Advanced Drug Delivery Reviews 61 (2009) 1203–1213

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Advanced Drug Delivery Reviews

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Combination therapy: Opportunities and challenges for polymer–drug conjugates asanticancer nanomedicines☆

Francesca Greco a,⁎, María J. Vicent b,⁎a School of Pharmacy, University of Reading, Whiteknights PO BOX 224 Reading RGD6 6AD Berkshire, UKb Centro de Investigación Príncipe Felipe, Polymer Therapeutics Laboratory, Medicinal Chemistry Dpt., E-46012 Valencia, Spain

☆ This review is part of the Advanced Drug Delivery Re⁎ Corresponding authors. Greco is to be contacted at

E-mail addresses: f.greco@rdg.ac.uk (F. Greco), mjvic

0169-409X/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.addr.2009.05.006

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 April 2009Accepted 14 May 2009Available online 20 August 2009

Keywords:Polymer-conjugatePolymer therapeuticsNanomedicineTargeted drug deliveryCombination therapyCancer

The discovery of new molecular targets and the subsequent development of novel anticancer agents areopening new possibilities for drug combination therapy as anticancer treatment. Polymer–drug conjugatesare well established for the delivery of a single therapeutic agent, but only in very recent years their use hasbeen extended to the delivery of multi-agent therapy. These early studies revealed the therapeutic potentialof this application but raised new challenges (namely, drug loading and drugs ratio, characterisation, anddevelopment of suitable carriers) that need to be addressed for a successful optimisation of the systemtowards clinical applications.

© 2009 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12042. Combination therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1204

2.1. Combination therapy in cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12042.1.1. Small molecule chemotherapy combinations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12042.1.2. Combinations based on endocrine therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12042.1.3. Combinations based on monoclonal antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12052.1.4. Preclinical studies on novel approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12052.1.5. Combination of different types of therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1205

3. Polymer–drug conjugates for combination therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12053.1. The concept of polymer–drug conjugates for combination therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1206

3.1.1. Polymer–drug conjugate+low molecular weight drug or other type of therapy . . . . . . . . . . . . . . . . . . . . . . 12063.1.2. Polymer–drug conjugate+polymer–drug conjugate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12073.1.3. Single polymeric carrier carrying a combination of drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12083.1.4. Polymer-directed enzyme prodrug therapy (PDEPT) and polymer–enzyme liposome therapy (PELT) . . . . . . . . . . . . 1209

4. Challenges associated with the use of polymer–drug conjugates for combination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12094.1. Identification of appropriate drug combinations and drug ratios . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12094.2. Kinetics of drug release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12094.3. Loading capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12104.4. Correlation of in vitro studies with behaviour in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12114.5. Physico-chemical characterisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12114.6. Clinical development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1211

5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1212Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1212References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1212

views theme issue on “Polymer Therapeutics: Clinical Applications and Challenges for Development”.Tel.: +44 118 378 8244; fax: +44 118 378 4703. Vicent, Tel.: +34 963289680; fax: +34 963289701.ent@cipf.es (M.J. Vicent).

ll rights reserved.

1204 F. Greco, M.J. Vicent / Advanced Drug Delivery Reviews 61 (2009) 1203–1213

1. Introduction

Several drug delivery systems (DDS) have successfully improvedthe therapeutic profile of conventional drugs, especially for cancertreatment. By increasing the selectivity towards a specific target,many of these technologies display decreased toxicity and thereforean improved therapeutic profile. Some systems, such as liposomes [1],microparticles [2] and PEGylated proteins [3,4], are well established,others, such as polymer–drug conjugates are expected to reach themarket in the near future [5].

DDS are commonly used for the delivery of single therapeuticagents, however, their application to deliver “cocktails” of drugs is stilllargely unexplored. This might seem unusual since combinationtherapy is routinely used in cancer treatment and indeed the com-bination of different therapeutic agents often improves therapeuticprofile [6].

In the last 5 years, a number of pioneering studies have beencarried out that highlight the suitability of polymer–drug conjugates todeliver drug combinations. The aim of this paper is to systematicallyreview these early works. We will first give an overview of the use ofcombination therapy with a focus on cancer treatment (Section 2). InSection 3, the phrase “polymer–drug conjugates for combinationtherapy” is defined and the current status of the field is analysed,with a particular attention to clinical studies. Finally, we discuss thedifficulties and challenges associated with this approach (Section 4)and we present some general conclusions (Section 5).

2. Combination therapy

Due to the molecular complexity of many diseases, combinationtherapy is becoming increasingly important for a better long-termprognosis and to decrease side effects. ‘Combination therapy’ forthe treatment of a disease generally refers to either the simultaneousadministration of two or more pharmacologically active agents or tothe combination of different types of therapy (e.g. chemotherapy andradiotherapy). Unlike single-agent therapy, multi-agent therapy canmodulate different signalling pathways in diseased cells, maximisingthe therapeutic effect and, possibly, overcoming mechanisms ofresistance [7].

Several diseases are routinely treated with combination therapy,including malaria, HIV/AIDS and cancer. For instance, the HighlyActive Antiretroviral Therapy (HAART), a strategy that combinesthree or more anti-HIV drugs, has become key for the treatmentof HIV since first introduced in 1999 [8]. In the case of malaria,combination therapy is the main strategy to control resistance toanti-malaria drugs. In this disease, resistance emerges as a resultof selection and then dissemination of mutated parasites withreduced susceptibility to drugs. Thus, by administering combinationsof drugs hitting different molecular targets the chances of successare maximised [9]. In other diseases, such as multiple sclerosis (MS),the combination approach is just beginning to be explored, but withgreat expectations for patients with relapsing–remitting MS. In thissense, a clinical trial promoted by Mount Sinai School of Medicine(CombiRx) is currently recruiting participants to determine thebenefits of combining interferon beta-1α (Avonex, Rebif®) andglatiramer acetate (Copaxone®) [10]. Finally, it is worth mentioningthat, even in diabetes some early studies have shown clear advan-tages of combination therapy against monotherapy. Suarez-Pinzonet al. demonstrated that combined treatment of glucagon-likepeptide-1 (GLP-1) and gastrin, but not GLP-1 or gastrin alone,restored normal glycemic levels in diabetic NOD mice by increasingthe pancreatic β-cell mass and downregulating the autoimmuneresponse [11].

Combination therapy applied to cancer is dealt below as a separatesection due to its remarkable therapeutic value and relevance to thisreview.

2.1. Combination therapy in cancer

The use of combination therapy for cancer treatment is wellestablished [6].Whereas chemotherapy drugs are normally associatedwith severe side-effects, administration of a combination of agentshitting different targets and displaying different toxicity profiles canimprove the therapeutic index either in the form of better efficacy orin the form of comparable efficacy and reduced toxicity.

2.1.1. Small molecule chemotherapy combinationsThe rational for combination chemotherapy have evolved from

the “total empiricism” in the 40s going through the “enlightenedempiricism”, with remarkable survival improvement in childhoodleukaemia and Hodgkin's disease, up to the current achievement of areasonably scientific treatment modality [12]. This evolution has beenbased on the application of several principles mainly developed onthe “enlightened empiricism” period, such as, biochemical synergy,tumour cell kinetics, non-over-lapping toxicity, increase of fractionalcell kill, non-cross-resistant agents or tumour cell resistance [12].For example, in acute nonlymphocytic leukaemia, the use of theanthracycline daunorubicin (DNA intercalator) with ara-C (inhibitorof DNA polymerase) is a successful example of complementaryinhibition as this combination interferes with DNA repair as well as itssynthesis. Another good example of biochemical synergy was theadministration of leucovorin (LV) prior to 5-fluorouracil (5-FU) incolorectal cancer as LV markedly enhance the ability of 5-FU to bindand consequently block the action of thymidilate synthetase.

Further traditional drug combinations for cancer therapy includeanthracyline-based combinations such as AC (adryamicin and cyclo-phosphamide), CAF (cyclophosphamide, adryamicin, 5-FU); metho-trexate-based combinations such as CMF (cyclophosphamide,methotrexate, 5-FU) and CMFVP (cyclophosphamide, methotrexate,5-fluorouracyl, vincristine and prednisone) [12,13]; and paclitaxelcontaining combinations, such as paclitaxel and carboplatin for ovaryand lung cancer or combined with vinorelbine for non-small cell lungcancer (NSCLC) [14,15]. Currently, new multi-agent therapies ormodifications of established regimes are being tested. In addition,variations of the administration patterns are also explored to improveresponse, decrease side effects and, ultimately maximise therapeuticbenefit. For instance, a Phase II study was carried out combiningpaclitaxel, 5-fluorouracil, folinic acid and cisplatin in patients withadvanced gastric cancer. In this study, weekly administration ofpaclitaxel was compared to threeweekly administration and the resultsshowedmaintenance of therapeutic efficacy and decrease of side effects[16].

Combination chemotherapy can also be used as palliative treat-ment (to reduce symptoms and prolong life expectancy rather thantreat the disease) or as adjuvant treatment pre- or post-surgery (toreduce tumourmass of advanced cancers prior to surgery or to removeunresectable metastasis or, post surgery, undetectable micrometas-tasis) [17].

