Advanced Drug Delivery Reviews 64 (2012) 629–639

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Advanced Drug Delivery Reviews

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Drug delivery to the brain in Alzheimer's disease: Consideration of theblood–brain barrier☆

William A. Banks ⁎Geriatric Research, Education, and Clinical Center, Veterans Affairs Puget Sound Health Care System, Seattle, WA, USADivision of Gerontology and Geriatric Medicine, Department of Internal Medicine, University of Washington School of Medicine, Seattle, WA, USA

☆ This review is part of the Advanced Drug Delivery Re⁎ Bldg 1/Rm 810A, 1660 S Columbian Way, Seattle, W

E-mail address: wabanks1@uw.edu.

0169-409X/$ – see front matter. Published by Elsevier Bdoi:10.1016/j.addr.2011.12.005

a b s t r a c t

a r t i c l e i n f o

Article history:Received 3 August 2011Accepted 9 December 2011Available online 17 December 2011

Keywords:Blood–brain barrierAlzheimer's diseaseDrug deliveryCerebrospinal fluidBiologicalsPeptidesRegulatory proteinsTransportTransmembrane diffusionP-glycoprotein

The successful treatment of Alzheimer's disease (AD) will require drugs that can negotiate the blood–brainbarrier (BBB). However, the BBB is not simply a physical barrier, but a complex interface that is in intimatecommunication with the rest of the central nervous system (CNS) and influenced by peripheral tissues.This review examines three aspects of the BBB in AD. First, it considers how the BBB may be contributingto the onset and progression of AD. In this regard, the BBB itself is a therapeutic target in the treatment ofAD. Second, it examines how the BBB restricts drugs that might otherwise be useful in the treatment of ADand examines strategies being developed to deliver drugs to the CNS for the treatment of AD. Third, it con-siders how drug penetration across the AD BBB may differ from the BBB of normal aging. In this case, thosedifferences can complicate the treatment of CNS diseases such as depression, delirium, psychoses, and paincontrol in the AD population.

Published by Elsevier B.V.

Contents

1. Introduction: the specter of Alzheimer's disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6302. Definitions of the BBB and its general relation to drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6303. The BBB as a cause of AD and therapeutic target . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 631

3.1. BBB disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6313.2. Decreased cerebrovascular blood flow and uptake of oxygen and glucose by the AD brain . . . . . . . . . . . . . . . . . . . . . . 6323.3. Vascular injury and a stroke/multi-infarct dementia (MID)/AD spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6323.4. CSF drainage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6333.5. Endothelial function/secretions/BBB cell inflammatory responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6333.6. Abeta transport: RAGE, LRP-1, and P-gp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6333.7. Other altered transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633

4. Strategies used to deliver AD drugs to the brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6344.1. BBB disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6344.2. Lipid solubility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6344.3. Antibodies as therapeutic agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6344.4. Transport systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 635

5. Alterations in the AD BBB: effects on drug delivery to the brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6355.1. BBB disruption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6355.2. Decreased CSF reabsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6365.3. Decreased cerebral blood flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6365.4. P-gp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 636

views theme issue on “Delivery of Therapeutics to the Central Nervous System”.A 98108, USA.

.V.

630 W.A. Banks / Advanced Drug Delivery Reviews 64 (2012) 629–639

6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 636Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 636References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 636

1. Introduction: the specter of Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disease that istypically characterized by its histological findings of neurofibrillarytangles and amyloid plaque, by increased levels of oxidative stressand neuroinflammation, and by greatly reduced levels of acetylcho-line. Chiefly characterized in its early stages by a decline in recentmemory, its later stages are characterized by a cognitive decline soprofound that its victims lose the abilities, interests, and skills to per-form even simple activities of daily living such as bathing, dressing,eating, and toileting. Long before these final stages, a person withAD can no longer recognize children, spouses, and siblings and losethe personality traits that had characterized them as individuals.These losses coupled with the appearance of other behavioral prob-lems that include violent behaviors, depression, delirium, variouspsychoses, and loss of judgment and social skills means that manypersons with Alzheimer's disease spend their last years institutional-ized and in a mental exile from others.

AD is epidemic with an estimated 33.9 million people worldwidehaving the disease [1]. The incidence rate increases exponentiallywith aging so that at age 90 about 12% of people have AD, but about40% of those over age 100 have it [2]. Several factors that put personsat increased risk of AD are a history of head injury, obesity, diabetesmellitus, hypertension, renal disease, and histories of smoking, trau-matic brain injury, or depression [1,3,4]. As the occurrence of manyof these factors is becoming more common, the incidence of ADmay increase even more. Clearly, interventions that prevent, stabilize,remediate, or cure AD are desperately needed.

To some extent, a fundamental difference in the approach to ther-apeutic discovery has arisen between the clinical and basic sciences.Clinical work has shown that AD begins years prior to onset of symp-toms as detected by commonly used cognitive instruments [5]. Giventhe assumption that cognitive decline and other symptoms in AD areprimarily caused by irreplaceable neuronal loss, it is also assumedthat AD drugs must be started as early as possible. This thinking hascontributed to the recent NIH-supported revision in Alzheimer's diag-nostic guidelines having a greater emphasis on preclinical diagnostictests, imaging, and biomarkers (http://www.nia.nih.gov/Alzheimers/Resources/diagnosticguidelines.htm). Such earlier diagnosis wouldbe needed in part to adequately test drugs at a time in the diseasewhen brain function can be largely preserved. In contrast, work in an-imal models of AD shows that the histopathological hallmarks andcognitive impairments can be reversed even in animals with long-standing disease [6–13]. The animal work, then, suggests that a re-versible neurotoxicity mediates the symptoms of AD to a significantdegree. This, in turn, offers hope that even well-advanced cases ofAD may be treatable and that preclinical diagnosis is not a prerequi-site to effective therapy. In short, clinical research posits that treat-ment must be started early to be effective, whereas basic scienceresearch posits that treatments can be effective even in well-establisheddisease.

