11Prodrug Approaches for Central Nervous System DeliveryQuentin R. Smith and Paul R. Lockman

11.1Blood–Brain Barrier in CNS Drug Development

The central nervous system (CNS) offers special opportunities and challenges forprodrug design and development. Critical among the opportunities is the wide rangeof neurotransmitter and neuromodulator systems in the CNS that provide attractivetargets for drug therapy. Some of these systems are present in different extents in theperipheral nervous system and other organs; hence, selective targeting to the CNS isbeneficial. Further, the brain is the site of a broad range of psychiatric, neurologic, andor neurodegenerative disorders,many ofwhich are chronic, have a component that isgenetic or inheritable, and currently have no curative therapy with treatment mainlypalliative to control symptoms. Based upon figures published by the NationalInstitutes of Health, one of three individuals suffers from a diagnosable CNSdisorder in any given year. CNS disorders represent 5 of the top 10 causes ofdisability and constitute >20% of total health care spending in the United States.Further, the number of individuals with CNS disorders is increasing in manycountries as populations age [1]. The CNS drug market (>$55 U.S. billion in2005) is second only to the cardiovascular drug market in total drug sales [2].However, CNS drugs take longer on average to get to market and have lower successrates relative to other drug classes.

Many factors contribute to the challenge of CNS drug development. Includedamong these are the unique properties of the �blood–brain barrier� (BBB). The brain,unlike most organs, has a highly selective and restrictive molecular permeabilityinterface between the circulation and the brain interstitial space, which impedes theexchange of most solutes between the blood and the CNS [3]. The BBB is formed atthe cellular level by the cerebral vascular endothelium that is joined together by highresistance tight junctions (>1500 ohmcm2) [4]. These tight junctions, formed by theproteins occludin and claudin, effectively seal off the aqueous paracellular channelsbetween brain endothelial cells, forcing most drugs and other solutes to cross by thetranscellular route [5, 6]. Thus, for a drug to pass readily into brain from the

Prodrugs and Targeted Delivery: Towards Better ADME Properties. Edited by Jarkko RautioCopyright � 2011 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 978-3-527-32603-7

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circulation, it must either have the appropriate physiochemical properties related tolipid solubility, polarity, ionic charge, and size to readily dissolve and diffuse acrossthe lipophilic cellmembranes of the brain vasculature [7] or be transported across theendothelium by one of >20 or more active or facilitated carrier systems that areexpressed in brain endothelial cells at high levels [8]. Diazepam is one drug thatreadily crosses the BBB by passive lipid diffusion across brain vascular endothelialcells. It has moderate lipophilicity (log Doctanol/saline pH 7.4¼ 3), neutral charge, andlowmolecularweight (<400), all factors favoring goodBBBpenetration [9]. Further, itdoes not appear to be a substrate of an active BBB drug efflux carrier, such asP-glycoprotein (Pgp), breast cancer resistance protein (BCRP), ormultidrug associateresistance proteins (MRP 1–7) or organic anion transporter 3 (OAT3). All of thesetransporters have been shown tomediate active drug efflux out of the CNS, leading tolower free drug levels in brain interstitial fluid than serum free drug concentration.As a consequence, diazepam equilibrates in brain rapidly (<15min) after systemicadministration at brain free levels equivalent to serum free drug concentration [10].The total brain concentration exceeds that free in serum at steady state by about 20-fold due to diazepam binding to CNS protein and membranes. Overall, fromcomparison of common properties of CNS active drugs, Lipinski recommendedthe �rule of five� for good CNS exposure [7]:

. 3 or fewer H-bond donors (OHs, NHs)

. 6 or fewer H-bond acceptors (Os and Ns)

. Molecular weight is <400

. c log P< 5

. Low active efflux transporter affinity

Other drugs with good BBB penetration and distribution properties includeethanol (alcohol), nicotine, caffeine, antipyrine, chlorpromazine, imipramine, pro-pranolol, naproxen, paroxetine, flunitrazepam, and valproate [9]. Most of these drugshave moderate lipophilicity (log Doctanol/saline pH 7.4¼ 0–3) and are either neutral orreversible anions or cations (with a measurable neutral fraction).