2.1.2. Combinations based on endocrine therapyHormone-dependent cancers (breast and prostate) are of particular

interest for combination therapy. Like any other type of solid tumours,prostate and breast cancer can be treated with surgery, radiotherapy,chemotherapy or a combination thereof, but can additionally be treatedwith hormone therapy. Indeed, clinical studies combining endocrineand chemotherapy have been reported already since the 1980s [18].More recently, new therapeutic agents have becomepart ofmulti-agentregimens. Recent trials have suggested the use of endocrine therapywith adjuvant bisphosphonate therapy (zoledronic acid) for thetreatment of breast cancer. The rationale for such a combination istwofold. First, zoledronic acid is expected to counterbalance the boneloss associated with oestrogen suppression. Indeed, the Z-FAST trialdemonstrated that zoledronic acid prevented aromatase inhibitor-

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associatedbone loss in postmenopausalwomenwithbreast cancer, thusrepresenting a cost-effective treatment for the prevention of bone lossand fractures in this cohort of patients [19]. Similar results were alsoobtained in the CALGB 79809 trial [20]. In addition to bone protection,other clinical trials aimed to confirm the antitumour activity ofzoledronic acid that had emerged in previous pre-clinical and clinicalstudies. A large clinical trial was carried out on 1803 premenopausalwomen with hormone-responsive breast cancer comparing the effectsof endocrine therapy (goserelin with either anastrozole or tamoxifen)with or without addition of zoledronic acid [21]. At 5 years since thebeginning of this trial patients treated with zoledronic acid andendocrine therapy displayed significantly prolonged disease freesurvival and relapse free survival. The authors attributed these positiveresults to the antimetastatic properties of zoledronic acid and highlightthe importance of such combination [22].

2.1.3. Combinations based on monoclonal antibodiesSince entering the marketplace, monoclonal antibodies have gained

an important role in cancer treatment aloneor in combinationwithothertherapeutic agents. Themonoclonal antibody trastuzumab(Herceptin®)is routinely combined with chemotherapy for the treatment of HER2-positive breast cancer [23]. Other monoclonal antibodies such asrituximab (Rituxan®) [24] or bevacizumab (Avastin®) [25] are usedfor the treatment of several metastatic cancers including colorectalcancer or NSCLC and have recently been assessed in combination withtraditional chemotherapy for the treatment of advanced breast tumours[26,27]. Bevacizumab is of particular interest as such molecule was thefirst antiangiogenic drug to be grantedUS Food andDrug Administration(FDA) approval to market (February 2004). At present, bevacizumab iscombinedwith chemotherapy, namely 5-fluorouracil based chemother-apy, carboplatin or paclitaxel for the treatment of metastatic colorectalcancer, NSCLC and metastatic breast cancer, respectively [26]. Forexample, a randomised Phase III trial compared the effect of paclitaxelwith or without bevacizumab as first-line therapy in metastatic breastcancer and showed that patients treated with this drug combinationdisplayed prolonged progression free survival [27]. In February 2008,Bevacizumab received accelerated approval from the FDA for use inmetastatic breast cancer in combination with paclitaxel chemotherapy.

Several clinical trials are assessing the efficacy of antiangiogenicdrugs in combination with traditional chemotherapy and with small-molecule inhibitors of specific molecular pathways such as erlotinib(Tarceva). Although the rationale for this type of combination is strong(i.e. target differentmolecular pathways tomaximise efficacy), severalPhase III trials have failed to improve overall survival [28]. Thesedisappointing results highlight the difficulties of optimising combina-tion therapy and can at least partially be attributed to the lack of a deepunderstanding of the molecular mechanisms underlying this disease.More multi-agents therapies are currently entering the clinicalpipeline in the hope of finding the most promising drug combinationsand/or identifying patient subpopulations for whom these therapiescan be advantageous, as was the case for trastuzumab and HER2positive cancers.

2.1.4. Preclinical studies on novel approachesIn addition to these clinical studies, exciting preclinical studies

exploring new combinations might in the future result into newtherapeutic protocols and, in parallel, they are expected to provideinsight into the cellularmechanisms regulating response to treatment.Loweand collaborators designed a two-drug combination therapy ableto reverse chemotherapy resistance in mice by re-establishing cellularmechanisms affecting growth and death patterns, often misregulatedin cancer cells [29]. Animalswere treatedwith rapamycin, doxorubicinor the two drugs in combination. In the case of drug combination,complete tumour remissionwas observedwhilst animals treatedwitheither drug alone (rapamycin or doxorubicin (Dox)) rarely experi-enced complete remission [29]. This combination relies on fine

regulation of specific molecular pathways, with rapamycin restoringprogrammed cell death in cancer cells andDox triggering suchprocess.

Finally, an interesting approach to rationally design drug combina-tions has been suggested [30]. The authors propose to identify and thentarget antigens whose expression is triggered by exposure to chemo-therapy. In this study, they exposed a xenograft model of colorectalcancer to the chemotherapeutic agent irinotecan (CPT-11), which led toinduction of LY6D/E46 antigen. Then, they prepared a monoclonalantibodyagainst this antigenandconjugated it tomonomethyl auristatin,a potent antitubulin drug, to form an immunoconjugate. Administrationof the combination of irinotecan and the immunoconjugate producedcomplete tumour regression but the single agents did not [30]. Althoughstill at preclinical development, this techniquepaves theway to rationallydesigned combinations and can expand the range of antibodies availablefor cancer treatment [31].

2.1.5. Combination of different types of therapyIn addition to the combination of two or more bioactive agents, the

combination of chemotherapy and radiotherapy has produced im-proved response and survival rates in cancer patients. Such combinationis well established and routinely used, however, recent studies areassessing the combination of radiation therapy with new drugs and/ornew therapeutic approaches. The National Cancer Institute haspromoted a randomised Phase II trial assessing the combination ofradiotherapy and a prostate specific antigen (PSA)-based vaccine inpatients affected by prostate cancer [32]. The idea was to maximise theeffect of radiotherapy by stimulating the patients' immune system andwas based on preclinical observations showing that radiation therapycan alter tumour cells and make themmore susceptible to the action ofthe body's immune system [33]. This clinical study confirmed that thevaccine was well tolerated and could be given safely to patientsundergoing radiation therapy for localized prostate cancer, with themajority of patients generating a PSA-specific cellular immune responseto the vaccine [32].

On the short term, combination therapy is more expensive thanmonotherapies, however as discussed above, appropriate drug combi-nations can produce significant benefits including a lower treatmentfailure, lower case-fatality ratios, slower development of drug resistanceand, in the long run, even savings [19]. Combination therapy alreadyplays a key role in cancer treatment and, if supported by an under-standing of the underlying molecular mechanisms, it is expected to doso evenmore in the future [6]. From this point of view, the application ofDDS for multi-agent therapy to ensure that such drug cocktails are trulysimultaneously delivered at the target site is of clear interest. In the nextsection, the use of polymer conjugates for this purpose is reviewedsystematically.

3. Polymer–drug conjugates for combination therapy

Polymer–drug conjugates are drugdelivery technologies inwhich adrug is covalently bound to a polymeric carrier, normally via abiodegradable linker. Such nanoconstructs were first proposed in the1970s [34], developed pre-clinically in the 1980s [35,36] and startedentering the clinical pipeline in the 1990s [37–40, reviewed in thisissue and in 41–44]. Currently, more than fourteen polymer–drugconjugates have undergone clinical evaluation and a polyglutamic acid(PGA)-paclitaxel conjugate (CT-2103, OPAXIO®, previously known asXyotax®) is expected to enter the market in the very near future[5,45,46]. In March 2008, Cell Therapeutics submitted a marketingauthorisation application for OPAXIO® for the treatment of patientswith NSCLC [5]. The main benefits of polymer–drug conjugatescompared to the parent free drug are: (a) passive tumour targetingby the enhanced permeability and retention (EPR) effect [47];(b) decreased toxicity [37]; (c) increased solubility in biological fluids[38] (d) ability to overpass some mechanisms of drug resistance [48]and (e) ability to elicit immunostimulatory effects [49,50]. Numerous

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reviews are available on polymer–drug conjugates carrying a singledrug [the reader is encouraged to refer to [41–43,51,52] and, spe-cifically on clinical studies 44], however, only very recently have theirapplications been extended to combination therapy.

3.1. The concept of polymer–drug conjugates for combination therapy

The term “polymer–drug conjugates for combination therapy” is ageneral phrase that encompasses at least four types of systems (Fig. 1),namely:

1) Type I: polymer–drug conjugate plus free drug. In this approach, apolymer–drug conjugate carrying a single drug is administered incombination with a low molecular weight drug or with anothertype of treatment (e.g. radiotherapy).

2) Type II: polymer–drug conjugate plus polymer–drug conjugate. In thesecond type, two polymer–drug conjugates, each carrying a singletherapeutic agent, are administered in combination.

3) Type III: Single polymeric carrier carrying a combination of drugs. Inthis approach two or more drugs are attached to a single polymercarrier.

4) Type IV: polymer-directed enzyme prodrug therapy (PDEPT) andpolymer enzyme liposome therapy (PELT). The fourth type of systemencompasses the combination of a polymer–drug conjugate carry-ing a single therapeutic agent with a polymer–enzyme conjugate(PDEPT) or the combination of a liposomal system with a polymer–phospholipase conjugate (PELT). The polymer–enzyme conjugate isresponsible for drug release following cleavage of the drug–polymerlinker (in PDEPT) or disruption of the liposomal system (in PELT).

Each system is discussed below and the key studies are sum-marised in Table 1.

