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121 Tumor-targeted Nanobullets for Anti-cancer Combination Therapy
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Wednesday 7 November 2012
Poster Session – Combinatorial Chemistry, Drug Delivery, Drug Design and Drug Synthesis
Conclusion: Combining novel chemotherapeutic agents with the current standard of chemotherapeutic care, TMZ, may not only inhibit tumor growth but also tumor recurrence. Our lab found that when a novel chemotherapeutic agent, JLK1486, was combined with TMZ, secondary sphere formation was inhibited, suggesting that dual treatment is more efficacious than single agent in inhibiting secondary sphere formation and may reduce tumor recurrence. Future direction: We are currently examining the mechanism of secondary sphere formation in dual versus single agent treated cells. Potential mechanisms include induced apoptosis as well as senescence. We are also examining the efficacies of single versus double treatments of TMZ+JLK1486 to inhibit tumor growth in vivo. 121 POSTER Tumor-targeted Nanobullets for Anti-cancer Combination Therapy R. van der Meel1 , S. Oliveira2 , I. Altintas1 , R. Haselberg3 , R.C. Roovers2 , P.M.P. van Bergen en Henegouwen2 , W.E. Hennink1 , W.E. Storm1 , R.M. Schiffelers1 , R.J. Kok1 . 1 Utrecht Institute of Pharmaceutical Sciences, Pharmaceutics, Utrecht, The Netherlands; 2 Cell Biology, Biology, Utrecht, The Netherlands; 3 Utrecht Institute of Pharmaceutical Sciences, Biomolecular Analysis, Utrecht, The Netherlands Background: The epidermal growth factor receptor (EGFR) is a well established target for anti-cancer therapy and several EGFR-targeted therapeutics are used in the clinic. However, crosstalk between EGFR and other receptor signaling networks can contribute to accelerated tumorigenesis and even induce resistance to EGFR-directed therapies. Combined inhibition of EGFR and insulin-like growth factor 1 receptor (IGF1R) signaling is a rational strategy to enhance anti-cancer treatment and possibly delay resistance development. We have encapsulated the antiIGF-1R kinase inhibitor AG538 in anti-EGFR nanobody-liposomes. The efficacy of this targeted dual-active nanomedicine was assessed on EGFR(over)expressing tumor cells in vitro. Material and Methods: Anti-EGFR nanobodies (EGa1) were coupled to PEGylated liposomes. AG538 was encapsulated in the EGa1-liposomes by remote loading with a calcium acetate gradient. EGa1-AG538-liposomes were characterized according to size, surface charge and encapsulation efficiency. Cellular assays were performed with EGFR-(over)expressing UM-SSC14C human head and neck cancer cells (14C) and EGFR−/− NIH 3T3 clone 2.2 murine fibroblasts (3T3 2.2). Cell association of rhodaminelabeled EGa1-AG538-liposomes was determined by flow cytometry and cell uptake was visualized by confocal microscopy. Inhibition of EGFR and IGF-1R signaling was investigated by Western Blotting with phosphospecific antibodies directed at the targeted pathways. Inhibition of tumor cell proliferation was determined by the sulforhodamine B (SRB) assay and the BrdU-colorimetric immunoassay. These assays were conducted upon continuous exposure to the nanomedicine for 48 h and short-term exposure for 4 h followed by a 44 h treatment-free period to mimic in vivo drug exposure. Results: EGa1-AG538-liposomes associated with EGFR-positive 14C cells in an EGFR-specific manner and were internalized by 14C cells but not by EGFR-negative 3T3 2.2 cells. EGa1-AG538-liposomes inhibited both EGFR and IGF-1R signaling activation and induced downregulation of EGFR. The dual-active nanomedicine strongly inhibited 14C proliferation in short-term exposure assays while control formulations did not. Similar inhibitory effects of the nanomedicine on cell proliferation were observed with EGFR(over)expressing MDA-MB-468 human breast cancer cells. Conclusions: Anti-EGFR nanobody liposomes loaded with anti-IGF1R kinase inhibitor simultaneously block activation of EGFR and IGF1R signaling. In short-term exposure assays, EGa1-AG538-L induces strong inhibition of tumor cell proliferation. This targeted nanomedicine is a promising anti-cancer therapy for tumors that are dependent on (over)expression of EGFR and IGF-1R and their in vivo applicability is currently under investigation. 