition–fragmentation chain transfer (RAFT) polymerization in acetate buffer [46]. RAFTcontrolled radical copolymerization of HPMA and N-(methacryloyloxy) succinimide [47] or pentafluorophenyl methacrylate [48] introducing the reactive groups into HPMA copolymer have enabled the synthesis of copolymers with Mn 3–135 kDa and polydispersity 1.1–1.3. Also
HPMA copolymer–aledronate conjugates with cathepsin K-controlled aledronate release were prepared by RAFT radical terpolymerization of HPMA, MA-G-G-P-Nle–alendronate, MA-TyrNH2 and MA-FITC (MA = methacryloyl) in a water–methanol mixture using S,S′bis(α,α'-dimethyl-α″-acetic acid)trithiocarbonate as chain transfer agent [49]. High polymerization yields and molecular weight distribution ranging from 1.1 to 1.8 (depending on molecular weight) were reported. Recently, a dexamethasone (DEX) conjugate with the drug bound to HPMA copolymer via pH-labile hydrazone bond was also synthesized by RAFT radical copolymerization of HPMA and MA–G–G–NHN=DEX in methanol/DMF using the trithiocarbonate as chain transfer agent. Molecular weight and polydispersity of the resulting conjugate (Mw) were 34 kDa and 1.3 respectively [50]. The RAFT copolymerization of HPMA seems to result in well-defined copolymers with narrow molecular weight distributions. This type of synthesis, after optimizing the initiation system and polymerization conditions, will certainly play an important role in the synthesis of polymer drug conjugates based on HPMA copolymers which are designed for in vivo testing and clinical evaluation. 2.2. Biodegradable branched and graft copolymers
Fig. 2. HPMA copolymer with reactive carbonyl thiazolidine-2-thione (–TT) groups.
As mentioned above, in conjugates intended for passive tumor targeting, molecular weight of the carrier should sufficiently exceed the threshold for glomerular filtration to prolong the persistence time of the conjugate in blood circulation and to achieve maximum tumor accumulation due to the EPR effect. At the same time, after the drug is released, the carrier has to be eliminated from the body. It is clear that in HPMA copolymers with nondegradable backbones this requires a
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proper design of the carrier using short linear polymer chains selfassembled in high-molecular-weight (HMW) micellar structure or chemically linked through biodegradable spacers to enable disintegration of the carrier to original short polymer chains with molecular weights allowing safe elimination of the polymer from the body, e.g. by glomerular filtration. HPMA copolymers, like some other polymers, accumulate in solid tumors due to the EPR effect, with the efficiency increasing with increasing molecular weight of the polymer carrier [29,51]. Unfortunately, the use of linear HPMA copolymers suitable as carriers for passive tumor targeting is limited; they cannot be degraded and thus easily excreted from the body if their molecular weight exceeds the glomerular filtration limit (∼ 50 kDa). Except for linear HPMA copolymer with Mw below 50 kDa [52], the branched high-molecular-weight HPMA copolymers with oligopeptide crosslinks susceptible to enzymatic (proteolytic) degradation were examined as promising candidates for drug carrier systems [12,14,15]. These conjugates, prepared by aminolysis with diamines of polymer precursors containing oligopeptide–ONp sequences, are characteristic of broad distribution of molecular weights. Direct copolymerization of HPMA with methacryloylated G-F-L-G-Dox and methyl N2,N5-bis[(N-methacryloylglycyl)phenylalanylleucylglycyl] ornithinate [53,54] resulted in HMW copolymers with narrower distribution. The HMW copolymers with molecular weight above the renal threshold were degraded by lysosomal enzymes to form short polymer chains excretable in urine. Except for difficult reproducibility of their syntheses also broad distribution of molecular weights was a drawback of such polymers. More sophisticated biodegradable HMW graft copolymer–Dox conjugates and star polymers with dendrimeric
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core structure and narrow distribution [55–57] have been developed and investigated (Fig. 3). In graft polymer conjugates [42,57], a multivalent HPMA copolymer was grafted with a similar but semitelechelic HPMA copolymer, both types of polymers containing Dox attached by hydrazone bonds enabling intracellular pH-controlled drug release. The polymer grafts were attached to the polymer backbone through spacers degradable enzymatically (G-F-L-G), reductively (disulfide bridges) or through spacers combining both biodegradable sequences [42]. The sequences facilitate, after drug release, intracellular degradation of the graft polymer carrier resulting in short polymer fragments excretable from the organism by glomerular filtration. In the star HPMA conjugates, the semitelechelic HPMA copolymers bearing Dox bound via a G-F-L-G spacer were grafted onto the PAMAM dendrimer. Dox was released from the system after incubation with the lysosomal enzymes but, unfortunately, biodegradation of the polymer skeleton was not reported and the problem of elimination of the polymer from a body solved [55,56]. 2.3. Micelle-forming copolymers Introduction of highly hydrophobic substituents (dodecyl, oleic acid or cholesteryl moieties) into a linear HPMA copolymer bearing Dox covalently bound as hydrazone [58] resulted in HPMA conjugates forming in aqueous solutions HMW supramolecular structures with hydrophobic core surrounded by a hydrophilic polymer shell bearing a covalently bound drug. Molecular weight of the copolymer was ∼30 kDa, and after self-assembling into micellar structure, it reached ∼100–200 kDa forming nanoparticles with diameters ca. 25 nm and narrow size distribution. It was shown that 65–75% of Dox was released
Fig. 3. Schematic structures of HPMA copolymer conjugates. (A) Linear conjugate; (B) branched conjugate; (C) grafted conjugate; (D) self-assembled micellar conjugate; (E) grafted dendritic star conjugate.
