U.S. patent application number 10/913800 was filed with the patent office on 2005-07-28 for orally administered small peptides synergize statin activity.
This patent application is currently assigned to The Regents of the University of California. Invention is credited to Anantharamaiah, Gattadahalli M., Fogelman, Alan M., Navab, Mohamad.
Application Number | 20050164950 10/913800 |
Document ID | / |
Family ID | 34197999 |
Filed Date | 2005-07-28 |
United States Patent
Application |
20050164950 |
Kind Code |
A1 |
Fogelman, Alan M. ; et
al. |
July 28, 2005 |
Orally administered small peptides synergize statin activity
Abstract
This invention provides novel peptides that ameliorate one or
more symptoms of atherosclerosis. The peptides are highly stable
and readily administered via an oral route. The peptides are
effective to stimulate the formation and cycling of pre-beta high
density lipoprotein-like particles and/or to promote lipid
transport and detoxification. This invention also provides a method
of tracking a peptide in a mammal. In addition, the peptides
inhibit osteoporosis. When administered with a statin, the peptides
enhance the activity of the statin permitting the statin to be used
at significantly lower dosages and/or cause the statins to be
significantly more anti-inflammatory at any given dose.
Inventors: |
Fogelman, Alan M.; (Beverly
Hills, CA) ; Anantharamaiah, Gattadahalli M.;
(Birmingham, AL) ; Navab, Mohamad; (Los Angeles,
CA) |
Correspondence
Address: |
QUINE INTELLECTUAL PROPERTY LAW GROUP, P.C.
P O BOX 458
ALAMEDA
CA
94501
US
|
Assignee: |
The Regents of the University of
California
|
Family ID: |
34197999 |
Appl. No.: |
10/913800 |
Filed: |
August 6, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10913800 |
Aug 6, 2004 |
|
|
|
10649378 |
Aug 26, 2003 |
|
|
|
60494449 |
Aug 11, 2003 |
|
|
|
Current U.S.
Class: |
514/1.9 ;
514/12.2; 514/7.4; 530/330; 530/331 |
Current CPC
Class: |
A61K 38/03 20130101;
A61P 11/00 20180101; A61P 43/00 20180101; A61P 19/10 20180101; C07K
5/1016 20130101; C07K 5/1021 20130101; C07K 7/02 20130101; C07K
5/0815 20130101; A61P 17/00 20180101; C07K 7/06 20130101; A61K
31/366 20130101; A61K 31/405 20130101; A61K 38/03 20130101; A61P
11/16 20180101; A61K 31/47 20130101; A61P 25/00 20180101; C07K
5/1019 20130101; C07K 14/775 20130101; A61P 21/00 20180101; A61P
11/06 20180101; A61P 9/00 20180101; A61P 25/28 20180101; A61K
31/366 20130101; A61P 29/00 20180101; A61P 9/10 20180101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 31/47 20130101; C07K 5/101 20130101; C07K 5/0808 20130101;
A61K 31/44 20130101; A61P 19/02 20180101; A61K 31/405 20130101;
A61P 3/10 20180101; C07K 5/0812 20130101; A61P 31/12 20180101; A61K
45/06 20130101; A61K 31/44 20130101; Y02P 20/55 20151101; A61K
31/505 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 31/505 20130101; A61P 19/00 20180101 |
Class at
Publication: |
514/017 ;
514/018; 530/330; 530/331 |
International
Class: |
A61K 038/06; A61K
038/05; C07K 005/06 |
Goverment Interests
[0002] This work was supported by United States Public Health
Service and National Heart, Lung, and Blood Institute Grants
HL30568 and HL34343. The Government of the United States of America
may have certain rights in this invention.
Claims
What is claimed is:
1. A peptide that ameliorates one or more symptoms of an
inflammatory condition, wherein said peptide: ranges in length from
3 to about 5 amino acids; is soluble in ethyl acetate at a
concentration greater than about 4mg/mL; is soluble in aqueous
buffer at pH 7.0; when contacted with a phospholipid in an aqueous
environment, forms particles with a diameter of approximately 7.5
nm and forms stacked bilayers with a bilayer dimension on the order
of 3.4 to 4.1 nm with spacing between the bilayers in the stack of
approximately 2 nm; has a molecular weight less than about 900
daltons; converts pro-inflammatory HDL to anti-inflammatory HDL or
makes anti-inflammatory HDL more anti-inflammatory; and does not
have the amino acid sequence Lys-Arg-Asp-Ser (SEQ ID NO:238) in
which Lys-Arg-Asp and Ser are all L amino acids.
2. The peptide of claim 1, wherein said peptide protects a
phospholipid against oxidation by an oxidizing agent
3. The peptide of claim 2, wherein said oxidizing agent is selected
from the group consisting of hydrogen peroxide, 13(S)--HPODE,
15(S)--HPETE, HPODE, HPETE, HODE, and HETE.
4. The peptide of claim 2, wherein said phospholipid is selected
from the group consisting of
1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcho- line (PAPC),
1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (SAPC)),
and 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphorylethanolamine
(SAPE).
5. A peptide that ameliorates one or more symptoms of an
inflammatory condition, said peptide having the formula:
18 X.sup.1-X.sup.2-X.sup.3.sub.n-X.sup.4
wherein: n is 0 or 1; X.sup.1 is a hydrophobic amino acid and/or
bears a hydrophobic protecting group; X.sup.4 is a hydrophobic
amino acid and/or bears a hydrophobic protecting group; and when n
is 0: X.sup.2 is an amino acid selected from the group consisting
of an acidic amino acid, a basic amino acid, and a histidine; when
n is 1: X.sup.2 and X.sup.3 are independently an acidic amino acid,
a basic amino acid, an aliphatic amino acid, or an aromatic amino
acid such that when X.sup.2 is an acidic amino acid; X.sup.3 is a
basic amino acid, an aliphatic amino acid, or an aromatic amino
acid; when X.sup.2 is a basic amino acid; X.sup.3 is an acidic
amino acid, an aliphatic amino acid, or an aromatic amino acid; and
when X.sup.2 is an aliphatic or aromatic amino acid, X.sup.3 is an
acidic amino acid, or a basic amino acid; said peptide converts
pro-inflammatory HDL to anti-inflammatory HDL or makes
anti-inflammatory HDL more anti-inflammatory; and said peptide does
not have the amino acid sequence Lys-Arg-Asp-Ser (SEQ ID NO:238) in
which Lys-Arg-Asp and Ser are all L amino acids.
6. The peptide of claim 5, wherein n is 0.
7. The peptide of claim 6, wherein wherein X.sup.1 and X.sup.4 are
independently selected from the group consisting of alanine (Ala),
valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro)
phenylalanine (Phe), tryptophan (Trp), methionine (Met), serine
(Ser) bearing a hydrophobic protecting group, beta-naphthyl
alanine, alpha-naphthyl alanine, norleucine, cyclohexylalanine,
threonine (Thr) bearing a hydrophobic protecting group, tyrosine
(Tyr) bearing a hydrophobic protecting group, lysine (Lys) bearing
a hydrophobic protecting group, arginine (Arg) bearing a
hydrophobic protecting group, omithine (Om) bearing a hydrophobic
protecting group, aspartic acid (Asp) bearing a hydrophobic
protecting group, cysteine (Cys) bearing a hydrophobic protecting
group, and glutamic acid (Glu) bearing a hydrophobic protecting
group.
8. The peptide of claim 7, wherein: X.sup.1 is is selected from the
group consisting of Glu, Leu, Lys, Orn, Phe, Trp, and norLeu;
X.sup.2 is selected from the group consisting of Asp, Arg, and Glu;
and X.sup.4 is selected from the group consisting of Ser, Thr, Ile,
Leu, Trp, Tyr, Phe, and norleu.
9. The peptide of claim 7, wherein X.sup.1 is is selected from the
group consisting of Glu, Leu, Lys, Orn, Phe, Trp, and norLeu;
X.sup.2 is selected from the group consisting of Lys, Arg, and His;
and X.sup.4 is selected from the group consisting of Asp, Arg, and
Glu.
10. The peptide of claim 6 wherein X.sup.1 bears a hydrophobic
protecting group.
11. The peptide of claim 10, wherein said hydrophobic protecting
group is selected from the group consisting of polyethylene glycol
(PEG), t-butoxycarbonyl (Boc), Fmoc, nicotinyl, OtBu, a benzoyl
group, an acetyl (Ac), a carbobenzoxy, methyl, ethyl, a propyl, a
butyl, a pentyl a hexyl ester, an N-methyl anthranilyl, and a 3 to
20 carbon alkyl, amide, a 3 to 20 carbon alkyl group,
9-fluoreneacetyl group, 1-fluorenecarboxylic group,
9-fluorenecarboxylic group, 9-fluorenone-1-carboxylic group,
benzyloxycarbonyl (is also called carbobenzoxy mentioned above),
Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl
(Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr),
Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl
(Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc),
4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO),
Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys),
1-(4,4-dimethyl-2,6-dioxocyclohexylid- ene)ethyl (Dde),
2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl
(2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), benzyloxymethyl (Bom),
cyclohexyloxy (cHxO),t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl
(tBu), trifluoroacetyl (TFA),
4[N-{1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyldibutyl)-amino}ben-
zyl ester (ODmab), .alpha.-allyl ester (OAll), 2-phenylisopropyl
ester (2-PhiPr), 1-[4,4-dimethyl-2,6-dioxycyclohex-1-yl-idene)ethyl
(Dde).
12. The peptide of claim 11, wherein said hydrophobic protecting
group is selected from the group consisting of Boc, Fmoc,
nicotinyl, and OtBu.
13. The peptide of claim 10, wherein X.sup.4 bears a hydrophobic
protecting group.
14. The peptide of claim 13, wherein said hydrophobic protecting
group is selected from the group consisting of polyethylene glycol
(PEG), t-butoxycarbonyl (Boc), Fmoc, nicotinyl, OtBu, a benzoyl
group, an acetyl (Ac), a carbobenzoxy, methyl, ethyl, a propyl, a
butyl, a pentyl a hexyl ester, an N-methyl anthranilyl, and a 3 to
20 carbon alkyl, amide, a 3 to 20 carbon alkyl group,
9-fluoreneacetyl group, 1-fluorenecarboxylic group,
9-fluorenecarboxylic group, 9-fluorenone-1-carboxylic group,
benzyloxycarbonyl (is also called carbobenzoxy mentioned above),
Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl
(Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr),
Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl
(Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc),
4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO),
Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys),
1-(4,4-dimethyl-2,6-dioxocyclohexylid- ene)ethyl (Dde),
2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl
(2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), benzyloxymethyl (Bom),
cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO),
t-Butyl (tBu), trifluoroacetyl (TFA),
4[N-{1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyldibutyl)-amino}ben-
zyl ester (ODmab), .alpha.-allyl ester (OAll), 2-phenylisopropyl
ester (2-PhiPr), 1-[4,4-dimethyl-2,6-dioxycyclohex-1-yl-idene)ethyl
(Dde).
15. The peptide of claim 14, wherein the N-terminus of said peptide
is blocked with a protecting group selected from the group
consisting of Boc-, Fmoc-, and Nicotinyl-.
16. The peptide of claim 14, wherein the C-terminus of said peptide
is blocked with a protecting group selected from the group
consisting of tBu, and OtBu.
17. The peptide of claim 6, wherein said peptide comprises the
amino acid sequence of a peptide in Table 3.
18. The peptide of claim 6, wherein said peptide is a peptide from
Table 3.
19. The peptide of claim 6, wherein said peptide comprises at least
one D-amino acid.
20. The peptide of claim 6, wherein said peptide comprises all
D-amino acids.
21. The peptide of claim 6, wherein said peptide comprises
alternating D- and L-amino acids.
22. The peptide of claim 6, wherein said peptide comprises all
L-amino acids.
23. The peptide of claim 6, wherein said peptide is mixed with a
pharmacologically acceptable excipient.
24. The peptide of claim 6, wherein said peptide is mixed with a
pharmacologically acceptable excipient suitable for oral
administration to a mammal.
25. The peptide of claim 6, wherein said polypeptide is provided as
a unit formulation in a pharmaceutically acceptable excipient.
26. The peptide of claim 6, wherein said polypeptide is provided as
a time release formulation.
27. The peptide of claim 6, wherein said peptide protects a
phospholipid against oxidation by an oxidizing agent
28. The peptide of claim 27, wherein said oxidizing agent is
selected from the group consisting of hydrogen peroxide,
13(S)--HPODE, 15(S)--HPETE, HPODE, HPETE, HODE, and HETE.
29. The peptide of claim 27, wherein said phospholipid is selected
from the group consisting of
1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphory- lcholine (PAPC),
1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (SAPC)),
1-stearoyl-2-arachidonyl-sn-glycero-3-phosphorylethanolamine
(SAPE).
30. The peptide of claim 6, wherein said peptide is coupled to a
biotin.
31. The peptide of claim 5, wherein: n is 1; and X.sup.2 and
X.sup.3 are independently an acidic amino acid or a basic amino
acid such that when X.sup.2 is an acidic amino acid, X.sup.3 is a
basic amino acid and when X.sup.2 is a basic amino acid, X.sup.3 is
an acidic amino acid.
32. The peptide of claim 31, wherein wherein X.sup.1 and X.sup.4
are independently selected from the group consisting of alanine
(Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline
(Pro), phenylalanine (Phe), tryptophan (Trp), methionine (Met),
serine (Ser) bearing a hydrophobic protecting group, beta-naphthyl
alanine, alpha-naphthyl alanine, norleucine, cyclohexylalanine,
threonine (Thr) bearing a hydrophobic protecting group, tyrosine
(Tyr) bearing a hydrophobic protecting group, lysine (Lys) bearing
a hydrophobic protecting group, arginine (Arg) bearing a
hydrophobic protecting group, omithine (Orn) bearing a hydrophobic
protecting group, aspartic acid (Asp) bearing a hydrophobic
protecting group, cysteine (Cys) bearing a hydrophobic protecting
group, and glutamic acid (Glu) bearing a hydrophobic protecting
group.
33. The peptide of claim 32, wherein X.sup.2 and X.sup.3 are
independently selected from the group consisting of Asp, Glu, Lys,
Arg, and His.
34. The peptide of claim 32, wherein X.sup.2 and X.sup.3 are
independently selected from the group consisting of Asp, Arg, and
Glu.
35. The peptide of claim 33 wherein X.sup.1 bears a hydrophobic
protecting group.
36. The peptide of claim 35, wherein said hydrophobic protecting
group is selected from the group consisting of polyethylene glycol
(PEG), t-butoxycarbonyl (Boc), Fmoc, nicotinyl, OtBu, a benzoyl
group, an acetyl (Ac), a carbobenzoxy, methyl, ethyl, a propyl, a
butyl, a pentyl a hexyl ester, an N-methyl anthranilyl, and a 3 to
20 carbon alkyl, amide, a 3 to 20 carbon alkyl group,
9-fluoreneacetyl group, 1-fluorenecarboxylic group,
9-fluorenecarboxylic group, 9-fluorenone-1-carboxylic group,
benzyloxycarbonyl (is also called carbobenzoxy mentioned above),
Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl
(Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr),
Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl
(Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc),
4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO),
Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys),
1-(4,4-dimethyl-2,6-dioxocyclohexylid- ene)ethyl (Dde),
2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl
(2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), benzyloxymethyl (Bom),
cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO),
t-Butyl (tBu), trifluoroacetyl (TFA),
4[N-{1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyldibutyl)-amino}ben-
zyl ester (ODmab), .alpha.-allyl ester (OAll), 2-phenylisopropyl
ester (2-PhiPr), 1-[4,4-dimethyl-2,6-dioxycyclohex-1-yl-idene)ethyl
(Dde).
37. The peptide of claim 35, wherein said said hydrophobic
protecting group is selected from the group consisting of Boc,
Fmoc, nicotinyl, and OtBu.
38. The peptide of claim 35, wherein X.sup.4 bears a hydrophobic
protecting group.
39. The peptide of claim 38, wherein said hydrophobic protecting
group is selected from the group consisting of polyethylene glycol
(PEG), t-butoxycarbonyl (Boc), Fmoc, nicotinyl, OtBu, a benzoyl
group, an acetyl (Ac), a carbobenzoxy, methyl, ethyl, a propyl, a
butyl, a pentyl a hexyl ester, an N-methyl anthranilyl, and a 3 to
20 carbon alkyl, amide, a 3 to 20 carbon alkyl group,
9-fluoreneacetyl group, 1-fluorenecarboxylic group,
9-fluorenecarboxylic group, 9-fluorenone-1-carboxylic group,
benzyloxycarbonyl (is also called carbobenzoxy mentioned above),
Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl
(Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr),
Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl
(Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc),
4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO),
Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys),
1-(4,4-dimethyl-2,6-dioxocyclohexylid- ene)ethyl (Dde),
2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl
(2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), benzyloxymethyl (Bom),
cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO),
t-Butyl (tBu), trifluoroacetyl (TFA),
4[N-{1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyldibutyl)-amino}ben-
zyl ester (ODmab), .alpha.-allyl ester (OAll), 2-phenylisopropyl
ester (2-PhiPr), 1-[4,4-dimethyl-2,6-dioxycyclohex-1-yl-idene)ethyl
(Dde).
40. The peptide of claim 35, wherein the N-terminus of said peptide
is blocked with a protecting group selected from the group
consisting of Boc-, Fmoc-, and Nicotinyl-.
41. The peptide of claim 35, wherein the C-terminus of said peptide
is blocked with a protecting group selected from the group
consisting of tBu, and OtBu.
42. The peptide of claim 31, wherein said peptide comprises the
amino acid sequence of a peptide in Table 4.
43. The peptide of claim 31, wherein said peptide is a peptide from
Table 4.
44. The peptide of claim 31, wherein said peptide comprises at
least one D-amino acid.
45. The peptide of claim 31, wherein said peptide comprises all
D-amino acids.
46. The peptide of claim 31, wherein said peptide comprises
alternating D- and L-amino acids.
47. The peptide of claim 31, wherein said peptide comprises all
L-amino acids.
48. The peptide of claim 31, wherein said peptide is mixed with a
pharmacologically acceptable excipient.
49. The peptide of claim 31, wherein said peptide is mixed with a
pharmacologically acceptable excipient suitable for oral
administration to a mammal.
50. The peptide of claim 31, wherein said polypeptide is provided
as a unit formulation in a pharmaceutically acceptable
excipient.
51. The peptide of claim 31, wherein said polypeptide is provided
as a time release formulation.
52. The peptide of claim 31, wherein said peptide protects a
phospholipid against oxidation by an oxidizing agent
53. The peptide of claim 31, wherein said peptide is coupled to a
biotin.
54. The peptide of claim 5, wherein: n is 1; and X.sup.2, X.sup.3
are independently an acidic, a basic, or a aliphatic amino acid
with one of X.sup.2 or X.sup.3 being an acidic or a basic amino
acid such that: when X.sup.2 is an acidic or a basic amino acid,
X.sup.3 is an aliphatic amino acid; and when X.sup.3 is an acid or
a basic amino acid, X.sup.2 is an aliphatic amino acid.
55. The peptide of claim 54, wherein wherein X.sup.1 and X.sup.4
are independently selected from the group consisting of alanine
(Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline
(Pro), phenylalanine (Phe), tryptophan (Trp), methionine (Met),
serine (Ser) bearing a hydrophobic protecting group, beta-naphthyl
alanine, alpha-naphthyl alanine, norleucine, cyclohexylalanine,
threonine (Thr) bearing a hydrophobic protecting group, tyrosine
(Tyr) bearing a hydrophobic protecting group, lysine (Lys) bearing
a hydrophobic protecting group, arginine (Arg) bearing a
hydrophobic protecting group, ornithine (Orn) bearing a hydrophobic
protecting group, aspartic acid (Asp) bearing a hydrophobic
protecting group, cysteine (Cys) bearing a hydrophobic protecting
group, and glutamic acid (Glu) bearing a hydrophobic protecting
group.
56. The peptide of claim 55, wherein X.sup.2 and X.sup.3 are
independently selected from the group consisting of Asp, Arg, Lys,
Leu, Ile, and Glu.
57. The peptide of claim 55, wherein Xl bears a hydrophobic
protecting group.
58. The peptide of claim 57, wherein said hydrophobic protecting
group is selected from the group consisting of polyethylene glycol
(PEG), t-butoxycarbonyl (Boc), Fmoc, nicotinyl, OtBu, a benzoyl
group, an acetyl (Ac), a carbobenzoxy, methyl, ethyl, a propyl, a
butyl, a pentyl a hexyl ester, an N-methyl anthranilyl, and a 3 to
20 carbon alkyl, amide, a 3 to 20 carbon alkyl group,
9-fluoreneacetyl group, 1-fluorenecarboxylic group,
9-fluorenecarboxylic group, 9-fluorenone-1-carboxylic group,
benzyloxycarbonyl (is also called carbobenzoxy mentioned above),
Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl
(Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr),
Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl
(Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc),
4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO),
Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys),
1-(4,4-dimethyl-2,6-dioxocyclohexylid- ene)ethyl (Dde),
2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl
(2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), benzyloxymethyl (Bom),
cyclohexyloxy (cHxO),t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl
(tBu), trifluoroacetyl (TFA),
4[N-{1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyldibutyl)-amino}ben-
zyl ester (ODmab), .alpha.-allyl ester (OAll), 2-phenylisopropyl
ester (2-PhiPr), 1-[4,4-dimethyl-2,6-dioxycyclohex-1-yl-idene)ethyl
(Dde).
59. The peptide of claim 57, wherein said said hydrophobic
protecting group is selected from the group consisting of Boc,
Fmoc, nicotinyl, and OtBu.
60. The peptide of claim 57, wherein X.sup.4 bears a hydrophobic
protecting group.
61. The peptide of claim 60, wherein said hydrophobic protecting
group is selected from the group consisting of polyethylene glycol
(PEG), t-butoxycarbonyl (Boc), Fmoc, nicotinyl, OtBu, a benzoyl
group, an acetyl (Ac), a carbobenzoxy, methyl, ethyl, a propyl, a
butyl, a pentyl a hexyl ester, an N-methyl anthranilyl, and a 3 to
20 carbon alkyl, amide, a 3 to 20 carbon alkyl group,
9-fluoreneacetyl group, 1-fluorenecarboxylic group,
9-fluorenecarboxylic group, 9-fluorenone-1-carboxylic group,
benzyloxycarbonyl (is also called carbobenzoxy mentioned above),
Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl
(Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr),
Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl
(Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc),
4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO),
Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys),
1-(4,4-dimethyl-2,6-dioxocyclohexylid- ene)ethyl (Dde),
2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl
(2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), benzyloxymethyl (Bom),
cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO),
t-Butyl (tBu), trifluoroacetyl (TFA),
4[N-{1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyldibutyl)-amino}ben-
zyl ester (ODmab), .alpha.-allyl ester (OAll), 2-phenylisopropyl
ester (2-PhiPr), 1-[4,4-dimethyl-2,6-dioxycyclohex-1-yl-idene)ethyl
(Dde).
62. The peptide of claim 57, wherein the N-terminus of said peptide
is blocked with a protecting group selected from the group
consisting of Boc-, Fmoc-, and Nicotinyl-.
63. The peptide of claim 57, wherein the C-terminus of said peptide
is blocked with a protecting group selected from the group
consisting of tBu, and OtBu.
64. The peptide of claim 54, wherein said peptide comprises the
amino acid sequence of a peptide in Table 5.
65. The peptide of claim 54, wherein said peptide is a peptide from
Table 5.
66. The peptide of claim 54, wherein said peptide comprises at
least one D-amino acid.
67. The peptide of claim 54, wherein said peptide comprises all
D-amino acids.
68. The peptide of claim 54, wherein said peptide comprises
alternating D- and L-amino acids.
69. The peptide of claim 54, wherein said peptide comprises all
L-amino acids.
70. The peptide of claim 54, wherein said peptide is mixed with a
pharmacologically acceptable excipient.
71. The peptide of claim 54, wherein said peptide is mixed with a
pharmacologically acceptable excipient suitable for oral
administration to a mammal.
72. The peptide of claim 54, wherein said polypeptide is provided
as a unit formulation in a pharmaceutically acceptable
excipient.
73. The peptide of claim 54, wherein said polypeptide is provided
as a time release formulation.
74. The peptide of claim 54, wherein said peptide protects a
phospholipid against oxidation by an oxidizing agent
75. The peptide of claim 54, wherein said peptide is coupled to a
biotin.
76. The peptide of claim 5, wherein: n is 1; and X.sup.2, X.sup.3
are independently an acidic, a basic, or an aromatic amino acid
with one of X.sup.2 or X.sup.3 being an acidic or a basic amino
acid such that: when X.sup.2 is an acidic or a basic amino acid,
X.sup.3 is an aromatic amino acid; and when X.sup.3 is an acid or a
basic amino acid, X.sup.2 is an aromatic amino acid.
77. The peptide of claim 76, wherein wherein X.sup.1 and X.sup.4
are independently selected from the group consisting of alanine
(Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline
(Pro), phenylalanine (Phe), tryptophan (Trp), methionine (Met),
serine (Ser) bearing a hydrophobic protecting group, beta-naphthyl
alanine, alpha-naphthyl alanine, norleucine, cyclohexylalanine,
threonine (Thr) bearing a hydrophobic protecting group, tyrosine
(Tyr) bearing a hydrophobic protecting group, lysine (Lys) bearing
a hydrophobic protecting group, arginine (Arg) bearing a
hydrophobic protecting group, ornithine (Orn) bearing a hydrophobic
protecting group, aspartic acid (Asp) bearing a hydrophobic
protecting group, cysteine (Cys) bearing a hydrophobic protecting
group, and glutamic acid (Glu) bearing a hydrophobic protecting
group.
78. The peptide of claim 77, wherein X.sup.2 and X.sup.3 are
independently is selected from the group consisting of Asp, Arg,
Glu, Trp, Tyr, Phe, and Lys.
79. The peptide of claim 76, wherein X.sup.1 bears a hydrophobic
protecting group.
80. The peptide of claim 79, wherein said hydrophobic protecting
group is selected from the group consisting of polyethylene glycol
(PEG), t-butoxycarbonyl (Boc), fmoc, nicotinyl, OtBu, a benzoyl
group, an acetyl (Ac), a carbobenzoxy, methyl, ethyl, a propyl, a
butyl, a pentyl a hexyl ester, an N-methyl anthranilyl, and a 3 to
20 carbon alkyl, amide, a 3 to 20 carbon alkyl group,
9-fluoreneacetyl group, 1-fluorenecarboxylic group,
9-fluorenecarboxylic group, 9-fluorenone-1-carboxylic group,
benzyloxycarbonyl (is also called carbobenzoxy mentioned above),
Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl
(Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr),
mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl
(Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc),
4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO),
Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys),
1-(4,4-dimethyl-2,6-dioxocyclohexylid- ene)ethyl (Dde),
2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl
(2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), benzyloxymethyl (Bom),
cyclohexyloxy (cHxO),t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl
(tBu), trifluoroacetyl (TFA),
4[N-{1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyldibutyl)-amino}ben-
zyl ester (ODmab), .alpha.-allyl ester (OAll), 2-phenylisopropyl
ester (2-PhiPr), 1-[4,4-dimethyl-2,6-dioxycyclohex-1-yl-idene)ethyl
(Dde).
81. The peptide of claim 79, wherein said said hydrophobic
protecting group is selected from the group consisting of Boc,
Fmoc, nicotinyl, and OtBu.
82. The peptide of claim 79, wherein X.sup.4 bears a hydrophobic
protecting group.
83. The peptide of claim 82, wherein said hydrophobic protecting
group is selected from the group consisting of polyethylene glycol
(PEG), t-butoxycarbonyl (Boc), Fmoc, nicotinyl, OtBu, a benzoyl
group, an acetyl (Ac), a carbobenzoxy, methyl, ethyl, a propyl, a
butyl, a pentyl a hexyl ester, an N-methyl anthranilyl, and a 3 to
20 carbon alkyl, amide, a 3 to 20 carbon alkyl group,
9-fluoreneacetyl group, 1-fluorenecarboxylic group,
9-fluorenecarboxylic group, 9-fluorenone-1-carboxylic group,
benzyloxycarbonyl (is also called carbobenzoxy mentioned above),
Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl
(Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr),
Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl
(Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc),
4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO),
Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys),
1-(4,4-dimethyl-2,6-dioxocyclohexylid- ene)ethyl (Dde),
2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl
(2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), benzyloxymethyl (Bom),
cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO),
t-Butyl (tBu), trifluoroacetyl (TFA),
4[N-{1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyldibutyl)-amino}ben-
zyl ester (ODmab), .alpha.-allyl ester (OAll), 2-phenylisopropyl
ester (2-PhiPr), 1-[4,4-dimethyl-2,6-dioxycyclohex-1-yl-idene)ethyl
(Dde).
84. The peptide of claim 79, wherein the N-terminus of said peptide
is blocked with a protecting group selected from the group
consisting of Boc-, Fmoc-, and Nicotinyl-.
85. The peptide of claim 79, wherein the C-terminus of said peptide
is blocked with a protecting group selected from the group
consisting of tBu, and OtBu.
86. The peptide of claim 76, wherein said peptide comprises the
amino acid sequence of a peptide in Table 6.
87. The peptide of claim 76, wherein said peptide is a peptide from
Table 6.
88. The peptide of claim 76, wherein said peptide comprises at
least one D-amino acid.
89. The peptide of claim 76, wherein said peptide comprises all
D-amino acids.
90. The peptide of claim 76, wherein said peptide comprises
alternating D- and L-amino acids.
91. The peptide of claim 76, wherein said peptide comprises all
L-amino acids.
92. The peptide of claim 76, wherein said peptide is mixed with a
pharmacologically acceptable excipient.
93. The peptide of claim 76, wherein said peptide is mixed with a
pharmacologically acceptable excipient suitable for oral
administration to a mammal.
94. The peptide of claim 76, wherein said polypeptide is provided
as a unit formulation in a pharmaceutically acceptable
excipient.
95. The peptide of claim 76, wherein said polypeptide is provided
as a time release formulation.
96. The peptide of claim 76, wherein said peptide protects a
phospholipid against oxidation by an oxidizing agent
97. The peptide of claim 76, wherein said peptide is coupled to a
biotin.
98. A peptide that ameliorates one or more symptoms of an
inflammatory condition, said peptide having the formula:
19 X.sup.1-X.sup.2-X.sup.3-X.sup.4-X.sup.5
wherein: X.sup.1 is a hydrophobic amino acid and/or bears a
hydrophobic protecting group; X.sup.5 is a hydrophobic amino acid
and/or bears a hydrophobic protecting group; and X.sup.2, X.sup.3,
and X.sup.4 are independently selected aromatic amino acids or
histidine; and said peptide converts pro-inflammatory HDL to
anti-inflammatory HDL or makes anti-inflammatory HDL more
anti-inflammatory.
99. The peptide of claim 98, wherein wherein X.sup.1 and X.sup.5
are independently selected from the group consisting of alanine
(Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline
(Pro), phenylalanine (Phe), tryptophan (Trp), methionine (Met),
phenylalanine (Phe), tryptophan (Trp), methionine (Met), serine
(Ser) bearing a hydrophobic protecting group, beta-naphthyl
alanine, alpha-naphthyl alanine, norleucine, cyclohexylalanine,
threonine (Thr) bearing a hydrophobic protecting group, tyrosine
(Tyr) bearing a hydrophobic protecting group, lysine (Lys) bearing
a hydrophobic protecting group, arginine (Arg) bearing a
hydrophobic protecting group, ornithine (Orn) bearing a hydrophobic
protecting group, aspartic acid (Asp) bearing a hydrophobic
protecting group, cysteine (Cys) bearing a hydrophobic protecting
group, and glutamic acid (Glu) bearing a hydrophobic protecting
group.
100. The peptide of claim 99, wherein X.sup.2, X.sup.3, and X.sup.4
are independently is selected from the group consisting of Phe,
Val, Trp, Tyr, and His.
101. The peptide of claim 98, wherein Xl bears a hydrophobic
protecting group.
102. The peptide of claim 101, wherein said hydrophobic protecting
group is selected from the group consisting of polyethylene glycol
(PEG), t-butoxycarbonyl (Boc), Fmoc, nicotinyl, OtBu, a benzoyl
group, an acetyl (Ac), a carbobenzoxy, methyl, ethyl, a propyl, a
butyl, a pentyl a hexyl ester, an N-methyl anthranilyl, and a 3 to
20 carbon alkyl, amide, a 3 to 20 carbon alkyl group,
9-fluoreneacetyl group, 1-fluorenecarboxylic group,
9-fluorenecarboxylic group, 9-fluorenone-1-carboxylic group,
benzyloxycarbonyl (is also called carbobenzoxy mentioned above),
Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl
(Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr),
Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl
(Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc),
4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO),
Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys),
1-(4,4-dimethyl-2,6-dioxocyclohexylid- ene)ethyl (Dde),
2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl
(2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), benzyloxymethyl (Bom),
cyclohexyloxy (cHxO),t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl
(tBu), trifluoroacetyl (TFA),
4[N-{1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyldibutyl)-amino}ben-
zyl ester (ODmab), .alpha.-allyl ester (OAll), 2-phenylisopropyl
ester (2-PhiPr), 1-[4,4-dimethyl-2,6-dioxycyclohex-1-yl-idene)ethyl
(Dde).
103. The peptide of claim 101, wherein said said hydrophobic
protecting group is selected from the group consisting of Boc,
Fmoc, nicotinyl, and OtBu.
104. The peptide of claim 101, wherein X.sup.5 bears a hydrophobic
protecting group.
105. The peptide of claim 104, wherein said hydrophobic protecting
group is selected from the group consisting of polyethylene glycol
(PEG), t-butoxycarbonyl (Boc), Fmoc, nicotinyl, OtBu, a benzoyl
group, an acetyl (Ac), a carbobenzoxy, methyl, ethyl, a propyl, a
butyl, a pentyl a hexyl ester, an N-methyl anthranilyl, and a 3 to
20 carbon alkyl, amide, a 3 to 20 carbon alkyl group,
9-fluoreneacetyl group, 1-fluorenecarboxylic group,
9-fluorenecarboxylic group, 9-fluorenone-1-carboxylic group,
benzyloxycarbonyl (is also called carbobenzoxy mentioned above),
Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl
(Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr),
Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl
(Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc),
4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO),
Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys),
1-(4,4-dimethyl-2,6-dioxocyclohexylid- ene)ethyl (Dde),
2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl
(2-CI-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), benzyloxymethyl (Bom),
cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO),
t-Butyl (tBu), trifluoroacetyl (TFA),
4[N-{1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyldibutyl)-amino}ben-
zyl ester (ODmab), .alpha.-allyl ester (OAll), 2-phenylisopropyl
ester (2-PhiPr), 1-[4,4-dimethyl-2,6-dioxycyclohex-1-yl-idene)ethyl
(Dde).
106. The peptide of claim 98, wherein the N-terminus of said
peptide is blocked with a protecting group selected from the group
consisting of Boc-, Fmoc-, and Nicotinyl-.
107. The peptide of claim 98, wherein the C-terminus of said
peptide is blocked with a protecting group selected from the group
consisting of tBu, and OtBu.
108. The peptide of claim 98, wherein said peptide comprises the
amino acid sequence of a peptide in Table 7.
109. The peptide of claim 98, wherein said peptide is a peptide
from Table 7.
110. The peptide of claim 98, wherein said peptide comprises at
least one D-amino acid.
111. The peptide of claim 98, wherein said peptide comprises all
D-amino acids.
112. The peptide of claim 98, wherein said peptide comprises
alternating D- and L-amino acids.
113. The peptide of claim 98, wherein said peptide comprises all
L-amino acids.
114. The peptide of claim 98, wherein said peptide is mixed with a
pharmacologically acceptable excipient.
115. The peptide of claim 98, wherein said peptide is coupled to a
biotin.
116. A peptide that ameliorates one or more symptoms of an
inflammatory condition, wherein said peptide: ranges in length from
5 to 11 amino acids; the terminal amino acids are hydrophobic amino
acids and/or bear hydrophobic protecting groups; the non-terminal
amino acids form at least one acidic domain and at least one basic
domain; and said peptide converts pro-inflammatory HDL to
anti-inflammatory HDL or makes anti-inflammatory HDL more
anti-inflammatory.
117. A peptide that ameliorates one or more symptoms of an
inflammatory condition, wherein said peptide: ranges in length from
5 to 11 amino acids; the terminal amino acids are hydrophobic amino
acids and/or bear hydrophobic protecting groups; the non-terminal
amino acids form at least one acidic domain or one basic domain and
at least one aliphatic domain; and said peptide converts
pro-inflammatory HDL to anti-inflammatory HDL or makes
anti-inflammatory HDL more anti-inflammatory.
