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Mohamad Navab, Ph.D.An Oral ApoJ Peptide Renders HDL

Anti-inflammatory in Mice and Monkeys and Dramatically Reduces

Atherosclerosis in ApoE Null Mice

ApoA-I Peptide mimetics- An emerging strategy for CVD prevention is the development of apoA-I mimetic peptides. This strategy is based on the hypothesis that apoA-I mimetic peptides could be capable of stimulating the first step in the RCT pathway in a manner similar to native apoA-I, which is 243 amino acid residues in length. In comparison, the apoA-I mimetic peptide D-4F comprises only 18 amino acids.

18 AA peptide

Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2

D-4F

-D-4F administered orally to mice led to the formation of pre-β HDL, increased cholesterol efflux from macrophages and enrichment of HDL with apoA-I and paraoxonase. -

18 AA peptideAc-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2

D-4F

-D-4F administered orally to mice led to the formation of pre-β HDL, increased cholesterol efflux from macrophages and enrichment of HDL with apoA-I and paraoxonase.

- It markedly reduced lesion formation on LDL r-/- mice on Western diet and in apoE-/- mice

18 AA peptideAc-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2

D-4F

D-4F

Similarly, when added to human plasma at nanomolar concentrations in an in vitro system, D-4F caused:- the movement of apoA-I to smaller particles, - reduction of lipid hydroperoxides, -increase of paraoxonase activity and -conversion of proinflammatory HDL to anti-inflammatory HDL.

-D-4F in humans.

D-4F

Similarly, when added to human plasma at nanomolar concentrations in an in vitro system, D-4F caused:- the movement of apoA-I to smaller particles, - reduction of lipid hydroperoxides, -increase of paraoxonase activity and -conversion of proinflammatory HDL to anti-inflammatory HDL.

-D-4F in humans: Phase 1, a, b, c

Negative Stain EM of D-4F in PBSD-4F, negative stain EM

Proposed Mechanism of Action of D-4F

LOOH & Ox-PAPC

and cholesterol Cholesterol Phospholipids

Pre- HDL

ABCA1

HDLApoA-I cycles

through pre- HDL

Chol

este

rol

ABCA

1

Phos

phol

ipid

sD-4F

Proposed Mechanism of Action of D-4F

LOOH & Ox-PAPC

and cholesterol

POVPC m/z 594.3

PGPC m/z 610.2

PEIPC m/z 828.6

CE-OOH

HPETEHPODE

MM-LDL

O

O

PAPC m/z 782.4

LDL

CH3(CH2 )14 CH2

Phosphorylcholine -

12-LO

Chol.18:2

CH2

HC

O

O -CH3(CH2 )14 CH2

Phosphorylcholine - CH2

HC

O

O -

CH3(CH2 )14 CH2

Phosphorylcholine CH2

HC

O

O

CH3(CH2 )14 CH2

Phosphorylcholine - CH2

HC

O

O -

O

O

OH

OO

O OH

O O

12-LOHPETEHPODEHPETEHPODE

O O

O

POVPC m/z 594.3

PGPC m/z 610.2

PEIPC m/z 828.6

Cholesterol Phospholipids

Pre- HDL

ABCA1

HDLApoA -I cycles through pre - HDL

Chol

este

rol

ABCA

1

Phos

phol

ipid

sD-4F

Proposed Mechanism of Action of D-4F

LOOH & Ox-PAPCand cholesterol

POVPC m/z 594.3

PGPC m/z 610.2

PEIPC m/z 828.6

CE-OOH

HPETEHPODE

MM-LDL

O

O

PAPC m/z 782.4

LDL

CH3(CH2 )14 CH2

Phosphorylcholine -

12-LO

Chol.18:2

CH2

HC

O

O -CH3(CH2 )14 CH2

Phosphorylcholine - CH2

HC

O

O -

CH3(CH2 )14 CH2

Phosphorylcholine CH2

HC

O

O

CH3(CH2 )14 CH2

Phosphorylcholine - CH2

HC

O

O -

O

O

OH

OO

O OH

O O

12-LOHPETEHPODEHPETEHPODE

O O

O

POVPC m/z 594.3

PGPC m/z 610.2

PEIPC m/z 828.6

Cholesterol Phospholipids

Pre- HDL

ABCA1

HDLApoA-I cycles

through pre- HDL

Chol

este

rol

ABCA

1

Phos

phol

ipid

sD-4F

Proposed Mechanism of Action of D-4FLOOH & Ox-PAPCinactivated and/or cleared and reverse cholesterol transport is also increased

