Non-camptothecin Topoisomerase Research Articles

Design and synthesis of Aza-boeravinone derivatives as potential novel topoisomerase I inhibitors

Based on the structural skeleton of natural products boeravinones, two types of 6H-chromeno[3,4-b]quinoline derivatives were designed and synthesized by nitrogen atom substitution strategy. Then, their cytotoxic activities were evaluated against six human tumor cell lines including HepG2 (hepatocellular carcinoma), A2780 (ovarian cancer), Hela (cervical cancer), HCT116 (colorectal cancer), SW1990 (pancreatic cancer), and MCF7 (breast cancer). The results showed that compounds ZML-8 and ZML-14 exhibited robust inhibitory activities against HepG2 cells with IC50 values of 0.58 and 1.94 μM, respectively. In addition, ZML-8 and ZML-14 showed higher selectivity against HepG2 and L-02 cells than Topotecan. Mechanistically, ZML-8 and ZML-14 not only induced cell cycle arrest in the G2/M phase and cell apoptosis, but also dose-dependently inhibited topoisomerase I activity and induced DNA damage in HepG2 cells. Molecular docking showed that ZML-8 and ZML-14 could interact with topoisomerase I-DNA complex with a similar binding mode to Topotecan. Inhibitory activities of these two compounds on topoisomerase I were then confirmed in both cell-free systems and in whole-cell lysates. Taken together, compounds ZML-8 and ZML-14 merit further development as a new generation of non-camptothecin topoisomerase I inhibitors for the treatment of cancer.

Engineered Human Nanoferritin Bearing the Drug Genz-644282 for Cancer Therapy.

Gastrointestinal tumors, including pancreatic and colorectal cancers, represent one of the greatest public health issues worldwide, leading to a million global deaths. Recent research demonstrated that the human heavy chain ferritin (HFt) can encapsulate different types of drugs in its cavity and can bind to its receptor, CD71, in several solid and hematological tumors, thus highlighting the potential use of ferritin for tumor-targeting therapies. Here, we describe the development and characterization of a novel nanomedicine based on the HFt that is named The-0504. In particular, this novel system is a nano-assembly comprising an engineered version of HFt that entraps about 80 molecules of a potent, wide-spectrum, non-camptothecin topoisomerase I inhibitor (Genz-644282). The-0504 can be produced by a standardized pre-industrial process as a pure and homogeneously formulated product with favourable lyophilization properties. The preliminary anticancer activity was evaluated in cultured cancer cells and in a mouse model of pancreatic cancer. Overall results reported here make The-0504 a candidate for further preclinical development against CD-71 expressing deadly tumors.

59P - Novel non-camptothecin compounds with antiproliferative activities against breast cancer cells

BackgroundEven though new molecular targeted agents are now being developed, they are still premature in application of clinical use for triple negative breast cancers (TNBCs). Therefore, it is highly demanded to explore the effective antitumor agents against TNBCs. Novel non-camptothecin topoisomerase I inhibitors are discovered and evaluated in this study. MethodsHuman MDA-MB-231 (BCRC-60425), BT-549 (ATCC® HTB-122™) and MCF-7 (BCRC-60436) were used in this study. Top1 assay was determined by gel mobility assay to validate the Top1-mediated DNA cleavages at different concentrations of non-camptothecin compounds. Cell viability analysis (MTT assay), comet assay and flow cytometry analysis were used to evaluate their growth inhibitory activity, DNA damage, and induction of cell arrest. Mechanistic pathways were studied and validated through western blot analysis, flow cytometry and confocal microscopy analysis. Binding interactions between top1 and non-camptothecin compound were analyzed by computational analysis (Discovery Studio). ResultsThe IC50 values (growth inhibitory activity) of these non-camptothecin compounds are in the micromolar to nanomolar range (1.8μm to 190nM) against MDA-MB-231, BT-549 and MCF-7 cell lines. These compounds significantly inhibited the process in which supercoiled DNA strand transforms into its relaxed state and showed antitumor spectra similar to camptothecin rather than doxorubicin. Comet tails were observed to increase significantly with various doses of compounds in a dose-dependent manner. In addition, their mode of actions were shown to involve G2/M arrest of the cell cycle along with a dose-dependent increase in protein levels of cleaved caspase-3 and cleavage of cPARP. Except apoptosis pathway, non-camptothecin compounds also induce necrosis, and autophagy. Favorable ADMET characteristics of non-camptothecin compounds were observed using ADMET Descriptors. ConclusionsOur results have provided evidence for therapeutic intervention in the treatment of TNBC using new top1 inhibitors, non-camptothecin compounds. Legal entity responsible for the studyWen-Shan Li. FundingMinistry of Science and Technology, Taiwan and Academia Sinica, Taiwan. DisclosureAll authors have declared no conflicts of interest.

