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Advanced Drug Delivery Reviews 61 (2009) 1131–1148

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Advanced Drug Delivery Reviews

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Development of HPMA copolymer–anticancer conjugates: Clinical experience andlessons learnt☆

Ruth Duncan ⁎School of Chemistry, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, UK

☆ This review is part of the Advanced Drug Delivery Re⁎ Tel.: +44 2920916160.

E-mail address: [email protected].

0169-409X/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.addr.2009.05.007

a b s t r a c t
a r t i c l e i n f o
Article history:Received 7 May 2009Accepted 11 May 2009Available online 20 August 2009

Keywords:HPMA copolymersPolymer therapeuticsNanomedicinesCancerPhase I/II

The concept of polymer–drug conjugates was proposed more than 30 years ago, and an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugate of doxorubicin covalently bound to the polymer backbone by aGly-Phe-Leu-Gly peptidyl linker (FCE28068) became thefirst synthetic polymer-based anticancer conjugate toenter clinical trial in 1994. This conjugate arose from rational design attempting to capitalise on passive tumourtargeting by the enhanced permeability and retention effect and, at the cellular level, lysosomotropic drugdelivery to improve therapeutic index. Early clinical results were promising, confirming activity in chemo-therapy refractory patients and the safety of HPMA as a new polymer platform. Subsequent Phase I/II trialshave investigated anHPMA copolymer-based conjugate containing a doxorubicin and additionally galactose asa targeting moiety to promote liver targeting (FCE28069), and also HPMA copolymer conjugates of paclitaxel(PNU 166945), camptothecin (PNU 166148) and two platinates (AP5280 and AP5346- ProLindac™). Thepreclinical and clinical observations made in these, and clinical studies with other polymer conjugates, shouldshape the development of next generation anticancer polymer therapeutics.

© 2009 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11321.1. General background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11321.2. HPMA copolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1133

2. Rationale for design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11332.1. Molecular weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11362.2. Linker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11362.3. Design in respect of drug loading, targeting residues and imaging agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1137

3. Preclinical development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11373.1. Preclinical toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11373.2. Validated characterisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1138

3.2.1. Identity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11383.2.2. Molecular weight and polydispersity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11383.2.3. Total drug and free drug content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1138

3.3. Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11394. Clinical experience . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1140

4.1. HPMA copolymer conjugates designed for passive targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11404.1.1. HPMA copolymer–doxorubicin (FCE28068) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11404.1.2. HPMA copolymer–paclitaxel (PNU 166945) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11414.1.3. HPMA copolymer–camptothecin (PNU 166148). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11414.1.4. HPMA copolymer–platinates (AP5280, AP5346). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1142

4.2. Conjugates designed for receptor-mediated targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11424.2.1. HPMA copolymer–doxorubicin–galactose (FCE28069) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1142

views theme issue on “Polymer Therapeutics: Clinical Applications and Challenges for Development”.

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1132 R. Duncan / Advanced Drug Delivery Reviews 61 (2009) 1131–1148

5. Conclusions and lessons learnt . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11435.1. Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11445.2. Design for optimum therapeutic index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11445.3. Clinical trial design. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1146

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1146References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1146

1. Introduction

1.1. General background

AlthoughHelmut Ringsdorf first proposed the concept of polymer–anticancer drug conjugates in 1975 [1] most early systems rarelyprogressed beyond in vitro testing. Lack of a sound biological rationalemeant that most conjugates tested in vivo were ineffective. For thisreason, and also uncertainly of the value of polymers as carriers per se(antibodies and proteins were muchmore favoured as a more ‘biolog-ically’ acceptable platform), the community was slow to embrace thevalue of this approach. Polymer–drug conjugateswere viewed as “nicescience” but an impractical mix of polymer and organic chemistry re-sulting in compounds so complicated that they would never be devel-oped clinically. These conceptual reservations were compounded bythe fact that polymers used pharmaceutically have many differentwell-known forms including implants, gels, and excipients (often astablets and capsules). Historically, and still today, this leads to con-fusion. Moreover, the use of water-soluble polymers as solutions forinjection was then much less appreciated. Over the last 20 years on-cologists have become increasingly familiar with ‘polymers’ in theirmany guises. Biodegradable polymeric implants are routinely usedboth as a subcutaneous (s.c.) depot to slowly release LHRH analogues(e.g. Zoladex®; Leupron Depot®) [2], for treatment of prostate andother hormone-dependent cancers, and for implantation post-glio-bastoma surgery to local delivery chemotherapy for treatment of re-sidual or recurrent disease (e.g. Gliadel®) [3]. Moreover, over the last15 years there have been a growing number of polymer conjugates,especially PEG-proteins that have come to market (reviewed else-where in this issue), and polymeric micelles entering clinical trials asanticancer agents and also, for micelles, as non-covalent drug deliverysystems (reviewed in [4]).

Early studies focused on both natural and synthetic polymers. Stilla popular question today — which is best? The appropriate answer isneither. In this field it is essential to choose the correct polymer foreach specific application/route of administration. Earlier on, polysac-charides were widely explored, dextran being particularly popularowing to its clinical approval for use as a plasma expander. A dextran–doxorubicin conjugate (AD-70) was the first polymer–drug conjugateto be tested clinically, the clinical formulation being supplied by AlphaTherapeutic GmbH. Anthracycline conjugation seemed by Schiff baseformation to oxidised dextran also modified with glycine as a pendantgroup [5]. The rationalewas to utilise tumour hypoxia to promote drugliberation. However, in a Phase I trial (13 patients) when AD-70 wasadministered every 21–28 days by a 30 min infusion unexpected toxi-cities (severe thrombocytopenia and hepatotoxicity) occurred, evenat the starting dose of 40mg/m2 (doxorubicin-equivalent) [5]. To notethis is approximately half the recommended dose for doxorubicin(also known as Adriamycin®), which used clinically at a dose of 60–75 mg/m2. Despite dose reduction to 12.5 mg/m2 toxicity was stillseen. AD-70 induced hepatotoxicity lasting for several weeks sug-gesting liver localisation with slow release of doxorubicin thereafter.This toxicity was attributed to reticuloendothelial (RE) cell uptake ofthe poly(glucose) and/or the fact that doxorubicin was conjugated tooxidised dextran so residual aldehyde groups would likely be presentafter drug conjugation. Phase II studies were neither reported for this

compound nor a carboxymethyldextran–camptothecin conjugate(DE-310) that comprised a camptothecin analogue DX-8951f wascovalently bound to the carbohydrate carrier via a Gly-Gly-Phe-Glypeptide linker [6]. Our research in the 1980s (supported by the CancerResearch Campaign (CRC UK) and Farmitalia Carlo Erba (becamePharmacia now Pfizer) in collaboration with the Institute of Macro-molecular Chemistry, Academy of Sciences of the Czech RepublicPrague) designed the first two synthetic polymer–drug conjugates toenter clinical trial as anticancer agents for intravenous (i.v.) injection.These were based on N-(2-hydroxypropyl)methacrylamide (HPMA)copolymers. This history has been well documented although mostlyfrom an academic viewpoint [7–13]. Here the rationale for design oflead compounds, key steps in preclinical development and the cur-rent clinical status of HPMA copolymer-based anticancer agents isreviewed. The challenges for effective clinical development of thesecomplex macromolecular prodrugs are also discussed.

All polymer–anticancer drug conjugates progressing throughclinical trials as anticancer agents are in effect macromolecular pro-drugs. They typically comprise a minimum of three components; anatural, synthetic or pseudosynthetic (e.g. poly(glutamic acid); PGA)water-soluble polymeric carrier usually of molecular weight 10,000–100,000 g/mol; a biodegradable polymer-drug linkage, and the bio-active antitumour agent (reviewed in [4,7,11]). It should be noted thatmany of the drugs so far attached (e.g. anthracyclines, taxanes, andcamptothecins) were chosen in the 1980s and 1990s when thesemoleculeswerefirst introduced into routine clinical practice. There arenow many more modern and interesting candidates. Most of thesependant drugs are extremely hydrophobic causing the conjugate toadopt a nanosized, unimolecular micelle conformation in aqueoussolution (typically 5–20 nm) [14]. As new chemical entities (NCEs)these conjugates have been rightly defined as polymer therapeuticsrather than (non-covalent) drug delivery systems such as liposomesand nanoparticles that simply entrap drugs (reviewed in [4,15]). Theyalso fall within the definition of “nanomedicines or nanopharmaceu-ticals” adopted by the European Science Foundation's Forward Look onNanomedicine (reviewed in [16]). In certain cases ligands have alsobeen introducedwith thehopeof promoting receptor-mediated tumourtargeting. For example, the HPMA copolymer–doxorubicin conjugatethat contains additionally galactosamine (FCE28069) designed in theearly 1980s [17] to target the hepatocyte asialoglycoprotein receptorwas the first synthetic, multivalent natural mimetic conjugate to enterclinical testing. To aid preclinical pharmacokinetic studies and facil-itate clinical imaging, imaging agents have also been incorporated intothe conjugate. This, togetherwith the recentmove towards conjugatescarrying combination therapy often results in highly complex, multi-functional, structures (Fig. 1). To note that schematic representationsthat show polymer conjugates as a “washing line” (see cartoon ofRingsdorf [1]) are outdated and indeed unhelpful. It has clearly beenshown that many polymer conjugates form a unimolecular micelle inaqueous solution. The ‘compactness’ and structure of these dynamicstructures (expected to change as pendant moieties are liberated) hasa significant effect of biological properties such as enzyme access,targeting ligand–receptor interaction and will also influence pharma-cokinetic properties. There is an urgent need to define and betterunderstand the structure–activity relationships of these complexarchitectures.

Fig. 1. Schematic showing the threemain components of polymer–drug conjugates. Thepolymer carrier, polymer–drug linker and drug payload (may be a combination therapy)are shown. In addition imaging moieties and also targeting residues are often addedgiving a very complex architecture.

