RESUMO
Neuropharmacokinetics considers cerebral drug distribution as a critical process for central nervous system drug action as well as for drug penetration through the CNS barriers. Brain distribution of small molecules obeys classical rules of drug partition, permeability, binding to fluid proteins or tissue components, and tissue perfusion. The biodistribution of all drugs, including both small molecules and biologics, may also be influenced by specific brain properties related to brain anatomy and physiological barriers, fluid dynamics, and cellular and biochemical composition, each of which can exhibit significant interspecies differences. All of these properties contribute to select optimal dosing paradigms and routes of drug delivery to reach brain targets for classical small molecule drugs as well as for biologics. The importance of these properties for brain delivery and exposure also highlights the need for efficient new analytical technologies to more comprehensively investigate drug distribution in the CNS, a complex multi-compartmentalized organ system.
Assuntos
Produtos Biológicos , Encéfalo , Produtos Biológicos/farmacocinética , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Sistemas de Liberação de Medicamentos , Humanos , Preparações Farmacêuticas/metabolismo , Especificidade da Espécie , Distribuição TecidualRESUMO
KEY POINTS: It is unclear precisely how macromolecules (e.g. endogenous proteins and exogenous immunotherapeutics) access brain tissue from the cerebrospinal fluid (CSF). We show that transport at the brain-CSF interface involves a balance between Fickian diffusion in the extracellular spaces at the brain surface and convective transport in perivascular spaces of cerebral blood vessels. Intrathecally-infused antibodies exhibited size-dependent access to the perivascular spaces and tunica media basement membranes of leptomeningeal arteries. Perivascular access and distribution of full-length IgG could be enhanced by intrathecal co-infusion of hyperosmolar mannitol. Pores or stomata present on CSF-facing leptomeningeal cells ensheathing blood vessels in the subarachnoid space may provide unique entry sites into the perivascular spaces from the CSF. These results illuminate new mechanisms likely to govern antibody trafficking at the brain-CSF interface with relevance for immune surveillance in the healthy brain and insights into the distribution of therapeutic antibodies. ABSTRACT: The precise mechanisms governing the central distribution of macromolecules from the cerebrospinal fluid (CSF) to the brain and spinal cord remain poorly understood, despite their importance for physiological processes such as antibody trafficking for central immune surveillance, as well as several ongoing intrathecal clinical trials. In the present study, we clarify how IgG and smaller single-domain antibodies (sdAb) distribute throughout the whole brain in a size-dependent manner after intrathecal infusion in rats using ex vivo fluorescence and in vivo three-dimensional magnetic resonance imaging. Antibody distribution was characterized by diffusion at the brain surface and widespread distribution to deep brain regions along the perivascular spaces of all vessel types, with sdAb accessing a four- to seven-fold greater brain area than IgG. Perivascular transport involved blood vessels of all caliber and putative smooth muscle and astroglial basement membrane compartments. Perivascular access to smooth muscle basement membrane compartments also exhibited size-dependence. Electron microscopy was used to show stomata on leptomeningeal coverings of blood vessels in the subarachnoid space as potential access points allowing substances in the CSF to enter the perivascular space. Osmolyte co-infusion significantly enhanced perivascular access of the larger antibody from the CSF, with intrathecal 0.75 m mannitol increasing the number of perivascular profiles per slice area accessed by IgG by â¼50%. The results of the present study reveal potential distribution mechanisms for endogenous IgG, which is one of the most abundant proteins in the CSF, as well as provide new insights with respect to understanding and improving the drug delivery of macromolecules to the central nervous system via the intrathecal route.
