selective delivery of therapeutic agents for the diagnosis and treatment of cancer

Review

10.1517/14712598.6.1.39 © 2006 Ashley Publications ISSN 1471-2598 39

Ashley Publicationswww.ashley-pub.com

Delivery

Selective delivery of therapeutic agents for the diagnosis and treatment of cancerGirja S Shukla† & David N Krag†Vermont Comprehensive Cancer Center, Department of Surgery, University of Vermont College of Medicine, 89 Beaumont Avenue, Burlington, VT 05405, USA

Research activity aimed towards achieving specific and targeted delivery ofcancer therapeutics has expanded tremendously in the last decade, resultingin new ways of directing drugs to tumours, as well as new types of drugs. Theavailable strategies exploit differences in the nature of normal and cancercells and their microenvironment. The discovery and validation ofcancer-associated markers, as well as corresponding ligands, is pivotal fordeveloping selective delivery technology for cancer. Although most currentclinical trials are either monoclonal antibody- or gene-based, methodologicaladvances in combinatorial libraries of peptides, single chain variablefragments and small organic molecules are expected to change this scenarioin the near future. Nanotechnology platforms today allow systematic andmodular combinations of therapeutic agents and tumour-binding moietiesthat may generate novel, personalised agents for selective delivery in cancer.This paper discusses recent developments and future prospects of targeteddelivery technologies in the management of cancer.

Keywords: ADEPT, cancer-specific targets, GDEPT, ligands, molecular diagnosis, prodrug therapy, targeted delivery, tumour imaging

Expert Opin. Biol. Ther. (2006) 6(1):39-54

1. Introduction

Delivery of anticancer drugs through veins is essentially uncontrolled delivery ofdrugs to all cells of the body, exposing normal cells as well as cancer cells to thenonspecific antiproliferative effects of anticancer drugs. The majority of existingcancer chemotherapeutics are delivered in this manner. What little selectivity thechemotherapeutics have for the cancer cells is due to the greater sensitivity of cancercells. The success of these cancer drugs is limited by concentration-dependentsystemic toxicity. The inability to deliver an adequate antiproliferative concentrationof drug results in failure to eliminate all of the cancer cells and the situation isworsened by the subsequent development of drug resistance [1]. Developingstrategies that help to restrict the action of therapeutic agents to cancer cells areexpected to decrease systemic toxicity and increase the efficacy of cancer cell killing.

The development of procedures for selective delivery of therapeutic agents for thediagnosis and treatment of cancer is an active area of ongoing investigations in bothexperimental and clinical trials. In broad terms, selective delivery of cancertherapeutic agents includes any methodology by which the functional concentrationof drug is higher at the cancer site than in normal tissue. A wide variety of methodsmay fall under the category of ‘selective delivery’, including interventions as simpleand mechanical as selective vascular administration in which the drug is physicallyisolated in a tumour-bearing area. An example is isolated limb perfusion, in whichthe injected drug is confined to a localised region of the body [2]. The drug

1. Introduction

2. Selective delivery by

passive targeting

3. Selective delivery through

cancer-specific antibodies

4. Peptide-mediated

selective delivery

5. Selective delivery through

nucleotide aptamers

6. Selective delivery by

prodrug activation

7. External energy-controlled

delivery

8. Organ-selective delivery

9. Expert opinion and conclusion

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Selective delivery of therapeutic agents for the diagnosis and treatment of cancer

40 Expert Opin. Biol. Ther. (2006) 6(1)

concentration can be considerably increased with correspond-ingly increased effectiveness of killing cancer cells. While thisapproach supports the notion that an increased concentrationof drug can lead to increased killing of cancer cells, any canceroutside the limb remains untreated. Most strategies, however,are pharmaceutical and are the subject of this review. In theseapproaches, the differences in the biochemical and physio-logical nature of normal and cancer cells and their micro-environment are exploited for selective delivery [3,4]. Not allpharmaceutical strategies of selective delivery fit into neatcategories, but a common feature is to increase the effectiveconcentration of bioactive molecules at the target site. Thesestrategies include:

• use of drug carriers that, due to their physical properties,accumulate preferentially at the tumour

• use of ligands that bind to overexpressed tumour-associatedantigens

• activation of anticancer drugs preferentially at the cancer site• external energy irradiation to release drugs at the cancer site

In recent years, the overexpressing cancer-specific antigens havebecome an important tool in developing different deliverytechnologies in cancer. The delivery by targeting cancer-specificantigens concentrates the therapeutics at the cancer site, andthis helps in diagnostic imaging and treatment of cancers. Forexample, ∼ 2,000,000 HER-2 receptors are present on anoverexpressing cancer cell surface, whereas a non-overexpress-ing normal cell has 10,000 – 20,000 molecules [5]. Thisrepresents ∼ 100-fold higher drug concentrations reaching tocancer cells than normal cells, when delivered through aHER-2-binding ligand.

A variety of technologies using combinations of differentapproaches are constantly being developed for selective deliv-ery of therapeutics in cancer diagnosis and treatment [6,7].These delivery systems employ different targets (e.g., cancercell and neovascular antigens, hypoxia, high osmoticpressure), targeting agents (e.g., monoclonal antibodies[mAbs], single chain variable fragments [scFvs], peptides,oligonucleotides), effectors (e.g., chemical or biologicaltoxins, radioisotopes, genes, enzymes, immunomodulators,oligonucleotides, imaging and diagnostic agents), vehicles(e.g., colloidal systems, including liposomes, emulsions,micelles, nanoparticles; polymer conjugates or implants;viruses; bacteria; modified allogenic cells), and drug-releasingswitches (e.g., thermal, radiation, ultrasound, magnetic field,visible or UV light, enzymes). This paper presents recentdevelopments in the field, with particular reference to deliverysystems that use cancer-targeting mechanisms for deliveringtherapeutics in cancer diagnosis and treatment.