Successful treatment of AD is likely to be pharmacologically basedand will in almost every case target the central nervous system (CNS).Drugs targeted to the CNS must negotiate the blood–brain barrier(BBB). This review will examine various aspects of the BBB that are rel-evant to drug delivery. This includes strategies for getting drugs acrossor in some cases bypassing the BBB. It also includesways inwhich alter-ations in the BBB can contribute to the onset and progression of AD; insuch cases the BBB itself is a therapeutic target. Finally, the review will

consider how alterations in the BBB of AD patients can alter uptake ofCNS drugs in general and so complicate the treatment of depression,psychoses, and other CNS diseases in the AD patient.

2. Definitions of the BBB and its general relation to drug delivery

The term BBB has been used to mean many things in the scientificliterature. It's roots began with experiments performed in the late19th and early 20th centuries that found that some dyes could stainthe brain after direct injection into the brain but not after peripheraladministration [14]. A parallel line of study found that bile salts in-duced seizures when injected directly into the brain but not afterperipheral administration. Explanation of the results of the dyestudy depends not only on a barrier separating the brain from theblood but also on the dye binding tightly to albumin in the blood.Thus, a conceptual definition of the BBB consistent with original ob-servations is that of a composite of processes that control the ex-change of substances between the blood and the fluids of the CNS(the brain interstitial fluid and the cerebrospinal fluid). This was ele-gantly stated by Roth and Barlow [15] over 50 years ago, a few yearsbefore the discovery of the ultrastructural basis of the BBB: “theblood–brain barrier is a complex anatomical, physiological and bio-chemical phenomenon, and no unitary hypothesis is adequate to em-brace all the observed events.”

A much more restrictive definition of the BBB is an anatomicallybased one that identifies the BBB as the microvasculature of thebrain. The arterioles, venules, and capillaries of the CNS are speciallymodified (intercellular tight junctions, decreased macropinocytosis,and greatly decreased number of intracellular fenestrea) so as to es-sentially eliminate the production of an ultrafiltrate [16]. This lackof production of an ultrafiltrate eliminates leakage, negates the needby the CNS for a well-defined lymphatic system, and largely accountsfor many unique characteristics of the CNS, including the historical al-though not entirely accurate idea that it is an immunoprivilegedspace [17,18].

However, the vascular BBB alone does not account for all of the as-pects of the conceptual BBB. The choroid plexus, often termed theblood-cerebrospinal fluid barrier, is the major site of production forthe cerebrospinal fluid (CSF) and is intimately involved in many as-pects of blood-CNS exchange, including those related to drugs[19,20]. The tanycytic barriers found at circumventricular organs[21], specialized barriers in the retina, and specialized barriers atthe cranial/spinal nerves offer additional complexity [22]. Anotherlevel of complexity is added in that all barrier cells are in intimatecommunication with cells on both sides of the barrier. The communi-cation provided by the resulting neurovascular unit acts to define theproperties of the BBB under changing physiologic conditions and like-ly has important roles in disease [22–25].

Without the production of an ultrafiltrate, the CNS must be nour-ished by othermechanisms. The BBB,whether defined conceptually, re-strictively, or broadly to include the choroid plexus and other barriersites is intimately involved in CNS nutrition. The BBB is endowed witha host of specific and selective transporters that supply the CNS withglucose, free fatty acids, amino acids, vitamins, minerals, and electro-lytes [26]. Brain-to-blood mechanisms such as reabsorption of CSF andefflux pumps such as p-glycoprotein (P-gp) aid the CNS in maintaininghomeostasis [27]. The BBB also plays roles in communication by trans-porting between the blood and CNS informational molecules such asregulatory proteins and peptides [28–30]. Enzymatic and secretory

Fig. 2. Three ways discussed in this review in which the BBB is relevant to Alzheimer'sdisease with examples. Endothelial cell #1: Dysfunctions of BBB can promote AD as ex-emplified here by impaired efflux of A-beta from brain. Endothelial cell #2: Drugsneeded to treat AD must cross the BBB as exemplified here by the ability of donepezilto be transported across the BBB. Endothelial cell #3: Alterations in BBB mean that de-livery of drugs to the CNS for non-AD conditions (pain control, depression, delirium) isdifferent than in non-AD patients as exemplified by impaired P-gp function.

631W.A. Banks / Advanced Drug Delivery Reviews 64 (2012) 629–639

functions of the cells that comprise the BBB contribute to the roles ofnutrition, homeostasis, and communication. The transporters of theBBB are not static, but vary with development and aging in responseto the changing needs of the CNS. This response is testimony to thecross-talk that is constantly going on between the cellswhich constitutethe blood–brain barriers and the cells of the CNS including pericytes,microglia, astrocytes, and neurons [22,31]. The BBB, then, is better con-ceived of as a regulatory interface between the CNS and blood than as arigid barrier.

Finally, there are many aspects that impact on specific drug deliv-ery strategies or that potentially affect disease progression in AD thatshould be considered in context with the BBB (Fig. 1). These includeCSF production and reabsorption, brain-CSF diffusion interactions,circulating binding proteins/soluble receptors, and neuroinflamma-tory mechanisms [17,19,31–35].