Assuntos
Encéfalo/fisiologia , Sistemas de Liberação de Medicamentos , Espaço Extracelular/metabolismo , Imunoglobulina G/metabolismo , Osmose , Anticorpos de Cadeia Única/farmacocinética , Animais , Transporte Biológico , Transporte Biológico Ativo , Barreira Hematoencefálica/metabolismo , Encéfalo/irrigação sanguínea , Difusão , Feminino , Injeções Espinhais , Imagem Óptica , Ratos , Ratos Sprague-Dawley , Anticorpos de Cadeia Única/administração & dosagem , Anticorpos de Cadeia Única/líquido cefalorraquidiano , Distribuição TecidualRESUMO
Passive immunotherapy, i.e., the administration of exogenous antibodies that recognize a specific target antigen, has gained significant momentum as a potential treatment strategy for several central nervous system (CNS) disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and brain cancer, among others. Advances in antibody engineering to create therapeutic antibody fragments or antibody conjugates have introduced new strategies that may also be applied to treat CNS disorders. However, drug delivery to the CNS for antibodies and other macromolecules has thus far proven challenging, due in large part to the blood-brain barrier and blood-cerebrospinal fluid barriers that greatly restrict transport of peripherally administered molecules from the systemic circulation into the CNS. Here, we summarize the various passive immunotherapy approaches under study for the treatment of CNS disorders, with a primary focus on disease-specific and target site-specific challenges to drug delivery and new, cutting edge methods.
Assuntos
Doenças do Sistema Nervoso Central/terapia , Sistemas de Liberação de Medicamentos/métodos , Imunização Passiva/métodos , HumanosRESUMO
Brain fluids are rigidly regulated to provide stable environments for neuronal function, e.g., low K+, Ca2+, and protein to optimise signalling and minimise neurotoxicity. At the same time, neuronal and astroglial waste must be promptly removed. The interstitial fluid (ISF) of the brain tissue and the cerebrospinal fluid (CSF) bathing the CNS are integral to this homeostasis and the idea of a glia-lymph or 'glymphatic' system for waste clearance from brain has developed over the last 5 years. This links bulk (convective) flow of CSF into brain along the outside of penetrating arteries, glia-mediated convective transport of fluid and solutes through the brain extracellular space (ECS) involving the aquaporin-4 (AQP4) water channel, and finally delivery of fluid to venules for clearance along peri-venous spaces. However, recent evidence favours important amendments to the 'glymphatic' hypothesis, particularly concerning the role of glia and transfer of solutes within the ECS. This review discusses studies which question the role of AQP4 in ISF flow and the lack of evidence for its ability to transport solutes; summarizes attributes of brain ECS that strongly favour the diffusion of small and large molecules without ISF flow; discusses work on hydraulic conductivity and the nature of the extracellular matrix which may impede fluid movement; and reconsiders the roles of the perivascular space (PVS) in CSF-ISF exchange and drainage. We also consider the extent to which CSF-ISF exchange is possible and desirable, the impact of neuropathology on fluid drainage, and why using CSF as a proxy measure of brain components or drug delivery is problematic. We propose that new work and key historical studies both support the concept of a perivascular fluid system, whereby CSF enters the brain via PVS convective flow or dispersion along larger caliber arteries/arterioles, diffusion predominantly regulates CSF/ISF exchange at the level of the neurovascular unit associated with CNS microvessels, and, finally, a mixture of CSF/ISF/waste products is normally cleared along the PVS of venules/veins as well as other pathways; such a system may or may not constitute a true 'circulation', but, at the least, suggests a comprehensive re-evaluation of the previously proposed 'glymphatic' concepts in favour of a new system better taking into account basic cerebrovascular physiology and fluid transport considerations.
Assuntos
Barreira Hematoencefálica/metabolismo , Líquido Cefalorraquidiano/metabolismo , Líquido Extracelular/metabolismo , Animais , Barreira Hematoencefálica/anatomia & histologia , Humanos , HidrodinâmicaRESUMO
Many neuroscientists use the term Blood-Brain Barrier (BBB) to emphasize restrictiveness, often equating or reducing the notion of BBB properties to tight junction molecules physically sealing cerebral endothelial cells, rather than pointing out the complexity of this biological interface with respect to its selectivity and variety of exchange between the general blood circulation and the central nervous tissue. Several authors in the field find it unfortunate that the exquisitely dynamic interfaces between blood and brain continue to be viewed primarily as obstructive barriers to transport. Although the term blood-brain interface is an excellent descriptor that does not convey the idea of a barrier, it is important and preferable for the spreading of an idea beyond specialist communities to try to appeal to well-chosen metaphors. Recent evidence reviewed here indicates that blood-brain interfaces are more than selective semi-permeable membranes in that they display many dynamic processes and complex mechanisms for communication. They are thus more like 'geopolitical borders'. Furthermore, some authors working on blood-brain interface-relevant issues have started to use the word border, for example in border-associated macrophages. Therefore, we suggest adopting the term Blood-Brain Border to better communicate the flexibility of and movement across blood-brain interfaces.
