Highly penetrative,drug-loaded nanocarriers improve treatment of glioblastoma

Current therapy for glioblastoma multiforme is insufficient, with nearly universal recurrence. Available drug therapies are unsuccessful because they fail to penetrate through the region of the brain containing tumor cells and they fail to kill the cells most responsible for tumor development and therapy resistance, brain cancer stem cells (BCSCs). To address these challenges, we combined two major advances in technology: (i) brain-penetrating polymeric nanoparticles that can be loaded with drugs and are optimized for intracranial convection-enhanced delivery and (ii) repurposed compounds, previously used in Food and Drug Administration-approved products, which were identified through library screening to target BCSCs. Using fluorescence imaging and positron emission tomography, we demonstrate that brain-penetrating nanoparticles can be delivered to large intracranial volumes in both rats and pigs. We identified several agents (from Food and Drug Administration-approved products) that potently inhibit proliferation and self-renewal of BCSCs. When loaded into brain-penetrating nanoparticles and administered by convection-enhanced delivery, one of these agents, dithiazanine iodide, significantly increased survival in rats bearing BCSC-derived xenografts. This unique approach to controlled delivery in the brain should have a significant impact on treatment of glioblastoma multiforme and suggests previously undescribed routes for drug and gene delivery to treat other diseases of the central nervous system.Of the ∼40,000 people diagnosed with primary brain tumors in the United States each year, an estimated 15,000 have glioblastoma multiforme (GBM), a World Health Organization grade IV malignant glioma (1). Despite considerable research efforts, the prognosis for GBM remains poor: median survival with standard-of-care therapy (surgery, systemic chemotherapy with temozolomide, and radiation) is 14.6 mo (2) and 5-y survival is 9.8% (3), with the vast majority of GBMs recurring within 2 cm of the original tumor focus (4). Histopathologically, GBM is characterized by its infiltrative nature and cellular heterogeneity, leading to a number of challenges that must be overcome by any presumptive therapy.The blood–brain barrier (BBB) is a major obstacle to treating GBM (5). It is estimated that over 98% of small-molecule drugs and ∼100% of large-molecule drugs or genes do not cross the BBB (6). Delivery of chemotherapeutics to the brain can be potentially achieved by using nanocarriers engineered for receptor-mediated transport across the BBB (7, 8), but the percentage of i.v. administered particles that enter the brain is low. It is not yet clear whether sufficient quantities of drug can be delivered by systemically administered nanoparticles to make this a useful method for treating tumors in the human brain. An alternate approach is to bypass the BBB: Clinical trials have demonstrated that the BBB can be bypassed with direct, locoregional delivery of therapeutic agents. For example, local implantation of a drug-loaded biodegradable polymer wafer (presently marketed as Gliadel), which slowly releases carmustine over a prolonged period, is a safe method for treating GBM. However, use of the Gliadel wafer results in only modest improvements in patient survival, typically 2 mo (9, 10). In prior work we showed that these wafers produce high interstitial drug concentrations in the tissue near the implant, but—because drugs move from the implant into the tissue by diffusion—penetration into tissue is limited to ∼1 mm, which could limit their efficacy (11, 12).We hypothesize that treatment of GBM can be improved by attention to three challenges: (i) enhancing the depth of penetration of locally delivered therapeutic agents, (ii) providing for long-term release of active agents, and (iii) delivering agents that are known to be effective against the cells that are most important in tumor recurrence. The first challenge can be addressed by convection-enhanced delivery (CED), in which agents are infused into the brain under a positive pressure gradient, creating bulk fluid movement in the brain interstitium (13). Recent clinical trials show that CED is safe and feasible (14–16), but CED alone is not sufficient to improve GBM treatment. For example, CED of a targeted toxin in aqueous suspension failed to show survival advantages over Gliadel wafers (14, 17). Although CED of drugs in solution results in increased penetration, most drugs have short half-lives in the brain and, as a result, they disappear soon after the infusion stops (17, 18). Loading of agents into nanocarriers—such as liposomes, micelles, dendrimers, or nanoparticles—can protect them from clearance. Significant progress has been made in CED of liposomes to the brain (19), although it is not clear that liposomes offer the advantage of long-term release. By contrast, CED of polymeric nanoparticles, such as nanoparticles made of poly(lactide-coglycolide) (PLGA), offers the possibility of controlled agent release. However, CED of PLGA nanoparticles, which are typically 100–200 nm in diameter, has been limited by the failure of particles to move by convection through the brain interstitial spaces (20–23), which are 38–64 nm in normal brain (24) and 7–100 nm in regions with tumor (25). Therefore, to overcome the first and second challenges, it is necessary to synthesize polymer nanocarriers that are much smaller than conventional particles and still capable of efficient drug loading and controlled release. We report here reliable methods for making PLGA nanoparticles with these characteristics.Drug developers have long been frustrated by the BBB, which severely limits the types of agents that can be tested for activity in the brain. We reasoned that creation of safe, versatile, brain-penetrating nanocarriers should enable direct testing of novel agents that address the complexity of GBM biology. For example, cells isolated from distinct regions of a given GBM bear grossly different expression signatures but seem to arise from a common progenitor (26): A small subpopulation of these progenitors drives tumor progression, promotes angiogenesis, and influences tumor cell migration (27–30). These cells have features of primitive neural stem cells and are called brain cancer stem cells (BCSCs) (29, 31–37). BCSCs, many of which are marked by CD133 (PROM1), are resistant to conventional drugs (28, 38), including carboplatin, cisplatin, paclitaxel, doxorubicin, vincristine, methotrexate, and temozolomide (39–42), as well as radiotherapy (29). These observations suggest that agents that affect BCSCs are more likely to lead to a cure for GBM (28, 38, 43, 44). Therefore, to illustrate the translational potential of brain-penetrating nanoparticles, we conducted a screen of ∼2,000 compounds that were previously used in Food and Drug Administration (FDA)-approved products for their ability to inhibit patient-derived BCSCs, encapsulated the best agents to emerge from the screen into brain-penetrating PLGA nanoparticles, and administered these nanocarriers by CED in a BCSC-derived xenograft model of GBM.     