This complexity has several implications for AD:

1) Dysfunction of the BBB, either from lack of adaption to CNS demandsor because of a primary defect in BBB dysfunction, can result in dis-ease. Specific alterations in the BBB may affect the onset or progres-sion of AD. In these cases, the BBB itself is a therapeutic target.

2) A myriad of strategies for delivering drugs to the AD brain havebeen proposed. This review will examine many of these strategiesin the context of BBB function.

3) AD-related alterations in the BBB have implications for treatmentof other CNS conditions in AD patients. For example, drugs used inthe treatment of depression, delirium, pain control, and psychosesmay access the brain differently in those with AD than in thosewithout AD. As a result, the dosages, efficacy, potency, therapeuticwindow, and side effect profiles of drugs may differ between ADand non-AD patients.

This review will consider drug delivery to the AD brain in thesethree contexts (Fig. 2): 1) The BBB as causal to AD and therefore itselfa therapeutic target; 2) selected strategies for delivering AD drugsacross the BBB; 3) how a BBB altered by AD affects the delivery ofdrugs to the brain for the treatment of other (non-AD) CNS diseases.

3. The BBB as a cause of AD and therapeutic target

Over a dozen mechanisms have implicated the cerebrovascula-ture, choroid plexus, or CSF drainage as playing a role in the onset

Fig. 1. The main functions of the BBB: Substances can enter the brain by extracellular, saturapedesis. The physical barrier formed by the capillary wall, saturable efflux systems, enzymstances by the CNS. The barrier cells also secrete a number of substances into brain and blo

or progression of AD (Table 1). These mechanisms are seldom mutu-ally exclusive and many could be interrelated or aspects of an under-lying process. This section will discuss the mechanisms that havebeen studied as therapeutic targets for the treatment of AD. Many ofthese mechanisms would also affect drug delivery to the brain in gen-eral and so are considered in that section as well.

3.1. BBB disruption

Some early observations reported that AD patients had increasedlevels of albumin in the CSF [36]. This was originally thought to repre-sent evidence that the BBB was disrupted in AD. However, later stud-ies have tended to find normal levels of albumin in the CSF [37,38] orto attribute increased albumin to a slower reabsorption of CSF backinto the blood stream, termed bulk flow [39]. Decreased bulk flow

able influx, and lipid solubility (transmembrane diffusion) pathways. Cells enter by di-atic activity at the barrier cells, and CSF reabsorption limit uptake and retention of sub-od.

Table 1Proposed mechanisms: involvement of the blood–brain barrier and cerebrovasculaturein Alzheimer's disease.

- Leaky BBB: toxins from the circulation enter the CNS- Tortuous capillary bed: rheological alterations impair nutrient uptake by the CNS- Defective glucose transport- Brain endothelium induces neuroinflammation- Brain endothelium releases neurotoxins- Decreased cerebral blood flow: deficient delivery results in hypoxia/deficientdelivery of nutrients

- Atherogenesis: stroke/MID/AD lie on a spectrum- A-beta induces ionophores in BBB cell membranes- Neurovascular hypothesis: defective brain-to-blood efflux allows accumulationof A-beta in brain

- Decreased CSF reabsorption: neurotoxic substances accumulate in CSF and brain- Decreased P-gp function: accumulation of xenobiotics, endogenous neurotoxins,and A-beta

- Altered expression/function at BBB of excitatory amino acid transporters:Glutamate and other neurotoxic substances accumulate in brain

- Oxidative damage induces BBB dysfunction- Altered metalloproteinase activity impairs BBB integrity- Increased blood-to-brain transport of A-beta

632 W.A. Banks / Advanced Drug Delivery Reviews 64 (2012) 629–639

has also been observed in a mouse model of AD [40]. Measures of in-creased albumin in the CSF correlate with disease progression [41]and are inversely related to CSF/serum ratios of folate [42]. Althoughthere is no evidence in humans or animal models for a massive dis-ruption of the BBB like that seen in multiple sclerosis or even instroke, there is evidence that microvascular leaks may be occurring.The “white matter changes” classically seen on AD imaging andthought to represent small vessel disease in the brain correlate withthe degree of microalbuminuria caused by microvascular leakage atanother very highly regulated interface: that of the kidney tubules[43]. Indeed, microalbuminuria, which is evidence of microvascularkidney disease, is a risk factor for cognitive decline [44]. This is consis-tent with ideas that AD has a systemic component and that it is part ofa spectrum of vascular disease. On the one hand, shared risk factorswith myocardial infarction and stroke support the idea of a spectrumof disease [45–47], whereas on the other hand a less robust protectionfrom statins and nonsteroidal anti-inflammatory drugs argues againstit [48–51]. Micropunctate lesions representing limited protein leak-age at capillaries have been demonstrated in some animal models ofAD [52]. Any BBB disruption in AD models seems to be too low to in-fluence influx of drugs [53].

Repair of BBB integrity, were disruption to exist, would represent avery tempting therapeutic target. A great deal has been discovered inrecent years about the construction and regulation of tight junctionsand about their dysfunction in ischemia, hypoxia, neuroAIDS, epilepsy,and other conditions. Little attention has been paid to the other,perhaps dominant, mechanisms of BBB disruption involving macropi-nocytosis and other vesicular mechanisms. Interestingly, one of thefew drugs available for the treatment of AD, the NMDA receptor antag-onist memantine, protects against BBB disruption [54–56], probably byblocking NMDA-mediated oxidative stress at the brain endothelial cells(BEC) [56]. Activated protein C has been proposed to protect BBB integ-rity through several mechanisms including decreased apoptosis of BEC(and hence decreased BBB disruption), decreased neuroinflammation,and neuroprotection [57,58]. Activated protein C binds to protease acti-vated receptor-1 on BECs to protect them against apoptosis. It is alsotransported across the BBB by the endothelial protein C receptor toaccess other cells of the neurovascular unit.

