Design of BET Inhibitor Bottlebrush Prodrugs with Superior Efficacy and Devoid of Systemic Toxicities.

Prodrugs engineered for preferential activation in diseased versus normal tissues offer immense potential to improve the therapeutic indexes (TIs) of preclinical and clinical-stage active pharmaceutical ingredients that either cannot be developed otherwise or whose efficacy or tolerability it is highly desirable to improve. Such approaches, however, often suffer from trial-and-error design, precluding predictive synthesis and optimization. Here, using bromodomain and extra-terminal (BET) protein inhibitors (BETi)-a class of epigenetic regulators with proven anticancer potential but clinical development hindered in large part by narrow TIs-we introduce a macromolecular prodrug platform that overcomes these challenges. Through tuning of traceless linkers appended to a "bottlebrush prodrug" scaffold, we demonstrate correlation of in vitro prodrug activation kinetics with in vivo tumor pharmacokinetics, enabling the predictive design of novel BETi prodrugs with enhanced antitumor efficacies and devoid of dose-limiting toxicities in a syngeneic triple-negative breast cancer murine model. This work may have immediate clinical implications, introducing a platform for predictive prodrug design and potentially overcoming hurdles in drug development.

Reduction of liver fibrosis by rationally designed macromolecular telmisartan prodrugs.

At present there are no drugs for the treatment of chronic liver fibrosis that have been approved by the Food and Drug administration of the United States. Telmisartan, a small-molecule antihypertensive drug, displays antifibrotic activity, but its clinical use is limited because it causes systemic hypotension. Here, we report the scalable and convergent synthesis of macromolecular telmisartan prodrugs optimized for preferential release in diseased liver tissue. We optimized the release of active telmisartan in fibrotic liver to be depot-like (that is, a constant therapeutic concentration) through the molecular design of telmisartan brush-arm star polymers, and show that these lead to improved efficacy and to the avoidance of dose-limiting hypotension in both metabolically and chemically induced mouse models of hepatic fibrosis, as determined by histopathology, enzyme levels in the liver, intact-tissue protein markers, hepatocyte necrosis protection, and gene-expression analyses. In rats and dogs, the prodrugs are retained long-term in liver tissue and have a well-tolerated safety profile. Our findings support the further development of telmisartan prodrugs that enable infrequent dosing in the treatment of liver fibrosis.

Publisher Correction: Reduction of liver fibrosis by rationally designed macromolecular telmisartan prodrugs.

In the version of this Article originally published, the author Peter Blume-Jensen was not denoted as a corresponding author; this has now been amended and the author's email address has been added. The 'Correspondence and requests for materials' statement was similarly affected and has now been updated with the author's initials 'P.B-J.'

PEGylated squalenoyl-gemcitabine nanoparticles for the treatment of glioblastoma.

New treatments for glioblastoma multiforme (GBM) are desperately needed, as GBM prognosis remains poor, mainly due to treatment resistance, poor distribution of therapeutics in the tumor tissue, and fast metabolism of chemotherapeutic drugs in the brain extracellular space. Convection-enhanced delivery (CED) of nanoparticles (NPs) has been shown to improve the delivery of chemotherapeutic drugs to the tumor bed, providing sustained release, and enhancing survival of animals with intracranial tumors. Here we administered gemcitabine, a nucleoside analog used as a first line treatment for a wide variety of extracranial solid tumors, within squalene-based NPs using CED, to overcome the above-mentioned challenges of GBM treatment. Small percentages of poly(ethylene) glycol (PEG) dramatically enhanced the distribution of squalene-gemcitabine nanoparticles (SQ-Gem NPs) in healthy animals and tumor-bearing animals after administration by CED. When tested in an orthotopic model of GBM, SQ-Gem-PEG NPs demonstrated significantly improved therapeutic efficacy compared to free gemcitabine, both as a chemotherapeutic drug and as a radiosensitizer. Furthermore, MR contrast agents were incorporated into the SQ-Gem-PEG NP formulation, providing a way to non-invasively track the NPs during infusion.

Distribution of polymer nanoparticles by convection-enhanced delivery to brain tumors.

