Sebastian G.
Spain
a and
Neil R.
Cameron
*b
aSchool of Pharmacy, University of Nottingham, University Park, Nottingham, NG7 2RD, UK. E-mail: sebastian.spain@nottingham.ac.uk; Tel: +44 (0)115 846 6241
bDepartment of Chemistry, University of Durham, University Science Laboratories, South Road, Durham, DH1 3LE, UK. E-mail: n.r.cameron@durham.ac.uk; Tel: +44 (0)191 3342008
First published on 9th July 2010
Glycopolymers, synthetic polymers displaying carbohydrate moieties, have been linked to many potential applications at the biology–chemistry interface. One area that holds particular promise is the employment of glycopolymers as vehicles for therapeutics or as therapeutics themselves. This review summarises some of the more prominent examples as well as those in the early stages of development.
![]() Sebastian G. Spain | Sebastian Spain was born in Gloucestershire, England, in 1981. He studied for an MChem degree at the University of Durham, graduating in July 2004. Subsequently, he remained at Durham where he undertook a PhD on the controlled synthesis and properties of glycopolymers under the supervision of Prof. Neil Cameron. Between March 2008 and February 2010 he was a postdoctoral research assistant for Prof. Len Seymour in the Department of Clinical Pharmacology, University of Oxford, investigating the polymeric modification of viruses for systemic gene delivery. Since March 2010 he has been a postdoctoral fellow for Prof. Cameron Alexander in the School of Pharmacy, University of Nottingham, developing self-assembling polymer–DNA hybrids as potential drug-delivery vehicles. |
![]() Neil R. Cameron | Prof. Neil Cameron undertook his BSc and PhD at the University of Strathclyde in Glasgow. Following two post-doctoral periods, he was appointed as a Lecturer in the Department of Chemistry at Durham University in 1997. He was promoted in 2005 to Reader and in 2008 he was appointed Professor of Bioactive Chemistry in the same department. His research is focused on the preparation of bioactive and/or bio-inspired macromolecules, including bioactive glycopolymers. His research to date has led to >90 publications and he has given >90 invited lectures. He was awarded the 2003 Young Researchers' Medal from the Macro Group UK and held a Durham University Christopherson/Knott Fellowship for 2008–09. |
Influenza infection is a multistep process: initially, the virus binds to N-acetyl neuraminic acid residues on the target cellvia lectin structures known as hæmagglutinin (HA) fingers on its surface membrane and then enters the cellviaendocytosis. The virus membrane then fuses with the endosome releasing a complex of RNA and proteins into the cytoplasm. These are transported into the nucleus and the process of virus replication begins.13 If initial binding of the virus to the cell can be prevented, subsequent uptake and replication can also be halted. A molecule that could block HA binding efficiently could prove a useful prophylactic during influenza breakouts, such as the recent H1N1 pandemic. Influenza hæmagglutinin, like most lectins, has a shallow binding site and its interaction with monovalent sialosides is typically weak (Kd ≈ 2 mM),14 thus multivalent ligands should provide improved avidity. Additionally, the virus surface presents a neuraminidase (NA) enzyme and consequently hæmagglutinin inhibitors need to be stable with respect to neuraminidase action.15,16
The first example of an influenza hæmagglutinin inhibitor (HAI) based around a glycopolymer was reported by Bovin et al. in 1990. Polymeric sialosides of varying carbohydrate densities were synthesised by the reaction between poly[4-nitrophenylacrylate] with monosialosides with amino-terminated linkers (Fig. 1). As would be expected, little or no inhibition was seen for monovalent sialosides, β-sialosides, or polymers carrying low quantities of α-sialoside residues (∼5%). Increasing the sialoside density from 10 to 30% indicated a maximum in inhibition at 20%, with 30% having a lower inhibitory effect than the 10% sialylated polymer.17
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Fig. 1 Synthesis of polymeric multivalent sialosides as used by Bovin et al.17 |
The main contributors to the field of glycopolymeric HAIs are Whitesides and collaborators. Throughout the 1990s Whitesides et al. published several studies on the inhibition activity of polymeric sialosides synthesised by both polymerisation of sialylated monomers and post-polymerisation functionalisation of reactive polymers.18–23
