Inorganica Chimica Acta 393 (2012) 64–74

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Inorganica Chimica Acta

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Review

Metallo-drugs in the treatment of malignant pleural mesothelioma

Ilaria Zanellato a, Ilaria Bonarrigo a, Elisabetta Gabano a, Mauro Ravera a, Nicola Margiotta b,Pier-Giacomo Betta c, Domenico Osella a,⇑a Dipartimento di Scienze e Innovazione Tecnologica, Sezione Ambiente-Vita, Università del Piemonte Orientale ‘‘Amedeo Avogadro’’, Viale T. Michel 11, 15121 Alessandria, Italyb Dipartimento Farmaco-Chimico, Università di Bari ‘‘Aldo Moro’’, Via E. Orabona 4, 70125 Bari, Italyc Lega Italiana per la Lotta ai Tumori (LILT), Sezione di Alessandria, Italy

a r t i c l e i n f o a b s t r a c t

Article history:Available online 15 June 2012

Metals in Medicine Special Issue

Keywords:Malignant mesotheliomaChemotherapyPlatinum drugsMetal drugsChemoresistance

Ilaria Zanellato receivedICRM-CNR in Milan for aOrientale (Alessandria, Itasupervision of Dr. Anne Vedrug discovery and medic

Ilaria Bonarrigo receivedmoved to Università del Pthe same University undemedicinal chemistry, in p

0020-1693/$ - see front matter � 2012 Elsevier B.V. Ahttp://dx.doi.org/10.1016/j.ica.2012.06.005

⇑ Corresponding author. Tel.: +39 0131 360266; faxE-mail address: domenico.osella@mfn.unipmn.it (D

Mesothelioma is a rare form of cancer that affects the membranous lining of the chest (pleura) and, lesscommonly, the lining of the abdomen and heart. The disease carries a poor prognosis and is typicallyassociated with exposure to asbestos, a mineral that has been widely used due to its fire resistanceand insulating properties. The standard non-surgical treatment for malignant pleural mesothelioma(MPM) is polychemotherapy combining cisplatin/carboplatin and a second agent with a different mech-anism of action. This review outlines the use of clinically approved platinum-drugs and the design ofmetal-drug candidates for the treatment of MPM.

� 2012 Elsevier B.V. All rights reserved.

her Laurea degree in Industrial Biotechnologies cum laude in 2005 from the University of Torino (Italy). Then she moved tofellowship concerning enzyme-based catalysis. In 2010 she earned her Ph.D in Chemistry at the University of Piemontely) working on metal-based chemotherapy of mesothelioma, and spending a research period in Paris at ENSCP (under thessières). She is completing her post-doc fellowship with Professor Domenico Osella. Her research interests are in the area ofinal chemistry, in particular on metal-based chemotherapeutics, cell-based assays, and pharmacology.

her Laurea degree in Pharmaceutical Chemistry and Technology in 2009 from the University of Pavia (Italy). In 2010 sheiemonte Orientale (Alessandria, Italy) for a CIRCMSB fellowship. She is currently enrolled as a Ph.D. student in Chemistry inr the guidance of Professor Domenico Osella. Her research interests are in the area of pharmacology, drug discovery andarticular on metal-based chemotherapeutics.

ll rights reserved.

: +39 0131 360250.. Osella).

Elisabetta Gabano received her Laurea degree in Chemistry cum laude in 2002 from the University of Piemonte Orientale (Alessandria, Italy). In 2005she got her PhD degree in Chemistry from the same University, where from 2005 to 2010 she was a postdoctoral fellow. In December 2010 she joinedthe Faculty of Science (now Department of Science and Technology, DiSIT) at the University of Piemonte Orientale, where she is Assistant Professor ofGeneral and Inorganic Chemistry. Her research interests focus on the synthesis and characterisation of coordination compounds as anti-cancer drugs.

Mauro Ravera received his Laurea in Chemistry cum laude from the University of Torino (Italy) in 1990. From 1991 to 1993 he was a postgraduatefellow in the Osella’s laboratory in Torino, also spending some research period at the University of Lausanne (under the supervision of the lateProfessor Carlo Floriani) and at the Australasian Microscale Chemistry Center (AMC2, Deakin University, Geelong, Australia). In 1993 he got a positionas Assistant Professor at the University of Piemonte Orientale, then he became Associate Professor of General and Inorganic Chemistry (2006) at theabove said University. His current research interests include the use of coordination compounds in medicine and biology, particularly as new anti-cancer drugs, and electrochemical studies of inorganic and bioinorganic compounds.

Nicola Margiotta joined Prof. Peter J. Sadler’s group at the Chemistry Department of the University of London in 1996 after receiving his degree inPharmaceutical Chemistry and Technology from the University of Bari (Italy). In 2000 he obtained his Ph.D. in Pharmaceutical Chemistry from theUniversity of Bari (Italy) where he is currently Assistant Professor of Chemistry. His main research interests are in the field of Medicinal InorganicChemistry and Organometallic Chemistry.

Pier-Giacomo Betta was born in 1949 in Alessandria (Italy) and obtained his Laurea in Medicine cum laude at the University of Torino in 1975,awarded with the Lepetit Doctoral Prize. He obtained specialization diplomas in Laboratory Medicine (1977), Anatomic Pathology (1979), ClinicalOncology (1981), and Experimental Pathology (1986). He was director of the Pathology Unit at the Alessandria National Hospital (1997–2011). Hecoordinated the National Environmental Carcinogenesis Commission of the Italian League against Cancer (LILT, 2002-2005). He has always beeninterested in asbestos-related pathology with special reference to malignant mesothelioma. He is currently a member of the American Society ofClinical Oncology (ASCO) and the New York Academy of Sciences.