Glioblastoma multiforme (GBM) is a fatal brain tumor characterized by infiltration beyond the margins of the main tumor mass and local recurrence after surgery. The blood-brain barrier (BBB) poses the most significant hurdle to brain tumor treatment. Convection-enhanced delivery (CED) allows for local administration of agents, overcoming the restrictions of the BBB. Recently, polymer nanoparticles have been demonstrated to penetrate readily through the healthy brain when delivered by CED, and size has been shown to be a critical factor for nanoparticle penetration. Because these brain-penetrating nanoparticles (BPNPs) have high potential for treatment of intracranial tumors since they offer the potential for cell targeting and controlled drug release after administration, here we investigated the intratumoral CED infusions of PLGA BPNPs in animals bearing either U87 or RG2 intracranial tumors. We demonstrate that the overall volume of distribution of these BPNPs was similar to that observed in healthy brains; however, the presence of tumors resulted in asymmetric and heterogeneous distribution patterns, with substantial leakage into the peritumoral tissue. Together, our results suggest that CED of BPNPs should be optimized by accounting for tumor geometry, in terms of location, size and presence of necrotic regions, to determine the ideal infusion site and parameters for individual tumors.

Systemic delivery of blood-brain barrier-targeted polymeric nanoparticles enhances delivery to brain tissue.

Delivery of therapeutic agents to the central nervous system is a significant challenge, hindering progress in the treatment of diseases such as glioblastoma. Due to the presence of the blood-brain barrier (BBB), therapeutic agents do not readily transverse the brain endothelium to enter the parenchyma. Previous reports suggest that surface modification of polymer nanoparticles (NPs) can improve their ability to cross the BBB, but it is unclear whether the observed enhancements in transport are large enough to enhance therapy. In this study, we synthesized two degradable polymer NP systems surface-modified with ligands previously suggested to improve BBB transport, and tested their ability to cross the BBB after intravenous injection in mice. All the NP preparations were able to cross the BBB, although generally in low amounts (<0.5% of the injected dose), which was consistent with prior reports. One NP produced significantly higher brain uptake (∼0.8% of the injected dose): a block copolymer of polylactic acid and hyperbranched polyglycerol, surface modified with adenosine (PLA-HPG-Ad). PLA-HPG-Ad NPs provided controlled release of camptothecin, killing U87 glioma cells in culture. When administered intravenously in mice with intracranial U87 tumors, they failed to increase survival. These results suggest that enhancing NP transport across the BBB does not necessarily yield proportional pharmacological effects.

A holistic approach to targeting disease with polymeric nanoparticles.

The primary goal of nanomedicine is to improve clinical outcomes. To this end, targeted nanoparticles are engineered to reduce non-productive distribution while improving diagnostic and therapeutic efficacy. Paradoxically, as this field has matured, the notion of targeting has been minimized to the concept of increasing the affinity of a nanoparticle for its target. This Opinion article outlines a holistic view of nanoparticle targeting, in which the route of administration, molecular characteristics and temporal control of the nanoparticles are potential design variables that must be considered simultaneously. This comprehensive vision for nanoparticle targeting will facilitate the integration of nanomedicines into clinical practice.

The effect of inflammatory cell-derived MCP-1 loss on neuronal survival during chronic neuroinflammation.

Intracranial implants elicit neurodegeneration via the foreign body response (FBR) that includes BBB leakage, macrophage/microglia accumulation, and reactive astrogliosis, in addition to neuronal degradation that limit their useful lifespan. Previously, monocyte chemoattractant protein 1 (MCP-1, also CCL2), which plays an important role in monocyte recruitment and propagation of inflammation, was shown to be critical for various aspects of the FBR in a tissue-specific manner. However, participation of MCP-1 in the brain FBR has not been evaluated. Here we examined the FBR to intracortical silicon implants in MCP-1 KO mice at 1, 2, and 8 weeks after implantation. MCP-1 KO mice had a diminished FBR compared to WT mice, characterized by reductions in BBB leakage, macrophage/microglia accumulation, and astrogliosis, and an increased neuronal density. Moreover, pharmacological inhibition of MCP-1 in implant-bearing WT mice maintained the increased neuronal density. To elucidate the relative contribution of microglia and macrophages, bone marrow chimeras were generated between MCP-1 KO and WT mice. Increased neuronal density was observed only in MCP-1 knockout mice transplanted with MCP-1 knockout marrow, which indicates that resident cells in the brain are major contributors. We hypothesized that these improvements are the result of a phenotypic switch of the macrophages/microglia polarization state, which we confirmed using PCR for common activation markers. Our observations suggest that MCP-1 influences neuronal loss, which is integral to the progression of neurological disorders like Alzheimer's and Parkinson disease, via BBB leakage and macrophage polarization.

