Pluronic based ?-cyclodextrin polyrotaxanes for treatment of Niemann-Pick Type C disease.
ABSTRACT: Niemann-Pick Type C disease (NPC) is a rare metabolic disorder characterized by disruption of normal cholesterol trafficking within the cells of the body. There are no FDA approved treatments available for NPC patients. Recently, the cycloheptaglucoside 2-hydroxypropyl-?-cyclodextrin (HP-?-CD) has shown efficacy as a potential NPC therapeutic by extending lifetime in NPC mice, delaying neurodegeneration, and decreasing visceral and neurological cholesterol burden. Although promising, systemic HP-?-CD treatment is limited by a pharmacokinetic profile characterized by rapid loss through renal filtration. To address these shortcomings, we sought to design a family of HP-?-CD pro-drug delivery vehicles, known as polyrotaxanes (PR), capable of increasing the efficacy of a given injected dose by improving both pharmacokinetic profile and bioavailability of the HP-?-CD agent. PR can effectively diminish the cholesterol pool within the liver, spleen, and kidney at molar concentrations 10-to-100-fold lower than monomeric HP-?-CD. In addition to this proof-of-concept, use of PR scaffolds with differing physiochemical properties reveal structure-activity relationships in which PR characteristics, including hydrophobicity, threading efficiency and surface charge, were found to both decisively and subtly effect therapeutic efficacy. PR scaffolds exhibit absorption, pharmacokinetics, and biodistribution patterns that are significantly altered from monomeric HP-?-CD. In all, PR scaffolds hold great promise as potential treatments for visceral disease in NPC patients.
Project description:Niemann-Pick type C (NPC) disease is characterized by the lysosomal accumulation of cholesterols and impaired autophagic flux due to the inhibited fusion of autophagosomes to lysosomes. We have recently developed ?-cyclodextrin (?-CD)-threaded biocleavable polyrotaxanes (PRXs), which can release threaded ?-CDs in response to intracellular environments as a therapeutic for NPC disease. The biocleavable PRXs exhibited effective cholesterol reduction ability and negligible toxic effect compared with hydroxypropyl-?-CD (HP-?-CD). In this study, we investigated the effect of biocleavable PRX and HP-?-CD on the impaired autophagy in NPC disease. The NPC patient-derived fibroblasts (NPC1 fibroblasts) showed an increase in the number of LC3-positive puncta compared with normal fibroblasts, even in the basal conditions; the HP-?-CD treatment markedly increased the number of LC3-positive puncta and the levels of p62 in NPC1 fibroblasts, indicating that autophagic flux was further perturbed. In sharp contrast, the biocleavable PRX reduced the number of LC3-positive puncta and the levels of p62 in NPC1 fibroblasts through an mTOR-independent mechanism. The mRFP-GFP-LC3 reporter gene expression experiments revealed that the biocleavable PRX facilitated the formation of autolysosomes to allow for autophagic protein degradation. Therefore, the ?-CD-threaded biocleavable PRXs may be promising therapeutics for ameliorating not only cholesterol accumulation but also autophagy impairment in NPC disease.
Project description:Five polyrotaxanes were synthesized by threading 2-hydroxypropyl-?-cyclodextrin (HP-?-CD) onto a variety of ?,?-ditriethylenediamino-N-carbamoyl-poly-(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (Pluronic) triblock copolymers using a two-pot strategy under heterogeneous, nonaqueous conditions. The threaded HP-?-CD units were retained on the pseudopolyrotaxane precursors by end-capping the branched diamine termini with sodium 2,4,6-trinitrobenzene sulfonate. Inclusion of the Pluronic copolymers within the HP-?-CD cavities was more favorable in nonpolar solvents, such as diethyl ether and n-hexane, both of which gave better coverage ratios than polar solvents. (1)H NMR and MALDI-TOF were used to estimate the average molecular weights of the purified polyrotaxane products. A globular morphology of aggregated polyrotaxanes was observed by tapping-mode AFM imaging of dried samples. Treatment of Niemann-Pick C (NPC) type 2-deficient fibroblasts with the polyrotaxane derivatives produced substantial reductions in sterol accumulation, as seen by diminished filipin staining in these cells, suggesting that Pluronic-based polyrotaxanes may be promising vehicles for delivery of HP-?-CD to cells with abnormal cholesterol accumulation.
