Marcel B. Bally

Drug interaction studies between paclitaxel (Taxol) and OC144-093 — A new modulator of MDR in cancer chemotherapy

SummaryThe MDR modulator, OC144-093, is a potential candidate for use in cancer therapy and exhibits potent biological activityin vitro andin vivo when combined with anticancer agents such as paclitaxel [1]. Its inhibitory interaction with P-glycoprotein (Pgp), the mdr1 gene product and a mechanistic participant in multidrug resistance [2], underlies its activity as a modulator of MDR. Having previously shown that OC144-093 is not a substrate for CYP3A [4] we first examined the effects of OC144-093 on paclitaxel metabolismin vitro. Using human liver microsomes, we have demonstrated that OC144-093 inhibited the CYP3A mediated metabolism of paclitaxel at high concentrations only (Ki=39.8±5.1 μM, n=3). Pharmacokinetic results also show that an oral dose of OC144-093, co-administered with paclitaxel caused negligible disturbance of the pharmacokinetic profile for paclitaxel when injected intravenously. In contrast, AUC values were elevated approximately 1.5-fold in all groups treated orally with paclitaxel and OC144-993. Cmax was enhanced approximately 2-fold in the co-dosed group. These characteristics are consistent with Pgp blockade in the gut enhancing oral bioavailability. Elimination properties of paclitaxel were affected only upon multiple dosing of OC144-093. These results warrant the further clinical assessment of OC144-093 as an MDR reversing agent.

CHAPTER 4.2 – Designing therapeutically optimized liposomal anticancer delivery systems: Lessons from conventional liposomes

This chapter describes liposomal anticancer drugs, and the dilemma faced when designing optimized liposomal anticancer drugs. Recent technological advances in the production, stability, and biological properties of liposomes have greatly increased the degree of sophistication that can be designed into liposomes to improve their therapeutic/toxic activity profile. Investigators designing liposomal anticancer drug carrier technology must contend with a dilemma of opposing goals in the different biological compartments that the formulations experience. Because the uptake of liposomes in tumors appears to be passive, extended circulation times appear necessary to facilitate liposome accumulation. It follows that drug leakage from the liposomes must be minimized to avoid toxicities associated with free drug. The inability to differentially control drug release rates in the plasma compartment and disease site is perhaps the most significant limitation of presently available liposomes. The processes that dictate the fate of liposomes after intravenous injection has increased and help to design formulations that will optimize the selectivity of action for encapsulated agents. Inclusion of additional components into conventional liposomes can now be done on the basis of extensive data describing the in vivo behavior of various liposome types.

Assessment of the involvement of CYP3A in the vitro metabolism of a new modulator of MDR in cancer chemotherapy, OC144-193, by human liver microsomes

SummaryThe novel substituted imidazole compound, OC144–093 exhibits potent biological activity in vitro and in vivo for reversal of P-glycoprotein (PgP) based resistance to cancer chemotherapy [1]. Its mechanism of action relies upon its inhibitory interaction with the mdr1 gene product, a known mediator of multidrug resistance (MDR) [2]. Overlapping substrate specificities and tissue distribution of cytochrome P450 3A (CYP3A) and PgP indicate the potential for drug-drug interactions when modulator and anticancer agent are coadministered [3]. We have examined the metabolism of OC144–093 in vitro using human liver microsomes to determine if CYP3A is involved. Our results show that OC144–093 is converted to one major metabolite (M1) in human liver microsomes which was identified by LCMS to be the O-deethylated derivative. Km and Vmax for O-deethylation were determined as 3.96±0.67 μM and 32.08±9.73 pmol/mg protein/min, respectively (n=3). Correlation studies conducted in a panel of human livers phenotyped for specific P450 enzyme activity showed a significant relationship between M1 formation and the activity of CYP2C9, CYP2B6, CYP2E1 and CYP3A4. Treatment of microsomes with carbon monoxide gas inhibited M1 formation and diethyldithiocarbamate and ketoconazole (>3 μM), non-specific CYP inhibitors, gave IC50 values of 124.4±21.6 μM and 25.3±3.2 μM respectively for the inhibition of O-deethylation also implicating the involvement of CYP enzymes. Specific CYP inhibitors of CYP3A4 were essentially non-inhibitory to M1 formation. We can conclude therefore that OC144–093 is not extensively metabolised in human liver microsomes although conversion to its O-deethylated derivative does occur. Our data indicates that this conversion is not mediated by CYP3A4.