Assuntos
Barreira Hematoencefálica , Sistema Cardiovascular , Células Endoteliais , Encéfalo , Junções ÍntimasRESUMO
Antisense oligonucleotides (ASOs) are promising therapeutics for treating various neurological disorders. However, ASOs are unable to readily cross the mammalian blood-brain barrier (BBB) and therefore need to be delivered intrathecally to the central nervous system (CNS). Here, we engineered a human transferrin receptor 1 (TfR1) binding molecule, the oligonucleotide transport vehicle (OTV), to transport a tool ASO across the BBB in human TfR knockin (TfRmu/hu KI) mice and nonhuman primates. Intravenous injection and systemic delivery of OTV to TfRmu/hu KI mice resulted in sustained knockdown of the ASO target RNA, Malat1, across multiple mouse CNS regions and cell types, including endothelial cells, neurons, astrocytes, microglia, and oligodendrocytes. In addition, systemic delivery of OTV enabled Malat1 RNA knockdown in mouse quadriceps and cardiac muscles, which are difficult to target with oligonucleotides alone. Systemically delivered OTV enabled a more uniform ASO biodistribution profile in the CNS of TfRmu/hu KI mice and greater knockdown of Malat1 RNA compared with a bivalent, high-affinity TfR antibody. In cynomolgus macaques, an OTV directed against MALAT1 displayed robust ASO delivery to the primate CNS and enabled more uniform biodistribution and RNA target knockdown compared with intrathecal dosing of the same unconjugated ASO. Our data support systemically delivered OTV as a potential platform for delivering therapeutic ASOs across the BBB.
Assuntos
Barreira Hematoencefálica , Oligonucleotídeos Antissenso , RNA Longo não Codificante , Receptores da Transferrina , Animais , Oligonucleotídeos Antissenso/farmacocinética , Oligonucleotídeos Antissenso/administração & dosagem , Barreira Hematoencefálica/metabolismo , Receptores da Transferrina/metabolismo , Humanos , RNA Longo não Codificante/metabolismo , RNA Longo não Codificante/genética , Camundongos , Transporte Biológico , Macaca fascicularis , Técnicas de Silenciamento de Genes , Distribuição TecidualRESUMO
Therapeutics must diffuse through the brain extracellular space (ECS) in order to distribute within the central nervous system (CNS) compartment; this requirement holds both for drugs that are directly placed within the CNS (i.e., central input) and for drugs that cross the barriers separating blood and brain following systemic administration. The diffusion of any substance within the CNS may be affected by a number of properties associated with the brain microenvironment, e.g., the volume fraction, geometry, width, and local viscosity of the ECS, as well as interactions with cell surfaces, the extracellular matrix, and components of the interstitial fluid. Here, we discuss ECS properties important in governing the distribution of macromolecules (e.g., antibodies and other protein therapeutics), nanoparticles and viral vectors within the CNS. We also provide an introduction to some of the methods commonly applied to measure diffusion of molecules in the brain ECS, with a particular emphasis on those used for determining the diffusion properties of macromolecules. Finally, we discuss how quantitative diffusion measurements can be used to better understand and potentially even improve upon CNS drug delivery by modeling delivery within and across species, screening drugs and drug conjugates, evaluating methods for altering drug distribution, and appreciating important changes in drug distribution that may occur with CNS disease or injury.
