Current Topics in Medicinal Chemistry, 2009, 9, 182-196 182 Recent Advances in Medicinal Chemistry and Pharmaceutical TechnologyStrategies for Drug Delivery to the Brain Nunzio Denora, Adriana Trapani, Valentino Laquintana, Angela Lopedota and Giuseppe Trapani* Dipartimento Farmaco-Chimico, Facoltà di Farmacia, Università degli Studi di Bari, Via Orabona 4, 70125 Bari, Italy Abstract: This paper provides a mini-review of some recent approaches for the treatment of brain pathologies examining both medicinal chemistry and pharmaceutical technology contributions. Medicinal chemistry-based strategies are essentially aimed at the chemical modification of low molecular weight drugs in order to increase their lipophilicity or the design of appropriate prodrugs, although this review will focus primarily on the use of prodrugs and not analog development. Recently, interest has been focused on the design and evaluation of prodrugs that are capable of exploiting one or more of the various endogenous transport systems at the level of the blood brain barrier (BBB). The technological strategies are essentially non-invasive methods of drug delivery to malignancies of the central nervous system (CNS) and are based on the use of nanosystems (colloidal carriers) such as liposomes, polymeric nanoparticles, solid lipid nanoparticles, polymeric micelles and dendrimers. The biodistribution of these nanocarriers can be manipulated by modifying their surface physico-chemical properties or by coating them with surfactants and polyethylene-glycols (PEGs). Liposomes, surfactant coated polymeric nanoparticles, and solid lipid nanoparticles are promising systems for delivery of drugs to tumors of the CNS. This mini-review discusses issues concerning the scope and limitations of both the medicinal chemistry and technological approaches. Based on the current findings, it can be concluded that crossing of the BBB and drug delivery to CNS is extremely complex and requires a multidisciplinary approach such as a close collaboration and common efforts among researchers of several scientific areas, particularly medicinal chemists, biologists and pharmaceutical technologists. Keywords: Blood-brain barrier, Drug targeting, Prodrugs, Carrier mediated transport, Liposomes, Nanoparticles. 1. INTRODUCTION Diseases of the central nervous system (CNS) are numerous and affect a large part of the world’s population. Stroke, ischemia, human immunodeficiency virus-1 (HIV-1) infection, epilepsy, and other psychiatric disorders such as anxiety, depression and schizophrenia are debilitating conditions that markedly affect the morbidity and mortality in modern society. The neurodegenerative diseases, such as Alzheimer’s (AD), Parkinson’s diseases (PD) and multiple sclerosis are characterized by symptoms related to movement, memory, and dementia due to the gradual loss of neurons. Brain tumors, including gliomas, astrocytomas and glioblastomas, constitute a relevant and unsolved clinical problem and the treatment of brain cancers are major challenges [1]. Unfortunately, few safe and effective methods are known for diagnosis and treatment of CNS disorders and this is mainly due to the anatomical characteristics of the CNS (see later discussion). The blood-brain barrier (BBB) represents an effective obstacle for the delivery of neuroactive agents to the central nervous system (CNS). The presence of the BBB makes treatment of many CNS diseases difficult to achieve, because the required therapeutic agents cannot be delivered across the barrier in sufficient amounts. It is estimated that more than 98% of small molecular weight drugs and practically *Address correspondence to this author at the Dipartimento FarmacoChimico, Facoltà di Farmacia, Università degli Studi di Bari, Via Orabona 4, 70125 Bari, Italy; Tel: (039) 080-5442764; Fax: (039) 0805442754; E-mail: trapani@farmchim.uniba.it 1568-0266/09 $55.00+.00 1015 100% of large molecular weight drugs (mainly peptides and proteins) developed for CNS pathologies do not readily cross the BBB [2]. To improve the brain penetration of potential therapeutic agents numerous medicinal chemistry- and pharmaceutical technology-based strategies have been explored and developed. The present mini-review deals with the various approaches, which have been recently established for the treatment of brain pathologies utilizing both the medicinal chemistry and technological contributions. Several reviews on specific topics have appeared in the literature and summarize the progress made in this area [3-12]. Here we build on the review of Ricci et al. in 2006 [13]. The aim of the present paper is to review the latest developments, evaluating both the scope and limitations of some strategies as well as the evidence supporting the importance of the therapeutic molecule features such as molecular weight and lipophilicity. The various barriers that impede the delivery of the drugs to the brain are reviewed. This is followed by a discussion of the use of both chemical modifications (i.e., medicinal chemistry approach) and nanocarriers (i.e., technological approach) for overcoming these barriers in order to effect delivery of drugs to sites in the CNS. 2. PHYSICAL BARRIERS TO THE PASSAGE OF MOLECULES FROM THE BLOOD COMPARTMENT TO THE BRAIN There are two physical barriers that separate the brain extracellular fluid from the blood. The first is constituted by © 2009 Bentham Science Publishers Ltd. Methodologies to Assess Brain Drug Delivery in Lead Optimization Current Topics in Medicinal Chemistry, 2009, Vol. 9, No. 2 the brain capillary endothelial cells that form the BBB. This physical barrier is characterized by tight junctions between endothelial cells, by the absence of fenestrations and low occurrence of pinocytic activity [14]. These features restrict the movement of compounds from the blood into the extracellular environment of the brain (Fig. (1)). The second barrier, located at the choroid plexus, is represented by the blood-cerebrospinal fluid barrier (BCSFB) that separates the blood from the cerebrospinal fluid (CSF) which, in turn, runs in the subarachnoid space surrounding the brain. Unlike the capillaries that form the BBB, the capillaries in the choroid plexus allow free movement of molecules via intracellular gaps and fenestrations [15]. The epithelial cells in the choroid plexus that form the BCSFB have complex tight junctions on the CSF (apical) side of the cells. These tight junctions of the epithelial cells in the choroid plexus are slightly more permeable than those found in the endothelial cells of the BBB [16] (Fig. (1)). In this context, it should be recognized that the BBB is interrupted in the case of brain tumors and some methods for exploiting it for therapeutic purposes have been suggested [17]. It is now well established that a tumor must develop its own vascular network to grow and the neovasculature within tumors consists of vessels with increased permeability due to the presence of large endothelial cell gaps compared with normal vessels [17]. Furthermore, recent studies highlighted Fig. (1). Schematic representation of the two main barriers in the CNS. 1016 183 the possibility to reach the brain following the nasal route of administration. In fact, it has been shown that, by using this pathway, the transport of drug across the olfactory region in the nasal cavity occurs thus reaching directly the brain tissue or the CSF. It is based on the connection existing between the nose and the brain, that is, the olfactory bulb. In fact, the olfactory epithelium is situated between the nasal septum and the lateral wall of each side of the two nasal cavities and just below the cribriform plate of the ethmoid bone separating the nasal cavity from the cranial cavity [18]. 