Getting into the Brain
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Being the most delicate organ of the body, the brain is protected against potentially toxic substances by the blood-brain barrier (BBB), which restricts the entry of most pharmaceuticals into the brain. The developmental process for new drugs for the treatment of CNS disorders has not kept pace with progress in molecular neurosciences because most of the new drugs discovered are unable to cross the BBB. The clinical failure of CNS drug delivery may be attributed largely to a lack of appropriate drug delivery systems. Localized and controlled delivery of drugs at their desired site of action is preferred because it reduces toxicity and increases treatment efficiency. The present review provides an insight into some of the recent advances made in the field of brain drug delivery.
The various strategies that have been explored to increase drug delivery into the brain include (i) chemical delivery systems, such as lipid-mediated transport, the prodrug approach and the lock-in system; (ii) biological delivery systems, in which pharmaceuticals are re-engineered to cross the BBB via specific endogenous transporters localized within the brain capillary endothelium; (iii) disruption of the BBB, for example by modification of tight junctions, which causes a controlled and transient increase in the permeability of brain capillaries; (iv) the use of molecular Trojan horses, such as peptidomimetic monoclonal antibodies totransport large molecules (e.g. antibodies, recombinant proteins, nonviral gene medicines or RNA interference drugs) across the BBB; and (v) particulate drug carrier systems. Receptor-mediated transport systems exist for certain endogenous peptides, such as insulin and transferrin, enabling these molecules to cross the BBB in vivo.
The use of polymers for local drug delivery has greatly expanded the spectrum of drugs available for the treatment of brain diseases, such as malignant tumours and Alzheimer’s disease. In addition, various drug delivery systems (e.g. liposomes, microspheres, nanoparticles, nanogels and bionanocapsules) have been used to enhance drug delivery to the brain. Recently, microchips and biodegradable polymers have become important in brain tumour therapy.
The intense search for alternative routes of drug delivery (e.g. intranasal drug delivery, convection-enhanced diffusion and intrathecal/intraventricular drug delivery systems) has been driven by the need to overcome the physiological barriers of the brain and to achieve high drug concentrations within the brain. For more than 30 years, considerable efforts have been made to enhance the delivery of therapeutic molecules across the vascular barriers of the CNS. The current challenge is to develop drug delivery strategies that will allow the passage of drug molecules through the BBB in a safe and effective manner.
KeywordsSolid Lipid Nanoparticles Ommaya Reservoir Nipecotic Acid Prodrug Approach Large Neutral Amino Acid Transporter
Despite considerable advances in research in the area of brain and CNS disorders, diseases of the CNS remain the world’s leading cause of disabilities, accounting for more hospitalizations and prolonged care than almost all other diseases combined. Being the most delicate organ of the body, the brain is protected against potentially toxic substances by the blood-brain barrier (BBB); however, the BBB also prevents the penetration of most molecules that have potential activity in the CNS. For drugs to be effective against CNS diseases, they must reach the brain by crossing the BBB. The BBB is a unique complex endothelial barrier that segregates the circulating blood compartment of the brain from the extracellular fluid (figure 1). The BBB consists of a continuous layer of endothelial cells joined by complex tight junctions. The luminal plasma membrane of the endothelial cells faces the blood compartment, whereas the abluminal membrane is directed towards the brain extracellular fluid.[1, 2, 3]
A common belief is that small molecules readily cross the BBB; however, findings suggest that 98% of small molecules are not able to cross the BBB and that 100% of large molecules cannot cross the BBB. Limited penetration of a drug into the brain is the rule rather than the exception. One study revealed that of 7000 drugs in the Comprehensive Medicinal Chemistry database, only 5% are used for the treatment of CNS diseases (i.e. mainly depression, schizophrenia and insomnia). Another study revealed that although 12% of drugs are active on the CNS, only 1% are active in the brain for diseases other than affective disorders.
Despite aggressive research, patients experiencing fatal and/or debilitating CNS diseases, such as brain tumours, HIV encephalopathy, epilepsy, cerebrovascular diseases and neurodegenerative disorders, far outnumber those dying of all types of systemic cancer or heart disease. The incidence of CNS disorders in humans increases with age; this, along with the fact that very few drugs can cross the BBB, exacerbates the problem of drug delivery to the CNS. It is predicted that by 2020, the number of people in the US aged ≥65 years will increase by 50%, which will result in a simultaneous increase in CNS disorders. This would result in an annual expenditure in the US for Alzheimer’s disease alone of $US0.5 trillion. Because one in every three people will experience a CNS disorder during his/her lifetime, the neuropharmaceutical market is predicted to become the largest sector of the pharmaceutical industry. Although the global market for CNS drugs compared with other classes of drugs has recently fallen considerably, a growth rate of approximately 500% would be required for the market for CNS drugs to be comparable to that for cardiovascular drugs. Again, the principal reason for this decrease is the difficulty in getting most CNS drugs to cross the BBB. Taking into the consideration the potential size of the global neuropharmaceutical market and the fact that only a few drugs can cross the BBB, one would predict that a priority for both the pharmaceutical industry and researchers would be the development of drug delivery technologies that enable drugs to cross the BBB.
Modern methods of CNS drug discovery consider expected pharmacological effects only after direct injection of the drug into the brain and not after systemic administration, because the transport of drugs across the BBB after systemic administration is negligible. The flow chart in figure 2 shows an approach made some years ago to the development of CNS drugs. There are two pathways by which CNS drugs may be developed: through trial and error or using rational drug design. Rational drug design is based on high-throughput screening of thousands of molecules and has a tendency to identify drugs with negligible ability to cross the BBB. Therefore, in the absence of parallel progress in the development of CNS drug delivery strategies, the CNS drug discovery programme invariably ends in failure. If effective CNS drug delivery systems were available, the drug discovery programme would be successful.
The present review deals mainly with the various approaches used to get drug molecules into the brain and will hopefully provide valuable insights for researchers interested in rational drug design of CNS drugs.
1. Approaches to Enhance Brain Drug Delivery
1.1 Chemical Delivery Systems
There have been various attempts to overcome the limited access of drugs to the brain by chemically modifying particular drugs. Chemical delivery systems can be divided into three main groups, namely lipid-mediated transport (lipidization of small molecules), a prodrug approach and a lock-in system (LIS).