Abstract 365: Indotecan (LMP400), indimitecan (LMP776) and LMP744, a new class of non-camptothecin topoisomerase I inhibitors selective for schlafen11-positive and BRCA-deficient cells that synergize with olaparib

Abstract To overcome the limitations of topoisomerase inhibitors, irinotecan and topotecan (chemical instability, drug efflux substrates, short half-life, dose-limiting bone marrow and gastrointestinal toxicity), we have developed the indenoisoquinolines (LMP400, LMP776 and LMP744). To rationally select patients for phase 2 clinical trials, we have taken two approaches. First, we mined the cancer cell lines genomic databases with CellMinerCDB and found that a dominant response determinant to the indenoisoquinolines is Schlafen 11 (SLFN11), a recently identified executor of cells undergoing replication stress. We validated this finding in isogenic cell lines. Second, because the major lesions generated by the trapping of TOP1 are replication-induced DNA double-strand breaks that are repaired by homologous recombination (HR), we determined whether the indenoisoquinolines exhibit a “synthetic lethality” in cells presenting BRCA1, BRCA2 or PALB2 deficiency. In addition, we tested wether this selectivity could be enhanced when combined with the PARP inhibitor, olaparib. Survival and cell cycle alterations were tested after treatment with the indenoisoquinolines as single agents in isogenic DT40, DLD1 and OVCAR cell lines, with BRCA1, BRCA2 or PALB2 deficiencies, and organoids cultured from patient-derived xenografts with BRCA2 loss, as well as in combination with olaparib. We found that BRCA1-, BRCA2- and PALB2-deficient cells are 3 to 5 times more sensitive to the indenoisoquinolines compared to isogenic cell lines. Moreover, combination treatments showed high synergy between all three indenoisoquinolines and olaparib. We also established the synergy between LMP400 (Indotecan) and olaparib in orthotopic allograft models derived from genetically engineered mouse models for serous epithelial ovarian cancer harboring BRCA1 loss. Better efficacy was observed with the combination over single agent treatments of LMP400 or olaparib. Our results provide a rationale for Phase 2 indenoisoquinoline clinical trials with the indenoisoquinolines in HR-deficient cancers as single agents and in combination with PARP inhibitors, and for measuring Schlafen 11 (SLFN11) as a clinical response determinant. Citation Format: Laetitia Marzi, Ludmila Szabova, Zoe Weaver Ohler, Shyam Sharan, Mike Beshiri, Junko Murai, Kathy Kelly, Yves Pommier. Indotecan (LMP400), indimitecan (LMP776) and LMP744, a new class of non-camptothecin topoisomerase I inhibitors selective for schlafen11-positive and BRCA-deficient cells that synergize with olaparib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 365.

NCI Comparative Oncology Program Testing of Non-Camptothecin Indenoisoquinoline Topoisomerase I Inhibitors in Naturally Occurring Canine Lymphoma.

Only one chemical class of topoisomerase I (TOP1) inhibitors is FDA approved, the camptothecins with irinotecan and topotecan widely used. Because of their limitations (chemical instability, drug efflux-mediated resistance, and diarrhea), novel TOP1 inhibitors are warranted. Indenoisoquinoline non-camptothecin topoisomerase I (TOP1) inhibitors overcome chemical instability and drug resistance that limit camptothecin use. Three indenoisoquinolines, LMP400 (indotecan), LMP776 (indimitecan), and LMP744, were examined in a phase I study for lymphoma-bearing dogs to evaluate differential efficacy, pharmacodynamics, toxicology, and pharmacokinetics. Eighty-four client-owned dogs with lymphomas were enrolled in dose-escalation cohorts for each indenoisoquinoline, with an expansion phase for LMP744. Efficacy, tolerability, pharmacokinetics, and target engagement were determined. The MTDs were 17.5 mg/m2 for LMP 776 and 100 mg/m2 for LMP744; bone marrow toxicity was dose-limiting; up to 65 mg/m2 LMP400 was well-tolerated and MTD was not reached. None of the drugs induced notable diarrhea. Sustained tumor accumulation was observed for LMP744; γH2AX induction was demonstrated in tumors 2 and 6 hours after treatment; a decrease in TOP1 protein was observed in most lymphoma samples across all compounds and dose levels, which is consistent with the fact that tumor response was also observed at low doses LMP744. Objective responses were documented for all indenoisoquinolines; efficacy (13/19 dogs) was greatest for LMP744. These results demonstrate proof-of-mechanism for indenoisoquinoline TOP1 inhibitors supporting their further clinical development. They also highlight the value of the NCI Comparative Oncology Program (https://ccr.cancer.gov/Comparative-Oncology-Program) for evaluating novel therapies in immunocompetent pets with cancers.