2.1. Approaches for Increasing Brain Penetration Generally, there are three approaches for increasing the penetration of drugs into the brain. The first is an invasive route that circumvents the obstacle of the BBB and/or BCSFB by direct administration of the drug into the brain. An alternative approach consists in generating a transient disruption of the BBB, allowing the therapeutic agents to enter into the brain from the blood through a more permeable BBB. The third approach concerns the chemical modification of the drug improving its penetration into the CNS. In addition to the previous mentioned approaches, a fourth option has been increasingly investigated in the last decades and consists in the use of formulation approaches. These technological strategies are essentially non-invasive methods and are based on the use of colloidal carriers 184 Current Topics in Medicinal Chemistry, 2009, Vol. 9, No. 2 Trapani et al. including mainly liposomes, polymeric- and solid lipidnanoparticles (see Section 4). However, work in this area has been primary limited to drug delivery to tumors of the brain. To bypass the BBB, direct injection of drugs into the brain involves intracerebral and intrathecal administration. This approach is invasive and requires a craniotomy. An advantage of this pathway is that a wide range of compounds including large-and small-molecules can be administered. As mentioned above, another option to by-pass the BBB is the intranasal administration based on the connection existing between the nose and the brain, that is the olfactory bulb [18]. This pathway provides a mean for the administration of various compounds into the CNS including toxic agents such as pathogens, viruses, and toxic metals as well as various therapeutic agents including small molecules and proteins. Small molecules such as cocaine and cephalexin as well as a number of protein therapeutic agents, such as insulin have been successfully delivered to the CNS using intranasal delivery [19-21] although for polar drug molecules this route is questionable with respect to the quantity of drug that can be delivered. This administration route has been found as a promising approach for rapid-onset delivery of some medications to the CNS bypassing the BBB. The strategy consisting in generating a transient disruption of the BBB includes the systemic administration of hyperosmotic solutions or vasoactive compounds such as bradykinin and related analogs, or various alkylglycerols. The use of osmotic agents such as mannitol or arabinose involves expansion of the blood volume caused by the addition of the hyperosmotic agent and disruption of the BBB. This barrier resumes its normal integrity and function returning the osmolarity of the blood to normal value. During this period when the tight cellular junctions between the brain capillary endothelial cells have been compromised, paracellular diffusion [Fig. (2)] of water-soluble drugs and solutes into the brain is increased [22]. The BBB can also be disrupted by pharmacological means. Several endogenous proinflammatory vasoactive agents, such as bradykinin, histamine, nitric oxide are known to induce increases in BBB permeability in a concentrationand time-dependent manner. However, although capable of producing increases in BBB permeability, these endogenous agents cannot be applied safely for CNS drug delivery. Thus, the effects of bradykinin on BBB permeability are shortlived, requiring carotid artery infusion. Better results were obtained with bradykynin analogs such as labradimil. The effect of labradimil is characterized by a significant greater plasma and tissue stability than seen with bradykinin [23]. The transient disruption of the BBB has also been observed by the systemic administration of various alkylglycerols. The extent of BBB disruption is seen to depend on the length of the alkyl group and the number of glycerols present in the structure. The exact mechanism(s) for the transient BBB disruption observed with the alkylglycerols is unknown [24]. Moreover, as with the bradykinin analogs, the disruption of the BBB by alkylglycerols is very short lived. The third strategy for improving the brain penetration of therapeutic agents utilizes the chemical modification of the drug to improve transcellular migration. As shown in Fig. 1017 (2), the transcellular routes available include passive diffusion, specific transport systems, and endocytic processes in brain capillary endothelial cells. Overall, this strategy should lead to lower neurotoxicity compared to that associated with BBB disruption. 2.2 Physiological Factors Affecting Drug Delivery to CNS Many transport mechanisms for the uptake of nutrients into CNS exist in the brain (Fig. (2)). These transport mechanisms may be exploited for brain drug delivery. These include: a) Passive Diffusion. The main factors affecting the passive diffusion of drugs across the BBB involve an adequate lipophilicity, neutral or uncharged nature, low hydrogen bonding potential and small molecular size (< 500 g/mol). Thus, the improvement of the passive diffusion of drugs across the BBB can often be achieved by either increasing lipophilicity or reducing molecular size. As lipophilicity is dependent on polarity and ionization, modification of functional groups on drugs provides a method for improving passive diffusion across the BBB. b) Carrier-mediated (Active) Transport. More than 20 carrier-mediated transporter proteins have been identified in cerebral capillaries of the BBB including transporters for glucose, amino acids, vitamins, and nucleotides. The carriers for the large amino acids (e.g., amino acid transporter of type 1, LAT 1) and glucose (e.g., glucose transporter of type 1 GLUT 1), especially, have a sufficiently high transport capacity [25]. Therefore, an approach for increasing the transcellular passage of drugs across BBB is to design drugs that structurally resemble or can be linked to endogenous compounds that are transported into the brain by the carriers or transporters expressed in the brain microvessel endothelial cells [26]. Transporters that have received the greatest attention are: i) Amino Acid Transporters. Together with the large neutral amino acid transporters, LA transporters, cationic-, anionic- and neutral-amino acid transporters have also been identified. LA transporters have been most exploited for drug delivery purposes [27]. L-Dopa is the most well-known example of a drug that is transported by LA transporters in the BBB. L-Dopa is an endogenous large amino acid and is a precursor of the neurotransmitter dopamine. LA transporters are also involved in the transport of other drugs such as L-melphalan, baclofen, and gabapentin across the BBB [28]. ii) Glucose Transporters. The most important glucose transporter present in the brain capillary endothelial cells is the type 1, glucose transporter, GLUT 1. Compared to other nutrient transport/carrier systems in the BBB, GLUT 1 has the highest transport capacity (more than 10-50 times greater than that of amino acid and carboxylic acid transporters) and therefore represents an attractive target for drug delivery to the CNS. Glycosylated analogs of various opioid compounds have shown increased CNS analgesic properties compared to the non-glycosylated compounds [29]. Methodologies to Assess Brain Drug Delivery in Lead Optimization Current Topics in Medicinal Chemistry, 2009, Vol. 9, No. 2 185 Fig. (2). Blood-brain barrier transport mechanisms. iii) Monocarboxylic Acid Transporter. The best-characterized organic acid transporter in the BBB is the monocarboxylic acid transporter (MCT). Examples of drugs entering the CNS through the MCT are salicylic acid and various cholesterol-lowering 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors [30]. iv) Nucleoside Transporters. There are two general types of nucleoside transporter expressed in the brain capillary endothelial cells: the facilitative nucleoside transporters that carry selective nucleosides either into or out of the cell and active and the sodium-dependent transporters that can move selective nueleosides into the cell against a concentration gradient. There are several examples of drugs that are substrates for nucleoside transport systems such as the anticancer agent, gemcitabine, the antiviral agents, 3'-azidodeoxythymidine (AZT) and 2',3'-dideoxycytidine (ddC) [31, 32]. v) Peptide Transport Systems. Peptide transport systems are present in the brain capillary endothelial cells forming the BBB. The exact molecular nature of these peptide transporters remains to be determined. However, specific saturable transport systems have been identified in the BBB for glutathione [33], peptide hormones [34] and growth factors [35]. 