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ligands do not penetrate the blood-brain barrier, while unwanted side effects ... the carrier systems and the adsorptive and receptor-mediated transports ... efflux transporters at the luminal membrane of brain endothelial cells limits the brain ...... 9-mer synthetic peptide analogue of bradykinin, increases intracellular Ca2+ and.
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Solubility, Delivery and ADME Problems of Drugs and Drug Candidates, 2011, 144-165

CHAPTER 8 Drug Transport and the Blood-Brain Barrier Mária A. Deli Biological Research Center, Hungarian Academy of Sciences; E-mail: [email protected] Abstract: The blood-brain barrier, a dynamic interface separating the brain from systemic circulation, is the major entry route for therapeutic compounds to the central nervous system. The blood-brain barrier phenotype of the endothelial cells of brain microvessels includes tight interendothelial junctions, the lack of pinocytosis and fenestrae, transendothelial transport pathways, and a metabolic barrier. The primary role of the blood-brain barrier is to create ionic homeostasis for neuronal functions, but it also provides the central nervous system with nutrients and protects it from toxic insults. The formation and maintenance of these organ-specific characteristics are based on cross talk between the cells of the neurovascular unit, such as brain endothelial cells, pericytes, astroglia, microglia and neurons. The problem of drug transport at the blood-brain barrier is two-fold: the great majority of neuropharmaceutical candidates, hydrophilic molecules, biopharmaceuticals and efflux transporter ligands do not penetrate the blood-brain barrier, while unwanted side effects develop if a drug with main peripheral action crosses the blood-brain barrier. Overcoming the major mechanisms restricting drug transport at the level of the blood-brain barrier, tight interendothelial junctions, efflux transporters and the enzymatic barrier can lead to better drug penetration to the brain. In addition, there are several physiological transport pathways – the carrier systems and the adsorptive and receptor-mediated transports – which can be exploited for drug targeting. Strategies for drug delivery and targeting to the brain include modification of the molecules, modification of the blood-brain barrier functions, and circumvention of the blood-brain barrier. Some of the techniques based on these strategies are already in clinical use, while others are promising new possibilities for improving the therapy of central nervous system diseases.

OVERVIEW OF THE STRUCTURE AND FUNCTIONS OF THE BLOOD-BRAIN BARRIER The blood-brain barrier (BBB), a dynamic interface separating the brain from systemic circulation, is the major entry route for therapeutic compounds to the central nervous system (CNS). The estimated total length of human brain capillaries is 650 km, with a total surface area of about 20 m2 [1, 2]. The complex tight junctions (TJs) between brain endothelial cells constitute the morphological basis of the BBB [3]. The primary role of the BBB is to create ionic homeostasis for neuronal functions [4]. It also provides the CNS with nutrients and protects it from toxic insults by sophisticated transport systems [5]. The low level of paracellular flux and transendothelial vesicular trafficking results in a transport barrier for drugs that are hydrophilic and have a molecular mass larger than 400 Da, while the presence of effective efflux transporters at the luminal membrane of brain endothelial cells limits the brain penetration of lipophilic xenobiotics and drugs. The BBB prevents 98% of potential neuropharmaceuticals, especially new biopharmacons, nucleic acids, peptide or protein drugs, from reaching their targets in the CNS [1]. For these reasons, the treatment of CNS diseases, including strokes, Alzheimer’s disease and brain tumours, remains unsatisfactory, and improving drug delivery to the CNS is considered essential for the future success of therapies for neurological disorders [6]. THE ANATOMICAL BASIS OF THE BLOOD-BRAIN BARRIER It was 125 years ago that Paul Ehrlich and his co-workers discovered, using dye studies, that the mammalian CNS has a unique compartment within the body. In the 1960s, the interendothelial junctions of cerebral capillaries were identified through electron microscopy as the anatomical basis of the vertebrate BBB [7,8]. Brain endothelial cells are characterized by their thin cytoplasm, their complex TJs, the lack of fenestrae, the small number of vesicles and the large number of mitochondria [9]. The freeze-fracture morphology of BBB TJs is unique: in addition to the correlation between the number of TJ strands and the tightness of the junctions, as measured by electrical resistance, the association of the particles with the inner (P-face) is as high as the association with the outer (E-face) lipidic leaflet of the plasma membrane. This high P-face/E-face ratio also reflects the quality of the barrier [9]. K. Tihanyi and M. Vastag (Eds) All rights reserved - © 2011 Bentham Science Publishers Ltd.

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Brain capillary endothelial cells have a dynamic interaction with their neighbouring cells, astroglia, pericytes, perivascular microglia and neurons. This cooperation contributes to their unique characteristics, displaying both endothelial and epithelial features [2, 5, 10, 11]. The cross talk between the cells of the neurovascular unit is crucial for the formation and maintenance of a functional BBB [5, 12] (Fig. 1).

Figure 1: The anatomical structure of the blood-brain barrier.

Astrocytes were the first to be recognized as regulators of brain endothelial characteristics and functions [4, 5, 13]. The astrocytic endfeet covering the surface of the brain capillaries allow both cross talk between the two cell types and transport between blood and the neural tissue [4]. Brain pericytes, the nearest neighbours of endothelial cells sharing a common basal membrane in cerebral capillaries, have a fundamental role in stabilizing the brain capillary structure in vivo [14, 15]. They also participate in the development, maintenance, and regulation of the BBB [12, 15]. Pericytes are also able to tighten the paracellular barrier in cultured brain endothelial cells and to increase their BBB properties in a way similar to that of to astrocytes [16, 17]. BLOOD-BRAIN PATHWAYS

BARRIER

PHYSIOLOGY:

SPECIFIC

CHARACTERISTICS,

TRANSPORT

The BBB phenotype of brain endothelial cells includes (i) TJs and the lack of pinocytosis and fenestrae, (ii) transendothelial transport pathways, and (iii) metabolic and detoxifying functions as listed in Table 1 [5, 6]. An additional line of defence is the highly negatively charged glycocalyx at the luminal surface of brain endothelial cells. Eleven percent of the genome of brain capillaries codes for transporters, underscoring their importance [18]. These transporters and carriers provide the CNS with nutrients, vitamins, minerals and metabolic precursors. The efflux transporters participate in the protection of the brain from potentially toxic molecules and xenobiotics, and in the regulation of the level of neurotransmitters and metabolites in the brain. Table 1: Major Elements of the Blood-Brain Barrier Phenotype Elements

Role

Anatomical barrier

Tight interendothelial junctions Absence of fenestrations and low number of pinocytotic vesicles Luminal glycocalyx

Restricting free exchange of solutes and cells between blood and the CNS

Transport pathways

Diffusion pathways Solute carriers Efflux pumps Adsorptive and receptor-mediated transendothelial transport

Supply of sugars, amino acids, lipids, vitamins, minerals, metabolic precursors, peptides, proteins Protection form xenobiotics Regulation of metabolites