A predictive microfluidic model of human glioblastoma to assess trafficking of blood–brain barrier-penetrant nanoparticles

The blood–brain barrier represents a significant challenge for the treatment of high-grade gliomas, and our understanding of drug transport across this critical biointerface remains limited. To advance preclinical therapeutic development for gliomas, there is an urgent need for predictive in vitro models with realistic blood–brain-barrier vasculature. Here, we report a vascularized human glioblastoma multiforme (GBM) model in a microfluidic device that accurately recapitulates brain tumor vasculature with self-assembled endothelial cells, astrocytes, and pericytes to investigate the transport of targeted nanotherapeutics across the blood–brain barrier and into GBM cells. Using modular layer-by-layer assembly, we functionalized the surface of nanoparticles with GBM-targeting motifs to improve trafficking to tumors. We directly compared nanoparticle transport in our in vitro platform with transport across mouse brain capillaries using intravital imaging, validating the ability of the platform to model in vivo blood–brain-barrier transport. We investigated the therapeutic potential of functionalized nanoparticles by encapsulating cisplatin and showed improved efficacy of these GBM-targeted nanoparticles both in vitro and in an in vivo orthotopic xenograft model. Our vascularized GBM model represents a significant biomaterials advance, enabling in-depth investigation of brain tumor vasculature and accelerating the development of targeted nanotherapeutics.

High-grade gliomas are the most common primary malignant brain tumors in adults (1). These include grade IV astrocytomas, commonly known as glioblastoma multiforme (GBM), which account for more than 50% of all primary brain cancers and have dismal prognoses, with a 5-y survival rate of less than 5% (2). Due to their infiltrative growth into the healthy brain tissue, surgery often fails to eradicate all tumor cells (3). While chemotherapy and radiation modestly improve median survival (4), most patients ultimately succumb to their tumors. This is primarily due to the presence of a highly selective and regulated endothelium between blood and brain parenchyma known as the blood–brain barrier (BBB) (5), which limits the entry of therapeutics into the brain tissue where tumors are located. The BBB, characterized by a unique cellular architecture of endothelial cells (ECs), pericytes (PCs), and astrocytes (ACs) (6, 7), displays up-regulated expression of junctional proteins and reduced paracellular and transcellular transports compared to other endothelia (8). While this barrier protects the brain from toxins and pathogens, it also severely restricts the transport of many therapeutics, as evidenced by the low cerebrospinal fluid (CSF)-to-plasma ratio of most chemotherapeutic agents (9). There is thus an important need to develop new delivery strategies to cross the BBB and target tumors, enabling sufficient drug exposure (10).Despite rigorous research efforts to develop effective therapies for high-grade gliomas, the majority of trialed therapeutics have failed to improve outcomes in the clinic, even though the agents in question are effective against tumor cells in preclinical models (11). This highlights the inability of current preclinical models to accurately predict the performance of therapeutics in human patients. To address these limitations, we developed an in vitro microfluidic model of vascularized GBM tumors embedded in a realistic human BBB vasculature. This BBB-GBM platform features brain microvascular networks (MVNs) in close contact with a GBM spheroid, recapitulating the infiltrative properties of gliomas observed in the clinic (12) and those of the brain tumor vasculature, with low permeability, small vessel diameter, and increased expression of relevant junctional and receptor proteins (7). This platform is well suited for quantifying vascular permeability of therapeutics and simultaneously investigating modes of transport across the BBB and into GBM tumor cells.There is strong rationale for developing therapeutic nanoparticles (NPs) for GBM and other brain tumors, as they can be used to deliver a diverse range of therapeutic agents and, with appropriate functionalization, can be designed to exploit active transport mechanisms across the BBB (13, 14). Liposomal NPs have been employed in the oncology clinic to improve drug half-life and decrease systemic toxicity (15), but, to date, no nanomedicines have been approved for therapeutic indications in brain tumors. We hypothesize that a realistic BBB-GBM model composed entirely of human cells can accelerate preclinical development of therapeutic NPs. Using our BBB-GBM model, we investigated the trafficking of layer-by-layer NPs (LbL-NPs) and ultimately designed a GBM-targeted NP. The LbL approach leverages electrostatic assembly to generate modular NP libraries with highly controlled architecture. We have used LbL-NPs to deliver a range of therapeutic cargos in preclinical tumor models (16, 17) and have recently demonstrated that liposomes functionalized with BBB-penetrating ligands improved drug delivery across the BBB to GBM tumors (18). Consistent with clinical data (19), we observed that the low-density lipoprotein receptor-related protein 1 (LRP1) was up-regulated in the vasculature near GBM spheroids in the BBB-GBM model and leveraged this information to design and iteratively test a library of NPs. We show that the incorporation of angiopep-2 (AP2) peptide moieties on the surface of LbL-NPs leads to increased BBB permeability near GBM tumors through LRP1-mediated transcytosis. With intravital imaging, we compared the vascular permeabilities of dextran and LbL-NPs in the BBB-GBM platform to those in mouse brain capillaries and validated the predictive potential of our in vitro model. Finally, we show the capability of the BBB-GBM platform to screen therapeutic NPs and predict in vivo efficacy, demonstrating improved efficacy of cisplatin (CDDP) when encapsulated in GBM-targeting LbL-NPs both in vitro and in vivo.     

Radionuclides as conditioning before stem cell transplantation.