1018 c) Vesicular Transport. Two types of vesicular transport processes are known: the fluid-phase endocytosis and the adsorptive endocytosis. However, only adsorptive endocytosis involves an initial binding or interaction with the plasma membrane of the cell. Vesicular transport due to adsorptive endocytosis is a saturable, ligand selective process. Several large macromolecules are transported from the blood into the brain through receptor-mediated endocytosis. Thus, these specific receptor-mediated transport processes represent another approach for enhancing transcellular permeability across the BBB. The most well-known processes are the transferrin- and insulin receptor-mediated vesicular transport. i) Transferrin is a glycoprotein that controls the transport of iron throughout the body. The brain capillary endothelial cells have a high density of transferrin receptors on their surface. Iron enters the cell as a complex with transferrin through an endocytic process that is initiated by the binding of transferrin to its receptor. Inside the brain endothelial cell, the iron is removed from the transferrin in the endosome. However, transferrin shows a limitation as a brain delivery vector because it is recycled back to the luminal surface of the brain capillary endothelial cell. As an alternative, a murine monoclonal antibody (MAb), OX-26 has been iden- 186 Current Topics in Medicinal Chemistry, 2009, Vol. 9, No. 2 Trapani et al. tified, which appears to be suitable for use as a drug carrier for this transport system. First, this MAb binds to the receptor and triggers endocytosis. Second, the OX26 MAb binds to an extracellular epitope on the transferrin receptor that is distinct from the transferrin ligand-binding site; thus, the OX-26 MAb does not interfere with transferrin binding to its receptor on the brain endothelial cells. The OX-26 antibody has proven to be an effective brain delivery vector, as it has been conjugated to a variety of drugs including methotrexate and nerve growth factor [36, 37]. ii) Insulin is a peptide hormone involved in glucose metabolism. The presence of insulin receptors in the CNS, suggest that insulin may have important functions also within the brain. Studies demonstrating the presence of high-affinity insulin receptors on the luminal plasma membrane of brain microvessel endothelial cells indicate that the peptide penetrates the BBB through a receptor-mediated transport process. Other studies support the potential use of insulin as brain delivery vector of therapeutic agents and macromolecules to the brain. For example, insulin has been used as a BBB transport vector for proteins [38]. Many other receptors such as insulin-like growth factor receptor, and a leptin receptor may be used for the same purpose. The corresponding ligands could be used as brain drug delivery vectors. In general, the strategy of using receptor-mediated transport consists in covalently binding drugs to peptides or MAbs, allowing the receptor recognition and transport into the brain of the drug-MAb complex. 2.3. Drug Efflux Transporter Systems in the BBB While it may seem simple to improve drug or prodrug transport to the CNS by changing lipophilicity or improving affinity for a transporter, attempts often fail. Several drugs are effluxed from brain to blood and the impact of this outwardly directed transport systems on CNS drug delivery is of great interest. In particular, several different efflux transporters are present in the BBB such as the Pglycoprotein (P-gp) and the multidrug resistance protein (MRP). These transporters are part of the larger ATP binding cassette (ABC) family of proteins that remove a wide variety of compounds from the cell through an ATP-dependent active transport process. In the past, these proteins were found to be over-expressed in various drug-resistant cancer cells; later on, they were also evidenced in normal cells such as intestinal and renal epithelial cells, hepatocytes, and brain capillary endothelial cells. Consequently, they influence the absorption, distribution, and elimination of many drugs. P-gp is a well-known drug efflux transporter. It has been demonstrated that the brain levels of drugs such as vincristine and ivermectin increased by 80-100-fold in the mice lacking P-gp. P-gp drug efflux in the BBB has been invoked to account for the reduced brain penetration of a number of structurally different drugs including digoxin, cyclosporin A (CSA), itraconazole, and opioid analgesics. To increase the CNS delivery of drugs that undergo active efflux, the co-administration of an inhibitor of the 1019 transporter or another substrate that can competitively saturate the efflux transporter has been explored. Thus, P-gp modulators such as verapamil diltiazem and CSA, have been used to increase the brain penetration of a number of compounds with low BBB permeability such as antiviral protease inhibitors, the anticancer agent paclitaxel, and the antifungal agent itraconazole. In addition, some polymers have been shown to inhibit drug efflux transporters. The mechanism by which polymers modulate drug efflux transporter activity involves alterations in membrane fluidity. For example, Pluronic block copolymer (P85) has been shown to enhance the permeability of a wide variety of drugs in an in vitro model of the BBB. These studies suggest that P85 can be used for improving drug permeability of the BBB through inhibition of drug efflux transporter system [39]. The practicability of such an approach is unknown. 3. MEDICINAL CHEMISTRY-BASED STRATEGIES Medicinal chemistry-based strategies are essentially aimed at the chemical modification of low molecular weight drugs in order to increase their lipophilicity [8]. This includes both an analog approach as well as a prodrug approach. Only the prodrug approach will be discussed here. For instance, the formation of an inactive prodrug is a way to increase the lipophilicity of a drug by attaching a lipophilic promoiety that can be cleaved to the parent drug on entering the CNS. Many of these examples involve ester-based prodrugs since by appropriate esterification of molecules containing -COOH, -OH, or -SH groups, it is possible to obtain derivatives with the desired lipophilicity. The classical example of such approach is heroin (i.e., the diacetyl ester of morphine) that rapidly crosses the BBB due to its high lipophilicity. Once in the brain, it is presumed to be hydrolyzed to morphine. The same approach has been employed with other therapeutic agents such as the anticancer agent chlorambucil and the neurotransmitters dopamine and gamma-aminobutyric acid (GABA) [3, 5, 8]. In recent years, notable interest has been focused on the design and evaluation of prodrugs that are capable of exploiting one or more of the various endogenous transport systems in the BBB. This is an attractive approach because it provides a more targeted approach to CNS drug delivery. The prodrug is designed to structurally resemble the endogeneous ligand of a specific transport system, which recognizes the prodrug as a substrate and transports it across the BBB. Thus, for example, the neurotransmitter dopamine is not able to cross the BBB due to its hydrophilic nature. However, the conversion of dopamine into its
