Viviane Brel

F14512, a Potent Antitumor Agent Targeting Topoisomerase II Vectored into Cancer Cells via the Polyamine Transport System

The polyamine transport system (PTS) is an energy-dependent machinery frequently overactivated in cancer cells with a high demand for polyamines. We have exploited the PTS to selectively deliver a polyamine-containing drug to cancer cells. F14512 combines an epipodophyllotoxin core-targeting topoisomerase II with a spermine moiety introduced as a cell delivery vector. The polyamine tail supports three complementary functions: (a) facilitate formulation of a water-soluble compound, (b) increase DNA binding to reinforce topoisomerase II inhibition, and (c) facilitate selective uptake by tumor cells via the PTS. F14512 is 73-fold more cytotoxic to Chinese hamster ovary cells compared with CHO-MG cells with a reduced PTS activity. A decreased sensitivity of L1210 leukemia cells to F14512 was observed in the presence of putrescine, spermidine, and spermine. In parallel, the spermine moiety considerably enhances the drug-DNA interaction, leading to a reinforced inhibition of topoisomerase II. The spermine tail of F14512 serves as a cell delivery vehicle as well as a DNA anchor, and this property translates at the cellular level into a distinct pharmacologic profile. Twenty-nine human solid or hematologic cell lines were used to characterize the high cytotoxic potential of F14512 (median IC50 of 0.18 micromol/L). Finally, the potent antitumor activity of F14512 in vivo was evidenced with a MX1 human breast tumor xenograft model, with partial and complete tumor regressions. This work supports the clinical development of F14512 as a novel targeted cytotoxic drug and sheds light on the concept of selective delivery of drugs to tumor cells expressing the PTS.

Synthesis of conjugated spermine derivatives with 7-nitrobenzoxadiazole (NBD), rhodamine and bodipy as new fluorescent probes for the polyamine transport system

The synthesis of a series of conjugated spermine derivatives with benzoxadiazole, phenylxanthene or bodipy fluorophores is described. These fluorescent probes were used to identify the activity of the polyamine transport system (PTS). N(1)-Methylspermine NBD conjugate 5 proved to have the optimal fluorescence characteristics and was used to show a selectivity for PTS-proficient CHO versus PTS-deficient CHO-MG cells. It can therefore be used as a tool for the selection of cells sensitive to cytotoxic compounds vectored through the PTS.

Cytotoxicity and cell death mechanisms induced by the polyamine-vectorized anti-cancer drug F14512 targeting topoisomerase II

The polyamines transport system (PTS) is usually enhanced in cancer cells and can be exploited to deliver anticancer drugs. The spermine-conjugated epipodophyllotoxin derivative F14512 is a topoisomerase II poison that exploits the PTS to target preferentially tumor cells. F14512 has been characterized as a potent anticancer drug candidate and is currently in phase 1 clinical trials. Here we have analyzed the mechanisms of cell death induced by F14512, compared to the parent drug etoposide lacking the polyamine tail. F14512 proved to be >30-fold more cytotoxic than etoposide against A549 non-small cell lung cancer cells and triggers less but unrecoverable DNA damages. The cytotoxic action of F14512 is extremely rapid (within 3 h) and does not lead to a marked accumulation in the S-phase of the cell cycle, unlike etoposide. Interestingly, A549 cells treated with F14512 were less prone to undergo apoptosis (neither caspases-dependent nor caspases-independent pathways) or autophagy but preferentially entered into senescence. Drug-induced senescence was characterized qualitatively and quantitatively by an increased β-galactosidase activity, both by cytochemical staining and by flow cytometry. A morphological analysis by electron microscopy revealed the presence of numerous multi-lamellar and vesicular bodies and large electron-lucent (methuosis-like) vacuoles in F14512-treated cell samples. The mechanism of drug-induced cell death is thus distinct for F14512 compared to etoposide, and this difference may account for their distinct pharmacological profiles and the markedly superior activity of F14512 in vivo. This study suggests that senescence markers should be considered as potential pharmacodynamic biomarkers of F14512 antitumor activity.