Initial studies involved copolymers of acrylamido α-O-sialoside (1, Fig. 2) with various N-substituted acrylamides. As Bovin et al. had described previously,17 a maximum level of inhibition was observed at intermediate levels of sialylation. This was rationalised by the competition between cooperative and efficient binding of the sialic acid groups: at low SA levels the groups are well separated and thus binding of one residue does not increase the likelihood of a subsequent group's binding; with high SA levels, binding may be limited as the steric bulk of the groups overcrowd one another. It was also noted that bulky or charged groups on the comonomer tended to reduce the binding efficiency and, in turn, inhibition.19,23 Although copolymers of 1 resulted in highly effective HAIs (inhibition constants (KiHAI) were typically 104–105 fold greater than monomeric equivalents on a per sugar basis), the O-linked SA made them susceptible to cleavage by neuraminidases. In order to alleviate this problem, acrylamido α-C-sialoside 2 was synthesised and copolymerised with acrylamide. Polymers with C-linked SA groups were found to have a maximum inhibitory effect comparable to that of their O-linked equivalents and had a far greater effect at low SA concentrations, probably due to their ability to interact with neuraminidase without deactivation.21 Despite high levels of inhibition displayed by polymers synthesised from sialylated monomers, they were still less efficient than either non-polymeric synthetic HAIs, such as sialylated liposomes,24 or naturally occurring HAIs, such as equine α2-macroglobulin,25KiHAI ≈ 100–200 nM. Consequently, Whitesides et al. turned their attention to the sialylation of reactive polymer backbones. The reasons for this are three-fold: firstly, due to differing monomer reactivities, it is unlikely that sialosides will be statistically placed along a polymer based upon feed ratio. If the comonomer is of higher reactivity it is likely that, especially at the low feed level of SA monomers, the result would be gradient copolymers. Secondly, as overcrowding was considered to be responsible for the reduction in activity of polymers containing greater quantities of SA, steric interactions should reduce over-functionalisation. Finally, polymers may be more directly compared; a single batch of a precursor polymer results in all derivative polymers having the same polydispersity and degree of polymerisation.
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Fig. 2 Sialic acid derived monomers and amines as used by Whitesides et al. in the synthesis of glycopolymeric influenza hæmagglutinin inhibitors. See Fig. 1 for structure of sialic acid (SA). |
The precursor polymers of choice were those featuring activated esters,26,27 such as poly[N-(acryloyloxy) succinimide] (pNAS) or poly[acrylic anhydride] (pAAn), which were reacted with amino-terminated sialosides including 3 and 4. pNAS was treated with a varying number of equivalents of 3 from 0.2–1.2 with respect to the number of succinimide groups; the level of sialoside incorporation was found to correlate directly with the number of sialoside equivalents up the maximum value of 1. After reaction of the sialoside, any remaining succinimide groups were functionalised by addition of an excess of a second amine or ammonia to yield copolymers of various N-substituted acrylamides. As was seen with the previous polymers, addition of charged groups had a negative effect on the inhibition, particularly positive charges where a singly positively charged side group has a more detrimental effect than a triply negatively charged side group; neutral, polar side groups also reduced inhibition with increasing steric bulk. Hydrophobic side groups increased or decreased inhibition depending on the steric bulk. Benzylamine, for example, was found to improve efficacy as its level of incorporation was increased; presumably through increased hydrophobic–hydrophobic interactions between the polymer and virus surface. Conversely, hexylamine resulted in reduced inhibition.20 Overall polymers with sub-nanomolar values of KiHAI could be produced. Polymers synthesised by similar methods from pAAn gave similar results.18 It was also determined that a synergistic treatment combining C-sialoside–acrylamide copolymers and low molecular weight monomeric neuraminidase inhibitors resulted in even greater inhibition of hæmagglutination. Although the mechanism of this synergy was not confirmed it is thought that the NA inhibitor displaces the polymer from the NA sites either allowing more SA residues to bind to HA sites or increasing the overall steric bulk of the polymer around the virus.28
In addition to hæmagglutinins, the neuraminidases are also targets for influenza treatment; in fact, the currently preferred influenza antivirals, such as oseltamivir (Tamiflu®, Hoffman-La Roche) and zanamivir (Relenza®, GlaxoSmithKline), act as transition state analogues of sialic acid cleavage.29 The presence of NAs on influenza at first seems counterproductive for the virus; NAs cleave sialic acid residues which would, in effect, reduce the chance of viral binding to the cell surface. In fact, the neuraminidases are essential for spread of the virus and infection of further host cells. Once a replicated virus has matured and budded from the cell, it can once again bind to the cell surfacevia the hæmagglutinin molecules. The neuraminidase cleaves the cell surfacesialic acid groups, releasing the new virus.16 Multivalent sialosides that are resistant to NAs have the potential of binding to these receptors and preventing the release of the virus from the host cell and therefore limiting the infection.