-amino acid, L-Dopa 1, enables the brain to uptake 1 via the large aminoacid transporter of type 1, LAT 1. L-Dopa 1 is then decarboxylated to dopamine by L-amino acid decarboxylase in the brain. This decarboxylation also occurs in peripheral tissues. Although approximately 95% of L-Dopa, 1, is metabolized to dopamine in the peripheral tissues, the percentage of remaining L-Dopa allows sufficient brain therapeutic activity [4, 5] (Fig. (3)). Also utilizing LAT 1 are 4-chlorokynurenine, a prodrug of 7-chlorokynurenic acid 2 [40], an N-methyl-D-aspartate antagonist, the anticonvulsant gabapentin, 3, and the anticancer agent melphalan, 4 [5] (Fig. (3)). A recent example of this strategy have been published by Gynther et al. who described the conjugation of a Methodologies to Assess Brain Drug Delivery in Lead Optimization Current Topics in Medicinal Chemistry, 2009, Vol. 9, No. 2 187 O O HO NH2 OH NH 2 OH HO Cl NH2 OH NH 2 OH O Cl O O 1 H2N 2 N 3 4 Cl O O O O NH 3+ClOH O S HOOC NH2 O N H N H 5 S O HN N H O COOH OH OH 6 HO HO NH NH HO O HO OH O O 7 HO O O O O OH HO O HO 8 OH HO OH CH3 O O O NH 2 N H H 3C O EtO Cl O O O HCl n N F CH 3 9 10 R2 R2 COOR N H 11 R = H 12 R = n-butyl N R1 N R3 N OH O R1 N R3 O HN OH R4 R 1 = R2 = R 3 = H, Cl R 4 = H, COOH, COOEt HN (CH2 )n COOR4 R 1 = R 2 = R3 = H, Cl; R4 = H, Et 14 n = 1 15 n = 3 13 Fig. (3). Chemical structures of low molecular weight drugs and prodrugs studied for delivery to the brain. hydrophilic drug, ketoprofen, to L-tyrosine [41]. Unlike ketoprofen itself, the amino acid L-tyrosine is a LAT 1substrate and, interestingly, is characterized by a phenolic hydroxyl group suitable for the conjugation with various drug molecules. The mechanism and the kinetics of the brain uptake of the ketoprofen-tyrosine prodrug 5 was studied using the in situ rat brain uptake model. The uptake of the prodrug was found to be concentration-dependent. In addition, a specific 1020 LAT 1 inhibitor significantly decreased the brain uptake of the prodrug. Therefore, the results indicate that a drugsubstrate conjugate is able to transport drugs into the brain LAT 1. Other transporters such as the peptide transport systems have been exploited for enhanced BBB penetration of dopamine. Thus, More and Vince [42] prepared and evaluated the compound 6 that can be considered a dopamine prodrug containing the peptide glutathione (
–glutamyl- 188 Current Topics in Medicinal Chemistry, 2009, Vol. 9, No. 2 Trapani et al. cysteinylglycine) as promoiety and mercaptopyruvate as a linker. The prodrug design rationale is based on the ability of 6 to cross the BBB through recognition by glutathione transporters and on its ability to release the active drug once inside the brain. These glutathione transporters are located on the luminal side of the BBB and show a broad substrate specificity. In vitro directional transport experiments using the Madin Darby canine kidney (MDCK II) cell line as BBB model, showed the ability of prodrug 6 to cross this model cell line. This data supports the possible use of glutathione as a carrier for drug targeting. A number of investigations have been performed with different drug molecules in order to determine the possibility of exploiting the GLUT 1 transporter system. Fernandez et al. [43] synthesized several glycosyl derivatives of dopamine and tested the affinity of the prodrugs to GLUT 1 in human erythrocytes. Dopamine was linked to glucose with different linkers at the C-1, C-3 and C-6 positions of glucose (compounds 7 and 8, respectively, in (Fig. (3))). The results of glucose uptake inhibition showed that the glucose derivatives that were conjugated at position C-6 showed better affinity for GLUT 1 in human erythrocytes than those substituted at C-1 or C-3 positions. The relevancy of these findings to BBB transport needs to be explored. Limitations for Carrier-Mediated Transport in the CNS Undoubtedly, the design of drugs that utilize endogenous transport systems at the BBB is an attractive approach for increasing drug delivery to the brain. By exploiting specific transport systems it is possible to provide a more targeted approach to CNS drug delivery than physicochemical alterations aimed at creating a more lipophilic therapeutic agent or prodrug. However, some criticism has been raised to this approach. The most relevant is that the transport systems are relatively selective systems and devoted to the passage of essential nutrients and metabolites inside and outside the brain. Thus, chemical modifications made to therapeutic agents to target specific transport systems in the BBB are much more restricted than those used to enhance lipophilicity. Furthermore, the resulting modifications often result in compounds with lower affinity for the transporter than the endogenous ligand. For example, the nucleosidebased antiviral agents, AZT and ddC are transported by the concentrative nucleoside transporter of type 1, CNT 1. However, the affinity of these agents for the transporter is approximately 25-fold lower than that of endogenous pyrimidine-based nucleosides. An additional often-overlooked obstacle is that one would prefer to give drugs via the oral route. If oral dosing is desired, conjugates or prodrugs such as those described above must be capable of being absorbed from the GIT and remain intact until they reach the brain. Designing prodrugs that can be efficiently absorbed, remain intact, cross the BBB and then be cleaved in brain tissue remains one of the great challenges both medicinal chemists and their drug delivery colleagues. New Trends to the Prodrug Approach The prodrugs described above essentially aimed to enhance the lipophilicity or to target specific transport 1021 systems. Recently new trends in the prodrug approach have been explored. An interesting application has been reported by Pignatello et al. [44] who studied amphiphilic prodrugs of the flurbiprofen containing lipoamino acids (LAA) as promoieties. LAA are
-amino acids bearing alkyl side chains, whose length and structure can be modified to achieve the desired physicochemical properties. Because of the presence of an alkyl chain and a polar amino acid head, LAA conjugation yields amphiphilic derivatives, with a membrane-like character that can favor interaction with and penetration through biological membranes and barriers. Due to its anti-inflammatory and analgesic activities, flurbiprofen can be considered as a potential neuroprotective agent in AD therapy. This drug, indeed, reduced the secretion of 
amyloid protein A42, the major component of senile plaques of AD brain, both in Neuro-2a cells and rat primary cortical neurons, as well as in the brain of tg2576 -amyloid transgenic mice. Flurbiprofen was modified with LAA residues to give compounds of general formula 9 (Fig. (3)) with the aim of increasing its availability in the brain. Biodistribution and pharmacokinetic studies showed that i.v. injection of the parent drug led to constant brain levels for 3 h; thereafter, the drug rapidly disappeared from this area. After injection of 9 (n = 9) the released flurbiprofen appeared in the brain after 1 h, and the intracerebral levels continued to rise up to the ninth hour, reaching higher values than those observed in the first 3 h after the injection of the parent drug. The findings suggest that using 9 (n = 9), the parent drug, flurbiprofen, is released and accumulated preferentially in the brain tissue. In addition, the results highlight the role played by the LAA residue on the biopharmaceutical profiles of prodrugs. In particular, relatively small differences in the chain length of the side alkyl chain of the LAA promoiety can led to a considerably different distribution profile. Further amphiphilic prodrugs containing LAA as promoieties were studied by the same authors using cloricromene, a coumarin derivative that possesses antithrombotic, antiplatlet actions and causes vasodilatation. It was found that the intraperitoneal administration of compound 10 (CLOR-C4) to rats was able to provide a slight but statistically significant higher concentration of the active metabolite (cloricromene acid) in the brain compared with the parent drug administered by the same way [45]. Based on the available data, it can be stated that these amphiphilic LAA containing prodrugs need to be further evaluated to establish their potential for improved brain delivery. Another interesting application of the prodrug approach is the chemical modification of drugs to enhance delivery across the nasal mucosa and to prevent their metabolic degradation. Nasal administration route allows rapid-onset delivery of drugs to the CNS bypassing the BBB. An example of such application has been provided by Wang et al.