Domenico Osella obtained his Laurea in chemistry summa cum laude (1974) and his abilitazione (1976) in industrial chemistry both at TurinUniversity. After a research period at the University Chemical Laboratory in Cambridge (RCS fellowship, supervisor Lord J. Lewis), he worked asresearch assistant professor and, then, as associate professor at Turin University. He moved to University of Basilicata (Potenza, Italy) as full professorin chemistry (1994) and, then, to the University ‘‘A. Avogadro’’ (Alessandria, Italy) (1996) serving as director of the Department of Environmental andLife Sciences (DiSAV), now Department of Science and Technology (DiSIT). His current research interests encompass the use of coordination com-pounds in the fields of medicine and biology, particularly the Pt-based anti-cancer drugs, as well as electrochemical and NMR studies of bioinorganiccompounds in the fields of food, environment and health. Osella has published about 220 papers in international (peer-reviewed) journals.

I. Zanellato et al. / Inorganica Chimica Acta 393 (2012) 64–74 65

66 I. Zanellato et al. / Inorganica Chimica Acta 393 (2012) 64–74

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 662. Polychemotherapy regimens and schedules with clinically used drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 673. Second and third generation Pt complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 684. Non-classical platinum complexes: cationic and multinuclear platinum complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 705. Pt(IV) complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 706. Drug targeting and delivery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 707. Bypassing the antioxidant armory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 718. Non Platinum drug candidates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 729. Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

1. Introduction

Malignant mesothelioma is a deadly tumor that affects the mes-othelial lining of the pleura and, less frequently, of the peritonealand pericardial cavities. Malignant pleural mesothelioma (MPM)is still a relatively rare form of cancer, but its incidence is increas-ing worldwide due to the extensive use of asbestos in insulationand construction materials over past decades [1]. The disease bur-den is still predominantly borne by the developed world, whereMPM rates are expected to decline after 2015–2020 following leg-islation aimed at reducing asbestos exposure in the workplace andin the environment. In contrast, since asbestos utilization has re-cently increased in developing countries, where safety regulationof asbestos mining and processing is poor, a corresponding shiftin MPM occurrence is expected.

MPM has a significantly long latency period of up to 40 years ormore. Prognosis is poor, and the median survival is around 1 yearfrom diagnosis. MPM is a difficult disease to deal with and sys-temic therapy is the only potential treatment option for the major-ity of patients, although rates of response to chemotherapy arelimited [2]. Advanced age, comorbidities, poor performance statusand locally advanced disease preclude aggressive surgery, i.e.,extrapleural pneumonectomy, in most patients. Chemotherapeuticregimens have included the alkylating agent cisplatin, 1 (Fig. 1)used alone or in combination with other antitumor drugs with dif-ferent mechanisms of action.

The current gold-standard frontline chemotherapy approved bythe Food and Drug Administration (FDA) for MPM consists of 1combined with the antifolate pemetrexed [3]. This chemotherapyregimen is given with vitamin supplementation (vitamin B12 andfolic acid) and improves the median overall survival for patientswith MPM (12.1 versus 9.3 months with 1 alone) [3]. If necessary,e.g., in elderly patients with comorbidities, carboplatin, 2, is em-ployed in lieu of 1, because of a lesser burden of toxicities.

Retrospective analysis of the ‘‘old’’ combination of 1 and theantimetabolite gemcitabine indicates comparable response andsurvival rates [4], so gemcitabine can still be useful for patientswho cannot receive pemetrexed treatment.

In spite of the increasing world incidence of MPM, the WorldHealth Organization (WHO) mortality database [1] reports only afew thousand new cases of the disease each year. MPM thus contin-ues to be considered a rare disease with a fairly small market size.This means that research into developing treatment options is notparticularly appealing to pharmaceutical companies, whose drugdevelopment programs are often dictated by market potential andopportunities for long-term profits to offset the time-consumingand costly clinical evaluation of new drugs and innovative thera-pies. Nonetheless, new drugs and tailored treatments are highlywarranted to improve the dire outcome of MPM patients. In suchdifficult circumstances, drugs developed for other neoplasms, such

as lung cancer, or even for other pathologies, are also being tenta-tively tested for the treatment of MPM. However, such attemptsat drug repositioning or repurposing often fail to take into accountthe biology of MPM. The peculiarity of MPM is that it arises from themesothelium, which represents undifferentiated pluripotent meso-derm persisting during the postembryonic phase with its multipo-tential properties of differentiation, thus accounting for theoutstanding microscopic phenotypic versatility of this tumor [5].Indeed, the histological appearance of MPM may range from theepithelioid (mostly composed of epithelial-shaped cells, 50–75%)to the sarcomatoid (mostly composed of spindle-shaped cells, 15–20%) pattern, with any combination of both (mixed or biphasicmesothelioma, 20–30%) [6]. Moreover, owing to their very patho-genesis (as described below), MPM cells are characterized by intrin-sic chemoresistance [7].

Complex interactions between asbestos fibers and mesothelialcells occur in vivo [8]. The most important chemico-physical prop-erties of asbestos fibers related to pathogenicity are surface chem-istry, surface area, fiber dimensions, and bio-persistence. Asbestosfibers may directly induce genotoxicity by catalyzing the genera-tion of reactive oxygen species (ROS) resulting in oxidized DNAbases and DNA strand breaks that can produce gene mutations, ifnot adequately repaired. Asbestos fibers can also physically inter-fere with the mitotic apparatus, which may result in aneuploidyor polyploidy and specific chromosomal alterations. Moreover,persistent inflammation and macrophage activation in tissue areascontaining asbestos deposits can secondarily generate additionalROS and reactive nitrogen species that can induce activation ofintracellular signaling pathways, stimulation of cell proliferationand survival, and epigenetic alterations [9]. Thus, the pathogenesisof mesothelioma is linked to the selection of cells characterized byelevated DNA repair activity [10],expression of several antioxidantenzymes [11] and strong activation of pro-survival/antiapoptoticpathways (such as NF-kB, mTOR and Bcl–XL) [12]. Each of thesemolecular pathways, originally activated to protect against danger-ous xenobiotics such as asbestos itself, can actually hinder the ac-tion of chemotherapeutics.

