Characteristics of CompoundsCharacteristics of CompoundsThat CrossThat Cross

The BloodThe Blood--Brain BarrierBrain Barrier

William A BanksWilliam A Banks

VAVA--GRECC/Saint Louis UniversityGRECC/Saint Louis University

Overview of the Blood-Brain Barrier

Substances That Cross

Substances That Should Not Cross, But Do

Median eminence

Inside of the cranium(the dura mater is outside the BBB)

Lepti

n

Ghrelin

PACAP

Extra-BarrierRoles of the Blood-Brain Barrier

NutritionGlucose, Amino Acids, FFA, Vitamins.

HomeostasisElectrolytes, HCO3, p-Glycoprotein...

CommunicationPeptides & Regulatory Proteins (Leptin, Enkephalins, Cytokines.)

Transmembrane Diffusion

-Non-Saturable- Lipid Solubility/ SQRT(MW)

-Drugs (e.g. Morphine)-Ethanol

Lipid Sol1/MW

SatTrnsprt(Influx)

SatTrnsprt(Efflux)

PK:Clearance/Tissue Uptake

Protein Binding

CBFBBB

(Greig, N. et al. Designing Drugs for Optimal Nervous System Activity. In: New Concepts of a Blood-Brain Barrier, Eds: J. Greenwood, D.J. Begley, M.B. Segal)

Sequestration

The Role of the Blood-Brain Barrier...

Banks Figure 2

Compound %Inj/g Ki (microl/g) Vd (mL) t1/2 (min) Ki(t1/2)/Vd

huIL-1alpha 0.07 0.236 2.6 2.06 0.188cycloHis-Pro 0.01 0.179 11.0 5.36 0.087PACAP38 0.12 2.86 6.6 2.90 1.26PACAP27 0.14 2.13 4.0 1.50 0.80IL-1ra 0.33 0.519 2.0 8.00 2.08Pan. Polypep. 0.07 1.15 5.4 3.40 0.733Amylin 0.11 0.899 3.0 3.00 0.899Leptin 0.17 0.587 2.43 5.46 1.32

0 1 2 30.0

0.1

0.2

0.3

0.4

Ki (microl/g-min)

%

I

n

j

e

c

t

e

d

D

o

s

e

p

e

r

g

o

f

B

r

a

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r2 = .001

0 1 2 30.0

0.1

0.2

0.3

0.4

Ki (t1/2/)/Vd

%

I

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j

e

c

t

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d

D

o

s

e

p

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g

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B

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r2 = 0.853

P X P X P = EFFECTRESENTATION

ENATRATION

OTENCY

Pharmacokinetics

domo

ic ac

idmo

rphin

eIL-

1PA

CAP3

8Le

ptin

IL-1ra

0.0

0.1

0.2

0.3

0.4

0.002

% of IV DoseTaken Upper g of Brain

%

I

n

j

/

g

Drugs for Alzheimers Disease

Chimeric VectorsLipid SolubilityGlycosylationNeutral PolyaminesCationizationNanoparticlesLiposomesAttach to a Substance Which Crosses (e.g., Peptides Penatrins)Nasal DeliveryIntrathecal DeliveryBBB Disruption (Hyperosmolar; Bradykinin Ag)Retroinverso PeptidesViral Vectors

Strategies for Drug Delivery

Drugs for Alzheimers Disease

Breaker Peptides

Antibodies

Antisense

Feeding Hormones: Ghrelin

Peptides and Cytokines Cross by Transmembrane Diffusion

Misperceptions about Lipid solubility

VA Levin and his 27 compounds4 MW >400

Cyclo(HisPro): MW 234; Reverses EtOH narcosis after Crossing BBB

: P-gp Substrates

Cytokine-Induced Neurotrophil Chemoattractant-1 (CINC1): MW 7.8 kD

Lipid Solubility vs BBB Penetration

Too Much of a Good Thing: Pitfalls

Increased uptake by Liver and other peripheral tissues

Gets caught in cell membranes

ALALABecomes a Pgp Substrate

Too powerful: morphine vs heroinF33-824 (D. Roemer Nature 77)

Breaker Peptides

Cross the BBB

Reduce ABP plaque number and formation &

Reverse cognitive impairments in transgenics overexpressing APP

Permanne,B. et al FASEB J, 16:860-862, 02Adessi,C. et al JBC 16:13905-13911, 03

Many Feeding Hormonesalso have

Neurotrophic & Cognitive Effects

Ghrelin controls hippocampal spine synapse density and memory performance

Diano,S. et al Nat Neurosci. March 06

octanoylatedmouseghrelin

des-octanoylmouse ghrelin

human ghrelin(2 AA's difference)

