Screening, Fabrication and Evaluation of Pharmaceutical Dosage Forms using P-Glycoprotein Modulators

2017 ◽  
Vol 10 (2) ◽  
pp. 3688-3699
Author(s):  
Joshi Vedamurthy ◽  
Shivakumar Inamdar ◽  
Ankit Acharya ◽  
Rajesh Kowti
Keyword(s):  
Cell Lines ◽  
Tinospora Cordifolia ◽  
Absorption Enhancement ◽  
Synergistic Activity ◽  
Capsicum Frutescens ◽  
Plumbago Zeylanica ◽  
Pharmaceutical Dosage Forms ◽  
Holarrhena Antidysenterica ◽  
Trachyspermum Ammi

In this project, in vitro absorption enhancement activity of P-gp substrates Fexofenadine (Fx) and Ciprofloxacin (Cp) were evaluated in everted rat gut sac model and Caco-2 cell lines. Verapamil was used as P-gp inhibitor. Piper betel, Trachyspermum ammi, Plumbago zeylanica, Trikatu, Moringaoleifera, Murraya koenigii,  Ferulafoitida  Zingiber officinale, Cheilocostus speciosus, Capsicum frutescens Operculina turpethum Holarrhena antidysenterica Mesuaferrea, Tinospora cordifolia,  and Picrorhiza kurroa, were selected and extracted with 99% alcohol and fresh juices of Citrus limon, Punica granatum seeds were also studied. In-vitro studies depicted that Fexofenadine and Ciprofloxacin absorption was increased greater than 20% in the presence of Operculinaturpethum, Capsicum frutescens, Holarrhena Antidysenterica, Tinospora cordifolia, Trikatu, Trachyspermum ammi, Plumbago zeylanica. The flux of the ciprofloxacin transport was in the range of 9-23 mcg/min and Papp         2.6 × 10-5 cm/sec to 4.1 × 10-5  cm/sec whereas Fexofenadine flux was in the range of 2-7.7 mcg/min and Papp 4.16 × 10–6 cm/sec to 1.62 ×       10-5 cm/sec.  In vitro antimicrobial activity of ciprofloxacin on selected microbes in presence of extracts also depicted synergistic activity. Histological studies revealed that there is no significant variation observed in the isolated sac in presence of the extracts. CaCo2 cell lines studies showed that, formulation enhanced the absorption of fexofenadine greater than 50%. Tablets were prepared and evaluated using the plant extracts which yielded >20% absorption enhancement of the substrates. In conclusion, tablet formulation containing the alcoholic extracts of Trachyspermum ammi, Plumbago zylanicum, Capsicum frutescens, Operculina turpethum, Holarrhena Antidysenterica, Tinospora cordifolia and Trikatu can act as an absorption enhancer for fexofenadine and ciprofloxacin. The mechanism of action of these herbs could be due to    P-gp inhibition. Further clinical studies are needed to prove its efficacy in humans.     

scholarly journals Antifungal Activity of Flocculosin, a Novel Glycolipid Isolated from Pseudozyma flocculosa

2005 ◽  
Vol 49 (4) ◽  
pp. 1597-1599 ◽  
Author(s):  
Benjamin Mimee ◽  
Caroline Labbé ◽  
René Pelletier ◽  
Richard R. Bélanger
Keyword(s):  
Antifungal Activity ◽  
Amphotericin B ◽  
Cell Lines ◽  
Human Cell ◽  
Synergistic Activity ◽  
Human Cell Lines ◽  
Antifungal Properties ◽  
Pseudozyma Flocculosa ◽  
In Vitro Antifungal Activity

ABSTRACT Flocculosin, a glycolipid isolated from the yeast-like fungus Pseudozyma flocculosa, was investigated for in vitro antifungal activity. The compound displayed antifungal properties against several pathogenic yeasts. Synergistic activity was observed between flocculosin and amphotericin B, and no significant cytotoxicity was demonstrated when tested against human cell lines.