1.1.1 Lipid-Mediated Transport (Lipidization of Small Molecules)
Lipidization of a drug mainly involves the addition of lipid-like molecules by modification of the hydrophilic moieties on the drug structure. Drug permeability across the BBB is favoured by lipophilicity, low molecular weight and lack of ionization at physiological pH. It is believed that lipid-soluble molecules (with a molecular weight <500 Da) may cross the BBB through the small pores that form transiently within the lipid bilayer.[11,12]
Drug transport across the BBB can also be increased by the addition of hydrophobic groups to a molecule, which improves drug transportation mainly by passive diffusion (figure 3). The best example of this sort of modification is the addition of methyl groups to various drugs in the barbiturate class, which results in increased lipophilicity and brain penetration in animals.[13,14] Morphine is converted to heroin by diacetylation of its two hydroxyl groups, which results in the removal of two hydrogen bonds between the drug and solvent (water). The transportation of heroin across the BBB is reported to be approximately 100-fold greater than for its parent drug morphine owing to the lipophilic nature of heroin. As a general rule, the BBB permeability of a drug decreases one log order in magnitude for each pair of hydrogen bonds added to the molecule as a polar functional group.[9,16]
Recently, with the emergence of liposomal drug delivery as an alternative to changing the lipophilicity of a drug, the ability of the drug to cross the less permeable and lipophilic BBB and to reach the brain, have improved. For example, improved drug delivery has been demonstrated for encapsulated doxorubicin in the treatment of experimental brain tumours.[17, 18, 19] Similar observations were made when liposomal doxorubicin was administered in phase I/II clinical trials to patients with brain tumours: patients exhibited stabilization of symptoms and increased survival rate.[20,21]
There are certain drawbacks to the lipidization of drugs. First, increasing the lipid solubility of a drug will increase the permeability of the lipidized molecule across all biological membranes in the body, not just the BBB. This will affect the plasma distribution of the drug, increasing plasma clearance and subsequently decreasing the plasma area under the concentration-time curve (AUC). In their study of lipidized chlorambucil, Greig et al. suggested that the plasma AUC of this formulation was reduced. Thus, although the pharmacological activity of a drug may be increased as a result of the greater transfer of drug across the BBB, the increased lipid solubility also results in increased plasma clearance, with a subsequent reduction in the plasma AUC, leading to a significant change in drug therapy.[22,23] Second, increased lipophilicity enhances plasma protein binding, which could neutralize any benefits associated with increased BBB permeability following lipidization. Third, lipidization will increase the molecular weight of a drug. Drugs that have a molecular weight >400–500 Da are unable to cross the BBB in a pharmacologically significant amount. Fischer et al. reported that BBB permeability decreases exponentially with an increase in the molecular size of a drug. If the size of a drug is increased from 52 Å (molecular weight, 200 Da) to 105 Å (molecular weight, 450 Da), its BBB permeability decreases 100-fold.
1.1.2 Prodrug Approach
The transport of drugs across the BBB can be increased using the prodrug approach. The design and use of prodrugs helps overcome any pharmaceutical and/or pharmacokinetic problems associated with the parent drug molecule. Modification of the properties of the prodrug can increase its affinity for the receptor target and improve its residence time. The prodrug is then converted at the site of action by either a chemical reaction or enzyme activity to a physiologically active compound. A typical example of the prodrug approach is the amidation or esterification of amino, carboxyl and hydroxyl groups, which increases their lipid solubility, thus causing higher uptake in the brain. Subsequent hydrolysis of these groups leads to the release of the active drug into the brain. Alternative prodrug formation could include the coupling of the active drug with lipid moieties, such as fatty acids, glycerides or phospholipids.
Prodrugs of acid-containing CNS drugs, such as niflumic acid, valproate, levodopa, vigabatrin and GABA, have been prepared by coupling these drugs with diglycerides or modified diglycerides. Although increased lipophilicity facilitates drug transport across the BBB, it also enhances the efflux process, which, in turn, results in poor tissue retention. Furthermore, if, in addition to its conversion to parent drug, the prodrug is metabolized by other pathways, there is the possibility of toxicity as a result of the production of these other metabolites.
1.1.3 Lock-In System
In addition to the lipidization or attachment of hydrophobic groups, chemical modification of a drug moiety can also increase its transport across the BBB. The LIS requires multichemical or enzymatic transformations for release of the active drug. In this system, two types of bioremovable moieties are usually introduced to convert the drug into an inactive precursor: a targetor moiety and a modifier function. The targetor moiety is responsible for targeting, site specificity and lock in, whereas the modifier function serves as a lipophilizer that protects against certain functions, or fine tunes which molecular properties are necessary to prevent premature, unwanted metabolic conversions. Once the lipophilic compound of the LIS enters the brain, it is converted into lipid-insoluble moieties and, hence, cannot leave the brain (i.e. it is locked in).
The LIS concept evolved from the prodrug concept and the only difference between the two systems is that although the prodrug contains one or more modifier function moieties, it does not contain targetor moieties. Another type of LIS is based on a dihydropyridine↔quaternary pyridinium ion redox system, which is chemically analogous to the NAD+↔NADH coenzyme system. The active drug is transformed to a 1,4-dihydropyridine moiety-containing conjugate, which is a type of LIS and is more lipophilic than the parent drug. When administered systemically in animals, the conjugated drug has been found to be distributed extensively throughout the body, including the brain. Upon oxidization in the body, the unstable dihydropyridine derivative forms a hydrophilic polar quaternary pyridinium salt, which, although eliminated from most tissues, is retained in the brain. Retention of the pyridinium salt in the brain is due to the ‘locking in’ of the ionized moiety by the BBB. Subsequently, cleavage of the carrier releases the free active drug.
During the past decade, targeted drug delivery to the brain through phosphonated derivatization has been explored and so-called anionic chemical delivery systems have been designed, synthesized and evaluated for testosterone and zidovudine.
1.2 Biological Delivery Systems
There are several mechanisms by which a molecule can cross the BBB (figure 3). Some of these have been used to enhance the transport of drug from the blood to the brain and include adsorptive-mediated transcytosis, carrier-mediated transport, receptor-mediated transport, active efflux transport and peptide vector strategies, each of which is discussed in greater detail below.