122 POSTER Drug-loaded Magnetically-responsive Nanoparticles: Validation of Anti-tumor Activity J. Klostergaard1 , E. Auzenne1 , C. Seeney2 . 1 University of Texas M.D., Molecular & Cellular Oncology, Houston, USA; 2 ViCorp Tech, Edmond, USA Background: Magnetically-responsive drug delivery platforms have typically used chemadsorptive strategies for non-covalent binding of chemotherapeutics to these carriers; a weakness of this approach is the inability to closely control subsequent desorption, which if it occurs before the platform arrives at the tumor site, results in toxicity from free drug, or if
it occurs incompletely at the tumor site, yields reduced anti-tumor efficacy. To mitigate these issues, we have developed a novel tumor-targeting platform in which the drug payload is covalently linked to magnetite-based, magnetically-responsive nanoparticles (MNP) via bioreversible bonds that will be cleaved predominantly in the tumor microenvironment. Materials and Methods: Paclitaxel (TXL) was covalently linked to silicacoated MNP via either ester or hydrazone bonds using silane-based linkers. Cytotoxic activity of each prodrug was first evaluated in vitro against MDA-MB-231 and MDA-MB-468 triple-negative breast carcinoma cells by exposure to MNP-TXL prodrug and control formulations for up to 120 hr before staining with MTT to determine survival. In vivo anti-tumor activity was evaluated using multiple cycles of intra-tumoral injections in orthotopic xenografts of these models in nude mice, with tracking by calculation of tumor volume. Results: A rank order of in vitro potency was observed among the TXL-loaded MNP formulations, with an ester-linked formulation being the weakest, and another ester-linked formulation which also had PEG attached to the MNP and a formulation with a pH-sensitive hydrazone bond both being 1−2 logs more potent. Evaluation in vivo revealed that tumors of mice given the control MNP-linker formulations grew steadily: more than doubling in tumor volume in 14 days. However, the TXL-loaded formulations demonstrated a rank order of ability to delay or reverse tumor growth following a multiple-dosing regimen, generally similar to their in vitro potency. A pilot toxicology study revealed no lesions in mice given the PEGMNPs loaded with TXL. Conclusion: We have validated the underlying concept and designed chemistry for Magnetic Vectoring and propose to further develop the lead MNP-TXL constructs for systemic administration followed by site-specific vectoring to tumor xenografts, on the path to clinical evaluation. 123 POSTER The Discovery of Potent and Selective Inhibitors of CYP17 Lyase A. Balog1 , L. Jayaraman2 , A. Fura3 , G.D. Vite1 , M. Gottardis2 , A. Huang1 , J.A. Newitt4 , T.E. Spires2 , M.T. Obermeier3 , S.M. Beyer5 . 1 Bristol-Myers Squibb Company, Oncology Chemistry, Princeton NJ, USA; 2 Bristol-Myers Squibb Company, Oncology Biology, Princeton NJ, USA; 3 Bristol-Myers Squibb Company, Metabolism and Pharmacokinetics, Princeton NJ, USA; 4 Bristol-Myers Squibb Company, Protein Science and Structure, Princeton NJ, USA; 5 Bristol-Myers Squibb Company, Veterinary Sciences, Princeton NJ, USA Background: Endogenous androgens promote the development and progression of prostate cancer (CaP). While surgical or chemical castration can reduce circulating androgen levels by 90% and are effective therapies, patients will eventually progress to castration-resistant prostate cancer (CRPC) where tumor growth can be driven by very low levels of androgens. Androgen precursors such as dehydroepiandrosterone (DHEA) and androstenedione (AdT) are produced in the adrenals by the action of the cytochrome P450 enzyme CYP17. These steroids are then converted peripherally