Metabolic barrier

Phase I and II enzymes

Protection from bioactive molecules

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Tight Interendothelial Junctions TJs sealing the paracellular clefts between brain endothelial cells are elaborate structures composed of integral membrane proteins, linker or adaptor proteins connecting them to the actin cytoskeleton, and signalling molecules enabling the dynamic regulation of the paracellular transport [19, 20]. In brain endothelial cells, tight and adherens junctions are intermingled [21]. Some of the junctional proteins are found in raft-like membrane microdomains [22]. These compartments can play an important role in the spatial organization of TJs, and probably in the regulation of paracellular permeability. The transmembrane TJ proteins identified in brain endothelial cells can be classified into several protein families. Occludin, the first integral membrane TJ protein described [23], and tricellulin [24] share homology. At least 15 members of the claudin family, the junctional adhesion molecules (JAM 1-3) and the endothelial-specific adhesion molecule (ESAM) are expressed in brain capillaries [25]. However, the expression and the paracellular gate function of these proteins can be very different. In the endothelial fraction of isolated brain capillaries, the mRNA expression of claudin-5 was 50-500 times higher than that of other claudins [25]. Functional experiments also support the role of claudin-5 in the gate function of the BBB. In claudin-5 knock-out mice, BBB permeability is selectively increased for markers with a molecular mass below 800 Dalton [26]. The next two highest mRNA levels in the microvessel fraction were measured for occludin and ESAM. Significant levels of mRNA were measured in the case of claudin10, -22, -23, -19, -17, -3, -8, -20, -12, and -15, although the exact role of these proteins in the barrier function of the BBB has not yet been elucidated. It is important to note that brain endothelial cells do not express claudin-4, -6, -7, 13, -14, -16, -18 [25], found in epithelial cells, indicating a different composition and regulation of the different barriers of the body [27]. TJs can be targeted by specific approaches to reversibly open barriers and improve drug delivery [27]. Transport Pathways As shown on (Fig. 2), six transport pathways can be distinguished at the level of the BBB.

Figure 2: Transport pathways at the BBB. 1, lipid-mediated diffusion; 2, paracellular diffusion; 3, carriers; 4, active efflux transport; 5, receptor-mediated transport; 6, adsorptive-mediated transcytosis. (Modified from [5])

1. Passive Transcellular Pathway for Lipophilic Molecules Lipophilic small molecules (MW less than 400-500) can enter the brain by lipid-mediated free diffusion [1, 28]. Drugs of abuse discovered by mankind, e.g. alcohol, caffeine, nicotine, heroin, etc., as well as all small molecule neurotherapeutics fall into this category. However, high lipid solubility increases uptake by peripheral tissues as well as sequestration in the brain microvasculature, both of which lead to decreased drug delivery to the brain [28]. 2. Passive Paracellular Pathway for Hydrophilic Molecules The restricted paracellular pathway by the tight interendothelial junctions of cerebral microvessels prevents hydrophilic molecules from entering the CNS freely. This is the reason why most proteins, peptides and polysaccharides cross the BBB poorly [1]. Drug candidates developed for CNS diseases are often hydrophilic, resulting in low BBB penetration.

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3. Carrier-Mediated Transport Saturable, bi-directional transport systems exist for nutrients, vitamins and minerals at the BBB [1, 29]. About 40 members of the solute carrier (SLC) transporter family were identified in brain microvessels by serial analysis of gene expression [18]. Transport proteins for glucose, the primary energy source for the CNS, GLUT-1 (SLC2A1) and GLUT-3 (SLC2A3) glucose transporters, and the sodium glucose co-transporter SGLT2 (SLC5A2) are of the utmost importance. GLUT-1 deficiency syndrome, a rare genetic disease, is characterized by infantile seizures, developmental delay, acquired microcephaly, spasticity, ataxia and hypoglycorrhachia, the low level of glucose in the cerebrospinal fluid [30]. In the absence of glucose, the transporters of monocarboxylic acids MCT-1, -2, and -6 (SLC16A1, 2, 6) are expressed in brain endothelial cells, and monocarboxylic acids or ketons like pyruvate and lactate are used as secondary energy sources, explaining the efficiency of a ketogenic diet for these patients. Amino acids are transported by carriers that also belong to the SLC family, like the cationic amino acid transporter CAT-1 (SLC7A1), the large neutral amino acid transporter LAT-1 (SLC7A5), system A amino acid transporters 1 and 2 SAT1 (SLC38A1), SAT2 (SLC38A2), and system N amino acid transporter SN1 (SLC38A3). Carriers at the BBB provide choline (SLC6A8), long chain fatty acids (FATP-1 or SLC27A1), and vitamins (SMVT or SLC5A6) to the brain. These transporters can be exploited for drug targeting. A good example is L-Dopa, used for the therapy of Parkinson’s disease, which crosses the BBB with LAT-1. Importantly, the uptake rate across the BBB for the endogenous ligand of a transporter is about 10 times higher than for the same molecule crossing by transmembrane diffusion [31]. Neuroactive peptides, like enkephalins and arginine-vasopressin, are transported by peptide transport systems (PTS-1-4) between the blood and the brain working in efflux or influx mode, or bi-directionally [32]. 4. Active Efflux Transport A growing number of active efflux transporters are being discovered at the BBB [18, 33, 34]. Several members of the ATP binding cassette or ABC transporter family, like P-glycoprotein (ABCB1), multidrug resistance proteins MRP-1, -4, -5 and -6 (ABCC1-6), and brain multidrug resistance proteins (BMDP/BCRP/ABCG2) have also been identified in brain capillaries. The specific expression of MRP-12 has also been demonstrated at the BBB [35]. Efflux transport plays a role in the regulation of the brain level of the excitatory neurotransmitter glutamate. Excitatory amino acid transporters EAAT 1-4 (SLC1A1-3, 6) described on brain endothelial cells [36], help to maintain the 2-3 order of magnitude difference between the concentration of glutamate in blood and the CNS, with a predominant efflux toward blood [37]. Organic anion-transporting polypeptides, such as OATP-1 and -2 (SLCO1C1, 2), belong to the SLC carrier family and participate in efflux mechanisms at the BBB. Efflux transporters are responsible for restricting drug penetration to the brain, resulting in inefficient treatment of CNS diseases such as stroke, neuroAIDS, brain tumours and neurodegenerative disorders [38]. An increasing amount of data is available on drug resistant epilepsy and the upregulation of efflux transporters, mainly P-glycoprotein [38]. 5. Receptor-Mediated Transcytosis Receptor-mediated transport is responsible for the brain penetration of peptides and proteins, as well as for their clearance [1, 39]. Transferrin, melanotransferrin, insulin, leptin, ghrelin, low density lipoprotein and many other important regulatory proteins are delivered through this mechanism [12]. It can be bidirectional, operating in both the blood-to-brain and brain-to-blood directions, such as for transferrin. An example for unidirectional transport is the BBB Fc receptor, which selectively transports IgG from the brain to the blood [1]. Receptor-mediated transcytosis has three major steps: receptor-mediated endocytosis at the luminal membrane of brain endothelial cells, transcytosis in vesicles through the very narrow cytoplasm of the cells, and exocytosis at the abluminal side of endothelium [1]. Caveolae, smooth plasmalemmal vesicles, also control transcellular permeability by regulating endocytosis, transcytosis and signalling in lipid-based microdomains of the BBB [12]. The caveolar membranes contain receptors for transferrin, insulin, ceruloplasmin, and low and high density lipoproteins [9]. The transcytotic pathway allows the specific targeting of large molecules, e.g. biopharmaceuticals or nanoparticles, to the CNS [40] (see Table 4). 6. Adsorptive-Mediated Transcytosis In physiological conditions, the restricted paracellular pathway, together with the lack of endothelial fenestrations and the reduced rate of pinocytosis, prevents the unregulated leakage of serum proteins into the CNS, which would be toxic to neurons [28]. Adsorptive-mediated transcytosis, which is present in peripheral endothelial cells and

