Trabectedin Research Articles

Abstract 3709: PARP1 expression (PARP1expr) drives synergy between PARP1 inhibitors (PARP1-Is) and trabectedin (TR)

Abstract Purpose of study. An attractive strategy to improve antitumor treatments is to inflict cytotoxic DNA damage with chemotherapy, and then impede DNA repair by molecular targeting. TR is a new drug characterized by a peculiar mechanism of action: TR traps DNA repair machinery leading to DNA damage, particularly in BRCA1/2-deficient tumors. We speculated that TR might activate PARP1, a key player in DNA-repair, and that subsequent PARP1 inhibition perpetuates TR-induced DNA damage leading to cell death. Experimental procedures and results. We developed a preclinical platform of 31 cell lines from different histotypes to explore the potential synergy between TR and the PARP1-Is olaparib (OL) and veliparib. We demonstrated that, regardless of BRCA1/2 status, PARP1-Is significantly increased TR activity, but a 15-fold range of sensitivity to the combination was observed. OL was proven the best PARP-I to combine with TR, probably due to its PARP1 trapping activity. In selected experiments, whole-genome expression profiling and GSEA analysis comparing cells displaying high vs. low synergism of the combination (HS-C vs. LS-C) revealed that DDR, G2/M cell cycle checkpoint, and DNA repair pathways were mechanistically involved in TR+OL synergy. TR induced PARP1 activation in 3/6 cell lines and PARP1-Is completely blocked both basal and TR-induced PARP1 activation. OL enhanced DNA damage response in 6/6 cell lines, but unrepairable DNA fragmentation was obtained in cells with high PARP1expr only. In two independent cell panels TR+OL synergism was directly related to PARP1expr both at mRNA (Pearson score r: 0.70, p = 0.00079) and protein level (r: 0.71, p = 0.015). Silencing and overexpression experiments validated the functional role of PARP1expr in determining TR+OL synergism: in HS-C the downmodulation of PARP1expr reduced sensitivity to TR+OL while the overexpression of PARP1 in LS-C rose TR+OL activity to levels observed in HS-C. Subcutaneous, intravenous and orthotopic xenografts of one HS-C (DMR) and one LS-C (SJSA-1) in NOD/SCID mice revealed OL significantly increased antitumor and antimetastatic activity of TR in cells with high PARP1expr only. Finally, we demonstrated that basal PARP1expr PARP1 activation by other cytotoxics with a stronger PARP1 activation observed in cells with high vs. low PARP1expr, regardless of the considered drug. Conclusions. OL enhances and potentially broaden TR cytotoxicity. TR+OL combination is particularly attractive in tumors harboring high PARP1expr and specific DDR-R gene signatures that might become predictive biomarkers of response. Future clinical validation of TR+OL combination may extend the use of PARP1-Is beyond BRCA1/2 defective tumors. Indeed, the crucial role of PARP1expr is confirmed regardless of tumor histotype and BRCA1/2 status. Further studies of combination between PARP1-Is and other cytotoxics should consider basal PARP1expr and activation after drug exposure. Citation Format: Ymera Pignochino, Federica Capozzi, Lorenzo D’ambrosio, Carmine Dell’aglio, Marco Basiricò, Paola Boccone, Erica Palesandro, Loretta Gammaitoni, Dario Sangiolo, Maria Serena Benassi, Massimo Aglietta, Giovanni Grignani. PARP1 expression (PARP1expr) drives synergy between PARP1 inhibitors (PARP1-Is) and trabectedin (TR). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3709.

Biosynthesis of Tetrahydroisoquinoline Antibiotics.

The tetrahydroisoquinoline (THIQ) alkaloids are naturally occurring antibiotics isolated from a variety of microorganisms and marine invertebrates. This family of natural products exhibit broad spectrum antimicrobial and strong antitumor activities, and the potency of clinical application has been validated by the marketing of ecteinascidin 743 (ET-743) as anticancer drug. In the past 20 years, the biosynthetic gene cluster of six THIQ antibiotics has been characterized including saframycin Mx1 from Myxococcus xanthus, safracin-B from Pseudomonas fluorescens, saframycin A, naphthyridinomycin, and quinocarcin from Streptomyces, as well as ET-743 from Ecteinascidia turbinata. This review gives a brief summary of the current status in understanding the molecular logic for the biosynthesis of these natural products, which provides new insights on the biosynthetic machinery involved in the nonribosomal peptide synthetase system. The proposal of the THIQ biosynthetic pathway not only shows nature's route to generate such complex molecules, but also set the stage to develop a different process for production of ET-743 by synthetic biology.

