Wide-spectrum Characterization of Trabectedin

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Review

ISSN 1462-2416Pharmacogenomics (2010) 11(6), 865–87810.2217/PGS.10.69 © 2010 Future Medicine Ltd

Wide-spectrum characterization of trabectedin: biology, clinical activity and future perspectives

After World War II, the US National Cancer Institute (MD, USA) carried out a wide-ranging program of screening marine plant and animals in order to find new and promising antineoplas-tic agents. Among several agents tested in clinical trials (Table 1), extracts from the Caribbean tuni-cate Ecteinascidia turbinata, later named ectein-ascidins (ETs), were shown to exert antitumor activity [1,2]. The small amount of ETs in E. tur-binata prevented their isolation for over a decade. The structure of one of these, ET-743, was finally determined in 1984 and chemically syn-thesized in 1996 [3,4]. Later, ET-743, or trabect-edin (Yondelis®, PharmaMar, Madrid, Spain), was the first representative of a marine natural product to receive marketing authorization [5].

Trabectedin has raised a growing interest in the last few years because of its proven effi-cacy in the treatment of advanced soft tissue sarcoma (STS). Given the recent introduction of this new compound into clinical use, a com-plete revision of its mechanisms of action (still not completely understood), the preclinical and clinical evidences that brought it to its approval in Europe, and its main side effects and their best management is needed.

Drug purification, biosynthesis & structureAs extensively reviewed by Cuevas and Francesch [5], the diff iculties faced when develop ing sufficient supplies of trabectedin have represented a severe limitation on its use for a long time.

The tunicate normally lives in coastal shal-low waters (0–15 m depth) and in lagoons. It is distributed throughout the Caribbean and in the temperate regions of the Atlantic and the Mediterranean.

Before 1997, PharmaMar started both a wide aquaculture program and a chemical syn-thesis program, in order to produce enough ET-743 for preclinical and clinical use.

For the aquaculture program, two main methodological approaches were developed: sea farms and inland facilities. In 2004, all of the Mediterranean aqua farms collectively produced approximately 100 metric tons of tunicate bio-mass. However, as a consequence of the isolation and purification procedures, the final yields of ET-743 were of very low concentrations. Thus, in order to achieve the future commercialization of the drug, it was necessary to develop a syn-thetic process to produce ET-743, so as to avoid the dependence on the natural source.

Since 1996, when the Corey group published the complete synthesis of ET-743 [4], several groups have improved the synthesis process, but the manufacture of ET-743 on an indus-trial scale was still not possible. Nevertheless, in 2003, PharmaMar successfully developed a short and straightforward semisynthetic process starting from cyanosafracin B, an anti biotic obtained by fermentation from the bacteria Pseudomonas fluorescens [6,7]. In addition, other synthetic or semisynthetic approaches have been reported from a large number of research groups [8–16].

Ecteinascidin-743 (trabectedin, Yondelis®; PharmaMar, Madrid, Spain), a 25-year-old antineoplastic alkylating agent, has recently shown unexpected and interesting mechanisms of action. Trabectedin causes perturbation in the transcription of inducible genes (e.g., the multidrug resistance gene MDR1) and interaction with DNA repair mechanisms (e.g., the nucleotide excision repair pathway) owing to drug-related DNA double strand breaks and adduct formation. Trabectedin was the first antineoplastic agent from a marine source (namely, the Caribbean tunicate Ecteinascidia turbinata) to receive marketing authorization. This article summarizes the mechanisms of action, the complex metabolism, the main toxicities, the preclinical and clinical evidences of its antineoplastic effects in different types of cancer and, finally, the future perspectives of this promising drug.

KEYWORDS: adducts n cancer n double strand breaks n ET-743 n MDR1 n NER n ovarian n sarcomas n therapy n trabectedin

Bruno Vincenzi†1, Andrea Napolitano1, Anna Maria Frezza1, Gaia Schiavon1, Daniele Santini1 & Giuseppe Tonini1

1University Campus Bio-Medico, Medical Oncology, Via Alvaro del Portillo 200, 00128 Rome, Italy †Author for correspondence: Tel.: +39 339 319 9912 [email protected]

For reprint orders, please contact: [email protected]

Pharmacogenomics (2010) 11(6)866 future science group

Review Vincenzi, Napolitano, Frezza, Schiavon, Santini & Tonini

Ecteinascidin-743 is composed of three fused tetrahydroisoquinoline rings (Figure 1) [17]: two of the rings (units A and B) recognize and bind covalently to the minor groove of the DNA double helix; the third ring (unit C) protrudes out of the minor groove and inter-acts directly with transcription factors, nota-bly the nucleotide excision repair (NER) endo nuclease, XPG [18]. Contrasting evidence currently exists regarding the necessity of this subunit for the specific biological responses induced by ET-743 [19,20].

Trabectedin mechanisms of action�n Trabectedin alkylating mechanisms

The antitumor mechanisms of action of ET-743 are various and not entirely understood. One of ET-743’s most studied properties is its abil-ity to interact with DNA. This property has been proposed on the basis of biochemistry, NMR spectro scopy, x-ray crystallography and molecular modeling data indicating structural similarities with other antibiotics containing tetrahydroisoquinoline [21–24].

