Raquel De Souza

Polymeric drug delivery systems for localized cancer chemotherapy

Cancer has become one of the most difficult health challenges of our time, accounting for millions of deaths yearly. Systemic chemotherapy is the most common therapeutic approach; however, considerable limitations exist including toxicities to healthy tissues and low achievable drug concentrations at tumor sites. More than 85% of human cancers are solid tumors, which can greatly benefit from localized delivery. This approach allows for high drug concentrations at the target site, lower systemic toxicity, and extended drug exposure which may be beneficial for cell cycle-specific drugs. Polymers have been widely considered in the development of localized delivery systems. This review focuses on both natural and synthetic biodegradable polymers that have been explored for localized chemotherapy, exploring their advantages, disadvantages, and clinical potential while citing examples of their use in pre-clinical development.

Biocompatibility of injectable chitosan-phospholipid implant systems.

Injectable biomaterials are desirable therapeutic platforms due to minimal invasiveness and improved patient compliance, and are applicable in such areas as compound delivery and tissue engineering. The present work examined the biocompatibility of injectable blends composed of chitosan, phospholipid and lauric aldehyde (PoLi(gel)-LA) or lauric chloride (PoLi(gel)-LCl). In vitro cytotoxicity was evaluated in L929 and HeLa cell lines. Both blends resulted in acceptable biocompatibility, although greater cell viability was seen with PoLi(gel)-LA. In vivo biocompatibility was investigated in healthy CD-1 mice. Subcutaneous injection of the PoLi(gel)-LA blend caused no local or systemic toxicities over a four-week period while the PoLi(gel)-LCl caused immediate local toxicity. Mice injected intraperitoneally with PoLi(gel)-LA did not show physical or behavioural alterations, and body weight changes did not differ from control animals. Furthermore, histological examination of spleen and liver showed unaltered morphology. Interleukin-6 levels in mice injected with PoLi(gel)-LA did not differ from levels of control animals (6.91+/-3.61 pg/mL versus 6.92+/-5.02 pg/mL, respectively). Biodegradation occurred progressively, with 7.4+/-5.02% of the original injected mass remaining after four weeks. Results obtained herein establish the biocompatibility of PoLi(gel)-LA and indicate its potential for use in various localized therapeutic applications.

Chemotherapy Dosing Schedule Influences Drug Resistance Development in Ovarian Cancer

Drug resistance leads to chemotherapy failure and is responsible for the death of a great majority of patients with metastatic, late-stage ovarian cancer. The present study addressed whether changes in the chemotherapy dosing schedule affect the development, further worsening, or circumvention of drug resistance in chemosensitive and chemoresistant ovarian cancer. Severe combined immunodeficient mice bearing HeyA8 and HeyA8-MDR xenografts were treated with docetaxel intermittently (1×/wk or 3×/wk) or continuously for 21 days. Tumor mRNA expression of genes implicated in docetaxel resistance was measured by quantitative real-time-PCR. Analyzed genes included those encoding for the drug efflux transporters mdr1 and mrp7 and for molecules that interfere with or overcome the effects of docetaxel, including β-tubulinIII, actinin4, stathmin1, bcl2, rpn2, thoredoxin, and akt2. In both models, continuous docetaxel resulted in greater antitumor efficacy than 1×/wk or 3×/wk dosing and did not induce upregulation of any analyzed genes. Once weekly dosing caused upregulation of various drug resistance–related genes, especially in chemoresistant xenografts. More frequent, 3×/wk dosing diminished this effect, although levels of various genes were higher than for continuous chemotherapy. Drug efflux transporter expression was further examined by Western blotting, confirming that intermittent, but not continuous, docetaxel induced significant upregulation. Overall, our results show that the presence and length of treatment-free intervals contribute to the development of drug resistance. Elimination of these intervals by continuous dosing resulted in superior antitumor efficacy and prevented drug resistance induction in chemosensitive and chemoresistant disease. These results encourage the clinical implementation of continuous chemotherapy to overcome and/or prevent drug resistance in newly diagnosed and recurrent, refractory ovarian cancer. Mol Cancer Ther; 10(7); 1289–99. ©2011 AACR.