[46] who studied the nasal administration of the nipecotic acid and n-butyl nipecotate (i.e., compounds 11 and 12, respectively, in (Fig. (3)) to rats. It was found that nasal dosing of the n-butyl ester of nipecotic acid provided a viable approach for delivering of nipecotic acid, a zwitterion, to the brain. A pharmacological response was observed only after dosing the ester, but not after i.v. nipecotic acid administration. There was no significant differences in Methodologies to Assess Brain Drug Delivery in Lead Optimization Current Topics in Medicinal Chemistry, 2009, Vol. 9, No. 2 189 nipecotic acid total brain levels after intravenous (i.v.) or nasal ester administration, suggesting the nasal route is as effective as the i.v. route for delivery of the acid to the brain thanks to the cleavable ester functional group. Rat brain disposition studies showed strong evidence that the ester hydrolysis was the rate limiting to nipecotic acid brain accumulation. This brain-targeted delivery system via nasal prodrugs seems a very promising approach for obtaining brain uptake of drugs. 13 is also that to reduce side effects. It should be noted that compounds 13 cannot be considered as simple co-drugs, i.e., prodrugs consisting of two pharmacologically active drugs that are coupled together in a single molecule so that each drug acts as a promoiety for the other; in fact, in compounds 13 the phenyl-imidazopyridine portion does not only play a pharmacodynamic role (i.e., the high affinity and selectivity for the GABA-BZR complex) but also a pharmacokinetic one (i.e., the BBB crossing ability). Prodrugs that utilize the chemical delivery systems (CDS) involving the conjugation between low weight drugs and molecules having specific targeting properties have been extensively evaluated [8]. The most studied CDS for brain delivery exploits linking a drug molecule to 1,4-dihydro-Nmethyl-nicotinic acid (i.e., dihydrotrigonelline). EstradiolCDS (Estredox) can be considered the most promising and practical application example to date and is undergoing Phase II clinical testing for the treatment of postmenopausal symptoms [47]. Derivatization of estradiol with the targetor, 1,4-dihydrotrigonelline, leads to an increase in lipophilicity thus enabling better transport across the BBB. By using estradiol-CDS, the concentration of estradiol in rat brain was elevated four to five times longer than after estradiol treatment alone. Evaluation of compounds such as 13 showed that all the prepared compounds were adequately stable to the chemical hydrolysis but unstable in physiological medium [54]. Receptor binding studies demonstrated that the examined compounds are essentially devoid of affinity for dopaminergic and benzodiazepine receptors. Bi-directional transport experiments across Madin–Darby Canine Kidney retrovirally transfected with the human MDR1 cDNA (MDCKII-MDR1) cell monolayers indicated that the compounds tested are not substrates of P-gp. Transport studies involving co-culture of bovine brain microvessel endothelial cell (BBMECs) and astrocytes monolayers (a well established in vitro method to estimate the BBB permeability) indicated that some of compounds 13 were able to cross the BBB. Interestingly, brain micro-dialysis experiments in rat showed that intraperitoneal acute administration of compounds 13 (R1 = R2 = R3 = Cl; R4 = COOEt; R5 = R6 = OH and R1 = R2 = R3 = Cl; R4 = H, R5 = R6 = OH) induced a dose- and time-dependent increase in the dopamine levels (up to 197%) in the rat medial prefrontal cortex. Based on these results, these compounds can be proposed as novel LDopa and dopamine prodrugs [54]. In the design of novel brain delivery systems by chemical modification, drug conjugation with receptor ligands represents another explored approach. In recent years, indeed, new useful potential cellular targets have been identified and characterized. Thus, for example, the peripheral benzodiazepine receptors (PBRs) have been identified in various peripheral tissues as well as in glial cells in the brain [48]. They are pharmacologically distinct from the central benzodiazepine receptors (CBRs) which are associated with GABA A receptors and mediate classical sedative, anxiolytic, and anticonvulsant properties of benzodiazepines. Among the different functions associated with the PBRs those associated with neurosteroid synthesis and the involvement in apoptosis processes [49, 50] are of particular interest. Evidence also indicates that PBRs are over-expressed in a number of tumor types, especially in the brain, and PBR expression appears to be related to the tumor malignancy grade. Based on all these observations, there are many potential clinical applications of PBR modulation, such as in oncologic, endocrine, neuropsychiatric and neurodegenerative diseases [51, 52]. Our research group showed that some 2-phenylimidazo[1,2-a]pyridine-3-acetamides are potent and selective ligands for PBRs [53]. Therefore, it seemed of interest to prepare compounds such as 13, which are characterized by an L-Dopa or dopamine moiety linked to appropriately substituted 2-phenyl-imidazopyridine-3-acetic acids. These conjugates were prepared in order to take advantage of i) the high affinity and selectivity for the GABA-benzodiazepine receptor (GABA-BZR) complex shown by most phenylimidazopyridine compounds; and ii) the high BBB crossing ability and lipophilicity shown by most phenyl-imidazopyridine derivatives. Moreover, since it is known that selective gabaergic agonists could be useful in patients who have complications associated with long-term L-Dopa treatment, a further expected advantage of using compounds 1022 A similar approach has been also applied to prepare compounds 14 and 15 which are characterized by a GABA or glycine moiety, respectively, linked to appropriately substituted 2-phenyl-imidazopyridine-3-acetic acids [55]. Again, stability, receptor binding, microdialysis and pharmacological studies were carried out on the compounds. The results demonstrated the feasibility of synthesizing useful anticonvulsants by coupling the amine function of the GABA with a phenylimidazopyridine portion [55,56]. Taking into account the functions of PBRs discussed above, it is clear that these receptors could also be the target to selectively increase anticancer drug delivery at brain level by using an appropriate PBR ligand-anticancer drug conjugate. Another possibility is PBR selective ligands that can serve as diagnostic imaging agents. The treatment of brain cancer is a formidable challenge in oncology. The failure of chemotherapy for brain tumors is due to the inability of intravenously administered anticancer agents to reach the brain parenchyma for the presence of the BBB although the capillaries that serve brain tumors are thought to be leakier. The first proof-of-concept of the potential of PBR ligandanticancer drug conjugate for brain delivery was reported by Guo et al. [57] who administered gemcitabine (GEM) and a PK11195 (a known PBR ligand)-GEM conjugate to nude rats bearing intracerebral tumors over-expressing PBRs. The pharmacokinetic and tissue distribution results showed a greater tumor selectivity and brain uptake by PK11195-GEM compared with GEM, attributable to receptor-mediated drug delivery and greater lipophilicity of the conjugate. These 190 Current Topics in Medicinal Chemistry, 2009, Vol. 9, No. 2 Trapani et al. findings prompted us to evaluate for the same purpose phenylimidazopyridine acetyl-melphalan conjugates in melphalan-sensitive human and rat glioma cell lines [58] as well as a cisplatin-like complex involving an imidazopyridin acetyl PBR ligand [59]. Moreover, we successfully used phenylimidazopyridine acetyl-fluorescein conjugates as a neurodiagnostic agent for activated microglia [60], an index of disease progression of several neurodegenerative disorders such as AD, PD, multiple sclerosis and HIV-associated dementia [61]. 