Sasmit Sarangi

Cholesterol-tethered platinum II-based supramolecular nanoparticle increases antitumor efficacy and reduces nephrotoxicity

Nanoscale drug delivery vehicles have been harnessed extensively as carriers for cancer chemotherapeutics. However, traditional pharmaceutical approaches for nanoformulation have been a challenge with molecules that exhibit incompatible physicochemical properties, such as platinum-based chemotherapeutics. Here we propose a paradigm based on rational design of active molecules that facilitate supramolecular assembly in the nanoscale dimension. Using cisplatin as a template, we describe the synthesis of a unique platinum (II) tethered to a cholesterol backbone via a unique monocarboxylato and O→Pt coordination environment that facilitates nanoparticle assembly with a fixed ratio of phosphatidylcholine and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000]. The nanoparticles formed exhibit lower IC50 values compared with carboplatin or cisplatin in vitro, and are active in cisplatin-resistant conditions. Additionally, the nanoparticles exhibit significantly enhanced in vivo antitumor efficacy in murine 4T1 breast cancer and in K-RasLSL/+/Ptenfl/fl ovarian cancer models with decreased systemic- and nephro-toxicity. Our results indicate that integrating rational drug design and supramolecular nanochemistry can emerge as a powerful strategy for drug development. Furthermore, given that platinum-based chemotherapeutics form the frontline therapy for a broad range of cancers, the increased efficacy and toxicity profile indicate the constructed nanostructure could translate into a next-generation platinum-based agent in the clinics.

Sequential application of a cytotoxic nanoparticle and a PI3K inhibitor enhances antitumor efficacy

Nanomedicines that preferentially deploy cytotoxic agents to tumors and molecular targeted therapeutics that inhibit specific aberrant oncogenic drivers are emerging as the new paradigm for the management of cancer. While combination therapies are a mainstay of cancer chemotherapy, few studies have addressed the combination of nanomedicines and molecular targeted therapeutics. Furthermore, limited knowledge exists on the impact of sequencing of such therapeutics and nanomedicines on the antitumor outcome. Here, we engineered a supramolecular cis-platinum nanoparticle, which induced apoptosis in breast cancer cells but also elicited prosurvival signaling via an EGF receptor/phosphoinositide 3-kinase (PI3K) pathway. A combination of mathematical modeling and in vitro and in vivo validation using a pharmacologic inhibitor of PI3K, PI828, demonstrate that administration of PI828 following treatment with the supramolecular cis-platinum nanoparticle results in enhanced antitumor efficacy in breast cancer as compared with when the sequence is reversed or when the two treatments are administered simultaneously. This study addresses, for the first time, the impact of drug sequencing in the case of a combination of a nanomedicine and a targeted therapeutic. Furthermore, our results indicate that a rational combination of cis-platinum nanoparticles and a PI3K-targeted therapeutic can emerge as a potential therapy for breast cancer.

P2Y12 receptor inhibition augments cytotoxic effects of cisplatin in breast cancer

Expression of P2Y12 receptors has been documented in some cancer cell lines like C6 glioma, renal carcinoma and colon carcinoma. However, its direct role in altering response to chemotherapeutics has not been studied. In this study, we characterize the expression of P2Y12 receptor in breast cancer cell lines and evaluate its role in enhancing the cytotoxic effects of cisplatin. We observed a significant upregulation in P2Y12 expression in 4T1 breast cancer cell line with cisplatin treatment. Co-administration of P2Y12 inhibitor with cisplatin resulted in significantly higher cytotoxic response in 4T1 cancer cell line. This was mediated by HIF1α-dependent upregulation of cellular apoptotic pathways. These findings identify P2Y12 receptor as a potential target to enhance antitumor efficacy of chemotherapeutic agents like cisplatin.

Anti-platelet agents augment cisplatin nanoparticle cytotoxicity by enhancing tumor vasculature permeability and drug delivery.

Tumor vasculature is critically dependent on platelet mediated hemostasis and disruption of the same can augment delivery of nano-formulation based chemotherapeutic agents which depend on enhanced permeability and retention for tumor penetration. Here, we evaluated the role of Clopidogrel, a well-known inhibitor of platelet aggregation, in potentiating the tumor cytotoxicity of cisplatin nano-formulation in a murine breast cancer model. In vivo studies in murine syngeneic 4T1 breast cancer model showed a significant greater penetration of macromolecular fluorescent nanoparticles after clopidogrel pretreatment. Compared to self-assembling cisplatin nanoparticles (SACNs), combination therapy with clopidogrel and SACN was associated with a 4 fold greater delivery of cisplatin to tumor tissue and a greater reduction in tumor growth as well as higher survival rate. Clopidogrel enhances therapeutic efficiency of novel cisplatin based nano-formulations agents by increasing tumor drug delivery and can be used as a potential targeting agent for novel nano-formulation based chemotherapeutics.