118. A peptide that ameliorates one or more symptoms of an
inflammatory condition, wherein said peptide: ranges in length from
5 to 11 amino acids; the terminal amino acids are hydrophobic amino
acids and/or bear hydrophobic protecting groups; the non-terminal
amino acids form at least one acidic domain or one basic domain and
at least one aromatic domain; and said peptide converts
pro-inflammatory HDL to anti-inflammatory HDL or makes
anti-inflammatory HDL more anti-inflammatory.
119. A peptide that ameliorates one or more symptoms of an
inflammatory condition, wherein said peptide: ranges in length from
6 to 11 amino acids; the terminal amino acids are hydrophobic amino
acids and/or bear hydrophobic protecting groups; the non-terminal
amino acids form at least one aromatic domain or two or more
aromatic domains separated by one or more histidines; and said
peptide converts pro-inflammatory HDL to anti-inflammatory HDL or
makes anti-inflammatory HDL more anti-inflammatory.
120. A pair of amino acids that ameliorates one or more symptoms of
an inflammatory condition, wherein said pair of amino aicds
comprise: a first amino acid bearing at least one protecting group;
and a second amino acid bearing at least one protecting group;
wherein said first amino acid and said second amino acid are
different species of amino acid, and wherein said pair of amino
acids converts pro-inflammatory HDL to anti-inflammatory HDL or
makes anti-inflammatory HDL more anti-inflammatory
121. The pair of amino acids of claim 120, wherein said pair of
amino acids, when contacted with a phospholipid in an aqueous
environment, forms particles with a diameter of approximately 7.5
nm and forms stacked bilayers with a bilayer dimension on the order
of 3.4 to 4.1 nm with spacing between the bilayers in the stack of
approximately 2 nm.
122. The pair of amino acids of claim 120, wherein said first and
second amino acids are independently selected from the group
consisting of an acidic amino acid, a basic amino acid, and a
non-polar amino acid.
123. The pair of amino acids of claim 122, wherein said first amino
acid is acidic or basic and said second amino acid is non-polar, or
said first amino acid is non-polar and said second amino acid is
acidic or basic.
124. The pair of amino acids of claim 122, wherein both amino acids
are acidic.
125. The pair of amino acids of claim 122, wherein both amino acids
are basic.
126. The pair of amino acids of claim 120, wherein said pair of
amino acids are covalently coupled together directly or through a
linker.
127. The pair of amino acids of claim 126, wherein the amino acids
are joined through a peptide linkage thereby forming a
dipeptide.
128. The pair of amino acids of claim 120, wherein said pair of
amino acids are mixed together, but not covalently linked.
129. The pair of amino acids of claim 120, wherein said protecting
group is selected from the group consisting of polyethylene glycol
(PEG), t-butoxycarbonyl (Boc), Fmoc, nicotinyl, OtBu, a benzoyl
group, an acetyl (Ac), a carbobenzoxy, methyl, ethyl, a propyl, a
butyl, a pentyl a hexyl ester, an N-methyl anthranilyl, and a 3 to
20 carbon alkyl, amide, a 3 to 20 carbon alkyl group,
9-fluoreneacetyl group, 1-fluorenecarboxylic group,
9-fluorenecarboxylic group, 9-fluorenone-1-carboxylic group,
benzyloxycarbonyl (is also called carbobenzoxy mentioned above),
Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl
(Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr),
Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl
(Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc),
4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO),
Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys),
1-(4,4-dimethyl-2,6-dioxocyclohexylid- ene)ethyl (Dde),
2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl
(2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), benzyloxymethyl (Bom),
cyclohexyloxy (cHxO),t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl
(tBu), trifluoroacetyl (TFA),
4[N-{1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyldibutyl)-amino}ben-
zyl ester (ODmab), .alpha.-allyl ester (OAll), 2-phenylisopropyl
ester (2-PhiPr), 1-[4,4-dimethyl-2,6-dioxycyclohex-1-yl-idene)ethyl
(Dde).
130. The pair of amino acids of claim 120, wherein the first amino
acid is blocked with a protecting group selected from the group
consisting of Boc-, Fmoc-, and nicotinyl-, and the second amino
acid is blocked with a protecting group selected from the group
consisting of tBu, and OtBu.
131. The pair of amino acids of claim 128, wherein each amino acid
bears at least two protecting groups.
132. The pair of amino acids where each amino acid is blocked with
a with a first protecting group selected from the group consisting
of Boc-, Fmoc-, and nicotinyl-, and a second protecting group
selected from the group consisting of tBu, and OtBu.
133. The pair of amino acids where each amino acid is blocked with
a Boc and an OtBu.
134. The pair of amino acids of claim 120, wherein the pair of
amino acids form a dipeptide selected from the group consisting of
Phe-Arg, Glu-Leu, and Arg-Glu.
135. The pair of amino acids of claim 120, wherein the pair of
amino acids form a dipeptide selected from the group consisting of
Boc-Arg-OtBu, Boc-Glu-OtBu, Boc-Phe-Arg-OtBu, Boc-Glu-Leu-OtBu, and
Boc-Arg-Glu-OtBu.
136. A pharmaceutical formulation comprising: one or more peptides
according to claims 1, 5, 6, 31, 54, 76, 98, 116, 117, and 119, or
a pair of amino acids according to claim 120; and a
pharmaceutically acceptable excipient.
137. The pharmaceutical formulation of claim 136, wherein the
peptide is present in an effective dose.
138. The pharmaceutical formulation of claim 136, wherein the
peptide is in a time release formulation.
139. The pharmaceutical formulation of claim 136, wherein the
formulation is formulated as a unit dosage formulation.
140. The pharmaceutical formulation of claim 136, wherein the
formulation is formulated for oral administration.
141. The pharmaceutical formulation of claim 136, wherein the
formulation is formulated for administration by a route selected
from the group consisting of oral administration, inhalation,
rectal administration, intraperitoneal injection, intravascular
injection, subcutaneous injection, transcutaneous administration,
inhalation administration, and intramuscular injection.
142. A kit comprising: a container containing one or more of the
peptides according to claims 1, 5, 6, 31, 54, 76, 98, 116, 117, and
119, or a pair of amino acids according to claim 120; and
instructional materials teaching the use of the peptide(s) or pairs
of amino acids in the treatment of a pathology characterized by
inflammation.
143. The kit of claim 142, wherein said pathology is a pathology
selected from the group consisting of atherosclerosis, rheumatoid
arthritis, lupus erythematous, polyarteritis nodosa, osteoporosis,
Altzheimer's disease, chronic obstructive pulmonary disease,
asthma, multiple sclerosis, diabetes, and a viral illnesses.
144. A method of mitigating one or more symptoms of atherosclerosis
in a mammal, said method comprising administering to said mammal an
effective amount of the peptide of claims 1, 5, 6, 31, 54, 76, 98,
116, 117, and 119, or a pair of amino acids according to claim
120.
145. The method of claim 144, wherein said peptide is in a
pharmaceutically acceptable excipient.
146. The method of claim 144, wherein said peptide is administered
in conjunction with a lipid.
147. The method of claim 144, wherein said peptide is in a
pharmaceutically acceptable excipient suitable for oral
administration.
148. The method of claim 144, wherein said peptide is administered
as a unit dosage formulation.
149. The method of claim 144, wherein said administering comprises
administering said peptide by a route selected from the group
consisting of oral administration, inhalation, rectal
administration, intraperitoneal injection, intravascular injection,
subcutaneous injection, transcutaneous administration, and
intramuscular injection.
150. The method of claim 144, wherein said mammal is a mammal
diagnosed as having one or more symptoms of atherosclerosis.
151. The method of claim 144, wherein said mammal is a mammal
diagnosed as at risk for stroke or atherosclerosis.
152. The method of claim 144, wherein said mammal is a human.
153. The method of claim 144, wherein said mammal is non-human
mammal.
154. A method of mitigating one or more symptoms of an inflammatory
pathology, , said method comprising administering to said mammal an
effective amount of the peptide of claims 1, 5, 6, 31, 54, 76, 98,
116, 117, and 119, or a pair of amino acids according to claim
120.
155. The method of claim 154, wherein said inflammatory pathology
is a pathology selected from the group consisting of
atherosclerosis, rheumatoid arthritis, lupus erythematous,
polyarteritis nodosa, osteoporosis, Altzheimer's disease, multiple
sclerosis, chronic obstructive pulmonary disease, asthma, diabetes,
and a viral illnesses.
156. The method of claim 154, wherein said peptide is in a
pharmaceutically acceptable excipient.
157. The method of claim 154, wherein said peptide is administered
in conjunction with a lipid.
158. The method of claim 154, wherein said peptide is in a
pharmaceutically acceptable excipient suitable for oral
administration.
159. The method of claim 154, wherein said peptide is administered
as a unit dosage formulation.
160. The method of claim 154, wherein said administering comprises
administering said peptide by a route selected from the group
consisting of oral administration, inhalation, rectal
administration, intraperitoneal injection, intravascular injection,
subcutaneous injection, transcutaneous administration, and
intramuscular injection.
161. The method of claim 154, wherein said mammal is a mammal
diagnosed as at risk for stroke.
162. The method of claim 154, wherein said mammal is a human.
163. The method of claim 154, wherein said mammal is non-human
mammal.
164. A method of enhancing the activity of a statin in a mammal,
said method comprising coadministering with said statin an
effective amount of the peptide of claims 1, 5, 6, 31, 54, 76, 98,
116, 117, and 119, or a pair of amino acids according to claim
120;
165. The method of claim 164, wherein said statin is selected from
the group consisting of cerivastatin, atorvastatin, simvastatin,
pravastatin, fluvastatin, lovastatin. rosuvastatin, and
pitavastatin.
166. The method of claim 164, wherein said peptide is administered
simultaneously with said statin.
167. The method of claim 164, wherein said peptide is administered
before said statin.
168. The method of claim 164, wherein said peptide is administered
after said statin.
169. The method of claim 164, wherein said peptide and/or said
statin are administered as a unit dosage formulation.
170. The method of claim 164, wherein said administering comprises
administering said peptide and/or said statin by a route selected
from the group consisting of oral administration, inhalation,
rectal administration, intraperitoneal injection, intravascular
injection, subcutaneous injection, transcutaneous administration,
and intramuscular injection.
171. The method of claim 164, wherein said mammal is a mammal
diagnosed as having one or more symptoms of atherosclerosis.
172. The method of claim 164, wherein said mammal is a mammal
diagnosed as at risk for stroke or atherosclerosis.
173. The method of claim 164, wherein said mammal is a human.
174. The method of claim 164, wherein said mammal is non-human
mammal.
175. A method of mitigating one or more symptoms associated with
atherosclerosis in a mammal, said method comprising: administering
to said mammal an effective amount of a statin; and an effective
amount of a peptide of claims 1, 5, 6, 31, 54, 76, 98, 116, 117,
and 119, or a pair of amino acids according to claim 120; wherein
the effective amount of the statin is lower than the effective
amount of a statin administered without said peptide.
176. The method of claim 175, wherein the effective amount of the
peptide is lower than the effective amount of the peptide
administered without said statin.
177. The method of claim 175, wherein said statin is selected from
the group consisting of cerivastatin, atorvastatin, simvastatin,
pravastatin, fluvastatin, lovastatin. rosuvastatin, and
pitavastatin.
178. The method of claim 175, wherein said peptide is administered
simultaneously with said statin.
179. The method of claim 175, wherein said peptide is administered
before said statin.
180. The method of claim 175, wherein said peptide is administered
after said statin.
181. The method of claim 175, wherein said peptide and/or said
statin are administered as a unit dosage formulation.
182. The method of claim 175, wherein said administering comprises
orally administering said composition.
183. The method of claim 175, wherein said administering is by a
route selected from the group consisting of oral administration,
inhalation, rectal administration, intraperitoneal injection,
intravascular injection, subcutaneous injection, transcutaneous
administration, inhalation administration, and intramuscular
injection.
184. The method of claim 175, wherein said mammal is a mammal
diagnosed as having one or more symptoms of atherosclerosis.
185. The method of claim 175, wherein said mammal is a mammal
diagnosed as at risk for stroke or atherosclerosis.
186. The method of claim 175, wherein said mammal is a human.
187. The method of claim 175, wherein said mammal is non-human
mammal.
188. A pharmaceutical formulation, the formulation comprising: a
statin and/or Ezetimibe; and a peptide or a concatamer of a peptide
according to any of claims 1, 5, 6, 31, 54, 76, 98, 116, 117, and
119, or a pair of amino acids according to claim 120.
189. The pharmaceutical formulation of claim 188, wherein the
peptide and/or the statin are present in an effective dose.
190. The pharmaceutical formulation of claim 189, wherein the
effective amount of the statin is lower than the effective amount
of the statin administered without the peptide.
191. The pharmaceutical formulation of claim 189, wherein the
effective amount of the peptide is lower than the effective amount
of the peptide administered without the statin.
192. The pharmaceutical formulation of claim 189, wherein the
effective amount of the Ezetimibe is lower than the effective
amount of the Ezetimibe administered without the peptide.
193. The pharmaceutical formulation of claim 189, wherein the
effective amount of the peptide is lower than the effective amount
of the peptide administered without the Ezetimibe.
194. The pharmaceutical formulation of claim 188, wherein the
statin is selected from the group consisting of cerivastatin,
atorvastatin, simvastatin, pravastatin, fluvastatin, lovastatin.
rosuvastatin, and pitavastatin.
195. The pharmaceutical formulation of claim 188, wherein the
Ezetimibe, the statin, and/or the peptide are in a time release
formulation.
196. The pharmaceutical formulation of claim 188, wherein the
formulation is formulated as a unit dosage formulation.
197. The pharmaceutical formulation of claim 188, wherein the
formulation is formulated for oral administration.
198. The pharmaceutical formulation of claim 188, wherein the
formulation is formulated for administration by a route selected
from the group consisting of oral administration, inhalation,
rectal administration, intraperitoneal injection, intravascular
injection, subcutaneous injection, transcutaneous administration,
inhalation administration, and intramuscular injection.
199. The pharmaceutical formulation of claim 188, wherein the
formulation further comprises one or more phospholipids.
200. A method of reducing or inhibiting one or more symptoms of
osteoporosis in a mammal, the method comprising administering to
the mammal one or more peptide according to claims 1, 5, 6, 31, 54,
76, 98, 116, 117, and 119, or a pair of amino acids according to
claim 120, wherein the peptide or pair of amino acids is
administered in a concentration sufficient to reduce or eliminate
one or more symptoms of osteoporosis.
201. The method of claim 200, wherein the peptide is administered
in a concentration sufficient to reduce or eliminate
decalcification of a bone.
202. The method of claim 200, wherein the peptide is administered
in a concentration sufficient to induce recalcification of a
bone.
203. The method of claim 200, wherein the peptide is mixed with a
pharmacologically acceptable excipient.
204. The method of claim 200, wherein the peptide is mixed with a
pharmacologically acceptable excipient suitable for oral
administration to a mammal.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
10/649,378, filed on Aug. 26, 2003, which claims benefit of and
priority to U.S. Ser. No. 60/494,449, filed on Aug. 11, 2003, all
of which are incorporated herein by reference in their entirety for
all purposes.
FIELD OF THE INVENTION
[0003] This invention relates to the field of atherosclerosis. In
particular, this invention pertains to the identification of a
class of peptides that are orally administrable and that ameliorate
one or more symptoms of atherosclerosis.
BACKGROUND OF THE INVENTION
[0004] Cardiovascular disease is a leading cause of morbidity and
mortality, particularly in the United States and in Western
European countries. Several causative factors are implicated in the
development of cardiovascular disease including hereditary
predisposition to the disease, gender, lifestyle factors such as
smoking and diet, age, hypertension, and hyperlipidemia, including
hypercholesterolemia. Several of these factors, particularly
hyperlipidemia and hypercholesteremia (high blood cholesterol
concentrations) provide a significant risk factor associated with
atherosclerosis.
[0005] Cholesterol is present in the blood as free and esterified
cholesterol within lipoprotein particles, commonly known as
chylomicrons, very low density lipoproteins (VLDLs), low density
lipoproteins (LDLs), and high density lipoproteins (HDLs).
Concentration of total cholesterol in the blood is influenced by
(1) absorption of cholesterol from the digestive tract, (2)
synthesis of cholesterol from dietary constituents such as
carbohydrates, proteins, fats and ethanol, and (3) removal of
cholesterol from blood by tissues, especially the liver, and
subsequent conversion of the cholesterol to bile acids, steroid
hormones, and biliary cholesterol.
[0006] Maintenance of blood cholesterol concentrations is
influenced by both genetic and environmental factors. Genetic
factors include concentration of rate-limiting enzymes in
cholesterol biosynthesis, concentration of receptors for low
density lipoproteins in the liver, concentration of rate-limiting
enzymes for conversion of cholesterols bile acids, rates of
synthesis and secretion of lipoproteins and gender of person.
Environmental factors influencing the hemostasis of blood
cholesterol concentration in humans include dietary composition,
incidence of smoking, physical activity, and use of a variety of
pharmaceutical agents. Dietary variables include amount and type of
fat (saturated and polyunsaturated fatty acids), amount of
cholesterol, amount and type of fiber, and perhaps amounts of
vitamins such as vitamin C and D and minerals such as calcium.
[0007] Epidemiological studies show an inverse correlation of high
density lipoprotein (HDL) and apolipoprotein (apo) A-I levels with
the occurrence of atherosclerotic events (Wilson et al. (1988)
Arteriosclerosis 8: 737-741). Injection of HDL into rabbits fed an
atherogenic diet has been shown to inhibit atherosclerotic lesion
formation (Badimon et al. (1990) J. Clin. Invest. 85:
1234-1241).
[0008] Human apo A-I has been a subject of intense study because of
its anti-atherogenic properties. Exchangeable apolipoproteins,
including apo A-I, possess lipid-associating domains (Brouillette
and Anantharamaiah (1995) Biochim. Biophys. Acta 1256:103-129;
Segrest et al. (1974) FEBS Lett. 38: :247-253). 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) FEBS Lett. 38: :247-253).
Characteristics of the class A amphipathic helix include the
presence of positively charged residues at the polar-nonpolar
interface and negatively charged residues at the center of the
polar face (Segrest et al. (1974) FEBS Lett. 38: 247-253; Segrest
et al. (1990) Proteins: Structure, Function, and Genetics 8:
103-117). Apo A-I has been shown to strongly associate 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.
SUMMARY OF THE INVENTION
[0009] This invention provides novel peptides and amino acid pairs,
administration of which mitigates one or more symptoms of
atherosclerosis and other inflammatory conditions such as
rheumatoid arthritis, lupus erythematous, polyarteritis nodosa,
osteoporosis, Alzheimer's disease, congestive heart failure,
endothelial dysfunction, viral illnesses such as influenza A, and
diseases such as multiple sclerosis. In certain embodiments, it was
a discovery of this invention that 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 readily taken up and delivered to
the serum, and are effective to mitigate one or more symptoms of
atherosclerosis. In certain embodiments, the peptides can be
formulated with all "L" amino acid residues and are still
effective, particular when administered by routes other than oral
administration.
[0010] It was also a discovery that "small" peptides (e.g., ranging
in length from about three amino acides to about 11 amino acids)
having hydrophobic terminal amino acids or terminal amino acids
rendered hydrophobic by one or more hydrophobic blocking goups and
having internal acidic and/or basic, and/or aliphatic, and/or
aromatic amino acids as described herin are also capable of
mitigating one or more symptoms of atherosclerosis or other
pathologies characterized by an inflammatory response.
[0011] The peptides, and/or amino acid pairs, of this invention are
typically effective to stimulate the formation and cycling of
pre-beta high density lipoprotein-like particles and/or to promote
lipid transport and detoxification.
[0012] The peptides, and/or amino acid pairs, described herein are
also effective for preventing the onset or inhibiting or
eliminating one or more symptoms of osteoporosis.
[0013] It was also a surprising discovery that the peptides, and/or
amino acid pairs, can be used to enhance (e.g., synergically
enhance) the activity of statins and/or Ezetimibe or other
cholesterol uptake inhibitors, thereby permitting the effective use
of statins or cholesterol uptake inhibitors at lower dosages and/or
cause the statins or cholesterol uptake inhibitors to be
significantly more anti-inflammatory at any given dose.
[0014] In certain embodiments, this invention provides peptides or
a combination of peptides, and/or amino acid pairs, that
ameliorates one or more symptoms of an inflammatory condition
(e.g., atherosclerosis atherosclerosis, rheumatoid arthritis, lupus
erythematous, polyarteritis nodosa, osteoporosis, Altzheimer's
disease, a viral illnesses, asthma, diabetes, etc.). Certain
preferred peptides range in length from 3 to about 5 amino acids;
are soluble in ethyl acetate at a concentration greater than about
4 mg/mL; are soluble in aqueous buffer at pH 7.0; when contacted
with a phospholipid in an aqueous environment, forms particles with
a diameter of approximately 7.5 nm and/or form stacked bilayers
with a bilayer dimension on the order of 3.4 to 4.1 nm with spacing
between the bilayers in the stack of approximately 2 nm; have a
molecular weight less than about 900 daltons; convert
pro-inflammatory HDL to anti-inflammatory HDL or makes
anti-inflammatory HDL more anti-inflammatory; and do not have the
amino acid sequence Lys-Arg-Asp-Ser (SEQ ID NO:238) in which
Lys-Arg-Asp and Ser are all L amino acids. In certain embodiments,
these peptides protects a phospholipid (e.g.,
1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcho- line (PAPC),
1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (SAPC)),
and 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphorylethanolamine
(SAPE). In certain embodiments, these peptides can include, but
need not be limited to any of the small peptides described
herein.
[0015] In certain embodiments, this invention provides peptides or
a combination of peptides, and/or amino acid pairs, that
ameliorates one or more symptoms of an inflammatory condition
(e.g., atherosclerosis atherosclerosis, rheumatoid arthritis, lupus
erythematous, polyarteritis nodosa, osteoporosis, Altzheimer's
disease, a viral illnesses, asthma, diabetes, etc.). Certain
preferred peptides are characterized by the formula:
X.sup.1--X.sup.2--X.sup.3.sub.n--.sup.4 where n is 0 or 1; X.sup.1
is a hydrophobic amino acid and/or bears a hydrophobic protecting
group; X.sup.4 is a hydrophobic amino acid and/or bears a
hydrophobic protecting group; and, when n is 0, X.sup.2 is an amino
acid selected from the group consisting of an acidic amino acid, a
basic amino acid, and a histidine; and, when when n is 1: X.sup.2
and X.sup.3 are independently an acidic amino acid, a basic amino
acid, an aliphatic amino acid, or an aromatic amino acid such that
when X.sup.2 is an acidic amino acid; X.sup.3 is a basic amino
acid, an aliphatic amino acid, or an aromatic amino acid; when
X.sup.2 is a basic amino acid; X.sup.3 is an acidic amino acid, an
aliphatic amino acid, or an aromatic amino acid; and when X.sup.2
is an aliphatic or aromatic amino acid, X.sup.3 is an acidic amino
acid, or a basic amino acid. Certain preferred peptides convert
pro-inflammatory HDL to anti-inflammatory HDL or make
anti-inflammatory HDL more anti-inflammatory. In certain
embodiments, the peptide does not have the amino acid sequence
Lys-Arg-Asp-Ser (SEQ ID NO:238) in which Lys, Arg, Asp, and Ser are
all L amino acids. Peptides of this invention include peptides
according to the formula above, and/or peptides comprising a
peptide of the formula above and/or concatamers of such
peptides.
[0016] In certain embodiments, X.sup.1 and X.sup.4 are
independently selected from the group consisting of alanine (Ala),
valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro),
phenylalanine (Phe), tryptophan (Trp), methionine (Met), serine
(Ser) bearing a hydrophobic protecting group, beta-naphthyl
alanine, alpha-naphthyl alanine, norleucine, cyclohexylalanine,
threonine (Thr) bearing a hydrophobic protecting group, tyrosine
(Tyr) bearing a hydrophobic protecting group, lysine (Lys) bearing
a hydrophobic protecting group, arginine (Arg) bearing a
hydrophobic protecting group, ornithine (Orn) bearing a hydrophobic
protecting group, aspartic acid (Asp) bearing a hydrophobic
protecting group, cysteine (Cys) bearing a hydrophobic protecting
group, and glutamic acid (Glu) bearing a hydrophobic protecting
group.
[0017] In certain embodiments, the peptide is a tri-mer (i.e., n is
0). In certain trimers, X.sup.1 is Glu, Leu, Lys, Orn, Phe, Trp, or
norLeu; X.sup.2 is acidic (e.g., aspartic acid, glutamic acid,
etc.), or basic (e.g., lysine, arginine, histidine, etc.) and
X.sup.4 is Ser, Thr, Ile, Leu, Trp, Tyr, Phe, or norleu. In certain
embodiments, the peptide comprises the amino acid sequence of a
peptide listed in Table 3. In certain embodiments, the peptide is a
protected trimer as shown in Table 3.
[0018] In certain embodiments, n is 1 and the peptide is or
comprises a tetramer in which X.sup.2 and X.sup.3 are independently
an acidic amino acid or a basic amino acid such that when X.sup.2
is an acidic amino acid, X.sup.3 is a basic amino acid and when
X.sup.2 is a basic amino acid, X.sup.3 is an acidic amino acid.
X.sup.1 and X.sup.4 can include independently selected amino acids,
e.g., as indicated above. In certain embodiments, X.sup.2 and
X.sup.3 are independently selected from Asp, Glu, Lys, Arg, and
His. In certain embodiments, the peptide comprises the amino acid
sequence of a peptide listed in Table 4. In certain embodiments,
the peptide is a protected tetramer as show in Table 4.
[0019] In still another embodiment, n is 1 and the peptide is or
comprises a tetramer in which X.sup.2 and X.sup.3 are independently
an acidic, a basic, or a aliphatic amino acid with one of X.sup.2
or X.sup.3 being an acidic or a basic amino acid such that when
X.sup.2 is an acidic or a basic amino acid, X.sup.3 is an aliphatic
amino acid; and when X.sup.3 is an acid or a basic amino acid,
X.sup.2 is an aliphatic amino acid. X.sup.1 and X.sup.4 can include
independently selected amino acids, e.g., as indicated above. In
certain embodiments, X.sup.2 and X.sup.3 are independently selected
from the group consisting of Asp, Glu, Lys, Arg, His, and Ile, more
preferably from the group consisting of Asp, Arg, Leu, and Glu. In
certain embodiments, the peptide comprises the amino acid sequence
of a peptide listed in Table 5. In certain embodiments, the peptide
is a protected tetramer as show in Table 5.
[0020] In another embodiment, n is 1 and the peptide is or
comprises a tetramer in which X.sup.2, X.sup.3 are independently an
acidic, a basic, or an aromatic amino acid with one of X.sup.2 or
X.sup.3 being an acidic or a basic amino acid such that when
X.sup.2 is an acidic or a basic amino acid, X.sup.3 is an aromatic
amino acid; and when X.sup.3 is an acid or a basic amino acid,
X.sup.2 is an aromatic amino acid. X.sup.1 and X.sup.4 can include
independently selected amino acids, e.g., as indicated above. In
certain embodiments, X.sup.2 and X.sup.3 are independently selected
from the group consisting of Asp, Arg, Glu, Trp, Tyr, Phe, and Lys.
In certain embodiments, the peptide comprises the amino acid
sequence of a peptide listed in Table 6. In certain embodiments,
the peptide is a protected tetramer as show in Table 6.
[0021] This invention also provides for peptides that are or
comprise a pentamer (5-mer) characterized by the formula:
X.sup.1--X.sup.2--X.sup.3-- -X.sup.4--X.sup.5, where X.sup.1 is a
hydrophobic amino acid and/or bears a hydrophobic protecting group;
X.sup.5 is a hydrophobic amino acid and/or bears a hydrophobic
protecting group; and X.sup.2, X.sup.3, and X.sup.4 are
independently selected aromatic amino acids or histidine; and the
peptide converts pro-inflammatory HDL to anti-inflammatory HDL or
makes anti-inflammatory HDL more anti-inflammatory. In certain
embodiments, X.sup.1 and X.sup.5 are independently selected from
the group consisting of alanine (Ala), valine (Val), leucine (Leu),
isoleucine (Ile), proline (Pro), phenylalanine (Phe), tryptophan
(Trp), methionine (Met), phenylalanine (Phe), tryptophan (Trp),
methionine (Met), serine (Ser) bearing a hydrophobic protecting
group, beta-naphthyl alanine, alpha-naphthyl alanine, norleucine,
cyclohexylalanine, threonine (Thr) bearing a hydrophobic protecting
group, tyrosine (Tyr) bearing a hydrophobic protecting group,
lysine (Lys) bearing a hydrophobic protecting group, arginine (Arg)
bearing a hydrophobic protecting group, ornithine (Orn) bearing a
hydrophobic protecting group, aspartic acid (Asp) bearing a
hydrophobic protecting group, cysteine (Cys) bearing a hydrophobic
protecting group, and glutamic acid (Glu) bearing a hydrophobic
protecting group. In certain embodiments X.sup.2, X.sup.3, and
X.sup.4 are independently is selected from the group consisting of
Phe, Val, Trp, Tyr, and His. In certain embodiments, the peptide
comprises the amino acid sequence of a peptide listed in Table 7.
In certain embodiments, the peptide is a protected tetramer as show
in Table 7.
[0022] This invention also provides for larger peptides that
ameliorate one or more symptoms of an inflammatory condition. In
certain embodiments, the peptide ranges in length from 5 to 11
amino acids; the terminal amino acids are hydrophobic amino acids
and/or bear hydrophobic protecting groups; the non-terminal amino
acids form at least one acidic domain and at least one basic
domain; and the peptide converts pro-inflammatory HDL to
anti-inflammatory HDL or makes anti-inflammatory HDL more
anti-inflammatory.
[0023] In certain embodiments, the peptide ranges in length from 5
to 11 amino acids; the terminal amino acids are hydrophobic amino
acids and/or bear hydrophobic protecting groups; the non-terminal
amino acids form at least one acidic domain or one basic domain and
at least one aliphatic domain; and the peptide converts
pro-inflammatory HDL to anti-inflammatory HDL or makes
anti-inflammatory HDL more anti-inflammatory.
[0024] In other embodiments, the peptide ranges in length from 5 to
11 amino acids; the terminal amino acids are hydrophobic amino
acids and/or bear hydrophobic protecting groups; the non-terminal
amino acids form at least one acidic domain or one basic domain and
at least one aromatic domain; and the peptide converts
pro-inflammatory HDL to anti-inflammatory HDL or makes
anti-inflammatory HDL more anti-inflammatory.
[0025] In still other embodiments, the peptide ranges in length
from 6 to 11 amino acids; the terminal amino acids are hydrophobic
amino acids and/or bear hydrophobic protecting groups; the
non-terminal amino acids form at least one aromatic domain or two
or more aromatic domains separated by one or more histidines; and
the peptide converts pro-inflammatory HDL to anti-inflammatory HDL
or makes anti-inflammatory HDL more anti-inflammatory.
[0026] This invention also provides for peptides that ameliorate
one or more symptoms of an inflammatory condition and that comprise
one or more amphipathic helices. Thus, this invention includes a
peptide or a concatamer of a peptide that ranges in length from
about 10 to about 30 amino acids, preferably from about 18 to about
30 amino acids; that comprises at least one class A amphipathic
helix; that comprises one or more aliphatic or aromatic amino acids
at the center of the non-polar face of said amphipathic helix; that
protects a phospholipid against oxidation by an oxidizing agent;
and that is not the D-18A peptide. In certain embodiments, the
peptide comprises the amino acid sequence of a peptide listed in
Table 2 or Table 12. In certain embodiments, the peptide is a
protected tetramer as show in Table 2 or Table 12.
[0027] In certain embodiments, the peptides of this invention
protect a phospholipid (e.g.,
1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcho- line (PAPC),
1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (SAPC)),
1-stearoyl-2-arachidonyl-sn-glycero-3-phosphorylethanolamine
(SAPE)) against oxidation by an oxidizing agent (e.g.,
13(S)--HPODE, 15(S)--HPETE, HPODE, HPETE, HODE, HETE, etc.).
[0028] Any of the peptides described herein can bear one or more
hydrophobic protecting groups on the amino terminal amino acid
(e.g., X.sup.1) and/or the carboxyl terminal amino acid (e.g.,
X.sup.4, X.sup.5, etc.). The protecting group(s) can be attached to
the amino or carboxyl terminus and/or to a side chain (R group) of
the amino acid. The protecting group(s) can be directly coupled
(e.g., through a covalent bond) or indirectly coupled (e.g.,
through a linker). Preferred hydrophobic protecting groups include,
but are not limited to t-butoxycarbonyl (Boc), Fmoc, nicotinyl,
OtBu, a benzoyl group, an acetyl (Ac), a carbobenzoxy, methyl,
ethyl, a propyl, a butyl, a pentyl a hexyl ester, an N-methyl
anthranilyl, and a 3 to 20 carbon alkyl, amide, a 3 to 20 carbon
alkyl group, 9-fluoreneacetyl group, 1-fluorenecarboxylic group,
9-fluorenecarboxylic group, 9-fluorenone-1-carboxylic group,
benzyloxycarbonyl (is also called carbobenzoxy mentioned above),
Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl
(Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr),
Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl
(Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc),
4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO),
Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys),
1-(4,4-dimethyl-2,6-dioxocyclohexylid- ene)ethyl (Dde),
2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl
(2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), benzyloxymethyl (Bom),
cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO),
t-Butyl (tBu), trifluoroacetyl (TFA),
4[N-{1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyldibutyl)-amino}ben-
zyl ester (ODmab), .alpha.-allyl ester (OAll), 2-phenylisopropyl
ester (2-PhiPr), 1-[4,4-dimethyl-2,6-dioxycyclohex-1-yl-idene)ethyl
(Dde), and the like. In certain embodiments, the said hydrophobic
protecting group is selected from the group consisting of Boc,
Fmoc, nicotinyl, and OtBu. In certain embodiments, the N-terminus
of the peptide is blocked with a protecting group selected from the
group consisting of Boc-, Fmoc-, and Nicotinyl- and/or the
C-terminus of the peptide is blocked with a protecting group
selected from the group consisting of tBu, and OtBu.
[0029] The peptides can also, optionally, include at least one D
amino acid. In certain embodiments, the peptides include a
plurality of D- amino acids or can even compirse all D-amino acids.
In certain embodiments, the peptide comprise alternating D- and
L-amino aicds. The peptides can also be all L-form amino acids. The
peptides can be isolated (e.g., substanitaly pure), dry or in
solution, and/or combined with a pharmacologically acceptable
excipient. In certain embodiments, the peptide is mixed with a
pharmacologically acceptable excipient suitable for oral
administration to a mammal (e.g., a human or a non-human mammal).
The peptide can be provided as a unit formulation in a
pharmaceutically acceptable excipient and/or as a time release
formulation.
[0030] The peptides can also be coupled to one or more biotins
(e.g., directly, through a linker, and/or through the amino acid
side chain). In certain embodiments, the biotin is coupled to a
lysine (Lys).
[0031] In certain embodiments, this invention also provides pairs
of amino acids that ameliorate one or more symptoms of an
inflammatory condition. The amino acid pair typically comprises a
first amino acid bearing at least one protecting group; and a
second amino acid bearing at least one protecting group; where the
first amino acid and the second amino acid are different species of
amino acid, and where the pair of amino acids converts
pro-inflammatory HDL to anti-inflammatory HDL or makes
anti-inflammatory HDL more anti-inflammatory. In various
embodiments the pair of amino acids, when contacted with a
phospholipid in an aqueous environment, forms particles with a
diameter of approximately 7.5 nm and forms stacked bilayers with a
bilayer dimension on the order of 3.4 to 4.1 nm with spacing
between the bilayers in the stack of approximately 2 nm. In certain
embodiments, the first and second amino acids are independently
selected from the group consisting of an acidic amino acid, a basic
amino acid, and a non-polar amino acid. In certain embodiments, the
first amino aicd is acidic or basic and the second amino acid is
non-polar, or the first amino acid is non-polar and said second
amino acid is acidic or basic. In certain embodiments, both amino
acids are acidic or basic. The first and second amino acid can,
optionaly, be covalently coupled together, e.g., directly or
through a linker. In certain embodiments, the amino acids are
joined through a peptide linkage thereby forming a dipeptide. In
certain embodiments, the first amino acid and the second amino acid
are mixed together, but not covalently linked. The protecting
groups include, but are not limited to any of the protecting groups
described herein. In certain embodiments, the first amino acid is
blocked with a protecting group selected from the group consisting
of Boc-, Fmoc-, and nicotinyl-, and the second amino acid is
blocked with a protecting group selected from the group consisting
of tBu, and OtBu. In certain embodiments, each amino acid bears at
least two protecting groups. In certain embodiments, each amino
acid is blocked with a with a first protecting group selected from
the group consisting of Boc-, Fmoc-, and nicotinyl-, and a second
protecting group selected from the group consisting of tBu, and
OtBu. In certain embodiments, each amino acid is blocked with a Boc
and an OtBu. In various embodiments the pair of amino acids form a
dipeptide selected from the group consisting of Phe-Arg, Glu-Leu,
and Arg-Glu. In certain embodiments, the pair of amino acids form a
dipeptide selected from the group consisting of Boc-Arg-OtBu,
Boc-Glu-OtBu, Boc-Phe-Arg-OtBu, Boc-Glu-Leu-OtBu, and
Boc-Arg-Glu-OtBu.