Assuntos
Encéfalo/fisiologia , Sistemas de Liberação de Medicamentos , Animais , Transporte Biológico , Barreira Hematoencefálica/fisiologia , Encéfalo/anatomia & histologia , Doenças do Sistema Nervoso Central/tratamento farmacológico , Doenças do Sistema Nervoso Central/metabolismo , Espaço Extracelular/metabolismo , Humanos , Substâncias Macromoleculares/administração & dosagem , Substâncias Macromoleculares/farmacocinética , Modelos NeurológicosRESUMO
Leucine-rich repeat kinase 2 (LRRK2) variants associated with Parkinson's disease (PD) and Crohn's disease lead to increased phosphorylation of its Rab substrates. While it has been recently shown that perturbations in cellular homeostasis including lysosomal damage can increase LRRK2 activity and localization to lysosomes, the molecular mechanisms by which LRRK2 activity is regulated have remained poorly defined. We performed a targeted siRNA screen to identify regulators of LRRK2 activity and identified Rab12 as a novel modulator of LRRK2-dependent phosphorylation of one of its substrates, Rab10. Using a combination of imaging and immunopurification methods to isolate lysosomes, we demonstrated that Rab12 is actively recruited to damaged lysosomes and leads to a local and LRRK2-dependent increase in Rab10 phosphorylation. PD-linked variants, including LRRK2 R1441G and VPS35 D620N, lead to increased recruitment of LRRK2 to the lysosome and a local elevation in lysosomal levels of pT73 Rab10. Together, these data suggest a conserved mechanism by which Rab12, in response to damage or expression of PD-associated variants, facilitates the recruitment of LRRK2 and phosphorylation of its Rab substrate(s) at the lysosome.
Lysosomes are cellular compartments tasked with breaking down large molecules such as lipids or proteins. They perform an essential role in helping cells dispose of obsolete or harmful components; in fact, defects in lysosome function are associated with a range of health conditions. For instance, many genes associated with an increased risk of developing Parkinson's disease code for proteins required for lysosomes to work properly, such as the kinase LRRK2. Previous work has shown that this enzyme gets recruited to the surface of damaged lysosomes, where it can modulate the function of another set of molecular actors by modifying them through a chemical process known as phosphorylation. Such activity is increased in harmful versions of LRRK2 linked to Parkinson's disease. However, the molecular mechanisms which control LRRK2 activity or its recruitment to lysosomes remain unclear. To examine this question, Wang, Bondar et al. first performed a targeted screen to identify proteins that can regulate LRRK2 activity. This revealed that Rab12, one of molecular actors that LRRK2 phosphorylates, can in turn modulate the activity of the enzyme. Further imaging and biochemical experiments then showed that Rab12 is recruited to damaged lysosomes and that this step was in fact necessary for LRRK2 to also relocate to these compartments. The data suggest that this Rab12-driven recruitment process increases the local concentration of LRRK2 near its Rab targets on the membrane of damaged lysosomes, and therefore leads to enhanced LRRK2 activity. Crucially, Wang, Bondar et al. showed that Rab12 also plays a role in the increased LRRK2 activity observed with two Parkinson's disease-linked mutations (one in LRRK2 itself and one in another lysosomal regulator, VPS35), suggesting that increased LRRK2 concentration on lysosomes may be a conserved mechanism that leads to increased LRRK2 activity in disease. Overall, these results highlight a new, Rab12-dependent mechanism that results in enhanced activity at the lysosomal membrane with variants associated with Parkinson's disease, and for LRRK2 in general when lysosomes are damaged. This knowledge will be helpful to develop therapeutic strategies that target LRRK2, and to better understand how increased LRRK2 activity and lysosomal injury may be linked to Parkinson's disease.
Assuntos
Fenômenos Biológicos , Lisossomos , Proteínas rab de Ligação ao GTP , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Lisossomos/metabolismo , Mutação , Fosforilação , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , HumanosRESUMO
Loss-of-function variants of TREM2 are associated with increased risk of Alzheimer's disease (AD), suggesting that activation of this innate immune receptor may be a useful therapeutic strategy. Here we describe a high-affinity human TREM2-activating antibody engineered with a monovalent transferrin receptor (TfR) binding site, termed antibody transport vehicle (ATV), to facilitate blood-brain barrier transcytosis. Upon peripheral delivery in mice, ATV:TREM2 showed improved brain biodistribution and enhanced signaling compared to a standard anti-TREM2 antibody. In human induced pluripotent stem cell (iPSC)-derived microglia, ATV:TREM2 induced proliferation and improved mitochondrial metabolism. Single-cell RNA sequencing and morphometry revealed that ATV:TREM2 shifted microglia to metabolically responsive states, which were distinct from those induced by amyloid pathology. In an AD mouse model, ATV:TREM2 boosted brain microglial activity and glucose metabolism. Thus, ATV:TREM2 represents a promising approach to improve microglial function and treat brain hypometabolism found in patients with AD.