3.1.1. Polymer–drug conjugate+low molecular weight drug or othertype of therapy

As anticancer regimens routinely involve the administration of drugcombinations (discussed in Section 2.1), the evaluation of polymer–drug conjugates in combination with free drugs was a logical step toundertake. Clinical studies assessed PGA–paclitaxel conjugate incombination with platinates. A Phase I study was carried out on forty-three patients with advanced solid tumours combining a fixed dose of

Fig. 1. Schematic representation of the different types of polymer-based

cis-platin (75 mg/m2) with escalating doses of PGA-paclitaxel. Asnormal for Phase I studies, the primary aim was to determine thetoxicity,maximumtolerateddose (MTD) and pharmacokinetics of PGA-paclitaxel. However, it was noticed that this combination showed goodactivity in refractory patients [53]. Another phase I studywas carriedouton twenty-two patients with advanced solid tumours testing thecombination of PGA-paclitaxel with carboplatin [54]. The MTD was225 mg/mL and three partial responses were observed. Interestingly,partial responses were observed in patients who had previously failedpaclitaxel therapy. Based on the promising results of these early studies,a Phase III clinical trial, named STELLAR 3 was carried out on 400patients with NSCLC cancer and poor performance status to assess andcompare PGA-paclitaxel plus carboplatin against paclitaxel pluscarboplatin.Whilst no improvement in patients' survival was observed,the combination containing the conjugate proved less toxic [54]. Inaddition, a retrospective data analysis suggested that the anticanceractivity of PGA-paclitaxel might be affected by oestrogen levels, thus,Cell Therapeutics Inc. is now enrolling female patients with advancedNSCLC and baseline oestradiol greater than 25 pg/mL, again comparingcarboplatin plus PGA-paclitaxel or plus paclitaxel [5]. These clinicalstudies highlighted thebenefits of combiningapolymer–drugconjugatewith another therapeutic agent administered as free drug. However,since no direct comparison was carried out against a single-agenttreatment (for instance, compared to the conjugate alone), the addedtherapeutic value of such combination compared to mono-therapy issomewhat difficult to quantify.

Other clinical studies have explored polymer–drug conjugatescombined with radiotherapy. A Phase I study was carried out ontwenty-one patientswith oesophageal and gastric cancer to assess PGA-paclitaxel and radiotherapy. The aim of the study was to establishthe safety and the MTD of this combination, which was found to be80 mg/m2. Interestingly, additional observations included a completeclinical response in 33% of patients with loco-regional disease [55]. Asdiscussed in Section 2.1 chemotherapy and radiotherapy are oftencombined in clinical practice. Polymer–drug conjugates are known topassively accumulate in the tumour tissue as a result of the leaky tumourvasculature (EPR effect) [47]. As radiotherapy can impact on tumourvasculature, possibly magnifying the EPR effect, the combination ofradiotherapy with polymer–drug conjugate is extremely interesting.Indeed, observations of increased activity if treatment with polymer–drug conjugates followed radiotherapy were reported already in early

combination therapy for targeted drug delivery by the EPR effect.

Table 1Representative examples of the different types of polymer-based combination and their status.

Name Polymer carrier Agent 1 Type (name)Agent 2 Type (name)

Status References

Type IPGA–paclitaxel+cisplatinum PGA Chemotherapy (paclitaxel)

Chemotherapy (Cisplatinum)Clinical (Phase I) [53]

PGA–paclitaxel+carboplatinum PGA Chemotherapy (paclitaxel)Chemotherapy (Carboplatinum)

Clinical (Phase III) [5,54]

PGA–paclitaxel+radiotherapy PGA Chemotherapy (paclitaxel)Radiotherapy

Clinical (Phase I) [55]

Type IIHPMA copolymer–Dox+HPMA copolymer–mesochlorin e6 HPMA copolymer Chemotherapy (Dox)

Phototherapy (Mscl e6)Preclinical [59,60]

PEG–(ZnPP) and PEG– (DAO) PEG Hemeoxigenase inhibitorEnzyme (DAO)

Preclinical [61]

CPT–PEG–LHRH+CPT–PEG–BH3 PEG Chemotherapy (CPT)Proapoptotic protein (BH3)Targeting residue (LHRH)

Preclinical (in vitro) [62,63]

Type IIIHPMA copolymer–Dox–AGM HPMA copolymer Chemotherapy (Dox)

Endocrine Therapy (AGM)Pre-clinical (in vitro) [66–68]

PEG–NO–EPI Branched PEG Chemotherapy (EPI)signalling molecule (NO)

Pre-clinical (in vivo) [69–71]

CPT–PEG–LHRH–BH3 Branched PEG Chemotherapy (CPT)Proapoptotic protein (BH3)Targeting residue (LHRH)

Pre-clinical (in vivo) [72]

HPMA–TNP–470–ALN HPMA copolymer Antiangiogenic agent (TNP-470)Bone targeting and antiangiogenicagent (ALN)

Pre-clinical (in vivo) [76]

HPMA–paclitaxel–ALN HPMA copolymer Chemotherapy (Paclitaxel)Bone targeting and antiangiogenicagent (ALN)

Pre-clinical (in vivo) [77]

HPMA–Gem–Dox HPMA copolymer Chemotherapy (Gemcitabine)Chemotherapy (Dox)

Pre-clinical (in vivo) [78]

HPMA–Dox–DEX HPMA copolymer Chemotherapy (Dox)Antiinflammatory drug (DEX)

In vitro [79]

PEG–poly(aspartate hydrazide) block copolymers–Dox–WOR PEG–poly(aspartate hydrazide)block copolymers

Chemotherapy (Dox)Phosphatidylinositol-3 kinaseinhibitor (WOR)

In vitro [80]

Type IVHPMA copolymer–Dox+HPMA copolymer–Cathepsin B HPMA copolymer Chemotherapy (Dox)

Proteolytic Enzyme (Cathepsin B)Pre-clinical (in vivo) [81]

HPMA copolymer–Dox+HPMA copolymer–β-lactamase HPMA copolymer Chemotherapy (Dox)Proteolytic enzyme/β-lactamase)

Pre-clinical (in vivo) [82]

PGA: poly-L-glutamic acid; Dox: doxorubicin; HPMA: N-hydroxypropyl methacrylamide; PEG: polyethylene glycol; EPI: epirubicin; CPT: camptotecin; LHRH: luteinizing-hormonerelease horme; ALN: alendronate; Gem: Gemcitabine.

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clinical trials [56]. Very recently, systematic preclinical studies werecarried out by Lammers et al. investigating the impact of radiotherapyon accumulation of macromolecules in tumours. In a first study theauthors looked at polyHPMA of different molecular weights and at anHPMA copolymer–Dox conjugate and observed that radiotherapypromoted passive tumour targeting of all these macromolecules [57].In a subsequent study, the same group assessed HPMA copolymerscontaining imaging agents and HPMA copolymer–drug conjugates andconfirmed that tumour targeting increased if mice were pre-treatedwith radiotherapy. Such increased tumour targeting ultimately resultedin an improved therapeutic index for both drugs assessed (polymerbound Dox and polymer bound gemcitabine) [58].

3.1.2. Polymer–drug conjugate+polymer–drug conjugateCombinations of two polymer–drug conjugates each carrying a

single therapeutic agent have also been suggested. To date, this strategyhas not been explored clinically, but pre-clinical reports are available.Kopeček and colleagues tested the combination of two polymer–drugconjugates containing the chemotherapeutic agent Dox and thephotoactivatable agent mesochlorin e6 (namely, HPMA copolymer–Dox and HPMA copolymer–mesochlorin e6) and showed that this

combination was more active than either conjugate alone. Even betteractivity was observedwhen the antibody OV-TL16was added for activetargeting [59]. Moreover, in a recent study, the authors developed thisstrategy further by exposing anovarian carcinoma cell line to sequentialadministration of two polymer conjugates, namely HPMA copolymer–SOS (i.e. 2,5-bis(5-hydroxymethyl-2-thienyl)furan), followed by HPMAcopolymer–mesochlorin e6 monoethylenediamine and observed syn-ergistic effects [60]. Another group evaluated the therapeutic effect oftwo PEG conjugates, namely PEG–zinc protoporphyrin (ZnPP, a hemeoxigenase inhibitor) and PEG–D-amino acid oxidase (DAO). Treatmentwith PEG–ZnPP followed by PEG–DAO and D-proline significantlyinhibited tumour growth in animal models, an effect that was notobserved for the conjugate alone [61]. Finally, Minko and colleaguesreported a targeted proapototic drug delivery system consisting oncamptotecin (CPT), BH3 domain peptide and Lutenizing HormoneRelease Hormone (LHRH) [62]. In order to test the efficacy of combiningsuch component this research group tested free CPT, CPT–PEG, CPT–PEG–BH3 or CPT–PEG–LHRH conjugates and the mixture of CPT–PEG–BH3 and CPT–PEG–LHRH conjugates in human ovarian carcinoma cells.An increased proapoptotic activitywas observedwhen the combinationCPT–PEG–BH3 plus CPT–PEG–LHRH was used [63].

Fig. 2. Representative examples of polymer combination therapy Type III. A) HPMA copolymer–AGM–Dox conjugate [66], B) EPI–PEG–NO8 [70] and C) PEG–poly(aspartatehydrazide) block copolymers–Dox–WOR [80].