1133R. Duncan / Advanced Drug Delivery Reviews 61 (2009) 1131–1148

1.2. HPMA copolymers

The first conjugate to enter clinical evaluation was HPMA copo-lymer–doxorubicin (FCE28068) in 1994 [18]. Since five other anti-cancer compounds and two gamma camera imaging agents derivedfrom this polymer have been evaluated clinically with administration(often as multiple cycles) to N250 patients. The homopolymer, poly(HPMA), was originally developed in the Czech Republic as a plasmaexpander [19]. It was non-toxic in preclinical tests at doses up to 30 g/kg, did not bind blood proteins and was not immunogenic. Moreoverlike PEG, grafting of HPMA copolymer chains to proteins reduces theirimmunogenicity [20]. From the outset there was likelihood that thishydrophilic polymer would display minimal inherent toxicity with itsmain limitation being lack of biodegradability of the polymer mainchain. To introduce the functionality needed for drug conjugation, andalso to insert biodegradable linkers for drug liberation, HPMA copo-lymers were prepared [21]. A full description of the evolution of thischemistry is beyond the scope of this article (see [7,12]), however, toenable comment on the challenges for manufacture and validation itis important to briefly describe the synthetic route here.

The synthesis of the HPMA copolymer intermediates used to createconjugates tested clinically involves free radical polymerisation usingHPMA and methacryloylated (MA)-peptidyl-nitrophenylester (ONp)as the comonomers (Fig. 2). Normally the monomer feed ratio ofHPMA:MA-peptide-ONp is 95:5, although this is changed to 90:10 incertain conjugates. Addition of MA-tyrosinamide specific cases as athird comonomer (1 mol%) has been used to prepare a ter-polymersuitable for 125I, 131I or 123I-radiolabelling thus enabling preclinicaland clinical gamma camera imaging [18,22–25]. Selection of optimalcomonomer chemistry/composition, and careful control of the re-action conditions to regulate polymerisation kinetics made it possibleto synthesise HPMA copolymer precursors of relatively narrow mole-cular weight distribution (Mw/Mn=1.2–1.5). (The final molecularweight distribution of large-scale batches for clinical trial can be nar-rowed further by fractionation methods if required.) Drugs (andwhere needed targeting residues) have been generally bound to such“key” polymeric intermediates using an aminolysis reaction (Fig. 2). Itis noteworthy— although full reviewbeyond the scope of this article—however, that general research studies have explored a much broaderrange of synthetic routes (e.g. use of drug-bearing comonomers,binding of drugs directly to poly(HPMA) homopolymer, and use ofwell characterised low molecular weight functionalised drug inter-mediates for conjugation). Each synthetic route brings a unique profile

of conjugate heterogeneity, challenges for purification and indeedimpurity characterisation (see [7,13]). Early studies showed that doxo-rubicin conjugation to the hydrophilic HPMA copolymer led to a N10-fold increase in its aqueous solubility. Since, this ‘solubilising’ oppor-tunity has also been used to improve the formulation properties ofother lipophilic drugs including paclitaxel. It should be emphasisedthat the –C–C–HPMA copolymer main chain is not biodegradable sothe conjugates developed clinically have been limited to a molecularweight of b40,000 g/mol to ensure eventual renal elimination.

2. Rationale for design

History has shown that effective conjugates have been the productof rational design. Synthesis of interesting chemical libraries, withoutdue attention to the complex chemical and biological landscape thatthe conjugate must circumvent in vivo, and use of polymers withinadequate biocompatibility for parenteral administration, and/orimpracticality for manufacture or ultimately clinical use invariablyleads to early failure (reviewed in [7–13]). Although we exploredmany different synthetic and natural polymers as potential carriersthrough the 1970s and 1980s, most were abandoned due to toxicity,immunogenicity, inadequate chemical functionality (or control ofchemical composition), unacceptable body distribution, and/or lackof biodegradability. Of all the polymers we examined HPMA copo-lymers offered, at that time, the greatest potential for developmentas an anticancer drug carrier platform. Appealing properties werethe prospect of a non-toxic and non-immunogenic polymer, multiplependant side chains to carry the drug/targeting residue payload (incontrast to PEG which has only two terminal functional groups), andnot least, the possibility to capitalise on the pioneering chemical syn-thetic methods introduced by Kopecek et al. in the 1970s (reviewedin [7,12,26]). The latter allowed the controlled synthesis of tailoredconjugate libraries by polymer analogous reaction for the in vitro andin vivo structure–activity studies needed to optimise polymer mole-cular weight, the peptidyl linkers used for drug attachment/releaseand, in the case of galactose for targeting, the degree of loading needsto accomplish cell selectivity.

From the biological viewpoint, the principal objectives of poly-mer–anticancer drug conjugation is to alter drug pharmacokinetics toachieve improved tumour targeting, reduced access to sites of non-specific toxicity (e.g. bone marrow, heart, brain, etc.) and to ensure, atthe cellular level, that an active drug can reach its pharmacologicaltarget. This might be an extracellular target but most usually it has anintracellular/nuclear location. Whichever, on arrival the drug must bereleased at a concentration and rate able to maximise its therapeuticaction. The current understanding of the complex mechanism of theaction of polymer–anticancer drug conjugates has been extensivelyreviewed [4,27]. Many factors appear to act in concert to produceantitumour activity (Fig. 3). Conjugate pharmacokinetics and the rate,and location of, drug liberation are of central importance to the thera-peutic benefit obtained. The use of hydrophilic polymers like HPMAcopolymers as the carrier can avoid rapid RE uptake (liver and spleen)that is commonly seen for classical nanoparticles and liposomes, andto a lesser extent their PEGylated forms (there is clinical proof of this).Moreover, the enhanced blood circulation time of polymer conjugat-ed, relative to free, drug brings an opportunity to promote tumourtargeting via the increased permeability of angiogenic tumour vascu-lature, i.e. enhanced permeability and retention (EPR) effect as firstdefined by Maeda and Matsumura [28]. Once in the tumour inter-stitium, all the quantitative evidences [29,30] suggest that HPMAcopolymer–doxorubicin conjugates will enter cells by the endocyticroute leading to lysosomotropic drug delivery [31].

The most effective polymer conjugates have been designed toinclude a polymer-drug linkage that is stable in the bloodstreamensuring very low levels of free drug in plasma and thus significantlyreducing bioactive drug access to sites of toxicity. This, together with

Fig. 2. Typical route to the synthesis of HPMA copolymer–anticancer agents evaluated clinically. Panel (a) polymer intermediate synthesised by copolymerisation and then drug (andtargeting residue not shown) binding by an aminolysis reaction. The conjugates FCE28068 HPMA copolymer–doxorubicin (panel b) and FCE28069 HPMA copolymer–doxorubicin–galactose (panel c) were synthesised in this way.


the relatively rapid renal elimination of non-galactose targeted HPMAcopolymer conjugates (compared to whole body clearance rates forfree drug which displays a high volume of distribution percolatingthroughout the body) explains the frequently seen reduction in con-jugate clinical toxicity with higher maximum tolerated doses (MTD)than seen for free drug. Since our first studies with HPMA copolymer–doxorubicin in preclinical in vivo models [32,33], EPR-mediatedtumour targeting with significantly increased tumour drug concentra-tions has been repeatedly demonstrated in in vivo tumour models

following HPMA copolymer conjugate administration. Notably, smal-ler tumours often display the highest uptake (up to 20% dose per gramtumour) [34].

Many HPMA copolymer conjugates tested clinically contain thepeptidyl linker Gly-Phe-Leu-Gly. This was designed specifically forcleavage by lysosomal thiol-dependent proteases [35] for polymerprodrug activation following lysosomotropic delivery [31]. Whetheror not polymer–drug conjugates display an altered pharmacologicalmechanism of action (compared to parent drug) in the in vivo/clinical

Fig. 3. Overview of the putative mechanism of action of HPMA copolymer–anticancer conjugates.


setting is still hotly debated. Hints suggest that HPMA copolymerconjugates can circumvent multidrug resistance (MDR) clinically[18,36]. This might be expected due to the uptake by endocytosisbypassing membrane-localised efflux proteins. However, an observa-tion that EPR targeting can producemuch higher tumour drug concen-tration could also explain this phenomenon as human tumours typicallydisplay ∼5–10 fold resistance index.

HPMA copolymer–anticancer conjugates frequently show superiorin vivo antitumour activity in rodent and xenograft tumour models(reviewed in [37]). Our experiments in large panel of tumour modelssuggested a drug release rate (i.e. variations in lysosomal thiol-pro-tease (cathepsin B) activity) maybe more important in determiningHPMA copolymer–doxorubicin activity than differences in the extent

of EPR-mediated targeting [34]. Moreover, important preclinical andclinical studies in non-small cell lung cancer (NSCLC) patients withthe PGA–paclitaxel conjugate (OPAXIO™ formerly XYOTAX™) haveindicated that oestrogen levels correlate with cathepsin B activity, andhence govern enzyme-mediated release paclitaxel from the conjugatewhich then impacts on efficacy/survival time (reviewed in [38–40]).These results underline the future importance of selection of themost appropriate patient population (in this case women with higheroestrogen levels) to achieve the best probability of a good clinical can-didate. It is also important to note that in March 2008, Cell Thera-peutics Inc. submitted a marketing authorization application (MAA)for OPAXIO™ to the EuropeanMedicines Agency (EMEA) that is underreview. They are seeking approval for on equivalent effectiveness


(non-inferiority) and improved safety basis as a single agent in first-line performance status 2 NSCLC patients.

Returning to the putative mechanism of action of HPMA copoly-mer anthracycline conjugates, from observations in preclinical studies(mainly in vitro) it has also been suggested that HPMA copolymer–doxorubicin conjugates act via different molecular mechanisms fromfree drug (including stronger activation of the apoptosis signalingpathways) compared with free doxorubicin [41,42]. However, otherstudies suggest that cell death induced by the same conjugates occursprimarily by necrosis. In vitro studies have also suggested that theyalso may act directly at the cell surface by interaction with mem-brane-localised death receptors (Fas-receptor pathway) to initiate cellkilling although these conclusions are also debated andmay simply bea result of the in vitro models, methodology or nature of the conju-gates used [43,44]. The kinetic complexities of in vitro experimentsinvolving conjugates (and use of different cell lines) make design andinterpretation of such studies particularly challenging.