The correlation between microproteinuria and white matterchanges raises the question of whether treatment with drugs directedat the renin–angiotensin–aldosterone axis would be as effective in pro-tecting the brain in AD as they have been in protecting renal function indiabetes mellitus. In this regard, angiotensin II has been shown to in-crease BBB permeability through both tight junction and vesicularmechanisms [59,60]. Some activities of the brain's renin–angiotensin

system have been reported to be increased in Alzheimer's disease [61]and treatment with angiotensin II blockers decreases brain inflamma-tion [62]. Patients with Alzheimer's disease and microalbuminuriathat were treated with agents that would either decrease angiotensinII levels or block its receptor were less likely to show mental status de-cline [44]. Several studies have found that angiotensin II receptor block-ade is associated with a decreased incidence of Alzheimer's disease,including a recent case control analysis from the UK [63]. The angioten-sin II blockers weremore protective than other antihypertensive agentsand more protective against Alzheimer's disease than against vasculardementias, suggesting that the effect was not attributable only to a de-crease in blood pressure. These results are consistentwith a therapeuticbenefit from protection of the brain's microvasculature.

3.2. Decreased cerebrovascular blood flow and uptake of oxygen andglucose by the AD brain

For substances that cross the BBB extremely well, cerebral bloodflow (CBF) dictates their rate of uptake by brain [64]. Glucose and ox-ygen are two substances vital to brain function with high extractionrates. CBF is not static but under the influence of the cells whose met-abolic demands are dependent on that CBF [23]. Cerebral blood flow(CBF) both at rest and to activated areas is decreased in the preclinicalstage of AD [5]. Whether the decreased CBF and decreased glucoseand oxygen use by the AD [65] brain is because of a defect in flowmechanisms or in response to a decreased demand by the CNS isunclear, but is a vital question as to their role in the pathogenesis ofAD and to therapeutic approaches [5]. If decreased CBF is in responseto a lower metabolic demand by the brain, then altering CBF or in-creasing oxygen and glucose uptake would not be expected to resultin a therapeutic benefit. If decreased CBF is a primary lesion, thenits impairment would be slowly starving the brain and improvedblood flow or improved oxygen and glucose delivery to the brainwould be expected to preserve brain function. An increasingly tortu-ous capillary bed results in rheological alterations that impair theability of nutrients to cross the BBB, thus magnifying the problem ofdecreased cerebral blood flow [66]. That “vascular starvation” canproduce severe CNS disease is exemplified in a family described byDe Vivo et al. [67]. These individuals have a 50% decrease in GLUT-1,the BBB transporter for glucose, and have mental retardation andseizures.

Restoring blood flow or enhancing oxygen and glucose delivery isnot likely to be easy. Simply increasing blood glucose will not greatlyincrease CNS levels of glucose because GLUT-1 is readily saturated;otherwise, diabetes mellitus would be expected to be a protectiverather than a risk factor for AD. A brain-derived peptide preparationtermed cerebrolysin increases GLUT-1 expression and glucose trans-port across the BBB by 60–90% in rats [68]. Transposition of the omen-tum as a means of improving CBF has been proposed and reported toreduce amyloid plaques although not neurofibrillary tangles [69]. Acase series of six AD patients treated with omentum transpositionshowed significantly less decline than predicted [70]. However, fat,including omentum, has now long been known to be a source of neu-rotropic and neuroprotective agents such as leptin [71]. As discussedbelow, the gastrointestinal hormones represent a large class of poten-tial therapeutics for the treatment of neurodegenerative diseases. Itmay be that the omentum is delivering neuroprotective gastrointesti-nal hormones to the brain.

3.3. Vascular injury and a stroke/multi-infarct dementia (MID)/ADspectrum

Consistent with this mechanism, hypertension and some of theother risk factors for stroke are also risk factors for AD; furthermore,treatment of hypertension and those risk factors is often associatedwith protection from AD. However, some anti-hypertensives may

633W.A. Banks / Advanced Drug Delivery Reviews 64 (2012) 629–639

exert their protective effects against AD through mechanisms otherthan blood pressure control. For example, as discussed above, angio-tensin II receptor blockers may protect against microvascular diseaseand, as discussed below, calcium channel inhibitors may improvebrain-to-blood efflux of amyloid beta peptide (A-beta). Similarly,the risk for AD imposed by dyslipidemias is not reversed by lipid low-ering drugs, suggesting that some other aspect of the metabolic syn-drome is contributing to AD [51].

3.4. CSF drainage

Reabsorption of the CSF back into the blood stream is impaired inaging and even more so in patients with AD [39]. As a result, toxinsmay be cleared more slowly from the CNS. Most of the CSF is drainedfrom the brain by way of the cribriform plate and is drained throughthe cervical lymphatics on the way to the blood [72,73]. Such drain-age likely modulates immune responses to CNS and peripheral anti-gens [17,18]. Increased levels of CNS toxins and altered immunefunctions mean that decreased CSF drainage could have a number ofnegative effects on cognition.

One therapeutic approach has been to shunt CSF from the brain toblood in AD patients. Although a pilot study initially showed encour-aging results for CSF shunting [74], a recent analysis of a randomized,double-blind, placebo-controlled trial of 215 patients failed to showbenefit to shunting [75].

Treatment of a mouse model of AD with antisense directed at APPhas been shown to reverse the defect in CSF reabsorption [40].