Precision medicine and personalized breast cancer: combination pertuzumab therapy

Trastuzumab (Herceptin), a monoclonal antibody directed against the human epidermal growth-factor receptor 2 (HER2), is the poster child for antibody-based targeted therapy in breast cancer. Pertuzumab, another humanized monoclonal antibody, binds to a different domain of HER2 and prevents the formation of HER2:HER3 dimers, which is the most potent heterodimer in the HER family. The combination of trastuzumab and pertuzumab has synergistic activity, and is associated with improved clinical outcomes. The US Food and Drug Administration (FDA) approved pertuzumab in combination with trastuzumab-based chemotherapy originally as first-line therapy for metastatic HER2-positive breast cancer in 2012, and more recently as neoadjuvant therapy for localized disease in 2013. Pertuzumab is the first neoadjuvant drug to receive accelerated approval by the FDA based on pathological complete response as the primary end point. In this article, we review the mechanism of action, pharmacokinetics, clinical efficacy, safety, and current role of pertuzumab in the management of breast cancer, as well as ongoing clinical trials and future directions regarding the utility of pertuzumab as a personalized therapeutic option for HER2-positive breast cancer. In the coming years, we anticipate increased utilization of neoadjuvant trials for drug development, biomarker discovery, and validation, and envision conduct of personalized breast cancer clinics in which therapies will be routinely selected based on genetic alterations in the tumor. Regardless of the targeted therapy combinations employed based on tumor genomic profile, trastuzumab and pertuzumab will likely continue to form the backbone of the personalized regimen for HER2-positive breast cancer.

PEGylated liposomal Gemcitabine: Insights into a potential breast cancer therapeutic

PurposeNanoencapsulation of chemotherapeutics is an established method to target breast tumors and has been shown to enhance the efficacy of therapy in various animal models. During the past two decades, the nucleoside analog Gemcitabine has been under investigation to treat both recalcitrant and localized breast cancer, often in combination with other chemotherapeutics. In this study, we investigated the chemotherapeutic efficacy of a novel Gemcitabine-encapsulated liposome previously formulated by our group, GemPo, on both sensitive (4T1) and recalcitrant (MDA-MB-231) breast cancer cell lines.MethodsGemcitabine free drug and liposomal Gemcitabine were compared both in vitro and in vivo using breast cancer models.ResultsWe demonstrated that GemPo differently hindered the growth, survival and migration of breast cancer cells, according to their drug sensitivities. Specifically, whereas GemPo was a more potent cytotoxic and apoptotic agent in sensitive breast cancer cells, it more potently inhibited cell migration in the resistant cell line. However, GemPo still acted as a more potent inhibitor of migration, in comparison with free Gemcitabine, irrespective of cell sensitivity. Administration of GemPo in a 4T1-bearing mouse model inhibited tumor growth while increasing mice survival, as compared with free Gemcitabine and a vehicle control. Interestingly, the inclusion of a mitotic inhibitor, Paclitaxel, synergized only with free Gemcitabine in this model, yet was as effective as GemPo alone. However, inclusion of Paclitaxel with GemPo significantly improved mouse survival.ConclusionsOur study is the first to demonstrate the pleiotropic effects of Gemcitabine and Gemcitabine-loaded nanoparticles in breast cancer, and opens the door for a novel treatment for breast cancer patients.

The Evolving Role of Circulating Tumor Cells in the Personalized Management of Breast Cancer: From Enumeration to Molecular Characterization

Circulating tumor cells (CTCs) represent tumor cells in the blood stream dislodged from the primary tumor. The presence of CTCs in the bloodstream provides a unique opportunity to sample cancer tissue by means of a relatively less-invasive “liquid biopsy.” Over the past decade, there has been a tremendous increase in the amount of research examining the potential clinical utility of CTCs in the management of cancer. A number of techniques to refine the sensitivity and range of CTC assays are also in development. In this article, we review the recent developments in the current and potential clinical applications of CTCs in breast cancer. CTC enumeration already has an established role as a prognostic biomarker in metastatic breast cancer, whereas molecular characterization of CTCs can serve as a potential predictive biomarker for therapy selection, pharmacodynamic evaluation, and identification of novel actionable targets for novel therapies. The role of CTCs in breast cancer screening and detection of recurrence is currently limited. Further development in techniques will be pivotal in enhancing the broad applicability of CTCs and advancing the field of personalized breast cancer therapy.