TOPOISOMERASES: FEATURES OF THE ACTION, CLASSIFICATION, CELL FUNCTIONS, INHIBITION, ANTHRAFURANDION

Introduction. Topoisomerases influence on DNA topology and are capable of running down their super spiraling molecules by importation of one- or two-chained ruptures with the subsequent restitution and also the negative super rounds or catenae’s. Topoisomerases are known to be targets for antineoplastic therapy. Inhibitors of these enzymes of various nature and chemical structure are widely used for the suppression of tumor Topoisomerase I and/or II activity with the blocking cells in the phase G2 and a delay of their introduction in mitosis. Such difficult curable tumors as colorectal cancer, carcinoma of the stomach, non-small cell lung cancer and so forth are the most sensitive to these drugs. The search of perspective antineoplastic inhibitors is implemented generally in ranks of the non-camptothecin agents among which heterocyclic condensed nitrogenous compounds, in particular, anthrafurandiones show the most significant results. The review of thematic literature from 2011 to 2018 is devoted to the description of properties of topoisomerase as targets and their inhibitors from perspective classes. Objectives: 1. The analysis of signal characteristics of topoisomerases as targets for anticancer non-camptothecin inhibitors. 2. Identification of structure-activity relationship in the ranks of potential inhibitors of topoisomerases. 3. The choice of the most perspective non-camptothecin topoisomerase inhibitors among heterocyclic condensed nitrogenous compounds on the basis of the comparative analysis of structure and properties. Material and methods. Materials of 79 scientific articles published in the leading biological, biochemical and chemical journals of the different countries within the 8 last years are subjected to the analysis. The structure of the review meets the purpose and tasks of the scientific analysis. Results. The analysis of the thematic literature showed topoisomerases to be relevant targets for antineoplastic therapy of severe oncological pathology. In this regard, intensive search of various pharmaceuticals among topoisomerase inhibitors is performed in recent years. Researchers modify the known basic structures as well as synthesize new compounds. The discovery of a top-directional effect of the known medicines expands the data on their mechanism of the action. To identify the topoisomerase inhibitory activity of the drug the methods with the use of plasmid DNA is applied. The cytotoxic activity, apoptosis induction, including the caspases activation, modification of mitochondrial potential, influence on p53 and others are examined in parallel studies. The research directed on the identification of new effective non-camptothecin oral topoisomerase inhibitors among the anthracyclines derivatives are of undoubted relevance. Such agents, in contrast to Doxorubicin (anthracycline antibiotic widely used for tumor therapy), have moderate toxicity and allow to control the growth of solid tumors and leukemia in mono-therapy mode. Conclusion. In terms of searching of original antineoplastic agents, a class of heterocyclic condensed nitrogenous compounds, first of all, the anthraquinones showing properties of topoisomerase inhibitors is one of the most promising. The results of chemical and biological research of the compounds of this series were laid in a basis of the design of medicinal substances and their drug formulations. Prognostically significant data obtained in preclinical testing allow us to hope that obtained antitumor agents will be highly effective on a clinical stage of trials.

Design, synthesis and evaluation of 4-substituted anthra[2,1-c][1,2,5]thiadiazole-6,11-dione derivatives as novel non-camptothecin topoisomerase I inhibitors

Previously, 4-tosylanthra[1,2-c][1,2,5]thiadiazole-6,11-dione (1) was identified as a novel non-camptothecin topoisomerase I (Top1) inhibitor by structure-based virtual screening. Herein, a series of 4-substituted derivatives were designed and synthesized. Most of them showed potent Top1 inhibitory activity. Their in vitro antiproliferative activity was also evaluated in A549, HCT-116 and ZR-75-30 human cancer cell lines. Compound 8s showed good antiproliferative activity with IC50 of 0.52μM and 0.42μM against HCT-116 and ZR-75-30 cell line, respectively. Top1 unwinding assay and molecular modeling studies rationalized the mode of action of this new class of inhibitors.

Design, synthesis and antitumor activity of non-camptothecin topoisomerase I inhibitors

Three groups of non-camptothecin compounds with four to five fused rings have been designed and synthesized. Their in vitro anti-proliferative activity has been evaluated with five different cancer cell lines (HCT116, PC3, U87MG, HepG2, SK-OV-3). Compounds B-2 and B-3 showed the most potent cell growth inhibition with IC50 of 169 nM and 325 nM against U87MG cell line correspondingly.

