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INTRODUCTION: Neurometabolic disorders remain challenging to treat, largely due to the limited availability of drugs that can cross the blood-brain barrier (BBB) and effectively target brain impairment. Key reasons for inadequate treatment include a lack of coordinated knowledge, few studies on BBB status in these diseases, and poorly designed therapies. AREAS COVERED: This paper provides an overview of current research on neurometabolic disorders and therapeutic options, focusing on the treatment of neurological involvement. It highlights the limitations of existing therapies, describes innovative protocols recently developed, and explores new opportunities for therapy design and testing, some of which are already under investigation. The goal is to guide researchers toward innovative and potentially more effective treatments. EXPERT OPINION: Advancing research on neurometabolic diseases is crucial for designing effective treatment strategies. The field suffers from a lack of collaboration, and a strong collective effort is needed to enhance synergy, increase knowledge, and develop a new therapeutic paradigm for neurometabolic disorders.
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Barrera Hematoencefálica , Sistemas de Liberación de Medicamentos , Humanos , Barrera Hematoencefálica/metabolismo , Animales , Diseño de Fármacos , Enzimas/metabolismo , Enzimas/administración & dosificación , Desarrollo de MedicamentosRESUMEN
The blood-brain barrier (BBB) hinders the distribution of therapeutics intended for treatment of diseases of the brain. Our previous studies demonstrated that that a soluble form of melanotransferrin (MTf; Uniprot P08582; also known as p97, MFI2, and CD228), a mammalian iron-transport protein, is an effective carrier for delivery of drug conjugates across the BBB into the brain and was the first BBB targeting delivery system to demonstrate therapeutic efficacy within the brain. Here, we performed a screen to identify peptides from MTf capable of traversing the BBB. We identified a highly conserved 12-amino acid peptide, termed MTfp, that retains the ability to cross the intact BBB intact, distributes throughout the parenchyma, and enter endosomes and lysosomes within neurons, astrocytes and microglia in the brain. This peptide may provide a platform for the transport of therapeutics to the CNS, and thereby offers new avenues for potential treatments of neuropathologies that are currently refractory to existing therapies.
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[This corrects the article DOI: 10.3389/fmolb.2021.611367.].
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The blood-brain barrier (BBB) hinders the distribution of therapeutics intended for treatment of neuroinflammation (NI) of the central nervous system. A twelve-amino acid peptide that transcytoses the BBB, termed MTfp, was chemically conjugated to siRNA to create a novel peptide-oligonucleotide conjugate (POC), directed to downregulate NOX4, a gene thought responsible for oxidative stress in ischemic stroke. The MTfp-NOX4 POC has the ability to cross the intact BBB and knockdown NOX4 expression in the brain. Following induction of ischemic stroke, animals pretreated with the POC exhibited significantly smaller infarcts; accompanied by increased protection against neurological deterioration and improved recovery. The data demonstrates that the MTfp can act as a nanomule to facilitate BBB transcytosis of siRNAs; where the NOX-4 specific siRNA moiety can elicit effective therapeutic knockdown of a gene responsible for oxidative stress in the central nervous system. This study is the first to conclusively demonstrate both siRNA-carrier delivery and therapeutic efficacy in any CNS disease model where the BBB remains intact and thus offers new avenues for potential treatments of oxidative stress underlying neuroinflammation in a variety of neuropathologies that are currently refractory to existing therapies.
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Delivery of biologic drugs across the blood-brain barrier is becoming a reality. However, the solutions often involve the assembly of complex multi-specific antibody molecules. Here we utilize a simple 12 amino-acid peptide originating from the melanotransferrin (MTf) protein that has shown improved brain delivery properties. 3D confocal fluorescence microscopic analysis demonstrated brain parenchymal localisation of a fluorescently labelled antibody (NIP228) when chemically conjugated to either the MTf peptide or full-length MTf protein. Measurement of plasma kinetics demonstrated the MTf peptide fusions had very similar kinetics to an unmodified NIP228 control antibody, whereas the fusion to MTf protein had significantly reduced plasma exposure most likely due to a higher tissue distribution in the periphery. Brain exposure for the MTf peptide fusions was significantly increased for the duration of the study, exceeding that of the fusions to full length MTf protein. Using a neuropathic pain model, we have demonstrated that fusions to interleukin-1 receptor antagonist (IL-1RA) are able to induce significant and durable analgesia following peripheral administration. These data demonstrate that recombinant and chemically conjugated MTf-based brain delivery vectors can deliver therapeutic levels of drug to the central nervous system.