Targeted radiotherapy using either radiolabeled monoclonal antibodies or bone-seeking radiopharmaceuticals can potentially result in delivery of high radiation doses to tumor or bone marrow with less toxicity to normal organs. High-dose radioimmunotherapy has produced encouraging results in lymphoma and leukemia. The use of alternative radionuclides and new combinations of radioimmunotherapy with chemotherapy may improve patient outcomes. Comparative trials will be necessary to confirm an improved therapeutic benefit with this approach.     

Engineering a lysosomal enzyme with a derivative of receptor-binding domain of apoE enables delivery across the blood–brain barrier

To realize the potential of large molecular weight substances to treat neurological disorders, novel approaches are required to surmount the blood–brain barrier (BBB). We investigated whether fusion of a receptor-binding peptide from apolipoprotein E (apoE) with a potentially therapeutic protein can bind to LDL receptors on the BBB and be transcytosed into the CNS. A lysosomal enzyme, α-L-iduronidase (IDUA), was used for biological and therapeutic evaluation in a mouse model of mucopolysaccharidosis (MPS) type I, one of the most common lysosomal storage disorders with CNS deficits. We identified two fusion candidates, IDUAe1 and IDUAe2, by in vitro screening, that exhibited desirable receptor-mediated binding, endocytosis, and transendothelial transport as well as appropriate lysosomal enzyme trafficking and biological function. Robust peripheral IDUAe1 or IDUAe2 generated by transient hepatic expression led to elevated enzyme levels in capillary-depleted, enzyme-deficient brain tissues and protein delivery into nonendothelium perivascular cells, neurons, and astrocytes within 2 d of treatment. Moreover, 5 mo after long-term delivery of moderate levels of IDUAe1 derived from maturing red blood cells, 2% to 3% of normal brain IDUA activities were obtained in MPS I mice, and IDUAe1 protein was detected in neurons and astrocytes throughout the brain. The therapeutic potential was demonstrated by normalization of brain glycosaminoglycan and β-hexosaminidase in MPS I mice 5 mo after moderate yet sustained delivery of IDUAe1. These findings provide a noninvasive and BBB-targeted procedure for the delivery of large-molecule therapeutic agents to treat neurological lysosomal storage disorders and potentially other diseases that involve the brain.     

Delivery of herpesvirus and adenovirus to nude rat intracerebral tumors after osmotic blood-brain barrier disruption.

The delivery of viral vectors to the brain for treatment of intracerebral tumors is most commonly accomplished by stereotaxic inoculation directly into the tumor. However, the small volume of distribution by inoculation may limit the efficacy of viral therapy of large or disseminated tumors. We have investigated mechanisms to increase vector delivery to intracerebral xenografts of human LX-1 small-cell lung carcinoma tumors in the nude rat. The distribution of Escherichia coli lacZ transgene expression from primary viral infection was assessed after delivery of recombinant virus by intratumor inoculation or intracarotid infusion with or without osmotic disruption of the blood-brain barrier (BBB). These studies used replication-compromised herpes simplex virus type 1 (HSV; vector RH105) and replication-defective adenovirus (AdRSVlacZ), which represent two of the most commonly proposed viral vectors for tumor therapy. Transvascular delivery of both viruses to intracerebral tumor was demonstrated when administered intraarterially (i.a.) after osmotic BBB disruption (n = 9 for adenovirus; n = 7 for HSV), while no virus infection was apparent after i.a. administration without BBB modification (n = 8 for adenovirus; n = 4 for HSV). The thymidine kinase-negative HSV vector infected clumps of tumor cells as a result of its ability to replicate selectively in dividing cells. Osmotic BBB disruption in combination with i.a. administration of viral vectors may offer a method of global delivery to treat disseminated brain tumors.     

Sustained Administration of Hormones Exploiting Nanoconfined Diffusion through Nanochannel Membranes

Implantable devices may provide a superior means for hormone delivery through maintaining serum levels within target therapeutic windows. Zero-order administration has been shown to reach an equilibrium with metabolic clearance, resulting in a constant serum concentration and bioavailability of released hormones. By exploiting surface-to-molecule interaction within nanochannel membranes, it is possible to achieve a long-term, constant diffusive release of agents from implantable reservoirs. In this study, we sought to demonstrate the controlled release of model hormones from a novel nanochannel system. We investigated the delivery of hormones through our nanochannel membrane over a period of 40 days. Levothyroxine, osteocalcin and testosterone were selected as representative hormones based on their different molecular properties and structures. The release mechanisms and transport behaviors of these hormones within 3, 5 and 40 nm channels were characterized. Results further supported the suitability of the nanochannels for sustained administration from implantable platforms.     

Vector-mediated delivery of 125I-labeled beta-amyloid peptide A beta 1-40 through the blood-brain barrier and binding to Alzheimer disease amyloid of the A beta 1-40/vector complex.

The brain amyloid of Alzheimer disease (AD) may potentially be imaged in patients with AD by using neuroimaging technology and a radiolabeled form of the 40-residue beta-amyloid peptide A beta 1-40 that is enabled to undergo transport through the brain capillary endothelial wall, which makes up the blood-brain barrier (BBB) in vivo. Transport of 125I-labeled A beta 1-40 (125I-A beta 1-40) through the BBB was found to be negligible by experiments with both an intravenous injection technique and an internal carotid artery perfusion method in anesthetized rats. In addition, 125I-A beta 1-40 was rapidly metabolized after either intravenous injection or internal carotid artery perfusion. BBB transport was increased and peripheral metabolism was decreased by conjugation of monobiotinylated 125I-A beta 1-40 to a vector-mediated drug delivery system, which consisted of a conjugate of streptavidin (SA) and the OX26 monoclonal antibody to the rat transferrin receptor, which undergoes receptor-mediated transcytosis through the BBB. The brain uptake, expressed as percent of injected dose delivered per gram of brain, of the 125I,bio-A beta 1-40/SA-OX26 conjugate was 0.15 +/- 0.01, a level that is 2-fold greater than the brain uptake of morphine. The binding of the 125I,bio-A beta 1-40/SA-OX26 conjugate to the amyloid of AD brain was demonstrated by both film and emulsion autoradiography performed on frozen sections of AD brain. Binding of the 125I,bio-A beta 1-40/SA-OX26 conjugate to the amyloid of AD brain was completely inhibited by high concentrations of unlabeled A beta 1-40. In conclusion, these studies show that BBB transport and access to amyloid within brain may be achieved by conjugation of A beta 1-40 to a vector-mediated BBB drug delivery system.     