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3.1.3. Single polymeric carrier carrying a combination of drugsAlthough several studies have developed polymer–drug conjugates,

which contained a targeting residue as well as a drug [e.g. 64 andreviewed in 65], only over the past 4 years two ormore drugs have beencombined within a single polymeric carrier (Fig. 2). This approach issubstantially different from polymer–drug conjugates containing atargeting group as both agents are expected to elicit a pharmacologicalresponse rather than one being the active ingredient and the otherincreasing tumour targeting. By conjugating both drugs to a singlecarrier, the simultaneous delivery of both drugs can be accomplished.

Thefirst conjugate of this typewas anHPMAcopolymer carrying thecombination of endocrine therapy (the aromatase inhibitor aminoglu-tethimide (AGM)) and chemotherapy (Dox), HPMA copolymer–AGM–

Dox conjugate [66]. The drug loading in this conjugate was approxi-mately 5%w/w for AGM and 7%w/w for Dox and the drugs were linkedvia a peptydil linker designed to be cleaved within the lysosomalcompartment of cancer cells. The anticancer activity of this conjugatewas assessed in vitro against breast cancer cell lines. Interestingly, theconjugate carrying both drugs wasmore active than the combination oftwo polymer conjugates each carrying a single drug [66]. A follow onstudy suggested that such increased activity could be due to a variety offactors, including drug release rate, conjugate conformation in solutionand possibly, activation of certain molecular pathways (induction ofapoptosis, e.g. downregulation of Bcl-2 protein) [67,68].

Others have used PEG to conjugate the chemotherapeutic agentepirubicin (EPI) and the diffusible messenger nitric oxide (NO). SinceunmodifiedPEGhas amaximum loading capacity of twodrugmoleculesper polymer chain, the authors elegantly addressed this issue by

building a dendronised structure to one terminus of the PEG chain[69,70]. Not only did this strategy allow to significantly increase NOloading (up to 8molecules per chain) but also to obtain two chemicallydistinct termini (a carboxylic acid used for NO conjugation and anhydroxyl group to conjugate EPI). This combination is of particularinterest as EPI and NO induce different pharmacological responsesthat are, in part, tissue-dependent. In cancer cells, EPI and NO actsynergistically, conversely, in cardiomyocytes NO counterbalances EPIinduced cardio-toxicity [70]. Conjugation of both agents onto a singlechain ensures that they undergo the same body distribution, thusmaximising the benefits of this combination. In vivo studies confirmedthat the PEG–NO–EPI conjugate displayed anticancer activity but wasless cardio-toxic [70,71].

A branched PEG polymer was also used by Minko et al. building onthe proapoptotic BH3 based PEG conjugate previously described andthe promising data from the combination of different conjugates [62].They synthesised a six-branched conjugate containing equimolecularamounts of CPT, BH3 and LHRH. In vitro studies showed that suchmulticomponent conjugate was almost 100 timesmore cytotoxic thanthe single conjugates and displayed enhanced antitumour activity invivo when compared with monotherapy [63,72].

Satchi-Fainaro et al. developed the first polymer–drug conjugatecontaining an antiangiogenic agent, TNP-470 (Caplostatin) [73,74],which is now under preclinical development by SynDevRx, Inc. [75]for various tumour models (melanoma, glioblastoma, colon, prostateand lung carcinomas). Building on this single drug system, the authorssubsequently developed an HPMA copolymer containing TNP-470and the aminobiisphosphonate alendronate [76 and in this issue]. In

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this combination, alendronate has the double function of targetingmoiety (to promote bone targeting) and of pharmacologically activeagent. In vitro evaluation of this combination conjugate confirmedits antiangiogenic and antitumour properties and in vivo assessmentfurther strengthened these positive results with almost completetumour regression observed in a human osteosarcoma model [76]. Ina complementary study, the same authors combined alendronate andpaclitaxel onto an HPMA copolymer carrier. In vitro studies againstprostate and breast cancer cells showed anticancer and antiangio-genic activity and suggest promising therapeutic applications for bonemetastasis [77 and in this issue].

In the last year, two more combination conjugates based on HPMAcopolymer have been developed. One conjugate, carrying two chemo-therapeutic drugs (gemcitabine (Gem) and Dox) was assessed in vivoand proved able to deliver the two drugs to tumour tissue [78]. As in thecase of HPMA–AGM–Dox [66], when tested in vivo in a tumour ratmodel, the combination conjugate HPMA–Gem–Dox was more activethan the combination of two polymer conjugates each carrying a singledrug, and even more than the combination of the free drugs.Furthermore, HPMA–Gem–Dox inhibited angiogenesis and inducedapoptosismore strongly than the controls [78]. The other conjugatewasan HPMA copolymer carrying the anticancer agent Dox and the anti-inflammatory agent dexamethasone (DEX)[79]. The authors prepared alibrary of conjugates containing only Dox, only DEX or the combinationof the two. Biological studies assessing the activity of the conjugate arewarranted to confirm the therapeutic benefit of this combination.

Finally, Kwon and collaborators developed an interesting systembased on polymer–drug conjugates and polymeric micelles [80]. Anamphiphilic polymer constituted by poly(ethylene glycol)–poly(aspartate hydrazide) block copolymers was prepared and Dox andthe phosphatidylinositol–3 kinase inhibitor wortmannin (WOR) wereattached alone or in combination, at different drug ratios. Chemico-physical studies confirmed that the conjugates assembled to formmicellar structures. It was observed that the delivery of both agentsvia the micellar system reduced the amount of drug necessary to elicitbiological activity [80].

3.1.4. Polymer-directed enzyme prodrug therapy (PDEPT) and polymer–enzyme liposome therapy (PELT)

Polymer-directed enzyme prodrug therapy is a two componentsstrategy based onpolymer conjugates. In this approach, a polymer–drugconjugate is combined with a polymer–enzyme conjugate with the aimof achieving selective release of the drug at the tumour site. Indeed, thelinker binding the drug to the polymer in the first conjugate is designedtobedegraded by the enzymeof the second conjugate. The initialmodelcompounds tested were HPMA copolymer–Dox, a conjugate that hasshown anticancer activity clinically in Phase II studies and HPMAcopolymer–cathepsin B (Fig. 3) [81]. Activation of HPMA copolymer–Dox would normally rely on its exposure to the lysosomal enzymecathepsin B and is therefore influenced by its cellular uptake rate,intracellular trafficking and, obviously, by the enzyme content encoun-tered. Administration of a polymer–cathepsin B conjugate ensuredappropriate drug release at the tumour site independently from thefactors listed above. Preclinical studies confirmed that the HPMA–cathepsin B conjugate was able to trigger Dox release in animalmodels,with an area under the curve (AUC) almost 4 fold higher than thatobtained with the HPMA copolymer–Dox alone [81]. The follow-upstudy extended and refined this strategy even further. Here, Dox wasconjugated toHPMAvia a Gly–Gly–cephalosporin linkage susceptible tothe action of the non-mammalian enzyme β-lactamase but not tocathepsins [82]. In this study, mice treated with the PDEPT combinationdisplayed increased survival and decreased tumour growth ratecompared to the control. Whilst no evidence of toxicity was found forthe PDEPT combination, immune responses are a possible issue whenusing non-human proteins. Polymer–protein conjugation is a well-known strategy for prolonging circulation time of proteins and

for decreasing their immunogenicity [4], which suggests that afteroptimisation, PDEPT has a great therapeutic potential and furtherstudies with catalytic antibodies have demonstrated its value [83].

Polymer–enzyme liposome therapy (PELT) [84] is a similarapproach but in this case a polymer–enzyme conjugate is adminis-tered to promote liposome degradation and subsequent drug releasefrom the liposomes. Initial studies with an HPMA copolymer–phospholipase C and the liposomal formulation Doxil confirmed thevalidity of this approach [85,86]. More recently, a dextrin–phospho-lipase A2 conjugate was developed. This conjugate was explored asanticancer agent itself [86] as well as part of a PELT strategy [87]. Invivo studies are awaited to confirm thepromise of this novel conjugate.

4. Challenges associated with the use of polymer–drug conjugatesfor combination

The presence of two or more therapeutic agents on a single poly-meric chain opens new therapeutic possibilities but at the same timeposes new challenges. In this section we identify the main issues asso-ciated with the development of this new type of therapeutic agents.

4.1. Identification of appropriate drug combinations and drug ratios

As discussed previously, most drug combinations are based on theassumption that by targeting different cellular pathways, therapeuticbenefit canbemaximised and toxicity reduced.Whilst several successfulcombinations confirmed the validity of this statement, other studies didnot meet the initial expectations [28]. Thus, identification of drugs thatare best delivered together is a key, and not trivial aspect. Thoroughbiological evaluation supported by a profound understanding of themolecular mechanisms involved is needed.

Another complex aspect of multiagent systems is the determinationof the optimal dose as the relative ratios between each component canimpact on activity. So far, a systematic research investigating the impactof different drug ratios on the biological activity of polymer–drugconjugates has yet to be carried out. A Canadian company, CelatorTechnologies Inc. has developed a methodical approach to assessdifferent drug ratios within their liposomal technology [88]. Thistechnology led to the development of different liposomal formulationsthat are now being assessed in Phase II clinical trials, namely CPX-1(irinotecan : floxuridine) and CPX-351 (cytarabine : daunorubicin). It ishoped that a similar approach will be applied to the development ofcombination polymer–drug conjugates in the future.