Ecteinascidin-743 contains a carbinolamine center at the N2 position. The elimination of the adjacent 21-OH group results in a Schiff base that is vulnerable to nucleophilic attack. Therefore, the binding of ET-743 to the exo-cyclic 2-amino group of guanine in the DNA minor groove leads to DNA alkylation.

Some important characteristics differenti-ate ET-743 from other minor groove mono-alkylating agents: a unique ability to bend DNA towards the major groove by alkylating the minor groove, duplex stabilization and extrahelical protrusion of the C-subunit [25].

Interestingly, the alkylation reaction is not random but DNA sequence specific (5´-PuGC, 5´-PyGG) [26]. This specificity seems to be mainly related to slower rates of the reverse dealkylation reaction in favored bonding sequences rather than in nonfavored ones (5´-AGT), probably owing to more stable conformational changes [27].

A very important point is the specificity of cellular proteins able to bind to ET-743-alkylated DNA sequences. Remarkably, the minor groove alkylation causes an interference in the DNA-topoisomerase I cleavage sites. At high concentrations (i.e., micromolar levels), ET-743 causes a topoisomerase I-induced for-mation of cleavage complexes – enzyme-linked and -mediated DNA strand breaks – which are the catalytic intermediates of topoisomer-ization reactions. On the other hand, it traps and stabilizes these complexes [28], functionally resembling ‘topo isomerase poisons’ [29]. Using pharmacologically reasonable concentrations of ET-743 (i.e., nanomolar levels) in sensitive cells, no DNA breakage or DNA/protein cross-links are seen [30], suggesting only an ancillary role of topoisomerase I poisoning in ET-743 cytotoxicity [31].

In a similar manner, the DNA binding of several transcription factors, notably SRF/TCF and NF-Y, is impaired at concentrations in the 50-µM range, which is still much higher than the concentrations that cause cellular cyto-toxicity. Nevertheless, the specificity of alkyl-ated DNA–protein interactions is confirmed by the evidence that other transcriptional factors (e.g., MYB and MYC) can bind to the DNA even at very high concentrations of ET-743 (300 µM), as alkylation does not involve the binding sequences of those specific transcription factors [32].

The main pharmacological activity of ET-743 is thus mostly independent of an alkylation-dependent inhibition of the DNA binding of proteins or transcription factors. In fact, on the basis of DNA–protein molecular modeling, it has been shown that ET-743 can target DNA with two or more molecules simultaneously and that the DNA–ET-743 covalent complex are virtually superimposable to the minor grooves of DNA when bound to the zinc fingers of transcription factors [33]. This structure is also strongly reminiscent of an RNA–DNA hybrid, and these striking similarities raise the inter-esting hypothesis that ET-743 preferentially targets the minor groove of DNA when bound to a zinc-finger-containing transcription factor by the exposition of a more readily accessible 2-amino group of guanine [34].

�n Trabectedin & perturbation of transcription & cell activitiesPreincubation of ET-743 with the transcription factor NF-Y causes inhibition of DNA bind-ing at lower concentrations compared with the

Table 1. Some Phase II marine natural products.

Compound Source organism Chemical class

Ecteinascidin-743 Ecteinascidia turbinata Tetrahydroisoquinolone alkaloid

Dolastatin 10 Dolabella auriculariaSymploca sp.

Linear peptide

Bryostatin 1 Bugula neritina Macrocyclic lactone

Kahalalide F Elysia rufescens/Bryopsis sp. Cyclic depsipeptide

Squalamine Squalus acanthias Aminosteroid

Dehydrodidemnin B Trididemnum solidum Cyclic depsipeptide

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Wide-spectrum characterization of trabectedin Review

pre incubation with DNA, suggesting that the protein rather than the minor groove is the target [31].

Furthermore, since the inhibition is extremely rapid (1 min) and the effect is observed at pharmaco logical concentrations, it is probably due to the C-subunit or the DNA bending to the major groove. Transcriptional activation of NF-Y-dependent genes is likely to be one of the main cytotoxic mechanisms of ET-743 action [35].

Among the inhibited genes, the most prom-ising targets code for MDR1 (P-glycoprotein [P-gp]), HSP79 and collagen I. ET-743 is the first pharmacologically relevant agent to prevent the activation of both MDR1 [36] and HSP79 [34] transcription by multiple stress inducers (i.e., his-tone deacetylase inhibitors and UV irradiation), without affecting their constitutive expression. ET-743 may thus selectively inhibit activation of MDR1 expression in tumor cells without affect-ing constitutive expression in normal cells, but acting as a specific antitumoral transcriptional regulator. Preclinical studies have also confirmed that ET-743 is an effective agent in MDR1-overexpressing cells [37], despite the hypothesis that P-gp overexpression contributes to ET-743 resistance in an ovarian cell line [38].

In a similar manner, ET-743 is responsible for a dose-dependent downregulation of COL1A1 (the gene coding for pro-a1 chain of collagen I) expression [39]. These data were confirmed by the particular ET-743 cytotoxicity in mesenchimal tumors (i.e., sarcomas) and by the ana lysis of a ET-743-resistant sarcoma cell line, in which pro-longed exposure to ET-743 caused changes in cell function through cytoskeleton rearrangement and/or modulation of collagen levels [40].

