Receptor-mediated Transport Systems Research Articles

The solute carrier SLC7A1 may act as a protein transporter at the blood-brain barrier

Despite extensive research, targeted delivery of substances to the brain still poses a great challenge due to the selectivity of the blood-brain barrier (BBB). Most molecules require either carrier- or receptor-mediated transport systems to reach the central nervous system (CNS). These transport systems form attractive routes for the delivery of therapeutics into the CNS, yet the number of known brain endothelium-enriched receptors allowing the transport of large molecules into the brain is scarce. Therefore, to identify novel BBB targets, we combined transcriptomic analysis of human and murine brain endothelium and performed a complex screening of BBB-enriched genes according to established selection criteria. As a result, we propose the high-affinity cationic amino acid transporter 1 (SLC7A1) as a novel candidate for transport of large molecules across the BBB. Using RNA sequencing and in situ hybridization assays, we demonstrated elevated SLC7A1 gene expression in both human and mouse brain endothelium. Moreover, we confirmed SLC7A1 protein expression in brain vasculature of both young and aged mice. To assess the potential of SLC7A1 as a transporter for larger proteins, we performed internalization and transcytosis studies using a radiolabelled or fluorophore-labelled anti-SLC7A1 antibody. Our results showed that SLC7A1 internalised a SLC7A1-specific antibody in human colorectal carcinoma (HCT116) cells. Moreover, transcytosis studies in both immortalised human brain endothelial (hCMEC/D3) cells and primary mouse brain endothelial cells clearly demonstrated that SLC7A1 effectively transported the SLC7A1-specific antibody from luminal to abluminal side. Therefore, here in this study, we present for the first time the SLC7A1 as a novel candidate for transport of larger molecules across the BBB.

Enhanced in vivo blood brain barrier transcytosis of macromolecular cargo using an engineered pH-sensitive mouse transferrin receptor binding nanobody

BackgroundThe blood brain barrier limits entry of macromolecular diagnostic and therapeutic cargos. Blood brain barrier transcytosis via receptor mediated transport systems, such as the transferrin receptor, can be used to carry macromolecular cargos with variable efficiency. Transcytosis involves trafficking through acidified intracellular vesicles, but it is not known whether pH-dependent unbinding of transport shuttles can be used to improve blood brain barrier transport efficiency.MethodsA mouse transferrin receptor binding nanobody, NIH-mTfR-M1, was engineered to confer greater unbinding at pH 5.5 vs 7.4 by introducing multiple histidine mutations. The histidine mutant nanobodies were coupled to neurotensin for in vivo functional blood brain barrier transcytosis testing via central neurotensin-mediated hypothermia in wild-type mice. Multi-nanobody constructs including the mutant M1R56H, P96H, Y102H and two copies of the P2X7 receptor-binding 13A7 nanobody were produced to test proof-of-concept macromolecular cargo transport in vivo using quantitatively verified capillary depleted brain lysates and in situ histology.ResultsThe most effective histidine mutant, M1R56H, P96H, Y102H-neurotensin, caused > 8 °C hypothermia after 25 nmol/kg intravenous injection. Levels of the heterotrimeric construct M1R56H, P96H, Y102H-13A7-13A7 in capillary depleted brain lysates peaked at 1 h and were 60% retained at 8 h. A control construct with no brain targets was only 15% retained at 8 h. Addition of the albumin-binding Nb80 nanobody to make M1R56H, P96H, Y102H-13A7-13A7-Nb80 extended blood half-life from 21 min to 2.6 h. At 30–60 min, biotinylated M1R56H, P96H, Y102H-13A7-13A7-Nb80 was visualized in capillaries using in situ histochemistry, whereas at 2–16 h it was detected in diffuse hippocampal and cortical cellular structures. Levels of M1R56H, P96H, Y102H-13A7-13A7-Nb80 reached more than 3.5 percent injected dose/gram of brain tissue after 30 nmol/kg intravenous injection. However, higher injected concentrations did not result in higher brain levels, compatible with saturation and an apparent substrate inhibitory effect.ConclusionThe pH-sensitive mouse transferrin receptor binding nanobody M1R56H, P96H, Y102H may be a useful tool for rapid and efficient modular transport of diagnostic and therapeutic macromolecular cargos across the blood brain barrier in mouse models. Additional development will be required to determine whether this nanobody-based shuttle system will be useful for imaging and fast-acting therapeutic applications.