4.2. Kinetics of drug release

It is generally accepted that drug release from the carrier is anessential requirement for polymer–drug conjugates to exert theiranticancer activity [reviewed in 41]. Therefore, the ideal linker shouldbe stable in the blood and enzymatically or chemically cleaved at thetumour site. Meticulous research carried out in the 1980s comparingpeptidyl linkers for selective cleavage in the lysosomal compartmentled to the development and clinical assessment of HPMA copolymer–GFLG–Dox [reviewed in 89 and references therein]. These early studiesshowed that different peptidyl linkers displayed different release rates,which in turn impacted on the conjugates activity [reviewed in 89 andreferences therein]. It was also observed that the biodegradability ofthe linker was dependent on the drug attached. For instance, thetetrapeptide GG was not degradable when Dox was linked but a time-dependent drug releasewas observedwhenmelphalanwas bound [90].For polymer–drug conjugates carrying more than one agent, drugrelease was affected by the presence of a second agent. This was trueeven for a single type of linker. For instance, Vicent et al. compared drugrelease rate fromHPMAcopolymer–GFLG–Dox–AGMwith that fromthecombination of HPMA copolymer–GFLG–Dox and HPMA copolymer–GFLG–AGM [66]. The release of Dox from HPMA copolymer–GFLG–Dox

Fig. 3. Schematic representation of the polymer-directed enzyme prodrug therapy (PDEPT) concept, combination therapy Type IV, showing HPMA copolymer–Dox (PK1) activatedby HPMA copolymer–Gly–Gly–cathepsin B as an example [81].

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was not affected by the presence of the HPMA copolymer–GFLG–AGMand similarly, the release of AGM from HPMA copolymer–GFLG–AGMwas not influenced by the presence of HPMA copolymer–GFLG–Dox.Conversely, the combination polymer HPMA copolymer–GFLG–Dox–AGMdisplayed a different drug release profile. The authors suggest thatsuch difference might at least partially explain the differences in thebiological behaviour [66]. A different scenariowas found by Ulbrich andcolleagues when comparing release of DEX from HPMA copolymers–DEX or from HPMA copolymer–Dox–DEX. The release of DEX wasaffected by the linkage used and by the incubation conditions (pH andenzymes), however the presence of Dox on the polymer did not affecteither DEX release rate or the amount of DEX released [79].

In vitro studies confirming drug release and quantifying amount ofdrug released and release rate are clearly important. For conjugatescombining more than one agent, relative drug release rate (whichdrug is released faster) and sequential drug release (which drug isreleased first) can further increase the complexity of the system andbecome key factors for activity [67,68].

4.3. Loading capacity

The need of a polymer carrier with a loading capacity adequate toensure delivery of sufficient amount of drugs to the tumour site hasalways been an important requirement for polymer–drug conjugates.

Table 2Examples of techniques typically employed to characterise polymer–drug conjugates(see [99] as general reference).

Property investigated Technique Reference

Covalent attachment of drug to the polymer NMRFT–IRMALDI–TOF

[102–105]

Total drug content HPLCUVNMR

[79,90,98,105]

Free drug content HPLCUVNMR

[79,90,98,105]

Molecular weight/polydispersity GPCMALDI–TOFLight scattering(QELS)

[79,90,106]

Size/Conformation of the conjugate in solutionFormation of supramolecular assemblies

SANSSAXSNMRLight scattering(DLS)

[66,100,101,105]

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Clearly, development of carriers with a high carrying capacity becomesevenmore vital if such carriers are to carry multi-agent therapies. Someof the linear polymers initially explored as drug carriers had a limitedcapacity. As discussedpreviously (in Section 3.1.3.), unmodifiedPEGcancarry a maximum of two drug molecules per chain, and the HPMAcopolymers explored so far have a relatively limited amount of mono-merswith appropriate functional groups for drug conjugation (normally,≤10%molar ratio) [89]. More recently, other linear polymers have beenexplored which display a better carrying capacity. For instance, PGA cantheoretically carry one drug molecule per monomer and indeed,conjugates based on PGA have a high drug loading (37 wt.% PGA–Paclitaxel [46] and 33–35 wt.% PGA–camptothecin [91]). In addition tolinear polymers, novel polymeric architectures such as dendrimers[92,93], hyperbranched polymers [94] or hybrid macromoleculararchitectures [95] are also being explored. Not only do these novelarchitectures display a good carrying capacity but they presentadditional advantages, including: (i) ability to display superficial end-group with tailored functionalities, (ii) monodispersed or quasi-monodispersednanoscale geometry, (iii) decreasedflexibility comparedto linear random coil polymers. Some issues have been raised in regardsto the toxicity and biocompatibility of these systems, which requirecareful consideration [96], however, it is clear that these newarchitectures have great potential in the context of drug delivery.

4.4. Correlation of in vitro studies with behaviour in vivo

Preliminary screening of the anticancer activity of newly synthe-sised polymer–drug conjugates is normally carried out in vitro againstcancer cells using standard cell viability assays. The usefulness of suchin vitro screening is debatable as, polymer–drug conjugates relay onaccumulation in the tumour tissue via the EPR effect, which can beobserved only in vivo models. In addition, polymer–drug conjugatesneed to be internalised by endocytosis, trafficked to the appropriatecellular compartment and enzymatically or chemically activated (i.e.drug released) before exerting their effect, which causes a significantdelay in the onset of action compared to the free drug. As a result, thefree drug is normally more active in vitro than the conjugated drugbut in vivo studies show opposite trends [89]. Based on theseconsiderations, the significance of in vitro tests and their relevanceto predict in vivo behaviour are difficult issues. Ethical considerationsand cost are obvious reasons in favour of in vitro pre-screening butthere are additional advantages, particularly in the case of polymerbased combination therapy. First, in vitro testing allows a comparisonof the relative activity of different polymer–drug conjugates, possiblebenefits of combining two agents within a single drug carrier can behighlighted at this early stage. Second, an extensive evaluation ofdifferent drug ratios can be carried out, which would not be feasible ata later stage (see also Section 4.1). Finally, specific experiments can bedesigned to elucidate the mechanism of action of these systemsincluding drug release mechanisms [66], their ability to trigger orblock specific cell process (e.g. apoptosis [97], or angiogenesis [73]).

4.5. Physico-chemical characterisation

Finally, another issue that needs careful consideration, notultimately as strict regulatory requirements need to be met, is thephysico-chemical characterisation of these nanoconstructs. Comparedto small molecules, polymer–drug conjugates are relatively complexsystems to fully characterise. These systems are intrinsically hetero-geneous due to several reasons. First, most polymeric carriers arepolydispersed. Second, covalent conjugation of a drug to the carrieris often a random process and, although optimisation of reactionconditions ensures a good degree of batch-to-batch reproducibility,the point of attachment of the drug within the chain remains in manycases not controllable and not directable. Attachment of a second drugto the same carrier complicates thematter even further. As a result, an

adequate physico-chemical characterisation is somewhat difficultto achieve. In contrast, for such compounds to be developed intomedicines and eventually reach the market, thorough characterisa-tion is needed. Moreover, an exhaustive chemico-physical character-isation of such compounds can underpin the understanding of theirbiological behaviour and contribute to the development of rationallydesigned subsequent generations.

A variety of techniques have been traditionally employed to char-acterise polymer–drug conjugates, including, NMR, IR, HPLC and GPC[reviewed in 98,99]. In recent years, sophisticated techniques have beenapplied for thefirst time topolymer–drug conjugates andare providing abetter understanding of these technologies (Table 2). For instance, static(QELS) and dynamic (DLS) light scattering [79], alone or in combinationwith SEC, are considered among the techniques to determine absoluteMw and size (hydrodynamic radius (Rh)) of these hybrid macromole-cules. Small Angle Neutron Scattering (SANS) has proved to be a usefultool to clarify their conformational properties in solution [66,100] andhow they affect their biological behaviour [101]. NMR techniques arealso useful tools to characterise macromolecular structures and theirintermolecular interactions with high spatial and temporal resolution.NMR spectroscopy techniques can provide information such as drugloading, sample heterogeneity and purity, molecular size, aggregation orbinding state. For example, the integrity of the drug and its covalentattachment to the polymer has been demonstratedwith the aid of 1D 1Hand 2D 1H NOESY and TOCSY measurements [102,103].

4.6. Clinical development

Transfer of these combination products into the clinic is extremelychallenging, since it calls for additionalmeasures to unequivocally provetheir clinical benefit. In particular, there is the need to demonstrate thatclinical benefits are due to the advanced drug delivery strategy ratherthan simply the additive/synergistic effects of the parent compoundsadministered as separate therapeutic entities. In other words, there isthe need to demonstrate that the combination of two or more agentswithin a single delivery system provides advantages over the simpleadministration of the free drugs combined. Due to the complexity indesigning such clinical trials and the consequent ethical issues, it isenvisaged that the development costs for such combination productsmight be significantly more than the development of current pharma-ceutical preparations. On the other hand, since these combinationtherapeutics are likely to be licensed as new therapeutic agents, theymight be perceived by pharmaceutical companies as opportunities toextend the patent lives of blockbuster drugs.

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5. Conclusions

The successful development of first generation polymer–drugconjugates in the mid 1980s and 1990s has inspired more recentstudies assessing their potential as drug delivery platforms for com-bination therapy. These early works unveiled unexpected therapeuticbenefits but raised new issues, in particular in relation to “systemdesign”. A better understanding of how drug combinations impact oncellular and molecular mechanisms is needed to rationally design newtherapeutics.