Proof of mechanism (POM) in the first-in-human trial of two novel indenoisoquinoline, non-camptothecin topoisomerase I (TOP1) inhibitors.

3031 Background: Indenoisoquinolines LMP 400 and LMP 776 form stable DNA-top1 cleavage complexes, have a preference for unique DNA cleavage sites, and are not substrates for ABC transporters compared to camptothecins. We conducted a randomized phase I trial of LMP 400 and LMP 776 in patients (pts) with refractory tumors to establish safety and MTD of LMP 400 and LMP 776 administered IV daily x 5 d, q 28d; determine PK; evaluate TOP1 and γH2AX levels in tumor biopsies (bx); and compare pharmacodynamic responses of LMP400 and LMP776. Methods: Pts had refractory malignancies; ≥ 18 yrs old; KPS > 70%; adequate organ function. DLT: drug-related gr ≥ 3 non-hem or gr 4 hem toxicities during C1. Pts assigned to receive either LMP 400 or LMP 776. Dose level (DL) in mg/m2/day for LMP 776: 1, 2, 3, 4.5 mg; LMP 400: 2.5, 5, 10, 20, 40, 80. Accelerated titration design with one pt per dose level (DL), 100% dose escalation until one DLT or 2 gr 2 toxicities, then 3+3 design. Tumor bx and circulating tumor cells (CTCs) were obtained at baseline and C1D3. Results: Twenty pts accrued to date [LMP 400 (11 pts); LMP 776 (9 pts)]; M/F 7/13; median age 59 (range 32-71 yrs); diagnosis (# of pts): colorectal (10), vaginal adenocarcinoma (1), head and neck (2), bladder (1), melanoma (1), pancreas (1), thyroid (1), small cell lung (1), sarcoma (1), lymphoma (1). DLTs: LMP 400- gr 4 myelosuppression, gr 3 fatigue at DL 6; DL 5 expanded to establish MTD; LMP 776 (DL2)- gr 4 hypercalcemia, no other DLTs, dose escalation continuing. One pt with irinotecan-refractory colon cancer had shrinkage of lung nodules post one cycle of LMP 400. Relative to baseline, 5 of 6 tumor bx demonstrated 20-50% reduction in TOP1 levels (LMP 776); an increase in γH2AX foci in tumor was observed in 2/6 LMP 776 and 1/1 LMP 400 pts. An increase in γH2AX was also observed in hair bulbs (3/3 pts) and CTCs (9/20 pts). PK for LMP 400 and LMP 776: mean T1/2 48 and 36 h; Cl 1.5 and 2.0 L/h/m2; Vc 30 and 31 L/m2, respectively. Conclusions: POM, as assessed by reduction in TOP 1 levels and increase in γH2AX in tumor and CTCs, and preliminary evidence of activity, has been demonstrated in this first-in-human trial of indenoisoquinoline-class of TOP1 inhibitors. Accrual is ongoing.

A first-in-human, phase I safety and pharmacokinetic study of genz-644282, a non-camptothecin topoisomerase I inhibitor, in patients with advanced solid tumors.

2534 Background: The approved topoisomerase I (TopI) inhibitors (irinotecan and topotecan) are camptothecin derivatives. The lactone ring structure of camptothecins negatively impacts their clinical potential.Genz-644282 is a novel non-camptothecin that induces TopI-DNA cleavage complexes at similar as well as unique genomic positions; that are more persistent. This study was designed using a pharmacokinetic-pharmacodynamic (PK-PD) model to predict the maximum tolerated dose (MTD). Methods: A PK-PD relationship of Genz-644282 to inhibit tumor growth was derived (Simeoni, Can Res 2004). Using an accelerated titration design, cohorts of 1, 3 or 6 patients received 3 weekly doses of Genz-644282 on a 28 day schedule starting with 0.5 mg/m2. After MTD on the 28 day schedule was exceeded, 21 day schedule was initiated. Results: 49 patients (N=44 data available: 26M :18F) were dosed. Tumor types were colorectal (15), breast (5), small cell lung (SCLC 3), non-small cell lung (4), others (17). As predicted from the PK-PD model 8 cohorts were evaluated on the 28 day schedule before defining the MTD. The MTD was 8 mg/m2 and 9 mg/m2 for the 28 day and 21 day schedules, respectively. Dose limiting toxicities that determined the MTD were: gr 4 thrombocytopenia (n=2); gr 2 thrombocytopenia (n=1) and gr 4 neutropenia (n=1) both of which resulted in ≥72h delays in initiating cycle 2. Treatment emergent adverse events (>10%) were nausea (45%), fatigue (39%), anorexia (32%), anemia (27%), vomiting (23%), thrombocytopenia (18%), diarrhea (16%), dehydration (16%), hyperglycemia (16%), cough (16%), dyspnea (14%), depression (11%) and hyponatremia (11%). Efficacy data suggest 2 responders with SCLC (1 minor response;1 complete response), and 2 patients (breast, gastric) with stable disease (≥ 6 months). PK data show a Genz-644282 half-life of approximately 50 h, a linear dose-exposure relationship, and no accumulation in between doses. Conclusions: Genz-644282 is a non-camptothecin TopI inhibitor in phase I development with ongoing expansion phase. The emerging pk, efficacy and safety data are suggestive of a distinct clinical profile of Genz-644282 from the camptothecins.