Abstract 2892: Enhancing anti cancer activity of PI103, a dual PI3K/mTOR inhibitor, by designing a self assembled Nanoformulation

The phosphoinositide 3-kinase (PI3K)/ mammalian target of rapamycin (mTOR) pathway plays a pivotal role in the growth and survival of various cancers. Small molecule inhibitors targeting PI3K/AKT/mTOR pathway (eg. Wortmannin, LY294002, PI-103, PI828) are promising anti cancer agents. Major drawbacks of these drugs are their poor water solubility and off-target toxicity. Nanoscale drug delivery systems have been harnessed as drug carriers to target tumours through enhanced permeation and retention effect. In this study, we developed a nanoformulation of PI103, a potent dual PI3K/mTOR inhibitor, to improve delivery and reduce side effects. PI103 was encapsulated in a homogeneous self-assembled mixture of phosphatidylcholine and cholesterol. DSPE-PEG protective shell was introduced to increase its circulation time. Thin-film-hydration technique followed by extrusion through 200 nm porous membranes developed nanoparticles of 110-150 nm size with 0.15-0.20 mM drug loading. Release kinetics study confirmed the stability of this formulation in PBS for several days as well as a sustained release property in in-vitro Cell Lysate system. In-vitro cell proliferation assay demonstrated improved cytotoxicity of nanoformulated PI103 in murine breast cancer cell line 4T1 and K-ras upregulated and PTEN suppressed ovarian cancer model cells. Fluorescent Activated Cell Sorting (FACS) analysis with FITC tagged Annexin-V, an early apoptosis marker, showed increased apoptosis with the nanoformulation than the free drug. Western blot analysis expressed more potent inhibition of PI3 kinase/Akt pathway by the nanoformulated PI103. These results indicate that nanoformulation can enhance the potential of PI103. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2892. doi:1538-7445.AM2012-2892

Abstract C187: Posttreatment with PI3-kinase inhibitor enhances the chemotherapeutic effect of cisplatin nanoparticles in breast cancer.

Nanoformulations of chemotherapeutic drugs are increasingly being studied for their potential to improve efficacy and safety. However, limited studies have been done on sequential dosing of nanochemotherapeutics and signal transduction inhibitors. In this study, pre-treatment and post-treatment dosing of PI-3 kinase inhibitor PI828 was used with cisplatin nanoparticles in murine breast cancer model. In vitro cell proliferation assay showed an enhanced cytotoxic capacity of cisplatin nanoparticle when used in combination with PI828. Moreover, post-treatment with the signal transduction inhibitors was found to be significantly more effective than pre-treatment. Western blot analysis showed a time dependent upregulation of phospho- AKT expression after administration of cisplatin nanoparticles, which was efficiently suppressed by PI828 in the post-treatment schedule. The upregulation of piAKT dependent signalling was mediated by both an EGFR dependent downstream activation of PI3 kinase pathway and an increased nuclear transcription of Akt gene. Post treatment with EGFR inhibitor (Erlotinib) showed similar enhancement in cytotoxicity of cisplatin nanoparticle. In a 4T1 syngenic murine cancer in-vivo model, post-treatment with PI828 and Erlotinib following cisplatin nanoparticles treatment significantly suppressed tumor growth as compared to the pre-treatment or nanoparticles alone. These results indicate that sequence of administration of signal transduction inhibitors can impact the outcome of treatment with cisplatin nanotherapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C187.

Abstract B221: Clopidogrel augments cisplatin cytotoxicity by enhancing delivery and direct receptor mediated effects.

Clopidogrel, a well known P2Y12 (purinergic receptor) inhibitor is widely used for its role in preventing thrombosis by platelet aggregation inhibition. Tumor vasculature is critically dependent on platelet mediated hemostasis and disruption of the same can promote its leakiness. This can increase the intra-tumoral delivery of chemotherapeutic nanoformulations, which depend on the Endothelial permeability and retention (EPR) effect. Also, Expression of P2Y12 receptors has been documented in some cancer cell lines like C6 glioma, kidney carcinoma and colon carcinoma. However, its direct role in altering response to chemotherapeutics has not been studied. In this project we evaluated the role of P2Y12 receptor inhibitors (Clopidogrel and 2-Methylthioadenosine 5-Monophosphate) in potentiating the tumor cytotoxicity of cisplatin based chemotherapeutics in vitro and in vivo. Western blot studies in 4T1 breast cancer cell lines showed a low basal expression of P2Y12 receptors with significant up regulation by cisplatin treatment in a time and dose dependent fashion. Co-administration of P2Y12 inhibitor (MeSAMP) with cisplatin resulted in significantly increased upregulation of caspase dependent apoptotic pathway when compared to cisplatin alone. This was mediated by downstream inhibition of PI3K-pAKT-mTOR pathway by the P2Y12 inhibitor. In-vitro cell proliferation assay showed that co-administration of 2-MeSAMP significantly reduced the IC-50 of cisplatin from 3.9 μM to 1.8μM. In-vivo studies in murine syngenic 4T1 breast cancer model showed significantly decreased tumour growth and increased survival in treatment groups using combination of clopidogrel and cisplatin (free and nanoformulation forms). This was more pronounced with cisplatin nano-formulation combination therapy. Further analysis of tumour tissue using Inductive Coupled Plasma-spectrophotometry confirmed that co-treatment with clopidogrel increased the delivery of free and nanoformulation form of cisplatin to tumor tissue by 1.8 and 4 folds respectively. Taken together our study findings suggest that clopidogrel can enhance therapeutic efficiency of cytotoxic agents by increasing delivery and by direct receptor level effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B221.