Mastoparan is a membranolytic anti-cancer peptide that works synergistically with gemcitabine in a mouse model of mammary carcinoma

Anti-cancer peptides (ACPs) are small cationic and hydrophobic peptides that are more toxic to cancer cells than normal cells. ACPs kill cancer cells by causing irreparable membrane damage and cell lysis, or by inducing apoptosis. Direct-acting ACPs do not bind to a unique receptor, but are rather attracted to several different molecules on the surface of cancer cells. Here we report that an amidated wasp venom peptide, Mastoparan, exhibited potent anti-cancer activities toward leukemia (IC50~8-9.2μM), myeloma (IC50~11μM), and breast cancer cells (IC50~20-24μM), including multidrug resistant and slow growing cancer cells. Importantly, the potency and mechanism of cancer cell killing was related to the amidation of the C-terminal carboxyl group. Mastoparan was less toxic to normal cells than it was to cancer cells (e.g., IC50 to PBMC=48μM). Mastoparan killed cancer cells by a lytic mechanism. Moreover, Mastoparan enhanced etoposide-induced cell death in vitro. Our data also suggest that Mastoparan and gemcitabine work synergistically in a mouse model of mammary carcinoma. Collectively, these data demonstrate that Mastoparan is a broad-spectrum, direct-acting ACP that warrants additional study as a new therapeutic agent for the treatment of various cancers.

The Development of Liposomes for Enhanced Delivery of Chemotherapeutics to Tumors

Liposomes were first discovered in the 1960s by Bangham, who observed that ordered spherical membranes spontaneously formed when dried lipids were hydrated into aqueous solutions (1). The potential utility of these carrier systems for the delivery of therapeutic agents to disease sites has continually evolved since this initial observation. In order for liposomes to be considered as a viable pharmaceutical-delivery system, many issues needed to be resolved including efficient drug encapsulation, liposome stability, and the production of homogeneous liposome populations. These obstacles were overcome in the 1980s with the development of various liposome-production procedures including extrusion (2), dialysis (3), homogenization (4), and dehydration/rehydration techniques (5). Currently, the most commonly used process is extrusion due to its ease of usage, simplicity, speed, and reproducibility.

A model approach for assessing liposome targeting in vivo

AbstractA procedure intended to facilitate characterization and optimization of liposomes designed for in vivo targeting to sites outside the blood compartment is described. The approach is based on a model consisting of administering streptavidin liposomes intravenously to mice previously injected intraperitoneally or intratumorally with biotinylated multilamellar vesicles (MLVs). In vivo targeting, therefore, is measured through the evaluation of streptavidin liposome accumulation and distribution within the site of MLV injection. In vitro studies suggested that optimal binding would be achieved when streptavidin liposomes, prepared with 2 mol% polyethylene glycol-modified phospholipids (PEG-SA-LUV), were incubated with multilamellar vesicles incorporating biotinoylaminohexanoyl DSPE (BAH-MLV). In vivo targeting studies were focused in three areas. The least stringent test determined PEG-SA-LUV binding to biotinylated MLVs in the peritoneal cavity after ip administration and resulted in a 17-fold increa...

Human gene therapy: principles and modern advances

Abstract The treatment of human diseases by gene therapy is logical, given the fact that it is possible to introduce nucleic acids into human cells. More importantly, genetic manipulations are able to elicit relevant biological responses. Despite the potential for gene therapy in the future, numerous technological problems still exist that limit its acceptance as a mainstream therapeutic option. Every problem, however, has a logical and practical solution. We focus this review on issues related to the fundamental problems of gene transfer. Given the progress and potential impact of this therapeutic technology on the treatment of a large number of human diseases, it is anticipated that gene therapy will revolutionize the way in which we treat disease in the next century.

Abstract 2902: The effectiveness of autophagy inhibition in sensitizing triple-negative breast cancer cells to chemotherapy

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAnnIntroduction: High recurrence rates, drug resistance after initial response to chemotherapy, and overall poor prognosis along with the limited treatment options make triple-negative breast cancers (TNBCs) a major clinical challenge. Autophagy, an evolutionary conserved degradation and recycling process, has been shown to function as an adaptive survival response to chemotherapy. Previous studies have indicated higher expression of autophagy markers in TNBCs compared to other breast cancer subtypes, as well as their dependence on autophagy for survival. Our laboratory has also shown in xenograft models an enhanced effectiveness of chemotherapy for the treatment of TNBC when given in combination with autophagy inhibition (AI). These results support TNBCs as a good candidate for AI to improve efficacy of existing therapeutic regimens. However, currently available agents for AI in cancer patients have limited effectiveness, and development of more potent autophagy inhibitors (AIs) is underway.nnObjective: Develop and test new tools for more potent AI in vivo.nnExperimental Design: We are employing in vitro models using TNBC lines MDA-MB-231 and SUM159PT, as well as their derivatives R8 and R75, resistant to Epirubicin (EPI) and other anthracyclines. We are evaluating effects of various AIs, including lysosomotropic agents HCQ and lys05, siRNAs and shRNAs targeting autophagy-related (Atg) proteins, and small molecule inhibitors (under development) of ATG4B protein. In vivo xenograft mouse models of MDA-MB-231 and R8 are being used to evaluate the effects of combinatorial therapy with EPI and AI.nnMethods: For the assessment of autophagy levels before and after AI we used autophagy flux (degradative completion of autophagy) assays. We evaluated the effects of chemotherapy alone and in combination with AIs on parent and resistant sub-lines by assessing their proliferation. For in vivo studies, TNBC cells are injected subcutaneously in Rag2M mice. Treatment with EPI, AI, or their combination is administered after tumor formation. Treatment efficacy is evaluated by tumor volume measurements; tumors are also assessed for the expression of autophagy markers.nnResults: Our in vitro experiments showed enhanced cytotoxicity of lys05 compared to HCQ either alone or in combination with EPI. However, the use of lys05 in vivo gives contradictory results and requires further evaluation. AI targeting ATG4B, using shRNA-inducible monoclonal cell lines derived from MDA-MB-231 cells and novel small molecule inhibitors of ATG4B, significantly affected cancer cell proliferation in vitro, and is currently being investigated in vivo.nnConclusion: Novel approaches to AI may serve as useful tools to assess the effects of AI in vitro and in vivo. Our preliminary results suggest that more potent AIs may improve the effectiveness of treatment of TNBC.nnSupported by CIHR GPG102167 and CIHR/AVON OBC127216.nnCitation Format: Svetlana Bortnik, Suganthi Chittaranjan, Jing Xu, Wieslawa H. Dragowska, Jianghong An, Adrienne Kyle, Nancy E. Go, Lubomir Vezenkov, Courtney Choutka, Amy Leung, Suzana Kovacic, Damien Bosc, Karen Gelmon, Marcel Bally, Steven Jones, Robert Young, Sharon Gorski. The effectiveness of autophagy inhibition in sensitizing triple-negative breast cancer cells to chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2902. doi:10.1158/1538-7445.AM2015-2902

