SHAPE Society

1. D-Stereoisomers of Apo A-I Mimetic Peptides
Given Orally
Reduce Atherosclerosis in Mice
Mohamad Navab
UCLA Cardiology

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

3. 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.

4. 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.

5. 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.

6. 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.

7. 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.

8. 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.

9. 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.

10. 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 face
Amino group

11. 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.

12. 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

13. ApoA-I mimetic D-peptides prevent
LDL induced monocyte chemotactic activityMigratedmonocytesperHPF
D-2F L-2F D-37pA L-37pA
Assay controls
h
HDL
+
h
LDL
h
LDL
LDL, NO CELLS
CELLS, N0 LDL
µg
250
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

14. 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
MigratedmonocytesperHPF
100 µg50
P<.01
100 µg50 100 µg50
N
o
Addition
h
LD
L
h
LD
L
+h
H
D
L
H
D
L
Assay Controls

15. 0
1
2
3
4
5
6
7
8
9
Saline L- 4F D - 4F
m VLDL+ m LDL
MigratedmonocytesperHPF
N
o
Addition
h
LD
L
h
LD
L
+
h
H
D
L
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

16. FPLC Fraction Number
0
2
4
6
8
6050403020100
60504030201000
2
4
6
8
L-4F
D-4F
24 hr .
5 min. 45 min.
60504030201000
2
4
6
8
6050403020100
0
2
4
6
8
L-4F
D-4F
6050403020100
D-4F
6050403020100
0
2
4
6
8
0
2
4
6
8
L-4F
3 hr.
FPLC Fraction Number
6050403020100
0
2
4
6
8
6050403020100
0
2
4
6
8
D-4F
L-4F
Cholesterol,
mg/dl
0
10
20 LDL HDL
125
I,CPMx10-3
5040302010
125
I,CPMx10-3
Plasma radioactivity following oral administration of 125
I- 4F peptide

17. cpm/mlplasma
0
2,000
4,000
6,000
8,000
10,000
12,000
Intact
18 mer
Free
counts
L - 4F
6040200 10 30 50
D - 4F
0
cpm/mlplasma
2,000
4,000
6,000
8,000
10,000
12,000
6040200 10 30 50
Intact D-4F in plasma at 4 hrs
HPLC Fractions

18. High levels of radioactivity in mouse urine
following oral administration of 125
I - L- 4F
cpm/50µlurine
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 - 4F
D - 4F

19. 20
30
40
50
60
70
80
90
100
110
120
130
Aorticrootlesionarea
µm2
x10-3
persection
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

20. 0
10
20
30
40
50
Saline D-5F
AorticsurfaceOROstain,µm2
8,800
21,500
59.06%
P<0.01

21. 0
10
20
30
40
50
60
P< .01
81 %
Saline D- 4F
Aorticrootlesionarea
µm2
x10-3
persection

22. -
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
Cholesterol(mg/dl)
FPLC Fractions
Control
D-4F
Plasma cholesterol in LDL R-/- mice gavaged with D-4F for 6 weeks

23. In Progress
0
5
10
15
20
25
30
35
40
13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Cholesterol(mg/dl)
Control
D-4F
Plasma cholesterol in apoE null mice on D-4F in drinking water for 5 weeks
FPLC Fractions

24. PON activity in apoE null mice on D-4F in drinking water for 5 weeks
ParaoxonaseActivity,units/mlplasma
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

25. 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

26. Plans
- Determine tissue distribution
- Investigate potential cytotoxic effects
- preparation for studies in humans