Delivery of hexosaminidase A to the cerebrum after osmotic modification of the blood--brain barrier.

The present studies were undertaken to evaluate the possibility that hexosaminidase A, the enzyme deficient in Tay--Sachs disease, could be effectively delivered to brain. Previous studies from our laboratory have shown that hypertonic mannitol can be used to osmotically produce reversible disruption of the blood--brain barrier in animals (rat and dog) and man without significant neurotoxicity and that such barrier modification significantly increases the delivery of cytoreductive chemotherapy agents to selected areas of brain. By using the rat model of blood--brain barrier modification and radiolabeled enzyme, increased hexosaminidase A delivery to brain has been demonstrated in more than 85 animals. The time of injection of hexosaminidase A after blood--brain barrier disruption is critical for maximum delivery. Rapid (over 30 sec) intra-arterial administration of hexosaminidase A immediately after blood--brain barrier disruption resulted in a marked increase in enzyme delivery to the brain when compared with controls without prior barrier disruption. When the enzyme was administered 15-20 min after barrier disruption, approximately 50% less hexosaminidase A was delivered; when given 60-120 min after barrier modification, the amount delivered was the same as in control animals. This critical time course is very different than that seen in trials of low molecular weight chemotherapeutic agents (methotrexate and adriamycin). These preliminary studies suggest that hexosaminidase A can be delivered to the brain by blood--brain barrier modification and may be indicative of the potential for enzyme replacement in patients who hae Tay--Sachs disease.     

Peritoneal seeding following potentially curative resection of colonic carcinoma: Implications for adjuvant therapy

Adjuvant therapeutic strategies for colon cancer are based on the knowledge of tumor recurrence patterns following potentially curative resection. Innovative methods for regional delivery of chemotherapy to the liver and peritoneal surfaces are now available to complement systemic treatment. We reviewed clinical, reoperation, and autopsy series to determine the incidence of peritoneal seeding following colon cancer resection. The data suggest a 25–35 percent peritoneal failure rate among patients that recur, indicating that intraperitoneal chemotherapy is a sensible adjuvant approach. The theory behind intraperitoneal chemotherapy and potential complications is discussed. We suggest initiation of clinical trials combining systemic and intraperitoneal chemotherapy.     

Delivery of Drugs with Ultrasound

In this article we discuss the potential role of microbubbles, traditionally used as ultrasound contrast agents, for site-specific drug delivery. To reach this goal, microbubbles capable of carrying a drug payload are being developed. To ensure that these microbubbles reach sufficient local concentration at disease sites, specific targeting for diseased tissues can be accomplished using several strategies. These strategies rely on either the intrinsic properties of microbubble shells or conjugation of monoclonal antibodies or other ligands to these shells that recognize antigens expressed in regions of disease. Site-specific delivery of antiinflammatory, antineoplastic, and thrombolytic drugs with microbubbles can be further enhanced by the ability to locally destroy microbubbles within an acoustic field, thereby releasing drugs and improving drug efficacy without systemic adverse effects. In the case of thrombi, ultrasound-mediated microbubble destruction also may facilitate the process of clot lysis. This review also will consider current limitations and technological advances required for the development of this field.     

血脑屏障ATP结合盒超家族转运蛋白与药物转运

血脑屏障ATP结合盒(ATP-bindingcassette,ABC)超家族转运蛋白对维护血脑屏障的完整性、保持脑内环境的稳定和药物转运等方面都起着重要作用。文章主要就ABC超家族转运蛋白,如P-糖蛋白、多药耐药蛋白和乳癌耐药蛋白在血脑屏障药物转运中的作用做了综述。     

Blood–brain barrier structure and function and the challenges for CNS drug delivery

The neurons of the central nervous system (CNS) require precise control of their bathing microenvironment for optimal function, and an important element in this control is the blood–brain barrier (BBB). The BBB is formed by the endothelial cells lining the brain microvessels, under the inductive influence of neighbouring cell types within the ‘neurovascular unit’ (NVU) including astrocytes and pericytes. The endothelium forms the major interface between the blood and the CNS, and by a combination of low passive permeability and presence of specific transport systems, enzymes and receptors regulates molecular and cellular traffic across the barrier layer. A number of methods and models are available for examining BBB permeation in vivo and in vitro, and can give valuable information on the mechanisms by which therapeutic agents and constructs permeate, ways to optimize permeation, and implications for drug discovery, delivery and toxicity. For treating lysosomal storage diseases (LSDs), models can be included that mimic aspects of the disease, including genetically-modified animals, and in vitro models can be used to examine the effects of cells of the NVU on the BBB under pathological conditions. For testing CNS drug delivery, several in vitro models now provide reliable prediction of penetration of drugs including large molecules and artificial constructs with promising potential in treating LSDs. For many of these diseases it is still not clear how best to deliver appropriate drugs to the CNS, and a concerted approach using a variety of models and methods can give critical insights and indicate practical solutions.     