1.2.1 Adsorptive-Mediated Transcytosis
The cellular uptake of proteins such as albumin and antibodies (IgG) can be enhanced by cationic modification. The cationized proteins mainly cross the BBB by adsorptive-mediated transcytosis (AMT). An electrostatic interaction exists between the cationized albumin and anionic charges on the BBB. This interaction also occurs further with sialic acid moieties on the luminal surface and heparin sulfate groups on the abluminal surface. AMT of cationized albumin is triggered by this electrostatic interaction and results in the transport of the moiety across the BBB. The use of cationized albumin for the transport of β-endorphin, a very large molecule that cannot cross the BBB, has been reported in rats.[34,35] After conjugation with cationized albumin, brain uptake of β-endorphin was increased.
When the isoelectric point of antibodies is raised from neutral to highly alkaline, cationized antibodies are formed. These antibodies are used mainly as neurodiagnostic and neuroimaging agents in various diseases, including brain tumours, Alzheimer’s disease and stroke.[34, 35, 36] For example, an antibody that recognizes a specific antigen on tumour cells can be used to detect a brain tumour.
1.2.2 Carrier-Mediated Transport
Carrier-mediated transport (CMT) involves the modification of a drug (small molecule) into a compound with a similar structure that mimics a nutrient and can thus make use of one of the several specialized CMT systems within the BBB that exist for the transport of essential compounds, such as amino acids, hexoses, vitamins and neuropeptides, into the brain. For example, dopamine, a water-soluble catecholamine, cannot cross the BBB, but once dopamine has been transformed to levodopa, a large neutral amino acid, it can cross the BBB making use of the large neutral amino acid transporter (LNAT). This transformation of dopamine has been used for approximately four decades to deliver dopamine into the brain for the treatment of Parkinson’s disease. Other drugs that cross the BBB via the LNAT include α-methyldopa, gabapentin and melphalan.[39, 40, 41] In addition to the LNAT, Na+-dependent vitamin C transporters are also involved in the transport of drugs across the BBB that would otherwise be unable to reach the brain, such as nipecotic acid and kynurenic acid. Table I lists the important transporters in the BBB and the molecules they transport.
The main goal of BBB biology is the molecular cloning of the specific transporters that function as CMT for drugs across the BBB. Many members of the glucose transporter (GLUT) gene family belong to the solute carrier (SLC) subgroups. SLC2, SLC7 and SLC16, among others, have been reported to be localized at the BBB. Studies investigating the number of GLUT1 sites have reported that GLUT1 constitutes >90% of BBB glucose transporters. By conjugating galactose and tyrosine chemically with glucose, Bonina et al.[46,47] have designed prodrug esters of two anticonvulsant drugs, namely nipecotic acid and 7-chloronokynurenic acid, that can be transported across the BBB by GLUT1. The major LNAT is LAT1, whereas the major cationic amino acid transporter is CAT1. In rats, CNT2 is the adenosine transporter in the BBB. It has been shown in vivo that adenosine flux from the blood to the brain via CNT2 is a sodium-dependent process in the rat species.[48,49] As indicated in table I, the choline transporter and the adenine nucleobase transporter in the BBB have not yet been cloned.
1.2.3 Receptor-Mediated Transport
Receptor-mediated transport (RMT) is how large endogenous molecules, such as neuropeptides (insulin, transferrin or leptin), cross the BBB. Table II lists some of the important RMT systems in the BBB. Insulin uptake into the brain is mediated by the insulin receptor, transferrin uptake is mediated by the transferrin receptor (TfR), insulin-like growth factor (IGF) uptake is mediated by the IGF receptor and leptin uptake is mediated by the leptin receptor. After intracerebral injection, IgG molecules are rapidly effluxed out of the brain by reverse transcytosis mediated by a BBB Fc receptor (FcR), also called FcRn (the neonatal Fc receptor) or the Fc fragment of IgG receptor transporter (FcGRT).[52,53] Brain endothelial cells also express scavenger receptors (scavenger receptor, class B, member 1 [SR-BI]), commonly known as acetylated low-density lipoprotein (LDL) receptors.
Designing genetically engineered fusion proteins to make use of RMT to transport molecules across the BBB is another approach for brain targeting. These proteins are prepared by fusing human transferrin to mouse-human chimeric IgG3 at three positions: at the end of heavy chain constant region 1, after the hinge, and after constant region 3. The resulting proteins reached the brain parenchyma after intravenous injection by IgG RMT.
Fusion proteins have also been designed to prevent cerebrovascular thrombosis. Low molecular weight, single-chain urokinase-type plasminogen activator has been fused with anti-platelet-endothelial cell adhesion molecule single-chain variable fragment and demonstrated to cross the BBB. When studied in mice, it was found that this fusion protein accumulated in the brain after intravascular injection and, without causing haemorrhagic complications, it lysed clots lodged in the cerebral arterial vasculature, resulting in rapid and stable cerebral reperfusion.
Recently, the use of nanoparticles to transport molecules via RMT has gained interest. Drugs in the nanoparticulate form that have been transported successfully to the brain via RMT include the hexapeptide dalargin, the dipeptide kytorphin, loperamide, tubocurarine, the NMDA receptor antagonist MRZ 2/576 and doxorubicin. It is believed that the nanoparticles mimic LDL particles and are subsequently transported by the LDL receptor.[57, 58, 59, 60, 61, 62, 63] In another study, translocation into the rat brain was compared for poly(methoxy-polyethyleneglycol cyanoacrylate-co-n-hexadecylcyanoacrylate) [PEG-PHDCA] nanoparticles and polyhexadecylcyanoacrylate (PHDCA) nanoparticles after intravenous injection using two-dimensional polyacrylamide gel electrophoresis, capillary electrophoresis and the Protein Lab-on-chip® (Johns Hopkins Whiting School of Engineering and the School of Medicine, Baltimore, MD, USA) method. In that study, it was demonstrated that PEG-PHDCA nanoparticles were able to translocate into the brain, whereas non-PEGylated PHDCA nanoparticles were not. Further studies were undertaken to determine the mechanism of transport into the endothelial cells in rat brain and it was found that transport was mediated by the LDL receptor and was the result of endocytosis of PEG-PHDCA nanoparticles into the cells.[65,66]
Advances in nanoparticle technology have resulted in the development of solid lipid nanoparticles (SLN), which consist of spherical solid lipid particles in the nanometre range, dispersed in water or in aqueous surfactant solution. These SLN, coated with hydrophilic polymer and specific ligands, are detected to a lesser degree in the reticuloendothelial system (RES) and gain access to the brain by RMT. For example, SLN coated with PEG and thiamine ligands bind to thiamine receptors and are transported into the brain via RMT.[68, 69, 70] Highly specific targeting of SLN can be achieved by using peptidomimetic antibodies to coat the SLN; these targeted PEGylated immunonanoparticles bind to a transcytotic receptor and cross the BBB without changing its permeability.