The effect of hyperbranched polyglycerol coatings on drug delivery using degradable polymer nanoparticles.

A key attribute for nanoparticles (NPs) that are used in medicine is the ability to avoid rapid uptake by phagocytic cells in the liver and other tissues. Poly(ethylene glycol) (PEG) coatings has been the gold standard in this regard for several decades. Here, we examined hyperbranched polyglycerols (HPG) as an alternate coating on NPs. In earlier work, HPG was modified with amines and subsequently conjugated to poly(lactic acid) (PLA), but that approach compromised the ability of HPG to resist non-specific adsorption of biomolecules. Instead, we synthesized a copolymer of PLA-HPG by a one-step esterification. NPs were produced from a single emulsion using PLA-HPG: fluorescent dye or the anti-tumor agent camptothecin (CPT) were encapsulated at high efficiency in the NPs. PLA-HPG NPs were quantitatively compared to PLA-PEG NPs, produced using approaches that have been extensively optimized for drug delivery in humans. Despite being similar in size, drug release profile and in vitro cytotoxicity, the PLA-HPG NPs showed significantly longer blood circulation and significantly less liver accumulation than PLA-PEG. CPT-loaded PLA-HPG NPs showed higher stability in suspension and better therapeutic effectiveness against tumors in vivo than CPT-loaded PLA-PEG NPs. Our results suggest that HPG is superior to PEG as a surface coating for NPs in drug delivery.

Optimization of stability, encapsulation, release, and cross-priming of tumor antigen-containing PLGA nanoparticles.

PURPOSE: In order to investigate Poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NP) as potential vehicles for efficient tumor antigen (TA) delivery to dendritic cells (DC), this study aimed to optimize encapsulation/release kinetics before determining immunogenicity of antigen-containing NP. METHODS: Various techniques were used to liberate TA from cell lines. Single (gp100) and multiple (B16-tumor lysate containing gp100) antigens were encapsulated within differing molecular weight PLGA co-polymers. Differences in morphology, encapsulation/release and biologic potency were studied. Findings were adopted to encapsulate fresh tumor lysate from patients with advanced tumors and compare stimulation of tumor infiltrating lymphocytes (TIL) against that achieved by soluble lysate. RESULTS: Four cycles of freeze-thaw + 15 s sonication resulted in antigen-rich lysates without the need for toxic detergents or protease inhibitors. The 80 KDa polymer resulted in maximal release of payload and favorable production of immunostimulatory IL-2 and IFN-γ. NP-mediated antigen delivery led to increased IFN-γ and decreased immunoinhibitory IL-10 synthesis when compared to soluble lysate. CONCLUSIONS: Four cycles of freeze-thaw followed by 15 s sonication is the ideal technique to obtain complex TA for encapsulation. The 80 KDa polymer has the most promising combination of release kinetics and biologic potency. Encapsulated antigens are immunogenic and evoke favorable TIL-mediated anti-tumor responses.

Convection-enhanced delivery of camptothecin-loaded polymer nanoparticles for treatment of intracranial tumors.

Direct delivery of chemotherapy agents to the brain via degradable polymer delivery systems-such as Gliadel®-is a clinically proven method for treatment of glioblastoma multiforme, but there are important limitations with the current technology-including the requirement for surgery, profound local tissue toxicity, and limitations in diffusional penetration of agents-that limit its application and effectiveness. Here, we demonstrate another technique for direct, controlled delivery of chemotherapy to the brain that provides therapeutic benefit with fewer limitations. In our new approach, camptothecin (CPT)-loaded poly(lacticco-glycolic acid) (PLGA) nanoparticles are infused via convection-enhanced delivery (CED) to a stereotactically defined location in the brain, allowing simultaneous control of location, spread, and duration of drug release. To test this approach, CPT-PLGA nanoparticles (~100 nm in diameter) were synthesized with 25% drug loading. When these nanoparticles were incubated in culture with 9L gliosarcoma cells, the IC50 of CPT-PLGA nanoparticles was 0.04 µM, compared to 0.3 µM for CPT alone. CPT-PLGA nanoparticles stereotactically delivered by CED improved survival in rats with intracranial 9L tumors: the median survival for rats treated with CPT-PLGA nanoparticles (22 days) was significantly longer than unloaded nanoparticles (15 days) and free CPT infusion (17 days). CPT-PLGA nanoparticle treatment also produced significantly more long-term survivors (30% of animals were free of disease at 60 days) than any other treatment. CPT was present in tissues harvested up to 53 days post-infusion, indicating prolonged residence at the local site of administration. These are the first results to demonstrate the effectiveness of combining polymer-controlled release nanoparticles with CED in treating fatal intracranial tumors.