Blood-brain barrier transport of cationized immunoglobulin G: enhanced delivery compared to native protein.

IgG molecules are potential neuropharmaceuticals that may be used for therapeutic or diagnostic purposes. However, IgG molecules are excluded from entering brain, owing to a lack of transport of these plasma proteins through the brain capillary wall, or blood-brain barrier (BBB). The possibility of enhanced IgG delivery through the BBB by cationization of the proteins was explored in the present studies. Native bovine IgG molecules were cationized by covalent coupling of hexamethylenediamine and the isoelectric point was raised to greater than 10.7 based on isoelectric focusing studies. Native and cationized IgG molecules were radiolabeled with 125I and chloramine T. Cationized IgG, but not native IgG, was rapidly taken up by isolated bovine brain microvessels, which were used as an in vitro model system of the BBB. Cationized IgG binding was time and temperature dependent and was saturated by increasing concentrations of unlabeled cationized IgG (dissociation constant of the high-affinity binding site, 0.90 +/- 0.37 microM; Bmax, 1.4 +/- 0.4 nmol per mg of protein). In vivo studies documented enhanced brain uptake of 125I-labeled cationized IgG relative to [3H]albumin, and complete transcytosis of the 125I-labeled cationized IgG molecule through the BBB and into brain parenchyma was demonstrated by thaw-mount autoradiography of frozen sections of rat brain obtained after carotid arterial infusions of 125I-labeled cationized IgG. These studies demonstrate that cationization of IgG molecules greatly facilitates the transport of these plasma proteins through the BBB in vivo, and this process may provide a new strategy for IgG delivery through the BBB.     

Cellular immunotherapy for high-grade glioma

The outcome for patients with the most common primary brain tumor, glioblastoma multiforme (GBM), remains poor. Several immunotherapeutic approaches are actively being pursued including antibodies and cell-based therapies. While the blood-brain barrier protects brain tumor cells from therapeutic antibodies, immune cells have the ability to traverse the blood-brain barrier and migrate into GBM tumors to exert their therapeutic function. Results of Phase I clinical studies with vaccines to induce GBM-specific T cells are encouraging and Phase II clinical trials are in progress. Nonvaccine-based cell therapy for GBM has been actively explored over the last four decades. Here we will review past clinical experience with adoptive cell therapies for GBM and summarize current strategies on how to improve these approaches.     

Iontophoretic device delivery for the localized treatment of pancreatic ductal adenocarcinoma

Poor delivery and systemic toxicity of many cytotoxic agents, such as the recent promising combination chemotherapy regimen of folinic acid (leucovorin), fluorouracil, irinotecan, and oxaliplatin (FOLFIRINOX), restrict their full utility in the treatment of pancreatic cancer. Local delivery of chemotherapies has become possible using iontophoretic devices that are implanted directly onto pancreatic tumors. We have fabricated implantable iontophoretic devices and tested the local iontophoretic delivery of FOLFIRINOX for the treatment of pancreatic cancer in an orthotopic patient-derived xenograft model. Iontophoretic delivery of FOLFIRINOX was found to increase tumor exposure by almost an order of magnitude compared with i.v. delivery with substantially lower plasma concentrations. Mice treated for 7 wk with device FOLFIRINOX experienced significantly greater tumor growth inhibition compared with i.v. FOLFIRINOX. A marker of cell proliferation, K_i-67, was stained, showing a significant reduction in tumor cell proliferation. These data capitalize on the unique ability of an implantable iontophoretic device to deliver much higher concentrations of drug to the tumor compared with i.v. delivery. Local iontophoretic delivery of cytotoxic agents should be considered for the treatment of patients with unresectable nonmetastatic disease and for patients with the need for palliation of local symptoms, and may be considered as a neoadjuvant approach to improve resection rates and outcome in patients with localized and locally advanced pancreatic cancer.Pancreatic cancer is among the most lethal malignancies because of its insidious onset and resistance to therapy. The overall 5-y survival rate for this disease is less than 5%, and estimates indicate that pancreatic cancer will be second only to non-small-cell lung cancer as the leading cause of cancer-related mortality in the United States by 2030 (1–2). Surgical resection is the only curative option, with 15% of patients having resectable disease at presentation. Complete resection of all gross and microscopic disease results in a median survival time of 22–23 mo (3, 4). However, nearly 40% of patients with pancreatic cancer have locally advanced, unresectable disease with a median overall survival time of 9.2–13.5 mo (5). Although the true effect of microscopic positive margins is not fully known, patients destined to have the longest survival are those for whom resection with curative intent is feasible (6).The efficacy of chemotherapy for pancreatic cancer is impaired by a unique desmoplastic response (7, 8). Pancreatic tumors have a dense desmoplastic stroma with fibrotic connective tissue that surrounds the tumor and may account for >80% of tumor volume (9). This leads to a microenvironment with low blood perfusion and hypoxia, serving as a barrier to diminish the delivery of anticancer drugs (10, 11). A local drug delivery device capable of overcoming this barrier could provide substantial benefit for patients with locally advanced pancreatic cancer.We have developed an implantable iontophoretic device capable of driving chemotherapies deep into solid tumors. The principle behind iontophoretic drug delivery is the movement of charged species under an applied electric field (12, 13). Iontophoresis has been previously evaluated for oncologic purposes (13–15). An iontophoretic Foley catheter was developed to deliver mitomycin C to bladder tumors. Significant clinical success was achieved using the treatment alone and in combination with Bacillus Calmette-Guérin therapy (15). In addition, Gratieri et al. explored the iontophoretic delivery of 5-fluorouracil and leucovorin in healthy pig buccal tissue for the treatment of head and neck cancer (14).One major benefit of this technology is the ability to deliver highly toxic agents limited by unwanted secondary effects. FOLFIRINOX, a promising mixture of cytotoxic agents including folinic acid (leucovorin), fluorouracil, irinotecan, and oxaliplatin, has limited use in many patients because of its high systemic toxicity (16, 17). Modified FOLFIRINOX regimens have been created to improve tolerability (5, 18). Given the ability of the iontophoretic device to deliver drugs locally with minimal systemic exposure, the iontophoretic delivery of FOLFIRINOX could further enhance the efficacy of this cytotoxic regimen by increasing the local drug concentration and decreasing systemic exposure. The aim of our study was to evaluate the iontophoretic delivery of FOLFIRINOX for the treatment of localized pancreatic cancer. We evaluated this therapy in xenografts derived from patients with pancreatic cancer, which have been shown to reflect recently defined RNA tumor subtypes in patients, mirror patient outcome, and be highly predictive of clinical response to many targeted agents (19, 20). We report the delivery of high levels of the FOLFIRINOX drugs to the tumor, a reduction in systemic exposure of the drugs, and potent tumor regression. This therapy has the potential to improve the resection rates and the outcome for patients with pancreatic cancer.     

