- Email: [email protected]
117 Exploring Novel Temozolomide Analogues to Overcome Glioblastoma Resistance
36
Wednesday 7 November 2012
Poster Session – Combinatorial Chemistry, Drug Delivery, Drug Design and Drug Synthesis
participation in the study receiving LY in combination with gemcitabine or pemetrexed. Conclusions: Despite the upper end of the 90% RCI for the desipramine Cmax LSM ratio being slightly greater than 1.25, it is not expected that this minimal change in Cmax (with no change in AUC ratios) would lead to any clinically significant CYP2D6 drug interactions. Therefore, no exclusion criteria for concomitant medications metabolized by CYP2D6 would likely be needed for Phase 3 studies. 115 POSTER Reversal of Drug Resistance to Chlorambucil by Its Conjugation to a Targeting Peptide M. Firer1 , G. Luboshits1 , S. Baskin2 , G. Gellerman2 . 1 Ariel University Center, Chemical Engineering and Biotechnology, Ariel, Israel; 2 Ariel University Center, Biological Chemistry, Ariel, Israel We previous demonstrated that Targeted drug delivery (TDD) via smal molecule carriers with high affinity to receptors on target cancer cells increases drug efficacy and does not induce the outgrowth of drug resistant clones of cancer cells. The ability to conjugate several and different anticancer drugs to a cell targeting carrier via appropriate linkers would further the development of carrier-drug conjugates and enhance the therapeutic efficacy of TDD. Moreover, the ability to engineer deviations in time release of different drugs from multi-conjugates suggests that employing dendrimeric linkers with multiple attachment and controlled release capabilities may significantly improve the architecture and properties of multi-drug conjugates. As a step towards preparing such conjugates, we describe the use the preparation of targeted multi-drug conjugates and tested their cytotoxic potential. A 13mer peptide derived from the Myelin Basic Protein (pMBP) was used to target the anti-cancer drug Chlorambucil (Chlor) to murine MBP B-cell leukemic cells bearing specific antibodies to pMBP. Initially, MBP cells as well as a non-relevant B-cell leukemic cell line (BCL-1) were resistant to Chlor. However exposure of the cells to a bioconjugate of pMBP-Chlor completely and specifically reversed the resistance of MBP cells but not that of BCL-1 cells or normal spleenocytes. These results highlight the possibility that bioconjugates comprising peptides targeted to B-cell leukemic cells could allow for more effective use of drugs with a proven clinical history in hematological cancer. We then compared dose response curves of MBP cells exposed to bioconjugates carrying single (pMBP-Chlor-1) or multiple (pMBP-Chlor2 or pMBP-Chlor-4) copies of Chlor. At equivalent doses of Chlor, there was a statistically significant enhancement in cytotoxic activity of multidrug copy conjugates versus single-drug copy conjugates. Updated results will also be presented on the efficacy of multi-drug conjugates versus free Chlor in reducing the growth of subcutaneous MBP tumors in a mouse model. These data suggest that the increased efficacy of multi-dose conjugates is not only related to the amount of delivered drug but also to the mode of delivery. Delivering the same dose of drug through receptor-mediated endocytosis is more efficient than delivery of free drug through non-specific cell uptake. Our study demonstrates that targeted multifunctional dendrone linkers bearing several copies of cytotoxic agents have enhanced efficacy for against cancer cells. 116 POSTER Transport of the Anticancer Agent, Dp44mT, via a Receptor-mediated Process A.M. Merlot1 , D.S. Kalinowski1 , N. Pantarat1 , S. Menezes1 , M. Doddareddy2 , D. Hibbs2 , D.R. Richardson1 . 1 University of Sydney, Pathology, Sydney NSW, Australia; 2 University of Sydney, Pharmacy, Sydney NSW, Australia Defective iron homeostasis has emerged as a feature of cancer cells (Cancer Res 2011;71:1511–1514). Hence, iron chelators may be a novel pharmacological intervention to target tumor cells that have disturbed iron metabolism. Several studies including clinical trials have demonstrated that certain classes of chelators possess potent anti-cancer activity. The chelator, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), demonstrates potent in vitro and in vivo anti-cancer activity (PNAS 2006;103:14901−6). Interestingly, recent investigations have shown that Dp44mT accumulates within the lysosomes, forming a redox-active complex, resulting in lysosomal permeabilization and apoptosis (Cancer Res 2011;71(17):5871−80). However, the mechanism by which this drug enters cells to induce cell death is unknown. Dp44mT was labeled with 14 C to assess membrane transport using human SK-N-MC neuroepithelioma cells. Studies were compared to the more lipophilic chelator, 14 C-2-benzoylpyridine 4-ethyl-3thiosemicarbazone (14 C-Bp4eT). Initial results demonstrated that 14 CBp4eT enters cells via passive diffusion (Mol Pharmacol 2010;78(4):675–