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from the micellar carrier within 24 h, after incubation in a buffer of pH 5 mimicking intracellular endosomal and lysosomal environments, while only ∼5% of Dox was released in saline phosphate buffer at physiological pH 7.4 modeling conditions during transport in blood circulation. Properties of these micellar systems have been improved by introduction of a biodegradable hydrazone linkage between polymer chain and cholesterol moiety by the reaction of a hydrazide group-bearing polymer precursor with cholest-4-en-3-one or cholestan-4-en-3-one [59] thus enabling intracellular degradation and disintegration of the carrier system resulting in polymer fragments which could be eliminated from the organism. Novel thermoresponsive polymer micelles prepared by selfassembling thermoresponsive poly[N-isopropylacrylamide-graftHPMA] copolymers with hydrolytically degradable glycosylamine groups between polymer blocks have been developed [60] for delivery of radiotherapeutics into solid tumors. The micelles forming biodegradable particles of ca. 130 nm in diameter were developed as carriers for radionuclides (131I and 90Y). They can be easily prepared from an aqueous polymer solution by heating to 37 °C.
3. Spacers To achieve satisfactory biological activity, most polymer-conjugated drugs should be released from the carrier at a site of their destination, mainly due to enzymatic or chemical hydrolysis. The target for anticancer drugs is the tumor tissue or, better, the cancer cell. Except for enzymatic release in lysosomes, a pH decrease after passing from blood circulation (pH 7.4) into intratumoral or intracellular environment (pH 5–6) or reductive environment in cytoplasm can be exploited to control the drug release. Clearly, there are also biologically active molecules not requiring release from the carrier to exhibit activity, like radionuclides [60], photosensitizers [61–63] or MRI contrast agents [64]. In such cases a simple nondegradable glycine, diglycine or other amino acid spacers have been used in synthesis of polymer–drug conjugates. Nevertheless, in most cases proper design of the spacer is a pre-requisite for drug release and activity of the conjugate in vivo.
3.1. Enzymatically degradable spacers Model studies of libraries of polymer conjugates containing oligopeptide spacers terminating in the drug model 4-nitroaniline incubated with serine or cysteine proteases showed that the length and detailed structure of the spacer control the rate of 4-nitroaniline release from the HPMA copolymer carrier [10,65–67]. Various structures of oligopeptide spacers stable in blood serum and plasma and degradable in the presence of lysosomal enzymes (tritosomes, cathepsins) were sorted out [68–71] with G-F-L-G spacer being the most suitable for synthesis of conjugates with satisfactory activity in vivo. Suitability of G-F-L-G spacer for attachment of a drug to the HPMA-based polymer carrier was verified in vitro and in vivo using anticancer drugs puromycin, daunomycin (DNM) and doxorubicin (Dox) [69,70,72–74]. Unexpectedly, the cytotoxicity and in vivo activity of Dox conjugates with G-F-L-G spacer were the highest even though conjugates with other spacers, e.g. G-L-F-G released Dox much faster either after incubation with cathepsin B or with tritosomes [74,75]. Surprisingly, configuration of amino acids in the spacer (L-Phe, D-Phe, DL-Phe) also significantly influenced in vivo activity of the respective conjugates with highest activity exhibited by conjugates containing Gly-DL-Phe-Leu-Gly spacer [33]. Unfortunately, detailed study of the effect of configuration of Phe and Leu in the G-F-L-G spacer on the in vivo activity has been never accomplished. Nevertheless, a wide variety of drugs have been conjugated with HPMA copolymer precursors, in most cases just using the G-F-L-G spacer. Thus aminoellipticin [76], derivatives of methotrexate [77], derivatives of cyclosporin A [78,79], geldanamycin [80–82], an 8-aminoquinoline analog NPC1161, {8-[(4-amino-1-methylbutyl)amino]-5-[3,4-dichlorophenoxy]-6-methoxy-4-methylquinoline} [83], an inhibitor of angiogenesis TNP-470 O-[N-(chlorocetyl)carbamoyl] fumagillol, CaplostatinTM) [84–86], or promising library of HPMA copolymers with cisplatin-like and carboplatin-like chelates releasing active platinum after hydrolysis of a spacer and exhibiting a significant anticancer activity even in clinical tests [87–90] and other drugs were conjugated to HPMA copolymer using a similar principle — aminolysis of reactive polymer precursors containing oligopeptide spacers. As mentioned above, most HPMA copolymer drug conjugates, including clinically tested conjugates PK1 and PK2 [91–93] (Fig. 4) were prepared by aminolysis of HPMA copolymers bearing
Fig. 4. Non-targeted PK1 (A) and galactose-targeted PK2 (B) HPMA copolymer–doxorubicin conjugates.