Identification of sensitivity markers for BMS-536924, an inhibitor for insulin-like growth factor-1 receptor

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 3506-3506 ◽  
Author(s):  
F. Huang ◽  
W. Hurlburt ◽  
R. Hafezi ◽  
X. Han ◽  
J. Chen ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Lines ◽  
Neuroblastoma Cell ◽  
Protein Profiling ◽  
Egfr Inhibitors ◽  
Synergistic Activity ◽  
Insulin Like Growth Factor ◽  
Molecule Inhibitor ◽  
Key Steps

3506 Background: Insulin-like growth factor-1 receptor (IGF-1R) signaling is an important regulator of mitogenesis, transformation to the oncogenic phenotype and anti-apoptotic effects in malignant cells. Over-expression of IGF-1R, seen in many tumors, may confer a growth advantage or drug resistance. A potent small-molecule inhibitor (BMS-536924) of IGF-1R tyrosine kinase showed anti-tumor activity in sarcoma, prostate, colon and pancreatic tumor models. One of the integral goals in the development of BMS-536924 as a cancer therapeutic is to identify molecular biomarkers predictive of response to the drug that ultimately will aid in selecting the patients who are most likely to benefit. Methods: The sensitivity (IC50) to BMS-536924 was determined for a panel of 29 pediatric sarcoma and neuroblastoma cell lines. Both microarray and LC/MS based protein profiling were utilized to analyze the baseline gene or protein expression level. Drug treatment studies were performed using two rhabdomyosarcoma cell lines, Rh41 (sensitive to BMS-536924) and Rh36 (resistant to the drug) to identify markers that are modulated by BMS-536924. Results: (1). Sixteen out of the 29 cell lines were highly sensitive to BMS-536924; candidate markers that correlated with the sensitivity to BMS-536924 were identified by gene expression and protein profiling. (2). Histological correlation was also discovered, with specific subtypes of sarcoma having a low IC50 to BMS-536924. (3). Pathway analysis noted that some major candidate markers are common key steps in the EGF-R pathway and the IGF1-R pathway. This observation of cross-talk between the two pathways led to the hypothesis of synergy with combined inhibition of both pathways. Combination studies of BMS-536924 and EGFR inhibitors were performed and synergism was observed. (4). Markers modulated by BMS-536924 in a sensitive cell line were identified. Conclusions: This work has identified candidate markers correlating to BMS-536924 sensitivity in vitro. The possible mechanism of synergistic activity of IGF1-R and EGFR inhibitors will be presented. No significant financial relationships to disclose.

Synergistic Activity of the Novel Src/Abl Inhibitor Bosutinib in Combination with Imatinib.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2930-2930 ◽  
Author(s):  
Sara Redaelli ◽  
Frank Boschelli ◽  
Michela Viltadi ◽  
Iris Meneghetti ◽  
Carlo Gambacorti
Keyword(s):  
Synergistic Effect ◽  
Cell Lines ◽  
Synergistic Effects ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cells ◽  
Synergistic Activity ◽  
Binding Modes ◽  
Imatinib Resistant ◽  
Wide Range

Abstract The oncogenic fusion protein Bcr-Abl is the underlying cause of Chronic Myeloid Leukemia (CML), and it is present in up to 35% of Acute Lymphoblastic Leukemia (ALL). The discovery of a selective Abl inhibitor, Imatinib mesylate, has revolutionized the treatment of CML. Recently, several new inhibitors have been developed with the aim of increasing both potency and selectivity against Abl. Bosutinib (SKI-606, Wyeth) is a dual Src/Abl inhibitor that showed an in vitro activity in the low nanomolar range on several BCR-ABL positive cells and it is, at present, in phase II clinical trials. Bosutinib is devoid of activity against some known “off-target” kinases blocked by imatinib, such as PDGFR and c-Kit. In addition structural and modelling data attribute to Bosutinib the ability to bind Bcr-Abl in the intermediate/active conformation, while Imatinib is able to bind only the inactive conformation of Bcr-Abl. In this study we analyzed in vitro the combination of Imatinib and Bosutinib in Bcr-Abl expressing cell lines to evaluate the possibility to decrease dosage of both drugs, increasing or maintaining the same efficacy but avoiding toxic effects. Combination effects were evaluated according to the method of Chou and Talalay, in which the combination index (CI) value is calculated for a combination of two drugs and allows the quantification of synergism: CI <1, =1 or >1 indicate synergistic, additive or antagonistic interactions, respectively. Proliferation assays on a panel of Imatinib-sensitive and Imatinib-resistant BCR-ABL positive cell lines were performed. Cells were treated with Imatinib and Bosutinib as single agents or in three ratio combinations (1:3, 1:10, 1:33 in favour to Imatinib) across a wide range of concentrations. Combination indexes (CI) calculated at IC50, IC75 and IC90 for K562 cells (Imatinib-sensitive), suggest a synergistic to very strong synergistic effect (CI= 0.01-0.53). Similarly, in KCl22, KU812 and Lama84 cells (Imatinib-sensitive) moderate to strong synergistic effects were observed. A slight to moderate synergism was also obtained in three Imatinib-resistant cell lines tested: Lama84R (CI=0.63-0.88), K562R (CI=0.63-0.82) and KCL22R (CI=0.62-0.92). Western blot analysis of the tyrosine phosphorylation status of K562S cells treated with a mixture of 100nM Imatinib and 10nM Bosutinib revealed a substantially more pronounced inhibition compared with either 100nM Imatinib or 10nM Bosutinib alone. The effect of the combination was also assessed in murine Ba/F3 cells transfected with either wild type (WT) or mutated forms of BCR-ABL. Parental Ba/F3 cells were not affected by the presence of both drugs, while in Ba/F3 BCR-ABL WT the CI ranged from 0.49 to 0.85, indicating moderate synergism. The combination of Imatinib and Bosutinib inhibited the growth of Ba/F3 BCR-ABL Y253F with a slight synergism (CI 0.77-0.87). No synergistic effect was observed on Ba/F3 BCR-ABL E255K and on the highly resistant T315I mutant. Fresh leukemic cells obtained from three CML patients were also studied. In these samples synergistic effects between Bosutinib and Imatinib were confirmed (CI=0.52, 0.73, 0.62). The different binding modes of Imatinib and Bosutinib may justify the synergistic effect observed in the CML lines. This results support a possible therapeutic advantage for the combination of Bosutinib and Imatinib against Philadelphia positive leukemias.