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transfers albumin, is highly downregulated at the BBB, but is reactivated in pathologies such as seizures and brain oedema and by pathological mediators such as histamine [10]. Enzymatic Barrier The endothelial cells of the BBB provide a metabolic barrier by expressing a number of enzymes that modify endogenous and exogenous molecules that could bypass the barrier and negatively affect neuronal functions. Specific phase I and phase II enzymes participate in the formation of this metabolic barrier, as well as in transport processes [12, 41]. Peptide transport across the BBB is also restricted by active metabolization by amino- and carboxypeptidases in brain endothelial cells [2]. Phase I enzymes at the BBB also include aldehyde and alcohol dehydrogenases, alkaline phosphatase, aminotransferases, aromatic L-amino decarboxylases, butyrylcholinesterase, catechol-O-methyl transferase, epoxy hydrolases, γ-glutamyl transpeptidase, ketone and alcohol oxidoreductases, monoamine oxidases A and B and transaminases. The best known example of the metabolic BBB is the exchange of dopamine between the blood and the brain. Dopamine cannot be taken up by brain endothelial cells and cannot reach the CNS. Its precursor, L-DOPA, uses the LAT-1 amino acid carrier to enter both the endothelial cells and the brain, where it is converted enzymatically to dopamine by DOPA-decarboxylase [42]. Recently, the phase I cytochrome P450 (CYP) enzyme profile has been described in isolated human brain capillaries and in an immortalized brain endothelial cell line [35, 43]. The presence of CYP1A1, 1B1, 2B6, 2D6, 2E1, 2J2, 2R1, 2S1 and 2U1 was confirmed in both models. The expression of Cyp1a2, 2b, 3a23, 7a1 and 27a1 was demonstrated in rat brain capillaries [44]. From phase II enzymes glutathione S-transferase-π (GSTπ), GSTα, Sult1a1, Sult1a2, and UDP-glucuronosyl-transferase Ugt1a1 are expressed at the BBB [44, 45]. GSTπ is coexpressed at the endothelial luminal membrane with the MRP-2 efflux pump, and a coordinated increase in the expression of both proteins could be achieved through the activation of the nuclear receptor PXR [44]. The CYP enzymes that have been discovered at the BBB so far participate primarily in the metabolism of endogenous molecules, while the major drug-metabolizing hepatic enzymes CYP3A4, CYP2C9 and CYP2D6 were absent from human brain capillaries. The metabolic enzymes, together with the efflux pumps, provide an important line of defence for the CNS. On the one hand, they metabolize xenobiotics to substrates of MRP pumps, while on the other hand they increase their efflux toward the blood by converting toxic endogenous CNS metabolites. STRATEGIES FOR TARGETING DRUGS TO THE CENTRAL NERVOUS SYSTEM The BBB is the main regulator of drug transport to the CNS by a strictly controlled and dynamic process via the specific transport pathways described above (Fig. 2). From a clinical perspective, the problem is twofold: an overwhelming proportion of potential neuropharmaceuticals do not penetrate the BBB [1], while unwanted CNS side effects develop if a drug with main peripheral action crosses the BBB [46]. While lipid soluble small molecules can enter the brain with lipid-mediated free diffusion, hydrophilic molecules, including large biopharmaceuticals or ligands of efflux transporters – the majority of medicaments – do not cross the BBB. The permeability of the BBB for a given molecule depends not only on its physico-chemical properties, but also on the presence or absence of influx and/or efflux transporters, receptors and drug metabolism. As discussed in the previous section, the major mechanisms at the level of BBB for controlling drug transport are interendothelial tight junctions, efflux transporters and the enzymatic barrier summarized in Table 2. Table 2: Major Mechanisms at the Level of the Blood-Brain Barrier to Restrict Drug Transport BBB feature

Limited pathway

Restricted transport of molecules

Strategies to overcome

Tight junctions

Paracellular diffusion

Hydrophilic Biopharmaceuticals

Tight junction modulators Absorption enhancers

Efflux transporters

Lipid-mediated diffusion

Lipophilic compounds Xenobiotics

Efflux transporter blockers

Enzymes

Transendothelial transport

Peptides Neurotransmitters

Prodrugs

compounds

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Overcoming the mechanisms listed in Table 2 can lead to better drug penetration to the brain. On the other hand, there are several physiological pathways, especially the carrier systems of the BBB listed in Table 3, which can be exploited for drug targeting across the BBB. Table 3: Physiological Pathways at the Blood-Brain Barrier to be Exploited for Drug Transport Pathway

Transport of molecules

Strategies to exploit the pathway

Lipid-mediated diffusion

Lipophilic small molecules

Increasing lipophilicity

Carrier-mediated transport

Ligands of carriers: amino glucose, and other nutrients

Receptor-mediated transport

Peptides, proteins

Drugs or nanoparticles conjugated to peptide or protein vectors (“molecular Trojan horses”)

Adsorptive-mediated transport

Serum proteins

Drugs or nanoparticles conjugated to cationic protein or peptide vectors

acids,

Development of ligand analogues Conjugation of drugs to ligands Ligandtargeted nanoparticles

The strategies for targeting drugs to the CNS can be classified into three major groups according to whether they are directed to the drug molecules, the BBB itself or to an alternative pathway. In the following sections, these major groups of strategies, namely (i) modification of the molecules, (ii) modification of the BBB functions and (iii) circumvention of the BBB will be discussed. Strategies for Modifying Molecules To estimate the solubility and permeability of a given drug molecule based on its characteristics, Lipinski’s rule of five [47] is often used, which postulates that molecules having not more than 5 hydrogen bond donors (OH and NH groups), not more than 10 hydrogen bond acceptors (N and O), a molecular weight under 500 and an octanol/water partition coefficient log P under 5 are eligible drug-like candidates. Even taking into account the fact that Lipinski’s rule of five does not apply to substrates for biological transporters, medicinal chemistry uses several methods to enhance the brain delivery of molecules by changing their physico-chemical properties, including techniques for increasing the lipid solubility or the cationic charge of molecules. More specific targeting can be achieved through exploiting the physiological transport pathways of the BBB, the carrier mediated transport, or the adsorptive- and receptor-mediated transcytotic systems (Table 4). Table 4: Examples for Molecule Modifications to Enhance Delivery of Drugs Across the Blood-Brain Barrier Method

Transport pathway

Drugs / molecules

Stage of development

Reference

INCREASED CATIONIC CHARGE AND TARGETING VIA ADSORPTIVE-MEDIATED TRANSCYTOSIS Cationization of the molecule