Moreover, it has been reported that ET-743 is responsible for a unique microtubule disorganization in vitro [41].

Further links between transcriptional inhibition and ET-743 action have not been investigated. ET-743 has been proved to also inhibit NF-Y-independent transcription (Sp1-mediated [42] or SXR-mediated transcription). SXR inhibition is particularly interesting because SXR regulates both drug efflux (by activating the expression of the MDR1 gene [43]) and metabo-lism (by activating the cytochrome P450 (CYP) CYP3A4 gene [44]). Moreover, gene-expression ana lysis through microarray technology has identified selected groups of genes inhibited by low doses of the drug in different cell lines [45,46].

Remarkably, another important indication of the unusual antiproliferative effect of ET-743 is its predominant activity in the G1 phase of the

cell cycle, eventually resulting in a G2/M block, a different mode of action to other alkylating agents, [24]. This effect has been confirmed by the ana lysis of differential ET-743 activity on G1/S promoters and nucleosomes [47].

The complex antiproliferative activity of ET-743 involves other mechanisms: an high-dose transcription-independent effect leading to rapid apoptosis involving mitochondria, JNK and caspase-3 [48], and a low-dose transcription-coupled block of the cell cycle involving the NER pathway [49].

�n Trabectedin & DNA repair mechanismsSince mammalian cell lines lacking NER gene products are resistant to ET-743, a novel mech-anism of cell-cycle block based on the NER pathway was recently hypothesized [48,50].

The mechanism of action is probably very similar to that of the topoisomerase I poison-ing: ET-743 traps an intermediate in the NER processing of DNA adducts. This trapped inter-mediate protein–ET-743–DNA adduct complex could be responsible for ET-743 cell toxicity [51]. In particular, the NER protein Rad13 seems to be crucial for ET-743 cytotoxic effect by the for-mation of a Rad13–DNA–ET-743 ternary com-plex, resulting in cell death [22]. The crucial role of this pathway is demonstrated by the evidence that restoration of NER function sensitizes cells to ET-743 treatment [52].

Investigating cell-cycle phase perturbation, the cells with functional NER appear to overcome the G1 block more easily, but are then blocked in the G2/M phase, suggesting that the NER mechanism itself leads to some sort of persistent DNA damage [53]. Unrepaired adducts probably mediate the damage by arresting and degrading

OO

N

N

O

NH

OH

OO

O

S

OH

OH

O

Subunit B

Subunit C

Subunit A

Figure 1. Ecteinascidin-743 and its three fused tetrahydroisoquinoline rings.



DNA polymerase II [54] and by forming DNA double strand breaks (DSBs), monitored by the activation of g-H2AX and Rad51 foci [55], even though several other studies showed no DNA breaks unless at micromolar concentrations of ET-743 [23,24].

In 2008, a flowchart of molecular pathways involved in ER-743 cytotoxicity was proposed. According to the authors, there are two types of DSBs produced by ET-743: trascription-coupled DSB involving the NER pathways and replication-coupled DSB involving the ATM pathway independently from trascription-cou-pled DSB [56]. Moreover, it has been recently shown that Von Hippel–Lindau tumor suppres-sor protein deficiency made the cells resistant to ET-743-induced cell death with a similar effect to NER deficiency. The Von Hippel–Lindau com-plex could act in promoting DNA polymerase II degradation in DNA damaged cells [53].

Finally, recent evidence suggests that the homologous recombination pathway – respon-sible for DNA break repair – is essential for ET-743 cytotoxicity, at least as much as the other previously mentioned pathways. In fact, cells lacking the homologous recombination path-way were extremely sensitive to the drug with an approximately 100-fold decrease in IC50 [57]. In a similar way, the Fanconi anemia pathway seems to be very important in the DNA repair of ET-743 DNA adducts that might functionally mimic an interstrand DNA crosslink (ICL) [33,58]. Indeed, cells with mutations in any of the tested Fanconi anemia genes are extremely sensitive to ET-743 [59], whose behavior mimics that of the ICL-forming drugs (e.g., mitomycin C) [60].

As previously described, ET-743 has a very complex mechanism of action via DNA alkyla-tion and protein interactions. Nevertheless, ET-743 is an alkylating agent, and its main pharmacological action seems to be due to both transcriptional regulation of inducible genes and to DNA DSB and ICL-like adducts (Figure 2).

Myxoid liposarcoma and could lead to the association of ET743 with compounds acting on lipogenic pathways.

�n Trabectedin & differentiation in myxoid liposarcoma tumorsAmong the different mechanisms of action ascribed to ET-743, one of the most fascinating is its potential ability to induce differentiation in specific histotypes of STS, such as myxoid liposarcoma. Myxoid liposarcoma is character-ized by a fusion between the cyclophosphamide, hydroxydaunorubicin (doxorubicin), Oncovin®

(vincristine; Genus Pharmaceuticals, Newbury, UK) and prednisone/prednisolone (CHOP) transcription factors and the FUS or EWS genes, which is probably one of the key points to its pathogenesis; ET743 has been proven to be par-ticularly effective in this subtype of sarcoma. One hypothesis is that the activity of trabectedin is related to the inactivation of the FUS–CHOP oncogene: Forni et al. reported that trabectedin causes detachment of the FUS–CHOP chimera from targeted promoters. Reverse transcription PCR and chromatin immunoprecipitation ana-lysis in a myxoid liposarcoma line and surgi-cal specimens of myxoid liposarcoma patients in vivo show activation of the CAAT/enhancer binding protein-mediated transcriptional pro-gram that leads to morphologic changes of terminal adipogenesis.