Phase I Clinical Pharmacology Study of F14512, a New Polyamine-Vectorized Anticancer Drug, in Naturally Occurring Canine Lymphoma

Purpose: F14512 is a new topoisomerase II inhibitor containing a spermine moiety that facilitates selective uptake by tumor cells and increases topoisomerase II poisoning. F14512 is currently in a phase I/II clinical trial in patients with acute myeloid leukemia. The aim of this study was to investigate F14512 potential in a new clinical indication. Because of the many similarities between human and dog lymphomas, we sought to determine the tolerance, efficacy, pharmacokinetic/pharmacodynamic (PK/PD) relationship of F14512 in this indication, and potential biomarkers that could be translated into human trials. Experimental Design: Twenty-three dogs with stage III–IV naturally occurring lymphomas were enrolled in the phase I dose-escalation trial, which consisted of three cycles of F14512 i.v. injections. Endpoints included safety and therapeutic efficacy. Serial blood samples and tumor biopsies were obtained for PK/PD and biomarker studies. Results: Five dose levels were evaluated to determine the recommended dose. F14512 was well tolerated, with the expected dose-dependent hematologic toxicity. F14512 induced an early decrease of tumoral lymph node cells, and a high response rate of 91% (21/23) with 10 complete responses, 11 partial responses, 1 stable disease, and 1 progressive disease. Phosphorylation of histone H2AX was studied as a potential PD biomarker of F14512. Conclusions: This trial demonstrated that F14512 can be safely administered to dogs with lymphoma resulting in strong therapeutic efficacy. Additional evaluation of F14512 is needed to compare its efficacy with standards of care in dogs, and to translate biomarker and efficacy findings into clinical trials in humans. Clin Cancer Res; 21(23); 5314–23. ©2015 AACR.

Antitumor activity of pyridoisoquinoline derivatives F91873 and F91874, novel multikinase inhibitors with activity against the anaplastic lymphoma kinase.

The anaplastic lymphoma kinase (ALK) is a validated target for the therapy of different malignancies. Aberrant expression of constitutively active ALK chimeric proteins has been implicated in the pathogenesis of anaplastic large-cell lymphoma (ALCL) and has been detected in other cancers such as inflammatory myofibroblastic tumors, diffuse large B-cell lymphomas, certain non-small-cell lung cancers, rhabdomyosarcomas, neuroblastomas and glioblastomas. In the course of a screening program aimed at identifying kinase inhibitors with novel scaffolds, the two pyridoisoquinoline derivatives F91873 and F91874, were identified as multikinase inhibitors with activity against ALK in a biochemical screen. F91873 and F91874 also inhibited nucleophosmin–ALK and signal transducer and activator of transcription 3 phosphorylation in the ALCL cell line COST with the same potency. Both F91873 and F91874 behaved as ATP noncompetitive inhibitors and inhibited cell proliferation of the ALK(+) ALCL cell lines COST, PIO, and Karpas299 ALCL. This growth inhibition effect was associated with a G1-phase cell cycle arrest. Furthermore, administration of F91874 to severe combined immunodeficient mice bearing COST tumor xenografts resulted in a significant antitumor efficacy at 15 mg/kg/day, illustrating the potential utility of such compounds in the treatment of ALK-related pathologies.

Semisynthetic neoboutomellerone derivatives as ubiquitin-proteasome pathway inhibitors

The interesting pharmacological properties of neoboutomellerones 1 and 2 were the basis for the assembly of a small library of analogues consisting of natural products isolated from the plant Neoboutonia melleri and of semisynthetic derivatives. As the two enone systems (C23-C24a and C1-C3) and the two hydroxyls groups (C22 and C26) of neoboutomellerones are required for activity, modifications were focused on these functional groups. Biological evaluation by using a cellular assay for proteasome activity provided clues regarding the mechanism of action of these natural products and synthetic derivatives. Certain neoboutomellerone derivatives inhibited the proliferation of human WM-266-4 melanoma tumor cells at submicromolar concentration and warrant evaluation as anticancer agents.