[0032] This invention also provides a pharmaceutical formulation
comprising one or more of the peptides, and/or amino acid pairs
described herein, and a pharmaceutically acceptable excipient.
Typically the peptide(s), and/or amino acid pairs, are present in
an effective dose. The peptide(s), and/or amino acid pairs, can
also be provided as a time release formulation and/or as a unit
dosage formulation. In certain embodiments, the formulation is
formulated for oral administration. In certain embodiments, the
formulation is formulated for administration by a route selected
from the group consisting of oral administration, inhalation (e.g.,
nasal administration, oral inhalation, etc.), rectal
administration, intraperitoneal injection, intravascular injection,
subcutaneous injection, transcutaneous administration, inhalation
administration, intramuscular injection, and the like.
[0033] Also provided is a kit comprising a container containing one
or more of the peptides, and/or amino acid pairs described herein,
and instructional materials teaching the use of the peptide(s),
and/or amino acid pairs, in the treatment of a pathology
characterized by inflammation (e.g., atherosclerosis
atherosclerosis, rheumatoid arthritis, lupus erythematous,
polyarteritis nodosa, asthma, osteoporosis, Altzheimer's disease, a
viral illnesses, etc.).
[0034] This invention also provides a method of mitigating (e.g.,
reducing or eliminating) one or more symptoms of atherosclerosis in
a mammal (human or non-human mammal). The method typically involves
administering to the mammal an effective amount of one or more of
the peptides, and/or amino acid pairs described herein. The
peptide, and/or amino acid pair, can be administered in a in a
pharmaceutically acceptable excipient (e.g., for oral
administration) and can, optionally be administered in conjunction
(e.g., before, after, or simultaneously) with a lipid. The
administering can comprise administering the peptide, and/or amino
acid pair, by a route selected from the group consisting of oral
administration, inhalation (e.g. nasal administration, oral
inhalation, etc.), rectal administration, intraperitoneal
injection, intravascular injection, subcutaneous injection,
transcutaneous administration, and intramuscular injection. In
certain embodiments, the mammal is a mammal diagnosed as having one
or more symptoms of atherosclerosis. In certain embodiments, the
mammal is a mammal diagnosed as at risk for stroke or
atherosclerosis.
[0035] In another embodiment, this invention provides method of
mitigating one or more symptoms of an inflammatory pathology (e.g.,
atherosclerosis, rheumatoid arthritis, lupus erythematous,
polyarteritis nodosa, osteoporosis, multiple sclerosis, diabetes,
asthma, Altzheimer's disease, a viral illnesses, etc.). The method
typically involves administering to the mammal an effective amount
of one or more of the peptides, and/or amino acid pairs, described
herein. The peptide, and/or amino acid pair, can be administered in
a in a pharmaceutically acceptable excipient (e.g., for oral
administration) and can, optionally be administered in conjunction
(e.g., before, after, or simultaneously) with a lipid. The
administering can comprise administering the peptide, and/or amino
acid pairs, by a route selected from the group consisting of oral
administration, inhalation, rectal administration, intraperitoneal
injection, intravascular injection, subcutaneous injection,
transcutaneous administration, and intramuscular injection. In
certain embodiments, the mammal is a mammal diagnosed as having one
or more symptoms of of the inflammatory pathology. In certain
embodiments, the mammal is a mammal diagnosed as at risk for the
inflammatory pathology.
[0036] The peptides, and/or amino acid pairs, of this invention
also act synergistically with statins and/or with a selective
cholesterol uptake inhibitor (e.g., Ezetimibe). The method
typically involves coadministering with the statin and/or
cholesterol uptake inhibitor an effective amount of one or more of
the peptides described herein. In certain embodiments, the statin
is selected from the group consisting of cerivastatin,
atorvastatin, simvastatin, pravastatin, fluvastatin, lovastatin.
rosuvastatin, and pitavastatin. The peptide can be administered
before, after, or simultaneously with the statin and/or the
cholesterol uptake inhibitor. The peptide and/or said statin and/or
cholesterol uptake inhibitor can be administered as a unit dosage
formulation. In certain embodiments, the administering comprises
administering said peptide and/or said statin by a route selected
from the group consisting of oral administration, nasal
administration, rectal administration, intraperitoneal injection,
intravascular injection, subcutaneous injection, transcutaneous
administration, and intramuscular injection. The mammal includes,
but is not limited to a mammal diagnosed as having one or more
symptoms of atherosclerosis or diagnosed as at risk for stroke or
atherosclerosis.
[0037] This invention also provides a method of mitigating one or
more symptoms associated with atherosclerosis in a mammal. The
method typically involves administering a statin and/or a selective
cholesterol uptake inhibitor; and an effective amount of one or
more peptides, and/or amino acid pairs, described herein, where the
the effective amount of the statin and/or cholesterol uptake
inhibitor is lower than the effective amount of a statin or a
cholesterol uptake inhibitor administered without the peptide(s),
and/or amino acid pairs. In certain embodiments, the effective
amount of the peptide(s), and/or amino acid pairs, is lower than
the effective amount of the peptide, and/or amino acid pairs,
administered without the statin and/or cholesterol uptake
inhibitor. In certain embodiments, the statin is selected from the
group consisting of cerivastatin, atorvastatin, simvastatin,
pravastatin, fluvastatin, lovastatin. rosuvastatin, and
pitavastatin. The peptide can be administered before, after, or
simultaneously with the statin and/or the cholesterol uptake
inhibitor. The peptide, and/or amino acid pair, and/or the statin
and/or cholesterol uptake inhibitor can be administered as a unit
dosage formulation. In certain embodiments, the administering
comprises administering the peptide, and/or amino acid pair, and/or
said statin by a route selected from the group consisting of oral
administration, inhalation, rectal administration, intraperitoneal
injection, intravascular injection, subcutaneous injection,
transcutaneous administration, and intramuscular injection. The
mammal includes, but is not limited to a mammal diagnosed as having
one or more symptoms of atherosclerosis or diagnosed as at risk for
stroke or atherosclerosis. The mammal includes, but is not limited
to a mammal diagnosed as having one or more symptoms of
atherosclerosis or diagnosed as at risk for stroke or
atherosclerosis.
[0038] In still another embodiment, this invention provides a
method of reducing or inhibiting one or more symptoms of
osteoporosis in a mammal. The method typically involves
administering to the mammal one or more peptide(s), and/or amino
acid pairs, described herein, where peptide, and/or amino acid
pair, is administered in a concentration sufficient to reduce or
eliminate one or more symptoms of osteoporosis. In certain
embodiments, the peptide(s), and/or amino acid pair(s), are
administered in a concentration sufficient to reduce or eliminate
decalcification of a bone. In certain embodiments, the peptide(s),
and/or amino acid pair(s), are administered in a concentration
sufficient to induce recalcification of a bone. The peptide(s),
and/or amino acid pairs, can be combined with a pharmacologically
acceptable excipient (e.g., an excipient suitable for oral
administration to a mammal).
[0039] In certain embodiments, the methods and/or peptides of this
invention exclude any one or more peptides disclosed in WO
97/36927, and/or U.S. Pat. Nos. 6,037,323, and/or 6,376,464, and/or
6753,313, and/or in Garber et al. (1992) Arteriosclerosis and
Thrombosis, 12: 886-894. In certain embodiments this invention
excludes any one or more peptides disclosed in U.S. Pat. No.
4,643,988 and/or in Garber et al (1992) that were synthesized with
all enantiomeric amino acids being L amino acids or synthesized
with D amino acids where the peptides are blocking groups. In
certain embodiments, this invention excludes peptides having the
formula A.sub.1-B.sub.1--B.sub.2--C.sub.1-D-B.sub.3--B.sub.4-A-
.sub.2-C.sub.2--B.sub.5--B.sub.6-A.sub.3-C.sub.3--B.sub.7--C.sub.4-A.sub.4-
-B.sub.8--B.sub.9 (SEQ ID NO:(SEQ ID NO:1) wherein A.sub.1,
A.sub.2, A.sub.3 and A.sub.4 are independently aspartic acid or
glutamic acid, or homologues or analogues thereof; B.sub.1,
B.sub.2, B.sub.3, B.sub.4, B.sub.5, B.sub.6, B.sub.7, B.sub.8 and
B.sub.9 are independently tryptophan, phenylalanine, alanine,
leucine, tyrosine, isoleucine, valine or .alpha.-naphthylalanine,
or homologues or analogues thereof; C.sub.1, C.sub.2, C.sub.3 and
C.sub.4 are independently lysine or arginine, and D is serine,
threonine, alanine, glycine, histidine, or homologues or analogues
thereof; provided that, when A.sub.1 and A.sub.2 are aspartic acid,
A.sub.3 and A.sub.4 are glutamic acid, B.sub.2 and B.sub.9 are
leucine, B.sub.3 and B.sub.7 are phenylalanine, B.sub.4 is
tyrosine, B.sub.5 is valine, B.sub.6, B.sub.8, and D are alanine,
and C.sub.1, C.sub.2, C.sub.3 and C.sub.4 are lysine, B.sub.1 is
not tryptophan. In certain embodiments, while this invention may
exclude one or more of the peptides described above, the peptide of
SEQ ID NO:8 (4F or D4F) will be expressly included.
[0040] In certain embodiments, this invention excludes any one or
more peptides in WO 97/36927 and/or D variants thereof. Particular
embodiments exclude one or more of the following: apoprotein A,
apoprotein A-1, apoprotein A-2, apoprotein A4, apoprotein B,
apoprotein B-48, apoprotein B-100, apoprotein C, apoprotein C-1,
apoprotein C-2, apoprotein C-3, apoprotein D, apoprotein E as
described in WO 97/36927.
[0041] In certain embodiments, also excluded are any one or more
peptides disclosed in U.S. Pat. No. 6,037,323 and/or D variants
thereof. Particular embodiments exclude apo A-I agonist compounds
comprising (i) an 18 to 22-residue peptide or peptide analogue that
forms an amphipathic .alpha.-helix in the presence of lipids and
that comprises the formula:
Z.sub.1--X.sub.1--X.sub.2--X.sub.3--X.sub.4--X.sub.5--X.sub.6--X.sub.7--X-
.sub.8--X.sub.9--X.sub.10--X.sub.11--X.sub.12--X.sub.13--X.sub.14--X.sub.1-
5--X.sub.16--X.sub.17--X.sub.18--Z.sub.2, (SEQ ID NO:2), where
X.sub.1 is Pro (P), Ala (A), Gly (G), Asn (N), Gln (Q) or D-Pro
(p); X.sub.2 is an aliphatic amino acid; X.sub.3 is Leu (L);
X.sub.4 is an acidic amino acid; X.sub.5 is Leu (L) or Phe (F);
X.sub.6 is Leu (L) or Phe (F); X.sub.7 is a basic amino acid;
X.sub.8 is an acidic amino acid; X.sub.9 is Leu (L) or Trp (W);
X.sub.10 is Leu (L) or Trp (W); X.sub.11 is an acidic amino acid or
Asn (N); X.sub.12 is an acidic amino acid; X.sub.13 is Leu (L), Trp
(W) or Phe (F); X.sub.14 is a basic amino acid or Leu (L); X.sub.15
is Gln (Q) or Asn (N); X.sub.16 is a basic amino acid; X.sub.17 is
Leu (L); X.sub.18 is a basic amino acid; Z.sub.1 is H.sub.2 N-- or
RC(O)NH--; Z.sub.2 is --C(O)NRR, --C(O)OR or --C(O)OH or a salt
thereof; each R is independently --H, (C.sub.1-C.sub.6) alkyl,
(C.sub.1-C.sub.6) alkenyl, (C.sub.1-C.sub.6) alkynyl,
(C.sub.5-C.sub.20) aryl, (C.sub.6-C.sub.26) alkaryl, 5-20 membered
heteroaryl or 6-26 membered alkheteroaryl or a 1 to 4-residue
peptide or peptide analogue in which one or more bonds between
residues 1-7 are independently a substituted amide, an isostere of
an amide or an amide mimetic; and each "-" between residues X.sub.1
through X.sub.18 independently designates an amide linkage, a
substituted amide linkage, an isostere of an amide or an amide
mimetic; or (ii) an altered form of formula (I) in which at least
one of residues X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5,
X.sub.6, X.sub.7, X.sub.8, X.sub.9, X.sub.10, X.sub.11, X.sub.12,
X.sub.13, X.sub.14, X.sub.15, X.sub.16, X.sub.17 or X.sub.18 is
conservatively substituted with another residue, and/or D variants
thereof.
[0042] In certain embodiments, this invention excludes peptides
having the sequence Lys-Arg-Asp-Ser (SEQ ID NO:238) and in certain
embodiments, this invention excludes peptides having the sequence
Lys-Arg-Asp-Ser (SEQ ID NO:238) in which Lys-Arg-Asp and Ser are
all L amino acids.
[0043] In certain embodiments the peptides of this invention show
less than 38%, preferably less than about 35%, more preferably less
than about 30% or less than about 25% LCAT activation activity as
measured by the assays provided in U.S. Pat. No. 6,376,464.
[0044] Definitions.
[0045] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residues is an artificial chemical analogue of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers.
[0046] The term "class A amphipathic helix" refers to a protein
structure that forms an .alpha.-helix producing a segregation of a
polar and nonpolar faces with the positively charged residues
residing at the polar-nonpolar interface and the negatively charged
residues residing at the center of the polar face (see, e.g.,
"Segrest et al. (1990) Proteins: Structure, Function, and Genetics
8: 103-117).
[0047] The term "ameliorating" when used with respect to
"ameliorating one or more symptoms of atherosclerosis" refers to a
reduction, prevention, or elimination of one or more symptoms
characteristic of atherosclerosis and/or associated pathologies.
Such a reduction includes, but is not limited to a reduction or
elimination of oxidized phospholipids, a reduction in
atherosclerotic plaque formation and rupture, a reduction in
clinical events such as heart attack, angina, or stroke, a decrease
in hypertension, a decrease in inflammatory protein biosynthesis,
reduction in plasma cholesterol, and the like. "Ameliorating one or
more symptoms of atherosclerosis" can also refer to improving blood
flow to vascular beds affected by atherosclerosis.
[0048] The term "enantiomeric amino acids" refers to amino acids
that can exist in at least two forms that are nonsuperimposable
mirror images of each other. Most amino acids (except glycine) are
enantiomeric and exist in a so-called L-form (L amino acid) or
D-form (D amino acid). Most naturally occurring amino acids are "L"
amino acids. The terms "D amino acid" and "L amino acid" are used
to refer to absolute configuration of the amino acid, rather than a
particular direction of rotation of plane-polarized light. The
usage herein is consistent with standard usage by those of skill in
the art.
[0049] The term "protecting group" refers to a chemical group that,
when attached to a functional group in an amino acid (e.g., a side
chain, an alpha amino group, an alpha carboxyl group, etc.) blocks
or masks the properties of that functional group. Preferred
amino-terminal protecting groups include, but are not limited to
acetyl, or amino groups. Other amino-terminal protecting groups
include, but are not limited to alkyl chains as in fatty acids,
propionyl, formyl and others. Preferred carboxyl terminal
protecting groups include, but are not limited to groups that form
amides or esters. The term "side chain protection groups" refers to
protecting groups that protect/block a side-chain (i.e. an R group)
of an amino acid. Side-chain protecting groups include, but are not
limited to amino protecting groups, carboxyl protecting groups and
hydroxyl protecting groups such as aryl ethers and guanidine
protecting groups such as nitro, tosyl etc.
[0050] The phrase "protect a phospholipid from oxidation by an
oxidizing agent" refers to the ability of a compound to reduce the
rate of oxidation of a phospholipid (or the amount of oxidized
phospholipid produced) when that phospholipid is contacted with an
oxidizing agent (e.g., hydrogen peroxide, 13-(S)--HPODE,
15-(S)--HPETE, HPODE, HPETE, HODE, HETE, etc.).
[0051] The terms "low density lipoprotein" or "LDL" is defined in
accordance with common usage of those of skill in the art.
Generally, LDL refers to the lipid-protein complex which when
isolated by ultracentrifugation is found in the density range
d=1.019 to d=1.063.
[0052] The terms "high density lipoprotein" or "HDL" is defined in
accordance with common usage of those of skill in the art.
Generally "HDL" refers to a lipid-protein complex which when
isolated by ultracentrifugation is found in the density range of
d=1.063 to d=1.21.
[0053] The term "Group I HDL" refers to a high density lipoprotein
or components thereof (e.g., apo A-I, paraoxonase, platelet
activating factor acetylhydrolase, etc.) that reduce oxidized
lipids (e.g., in low density lipoproteins) or that protect oxidized
lipids from oxidation by oxidizing agents.
[0054] The term "Group II HDL" refers to an HDL that offers reduced
activity or no activity in protecting lipids from oxidation or in
repairing (e.g., reducing) oxidized lipids.
[0055] The term "HDL component" refers to a component (e.g.,
molecules) that comprises a high density lipoprotein (HDL). Assays
for HDL that protect lipids from oxidation or that repair (e.g.,
reduce oxidized lipids) also include assays for components of HDL
(e.g., apo A-I, paraoxonase, platelet activating factor
acetylhydrolase, etc.) that display such activity.
[0056] The term "human apo A-I peptide" refers to a full-length
human apo A-I peptide or to a fragment or domain thereof comprising
a class A amphipathic helix.
[0057] A "monocytic reaction" as used herein refers to monocyte
activity characteristic of the "inflammatory response" associated
with atherosclerotic plaque formation. The monocytic reaction is
characterized by monocyte adhesion to cells of the vascular wall
(e.g., cells of the vascular endothelium), and/or chemotaxis into
the subendothelial space, and/or differentiation of monocytes into
macrophages, and/or monocyte chemotaxis as measured in vitro (e.g.,
utilizing a neuroprobe chamber).
[0058] The term "absence of change" when referring to the amount of
oxidized phospholipid refers to the lack of a detectable change,
more preferably the lack of a statistically significant change
(e.g., at least at the 85%, preferably at least at the 90%, more
preferably at least at the 95%, and most preferably at least at the
98% or 99% confidence level). The absence of a detectable change
can also refer to assays in which oxidized phospholipid level
changes, but not as much as in the absence of the protein(s)
described herein or with reference to other positive or negative
controls.
[0059] The following abbreviations are used herein: PAPC:
L-.alpha.-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine;
POVPC: 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine;
PGPC: 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine; PEIPC:
1-palmitoyl-2-(5,6-epoxyisoprostane
E.sub.2)-sn-glycero-3-phsophocholine; ChC18:2: cholesteryl
linoleate; ChC18:2-OOH: cholesteryl linoleate hydroperoxide; DMPC:
1,2-ditetradecanoyl-rac-glycerol-3-phosphocholine; PON:
paraoxonase; HPF: Standardized high power field; PON: paraoxonase;
BL/6: C57BL/6J; C3H:C3H/HeJ.
[0060] The term "conservative substitution" is used in reference to
proteins or peptides to reflect amino acid substitutions that do
not substantially alter the activity (specificity (e.g., for
lipoproteins))or binding affinity (e.g., for lipids or
lipoproteins)) of the molecule. Typically conservative amino acid
substitutions involve substitution one amino acid for another amino
acid with similar chemical properties (e.g., charge or
hydrophobicity). The following six groups each contain amino acids
that are typical conservative substitutions for one another: 1)
Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D),
Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine
(R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M),
Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan
(W).
[0061] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same, when compared and aligned for maximum correspondence, as
measured using one of the following sequence comparison algorithms
or by visual inspection. With respect to the peptides of this
invention sequence identity is determined over the full length of
the peptide.
[0062] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are input into a computer, subsequence coordinates are designated,
if necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0063] Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman (1988)
Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations
of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the
Wisconsin Genetics Software Package, Genetics Computer Group, 575
Science Dr., Madison, Wis.), or by visual inspection (see generally
Ausubel et al., supra).
[0064] One example of a useful algorithm is PILEUP. PILEUP creates
a multiple sequence alignment from a group of related sequences
using progressive, pairwise alignments to show relationship and
percent sequence identity. It also plots a tree or dendogram
showing the clustering relationships used to create the alignment.
PILEUP uses a simplification of the progressive alignment method of
Feng & Doolittle (1987) J. Mol. Evol. 35:351-360. The method
used is similar to the method described by Higgins & Sharp
(1989) CABIOS 5: 151-153. The program can align up to 300
sequences, each of a maximum length of 5,000 nucleotides or amino
acids. The multiple alignment procedure begins with the pairwise
alignment of the two most similar sequences, producing a cluster of
two aligned sequences. This cluster is then aligned to the next
most related sequence or cluster of aligned sequences. Two clusters
of sequences are aligned by a simple extension of the pairwise
alignment of two individual sequences. The final alignment is
achieved by a series of progressive, pairwise alignments. The
program is run by designating specific sequences and their amino
acid or nucleotide coordinates for regions of sequence comparison
and by designating the program parameters. For example, a reference
sequence can be compared to other test sequences to determine the
percent sequence identity relationship using the following
parameters: default gap weight (3.00), default gap length weight
(0.10), and weighted end gaps.
[0065] Another example of algorithm that is suitable for
determining percent sequence identity and sequence similarity is
the BLAST algorithm, which is described in Altschul et al. (1990)
J. Mol. Biol. 215: 403-410. Software for performing BLAST analyses
is publicly available through the National Center for Biotechnology
Information (http://www.ncbi.nlm.nih.go- v/). This algorithm
involves first identifying high scoring sequence pairs (HSPs) by
identifying short words of length W in the query sequence, which
either match or satisfy some positive-valued threshold score T when
aligned with a word of the same length in a database sequence. T is
referred to as the neighborhood word score threshold (Altschul et
al, supra). These initial neighborhood word hits act as seeds for
initiating searches to find longer HSPs containing them. The word
hits are then extended in both directions along each sequence for
as far as the cumulative alignment score can be increased.
Cumulative scores are calculated using, for nucleotide sequences,
the parameters M (reward score for a pair of matching residues;
always>0) and N (penalty score for mismatching residues;
always<0). For amino acid sequences, a scoring matrix is used to
calculate the cumulative score. Extension of the word hits in each
direction are halted when: the cumulative alignment score falls off
by the quantity X from its maximum achieved value; the cumulative
score goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) of 10, M=5, N=-4, and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.
Acad. Sci. USA 89:10915).
[0066] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin & Altschul
(1993) Proc. Natl. Acad. Sci. USA, 90: 5873-5787). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.1, more preferably less than about 0.01, and
most preferably less than about 0.001.
[0067] The term "D-18A peptide" refers to a peptide having the
sequence: D-W-L-K-A-F--Y-D-K--V-A-E-K-L-K-E-A-F (SEQ ID NO:3) where
all of the enantiomeric amino acids are D form amino acids.
[0068] The term "coadministering" or "concurrent administration",
when used, for example with respect to a peptide of this invention
and another active agent (e.g., a statin), refers to administration
of the peptide and the active agent such that both can
simultaneously achieve a physiological effect. The two agents,
however, need not be administered together. In certain embodiments,
administration of one agent can precede administration of the
other, however, such coadministering typically results in both
agents being simultaneously present in the body (e.g., in the
plasma) at a significant fraction (e.g., 20% or greater, preferably
30% or 40% or greater, more preferably 50% or 60% or greater, most
preferably 70% or 80% or 90% or greater) of their maximum serum
concentration for any given dose.
[0069] The term "detoxify" when used with respect to lipids, LDL,
or HDL refers the removal of some or all oxidizing lipids and/or
oxidized lipids. Thus, for example, the uptake of all or some HPODE
and/or HPETE (both hydroperoxides on fatty acids) will prevent or
reduce entrance of these peroxides into LDLs and thus prevent or
reduce LDL oxidation.
[0070] The term "pre-beta high density lipoprotein-like particles"
typically refers to cholesterol containing particles that also
contain apoA-I and which are smaller and relatively lipid-poor
compared to the lipid: protein ratio in the majority of HDL
particles. When plasma is separated by FPLC, these "pre-beta high
density lipoprotein-like particles" are found in the FPLC fractions
containing particles smaller than those in the main HDL peak and
are located to the right of HDL in an FPLC chromatogram as shown in
related application U.S. Ser. No. 10/423,830.
[0071] The phrase "reverse lipid transport and detoxification"
refers to the removal of lipids including cholesterol, other
sterols including oxidized sterols, phospholipids, oxidizing
agents, and oxidized phospholipids from tissues such as arteries
and transport out of these peripheral tissues to organs where they
can be detoxified and excreted such as excretion by the liver into
bile and excretion by the kidneys into urine. Detoxification also
refers to preventing the formation and/or destroying oxidized
phospholipids as explained herein.
[0072] The term "biological sample" as used herein refers to any
sample obtained from a living organism or from an organism that has
died. Examples of biological samples include body fluids, tissue
specimens, cells and cell lines taken from an organism (e.g., a
human or non-human mammal).
[0073] The term "amide" when referring to a hydrophobic protecting
group or a hydrophobic blocking group includes a simple amide to
methylamide or ethylamide. The term also includes alkyl amides such
as CO--NH--R where R is methyl, ethyl, etc. (e.g., up to 7,
preferably 9, more preferably 11 or 13 carbons).
[0074] The term "D-peptide" refers to a peptide in which one or
more of the enantiiomeric amino acids comprising the peptide are D
form amino acids. In certain embodiments, a plurality of the
enantiomeric amino acids are D form amino acids. In certain
embodiments, at least half of the enantiomeric amino acids are D
form amino acids. In certain embodiments, the peptide comprises
alternating D- and L-form amino acids. In certain embodiments, all
of the enantiomeric amino acids are D form amino acids.
[0075] The term "L-peptide" refers to a peptide in which all of the
amino acids (enantiomeric amino acids) are L-form amino acids.
[0076] A peptide that "converts pro-inflammatory HDL to
anti-inflammatory HDL or makes anti-inflammatory HDL more
anti-inflammatory" refers to a peptide that when administered to a
mammal (e.g., a human, a rat, a mouse, etc.), or that when used in
an appropriate ex vivo assay (e.g., as described herein), converts
HDL to an HDL that reduces or blocks lipid oxidation by an
oxidizing agent (e.g., as described in U.S. Ser. No. 6,596,544),
and/or that has increased paraoxonase activity, and/or that
decreases LDL-induced monocyte chemotactic activity generated by
artery wall cells as compared to HDL in a control assay (e.g., HDL
from a control animal or assay administered a lower dose of the
peptide or a negative control animal or assay lacking the peptide).
The alteration of HDL (conversion from non-protective to protective
or increase in protective activity) is preferably a detectable
change. In preferred embodiments, the change is a statistically
significant change, e.g., as determined using any statistical test
suited for the data set provided (e.g., t-test, analysis of
variance (ANOVA), semiparametric techniques, non-parametric
techniques (e.g., Wilcoxon Mann-Whitney Test, Wilcoxon Signed Ranks
Test, Sign Test, Kruskal-Wallis Test, etc.). Preferably the
statistically significant change is significant at least at the
85%, more preferably at least at the 90%, still more preferably at
least at the 95%, and most preferably at least at the 98% or 99%
confidence level. In certain embodiments, the change is at least a
10% change, preferably at least a 20% change, more preferably at
least a 50% change and most preferably at least a 90% change.
[0077] The phrase "in conjunction with" when used herein, e.g. in
reference to the administration of two amino acids comprising an
amino acid pair, in reference to the use of combinations of
peptides of this invention, in reference to the use of
peptides/amino acid pairs of this invention with other
pharmacologically active agent(s) (e.g., one or more statins), and
the like, indicates that the two (or more) agents are administered
so that there is at least some chronological overlap in their
physiological activity on the organism. Thus the two or more agents
can be administered simultaneously and/or sequentially. In
sequential administration there may even be some substantial delay
(e.g., minutes or even hours or days) before administration of the
second agent as long as the first administered agent has exerted
some physiological alteration on the organism when the second
administered agent is administered or becomes active in the
organism.
BRIEF DESCRIPTION OF THE DRAWINGS
[0078] FIG. 1 illustrates a synthesis scheme for the solution phase
synthesis of peptides according to this invention.
[0079] FIG. 2 illustrates the process for synthesizing a
tetrapeptide using the process outlined in FIG. 1.
[0080] FIG. 3 shows that pre-incubation (pre-treatment) but not
co-incubation (Co-inc) of Boc-Lys(Boc)-Arg-Asp-Ser(tBu)-OtBu
(synthesized from all D-amino acids) (SEQ ID NO:238 in Table 4)
inhibited LDL-induced monocyte chemotactic activity produced by
human artery wall cells (HAEC). The cells were either pre-incubated
with 125 .mu.g/ml, 250 .mu.g/ml, or 500 .mu.g/ml of the peptide,
the peptide was then removed and LDL at 100 .mu.g/ml cholesterol
with fresh medium was added or the same concentrations of peptide
were added together with the LDL and monocyte chemotactic activity
determined.
[0081] FIG. 4 shows that the addition of the tetrapeptide described
in FIG. 3 to the drinking water of apoE null mice converted HDL and
the post-HDL FPLC fractions from pro-inflammatory to
anti-inflammatory similar to D-4F. The tetrapeptide or D-4F were
added to the drinking water of the mice (n=4 for each condition) at
a concentration of 5 .mu.g/ml for 18 hours. The mice were bled and
their lipoproteins were separated by FPLC. A control human LDL at
100 .mu.g/ml of Cholesterol was added (LDL) or not added (No
Addition) to human artery wall cocultures or was added together
with HDL at 50 .mu.g/ml from a normal human control subject
(+Control HDL) or HDL at 50 .mu.g/ml from apoE null mice that
received drinking water without peptide (+Water Control HDL) or
received the tetrapeptide (+D-Tetra HDL) or D-4F (+D4F HDL) or the
post-HDL FPLC fractions from apoE null mice that did not receive
the peptide (+Water Control post HDL) or from mice that did receive
the tetrapeptide (+D-Tetra post HDL) or received D-4F (+D4F post
HDL)were added at 20 .mu.g/ml together with the control human LDL
at 100 .mu.g/ml of Cholesterol. After 8 hours the supernatants were
assayed for monocyte chemotactic activity.
[0082] FIG. 5 shows that apoE null mice receiving D-tetrapeptide or
D-4F in their drinking water have LDL that induces less monocyte
chemotactic activity. The LDL from the FPLC fractions of the mice
described in FIG. 4 was added to the cocultures at 100 .mu.g/ml.
After 8 hours the supernatants were assayed for monocyte
chemotactic activity.
[0083] FIG. 6 shows that SEQ ID NO:258 from Table 4 (designated
D-11 in the figure) when synthesized from all D-amino acids or D-4F
given orally renders HDL anti-inflammatory in apoE null mice but a
peptide containing the same D-amino acids as in D-4F but arranged
in a scrambled sequence that prevents lipid binding did not. Five
hundred micrograms of SEQ ID NO:258 synthesized from D-amino acids
(D-11) or 500 .mu.g of D-4F (D-4F) or 500 .mu.g of scrambled D-4F
(Scramb. Pept.) were instilled via a tube into the stomachs of
female, 3 month old apoE null mice, (n=4) and the mice were bled 20
min (20 min after gavage) or 6 hours later (6 hr after gavage).
Plasma was separated and HDL was isolated by FPLC. Cultures of
human aortic endothelial cells received medium alone (No
Addition/Assay Controls), standard normal human LDL at 100 .mu.m/mL
cholesterol without (LDL/Assay Controls) or together with standard
control human HDL (LDL+Control HDL/Assay Controls) at 50 .mu.m/mL
cholesterol, or control human LDL at 100 .mu.gm/mL cholesterol was
added with mouse HDL at 50 .mu.gm/mL cholesterol obtained from mice
that received the scrambled D-4F peptide (LDL+Scramb.Pept. HDL), or
D-4F (LDL+D-4F HDL) or SEQ ID NO:258 made from all D-amino acids
(LDL+D-11 HDL). The cultures were incubated for 8 hrs. The
supernatants were then assayed for monocyte chemotactic activity.
The values are mean.+-.SD of the number of migrated monocytes in 9
high power fields. *indicates p<0.001.
[0084] FIG. 7 shows that apoE null mice receiving D-4F or SEQ ID
NO:258 from Table 4 synthesized from D-amino acids (designated
D-11) (but not from mice that received scrambled D-4F) have LDL
that induces less monocyte chemotactic activity. The LDL from the
FPLC fractions of the mice described in FIG. 6 was added to the
cultures at 100 .mu.g/ml. After 8 hours the supernatants were
assayed for monocyte chemotactic activity. *indicates p<0.001,
**indicates p<0.01.
[0085] FIG. 8 shows that HDL was converted from pro-inflammatory to
anti-inflammatory after addition of SEQ ID NO:238 in Table 4
synthesized from D amino aicids (designated D-1)_to the chow of
apoE null mice (200 .mu.g/gm chow for 18 hours). Assay Controls: No
Addition, no addition to the cocultures; LDL ,a standard control
human LDL was added to the cocultures; +Control HDL, a control
normal human HDL was added to the cocultures. Chow LDL, LDL from
mice that received chow alone; +Chow Autolog. HDL, HDL from the
mice that received Chow alone was added together with the LDL from
these mice; +D-1 Autolog. HDL, HDL from the mice receiving the
peptide was added together with the LDL from these mice to the
cocultures and monocyte chemotactic activity was determined.
[0086] FIG. 9 shows that the tetrapeptide (SEQ ID NO:258 in Table
4) was ten times more potent than SEQ ID NO:238 in vitro. The
tetrapeptide was added or not added in a pre-incubation to human
artery wall cell cocultures at 100, 50, 25 or 12.5 .mu.m/mL and
incubated for 2 hrs. The cultures were then washed. Some wells then
received medium alone (No Addition). The other wells either
received standard normal human LDL at 100 .mu.gm/mL cholesterol
(LDL) or received this LDL together with a standard control human
HDL (LDL+Control HDL) at 50 .mu.gm/mL cholesterol and were
incubated for 8 hrs. Culture supernatants were then assayed for
monocyte chemotactic activity. The values are mean.+-.SD of the
number of migrated monocytes in 9 high power fields. The wells that
received the tetrapeptide in the 2 hr pre-incubation at the
concentrations noted above followed by the addition of LDL at 100
.mu.gm/mL cholesterol are indicated in the figure
(LDL+tetrapeptide, in .mu.gm/ml).
[0087] FIG. 10 shows that SEQ ID NOs:243, 242, and 256 from Table 4
(designated Seq No.5, Seq No.6, and Seq No. 9, respectively in the
figure) convert pro-inflammatory HDL from apoE null mice to
anti-inflammatory HDL. Two month old female apo E null mice (n=4
per treatment) fasted for 18 hrs, were injected intraperitoneally
with L-tetrapeptides at 20 .mu.gm peptide/mouse or were injected
with the saline vehicle (Saline Vehicle). Two hours later, blood
was collected from the retroorbital sinus under mild anesthesia
with Isofluorine. Plasma was separated and HDL was isolated by
FPLC. HDL inflammatory/anti-inflammatory properties were then
determined. Cultures of human aortic endothelial cells received
medium alone (No Addition), standard normal human LDL at 100
.mu.gm/mL cholesterol without (LDL) or together with standard
control human HDL (LDL+Control HDL) at 50 .mu.gm/mL cholesterol, or
standard control human LDL at 100 .mu.gm/mL cholesterol with mouse
HDL at 50 .mu.gm/mL cholesterol obtained from mice that received
the tetrapeptides or the saline vehicle (LDL+HDL from mice injected
intraperitoneally). The cultures were incubated for 8 hrs. The
supernatants were then assayed for monocyte chemotactic activity.