2. Selective delivery by passive targeting

A therapeutic agent stably associated with sustained-release drugcarriers, such as liposomes, polymers and other nanoparticles,has been used for selective delivery of therapeutics for diagnosis

and treatment in cancer. These carriers have been shown toextravasate and localise to areas of increased vascular permeabil-ity, such as those found in angiogenic tumours, in a processreferred to as passive targeting. Of these drug delivery systems,liposome-based formulations of the anthracycline drugs havehad a great impact on the treatment of cancer patients to date.Tumour vessels have an increased permeability to macromole-cules owing to a lack of tight junctions that are normally presentbetween adjacent vasculature endothelial cells of other vessels.The size of the gaps between the cells in tumour vessels is suffi-ciently large to allow the extravasation of most targetedtherapeutics from vessels into the tumour interstitial space [6-8].The therapeutic agents retained in these carriers while in circula-tion are not freely available, thus minimising the uptake bynormal tissues. Surface modification of carriers with polyethyl-ene glycol (PEG), commonly referred to as the pegylationprocess, to produce sterically hindered or STEALTH liposomes,has been shown to protect carriers from reticuloendothelialsystem uptake [7]. This allows extended circulation time, whichin turn maximises carrier uptake by tumour. The limitedlymphatic drainage in tumours helps the carrier-bound drug toaccumulate in tumour tissue. The phenomenon of thecombined effect of these factors is termed the enhanced permea-tion and retention (EPR) effect. The pegylated liposomal doxo-rubicin is in the market and currently being used for cancertreatment. Recently, composite vehicle particles of 80 − 120 nm,known as ‘nanocells’, consisting of a solid biodegradable polymercore surrounded by a pegylated-lipid membrane, were used forstaged delivery of two drugs in the treatment of cancer [9]. Theantiangiogenic drug combretastatin dissolved in a lipid envelopewas released first, followed by a slow release of the chemothera-peutic drug doxorubicin from polymer degradation. The firstdelivery of the antiangiogenic factor led to tumour vasculatureshut down, trapping in the tumour the inner particle, withsecond payload of chemotherapeutic drug. The subsequentrelease of the chemotherapy in the tumour killed the cancer cells.This is an example of next-generation colloidal delivery systemsaiming for an integrative approach in cancer treatment. Table 1shows some of the colloidal system-based anticancer drugsavailable in the market for clinical management of cancer.

Passive targeting by extravasation from tumour vesselsallows drug-encapsulating carriers to accumulate withintumour stroma and release the bioactive drug after enzymaticdegradation and/or phagocytic attack, with subsequent diffu-sion into the cancer cells. Recent developments in this fieldhave generated systems that deliver the therapeutic directly tocancer cells through active tumour targeting. This has beenachieved by linking therapeutic moieties to high-affinityligands directed against cancer-associated targets.

3. Selective delivery through cancer-specific antibodies

The advent of mAb technology in the 1970s and the develop-ment of genetically engineered derivatives in the 1980s, along

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Shukla & Krag

Expert Opin. Biol. Ther. (2006) 6(1) 41

with technological advances in the bulk production of mAbs,have led to a number of clinical studies to evaluate the efficacyof cancer-specific mAbs in the targeted delivery in cancer.Several mAbs have been approved in recent years and are inthe market for their use in cancer management (Table 2). Theexquisite specificity of mAbs makes them ideal vehicles forselective delivery of a variety of diagnostic/imaging agents orbiologically active molecules, such as chemotherapy, radioiso-topes, immunomodulators, chemical and biological toxins,and fluorescence molecules [10-17]. Gemtuzumab ozogamicinis the first mAb that was approved for selective delivery of asmall molecule cytotoxin to cancer cells. The antibody islinked with the highly potent DNA-cleaving agent calicheam-icin through an acid-labile hydrazone moiety, which, follow-ing internalisation into low pH endosomes, breaks andreleases a free cytotoxic agent. Several recombinant immuno-toxin products carrying Pseudomonas exotoxin, such asLMB-2 (anti-CD25) and BL22 (anti-CD22), are in cancerclinical trials. Ibritumomab tiuxetan and tositumomabantibodies that deliver radiotoxic agents to cancer cells areapproved for the treatment of non-Hodgkin’s lymphoma.Another mAb delivering radioisotopes, labetuzumab(CEA-Cide™, anticarcinoembronic antigen), is in clinicaltrials in colorectal and pancreatic cancer patients. Antibodieshave also been employed for delivering magnetic resonanceimaging (MRI) contrast agents [18-20], radionuclides [21,22],quantum dots [8] and metal-based nanoshells [23] for cancerimaging. The naked or unlabelled antibodies that are already inthe market for cancer treatment (Table 2) and those being evalu-ated in cancer clinical trials at present(e.g., 17-1A [edrecolomab,anti-EpCAM]; B43.13 [oregovomab, anti-CA125]; epratuzu-mab [anti-CD22]; and Hu1D10 [apolizumab, anti-1D10])have potential for use as a vehicles for selective delivery oftherapeutic agents in cancer diagnosis and treatment.

Although encouraging results with antibody conjugates inthe management of haematopoietic malignancies, especiallynon-Hodgkin’s lymphoma, have generated considerableinterest in such therapy, experience with solid tumours hasbeen less enthusiastic [10,24]. This is due, at least in part, to avariety of factors, such as poor tumour vascularisation, barriersto antibody penetration and high intratumoural pressure, thatlimit the antibody conjugate delivery in solid tumours incomparison to the relative accessibility of the malignant cells inblood and bone marrow. The major limitations in antibody-targeted delivery are related to large antibody size, nonspecificuptake of antibodies by the liver and the reticuloendothelialsystem, and immunogenicity [24]. The development of humanor chimeric antibodies has largely overcome the host immuneresponses to rodent-derived mAbs [25]. The use of antibodyfragments, such as Fab, F(ab’)2 and scFv, has been helpful toresolve certain size-related issues [24,26,27]. In a clinical trial, thescFv MFE-23 against carcinoembryonic antigen has been usedfor selective delivery of a radioactive tracer for gamma-cameraimaging of tumour [28]. The development of phage displaytechnology for the selection of target-specific scFvs has openedenormous possibilities in this field [29,30].

