Biodegradable, injectable depot formulations for long-term controlled drug release have improved therapy for a number of drug molecules and led to over a dozen highly successful pharmaceutical products. Until now, success has been limited to several small molecules and peptides, although remarkable improvements have been accomplished in some of these cases. For example, twice-a-year depot injections with leuprolide are available compared to the once-a-day injection of the solution dosage form. Injectable depots are typically prepared by encapsulation of the drug in poly(lactic-co-glycolic acid) (PLGA), a polymer that is used in children every day as a resorbable suture material, and therefore, highly biocompatible. PLGAs remain today as one of the few “real world” biodegradable synthetic biomaterials used in US FDA-approved parenteral long-acting-release (LAR) products. Despite their success, there remain critical barriers to the more widespread use of PLGA LAR products, particularly for delivery of more peptides and other large molecular drugs, namely proteins. In this review, we describe key concepts in the development of injectable PLGA controlled-release depots for peptides and proteins, and then use this information to identify key issues impeding greater widespread use of PLGA depots for this class of drugs. Finally, we examine important approaches, particularly those developed in our research laboratory, toward overcoming these barriers to advance commercial LAR development.

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A biomimetic approach to organic solvent-free microencapsulation of proteins based on the self-healing capacity of poly (DL)-lactic-co-glycolic acid (PLGA) microspheres containing glycosaminoglycan-like biopolymers (BPs), was examined. To screen BPs, aqueous solutions of BP [high molecular weight dextran sulfate (HDS), low molecular weight dextran sulfate (LDS), chondroitin sulfate (CS), heparin (HP), hyaluronic acid (HA), chitosan (CH)] and model protein lysozyme (LYZ) were combined in different molar and mass ratios, at 37 °C and pH 7. The BP-PLGA microspheres (20–63 µm) were prepared by a double water-oil-water emulsion method with a range of BP content, and trehalose and MgCO3 to control microclimate pH and to create percolating pores for protein. Biomimetic active self-encapsulation (ASE) of proteins [LYZ, vascular endothelial growth factor165 (VEGF) and fibroblast growth factor (FgF-20)] was accomplished by incubating blank BP-PLGA microspheres in low concentration protein solutions at ~24 °C, for 48 h. Pore closure was induced at 42.5 °C under mild agitation for 42 h. Formulation parameters of BP-PLGA microspheres and loading conditions were studied to optimize protein loading and subsequent release. LDS and HP were found to bind >95% LYZ at BP:LYZ >0.125 w/w, whereas HDS and CS bound > 80% LYZ at BP:LYZ of 0.25–1 and < 0.33, respectively. HA-PLGA microspheres were found to be not ideal for obtaining high protein loading (>2% w/w of LYZ). Sulfated BP-PLGA microspheres were capable of loading LYZ (~2–7 % w/w), VEGF (~ 4% w/w), and FgF-20 (~2% w/w) with high efficiency. Protein loading was found to be dependent on the loading solution concentration, with higher protein loading obtained at higher loading solution concentration within the range investigated. Loading also increased with content of sulfated BP in microspheres. Release kinetics of proteins was evaluated in-vitro with complete release media replacement. Rate and extent of release were found to depend upon volume of release (with non-sink conditions observed < 5ml release volume for ~18mg loaded BP-PLGA microspheres), ionic strength of release media and loading solution concentration. HDS-PLGA formulations were identified as having ideal loading and release characteristics. These optimal microspheres released ~ 73–80 % of the encapsulated LYZ over 60 days, with > 90 % of protein being enzymatically active. Nearly 72% of immunoreactive VEGF was similarly released over 42 days, without significant losses in heparin binding affinity in the release medium.

