U.S. patent application number 11/333889 was filed with the patent office on 2007-08-23 for compounds for control of appetite.
This patent application is currently assigned to University of Cincinnati. Invention is credited to Ambikaipakan Balasubramaniam.
Application Number | 20070197445 11/333889 |
Document ID | / |
Family ID | 38429007 |
Filed Date | 2007-08-23 |
United States Patent
Application |
20070197445 |
Kind Code |
A1 |
Balasubramaniam;
Ambikaipakan |
August 23, 2007 |
Compounds for control of appetite
Abstract
This invention relates generally to neuropeptide Y ("NPY")
Y.sub.4 receptor agonists including pancreatic polypeptide (PP),
analogs thereof, and peptide fragments of PP, e.g. PP(32-36), and
analogs thereof, to pharmaceutical compositions containing such
Y.sub.4 receptor agonists, and to methods for treatment of mammals
using the same. The NPY Y.sub.4 receptor agonists may be
administered to mammals either alone or in combination with NPY
Y.sub.2 receptor agonists including peptide YY (PYY) (3-36),
analogs thereof, and to peptide fragments of PYY(3-36), e.g.
PYY(22-36) and PYY(25-36), and analogs thereof, such as to control
food intake in mammals, blood pressure, cardiovascular response,
libido, circadian rhythm, hyperlipidimia, chronic pancreatitis, and
nonalcoholic fatty liver disease including nonalcoholic
steatohepatitis.
Inventors: |
Balasubramaniam; Ambikaipakan;
(Cincinnati, OH) |
Correspondence
Address: |
WOOD, HERRON & EVANS, LLP
2700 CAREW TOWER
441 VINE STREET
CINCINNATI
OH
45202
US
|
Assignee: |
University of Cincinnati
Cincinnati
OH
|
Family ID: |
38429007 |
Appl. No.: |
11/333889 |
Filed: |
January 18, 2006 |
Current U.S.
Class: |
514/4.9 ;
514/15.6; 514/16.4; 514/5.2; 514/7.4; 530/327; 530/328 |
Current CPC
Class: |
A61P 3/04 20180101; C07K
7/06 20130101; C07K 7/08 20130101; A61K 38/00 20130101; C07K
14/57545 20130101 |
Class at
Publication: |
514/014 ;
530/327; 530/328 |
International
Class: |
A61K 38/10 20060101
A61K038/10; C07K 7/08 20060101 C07K007/08 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0001] The U.S. Government has a paid-up license in this invention
and the right in limited circumstances to require the patent owner
to license others on reasonable terms as provided for by the terms
of Grant/Contract No. GM47122-08S1 awarded by the National
Institutes of Health.
Claims
1. A compound having the formula: ##STR26## wherein: each X,
independently, is an aromatic amino acid; each Y, independently, is
an amino acid having a guanidino group; each Z, independently, is
an aliphatic amino acid; n=1, 2, 3 or 4; and wherein the compound
optionally includes one or two pseudopeptide bonds where each
pseudopeptide bond is independently selected from --CH.sub.2--NH--,
--CH.sub.2--S--, --CH.sub.2--CH.sub.2--, --CH.sub.2--O-- and
CH.sub.2--CO--.
2. The compound of claim 1 wherein the compound includes one or two
pseudopeptide bonds where each pseudopeptide bond is
--CH.sub.2--NH--.
3. The compound of claim 1 wherein the aromatic amino acid is
selected from the group consisting of Phe, Trp, and Tyr and the
aliphatic amino acid is selected from the group consisting of Ile,
Leu, Val, Ala, Gly, and Nva.
4. The compound of claim 1 wherein the aromatic amino acid is a
hydroxyl aromatic amino acid.
5. The compound of claim 1, wherein said compound is ##STR27##
6. A pharmaceutically acceptable salt of the compound of claim
1.
7. A method for controlling an NPY mediated physiological response
in a subject comprising administering to said subject the compound
of claim 1.
8. The method of claim 7 wherein the composition is capable of
suppressing appetite.
9. The method of claim 7 wherein the step of administering to said
subject the compound of claim 1 includes administering to said
subject a mixture of the compound of claim 1 and a second compound
having the formula: ##STR28## wherein X is a chain of 0-5 amino
acids, inclusive, where the N-terminal amino acid is bonded to
R.sup.1 and R.sup.2 by the nitrogen of the amino group of the
N-terminal amino acid; Y is a chain of 0-4 amino acids, inclusive,
where the C-terminal amino acid has a carboxylamide group, which is
independently bonded to R.sup.3 and R.sup.4; R.sup.1 and R.sup.2
are each independently bonded to the amino group of the N-terminal
amino acid and selected from H, (C.sub.1-C.sub.12)alkyl,
(C.sub.6-C.sub.18)aryl, (C.sub.1-C.sub.12)acyl, (C.sub.7-C.sub.18)
aralkyl, and (C.sub.7-C.sub.18)alkaryl; R.sup.3 and R.sup.4 are
each independently bonded to the amide group of the C-terminus
amino acid and selected from H, (C.sub.1-C.sub.12)alkyl,
(C.sub.6-C.sub.18)aryl, (C.sub.1-C.sub.12)acyl, (C.sub.7-C.sub.18)
aralkyl, and (C.sub.7-C.sub.18)alkaryl; A.sup.22 is an aromatic
amino acid, Ala, Aib, Anb, N-Me-Ala or is deleted; A.sup.23 is Ser,
Thr, Ala, Aib, N-Me-Ser, N-Me-Thr, N-Me-Ala or is deleted; A.sup.24
is Leu, Ile, Nle, Val, Trp, Gly, Nva, Aib, Anb, N-Me-Leu or is
deleted; A.sup.25 is Arg, Lys, homo-Arg, diethyl-homo-Arg,
Lys-p.epsilon.-NH--R (where R is H, a branched or straight chain
(C.sub.1-C.sub.10) alkyl group, or an aryl group), Orn or is
deleted; A.sup.26 is Ala, His, Thr, 3-Me-His, 1-Me-His,
.beta.-pyrazolylalaline, N-Me-His, Arg, Lys, homo-Arg,
diethyl-homo-Arg, Lys-.epsilon.-NH--R (where R is H, a branched or
straight chain (C.sub.1-C.sub.10) alkyl group, an aryl group, or a
pharmaceutically acceptable salt thereof), Orn or is deleted;
A.sup.27 is an aromatic amino acid; A.sup.28 is Leu, Ile, Nle, Val,
Trp, Aib, Anb or N-Me-Leu; A.sup.29 is Asn, Ala, Gln, Gly, Trp or
N-Me-Asn; A.sup.30 is Leu, Ile, Nle, Nva, Fla, Val, Trp, Aib, Anb
or N-Me-Leu; A.sup.31 is Val, Leu, Nle, Nva, Ile, Trp, Aib, Anb or
N-Me-Val; A.sup.32 is Thr, Ser, D-Trp, N-Me-Ser or N-Me-Thr; and
wherein the compound optionally includes one or two pseudopeptide
bonds where each pseudopeptide bond is independently selected from
--CH.sub.2--NH--, --CH.sub.2--S--, --CH.sub.2--CH.sub.2--,
--CH.sub.2--O-- and CH.sub.2--CO--.
10. The method of claim 9 wherein the second compound is
N-.alpha.-Ac[Nle.sup.24,28, Trp.sup.30, Nva.sup.31,
.psi..sup.35/36]PYY(22-36)-NH.sub.2 (SEQ. ID. NO. 7) wherein .psi.
is --CH2-NH--.
11. The method of claim 10 wherein the compound of claim 1 is
##STR29##
12. The method of claim 7 wherein the step of administering to said
subject the compound of claim 1 includes administering to said
subject a mixture of the compound of claim 1 and a second compound
having the formula: ##STR30## wherein: the N-terminal amino acid is
bonded to R.sup.1 and R.sup.2; Y is a chain of 0-4 amino acids,
inclusive, where the C-terminal amino acid is bonded to R.sup.3 and
R.sup.4 by the side chain of the C-terminal amino acid or by the
carbon of the carboxyl group of the C-terminal amino acid; R.sup.1
and R.sup.2 are each independently bonded to the amino group of the
N-terminal amino acid and selected from H, (C.sub.1-C.sub.12)alkyl,
(C.sub.6-C.sub.18)aryl, (C.sub.1-C.sub.12)acyl, (C.sub.7-C.sub.18)
aralkyl, and (C.sub.7-C.sub.18)alkaryl; R.sup.3 and R.sup.4 are
each independently bonded to the amide group of the C-terminus
amino acid and are selected from H, (C.sub.1-C.sub.12)alkyl,
(C.sub.6-C.sub.18)aryl, (C.sub.1-C.sub.12)acyl,
(C.sub.7-C.sub.18)aralkyl, and (C.sub.7-C.sub.18)alkaryl; A.sup.25
is Arg, Lys, homo-Arg, diethyl-homo-Arg, lys-.epsilon.-NH--R where
R is H, a branched or straight chain C.sub.1-C.sub.10 alkyl group,
or an aryl group, Orn or is deleted; A.sup.26 is Ala, His, Thr,
3-Me-His, 1-Me-His, .beta.-pyrozolylalanin, N-Me-His, Arg, Lys,
homo-Arg, diethyl-homo-Arg, Lys-e-NH--R where R is H, a branched or
straight chain (C.sub.1-C.sub.10) alkyl group, or an aryl group,
Orn or is deleted; A.sup.27 is an aromatic amino acid: A.sup.28 is
Leu, Ile, Val, Trp Nle, Nva, Aib, Anb, or N-Me-Leu; A.sup.29 is
Asn, Ala, Gin, Fly, Trp, or N-Me-Asn; A.sup.30 is Leu, Ile, Val,
Trp, Nle, Nva, Aib, Anb, or N-Me-Leu; A.sup.31 is Val, Ile, Trp,
Nva, Aib, Anb, or N-Me-Val; A.sup.32 is Thr, Ser, N-Me-Ser,
N-Me-Thr, or D-Trp; and wherein the compound optionally includes
one or two pseudopeptide bonds where each pseudopeptide bond is
independently selected from --CH.sub.2--NH--, --CH.sub.2--S--,
--CH.sub.2--CH.sub.2--, --CH.sub.2--O-- and CH.sub.2--CO--.
13. A therapeutic composition capable of controlling an NPY
mediated physiological response comprising a therapeutically
effective amount of the compound of claim 1 together with a
pharmaceutically acceptable carrier substance.
14. The composition of claim 13, wherein the therapeutic
composition is capable of suppressing appetite.
15. The composition of claim 13, wherein said composition is in the
form of a liquid, pill, tablet, or capsule for oral administration
to a subject.
16. The composition of claim 13, wherein said composition is in the
form of a liquid for nasal, intravenous, subcutaneous, parenteral,
or intraperitoneal administration to a subject.
