Proton Pump Inhibitors Augment Nilotinib and Dasatinib Mediated Bcr-Abl Kinase Inhibition

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.

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In Contrast to Imatinib, Dasatinib Intracellular Concentration In CML-CD34+ Progenitors Is Not Significantly Different Than That Observed In CD34- Mature Cells.

Blood ◽

10.1182/blood.v116.21.1205.1205 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1205-1205

Author(s):

Devendra K Hiwase ◽

Jane Engler ◽

Verity Saunders ◽

Deborah L. White ◽

Timothy Hughes

Keyword(s):

Mrna Expression ◽

Research Funding ◽

Kinase Activity ◽

Intracellular Uptake ◽

Leukemic Stem Cells ◽

Kinase Inhibition ◽

Abl Kinase ◽

Psc 833 ◽

Cd34 Cells ◽

Expression And Activity

Abstract Abstract 1205 Long term follow up of imatinib (IM) clinical studies and in vitro studies suggest that tyrosine kinase inhibitors (TKI) do not eradicate leukemic stem cells. Refractoriness of leukemic stem cells is postulated to be due to inadequate Bcr-Abl kinase inhibition which in turn could be due to low intracellular uptake and retention (IUR) of TKI. We have previously demonstrated that IM cellular uptake is predominantly mediated by the organic cation transporter protein (OCT-1) and patients with low OCT-1 activity have suboptimal response as compared to patients with high OCT-1 activity. More recently Engler et al (Leukemia 2010) demonstrated that IM IUR is significantly lower in CML-CD34+ cells compared to CD34- cells. This could be due to low expression and activity of the OCT-1 protein and/or high expression of ABCB1 and/or ABCG2 (Jiang et al Leukemia 2007). Although dasatinib is clinically available there are no published data assessing dasatinib IUR in CML-CD34+ progenitors. We and others have previously demonstrated that dasatinib cellular uptake is predominantly OCT-1 independent and dasatinib is a substrate of ABCB1 and ABCG2. We hypothesized that dasatinib IUR would be lower in CML-CD34+ cells compared to CD34- cells. In this study we compare dasatinib and IM IUR; and OCT-1 and ABCB1 mRNA expression in CML-CD34+ and CD34- cells of newly diagnosed CML-CP patients. CD34+ and CD34- cells were incubated with 14C-dasatinib (100 nM and 1 μM) or 14C-IM (2 μM) for 2h and IUR was assessed as described previously (Hiwase et al Clin Cancer Res. 2008). As shown previously, the OCT-1 expression and activity was lower in CML-CD34+ cells, and resulted in lower IM IUR in CML-CD34+ cells compared to CML-CD34- cells (15±5 vs. 27±5; p=0.04; Fig 1A and C). However at a therapeutically achievable concentration (100 nM dasatinib) and at higher concentration (1 μM dasatinib), there was no significant difference in dasatinib IUR in CML-CD34+ and CD34- cells (Fig 1B). Low OCT-1 expression and activity in CML-CD34+ cells did not influence the dasatinib IUR; further confirming that dasatinib cellular influx is predominantly OCT-1 independent. Despite higher ABCB1 mRNA expression in CML-CD34+ cells, the dasatinib IUR was not lower in CML-CD34+ cells compared to CD34- cells. High ABCB1 mRNA expression may not necessarily translate into high ABCB1 activity. To this end, we assessed the effect of PSC-833, an ABCB1 inhibitor, on dasatinib IUR and dasatinib mediated Bcr-Abl kinase inhibition in CML-CD34+ cells. The baseline p-Crkl, a surrogate marker of Bcr-Abl kinase activity, was significantly higher in CML-CD34+ compared to CML-CD34- cells (67±5% vs. 55±8%; p=0.002; n=9). PSC-833 neither increased dasatinib IUR, nor enhanced dasatinib mediated Bcr-Abl kinase inhibition in CML-CD34+ cells (% p-Crkl at 10 nM dasatinib: 20±6 vs. 27±10). Similarly, Ko143, an ABCG2 inhibitor, did not significantly change dasatinib IUR or Bcr-Abl kinase inhibition (% p-Crkl at 10 nM dasatinib: 21±3 vs. 27±10). In summary, although dasatinib is an ABCB1 and ABCG2 substrate, ABCB1 and ABCG2 inhibitors neither increase dasatinib IUR, nor enhance dasatinib mediated Bcr-Abl kinase inhibition in CML-CD34+ cells. This data suggest that dasatinib IUR in CML-CD34+ cells is not influenced by ABCB1 and ABCG2. Hatziieremia et al (Exp. Hematology, 2009) reported that ABCB1 activity is low in CML-CD34+ cells and suggested that it did not influence IM level in CML-CD34+ cells. We further demonstrated that 100 nM dasatinib inhibited ≥95% Bcr-Abl kinase activity in CML-CD34+ and CD34- cells. In summary our data demonstrates that in contrast to IM, the intracellular concentration of dasatinib is equivalent in mature and immature CML cell compartments which may contribute to better targeting of early CML progenitors with dasatinib. Fig. 1: In contrast to IM, dasatinib intracellular uptake and retention (IUR) is not significantly different in CML-CD34+and mature CD34-cells: (A) OCT-1 activity and IM IUR is significantly lower in CML-CD34+ than CD34- cells (p=0.04). (B) However, dasatinib IUR is not significantly different in CD34+ and CD34- cells (p=0.8) (C) OCT-1 mRNA expression is lower in CML-CD34+ cells than CD34- cells. While ABCB1 expression is significantly higher in CML-CD34+ compared to CD34- cells (p=0.007). Fig. 1:. In contrast to IM, dasatinib intracellular uptake and retention (IUR) is not significantly different in CML-CD34+ and mature CD34- cells: (A) OCT-1 activity and IM IUR is significantly lower in CML-CD34+ than CD34- cells (p=0.04). (B) However, dasatinib IUR is not significantly different in CD34+ and CD34- cells (p=0.8) (C) OCT-1 mRNA expression is lower in CML-CD34+ cells than CD34- cells. While ABCB1 expression is significantly higher in CML-CD34+ compared to CD34- cells (p=0.007). Disclosures: White: Novartis: Honoraria, Research Funding; BMS: Research Funding. Hughes:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding.