A Historical Review of Brain Drug Delivery.

The history of brain drug delivery is reviewed beginning with the first demonstration, in 1914, that a drug for syphilis, salvarsan, did not enter the brain, due to the presence of a blood–brain barrier (BBB). Owing to restricted transport across the BBB, FDA-approved drugs for the CNS have been generally limited to lipid-soluble small molecules. Drugs that do not cross the BBB can be re-engineered for transport on endogenous BBB carrier-mediated transport and receptor-mediated transport systems, which were identified during the 1970s–1980s. By the 1990s, a multitude of brain drug delivery technologies emerged, including trans-cranial delivery, CSF delivery, BBB disruption, lipid carriers, prodrugs, stem cells, exosomes, nanoparticles, gene therapy, and biologics. The advantages and limitations of each of these brain drug delivery technologies are critically reviewed.

Identification of lamprey variable lymphocyte receptors that target the brain vasculature

The blood–brain barrier (BBB) represents a significant bottleneck for the delivery of therapeutics to the central nervous system. In recent years, the promise of coopting BBB receptor-mediated transport systems for brain drug delivery has increased in large part due to the discovery and engineering of BBB-targeting antibodies. Here we describe an innovative screening platform for identification of new BBB targeting molecules from a class of lamprey antigen recognition proteins known as variable lymphocyte receptors (VLRs). Lamprey were immunized with murine brain microvessel plasma membranes, and the resultant repertoire cloned into the yeast surface display system. The library was screened via a unique workflow that identified 16 VLR clones that target extracellular epitopes of in vivo-relevant BBB membrane proteins. Of these, three lead VLR candidates, VLR-Fc-11, VLR-Fc-30, and VLR-Fc-46 selectively target the brain vasculature and traffic within brain microvascular endothelial cells after intravenous administration in mice, with VLR-Fc-30 being confirmed as trafficking into the brain parenchyma. Epitope characterization indicates that the VLRs, in part, recognize sialylated glycostructures. These promising new targeting molecules have the potential for brain targeting and drug delivery with improved brain vascular specificity.

The Isolated Brain Microvessel: A Versatile Experimental Model of the Blood-Brain Barrier.

A versatile experimental model for the investigation of the blood-brain barrier (BBB), including the neuro-vascular unit, is the isolated brain microvessel preparation. Brain microvessels are primarily comprised of endothelial cells, but also include pericytes, pre-capillary arteriolar smooth muscle cells, astrocyte foot processes, and occasional nerve endings. These microvessels can be isolated from brain with a 3 h procedure, and the microvessels are free of brain parenchyma. Brain microvessels have been isolated from fresh animal brain, fresh human brain obtained at neurosurgery, as well as fresh or frozen autopsy human brain. Brain microvessels are the starting point for isolation of brain microvessel RNA, which then enables the production of BBB cDNA libraries and a genomics analysis of the brain microvasculature. Brain microvessels, combined with quantitative targeted absolute proteomics, allow for the quantitation of specific transporters or receptors expressed at the brain microvasculature. Brain microvessels, combined with specific antibodies and immune labeling of isolated capillaries, allow for the cellular location of proteins expressed within the neuro-vascular unit. Isolated brain microvessels can be used as an “in vitro” preparation of the BBB for the study of the kinetic parameters of BBB carrier-mediated transport (CMT) systems, or for the determination of dissociation constants of peptide binding to BBB receptor-mediated transport (RMT) systems expressed at either the animal or the human BBB. This review will discuss how the isolated brain microvessel model system has advanced our understanding of the organization and functional properties of the BBB, and highlight recent renewed interest in this 50 year old model of the BBB.

A review on graphene nanoribbons for advanced biomedical applications

Graphene nanoribbons materialize as a next-generation carrier for development of nanodimensional diagnostic devices and drug delivery systems due to the unique and cutting-edge electronic, thermal, mechanical and optical properties associated with graphene. This review article focuses on the important applications of GNRs in the field of biomedicine and biosensing. Graphene nanoribbons are highly developed form of graphene with a wide importance due to their distinctive properties such as large surface area, enhanced mechanical strength and improved electro-conductivity. GNRs are effective substitutes for conventional silicon-based transistors used in biochemical reactions and exploited in the fields of biomedicine and diagnostics due to their effective uptake by mammalian cells. The cellular interactions of GNRs consist of highly specific receptor-mediated transport, phagocytosis and non-specific transport systems involving copious forces of adhesion. The presence of quantum chains in GNRs increases their potential for fabrication of technically challenging sensing devices in the future.