17. The composition of claim 13 wherein the therapeutically
effective amount of the compound of claim 1 includes a
therapeutically effective amount of a mixture of the compound of
claim 1 and a second compound having the formula: ##STR31## wherein
X is a chain of 0-5 amino acids, inclusive, where the N-terminal
amino acid is bonded to R.sup.1 and R.sup.2 by the nitrogen of the
amino group of the N-terminal amino acid; Y is a chain of 0-4 amino
acids, inclusive, where the C-terminal amino acid has a
carboxylamide group, which is independently bonded to R.sup.3 and
R.sup.4; R.sup.1 and R.sup.2 are each independently bonded to the
amino group of the N-terminal amino acid and selected from H,
(C.sub.1-C.sub.12)alkyl, (C.sub.6-C.sub.18)aryl,
(C.sub.1-C.sub.12)acyl, (C.sub.7-C.sub.18) aralkyl, and
(C.sub.7-C.sub.18)alkaryl; R.sup.3 and R.sup.4 are each
independently bonded to the amide group of the C-terminus amino
acid and selected from H, (C.sub.1-C.sub.12)alkyl,
(C.sub.6-C.sub.18)aryl, (C.sub.1-C.sub.12)acyl,
(C.sub.7-C.sub.18)aralkyl, and (C.sub.7-C.sub.18)alkaryl; A.sup.22
is an aromatic amino acid, Ala, Aib, Anb, N-Me-Ala or is deleted;
A.sup.23 is Ser, Thr, Ala, Aib, N-Me-Ser, N-Me-Thr, N-Me-Ala or is
deleted; A.sup.24 is Leu, Ile, Nle, Val, Trp, Gly, Nva, Aib, Anb,
N-Me-Leu or is deleted; A.sup.25 is Arg, Lys, homo-Arg,
diethyl-homo-Arg, Lys-p.epsilon.-NH--R (where R is H, a branched or
straight chain (C.sub.1-C.sub.10) alkyl group, or an aryl group),
Orn or is deleted; A.sup.26 is Ala, His, Thr, 3-Me-His, 1-Me-His,
.beta.-pyrazolylalaline, N-Me-His, Arg, Lys, homo-Arg,
diethyl-homo-Arg, Lys-.epsilon.-NH--R (where R is H, a branched or
straight chain (C.sub.1-C.sub.10) alkyl group, an aryl group, or a
pharmaceutically acceptable salt thereof), Orn or is deleted;
A.sup.27 is an aromatic amino acid; A.sup.28 is Leu, Ile, Nle, Val,
Trp, Aib, Anb or N-Me-Leu; A.sup.29 is Asn, Ala, Gln, Gly, Trp or
N-Me-Asn; A.sup.30 is Leu, Ile, Nle, Nva, Fla, Val, Trp, Aib, Anb
or N-Me-Leu; A.sup.31 is Val, Leu, Nle, Nva, Ile, Trp, Aib, Anb or
N-Me-Val; A.sup.32 is Thr, Ser, D-Trp, N-Me-Ser or N-Me-Thr; and
wherein the compound optionally includes one or two pseudopeptide
bonds where each pseudopeptide bond is independently selected from
--CH.sub.2--NH--, --CH.sub.2--S--, --CH.sub.2--CH.sub.2--,
--CH.sub.2--O-- and CH.sub.2--CO--.
18. The composition of claim 17 wherein the second compound is
N-.alpha.-Ac[Nle.sup.24,28, Trp.sup.30, Nva.sup.31,
.psi..sup.35/36]PYY(22-36)-NH.sub.2 (SEQ. ID. NO. 7) wherein .psi.
is --CH2-NH--.
19. The composition of claim 18 wherein the compound of claim 1 is
##STR32##
20. The composition of claim 13 wherein the therapeutically
effective amount of the compound of claim 1 includes a
therapeutically effective amount of a mixture of the compound of
claim 1 and a second compound having the formula: ##STR33##
wherein: the N-terminal amino acid is bonded to R.sup.1 and
R.sup.2; Y is a chain of 0-4 amino acids, inclusive, where the
C-terminal amino acid has a carboxylamide group, which is
independently bonded to R.sup.3 and R.sup.4; R.sup.1 and R.sup.2
are each independently bonded to the amino group of the N-terminal
amino acid and selected from H, (C.sub.1-C.sub.12)alkyl,
(C.sub.6-C.sub.18)aryl, (C.sub.1-C.sub.12)acyl, (C.sub.7-C.sub.18)
aralkyl, and (C.sub.7-C.sub.18)alkaryl; R.sup.3 and R.sup.4 are
each independently bonded to the amide group of the C-terminus
amino acid and are selected from H, (C.sub.1-C.sub.12)alkyl,
(C.sub.6-C.sub.18)aryl, (C.sub.1-C.sub.12)acyl,
(C.sub.7-C.sub.18)aralkyl, and (C.sub.7-C.sub.18)alkaryl; A.sup.25
is Arg, Lys, homo-Arg, diethyl-homo-Arg, lys-.epsilon.-NH--R where
R is H, a branched or straight chain C.sub.1-C.sub.10 alkyl group,
or an aryl group, Orn or is deleted; A.sup.26 is Ala, His, Thr,
3-Me-His, 1-Me-His, .beta.-pyrozolylalanin, N-Me-His, Arg, Lys,
homo-Arg, diethyl-homo-Arg, Lys-e-NH--R where R is H, a branched or
straight chain (C.sub.1-C.sub.10) alkyl group, or an aryl group,
Orn or is deleted; A.sup.27 is an aromatic amino acid: A.sup.28 is
Leu, Ile, Val, Trp Nle, Nva, Aib, Anb, or N-Me-Leu; A.sup.29 is
Asn, Ala, Gin, Fly, Trp, or N-Me-Asn; A.sup.30 is Leu, Ile, Val,
Trp, Nle, Nva, Aib, Anb, or N-Me-Leu; A.sup.31 is Val, Ile, Trp,
Nva, Aib, Anb, or N-Me-Val; A.sup.32 is Thr, Ser, N-Me-Ser,
N-Me-Thr, or D-Trp; and wherein the compound optionally includes
one or two pseudopeptide bonds where each pseudopeptide bond is
independently selected from --CH.sub.2--NH--, --CH.sub.2--S--,
--CH.sub.2--CH.sub.2--, --CH.sub.2--O-- and CH.sub.2--CO--.
21. A compound having the formula: ##STR34## wherein: each X,
independently, is an aromatic amino acid; each Y, independently, is
an amino acid having a guanidino group; each Z, independently, is
an aliphatic amino acid; A1 and A2, independently, are selected
from Cys, Pen, Glu, Asp, Lys, and Dpr; and wherein the compound
optionally includes one or two pseudopeptide bonds where each
pseudopeptide bond is independently selected from --CH.sub.2--NH--,
--CH.sub.2--S--, --CH.sub.2--CH.sub.2--, --CH.sub.2--O-- and
CH.sub.2--CO--.
22. The compound of claim 21 wherein the compound includes one or
two pseudopeptide bonds where each pseudopeptide bond is
--CH.sub.2--NH--.
23. The compound of claim 21 wherein the aromatic amino acid is
selected from the group consisting of Phe, Trp, and Tyr and the
aliphatic amino acid is selected from the group consisting of Ile,
Leu, Val, Ala, Gly, and Nva.
24. The compound of claim 21 wherein the aromatic amino acid is a
hydroxyl aromatic amino acid.
25. The compound of claim 21, wherein said compound is
##STR35##
26. A pharmaceutically acceptable salt of the compound of claim
21.
27. A method for controlling an NPY mediated physiological response
in a subject comprising administering to said subject the compound
of claim 21.
28. The method of claim 27 wherein the compound is capable of
suppressing appetite.
29. The method of claim 27 wherein the step of administering to
said subject the compound of claim 21 includes administering to
said subject a mixture of the compound of claim 21 and a second
compound having the formula: ##STR36## wherein X is a chain of 0-5
amino acids, inclusive, where the N-terminal amino acid is bonded
to R.sup.1 and R.sup.2 by the nitrogen of the amino group of the
N-terminal amino acid; Y is a chain of 0-4 amino acids, inclusive,
where the C-terminal amino acid has a carboxylamide group, which is
independently bonded to R.sup.3 and R.sup.4; R.sup.1 and R.sup.2
are each independently bonded to the amino group of the N-terminal
amino acid and selected from H, (C.sub.1-C.sub.12)alkyl,
(C.sub.6-C.sub.18)aryl, (C.sub.1-C.sub.12)acyl, (C.sub.7-C.sub.18)
aralkyl, and (C.sub.7-C.sub.18)alkaryl; R.sup.3 and R.sup.4 are
each independently bonded to the amide group of the C-terminus
amino acid and selected from H, (C.sub.1-C.sub.12)alkyl,
(C.sub.6-C.sub.18)aryl, (C.sub.1-C.sub.12)acyl, (C.sub.7-C.sub.18)
aralkyl, and (C.sub.7-C.sub.18)alkaryl; A.sup.22 is an aromatic
amino acid, Ala, Aib, Anb, N-Me-Ala or is deleted; A.sup.23 is Ser,
Thr, Ala, Aib, N-Me-Ser, N-Me-Thr, N-Me-Ala or is deleted; A.sup.24
is Leu, Ile, Nle, Val, Trp, Gly, Nva, Aib, Anb, N-Me-Leu or is
deleted; A.sup.25 is Arg, Lys, homo-Arg, diethyl-homo-Arg,
Lys-p.epsilon.-NH--R (where R is H, a branched or straight chain
(C.sub.1-C.sub.10) alkyl group, or an aryl group), Orn or is
deleted; A.sup.26 is Ala, His, Thr, 3-Me-His, 1-Me-His,
.beta.-pyrazolylalaline, N-Me-His, Arg, Lys, homo-Arg,
diethyl-homo-Arg, Lys-.epsilon.-NH--R (where R is H, a branched or
straight chain (C.sub.1-C.sub.10) alkyl group, an aryl group, or a
pharmaceutically acceptable salt thereof), Orn or is deleted;
A.sup.27 is an aromatic amino acid; A.sup.28 is Leu, Ile, Nle, Val,
Trp, Aib, Anb or N-Me-Leu; A.sup.29 is Asn, Ala, Gln, Gly, Trp or
N-Me-Asn; A.sup.30 is Leu, Ile, Nle, Nva, Fla, Val, Trp, Aib, Anb
or N-Me-Leu; A.sup.31 is Val, Leu, Nle, Nva, Ile, Trp, Aib, Anb or
N-Me-Val; A.sup.32 is Thr, Ser, D-Trp, N-Me-Ser or N-Me-Thr; and
wherein the compound optionally includes one or two pseudopeptide
bonds where each pseudopeptide bond is independently selected from
--CH.sub.2--NH--, --CH.sub.2--S--, --CH.sub.2--CH.sub.2--,
--CH.sub.2--O-- and CH.sub.2--CO--.
30. The method of claim 29 wherein the second compound is
N-.alpha.-Ac[Nle.sup.24,28, Trp.sup.30, Nva.sup.31,
.psi..sup.35/36]PYY(22-36)-NH.sub.2(SEQ. ID. NO. 7) wherein .psi.
is --CH2-NH--.