Cyclophosphamide-Mediated Tumor Priming for Enhanced Delivery and Antitumor Activity of HER2-Targeted Liposomal Doxorubicin (MM-302)

Given the bulky nature of nanotherapeutics relative to small molecules, it is hypothesized that effective tumor delivery and penetration are critical barriers to their clinical activity. HER2-targeted PEGylated liposomal doxorubicin (MM-302, HER2-tPLD) is an antibody–liposomal drug conjugate designed to deliver doxorubicin to HER2-overexpressing cancer cells while limiting uptake into nontarget cells. In this work, we demonstrate that the administration and appropriate dose sequencing of cyclophosphamide can improve subsequent MM-302 delivery and enhance antitumor activity in preclinical models without negatively affecting nontarget tissues, such as the heart and skin. We demonstrate that this effect is critically dependent on the timing of cyclophosphamide administration. Furthermore, the effect was found to be unique to cyclophosphamide and related analogues, and not shared by other agents, such as taxanes or eribulin, under the conditions examined. Analysis of the cyclophosphamide-treated tumors suggests that the mechanism for improved MM-302 delivery involves the induction of tumor cell apoptosis, reduction of overall tumor cell density, substantial lowering of interstitial fluid pressure, and increasing vascular perfusion. The novel dosing strategy for cyclophosphamide described herein is readily translatable to standard clinical regimens, represents a potentially significant advance in addressing the drug delivery challenge, and may have broad applicability for nanomedicines. This work formed the basis for clinical evaluation of cyclophosphamide for improving liposome deposition as part of an ongoing phase I clinical trial of MM-302 in HER2-positive metastatic breast cancer. Mol Cancer Ther; 14(9); 2060–71. ©2015 AACR.

Continuous Docetaxel Chemotherapy Improves Therapeutic Efficacy in Murine Models of Ovarian Cancer

Ovarian cancer is known as the silent killer for being asymptomatic until late stages. Current first-line treatment consists of debulking surgery followed by i.v. chemotherapeutics administered intermittently, which leads to insufficient drug concentrations at tumor sites, accelerated tumor proliferation rates, and drug resistance, resulting in an overall median survival of only 2 to 4 years. For these reasons, more effective treatment strategies must be developed. We have investigated a localized, continuous chemotherapy approach in tumor models of human and murine ovarian cancers using the antineoplastic agent docetaxel. We show here that continuous docetaxel therapy is considerably more efficacious than intermittent therapy, resulting in a greater decrease in tumor burden and ascites fluid accumulation. Immunohistochemical analyses show that continuous chemotherapy abrogates tumor cell proliferation and angiogenesis to the tumor microenvironment, leading to greater tumor cell death than intermittent docetaxel therapy. Overall, our results show greater therapeutic advantages of continuous over intermittent chemotherapy in the treatment of ovarian cancer. Mol Cancer Ther; 9(6); 1820–30. ©2010 AACR.

Combination drug delivery strategy for the treatment of multidrug resistant ovarian cancer.

The onset of multidrug resistance (MDR) in ovarian cancer is one of the main causes of treatment failure and low survival rates. Inadequate drug exposure and treatment-free periods due to intermittent chemotherapy select for cancer cells overexpressing drug efflux transporters, resulting in resistant disease. The present study examines the sustained administration of the chemotherapeutic agent docetaxel (DTX) alone and in combination with cepharanthine (CEP), a potent drug efflux transporter inhibitor. DTX and CEP were delivered via the intraperitoneal route in a sustained manner using an injectable polymer-lipid formulation. In vitro, the combination strategy resulted in significantly (p < 0.05) more apoptosis, greater intracellular accumulation of DTX, and lower DTX efflux in ovarian cancer cells showing the MDR phenotype. In vivo, sustained treatment with DTX and CEP showed significantly greater (p < 0.05) tumor inhibition (91 ± 4%) in a murine model of multidrug resistant ovarian cancer compared to sustained DTX treatment (76 ± 6%) and was more than twice as efficacious as intermittent DTX treatment. Overall findings from these studies highlight the impact of sustained delivery of monotherapy and combination therapy in the management of refractory ovarian cancer displaying the MDR phenotype.

An injectable depot system for sustained intraperitoneal chemotherapy of ovarian cancer results in favorable drug distribution at the whole body, peritoneal and intratumoral levels.

The current study characterizes the impact of docetaxel (DTX) distribution on efficacy following sustained intraperitoneal (IP) chemotherapy in murine models of ovarian cancer. A polymer-lipid biodegradable depot (PoLigel) was used to deliver DTX in a sustained manner over 21-days following IP administration. Distribution and efficacy studies were carried out in SCID mice bearing SKOV3 IP solid tumors or C57BL/6 mice with ID8 IP ascites fluid. In addition, a subcutaneous (SC) SKOV3 model was used to determine whether systemic drug levels that result from IP administration of the PoLigel influence antitumor efficacy. Immunostained IP and SC SKOV3 tumor sections were used to study cell death, intratumoral drug distribution and tumor penetration. Sustained concentrations of DTX were observed in plasma, tissue, tumor and ascites over the entire study period. Drug accumulation was several fold greater in tumors and ascites when compared to plasma levels. Sustained chemotherapy resulted in significant reduction in tumor burden and ascites volume. IP tumors showed greater cell death compared to the SC tumors as seen by higher TUNEL and caspase-3 expression. At the intratumoral level, DTX distributed more towards the core of IP tumors compared to the SC tumors. Tumor penetration of drug from nearest blood vessel was 1.5 fold greater in the IP tumors than the SC tumors. Overall, favorable drug distribution at the whole-body, peritoneal and intratumoral levels in combination with local and systemic sustained drug exposure contribute to the high efficacy observed. These results encourage the clinical use of IP sustained chemotherapy for ovarian cancer.

