Yves Guminski

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.

Preclinical activity of F14512, designed to target tumors expressing an active polyamine transport system

SummaryWe have exploited the polyamine transport system (PTS) to deliver selectively a spermine-drug conjugate, F14512 to cancer cells. This study was aimed to define F14512 anticancer efficacy against tumor models and to investigate whether fluorophor-labeled polyamine probes could be used to identify tumors expressing a highly active PTS and that might be sensitive to F14512 treatments. Eighteen tumor models were used to assess F14512 antitumor activity. Cellular uptake of spermine-based fluorescent probes was measured by flow cytometry in cells sampled from tumor xenografts by needle biopsy. The accumulation of the fluorescent probe within B16 tumors in vivo was assessed using infrared fluorescence imaging. This study has provided evidence of a major antitumor activity for F14512. Significant responses were obtained in 67% of the tumor models evaluated, with a high level of activity recorded in 33% of the responsive models. Complete tumor regressions were observed after i.v., i.p. or oral administrations of F14512 and its antitumor activity was demonstrated over a range of 2–5 dose levels, providing evidence of its good tolerance. The level of cellular fluorescence emitted by the fluorescent probes was higher in cells sampled from tumors sensitive to F14512 treatments than from F14512-refractory tumors. We suggest that these probes could be used to identify tumors expressing a highly active PTS and guide the selection of patients that might be treated with F14512. These results emphasize the preclinical interest of this novel molecule and support its further clinical development.

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.

F 11782, a dual inhibitor of topoisomerases I and II with an original mechanism of action in vitro, and markedly superior in vivo antitumour activity, relative to three other dual topoisomerase inhibitors, intoplicin, aclarubicin and TAS-103

Purpose: F 11782 (2′′,3′′-bis pentafluorophenoxyacetyl-4′′,6′′-ethylidene-β-d-glucoside of 4′-phosphate-4′-dimethylepipodophyllotoxin, di-N-methyl glucamine salt) is a newly synthesized dual catalytic inhibitor of topoisomerases I and II with major in vivo antitumour activity. In this study, we compared and contrasted F 11782 with three other known inhibitors of both these nuclear enzymes, namely aclarubicin, intoplicin and TAS-103, and established its novel mechanism of action. Methods: In vitro growth-inhibitory effects against a panel of murine and tumour cell lines were measured by cell counting, clonogenicity or tetrazolium metabolic dye (MTT) assays. In vivo antitumour activities were evaluated against two murine tumour models (i.v. P388 leukaemia and s.c. B16 melanoma). Finally, interactions with either DNA or DNA-topoisomerases were determined using various methodologies: DNA-intercalator displacement, pBR322 DNA relaxation, kDNA decatenation, topoisomerase II extractability measurements, stabilization of topoisomerase-induced cleavable complexes (CC) in vitro and in cells, and gel retardation assays. Results: F 11782 had a different profile of sensitivities and proved generally less cytotoxic than the other dual inhibitors tested in vitro, while showing significantly superior antitumour activity in vivo. F 11782, which did not stabilize CC either in vitro or in cells, was the only compound of this series capable of inhibiting the catalytic activity of both DNA-topoisomerases without interacting with DNA, and of completely impairing the binding of these nuclear proteins to DNA. Moreover, only cotreatment of cells in vitro with F 11782 enhanced the cytotoxic activity of etoposide. Conclusion: These results emphasize the novel mechanism of action of F 11782 vis-à-vis the other dual inhibitors of topoisomerases I and II and so augur well for its future clinical development.

99mTc-HYNIC-spermine for imaging polyamine transport system-positive tumours: preclinical evaluation