4. Peptide-mediated selective delivery

Carrier peptides, acting as shuttles for the selective delivery ofanticancer compounds, are much smaller than antibodies, butstill retain selective binding affinity to cancer target molecules,and may overcome several of the limitations of antibodytherapy [31-33]. One of the great strengths of peptidesas potential delivery agents lies in the recently developedpowerful approaches in phage display and combinatoriallibrary technologies for discovering and screening newcancer-specific peptides [34-37]. Cancer target-specific peptides

Table 1. Examples of colloidal delivery system-based drugs available for cancer treatment.

Drug name Delivery system Mode of action Trade name and company

Doxorubicin citrate Liposome Anthracyclin,DNA intercalator

DaunoXome® – Gilead SciencesMyocet™ – Zeneus Pharma, Elan Corporation

Doxorubicin HCl Liposome Anthracyclin,DNA intercalator

Liposome encapsulated doxorubicin – Neopharm

Doxorubicin HCl Pegylatedliposome

Anthracyclin,DNA intercalator

Doxil® – Tibotec Therapeutics;Caelyx® – Schering-Plough

Lurtotecan Liposome Topoisomerase inhibitor NX211 – OSI Pharmaceuticals

Vincristine Liposome Antimicrotubule agent Marqibo™ – INEX Pharmaceuticals

Carmustine (BCNU) Polymer conjugate Alkylating agents Gliadel® – Guilford Pharmaceuticals

Cytarabine Lipid vesicles Cytosine arabinoside, Antimetabolite

Depocyt® – Skyepharma/Enzon, Pharmacia Coporation, Paladin

Triptorelin pamoate Biodegradable microgranules Gonadotropin inhibitor Trelstar depot® – Debiopharm

Pegylated liposome: Polyethyleneglycol-coated liposome

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have been used to deliver a variety of therapeutic agents tocancer cells for diagnosis and treatment [38-43]. Peptides havebeen reported to selectively deliver semiconductor quantumdots [44], fluorescence imaging agents, or radioisotopes [43,45]

to cell-specific normal and cancer targets in animals. Recently,experimental molecular diagnostic imaging of oncogenemRNAs has been performed with the help of insulin-likegrowth factor (IGF)1, a receptor-binding peptide in nudemice bearing human IGF1 receptor-overexpressing MCF7breast cancer xenografts. MYC peptide nucleic acid probesconstructed of an IGF1 peptide loop on the C-terminus and a[99mTc]chelator peptide on the N-terminus were visualisedin the tumours using scintigraphic detection technique [46].

5. Selective delivery through nucleotide aptamers

High-affinity aptamers (short DNA or RNA oligonucleotideligands) as targeted therapeutics for the diagnosis and treatmentof cancers are also in development [47-49]. Combinatorialoligonucleotide libraries have been used for the selection ofaptamers that bind to a well-characterised and established

cancer marker selectively and with high affinity [50-53]. Theaptamers in conjugation with appropriate molecules may beuseful for imaging and treatment of cancer [54].

6. Selective delivery by prodrug activation

In order to diminish severe side effects of systemicchemotherapeutic treatment, several prodrug strategies havebeen evolved. Selective delivery of cancer therapeutics is doneby administering a less toxic prodrug form that can beconverted into an active drug at the cancer site. Most of theprodrugs are designed to have a ‘trigger’, ‘linker’ and ‘effector’.The ‘trigger’, following cancer-specific metabolism, modifiesthe ‘linker’, resulting in an activation of the ‘effector’. There areseveral mechanisms potentially exploitable for selective activa-tion. Some utilise unique aspects of tumour physiology (such asselective enzyme expression or hypoxia), whereas others arebased on tumour antigen-specific delivery techniques.

6.1 Targeting endogenous enzymes of cancer cellsIn this approach, systemically administered non-toxic prodrugsare transformed into active drugs at the cancer site as a result of

Table 2. A list of the approved monoclonal antibodies for cancer treatment. Some are already being used for selective delivery of therapeutics in cancer patients and others have a potential for their use as delivery vehicles in future.

mAb name(Approval year)

Target Treatment of Naked/conjugate Trade name Manufacturer

Gemtuzumab ozogamicin(2000)

CD33 Acute myelogenous leukaemia

Calicheamicin, cytotoxic antibiotic

Mylotarg Wyeth, Madison, NJ

Ibritumomab tiuxetan (2002)

CD20 Non-Hodgkin’s lymphoma

Radioisotopes In-111 or Y-90

Zevalin IDEC Pharmaceuticals Corp, San Diego, CA,USA

Iodine I 131 Tositumomab (2003)

CD20 Non-Hodgkin’s lymphoma

Radioisotope I-131 Bexxar® GlaxoSmithKline, Research Triangle Park, NC, USA

Rituximab (1997) CD20 Non-Hodgkin’s lymphoma

Naked* Rituxan® Genentech, Inc., South San Francisco, CA, USA

Trastuzumab (1998) HER-2 Breast cancer Naked* Herceptin® Genentech, Inc., South San Francisco, CA, USA

Alemtuzumab (2001)

CD52 Chronic lymphocytic leukaemia

Naked* Campath® Millennium and ILEX Partners, Cambridge, MA, USA

Cetuximab (2004) EGFR Colorectal cancer Naked* Erbitux™ ImClone Systems, Inc., Branchburg, NJ, USA

Bevacizumab (2004) VEGF Colorectal cancer Naked* Avastin™ Genentech, Inc., South San Francisco, CA, USA

*Antibodies that are approved for cancer treatment and have potential to be used in the future as vehicles for selective delivery of therapeutics in cancer diagnosis and treatment.EGFR: Epidermal growth factor receptor; VEGF: Vascular endothelial growth factor.