Hemisynthesis of Anisomycin Derivatives as Antitumor Agents

The antibiotic anisomycin, secreted by Streptomyces griseolus, can induce tumor cell death and it displays antimetastatic activity coupled with induction of apoptosis. Herein we report the hemi-synthesis of 16 novel anisomycin derivatives and their biological activity. The protein synthesis inhibition and the effects on cancer cell proliferation and migration were assessed for two series of molecules to determine structure-activity relationships. The secondary amino group of anisomycin is essential to preserve the bioactivity. Although, the natural product is the most active component of the series but an active derivative has been identified

Abstract 5565: Tumor targeting and enhanced efficacy of novel polyamine-cytotoxic conjugates.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Rapidly proliferating cancer cells have a higher demand on polyamines resulting in an over-activated Polyamines Transport System (PTS)1. Polyamines entry can be exploited as a selective anticancer drug delivery system. We synthesized polyamines-epipodophylotoxin conjugates with improved physico-chemical and pharmacological properties. As proof of concept, we demonstrated that the conjugation of epipodophylotoxin with spermine changes the pharmacological profile of the cytotoxic moiety by increasing the solubility, enhances the cellular distribution through the PTS, and increases the capacity to inhibit topoisomerase II due to stronger DNA interaction, as compared to the closest structurally related compound etoposide. Among several conjugates, F145122 was selected as a drug candidate and is currently undergoing a phase 1/2 clinical evaluation in acute myeloid leukaemia (AML). Based on this clinically validated objective to target cancer cells with the PTS, we set up a chemical platform of conjugation of polyamine moieties. We extended our approach to 3 natural products of interest in oncology: (i) an inhibitor of elongation phase in protein translation, a modulator of redox cell regulation and an inhibitor of polyADP-ribose polymerase. This approach led to the synthesis of novel spermine-pancratistatine, -artemisinine and -PARPi conjugates and their biochemical and biological characterization in terms of protein synthesis, PARP inhibition, ROS induction, anti-proliferative effects on cancer cells and their capacity to be imported via the PTS. Cell internalisation through the PTS was measured by differential cytotoxicity on PTS+ vs. PTS− cells, or by competition with a fluorescent probe. We also evaluated selected compounds in vivo and investigated whether their modified properties translate into a higher level of antitumor activity associated with an enlarged therapeutic index in a PTS+ tumor model. For the PARP inhibitor-spermine conjugates, an increase in cellular uptake was confirmed, without impairing the cytotoxic properties. The dedicated polyamine conjugation platform presented here can be adapted to many cytotoxic scaffolds to enhance their solubility, preferential incorporation into cancer cells through the PTS, and their pharmacological activity in vitro and in vivo. The design of polyamine-vectorized cytotoxic agents will be presented. 1 Delcros J.G. et al., Biochem. J., 1993, 269 2 PCT Int. Appl. WO2005/100363 (Pierre Fabre Medicament); Barret J.-M. et al., Cancer Res. 2008, 68, 9845 Citation Format: Frederic Lieby-Muller, Jean-Philippe Annereau, Viviane Brel, Frederic Marion, Yves Guminski, Florence Redoules, Nathalie de Saint Jores, Karine Andre, Anna Kruczynski, Christian Bailly, Nicolas Guilbaud. Tumor targeting and enhanced efficacy of novel polyamine-cytotoxic conjugates. [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 5565. doi:10.1158/1538-7445.AM2013-5565

