Applications of biotechnology in neurology


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Applications of biotechnology in neurology

  1. 1. 477K.K. Jain, Applications of Biotechnology in Neurology,DOI 10.1007/978-1-62703-272-8_13, © Springer Science+Business Media, LLC 2013IntroductionThe delivery of drugs to the brain is a challenge in the treatment of CNS disorders(Jain 2010). The major obstruction to CNS drug delivery is the blood–brain barrier(BBB), which limits the access of drugs to the brain substance. In the past, treat-ment of CNS disease was done mostly with systemically administered drugs. Thistrend continues. Most CNS-disorder research is directed toward the discovery ofdrugs and formulations for controlled release; little attention has been paid to themethod of delivery of these drugs to the brain. Now biotechnology is making asignificant contribution to drug delivery in disorders of the CNS.Other neural barriers are the blood-CSF barrier, the blood-retinal barrier, theblood-labyrinth barrier, and the blood-nerve barrier, which is applicable only tothe peripheral nervous system. The BBB has been much more extensively inves-tigated than the blood-nerve barrier. A number of enzymes, transporters, andreceptors have been investigated at both the blood-nerve barrier and BBB, aswell as in the perineurium of peripheral nerves, which is also a metabolicallyactive diffusion barrier. Knowledge of the BBB is important in neurology for thefollowing reasons:Understanding of brain function•Pathophysiology of neurologic disorders•Drug delivery to the brain•An understanding of specific interactions between the brain endothelium, astro-cytes, and neurons that may regulate BBB function and how these interactions aredisturbed in pathological conditions could lead to the development of neuroprotec-tive and restorative therapies.Chapter 13Role of Biotechnology in Drug Deliveryto the Nervous System
  2. 2. 478 13 Role of Biotechnology in Drug Delivery to the Nervous SystemHistorical Evolution of Drug Delivery for CNS DisordersLandmarks in the historical evolution of drug delivery technology to the brain areshown in Table 13.1. They are related to the history of the blood–brain barrier (BBB).Although the cerebral ventricles were tapped for hydrocephalus in ancient times,the first perforation of subarachnoid space by lumbar puncture was made in 1885 toadminister cocaine for anesthesia (Corning 1885). Paul Ehrlich described the con-cept of the blood–brain barrier in the same year when he observed that dyes injectedinto the vascular system were rapidly taken up by all the organs except the brain(Ehrlich 1885). Later, research showed that dyes injected into the cerebrospinal fluidhave free access to the neural tissues but do not enter the blood supply of the brain.Coining of the term “blood–brain barrier” to describe this phenomenon is attributedto Lewandowski in 1900. Despite the BBB, intracerebral distribution of varioussubstances was observed. “Barrière hémato-encéphalique” was defined as a cerebralblood vessel compartment in which choroid plexus was semipermeable, facilitatingthe flow of substances from the blood into the CSF (Stern and Gautier 1921). BBBpermeability to hexoses, amino acids, amines, and neurotransmitters was demon-strated 50 years later by radiolabeled substances (Oldendorf 1971).Broman first observed the transient opening or disruption of the BBB after intraca-rotid arterial administration of hypertonic solutions in 1941 (Broman 1941). The firstinjections into the cerebral circulation were of contrast materials for cerebral angiog-raphy (Moniz 1927). The injection of a therapeutic substance (diazepam) into thecarotid arteries was not reported until almost a half-century later (Doppman 1973).The advent of stereotactic surgery more than 50 years ago opened the way forplacing instruments at selected targets in the depths of the brain for the treatmentof movement disorders (Spiegel et al. 1947). This approach was used some yearslater to perform chemopallidectomy by injection of a mixture of procaine and alco-hol into the globus pallidus (Cooper 1954). The techniques of creating lesions inbasal ganglia have been refined, but these principles of localization and injectioncontinue to be used for the introduction of novel therapeutic agents into the brainfor the treatment of movement disorders. The first implantable pump for intrathecaland intraventricular injection of morphine for the treatment of cancer pain wasdescribed in 1978 (Lazorthes et al. 1991). During the past 25 years, further progresshas taken place with the development of intra-arterial chemotherapy, direct injec-tions of therapeutic substances into intracranial lesions, and strategies to overcomethe BBB. Further advances have taken place with the development of cell and genetherapies as well as nanobiotechnology.The Neurovascular UnitA neurovascular unit, consisting of endothelial cells, neurons, and glia, regulates theBBB. The fact that endothelial cells of brain capillaries differ greatly from those inthe periphery confers on the BBB its discriminatory characteristics. Brain endothelialcells display tight junctions, absence of intercellular clefts and fenestrations, minor
  3. 3. 479The Neurovascular UnitTable 13.1 Landmarks in the development of drug delivery to the CNSYear Observation/concept/comment1885 First lumbar puncture to administer cocaine for anesthesia (Corning 1885)1885 Concept of BBB indicated by the observation that dyes injected into the vascularsystem were rapidly taken up by all the organs except the brain (Ehrlich1885)1900 Coining of the term “blood–brain barrier” to describe the phenomenon(Lewandowski 1900)1913 BBB observed to be decreased in the choroid plexus (Goldmann 1913)1920 Intracerebral distribution of various substances administered systematically wasobserved1921–1922 Intracerebral distribution of various substances was observed. “Barriére hémato-encephalique” was defined as a cerebral blood vessel compartment in whichchoroid plexus was semipermeable, facilitating the flow of substances fromthe blood into the CSF (Stern and Gautier 1921, 1922)1927 First injections into the cerebral circulation: contrast materials for cerebralangiography1940 Description of vertebral venous plexus and its connection to blood vessels of thebrain laid the anatomical basis for use of epidural venous injection for drugdelivery to the CNS (Batson 1940)1940s Tor Broman of Goteborg, Sweden showed that the anatomical substrate ofthe BBB was the brain capillary wall. This was confirmed by electronmicroscopic studies a quarter of a century later1941 Opening of the BBB by pharmacological means1947 Stereotactic equipment for guided placement of instruments at selected targets inthe depth of the brain for the treatment of movement disorders1950s Electron microscopy used to demonstrate lack of extracellular fluid compartmentbetween glia and neurons in the brain and this was given as an explanation offailure of substances to enter the brain1954 Injection of a mixture of procaine and alcohol into the globus pallidus of thebrain for treatment of movement disorders1967 Electron microscopy confirmed brain capillary wall to be the BBB (Reese andKarnovsky 1967)1973 First injection of a therapeutic substance (diazepam) into the carotid arteries1978 First implantable pump for intrathecal and intraventricular injection of morphine1980s Studies in molecular biology of the BBB. Cloning and sequencing of glucosetransporter gene (Weiler-Guttler et al. 1989)1990s Further development of direct injections of therapeutic substances, includingbiologicals (e.g. gene therapy), into the brain or intracranial lesions, anddevelopment of strategies to overcome the BBB1995 Use of nanoparticles for drug delivery across BBB© Jain PharmaBiotechpinocytic activity, and a high electrical resistance. These cells have a continuousbasal membrane and are surrounded by pericytes and the end feet of astrocytes,which are part of the BBB and control its permeability (Abbott et al. 2010). Variousproteins expressed in the neurovascular unit are listed in Table 13.2.
  4. 4. 480 13 Role of Biotechnology in Drug Delivery to the Nervous SystemGenes that regulate the expression of drug transporters and drug metabolizingenzymes (DMEs) in the BBB have been shown to be polymorphic, resulting in thesynthesis of proteins with impaired or increased activities (Johansson and Ingelman-Sundberg 2011). These polymorphisms may thus profoundly affect the blood levelsof drugs and chemical toxins. Using transcriptomics and proteomics approaches,the presence of these proteins has been demonstrated in isolated human brainmicrovessels and cortex biopsies (Shawahna et al. 2011). DTs and DMEs controlthe access to the brain and local concentration of both endobiotics and xenobiotics.Transport proteins enable passage of those substances required by the CNS, suchas glucose, essential amino acids, and neurotransmitter precursors. There are alsotransporters and metabolic enzymes that function in the opposite direction, therebypreventing access to the brain of some lipid-soluble drugs and potentially toxicsubstances, including metabolites, which might otherwise be able to diffuse into thebrain and cause damage. Unfortunately, this means that the BBB can also preventotherwise effective drugs from entering the brain.Passage of Substances Across the Blood–Brain BarrierSeveral carrier or transport systems, enzymes, and receptors that control the pene-tration of molecules have been identified in the BBB endothelium on the basis ofphysiological and biochemical studies. Passage of substances across the bloodbrain barrier is shown in Fig. 13.1.Various transporters localized in the BBB that control penetration of moleculesacross the BBB include the following:Energy transporters such as glucose transporter•Amino acid transporters•Table 13.2 Proteins expressed at the neurovascular unitProteins Function/cellular expressionavb8 integrin Cell adhesion/neurons, gliaAquaporin-4 Water transport/astrocyteClaudins Tight junctions/endothelial cellJunctional adhesion molecule Tight junction endothelial cellOccludins Tight junctions/endothelial cellPlatelet-derived growth factor receptor b Tyrosine kinase/pericyteSphingosine-1-phosphate G protein-coupled receptor/endothelialcell, pericytesSrc-suppressed C-kinase substrate A-kinase anchoring scaffold protein/endothelial cellTie2 Tyrosine kinase/endothelial cellZona occludens Tight junction signaling, membrane-associatedguanylate kinase/endothelial cell© Jain PharmaBiotech
  5. 5. 481Passage of Substances Across the Blood–Brain BarrierFig. 13.1 Various forms of passage of substances across the blood brain barrier. (1) Passivediffusion. Fat-soluble substances dissolve in the cell membrane and cross the barrier, e.g. alcohol,nicotine, and caffeine. Water-soluble substances like penicillin have difficulty in getting through.(2) Active transport. Substances that the brain needs such as glucose and amino acids are carriedacross by special transport proteins. (3) Receptor-mediated transport. Molecules link up to recep-tors on the surface of the brain and are escorted through, e.g. insulin (© Jain PharmaBiotech)Neurotransmitter transporters•Organic anions and cation transporters•ABC transporters•Efflux systems such as P-glycoprotein efflux system•Miscellaneous transporters such as some hormones and vitamins.•Among these, the action of efflux transporters at the BBB may have clinicalsignificance by reducing the effectiveness of drugs targeted at CNS disorders.Therefore, modulation of these efflux transporters by design of inhibitors and/ordesign of compounds that have minimal affinity for these transporters may wellenhance the treatment of intractable CNS disorders.Various enzymes control the penetration of molecules across the BBB. Anexample is monoamineoxidase, which provides an enzymatic barrier and hindersthe influx of monoamine precursors into the brain. After their entry into theendothelial cells, monoamines are decarboxylated by cytoplasmic monoamineoxi-dase, thus, effectively preventing a flood of peripheral monoaminergic neurotrans-mitters in the neuronal environments