Acknowledgements

The authors thank Ruth Duncan and Helmut Ringsdorf for con-tinuous encouragement and fruitful discussions; Philipp Seib forunvaluable scientific feedback and for proof reading this manuscriptand Cristina Fante and JoséM. Plaja for their contribution to the figures.MJV is a Ramóny Cajal researcher. Acknowledgment to SpanishMICINNfor grant CTQ2007-60601.

References

[1] V.P. Torchilin, Recent advances with liposomes as pharmaceutical carriers, Nat.Rev. Drug Discov. 4 (2005) 145–160.

[2] C. Amin, N. Mackman, N.S. Key, Microparticles and cancer, Pathophysiol.Haemost. Thromb. 36 (2008) 177–183.

[3] J.M. Harris, R.B. Chess, Effect of pegylation on pharmaceuticals, Nat. Rev. DrugDiscov. 2 (2003) 214–221.

[4] F.M. Veronese, J.M.E. Harris, Peptide and Protein PEGylation III: advances inchemistry and clinical applications, Adv. Drug Deliv. Rev. 60 (2008) 1–88 (wholevolume).

[5] http://www.ctiseattle.com.[6] J.M.W. Gee, A. Howell, W.J. Gullick, C.C. Benz, R.L. Sutherland, R.J. Santen, L.A.

Martin, F. Ciardiello, W.R. Miller, M. Dowsett, P. Barrett-Lee, J.F.R. Robertson, S.R.Johnston, H.E. Jones, A.E. Wakeling, R. Duncan, R.I. Nicholson, Consensusstatement Endocrin, Rel. Cancer 12 (2005) S1–S7.

[7] H.J. Broxterman, N.H. Georgopapadakou, Anticancer therapeutics: “addictive”targets, multi-targeted drugs, new drug combinations, Drug Resis. Updat. 8 (2005)183–197.

[8] K. de Gaetano Donati, R. Rabagliati, L. Iacoviello, R. Cauda, HIV infection, HAART,and endothelial adhesion molecules: current perspectives, Lancet Infect. Dis. 4(2004) 213–222.

[9] P. Orjuela, I. Gonzalez, L. Osorio, Combination therapy as a strategy to preventantimalarial drug resistance, Biomédica 24 (2004) 423–437.

[10] http://clinicaltrials.gov.[11] W.L. Suarez-Pinzon, R.F. Power, Y. Yan, C. Wasserfall, M. Atkinson, A. Rabinovitch,

Combination therapy with glucagon-like peptide-1 and gastrin restores normo-glycemia in diabetic NOD mice, Diabetes 57 (2008) 3281–3288.

[12] M.C. Perry (Ed.), The Chemotherapy Source Book, Williams & Wilkins, MarylandUSA, 1992.

[13] M. Tanabe, Y. Ito, N. Tokudome, T. Sugihara, H.Miura, S. Takahashi, Y. Seto, T. Iwase,K. Hatake, Possible use of combination chemotherapy with mitomycin C andmethotrexate for metastatic breast cancer pretreated with anthracycline andtaxanes, Breast Cancer 12 (2009) 1–6.

[14] F. Muggia, Platinum compounds 30 years after the introduction of cisplatin:implications for the treatment of ovarian cancer act, Gyn.Oncol. 112 (2009)275–281.

[15] M.Berhoune, E. Banu, F. Scotte, P. Prognon, S.Oudard, B. Bonan, Therapeutic strategyfor treatment of metastatic non-small cell lung cancer, Annals Pharmacother. 42(2008) 1640–1652.

[16] F. Honecker, C. Kollmannsberger, D. Quietzsch, C. Haag, M. Schroeder, C. Spott,J.T. Hartmann, W. Baronius, V. Hempel, L. Kanz, C. Bokemeyer, Phase II study ofweekly paclitaxel plus 24-h continuous infusion 5-fluorouracil, folinic acid and3-weekly cisplatin for the treatment of patients with advanced gastric cancer,Anticancer Drugs 13 (2002) 497–503.

[17] M. Akeson, B.-M. Zetterqvist, K. Dahllöf, M. Brännström, G. Horvath, Effect ofadjuvant paclitaxel and carboplatin for advanced stage epithelial ovarian cancer:a population-based cohort study of all patients in western Sweden with long-term follow-up, Acta Obstet. Gynecol. Scand. 87 (2008) 1343–1352.

[18] O.H. Pearson, C.A. Hubay, N.H. Gordon, J.S. Marshall, J.P. Crowe, B.M. Arafah,W. McGuire, Endocrine versus endocrine plus five-drug chemotherapy inpostmenopausal women with stage II estrogen receptor–positive breast cancer,Cancer 64 (1989) 1819–1823.

[19] J.F. Logman, B.M. Heeg, M.F. Botteman, S. Kaura, B.A. van Hout, Economicevaluation of zoledronic acid for the prevention of fractures in postmenopausalwomen with early-stage breast cancer receiving aromatase inhibitors in theUnited Kingdom, ASCO, Breast Cancer Symp., vol. 177, (2008).

[20] C.L. Shapiro, S. Halabi, G. Gibson, D.J. Weckstein, J. Kirshner, W.M. Sikov, E.P. Winer,C.A. Hudis, C. Isaacs, D. Weckstein, R.L. Schilsky, E. Paskett, Effect of zoledronic acid(ZA) on bone mineral density (BMD) in premenopausal women who develop

ovarian failure (OF) due to adjuvant chemotherapy (AdC): first results from CALGBtrial 79809, J. Clin. Oncol. 26 (2008) (May 20 suppl, abstr 512).

[21] M. Gnant, B. Mlineritsch, W. Schippinger, G. Luschin-Ebengreuth, S. Poestlberger,C. Menzel, R. Jakesz, E. Kubista, C. Marth, R. Greil, On behalf of the ABCSG,Adjuvant ovarian suppression combined with tamoxifen or anastrozole, alone orin combination with zoledronic acid, in premenopausal women with hormone-responsive, stage I and II breast cancer: first efficacy results from ABCSG-12,J. Clin. Oncol. 26 (2008) (May 20 suppl., abstr LBA4).

[22] M. Gnant, B. Mlineritsch, W. Schippinger, G. Luschin-Ebengreuth, S. Poestlberger,C. Menzel, R. Jakesz, E. Kubista, C. Marth, R. Greil, On behalf of the ABCSG,Adjuvant endocrine therapy plus zoledronic acid in premenopausal womenwith early-stage breast cancer: 5-year follow-up of the ABCSG-12 bone-mineraldensity substudy, Lancet Oncol. 9 (2008) 840–849.

[23] G.Metro,M.Mottolese, A. Fabi, HER-2-positivemetastatic breast cancer: trastuzumaband beyond, Expert Opin. Pharmacother. 9 (2008) 2583–2601.

[24] A. Bharthuar, L. Egloff, J. Becker, M. George, J.W. Lohr, G. Deeb, R.V. Iyer,Rituximab-based therapy for gemcitabine-induced hemolytic uremic syndromein a patient with metastatic pancreatic adenocarcinoma: a case report, CancerChemother. Pharmacol. 62 (2008) 1–5.

[25] H. Van Poppel, Treatment of advanced and metastatic renal cancer: a revolution?Eur. Urol. Suppl. 8 (2009) 483–488.

[26] H.S. Rugo, Bevacizumab in the treatment of breast cancer: rationale and currentdata, Oncologist 9 (Suppl 1) (2004) 43–49.

[27] K.Miller,M.Wang, J. Gralow,M.Dickler,M. Cobleigh, E.A. Perez, T. Shenkier, D. Cella,N.E. Davidson, Paclitaxel plus Bevacizumab versus paclitaxel alone for metastaticbreast cancer, N. Engl. J. Med. 357 (2007) 2666–2676.

[28] D. Jones, Avastin–Tarceva combination fails in lung cancer, Nat. Biotechnol. 27(2009) 108–109.

[29] H.-G. Wendel, E. Stanchina, J.S. Fridman, A. Malina, S. Ray, S. Kogan, C. Cordon-Cardo, J. Pelletier, S.W. Lowe, Survival signalling by Akt and eIF4E in oncogenesisand cancer therapy, Nature 482 (2004) 332–337.

[30] D. Lane,Designer combination therapy for cancer,Nat. Biotechnol. 24 (2006)163–164.[31] G.P. Adams, L.M.Weiner, Monoclonal antibody therapy of cancer, Nat. Biotechnol.

23 (2005) 1147–1157.[32] J.L. Gulley, P.M. Arlen, A. Bastian, S. Morin, J. Marte, P. Beetham, K.-Y. Tsang,

J. Yokokawa, J.W. Hodge, C. Menard, K. Camphausen, N. Coleman, F. Sullivan,S.M. Steinberg, J. Schlom, William, Combining a recombinant cancer vaccinewith standard definitive radiotherapy in patients with localized prostate cancer,Clin. Cancer Res. 11 (2005) 3353–3362.

[33] M. Chakraborty, S.I. Abrams, C.N. Coleman, K. Camphausen, J. Schlom, J.W. Hodge,External beam radiation of tumors alters phenotype of tumor cells to render themsusceptible to vaccine mediated T-cell killing, Cancer Res. 64 (2004) 4328–4337.

[34] H. Ringsdorf, Structure and propreties of pharmacologically active polymers,J. Polym. Sci., Polym. Symp. 51 (1975) 135–153.