In Italy, one of the highest incidence of death from pleuralmesothelioma [13] is observed in Casale Monferrato in the Prov-ince of Alessandria. This is the sad legacy of Eternit, which wasthe oldest and largest asbestos cement plant in the country. Asour University (Amedeo Avogadro) is located nearby in the sameprovince, we were able to perform several in vitro pharmacologicaltests exploiting new combinations or new schedules of establishedantitumor drugs and newly synthesized Pt drug candidates.

The cell lines tested, derived from pleural effusions of untreatedMPM patients obtained from the Mesothelioma Bio-Bank (NationalHospital, Alessandria), were BR95 (epithelioid), MG06 (mixed-predominantly epithelioid), MM98 (sarcomatoid) and MM98R, acisplatin-resistant cell line derived, in our laboratory, from wild

Fig. 1. Sketch of the compounds described in the text.

I. Zanellato et al. / Inorganica Chimica Acta 393 (2012) 64–74 67

type MM98 by exposure to sub-lethal concentrations of 1 for sev-eral months [14]. In some of the tests, we also employed humanmesothelial cells (HMC) isolated from patients with no history ofmalignant disease [15] as a non-tumor control.

2. Polychemotherapy regimens and schedules with clinicallyused drugs

Cisplatin is known to exert its cytotoxic activity mainly throughthe formation of intra-strand DNA–platinum adducts [16]. Gemcit-abine, a cytidine analog, acts by blocking nucleic acid synthesis aswell as several enzymes in the nucleotide biosynthesis pathway[17,18], most notably ribonucleotide reductase. This leads to areduction in deoxynucleoside triphosphate (dNTP) pools, whichcontributes to inhibition of DNA synthesis and repair [19].Cisplatin/gemcitabine combinations have been extensively evalu-ated both in vitro and in vivo in several tumor types, includinghead/neck and ovarian carcinomas and non-small cell lung cancers

(NSCLC), showing antagonistic, additive or synergistic effects,depending on the tumor type and treatment schedule [20,21].We tried to assess the relationship between treatment scheduleand anti-mesothelioma activity, taking into account the peculiarhistology of the tumor. We found that pre-treatment with gemcit-abine resulted in a synergistic effect in all MPM cell lines, inducinga strong S-phase arrest that correlated with accumulation of dou-ble strand breaks [14], the most lethal DNA lesions [22,23]. Whencells are exposed to gemcitabine before 1, dNTP-depleted cells areable to remove intra- and inter-strand Pt-adducts, but cannot fillthe gaps created by the DNA repair machinery, leading to singlestrand and double strand break accumulation.

Histone deacetylase (HDAC) inhibitors have been suggested as apromising potential component of polychemotherapeutic protocol,as it has been shown to enhance the antitumor effects of bothDNA-damaging drugs, such as 1 [24], and antimetabolites, suchas gemcitabine [25]. More specifically, valproic acid (VPA, a wellknown anti-epileptic drug) has HDAC inhibiting activity [26]; itexhibits synergistic cytotoxicity with 1 in ovarian carcinoma cells,

Fig. 1 (continued)

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and can also resensitize cells that have acquired resistance against1 [27]. VPA has been found to increase the efficacy of the cisplatin/pemetrexed combination on mesothelioma cell lines and in tumortissues from patients’ biopsies [28]. A recent phase II study showedthat VPA plus doxorubicin may be a promising chemotherapy reg-imen in patients with refractory or recurrent MM, for which nostandard therapy is available [29].

As a possible second-line therapy on cisplatin-resistant cells, wedetermined the pharmacological interaction of VPA in conjunctionwith either 1 or gemcitabine using the combination index and thedose reduction index [30,31]. Briefly, the combination index (CI) isa numerical value, calculated as described below (see Table 1 foot-note), providing a quantitative measure of the extent of drug com-bination. This is a parameter that indicates whether the combinedeffect of two drugs is synergistic (CI < 1), additive (CI = 1) or antag-onistic (CI > 1). Rearrangement of the CI equation provides thedose-reduction index (DRI), a parameter that determines the mag-nitude of dose reduction allowed for each drug when given in syn-ergistic combination, as compared with the concentration of asingle agent needed for achieving the same effect. A favorableDRI (>1) allows dose-reduction that leads to toxicity reduction inthe therapeutic applications (Table 1).

VPA was synergistic with gemcitabine in the wild cell linesBR95 and MM98, but additive only in the cisplatin-resistant sub-line MM98R.

The association between 1 and VPA showed synergism in BR95and in MM98R, but was additive in MM98, giving DRI values rang-ing from 2.4 in the sarcomatoid cell lines to 9.6 in the epithelioidone.

When gemcitabine treatment precedes VPA challenge, a modestenhancement of antitumor activity is observed. When gemcitabinetreatment occurs before the challenge of the combination 1/VPA,the overall effect is additive on MM98 and synergistic on BR95.This schedule was characterized by more than a tenfold dosereduction for 1; this might mitigate the side effects of this alkylat-ing drug.