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Blocking of Cytokine Survival Signals along with Intense Bcr-Abl Kinase Inhibition May Eradicate CML Progenitor Cells.

Blood ◽

10.1182/blood.v114.22.3250.3250 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3250-3250

Author(s):

Devendra K Hiwase ◽

Deborah L White ◽

Jason A Powell ◽

Verity A Saunders ◽

Stephanie Zrim ◽

...

Keyword(s):

Cell Death ◽

Cell Lines ◽

Research Funding ◽

Kinase Activity ◽

Short Term ◽

Kinase Inhibition ◽

Gm Csf ◽

Abl Kinase ◽

Cd34 Cells ◽

Short Term Culture

Abstract Abstract 3250 Poster Board III-1 Preclinical studies of imatinib set the paradigm of continuous Bcr-Abl kinase inhibition for optimal response in chronic myeloid leukemia (CML). However, the clinical success of once daily dasatinib, despite its short serum half life, implies that intermittent inhibition of Bcr-Abl kinase activity is sufficient for clinical response. In vitro studies also demonstrated that short-term intense (≥90%) Bcr-Abl kinase inhibition triggers cell death in BCR-ABL + cell lines, demonstrating their oncogene addiction. However, the effect of short-term intense kinase inhibition on CD34+ CML progenitors is not studied. Clinical, mathematical modelling and in vitro studies suggest that leukemic stem cells (LSC) are difficult to eradicate and hence the majority of CML patients may not be cured with tyrosine kinase inhibitors (TKI). Inadequate Bcr-Abl kinase inhibition has been postulated to cause refractoriness of LSC to TKI's. This may be due to increased expression of ABCB1 and ABCG2 efflux proteins, or the quiescent state of LSC. However, the phenomenon could be independent of Bcr-Abl kinase activity. In vivo leukemic progenitors live in a cytokine rich environment which may be providing a mechanism for Bcr-Abl independent resistance. We have assessed the impact of short-term intense Bcr-Abl kinase inhibition on CML cell lines and CML CD34+ primary cells in the presence and absence of cytokines. In CML cell lines, short-term (cells were cultured with dasatinib for 30 min and following thorough drug washout, cells were recultured in drug free media for 72 hr) intense Bcr-Abl kinase inhibition with 100 nM dasatinib triggers cell death. In CML-CD34+ cells 30 min of culture with 100 nM dasatinib (n=13) or 30 μM IM (n=7) reduced the level of p-Crkl (surrogate marker of Bcr-Abl kinase activity) by 97±3% and 96±4% respectively. In the presence of either a six growth factors cocktail (6-GF; n=10) or GM-CSF (n=11) or G-CSF (n=4) alone, despite 97% inhibition of p-Crkl, short-term culture with 100 nM dasatinib (D100ST) reduced colony forming cells (CFC) by only 24%, 32% or 5%, respectively. However without cytokines, D100ST reduced CML-CD34+ CFCs by 70%. Consistent with the results observed with dasatinib, short-term culture with 30 μM imatinib (IM) (n=3) also reduced 90% CFC in the absence of cytokines but by only 38% in the presence of 6-GF. These results suggest that in CML-CD34+ cells, GM-CSF, G-CSF or 6-GF mediate Bcr-Abl independent TKI resistance. It is possible that cytokines may be promoting cell survival via signalling pathways that are refractory to dasatinib. To examine this possibility, we assessed the effect of D100ST on p-STAT5 signalling in CML-CD34+ cells, in the presence and absence of GM-CSF, G-CSF or 6-GF. STAT5 was constitutively phosphorylated in CML-CD34+ cells, and in the absence of cytokines, D100ST reduced the p-STAT 5. STAT5 phosphorylation was not inhibited by D100ST when cells were cultured with 6-GFs or GM-CSF however, the combination of D100ST and a Janus kinase (Jak) inhibitor dramatically reduced p-STAT5. Similarly, in the presence of GM-CSF (32.35±5.16% vs. 68.33±14.90%) or G-CSF (58.13±13 vs. 94.68±21.12) combination of D100ST and JAK inhibitor significantly reduced CFC compared to D100ST only. Thus our data suggest that in contrast to CML cell lines, primary CML progenitors may not be completely dependent on the BCR-ABL oncogene and that activation of the cytokine mediated JAK-2/STAT-5 pathway may circumvent the need for BCR-ABL signalling for maintenance of survival. Thus a therapeutic strategy based on short-term intense kinase inhibition may have limited success unless critical redundant cytokine-induced survival pathways are also inhibited. We postulate that blockade of cytokine signalling along with short-term intense Bcr-Abl kinase inhibition with a potent second generation TKI may provide a novel strategy to eradicate primitive CML cells. Fig 1 In CML-CD34+ cells, Jak kinase inhibition abrogates the rescuing effect of cytokines on cell death induced by BCR-ABL blockade: In the absence of cytokines (No GF, n=11) short-term culture with 100 nM dasatinib (D100ST) reduced CFCs by 67% of control, however in the presence of 6-GFs (n=10), GM-CSF (n=10) or G-CSF (n=4) it could reduce CFCs by only 24%, 32% or 5% of control respectively (B) In the presence of GM-CSF (n= 4) or G-CSF (n= 4), combination of Jak inhibition and D100ST reduced CFC compared to dasatinib alone. Fig 1. In CML-CD34+ cells, Jak kinase inhibition abrogates the rescuing effect of cytokines on cell death induced by BCR-ABL blockade: In the absence of cytokines (No GF, n=11) short-term culture with 100 nM dasatinib (D100ST) reduced CFCs by 67% of control, however in the presence of 6-GFs (n=10), GM-CSF (n=10) or G-CSF (n=4) it could reduce CFCs by only 24%, 32% or 5% of control respectively (B) In the presence of GM-CSF (n= 4) or G-CSF (n= 4), combination of Jak inhibition and D100ST reduced CFC compared to dasatinib alone. Disclosures: White: Novartis and Britol-Myers Squibb: Research Funding. Hughes:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Lyn Kinase Alters Gab2 Phosphorylation and c-Cbl Protein Levels To Regulate Imatinib Sensitivity and Survival of Chronic Myelogenous Leukemia Cells.