Project description:Niemann-Pick Type C1 disorder (NPC) is a rare lysosomal storage disease characterized by the accumulation of cholesterol in lysosomes. NPC has no FDA approved treatments yet, however 2-hydroxypropyl-?-cyclodextrin (HP?CD) has shown efficacy for treating the disease in both mouse and feline NPC models and is currently being investigated in late stage clinical trials. Despite promising results, therapeutic use of HP?CD is limited by the need for high doses, ototoxicity and intrathecal administration. These limitations can be attributed to its poor pharmacokinetic profile. In the attempt to overcome these limitations, we have designed a ?-cyclodextrin (?CD) based polymer prodrugs (ORX-301) for an enhanced pharmacokinetic and biodistribution profile, which in turn can potentially provide an improved efficacy at lower doses. We demonstrated that subcutaneously injected ORX-301 extended the mean lifespan of NPC mice at a dosage 5-fold lower (800?mg/kg, body weight) the HP?CD dose proven efficacious (4000?mg/kg). We also show that ORX-301 penetrates the blood brain barrier and counteracts neurological impairment. These properties represent a substantial improvement and appear to overcome major limitations of presently available ?CD-based therapy, demonstrating that this novel prodrug is a valuable alternative/complement for existing therapies.
Project description:Niemann-Pick type C (NPC) disease is a fatal hereditary neurodegenerative disorder characterized by a massive accumulation of cholesterol in lysosomes and late endosomes due to a defect in intracellular cholesterol trafficking. Dysfunction in intracellular cholesterol trafficking is responsible for about 50 rare inherited lysosomal storage disorders including NPC. The lysosomal proteins NPC1 and NPC2 play a crucial role in trafficking of cholesterol from late endosomes and lysosomes to other cellular compartments. However, the detailed mechanisms of cholesterol trafficking at the late endosomes/lysosomes (LE/LY) are poorly understood. Studies showed that 2-hydroxypropyl-?-cyclodextrin (HP?CD) alleviates the cholesterol accumulation defect in animal model and has been approved for a phase 2b/3 clinical trial for NPC. HP?CD is known to bind cholesterol; however, the mechanisms how HP?CD mediates the exit of cholesterol from the LE/LY compartments are still unknown. Further, another cyclodextrin (CD) derivative, 2-hydroxypropyl-?-cyclodextrin (HP?CD), was shown to reduce intracellular cholesterol accumulation in NPC patient cells and NPC mice model. Herein, we identified a number of candidate proteins differentially expressed in NPC patient-derived cells compared to cells derived from a healthy donor using a proteomic approach. Interestingly, both HP?CD and HP?CD treatments modulated the expression of most of these NPC-specific proteins. Data showed that treatment with both CDs induces the expression of the lysosome-associated membrane protein 1 (LAMP-1) in NPC patient-derived cells. Remarkably, LAMP-1 overexpression in HeLa cells rescued U18666A-induced cholesterol accumulation suggesting a role of LAMP-1 in cholesterol trafficking. We propose that HP?CD and HP?CD facilitate cholesterol export from the LE/LY compartments via the LAMP-1 protein, which may play a crucial role in cholesterol trafficking at the LE/LY compartments when there is no functional NPC1 protein. Together, this study uncovers new cellular mechanisms for cholesterol trafficking, which will contribute to development of novel therapeutic approaches for lysosomal storage diseases.