There is, however, growing evidence that HPMA copolymer–doxo-rubicin conjugates can exert an immunostimulatory effect (Rihovaet al. have conducted numerous studies with important clinical rel-evance and Rihova provides persuasive evidence supporting thismech-anism elsewhere in this issue also see [45–47]). It is suggested that theearly antitumour activity of HPMA copolymer–anthracyclines in vivooccurs via cytotoxic or cytostatic drug action, but that secondary immu-nostimulatory action of circulating low levels of conjugate augmentsthis effect.

The key structural features of HPMA copolymer conjugates thatgovern clinical potential are briefly reviewed below to illustrate someimportant considerations for product specification definition.

2.1. Molecular weight

One of the biggest challenges for development of polymer–drugconjugates is heterogeneity of structure. This is particularly importantin the context of polydispersity (the range of molecular polymer chainweights present) as molecular size has such an impact on whole bodyand cellular pharmacokinetics with consequent implications for ef-ficacy and toxicity. Our early studies on endocytosis using poly(vinyl-pyrolidone) (PVP) as a probe in different cell types showed that uptakeof 125I-labelled PVP by epithelial cells was reduced when conjugateshad a molecular weight of N100,000 g/mol [48]. The effect of size onuptake was cell type-dependent and clearly conjugates designed forlysosomotropic delivery must be small enough to ensure internali-sation and consequently exposure to the endosomal/lysosomal envi-ronment (pH or enzymes) that mediates effective drug release. Thereis growing awareness that the particular route of endocytic uptake(and also contribution of the exocytosis pathways) can influence intra-cellular trafficking and hence will affect bioactivity. This is anotherimportant matter and beyond the scope of this review.

As mentioned above, the lack of HPMA copolymer main chaindegradability limits safe use to those polymers of molecular weightb40,000 g/mol amenable to renal elimination. (Our early studies andthose of others showed that higher molecular weight HPMA copoly-mers accumulate in skin as well as the RE system [49], and this is apotentially deleterious effect that would be compounded by chronicadministration.) It soon became clear that high early phase plasmaconcentration is a pivotal driving force for EPR-mediated targeting[50,51]. Moreover, conjugates with the longest plasma circulationdisplayed greatest tumour accumulation. In preclinical models, 125I-labelledHPMAcopolymer fractions covering a broadmolecularweightrange (up to 800,000 g/mol) demonstrated equivalent tumourtargeting indicating a wide size tolerance for extravasation [50]. Thiswas not surprising given their relatively small diameter [14] comparedto many other nanoparticles that display EPR-mediated targeting(typically 80–150 nm in size). However, it should be stressed thatangiogenesis is a dynamic process with the gaps in a vessel wall con-

tinuing to close as the vessel matures. This restricts efficient extra-vasation to those tumour regions where angiogenesis is ongoing.Although the efficiency of EPR-mediated tumour targeting in all clin-ical settings is still a matter of some debate (see Section 4), this mech-anism of passive targeting has been observed clinically for a growingnumber of ‘nanomedicines’. Moreover, angiogenesis is established asan interesting therapeutic target, in its own right, and the first HPMAcopolymer conjugates designed to inhibit angiogenesis [52] are de-scribed elsewhere in this issue by Satchi-Fainaro.

2.2. Linker

Our early systematic studies led to the design of the Gly-Phe-Leu-Gly linker still widely used today in both HPMA copolymer and otherconjugates. This tetrapeptide was designed for stability in the circu-lation [53], and for degradation by the lysosomal thiol-dependantproteases, particularly cathepsin B [35]. It liberates 100% of conju-gated doxorubicin over a 24–48 h period in vitro, in animals andapparently also in man. The recent unexpected observation that PGA–paclitaxel efficacy is different in male and female NSCLC patientsprobably due to an oestrogen-related influence on cathepsin B levelshas significant implications for the choice of the patient cohort for allconjugates requiring cathepsin B activation.

It soon became clear that optimisation of drug release kineticsin the biological environments that a conjugate may encounter iskey to successful lead candidate development (e.g. blood+esterases(pH 7.4); lysosomes+lysosomal enzymes (pH 5.5) and urine (usually∼pH 6 but varies from 4.5–8.0)). Rapid drug liberation in the circu-lation inevitably eradicates the pharmacokinetic advantage of conju-gation. (Similarly any extracellular release into tissue culture mediamakes in vitro cytotoxicity testing meaningless in terms of optimalcandidate selection.) Releasing drug too slowly at the target usuallyminimises/eliminates activity, and additionally selective drug libera-tion in, or during passage through normal tissues can bring new/unexpected harmful effects (e.g. see HPMA copolymer–camptothecinbladder toxicity described later [54–56]).

HPMA copolymer–platinates (Pt) brought new challenges forlinker design [57,58]. For optimal activity there is a need to liberatethe biactive di-aqua Pt species in the vicinity of tumour cell DNA tomaximise cross-linking. Premature protein binding and/or furtherhydrolysis to the inactive di-hydroxy Pt results in total loss ofbioactivity. Pendant side chains bearing terminal ligands were neededfor platination, with an option also to introduce peptidyl spacers thatwould require enzymatic cleavage as a first step allowing subse-quent hydrolytic release of active Pt. First a library of conjugates weresynthesised to aid lead candidate selection for both cisplatin andcarboplatin analogues. They were prepared from HPMA copolymerintermediates containing either –Gly-Gly-ONp or Gly-Phe-Leu-Gly-ONp side-chains (5 or 10 mol%). To enable platination these linkerswere initially modified by hydrolysis to give a terminal –COOH, oraminolysis with ethylenediamine (en) (cisplatin analogues), amino-malonate or aminoaspartate (carboplatin analogues) to provide theligand for platination. The products had amolecular weight of 25,000–31,000 g/mol with a Pt loading of 3–10 wt.%. The HPMA copolymerGly-Phe-Leu-Gly-en-Pt requires lysosomal enzyme activation torelease active Pt species, confirmed in vivo by lack of antitumouractivity of conjugates containing the non-degradable linker –Gly-Gly-en-Pt [59]. The –COO–Pt analogues released platinum species muchtoo rapidly for useful delivery [59]. The carboplatin analogues showedhydrolytic release of Pt in vitro at pH 7.4 that correlated with theexpected stability of the 6 and 7 membered chelate rings; 14%/24 h Ptrelease from the malonate and 68%/24 h release in the case of theaspartate [58]. The more recently described diaminocyclohexyl(DACH) platinate AP5346 showed pH-sensitive Pt release with only3.5%/24 h of the total Pt liberated in buffer at pH 7.4, but showed a 7-fold higher release rate at pH 5.4 [59].


2.3. Design in respect of drug loading, targeting residues and imagingagents

A few comments are needed to underline design considerationsrelating to drug loading, incorporation of targeting residues, and alsoinclusion of functionality for gamma camera or MRI imaging. Per-fectionist synthetic chemistry often seeks to achieve the highest drugloading possible — one drug molecule per momoner may seem anattractive goal, but this is not necessarily the case. The hydrophobi-city of many drugs usually leads to decreased solubility as loadingincreases, and there is a significant risk of forming particulates thatwould be dangerous on injection. Moreover, there is an optimumdrugloading that can ensure external disposition of the hydrophilic HPMAcopolymer chain (‘covering’ a more hydrophobic interior) (see Fig. 1)to guarantee minimal non-specific membrane binding. Early in vitroendocytic uptake studies [60] using libraries of HPMA bearing-TyrNH2

or Gly-Gly-TyrNH2 copolymers (side chains 1–20 mol%) nicely illus-trated this fact. Non-specific adsorptive endocytosis was only trig-gered at a loading of N10 mol%. Drug loading must be optimised toensure that a pharmacologically relevant dose can be delivered inrelation to drug potency, frequency of dosing needed (daily dosing oflarge quantities of a non-degradable polymer carrier are impracticalfrom both a safety or infusion volume perspective), and themaximumtumour targeting achievable (% dose/g). Sometimes it is evident fromthe outset that these issues will ultimately limit the creation of acredible clinical candidate.

There is a need also to carefully optimise the loading of any tar-geting residue used. This can be challenging if a compromise is neededbetween the achievable drug loading and targeting residue loadingdue to total carrying capacity. Without careful optimisation of compo-sition the realisation of efficient targeting and activity would be futile.TheHPMAcopolymer–doxorubicin galactose conjugate, initially calledPrague–Keele (PK2), that became the clinical candidate FCE28069was designed to target the hepatocyte asialoglycoprotein receptorvia multivalent interaction of the pendant galactose (mimicking anasialoglycoprotein). Preclinical studies confirmed that incorporation(aminolysis of HPMA copolymer–ONp intermediates using galactos-amine) promoted significant hepatocyte targeting after i.v. injectionby up ∼80% of dose administered [61] providing the galactose loadingwas ≥4 mol % [61]. Preclinical studies also illustrated the challenge ofdefining a clinical dosing strategy. The magnitude of liver targeting (%dose) achieved is markedly dose-dependent with asialoglycoproteinreceptor saturation at a relatively lowdose in rodentmodels [62]. Suchreceptor saturation questions the value of classical bolus dose esca-lation in Phase I studies for all targeted therapeutics/nanomedicines.It also underlines the importance of early determination of receptorsaturation in the patient setting. The need for early clinical pharma-cokinetics, preferably using a non-invasive method, led to the designof the first polymeric gamma camera imaging agents [18,22–24]. Tominimise potential artefacts due to inclusion of the MA-Tyr-NH2 forradiolabelling a low monomer loading (∼1 mol%) was used, and forclinically used probes it was necessary to carefully validate the chem-ical composition of these complex multi-component imaging analo-gues. To ensure that the early phase pharmacokinetic profile of HPMAcopolymer conjugates was consistent if monitored by radioactivity(the labelled polymer backbone) and HPLC to monitor bound and freedrug preclinical in vivo experiments were conducted [24,33]. For poly-mer conjugates it is also possible to develop HPLC techniques that areable to follow drug release in vivo with time.