Increased brain uptake of targeted nanoparticles by adding an acid-cleavable linkage between transferrin and the nanoparticle core

Most therapeutic agents are excluded from entering the central nervous system by the blood–brain barrier (BBB). Receptor mediated transcytosis (RMT) is a common mechanism used by proteins, including transferrin (Tf), to traverse the BBB. Here, we prepared Tf-containing, 80-nm gold nanoparticles with an acid-cleavable linkage between the Tf and the nanoparticle core to facilitate nanoparticle RMT across the BBB. These nanoparticles are designed to bind to Tf receptors (TfRs) with high avidity on the blood side of the BBB, but separate from their multidentate Tf–TfR interactions upon acidification during the transcytosis process to allow release of the nanoparticle into the brain. These targeted nanoparticles show increased ability to cross an in vitro model of the BBB and, most important, enter the brain parenchyma of mice in greater amounts in vivo after systemic administration compared with similar high-avidity nanoparticles containing noncleavable Tf. In addition, we investigated this design with nanoparticles containing high-affinity antibodies (Abs) to TfR. With the Abs, the addition of the acid-cleavable linkage provided no improvement to in vivo brain uptake for Ab-containing nanoparticles, and overall brain uptake was decreased for all Ab-containing nanoparticles compared with Tf-containing ones. These results are consistent with recent reports of high-affinity anti-TfR Abs trafficking to the lysosome within BBB endothelium. In contrast, high-avidity, Tf-containing nanoparticles with the acid-cleavable linkage avoid major endothelium retention by shedding surface Tf during their transcytosis.The inability of drugs to cross the blood–brain barrier (BBB) is one of the major impairments to developing treatments for neurological diseases. This highly restrictive, physiologic barrier excludes 98% of small-molecule drugs and ∼100% of large-molecule drugs from reaching the central nervous system (CNS) from blood circulation (1). Many methods to bypass the BBB have been investigated, such as transient disruption of the BBB, inhibition of efflux pumps, or transport using endogenous transcytosis systems, including receptor-mediated transcytosis (2–4).Transferrin receptor (TfR) has been one of the primary targets investigated for receptor-mediated transcytosis across the BBB because of its high expression on BBB endothelium (5). Anti-TfR antibody–drug conjugates have received the most attention because of their ability to bind TfR with high affinity without interfering with endogenous transferrin (Tf) (6–8). Despite the perceived potential of anti-TfR antibody–drug conjugates, a BBB-permeable drug using this approach has yet to reach the clinic. Yu et al. showed that anti-TfR Abs enter the brain in greater numbers when their affinity to TfR is reduced (9). Follow-up work from the same group showed that high-affinity, bispecific anti-TfR Abs preferentially trafficked to the lysosome within BBB endothelium, rather than transcytosing, whereas low-affinity Abs did not (10). A similar effect was seen with a divalent anti-TfR Ab, which entered the lysosome in significantly greater amounts than the monovalent variant (11).Recently, our group demonstrated that Tf-containing, 80-nm gold nanoparticles (AuNP) with near-neutral zeta potentials are capable of accessing the brain parenchyma from the blood when their avidity to TfR is appropriately tuned (12). If the avidity is too high, the nanoparticles remain strongly associated with the endothelial cells of the BBB, whereas nanoparticles of lower avidity are able to release into the brain after transcytosis. Although the lower-avidity nanoparticles showed the greatest ability to enter the brain, the higher-avidity nanoparticles still were able to cross the BBB in greater amounts than non-Tf-containing nanoparticles.As with Ab BBB transcytosis, the nanoparticles with reduced avidity to TfR showed the greatest ability to cross the BBB. A major obstacle to translating these agents to viable therapeutics is the need to dose very high quantities in the blood for an appreciable amount of drug to reach the CNS (6, 9, 12). We attempted to increase the ability of Tf-containing nanoparticles to reach the brain parenchyma by incorporating a small chemical linker between the Tf and AuNP cores that cleaves at mildly acidic pH. This design provides for high-avidity interactions with TfR at the blood side of the BBB to enable practical, systemic dosing amounts. Then, as the targeted nanoparticles transcytose, we use the drop in pH (13, 14) the bound nanoparticles would experience during the transcytosis process to trigger the cleavage of the linkage between the Tf and the nanoparticle core. Thus, when the transcytosing vesicle reaches the brain, the nanoparticles will no longer be bound and can be released into the parenchyma. With this design, the nanoparticle will retain high-avidity interactions with TfR on the blood side of the BBB, but not be restricted once within the endothelium (Fig. 1A). Recently, in vitro results using an anti-TfR Ab with reduced affinity at pH 5.5 showed the ability to transcytose across hCMEC/D3 cells, whereas Abs with high affinity independent of pH were trafficked to the lysosome (15), suggesting vesicle trafficking may be affected by a particular targeting ligand. Thus, we also investigated whether Tf, the natural ligand for the TfR, and anti-TfR Abs behaved differently when used as the targeting agents for the nanoparticles. Our results show a nearly threefold increase in the ability of high-avidity nanoparticles to reach the brain parenchyma in vivo after incorporation of an acid-cleavable, diamino ketal (DAK) linker. We also observed a direct relationship between brain penetration of nanoparticles and surface Tf-DAK content. Furthermore, no improvement was seen in the ability for anti-TfR Ab-containing nanoparticles to cross the BBB with addition of the DAK linker, suggesting there are significant differences in their intracellular trafficking compared with that of Tf-containing nanoparticles.Open in a separate windowFig. 1.(A) Proposed mechanism of transcytosis for Tf-containing nanoparticles with an acid-cleavable linkage. After endocytosis, rapid acidification of the endosome causes separation of the Tf ligand from the nanoparticle core, allowing free movement of the nanoparticle into the brain parenchyma once transcytosis is complete. (B) Preparation of acid-cleavable DSS-DAK-PEG-OPSS and addition to the targeting ligand (Tf/Ab) to create the cleavable conjugate. (C) Addition of the Tf/Ab-DAK-PEG-OPSS ligand followed by excess mPEG-SH to prepare targeted gold nanoparticles. n ∼ 120 for 5-kDa PEG.     