Abstract 988: F14512, a novel vectorized topoiserase II inhibitor, bypasses MDR1 mediated resistance.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC F14512 is a novel polyamine-vectorized topoisomerase II inhibitor currently in phase 1/2 clinical trial in acute myeloid leukemia (AML). Since ABCB1 (also called P-glycoprotein or MDR1) has been reported to be expressed at high levels in patients with resistant leukaemia cells, and constitutes a pejorative marker of therapy, we decided to investigate whether F14512 could be exported by ABCB1 or other ABC transporters susceptible to confer drug resistance to chemotherapy, such as MRP1. In addition, we established a F14512-resistant cell line to study the mechanism of resistance to the drug. With membrane preparation enriched in functional MDR1, we found that [F14512][1] was unable to stimulate the ATPase activity of this transporter. Using a cellular assay over expressing MDR1 transporter by recombinant transfection of MDCKII cells, [F14512][1] was detectable, but the intracellular level was unchanged regardless of the MDR1 status or in presence of MDR1 competitors. Consistently, iterative selection of a [F14512][1]-resistant A-549 cell line with 36 cycles of [F14512][1] at cytotoxic doses (0.5 EC50) over 9 months, led to only moderate shift of resistance (9 fold). In the resulting selected A-549 subclones, MDR1, MRP1 and ABCG2 were detectable at the basal level. The ability of [F14512][1] to bypass MDR1 was finally assessed in a vinorelbine-resistant P388 model overexpressing high level of functional ABCB1 as indicated with the positive rhodamine export assay and cross resistance to MDR1 substrate. Moreover, [F14512][1] displays strong antileukemic activity in MDR1-positive P388 cells in vitro and additional in vivo measurements are in progress. In conclusion, the data concur to show that the antitumor activity of the targeted cytotoxic agent [F14512][1] is not impacted by the MDR1 status of cancer cells. MDR1 clearly does not affect the potency of [F14512][1] and this characteristic augurs well for the ongoing development of the drug in AML patients. Citation Format: Jean Philippe Annereau, Viviane Brel, William Riquet, Laurent Creancier, Isabelle Vandenberghe, Emmanuel Fournier, Celine Robichon, Aline Stennevin, Vanessa Offrete, Laurence Lacastaigneratte, Bruno Gomes, Anna Kruczynski, Christian Bailly, Nicolas Guilbaud. F14512, a novel vectorized topoiserase II inhibitor, bypasses MDR1 mediated resistance. [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 988. doi:10.1158/1538-7445.AM2013-988 [1]: /lookup/external-ref?link_type=GEN&access_num=F14512&atom=%2Fcanres%2F73%2F8_Supplement%2F988.atom

Abstract A51: Antiproliferative activity of F14512, a novel polyamine-vectorized drug, on multidrug resistant cancer cells

F14512 is a novel vectorized topoisomerase II inhibitor currently in phase I clinical study in AML. Briefly, F14512 combines a polyamine (spermine) chain with an epipodophyllotoxin warhead. An enhanced antitumor effect associated with a large therapeutic index results from this specific targeting of cancer cells through polyamine transport system. One of the most important limitations in chemotherapeutic treatment is acquired or innate drug resistance. To investigate this phenomenon for F14152, we selected A549 NSCLC cell lines upon F14512 treatment by increasing doses selection, and characterized the profile of resistance in term of ABC transporter expression and modification of topoisomerase II expression. As results, resistant cell lines were selected after 46 passages and 32 treatment of F14512 with doses ranging 0.25 to 4 IC50 of inhibition of proliferation, over a period of 9 months. As key observation, the intense selection pressure led solely to a moderated shift of EC50 of proliferation, not mediated through MDR1 nor MRP1, but more likely attributable to reduction of topoisomerase II expression. Moreover, we demonstrated that F14512 retains significant antiproliferative activity of F14512 on cell line overexpressing MDR1: CEM selected in vinblastine, A-549 in vinflunine, and P388 in vinorelbine, with a striking enhanced impact on the later model. According to these first investigations, we conclude that MDR1 would not be involved in the resistance mechanism against F14512. We are investigating hypotheses potentially explaining the collateral sensitivity to this novel polyamine derivative on MDR1 expressing P-388 cells.