Blood ◽

10.1182/blood.v108.11.2132.2132 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2132-2132

Author(s):

Ji Wu ◽

Feng Meng ◽

Moshe Talpaz ◽

Nicholas J. Donato

Keyword(s):

Tyrosine Phosphorylation ◽

Kinase Inhibitor ◽

K562 Cells ◽

Imatinib Resistance ◽

Kinase Inhibition ◽

Protein Levels ◽

Abl Kinase ◽

Lyn Kinase ◽

Imatinib Resistant ◽

Cbl Protein

Abstract The tyrosine kinase inhibitor imatinib mesylate (Gleevec) is effective in controlling BCR-ABL expressing leukemias but resistance occurs in some early phase patients while it is more common in advanced disease. Resistance has been generally associated with mutations in the BCR-ABL kinase that effect drug affinity. However patients are also increasingly reported to fail imatinib therapy while retaining wild-type BCR-ABL expression. Our previous studies suggested a role for Lyn, a Src-related kinase, in imatinib resistance. K562 cells selected for imatinib resistance (K562R) overexpress Lyn kinase and its targeted silencing overcomes imatinib resistance and engages apoptosis. Overexpression of Lyn in K562 cells reduces imatinib sensitivity (3-fold) and patients that fail imatinib therapy in the absence of BCR-ABL mutations express a highly activated Lyn kinase that is not suppressed by imatinib. Silencing Lyn expression in patient specimens induces changes in cell survival that are proportional to the level of Lyn protein reduction. To understand the role of Lyn kinase in imatinib resistance and apoptosis we examined proteins associated with this kinase in imatinib resistant cell lines, leukemic cells overexpressing Lyn and specimens derived from imatinib resistant patients. Lyn overexpression blocked complete suppression of BCR-ABL tyrosine phosphorylation by imatinib and affected BCR-ABL signaling adaptors. Although BCR-ABL forms a stable complex with the leukemogenic-critical adaptor protein Gab2 in imatinib sensitive cells, Lyn overexpression resulted in the formation of Lyn:Gab2 complexed in resistant cells. BCR-ABL kinase inhibition failed to reduce tyrosine phosphorylation of Gab2 in these cells while Lyn silencing or kinase inhibition (with dasatinib) completely suppressed Gab2 tyrosine phosphorylation and correlated with the induction of apoptosis. Lyn silencing in K562R cells also lead to a reciprocal increase in the tyrosine phosphorylation and association with a protein of ~120kDa, identified as the E3 ligase, c-Cbl. Lyn overexpression in K562 cells reduced their imatinib sensitivity and reduced c-Cbl protein levels. Kinase inhibitor and co-transfection studies demonstrated that tyrosine phosphorylation of c-Cbl at a critical signaling site (Y774) is primarily controlled by BCR-ABL and deletion or mutation of the c-Cbl RING domain altered its BCR-ABL phosphorylation. These results suggest that c-Cbl complexes are regulated at both the protein and phosphorylation level by Lyn and BCR-ABL kinase activities, respectively. Overexpression and/or activation of Lyn may disrupt the balance and regulation of critical regulators of leukemogenic signaling (Gab2) or protein trafficking and stability (c-Cbl), resulting in increased cell survival and reduced responsiveness to BCR-ABL kinase inhibition. We conclude that Lyn alters the level and function of critical signaling adaptor proteins in CML cells.

