Proton Pump Inhibitors Augment Nilotinib and Dasatinib Mediated Bcr-Abl Kinase Inhibition
Authors: Hiwase Devendra K , Eadie Laura , Saunders Verity , Hughes Timothy , White Deborah L. 
Published: October 2019

Abstract
Abstract 3991
Proton pump inhibitors (PPIs) are widely used for the treatment of gastro-oesophageal reflux and peptic ulcer disease. PPIs may not only alter absorption and metabolism of tyrosine kinase inhibitors (TKIs) but could also alter intracellular uptake and retention (IUR) into leukemic cells by interacting with ABCB1 and ABCG2 pump. There is limited literature assessing the interaction of PPI with TKI's at the cellular level. Here we compare the interaction of PPI with dasatinib, imatinib and nilotinib. Mononuclear cells (MNC) of CML-CP patients, K562 and K562-Dox (ABCB1 overexpressing), K562-ABCG2 cells were cultured with 14C-labelled dasatinib, imatinib and nilotinib with or without PPI for 2h and IUR were assessed. The effect of combining PPI with TKI's on Bcr-Abl kinase activity was assessed by measuring p-Crkl (surrogate marker of Bcr-Abl kinase activity). High concentrations of pantoprazole (1 and 2 mM) significantly increased dasatinib IUR in K562-Dox cells (p=0.004 and p=0.0006 respectively) but not in K562 cells (p=0.7 and p=0.1). Similarly, 1 mM (p=0.01) and 2 mM (p=0.007) esomeprazole significantly increased dasatinib IUR in K562-Dox cells but not in K562 cells. These data suggest that high concentration of PPI inhibit ABCB1 mediated dasatinib efflux. This was further supported by significant reduction in IC50dasatinib in K562-Dox cells by 1 mM pantoprazole (112±37 nM to 28±6; p=0.02; n=4). Similarly, 1 mM esomeprazole reduced IC50dasatinib (112±37 nM to 12±3 nM; n=2). This demonstrates that PPI mediated increase in IUR translates to increased kinase inhibition and lower IC50dasatinib. At lower concentrations (100 to 400 μM), neither pantoprazole nor esomeprazole significantly changed the dasatinib IUR or the IC50dasatinib in K562-Dox cells. In K562-ABCG2 cell line 100, 200, 400, 500, 1000 μM pantoprazole reduced IC50dasatinib from 27 nM to 15, 7, 11, 9.5 and 6.5 nM respectively. Similarly, 100, 200, 400, 500 and 1000 μM esomeprazole reduced IC50dasatinib from 21 nM to 15, 11, 8.5, 4.5 and 3.5 nM respectively. These data suggest that PPI enhances dasatinib mediated Bcr-Abl kinase inhibition in ABCG2-overexpressing cells. Although PPI did not change dasatinib IUR significantly in primary CML-MNC (n=10, Table I), it reduced IC50dasatinib (n=4, Table II).
Similarly, pantoprazole and esomeprazole (5 to 400 μM) significantly increased nilotinib IUR (n=10, Table I) and significantly reduced IC50nilotinib in CML-MNC (n=3, Table II). Pantoprazole also increased the nilotinib IUR in K562 and K562-Dox cells, and reduced the IC50nilotinib in K562 (500 vs. 250 nM) and K562-Dox (600 vs. 230 nM) cells. Similarly esomeprazole reduced the IC50nilotinib in K562 (500 vs. 250 nM) and K562-Dox (600 vs. 150 nM) cells. The effect of PPI on IC50nilotinib was dose dependent. Pantoprazole and esomeprazole reduced imatinib IUR in K562, K562-Dox and CML-MNC. Pantoprazole increased IC50imatinib in K562 (3.8 to 4 μM) and K562-Dox (6.5 to 8 μM) cells. Similarly, 200 μM of pantoprazole and esomeprazole significantly reduced IM IUR in CML-MNC. However, the effect of pantoprazole on IC50imatinib in CML-MNC was variable and modest (Table II).
Our data provide evidence that PPI might interfere with TKI activity. PPI's can enhance the dasatinib and nilotinib mediated Bcr-Abl kinase inhibition in primary CML cells.
Table I: Effect of PPI on dasatinib, imatinib and nilotinib intracellular uptake and retention (IUR) in CML-MNC (n=10) TKI at 1μM TKI at 2μM Pantoprazole 0 μM 100μM 200μM 400μM 0uM 100μM 200μM 400μM Imatinib 13.3 10.9 9.9 8.8 23.7 20.4 18.9 16.3 p-value 0.06 0.01 0.004 0.2 0.09 0.01 Nilotinib 13.0 16.4 16.6 17.4 30.5 56.9 66.2 74.7 p-value 0.04 0.02 0.03 0.004 0.001 <0.001 Dasatinib 10.3 11.1 9.4 7.9 18.9 18.3 17.7 18.9 p-value 0.6 0.1 0.08 0.4 0.4 0.2 Table II: Effect of PPI on IC50 of nilotinib, dasatinib and imatinib in CML-MNC Nilotinib Nilotinib + 200 μM pantoprazole (% change) Nilotinib + 200 μM esomeprazole (% change) CML1 62.5 nM 30 nM (−52) 55 nM (−12) CML2 80 nM 32 nM (−60) 17 nM (−78) CML3 75 nM 68 nM (−9.4) 27 nM (−64) Dasatinib Dasatinib + 200 μM pantoprazole (% change) Dasatinib + 200 μM esomeprazole (% change) CML1 1.8 nM 0.7 nM (−61) 1.2 nM (−33) CML2 3 nM 2.6 nM (−13) – CML3 7 nM 2.5 nM (−64) – CML4 2.25 nM 1.7 nM (−25) 1.5 nM (−33) Imatinib Imatinib + 200 μM pantoprazole (% change) Imatinib + 200 μM esomeprazole (% change) CML1 1.05 μM 0.325 μM (−69) 0.8 μM (−23) CML2 1.8 μM 1.35 μM (−25) CML3 0.83 μM 1.15 μM (+38) 0.65 μM (−21)

Disclosures:
Hughes: Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. White:Novartis: Honoraria, Research Funding; BMS: Research Funding.