Arnaud Pillon

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.

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.

Activity of the polyamine-vectorized anti-cancer drug F14512 against pediatric glioma and neuroblastoma cell lines.

SummaryThe poor prognosis of children with high-grade glioma (HGG) and high-risk neuroblastoma, despite multidisciplinary therapeutic approaches, demands new treatments for these indications. F14512 is a topoisomerase II inhibitor containing a spermine moiety that facilitates selective uptake by tumor cells via the Polyamine Transport System (PTS) and increases topoisomerase II poisoning. Here, F14512 was evaluated in pediatric HGG and neuroblastoma cell lines. PTS activity and specificity were evaluated using a fluorescent spermine-coupled probe. The cytotoxicity of F14512, alone or in combination with ionizing radiation and chemotherapeutic agents, was investigated in vitro. The antitumor activity of F14512 was assessed in vivo using a liver-metastatic model of neuroblastoma. An active PTS was evidenced in all tested cell lines, providing a specific and rapid transfer of spermine-coupled compounds into cell nuclei. Competition experiments confirmed the essential role of PTS in the cell uptake and cytotoxicity of F14512. This cytotoxicity appeared greater in neuroblastoma cells compared with HGG cells but appeared independent of PTS activity levels. In vivo evaluation confirmed a marked and prolonged antitumoral effect in neuroblastoma cells. The combinations of F14512 with cisplatin and carboplatin were often found to be synergistic, and we demonstrated the significant radiosensitizing potential of F14512 in the MYCN-amplified Kelly cell line. Thus, F14512 appears more effective than etoposide in pediatric tumor cell lines, with greater efficacy in neuroblastoma cells compared with HGG cells. The synergistic effects observed with platinum compounds and the radiosensitizing effect could lead to a clinical development of the drug in pediatric oncology.

Optical Imaging of Disseminated Leukemia Models in Mice with Near-Infrared Probe Conjugated to a Monoclonal Antibody

Background The assessment of anticancer agents to treat leukemia needs to have animal models closer to the human pathology such as implantation in immunodeficient mice of leukemic cells from patient samples. A sensitive and early detection of tumor cells in these orthotopic models is a prerequisite for monitoring engraftment of leukemic cells and their dissemination in mice. Therefore, we developed a fluorescent antibody based strategy to detect leukemic foci in mice bearing patient-derived leukemic cells using fluorescence reflectance imaging (FRI) to determine when to start treatments with novel antitumor agents. Methods Two mAbs against the CD44 human myeloid marker or the CD45 human leukocyte marker were labeled with Alexa Fluor 750 and administered to leukemia-bearing mice after having verified the immunoreactivity in vitro. Bioluminescent leukemic cells (HL60-Luc) were used to compare the colocalization of the fluorescent mAb with these cells. The impact of the labeled antibodies on disease progression was further determined. Finally, the fluorescent hCD45 mAb was tested in mice engrafted with human leukemic cells. Results The probe labeling did not modify the immunoreactivity of the mAbs. There was a satisfactory correlation between bioluminescence imaging (BLI) and FRI and low doses of mAb were sufficient to detect leukemic foci. However, anti-hCD44 mAb had a strong impact on the tumor proliferation contrary to anti-hCD45 mAb. The use of anti-hCD45 mAb allowed the detection of leukemic patient cells engrafted onto NOD/SCID mice. Conclusions A mAb labeled with a near infrared fluorochrome is useful to detect leukemic foci in disseminated models provided that its potential impact on tumor proliferation has been thoroughly documented.

Discovery bioanalysis and in vivo pharmacology as an integrated process: a case study in oncology drug discovery

BACKGROUND A bioanalytical team dedicated to in vivo pharmacology was set up to accelerate the selection and characterization of compounds to be evaluated in animal models in oncology. RESULTS A DBS-based serial microsampling procedure was optimized from sample collection to extraction to obtain a generic procedure. UHPLC-high-resolution mass spectrometer configuration allowed for fast quantitative and qualitative analysis. Using an optimized lead compound, we show how bioanalysis supported in vivo pharmacology by generating blood and tumor exposure, drug monitoring and PK/PD data. CONCLUSION This process provided unique opportunities for the characterization of drug properties, selection and assessment of compounds in animal models and to support and expedite proof-of-concept studies in oncology.

