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BACKGROUND: External ventricular drainage (EVD) carries a high risk of ventriculitis, increasingly caused by MDR Gram-negative bacteria such as Escherichia coli and Acinetobacter baumannii. Existing antimicrobial EVD catheters are not effective against these, and we have developed a catheter with activity against MDR bacteria and demonstrated the safety of the new formulation for use in the brain. OBJECTIVES: Our aim was to determine the ability of a newly formulated impregnated EVD catheters to withstand challenge with MDR Gram-negative bacteria and to obtain information about its safety for use in the CNS. METHODS: Catheters impregnated with three antimicrobials (rifampicin, trimethoprim and triclosan) were challenged in flow conditions at four weekly timepoints with high doses of MDR bacteria, including MRSA and Acinetobacter, and monitored for bacterial colonization. Catheter segments were also inserted intracerebrally into Wistar rats, which were monitored for clinical and behavioural change, and weight loss. Brains were removed after either 1 week or 4 weeks, and examined for evidence of inflammation and toxicity. RESULTS: Control catheters colonized quickly after the first challenge, while no colonization occurred in the impregnated catheters even after the 4 week challenge. Animals receiving the antimicrobial segments behaved normally and gained weight as expected. Neurohistochemistry revealed only surgical trauma and no evidence of neurotoxicity. CONCLUSIONS: The antimicrobial catheter appears to withstand bacterial challenge for at least 4 weeks, suggesting that it might offer protection against infection with MDR Gram-negative bacteria in patients undergoing EVD. It also appears to be safe for use in the CNS.
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Antibacterianos/administración & dosificación , Infecciones Bacterianas/prevención & control , Infecciones Relacionadas con Catéteres/prevención & control , Cateterismo/efectos adversos , Cateterismo/métodos , Ventriculitis Cerebral/prevención & control , Animales , Catéteres/microbiología , Pérdida de Líquido Cefalorraquídeo , Modelos Animales de Enfermedad , Humanos , Masculino , Modelos Teóricos , Ratas Wistar , Rifampin/administración & dosificación , Resultado del Tratamiento , Triclosán/administración & dosificación , Trimetoprim/administración & dosificaciónRESUMEN
BACKGROUND: The design and use of convection-enhanced delivery catheters remains an active field as clinical trials have highlighted suboptimal distribution as a contributory factor to the failure of those studies. Recent studies indicate limitations and challenges in achieving target coverage using conventional point source delivery. NEW METHOD: The recessed step catheter(RSC), developed by this group, does not function as a point source delivery device, but instead uses 'controlled reflux' of the infusate to a flow inhibiting recess feature. Here we investigate a range of clinically useful step lengths in agarose gel and investigate proof-of-principle in vivo(n = 5). Infusion morphology was characterised in terms of length, width and distribution volume over a range of flow rates. RESULTS: For a fixed infusion volume, increases in catheter step length strongly correlated with increases in the length and volume of distribution (r>0.90, p < 0.001) whilst there were small reductions in the width of distribution (r<-0.62, p < 0.001). Step lengths below 6 mm produced spherical distributions while steps above 12 mm produced elongated distributions. Increasing peak flow rates resulted in significant reductions in distribution volume at each step length, and an increased risk of reflux beyond the step. Modifications to the infusion morphology using changes in step length were confirmed in vivo. CONCLUSIONS: The combination of the recessed step and the ability to adjust the step length with this catheter design make it highly suitable for tailoring the distribution volume of the infusate to meet specific morphological target volumes in the brain.