C-reactive protein (CRP) and peripheral blood monocyte count (PBMC) in patients (pts) with advanced soft tissue sarcomas (ASTS) treated with trabectedin (TR): correlation with progression-free survival (PFS).

e21517 Background: Several evidences showed systemic inflammation is directly implicated in cancer development and progression. Accordingly, a relationship between inflammatory features (CRP, neutr...

Predictive biomarkers of trabectedin (TR) and olaparib (OL) synergism in preclinical models of bone and soft tissue sarcoma (BSTS).

10569 Background: TR is a DNA damaging agent as 2nd line-therapy in STS. We speculated that its antitumor activity could be potentiated by a “synthetic lethality” approach targeting key molecules i...

Pegylated liposomal doxorubicin for relapsed epithelial ovarian cancer.

Ovarian cancer is the eighth most common cancer in women and it is usually diagnosed at an advanced stage. The majority of ovarian tumours are epithelial in origin. Women with relapsed epithelial ovarian cancer (EOC) often have a reduced performance status with a limited life expectancy, therefore maintaining quality of life with effective symptom control is the main purpose of treatment. Drug treatment of relapsed disease is directed by the platinum-free interval: relapsed platinum-sensitive disease is usually re-treated with platinum-based therapy and platinum-resistant disease challenged with non-platinum drugs. However, the side-effects of chemotherapy agents may be severe and optimal treatment regimens are unclear. Pegylated liposomal doxorubicin (PLD), which contains a cytotoxic drug called doxorubicin hydrochloride is one of several treatment modalities that may be considered for single-agent treatment of relapsed EOC, or used in combination with other drugs. To assess the efficacy and safety of PLD in women with relapsed epithelial ovarian cancer (EOC). We searched the Cochrane Gynaecological Cancer Group (CGCG) trials register, CENTRAL, MEDLINE and EMBASE from 1990 to February 2013. We also searched online registers of clinical trials, abstracts of scientific meetings and reference lists of included studies. Randomised controlled trials (RCTs) that evaluated PLD in women diagnosed with relapsed epithelial ovarian cancer. Two review authors independently abstracted data to a pre-designed data collection form and assessed the risk of bias according to the Cochrane Handbook for Systematic Reviews of Interventions guidelines. Where possible, we pooled collected data in meta-analyses using RevMan 5.2 software. We included 14 RCTs that evaluated PLD alone or in combination with other drugs. Four RCTs contributed no data to the meta-analyses. Two studies compared PLD plus carboplatin (carbo) to paclitaxel (PAC)/carbo in women with platinum-sensitive relapsed EOC. Overall survival (OS) was similar for these treatments, however progression-free survival (PFS) was longer with PLD/carbo (1164 participants; hazard ratio (HR) 0.85, 95% confidence interval (CI) 0.74 to 0.97; I² = 7%; P value 0.01). PLD/carbo was associated with significantly more anaemia and thrombocytopenia than PAC/carbo, whereas PAC/carbo was associated with significantly more alopecia, neuropathies, hypersensitivity reactions and arthralgias/myalgias. PLD/carbo was well-tolerated and women receiving this treatment were significantly less likely to discontinue treatment than those receiving PAC/carbo (two studies, 1150 participants; risk ratio (RR) 0.38, 95% CI 0.26 to 0.57; I² = 0%; P < 0.00001).Five studies compared other agents to PLD alone. None of these agents were associated with significantly better survival or severe adverse-event profiles than PLD. Topotecan and gemcitabine were associated with significantly more haematological severe adverse events than PLD, and patupilone was associated with significantly more severe neuropathies and diarrhoea. Severe hand-foot syndrome (HFS) occurred consistently more frequently with PLD than the other drugs.Three studies compared PLD combination treatment to PLD alone. Two combinations resulted in a significantly longer PFS compared with PLD alone: trabectedin (TBD)/PLD (one study, 672 women; HR 0.79, 95% CI 0.65 to 0.96; P value 0.02) and vintafolide (EC145)/PLD (one study, 149 women; HR 0.63, 95% CI 0.41 to 0.97; P value 0.04). TBD/PLD appeared to benefit the partially platinum-sensitive subgroup only. Further studies are likely to have an important impact on our confidence in these estimates. TBD/PLD was associated with significantly more haematological and gastrointestinal severe adverse events than PLD alone, whereas EC145/PLD appeared to be well-tolerated.For platinum-resistant relapsed EOC, the median PFS and OS for single-agent PLD across seven included studies was 15 weeks and 54 weeks, respectively. Severe HFS occurred significantly more frequently in women receiving a 50 mg/m² dose of PLD than those receiving less than 50 mg/m² (17% versus 2%, respectively; P value 0.01). In platinum-sensitive relapsed epithelial ovarian cancer, PLD/carbo is more effective than PAC/carbo and is better tolerated; PLD/carbo should therefore be considered as first-line treatment in women with platinum-sensitive relapsed EOC. PLD alone is a useful agent for platinum-resistant relapsed EOC, however it remains unclear how it compares with other single agents for this subgroup and in what order these agents should be used. There is insufficient evidence to support the use of PLD in combination with other agents in platinum-resistant relapsed EOC.