The cytotoxicity of some bifunctional Pt(II) derivatives thathave two VPA ligands as leaving groups has recently been studiedon different cell lines by Marmion and co-workers [32]. The in-crease in activity with respect to the parent Pt(II) complex wasrather modest. In this context, it is important to notice that VPAacts in a mM range (Table 2), while the Pt(II) antitumor derivativesare employed in lM range.

3. Second and third generation Pt complexes

A limited number of papers dealing with the use of Pt com-plexes other than 1 in the treatment of MPM have appeared inthe literature.

Table 1Combination index (CI) and dose reduction index (DRI).a

CI BR95 (DRI) CI MM98 (DRI) CI MM98R (DRI)

VPA:IC50 (±SD) 1.5 ± 0.2 mM 3.9 ± 0.9 mM 3.5 ± 0.7 mMVPA + GEM 0.4 ± 0.1 0.4 ± 0.3 1.5 ± 0.7VPA + 1 0.5 ± 0.1 (9.6) 1.1 ± 0.5 (3.2) 0.6 ± 0.1 (2.4)GEM ? VPA 0.9 ± 0.3 1.1 ± 0.1 –GEM ? VPA + 1 0.6 ± 0.1 (12.8) 1.2 ± 0.3(11.4) –

a CI and DRI were calculated for 50% residual viability according to the non-independent model of interaction for two drugs with the following equations:CI ¼ CA

IC50;Aþ CB

IC50;Bþ CA�CB

IC50;A�IC50;B, and DRI ¼ IC50;A

CA, where IC50,A and IC50,B are the concen-

trations for single agents A and B, respectively, to achieve 50% residual viability,while CA and CB are the concentrations of drug A and drug B used in combination toachieve the same effect. For the combination of three drugs, the equation for threemutually non-exclusive inhibitors was used: CI ¼ CA

IC50;Aþ CB

IC50;Bþ CC

IC50;Cþ

CA CBIC50;A IC50;B

þ CA CCIC50;A IC50;C

þ CB CCIC50;B IC50;C

þ CA CB CCIC50;AIC50;BIC50;C

. In all cases CI < 1 = synergism,

CI � 1 = additivity, CI > 1 = antagonism.

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Among the ‘‘second generation’’ Pt complexes which have leav-ing groups other than ammonia, carboplatin, 2, is widely used inMPM chemotherapy in lieu of 1, as stated in the introduction [33].

Taking into account the slower activation by hydrolysis that im-plies higher IC50 values (the half maximal inhibitory concentrationof a drug), our model cell lines show that the anti-mesotheliomaactivity profile is almost the same as the cisplatin-resistant lineMM98R, This is to be expected, since the electrophilic agent[Pt(NH3)2]2+ is the same in terms of selectivity index (SI, the ratiobetween IC50 (HMC) and the mean of IC50 on BR95, MG06, andMM98) and of resistance factor (RF = (IC50 MM98R)/(IC50 MM98).

‘‘Third generation’’ platinum complexes with different (gener-ally bulkier) carrier groups give different DNA adducts that aresometimes processed by different DNA repair pathways [34] andmay thus overcome cisplatin resistance. We preliminarily testedcis-amminedichlorido(cyclohexylamine)platinum(II) (JM118, 4,Fig. 1 [35]) on our MPM cell lines and found activity comparable

Table 2IC50 values [lM] ± S.D. obtained after 72 h of treatment with the Pt complexes tested in m

Compound IC50 [lM] ± S.D.

HMC (SI) MG06

Cisplatin, 1 6.7 ± 1.2 (1.5)d 4.1 ± 1.5d

Carboplatin, 2 96.9 ± 10.5(2.1)a 47.0 ± 5.7a

Oxaliplatin, 3 4.9 ± 0.5(1.5)a 3.0 ± 0.9a

4 – –6 0.8 ± 0.1 (0.7)a 0.8 ± 0.2a

7 – 2.9 ± 0.3a

8 13.1 ± 1.2 (2.2)a 8.0 ± 0.9a

9 – –10 – –11 – –12 – –13 – –14 – –16a – –17a – –18a – –16b – –17b – –18b – –19 – 26.9 ± 6.3c

20 – 17.6 ± 2.3c

21 – 42.9 ± 4.7a

22 22.8 ± 2.0d(1.6) 13.2 ± 4d

23 21.3 ± 1.9d(1.3) 11.1 ± 0.6d

24 18.9 ± 3.1d(1.7) 7.2 ± 0.8d

– = Not determined. Selectivity index, SI, is the ratio between IC50 (HMC) and the mean ofa unpublished results, see Section 9 for further details.b Ref. [53].c Ref. [54].d Ref. [55].

to that of 1 (Table 2). A similar carrier group characterizes cyclo-platam, namely cis-ammine(cyclopentanamine)[hydroxybutane-dioato(2-)-O1,O4]platinum(II), 5, a drug developed in Russia inthe early 1980s [36]. In a Russian-language paper it was claimedthat cyclopatam, had passed phase I and moved to phase II clinicaltrials for treatment of pleural mesothelioma, alone and in combi-nation with other drugs. Cyclopatam was recommended both fororal and intracavitary administration; the only reported side effectwas moderate leukothrombocytopenia [37].

The world-wide approved third generation platinum complexis oxaliplatin ((1R,2R-DACH)(ethanedioato-O,O’)platinum(II), 3,where DACH = cyclohexanediamine). Compound 3 exhibits clinicalactivity in the treatment of cisplatin/carboplatin refractory dis-eases such as colon cancer [38] and has seldom been employedin MPM chemotherapy, e.g., in combination with raltitrexed, vino-relbine or gemcitabine [39]. When 3 was administered with raltitr-exed, the combination was considered promising in several phaseI–III studies, with an acceptable tolerability profile, and thoughtworthy of further study [40–45]. In another case study, however,the trial was interrupted because, although well tolerated, the che-motherapy yielded no objective responses [46]. The combination ofvinolrebine with 3 appeared to be no more effective than vinorel-bine monotherapy [47]. Schutte and co-workers have tested a 3and gemcitabine protocol as an active first line therapy in MPM,producing a response rate of 40% and a median survival of13 months in a 25-patient study [48]. More recently, it has beendemonstrated that patients with relapsed MPM refractory to a pre-vious pemetrexed/platinum treatment may benefit from consecu-tive therapy with 3 and gemcitabine, resulting in a diseasecontrol rate of 45% in the absence of important side-toxicities [49].