Targeting DNA Topoisomerase I with Non-Camptothecin Poisons

DNA topoisomerase I is required for DNA relaxation during a variety of cellular functions. The identification of camptothecins as specific enzyme poisons and their clinical efficacy have stimulated extensive efforts to exploit topoisomerase I as a therapeutic target for cancer. However, several limitations of camptothecins, such as low solubility and stability, high toxicity, and the occurrence of resistance, have encouraged the development of non-camptothecin topoisomerase I inhibitors. Different natural and synthetic compounds (e.g., indolocarbazoles, dibenzonaphthyridine and indenoisoquinoline) have been extensively studied as alternatives to camptothecins and have been proved to be promising therapeutic agents. In this review, we comparatively evaluate the preclinical results obtained with the different non-camptothecin poisons proposed thus far as topoisomerase I inhibitors, with special reference to cellular pharmacology, and discuss the perspective for their use in the clinical setting.

Abstract C204: Potentiation of the novel topoisomerase I inhibitor indenoisoquinoline LMP-400 by the Chk1/2 inhibitor, AZD7762.

Abstract Novel topoisomerase I (Top1) inhibitors are in clinical development to circumvent the drawbacks of camptothecins (1–3). Here we report molecular investigations of the clinical indenoisoquinoline Top1 inhibitor, LMP-400 by itself and in combination with the cell cycle checkpoint inhibitor, AZD7762 (4). Drug effects on DNA replication and killing of cancer cells were examined. LMP-400 shows synergistic antiproliferative activity when combined with AZD7762 in human colon carcinoma HT-29 and HCT-15 cells. Inhibition of S-phase progression and bromodeoxyuridine incorporation was similarly induced by LMP-400 and CPT and was abrogated by AZD7762. Replication studied by single DNA molecule analyses and immunofluorescence microscopy (molecular combing) (5) showed rapid inhibition of fork progression in response to LMP-400 treatment with subsequent recapitulation after AZD7762 addition. Consistent with cell cycle checkpoint abrogation, AZD7762 inhibited both the activation of Chk1 and Chk2 autophosphorylations in LMP-400-treated cells. In spite of the potent dual inhibition of Chk1 and Chk2 at nanomolar concentrations of AZD7762, cell cycle inhibition and synergism with LMP-400 were only observed at higher concentrations and independently of Chk2 both in Chk2-complemented HT-15 and Chk2 knockout HCT-116 cells. Our study demonstrates the rationale for combining checkpoint kinase inhibitors and the novel non-camptothecin indenoisoquinoline inhibitors.

Synthesis of novel benzoxanthone analogues as non-Camptothecin topoisomerase I inhibitors

Structure modification of the side chain of the lead compound benzoxanthone provided a series of benzoxanthone analogues and 12 of them were first reported. The results showed that most of these compounds had moderate cytotoxicity against tumour cells with the 50% inhibition concentration in the micromolar range. Furthermore, benzoxanthone derivatives 5, 6c, 7a and 7e, showed potent topoisomerase I (Topo I) inhibitory effect and the results indicated that some compounds had potential for development as non-Camptothecin (CPT) topoisomerase I inhibitors.