Abstract 1684: Autophagy inhibition as an effective strategy for sensitizing triple-negative breast cancer cells to chemotherapy.

Introduction: Triple-negative breast cancer (TNBC), defined by a lack of expression of the estrogen, progesterone and HER-2 receptors, remains a major clinical challenge due to higher recurrence rates and poorer prognosis compared to other subtypes of breast cancer. Tumors that initially respond to chemotherapy - the core treatment option for the patients with an advanced disease - eventually develop resistance. New therapeutic options are urgently required for TNBC. Autophagy, a lysosome-mediated degradation and recycling process, has been shown to function as an adaptive survival response during chemotherapy. Previous studies in other cancer subtypes have indicated that autophagy inhibition can restore chemotherapeutic sensitivity and enhance treatment response. Objective: Generate proof-of-principle evidence for autophagy inhibition as an effective treatment strategy for TNBC. Experimental Design: We are employing in vitro models using TNBC lines MDA-MB-231 and SUM159PT, as well as their derivative lines (R8 and R75, respectively) resistant to Epirubicin (EPI) and other anthracyclines. In vivo xenograft mouse models of MDA-MB-231 and R8 are being used to evaluate the effects of combinatorial therapy with EPI and autophagy inhibitor hydroxychloroquine (HCQ). Methods: We assessed levels of autophagy in TNBC cell lines treated with EPI, developed EPI- resistant sub-lines, and compared basal autophagy levels in parental and resistant lines, using autophagy flux (degradative completion of autophagy) assays. We evaluated the effects of chemotherapy alone and in combination with autophagy inhibitors (HCQ or siRNAs targeting autophagy-related (Atg) proteins) on both parent and resistant sub-lines by assessing their viability. For in vivo studies, MDA-MB-231 cells were injected subcutaneously in Rag2M mice. After tumor formation, mice were treated with EPI, HCQ or their combination, and treatment efficacy was evaluated by tumor volume measurements. Autophagy levels in tumors were also assessed. Results: TNBC cells demonstrated increased autophagy in response to EPI treatment in vitro and in vivo. EPI- resistant lines showed at least 1.5 fold increased basal autophagy levels compared to their parental lines suggesting a possible adaptive role for autophagy in development of chemoresistance. Knock-down of Atg proteins by siRNA dramatically reduced the viability of EPI-resistant sub-lines, which indicates dependence of drug-resistant cells on autophagy for survival. Resistance of MDA-MB-231-R8 cells to EPI was reverted by autophagy inhibition in vitro. Combination of EPI with HCQ in vivo showed an enhanced tumor response to treatment compared to monotherapy with EPI. Additional in vivo studies are in progress. Conclusion: Our preliminary results suggest that autophagy inhibition may be an effective strategy for the treatment of chemo-refractory TNBC cells. Citation Format: Svetlana Bortnik, Suganthi Chittaranjan, Wieslawa H. Dragowska, Namal Abeysundara, Amy Chen, Lindsay DeVorkin, Nancy Dos Santos, Nancy Erro Go, Amy Leung, Dana Masin, Maria Rizza, Dita Strutt, Sherry Weppler, Jing Xu, Hong Yan, Karen Gelmon, Donald Yapp, Marcel Bally, Sharon M. Gorski. Autophagy inhibition as an effective strategy for sensitizing triple-negative breast cancer cells to chemotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1684. doi:10.1158/1538-7445.AM2013-1684