to the androgens testosterone (T) and dihydrotestosterone (DHT), even in castrate patients. One approach to decrease androgen levels further is to inhibit CYP17 lyase in both the adrenals and tumor. The CYP17 enzyme has both 17,20-lyase and 17a-hydroxylase activities. Specific inhibition of the lyase function of CYP17 would be desirable to avoid mineralocorticoid excess resulting from hydroxylase inhibition. Targeting CYP17 with agents such as abiraterone acetate has been shown to reduce circulating androgens to levels well below castration, leading to significant clinical benefit for CRPC patients. However, concomitant steroids are usually required with abiraterone to address increased levels of mineralocorticoids, potentially due to inhibition of CYP17 hydroxylase. Our program objective was to find a highly lyase-specific and selective inhibitor of CYP17 with minimal glucocorticoid and mineralocorticoid perturbation. Methods: Iterative design and molecular modeling were used to discover novel inhibitors of CYP17 lyase. Scintillation proximity assays (SPA) were developed for both cynomolgus monkey and human CYP17 lyase and hydroxylase. Assays were also developed for cyno and human CYP11B1 and CYP21 as additional measures of selectivity. Compounds with the desired in vitro profile were dosed in both gonadally-intact and chemically-castrated cynomolgus monkeys. In these studies, levels of T, progesterone, hydroxy-progesterone and cortisol were monitored to gauge the pharmacodynamic (PD) effect and level of selectivity associated with lead compounds. Results: Numerous compounds with robust lyase specificity and overall CYP17 selectivity were identified. In the cyno PD model, many compounds exhibited excellent reduction of T with minimal fluctuations in progesterone and cortisol levels, suggesting good overall selectivity. The benzimidazole, BMS-916351, was identified as the lead compound and considered for clinical development. Conclusion: We identified BMS-916351 as a highly potent and selective inhibitor of CYP17 lyase. This compound demonstrated robust decreases
Wednesday 7 November 2012
Poster Session – Combinatorial Chemistry, Drug Delivery, Drug Design and Drug Synthesis
Conclusion: Combining novel chemotherapeutic agents with the current standard of chemotherapeutic care, TMZ, may not only inhibit tumor growth but also tumor recurrence. Our lab found that when a novel chemotherapeutic agent, JLK1486, was combined with TMZ, secondary sphere formation was inhibited, suggesting that dual treatment is more efficacious than single agent in inhibiting secondary sphere formation and may reduce tumor recurrence. Future direction: We are currently examining the mechanism of secondary sphere formation in dual versus single agent treated cells. Potential mechanisms include induced apoptosis as well as senescence. We are also examining the efficacies of single versus double treatments of TMZ+JLK1486 to inhibit tumor growth in vivo. 121 POSTER Tumor-targeted Nanobullets for Anti-cancer Combination Therapy R. van der Meel1 , S. Oliveira2 , I. Altintas1 , R. Haselberg3 , R.C. Roovers2 , P.M.P. van Bergen en Henegouwen2 , W.E. Hennink1 , W.E. Storm1 , R.M. Schiffelers1 , R.J. Kok1 . 1 Utrecht Institute of Pharmaceutical Sciences, Pharmaceutics, Utrecht, The Netherlands; 2 Cell Biology, Biology, Utrecht, The Netherlands; 3 Utrecht Institute of Pharmaceutical Sciences, Biomolecular Analysis, Utrecht, The Netherlands Background: The epidermal growth factor receptor (EGFR) is a well established target for anti-cancer therapy and several EGFR-targeted therapeutics are used in the clinic. However, crosstalk between EGFR and other receptor signaling networks can contribute to accelerated tumorigenesis and even induce resistance to EGFR-directed therapies. Combined inhibition of EGFR and insulin-like growth factor 1 receptor (IGF1R) signaling is a rational strategy to enhance anti-cancer treatment and possibly delay resistance development. We have encapsulated the antiIGF-1R