  6. 6. 482 13 Role of Biotechnology in Drug Delivery to the Nervous SystemReceptor-Mediated Peptide and Protein TranscytosisThe transport of peptides and proteins across cellular barriers- transcytosis- hasbeen documented in a number of systems. Examples include the transport of IgGacross the intestinal epithelium and human placenta, the transport of insulin andinsulin-like growth factors across the aortic endothelium, and the transport of epi-dermal growth factor across the kidney epithelium. It is not surprising that transcy-tosis occurs across the BBB. In addition to the unidirectional and bidirectionaltransport of small molecules, other macromolecules are able to enter the brain tis-sue from the blood by a receptor-mediated process. An example of this is the trans-port of transferrin across the BBB. Brain cells require a constant supply of iron tomaintain their function and brain may substitute its iron through transcytosis ofiron-loaded transferrin across the brain microvasculature. Other biologically activeproteins such as insulin and immunoglobulin G are actively transcytosed throughBBB endothelia. The presence of receptors involved in the transcytosis of ligandsfrom the blood to the brain offers opportunities for developing new approaches tothe delivery of therapeutic compounds across the BBB.Molecular Biology of the BBBThe molecular composition of the BBB has been studied by immunocytochemistry,and the results of these studies show that the BBB exhibits a specific collection ofstructural and metabolic properties that are also found in the tight-transporting epi-thelia. These conclusions are substantiated by the use of antibodies that recognizeproteins of nonBBB origin and BBB-specific proteins. BBB-specific immunoprobeshave a potential application for investigating the pathomechanisms that lead to thebreakdown of BBB. Different patterns of BBB disintegration are anticipated underdifferent pathological conditions, e.g., inflammatory reactions versus tumors.Genes that are selectively expressed at the BBB have been cloned. These includeGLUT-1 (glucose transporter) and GGTP (gamma-glutamyl transpeptidase). TheBBB GLUT-1 transporter maintains the availability in the brain of glucose and theregulation of the protein is mediated at the levels of the gene transcription, mRNAtranslation and stability, and posttranscriptional processes. GLUT-1 plays a role inthe development of the cerebral endothelial cells with BBB properties in vivo.Knockdown of GLUT-1 in an animal model was shown to produce loss of the cere-bral endothelial cells and downregulation of the junctional proteins important forintactness of the tight junctions with resulting leaky BBB and vasogenic cerebraledema (Zheng et al. 2010). This finding suggests that research into modulation ofGLUT-1 expression may lead to therapeutic strategies for preventing vasogeniccerebral edema.Molecular mechanisms of the “tight junctions” of the BBB are just being unrav-eled. In addition to occludens, other molecules (such as claudins) may be respon-sible for the integrity of the tight junctions. A molecular analysis of the BBB has
  7. 7. 483Passage of Substances Across the Blood–Brain Barrierclinical relevance for the development of new therapeutic strategies for neurologicdisorders.Genomics of BBBGenomic and proteomic analyses have been used to study the BBB and how itrelates to the pathogenesis of major neurologic diseases. Shear stress associatedwith blood flow in arteries has variable effects on endothelial cells, which aremodulated by induction or suppression of genes regulating endothelial physiology,e.g., formation of inter-endothelial tight junction and expression of specific carrier-mediated transporters (Cucullo et al. 2011). These findings can form the basis ofdeveloping innovative therapeutic strategies to improve the management of BBB-related diseases.Many aspects of how the BBB functions at the molecular level remain unre-solved; therefore, a variety of genomic and proteomic techniques have been used inBBB research. Genomic methods include gene microarray technologies, serialanalysis of gene expression (SAGE) and suppression subtractive hybridization(SSH). Gene microarray technologies are useful for generating semiquantitativedata regarding gene expression across an entire genome. SAGE generates informa-tion about the entire gene expression profile by parsing mRNAs into short nucle-otide fragments called tags, which allow for the quantitative cataloging of allexpressed genes in cells or tissues. Finally, SSH is a PCR-based method for identi-fying differentially regulated (up- or down-regulated) and tissue-specific (not detect-able in compared samples) gene transcripts.A comprehensive gene expression profile of rat brain microvessels using SAGEhas been reported (Enerson and Drewes 2006). This resulted in identification of 864genes, including several known for their abundant expression at the BBB, such asthe transferrin receptor. Sorting enriched genes based on function revealed groupsthat encode transporters (11%), receptors (5%), proteins involved in vesicletrafficking (4%), structural proteins (10%), and components of signal transductionpathways (17%). This genomic repertoire emphasizes the unique cellular pheno-type existing within the brain and further implicates the BBB as a mediator betweenthe brain and periphery. These results may provide a useful resource and referencepoint from which to determine the effects of different physiological, developmental,and disease processes on BBB gene expression. Currently, some of the researchpriorities include examination of the genes and proteins that are uniquely expressedby the intact BBB and mechanisms by which brain cells regulate endothelial cellgene expression.Proteomics of BBBProteomics analyses are currently being used to examine BBB function in healthyand diseased brain to better characterize this dynamic interface. Because the levelsof mRNA and protein in cells do not always correlate, proteomic methods have beendeveloped to examine proteins, the real actors in many cell functions. A widely used
  8. 8. 484 13 Role of Biotechnology in Drug Delivery to the Nervous Systemtechnique for creating differential proteomic profiles is 2D polyacrylamide gel elec-trophoresis (2D PAGE). This technique separates proteins according to charge andmass allowing for the resolution of up to 10,000 individual protein spots on a singlegel. Mass spectrometry (MS) is often used in conjunction with this method to iden-tify the resolved proteins. Another proteomic technique employed in BBB researchuses isotope coded affinity tags (ICAT). Labeling of protein samples with discreteisotope tags allows for a semiquantitative comparison of protein expression usingMS. Finally, similar to genomic arrays, proteomic arrays can be used to evaluatedifferential protein expression. One type of protein array used in BBB research is theantibody array. In this technology, antibodies are immobilized at high concentrationson a substrate to capture antigens from protein mixtures such as cell lysates.It is becoming increasingly evident that proteomic approaches have the potential toclarify the unique attributes of a healthy BBB, to identify therapeutic targets in dis-eased brain, and to identify novel conduits for noninvasive delivery of drugs againstthese targets. It has been estimated that approximately 3% of the proteins encoded bythe human genome function as molecular transporters. It is also likely that a significantportion of the genes encoding proteins with unknown functional roles are also molecu-lar transporters. These can be discovered through genomic/proteomic approaches.Membrane proteins are somewhat difficult to study with conventional proteomic tech-niques. It is now possible to perform differential membrane protein expressionprofiling of the BBB as a complement to differential gene expression profiling.Genomics and proteomics approaches have also been applied to analyze other func-tions of the BBB including immunological response, bacterial invasion, and trans-porter expression in epilepsy. The effects of TNFa on human cerebral endothelial cellswere profiled using microarray and two-dimensional gel electrophoresis. It was dis-covered that cell adhesion, apoptosis, and chemotaxis genes were differentiallyexpressed, and these findings were corroborated by proteomic analysis.Damage to BBB Manifested as Increased PermeabilityBBB is damaged in several neurological disorders such as stroke, TBI, brain tumors,infections, multiple sclerosis, and neurodegenerative disorders. The impairment ofpermeability of BBB is variable and cannot be used to improve drug delivery fortreatment of these disorders. BBB is also damaged due to neurotoxicity of drugs.Methods used for opening the BBB usually produce only transient increase of per-meability but excessive force used may damage the BBB.Brain Imaging for Testing Permeability of the BBBIn animal studies, BBB permeability can be quantitatively evaluated by measuringthe concentration of non-permeable radioactive materials, traceable macromoleculesor dyes in the brain. However, this approach is not applicable in humans due to itsinvasiveness and the potential dangers. Therefore, in most human studies, BBB per-
  9. 9. 485Passage of Substances Across the Blood–Brain Barriermeability is usually qualitatively evaluated using brain imaging techniques. Twomain approaches are used for studying the integrity of human BBB in vivo: (1)structural imaging employs contrast agents that only penetrate the BBB at sites ofdamage, and (2) functional imaging is used to study the transport of substancesacross the BBB − both intact and damaged. Structural imaging employs contrastagents with CT scanning and is relatively insensitive. MRI with the contrast agentgadolinium is more sensitive. Functional imaging is done with PET and can quantifycerebral uptake of therapeutic agents, such as cytotoxic agents and MAbs. SPECTis less versatile than PET, but can provide semiquantitative measurement of BBBleakage of albumin or red blood cells. Quantitative approaches are available to mea-sure BBB permeability using differentiated images and statistical analyses of CT orMRI images following the administration of standard contrast agents. This enablesquantification of the spatial characteristics of BBB-disruption and behavior of con-trast agents with time under different neurological conditions. This method mayenable assessment of the functional implications of the BBB integrity in variousCNS diseases as well as matching the required drug dose with BBB penetrability inspecific patients. PET can also be used to image regional P-glycoprotein activity andinhibition at the human BBB as it affects the distribution of drugs in the variousbrain regions protected by the barrier (Eyal et al. 2010).Biomarkers of Disruption of BBBThere is a need for biomarkers to detect early changes in BBB in various condi-tions. Loss of integrity of the BBB resulting from ischemia/reperfusion is believedto be a precursor to hemorrhagic transformation (HT) and poor outcome in acutestroke patients. A novel MRI biomarker has been used to characterize early BBBdisruption in human focal brain ischemia and its association with reperfusion, HT,and poor outcome (Latour et al. 2004). Reperfusion was found to be the most pow-erful independent predictor of early BBB disruption and thus of HT and is impor-tant for the decision for acute thrombolytic therapy. Early BBB disruption asdefined by this imaging biomarker may be a promising target for adjunctive therapyto reduce the complications associated with thrombolytic therapy, broaden thetherapeutic window, and improve clinical outcome in acute stroke.The astrocytic protein S-100beta is a potentially useful peripheral biomarker ofBBB permeability. Other biomarkers of BBB have been recently discovered byproteomic approaches. These proteins are virtually absent in normal blood, appearin serum from patients with cerebral lesions, and can be easily detected.Peripheral assessment of BBB opening can be achieved by detection in blood ofbrain-specific proteins that extravasate when these endothelial junctions arebreached. A proteomic approach was used to discover clusters of CNS-specificproteins with extravasation into serum that correlates with BBB openings. Proteinprofiles from blood samples obtained from patients undergoing BBB disruptionwith intra-arterial hyperosmotic mannitol are compared with pre-BBB openingserum. A low molecular weight protein (14 kDa) was identified by MS as transthy-
  10. 10. 486 13 Role of Biotechnology in Drug Delivery to the Nervous Systemretin (TTR), which consistently correlates with BBB disruption. S-100beta, pre-sumably originating from perivascular astrocytic end feet, precedes extravasation ofTTR by several minutes. Because TTR is localized primarily in choroid plexus and,as found in CSF, it may be a peripheral tracer of blood-to-CSF although S-100betais a marker of BBB integrity. TTR could be used to detect disease and to determinewhen the body may be more or less receptive to medications.Strategies to Cross the BBBVarious strategies that have been used for manipulating the BBB for drug deliveryto the brain include osmotic and chemical opening of the BBB as well as the use oftransport/carrier systems. Other strategies for drug delivery to the brain involvebypassing the BBB. The drawback of strategies to open the BBB are damage to thebarrier as well as uncontrolled passage of drugs into the brain. The ideal method fortransporting drugs across the BBB should be controlled and not damage the barrier.Biotechnology-based strategies for crossing the BBB are shown in Table 13.3.Potential therapeutic applications of manipulation of the BBB are mostly in useto facilitate drug delivery to brain tumors in clinical trials. Methods of focal deliv-ery of therapeutic and diagnostic substances to the brain across the BBB are inexperimental stages for infectious, genetic, and neurodegenerative disorders. A par-ticular advantage will be the delivery of the genes to the required site as opposedTable 13.3 Biotechnology-based strategies for drug delivery to the CNSStrategies for crossing the BBBBiotechnology-based modification of the drug to enhance its lipid solubilityChimeric peptidesGlycosylation Independent Lysosomal TargetingInhibition of P-glycoproteinMonoclonal antibody fusion proteinsNanoparticle-based technologiesNeuroimmunophilinsNO donors for opening the BBBTrojan horse approachUse of carrier systemsUse of receptor-mediated transocytosis to cross the BBBUse of transport systems: 2B-Trans™ technology, ABC afflux transporters, G-Technology®Microorganisms-based drug delivery to the brainBacteriophages for brain penetrationBacterial vectorsCell therapyGene therapy© Jain PharmaBiotech
