Ex Vivo modeling of the apoptotic effects of Vivia009 and its metabolite in patients with Chronic Lymphocytic Leukemia
Matoses-Osborne M (1), Primo D (2), Hernandez P (2), Bennett TA (2), Ballesteros J (2), Caveda L (2), Trocˇniz IF (1)
(1) Department of Pharmacy and Pharmaceutical Technology; School of Pharmacy; University of Navarra; Spain. (2) Vivia Biotech S.L.; Madrid; Spain
Objectives: Vivia009 is a patented new drug indication for B-cell chronic lymphocytic leukaemia (B-CLL), a cancer of white blood cells that originates in bone marrow and develops in lymph nodes. Both, Vivia009 and the metabolite have shown in vitro and ex vivo activity. The aim of this study is to develop a pharmacodynamic model describing the apoptotic effects of the two compounds in combination characterizing the type of interaction (additivity, antagonistic, or synergistic).
Methods: Samples were extracted from patients diagnosed with B-CLL. Blood was then diluted with RPMI and 45ul of the suspension is added to each well of a 96-well plate that contains the pharmacological agents. The plate is divided in rows containing Vivia009, its metabolite or the combination of both. Drugs alone are set up in dose-response, starting at 100 uM with a 1 to 3 dilution, and for combinations, a fix amount of Vivia009 is added to a dose-response of the metabolite. The compound plates are then incubated for 6, 12 or 24 hours at 37║C with 5% CO2. After incubation, the erythrocytes are lysed and Annexin V-FITC, monoclonal antibodies anti-CD45-APC and anti-CD19-PE, are added to each well. The plates are then transferred to an automated flow cytometry system where the contents of each well is aspirated and analyzed by a CyAn flow cytometer. All data generated were analyzed simultaneously. The relationship between concentration and time of exposure and the percentage of apoptotic cells was described using the population approach with NONMEM VII.
Results: Visual inspection of the response vs. concentration profiles revealed that time of drug exposure, in addition to concentration was an important factor in the apoptotic effect, which was taken into account in the model including a delayed compartment for the parent drug and the metabolite. Isobolograms indicated a synergistic interaction which was modeled using an empirical model for synergism. Vivia009 and the metabolite showed similar potencies and mean apoptotic signal transduction times. The interaction parameter alpha was estimated with a value greater than 0 (0.43) indicating synergistic effects between Vivia009 and its main metabolite.
Conclusions: The results of this modeling exercise together with an integrated parent drug and metabolite pharmacokinetic model will help to optimize the dose to achieve a desirable active concentration (Vivi009 + metabolite) in patients diagnosed with B-CLL.
 Krutzik PO, Nolan GP (2006).Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling. Nat Methods.May;3(5):361-8..
 Fidler M, Kern SE (2006). Flexible interaction model for complex interactions of multiple anesthetics. Anesthesiology. Aug;105(2):286-96.