kinase inhibitor AG538 in anti-EGFR nanobody-liposomes. The efficacy of this targeted dual-active nanomedicine was assessed on EGFR(over)expressing tumor cells in vitro. Material and Methods: Anti-EGFR nanobodies (EGa1) were coupled to PEGylated liposomes. AG538 was encapsulated in the EGa1-liposomes by remote loading with a calcium acetate gradient. EGa1-AG538-liposomes were characterized according to size, surface charge and encapsulation efficiency. Cellular assays were performed with EGFR-(over)expressing UM-SSC14C human head and neck cancer cells (14C) and EGFR−/− NIH 3T3 clone 2.2 murine fibroblasts (3T3 2.2). Cell association of rhodaminelabeled EGa1-AG538-liposomes was determined by flow cytometry and cell uptake was visualized by confocal microscopy. Inhibition of EGFR and IGF-1R signaling was investigated by Western Blotting with phosphospecific antibodies directed at the targeted pathways. Inhibition of tumor cell proliferation was determined by the sulforhodamine B (SRB) assay and the BrdU-colorimetric immunoassay. These assays were conducted upon continuous exposure to the nanomedicine for 48 h and short-term exposure for 4 h followed by a 44 h treatment-free period to mimic in vivo drug exposure. Results: EGa1-AG538-liposomes associated with EGFR-positive 14C cells in an EGFR-specific manner and were internalized by 14C cells but not by EGFR-negative 3T3 2.2 cells. EGa1-AG538-liposomes inhibited both EGFR and IGF-1R signaling activation and induced downregulation of EGFR. The dual-active nanomedicine strongly inhibited 14C proliferation in short-term exposure assays while control formulations did not. Similar inhibitory effects of the nanomedicine on cell proliferation were observed with EGFR(over)expressing MDA-MB-468 human breast cancer cells. Conclusions: Anti-EGFR nanobody liposomes loaded with anti-IGF1R kinase inhibitor simultaneously block activation of EGFR and IGF1R signaling. In short-term exposure assays, EGa1-AG538-L induces strong inhibition of tumor cell proliferation. This targeted nanomedicine is a promising anti-cancer therapy for tumors that are dependent on (over)expression of EGFR and IGF-1R and their in vivo applicability is currently under investigation. 122 POSTER Drug-loaded Magnetically-responsive Nanoparticles: Validation of Anti-tumor Activity J. Klostergaard1 , E. Auzenne1 , C. Seeney2 . 1 University of Texas M.D., Molecular & Cellular Oncology, Houston, USA; 2 ViCorp Tech, Edmond, USA Background: Magnetically-responsive drug delivery platforms have typically used chemadsorptive strategies for non-covalent binding of chemotherapeutics to these carriers; a weakness of this approach is the inability to closely control subsequent desorption, which if it occurs before the platform arrives at the tumor site, results in toxicity from free drug, or if
it occurs incompletely at the tumor site, yields reduced anti-tumor efficacy. To mitigate these issues, we have developed a novel tumor-targeting platform in which the drug payload is covalently linked to magnetite-based, magnetically-responsive nanoparticles (MNP) via bioreversible bonds that will be cleaved predominantly in the tumor microenvironment. Materials and Methods: Paclitaxel (TXL) was covalently linked to silicacoated MNP via either ester or hydrazone bonds using silane-based linkers. Cytotoxic activity of each prodrug was first evaluated in vitro against MDA-MB-231 and MDA-MB-468 triple-negative breast carcinoma cells by exposure to MNP-TXL prodrug and control formulations for up to 120 hr before staining with MTT to determine survival. In vivo anti-tumor activity was evaluated using multiple cycles of intra-tumoral injections in orthotopic xenografts of these models in nude mice, with tracking by calculation of tumor volume. Results: A rank order of in vitro potency was observed among the TXL-loaded MNP formulations, with an ester-linked formulation being the weakest, and another ester-linked formulation which also had PEG attached to the MNP and a formulation with