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cancer cell-specific metabolism. Consistently overexpressedendogenous enzymes of tumour cells have been used for theactivation of prodrugs in experimental studies [55]. Clinicaltrials with alkylating indoloquinone EO9 in 38 patients withadvanced non-small cell lung cancer [56], 22 patients withbreast cancer, 26 patients with colon cancer, 20 patients withgastric cancer and 24 patients with pancreatic cancer [57] havebeen reported. However, the results of these clinical studieshave been disappointing and largely attributed to varying levelsof endogenous enzyme activities in tumours [58].

6.2 Targeting secreted enzymes from cancer cellsAnother approach uses prodrugs that are excluded from thecells until cleaved by an enzyme produced and secretedpeferentially by the tumour cells. The peptide–doxorubicinprodrug, L-377202, activated by serine proteaseprostate-specific antigen (PSA) has been shown to selectivelykill prostate tumour cells positive for PSA in an animalmodel [59]. The L-377202 prodrug was ∼ 15 times moreeffective than was doxorubicin at maximally tolerated doses ininhibiting the growth of human prostate cancer tumours innude mice. In a Phase I clinical study in 9 patients withadvanced hormone-resistant prostate cancer, the doxorubicinconjugate of this heptapeptide was well tolerated and found todecrease PSA levels [60].

6.3 Targeting tumour hypoxiaHypoxia is an inherent and potentially exploitable feature ofmost solid tumour types [61]. Hypoxia may occur due toimperfect neovascularisation and compression or obstruction of

blood vessels due to high interstiatial pressures. These featuresare commonly observed during rapid growth of tumours [62].Advances in the chemistry of bioreductive drug activation haveled to the design of hypoxia-selective drug delivery systems.These prodrugs initially undergo one-electron reduction byreductases to give the radical anion, which in normal cells arereoxidised to the parent compound, but in hypoxic tumourcells they are further reduced to more hydrophilic species andtrapped inside. These drugs can be selectively delivered totumours with defined hypoxic fractions rich in the requiredactivating enzymes. Some of the bioreductive prodrugs havenow reached advanced stages of development, including clinicalevaluation; for example, tirapazamine [63-66], AQ4N [67-69] andporfiromycin [70]. The results of these clinical studies were veryencouraging, particularly in cases where bioreductive prodrugswere used in conjunction with radiation and conventionalchemotherapeutic agents. Certain metal-based hypoxic-selec-tive agents for therapy and imaging are under development atpresent [71]. Recently, attempts have been made to targettumour hypoxia for imaging purposes [72], which may help indeveloping tumour profile and treatment decision.

Some examples of the clinical and experimental studies thatutilised tumour enzymes for prodrug therapy are summarisedin Table 3.

6.4 Antibody-directed enzyme prodrug therapyAntibody-directed prodrug therapy (ADEPT) is a 2-stepapproach in which first the antibody–enzyme construct isadministered intravenously. This is composed of an antibodyagainst a tumour-specific target linked to an enzyme that

Table 3. Examples of exploiting tumour enzymes in prodrug cancer therapy in various experimental and clinical studies.

Enzymes Prodrugs Model systems

Endogenous enzymesDT-Diphorase Quinone E09

Tiazofurin

Phase II clinical trial in 92 patients with breast, gastric, pancreatic and colorectal cancer [56]; Multi-centre randomised trial in 38 patients with non-small cell lung cancer [57].Experimental studies [55].

Secreted enzymes

Serine protease PSA L-377202 (DOX–peptide conjugate)

Safety and pharmacokinetics studies in 9 patients with prostate cancer [60]; Experimental studies [59].

Hypoxia-targeted

Cy P450/Cy P450 reductase

NADPH: Cy P450 reductase (CYP3A)

Tirapazamine

AQ4NPorfiromycin

Metal-based prodrugs

Phase I study in 13 patients with lung cancer [65]; 16 patients with oropharyngeal tumours [66]; Phase I/II study in 72 patients with non-small cell lung cancer [63].Experimental studies and clinical trials [67-69].Phase I toxicity trial in 21 patients and a Phase III trial in 34 patients with head and neck squamous cell carcinoma [70].Experimental studies [71].

AQ4n: 1,4-Bis([2-(dimethylamino-N-oxide)ethyl]amino)5,8-dihydroxy- anthracene-9,10-dione), an alkylaminoanthraquinone N-oxide; L-377202: Peptide N-glutaryl-(4-hydroxyprolyl)AlaSer-cyclohexylglycyl-GlnSerLeu-CO2H linked to the aminoglycoside portion of doxorubicin; Porfiromycin: 6-Amino-1,1a,2,8,8a,8b-hexahydro-8-(hydroxymethyl)-8a-methoxy-1,5-dimethyl-azirino[2’,3’:3,4]pyrrolo[1,2-a]indole-4,7-dione, carbamate ester; PSA: Prostate-specific antigen; Quinone E09: Synthetic indoloquinone, structurally related to mytocin C; Tiazofurin: 2-β-D-Ribofuranosyl-4-thiazolecarboxamide; Tirapazamine: 3-Amino-1,2,4-benzotriazine-1,4-dioxide (SR-4233).

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activates a prodrug. In the second step, after the unboundantibody–enzyme conjugate construct is cleared from thecirculation, a prodrug is administered intravenously. Theprodrug is an anticancer drug that has been rendered lessactive by chemical addition of enzyme-cleavable moieties. Theprodrug is converted to an active form by the tumour-boundantibody–enzyme, which results in local accumulation of theactive form of the anticancer drug [73]. Due to the extracellu-lar site of enzyme action, most of the enzymes used inADEPT studies are hydrolysing enzymes that do not requirecofactors. A variety of enzymes, such as β-lactamase [74-80],cytosine deaminase [81], β-glucuronidase [82-85], carboxypepti-dase A1 [86] and alkaline phosphatase [87-89], and theircounterpart prodrugs have been used in many experimentalstudies. The choice of using non-mammalian enzymeswithout a human homologue helps to achieve high selectivityin ADEPT, but these enzymes raise an immunogenic responsethat can limit duration of the therapy. Table 4 presentsexamples of enzymes and their prodrug substrates used inADEPT clinical trials and experimental studies.