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Portadores de Fármacos/metabolismo , Proteína Antagonista del Receptor de Interleucina 1/uso terapéutico , Glicoproteínas de Membrana/metabolismo , Neuralgia/tratamiento farmacológico , Péptidos/metabolismo , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Portadores de Fármacos/química , Humanos , Proteína Antagonista del Receptor de Interleucina 1/administración & dosificación , Proteína Antagonista del Receptor de Interleucina 1/farmacocinética , Masculino , Glicoproteínas de Membrana/química , Ratones Endogámicos C57BL , Neuralgia/metabolismo , Péptidos/químicaRESUMEN
PURPOSE: The ability of human melanotransferrin (hMTf) to carry a therapeutic concentration of trastuzumab (BTA) in the brain after conjugation (in the form of trastuzumab-melanotransferrin conjugate, BT2111 conjugate) was investigated by measuring the reduction of the number and size of metastatic human HER2+ breast cancer tumors in a preclinical model of brain metastases of breast cancer. METHODS: Human metastatic brain seeking breast cancer cells were injected in NuNu mice (n = 6-12 per group) which then developed experimental brain metastases. Drug uptake was analyzed in relation to metastasis size and blood-tumor barrier permeability. To investigate in-vivo activity against brain metastases, equimolar doses of the conjugate, and relevant controls (hMTf and BTA) in separate groups were administered biweekly after intracardiac injection of the metastatic cancer cells. RESULTS: The trastuzumab-melanotransferrin conjugate (BT2111) reduced the number of preclinical human HER2+ breast cancer metastases in the brain by 68% compared to control groups. Tumors which remained after treatment were 46% smaller than the control groups. In contrast, BTA alone had no effect on reducing number of metastases, and was associated with only a minimal reduction in metastasis size. CONCLUSIONS: The results suggest the novel trastuzumab-melanotransferrin conjugate (BT2111) may have utility in treating brain metastasis and validate hMTf as a potential vector for antibody transport across the Blood Brain Barrier (BBB).
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Antineoplásicos/administración & dosificación , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias de la Mama/tratamiento farmacológico , Glicoproteínas de Membrana/química , Trastuzumab/administración & dosificación , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Barrera Hematoencefálica/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/secundario , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Colorantes Fluorescentes/química , Xenoinjertos , Humanos , Ratones Desnudos , Imagen Óptica , Permeabilidad , Unión Proteica , Receptor ErbB-2/metabolismo , Trastuzumab/química , Trastuzumab/farmacocinéticaRESUMEN
The central nervous system is a sanctuary protected by barriers, among which the blood-brain barrier (BBB). The BBB is formed by the specific nature of the endothelial cells of the brain capillaries, that allow brain access only to nutrients necessary for brain cell survival and function. These properties of the BBB result in the incapacity of therapeutic compounds, both small and large, to reach the brain at therapeutic concentrations. Various strategies are now being developed to enhance the amount and concentration of these compounds in the brain parenchyma. The development of new technologies such as peptide vectors will achieve the delivery of active agents in therapeutic concentration across the BBB to treat brain diseases such as cancer or neurodegenerative disorders. In this paper, design of new active peptides and the development of new protein, peptide, vectors for drug brain delivery using physiological approaches will be addressed. In addition to the Angiopep platform technology which is in development at Angiochem Inc. and is to date the most advanced in human clinical trials, the new Transcend technology using a protein melanotransferrin for the transport of biologics such as lysosomal enzymes and antibodies will be discussed.