Cationic ferritin, albumin, immunglobulin

Animal experiments

[1,48-52]

Cationic cell penetrating peptide vector (SynB)

Vectorized paclitaxel (SYN 2001)

Phase I trial (glioma)

[53]

Dalargin, doxorubicin, morphin-6-glucuronide

Animal experiments

[54-57]

carboxy-fluorescein, rhodamine-dipalmitoylphosphatidylethanolamine

In vitro experiments

[58]

6-coumarin

Animal experiments

[59,60]

Phospholipid linked valproic acid (DP-VPA)

Phase II trial (epilepsy)

[61]

Cationic albumin coupled liposome

Adsorptive-mediated transcytosis

Cationic albumin coupled nanoparticle INCREASED LIPOPHILICITY Lipophilic analogues

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Estradiol-chemical delivery system (Estredox)

Phase II trial (postmenopausal symptoms)

[62]

Thyrotropin-releasing hormone prodrug

Animal experiments

[63]

Non-steroidal antiinflammatory prodrugs

In silico, in vitro experiments

[64]

Liposomes

enkephalins

Animal experiments

[65]

Lipid nanoparticle

superparamagnetic iron oxide

Animal experiment

[66]

L-Dopa,

melphalan, L-4chlorokinurenine, gabapentin, baclofen

Clinical use

[67]

Glycosylated opiates

Animal experiments

[68]

Vasoactive intestinal peptide in glucose-targeted niosomes

Animal experiment

[69]

Glucose-targeted PEGylated paramagnetic niosome

Animal experiment

[70]

doxorubicin glutathionePEG liposomes

Preclinical development (brain tumour)

[71]

Fusion proteins and immunoliposomes targeting mouse transferrin receptors

Animal experiments

[72]

DNA loaded pegylated immunoliposome targeting human insulin receptor

Animal experiments

[73]

IgG fusion proteins with neurotrophic factors, decoy receptors, therapeutic enzymes, single chain Fv antibodies

Animal experiments

[74]

Paclitaxel-, doxorubicin-, etoposide-AngioPep conjugates

Phase I (brain tumour), Preclinical

[75-77]

Receptor-associated peptide (RAP) and protein drug conjugates

Preclinical development

[78, 79]

Paclitaxel, adriamycin melanotransferrin (p97) conjugates

Preclinical development

[80]

LDL receptor

Loperamide, dalargin loaded ApoE-linked nanoparticles

Animal experiment

[81, 82]

Diphtheria toxin /HBEGF receptor

Diphteria toxin (CRM197)peroxidase conjugate

Animal experiment

[40]

Lipid-mediated free diffusion

TARGETED TRANSPORT THROUGH BBB CARRIERS LAT1

GLUT1 Carrier-mediated transport

Glutathione transporter

TARGETED TRANSPORT THROUGH BBB RECEPTORS Transferrin receptor Receptor-mediated transport Insulin receptor

LRP1 and LRP2 receptor

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1. Targeting by Cationization and Adsorptive-Mediated Transcytosis The luminal surface of brain endothelial cells contains glycocalyx residues that establish a strong negative charge that contributes to the barrier phenotype [83]. Negatively charged molecules, e.g. FITC-dextrans or nanoparticles, cross the barrier in significantly lower amounts than neutral or cationic ones [48, 49]. Cationization of ferritin [50], albumin [51], or anti-tetanus immunoglobulin fragments [52] produces better transcytosis without changing the barrier integrity. Linear peptide fragments derived from antimicrobial peptide protegrin 1, called SynB vectors, belong to the large and heterogenous family of cell-penetrating peptides. The most important property of these peptide sequences is that they possess multiple positive charges [84]. SynB vectors were shown to be able to enhance the brain delivery of analgesic compounds (dalargin and morphin-6-glucuronide), anticancer agents (doxorubicin) and antibiotics (benzylpenicillin) in animal experiments [54-57, 85]. The vectorized paclitaxel is being tested for the treatment of brain glioma in phase I clinical trials [53]. It has been demonstrated that SynB vectors use the adsorptive-mediated endocytotic transport process at the BBB, and the net positive charge of the peptide vector plays a role in its binding to the endothelial surface and its subsequent internalization [84]. Cationization can be also used for nanoparticle targeting. Albumin loaded in lipid coated cationic nanoparticles had a 27-fold enhanced transport across brain endothelial monolayers [49]. Cationized albumin-coupled liposomes were also effective in targeting cultured brain endothelial cells and isolated brain capillaries [58], while cationic albuminconjugated solid polymeric nanoparticles showed enhanced brain penetration in animal studies [59, 60]. 2. Lipophilic Analogues, Liposomes, Lipid Nanoparticles Increasing lipid solubility is one of the techniques for increasing the BBB transport of a molecule. The best known example is heroin, the diacetyl derivative of morphine, which penetrates the BBB better than the parental compound [86]. This approach can be applied not only to small molecules, but also to proteins and other large biomolecules. Monoacylation of ribonuclease A facilitates the transport of the enzyme through the BBB; palmitoylated and stearoylated, but not myristoylated, derivatives show increased BBB transport without degradation or modification of barrier permeability [87]. A minimal length of 16 carbon atoms is required for a translocation of ribonuclease A across brain endothelial cells. Prodrug design for brain delivery of small- and medium-sized neuropeptides is achieved by the chemical modification of the bioactive molecule, which is attached to a lipid vector, like a fatty acid, a glyceride or phospholipid. The pharmacologically inactive molecule, which penetrates through the BBB, will be released enzymatically only at the site of action. This retrometabolic drug design was successfully applied to target thyrotropin-releasing hormone analogues to the brain [63]. A lipophilic analogue of valproate, DP-VPA, has entered phase II trials for the treatment of epilepsy and depression [61]. Estredox is a novel brain-targeted delivery system for estradiol in phase II clinical trials, which has more favourable pharmacological properties than estrogen replacement therapy [62]. The transport of ionically charged solid polymeric nanoparticles is enhanced several fold compared to neutral nanoparticles as a result of lipid coating [49]. Liposome encapsulation increases the brain delivery of enkephalins [65]. Lipid nanoparticle-containing superparamagnetic iron particles are able to cross the BBB, and CNS uptake has been demonstrated by in vivo magnetic resonance imaging [66]. While high lipid solubility can increase the transport of a given drug across the BBB, it can also enhance uptake by peripheral tissues as well as sequestration in the capillary bed, resulting in decreased concentration in blood and a decreased amount of the drug presented to the BBB [28]. 3. Targeting by Carrier Mediated Transport Despite the abundance of saturable, carrier-mediated transport systems at the BBB, this physiological pathway is still not fully exploited for drug delivery (Table 3). The usefulness of this approach is clearly demonstrated by the number of clinically used drugs, such as L-Dopa, baclofen and melphalan, that cross the BBB via one of the amino acid transporters, LAT1 [28, 67]. Having chemical similarities to L-phenylalanine, these drugs can use the