Photo-physicochemical properties and in vitro photodynamic therapy activity of morpholine-substituted Zinc(II)-Phthalocyanines π-π stacked on biotinylated graphene quantum dots

Two morpholine-substituted Zn(II) phthalocyanines, complex 4 and the cationic 5, were synthesized and conjugated to graphene quantum dots (GQDs) and biotinylated GQDs (GQDs-biotin) by non-covalent π-π interactions. The GQDs-biotin are prepared as potential nanoparticle-based Pc delivery vector combined with a receptor-mediated transport system using biotin. The photo-physicochemical properties of the Pc complexes and their corresponding conjugates were studied. Upon conjugation, the fluorescence quantum yields decrease for 4 and 5, however, the triplet quantum yields were increased for the conjugates. All samples demonstrated singlet oxygen generation. For conjugated complexes, the singlet quantum yields decreased due to the screening effect in some cases. An increase in the photodynamic therapy activities upon quaternization was observed for the conjugates, with the cell viability decreasing from 66.2% to 51.2% after treatment for 4-GQDs and 5-GQDs, respectively. A relatively better performance was also observed for the cationic complex in combination with the biotin functionalized GQDs, 5-GQDs-biotin, where the cell viability was 34.9% after treatment. Moreover, the cellular uptake of 5-GQDs-biotin over 24 h was relatively higher compared to Pc complex alone and other Pcs-GQDs conjugates.

Blood-brain barrier transport machineries and targeted therapy of brain diseases.

Introduction: Desired clinical outcome of pharmacotherapy of brain diseases largely depends upon the safe drug delivery into the brain parenchyma. However, due to the robust blockade function of the blood-brain barrier (BBB), drug transport into the brain is selectively controlled by the BBB formed by brain capillary endothelial cells and supported by astrocytes and pericytes. Methods: In the current study, we have reviewed the most recent literature on the subject to provide an insight upon the role and impacts of BBB on brain drug delivery and targeting. Results: All drugs, either small molecules or macromolecules, designated to treat brain diseases must adequately cross the BBB to provide their therapeutic properties on biological targets within the central nervous system (CNS). However, most of these pharmaceuticals do not sufficiently penetrate into CNS, failing to meet the intended therapeutic outcomes. Most lipophilic drugs capable of penetrating BBB are prone to the efflux functionality of BBB. In contrast, all hydrophilic drugs are facing severe infiltration blockage imposed by the tight cellular junctions of the BBB. Hence, a number of strategies have been devised to improve the efficiency of brain drug delivery and targeted therapy of CNS disorders using multimodal nanosystems (NSs). Conclusions: In order to improve the therapeutic outcomes of CNS drug transfer and targeted delivery, the discriminatory permeability of BBB needs to be taken under control. The carrier-mediated transport machineries of brain capillary endothelial cells (BCECs) can be exploited for the discovery, development and delivery of small molecules into the brain. Further, the receptor-mediated transport systems can be recruited for the delivery of macromolecular biologics and multimodal NSs into the brain.

Overcoming the blood-brain tumor barrier for effective glioblastoma treatment.