a pH-sensitive hydrazone bond both being 1−2 logs more potent. Evaluation in vivo revealed that tumors of mice given the control MNP-linker formulations grew steadily: more than doubling in tumor volume in 14 days. However, the TXL-loaded formulations demonstrated a rank order of ability to delay or reverse tumor growth following a multiple-dosing regimen, generally similar to their in vitro potency. A pilot toxicology study revealed no lesions in mice given the PEGMNPs loaded with TXL. Conclusion: We have validated the underlying concept and designed chemistry for Magnetic Vectoring and propose to further develop the lead MNP-TXL constructs for systemic administration followed by site-specific vectoring to tumor xenografts, on the path to clinical evaluation. 123 POSTER The Discovery of Potent and Selective Inhibitors of CYP17 Lyase A. Balog1 , L. Jayaraman2 , A. Fura3 , G.D. Vite1 , M. Gottardis2 , A. Huang1 , J.A. Newitt4 , T.E. Spires2 , M.T. Obermeier3 , S.M. Beyer5 . 1 Bristol-Myers Squibb Company, Oncology Chemistry, Princeton NJ, USA; 2 Bristol-Myers Squibb Company, Oncology Biology, Princeton NJ, USA; 3 Bristol-Myers Squibb Company, Metabolism and Pharmacokinetics, Princeton NJ, USA; 4 Bristol-Myers Squibb Company, Protein Science and Structure, Princeton NJ, USA; 5 Bristol-Myers Squibb Company, Veterinary Sciences, Princeton NJ, USA Background: Endogenous androgens promote the development and progression of prostate cancer (CaP). While surgical or chemical castration can reduce circulating androgen levels by 90% and are effective therapies, patients will eventually progress to castration-resistant prostate cancer (CRPC) where tumor growth can be driven by very low levels of androgens. Androgen precursors such as dehydroepiandrosterone (DHEA) and androstenedione (AdT) are produced in the adrenals by the action of the cytochrome P450 enzyme CYP17. These steroids are then converted peripherally to the androgens testosterone (T) and dihydrotestosterone (DHT), even in castrate patients. One approach to decrease androgen levels further is to inhibit CYP17 lyase in both the adrenals and tumor. The CYP17 enzyme has both 17,20-lyase and 17a-hydroxylase activities. Specific inhibition of the lyase function of CYP17 would be desirable to avoid mineralocorticoid excess resulting from hydroxylase inhibition. Targeting CYP17 with agents such as abiraterone acetate has been shown to reduce circulating androgens to levels well below castration, leading to significant clinical benefit for CRPC patients. However, concomitant steroids are usually required with abiraterone to address increased levels of mineralocorticoids, potentially due to inhibition of CYP17 hydroxylase. Our program objective was to find a highly lyase-specific and selective inhibitor of CYP17 with minimal glucocorticoid and mineralocorticoid perturbation. Methods: Iterative design and molecular modeling were used to discover novel inhibitors of CYP17 lyase. Scintillation proximity assays (SPA) were developed for both cynomolgus monkey and human CYP17 lyase and hydroxylase. Assays were also developed for cyno and human CYP11B1 and CYP21 as additional measures of selectivity. Compounds with the desired in vitro profile were dosed in both gonadally-intact and chemically-castrated cynomolgus monkeys. In these studies, levels of T, progesterone, hydroxy-progesterone and cortisol were monitored to gauge the pharmacodynamic (PD) effect and level of selectivity associated with lead compounds. Results: Numerous compounds with robust lyase specificity and overall CYP17 selectivity were identified. In the cyno PD model, many compounds exhibited excellent reduction of T with minimal fluctuations in progesterone and cortisol levels, suggesting good overall selectivity. The benzimidazole, BMS-916351, was identified as the lead compound and considered for clinical development. Conclusion: We identified BMS-916351 as a highly potent and selective inhibitor of CYP17 lyase. This compound demonstrated robust decreases