D-Stereoisomers of Apo A-I Mimetic Peptides

Given Orally

Reduce Atherosclerosis in Mice

Mohamad Navab

UCLA Cardiology

Objective:

Identification of a class of peptides that are orally administrable and that ameliorate one or more symptoms of atherosclerosis.

Introduction:We have reported that when LDL was incubated with apoA-I or apo A-I peptide mimetics, LDL became resistant to oxidation by artery wall cells in coculture.

Similarly, when the artery wall cells in culture were pretreated with apoA-I, or the peptide mimetics, the cells were no longer capable of oxidizing LDL.

Peripheral human monocytes pre-incubated with apoA-I became similarly incapable of oxidizing LDL.

We showed that apoA-I removed, from LDL, the “seeding molecules” HPETEs and HPODEs that are required for LDL lipid oxidation.

ApoA-I and other exchangeable apolipoproteins, possess lipid-associating domains.

Brouillette & Anantharamaiah (1995), Segrest et al. (1974) Apo A-I has been postulated to possess eight tandem repeating 22mer sequences, most of which have the potential to form class A amphipathic helical structures.

(Segrest et al. 1974)

Apo A-I strongly associates with phospholipids to form complexes and to promote cholesterol efflux from cholesterol-enriched cells.

The delivery and maintenance of serum levels of apo A-I to effectively mitigate one or more symptoms of atherosclerosis has heretofore proven elusive.

Peptides comprising a class A amphipathic helix when formulated with "D" amino acid residue(s) and/or having protected amino and carboxyl termini can be: - orally administered to an organism, - are taken up and delivered to serum

- are effective to mitigate symptoms of atherosclerosis.

Novel peptide administration of which mitigate one or more symptoms of atherosclerosis.

The peptide ranges in length from about 10 to about 30 amino acids, at least one class A amphipathic helix,at least one "D" amino acid residue, protects a phospholipid against oxidation by an oxidizing agent,

Protecting groups include, acetyl, and amide groups coupled to the amino terminus and the carboxyl terminus.

Particularly preferred peptides comprise greater than about 50% amino acid sequence identity with human or mouse apo A-1 or with the polypeptide encoded by the exon encoding a class A amphipathic helix of human or mouse apo A-1.

Peptides formulated using D amino acids, the peptides show dramatically elevated serum half-lives and, particularly when the amino and/or carboxy termini are blocked, can be orally administered.

Such D-form peptides retain the biological activity of the corresponding L-form peptide. In vivo animal studies using such D-form peptides showed effective oral delivery, elevated serum half-life, and the ability to mitigate or prevent/inhibit symptoms of atherosclerosis.

Normal HDL inhibits three steps in the formation of mildly oxidized LDL.

In those studies treating human LDL in vitro with apo A-I or an apo A-I mimetic peptide removed seeding molecules from the LDL that included HPODE and HPETE. These “seeding” molecules were required for cocultures of human artery wall cells to be able to oxidize LDL and for the LDL to induce the artery wall cells to produce monocyte chemotactic activity. We also demonstrated that after injection of apo A-I into mice or infusion into humans, the LDL isolated from the mice or human volunteers after injection/infusion of apo A-I was resistant to oxidation by human artery wall cells and did not induce monocyte chemotactic activity in the artery wall cell cocultures.

It is noted that the fourth exon of apo A-I, when folded into 3.667 residues/turn produces a class A amphipathic helical structure.

One particularly preferred class A peptide, designated 18A (Anantharamaiah, 1986) was modified to produce peptides orally administrable and highly effective at inhibiting or preventing one or more symptoms of atherosclerosis.

The peptides may act in vivo by picking up “seeding molecule(s)” that mitigate oxidation of LDL.

Anantharamaiah’s group determined that increasing the number of Phe residues on the hydrophobic face of the 18A would theoretically increase lipid affinity as determined by computation. Palgunachari et al. (1996)

Apo A-I Peptide Mimetic 2F Hydrophobic face

Hydrophilic faceAmino group

Therefore, initially 5 additional Phe was chosen and hence the peptides designation as 5F.

The 5F peptide was blocked in that the amino terminal residue was acetylated and the carboxyl terminal residue was amidated.

The new class A peptide analog, 5F inhibited , lesion development in atherosclerosis-susceptible mice.

Garber et al. 2000

The peptides used are chemically synthesized using standard chemical peptide synthesis techniques.

Peptides with an additional 2, 3 and 4 Phe would have higher theoretical lipid affinity.

Theoretically, a systematic substitution of residues in the nonpolar face of 18A with Phe could yield six peptides.

ApoA-1 mimetic peptides.