Gliomas are the most common primary brain tumors. Particularly in adult patients, the vast majority of gliomas belongs to the heterogeneous group of diffuse gliomas, i.e. glial tumors characterized by diffuse infiltrative growth in the preexistent brain tissue. Unfortunately, glioblastoma, the most aggressive (WHO grade IV) diffuse glioma is also by far the most frequent one. After standard treatment, the 2-year overall survival of glioblastoma patients is approximately only 25%. Advanced knowledge in the molecular pathology underlying malignant transformation has offered new handles and better treatments for several cancer types. Unfortunately, glioblastoma multiforme (GBM) patients have not yet profited as although numerous experimental drugs have been tested in clinical trials, all failed miserably. This grim prognosis for GBM is at least partly due to the lack of successful drug delivery across the blood-brain tumor barrier (BBTB). The human brain comprises over 100 billion capillaries with a total length of 400 miles, a total surface area of 20 m(2) and a median inter-capillary distance of about 50 μm, making it the best perfused organ in the body. The BBTB encompasses existing and newly formed blood vessels that contribute to the delivery of nutrients and oxygen to the tumor and facilitate glioma cell migration to other parts of the brain. The high metabolic demands of high-grade glioma create hypoxic areas that trigger increased expression of VEGF and angiogenesis, leading to the formation of abnormal vessels and a dysfunctional BBTB. Even though the BBTB is considered 'leaky' in the core part of glioblastomas, in large parts of glioblastomas and, even more so, in lower grade diffuse gliomas the BBTB more closely resembles the intact blood-brain barrier (BBB) and prevents efficient passage of cancer therapeutics, including small molecules and antibodies. Thus, many drugs can still be blocked from reaching the many infiltrative glioblastoma cells that demonstrate 'within-organ-metastasis' away from the core part to brain areas displaying a more organized and less leaky BBTB. Hence, drug delivery in glioblastoma deserves explicit attention as otherwise new experimental therapies will continue to fail. In the current review we highlight different aspects of the BBTB in glioma patients and preclinical models and discuss the advantages and drawbacks of drug delivery approaches for the treatment of glioma patients. We provide an overview on methods to overcome the BBTB, including osmotic blood-brain barrier disruption (BBBD), bradykinin receptor-mediated BBTB opening, inhibition of multidrug efflux transporters, receptor-mediated transport systems and physiological circumvention of the BBTB. While our knowledge about the molecular biology of glioma cells is rapidly expanding and is, to some extent, already assisting us in the design of tumor-tailored therapeutics, we are still struggling to develop modalities to expose the entire tumor to such therapeutics at pharmacologically meaningful quantities. Therefore, we must expand our knowledge about the fundamentals of the BBTB as a step toward the design of practical and safe devices and approaches for enhanced drug delivery into the diseased brain area.

Targeting receptor-mediated transport for delivery of biologics across the blood-brain barrier.

Biologics are an emerging class of medicines with substantial promise to treat neurological disorders such as Alzheimer's disease, stroke, and multiple sclerosis. However, the blood-brain barrier (BBB) presents a formidable obstacle that appreciably limits brain uptake and hence the therapeutic potential of biologics following intravenous administration. One promising strategy for overcoming the BBB to deliver biologics is the targeting of endogenous receptor-mediated transport (RMT) systems that employ vesicular trafficking to transport ligands across the BBB endothelium. If a biologic is modified with an appropriate targeting ligand, it can gain improved access to the brain via RMT. Various RMT-targeting strategies have been developed over the past 20 years, and this review explores exciting recent advances, emphasizing studies that show brain targeting in vivo.

Mechanisms of action of brain insulin against neurodegenerative diseases

Insulin, a pancreatic hormone, is best known for its peripheral effects on the metabolism of glucose, fats and proteins. There is a growing body of evidence linking insulin action in the brain to neurodegenerative diseases. Insulin present in central nervous system is a regulator of central glucose metabolism nevertheless this glucoregulation is not the main function of insulin in the brain. Brain is known to be specifically vulnerable to oxidative products relative to other organs and altered brain insulin signaling may cause or promote neurodegenerative diseases which invalidates and reduces the quality of life. Insulin located within the brain is mostly of pancreatic origin or is produced in the brain itself crosses the blood-brain barrier and enters the brain via a receptor-mediated active transport system. Brain Insulin, insulin receptor and insulin receptor substrate-mediated signaling pathways play important roles in the regulation of peripheral metabolism, feeding behavior, memory and maintenance of neural functions such as neuronal growth and differentiation, neuromodulation and neuroprotection. In the present review, we would like to summarize the novel biological and pathophysiological roles of neuronal insulin in neurodegenerative diseases and describe the main signaling pathways in use for therapeutic strategies in the use of insulin to the cerebral tissues and their biological applications to neurodegenerative diseases.