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Hypomagnesemia in hemodialysis patients taking proton pump inhibitors

Journal of Fatima Jinnah Medical University ◽

10.37018/dezk2659 ◽

2020 ◽

Vol 14 (2) ◽

pp. 55-58

Author(s):

Abdul Malik ◽

Syed Mohkumuddin ◽

Humaira Rahim ◽

Shamima Hanif

Keyword(s):

Proton Pump Inhibitors ◽

Proton Pump ◽

Serum Magnesium ◽

Cross Sectional Study ◽

Hemodialysis Patients ◽

Mean Value ◽

Female Dominance ◽

P Value ◽

Cross Sectional ◽

The Mean

Background: Proton pump inhibitors (PPIs) are in routine widely prescribed to hemodialysis patients. Recent studies have reported the association of PPIs use with hypomagnesemia in patients with long term hemodialysis. This study aims to determine the frequency of hypomagnesemia in patients of hemodialysis taking proton pump inhibitors. Patients and methods: This cross-sectional study was conducted in the Department of Nephrology of Sandman Provincial Hospital Quetta from 01-6-2019 till 01-9-2019. A total of 120 patients (52 PPI users and 68 non-PPI users) who were on HD for more than 06 months were included. Data regarding age, gender, duration of hemodialysis and taking PPIs were collected. Determination of serum magnesium was made by taking 3 different samples at 2 weeks’ interval and the mean value of serum magnesium was calculated. Serum Mg2+ levels <2.0 mg/dL was taken as hypomagnesemia. A Chi-square test was applied to determine the association of PPI use with hypomagnesemia. Results: Demographic variables such as age and gender were not significantly different between the groups. There was female dominance in both groups (73% in PPI groups and 66.1% in the non-PPI group (p-value 0.65). The mean duration of dialysis was 45.3±13.8 months in PPI users versus 48.9±12.9 months in non-PPI users (p-value 0.14). There was a significantly higher frequency of hypomagnesemia in PPI users; 36 (69.3%) versus 27 (39.7%) in non-PPI users (p-value 0.001). Conclusion: The use of PPI is associated with a significant reduction in serum magnesium levels. So serum magnesium levels should be advised as routine monitoring in patients of hemodialysis taking PPIs.

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A Prospective Study of Clinical Outcomes in Laryngopharyngeal Reflux Diseases Following Treatment with Proton Pump Inhibitors in a Tertiary Care Hospital in Kerala

Journal of Evidence Based Medicine and Healthcare ◽

10.18410/jebmh/2021/469 ◽

2021 ◽

Vol 8 (28) ◽

pp. 2538-2543

Author(s):

Binu Raju George ◽

Ajayan P.V ◽

Saify Samad

Keyword(s):