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reactive –ONp groups at the end of the oligopeptide spacer. A different strategy of synthesis of the conjugates with oligopeptides as spacers has been employed [94–98] in some cases. The amino (Dox, melphalan) or hydroxy group-containing drug (paclitaxel, campthotecin) was acylated with one or more amino acids and then amino acid drug derivative was used for aminolysis of a polymer precursor with a spacer consisting of remaining amino acid(s) to form a conjugate in which the drug was coupled with the oligopeptide spacer of a required structure (G-F-L-G, MAG polymer) [68,94]. In both cases the strategy of subsequent modification of the reactive HPMA copolymer precursor resulted in less defined copolymers containing, except for spacers terminating in Dox, also certain amounts of spacers terminating in carboxyl or 2-hydroxypropyl-1-amide groups. The effect of these changes in a structure of the PK1 type polymer on its biological behavior was studied in detail [99]. With the aim of preparing conjugates with well-defined structures, the polymer conjugates of Dox have been synthesized by direct copolymerization of HPMA with a drug monomer (e.g., methacryloylated oligopeptide terminating in Dox) [33,100,101]. This strategy has been used for the synthesis of polymer–Dox conjugates, but also for the synthesis of their targeted derivatives. In this case polymerizable monomeric antibody fragment (methacryloylated Fab′) was synthesized and copolymerized with monomeric drug mesochlorin e6 [102]. This strategy was also used for the synthesis of HPMA-based conjugates bearing two different drugs or drug and targeting moiety, each of them being coupled to the carrier via a spacer of different composition [75]. Also synthesis of other polymer prodrugs with dual activity (e.g., a conjugate bearing aromatase inhibitor, aminoglutethimide, and Dox bound to the same carrier via oligopeptide spacer) was reported [103] recently. In addition, also oligopeptide spacer G-G-P-Nle was used for synthesis of prostaglandin E1 (PGE1) and aledronate HPMA copolymer conjugates releasing the active compound by cleavage of the spacer in the presence of cathepsin K [49,104]. For the study of specific colon release of drugs the conjugates containing azo bonds in the spacer have been synthesized (see below). 3.2. Spacers susceptible to chemical hydrolysis Dox-bearing HPMA copolymer conjugates in which Dox was bound to the polymer via pH-sensitive hydrazone bond or cis-aconityl spacer have been developed [105,106]. In the hydrazone-bond-containing
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conjugates (hydrazones), Dox is bound to the polymer precursor bearing hydrazide groups by the hydrazone bond formed by the reaction of polymer hydrazides with the C13 oxo group of Dox. This method of Dox attachment allows much higher loading of the carrier (up to 18 wt.% Dox) keeping full solubility of the product when compared with similar conjugates PK1 or PK2 with amide bond-bound Dox in which loading higher than ∼8 wt.% results in aggregation and separation of a polymer from aqueous solutions. Moreover, hydrazide precursors can be easily prepared with a wide range of molecular weights. In the precursors single amino acid or short oligopeptide residues terminating in hydrazide group form spacers that are quite stable during transport of the polymer–Dox conjugate in blood (pH 7.4) and release Dox in mildly acid environment (pH ∼ 5) at a rate only partly controlled by the spacer structure [107]. Surprisingly, detailed structure of the carrier (linear, grafted, and micellar) does not significantly influence the rate of hydrolysis of the hydrazone bond (Dox release) [57,58,107]. A HPMA copolymer–Dox conjugate with a drug bound via amide bond and cis-aconityl spacer was synthesized and cytotoxicity for A2780 sensitive and A2780/AD resistant human ovarian carcinoma cells was determined [105,108]. Acylation of a drug with cis-aconityl anhydride in the first step of the synthesis is not completely selective and the subsequent conjugation with a polymer is complicated by low selectivity of the remaining carboxylic groups, which results in a mixture of spacer structures differing in the rate of drug release [109]. 3.3. Other biodegradable spacers Linear conjugates releasing the drug after two-step degradation of the oligopeptide spacer were developed and studied. In the conjugate designed for 5-fluorouracil (5-FU) delivery α-glycine derivative of 5fluorouracil (α-Gly-5-FU) was attached to the HPMA copolymer via oligopeptide spacer. In the first step α-Gly-5-FU was released by enzymatic degradation of an oligopeptide spacer and in the second step the glycine derivative was hydrolysed releasing free 5-FU (Fig. 5) [110,111]. The rate of drug release was controlled by the oligopeptide spacer length and by hydrophobicity of the penultimate amino acid residue in the spacer. Interesting HPMA conjugates have been designed enabling specific drug release in colon due to combination of reductive and enzymatic degradation of azo groups and oligopeptide sequence-containing spacer.
Fig. 5. HPMA copolymer conjugate with 5-fluorouracil (5-FU) and its two-step release.
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Fig. 6. HPMA copolymer conjugate with 5-aminosalicylic acid susceptible to specific azoreductase degradation (in colon).
The conjugates containing 5-aminosalicylic acid or 9-aminocamptothecin (9-AC) bound to the HPMA copolymer via biodegradable spacer (Fig. 6) enabled selective release of the drugs after incubation with azoreductases [32,112]. A combination of azoreductase and proteolytic activity [113] was responsible for selective drug release in colon while the conjugate was stable in stomach and small intestine. Absorption of the drug in vivo after oral administration was proved in rats [114]. Similar HPMA copolymer conjugates designed for colon-specific delivery of 9-AC bound to the carrier via the same (oligopeptide, azo group-containing) or N-(4-aminobenzyl)carbamate group-containing spacers were synthesized. The design of the spacers ensured an efficient two-step release of 9-AC from the conjugates in the colon, starting by azoreductase cleavage followed by enzymatic cleavage of amino acid residue or 1,6-elimination (Fig. 7) [112–116]. A biodegradable spacer consisting of 4-aminobenzyl alcohol group enabling 1,6-elimination and a cathepsin K-sensitive tetrapeptide (G-G-P-Nle) was used in the structure of prostaglandin E1 (PGE1)-HPMA copolymer conjugate designed for treatment of osteoporosis. Here PGE1 was attached to the HPMA copolymer via a spacer and an ester bond [104,117,118]. An interesting disulfide linkage susceptible to reductive degradation was used for attachment of photosensitizer mesochlorin e6 to a HPMA copolymer to overcome problems of its systemic cytotoxicity. Spacer degradation and fast release of the drug after incubation of the conjugate with SKOV-3 ovarian carcinoma cells was reported demonstrating the disulfide linkage potential for drug attachment [119]. Another interesting dendritic oligopeptide spacer containing G-F-LG-F-K-4-aminobenzyl sequence was used for the synthesis of the HPMA copolymer–paclitaxel conjugate. The spacer enabled triple loading of the hydrophobic drug paclitaxel and its release as a result of cleavage by the endogenous enzyme cathepsin B. The polymer–drug conjugate exhibited enhanced cytotoxicity to murine prostate adenocarcinoma (TRAMP C2) cells in comparison with a classic conjugate [120]. 4. Polymer-conjugated biologically active molecules A variety of biologically active molecules have been used for conjugation with HPMA copolymers, starting with low-molecularweight (LMW) antibiotics and anticancer drugs and ending with HMW proteins and glycoproteins. Anticancer drugs were the active molecules