Abstract LB-213: The antitumor effect of trabectedin (TR) is potentiated by olaparib (OL) in preclinical models of bone and soft tissue sarcomas (STS).

Abstract Background. TR is a DNA-binding alkaloid approved in Europe for the treatment STS as second/third line therapy. TR interferes with gene transcription and nucleotide excision repair, inducing DNA double strand breaks (DSBs). There is a strong clinical interest to increase TR activity by combination with other anti-cancer drugs. PARP-1 inhibitors (PARPi) which disable DNA base-excision repair mechanism causing the accumulation of DSBs, look like a worth to explore TR therapeutic partners. Our preclinical studies were focused on the effects of the combination of TR with the PARPi (OL and veliparib). Methods: We explored DNA damage by comet assay and P-H2AX determination, PARP-1 activity by polyADPribosylation assay in a panel of 18 sarcoma cell lines, evaluating cell viability after 72h treatment with escalating doses of TR, PARPi, and their constant combination. Colony growth, cell cycle, apoptosis and DNA damage were evaluated. Antitumoral activity was checked after 4 weeks of treatment with 3 endovenous injection of TR once a week and OL intraperitoneally 5 days/week, and compared to control and single agents in bone (SJSA-1) and soft tissue sarcoma (DMR) xenografted NOD/SCID mice. TUNEL assay, P-H2AX, PCNA and CD31 immunohistochemistry was done on explanted xenografts. Results: We showed TR-induced DNA damage and PARP-1 activation in sarcoma sensitive cell line. The addition of PARPi completely blocked basal and TR-induced PARP-1 activation. DNA damage response and checkpoints (ATM, ATR, BRCA1, CHK1, CHK2, p53) were activated after single agents and combination treatment inducing cell cycle arrest in S/G2 phase. This block was released after 48h in single agent-treated cells but not in combination-treated cells leading to apoptotic cell death. Colony growth assay and viability tests revealed a strong synergism of the two drugs (Combination Index &amp;lt; 1, based on Chou-Talalay method). Different sarcoma histotypes displayed differences in sensitivity to both drugs and combination (Ewing's sarcoma the most sensitive&amp;gt; myxoid liposarcoma &amp;gt; leiomyosarcoma &amp;gt; osteosarcoma ≥ undifferentiated pleomorphic &amp;gt; fibrosarcoma). The antitumor effect of combined TR and OL was shown after in vivo treatment of SJSA-1 and DMR xenografted mice. A significant tumor volume reduction was observed if compared to both control and single agent and was attributed to increased DNA damage, apoptosis and reduced proliferation, but not to anti-angiogenic potentiation. In fact, a direct effect on tumor vasculature was shown in TR and combination treated group vs control. Conclusions: Our results validate the biological rationale to combine TR and PARPi in sarcoma and suggest exploring this pharmacological approach in the clinical setting. Citation Format: Ymera Pignochino, Federica Capozzi, Carmine Dell'aglio, Marco Basiricò, Lorenzo D'ambrosio, Danilo Galizia, Erica Palesandro, Maria Serena Benassi, Massimo Aglietta, Giovanni Grignani. The antitumor effect of trabectedin (TR) is potentiated by olaparib (OL) in preclinical models of bone and soft tissue sarcomas (STS). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-213. doi:10.1158/1538-7445.AM2013-LB-213

Preclinical evidence of ET-743 as a potential chemotherapy option for the treatment of biliary carcinoma.