Proton Pump Inhibitors ◽

Proton Pump ◽

Laryngopharyngeal Reflux ◽

Cost Effective ◽

Reflux Symptom ◽

P Value ◽

Reflux Symptom Index ◽

Symptom Index ◽

A Prospective Study

BACKGROUND Laryngopharyngeal reflux (LPR) is found to be a common disease encountered in Otolaryngology practice. LPR presents clinically with symptoms of laryngeal irritation, frequent throat clearing, cough, and hoarseness of voice. The main diagnostic methods currently used are Fiber-optic laryngoscopy and in some centers pH monitoring. Proton pump inhibitors (PPIs) are used and found to be cost-effective and useful for the treatment of LPR. The main objective of this study was to study the effectiveness of PPIs in alleviating the symptoms assessed using Reflux Symptom Index (RSI) score and Reflux Finding Scores (RFS). METHODS A prospective study was carried out on 100 patients attending the ENT OPD of Government Medical College and Hospital, Thrissur, Kerala. Patients were evaluated for improvement in symptoms of Laryngopharyngeal reflux disease following use of proton pump inhibitors, using Reflux symptom index and Reflux finding scores using 70 degree / flexible nasopharyngolaryngoscopy. Patients with clinical findings of LPRD with RSI score > 13 and RFS score > 7 were given a standard treatment protocol followed in our ENT department using Tab. Pantoprazole 40 mg twice daily before food and the treatment response was assessed by proper follow up at 6 weeks and 12 weeks. On each follow up visit, improvement in RSI and RFS scores with Proton pump inhibitor therapy was assessed. Data collected was then tabulated and analysed. RESULTS The study was conducted in 100 patients, 59 % of whom were females and 41 % males. Mean RSI score changed from 18.9 at the beginning to 14.5 at 6 weeks of treatment and 9.0 at 12 weeks of treatment with Proton pump inhibitor. Mean RFS score changed from 10.7 at the beginning to 8.7 at 6 weeks of treatment and to 5.9 at 12 weeks of treatment. Comparison of mean Reflux Symptom Index and mean Reflux Finding Scores before and after treatment revealed improvement and the result was statistically significant (p value < 0.001). CONCLUSIONS The use of RSI and RFS scores in the assessment of PPIs at fixed intervals is cost effective and avoids time consuming and cost intensive examinations. These scores also help in early diagnosis and long term follow up of LPR patient. Fixed time interval PPI treatment significantly improved RSI and RFS scores in LPR patients. The mean RSI score changed from 18.9 at the beginning of treatment to 14.5 at 6 weeks after treatment (p value < 0.001) and 9.0 after 12 weeks of treatment; (p value < 0.001) The mean RFS score changed from 10.7 at the beginning of treatment to 8.7 at 6 weeks after treatment (p value < 0.001) and 5.9 after 12 weeks of treatment; (p value < 0.001). KEYWORDS Laryngopharyngeal Reflux, Reflux Symptom Index, Reflux Finding Score

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Potent Transient Inhibition of BCR-ABL by Dasatinib Leads to Complete Cytogenetic Remissions in Patients with Chronic Myeloid Leukemia: Implications for Patient Management and Drug Development.

Blood ◽

10.1182/blood.v108.11.2166.2166 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2166-2166 ◽

Cited By ~ 6

Author(s):

Neil P. Shah ◽

John M. Nicoll ◽

Eric Bleickardt ◽

Claude Nicaise ◽

Ronald L. Paquette ◽

...

Keyword(s):

Tyrosine Kinase ◽

Cell Line ◽

Cell Biology ◽

Kinase Activity ◽

Kinase Inhibition ◽

Once Daily ◽

Minute Exposure ◽

Abl Kinase ◽

Short Term Exposure ◽

Dose Modifications

Abstract Dasatinib (Sprycel; formerly BMS-354825) is an oral multi-targeted SRC/ABL tyrosine kinase inhibitor that is approximately 325-fold more potent than imatinib, and has been recently approved by the US FDA for the treatment of imatinib-resistant and -intolerant Philadelphia chromosome-associated leukemias. In contrast to other tyrosine kinase inhibitors, pharmacokinetic analyses reveal that dasatinib has a short half-life (5–6 hours), with near-complete loss of BCR-ABL kinase activity inhibition eight hours after drug administration in patients with CML. Remarkably, patients treated with dasatinib as infrequently as once daily, five days per week, have achieved complete cytogenetic remission (CCyR). We tested transient BCR-ABL inhibition in vitro by assessing the sensitivity of the CML cell line K562 to short-term exposure of dasatinib. After as short as a 20-minute exposure to a clinically-relevant dasatinib concentration (100 nM), the majority of cells undergo apoptosis when analyzed after 48 hours, whereas a clinically-relevant concentration of imatinib (5 uM) failed to result in substantial cytotoxicity under these conditions. When higher concentrations (>=12.5 uM) of imatinib were used that correct for differences in potency between imatinib and dasatinib, cytotoxicity at 48 hours after a 20-minute exposure was similar to that observed with dasatinib. These results exclude SRC family inhibition as a potential mediator of this effect, and were reproducible in a second CML cell line, KU-812. Importantly, cytotoxicity was not observed in BCR-ABL-negative leukemia cell lines under these conditions. Signiificantly, similar phenomenon was observed when an erlotinib-sensitive non-small cell lung cancer was exposed to high concentrations of erlotinib for 20 minutes. We assessed the degree of BCR-ABL kinase inhibition achieved in 20 patients with chronic phase CML who were treated with dasatinib once daily as part of a phase I clinical trial by quantifying the ratio of phospho-CRKL to CRKL (a BCR-ABL substrate) achieved in PBMCs harvested four hours after the first dose of dasatinib. We found a strong correlation between the magnitude of BCR-ABL kinase inhibition and the depth of response achieved. Notably, CCyR was achieved exclusively in four patients who experienced the deepest inhibition of BCR-ABL kinase activity activity. The current medical management of CML involves assessing the degree of cytogenetic response after 6–12 months of imatinib therapy before considering dose modifications of imatinib. While our findings will need to be validated prospectively in larger cohorts of patients, they suggest that it may be feasible to make early dose modifications based upon the degree of target inhibition following the initial dose of dasatinib, and thus maximize the likelihood of clinical benefit. Together, these findings have potentially significant implications not only for the optimal clinical management of CML patients, but also for the rational development of small molecule inhibitors of other cancer-associated tyrosine kinases, as well as our global understanding of cancer cell biology.

