The Blood-Brain Barrier and CNS Drug Development Dr ...and CNS Drug Development Dr....

The Blood-Brain Barrier

and CNS Drug Development

Dr. Danica Stanimirovic

The screen versions of these slides have full details of copyright and acknowledgements 1

The Blood-Brain Barrier and CNS Drug Development


Director, Neurobiology Program

National Research Council of Canada

Institute for Biological Sciences

"The Program is committed to understanding the molecular nature

of neurodegenerative diseases… Our hope is that our discoveries will one day

reduce disability and improve the quality of life of those affected by brain diseases"

1

The Gap

� Neurodegener ati ve diseases

(NDDs) are estimated

to contribute as much as 35%

of the disease burden

in the seven major

pharmaceuti cal markets

� By the year 2040, NDDs will

become the world’s second

leading cause of death, overtaking

cancer (WHO)

� Despite these statistics,

there are few effective treatments

for the majority of the Central

Nervous System (CNS) disorders

� Lack of translation

of promising therapies

from experimental models

into clinical applications

The blood-brain barrier is a major impediment

in the CNS drug development

2

Medicinal

chemistryStrategies to overcome

the blood-br ain barrierDirect injection

BBB

CNS Drug Development

More that 98% of small drugs and virtually all biologics

(peptides, antibodies, genes) do not cross the BBB

in pharmacologically relevant concentrations

3





BBB Disruption

Sledge and hammer approachBrick wall

Blood-Brain Barrier

Ligands

Pumps

Channels

Pores

‘Intelligent’ drug deliveryFluid, specialized membrane

Brain endothelial cells

The Blood-Brain Barrier –

Evolving Concept

4

Neurovascular Unit –

an Integrated Concept

The BBB properties result from interactions and functional

integration of brain endothelial cells and other cellular

and acellular components of the neurovascular unit

Endothelialcells

Pericyte

Tight Junction (TJ)

Astrocyte footprocesses

Neuron

Basementmembrane

5

Tight Junctions – the ‘Physical’ Barrier

Signaling roles of TJ proteins

Break Modulate

Cadherins Cadherins

ZO1

ZO2

Actin

Claudins

Occludin

Junctional adhesion molecule

Tight junction

Adherens junction

Apical plasma membrane

Basal plasma membrane

Paracellular Space

Shin K et al., Annu Rev Cell Dev Biol 22:207-35 20066





Zhang W, Stanimirovic D; (2005) The Transport Systems of the Blood-Brain Barrier, In: The Blood-Brain Barrier and its Microenvironment: Basic Physiology to Neurological Disease (deVries E and Prat A, eds), Taylor & Francis Group, NY

Transporters: Dynamic Blood-Brain Barrier

Endothelial cells

Pericyte

Tight Junction (TJ)

Transporters

for glucose, amino acids,nucleosides

Oatp2MCT1MRP-1?MRP-5?

Transportersfor glucose, amino acids,nucleosides,MCT1, Oatp2,Oatp14, OCTN2

Physical barrier (TJ):passive diffusion

for water, fluorescein,lipid soluble small

molecules, Amphiphilic drugs

AME

RME

Efflux pumps:MDR-1 Pgp

MRP-1, ABCG2,OAT3

Neurotransmittertransporters

Circulation

Brain

EAAT1-3Oatp2ATA2

GAT2/BGT-1OAT

Astrocyte foot processes

Neuron

Basement membrane

Transferrin receptor

Insulin receptorLRP,SR

RAGE(-)(+)

7

Strategies to Deliver Drugs to the Brain

Trans-cranial

� Intra-cerebroventricular (ICV) or intrathecal injection

� Intra-cerebral (IC) injection/implantation

� Convection-enhanced diffusion (CED)

Non-Vascular Routes

Some advantages

� Region selective delivery

� Cells, biologics

� No systemic toxicity

Disadvantages

� Invasive

� Side effects

� Limited transfer/diffusion

8

The subarachnoid space (D) is continuous with the extracellular space of the olfactory mucosa; From Mathison et al., (1998)

� Olfactory (trans-nasal) route


Non-Vascular Routes

� The capacit y of the system in humans?

9





� 100 billion capillaries in human brain

� Surface area – 20 m2

� Diffusion to target – 10-25 m

Trans-vascular (BBB) routes


10

Routes for Drug Delivery Across the BBB

De Boer AG, Gaillard PJ. 2007, Annu rev pharmacol, Toxicol 47:323-55 11

1. Transcellular Lipophilic Diffusion

For many drugs/solutes

(open points), there is a clear

correlation between lipid

solubility and BBB penetration

Octanol/wat er partition coefficient

Facilitated transport

Efflux transporters

Adapted from Levin (1980)

Lipidization strategies

� Increase of non-specific

distribution in all organs

12





2. Inhibition of Efflux Pumps

50%-70% of drugs are substrates of MDR-1 Pgp and BCRP

[11C]Carasolol

+Cyclosporin Control

Elsinga P et al., Mol Imaging Biol (2005) 7:37Y44

Examples of increased

CNS drug deliv ery

with Pgp/BCRP inhibitors:

