Rhizen Pharmaceuticals (Switzerland)

Introduction: Calcium release activated calcium channels inhibitors have a potent role in treatment of autoimmune disorders. RP3128 is a novel and potent inhibitor of Calcium Release Activated Calcium (CRAC) channel activity with demonstrated efficacy in several models of immune-inflammatory disorders. The objective of this study was to assess the effect of RP3128 and combinations in attenuating inflammatory responses associated with respiratory diseases. Methods: Inhibition of antigen-induced cytokine release in primary T-lymphocytes was estimated. Peripheral blood neutrophils or mononuclear cells were isolated from COPD or asthmatic patients, pre-incubated with RP3128 for 0.5 h, and stimulated with 5% cigarette smoke extract (CSE) or 1 μg/ml LPS for 6 h for measurement of cytokine release. Effect of the combination of 1 μM RP3128 with 0.1 nM Fluticasone on IL-1β, IL-6, and GM-CSF release in mononuclear cells from asthmatic patients was also assessed. Results: Incubation with 1 nM or 1 μM RP3128 reduced IL-8 secretion by 25% and 50% respectively, in COPD patient-derived neutrophils. In asthmatic patient-derived cells, RP3128 inhibited GM-CSF, IL6 and IL1β release induced by LPS with IC50 of 3.9 nM, 1.3 nM, and 10 nM, respectively. Further, addition of 1 μM RP3128 significantly (P Conclusions: Results demonstrate the anti-inflammatory effects of RP3128 in COPD and asthmatic patient-derived primary cells thereby indicating the therapeutic potential of this molecule in respiratory disorders.

Abstract Background: Multiple Myeloma (MM) accounts for approximately 2.1% of all cancer deaths with a 5-year median survival rate of 49.6%. Besides chemotherapy, treatment options include monoclonal antibodies, immunomodulatory agents, and proteasome inhibitors. Carfilzomib is a selective proteasome inhibitor administered intravenously in patients with relapsed or refractory MM. With cancer cell metabolism emerging as a critical regulator of tumor progression, combining carfilzomib with an inhibitor of the metabolic machinery represents a novel therapeutic strategy to prevent MM progression. RP10107 is a potent, and selective glutaminase (GLS-1) inhibitor that demonstrated high potency against mouse (IC50=21.2 nM), rat (IC50=18.2 nM) and human (IC50=26.4 nM) enzymes with selectivity over GLS-2 (>380-fold). The objective of this study was to evaluate the effect of a combination of carfilzomib and RP10107 in MM cell lines. Methods: Glutamate concentrations in MM cell lines (MM-1S and RPMI-8226) following treatment with RP10107 was estimated by LC-MS/MS. Synergism between RP10107 and carfilzomib was determined using different concentrations of the compounds in a 5 x 5 grid. Synergism, additivity, or antagonism was calculated based on the BLISS score. Apoptosis was determined by Annexin V/7AAD staining using a MUSE® Annexin V and dead cell assay kit (Millipore) while the effect on cell cycle was estimated using Guava Cell Cycle Reagent (Millipore). Expression of cleaved PARP and cleaved caspase-8 were determined by Western Blotting. Results: Treatment of MM cell lines with RP10107 resulted in an increased ratio of glutamine to glutamate with a doubling noticed at concentrations as low as 30 nM. A high degree of synergism between RP10107 and carfilzomib was noticed in both the MM cell lines. Addition of 3 nM Carfilzomib to 30 or 500 nM RP10107 resulted in near complete inhibition of RPMI-8226 and MM-1S cell growth, respectively. Additionally, incubation with the combination (1 μM RP10107 + 15 nM Carfilzomib) for 72 h caused a G0/G1 arrest with a corresponding increase in the percent of apoptotic cells (2.5-fold) compared to the individual agents. Incubating RPMI-8226 cells with a combination of RP10107 and Carfilzomib for 48 h resulted in a 2.2- and 13.6-fold increase in expression of cleaved caspase-8 and cleaved PARP, respectively. Conclusions: Addition of RP10107, a glutaminase inhibitor, potentiated Carfilzomib activity in MM cells. Findings provide a rationale for use of the combination in future clinical trials involving MM patients thereby providing a safer and more effective alternative to currently available therapy. Citation Format: Srikant Viswanadha, Satyanarayana Eleswarapu, Kumar V. Penmetsa, Swaroop Vakkalanka. Synergistic effects of glutaminase and proteasome inhibition in multiple myeloma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1889.

7510Background: Tenalisib is a novel, next generation, highly specific, dual equi-potent PI3K δ/γ inhibitor. Early results demonstrated an acceptable safety profile with encouraging clinical activi...