3.5. Endothelial function/secretions/BBB cell inflammatory responses

The cells that comprise the BBB, including BECs and the epithelialcells of the choroid plexus, have secretory capacity. Some studieshave shown that BECs from AD patients secrete a neurotoxin [76].Other studies have shown that they secrete cytokines, prostaglandins,nitric oxide and other substances both constitutively and in responseto viral, bacterial, and hormonal substances [77–79]. BECs themselvesare likely responding to various aspects of the AD environment. BECsrespond to a wide range of substances in the blood and CNS. For ex-ample, insulin alters BEC alkaline phosphatase levels and the BBBtransport rates of tryptophan and leptin [80–82].

One substance that BECs could be responding to is A-beta. BECs bind,internalize, and transport A-beta1-40 and A-beta1-42. A-beta1-40 takenup from the blood is not well transported across the BBB but mostlyremains adhered to or internalized by the BEC, whereas A-beta1-42 istransported across the BBB [83,84]. A-beta acts on BECs to induce chemo-kine secretion,monocyte trafficking, decreased proliferation, altered gly-coprotein expression, altered permeability, and altered nitric oxidesynthase activity [85–90]. Tau proteins also lead to disruption of theBBB through the release of cytokines from activated microglia [91].These findings are consistentwith the AD environment promoting barri-er cell secretions that have detrimental effects on cognition by affectingneuronal, glial, and pericyte functions [92].

Treatments directed at disrupting the BEC/A-beta interactionscould alter or control the toxic effects that result from BECs respond-ing to A-beta. Because the BEC is exposed to peripherally generatedA-beta on its luminal side and to CNS-generated A-beta on its ablum-inal side, treatments directed at A-beta within the brain may have dif-ferent effects on BEC function than those directed towards peripheralsources of A-beta.

3.6. Abeta transport: RAGE, LRP-1, and P-gp

A-beta is transported bidirectionally across the BBB; that is, bothin the brain-to-blood (efflux) and the blood-to-brain (influx) direc-tions. Separate transporters are responsible with blood-to-braintransport being primarily mediated by RAGE and the brain-to-blood

transport being primarily meditated by LRP-1 [93]. P-gp also seemsto have an effect on the brain-to-blood transport of A-beta [94–96]as may other related transporters [97,98]. Increased blood-to-braintransport by RAGE and decreased efflux by LRP-1 and P-gp all act toenhance the uptake or retention of A-beta in AD [94,95,99,100].

The evidence for efflux being important in A-beta accumulation inbrain and for cognitive impairment is especially convincing. Knock-down of LRP-1 with antisense results in decreased A-beta efflux, in-creased brain levels of A-beta1-42, and cognitive impairment [101].Mutations in LRP-1 result in decreased A-beta efflux [100] and P-gpknockout mice have accumulations in brain A-beta and cognitive def-icits [102]. It is unclear how P-gp interacts with LRP-1 in the efflux ofA-beta. One possibility is that P-gp primarily prevents the uptake bythe BEC of A-beta from the circulation; another possibility is thatLRP-1 and P-gp somehow interact in a two-step process to removeA-beta from the brain. Why A-beta has both influx and efflux trans-porters is not clear. However, A-beta has the ability to promote mem-ory at lower concentrations [102] than that at which it impairs it[103]. As illustrated for potassium, the presence of influx and effluxtransporters at the BBB allows for a very precise control of CNS levels[104]. It may be that efflux/influx transporters act in tandem to main-tain the CNS levels of A-beta at its most optimal concentration.

The transporters are differentially regulated with regards to A-betatransport. For example, A-beta influx is altered by A-beta binding toapolipoproteins [105–107]. Transport in both directions is likely influ-enced by the primary structure of A-beta as exemplified by A-beta mu-tations being less well transported and by human A-beta being effluxedless well than murine A-beta in mice [108,109]. Secondary structure isalso likely importantwith the assumption that monomers are preferredligands. Finally, inflammation induces an increased influx and de-creased efflux of A-beta across the BBB [110], changes which may inpart be mediated by inhibition of P-gp [111,112].

Several therapeutic options are suggested by the alterations in A-betatransport; someof these have been tested and seem tohave beneficial ef-fects. Knocking downAPP expression results in recovery of A-beta efflux,both suggesting that antisense directed against APP could be used ther-apeutically to correct efflux and also that A-beta somehow poisons itsown efflux systems [101]. It could be that A-beta induces the oxidationof LRP-1 shown to occur in AD patients and thus impairs LRP-1 function[113]. In an AD mouse model with impaired A-beta efflux, treatmentwith APP-directed antisense reduces oxidative stress [114] and treat-ment with antioxidants lead to improved cognition [115]. Treatmentwith the nonsteroidal anti-inflammatory drug indomethacin restoresthe inflammation-induced inhibition of efflux, but not the enhancementof influx [110]. Indomethacin, but not necessarily other nonsteroidals,has been associated epidemiologically with protection against AD [116].Vitamin D enhances A-beta efflux [117] and the calcium channel blockernilvadipine increasesA-beta efflux, reduces brain levels of A-beta, and im-proves cognition in an animal model of AD [118]; calcium channelblockers commonly used in the US such as amlodipine and nifedipinedo not affect A-beta efflux. Thus, evidence exists that treatment withAPP antisense, antioxidants, vitamin D, nonsteroidal anti-inflammatorydrugs, and calcium channel blockers can restore the deficit in A-betaclearance from brain.