684). Whereas, the saturable and temperature-dependent uptake process of Dp44mT indicated the existence of a receptor-mediated transport mechanism. Moreover, the uptake of 14 C-labeled Dp44mT decreased with increasing concentrations of unlabeled Dp44mT, also indicating a saturable transport system. In contrast, raising the concentration of the unlabeled chelators, Bp4eT and pyridoxal isonicotinoyl hydrazone (PIH), which share some structural features, had little effect on 14 C-Dp44mT uptake. The protein, human serum albumin (HSA; 40 mg/mL), that is well known to bind drugs and affect their delivery, markedly (p < 0.01) increased 14 CDp44mT cellular uptake. However, this effect was inhibited in the presence of excess HSA, suggesting an albumin receptor could be involved. Uptake experiments were also performed in the presence of serum transferrin (Tf) and the structurally-related protein, bovine serum albumin (BSA). 14 CDp44mT uptake was not significantly altered in the presence of Tf, while it was considerably (p < 0.01) reduced in the presence of BSA. Thus, the HSA-induced increase in 14 C-Dp44mT uptake appeared to be specific for this protein. The augmented accumulation of 14 C-Dp44mT in the presence of HSA was observed in six different cell lines, demonstrating that this transport mechanism was not cell-type specific. Subsequently, drug-protein binding studies were performed to suggest that Dp44mT binds directly to HSA and BSA presumably at Sudlow’s Site I. In conclusion, the interaction of Dp44mT with HSA enhances its uptake into human cancer cells by a saturable, receptor-mediated mechanism. 117 POSTER Exploring Novel Temozolomide Analogues to Overcome Glioblastoma Resistance A. Ross1 , Y.P. Ramirez1 , R.M. Phillips2 , R.T. Wheelhouse3 . 1 University of Massachusetts Medical School, Biochemistry and Molecular Pharmacology, Worcester, USA; 2 University of Bradford, Institute of Cancer Therapeutics, Worcester, USA; 3 University of Bradford, Department of Medicinal Chemistry, Worcester, USA Background: Glioblastoma multiforme (GBM) tumors are highly malignant in nature and despite an aggressive therapy regimen, long-term survival for glioma patients is uncommon, as cells with intrinsic or acquired resistance can repopulate the tumor. We need to investigate new therapies for enhancing GBM treatment. Using neurosphere glioma lines, we are analyzing responses following treatment with novel Temozolomide (TMZ) analogues DP86 (monofunctional agent) and DP68 (bifunctional agent) and, in particular, self-renewal and secondary sphere formation. Materials and Methods: Sensitivity to TMZ and TMZ analogues, DP86 and DP68, in a panel of lines with varying MGMT expression, were analyzed by calculating the sphere IC50, which is the concentration of drug that inhibits sphere formation by 50%. Cells were exposed to TMZ drugs and incubated for seven days, at which point spheres were counted. Twentyone day neurosphere assays were carried out to assess the initial response to therapy as well the capacity to self-renew and repopulate the culture. For neurosphere assays GBM neurospheres were plated at clonal density, treated with: DMSO, TMZ alone, GSI alone, or a combination of TMZ and GSI. Cells are analyzed and fed at day seven, pH dissociated to single cells and re-plated at day 14, and allowed to grow until day 21. Cell cycle expression was analyzed by flow cytometry after treatment with TMZ and TMZ analogues. Results: Treatment with TMZ significantly decreases initial neurosphere formation as we observed a five to ten fold difference in DMSO versus TMZ treated cultures; however, after a short recovery period, a small number of cells resume neurosphere formation and repopulate both MGMT positive and negative lines. Glioma lines treated with DP86 showed increased sensitivity, inhibiting secondary sphere formation. All cell lines treated with DP68 showed a dramatic decrease in neurospheres after treatment at doses 20-fold lower than TMZ and 10-fold lower than DP86. Secondary sphere formation was completely inhibited at low doses of DP68. Cells treated with DP86 and DP68 exhibit a G2/M arrest as demonstrated by flow cytometry. Conclusion: Our preliminary data indicates that these novel TMZ analogues, DP86 and DP68, not only inhibit neurosphere formation at lowers doses than TMZ but also completely inhibit secondary sphere formation. These effects are independent of MGMT expression in glioma cells, suggesting that these novel TMZ analogues will be more efficacious than TMZ for the treatment of glioblastoma.