Linhardt et al. synthesised C-linked glycopolymer 5 (Fig. 3) and tested its ability to inhibit neuraminidase from Clostridium perfringens, a common bacterium. Compound 5 was synthesised by enzymatic polymerisation of the aromatic monomer by soybean peroxidase in the presence of hydrogen peroxide. Compound 5 was seen to inhibit neuraminidase 10-fold greater than monomeric equivalents.30 Matsuoka et al. synthesised 6 as a copolymer by radical polymerisation of an acetate-protected vinyl precursor with vinyl acetate; after treatment with NaOH, sialylated poly[vinyl alcohol] was isolated. In preliminary tests, polymers were shown to have an inhibitory effect against influenza neuraminidases in the millimolar range.31 Dendritic sialosides synthesised by the same group display similar levels of inhibition.32
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Fig. 3 Polymeric neuraminidase inhibitors as synthesised by Linhardt et al. (5) and Matsuoka et al. (6). SA = α-sialoside, structure given in Fig. 1. |
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Fig. 4 Sulfated maltoheptaose derived methacrylate glycopolymers as synthesised by Yoshida et al. |
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Fig. 5 Glucosamine based glycomonomers as synthesised by Miura et al.47 |
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Fig. 6 Autoradiogram of an adult mouse 30 min after intravenous injection of radiolabelled histamine. The dark regions show where the histamine is located—none is detected in the brain and spinal cord regions. Reprinted from W. M. Pardridge, The Blood–Brain Barrier: Bottleneck in Brain Drug Development, NeuroRx, 2005, 2, 3–14, Copyright (2005), with permission from The American Society for Experimental NeuroTherapeutics, Inc. Published by Elsevier Inc. |
An example of such a treatment has been demonstrated by Hashida et al. in mice. The K vitamins are a family of hydrophobic molecules required for the synthesis of the proteins involved in blood coagulation.55 The denotation of ‘K’ vitamins derives from the German naming koagulations vitamin,56 consequently vitamin K deficiency may lead to hæmorrhaging. It is common for expectant mothers and newborns to be administered vitamin K as a prophylactic but this has found controversy due to its administration having been weakly linked to childhood cancers and other side effects.57,58 The majority of coagulating proteins are synthesised in the liver and thus targeted delivery of vitamin K to the liver may allow suitable prophylaxis with reduction of potential side effects. Hepatic (liver) cells are known to express the galactoside-binding asialoglycoprotein receptor (ASGPR) on their surface; on binding, the galactoside-conjugate is internalised by the cell.59 Hashida et al. synthesised terpolymers based upon a poly[L-glutamic acid] (PLGA) backbone (Fig. 8) by reaction with ethylenediamine followed by 2-imino-2-methoxyethyl 1-thiogalactoside to produce galactosylated PLGA. In turn, this was reacted with vitamin K5, a synthetic K vitamin analogue, to yield a galactosyl–PLGA–vitamin K conjugate. The anti-hæmorrhagic effect of such polymers was determined in mice models by comparison of the prothrombin time, a measure of coagulation efficiency, after systemic treatment with warfarin. As expected, in all cases prothrombin time was increased for warfarin treated mice compared with untreated. Warfarin treated mice that received intravenous (IV), unconjugated vitamin K only had a statistically significant reduction in prothrombin time 4 h after treatment, those receiving IV galactose–PLGA–K conjugate had a significant reduction at 2, 3 and 4 h time points.60
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Fig. 8 Anti-hæmorrhagic, poly[L-glutamic acid] based terpolymers as synthesised by Hashida et al. |