A multimodal nano agent for image-guided cancer surgery

Intraoperative imaging technologies including computed tomography and fluorescence optical imaging are becoming routine tools in the cancer surgery operating room. They constitute an enabling platform for high performance surgical resections that assure local control while minimizing morbidity. New contrast agents that can increase the sensitivity and visualization power of existing intraoperative imaging techniques will further enhance their clinical benefit. We report here the development, detection and visualization of a dual-modality computed tomography and near-infrared fluorescence nano liposomal agent (CF800) in multiple preclinical animal models of cancer. We describe the successful application of this agent for combined preoperative computed tomography based three-dimensional surgical planning and intraoperative target mapping (>200 Hounsfield Units enhancement), as well as near-infrared fluorescence guided resection (>5-fold tumor-to-background ratio). These results strongly support the clinical advancement of this agent for image-guided surgery with potential to improve lesion localization, margin delineation and metastatic lymph node detection.

Effects of Doxorubicin Delivery Systems and Mild Hyperthermia on Tissue Penetration in 3D Cell Culture Models of Ovarian Cancer Residual Disease

Current chemotherapy strategies for second-line treatment of relapsed ovarian cancer are unable to effectively treat residual disease post-cytoreduction. The findings presented herein suggest that tissue penetration of drug is not only an issue for large, unresectable tumors, but also for invisible, microscopic lesions. The present study sought to investigate the potential of a block copolymer micelle (BCM) formulation, which may reduce toxicities of doxorubicin (DOX) in a similar way to pegylated liposomal doxorubicin (PLD, Doxil/Caelyx), while enhancing penetration into tumor tissue and improving intratumoral availability of drug. To achieve this goal, 50 nm-sized BCMs capable of high DOX encapsulation (BCM-DOX) at drug levels ranging from 2 to 7.6 mg/mL were formulated using an ultrafiltration technique. BCM-DOX was evaluated in 2D and 3D cell culture of the human ovarian cancer cell lines HEYA8, OV-90, and SKOV3. Additionally, the current study examines the impact of mild hyperthermia (MHT) on the cytotoxicity of DOX. The BCM-DOX formulation fulfilled the goal of controlling drug release while providing up to 9-fold greater cell monolayer cytotoxicity in comparison to PLD. In 3D cell culture, using multicellular tumor spheroids (MCTS) as a model of residual disease postsurgery, BCM-DOX achieved the benefits of an extended release formulation of DOX and resulted in improvements in drug accumulation over PLD, while yielding drug levels approaching that achievable by exposure to DOX alone. In comparison to PLD, this translated into superior MCTS growth inhibition in the short term and comparable inhibition in the long term. Overall, although MHT appeared to enhance drug accumulation in HEYA8 MCTS treated with BCM-DOX and DOX alone in the short term, improved growth inhibition of MCTS by MHT was not observed after 48 h of drug treatment. Evaluation of BCM-DOX in comparison to PLD as well as the effects of MHT is warranted in vivo.

The impact of sustained and intermittent docetaxel chemotherapy regimens on cognition and neural morphology in healthy mice

RationaleA subset of cancer survivors demonstrates impairments in cognition long after chemotherapy completion. At present, it is unclear whether these changes are due to direct neurotoxic effects of chemotherapy.ObjectivesThis study examined the impact of variable docetaxel (DTX) chemotherapy dosing on brain DTX exposure via analyses of neural morphology and changes in cognition.MethodsMale CD-1 mice were treated with DTX either intermittently (8 mg/kg i.p. weekly) or via a sustained delivery system (DTX-PoLigel), which continuously releases DTX. Both groups received total DTX doses of 32 mg/kg. Mice were assessed on the novel object recognition (NOR) task and the Morris water maze (MWM) shortly after treatment.ResultsPost-treatment behavioral testing demonstrated impaired NOR in mice treated with either dosing schedule relative to controls. No differences were observed between groups in MWM training and initial testing, though control mice performed better than chance while DTX-treated mice did not. Appreciable amounts of DTX were found in the brain after both treatment regimens. DTX treatment did not significantly increase levels of apoptosis within the CNS. However, some elevation in neural autophagy was observed following DTX treatment. Analysis of astrocytic activation demonstrated that intermittent DTX treatment resulted in an elevation of GFAP-positive astrocytes for 48 h after administration. Sustained chemotherapy demonstrated prolonged but lower levels of astrocyte activation over 9 days following implantation.ConclusionsDTX treatment induced cognitive impairment shortly after treatment. Further, these findings suggest an association between DTX dosing, neurotoxicity, and cognitive effects.