The values are mean.+-.SD of the number of migrated monocytes in 9
high power fields.
[0088] FIG. 11 shows that SEQ ID NO:258 from Table 4 (designated
S-11 in the Figure) converts pro-inflammatory HDL from apoE null
mice to anti-inflammatory HDL better than SEQ ID NO:254 and SEQ ID
NO:282 (designated S-7 and S-35, respectively in the Figure).
Two-month-old female apo E null mice (n=4 per treatment) fasted for
18 hrs, were injected intraperitoneally with S-7 or S-11 or S-35,
at 20 .mu.gm peptide/mouse or were injected with the saline vehicle
(Saline Vehicle). Two hours later, blood was collected from the
retroorbital sinus under mild anesthesia with Isofluorine. Plasma
was separated and LDL and HDL were isolated by FPLC. HDL
inflammatory/anti-inflammatory properties were then determined.
Cultures of human aortic endothelial cells received medium alone
(No Addition/Assay Controls), standard normal human LDL at 100
.mu.gm/mL cholesterol without (LDL/Assay Controls) or together with
standard control human HDL (+Control HDL/Assay Controls) at 50
.mu.gm/mL cholesterol, or mouse LDL at 100 .mu.gm/mL cholesterol
with mouse HDL at 50 .mu.gm/mL cholesterol obtained from mice that
received S-7, or S-11 or S-35 (LDL+S-7 HDL. LDL+S-11 HDL, LDL+S-35
HDL, respectively) or the saline vehicle (LDL+Saline HDL)). The
cultures were incubated for 8 hrs. The supernatants were then
assayed for monocyte chemotactic activity. The values are
mean.+-.SD of the number of migrated monocytes in 9 high power
fields. *p<0.001.
[0089] FIG. 12. The LDL from the FPLC fractions of the mice
described in Figure 11 was added to the cells at 100 .mu.g/ml.
After 8 hours the supernatants were assayed for monocyte
chemotactic activity. Assay Controls are as described in FIG. 11.
Saline LDL, LDL from mice injected with the saline vehicle; S-7
LDL, LDL from mice injected with SEQ ID NO:254 from Table 4 as
described in FIG. 11; S-11 LDL, LDL from mice injected with SEQ ID
NO:258 from Table 4 as described in FIG. 11; S-35, LDL from mice
injected with SEQ ID NO:282 as described in FIG. 9. #
p<0.001.
[0090] FIG. 13 shows serum Amyloid A (SAA) plasma levels after
injection of peptides. SAA levels in plasma were measured 24 hours
after injection of the peptides described in FIGS. 11 and 12.
*p<0.001.
[0091] FIG. 14 shows that SEQ ID NO:258 from Table 4 when
synthesized from all L-amino acids and given orally converts
pro-inflammatory HDL from apoE null mice to anti-inflammatory HDL.
Female, 3 month old apoE null mice, (n=4), were given 200
micrograms in water of the peptide described as SEQ ID NO:258 from
Table 4, which was synthesized from all L-amino acids (designated
S-11 in the figure). The peptide or water without peptide was
administered by stomach tube and the mice were bled 4 hours later.
A second group of four mice were given access to standard mouse
chow in powdered form and containing 200 micrograms of the S-11,
which was synthesized from all L-amino acids and added per1.0 gram
of powdered mouse chow in a total of 4 grams of powdered mouse chow
containing a total of 800 micrograms of the peptide for the cage of
four mice or they were given the same powdered mouse chow without
peptide. The chow was available to the mice overnight and by
morning the chow was consumed and the mice were bled. Plasma was
separated and HDL was isolated by FPLC. HDL
inflammatory/anti-inflammatory properties were then determined.
Cultures of human aortic endothelial cells received medium alone
(No Addition/Assay Controls), standard normal human LDL at 100
.mu.gm/mL cholesterol without (LDL/Assay Controls) or together with
standard control human HDL (LDL+Cont.HDUAssay Controls) at 50
.mu.gm/mL cholesterol, or control human LDL at 100 .mu.gm/mL
cholesterol with mouse HDL at 50 .mu.gm/mL cholesterol obtained
from mice that received no peptide (LDL+No Peptide HDL) or L-S-11
(LDL+L-S-11 HDL) by stomach tube (By gastric gavage) or in the
mouse chow (Powdered diet). The cultures were incubated for 8 hrs.
The supernatants were then assayed for monocyte chemotactic
activity. The values are mean.+-.SD of the number of migrated
monocytes in 9 high power fields. p<0.001.
[0092] FIG. 15 shows that L-S-11, when synthesized from all L-amino
acids and given orally increased plasma paraoxonase activity. The
plasma from the mice described in FIG. 14 was assayed for
paraoxonase activity (PON Activity, which is shown in the figure as
Units per 500 .mu.l of plasma). No peptide, mice that received
water or food alone without peptide. L-S-11, mice given 200
micrograms in water or food of the peptide described as SEQ ID
NO:256 from Table 4 as described in FIG. 14. P<0.001.
[0093] FIG. 16 shows that SEQ ID NO:238 (designated D-1) and SEQ ID
NO:258 (designated D-11) from Table 4 when synthesized from all
D-amino acids and given orally renders HDL anti-inflammatory in
apoE null mice but SEQ ID NO:238, when synthesized from all L-amino
acids (L-1) and given orally did not. Female, 3 month old apoE null
mice, (n=4), were given access to standard mouse chow in powdered
form and containing 0.5 milligram of each peptide added per 1.0
gram of powdered mouse chow in a total of 4 grams of powdered mouse
chow containing a total of 2.0 milligrams of the peptide for the
cage of four mice or they were given the same powdered mouse chow
without peptide. The chow was available to the mice for 24 hrs at
which time the chow was consumed and the mice were bled. Plasma was
separated and HDL was isolated by FPLC. Cultures of human aortic
endothelial cells received medium alone (No Addition/Assay
Controls), standard normal human LDL at 100 .mu.gm/mL cholesterol
without (LDL/Assay Controls) or together with standard control
human HDL (LDL+Control HDL/Assay Controls) at 50 .mu.gm/mL
cholesterol, or control human LDL at 100 .mu.gm/mL cholesterol was
added with mouse HDL at 50 .mu.gm/mL cholesterol obtained from mice
that received no peptide (LDL+No Pep. HDL), or SEQ ID NO:238 made
from all L-amino acids (LDL+L-1 HDL), or SEQ ID NO:238 made from
all D-amino acids (LDL+D-1 HDL) or SEQ ID NO:258 made from all
D-amino acids (LDL+D-11 HDL). The cultures were incubated for 8
hrs. The supernatants were then assayed for monocyte chemotactic
activity. The values are mean.+-.SD of the number of migrated
monocytes in 9 high power fields. *indicates p<0.01 and
**indicates p<0.001.
[0094] FIG. 17 shows that SEQ ID NO:238 (D-1) and SEQ ID NO:258
(D-11) from Table 4 when synthesized from all D-amino acids and
given orally renders HDL anti-inflammatory and reduces LDL-induced
monocyte chemotactic activity in apoE null mice but SEQ ID NO:238,
when synthesized from all L-amino acids and given orally, did not.
Plasma from the mice described in FIG. 16 was separated and HDL and
LDL were isolated by FPLC. Cultures of human aortic endothelial
cells received medium alone (No Addition/Assay Controls), standard
normal human LDL at 100 .mu.gm/mL cholesterol without (LDL/Assay
Controls) or together with standard control human HDL (LDL+Control
HDL/Assay Controls) at 50 .mu.gm/mL cholesterol, or autologous
mouse LDL at 100 .mu.gm/mL cholesterol alone (mLDL) or with mouse
HDL at 50 .mu.gm/mL cholesterol obtained from mice that received no
peptide (mLDL+No Pep. HDL), or SEQ ID NO:238 made from all L-amino
acids (mLDL+L-1 HDL), or SEQ ID NO:238 made from all D-amino acids
(mLDL+D-1 HDL) or SEQ ID NO:258 made from all D-amino acids
(mLDL+D-11 HDL). The cultures were incubated for 8 hrs. The
supernatants were then assayed for monocyte chemotactic activity.
The values are mean.+-.SD of the number of migrated monocytes in 9
high power fields. *indicates p<0.05, **indicates p<0.01 and
***indicates p<0.001.
[0095] FIG. 18 shows that SEQ ID NO:258 from Table 4 synthesized
from all D-amino acids (D-11), when given orally to mice, raised
HDL cholesterol concentrations while giving SEQ ID NO:238
synthesized from either L- or D-amino acids (L-1 or D-1,
respectively) orally did not. Plasma HDL-cholesterol concentrations
from the mice that are described in FIGS. 16 and 17 were
determined. No Peptide HDL, plasma HDL-cholesterol in mice that
received no peptide; L-1 HDL, plasma HDL-cholesterol in mice that
received SEQ ID NO:238 synthesized from L-amino acids; D-1 HDL,
plasma HDL-cholesterol in mice that received SEQ ID NO:238
synthesized from D-amino acids; D-11 HDL, plasma HDL-cholesterol in
mice that received SEQ ID NO:258 synthesized from D-amino acids.
*indicates p<0.001.
[0096] FIG. 19 shows that SEQ ID NO:258 from Table 4 synthesized
from all D-amino acids (D-11) when given orally to mice raised HDL
paraoxonase (PON) activity while giving SEQ ID NO:238 synthesized
from either L- or D-amino acids (L-1, D-1, respectively) orally did
not. Paraoxonase activity in the HDL described in FIG. 18 was
determined. The values are activity per 500 microliters of plasma.
*indicates p<0.001.
[0097] FIG. 20 shows that pravastatin and D-4F act synergistically
to reduce aortic lesions as determine in en face preparations in
apoE null mice. Five week old female apoE null mice were given in
their drinking water either no additions (water control),
pravastatin 50 .mu.g/ml, pravastatin 20 .mu.g/ml or D-4F 2
.mu.g/ml, or D-4F 5 .mu.g/ml, or pravastatin (PRAVA.) 20 .mu.g/ml
together with D-4F 2 .mu.g/ml, or pravastatin(PRAVA.) 50 .mu.g/ml
together with D-4F 5 .mu.g/ml. After 11 weeks the mice were
sacrificed and lesions determined in en face aortic
preparations.
[0098] FIG. 21 shows that pravastatin and D-4F act synergistically
to reduce aortic sinus lesions in apoE null mice. Five week old
female apoE null mice were given in their drinking water either no
additions (water control), pravastatin 50 .mu.g/ml, pravastatin 20
.mu.g/ml or D-4F 2 .mu.g/ml, or D-4F 5 .mu.g/ml, or pravastatin(P)
50 .mu.g/ml together with D-4F 5 .mu.g/ml, or pravastatin(P) 20
.mu.g/ml together with D-4F 2 .mu.g/ml. After 11 weeks the mice
were sacrificed and aortic sinus lesions were determined.
[0099] FIG. 22 shows that D-4F and SEQ ID NO:242 and SEQ ID NO:258
from Table 4 dramatically reduce lipoprotein lipid hydroperoxides
in apoE null mice. Fifty .mu.g/gm of SEQ ID NO:242 (D-198 in the
drawing) or SEQ ID NO:258 (D-203 in the drawing) or D-4F (the
peptides were synthesized from all D-amino acids) were added to the
chow of apoE null mice or the mice were continued on chow without
additions (None). Eighteen hours later the mice were bled, their
plasma fractionated by FPLC and the lipid hydroperoxide (LOOH)
content of their low density lipoproteins (LDL) and high density
lipoproteins (HDL) were determined. *indicates p<0.01.
[0100] FIG. 23 shows the solubility of peptides in ethyl acetate.
SEQ ID NO 254: Boc-Lys(.epsilon.Boc)-Glu-Arg-Ser(tBu)-OtBu; and SEQ
ID NO 258: Boc-Lys(.epsilon.Boc)-Arg-Glu-Ser(tBu)-OtBu. Also shown
is the solubility in ethyl acetate of SEO ID NO: 250.
[0101] FIG. 24 SEQ ID NO:258 forms 7.5 nm particles when mixed with
DMPC in an aqueous environment. To 1 mg/ml of DMPC suspension in
phosphate buffered saline (PBS) was added 10% deoxycholate until
the DMPC was dissolved. SEQ ID NO:258 or SEQ ID NO:254 were added
(DMPC: peptide; 1:10; wt:wt) and the reaction mixture dialyzed.
After dialysis the solution remained clear with SEQ ID NO:258 but
was turbid after the deoxycholate was removed by dialysis in the
case of SEQ ID NO:254. The figure is an electron micrograph
prepared with negative staining and at 147,420.times.
magnification. The arrows indicate SEQ ID NO:258 particles
measuring 7.5 nm (they appear as small white particles).
[0102] FIG. 25 SEQ ID NO:258 added to DMPC in an aqueous
environment forms particles with a diameter of approximately 7.5 nm
(large open), and stacked lipid-peptide bilayers (large striped
arrow) (small arrows pointing to the white lines in the cylindrical
stack of disks) with a bilayer dimension on the order of 3.4 to 4.1
nm with spacing between the bilayers (black lines between white
lines in the stack of disks) of approximately 2 nm. The conditions
and magnifications are the same as described in FIG. 24.
[0103] FIG. 26 shows that the peptide of SEQ ID NO:258added to DMPC
in an aqueous environment forms stacked lipid-peptide bilayers
(striped arrow) and vesicular structures of approximately 38 nm
white arrows).
[0104] FIG. 27 shows that DMPC in an aqueous environment without
SEQ ID NO:258 does not form particles with a diameter of
approximately 7.5 nm, or stacked lipid-peptide bilayers, nor
vesicular structures of approximately 38 nm. The DMPC vesicles
shown are 12.5-14 nm. The conditions and magnifications are the
same as described in FIG. 24.
[0105] FIG. 28 shows a molecular model of the peptide of SEQ ID
NO:254 compared to the peptide of SEQ ID NO:258. Red represents
oxygen, blue represents nitrogen, gray represents carbon, and white
represents hydrogen molecules.
[0106] FIG. 29 shows a space-filling molecule model of SEQ ID
NO:254 compared to SEQ ID NO:258. The arrows in this space filling
molecular model identify the polar and non-polar portions of the
molecules. The color code is the same as in FIG. 28.
[0107] FIG. 30 illustrates peptide backbones (in the bottom panels)
for the orientations given in the top panels.
[0108] FIG. 31 shows molecular models of SEQ ID NO:254 compared to
SEQ ID NO:258 identifying the Ser(tBu)-OtBu groups. The color code
is as in FIG. 28.
[0109] FIG. 32 shows molecular models of SEQ ID NO:254 compared to
SEQ ID NO 258 identifying various blocking groups. The color code
is as in FIG. 28.
[0110] FIG. 33 shows that SEQ ID NO:258 (but not SEQ ID NO:254)
renders apoE null HDL anti-inflammatory.
[0111] FIG. 34 shows that SEQ ID NO:258 but not SEQ ID NO:254,
significantly decreases aortic root atherosclerosis in apoE null
mice. The aortic root (aortic sinus) lesion score was determined in
the apoE null mice described in FIG. 33. The number of mice in each
group is shown (n=) at the bottom of the figure and a
representative section for each group is shown at the top of the
figure.
[0112] FIG. 35 shows that SEQ ID NO:258 but not SEQ ID NO:254
significantly decreases aortic atherosclerosis in en face
preparations in apoE null mice. The percent aortic surface
containing atherosclerotic lesions was determined in en face
preparations in the apoE null mice described in FIG. 33. The number
of mice in each group is shown (n=) at the bottom of the left panel
and a representative aorta for mice fed chow alone or chow
supplemented with. SEQ ID NO:258 is shown in the right panel.
[0113] FIG. 36 shows that SEQ ID NO:250 synthesized from all
L-amino acids significantly decreases atherosclerosis. ApoE null
mice (20 per group) were maintained on a chow diet (Chow) or on
chow supplemented with 200 .mu.g/gm chow of SEQ ID NO:250 (250)
synthesized from all L-amino acids. After 12 weeks the mice were
sacrificed and the % Aortic Surface Area with Lesions was
determined in en face preparations. *p=0.012
DETAILED DESCRIPTION
[0114] This invention pertains to the discovery that synthetic
peptides designed to mimic the class A amphipathic helical motif
(Segrest et al. (1990) Proteins: Structure, Function, and Genetics
8: 103-117) are able to associate with phospholipids and exhibit
many biological properties similar to human apo-A-I. In particular,
it was a discovery of this invention that when such peptides are
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 even be orally administered.
[0115] It was also a surprising discovery that these peptides can
stimulate the formation and cycling of pre-beta high density
lipoprotein-like particles. In addition, the peptides are capable
of enhancing/synergizing the effect of statins allowing statins to
be administered as significantly lower dosages or to be
significantly more anti-inflammatory at any given dose. It was also
discovered that the peptides described herein can inhibit and/or
prevent and/or treat one or more symptoms of osteoporosis. The
peptides can also increase pre-beta HDL; and/or increase HDL
paroxynase activity.
[0116] Moreover, it was a surprising discovery of this invention
that 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 one or
more symptoms of atherosclerosis.
[0117] It was also a surprising discovery that certain small
peptides consisting of a minimum of two amino acids, or pairs of
single amino acids, preferentially (but not necessarily) with one
or more of the amino acids being the D-sterioisomer of the amino
acid, and possessing hydrophobic domains to permit lipid protein
interactions, and hydrophilic domains to permit a degree of water
solubility also possess significant anti-inflammatory properties.
Without being bound to a particular theory, it is believed that the
peptides, or pairs of amino acids, described herein bind the
"seeding molecules" required for the formation of pro-inflammatory
oxidized phospholipids such as Ox-PAPC, POVPC, PGPC, and PEIPC.
Since many inflammatory conditions are mediated at least in part by
oxidized lipids, we believe that the peptides, or pairs of amino
acids, of this invention are effective in ameliorating conditions
that are known or suspected to be due to the formation of
biologically active oxidized lipids. These include, but are not
limited to atherosclerosis, rheumatoid arthritis, lupus
erythematous, polyarteritis nodosa, multiple sclerosis, asthma,
diabetes, Alzheimer's disease, and osteoporosis. The "small
peptides" typically range in length from 2 or 3 amino acids to
about 15 amino acids, more preferably from about 4 amino acids to
about 10 or 11 amino acids, and most preferably from about 4 to
about 8 or 10 amino acids. The peptides are typically characterized
by having hydrophobic terminal amino acids or terminal amino acids
rendered hydrophobic by the attachment of one or more hydrophobic
"protecting" groups. The internal structures of the peptides are
described in more detail herein.
[0118] In addition, it was a surprising finding of this invention
that a number of physical properties predict the ability of the
small peptides (e.g., less than 10 amino acids, perferably less
than 8 amino acids, more preferably from about 2 or 3 to about 5 or
6 amino acids), or pairs of amino acids, of this invention to
render HDL more anti-inflammatory and to mitigate atherosclerosis
and/or other pathologies characterized by an inflammatory response
in a mammal. The physical properties include high solubility in
ethyl acetate (e.g., greater than about 4 mg/mL), and solubility in
aqueous buffer at pH 7.0. Upon contacting phospholipids such as
1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), in an aqueous
environment, the particularly effective small peptides form
particles with a diameter of approximately 7.5 nm (.+-.0.1 nm),
and/or form stacked bilayers with a bilayer dimension on the order
of 3.4 to 4.1 nm with spacing between the bilayers in the stack of
approximately 2 nm, and/or also form vesicular structures of
approximately 38 nm). In certain preferred embodiments, the small
peptides, or pairs of amino acids, have a molecular weight of less
than about 900 Da.
[0119] I. Stimulating the Formation and Cycling of Pre-Beta High
Density Lipoprotein-Like Particles.
[0120] Reverse cholesterol transport is considered to be important
in preventing the build up of lipids that predisposes to
atherosclerosis (Shah et al. (2001) Circulation, 103: 3047-3050.)
Many have believed the lipid of consequence is cholesterol. Our
laboratory has shown that the key lipids are oxidized phospholipids
that initiate the inflammatory response in atherosclerosis (Navab
et al. (2001) Arterioscler Thromb Vasc Biol., 21(4): 481-488; Van
Lenten et al. (001) Trends Cardiovasc Med, 11: 155-161; Navab M et
al. (2001) Circulation, 104: 2386-2387).
[0121] This inflammatory response is also likely responsible for
plaque erosion or rupture that leads to heart attack and stroke.
HDL-cholesterol levels are inversely correlated with risk for heart
attack and stroke (Downs et al. (1998) JAMA 279: 1615-1622; Gordon
et al. (1977) Am J Med., 62: 707-714; Castelli et al. (1986) JAMA,
256: 2835-2838).
[0122] Pre-beta HDL is generally considered to be the most active
HDL fraction in promoting reverse cholesterol transport (e.g.,
picking up cholesterol from peripheral tissues such as arteries and
carrying it to the liver for excretion into the bile; see, Fielding
and Fielding (2001) Biochim Biophys Acta, 1533(3): 175-189).
However, levels of pre-beta HDL can be increased because of a
failure of the pre-beta HDL to be cycled into mature
alpha-migrating HDL e.g., LCAT deficiency or inhibition (O'Connor
et al. (1998) J Lipid Res, 39: 670-678). High levels of pre-beta
HDL have been reported in coronary artery disease patients (Miida
et al. (1996) Clin Chem., 42: 1992-1995).
[0123] Moreover, men have been found to have higher levels of
pre-beta HDL than women but the risk of men for coronary heart
disease is greater than for women (O'Connor et al. (1998) J Lipid
Res., 39: 670-678). Thus, static measurements of pre-beta HDL
levels themselves are not necessarily predictive of risk for
coronary artery disease. The cycling, however, of cholesterol
through pre-beta HDL into mature HDL is universally considered to
be protective against atherosclerosis (Fielding and Fielding (2001)
Biochim Biophys Acta, 1533(3): 175-189). Moreover, we have
demonstrated that the removal of oxidized lipids from artery wall
cells through this pathway protects against LDL oxidation.
[0124] Despite relatively low absorption rates when orally
administered, the peptides of this invention (e.g., D-4F) were
highly active.
[0125] In studies of Apo-E null mice orally administered D-4F, we
determined that 20 min after absorption from the intestine, D-4F
forms small pre-beta HDL-like particles that contain relatively
high amounts of apoA-I and paraoxonase. Indeed, estimating the
amount of apoA-I in these pre-beta HDL-like particles from Western
blots and comparing the amount of apoA-I to the amount of D-4F in
these particles (determined by radioactivity or LC-MRM) suggests
that as D-4F is absorbed from the intestine, it acts as a catalyst
causing the formation of these pre-beta HDL-like particles. This
small amount of intestinally derived D-4F appears to recruit
amounts of apoA-I, paraoxonase, and cholesterol into these
particles that are orders of magnitude more than the amount of D-4F
(see, e.g., Navab et al. (2004) Circulation, 109: r120-r125).
[0126] Thus, following absorption, D-4F, and other peptides, or
pairs of amino acids, of this invention, rapidly recruit relatively
large amounts of apoA-I and paraoxonase to form pre-beta HDL-like
particles which are very likely the most potent particles for both
promoting reverse cholesterol transport and for destroying
biologically active oxidized lipids. We believe that the formation
of these particles and their subsequent rapid incorporation into
mature HDL likely explains the dramatic reduction in
atherosclerosis that we observed in LDL receptor null mice on a
Western diet and in apoE-null mice on a chow diet independent of
changes in plasma cholesterol or HDL-cholesterol (Id.).
[0127] Thus, in one embodiment, this invention provides methods of
stimulating the formation and cycling of pre-beta high density
lipoprotein-like particles by administration of one or more
peptides, or pairs of amino acids, as described herein. The
peptides, or pairs of amino acids, can thereby promote lipid
transport and detoxification.
[0128] II. Mitigation of a Symptom of Atherosclerosis.
[0129] We discovered that normal HDL inhibits three steps in the
formation of mildly oxidized LDL. In those studies (see, copending
application U.S. Ser. No. 09/541,468, filed on Mar. 31, 2000) we
demonstrated that treating human LDL in vitro with apo A-I or an
apo A-I mimetic peptide (37 pA) 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.
[0130] The protective function of certain peptides of this
invention is illustrated in the parent applications (Ser. No.
09/645,454, filed Aug. 24, 2000, Ser. No. 09/896,841, filed Jun.
29, 2001, and WO 02/15923 (PCT/US01/26497), filed Jun. 29, 2001,
see, e.g., FIGS. 1-5 in WO 02/15923. FIG. 1, panels A, B, C, and D
in WO 02/15923 show the association of .sup.14C-D-5F with blood
components in an ApoE null mouse. It is also demonstrated that HDL
from mice that were fed an atherogenic diet and injected with PBS
failed to inhibit the oxidation of human LDL and failed to inhibit
LDL-induced monocyte chemotactic activity in human artery wall
coculures. In contrast, HDL from mice fed an atherogenic diet and
injected daily with peptides described herein was as effective in
inhibiting human LDL oxidation and preventing LDL-induced monocyte
chemotactic activity in the cocultures as was normal human HDL
(FIGS. 2A and 2B in WO 02/15923). In addition, LDL taken from mice
fed the atherogenic diet and injected daily with PBS was more
readily oxidized and more readily induced monocyte chemotactic
activity than LDL taken from mice fed the same diet but injected
with 20 .mu.g daily of peptide 5F. The D peptide did not appear to
be immunogenic (FIG. 4 in WO 02/15923).
[0131] The in vitro responses of human artery wall cells to HDL and
LDL from mice fed the atherogenic diet and injected with a peptide
according to this invention are consistent with the protective
action shown by such peptides in vivo. Despite, similar levels of
total cholesterol, LDL-cholesterol, IDL+VLDL-cholesterol, and lower
HDL-cholesterol as a percent of total cholesterol, the animals fed
the atherogenic diet and injected with the peptide had
significantly lower lesion scores (FIG. 5 in WO 02/15923). The
peptides thus prevented progression of atherosclerotic lesions in
mice fed an atherogenic diet.
[0132] Thus, in one embodiment, this invention provides methods for
ameliorating and/or preventing one or more symptoms of
atherosclerosis and/or other conditions characterized by an
inflammatory response.
[0133] III. Mitigation of a Symptom of Atheroscloerosis Associated
with an Acute Inflammatory Response.
[0134] The peptides, or pairs of amino acids, of this invention are
also useful in a number of contexts. For example, we have observed
that cardiovascular complications (e.g., atherosclerosis, stroke,
etc.) frequently accompany or follow the onset of an acute phase
inflammatory response. Such an acute phase inflammatory response is
often associated with a recurrent inflammatory disease (e.g.,
leprosy, tuberculosis, systemic lupus erythematosus, and rheumatoid
arthritis), a viral infection (e.g., influenza), a bacterial
infection, a fungal infection, an organ transplant, a wound or
other trauma, an implanted prosthesis, a biofilm, and the like.
[0135] It was a surprising discovery of this invention that
administration of one or more of the peptides described herein, can
reduce or prevent the formation of oxidized phospholipids during or
following an acute phase response and thereby mitigate or eliminate
cardiovascular complications associated with such a condition.
[0136] Thus, for example, we have demonstrated that a consequence
of influenza infection is the diminution in paraoxonase and
platelet activating acetylhydrolase activity in the HDL. Without
being bound by a particular theory, we believe that, as a result of
the loss of these HDL enzymatic activities and also as a result of
the association of pro-oxidant proteins with HDL during the acute
phase response, HDL is no longer able to prevent LDL oxidation and
was no longer able to prevent the LDL-induced production of
monocyte chemotactic activity by endothelial cells.
[0137] We observed that in a subject injected with very low dosages
of the polypeptides of this invention (e.g., 20 micrograms for
mice) daily after infection with the influenza A virus paraoxonase
levels did not fall and the biologically active oxidized
phospholipids were not generated beyond background. This indicates
that D-4F (and/or other peptides of this invention) can be
administered (e.g., orally or by injection) to patients with known
coronary artery disease during influenza infection or other events
that can generate an acute phase inflammatory response (e.g., due
to viral infection, bacterial infection, trauma, transplant,
various autoimmune conditions, etc.) and thus we can prevent by
this short term treatment the increased incidence of heart attack
and stroke associated with pathologies that generate such
inflammatory states.
[0138] Thus, in certain embodiments, this invention contemplates
administering one or more of the peptides, or pairs of amino acids,
of this invention to a subject at risk for, or incurring, an acute
inflammatory response and/or at risk for or incurring a symptom of
atherosclerosis.
[0139] Thus, for example, a person having or at risk for coronary
disease may prophylactically be administered a polypeptide, or pair
of amino acids, of this invention during flu season. A person (or
animal) subject to a recurrent inflammatory condition, e.g.,
rheumatoid arthritis, various autoimmune diseases, etc., can be
treated with a polypeptide of this invention to mitigate or prevent
the development of atherosclerosis or stroke. A person (or animal)
subject to trauma, e.g., acute injury, tissue transplant, etc. can
be treated with a polypeptide of this invention to mitigate the
development of atherosclerosis or stroke.
[0140] In certain instances such methods will entail a diagnosis of
the occurrence or risk of an acute inflammatory response. The acute
inflammatory response typically involves alterations in metabolism
and gene regulation in the liver. It is a dynamic homeostatic
process that involves all of the major systems of the body, in
addition to the immune, cardiovascular and central nervous system.
Normally, the acute phase response lasts only a few days; however,
in cases of chronic or recurring inflammation, an aberrant
continuation of some aspects of the acute phase response may
contribute to the underlying tissue damage that accompanies the
disease, and may also lead to further complications, for example
cardiovascular diseases or protein deposition diseases such as
amyloidosis.
[0141] An important aspect of the acute phase response is the
radically altered biosynthetic profile of the liver. Under normal
circumstances, the liver synthesizes a characteristic range of
plasma proteins at steady state concentrations. Many of these
proteins have important functions and higher plasma levels of these
acute phase reactants (APRs) or acute phase proteins (APPs) are
required during the acute phase response following an inflammatory
stimulus. Although most APRs are synthesized by hepatocytes, some
are produced by other cell types, including monocytes, endothelial
cells, fibroblasts and adipocytes. Most APRs are induced between
50% and several-fold over normal levels. In contrast, the major
APRs can increase to 1000-fold over normal levels. This group
includes serum amyloid A (SAA) and either C-reactive protein (CRP)
in humans or its homologue in mice, serum amyloid P component
(SAP). So-called negative APRs are decreased in plasma
concentration during the acute phase response to allow an increase
in the capacity of the liver to synthesize the induced APRs.
[0142] In certain embodiments, the acute phase response, or risk
therefore is evaluated by measuring one or more APPs. Measuring
such markers is well known to those of skill in the art, and
commercial companies exist that provide such measurement (e.g., AGP
measured by Cardiotech Services, Louisville, Ky.).
[0143] IV. Synergizing the Activity of Statins.
[0144] It was also discovered that, adding a low dosage of D-4F (1
.mu.g/ml) to the drinking water of apoE null mice for 24 hours did
not significantly improve HDL function (see, e.g., related
application U.S. Ser. No. 10/423,830). In addition, adding 0.05
mg/ml of atorvastatin or pravastatin alone to the drinking water of
the apoE null mice for 24 hours did not improve HDL function.
However, when D-4F 1 .mu.g/ml was added to the drinking water
together with 0.05 mg/ml of atorvastatin or pravastatin there was a
significant improvement in HDL function). Indeed the
pro-inflammatory apoE null HDL became as anti-inflammatory as 350
.mu.g/ml of normal human HDL (h, HDL see, e.g., related application
U.S. Ser. No. 10/423,830).
[0145] Thus, doses of D-4F alone, or statins alone, which by
themselves had no effect on HDL function when given together acted
synergistically. When D-4F and a statin were given together to apo
E null mice, their pro-inflammatory HDL at 50 .mu.g/ml of
HDL-cholesterol became as effective as normal human HDL at 350
.mu.g/ml of HDL-cholesterol in preventing the inflammatory response
induced by the action of HPODE oxidizing PAPC in cocultures of
human artery wall cells.
[0146] Thus, in certain embodiments this invention provides methods
for enhancing the activity of statins. The methods generally
involve administering one or more peptides, or pairs of amino
acids, as described herein concurrently with one or more statins.
The D-4F or other similar peptides as described herein achieve
synergistic action between the statin and the orally peptide(s) to
ameliorate atherosclerosis. In this context statins can be
administered at significantly lower dosages thereby avoiding
various harmful side effects (e.g., muscle wasting) associated with
high dosage statin use and/or the anti-inflammatory properties of
statins at any given dose are significantly enhanced.
[0147] V. Inhibiting/Treating Osteoporosis.7
[0148] Vascular calcification and osteoporosis often co-exist in
the same subjects (Ouchi et al. (1993) Ann NY Acad Sci., 676:
297-307; Boukhris and Becker ('1972) JAMA, 219: 1307-1311; Banks et
al. (1994) Eur J Clin Invest., 24: 813-817; Laroche et al. (1994)
Clin Rheumatol., 13: 611-614; Broulik and Kapitola (1993) Endocr
Regul., 27: 57-60; Frye et al. (1992) Bone Mine., 19: 185-194;
Barengolts et al. (1998) Calcif Tissue Int., 62: 209-213; Burnett
and Vasikaran (2002) Ann Clin Biochem., 39: 203-210. Parhami et al.
(1997) Arterioscl Thromb Vasc Biol., 17: 680-687, demonstrated that
mildly oxidized LDL (MM-LDL) and the biologically active lipids in
MM-LDL [i.e. oxidized 1-palmitoyl-2-arachidonoyl-sn-glyc-
ero-3-phosphorylcholine) (Ox-PAPC)], as well as the isoprostane,
8-iso prostaglandin E.sub.2, but not the unoxidized
phospholipid(PAPC) or isoprostane 8-iso progstaglandin
F.sub.2.alpha. induced alkaline phosphatase activity and
osteoblastic differentiation of calcifying vascular cells (CVCs) in
vitro, but inhibited the differentiation of MC3T3-E1 bone
cells.
[0149] The osteon resembles the artery wall in that the osteon is
centered on an endothelial cell-lined lumen surrounded by a
subendothelial space containing matrix and fibroblast-like cells,
which is in turn surrounded by preosteoblasts and osteoblasts
occupying a position analogous to smooth muscle cells in the artery
wall (Id.). Trabecular bone osteoblasts also interface with bone
marrow subendothelial spaces (Id.). Parhami et al. postulated that
lipoproteins could cross the endothelium of bone arteries and be
deposited in the subendothelial space where they could undergo
oxidation as in coronary arteries (Id.). Based on their in vitro
data they predicted that LDL oxidation in the subendothelial space
of bone arteries and in bone marrow would lead to reduced
osteoblastic differentiation and mineralization which would
contribute to osteoporosis (Id.). Their hypothesis further
predicted that LDL levels would be positively correlated with
osteoporosis as they are with coronary calcification (Pohle et al.
(2001) Circulation, 104: 1927-1932), but HDL levels would be
negatively correlated with osteoporosis (Parhami et al. (1997)
Arterioscl Thromb Vasc Biol., 17: 680-687).
[0150] In vitro, the osteoblastic differentiation of the marrow
stromal cell line M2-10B4 was inhibited by MM-LDL but not native
LDL (Parhami et al. (1999) J Bone Miner Res., 14: 2067-2078). When
marrow stromal cells from atherosclerosis susceptible C57BL/6 (BL6)
mice fed a low fat chow diet were cultured there was robust
osteogenic differentiation (Id.). In contrast, when the marrow
stromal cells taken from the mice after a high fat, atherogenic
diet were cultured they did not undergo osteogenic differentiation
(Id.). This observation is particularly important since it provides
a possible explanation for the decreased osteogenic potential of
marrow stromal cells in the development of osteoporosis (Nuttall
and Gimble (2000) Bone, 27: 177-184). In vivo the decrease in
osteogenic potential is accompanied by an increase in adipogenesis
in osteoporotic bone (Id.).
[0151] It was found that adding D-4F to the drinking water of apoE
null mice for 6 weeks dramatically increased trabecular bone
mineral density and it is believed that the other peptides of this
invention will act similarly.
[0152] Our data indicate that osteoporosis can be regarded as an
"atherosclerosis of bone". It appears to be a result of the action
of oxidized lipids. HDL destroys these oxidized lipids and promotes
osteoblastic differentiation. Our datat indicate that administering
peptide(s) of this invention to a mammal (e.g., in the drinking
water of apoE null mice) dramatically increases trabecular bone in
just a matter of weeks.