Trabectedin metabolism & toxicities�n Trabectedin metabolism

Data regarding ET-743 metabolism are still limited. In vitro experiments consisting of drug incubation with a human lymphoblast-expressed CYP3A4 isoform suggested the presence of at least three metabolites [301].

With an initial in vivo evaluation, no metab-olites were observed in urine, serum or bile samples from patients treated with intravenous ET-743, whereas deacetylated ET-743 was found after plasma incubation [61]. Recent evidence obtained by using [14C] ET-743 proved that the drug is quickly metabolically converted by the liver to a large number of compounds excreted in both urine (5–10% of total) and feces (major excretory pathway) [62,63].

The presence of O-glucuronidated and glutathione-conjugated metabolites is likely [64], although the inhibition of phase II enzy mes, such as UDP-glucuronosyltransferase, N-acetyltransferase, sulfotransferase and gluta-thione S-transferase, did not show any significant influence on the cytotoxicity of ET-743 [65].

The most important ET-743 biotransforma-tion pathway is the enzymatic conversion by liver microsomal proteins (~50% of the total conver-sion). Two main chemical products are formed during the microsomal breakdown: ET-743S1 and ET-743S2. Two other molecules, ET-743M5 and ET-743M6, are undoubtedly metabolites of ET-743S2 [301]. ET-743 biotransformation at the microsomal level is mainly due to CYP3A4, 2C9, 2C19, 2D6 and, to a minor extent, 2E1 [66]. These data have been confirmed in vitro by the increased cytotoxicity of ET-743 (measured as IC50 decrease) observed in a HepG2 cell line after



incubation with CYP inhibitors [63]. Interestingly, male microsomes showed a significantly lower ET-743 biotransformation rate, probably owing to the different amounts of CYP3A4 and other CYPs in the microsomal preparations. Typically, higher quantities of CYP3A4 and faster conver-sion of ET-743 were observed for female micro-somes compared with male microsomes, with CYP3A4 as the major isozyme involved in ET-743 biotransformation (~95%) [64].

Therefore, since CYP3A4 has an important role in ET-743 metabolism, the risk of in vivo drug–drug interactions must be considered when the agent is combined with other CYP3A4 substrates [67].

Finally, given the complexities of ET-743 metabolism, it is unlikely that a single poly-morphism or the inhibition of a single metabolic pathway can change its metabolic profile.

�n Trabectedin toxicitiesThe liver is a key organ not only for ET-743 metabolism but also for its collateral effects. Preclinical studies in animals demonstrated liver toxicity as an important side effect of ET-743. In these studies, the female rat was identified as the species with the highest risk of hepatotoxicity from ET-743 [68]. Repeated treatment cycles were able to increase the extent of clinical chemistry changes, suggesting cumulative toxicity [69].

The unusual form of ET-743 hepatotoxicity in female rats consists of damaged biliary duct epi-thelia accompanied by inflammation followed by peribiliary fibrosis. The pathological alterations were accompanied by defects of liver function, such as dramatic elevation of plasma bilirubin lev-els, moderate increases in plasma levels of alkaline phosphatase (ALP) and aspartate transaminase (AST) and a decrease in hepatic CYP activity [70].

In fact, together with dose-limiting myelo-suppression (i.e., neutropenia and thrombo-cytopenia), the main ET-743 toxicities registered during clinical trials were acute (but revers-ible) transaminitis and subclinical cholangitis, characterized by increases in ALP and/or bilirubin [71].

Interestingly, it has been shown that high-dose dexamethasone administered 24 h before ET-743 protects rats from hepatotoxicity with-out compromising antitumor activity. This effect has been firstly assigned to the dramatic dexa-methasone-induced reduction of ET-743 hepatic levels via the induction of the CYP enzyme CYP3A [72]. Considering the side effects of high-dose dexamethasone [73,74], the hepatoprotective effect of other CYP3A inducers have been tested

for a potential use in humans. Indole-3-carbinole (I3C), a potent inducer of CYP enzymes [75], is the hydrolysis product of glucosinolates present in cruciferous vegetables, such as broccoli and Brussels sprouts. As expected, the food intake of I3C protected against the detrimental hepatic effects of ET-743 without interfering with its antitumor activity in a model of mammary carci noma. Surprisingly, ingestion of I3C did not decrease hepatic levels of ET-743 in comparison with animals who received ET-743 alone, sug-gesting that CYP induction is not the main hepa-toprotective event. Thus, the interference with NF-kB transcriptional activity is the most attrac-tive candidate mechanism to explain hepatopro-tection, as it is common to ET-743 antidotes, I3C and dexamethasone [76]. These data were con-firmed by the evidence that the protection pro-vided by a pretreatment with b-naphthoflavone and phenobarbitone, two other CYP inducers, was not as effective in vivo as dexamethasone. Pretreatment with N-acetylcysteine, a non-CYP-inducing hepatoprotective drug, did not protect, from ET-743-induced liver changes [77]. Recent studies assessed the capability of sandwich-cultured primary rat hepatocytes to predict the hepatoprotective effect of dexamethasone, attrib-uted, at least in part, to enhanced multidrug resistance-associated proteins biliary excretion and increased metabolism by CYP3A1/2 [78].