Early ADEPT clinical trials were conducted using theenzyme carboxypeptidase G2 linked to F(ab’)2 fragment ofmurine A5B7 mAb and the benzoic acid mustard prodrugCMDA [90-93]. The infusion of galactosylated second clearingantibody against carboxypeptidase G2 enzyme was done tolower the conjugate levels in the circulation and othernon-tumour tissues before prodrug administration in theseclinical trials. The results were mixed, ranging from no responseto reduction or disappearances of colon tumour deposit andliver metastases. In another Phase I clinical trial, ADEPT wasgiven to 27 patients using A5CP antibody–carboxypeptidase G2conjugate and carbamate prodrug bis-iodo phenol mustard

(ZD2767P) without the treatment with galactosylatedclearing antibody [94]. There were no clinical or radiologicalresponses seen in this study, but 3 patients had stable diseaseat day 56. In the ADEPT clinical trials mentioned above, allthe patients developed antibodies to mouse IgG and enzyme,and cyclosporin treatment was given to suppress the immuneresponse for multiple rounds of therapy. ADEPT faces somevery important challenges related to the large size oftumour-targeting antibody, which include a slow conjugateclearance from blood and normal tissues, limited delivery inpoorly vascularised tumours, and immunogenicity [93,95,96].Studies have shown that certain modifications in fusionproteins could be helpful in reducing some of these problems.Recently, an accelerated clearance of a multifunctionalmannosylated fusion protein from circulation, via hepaticmannose receptors, has been reported in a preclinical study [97].The identification and silencing of epitopes recognised by thehuman immune system have also produced some success inreducing antibody response to mAb conjugates in ADEPT [98].Furthermore, animal studies with xenograft models have shownthat enzyme conjugates of smaller size, in which the ligand unitis a scFv or peptide, may overcome some of the limitationsrelated to antibody molecules [75,84,85,99-101].

6.5 Gene-directed enzyme prodrug therapyGene-directed enzyme prodrug therapy (GDEPT), alsoknown as suicide gene therapy, delivers and expresses the genecoding for an enzyme that is capable of activating a systemi-cally administered prodrug within cancer cells. This can resultin high concentrations of cytotoxic drug at cancer siteswithout harming the rest of the body [102]. In experimentalstudies, genes of several of the enzymes employed for ADEPT

Table 4. Examples of exogenous enzymes used in antibody-directed enzyme prodrug therapy in various experimental and clinical studies.

Enzymes Prodrugs Model systems

Non-mammalian enzymes with no human homologueCarboxypeptidase G2

β-LactamaseCytosine deaminase

Glutamated prodrug: benzoic acidmustard (CMDA)

Bis-iodo phenol mustard (ZD2767P)

Cephalosporin prodrugs5-Fluorocytosine

Clinical trials in 17 patients with colorectal cancer [90,91]; Ten patients with colorectal cancer [92]; Pharmacokinetic study [93]. These used clearing Ab to effectively remove mAb–enzyme before prodrug treatment.Clinical trial in 27 patients, without second clearing Ab [94].Experimental studies [74-80].Experimental studies [81].

Mammalian enzymes, including human enzymesβ-GlucuronidaseCarbxypeptidase A1Alkaline phosphatase

Glucuronide prodrugsα-Peptide prodrugsPhosphate prodrugs

Experimental studies [82-85].Experimental studies [86].Experimental studies [87-89].

α-Peptide prodrugs: For example, methotrexate alpha-peptides; Cephalosporin prodrugs: 7-(4-Carboxybutanamido)cephalosporin conjugates of chemotoxic drugs, such as paclitaxel, doxorubicin, mustard; CMDA: 4-[(2-Chloroethyl) (2-mesyloxyethyl)amino]-benzoyl-L-glutamic acid; Glucuronide prodrugs: For example, N-[4-(daunorubicin or doxorubicin-N-carbonyl(-oxymethyl) phenyl] O-beta-glucuronyl carbamate; Phosphate prodrugs: For example, 4’-Demethylepipodophyllotoxin 9-[4,6-O-(R)-ethylidene-(β)-D-glucopyranoside] with 4’-(dihydrogen phosphate) also called etoposide phosphate, p-[N,N-bis(2-chloroethyl)amino]phenyl phosphate, mitocin phosphate; ZD2767P: [4-[bis(2-iodoethyl)-amino]phenyl oxy carbonyl]-L-glutamic acid.

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were used for GDEPT [103]. This approach uses both biologi-cal and chemical vector systems available for gene therapy,such as replicable and non-replicable viruses, bacteria andcolloidal systems. When using a viral vector for gene deliveryand expression, GDEPT is sometimes referred to asvirus-directed enzyme prodrug therapy.

Several clinical trials using either adenoviral or retroviraltransduction of herpes simplex virus thymidine kinase(HSV-tk) and ganciclovir prodrug have been conducted incancer patients [104-110]. The GDEPT with HSV-tk has alsobeen reported for positron emission tomography (PET) inglioblastoma patients using prodrug 2’-fluoro-2’-deoxy-1β-D-arabino-furanosyl-5-[124I]iodo-uracil [111]. Several othergenes, such as cytochrome P4502B1 [112], nitroreductase [113]

and cytosine deaminase [114], have been employed for enzymeprodrug therapy trials in cancer patients. Genetically modi-fied, non-pathogenic bacteria [115-119] and allogenic cells [112]

have also been investigated as potential vectors in GDEPT.Different strategies have been adopted to direct these vectorsto cancer sites. These include direct intratumoural inocula-tion or ligand-mediated targeting to cancer-specific antigen.These ligands are chemically conjugated to vectors orexpressed on the coat protein of viral vectors. The use ofbifunctional bridging agents recognising both the virus andthe cancer cell antigen is also common [120]. Viruses aremodified to diminish the binding to their natural receptors onthe cells. The cancer cell-specific ligands, as in other tumour-selective systems, include mAbs, scFvs, peptides or nucleotideaptamers. Another variation of GDEPT that exploits knowntranscriptional differences between normal and tumour cells

to drive selective expression of a prodrug metabolising enzymeis called genetic prodrug activation therapy. It is similar totranscriptional targeting in gene therapy. In this approach, thetrans-response element of a known overexpressed cancer geneis placed upstream of the enzyme gene, resulting in a robustexpression of exogenous enzyme in cancer cells in comparisonto other cells. A clinical trial with cytosine deaminaseexpressed by erbB2-promoter followed by 5-fluorocytosineprodrug treatment has been conducted in breast cancerpatients [114]. Table 5 presents examples of GDEPT clinicaltrials using different enzymes, prodrugs and vectors.