Drugs with limited brain distribution often exhibit one or more of the followingproperties:

. High molecular weight (>1000–1 000 000 Da)

. Permanent charge (e.g., quaternary nitrogen)

. Polar with reduced lipophilicity and enhanced hydrogen bonding (e.g., sucrose)

. Substrate for one or more active efflux transporters

Strongly polar compounds, such as sucrose, inulin (3–5 kDa) or dextrans(3–2000 kDa), cross the BBB to only a limited degree and are rapidly removed frombrain interstitial fluid by the CSF sink effect [11]. Therefore, their brain levels fail toreach that of plasma even after long exposure periods. Among active efflux transpor-ters,Pgp,BCRP,MRP4,andOAT3allhavebeenshowntobeexpressedathigh levels attheBBB[12]andtohavecritical roles inlimitingbrainexposureformanyagents [8].Forexample, BBB active efflux transport has been shown to restrict brain exposure ofcardiovascular (e.g., digoxin, quinidine, verapamil, and lovastatin), immunologic

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(e.g., cortisol, dexamethasone, and cyclosporine), anticancer (e.g., paclitaxel,methotrexate, vinblastine, doxorubicin, and lapatinib), anti-infective (ivermectin,erythromycin, and aprenavir), opioid (morphine, methadone, fentanyl, and lopera-mide), and antiepileptic (phenytoin) compounds [13]. Pgp transports a broad array ofcompounds, favoring agents with >8 (NþO)s, >400MW, and pKa> 4 [14]. TheMRPs are a family of transporters that mediate an equally broad array of substrates,including anions, drug conjugates (with glutathione, glucuronic acid, or sulfate), andnucleosides. For Pgp, BCRP, andMRP, active efflux is driven by hydrolysis of ATP toform ADP, whereas for other transporters (e.g., OAT3) net transport is driven byexchange with ions or other compounds.

As a result of low passive permeability and active efflux transport at the BBB>98%of new chemical entities have been reported to show poor penetration into brain [15].Among critical new biologic agents (i.e., new therapeutic proteins, peptides, andnucleic acids withMW> 10 kDa), the percentage that shows poor delivery to brain isevenhigher (>99%).As a result, theBBBhas been referred to as �the problem lurkingbehind the problem of CNS drug delivery� [16].

11.2Prodrug Strategies

A primary focus of CNS prodrug strategies has been to improve drug uptake andtargeting across the BBB followed by spontaneous ormetabolic breakdown to releaseactive agent that is preferentially retained in brain (Figure 11.1). In thismanner, drugexposure of active agent is enhanced in theCNSand reduced in other organs thatmaybe susceptible to toxic effects. In some instances, the primary problem has beengetting active drug to the site of actionwithin the CNS across the BBB. In other cases,selective expression of CNS or disease state enzymes has been utilized to catalyzelocal release of active agent.

Prodrugs are common in somefields, such as oncology, where a number of the keydrugs are antimetabolites. These agents are taken up into cells and converted to activespecies by enzymes within the cytoplasm and nucleus. Classic examples include5-fluorouracil, 6-mercaptopurine, and 6-thioguanine, which are converted withincells to antimetabolite nucleosides and nucleotides that interfere with nucleic acidand protein synthesis and function. They target cancer cells with elevated rates ofprotein synthesis and mitosis. Capecitabine is a more recently developed prodrug of5-fluorouracil, which relies on two additional steps, one of which is preferentiallyupregulated in tumor cells, to target 5-fluorouracil formation in cancer cells and limitexposure to normal cells.

Anticancer drug delivery is a particular problem for therapy of primary braintumors and for brain metastases of systemic tumors, as the BBB expresses multipleefflux transporters that limit CNS exposure of nucleobases, nucleosides, andnucleotides used in cancer therapy, aswell asmost natural product chemotherapeuticagents, such as paclitaxel, doxorubicin, vincristine, methotrexate, and etoposide.Therefore, creative prodrug strategies are being explored to improve brain delivery of

11.2 Prodrug Strategies j255

chemotherapeutic drugs, as well as drugs from other classes, which show restricteddelivery to the CNS. Included within this are antibiotic and antiviral agents, many ofwhich show restricted distribution to brain for the treatment of CNS and meningealinfections.