ET-743 RAD13 NF-Y

DNA breaks

DNA alkylationand bending

Transcriptionalregulation

Figure 2. Ecteinascidin-743 mechanism of action.ET-743: Ecteinascidin-743.



There are poor data regarding the mechanisms of hepatoxicity and hepatoprotection in humans. A recent meta-ana lysis showed that dexametha-sone pretreatment increases ET-743 systemic clearance by 19% by increasing bile flow [79]. A recent pharmacokinetic/pharmacodynamic ana-lysis predicted an enhancement of ET-743 safety and efficacy after administration of dexametha-sone and a dose-reduction strategy based on the serum concentration of liver enzymes [80].

As mentioned earlier, the other important and dose-limiting ET-743 toxicity is represented by myelosuppression. Reversible dose-dependent neutropenia was the most frequently reported toxicity in Phase II studies, with median nadir values observed at approximately 14 days [81]. ET-743-induced neutropenia is largely dependent on the intensity of dose administered and inter-dose intervals, but not the duration of intravenous infusion [82].

A pharmacokinetic/pharmacodynamic meta-ana lysis showed that the pharmacological effect of ET-743 on absolute neutrophil count was more closely related to drug concentrations in a hypothetical effect compartment, representa-tive of bone marrow, rather than from the central plasma compartment, with ET-743 reducing the proliferation rate and stimulating the killing rate of progenitor cells [81]. In mice, ET-743 toxic-ity was lower in stem cells than in committed progenitors, suggesting an unlikely long-term myelosuppression as a consequence of ET-743 treatment [83]. Other in vitro evidence suggests a direct immuno modulatory effect of ET-743 through inhibited differentiation of monocytes to macrophages and reduced production of CCL2 and IL-6 [84].

Interestingly, advanced sarcoma patients receiving dexamethasone before starting ET-743 experienced lower liver and bone marrow toxici-ties than patients not receiving dexamethasone, even though any relationship between the two toxicities remains to be elucidated [85].

Moreover, several case reports described another rare toxicity of ET-743 treatment: rhabdo myolysis. In a Phase I trial, one patient experienced grade 4 rhabdomyolysis, renal failure requiring dialy-sis, grade 4 neutropenia and grade 3 thrombo-cytopenia. Hepatic toxicity in this patient was not reported [86]. Two cases of rhabdomyolysis were reported in a subsequent French Phase II trial [87], as well as in another European Phase II study [88].

It has been shown that elevated intercycle peaks of ALP and AST, as well as increased baseline bili-rubin, are independent predictors of multiorgan toxicities [89]. For this reason, the differences in

toxicities between European and American trials may be due to a greater degree of dose adjustment in the USA based on liver enzymes. However, a fatal rhabdomyolysis case has been reported by a US group [90].

Finally, skin and soft tissue damage have been reported after ET-743 extravasation from a central venous access device [91].

Preclinical & clinical evidencesThe first preclinical evidence of ET-743 antitu-mor activity is relatively recent. Approximately 10 years ago, the effect of ET-743 was dem-onstrated in human tumors explanted from patients [92], ovarian carcinoma [93,94], melanoma and non-small-cell lung cancer [93] xenografts. In human STS cell lines explanted from chemonaive patients, gene-expression profile ana lysis revealed the upregulation of 86 genes and the down-regulation of 244 genes in response to ET-743, suggesting an important impact in tumor cell biology [95]. Interestingly, ET-743 promoted dif-ferentiation in myxoid liposarcoma tumors by detachment of the pathogenic FUS–CHOP chimera protein from its promoters [96].

Ecteinascidin-743 also showed sequence-dependent synergistic cytotoxicity with pacli-taxel (administered before ET-743) in human breast cancer cell lines in vitro and in vivo [97], and with paclitaxel (administered before ET-743) or doxorubicin (administered after ET-743) in STS cells [98].

An additive or additive-to-synergistic effect was seen in vitro with ET-743 simultaneously or after cisplatin [99], irinotecan [100] or doxorubicin [101] in human rabdomyosarcoma or ovarian cancer cell lines and in a range of human tumor xenografts, including rhabdo-myosarcoma, fibrosarcoma, non-small-cell lung cancer and ovarian cancer. Relevant effects have been also seen both in drug-sensitive and drug-resistant bone tumor cells [102]. Moreover, com-bination therapy with ET-743 and plasminogen-related protein B caused increase in human chondro sarcoma necrosis by antagonizing tumor-associated microvessel formation [103].

Finally, ET-743 exerts cell line-dependent radiosensitizing properties. Initial evidence sug-gested a moderate effect only with cytotoxic con-centrations of ET-743 [104], but more recent find-ings showed a significant in vitro radiosensitizing effect and the induction of cell-cycle changes and apoptosis in several human cancer cell lines in the presence of pharmacologically appropriate (within the nanomolar range) concentrations of the drug [105].