GDEPT has the advantage of using those enzymes that alsorequire endogenous cofactors, but it faces most of thechallenges of gene therapy – right from vector delivery toenzyme expression. Many of the enzymes used in GDEPT actintracellularly by converting prodrugs to active drugs withincells [103]; however, an extracellular effector system comprisedof the secreted form of lysosomal β-glucuronidase andglucuronidated doxorubicin prodrug has also beenreported [121]. In another development, the use of non-invasive19F magnetic resonance spectroscopy and diffusion-weightedMRI as tools to assess gene function and GDEPT efficacy hasbeen demonstrated following administration of a construct ofbifunctional fusion gene between cytosine deaminase anduracil phosphoribosyltransferase [122].

7. External energy-controlled delivery

Some selective delivery strategies involve focusing externalenergy for concentrating or delivering therapeutics at the

Table 5. Examples of clinical studies on gene-directed enzyme prodrug therapy.

Gene Prodrug Vector Study details

HSV-tk

CYP2B1

124I-FIAU

Ifosfamide

Liposome

Allogenic cells

Positron-emission tomography studies with 5 glioblastoma patients in Phase I/II clinical trial [111].Phase I/II clinical trial in 14 pancreatic cancer patients given cells to tumour vessels [112].

Bacterial nitroreductaseHSV-tk

HSV-tkHSV-tkHSV-tk

HSV-tk

HSV-tk

HSV-tk

CB1954

GCV

GCV/VCVVCVGCV

GCV

GCV/VCV

GCV

RD-ADV

RD-ADV

RD-ADVRD-ADVRD-REV

RD-ADV

RD-ADV

RC-REV

Clinical trial in which 8 patients with primary or secondary (colorectal) liver cancer received direct intratumoural inoculation [113].Phase I clinical trial with 18 prostate cancer patients receiving injection directly into the prostate [105].Multi-centre clinical trial in 85 prostate cancer patients [106].Phase I/II clinical trial with 30 prostate cancer patients [107].Phase III multi-centre clinical trial in which 248 glioblastoma multiforme patients received intracerebral injection [110].Clinical trial in 21 malignant pleural mesothelioma receivingpleural injection [108].Clinical trial in 52 prostate cancer patients receiving injection into the prostate [109].Clinical trial in which 48 glioblastoma multiforme patients received intracerebral injection of suspension of retroviral vector-producing cells [104].

Cytosine deaminase

5-Fluorocytosine Directed by erbB2 promoter

Phase I clinical trial using direct intratumoural injection of plasmid construct in 12 breast cancer patients [114].

124I-FIAU: 2’-Fluoro-2’-deoxy-1β-D-arabino-furanosyl-5-[124I]iodo-uracil; Allogenic cells: CYP2B1 enzyme-expressing genetically modified cells; CB1954: 5-(Aziridin-1-yl)-2,4-dinitrobenzamide; CYP2B1: Cytochrome P4502B1; GCV: Ganciclovir prodrug; HSV-tk: Herpes simplex virus thymidine kinase gene; RD-ADV: Replication-deficient adenovirus; RC-REV: Replication-competent retrovirus; RD-REV: Replication-deficient retrovirus; VCV: Valacyclovir prodrug.

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cancer site. A variety of delivery systems in this category are inthe experimental stage, although some have been used inclinical trials as well.

7.1 Selective delivery through photodynamic therapyPhotodynamic therapy or photochemotherapy destroyscancer cells through the use of a wavelength-specific laser lightin combination with a photosensitising agent. Depending onthe part of the body being treated, the drug is either injectedinto the bloodstream or applied to the skin. The fibre-optic isplaced close to the cancer to deliver the proper amount oflight. After light exposure, the photosensitising agentsproduce radical oxygen species that kill cancer cells. Attemptshave been made to increase the uptake of photosensitisingdrugs by the cancer cells and precise delivery of light to cancersite [123-126]. The fibre-optic can be directed through abronchoscope into the lungs for the treatment of lung cancer,or through an endoscope into the oesophagus for thetreatment of oesophageal cancer [127].

Another modality that uses near-infrared light forlocalised therapeutic thermal ablation and imaging oftumour cells following treatment with gold-coatednanoshells has also been reported [128,129].

7.2 Magnetically targeted deliveryMagnetic-targeted carriers bound to chemotherapeutic orradiological agents in blood circulation can be selectivelyconcentrated at a tumour site using an external constantmagnetic field [130]. Furthermore, thermal activation of aninactive prodrug entrapped in magnetoliposomes using ahigh-frequency magnetic field has been proposed [131].Site-directed thermal ablation of tumour triggered by externalmagnetic field has also been reported [132-135]. The first clini-cal trial using magnetic microspheres loaded with cytotoxicagent was performed in Germany in 14 patients with solidtumours [136]. In a clinical trial with 4 hepatocellularcarcinoma patients, an external magnet was used successfullyto restrict the doxorubicin bound to metallic iron-activatedparticles at the cancer site following MRI-guided transcatheterdelivery to hepatic artery [137].

7.3 Selective delivery through X-ray exposureIn experimental studies with animal models, another class oftargeting methods uses external energy as a trigger forlocalised activation of cytotoxicity. X-rays were used toactivate therapeutic genes driven by a radiation-induciblepromoter of the Egr-1 gene [138]. The Egr–TNF construct wasdelivered through a replication-deficient adenovirus to humantumours growing in nude mice. Exposure of tumours toX-rays resulted in increased TNF-α production and tumourgrowth control without normal tissue damage.