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Encéfalo/fisiología , Sistemas de Liberación de Medicamentos/instrumentación , Sistemas de Liberación de Medicamentos/métodos , Animales , Cateterismo/instrumentación , Cateterismo/métodos , Catéteres , Convección , Sus scrofaRESUMEN
BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is a lethal type of pediatric brain tumor that is resistant to conventional chemotherapies. Palbociclib is a putative novel DIPG treatment that restricts the proliferation of rapidly dividing cancer cells via selective inhibition of cyclin-dependent kinase (CDK) 4 and CDK6. However, implementing palbociclib as a monotherapy for DIPG is unfeasible, as CDK4/6 inhibitor resistance is commonplace and palbociclib does not readily cross the blood-brain barrier (BBB) or persist in the central nervous system. To inhibit the growth of DIPG cells, we aimed to use palbociclib in combination with the rapamycin analog temsirolimus, which is known to ameliorate resistance to CDK4/6 inhibitors and inhibit BBB efflux. MATERIALS AND METHODS: We tested palbociclib and temsirolimus in three patient-derived DIPG cell lines. The expression profiles of key proteins in the CDK4/6 and mammalian target of rapamycin (mTOR) signaling pathways were assessed, respectively, to determine feasibility against DIPG. Moreover, we investigated effects on cell viability and examined in vivo drug toxicity. RESULTS: Immunoblot analyses revealed palbociclib and temsirolimus inhibited CDK4/6 and mTOR signaling through canonical perturbation of phosphorylation of the retinoblastoma (RB) and mTOR proteins, respectively; however, we observed noncanonical downregulation of mTOR by palbociclib. We demonstrated that palbociclib and temsirolimus inhibited cell proliferation in all three DIPG cell lines, acting synergistically in combination to further restrict cell growth. Flow cytometric analyses revealed both drugs caused G1 cell cycle arrest, and clonogenic assays showed irreversible effects on cell proliferation. Palbociclib did not elicit neurotoxicity in primary cultures of normal rat hippocampi or when infused into rat brains. CONCLUSION: These data illustrate the in vitro antiproliferative effects of CDK4/6 and mTOR inhibitors in DIPG cells. Direct infusion of palbociclib into the brain, in combination with systemic delivery of temsirolimus, represents a promising new approach to developing a much-needed treatment for DIPG.
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OBJECTIVE The pan-histone deacetylase inhibitor panobinostat has preclinical efficacy against diffuse intrinsic pontine glioma (DIPG), and the oral formulation has entered a Phase I clinical trial. However, panobinostat does not cross the blood-brain barrier in humans. Convection-enhanced delivery (CED) is a novel neurosurgical drug delivery technique that bypasses the blood-brain barrier and is of considerable clinical interest in the treatment of DIPG. METHODS The authors investigated the toxicity, distribution, and clearance of a water-soluble formulation of panobinostat (MTX110) in a small- and large-animal model of CED. Juvenile male Wistar rats (n = 24) received panobinostat administered to the pons by CED at increasing concentrations and findings were compared to those in animals that received vehicle alone (n = 12). Clinical observation continued for 2 weeks. Animals were sacrificed at 72 hours or 2 weeks following treatment, and the brains were subjected to neuropathological analysis. A further 8 animals received panobinostat by CED to the striatum and were sacrificed 0, 2, 6, or 24 hours after infusion, and their brains explanted and snap-frozen. Tissue-drug concentration was determined by liquid chromatography tandem mass spectrometry (LC-MS/MS). Large-animal toxicity was investigated using a clinically relevant MRI-guided translational porcine model of CED in which a drug delivery system designed for humans was used. Panobinostat was administered at 30 µM to the ventral pons of 2 juvenile Large White-Landrace cross pigs. The animals were subjected to clinical and neuropathological analysis, and findings were compared to those obtained in controls after either 1 or 2 weeks. Drug distribution was determined by LC-MS/MS in porcine white and gray matter immediately after CED. RESULTS There were no clinical or neuropathological signs of toxicity up to an infused concentration of 30 µM in both small- and large-animal models. The half-life of panobinostat in rat brain after CED was 2.9 hours, and the drug was observed to be distributed in porcine white and gray matter with a volume infusion/distribution ratio of 2 and 3, respectively. CONCLUSIONS CED of water-soluble panobinostat, up to a concentration of 30 µM, was not toxic and was distributed effectively in normal brain. CED of panobinostat warrants clinical investigation in patients with DIPG.
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Antineoplásicos/administración & dosificación , Neoplasias del Tronco Encefálico/tratamiento farmacológico , Convección , Glioma/tratamiento farmacológico , Panobinostat/administración & dosificación , Animales , Antineoplásicos/farmacocinética , Neoplasias del Tronco Encefálico/diagnóstico por imagen , Proteínas de Unión al Calcio/metabolismo , Cromatografía Liquida , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Proteína Ácida Fibrilar de la Glía/metabolismo , Glioma/diagnóstico por imagen , Imagen por Resonancia Magnética , Masculino , Proteínas de Microfilamentos/metabolismo , Panobinostat/farmacocinética , Fosfopiruvato Hidratasa/metabolismo , Ratas , Ratas Wistar , Porcinos , Espectrometría de Masas en Tándem , Factores de Tiempo , Distribución Tisular/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Targeting epigenetic changes in diffuse intrinsic pontine glioma (DIPG) may provide a novel treatment option for patients. This report demonstrates that sodium valproate, a histone deacetylase inhibitor (HDACi), can increase the cytotoxicity of carboplatin in an additive and synergistic manner in DIPG cells in vitro. Sodium valproate causes a dose-dependent decrease in DIPG cell viability in three independent ex vivo cell lines. Furthermore, sodium valproate caused an increase in acetylation of histone H3. Changes in cell viability were consistent with an induction of apoptosis in DIPG cells in vitro, determined by flow cytometric analysis of Annexin V staining and assessment of apoptotic markers by western blotting. Subsequently, immunofluorescent staining of neuronal and glial markers was used to determine toxicity in normal rat hippocampal cells. Pre-treatment of cells with sodium valproate enhanced the cytotoxic effects of carboplatin, in three DIPG cell lines tested. These results demonstrate that sodium valproate causes increased histone H3 acetylation indicative of HDAC inhibition, which is inversely correlated with a reduction in cell viability. Cell viability is reduced through an induction of apoptosis in DIPG cells. Sodium valproate potentiates carboplatin cytotoxicity and prompts further work to define the mechanism responsible for the synergy between these two drugs and determine in vivo efficacy. These findings support the use of sodium valproate as an adjuvant treatment for DIPG.