Blood-Brain Barrier Molecular Trojan Horse Enables Imaging of Brain Uptake of Radioiodinated Recombinant Protein in the Rhesus Monkey

Recombinant proteins are large molecule drugs that do not cross the blood-brain barrier (BBB). However, BBB-penetration of protein therapeutics is enabled by re-engineering the recombinant protein as IgG fusion proteins. The IgG domain is a monoclonal antibody (mAb) against an endogenous BBB receptor-mediated transport system, such as the human insulin receptor (HIR), and acts as a molecular Trojan horse to ferry the fused protein across the BBB. In the present study, a recombinant lysosomal enzyme, iduronate 2-sulfatase (IDS), is fused to the HIRMAb, and BBB penetration of the IDS alone vs the HIRMAb-IDS fusion protein is compared in the Rhesus monkey. Recombinant IDS and the HIRMAb-IDS fusion protein were radiolabeled with indirect iodination with the [(125)I]-Bolton-Hunter reagent and with direct iodination with Iodogen/[(125)I]-idodine. IDS and the HIRMAb-IDS fusion protein have comparable plasma pharmacokinetics and uptake by peripheral organs. IDS does not cross the BBB. The HIRMAb-IDS fusion protein crosses the BBB and the brain uptake is 1% of injected dose/brain. Brain imaging shows HIRMAb-IDS penetration to all parts of brain, and immunoprecipitation of brain radioactivity shows intact fusion protein in brain. The use of BBB molecular Trojan horses enables brain imaging of recombinant proteins that are re-engineered for BBB transport.

Study of the transcytosis of an anti-transferrin receptor antibody with a Fab′ cargo across the blood–brain barrier in mice

One strategy used to transport pharmacologically active substances across the blood–brain barrier (BBB) is to link the substance to a molecule capable of crossing the BBB using a receptor-mediated transcellular transport system in brain capillary endothelial cells. The transferrin receptor (TfR) is related to a transcytosis process in these cells, and the 8D3 antibody, directed against mouse TfR, is able to induce a receptor response. In this work, the potential of 8D3 to carry molecules across the BBB was investigated. This antibody (either unlabeled or FITC-labeled) was intravenously administered to ICR-CD1 mice. Immunocomplexes (ICs) consisting of 8D3 antibody (carrier) and Fab′ fragments (simulated cargo), in some cases directly fluorolabeled with FITC, were also administered to ICR-CD1 mice. At different time-points of recirculation, the IC components were studied using immunohistochemical procedures and fluorescence resonance energy transfer (FRET). The results suggested that 8D3 alone or in an IC with Fab′ fragments entered the endothelial cells. FRET analysis indicated that the colocalization of their fluorescent signals inside the endothelial cells decreased with time, indicating that ICs can be processed and Fab′ fragments probably separated from 8D3. However, neither 8D3 alone nor the IC components crossed the BBB.

Drug Transport across the Blood–Brain Barrier

The blood-brain barrier (BBB) prevents the brain uptake of most pharmaceuticals. This property arises from the epithelial-like tight junctions within the brain capillary endothelium. The BBB is anatomically and functionally distinct from the blood-cerebrospinal fluid barrier at the choroid plexus. Certain small molecule drugs may cross the BBB via lipid-mediated free diffusion, providing the drug has a molecular weight <400 Da and forms <8 hydrogen bonds. These chemical properties are lacking in the majority of small molecule drugs, and all large molecule drugs. Nevertheless, drugs can be reengineered for BBB transport, based on the knowledge of the endogenous transport systems within the BBB. Small molecule drugs can be synthesized that access carrier-mediated transport (CMT) systems within the BBB. Large molecule drugs can be reengineered with molecular Trojan horse delivery systems to access receptor-mediated transport (RMT) systems within the BBB. Peptide and antisense radiopharmaceuticals are made brain-penetrating with the combined use of RMT-based delivery systems and avidin-biotin technology. Knowledge on the endogenous CMT and RMT systems expressed at the BBB enable new solutions to the problem of BBB drug transport.