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Barrera Hematoencefálica/metabolismo , Encefalopatías/tratamiento farmacológico , Encéfalo/metabolismo , Portadores de Fármacos , Péptidos , Encéfalo/efectos de los fármacos , Diseño de Fármacos , Humanos , Enfermedades Neurodegenerativas/tratamiento farmacológico , Farmacocinética , Receptor de Insulina , Receptores de LDL , Receptores de TransferrinaRESUMEN
The blood-brain barrier (BBB) is formed by the special nature of the endothelial cells of the brain capillaries characterized by tight junctions between cells and a high expression of efflux pumps only allowing the brain access to nutrients necessary for cell survival and function. These properties of the BBB result in the incapacity of small and large therapeutic compounds to reach the brain at therapeutic concentrations. Various strategies are now being developed to enhance the amount and concentration of these compounds in the brain parenchyma. The development of new technologies such as peptide vectors has the potential to achieve the delivery of active agents in therapeutic concentrations across the BBB to treat brain diseases such as brain primary and metastatic cancers and neurodegenerative disorders. In this review, the design of new active peptides and development of new peptide vectors for drug brain delivery using physiological approaches will be addressed. A new chemical entity incorporating angiopep peptide in a small anticancer agent (paclitaxel) is now in clinical trials. It is the first of such designed agents to be validated for the treatment of human brain cancers and opens the door for such approaches.
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Barrera Hematoencefálica , Encéfalo/metabolismo , Sistemas de Liberación de Medicamentos , Péptidos/administración & dosificación , Animales , Transporte Biológico , Humanos , Nanopartículas/administración & dosificación , TranscitosisRESUMEN
The central nervous system is protected by barriers which control the entry of compounds into the brain, thereby regulating brain homeostasis. The blood-brain barrier, formed by the endothelial cells of the brain capillaries, restricts access to brain cells of blood-borne compounds and facilitates nutrients essential for normal metabolism to reach brain cells. This very tight regulation of the brain homeostasis results in the inability of some small and large therapeutic compounds to cross the blood-brain barrier (BBB). Therefore, various strategies are being developed to enhance the amount and concentration of therapeutic compounds in the brain. In this review, we will address the different approaches used to increase the transport of therapeutics from blood into the brain parenchyma. We will mainly concentrate on the physiologic approach which takes advantage of specific receptors already expressed on the capillary endothelial cells forming the BBB and necessary for the survival of brain cells. Among all the approaches used for increasing brain delivery of therapeutics, the most accepted method is the use of the physiological approach which takes advantage of the transcytosis capacity of specific receptors expressed at the BBB. The low density lipoprotein receptor related protein (LRP) is the most adapted for such use with the engineered peptide compound (EPiC) platform incorporating the Angiopep peptide in new therapeutics the most advanced with promising data in the clinic.
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Barrera Hematoencefálica/efectos de los fármacos , Encefalopatías/tratamiento farmacológico , Encéfalo/efectos de los fármacos , Fármacos del Sistema Nervioso Central/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Animales , Barrera Hematoencefálica/fisiopatología , Encéfalo/fisiopatología , Encefalopatías/fisiopatología , Permeabilidad Capilar/efectos de los fármacos , Permeabilidad Capilar/fisiología , Fármacos del Sistema Nervioso Central/uso terapéutico , Vías de Administración de Medicamentos , HumanosRESUMEN
New and effective therapeutics that cross the blood-brain barrier (BBB) are critically needed for treatment of many brain diseases. We characterize here a novel drug development platform that is broadly applicable for the development of new therapeutics with increased brain penetration. The platform is based on the Angiopep-2 peptide, a sequence derived from ligands that bind to low-density lipoprotein receptor-related protein-1 (LRP-1), a receptor expressed on the BBB. Fluorescent imaging studies of a Cy5.5Angiopep-2 conjugate and immunohistochemical studies of injected Angiopep-2 in mice demonstrated efficient transport across the BBB into brain parenchyma and subsequent co-localization with the neuronal nuclei-selective marker NeuN and the glial marker glial fibrillary acidic protein (GFAP). Uptake of [(¹²5I]-Angiopep-2 into brain endothelial cells occurred by a saturable mechanism involving LRP-1. The primary sequence and charge of Angiopep-2 were crucial for its passage across the BBB. Overall, the results demonstrate the significant potential of this platform for the development of novel neurotherapeutics.