转铁蛋白受体介导的药物转运在急性脑梗死治疗中的应用

在急性脑缺血的超早期,完整的血脑屏障(BBB)使许多治疗药物无法在有效治疗时间窗内进入脑组织发挥作用。转铁蛋白受体(TfR)单克隆抗体能有效通过受体介导的细胞内吞作用使结合的药物通过BBB,从而大大增加了治疗药物的定向选择性,使之更好地发挥治疗作用。     

Treating atherosclerosis: local drug delivery from laboratory studies to clinical trials

Treating only the specific section of the vascular bed that is diseased appears to make sense. Giving drugs systematically to treat perhaps only a few centimetres of affected artery carries with it the risk of systemic side effects and reduced efficacy consequent on low concentrations of agent at the site of the problem. There has thus been great interest since the early 1990s in local drug delivery. Initial targets were the thrombotic response to plaque disruption but the problems arising from the incidental damage inflicted by devices used in interventional cardiology and the pathological consequences of this, namely smooth muscle cell initiated intimal hyperplasia, soon became the focus of pre-clinical studies. Problems to be overcome were the low efficiency of delivery of drugs and the low retention rates. Solutions to these problems included the development of strategies to target drugs, through the use of antibodies directed at antigens newly released at the site of damage. As it became clear that stents were becoming central to the attainment of a better clinical response to intervention by their inherent physical properties, it also became obvious that stents could be used to deliver agents. Issues such as which stent, how to load the drug onto the stent and what drug to use to inhibit the unwanted pathobiological response are ongoing issues.     

Overview of randomized trials of antiarrhythmic drugs and devices for the prevention of sudden cardiac death

Background Sudden cardiac death is a prominent feature of the natural history of heart disease. The efficacy of antiarrhythmic drugs and devices in preventing sudden death and reducing total mortality is uncertain. Methods We reviewed randomized trials and quantitative overviews of type I and type III antiarrhythmic drugs. We also reviewed the randomized trials of implantable cardioverter defibrillators and combined these outcomes in a quantitative overview. Results Randomized trials of type I antiarrhythmic agents used as secondary prevention after myocardial infarction show an overall 21% increase in mortality rate. Randomized trials of amiodarone suggest a 13% to 19% decrease in mortality rate, and sotalol has been effective in several small trials. Trials of pure type III agents, however, have shown no mortality benefit. An overview of implantable defibrillator trials shows a 24% reduction in mortality rate (CI 15%-33%) compared with alternative therapy, most often amiodarone. Conclusion Amiodarone is effective in reducing the total mortality rate by 13% to 19%, and the implantable defibrillator reduces the mortality rate by a further 24%. (Am Heart J 2002;144:422-30.)     

Intracranial microcapsule chemotherapy delivery for the localized treatment of rodent metastatic breast adenocarcinoma in the brain