18A D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F

2F Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH2

3F Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH2

3F14 Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2

4F Ac-D-W-F-K-A-F-Y-E-K-V-A-E-K-L-K-E-F-F-NH2

5F Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2

6F Ac-D-W-L-K-A-F-Y-D-K-F- F-E-K-F-K-E-F-F-NH2

7F Ac-D-W-F-K-A-F-Y-D-K-F-F- E-K-F-K-E-F-F-NH2

ApoA-I mimetic D-peptides prevent LDL induced monocyte chemotactic activity

Mig

rate

d m

onoc

ytes

per

HPF

D-2F L-2F D-37pA L-37pA

Assay controls

h HDL +

h LD

L

h LDL LDL, NO CELLS

CELLS, N0 LDL µg25

0

125 62 31

+ h LDL

250

125 62 31

0

1

2

3

4

5

6

7

8

9

10

250

125 62 31 250

125 62 31

Increased HDL protective capacity after oral peptide, D-4F in LDL R-/- mice

0

1

2

3

4

5

6

7

8

9

m HDL + LDL

Saline D - 4 FL- 4 F

Mig

rate

d m

onoc

ytes

per

HPF

100 µg50

P<.01

100 µg50 100 µg50

No Addition

h LDL

h LDL +h HDL HDL

Assay Controls

0

1

2

3

4

5

6

7

8

9

Saline L- 4F D - 4F

m VLDL+ m LDL

Mig

rate

d m

onoc

ytes

per

HPF

No Addition

h LDL

h LDL + h HDL

Assay Controls

m LDL

Saline L- 4F D-4F

P<.001

P<.001

Increased resistance to oxidation for LDL after oral peptide, D - 4F in LDL R-/- mice

FPLC Fraction Number

02468

6050403020100

605040302010002468

L-4F

D-4F 24 hr .

5 min. 45 min.

60504030201000

2 4

6

8

60504030201000

2

4

6

8L-4F

D-4F

6050403020100

D-4F

6050403020100

0

2

4

6

8

02468

L-4F

3 hr.

FPLC Fraction Number

60504030201000

2

4

68

60504030201000

24

6

8

D-4F

L-4F

Cho

lest

erol

, m

g/dl

0

10

20 LDL HDL

125 I

, CPM

x 1

0-3

5040302010

125 I

, CPM

x 1

0-3

Plasma radioactivity following oral administration of 125I- 4F peptide

cpm

/ml p

lasm

a

0

2,000

4,000

6,000

8,000

10,000

12,000

Intact18 mer

Free counts L - 4F

6040200 10 30 50

D - 4F

0

cpm

/ml p

lasm

a2,000

4,000

6,000

8,000

10,000

12,000

6040200 10 30 50

Intact D-4F in plasma at 4 hrs

HPLC Fractions

High levels of radioactivity in mouse urine following oral administration of 125 I - L- 4F

cpm

/ 50

µl u

rine

30 60 120 240 min

0

2 000 000

4 000 000

6 000 000

8 000 000

Time after gavage with 125 I-4F

L - 4FD - 4F

20

30

40

50

60

70

80

90

100

110

120

130

Aort

ic r

oot

lesi

on a

rea

µm 2

x 1

0 -3 p

er s

ecti

on

D - 5 FSaline

84,495

50,100

41%

Reduction by D-5F of lesion scores in LDL receptor deficient mice on a Western type diet.

P<0.026

0

10

20

30

40

50

Saline D-5F

Aor

tic s

urfa

ce O

RO

sta

in, µ

m2

8,800

21,500

59.06%

P<0.01

0

10

20

30

40

50

60

P< .01

81 %

Saline D- 4F

Aort

ic r

oot

lesi

on a

rea

µm 2

x 1

0 -3 p

er s

ecti

on

-

0

5

5

0

0

5

1

1

20

2

3

3 5

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

Cho

lest

erol

(mg/

dl)

FPLC Fractions

ControlD-4F

Plasma cholesterol in LDL R-/- mice gavaged with D-4F for 6 weeks

In Progress

0

5

10

15

20

25

30

35

40

13 1415 1617 18 1920 2122 23 2425 26 2728 2930 31 3233 3435 36

Chol

este

rol

(mg/

dl)

ControlD-4F

Plasma cholesterol in apoE null mice on D-4F in drinking water for 5 weeks

FPLC Fractions

PON activity in apoE null mice on D-4F in drinking water for 5 weeksPa

raox

onas

e Ac

tivi

ty ,

unit

s/m

l pla

sma

0

10

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

Control+ D-4F

2

4

6

8

FPLC Fractions

Peptides formulated using D amino acids show dramatically elevated serum half-lives and, particularly when the amino and/or carboxy termini are blocked, can be orally administered.

Such D-form peptides retain the biological activity of the corresponding L-form peptide. In vivo animal studies using such D-form peptides showed effective oral delivery, elevated serum half-life, and the ability to markedly reduce atherosclerotic lesions.

Conclusion

Plans

- Determine tissue distribution

- Investigate potential cytotoxic effects

- preparation for studies in humans