LOOH & Ox-PAPCand cholesterol

POVPC m/z 594.3

PGPC m/z 610.2

PEIPC m/z 828.6

CE-OOH

HPETEHPODE

MM-LDL

O

O

PAPC m/z 782.4

LDL

CH3(CH2 )14 CH2

Phosphorylcholine -

12-LO

Chol.18:2

CH2

HC

O

O -CH3(CH2 )14 CH2

Phosphorylcholine - CH2

HC

O

O -

CH3(CH2 )14 CH2

Phosphorylcholine CH2

HC

O

O

CH3(CH2 )14 CH2

Phosphorylcholine - CH2

HC

O

O -

O

O

OH

OO

O OH

O O

12-LOHPETEHPODEHPETEHPODE

O O

O

POVPC m/z 594.3

PGPC m/z 610.2

PEIPC m/z 828.6

Cholesterol Phospholipids

Pre- HDL

ABCA1

HDLApoA-I cycles

through pre- HDL

Chol

este

rol

ABCA

1

Phos

phol

ipid

sD-4F

D-4F Plus Pravastatin Increased ApoA-I in both Alpha and Pre-beta HDL Particles

CHOW D-4F

PRAVASTATIN D-4F+PRAVASTATINPre- alpha

Lesion Prevention

0

50,000

100,000

150,000

200,000

250,000

300,000

m2 /

Sect

ion

CHOW D-4F PRAVASTATIND-4F+

PRAVASTATIN

A Comparison of 4F and [113-122]apoJ

Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 4F (18 aa)

Ac- L-V-G-R-Q-L-E-E-F-L-NH2 [113-122]apoJ (10aa)

D-[113-122]apoJ Increases Paraoxonase 1 Activity in ApoE Null Mice

PON Activity per unit

Cholesterol

0.00

0.50

1.00

1.50

2.00

2.50

3.00

CHOW HDL

D-[113-122]apoJ HDL

0.00

0.50

1.00

1.50

2.00

2.50

3.00

CHOW D-[113-122]apoJ HDL

p=0.0001

D-[113-122]apoJ Renders HDL Anti-

inflammatory in ApoE Null Mice

NoAddition

Cont.LDL

Cont. LDL +

HDL

0

2

4

6

8

Water

Mig

rate

dM

onoc

ytes

/Fie

ld

Autologous HDL+ LDL from treated ApoE Null Mice

*

Assay Controls

D [113-122] apoJNoAddition

Cont.LDL

Cont. LDL +

HDL

0

2

4

6

8

Assay Controls

- -

Comparison of D-4F and D-[113-122]apoJ

8 Br - cAMP-Induced Efflux (ABCA1-Mediated)

0

10

20

30

40

50

% E

fflux

HDL D-[113-122]apoJ

8 Br - -Induced Efflux (ABCA1-Mediated)

VehicleStd.

(5% plasma)

D-4FD-4F

D-[113-122]apoJ Decreases Lesions in ApoE Null Mice (Aortic Root Sinus)

0

100,000

200,000

300,000

400,000

500,000

CHOWn=21

CHOW+D-[113-122]apoJ

n=23

395893+63371

117685+107571

p=4.3x 10-13

70.2%

Aorti

c sin

us le

sion

area

CHOWn=21

CHOW+D-[113-122]apoJ

n=23

395893+63371

117685+107571

p=4.3x 10-13

m2/section

D-[113-122]apoJ Decreases Lesions in ApoE Null Mice (En Face)

0

1.0

2.0

3.0

4.0

CHOWn=27

CHOW +D-[113-122]apoJ

n=23

% S

URFA

CE A

REA

WIT

H LE

SION

S

1.7+/-0.8

0.5+/-0.5

70.5%

p=1.5x10-6

D-[113-122]apoJ Decreases Lipoprotein Lipid Hydroperoxides in Monkeys

0

100

200

300

400

Time 0 3 hrs

HDL

0

400

800

1200

Time 0 3 hrs

LDLp=0.001

ng L

OO

H p

er 5

0 g

cho

lest

erol

ng L

OO

H p

er 5

0 g

cho

lest

erol

p=0.001

0

100

200

300

400

0

100

200

300

400

Time 0 3 hrs

HDL

0

400

800

1200

0

400

800

1200

Time 0 3 hrs

LDL

g ch

oles

tero

l

g ch

oles

tero

l

D-[113-122]apoJ Renders HDL Anti-inflammatory in Monkeys

Mig

rate

d M

onoc

ytes

/Fie

ld

0

5

10

15

20

0

5

10

15

20

No Addition

LDL+ Monkey HDL at Time 0

Monkey # 1 2 3 4 1 2 3 4 Std. LDL

LDL+ Std. HDL

Assay ControlsLDL+ Monkey HDL at 3 hrs

P<0.001

A Comparison of ApoA-I, 4F, [113-122]apoJ, and Tetrapeptides

ApoA-I (243 aa)

Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 4F (18 aa)

Ac- L-V-G-R-Q-L-E-E-F-L-NH2 [113-122]apoJ (10aa)

Boc-K(eBoc)-R-E-S(tBu)-OtBu KRES (4aa)

Boc-F-R-E-L-OtBu FREL (4aa)

Making the Good Cholesterol Better TM

Oral Amphipathic Peptides with Anti-atherosclerotic Properties

•D-[113-122]apoJ is one of three classes of peptides that associate with HDL after an oral dose

•Peptides with a Class A amphipathic helix (D-4F)•Peptides with a G* amphipathic helix (D-[113-122]apoJ)•Tetrapeptides that are too small to form a helix (KRES; FREL)

14C-ApoJ 125I-D-4F

Biologically Active

Biologically Inactive

14C-FREL

Exploring the Differences in the Association

of the Peptides with HDL

•We hypothesized that given the differing temporal association of the peptids with HDL a combination of the three compounds ( D-4F and D-[113-122]apoJ and FREL) might extend the anti-inflammatory properties of HDL after a single oral dose.

HDL Inflammatory Properties 6 Hours After Dosing

0

5

10

15

20

25

F+J F+J

0.5 D- 4F

Mig

rate

d M

onoc

ytes

/Fie

ld

F+J 1.0 D- 4F

p<0.001p<0.001

LDL+HDLNo Addition

Std LDL

+Std HDL

24 Hours After Dosing

0

5

10

15

Mig

rate

d M

onoc

ytes

/Fie

ld

No Addition

Std LDL

+Std HDL F+J

F+J

0.5 D -4F

F+J

1.0 D - 4F

p<0.001p<0.001

LDL+HDL

48 Hours After Dosing

0

5

10

15

20

25

30

Mig

rate

d M

onoc

ytes

/Fie

ld p<0.001p<0.01

No Addition

Std LDL

+Std HDL F+J

F+J

0.5 D- 4F

F+J

1.0 D- 4F

LDL+HDL

72 Hours After Dosing

0

5

10

15

20

25

30

Mig

rate

d M

onoc

ytes

/Fie

ld

No + HDL + HDL + HDL+F+J + HDL + HDL+F+J

p<0.001 p<0.001

No Addition

Std LDL

+Std HDL F+J

F+J

0.5 D- 4F

F+J

1.0 D- 4F

D-4F versus D-[113-122]apoJ versus Tetrapeptides

Property D-4F 113-122 TetrapeptidesStimulatePre- HDL Yes No NoStim HDLChol Efflux Yes Yes NoStim. ReverseChol Transport Yes ND NoReduceLOOH Yes Yes YesIncrease PON Yes Yes YesImprove HDL Yes Yes YesInflammatoryProperties

D-4F versus D-[113-122]apoJ versus TetrapeptidesProperty D-4F 113-122 TetrapeptidesImproveLDL-MCA Yes Yes YesPrevent Lesions ~75% ~70% ~50%L-Active Orally No Not likely YesStatin Synergy Yes ND NDRegress Lesions Yes ND ND

Summary

•Oral amphipathic peptides from different classes associate with HDL differently but share certain common properties:

•Reduce HDL-lipid hydroperoxides•Increase HDL-anti-oxidant enzymes (e.g. Paraoxonase)•Render HDL anti-inflammatory

•These peptides provide unique tools for dissecting and understanding HDL metabolism and its relationship to the innate immune system.

UCLA UAB

Alan M. Fogelman G.M. AnantharamaiahSrinivasa T. Reddy David W. GarberBrian J. Van Lenten Manjula ChaddhaGreg P. Hough Geeta DattaSusan Y. Hama Shaila Handattu Alan Wagner Vinod K. Mishra Joy Frank Mayakonda N. Palgunachari Victor R. Grijalva Vannakambadi K. Ganesh Jan Danciger