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Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Sistemas de Liberación de Medicamentos , Péptidos/metabolismo , Animales , Antígenos Nucleares/análisis , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/irrigación sanguínea , Encéfalo/efectos de los fármacos , Carbocianinas , Células Endoteliales/metabolismo , Proteína Ácida Fibrilar de la Glía/análisis , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Microscopía Fluorescente , Proteínas del Tejido Nervioso/análisis , Transporte de Proteínas , Ensayo de Unión Radioligante , Ratas , TranscitosisRESUMEN
The central nervous system is a sanctuary protected by barriers that regulate brain homeostasis and control the transport of endogenous compounds into the brain. The blood-brain barrier, formed by endothelial cells of the brain capillaries, restricts access to brain cells allowing entry only to amino acids, glucose and hormones needed for normal brain cell function and metabolism. This very tight regulation of brain cell access is essential for the survival of neurons which do not have a significant capacity to regenerate, but also prevents therapeutic compounds, small and large, from reaching the brain. As a result, various strategies are being developed to enhance access of drugs to the brain parenchyma at therapeutically meaningful concentrations to effectively manage disease.
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Barrera Hematoencefálica/metabolismo , Encefalopatías/metabolismo , Preparaciones Farmacéuticas/metabolismo , Animales , Transporte Biológico Activo/efectos de los fármacos , Transporte Biológico Activo/fisiología , Barrera Hematoencefálica/efectos de los fármacos , Encefalopatías/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/métodos , Humanos , Preparaciones Farmacéuticas/administración & dosificación , Resultado del TratamientoRESUMEN
BACKGROUND: Therapeutic intervention in many neurological diseases is thwarted by the physical obstacle formed by the blood-brain barrier (BBB) that excludes most drugs from entering the brain from the blood. Thus, identifying efficacious modes of drug delivery to the brain remains a "holy grail" in molecular medicine and nanobiotechnology. Brain capillaries, that comprise the BBB, possess an endogenous receptor that ferries an iron-transport protein, termed p97 (melanotransferrin), across the BBB. Here, we explored the hypothesis that therapeutic drugs "piggybacked" as conjugates of p97 can be shuttled across the BBB for treatment of otherwise inoperable brain tumors. APPROACH: Human p97 was covalently linked with the chemotherapeutic agents paclitaxel (PTAX) or adriamycin (ADR) and following intravenous injection, measured their penetration into brain tissue and other organs using radiolabeled and fluorescent derivatives of the drugs. In order to establish efficacy of the conjugates, we used nude mouse models to assess p97-drug conjugate activity towards glioma and mammary tumors growing subcutaneously compared to those growing intracranially. PRINCIPAL FINDINGS: Bolus-injected p97-drug conjugates and unconjugated p97 traversed brain capillary endothelium within a few minutes and accumulated to 1-2% of the injected by 24 hours. Brain delivery with p97-drug conjugates was quantitatively 10 fold higher than with free drug controls. Furthermore, both free-ADR and p97-ADR conjugates equally inhibited the subcutaneous growth of gliomas growing outside the brain. Evocatively, only p97-ADR conjugates significantly prolonged the survival of animals bearing intracranial gliomas or mammary tumors when compared to similar cumulated doses of free-ADR. SIGNIFICANCE: This study provides the initial proof of concept for p97 as a carrier capable of shuttling therapeutic levels of drugs from the blood to the brain for the treatment of neurological disorders, including classes of resident and metastatic brain tumors. It may be prudent, therefore, to consider implementation of this novel delivery platform in various clinical settings for therapeutic intervention in acute and chronic neurological diseases.