scholarly journals Targeting XIAP Via Degradation of MDM-2 By MX69 in Aggressive Lymphomas

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5379-5379
Author(s):  
Sumera Khan ◽  
Kyle Runckel ◽  
Cory Mavis ◽  
Matthew J. Barth ◽  
Francisco J. Hernandez-Ilizaliturri
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
Single Agent ◽  
B Cell Lymphoma ◽  
Parp Cleavage ◽  
Synergistic Activity ◽  
In Vitro Exposure ◽  
Induction Of Apoptosis

Abstract Background: The addition of Rituximab to front-line therapy has improved clinical outcomes in diffuse large B-cell lymphoma (DLBCL), but it has also altered the biology of relapsed/refractory disease. To better understand the mechanisms responsible for Rituximab associated chemotherapy cross-resistance our group developed and characterized several Rituximab resistance cell lines (RRCL). We previously demonstrated using SiRNA interference, that X-linked inhibitor of apoptosis (XIAP) is critical for chemotherapy sensitivity and survival in RRCL. MX69, a dual inhibitor of Mdm2 and XIAP that indirectly downregulates XIAP, is undergoing pre-clinical testing. MX69 affects XIAP levels by its effects on the ubiquitination and degradation of endogenous MDM-2, resulting in decrease XIAP translation and activation of caspase 3, 7 and 9 as well as PARP cleavage leading to apoptosis of cancer cells. In our current work, we pharmacologically inhibited XIAP in lymphoma pre-clinical models using MX69. Materials and Methods: A panel of Burkitt's Lymphoma (BL, including RRCL), germinal center B-cell (GCB)-DLBCL (including RRCL), activated B-cell (ABC)-DLBCL, Mantle cell Lymphoma (MCL) and Pre-B cell Leukemia cell lines were exposed to MX69 as a single agent (0-80uM) over 24, 48, 72 hrs and IC50 concentrations were calculated for each cell line. Changes in Mdm2, p53, XIAP and PARP expressions were determined following MX69 exposure (at IC50 doses) for 24 hrs. Induction of apoptosis was evaluated by Annexin V/propidium iodine staining. Subsequently, cell lines were exposed to MX69 (0-80 uM), in combination with Doxorubicin (0-1uM), Cytarabine(0-50uM), Vincristine (0-10nM), Etoposide(0-50uM), Carboplatin (0-20uM), Ixazomib (0-1.5uM), Ibrutinib (0-20uM) and Venetoclax (0-10uM) for 48 hours. Cell viability was determined by Cell Titerglo. Coefficient of synergy was calculated using CalcuSyn. Results: In vitro, MX69 single agent exposure induced cell death in a dose and time-dependent manner in all cell lines tested. Western blotting studies confirmed downregulation of Mdm2, XIAP and changes in P53 and PARP, following in vitro exposure to MX69. Induction of apoptosis was observed by flow cytometry in all cell lines tested. The combination of MX69 with Doxorubicin, Cytarabine, Vincristine, Ixazomib, Carboplatin, Etoposide, Ibrutinib, and Venetoclax resulted in significant synergistic activity. The strongest CI of synergy was observed when cell lines were exposed to MX69 and Venetoclax, Ixazomib, Etoposide or Ibrutinib. Conclusion: Our data suggests that in vitro exposure of a wide variety of B-cell lymphoma cell lines (including BL, DLBCL, MCL or RRCL) to MX69 resulted in anti-tumor activity. Perhaps related to its anti-tumor effects, MX69 inhibited XIAP levels. These findings are similar to prior SiRNA XIAP knockdown experiments. Strong synergistic activity was observed when XIAP was combined with various chemotherapy agents and small molecules inhibitors (such as Venetoclax, ixazomib or ibrutinib). Ex vivo experiments using primary tumor cells isolated from lymphoma patients and lymphoma mouse models are been planned. Targeting Mdm2 and XIAP can be an attractive therapeutic strategy in patients with Rituximab-sensitive or -resistant B-cell lymphoma. Disclosures No relevant conflicts of interest to declare.