7.4 Radiation-induced selective deliveryRadiation has been used to selectively deliver the drugs tospecific sites such as neoplasms or aberrant blood vessels. It is

based on the fact that blood vessels following exposure toionising radiation express a number of cell adhesion moleculesand receptors, such as intercellular adhesion molecule(ICAM)-1, E-selectin, P-selectin and the β3 integrin, whichcan be used as a target for specific delivery of therapeutics.The experimental studies with animal models havedemonstrated that the ligand-conjugated therapeutics againstthese radiation-induced targets can selectively deliver drugsand imaging agents to tumours following focusedradiation [139,140]. Phase I pharmacokinetic studies onradiation-controlled drug delivery systems have shown thatthe schedule of administration of the therapeutic conjugate inrelation to radiation exposure is crucial [141].

7.5 Ultrasound-guided deliveryThe technique is based on encapsulation of therapeutic agentsin polymeric micelles, passive trapping of the micelles in atumour, and local release of the drug at the target site. The gasesentrapped within lipid coatings make microbubbles sufficientlystable for circulation, and at the same time they can be cavitatedwith focused ultrasound energy for site-specific local delivery ofbioactive materials. It permits using lower concentrations ofdrugs systemically and concentrates the drug at the ultrasound-irradiated site. This technique can be used for both diagnosticand therapeutic applications [142]. There is also a possibility ofattaching microbubbles to site-specific ligands for moleculartargeting. The lipid-coated perfluorocarbon gas-based micro-bubble products are currently in market for diagnostic ultra-sound imaging. Studies in animal tumour models havedemonstrated the selective delivery of chemotherapeutics, genesand imaging agents from acoustically active micelles at thetumour site following exposure to focused ultrasound [143,144].

8. Organ-selective delivery

Attempts have also been made for organ-specific delivery ofcancer therapeutics. A methotrexate–bisphosphonate conju-gate containing a peptide bond has been found to possess > 5times more antineoplastic activity against osteosarcoma inexperimental animal models than methotrexate alone [145].This approach is based on the high affinity of P-C-P portionof the bisphosphonate to hydroxyapatite, which is a majorbone constituent. These developments which are being testedfor bone-selective delivery of a variety of agents [146] areimportant because drug delivery to bone is limited by itsunique anatomical features. Organ-specific pharmaceuticalapproaches have also been used for the colon and liver [147,148].

In addition to tumour targeting, developing successfultechnology for a selective delivery requires an appropriateformulation that helps to avoid sequestration of the targetingmolecules by the reticuloendothelium system, to reduce theclearance of small drugs, to provide protection of active agentsfrom degradation, to overcome the biological barriers thatprevent it reaching the target, and to tackle the augmentedosmotic pressure state in cancer lesions.

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9. Expert opinion and conclusion

Despite a number of advances in the past few decades,systemic cancer therapy is hampered by problems of severeunwanted side effects and the development of drug resistance.Research activity aimed towards achieving specific andtargeted delivery of cancer therapeutics has expanded tremen-dously in the last decade with new methods of directing drugsto tumours, as well as drugs with new mechanisms of action.At this moment, it is not clear whether some of the cancertargeting therapeutic strategies presented in this review, inspite of showing encouraging results in animal models, will besuccessful in the clinic. The success in this field will rely onthe vector system and delivery strategy, exploration of newtargets and their high-affinity ligands, better understanding ofthe biology of cancer, and the invention of more clinicallyrelevant models for human cancer research. Ultimately, muchof what we will learn about tumour-selective delivery in thenext few years will depend on how rapidly clinical-gradereagents will become available, and on the determination ofboth industrial and academic groups to perform translationalclinical research on these strategies.

For the success of ligand-mediated selective deliverystrategies, it is essential to develop high-affinity ligands againstvalidated cancer targets. The discovery and validation oftumour-associated markers as well as corresponding ligandsremain an important challenge for the development oftechnology for the biomarker-targeted delivery of therapeuticagents for the treatment and imaging of solidtumours [3,4,149,150]. Although mAbs offer excellent specificityand affinity, and their use in the haematological malignancieshas shown great promise, poor barrier penetration is animportant issue in solid tumour treatment. It has beenrecognised that only 0.001 – 0.01% of intravenously adminis-tered mAbs reach their parenchymal targets in vivo [151].Targeting cancer-specific vascular targets for selective deliveryof mAb-conjugated therapeutics to tumour sites shouldminimise the problems associated with physiological barriersto the delivery of macromolecules to parenchymal targets [41].This strategy could be especially useful in ADEPT because ofa bystander effect on cancer cells owing to enzymatic genera-tion of small diffusible active drug molecules at tumour site.Advances in genetic engineering of antibodies have generatedbispecific antibodies that have one targeting arm to recognisetumour antigens and another to bind effectors. Pretargetingwith such antibodies allows time for tumour-target bindingand clearance from circulation, before toxic effectors areadministered [21]. The technology also led to the developmentof catalytic antibodies or abzymes that may find use inADEPT [152,153]. Clinical trials with targeted delivery of drugshave shown promise with prodrug therapy. Two amplifica-tions steps are obtained with prodrug therapy. The first isthat the activating ligand–enzyme conjugate is present athigher concentration in the tumour target. The secondamplification step is the step up achieved by enzyme catalysis

of one mAb–enzyme conjugate molecule activating manyprodrug molecules to an active cytotoxic state.

In the near future, antibody fragments and peptide ligandsgenerated by molecular methods may lead to improvementsover antibodies generated in an animal. Using phage displaytechnology, rapid identification of such ligands againstcancer-specific targets should facilitate the development oftumour-specific reagents and may even lead to individualisedtherapy. The economical bulk production of such proteins inbacterial hosts is an added advantage over the difficulties ofproducing large quantities of monoclonal antibodies requiredfor clinical use. Recently, we screened a phage-displayedβ-lactamase scaffold library in which an outer loop of theenzyme was randomised, and selected a number ofhigh-affinity cancer-binding clones (under preparation).These targeted enzymes, which bring together in onemolecule a tumour-binding module and an effector module,are much smaller than an antibody–enzyme construct. Severalphage-derived products are in the clinical pipeline and somehave been approved by the US Food and Drug Administrationfor human use [3,154].