1.2.4 Active Efflux Transport
P-glycoprotein is the most widely researched active efflux transporter at the BBB. It is a product of adenosine triphosphate-binding cassette (ABC) transporter, subfamily B, member 1, also called P-glycoprotein gene. Recently, it has been reported that the active efflux transport of drug from the brain to the blood is mediated by two types of transporters: energy-dependent and energy-independent transporters. The energy-dependent transporter is a member of the ABC gene family and is present at the luminal membrane, whereas the energy-independent transporter is present at the abluminal membrane and belongs to the SLC gene family. Members of the SLC gene family include the organic anion transporter (OAT) or OAT polypeptide, acidic amino acid transporters, such as members of the glutamic acid amino acid transporter family, and active efflux transporters, such as the taurine transporter. It is also possible that the energy-independent transporter is located at the luminal membrane and that the energy-dependent transporter is present at the abluminal membrane.
1.2.5 Peptide Vector Strategies
Using small synthetic peptides that can cross cell membranes is another approach for the delivery of neuropharmaceuticals. Pegelin and penetratin (18 and 16 amino acids, respectively) are small synthetic peptides that can cross the BBB efficiently and have been used effectively to deliver biologically active substances to cells.[13,74] Rousselle et al. coupled doxorubicin, an anticancer agent, with the small peptide vectors (SynB1) using a chemical linker (succinate) and investigated the ability of the complex to cross the BBB in an in situ cerebral perfusion model in rats (figure 4). The results of that study showed that the brain uptake of doxorubicin alone is very low because of efficient efflux by the P-glycoprotein pump within the BBB. However, when doxorubicin was coupled with the peptide vectors, its uptake was increased significantly.
In another study, Schwarze et al. conjugated the β-galactosidase protein with a peptide derived from transactivating regulatory proteins. Intraperitoneal injection of the peptide-fused proteins in mice resulted in their transport to all biological tissues, including the brain, whereas there was no transport of unfused proteins into the brain.
1.3 Disruption of the Blood Brain-Barrier (BBB)
The presence of endothelial tight junctions prevents the passage of drugs across the BBB. Disruption of these tight junctions can lead to increased drug delivery to the brain. The disruption of the BBB must be transient and reversible to facilitate the delivery of therapeutic molecules. Disruption of the BBB leads to opening of either a paracellular route through the endothelium by opening the tight junctions or a transcellular route through the endothelium. The extent of the disruption of the BBB is important because this intrusion may be highly toxic and may lead to chronic neuropathological changes, cerebral vasculopathy and seizures.[77,78] Disruption of the BBB can be achieved by osmotic disruption (e.g. hyperosmotic shock), biochemical disruption (e.g. by the administration of vasoactive substances) or by alkylglycerols, as discussed below.
1.3.1 Osmotic Disruption of the BBB (Hyperosmotic Shock)
Osmotic disruption of the BBB increases the brain delivery of water-soluble drugs. It occurs as a result of shrinkage of endothelial cells because of the hypertonic environment and the opening of the endothelial tight junctions that constitute the anatomical basis of the BBB.[79,80] Mannitol, a hyperosmolar agent that has been approved for use in patients, has been investigated in both preclinical and clinical trials. Mannitol is used mainly for the administration of anticancer agents, such as cyclophosphamide, procarbazine and methotrexate, in the treatment of brain tumours. However, the use of mannitol to improve drug uptake into the brain should proceed with caution, because Salahuddin et al.[77,82] reported that when hypertonic mannitol was infused into the carotid artery of rats, structural brain damage was observed within regions showing BBB disruption. Other risk factors associated with the use of mannitol include the passage of plasma proteins across the BBB, altered glucose uptake, expression of heat shock proteins and microembolism or abnormal neuronal function.
1.3.2 Biochemical Disruption of the BBB (Administration of Vasoactive Substances)
It has been reported that infusion of vasoactive amines such as histamine, bradykinin and the bradykinin analogue receptor-mediated permeabilizer (RMP)-7 selectively opens the blood-tumour barrier in experimental animals. Bradykinin increases tight junction permeability by activating bradykinin B2 receptors on endothelial cells. Matsukado et al. reported that RMP-7 increases drug delivery to tumours and the survival of glioma-bearing rats. Based on these findings, clinical trials were initiated using RMP-7 to enhance the brain delivery of antitumour agents.[85,86] In addition, RMP-7 was found to be more stable and more potent than bradykinin in mice. RMP-7 has been administered with carboplatin to patients with gliomas in a phase II clinical trial.[85,86] However, the trial was abandoned because of the potential for structural brain damage in areas in which the BBB had been disrupted.
It has been observed that the solvents, stabilizers or adjuvants present in some drug formulations can inadvertently disrupt the BBB. Solvents such as sodium laurilsulfate (sodium dodecyl sulfate), ethanol, dimethyl-sulfoxide, glycerol and polysorbate-80 are commonly used in drug formulations and are administered in doses that, in animals, have been shown to disrupt the BBB.[87, 88, 89, 90]
1.3.3 BBB Disruption by Alkylglycerols
A relatively new approach for the transient disruption of the BBB involves the systemic administration of various alkylglycerols.[91, 92, 93] Erdlenbruch et al. reported reversible and concentration-dependent increases in BBB permeability to several anticancer and antibacterial agents. In addition, the permeability of the BBB to methotrexate has been reported to increase from 2- to 200-fold using this approach. The degree of disruption of the BBB depends on the length of the alkyl group and the number of glycerols present in the structure. Recent studies[91, 92, 93] examining the effect of alkylglycerols on the permeability of isolated brain capillaries to large macromolecules suggest that the increase in permeability is due to temporary breakdown of tight junctions between cells. Although the exact mechanism responsible for the transient disruption in the BBB observed with alkylglycerols remains unknown, the fact that the response is concentration and structure dependent suggests an interaction with receptor sites within the brain microvasculature.