Metastases represent the most common brain tumors in adults. Surgical resection alone results in 45% recurrence and is usually accompanied by radiation and chemotherapy. Adequate chemotherapy delivery to the CNS is hindered by the blood–brain barrier. Efforts at delivering chemotherapy locally to gliomas have shown modest increases in survival, likely limited by the infiltrative nature of the tumor. Temozolomide (TMZ) is first-line treatment for gliomas and recurrent brain metastases. Doxorubicin (DOX) is used in treating many types of breast cancer, although its use is limited by severe cardiac toxicity. Intracranially implanted DOX and TMZ microcapsules are compared with systemic administration of the same treatments in a rodent model of breast adenocarcinoma brain metastases. Outcomes were animal survival, quantified drug exposure, and distribution of cleaved caspase 3. Intracranial delivery of TMZ and systemic DOX administration prolong survival more than intracranial DOX or systemic TMZ. Intracranial TMZ generates the more robust induction of apoptotic pathways. We postulate that these differences may be explained by distribution profiles of each drug when administered intracranially: TMZ displays a broader distribution profile than DOX. These microcapsule devices provide a safe, reliable vehicle for intracranial chemotherapy delivery and have the capacity to be efficacious and superior to systemic delivery of chemotherapy. Future work should include strategies to improve the distribution profile. These findings also have broader implications in localized drug delivery to all tissue, because the efficacy of a drug will always be limited by its ability to diffuse into surrounding tissue past its delivery source.The rationale behind localized drug delivery is that high concentrations of drug may be reached in target tissue while minimizing systemic exposure to the large quantities of drug that might otherwise be necessary to achieve the desired biologic effect. Localized drug delivery has been used, such as in drug-eluting stents for coronary artery disease, metered dose inhalers for the treatment of asthma, and intracranial chemotherapy wafers for brain tumors. This premise is particularly applicable to conditions affecting tissue that is sequestered from systemic circulation, such as conditions affecting the CNS.Adequate and efficacious drug delivery to the CNS has proved challenging. Systemic delivery methods are hindered by low permeability at the blood–brain barrier (BBB) to nonlipophilic drugs, which requires high systemic doses to be administered, thereby increasing systemic toxicity (1, 2). One method of circumventing the BBB is to place polymers that can release drug in a time-controlled manner at the site of tumor resection (3–6). Localized drug delivery devices provide high drug concentrations to the tumor site while preventing drug degradation and clearance until its release (7, 8). The efficacy of current intracranial drug delivery, such as Carmustine wafers, has promise, with a doubling of median survival from 9 to 20 mo in the case of Carmustine wafers (6, 9, 10). Phase III trial data indicated a 2-to 3-mo survival advantage in Carmustine wafers-treated patients (11), and Carmustine wafers continue to have a role combined with radiation and oral temozolomide (TMZ). However, nearly all patients still succumb to tumor progression.The clinical focus of CNS drug delivery has been high-grade glioma (12). The modest increases in survival seen with such strategies have been attributed, in part, to the limited efficacy of the chemotherapies used and the disease target itself, because these tumors are among the most infiltrative and aggressive tumors studied (6, 8). It has also been postulated that the chemotherapy that has been used (Carmustine) does not diffuse well through brain tissue to produce tissue effects away from the implantation site. Such strategies have not been widely tested in more focal, noninfiltrative lesions of the brain, such as brain metastases. Several experiments have been conducted with polymer-based vehicles, which locally delivery chemotherapy, that suggest their efficacy in brain metastases (13). The microcapsules described here were studied in a rodent glioma model and showed tunable release kinetics and efficacy (12). These vehicles have, however, not been tested in brain metastases.Metastases to the brain are the most commonly encountered tumors in the brain (14–17). It is estimated that 100,000–200,000 new cases are identified in the United States each year. Furthermore, 20% of patients with disseminated cancer have evidence of CNS involvement at autopsy (14). Certain tumors have a predilection to metastasize to the brain with nonsmall cell lung cancer, breast carcinoma, small cell lung carcinoma, malignant melanoma, renal cell carcinoma, gastrointestinal carcinoma, uterine carcinoma, and unknown primary carcinoma in decreasing frequency (14, 15). Cell surface markers seem to predict metastatic potential; for example, human epidermal growth factor receptor 2-positive breast carcinoma is more likely to present with CNS lesions (16). Metastases are focal in nature, with no true invasion of brain tissue in contrast to high-grade gliomas.The treatment of brain metastases is often multimodal, with surgery, whole-brain radiotherapy (WBRT), stereotactic radiosurgery, and chemotherapy all playing a role (18, 19). The selection of specific modalities of treatment is dependent on patient-specific factors, such as number, size, and location of metastases, patient’s overall health and prognosis, and degree of symptoms attributable to these metastases. Surgery remains a standard treatment for metastatic lesions, particularly those that are symptomatic for patients with good functional status and well-controlled extracranial disease (19). Metastases tend to be noninfiltrative histologically, suggesting that surgical treatment should reduce or eliminate tumor burden and be beneficial. Multiple retrospective analyses have shown survival benefit from resection of single brain metastases, particularly combined with WBRT (19–22). More recent data from 2005 suggest that patients with two or three metastases also show increases in overall survival when the dominant symptomatic lesion is surgically resected (23). The most promising trial of combination chemotherapy showed response rates of 40–59% in breast cancer metastases to the brain (24), although more recent studies show a wide range of response from 10% to 45%, with variability based on the primary tumor (19). Systemic chemotherapy delivery has played a limited role in the treatment of brain metastases, in part because of the poor penetration through the BBB (19). More recent studies suggest that chemotherapeutic agents that have better penetration of the BBB, such as TMZ, may have a role in newly diagnosed metastases, not just as salvage therapy (25, 26). The agents that have shown promise include cyclophosphamide, 5-fluorouracil, methotrextate and vincristine combined, and TMZ, although the use of TMZ has been restricted to recurrent breast metastases (19, 27, 28). The use of TMZ in recurrent breast cancer brain metastases has shown modest increases in survival, although this patient population represents a selection of the most aggressive and refractory cancers that have withstood surgery, WBRT, and multiple other systemic chemotherapies.Doxorubicin (DOX) has been used to treat early-stage, node-positive breast cancer, particularly HER2-positive tumors, although it is more commonly used for treatment of non-Hodgkins lymphoma and certain leukemias (29). Its clinical use in breast metastases to the brain has largely been limited by the resistance of the tumor to the chemotherapy as well as its poor penetration of the BBB and its dose-related adverse effects. The risk of developing potentially lethal cardiotoxicity increased significantly when doses of DOX reach 500 mg/m² (30).Microcapsules loaded with DOX or TMZ were administered intracranially in an experimental model of metastases to the brain in the following set of experiments. The CRL1666 cell line has been used in a metastatic spine model. This body of work represents the first time that it has been successfully used in a brain metastasis model to the best of our knowledge (31). Their efficacy was compared with systemic administration of DOX and TMZ. Tissue harvested from these animals was then processed for evidence of induction of apoptosis, and the distribution of drug released from the devices was also examined to test the relationship between drug distribution and biologic activity and efficacy.