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Adyuvantes Farmacéuticos/farmacología , Anticonvulsivantes/farmacología , Neoplasias del Tronco Encefálico/tratamiento farmacológico , Glioma/tratamiento farmacológico , Ácido Valproico/farmacología , Acetilación/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Neoplasias del Tronco Encefálico/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Reposicionamiento de Medicamentos/métodos , Epigénesis Genética/efectos de los fármacos , Glioma/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Humanos , RatasRESUMEN
BACKGROUND: Intraparenchymal convection-enhanced delivery (CED) of therapeutics directly into the brain has long been endorsed as a medium through which meaningful concentrations of drug can be administered to patients, bypassing the blood brain barrier. The translation of the technology to clinic has been hindered by poor distribution not previously observed in smaller pre-clinical models. In part this was due to the larger volumes of target structures found in humans but principally the poor outcome was linked to reflux (backflow) of infusate proximally along the catheter track. Over the past 10 years, improvements have been made to the technology in the field which has led to a small number of commercially available devices containing reflux inhibiting features. NEW METHOD: While these devices are currently suitable for acute or short term use, several indications would benefit from longer term repeated, intermittent administration of therapeutics (Parkinson's, Alzheimer's, Amyotrophic lateral sclerosis, Brain tumours such as Glioblastoma Multiforme (GBM) and Diffuse intrinsic Pontine Glioma (DIPG), etc.). RESULTS: Despite the need for a chronically accessible platform for such indications, limited experience exists in this part of the field. COMPARISON WITH EXISTING METHOD(S): At the time of writing no commercially available clinical platform, indicated for chronic, intermittent or continuous delivery to the brain exists. CONCLUSIONS: Here we review the improvements that have been made to CED devices over recent years and current state of the art for chronic infusion systems.
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Encéfalo , Catéteres , Sistemas de Liberación de Medicamentos/métodos , Convección , HumanosRESUMEN
CONTEXT: Inadequate penetration of the blood-brain barrier (BBB) by systemically administered chemotherapies including carboplatin is implicated in their failure to improve prognosis for patients with glioblastoma. Convection-enhanced delivery (CED) of carboplatin has the potential to improve outcomes by facilitating bypass of the BBB. OBJECTIVE: We report the first use of an implantable CED system incorporating a novel transcutaneous bone-anchored port (TBAP) for intermittent CED of carboplatin in a patient with recurrent glioblastoma. MATERIALS AND METHODS: The CED catheter system was implanted using a robot-assisted surgical method. Catheter targeting accuracy was verified by performing intra-operative O-arm imaging. The TBAP was implanted using a skin-flap dermatome technique modeled on bone-anchored hearing aid surgery. Repeated infusions were performed by attaching a needle administration set to the TBAP. Drug distribution was monitored with serial real-time T2-weighted magnetic resonance imaging (MRI). RESULTS: All catheters were implanted to within 1.5 mm of their planned target. Intermittent infusions of carboplatin were performed on three consecutive days and repeated after one month without the need for further surgical intervention. Infused volumes of 27.9 ml per day were well tolerated, with the exception of a single seizure episode. Follow-up MRI at eight weeks demonstrated a significant reduction in the volume of tumor enhancement from 42.6 ml to 24.6 ml, and was associated with stability of the patient's clinical condition. CONCLUSION: Reduction in the volume of tumor enhancement indicates that intermittent CED of carboplatin has the potential to improve outcomes in glioblastoma. The novel technology described in this report make intermittent CED infusion regimes an achievable treatment strategy.