31. The method of claim 30 wherein the compound of claim 21 is
##STR37##
32. The method of claim 27 wherein the step of administering to
said subject the compound of claim 21 includes administering to
said subject a mixture of the compound of claim 21 and a second
compound having the formula: ##STR38## wherein: the N-terminal
amino acid is bonded to R.sup.1 and R.sup.2; Y is a chain of 0-4
amino acids, inclusive, where the C-terminal amino acid has a
carboxylamide group, which is independently bonded to R.sup.3 and
R.sup.4; R.sup.1 and R.sup.2 are each independently bonded to the
amino group of the N-terminal amino acid and selected from H,
(C.sub.1-C.sub.12)alkyl, (C.sub.6-C.sub.18)aryl,
(C.sub.1-C.sub.12)acyl, (C.sub.7-C.sub.18) aralkyl, and
(C.sub.7-C.sub.18)alkaryl; R.sup.3 and R.sup.4 are each
independently bonded to the amide group of the C-terminus amino
acid and are selected from H, (C.sub.1-C.sub.12)alkyl,
(C.sub.6-C.sub.18)aryl, (C.sub.1-C.sub.12)acyl, (C.sub.7-C.sub.18)
aralkyl, and (C.sub.7-C.sub.18)alkaryl; A.sup.25 is Arg, Lys,
homo-Arg, diethyl-homo-Arg, lys-.epsilon.-NH--R where R is H, a
branched or straight chain C.sub.1-C.sub.10 alkyl group, or an aryl
group, Orn or is deleted; A.sup.26 is Ala, His, Thr, 3-Me-His,
1-Me-His, .beta.-pyrozolylalanin, N-Me-His, Arg, Lys, homo-Arg,
diethyl-homo-Arg, Lys-e-NH--R where R is H, a branched or straight
chain (C.sub.1-C.sub.10) alkyl group, or an aryl group, Orn or is
deleted; A.sup.27 is an aromatic amino acid: A.sup.28 is Leu, Ile,
Val, Trp Nle, Nva, Aib, Anb, or N-Me-Leu; A.sup.29 is Asn, Ala,
Gin, Fly, Trp, or N-Me-Asn; A.sup.30 is Leu, Ile, Val, Trp, Nle,
Nva, Aib, Anb, or N-Me-Leu; A.sup.31 is Val, Ile, Trp, Nva, Aib,
Anb, or N-Me-Val; A.sup.32 is Thr, Ser, N-Me-Ser, N-Me-Thr, or
D-Trp; and wherein the compound optionally includes one or two
pseudopeptide bonds where each pseudopeptide bond is independently
selected from --CH.sub.2--NH--, --CH.sub.2--S--,
--CH.sub.2--CH.sub.2--, --CH.sub.2--O-- and CH.sub.2--CO--.
33. A therapeutic composition capable of controlling an NPY
mediated physiological response comprising a therapeutically
effective amount of the compound of claim 21 together with a
pharmaceutically acceptable carrier substance.
34. The composition of claim 33 wherein the therapeutic composition
is capable of suppressing appetite.
35. The composition of claim 33 wherein said composition is in the
form of a liquid, pill, tablet, or capsule for oral administration
to a subject.
36. The composition of claim 33 wherein said composition is in the
form of a liquid for nasal, intravenous, subcutaneous, parenteral,
or intraperitoneal administration to a subject to a subject.
37. The composition of claim 33 wherein the therapeutically
effective amount of the compound of claim 21 includes a
therapeutically effective amount of a mixture of the compound of
claim 21 and a second compound having the formula: ##STR39##
wherein X is a chain of 0-5 amino acids, inclusive, where the
N-terminal amino acid is bonded to R.sup.1 and R.sup.2 by the
nitrogen of the amino group of the N-terminal amino acid; Y is a
chain of 0-4 amino acids, inclusive, where the C-terminal amino
acid has a carboxylamide group, which is independently bonded to
R.sup.3 and R.sup.4; R.sup.1 and R.sup.2 are each independently
bonded to the amino group of the N-terminal amino acid and selected
from H, (C.sub.1-C.sub.12)alkyl, (C.sub.6-C.sub.18)aryl,
(C.sub.1-C.sub.12)acyl, (C.sub.7-C.sub.18) aralkyl, and
(C.sub.7-C.sub.18)alkaryl; R.sup.3 and R.sup.4 are each
independently bonded to the amide group of the C-terminus amino
acid and selected from H, (C.sub.1-C.sub.12)alkyl,
(C.sub.6-C.sub.18)aryl, (C.sub.1-C.sub.12)acyl, (C.sub.7-C.sub.18)
aralkyl, and (C.sub.7-C.sub.18)alkaryl; A.sup.22 is an aromatic
amino acid, Ala, Aib, Anb, N-Me-Ala or is deleted; A.sup.23 is Ser,
Thr, Ala, Aib, N-Me-Ser, N-Me-Thr, N-Me-Ala or is deleted; A.sup.24
is Leu, Ile, Nle, Val, Trp, Gly, Nva, Aib, Anb, N-Me-Leu or is
deleted; A.sup.25 is Arg, Lys, homo-Arg, diethyl-homo-Arg,
Lys-p.epsilon.-NH--R (where R is H, a branched or straight chain
(C.sub.1-C.sub.10) alkyl group, or an aryl group), Orn or is
deleted; A.sup.26 is Ala, His, Thr, 3-Me-His, .beta.-Me-His,
.beta.-pyrazolylalaline, N-Me-His, Arg, Lys, homo-Arg,
diethyl-homo-Arg, Lys-.epsilon.--NH--R (where R is H, a branched or
straight chain (C.sub.1-C.sub.10) alkyl group, an aryl group, or a
pharmaceutically acceptable salt thereof), Orn or is deleted;
A.sup.27 is an aromatic amino acid; A.sup.28 is Leu, Ile, Nle, Val,
Trp, Aib, Anb or N-Me-Leu; A.sup.29 is Asn, Ala, Gln, Gly, Trp or
N-Me-Asn; A.sup.30 is Leu, Ile, Nle, Nva, Fla, Val, Trp, Aib, Anb
or N-Me-Leu; A.sup.31 is Val, Leu, Nle, Nva, Ile, Trp, Aib, Anb or
N-Me-Val; A.sup.32 is Thr, Ser, D-Trp, N-Me-Ser or N-Me-Thr; and
wherein the compound optionally includes one or two pseudopeptide
bonds where each pseudopeptide bond is independently selected from
--CH.sub.2--NH--, --CH.sub.2--S--, --CH.sub.2--CH.sub.2--,
--CH.sub.2--O-- and CH.sub.2--CO--.
38. The composition of claim 36 wherein the second compound is
N-.alpha.-Ac[Nle.sup.24,28, Trp.sup.30, Nva.sup.31,
.psi..sup.35/36]PYY(22-36)-NH.sub.2 (SEQ. ID. NO. 7) wherein .psi.
is --CH2-NH--.
39. The composition of claim 38 wherein the compound of claim 21 is
##STR40##
40. The composition of claim 33 wherein the therapeutically
effective amount of the compound of claim 21 includes a
therapeutically effective amount of a mixture of the compound of
claim 21 and a second compound having the formula: ##STR41##
wherein: the N-terminal amino acid is bonded to R.sup.1 and
R.sup.2; Y is a chain of 0-4 amino acids, inclusive, where the
C-terminal amino acid has a carboxylamide group, which is
independently bonded to R.sup.3 and R.sup.4; R.sup.1 and R.sup.2
are each independently bonded to the amino group of the N-terminal
amino acid and selected from H, (C.sub.1-C.sub.12)alkyl,
(C.sub.6-C.sub.18)aryl, (C.sub.1-C.sub.12)acyl, (C.sub.7-C.sub.18)
aralkyl, and (C.sub.7-C.sub.18)alkaryl; R.sup.3 and R.sup.4 are
each independently bonded to the amide group of the C-terminus
amino acid and are selected from H, (C.sub.1-C.sub.12)alkyl,
(C.sub.6-C.sub.18)aryl, (C.sub.1-C.sub.12)acyl, (C.sub.7-C.sub.18)
aralkyl, and (C.sub.7-C.sub.18)alkaryl; A.sup.25 is Arg, Lys,
homo-Arg, diethyl-homo-Arg, lys-.epsilon.-NH--R where R is H, a
branched or straight chain (C.sub.1-C.sub.10) alkyl group, or an
aryl group, Orn or is deleted; A.sup.26 is Ala, His, Thr, 3-Me-His,
1-Me-His, .beta.-pyrozolylalanin, N-Me-His, Arg, Lys, homo-Arg,
diethyl-homo-Arg, Lys-e-NH--R where R is H, a branched or straight
chain (C.sub.1-C.sub.10) alkyl group, or an aryl group, Orn or is
deleted; A.sup.27 is an aromatic amino acid: A.sup.28 is Leu, Ile,
Val, Trp Nle, Nva, Aib, Anb, or N-Me-Leu; A.sup.29 is Asn, Ala,
Gin, Fly, Trp, or N-Me-Asn; A.sup.30 is Leu, Ile, Val, Trp, Nle,
Nva, Aib, Anb, or N-Me-Leu; A.sup.31 is Val, Ile, Trp, Nva, Aib,
Anb, or N-Me-Val; A.sup.32 is Thr, Ser, N-Me-Ser, N-Me-Thr, or
D-Trp; and wherein the compound optionally includes one or two
pseudopeptide bonds where each pseudopeptide bond is independently
selected from --CH.sub.2--NH--, --CH.sub.2--S--,
--CH.sub.2--CH.sub.2--, --CH.sub.2--O-- and CH.sub.2--CO--.
41. A compound having the formula: H--[X--Y-Z-Y--X].sub.n--NH.sub.2
wherein: each X, independently, is an aromatic amino acid; each Y,
independently, is an amino acid having a guanidino group; each Z,
independently, is an aliphatic amino acid; n=1, 2, 3 or 4; and
wherein the compound optionally includes one or two pseudopeptide
bonds where each pseudopeptide bond is independently selected from
--CH.sub.2--NH--, --CH.sub.2--S--, --CH.sub.2--CH.sub.2--,
--CH.sub.2--O-- and CH.sub.2--CO--.
42. The compound of claim 41 wherein the compound includes one or
two pseudopeptide bonds where each pseudopeptide bond is
--CH.sub.2--NH--.
43. The compound of claim 41 wherein the aromatic amino acid is
selected from the group consisting of Phe, Trp, and Tyr and the
aliphatic amino acid is selected from the group consisting of Ile,
Leu, Val, Ala, Gly, and Nva.
44. The compound of claim 41 wherein the aromatic amino acid is a
hydroxyl aromatic amino acid.
45. The compound of claim 41, wherein said compound is either
H-Tyr-Arg-Leu-Arg-Tyr-Tyr-Arg-Leu-Arg-Tyr-NH.sub.2 (SEQ. ID. NO. 9)
or
H-Tyr-Arg-Leu-Arg-Tyr-Tyr-Arg-Leu-Arg-Tyr-Tyr-Arg-Leu-Arg-Tyr-NH.sub.2
(SEQ. ID. NO. 10).
46. A pharmaceutically acceptable salt of the compound of claim
41.
47. A method for controlling an NPY mediated physiological response
in a subject comprising administering to said subject the compound
of claim 41.
48. The method of claim 47 wherein the compound is capable of
suppressing appetite.