Testing of the topoisomerase 1 inhibitor Genz‐644282 by the pediatric preclinical testing program

Genz-644282 is a novel non-camptothecin topoisomerase I poison that is in clinical development. Genz-644282 was tested against the PPTP in vitro panel (0.1 nM to 1 µM), and in vivo using three times per week × 2 schedule repeated at day 21 at its maximum tolerated dose (MTD) of 4 mg/kg. Subsequently Genz-644282 was tested at 4, 3, 2, and 1 mg/kg in 3 models to assess the dose-response relationship. mRNA gene signatures predictive for Genz-644282 response in vitro were applied to select 15 tumor models that were evaluated prospectively. In vitro, Genz-644282 demonstrated potent cytotoxic activity with a median IC(50) of 1.2 nM (range 0.2-21.9 nM). In vivo, Genz-644282 at its MTD (4 mg/kg) induced maintained complete responses (MCR) in 6/6 evaluable solid tumor models. At 2 mg/kg Genz-644282 induced CR or MCR in 3/3 tumor models relatively insensitive to topotecan, but there were no objective responses at 1 mg/kg. Further testing at 2 mg/kg showed that Genz-644282 induced objective regressions in 7 of 17 (41%) models. There was a significant correlation between predictive response scores based on Affymetrix U133Plus2 baseline tumor expression profiles and the observed in vivo responses to Genz-644282. Genz-644282 was highly active within a narrow dose range (2-4 mg/kg), typical of other topoisomerase I poisons. As with other topoisomerase I poisons, how accurately these data will translate to clinical activity will depend upon the drug exposures that can be achieved in children treated with this agent.

Abstract 3525: Molecular determinants of the anticancer efficacy of indenoisoquinolines

Abstract Topoisomerase I (top1) is a validated target for the anticancer alkaloid camptothecin and its analogs (CPTs). CPTs are the only class of topoisomerase I (top1) inhibitors approved for cancer treatment in clinic. However, CPTs have pharmacologic and clinical limitations. This leads to the development of noncamptothecins, such as indenoisoquinolines (INDs), as novel top1 inhibitors. Currently three INDs have been selected as leads for clinical development by the National Cancer Institute [1]. INDs are endowed with favorable characteristics. Persistence of top1 cleavage complexes induced by INDs is higher than that of induced by CPTs. The top1 cleavage sites trapped by the INDs have different genomic locations, implying differential targeting of cancer cell genomes. Presently, we demonstrate that molecular dynamics and bioinformatics are valuable tools for the study of molecular determinants of anticancer drugs. Our previous molecular dynamics studies revealed that the most persistent CPTs have higher calculated free-energy barriers for drug dissociation from the flanking base pairs [2]. We have recently extended our studies to INDs. We found the INDs have higher calculated free-energy barriers when compared with that of CPTs. This may explain why INDs experimentally produce more persistent top1 cleavage complexes when compared with that of CPTs [3]. The National Cancer Institute (NCI)-60 panel consists of 60 human tumor cell lines established for screening anticancer efficacy of a larger number of molecules, including INDs. To deciphering the relationships between anticancer efficacy of INDs and the characteristics of the cancer cells, the molecular profile data on the NCI-60 were downloaded from the CellMiner database [4]. Integromic and bioinformatics analyses were carried out on the markers that correlated with the responses of INDs-treatments. Same and unique markers, when compared to that of CPTs, were identified for INDs. Further studies are required for the validation of these predictive markers.

Abstract C217: Genz-644282, a novel non-camptothecin topoisomerase I inhibitor for cancer treatment