The use of alkylglycerols to increase the permeability of the BBB to anticancer agents has been examined in a rat glioma tumour model.[92,94,95] In one of these studies, several different alkylglycerols were examined for their ability to increase the CNS delivery of methotrexate in C6 glioma-bearing rats. The results indicate a significant increase in methotrexate accumulation at both tumour and nontumour sites within the brain. The effects of the alkylglycerols were concentration dependent. However, the magnitude of the increase in methotrexate delivery to the tumour site detected with the highest doses of alkylglycerols examined was comparable to that observed following osmotic disruption of the BBB, but was significantly greater than that observed with the bradykinin analogue RMP-7 (labradimil)[94,95]
1.4 Molecular Trojan Horses for Brain Drug Delivery
Drugs that cannot be modified by lipidization or are too large to diffuse across the BBB require other approaches to gain entry to the brain. When a vector that can access a specific catalysed transport mechanism is attached to an active moiety, it can ferry the drug, like a ‘Trojan horse’, across the BBB.
Pardridge reported that certain peptidomimetic monoclonal antibodies (mAbs), along with endogenous ligands, undergo RMT across the BBB via endogenous peptide receptor transporters. The receptor-specific mAb binds to an exofacial epitope on the receptor that is spatially removed from the binding site of the endogenous ligand, and this receptor binding allows the mAb to be ‘piggy backed’ across the BBB through the endogenous RMT system. When attached to a drug or nonviral plasmid DNA, the receptor-specific mAb assists the moiety to cross the BBB, thus acting as a molecular trojan horse. The human insulin receptor (HIR), the most potent molecular trojan horse in the BBB, is active in humans and rhesus monkeys. Brain drug delivery in the rhesus monkey was achieved by attaching a murine 83-14 mAb to the HIR.[96,97] A genetically engineered fusion protein for HIRs was prepared by fusing human brain-derived neurotrophic factor (BDNF) to the chimeric mAb and, after intravenous administration to a rhesus monkey, it was found that this fusion protein was transported into the brain. In mice, the rat 8D3 mAb to the mouse TfR is used to cross the BBB, whereas in rats the murine OX26 mAb to the rat TfR is used; the OX26 mAb is not active in mice or other species.
The complex formed by a trojan horse and a nontransportable drug is called a chimeric peptide because the molecule is bifunctional. When administered systemically, the trojan horse part of the chimeric complex binds to specific receptors (Rt1) on brain capillaries, which are part of the BBB, thus enabling transport of the drug across the BBB. Once the chimeric peptide has penetrated the brain, the drug part of the chimeric complex can bind to its receptor (Rt2) on brain cells to initiate its pharmacological effect (figure 5). However, when the drug is administered alone, it is not pharmacologically active because it cannot cross the BBB.
A definitive test to determine whether a given BBB delivery system is effective is to examine the CNS pharmacological effects of the drug attached to the delivery system in vivo following intravenous administration of low doses of the drug-delivery system complex. It is assumed that an effective delivery system will mediate drug transport across the BBB without disrupting it.[77,78,101] Large pharmaceutical molecules that can cross the BBB in vivo complexed with a molecular trojan horse to produce in vivo CNS pharmacological effects are listed in table III.
1.5 Drug Delivery Approach
1.5.1 Drug Delivery via Catheters and Pumps
The simplest method to overcome the BBB is to deliver drugs by direct infusion into the brain interstitium via a catheter. The Ommaya reservoir or implantable pump was developed for this purpose and has been used for many years to deliver various anticancer agents, such as methotrexate, doxorubicin, bleomycin and cisplatin, and biological agents such as interleukin (IL)-2 and interferon-γ, to brain tumours. The Ommaya reservoir may be used in several ways. Its primary function is to facilitate the uniform delivery of intrathecal chemotherapy. By implanting the Ommaya reservoir, multiple rounds of chemotherapy may be given through a single access site, thereby increasing patient comfort and reducing the stress and pain associated with repeated spinal injections. The Ommaya reservoir also serves as a sampling site for the removal of CSF. Samples are withdrawn and analysed for the presence of abnormal cells. Some physicians use the reservoir to deliver pain medication and, more recently, trials have been conducted to test the efficacy of the Ommaya reservoir in delivering gene therapy to cancer patients (i.e. treating a disease caused by a malfunctioning gene by the introduction of a normal gene back into the patient).[115, 116, 117, 118, 119, 120]
Recently, newer types of implantable pumps, such as the MiniMed PIMS pumps (MiniMed, Sylmor, CA, USA), Medtronic SynchroMed system (Medtronic, Minneapolis, MN, USA) and Infusaid pump (Infusaid, Norwood, MA, USA), have been developed to overcome the drawbacks associated with the Ommaya reservoir, i.e. slow rate of drug distribution within the CSF and increase in intracranial pressure associated with fluid injection or infusion into small ventricular volumes, resulting in high clinical incidence of haemorrhage, CSF leaks, neurotoxicity and CNS infections. These newer systems deliver drugs at a constant rate and for an extended period of time. These pumps have been demonstrated to be of use in experimental brain tumour models and are currently being used in many phase III brain tumour clinical trials.
Although these catheters and pumps appear to be a promising strategy for brain delivery, clinical studies of the delivery of glial cell line-derived neurotrophic factor (GDNF) using these catheters and pumps have not been successful. In an initial study in rhesus monkeys, Grondin et al. reported that the prolonged and controlled delivery of GDNF into the brain had beneficial effects in long-term neurodegenerative disease processes, such as Parkinson’s disease. However, the results of subsequent clinical trials were mixed because although two phase I trials suggested a beneficial effect of GDNF infused intraputaminally through implanted programmable catheters and pumps in patients with Parkinson’s disease,[124, 125, 126] another two multicentre trials reported no significant improvement or the development of major adverse events when patients were implanted with catheters/pumps or were given intracerebroventricular injections of GDNF, respectively. Thus, the drug was withdrawn. It has been suggested by Salvatore et al. that the failure of GDNF in these studies was due to inadequate bioavailability and brain targeting (i.e. GDNF was unable to reach the putamen and substantia nigra to exert its effects).