Triple negative breast cancer (TNBC) (ER-, PR-, Her2-), constituting 10-20% of all breast cancers, is a heterogeneous disease with limited treatment options and poor prognosis. TNBCs exhibit rapid progression with the duration of response to first line palliative chemotherapy typically less than 12 weeks, and overall five year survival of patients with metastatic TNBC of 22%. The cancer stem cell (CSC) model provides an attractive explanation for relapse of TNBC after primary therapy since these cells demonstrate resistance to conventional chemotherapy. CSCs which survive primary treatments, such as docetaxel, may self-renew and differentiate into the heterogeneous tumor bulk resulting in local recurrence and distant metastasis. Docetaxel has been demonstrated to not only fail to eliminate CSCs but expands this population in preclinical models. Further, docetaxel increases circulating IL-6 in patients following therapy, a cytokine reported to expand breast CSCs. Therefore, we sought to combine docetaxel with a small molecule CSC inhibitor capable of reducing IL-6 production (sulforaphane, SF) for the effective treatment of TNBCs. Our results in vitro demonstrate that docetaxel treatment (5 nM) increases the proportion of CSCs in TNBC cell lines (SUM149 and SUM159) as evident by flow cytometry analysis using the ALDEFLUOR assay (70.6±22.0%) and cells which are CD44+/CD24-/EpCAM+ (2.9 fold). Mammosphere formation assay reveals 1 nM docetaxel increases secondary sphere formation rate by 75.8±29%. As determined by ELISA, 5 nM docetaxel treatment for 72 hours induces 3.5 fold increase in IL-6 production. Conversely, SF (2.5 μM) selectively reduces the proportion of ALDEFLUOR positive cells (51.5±15.0%) and mammosphere formation (39.2±3.8%) while reducing IL-6 (55.6±5.0%) through regulation of NF-kB activity. In combination docetaxel and SF synergize to effectively reduce bulk cell line proliferation (combination index range 1-0.093). Further, SF prevents docetaxel mediated CSC expansion and IL-6 production. Using a mouse xenograft model docetaxel (10 mg/kg weekly) reduces tumor growth of established tumors by 83.2±6.0% whereas SF (50 mg/kg daily) inhibits primary tumor growth by 37.4±14.6%. In addition, secondary reimplantation assays with limiting dilution analysis reveals docetaxel increases the frequency of the tumor initiating CSCs (1/1514 control cells vs 1/330 docetaxel treated cells) while SF reduces the frequency to 1/3181 cells. In vivo, the combination of docetaxel and SF exhibits a greater reduction in primary tumor volume (92.5±2.1% reduction relative to control), and synergistically inhibit the CSC population (1 in 4245 cells). These results suggest that SF mediated inhibition of breast CSCs and IL-6 provide a scientific rationale for using this agent in combination with docetaxel for TNBC. Citation Format: Joseph P. Burnett, Ronack B. Shah, Hayley J. Paholak, Sean P. McDermott, Yasuhiro Tsume, Max W. Wicha, Duxin Sun. Combination of docetaxel with sulforaphane synergistically inhibits triple negative breast cancer and cancer stem cells. [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 4076. doi:10.1158/1538-7445.AM2015-4076

Background: In both breast cancer patients and healthy women, we have previously demonstrated that select neutrophils found in breast cancer patients as opposed to healthy women are cytotoxic to breast cancer cell lines. (Granot Z et al. Cancer Cell. 2010) This work stemmed from our prior research in murine breast cancer models indicating that primary breast tumors can mobilize select neutrophils, termed Tumor Entrained Neutrophils (“TENS”); these entrained neutrophils have the unique capacity to inhibit metastatic seeding in the lung through cell-kill mechanisms. (Granot Z et al. Cancer Cell. 2010) In this study, we evaluated the relationship between select chemokines and neutrophil cytotoxicity in breast cancer patients versus healthy volunteers. Methods: Neutrophils were purified from the blood of 75 randomly selected newly diagnosed pre-operative breast cancer patients without evidence of metastatic disease, and 47 healthy female volunteers with no history of cancer. Cytotoxicity was evaluated by incubating neutrophils with luciferase labeled MDA-MB-231 cells. Luciferase activity was measured as a reflection of% cytotoxicity. Serum was also isolated from breast cancer patients and healthy volunteers. Based on prior experiments, we used the Millipore® Milliplex Human Cytokine Plex Kit to evaluate Il-1Ra, MCP-1 and TNFa in our serum samples in 50/75 of the cancer patients and 25/47 controls. We used multiple linear regression to develop a model to predict cytotoxicity as a function of the chemokines. Results: In comparison to healthy volunteers whose mean neutrophil cytotoxicity to MDA-MB-231 cells was 6.5%, pre-operative breast cancer patients demonstrated a mean neutrophil cytotoxicity of 12.7%, p&lt;0.0001. We then evaluated the serum chemokine levels of 50 of the breast cancer patients; 31/50 had high neutrophil cytotoxicity (&gt;10%), and 19/50 had low neutrophil cytotoxicity (&lt;10%). We compared the serum chemokine levels in the patients to the levels found in the 25 controls. Using a multiple linear regression model, we found that the levels of these three chemokines are associated with cytotoxicity (R2 = 0.126, p = 0.022). An ANOVA decomposition of the model suggested that Il.1RA was the most predictive (p = 0.018) followed by MCP.1 (p = 0.088) and TNF.alpha (p = 0.245). Conclusion: Our work demonstrates the cytotoxic role of select neutrophils in the peripheral blood of breast cancer patients as contrasted with neutrophils from healthy women. We further demonstrate that select chemokines appear to be correlated with neutrophil cytotoxicity. We are currently evaluating the prognostic and therapeutic roles of cytotoxic neutrophils and their related chemokines in breast cancer patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-01-07.