[35] R. Duncan, L.C.W. Seymour, L. Scarlett, J.B. Lloyd, P. Rejmanova, J. Kopeček, Fate ofN-(2-hydroxypropyl)methacrylamide copolymers with pendant galactosamineresidues after intravenous administration to rats, Biochim. Biophys. Acta 880(1986) 62–71.

[36] R. Duncan, L.W. Seymour, K.B. O'Hare, P. Flanagan, S.Wedge, K. Ulbrich, J. Strohalm,V. Subr, F. Spreafico, M. Grandi, M. Ripamonti, M. Farao, A. Suarato, Preclinicalevaluation of polymer-bound doxorubicin, J. Control. Release 19 (1992) 331–346.

[37] P.A. Vasey, S.B. Kaye, R. MorrisonC, P.Wilson Twelves, R. Duncan, A.H. Thomson, L.S.Murray, T.E. Hilditch, T. Murray, S. Burtless, D. Fraier, E. Frigerio, J. Cassidy, Phase Iclinical and pharmacokinetic study of PK1 [N-(2-hydroxypropyl)methacrylamidecopolymer doxorubicin]: first member of a new class of chemotherapeutic agents–drug–polymer conjugates. Cancer Research Campaign Phase I/II Committee, Clin.Cancer Res. 5 (1999) 83–94.

[38] J.M. Meerum Terwogt, W.W. ten Bokkel Huinink, J.H.M. Schellens, M. Schot,I. Mandjes, M. Zurlo, M. Rocchetti, H. Rosing, K.J.H. Beijnen, Phase I clinicaland pharmacokinetic study of PNU166945, a novel water soluble polymerconjugated prodrug of paclitaxel, Anticancer Drug Des. 12 (2001) 315–323.

[39] P. Stood, R. Zarzycki, J. Erberich, R. Mazanet, S. Howell, D. Nowotnick, Resultsof preclinical and clinical studies on ProlindacTM (AP5346), a DACH–platinumHPMA polmer prodrug, Proc. Int Symp. Polymer Therapeutics (2007) 41.

[40] http://www.accesspharma.com.[41] R. Duncan, Polymer conjugates as anticancer nanomedicines, Nat. Rev. Cancer 6

(2006) 688–701.[42] M.J. Vicent, R. Duncan, Polymer conjugates: nanosized medicines for treating

cancer, Trends Biotechnol. 24 (2006) 39–47.[43] R. Satchi-Fainaro, R. Duncan, C.M. Barnes, Polymer therapeutics for cancer:

current status and future challenges, Adv. Poym. Sci. 193 (2006) 1–65.[44] C. Li, S.Wallace, Polymer–drug conjugates: recent development in clinical oncology,

Adv. Drug Deliv. Rev. 60 (2008) 886–898.[45] S.D. Chipman, F.B. Oldham, G. Pezzoni, J.W. Singer, Biological and clinical character-

ization of paclitaxel poliglumex (PPX, CT-2103), a macromolecular polymer–drugconjugate, Int. J. Nanomed. 1 (2006) 375–383.

[46] J.W. Singer, Paclitaxel poliglumex (XYOTAX™, CT-2103): a macromoleculartaxane, J. Control. Release 109 (2005) 120–126.

[47] H. Maeda, Polymer conjugated macromolecular drugs for tumor-specifictargeting, in: A.J. Domb (Ed.), Polymeric Site-specific Pharmacotherapy, JohnWiley and Sons Ltd, USA, 1994, pp. 95–116.

[48] T.Minko, P.Kopečková, V.Pozharov, J.Kopeček,HPMAcopolymer boundadriamycinovercomes MDR1 gene encoded resistance in a human ovarian carcinoma cell line,J. Control. Release 54 (1998) 223–233.

[49] B. Ríhová, J. Strohalm, J. Prausová, K. Kubácková, M. Jelínková, L. Rozprimová,M. Sírová, D. Plocová, T. Etrych, V. Subr, T. Mrkvan, M. Kovár, K. Ulbrich,

1213F. Greco, M.J. Vicent / Advanced Drug Delivery Reviews 61 (2009) 1203–1213

Cytostatic and immunomobilizing activities of polymer-bound drugs: exper-imental and first clinical data, J. Control. Release 28 (2003) 1–16.

[50] M. Sirova, J. Strohalm, V. Subr, D. Plocova, P. Rossmann, T. Mrkvan, K. Ulbrich,B. Rihova, Treatment with HPMA copolymer-based doxorubicin conjugatecontaining human immunoglobulin induces long-lasting systemic anti-tumourimmunity in mice, Cancer Immunol. Immunother. 56 (2007) 35–47.

[51] F. Greco, M.J. Vicent, Polymer–drug conjugates: current status and future trends,Frontiers Biosci. 13 (2008) 2744–2756.

[52] R. Haag, F. Kratz, Polymer therapeutics: concepts and applications, Angew. Chem.Int. Ed. 45 (2006) 1198–1215.

[53] C.F. Verschraegen, K. Skubitz, A. Daud, A.P. Kudelka, I. Rabinowitz, C. Allievi,A. Eisenfeld, J.W. Singer, F.B. Oldham, A phase I and pharmacokinetic study ofpaclitaxel poliglumex and cisplatin in patients with advanced solid tumors,Cancer Chemother. Pharmacol. 63 (2009) 903–910.

[54] C.J. Langer, K.J. O'Byrne,M.A. Socinski, S.M.Mikhailov, K. Lesniewski-Kmak, Phase IIItrial comparing paclitaxel poliglumex (CT-2103, PPX) in combination withcarboplatin versus standard paclitaxel and carboplatin in the treatment of PS 2patients with chemotherapy–naive advanced non-small cell lung cancer, J. Thorac.Oncol. 3 (2008) 623–630.

[55] T.A. Dipetrillo, L.Milas, D. Evans, Paclitaxel poliglumex(PPX–xyotax) andconcurrentradiation for esophageal andgastric cancer— aphase I study, J. Clin. Oncol. 29 (2006)376–379.

[56] S. Ke, L. Milas, C. Charnsangavej, S.Wallace, C. Li, Potentiation of radioresponse bypolymer–drug conjugates, J. Control. Release 74 (2001) 237–242.

[57] T. Lammers, P. Peschke, R.Kühnlein, V. Subr,K.Ulbrich, J. Debus, P.Huber,W.Hennink,G. Storm, Effect of radiotherapy and hyperthermia on the tumor accumulation ofHPMA copolymer-based drug delivery systems, J. Control. Release 117 (2007)333–341.

[58] T. Lammers, V. Subr, P. Peschke, R. Kühnlein,W.E. Hennink, K. Ulbrich, F. Kiessling,M. Heilmann, J. Debus, P.E. Huber, G. Storm, Image-guided and passively tumour-targeted polymeric nanomedicines for radiochemotherapy, Br. J. Cancer 99(2008) 900–910.

[59] J.G. Shiah, Y. Sun, P. Kopečková, C.M. Peterson, R.C. Straight, J. Kopeček, Combinationchemotherapy and photodynamic therapy of targetable N-(2-hydroxypropyl)methacrylamide copolymer–doxorubicin/mesochlorin e(6)–OV–TL 16 antibodyimmunoconjugates, J. Control. Release 74 (2001) 249–253.

[60] J. Hongrapipat, P. Kopečková, J. Liu, S. Prakongpan, J. Kopeček, Combinationchemotherapy and photodynamic therapy with fab' fragment targeted HPMAcopolymer conjugates in human ovarian carcinoma cells, Mol. Pharmacol. 5 (2008)696–709.

[61] J. Fang, T. Sawa, T. Akaike, K. Greish, H. Maeda, Enhancement of chemothera-peutic response of tumor cells by a heme oxygenase inhibitor, pegylated zincprotoporphyrin, Int. J. Cancer 109 (2004) 1–8.

[62] J.J. Khandare, P. Chandna, Y. Wang, V.P. Pozharov, T. Minko, Novel polymericprodrug with multivalent components for cancer therapy, J. Pharmacol. Exp.Ther. 317 (2006) 929–937.

[63] J. Khandare, T. Minko, Polymer–drug conjugates: progress in polymeric prodrugsProg, Polym. Sci. 31 (2006) 359–397.

[64] L.W. Seymour, D. Ferry, D. Anderson, P. Julyan, R. Poyner, J. Doran, A. Young,S. Burtles, D. Kerr, Hepatic drug targeting: phase I evaluation of polymer-bounddoxorubicin, J. Clin. Oncol. 20 (2002) 1668–1676.

[65] J.G.W. Kosternik, W. Helfrich, L.F.M.H. De Leij, Strategies for specific drugtargeting to tumour cells, in: Drug targeting. organ-specific strategies, Weinheim,Germany: Wiley-VCH (Eds) 2001 pp. 199–231.

[66] M.J. Vicent, F. Greco, A. Paul, P.C. Griffiths, R.I. Nicholson, R. Duncan, Polymertherapeutics designed for a combination therapy of hormone-dependent cancer,Angew. Chem. Int. Ed. 44 (2005) 4061–4066.

[67] F. Greco, M.J. Vicent, N.A. Penning, R.I. Nicholson, R. Duncan, HPMA copolymer–aminoglutethimide conjugates inhibit aromatase in MCF-7 cell lines, J. DrugTarget. 13 (2005) 459–470.

