REVISITING METRONOMICS: MODEL-‐DRIVEN GEMCITABINE IN MICE WITH RESISTANT NEUROBLASTOMA Aurelie Lombard SMARTc UMR S_911 CRO2, Marseille 1 Metronomics •  New, alterna,ve dosing schedules can help to improve the eﬃcacy/toxicity balance of an,cancer treatments. •  Metronomics is based upon the repeated administra,on of low-‐dose cytotoxics over a long period of ,me. •  Beyond reducing toxici,es, higher eﬃcacy can be achieved through novel mechanisms of ac,on (i.e., an,angiogenic + immuno-‐s,mula,ng proper,es). 2 André et al. 2012 Metronomics •  The very concept of “repeated administra,on of low-‐dose cytotoxics over a long period of ,me” covers numerous possibili,es: •  What is a small dose? •  What is a repeated administra,on? •  What is a long period of ,me? •  Empirical search for op,mal condi,ons would require costly , unethical and ,me-‐consuming animal experiments. 3 Metronomics Gemcitabine •  Doses: 1, 2, 3.3, 5, 10, 30 mg/kg… •  Schedule: daily, 1-day on/2-days off, 3-weeks on/1-week off … •  Duration: 3 weeks, 4 weeks, continuous… 4 Modeling & Simulation •  Mathema,cal modeling can help to iden,fy ann op,mal solu,on among numerous combina,ons. •  This strategy is based upon the use of any data made available which are likely to help build a mathema,cal model and iden,fy its parameters. + •  Once built and validated, the model can generate in silico an inﬁnity of simula,ons ‘,ll a solu,on to a given problem (e.g.: “what is the best dosing to ensure maximum eﬃcacy?”) is found. 5 Computational Oncology & Metronomics 6 Barbolosi D et al. 2013, 2014 MetroGem Study Can metronomics help to improve the eﬃcacy of gemcitabine on a resistant neuroblastoma (GI-‐MEN, p53-‐)? Standard Model-‐driven GMZR 100 mg/kg i.p. QW for 4 weeks METROGEM1 1 mg/kg or 0.5 mg/kg SSC QD for 4 weeks VS. 7 MetroGem Study 1. 1I. Neuroblastome III. Traitement Treatment métro VS. std GI-ME-N transfection Transfection stable Luc + Luc+ par lentivirus 1V. PK de la Drug gemcitabine Monitoring 100 000 cells Xenogreffe s.c. xenograft 100 000 cellules Luc+ V. VI1. Suivi Vascular densité vasculaire density V1. Mesure de la masse tumorale Tumor Mass Monitoring parbybioluminescence bioluminescence Inflammation Suivi Grade d’inflammation 8 1. Contrôle 2.Administration METROGEM 0.5 2.Administration METROGEM 1 4.Administration Standard 100 MetroGem Study •  Transfection GI-ME-N Sélection des selected transfectants Stable clones with résistants puromycinà after lentivirus infection (Luc+ cells). puromycine : cellules LUC+. Cell/light correlation: Calcul nombre photons/cellule 4,5E+09 4E+09 3,5E+09 3E+09 2,5E+09 2E+09 1,5E+09 1E+09 500000000 0 photons émis/sec y = 4204,7x R² = 0,9996 0 200000 400000 600000 800000 Nombre de Cellules 1000000 1200000 Chaque cellule GI-ME-N-Luc+ émet 4205 photos/sec Each GI-ME-N-Luc+ cell emits 4205 photons/sec 9 MetroGem Study •  s.s.c. xenograft, treatment and tumor growth monitoring. 100 000 GI-ME-N Luc+ cells in 60% matrigel. Metronomic gemcitabine is administered using an osmotic pump. Pump has been calibrated to release: Ø  1 mg/kg/d (MetroGem1) Ø  0.5 mg/kg/d (MetroGem0.5) 10 MetroGem Study •  s.s.c. xenograft, treatment and tumor growth monitoring. Tumor growth is monitored by 2D + 3D bioluminescence. Main endpoint: search for a significant difference between the groups after treatment completion (i.e., 4 weeks). 11 MetroGem Study •  Drug Monitoring (LC-MS/MS). 40 35 GEM 10000 1000 100 dFdU 30 dFdU (ng/ml) Log gemcitabine (ng/ml) 100000 25 20 15 10 10 5 0 1 Standard Standard MetroGem1 MetroGem1 100000 Whereas standard Gem leads to Cmax around 80 µg/ml, 0.1 µg/ml steady state concentrations are observed with MetroGem1. 10000 1000 100 12 10 1 WK1 WK2 WK3 WK4 MetroGem Study •  Vascular Density-1(AngioSense 780) 9E+09 8E+09 Fluorescence 7E+09 6E+09 5E+09 4E+09 3E+09 2E+09 1E+09 0 Control 100 mg/kg MetroGem1 MetroGem0.5 MetroGem leads to lower vascular density 13 MetroGem Study •  Tumoral Inflammation (Prosense 680). 1200 photons émis/sec/100000000 1000 800 600 400 200 0 CONTROL STANDARD METRONOMIQUE A trend towards lower cathepsin expression is observed with MetroGem 14 MetroGem Study •  Antiproliferative efficacy. 9,00E+02 Control Gemcitabine 100 mg/kg 8,00E+02 Tumor size (10^6 p/sec) 7,00E+02 MetroGem-1 6,00E+02 MetroGem-0.5 5,00E+02 4,00E+02 3,00E+02 * * 2,00E+02 1,00E+02 *: p<0.05, Anova 0,00E+00 0 10 20 30 40 50 60 70 80 90 100 Time (days) Métronomique 1 mg/kg/j Métronomique 0.5 mg/kg/j 100 mg/kg 100 mg/kg 100 mg/kg 100 mg/kg Standard Gem shows liVle eﬃcacy at 100 mg/kg/w. MetroGem achieves a signiﬁcant reduc\on in tumor growth (-‐60%) a_er treatment comple\on (p<0.05 ANOVA). Stabilized disease is maintained at least 40 days a_er stopping treatment (p<0.05) 15 Conclusions •  Mathema,cal modeling can help to op,mize dose and scheduling of a complex regimen. •  When applied to metronomics gemcitabine, model-‐driven regimen led to a signiﬁcant reduc,on of tumor growth plus a stable disease for at least 90 days, whereas standard GEM proved to be ineﬀec,ve. •  Reducing tumor perfusion and an,-‐angiogenic eﬀect could, at least partly, explain this increase in eﬃcacy. •  Why controlled disease was achieved up to 50 days a_er stopping all treatment, remains to be elucidated. •  Although promising, these preliminary results will have to be conﬁrmed on more aggressive neuroblastoma models. 16 Thanks for Listening ! 17