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The Protein Tyrosine Phosphatase STS-1 Interacts with Bcr-Abl and Impairs the Response of Philadelphia-Positive Acute Lymphoblastic Leukemia (ALL) to Tyrosine Kinase Inhibitors

Blood ◽

10.1182/blood.v116.21.3236.3236 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3236-3236

Author(s):

Marcus Liebermann ◽

Daniela Hoeller ◽

Susanne Badura ◽

Tamara Tesanovic ◽

Hubert Serve ◽

...

Keyword(s):

Tyrosine Kinase ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Research Funding ◽

Kinase Activity ◽

Kinase Inhibitors ◽

Signalling Pathways ◽

Abl Kinase ◽

Clinical Resistance

Abstract Abstract 3236 Bcr-Abl is a leukemogenic fusion gene that by itself is sufficient for cellular transformation (Daley et al.) and is the hallmark of chronic myeloid leukemia and Philadelphia chromosome positive (Ph+) ALL. The Bcr-Abl fusion protein is a constitutively active tyrosine kinase (TK) which disrupts multiple cellular signalling pathways controlling apoptosis, cell cycle, proliferation and DNA repair. In Ph+ ALL, a subtype of ALL with a particularly poor prognosis, targeted inhibition of Bcr-Abl activity by Abl kinase inhibitors such as imatinib has improved treatment outcome but has not abrogated the frequent development of clinical resistance. In addition to mutations in the Bcr-Abl tyrosine kinase domain (TKD), it has become apparent that other resistance mechanisms contribute to disease progression. The activity of proteins involved in the above-mentioned signalling pathways and possibly resistance to TK inhibitors (TKI) is controlled at least partially by posttranslational modifications such as phosphorylation, which is regulated by the balance between kinases and protein tyrosine phosphatases (PTP). We previously showed that PTP1B is a negative regulator of Bcr-Abl-mediated transformation and modulates sensitivity to the TKI imatinib (Koyama et al). We hypothesized that other phosphatases for which Bcr-Abl is a substrate may also contribute to resistance, one candidate being Suppressor of T-cell receptor Signalling 1 (STS-1), which negatively regulates the endocytosis of receptor TK involved in a variety of hematologic malignancies. It was the aim of this study to determine whether: i) Bcr-Abl is a substrate of STS-1 ii) STS-1 is able to dephosphorylate Bcr-Abl iii) expression of STS-1 reduces the proliferation of Bcr-Abl expressing cells by inhibiting Bcr-Abl kinase activity iv) the level of STS-1 expression modulates the sensitivity of Bcr-Abl positive cells to TKI In order to answer these questions, we used 293T cells, a human primary embryonal kidney cell line, and the IL3-dependent murine pro B cell line Ba/F3. Both cell lines were modified with constructs encoding both forms of Bcr-Abl (p185/p210) and Sts-1. For experiments with endogenous Bcr-Abl (p185) and Sts-1 we used Sup B15 cells, a human B cell precursor leukemia, and its TKI-resistant subline (Sup B15 RT), which was generated in our lab and is highly resistant not only to imatinib but also to 2nd generation TKIs (Nilotinib & Dasatinib), with no evidence of TKD mutations or transcriptional up-regulation of Bcr-Abl. In all above described cell lines the interaction between Bcr-Abl and Sts-1 could be shown in an overexpressed system (293T & Ba/F3) and on an endogenous level (Sup B15 & Sup B15 RT) by using co-IPs followed by SDS-PAGE and Western blotting. The functional relevance was examined by testing the ability of Sts-1 to dephosphorylate Bcr-Abl. Complete dephosphorylation of Bcr-Abl was shown for p185bcr-abl and p210bcr-abl in 293T cells. To verify that the functional activity was also present in hematopoietic cells, we analyzed the ability of Sts-1 to dephosphorylate Bcr-Abl in Ba/F3 and Sup B15 cells. Dephosphorylation was observed in both cell lines but was less pronounced than in 293T cells. We therefore more closely examined the most important tyrosine (Tyr) residues of Bcr-Abl and identified Tyr245 and Tyr412 as the major targets of Sts-1. Phosphorylation of Tyr245 and Tyr412 was decreased by ∼60% in Ba/F3 cells and ∼39% in Sup B15 cells. These two residues are known to be important for regulating cell proliferation, survival and cell motility. In a competitive proliferation assay in the absence of IL3, the proliferation rate of BA/F3 cells infected with Bcr-Abl and Sts – 1 was reduced compared to a Bcr-Abl infected control population. When treated with imatinib the Sts-1 expressing cells showed an approximately 5-fold reduced proliferation rate compared to cells lacking Sts-1, or to imatinib-resistant cells harbouring the Bcr-Abl “gatekeeper mutation” T315I. The expression level of Sts-1 was found to be approximately 3-fold lower in the Sup B15 RT compared to the WT cell line. Regulation appeared to occur at the transcriptional level as shown by quantitive RT-PCR. These results show that Bcr-Abl is a substrate of Sts-1, that this phosphatase modulates Bcr-Abl kinase activity and may abrogate the response to TKI. This suggests that phosphatases may contribute to the development of clinical resistance of Ph+ leukemias to TKIs. Disclosures: Ottmann: Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding.