Abstract Topoisomerase I (Top1), a well-established anticancer target, is a nuclear enzyme producing a single-strand DNA break to allow transcription. Camptothecin Top1 inhibitors are important drugs for the treatment of a variety of cancers including colon cancer, leukemias and ovarian cancer. Potent non-camptothecin Top1 inhibitors were identified. One of these, Genz-644282, binds tightly and selectively to the DNA:Top1 complex. The effects of Genz-644282, topotecan and irinotecan/SN38 in cell-based assays were compared. The colony formation assay is the gold-standard for determination of the cytotoxicity of compounds toward bone marrow and malignant cells as it measures the survival/killing of cells capable of self-renewal. Mouse and human bone marrow were subjected to colony forming unit -granulocyte/macrophage (CFU-GM) assays over a 5-log concentration range with continuous exposure to the compounds for 13–15 days. The IC90s for topotecan and SN38 for mouse CFU-GM were 519 nM and 331 nM, respectively. For human CFU-GM, the IC90s for topotecan and SN38 were 19 nM and 26 nM, resulting in mouse to human differentials of 28- and 13-fold. Genz-644282 was more potent, with IC90s of 8.4nM for mouse CFU-GM and 1.2 nM for human CFU-GM; thus only a 7-fold differential between species. The cytotoxicities of topotecan, SN38 and Genz-644282 were compared in the ability of seven human tumor cell lines of varied histologies to form colonies after a week of exposure to a 5-log concentration range of each compound. Human bone marrow CFU-GM was much more sensitive to topotecan than were RPMI-8226 multiple myeloma, KBV-1 cervical carcinoma or NCI-H460 non-small cell lung carcinoma cells. The IC90s for topotecan ranged between 11 and 257 nM. SN38 cytotoxicity to human bone marrow CFU-GM and to several human tumor cell lines was very similar with IC90s in the cancer lines ranging between 2 and 55 nM. HT29 human colon cancer cells, however, were markedly sensitive to SN38 (IC90 = 2 nM). Genz-644282 was a more potent cytotoxic agent than topotecan or SN38 with IC90 values ranging between 0.7 and 9.1 nM across the seven cell lines. MDA-MB-231 human breast carcinoma, HT29 colon carcinoma and RPMI-8226 cells were most sensitive to Genz-644282 with IC90s ranging between 0.7 and 7.0 nM. The growth inhibition produced by 72-hr exposure to Genz-644282 was examined in 26 established human tumor cell lines representing pancreatic carcinoma, melanoma, non-small cell lung cancer, breast carcinoma, ovarian carcinoma and renal cell carcinoma using a Cell TiterGlo ATP content endpoint. The IC90s for Genz-644282 ranged between 0.054 micromolar and 5 micromolar in this assay. The most sensitive tumor lines were 4 of 6 pancreatic cancer lines and the least sensitive lines included 3 of 4 melanoma lines. Colony formation data were analyzed by non-linear regression using SAS version 8.2. Based upon these findings and other data, Genz-644282 was selected as a development candidate. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C217.

Abstract C219: Molecular and cellular pharmacology of the novel non-camptothecin topoisomerase I inhibitor, GENZ644282

Abstract Camptothecin (CPT) derivatives are powerful anticancer drugs because of their ability to trap topoisomerase I (Top1)-DNA cleavage complexes. However, they exhibit clinical limitations due to the instability of their α-hydroxylactone six-membered E-ring structure, which is essential for trapping Top1-DNA cleavage complexes. Additionally, they exhibit bone marrow and intestinal toxicity in adults and are drug efflux substrates. Here, we report a Top1 inhibitor, Genz644282. In the cell lines studied, we find that Genz644282 as well as its metabolites induce Top1 cleavage at similar, as well as unique genomic positions, compared with CPT. The compound also induces protein-linked DNA breaks and Top1-DNA cleavage complexes that persist longer after drug removal than CPT. Concentration-dependent and persistent H2AX formation was readily observed in cells treated with Genz644282, and was present in greater than 50% of the cell population following 24 hours drug treatment. The compound shows partial cross-resistance in cell lines resistant to CPT. These cell lines include the prostate cancer cell line (DU145RC0.1), and human leukemic (CEM/C2) cells. Genz644282 demonstrated limited cross-resistance in Top1-knockdown colon cancer (HCT116) and breast cancer (MCF7) cells. The compound also demonstrated limited resistance in human lung cancer cells (H460/MX20). These lung cancer cells express (BCRP, ABCG2), a member of the ATP-binding cassette family of cell surface transport proteins known to have ABCG2 drug-efflux pumps. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C219.

Abstract C220: Genz-644282, a non-camptothecin topoisomerase one inhibitor, demonstrates a wide spectrum of in vitro and in vivo antitumor activity