193 Background: The standard chemotherapy for unresectablebiliary tract carcinoma (BTC) is based on gemcitabine and platinum compounds. However, these combinations have not been shown to be effective in improving long-term survival. Thus, there is a real need to find new strategies that would impact in a significant way on clinical outcome. Ecteinascidin-743 (ET-743), a compound isolated from the marine tunicate Ecteinascidia turbinata. ET-743, is approved for the treatment of ovarian cancer and soft tissue sarcoma. Phase II and III clinical trials are ongoing for the treatment of different solid tumors. No preclinical data are available about the efficacy of ET-743 in BTC. In a phase I study, one patient received ET-743 plus capecitabine and experienced a long lasting complete metabolic response. Here, we investigated the antitumor activity of ET-743 in preclinical BTC models. Methods: Four BTC cell lines TFK1, EGI-1, HuH28 and TGBC1 were used to evaluate the effect of ET-743 on proliferation, cell cycle, apoptosis and on the activation of DNA damage proteins. The effect on proliferation was also investigated on a primary cell culture of a gallbladder carcinoma (GBC) resistant to gemcitabine and oxaliplatin. On the same cells, the inhibition of VEGF secretion mediated by ET-743 was analyzed by ELISA. The anti-tumor activity of ET-743 was tested on EGI-1 xenografts in NOD/SCID mice. Results: In vitro, ET-743 is able to markedly reduce cell proliferation of BTC cell lines through cell cycle blockage on G0/G1 phase and to inhibit the growth of primary cell culture derived from GBC patient. Moreover, ET-743 promotes apoptosis by caspase 3 activation, activates proteins involved in DNA damage and reduces VEGF secretion. In the in vivo model, ET-743 is able to slow tumor growth in BTC xenograft. The mechanism of anti-tumor activity involves DNA damage, the induction of hypoxia transcription factor-1, and angiogenesis inhibition. ET-743 has no significant effect on apoptosis in vivo. Conclusions: These data suggest that ET-743 could represent an alternative chemotherapy for BTC treatment and encourage the development of clinical trials of ET-743 in BTC patients.

350 Poly (ADP-ribose) Polymerase-1 (PARP-1) Inhibitors Potentiate Trabectedin Activity in Preclinical Models of Bone and Soft Tissue Sarcomas

Background: Trabectedin (TR) is a DNA-binding alkaloid approved in Europe for the treatment of soft tissue sarcoma as second/third line therapy. TR binds to the minor groove of DNA and interferes with gene transcription and nucleotide excision repair, inducing DNA double strand breaks (DSBs), and S/G2 cell cycle arrest. There is a strong clinical interest to increase TR activity by combination with other anti-cancer drugs. PARP-1 inhibitors (PARP1-i) which disable DNA base-excision repair mechanism causing the accumulation of DSBs, look like a worth to explore TR therapeutic partners. We focused our in vitro studies on the effects of the combination of TR with the PARP1-i olaparib (OL) and veliparib (VEL). Methods:We explored DNA damage by comet assay and phospho-histone H2AX determination and PARP-1 activity by polyADPribosylation assay in a panel of sarcoma cell lines treated with TR, OL,VEL and combinations. We evaluated cell viability after 72h treatment with escalating doses of TR (0−2nM), PARP1-i (VEL:0−80mcM; OL:0−20mcM), and their constant combinations. Following colony formation, cell cycle, apoptosis and DNA damage response were checked. Results: We demonstrated that TR-induced DNA damage and PARP1 activation in sarcoma sensitive cell line. The addition of PARP1i completely blocked basal and TR-induced PARP-1 activation. DNA damage response and checkpoints (ATM, ATR, BRCA1, CHK1, CHK2, p53) are activated after treatments with single agents and combinations inducing cell cycle arrest in S/G2 phase. This block was release after 48h in single agents-treated cells but not in combinations-treated cells leading to apoptotic cell death. Colony growth assay and viability tests revealed a strong synergism of the two drugs (Combination Index leyomiosarcoma > osteosarcoma undifferentiated pleomorphic > fibrosarcoma the most resistant). Determination of molecular markers of sensitivity and in vivo antitumoral activity studies are ongoing. Conclusions: Our results validate the biological rationale to combine TR and PARP1-i in sarcomas and suggest exploring this pharmacological approach in the clinical setting.