Several in vitro studies attempted to elucidate the mechanisms of cellular resistance to ET-743. MDR1 seems not to be crucial, since ET-743 is clearly a P-gp substrate, but it makes the cells resistant to ET-743 only when expressed at very high levels [106]. Conversely, other pathways seem to be important, for example IGF-1 recep-tor for Ewing’s sarcoma cell line TC-71 [107], and collagen I for chondrosarcoma cell line CS-1 [39].

A further confirmation of this hypothesis is that resistance to ET-743 did not correlate with resistance to doxorubicin [108], indicating that the two drugs may act through different mecha-nisms. For example, p53 mutations and deletions in sarcoma cells correlate with extreme sensitivity to ET-743 [109], whereas wild-type p53 sensitizes cells to doxorubicin by downregulating MDR1 expression [110]. Moreover, in vitro ET-743 pre-vented MDR1 gene expression, showing a syn-ergistic cytotoxic effect with doxorubicin (a well-known MDR1 substrate) [98].

Finally, a human chondrosarcoma cell line resistant to ET-743 exhibited crossresistance to cisplatin and methotrexate but not to doxoru-bicin. Zinc finger proteins, especially ZNF93, are thought to be involved in mechanisms of resistance, probably because of their role in DNA damage repair [111].

Following the preclinical studies, a large number of clinical trials have been carried out in order to evaluate the in vivo effect of ET-743. A large number of Phase I trials included patients with any solid malignancies, without focusing on specific diseases, who were preclinically more sensitive to ET-743. Table 2 shows maxi-mum tolerated doses, dose-limiting toxicities and synergies data.

These results suggested that maximum tol-erated dose slightly changes with the sched-ule (1100–1800 µg/m2) and the dose-limiting toxicities are mainly hematological toxicities (i.e., neutro penia and thrombocytopenia) and asthenia rather than transient transaminase ele-vation. It should be highlighted that ET-743 does not interact with other common chemotherapeu-tic agents (e.g., doxorubicin, gemcitabine and cisplatin) from a pharmacodynamic/pharmaco-kinetic point of view. Therefore, combination therapies could be suitable and useful in the management of advanced diseases.

Several Phase II trials have been carried out and are currently ongoing in patients with cer-tain tumors, leading to more specific and clini-cally useful results. ET-743 seems to be a very promising drug indeed for the treatment of a wide spectrum of tumors.

Nevertheless, ET-743 failed to show signifi-cant cytotoxicity against gastrointestinal stromal tumors [112,113] and pretreated advanced colorectal cancer [114].

However, several studies reported interesting rates of responses or disease stabilization during ET-743 therapies with Phase II toxicities over-lapping Phase I ones, thus confirming the clinical impact of the drug. The most promising results have been achieved against STS (especially leio-myosarcomas and myxoid liposarcoma) and ovar-ian cancers (Table 3). Retrospective studies con-firmed ET-743 efficacy in advanced pretreated STSs [115] and advanced pretreated myxoid lipo-sarcomas [116,117]. A case report showed a durable objective response lasting at least 8 months in a case of advanced, recurrent and refractory uter-ine leiomyosarcoma treated with 1200 µg/m2

intravenous ET-743 over 24 h every 3 weeks [118].In evaluating clinical trials results, it should

be noted that a recent report suggested the inad-equacy of size-based response criteria (Response Evaluation Criteria In Solid Tumors) to assess the efficacy of ET-743 in metastatic sarcoma patients [119].

More recent trials (recently completed, active or recruiting) will give further information on ET-743 cytotoxicity in several types of cancer (advanced prostate cancer, STS, ovarian cancer, breast cancer, mesothelioma, endometrial carci-noma, primary peritoneal cavity cancer and fal-lopian tube cancer) and on ET-743 potential heart toxicity (altered ECG QT intervals)[201].

In Europe, ET-743 in monotherapy (1.5 mg/m2 as a 24-h continuous infusion every 3 weeks) is currently the only agent approved as a second-line treatment in the treatment of advanced STS after the failure of anthra cycline- and ifosfamide-based regimens. The drug received orphan drug status from the European Commission for the treatment of ovarian can-cer in October 2003. The US FDA granted ET-743 orphan drug status for both STS and ovarian cancer.

As for trabectedin hepatic effects, plasma liver enzymes level (transaminases, bilirubin, alkalin phosphatases and 5´-nucleotidase) should be checked before each course, because patients with any baseline alterations have a significantly higher probability of developing severe liver tox-icity. The risk of developing grade 3–4 toxicities seems to be strongly reduced through premedi-cation with dexamethasone (4 mg orally twice daily 24 h before therapy). A study by Grosso et al. showed how elevation of transaminases, neutropenia and thrombocytopenia incidence



are significantly lower in patients pretreated with dexamethasone (2%, 2% and 0) then in not premedicated patients (34, 24 and 25%, respectively) [84].

Putative predictive factors of activityAs soon as the first promising effects of ET-743 appeared, strong efforts were made to identify putative predictive factors of activity and to reli-ably select subpopulations of patients who may benefit from ET-743 therapy.

The complex ET-743 metabolism seems to exclude CYP genes as potential candidates. NER protein levels rather than mRNA levels seem to be more promising [142]. Moreover, it has been recently proposed that screening for mutations in Fanconi anemia genes may facili-tate the identification of tumors displaying enhanced sensitivity to ET-743 [59].