Abstract Genz-644282 (GZ) is a novel non-camptothecin topoisomerase I (Top1) inhibitor. The in vitro and in vivo activity of GZ and its M1 and M2 metabolites were explored and compared with the activity of camptothecin Top1 inhibitors. In vitro in mouse, rat, dog, and human GZ exhibited high metabolic stability, plasma binding of 88–93% and exhibits concentration dependent partitioning into red blood cells. In vivo, GZ has a large volume of distribution and low-to-moderate clearance in mouse, rat and dog. In nude mice, the t1/2 for GZ is 3.6 h (po), 10.4 h (ip) and 5.1h (iv) and longer in tumor-bearing mice. In human HCT-116 colon ca, HT-29 colon ca and NCI-H460 NSCLC cells the concentration response for Genz-6244282, M1 and M2 are the same. Upon 72h exposure of the cells to GZ, M1 or M2 the IC50 concentrations were 0.5-0.65 nM and the IC90 concentrations were 1.8–2 nM. In order to evaluate the antitumor activity of GZ as compared to several approved anticancer agents, the compound was tested in seven xenograft models: LOX-IMVI melanoma, DLD-1 and HCT-15 colon, MDA-MB-231 breast, NCI-H292 and NCI-H1299 lung ca. GZ was compared against two of its metabolites, Genz-649974 (GZ-74) and Genz-649975 in the HCT-116 colon ca resulting in comparable activity with GZ-74. GZ was administered intravenously on a QODx3 schedule for 2 cycles. The tumor growth delay, TGD, (T-C) and increase in lifespan, ILS, (T/C) for each study are listed in the table below. All of the GZ dosages were well tolerated resulting in a maximum body weight loss of ≤20%, except for the high dosages in the HCT-15 and NCI-H292 in which there was a maximum body weight loss of 25.7 and 20.9%, respectively. Based on these findings and other data, GZ was selected to be a development candidate. Treatments Dose (mg/kg/day) Route/Schedule TGD (T-C) ILS (T/C) Tumor Line GZ 1 IV/QODx3 2 days 1.1x for 2 cycles GZ 4 IV/QODx3 25 days 2.8x LOX-IMVI Dacarbazine 90 IP/QDx5 14 days 2.0x GZ 4 IV/QODx3 8 days 1.2x for 2 cycles CPT-11 60 IV/Q4Dx3 5 days 1.1x DLD-1 for 2 cycles GZ 1 IV/QODx3 14 days 1.3x for 2 cycles GZ 2 IV/QODx3 35 days 1.8x HCT-15 for 2 cycles CPT-11 60 IV/Q4Dx3 28 days 1.7x GZ 1 IV/QODx3 21 days 1.7x for 2 cycles GZ 1.36 IV/QODx3 >47 days >2.3x MDA-MB-231 for 2 cycles GZ 1.7 IV/QODx3 35 days 2.0x for 2 cycles Docetaxel 20 IV/QODx3 >47 days >2.3x GZ 1 IV/QODx3 18 days 1.5x for 2 cycles GZ 1.36 IV/QODx3 21 days 1.6x for 2 cycles NCI-H292 GZ 1.7 IV/QODx3 21 days 1.6x for 2 cycles Docetaxel 20 IV/QODx3 39 days 2.1x GZ 1 IV/QODx3 20 days 1.7x for 2 cycles GZ 1.36 IV/QODx3 24 days 1.8x for 2 cycles NCI-H1299 GZ 1.7 IV/QODx3 34 days 2.1x for 2 cycles Docetaxel 20 IV/QODx3 17 days 1.6x GZ-74 4 IV/QODx3 25 days 1.8x for 2 cycles GZ-74 6 IV/QODx3 28 days 1.9x for 2 cycles HCT-116 GZ-74 8 IV/QODx3 32 days 2.0x for 2 cycles GZ 1.7 IV/QODx3 28 days 1.9x for 2 cycles Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C220.

Synthesis of N-substituted benzo[ c][1,7]- and benzo[ c][1,8] phenanthrolin-(5 H)-6-ones through a Pd-mediated Suzuki–Miyaura heteroaryl-aryl coupling reaction

In the course of the search for non-camptothecin topoisomerase I inhibitors we have undertaken the synthesis of N-substituted benzo[ c][1,7]- and benzo[ c][1,8]phenanthrolinone derivatives. An intermolecular Suzuki–Miyaura heteroaryl-aryl coupling reaction was planned as the key step. Then a nitro reduction followed by a concomitant lactamization achieved the construction of the tetracycle structures. This methodology permitted a rapid and efficient elaboration of biologically potent compounds.

Activity of Indenoisoquinolines against African Trypanosomes

African trypanosomiasis (sleeping sickness), caused by protozoan Trypanosoma brucei species, is a debilitating disease that is lethal if untreated. Available drugs are antiquated, toxic, and compromised by emerging resistance. The indenoisoquinolines are a class of noncamptothecin topoisomerase IB poisons that are under development as anticancer agents. We tested a variety of indenoisoquinolines for their ability to kill T. brucei. Indenoisoquinolines proved trypanocidal at submicromolar concentrations in vitro. Structure-activity analysis yielded motifs that enhanced potency, including alkylamino substitutions on N-6, methoxy groups on C-2 and C-3, and a methylenedioxy bridge between C-8 and C-9. Detailed analysis of eight water-soluble indenoisoquinolines demonstrated that in trypanosomes the compounds inhibited DNA synthesis and acted as topoisomerase poisons. Testing these compounds on L1210 mouse leukemia cells revealed that all eight were more effective against trypanosomes than against mammalian cells. In preliminary in vivo experiments one compound delayed parasitemia and extended survival in mice subjected to a lethal trypanosome challenge. The indenoisoquinolines provide a promising lead for the development of drugs against sleeping sickness.