1.5.2 Drug Delivery from Microspheres
Recent attention has focused on the use of lipid-based polymeric devices, such as microspheres, for the delivery of anticancer agents. When a polymeric-based chemotherapeutic agent is conjugated with a water-soluble macromolecule, drug penetration into the brain is improved as a result of longer retention of the drug in brain tissue. Recently, IL-2-loaded microspheres have been developed for the local delivery of the cytokine in the treatment of brain tumours. The major advantage of the microparticulate system is that it can be implanted easily by stereotaxy in discrete, precise and functional areas of the brain without damaging surrounding tissue. This type of implantation is more convenient than the insertion of catheters and pumps via open surgery, and can be repeated if required.
Menei et al. developed a new method of drug delivery into the brain using implantable, biodegradable microspheres. A phase I study of the stereotaxic implantation of fluorouracil-releasing microspheres was performed in patients with malignant glioma. In this study, the authors demonstrated that the biodegradable microspheres could be implanted by stereotaxy and were efficient systems for drug delivery into brain tumours. This method also has potential applications in the treatment of patients with other malignancies.
1.5.3 Drug Delivery from Biodegradable Wafers
The development of biodegradable polymers is a major step in polymer technology and its clinical application. The polyanhydride poly[bis(p-carboxyphenoxy) propane : sebacic acid] (PCPP : SA) polymer is one example of a biodegradable polymer that releases drugs following polymer diffusion and degradation. Changing the ratio of carboxyphenoxy propane to sebacic acid changes the degradation properties of the polymer from days to years. PCPP-SA polymers can be fabricated into different shapes (e.g. rods, wafers or microspheres) and can be used to deliver various compounds, including hormones, neurotransmitters, enzymes and antineoplastics. Because of the biodegradable nature of the polymer, no foreign residue is left behind and the biodegradable products are not mutagenic, cytotoxic or teratogenic.
The Gliadel® (carmustine [BCNU]-PCPP : SA polymer) [MGI Pharma, Bloomington, MN, USA] wafer is the only US FDA-approved polymeric system for the local delivery of drugs to brain tumours. Using this system, an improvement in the survival of patients with glioblastoma multiforme brain tumours has been demonstrated. However, owing to insufficient diffusion of the therapeutic agent, it is likely to only reach adjacent sites.
1.5.4 Drug Delivery from Colloidal Drug Carrier Systems
Liposomes have been used extensively to improve drug delivery across the BBB.[138, 139, 140, 141, 142] Thermosensitive liposomes containing doxorubicin have been used for the treatment of malignant gliomas. Drug release was achieved when the tumour core was heated to 40°C by a brain heating system. Unfortunately, conventional liposomes are eliminated from the circulation by the RES. To overcome this problem, a water-soluble polymer-like PEG is attached to the surface of the liposomes and they are converted to stealth liposomes, which have been reported to have longer circulation in the body and reduced clearance by the RES system. Novel, highly stable nanoliposomes containing the anticancer drug irinotecan (CPT-11) were demonstrated to prolong tissue retention of the drug and enhance its antitumour effects in an intracranial U87 glioma xenograft model. In addition, large doses of glucocorticosteroids have been delivered using long-circulating PEG-liposomes for the treatment of multiple sclerosis. These types of liposomes have also been created containing doxorubicin and have been shown in clinical trials to be effective in the treatment of glioblastomas and metastatic tumours.[146, 147, 148] Table IV summarizes the application of liposomes in the treatment of CNS disorders.
Immunoreactive moieties can also be targeted to the BBB by attaching them to PEGylated liposomes. PEGylated liposomes conjugated with transferrin have been delivered successfully to the post-ischaemic cerebral endothelium in rats. PEGylated immunoliposomes conjugated with the mAb OX26 were used to target TfR. When encapsulated into liposomes, many compounds, including doxorubicin, digoxin, β-galactosidase gene, hydroxylase plasmid, biotinylated oligonuleotides, neutrophin peptides and striatal tyrosine hydroxylase, have been delivered successfully into the brain.[142,155, 156, 157, 158, 159] PEGylated (stealth) BBB-targeted immunoliposomes have been prepared by coupling with thiolated monoclonal anti-gliofibrillary acidic protein (GFAP) antibodies and used against human GFAP. Because of the problems these liposomes have penetrating an intact BBB, it has been suggested that they could be used to deliver drugs to glial brain tumours (which continue to express GFAP) or to other areas in the brain with a partially disrupted BBB (i.e. pathological loci in the brain).
Genetic material, when encapsulated into cationic liposomes, is protected from the extracellular environment and is thus transferred to target cells. There are many mono- or multivalent cationic lipids currently available for gene transfer, including 1,2-dioleoyl-3-trimethylammonium-propane or N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride. Matsuo et al. used haemagglutatinin virus of Japan liposomes with fusogenic activity to introduce oligodeoxynucleotides into mouse brain capillary endothelial C4 cells. Unfortunately, gene transfer into the brain via cationic liposomes requires an invasive route of administration.
By complexing liposomes with an antibody or a ligand that will be recognized by cell surface receptors in the targeted tissue, active targeting can be achieved. PEGylated liposomes can be targeted to the brain using mAbs, because they are able to attach to a receptor expressed on the BBB and to trigger RMT across the BBB. Thus, targeted mAbs can act as molecular trojan horses to transport liposomes across the biological barriers in the brain using endogenous transport systems.
The term nanoparticles refers to well defined particles ranging in size from approximately 10 to 1000 nm (1 µm) with a core shell structure (nanocapsules) or a continuous matrix structure (nanospheres). Similar to liposomes, the surface of nanoparticles can be modified by PEGylation to reduce their clearance by the RES system.[163,164] When administered intravenously, doxorubicin-loaded polysorbate 80-coated nanoparticles cured 40% of intracranially transplanted glioblastomas in rats. In another study, valproic acid-loaded nanoparticles were demonstrated to reduce the toxicity normally associated with this therapy, not only because of a reduction in the dose necessary, but also because of the inhibition of the formation of toxic metabolites.
Antineoplastic agents (doxorubicin), the NMDA receptor antagonist MRZ 2/576, peptides (dalargin and kytorphin) and analgesics (dalargin and loperamide) have all been delivered to the CNS following either their incorporation into or absorption onto the surface of poly(butyl)cyanoacrylate (PBCA) nanoparticles. Another chelator, namely penicillamine, has been formulated as a nanoparticle and investigated as a therapy for Alzheimer’s and other CNS diseases.