most frequently used in the synthesis of conjugates and for evaluation of biological activity in vitro and in vivo. 4.1. Cancerostatics and other low-molecular-weight drugs In most HPMA copolymer conjugates the drug was attached to the end of the spacer via amide bond. These conjugates were prepared by aminolysis of polymer precursors containing –ONp or –TT reactive groups with the drugs or their amino derivatives. The reaction was carried out preferably in organic solvents but also in water [121]; best yields were obtained using a –TT precursor [36]. In the case of Dox, primary amino group was employed for synthesis of the conjugates releasing the drug in an enzymatically catalyzed reaction (oligopeptide spacer) while the reaction of Dox keto group with hydrazide groups of the polymer precursor resulted in the conjugates releasing Dox due to pH-sensitive chemical hydrolysis (see above). Analogous to doxorubicin, aminolytic reaction was employed for example in the synthesis of HPMA copolymer conjugates with sarcolysin [122], daunomycin and puromycin [69], glycyl derivative of 5-fluorouracil [110,111,123], emetine [124], some cisplatin and other Pt derivatives like diaminocyclohexaneplatinum [89,125–127], 9-aminocamptothecin [112–116] or with 6-(3aminopropyl)ellipticine [76]. An aminolytic reaction was also employed in the synthesis of TNP-470 (Caplostatin) [84–86], the drug with significant antiangiogenic activity and in the synthesis of conjugates with other drugs like 8-aminoquinoline analog NPC1161 [142] or amphotericin B [143]. If the drug did not contain amino group the drug was first modified and then attached to the carrier by aminolysis as it was described, e.g., for methotrexate [77] or for 1,5-diazaanthraquinone [128]. In some cases primary hydroxy group in the drug structure was acylated and thus the drug was attached to a polymer via ester bond as in the case of mitoxanthrone [131–133] or camptothecin [129,130] and paclitaxel, where G-F-L-G-F-K-4-aminobenzyl linker was used [97,98,120]. In other cases polymer precursor bearing carboxylic groups was conjugated with an OH-group-containing drug (5-fluoro-1,3-bis (hydroxymethyluracil) using the carbodiimide method [134,135]. The strategy using methacryloylated drug derivatives and their copolymerization with HPMA was employed in the synthesis of polymer–drug conjugates, namely doxorubicin [33] or aledronate [49]. This strategy
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Fig. 7. HPMA copolymer conjugate with 9-aminocamptothecin and the drug release by azoreductase degradation followed by 1,6-elimination.
resulted in better defined conjugate structure. Similar strategy has been also used in the synthesis of the conjugate containing dexamethasone (Dex) bound via pH-sensitive hydrazone bond [50,136]. In this case Dex-containing monomer (MA–G–G–NHN=Dex) was synthesized and copolymerized with HPMA by reversible addition–fragmentation transfer (RAFT) polymerization. Except for well-defined structure also narrow molecular weight distribution has been achieved. In addition to cancerostatics, polymer bound antibiotics are a group of promising polymer therapeutics even if not yet at the stage of clinical tests. In their synthesis, aminolytic reaction and amide group were mostly used for drug attachment, starting with the conjugates based on 6-aminopenicillanic acid and ampicilin [21] and, later on, dealing with chloramphenicol [137], kanamycin, gentamicin [138,139] and gramicidin S [140]. In comparison with parent antibiotics the conjugates with various oligopeptide spacers showed superior antibacterial activity in vivo with the highest activity of the conjugate with G-F-L-G spacer. With the aim to promote bone formation, the potent anabolic agent prostaglandin E1 was conjugated with HPMA copolymer targeted with aspartic acid octamer. 4-Aminobenzyl ester group and a cathepsin K-sensitive tetrapeptide G-G-P-Nle [117,141] were used as a spacer. A more sophisticated method had to be developed for conjugation of HPMA copolymers with immunosuppressant cyclosporin A [144]. Unsaturated aliphatic substituent in this N-methylated cyclic undecapeptide was hydroformylated in the first step and then either conjugated through the introduced aldehyde group to amino groupbearing copolymer via azomethine (CH=N) groups or, after hydrazinolysis, conjugated with reactive –ONp polymer precursors forming a conjugate bearing the drug bound via pH-sensitive hydrazone bond. A different strategy was used in the synthesis of the conjugate designed for colon delivery [78,145]. In this case an amino group was
introduced into cyclosporin A derivative after epoxidation and aminolysis with ethylenediamine. An aromatic azo bond which can be specifically cleaved by azoreductase in colon to release the drug effective in the treatment of colon diseases was employed in this case. A similar strategy, i.e. azo bond degradable by azoreductases, was chosen for the synthesis of a 5-aminosalicylic acid conjugate [146]. Drugs with antiviral and cytostatic activity, 9-[2-(phosphonomethoxy)ethyl] derivatives of adenine, 2,6-diaminopurine and 2,6diaminoguanine, were conjugated with a HPMA copolymer precursor bearing primary amino groups. The phosphonamide link between the polymer and nucleotide proved to be stable in a buffer of pH 7.4 but undergoing slow hydrolysis at pH 5.0 enabling controlled drug release. The rate of drug release depends on the detailed structure of heterocyclic base [147]. 4.2. Proteins and enzymes Selected therapeutically active enzymes have been conjugated with linear semitelechelic poly(HPMA) (star conjugates) and multivalent HPMA (classic conjugates) copolymer precursors with the aim to improve the enzyme stability in vivo, prolong their blood circulation, reduce their immunogenicity and antigenicity and improve their bioavailability. In early stages of development the conjugation reactions were studied using a model protein α-chymotrypsin [148] and a multivalent HPMA copolymer precursor (classic conjugate). Later on, semitelechelic poly (HPMA) with various end groups were used for synthesis of the star conjugates of this enzyme [37]. It was shown that polymer modification did not significantly influence the enzyme activity in LMW and even HMW substrates. Classic and star conjugates with enzymes exhibiting anticancer activity, bovine seminal ribonuclease (BS-RNAse) [39,149,150] and RNAse A [151,152] were synthesized and their remarkable antitumor
