IN VITRO COMPARATIVE STUDY OF THE PHOSPHORYLATION INHIBITION AND APOPTOTIC ACTIVITY INDUCED BY THE ORIGINAL AND A BIOEQUIVALENT PHARMACOLOGICAL IMATINIB

PALMITTELLI M; LARRIPA I; DE CAMPOS NEBEL; GUTIERREZ L; GONZALEZ CID M

Mar del Plata

Congreso; LXI REUNIÓN ANUAL SAIC; 2016

Soc. Arg. de Investigación Clínica

The development of tyrosine kinase inhibitors (TKI) marked the biggest advance in the treatment of chronic myeloid leukemia (CML). The TKIs block the ATP binding site of the onco-protein BCR/ABL1 with high affinity and specificity. Thereby, the tumor cells lose their proliferative advantage and the apoptotic process is induced.The aim of this study was to compare the biological effectiveness of generic TKI (Imatinova®, Celnova Pharma) versus the original inhibitor (Glivec®, Novartis) in vitro, through an induction of apoptosis and inhibition of phosphorylation of BCR/ABL1 in K562, MEG01 (BCR/ABL1 p210) and SUB15 (BCR/ABL1 p190) cell lines.Early apoptosis was assessed by flow cytometry with Annexin V-FITC in K562 and MEG01 cells subjected to 2.5 µM of each TKIs and evaluated at 24, 48 and 72 hs. This analysis showed a maximum level of apoptosis at 72 hs, with values higher than 90% in both cell lines. Late apoptosis was evaluated by acridine orange/ethidium bromide staining. K562 and MEG01 cells were treated with increasing concentrations of each TKIs (0.25 ? 5.0 uM) and analyzed by fluorescence microscopy at 72hs. Both TKIs showed values of apoptosis higher than 83% for both cell lines. Therefore no significant differences were observed between both TKIs regarding apoptosis.We also investigate the inhibition on BCR-ABL1 tyrosine kinase activity through the effect on phosphorylation status of CRKL (Crk-like protein, sustrate of BCR-ABL1) in K562, MEG01 and SUB15 cell lines by western blot (WB) using the antibody p-CRKL. Our results showed that both drugs induced similar reduction in p-CRKL with the doses tested (5-20µM). Higher doses than 20µM induced elevated levels of apoptosis preventing the WB analysis.These studies in cell lines BCR/ABL1 positive (p210 and p190) showed that induced apoptosis and the reduction of levels p-CRKL were similar, indicating that, in vitro, Imatinova® and Glivec® show the same behavior which would indicate an analog biological response.