A number of reviews have covered lipophilic CNS prodrugs that are convertedpreferentially within the CNS into trapped or active species based upon oxidation/reduction, esterase, or other enzymatic mechanisms [17–20]. Such approaches havesome potential for targeting as the brain receives 15% of the cardiac output eventhough it comprises only 2% of body weight in humans. Thus, if the compound hashigh CNS extraction (>75% per single pass through the vascular bed) followed byrapid conversion to trapped or active species, the potential exists for selective CNSaccumulation above that expected for distribution based upon organ size. However,enhanced lipophilicity, although it may improve passage across the BBB, is alsoassociated with other problems, including rapid metabolic clearance, enhancedbinding to plasma and tissue proteins, elevated volume of distribution, and increasedaffinity for active efflux transporters. Hence, in many cases, it may not improve thefraction of drug that distributes to brain, and in some cases, may actually beassociated with marked reductions.

Therefore, this chapter will focus on newer prodrug strategies focused onpreferential brain targeting based upon BBB carrier- or receptor-mediated transport.Over 20 carrier transporters have been identified at the cerebral capillaries of the BBBthat mediate the brain uptake of various essential nutrients, vitamins, peptides, andhormones. This includes GLUT1 that transports D-glucose into brain for cerebralenergy metabolism, as well as LAT1 and CAT1, which, respectively, transportessential large neutral and basic (cationic) amino acids into brain for proteinsynthesis and neurotransmitter (e.g., serotonin, dopamine, norepinephrine, andhistamine) formation [8]. Further, the low-affinity choline transporter is also highly

Figure 11.1 Schematic model of prodrugstrategy for drug delivery to the CNS utilizing aprodrug structure to facilitate transfer acrossthe BBB. Once within the CNS compartment,

the prodrug is then either enzymatically ornonenzymatically converted to active drugspecies that interacts at the active site.

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expressed at theBBBandmediates brain entry of the quarternary amine, choline, intobrain for phospholipid and acetylcholine synthesis. Similarly, the BBB also expressesa large number of protein receptors, including low-density lipoprotein receptor-related protein (LRP1), insulin receptor, transferrin receptor, and diphtheria toxinreceptor [21]. Drug delivery vectors have been created for each of these receptorsusing antibodies or selective substrates that bind and are transcytosed into brainacross the BBB. Due to preferential BBB expression of these receptors and trans-porters, the potential exists for enhanced drug delivery and trapping by brain withreduced exposure and toxicity to other organs.

11.3Prodrug Strategies Based Upon BBB Nutrient Transporters

Most plasma large neutral amino acids are taken up into brain by the sodium-independent, System LAT1 exchange transporter. This carrier was characterizedinitially by Oxender and Christensen [22] and was subsequently cloned by Kanai [23].It mediates the brain uptake of >10 primary neutral amino acids in plasma [24] viasaturable, stereospecific (preferring L > D), bidirectional and sodium/energy inde-pendent transport. Of the carriers at the BBB, System LAT1 displays severalproperties that make it well suited as a brain drug delivery vector. First, it has botha large maximal transport capacity (Vmax �40–60 nmol/min/g) and an appreciablebinding affinity (affinity� 1/Km; Km¼ 10–200mM) [25] so that rapid rates of blood–brain barrier exchange can be obtained (Kin> 10�3 ml/s/g) with half times for brainequilibration of <15min for high-affinity substrates. Second, it possesses fairlysimple structural requirements for binding and accepts a wide variety of amino acidsubstrates [26]. And third, although the carriermediates the brain uptake of essentialamino acids that are required for brain metabolism, transient disruption in brainsupply, as might occur with drug overdose, does not produce irreversible braindamage. This latter possibility may limit use of the cerebrovascular glucose trans-porter (GLUT1) as a drug delivery vector because of the critical need of the brain for acontinuous supply of D-glucose for brain energy metabolism.

BBB System LAT1 has been shown to mediate brain delivery of L-DOPA as adopamine precursor for the treatment of Parkinson�s disease [27]. Dopaminenormally exhibits very low uptake into brain across the BBB. The dopaminedeficiency that occurs in Parkinson�s disease can be treated with dopamine agoniststhat cross the BBB, but not with dopamine itself due to restricted diffusion ofdopamine across the BBB. This dilemma was solved by administration of L-DOPA,which is shuttled across the BBB by System LAT1 and subsequently decarboxylatedinto dopamine by aromatic amino acid decarboxylase to create a successful therapy.Subsequently, this same carrier has been used for brain delivery of gabapentin andpregabalin.