4. PHARMACEUTICAL STRATEGIES TECHNOLOGY-BASED The technological strategies are essentially non-invasive methods of drug delivery to the CNS and represent valuable approaches for enhancing transcellular permeability of therapeutic agents and biomacromolecules across the BBB. They are based on the use of nanosystems (colloidal carriers), mainly liposomes and polymeric nanoparticles even though other systems such as solid lipid nanoparticles, polymeric micelles and dendrimers are also being tried. Following intravenous administration, the colloidal systems can extravasate only in tissues with a discontinuous capillary endothelium, such as the liver, spleen, and bone marrow, as well as into solid tumors and inflammed tissues where the endothelial cells are not closely joined together and increased vascular permeability occurs. An important requirement of the systemic intravenous use of these nanocarriers is their ability to circulate in the bloodstream for a prolonged period of time. However, after intravenous administration, they interact with the reticuloendothelial system (RES) which removes them from the blood stream [62]. This process mainly depends on particle size, charge and surface Fig. (4). Capture mechanisms of nanocarriers by cells. 1023 properties of the nanocarrier [63]. To prevent the uptake by the RES, poly(ethylene glycol) (PEG) coating or direct chemical linking of PEG to the particle surface provides relatively long plasma residence times. However, PEGylated carriers are characterized by a low affinity for brain tissue as they are not transported through the BBB. Nevertheless, the nanosystems may represent useful tools for non-invasive drug delivery to brain utilizing active targeting. In fact, these nanocarriers can be taken up actively by carrier–mediated transport (CMT), receptor-mediated endocytosis (RME) and adsorptive-endocytosis (AME) (Fig. (4)) and hence reach the cerebral parenchyma, or are degradated within lysosomes leading to the drug being released into the brain tissue. Liposomes Liposomes have long been used as carrier systems for the delivery of therapeutic agents because of their easy preparation, good biocompatibility, low toxicity and commercial availability. They are vesicles composed of lipid bilayers surrounding internal aqueous compartments. Relatively large amounts of drug molecules can be incorporated into the aqueous compartment (water soluble compounds) or lipid bilayers (lipophilic compounds). Conventional liposomes are rapidly cleared from circulation by macrophages of the RES and this limits their usefulness as drug delivery systems. Extended circulation time can be accomplished by decreasing the particle size (<100 nm) and by liposome-surface modification with PEG (Stealth liposomes). To target PEGylated liposomes to the brain, they can be additionally modified with monoclonal antibodies to glial fibrillary acidic proteins, transferrin receptors (OX-26), or human insulin receptors [64]. Methodologies to Assess Brain Drug Delivery in Lead Optimization Current Topics in Medicinal Chemistry, 2009, Vol. 9, No. 2 A recent application of transferrin surface-conjugated liposomes includes the delivery of the anticancer drug 5fluorouracil (5-FU) to brain. 5-FU is one of the most powerful anticancer agents, but cannot reach an effective concentration in the brain tumor cells when administered systemically. Soni et al. prepared transferrin coupled liposomes by utilizing the -NH2 groups present on the surface of stearylamine containing liposomes with the COOH groups of transferrin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride as a coupling agent [65]. The biodistribution of free 5-FU, non-coupled and coupled liposomes bearing 5-FU was determined following a single intravenous injection in rats. To measure the fraction of 5-FU reaching the brain and other organs the anticancer drug was coupled with 99mTc-DTPA and the radioactivity using a gamma scintillation apparatus was measured in various organs. The results of in vivo studies showed an apparent selective uptake of the transferrin-coupled liposomes from the brain capillary endothelial cells. An average of 10-fold increase in the brain uptake of the drug was observed after the liposomal delivery of 5-FU, while the transferrin-coupled liposomes caused a 17-fold increase in the brain uptake of 5-FU. This result could be related to the presence of transferrin receptors on BBB and consequently to a greater access of the transferrin-coupled liposomes across the barrier through a RME mechanism. Doi et al. proposed transferrin-conjugated PEGylated liposomes to achieve tumor-specific delivery of sodium borocaptate (Na210B12H11SH, BSH) to malignant glioma, as an application of boron neutron capture therapy (BNCT) to brain cancer [66]. BCNT is based on the nuclear reactions between 10B and thermal neutrons to give high linear energy transfer alpha particles (4He) and lithium-7 (7Li) nuclei (10B + 1n
7Li + 4He). The resulting lithium ions and alpha particles are high linear energy transfer particles which give a high biological effect. Their short range in tissue (5-9 mm) limits radiation damage to those cells in which boron atoms are located. The successful treatment of cancer by BNCT requires the selective delivery of a sufficient number of 10B atoms to tumor cells [67]. The 10B concentrations at level of U87D human glioma cells from three boron delivery systems (bare BSH, PEGBSH, and transferrin-conjugated PEGylated liposomes, TFPEG-BSH) were determined in vitro and in vivo by using inductively coupled plasma-atomic emission spectrometry (ICP-AES). The tumor-to-blood and the tumor-to-normal brain ratios were also evaluated. The TF-PEG-BSH showed the most prominent effects by neutron irradiation among the three delivery systems examined. In fact, TF-PEG-BSH showed highly selective and efficient 10B delivery in tumor tissue. Moreover, the survival rate in tumor-bearing mice after BNCT was best in the TF-PEG-BSH group. These findings demonstrate that TF-PEG-BSH is a potent Boron delivery system for BNCT not only because it delivered a high concentration of 10B to the tumor tissue, but also because it was highly selective for the tumor cells [66]. Modified liposomes have also been used for enhanced gene delivery to brain tumors. Torchilin and coworkers investigated the potential of trans-activating transcriptional peptide (TATp)-modified liposomes to enhance the delivery 1024 191 of a model gene, (i.e., the plasmid encoding for the green fluorescent protein (pEGFP-N1)), to human brain tumor U87 MG cells in an intracranial model in nude mice. TATpliposomes demonstrated an enhanced delivery of pEGFP-N1 both to U-87 MG cells and in vivo selectively to tumor cells (and subsequent effective transfection) compared to plasmidloaded liposomes [68]. Moreover, no transfection was noted in the normal brain adjacent to tumor. As demonstrated by these promising results, TATp-liposomes could serve as potential delivery systems for the transfer of genes to human brain tumors in vivo. Surface modified RGD peptide-liposomes to allow sitespecific drug delivery to