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Antineoplásicos/administración & dosificación , Barrera Hematoencefálica , Portadores de Fármacos , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacocinética , Doxorrubicina/uso terapéutico , Colorantes Fluorescentes , Humanos , Ratones , Ratones Desnudos , Paclitaxel/administración & dosificación , Paclitaxel/farmacocinética , Paclitaxel/uso terapéuticoRESUMEN
The blood-brain barrier (BBB) restricts the entry of proteins as well as potential drugs to cerebral tissues. We previously reported that a family of Kunitz domain-derived peptides called Angiopeps can be used as a drug delivery system for the brain. Here, we further characterize the transcytosis ability of these peptides using an in vitro model of the BBB and in situ brain perfusion. These peptides, and in particular Angiopep-2, exhibited higher transcytosis capacity and parenchymal accumulation than do transferrin, lactoferrin, and avidin. Angiopep-2 transport and accumulation in brain endothelial cells were unaffected by the P-glycoprotein inhibitor, cyclosporin A, indicating that this peptide is not a substrate for the efflux pump P-glycoprotein. However, competition studies show that activated alpha(2)-macroglobulin, a specific ligand for the low-density lipoprotein receptor-related protein-1 (LRP1) and Angiopep-2 can share the same receptor. In addition, LRP1 was detected in glioblastomas and brain metastases from lung and skin cancers. Fluorescent microscopy also revealed that Alexa488-Angiopep-2 co-localized with LRP1 in brain endothelial cell monolayers. Overall, these results suggest that Angiopep-2 transport across the BBB is, in part, mediated by LRP1.
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Barrera Hematoencefálica/fisiología , Encéfalo/fisiología , Sistemas de Liberación de Medicamentos/métodos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/fisiología , Oligopéptidos/fisiología , Fragmentos de Péptidos/metabolismo , Somatostatina/análogos & derivados , Animales , Barrera Hematoencefálica/metabolismo , Bovinos , Línea Celular Tumoral , Células Cultivadas , Vectores Genéticos/administración & dosificación , Vectores Genéticos/metabolismo , Humanos , Fragmentos de Péptidos/administración & dosificación , Péptidos Cíclicos , Transporte de Proteínas/fisiología , Ratas , Somatostatina/fisiologíaRESUMEN
By controlling access to the brain, the blood-brain barrier (BBB) restricts the entry of proteins and potential drugs to cerebral tissues. We demonstrate here the transcytosis ability of aprotinin and peptides derived from Kunitz domains using an in vitro model of the BBB and in situ brain perfusion. Aprotinin transcytosis across bovine brain capillary endothelial cell (BBCEC) monolayers is at least 10-fold greater than that of holo-transferrin. Sucrose permeability was unaffected by high concentrations of aprotinin, indicating that transcytosis of aprotinin was unrelated to changes in the BBCEC monolayer integrity. Alignment of the amino acid sequence of aprotinin with the Kunitz domains of human proteins allowed the identification and design of a family of peptides, named Angiopeps. These peptides, and in particular Angiopep-2, exhibit higher transcytosis capacity and parenchyma accumulation than aprotinin. Overall, these results suggest that these Kunitz-derived peptides could be advantageously used as a new brain delivery system for pharmacological agents that do not readily enter the brain.