In our model cell lines 3 is slightly more active than 1, but onthe MM98R cell line it shows a resistance factor of 0.9 (instead ofthe original 6.1 of 1), thus bypassing cisplatin resistance (Table 2),although it retains an SI as low as 1. The dichlorido analog[PtCl2(1R,2R-DACH)], 6, whose carrier ligand is also DACH, pro-

odel MPM cell lines.

BR95 MM98 MM98R (RF)

6.2 ± 0.9d 3.2 ± 1.0d 19.4 ± 2.8 (6.1)d

32.2 ± 6.8b 57.5 ± 11.6b –c

2.4 ± 0.7a 4.4 ± 0.8a 3.8 ± 0.9 (0.9)a

4.0 ± 0.4a 2.3 ± 0.1a –1.0 ± 0.2a 2.2 ± 0.4a 2.5 ± 0.6 (1.1)a

3.0 ± 0.5a 1.1 ± 0.2a 5.3 ± 0.7 (3.7)a

4.2 ± 0.6a 1.0 ± 0.2a 2.0 ± 0.2 (2)a

37.5 ± 2.4a 82.3 ± 7.9a –20.3 ± 5.4a 14.3 ± 0.3a –11.7 ± 0.9a 2.3 ± 0.8a –0.023 ± 0.003a 0.032 ± 0.008a –6.9 ± 1.7a 1.5 ± 0.3a –0.008 ± 0.002a 0.028 ± 0.007a –27.9 ± 4.8b >100b –17.4 ± 10b 25.8 ± 7b –>50b 25.8 ± 7b –30.4 ± 8.5b 8.2 ± 2.2b –18 ± 10b 2.9 ± 0.6b –14.4 ± 0.2b 3.0 ± 0.8b –29.4 ± 7.6c 20.7 ± 6.5c 60.5 ± 8.2 (2.9)c

10.9 ± 1.2c 11.8 ± 3.5c 17.3 ± 3.4 (1.5)c

42.8 ± 2.6a 30.2 ± 3.6a 34.4 ± 3.7 (1.1)a

17.5 ± 4.9d 12.7 ± 3d 12.1 ± 1.9 (1.0)d

15.2 ± 2.1d 10.1 ± 2.6d 13.2 ± 2.3 (1.3)d

5.6 ± 0.2d 5.6 ± 0.5d 9.4 ± 0.9 (1.7)d

IC50 on BR95, MG06, and MM98; resistance factor, RF = (IC50 MM98R)/(IC50 MM98).

70 I. Zanellato et al. / Inorganica Chimica Acta 393 (2012) 64–74

duced a lower IC50 (higher activity) as expected, since it hydrolizeschlorides faster than oxalate, but gave similar outcomes (RF and SI)in our model cell lines, since it forms identical adducts with DNA.

The congeners [PtCl2(cis-1,4-DACH)], 7, and [Pt(oxalato)(cis-1,4-DACH)], 8, contain an isomeric form of the DACH carrier ligand pres-ent in 3, that confers high water solubility to the corresponding Ptcomplexes. As such, they have been widely investigated as potentialplatinum anticancer drugs [50] (Table 2). In particular, 7 was as ac-tive as its isomer 6 on MM98R, but its RF was higher than that of 3(3.7 vs. 0.9). In particular, the in vitro and in vivo performances of7 suggested that its spectrum of activity is different from that of 1,and that 7 is not recognized by cells made resistant to 3 [51].

The experimental data indicate that differences in shape or con-formation of the cyclohexane ring play an important role in elicit-ing the differential antitumor activity of 1,4-DACH with respect to1,2-DACH complexes [52].

4. Non-classical platinum complexes: cationic and multinuclearplatinum complexes

Cationic platinum(II) complexes represent a non-traditionalclass of compounds with antitumor properties against some typesof tumors expressing high levels of organic cation and coppertransporters [56]. Although no data concerning the expression ofthese transporters are available for MPM, we assayed the actionof two cationic complexes, namely cis-diamminechloridopyridine-platinum(II)]+, 9, and (l-1,4-diaminobutane)bis{trans-diammine-chloridoplatinum(II)}2+, 10, on MPM cell lines.

According to the number of platinum units, cationic complexescould form monofunctional or bifunctional (mainly interstrand)adducts with DNA [57,58]. Such drugs arouse interest becausetheir anticancer activity is linked to increased intracellular accu-mulation involving polyamine and membrane cationic transport-ers, as has been demonstrated for 9 [59,60]. This contradicts theoriginal assumption by Cleare and Hoeschele [61,62] that chargedPt-compounds cannot cross the cellular membrane. Once insidethe cell, their cationic nature allows them to interact with DNAmore rapidly than 1. They are also more water-soluble than theirneutral counterparts, thus possessing greater bioavailability [56].

We assayed the monoplatinum complex 9 and the diplatinumcomplex 10 [63] in our model MPM cell lines, but the results weredisappointing (Table 2).

