ABSTRACT
The methylthioadenosine phosphorylase (MTAP) gene is located adjacent to the cyclin-dependent kinase inhibitor 2A (CDKN2A) tumor-suppressor gene and is co-deleted with CDKN2A in approximately 15% of all cancers. This co-deletion leads to aggressive tumors with poor prognosis that lack effective, molecularly targeted therapies. The metabolic enzyme methionine adenosyltransferase 2α (MAT2A) was identified as a synthetic lethal target in MTAP-deleted cancers. We report the characterization of potent MAT2A inhibitors that substantially reduce levels of S-adenosylmethionine (SAM) and demonstrate antiproliferative activity in MTAP-deleted cancer cells and tumors. Using RNA sequencing and proteomics, we demonstrate that MAT2A inhibition is mechanistically linked to reduced protein arginine methyltransferase 5 (PRMT5) activity and splicing perturbations. We further show that DNA damage and mitotic defects ensue upon MAT2A inhibition in HCT116 MTAP-/- cells, providing a rationale for combining the MAT2A clinical candidate AG-270 with antimitotic taxanes.
Subject(s)
DNA Damage/drug effects , Enzyme Inhibitors/pharmacology , Methionine Adenosyltransferase/antagonists & inhibitors , Protein-Arginine N-Methyltransferases/genetics , Purine-Nucleoside Phosphorylase/genetics , RNA Splicing/drug effects , RNA, Messenger/genetics , Animals , Cell Line , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p16 , DNA Damage/genetics , Gene Deletion , HCT116 Cells , HEK293 Cells , Humans , Methionine Adenosyltransferase/genetics , Mice, Inbred NOD , Mice, Nude , Mice, SCID , Neoplasms/drug therapy , Neoplasms/genetics , RNA Splicing/genetics , S-Adenosylmethionine/metabolismABSTRACT
The endothelium is a target for therapeutic and diagnostic interventions in a plethora of human disease conditions including ischemia, inflammation, edema, oxidative stress, thrombosis and hemorrhage, and metabolic and oncological diseases. Unfortunately, drugs have no affinity to the endothelium, thereby limiting the localization, timing, specificity, safety, and effectiveness of therapeutic interventions. Molecular determinants on the surface of resting and pathologically altered endothelial cells, including cell adhesion molecules, peptidases, and receptors involved in endocytosis, can be used for drug delivery to the endothelial surface and into intracellular compartments. Drug delivery platforms such as protein conjugates, recombinant fusion constructs, targeted liposomes, and stealth polymer carriers have been designed to target drugs and imaging agents to these determinants. We review endothelial target determinants and drug delivery systems, describe parameters that control the binding of drug carriers to the endothelium, and provide examples of the endothelial targeting of therapeutic enzymes designed for the treatment of acute vascular disorders including ischemia, oxidative stress, inflammation, and thrombosis.
Subject(s)
Drug Carriers/therapeutic use , Endothelium, Vascular , Ischemia/drug therapy , Oxidative Stress/drug effects , Thrombosis/drug therapy , Animals , Antigens, Differentiation/metabolism , Drug Carriers/chemistry , Edema/drug therapy , Edema/metabolism , Edema/pathology , Endothelium, Vascular/metabolism , Endothelium, Vascular/physiology , Hemorrhage/drug therapy , Hemorrhage/metabolism , Hemorrhage/pathology , Humans , Ischemia/metabolism , Ischemia/pathology , Metabolic Diseases/drug therapy , Metabolic Diseases/metabolism , Metabolic Diseases/pathology , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , Thrombosis/metabolism , Thrombosis/pathologyABSTRACT
Therapeutic proteins are prone to inactivation by aggregation, proteases and natural inhibitors, motivating development of protective delivery systems. Here we focus on protective encapsulation of the potent antioxidant enzyme, catalase, by filamentous polymer nanocarriers (f-PNC), with the specific goal of addressing whether polymer molecular weight (MW) controls formation and structural properties such as size and stiffness. While maintaining the same MW ratio of polyethylene glycol to polylactic acid, a series of PEG-b-PLA diblock copolymers were synthesized, with total MW ranging from about 10 kg/mol to 100 kg/mol. All diblocks formed f-PNC upon processing, which encapsulated active enzyme that proved resistant to protease degradation. Further, f-PNC stiffness, length, and thickness increased with increasing MW. Interestingly, heating above a polymer's glass transition temperature (<30 degrees C) increased f-PNC flexibility. Thus, we report here for the first time f-PNC that encapsulate an active enzyme with polymer MW-tunable flexibility, offering several potential therapeutic applications.