The Combination of the mTOR Inhibitor Rapamycin and Proteasome Inhibitor Bortezomib Is Synergistic In Vitro in Multiple Myeloma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3495-3495 ◽  
Author(s):  
Garrett O’Sullivan ◽  
Xavier Leleu ◽  
Xiaoying Jia ◽  
Evdoxia Hatjiharisi ◽  
Hai Ngo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Cell Lines ◽  
Stromal Cells ◽  
Mtor Inhibitor ◽  
Significant Inhibition ◽  
Synergistic Activity ◽  
Higher Doses

Abstract Background: The PI3K and NFk-B/proteasome pathways are major regulators of survival in Multiple Myeloma (MM). Previous studies have demonstrated clinical efficacy of bortezomib in MM; however, not all patients responded to this agent. mTOR inhibitors have demonstrated significant in vitro and in vivo activity in MM, specifically clinical trials with the mTOR inhibitor CCI-779 (Wyeth) in MM. Therefore, we examined whether inhibition of the PI3K pathway by mTOR and proteasome inhibitors may lead to synergistic activity in MM. Methods: MM cell lines (MM.1S, RPMI, U266, OPM2) were treated with rapamycin 1–5nM (Sigma Aldrich), bortezomib 2.5–10nM (Millenium, MA), or the combination. Cytotoxicity was measured by the MTT assay at 48 hrs; DNA synthesis was measured using thymidine uptake assay; apoptosis was studied using Apo2.7 by flow cytometry, and cell cycle regulation was determined using flow cytometry. To determine whether these agents can overcome the growth advantage conferred by bone marrow stromal cells (BMSCs), we co-cultured cell lines with stromal cells. Normal peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers. Determination of the additive or synergistic effect of the combination was calculated using the CalcuSyn software (Biosoft, MO) based on the Chou-Talalay method, with synergistic activity determined as a combination index (CI) of <1.0. Results: Rapamycin induced dose-dependent cytotoxicity from 0.1nM to 1nM, with an IC50 of 5nM in MM.1S and OPM2. Interestingly, higher doses did not induce further cytotoxicity, confirming that low doses of rapamycin are as effective as higher doses. RPMI and U266 MM cell lines were less sensitive to rapamycin, with 5nM inducing 40% and 20% decrease in survival, respectively. Bortezomib induced significant inhibition of survival in all MM cell lines with an IC50 of 2.5nM, as previously reported. The combination of agents induced significant inhibition of proliferation as compared to each agent alone, specifically with the combination of 5nM rapamycin with 5nM of bortezomib. In the DNA synthesis assay, the combination of bortezomib and rapamycin was significantly cytotoxic compared to each agent alone, specifically at the dose of 5nM rapamycin and bortezomib 2.5nM. The combination of rapamycin 1 to 5 nM and bortezomib 5 to 10 nM were synergistic with a CI index less than 1.0, as in RPMI (CI=0.4) and U266 (CI=0.2) cell lines. The combination of rapamycin and bortezomib at serial concentrations did not trigger cytotoxicity in PBMCs from normal volunteers, indicating significant cytotoxicity in malignant cells, with lack of toxicity in normal PBMCs and suggesting a therapeutic index. The combination of bortezomib and rapamycin demonstrated a significant inhibitory effect on the growth of MM cell lines even in coculture with stromal cells. Cell cycle analysis demonstrated G1 arrest at 24 and 48 hrs in MM.1S cells. Similar results were obtained using primary CD138+ myeloma cells from patients. Conclusion: The combination of rapamycin and bortezomib resulted in synergistic in vitro cytotoxicity in MM cells. These results provide the framework for clinical trials evaluating the combination of CCI-779 and bortezomib in MM.

scholarly journals Enhanced Antitumor Effect of Trastuzumab and Duligotuzumab or Ipatasertib Combination in HER-2 Positive Gastric Cancer Cells