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activity in vivo in mice was demonstrated. Using similar techniques, also poly(HPMA) conjugates with superoxide dismutase [153] or trypsin [154] were synthesized and their biological properties evaluated. In synthesis of star conjugates the semitelechelic HPMA polymers prepared by radical chain-transfer polymerization or copolymerization of HPMA in the presence of 3-sulfanylpropanoic acid as chain transfer agent followed by activation of a terminal carboxyl group by esterification with Nhydroxysuccinimide [37,39,154] were employed for aminolytic grafting of the central enzyme molecule. Conjugates with classic structure were synthesized by aminolysis of multivalent HPMA copolymers bearing -ONp groups. The highest in vivo antitumor activity in mice was obtained after treatment of animals with the conjugate combining activity of the anticancer drug Dox with that of BS-RNAse [149]. Conjugates of HPMA copolymers with enzymes also showed a potential for exploitation in the PDEPT (polymer-directed enzyme prodrug therapy) approach, where combination of HPMA copolymer bearing Dox prodrug, PK1, with the poly(HPMA)-cathepsin B conjugate or combination of PK1 analog with poly(HPMA)-G-G-βlactamase [155] generates the cytotoxic drug selectively inside the tumor [156,157]. 4.3. Imaging agents and diagnostics Although HPMA copolymers as carriers of anticancer drugs have been widely studied and there is an increasing need for clinically appropriate labeling of polymer delivery systems to study their in vivo pharmacokinetics, only few examples of HPMA conjugates suitable for diagnostic purposes are described. Diagnostics based on nondegradable HPMA conjugates of molecular weights ca. 25 kDa enabling formation of chelate complexes with radionuclides or other metals (gadolinium) and, in some cases, also a LMW targeting moiety have been described. Scintigraphic methods have been employed to monitor the biodistribution of 123I-labeled HPMA copolymer–Dox and HPMA copolymer–Dox–galactosamine conjugates in tumor-bearing mice [158,159] as well as radioiodinated imaging agent in humans [91,93,160]. Nevertheless, radioiodinated compounds are not often used in patients due to high cost (123I), suboptimal energy (125I) and the high radiation dose and poor image quality (131I). Alternatively, technetium-99 m (99mTc) is an ideal radiolabel to visualize the time-dependent biodistribution of polymer–drug conjugates in patients. Neutral poly[HPMA-co-MA-G-G-(N-ω-bis(2pyridylmethyl)-L-lysine)-co-MA-TyrNH2] and electronegative poly [HPMA-co-MA-G-G-OH-co-MA-G-G-(N-ω-bis(2-pyridylmethyl)-Llysine)-co-MA-TyrNH2] were synthesized, radiolabelled with 99mTc (CO)3 and used for a study of dependence of in vivo biodistribution on polymer molecular weight and charge [161]. Similar RGD4Ctargeted 99mTc-radiolabeled HPMA copolymer conjugate prepared from poly[HPMA-co-MA-G-G-(N-ω-bis(2-pyridylmethyl)-L-lysine)co-MA-G-G-ONp-co-MA-TyrNH2] precursor was used for evaluation of selectivity of binding to the αVβ3 integrin as a marker of tumor neovasculature. Scintigraphy of human prostate carcinoma xenografts DU145-bearing mice 24 h after intravenous injection of 99mTclabeled HPMA copolymer targeted with RGD4C conjugate showed a marked localization in tumor [162,163]. Multicomponent HPMA copolymer bearing (N-ω-bis(2-pyridylmethyl)-L-lysine), 2-amino3-(isothiourea-phenyl)propyl-cyclohexane-1,2-diamine-N,N-N′,N″, N″-pentaacetic acid and tyrosinamide groups were used for the synthesis of RGD4C-targeted HPMA conjugate combining radiodiagnostic and radiotherapeutic properties. The side chains incorporated into copolymer enabled chelation of both 99mTc and 90Y. The effect of 90Y radiotherapy targeted on angiogenic vasculature on tumor growth was assessed in SCID mice bearing DU145 human prostate carcinoma [164]. Likewise, 188Re radiotherapeutics complexed with poly[HPMA-co-bis(2-pyridylmethyl)-4-vinylbenzylamine] was synthesized and studied as a water-soluble polymer radiotherapeutics [165]. Even though there is a serious interest in using polymer radio-
therapeutics in human medicine, the application of polymer radionuclides is rather directed into the field of imaging agents. Several HPMA copolymers were designed as polymer contrast agents recommended for magnetic resonance imaging (MRI). HPMA copolymer-linked nitroxides have been prepared by aminolysis of poly(HPMA-co-MA-G-G-ONp) precursor with 3-(aminomethyl)-2,2,5,5-tetramethylpyrrolidin-1-oxyl. Relaxivity of HPMA copolymer-linked nitroxides increased with increasing content of nitroxide groups in a conjugate, showing a higher relaxivity than the commercially used Gd chelate with diethylenetriaminepentaacetic acid (Gd–DTPA) [166]. For the Gd chelate of the copolymer poly{HPMA-coMA-Acap-Asp-[Asp(OH)2]2} the T1 and T2 relaxation times were determined and the complex was used in vivo as a contrast agent in micro-MRI angiography [64]. Another Gd complex was synthesized from poly{HPMA-co-MA-G-G-mannosamine-co-aminopropylmethacrylamide-benzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-co-5-[3-(methacryloylaminopropyl)thioureidyl]fluorescein} and used for magnetic resonance imaging of macrophage-mediated malignancies [167]. Later on, the same team described the use of Gd complex prepared from poly(HPMA-co-MA-G-F-L-G-Dox-co-aminopropylmethacrylamide-benzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid) precursor for MR monitoring the fate of poly(HPMA)based drug delivery systems in vivo [168] and the use of a similar system targeted with RGDfK sequence for monitoring polymer interaction with αVβ3 integrin over expressed on both endothelial and tumor cells [169]. For examples of Gd-based diagnostics see Fig. 8. Recently random and block copolymers prepared by RAFT polymerization were used for radiolabelling with 18F to form a copolymer suitable for positron emission tomography (PET) [48]. Tyramine ligands were fluoroalkylated with 2-[18F]fluoroethyl-1-tosylate in high yield and the conjugate proved its suitability for imaging in small animal PET experiments. 5. Targeting moieties Multivalent reactive HPMA copolymers facilitate attachment or incorporation of various ligands to the polymer backbone, either those serving as efficient targeting moieties or those changing the carrier characteristics and thus influencing its interactions with the body compartments. Various targeting moieties have been employed in the synthesis of HPMA copolymer–drug conjugates to achieve their specific delivery, a majority of them being designed for treatment of cancer. 5.1. Antibodies and their fragments Antibodies (Ab) are glycoproteins with high specificity for cell membrane antigens thus offering the possibility of being used as targeting moiety for receptor-mediated targeting of polymer–drug conjugates. Ab contain various functional groups (primary and secondary amino, carboxyl and thiol groups) enabling direct conjugation with reactive polymer precursors, or such groups can be easily incorporated into antibody molecule, e.g., aldehyde groups by oxidation of saccharide units in the FC part of Ab or thiol groups by selective reduction. Most of the antibody-targeted HPMA copolymer–drug conjugates have been prepared by consecutive aminolysis of a multivalent polymer precursor bearing –ONp groups (classic conjugates) [61–63,170–173] with attachment of a drug in the first step, followed by aminolytic conjugation with Ab or its fragments. In most cases Dox, cyclosporin A or chlorine e6 were conjugated with the polymer in organic solvent (DMSO). After separation of the unreacted drug the remaining reactive groups in the precursor were aminolysed by antibody in aqueous solution. For proper conjugation, careful selection of reaction conditions like pH, concentrations of reactants or temperature are the most important factors influencing the conjugate quality and, preferably, preservation of the ability of the conjugated antibody to bind to cell
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Fig. 8. HPMA copolymer Gd chelate for MR imaging. (A) Gd chelate with HPMA copolymer bearing aspartic acid residues; (B) RGDfK targeted Gd chelate with HPMA-DOTA copolymer.