brain were described by Qin et al. [69]. RGD peptide (i.e., Arg-Gly-Asp) can combine with integrin receptors that are expressed on the surface of leukocytes (neutrophils and monocytes). Taking into account that in many neurological diseases, leukocytes can across an intact BBB, it is possible to exploit these inflammatory cells as targeted delivery systems. RGD peptide was conjugated to liposomes and these surface modified nanocarriers were then loaded with ferulic acid (4-hydroxy-3-methoxycinnamic (FA)). FA is known to have a wide range of pharmacological effects including antioxidant, radical scavenging, antiapoptotic, antiinflammatory, neuroprotective properties and hence, it can be used as a promising protective agent for the treatment of neurodegenerative disorders such as Parkinson’s, Alzheimer’s, and stroke. However, its poor penetration into brain limits its application to neurodegenerative diseases. In the study of Qin et al. [69] it was showed that i) RGD-liposomes could bind to monocytes/ neutrophils efficiently; and ii) following the administration of RGD-liposomes to brain inflammation-bearing rats the concentration of FA in brain was 6-fold higher than that of FA solution and 3-fold higher than that of uncoated liposomes. Moreover, in pharmacodynamic studies it was found that FA containing liposomes exhibited greater antioxidant activity to FA solution on U937 cells which is a well-characterized model of oxidative cell injury. Hence, the strategy of exploiting inflammatory cells is a promising approach because it can deliver drug directly to the inflammatory site in the brain. Nanoparticles Nanoparticles (NPs) are solid colloidal particles made of polymeric materials ranging in size from 1-1000 nm. This definition includes both nanocapsules, with a core-shell structure (a reservoir system), and nanospheres (a matrix system). Since it is often difficult to prove whether these particles have a continuous matrix or a shell-like wall, the more general term NPs is thus usually accepted [11]. NPs are used as carrier systems in which the drug is dissolved, entrapped, encapsulated, adsorbed or chemically linked to the surface [10]. NPs possess the advantage of a high drugloading capacity and can provide protection against chemical and enzymatic degradation. Examples of synthetic polymers used to prepare NPs are poly(alkylcyanoacrylate) (PACA), acrylic copolymers, poly(D,L-lactide-co-glycolide), and poly(lactide). NPs have also been prepared from natural proteins (albumin and gelatin) and polysaccharides, starch and chitosan). 192 Current Topics in Medicinal Chemistry, 2009, Vol. 9, No. 2 Trapani et al. Like liposomes, NPs are rapidly cleared from the blood following intravenous administration. As carriers for drug delivery to the brain, NPs need to be small (<100 nm) to avoid the RES, neutrophil activation, platelet aggregation, and inflammation. Several attempts have been made to change the biodistribution of NPs. The most promising results were obtained by coating NPs with hydrophilic surfactants or by covalently linking PEG- (PEGylation) or polyethylene oxide-chains on their surface. In vivo studies performed by Kreuter et al. [70], unequivocally demonstrated that a successful targeting to the brain of the hexapeptide dalargin, a Leu-enkephalin analogue with opioid activity, was observed only by overcoating drug adsorbed PACA NPs with different polysorbates such as Tween 80. These fíndings suggested that the overcoating of the NPs with the polysorbates leads to a characteristic alteration of the NP surface properties, responsible for the uptake by the endothelial cells. Various NPs formulations have been used for delivery of drugs including analgesics, antiepileptics, antibiotics, and antitumor drugs to the CNS. Benoit and coworkers [71] provided an interesting application of modified nanocapsules. These authors prepared lipid-based nanocapsules (LNC) using the non-ionic hydrophilic surfactant Solutol® HS15 (hydroxystearate of poly (ethylene glycol) which confers extended circulation times and P-gp efflux pump inhibiting properties. These LNCs were conjugated to OX-26 monoclonal antibodies (OX-26 MAb) and Fab' fragments giving rise to immunonanocapsules that provided delivery of lipophilic radionuclides and therapeutic molecules to the brain. Biodistribution of the immunonanocapsules, labeled with a 188Re lipophilic complex, was determined in healthy rats. At 24 h post-injection, the brain concentrations of Fab'immunonanocapsules and OX-26-immunonanocapsules were, respectively, 1.5 and 2-fold higher than non-targeted nanocapsules. In addition, it was found that the targeting of cerebral tissue with the OX-26-immunonanocapsules was more efficient than that with the Fab'-immunonanocapsules. These targeted nanocarriers appear to be a promising alternative to liposomal systems already employed for active targeting. Besides solid polymeric nanoparticles (NPs), solid lipid nanoparticles (SLN) have also been employed for delivering drugs to the CNS. SLN are dispersions of solid lipids stabilized with emulsifier or emulsifier/co-emulsifier complex in water. Solid lipids employed to prepare SLN are widely used food lipids and commonly used emulsifiers including poloxamers, polysorbates and bile salts. Like liposomes and NPs, the biodistribution of SLN can be manipulated by modifying the surface physico-chemical properties of SLN to address them to the target tissue. In recent years, the potential use of SLN for brain drug targeting purposes has been widely investigated and interesting reviews on this topic has been published [11, 12]. In particular, delivery to the brain of antitumor drugs, including camptothecin, doxorubicin and paclitaxel, incorporated into SLNs and PEGylated SLNs was studied [72-74]. In all of the studies it has been found that significantly higher drug concentrations were detected in the brain when the antitumor drugs were encapsulated and delivered in a SLN. It is 1025 therefore a noteworthy finding that SLNs appear by their nature to be capable of overcoming the BBB.In comparison with surfactant coated polymeric NPs (specifically useful in bypassing BBB), SLN have some potential advantages such as low intrinsic cytotoxicity, physical stability, protection of labile drugs from degradation, controlled release, and easy preparation. Actually, the very low cytotoxicity of SLN and biodegradability of lipids used in their preparation makes them very attractive candidates for brain delivery and particularly for the treatment of brain tumors. In this regard, the potential of SLN as carriers for delivering anticancer drugs has been further underlined in the recent study of Brioschi et al.[75]. In this study the efficacy of SLN as carriers of different types of antineoplastic agents (such as doxorubicin, paclitaxel and the prodrug Cholesteryl butyrate) in brain tumor therapy has been shown in vivo in an experimental rat brain glioma model. It was found that doxorubicin vehiculated by SLN achieved intratumoural concentrations ranging from 12- (after 30 min) to 50- (after 24 hours) folds higher compared to free solutions. In addition, in the contralateral healthy hemisphere in which BBB was not disrupted, doxorubicin-SLN achieved subtherapeutic concentrations, while the free drug did not reach significant levels. On the other hand, i.v. administration of paclitaxel incorporated in SLN to normal rabbits produced drug concentrations in brain tissue ten-folds higher than after administration of paclitaxel free solutions. These results clearly showed that SLN are able to successfully vehiculate cytotoxic drugs into the brain and to induce effective antitumoral response. SLN have also been evaluated a for brain delivery of the potent and frequently used HIV protease inhibitor (PI), atazanavir, that, like other PIs exhibits low brain permeability [76]. Thus, spherical SLNs with an average particle size of about 