Assuntos
Doença de Alzheimer , Células-Tronco Pluripotentes Induzidas , Humanos , Animais , Camundongos , Microglia , Barreira Hematoencefálica , Distribuição Tecidual , Anticorpos , Encéfalo , Modelos Animais de Doenças , Glicoproteínas de Membrana , Receptores Imunológicos/genéticaRESUMO
Brain exposure of systemically administered biotherapeutics is highly restricted by the blood-brain barrier (BBB). Here, we report the engineering and characterization of a BBB transport vehicle targeting the CD98 heavy chain (CD98hc or SLC3A2) of heterodimeric amino acid transporters (TVCD98hc). The pharmacokinetic and biodistribution properties of a CD98hc antibody transport vehicle (ATVCD98hc) are assessed in humanized CD98hc knock-in mice and cynomolgus monkeys. Compared to most existing BBB platforms targeting the transferrin receptor, peripherally administered ATVCD98hc demonstrates differentiated brain delivery with markedly slower and more prolonged kinetic properties. Specific biodistribution profiles within the brain parenchyma can be modulated by introducing Fc mutations on ATVCD98hc that impact FcγR engagement, changing the valency of CD98hc binding, and by altering the extent of target engagement with Fabs. Our study establishes TVCD98hc as a modular brain delivery platform with favorable kinetic, biodistribution, and safety properties distinct from previously reported BBB platforms.
Assuntos
Barreira Hematoencefálica , Encéfalo , Animais , Camundongos , Distribuição Tecidual , Anticorpos , Engenharia , Macaca fascicularisRESUMO
Delivery of biotherapeutics across the blood-brain barrier (BBB) is a challenge. Many approaches fuse biotherapeutics to platforms that bind the transferrin receptor (TfR), a brain endothelial cell target, to facilitate receptor-mediated transcytosis across the BBB. Here, we characterized the pharmacological behavior of two distinct TfR-targeted platforms fused to iduronate 2-sulfatase (IDS), a lysosomal enzyme deficient in mucopolysaccharidosis type II (MPS II), and compared the relative brain exposures and functional activities of both approaches in mouse models. IDS fused to a moderate-affinity, monovalent TfR-binding enzyme transport vehicle (ETV:IDS) resulted in widespread brain exposure, internalization by parenchymal cells, and significant substrate reduction in the CNS of an MPS II mouse model. In contrast, IDS fused to a standard high-affinity bivalent antibody (IgG:IDS) resulted in lower brain uptake, limited biodistribution beyond brain endothelial cells, and reduced brain substrate reduction. These results highlight important features likely to impact the clinical development of TfR-targeting platforms in MPS II and potentially other CNS diseases.