Subject(s)
Drug Carriers , Enzymes/chemistry , Lactic Acid/chemistry , Nanoparticles , Polyethylene Glycols/chemistry , Polymers/chemistry , Biocatalysis , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Polyesters , TemperatureABSTRACT
The use of continuous manufacturing has been increasing within the pharmaceutical industry over the last few years. Continuous direct compression has been the focus of publications on the topic to date. The use of wet granulation can improve segregation resistance, uniformity, enhance density, and flow properties for improved tabletability, or improve stability of products that cannot be manufactured by using a direction compression process. This article focuses on development of appropriate control strategies for continuous wet granulation (especially twin screw wet granulation) through equipment design, material properties and manufacturing process along with areas where additional understanding is required. The article also discusses the use of process analytical technologies as part of the control and automation approach to ensure a higher assurance of product quality. Increased understanding of continuous wet granulation should result in increased utilization of the technique, thereby allowing for an increase in diversity of products manufactured by continuous manufacturing and the benefits that comes with a more complex process such as wet granulation compared with direct compression process.
Subject(s)
Drug Compounding/methods , Tablets/chemistry , Drug Industry/methods , Equipment Design/methodsABSTRACT
Type B Niemann-Pick disease (NPD) is a multiorgan system disorder caused by a genetic deficiency of acid sphingomyelinase (ASM), for which lung is an important and challenging therapeutic target. In this study, we designed and evaluated new delivery vehicles for enzyme replacement therapy of type B NPD, consisting of polystyrene and poly(lactic-coglycolic) acid polymer nanocarriers targeted to intercellular adhesion molecule (ICAM)-1, an endothelial surface protein up-regulated in many pathologies, including type B NPD. Real-time vascular imaging using intravital microscopy and postmortem imaging of mouse organs showed rapid, uniform, and efficient binding of fluorescently labeled ICAM-1-targeted ASM nanocarriers (anti-ICAM/ASM nanocarriers) to endothelium after i.v. injection in mice. Fluorescence microscopy of lung alveoli actin, tissue histology, and 125I-albumin blood-to-lung transport showed that anti-ICAM nanocarriers cause neither detectable lung injury, nor abnormal vascular permeability in animals. Radioisotope tracing showed rapid disappearance from the circulation and enhanced accumulation of anti-ICAM/125I-ASM nanocarriers over the nontargeted naked enzyme in kidney, heart, liver, spleen, and primarily lung, both in wild-type and ASM knockout mice. These data demonstrate that ICAM-1-targeted nanocarriers may enhance enzyme replacement therapy for type B NPD and perhaps other lysosomal storage disorders.
Subject(s)
Drug Carriers/administration & dosage , Intercellular Adhesion Molecule-1/metabolism , Nanostructures/administration & dosage , Niemann-Pick Disease, Type B/metabolism , Sphingomyelin Phosphodiesterase/administration & dosage , Abdominal Muscles/metabolism , Animals , Drug Carriers/pharmacokinetics , Endothelium, Vascular/metabolism , Kidney/metabolism , Lactic Acid/administration & dosage , Lactic Acid/pharmacokinetics , Liver/metabolism , Lung/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Myocardium/metabolism , Polyglycolic Acid/administration & dosage , Polyglycolic Acid/pharmacokinetics , Polylactic Acid-Polyglycolic Acid Copolymer , Polymers/administration & dosage , Polymers/pharmacokinetics , Polystyrenes/administration & dosage , Polystyrenes/pharmacokinetics , Recombinant Proteins/administration & dosage , Recombinant Proteins/genetics , Recombinant Proteins/pharmacokinetics , Sphingomyelin Phosphodiesterase/genetics , Sphingomyelin Phosphodiesterase/pharmacokinetics , Spleen/metabolismABSTRACT
The medical utility of proteins, e.g. therapeutic enzymes, is greatly restricted by their labile nature and inadequate delivery. Most therapeutic enzymes do not accumulate in their targets and are inactivated by proteases. Targeting of enzymes encapsulated into substrate-permeable polymer nano-carriers (PNC) impermeable for proteases might overcome these limitations. To test this hypothesis, we designed endothelial targeted PNC loaded with catalase, an H(2)O(2)-detoxifying enzyme, and tested if this approach protects against vascular oxidative stress, a pathological process implicated in ischemia-reperfusion and other disease conditions. Encapsulation of catalase (MW 247 kD), peroxidase (MW 42 kD) and xanthine oxidase (XO, MW 300 kD) into approximately 300 nm diameter PNC composed of co-polymers of polyethylene glycol and poly-lactic/poly-glycolic acid (PEG-PLGA) was in the range approximately 10% for all enzymes. PNC/catalase and PNC/peroxidase were protected from external proteolysis and exerted enzymatic activity on their PNC diffusible substrates, H(2)O(2) and ortho-phenylendiamine, whereas activity of encapsulated XO was negligible due to polymer impermeability to the substrate. PNC targeted to platelet-endothelial cell (EC) adhesion molecule-1 delivered active encapsulated catalase to ECs and protected the endothelium against oxidative stress in cell culture and animal studies. Vascular targeting of PNC-loaded detoxifying enzymes may find wide medical applications including management of oxidative stress and other toxicities.