167 nm were formulated and loaded with [(3)H]-atazanavir. The results showed a significantly higher accumulation in the human brain microvessel endothelial cell line (representative of the blood-brain barrier) as compared to the drug aqueous solution. These data suggest that SLNs could be a promising drug delivery system to enhance brain uptake of atazanavir and potentially other PIs. Polymeric Micelles Polymeric micelles are formed spontaneously in aqueous solutions of amphiphilic block copolymers and have coreshell architecture. Self-assembly occurs when the copolymer concentration reaches a threshold value known as the critical micelle concentration (CMC). The size of polymeric micelles usually varies from ca. 10 to 100 nm. The core is composed of hydrophobic polymer blocks [e.g., poly (propylene glycol) (PPG), poly(D,L-lactide), poly(caprolactone), etc.] and a shell of hydrophilic polymer blocks (e.g., PEG). Most of them are biodegradable and biocompatible. Of particular interest are Pluronic block copolymers that contain two hydrophilic PEG and one hydrophobic PPG blocks (PEG-PPG-PEG). They were shown to cross the membranes of cultured brain microvessel endothelial cells and to inhibit a drug efflux transport protein, P-gp [77]. Therefore, it is expected that the bioavailability at brain level of various P-gp-substrate drugs should increase by using polymeric micelles with Pluronic block copolymers. Another Methodologies to Assess Brain Drug Delivery in Lead Optimization Current Topics in Medicinal Chemistry, 2009, Vol. 9, No. 2 recent study has shown that polymeric micelles selfassembled from the transcriptional activator peptide TATpoly(ethylene glycol) (PEG)-block-cholesterol (TAT-PEG-bChol) can be used as carrier for drug delivery across BBB [78,79]. Ciprofloxacin as a model drug was efficiently loaded into the micelles having spherical nature and average size lower than 200 nm. The uptake of micelles with TAT by human brain endothelial cells was greater than those without TAT. The micelles with TAT may provide a promising carrier for delivering antibiotics across the BBB to treat brain infections. However, it should be noted that differently formulated polymeric micelles were ineffective and unable to deliver antibiotics to CNS. In fact, in the study by Yáñez et al. [80] aimed at determining the tissue distribution in rats of the macrolide antibiotic rapamycin incorporated in PEGblock-poly(-caprolactone) (PEG-b-PCL) micelles formulated with and without the addition of
-tocopherol, it was observed that the uptake by the brain was dramatically reduced compared to control rapamycin dissolved in Tween 80/PEG 400/N,N-dimethylacetamide. These findings, taken together, leads one to conclude that polymeric micelles with Pluronic and those with TAT-PEGb-Chol may open new opportunities for the treatment of some brain diseases, however, more extensive studies are needed. Dendrimers Dendrimers are highly branched polymer molecules formed by a central core to which the branches are attached, the shell of the branches surrounding the core, and the surface formed by the branches termini. They have a size comparable with that of polymeric micelles or nanoparticles of small dimensions. Thus, for instance, a typical dendrimer molecule, such as poly(amidoamine) (PAMAM) dendrimer, has a diameter ranging from 1.5 to 14.5 nm. Various drugs can be entrapped within the dendrimer interior cavities known as “dendritic boxes” and the drug can be released following the shell degradation under physiological conditions. Hydrophobic and hydrophilic polymer blocks can be grafted to the surface resulting in formation of more stable systems that are characterized by longer circulation in the body. As carriers for drug delivery to the brain, dendrimers conjugates with anti-cancer agents have been studied for the treatment of tumors at CNS level [81]. In addition, gene delivery into brain has been also shown using a transferrinconjugated PEGmodified PAMAM dendrimer [82]. A smart development of using dendrimers for brain delivery has been recently reported by Hildgen and coworkers [83]. These authors synthesized polyether-copolyester (PEPE) dendrimers loaded with methotrexate (MTX) and conjugated to Dglucosamine. Glucose conjugation to the dendrimers confers not only enhanced delivery across the BBB but also tumortargeting property through facilitative glucose metabolism by the glucose transporters (GLUT) in the tumors. The antitumor activity of these MTX loaded dendrimers was evaluated against glioma cells and avascular human glioma tumor spheroids. Glucosylated dendrimers were found to be endocytosed in significantly higher amounts than nonglucosylated dendrimers by both the cell lines. IC50 of 1026 193 MTX after loading in dendrimers was lower than that of the free MTX, suggesting that loading MTX in PEPE dendrimers increased its potency. It was found that these targeted dendrimers were able to kill even MTX-resistant cells highlighting their ability to overcome MTX resistance. In addition, by using cocultures of bEnd.3 and U373 MG cells as model for the BBB, the amount of MTX-transported across BBB by glucosylated PEPE dendrimers resulted three to five times more than that in dendrimers alone. The results show that glucosamine can be used as an effective ligand not only for targeting glial tumors but also for enhanced permeability across BBB. 5. CONCLUSIONS Based on the findings presented above, it is increasingly clear that crossing of BBB and drug delivery to CNS is a complex and challenging task requiring close collaboration and common efforts among researchers of several scientific areas including pharmaceutical sciences, biological chemistry, physiology and pharmacology. In this scenario, the role played by the medicinal chemists and pharmaceutical technologists is surely relevant. Medicinal chemistry-based strategies are mainly applicable to low molecular weight drugs. The most followed and efficient medicinal chemistry-based approaches are i) the lipidization (prodrug) approach i.e., the optimization of physicochemical characteristics that allow for passive diffusion through the BBB via the transcellular route, ii) structural modifications resulting in features necessary to serve as a substrate for endogenous influx transport systems of the BBB. However, the latter type of approach seems to possess some intrinsic limitations because the transport systems existing in the BBB are relatively selective systems and devoted to the passage of essential nutrients and metabolites. Therefore, compounds designed to target specific transport systems often result with lower affinity for the transporter than the endogenous ligand. A better understanding of transport mechanisms and those modulating drug efflux transporter activity and drug resistance at the BBB level may be of crucial importance for overcoming, at least in part, these limitations. As for the lipidization through prodrugs, there is a need for new chemical modification approach such as the design and development of dual action prodrugs, for example prodrugs whose promoieties allow not only enhanced BBB permeability but also targeting to specific sites. The use of appropriate nanocarrier systems for low and high molecular weight drugs may be advantageous because they are non-invasive systems, allow good drug loading capacity and protect the molecules to be delivered against inactivation mechanisms and clearance. Liposomes, surfactant coated polymeric nanoparticles, and solid lipid nanoparticles are promising systems for delivery of drugs to CNS. At present, it seems that particulate systems can be successfully exploited for delivering drugs to the CNS in the case of brain tumors with disorganized vasculature and leakier capillaries. However, there are other limiting factors for the use of nanocarriers-based CNS delivery systems. 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