  11. 11. 487Passage of Substances Across the Blood–Brain Barrierto the exposure of the whole brain to the therapeutic agent. Genetic and otherdefects leading to brain changes in Down’s syndrome, Alzheimer’s disease, amyo-trophic lateral sclerosis, Huntington’s disease, Gaucher disease, hypertension, andother disorders are rapidly being identified. Several effective therapeutic agents areavailable but their use is limited pending improvement of drug delivery across theBBB. In silico methods are available to predict BBB penetration by drugs(Lanevskij et al. 2010).2B-Trans™ TechnologySystems directed at endogenous receptor-mediated uptake mechanisms have beenshown to be effective in animal models including primates. Various systems use thelow-density lipoprotein-related protein 1 receptor, the low-density lipoprotein-related protein 2 receptor (also known as megalin and glycoprotein 330) or thediphtheria toxin receptor, which is the membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor.The 2B-Trans™ technology (to-BBB) uses a well characterized and effectivetransport system with a specific carrier protein that has an excellent proven safetyprofile in human (Gaillard and de Boer 2008). Advantages of 2B-Trans™ receptorare as follows:It uses receptor-mediated endocytosis, an effective and safe transport mecha-•nism, for delivery of large proteins and liposomes containing drugs and genesacross the BBB.Because the receptor has no endogenous ligands, there is no competition from•endogenous ligands, or blockade of transport to the brain of essential nutrients.It is constitutively expressed on the BBB, neurons and glial cells•The receptor expression is highly amplified in disease conditions and thus•allows for site-specific disease targetingto-BBB holds a patent claim on the use of all known ligands/carrier proteins for•the receptor to deliver drugs to the brain.ABC Afflux Transporters and Penetration of the BBBA significant number of lipid soluble molecules, among them many useful thera-peutic drugs have lower brain permeability than would be predicted from adetermination of their lipid solubility. These molecules are substrates for the ABCefflux transporters, which are present in the BBB and BCSB, and the activity ofthese transporters very efficiently removes the drug from the CNS, thus limitingbrain uptake. P-glycoprotein (Pgp) was the first of these ABC transporters to bedescribed, followed by the multidrug resistance-associated proteins and morerecently breast cancer resistance protein. All are expressed in the BBB and BCSFBand combine to reduce the brain penetration of many drugs. This phenomenon of“multidrug resistance” is a major hurdle when it comes to the delivery of therapeu-tics to the brain. Therefore, the development of strategies for bypassing the
  12. 12. 488 13 Role of Biotechnology in Drug Delivery to the Nervous Systeminfluence of these ABC transporters and for the design of effective drugs that arenot substrates and the development of inhibitors for the ABC transporters becomesa high imperative for the pharmaceutical industry (Begley 2004).Another potentially promising approach to enhancing the delivery of otherwisenon-permeating drugs to the brain is the use of excipients, which are able to interactwith ABC transporters and modify function. Certain Pluronic block co-polymersappear to target pgp by two mechanisms: depletion of cellular ATP and altering thephysicochemical properties of the membrane lipid phase. ABC transporters may beof therapeutic benefit in situations where acute dosing is indicated. However, it isuncertain whether chronic administration of blocking agents is feasible given theprotective role of these transporters in the BBB and other organs. Several strategiesdesigned to bypass pgp at the BBB without direct inhibition have been described andtested including a system that uses antibody-coupled immunoliposomes to transportpgp substrates. The strategy is to move the encapsulated drug through the lumenalplasma membrane of capillary endothelial cells avoiding direct interaction with pgp.Immunoliposomes, which are coupled to an antitransferrin receptor antibody, havebeen shown in vivo and in vitro to be internalized at the BBB by means of receptor-mediated endocytosis and to deliver p-gp substrates efficiently to the brain. Similarresults have been obtained with liposomes coupled to cationized albumin.The discovery of active carrier proteins and cytotic mechanisms and their con-tribution to drug permeation across the barrier would promote the successful devel-opment of efficient CNS drugs and to an understanding of unwanted CNS sideeffects of non-CNS drugs. From the molecular biology and pharmacology of theproteins involved, it might be possible to identify specific probes to distinguishtransporter subtypes as well as tools to transiently modify barriers to drug absorp-tion through competition. An understanding of the mechanisms by which expres-sion and function of drug transporters is regulated would help in improving drugdelivery to the CNS.Carrier-Mediated Drug Delivery Across the BBBOf the various carrier systems, those for glucose and neutral amino acids havehigh enough transport capacity to hold promise of significant drug delivery to thebrain. Glucose transporter has the limitation that only molecules closely resem-bling D-glucose are transported. Neutral amino acids are less specific. Entry viathis carrier may explain the central effects of the muscle relaxant baclofen.Transport systems for peptides may prove to be effective targets for peptide drugsrequired to control natural peptide hormones.Drugs used to treat neurologic disorders appear to cross the BBB more easilywhen an ascorbic acid molecule is attached. Ascorbic acid works like a shuttle and,theoretically, could transport any compound into the brain. The ascorbic acidSVCT2 transporter, which is believed to play a major role in regulating the transportof ascorbic acid into the brain, provides a targeted delivery to the brain. Potentialapplications include drugs to treat neurodegenerative diseases, e.g. AD and PD.
  13. 13. 489Passage of Substances Across the Blood–Brain BarrierA feasible method to achieve carrier-mediated drug transport into the rat brainwas shown to be via the specific, large neutral amino acid transporter (LAT1) byconjugating a model compound to L-tyrosine (Gynther et al. 2008). A hydrophilicdrug, ketoprofen, that is not a substrate for LAT1 was chosen as a model compound.The mechanism and the kinetics of the brain uptake of the prodrug were determinedwith an in situ rat brain perfusion technique and found to be concentration-depen-dent. Moreover, a specific LAT1 inhibitor significantly decreased the brain uptakeof the prodrug.The iron binding protein p97 (melanotransferrin) is closely related to Tf andlactoferrin and as a result of alternative splicing, it exists in both a soluble form anda cell surface GPI-linked form. However, in normal brain it appears to discretelylocalize on the surface of endothelial cells and transiting through brain capillaryendothelium. Studies on the structure and function of p97 suggest it might be anideal carrier for transport of drug conjugates into the brain. A study provides theinitial proof of concept for p97 as a carrier capable of shuttling therapeutic levelsof drugs from the blood to the brain for the treatment of neurological disorders,including classes of resident and metastatic brain tumors (Karkan et al. 2008). Thisnovel delivery platform may be useful in various clinical settings for therapeuticintervention in acute and chronic neurological diseases and is in commercial devel-opment for CNS drug delivery.G-Technology®Glutathione, an endogenous tripepeptide transporter, is highly expressed on theBBB. Glutathione is found in high levels in the brain and cerebral vasculature.Glutathione has favorable antioxidant-like properties and plays a central role indetoxification of intracellular metabolites. Glutathione transporters are conservedacross all mammalian species, including humans. Glutathione is considered to besafe to administer to humans for a prolonged period of time. Glutathione is mar-keted as functional food ingredient and antioxidant, and applied as supportivetherapy in cancer and HIV treatments and as excipient in parenteral formulations.Glutathione coated on the surface of nanosized liposomes was shown to be welltolerated and to effectively deliver several classes of drugs to the brain in a rangeof experimental studies reproducibly performed by independent laboratories. Thisis the basis of G-Technology® (to-BBB), which uses pegylated liposomes coatedwith glutathione, an endogenous tripepeptide transporter expressed on the BBB, tofacilitate delivery of drugs to the brain (Gaillard 2011). Applied to anticancer drugs,it improves targeted delivery to the brain tumors after systemic administration andreduces adverse effects. Glutathione pegylated liposomal doxorubicin (2B3-101) isin phase I/II clinical trials in patients with brain cancer. G-Technology has also beenapplied for delivery of methylprednisolone, which is used for several diseases witha neuroinflammatory component. A product, 2B3-201(to-BBB), is in preclinicalstudies for potential applications in multiple sclerosis, acute spinal cord injury andlupus erythematosus involving the CNS (Gaillard et al. 2012).
  14. 14. 490 13 Role of Biotechnology in Drug Delivery to the Nervous SystemGlycosylation Independent Lysosomal TargetingEnzyme replacement therapies use a novel, proprietary technology, known asGlycosylation Independent Lysosomal Targeting (GILT), which improves thedelivery of lysosomal enzymes to clinically significant tissues (LeBowitz 2005).A target directing molecule (the tag) is embedded within the therapeutic enzyme topromote internalization into cells. The internalization process is accomplished bythe binding of the tag to a receptor found on the surface of the target cell thereforefacilitating endocytosis. GILT technology might be used to deliver drugs across theBBB. This would be an important application because several lysosomal storagediseases have profound neurological components and conventionally glycosylateddrugs are unable to address this problem.Inhibition of P-glycoprotein to Enhance Drug Delivery Across the BBBP-glycoprotein (P-gp) drug efflux transporter is present at high densities in the lumi-nal membranes of brain endothelium. It limits entry into the CNS for a large numberof prescribed drugs, contributes to the poor success rate of CNS drug candidates, andpatient-to-patient variability in response to CNS pharmacotherapy. It pumps outsome cytotoxic agents used to treat brain tumors and excludes them from the brain.Recent studies focused on understanding the mechanisms by which P-gp activity inthe BBB can be modulated to improve drug delivery into the brain.Using intact brain capillaries from rats and mice, scientists have identified mul-tiple extracellular/intracellular signals that regulate this transporter and severalsignaling pathways have been mapped (Miller et al. 2008). Three pathways that aretriggered by elements of the brain’s innate immune response are: (1) by glutamate;(2) by xenobiotic-nuclear receptor (pregnane X receptor) interactions; and (3) byelevated Ab levels. Signaling is complex, with several pathways sharing commonsignaling elements − TNFR- 1, endothelin B receptor, PKC, and NOS − suggestinga regulatory network. Several pathways include autocrine/paracrine elements,involving release of the proinflammatory cytokine, TNF-a, and the polypeptidehormone, endothelin-1. Several steps in signaling are potential therapeutic targetsthat could be used to modulate P-gp activity in the clinic. Strategies for P-gp modu-lation include (1) direct inhibition by specific competitors, (2) functional modula-tion, and (3) transcriptional modulation. Each has the potential to specificallyreduce P-gp function and thus selectively increase brain permeability of P-gpsubstrates. A crosslinked dimer of galantamine, Gal-2, inhibits P-gp mediatedefflux mechanism at the BBB by competing for the substrate binding sites (Namanjaet al. 2009). Several specific inhibitors of P-gp as efflux transporters are in clinicaldevelopment.LipoBridge™ TechnologyLipoBridge™ (Genzyme Pharmaceuticals) temporarily and reversibly opens tightjunctions to facilitate transport of drugs across the BBB and into the CNS.