[0153] This indicates that the peptides, or pairs of amino acids,
described herein are useful for mitigation one or more symptoms of
osteoporosis (e.g., for inhibiting decalcification) or for inducing
recalcification of osteoporotic bone. The peptides are also useful
as prophylactics to prevent the onset of symptom(s) of osteoporosis
in a mammal (e.g., a patient at risk for osteoporosis).
[0154] We believe similar mechanisms are a cause of coronary
calcification, e.g., calcific aortic stenosis. Thus, in certain
embodiments, this invention contemplates the use of the peptides,
or pairs of amino acids, described herein to inhibit or prevent a
symptom of a disease such as coronary calcification, calcific
aortic stenosis, osteoporosis, and the like.
[0155] VI. Other Indications.
[0156] Without being bound to a particular theory, we also belive
the peptides, or pairs of amino acids, described herein are useful,
prophylactically or therapeutically, to mitigate the onset and/or
more or more symptoms of a variety of other conditions including,
but not limited to polymyalgia rheumatica, polyarteritis nodosa,
scleroderma, lupus erythematosus, multiple sclerosis, idiopathic
pulmonary fibrosis, chronic obstructive pulmonary disease (e.g.,
asthma), Alzheimers Disease, AIDS, and diabetes. Typically, the
peptides will be useful in mitigation a symptom caused by or
associated with an inflammatory response in these conditions.
[0157] VII. Peptide/Amino Acid Pair Administration.
[0158] The methods of this invention typically involve
administering to an organism, preferably a mammal, more preferably
a human one or more of the peptides, or pairs of amino acids, of
this invention (or mimetics of such peptides, or pairs of amino
acids). The peptide(s), or pairs of amino acids, can be
administered, as described herein, according to any of a number of
standard methods including, but not limited to injection,
suppository, inhalation (e.g., nasal spray, oral inhalation, etc.),
time-release implant, transdermal patch, and the like. In one
particularly preferred embodiment, the peptide(s) are administered
orally (e.g., as a syrup, capsule, powder, gelcap, or tablet).
[0159] The methods can involve the administration of a single
peptide or pair of amino acids of this invention or the
administration of two or more different peptides or or pairs of
amino acids. The peptides, or pairs of amino acids, can be provided
as monomers or in dimeric, oligomeric or polymeric forms. In
certain embodiments, the multimeric forms may comprise associated
monomers (e.g., ionically or hydrophobically linked) while certain
other multimeric forms comprise covalently linked monomers
(directly linked or through a linker).
[0160] While the invention is described with respect to use in
humans, it is also suitable for animal, e.g., veterinary use. Thus
preferred organisms include, but are not limited to humans,
non-human primates, canines, equines, felines, porcines, ungulates,
largomorphs, and the like.
[0161] The methods of this invention are not limited to humans or
non-human animals showing one or more symptom(s) of atherosclerosis
(e.g., hypertension, plaque formation and rupture, reduction in
clinical events such as heart attack, angina, or stroke, high
levels of plasma cholesterol, high levels of low density
lipoprotein, high levels of very low density lipoprotein, or
inflammatory proteins such as CRP, etc.), but are useful in a
prophylactic context. Thus, the peptides of this invention, or
pairs of amino acids, (or mimetics thereof) can be administered to
organisms to prevent the onset/development of one or more symptoms
of atherosclerosis and/or one of the other indications described
herein. Particularly preferred subjects in this context are
subjects showing one or more risk factors for atherosclerosis
(e.g., family history, hypertension, obesity, high alcohol
consumption, smoking, high blood cholesterol, high blood
triglycerides, elevated blood LDL, VLDL, EDL, or low HDL, diabetes,
or a family history of diabetes, high blood lipids, heart attack,
angina or stroke, etc.) and/or one of the other conditions
described herein.
[0162] In certain embodiments, the peptides, or pairs of amino
acids, of this invention can also be administered to stimulate the
formation and cycling of pre-beta high density lipoprotein-like
particles and/or to promote reverse lipid transport and
detoxification.
[0163] The peptides, or pairs of amino acids, are also useful for
administration in conjunction with statins where they enhance
(e.g., synergize) the activity of the statin at typically
administered dosages and/or permit the statin(s) to be administered
at lower dosages.
[0164] In addition, the peptides, or pairs of amino acids, can be
administered to reduce or eliminate one or more symptoms of
osteoporosis and/or diabetes, and/or any of the other conditions
described herein, and/or to prevent/inhibit the onset of one or
more symptoms of osteoporosis and/or any of the other indications
described herein.
[0165] VIII. Certain Preferred Peptides and Their Preparation.
[0166] A) Class A Amphipathic Helical Peptides.
[0167] It was a discovery of this invention that peptides
comprising a class A amphipathic helix ("class A peptides"), are
capable of mitigating one or more symptoms of atherosclerosis.
Class A peptides are characterized by formation of an .alpha.-helix
that produces a segregation of polar and non-polar residues thereby
forming a polar and a nonpolar face with the positively charged
residues residing at the polar-nonpolar interface and the
negatively charged residues residing at the center of the polar
face (see, e.g., Anantharamaiah (1986) Meth. Enzymol, 128:
626-668). 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.
[0168] One particularly preferred class A peptide, designated 18A
(see, e.g., Anantharamaiah (1986) Meth. Enzymol, 128: 626-668) was
modified as described herein to produce peptides orally
administratable and highly effective at inhibiting or preventing
one or more symptoms of atherosclerosis. Without being bound by a
particular theory, it is believed that the peptides of this
invention may act in vivo may by picking up seeding molecule(s)
that mitigate oxidation of LDL.
[0169] We determined that increasing the number of Phe residues on
the hydrophobic face of 18A would theoretically increase lipid
affinity as determined by the computation described by Palgunachari
et al. (1996) Arteriosclerosis, Thrombosis, & Vascular Biology
16: 328-338. Theoretically, a systematic substitution of residues
in the nonpolar face of 18A with Phe could yield six peptides.
Peptides with an additional 2, 3 and 4 Phe would have theoretical
lipid affinity (.lambda.) values of 13, 14 and 15 units,
respectively. However, the .lambda. values jumped four units if the
additional Phe were increased from 4 to 5 (to 19 .lambda. units).
Increasing to 6 or 7 Phe would produce a less dramatic increase (to
20 and 21 .lambda. units, respectively). Therefore, we chose 5
additional Phe (and hence the peptides designation as 5F). In one
particularly preferred embodiment, the 5F peptide was blocked in
that the amino terminal residue was acetylated and the carboxyl
terminal residue was amidated.
[0170] The new class A peptide analog, 5F, inhibited lesion
development in atherosclerosis-susceptible mice. The new peptide
analog, 5F, was compared with mouse apo A-I (MoA-I) for efficacy in
inhibiting diet-induced atherosclerosis in these mice using peptide
dosages based on the study by Levine et al. (Levine et al. (1993)
Proc. Natl. Acad. Sci. USA 90:12040-12044).
[0171] A number of other class A peptides were also produced and
showed varying, but significant degrees of efficacy in mitigating
one or more symptoms of atherosclerosis. A number of such peptides
are illustrated in Table 1.
1TABLE 1 Illustrative mimetics of the amphipathic helix of Apo A-I
for use in this invention. Peptide SEQ ID Name Amino Acid Sequence
NO. 18A D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F 4 2F
Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH.sub.2 5 3F
Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH.sub.2 6 3F14
Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH.sub.2 7 4F
Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH.sub.2 8 5F
Ac-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH.sub.2 9 6F
Ac-D-W-L-K-A-F-Y-D-K-F-F-E-K-F-K-E-F-F-NH.sub.2 10 7F
Ac-D-W-F-K-A-F-Y-D-K-F-F-E-K-F-K-E-F-F-NH.sub.2 11
Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-F-F-NH.sub.2 12
Ac-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-A-F-NH.sub.2 13
Ac-D-W-L-K-A-F-Y-D-K-V-F-E-K-L-K-E-F-F-NH.sub.2 14
Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-F-F-NH.sub.2 15
Ac-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH.sub.2 16
Ac-E-W-L-K-L-F-Y-E-K-V-L-E-K-F-K-E-A-F-NH.sub.2 17
Ac-E-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH.sub.2 18
Ac-E-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-F-F-NH.sub.2 19
Ac-E-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-A-F-NH.sub.2 20
Ac-E-W-L-K-A-F-Y-D-K-V-F-E-K-L-K-E-F-F-NH.sub.2 21
Ac-E-W-L-K-A-F-Y-D-K-V-A-E-K-F-K-E-F-F-NH.sub.2 22
Ac-E-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH.sub.2 23
AC-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH.sub.2 24
Ac-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH.sub.2 25
Ac-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH.sub.2 26
Ac-A-F-Y-D-K-F-F-E-K-F-K-E-F-F-NH.sub.2 27
Ac-A-F-Y-D-K-F-F-E-K-F-K-E-F-F-NH.sub.2 28
Ac-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH.sub.2 29
Ac-A-F-Y-D-K-V-A-E-K-L-K-E-F-F-NH.sub.2 30
Ac-A-F-Y-D-K-V-F-E-K-F-K-E-A-F-NH.sub.2 31
Ac-A-F-Y-D-K-V-F-E-K-L-K-E-F-F-NH.sub.2 32
Ac-A-F-Y-D-K-V-A-E-K-F-K-E-F-F-NH.sub.2 33
Ac-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-NH.sub.2 34
Ac-L-F-Y-E-K-V-L-E-K-F-K-E-A-F-NH.sub.2 35
Ac-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH.sub.2 36
Ac-A-F-Y-D-K-V-A-E-K-L-K-E-F-F-NH.sub.2 37
Ac-A-F-Y-D-K-V-F-E-K-F-K-E-A-F-NH.sub.2 38
Ac-A-F-Y-D-K-V-F-E-K-L-K-E-F-F-NH.sub.2 39
Ac-A-F-Y-D-K-V-A-E-K-F-K-E-F-F-NH.sub.2 40
Ac-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH.sub.2 41
Ac-D-W-L-K-A-L-Y-D-K-V-A-E-K-L-K-E-A-L-NH.sub.2 42
Ac-D-W-F-K-A-F-Y-E-K-V-A-E-K-L-K-E-F-F-NH.sub.2 43
Ac-D-W-F-K-A-F-Y-E-K-F-F-E-K-F-K-E-F-F-NH.sub.2 44
Ac-E-W-L-K-A-L-Y-E-K-V-A-E-K-L-K-E-A-L-NH.sub.2 45
Ac-E-W-L-K-A-F-Y-E-K-V-A-E-K-L-K-E-A-F-NH.sub.2 46
Ac-E-W-F-K-A-F-Y-E-K-V-A-E-K-L-K-E-F-F-NH.sub.2 47
Ac-E-W-L-K-A-F-Y-E-K-V-F-E-K-F-K-E-F-F-NH.sub.2 48
Ac-E-W-L-K-A-F-Y-E-K-F-F-E-K-F-K-E-F-F-NH.sub.2 49
Ac-E-W-F-K-A-F-Y-E-K-F-F-E-K-F-K-E-F-F-NH.sub.2 50
Ac-D-F-L-K-A-W-Y-D-K-V-A-E-K-L-K-E-A-W-NH.sub.2 51
Ac-E-F-L-K-A-W-Y-E-K-V-A-E-K-L-K-E-A-W-NH.sub.2 52
Ac-D-F-W-K-A-W-Y-D-K-V-A-E-K-L-K-E-W-W-NH.sub.2 53
Ac-E-F-W-K-A-W-Y-E-K-V-A-E-K-L-K-E-W-W-NH.sub.2 54
Ac-D-K-L-K-A-F-Y-D-K-V-F-E-W-A-K-E-A-F-NH.sub.2 55
Ac-D-K-W-K-A-V-Y-D-K-F-A-E-A-F-K-E-F-L-NH.sub.2 56
Ac-E-K-L-K-A-F-Y-E-K-V-F-E-W-A-K-E-A-F-NH.sub.2 57
Ac-E-K-W-K-A-V-Y-E-K-F-A-E-A-F-K-E-F-L-NH.sub.2 58
Ac-D-W-L-K-A-F-V-D-K-F-A-E-K-F-K-E-A-Y-NH.sub.2 59
Ac-E-K-W-K-A-V-Y-E-K-F-A-E-A-F-K-E-F-L-NH.sub.2 60
Ac-D-W-L-K-A-F-V-Y-D-K-V-F-K-L-K-E-F-F-NH.sub.2 61
Ac-E-W-L-K-A-F-V-Y-E-K-V-F-K-L-K-E-F-F-NH.sub.2 62
Ac-D-W-L-R-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-NH.sub.2 63
Ac-E-W-L-R-A-F-Y-E-K-V-A-E-K-L-K-E-A-F-NH.sub.2 64
Ac-D-W-L-K-A-F-Y-D-R-V-A-E-K-L-K-E-A-F-NH.sub.2 65
Ac-E-W-L-K-A-F-Y-E-R-V-A-E-K-L-K-E-A-F-NH.sub.2 66
Ac-D-W-L-K-A-F-Y-D-K-V-A-E-R-L-K-E-A-F-NH.sub.2 67
Ac-E-W-L-K-A-F-Y-E-K-V-A-E-R-L-K-E-A-F-NH.sub.2 68
Ac-D-W-L-K-A-F-Y-D-K-V-A-E-K-L-R-E-A-F-NH.sub.2 69
Ac-E-W-L-K-A-F-Y-E-K-V-A-E-K-L-R-E-A-F-NH.sub.2 70
Ac-D-W-L-K-A-F-Y-D-R-V-A-E-R-L-K-E-A-F-NH.sub.2 71
Ac-E-W-L-K-A-F-Y-E-R-V-A-E-R-L-K-E-A-F-NH.sub.2 72
Ac-D-W-L-R-A-F-Y-D-K-V-A-E-K-L-R-E-A-F-NH.sub.2 73
Ac-E-W-L-R-A-F-Y-E-K-V-A-E-K-L-R-E-A-F-NH.sub.2 74
Ac-D-W-L-R-A-F-Y-D-R-V-A-E-K-L-K-E-A-F-NH.sub.2 75
Ac-E-W-L-R-A-F-Y-E-R-V-A-E-K-L-K-E-A-F-NH.sub.2 76
Ac-D-W-L-K-A-F-Y-D-K-V-A-E-R-L-R-E-A-F-NH.sub.2 77
Ac-E-W-L-K-A-F-Y-E-K-V-A-E-R-L-R-E-A-F-NH.sub.2 78
Ac-D-W-L-R-A-F-Y-D-K-V-A-E-R-L-K-E-A-F-NH.sub.2 79
Ac-E-W-L-R-A-F-Y-E-K-V-A-E-R-L-K-E-A-F-NH.sub.2 80
D-W-L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-P-D-W- 81
L-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F D-W-L-K-A-F-Y-D-K-V-A-E--
K-L-K-E-F-F-P-D-W- 82 L-K-A-F-Y-D-K-V-A-E-K-L-K-E-F-F
D-W-F-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F-P-D-W- 83
F-K-A-F-Y-D-K-V-A-E-K-L-K-E-A-F
D-K-L-K-A-F-Y-D-K-V-F-E-W-A-K-E-A-F-P-D-K- 84
L-K-A-F-Y-D-K-V-F-E-W-L-K-E-A-F D-K-W-K-A-V-Y-D-K-F-A-E--
A-F-K-E-F-L-P-D-K- 85 W-K-A-V-Y-D-K-F-A-E-A-F-K-E-F-L
D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-P-D-W- 86
F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F
D-W-L-K-A-F-V-Y-D-K-V-F-K-L-K-E-F-F-P-D-W- 87
L-K-A-F-V-Y-D-K-V-F-K-L-K-E-F-F D-W-L-K-A-F-Y-D-K-F-A-E--
K-F-K-E-F-F-P-D-W- 88 L-K-A-F-Y-D-K-F-A-E-K-F-K-E-F-F
Ac-E-W-F-K-A-F-Y-E-K-V-A-E-K-F-K-E-A-F-NH.sub.2 89
Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-NH.sub.2 90
Ac-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-NH.sub.2 91
Ac-F-K-A-F-Y-E-K-V-A-E-K-F-K-E-NH.sub.2 92
NMA-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-NH.sub.2 93
NMA-F-K-A-F-Y-E-K-V-A-E-K-F-K-E-NH.sub.2 94
NMA-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH.sub.2 95
NMA-E-W-F-K-A-F-Y-E-K-V-A-E-K-F-K-E-A-F-NH.sub.2 96
NMA-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH.sub.2 97
NMA-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-NH.sub.2 98
Ac-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH.sub.2 99
NMA-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH.sub.2
Ac-E-W-L-K-A-F-Y-E-K-V-F-E-K-F-K-E-F-F-NH.sub.2 100
NMA-E-W-L-K-A-F-Y-E-K-V-F-E-K-F-K-E-F-F-NH.sub.2
Ac-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH.sub.2 101
NMA-A-F-Y-D-K-V-F-E-K-F-K-E-F-F-NH.sub.2
Ac-A-F-Y-E-K-V-F-E-K-F-K-E-F-F-NH.sub.2 102
NMA-A-F-Y-E-K-V-F-E-K-F-K-E-F-F-NH.sub.2
Ac-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-NH.sub.2 103
NMA-D-W-L-K-A-F-Y-D-K-V-F-E-K-F-NH.sub.2
Ac-E-W-L-K-A-F-Y-E-K-V-F-E-K-F-NH.sub.2 104
NMA-E-W-L-K-A-F-Y-E-K-V-F-E-K-F-NH.sub.2
Ac-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-NH.sub.2 105
NMA-L-K-A-F-Y-D-K-V-F-E-K-F-K-E-NH.sub.2
Ac-L-K-A-F-Y-E-K-V-F-E-K-F-K-E-NH.sub.2 106
NMA-L-K-A-F-Y-E-K-V-F-E-K-F-K-E-NH.sub.2 .sup.1Linkers are
underlined. NMA is N-Methyl Anthranilyl.
[0172] In certain preferred embodiments, the peptides include
variations of 4F (SEQ ID NO:8 in Table 1) or D-4F where one or both
aspartic acids (D) are replaced by glutamic acid (E). Also
contemplated are peptides (e.g., 4F or D-4F) where 1, 2, 3, or 4
amino acids are deleted from the carboxyl terminus and/or 1, 2, 3,
or 4 amino acids are deleted from the carboxyl terminus and/or one
or both aspartic acids (D) are replaced by glutamic acid (E). In
any of the peptides described herein, the N-terminus can be blocked
and labeled using a mantyl moiety (e.g., N-methylanthranilyl).
[0173] While various peptides of Table 1, are illustrated with an
acetyl group or an N-methylanthranilyl group protecting the amino
terminus and an amide group protecting the carboxyl terminus, any
of these protecting groups may be eliminated and/or substituted
with another protecting group as described herein. In particularly
preferred embodiments, the peptides comprise one or more D-form
amino acids as described herein. In certain embodiments, every
amino acid (e.g., every enantiomeric amino acid) of the peptides of
Table 1 is a D-form amino acid.
[0174] It is also noted that Table Table 1 is not fully inclusive.
Using the teaching provided herein, other suitable class A
amphipathic helical peptides can routinely be produced (e.g., by
conservative or semi-conservative substitutions (e.g., D replaced
by E), extensions, deletions, and the like). Thus, for example, one
embodiment utilizes truncations of any one or more of peptides
shown hwerein (e.g., peptides identified by SEQ ID Nos:5-23 and
42--in Table 1). Thus, for example, SEQ ID NO:24 illustrates a
peptide comprising 14 amino acids from the C-terminus of 18A
comprising one or more D amino acids, while SEQ ID NOS:25-41
illustrate other truncations.
[0175] Longer peptides are also suitable. Such longer peptides may
entirely form a class A amphipathic helix, or the class A
amphipathic helix (helices) can form one or more domains of the
peptide. In addition, this invention contemplates multimeric
versions of the peptides. Thus, for example, the peptides
illustrated heren can be coupled together (directly or through a
linker (e.g., a carbon linker, or one or more amino acids) with one
or more intervening amino acids). Illustrative polymeric peptides
include 18A-Pro-18A and the peptides of SEQ ID NOs:81-88, in
certain embodiments comprising one or more D amino acids, more
preferably with every amino acid a D amino acid as described herein
and/or having one or both termini protected.
[0176] B) Other Class A Amphipathic Helical Peptide Mimetics of
apoA-I Having Aromatic or Aliphatic Residues in the Non-Polar
Face.
[0177] In certain embodiments, this invention also provides
modified class A amphiphathic helix peptides. Certain preferred
peptides incorporate one or more aromatic residues at the center of
the nonpolar face, e.g., 3F.sup.C.pi., (as present in 4F), or with
one or more aliphatic residues at the center of the nonpolar face,
e.g., 3F.sup.I.pi.. Without being bound to a particular theory, we
believe the central aromatic residues on the nonpolar face of the
peptide 3F.sup.C.pi., due to the presence of .pi. electrons at the
center of the nonpolar face, allow water molecules to penetrate
near the hydrophobic lipid alkyl chains of the peptide-lipid
complex, which in turn would enable the entry of reactive oxygen
species (such as lipid hydroperoxides) shielding them from the cell
surface. Similarly, we also believe the peptides with aliphatic
residues at the center of the nonpolar face, e.g., 3F.sup.I.pi.,
will act similarly but not quite as effectively as
3F.sup.C.pi..
[0178] Preferred peptides will convert pro-inflammatory HDL to
anti-inflammatory HDL or make anti-inflammatory HDL more
anti-inflammatory, and/or decrease LDL-induced monocyte chemotactic
activity generated by artery wall cells equal to or greater than
D4F or other peptides shown in Table 1. Peptides showing this
activity are useful in ameliorating atherosclerosis and other
inflammatory conditions such as rheumatoid arthritis, lupus
erythematous, polyarteritis nodosa, osteoporosis, Alzheimer's
disease, congestive heart failure, endothelial dysfunction, and
viral illnesses such as influenza A and diseases such as multiple
sclerosis.
2TABLE 2 Examples of certain preferred peptides. Name Sequence SEQ
ID NO (3F.sup.C.pi.) Ac-DKWKAVYDKFAEAFKEFL-NH.sub.2 107
(3F.sup.I.pi.) Ac-DKLKAFYDKVFEWAKEAF-NH.sub.2 108
[0179] C) Smaller Peptides.
[0180] It was also a surprising discovery that certain small
peptides consisting of a minimum of three amino acids
preferentially (but not necessarily) with one or more of the amino
acids being the D-sterioisomer of the amino acid, and possessing
hydrophobic domains to permit lipid protein interactions, and
hydrophilic domains to permit a degree of water solubility also
possess significant anti-inflammatory properties. Without being
bound to a particular theory, it is believed that the peptides bind
the "seeding molecules" required for the formation of
pro-inflammatory oxidized phospholipids such as Ox-PAPC, POVPC,
PGPC, and PEIPC. Since many inflammatory conditions are mediated at
least in part by oxidized lipids, we believe that the peptides of
this invention are effective in ameliorating conditions that are
known or suspected to be due to the formation of biologically
active oxidized lipids. These include, but are not limited to
atherosclerosis, rheumatoid arthritis, lupus erythematous,
polyarteritis nodosa, pulmonary disease, asthma, multiple
sclerosis, Alzheime's disease, diabetes, and osteoporosis. The
"small peptides" typically range in length from 3 amino acids to
about 15 amino acids, more preferably from about 4 amino acids to
about 10 or 11 amino acids, and most preferably from about 4 to
about 8 or 10 amino acids. The peptides are typically characterized
by having hydrophobic terminal amino acids or terminal amino acids
rendered hydrophobic by the attachment of one or more hydrophobic
"protecting" groups.
[0181] In certain embodiments, the peptides can be characterized by
Formula I, below:
3 X.sup.1-X.sup.2-X.sup.3.sub.n-X.sup.4 I
[0182] where, n is 0 or 1, X.sup.1 is a hydrophobic amino acid
and/or bears a hydrophobic protecting group, X.sup.4 is a
hydrophobic amino acid and/or bears a hydrophobic protecting group;
and when n is 0 X.sup.2 is an acidic or a basic amino acid; when n
is 1: X.sup.2 and X.sup.3 are independently an acidic amino acid, a
basic amino acid, an aliphatic amino acid, or an aromatic amino
acid such that when X.sup.2 is an acidic amino acid; X.sup.3 is a
basic amino acid, an aliphatic amino acid, or an aromatic amino
acid; when X.sup.2 is a basic amino acid; X.sup.3 is an acidic
amino acid, an aliphatic amino acid, or an aromatic amino acid; and
when X.sup.2 is an aliphatic or aromatic amino acid, X.sup.3 is an
acidic amino acid, or a basic amino acid.
[0183] Longer peptides (e.g., up to 10, 11, or 15 amino acids)are
also contemplated within the scope of this invention. Typcially
where the shorter peptides (e.g., peptides according to formula I)
are characterized by an acidic, basic, aliphatic, or aromatic amino
acid, the longer peptides are characterized by acidic, basic,
aliphatic, or aromatic domains comprising two or more amino acids
of that type.
[0184] 1) Functional Properties of Active Small Peptides.
[0185] It was a surprising finding of this invention that a number
of physical properties predict the ability of small peptides (e.g.,
less than 10 amino acids, preferably less than 8 amino acids, more
preferably from about 3 to about 5 or 6 amino acids) of this
invention to render HDL more anti-inflammatory and to mitigate
atherosclerosis and/or other pathologies characterized by an
inflammatory response in a mammal. The physical properties include
high solubility in ethyl acetate (e.g., greater than about 4
mg/mL), and solubility in aqueous buffer at pH 7.0. Upon contacting
phospholipids such as 1,2-Dimyristoyl-sn-glycero-3-phosph- ocholine
(DMPC), in an aqueous environment, the particularly effective small
peptides induce or participate in the formation of particles with a
diameter of approximately 7.5 nm (.+-.0.1 nm), and/or induce or
participate in the formation of stacked bilayers with a bilayer
dimension on the order of 3.4 to 4.1 nm with spacing between the
bilayers in the stack of approximately 2 nm, and/or also induce or
participate in the formation of vesicular structures of
approximately 38 nm). In certain preferred embodiments, the small
peptides have a molecular weight of less than about 900 Da.
[0186] Thus, in certain embodimements, this invention contemplates
small peptides that ameliorate one or more symptoms of an
inflammatory condition, where said peptide(s): ranges in length
from about 3 to about 8 amino acids, preferably from about 3 to
about 6, or 7 amino acids, and more preferably from about 3 to
about 5 amino acids; are soluble in ethyl acetate at a
concentration greater than about 4 mg/mL; are soluble in aqueous
buffer at pH 7.0; when contacted with a phospholipid in an aqueous
environment, form particles with a diameter of approximately 7.5 nm
and/or form stacked bilayers with a bilayer dimension on the order
of 3.4 to 4.1 nm with spacing between the bilayers in the stack of
approximately 2 nm; have a molecular weight less than about 900
daltons; convert pro-inflammatory HDL to anti-inflammatory HDL or
make anti-inflammatory HDL more anti-inflammatory; and do not have
the amino acid sequence Lys-Arg-Asp-Ser (SEQ ID NO:238) in which
Lys-Arg-Asp and Ser are all L amino acids. In certain embodiments,
these small peptides protect a phospholipid against oxidation by an
oxidizing agent.
[0187] While these small peptides need not be so limited, in
certain embodiments, these small peptides can include the small
peptides described below.
[0188] 2) Tripeptides.
[0189] It was discovered that certain tripeptides (3 amino acid
peptides) can be synthesized that show desirable properties as
described herein (e.g., the ability to convert pro-inflammatory HDL
to anti-inflammatory HDL, the ability to decrease LDL-induced
monocyte chemotactic activity generated by artery wall cells, the
ability to increase pre-beta HDL, etc.). In certain embodiments,
the peptides are characterized by formula I, wherein N is zero,
shown below as Formula II:
4 X.sup.1-X.sup.2-X.sup.4 II
[0190] where the end amino acids (X.sup.1 and X.sup.4) are
hydrophobic either because of a hydrophobic side chain or because
the side chain or the C and/or N terminus is blocked with one or
more hydrophobic protecting group(s) (e.g., the N-terminus is
blocked with Boc-, Fmoc-, Nicotinyl-, etc., and the C-terminus
blocked with (tBu)-OtBu, etc.). In certain embodiments, the X.sup.2
amino acid is either acidic (e.g., aspartic acid, glutamic acid,
etc.) or basic (e.g., histidine, arginine, lysine, etc.). The
peptide can be all L-amino acids or include one or more or all
D-amino acids.
[0191] Certain preferred tripeptides of this invention include, but
are not limited to the peptides shown in Table 3.
5TABLE 3 Examples of certain preferred tripeptides bearing
hydrophobic blocking groups and acidic, basic, or histidine central
amino acids. SEQ ID X.sup.1 X.sup.2 X.sup.3 X.sup.4 NO
Boc-Lys(.epsilon.Boc) Arg Ser(tBu)-OtBu 109 Boc-Lys(.epsilon.Boc)
Arg Thr(tBu)-OtBu 110 Boc-Trp Arg Ile-OtBu 111 Boc-Trp Arg Leu-OtBu
112 Boc-Phe Arg Ile-OtBu 113 Boc-Phe Arg Leu-OtBu 114
Boc-Lys(.epsilon.Boc) Glu Ser(tBu)-OtBu 115 Boc-Lys(.epsilon.Boc)
Glu Thr(tBu)-OtBu 116 Boc-Lys(.epsilon.Boc) Asp Ser(tBu)-OtBu 117
Boc-Lys(.epsilon.Boc) Asp Thr(tBu)-OtBu 118 Boc-Lys(.epsilon.Boc)
Arg Ser(tBu)-OtBu 119 Boc-Lys(.epsilon.Boc) Arg Thr(tBu)-OtBu 120
Boc-Leu Glu Ser(tBu)-OtBu 121 Boc-Leu Glu Thr(tBu)-OtBu 122
Fmoc-Trp Arg Ser(tBu)-OtBu 123 Fmoc-Trp Asp Ser(tBu)-OtBu 124
Fmoc-Trp Glu Ser(tBu)-OtBu 125 Fmoc-Trp Arg Ser(tBu)-OtBu 126
Boc-Lys(.epsilon.Boc) Glu Leu-OtBu 127 Fmoc-Leu Arg Ser(tBu)-OtBu
128 Fmoc-Leu Asp Ser(tBu)-OtBu 129 Fmoc-Leu Glu Ser(tBu)-OtBu 130
Fmoc-Leu Arg Ser(tBu)-OtBu 131 Fmoc-Leu Arg Thr(tBu)-OtBu 132
Boc-Glu Asp Tyr(tBu)-OtBu 133 Fmoc-Lys(.epsilon.Fmoc) Arg
Ser(tBu)-OtBu 134 Fmoc-Trp Arg Ile-OtBu 135 Fmoc-Trp Arg Leu-OtBu
136 Fmoc-Phe Arg Ile-OtBu 137 Fmoc-Phe Arg Leu-OtBu 138 Boc-Trp Arg
Phe-OtBu 139 Boc-Trp Arg Tyr-OtBu 140 Fmoc-Trp Arg Phe-OtBu 141
Fmoc-Trp Arg Tyr-OtBu 142 Boc-Orn(.delta.Boc) Arg Ser(tBu)-OtBu 143
Nicotinyl Lys(.epsilon.Boc) Arg Ser(tBu)-OtBu 144 Nicotinyl
Lys(.epsilon.Boc) Arg Thr(tBu)-OtBu 145 Fmoc-Leu Asp Thr(tBu)-OtBu
146 Fmoc-Leu Glu Thr(tBu)-OtBu 147 Fmoc-Leu Arg Thr(tBu)-OtBu 148
Fmoc-norLeu Arg Ser(tBu)-OtBu 149 Fmoc-norLeu Asp Ser(tBu)-OtBu 150
Fmoc-norLeu Glu Ser(tBu)-OtBu 151 Fmoc-Lys(.epsilon.Boc) Arg
Ser(tBu)-OtBu 152 Fmoc-Lys(.epsilon.Boc) Arg Thr(tBu)-OtBu 153
Fmoc-Lys(.epsilon.Boc) Glu Ser(tBu)-OtBu 154 Fmoc-Lys(.epsilon.Boc)
Glu Thr(tBu)-OtBu 155 Fmoc-Lys(.epsilon.Boc) Asp Ser(tBu)-OtBu 156
Fmoc-Lys(.epsilon.Boc) Asp Thr(tBu)-OtBu 157 Fmoc-Lys(.epsilon.Boc)
Glu Leu-OtBu 158 Fmoc-Lys(.epsilon.Boc) Arg Leu-OtBu 159
Fmoc-Lys(.epsilon.Fmoc) Arg Thr(tBu)-OtBu 160
Fmoc-Lys(.epsilon.Fmoc) Glu Ser(tBu)-OtBu 161
Fmoc-Lys(.epsilon.Fmoc) Glu Thr(tBu)-OtBu 162
Fmoc-Lys(.epsilon.Fmoc) Asp Ser(tBu)-OtBu 163
Fmoc-Lys(.epsilon.Fmoc) Asp Thr(tBu)-OtBu 164
Fmoc-Lys(.epsilon.Fmoc) Arg Ser(tBu)-OtBu 165
Fmoc-Lys(.epsilon.Fmoc)) Glu Leu-OtBu 166 Boc-Lys(.epsilon.Fmoc)
Asp Ser(tBu)-OtBu 167 Boc-Lys(.epsilon.Fmoc) Asp Thr(tBu)-OtBu 168
Boc-Lys(.epsilon.Fmoc) Arg Thr(tBu)-OtBu 169 Boc-Lys(.epsilon.Fmoc)
Glu Leu-OtBu 170 Boc-Orn(.delta.Fmoc) Glu Ser(tBu)-OtBu 171
Boc-Orn(.delta.Fmoc) Asp Ser(tBu)-OtBu 172 Boc-Orn(.delta.Fmoc) Asp
Thr(tBu)-OtBu 173 Boc-Orn(.delta.Fmoc) Arg Thr(tBu)-OtBu 174
Boc-Orn(.delta.Fmoc) Glu Thr(tBu)-OtBu 175 Fmoc-Trp Asp Ile-OtBu
176 Fmoc-Trp Arg Ile-OtBu 177 Fmoc-Trp Glu Ile-OtBu 178 Fmoc-Trp
Asp Leu-OtBu 179 Fmoc-Trp Glu Leu-OtBu 180 Fmoc-Phe Asp Ile-OtBu
181 Fmoc-Phe Asp Leu-OtBu 182 Fmoc-Phe Glu Leu-OtBu 183 Fmoc-Trp
Arg Phe-OtBu 184 Fmoc-Trp Glu Phe-OtBu 185 Fmoc-Trp Asp Phe-OtBu
186 Fmoc-Trp Asp Tyr-OtBu 187 Fmoc-Trp Arg Tyr-OtBu 188 Fmoc-Trp
Glu Tyr-OtBu 189 Fmoc-Trp Arg Thr(tBu)-OtBu 190 Fmoc-Trp Asp
Thr(tBu)-OtBu 191 Fmoc-Trp Glu Thr(tBu)-OtBu 192 Boc-Phe Arg
norLeu-OtBu 193 Boc-Phe Glu norLeu-OtBu 194 Fmoc-Phe Asp
norLeu-OtBu 195 Boc-Glu His Tyr(tBu)-OtBu 196 Boc-Leu His
Ser(tBu)-OtBu 197 Boc-Leu His Thr(tBu)-OtBu 198
Boc-Lys(.epsilon.Boc) His Ser(tBu)-OtBu 199 Boc-Lys(.epsilon.Boc)
His Thr(tBu)-OtBu 200 Boc-Lys(.epsilon.Boc) His Leu-OtBu 201
Boc-Lys(.epsilon.Fmoc) His Ser(tBu)-OtBu 202 Boc-Lys(.epsilon.Fmoc)
His Thr(tBu)-OtBu 203 Boc-Lys(.epsilon.Fmoc) His Leu-OtBu 204
Boc-Orn(.delta.Boc) His Ser(tBu)-OtBu 205 Boc-Orn(.delta.Fmoc) His
Thr(tBu)-OtBu 206 Boc-Phe His Ile-OtBu 207 Boc-Phe His Leu-OtBu 208
Boc-Phe His norLeu-OtBu 209 Boc-Phe Lys Leu-OtBu 210 Boc-Trp His
Ile-OtBu 211 Boc-Trp His Leu-OtBu 212 Boc-Trp His Phe-OtBu 213
Boc-Trp His Tyr-OtBu 214 Boc-Phe Lys Leu-OtBu 215
Fmoc-Lys(.epsilon.Fmoc) His Ser(tBu)-OtBu 216
Fmoc-Lys(.epsilon.Fmoc) His Thr(tBu)-OtBu 217
Fmoc-Lys(.epsilon.Fmoc)) His Leu-OtBu 218 Fmoc-Leu His
Ser(tBu)-OtBu 219 Fmoc-Leu His Thr(tBu)-OtBu 220
Fmoc-Lys(.epsilon.Boc) His Ser(tBu)-OtBu 221 Fmoc-Lys(.epsilon.Boc)
His Thr(tBu)-OtBu 222 Fmoc-Lys(.epsilon.Boc) His Leu-OtBu 223
Fmoc-Lys(.epsilon.Fmoc) His Ser(tBu)-OtBu 224
Fmoc-Lys(.epsilon.Fmoc) His Thr(tBu)-OtBu 225 Fmoc-norLeu His
Ser(tBu)-OtBu 226 Fmoc-Phe His Ile-OtBu 227 Fmoc-Phe His Leu-OtBu
228 Fmoc-Phe His norLeu-OtBu 229 Fmoc-Trp His Ser(tBu)-OtBu 230
Fmoc-Trp His Ile-OtBu 231 Fmoc-Trp His Leu-OtBu 232 Fmoc-Trp His
Phe-OtBu 233 Fmoc-Trp His Tyr-OtBu 234 Fmoc-Trp His Thr(tBu)-OtBu
235 Nicotinyl Lys(.epsilon.Boc) His Ser(tBu)-OtBu 236 Nicotinyl
Lys(.epsilon.Boc) His Thr(tBu)-OtBu 237
[0192] While the pepides of Table 3 are illustrated with particular
protecting groups, it is noted that these groups may be substituted
with other protecting groups as described herein and/or one or more
of the shown protecting group can be eliminated.