PSC833: Vinblastine

Paclitaxel

GF120918: Colchicine

Vinblastin

Amprenivir

LY335979: Paclitaxel

Nelfinavir

13

3. Paracellular Hydrophilic Diffusion

BBB disruption

BBB disruption has serious long-term consequences

Osmotic BBB disruption

� Intracarotid injection of hy pertonic solution of manitol

�Hospitalization

Focal ultrasound or electromagnetic f ield

�Focused, local BBB disruption

Transient chemical modulation of TJ tightness

�Bradiky nin B(2) receptor agonist, RMP-7 (Cereport) [400D 1kD]

�Alky lgly cerols

14

4. Carrier-Mediated Transport

�The GLUT1 glucose transporter transports glucose and other hexoses,

including 2-deoxyglucose and fluoro-deoxyglucose used in (PET) scanning

�LAT1 transports large and small neutral amino acids, as well as certain amino

acid drugs including l-dopa, α-methyl-dopa, α-methyl-para-tyrosine or gabapentin

�The cationic amino acid transporter CAT1 transports basic amino acids,

such as arginine or lysine

�The monocarboxylic acid transporter MCT1 transports lactate, pyruvate,

ketone bodies and certain monocarboxylic acid drugs such as probenecid

�The concentrative nucleoside transporter CNT2 transports purine nucleosides,

and certain pyrimidine nucleosides such as uridine

�Choline undergoes carrier-mediated transport across the BBB

via a sodium-independent process

�The purine bases, but not the pyrimidine bases, undergo carrier-mediated

transport across the BBB

The various CMT systems provide a diverse space of molecular

structure that could be mimicked with medicinal chemistry

modifications of drugs that normally do not cross the BBB

15





RCA-1

5. Adsorptive Endocytosis

� Cationized IgG

� Cationized liposomes

� Cell-penetrating peptide v ectors (Sy nB1 and penetratin)

Glycocalyx

++

+Drug

16

6. Receptor-Mediated Transcytosis

ReceptorLigand

� Clathrin-coated v esicles

� Cav eolae

17

Molecular ‘Trojan Horses’ Concept

Pardridge WM: Curr Opin Pharmacol 2006 Oct;6(5):494-500 18





‘Trojan Horses’

‘Receptors-targets’

Transferrin receptor

Insulin receptor (IGFR1 & 2)

LRP1/2

Targeting vectors

OX-26 (IgG – rat; mouse)

IR-Ab (mouse, humanized)

ApoE (nanocarriers)

Angio-pepTM

Mellanotransf err in (p97)

19

Pardridge WM: Pharmaceutical Research (2007)

Human Insulin Receptor (HIR)

as a ‘Trojan Horse’ for Drug Delivery to Brain

20

Poly(butyl cyanoacrylate) nanoparticles coated with polysorbate 80 (Tween 80), which further binds Apo-E in the bloodstream

‘Trojan Horses’ and Nanocarriers

LRP

Kreuter J (2001) Adv Drug Deliv Rev 47(1):65-81 21





‘Trojan Horses’ - Considerations

�Selectiv ity of receptor

f or brain v essels

�Capacity /saturation

�Regulation/recy cling

�Distribution in brain v ascular beds

�Modulation in disease

Receptor Targeting vectors

� Selectiv ity to receptor

� Binding af f inity

� Versatility – link

and release strategies

� Brain ef f lux (FcRn)

� Pharmacokinetics, stability,

non-specif ic clearance

22

Discovery and Development of Novel

‘Trojan Horses’

Discovery Platforms

� Genomics/proteomics –

selective ‘biomarkers’ of brain vessels

� Phage-display platforms

single-domain antibodies

single chain antibodies

peptides

23

IgG

150 kDa

VHH

13 kDa

scFv

25 kDa

Fab

50 kDa

Single Domain Antibodies

� Recognition of uncommon and hidden epitopes

� Cav ity binding – enzy mes and receptors

� Innov ativ e drug f ormats

� Ease of manuf acture and f ormulation

� Solubility and stability

24





Single-Domain Antibody

Phage Display Libraries

Platform technology for selecting ‘targeting’ antibodies

against diseased tissues (e.g., cancer, vasculature, etc.)