  15. 15. 491Passage of Substances Across the Blood–Brain BarrierLipoBridge itself forms a clear suspension of nanoparticles in water and can solu-bilize or stabilize some drugs, is non-immunogenic and is excreted unmetabolized.It has been demonstrated in several laboratories that intracarotid injections of asimple mixture of Lipobridge™ and model compounds or pharmaceutical activescan deliver these actives into one or both hemispheres of the brain allowing forincreased concentration in a selected hemisphere. It can be administered orally aswell as intravenously. LipoBridge has been used to administer anticancer drugs forbrain cancer in animals. Safety clinical studies in humans are in progress.Modification of the Drug to Enhance Its Lipid SolubilityThere is a good correlation between the lipid solubility of a drug and the BBBpenetration in vivo. The lipophilic pathway also provides a large surface area fordrug delivery. It is approximately 12 m2in an average human brain. Therefore,addition of hydrophobic groups to molecules increases their ability to penetrate theBBB. Addition of methyl groups in a series of barbiturates improves lipophilicityand brain penetration, leading to increased hypnotic action. It is also possible togenerate a lipophilic prodrug that is broken down to release the more active drugwithin the brain. An example of this is heroin, which enters the brain readily due toits lipophilicity but, after entry, hydrolyses to morphine, which is less lipophilic andless likely to diffuse back across the BBB, leading to prolongation of duration ofits action on the brain. Ester formation is another approach for increasing the lipo-philicity of polar molecules exhibiting poor CNS penetration. A number of investi-gators have explored the lipophilic-ester concept for improving the CNS deliveryof antiviral agents. An example of this is improvement of the BBB penetration ofGABA (gamma amino butyric acid), an anticonvulsant agent with poor CNS pen-etration, by use of lipophilic esters.Although increasing lipophilicity generally increases penetration across theBBB, it may also result in reduction of biological action due to drug-receptor inter-action, drug metabolism, or binding to plasma proteins as in the case of barbitu-rates. Therefore, optimization rather than maximization of both lipophilicity andrate of bioconversion are required.Lipid-binding carriers may reduce the binding of neurotrophic factors to serumlipids and increase transport across the BBB. Liposomes have been considered, buttheir size is too large to cross the BBB.Monoclonal Antibody Fusion ProteinsThese involve conjugation of a drug to a transport vector. These have diagnostic andtherapeutic applications for the treatment of brain tumors. Nontransportablespecific antigen-binding monoclonal antibodies such as IgG3 have been attached toa transport vector such as insulin-like growth factor. The bifunctional molecule cancross the BBB through interaction with the receptor for insulin-like growth factor.
  16. 16. 492 13 Role of Biotechnology in Drug Delivery to the Nervous SystemTransferrin is a specific receptor for molecules that are not synthesized in thebrain but play an essential biological role. This transfer mechanism can be exploitedin an approach in which antiferritin receptor antibodies are covalently linked toNGF, resulting in a substantial transfer of biologically active nerve growth factoracross the BBB into the CNS. NGF can be transported across the BBB by conjugat-ing with OX-2, an antibody directed against the transferrin receptor.One suitable transport vector is a peptidomimetic MAb that is transported by anendogenous BBB receptor-mediated transcytosis system, such as the transferrinreceptor. The MAb carries any drug attached to it across the BBB. However, thenumber of small molecules that can be conjugated to monoclonal antibodies vectorsis limited. The carrying capacity of the vector can be greatly expanded by attachingliposomes to the vector.A new generation of multifunctional fusion proteins are being engineered atArmaGen Technologies to cross the BBB following intravenous administration andto produce a therapeutic effect on brain disorders (Boado 2008). These fusion pro-teins are comprised of both a transport and a therapeutic domain. The transportdomain is a MAb directed to an exofacial epitope of the BBB human insulin recep-tor (HIR), which uses the BBB endogenous insulin transport system to gain accessto the brain via receptor-mediated transcytosis without interfering with the normaltransport of insulin. Both human-chimeric and fully humanized versions of the anti-human HIRMAb have already been produced. The therapeutic domain of thesefusion proteins consists of the peptide or protein of interest fused to the carboxylterminus of the CH3 region of the heavy chain of the anti-human HIRMAb. Avariety of HIRMAb fusion proteins were engineered aiming at the development oftherapeutics for stroke and PD, as in the case of HIRMAb-BDNF and HIRMAb-GDNF, respectively, HIRMAb-IDUA for the treatment of Hurler’s disease,HIRMAb-Ab single chain antibody for passive immunotherapy of AD, andHIRMAb-avidin as delivery system for biotinylated drugs, like siRNAs. The mul-tifunctionality of these fusion proteins has been validated in preclinical work,including brain update in primates. Pending further development into pharmaco-logical and toxicological studies, and clinical trials, members of the biotherapeuticfamily discussed in the present review, designed to overcome the brain drug deliv-ery hurdle, are positioned to become a new generation of neuropharmaceuticaldrugs for the treatment of human CNS disorders.NeuroimmunophilinsNeuroimmunophilin ligands are small molecules that in can repair and regeneratedamaged nerves without affecting normal, healthy nerves. Neuroimmunophilin ligandsmay have application in the treatment of a broad range of diseases, including PD,spinal cord injury, brain trauma, and peripheral nerve injuries. The immunosuppres-sants tacrolismus (FK-506) and cyclosporin are in clinical use for the treatment ofallograft rejection following organ transplantation. Immunophilins can regulate neu-ronal survival and nerve regeneration although the molecular mechanisms are poorlyunderstood. Neuroimmunophilin can be administered orally and can cross the BBB.
  17. 17. 493Passage of Substances Across the Blood–Brain BarrierPeptide-Mediated Transport Across the BBBUnder normal conditions, vesicular transport that involves receptor-mediated endocy-tosis is responsible for only a small amount of molecular trafficking across the BBB,but it may be suitable for the delivery of agents that are too large to use other routes.A number of different peptide families with the ability to cross the cell mem-branes have been identified. Certain of these families enter the cells by a receptor-independent mechanism, are short (10–27 amino acid residues), and can deliversuccessfully various cargoes across the cell membrane into the cytoplasm ornucleus. Some of these vectors have also shown the ability to deliver hydrophilicmolecules across the BBB.Another approach involves forming a chimeric peptide by coupling an otherwisenontransportable drug to a BBB transporter vector by a disulfide bond. The chime-ric peptide is then endocytosed by the capillary endothelial cells and transported tothe brain where it can be cleaved by disulfide reductase to release the pharmaco-logically active compound. BBB peptide receptor systems include those for insulin,insulin-like growth factor, transferrin, and leptin. Conjugation of doxorubicin orpenicillin to peptide vectors significantly enhances their brain uptake. Peptide-mediated strategies can improve the availability and efficacy of CNS drugs.Transport of Small Molecules Across the BBBLipid-soluble small molecules with a molecular mass less than 400 Da are trans-ported readily through the BBB in vivo via lipid-mediated transport. However, othersmall molecules lacking these molecular properties, antisense drugs, and peptide-based pharmaceuticals ordinarily undergo negligible transport through the BBB inpharmacologically significant amounts. Some small-molecule neuroprotectiveagents have failed in human trials due to poor transport of these agents across theBBB. Strategies that enable drug transport through the BBB arise from knowledgeof the molecular and cellular biology of BBB transport processes. As biology-drivendrug discovery progresses, more large molecules are being discovered as potentialtherapeutics. Some of the strategies for samll molecules transport may be used fortransporting larger molecules such as gene medicine and recombinant proteins.Trojan Horse ApproachAttaching an active drug molecule to a vector that accesses a specific catalyzedtransporter mechanism creates a Trojan horse-like deception that tricks the BBBinto welcoming the drug through its gates. Transport vectors, such as endogenouspeptides, modified proteins, or peptidomimetic MAbs are one way of tricking thebrain into allowing these molecules to pass. Intravenously administered molecules,attached to Trojan horses for CNS effect in experimental animals, are shown inTable 13.4.Biopharmaceuticals, including recombinant proteins, MAb therapeutics, andantisense or siRNAs, cannot be developed as drugs for the brain, because theselarge molecules do not cross the BBB. Biopharmaceuticals must be re-engineered
  18. 18. 494 13 Role of Biotechnology in Drug Delivery to the Nervous Systemto cross the BBB, and this is possible with genetically engineered molecular Trojanhorses (Pardridge 2008). A molecular Trojan horse is an endogenous peptide, orpeptidomimetic MAb, which enters brain from blood via receptor-mediated trans-port on endogenous BBB transporters. Recombinant neurotrophins, single chain Fvantibodies, or therapeutic enzymes may be re-engineered as IgG fusion proteins.The engineering of IgG-avidin fusion proteins enables the BBB delivery of bioti-nylated drugs. The IgG fusion proteins are new chemical entities that are dual ortriple function molecules that bind multiple receptors. The fusion proteins are ableboth to enter the brain, by binding an endogenous BBB receptor, and to induce thedesired pharmacologic effect in brain, by binding target receptors in the brainbehind the BBB. The development of molecular Trojan horses for BBB drug deliv-ery allows the re-engineering of biopharmaceuticals that, owing to the BBB prob-lem, could not otherwise be developed as new drugs for the human brain.The BDNF chimeric peptide is formed by conjugation of BDNF to a MAb to theBBB transferrin receptor, and the MAb acts as a molecular Trojan horse to ferry theBDNF across the BBB via transport on the endogenous BBB transferrin receptor. Highdegrees of neuroprotection in transient forebrain ischemia, permanent middle cere-bral artery occlusion, or reversible middle cerebral artery occlusion are achieved withthe delayed intravenous administration of BDNF chimeric peptides. In contrast, noneuroprotection is observed following the intravenous administration of unconjugatedBDNF, because the neurotrophin does not cross the BBB in vivo.Such CNS targeting polypeptides have the advantage in that they can be admin-istered directly to an individual, or they can be expressed via an encoding nucleicacid by non-target cells, and they will travel to and concentrate in the CNS. Thisapproach will be applicable to lysosomal storage diseases such as Gaucher’s disease,Hunter’s disease and Fabry Syndrome. In addition the fusion of ligands to facilitateTable 13.4 Molecules attached to Trojan horses injected intravenously for CNS effectMolecules Type CNS effectAb1–40 Peptide Imaging brain amyloid in vivo withpeptide radiopharmaceuticalBrain-derived neurotrophic factor Peptide Neuroprotection in cerebral ischemiaEGF receptor RNAi/antisense Increase in survival time of animalmodels of human brain cancerEpidermal growth factor Peptide Early detection of brain cancer in vivowith peptide radiopharmaceuticalFibroblast growth factor-2 Peptide Reduction of cerebral infarction in middlecerebral artery occlusion modelPeptide nucleic acid Peptide Imaging gene expression in vivo withantisense radiopharmaceuticalTyrosine hydroxylase Gene therapy Normalization of striatal enzyme activityin models of Parkinson’s diseaseVasoactive intestinal peptide Peptide Increase in cerebral blood flow© Jain PharmaBiotech
  19. 19. 495Passage of Substances Across the Blood–Brain Barrierpassage of proteins across the BBB may be a general method for delivering thera-peutic proteins to the CNS for neurodegenerative diseases such as AD and PD.Use of Receptor-Mediated Transocytosis to Cross the BBBThe concept of using receptor-mediated endocytosis to deliver peptides into thebrain was initially described with the findings on the transendothelial transport ofinsulin across the BBB. Subsequent studies demonstrated that a neuropeptide couldbe delivered into the CNS using receptor-mediated endocytosis by targeting thetransferrin receptor with the MAb OX-26. The development of chimeric proteinscontaining this MAb, specific linkers and a neurotropic peptide has enabled deliv-ery into the brain of significant levels of this peptide. In addition, the transendothe-lial transport of MAb OX-26 is similar to the transport of human transferrin acrossthe BBB. This process is more adequately described as transcytosis and is depictedin Fig. 13.2. The non-transportable drug is attached to a protein or peptide vector,which is accepted by the receptor at the luminal side of the BBB and endocytosed.After exocytosis in the brain interstitial fluid, the chemical link binding the drug tothe peptide is cleaved and the drug binds to a receptor at the neuron.Antibodies against receptors that undergo transcytosis across the BBB have beenused as vectors to target drugs or therapeutic peptides into the brain. A novel singledomain antibody, FC5, transmigrates across human cerebral endothelial cellsin vitro and the BBB in vivo. The transport of FC5 across human brain endothelialcells is polarized, charge independent and temperature dependent, suggesting areceptor-mediated process. FC5 failed to recognize brain endothelial cells-derivedlipids, suggesting that it binds luminal alpha(2,3)-sialoglycoprotein receptor whichtriggers clathrin-mediated endocytosis (Abulrob et al. 2005). This putative receptormay be a new target for developing brain-targeting drug delivery vectors.BLOOD-BRAINBARRIERBlood BrainExocytosisEndocytosisCleavageReceptorbindingDrug VectorRV RVVector DrugNeuronDrugVectorRV=receptor for the vectorFig. 13.2 Use of receptor-mediated transcytosis to cross the BBB (© Jain PharmaBiotech)