5. Pt(IV) complexes

Satraplatin (JM216, trans,cis,cis-diacetatoamminedichlori-do(cyclohexanamine)platinum(IV), 11) the Pt(IV) derivative of 4,was tested in a phase I trial for MPM [64]. In vitro 11 gave resultssimilar to 1 and 4 in our model cell lines (Table 2). This is not sur-prising, since the DNA adducts found after treatment with 11 aresimilar to those of 1 [65] and can be removed by the same DNA re-pair pathway [66].

The DACH analog trans,cis,cis-diacetatodichlorido(cyclohexane-1R,2R-diamine)platinum(IV), 13, proved to be more active than11 thanks to its carrier group, as already observed above for thecorresponding Pt(II) complexes (Table 2). The same was true forthe two corresponding long-chain (eptanoato) Pt(IV) complexes12 and 14, but their IC50 values were dramatically lower (fromlM to nM range; Table 2). Interestingly, in combination experi-ments, the IC50 values of cells treated with a mixture of 6 and hep-tanoic acid in a molar ratio 1:2 did not differ significantly fromthose obtained for the Pt(II) complex alone (IC50 values for hepta-noic acid are in the mM range), with a CI > 1.

6. Drug targeting and delivery

Active and passive drug targeting and delivery (DTD) strategiesare designed to selectively direct drugs against malignant tissuesusing biomolecular (active) or macromolecular (passive) vectors[67].

Passive DTD relies on the enhanced permeability and retention(EPR) effect due to vascular permeability and inefficient lymphaticdrainage of the tumor mass [68]. In this situation, macromolecules(bearing the active drug) with opportune dimensions and chemico-physical properties may be accumulated at the tumor site [69].Macromolecular vectors include liposomes, microspheres, nano-particles, polymeric micelles, modified plasma proteins, polysac-charides, and artificial biodegradable polymers.

Aroplatin™ (or L-NDDP) is an example of passive DTD. It isbased on cis-(cyclohexane-1R,2R-diamine)bis(neodecanoato)plati-num(II), 15, a structural analog of 3, formulated in multilamellarliposomes ranging from 1 to 3 lm in diameter [70,71]. Preclinicalstudies in rats treated with intravenous and intraperitoneal aropl-atin [72] demonstrated that the peritoneal fluid levels of aroplatinwere several times higher than those of 1. The evidence that aropl-atin may have a more favorable pharmacokinetic profile for intra-peritoneal therapy stimulated clinical and pharmacological studiesof aroplatin in MPM patients [73,74]. The results showed thatintrapleural aroplatin therapy is feasible with significant but man-ageable toxicity. Although local responses were highly encourag-ing, areas of mesothelioma that are not in direct communicationwith the pleural space evade drug exposure.

Active DTD involves ligands for tumor-related transporters,membrane cancer-specific antigens or over-expressed receptors.Vectors suitable for active DTD include hormones, monoclonalantibodies, bile acids, amino acids, sugars etc., i.e., any moleculethat allows selective conveyance by transporters over/expressedin cancer cells or is typically tropic for the specific tumor tissue[67].

Bisphosphonates (BPs) are clinically used as inhibitors of osteo-clasts, the cells responsible for bone resorption [75]. More recently,it has been shown that BPs can inhibit tumor cell proliferation, in-duce apoptosis, and decrease cell invasion as well as cell adhesionon bone matrix [76–78]. Depending on their molecular structures,these drugs can be divided into pyrophosphate-resembling (p-BPs,such as clodronate and medronate) and nitrogen-containing bis-phosphonates (n-BPs, such as risedronate and pamidronate).Because of their high calcium chelating potentialities, they are alsoused as technetium complexes for bone scintigraphy. The uptake ofthe bone scan agent 99mTc-medronate (99mTc-MDP, medronatestands for methylene diphosphonate) in MPM effusions [79–81]was found serendipitously during clinical investigations, but thistropism is now well established and experimentally verified.Furthermore, n-BPs effectively inhibit the proliferation of mesothe-lioma cells in vitro and in vivo and significantly extend the survivaltime of mice bearing experimental models of mesothelioma[82,83].

Thus, we speculated that platinum complexes containing n-BPscould target MPM and exert a cytostatic/cytotoxic effect that sumsthe activities of the two constituting moieties (bifunctional drugs).Some (Pt–n-BP)s have already been successfully investigated asspecific bone targeted antineoplastic drug candidates [84–89].We have reported on the synthesis and in vitro tests of diplatinumcomplexes containing am(m)ine ligands (A) and a n-BP moiety as abridging ligand (16–18/a-b) [53].

Unfortunately, the Pt-n-BP complexes showed only modestactivity depending upon the type of n-BP bridging the two plati-num subunits and upon the type of amine carrier ligands complet-ing the coordination sphere of platinum. The sarcomatoid cell line

Fig. 2. Scheme of the aquation process of compounds 16b–18b in physiological-like conditions (100 mM phosphate buffer, pH = 7.4, 120 mM NaCl, 37 �C) (adapted from Ref.[53]).

I. Zanellato et al. / Inorganica Chimica Acta 393 (2012) 64–74 71

MM98 resulted somewhat more sensitive to the compounds underinvestigation than the epithelial BR95 one; interestingly, the re-verse is true for the clinically employed drugs 1 and 2.

To test whether the minimal cytotoxic activity of the Pt–n-BPcompounds could be attributed to their slow hydrolysis, whichcurtails the splitting of the active components, we monitored thestability of compounds 16b–18b in physiological-like conditionsby 31P and 195Pt NMR spectroscopy and ESI-MS spectrometry.