[0193] 3) Small Peptides with Central Acidic and Basic Amino
Acids.
[0194] In certain embodiments, the peptides of this invention range
from four amino acids to about ten amino acids. The terminal amino
acids are typically hydrophobic either because of a hydrophobic
side chain or because the terminal amino acids bear one or more
hydrophobic protecting groups end amino acids (X.sup.1 and X.sup.4)
are hydrophobic either because of a hydrophobic side chain or
because the side chain or the C and/or N terminus is blocked with
one or more hydrophobic protecting group(s) (e.g., the N-terminus
is blocked with Boc-, Fmoc-, Nicotinyl-, etc., and the C-terminus
blocked with (tBu)-OtBu, etc.). Typically, the central portion of
the peptide comprises a basic amino acid and an acidic amino acid
(e.g., in a 4 mer) or a basic domain and/or an acidic domain in a
longer molecule.
[0195] These four-mers can be represented by Formula I in which
X.sup.1 and X.sup.4 are hydrophobic and/or bear hydrophobic
protecting group(s) as described herein and X.sup.2 is acidic while
X.sup.3 is basic or X.sup.2 is basic while X.sup.3 is acidic. The
peptide can be all L-amino acids or include one or more or all
D-amino acids.
[0196] Certain preferred of this invention include, but are not
limited to the peptides shown in Table 4.
6TABLE 4 Illustrative examples of small peptides with central
acidic and basic amino acids. SEQ ID X.sup.1 X.sup.2 X.sup.3
X.sup.4 NO Boc-Lys(.epsilon.Boc) Arg Asp Ser(tBu)-OtBu 238
Boc-Lys(.epsilon.Boc) Arg Asp Thr(tBu)-OtBu 239 Boc-Trp Arg Asp
Ile-OtBu 240 Boc-Trp Arg Asp Leu-OtBu 241 Boc-Phe Arg Asp Leu-OtBu
242 Boc-Phe Arg Asp Ile-OtBu 243 Boc-Phe Arg Asp norLeu-OtBu 244
Boc-Phe Arg Glu norLeu-OtBu 245 Boc-Phe Arg Glu Ile-OtBu 246
Boc-Phe Asp Arg Ile-OtBu 247 Boc-Phe Glu Arg Ile-OtBu 248 Boc-Phe
Asp Arg Leu-OtBu 249 Boc-Phe Arg Glu Leu-OtBu 250 Boc-Phe Glu Arg
Leu-OtBu 251 Boc-Phe Asp Arg norLeu-OtBu 252 Boc-Phe Glu Arg
norLeu-OtBu 253 Boc-Lys(.epsilon.Boc) Glu Arg Ser(tBu)-OtBu 254
Boc-Lys(.epsilon.Boc) Glu Arg Thr(tBu)-OtBu 255
Boc-Lys(.epsilon.Boc) Asp Arg Ser(tBu)-OtBu 256
Boc-Lys(.epsilon.Boc) Asp Arg Thr(tBu)-OtBu 257
Boc-Lys(.epsilon.Boc) Arg Glu Ser(tBu)-OtBu 258
Boc-Lys(.epsilon.Boc) Arg Glu Thr(tBu)-OtBu 259 Boc-Leu Glu Arg
Ser(tBu)-OtBu 260 Boc-Leu Glu Arg Thr(tBu)-OtBu 261 Fmoc-Trp Arg
Asp Ser(tBu)-OtBu 262 Fmoc-Trp Asp Arg Ser(tBu)-OtBu 263 Fmoc-Trp
Glu Arg Ser(tBu)-OtBu 264 Fmoc-Trp Arg Glu Ser(tBu)-OtBu 265
Boc-Lys(.epsilon.Boc) Glu Arg Leu-OtBu 266 Fmoc-Leu Arg Asp
Ser(tBu)-OtBu 267 Fmoc-Leu Asp Arg Ser(tBu)-OtBu 268 Fmoc-Leu Glu
Arg Ser(tBu)-OtBu 269 Fmoc-Leu Arg Glu Ser(tBu)-OtBu 270 Fmoc-Leu
Arg Asp Thr(tBu)-OtBu 271 Boc-Glu Asp Arg Tyr(tBu)-OtBu 272
Fmoc-Lys(.epsilon.Fmoc) Arg Asp Ser(tBu)-OtBu 273 Fmoc-Trp Arg Asp
Ile-OtBu 274 Fmoc-Trp Arg Asp Leu-OtBu 275 Fmoc-Phe Arg Asp
Ile-OtBu 276 Fmoc-Phe Arg Asp Leu-OtBu 277 Boc-Trp Arg Asp Phe-OtBu
278 Boc-Trp Arg Asp Tyr-OtBu 279 Fmoc-Trp Arg Asp Phe-OtBu 280
Fmoc-Trp Arg Asp Tyr-OtBu 281 Boc-Orn(.delta.Boc) Arg Glu
Ser(tBu)-OtBu 282 Nicotinyl Lys(.epsilon.Boc) Arg Asp Ser(tBu)-OtBu
283 Nicotinyl Lys(.epsilon.Boc) Arg Asp Thr(tBu)-OtBu 284 Fmoc-Leu
Asp Arg Thr(tBu)-OtBu 285 Fmoc-Leu Glu Arg Thr(tBu)-OtBu 286
Fmoc-Leu Arg Glu Thr(tBu)-OtBu 287 Fmoc-norLeu Arg Asp
Ser(tBu)-OtBu 288 Fmoc-norLeu Asp Arg Ser(tBu)-OtBu 289 Fmoc-norLeu
Glu Arg Ser(tBu)-OtBu 290 Fmoc-norLeu Arg Glu Ser(tBu)-OtBu 291
Fmoc-Lys(.epsilon.Boc) Arg Asp Ser(tBu)-OtBu 292
Fmoc-Lys(.epsilon.Boc) Arg Asp Thr(tBu)-OtBu 293
Fmoc-Lys(.epsilon.Boc) Glu Arg Ser(tBu)-OtBu 294
Fmoc-Lys(.epsilon.Boc) Glu Arg Thr(tBu)-OtBu 295
Fmoc-Lys(.epsilon.Boc) Asp Arg Ser(tBu)-OtBu 296
Fmoc-Lys(.epsilon.Boc) Asp Arg Thr(tBu)-OtBu 297
Fmoc-Lys(.epsilon.Boc) Arg Glu Ser(tBu)-OtBu 298
Fmoc-Lys(.epsilon.Boc) Arg Glu Thr(tBu)-OtBu 299
Fmoc-Lys(.epsilon.Boc) Glu Arg Leu-OtBu 300 Fmoc-Lys(.epsilon.Boc)
Arg Glu Leu-OtBu 301 Fmoc-Lys(.epsilon.Fmoc) Arg Asp Thr(tBu)-OtBu
302 Fmoc-Lys(.epsilon.Fmoc) Glu Arg Ser(tBu)-OtBu 303
Fmoc-Lys(.epsilon.Fmoc) Glu Arg Thr(tBu)-OtBu 304
Fmoc-Lys(.epsilon.Fmoc) Asp Arg Ser(tBu)-OtBu 305
Fmoc-Lys(.epsilon.Fmoc) Asp Arg Thr(tBu)-OtBu 306
Fmoc-Lys(.epsilon.Fmoc) Arg Glu Ser(tBu)-OtBu 307
Fmoc-Lys(.epsilon.Fmoc) Arg Glu Thr(tBu)-OtBu 308
Fmoc-Lys(.epsilon.Fmoc)) Glu Arg Leu-OtBu 309
Boc-Lys(.epsilon.Fmoc) Arg Asp Ser(tBu)-OtBu 310
Boc-Lys(.epsilon.Fmoc) Arg Asp Thr(tBu)-OtBu 311
Boc-Lys(.epsilon.Fmoc) Glu Arg Ser(tBu)-OtBu 312
Boc-Lys(.epsilon.Fmoc) Glu Arg Thr(tBu)-OtBu 313
Boc-Lys(.epsilon.Fmoc) Asp Arg Ser(tBu)-OtBu 314
Boc-Lys(.epsilon.Fmoc) Asp Arg Thr(tBu)-OtBu 315
Boc-Lys(.epsilon.Fmoc) Arg Glu Ser(tBu)-OtBu 316
Boc-Lys(.epsilon.Fmoc) Arg Glu Thr(tBu)-OtBu 317
Boc-Lys(.epsilon.Fmoc) Glu Arg Leu-OtBu 318 Boc-Orn(.delta.Fmoc)
Arg Glu Ser(tBu)-OtBu 319 Boc-Orn(.delta.Fmoc) Glu Arg
Ser(tBu)-OtBu 320 Boc-Orn(.delta.Fmoc) Arg Asp Ser(tBu)-OtBu 321
Boc-Orn(.delta.Fmoc) Asp Arg Ser(tBu)-OtBu 322 Boc-Orn(.delta.Fmoc)
Asp Arg Thr(tBu)-OtBu 323 Boc-Orn(.delta.Fmoc) Arg Asp
Thr(tBu)-OtBu 324 Boc-Orn(.delta.Fmoc) Glu Arg Thr(tBu)-OtBu 325
Boc-Orn(.delta.Fmoc) Arg Glu Thr(tBu)-OtBu 326 Fmoc-Trp Asp Arg
Ile-OtBu 327 Fmoc-Trp Arg Glu Ile-OtBu 328 Fmoc-Trp Glu Arg
Ile-OtBu 329 Fmoc-Trp Asp Arg Leu-OtBu 330 Fmoc-Trp Arg Glu
Leu-OtBu 331 Fmoc-Trp Glu Arg Leu-OtBu 332 Fmoc-Phe Asp Arg
Ile-OtBu 333 Fmoc-Phe Arg Glu Ile-OtBu 334 Fmoc-Phe Glu Arg
Ile-OtBu 335 Fmoc-Phe Asp Arg Leu-OtBu 336 Fmoc-Phe Arg Glu
Leu-OtBu 337 Fmoc-Phe Glu Arg Leu-OtBu 338 Fmoc-Trp Arg Asp
Phe-OtBu 339 Fmoc-Trp Arg Glu Phe-OtBu 340 Fmoc-Trp Glu Arg
Phe-OtBu 341 Fmoc-Trp Asp Arg Tyr-OtBu 342 Fmoc-Trp Arg Glu
Tyr-OtBu 343 Fmoc-Trp Glu Arg Tyr-OtBu 344 Fmoc-Trp Arg Asp
Thr(tBu)-OtBu 345 Fmoc-Trp Asp Arg Thr(tBu)-OtBu 346 Fmoc-Trp Arg
Glu Thr(tBu)-OtBu 347 Fmoc-Trp Glu Arg Thr(tBu)-OtBu 348 Fmoc-Phe
Arg Asp norLeu-OtBu 349 Fmoc-Phe Arg Glu norLeu-OtBu 350 Boc-Phe
Lys Asp Leu-OtBu 351 Boc-Phe Asp Lys Leu-OtBu 352 Boc-Phe Lys Glu
Leu-OtBu 353 Boc-Phe Glu Lys Leu-OtBu 354 Boc-Phe Lys Asp Ile-OtBu
355 Boc-Phe Asp Lys Ile-OtBu 356 Boc-Phe Lys Glu Ile-OtBu 357
Boc-Phe Glu Lys Ile-OtBu 358 Boc-Phe Lys Asp norLeu-OtBu 359
Boc-Phe Asp Lys norLeu-OtBu 360 Boc-Phe Lys Glu norLeu-OtBu 361
Boc-Phe Glu Lys norLeu-OtBu 362 Boc-Phe His Asp Leu-OtBu 363
Boc-Phe Asp His Leu-OtBu 364 Boc-Phe His Glu Leu-OtBu 365 Boc-Phe
Glu His Leu-OtBu 366 Boc-Phe His Asp Ile-OtBu 367 Boc-Phe Asp His
Ile-OtBu 368 Boc-Phe His Glu Ile-OtBu 369 Boc-Phe Glu His Ile-OtBu
370 Boc-Phe His Asp norLeu-OtBu 371 Boc-Phe Asp His norLeu-OtBu 372
Boc-Phe His Glu norLeu-OtBu 373 Boc-Phe Glu His norLeu-OtBu 374
Boc-Lys(.epsilon.Boc) Lys Asp Ser(tBu)-OtBu 375
Boc-Lys(.epsilon.Boc) Asp Lys Ser(tBu)-OtBu 376
Boc-Lys(.epsilon.Boc) Lys Glu Ser(tBu)-OtBu 377
Boc-Lys(.epsilon.Boc) Glu Lys Ser(tBu)-OtBu 378
Boc-Lys(.epsilon.Boc) His Asp Ser(tBu)-OtBu 379
Boc-Lys(.epsilon.Boc) Asp His Ser(tBu)-OtBu 380
Boc-Lys(.epsilon.Boc) His Glu Ser(tBu)-OtBu 381
Boc-Lys(.epsilon.Boc) Glu His Ser(tBu)-OtBu 382
[0197] While the pepides of Table 4 are illustrated with particular
protecting groups, it is noted that these groups may be substituted
with other protecting groups as described herein and/or one or more
of the shown protecting group can be eliminated.
[0198] 4) Small Peptides Having Either an Acidic or Basic Amino
Acid in the Center Together with a Central Aliphatic Amino
Acid.
[0199] In certain embodiments, the peptides of this invention range
from four amino acids to about ten amino acids. The terminal amino
acids are typically hydrophobic either because of a hydrophobic
side chain or because the terminal amino acids bear one or more
hydrophobic protecting groups. End amino acids (X.sup.1 and
X.sup.4) are hydrophobic either because of a hydrophobic side chain
or because the side chain or the C and/or N terminus is blocked
with one or more hydrophobic protecting group(s) (e.g., the
N-terminus is blocked with Boc-, Fmoc-, Nicotinyl-, etc., and the
C-terminus blocked with (tBu)-OtBu, etc.). Typically, the central
portion of the peptide comprises a basic or acidic amino acid and
an aliphatic amino acid (e.g., in a 4 mer) or a basic domain or an
acidic domain and an aliphatic domain in a longer molecule.
[0200] These four-mers can be represented by Formula I in which
X.sup.1 and X.sup.4 are hydrophobic and/or bear hydrophobic
protecting group(s) as described herein and X.sup.2 is acidic or
basic while X.sup.3 is aliphatic or X.sup.2 is aliphatic while
X.sup.3 is acidic or basic. The peptide can be all L-amino acids or
include one, or more, or all D-amino acids.
[0201] Certain preferred of this invention include, but are not
limited to the peptides shown in Table 5.
7TABLE 5 Examples of certain preferred peptides having either an
acidic or basic amino acid in the center together with a central
aliphatic amino acid. SEQ ID X.sup.1 X.sup.2 X.sup.3 X.sup.4 NO
Fmoc-Lys(.epsilon.Boc) Leu Arg Ser(tBu)-OtBu 383
Fmoc-Lys(.epsilon.Boc) Arg Leu Ser(tBu)-OtBu 384
Fmoc-Lys(.epsilon.Boc) Leu Arg Thr(tBu)-OtBu 385
Fmoc-Lys(.epsilon.Boc) Arg Leu Thr(tBu)-OtBu 386
Fmoc-Lys(.epsilon.Boc) Glu Leu Ser(tBu)-OtBu 387
Fmoc-Lys(.epsilon.Boc) Leu Glu Ser(tBu)-OtBu 388
Fmoc-Lys(.epsilon.Boc) Glu Leu Thr(tBu)-OtBu 389
Fmoc-Lys(.epsilon.Boc) Leu Glu Thr(tBu)-OtBu 390
Fmoc-Lys(.epsilon.Fmoc) Leu Arg Ser(tBu)-OtBu 391
Fmoc-Lys(.epsilon.Fmoc) Leu Arg Thr(tBu)-OtBu 392
Fmoc-Lys(.epsilon.Fmoc) Glu Leu Ser(tBu)-OtBu 393
Fmoc-Lys(.epsilon.Fmoc) Glu Leu Thr(tBu)-OtBu 394 Boc-Lys(Fmoc) Glu
Ile Thr(tBu)-OtBu 395 Boc-Lys(.epsilon.Fmoc) Leu Arg Ser(tBu)-OtBu
396 Boc-Lys(.epsilon.Fmoc) Leu Arg Thr(tBu)-OtBu 397
Boc-Lys(.epsilon.Fmoc) Glu Leu Ser(tBu)-OtBu 398
Boc-Lys(.epsilon.Fmoc) Glu Leu Thr(tBu)-OtBu 399
Boc-Lys(.epsilon.Boc) Leu Arg Ser(tBu)-OtBu 400
Boc-Lys(.epsilon.Boc) Arg Phe Thr(tBu)-OtBu 401
Boc-Lys(.epsilon.Boc) Leu Arg Thr(tBu)-OtBu 402
Boc-Lys(.epsilon.Boc) Glu Ile Thr(tBu) 403 Boc-Lys(.epsilon.Boc)
Glu Val Thr(tBu) 404 Boc-Lys(.epsilon.Boc) Glu Ala Thr(tBu) 405
Boc-Lys(.epsilon.Boc) Glu Gly Thr(tBu) 406 Boc--Lys(.epsilon.Boc)
Glu Leu Ser(tBu)-OtBu 407 Boc-Lys(.epsilon.Boc) Glu Leu
Thr(tBu)-OtBu 408
[0202] While the pepides of Table 5 are illustrated with particular
protecting groups, it is noted that these groups may be substituted
with other protecting groups as described herein and/or one or more
of the shown protecting group can be eliminated.
[0203] 5) Small Peptides Having Either an Acidic or Basic Amino
Acid in the Center Together with a Central Aromatic Amino Acid.
[0204] In certain embodiments, the peptides of this invention range
from four amino acids to about ten amino acids. The terminal amino
acids are typically hydrophobic either because of a hydrophobic
side chain or because the terminal amino acids bear one or more
hydrophobic protecting groups end amino acids (X.sup.1 and X.sup.4)
are hydrophobic either because of a hydrophobic side chain or
because the side chain or the C and/or N terminus is blocked with
one or more hydrophobic protecting group(s) (e.g., the N-terminus
is blocked with Boc-, Fmoc-, Nicotinyl-, etc., and the C-terminus
blocked with (tBu)-OtBu, etc.). Typically, the central portion of
the peptide comprises a basic or acidic amino acid and an aromatic
amino acid (e.g., in a 4 mer) or a basic domain or an acidic domain
and an aromatic domain in a longer molecule.
[0205] These four-mers can be represented by Formula I in which
X.sup.1 and X.sup.4 are hydrophobic and/or bear hydrophobic
protecting group(s) as described herein and X.sup.2 is acidic or
basic while X.sup.3 is aromatic or X.sup.2 is aromatic while
X.sup.3 is acidic or basic. The peptide can be all L-amino acids or
include one, or more, or all D-amino acids. Five-mers can be
represented by a minor modification of Formula I in which X.sup.5
is inserted as shown in Table 6 and in which X.sup.5 is typically
an aromatic amino acid.
[0206] Certain preferred of this invention include, but are not
limited to the peptides shown in Table 6.
8TABLE 6 Examples of certain preferred peptides having either an
acidic or basic amino acid in the center together with a central
aromatic amino acid. SEQ ID X.sup.1 X.sup.2 X.sup.3 X.sup.5 X.sup.4
NO Fmoc-Lys(.epsilon.Boc) Arg Trp Tyr(tBu)-OtBu 409
Fmoc-Lys(.epsilon.Boc) Trp Arg Tyr(tBu)-OtBu 410
Fmoc-Lys(.epsilon.Boc) Arg Tyr Trp-OtBu 411 Fmoc-Lys(.epsilon.Boc)
Tyr Arg Trp-OtBu 412 Fmoc-Lys(.epsilon.Boc) Arg Tyr Trp
Thr(tBu)-OtBu 413 Fmoc-Lys(.epsilon.Boc) Arg Tyr Thr(tBu)-OtBu 414
Fmoc-Lys(.epsilon.Boc) Arg Trp Thr(tBu)-OtBu 415
Fmoc-Lys(.epsilon.Fmoc) Arg Trp Tyr(tBu)-OtBu 416
Fmoc-Lys(.epsilon.Fmoc) Arg Tyr Trp-OtBu 417
Fmoc-Lys(.epsilon.Fmoc) Arg Tyr Trp Thr(tBu)-OtBu 418
Fmoc-Lys(.epsilon.Fmoc) Arg Tyr Thr(tBu)-OtBu 419
Fmoc-Lys(.epsilon.Fmoc) Arg Trp Thr(tBu)-OtBu 420
Boc-Lys(.epsilon.Fmoc) Arg Trp Tyr(tBu)-OtBu 421
Boc-Lys(.epsilon.Fmoc) Arg Tyr Trp-OtBu 422 Boc-Lys(.epsilon.Fmoc)
Arg Tyr Trp Thr(tBu)-OtBu 423 Boc-Lys(.epsilon.Fmoc) Arg Tyr
Thr(tBu)-OtBu 424 Boc-Lys(.epsilon.Fmoc) Arg Trp Thr(tBu)-OtBu 425
Boc-Glu Lys(.epsilon.Fmoc) Arg Tyr(tBu)-OtBu 426
Boc-Lys(.epsilon.Boc) Arg Trp Tyr(tBu)-OtBu 427
Boc-Lys(.epsilon.Boc) Arg Tyr Trp-OtBu 428 Boc-Lys(.epsilon.Boc)
Arg Tyr Trp Thr(tBu)-OtBu 429 Boc-Lys(.epsilon.Boc) Arg Tyr
Thr(tBu)-OtBu 430 Boc-Lys(.epsilon.Boc) Arg Phe Thr(tBu)-OtBu 431
Boc-Lys(.epsilon.Boc) Arg Trp Thr(tBu)-OtBu 432
[0207] While the pepides of Table 6 are illustrated with particular
protecting groups, it is noted that these groups may be substituted
with other protecting groups as described herein and/or one or more
of the shown protecting group can be eliminated.
[0208] 6) Small Peptides Having Aromatic Amino Acids or Aromatic
Amino Acids Separated by Histidine(s) at the Center.
[0209] In certain embodiments, the peptides of this invention are
characterized by .pi. electrons that are exposed in the center of
the molecule which allow hydration of the particle and that allow
the peptide particles to trap pro-inflammatory oxidized lipids such
as fatty acid hydroperoxides and phospholipids that contain an
oxidation product of arachidonic acid at the sn-2 position.
[0210] In certain embodiments, these peptides consist of a minimum
of 4 amino acids and a maximum of about 10 amino acids,
preferentially (but not necessarily) with one or more of the amino
acids being the D-sterioisomer of the amino acid, with the end
amino acids being hydrophobic either because of a hydrophobic side
chain or because the terminal amino acid(s) bear one or more
hydrophobic blocking group(s), (e.g., an N-terminus blocked with
Boc-, Fmoc-, Nicotinyl-, and the like, and a C-terminus blocked
with (tBu)-OtBu groups and the like). Instead of having an acidic
or basic amino acid in the center, these peptides generally have an
aromatic amino acid at the center or have aromatic amino acids
separated by histidine in the center of the peptide.
[0211] Certain preferred of this invention include, but are not
limited to the peptides shown in Table 7.
9TABLE 7 Examples of peptides having aromatic amino acids in the
center or aromatic amino acids or aromatic domains separated by one
or more histidines. SEQ ID X.sup.1 X.sup.2 X.sup.3 X.sup.4 X.sup.5
NO Boc-Lys(.epsilon.Boc) Phe Trp Phe Ser(tBu)-OtBu 433
Boc-Lys(.epsilon.Boc) Phe Trp Phe Thr(tBu)-OtBu 434
Boc-Lys(.epsilon.Boc) Phe Tyr Phe Ser(tBu)-OtBu 435
Boc-Lys(.epsilon.Boc) Phe Tyr Phe Thr(tBu)-OtBu 436
Boc-Lys(.epsilon.Boc) Phe His Phe Ser(tBu)-OtBu 437
Boc-Lys(.epsilon.Boc) Phe His Phe Thr(tBu)-OtBu 438
Boc-Lys(.epsilon.Boc) Val Phe Phe-Tyr Ser(tBu)-OtBu 439
Nicotinyl-Lys(.epsilon.Boc) Phe Trp Phe Ser(tBu)-OtBu 440
Nicotinyl-Lys(.epsilon.Boc) Phe Trp Phe Thr(tBu)-OtBu 441
Nicotinyl-Lys(.epsilon.Boc) Phe Tyr Phe Ser(tBu)-OtBu 442
Nicotinyl-Lys(.epsilon.Boc) Phe Tyr Phe Thr(tBu)-OtBu 443
Nicotinyl-Lys(.epsilon.Boc) Phe His Phe Ser(tBu)-OtBu 444
Nicotinyl-Lys(.epsilon.Boc) Phe His Phe Thr(tBu)-OtBu 445 Boc-Leu
Phe Trp Phe Thr(tBu)-OtBu 446 Boc-Leu Phe Trp Phe Ser(tBu)-OtBu
447
[0212] While the pepides of Table 7 are illustrated with particular
protecting groups, it is noted that these groups may be substituted
with other protecting groups as described herein and/or one or more
of the shown protecting group can be eliminated.
[0213] 7) Summary of Tripeptides and Tetrapeptides.
[0214] For the sake of clarity, a number of tripeptides and
tetrapeptides of this invention are generally summarized below in
Table 8.
10TABLE 8 General structure of certain peptides of this invention.
X.sup.1 X.sup.2 X.sup.3 X.sup.4 hydrophobic side chain Acidic or --
hydrophobic side or hydrophobic Basic chain or protecting group(s)
hydrophobic protecting group(s) hydrophobic side chain Basic Acidic
hydrophobic side or hydrophobic chain or protecting group(s)
hydrophobic protecting group(s) hydrophobic side chain Acidic Basic
hydrophobic side or hydrophobic chain or protecting group(s)
hydrophobic protecting group(s) hydrophobic side chain Acidic
Aliphatic hydrophobic side or hydrophobic or Basic chain or
protecting group(s) hydrophobic protecting group(s) hydrophobic
side chain Aliphatic Acidic or Basic hydrophobic side or
hydrophobic chain or protecting group(s) hydrophobic protecting
group(s) hydrophobic side chain Acidic Aromatic hydrophobic side or
hydrophobic or Basic chain or protecting group(s) hydrophobic
protecting group(s) hydrophobic side chain Aromatic Acidic or Basic
hydrophobic side or hydrophobic chain or protecting group(s)
hydrophobic protecting group(s) hydrophobic side chain Aromatic His
Aromatic hydrophobic side or hydrophobic chain or protecting
group(s) hydrophobic protecting group(s)
[0215] Where longer peptides are desired, X.sup.2 and X.sup.3 can
represent domains (e.g., regions of two or more amino acids of the
specified type) rather than individual amino acids. Table 8. is
intended to be illustrative and not limiting. Using the teaching
provided herein, other suitable peptides can readily be
identified.
[0216] 8) Paired Amino Acids and Dipeptides.
[0217] In certain embodiments, this invention pertains to the
discovery that certain pairs of amino acids, administered in
conjunction with each other or linked to form a dipeptide have one
or more of the properties described herein. Thus, without being
bound to a particular theory, it is believed that when the pairs of
amino acids are administered in conjunction with each other, as
described herein, they are capable participating in or inducing the
formation of micelles in vivo.
[0218] Similar to the other small peptides described herein, it is
belived that the pairs of peptides will associate in vivo, and
demonstrate physical properties including high solubility in ethyl
acetate (e.g., greater than about 4 mg/mL), solubility in aqueous
buffer at pH 7.0. Upon contacting phospholipids such as
1,2-Dimyristoyl-sn-glycero-3-phosphochol- ine (DMPC), in an aqueous
environment, it is believed the pairs of amino acids induce or
participate in the formation of particles with a diameter of
approximately 7.5 nm (.+-.0.1 nm), and/or induce or participate in
the formation of stacked bilayers with a bilayer dimension on the
order of 3.4 to 4.1 nm with spacing between the bilayers in the
stack of approximately 2 nm, and/or also induce or participate in
the formation of vesicular structures of approximately 38 nm).
[0219] Moreover, it is further believed that the pairs of amino
acids can display one or more of the following physiologically
relevant properties:
[0220] 1. They convert pro-inflammatory HDL to anti-inflammatory
HDL or make anti-inflammatory HDL more anti-inflammatory;
[0221] 2. They decrease LDL-induced monocyte chemotactic activity
generated by artery wall cells;
[0222] 3. They stimulate the formation and cycling of pre-62
HDL;
[0223] 4. They raise HDL cholesterol; and/or
[0224] 5. They increase HDL paraoxonase activity.
[0225] The pairs of amino acids can be administered as separate
amino acids (administered sequentially or simulataneously, e.g. in
a combined formulation) or they can be covalently coupled directly
or through a linker (e.g. a PEG linker, a carbon linker, a branched
linker, a straight chain linker, a heterocyclic linker, a linker
formed of derivatized lipid, etc.). In certain embodiments, the
pairs of amino acids are covalently linked through a peptide bond
to form a dipeptide. In various embodiments while the dipeptides
will typically comprise two amino acids each bearing an attached
protecting group, this invention also contemplates dipeptides
wheren only one of the amino acids bears one or more protecting
groups.
[0226] The pairs of amino acids typically comprise amino acids
where each amino acid is attached to at least one protecting group
(e.g., a hydrophobic protecting group as described herein). The
amino acids can be in the D or the L form. In certain embodiments,
where the amino acids comprising the pairs are not attached to each
other, each amino acid bears two protecting groups (e.g., such as
molecules 1 and 2 in Table 9).
11TABLE 9 Illustrative amino acid pairs of this invention. Amino
Acid Pair/dipeptide 1. Boc-Arg-OtBu* 2. Boc-Glu-OtBu* 3.
Boc-Phe-Arg-OtBu** 4. Boc-Glu-Leu-OtBu** 5. Boc-Arg-Glu-OtBu***
*This would typically be administered in conjunciton with a second
amino acid. **In certain embodiments, these dipeptides would be
administered in conjunction with each other. ***In certain
embodiments, this peptide would be administered either alone or in
combination with one of the other peptides described herein . .
.
[0227] Suitable pairs of amino acids can readily be identified by
providing the pair of protected amino acids and/or a dipeptide and
then screening the pair of amino acids/dipeptide for one or more of
the physical and/or physiological properties described above. In
certain embodiments, this invention excludes pairs of amino acids
and/or dipeptides comprising aspartic acid and phenylalanine. In
certain embodiments, this invention excludes pairs of amino acids
and/or dipeptides in which one amino acid is
(-)-N-[(trans-4-isopropylcyclohexan-
e)carbonyl]-D-phenylalanine(nateglinide).
[0228] In certain embodiments, the amino acids comprising the pair
are independently selected from the group consisting of an acidic
amino acid (e.g., aspartic acid, glutamic acid, etc.), a basic
amino acid (e.g., lysine, arginine, histidine, etc.), and a
non-polar amino acid (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, tryptophan, methionine, etc.). In certain
embodiments, where the first amino acid is acidic or basic, the
second amino acid is non-polar and where the second amino acid is
acidic or basic, the first amino acid is non-polar. In certain
embodiments, where the first amino acid is acidic, the second amino
acid is basic, and vice versa. (see, e.g., Table 10).
[0229] Similar combinations can be obtained by administering pairs
of dipeptides. Thus, for example in certain embodiments, molecules
3 and 4 in Table 9 would be administered in conjunction with each
other.
12TABLE 10 Certain generalized pepide pairs. First Amino acid
Second Amino acid 1. Acidic Basic 2. Basic Acidic 3. Acidic
Non-polar 4. Non-polar Acidic 5. Basic Non-polar 6. Non-polar
Basic
[0230] It is noted that these amino acid pairs/dipeptides are
intended to be illustrative and not limiting. Using the teaching
provided herein other suitable amino acid pairs/dipeptides can
readily be determined.
[0231] D) Other Peptide Modifications.
[0232] It was a surprising discovery that the peptides described
herein, particular when they incorporated one or more D-amino
acids, they retained their activity and could also be administered
orally. Moreover this oral administration resulted in relatively
efficient uptake and significant serum half-life thereby providing
an efficacious method of mitigating one or more symptoms of
atherosclerosis or other pathologies characterized by an
inflammatory process.
[0233] Using the teaching provided herein, one of skill can
routinely modify the illustrated peptides to produce other similar
peptides of this invention. For example, routine conservative or
semi-conservative substitutions (e.g., E for D) can be made of the
existing amino acids. The effect of various substitutions on lipid
affinity of the resulting peptide can be predicted using the
computational method described by Palgunachari et al. (1996)
Arteriosclerosis, Thrombosis, & Vascular Biology 16: 328-338.
The peptides can be lengthened or shortened as long as the class A
.alpha.-helix structure is preserved. In addition, substitutions
can be made to render the resulting peptide more similar to
peptide(s) endogenously produced by the subject species.
[0234] In certain embodiments, the peptides of this invention
comprise "D" forms of the peptides described in U.S. Pat. No.
4,643,988, more preferably "D" forms having one or both termini
coupled to protecting groups. In certain embodiments, at least 50%
of the enantiomeric amino acids are "D" form, more preferably at
least 80% of the enantiomeric amino acids are "D" form, and most
preferably at least 90% or even all of the enantiomeric amino acids
are "D" form amino acids.
[0235] While, in certain embodiments, the peptides of this
invention utilize naturally-occurring amino acids or D forms of
naturally occurring amino acids, substitutions with non-naturally
occurring amino acids (e.g., methionine sulfoxide, methionine
methylsulfonium, norleucine, episilon-aminocaproic acid,
4-aminobutanoic acid, tetrahydroisoquinoline-- 3-carboxylic acid,
8-aminocaprylic acid, 4-aminobutyric acid,
Lys(N(epsilon)-trifluoroacetyl), .alpha.-aminoisobutyric acid, and
the like) are also contemplated.
[0236] In addition to the peptides described herein,
peptidomimetics are also contemplated herein. Peptide analogs are
commonly used in the pharmaceutical industry as non-peptide drugs
with properties analogous to those of the template peptide. These
types of non-peptide compound are termed "peptide mimetics" or
"peptidomimetics" (Fauchere (1986) Adv. Drug Res. 15: 29; Veber and
Freidinger (1985) TINS p.392; and Evans et al. (1987) J. Med. Chem.
30: 1229) and are usually developed with the aid of computerized
molecular modeling. Peptide mimetics that are structurally similar
to therapeutically useful peptides may be used to produce an
equivalent therapeutic or prophylactic effect.
[0237] Generally, peptidomimetics are structurally similar to a
paradigm polypeptide (e.g, 4F, SEQ ID NO: 258 described herein),
but have one or more peptide linkages optionally replaced by a
linkage selected from the group consisting of: --CH.sub.2NH--,
--CH.sub.2S--, --CH.sub.2--CH.sub.2--, --CH.dbd.CH-- (cis and
trans), --COCH.sub.2--, --CH(OH)CH.sub.2--, --CH.sub.2SO--, etc. by
methods known in the art and further described in the following
references: Spatola (1983) p. 267 in Chemistry and Biochemistry of
Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel
Dekker, New York; Spatola (1983) Vega Data 1(3) Peptide Backbone
Modifications. (general review); Morley (1980) Trends Pharm Sci pp.