Cancers ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2339
Author(s):  
Maria Maddalena Laterza ◽  
Vincenza Ciaramella ◽  
Bianca Arianna Facchini ◽  
Elisena Franzese ◽  
Carmela Liguori ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cancer Cells ◽  
Cell Lines ◽  
Antitumor Effect ◽  
Combined Treatment ◽  
Human Gastric Cancer ◽  
Synergistic Activity ◽  
Her2 Positive ◽  
Gastric Cancer Cells

The anti-HER2 monoclonal antibody trastuzumab is a key drug for the treatment of HER2-positive gastric cancer (GC); however, its activity is often limited by the onset of resistance and mechanisms of resistance are still poorly understood. Several targeted agents showed synergistic activity by concomitant use with trastuzumab in vitro and are under clinical investigation. The aim of this study was to assess the antitumor activity of duligotuzumab, an anti HER3/EGFR antibody or ipatasertib, an AKT inhibitor, combined with trastuzumab in a panel of HER2-positive human gastric cancer cells (GCC), and the efficacy of such combinations in HER2-resistant cells. We have assessed the efficacy of duligotuzumab or ipatasertib and trastuzumab in combination, analyzing proliferation, migration and apoptosis and downstream intracellular signaling in vitro on human HER2-positive GCC (NCI-N87, OE33, OE19) and in negative HER2 GCC (MKN28). We observed a reduction of proliferation, migration and apoptotic rate in HER2-positive OE33, OE19 and N87 cell lines with the combination of duligotuzumab or ipatasertib plus trastuzumab. In particular, in OE33 and OE19 cell lines, the same combined treatment inhibited the activation of proteins downstream of HER2, HER3, AKT and MAPK pathways. Targeting both HER2 and HER3, or HER2 and AKT, results in an improved antitumor effect on HER2-positive GCC.

Combination of DNA Methylation Inhibitor 5-Azacytidine and Arsenic Trioxide Has Synergistic Activity in Multiple Myeloma

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5173-5173
Author(s):  
Guanghua Chen ◽  
Fengru Lin ◽  
Wu Depei ◽  
Yi Wang ◽  
Haiwen Huang ◽  
...  
Keyword(s):  
Arsenic Trioxide ◽  
Cell Lines ◽  
Methylation Status ◽  
Cpg Islands ◽  
Full Length ◽  
Synergistic Activity ◽  
Specific Pcr ◽  
Rpmi 8226

Abstract This study was undertaken to study the effect of 5-azacytidine on XAF1 expression in myeloma cells and efficacy of 5-azacytidine and arsenic trioxide combination treatment in myeloma in vivo and in vitro. XAF1 expression was analyzed by semi-quantitative PCR and Western blotting. Methylation specific PCR (MSP) was used to detect methylation status of XAF1 promoter CpG islands. RPMI 8226 and XG-7 cells were treated with various concentrations of arsenic trioxide and 5-azacytidine. Expression of XAF1 mRNA variants were confirmed by gel electrophoresis and sequencing. Untreated RPMI 8226 cell expresses two variants of XAF1 mRNA. Untreated XG-7 cell has no expression of XAF1. Hypermethylation of XAF1 promoter CpG islands was detected in both cell lines. Both cell lines express full-length XAF1 mRNA transcript after treated with 2.5 μmol/L 5-azacytidine for 72 h. Our studies demonstrated that 5-azacytidine exhibits antimyeloma synergy with arsenic trioxide. 5-azacytidine treatment reversed XAF1 promoter region hypermethylation status, leading to expression of full-length XAF1 transcript. In addition, combination of 5-azacytidine and arsenic trioxide was effective in slowing myeloma growth and prolonging survival of myeloma-loaded nude mice. The findings suggested that 5-azacytidine and arsenic trioxide may be an effective combination in the therapy of myeloma patients.

scholarly journals MCL-1 Inhibition By the Selective MCL-1 Inhibitor AMG-176 Induces in Vitro Activity Against Burkitt Lymphoma Cell Lines and Synergistically Enhances the Cytotoxic Effect of Chemotherapy and BH3 Mimetics

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5303-5303 ◽  
Author(s):  
Tara Daly ◽  
Thomas Ippolito ◽  
Juan J. Gu ◽  
Cory Mavis ◽  
Pallawi Torka ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Western Blotting ◽  
Single Agent ◽  
Cytotoxic Chemotherapy ◽  
Time Dependent ◽  
Synergistic Activity ◽  
Minimal Effect ◽  
Bh3 Mimetic