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Encéfalo/metabolismo , Química Farmacéutica/métodos , Sistemas de Liberación de Medicamentos/métodos , Péptidos/administración & dosificación , Péptidos/genética , Secuencia de Aminoácidos/genética , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Encéfalo/efectos de los fármacos , Técnicas de Cocultivo , Humanos , Ratones , Datos de Secuencia Molecular , Péptidos/farmacocinética , RatasRESUMEN
The E3 region of adenovirus codes for several membrane proteins, most of which are involved in immune evasion and prevention of host cell apoptosis. We explored the topology and targeting mechanisms of E3-6.7K, the most recently described member of this group, by using an in vitro translation system supplemented with microsomes. Here, we present evidence that E3-6.7K, one of the smallest signal-anchor proteins known, translocates across the membrane of the endoplasmic reticulum in a posttranslational, ribosome-independent, yet ATP-dependent manner, reminiscent of the translocation of tail-anchored proteins. Our analysis also demonstrated that E3-6.7K could achieve several distinct topological fates. In addition to the previously postulated type III orientation (N-luminal/C-cytoplasmic, termed NtmE3-6.7K), we detected a tail-anchored form adopting the opposite orientation (N-cytoplasmic/C-luminal, termed CtmE3-6.7K) as well as the possibility of a fully translocated form (N and C termini are both translocated, termed NCE3-6.7K). Due to the translocation of a positively charged domain, both the CtmE3-6.7K and NCE3-6.7K topologies of E3-6.7K constitute exceptions to the "positive inside" rule. The NtmE3-6.7K and NCE3-6.7K are the first examples of posttranslationally translocated proteins in higher eukaryotes that are not tail anchored. Distinct topological forms were also found in transfected cells, as both N and C termini of E3-6.7K were detected on the extracellular surface of transfected cells. The demonstration of unexpected topological forms and translocation mechanisms for E3-6.7K defies conventional thinking about membrane protein topogenesis and advises that both the mode of targeting and topology of signal-anchor proteins should be determined experimentally.
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Proteínas E3 de Adenovirus/química , Proteínas E3 de Adenovirus/metabolismo , Retículo Endoplásmico/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Procesamiento Proteico-Postraduccional , Adenosina Trifosfato/metabolismo , Proteínas E3 de Adenovirus/genética , Secuencia de Aminoácidos , Células HeLa , Humanos , Proteínas de la Membrana/genética , Microsomas/metabolismo , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
Levels of soluble melanotransferrin in serum have been reported to be higher in patients with Alzheimer's disease than in control subjects. The present study investigated melanotransferrin in human body fluids in the light of these findings. To clarify the correlation between melanotransferrin and Alzheimer's disease, the melanotransferrin content was determined by non-reducing, denaturing SDS/PAGE and Western blotting. Under these conditions, serum melanotransferrin migrated at 79 and 82 kDa. Melanotransferrin antigenicity and the relative proportions of the two forms were very sensitive to factors that altered its conformation, including disulphide bridges, pH and bivalent cations. Serum melanotransferrin levels were not significantly different between control subjects and patients with Alzheimer's disease using whole serum, EDTA-supplemented serum or serum immunoglobulin-depleted by Protein G-Sepharose and enriched by affinity precipitation with the lectin from Asparagus pea. Glycosylated forms of serum melanotransferrin bound to Asparagus lectin manifested similar patterns on two-dimensional gel electrophoresis in samples from controls and Alzheimer's disease subjects. Melanotransferrin was also present in saliva and at a high level in urine, but contents were similar in controls and patients with Alzheimer's disease. Together, these results demonstrate that serum melanotransferrin exists in various conformations depending on the binding of bivalent cations or following post-translational modification. These data also indicate that human serum melanotransferrin levels are unchanged in subjects with Alzheimer's disease.