Project description:Niemann-Pick disease type C (NPC) is an autosomal recessive disorder characterized by abnormal accumulation of free cholesterol and sphingolipids in lysosomes. The iminosugar miglustat, which inhibits hexosylceramide synthesis, is used for NPC treatment, and 2-hydroxypropyl-?-cyclodextrin (HP-?-CD), a cyclic oligosaccharide derivative, is being developed to treat NPC. Moreover, therapeutic potential of 2-hydroxypropyl-?-cyclodextrin (HP-?-CD) was shown in NPC models, although its mechanism of action remains unclear. Here, we investigated the effects of HP-?-CD, HP-?-CD, and their homolog 2-hydroxypropyl-?-cyclodextrin (HP-?-CD) on lipid accumulation in Npc1-null Chinese hamster ovary (CHO) cells compared with those of miglustat. HP-?-CD and HP-?-CD, unlike HP-?-CD, reduced intracellular free cholesterol levels and normalized the lysosome changes in Npc1-null cells but not in wild-type CHO cells. In contrast, miglustat did not normalize intracellular free cholesterol accumulation or lysosome changes in Npc1-null cells. However, miglustat decreased the levels of hexosylceramide and tended to increase those of sphingomyelins in line with its action as a glucosylceramide synthase inhibitor in both Npc1-null and wild-type CHO cells. Interestingly, HP-?-CD and HP-?-CD, unlike HP-?-CD, reduced sphingomyelins in Npc1-null, but not wild-type, cells. In conclusion, HP-?-CD and HP-?-CD reduce the accumulation of sphingolipids, mainly sphingomyelins, and free cholesterol as well as lysosome changes in Npc1-null, but not in wild-type, CHO cells.
Project description:BACKGROUND:Niemann-Pick Disease Type C (NPC) is an inherited, often fatal neurovisceral lysosomal storage disease characterized by cholesterol accumulation in every cell with few known treatments. Defects in cholesterol transport cause sequestration of unesterified cholesterol within the endolysosomal system. The discovery that systemic administration of hydroxypropyl-beta cyclodextrin (HP?PD) to NPC mice could release trapped cholesterol from lysosomes, normalize cholesterol levels in the liver, and prolong life, led to expanded access use in NPC patients. HP?CD has been administered to NPC patients with approved INDs globally since 2009. RESULTS:Here we present safety, tolerability and efficacy data from 12 patients treated intravenously (IV) for over 7?years with HP?CD in the US and Brazil. Some patients subsequently received intrathecal (IT) treatment with HP?CD following on average 13?months of IV HP?CD. Several patients transitioned to an alternate HP?CD. Moderately affected NPC patients treated with HP?CD showed slowing of disease progression. Severely affected patients demonstrated periods of stability but eventually showed progression of disease. Neurologic and neurocognitive benefits were seen in most patients with IV alone, independent of the addition of IT administration. Physicians and caregivers reported improvements in quality of life for the patients on IV therapy. There were no safety issues, and the drug was well tolerated and easy to administer. CONCLUSIONS:These expanded access data support the safety and potential benefit of systemic IV administration of HP?CD and provide a platform for two clinical trials to study the effect of intravenous administration of HP?CD in NPC patients.
Project description:Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder caused by mutations in NPC1 and NPC2 genes that result in an accumulation of cholesterol in lysosomes. The majority of children with NPC die in adolescence. Currently, no FDA-approved therapies exist for NPC and the mechanisms of NPC disease are not fully understood. Our recent study and the reports from other laboratories showed that 2-hydroxypropyl-?-cyclodextrin (HP?CD) alleviates cholesterol accumulation in NPC1-deficient cells in spite of its low binding affinity for cholesterol. In this study, we explored the cellular changes that are induced upon HP?CD treatment in NPC1 patient-derived fibroblasts. We show that HP?CD treatment increases lysosome-ER association and enhances autophagic activity. Our study indicates that HP?CD induces an activation of the transcription factor EB (TFEB), a master regulator of lysosomal functions and autophagy. Lysosome-ER association could potentially function as conduits for cholesterol transport from lysosomes to the ER. Accumulating evidence suggests a role for autophagy in rescuing the cholesterol accumulation in NPC and other degenerative diseases. Collectively, our findings suggest that HP?CD restores cellular homeostasis in NPC1-deficient cells via enhancing lysosomal dynamics and functions. Understanding the mechanisms of HP?CD-induced cellular pathways could contribute to effective NPC therapies.