Drug delivery to the CNS

Effective delivery of biological products to the central nervous system remains on the frontier of modern medicine. The blood–brain barrier (BBB) is an efficient and elegant system for regulating the influx and efflux of molecular entities. The selectivity of this endothelial layer is essential for maintaining a homeostatic CNS environment and protecting neuronal tissues from foreign or harmful blood born compounds. Therapeutic interventions for CNS diseases, to be effective, must contend with traversing this barrier and reaching their targets. Although there have been substantial advances in our understanding of the molecular biology of the BBB, we have yet to translate this knowledge into consistent and reliable modalities for delivering a diverse array of therapeutic biological agents. Circumventing rather than traversing the BBB has been successfully used to deliver small molecules to the CNS for pain management, treatment of spasticity, and cancer chemotherapy. Typically, this requires the use of mechanical strategies, such as ports and pumps, to deliver drugs directly into the CSF. This approach has an inherent risk of infection, and is also limited by the physical and physiological factors governing tissue penetration and distribution. A deeper understanding of receptor-mediated transport systems and CSF dynamics is needed in order to generalize this approach to treat a broader spectrum of CNS diseases. In July 2011, the Controlled Release Society held a workshop designed to bring together leading scientists in the fields of drug delivery, vascular biology, imaging, animal model development, and clinical research (1), with the goal of engaging in multidisciplinary discussions of the strategies needed to develop drug and biological products targeted to the CNS, from drug discovery through to clinical evaluation. The presenters were invited to participate in this special issue of Drug Delivery and Translational Research to capture the wealth of information and thought provoking material that was presented in this unique setting. Patricia Dickson, the workshop facilitator, describes the current state of CNS delivery and provides some insight into the challenges associated with conducting clinical trials, particularly in rare CNS diseases. William Banks provides a synopsis of the molecular interactions related to the influx/efflux modalities of the BBB and how they could be adapted to facilitate brain delivery from systemic administration. Recent work has shown that intranasal administration is a potential access point for the BBB. Leah Hanson et al. demonstrate that intranasal administration of an iron chelator reduces spatial memory loss in a mouse model of Alzheimer’s disease, suggesting that this approach may be a viable, noninvasive approach for CNS delivery. An area of intense research in CNS delivery of large molecule biologics is in the treatment of lysosomal storage diseases (LSD), a collection of ~50 different rare genetic disorders characterized by a deficiency in the activity of one of the lysosomal enzymes. In the majority of the LSDs, uncontrolled accumulation of substrate results in progressive CNS impairment and premature death. SilviaMuro provides a review of the underlying physiology of this class of diseases, and approaches to deliver protein therapeutics across the BBB. Another approach under investigation for the treatment of LSDs is the direct administration of enzyme to the CNS. Richard Pfeifer et al. provide evidence in nonhuman primates that lumbar administration is safe, well tolerated, and likely to deliver sufficient enzyme to the relevant anatomical sites within the brain. Another major challenge in developing delivery modalities for CNS therapeutics is accurately assessing where and how much product is delivered to the targeted tissue. Immunohistochemistry, the standard method for describing biodistribution in nonclinical studies, requires a large number of animals and is not applicable in the clinical setting. Mikhail Papisov et In addition to the contributing authors, I would like to thank Anne Pariser for her significant contributions to the CNS Workshop and her thoughtful review of this article.

Gd(iii) chelates for MRI contrast agents: from high relaxivity to “smart”, from blood pool to blood–brain barrier permeable

Paramagnetic Gd(III) chelate contrast agents have been extensively used to enhance the signal of MRI scans for the last three decades. The use of Gd(III) chelate contrast agents is projected to increase as new agents and applications arise, thanks to the favorable combination of a large magnetic moment and long electron spin relaxation time of the Gd(III) ion. The relaxivity and stability of Gd(III) chelates are the primary requisites for the development of contrast agents as both small doses and low release of free Gd(III) ions will reduce the toxicity. The physico-chemical parameters and structure-related relaxation mechanisms provide the strategies for chelate design. The higher relaxivity and efficacy of the contrast agent can be improved by designing pH-, metal ion-, enzyme- or small biomolecule-dependent “smart” contrast agents. Through conjugation to biomacromolecules such as polymers, dendrimers or non-covalent binding to plasma proteins, contrast agents could increase the blood half-life and can be used for contrast-enhanced MRI. When conjugated to certain diagnostic or therapeutic proteins, low molecular weight Gd(III) chelates could cross the blood–brain barrier (BBB) by means of a receptor-mediated transport system or receptor-mediated transcytosis.