351 Temozolomide-sensitizing Effects of ABT-888 Are Limited to Inherently TMZ-sensitive MGMT Hypermethylated GBM Xenografts

Background: Trabectedin (TR) is a DNA-binding alkaloid approved in Europe for the treatment of soft tissue sarcoma as second/third line therapy. TR binds to the minor groove of DNA and interferes with gene transcription and nucleotide excision repair, inducing DNA double strand breaks (DSBs), and S/G2 cell cycle arrest. There is a strong clinical interest to increase TR activity by combination with other anti-cancer drugs. PARP-1 inhibitors (PARP1-i) which disable DNA base-excision repair mechanism causing the accumulation of DSBs, look like a worth to explore TR therapeutic partners. We focused our in vitro studies on the effects of the combination of TR with the PARP1-i olaparib (OL) and veliparib (VEL). Methods:We explored DNA damage by comet assay and phospho-histone H2AX determination and PARP-1 activity by polyADPribosylation assay in a panel of sarcoma cell lines treated with TR, OL,VEL and combinations. We evaluated cell viability after 72h treatment with escalating doses of TR (0−2nM), PARP1-i (VEL:0−80mcM; OL:0−20mcM), and their constant combinations. Following colony formation, cell cycle, apoptosis and DNA damage response were checked. Results: We demonstrated that TR-induced DNA damage and PARP1 activation in sarcoma sensitive cell line. The addition of PARP1i completely blocked basal and TR-induced PARP-1 activation. DNA damage response and checkpoints (ATM, ATR, BRCA1, CHK1, CHK2, p53) are activated after treatments with single agents and combinations inducing cell cycle arrest in S/G2 phase. This block was release after 48h in single agents-treated cells but not in combinations-treated cells leading to apoptotic cell death. Colony growth assay and viability tests revealed a strong synergism of the two drugs (Combination Index leyomiosarcoma > osteosarcoma undifferentiated pleomorphic > fibrosarcoma the most resistant). Determination of molecular markers of sensitivity and in vivo antitumoral activity studies are ongoing. Conclusions: Our results validate the biological rationale to combine TR and PARP1-i in sarcomas and suggest exploring this pharmacological approach in the clinical setting.

Preclinical study of trabectedin (TR) and poly(ADP-ribose)polymerase 1 (PARP-1) inhibitor combination in soft tissue sarcoma (STS).

10066 Background: TR is an alkylating agent approved in Europe for the treatment of advanced STS as second/third line therapy. TR binds to the minor groove of DNA and interferes with gene transcription and nucleotide excision repair mechanism, inducing DNA double strand breaks (DSBs), and S/G2 cell cycle arrest. There is a strong clinical interest to increase TR activity combining it with other anti-cancer drugs. PARP-1 inhibitors disable DNA base-excision repair mechanism causing the accumulation of DSBs and look like a reasonable TR partner to be explored. We focused our in vitro studies on the effects of the combination of TR with the PARP-1 inhibitor Olaparib (OL). Methods: We explored the activity of TR-OL combination against a panel of different histotypes of STS cell lines, evaluating cell viability after 72h treatment with escalating doses of TR (0-2 nM), OL (0-20 µM), and their constant combination. Following colony formation, cell cycle, apoptosis (annexin V+/PI+) and DNA damage (phospho-histone H2AX - Ser139) were checked. Results: The TR-OL combination strongly affects STS cell viability, showing synergism (Combination Index &lt; 1, based on Chou–Talalay method) on 8 out of 13 cell lines tested. We observed a strong synergism, as a massive reduction of colony growth (402.91, MES-SA, and DMR-SN-8.4.98 lines) induced by the combination if compared with each single agent (78% vs. ~27% : p&lt;0.05). OL potentiates the S/G2 cell-cycle arrest caused by TR at 48h (Control= 29%, TR= 33.4%, OL= 31.5%, TR-OL= 80.9%), and induces a strong increase of the apoptotic cells at 72h (Control= 17.4%, TR= 28.3%, OL= 33.5%, TR-OL= 56.8%). Furthermore, the TR-OL synergism on DNA damage is confirmed by a significant increase of the DSBs marker (Control= 8.7%, TR= 59.5%, OL= 23.8%, TR-OL= 76.5%). Conclusions: These results validate the biological rationale to combine TR and PARP-1 inhibitors in STS and suggest assessing this drug combination in the clinical setting.