Project description:Niemann-Pick type C disease (NPC) is a lysosomal storage disease that is characterized by a progressive accumulation of unesterified cholesterol in the lysosomes leading to organ damage from cell dysfunction. Hydroxypropyl-?-cyclodextrin (HP-?-CD) is an attractive drug candidate for treating NPC, as it diminishes cholesterol accumulation in NPC cells. Systemic HP-?-CD treatment, however, is limited by rapid renal clearance. We designed a new anionic HP-?-CD polyrotaxane to act as a slow release formulation based on a polyalkylene phosphate core to improve the pharmacokinetics. The polyalkylene phosphate comprises hydrophobic decamethylene spacers linked by biodegradable anionic phosphodiester bonds. HP-?-CD was threaded onto this polymer first and ?-CD afterwards to prevent burst release of the threaded HP-?-CD. Our findings show that HP-?-CD was slowly released from the watersoluble polyrotaxane over a 30 days period. The polyrotaxane provided persistently diminished cholesterol levels in NPC1 cells by 20% relative to untreated cells. These results demonstrate the therapeutic potential of this novel HP-?-CD polyrotaxane for the mobilization of aberrantly stored cholesterol in NPC1 cells.
Project description:The drug 2-hydroxypropyl-?-cyclodextrin (HP?CD) reduces lysosomal cholesterol accumulation in Niemann-Pick disease, type C (NPC) and has been advanced to human clinical trials. However, its mechanism of action for reducing cholesterol accumulation in NPC cells is uncertain and its molecular target is unknown. We found that methyl-?-cyclodextrin (M?CD), a potent analog of HP?CD, restored impaired macroautophagy/autophagy flux in Niemann-Pick disease, type C1 (NPC1) cells. This effect was mediated by a direct activation of AMP-activated protein kinase (AMPK), an upstream kinase in the autophagy pathway, through M?CD binding to its ?-subunits. Knockdown of PRKAB1 or PRKAB2 (encoding the AMPK ?1 or ?2 subunit) expression and an AMPK inhibitor abolished M?CD-mediated reduction of cholesterol storage in NPC1 cells. The results demonstrate that AMPK is the molecular target of M?CD and its activation enhances autophagy flux, thereby mitigating cholesterol accumulation in NPC1 cells. The results identify AMPK as an attractive target for drug development to treat NPC.
Project description:Several lines of evidence suggest that ?-cyclodextrin (?-CD) derivatives initiate the efflux of accumulated, unesterified cholesterol from the late endosomal/lysosomal compartment in Niemann Pick C (NPC) disease models. Unfortunately, repeated injections or continuous infusions of current ?-CD therapies are required to sustain suppression of symptoms and prolong life. In an effort to make CD treatment a more viable option by boosting efficacy and improving pharmacokinetics, a library of Pluronic surfactant-based ?-CD polyrotaxanes has been developed using biocompatible poly(ethylene glycol) (PEG)-polypropylene glycol (PPG)-PEG triblock copolymers. These compounds carry multiple copies of ?-CD as shown by (1)H NMR, 2D nuclear Overhouser effect spectroscopy, gel permeation chromatography/multiangle light scattering, analytical ultracentrifugation analysis, matrix assisted laser desorption/ionization mass spectrometry, and diffusion-ordered spectroscopy. Analyses of free ?-cyclodextrin contamination in the compounds were made by reverse phase high pressure liquid chromatography and hydrophilic interaction liquid chromatography. Dethreading kinetics were studied by reverse phase high pressure liquid chromatography, UV/vis, and (1)H NMR analysis. Filipin staining studies using npc2(-/-) fibroblasts show significant reversal of cholesterol accumulation after treatment with polyrotaxane compounds. The rate and efficacy of reversal is similar to that achieved by equivalent amounts of monomeric ?-CD alone.