Subject(s)
Catalase/metabolism , Cell Membrane Permeability , Drug Delivery Systems , Endothelial Cells/metabolism , Nanostructures/chemistry , Polymers/chemistry , Polymers/metabolism , Animals , Antioxidants/metabolism , Catalase/chemistry , Catalase/therapeutic use , Cell Adhesion Molecules/metabolism , Cells, Cultured , Humans , Male , Mice , Mice, Inbred C57BLABSTRACT
The unique properties of synthetic nanostructures promise a diverse set of applications as carriers for drug delivery, which are advantageous in terms of biocompatibility, pharmacokinetics, targeting and controlled drug release. Historically, more traditional drug delivery systems have focused on spherical carriers. However, there is a growing interest in pursuing non-spherical carriers, such as elongated or filamentous morphologies, now available due to novel formulation strategies. Unique physiochemical properties of these supramolecular structures offer distinct advantages as drug delivery systems. In particular, results of recent studies in cell cultures and lab animals indicate that rational design of carriers of a given geometry (size and shape) offers an unprecedented control of their longevity in circulation and targeting to selected cellular and subcellular locations. This article reviews drug delivery aspects of non-spherical drug delivery systems, including material selection and formulation, drug loading and release, biocompatibility, circulation behavior, targeting and subcellular addressing.
Subject(s)
Drug Delivery Systems/methods , Drug Design , Polymers/chemistry , Chemistry, Pharmaceutical , Drug Compounding , Liposomes/chemistry , Micelles , Nanostructures/chemistryABSTRACT
Rapid clearance and proteolysis limit delivery and efficacy of protein therapeutics. Loading into biodegradable polymer nanocarriers (PNC) might protect proteins, extending therapeutic duration, but loading can be complicated by protein unfolding and inactivation. We encapsulated active enzymes into methoxy-poly(ethylene glycol- block-lactic acid) (mPEG-PLA) PNC with a freeze-thaw double emulsion ( J. Controlled Release 2005, 102 (2), 427-439). On the basis of concepts of amphiphile self-assembly, we hypothesized that the copolymer block ratio that controls spontaneous curvature would influence PNC morphology and loading. We examined PNC yield, shape, stability, loading, activity, and protease resistance of the antioxidant enzyme, catalase. PNC transitioned from spherical to filamentous shapes with increasing hydrophobic polymer fraction, consistent with trends for self-assembly of lower MW amphiphiles. Importantly, one diblock copolymer formed filamentous particles loaded with significant levels of protease-resistant enzyme, demonstrating for the first time encapsulation of an active therapeutic enzyme into filamentous carriers. PNC morphology also greatly influenced its degradation, offering a new means of controlled delivery.
Subject(s)
Drug Carriers/chemistry , Enzymes/chemistry , Nanospheres/chemistry , Polymers/chemistry , Animals , Cattle , Drug Carriers/administration & dosage , Enzymes/administration & dosage , Nanospheres/administration & dosage , Polymers/administration & dosageABSTRACT
Vascular drug targeting may improve therapies, yet a thorough understanding of the factors that regulate effects of drugs directed to the endothelium is needed to translate this approach into the clinical domain. To define factors modulating the efficacy and effects of endothelial targeting, we used a model enzyme (glucose oxidase, GOX) coupled with monoclonal antibodies (anti-TM(34) or anti-TM(201)) to distinct epitopes of thrombomodulin, a surface determinant enriched in the pulmonary endothelium. GOX delivery results in conversion of glucose and oxygen into H(2)O(2) leading to lung damage, a clear physiologic endpoint. Results of in vivo studies in mice showed that the efficiency of cargo delivery and its effect are influenced by a number of factors including: 1) The level of pulmonary uptake of the targeting antibody (anti-TM(201) was more efficient than anti-TM(34)); 2) The amount of an active drug delivered to the target; 3) The amount of target antigen on the endothelium (animals with suppressed TM levels showed less targeting); and, 4) The substrate availability for the enzyme cargo in the target tissue (hyperoxia augmented GOX-induced injury). Therefore, both activities of the conjugates and biological factors control targeting and effects of enzymatic cargo. Understanding the nature of such "modulating biological factors" will hopefully allow optimization and ultimately applications of drug targeting for "individualized" pharmacotherapy.