Costantino et al. synthesized nanoparticles by conjugating the biodegradable copolymer poly(D,L-lactide-co-glycolide) [PLGA] with five short peptides by means of an amidic linkage. These peptides, which are similar to synthetic opioid peptides, were synthesized in turn by means of 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis. The ability of these nanoparticles to cross the BBB was assessed in vivo using the rat brain perfusion technique and, in one case, after systemic administration (injection into the rat femoral vein). Fluorescent and confocal microscopy studies showed that although PLGA nanoparticles were unable to cross the BBB, for the first time these solid nanoparticles, surface modified with peptides, were shown to cross the BBB.
Similarly, Aktas et al., designed nanoparticles of the peptide Z-DEVD-FMK, which is a specific caspase inhibitor that reduces vulnerability to neuronal cell death after cerebral ischaemia. Nanoparticles were prepared by conjugating poliglusam (chitosan) with PEG. This PEG was conjugated with the OX26 mAb, thus finally giving a chitosan-PEG-OX26 nanoparticle loaded with the Z-DEVD-FMK peptide. After fluorescent labelling, these nanoparticles were administered intravenously to mice. Electron microscopy revealed that the nanoparticles were able to reach brain areas outside the intravascular compartment. Consequently, these novel nanoparticles appear to be promising carriers for the transport of the anticaspase peptide Z-DEVD-FMK into the brain. In another study, Tosi et al. prepared nanoparticles of the polyester PLGA conjugated with the glycosylated heptapeptide H2N-Gly-L-Phe-D-Thr-Gly-L-Phe-L-Leu-L-Ser(O-β-D-Glucose)-CONH2 loaded with loperamide. Following intravenous administration, it was found that the nanoparticles were able to cross the BBB and deliver loperamide into the brain.
A recent investigation has indicated that the delivery of doxorubicin to the brain by means of PBCA nanoparticles coated with poloxamer-188 and polysorbate-80 may be augmented by an interaction between apolipoprotein A-I adsorbed on the surface of nanoparticles in plasma and the scavenger receptor SR-BI located at the BBB. This is the first study showing a significant correlation between the adsorption of apolipoprotein A-I on the nanoparticle surface and drug delivery across the BBB.
As mentioned in section 1.2.3, SLN are a potential drug delivery system for brain targeting, and modification of the surface properties of these SLN can decrease their uptake by the RES.[172, 173, 174] When camptothecin and doxorubicin were loaded into SLN, drug accumulation was observed in the brain after both oral and intravenous administration.[175,176] However, when the surface characteristics of SLN were modified by means of PEG derivatives or PEG-containing surfactants, better results were achieved in terms of brain targeting.[175,177, 178, 179] Two new SLN formulations made with biocompatible materials, namely emulsifying wax and Brij® 72, stabilized by polysorbate-80 and Brij® 78 (Sigma-Aldrich Corporation, St Louis, MO, USA), showed significant brain uptake, as measured during a short-term in situ rat brain perfusion experiment.[180, 181, 182] SLN loaded with superparamagnetic iron oxide is a new type of nuclear magnetic resonance contrast agent, which is able to cross the BBB and accumulate in the brain parenchyma after intravenous injection in rats.
Yang et al. studied the in vivo transport of carboxylated polystyrene nanospheres (20 nm) across the BBB following cerebral ischaemia and reperfusion. A transient increase in fluorescence intensity induced by cerebral ischaemia and reperfusion was observed, indicating accumulation of the nanospheres in the brain as a result of the opening of tight junctions.
Yumi et al. developed bionanocapsules selectively targeted to brain tumours. Epidermal growth factor receptor (EGFR), specifically a constitutively active genomic sequence deletion variant of the EGFR (EGFRvIII), is overexpressed in human glioblastoma. In their study, Yumi et al. replaced the pre-S1 peptide with the antibody affinity motif of protein A and made hybrid bionanocapsules conjugated with anti-human EGFR antibody recognizing EGFRvIII. The hybrid bionanocapsules were efficiently delivered to glioma cells, but not normal glial cells. Moreover, delivery of the hybrid bionanocapsules to brain tumours in an in vivo brain tumour model suggests that this approach using bionanocapsules has considerable promise in brain tumour-targeted drug delivery.
Vinogradov et al.[186, 187, 188, 189] have developed nanogels as a new class of carrier systems for the delivery of drugs and biomacromolecules to the CNS. Nanogels are prepared from a network of cross-linked ionic polyethyleneimine and non-ionic PEG chains (PEG-cl-polyethyleneimine). The effect of nanogels on the receptor-mediated delivery of oligonucleotides across polarized monolayers of bovine brain microvessel endothelial cells has been investigated. Following the incorporation of the oligonucleotides into nanogels, there was an increase in their transport across the cell monolayers. Kabanov and Batrakova reported a further increase in oligonucleotide transport following modification of nanogel carriers by insulin or transferrin ligands.
1.5.5 Drug Delivery from Biological Tissues
Drug delivery from biological tissues involves implantation of a tissue into the brain that secretes the desired therapeutic substance. This approach has been used extensively for the treatment of Parkinson’s disease. Owing to the lack of neurovascular innervation, the chances of the transplanted tissue surviving are limited. In attempting to overcome this problem, Leigh et al. demonstrated enhanced vascularization and microvascular permeability in cell suspension embryonic neural grafts compared with solid grafts. Gene therapy has been used as an alternative method to develop an optimized biological tissue for interstitial drug delivery. In this technique, cells are genetically modified prior to implantation to release the desired therapeutic agent.
Recently, the therapeutic delivery of proteins has been reviewed using non-neural cells. The duration of survival of the biological tissue can be improved by co-grafting the cells engineered to release neurotropic factors with cells engineered to release therapeutic proteins.
1.5.6 Drug Delivery from Microchips
Microchips are a promising and novel method to deliver either single or multiple chemical substances into the brain. This type of drug delivery uses a solid-state silicon microchip from which controlled drug release can be obtained. The mechanism of drug release depends on the electrochemical dissolution of a thin anode membrane covering multiple microreservoirs filled with solids, liquids or gels. By selecting a biocompatible device, a microchip can be used to deliver up to 1000 different drugs.