Wednesday 7 November 2012
Poster Session – Combinatorial Chemistry, Drug Delivery, Drug Design and Drug Synthesis
participation in the study receiving LY in combination with gemcitabine or pemetrexed. Conclusions: Despite the upper end of the 90% RCI for the desipramine Cmax LSM ratio being slightly greater than 1.25, it is not expected that this minimal change in Cmax (with no change in AUC ratios) would lead to any clinically significant CYP2D6 drug interactions. Therefore, no exclusion criteria for concomitant medications metabolized by CYP2D6 would likely be needed for Phase 3 studies. 115 POSTER Reversal of Drug Resistance to Chlorambucil by Its Conjugation to a Targeting Peptide M. Firer1 , G. Luboshits1 , S. Baskin2 , G. Gellerman2 . 1 Ariel University Center, Chemical Engineering and Biotechnology, Ariel, Israel; 2 Ariel University Center, Biological Chemistry, Ariel, Israel We previous demonstrated that Targeted drug delivery (TDD) via smal molecule carriers with high affinity to receptors on target cancer cells increases drug efficacy and does not induce the outgrowth of drug resistant clones of cancer cells. The ability to conjugate several and different anticancer drugs to a cell targeting carrier via appropriate linkers would further the development of carrier-drug conjugates and enhance the therapeutic efficacy of TDD. Moreover, the ability to engineer deviations in time release of different drugs from multi-conjugates suggests that employing dendrimeric linkers with multiple attachment and controlled release capabilities may significantly improve the architecture and properties of multi-drug conjugates. As a step towards preparing such conjugates, we describe the use the preparation of targeted multi-drug conjugates and tested their cytotoxic potential. A 13mer peptide derived from the Myelin Basic Protein (pMBP) was used to target the anti-cancer drug Chlorambucil (Chlor) to murine MBP B-cell leukemic cells bearing specific antibodies to pMBP. Initially, MBP cells as well as a non-relevant B-cell leukemic cell line (BCL-1) were resistant to Chlor. However exposure of the cells to a bioconjugate of pMBP-Chlor completely and specifically reversed the resistance of MBP cells but not that of BCL-1 cells or normal spleenocytes. These results highlight the possibility that bioconjugates comprising peptides targeted to B-cell leukemic cells could allow for more effective use of drugs with a proven clinical history in hematological cancer. We then compared dose response curves of MBP cells exposed to bioconjugates carrying single (pMBP-Chlor-1) or multiple (pMBP-Chlor2 or pMBP-Chlor-4) copies of Chlor. At equivalent doses of Chlor, there was a statistically significant enhancement in cytotoxic activity of multidrug copy conjugates versus single-drug copy conjugates. Updated results will also be presented on the efficacy of multi-drug conjugates versus free Chlor in reducing the growth of subcutaneous MBP tumors in a mouse model. These data suggest that the increased efficacy of multi-dose conjugates is not only related to the amount of delivered drug but also to the mode of delivery. Delivering the same dose of drug through receptor-mediated endocytosis is more efficient than delivery of free drug through non-specific cell uptake. Our study demonstrates that targeted multifunctional dendrone linkers bearing several copies of cytotoxic agents have enhanced efficacy for against cancer cells. 116 POSTER Transport of the Anticancer Agent, Dp44mT, via a Receptor-mediated Process A.M. Merlot1 , D.S. Kalinowski1 , N. Pantarat1 , S. Menezes1 , M. Doddareddy2 , D. Hibbs2 , D.R. Richardson1 . 1 University of Sydney, Pathology, Sydney NSW, Australia; 2 University of Sydney, Pharmacy, Sydney NSW, Australia Defective iron homeostasis has emerged as a feature of cancer cells (Cancer Res 2011;71:1511–1514). Hence, iron chelators may be a novel pharmacological intervention to target tumor cells that have disturbed iron metabolism. Several studies including clinical trials have demonstrated that certain classes of chelators possess potent anti-cancer activity. The chelator, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), demonstrates potent in vitro and in vivo anti-cancer activity (PNAS 2006;103:14901−6). Interestingly, recent investigations have shown that Dp44mT accumulates within the lysosomes, forming a redox-active complex, resulting in lysosomal permeabilization and apoptosis (Cancer Res 2011;71(17):5871−80). However, the mechanism by which this drug enters cells to induce cell death is unknown. Dp44mT was labeled with 14 C to assess membrane transport using human SK-N-MC neuroepithelioma cells. Studies were compared to the more lipophilic chelator, 14 C-2-benzoylpyridine 4-ethyl-3thiosemicarbazone (14 C-Bp4eT). Initial results demonstrated that 14 CBp4eT enters cells via passive diffusion (Mol Pharmacol 2010;78(4):675–