Subject(s)
Antibodies, Monoclonal/metabolism , Drug Carriers , Endothelium, Vascular/metabolism , Enzymes/metabolism , Lung/blood supply , Thrombomodulin/metabolism , Animals , Antibody Affinity , Chemistry, Pharmaceutical , Dose-Response Relationship, Drug , Drug Delivery Systems , Endothelium, Vascular/immunology , Enzymes/administration & dosage , Enzymes/chemistry , Enzymes/toxicity , Glucose/metabolism , Glucose Oxidase/metabolism , Hydrogen Peroxide/metabolism , Hyperoxia/metabolism , Injections, Intravenous , Lung/drug effects , Lung/metabolism , Lung/pathology , Mice , Mice, Inbred C57BL , Microspheres , Oxygen/metabolism , Polystyrenes/chemistry , Thrombomodulin/immunology , Time FactorsABSTRACT
Targeting of therapeutics or imaging agents to the endothelium has the potential to improve specificity and effectiveness of treatment for many diseases. One strategy to achieve this goal is the use of nanoparticles (NPs) targeted to the endothelium by ligands of protein determinants present on this tissue, including cell adhesion molecules, peptidases, and cell receptors. However, detachment of the radiolabel probes from NPs poses a significant problem. In this study, we devised polymeric NPs directly labeled with radioiodine isotopes including the positron emission tomography (PET) isotope (124)I, and characterized their targeting to specific endothelial determinants. This approach provided sizable, targetable probes for specific detection of endothelial surface determinants non-invasively in live animals. Direct conjugation of radiolabel to NPs allowed for stable longitudinal tracking of tissue distribution without label detachment even in an aggressive proteolytic environment. Further, this approach permits tracking of NP pharmacokinetics in real-time and non-invasive imaging of the lung in mice using micro-PET imaging. The use of this strategy will considerably improve investigation of NP interactions with target cells and PET imaging in small animals, which ultimately can aid in the optimization of targeted drug delivery.
Subject(s)
Drug Delivery Systems/methods , Endothelial Cells/diagnostic imaging , Nanoparticles , Polyvinyls , Positron-Emission Tomography/methods , Staining and Labeling , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Endothelial Cells/drug effects , Female , Iodine Radioisotopes , Lung/diagnostic imaging , Mice , Mice, Inbred C57BL , Nanoparticles/chemistry , Nanoparticles/ultrastructure , Particle Size , Polyvinyls/chemical synthesis , Polyvinyls/chemistry , Time FactorsABSTRACT
Antioxidant enzymes (AOEs) catalase and superoxide dismutase (SOD) detoxify harmful reactive oxygen species, but the therapeutic utility of AOEs is hindered by inadequate delivery. AOE modification by poly-ethylene glycol (PEG) and encapsulation in PEG-coated liposomes increases the AOE bioavailability and enhances protective effects in animal models. Pluronic-based micelles formed with AOEs show even more potent protective effects. Furthermore, polymeric nanocarriers (PNCs) based on PEG-copolymers protect encapsulated AOEs from proteolysis and improve delivery to the target cells, such as the endothelium lining the vascular lumen. Antibodies to endothelial determinants conjugated to AOEs or AOE carriers provide targeting and intracellular delivery. Targeted liposomes, protein conjugates and magnetic nanoparticles deliver AOEs to sites of vascular oxidative stress in the cardiovascular, pulmonary and nervous systems. Further advances in nanodevices for AOE delivery will provide a basis for the translation of this approach in the clinical domain.