3. Preclinical development

The unique features of HPMA copolymer–drug conjugates, moreclosely related to protein- and immunoconjugates than small mole-cule drugs, required a paradigm shift in the techniques adopted forpreclinical screening and development. Approximately 7 years lapsed

from the “in principle” acceptance of an HPMA copolymer–doxoru-bicin conjugate as a clinical candidate (by the CRC Phase I/II Com-mittee in 1987) to recruitment of the first patient on study in 1994.During this time a meticulous preclinical development approach(without Federico Spreafico and the considerable expertise in Farm-italia Carlo Erba/Pharmacia and the manufacturing expertise of Poly-mer Laboratories Inc. this transfer would never have been realised)paved the way for passage of future conjugates from laboratory toclinic. With an experienced team it is now possible to initiate ananticancer polymer–drug project, identify a lead compound, and prog-ress to first clinical entry within 3–5 years. This timeframe is com-parable to that common for low molecular weight chemical entities,and was achieved for our more recent HPMA copolymer–platinateproject (licensed to Access Pharmaceuticals Inc.), and the OPAXIO™conjugate of Cell Therapeutics Inc. There are three areas of particularimportance in preclinical development; toxicology, the developmentof techniques for validated characterisation, and formulation devel-opment. These issues are briefly reviewed here (see [27] for full back-ground bibliography).

3.1. Preclinical toxicology

Any polymer proposed as a carrier must be biocompatible in thecontext of its particular route of administration, dose, and frequencyof use. Through the 1980s our work evolved a library of lab-basedtechniques to aid us select polymers we felt might be a suitable de-velopment towards clinical testing. (This subject is reviewed in fullelsewhere in this issue by Duncan and Gaspar.) This preliminaryscreen included assessment of polymer cytotoxicity in vitro (MTT test),haematotoxicity (by red bood cell lysis), and rate of enzymatic orhydrolytic degradation. When deemed ethically acceptable to prog-ress to in vivo animal studies with a candidate polymer, preliminarybiodistribution studies were undertaken to ensure that the polymeritself was not inherently hepatotropic (or would target other normaltissues), and also studies were undertaken to verify the polymer itselfwould not be immunogenic using ELISA assays to define the IgG/IgMresponse and othermethods to investigate a potential cellular immuneand humoral response. These early studies on HPMA copolymers havebeen reviewed [27]. (It should be noted that Blanka Rihova has beenthe pioneer in defining/understanding immunological properties ofwater-soluble polymers in general (see reviews [63,64]) From theoutset, it was clear that the HPMA polymer chain, an importantprimarymetabolite arising from these conjugates, was likely to displaya good safety profile in these respects.

Having designed a promising drug conjugate, it was still necessaryto conduct quantitative preliminary in vivo biodistribution studies(usually at 1 h but again not to Good Laboratory Practice (GLP), but labstandards) to ensure that drug coupling to the polymer would notprovoke RE (or other potentially disadvantageous) biodistribution.With a positive profile, whole body distribution was then carefullyassessed with time (typically 72 h) in mice bearing s.c. tumours bothto quantify tumour targeting and also identify any “potential” normaltissue redistribution that might have toxicological implications.Quantitation methods used included 125I-labelled polymer backbone,HPLC determination of free and conjugated drug in the circulationtissues, and more subjectively whole body autoradiography was alsoused. Before progression into clinical trials, our laboratory-based HPLCmethodology for doxorubicin conjugates was validated to GLP stan-dards [65], and then pharmaco- and toxico-kinetics re-determined inappropriate animal models (rodent and dog).

Selection of a specific clinical candidate triggered a conventionalGLP programme of preclinical toxicology. This was used to establishboth safety profile and the starting dose for clinical studies. Rarelydo such studies enter the academic literature, but the single and mul-tiple dose studies conducted in two rodent species for HPMA copoly-mer–doxorubicin (FCE28068) have been published [66]. (The studies


undertaken in dogs are still unpublished.) In preliminary rodentstudies the LD50 inMF1mice after a single i.v. injectionwas∼63mg/kg(doxorubicin-equiv.). Subsequently the conjugate was administeredto MF1 mice (22.5 or 45 mg/kg) in a single dose study, and was alsoadministered to MF1 mice or Wistar rats weekly for 5 consecutiveweeks at 12.0 or 22.5 mg/kg (mice) or 3 and 5 mg/kg (rats) in a mul-tiple dose study. FCE28028 induced haematological changes shortlyafter treatment, and in the single dose study alanine and aspartateaminotransferase levels were elevated at higher doses. Liver damagewas seen only in rat tissue during histological examination. Otherhistological changes induced by FCE28068 included thymic and tes-ticular atrophy, bonemarrow depletion, gastrointestinal tract changesand in the multiple dose study an increase in nuclear size in the proxi-mal tubules of the kidney was noted although no changes in urinewere seen. Recovery from these effects was seen in rats at 59 days.These toxicities are generally typical of anthracyclines, and a FCE28068dose of 20 mg/m2 (doxorubicin-equiv.) was recommended as a safestarting dose for Phase I clinical trials [66].

For the anthracycline conjugates FCE28068 and FCE28069, addi-tional studies were requested reflecting the macromolecular natureof the compound and also the known toxicological profile of doxo-rubicin. It was shown that HPMA copolymer–doxorubicin was not im-munogenic [67] and also that drug conjugation significantly reducedanthracycline-related cardiotoxicity as measured by both histologicalexamination and measurement of left ventricular ejection fraction inrats [68,69].

3.2. Validated characterisation

Typically several kilograms of product must be manufacturedusing a carefully controlled process (good manufacturing practice;GMP) to advance a polymer–drug conjugate into Phase I/II trial. Thisproduct must have a well-defined chemical specification ensuringreproducible batches. It must be characterised by GLP validated ana-lytical techniques, a formulation and validated assays for identity,purity and stability assays must be developed. These batches must beused in preclinical toxicological tests, and the same material used toprepare sufficient vials for the first clinical studies.

Methodology used to characterise many of the HPMA copolymer-based clinical candidates has been described (reviewed [27]). Themost salient points are noted here. It is important to stress that inaddition to likely residual impurities (solvents etc.) that are typical ofany manufactured drug substance, polymer conjugates bring risk ofother side products including residual monomer, side reactions alongthe polymer chain and/or side products arising from polymer cross-linking/particle formation. For example, during drug conjugation toHPMA copolymers by aminolysis reactions non-specific hydrolysiscan occur leading to the introduction of a small number of monomersterminating in –COOH, and the final stage aminolysis with 2-propano-lamine leads to the introduction of 2-hydroxypropylamide terminat-ing units. These co-monomers are rarely acknowledged in academicformulae (Fig. 4). The following methods have been used to char-acterise HPMA copolymer–anthracycline conjugates in respect ofidentity, molecular weight and polydispersity, content of the activeprinciple (free and bound drug) and in specific cases, the content oftargeting ligand.

3.2.1. IdentityHigh field (600 MHz) 2D NOESY and TOCSY 1H NMR techniques

were developed to define the identity of HPMA copolymer–anthracy-line conjugates (FCE28068 and FCE28069 [70]). Proton assignmentswere made by comparison with reference compounds and this en-abled definition any peptidyl (Phe) isomerisation [70]. The diagnosticsignals for side-chains terminating in –COOH and 2-hydroxyproyla-mide were also visible. High field NMR was also be used to detectcontaminating solvents, free 2-propanolamine, free doxorubicin and

in the case of FCE28069 free galactosamine with great sensitivity;b0.1% in some cases. The bound galactosamine present in FCE28069was present in the 4 isomeric α- and β-pyranose and furanose formswith the α-pyranose form predominating [70].

3.2.2. Molecular weight and polydispersityFor reasons outlined above, the definition of an acceptable speci-

fication for molecular weight and polydispersity are crucial to ensuresafety/activity. These parameters are particularly important to definecarefully for a non-biodegradable polymer backbone where there isa need to carefully define the fraction (%) in any batch that mightrepresent a molecular weight higher than the renal threshold andthus with a potential for cumulative retention in the body. Size ex-clusion chromatography (SEC) using commercial columns and con-jugate molecular weight fractions of narrow polydispersity were usedto construct compound-specific universal calibration curves [71,72].For example, in the case of the HPMA copolymer–paclitaxel conjugate(PNU166945) thirteen fractions were prepared together with 6 frac-tions of the homopolymer polyHPMA and these analysed by SEC, vis-cometry, and light scattering to give the molar mass distribution,intrinsic viscosity and size dimensions [73]. Such conjugate molecularweight fractions have also been tested biologically in efficacy andtoxicity studies to justify both in context of the product specificationset.

3.2.3. Total drug and free drug contentGLP standardised HPLC methods have been routinely used to

determine total and free drug in HPMA copolymer conjugates. Freedrug can be extracted from an aqueous solution of the conjugate. ForFCE28068 the sensitivity of this method was ∼0.01% and the recoveryof free doxorubicin was 97.7% [74]. Total drug content can be quan-tified by mild conjugate hydrolysis (optimisation of hydrolysis condi-tions using a model compounds, and definition of the release kineticsis required) to liberate free drug derivatives for determination byHPLC. Total doxorubicin in FCE28068 and FCE28069 which was esti-mated following mild hydrolysis using 1 N HCl at 50 °C for 1.5 h gaveN99% cleavage of the glycosidic bond, with a 96.9% recovery in respectof the total doxorubicin content. The need for such validated method-ology was highlighted for the doxorubicin conjugates where it wasshownthat theUVextinctioncoefficient forbounddoxorubicin (method-ology often used in lab assays) gave an over estimate (∼10%) of the drugcontent. Obviously this is an unacceptable error where an accurateclinical dose is vital to safety/efficacy.

Similarly, validated HPLC techniques were used to assess galactosecontent of FCE28069 [74]. Derivatisation with OPA led to irreproduc-ible HPLC results without pre-treatment with sodium borohydride toeliminate anomeric equilibrium and inter-conversion between thefuranose, pyranose and open forms of the sugar. For quantitation thebound galactosamine of FCE28069 was released from the polymerwith heating under strongly acidic conditions (6 N HCl), and themethod was optimised using N-acetyl-D-galactosamine as a model.The challenges, and often total impracticality, of validation of com-position of complexmulti-component conjugates frequently proposedas new “nanomedicines” in many research publications is habituallyoverlooked. In general, earlier consideration would better guide theproposal of “nanomedicines” that have a real chance of later clinicaldevelopment.