No clear putative predictive factors of ET-743 activity have been identified so far. Therefore, greater efforts are needed in studies investigating such aspects.

Conclusion & future perspectiveEcteinascidin-743 is a novel natural anti-cancer agent, now synthetically produced, with unique mechanisms of action involving DNA alkylation, transcriptional repression and NER/homologous recombination-dependent cell-cycle blocking. The metabolism of ET-743 is quite complex amd is mainly achieved through liver microsomal CYPs.

Preclinical and clinical evidence has dem-onstrated how trabectedin combines a favor-able tolerability profile with proven efficacy in the treatment of advanced STS and ovar-ian cancer. Moreover, ET-743 does not seem

Table 2. Phase I concluded clinical trials.

Drug(s) Tumors Administration Results Ref.

ET-743 Resistant solid tumors

24 h intravenous q21 MTD: 1800 µg/m2

1500 µg/m2 dose is clinically feasibleDLTs: severe thrombocytopenia and neutropenia

[121,122]

ET-743 STSs 24 h intravenous q21 1500 µg/m2

Risk of severe toxicity enhanced in patients with hepatic dysfunctionDexamethasone co-treatment decreased incidence of severe toxicity and AUC

[123]

ET-743 Solid tumors 72 h intravenous q21 MTD: 1200 µg/m2

DLTs: neutropenia, thrombocytopenia and fatigue

[86]

ET-743 Solid tumors 1 h intravenous q21 vs 3 h intravenous q21

1 h MTD: 1100 µg/m2

1 h DLTs: thrombocytopenia and fatigue3 h MTD: 1800 µg/m2

3 h DLTs: pancytopenia and fatigue1650 µg/m2 dose is clinically feasible

[124]

ET-743 Refractory solid tumors in children

3 h intravenous q21 Premedication with dexamethasone followed by 1100 µg/m2 dose

[125]

ET-743 Advanced solid tumors

1 h intravenous for 3 wks q28 vs 3 h intravenous for 3 wks q28

1 h MTD: 610 µg/m2 3 h MTD: 580 µg/m2 DLTs: febrile neutropenia and fatigue

[126]

PLD plus ET-743 Advanced solid tumors

1 h intravenous PLD (30 mg/m2), then 3 h intravenous ET-743 q21

MTDET-743: 1100 µg/m2

ET-743 plus PLD generally well tolerated

[127]

Gemcitabine plus ET-743

Advanced solid tumors

Intravenous on days 1–8-15 of q28 cycle

Study closed for unacceptable frequency of adjustments to the weekly dosing schedule for hepatic toxicityNo DLTs observed

[128]

Doxorubicin plus ET-743

STSs 10’/15´ intravenous doxorubicin (60 mg/m2) followed by 3 h intravenous on day 1 q21

MTDET-743: 1100 µg/m2

DLT: neutropenia

[129]

Cisplatin plus ET-743

Solid tumors Intravenous day 1 and 8 q21 MTDET-743: 600 µg/m2

MTDCDDP: 40 mg/m2

DLT: febrile neutropenia

[130]

Doxorubicin plus ET-743

Advanced STS and breast cancer

Intravenous q21 MTDET-743: 800 µg/m2

MTDDOXO: 60 mg/m2

DLTs: febrile neutropenia and asthenia

[131]

AUC: Area under curve; DLT: Dose-limiting toxicity; MTD: Maximum tolerated dose; PLD: Pegylated liposomal doxorubicin; STS: Soft tissue sarcoma; wk: Week.



to pharmacologically interact with other anti-neoplastic drugs, demonstrating additive or additive-to-synergic effects with common chemotherapeutic agents. Further studies are needed to assess its activity in combination with standard chemotherapeutic agents and with new biological compounds (i.e., monoclonal antibodies or small-molecule tyrosine kinase inhibitors) in STS and also in other human neoplasia of different histological origins.

Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a finan-cial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Table 3. Conducted Phase II clinical trials.

Tumors Administration Results Toxicities Ref.

STS: 25 OS: 3Ewing’s sarcoma: 1 PD at accrual

24 h intravenous q21–28 1200–1800 µg/m2

STS: 2 PRs OS: 2 PRs2 MRs, 10 SDs

Transient transaminitis G3 (24%) and G4 (5%), neutropenia G3–4 (32%) with concomitant G3–4 thrombocytopenia (5.1%), asthenia G2–3 (21%)

[132]

Advanced pretreated STS: 54 (41% leiomyosarcoma)

24 h intravenous q211500 µg/m2

2 PRs, 4 MRs,9 SDs, 3 CRs after surgery

Transient transaminitis G3–4 (50%), neutropenia G3–4 (61%), nausea, vomiting and asthenia

[88]

Advanced anthracycline-pretreated STS: 54


3 PRs, 4 MRs22 SDs

Transient transaminitis G3–4 (~60%), neutropenia G3–4 (~60%)

[87]

Progressive refractory SFS: 36


1 CR, 2 PRs,2 MRs

Neutropenia G3–4 (34%), transient transaminitis G3–4 (26%)

[133]