3.7. Other altered transporters

Transporters in addition to those for glucose and A-beta may be al-tered in AD or models of AD. Lower levels in brain, CSF, or CSF/bloodratios for insulin, vitamin B12, and folate suggest that transporters forthese substances may be defective in AD [42,119,120]. In animalmodels of AD, regional transport of the cytokines tumor necrosisfactor-alpha and interleukin-1 are altered [121,122]. Examples ofthese have been provided in the SAMP8 mouse, a natural mutationthat has an age-dependent accumulation of A-beta and age-dependent impairments in learning and memory [6,12]. The SAMP8

634 W.A. Banks / Advanced Drug Delivery Reviews 64 (2012) 629–639

mouse develops many of the findings of AD brains including choliner-gic deficits, oxidative changes, impaired CSF reabsorption, and im-paired A-beta efflux, all of which are reversed by treatment eitherwith antibody directed against A-beta or antisense directed againstamyloid precursor protein (APP) [8,40,115,123–128]. IL-1 is nottransported into the hippocampus, thalamus, hypothalamus, pons-medulla, or cerebellum of SAMP8 mice whereas it is transportedinto these regions of non-SAMP8 mice. How alterations in thesetransporters might affect the brain are largely unexplored.

4. Strategies used to deliver AD drugs to the brain

It is estimated that over 400 drugs for AD are being investigated inabout 830 clinical trials (ClinicalTrials.gov). Many more substancesare being investigated in animal models of AD. Drugs that mustreach deep brain targets as is the case in ADmust cross the BBB. How-ever, many drug trials fail because of inadequate trial design with oneof the chief flaws being a neglect regarding BBB penetration [129].The BBB represents one of the greatest challenges for drug deliveryto the CNS and many strategies have been devised to meet that chal-lenge. Here, the mechanisms by which potential AD drugs cross theBBB are reviewed.

4.1. BBB disruption

At first it seems obvious that any disruption in the BBB would im-prove drug delivery to the brain. This has tempted many to proposedisrupting the BBB for the purposes of drug delivery, despite the ob-vious problem that many of the endogenous substances that willthen enter the brain from the blood are neurotoxic [130]. For this rea-son, disruption of the BBB in the delivery of therapeutics must becarefully controlled [131]. Studies in stroke models and with osmoticopening show that the resulting disruption of the BBB is sufficient toallow therapeutic levels of drug to accumulate in the disrupted region[131,132]. However, other studies suggest that the increase in influxrate resulting from most approaches to BBB disruption is insignificantcompared to the other dynamics that determine the equilibrium be-tween brain and blood for a solute. For example, the efflux transport-er for potassium is so robust that BBB disruption does not alter itsconcentration in the CSF [133]. The proposed micropunctate disrup-tions of the BBB proposed in AD and seen in some animal modelsmay not be sufficient to allow drugs to reach therapeutic levels. Thisis because even a disrupted BBB is usually still very restrictive in com-parison to peripheral tissue beds. Additionally, the poor diffusionwithin brain tissue would prevent drug from reaching areas of thebrain more than a few hundred microns from the lesion. Recently,Cheng et al. found that BBB disruptions in animal models of AD andmultiple sclerosis were not sufficient to alter small molecule uptakeby brain [53]. Thus, for chronic diseases like AD, current pharmacologicmethods of BBB disruption do not offer an acceptable cost/benefit ratiofor drug delivery.

4.2. Lipid solubility

Most CNS drugs used in the clinic are small, lipid soluble mole-cules. As such, drug development in this area continues to be dynam-ic, as exemplified by the work on the cholinesterase inhibitorsphenserine and posiphen [134]. The presence of brain-to-blood trans-porters such as P-gp impede the ability of many small, lipid solubleplanar molecules from accumulating in the CNS to therapeutic levels[135]. However, P-gp activity is decreased in AD and so the AD brainmay be exposed to much higher concentrations and to many moredrugs than is the non-AD patient [94,95]. A common misinterpreta-tion of the literature [136] is that only molecules less than 400–500Daltons can cross the BBB by lipid solubility/transmembrane diffu-sion, but in fact larger molecules can cross the BBB in amounts

sufficient to affect the CNS in ADmodels. For example, the nonpeptidesomatostatin agonist NNC 26–9100 (MW=556 Da) exerts positiveeffects on cognition in an animal model of AD [137]. In particular,the rules derived from small molecules to predict non-saturable pas-sage across cell membranes [138] do not apply very well to biologi-cals such as peptides and proteins. Breaker peptides, small peptidesof 600–700 Da that attach to the termini of AB and so prevent or re-verse fibrillation, cross the BBB sufficiently to reverse plaques and im-prove cognition in an AD mouse model [13,139]. The 27 amino acidform of pituitary adenylate cyclase activating polypeptide (PACAP;MW=3148 Da) crosses the BBB by transmembrane diffusion in suffi-cient amounts to improve cognition in an AD mouse model when itsefflux from brain is inhibited [140].

4.3. Antibodies as therapeutic agents

Antibodies as therapeutics have been used in two main ways in AD:1) as directly targeting pathologic agents and 2) as delivery vehicles.The strategies and the BBB interactions are very different for thesetwo approaches. This section will consider the BBB aspects for anti-bodies directly targeting pathologic agents. The section on saturabletransporters below will consider antibodies used as delivery vehicles.

The antibody target in AD has usually been A-beta. Animal studiesshow that active or passive immunization against A-beta can decreaseplaque number and improve cognition. A significant number of pa-tients actively immunized against A-beta developed problems relatedto the cerebrovaculature such as stroke and encephalitis. Phase 3 tri-als of passive immunization are ongoing.