Background: Although >90% of Burkitt Lymphoma (BL) patients will be cured by intensive multi-agent immunochemotherapy, relapsed and refractory BL patients have poor prognoses demonstrating the need to identify novel therapies. Myeloid cell leukemia-1 (MCL-1) is a pro-survival protein within the BCL-2 family that has been investigated as a therapeutic target in several leukemia and lymphoma pre-clinical models and clinical studies. Analysis of pediatric BL tumors has implicated MCL-1 in BL pathogenesis (Giulino-Roth et al., Blood 2012) and targeting MCL-1 may be effective in MYC-driven lymphomas (Kelly et al., Genes Dev 2014). High MCL-1 expression has also been associated with therapy resistance and can confer resistance to BH3 mimetic agents (Carrington et al., Cell Death Differ 2017). AMG176, a selective MCL-1 inhibitor, is an effective promoter of apoptosis and is currently under investigation in clinical trials for relapsed and refractory multiple myeloma, lymphoma, and acute myeloid leukemia. Hypothesis: We hypothesized that MCL-1 inhibition with AMG176 will promote apoptosis and increase sensitivity of BL therapy-sensitive (TSCL) and therapy-resistant (TRCL) cell lines to other chemotherapy drugs. Methods: A panel of TSCLs (Raji, Ramos and Daudi) and one TRCL (Raji 4RH) BL cell lines were exposed to increasing concentrations of AMG176 as a single agent and in combination with Doxorubicin or Venetoclax at different time points. Cell viability was analyzed using PrestoBlue. Synergy in drug combination exposures was determined by Calcusyn software and reported as a combination index (CI) value with values increasingly <1 indicating increasing synergistic activity. Apoptosis induction was analyzed by flow cytometry for Annexin V (AV) and Sytox-7AAD and by western blotting for PARP. Effects of MCL-1 inhibition on the expression of other pro- and anti-apoptotic BCL-2 family member proteins was assessed by western blotting. AMG176 mechanism(s) of action was evaluated by co-immunoprecipitation followed by western blotting for pro-apoptotic BCL-2 family members known to be bound by MCL-1. Results: Single agent AMG176 exposure of BL cell lines exhibited a dose and time-dependent decrease in viable cells. 48hr IC50 values of Ramos (0.3097uM) and Raji (1.652uM) showed an increased sensitivity to AMG176 as compared to Daudi (9.616uM) and Raji 4RH (12.45uM). Exposure of cells to AMG176 also resulted in altered expression levels of pro-survival and pro-apoptotic proteins of the BCL-2 family proteins. Co-immunoprecipitation performed after 48hrs of AMG176 exposure exhibited a decrease in MCL-1:BIM and MCL-1:BAK complexes in Raji and Ramos cells promoting a pro-apoptotic environment. After 48hr exposure to AMG176, induction of apoptosis was observed in a dose-dependent manner in all cell lines as evidenced by increased PARP cleavage on western blot and AV-Sytox positivity on flow cytometry. To investigate the ability of MCL-1 inhibition to enhance the activity of cytotoxic chemotherapy, BL cells were exposed to AMG176 in combination with Doxorubicin. Daudi and Raji exhibited strong synergy with peak synergistic activity observed at 72hrs (Daudi CI values <0.1 at all AGM176 concentrations with 0.1-0.2uM Doxorubicin; Raji CI ranged from 0.233-0.295 at multiple AMG176 concentrations with <0.4uM Doxorubicin) while a minimal effect was observed in Ramos and Raji 4RH. As high MCL-1 is associated with resistance to Venetoclax by sequestering free BIM following Venetoclax exposure, the combination of AMG176 and Venetoclax was investigated and also showed significant synergy in BL cells. A minimal effect was observed in Daudi and Raji 4RH in Venetoclax combination but Ramos and Raji, the more AMG176 sensitive cell lines, showed peak synergy at 24hrs at high doses of Venetoclax (Ramos CI values ranged from 0.3-0.478; Raji CI ranged from 0.511 to 0.67). Conclusion: MCL-1 inhibition with AMG176 demonstrates dose- and time-dependent anti-lymphoma activity impairing in vitro proliferation and inducing apoptosis in BL cells. AMG176 exhibits synergistic activity in combination with cytotoxic chemotherapy in both therapy-sensitive and therapy-resistant BL cell lines and synergistically enhances response to the BH3 mimetic agent Venetoclax. These findings highlight the potential of MCL-1 inhibition as a therapeutic option in BL. This work was supported by T35 NIH training grant (T35AI089693). Disclosures No relevant conflicts of interest to declare.

scholarly journals Effects of Proteasome Inhibition in the Anti-Tumor Activity of Venetoclax in Mantle Cell Lymphoma Pre-Clinical Models