System LAT1 offers special promise as a brain delivery vector for chemothera-peutic drugs as it is upregulated at the BBB in brain tumors [28, 29]. One prodrugtherapy initially targeted in this direction is L-melphalan, the para-nitrogen mustard

11.3 Prodrug Strategies Based Upon BBB Nutrient Transporters j257

analogueof L-phenylalanine (Figure11.2). L-Phenylalanine isanexcellent substrate forBBB System LAT1 with a Km¼ 11� 1 mMand a Vmax of 41� 2 nmol/min/g [25, 30].However, melphalan is a weak substrate for System LAT1, with >10-fold-reducedaffinity and 8-fold lower Vmax than L-phenylalanine. As a consequence, BBB�permeability� to melphalan is only 1% that of L-phenylalanine, leading to reducedbrain accumulation and anticancer drug exposure [31].

Various strategies have been explored to enhance BBBmelphalan transport acrossthe BBB, including intra-arterial administration and amino acid lowering diets andenzymes [32, 33]. However, the poor transport affinity and capacity of System LAT1for melphalan have proved limiting. In contrast, Haines et al. [34] proposed a tetralinanalogue of melphalan with improved affinity for System LAT1. This compound,DL-2-NAM-7 (Figure 11.2), has 50-fold greater affinity (Km¼ 0.2� 0.02 mM) forSystem LAT1 than melphalan with an effective BBB �permeability� within 2–3-foldof that of L-phenylalanine. It equilibrates in brain in 8–10min, as compared to a2 h t1/2 for melphalan [30, 31].

Both melphalan and DL-2-NAM-7 are prodrugs, which enter brain and areconverted sequentially into azridinium and carbonium ions on the nitrogenmustard that alkylate nucleic acids and proteins (Figure 11.2). Similarly, Gyntheret al. [35] created a LAT1 prodrug of theNSAID ketoprofen (Figure 11.3) based uponL-tyrosine with a BBB Km of 22� 9 mM and a Vmax of 1.4� 0.15 nmol/min/g. Thecompound was transported into rat brain by System LAT1 and subsequently

Figure 11.2 Left: Structure of L-phenylalanineand L-phenylalanine nitrogen mustardanalogues, melphalan and DL-2-NAM-7. Right:Schematic showing conversion of the nitrogen

mustard to the active azridinium ionsintermediate that alkylates electron-rich speciesincluding proteins and nucleic acids.

258j 11 Prodrug Approaches for Central Nervous System Delivery

hydrolyzed in brain. The enzymatic hydrolysis followed an in vitro half-life of4.4� 1.8min in brain homogenate and 10.2� 0.4min in serum. Similarly, Gyntheret al. [36] prepared glucose analogues of ketoprofen and indomethacin thatcompetitively reduced brain D-glucose uptake by GLUT1 and showed measurableuptake into brain.

BBB System LAT1 has also been used to achieve brain delivery of N-methyl-D-aspartate (NMDA) receptor antagonists, 7-chlorokynurenic acid and 5,7-dichloroky-nurenic acid (Figure 11.4) [37]. These agents are the prototypic antagonists thathelped define the glycine coagonist site of theNMDAreceptor and show� two ordersof magnitude better potency and affinity (1/Ki) for the receptor than the nonspecificexcitatory amino acid antagonist kynurenic acid [38, 39]. NMDA receptors playimportant roles in neuronal damage induced by excitatory amino acids followingstroke, head injuries, and seizures, and have been suggested to contribute to celldeath in several neurodegenerative diseases [40]. When administered directly intothe CNS, 7-chlorokynurenic acid and 5,7-dichlorokynurenic acid are neuroprotectivein models of excitotoxic injury and neurodegeneration. However, due to poortransport across the BBB, they show minimal passage into brain following systemicadministration. Hokari et al. [41] demonstrated that systemic administration ofkynurenic acid precursors, 4-chlorokynurenine and 4,6-dichlorokynurenine resultedin System LAT1 transport into brain followed by enzymatic conversion to thekynurenic acid products (Figure 11.5). Previous studies by the same group hadshown saturable transport of L-kynurenine across the BBB [42]. Similar studiesshowed that transport of the chlorokynurenine precursors was self-saturable andinhibitable by L-leucine, characteristics consistent with BBB System LAT1 transport

Figure 11.3 L-Tyrosine and D-glucose analogues of ketoprofen.