  20. 20. 496 13 Role of Biotechnology in Drug Delivery to the Nervous SystemThe high level of expression of transferrin receptors (TfR) on the surface ofendothelial cells of the BBB have been widely utilized to deliver drugs to the brain.This approach has been explored for the delivery of citicoline, a neuroprotectivedrug for stroke that does not readily cross the BBB because of its strong polarnature. Low concentrations of citicoline encapsulated in transferrin-coupled lipo-somes could offer therapeutic benefit in treating stroke compared to administrationof free citicoline (Suresh Reddy et al. 2006). This is likely due to the entry of citi-coline into cells via TfR-mediated endocytosis.NeuroTrans™, a proprietary technology based on receptor-associated protein(RAP) is being developed for the delivery of therapeutics across the BBB. In pre-clinical studies, NeuroTrans™ has been conjugated to a variety of protein drugs,including enzymes and growth factors, without interfering with the function ofeither fusion partner (Prince et al. 2004). Studies indicate that radio-labeledNeuroTrans™ may be transcytosed across the BBB and, that fusions betweenNeuroTrans™ and therapeutic proteins may be manufactured economically (Panet al. 2004). Scanning electron microscopy imaging is being used to determinewhether the NeuroTrans™ peptide is able to enter the brain tissue through the pro-cess of transcytosis. This will also help the assessment of the time frame of trans-port, the extent or amount of NeuroTrans™ transported, and the biodistribution ofNeuroTrans™ within various brain compartments.Use of Nanobiotechnology for Therapeutic DeliveryAcross the BBBAmong the various approaches that are available, nanobiotechnology-based deliv-ery methods provide the best prospects for achieving this ideal. This topic is dis-cussed in more detail in the chapter on “Nanomedicine”. Various nanoparticles(NPs) used for drug delivery to the brain and their known mechanism of action arereviewed elsewhere (Jain 2012). Some strategies use multifunctional NPs. Animportant application of nanobiotechnology is delivery of therapy for brain tumorsacross the BBB as well as combination of diagnostics with therapeutics. Despitesome current limitations, future prospects for NP-based therapeutic delivery to thebrain are excellent.Delivery of Cell Therapy to the BrainCell therapy is described in Chap. 12. Although cells may deliver therapeuticsthemselves, there is also a need for drug delivery systems for cell therapies. Variousmethods of delivery of cells for therapeutic purposes are listed in Table 13.5.
  21. 21. 497Delivery of Cell Therapy to the BrainIntravenous Delivery of Stem CellsIntravenous (IV) stem cell delivery for regenerative tissue therapy has been increas-ingly used in both experimental and clinical trials. However, recent data suggestthat the majority of administered stem cells are initially trapped in the lungs. Anexperimental study with labeled stem has shown that the majority of menchymalstem cells (MSCs) are trapped inside the lungs following intravenous infusion,whereas pulmonary passage of neural stem cells (NSCs) and multipotent adultprogenitor cells (MAPCs) was increased twofold and bone marrow-derived mono-nuclear cells (BMMC) was increased 30-fold as compared to MSCs (Fischer et al.2009). Inhibition of MSC CD49d significantly increased MSC pulmonary passage.Infusion via two boluses increased pulmonary MSC passage as compared to singlebolus administration. Infrared imaging revealed stem cells evenly distributed overall lung fields. It is concluded that larger stem and progenitor cells are initiallytrapped inside the lungs following intravenous administration with a therapeuticallyquestionable number of cells reaching the arterial system acutely.Intraarterial Delivery of Stem CellsOver the past 10 years, intra-arterial (IA) delivery has been under investigation inpatients with cardiac and peripheral vascular disease and safety has been demon-strated in clinical trials. IA delivery also has the potential advantage of selectivelytargeting cell therapies to the ischemic brain tissue (Misra et al. 2012). IA injectionof NSCs using a microneedle technique does not cause microembolic strokes (Chuaet al. 2011).Pharmacologically Active MicrocarriersPharmacologically active microcarriers (PAM) were developed at INSERM inFrance to overcome certain problems encountered in cell therapy, particularly cellsurvival, lack of cell differentiation and integration in the host tissue (Tatard et al.Table 13.5 Methods of delivery of cell therapy to the CNSInjections: intramuscular, intravenous, intraarterial, intrathecalEngineering of cells for targeted delivery to CNS lesions via systemic circulationImplantation into the CNS by surgical proceduresOral intake of encapsulated cellsPharmacologically active microcarriersUse of special devices for delivery of cells© Jain PharmaBiotech
  22. 22. 498 13 Role of Biotechnology in Drug Delivery to the Nervous System2005). These biodegradable spherical microparticles are made with poly(D,L-lactic-co-glycolic acid) (PLGA) and coated with adhesion molecules. Their diam-eter may vary, depending on the type of transported cell, from 10 to 500 mm. Thepreferential cell adhesion of the cells to be grafted on the microcarriers permitstheir preparation or transformation in vitro without the use of enzymes of animalorigin. The cell adhesion molecules as well as the growth factors may induce thesurvival and differentiation of stem cells towards a determined phenotype. Themicrospheres are spontaneously degraded, without toxicity and without interferingwith the activity or integration of the grafted cells, in a few weeks or months afterimplantation, depending on the composition of the polymer. PAM may serve as asupport for cell culture and may be used as cell carriers presenting a controlleddelivery of active protein. They can thus support the survival and differentiation ofthe transported cells as well as their microenvironment. They reduce the hostimmune reaction and favor the tissue integration of the grafted cells.To develop this tool, nerve growth factor (NGF)-releasing PAM, conveyingPC12 cells, were produced and characterized. These cells have the ability to dif-ferentiate into sympathetic-like neurons after adhering to a substrate, in the pres-ence of NGF, and can then release large amounts of dopamine. Certain parameterssuch as the size of the microcarriers, the conditions enabling the coating of themicroparticles and the subsequent adhesion of cells were thus studied to produceoptimized PAM. NGF-releasing PAM coated with fibronectin plus polylysine andtransporting PC12 cells were evaluated in an animal model of PD. After transplan-tation, the PAM induced the differentiation, reduced cell death and proliferation ofthe PC12 cells and the animals presented an ameliorated behavior.PAM may be used in any type of cell therapy: (1) tissue reconstruction byimplanting embryonic cells, cell lines, genetically modified cells or stem cells;(2) for the grafting of cells for nd central nervous system (3) cell therapy for genetransfer; and (4) anticancer vaccination as PAM may present tumor cells or frag-ments of these cells to immunocompetent cells while delivering immunostimulat-ing cytokines.Targeted Delivery of Engineered Cells to SpecificTissues via CirculationMinimally invasive delivery of a large quantity of viable cells to a tissue of interestwith high engraftment efficiency is a challenge in cell therapy. Low and inefficienthoming of systemically delivered MSCs, e.g., is considered to be a major limita-tion of existing MSC-based therapeutic approaches, caused predominantly byinadequate expression of cell surface adhesion receptors. The surface of MSCswas modified without genetic manipulation with a nanometer-scale polymer con-struct containing sialyl Lewisx (sLex) that is found on the surface of leukocytesand mediates cell rolling within inflamed tissue (Sarkar et al. 2011). The sLexengineered MSCs exhibited a robust rolling response on inflamed endothelium
  23. 23. 499Delivery of Cell Therapy to the Brainin vivo and homed to inflamed tissue with higher efficiency compared with nativeMSCs. This is a simple method to potentially target any cell type to specific tissuesvia the circulation.Devices for Delivery of Cell TherapyA self-assembling cube-shaped perforated container, no larger than a dust speck,has been devised to could serve as a delivery system for medications and celltherapy (Gimi et al. 2005). Because of their metallic nature, the location of cubiccontainers in the body could easily be tracked by MRI. The microcontainers couldsomeday incorporate electronic components that would allow the cubes to act asbiosensors to release medication on demand in response to a remote-controlledradio frequency signal. Biohybrid implants represent a new class of medicaldevice in which living cells, supported by hydrogel matrix and surrounded by asemipermeable membrane, produce and deliver therapeutic reagents to specificsites within a host.Cell EncapsulationModern encapsulation techniques involve surrounding the cells with selectivelypermeable membranes. The pores of the membranes should be small enough toblock entry of immune mediators but large enough to allow inward diffusion ofoxygen and nutrients required for the survival of cells and for outward diffusion ofactive molecules produced by the cells. Encapsulation avoids some of the compli-cations of free cell transplants including local reaction at the site of transplantationand tumor formation. The required characteristics of a membrane device are:The material should be biologically inert and nontoxic to the tissues•It should be nonimmunogenic•It should be sturdy enough to withstand a considerable amount of•manipulationPore size should be adequate to allow the passage of oxygen and nutrients for•the cellsIt should be possible to vary pore size according to need•Availability of an economical large-scale process for production•It is difficult to encapsulate living cells using polymers because of toxic interactionsbetween solvents and cells during the formation of thermoplastic-based microcap-sules. Biodegradable materials can be synthetic or natural and they are degradedin vivo both enzymatically as well as nonenzymatically. Their by-products are usu-ally nontoxic and excreted via physiological pathways. Examples of natural biode-
  24. 24. 500 13 Role of Biotechnology in Drug Delivery to the Nervous Systemgradable materials are human serum albumin and collagen. Because of their costand the possibility of contamination, several synthetic biodegradable polymershave been developed. Polyelectrolyte microcapsules are fragile, both physically andchemically. Great care is required in handling during transplantation and in vivofailure may occur 3 months post-transplantation. Thermoplastic-based microcap-sules are mechanically more durable.Another method of encapsulating cells is use of permeable alginate shells thatcan maintain structural integrity in vivo for extended periods of time. It is prefera-ble to most of the currently used techniques for capsule generation that yield micro-spheres prone to wall degradation. Shell properties may be modified to regulatepermeability, providing controlled delivery of desired substances.Encapsulated Cell BiodeliveryThe encapsulated cell (EC)-biodelivery (NsGene) is a general biodelivery systemof cell-derived substances to the CNS that provides a controlled, site-specific andsafe delivery of a variety of therapeutic substances. For CNS indications one ormultiple EC-biodelivery devices can be implanted in defined regions of the brain todeliver any proteins or neurotransmitters across the BBB. EC-biodelivery systemconsists of a catheter-like device that in the active portion contains a geneticallymodified human cell line enclosed behind a semi-permeable hollow fiber mem-brane. The membrane allows for the influx of nutrients and outflow of the therapeu-tic factor(s) but does not allow for the direct contact between the therapeutic cellsand the host tissue. The encapsulated cells provide for long-term (>12 months)secretion of a therapeutic factor from the implanted device. This offers great safetyadvantages over direct cell/gene therapy approaches and technical and functionaladvantages over pump technologies. The device is compatible with stereotacticneurosurgical techniques and instrumentation adapters to common stereotacticframes have been made. EC-biodelivery devices are suitable for intraparenchymal,intracerebroventricular, or intrathecal placement.Therapeutic Applications of Encapsulated Cellsin Neurological DisordersTable 13.6 lists therapeutic applications of encapsulated cells in neurologicaldisorders.Advantages of encapsulated technology include the following:Continuous delivery of therapeutic proteins and peptides in the treatment of•chronic diseases. Reduction of side effects associated with systemic administra-tion of proteins and peptides, which can cause peaks in serum protein levels.