49. The method of claim 47 wherein the step of administering to
said subject the compound of claim 1 includes administering to said
subject a mixture of the compound of claim 1 and a second compound
having the formula: ##STR42## wherein X is a chain of 0-5 amino
acids, inclusive, where the N-terminal amino acid is bonded to
R.sup.1 and R.sup.2 by the nitrogen of the amino group of the
N-terminal amino acid; Y is a chain of 0-4 amino acids, inclusive,
where the C-terminal amino acid has a carboxylamide group, which is
independently bonded to R.sup.3 and R.sup.4; R.sup.1 and R.sup.2
are each independently bonded to the amino group of the N-terminal
amino acid and selected from H, (C.sub.1-C.sub.12)alkyl,
(C.sub.6-C.sub.18)aryl, (C.sub.1-C.sub.12)acyl, (C.sub.7-C.sub.18)
aralkyl, and (C.sub.7-C.sub.18)alkaryl; R.sup.3 and R.sup.4 are
each independently bonded to the amide group of the C-terminus
amino acid and selected from H, (C.sub.1-C.sub.12)alkyl,
(C.sub.6-C.sub.18)aryl, (C.sub.1-C.sub.12)acyl, (C.sub.7-C.sub.18)
aralkyl, and (C.sub.7-C.sub.18)alkaryl; A.sup.22 is an aromatic
amino acid, Ala, Aib, Anb, N-Me-Ala or is deleted; A.sup.23 is Ser,
Thr, Ala, Aib, N-Me-Ser, N-Me-Thr, N-Me-Ala or is deleted; A.sup.24
is Leu, Ile, Nle, Val, Trp, Gly, Nva, Aib, Anb, N-Me-Leu or is
deleted; A.sup.25 is Arg, Lys, homo-Arg, diethyl-homo-Arg,
Lys-p.epsilon.-NH--R (where R is H, a branched or straight chain
(C.sub.1-C.sub.10) lkyl group, or an aryl group), Orn or is
deleted; A.sup.26 is Ala, His, Thr, 3-Me-His, 1-Me-His,
.beta.-pyrazolylalaline, N-Me-His, Arg, Lys, homo-Arg,
diethyl-homo-Arg, Lys-.epsilon.-NH--R (where R is H, a branched or
straight chain (C.sub.1-C.sub.10) alkyl group, an aryl group, or a
pharmaceutically acceptable salt thereof), Orn or is deleted;
A.sup.27 is an aromatic amino acid; A.sup.28 is Leu, Ile, Nle, Val,
Trp, Aib, Anb or N-Me-Leu; A.sup.29 is Asn, Ala, Gln, Gly, Trp or
N-Me-Asn; A.sup.30 is Leu, Ile, Nle, Nva, Fla, Val, Trp, Aib, Anb
or N-Me-Leu; A.sup.31 is Val, Leu, Nle, Nva, Ile, Trp, Aib, Anb or
N-Me-Val; A.sup.32 is Thr, Ser, D-Trp, N-Me-Ser or N-Me-Thr; and
wherein the compound optionally includes one or two pseudopeptide
bonds where each pseudopeptide bond is independently selected from
--CH.sub.2--NH--, --CH.sub.2--S--, --CH.sub.2--CH.sub.2--,
--CH.sub.2--O-- and CH.sub.2--CO--.
50. The method of claim 49 wherein the second compound is
N-.alpha.-Ac[Nle.sup.24,28, Trp.sup.30, Nva.sup.31,
.psi..sup.35/36]PYY(22-36)-NH.sub.2 (SEQ. ID. NO. 7) wherein .psi.
is --CH2-NH--.
51. The method of claim 50 wherein the compound of claim 41 is
either H-Tyr-Arg-Leu-Arg-Tyr-Tyr-Arg-Leu-Arg-Tyr-NH.sub.2 (SEQ. ID.
NO. 9) or
H-Tyr-Arg-Leu-Arg-Tyr-Tyr-Arg-Leu-Arg-Tyr-Tyr-Arg-Leu-Arg-Tyr-NH.sub.2
(SEQ. ID. NO. 10).
52. The method of claim 47 wherein the step of administering to
said subject the compound of claim 1 includes administering to said
subject a mixture of the compound of claim 1 and a second compound
having the formula: ##STR43## wherein: the N-terminal amino acid is
bonded to R.sup.1 and R.sup.2; Y is a chain of 0-4 amino acids,
inclusive, where the C-terminal amino acid has a carboxylamide
group, which is independently bonded to R.sup.3 and R.sup.4;
R.sup.1 and R.sup.2 are each independently bonded to the amino
group of the N-terminal amino acid and selected from H,
(C.sub.1-C.sub.12)alkyl, (C.sub.6-C.sub.18)aryl,
(C.sub.1-C.sub.12)acyl, (C.sub.7-C.sub.18) aralkyl, and
(C.sub.7-C.sub.18)alkaryl; R.sup.3 and R.sup.4 are each
independently bonded to the amide group of the C-terminus amino
acid and are selected from H, (C.sub.1-C.sub.12)alkyl,
(C.sub.6-C.sub.18)aryl, (C.sub.1-C.sub.12)acyl, (C.sub.7-C.sub.18)
aralkyl, and (C.sub.7-C.sub.18)alkaryl; A.sup.25 is Arg, Lys,
homo-Arg, diethyl-homo-Arg, lys-.epsilon.-NH--R where R is H, a
branched or straight chain (C.sub.1-C.sub.10) alkyl group, or an
aryl group, Orn or is deleted; A.sup.26 is Ala, His, Thr, 3-Me-His,
1-Me-His, .beta.-pyrozolylalanin, N-Me-His, Arg, Lys, homo-Arg,
diethyl-homo-Arg, Lys-e-NH--R where R is H, a branched or straight
chain (C.sub.1-C.sub.10) alkyl group, or an aryl group, Orn or is
deleted; A.sup.27 is an aromatic amino acid: A.sup.28 is Leu, Ile,
Val, Trp Nle, Nva, Aib, Anb, or N-Me-Leu; A.sup.29 is Asn, Ala,
Gin, Fly, Trp, or N-Me-Asn; A.sup.30 is Leu, Ile, Val, Trp, Nle,
Nva, Aib, Anb, or N-Me-Leu; A.sup.31 is Val, Ile, Trp, Nva, Aib,
Anb, or N-Me-Val; A.sup.32 is Thr, Ser, N-Me-Ser, N-Me-Thr, or
D-Trp; and wherein the compound optionally includes one or two
pseudopeptide bonds where each pseudopeptide bond is independently
selected from --CH.sub.2--NH--, --CH.sub.2--S--,
--CH.sub.2--CH.sub.2--, --CH.sub.2--O-- and CH.sub.2--CO--.
53. A therapeutic composition capable of controlling an NPY
mediated physiological response comprising a therapeutically
effective amount of the compound of claim 41 together with a
pharmaceutically acceptable carrier substance.
54. The composition of claim 53 wherein the therapeutic composition
is capable of suppressing appetite.
55. The composition of claim 53 wherein said composition is in the
form of a liquid, pill, tablet, or capsule for oral administration
to a subject.
56. The composition of claim 53 wherein said composition is in the
form of a liquid for nasal, intravenous, subcutaneous, parenteral,
or intraperitoneal administration to a subject to a subject.
57. The composition of claim 53 wherein the therapeutically
effective amount of the compound of claim 41 includes a
therapeutically effective amount of a mixture of the compound of
claim and a second compound having the formula: ##STR44## wherein X
is a chain of 0-5 amino acids, inclusive, where the N-terminal
amino acid is bonded to R.sup.1 and R.sup.2 by the nitrogen of the
amino group of the N-terminal amino acid; Y is a chain of 0-4 amino
acids, inclusive, where the C-terminal amino acid has a
carboxylamide group, which is independently bonded to R.sup.3 and
R.sup.4; R.sup.1 and R.sup.2 are each independently bonded to the
amino group of the N-terminal amino acid and selected from H,
(C.sub.1-C.sub.12)alkyl, (C.sub.6-C.sub.18)aryl,
(C.sub.1-C.sub.12)acyl, (C.sub.7-C.sub.18) aralkyl, and
(C.sub.7-C.sub.18)alkaryl; R.sup.3 and R.sup.4 are each
independently bonded to the amide group of the C-terminus amino
acid and selected from H, (C.sub.1-C.sub.12)alkyl,
(C.sub.6-C.sub.18)aryl, (C.sub.1-C.sub.12)acyl, (C.sub.7-C.sub.18)
aralkyl, and (C.sub.7-C.sub.18)alkaryl; A.sup.22 is an aromatic
amino acid, Ala, Aib, Anb, N-Me-Ala or is deleted; A.sup.23 is Ser,
Thr, Ala, Aib, N-Me-Ser, N-Me-Thr, N-Me-Ala or is deleted; A.sup.24
is Leu, Ile, Nle, Val, Trp, Gly, Nva, Aib, Anb, N-Me-Leu or is
deleted; A.sup.25 is Arg, Lys, homo-Arg, diethyl-homo-Arg,
Lys-p.epsilon.-NH--R (where R is H, a branched or straight chain
(C.sub.1-C.sub.10) alkyl group, or an aryl group), Orn or is
deleted; A.sup.26 is Ala, His, Thr, 3-Me-His, 1-Me-His,
.beta.-pyrazolylalaline, N-Me-His, Arg, Lys, homo-Arg,
diethyl-homo-Arg, Lys-.epsilon.-NH--R (where R is H, a branched or
straight chain (C.sub.1-C.sub.10) alkyl group, an aryl group, or a
pharmaceutically acceptable salt thereof), Orn or is deleted;
A.sup.27 is an aromatic amino acid; A.sup.28 is Leu, Ile, Nle, Val,
Trp, Aib, Anb or N-Me-Leu; A.sup.29 is Asn, Ala, Gln, Gly, Trp or
N-Me-Asn; A.sup.30 is Leu, Ile, Nle, Nva, Fla, Val, Trp, Aib, Anb
or N-Me-Leu; A.sup.31 is Val, Leu, Nle, Nva, Ile, Trp, Aib, Anb or
N-Me-Val; A.sup.32 is Thr, Ser, D-Trp, N-Me-Ser or N-Me-Thr; and
wherein the compound optionally includes one or two pseudopeptide
bonds where each pseudopeptide bond is independently selected from
--CH.sub.2--NH--, --CH.sub.2--S--, --CH.sub.2--CH.sub.2--,
--CH.sub.2--O-- and CH.sub.2--CO--.
58. The composition of claim 57 wherein the second compound is
N-.alpha.-Ac[Nle.sup.24,28, Trp.sup.30, Nva.sup.31,
.psi..sup.35/36]PYY(22-36)-NH.sub.2 (SEQ. ID. NO. 7) wherein .psi.
is --CH2-NH--.
59. The composition of claim 58 wherein the compound of claim 41 is
either H-Tyr-Arg-Leu-Arg-Tyr-Tyr-Arg-Leu-Arg-Tyr-NH.sub.2 (SEQ. ID.
NO. 9) or
H-Tyr-Arg-Leu-Arg-Tyr-Tyr-Arg-Leu-Arg-Tyr-Tyr-Arg-Leu-Arg-Tyr-NH.su-
b.2 (SEQ. ID. NO. 10).