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Enfermedad de Alzheimer/sangre , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/sangre , Enfermedad de Alzheimer/diagnóstico , Animales , Anticuerpos Monoclonales/metabolismo , Antígenos de Neoplasias , Tampones (Química) , Cationes Bivalentes/química , Línea Celular , Precipitación Química , Cricetinae , Electroforesis en Gel Bidimensional , Glicosilación , Humanos , Inmunoglobulinas/metabolismo , Lectinas/química , Antígenos Específicos del Melanoma , Proteínas de Neoplasias/química , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/orina , Proteínas del Tejido Nervioso/metabolismo , Lectinas de Plantas/química , Conformación Proteica/efectos de los fármacos , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/sangre , Isoformas de Proteínas/química , Isoformas de Proteínas/inmunología , Procesamiento Proteico-Postraduccional , Proteínas y Péptidos Salivales/química , Sensibilidad y Especificidad , Proteína Estafilocócica A/metabolismoRESUMEN
We recently reported that human recombinant melanotransferrin (p97) presents a high transport rate across the blood-brain barrier that might involve the low-density lipoprotein receptor-related protein (LRP). We now report new interactions between p97 and another LRP ligand, the urokinase plasminogen activator (uPA) complex. By using biospecific interaction analysis, both pro-uPA and plasminogen are shown to interact with immobilized p97. Moreover, the activation of plasminogen by pro-uPA is increased by soluble p97. Because the uPA system plays a crucial role in cell migration, both in cancer and in angiogenesis, we also measured the impact of both endogenous membrane-bound and exogenous p97 on cell migration. The monoclonal antibody L235 (which recognizes a conformational epitope on p97) inhibited the migration of human microvascular endothelial cells (HMECs-1) and of human melanoma SK-MEL-28 cells, indicating that endogenous membrane-bound p97 could be associated with this process. In addition, low concentrations of exogenous p97 (10 and 100 nM) inhibited HMEC-1 and SK-MEL28 cell migration by more than 50%. These results indicate that membrane-bound and soluble p97 affect the migration capacity of endothelial and melanoma cells and suggest that p97 could be involved in the regulation of plasminogen activation by interacting with pro-uPA and plasminogen.
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Movimiento Celular/fisiología , Proteínas de Neoplasias/farmacocinética , Plasminógeno/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , Antígenos de Neoplasias , Astrocitos/citología , Barrera Hematoencefálica/fisiología , Bovinos , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/citología , Humanos , Melanoma , Antígenos Específicos del Melanoma , Proteínas de Neoplasias/metabolismo , Neovascularización Patológica/metabolismo , Ratas , Solubilidad , Células Tumorales Cultivadas/citología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
OBJECTIVE: This study was undertaken to assess the role of p97 (also known as melanotransferrin) in the transfer of iron into the brain, because the passage of most large molecules is limited by the presence of the blood-brain barrier, including that of the serum iron transporter transferrin. METHODS: To study the function of the soluble form of p97, we followed the uptake of radioiodinated and 55Fe loaded p97 and transferrin by the brain during a 24-hour period. RESULTS: We show that the soluble form of p97 has the ability to transcytose across the murine blood-brain barrier, and its transcytosis can be inhibited in a specific manner. We also provide evidence that p97 transports iron into the brain more efficiently than transferrin. CONCLUSIONS: These data support the idea that p97 is an important iron transporter across the blood-brain barrier in normal physiology and possibly in neurodegenerative diseases, such as Alzheimer disease, in which iron homeostasis in the brain becomes disrupted.
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Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Hierro/metabolismo , Proteínas de Neoplasias/farmacocinética , Animales , Antígenos de Neoplasias , Transporte Biológico , Humanos , Radioisótopos de Yodo , Radioisótopos de Hierro , Antígenos Específicos del Melanoma , Ratones , Ratones Endogámicos C57BL , Transferrina/farmacocinéticaRESUMEN
The blood-brain barrier (BBB) performs a neuroprotective function by tightly controlling access to the brain; consequently it also impedes access of proteins as well as pharmacological agents to cerebral tissues. We demonstrate here that recombinant human melanotransferrin (P97) is highly accumulated into the mouse brain following intravenous injection and in situ brain perfusion. Moreover, P97 transcytosis across bovine brain capillary endothelial cell (BBCEC) monolayers is at least 14-fold higher than that of holo-transferrin, with no apparent intra-endothelial degradation. This high transcytosis of P97 was not related to changes in the BBCEC monolayer integrity. In addition, the transendothelial transport of P97 was sensitive to temperature and was both concentration- and conformation-dependent, suggesting that the transport of P97 is due to receptor-mediated endocytosis. In spite of the high degree of sequence identity between P97 and transferrin, a different receptor than the one for transferrin is involved in P97 transendothelial transport. A member of the low-density lipoprotein receptor protein family, likely LRP, seems to be involved in P97 transendothelial transport. The brain accumulation, high rate of P97 transcytosis and its very low level in the blood suggest that P97 could be advantageously employed as a new delivery system to target drugs directly to the brain.