1.6 Other Alternative Routes/Methods
1.6.1 Intranasal Drug Delivery to the Brain
When administered intranasally, a significant amount of drug can reach the CSF and olfactory bulb via olfactory sensory neurons. Theoretically, this approach could be useful for the delivery of therapeutic proteins, such as the delivery of BDNF to the olfactory bulb for the treatment of Alzheimer’s disease. After intranasal administration, it is believed that drug delivery to the CNS is either through intra- or extraneuronal routes.[199,200] Thorne et al. investigated the CNS drug delivery of IGF-I, a 7.65 kDa neurotrophic factor, following its intranasal administration and the possible pathways and mechanisms underlying its transport from the nasal passage to the CNS. The results of that study suggested that intranasally delivered IGF-I bypasses the BBB via olfactory and trigeminal-associated extracellular pathways to rapidly elicit biological effects at multiple sites within the brain and spinal cord.
Recent studies in animal models, as well as in humans,[203, 204, 205, 206, 207] revealed that drug uptake in the CSF and brain depends on the molecular weight and lipophilicity of the drug molecule. Figure 6 shows the route by which intranasally administered drugs reach the CSF and brain tissues. In a recent study, after nasal administration into the right nostril of mice, [3H]-dopamine was found in the right olfactory bulb and, after 4 hours, its concentration in the right olfactory bulb was 27-fold greater than that in the left bulb; the concentration of [3H]-dopamine reached in the right olfactory bulb was higher than that following its intravenous administration and subsequent uptake into the brain. A similar study was performed using radiolabelled morphine, which found that morphine was transferred along the olfactory pathway to the CNS. Some of the latest studies[204,205,210] performed in humans have revealed that insulin, apomorphine and melatonin/hydroxocobalamin are delivered to the brain after intranasal administration.
Xiaomei et al. investigated whether direct nose-to-brain transport of estradiol exists by measuring the uptake of estradiol into the CSF after its intranasal and intravenous administration to rats. These authors found that estradiol is transported into the CSF via olfactory neurons. Similarly, the effectiveness of the intranasal administration of ergoloid mesylate, which is subject to first-pass metabolism after oral administration, for intrabrain delivery was investigated. The results of that study were promising. Gao et al.[213, 214, 215] demonstrated enhanced intrabrain delivery after the intranasal administration of vasoactive intestinal peptide in PEG-poly (lactic acid) nanoparticles modified with wheat germ agglutinin and Ulex europaeus agglutinin-I. There are still some difficulties that need to be overcome with this technique, such as the possibility of mucosal irritation, the enzymatically active and low-pH nasal epithelium and the possibility of considerable variability in results because of nasal pathology (e.g. the common cold). However, despite these inherent difficulties, direct nose-to-brain transport of three neuropeptides and direct access of these neuropeptides to the CSF, bypassing the bloodstream, has recently been observed in human trials. One obvious advantage of this technique is that it is non-invasive.
1.6.2 Convection-Enhanced Diffusion
In convection-enhanced diffusion, drug distribution takes place via bulk flow. A catheter is implanted in the brain and is connected to a pump that drives fluid flow to the brain at a prescribed rate of infusion. This type of delivery system is being investigated in clinical trials of high-grade gliomas.[218, 219, 220, 221] One of the trials uses IL-13 PE38QQR, a recombinant toxin composed of the enzymatically active portion of Pseudomonas Exotoxin A (Neopharm Ltd, Petach-Tiqva, Israel) conjugated with human IL-13. IL-13 PE38QQR binds to the IL-13 receptor, which is overexpressed in malignant gliomas. By binding to the IL-13 receptor, the recombinant toxin is able to gain entry to the glioma cells; however, at nanomolar concentrations, selective and potent cytotoxicity may develop.[218, 219, 220, 221] Another phase I/II study investigated IL-13 PE38QQR as an antitumour agent for the treatment of patients with recurrent malignant gliomas. These trials reported safe local delivery of IL-13 PE38QQR with an increased survival rate of patients (70.3 weeks) compared with those patients treated using an optimal catheter (41.4 weeks). The median survival for this same patient population on the basis of a report of clinical trials with other treatments is only 20–26 weeks. Another phase III clinical trial[223,224] investigated the convection-enhanced diffusion of the targeted toxin TransMID™ (Xenova Group Ltd, Berkshire, United Kingdom). TransMID™ is a conjugate of human transferrin and a diphtheria toxin containing a point mutation (CRM107). To achieve tumour specificity, the transferrin portion of the conjugate binds to TfR present on metabolically active cells and, after binding, the CRM107 portion acts intracellularly to exert its cytotoxic effects.[223,224]
1.6.3 Intrathecal/Intraventricular Drug Delivery to the Brain
An intrathecal/intraventricular approach delivers a drug into the CSF, bypassing the brain-CSF barrier. Because molecules can be exchanged freely between the CSF and extracellular fluids of the brain parenchyma, delivering drugs to CSF could theoretically result in increased therapeutic drug concentrations. Drug diffusion through the brain parenchyma is very slow and inversely proportional to the molecular weight of the drug. Following intra-CSF administration, the concentration of macromolecules (e.g. proteins) in the brain parenchyma was found to be negligible.[137,226] For this reason, treatment of intraparenchymal CNS tumours with chemotherapy administered into the CSF has not been proven effective. However, using the intrathecal route of administration for drug delivery to the brain has proven successful. For example, intrathecal injection of baclofen has been used to treat spasticity, opioids have been infused intrathecally for the treatment of severe chronic pain and glycopeptide and aminoglycoside antibacterials have been administered intracerebroventricularly for the treatment of meningitis and the intraventricular treatment of meningeal metastasis. In all cases, the intrathecal/intraventricular approach has delivered the drugs near to ventricular surfaces.[230,231]
The delivery of drugs to the CNS has been a challenging area of research for several decades. Considerable efforts have been made to develop various CNS drug delivery systems, revealing the unlimited potential of these methods. The investigation of a variety of strategies reflects the inherent difficulty in transporting therapeutic and imaging agents across the BBB. The lipidization of molecules has shown considerable potential for the transfer of drugs across the BBB; however, more studies are required before the advantages of this approach are fully defined. The use of endogenous transporters also has considerable potential in transporting of a variety of molecules across the BBB. Various drug delivery strategies using particulate carriers have been explored, with some currently under clinical trial and others already available on the market. Substantial progress will be achieved only if continuous vigorous research efforts to develop less toxic and more therapeutic drug molecules are paralleled with more effective mechanisms for delivering these drugs to their CNS targets.
No sources of funding were used to assist in the preparation of this review. The authors have no conflicts of interest that are directly relevant to the content of this review.
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