[68] F. Greco, M.J. Vicent, S. Gee, A.T. Jones, J. Gee, R.I. Nicholson, R. Duncan, Themechanism of enhanced cytotoxicity of HPMA copolymer–DOX–AGM in breastcancer cells, J. Control. Release 117 (2007) 28–39.

[69] G. Pasut, S. Scaramuzza, O. Schiavon, R. Mendichi, F.M. Veronese, PEG–epirubicinconjugates with high drug loading, J. Bioact. Compat. Polym. 20 (2005) 213–230.

[70] L. Santucci, A. Mencarelli, B. Renga, Nitric oxide modulates proapoptotic andantiapoptotic properties of chemotherapy agents: the case of NO–pegylatedepirubicin, FASEB J. 20 (2006) 765–773.

[71] L. Santucci,A.Mencarelli, B. Renga,D.Ceccobelli,G. Pasut, F.Veronese, E.Distrutti,M.D.Fiorucci, Cardiac safety and anti-tumoral activity of a new nitric oxide derivative ofpegylated epirubicin in mice, Anticancer Drugs 18 (2007) 1081–1091.

[72] P. Chadna, Y. Wang, E. Ver, J. Khandare, A.A. Vetcher, V.A. Soldatenkov, T. Minko,A novel targeted proapoptotic drug delivery system for efficient anticancertherapy, Proc. 35th Annual Mtg. Control. Rel. Soc. (2008) p.35.

[73] R. Satchi-Fainaro, M. Puder, J.W. Davies, H.T. Tran, D.A. Sampson, A.K. Greene,G. Corfas, J. Folkman, Targeting angiogenesis with a conjugate of HPMAcopolymer and TNP-470, Nat. Med. 10 (2004) 255–261.

[74] R. Satchi-Fainaro, R. Mamluk, L. Wang, S.M. Short, J.A. Nagy, D. Feng, A.M. Dvorak,H.F. Dvorak, M. Puder, D. Mukhopadhyay, J. Folkman, Inhibition of vesselpermeability by TNP-470 and its polymer conjugate, caplostatin, Cancer Cell 7(2005) 251–261.

[75] http://www.syndevrx.com.[76] E. Segal, H. Pan, P. Ofek, T. Udagawa, P. Kopečková, J. Kopeček, R. Satchi-Fainaro,

Targeting Angiogenesis-Dependent Calcified Neoplasms Using Combined Poly-mer Therapeutics, PLoS ONE 4 (4) (2009) e5233.

[77] K. Miller, R. Erez, E. Segal, D. Shabat, R. Satchi-Fainaro Targeting Bone Metastaseswith a Novel Bi-Specific Anticancer and Anti-Angiogenic Polymer–Alendronate–Taxane Conjugate, Angew. Chem. Int. Ed. 48 (2009) 2949–2954.

[78] T. Lammers, V. Subr, K. Ulbrich, P. Peschke, P.E. Huber, W.E. Hennink, G. Storm,Simultaneous delivery of doxorubicin and gemcitabine to tumors in vivo usingprototypic polymeric drug carriers, Biomaterials 30 (2009) 3466–3475.

[79] H. Krakovicova, T. Etrych, K. Ulbrich, HPMA-based polymer conjugates with drugcombination, Eur. J. Pharm. Sci. 37 (2009) 405–412.

[80] Y. Bae, T.A. Diezi, A. Zhao, G.S. Kwon, Mixed polymeric micelles for combinationcancer chemotherapy through the concurrent delivery of multiple chemother-apeutic agents, J. Control. Release 122 (2007) 324–330.

[81] R. Satchi, T.A. Connors, R. Duncan, PDEPT: polymer-directed enzyme prodrugtherapy I. HPMA copolymer–cathepsin B and PK1 as a model combination, Br. J.Cancer 85 (2001) 1070–1076.

[82] R. Satchi-Fainaro, H. Hailu, J.W. Davies, C. Summerford, R. Duncan, PDEPT:Polymer-directed enzyme prodrug therapy. 2. HPMA copolymer–beta-lactamaseand HPMA copolymer–C–Dox as a model combination, Bioconjug. Chem 14(2003) 797–804.

[83] R. Satchi-Fainaro, W. Wrasidlo, H.N. Lode, D. Shabat, Synthesis and character-ization of a catalytic antibody-HPMA copolymer-conjugate as a tool for tumorselective prodrug activation, Bioorg. Med. Chem. 10 (2002) 3023–3029.

[84] R. Duncan, S. Gac-Breton, R. Keane, R. Musila, Y.N. Sat, R. Satchi, F. Searle,Polymer–drug conjugates, PDEPT and PELT: basic principles for design andtransfer from the laboratory to clinic, J. Control. Release 74 (2001) 135–146.

[85] R. Satchi-Fainaro, R. Duncan, PELT: Polymer enzyme liposome therapy. HPMAcopolymer–phospholipase C and Doxil/ Daunoxomes as a novel combination.(unpublished data).

[86] E.L. Ferguson, D. Schmaljohann, R. Duncan, Polymer–phospholipase conjugatesas novel anti-cancer agents: dextrin–phospholipase A2, Proc. 33rd Annual Mtg,Control. Release Soc. 33 (2006) 660.

[87] E.L. Ferguson, R. Duncan, Investigating a dextrin–phospholipase A2 conjugate asa trigger for polymer enzyme liposome therapy (PELT), Proc. 7th Int. Symp.Polymer Therapeutics: From Lab to clinic, (2008), p. 96.

[88] http://www.celator.ca.[89] R. Duncan, N-(2-Hydroxypropyl) methacrylamide copolymer conjugates, in: G.S.

Kwon (Ed.), Polymeric Drug Delivery System, Marcel Dekker, Inc, New York,2005, pp. 1–92.

[90] R. Duncan, I.C. Hume, H.J. Yardley, P.A. Flanagan, K. Ulbrich, V. Subr, J. Strohalm,Macromolecular prodrugs for use in targeted cancer chemotherapy: melphalancovalently coupled to N-(2-hydroxypropyl) methacrylamide copolymers, J.Control. Release 16 (1991) 121–136.

[91] R. Bhatt, P. de Vries, J. Tulinsky, G. Bellamy, B. Baker, J.W. Singer, P. Klein,Synthesis and in vivo antitumor activity of poly(L-glutamic acid) conjugates of 20(S)-camptothecin, J. Med. Chem. 46 (2003) 190–193.

[92] N.K. Jain, A. Asthana, Dendritic systems in drug delivery applications, ExpertOpin. Drug. Deliv. 4 (2007) 495–512.

[93] C.C. Lee, J.A. MacKay, J.M.J. Frechet, Designing dendrimers for biologicalapplications, Nat. Biotechnol. 23 (2005) 1517–1526.

[94] R.K. Kainthan, S.R. Hester, E. Levin, In vitro biological evaluation of highmolecularweight hyperbranched polyglycerols, Biomaterials 28 (2007) 4581–4590.

[95] M.K. Mishra, S. Kobayashi (Eds.), Star and Hyperbranched Polymers, MarcelDekker, New York, 1999.

[96] R. Duncan, L. Izzo, Dendrimer biocompatibility and toxicity, Adv. Drug Deliv. Rev.57 (2005) 2215–2237.

[97] M.J. Vicent MJ, Polymer–drug conjugates as modulators of cellular apoptosis,AAPS J. 9 (2007) E200–E207.

[98] A.D. Schluter, A covalent chemistry approach to giant macromolecules withcylindrical shape and an engineerable interior and surface, Top. Curr. Chem. 245(2005) 151–191.

[99] M.J. Vicent, L. Dieudonne, R.J. Carbajo, A. Pineda-Lucena, Polymer conjugates astherapeutics: future trends, challenges and opportunities, Expert Opin. Drug.Deliv. 5 (2008) 593–614.

[100] P.C. Griffiths, A. Paul, Z. Khayat, K.W. Wan, S.M. King, I. Grillo, R. Schweins,P. Ferruti, J franchini, R. Duncan, Understanding the mechanism of action ofpoly(amidoamine)s as endosomolytic polymers: correlation of physicochem-ical and biological properties, Biomacromolecules 5 (2004) 1422–1427.

[101] A. Paul, M.J. Vicent, R. Duncan, Using small-angle neutron scattering to study thesolution conformation of N-(2-hydroxypropyl)methacrylamide copolymer–doxorubicin conjugates, Biomacromolecules 8 (2007) 1573–1579.

[102] M.J. Vicent, E. Pérez-Payá, Poly-L-glutamic acid (PGA) aided inhibitors of apoptoticprotease activating factor 1 (Apaf-1): an antiapoptotic polymeric nanomedicine,J. Med. Chem. 49 (2006) 3763–3765.

[103] V. Pinciroli, V. Rizzo, F. Angelucci, M. Tato, A. Vigevani, 1H-NMR of polymerconjugates with the anticancer drug doxorubicin, Magn. Reson. Chem. 35 (1997)2–8.

[104] J. Hentschel, M.G.J. ten Cate, H.G. Borner, Peptide-guided organization ofpeptide–polymer conjugates: expanding the approach from oligo- to polymers,Macromolecules 40 (2007) 9224–9232.

[105] J.E. Klee, Review: mass spectrometry of step-growth polymers, Eur. J. MassSpectrom. 11 (2005) 591–610.

[106] R.Mendichi, V. Rizzo,M. Gigli, A.G. Schieroni, Fractionation and characterization of aconjugate between a polymeric drug-carrier and the antitumor drug camptothecin,Bioconjug. Chem. 13 (2002) 1253–1258.