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transporter to enter the CNS. Another good example of a drug to use LAT1 as a carrier to penetrate to brain is L-4chlorokinurenin, which is metabolised in the brain tissue to the active neuroprotective drug 7-chlorokinurenic acid, which cannot cross the BBB [67]. Another amino acid transport system for L-glutamate was utilized to facilitate a non-permeable drug, p-di(hydroxyethyl)-amino-D-phenylalanine, across bovine brain microvessel endothelial monolayers by conjugating the drug with L-glutamate amino acid [88]. As mentioned previously, three SLC transporters for glucose have so far been described at the BBB. This group of transporters can be targeted by glucose as a ligand. Vasoactive intestinal peptide has been delivered to the CNS by glucose-labelled niosomes, vesicular nanoparticles made from non-ionic surfactants [69]. Glucose-targeted PEGylated paramagnetic niosomes have also been used successfully to detect brain tumours in mice by magnetic resonance imaging [70]. While serum proteins do not cross the BBB in physiological conditions, glycosylation of albumin results in an 8-fold elevation of transendothelial transport [51]. Glycosylated opiates and glycodermorphins both have prolonged analgesic activity and enhanced BBB penetration [68]. It should be noted that in the transport of glycosylated molecules, the receptor for advanced glycation endproducts (RAGE) may also participate via receptor-mediated transport [29]. Recently, the saturable glutathione transporter at the BBB has been tested for drug delivery. Glutathione-conjugated PEGylated liposomes containing doxorubicin have been successful in preclinical studies and are expected to enter clinical trials soon [71]. 4. Targeting by Receptor-Mediated Transport Receptor-mediated transport is an important pathway for peptides and proteins to reach the CNS, and the receptors for these specific ligands, such as insulin and transferring, are highly expressed at the BBB [1, 86]. The receptor-mediated transport systems of the BBB have been extensively characterized and tested for targeting large biomolecules as neurodiagnostics or neurotherapeutics to the brain via molecular Trojan horses, i.e. brain transport vectors, such as endogenous or modified peptide ligands or peptidomimetic antibodies specific to these transporters (Table 4) [1, 86]. One of the most studied receptors for the transendothelial pathway is the transferrin receptor, which physiologically takes part in the iron transport across the BBB. A receptor-specific peptidomimetic antibody for the mouse transferrin receptor has been successfully used to target biopharmaceuticals to the brain, including the nerve growth factor and the brainderived neurotrophic factor, enzymes for enzyme replacement therapy, neurodiagnostics such as the radio labelled epidermal growth factor, and non-viral genes encapsulated in PEGylated immunoliposomes [1, 40, 72, 86]. Targeting approaches using the insulin receptor at the BBB are also successful. Global non-viral gene transfers to primate brains were achieved through DNA-loaded PEGylated immunoliposome-targeting human insulin receptors [73]. IgG fusion proteins have been genetically engineered to contain neurotrophic factors, decoy receptors, therapeutic enzymes and single-chain Fv antibodies, which are targeted to the brain via transport by the BBB insulin receptor, and exert pharmacological effects in the brain at the cognate receptor, ligand, or enzyme substrate in animal studies [74]. The genetically-engineered human insulin receptor monoclonal antibody as a brain transport vector can potentially be used to target drugs or genes to the human brain [1, 74]. One of the most successful groups of brain transport vectors targets low density lipoprotein receptor-related protein 1 or 2 (LRP-1, -2) [75-80, 89]. Aprotinin is a ligand of LRP-2, and its functional peptide derivatives, called angiopep vectors, were designed to mediate transcytosis across the BBB via LRP [89]. Angiopep-2, a 19-amino-acid peptide vector conjugated to paclitaxel, doxorubicin and etoposide, was successfully tested in preclinical development to cross the BBB [76, 77, 90]. Paclitaxel-angiopep (ANG1005) is currently being evaluated in phase 1/2 multicenter studies in brain tumour patients [75]. An efficient transfer of receptor-associated peptide (RAP) conjugated to therapeutic proteins across the BBB via LRPs has also been demonstrated [78, 79]. Melanotransferrin (p97), another ligand of LRP, conjugated to paclitaxel or adriamycin could effectively shuttle these chemotherapeutics to the brain in animal studies [80]. Apolipoprotein E conjugated to nanoparticles loaded with loperamide and dalargin strongly enhanced drug transport into the brain, most probably through interaction with lipoprotein receptors, such as low density lipoprotein receptors

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[81, 82]. A BBB receptor for a membrane-bound precursor of heparin binding epidermal growth factor-like growth factor (HB-EGF) also acts as a receptor for the diphtheria toxin [40, 89]. A safe, clinically used ligand of this BBB receptor, CRM197, has been tested as an effective uptake receptor for the targeting of drugs to the brain [40]. Using antibody phage display technology, single domain antibodies could be selected to effectively cross human in vitro BBB models and the BBB in vivo, with the potential for use in macromolecule delivery as brain targeting vectors [91]. The transport of FC5, one of the antibodies, across human brain endothelial cells was polarized, charge-independent and temperature-dependent, suggesting a receptor-mediated process probably involving the α(2,3)-sialoglycoprotein receptor, which triggers clathrin-mediated endocytosis [92]. Strategies for Modifying BBB Functions The efflux pumps and the strictly regulated paracellular barrier are the two major BBB functions under focus in attempts to enhance drug delivery by modulation of the BBB. A safe, rapid, and transient increase of BBB permeability pathways and blocking of efflux pumps without systemic and CNS side-effects would be optimal for clinical use to increase drug delivery to the brain. Examples of clinical and experimental approaches for achieving these two goals are listed in Table 5, with emphasis on applicability in human therapy. Table 5: Modifications of Blood-Brain Barrier Functions to Enhance Delivery of Drugs to the CNS Method

Drugs / molecules

Stage of development

Reference

Osmotic BBB disruption by intraarterial mannitol

Anticancer drugs

Clinical trials

[6,93]

Cereport (lobradimil)

Carboplatin

Phase I-II clinical trials terminated

[94]

Short-chain alkylglycerols and oligoglycerolipids (LipoBridge)

Fluorescein, albumin, anticancer drugs, antibiotics

Preclinical development, animal experiments

[95-97]

Zonula occludens toxin

Methotrexate, paclitaxel

Animal experiment (intracarotid injection)

[98]

Fatty acids, modified fatty acids, phospholipids

γ-aminobutyric acid, gadolinium, mannitol

Animal experiments (intracarotid injection)

[99,100]

Surfactants (bile salts)

Fluorescein, albumin, mannitol

Animal experiments (intracarotid injection, in situ brain perfusion)

[101-103]

Cationic polymers

Horse radish peroxidase, albumin, nobiletin

Animal experiments (intracarotid injection, in situ brain perfusion)

[104-106]

Nitric oxide donors

Sucrose, γ-aminobutyric acid

Animal experiments (intracarotid injection, in situ brain perfusion)

[107,108]

High frequency focused ultrasound

Antibodies, doxorubicin

Animal experiments

[109-111]

P-glycoprotein inhibition by cyclosporine

Verapamil

Human PET study

[112,113]

P-glycoprotein inhibition by cyclosporine A

Verapamil, altanserine, GR205171

Animal and human PET study

[114]