11.3 Prodrug Strategies Based Upon BBB Nutrient Transporters j259

(Figure 11.6). Best-fit kinetic estimates of BBB transport for 4-chlorokynureninewereKm¼ 105� 14 mMandVmax¼ 17� 2 nmol/min/g [41]. 4,6-Dichlorokynurenine alsoshowed concentration dependent inhibition of BBB System LAT1 L-leucine transportwith a Ki¼ 410� 18mM. Systemic administration of 4-chlorokynurenine resultedin measurable brain levels of both 4-chlorokynurenine and its metabolic product,7-chlorokynurenic acid [41] (Figure 11.6). Subsequent studies have confirmed thesefindings under disease conditions, which augment the quantity of kynurenic acidproduct formed [43, 44].

Thus, these studies have demonstrated the efficacy of System LAT1 prodrugdelivery across the BBB for the treatment of CNS diseases. Beyond L-DOPA, thestrategy has been utilized for anticancer amino acid prodrugs (e.g., melphalan/NAM), and amino acid-NSAID conjugates, and precursors of neuroprotectivekynurenic acids. Battaglia et al. [45] explored brain delivery of 7-chlorokynurenicacid using systemically administered 7-chlorokynurenic acid-D-glucose conjugatestargeted toward theBBBGLUT1 transporter This strategy avoidedCNS conversion ofchlorokynurenine to the kynurenic acid active species. Further, the BBB GLUT1transporter has greater capacity by>30-fold than the BBB LAT1 carrier. The authorsdemonstrated uptake of the prodrug and release of the free drug using the GLUT1targeted strategy. However, as previously noted, the BBB GLUT1 transport systemplays a critical role in brain delivery of glucose for cerebral energy metabolism.Therefore, this may not be the best BBB transport system to target for brain delivery,as overdose of the prodrug agent may compromise brain glucose supply, leading tocerebral hypoglycemia, seizures, or coma.

Similar to LAT1, the BBB choline transporter (CHT) has the ability to deliverboth cationic prodrugs and its primary substrate choline to the CNS concurrently.

Figure 11.4 Schematicmodel of L-kynurenine prodrug strategy showing 4-Cl-kynurenine transportacross the BBB by system LAT1 and conversion within brain by the enzyme kynurenineaminotransferase to the active, neuroprotective species, 7-Cl-kynurenic acid.

260j 11 Prodrug Approaches for Central Nervous System Delivery

This is possible since endogenous choline plasma concentrations are only �25%of the Km of the CHT [46, 47] and that the CHT is rather promiscuous in bindingmolecules that bear cationic charges. It was shown the BBB CHT translocated twonicotinic antagonists, which are preclinical leads for smoking cessation therapy,across the BBB to achieve 10-fold greater brain distribution than that predicted bypassive permeation. These compounds are the bis-quaternary ammonium, N,N0-dodecyl-bis-picolinium bromide [48] and the N-n-alkylnicotinium analogue, N-n-octylnicotinium iodide (NONI)(Figure 11.7) [46]. Further work demonstrated thatafter crossing the BBB, these two nicotinic antagonists inhibit nAChR-mediatednicotine-evoked [3H]-dopamine release with an IC50 of 2–5 nM [49]. While theCHT transporter may have a unique ability to distribute a wide variety of chargedcations into brain [50], unlike LAT1, it has not been cloned, which limits drugdesign and discovery. To overcome this deficit, molecular modeling has been usedto outline structural binding and transport requirements (Figure 11.7) [50], whichinclude (1) a significant need for the presence of a cationic nitrogen center in the

Figure 11.5 Structures of kynurenine, 4-chlorokynurenine and 4,6-dichlorokynureninetogether with matching neuroprotectivemetabolites, kynurenic acid, 7-chlorokynurenic

acid and 5,7-dichlorokynurenic acid. Kivalues [38, 39] represent the inhibition constantat the glycine binding site of the NMDAreceptor.

11.3 Prodrug Strategies Based Upon BBB Nutrient Transporters j261

0

10

20

30Control

1mM LEU

7-CI-KYNAB

rain

Co

nc

en

tra

tio

n (

pm

ol/g

)

0

1

2

3

4

5

Transport Km

105 ± 14µM

4-CI-KYN

Bra

in C

on

ce

ntr

ati

on

(n

mo

l/g

)

Figure 11.6 Graphs showing reduction in brain uptake of 4-chlorokynurenine by competition fromL-leucine (1mM) and matching reduction in amounts of 7-chlorokynurenic acid formed within theCNS. Data are from Ref. [41].