Analysis of Hohn (HC) and porth-a-cath (PAC) central venous catheters (CVC) adverse events (AEs) and related costs in advanced soft tissue sarcomas (STS) treated with trabectedin (TR) 24-hour (24-h) infusion therapy.

e20512 Background: TR is registered as second line in STS. The 24-h infusion schedule showed a better activity profile but a CVC is mandatory to administer TR with related costs and possible complications. The HC (Bard Access System, USA) is a silicone, open-ended, non-tunneled CVC characterized by low cost and easy placement and removal. We performed a retrospective analysis of STS patients (pts) treated with TR through a CVC to evaluate the incidence of CVC AEs (malfunctioning, thrombosis, infection or extravasation) in pts carrying HC or PAC. Methods: Eligible criteria were: at least 1 course of TR via CVC, adequate premedication (steroid and 4000 units/die of low molecular weight heparin), absence of previous CVC alterations or any underlying coagulation disorder. All CVC were placed in our Institution under radiological control and full medical history was available for all pts included into the study. Average total costs (placement, hospital admission, device, and medications) are 255 and 560 $ for HC and PAC, respectively. Results: Between March 2008 and October 2011 42 pts, median age 58 (20-74) yrs, received TR after placement of a CVC: 14 (33%) HC and 28 (67%) PAC. At the time of TR administration, pts had received a median of 2 (range 1-4) chemotherapeutic lines. A total of 185 courses of TR (44 via HC / 141 via PAC) were administered at a median dose of 1.3 mg/m2 with a median of 3 (1-22) courses per pts. AEs occurred after a median of 2 (range 1-6) TR courses. We recorded 13 CVC-related AEs: 7 infection (2 HC / 5 PAC), 4 thrombosis (2 HC / 2 PAC), 2 malfunctioning (1 HC / 1 PAC,) no extravasation. CVC had to be removed and substituted in 10 pts (5 HC/5 PAC) with an estimated total cost of 280 and 680 $ for HC and PAC, respectively. In PAC pts, AEs determined 5 dose delays, only. Conclusions: CVC AEs should be considered in the management of pts treated with TR. HC showed a favorable safety profile and its AEs management is easier compared to implanted CVC. If disease does not progress after 2 cycles, HC can be converted in an implanted CVC. However, HC is also an adequate and cheaper device when best supportive care is the only useful therapy left.

Hijacking a hydroxyethyl unit from a central metabolic ketose into a nonribosomal peptide assembly line

Nonribosomal peptide synthetases (NRPSs) usually catalyze the biosynthesis of peptide natural products by sequential selection, activation, and condensation of amino acid precursors. It was reported that some fatty acids, α-ketoacids, and α-hydroxyacids originating from amino acid metabolism as well as polyketide-derived units can also be used by NRPS assembly lines as an alternative to amino acids. Ecteinascidin 743 (ET-743), naphthyridinomycin (NDM), and quinocarcin (QNC) are three important antitumor natural products belonging to the tetrahydroisoquinoline family. Although ET-743 has been approved as an anticancer drug, the origin of an identical two-carbon (C(2)) fragment among these three antibiotics has not been elucidated despite much effort in the biosynthetic research in the past 30 y. Here we report that two unexpected two-component transketolases (TKases), NapB/NapD in the NDM biosynthetic pathway and QncN/QncL in QNC biosynthesis, catalyze the transfer of a glycolaldehyde unit from ketose to the lipoyl group to yield the glycolicacyl lipoic acid intermediate and then transfer the C(2) unit to an acyl carrier protein (ACP) to form glycolicacyl-S-ACP as an extender unit for NRPS. Our results demonstrate a unique NRPS extender unit directly derived from ketose phosphates through (α,β-dihydroxyethyl)-thiamin diphosphate and a lipoyl group-tethered ester intermediate catalyzed by the TKase-ACP platform in the context of NDM and QNC biosynthesis, all of which also highlights the biosynthesis of ET-743. This hybrid system and precursor are distinct from the previously described universal modes involving the NRPS machinery. They exemplify an alternate strategy in hybrid NRPS biochemistry and enrich the diversity of precursors for NRPS combinatorial biosynthesis.