Compound 16b proved to be very stable and no release of freebisphosphonate or 7 was observed; only one oxygen atom of thebridging bisphosphonate ligand was displaced by either a chlorideor the bisphosphonate-bound aminic group. In contrast, the lowertendency of the aminic group in ligand 17 to coordinate to Pt dis-placing one oxygen atom of the bridging bisphosphonate resultedin the partial release in solution of the cytotoxic compound 7 (to-gether with monomeric Pt–n-BP derivatives). This explains why17b was more cytotoxic than 16b. In the case of complex 18b, boththe simple displacement of one oxygen atom of the bridging bis-phosphonate ligand by a chloride and the limited formation of 7was observed (Fig. 2). In conclusion, the study of the aquation pro-cess indicates that the two building blocks (Pt-core and n-BP) donot split, explaining the low cytotoxic activity of this series ofcomplexes.

7. Bypassing the antioxidant armory

Among the factors involved in chemoresistance against plati-num drugs (a multifactorial phenomenon), important is their inac-tivation by S-based nucleophiles [90], mainly glutathione, GSH(present in mM concentrations) or metallothioneins (MTs, smallproteins having 20 Cys groups per molecule), the most importantcellular detoxifying agents [91–93]. In the case of MPM, the aber-rant induction of signaling responses by redox stimuli caused byasbestos exposure leads to altered gene expression, causing, interalia, an increase in anti-oxidant enzyme level and GSH metabolism[11,94–96]. Cisplatin-induced cytotoxicity may be significantlypotentiated in MPM by inhibiting -glutamylcysteine synthetase(-GCS), the rate-limiting enzyme in GSH biosynthesis, by buthio-

nine sulfoximine (BSO), which is, however, too toxic a derivativeto be employed in clinical treatment [97].

Another GSH-associated enzyme involved in cisplatin resistanceis glutathione-S-transferase (GST). The role of GST in the detoxifi-cation of the electrophilic agent 1 is well established, as it has beenproven to catalyze the conjugation of this drug with GSH [98–101].Thus, targeting GST might lead to overcome intrinsic and/or in-duced MPM chemoresistance, since GST has been reported to be of-ten expressed in MPM.

Ethacrynic acid (2-[2,3-dichloro-4-(2-methylidenebutanoyl)phenoxo]acetic acid, EA), developed as a diuretic, is an inhibitorof GST and has been explored as a chemo-adjuvant targeted toGSH-dependent pathways [102,103].

In this context, Dyson and co-workers [104,105] developed aPt(IV) derivative, namely cis,cis,trans-diamminodichloridobis(eth-acrynato)platinum(IV) (ethacraplatin, 20), potentially able to re-lease the antitumor drug 1 and two equivalents of the axiallycoordinated GST-inhibitor EA upon a 2e reduction. In order to bet-ter understand the role of EA on the pharmacological effects ofPt-drugs applied to MPM, a Pt(II) analog of compound 20 was syn-thesized, namely cis-diamminobis(ethacrynato)platinum(II), 19,where two ethacrynato moieties act as ‘‘classical’’ leaving groupswithin the square-planar coordination of Pt(II) [54]. Both com-plexes 19 (after simple hydrolysis) and 20 (after reduction andhydrolysis of the cisplatin metabolite) can share the formation ofthe same cytotoxic [Pt(NH3)2]2+ agent. The Pt(IV) complex is al-ways more potent than the Pt(II) analog, and both are more cyto-toxic than 2. Instead, 19 and 20 perform less well than 1, albeitboth compounds show much better resistance factors.

Complex 20 acts as a GST inhibitor per se, according to Dysonand co-workers [106]. In fact, in an a-cellular enzymatic assay, itexhibited a higher GST-inhibition level than that of free EA,whereas 19 was able to inhibit GST at a level comparable withEA. Nonetheless, the overall GST activity, performed on cell lysatesafter treatment with the compounds under investigation, did notchange. As a consequence, enzyme inhibition by EA, 19 or 20was overcome by the cellular machinery possibly increasing GSH,the enzyme substrate [107,108]. In fact, the intracellular levels of

72 I. Zanellato et al. / Inorganica Chimica Acta 393 (2012) 64–74

GSH, after treatment with sublethal concentration of drugs,showed a significant increase.

Another way of reducing the level of undesired (deactivating)substitution reactions carried out by GSH in Pt(II) square-planarcomplexes is to use bulky carrier ligands [109]. This realizationprompted the design, synthesis, preclinical tests and clinical trialsof cis-amminedichlorido(2-methylpyridine)platinum(II) (picopla-tin, ZD0437 or AMD-437, 21), [34]. The presence of the stericallydemanding 2-methylpyridine hinders the axial approach of nucle-ophiles to the platinum core without detriment to the level of DNAplatination [110–112]. Picoplatin has been granted orphan drugdesignation (EU/3/07/502, EMEA/OD/055/07) for the treatment ofsmall cell lung cancer.

Picoplatin was used in a phase II trial as second-line therapy forMPM to evaluate its activity and tolerability in patients with re-lapsed or progressive disease who had received one prior cycle ofchemotherapy. In this trial, no complete responses were achieved,but picoplatin demonstrated a manageable tolerability profile. Med-ian time to progression was less than 3 months and median overallsurvival was about 7 months from the time of enrollment. Althoughthe study did not find significant clinical benefit, it demonstrated thefeasibility of conducting clinical trials in mesothelioma patients whosuffer from disease relapse after prior chemotherapy [113].