Figure 11.7 Structures of choline(endogenous substrate) and the two nicotinicantagonists N,N0-dodecyl-bis-picolinium, andN-n-octylnicotinium, which are shuttled fromblood to brain via the BBB CHT. Structuralrequirements of a molecule to use this

transporter include a requisite cationic nitrogen(square); a hydrophobic interaction adjacent tothe quaternary ammonium center (circle); and,less significantly, a hydroxyl groupapproximately from3.26 to 3.30A

�away from the

cationic nitrogen (triangle).

262j 11 Prodrug Approaches for Central Nervous System Delivery

molecule [51], (2) a molecule that provides a hydrophobic interaction around theanionic binding site that accommodates the quaternary ammonium center, and (3)to a lesser degree the presence of at least one hydroxyl group located approximatelyfrom 3.26 to 3.30 A

�away from the cationic nitrogen center [52–54]. Further work

on utilizing this nutrient transporter as a vector for the delivery of cationicprodrugs may be beneficial since it may improve the less than 1% blood-to-brainextraction (related to diffusion) normally observed at the BBB with chargedcations [53, 55, 56].

11.4Prodrug Strategies Based Upon BBB Receptors

In amanner comparable to the BBB carrier-mediated approach, significant work hasalso focused on brain drug delivery using BBB receptor-mediated transport strate-gies. Pardridge [57] has pioneered BBB transcytosis of antibodies to the transferrinand insulin receptors for brain delivery of small drugs, peptides, proteins, nano-particles and liposomes. Similarly, Karkan et al. [58] have explored brain deliveryusing drug conjugates of the iron transport protein, melanotransferrin. With thisapproach, they found enhanced survival in mice bearing intracranial mammarytumors or gliomas after treatmentwith themelanotransferrin-doxorubicin conjugatethan with free doxorubicin alone.

In similar manner, a number of investigators have explored BBB prodrugdelivery using absorptive-mediated transcytosis strategies based upon cationicpeptides and proteins – that is, �cell-penetrating peptides� such as penetratin,transportan, HIV Tat peptide and homoarginine peptides (Arg 7 and 9) [59]. Forexample, Tanabe et al. [60] found 3–4-fold enhanced brain uptake of a cationicarginine-vasopressin peptide via absorptive-mediated endocytosis than of thematching parent peptide. The cationic peptide was specifically tailored withadditional arginine, histidine, and proline residues that would be cleaved off bypostproline cleaving enzyme once peptide entry was gained to the CNS. Similarly,Rousselle et al. [61] showed markedly enhanced (30-fold) brain delivery of theanticancer drug doxorubicin into brain following conjugation to the cationicpeptides, penetratin and SynB using the in situ brain perfusion technique. BBBtransport matched that expected for an absorptive-mediated transport process,showing inhibition with added cationic peptide or polylysine. However, followingintravenous administration, brain uptake of the doxorubicin-SynB conjugate onlyexceeded that of matching free doxorubicin for 10–30min and at the highest pointonly achieved a 2.5-fold gradient, suggesting that the conjugate may efflux out ofbrain rapidly or that other factors conspire to limit brain accumulation via thisstrategy. One hypothesis put forward to explain this discrepancy is that, becausecationic peptides increase uptake intomost cells, there is little preferential increasein individual tissues, such as the percent injected dose that goes to brain.

A matching prodrug strategy based upon BBB receptor-mediated transport usesLRP1. LRP1 is one member of a family of LDL receptor proteins [62]. It is

11.4 Prodrug Strategies Based Upon BBB Receptors j263

preferentially expressed at the endothelial cells of the BBB and mediates rapid celluptake of a wide variety of ligands, including RAP, a2-macroglobulin, aprotinin,lactoferrin, and tPA, among others. LRP transport has been utilized to delivercyanoacrylate nanoparticles bearing drugs to the CNS [63]. More recently, LRP1 hasbeen targeted via the 19 amino acid AngioPep peptides (e.g., AngioPep-2) that bind toLRP1 and are transcytosed across the BBB in vitro and in vivo [64]. Transport via thispathway is competitively reduced by self-peptide as well as LRP1 ligands, includingRAP. Further, LRP1mediated transport of RAP anda2-macroglobulin is inhibited bycoadministration of AngioPep-2 [65].