Abstract Background: Esophageal cancer is the 6th leading cause of cancer mortality worldwide with poor prognosis and 5-year survival rate as low as 18%. It is an unmet need to develop more effective chemotherapeutic drugs to improve the treatment outcome. We previously reported that the expression of Orai1, a calcium channel, was elevated in esophageal squamous cell carcinoma (ESCC), a major form of esophageal cancer, and the high expression is associated with poor survival rate in patients. Orai1-mediated store-operated calcium entry (SOCE) has been demonstrated to contribute to cancer cell proliferation, migration, and invasion in ESCC and other cancers. RP4010 is a potent inhibitor for Orai1 channel (IC50=60 nM) with demonstrated efficacy across a range of cancer cell lines in vitro. Here we evaluated the anticancer effect of RP4010 in cultured ESCC cells and xenograft animals. Materials and Method: Orai channel inhibitory function was analyzed in ESCC cells loaded with fluorescent calcium indicator Fura-2. The antiproliferative effect was evaluated by MTT assay in various human ESCC cell lines, e.g., KYSE-30, KYSE-150, KYSE-790, and KYSE-70. Quantitative real-time RT-PCR, Western blot, and flow cytometry were used to analyze the changes in cell cycle, changes in mRNAs and proteins of some key molecules in calcium signaling pathway. The antitumor effects of RP4010 were monitored in ESCC xenografted nude mice. Result: Compared to commonly used Orai1 channel blockers, such as BTP-2, RP4010 is more potent in blocking Orai1-mediated SOCE and cell proliferation in ESCC cells. The IC50 in cell viability analysis for RP4010 were calculated as 1.2, 1.4, 1.4, and 5.5 μM for KYSE-30, KYSE-70, KYSE-150, and KYSE-790, respectively, while IC50 for BTP-2 was 17.7-17.8 μM for these cells. Treatment of RP4010 (10 μM) halted intracellular calcium oscillations, inhibited calcium-dependent activation of Akt/NF-κB and ERK1/2 pathways, and resulted in cell cycles arrested at G0/G1 phase. Interestingly, RP4010 did not induce significant cell death even at rather high concentrations. In vivo study revealed that the mice tolerated daily I.P. injection of 20 mg/kg RP4010 without body weight loss or noticeable adverse effects. At this dose, RP4010 significantly reduced tumor growth compared to vehicle treated control group. Conclusion: R4010 is a novel potent inhibitor for Orai1-mediated SOCE with demonstrated potential in animal models of esophageal cancer. Data indicate a therapeutic potential of the molecule in ESCC. Citation Format: Chaochu Cui, Yan Chang, Xiaoli Zhang, Kumar V. Penmetsa, Srikant Viswanadha, Liwu Fu, Zui Pan. Targeting Orai1-mediated store-operated Ca2+ entry by a novel compound RP4010 for antiproliferative activity against esophagus squamous cell carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B062.

Abstract Background: Breast cancer is the leading cause of cancer-related death among women worldwide. While early detection techniques, adjuvant therapy, and hormone replacement therapy, have led to a substantial decrease in mortality rates, breast cancer, especially triple negative breast cancer (TNBC) remains an important unmet clinical challenge. Although, Taxol, a mitotic inhibitor, is widely used in conjunction with other chemotherapeutic drugs for both early and late stage breast cancer, it is fraught with adverse effects. With cancer cell metabolism emerging as a critical regulator of tumor progression, combining Taxol with an inhibitor of the metabolic machinery represents a potential therapeutic strategy to prevent breast cancer progression. RP10107 is a novel, potent, and selective glutaminase (GLS-1) inhibitor that demonstrated high potency against mouse (IC50 = 21.2 nM), rat (IC50 = 18.2 nM) and human (IC50 = 26.4 nM) enzymes with selectivity over GLS-2 (>380-fold). The objective of this study was to evaluate the effect of a combination of Taxol and RP10107 in breast cancer cell lines. Methods: Half-maximal growth inhibition of MDA-MB-231 (TNBC), MCF-7 (ER+, PR+), and ZR 75.1 (ER+) cells was assessed using the colorimetric MTT reagent following incubation with Taxol or in combination with 30 nM RP10107 for 72 h. Additionally, synergism between Taxol and RP10107 was determined using different concentrations of the compounds in a 5 × 5 grid. Synergism, additivity, or antagonism was calculated based on the BLISS score. Apoptosis was determined by Annexin V/7AAD staining using a MUSE® Annexin V and dead cell assay kit (Millipore). Results: Taxol inhibited growth of MDA-MB-231, MCF-7, and ZR 75.1 cells with GI50 of 48.9, 2.9, and 1.6 nM, respectively. Although the shift in GI50 upon addition of 30 nM RP10107 in MCF-7 and ZR 75.1 was modest, a >480-fold reduction was observed in the TNBC cell line, MDA-MB-231 (GI50 = 0.9 nM). Similarly, while synergism (BLISS score >10) between Taxol and RP10107 was noticed in all the cell lines tested, effect was most pronounced in MDA-MB-231 cells with >70% growth inhibition seen upon combining of 3 nM Taxol with 10 nM RP10107. Additionally, incubation with the combination (1 nM Taxol + 1 nM RP10107) for 72 h increased the percent of apoptotic cells in MDA-MB-231 cells (35%) compared to the individual agents. Conclusions: Addition of RP10107, a potent and selective GLS-1 inhibitor, potentiated Taxol activity in breast cancer cells with remarkable synergy evident in the TNBC cell line, MDA-MB-231. Findings provide a rationale for use of the combination in future clinical trials involving hard-to-treat TNBC patients thereby providing a safer and much effective option compared to currently available therapy. Citation Format: Kumar V. Penmetsa, Satyanarayana Eleswarapu, Saketh Sriram Dinavahi, Srikant Viswanadha, Swaroop Vakkalanka. RP10107, a novel and potent glutaminase inhibitor, potentiates taxol activity in breast cancer cell lines. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B172.