Assuntos
Iduronato Sulfatase , Mucopolissacaridose II , Receptores da Transferrina , Proteínas Recombinantes de Fusão , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Iduronato Sulfatase/metabolismo , Iduronato Sulfatase/farmacologia , Lisossomos/metabolismo , Camundongos , Mucopolissacaridose II/metabolismo , Receptores da Transferrina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Distribuição TecidualRESUMO
BACKGROUND: Genetic mutations underlying familial Alzheimer's disease (AD) were identified decades ago, but the field is still in search of transformative therapies for patients. While mouse models based on overexpression of mutated transgenes have yielded key insights in mechanisms of disease, those models are subject to artifacts, including random genetic integration of the transgene, ectopic expression and non-physiological protein levels. The genetic engineering of novel mouse models using knock-in approaches addresses some of those limitations. With mounting evidence of the role played by microglia in AD, high-dimensional approaches to phenotype microglia in those models are critical to refine our understanding of the immune response in the brain. METHODS: We engineered a novel App knock-in mouse model (AppSAA) using homologous recombination to introduce three disease-causing coding mutations (Swedish, Arctic and Austrian) to the mouse App gene. Amyloid-ß pathology, neurodegeneration, glial responses, brain metabolism and behavioral phenotypes were characterized in heterozygous and homozygous AppSAA mice at different ages in brain and/ or biofluids. Wild type littermate mice were used as experimental controls. We used in situ imaging technologies to define the whole-brain distribution of amyloid plaques and compare it to other AD mouse models and human brain pathology. To further explore the microglial response to AD relevant pathology, we isolated microglia with fibrillar Aß content from the brain and performed transcriptomics and metabolomics analyses and in vivo brain imaging to measure energy metabolism and microglial response. Finally, we also characterized the mice in various behavioral assays. RESULTS: Leveraging multi-omics approaches, we discovered profound alteration of diverse lipids and metabolites as well as an exacerbated disease-associated transcriptomic response in microglia with high intracellular Aß content. The AppSAA knock-in mouse model recapitulates key pathological features of AD such as a progressive accumulation of parenchymal amyloid plaques and vascular amyloid deposits, altered astroglial and microglial responses and elevation of CSF markers of neurodegeneration. Those observations were associated with increased TSPO and FDG-PET brain signals and a hyperactivity phenotype as the animals aged. DISCUSSION: Our findings demonstrate that fibrillar Aß in microglia is associated with lipid dyshomeostasis consistent with lysosomal dysfunction and foam cell phenotypes as well as profound immuno-metabolic perturbations, opening new avenues to further investigate metabolic pathways at play in microglia responding to AD-relevant pathogenesis. The in-depth characterization of pathological hallmarks of AD in this novel and open-access mouse model should serve as a resource for the scientific community to investigate disease-relevant biology.
Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Amiloidose/metabolismo , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Microglia/metabolismo , Placa Amiloide/patologia , Receptores de GABA/metabolismoRESUMO
The intercellular spaces between neurons and glia contain an amorphous, negatively charged extracellular matrix (ECM) with the potential to shape and regulate the distribution of many diffusing ions, proteins and drugs. However, little evidence exists for direct regulation of extracellular diffusion by the ECM in living tissue. Here, we demonstrate macromolecule sequestration by an ECM component in vivo, using quantitative diffusion measurements from integrative optical imaging. Diffusion measurements in free solution, supported by confocal imaging and binding assays with cultured cells, were used to characterize the properties of a fluorescently labeled protein, lactoferrin (Lf), and its association with heparin and heparan sulfate in vitro. In vivo diffusion measurements were then performed through an open cranial window over rat somatosensory cortex to measure effective diffusion coefficients (D*) under different conditions, revealing that D* for Lf was reduced approximately 60% by binding to heparan sulfate proteoglycans, a prominent component of the ECM and cell surfaces in brain. Finally, we describe a method for quantifying heparan sulfate binding site density from data for Lf and the structurally similar protein transferrin, allowing us to predict a low micromolar concentration of these binding sites in neocortex, the first estimate in living tissue. Our results have significance for many tissues, because heparan sulfate is synthesized by almost every type of cell in the body. Quantifying ECM effects on diffusion will also aid in the modeling and design of drug delivery strategies for growth factors and viral vectors, some of which are likely to interact with heparan sulfate.
