ABSTRACT
Our group has recently shown that brain-penetrant ataxia telangiectasia-mutated (ATM) kinase inhibitors may have potential as novel therapeutics for the treatment of Huntington's disease (HD). However, the previously described pyranone-thioxanthenes (e.g., 4) failed to afford selectivity over a vacuolar protein sorting 34 (Vps34) kinase, an important kinase involved with autophagy. Given that impaired autophagy has been proposed as a pathogenic mechanism of neurodegenerative diseases such as HD, achieving selectivity over Vps34 became an important objective for our program. Here, we report the successful selectivity optimization of ATM over Vps34 by using X-ray crystal structures of a Vps34-ATM protein chimera where the Vps34 ATP-binding site was mutated to approximate that of an ATM kinase. The morpholino-pyridone and morpholino-pyrimidinone series that resulted as a consequence of this selectivity optimization process have high ATM potency and good oral bioavailability and have lower molecular weight, reduced lipophilicity, higher aqueous solubility, and greater synthetic tractability compared to the pyranone-thioxanthenes.
Subject(s)
Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , Pyridones/chemistry , Pyrimidinones/chemistry , Animals , Ataxia Telangiectasia Mutated Proteins/metabolism , Binding Sites , Brain/metabolism , Class III Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Class III Phosphatidylinositol 3-Kinases/metabolism , Crystallography, X-Ray , Drug Design , Half-Life , Humans , Huntington Disease/drug therapy , Male , Mice , Mice, Inbred C57BL , Molecular Dynamics Simulation , Morpholinos/chemistry , Pyridones/metabolism , Pyridones/therapeutic use , Pyrimidinones/metabolism , Pyrimidinones/therapeutic use , Structure-Activity RelationshipABSTRACT
Genetic and pharmacological evidence indicates that the reduction of ataxia telangiectasia-mutated (ATM) kinase activity can ameliorate mutant huntingtin (mHTT) toxicity in cellular and animal models of Huntington's disease (HD), suggesting that selective inhibition of ATM could provide a novel clinical intervention to treat HD. Here, we describe the development and characterization of ATM inhibitor molecules to enable in vivo proof-of-concept studies in HD animal models. Starting from previously reported ATM inhibitors, we aimed with few modifications to increase brain exposure by decreasing P-glycoprotein liability while maintaining potency and selectivity. Here, we report brain-penetrant ATM inhibitors that have robust pharmacodynamic (PD) effects consistent with ATM kinase inhibition in the mouse brain and an understandable pharmacokinetic/PD (PK/PD) relationship. Compound 17 engages ATM kinase and shows robust dose-dependent inhibition of X-ray irradiation-induced KAP1 phosphorylation in the mouse brain. Furthermore, compound 17 protects against mHTT (Q73)-induced cytotoxicity in a cortical-striatal cell model of HD.
Subject(s)
Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors , Huntington Disease/drug therapy , Neuroprotective Agents/therapeutic use , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Animals , Disease Models, Animal , Dogs , Humans , Madin Darby Canine Kidney Cells , Mice , Neuroprotective Agents/metabolism , Neuroprotective Agents/pharmacokinetics , Proof of Concept StudyABSTRACT
In this study we present an approach to pre-program lysozyme release from large size (100-300 µm) poly(DL-lactic acid-co-glycolic acid) (PLGA) microparticles. This approach involved blending in-house synthesized triblock copolymers with a PLGA 85:15. In this work it is demonstrated that the lysozyme release rate and the total release are related to the mass of triblock copolymer present in polymer formulation. Two triblock copolymers (PLGA-PEG1500-PLGA and PLGA-PEG1000-PLGA) were synthesized and used in this study. In a like-for-like comparison, these two triblock copolymers appeared to have similar effects on the release of lysozyme. It was shown that blending resulted in the increase of the total lysozyme release and shortened the release period (70% release within 30 days). These results demonstrated that blending PLGA-PEG-PLGA triblock copolymer with PLGA 85:15 can be used as a method to pre-program protein release from microparticles. These microparticles with modulated protein release properties may be used to create microparticle-based tissue engineering constructs with pre-programmed release properties.
Subject(s)
Lactic Acid/chemistry , Polyglycolic Acid/chemistry , Proteins/chemistry , Chemistry, Pharmaceutical/methods , Drug Carriers/chemistry , Polyethylene Glycols/chemistry , Polyglactin 910/chemistry , Polylactic Acid-Polyglycolic Acid Copolymer , Polymers/chemistry , Tissue Engineering/methodsABSTRACT
There is a need to control the spatio-temporal release kinetics of growth factors in order to mitigate current usage of high doses. A novel delivery system, capable of providing both structural support and controlled release kinetics, has been developed from PLGA microparticles. The inclusion of a hydrophilic PLGA-PEG-PLGA triblock copolymer altered release kinetics such that they were decoupled from polymer degradation. A quasi zero order release profile over four weeks was produced using 10% w/w PLGA-PEG-PLGA with 50:50 PLGA whereas complete and sustained release was achieved over ten days using 30% w/w PLGA-PEG-PLGA with 85:15 PLGA and over four days using 30% w/w PLGA-PEG-PLGA with 50:50 PLGA. These three formulations are promising candidates for delivery of growth factors such as BMP-2, PDGF and VEGF. Release profiles were also modified by mixing microparticles of two different formulations providing another route, not previously reported, for controlling release kinetics. This system provides customisable, localised and controlled delivery with adjustable release profiles, which will improve the efficacy and safety of recombinant growth factor delivery.
Subject(s)
Microspheres , Proteins/metabolism , Regenerative Medicine , Lactic Acid/chemistry , Microscopy, Electron, Scanning , Polyethylene Glycols/chemistry , Polyglycolic Acid/chemistry , Polylactic Acid-Polyglycolic Acid CopolymerABSTRACT
Biodegradable polymer scaffolds have great potential for regenerative medicine applications such as the repair of musculoskeletal tissues. Here, we describe the development of scaffolds that blend hydrogel components with thermoplastic materials, combining the unique properties of both components to create mouldable formulations. This study focuses on the structural and mechanical properties of the composite scaffolds, produced by combining temperature-sensitive poly(DL-lactic acid-co-glycolic acid) (PLGA)/poly(ethylene glycol) (PEG) particles with a hydrogel component [Pluronic F127, fibrin or hyaluronic acid (HyA)]. The composite formulations solidified over time at 37°C, with a significant increase (p ≤ 0.05) in compressive strength observed from 15 min to 2 h at this temperature. The maximum compressive strength was 1.2 MPa for PLGA/PEG-Pluronic F127 scaffolds, 2.4 MPa for PLGA/PEG-HyA scaffolds and 0.6 MPa for PLGA/PEG-fibrin scaffolds. Porosity for each of the PLGA/PEG-hydrogel formulations tested was between 50 and 51%. This study illustrates the ability to combine this thermoplastic PLGA/PEG system with hydrogels to fabricate composite scaffolds, and demonstrates that altering the particle to hydrogel ratio produces scaffolds with varying mechanical properties.