In vivo investigation of the role of SfmO2 in saframycin A biosynthesis by structural characterization of the analogue saframycin O

Saframycin A (SFM-A), a tetrahydroisoquinoline antibiotic isolated from Streptomyces lavendulae , shows potent anti-proliferation activities against a variety of tumor cell lines, and shares the core structure with ecteinascidin 743 (ET-743), the anticancer drug for soft-tissue sarcoma. Characterization of the SFM-A biosynthetic gene cluster revealed three nonribosomal peptide synthetase genes and a series of genes encoding oxygenases. To investigate the function of sfmO2 gene, encoding a FAD-dependent monooxygenase/hydroxylase, we constructed the gene replacement mutant (Δ sfmO2 ) strain S. lavendulae TL2007 and the corresponding gene complementation mutant strain S. lavendulae TL2008. A novel compound, SFM-O, was isolated from the Δ sfmO2 replacement mutant strain and its structure was characterized by comparison to the HRMS and NMR spectra of SFM-A. These findings indicated that sfmO2 is responsible for the oxidation of ring A in the biosynthetic pathway of SFM-A, and the new compound SFM-O could be considered as an advanced intermediate in the semisynthesis of ET-743.

Administration of 24-hour intravenous infusions of trabectedin every 3 weeks in ambulatory patients with mesenchymal tumors via disposable elastomeric pumps

e13530 Background: Patients (pts) with sarcoma whose disease progresses after standard chemotherapy have poor outcome. In this setting, DNA- transcription-interacting cytotoxic agent trabectedin (TRA) is efficacious and marketed in Europe. It is administered as 24-h i.v. infusion q3w with steroid co-medication. To overcome the inconvenience of hospitalization for drug delivery TRA is now given in Leuven via disposable elastomeric pumps, which facilitate ambulatory treatment and are compatible with the drug. Material and Methods: Heavily pre-treated pts with sarcoma were offered chemotherapy with TRA 1.5 mg/m2 as 24-h i.v. infusion via port catheter, either during hospitalization using electronic pumps or as outpatients using the Baxter LV10 disposable pump (drug dissolved in 267 ml NaCl 0.9%). Co-medication consisted of antiemetics and dexamethasone 2x4 mg days -1,1,2,3. Results: Between 09/07–12/08 28 pts were treated, and 21 (75%) elected outpatient therapy (9 F, 12 M, med. age 49 yrs, range 19–68). Common diagnoses included leiomyo- (5), lipo- (4), synovial (2) and myxofibrosarcomas. Pts had previous primary surgery (17), adjuvant RT (4) and surgery for relapse/metastasis (7). They had local relapse (2), distant metastasis (12) or both (7) when starting TRA, 19 had received previous chemotherapy with a med. number of 2 prior lines (range, 0–5). We administered 130 cycles of TRA in 21 pts, with a med. number of 3 cycles/patient (range, 1–24). Dose reductions were done in 60 cycles, mainly due to laborabory events. Best response (RECIST) was 3 confirmed PR, 1 PR pending confirmation, 6 SD, 11 PD. Grade 3/4 (CTC) AEs were limited to one case each of hemorrhage and lung embolism, other AEs were in line with published TRA experience. One port catheter contamination required replacement, one catheter tip thrombosis occurred and one extravasation due to needle dislocation was observed. Conclusions: Outpatient administration of TRA as 24-h infusion via port catheter using Baxter LV10 pumps is preferred by 3/4 pts, is feasible, safe, effective, cost-efficient and should be considered routine practice in this clinical setting. [Table: see text]

Cytotoxic and Apoptogenic Activity of a Methanolic Extract from the Marine Invertebrate Ciona intestinalis on Malignant Cell Lines

Marine invertebrates provide a series of natural products with different biological activities. Several of these compounds and their derivatives showed a potent anticancer effect. Tunicates represent an important source of bioactive agents, leading to the isolation of ecteinascidin-743 (ET-743), a compound isolated from the Caribbean sea squirt Ecteinascidia turbinata with a potent cytotoxic activity against a variety of tumours in vitro and in vivo. Current phase II clinical trials against soft tissue sarcomas in Europe and the United States indicate that ET-743 represents a highly promising anticancer agent. Another example is aplidine from the Mediterranean tunicate Aplidium albicans, with a broad spectrum activity against various types of cancers, such as colorectal, lymphoma, thyroid and renal cancers. In the present work, we reported, for the first time, that a partially purified methanolic extract prepared from the ascidian Ciona intestinalis inhibited cell proliferation in human cell lines of different origin, including Caco2, HPB-ALL, U-937 and HL-60 and induced early apoptotic events, such as caspase-3 activation and internucleosomal DNA degradation. We suggest the presence in the Ciona intestinalis extract of bioactive compounds possessing anticancer activity.