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2942-2942
Author(s):  
Shalin K. Kothari ◽  
Cory Mavis ◽  
Juan Gu ◽  
Francisco J. Hernandez-Ilizaliturri
Keyword(s):  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Scid Mice ◽  
Parp Cleavage ◽  
Synergistic Activity ◽  
Clinical Models ◽  
Anti Tumor Activity

Abstract Background: At the molecular level, mantle cell lymphoma (MCL) is characterized by the deregulation of Bcl-2 family members (Mcl-1, BIM) and cell cycle (cyclin D1) regulatory proteins. Perhaps related to this, the clinical outcome of MCL continues to be poor specially for those patients with disease progression after high dose chemotherapy and autologous stem cell rescue and/or BTK inhibitors, stressing the need to develop novel therapeutic strategies or optimize current available options. Venetoclax (V), a highly selective Bcl-2 inhibitor, has shown modest activity against relapsed/refractory MCL. Over-expression of Mcl-1 has been postulated to be a mechanism of resistance to V limiting its anti-tumor activity in subtypes of lymphoma including MCL. The lethality by proteasome inhibitors (PIs) has been associated with changes in the Bcl-2 family members (Bax, Noxa, Mcl-1 and Bcl-XL) in lymphoma pre-clinical models, making them ideal agents to combine with V. To this end, we studied the anti-tumor activity of combining PIs with V in MCL pre-clinical models. Materials and Methods: A panel cytarabine sensitive (Rec-1, Jeko, Granta, HBL-2, Z-138 and Mino) and resistant (araC) cell lines (Jeko araC, HBL-2 araC, and Mino araC) were exposed to V, Bortezomib (BTZ), carfilzomib (CFZ), or ixazomib (IXZ) for 24, 48 and 72 hours. Cell viability was calculated measuring the ATP content. IC50 drug concentrations were calculated for each agent. Subsequently, MCL cell lines were exposed to escalating doses of V (0.001uM-5uM) and CFZ (1.5625nM-50nM), BTZ (3.125nM-100nM) or IXZ (3.125nM-100nM). In addition, primary tumor cells isolated from B-cell lymphoma patients (N=21) including MCL patients were exposed to V +/- BTZ or CFZ for 48 hrs. Cell viability was determined by Cell Titerglo. Coefficient of synergy were calculated using CalcuSyn software program. Induction of apoptosis was detected by Annexin V/Propidium iodine staining and PARP cleavage. Changes in Bcl-2 and cell cycle regulatory proteins were evaluated by Western blotting in HBL-2 cells. For in vivo experiments, 6-8 weeks old severe combined immunodeficiency (SCID) mice were inoculated with 10x106 HBL-2 cells via tail vein injection (IV). Subsequently, SCID mice were treated with V (100mg/kg/dose via gastric lavage on days 3-7, 10-14 and 17-21) or IXZ (6mg/kg/dose IV days 3, 6, 10, 13, 17 and 20) or combination of both agents. A group of untreated animals was used as a control. Differences in survival were evaluated between treatment groups. Results: In vitro exposure of MCL cell lines to either V, BTZ, CFZ, and IXZ induced cell death in a dose- and time-dependent manner. Significant synergistic activity was observed by combining both V with CFZ or IXZ at known sub-therapeutic and therapeutic doses of individual agents measured by ATP content and apoptosis potential. Anti-tumor activity was observed in cytarabine sensitive and resistant cell lines. Similar findings were observed in primary tumor cells isolated from B-cell lymphoma patients. In vitro exposure of MCL cell lines with the lowest IC50 (HBL-2) to V and PIs (BTZ, CFZ, or IXZ) resulted in the upregulation of Noxa, BIM, Mcl-1 cleavage form (pro-apoptotic) and downregulation of Bcl-XL leading to PARP cleavage and apoptosis. In vivo treatment of MCL bearing SCID mice with V resulted in significant anti-tumor activity when compared to single agent IXZ treated or control animals. Of interest, MCL bearing SCID animals treated with V and IXZ exhibited a better disease control and the survival was longer than SCID animals treated with V or IXZ single agent (P<0.05). Conclusion: Our data suggests that V exhibits strong synergistic activity with PIs, especially with CFZ (in vitro) or IXZ (in vitro and in vivo). Together, our data supports the evaluation of V in combination with readily available novel PIs (IXZ or CFZ) in relapsed/refractory MCL. (Supported by LRF grant 555463, an NIH grant R01 CA136907-01A1 and a grant from The Roswell Park Cancer Institute Alliance Foundation) Disclosures No relevant conflicts of interest to declare.