25

Selection of the BBB – Targeting sdAbs

Biopanning & Enrichment

Subtractive panning

between human lung

and human brain

endothelial cells

Selecting species

from enriched library

that transmigrate across

the BBB in vitro

Test BBB permeability

in vivo

Tanha et al., Methods in Mol Medicine, 2003Muruganandam et al., FASEB J 16:240 26

Brain-Targeting Single-Domain Antibodies

Two sdAb that cross the BBB model in vitro, FC5 and FC44,

were selected from non-immunized llama sdAb library

Muruganandam et al., FASEB J 16:240 27





Mechanisms of Transport

� Mediated by clathrin-coated v esicles

� Saturable and energy dependent

� Triggered by FC5 binding

to the sialogly coprotein polarized

to the luminal surf ace of HCEC

FC5/Clathrin

Receptor-Mediated Transcytosis (RMT)

Abulrob et al., J Neurochemistry, 2005

FC5 co-localizes with clathrin-coated vesicles

Human brain endothelial cells

28

HRP

HRP

Linking Molecules to FC5

Develop chemistries to link moieties to sdAbs

0 10 20 30 40 50 60 705

10

15

20

25

30

35

Transport across BBB in vitro

Time [min]

Clearance [ml/] HRP-IgG-cysFC5

HRP-IgG

cysFC5

IgG cysFC5

IgG

+

Abulrob et al., Drug Transporters and the Diseased Brain, ICS 1277 (2005) 29

In Vivo Biodistribution of FC5

eXplore Optix shown with optional user workstation

� ‘Real-time’

observ ation of drug

biodistribution in vivo

Time-domain, near infra-red in vivo optical imaging

+

Cy5.5

FC5-

Conjugation of FC5 with NIR probe Cy5.5

30





Improving Pharmacokinetics

PEGylation increased plasma

half-life of FC5 from ~5 min to ~4 h !

� PEG-ylat ed di-FC5

cysactivated

cys

PEG

Maleimide

FC5 Cy5.5lys

FC5 lys Cy5.5

Site-specific PEGylation

� Pentameric FC5 (P5)

Oligomerization

Pentamerization increases

efficiency of transcytosis

31

NC11

FC5

P5

diFC5-PEG

Cy5.5

Brain Accumulation of FC5 Formulations

Head Imaging, 6h

Grids - 5 mm

Volume concentration slices

Volume concentration planes

Dose: 3 nM of sdAb i.v. 32

Detection of FC5 in Brain Tissues

� At the end of imaging protocol – animals injected

with Tomato lectin (green) to stain brain vessels

� Brain and other organs dissected and sectioned

� Sections stained for cell-selective antigens

(GFAP-astrocytes; NeuN-neurons, CD31-endothelial

cells; laminin – basement membrane)

� Cy5.5-FC5 co-localized with above markers

by confocal microscopy

33





Detection of FC5 in the Brain

by Confocal Microscopy

FCFCFCFC5555 NCNCNCNC11111111

Green – vessels

Red – Cy5.5 (FC5 or NC11)

Ve sselVe sselVe sselVe ssel

Laminin (BM)

CD31

FC5

Dose: 3 nM of sdAb i.v.Immunostaining – 6 h after injection 34

FC5 (green) NeuN (red) Cell nuclei (blue)

FC5

Frontal cortex

FC5, double injection (0, 2 h)

immunostaining – 4 h after last injection

Brain vessel

Neurons

Detection of FC5 in the Brain

by Confocal Microscopy

35

Summary

� FC5 transmigrates the BBB after intravenous injection

� FC5 is detected by confocal microscopy in both brain

vessels and brain parenchyma

� PEGylation and oligomerization of FC5 improves

its brain biodistribution

� FC5 is a novel ‘Trojan horse’ for BBB drug delivery

Can FC5 carry therapeutic into the brain?

36





FC5-Functionalized Liposomes

Mol ratio

Lecithin (758) 2

Cholesterol (387) 1

DOGS-NTA (1057.02) 0.06

dode-PE (889.29) 0.01

mPEG-DSPE (2805.54) 0.08

Composition of liposomes

for doxorubicin encapsulation

Lecithin

Cholesterol

DOGS-NTA

DSPE-PEG2000

FC5 or P5

Dox

Size 137 nm, SD = 59 nm (41%)

Doxorubicin is substrate for P-glycoprotein

37

Delivery of Doxorubicin to the Brain

Using FC5-Functionalized Liposomes

Optical imaging 24h after injection

Lipo- FC5

Lipo- P5

Lipo- NC11

38

Separate capillaries

and brain parenchy ma

Measure Dox

concentrations using

f luorometric methods

Integrated imaging/delivery platform

Delivery of Doxorubicin to the Brain

Using FC5-Functionalized Liposomes

Af ter imaging - perf usion

Dissect brains

*

*

# *

# *

*

Capillaries

Brain

0

50

100

150

200

250

300

350

400

Dox-lipo-

FC5

Dox-lipo-P5 Dox-lipo Free Dox

ng doxorubicin/ g tissue

39





‘Tailoring’ BBB Delivery Strategies

to Specific Brain Diseases

� The immature or aged BBB

� The local vs. generalized brain delivery

� Acute (trauma, stroke) vs. chronic brain diseases

(AD, neurodegeneration)

� The level and timing of the BBB disruption

in various diseases

� Molecular and cellular targets within brain

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� Side effects and acceptability of protocols

� Pharmacologically-relevant doses

� Clinical and commercial viability

� Tailoring both delivery system and delivered therapeutic

‘Tailoring’ BBB Delivery Strategies

to Specific Brain Diseases

The brain vasculature and the BBB

are still vastly unexploited ‘targets’

for the development of CNS therapies

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