Similarly Fleming et al. have targeted boar spermatozoa, which are known to display a galactose-binding lectin with great similarity to the hepatic ASGPR, with galactosyl polymersin vitro. They synthesised terpolymers of 2-(β-D-galactosyloxy)ethyl methacrylate, 2-(dimethylamino)ethyl methacrylate (DMAEMA) and a methacrylate featuring an α-tocopherol functionality. The resulting polymers were incubated with spermatozoa in an attempt to deliver the α-tocopherol, an antioxidant, to the cells to reduce oxidative damage during storage. Although the polymers appeared to have some protective effect to confirm their entrance into the cells, rather than acting as an extra-cellular protectant, the α-tocopherol monomer was replaced by a fluorescent monomer, hostasol methacrylate, and the polymer inside the cell visualised by confocal microscopy (Fig. 9).61
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Fig. 9 Confocal micrograph of boar spermatozoa after incubation with a poly[GalEMA-DMAEMA-hostasol methacrylate] terpolymer. Image provided by the author. |
A polymer based upon this model, PK2 (FCE28069, Fig. 10), with the majority of tyrosinamide replaced with the anti-tumour drug doxorubicin (DOX) has been evaluated in a Phase I clinical trial for hepatoma. Trace levels of tyrosinamide were maintained to facilitate radiolabelling and subsequent imaging. Doxorubicin is a highly effective chemotherapy drug, however, off-target cardiotoxicity limits its use.65,66 A preclinical study in a rat model was used to determine the level of cardiotoxicity cf. free DOX. PK2 and free DOX were administered by both IV and intraperitoneal (IP) injection. Acute and cardiovascular toxicities were monitored by weight loss and cardiac output respectively. Animals receiving free DOX IV displayed acute toxicity at doses greater than 2 mg kg−1, with significant (>20%) weight loss 8–12 weeks after administration. By comparison, animals treated with PK2 at up to 12 mg kg−1 (DOX equivalent) were seen to gain weight, albeit at a reduced rate compared to the saline control. IP administration was considerably less toxic for both PK2 and free DOX, with no mean weight loss after 12 weeks. Animals administered 12 and 18 mg kg−1DOX equivalents of PK2 gained weight at a comparable rate to the saline controls. Cardiotoxicity, measured by relative cardiac output, was found to be insignificant in IV doses of PK2 up to 12 mg kg−1 and free DOX at 2 mg kg−1. 3 mg kg−1DOX yielded significant decreases in cardiac output (>35%, p < 0.0005) after 12 weeks. No animals survived to the 12 week end-point when administered 4 mg kg−1DOX IV. Overall survival curves showed that all animals receiving IV PK2 survived to 12 weeks post-administration. IP administration was, again, seen to be far less toxic with only doses of 5 and 6 mg kg−1 of DOX or 36 mg kg−1PK2 resulting in less than 80% survival at 12 weeks.67
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Fig. 10 Structure of doxorubicin-conjugated polymer PK2. The trace tyrosinamide modifications are omitted for clarity. |
PK2 was assessed in a Phase I clinical trial involving patients with confirmed primary or secondary solid hepatic tumours. Patients were administered PK2 IV at 3 week intervals for a maximum of 6 treatment cycles. Doses administered ranged from 20–160 mg m−2DOX equivalent. Blood and urine samples were collected at various times up to 8 days after each treatment. Blood and urine samples were analysed to determine the level of polymer-bound and free doxorubicin and metabolites; distribution of the polymer was determined by full body imaging of 123I using single photon emission computed tomography. The galactosamine–polymer–doxorubicin conjugate was seen to be rapidly cleared from the bloodstream with 15–20% of the administered dose accumulating in the liver after 24 h. A control polymer, identical except for the absence of galactosamine residues, was seen to remain in the bloodstream for longer and was found to have a general body distribution with no organ specificity. Despite a large accumulation of the polymer in the liver the majority was found in healthy hepatic cells rather than in the tumours themselves, although the accumulation was still significant compared to background. The reduced uptake to cancerous cells was rationalised by the reduced levels of ASGPR that hepatoma cells are known to express compared to their healthy counterparts.68–71 The increased uptake compared to other tissues may instead be due to the enhanced permeability and retention effect and not a result of lectin targeting.72