60. The composition of claim 53 wherein the therapeutically
effective amount of the compound of claim 41 includes a
therapeutically effective amount of a mixture of the compound of
claim and a second compound having the formula: ##STR45## wherein:
the N-terminal amino acid is bonded to R.sup.1 and R.sup.2; Y is a
chain of 0-4 amino acids, inclusive, where the C-terminal amino
acid has a carboxylamide group, which is independently bonded to
R.sup.3 and R.sup.4; R.sup.1 and R.sup.2 are each independently
bonded to the amino group of the N-terminal amino acid and selected
from H, (C.sub.1-C.sub.12)alkyl, (C.sub.6-C.sub.18)aryl,
(C.sub.1-C.sub.12)acyl, (C.sub.7-C.sub.18) aralkyl, and
(C.sub.7-C.sub.18)alkaryl; R.sup.3 and R.sup.4 are each
independently bonded to the amide group of the C-terminus amino
acid and are selected from H, (C.sub.1-C.sub.12)alkyl,
(C.sub.6-C.sub.18)aryl, (C.sub.1-C.sub.12)acyl,
(C.sub.7-C.sub.18)aralkyl, and (C.sub.7-C.sub.18)alkaryl; A.sup.25
is Arg, Lys, homo-Arg, diethyl-homo-Arg, lys-.epsilon.-NH--R where
R is H, a branched or straight chain (C.sub.1-C.sub.10) alkyl
group, or an aryl group, Orn or is deleted; A.sup.26 is Ala, His,
Thr, 3-Me-His, 1-Me-His, .beta.-pyrozolylalanin, N-Me-His, Arg,
Lys, homo-Arg, diethyl-homo-Arg, Lys-e-NH--R where R is H, a
branched or straight chain (C.sub.1-C.sub.10) alkyl group, or an
aryl group, Orn or is deleted; A.sup.27 is an aromatic amino acid:
A.sup.28 is Leu, Ile, Val, Trp Nle, Nva, Aib, Anb, or N-Me-Leu;
A.sup.29 is Asn, Ala, Gin, Fly, Trp, or N-Me-Asn; A.sup.30 is Leu,
Ile, Val, Trp, Nle, Nva, Aib, Anb, or N-Me-Leu; A.sup.31 is Val,
Ile, Trp, Nva, Aib, Anb, or N-Me-Val; A.sup.32 is Thr, Ser,
N-Me-Ser, N-Me-Thr, or D-Trp; and wherein the compound optionally
includes one or two pseudopeptide bonds where each pseudopeptide
bond is independently selected from --CH.sub.2--NH--,
--CH.sub.2--S--, --CH.sub.2--CH.sub.2--, --CH.sub.2--O-- and
CH.sub.2--CO--.
61. A method for controlling an NPY mediated physiological response
in a subject comprising administering to said subject a mixture of
NPY Y.sub.4 and Y.sub.2 selective receptor agonists.
62. The method of claim 61 wherein the Y.sub.4 receptor agonist is
PP, a peptide fragment thereof, or an analog of PP or the peptide
fragment, and the Y.sub.2 receptor agonist is PYY(3-36), a peptide
fragment thereof, or an analog of PYY(3-36) or the peptide
fragment.
63. The method of claim 62 wherein the Y.sub.4 receptor agonist is
the peptide fragment PP(32-36) or an analog thereof and the Y.sub.2
receptor agonist is the peptide fragment PYY(22-36), or an analog
thereof.
64. The method of claim 61, wherein the mixture is capable of
suppressing appetite.
65. A method for controlling an NPY mediated physiological response
in a subject comprising administering to said subject, together
with a pharmaceutically acceptable carrier substance, a therapeutic
composition comprising a therapeutically effective amount of a
mixture of NPY Y.sub.2 and Y.sub.4 receptor agonists.
66. The method of claim 65 wherein the Y.sub.4 receptor agonist is
PP, a peptide fragment thereof, or an analog of PP or the peptide
fragment, and the Y.sub.2 receptor agonist is PYY(3-36), a peptide
fragment thereof, or an analog of PYY(3-36) or the peptide
fragment.
67. The method of claim 66 wherein the Y.sub.4 receptor agonist is
the peptide fragment PP(32-36) or an analog thereof and the Y.sub.2
receptor agonist is the peptide fragment PYY(22-36), or an analog
thereof.
68. The method of claim 67 wherein the analog of PYY(22-36) is
N-.alpha.-Ac[Nle.sup.24,28, Trp.sup.30, Nva.sup.31,
.psi..sup.35/36]PYY(22-36)-NH.sub.2 (SEQ. ID. NO. 7) wherein .psi.
is --CH2-NH-- and the analog of PP(32-36) is either ##STR46##
69. The method of claim 65, wherein the mixture is capable of
suppressing appetite.
70. The method of claim 65 wherein said composition is in the form
of a liquid, pill, tablet, or capsule for oral administration to a
subject.
71. The method of claim 65 wherein said composition is in the form
of a liquid for nasal, intravenous, subcutaneous, parenteral, or
intraperitoneal administration to a subject.
Description
FIELD OF THE INVENTION
[0002] This invention relates generally to neuropeptide Y ("NPY")
Y.sub.4 receptor agonists including pancreatic polypeptide (PP),
analogs thereof, and to peptide fragments of PP, and analogs
thereof, and to methods for treatment of mammals using the same
and, more specifically, the invention is directed to NPY Y.sub.4
receptor agonists including PP(32-36), and analogs thereof, to
pharmaceutical compositions containing such pentapeptides, and to
methods of treatment of mammals using such pentapeptides. The NPY
Y.sub.4 receptor agonists may be administered to mammals either
alone or in combination with NPY Y.sub.2 receptor agonists
including peptide YY (PYY) (3-36), analogs thereof, and to peptide
fragments of PYY(3-36), and analogs thereof, such as to control
food intake in mammals.
BACKGROUND OF THE INVENTION
[0003] Obesity is a major disorder affecting as much as one third
of the North American population. Several studies have shown that
such individuals are at increased risk in developing cardiovascular
disease (hypertension and hyper-cholesterolemia), diabetes and
several types of cancer. The effective treatment of obesity,
however, remains a largely unachieved goal. Existing
pharmaco-therapeutic approaches to weight loss involve the use of
amphetamine-based agents such as amphetamine, diethylpropion,
mazindol and fenfluramine which act directly on the central nervous
system ("CNS") to lower food intake by modulating dopaminergic,
adrenergic and/or serotonergic mechanisms. Although weight loss can
be achieved with such agents, their use is restricted due to CNS
side-effects, potential addiction liability and the production of
tolerance to their actions, with chronic administration leading to
potential depression, vestibular disturbances, hallucinations and
addiction, as well as interference with the actions of other drugs,
such as MAO inhibitors and antihypertensives. There is also a
subpopulation of obese patients that is refractory to present
anorectic drug treatments. The medical need is high for an
effective anorectic agent that overcomes the above disadvantages of
existing therapies. Of particular need are agents which act by
alternative mechanisms to modulate food intake and/or
metabolism.
[0004] Throughout this application, various publications are
referenced. The disclosure of these publications is hereby
incorporated by reference into this application to describe more
fully the art to which this invention pertains.
[0005] Neuropeptide Y ("NPY")
[0006] Neuropeptides are small peptides originating from large
precursor proteins synthesized by peptidergic neurons and
endocrine/paracrine cells. They hold promise for treatment of
neurological, psychiatric, and endocrine disorders (De Wied, D. In:
Neuropeptides: Basics and Perspectives (Elsevier, Amsterdam-New
York-Oxford), 1990). Often the precursors contain multiple
biologically active peptides. There is great diversity of
neuropeptides in the brain caused by alternative splicing of
primary gene transcripts and differential precursor processing. The
neuropeptide receptors serve to discriminate between ligands and to
activate the appropriate signals. Thus, it is expected that the
receptors for neuropeptides consist of a large number of
members.
[0007] Neuropeptide Y (NPY), a 36-amino acid peptide, is the most
abundant neuropeptide to be identified in mammalian brain. Human
NPY has the formula:
H-Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Met-Ala-
-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-N-
H2 (SEQ. ID. NO. 1). Porcine and rat NPY have the same sequence
except for Leu instead of Met in the 17-position.
[0008] NPY is an important regulator in both the central and
peripheral nervous systems (Heilig, M. and E. Widerlov.
Neuropeptide Y: an overview of central distribution, functional
aspects, and possible involvement of neuropsychiatric illnesses.
Acta Psychiatr. Scand. 82:95-114 (1990)) and influences a diverse
range of physiological parameters, including effects on psychomotor
activity, food intake, central endocrine secretion, and
vasoactivity in the cardiovascular system. High concentrations of
NPY are found in the sympathetic nerves supplying the coronary,
cerebral, and renal vasculature and has contributed to
vasoconstriction. NPY binding sites have been identified in a
variety of tissues, including spleen (Lundberg, J. M., A. Hemsen,
O. Larsson, A. Rudehill, A. Saria, and B. Fredholm. Neuropeptide Y
receptor in pig spleen: binding characteristics, reduction of
cyclic AMP formation and calcium antagonist inhibition of
vasoconstriction. Eur. J. Pharmacol. 145:21-29 (1988)), intestinal
membranes, brain (Hinson, J., C. Rauh, and J. Coupet. Neuropeptide
Y stimulates inositol phospholipid hydrolysis in rat brain
microprisms. Brain Response. 446:379-382 (1988)), aortic smooth
muscle (Mihara, S., Y. Shigeri, and M. Fujimoto. Neuropeptide
Y-induced intracellular Ca2+ increase in vascular smooth muscle
cells. FEBS Lett. 259: 79-82 (1989)), kidney, testis, and placenta
(Dumont, Y., J. C. Martel, A. Fournier, S. St.-Pierre, and R.
Quiron. Neuropeptide Y and neuropeptide Y receptor subtypes in
brain and peripheral tissues. Prog. Neurobiol. 38:125-167 (1992)).
In addition, binding sites have been reported in a number of rat
and human cell lines (e.g. Y1 in SK-N-MC, MC-IXC, CHP-212, and PC12
cells; Y2 in SK-N--Be(2), CHP-234, and SMS-MSN)(Grundemar, L., S.
P. Sheikh, and C. Wahlestedt, In: The Biology of Neuropeptide Y and
Related Peptides. (Humana Press, Inc., Totawa, N.J.), (1992)).
[0009] NPY forms a family (called the pancreatic polypeptide
family) together with pancreatic polypeptide (PP) and peptide YY
(PYY) which all consist of 36 amino acids and have a common
tertiary structure, the so-called PP-fold (Glover, I. D., D. J.
Barlow, J. E. Pitts, S. P. Wood, I. J. Tickle, T. L. Blundell, K.
Tatemoto, J. R. Kimmel, A. Wollmer, W. Strassburger, and Y.-S.
Zhang. Conformational studies of the pancreatic polypeptide hormone
family. Eur. J. Biochem. 142:379-385 (1985)). Specific features of
this family include a polyproline helix in residues 1 through 8,
beta-turn in residues 9 through 14, an alpha-helix in residues 15
through 30, an outward-projecting C-terminus in residues 30 through
36, and a carboxy terminal amide (Schwartz, T. W., J. Fuhlendorff,
L. L. Kjems, M. S. Kristensen, M. Vervelde, M. O'Hare, J. L.
Krstenansky, and B. Bjornholm. Signal epitopes in the
three-dimensional structure of neuropeptide Y. Ann. N.Y. Acad. Sci.
611:35-47 (1990)). The C-terminal amidated residue of these
peptides appears to be essential for biological activity
(Wahlestedt et al., 1986). Studies with peptide fragments of NPY
have indicated that multiple NPY receptor subtypes exist
(Wahlestedt, C., N. Yanaihara, and R. Hakanson. Evidence for
different pre- and postjunctional receptors for neuropeptide Y and
related peptides. Regul. Pept. 13:307-318 (1986)). Specifically,
six receptor subtypes, denoted as Y1, Y2, Y3, Y4, Y5, and Y6, are
understood to mediate the actions of NPY with each to-date, except
for Y3, having been cloned.
[0010] The Y1, Y2, Y4, and Y5 receptors have been proposed to
regulate feeding behavior, i.e. food intake, in subjects. A key
pharmacological feature which distinguishes Y1 from Y2 is the fact
that the Y1 receptor (and not the Y2 receptor) responds to an
analog of NPY modified at residues 31 and 34 ([Leu31,Pro34]NPY),
whereas the Y2 receptor (and not the Y1 receptor) has high affinity
for the NPY peptide carboxyl-terminal fragment
NPY-(13-36)(Fuhlendorff, J., U. Gether, L. Aakerlund, N.