Validated assays developed prior to clinical evaluation of theHPMAcopolymer–platinates carboplatin (AP 5280) and DACH (AP5346)analogues have been described [75–77]. To define identity and purity(in respect of low molecular weight impurities to b0.1%) 1H NMRand infrared (IR) spectroscopy were used. SEC was used to determinemolecular weight and polydispersity, and 195Pt NMR to verify the co-ordination environment of the Pt and ICP-AES to determine platinumcontent. A scale-up process for the cost-effective manufacture ofHPMA copolymer–DACH platinate has been described [77], together

Fig. 4. Comprehensive representation of HPMA copolymer conjugates showing typical monomer structures that might be present as impurities. During synthesis it is possible tointroducemonomer impurities (exact chemistry will depend on route of synthesis), albeit in low level (panel a). These are often ignoredwhen drawing conjugate chemical structure.However, when entering clinical development it is important to define conjugates accurately to avoid confusion. Panels b and c show structures given for the HPMA copolymer–camptothecin MAG-CPT in two different Phase I clinical trials papers, Bisset et al. [55] and Schoemaker et al. [54], to illustrate the problem. It is also suggested in [55] that thestructure in panel (b) degrades from B to A which is incorrect.


with a technique for atomic-absorption spectrometry quantitation oftotal Pt in patient plasma samples [77].

3.3. Formulation

Relatively few studies discuss formulation issues relating topolymer-drug conjugates and indeed “nanomedicines in general”.The conjugate is in effect the active drug principle. The hydrophilicpolymer component can increase drug solubility, aids creation of asuitable lyophilised formulation, and also ‘enhance’ drug stability.However, as for any other medicine there is a need to formulate as the

final product that is sterile, practical to reconstitute for patientadministration, and also has the desired shelf-life stability at a definedstorage temperature. Furthermore, additional GLP validated analyticaltechniques are required to monitor conjugate identity/stability in thepresence of any excipients used andwhen placed in themanufacturedcontainers used and/or any infusion devices. Despite the high solu-bility (∼5%) of FCE28068 and FCE28069 relative to doxorubicin, thedissolution rate is relatively slow (N30 min). This is impractical forreconstitution in a hospital setting prior to injection. Optimised con-jugate formulations [78] were shown to contain surfactant (polysor-bate 80) as a dissolution enhancer, a soluble filler lactose and a small

Table 1Characteristics of the HPMA copolymer conjugates that have entered early clinical trials as anticancer agents.

Code Composition Status ∼Mw(g/mol)

∼Total drugContent (wt.%)

Free Drug (% total) Principal mechanism ofdrug release

FCE 28068 HPMA copolymer–GFLG–doxorubicin Phase II 30,000 8.5 b2 Enzymatic (cathepsin B)FCE 28069 HPMA copolymer–GFLG–doxorubicin–galactosamine Phase I/II 25,000 7 b2 Enzymatic (cathepsin B)PNU166945 HPMA copolymer–paclitaxel Phase Ia NSb 5 NSb HydrolysisMAG-CPT, PCNU166148 HPMA copolymer–camptothecin Phase Ia 18,000 10 b0.01 (wt.%) HydrolysisAP5280 HPMA copolymer–carboplatinate analogue Phase I/II 25,000 8.5 NSb HydrolysisAP5346 HPMA copolymer–DACH platinate analogue Phase II 25,000 10 NSb Hydrolysis

a Stopped after Phase I trial.b NS - not stated.


amount of ethanol. Typically solutions of FCE28068 and FCE28069(concentration ranging from 30–50 mg/mL conjugates) were filtersterilised and then freeze dried resulting in a lyophilised cake thatcould easily be reconstituted with water or NaCl for injection. In thisform dissolution occurs within ∼2 min.

For clinical use AP5280 (a 200mg unit) and AP5346were preparedas lyophilised formulations and stability characterised in various solu-tions with potential for reconstitution [79,80]. The AP5280 lyophilisedproduct in glass vials was stable N12 months at 2–8 °C in the dark.When reconstituted low molecular weight Pt species were slowlyreleased from AP5280 in all solutions tested, but fastest release oc-curred in normal saline. Therefore a 5% dextrose in water solution wasselected for reconstitution as there was b1.0% the total Pt present infree form if used within 8 h after preparation was proposed. It wasshown that these infusion solutions were compatible with a PVC anddid not cause haemolysis.

Table 1 summarises the physico-chemical properties and analyt-ical information relating to HPMA copolymer conjugates that haveentered clinical trials.

4. Clinical experience

To aid comparisons, here the clinical results and pharmacokineticdata obtained for HPMA copolymer conjugates in those Phase I/IIclinical trials carried out to guidelines of good clinical practice (GCP)are summarised. It should be noted that the clinical results have alsobeen obtained in six patients (resistant metastatic cancer) treatedwith HPMA copolymer carrier targeted with autologous or commer-cial human immunoglobulin and these investigations are reported infull elsewhere in this volume by Rihova.

4.1. HPMA copolymer conjugates designed for passive targeting

4.1.1. HPMA copolymer–doxorubicin (FCE28068)FCE28068 was designed in our early studies and the product

licensed to Farmitalia Carlo Erba/Pharmacia (see [4,27,37]). Synthe-sised by aminolysis of an HPMA copolymer–Gly-(D,L)Phe-Leu-Gly-ONp precursor (HPMA:MA-peptide-ONp 95:5) with doxorubicin(Fig. 2b), FCE28068 had a molecular weight of ∼30,000 g/mol, adoxorubicin content ∼8.5 wt.% and free doxorubicin content b2% ofthe total doxorubicin. Containing ∼4 molecules of doxorubicin perpolymer chain, this conjugate forms a unimolecularmicelle in aqueoussolution with a diameter of ∼6 nm [14]. Different molecular weightfractions of FCE28068 have slightly different average drug loadingand this heterogeneity in itself can influence micellar structure, acti-vating enzyme access, and thus potentially activity and toxicity. Thisunderlines the need for a carefully defined specification of the manu-factured/clinical batches used.

During Phase I clinical trial, FCE28068 was administered as a shortinfusion every 3 weeks [18]. To minimise potential osmotic effects theinfusion rate (4.16 mL/min) and concentration (2 mg/mL doxorubi-cin-equiv.) were kept constant during dose escalation. Thus infusiontime was gradually extended. Dose escalation progressed cautiously

as neither polyHPMA nor any HPMA copolymers had previously beenadministered to humans. Classical Phase I entry criteria were used forpatient selection [18], however, as vascular permeability was con-sidered not only an opportunity for tumour targeting but also potentialtoxicity, patients with known brain metastases were excluded fromthis trial. The starting dose of 20mg/m2 (doxorubicin-equiv.)— chosenfrom rodent preclinical studies [66] — was escalated to a maximumtolerated dose (MTD) of 320 mg/m2 (doxorubicin-equiv). Nopolymer-related toxicity or immunogenicity was observed withdose-limiting toxicity (DLT) typical of anthracyclines (including feb-rile neutropenia andmucositis). The time to nadir of neutrophil countswas typically 15–21 days. No alopecia was seen until doses greaterthan180 mg/m2 and nausea was mild without need for anti-emeticsuntil doses greater than 240 mg/m2 Anthracycline-related cardiotoxi-city was absent despite individual cumulative FCE28068 doses of up to1680mg/m2 (doxorubicin-equiv.). (These cumulative doses representN20 g/m2 of HPMA copolymer). In terms of activity, FCE28068produced 2 partial and 2 minor responses in the cohort of 36 patientsenrolled and these were in non-small cell lung cancer (NSCLC), colo-rectal cancer and anthracycline-resistant breast cancer at 80 mg/m2

(doxorubicin-equiv.) and anthracycline-naive breast cancer [18]. Theobservation of antitumour activity in cancers considered resistant/refractory to conventional chemotherapy at lower doxorubicin doses(80–180 mg/m2) was consistent with EPR-mediated targeting andactivity in an epirubicin-resistant patient was particularly interesting.Imaging with a 131I-labelled FCE28068 analogue generally gave poorresolution, but uptake was seen in tumour sites in 6 of the 21 patientsstudied — particularly visible in a head and neck primary tumourwhere the tumour radioactivity was 2.2% dose at 2–3 h 1.3% dose at24 h and 0.5% dose after 8 days [18].

Clinical pharmacokinetics assessed by HPLC and using the 131I-labelled analogue for gamma camera imaging showed a profile inhumans similar to that reported in the preclinical studies. Prolongedplasma circulation compared to free doxorubicin, the absence of liveraccumulation and significant renal elimination (50–75% over 24 h)over time were seen. FCE28068 had a t1 / 2α=1.8 h and t1 / 2β=93 hand there was no evidence of dose-dependency of pharmacokinetics[18].

Phase II studies with FCE28068 were conducted in patients withbreast (17 patients), NSCLC (29 patients) and colorectal (16 patients)cancer [24]. Up to 8 courses at a dose of 280 mg/m2 doxorubicin-equiv. were administered i.v., and in this case a 123I-labelled imaginganalogue was also used for patient imaging. Toxicity was predomi-nantly grade 3 neutropenia (5/62; 8.1% overall) with a higher inci-dence in breast cancer patients (4/17; 23.5%). Of the 14 evaluablepatients with breast cancer 3 had partial responses, and these were allanthracycline-naive patients. In 26 evaluable patients with NSCLC, 3chemotherapy-naive patients had a partial response. In contrast, noneof the 16 evaluable patientswith colorectal cancer responded. Imagingshowed tumour accumulation in metastatic breast cancer. Given thatthis is also activated by cathepsin B (cf. Opaxio™ [40]) it would behelpful to revisit analysis of these data in the context of gender. Thesestudies also confirmed the pharmacokinetics seen for FCE28028 in


Phase I [18], and also the improved imaging showed obvious tumouraccumulation in two metastatic breast cancer patients.