Trabectedin—a targeted chemotherapy?

Trabectedin—a targeted chemotherapy?

DNA and Non-DNA Targets in the Mechanism of Action of the Antitumor Drug Trabectedin

We have analyzed the DNA binding properties of the antitumor agent trabectedin (ET-743, Yondelis) and different analogs, namely, ET-745, lacking the C21-hydroxyl group, and ET-637, ET-594, ET-637-OBu, with modifications at the trabectedin C domain, versus their effects on cell cycle, apoptosis, and gene expression. ET-745 failed to bind DNA, highlighting the importance of the C21-hydroxyl group for DNA binding. Analogs ranked trabectedin >> ET-637 approximately ET-594 > ET-637-OBu >> ET-745 for their DNA binding capacity; ET-637 and ET-594 display very different biological activities. Drugs were clustered in three major groups showing high (trabectedin, ET-637), intermediate (ET-637-OBu), and low (ET-594, ET-745) cytotoxic activity and similar transcriptional profiling responses. C21-hydroxyl-deficient analogs of the above-mentioned compounds showed a dramatic decrease in biological activity. Our data suggest that trabectedin interacts with an additional non-DNA target to raise an effective antitumor response, and that this interaction is favored through trabectedin-DNA complexes.

Ecteinascidin-743 (ET-743) for Chemotherapy-Naive Patients With Advanced Soft Tissue Sarcomas: Multicenter Phase II and Pharmacokinetic Study

To evaluate the response rate, toxicity profile, and pharmacokinetics of ecteinascidin-743 (ET-743) as first-line therapy in patients with unresectable advanced soft tissue sarcoma (STS). Thirty-six patients with STS were enrolled onto the study between September 1999 and August 2000. Patients were treated with 1.5 mg/m2 of ET-743 given as a 24-hour continuous intravenous (IV) infusion every 21 days. Pharmacokinetic sampling was performed in 23 patients. One complete and five partial responses were achieved in 35 assessable patients for an overall response rate of 17.1% (95% CI, 6.6% to 33.6%). In addition, one patient had a minor response, leading to an overall clinical benefit of 20%. Neutropenia and transaminitis were the main grade 3 to 4 toxicities, which occurred in 33% and 36% of the patients. The estimated 1-year progression-free and overall survival rates were 21% (95% CI, 11% to 41%) and 72% (95% CI, 59% to 88%), respectively. Total body clearance (L/h) was not significantly correlated with body-surface area (r = -0.28; P = .21). Mild hepatic impairment or the extent of prior cytotoxic therapy does not seem to contribute significantly to the high interpatient variability (49%) in the clearance of this drug. Severity of treatment-related toxicity was not correlated with pharmacokinetic variables. ET-743 demonstrates clinical activity as first-line therapy against STS with acceptable toxicity. Additional studies to establish empirical dosing guidelines may be necessary to improve the safety of the drug in patients with varying degrees of hepatic dysfunction and definitively establish the role of ET-743 for patients with these malignancies.

Tumor control and objective responses: single-center experience with ecteinascidin-743 (ET-743), an active compound for the treatment of patients with advanced soft tissue and bone sarcomas

9027 Background: ET-743 (Yondelis) is a marine-derived minor-groove binder which interferes with gene activation and nucleotide excision repair, modulates drug-resistance, induces DNA strand breaks...

Ecteinascidin-743 is a potent inhibitor of P450 3A4 enzyme and accumulates cytoplasmic PXR to inhibit transcription of P450 3A4 and MDR1: Implications for the enhancement of cytotoxicity to chemotherapeutic agents in osteosarcoma

9026 Background: Ecteinascidin 743 (ET-743) is a marine product that has been suggested to inhibit pregnane xenobiotic receptor (PXR) transcriptional activity based on its ability to down-regulate ...