P-glycoprotein inhibition by verapamil

Quinacrine

Human study

[115]

P-glycoprotein inhibition by tariquidar

Serotonin 5-HT1A antagonist MPPF

microPET animal experiment

[116]

Transcriptional regulation of Pglycoprotein by COX-2 inhibition

Antiepileptic drugs

Rodent model of epilepsy

[117]

MODULATION OF BBB PERMEABILITY

MODULATION OF BBB TRANSPORTERS

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MODULATION OF BBB PERMEABILITY FOR DRUG DELIVERY Osmotic stress induced by mannitol or arabinose causes a rapid and reversible increase in paracellular permeability through brain endothelial monolayers [118], and in animal models [119]. Dilatation of junctional clefts was observed in rats after intracarotid infusion of hyperosmotic arabinose, and osmotic shock altered the localization and expression of TJ proteins [120]. Src kinase-mediated phosphorylation of β-catenin may play a role in the mechanism of mannitol-induced TJ opening [121]. BBB disruption using hyperosmotic mannitol is one of the safest and most effective methods in the last 20 years of inducing a reversible increase of BBB permeability for cytostatic drug delivery to treat brain tumours clinically [6]. Multicenter clinical studies suggest that BBB disruption by intraarterial hyperosmotic mannitol can enhance the penetration of anticancer drugs and prolong survival in patients with malignant brain tumours [93]. However, the method has not been widely used, because the procedure is difficult, invasive, and neurotoxicity may develop after the treatment. Cereport (RMP-7, labradimil), a 9-mer synthetic peptide analogue of bradykinin, increases intracellular Ca2+ and cyclic GMP levels through bradykinin B2 receptors in brain endothelial cells [122, 123]. Cereport induces a rapid and transient elevation in the paracellular brain endothelial permeability in both cultured cells and mice [122, 124]. Cereport enhanced the CNS delivery of carboplatin, loperamide and cyclosporin-A, and increased the chemotherapeutic, analgesic and neuroprotective effects, respectively [125-127]. It also improved the transport of horseradish peroxidase across an in vitro BBB model, and attached to liposomes it facilitated Evans blue penetration to the brain in rats [128]. Cereport augmented the permeability of chemotherapeutic agents in tumour vessels and increased the survival in rodent models of glioma and metastatic brain tumours [127, 129]. However, intravenouslygiven adjuvant Cereport did not have a significant beneficial effect in a Phase II study in childhood high-grade and brainstem gliomas [94]. Although Cereport could not potentiate chemotherapy of brain tumours due to the high level of P-glycoprotein on the tumour cells, it can still be useful in enhancing drug delivery across the BBB in other diseases [125-128]. Short-chain alkylglycerol 1-O-pentylglycerol administered to the carotid artery can rapidly and reversibly enhance brain penetration of antineoplastic agents and antibiotics in rats [130]. It could also increase the delivery of erucylphosphocholine and methotrexate to implanted tumours in rats [95, 96, 130, 131]. 1-O-pentylglycerol-induced extravasation of fluorescein, albumin and methotrexate in the ipsilateral brain and to implanted brain tumour in rats and mice was mediated by enhanced paracellular permeability of TJs [96]. The effect of 1-O-pentylglycerol was lower than that of hyperosmotic mannitol, but in contrast to mannitol it did not enhance the permeability of the contralateral brain hemisphere or the cerebellum and brain stem for antineoplastic drugs [95]. The alkylglycerolinduced BBB-opening was more rapid and the effect was localised to the site of injection, with no in vitro and longterm in vivo toxicity or neuropathological changes [95]. Therefore short-chain alkylglycerols and oligoglycerolipids are considered as new and safer alternatives for BBB disruption. Integral membrane TJ proteins could be potential targets for modulation of paracellular permeability in brain endothelial cells (27). Zonulin/zonula occludens toxin (Zot) is an active TJ modulator at the BBB. It induces a reversible, concentration-dependent increase in the paracellular transport of permeability markers and chemotherapeutic agents doxorubicin and paclitaxel, without short-term toxicity [132]. A Zot-binding glycoprotein was also purified from the brain [133]. The Zot-active fragment ΔG, given into the carotid artery of rats, increased the brain penetration of sucrose, paclitaxel, and the hydrophilic methotrexate several fold [98]. There are no data available on the effect of other new epithelial TJ modulator peptides on the BBB [27]. Vasoactive compounds, such as histamine, bradykinin, or leukotrienes, increase BBB permeability and are wellknown mediators of brain oedema formation [10, 11, 134]. Blood vessels in brain tumour tissue are more sensitive to the permeability-enhancing effects of these mediators than normal vasculature [134]. Histamine increases the paracellular permeability in both in vitro and in vivo BBB models [10, 11]. Histamine injected to the carotid artery results in extravasation of Evans blue-albumin and γ-aminoisobutyric acid in tumour and peritumoural brain tissue [135, 136]. Signalling pathways contributing to histamine-induced increases in BBB permeability include histamine H2 receptors, nitric oxide and cyclic GMP production [137, 138]. The effect of some absorption enhancers, such as fatty acids, modified fatty acids, and ionic surfactant bile salts has been also tested on BBB permeability. Reversible opening of the BBB to Evans blue and γ-aminoisobutyric acid

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Solubility, Delivery and ADME Problems of Drugs and Drug Candidates 155