Langeland-Johansen, H. Thogersen, S. G. Melberg, U. B. Olsen, O.
Thastrup, and T. W. Schwartz. [Leu31,Pro34]Neuropeptide Y: A
specific Y1 receptor agonist. Proc. Natl. Acad. Sci. USA 87:182-186
(1990)). NPY analogs and N-terminally-shortened fragments, e.g.
NPY(18-36), which contain one or more specific D-isomer
substitutions for the naturally occurring residues (as well as
pharmaceutically acceptable salts thereof), dispersed in a
pharmaceutically acceptable liquid or solid carrier, can be
administered to mammals, including humans, to substantially lower
blood pressure over an extended period of time or to counteract
hypertension.
[0011] Experimental and clinical observations also have supported
the concept that neuropeptides play central roles in
neurotransmission as well as the regulation of secretory functions
of adenohypophysial, pancreatic, adrenalcortical and gut cells.
Among the thirty or so neuropeptides that have been implicated in
neuronal function in the mammalian central nervous system, several
have also been suggested to function as neurotransmitters or
neuromodulators primarily in afferent neurons.
[0012] An additional action of NPY is to decrease cardiac
contractility (inotropy). This is an extremely important action of
NPY, because it is known that, under many circumstances in which
inotropy is decreased, diseases of life-threatening importance,
e.g. congestive heart failure and cardiogenic shock, are associated
with probable increased release of NPY into the blood. Prevention
of NPY release, using a presynaptic NPY agonist, or NPY's action,
using a postsynaptic NPY antagonist, may be beneficial in these
disease states.
[0013] NPY has also been reported to produce coronary artery
vasoconstriction and thereby may decrease myocardial blood flow
resulting in myocardial ischemia. Such a circumstance can result in
angina pectoris or, under more severe circumstances, may result in
myocardial infarction and death. In recent years, several classes
of drugs have proven effective in dilating coronary arteries to
prevent such events.
[0014] Peptide YY ("PYY")
[0015] Peptide YY (PYY) is a 36-residue peptide amide isolated
originally from porcine intestine, and localized in the endocrine
cells of the gastrointestinal tract and pancreas (Tatemoto et al.
Proc. Natl. Acad. Sci. 79:2514, 1982). Peptide YY has N-terminal
and C-terminal tyrosine amides; accordingly, these two tyrosines
give PYY its name (Y represents the amino acid tyrosine in peptide
nomenclature). In addition, PYY shares a number of central and
peripheral regulatory roles with its homologous peptide
Neuropeptide Y (NPY), which was originally isolated from porcine
brain (Tatemoto, Proc. Natl. Acad. Sci. 79:5485, 1982). PYY is
localized in intestinal cells; NPY, in contrast, is present in the
submucous and myenteric neurons that innervate the mucosal and
smooth muscle layers, respectively (Ekblad et al. Neuroscience
20:169, 1987). Both PYY and NPY are believed to inhibit gut
motility and blood flow (Laburthe, Trends Endocrinol. Metab. 1:168,
1990), and they are also thought to attenuate basal (Cox et al. Br.
J. Pharmacol. 101:247, 1990; Cox et al. J. Physiol. 398:65, 1988;
Cox et al. Peptides 12:323, 1991; Friel et al. Br. J. Pharmacol.
88:425, 1986) and secretatogue-induced intestinal secretion in rats
(Lundberg et al. Proc. Natl. Acad. Sci USA 79:4471, 1982; Playford
et al. Lancet 335: 1555, 1990) and humans (Playford et al., supra),
as well as stimulate net absorption (MacFadyen et al. Neuropeptides
7:219, 1986). Elevated plasma PYY levels have been reported in
individuals suffering from several conditions that cause diarrhea
(Adrian et al. Gastroenterology 89:1070, 1985). Taken together,
these observations suggest that PYY and NPY are released into the
circulation after a meal (Adrian et al. Gastroenterology 89:1070,
1985: Balasubramaniam et al. Neuropeptides 14:209, 1989), and, thus
may play a physiological role in regulating intestinal secretion
and absorption, serving as natural inhibitors of diarrhea.
[0016] A high affinity PYY receptor system that exhibits a slightly
higher affinity for PYY than NPY has been characterized in rat
intestinal epithelia (Laburthe et al. Endocrinology 118:1910, 1986;
Laburthe, Trends Endocrinol. Metabl. supra) and shown to be
negatively coupled to adenylate cyclase (Servin et al.
Endocrinology 124:692, 1989). Consistently, PYY exhibited greater
antisecretory potency than NPY in voltage clamped preparations of
rat small intestine (Cox et al. J. Physiol. supra), while
C-terminal fragments of NPY were found to be less effective in
their antisecretory potency than PYY (Cox et al. Br. J. Pharmacol,
supra). Structure-activity studies using several partial sequences
have led to the identification of PYY(22-36) as the active site for
interacting with intestinal PYY receptors (Balasumbramaniam et al.
Pept. Res. 1:32, 1988). This intestinal PYY-preferring receptor has
now been cloned and shown to be identical to the Y.sub.2 receptors
cloned from the brain (Goumain et al. Mol Pharmacol 60:124-134,
2001).
[0017] In addition, PYY has been implicated in a number of
physiological activities including nutrient uptake (see, e.g.,
Bilcheik et al. Digestive Disease Week 506:623, 1993), cell
proliferation (see, e.g., Laburthe, Trends Endocrinol. Metab.
1:168, 1990; Voisin et al. J. Bio. Chem, 1993), lipolysis (see,
e.g., Valet et al. J. Clin. Invest. 85:291, 1990), and
vasoconstriction (see, e.g., Lundberg et al., Proc. Natl. Acad.
Sci, USA 79:4471, 1982).
[0018] The amino acid sequences of porcine and human PYY are as
follows: TABLE-US-00001 porcine PYY:
YPAKPEAPGEDASPEELSRYYASLRHYLNLVTR (SEQ. ID. NO. 2) QRY, human PYY:
YPIKPEAPGEDASPEELNRYYASLRHYLNLVTR (SEQ. ID. NO. 3) QRY.
The amino acid sequences for dog PYY and for rat PYY are the same
as that of porcine PYY.
[0019] With respect to PYY, it has been reported previously that
peripheral administration of PYY(3-36), a NPY Y.sub.2-preferring
ligand, can on peripheral administration attenuate food intake in
normal and fasted mice and rats as well as in normal and obese
humans (Nature 418:650-654; 2002, N Engl J Med 349:941-948; 2003).
The anorexigenic actions of PYY(3-36) are suggested to be mediated
by arcuate nucleus Y.sub.2 receptors. One advantage of using Y2
selective ligands is that they can suppress the food intake on
peripheral administration, whereas Y1 and Y5 selective ligands, as
described above, have to penetrate the BBB to modulate food
intake.
[0020] In addition to interacting with the Y2 receptor, PYY(3-36)
can potently activate Y4 and Y5 receptors. Consequently, the
inventor previously developed Y2 receptor selective agonists that
are based on PYY(22-36) and PYY(25-36) (See U.S. Pat. Nos.
5,604,203, and 6,046,167 to Balasubramaniam) which are devoid of
activities at the other NPY receptors including Y1, Y4, and Y5 at
concentrations up to 20,000 nM. Preferred PYY(25-36) analogs
include N-.alpha.-Ac-[Trp.sup.30]PYY(25-36)-NH.sub.2 (SEQ. ID. NO.
4), referred to as BWX-115, N-.alpha.-Ac-[Trp.sup.27,
.psi..sup.35/36]PYY(25-36)-NH.sub.2 (SEQ. ID. NO. 5), referred to
as BT-56, and N-.alpha.-Ac-[Trp.sup.30,
.psi..sup.35/36]PYY(25-36)-NH.sub.2 (SEQ. ID. NO. 6), referred to
as BT-123, and the PYY(22-36) analog N-.alpha.-Ac[Nle.sup.24,28,
Trp.sup.30, Nva.sup.31, .psi..sup.35/36]PYY(22-36)-NH.sub.2 (SEQ.
ID. NO. 7), referred to as B-48, [wherein .psi. in the foregoing
formulas is --CH2-NH--] which can be used to control food intake in
animals and humans.
[0021] Pancreatic Peptide ("PP")
[0022] Pancreatic peptide (PP) is a 36-amino-acid secretory peptide
that is predominantly produced by the pancreas and released in
response to nutrient stimuli.
[0023] The amino acid sequence of human PP is as follows:
TABLE-US-00002 APLEPVYPGDNATPEQMAQYAADLRRYINMLTR (SEQ. ID. NO. 8)
PRY.
[0024] PP, like PYY, shares a number of central and peripheral
regulatory roles with its homologous peptide NPY. It has been
reported recently that PP can attenuate body weight increase in
mice through inhibiting food intake and increasing energy
expenditures (Gastroenterology 124:1325-1336; 2003). These actions
are mediated by altering the expression of orexigenic (NPY, orexin
& ghrelin downregulated) and anorexigenic (urocortin
upregulated) peptides, and decreasing gastric emptying and activity
of the vagovagal or vago-sympathetic reflex arc. In addition, the
anorectic effects of PP have been suggested to be mediated by the
Y.sub.4 receptors in the area postrema (AP) because: a)
autoradiography following peripheral administration of PP revealed
high accumulation of PP in AP; b) expression of c-fos is seen in AP
following iv administration of PP; and c) high density of Y.sub.4
receptors are present in AP (Brain Res 760:137-149; 1997).
[0025] One advantage of using Y4 selective ligands, like the Y2
selective ligand, is that they can suppress the food intake on
peripheral administration, whereas Y1 and Y5 selective ligands, as
described above, have to penetrate the BBB to modulate food intake.
Native PP may not be an ideal candidate for suppressing food intake
because of its short half-life of about six minutes and its
interactions with Y5 receptors.
[0026] Accordingly, it would be desirable to develop NPY Y.sub.4
receptor agonists that can be used alone or mixed with NPY Y.sub.2
receptor agonists, as disclosed in U.S. Pat. Nos. 5,604,203, and
6,046,167 to Balasubramaniam, which are herein incorporated by
reference, for controlling an NPY mediated physiological response
in a subject, such as to control food intake in mammals, and that
are expected to prove useful in the treatment of weight problems
(e.g. obesity and diabetes), eating disorders, and such.
SUMMARY OF THE INVENTION
[0027] This invention is directed to neuropeptide Y ("NPY") Y.sub.4
receptor agonists including pancreatic polypeptide (PP), analogs
thereof, and peptide fragments of PP, e.g. PP(32-36), and analogs
thereof, to pharmaceutical compositions containing such Y.sub.4
receptor agonists, and to methods for treatment of mammals using
the same. The NPY Y.sub.4 receptor agonists may be administered to
mammals either alone or in combination with NPY Y.sub.2 receptor
agonists including peptide YY (PYY) (3-36), analogs thereof, and to
peptide fragments of PYY(3-36), e.g. PYY(22-36) and PYY(25-36), and
analogs thereof, as disclosed in U.S. Pat. Nos. 5,604,203, and
6,046,167 to Balasubramaniam, which are herein incorporated by
reference, such as to control food intake in mammals, blood
pressure, cardiovascular response, libido, circadian rhythm,
hyperlipidimia, chronic pancreatitis, and nonalcoholic fatty liver
disease including nonalcoholic steatohepatitis.