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Antineoplásicos/administración & dosificación , Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Carboplatino/administración & dosificación , Carboplatino/uso terapéutico , Sistemas de Liberación de Medicamentos/métodos , Glioblastoma/tratamiento farmacológico , Administración Cutánea , Antineoplásicos/efectos adversos , Carboplatino/efectos adversos , Catéteres de Permanencia , Convección , Epilepsia Generalizada/complicaciones , Femenino , Humanos , Infusiones Intravenosas , Imagen por Resonancia Magnética , Persona de Mediana Edad , RobóticaRESUMEN
We currently use Convection-Enhanced Delivery (CED) of the platinum-based drug, carboplatin as a novel treatment strategy for high grade glioblastoma in adults and children. Although initial results show promise, carboplatin is not specifically toxic to tumour cells and has been associated with neurotoxicity at high infused concentrations in pre-clinical studies. Our treatment strategy requires intermittent infusions due to rapid clearance of carboplatin from the brain. In this study, carboplatin was encapsulated in lactic acid-glycolic acid copolymer (PLGA) to develop a novel drug delivery system. Neuronal and tumour cytotoxicity were assessed in primary neuronal and glioblastoma cell cultures. Distribution, tissue clearance and toxicity of carboplatin nanoparticles following CED was assessed in rat and porcine models. Carboplatin nanoparticles conferred greater tumour cytotoxicity, reduced neuronal toxicity and prolonged tissue half-life. In conclusion, this drug delivery system has the potential to improve the prognosis for patients with glioblastomas.
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Carboplatino/uso terapéutico , Convección , Sistemas de Liberación de Medicamentos , Glioblastoma/tratamiento farmacológico , Ácido Láctico/química , Nanopartículas/química , Ácido Poliglicólico/química , Animales , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Células Cultivadas , Endocitosis/efectos de los fármacos , Glioblastoma/patología , Hipocampo/patología , Humanos , Masculino , Nanopartículas/toxicidad , Neurotoxinas/toxicidad , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Ratas Wistar , Sus scrofaRESUMEN
Formulations of cationic liposomes and polymers readily self-assemble by electrostatic interactions with siRNA to form cationic nanoparticles which achieve efficient transfection and silencing in vitro. However, the utility of cationic formulations in vivo is limited due to rapid clearance from the circulation, due to their association with serum proteins, as well as systemic and cellular toxicity. These problems may be overcome with anionic formulations but they provide challenges of self-assembly and transfection efficiency. We have developed anionic, siRNA nanocomplexes utilizing anionic PEGylated liposomes and cationic targeting peptides that overcome these problems. Biophysical measurements indicated that at optimal ratios of components, anionic PEGylated nanocomplexes formed spherical particles and that, unlike cationic nanocomplexes, were resistant to aggregation in the presence of serum, and achieved significant gene silencing although their non-PEGylated anionic counterparts were less efficient. We have evaluated the utility of anionic nanoparticles for the treatment of neuronal diseases by administration to rat brains of siRNA to BACE1, a key enzyme involved in the formation of amyloid plaques. Silencing of BACE1 was achieved in vivo following a single injection of anionic nanoparticles by convection enhanced delivery and specificity of RNA interference verified by 5' RACE-PCR and Western blot analysis of protein.
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Secretasas de la Proteína Precursora del Amiloide/genética , Ácido Aspártico Endopeptidasas/genética , Liposomas/química , Nanopartículas/química , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Animales , Aniones/química , Encéfalo/metabolismo , Encefalopatías/genética , Encefalopatías/terapia , Línea Celular , Terapia Genética , Humanos , Lípidos/química , Masculino , Polietilenglicoles/química , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/uso terapéutico , Ratas , Ratas Wistar , Transfección/métodosRESUMEN
The main determinant of glioblastoma (GBM) resistance to temozolomide (TMZ) is thought to be O(6)-methylguanine-DNA methyltransferase (MGMT), which is a DNA-repair enzyme that removes alkyl groups from the O(6)-position of guanine. Previously, we reported that a MGMT-siRNA/cationic liposome complex exerted a clear synergistic antitumor effect in combination with TMZ. Translation to a clinical setting might be desirable for reinforcing the efficacy of TMZ therapy for GBM. In this study, we aim to evaluate the safety of MGMT-siRNA/cationic liposome complexes and determine whether the convection-enhanced delivery of these complexes is suitable for clinical use by undertaking preclinical testing in laboratory animals. No significant adverse events were observed in rats receiving infusions of MGMT-siRNA/cationic liposome complex directly into the brain with or without TMZ administration. A pig which received the complex administered by CED also showed no evidence of neurological dysfunction or histological abnormalities. However, the complex did not appear to achieve effective distribution by CED in either the rat or the porcine brain tissue. Considering these results together, we concluded that insufficient distribution of cationic liposomes was achieved for tumor treatment by CED.