  25. 25. 501Delivery of Cell Therapy to the BrainSite-specific delivery of therapeutic proteins.•Optimal patient compliance by avoiding painful injections•De novo production of proteins by encapsulated cells is important for proteins•that are difficult to manufacture and purify due to stability problems.Optimal therapeutic outcome by concomitant administration of several proteins.•Controllable dosing by adjusting the number of cells implanted and using self-•regulating drug delivery system such as gene switches and oxygenation regulation.Implantation of Microencapulated Genetically Modified CellsMicroencapsulation of recombinant cells is a novel and potentially cost-effectivemethod of heterologous protein delivery. A ‘universal’ cell line, geneticallymodified to secrete any desired protein, is immunologically protected from tissuerejection by enclosure in microcapsules. The microcapsule can then be implantedin different recipients to deliver recombinant proteins in vivo.There is increasing research on using artificial cells to microencapsulate geneticallyengineered cells for gene therapy. There are several methods to achieve this. OneTable 13.6 Therapeutic applications of encapsulated cells in neurological disordersDisease Method Results/referenceAlzheimer’sdiseaseEC-biodelivery secreting NGF Protection of cholinergicneurons and improvedmemoryAmyotropic lateralsclerosisCNTF cells intrathecally in humanpatientsSafety and sustained deliverydemonstratedEpilepsy EC-biodelivery technology to deliver theactive compound into the rat brainFor suppression of epilepticseizuresGlioblastomamultiformeEncapsulated cells producing endostatinin rat tumor modelTumor cell migration andinvasion is greatly reducedHuntington’sdisease (HD)Injection of encapsulated CNTFsecreting cells into the striatum of ratmodel of HDNeuroprotective effectobserved (Emerich andThanos 2006)Pain Implantation of chromaffin cells insubarachnoid space in humansProlonged cell survival but nopain reduction in phase IIParkinson’sdiseaseCatecholamine and GDNF cells in ratand primate brainImproved behavior, protectionof dopaminergic neuronsRetinitispigmentosaEncapsulated cells transfected with thehuman CNTF gene were implantedinto the vitreous of the eyePhase I trial indicated thatCNTF is safe for thehuman retina© Jain PharmaBiotechCNTF ciliary neurotrophic factor, GDNF Glial-derived neurotrophic factor
  26. 26. 502 13 Role of Biotechnology in Drug Delivery to the Nervous Systemapproach is to encapsulate genetically engineered cells and implant them into thebody, e.g., beta-endorphin secreting cells for pain treatment and neurotrophic factorsecreting cells as trophic factors for neurodegenerative diseases. There are someproblems related to implantation including the following:Safety problems related to the introduction of genetically engineered material•into the bodyAlthough the cells are protected from rejection by leucocytes and antibodies,•there is potential rejection by complements and cytokines.Ferrofluid Microcapsules for Tracking with MRIImplanting recombinant cells encapsulated in alginate microcapsules to expresstherapeutic proteins has been proven effective in treating several mouse models ofhuman neurological disorders. In anticipation of clinical application, magnetizedferrofluid alginate microcapsules have been synthesized, which can be trackedin vivo by MRI (Shen et al. 2005). Ferrofluid-enhanced alginate microcapsules arecomparable to classic alginate microcapsules in permeability and biocompatibility.Their visibility and stability to MRI monitoring permits qualitative and quantitativetracking of the implanted microcapsules without invasive surgery. These propertiesare important advantages for the application of immunoisolation devices in humancell/gene therapy.Delivery of Gene Therapy to the BrainGene therapy, a sophisticated method of delivery of therapeutics, is described inChap. 13. Various methods of delivery of DNA and genes for therapeutic purposesthat are relevant to neurological disorders are listed in Table 13.7.Clinical Applications of Biotechnology for CNS Drug DeliverySeveral techniques are used for drug delivery in CNS disorders (Jain 2012). Thischapter will emphasize the role of biotechnology. Of the various approachesdescribed, the following have been used clinically.
  27. 27. 503Clinical Applications of Biotechnology for CNS Drug DeliveryIntroduction of Therapeutic Substances into the CSFTherapeutic recombinant proteins can be introduced into the brain via this route.Implantation of genetically engineered encapsulated cells producing CNTF in thespinal subarachnoid space is an example of gene delivery into the CNS via this route.Deposition of transgenic constructs into the subarachnoid space or the ventricularsystem is likely to be an efficient route for transducing not only the subependymalregion but also for disseminating products of gene expression into the brain.Osmotic Opening of the Blood–Brain BarrierThe clinical experience with osmotic opening of the BBB is based on intracarotidinjections of an inert hypertonic solution (generally mannitol). The osmotic methodhas been shown to be clinically effective in humans and has been used to facilitateTable 13.7 Methods for delivery of gene therapy in neurological disordersRoutes of delivery of DNA/genesIntramuscular DNA injection, e.g. muscular dystrophiesDirect injection into lesions such as brain tumorsIntravenous DNA injection: for systemic administration targeted to a lesion in specificlocation in CNSIntraarterial delivery by catheter for delivery to cerebral circulationIntracerebral DNA injectionIntroduction of genes into the CSF pathways: intrathecal, intraventricularTargeting of CNS by retrograde axonal transportIntranasal instillation for introduction into the brain along the olfactory tractVector-mediated gene transfer: viral or nonviralTransplantation of genetically engineered cells for in vivo production of proteins orantibodiesTargeted gene therapy applicable to the nervous systemTargeted adenoviral vectors using molecular conjugatesTargeted nonviral gene therapy: locus control regionLiposome targetingAntibody-mediated gene targetingCell-targeted gene therapy using cell-binding peptides as vectorsNeuronal cell targetingControlling gene expression in gene therapyPharmacological control of gene expression: antibiotics such as tetracycline, small moleculesBinary system for toxin gene therapyManipulation of gene regulatory factors: e.g. heat shock proteins, cytokinesGene switch system to control in vivo expression: e.g. dumbbell shaped molecules aspharmacological rheostatEngineered zinc finger DNA binding proteins for gene correction© Jain PharmaBiotech
  28. 28. 504 13 Role of Biotechnology in Drug Delivery to the Nervous Systemthe entry of anticancer agents into brain tumors in phase III trials. Although thisapproach increases the efficacy of the cytotoxic drugs, it also increases their neuro-toxicity by increasing the permeability of the BBB of the normal brain. Thisapproach has also been used to facilitate the delivery of adenoviral vectors for genetherapy of brain tumors and for the administration of bifunctional fusion proteinsof tumor-specific MAbs for the treatment of brain tumors. Opening of the BBBfacilitates the entry of superparamagnetic iron oxide conjugates used as adjuncts toMRI for diagnosis of brain metastases.Intraarterial Administration of TherapeuticSubstances for CNS DisordersAcrylic and thrombosis inducing material for occluding arteriovenous malforma-tions are administered intraarterilly in neuroradiology and neurosurgery. A numberof drugs have also been administered by this route. This route is used for directinjection of gene vectors into the arterial circulation of the brain. The approvedmethod of administration of thrombolytic agents such as recombinant tissue plas-minogen activator, a biotechnology product, is intravenous but considerable experi-ence exists with the use of intraarterial thrombolysis in patients in whom the lesionshave been demonstrated by angiography prior to thrombolysis.The BBB hinders the penetration of anti-HIV drugs into the brain for treatmentof AIDS encephalopathy, promoting viral replication, the development of drugresistance, and, ultimately, subtherapeutic concentrations of drugs reaching thebrain, leading to therapeutic failure. The specificity and efficiency of anti-HIV drugdelivery can be enhanced by using nanocarriers with specific brain-targeting, cell-penetrating ligands (Wong et al. 2010).Drug Delivery to the Brain in PDGDNF is potentially useful in the treatment of PD (PD), but penetration into braintissue from either the blood or the CSF is limited. GDNF was delivered directly intothe putamen of a patient with AD in a phase 1 safety trial (Gill et al. 2003). The treat-ment was effective with no serious complications. An open-label study has demon-strated the safety and potential efficacy of unilateral intraputaminal GDNF infusionfor 6 months via a catheter in patients with advanced PD (Slevin et al. 2005).Drug Delivery to the Brain in ADSeveral routes of drug delivery other than oral have been explored for the manage-ment of AD (AD). Transdermal rivastigmine maintains steady drug levels in the
  29. 29. 505Clinical Applications of Biotechnology for CNS Drug Deliverybloodstream, improving tolerability and allowing a higher proportion of patients toreceive therapeutic doses compared to the capsule form of the medication. A num-ber of studies are exploring the nasal route of drug delivery for AD.Biological therapies for the treatment of AD that exploit mechanisms of penetra-tion of the BBB include peptides, vaccines, antibodies, and antisense oligonucle-otides (Banks 2008).An experimental study demonstrated that the brain concentration of intrave-nously injected rivastigmine can be enhanced over 3.82-fold by binding to poly(n-butylcyanoacrylate) nanoparticles coated with 1% nonionic surfactant polysorbate80 (Wilson et al. 2008).Drug Delivery in EpilepsySpecial methods of drug delivery would improve the control of seizures, reducetoxic effects, and increase compliance in patients with epilepsy, such as by use oflong-acting formulations and subcutaneous implants. Overexpression ofP-glycoprotein and other efflux transporters in the cerebrovascular endothelium, inthe region of the epileptic focus, may also lead to drug resistance in epilepsy. Thishypothesis is supported by the findings of elevated expression of efflux transportersin epileptic foci in patients with drug-resistant epilepsy, induction of expression byseizures in animal models, and experimental evidence that some commonly usedantiepileptic drugs are substrates. Further studies to delineate the exact role, if any,of P-glycoprotein and other efflux transporters in drug-resistant epilepsy are war-ranted (Kwan and Brodie 2005).Innovative Methods of Drug Delivery for GlioblastomaMultiformeSeveral innovative therapies inclusing biological are being investigated for treat-ment of glioblastoma multiforme. Methods relevant to biotechnology are shown inTable 13.8.Gene therapy for GBM is described in Chap. 12. Examples of other strategiesare as follows:Biodegradable polymer implants containing anticancer drugs. Polymer-baseddrug delivery to the brain has special applications for the delivery of anticanceragents to malignant brain tumors. One example is the use of carmustine implants.One of the problems with surgical excision of GBM is local recurrence within 2 cmof the primary lesion. Strategies to prevent local recurrence include implantation ofdelivery devices containing chemotherapeutic agents. Biodegradable polymerimpregnated with carmustine (Gliadel), an approved product, is implanted into thetumor cavity after surgery improves the survival of patients.