[0028] Accordingly, in one aspect, the present invention features
NPY Y.sub.4 receptor agonists of PP(32-36) and analogs thereof,
such pentapeptide analogs having the formula: ##STR1##
[0029] wherein: [0030] each X, independently, is an aromatic amino
acid; [0031] each Y, independently, is an amino acid having a
guanidino group; [0032] each Z, independently, is an aliphatic
amino acid; [0033] n=1, 2, 3 or 4; and [0034] wherein the compound
optionally includes one or two pseudopeptide bonds where each
pseudopeptide bond is independently selected from --CH.sub.2--NH--,
--CH.sub.2--S--, --CH.sub.2--CH.sub.2--, --CH.sub.2--O-- and
CH.sub.2--CO--.
[0035] A preferred compound of formula (I) includes, ##STR2##
(which is referred to as B-74).
[0036] In another aspect, the present invention features
pentapeptide analogs of PP(32-36) having the formula: ##STR3##
[0037] wherein: [0038] each X, independently, is an aromatic amino
acid; [0039] each Y, independently, is an amino acid having a
guanidino group; [0040] each Z, independently, is an aliphatic
amino acid; [0041] A1 and A2, independently, are selected from Cys,
Pen, Glu, Asp, Lys, and Dpr; and [0042] wherein the compound
optionally includes one or two pseudopeptide bonds where each
pseudopeptide bond is independently selected from --CH.sub.2--NH--,
--CH.sub.2--S--, --CH.sub.2--CH.sub.2--, --CH.sub.2--O-- and
CH.sub.2--CO--.
[0043] A preferred compound of formula (II) includes, ##STR4##
[0044] In another aspect, the present invention features
pentapeptide analogs of PP(32-36) having the formula:
H--[X--Y-Z-Y--X].sub.n--NH.sub.2 (III)
[0045] wherein: [0046] each X, independently, is an aromatic amino
acid; [0047] each Y, independently, is an amino acid having a
guanidino group; [0048] each Z, independently, is an aliphatic
amino acid; [0049] n=1, 2, 3 or 4; and [0050] wherein the compound
optionally includes one or two pseudopeptide bonds where each
pseudopeptide bond is independently selected from --CH.sub.2--NH--,
--CH.sub.2--S--, --CH.sub.2--CH.sub.2--, --CH.sub.2--O-- and
CH.sub.2--CO--.
[0051] Preferred compounds of formula (III) include
H-Tyr-Arg-Leu-Arg-Tyr-Tyr-Arg-Leu-Arg-Tyr-NH.sub.2 (SEQ. ID. NO. 9)
or
H-Tyr-Arg-Leu-Arg-Tyr-Tyr-Arg-Leu-Arg-Tyr-Tyr-Arg-Leu-Arg-Tyr-NH.sub.2
(SEQ. ID. NO. 10).
[0052] In another aspect, NPY Y.sub.4 receptor agonists, such as
those of formulas (I-III), are mixed or combined with NPY Y.sub.2
receptor agonists, such as analogs of PYY(25-36) or PYY(22-36), for
controlling an NPY mediated physiological response in a subject,
e.g. to suppress appetite, such analogs of PYY(25-36) and of
PYY(22-36), respectively, having formulas (IV) and (V) below:
##STR5##
[0053] wherein: [0054] the N-terminal amino acid is bonded to
R.sup.1 and R.sup.2; [0055] Y is a chain of 0-4 amino acids,
inclusive, where the C-terminal amino acid has a carboxylamide
group; which is independently bonded to R.sup.3 and R.sup.4, e.g.,
##STR6## [0056] R.sup.1 and R.sup.2 are each independently bonded
to the amino group of the N-terminal amino acid and selected from
H, (C.sub.1-C.sub.12)alkyl, (C.sub.6-C.sub.8)aryl,
(C.sub.1-C.sub.12)acyl, (C.sub.7-C.sub.18) aralkyl, and
(C.sub.7-C.sub.18)alkaryl; [0057] R.sup.3 and R.sup.4 are each
independently bonded to the amide group of the C-terminus amino
acid, e.g., ##STR7## [0058] (where R denotes the side chain group
of the amino acid, e.g. R.dbd.H in Gly, etc.), and selected from H,
(C.sub.1-C.sub.12)alkyl (e.g. methyl), (C.sub.6-C.sub.18)aryl (e.g.
phenyl), (C.sub.1-C.sub.12)acyl (formyl, acetyl, napthaleneacetyl,
and myristoyl), (C.sub.7-C.sub.18) aralkyl (e.g. benzyl), and
(C.sub.7-C.sub.18)alkaryl (e.g. p-methlyphenyl); [0059] A.sup.25 is
Arg, Lys, homo-Arg, diethyl-homo-Arg, lys-.epsilon.-NH--R where R
is H, a branched or straight chain (C.sub.1-C.sub.10) alkyl group,
or an aryl group, Orn or is deleted; [0060] A.sup.26 is Ala, His,
Thr, 3-Me-His, 1-Me-His, .beta.-pyrozolylalanin, N-Me-His, Arg,
Lys, homo-Arg, diethyl-homo-Arg, Lys-e-NH--R where R is H, a
branched or straight chain (C.sub.1-C.sub.10) alkyl group, or an
aryl group, Orn or is deleted; [0061] A.sup.27 is an aromatic amino
acid: [0062] A.sup.28 is Leu, Ile, Val, Trp Nle, Nva, Aib, Anb, or
N-Me-Leu; [0063] A.sup.29 is Asn, Ala, Gin, Fly, Trp, or N-Me-Asn;
[0064] A.sup.30 is Leu, Ile, Val, Trp, Nle, Nva, Aib, Anb, or
N-Me-Leu; [0065] A.sup.31 is Val, Ile, Trp, Nva, Aib, Anb, or
N-Me-Val; [0066] A.sup.32 is Thr, Ser, N-Me-Ser, N-Me-Thr, or
D-Trp; and [0067] wherein the compound optionally includes one or
two pseudopeptide bonds where each pseudopeptide bond is
independently selected from --CH.sub.2--NH--, --CH.sub.2--S--,
--CH.sub.2CH.sub.2--, --CH.sub.2--O-- and CH.sub.2--CO--; ##STR8##
wherein [0068] X is a chain of 0-5 amino acids, inclusive, where
the N-terminal amino acid is bonded to R.sup.1 and R.sup.2 by the
nitrogen of the amino group of the N-terminal amino acid; [0069] Y
is a chain of 0-4 amino acids, inclusive, where the C-terminal
amino acid has a carboxylamide group; which is independently bonded
to R.sup.3 and R.sup.4, e.g., ##STR9## [0070] R.sup.1 and R.sup.2
are each independently bonded to the amino group of the N-terminal
amino acid and selected from H, (C.sub.1-C.sub.12)alkyl,
(C.sub.6-C.sub.18)aryl, (C.sub.1-C.sub.12)acyl, (C.sub.7-C.sub.18)
aralkyl, and (C.sub.7-C.sub.18)alkaryl; [0071] R.sup.3 and R.sup.4
are each independently bonded to the amide group of the C-terminus
amino acid, e.g., ##STR10## [0072] (where R denotes the side chain
group of the amino acid, e.g. R.dbd.H in Gly, etc.), and selected
from H, (C.sub.1-C.sub.12)alkyl (e.g. methyl),
(C.sub.6-C.sub.18)aryl (e.g. phenyl), (C.sub.1-C.sub.12)acyl
(formyl, acetyl, napthaleneacetyl, and myristoyl),
(C.sub.7-C.sub.18)aralkyl (e.g. benzyl), and
(C.sub.7-C.sub.18)alkaryl (e.g. p-methlyphenyl); [0073] A.sup.22 is
an aromatic amino acid, Ala, Aib, Anb, N-Me-Ala or is deleted;
[0074] A.sup.23 is Ser, Thr, Ala, Aib, N-Me-Ser, N-Me-Thr, N-Me-Ala
or is deleted; [0075] A.sup.24 is Leu, Ile, Nle, Val, Trp, Gly,
Nva, Aib, Anb, N-Me-Leu or is deleted; [0076] A.sup.25 is Arg, Lys,
homo-Arg, diethyl-homo-Arg, Lys-p.epsilon.-NH--R (where R is H, a
branched or straight chain (C.sub.1-C.sub.10) alkyl group, or an
aryl group), Orn or is deleted; [0077] A.sup.26 is Ala, His, Thr,
3-Me-His, 1-Me-His, .beta.-pyrazolylalaline, N-Me-His, Arg, Lys,
homo-Arg, diethyl-homo-Arg, Lys-.epsilon.-NH--R (where R is H, a
branched or straight chain (C.sub.1-C.sub.10) alkyl group, an aryl
group, or a pharmaceutically acceptable salt thereof), Orn or is
deleted; [0078] A.sup.27 is an aromatic amino acid; [0079] A.sup.28
is Leu, Ile, Nle, Val, Trp, Aib, Anb or N-Me-Leu; [0080] A.sup.29
is Asn, Ala, Gln, Gly, Trp or N-Me-Asn; [0081] A.sup.30 is Leu,
Ile, Nle, Nva, Fla, Val, Trp, Aib, Anb or N-Me-Leu; [0082] A.sup.31
is Val, Leu, Nle, Nva, Ile, Trp, Aib, Anb or N-Me-Val; [0083]
A.sup.32 is Thr, Ser, D-Trp, N-Me-Ser or N-Me-Thr; and wherein the
compound optionally includes one or two pseudopeptide bonds where
each pseudopeptide bond is independently selected from
--CH.sub.2--NH--, --CH.sub.2--S--, --CH.sub.2--CH.sub.2--,
--CH.sub.2--O-- and CH.sub.2--CO--.
[0084] A preferred compound of formula (IV) includes
N-.alpha.-Ac[Trp.sup.27, .psi..sup.35/36]PYY(25-36)-NH.sub.2 (SEQ.
ID. NO. 5), referred to as BT-56,
N-.alpha.-Ac[Trp.sup.30]PYY(25-36)-NH.sub.2 (SEQ. ID. NO. 4),
referred to as BWX-115, and N-.alpha.-Ac-[Trp.sup.30,
.psi..sup.35/36]PYY(25-36)-NH.sub.2 (SEQ. ID. NO. 6), referred to
as BT-123, wherein .psi. in the foregoing formulas is --CH2-NH--. A
preferred compound of formula (V) includes
N-.alpha.-Ac[Nle.sup.24,28, Trp.sup.30, Nva.sup.31,
.psi..sup.35/36]PYY(22-36)-NH.sub.2 (SEQ. ID. NO. 7), referred to
as B-48, wherein .psi. is --CH2-NH--.
[0085] The formulas of I-V, as indicated above, optionally include
at least one pseudopeptide bond between amino acid residues. By
"psuedopeptide bond" is meant that the carbon atom participating in
the bond between two residues is reduced from a carbonyl carbon to
a methylene carbon, i.e., CH.sub.2--NH; or less preferably that of
CO--NH is replaced with any of CH.sub.2--S, CH.sub.2--CH.sub.2,
CH.sub.2--O, or CH.sub.2--CO. A psuedopeptide bond may be
symbolized herein by ".psi.". The psuedopeptide bond analogs can be
used to form dimeric analogs. A detailed discussion of
psuedopeptide bonds is given in Coy et al. (1998) Tetrahedon
44:835-841.
[0086] In another aspect, the invention features a method of
controlling the food intake, i.e. appetite, of a subject comprising
administering to said subject the compound of formula I, II, or III
alone or in combination with formulas IV or V.
[0087] In other preferred embodiments, a therapeutically effective
amount of a compound of formula I, II, or III alone, or in
combination with formulas IV or V, and a pharmaceutically
acceptable carrier substance together form a therapeutic
composition capable of suppressing an NPY mediated physiological
response.