Transporters in the Brain Endothelial Barrier

The blood-brain barrier (BBB) not only impedes the influx of intravascular substances from blood to brain, but also promotes transport of substances from blood to brain or from brain to blood through several transport systems such as carrier-mediated transport, active efflux transport, and receptor-mediated transport systems. The multidrug resistance transporter P-glycoprotein (P-gp) is an ATP-dependent efflux pump and contributes to efflux of undesirable substances such as amyloid-beta:(Abeta) proteins from the brain into the blood as well as many drugs such as anti-cancer drugs. The inhibition of P-gp has favorable and unfavorable effects on living bodies. P-gp deficiency at the BBB induces the increase of Abeta:deposition in the brain of an Alzheimer disease mouse model. It is also known that the Abeta:deposition is inversely correlated with P-gp expression in the brains of elderly non-demented humans. However, the transient inhibition of P-gp by antidepressants enables medicines such as anti-cancer drugs to enter the brain. Concerning Abeta:clearance in the brain, the low-density lipoprotein receptor-related protein 1 (LRP1) is a major efflux transporter for Abeta, while the receptor for advanced glycation end products (RAGE) is a major influx transporter for Abeta:across the BBB. Dysfunction of the BBB with efflux and influx transporters may contribute to the pathogenesis of some degenerative neuronal disorders. This review will focus on several transporters and discuss how medicines pass the BBB to reach the brain parenchyma.

Getting into the Brain

Being the most delicate organ of the body, the brain is protected against potentially toxic substances by the blood-brain barrier (BBB), which restricts the entry of most pharmaceuticals into the brain. The developmental process for new drugs for the treatment of CNS disorders has not kept pace with progress in molecular neurosciences because most of the new drugs discovered are unable to cross the BBB. The clinical failure of CNS drug delivery may be attributed largely to a lack of appropriate drug delivery systems. Localized and controlled delivery of drugs at their desired site of action is preferred because it reduces toxicity and increases treatment efficiency. The present review provides an insight into some of the recent advances made in the field of brain drug delivery.The various strategies that have been explored to increase drug delivery into the brain include (i) chemical delivery systems, such as lipid-mediated transport, the prodrug approach and the lock-in system; (ii) biological delivery systems, in which pharmaceuticals are re-engineered to cross the BBB via specific endogenous transporters localized within the brain capillary endothelium; (iii) disruption of the BBB, for example by modification of tight junctions, which causes a controlled and transient increase in the permeability of brain capillaries; (iv) the use of molecular Trojan horses, such as peptidomimetic monoclonal antibodies to transport large molecules (e.g. antibodies, recombinant proteins, nonviral gene medicines or RNA interference drugs) across the BBB; and (v) particulate drug carrier systems. Receptor-mediated transport systems exist for certain endogenous peptides, such as insulin and transferrin, enabling these molecules to cross the BBB in vivo.The use of polymers for local drug delivery has greatly expanded the spectrum of drugs available for the treatment of brain diseases, such as malignant tumours and Alzheimer's disease. In addition, various drug delivery systems (e.g. liposomes, microspheres, nanoparticles, nanogels and bionanocapsules) have been used to enhance drug delivery to the brain. Recently, microchips and biodegradable polymers have become important in brain tumour therapy.The intense search for alternative routes of drug delivery (e.g. intranasal drug delivery, convection-enhanced diffusion and intrathecal/intraventricular drug delivery systems) has been driven by the need to overcome the physiological barriers of the brain and to achieve high drug concentrations within the brain. For more than 30 years, considerable efforts have been made to enhance the delivery of therapeutic molecules across the vascular barriers of the CNS. The current challenge is to develop drug delivery strategies that will allow the passage of drug molecules through the BBB in a safe and effective manner.