Polymer nanoparticles containing tumor lysates as antigen delivery vehicles for dendritic cell-based antitumor immunotherapy.

Encapsulation of tumor-associated antigens in polymer nanoparticles (NP) is a promising approach to enhance efficiency of antigen delivery for anti-tumor vaccines. Head and neck squamous carcinoma (HNSCC) cell lines were initially used to generate tumor-associated antigens (TAA)-containing poly (lactic-co-glycolic acid) (PLGA) NP; encapsulation efficiency and release kinetics were profiled. Findings were adopted to entrap fresh tumor lysate from five patients with advanced HNSCC. To test the hypothesis that NP enhance antigen presentation, dendritic cell (DC) produced from patient blood monocyte precursors were loaded with either the un-encapsulated or NP-encapsulated versions of tumor lysates. These were used to stimulate freshly-isolated autologous CD8+ T cells. In four of five patients, anti-tumor CD8+ T cells showed significantly increased immunostimulatory IFN-γ (p=0.071) or decreased immmunoinhibitory IL-10 production (p=0.0004) associated with NP-mediated antigen delivery. The observations represent an enabling step in the production of clinically-translatable, inexpensive, highly-efficient, and personalized polymer-based immunotherapy for solid organ malignancies. FROM THE CLINICAL EDITOR: Enhancing the antigen presentation may be a viable approach to increase the efficiency of tumor cell directed cytotoxicity via immune mechanisms. This study presents an example for this using head and neck cancer cell lines and nanotechnology-based encapsulated antigen presentation to dendritic cells. The observed CD8+ T-cell response was significantly enhanced. This method may pave the way to a highly efficient cancer cell elimination method with minimal to no toxicity.

Intravaginal gene silencing using biodegradable polymer nanoparticles densely loaded with small-interfering RNA.

Vaginal instillation of small-interfering RNA (siRNA) using liposomes has led to silencing of endogenous genes in the genital tract and protection against challenge from infectious disease. Although siRNA lipoplexes are easily formulated, several of the most effective transfection agents available commercially may be toxic to the mucosal epithelia and none are able to provide controlled or sustained release. Here, we demonstrate an alternative approach using nanoparticles composed entirely of FDA-approved materials. To render these materials effective for gene silencing, we developed novel approaches to load them with high amounts of siRNA. A single dose of siRNA-loaded nanoparticles to the mouse female reproductive tract caused efficient and sustained gene silencing. Knockdown of gene expression was observed proximal (in the vaginal lumen) and distal (in the uterine horns) to the site of topical delivery. In addition, nanoparticles penetrated deep into the epithelial tissue. This is the first report demonstrating that biodegradable polymer nanoparticles are effective delivery vehicles for siRNA to the vaginal mucosa.

Biodegradable meshes printed with extracellular matrix proteins support micropatterned hepatocyte cultures.

The spatial organization of cells of different phenotypes is an important and often defining determinant of tissue function. In tissue engineering, which attempts to rebuild functional tissues from cellular and synthetic components, spatial patterning of cells onto biomaterials is likely to be equally important. We have printed combinatorial arrays of extracellular matrix (ECM) and screened them for attachment by HepG2 hepatocytes, LX-2 hepatic stellate cells, primary portal fibroblasts, and bovine aortic endothelial cells-cells selected as representative phenotypes found in adult liver. Differential cell attachment to the underlying matrix proteins allowed us to establish two-dimensional co-cultures of HepG2 with these non-parenchymal cell types. These general approaches were then translated to tissue engineering scaffolds where deposition of ECM proteins onto electrospun polylactide meshes resulted in patterned HepG2 cultures. We observed that the spatial organization of fibronectin deposits influenced HepG2 attachment and the establishment of co-cultures on our arrays. These micropatterned co-culture systems should serve as valuable tools for studying the soluble and insoluble signals involved in liver development, function, and disease.