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Non-viral vector formulations comprise typically complexes of nucleic acids with cationic polymers or lipids. However, for in vivo applications cationic formulations suffer from problems of poor tissue penetration, non-specific binding to cells, interaction with serum proteins and cell adhesion molecules and can lead to inflammatory responses. Anionic formulations may provide a solution to these problems but they have not been developed to the same extent as cationic formulations due to difficulties of nucleic acid packaging and poor transfection efficiency. We have developed novel PEGylated, anionic nanocomplexes containing cationic targeting peptides that act as a bridge between PEGylated anionic liposomes and plasmid DNA. At optimized ratios, the components self-assemble into anionic nanocomplexes with a high packaging efficiency of plasmid DNA. Anionic PEGylated nanocomplexes were resistant to aggregation in serum and transfected cells with a far higher degree of receptor-targeted specificity than their homologous non-PEGylated anionic and cationic counterparts. Gadolinium-labeled, anionic nanoparticles, administered directly to the brain by convection-enhanced delivery displayed improved tissue penetration and dispersal as well as more widespread cellular transfection than cationic formulations. Anionic PEGylated nanocomplexes have widespread potential for in vivo gene therapy due to their targeted transfection efficiency and ability to penetrate tissues.
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ADN/administración & dosificación , Nanopartículas/administración & dosificación , Péptidos/metabolismo , Polietilenglicoles/química , Transfección/métodos , Animales , Encéfalo/metabolismo , Línea Celular , Línea Celular Tumoral , Medios de Contraste/química , ADN/química , Colorantes Fluorescentes/química , Gadolinio/química , Humanos , Lípidos/química , Liposomas , Masculino , Ratones , Nanopartículas/química , Ratas , Ratas Wistar , Rodaminas/químicaRESUMEN
Convection enhanced delivery (CED) is a method of direct injection to the brain that can achieve widespread dispersal of therapeutics, including gene therapies, from a single dose. Non-viral, nanocomplexes are of interest as vectors for gene therapy in the brain, but it is essential that administration should achieve maximal dispersal to minimise the number of injections required. We hypothesised that anionic nanocomplexes administered by CED should disperse more widely in rat brains than cationics of similar size, which bind electrostatically to cell-surface anionic moieties such as proteoglycans, limiting their spread. Anionic, receptor-targeted nanocomplexes (RTN) containing a neurotensin-targeting peptide were prepared with plasmid DNA and compared with cationic RTNs for dispersal and transfection efficiency. Both RTNs were labelled with gadolinium for localisation in the brain by MRI and in brain sections by LA-ICP-MS, as well as with rhodamine fluorophore for detection by fluorescence microscopy. MRI distribution studies confirmed that the anionic RTNs dispersed more widely than cationic RTNs, particularly in the corpus callosum. Gene expression levels from anionic formulations were similar to those of cationic RTNs. Thus, anionic RTN formulations can achieve both widespread dispersal and effective gene expression in brains after administration of a single dose by CED.
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Encéfalo/metabolismo , Técnicas de Transferencia de Gen , Nanopartículas/química , Ácidos Nucleicos/uso terapéutico , Receptores de Superficie Celular/metabolismo , Animales , Encéfalo/efectos de los fármacos , Línea Celular Tumoral , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Liposomas/química , Imagen por Resonancia Magnética , Masculino , Ratones , Nanosferas , Ácidos Nucleicos/farmacología , Péptidos/metabolismo , Plásmidos/metabolismo , Ratas , Ratas Wistar , Espectrofotometría Atómica , Distribución Tisular/efectos de los fármacos , TransfecciónRESUMEN
Enzymatic degradation contributes to the control of intracerebral amyloid-ß (Aß) peptide levels. Previous studies have demonstrated the therapeutic potential of viral vector-mediated neprilysin (NEP) gene therapy in mouse models of Alzheimer's disease (AD). However, clinical translation of NEP gene therapy is limited by ethical and practical considerations. In this study we have assessed the potential of convection-enhanced delivery (CED) as a means of elevating intracerebral NEP level and activity and degrading endogenous Aß. We analyzed the interstitial and perivascular distribution of NEP following CED into rat striatum. We measured NEP protein level, clearance, activity, and toxicity by ELISA for NEP and synaptophysin, NEP-specific activity assay, and immunohistochemistry for NEP, NeuN, glial fibrillary acidic protein and Iba1. We subsequently performed CED of NEP in normal aged rats and measured endogenous Aß by ELISA. CED resulted in widespread distribution of NEP, and a 20-fold elevation of NEP protein level with preservation of enzyme activity and without evidence of toxicity. CED in normal, aged rats resulted in a significant reduction in endogenous Aß(40) (p = 0.04), despite rapid NEP clearance from the brain (half-life ~3 h). CED of NEP has therapeutic potential as a dynamically controllable Aß(40)-degrading therapeutic strategy for AD. Further studies are required to determine the longer term effects on Aß (including Aß(42)) and on cognitive function.