684). Whereas, the saturable and temperature-dependent uptake process of Dp44mT indicated the existence of a receptor-mediated transport mechanism. Moreover, the uptake of 14 C-labeled Dp44mT decreased with increasing concentrations of unlabeled Dp44mT, also indicating a saturable transport system. In contrast, raising the concentration of the unlabeled chelators, Bp4eT and pyridoxal isonicotinoyl hydrazone (PIH), which share some structural features, had little effect on 14 C-Dp44mT uptake. The protein, human serum albumin (HSA; 40 mg/mL), that is well known to bind drugs and affect their delivery, markedly (p < 0.01) increased 14 CDp44mT cellular uptake. However, this effect was inhibited in the presence of excess HSA, suggesting an albumin receptor could be involved. Uptake experiments were also performed in the presence of serum transferrin (Tf) and the structurally-related protein, bovine serum albumin (BSA). 14 CDp44mT uptake was not significantly altered in the presence of Tf, while it was considerably (p < 0.01) reduced in the presence of BSA. Thus, the HSA-induced increase in 14 C-Dp44mT uptake appeared to be specific for this protein. The augmented accumulation of 14 C-Dp44mT in the presence of HSA was observed in six different cell lines, demonstrating that this transport mechanism was not cell-type specific. Subsequently, drug-protein binding studies were performed to suggest that Dp44mT binds directly to HSA and BSA presumably at Sudlow’s Site I. In conclusion, the interaction of Dp44mT with HSA enhances its uptake into human cancer cells by a saturable, receptor-mediated mechanism. 117 POSTER Exploring Novel Temozolomide Analogues to Overcome Glioblastoma Resistance A. Ross1 , Y.P. Ramirez1 , R.M. Phillips2 , R.T. Wheelhouse3 . 1 University of Massachusetts Medical School, Biochemistry and Molecular Pharmacology, Worcester, USA; 2 University of Bradford, Institute of Cancer Therapeutics, Worcester, USA; 3 University of Bradford, Department of Medicinal Chemistry, Worcester, USA Background: Glioblastoma multiforme (GBM) tumors are highly malignant in nature and despite an aggressive therapy regimen, long-term survival for glioma patients is uncommon, as cells with intrinsic or acquired resistance can repopulate the tumor. We need to investigate new therapies for enhancing GBM treatment. Using neurosphere glioma lines, we are analyzing responses following treatment with novel Temozolomide (TMZ) analogues DP86 (monofunctional agent) and DP68 (bifunctional agent) and, in particular, self-renewal and secondary sphere formation. Materials and Methods: Sensitivity to TMZ and TMZ analogues, DP86 and DP68, in a panel of lines with varying MGMT expression, were analyzed by calculating the sphere IC50, which is the concentration of drug that inhibits sphere formation by 50%. Cells were exposed to TMZ drugs and incubated for seven days, at which point spheres were counted. Twentyone day neurosphere assays were carried out to assess the initial response to therapy as well the capacity to self-renew and repopulate the culture. For neurosphere assays GBM neurospheres were plated at clonal density, treated with: DMSO, TMZ alone, GSI alone, or a combination of TMZ and GSI. Cells are analyzed and fed at day seven, pH dissociated to single cells and re-plated at day 14, and allowed to grow until day 21. Cell cycle expression was analyzed by flow cytometry after treatment with TMZ and TMZ analogues. Results: Treatment with TMZ significantly decreases initial neurosphere formation as we observed a five to ten fold difference in DMSO versus TMZ treated cultures; however, after a short recovery period, a small number of cells resume neurosphere formation and repopulate both MGMT positive and negative lines. Glioma lines treated with DP86 showed increased sensitivity, inhibiting secondary sphere formation. All cell lines treated with DP68 showed a dramatic decrease in neurospheres after treatment at doses 20-fold lower than TMZ and 10-fold lower than DP86. Secondary sphere formation was completely inhibited at low doses of DP68. Cells treated with DP86 and DP68 exhibit a G2/M arrest as demonstrated by flow cytometry. Conclusion: Our preliminary data indicates that these novel TMZ analogues, DP86 and DP68, not only inhibit neurosphere formation at lowers doses than TMZ but also completely inhibit secondary sphere formation. These effects are independent of MGMT expression in glioma cells, suggesting that these novel TMZ analogues will be more efficacious than TMZ for the treatment of glioblastoma.