PurposeF14512 exploiting the polyamine transport system (PTS) for tumour cell delivery has been described as a potent antitumour agent. The optimal use of this compound will require a probe to identify tumour cells expressing a highly active PTS that might be more sensitive to the treatment. The aim of this study was to design and characterize a scintigraphic probe to evaluate its uptake in cancer cells expressing the PTS.MethodsThree polyamines coupled to a hydrazinonicotinamide (HYNIC) moiety were synthesized and labelled with 99mTc. Their radiochemical purity was determined by HPLC. The plasma stability of the 99mTc-HYNIC-spermine probe and its capacity to accumulate into PTS-active cells were also evaluated. In vitro internalization was tested using murine melanoma B16/F10 cells and human lung carcinoma A549 cells. Biodistribution was determined in healthy mice and tumour uptake was studied in B16/F10 tumour-bearing mice. A HL-60-Luc human leukaemia model was used to confront single photon emission computed tomography (SPECT) images obtained with the 99mTc-labelled probe with those obtained by bioluminescence.ResultsThe 99mTc-HYNIC-spermine probe was selected for its capacity to accumulate into PTS-active cells and its stability in plasma. In vitro studies demonstrated that the probe was internalized in the cells via the PTS. In vivo measurements indicated a tumour to muscle scintigraphic ratio of 7.9±2.8. The combined bioluminescence and scintigraphic analyses with the leukaemia model demonstrated that the spermine conjugate accumulates into the tumour cells.ConclusionThe 99mTc-HYNIC-spermine scintigraphic probe is potentially useful to characterize the PTS activity of tumours. Additional work is needed to determine if this novel conjugate may be useful to analyse the PTS status of patients with solid tumours.

Abstract A87: Synthesis and structure‐activity relationships of a series of epipodophyllotoxin polyamine conjugated derivatives vectorized for active polyamine transporter system in tumor cells, leading to the selection of F14512 for clinical trials

One of the major concerns for chemotherapy is the selective targeting of drugs into highly proliferative cancer cells. Natural polyamines (spermine, spermidine, putrescine) are essential for the regulation of cellular growth and differentiation. Due to their highly proliferative nature, cancer cells have a pronounced need to import polyamines from their external environment, through the polyamine transporter system (PTS). On the basis of this biological mechanism, we vectorized the new cytotoxic anticancer compound F14512, a epipodophyllotoxin — spermine conjugate, into tumor cells.1 Here we present the synthesis and the structure‐activity relationships of a new series of compounds constituted by an podophyllotoxin core tethered with a polyamine moiety with a variable spacer. Two synthetic strategies with protected polyamines, and a direct 3 steps synthesis of F14512 from natural podophyllotoxin and spermine without any protection are presented.2 This series of topoisomerase II inhibitors were checked for their cytotoxicity on A549 lung cancer cell line, displaying marked potency up to nM range. Cancer cell internalization through PTS was assessed by selective cytotoxicity on different PTS expressing cell lines, and by competition experiments. Our results displayed a potent specificity for the conjugated tetramine (spermine) compounds, which were more recognized than the triamine (spermidine) ones, while mono and diamines showed no selectivity. Lead compounds were also tested in vivo and proved potent antitumor activity. This series of new water‐soluble cytotoxic compounds culminates with the selection of F14512 for clinical trials. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A87.

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

Practical Demethylation of Podophyllotoxin and Efficient Preparation of 4-Amino-4-deoxy-4′-demethylepipodophyllotoxin

Abstract 4′-Demethylepipodophyllotoxin (4′-DMEP) was readily available through demethylation of podophyllotoxin using methionine in methanesulfonic acid in the presence of TFA (or acetone/water). Thus, 4-amino-4-deoxy-4′-demethylepipodophyllotoxin was obtained in three steps in excellent yield by a Ritter reaction on 4′-DMEP, followed by treatment with thiourea in AcOH. GRAPHICAL ABSTRACT

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.

Abstract 2747: Synergistic antileukemic activity of F14512 in combination with AraC

New antileukemic agents are urgently needed to achieve improvement of the survival of patients suffering from Acute Myelogenous Leukemia (AML). F14512 combines an epipodophyllotoxin core with a spermine moiety that targets cells with a high demand for polyamines such as cancer cells. F14512 exhibits a high level of in vivo anti-tumor activity in a series of experimental murine and human solid tumor models and has entered into clinical phase one. In this study, we investigated the in vivo antileukemic activity of F14512 against cell line or primary human AML models and evaluated its potential in combination with the reference antileukemic agent, AraC. HL60 cells or AML cells collected from different patients were engrafted onto NSG mice. Using flow cytometry and q-PCR analysis, we demonstrated that multiple administrations of F14512 for 3 weeks resulted in an extensive reduction of AML cell number in the bone marrow and blood of treated mice. We also showed in vitro and in vivo synergistic activity of F14512 in combination with AraC in HL-60 and primary AML models when both compounds were used at suboptimal doses. The mechanisms triggering primary leukemic cell death were investigated and our results indicated that senescence could be involved. Collectively, these results demonstrate that F14512 exhibits a marked in vivo antileukemic activity, supporting the Phase I clinical trials of this novel promising drug candidate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2747. doi:1538-7445.AM2012-2747