The AngioPep-2 peptide has been conjugated to anticancer drugs, includingpaclitaxel, doxorubicin, and etoposide [66, 67]. Figure 11.8 illustrates the Angio-Pep-2 prodrug strategy for paclitaxel. Paclitaxel is a large, highly hydrophobicnatural product anticancer compound that normally crosses the BBB very poorlydue to BBB active efflux transport. Conjugation of three paclitaxel residues toAngioPep-2 leads to the ANG1005 prodrug that shows 100-fold greater brainaccumulation than paclitaxel by in situ rat brain perfusion (Figure 11.8) [68].ANG1005 is taken up into brain and is not simply bound to the vascular luminalmembrane, as it cannot be washed out of the vasculature by short perfusion withdrug free saline and is present mostly in the brain parenchyma fraction afterremoval of cerebral endothelial cells by capillary depletion. ANG1005 exhibits4–50-fold improved delivery to brain and brain metastases of breast cancer whenadministered in vivo to mice [68]. Conjugation of paclitaxel to AngioPep-2 not onlyallows receptor-mediated transcytosis of the conjugate prodrug across the BBB butalso avoids active efflux drug transport of paclitaxel back across the BBB into thecirculation. Experiments by Regina et al. [67] showed markedly enhanced brainpaclitaxel accumulation after IV administration of paclitaxel in Pgp knockout mice,whereas brain ANG1005 uptake is unaffected in matching Pgp transporterknockout animals. For anticancer activity, ANG1005 must be cleaved by esterasesto release free paclitaxel. Consistent with improved brain accumulation, theANG1005 conjugate demonstrated improved in vivo anticancer activity againstprimary brain tumors and against brain metastases in mice [67]). ANG1005 iscurrently in Phase 1 clinical trials in humans as a new brain tumor anticanceragent with improved delivery across the BBB. The AngioPep-2 peptide has alsoshown promise for promoting brain uptake of biodegradable nanoparticles, asdemonstrated by Ke et al. [69].

11.5CNS Prodrug Summary

In conclusion, a number of prodrug vectors have been developed to promote drugdistribution to the CNS to achieve therapeutic concentrations at key brain sites.Most studies have recently focused upon carrier-mediated and receptor-mediatedBBB transport strategies to promote selective brain targeting and retention, basedupon the concept that the BBB shows enhanced levels of critical nutrient

264j 11 Prodrug Approaches for Central Nervous System Delivery

transporters (e.g., LAT1 and GLUT1) and receptors (e.g., insulin receptor andLRP1) than other organs. Given that the brain represents 1–3% of body weightin animals, brain distribution for a drug that shows equal distribution amongtissues would be expected to be only 1–3%. In fact, most drugs show muchless brain distribution than 1–3% of injected dose by one to three orders ofmagnitude because of the presence of BBB. The hope is that with selective prodrug

Figure 11.8 (a) Schematic showing thestructure of ANG1005, the 3 paclitaxel þ 1AngioPep-2 peptide conjugate. (b) Timecourse of AngioPep-2 uptake into brain byin situ rat brain perfusion. Inulin is a markerof vascular volume, which normally does notcross in short experiments (<15min). Thelow values for inulin space (�1%) indicatethat AngioPep-2 does not alter the passivepermeability of the BBB. (c) Capillarydepletion experiment showing that themajority of ANG1005 tracer is found in the

vascular-free, parenchymal fraction.AngioPep-2 was perfused through the brainvasculature for 2min prior to post perfusionwashout followed by capillary depletion usingdextran. (d) Mean BBB permeability–surfacearea product (PS) values for initial brainuptake of ANG1005, AngioPep-2, andpaclitaxel during in situ brain perfusion.Points and bars equal mean� SD or SEMfor n¼ 3–12 perfusions. Data are fromRef. [68].

11.5 CNS Prodrug Summary j265

targeting and retention, this percentage can be increased above 1–3% to achievetrue preferential distribution to the CNS.

Acknowledgments

We would like to acknowledge the contributions of Chris Adkins and RajenderMittapalli to the preparation of this paper. This research was supported by grant5R01NS52484 from NIH/NINDS and grant W81XWH-062-0033 from the Depart-ment of Defense Breast Cancer Research Program.

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