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Fig. 11 Amphiphilic polycarbonate block copolymers as used by Hedrick et al. for the delivery of doxorubicin. |
The use of glycopolymers as molecular chaperones for proteins has been demonstrated by Chaikof et al. They utilised cyanoxyl-mediated polymerisation75,76 for the synthesis of several biomimetic glycopolymer species from alkenyl, acryloyl and acrylamido glycomonomers, often in their sulfated form (Fig. 12).75,77–79 Several of these polymers were tested with respect to their ability to act as mimics of heparan sulfates.78In vivo, fibroblast growth factor 2 (FGF-2) is bound by heparan sulfate, an anionic polysaccharide, which acts as a molecular chaperone protecting FGF-2 from deactivation and facilitating its binding to FGF receptor-1 (FGFR-1).80 Binding assays found that polymers featuring N-acetylglucosamine residues did bind, but weakly compared to heparan sulfate, the linker length was seen to have little effect.78 Sulfated sugars bound more strongly than their non-sulfated equivalents, particularly for polymers featuring pendant lactose groups. Further investigation into the FGF-2 chaperone role of glycopolymers featuring lactose sulfate residues found that low molecular weight (∼10 kDa) polymers containing ca. 10% of the glycomonomer were nearly as effective as heparin in dimerising FGF-2 and binding it to FGFR-1. The chaperone qualities of the polymer were also demonstrated by the extra stability that it gave FGF-2 with respect to degradation by acid, heat and trypsin.81 The glycosyl functionalised polymers were also compared to heparin in anticoagulant activity assays. Polymers featuring monosaccharides had no anticoagulant activity, those featuring lactosyl groups displayed interesting anticoagulant activity dependent on polymer composition and functionalisation. Non-sulfated polymers, like the monosaccharide polymers, had no activity, sulfated polymers were active but homopolymers less so than copolymers with acrylamide.
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Fig. 12 Glycosylated monomers as used by Chaikof et al. for the synthesis of heparin mimics viacyanoxyl-mediated polymerisation. |
Wang et al. have produced glycohydrogels for the treatment of norovirus infection, one of the main causes of gastroenteritis. Noroviruses have been demonstrated to bind to the surface glycans on erythrocytes that determine the blood group with a particular affinity to the B group epitope. They synthesised the monomer 1-acrylamido-3,6-dioxa-8-octyl-O-(α-L-fucopyranosyl)-(12)-O-(α-D-galactopyranosyl)-(13)-β-D-galactoside (10) and subsequently polymerised it in the presence of acrylamide, diallyldimethylammonium chloride and N,N′-methylene bisacrylamide to yield hydrogels of the type shown in Fig. 13. The entrapment ability of the glycohydrogels with respect to norovirus was determined using recombinant virus-like particles, virus particles that carry no payload, and ELISA assays. Glycohydrogels were seen to reduce dramatically the solution virus concentration compared to hydrogels prepared in the absence of the glycosylated monomer.84
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Fig. 13 Synthesis of glycohydrogels capable of binding norovirus as synthesised by Wang et al. |
The most advanced example, that of PK2, was found to be unsatisfactory for its planned target but has demonstrated that it is possible to produce a relatively complex drug-conjugate to a standard that permits clinical evaluation, that is cGMP. PK2 was synthesised using methodologies that much of the synthetic polymer community would now consider archaic and ill-defined but their simplicity may be an advantage in seeking FDA, or equivalent, approval. Combined with the advances in polymerisation technologies that have occurred over recent decades the production of materials of consistent, defined quality may allow translation of some of aforementioned examples to clinical development.
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