  30. 30. 506 13 Role of Biotechnology in Drug Delivery to the Nervous SystemImmunoliposomes are antibody-directed liposomes that have been used for deliveryof the antineoplastic agent daunomycin to the rat brain. PEG-conjugated immunoli-posomes increase the drug-carrying capacity of the MAb by up to 4 logarithmicorders in magnitude. Specific MAb-mediated targeting of daunomycin to the ratbrain has been achieved by the use of an immunoliposome-based drug delivery sys-tem. This has potential applications for the chemotherapy of brain neoplasms.Thermosensitive liposomes are microscopic vesicles that can contain drugs andrelease them in response to hyperthermia. Thermosensitive liposomes have beenused to deliver the anticancer agent cis-platinum in conjunction with localized brainheating in experimental malignant gliomas in rats.Chemotherapy is given by intraventricular and intrathecal injection for menin-geal cancer, but has serious toxic effects on the brain. A randomized study con-cluded that there was no difference for patients treated with sustained-releasecytarabine, whether the route used was intrathecal or intraventricular, but forpatients treated with short-acting drugs such as methotrexate, there was a statisti-cally significant difference favoring patients receiving intraventricular therapy(Glantz et al. 2010).The development of MAbs that recognize tumor-associated antigens has raised thepossibility of creating more tumor-specific therapeutic agents. Antibodies have beenconjugated with radionucleotides and various peptide toxins to create new drugs withhigh tumor selectivity in vitro. Attempts to develop these compounds for clinical useare limited by transcapillary and interstitial barriers encountered during delivery tosolid tumors. In meningeal cancer, the tumor cells often grow as thin sheets based inCSF, which reduces the problem of drug delivery and tissue penetration.Table 13.8 Biotechnology-based methods of drug delivery for glioblastoma multiformeDesign of anticancer drugs with higher penetration across the BBBPegylated liposomal doxorubicinIntravascular delivery of chemotherapeutic agentsLipid-coated microbubbles as a delivery vehicle for taxolLocal delivery of chemotherapeutic agentsBiodegradable polymer wafers: carmustine implantsBiodegradable nanoparticles containing 5-fluorouracilNanoparticle-based targeted delivery of chemotherapy across the BBBChemotherapy sensitizationUse of thermosensitive liposomes and localized hyperthermiaTargeted monoclonal antibodies (MAbs)MAbs conjugated with liposomesMAbs conjugated with a toxin against cell-surface antigens on glioma cellsSystemically administered anticancer therapy targeted to GBM: Trojan horseGene therapy/antisense therapyCell therapyMesenchymal stem cells to deliver treatment for gliomasNeural stem cells for drug delivery to brain tumorsTargeting cancer stem cells© Jain PharmaBiotech
  31. 31. 507Clinical Applications of Biotechnology for CNS Drug DeliveryIn a phase I clinical trial, intraventricular iodine-131-labeled MAb 3F8 (131I-3F8)targeting leptomeningeal cancer and was generally well tolerated and a high CSF-to-blood ratio was achieved (Kramer et al. 2007). This technique may have clinicalutility in the treatment of leptomeningeal malignancies.Calcium-activated potassium channels are overexpressed in brain tumorendothelial cells compared with normal brain tissue and play a pivotal role inblood–brain tumor barrier permeability regulation. Intravenous infusion of NS1619,a potassium channel agonist, and bradykinin selectively enhance blood–braintumor barrier permeability and enhance selective delivery of chemotherapeuticdrugs to metastatic brain tumors in a rat model (Hu et al. 2007). However, thismethod has not been used clinically.Nanoparticle-Based Targeted Delivery of Chemotherapy Across the BBBSome of techniques used for facilitating transport of therapeutic substances acrossthe BBB involve damage to the BBB, which is not desirable. Technologies basedon nanoparticles targeted delivery of anticancer drugs across the BBB. A conceptof targeted drug delivery to GBM across the BBB is shown in Fig. 13.3.Nanoparticles made of poly(butyl cyanoacrylate) (PBCA) or poly(lactic-co-glycolicacid) (PLGA) coated with polysorbate 80 or poloxamer 188 enable the transport ofdoxorubicin across the BBB. Following intravenous injection to rats with intracranialglioblastoma, these particles loaded with doxorubicin significantly increased thesurvival times and led to a complete tumor remission in 20–40% of the animals(Kreuter and Gelperina 2008). Nanoparticles considerably reduced the dose-limitingFig. 13.3 A concept of targeted drug delivery to GBM across the BBB. Nanoparticle (N) com-bined with a monoclonal antibody (MAb) for receptor (R) crosses the blood brain barrier (BBB)into brain by Trojan horse approach. N with a ligand targeting BBB▼traverses the BBB byreceptor-mediated transcytosis. Ligand)docks on a cancer cell receptor and N delivers antican-cer payload to the cancer cell in glioblastoma multiforme (GBM) (© Jain PharmaBiotech)
  32. 32. 508 13 Role of Biotechnology in Drug Delivery to the Nervous Systemcardiotoxicity and also the testicular toxicity of this drug. The drug transport acrossthe BBB by nanoparticles is due to a receptor-mediated interaction with the braincapillary endothelial cells, which is facilitated by certain plasma apolipoproteinsadsorbed by nanoparticles in the blood.A polymeric nanobioconjugate drug based on biodegradable, nontoxic, andnonimmunogenic polymalic acid as a universal delivery nanoplatform is used fordesign of a nanomedicine for intravenous treatment of brain tumors (Ding et al.2010). The polymeric drug passes through the BTB and tumor cell membrane usingtandem monoclonal antibodies targeting the BTB and tumor cells. The next step forpolymeric drug action is inhibition of tumor angiogenesis by specifically blockingthe synthesis of a tumor neovascular trimer protein, laminin-411, by attached anti-sense oligonucleotides, which are released into the target cell cytoplasm via pH-activated trileucine, an endosomal escape moiety. Introduction of a trileucineendosome escape unit results in significantly increased antisense oligonucleotidedelivery to tumor cells, inhibition of laminin-411 synthesis, specific accumulationin brain tumors, and suppression of intracranial glioma growth compared with pH-independent leucine ester. The availability of a systemically active polymeric drugdelivery system that crosses BTB, targets tumor cells, and inhibits tumor growth isa promising strategy of glioma treatment.In vivo application of nanoparticle-based platforms in brain tumors is limited byinsufficient accumulation and retention within tumors due to limited specificity forthe target, and an inability to traverse the BBB. A nanoprobe has been designed thatcan cross the BBB and specifically target brain tumors in a genetically engineeredmouse model, by using in vivo magnetic resonance and biophotonic imaging, aswell as histologic and biodistribution analyses (Veiseh et al. 2009). The nanoprobeis made of an iron oxide nanoparticle coated with biocompatible PEG-grafted chi-tosan copolymer, to which a tumor-targeting agent, chlorotoxin (a small peptideisolated from scorpion venom), and a near-IR fluorophore are conjugated. Theparticle was about 33 nm in diameter when wet, i.e. about a third the size of similarparticles used in other parts of the body. The nanoprobe shows an innocuous toxic-ity profile and sustained retention in tumors. The nanoparticles remained in mousetumors for up to 5 days and did not show any evidence of damaging the BBB. Withthe versatile affinity of the targeting ligand and the flexible conjugation chemistryfor alternative diagnostic and therapeutic agents, this nanoparticle platform can bepotentially used for the diagnosis and treatment of a variety of brain tumors. Thefluorescent nanoparticles improved the contrast between the tumor tissue and thenormal tissue in both MRI and optical imaging, which are used during surgery tosee the tumor boundary more precisely. Precise imaging of brain tumor margins isimportant because patient survival for brain tumors is directly related to the amountof tumor that can be resected.Nano-imaging could also help with early detection of brain tumors. Currentimaging techniques have a maximum resolution of 1 mm. Nanoparticles couldimprove the resolution by a factor of 10 or more, allowing detection of smallertumors and earlier treatment. Future research will evaluate this nanoparticle’spotential for treating tumors. It has already been shown that chlorotoxin combined