receptors [75]. In addition to synthesis by aminolysis, the synthesis by copolymerization of HPMA with monomeric drug and methacryloylated reactive ester has been employed [33]. In this case a rather difficult removal of free drug from the reaction mixture can be dropped. Moreover, this method allows attachment of a drug and targeting moiety with spacers of different composition. However, the second step of synthesis, aminolysis of the multivalent polymer with a multivalent antibody resulting in formation of HMW (∼1000 kDa) branched structures with broad distributions of molecular weights and a decreased binding activity of the antibody, remains a crucial reaction step leading to conjugation with Ab. Even though some problems arise with the conjugate structure, the classic Dox conjugates can be relatively easily synthesized and their anticancer activity is notable even in humans [174–177]. As some of the amino groups of the Ab at the binding site for antigen are fairly reactive and can be involved in conjugation reaction with a polymer, the binding activity of Ab can be preserved by protection of the amino groups by reaction with dimethylmaleic anhydride [33] prior to conjugation with the polymer. If 50–70% of antibody amino groups were protected during conjugation, the conjugate (after deprotection) exhibited a higher binding activity in vitro and even a better anticancer activity in vivo than that synthesized without antibody protection. A further improvement of the synthesis of classic conjugates was achieved by using –TT polymer precursors. Better stability of –TT groups in aqueous solutions allowed one-pot aminolysis of a polymer precursor with both Dox and Ab carried out at constant pH and temperature [121]. The obtained conjugate contained only very small amounts of free Dox and unreacted antibody. Further improvement of properties of antibody-targeted conjugates was achieved with star conjugates (Fig. 9). In these conjugates, more semitelechelic HPMA copolymer–Dox chains with Dox attached through a biodegradable oligopeptide spacer or hydrazone bonds were linked to the central antibody molecule through amide bond [178]. The semi-
telechelic polymers were prepared by radical polymerization in the presence of 3-sulfanylpropanoic acid as a chain transfer agent [179] or using an azo initiator containing thiazolidine-2-thione (–TT) or 2pyridyldisulfanyl groups [44]. Reactions of semitelechelic (monovalent) copolymers with Ab avoided branching in the conjugate structure typical of classic conjugates. This is why the star conjugates were better defined than the classic conjugates. Molecular weight of the star conjugates was lower (∼500 kDa versus 1000 kDa of classic conjugates) and molecular weight distribution more narrow [33,178]. It seems that further improvement of properties of the star and classic conjugates can be achieved by attaching Dox to a polymer with a pH-sensitive hydrazone bonds and by conjugation with Ab via its oxidized FC fragment or via a sulfide or a reducible disulfide bridges [180]. Such conjugates can be prepared by the reaction of thiol-containing antibody (the –SH groups introduced by the reaction with 2-iminothiolane) with semitelechelic or multivalent polymer precursor bearing Dox and containing maleimide (forming stable sulfide bridge) or 2-pyridyldisulfanyl groups (forming reducible disulfide bridge) [44,108,181]. Depending on the selected precursor (semitelechelic or multivalent) the classic or star structure of the conjugates can be obtained. Simplification of the conjugate structure was achieved by replacement of Ab with its Fab fragment. The polymer–mesochlorin e6 conjugate targeted with Fab antibody fragment was prepared by copolymerization of HPMA with monomeric drug and methacryloylated antibody fragment (MA-Fab) prepared from OV-TL 16 antibody [182,183]. The MA-Fab comonomer contained PEG spacer and exhibited a high reactivity in copolymerization with HPMA enabling incorporation of about two Fab fragments in every polymer chain. Selective oxidation of saccharide units in the FC part of the antibody and the use of the aldehyde groups for conjugation with a polymer precursor brought even a better protection of the Ab binding activity. HPMA copolymer precursors bearing amino, hydrazine or hydrazide
160 K. Ulbrich, V. Šubr / Advanced Drug Delivery Reviews 62 (2010) 150–166 Fig. 9. HPMA copolymer–drug–antibody conjugates. (A) Classic conjugate prepared by aminolysis; (B) star conjugate prepared by aminolysis; (C) star conjugate prepared from Ab modified by 2-iminothiolane; (D) classic conjugate prepared from Ab modified by 2-iminothiolane; (E) star conjugate prepared from Ab reduced with dithiothreitol; (F) conjugate prepared by oxidation of Ab with periodate and polymer hydrazide precursor (G) conjugate prepared by oxidation of Ab with periodate and amino polymer precursor, azomethine bond was reduced with cyanoborohydride; (H) classic conjugate prepared by aminolysis after protection of a part of amino groups with dimethylmaleicanhydride.