Abstract 1219: Patient-derived tumor models of resistant metastatic melanoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Despite recent advances in the therapy of metastatic melanoma, patients have a poor prognosis and die from their disease, with a 5-year survival rate of less than 20%. Metastatic melanoma remains mainly chemoresistant, therefore novel therapeutic strategies to overcome primary or acquired resistance are required. In order to more reliably predict clinical activity of novel compounds in melanoma patients, we have established a series of patient-derived tumor xenograft models (PDTX). Surgically resected tumors samples were obtained from patients with metastatic melanoma before or after treatment. Twenty-three samples from 9 patients and representative of various stages of disease progression were implanted onto immunocompromised NSG mice. A high take rate of 56% (13/23 samples) was observed, corresponding to the samples from 8/9 patients. Tumor take was independent of tumor size, histologic parameters and B-RAF status. The first tumor generation (graft) harbouring the patient-derived sample (G1), as well as the subsequent generations were characterized for each PDTX. The pattern of expression of the melanoma differentiation markers S100, melanosome, tyrosinase and melan-A were maintained over the serial transplantations. These models also closely recapitulated the heterogeneity of patient tumors in terms of cell morphology over the first generations (G1-G5), but this heterogeneity tended to decrease with the number of generations. One of these primary PDTXs, MEL-11 exhibited metastatic spread in axillary lymph nodes. Furthermore 2 other primary melanoma PDTXs, MEL-1 and MEL-3, established from primary refractory patient metastatic melanoma retained complete resistance to temozolomide and vemurafenib, the two reference drugs for melanoma. The MEL-3 model was selected to assess the anti-melanoma activity of F-RK-4, a novel multi-kinases inhibitor (including inhibition of mutated B-RAF). Multiple i.p. administrations of F-RK-4 resulted in a significant antitumor activity, as reflected by a tumor growth inhibition of 50%. Overall these results suggest that these patient-derived melanoma xenografts represent a useful preclinical tool to identify novel anti-melanoma therapeutics. Citation Format: Bruno Gomes, Celine ROBICHON, Arnaud Pillon, Jean-Philippe Annereau, Sandrine Pourtau, Jean-Christophe Blanchet, Aline Stennevin, Karim Bedjeguelal, Philippe Rochaix, Ignacio Garrido-Stowhas, Laurence Lamant, Nicolas Meyer, Nicolas Guilbaud, Christian Bailly, Anna Kruczynski. Patient-derived tumor models of resistant metastatic melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1219. doi:10.1158/1538-7445.AM2014-1219

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

Abstract A214: F14512, a novel targeted cytotoxic agent exhibits a marked antileukemic activity, alone and in combination with AraC.

Chemotherapy remains mainly used for the treatment of Acute Myelogenous Leukemia (AML). However, in the past 3 decades limited progress has been achieved in improving the long-term disease-free survival. Therefore the development of more effective drugs for AML represents a high level of priority. F14512 combines an epipodophyllotoxin core targeting topoisomerase II with a spermine moiety introduced as a cell delivery vector. In fact the polyamine moiety facilitates F14512 selective uptake by tumor cells via the polyamine transport system, a machinery frequently overactivated in cancer cells. In this study, we report the in vivo antileukemic activity of F14512 against human AML models, established from patient samples. AML cells, collected from 3 different patients, were established onto NSG mice (LAM-2, LAM-7 and LAM-18). These 3 AML samples exhibited a normal karyotype, with FLT3-ITD, NPM1 and DNMTA3 mutations which proved stable over serial transplantations in vivo. After multiple i.v. administrations of F14512, 3 times a week for 3 weeks, an extensive reduction of AML cell number (98–99%) was observed in LAM-2 and LAM-7 - bearing mice. This antileukemic activity was recorded on the basis of flow cytometry, q-PCR and histology assessments. The effects of F14512 on LAM-18 bearing mice were marginal with an inhibition of AML cell growth of 42%. We also show in vitro and in vivo synergistic effects of F14512 in combination with AraC, one of the frontline chemotherapeutic agents for AML. These results were obtained using the HL-60 cell line. The activity of F14512 in combination with AraC will be further investigated in patient AML models. Collectively, these results demonstrate that F14512 exhibits a marked in vivo antileukemic activity, supporting its clinical development. Phase I clinical trials in onco-hematology are on going with this novel promising drug candidate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A214.

Abstract B186: Preclinical antileukemic activity of F14512, a novel targeted cytotoxic agent

Despite advances in the therapy of acute myeloid leukemia (AML), the majority of patients die from their disease. Therefore, the lack of effective therapy mandates the development of novel compounds to improve the outcome of patients with relapsed and refractory leukemias. F14512 is a potent spermine‐epipodophyllotoxin conjugate exploiting the polyamine transport system for tumor cell delivery. In this study, we report the in vivo antitumor activity of F14512 against experimental models of AML cell lines and of patient AML samples. F14512 markedly reduced the growth of HL‐60 and U937 cell lines in an in vivo xenotransplantation model, resulting in a highly significant increase of survival of leukemia‐bearing mice. Etoposide evaluated concurrently demonstrated only moderate in vivo activity against these models. F14512 induced in vivo apoptosis of HL‐60 cells, as shown by caspase‐3 activation and PARP cleavage. In an effort to mimic the human disease, we injected approximately 106 AML cells collected from a patient into NOD/SCID mice and allowed them to establish as xenografts for 8 weeks. Subsequent treatment with F14512 was carried out for 2 or 3 weeks followed by the analysis at the end of treatment and 1 week after the end of treatment. Two human AML samples were analyzed. Multiple i.v. administrations of F14512 at 0.32 mg/kg, induced an extensive reduction of the number of leukemic cells in mouse bone marrow and blood (97–99%), assessed by flow cytometry analysis, quantitative RT‐PCR and histology. To identify leukemic cells expressing an active polyamine transport system, we developed a functional method based on the measurement of the cellular uptake of a nitrobenzoxadiazole fluorescent probe (F96982) combining the same spermine moiety as F14512. The level of fluorescence emitted by the probe F96982 was high in HL‐60 cells as well in the 2 patient AML samples that proved to be sensitive to F14512 in vivo. Collectively, these results demonstrated that F14512 exhibits a marked in vivo antileukemic activity, supporting its clinical development. Phase I clinical trials in onco‐hematology are now initiated with this novel promising drug candidate. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B186.