Subject(s)
Antioxidants/administration & dosage , Catalase/administration & dosage , Drug Delivery Systems/methods , Endothelium, Vascular/metabolism , Nanostructures/chemistry , Superoxide Dismutase/administration & dosage , Animals , HumansABSTRACT
The endothelial lining of the lumen of blood vessels is a key therapeutic target for many human diseases. Polymeric filomicelles that self-assemble from polyethylene oxide (PEO)-based diblock copolymers are long and flexible rather than small or rigid, can be loaded with drugs, and--most importantly--they circulate for a prolonged period of time in the bloodstream due in part to flow alignment. Filomicelles seem promising for targeted drug delivery to endothelial cells because they can in principle adhere strongly, length-wise to specific cell surface determinants. In order to achieve such a goal of vascular drug delivery, two fundamental questions needed to be addressed: (i) whether these supramolecular filomicelles retain structural integrity and dynamic flexibility after attachment of targeting molecules such as antibodies, and (ii) whether the avidity of antibody-carrying filomicelles is sufficient to anchor the carrier to the endothelial surface despite the effects of flow that oppose adhesive interactions. Here we make targeted filomicelles that bear antibodies which recognize distinct endothelial surface molecules. We characterize these antibody targeted filomicelles and prove that (i) they retain structural integrity and dynamic flexibility and (ii) they adhere to endothelium with high specificity both in vitro and in vivo. These results provide the basis for a new drug delivery approach employing antibody-targeted filomicelles that circulate for a prolonged time yet bind to endothelial cells in vascular beds expressing select markers.
Subject(s)
Antibodies/metabolism , Drug Delivery Systems , Endothelium, Vascular/metabolism , Micelles , Polymers , Humans , NanoparticlesABSTRACT
Protein drugs, such as recombinant enzymes useful for detoxification and replacement therapies, have extraordinary specificity and potency. However, inherently inadequate delivery to target sites and rapid inactivation limit their medical utility. Using chaperone polymeric particles designed within an injectible size range (sub-micron) may help solve these shortcomings. Such nanocarriers would (i) prevent premature inactivation of encapsulated therapeutic protein cargoes, (ii) provide a carrier that can be surface decorated by targeting ligands, and (iii) optimize sub-cellular localization of the drug. This chapter describes the techniques successfully employed for the preparation of polymer nanocarriers (PNC) loaded with the antioxidant enzyme, catalase, and targeted to endothelial cells. Methods of PNC synthesis, loading with catalase, characterization, coupling of a targeting moiety, and in vitro testing of the enzymatic and targeting activities are provided here. Advantages and disadvantages of specific designs are discussed. Due to the modular nature of the targeting methodology employed, it is believed that these protocols will provide a solid foundation for the formulation of a wide variety of enzymatic drug targeting strategies.
Subject(s)
Catalase , Drug Carriers , Drug Delivery Systems/methods , Nanostructures , Polymers , Animals , Catalase/metabolism , Catalase/therapeutic use , Cell Adhesion Molecules/metabolism , Cell Line , Drug Carriers/chemical synthesis , Drug Carriers/chemistry , Drug Carriers/metabolism , Endothelial Cells/cytology , Endothelial Cells/metabolism , Humans , Nanostructures/chemistry , Particle Size , Polymers/chemical synthesis , Polymers/chemistry , Polymers/metabolismABSTRACT
PURPOSE: Based on a unique phase alignment that occurs during formulation, we postulated that PEG-ylation of the cargo enzyme would enhance its encapsulation within diblock copolymer nanocarriers and thus resistance to proteases. METHODS: A freeze-thaw modified double emulsion technique was utilized to encapsulate either the catalytically active enzyme catalase (MW approximately 250 kDa) or PEG-catalase in PEG-PLA polymer nanocarriers (PNC). Spectrophotometer measurement of substrate depletion was utilized to monitor enzyme activity. Isotope labeling of the enzyme was used in conjunction with activity measurements to determine PNC loading efficiency and PNC-enzyme resistance to proteases. This labeling also enabled blood clearance measurements of PNC-loaded and non-loaded enzymes in mice. RESULTS: Non-loaded PEG-catalase exhibited longer circulation times than catalase, but was equally susceptible to proteolysis. Modulation of the ratio of relatively hydrophilic to hydrophobic domains in the diblock PEG-PLA copolymer provided either filamentous or spherical PNC loaded with PEG-catalase. For both PNC geometries, encapsulation and resistance to proteases of the resultant PNC-loaded enzyme were more effective for PEG-catalase than catalase. Isotope tracing showed similar blood levels of PNC-loaded and free PEG-catalase in mice. CONCLUSIONS: PEGylation enhances active catalase loading within PNC and resistance to protease degradation, relative to unloaded PEG-catalase.