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Identification of TKI-Sensitive Point Mutations That Activate c-ABL Kinase Activity and Transformation Potential and Confer in Vitro Resistance to the Allosteric ABL Inhibitor GNF-5

Blood ◽

10.1182/blood.v126.23.17.17 ◽

2015 ◽

Vol 126 (23) ◽

pp. 17-17

Author(s):

Bianca J Lee ◽

Neil P. Shah

Keyword(s):

Research Funding ◽

Kinase Activity ◽

Point Mutations ◽

Therapeutic Targets ◽

Chromosome 9 ◽

Fusion Partner ◽

Allosteric Site ◽

Abl Kinase ◽

T315i Mutation

Abstract Introduction: Pathologically activated tyrosine kinases represent attractive therapeutic targets, with deregulated ABL kinase representing one of the best-validated examples.While physiologic activation of c-ABL is tightly controlled by intramolecular interactions, ABL fusion proteins are constitutively activated by oligomerization driven by the fusion partner, as well as by loss of kinase autoinhibition due to removal of an autoinhibitory myristate site at the N-terminus of c-ABL (Nagar et al, Cell, 2003). Recent routine genomic tumor sequencing of various human malignancies has identified point mutations of unknown significance in numerous kinases, including c-ABL, and it is expected that the increasingly common practice of tumor genome sequencing will provide new opportunities for tailored targeted therapy. To maximize therapeutic benefit and avoid unnecessary cost and toxicity, it will be critical to distinguish "driver" from "passenger" mutations. We therefore sought to: (i) test the transforming potential of select clinically detected c-ABL mutants, (ii) prospectively identify novel additional activating c-ABL point mutations, and (iii) determine if active mutant c-ABL isoforms retain sensitivity to ABL TKIs. Results: Two of four clinically identified c-ABL mutations tested caused a dramatic increase in ABL kinase activity and transformed Ba/F3 cells to growth factor independence. A mutagenesis screen of c-ABL identified six additional transforming point mutations in the SH3 and kinase domains, including one that has been reported clinically in breast invasive carcinoma (cBioPortal.org). Relative to BCR-ABL, all c-ABL mutants showed heightened sensitivity to imatinib. Allosteric site inhibitors targeting the myristate-binding pocket in the BCR-ABL kinase domain (KD) have been shown to inhibit BCR-ABL kinase activity, purportedly by mimicking binding of the c-ABL myristate group and establishing c-ABL-like autoinhibitory constraints (Hantschel, Haematologica, 2012). While only two c-ABL mutations from the screen were near the allosteric site, all mutants exhibited decreased sensitivity to the allosteric inhibitor GNF-5. Recent studies have shown that derivative compounds of GNF-5 targeting this site in BCR-ABL are subvertable by on-target resistance mutations. We tested whether three BCR-ABL mutations described to confer in vitro, and in one case clinical, resistance to ABL001 (currently undergoing clinical trial evaluation) could impact c-ABL kinase activity presumably by perturbing this autoinhibitory pocket. In the context of c-ABL, all three ABL001-resistant mutations activated ABL kinase, transformed Ba/F3 cells, and conferred substantial resistance to allosteric inhibition relative to native BCR-ABL. These findings raise the possibility that c-ABL mutations may play a role in resistance to emerging allosteric ABL inhibitors. Importantly, previous work suggested that the genomic breakpoint on chromosome 9 in one third of CML cases is located 5' to ABL exon 1b, which would be predicted to result in simultaneous KD mutation expression in BCR-ABL and c-ABL (Uphoff et al, Leuk Res, 1999). To definitively demonstrate that co-expression of KD mutations in c-ABL and BCR-ABL can occur in primary patient samples, we analyzed RNA from five CML patients harboring a dominant BCR-ABL T315I mutation, and identified a heterozygous c-ABL 1b T315I mutation in one case. Conclusions: We have identified eight novel activating point mutations in c-ABL (three found in clinical isolates) that are potential therapeutic targets of ABL TKIs. Activating c-ABL mutants, including T315I, confer substantial resistance to the allosteric ABL inhibitor GNF-5 regardless of proximity to the inhibitor-binding site, implying resistance mechanisms beyond mere steric hindrance. Thus, mutations may induce distal conformational changes by disrupting a kinase conformation required for allosteric compound inhibition. In a subset of CML cases, c-ABL point mutants are co-expressed from the same allele as BCR-ABL, suggesting that the location of the chromosome 9 breakpoint may negatively impact clinical responsiveness to allosteric ABL inhibitors. Sequence analysis of c-ABL in patients with resistance to allosteric ABL inhibitors is required to validate c-ABL point mutants as a clinically important mechanism of resistance to this emerging class of targeted therapeutics. Disclosures Off Label Use: the use of ABL TKIs for malignancies associated with point mutations in c-ABL. Shah:Pfizer: Research Funding; Bristol-Myers Squibb: Research Funding; Plexxikon Inc.: Research Funding.