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groups were used in the synthesis of antibody-targeted conjugates with Dox, immunosuppressant cyclosporin A [144] or chlorin (mesochlorin) e6 [62,172]. The conjugates showed much better binding to specific cell receptors than those prepared by aminolysis. Unfortunately, a major drawback restricting wider exploitation of such promising carrier systems consists in a lower content (∼30 wt.%) of the drug-bearing polymer in the conjugate due to a low number of aldehyde groups available for conjugation with a polymer after Ab oxidation (5–7 per molecule). Hence, the content of polymer chains conjugated with the antibody was lower. Specific conjugation of the dithiothreitol-reduced antibody (antiCD20, containing –SH groups) with a semitelechelic HPMA copolymer precursor avoiding changes of the Ab binding site for antigen [184] resulted in a star structure with a narrow molecular weight distribution. In the conjugates, Dox was attached to the carrier via enzymatically (G-F-L-G) or hydrolytically (hydrazone bonds) degradable spacers. Preliminary tests of biological activity confirmed a considerable targeting capacity of monoclonal Ab after conjugation with the copolymer. This method of conjugation seems to open new perspectives for development of antibody-targeted polymer drug delivery systems. For selected structures of polymer-antibody conjugates, see the scheme in Fig. 8. Except for antibodies, also HPMA copolymers bearing Dox targeted with plant lectines wheat germ agglutinin (WGA) or peanut agglutinin (PNA) were synthesized by two-step aminolysis. The conjugates were employed for treatment of human colorectal SW 620 carcinoma in mice [185]. A detailed study of fluorescence labeled HPMA copolymer–WGA or HPMA copolymer–PNA synthesized in similar way and targeted on goblet cells or other cells of the gastrointestinal tract was published [186,187]. Problems with reproducibility of synthesis, a rather complex structure of the conjugates targeted with large proteins or glycoproteins and potential immunogenicity of the conjugates limit their exploitation as polymer therapeutics. This is why oligopeptides recognized as an active sequence responsible for interaction of glycoproteins with their receptors were selected and their potential as targeting moieties has been tested. 5.2. Peptides and oligopeptides Well-defined peptides and oligopeptides selectively interacting with cell membrane receptors are promising candidates for efficient targeting moieties in drug delivery systems. Their defined composition and known reactivity of individual amino acid residues allow specific attachment to polymer precursors using standard methods of peptide and polymer chemistry yielding a well-defined product. The structure of appropriate peptides can be sorted out using the knowledge of binding domains of natural proteins, such as proteins of extracellular matrix like fibronectin or laminin or by combinatorial methods as phage display [188]. An advantage of the use of synthetic oligopeptides as targeting moieties consists not only in their defined structure, but also in their availability. They can be produced in much larger quantities and at much lower costs than the monoclonal antibodies or their fragments. Active targeting of the conjugates using oligopeptide sequences can be directed straight to the receptors of malignant cells or can be aimed at normal cells of tumor endothelium. Also, combination of targeting ability of the peptides and their antiangiogenic inhibitory effect can be employed in designing polymer therapeutics for treatment of cancer. The melanocyte stimulating hormone (α-MSH), an oligopeptide composed of thirteen amino acids, was selected for targeting of HPMA copolymer–Dox conjugates designed for treatment of B16F10 melanoma [189]. The conjugates were prepared by consecutive aminolysis of polymer precursors bearing G-G-ONp or G-F-L-G-ONp ligands (Mw ∼ 20 kDa) with Dox and α-MSH in DMSO. Only negligible branching was observed after conjugation of multivalent HPMA
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copolymer with α-MSH indicating that the Lys ε-amino group was preferably involved in the binding reaction. The Epstein–Barr virus nonapeptide E-D-P-G-F-F-N-V-E promoting receptor-mediated targeting on T- and B-cell lymphoma were also used for synthesis of HPMA copolymer–Dox conjugates. Also here, the conjugates were prepared by consecutive aminolysis of polymer precursors containing G-G and G-F-L-G spacers. The authors ascribed the better binding efficiency and higher cytotoxicity of the conjugate with G-F-L-G spacer to a larger distance between a polymer and peptide in the case of G-F-L-G than G-G spacer [190]. Two 111In-bearing HPMA conjugates targeted with cyclic oligopeptides RGDfK or RGD4C, the peptides with high affinity to αVß3 integrin, were designed for targeting a diagnostic or therapeutic radionuclide on tumor angiogenic vasculature [191,192]. The conjugates were prepared by copolymerization of HPMA with reactive MA-G-G-ONp and chelating monomer followed by aminolysis with the respective oligopeptide. Similar radionuclide-bearing conjugates (99mTc, 90Y) targeted with RGD4C were synthesized using the same synthetic strategy but a specific chelator for 99mTc and 90Y in place of DTPA (diethylenetriaminepentaacetic acid) used for indium chelation [162–164]. The hydrophilic undeca(ethylene oxide) spacer between a polymer and targeting C-P-L-H-Q-R-P-M-C nonapeptide was used in the synthesis of HPMA copolymer–Dox conjugate with Dox bound by hydrolytically cleavable hydrazone bonds. The conjugate was tested for binding to receptors of human metastatic cancer cell line PC3MM2. An efficient cell binding and significant antiproliferative activity of the conjugate were reported [193]. With the aim to accomplish intracellular localization of a drug at specific subcellular target such as nucleus, HPMA copolymer conjugates containing Tat peptide (G-R-K-K-R-R-Q-R-R-R) as nuclear localization system [194] have been synthesized and their targeting efficiency studied [195–197]. In the conjugates bearing Dox and fluorescent label, the Tat peptide was attached to the polymer via a spacer providing fluorescent labeling facilitating the study of intracellular fate of the conjugate. As last example of conjugates targeted with oligopeptide moiety, the conjugates of aspartic acid octamer with HPMA copolymer bearing therapeutic (prostaglandin E1) or MRI contrast agent (Gd) should be mentioned. The conjugate can be targeted on bone surfaces and used for therapy or diagnostics [141,198]. Also in this synthesis, aminolysis of the reactive HPMA copolymer with an Asp derivative was employed. 