Advanced STS: 99 24 h intravenous q211500 µg/m2

8 PRs, 45 SDs Neutropenia G3–4 (52%),transient transaminitis G3–4 (40%)

[134]

Unresectable advanced chemonaive STS


1 CR, 5 PRs, 1 MR Neutropenia G3–4 (33%), transient transaminitis G3–4 (36%)

[135]

Liposarcoma and leiomyosarcoma: 270

24 h intravenous q211500 µg/m2 vs3 h intravenous every wk for 3 wks q28 580 µg/m2

Reduction in the relative risk of progression for patients treated in the q21

Neutropenia G3–4 (21% in 24 h arm vs 2% in 3 h arm), transient transaminitis G3–4 (32% in 24 h arm vs 3% in 3 h arm)

[136]

Pretreated advanced sarcoma: 21

24 h intravenous q21900–1500 µg/m2

3 PRs, 8 SDs Neutropenia G3–4 (3), transient transaminitis G3–4 (8)

[137]

Ovarian carcinoma: platinum/taxane resistant (30) or sensitive (29)


Platinum sensitive: 1 CR, 9 PRs, 9 SDsPlatinum resistant: 2 PRs, 8 SDs

Transient transaminitis, asthenia, neutropenia

[138]

Ovarian carcinoma: platinum resistant (79) or sensitive (62)

3 h intravenous q28580 µg/m2 once weekly for 3 wks

Platinum sensitive: 4 CR, 14 PRs, 22 SDsPlatinum resistant: 5 PRs, 36 SDs

Neutropenia G3–4 (8%), transient transaminitis G3–4 (12%)

[139]

Relapsed, platinum-sensitive, advanced ovarian cancer: 107

Arm A: 24 h intravenous q21 1500 µg/m2 vs Arm B: 3h intravenous q21 1300 µg/m2

Arm A: ORR 38.9%Arm B: ORR 35.8%

Neutropenia 55% arm A and 37% arm B, transient transaminitis 55% arm A and 59% arm B, nausea/vomiting and asthenia

[140]

Persistent or recurrent endometrial carcinoma: 50

3 h intravenous q21 1300 µg/m2

1 CR, 18 SDs Transient transaminitis G3–4 (40%), neutropenia G3–4 (13%), asthenia G3–4 (14%)

[141]

Pretreated advanced breast cancer: 21


4 PRs, 2 MRs, 6 SDs Transient transaminitis [142]

GIST: 20 24 h intravenous q211500 µg/m2

No ORR, 2 SDs Transient transaminitis G3 (10 patients), asthenia G3 (1 patient)

[113]

GIST: 27 24 h intravenous q211500 µg/m2

No ORR, 9 SDs Transient transaminitis G3–4 (47%), neutropenia G3–4 (48%)

[114]

Pretreated advanced colorectal cancer: 21


No ORR, 4 SDs Transient transaminitis G3–4 (62%), neutropenia G3–4 (42.8%)

[115]

CR: Complete response; GIST: Gastrointestinal stromal tumor; MR: Minimal response; ORR: Overall response rate; OS: Overall survival; PD: Progressive disease; PR: Partial response; SD: Stable disease; STS: Soft tissue sarcoma; wk: Week.



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Executive summary

Trabectedin in the past: from discovery to industrial synthesis � Since the late 1960s, extracts from the Caribbean tunicate Ecteinascidia turbinata have shown promising anticancer activity. � In 1984, the structure of Ecteinascidin (ET)-743 was finally determined. � Since 1996, several chemical processes for synthesis have been achieved.

Pleiotropic & complex mechanisms of action � ET-743 acts as a DNA-alkylating drug, but functionally resembling classical ‘topoisomerase poisons’. � ET-743 inhibits the transcription of inducible genes (notably MDR1) via different pathways (e.g., NF-Y mediated, Sp1 mediated and

SXR mediated). � ET-743 interacts with DNA-repairing pathways (notably nucleotide excision repair and homologous recombination) owing to its ability to

form adducts and induce DNA breaks.

Metabolism & toxicities � The liver is the key organ for both metabolism and specific (i.e., nonhematological) toxicities. � In vivo, dexamethasone reduces both liver and hematological toxicities, probably via interference with NF-kB transcriptional activity.

Preclinical/clinical evidences � ET-743 shows pronounced antineoplastic activity in very different cancer line cells, both alone and in combination with common

chemotherapeutic agents. � ET-743 in monotherapy (1.5 mg/m2 as a 24-h continuous infusion every 3 weeks) is the only agent approved as a second-line therapy in

the treatment of advanced soft tissue sarcoma (Europe). � ET-743 has received the orphan drug status for the treatment of ovarian cancer (Europe and USA) and for soft tissue sarcoma (USA). � The risk of developing grade 3–4 toxicities seems to be strongly reduced after premedication with dexamethasone.

Trabectedin in the future � Further studies are needed to understand specific predictive factors of response to trabectedin. � Future studies are needed to study trabectedin-based schedules in combination with chemotherapeutic and biological agents

(i.e., monoclonal antibodies and small inhibitors).



875www.futuremedicine.com

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�n Website201 Ongoing and unpublished clinical trials

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�n Patent301 Metabolites of Ecteinascidin-743

WO 99/58125 (1999).

Wide-spectrum Characterization of Trabectedin

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