Insertion of the picoplatin square-planar structure in a Pt(IV)octahedral scaffold should provide an ideal pro-drug candidatethat would be easily reduced, but hardly deactivated by GSH, oftenover-expressed by resistant tumor cells. Interestingly enough is toconsider the dual role of GSH, that on the one hand promotes thereduction Pt(IV) to Pt(II) activating the pro-drug in cell, on theother hand hinders the action of the resulting Pt(II) metabolite.We synthesized a series of picoplatin-based Pt(IV) complexes withcarboxylic acids containing carbon chains of increasing length asaxial ligands (22–24) [55]. On MPM cells, complexes 22–24 showincreased activity as the carbon chain length increases (Table 2),as previously observed for other series of homologous Pt(IV) com-plexes [114–116], approaching or in one case bypassing that of 1itself. All of the complexes are more potent than 21. Moreover, theyalways exhibited a lower RF than 1. Longer chains means havingboth a higher lipophilicity (and in turn likely higher uptake) andhigher (less negative, i.e., easier reduction) reduction peak poten-tials. Therefore, these data are consistent with the general findingthat the cytotoxicity of Pt(IV) complexes depends on both lipo-philic and electronic features [116,117].

A similar observation was made regarding the ability to dis-criminate between normal and malignant cells, which is of para-mount importance for developing clinically applicablechemotherapeutics. SI increases with axial chain length for 22–24, whereas 1, as expected, exhibits low selectivity (SI = 1.5).

When complexes 22–24 are reacted with GSH, formation of thecorresponding Pt(II) species is observed, supporting the generalview of an ‘‘activation by reduction’’ mechanism. Interestingly, allthe Pt(II) metabolites contain the picoline ligand, thus guarantee-ing low inactivation of the complex by GSH. This explains thestrong antiproliferative activity of 22–24 observed in the cisplatinresistant MM98R cell line.

The insertion of the picoplatin moiety into the octahedral struc-ture of Pt(IV) with additional axial carboxylato ligands thus affordspromising complexes for the treatment of tumors, such as MPM,whose chemoresistance is mainly based on overexpression of GSH.

8. Non Platinum drug candidates

In an attempt to bypass the chemoresistance of MPM against 1,we also tested the biological activity of very promising non-platinum metal-drug candidates, synthesized and studied by other

European groups linked with us within the European Union COSTD39 network.

We tested two hydroxyferrocifens (25 and 26) developed by theJaouen group in Paris. Hydroxyferrocifens are ferrocenyl derivativesof 4-OH-tamoxifen, the active metabolite of the non-steroidal selec-tive estrogen receptor modulator (SERM) tamoxifen, which is widelyprescribed for the treatment of hormone-dependent breast cancer.In these molecules, an aromatic ring of hydroxytamoxifen was re-placed with a ferrocene moiety [118]. Originally designed for fight-ing breast cancers overexpressing estrogen receptors (ER(+)), thesederivatives proved to be very active in ER(�) breast tumors as wellas in several different neoplasms, and some showed promising per-formances in MPM. The two bio-organometallic compounds 25–26were more potent in inhibiting cell proliferation than 1. The mecha-nism of action is still under investigation, although senescenceseems to be involved in the antiproliferative process [119,120].

We are also actively studying the action of Co-ASS (hexacar-bonyl[2-propinylacetylsalicylate]dicobalt, 27), which was devel-oped by the Gust group in Innsbruck. Celecoxib, a non-steroidalanti-inflammatory drug acting as a selective cyclooxygenase-2(COX-2) inhibitor, has been reported to exert potent antiprolifera-tive activity on MPM cell lines [121,122] as well as on nude micebearing intraperitoneal MPM [123]. Because 27 has been shownto act as a COX-2 inhibitor as well as an antiangiogenic agent[124,125], it might provide an interesting approach in the treat-ment of MPM.

9. Experimental

Syntheses, characterization and biological evaluation of manyof the compounds reported in Table 2 have been previously pub-lished (see references in the text). On the contrary, for carboplatin,oxaliplatin, complexes 4, 6–14, and 21 the cytotoxicity on MPMcell lines has been reported here for the first time and it has beenevaluated following already published procedures [55]. Briefly,compounds under investigation were dissolved in DMSO and thesemother solutions were serially diluted in complete medium (Ham’sF10, DMEM or RPMI-1640 media, depending on the cell lines, sup-plemented with L-glutamine, penicillin, streptomycin and fetal bo-vine serum). The total DMSO concentration never exceeded 0.5%, avalue that was found to be non-toxic to the cell tested. Challengewith the compounds was performed at 37 �C in a 5% CO2 humidi-fied chamber for 72 h continuous treatment.

At the end of the experiment, the [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium]inner salt (MTS) assay was performed using a commercial kit;absorbance values were recorded at 490/620 nm by a spectropho-tometric plate reader and corrected by subtraction of the absor-bance of MTS alone. Residual cell viability was also evaluated bymeans of the resazurin reduction assay, added at the end of thetreatment, and the amount of the reduced product (resorufin)was measured by means of fluorescence using an excitation wave-length of 550 nm and an emission wavelength of 585 nm.

In each experiment, the cells were challenged with the drugcandidates at different concentrations and the final data were cal-culated from at least three replicates of the same experiment car-ried out in triplicate. The half inhibiting concentration (IC50),defined as the concentration of the drug reducing cell viability by50%, was obtained from the dose–response sigmoid by using stan-dard curve-fitting packages.

Acknowledgments

We thank the Regione Piemonte (Ricerca Sanitaria 2008,Research Project on Rare Diseases), the Provincia di Alessandria

I. Zanellato et al. / Inorganica Chimica Acta 393 (2012) 64–74 73

(Research Project 2010-0081200) and the CRT Foundation (Re-search Project ‘‘Improvement of Mesothelioma Treatment’’ 2010)for financial support. The research was carried out within theframework of the European Cooperation COST Action CM1105(Functional Metal Complexes That Bind To Biomolecules). The In-ter-University Consortium for Research on the Chemistry of MetalIons in Biological Systems (CIRCMSB, Bari, Italy) is gratefullyacknowledged for a grant to I.B.

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