4.1.2. HPMA copolymer–paclitaxel (PNU 166945)The HPMA copolymer–paclitaxel PNU 166945 was developed by

Pharmacia with the specific aim of improving drug solubility to enablemore convenient administration with subsequent “controlled release”of paclitaxel [36]. It was also hoped to capitalise on the EPR effect fortumour targeting. First Gly–paclitaxel was synthesised (conjugatedvia the 2′ position of paclitaxel) before attachment to an HPMA copo-lymer precursor containing Gly-Phe-Leu side-chains to give a tetra-peptide spacerwith drug attachment via a terminal ester bond (Fig. 5).PNU166945 was more soluble than paclitaxel (N2 mg/mL conjugatecompared to 0.0001 mg/mL paclitaxel) and had a drug content of∼5 wt.%. This loading is relatively low considering the decreasedpotency of paclitaxel compared todoxorubicin, andmoreover in compar-ison toPGA–paclitaxel (OPAXIO™)wherepaclitaxel is also linked throughthe 2′ position but to the biodegradable PGA (Mw ∼17,000 g/mol) chainwith a loading of ∼37 wt.%. Although paclitaxel can theoretically bereleased from from both OPAXIO™ and PNU166945 by hydrolysis,enzymatic cleavage or a combination of both mechanisms, it has beenshown that enzymatic cleavage of the backbone is a prerequisite forrelease from the PGA polymer. In the case of the non-degradableHPMA copolymer carrier, with its low drug loading drug release byhydrolysis is likely to predominate.

In the Phase I study, PNU166945 was administered by a 1 h infu-sion every 3 weeks to a total of 12 patients [36]. The starting doseof 80 mg/m2 (paclitaxel-equiv.) was one-third the MTD seen in dogs,and this was escalated to 196 mg/m2 (paclitaxel-equiv.). Howeverthis trial was discontinued before reaching dose-limiting toxicity(DLT). The reason stated was a concern of potential clinical neuro-toxicity at higher doses following observations that had been made inpreclinical animal studies (these studies have never been published).Haematological toxicity was mild and dose-independent, and othertoxicities were consistent with paclitaxel toxicity including flu-likesymptoms, mild nausea and vomiting, and neuropathy. Neurotoxicitygrade 2 occurred in two patients at a dose of 140 mg/m2 (although

Fig. 5. HPMA copolymer–paclitaxel.

grade 1was pre-existing on their entry) and one patient at 196mg/m2

had grade 3 neuropathy after the fourth cycle. Alopecia was absentthroughout [36]. Even though this trial involved small patient num-bers, antitumour activity was observed. A paclitaxel-refractory breastcancer patient showed remission of skin metastases after 2 coursesat 100 mg/m2, and two other patients had stable disease at a dose of140 mg/m2.

Plasma pharmacokinetics were measured over 48 h using HPLCandwere linear with dose both for PNU166945 and the released pacli-taxel. The conjugate had a t1 / 2∼6.5 h and its volume of distributionindicated plasma circulation. Free paclitaxel released from the con-jugate had a t1 / 2∼1.2 h and free drug levels were low, ∼1% of thepaclitaxel present in plasma as conjugate. It is noteworthy, that as forFCE28068, antitumour activity was seen at a relatively low paclitaxeldose of 100 mg/m2.

4.1.3. HPMA copolymer–camptothecin (PNU 166148)Pharmacia also developed a series of HPMA copolymer conjugates

containing the topoisomerase inhibitor, camptothecin [81,82]. Thesederivatives were synthesised from an HPMA copolymer precursorcomposed of HPMA: methacryloyl-glycine (MA-Gly)-ONp 95:5 or90:10; hence, the MAG acronym. It was stated that these conjugateswere also designed primarily to improve the clinical delivery of CPT,capitalise on EPR-mediated targeting and utilise a hydrolytically labilespacer able to liberate drug intratumourally in a pH and enzyme-dependant manner. Modified at the C-20 α-hydroxy with glycine,a Gly-camptothecin was conjugated via different linkers to theMAG polymeric intermediate giving conjugates of Mw of ∼20,000–30,000 g/molwith a camptothecin loading 5–10wt.%. Drugwas shownto be released either by chemical or esterase-mediated hydrolysis[81,82]. In isotonic solutions that these conjugates formed aggregates(with association number higher than 2 in more concentratedsolutions — up to 10 mg/mL), but no significant aggregation occurredin plasma, and conjugates interactedwith serumalbuminmoreweaklythan free camptothecin [83]. Pharmacokinetic studies showing signif-icant renal elimination that was consistent with this conclusion [81].

From the library of different linkers studied, a clinical candidatePNU166148 (often in papers this is called MAG–CPT) containing aGly-C6-Gly-linkage was selected for Phase I studies, having an averagemolecular weight of ∼18,000 g/mol (polydispersity ∼1.4), a total drugcontent of ∼10 wt.% and a free drug content of b0.1% total (structurespublished are shown in Fig. 4b,c). In an attempt to find the optimum,several alternative dosing regimes were employed in the Phase Istudies. In all cases the conjugate was administered to patients withsolid tumours. First studies used an i.v. infusionover 30minwithMAG–CPT given every 28 days [55,84]. The starting dose was 30 mg/m2

(camptothecin-equiv.) and escalation progressed in 23 patientsthrough 47 courses to a dose of 240 mg/m2 that proved to be wellabove theMTD.All patients treatedat this dose showed life-threateningtoxicity and one patient died on study. A manageable dose for Phase IIstudies was recommended as 200 mg/m2. The DLTs included grade 4myelosupression (neutropenia and thrombocytopenia), and grade 3diarrhoea. Severe and unpredictable cystitis was also seen in somecases.

In a second Phase I trial, MAG–CPT was administered daily for3 days repeated every 4 weeks [54]. For this regime the starting dosewas 17 mg/m2/day with escalation to 130 mg/m2/day (i.e. total doseper cycle=390 mg/m2). The 16 patients received 39 courses at sevendose levels. Although haematological toxicity was mild and relatedto theadministereddose,bladder toxicity (includingdysuriaandmicro-scopic and sometimes macroscopic haematuria) was the dose-limitingat 68mg/m2/day and higher. It could only be resolved bywithdrawal oftreatment. A third schedule was investigated in a Phase I trial whereMAG–CPT was administered i.v. weekly for 3 weeks in a 4 week cycle[56]. The starting dose of 80 mg/m2/week used produced no DLTduring the first cycle (initially in 3 patients, and then the cohort


enlarged to 6 patients). However, 2/3 of these original patients exper-ienced cumulative bladder toxicity during the second and third cycles.Also three patients were enrolled at a second dose level of 120 mg/m2/week accrual at this levelwas stopped immediately it became apparentthat cumulative bladder toxicity was dose-limiting at the lower dose.Obviously this weekly schedule of MAG–CPT was unsuitable. Thesewere the first Phase I studies involving an HPMA copolymer conjugatewhere no objective clinical responses were seen. (One patient withrenal cell carcinoma had tumour shrinkage and a colon patient hadstable disease for 62 days.) The differences seen in toxicity underlinethe importance of a carefully optimising dosing schedule in respect ofconjugate pharmacokinetics and rate and location of the drug release.

A validatedHPLC assay [85]was established to determine the phar-macokinetics of polymer-bound and free CPT in these clinical studies.No dose-dependency of plasma clearance was seen for either MAG–CPT or the released CPT [54–56]. Again plasma levels of MAG–CPTwere ∼100 times higher than free CPT, not surprising as free drug willrapidly exit the circulation and conjugate cannot other than by renalelimination or tissue extravasation. Conjugated and free CPT was stillseen in plasma 4 weeks after administration, although it is unclear ifthis is protein bound, or for conjugates polymers of molecular weighthigher than the renal threshold. Patients receiving multiple cycles didnot, however, show progressive accumulation.

Preclinical studies in rodents, and the clinical studieswith FCE28068(including imaging) had already well established the rapid renalexcretion of HPMA copolymer conjugates when polymer molecularweight was lower than the renal threshold [18,50]. As would havebeen predicted, MAG-CPT urinary excretion was also very high (50–90% at 24 h) and the conjugate was still detectable in urine after4 weeks [54–56]. Notably, free CPT in urine at 3 days represented only1–4%. The observed bladder toxicity, exacerbated by repeated dosingduring each cycle, and a urine labile polymer–drug linker, is thus notsurprising. Lack of similar toxicity in the case of FC28608 is explicableas the Gly-Phe-Leu-Gly linker is stable during excretion, neverthe-less in the first clinical trials with this conjugate it was evident thatcareful monitoring of renal function (not typically undertaken foranthracyclines) was needed in case tubule reabsorption might resultin unexpected kidney toxicity. Pharmacokinetic–pharmacodynamicinvestigations for MAG–CPT clearly showed a positive correlation be-tween the total CPT excreted with (i) urine production (0–24 h) and(ii) the worst bladder toxicity.

To seek evidence of MAG–CPT tumour targeting, a pilot clinicalstudy was conducted involving 10 patients with localised colorectalcancer. They were given MAG–CPT (60 mg/m2 camptothecin-equiv.)at 24 h, 3 days or 7 days before surgical removal of the primary tumour[86], and then plasma, tumour, and adjacent normal tissue samplesanalysed for free and polymer-bound drug. At 24 h after dosing nosignificant MAG–CPT tumour targeting was observed (861±216 ng/gin tumour and 751±215 ng/g normal; equivalent to ∼0.0007% dose/g). Moreover, free CPT levels were lower in tumour (12.2±4.7 ng/g)than in normal tissue (21.9±6.7 ng/g). It is noteworthy that theseresults differ from data obtained in murine tumour models wherehigher MAG–CPT levels were seen in tumours after a single i.v. dose.Moreover, they are not consistent with the clinical gamma cameraimaging reported for FCE28068 [18] which showed that the highesttumour levels of conjugate (2.2% at 2–3 h) significantly decreasedwithtime thereafter. Based on these Phase I clinical results, and the lack ofevidence for tumour targeting, the clinical development of MAG–CPTwas abandoned.

4.1.4. HPMA copolymer–platinates (AP5280, AP5346)With the hope of designing an HPMA copolymer–platinate (Pt)

suitable for clinical development in the 1990s we prepared a libraryof conjugates containing Pt ligands (Fig. 6a) synthesised as ‘cisplatin’or ‘carboplatin’ mimetics [57,58]. This technology licensed to AccessPharmaceuticals Inc. who have subsequently transferred two com-

pounds into clinical trials which are ongoing. These compounds area carboplatinum analogue AP5280 (Fig. 6b) and an oxaliplatin (1,2-diaminocyclohexyl (DACH) platinum) analogue AP5346 (Fig. 6b).As this programme is comprehensively reviewed elsewhere in thisvolume (Nowotnik and Cvitkovic) a brief overview is given to enablecomparison with other studies here.