Future prospects of nanoparticles on brain targeted drug delivery

To the editor Human brain is restricted and separated from circulatory network by a highly efficient blood brain barrier. This is constituted by relatively impermeable endothelial cells with tight junctions, enzymatic activity and active efflux transport systems. Physiologically, blood-brain barrier is designed in such a manner that it can only permit the transport of molecules essential for functional activity of brain. It efficiently prevents flow of water-soluble molecules from blood circulation into central nervous system, and can also decrease concentration of lipid-soluble molecules by the action of enzymes or efflux pumps [1]. Presently, drug delivery to central nervous system is a major menace as multiple cerebral diseases like Alzheimer’s, brain tumors, prion diseases are cropping up. The blood brain barrier (BBB) represents an insurmountable obstacle for a large number of drugs including antibiotics, antineoplastic agents and a variety of central nervous system (CNS)-active drugs like neuropeptides [1, 2]. This creates a considerable threat for the therapy of cerebral diseases. Currently, nanoparticles are utilized as a drug delivery vehicle. The use of nanoparticles to deliver drugs to the brain by infiltrating blood brain barrier (BBB) may provide a significant strategy to break this impasse. The primary advantage of nanoparticle carrier technology is that it can cross blood brain barrier entrapping the original characteristics of the therapeutic drug molecule. Furthermore, this system may reduce drug leaching in the brain and decrease peripheral toxicity [3]. Drugs that have successfully been transported into the brain using nanoparticle carrier include the hexapeptide dalargin, the dipeptide kytorphin, loperamide, tubocurarine, the NMDA receptor antagonist MRZ 2/ 576, doxorubicin, etc. The nanoparticles may be special vehicle for the treatment of the brain tumors [2] especially for primary and metastatic brain tumors [4]. Currently, nanoparticle iron chelators have been used to treat Alzheimer disease and other neurologic disorders associated with trace metal imbalance [5]. It has been reported that PEGylated polymeric nanoparticles are efficient drug carrier for the delivery of active therapeutic molecules in prion diseases [6]. Strategies for nanoparticle targeting to the brain rely on the presence of its interaction with specific receptor-mediated transport systems in the BBB. For example, polysorbate 80/LDL, transferrin receptor binding antibody (such as OX26), lactoferrin, cellpenetrating peptides and melanotransferrin have been shown its efficacy of delivering a self non-transportable drug into the brain via chimeric construct that can undergo receptor-mediated transcytosis [7–11]. It has been reported that poly (butylcyanoacrylate) nanoparticles is able to deliver hexapeptide dalargin, doxorubicin and other agents into the brain which C. Chakraborty Department of Biotechnology, College of Engineering and Technology, IILM Academy of Higher Learning, Knowledge Park-II, Greater Noida, India

Effects of intranasal insulin on cognitive function and body composition

Systemic insulin has rapid access to the brain by a receptor-mediated transport system located in endothelial cells of brain microvessels. In several species, including man, parallel increases in the concentration of insulin in the cerebrospinal fluid (CSF) after acute elevation of plasma insulin levels are well documented. In animal studies central nervous insulin has been shown to play an important role in body weight regulation and memory function. Direct intracerebroventricular administration of insulin reduced food intake and caused weight loss. Moreover, insulin in the brain has been shown to improve memory function. However, in humans the investigation of central nervous effects of insulin has been hampered by the fact, that infusion of insulin and glucose is not practicable for a longer time to obtain changes in body weight. Recently, we provided evidence, that insulin enters the CSF compartment after intranasal administration without absorption into the blood stream. With this approach, we investigated the effect of long-term intranasal administration on body weight regulation in healthy subjects. After a baseline of 2 weeks 40 lean subjects (16 females, 24 males) were treated for 8 weeks with intranasal insulin (4×40 IU/day) or placebo in a double blind, between subject comparison. Body weight and body composition were measured weekly by bioimpedance analysis. Blood glucose and plasma insulin levels were identical during intranasal administration of insulin and placebo, indicating that no relevant amount of insulin entered the blood stream. There was no decrease in body weight and body fat mass during intranasal insulin administration in woman as compared to placebo. However, in contrast to women, insulin treated men continuously lost body weight compared to the placebo condition (baseline adjusted body weight at the end of the study, placebo: 79.84±0.57kg; insulin: 78.12±0.57kg, p<0.05). Most important, this weight loss was mainly a result of a reduction in body fat mass (placebo: 14.12±0.61kg, insulin: 12.25 0.61g, p<0.05). With the same approach, effects of intranasal administration of insulin (4×40 IU/day) or placebo for 8 weeks on memory function were investigated in 38 healthy subjects (14 females, 24 males). Every week subjects were presented with a word list containing 30 words. One week later they had to tell the words they remembered. Compared to the baseline condition intranasal administration of insulin significantly improved memory function (words recalled, placebo: 2.92±1.00, insulin: 6.20±1.03, p<0.05) at the end of the treatment period. There was no difference in the insulin effects on memory function in males and females. With the same approach, effects of intranasal insulin aspart even exceeded the beneficial effects of human insulin on body fat mass (placebo: +0.45±0.63kg, human insulin: -1.38±0.63kg, insulin aspart: -2.67±0.63kg, p<0.08) and memory function (words recalled, placebo: 1.83±0.75, human insulin: 5.17±1.56, insulin aspart: 8.35±1.09, p<0.05) in 36 healthy men. These results indicate an important role for insulin in body weight regulation and memory function in humans. Concerning recent findings of a reduced transport of insulin across the blood brain barrier in obese subjects and in Alzheimer patients, these findings could be of considerable clinical relevance.