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Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/metabolismo , Neprilisina/administración & dosificación , Neprilisina/uso terapéutico , Envejecimiento/fisiología , Enfermedad de Alzheimer/metabolismo , Animales , Antígenos Nucleares/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Proteínas de Unión al Calcio/metabolismo , Cateterismo , Relación Dosis-Respuesta a Droga , Sistemas de Liberación de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica , Masculino , Proteínas de Microfilamentos/metabolismo , Neprilisina/farmacocinética , Proteínas del Tejido Nervioso/metabolismo , Neuroimagen , Nimodipina/farmacología , Vehículos Farmacéuticos , Polietilenglicoles , Ratas , Ratas Wistar , Sinaptofisina/metabolismoRESUMEN
Gadolinium-labelled nanocomplexes offer prospects for the development of real-time, non-invasive imaging strategies to visualise the location of gene delivery by MRI. In this study, targeted nanoparticle formulations were prepared comprising a cationic liposome (L) containing a Gd-chelated lipid at 10, 15 and 20% by weight of total lipid, a receptor-targeted, DNA-binding peptide (P) and plasmid DNA (D), which electrostatically self-assembled into LPD nanocomplexes. The LPD formulation containing the liposome with 15% Gd-chelated lipid displayed optimal peptide-targeted, transfection efficiency. MRI conspicuity peaked at 4h after incubation of the nanocomplexes with cells, suggesting enhancement by cellular uptake and trafficking. This was supported by time course confocal microscopy analysis of transfections with fluorescently-labelled LPD nanocomplexes. Gd-LPD nanocomplexes delivered to rat brains by convection-enhanced delivery were visible by MRI at 6 h, 24 h and 48 h after administration. Histological brain sections analysed by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) confirmed that the MRI signal was associated with the distribution of Gd(3+) moieties and differentiated MRI signals due to haemorrhage. The transfected brain cells near the injection site appeared to be mostly microglial. This study shows the potential of Gd-LPD nanocomplexes for simultaneous delivery of contrast agents and genes for real-time monitoring of gene therapy in the brain.
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Medios de Contraste/administración & dosificación , ADN/administración & dosificación , Gadolinio/administración & dosificación , Glicosiltransferasas/administración & dosificación , Nanopartículas/administración & dosificación , Animales , Encéfalo/metabolismo , Línea Celular Tumoral , Medios de Contraste/química , Medios de Contraste/farmacocinética , ADN/química , Ácidos Grasos Monoinsaturados/química , Gadolinio/química , Gadolinio/farmacocinética , Glicosiltransferasas/química , Humanos , Imagen por Resonancia Magnética/métodos , Masculino , Nanopartículas/química , Péptidos , Fosfatidiletanolaminas/química , Compuestos de Amonio Cuaternario/química , Ratas , Ratas Wistar , Transfección/métodosRESUMEN
BACKGROUND: Convection-enhanced delivery (CED), a direct method for drug delivery to the brain through intraparenchymal microcatheters, is a promising strategy for intracerebral pharmacological therapy. By establishing a pressure gradient at the tip of the catheter, drugs can be delivered in uniform concentration throughout a large volume of interstitial fluid. However, the variables affecting perivascular distribution of drugs delivered by CED are not fully understood. The aim of this study was to determine whether the perivascular distribution of solutes delivered by CED into the striatum of rats is affected by the molecular weight of the infused agent, by co-infusion of vasodilator, alteration of infusion rates or use of a ramping regime. We also wanted to make a preliminary comparison of the distribution of solutes with that of nanoparticles. METHODS: We analysed the perivascular distribution of 4, 10, 20, 70, 150 kDa fluorescein-labelled dextran and fluorescent nanoparticles at 10 min and 3 h following CED into rat striatum. We investigated the effect of local vasodilatation, slow infusion rates and ramping on the perivascular distribution of solutes. Co-localisation with perivascular basement membranes and vascular endothelial cells was identified by immunohistochemistry. The uptake of infusates by perivascular macrophages was quantified using stereological methods. RESULTS: Widespread perivascular distribution and macrophage uptake of fluorescein-labelled dextran was visible 10 min after cessation of CED irrespective of molecular weight. However, a significantly higher proportion of perivascular macrophages had taken up 4, 10 and 20 kDa fluorescein-labelled dextran than 150 kDa dextran (p < 0.05, ANOVA). Co-infusion with vasodilator, slow infusion rates and use of a ramping regime did not alter the perivascular distribution. CED of fluorescent nanoparticles indicated that particles co-localise with perivascular basement membranes throughout the striatum but, unlike soluble dextrans, are not taken up by perivascular macrophages after 3 h. CONCLUSIONS: This study suggests that widespread perivascular distribution and interaction with perivascular macrophages is likely to be an inevitable consequence of CED of solutes. The potential consequences of perivascular distribution of therapeutic agents, and in particular cytotoxic chemotherapies, delivered by CED must be carefully considered to ensure safe and effective translation to clinical trials.