AP5280 has a Mw ∼25,000 g/mol (polydispersity of 1.7), and a Ptcontent of ∼7 wt.%. It accumulates in solid tumours by the EPR effect[58,87], and in preclinical studies its MTDwas 6-fold greater than thatof carboplatin, and also it displayed a higher therapeutic index thancarboplatin in a number of tumour models [87]. On this basis, AP5280entered Phase I studies [88] using a 1 h infusion administered every21 days. The starting dose of 90 mg/m2 was escalated to 4500 mg/m2

(Pt-equivalent). The DLT was grade 3 vomiting at 4500mg Pt/m2 seenin 2/6 patients, but interestingly both renal toxicity and myelosup-pression, commonly seen for cisplatin and carboplatin were minimalusing this schedule. A dose of 3300 mg Pt/m2 was recommended forPhase II studies.

During the development of AP5280, oxaliplatin received Regula-tory Authority approval making it timely to investigate a secondHPMA copolymer–platinate (AP5346) designed as a DACH platinumanalogue. In preclinical studies this compound (AP5346 also calledProLindac™) showed superior antitumour activity compared to oxa-liplatin (both at their MTD) in B16 melanoma and human ovariancarcinoma models [89]. The conjugate was also more effective thancisplatin in both cisplatin-sensitive and cisplatin-resistant variantsof the M5076 tumour. When injected at equitoxic doses, AP5346 gavea 16.3 fold higher Pt AUC in the tumour compared to oxaliplatin,and this resulted in a 14.2 fold increase in the tumour–Pt DNA levelachieved [89]. A Phase I study (26 Patients) with AP5346 [90] used aprotocol where the compound was given as a 1 h i.v. infusion on days1, 8, 15 of the 4 week cycle. Doses were escalated from 40–1280 mg/m2 Pt equiv. and the reported DLT included vomiting, fatigue and, inthis case renal insufficiency. Antitumour activity was seen with 2partial responses in metastatic melanoma and ovarian cancer, and CA-125 normalisation in a suspected ovarian cancer patient.

4.2. Conjugates designed for receptor-mediated targeting

Although intellectually attractive, the ability to realise receptor-mediated tumour targeting in vivo and/or the clinical setting hasproved very difficult (discussed at more length elsewhere [4]). Ofthe many HPMA copolymer conjugates explored in the preclinicalliterature only one has been transferred into the clinic.

4.2.1. HPMA copolymer–doxorubicin–galactose (FCE28069)The second compound arising from our CRC-funded research

licensed to Farmitalia Carlo Erba/Pharmacia (FCE28069) (Fig. 2c) con-tained doxorubicin and additionally galactosamine bound to theHPMA copolymer backbone via a Gly-(D,L)Phe-Leu-Gly linker. Theconjugate had aMw ∼25,000 g/mol, a doxorubicin content of ∼7.5 wt.%, and a free doxorubicin content b2% total doxorubicin. The galacto-samine content was 1.5–2.5 mol%. FCE28069 was less soluble thanFCE28068 due to the increased content of relatively hydrophobic side-chains. Phase I evaluation of FCE28069was conducted in 31 patients ofwhich 23 had primary hepatoma [25], and a 123I-labelled FCE28069gamma camera imaging analogue was used to follow distribution.Initially, an i.v. infusion rate of 4.16 mL/min (2 mg/mL doxorubicin-equiv.) was given every 3 weeks, but due to pain the infusion rate wasreduced to 2 mL/min using a 1.0 mg/mL solution. The starting dose of20 mg/m2 (doxorubicin-equiv.) was escalated to the MTD of 160 mg/m2. As for FCE28068 the DLTs were typical of anthracyclines, prin-cipally myelosuppression and mucositis. Interestingly, this MTD wassignificantly lower than seen for FCE28068. Whilst the authors specu-late that this might be due to the presence of extra-hepatic galactosereceptors [25], it is known that FCE28069 has a lower solubility and the

Fig. 6. HPMA copolymer–platinates. Panel (a) shows the HPMA copolymer intermediates and platimum ligands used to generate a library of first generation compounds [57,58] andpanel (b) shows the compounds that have subsequently entered clinical trial.


hint of lung localisation at early times by gamma camera imagingmight be a result of solubility issues. Furthermore, small angle neutronscattering (SANS) has also shown that FCE28069 has a different struc-ture in solution [14].

Of the 23 hepatocellular carcinoma patients 2 showed a measur-able partial response which at the time of report was lasting N26 andN47 months respectively [25]. A third patient showed a reductionin tumour volume and 11 had stable disease. FCE28069 plasma levelsdetermined by HPLC were indistinguishable from those measuredusing the radiolabelled analogue and pharmacokinetics were linearwith increasing dose. Less than 0.1% of the plasma levels were freedoxorubicin, and in this case urinary excretion at 24 h was only 5%.(Preclinical animal studies showed hepatobiliary excretion to be themain route of elimination in this case.) SPECT gamma camera imagingindicated FCE28069 liver levels of 15–20% dose at 24 h. The majorityof radioactivity was associated with normal liver (after 24 h 16.9%)with lower accumulation in hepatic tumour (3.2% dose)— not surpris-ing as hepatoma tends to lose the target asialoglycoprotein receptor asthe disease progresses. Nevertheless the doxorubicin concentration inhepatoma was still estimated as 12–50 fold higher than would seen

following the administration of free doxorubicin. In this study a clavi-cular metastasis arising in a hepatoma patient showed clear FCE28069localisation using gamma camera imaging.

5. Conclusions and lessons learnt

Although several polymer–anticancer drug conjugates have shownconsiderable promise in clinical trials over the last 15 years, the firstmarketed product is still awaited. This is a crucial milestone to achievequickly for this concept to gain acceptance andmove forward. There isuniversal hope that OPAXIO™ will take that step. The HPMA copo-lymer–platinates currently in Phase II clinical trials are the nearestin this family to progress. Access Pharmaceuticals Inc. have recentlyannounced (March 2009; www.accesspharma.com) positive safetyand efficacy results from its Phase II single agent clinical study ofProLindac™ where 66% of late-stage, heavily pre-treated ovariancancer patients who received the highest dose achieved clinicallymeaningful disease stabilisation. It was also announced that a numberof combination trials (combining with other cancer agents, such aspaclitaxel and gemcitabine) in multiple solid tumour indications

http://www.accesspharma.com


including colorectal and ovarian cancer will follow. In addition, Pro-Lindac™ has been licensed to Jiangsu Aosaikang Pharmaceutical Co.,Ltd. for development in the Greater China Region and to JCOM, Ltd fordevelopment in South Korea. Both companies will conduct furtherPhase II combination studies in specific tumour types.

Preclinical research continues to design novel HPMA copolymerconjugates with the ambition of identifying the next anticancer can-didates for clinical evaluation. For example, there are conjugates con-taining new anticancer agents (e.g. geldanamycin [91], wortmannin[92], diazaanthraquinone [93]), containing drug combinations (e.g. ofendocrine and chemotherapy [94–96], combination chemotherapy ofdoxorubicin and gemcitabine [97], and for combination with radio-therapy [98]. Also there has been a move away from the popular Gly-Phe-Leu-Gly linker to novel linkers designed for pH-dependant cleavage[99]. (There is a growing realisation importance of combination therapyin the context of polymer therapeutics and this is reviewed elsewhere inthis issue by Greco and Vicent [105].).

What lessons have been learnt over the last 30 years that will beuseful to (i) guide optimum design of second generation candidateswith a ‘real’ potential for clinical development, and (ii) address thekey issues needed to maximise the possibility of successful transfer ofsuch candidate(s) from lab to clinical use? The following summarisethe most important key issues:

• There is an urgent need for improved synthetic chemistry, linkerdesign and for researchers to understand the concept of validatedcharacterisation of product identity/purity [100]. The complexity ofpolymer heterogeneity should be carefully addressed if conjugatesare to satisfy “Regulatory Requirements” [101].

• Adoption of appropriate preclinical screening methodology for can-didate selection. Classical in vitro screening of cytotoxicity is notuseful. The key milestones are appropriate linker design (releaserate), appropriate whole body distribution (and route of eliminationof the non-biodegradable carrier) and confirmation of improvedtherapeutic index in vivo (reviewed in [24]).

• Appropriate clinical trial design.• Some of the most important issues relate to practicality of industrialdevelopment and ability to satisfy Regulatory Authority require-ments regarding product manufacture, characterisation, safety andefficacy. There is still a need for consensus regarding terminology,optimal routes to synthesis with minimal heterogeneity and stan-dardised in vitro and in vivo screening methodology suitable to giveoptimal lead candidates.

5.1. Terminology

Still relatively a few individuals (relative to medicinal chemistry)have bridging expertise relating to polymer chemistry, organic andpeptide chemistry, cell andmolecular biology, pharmaceutical sciencesand the clinical development of polymer–drug conjugates. Even fewerhave industrial development experience with such compounds. Suc-cessful (and safe) research and development in this field can only oc-cur with consistent terminology. The current literature is pepperedwith inconsistencies and inaccuracies. For example, it is misleading tocall polymer–drug conjugates a “formulation”. This shows a lack ofappreciation of what a pharmaceutical formulation is. Moreover, it isessential that conjugates are carefully defined, and that the descrip-tions are well understood by clinical end-users and regulatory asses-sors. Often the terminology used confuses a homopolymer (to whichdrug can be theoretically bound)with a co- or ter-polymer. Sometimesit is impossible to understand subtle, but important issues relating toidentity, route of degradation and drug loading (dosage) from the textwritten.

All studies (preclinical and clinical) must adequately define con-jugate molecular weight and polydispersity, total drug content (wt.%)and the free drug impurity (% total) as a minimum characterisation.