Assuntos
Matriz Extracelular/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Lactoferrina/metabolismo , Córtex Somatossensorial/metabolismo , Animais , Sítios de Ligação , Células Cultivadas , Fluoresceínas/química , Fluoresceínas/metabolismo , Humanos , Lactoferrina/química , Ratos , Ratos Sprague-Dawley , Córtex Somatossensorial/ultraestrutura , Transferrina/química , Transferrina/metabolismoRESUMO
Perillyl alcohol (POH) has been extensively studied for the treatment of peripheral and primary brain tumors. The intranasal route of administration has been preferred for dosing POH in early-stage clinical trials associated with promising outcomes in primary brain cancer. However, it is unclear how intranasal POH targets brain tumors in these patients. Multiple studies indicate that intranasally applied large molecules may enter the brain and cerebrospinal fluid (CSF) through direct olfactory and trigeminal nerve-associated pathways originating in the nasal mucosa that bypass the blood-brain barrier. It is unknown whether POH, a small molecule subject to extensive nasal metabolism and systemic absorption, may also undergo direct transport to brain or CSF from the nasal mucosa. Here, we compared CSF and plasma concentrations of POH and its metabolite, perillic acid (PA), following intranasal or intravascular POH application. Samples were collected over 70 min and assayed by high-performance liquid chromatography. Intranasal administration resulted in tenfold higher CSF-to-plasma ratios for POH and tenfold higher CSF levels for PA compared to equal dose intravascular administration. Our preclinical results demonstrate POH undergoes direct transport from the nasal mucosa to the CSF, a finding with potential significance for its efficacy as an intranasal chemotherapeutic for brain cancer.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Monoterpenos/farmacologia , Mucosa Nasal/efeitos dos fármacos , Administração Intranasal , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/patologia , Neoplasias Encefálicas/líquido cefalorraquidiano , Neoplasias Encefálicas/patologia , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Humanos , Ratos , Nervo Trigêmeo/efeitos dos fármacosRESUMO
Effective delivery of protein therapeutics to the central nervous system (CNS) has been greatly restricted by the blood-brain barrier (BBB). We describe the development of a BBB transport vehicle (TV) comprising an engineered Fc fragment that exploits receptor-mediated transcytosis for CNS delivery of biotherapeutics by binding a highly expressed brain endothelial cell target. TVs were engineered using directed evolution to bind the apical domain of the human transferrin receptor (hTfR) without the use of amino acid insertions, deletions, or unnatural appendages. A crystal structure of the TV-TfR complex revealed the TV binding site to be away from transferrin and FcRn binding sites, which was further confirmed experimentally in vitro and in vivo. Recombinant expression of TVs fused to anti-ß-secretase (BACE1) Fabs yielded antibody transport vehicle (ATV) molecules with native immunoglobulin G (IgG) structure and stability. Peripheral administration of anti-BACE1 ATVs to hTfR-engineered mice and cynomolgus monkeys resulted in substantially improved CNS uptake and sustained pharmacodynamic responses. The TV platform readily accommodates numerous additional configurations, including bispecific antibodies and protein fusions, yielding a highly modular CNS delivery platform.
Assuntos
Secretases da Proteína Precursora do Amiloide , Barreira Hematoencefálica , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Ácido Aspártico Endopeptidases/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Haplorrinos/metabolismo , Fragmentos Fc das Imunoglobulinas , Camundongos , Receptores da Transferrina/metabolismoRESUMO
Most lysosomal storage diseases (LSDs) involve progressive central nervous system (CNS) impairment, resulting from deficiency of a lysosomal enzyme. Treatment of neuronopathic LSDs remains a considerable challenge, as approved intravenously administered enzyme therapies are ineffective in modifying CNS disease because they do not effectively cross the blood-brain barrier (BBB). We describe a therapeutic platform for increasing the brain exposure of enzyme replacement therapies. The enzyme transport vehicle (ETV) is a lysosomal enzyme fused to an Fc domain that has been engineered to bind to the transferrin receptor, which facilitates receptor-mediated transcytosis across the BBB. We demonstrate that ETV fusions containing iduronate 2-sulfatase (ETV:IDS), the lysosomal enzyme deficient in mucopolysaccharidosis type II, exhibited high intrinsic activity and degraded accumulated substrates in both IDS-deficient cell and in vivo models. ETV substantially improved brain delivery of IDS in a preclinical model of disease, enabling enhanced cellular distribution to neurons, astrocytes, and microglia throughout the brain. Improved brain exposure for ETV:IDS translated to a reduction in accumulated substrates in these CNS cell types and peripheral tissues and resulted in a complete correction of downstream disease-relevant pathologies in the brain, including secondary accumulation of lysosomal lipids, perturbed gene expression, neuroinflammation, and neuroaxonal damage. These data highlight the therapeutic potential of the ETV platform for LSDs and provide preclinical proof of concept for TV-enabled therapeutics to treat CNS diseases more broadly.