Blood Brain Barrier

Blood Brain Barrier

CNS-derived ghrelin?

hGhrelin: Stomach Peptide thatCrosses the BBB to Induce Feeding

Ghrelin has a high uptake across hippocampus BBB

Ghrelin Promotes LTP Generation

Ghrelin IncreasesHippocampalSpine SynapseDensity

Ghrelin Enhances Learning and Memory

T-MazeActiveAvoidance

Step Down T-MazeSAMP8(AD Model)

Conclusion I: Blood to Brain

Ghrelin is Transported intoBrain by a Saturable System

Ghrelin Improves CognitionIn Models of AD

Across the BBB

Passage of

0.0 0.5 1.0 1.50

1

2

3

4

5

131 Antibody

125 Albumin

Early Uptake

Expt (h)

B

r

a

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n

/

S

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R

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Extracellular Pathways

Functional LeaksPial Surface Circumventricular Organs

AlbuminHorseradish PeroxidaseAntibodyErythropoietin

0 100 200 300 4000

10

20

131 Antibody

125 Albumin

Late Uptake

Expt (hr)

B

r

a

i

n

/

S

e

r

u

m

R

a

t

i

o

0 2 4 60.00

0.05

0.10

0.15

24 48 72

Time (h)

%

I

n

j

/

g

Conclusion II:

Early Phase: Antibody Slowly Enters Brain By Extracellular Pathways

Late Phase: Slower than Albumin: Suggests Efflux Mechanism

?Does Enough Antibody Enter CNS to Remove ABP?

Passage of Antisense Across the BBB

A Phosphorothioate 42merAntisense

Directed At Mid Region of ABP

Effective in Reversing Cognitive Impairmentsin Aged SAMP8 Mice After CNS Injection

OP

O

A

ORB

RB

A = O(-): phosphodiester= S(-): phorphorothioate= CH3: methylphosphonate

R = Nucleoside

B = O

R

4 mo 8 mo 12 mo0.0

0.1

0.2

0.3 **

*

Age

A

m

y

l

o

i

d

(

p

g

/

m

g

p

r

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t

e

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)

SAMP8 MouseSAMP8 Mouse

Amygdala Septum Hippocamp0

1

2

3

4

5

6

** *

AntisenseNS

Amyloid Beta ProteinO

D

x

m

m

2

ICV Antisense

0 100 200 300 4000

50

100

150

200

Expt (min)

B

r

a

i

n

/

S

e

r

u

m

R

a

t

i

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(

l

/

g

)

0 10 20 300

25

50

B

r

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/

S

e

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m

R

a

t

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(

l

/

g

)

Ki = 1.4 microl/g-min

0 100 2000

10000

20000

30000

Time (min)

C

P

M

/

m

l

s

e

r

u

m

0 5 103.5

4.0

4.5

Time (min)l

o

g

(

C

P

M

/

m

l

s

e

r

u

m

)

0 500 1000 15000.0

0.1

0.2

0.3

Time (min)

%

I

n

j

/

g

-

b

r

a

i

n

P-Ol

g Only

+10

g

+200

g

50

75

100

* *

Unlabeled Olg

%

C

o

n

t

r

o

l

(

P

-

O

l

g

)

Antisense: Competition

Whole

Brai

n

Corte

xHi

ppoc

ampu

s

0.00

0.25

0.50

0.75

1.00 IV%

I

n

j

/

g

o

f

B

r

a

i

n

R

e

g

i

o

n

IV/ICV = 100:160 ng6 microg

Control Antisense0

5

10

15

*

M

e

a

n

T

r

i

a

l

s

t

o

F

i

r

s

t

A

v

o

i

d

a

n

c

e

Acquisition

Control Antisense0

10

20

*

RetentionM

e

a

n

T

r

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l

s

t

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C

r

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Conclusion III

Oligophosphorothioate42mer Antisense Directed Against

Amyloid Beta Protein Crosses the BBB by a Saturable System

To Reverse Cognitive Impairments

ConclusionsTx Cross the BBB by Sat and Non-Sat Mxn

Non-Barrier Causes of Dec Accumulatn in CNSUnfavorable PharmacokineticsSerum Binding ProteinsBBB Enzymes, Efflux Systems

Peptides, Antibodies, Antisense as Tx

Targeting the BBB ItselfBypass of the BBB for Selected Scenarios

Intrathecal/Nasal