These factors have a significant impact on the in vitro, in vivo bio-logical or clinical profile reported and conclusions drawn. For example,two papers reporting the Phase I clinical studies for MAG-CPT showconjugate structures which are different-incorrect- (Fig. 4b,c [54,55]),and indeed in [55] the statement that “the fact that the Cmax andAUC0–168 h of free CPT in patients treated with 80 vs 120 mg/m2

appeared similar might be explained by the slow release rate of MAG–CPT” (i.e. conversion frommonomer B to A in Fig. 4b) ismisguided. Thepolymer backbone does not degrade to release this monomer.

It is essential that clinicians and industrial researchers involved inthe development of complex polymer therapeutics are aware of struc-ture, pharmacokinetics and biological basis for efficacy/toxicity. More-over, in clinical studies it can be very confusing to quote polymer–drug doses as mg/kg (preclinical studies) or mg/m2 (clinical trial)without clearly definingwhether this is drug content or conjugate dose.It is essential that the doses specify the amount of bioactive admin-istered. Qualification with definition of the wt.% drug in the conjugateallows also a clear appreciation of the amount of polymer administeredto the patient. For example, in the case of HPMA copolymers thosetrials that reached anMTDof N1000mg/m2 in respect of bioactive drugequiv. require administration of N10 g HPMA copolymer (if the activeis 10wt.%) at each dosing cycle of administration.We often discuss theneed for improved interdisciplinary communication — at the preclin-ical–clinical interface as this is vitally important to define theterminology and use of it accurately.

5.2. Design for optimum therapeutic index

Unequivocally, and returning to the original hypothesis, it hasbeen shown that drug conjugation dramatically alters biodistributionin vivo and clinical studies have underlined the importance of linkerstability/site and rate/site of drug liberation. It should be emphasisedthat such conjugates were born on the basis of a pharmacokineticrationale for design. Understanding pharmacokinetics in vivo and inthe clinical setting is pivotal.

In respect of polymer–drug linkers, the clinical observations do,however, raise a dilemma. It can be argued that, in the case of HPMAcopolymer conjugates where the maximum achievable drug loadingis relatively modest (∼10 wt.% compared to the biodegradable PGA(∼35 wt.%), drug conjugation via an ester linkage (paclitaxel and CPT)has failed. In contrast, the peptidyl linkers originally designed for en-zymatic activation (doxorubicin) produced conjugates showing goodcorrelation between the preclinical and clinical reduction in toxicity,toxicity profile and pharmacokinetics observed. Yet, given the emerg-ing complication of potential patient differences in cathepsin B levels,there is a real need to reflect on optimal linkers for second generationconjugates. Will the pH-sensitive hydrazone linkers proposed byUlbrich et al. [99] bring improved therapeutic index or will they, likeother hydrolytically labile linkers, bring new clinical toxicities/chal-lenges? The fact that the thiol-proteases are raised in certain tumours,often in relation to metastatic potential, continues to make them anattractive target, but there is a clear need to type/select patients forpolymer therapeutic treatment if they are to be used to best effect.Moreover, preclinical experiments specifically directed towards abetter understanding of the cell/animal models used in terms ofenzymatic activation mechanisms are urgently needed (see effortsSinger et al. who have also used cathepsin B-knock out mouse models[102] in this context).

From the first clinical pharmacokinetic studies (HPLC and gammacamera imaging [18,24,25,55,56,85,86]) it has been possible to gain anappreciation of the biodistribution of HPMA copolymer conjugates.However, for such studies to be useful in guiding clinical developmentof second generation conjugates there is a need to better considersampling time, probes used and also data expression. Phase I gammacamera imaging studies of FCE28068 used a 131I-labelled analoguewhich gave poor tumour resolution. This was improved in the Phase II


trials where a 123I-labelled analogue and SPECT were used, howeverthere is still an urgent need for improved gamma camera and PETimaging techniques to enable visualisation of biodistribution in realtime. The ethical constraints of clinical trial design often make itimpractical to undertake pharmacokinetic analyses at all the timepoints that would be scientifically optimal. However, it would be wiseto (i) use the time-dependant-pharmacokinetics seen in animal studiesto optimise selection of time points for clinical pharmacokinetic studiesin relation to the conjugate properties and (ii), in addition to the clas-sical pharmacokinetic parameters, express biodistribution in terms oft1 /2α, percentage dose administered and the recovered dose at eachtime point. It is known that the early phase clearance rate correlatespositively with EPR-mediated targeting, but the classical pharmacoki-netic parameters quoted in clinical studies (e.g. terminal t1 /2, AUC, CL)do not best describe the important issues for polymer conjugates.

The authors of one of the MAG–CPT studies [55] suggest that theirpharmacokinetic approach might be employed to guide the clinicaldevelopment of other polymeric anticancer drugs. This is arguably notthe case. The high anticancer drug attrition rate (between 95% [103]and 82% [104]) seen for all compounds entering clinical trial may, inmany cases, be due to limited appreciation of pharmacokinetics interms of whole body distribution and intracellular fate (leading to

Fig. 7. Summary of the complex pharmacokinetics of pol

poor efficacy and toxicity). The classically quoted pharmacokineticparameters are estimated from drug/metabolite concentrations inaccessible body fluids (i.e. plasma and urine) and tissues, but inev-itably this gives very limited picture. To the future it is essential todevelop methodology for polymer conjugates that will better quan-titate whole body and target tissue (subcellular) drug levels andmetabolism monitoring the changes taking place in real time. At thetissue level only non-invasive imaging can begin to provide suchinformation, but today techniques still lack adequate sensitivity. Forsmall molecules it is usual that the drug and its metabolites distributein a similar way, however, it is essential to remember the pharma-cokinetic differences between the macromolecular prodrug (endocy-tic uptake) and low molecular bioactive species released (randomdistribution).

Fig. 7 illustrates some of the complex issues that need consider-ation when trying to define the pharmacokinetics of polymer conju-gates and their metabolites to enable correlation with an antitumouractivity. Samples analysed in the HPMA copolymer conjugate clinicalstudies include plasma (A), urine (B) and in a few patients tumourtissue (C) — see Fig. 7 for key. Plasma levels of conjugated and freedrug need to be considered and bothmay have the capacity to protein-bind. It is known that plasma levels of conjugate drive the EPR effect,

ymer–drug conjugates following i.v. administration.


but the rapid renal elimination (needed to ensure excretion of the non-degradable HPMA copolymeric carrier) leads to fast plasma clearanceabolishing the driving force for EPR-mediated targeting after the first1–3 h. Clinical pharmacokinetic studies confirmed the initial volumeofdistribution of conjugated drug to be in the circulation, coupled with arelatively rapid phase of renal excretion. It seems likely that HPMAconjugates containing paclitaxel and camptothecin release drug inplasma relatively quickly, and this provides an opportunity for proteinbinding of free drug (difficult to distinguish from polymer-bound drugunless assays are especially designed to answer that question) andmost importantly from a quantitative viewpoint rapid redistributioninto all tissues with the same kinetics as would be expected for theparent drug.

Although EPR-mediated targeting is still believed of fundamentalimportance to the passive tumour targeting of many polymer conju-gates andmany other nanomedicines already in clinical use and underdevelopment, there is still a clear need to define its efficiency in spe-cific tumour types in patients, and at specific disease stages. The fail-ure to gain evidence of drug targeting in colon cancer patients withtheMAG–CPT conjugate [86] (even given the small numbers of patientsstudied) shows the importance of such investigations using moresensitive methodology to validate this approach clinically.

5.3. Clinical trial design

With the exception of MAG–CPT, all the HPMA copolymer–chemo-therapy conjugates so far tested clinically have shown activity (in theform of stable disease and partial responses) in heavily pre-treatedchemotherapy-resistant patients. The Phase I clinical studies reportedhere typically used the optimumdosing schedule relating to the parentdrug (e.g. the doxorubicin conjugate FCE28068 was only tested in thisway). Dose was increased to the MTD using the classically acceptedapproach for new anticancer chemotherapeutic agents. SubsequentlyPhase II studies were undertaken at the Phase I-recommended MTDeven though several conjugates had shown anti-tumour activity atlower doses (e.g. FCE28068 [18])where therewas significantly reduceddrug toxicity (life threatening and inconvenient— nausea, vomiting,and hair loss) compared to the parent drug. Although the MAG–CPTPhase I studies evaluated several different dosing regimes these studiesserved only to define the optimum in terms of toxicity reduction.

Future clinical studies must optimise clinical trial design on a con-jugate-by-conjugate basis in terms of their pharmacokinetic–phar-macodynamic properties (this will be different from that of the agentconjugated). Patient/target disease selection should be based on tumourtype, potential for EPR-mediated targeting, status regarding activat-ing enzymes e.g. cathepsin B, with exclusion of patients with higherrisk of adverse reactions e.g. poor renal function; non-specific sites forundesirable extravasation e.g. brain metasases; and sites of inflam-mation. Finally, there is a pressing need for increased understandingof potential mechanisms of resistance to such polymeric anticancerconjugates, and indeed other nano-sizedmedicines designed for endo-cytic internalisation/activation as the field is still in its infancy. Plainly,inadequate cathespin B levels could have been predicted to influenceactivity of those conjugates requiring their activation. The uniquecellular mechanism(s) of delivery of conjugates are still frequentlyoverlooked. Tumour cellular characteristics such as rate of endocy-tosis/exocytosis, trafficking pathways used, vesicular pH, vesicularmembrane permeability to released drugs etc. are all potentially fea-tures that will influence the therapeutic potency/resistance to con-jugate therapy. Preclinical and clinical investigations should be given ahigher priority to enable the definition of those keymarkers of primaryimportance to the “nanomedicine therapeutic index” in advance ofclinical studies. Only in this way will the community be able to buildon past experience and ensure choice of the appropriate patients fortreatment who will have the best chance of response to these inno-vative medicines.

Acknowledgements

I would like to acknowledge themany colleagues and collaborators(inmy own group and those of Jindrich Kopecek, Karel Ulbrich, BlankaRihova and importantly Farmitalia Carlo Erba, Milan) who gave birthto this field and brought the HPMA copolymer programme into clinicaldevelopment. Also thanks to the UK Cancer Research Campaign, whohad the vision to support a young scientist, and this “ahead of its time”programme. Without the inspiration and support of Tom Connors,Helmut Ringsdorf, and Federico Spreafico (Farmitalia Carlo Erba/Pharmacia) this family of anticancer agentswould not have been born.

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