was observed after an intracarotid artery infusion of oleic acid in rats [139]. Emulsions made from oleic acid or linoleic acid injected to the carotid artery increased BBB permeability in a reversible manner in cats [99]. Intracarotid infusion of sodium caprate, a clinically used absorption enhancer in the gastrointestinal tract, produced reversible, dose-related BBB opening for mannitol in rats [100]. However, high doses were toxic and caused severe brain oedema and cardio-respiratory failure [100]. Intracarotid infusion of the anionic surfactant sodium dodecyl sulfate, a solubilizer and stabilizer in pharmaceutical preparations, also causes an extensive, dose-dependent and reversible BBB opening in rats [140]. Sodium dehydrocholate infusion to the carotid artery of rats disrupts the BBB in a concentration-dependent manner [101, 102]. Sodium deoxycholate and taurochenodeoxycholate have the same effect using in situ rat brain perfusion [103]. Sodium dihydroxy-oxo-cholanate, a modified bile salt, increased BBB permeability and enhanced the brain uptake of quinine and the analgesic action of morphine [141]. However, dehydrocholate and deoxycholate are strong detergents, and high doses induce lytic action on endothelial cell membranes with the development of EEG changes and epileptiform activity [103, 142, 143], limiting their possible clinical application. The negative surface charge of the luminal endothelial membrane of brain vessels contributes to the barrier phenotype, and cationic molecules show better BBB permeability, as discussed in previous sections [11]. Intracarotid infusion of the polycations protamine, poly-L-lysine and poly-L-arginine in rats results in a permeability increase for albumin [105]. Protamine decreased the negative luminal surface charge in brain vessels and increased the extravasation of peroxidase in a rat brain perfusion model [104]. Peroxidase extravasation could be demonstrated in endothelial TJs, suggesting a paracellular route of the BBB opening [104]. The cationic polymer chitosan, applied in microemulsion, improved nobiletin transport to the brain in mice [106]. Release of excess nitric oxide (NO) is involved in the signalling pathways activated by bradykinin and histamine, leading to increased BBB permeability in both tumour and non-tumour brain tissue [108]. While basal NO production is necessary for BBB integrity, excessive NO release by either inducible NO synthase or by NO donors leads to increased permeability in BBB models [11]. The NO donor molecule SNAP enhanced BBB permeability for sucrose about 5-fold in all forebrain regions, using an in situ rodent brain perfusion method [107]. Intracarotid infusion of shortacting NO donors led to increased transport of γ-aminoisobutyric acid to tumour tissue in rats [108]. Experimental BBB opening by high intensity focused ultrasound is a novel method [144]. Low frequency magnetic resonance imaging-guided ultrasound bursts can induce local, reversible disruption of the BBB in rabbits [145]. It can also increase the brain delivery of the dopamine D4 receptor antibody, and Herceptin, a humanized anti-human epidermal growth factor receptor 2 monoclonal antibody, in mice [109, 110], and doxorubicin in rats [111]. The mechanisms of transport of molecules by sonication can involve transcytosis, endothelial fenestration and channel formation, partial opening of TJs and free passage through the injured endothelium [146]. After the necessary safety and efficacy tests in more experimental models, focused ultrasound treatment may have therapeutic potential for brain tumours [93]. Modulation of BBB Transporters for Drug Delivery Efflux pumps localized at the BBB are also in the focus of attempts to enhance drug delivery through modulation of the BBB. P-glycoprotein, the most well known efflux pump at the BBB, prevents entry of a wide variety of drugs to the brain, including antibiotics, antineoplastic agents and drugs to treat epilepsy and AIDS [147]. P-glycoprotein blockers are therefore extensively studied to increase the levels of therapeutic and diagnostic compounds in the brain [148] (Table 5). The delivery of drugs to the brain can be enhanced by substrates and competitive inhibitors of P- glycoprotein, such as cyclosporine A and verapamil. Cyclosporine increases the brain uptake of verapamil and other P-glycoprotein ligands in brain tissue in animal and human PET studies [112-114]. Selective inhibitors of P-glycoprotein, such as valsprodar and elacridar, elevate both CNS anticancer drug delivery and therapeutic efficacy in animal models [149]. Tariquidar is a potent, specific and non-competitive third-generation P-glycoprotein inhibitor. In a recent study, it enhanced the brain uptake and binding of a serotonin 5-HT1A receptor antagonist in rats, measured by microPET [116]. These data are promising from a therapeutic point of view and are supported by clinical observations. Combination therapy of low-dose quinacrine and the P-glycoprotein inhibitor verapamil for patients with Creutzfeldt-Jakob disease has better efficacy and fewer adverse effects [115].

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The major limitation to the successful use of these inhibitors in human therapy is their toxicity. The first- and second-generation P-glycoprotein modulators have failed in clinical trials due to toxic side effects [150]. As an example, the lack of respiratory depression produced by loperamide, which allows it to be safely used therapeutically, can be reversed by the P-glycoprotein inhibitor quinidine in humans, resulting in serious toxic and abuse potential. [151]. The use of P-glycoprotein blockers with cytostatic drugs should be carefully planned and monitored. P-glycoprotein protects the BBB against the disruptive effects of the anti-microtubule drugs vinblastine, colchicine and paclitaxel. When anti-microtubule drugs are used in combination with potent P-glycoprotein modulators, the integrity of the brain endothelial cell monolayers becomes damaged [152]. Experimental data indicate that excipients can also modify brain endothelial P-glycoprotein activity. Cyclodextrins can also increase the transport of doxorubicin through a BBB model [153] by cholesterol extraction from the brain capillary endothelial cell membrane, leading to a modulation of P-glycoprotein activity. Pluronic block copolymer P85 enhanced BBB transport of digoxin both in vitro and in vivo [154]. Energy depletion and membrane fluidization are critical factors contributing to the activity of P85 block copolymer at the BBB [155]. The identification of a glutamate/NMDA-R/cyclooxygenase-2 signalling pathway in the upregulation of Pglycoprotein and subsequent pharmacoresistance in epilepsy led to the hypothesis that targeting this pathway can prevent seizure-induced P-glycoprotein over-expression in the epileptic brain [148]. The highly selective cyclooxygenase-2 inhibitors SC-58236 and NS-398 kept P-glycoprotein expression at control levels and increased brain delivery of phenytoin in rodent models of epilepsy [117]. As no impact of cyclooxygenase inhibition on basal P-glycoprotein expression and transport activity was observed, the physiologically relevant transport function of Pglycoprotein can be preserved. This innovative approach may represent a new possibility in the treatment of epilepsy. Transcriptional regulation of P-glycoprotein can also have an impact in the treatment of other CNS diseases. In Alzheimer’s disease, where down-regulation of efflux transporters such as P-glycoprotein is associated with disease progression [12], P-glycoprotein inducers could be beneficial [148]. Strategies to Circumvent the Blood-Brain Barrier, Unconventional Routes Circumvention of the BBB is used as a third major strategy to enhance drug penetration to the brain. Drug delivery possibilities include the direct, invasive administration of pharmacons to the brain tissue or to the cerebrospinal fluid (CSF) space to treat CNS diseases, or the use of non-invasive alternative nasal and ocular pathways, as summarized in Table 6. Table 6: Circumvention of the Blood-Brain Barrier to Enhance Delivery of Drugs to the CNS Indications

Examples for drugs / molecules

Stage of development

Reference

INTRATHECAL DRUG ADMINISTRATION Anesthesia, pain

Extended-release (DepoDur)

morphine

Clinically used

[156]

Lymphomatous meningitis

Extended-release (DepoCyte)

cytarabine

Clinically used

[157]

Spasticity, pain

Baclofen (by implanted pump)

Clinically used

[158]

INTRACEREBROVENTRICULAR DRUG ADMINISTRATION CNS infections

Antimicrobial drugs

Clinically used

[159]

Parkinson’s disease

Glial cell line-derived neurotrophic factor

Human study, ineffective

[160]

Niemann-Pick A disease

acid sphingomyelinase

Animal experiment

[161]

INTRAPARENCHYMAL DRUG OR CELL ADMINISTRATION Gliomas

Polymer-controlled carmustin (Gliadel)

release

of

Parkinson’s disease

Intraputamenal glial cell linederived neurotrophic factor infusion

Clinically used

[162]

Open label human study, controlled clinical trial

[160]

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Parkinson’s disease

Solubility, Delivery and ADME Problems of Drugs and Drug Candidates 157

Fetal ventral mesencephalic tissue transplants, embryonic and induced pluripotent stem cells

Animal experiments human studies

and

[163]

CONVECTION-ENHANCED DELIVERY OF DRUGS TO BRAIN Gliomas

Antineoplastic drugs

Clinical trials

[164]

Glioblastoma multiforme

Diphtheria toxin –transferrin conjugate (TransMID)

Phase III trial terminated

[165]