Assuntos
Barreira Hematoencefálica , Iduronato Sulfatase , Animais , Encéfalo , Modelos Animais de Doenças , Terapia de Reposição de Enzimas , Lisossomos , CamundongosRESUMO
This article highlights the scientific achievements, professional career, and personal interactions of Malcolm B. Segal who passed away in July this year. Born in 1937 in Goodmayes, Essex, UK, Segal rose to the Chairman position in the Division of Physiology at United Medical and Dental School of Guy's and St. Thomas' Hospitals, retiring in 2006 after his long professional career in biomedical science. Being trained in Hugh Davson's laboratory, Segal became one of the pioneers in research on cerebrospinal fluid physiology and the choroid plexus. During the course of his career, Segal himself trained a number of young scientists and collaborated with many colleagues around the world, making long-lasting friendships along the way. In addition to his professional accomplishments as a researcher and educator, Segal was an avid sailor and wine connoisseur, and enjoyed teaching classes on navigation and wine tasting.
Assuntos
Líquido Cefalorraquidiano/fisiologia , Plexo Corióideo/fisiologia , Fisiologia/história , História do Século XX , História do Século XXIRESUMO
The intranasal route has been hypothesized to circumvent the blood-brain and blood-cerebrospinal fluid barriers, allowing entry into the brain via extracellular pathways along olfactory and trigeminal nerves and the perivascular spaces (PVS) of cerebral blood vessels. We investigated the potential of the intranasal route to non-invasively deliver antibodies to the brain 30â¯min following administration by characterizing distribution, dose-response, and mechanisms of antibody transport to and within the brain after administering non-targeted radiolabeled or fluorescently-labeled full length immunoglobulin G (IgG) to normal adult female rats. Intranasal [125I]-IgG consistently yielded highest concentrations in the olfactory bulbs, trigeminal nerves, and leptomeningeal blood vessels with their associated PVS. Intranasal delivery also resulted in significantly higher [125I]-IgG concentrations in the CNS than systemic (intra-arterial) delivery for doses producing similar endpoint blood concentrations. Importantly, CNS targeting significantly increased with increasing dose only with intranasal administration, yielding brain concentrations that ranged from the low-to-mid picomolar range with tracer dosing (50⯵g) up to the low nanomolar range at higher doses (1â¯mg and 2.5â¯mg). Finally, intranasal pre-treatment with a previously identified nasal permeation enhancer, matrix metalloproteinase-9, significantly improved intranasal [125I]-IgG delivery to multiple brain regions and further allowed us to elucidate IgG transport pathways extending from the nasal epithelia into the brain using fluorescence microscopy. The results show that it may be feasible to achieve therapeutic levels of IgG in the CNS, particularly at higher intranasal doses, and clarify the likely cranial nerve and perivascular distribution pathways taken by antibodies to reach the brain from the nasal mucosae.
Assuntos
Encéfalo/metabolismo , Imunoglobulina G/administração & dosagem , Administração Intranasal , Animais , Encéfalo/irrigação sanguínea , Feminino , Imunoglobulina G/análise , Imunoglobulina G/sangue , Injeções Intra-Arteriais , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual , Nervo Trigêmeo/metabolismoRESUMO
Perivascular compartments surrounding central nervous system (CNS) vessels have been proposed to serve key roles in facilitating cerebrospinal fluid flow into the brain, CNS waste transfer, and immune cell trafficking. Traditionally, these compartments were identified by electron microscopy with limited molecular characterization. Using cellular markers and knowledge on cellular sources of basement membrane laminins, we here describe molecularly distinct compartments surrounding different vessel types and provide a comprehensive characterization of the arachnoid and pial compartments and their connection to CNS vessels and perivascular pathways. We show that differential expression of plectin, E-cadherin and laminins α1, α2, and α5 distinguishes pial and arachnoid layers at the brain surface, while endothelial and smooth muscle laminins α4 and α5 and smooth muscle actin differentiate between arterioles and venules. Tracer studies reveal that interconnected perivascular compartments exist from arterioles through to veins, potentially providing a route for fluid flow as well as the transport of large and small molecules.