463-468 (general review); Hudson et al. (1979) Int J Pept Prot Res
14:177-185 (--CH.sub.2NH--, CH.sub.2CH.sub.2--); Spatola et al.
(1986) Life Sci 38:1243-1249 (--CH.sub.2--S); Hann, (1982) J Chem
Soc Perkin Trans I 307-314 (--CH--CH--, cis and trans); Almquist et
al. (1980) J Med Chem. 23:1392-1398 (--COCH.sub.2--);
Jennings-White et al. (1982) Tetrahedron Lett. 23:2533
(--COCH.sub.2--); Szelke, M. et al., European Appln. EP 45665
(1982) CA: 97:39405 (1982) (--CH(OH)CH2--); Holladay et al. (1983)
Tetrahedron Lett 24:4401-4404 (--C(OH)CH.sub.2--); and Hruby (1982)
Life Sci., 31:189-199 (--CH.sub.2--S--)).
[0238] A particularly preferred non-peptide linkage is
--CH.sub.2NH--. Such peptide mimetics may have significant
advantages over polypeptide embodiments, including, for example:
more economical production, greater chemical stability, enhanced
pharmacological properties (half-life, absorption, potency,
efficacy, etc.), reduced antigenicity, and others.
[0239] In addition, circular permutations of the peptides described
herein or constrained peptides (including cyclized peptides)
comprising a consensus sequence or a substantially identical
consensus sequence variation may be generated by methods known in
the art (Rizo and Gierasch (1992) Ann. Rev. Biochem. 61: 387); for
example, by adding internal cysteine residues capable of forming
intramolecular disulfide bridges which cyclize the peptide.
[0240] IX. Functional Assays of Peptides.
[0241] Certain peptides of this invention are desctribed herein by
various formulas (e.g., Formula I, above) and/or by particular
sequences. In certain embodiments, however, preferred peptides of
this invention are characterized by one or more of the following
functional properties:
[0242] 1. They convert pro-inflammatory HDL to anti-inflammatory
HDL or make anti-inflammatory HDL more anti-inflammatory;
[0243] 2. They decrease LDL-induced monocyte chemotactic activity
generated by artery wall cells;
[0244] 3. They stimulate the formation and cycling of pre-.beta.
HDL;
[0245] 4. They raise HDL cholesterol; and/or
[0246] 5. They increase HDL paraoxonase activity.
[0247] The specific peptides disclosed herein, and/or peptides
corresponding to the various formulas described herein can readily
be tested for one or more of these activities as desired.
[0248] Methods of screening for each of these functional properties
are well known to those of skill in the art. In addition, such
screens are illustrated herein in the Examples. In particular, it
is noted that assays for monocyte chemotactic activity, HDL
cholesterol, and HDL HDL paraoxonase activity are illustrated in
PCT/US01/26497 (WO 02/15923). Assays for determining HDL
inflammatory and/or anti-inflammatory properties were performed as
described below.
[0249] A) Determination of HDL Inflammatorv/Anti-Inflammatory
Properties
[0250] 1) Monocyte Chemotactic Activity (MCA) Assay
[0251] Lipoproteins, human artery wall cocultures, and monocytes
were prepared and monocyte chemotactic activity (MCA) was
determined as previously described (Van Lenten et al. (2002)
Circulation, 106: 1127-1132). Induction of MCA by a standard
control LDL was determined in the absence or presence of the
subject's HDL. Values in the absence of HDL were normalized to 1.0.
Values greater than 1.0 after the addition of HDL indicated
pro-inflammatory HDL; values less than 1.0 indicated
anti-inflammatory HDL.
[0252] 2) Cell-Free Assay
[0253] The cell-free assay was a modification of a previously
published method.sup.9 using PEIPC as the fluorescence-inducing
agent. Briefly, HDL was isolated by dextran sulfate method. Sigma
"HDL cholesterol reagent" (Catalog No. 352-3) containing dextran
sulfate and magnesium ions was dissolved in distilled water (10.0
mg/ml). Fifty microliters of dextran sulfate solution was mixed
with 500 .mu.l of the test plasma and incubated at room temperature
for 5 min and subsequently centrifuged at 8,000 g for 10 min. The
supernatant containing HDL was used in the experiments after
cholesterol determination using a cholesterol assay kit (Cat. No.
2340-200, Thermo DMA Company, Arlington, Tex.). We have previously
reported (Navab et al. (2001) J Lipid Res, 1308-1317) that HDL
isolated by this method inactivates bioactive phospholipids to a
similar extent as compared with HDL that has been isolated by
conventional ultracentrifuge methods. To determine the
inflammatory/anti-inflammatory properties of HDL samples from
patients and controls, the change in fluorescence intensity as a
result of the oxidation of DCFH by PEIPC in the absence or presence
of the test HDL was used. DCFH-DA was dissolved in fresh methanol
at 2.0 mg/ml and was incubated at room temperature and protected
from light for 30 min. resulting in the release of DCFH. The assay
was adapted for analyzing a large number of samples with a plate
reader. Flat-bottom, black, polystyrene microtiter plates
(Microfluor2, Cat. No. 14-245-176, Fisher) were utilized for this
purpose. Ten .mu.l of PEIPC solution (final concentration of 50
.mu.g/ml), and 90 .mu.l of HDL-containing dextran sulfate
supernatant (final concentration of 10 .mu.g/ml cholesterol), were
aliquoted into microtiter plates and mixed. The plates were then
incubated at 37.degree. C. on a rotator for 1.0 hr. Ten .mu.l of
DCFH solution (0.2 mg/ml) was then added to each well, mixed and
incubated for an additional 2 hrs at 37.degree. C. with rotation.
The fluorescence was subsequently determined with a plate reader
(Spectra Max, Gemini XS; Molecular Devices) at an excitation
wavelength of 485 nm and emission wavelength of 530 nm and cutoff
of 515 nm with the photomultiplier sensitivity set at "medium".
Values for intra- and interassay variability were 5.3.+-.1.7% and
7.1.+-.3.2%, respectively. Values in the absence of HDL were
normalized to 1.0. Values greater than 1.0 after the addition of
the test HDL indicated pro-inflammatory HDL; values less than 1.0
indicated anti-inflammatory HDL.
[0254] 3) Other Procedures
[0255] Plasma levels of interleukin-6 (IL-6) and tumor necrosis
factor-.alpha. (TNF-.alpha.) were determined by previously
published methods (Scheidt-Nave et al. (2001) J Clin Endocrinol
Metab., 86:2032-2042; Piguet et al. (1987) J Experiment Med., 166,
1280-1289). Plasma total cholesterol, triglycerides,
LDL-cholesterol, HDL-cholesterol and glucose were determined as
previously described (Navab et al. (1997) J Clin Invest,
99:2005-2019) using kits (Sigma), and hs-CRP levels (Rifai et al.
(1999) Clin Chem., 45:2136-2141) were determined using a sandwich
enzyme immunoassay from Immunodiagnostik (ALPCO Diagnostics,
Windham, N.H.). Statistical significance was determined with model
I ANOVA, and significance was defined as a value of p<0.05.
[0256] 4) Screening Physical Properties of Small Peptides.
[0257] It was a surprising finding of this invention that a number
of physical properties predict the ability of the small peptides
(e.g., less than 10 amino acids, preferably less than 8 amino
acids, more preferably from about 3 to about 5 or 6 amino acids) of
this invention to render HDL more anti-inflammatory and to mitigate
atherosclerosis and/or other pathologies characterized by an
inflammatory response in a mammal. As explained herein, the
physical properties include high solubility in ethyl acetate (e.g.,
greater than about 4 mg/mL), and solubility in aqueous buffer at pH
7.0. Upon contacting phospholipids such as
1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), in an aqueous
environment, the particularly effective small peptides form
particles with a diameter of approximately 7.5 nm (.+-.0.1 nm),
and/or form stacked bilayers with a bilayer dimension on the order
of 3.4 to 4.1 nm with spacing between the bilayers in the stack of
approximately 2 nm, and/or also form vesicular structures of
approximately 38 nm). In certain preferred embodiments, the small
peptides have a molecular weight of less than about 900 Da.
[0258] Virtually any small peptide can readily be screened for one
or more of these properties, e.g., as described herein in Example
3. Indeed combinatorial libraries of small peptides containing
greater than about 10.sup.4, or 10.sup.5, more preferably greater
than about 10.sup.6 or 10.sup.7, and most preferably greater than
about 10.sup.8 or 10.sup.9 small peptides can readily be produced
using methods well known to those of skill the art. The peptide
libraries can be random libraries, or, alternatively, in certain
embodiments, the libraries will comprise small peptides made in
accordance with one or more of the formulas provided herein.
[0259] The peptide libraries can then readily be screened, e.g.,
using high throughput screening methods for one more of the
physical properties described above. Peptides that test positive in
these assays are likely to have the ability to render HDL more
anti-inflammatory and to mitigate atherosclerosis and/or other
pathologies characterized by an inflammatory response in a
mammal.
[0260] It is noted that the foregoing screening methods are merely
illustrative and not intended to be limiting. Using the teachings
provided herein, other assays for the desired functional properties
of the peptides can readily be provided.
[0261] X. Peptide Preparation.
[0262] A) General Synthesis Methods.
[0263] The peptides used in this invention can be chemically
synthesized using standard chemical peptide synthesis techniques
or, particularly where the peptide does not comprise "D" amino acid
residues, the peptide can readily be recombinantly expressed. Where
the "D" polypeptides are recombinantly expressed, a host organism
(e.g., bacteria, plant, fungal cells, etc.) can be cultured in an
environment where one or more of the amino acids is provided to the
organism exclusively in a D form. Recombinantly expressed peptides
in such a system then incorporate those D amino acids.
[0264] In certain embodiments, D amino acids can be incorporated in
recombinantly expressed peptides using modified amino acyl-tRNA
synthetases that recognize D-amino acids.
[0265] In certain preferred embodiments the peptides are chemically
synthesized by any of a number of fluid or solid phase peptide
synthesis techniques known to those of skill in the art. Solid
phase synthesis in which the C-terminal amino acid of the sequence
is attached to an insoluble support followed by sequential addition
of the remaining amino acids in the sequence is a preferred method
for the chemical synthesis of the polypeptides of this invention.
Techniques for solid phase synthesis are well known to those of
skill in the art and are described, for example, by Barany and
Merrifield (1963) Solid-Phase Peptide Synthesis; pp. 3-284 in The
Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in
Peptide Synthesis, Part A.; Merrifield et al. (1963) J. Am. Chem.
Soc., 85: 2149-2156, and Stewart et al. (1984) Solid Phase Peptide
Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill.
[0266] In one embodiment, the peptides are synthesized by the solid
phase peptide synthesis procedure using a benzhyderylamine resin
(Beckman Bioproducts, 0.59 mmol of NH.sub.2/g of resin) as the
solid support. The COOH terminal amino acid (e.g.,
t-butylcarbonyl-Phe) is attached to the solid support through a
4-(oxymethyl)phenacetyl group. This is a more stable linkage than
the conventional benzyl ester linkage, yet the finished peptide can
still be cleaved by hydrogenation. Transfer hydrogenation using
formic acid as the hydrogen donor is used for this purpose.
Detailed protocols used for peptide synthesis and analysis of
synthesized peptides are describe in a miniprint supplement
accompanying Anantharamaiah et al. (1985) J. Biol. Chem., 260(16):
10248-10255.
[0267] It is noted that in the chemical synthesis of peptides,
particularly peptides comprising D amino acids, the synthesis
usually produces a number of truncated peptides in addition to the
desired full-length product. The purification process (e.g., HPLC)
typically results in the loss of a significant amount of the
full-length product.
[0268] It was a discovery of this invention that, particularly in
the synthesis of a D peptide (e.g., D-4), in order to prevent loss
in purifying the longest form one can dialyze and use the mixture
and thereby eliminate the last HPLC purification. Such a mixture
loses about 50% of the potency of the highly purified product
(e.g., per wt of protein product), but the mixture contains about 6
times more peptide and thus greater total activity.
[0269] B) Incorporating D-Form Amino Acids.
[0270] D-amino acids can be incorporated at one or more positions
in the peptide simply by using a D-form derivatized amino acid
residue in the chemical synthesis. D-form residues for solid phase
peptide synthesis are commercially available from a number of
suppliers (see, e.g., Advanced Chem Tech, Louisville; Nova Biochem,
San Diego; Sigma, St Louis; Bachem California Inc., Torrance,
etc.). The D-form amino acids can be completely omitted or
incorporated at any position in the peptide as desired. Thus, for
example, in certain embodiments, the peptide can comprise a single
D-amino acid, while in other embodiments, the peptide comprises at
least two, generally at least three, more generally at least four,
most generally at least five, preferably at least six, more
preferably at least seven and most preferably at least eight D
amino acids. In particularly preferred embodiments, essentially
every other (enantiomeric) amino acid is a D-form amino acid. In
certain embodiments at least 90%, preferably at least 90%, more
preferably at least 95% of the enantiomeric amino acids are D-form
amino acids. In one particularly preferred embodiment, essentially
every enantiomeric amino acid is a D-form amino acid.
[0271] C) Solution Phase Synthesis Methods.
[0272] In certain embodiments, the peptides of this inventioin can
readily be synthesized using solution phase methods. One such
synthesis scheme is illustrated in FIGS. 1 and 2.
[0273] In this scheme, A,B, C and D represent amino acids in the
desired peptide. X-represents a permanent .alpha.-amino protecting
group. Y-represents a permanent .alpha.-carboxyl protecting group.
Letters m and n represent side chain protecting groups if the N-
and C-terminal amino acids possess side chain functional groups.
Side chain protecting groups o and p are protecting groups that can
be removed by a treatment such as catalytic transfer hydrogenation
using ammonium formate as the hydrogen donor (Anantharamaiah and
Sivanandaiah (1977) Chem Soc. Perkin Trans. 490: 1-5; and Babiker
et al. (1978) J. Org. Chem. 44: 3442-3444) under the (neutral)
conditions in which side chain protecting groups m and p and
.alpha.-amino and .alpha.-carboxyl protecting groups are stable.
HOBT-HBTU represents condensing reagents under which minimum
reacimization is observed.
[0274] To the activated amino acid X-A(m) in presence of
1-hydroxybenzotriazole-2
(H-Benzotriazole-1-yl)-1,1,3,3-tetramethylammoni- um
hexafluorophosphate (HOBT-HBTU) and a small amount of tertiary
amine such diisopropylethylamine (DIEA) in DMF is added 2
equivalents of DIEA salt of H.sub.2N--B(n)--COO.sup.- and stirred
overnight at room temperature. The reaction is allowed to go to
completion with respect to activated carboxylic acid using excess
of amino acid in which .alpha.-amino is free and carboxyl is
temporarily protected as DIEA salt. The reaction mixture is
acidified using aqueous citric acid (10%) and extracted with ethyl
acetate. In this process the free amino acid remains in citric
acid. After washing ethyl acetate with water, the N-terminal
protected dipeptide free acid is extracted with 5% sodium
bicarbonate solution and acidified. The dipeptide free acid was
extracted with ethyl acetate, the organic layer is dried
(Na.sub.2SO.sub.4) and solvent evaporated to obtain the dipeptide
free acid. The tripeptide is also obtained in a similar manner by
reacting the dipeptide free acid with the suitably protected amino
acid in which the .alpha.-amino is free and the carboxyl is
temporarily protected as a DIEA salt. To obtain the tetrapeptide,
the suitably carboxyl protected amino acid was condensed using
HOBT-HBTU. Since the final tetrapeptide is a protected peptide, the
reaction mixture after the condensation was taken in ethyl acetate
and washed extensively with both aqueous bicarbonate (5%) and
citric acid (5%) and then with water. These washings will remove
excess of free acid and free base and the condensing reagents. The
protected peptide is then reprecipitated using ethyl acetate (or
ether) and petroleum ether. The protected free peptide is then
subjected to catalytic transfer hydrogenation in presence of
freshly prepared palladium black (Pd black) using ammonium formate
as the hydrogen donor. This reaction can be carried out in almost
neutral condition thus not affecting the acid sensitive side chain
protecting groups. This process will remove the protecting groups
on amino acids B and C. An example of this procedure is given below
using the synthesis of SEQ ID NO:256.
[0275] It is noted that this reaction scheme is intended to be
illustrative and not limiting. Using the teachings provided herein,
other suitable reactions schemes will be known to those of skill in
the art.
[0276] D) Protecting Groups.
[0277] In certain embodiments, the one or more R-groups on the
constituent amino acids and/or the terminal amino acids are blocked
with a protecting group, most preferably a hydrophobic protecting
group. Without being bound by a particular theory, it was a
discovery of this invention that blockage, particularly of the
amino and/or carboxyl termini of the subject peptides of this
invention greatly improves oral delivery and significantly
increases serum half-life.
[0278] A wide number of protecting groups are suitable for this
purpose. Such groups include, but are not limited to acetyl, amide,
and alkyl groups with acetyl and alkyl groups being particularly
preferred for N-terminal protection and amide groups being
preferred for carboxyl terminal protection. In certain embodiments,
the blocking groups can additionally act as a detectable label
(e.g., N-methyl anthranilyl).
[0279] In certain particularly preferred embodiments, the
protecting groups include, but are not limited to alkyl chains as
in fatty acids, propionyl, formyl, and others. Particularly
preferred carboxyl protecting groups include amides, esters, and
ether-forming protecting groups. In one preferred embodiment, an
acetyl group is used to protect the amino terminus and an amide
group is used to protect the carboxyl terminus. These blocking
groups enhance the helix-forming tendencies of the peptides.
Certain particularly preferred blocking groups include alkyl groups
of various lengths, e.g., groups having the formula:
CH.sub.3--(CH.sub.2).sub.n--CO-- where n ranges from about 3 to
about 20, preferably from about 3 to about 16, more preferably from
about 3 to about 13, and most preferably from about 3 to about
10.
[0280] Other protecting groups include, but are not limited to
N-methyl anthranilyl, Fmoc, t-butoxycarbonyl (t-BOC),
9-fluoreneacetyl group, 1-fluorenecarboxylic group,
9-florenecarboxylic group, 9-fluorenone-1-carboxylic group,
benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl
(Mtt), 4-methoxytrityl (Mmt),
4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr),
Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl
(Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc),
4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO),
Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys),
1-(4,4-dimentyl-2,6-diaxocyclohexylid- ene)ethyl (Dde),
2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-clorobenzyloxycarbonyl
(2-Cl-Z), 2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom),
cyclohexyloxy (cHxO),t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl
(tBu), Acetyl (Ac), and Trifluoroacetyl (TFA).
[0281] Protecting/blocking groups are well known to those of skill
as are methods of coupling such groups to the appropriate
residue(s) comprising the peptides of this invention (see, e.g.,
Greene et al., (1991) Protective Groups in Organic Synthesis, 2nd
ed., John Wiley & Sons, Inc. Somerset, N.J.). In one preferred
embodiment, for example, acetylation is accomplished during the
synthesis when the peptide is on the resin using acetic anhydride.
Amide protection can be achieved by the selection of a proper resin
for the synthesis. During the synthesis of the peptides described
herein in the examples, rink amide resin was used. After the
completion of the synthesis, the semipermanent protecting groups on
acidic bifunctional amino acids such as Asp and Glu and basic amino
acid Lys, hydroxyl of Tyr are all simultaneously removed. The
peptides released from such a resin using acidic treatment comes
out with the n-terminal protected as acetyl and the carboxyl
protected as NH.sub.2 and with the simultaneous removal of all of
the other protecting groups.
[0282] XI. Enhancing Peptide Uptake/Oral Availability.
[0283] A) Use of D-Amino Acids.
[0284] It was also a surprising discovery of this invention that
when an all L amino acid peptide (e.g., otherwise having the
sequence of the peptides of this invention) is administered in
conjunction with the D-form (i.e. a peptide of this invention) the
uptake of the D-form peptide is increased. Thus, in certain
embodiments, this invention contemplates the use of combinations of
D-form and L-form peptides in the methods of this invention. The
D-form peptide and the L-form peptide can have different amino acid
sequences, however, in preferred embodiments, they both have amino
acid sequences of peptides described herein, and in still more
preferred embodiments, they have the same amino acid sequence.
[0285] It was also a discovery of this invention that concatamers
of the class A amphipathic helix peptides of this invention are
also effective in mitigating one or more symptoms of
atherosclerosis. The monomers comprising the concatamers can be
coupled directly together or joined by a linker. In certain
embodiments, the linker is an amino acid linker (e.g., a proline),
or a peptide linker (e.g., Gly.sub.4Ser.sub.3) (SEQ ID NO:448). In
certain embodiments, the concatamer is a 2 mer, more preferably a 3
mer, still more preferably a 4 mer, and most preferably 5 mer, 8
mer, 10 mer, or 15 mer.
[0286] B) Alternating D- and L-Amino Acids.
[0287] It was discovered that alternating the sterioisoforms of the
amino acids at the center of the peptide will allow hydration of
the particle and will better allow the peptide particles to trap
pro-inflammatory oxidized lipids such as fatty acid hydroperoxides
and phospholipids that contain an oxidation product of arachidonic
acid at the sn-2 position.
[0288] Thus, in certain embodiments, the peptides described herein
can be synthesized to comprise from 4 amino acids to 10-15 amino
acids, preferentially (but not necessarily) with the center
(non-terminal) amino acids being alternating D and L sterioisomers
of the amino acids. The terminal amino acids can be hydrophobic
either because of a hydrophobic side chain or because the amino
acids bear hydrophobic blocking groups as described herein (e.g.,
an N-terminus is blocked with Boc-, Fmoc-, Nicotinyl-, and the like
and the C-terminus blocked with (tBu)-OtBu and the like.
[0289] Examples of such peptides are illustrated in Table 11.
13TABLE 11 Certain examples of peptides containing alter- nating D-
and L- residues in the central region. Sequence SEQ ID NO
Boc-Lys(.epsilon.Boc)-D-Ar- g-L-Asp-Ser(tBu)-OtBu 449
Boc-Lys(.epsilon.Boc)-L-Arg-D-A- sp-Ser(tBu)-OtBu/ 450
[0290] It is noted that while specific amino acid sequences are
illustrated in Table 11, alternating D- and L-amino acids can be
used in any of the peptides described herein.
[0291] C) Biotin-Derivatized Peptides.
[0292] In certain embodiments, any of the peptides described herein
can be attached (covalently coupled directly or indirectly through
a linker) to one or more biotins. The biotin interacts with the
intestinal sodium-dependent multivitamin transporter and thereby
facilitates uptake and bioavailability of orally administered
peptides.
[0293] The biotin can be directly coupled or coupled through a
linker or through a side chain of an amino acid by any of a number
of convenient means known to those of skill in the art. In certain
embodiments, the biotin is attached to the amino groups of
lysine.
[0294] A number of biotin-coupled peptides are illustrated in Table
12.
14TABLE 12 Examples of certain preferred peptides: SEQ ID Sequence
NO Ac-Asp-Trp-Phe-Lys(.epsilon.-biotin)-Ala-Phe-Tyr- 451
Asp-Lys(.epsilon.-biotin)-Val-Ala-Glu-Lys(.epsilon.-biotin)-
Phe-Lys(.epsilon.-biotin)-Glu-Ala-Phe-NH.sub.2
Ac-Asp-Trp-Phe-Lys(.epsilon.-biotin)-Ala-Phe-Tyr- 452
Asp-Lys(.epsilon.-biotin)-Val-Ala-Glu-Lys(.epsilon.-biotin)-
Phe-Lys-Glu-Ala-Phe-NH.sub.2 Ac-Asp-Trp-Phe-Lys-Ala-Ph-
e-Tyr-Asp-Lys(.epsilon.- 453 biotin)-Val-Ala-Glu-Lys(.epsi-
lon.-biotin)-Phe-Lys(.epsilon.- biotin)-Glu-Ala-Phe-NH.sub- .2
Ac-Asp-Trp-Phe-Lys(.epsilon.-biotin)-Ala-Phe-Tyr- 454
Asp-Lys-Val-Ala-Glu-Lys(.epsilon.-biotin)-Phe-Lys(.epsilon.- -
biotin)-Glu-Ala-Phe-NH.sub.2
Ac-Asp-Trp-Phe-Lys(.epsilon.-biotin)-Ala-Phe-Tyr- 455
Asp-Lys(.epsilon.-biotin)-Val-Ala-Glu-Lys-Phe-Lys(.epsilon.-
biotin)-Glu-Ala-Phe-NH.sub.2 Ac-Asp-Trp-Phe-Lys(.epsil-
on.-biotin)-Ala-Phe-Tyr- 456 Asp-Lys-Val-Ala-Glu-Lys-Phe-L-
ys(.epsilon.-biotin)- Glu-Ala-Phe-NH.sub.2
Ac-Asp-Trp-Phe-Lys(.epsilon.-biotin)-Ala-Phe-Tyr- 457
Asp-Lys(.epsilon.-biotin)-Val-Ala-Glu-Lys-Phe-Lys-
Glu-Ala-Phe-NH.sub.2 Ac-Asp-Trp-Phe-Lys-Ala-Phe-Tyr-Asp-L- ys-Val-
458 Ala-Glu-Lys(.epsilon.-biotin)-Phe-Lys(.epsilon- .-biotin)-Glu-
Ala-Phe-NH.sub.2 Ac-Asp-Trp-Phe-Lys-Ala-Phe-Tyr-Asp-Lys(.epsilon.-
459 biotin)-Val-Ala-Glu-Lys-Phe-Lys(.epsilon.-biotin)-
Glu-Ala-Phe-NH.sub.2 Ac-Asp-Trp-Phe-Lys-Ala-Phe-Tyr-Asp-L-
ys(.epsilon.- 460 biotin)-Val-Ala-Glu-Lys(.epsilon.-biotin-
)-Phe-Lys- Glu-Ala-Phe-NH.sub.2
Ac-Asp-Trp-Phe-Lys(.epsilon.-biotin)-Ala-Phe-Tyr- 461
Asp-Lys-Val-Ala-Glu-Lys(.epsilon.-biotin)-Phe-Lys-
Glu-Ala-Phe-NH.sub.2 Ac-Asp-Trp-Phe-Lys(.epsilon.-biotin)-
-Ala-Phe-Tyr- 462 Asp-Lys-Val-Ala-Glu-Lys-Phe-Lys-Glu-Ala-- Phe-
NH.sub.2 Ac-Asp-Trp-Phe-Lys-Ala-Phe-T- yr-Asp-Lys(.epsilon.- 463
biotin)-Val-Ala-Glu-Lys-Phe-Lys-- Glu-Ala-Phe- NH.sub.2
Ac-Asp-Trp-Phe-Lys-Ala-Phe-Tyr-Asp-Lys-Val- 464
Ala-Glu-Lys(.epsilon.-biotin)-Phe-Lys-Glu-Ala-Phe- NH.sub.2
Ac-Asp-Trp-Phe-Lys-Ala-Phe-Tyr-Asp-Lys-Val- 465
Ala-Glu-Lys-Phe-Lys(.epsilon.- biotin)-Glu-Ala-Phe-NH.sub.2
[0295] XII. Pharmaceutical Formulations.
[0296] In order to carry out the methods of the invention, one or
more peptides, or pairs of amino acids, or peptide mimetics of this
invention are administered, e.g., to an individual diagnosed as
having one or more symptoms of atherosclerosis, or as being at risk
for atherosclerosis. The peptides, or pairs of amino acids, or
peptide mimetics can be administered in the "native" form or, if
desired, in the form of salts, esters, amides, prodrugs,
derivatives, and the like, provided the salt, ester, amide, prodrug
or derivative is suitable pharmacologically, i.e., effective in the
present method. Salts, esters, amides, prodrugs and other
derivatives of the active agents may be prepared using standard
procedures known to those skilled in the art of synthetic organic
chemistry and described, for example, by March (1992) Advanced
Organic Chemistry; Reactions, Mechanisms and Structure, 4th Ed.
N.Y. Wiley-Interscience.
[0297] For example, acid addition salts are prepared from the free
base using conventional methods, that typically involve reaction
with a suitable acid. Generally, the base form of the drug is
dissolved in a polar organic solvent such as methanol or ethanol
and the acid is added thereto. The resulting salt either
precipitates or may be brought out of solution by addition of a
less polar solvent. Suitable acids for preparing acid addition
salts include both organic acids, e.g., acetic acid, propionic
acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic
acid, succinic acid, maleic acid, fumaric acid, tartaric acid,
citric acid, benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,
salicylic acid, and the like, as well as inorganic acids, e.g.,
hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,
phosphoric acid, and the like. An acid addition salt may be
reconverted to the free base by treatment with a suitable base.
Particularly preferred acid addition salts of the active agents
herein are halide salts, such as may be prepared using hydrochloric
or hydrobromic acids. Conversely, preparation of basic salts of the
peptides or mimetics are prepared in a similar manner using a
pharmaceutically acceptable base such as sodium hydroxide,
potassium hydroxide, ammonium hydroxide, calcium hydroxide,
trimethylamine, or the like. Particularly preferred basic salts
include alkali metal salts, e.g., the sodium salt, and copper
salts.
[0298] Preparation of esters typically involves functionalization
of hydroxyl and/or carboxyl groups, that can be present within the
molecular structure of the drug. The esters are typically
acyl-substituted derivatives of free alcohol groups, i.e., moieties
that are derived from carboxylic acids of the formula RCOOH where R
is alky, and preferably is lower alkyl. Esters can be reconverted
to the free acids, if desired, by using conventional hydrogenolysis
or hydrolysis procedures.
[0299] Amides and prodrugs may also be prepared using techniques
known to those skilled in the art or described in the pertinent
literature. For example, amides may be prepared from esters, using
suitable amine reactants, or they may be prepared from an anhydride
or an acid chloride by reaction with ammonia or a lower alkyl
amine. Prodrugs are typically prepared by covalent attachment of a
moiety that results in a compound that is therapeutically inactive
until modified by an individual's metabolic system.
[0300] The peptides, or pairs of amino acids, or mimetics
identified herein are useful for parenteral, topical, oral, nasal
(or otherwise inhaled), rectal, or local administration, such as by
aerosol or transdermally, for prophylactic and/or therapeutic
treatment of atherosclerosis and/or symptoms thereof and/or for one
or more of the other indications identified herein. The
pharmaceutical compositions can be administered in a variety of
unit dosage forms depending upon the method of administration.
Suitable unit dosage forms, include, but are not limited to
powders, tablets, pills, capsules, lozenges, suppositories,
patches, nasal sprays, injectibles, implantable sustained-release
formulations, lipid complexes, etc.
[0301] The peptides, and/or pairs of amino acids, and/or peptide
mimetics of this invention are typically combined with a
pharmaceutically acceptable carrier (excipient) to form a
pharmacological composition. Pharmaceutically acceptable carriers
can contain one or more physiologically acceptable compound(s) that
act, for example, to stabilize the composition or to increase or
decrease the absorption of the active agent(s). Physiologically
acceptable compounds can include, for example, carbohydrates, such
as glucose, sucrose, or dextrans, antioxidants, such as ascorbic
acid or glutathione, chelating agents, low molecular weight
proteins, protection and uptake enhancers such as lipids,
compositions that reduce the clearance or hydrolysis of the active
agents, or excipients or other stabilizers and/or buffers.
[0302] Other physiologically acceptable compounds include wetting
agents, emulsifying agents, dispersing agents or preservatives that
are particularly useful for preventing the growth or action of
microorganisms. Various preservatives are well known and include,
for example, phenol and ascorbic acid. One skilled in the art would
appreciate that the choice of pharmaceutically acceptable
carrier(s), including a physiologically acceptable compound
depends, for example, on the route of administration of the active
agent(s) and on the particular physio-chemical characteristics of
the active agent(s).
[0303] The excipients are preferably sterile and generally free of
undesirable matter. These compositions may be sterilized by
conventional, well-known sterilization techniques.
[0304] In therapeutic applications, the compositions of this
invention are administered to a patient suffering from one or more
symptoms of atherosclerosis or at risk for atherosclerosis in an
amount sufficient to cure or at least partially prevent or arrest
the disease and/or its complications. An amount adequate to
accomplish this is defined as a "therapeutically effective dose."
Amounts effective for this use will depend upon the severity of the
disease and the general state of the patient's health. Single or
multiple administrations of the compositions may be administered
depending on the dosage and frequency as required and tolerated by
the patient. In any event, the composition should provide a
sufficient quantity of the active agents of the formulations of
this invention to effectively treat (ameliorate one or more
symptoms) the patient.
[0305] The concentration of peptide, or pair of amino acids, or
mimetic can vary widely, and will be selected primarily based on
fluid volumes, viscosities, body weight and the like in accordance
with the particular mode of administration selected and the
patient's needs. Concentrations, however, will typically be
selected to provide dosages ranging from about 0.1 or 1 mg/kg/day
to about 50 mg/kg/day and sometimes higher. Typical dosages range
from about 3 mg/kg/day to about 3.5 mg/kg/day, preferably from
about 3.5 mg/kg/day to about 7.2 mg/kg/day, more preferably from
about 7.2 mg/kg/day to about 11.0 mg/kg/day, and most preferably
from about 11.0 mg/kg/day to about 15.0 mg/kg/day. In certain
preferred embodiments, dosages range from about 10 mg/kg/day to
about 50 mg/kg/day. It will be appreciated that such dosages may be
varied to optimize a therapeutic regimen in a particular subject or
group of subjects.
[0306] In certain preferred embodiments, the peptides, and/or pairs
of amino acids, and/or peptide mimetics of this invention are
administered orally (e.g., via a tablet) or as an injectable in
accordance with standard methods well known to those of skill in
the art. In other preferred embodiments, the peptides, or pairs of
amino acids, can also be delivered through the skin using
conventional transdermal drug delivery systems, i.e., transdermal
"patches" wherein the active agent(s) are typically contained
within a laminated structure that serves as a drug delivery device
to be affixed to the skin. In such a structure, the drug
composition is typically contained in a layer, or "reservoir,"
underlying an upper backing layer. It will be appreciated that the
term "reservoir" in this context refers to a quantity of "active
ingredient(s)" that is ultimately available for delivery to the
surface of the skin. Thus, for example, the "reservoir" may include
the active ingredient(s) in an adhesive on a backing layer of the
patch, or in any of a variety of different matrix formulations
known to those of skill in the art. The patch may contain a single
reservoir, or it may contain multiple reservoirs.
[0307] In one embodiment, the reservoir comprises a polymeric
matrix of a pharmaceutically acceptable contact adhesive material
that serves to affix the system to the skin during drug delivery.
Examples of suitable skin contact adhesive materials include, but
are not limited to, polyethylenes, polysiloxanes, polyisobutylenes,
polyacrylates, polyurethanes, and the like. Alternatively, the
drug-containing reservoir and skin contact adhesive are present as
separate and distinct layers, with the adhesive underlying the
reservoir which, in this case, may be either a polymeric matrix as
described above, or it may be a liquid or hydrogel reservoir, or
may take some other form. The backing layer in these laminates,
which serves as the upper surface of the device, preferably
functions as a primary structural element of the "patch" and
provides the device with much of its flexibility. The material
selected for the backing layer is preferably substantially
impermeable to the active agent(s) and any other materials that are
present.
[0308] Other preferred formulations for topical drug delivery
include, but are not limited to, ointments and creams. Ointments
are semisolid preparations, that are typically based on petrolatum
or other petroleum derivatives. Creams containing the selected
active agent are typically viscous liquid or semisolid emulsions,
often either oil-in-water or water-in-oil. Cream bases are
typically water-washable, and contain an oil phase, an emulsifier
and an aqueous phase. The oil phase, also sometimes called the
"internal" phase, is generally comprised of petrolatum and a fatty
alcohol such as cetyl or stearyl alcohol; the aqueous phase
usually, although not necessarily, exceeds the oil phase in volume,
and generally contains a humectant. The emulsifier in a cream
formulation is generally a nonionic, anionic, cationic or
amphoteric surfactant. The specific ointment or cream base to be
used, as will be appreciated by those skilled in the art, is one
that will provide for optimum drug delivery. As with other carriers
or vehicles, an ointment base should be inert, stable,
nonirritating and nonsensitizing.