The mechanisms proposed by which antibodies act all involve theBBB but in radically different ways. One proposal is that antibodiescross the BBB to directly interact with AB in the brain [141]. Anti-bodies given directly into the brain can indeed rapidly restore BBBdeficits and reverse cognitive impairments in AD mouse models[52,124,125,142,143]. Antibodies cross the BBB slowly and in smallamounts by the mechanism of the extracellular pathways [142,144].IgG molecules are transported out of the brain by a saturable effluxsystem as well as with the reabsorption of CSF [141], whereas IgMmolecules return to the blood only with CSF reabsorption [105]. Itwas once thought that the saturable efflux system for IgG was medi-ated by FcRn [145], but recent work has shown that transport func-tion occurs in FcRn knockout mice [146]. The lack of a saturableefflux component for IgM means that after a single intravenous injec-tion the amount of IgM accumulating in the CNS is about twice that ofIgG molecules [142]. Thus, the ability to cross the BBB, accumulate inbrain, and to reverse disease means that it is possible that antibodiesact in this manner.

Another proposal is that antibodies act by binding A-beta in thecirculation [147]. This would prevent circulating A-beta from contrib-uting to brain levels of A-beta. It is well established that A-beta istransported from blood into the brain by RAGE and that transport isenhanced in AD mouse models and with activation of the innate im-mune system [93,110,148]. Thus, circulating antibodies could preventA-beta produced in peripheral tissues from entering the CNS as wellas prevent A-beta that had been effluxed from the CNS by LRP-1and P-gp from reentering the CNS. However, it is unclear whether cir-culating A-beta contributes to the A-beta load in brain. The evidencethat blood levels of A-beta contribute to brain levels is exemplifiedby the paper of Sutcliffe et al. [149]. In that study, animals treatedwith STI571, a cancer therapeutic that decreases A-beta production,had a decrease in both their brain and blood levels of A-beta. Giventhat STI571 does not cross the BBB, it was argued that a reduction inA-beta production occurred at peripheral but not at CNS sites; there-fore, brain levels of A-beta fell because of the decreased contributionof A-beta from blood. However, this paper did not rigorously showthat STI571 does not cross the BBB. If it does cross the BBB, then itmay have been decreasing A-beta production within the CNS. More

Table 2Effects of changes in the AD BBB on drug uptake.

Alteration Directional effect Drugs affected

BBB disruption Increased local uptake All drugs*Decreased CSF reabsorption Increased residence All-to-most drugs@

time in CSF and braininterstitial fluid

Decreased cerebral blood flow Decreased uptake Flow-dependent drugsDecreased P-gp activity# Increased uptake P-gp substrates

Additional considerations: *A molecular weight limit may occur depending oncharacteristics of disruption; induction of local events related to neuroinflammationmay alter many aspects that impact on drug action such as degradation,sequestration, and receptor activity. @Drugs entering the CNS at the vascular BBBthat are small and highly lipid soluble, that have robust efflux systems, or that areavidly sequestered or degraded are less likely to be affected. #Inflammatoryconditions have also been proposed to result in increased P-gp activity.

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convincing is the paper of Atwal et al. who used a BACE1 inhibitor todecrease peripheral and brain production of A-beta [150]. Theyshowed that when production of A-beta is decreased in the CNS,brain A-beta levels decrease rapidly. However, at a concentrationthat inhibited only peripheral production, brain levels of A-betaremained unchanged. This work strongly suggests that peripherallevels of A-beta contribute little or nothing to CNS levels of A-beta.Thus, a controversy exits in the literature as to whether circulatingA-beta contributes to levels of A-beta in the brain; in turn, thismeans that it is unclear whether sequestration of A-beta in theblood is the mechanism by which antibodies affect AD.

4.4. Transport systems

Use of endogenous transport systems is the great, untapped strat-egy in drug delivery to the brain. The vascular BBB and blood-CSF bar-rier are both richly endowed with known transporters; yet it isestimated that the majority of BBB transporters have yet to be discov-ered [151]. Transporters for many of the peptides and regulatory pro-teins typically have the added complexity of having a heterogenousdistribution [152]; this characteristic could be used to target drugdelivery to specific areas of the brain. For example, the BBB trans-porter for interleukin-1 is especially concentrated at the posteriordivision of the septum, the leptin transporter at the arcuate nucleusof the hypothalamus, and the transporter for APP-directed antisenseat the hippocampus [7,121,153,154].

From clinical use to preclinical study, a few drugs used in AD areknown to take advantage of transport systems. Donepezil and proba-bly other cholinesterase inhibitors, one of only two classes of drugsapproved for the treatment of AD, is transported across the BBB byan organic cation transporter, most likely that for choline [155,156].The antioxidants N-acetylcysteine and alpha-lipoic acid, the B vita-mins, and to a large extent the vitamin E's have their CNS levels reg-ulated by BBB transporters [157]. Caffeine, proposed to reduceamyloid burden in an AD mouse model [158], is transported acrossthe BBB by the adenosine transporter [159]. Oligophosphorothioateantisense molecules cross the BBB by a saturable transporter [7]. Anoligophosphorothioate antisense that targets APP is effective in re-ducing levels of APP in brain, stimulating A-beta efflux from brain, re-ducing oxidative stress, and improving cognition in the aged SAMP8mouse [7,8,114,160]. Another oliogophosphorothioate antisense di-rected at an efflux component of peptide transport system-6 [140]decreases the brain-to-blood transport of the neuroprotectant pitui-tary adenylate cylcase activating polypeptide (PACAP). This, in turn,allows blood-borne PACAP to accumulate in brain and to improvecognition in an animal model of AD.