  33. 33. 509Referenceswith nanoparticles dramatically slows tumors’spread. It remains to be seen whetherthat ability could extend to medulloblastoma, the most common malignant solidtumor to affect children.ReferencesAbbott NJ, Patabendige AA, Dolman DE, et al. Structure and function of the blood–brain barrier.Neurobiol Dis 2010;37:13–25.Abulrob A, Sprong H, Van Bergen en Henegouwen P, Stanimirovic D. The blood–brain barriertransmigrating single domain antibody: mechanisms of transport and antigenic epitopes inhuman brain endothelial cells. J Neurochem 2005;95:1201–14.Banks WA. Developing drugs that can cross the blood–brain barrier: applications to Alzheimer’sdisease. BMC Neurosci 2008;9(Suppl 3):S2.Batson OV. The function of the vertebral veins and their role in the spread of metastases, Ann Surg1940;112:138–49.Begley DJ. ABC transporters and the blood–brain barrier. Curr Pharm Des 2004;10:1295–312.Boado RJ. A new generation of neurobiological drugs engineered to overcome the challenges ofbrain drug delivery. Drug News Perspect 2008;21:489–503.Broman T. The possibilities of the passage of substances from the blood to the central nervoussystem. Acta Psych Neurol 1941;16:1–25.Chua JY, Pendharkar AV, Wang N, et al. Intra-arterial injection of neural stem cells using amicroneedle technique does not cause microembolic strokes. J Cereb Blood Flow Metab2011;31:1263–71.Cooper IS. Intracerebral injection of procaine into the globus pallidus in hyperkinetic disorders.Science 1954;119:417–8.Corning JL. Spinal anesthesia and local medication of the cord. NY Med J 1885;42:483–5.Cucullo L, Hossain M, Puvenna V, Marchi N, Janigro D. The role of shear stress in blood–brainbarrier endothelial physiology. BMC Neuroscience 2011;12:40.Ding H, Inoue S, Ljubimov AV, et al. Inhibition of brain tumor growth by intravenous poly(b-Lmalic acid) nanobioconjugate with pH-dependent drug release. PNAS 2010;107:18143–8.Doppman JL. Intra-arterial valium--its safety and effectiveness. Radiology 1973;106:335–8.Ehrlich P. “Das sauerstoffbedürfnis des organismus,” eine farbanalytische studie. Berlin:Hirschwald, 1885.Emerich DF, Thanos CG. In vitro culture duration does not impact the ability of encapsulatedchoroid plexus transplants to prevent neurological deficits in an excitotoxin-lesioned rat modelof Huntington’s disease. Cell Transplant 2006;15:595–602.Enerson BE, Drewes LR. The rat blood–brain barrier transcriptome. J Cereb Blood Flow Metab2006;26:959–73.Eyal S, Ke B, Muzi M, et al. Regional P-glycoprotein activity and inhibition at the human blood–brain barrier as imaged by positron emission tomography. Clin Pharmacol Ther2010;87:579–85.Fischer UM, Harting MT, Jimenez F, et al. Pulmonary Passage is a Major Obstacle for IntravenousStem Cell Delivery: The Pulmonary First-Pass Effect. Stem Cells and Development 2009;18:683–92.Gaillard PJ, Appeldoorn CC, Rip J, et al. Enhanced brain delivery of liposomal methylpredniso-lone improved therapeutic efficacy in a model of neuroinflammation. J Control Release 2012Jun 23. [Epub ahead of print].Gaillard PJ, de Boer AG. 2B-Trans-Technology: targeted delivery across the blood–brain barrier.In, Jain KK (ed) Drug Delivery Systems, Springer/Humana Press, 2008:161–175.Gaillard PJ. Case Study: to-BBB’s G-Technology®, getting the best from drug-delivery researchwith industry-academia partnerships. Therapeutic Delivery 2011;2:1391–94.
  34. 34. 510 13 Role of Biotechnology in Drug Delivery to the Nervous SystemGill SS, Patel NK, Hotton GR, et al. Direct brain infusion of glial cell line-derived neurotrophicfactor in PD. Nat Med 2003;9:589–95.Gimi B, Leong T, Gu Z, et al. Self-assembled 3D radiofrequency-shielded (RS) containers for cellencapsulation. Biomedical Microdevices 2005;7:341–5.Glantz MJ, Van Horn A, Fisher R, Chamberlain MC. Route of intracerebrospinal fluid chemo-therapy administration and efficacy of therapy in neoplastic meningitis. Cancer2010;116:1947–52.Goldmann E. Vitalfarbungen am Zentralnervensystem. Beitrag zur Physio-Pathologie des PlexusChoroideus und der Hirnhaute (Intravital labeling of the central nervous system. A study on thepathophysiology of the choroid plexus and the meninges). Abhandlungen der konigliche preus-sischen Akademie der Wissenshaften, Physikalisch-Mathematische Klasse 1913;1:1–64.Gynther M, Laine K, Ropponen J, et al. Large neutral amino acid transporter enables brain drugdelivery via prodrugs. J Med Chem 2008;51:932–6.Hu J, Yuan X, Ko MK, et al. Calcium-activated potassium channels mediated blood–brain tumorbarrier opening in a rat metastatic brain tumor model. Mol Cancer 2007;6:22.Jain KK. Nanobiotechnology-based strategies for crossing the blood–brain barrier. Nanomedicine2012;7:1225–33.Jain KK. An overview of drug delivery to the central nervous system. Neuromethods 2010;45:1–13.Johansson I, Ingelman-Sundberg M. Genetic polymorphism and toxicology--with emphasis oncytochrome p450. Toxicol Sci 2011;120:1–13.Karkan D, Pfeifer C, Vitalis TZ, et al. A Unique Carrier for Delivery of Therapeutic Compoundsbeyond the Blood–brain Barrier. PLoS ONE 2008;3:e2469.Kramer K, Humm JL, Souweidane MM, et al. Phase I study of targeted radioimmunotherapy forleptomeningeal cancers using intra-Ommaya 131-I-3F8. J Clin Oncol 2007;25:5465–70.Kreuter J, Gelperina S. Use of nanoparticles for cerebral cancer. Tumori 2008;94:271–7.Kwan P, Brodie MJ. Potential role of drug transporters in the pathogenesis of medically intractableepilepsy. Epilepsia 2005;46:224–35.Lanevskij K, Japertas P, Didziapetris R, Petrauskas A. Prediction of blood–brain barrier penetra-tion by drugs. In: Jain KK, editor. Drug Delivery to the Central Nervous System. New York:Humana/Springer, 2010:63–83.Latour LL, Kang DW, Ezzeddine MA, et al. Early blood–brain barrier disruption in human focalbrain ischemia. Ann Neurol 2004;56:468–77.Lazorthes Y, Sallerin-Caute B, Verdie JC, Bastide R. Advances in drug delivery systems andapplications in neurosurgery. Adv Tech Stand Neurosurg 1991;18:143–92.LeBowitz JH. A breach in the blood–brain barrier. Proc Natl Acad Sci USA 2005;102:14485–6.Lewandowski M. Zur Lehre der Cerebrospinalflüssigkeit. Z Klin Med 1900;40:480–94.Miller DS, Bauer B, Hartz AM. Modulation of P-glycoprotein at the blood–brain barrier: oppor-tunities to improve central nervous system pharmacotherapy. Pharmacol Rev 2008;60:196–209.Misra V, Lal A, El Khoury R, et al. Intra-Arterial Delivery of Cell Therapies for Stroke. Stem CellsDev 2012;21:1007–15.Moniz E. L’encéphalographie artérielle, son importance dans la localisation des tumors cérébrales.Rev Neurol 1927;2:72–90.Namanja HA, Emmert D, Pires MM, et al. Inhibition of human P-glycoprotein transport andsubstrate binding using a galantamine dimer. Biochem Biophys Res Commun 2009;388:672–6.Oldendorf WH. Brain uptake of radiolabelled amino acids, amines and hexoses after arterial injec-tion. Am J Physiol 1971;221:1629–39.Pan W, Kastin AJ, Zankel TC, et al. Efficient transfer of RAP across the blood–brain barrier. J CellSci 2004;117:5071–78.Pardridge WM. Re-engineering biopharmaceuticals for delivery to brain with molecular Trojanhorses. Bioconjug Chem 2008;19:1327–38.
  35. 35. 511ReferencesPrince WS, McCormick LM, Wendt DJ, et al. Lipoprotein receptor binding, cellular uptake, andlysosomal delivery of fusions between the receptor-associated protein (RAP) and alpha-L-iduronidase or acid alpha-glucosidase. J Biol Chem 2004;279:35037–46.Reese TS, Karnovsky MJ. Fine structural localization of a blood–brain barrier to exogenousperoxidase. J Cell Biol 1967;34:207–17.Sarkar D, Spencer JA, Phillips JA, et al. Engineered cell homing. Blood 2011;118:e184–91.Shawahna R, Uchida Y, Decleves X, et al. Transcriptomic and Quantitative Proteomic Analysis ofTransporters and Drug Metabolizing Enzymes in Freshly Isolated Human Brain Microvessels.Mol Pharm 2011;8:1332–41.Shen F, Li AA, Gong YK, et al. Encapsulation of Recombinant Cells with a Novel MagnetizedAlginate for Magnetic Resonance Imaging. Human Gene Therapy 2005;16:971–84.Slevin JT, Gerhardt GA, Smith CD, Gash DM, Kryscio R, Young B. Improvement of bilateralmotor functions in patients with PD through the unilateral intraputaminal infusion of glial cellline-derived neurotrophic factor. J Neurosurg 2005;102:216–22.Spiegel EA, Wycis HA, Marks M, Lee AJ. Stereoscopic apparatus for operations on the humanbrain. Science 1947;106:349–50.Stern L, Gautier R. Les rapports entre le liquide céphalo-rachidien et al. circulation sanguine. ArchInt Physiol 1922;17:391–448.Stern L, Gautier R. Rapports entre le liquide céphalo-rachidien et al. circulation sanguine. ArchInt Physiol 1921;17:138–92.Suresh Reddy J, Venkateswarlu V, Koning GA. Radioprotective effect of transferrin targeted citi-coline liposomes. J Drug Target 2006;14:13–9.Tatard VM, Venier-Julienne MC, Saulnier P, et al. Pharmacologically active microcarriers: a toolfor cell therapy. Biomaterials 2005;26:3727–37.Veiseh O, Sun C, Fang C, et al. Specific targeting of brain tumors with an optical/magnetic reso-nance imaging nanoprobe across the blood–brain barrier. Cancer Res 2009;69:6200–7.Weiler-Guttler H, Zinke H, Mockel B, Frey A, Gassen HG. cDNA cloning and sequence analysisof the glucose transporter from porcine blood–brain barrier. Biol Chem Hoppe Seyler1989;370:467–73.Wilson B, Samanta MK, Santhi K, Kumar KP, Paramakrishnan N, Suresh B. Poly(n-butylcyanoacrylate) nanoparticles coated with polysorbate 80 for the targeted delivery ofrivastigmine into the brain to treat Alzheimer’s disease. Brain Res 2008;1200:159–68.Wong HL, Chattopadhyay N, Wu XY, Bendayan R. Nanotechnology applications for improveddelivery of antiretroviral drugs to the brain. Adv Drug Deliv Rev 2010;62:503–17.Zheng PP, Romme E, van der Spek PJ, Dirven CM, Willemsen R, Kros JM. Glut1/SLC2A1 iscrucial for the development of the blood–brain barrier in vivo. Ann Neurol 2010;68:835–44.