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Frequency of spontaneous bacterial peritonintis in chronic liver disease patients using proton pump inhibitors.

The Professional Medical Journal ◽

10.29309/tpmj/2020.27.03.968 ◽

2020 ◽

Vol 27 (03) ◽

pp. 455-460

Author(s):

Tahir Ullah Khan ◽

Wali Khan ◽

Sajjad Iqbal

Keyword(s):

Liver Disease ◽

Chronic Liver Disease ◽

Proton Pump Inhibitors ◽

Proton Pump ◽

Spontaneous Bacterial Peritonitis ◽

Chronic Liver ◽

P Value ◽

Exclusion Criteria ◽

Bacterial Peritonitis ◽

Study Setting

Objectives: To determine the frequency of spontaneous bacterial peritonitis (SBP) in chronic liver disease patients with ascites using proton pump inhibitors (PPI). Study Design: Prospective cohort study. Setting: Medicine Unit-II, Shalamar Hospital Lahore. Period: From 1st January to 30thJune 2017. Materials & Methods: A total of 380 patients enrolled in present study were divided into two groups; PPI group and non PPI group. Each group had 190 patients. We performed an extensive matching in both groups to minimize selection bias and SBP incidence was compared in both groups. Multivariate analysis was applied to sort out the causal relationship between PPI use and SBP. Results: Applying strict exclusion criteria, SBP incidence in chronic liver disease patients with ascites was found significantly high in PPI group than non-PPI group (12.63% vs. 6.84%, P Value 0.002). Moreover, a large no of patients (39.47%) were using PPI for inappropriate indications while acid peptic disease was the most common indication for its use (60.53%). Conclusion: Use of proton pump inhibitors in chronic liver disease patients with ascites significantly increases the risk of spontaneous bacterial peritonitis.

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Use of Proton Pump Inhibitors in Saudi Arabia: A Cross-Sectional Retrospective Drug Utilization Study

Journal of Pharmaceutical Research International ◽

10.9734/jpri/2020/v32i3130918 ◽

2020 ◽

pp. 48-54

Author(s):

Mohammad Daud Ali ◽

Ayaz Ahmad ◽

Nuzhat Banu ◽

Latha S. Kannan

Keyword(s):

Saudi Arabia ◽

Drug Utilization ◽

Proton Pump Inhibitors ◽

Proton Pump ◽

P Value ◽

Prescribing Pattern ◽

Cross Sectional ◽

Nice Guideline ◽

Number Of Patients ◽

One Year

Objective: Chief aim of the current study was to draw attention in the prescribing pattern and utilization of PPIs in one year at a single private hospital of Saudi Arabia. Methods: This is a cross-sectional, retrospective drug utilization research on Proton pump inhibitors (PPIs). The PPI usage pattern of in- and out- patients of Al-Mana Group of Hospital (AGH) Al-Khobar between January 1, 2019 and December 31, 2019 were investigated, including incidence, prevalence, and duration. Results: We observed 27229 items of PPI were dispensed in the inpatient and outpatient pharmacy department of AGH-Al-khobar. Among all the PPI user more than the half {(52.98%, n = 14426), 95%CI (52.0-53.5)} were male. Nearly equal number of PPI users belongs between 18-40 years {(39.22%, n =10680), 95%CI (38.64-39.80)} and 41-60 years {(39.15%, n =10662), 95%CI (38.6-39.75)}. Among all the PPI users 61.46% (n=16736) were from community of Saudi Arabia while 38.53% (n=10493) from Non-Saudi. Among all the dispensed PPIs drugs Pantoprazole is dispensed to the highest number of patients {79% (95%CI, 78.53-79.50) (n=21515), p<0.05} while their average duration of therapy was 18.78 days. All the PPI prescribed to the AGH-Al-khobar patients adhere to the NICE guideline (p-value <0.05). Conclusions: We also observed that PPIs was prescribed in AGH Al-khobar adhere to clinical guidelines. In our study among all the PPI Pantoprazole was prescribed to the highest number of patients, hence their safe and effective use must be warranted.

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