Many gastrointestinal hormones have effects on cognition and arebeing explored as treatments for AD. These include leptin [161–164], in-sulin [165,166], ghrelin [167], glucagon-like peptide [168–171], vasoac-tive intestinal peptide and the closely related PACAP [172–174], andsubstance P [175,176] . Most of these hormones cross the BBB by wayof saturable transportmechanisms [154,177–180] and likely have phys-iological roles in neural development, neuroprotection, and cognition.However, the short half-life in blood and peripheral effects of these hor-mones complicate their use for brain effects and so alternative strate-gies have been tried to improve brain delivery. For example, toovercome the very short circulation half-life of glucagon-like peptide-1 [178,181], it and its longer acting homolog exendin has been adminis-tered by the intranasal route [170]. Intranasal administration at thelevel of the cribriform plate has been shown to facilitate entry into theCNS for this and other peptides [182–186]. To overcome its hypoglyce-mic effect when given peripherally, insulin has also been administeredby the intranasal route and shown to have positive effects in AD[187–189].

Trojan horse approaches attempt to harness transporters not todeliver their endogenous ligands, but to deliver attached therapeutic

agents. A number of substances have been used as the endogenous li-gand, including glucose. Antibodies with attached therapeutic cargosdirected at BBB transporters such as transferrin and melanotransfer-rin have been widely investigated, including in the delivery of agentsfor the treatment of AD [190,191]. The proposed mechanism is that anantibody is directed at a target, often a transporter, on the luminalsurface of the brain endothelial cell. In theory, the antibody will betransported across the BEC by a vesicular mechanism so that it andany attached drug is delivered into the brain. This strategy has hadsome unforeseen complications, such as routing of the induced vesi-cles to the lysosomal compartment with subsequent return of thevesicle to the luminal (not the abluminal) membrane. Recently, Yuet al. [192] have demonstrated that using high doses an antibodywith a low affinity for the transferrin receptor can indeed deliverlarge amounts of drug to the CNS. Cognitive effects were not deter-mined in that study.

5. Alterations in the AD BBB: effects on drug delivery to the brain

The idea that drugs in common use may be taken up differently bythe brain of the AD patient than in the non-AD patient has rarely beenconsidered. Yet many of the properties of the BBB that determine theextent to which currently used drugs are taken up by the brain areknown to be altered in AD: CSF reabsorption, cerebral blood flow,and P-gp activity are the best studied examples [39,94,95]. Not all ofthese changes are relevant to all drugs and some of these effectswill tend to increase and other effects will tend to decrease the equi-librium of drugs in the brain (Table 2). Therefore, it is hard to predictwhat the net effect of these changes will be on any particular drug.However, overall these differences could cause shifts in the efficacy,potency, therapeutic window, side-effect profiles, and dosage of com-monly used drugs. These shifts could explain in part why AD patientsseem at times at risk for certain side effects from CNS drugs such asdrug-induced delirium. A better understanding of how brain pharma-cokinetics differ in AD patients would allow appropriate adjustmentsin drug dosages and fewer CNS side effects.

5.1. BBB disruption

Micro-disruptions of the BBB could allow increased access of somedrugs to the immediate environment around those disruptions. Diffu-sion within brain tissue is poor and so it is likely that drug would belimited to within a few 100 μm of the disruption.

Paradoxically, disruptions of the BBB could actually retard thebrain retention of some drugs. Even modest disruptions of the BBBare pathologically significant and induce inflammatory responses.Tumor necrosis factor-alpha can increase Pgp activity leading to a fur-ther reduction of accumulation of Pgp substrates by brain [193–195].

636 W.A. Banks / Advanced Drug Delivery Reviews 64 (2012) 629–639

5.2. Decreased CSF reabsorption

This would be expected to increase the residence time of drugswithin the CNS. This is true for drugs entering the CNS at the vascularbarrier as well as at the choroid plexus.

5.3. Decreased cerebral blood flow

Only drugs that rapidly enter the CNS are flow-dependent; drugswith lower rates of entry will not have their uptake by brain substan-tially altered by a decrease in CBF. Donepezil, for example, is likely aflow dependent drug, whereas the oligophosphorothioate antisensemolecules are not. Therefore, a decreased cerebral blood flow wouldbe expected to decrease donepezil uptake and its therapeutic effect,but not that of the oligophosphorothioate antisenses.

5.4. P-gp

P-gp function is decreased in AD [94,95]. The decreased activity ofP-gp may be mediated by the neuroinflammatory state of AD[111,112]. A decrease in P-gp activity would have widespread impli-cations for the use of CNS drugs in AD as so many commonly useddrugs are P-gp substrates. Alternatively, tumor necrosis factor alphacan increase P-gp activity and so decrease drug access to the brain[193,195,196]. It may be that the increased access of these drugs tothe brain or the sudden inhibition of drug transport into brain con-tributes to the increased vulnerability of AD patients to the develop-ment of delirium.

6. Conclusions

The BBB acts as a dynamic interface between the CNS and the pe-ripheral tissues. Many aspects of the BBB are affected and thesechanges in turn have implications for the onset, progression, control,and treatment of AD. Changes in the BBB itself may contribute to theonset and progression of AD. In this case, the BBB itself becomes atherapeutic target. The BBB is also a formidable barrier for the deliv-ery of drugs to brain tissue in the treatment of AD. Several strategieshave been applied to drug delivery including development of lipidsoluble drugs, BBB disruption, and use of transport systems. Drugsthat normally cross the BBB may be taken up differently by the ADbrain than by the normal BBB, thus complicating the treatment ofCNS conditions such as pain, depression, psychoses, and delirium inthe AD population.

Acknowledgements

This works was supported by VA Merit Review and RO1 AG029839.

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