Glioblastoma multiforme

targeted Pseudomonas aeruginosa exotoxins

Phase I-III trials

[166]

Epilepsy

ω-conotoxins, neurotoxins

Animal experiments

[167]

botulinum

NASAL PATHWAY TO BRAIN Stroke

Osteopontin peptide mimetics

Animal experiment

[168]

Neurodegeneration

Nerve growth factor

Animal experiment

[169, 170]

To enhance eventrelated potentials in attention tasks

Cholecystokinin, vasopressin

Placebo-controlled, doubleblind within-subject crossover human study

[171, 172]

OCULAR PATHWAY TO BRAIN Cholinergic brain injury

Nerve growth factor

Animal experiment

[173]

Neurodegeneration

Nerve growth factor

Animal experiment

[170]

Intrathecal and intracerebroventricular administration of drugs is widely and effectively used clinically to treat chronic pain, spasticity, CNS infections or to induce local anesthesia for surgery (Table 6). However, the slow and ineffective diffusion between the CSF space and the brain parenchyma and within the intracellular space of the brain [174] limits the efficacy of this approach in the case of neurodegenerative diseases [160] or tumours. To overcome both the BBB and the limited drug diffusion from the CSF, intraparenchymal delivery of antineoplastic drugs by locally implanted drug-targeting wafers (e.g. Gliadel) [162] or by implanted catheters and subcutaneous pumps are used clinically. The direct local injection of neurotrophic growth factors or transplanted cells may represent a new therapeutic possibility for neurodegenerative diseases such as Parkinson’s disease [160, 163]. Convection-enhanced delivery (CED) is a novel drug-delivery technique that uses positive hydrostatic pressure to deliver a fluid containing a therapeutic substance by bulk flow directly into the interstitial space within a localized region of the brain parenchyma. CED provides a wider, more homogenous distribution than bolus deposition (focal injection) or other diffusion-based delivery approaches. It is estimated that more than 1000 patients have received CED infusions in clinical trials of malignant glioma [164]. CED can be used to deliver not only cytostatic drugs, but also bacterial toxins conjugated to the ligands of tumour cell receptors [166]. Experimental data indicate that the method may be used for the treatment of intractable epilepsy instead of surgery [167]. Nasal and Ocular Routes In recent years, the nasal route for delivery of drugs to the brain via the olfactory region has received considerable attention [175, 176]. In contrast to invasive strategies for circumventing the BBB, the nasal pathway can be exploited for the non-invasive delivery of drugs to the CNS. The nasal epithelial surface area is small, about 150 cm2, and consists of respiratory and olfactory regions. Although the olfactory system possesses elaborate epithelial, endothelial and glial barriers expressing occludin, claudins and other TJ proteins [177], due to its special anatomical localization it provides a direct access to brain, as shown on (Fig. 3) [178]. In animal studies, a large number of low molecular weight drugs and peptides, such as estradiol, progesterone, dihydroergotamine and cocaine, have been shown to reach the CSF, the olfactory bulb and in some cases other parts of the brain after nasal administration. Even large molecules, such as protein nerve growth factor, insulin-like growth factor and fibroblast growth factor, could be transported to the CNS [177, 178, 179]. Most of the studies

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evaluating nose to brain delivery of drugs in humans measured the pharmacological effects of drugs on the CNS indirectly, but there are data on the direct measurement of nose to brain transport, confirming the likely existence of the nasal route to the CNS in humans [179]. The nasal route could be important for drugs that are used in crisis treatments, such as for pain, and for centrally acting drugs where the putative pathway from nose to brain might provide a faster and more specific therapeutic effect [180].

Figure 3: Transport pathways between the nasal cavity and the CNS. Possible transport routes via the olfactory (green) and the trigeminal (red) nerves, or in the CSF (blue) (modified from [178]).

To formulate effective dosage forms for intranasal application, several aspects should be taken into consideration. Nasally administered drugs are cleared rapidly from the nasal cavity into the gastrointestinal tract by the mucociliary clearance system [176]. Other factors limiting the nasal absorption of large molecular weight or polar drugs are the low membrane permeability and the interepithelial junctional complexes, which hinder trans- and paracellular transport, respectively [176]. For these reasons, application of mucoadhesive agents to increase the residence time of formulations on the nasal mucosa, and absorption enhancers to elevate drug transport across the nasal mucosa are crucial in nasal vehicles. We have successfully developed nasal vehicles containing hyaluronate or Klucel as mucoadhesive components and Cremophor as an absorption enhancer to deliver the hydrophilic 4 kDa dextran [181] and the bioactive peptide amyloid-β 1-42 to different brain regions in rats [182]. A new alternative route to the brain across the epithelial barriers of the eye is emerging, which also bypasses the BBB. Recent experimental results indicate the possibility of drug delivery to the CNS following ocular application through non-systemic routes [170, 173]. CONCLUSION/PERSPECTIVES For a long time, the only clinically used approaches for targeting drugs to the brain were the osmotic disruption of the BBB and the circumvention of BBB by intrathecal, intraparenchymal or intracerebrocentricular drug administration (Tables 5-6). In the last decade, new data on BBB transport mechanisms has resulted in the rapid development of novel strategies for increasing drug delivery to the CNS by exploiting physiological pathways such as carrier-, receptor- and adsorptive mediated transport processes at the BBB (Table 3). The greatest progress has been seen in the field of brain tumor treatment, where new vectorized transport systems – SynB and Angiopep conjugated chemotherapeutics – for the treatment of gliomas and brain metastases have entered clinical trials (Table 4). Several other transport platforms targeting BBB carriers and receptors are already in preclinical development, and may be ready to be used in clinical studies soon. As the two major mechanisms for limiting drug transport at the BBB are intercellular tight junctions and drug efflux pumps, several strategies are focused on modulating them. Tight junction modulators are already being used in clinical studies for different epithelial barriers [27]. However, only two methods for transiently and reversibly opening the BBB for the delivery of anticancer drugs have been used in patients: intraarterial hyperosmotic mannitol

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Solubility, Delivery and ADME Problems of Drugs and Drug Candidates 159

and Cereport (Table 5). If these prove to be a safer and more controllable way of opening the BBB, short chain alkylglycerols may represent an alternative to these methods. Among the experimental approaches, high frequency focused ultrasound seems to be a promising and non-invasive way of opening the BBB. Efflux transporter inhibitors as co-drugs failed in several clinical trials due to safety and toxicity issues. An innovative approach to modulating Pglycoprotein by transcriptional regulation in the treatment of epilepsy [148] may have an impact in the treatment of several CNS diseases. Due to the diversity of neurotherapeutics and the complexity of the functions of the BBB, especially the regulation of transport systems, no single “magic bullet” can be developed to enable drugs to cross the BBB. Nevertheless, great progress in the treatment of several groups of CNS diseases, including brain tumours and epilepsy, can be expected in the near future, based on a better understanding of the transport processes at the BBB. Hopefully, these new therapeutic approaches will soon be followed by other innovative transport platforms that will help to treat an ever wider range of neurological diseases. ACKNOWLEDGEMENT Supported by TÁMOP-4.2.2-08/1/2008-0002. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19]

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