5.3. Saccharides Aminosaccharides were suggested as first efficient targeting moieties in HPMA copolymers in the early eighties [16,199]. The conjugates bearing Dox or daunomycin were prepared by consecutive aminolysis of biodegradable G-F-L-G-ONp and nondegradable G-G-ONp ligandcontaining precursors with a drug and galactosamine (targeting on liver hepatocytes), mannosamine (targeting on macrophages) or fucosamine (targeting on L1210 leukemia cells or colon mucosa) and their in vitro and in vivo activity was verified [16,200–203]. Fucosaminetargeted conjugates exhibited a significant antitumor activity in mice inoculated with L1210 leukemia cells [203] while conjugates targeted with mannosamine showed an increased affinity to macrophages [204]. In some cases also a small amount (∼ 1 mol.%) of N-methacryloyltyrosinamide was incorporated in the copolymer structure allowing radiolabelling of the copolymers for detailed pharmacokinetic study [158]. The doxorubicin conjugate targeted with galactosamine (PK2) on asialoglycoprotein receptor was the first actively targeted polymer conjugate undergoing phase I/II clinical evaluation [93,160,205]. An interesting polymer system containing galactose and designed for liver targeting was prepared by direct copolymerization of HPMA with 6-O-methacryloyl-D-galactose [189,206]. Enhanced accumulation of
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the conjugates in liver and their high affinity to hepatoma HepG2 cells in rats were documented. Monomeric mannose derivatives (MA-G-G-ManN) were also employed in the synthesis of anti-leishmanial drug conjugates using precipitation radical copolymerization with HPMA [83,142]. Even though some authors mention N-acetylmannosamine as a targeting moiety on macrophages, they used rather acylated mannosamine than N-acetylmannosamine in their conjugates. Aminosaccharides, in particular fucosamine, were evaluated as effective targeting moieties adhering to colon mucosa for delivery to colon of HPMA copolymer conjugates bearing 5-aminosalicylic acid [30,32,146,207]. It was reported that saccharides attached to polymer carrier in clusters of more carbohydrate residues could improve the targeting of HPMA copolymer–Dox conjugates on human colon adenocarcinoma cells [208–210]. Conjugates targeted with galactosamine, lactose, or triplicate galactose cluster showed specific adhesion to Colo-205, SW-480 and SW-620 adenocarcinoma cells with preferential binding of the trivalent galactose conjugate. Also, a high affinity of the galactose-cluster-targeted conjugate and lactosecontaining copolymers to HepG2 cells was reported. The binding depended on the content of saccharide moieties in HPMA copolymers.
defined biodegradable passively targeted high-molecular-weight systems designed for solid tumor therapy as well as conjugates specifically targeted with monoclonal antibodies, specific peptides and other moieties will be developed in near future as single therapeutics or, better, as highly specific chemo- and radiotherapeutics combining specific activity of two or more chemotherapeutics, chemo- and radiotherapeutic agents or enable combination of diagnostics and therapeutics conjugated to the same carrier system. Also treatment of cancer and other diseases with “coctails” of polymeric conjugates differing in mechanism of action remains open for further discovery. Last but not least, the use of HPMA copolymers for coating and further modification of viral and non-viral gene delivery vectors opens new horizons for wide exploitation of this type of hydrophilic synthetic polymers for biomedicinal applications. Acknowledgements This work was supported by grant of Academy of Sciences of the Czech Republic (KAN 200200651), grant Praemium Academiae 2008 and Grant Agency of Academy of Sciences of the Czech Republic (IAAX 00500803).
5.4. Other targeting moieties References Except for conjugates mentioned earlier, HPMA copolymer conjugates targeted with folic acid, a derivative of glutamic acid showing high affinity to fast-dividing cells, have also been synthesized and tested for their antitumor activity [211]. Due to two carboxylic groups in a folic acid molecule the conjugation reaction have to be carefully designed. Attachment of vitamin B12 or folic acid targeting ligands to HPMA copolymer–G-F-L-G–methotrexate or platinum complexes conjugates gave a superior tumor growth inhibition compared with the analogous but untargeted polymer conjugates [211]. As a last example of targeting HPMA copolymer conjugates using LMW moieties, bisphosphonate ligands suitable for targeting of HPMA copolymers on hard tissue (bone), should be mentioned. A HPMA copolymer targeted with alendronate bound to the carrier through a diglycine spacer [212] formed a very strong and stable complex with a bone model hydroxyapatite in vitro and also showed accumulation in bones in vivo. Similar HPMA conjugates bearing radionuclides 125I or 111In and Dox [213] proved to be efficient in binding to hydroxyapatite as a bone model. In all syntheses aminolysis of polymer precursors was used. 6. Outlook and concluding remarks A wide variety of structures of HPMA copolymer-based drug carriers have been designed and the drug conjugates synthesized and tested in the last two decades. Although many of them showed promising therapeutic activity or imaging properties in animal models, only Dox, taxols, camptothecin and Pt complex conjugates were clinically evaluated. Unfortunately, so far none of them have been approved for clinical application and commercialized yet. Nevertheless, development of new synthetic routes like RAFT polymerization, design of new structures resulting in very narrow distribution of molecular weights of polymer precursors and conjugates including rather complex structures (biodegradable polymer-grafted dendritic structures) together with development of a great variety of new spacer structures and conjugation techniques allow to exploit variability of structures of multivalent HPMA copolymers. Thus, the design of tailor-made polymer therapeutics and achievement of organ- or cell-specific drug delivery are promising. Advances in cell biology offer new possibilities in identification of specific targets and selection of proper targeting moieties and biologically active compounds as well as new methods of study of cellular and subcellular fate and the mechanisms of action of newly designed polymer therapeutics. With high probability, well-
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