scholarly journals Targeting BET Bromodomains in Pre-Clinical Models of Burkitt Lymphoma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5381-5381 ◽  
Author(s):  
Thomas Ippolito ◽  
Juan J Gu ◽  
Gregory Tang ◽  
Cory Mavis ◽  
Rodney R. Miles ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Viability ◽  
Cell Line ◽  
Cell Lines ◽  
Burkitt Lymphoma ◽  
Mtor Pathway ◽  
Synergistic Activity ◽  
Pi3k Activation ◽  
Induction Of Apoptosis

Abstract Background: Burkitt lymphoma (BL) is the most common NHL type in children. Although treatment for pediatric BL has improved significantly, there is an urgent need for novel therapies that reduce the toxicity of modern treatment regimens and improve on the dismal survival observed in the relapsed/refractory setting where only about 20-30% of patients survive their disease. Recent reports have implicated co-activation of c-Myc and PI3K in Burkitt lymphomagenesis. Genomic analysis of recurrent oncogenic mutations in BL have identified tonic B-cell receptor signaling and the over-expression of Myc induced microRNAs from the MIR17-92 family, e.g. mir17 and mir19, as possible mechanisms of PI3K activation in BL. Mir17 and mir19, have been implicated in Burkitt lymphomagenesis and their overexpression may be associated with a higher risk of relapse. The protein phosphatase and tensin homolog (PTEN) is a major regulator of PI3K pathway activation. MIR17-92 cluster members have been shown to target PTEN leading to increased PI3K activation. We have previously identified increased expression of mir17 and mir19 along with increased activation of AKT in cell line models of chemotherapy resistant BL suggesting a potential mechanism for increased resistance. BET bromodomains interact with chromatin and enhance transcriptional activation of numerous genes including c-Myc. Thus, BET bromodomains represent a promising target in BL. To investigate the effects of inhibition of c-Myc driven activation of the PI3K/AKT/mTOR pathway, we characterized the activity of the highly potent small molecule bromodomain inhibitor JQ1 in chemotherapy sensitive and resistant BL cell lines. Additionally, we analyzed the ability to enhance anti-lymphoma activity of PI3K/Akt/mTOR pathway inhibition in BL by the combination of BET bromodomain inhibition and targeted inhibition of the PI3K/AKT/mTOR pathway. Methods: The in vitro activity of JQ1 was investigated in the BL cell lines Raji, Raji 4RH (chemotherapy-rituximab resistant), Raji 8RH (rituximab resistant), Ramos, and Daudi. Cell Viability following exposure to JQ1 alone and in combination with the PI3K/mTOR inhibitor omipalisib (GSK458)) was analyzed using Cell-Titer Glo or Alamar Blue assays following 24, 48, and 72 hour exposure over a range of inhibitor concentrations. Induction of apoptosis was analyzed using western blotting for cleaved PARP. C-Myc expression following JQ1 exposure was determined by western blot following 48 hour JQ1 exposure. The effect of JQ1 exposure on the expression of c-Myc induced microRNA expression was determined by qRT-PCR in cells exposed to JQ1 for 48hours. Synergy of combination exposures was determined using CalcuSyn to generate combination index (CI) values. Results: In vitro exposure of BL cell lines to JQ1 for 24, 48, and 72 hours resulted in a significant dose and time dependent decrease in viable cells (72 hour IC50 values: Raji 0.12µM, Raji 4RH 1.7µM, Raji 8RH 0.7µM, Ramos 0.22µM and Daudi 4µM). There was an increase in cleaved PARP after 72 hour exposure indicating induction of apoptosis. While single agent effect was seen in the resistant Raji 4RH cell line, activity was noted to primarily occur at the higher end of the dosing. Western blot analysis demonstrated a reduction in c-Myc expression following exposure to JQ1 1µM for 24 hours (relative band intensity normalized to control: Raji=0.12, Raji 4RH=0.18, Raji 8RH=0.11). qPCR showed a reduction in Mir17 relative transcription levels after 48 hours of exposure to JQ1 2.5mM (relative expression compared to control: Raji=0.72, Raji 4RH=0.98, Raji 8RH=0.83, Ramos=0.57, Daudi=0.46). When combined with omipalisib, an increased effect on cell viability was noted. The combination effect was noted to be synergistic (CI<0.9) at multiple dose combinations while other combinations exhibited primarily additive effects. Conclusion: In vitro inhibition of BET bromodomains with JQ1 results in a decrease in c-Myc expression and a decrease in c-Myc dependent miR expression with impaired proliferation and induction of apoptosis in chemotherapy-sensitive and -resistant BL cell lines. Augmented, and in some cases synergistic, activity is also noted with dual inhibition of BET bromodomains and the PI3K/Akt/mTOR pathway in BL cell lines. Disclosures No relevant conflicts of interest to declare.

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