Nucleotide aptamers are also very promising candidates astumour-targeting ligands because of their high affinity andease in selection against numerous targets. The new genera-tion of aptamers or ‘spiegelmers’ are nuclease-resistant, whichmakes them more stable under biological conditions [155,156].Small molecular weight molecules are an attractive alternativeto peptides and nucleotides as tumour-specific deliveryvehicles. They have good penetrative characteristics, can bereadily conjugated to effectors and can have a high affinity forantigens expressed on tumour cell surface. Such small organiccompounds are expected to be non-immunogenic and shoulddisplay an improved tumour penetration compared withantibodies. Folic acid is an example of a small moleculeligand that binds with high-affinity to membrane-boundfolate receptors known to be overexpressed on a variety ofhuman cancers. Folic acid has been reported to selectivelydeliver a wide array of imaging and toxic agents to suchcancers [157]. The developments in the generation of syntheticcombinatorial libraries of a class of organic chemicals,commonly referred to as ‘small molecules’, and theirhigh-throughput screening have paved the way for develop-ing this new class of cancer target-specific ligands for selectivedelivery in cancer imaging and treatment. It is expected toprovide high affinity ligands for antigens with cavities (e.g.,enzymes), but isolation of high-affinity ligands for flatproteins could be a challenge. Improvements in the technolo-gies for isolating high-affinity and high-specificity smallmolecules will allow cancer-targeting experiments to becarried out with classes of small molecules having differentpharmacokinetic properties [158-160].

Although several GDEPT clinical trials have shown someencouraging results, the strategies that require a gene transferstep in the selective delivery of therapeutics have to deal withall the challenges that gene therapy faces today. These include

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poor gene transfer, degradation and immune attack of vectors,control and regulation of gene expression, environmentalsafety and, above all, patient toxicity [161]. According to TheJournal of Gene Medicine website ‘Gene Therapy ClinicalTrials Worldwide’ [201], 7.6% of the trials are in the field ofsuicide gene therapy. Vehicles such as retroviruses, adeno-viruses, and non-viral systems adapted for clinical trialsemploying different strategies of selective delivery haveproduced mixed results. The future of such strategies will behighly dependent on the development of safe and effectivevectors for gene transfer.

The use of external energy-guided delivery technologiesdepends on the exposure modality and the location oftumours; therefore, their application is limited only tospecific situations. For example, photodynamic therapy issuitable only for tumours that can be exposed to focusedlight. However, for obstruction of the gut or airway byadvanced tumours, such directed treatment, albeit limitedand non-systemic, may provide substantial benefit. In thenear future, the external energy-guided delivery systems areexpected to be more often coupled with other molecularlytargeted delivery systems, such as those employinghigh-affinity ligands.

The bioavailability of the selectively delivered agents at thedesired site depends on several characteristics, includingmolecular size; hydrophilicity; charge; stability; rate of elimi-nation through degradation, secretion and nonspecificbinding; and tissue specific factors such as interstitial pressuresand vascular permeability. Advances in the field of nanotech-nology have helped create a new generation of nanovectors.These not only avoid biobarriers, but also allow placement ofdesired targeting moieties on the surface of the particles, aswell as loading the particles with different imaging and/ortherapeutic agents [7]. Such nanovectors may significantlyincrease the number of therapeutic molecules that can be

delivered per tumour target molecule and can allow sustainedrelease of the therapy over time. Recently, the development ofa delivery vehicle ‘nanocell’ for the sequential delivery of twodrugs makes it possible to target distinct tumour compart-ments or multiple signalling pathways [9]. By systematicallycombining preferred therapeutic agents and cancer-targetingmoieties, nanotechnology platform may help to generatenovel, personalised agents for selective delivery in the diagnosisand treatment of cancer.

The success of biomarker-targeted delivery technology willdepend on the identification of suitable markers of neoplasticdisease and understanding target evolution over time. Deploy-ment of these markers in advanced drug delivery systems willfacilitate diagnostic screening and effective treatment ofcancer. Advanced imaging modalities in combination withcancer target-specific probes provide a powerful non-invasivediagnostic tool. Molecular diagnostic imaging is an emergingtechnology that may distinguish patient populations thatwould respond to targeted therapy from non-responders. Theconvergence of genomic technologies and the development ofdrugs designed against specific molecular targets are expectedto provide many opportunities for cancer diagnosis andtreatment in near future. The development of suitabletargeted delivery systems for these new generations of drugsand their successful translation into clinical practice is one ofthe most promising but significant challenges in the field ofoncology today.

Acknowledgements

The work was supported by The National Cancer Institute(R01 CA080790 & R01 CA112091) and US Army MedicalResearch (W81XWH0510237), and in part by the VermontCancer Center core grant (PHS CA22435) and SD IrelandCancer Research fund.

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161. EDELSTEIN ML, ABEDI MR, WIXON J, EDELSTEIN RM: Gene therapy clinical trials worldwide 1989-2004-an overview. J. Gene Med. (2004) 6(6):597-602.

Website201. http://www.wiley.co.uk/wileychi/genmed/

clinical/Gene Therapy Clinical Trials Worldwide, website provided by The Journal of Gene Medicine, updated July 2005.

AffiliationGirja S Shukla†1 PhD & David N Krag1 MD†Author for correspondence1Vermont Comprehensive Cancer Center, Department of Surgery, University of Vermont College of Medicine, 89 Beaumont Avenue, Burlington, VT 05405, USATel: +1 802 656 9488; Fax: +1 802 656 5833;E-mail: [email protected]

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selective delivery of therapeutic agents for the diagnosis and treatment of cancer

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