[0309] Unlike typical peptide formulations, the peptides, or pairs
of amino acids, of this invention comprising D-form amino acids can
be administered, even orally, without protection against
proteolysis by stomach acid, etc. Nevertheless, in certain
embodiments, peptide delivery can be enhanced by the use of
protective excipients. This is typically accomplished either by
complexing the polypeptide with a composition to render it
resistant to acidic and enzymatic hydrolysis or by packaging the
polypeptide in an appropriately resistant carrier such as a
liposome. Means of protecting polypeptides for oral delivery are
well known in the art (see, e.g., U.S. Pat. No. 5,391,377
describing lipid compositions for oral delivery of therapeutic
agents).
[0310] A) Sustained Release Formulations.
[0311] Elevated serum half-life can be maintained by the use of
sustained-release protein "packaging" systems. Such sustained
release systems are well known to those of skill in the art. In one
preferred embodiment, the ProLease biodegradable microsphere
delivery system for proteins and peptides (Tracy (1998) Biotechnol.
Prog. 14: 108; Johnson et al. (1996), Nature Med. 2: 795; Herbert
et al. (1998), Phannaceut. Res. 15, 357) a dry powder composed of
biodegradable polymeric microspheres containing the protein in a
polymer matrix that can be compounded as a dry formulation with or
without other agents.
[0312] The ProLease microsphere fabrication process was
specifically designed to achieve a high protein encapsulation
efficiency while maintaining protein integrity. The process
consists of (i) preparation of freeze-dried protein particles from
bulk protein by spray freeze-drying the drug solution with
stabilizing excipients, (ii) preparation of a drug-polymer
suspension followed by sonication or homogenization to reduce the
drug particle size, (iii) production of frozen drug-polymer
microspheres by atomization into liquid nitrogen, (iv) extraction
of the polymer solvent with ethanol, and (v) filtration and vacuum
drying to produce the final dry-powder product. The resulting
powder contains the solid form of the protein, which is
homogeneously and rigidly dispersed within porous polymer
particles. The polymer most commonly used in the process,
poly(lactide-co-glycolide) (PLG), is both biocompatible and
biodegradable.
[0313] Encapsulation can be achieved at low temperatures (e.g.,
-40.degree. C). During encapsulation, the protein is maintained in
the solid state in the absence of water, thus minimizing
water-induced conformational mobility of the protein, preventing
protein degradation reactions that include water as a reactant, and
avoiding organic-aqueous interfaces where proteins may undergo
denaturation. A preferred process uses solvents in which most
proteins are insoluble, thus yielding high encapsulation
efficiencies (e.g., greater than 95%).
[0314] In another embodiment, one or more components of the
solution can be provided as a "concentrate", e.g., in a storage
container (e.g., in a premeasured volume) ready for dilution, or in
a soluble capsule ready for addition to a volume of water.
[0315] B) Combined Formulations.
[0316] In certain instances, one or more peptides, and/or pairs of
amino acids, of this invention are administered in conjunction with
one or more active agents (e.g., statins, beta blockers, ACE
inhibitors, lipids, etc.). The two agents (e.g., peptide and
statin) can be administered simultaneously or sequentially. When
administered sequentially the two agents are administered so that
both achieve a physiologically relevant concentration over a
similar time period (e.g., so that both agents are active at some
common time).
[0317] In certain embodiments, both agents are administered
simultaneously. In such instances it can be convenient to provide
both agents in a single combined formulation. This can be achieved
by a variety of methods well known to those of skill in the art.
For example, in a tablet formulation the tablet can comprise two
layers one layer comprising, e.g., the statin(s), and the other
layer comprising e.g., the peptide(s). In a time release capsule,
the capsule can comprise two time release bead sets, one for the
peptide(s) and one containing the statin(s).
[0318] The foregoing formulations and administration methods are
intended to be illustrative and not limiting. It will be
appreciated that, using the teaching provided herein, other
suitable formulations and modes of administration can be readily
devised.
[0319] XIII. Additional Pharmacologically Active Agents.
[0320] Additional pharmacologically active agents may be delivered
along with the primary active agents, e.g., the peptides, or pairs
of amino acids, of this invention. In one embodiment, such agents
include, but are not limited to agents that reduce the risk of
atherosclerotic events and/or complications thereof. Such agents
include, but are not limited to beta blockers, beta blockers and
thiazide diuretic combinations, statins, aspirin, ace inhibitors,
ace receptor inhibitors (ARBs), and the like.
[0321] A) Statins.
[0322] It was a surprising discovery that administration of one or
more peptides of this invention "concurrently" with one or more
statins synergistically enhances the effect of the statin(s). That
is, the statins can achieve a similar efficacy at lower dosage
thereby obviating potential adverse side effects (e.g., muscle
wasting) associated with these drugs and/or cause the statins to be
significantly more anti-inflammatory at any given dose.
[0323] The major effect of the statins is to lower LDL-cholesterol
levels, and they lower LDL-cholesterol more than many other types
of drugs. Statins generally inhibit an enzyme, HMG-CoA reductase,
which controls the rate of cholesterol production in the body.
These drugs typically lower cholesterol by slowing down the
production of cholesterol and by increasing the liver's ability to
remove the LDL-cholesterol already in the blood.
[0324] The large reductions in total and LDL-cholesterol produced
by these drugs appears to result in large reductions in heart
attacks and heart disease deaths. Thanks to their track record in
these studies and their ability to lower LDL-cholesterol, statins
have become the drugs most often prescribed when a person needs a
cholesterol-lowering medicine. Studies using statins have reported
20 to 60 percent lower LDL-cholesterol levels in patients on these
drugs. Statins also reduce elevated triglyceride levels and produce
a modest increase in HDL-cholesterol. Recently it has been
appreciated that statins have anti-inflammatory properties that may
not be directly related to the degree of lipid lowering achieved.
For example it has been found that statins decrease the plasma
levels of the inflammatory marker CRP relatively independent of
changes in plasma lipid levels. This anti-inflammatory activity of
statins has been found to be as or more important in predicting the
reduction in clinical events induced by statins than is the degree
of LDL lowering.
[0325] The statins are usually given in a single dose at the
evening meal or at bedtime. These medications are often given in
the evening to take advantage of the fact that the body makes more
cholesterol at night than during the day. When combined with the
peptides described herein, the combined peptide/statin treatment
regimen will also typically be given in the evening.
[0326] Suitable statins are well known to those of skill in the
art. Such statins include, but are not limited to atorvastatin
(Lipitor.RTM., Pfizer), simvastatin (Zocor.RTM., Merck0,
pravastatin (Pravachol.RTM., Bristol-Myers Squibb.RTM., fluvastatin
(Lescol.RTM., Novartis), lovastatin (Mevacor.RTM., Merck),
rosuvastatin (Crestor.RTM., Astra Zeneca), and Pitavastatin
(Sankyo), and the like.
[0327] The combined statin/peptide dosage can be routinely
optimized for each patient. Typically statins show results after
several weeks, with a maximum effect in 4 to 6 weeks. Prior to
combined treatment with a statin and one of the peptides described
herein, the physician would obtain routine tests for starting a
statin including LDL-cholesterol and HDL-cholesterol levels.
Additionally, the physician would also measure the
anti-inflammatory properties of the patient's HDL and determine CRP
levels with a high sensitivity assay. After about 4 to 6 weeks of
combined treatment, the physician would typically repeat these
tests and adjust the dosage of the medications to achieve maximum
lipid lowering and maximum anti-inflammatory activity.
[0328] B) Cholesterol Absorption Inhibitors.
[0329] In certain embodiments, one or more peptides, and/or pairs
of amino acids, of this invention are administered to a subject in
conjunction with one or more cholesterol absorption inhibitors. The
peptide(s) can be administered before, after, or simultaneously
with the cholesterol absorption inhibitor. In the latter case, the
cholesterol absorption inhibitor can be provided as a separate
formulation or as a combined formulation with one or more of the
peptide(s).
[0330] Cholesterol absorption inhibitors are well known to those of
skill in the art. One important cholesterol absorption inhibitor is
Ezetimibe, also known as
1-(4-fluorophenyl)-3(R)-[3-(4-fluorophenyl)-3(S)-hydroxypro-
pyl]-4(S)-(4-hydroxyphenyl)-2-azetidinone (available from Merck).
Ezetimibe reduces blood cholesterol by inhibiting the absorption of
cholesterol by the small intestine.
[0331] C) Beta Blocers.
[0332] Suitable beta blockers include, but are not limited to
cardioselective (selective beta 1 blockers), e.g., acebutolol
(Sectral.TM.), atenolol (Tenormin.TM.), betaxolol (Kerlone.TM.),
bisoprolol (Zebeta.TM.), metoprolol (Lopressor.TM.), and the like.
Suitable non-selective blockers (block beta 1 and beta 2 equally)
include, but are not limited to carteolol (Cartrol.TM.), nadolol
(Corgard.TM.), penbutolol (Levatol.TM.), pindolol (Visken.TM.),
carvedilol, (Coreg.TM.), propranolol (Inderal.TM.), timolol
(Blockadren.TM.), labetalol (Normodyne.TM., Trandate.TM.), and the
like.
[0333] Suitable beta blocker thiazide diuretic combinations
include, but are not limited to Lopressor HCT, ZIAC, Tenoretic,
Corzide, Timolide, Inderal LA 40/25, Inderide, Normozide, and the
like.
[0334] D) ACE Inhibitors.
[0335] Suitable ace inhibitors include, but are not limited to
captopril (e.g., Capoten.TM. by Squibb), benazepril (e.g.,
Lotensin.TM. by Novartis), enalapril (e.g., Vasotec.TM. by Merck),
fosinopril (e.g., Monopril.TM. by Bristol-Myers), lisinopril (e.g.,
Prinivil.TM. by Merck or Zestril.TM. by Astra-Zeneca), quinapril
(e.g., Accupril.TM. by Parke-Davis), ramipril (e.g., Altace.TM. by
Hoechst Marion Roussel, King Pharmaceuticals), imidapril,
perindopril erbumine (e.g., Aceon.TM. by Rhone-Polenc Rorer),
trandolapril (e.g., Mavik.TM. by Knoll Pharmaceutical), and the
like. Suitable ARBS (Ace Receptor Blockers) include but are not
limited to losartan (e.g., Cozaar.TM. by Merck), irbesartan (e.g.,
Avapro.TM. by Sanofi), candesartan (e.g., Atacand.TM. by Astra
Merck), valsartan (e.g., Diovan.TM. by Novartis), and the like.
[0336] E) Lipid-Based Formulations.
[0337] In certain embodiments, the peptides, and/or pairs of amino
acids, of this invention are administered in conjunction with one
or more lipids. The lipids can be formulated as an active agent,
and/or as an excipient to protect and/or enhance transport/uptake
of the peptides, or they can be administered separately.
[0338] Without being bound by a particular theory, it was
discovered of this invention that administration (e.g., oral
administration) of certain phospholipids can significantly increase
HDL/LDL ratios. In addition, it is believed that certain
medium-length phospholipids are transported by a process different
than that involved in general lipid transport. Thus,
co-administration of certain medium-length phospholipids with the
peptides of this invention confer a number of advantages: They
protect the phospholipids from digestion or hydrolysis, they
improve peptide uptake, and they improve HDL/LDL ratios.
[0339] The lipids can be formed into liposomes that encapsulate the
polypeptides of this invention and/or they can be simply
complexed/admixed with the polypeptides. Methods of making
liposomes and encapsulating reagents are well known to those of
skill in the art (see, e.g., Martin and Papahadjopoulos (1982) J.
Biol. Chem., 257: 286-288; Papahadjopoulos et al. (1991) Proc.
Natl. Acad. Sci. USA, 88: 11460-11464; Huang et al. (1992) Cancer
Res., 52:6774-6781; Lasic et al. (1992) FEBS Lett., 312: 255-258.,
and the like).
[0340] Preferred phospholipids for use in these methods have fatty
acids ranging from about 4 carbons to about 24 carbons in the sn-1
and sn-2 positions. In certain preferred embodiments, the fatty
acids are saturated. In other preferred embodiments, the fatty
acids can be unsaturated. Various preferred fatty acids are
illustrated in Table 13.
15TABLE 13 Preferred fatty acids in the sn-1 and/or sn-2 position
of the preferred phospholipids for administration of D
polypeptides. Carbon No. Common Name IUPAC Name 3:0 Propionoyl
Trianoic 4:0 Butanoyl Tetranoic 5:0 Pentanoyl Pentanoic 6:0 Caproyl
Hexanoic 7:0 Heptanoyl Heptanoic 8:0 Capryloyl Octanoic 9:0
Nonanoyl Nonanoic 10:0 Capryl Decanoic 11:0 Undcanoyl Undecanoic
12:0 Lauroyl Dodecanoic 13:0 Tridecanoyl Tridecanoic 14:0 Myristoyl
Tetradecanoic 15:0 Pentadecanoyl Pentadecanoic 16:0 Palmitoyl
Hexadecanoic 17:0 Heptadecanoyl Heptadecanoic 18:0 Stearoyl
Octadecanoic 19:0 Nonadecanoyl Nonadecanoic 20:0 Arachidoyl
Eicosanoic 21:0 Heniecosanoyl Heniecosanoic 22:0 Behenoyl
Docosanoic 23:0 Trucisanoyl Trocosanoic 24:0 Lignoceroyl
Tetracosanoic 14:1 Myristoleoyl (9-cis) 14:1 Myristelaidoyl
(9-trans) 16:1 Palmitoleoyl (9-cis) 16:1 Palmitelaidoyl
(9-trans)
[0341] The fatty acids in these positions can be the same or
different. Particularly preferred phospholipids have
phosphorylcholine at the sn-3 position.
[0342] XIV. Kits.
[0343] In another embodiment this invention provides kits for
amelioration of one or more symptoms of atherosclerosis and/or for
the prophylactic treatment of a subject (human or animal) at risk
for atherosclerosis and/or for stimulating the formation and
cycling of pre-beta high density lipoprotein-like particles and/or
for inhibiting one or more symptoms of osteoporosis. The kits
preferably comprise a container containing one or more of the
peptides, and/or pairs of amino acids, and/or peptide mimetics of
this invention. The peptide, and/or pairs of amino acids, and/or
peptide mimetic can be provided in a unit dosage formulation (e.g.,
suppository, tablet, caplet, patch, etc.) and/or may be optionally
combined with one or more pharmaceutically acceptable
excipients.
[0344] The kit can, optionally, further comprise one or more other
agents used in the treatment of heart disease and/or
atherosclerosis. Such agents include, but are not limited to, beta
blockers, vasodilators, aspirin, statins, ace inhibitors or ace
receptor inhibitors (ARBs) and the like, e.g., as described
above.
[0345] In certain preferred embodiments, the kits additionally
include a statin (e.g., cerivastatin, atorvastatin, simvastatin,
pravastatin, fluvastatin, lovastatin. rosuvastatin, pitavastatin,
etc.) either formulated separately or in a combined formulation
with the peptide(s). Typically the dosage of a statin in such a
formulation can be lower than the dosage of a statin typically
presecribed without the synergistic peptide.
[0346] In addition, the kits optionally include labeling and/or
instructional materials providing directions (i.e., protocols) for
the practice of the methods or use of the "therapeutics" or
"prophylactics" of this invention. Preferred instructional
materials describe the use of one or more polypeptides, and/or
pairs of amino acids, of this invention to mitigate one or more
symptoms of atherosclerosis and/or to prevent the onset or increase
of one or more of such symptoms in an individual at risk for
atherosclerosis and/or to stimulate the formation and cycling of
pre-beta high density lipoprotein-like particles and/or to inhibit
one or more symptoms of osteoporosis and/or to mitigate one or more
symptoms of a pathology characterized by an inflammatory response.
The instructional materials may also, optionally, teach preferred
dosages/therapeutic regiment, counter indications and the like.
[0347] While the instructional materials typically comprise written
or printed materials they are not limited to such. Any medium
capable of storing such instructions and communicating them to an
end user is contemplated by this invention. Such media include, but
are not limited to electronic storage media (e.g., magnetic discs,
tapes, cartridges, chips), optical media (e.g., CD ROM), and the
like. Such media may include addresses to internet sites that
provide such instructional materials.
EXAMPLES
[0348] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example 1
Evaluation of Small Peptides to Mediate Symptoms of Atherosclerosis
and Other Inflammatory Pathologies.
[0349] The apo A-I mimetic peptides described herein (see, e.g.,
Table 1) exhibit antiatherogenic properties similar to apo A-I in
that they remove the "seeding molecules" (e.g., oxidized
phospholipids such as Ox-PAPC, POVPC, PGPC, and PEIPC, etc.)
necessary for artery wall cells to oxidized IDL and are similar to
apo A-I in that they ameliorated atherosclerosis in mouse
models.
[0350] The apo A-I mimetic peptides (e.g., D-4F, SEQ ID NO:8),
differ from apo A-I in that they are also active in a co-incubation
similar to apo J (see, e.g., U.S. Ser. No. 10/120,508 and
PCT/US03/09988). These peptides generally do not have substantial
sequence homology to apo A-I, but have homology in their helical
structure and in their ability to bind lipids.
[0351] The smaller peptides described herein (see, e.g., Tables 4-7
herein) are similar to native apoA-I in that they prevent LDL
oxidation and LDL-induced monocyte chemotactic activity in a
pre-incubation with artery wall cells but not in a co-incubation
(see, e.g., FIG. 3).
[0352] The peptide described in FIG. 3 was also active in vivo
(FIG. 4). The tetrapeptide or D-4F (SEQ ID NO:8) were added at 5
.mu.g/ml to the drinking water or not added to the drinking water
of apoE null mice (a mouse model of human atherosclerosis). After
18 hours the mice were bled and their lipoproteins isolated by
FPLC. Adding the fractions containing mature HDL or the FPLC
fractions after these fractions where pre-beta HDL would be
expected (particles that come off the FPLC column just after the
main HDL peak; post HDL) from mice that received drinking water
without peptide increased the monocyte chemotactic activity induced
by a control LDL added to a human artery wall cell coculture (FIG.
4). In contrast, adding HDL or the post HDL FPLC fractions from the
mice that received the tetrapeptide or D-4F in their drinking water
significantly decreased the LDL-induced monocyte chemotactic
activity indicating that the tetrapeptide and D-4F converted these
lipoproteins from a pro-inflammatory to an anti-inflammatory state
(FIG. 4).
[0353] As shown in FIG. 5, LDL taken from the mice that received
the tetrapeptide or D-4F induced significantly less monocyte
chemotactic activity than did LDL from mice that did not receive
the peptides confirming the biologic activity of the orally
administered D-tetrapeptide.
[0354] FIG. 6 demonstrates that HDL taken 20 min or 6 hours after
SEQ ID NO:258 from Table 4 synthesized from D-amino acids was
instilled into the stomachs of apoE null mice by stomach tube, was
converted from pro-inflammatory to anti-inflammatory and was
similar to that from mice that received D-4F and quite different
from mice that received a peptide with the same D-amino acids as in
D-4F but arranged in such a way as to prevent the formation of a
class A amphipathic helix and hence rendering the peptide unable to
bind lipids (scrambled D-4F).
[0355] FIG. 7 demonstrates that at both 20 min and 6 hours after
oral administration of D-4F or SEQ ID NO:258 synthesized from
D-amino acids the mouse LDL was significantly less able to induce
monocyte chemotactic activity compared to LDL taken from mice that
received the scrambled D-4F peptide.
[0356] FIG. 8 demonstrates that adding SEQ ID NO:238 in Table 4
(synthesized from all D-amino acids) to the food of apoE null mice
for 18 hours converted the pro-inflammatory HDL of apoE null mice
to anti-inflammatory HDL.
[0357] FIG. 9 demonstrates that in vitro SEQ ID NO:258 in Table 4
was ten times more potent than SEQ ID NO:238.
[0358] As shown in FIG. 3 SEQ ID NO:238 at 125 .mu.g/ml was only
mildly effective while as shown in FIG. 9, SEQ ID NO:258 was highly
active at 12.5 .mu.g/ml in a pre-incubation in vitro.
[0359] The experiments shown in FIG. 10 demonstrate that SEQ ID
NO:243, SEQ ID NO: 242, and SEQ ID NO:256 from Table 4 were also
able to convert the pro-inflammatory HDL of apoE null mice to
anti-inflammatory HDL.
[0360] The activity of particular peptides of this invention is
dependent on particular amino acid substitutions as shown in FIGS.
11, 12, and 13. SEQ ID NO:254 is identical with SEQ ID NO:258
except that the positions of the arginine and glutamic acid amino
acids are reversed in the sequence (i.e. SEQ ID NO:254 is
Boc-Lys(eBoc)-Glu-Arg-Ser(tBu)-OtBu, while SEQ ID NO:258 is
Boc-Lys(FBoc)-Arg-Glu-Ser(tBu)-OtBu). As a result of this seemingly
minor change, SEQ ID NO: 254 is substantially less effective in
these assays than SEQ ID NO:258.
[0361] The experiments described in FIGS. 11 and 12 demonstrate
that SEQ ID NO:258 from Table 4 was more effective in converting
pro-inflammatory HDL to anti-inflammatory HDL and rendering LDL
less able to induce monocyte chemotactic activity than was either
SEQ ID NO:254 or SEQ ID NO:282.
[0362] Serum Amyloid A (SAA) is a positive acute phase reactant in
mice that is similar to C-Reactive Protein (CRP) in humans. The
data in FIG. 13 indicate that this acute phase reactant was
significantly reduced in plasma after injection of SEQ ID NO:258
and to a lesser, non-significant degree after injection of SEQ ID
NO:254 and 282.
[0363] FIG. 14 demonstrates that the peptide described in Table 4
as SEQ ID NO:258, when synthesized from all L-amino acids and given
to apoE null mice orally converted pro-inflammatory HDL to
anti-inflammatory and increased plasma paraoxonase activity (FIG.
15).
[0364] FIGS. 16, 17, 18, and 19 demonstrate that the peptide
described in Table 4 as SEQ ID NO:258 when synthesized from all
D-amino acids and given orally to apoE null mice rendered HDL
anti-inflammatory (FIGS. 16 and 17), reducing LDL-induced monocyte
chemotactic activity (FIG. 17) and increasing plasma
HDL-cholesterol (FIG. 18) and increasing HDL paraoxonase activity
(FIG. 19). These data also show that SEQ ID NO:238, when
synthesized from all L-amino acids and given orally to apoE null
mice, did not significantly alter HDL inflammatory properties
(FIGS. 16 and 17) nor did it significantly alter LDL-induced
monocyte chemotactic activity (FIG. 17) nor did it significantly
alter plasma HDL-cholesterol concentrations (FIG. 18), nor did it
significantly alter HDL paraoxonase activity (FIG. 19).
Additionally these data show that when SEQ ID NO:238 from Table 4
was synthesized from all D-amino acids and was given orally to apoE
null mice, HDL was rendered anti-inflammatory (FIGS. 16 and 17),
and reduced LDL-induced monocyte chemotactic activity (FIG. 17),
but neither change was as dramatic as with SEQ ID NO:258. Moreover,
unlike SEQ ID NO:258, SEQ ID NO:238 from Table 4 when synthesized
from all D-amino acids did not raise plasma HDL-cholesterol
concentrations (FIG. 18) and did not increase HDL paraoxonase
activity (FIG. 19). We conclude that SEQ ID NO:238 from Table 4
when synthesized from L-amino acids is not effective when given
orally but is effective when synthesized from D-amino acids, but is
substantially less effective than SEQ ID NO:258.
[0365] The data presented herein demonstrate that SEQ ID NO:238
when synthesized from all L-arnino acids and given orally is
generally ineffective, and when synthesized from all D-amino acids,
while effective, is substantially less effective than the same dose
of SEQ ID NO:258 synthesized from all D-amino acids when
administered orally.
Example 2
Peptides Synergize Statin Activity
[0366] FIGS. 20 and 21 show the very dramatic synergy between a
statin (pravastatin) and D-4F in ameliorating atherosclerosis in
apoE null mice. Mice are known to be resistant to statins. The mice
that received pravastatin in their drinking water at 20 .mu.g/ml
consumed a dose of pravastatin equal to 175 mg per day for a 70 Kg
human and the mice that received pravastatin in their drinking
water at 50 .mu.g/ml consumed a dose of pravastatin equal to 437.5
mg per day for a 70 Kg human. As shown in FIGS. 20 and 21, these
very high doses of pravastatin were not effective in ameliorating
atherosclerotic lesions in apoE null mice. As shown in FIGS. 20 and
21, adding D-4F alone to the drinking water of the apoE null mice
at concentrations of 2 .mu.g/ml or 5 .mu.g/ml did not reduce
atherosclerotic lesions. These doses of D-4F would be equivalent to
doses of 17.5 mg per day, and 43.75 mg per day, respectively, for a
70 Kg human. Remarkably, as shown in FIGS. 20 and 21, adding the
same concentrations of pravastatin and D-4F together to the
drinking water of the apoE null mice essentially abolished
atherosclerosis in these mice. This indicates a very high degree of
synergy between a statin (pravastatin) and D-4F.
[0367] FIG. 22 shows that SEQ ID NO.198 and SEQ ID NO. 203 from
Table 4 were equally effective or even more effective than D-4F in
reducing the lipid hydroperoxide content of both LDL and HDL in
apoE null mice. These data are consistent with D-4F and the
peptides described in this application acting in part by
sequestering the "seeding molecules" necessary for LDL to induce
the inflammatory atherosclerotic reaction. Taken together with the
data shown in FIGS. 3 to 19 it is very likely that the peptides
described in this application (e.g., SEQ ID NO:250 198 and SEQ ID
NO: 258 from Table 4) will be as or more effective than D-4F in
ameliorating atherosclerosis.
Example 3
Physical Properties of Novel Small Organic Molecules (Molecular
Weight<900 Daltons) that Predict Ability to Render HDL More
Anti-Inflammatory and Mitigate Atherosclerosis in a Mammal
[0368] It was a surprising finding of this invention that a number
of physical properties predict the ability of the small peptides of
this invention to render HDL more anti-inflammatory and to mitigate
atherosclerosis and/or other pathologies characterized by an
inflammatory response in a mammal. The physical properties include
high solubility in ethyl acetate (e.g., greater than about 4
mg/mL), and solubility in aqueous buffer at pH 7.0. Upon contacting
phospholipids such as 1,2-Dimyristoyl-sn-glycero-3-phosphocholine
(DMPC), in an aqueous environment, the particularly effective small
peptides form particles with a diameter of approximately 7.5 nm
(.+-.0.1 nm), and/or form stacked bilayers with a bilayer dimension
on the order of 3.4 to 4.1 nm with spacing between the bilayers in
the stack of approximately 2 nm, and/or also form vesicular
structures of approximately 38 nm). In certain preferred
embodiments, the small peptides have a molecular weight of less
than about 900 Da.
[0369] The predictive effect of these physical properties is
illustrated by a comparison of two sequences:
16 SEQ ID NO 254: Boc-Lys(.epsilon.Boc)-Glu-Arg-Ser(tBu)-OtBu; and
SEQ ID NO 258: Boc-Lys(.epsilon.Boc)-Arg-Glu-Ser(t- Bu)-OtBu
[0370] To evaluate solubility in ethyl acetate, each peptide was
weighed and added to a centrifuge tube and ethyl acetate (HPLC
grade; residue after evaporation <0.0001%) was added to give a
concentration of 10 mg/mL. The tubes were sealed, vortexed and kept
at room temperature for 30 minutes with vortexing every 10 minutes.
The tubes were then centrifuged for 5 minutes at 10,000 rpm and the
supernatant was removed to a previously weighed tube. The ethyl
acetate was evaporated under argon and the tubes weighed to
determine the amount of peptide that had been contained in the
supernatant. The percent of the originally added peptide that was
dissolved in the supernatant is shown on the Y-axis. The data are
mean.+-.S.D. Control represents sham treated tubes; SEQ ID NO 254
and SEQ ID NO 258 were both synthesized from all D-amino acids; SEQ
ID NO 250 was synthesized from all L-amino acids.
[0371] As shown in FIG. 23, SEQ ID NO 258 is very soluble in ethyl
acetate while SEQ ID NO 254 is not (both synthesized from all
D-amino acids). Additionally the data in FIG. 23 demonstrate that
SEQ ID NO 250 [Boc-Phe-Arg-Glu-Leu-OtBu] (synthesized from all
L-amino acids) is also very soluble in ethyl acetate.
[0372] To 1 mg/ml of DMPC suspension in phosphate buffered saline
(PBS) was added 10% deoxycholate until the DMPC was dissolved.
Peptides, SEQ ID NO 258 or SEQ ID NO 254, were added (DMPC:
peptide; 1:10; wt:wt) and the reaction mixture dialyzed. After
dialysis the solution remained clear with SEQ ID NO 258 but was
turbid after the deoxycholate was removed by dialysis in the case
of SEQ ID NO 254.
[0373] FIGS. 24-26--demonstrate that when SEQ ID NO 258 was added
to DMPC in an aqueous environment particles with a diameter of
approximately 7.5 nm formed, stacked lipid bilayers with a bilayer
dimension on the order of 3.4 to 4.1 nm with spacing between the
bilayers in the stack of approximately 2 nm formed, and vesicular
structures of approximately 38 nm also formed.
[0374] In particular, FIG. 24 shows an electron micrograph prepared
with negative staining and at 147,420.times. magnification. The
arrows indicate SEQ ID NO 258 particles measuring 7.5 nm (they
appear as small white particles).
[0375] As illustrated in FIG. 25 a peptide comprising SEQ ID NO 258
added to DMPC in an aqueous environment forms particles with a
diameter of approximately 7.5 nm (white arrows), and stacked
lipid-peptide bilayers (striped arrows pointing to the white lines
in the cylindrical stack of disks) with a bilayer dimension on the
order of 3.4 to 4.1 nm with spacing between the bilayers (black
lines between white lines in the stack of disks) of approximately 2
nm.
[0376] FIG. 26 shows that the peptide of SEQ ID NO 258 added to
DMPC in an aqueous environment forms stacked lipid-peptide bilayers
(striped arrow) and vesicular structures of approximately 38 nm
white arrows).
[0377] FIG. 27 shows that DMPC in an aqueous environment without
SEQ ID NO 258 does not form particles with a diameter of
approximately 7.5 nm, or stacked lipid-petide bilayers, nor
vesicular structures of approximately 38 nm.
[0378] The peptide of SEQ ID NO 254 (which differs from the peptide
of SEQ ID NO 258 only in the order of arginine and glutamic acid in
regard to the amino and carboxy termini of the peptide) did not
form particles with a diameter of approximately 7.5 nm, or stacked
lipid-peptide bilayers, nor vesicular structures of approximately
38 nm under the conditions as described in FIG. 24 (data not
shown). Thus, the order of arginine and glutamic acid in the
peptide dramatically altered its ability to interact with DMPC and
this was predicted by the solubility in ethyl acetate (i.e., the
peptide of SEQ ID NO 258 was highly soluble in ethyl acetate and
formed particles with a diameter of approximately 7.5 nm, and
stacked lipid-peptide bilayers, as well as vesicular structures of
approximately 38 nm, while the peptide of SEQ ID NO 254 was poorly
soluble in ethyl acetate and did not form these structures under
the conditions described in FIG. 24). In addition to the protocol
described in FIG. 24, similar results were also obtained if the
DMPC suspension in PBS was added to the peptide of SEQ ID NO 258
(DMPC:peptide; 1:10; wt:wt) or to the peptide of SEQ ID NO 254
(DMPC:peptide; 1:10; wt:wt) and the mixture recycled between just
above the transition temperature of DMPC (just above 50.degree. C.)
and room temperature each hour for several cycles and then left at
room temperature for 48 hours (data not shown).
[0379] The physical properties of the peptide of SEQ ID NO 258 (but
not the peptide of SEQ ID NO 254) indicate that this peptide has
amphipathic properties (i.e., it is highly soluble in ethyl
acetate, it is also soluble in aqueous buffer at pH 7.0 [data not
shown], and it interacts with DMPC as described above). It was a
surprising finding of this invention that the peptides that are
highly soluble in ethyl acetate, and are also soluble in aqueous
buffer at pH 7.0, interacted with DMPC to form lipid-peptide
complexes that are remarkably similar to the nascent HDL particles
formed by the interaction of apoA-I with cells (Forte, et al.
(1993) J. Lipid Res. 34: 317-324).
[0380] Table 13 compares the interaction of lipid-free human apoA-I
with CHO--C19 cells in vitro with the interaction of SEQ ID NO 258
with DMPC as indicated in FIGS. 4-7 above.
17TABLE 13 Comparison of the interaction of the peptide of SEQ ID
NO 258 with DMPC as indicated in FIGS. 24-27 above with the
interaction of lipid-free human apoA-I interacting with CHO-C-19
cells as described in Forteet al. (1993) J. Lipid Res. 34: 317-324.
SEQ ID NO Property ApoA-I/Cells 258/DMPC Prominent Feature
Discoidal particles Stacked bilayers in stacked in rouleaux
cylindrical form formation Bilayer dimension 4.6 nm 3.4-4.1 nm
Spacing between discoidal 1.9 nm 2.0 nm particles/bilayers Size
"Nascent HDL Particles" 7.3 nm 7.5 nm Vesicular structures 34.7 nm
38 nm
[0381] Thus, the small peptides described here that are highly
soluble in ethyl acetate and are also soluble in aqueous buffers at
pH 7.0 interact with lipids (DMPC) similar to apoA-I, which has a
molecular weight of 28,000 Daltons.
[0382] The molecular models shown in FIGS. 28-32 demonstrate the
spatial characteristics of SEQ ID NO 254 compared to SEQ ID NO
258.
[0383] The molecular models shown in FIGS. 28-32 indicate that both
the peptide of SEQ ID NO 254 and the peptide of SEQ ID NO 258
contain polar and non-polar portions in each molecule but there are
spatial differences in the arrangement of the polar and non-polar
components of the two molecules. As a result of the differences in
the spatial arrangement of the molecules there are differences in
the solubility of the two molecules in ethyl acetate (FIG. 23) and
in their interaction with DMPC (FIGS. 24-27).
[0384] The data in FIGS. 33-35 demonstrate that the physical
properties of the peptide of SEQ ID NO 254 versus the peptide of
SEQ ID NO 258 predict the ability of these molecules to render HDL
anti-inflammatory and mitigate atherosclerosis when given orally to
a mammal.
[0385] Female apoE null mice at age 8 weeks were given no additions
to their diet (Chow) or received 200 .mu.g/gm chow of SEQ ID NO 254
(+254) or 200 .mu.g/gm chow of SEQ ID NO 258 (+258), both
synthesized from all D-amino acids. After 15 weeks the mice were
bled and their plasma fractionated by FPLC and their HDL (MHDL)
tested in a human artery wall cell coculture. A standard human LDL
(at 100 .mu.g/mL of LDL-cholesterol) was added alone (LDL) or not
added (no addition) or was added with 50 .mu.g/mL of normal human
HDL (hHDL) or 50 .mu.g/mL of mouse HDL (MHDL) to human artery wall
cocultures and the resulting monocyte chemotactic activity was
determined and plotted on the Y-axis. FIG. 33 shows that the HDL
from apoE null mice was rendered anti-inflammatory after the mice
were fed SEQ ID NO 258 but not after SEQ ID NO 254.
[0386] As shown in FIG. 34 the peptide of SEQ ID NO 258 but not the
peptide of SEQ ID NO 254 significantly reduced atherosclerosis in
the aortic root (aortic sinus) of the apoE null mice described
above. FIG. 35 demonstrates that SEQ ID NO 258 but not SEQ ID NO
254 also significantly decreased atherosclerosis in en face
preparations of the aortas. FIG. 23 demonstrates that the
solubility in ethyl acetate of SEQ ID NO 250 synthesized from all
L-amino acids (see FIG. 23 above) accurately predicts the ability
of this molecule to ameliorate atherosclerosis in apoE null
mice.
[0387] Thus, the physical properties of these small peptides
accurately predicted the ability of the peptides to ameliorate
atherosclerosis in apoE null mice.
[0388] We thus teach that small peptides, typically with molecular
weights of less than about 900 Daltons that are highly soluble in
ethyl acetate (greater than about 4 mg/mL), and also are soluble in
aqueous buffer at pH 7.0, and that when contacted with
phospholipids such as 1,2-Dimyristoyl-sn-glycero-3-phosphocholine
(DMPC), in an aqueous environment, form particles with a diameter
of approximately 7.5 nm, and/or form stacked bilayers with a
bilayer dimension on the order of 3.4 to 4.1 nm with spacing
between the bilayers in the stack of approximately 2 nm, and/or
they also form vesicular structures of approximately 38 nm, when
administered to a mammal render HDL more anti-inflammatory and
mitigate one or more symptoms of atherosclerosis and other
pathologies characterized by an inflammatory response.
[0389] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
* * * * *
References