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Nutrient deprivation during ischemia leads to severe insult to neurons causing widespread excitotoxic damage in specific brain regions such as the hippocampus. One possible strategy for preventing neurodegeneration is to express therapeutic proteins in the brain to protect against excitotoxicity. We investigated the utility of equine infectious anemia virus (EIAV)-based vectors as genetic tools for delivery of therapeutic proteins in an in vivo excitotoxicity model. The efficacy of these vectors at preventing cellular loss in target brain areas following excitotoxic insult was also assessed. EIAV vectors generated to overexpress the human antiapoptotic Bcl-2 or growth factor glial-derived neurotrophic factor (GDNF) genes protected against glutamate-induced toxicity in cultured hippocampal neurons. In an in vivo excitotoxicity model, adult Wistar rats received a unilateral dose of the glutamate receptor agonist N-methyl-D-aspartate to the hippocampus that induced a large lesion in the CA1 region. Neuronal loss could not be protected by prior transduction of a control vector expressing beta-galactosidase. In contrast, EIAV-mediated expression of Bcl-2 and GDNF significantly reduced lesion size thus protecting the hippocampus from excitotoxic damage. These results demonstrate that EIAV vectors can be effectively used to deliver putative neuroprotective genes to target brain areas and prevent cellular loss in the event of a neurological insult. Therefore these lentiviral vectors provide potential therapeutic tools for use in cases of acute neurotrauma such as cerebral ischemia.
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Hipocampo/efectos de los fármacos , Hipocampo/fisiopatología , Lentivirus/genética , Factores de Crecimiento Nervioso/metabolismo , Fármacos Neuroprotectores/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Línea Celular , Vectores Genéticos/genética , Factor Neurotrófico Derivado de la Línea Celular Glial , Hipocampo/metabolismo , Hipocampo/patología , Humanos , N-Metilaspartato/toxicidad , Factores de Crecimiento Nervioso/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Ratas , Técnicas de Cultivo de TejidosRESUMEN
In this study we have used a molecular approach to manipulate CREB gene expression to study its role in the regulation of neuronal cell death. To achieve this, adenoviral (Ad) vectors encoding EGFP, CREB, and a powerful CREB dominant-negative, known as A-CREB were constructed. The over-expression of CREB but not A-CREB was found to protect primary hippocampal neurons from staurosporine-induced apoptosis, glutamate induced excitotoxicity and exposure to an in vitro ischaemic stress. Hence, manipulating CREB-regulated pathways may provide a means of delaying or preventing the neuronal cell death associated with ischaemic related injury, and in neurodegenerative diseases such as Huntington's and Alzheimer's disease.
Asunto(s)
Apoptosis/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/biosíntesis , Agonistas de Aminoácidos Excitadores/farmacología , Neuronas/metabolismo , Estrés Fisiológico/metabolismo , Adenoviridae/genética , Animales , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Células HeLa , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Neuronas/efectos de los fármacos , Ratas , Ratas Wistar , Estrés Fisiológico/genéticaRESUMEN
BACKGROUND: Adenoviral (Ad) vectors are one of the most widely used tools for modelling gene therapy strategies. However, they have not been used in long-term models of neurological disease, as the period of time for which they mediate strong transgene expression is limited and/or variable. In this study we investigated the longevity of transgene expression in the brain when the powerful neuron-specific Ad-synapsin (Sy)-EGFP-woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) vector cassette is used at titres that do not elicit an immune response. METHODS: Adenoviral vectors expressing enhanced green fluorescent protein (EGFP) under the control of either the hCMV, hCMV-WPRE, Sy or Sy-WPRE promoter were constructed. These vectors were injected into the dentate gyrus region of hippocampus and transgene expression and immune cell infiltration assessed by fluorescence microscopy and immunocytochemical techniques, respectively. RESULTS: The quantitative analysis of EGFP expression showed that there was no significant change in synapsin or synapsin-WPRE driven transcription 9 months after injection when compared with expression levels obtained 3 days after injection. However, when the hCMV promoter or the hCMV-WPRE promoter cassette drove transgene expression, there was a dramatic fall in expression levels and very little expression was seen 9 months post-transfection. CONCLUSIONS: This study shows that non-integrating vectors can be used to mediate powerful, long-term episomal transgene expression in neurones. This work has important implications for neuronal gene therapy and is of relevance to studies investigating memory, behaviour and neuronal gene function.
Asunto(s)
Adenoviridae/genética , Encéfalo/metabolismo , Vectores Genéticos , Hipocampo/metabolismo , Neuronas/metabolismo , Regiones Promotoras Genéticas , Sinapsinas/genética , Animales , Encéfalo/virología , Línea Celular , Expresión Génica , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes , Virus de la Hepatitis B de la Marmota/genética , Humanos , Proteínas Luminiscentes , Masculino , Neuronas/virología , Procesamiento Postranscripcional del ARN , Ratas , Ratas Wistar , Sinapsinas/metabolismo , TransgenesRESUMEN
Due to the complexity of brain function and the difficulty in monitoring alterations in neuronal gene expression, the potential of lentiviral gene therapy vectors to treat disorders of the CNS has been difficult to fully assess. In this study, we have assessed the utility of a third-generation equine infectious anemia virus (EIAV) in the Brattleboro rat model of diabetes insipidus, in which a mutation in the arginine vasopressin (AVP) gene results in the production of nonfunctional mutant AVP precursor protein. Importantly, by using this model it is possible to monitor the success of the gene therapy treatment by noninvasive assays. Injection of an EIAV-CMV-AVP vector into the supraoptic nuclei of the hypothalamus resulted in expression of functional AVP peptide in magnocellular neurons. This was accompanied by a 100% recovery in water homeostasis as assessed by daily water intake, urine production, and urine osmolality lasting for a 1-year measurement period. These data show that a single gene defect leading to a neurological disorder can be corrected with a lentiviral-based strategy. This study highlights the potential of using viral gene therapy for the long-term treatment of disorders of the CNS.
Asunto(s)
Arginina Vasopresina/genética , Diabetes Insípida Neurogénica/terapia , Terapia Genética , Vectores Genéticos , Virus de la Anemia Infecciosa Equina/genética , Núcleo Supraóptico/metabolismo , Animales , Arginina Vasopresina/metabolismo , Diabetes Insípida Neurogénica/metabolismo , Diabetes Insípida Neurogénica/patología , Homeostasis , Humanos , Hibridación in Situ , Masculino , Ratas , Ratas Brattleboro , Ratas Endogámicas WKY , Núcleo Supraóptico/patología , Factores de Tiempo , Vasoconstrictores/metabolismo , Agua/metabolismoRESUMEN
Viral vectors are excellent tools for studying gene function in the brain, although a limitation has been the ability to effectively target transgene expression to specific neuronal populations. This generally cannot be overcome by the use of neuron-specific promoters, as most are too large to be used with current viral vectors and expression from these promoters is often relatively weak. We therefore developed a composite expression cassette, comprising 495 bp of the weak human SYN1 (synapsin-1) promoter and 800 bp of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Studies in hippocampal cultures, organotypic cultures, and in vivo showed that the 3' addition of the WPRE to the SYN1 element greatly increased enhanced green fluorescent protein expression levels with no loss of neuronal specificity. In vivo studies also showed that transgene expression was enhanced with no loss of neuronal specificity in dentate-gyrus neurons for at least 6 weeks following transfection. Therefore, unlike most powerful promoter systems, which mediate expression in neurons and glia, this SYN1-WPRE cassette can target powerful long-term transgene expression to central nervous system neurons when delivered at relatively low titers of adenovirus. Its use should therefore facilitate both gene therapy studies and investigations of neuronal gene function.