BRIEF DESCRIPTION OF THE FIGURES
[0088] FIG. 1 is a graph representing the feeding patterns of
animals treated with a PP(32-36) analog by intraperitoneal
injection. The compounds tested include a control (saline), hPP,
and ##STR11##
[0089] FIG. 2 is a graph representing the feeding patterns of
animals treated with a PP(32-36) analog (Y4 receptor agonist), and
a mixture of the PP(32-36) analog and a PYY(22-36) analog (Y2
receptor agonist), by intraperitoneal injection. The compounds
tested include a control (saline), ##STR12## and a combination of
B-74 and N-.alpha.-Ac[Nle.sup.24,28, Trp.sup.30, Nva.sup.31,
.psi..sup.35/36]PYY(22-36)-NH.sub.2 (SEQ. ID. NO. 7) (B-48) wherein
.psi. is --CH2-NH--; and
[0090] FIG. 3 is a graph representing the feeding patterns of
animals treated with a PYY(22-36) analog (Y2 agonist receptor), and
a mixture of the PYY(22-36) analog and a PP(32-36) analog (Y4
receptor agonist), by intraperitoneal injection. The compounds
tested include a control (saline), N-.alpha.-Ac[Nle.sup.24,28,
Trp.sup.30, Nva.sup.31, .psi..sup.35/36]PYY(22-36)-NH.sub.2 (SEQ.
ID. NO. 7) (B-48) wherein .psi. is --CH2-NH--, and a combination of
B-48 and ##STR13##
DETAILED DESCRIPTION OF THE INVENTION
[0091] The present invention is directed to neuropeptide Y ("NPY")
Y.sub.4 receptor agonists including pancreatic polypeptide (PP),
analogs thereof, and peptide fragments of PP, e.g. PP(32-36), and
analogs thereof, to pharmaceutical compositions containing such
Y.sub.4 receptor agonists, and to methods for treatment of mammals
using the same. The NPY Y.sub.4 receptor agonists may be
administered to mammals either alone or in combination with NPY
Y.sub.2 receptor agonists including peptide YY (PYY) (3-36),
analogs thereof, and to peptide fragments of PYY(3-36), e.g.
PYY(22-36) and PYY(25-36), and analogs thereof, such as to control
food intake in mammals, blood pressure, cardiovascular response,
libido, circadian rhythm, hyperlipidimia, chronic pancreatitis, and
nonalcoholic fatty liver disease including nonalcoholic
steatohepatitis.
[0092] One advantage of using the Y4 selective ligands of the
present invention is that they can suppress food intake on
peripheral administration, whereas Y1 and Y5 selective ligands, as
described above, have to penetrate the BBB to modulate food intake.
Additionally, an advantage of using Y2 selective ligands is that
they also can suppress the food intake on peripheral
administration. Therefore, the inventor has developed NPY Y.sub.4
receptor agonists that can be used alone or mixed with NPY Y.sub.2
receptor agonists, as are disclosed in U.S. Pat. Nos. 5,604,203,
and 6,046,167 to Balasubramaniam, which are herein incorporated by
reference, for controlling an NPY mediated physiological response
in a subject, such as to control food intake in mammals by
suppressing appetite.
[0093] Accordingly, in one aspect, the present invention features
NPY Y.sub.4 receptor agonists of PP(32-36) and analogs thereof,
such pentapeptide analogs having the formula: ##STR14##
[0094] wherein: [0095] each X, independently, is an aromatic amino
acid; [0096] each Y, independently, is an amino acid having a
guanidino group; [0097] each Z, independently, is an aliphatic
amino acid; [0098] n=1, 2, 3 or 4; and [0099] wherein the compound
optionally includes one or two pseudopeptide bonds where each
pseudopeptide bond is independently selected from --CH.sub.2--NH--,
--CH.sub.2--S--, --CH.sub.2--CH.sub.2--, --CH.sub.2--O-- and
CH.sub.2--CO--.
[0100] A preferred compound of formula (I) includes, ##STR15##
(which is referred to as B-74).
[0101] In another aspect, the present invention features NPY
Y.sub.4 receptor agonists or pentapeptide analogs of PP(32-36)
having the formula: ##STR16##
[0102] wherein: [0103] each X, independently, is an aromatic amino
acid; [0104] each Y, independently, is an amino acid having a
guanidino group; [0105] each Z, independently, is an aliphatic
amino acid; [0106] A1 and A2, independently, are selected from Cys,
Pen, Glu, Asp, Lys, and Dpr; and [0107] wherein the compound
optionally includes one or two pseudopeptide bonds where each
pseudopeptide bond is independently selected from --CH.sub.2--NH--,
--CH.sub.2--S--, --CH.sub.2--CH.sub.2--, --CH.sub.2--O-- and
CH.sub.2--CO--.
[0108] A preferred compound of formula (II) includes, ##STR17##
[0109] In another aspect, the present invention features NPY
Y.sub.4 receptor agonists or pentapeptide analogs of PP(32-36)
having the formula: H--[X--Y-Z-Y--X].sub.n--NH.sub.2 (III)
[0110] wherein: [0111] each X, independently, is an aromatic amino
acid; [0112] each Y, independently, is an amino acid having a
guanidino group; [0113] each Z, independently, is an aliphatic
amino acid; [0114] n=1, 2, 3 or 4; and [0115] wherein the compound
optionally includes one or two pseudopeptide bonds where each
pseudopeptide bond is independently selected from --CH.sub.2--NH--,
--CH.sub.2--S--, --CH.sub.2--CH.sub.2--, --CH.sub.2--O-- and
CH.sub.2--CO--.
[0116] Preferred compounds of formula (III) include
H-Tyr-Arg-Leu-Arg-Tyr-Tyr-Arg-Leu-Arg-Tyr-NH.sub.2 (SEQ. ID. NO. 9)
or
H-Tyr-Arg-Leu-Arg-Tyr-Tyr-Arg-Leu-Arg-Tyr-Tyr-Arg-Leu-Arg-Tyr-NH.sub.2
(SEQ. ID. NO. 10).
[0117] For formulas I-III, aromatic amino acids can include, for
example, phenylalanine, tryptophan, or tyrosine, which is a
hydroxyl aromatic amino acid; aliphatic amino acids can include,
for example, isoleucine, leucine, norvaline, glycine, alanine, or
valine; and amino acids that have a guanidino group may include,
for example, arginine. In addition, the C-terminal of the compounds
of formulas (I-III) are amidated.
[0118] In another aspect, NPY Y.sub.4 receptor agonists, such as
those of formulas (III), are mixed or combined with NPY Y.sub.2
receptor agonists, such as analogs of PYY(25-36), for controlling
an NPY mediated physiological response in a subject, e.g. to
suppress appetite, such analogs of PYY(25-36) having the formula:
##STR18##
[0119] wherein: [0120] the N-terminal amino acid is bonded to
R.sup.1 and R.sup.2; [0121] Y is a chain of 0-4 amino acids,
inclusive, where the C-terminal amino acid has a carboxylamide
group; which is independently bonded to R.sup.3 and R.sup.4, e.g.,
##STR19## [0122] R.sup.1 and R.sup.2 are each independently bonded
to the amino group of the N-terminal amino acid and selected from
H, (C.sub.1-C.sub.12)alkyl (e.g. methyl), (C.sub.6-C.sub.18)aryl
(e.g. phenyl), (C.sub.1-C.sub.12)acyl (formyl, acetyl,
napthaleneacetyl, and myristoyl), (C.sub.7-C.sub.18)aralkyl (e.g.
benzyl), and (C.sub.7-C.sub.18)alkaryl (e.g. p-methlyphenyl);
[0123] R.sup.3 and R.sup.4 are each independently bonded to the
amide group of the C-terminus amino acid, e.g., ##STR20## [0124]
(where R denotes the side chain group of the amino acid, e.g.
R.dbd.H in Gly, etc.), and selected from H, (C.sub.1-C.sub.12)alkyl
(e.g. methyl), (C.sub.6-C.sub.18)aryl (e.g. phenyl),
(C.sub.1-C.sub.12)acyl (formyl, acetyl, napthaleneacetyl, and
myristoyl), (C.sub.7-C.sub.18)aralkyl (e.g. benzyl), and
(C.sub.7-C.sub.18)alkaryl (e.g. p-methlyphenyl); [0125] A.sup.25 is
Arg, Lys, homo-Arg, diethyl-homo-Arg, lys-.epsilon.-NH--R (where R
is H, a branched or straight chain (C.sub.1-C.sub.10) alkyl group,
or an aryl group), Orn or is deleted; [0126] A.sup.26 is Ala, His,
Thr, 3-Me-His, 1-Me-His, .beta.-pyrozolylalanin, N-Me-His, Arg,
Lys, homo-Arg, diethyl-homo-Arg, Lys-e-NH--R (where R is H, a
branched or straight chain (C.sub.1-C.sub.10) alkyl group, or an
aryl group), Orn or is deleted; [0127] A.sup.27 is an aromatic
amino acid: [0128] A.sup.28 is Leu, Ile, Val, Trp Nle, Nva, Aib,
Anb, or N-Me-Leu; [0129] A.sup.29 is Asn, Ala, Gin, Fly, Trp, or
N-Me-Asn; [0130] A.sup.30 is Leu, Ile, Val, Trp, Nle, Nva, Aib,
Anb, or N-Me-Leu; [0131] A.sup.31 is Val, Ile, Trp, Nva, Aib, Anb,
or N-Me-Val; [0132] A.sup.32 is Thr, Ser, N-Me-Ser, N-Me-Thr, or
D-Trp.
[0133] In preferred embodiments Y is
A.sup.33-A.sup.34-A.sup.35-A.sup.36 wherein
[0134] A.sup.33 is Arg, Lys, homo-Arg, diethyl-homo-Arg,
Lys-.epsilon.-NH--R (where R is H, a branched or straight chain
(C.sub.1-C.sub.10) alkyl group, or (C.sub.6-C.sub.18) aryl group),
Cys, or Orn
[0135] A.sup.34 is Gln, Asn, Ala, Gly, N-Me-Gin, Aib, Cys, or
Anb;
[0136] A.sup.35 is Arg, Lys, homo-Arg, diethyl-homo-Arg,
Lys-.epsilon.-NH--R (where R is H, a branched or straight chain
(C.sub.1-C.sub.10) alkyl group, or (C.sub.6-C.sub.18) aryl group),
Cys, or Orn; and
[0137] A.sup.36 is an aromatic amino acid, Cys, or a
pharmaceutically acceptable salt thereof.
[0138] Most preferably, the compound of formula (IV) includes
N-.alpha.-Ac[Trp.sup.27, .psi..sup.35/36]PYY(25-36)-NH.sub.2 (SEQ.
ID . NO. 5), referred to as BT-56, and
N-.alpha.-Ac[Trp.sup.30]PYY(25-36)-NH.sub.2 (SEQ. ID. NO. 4),
referred to as BWX-115, and N-.alpha.-Ac-[Trp.sup.30,
.psi..sup.35/36]PYY(25-36)-NH.sub.2 (SEQ. ID. NO. 6), referred to
as BT-123, wherein .psi. in the foregoing formulas is
--CH2-NH--.
[0139] In yet another aspect, NPY Y.sub.4 receptor agonists, such
as those of formulas (I-III), are mixed with NPY Y.sub.2 receptor
agonists, such as analogs of PYY(22-36), for controlling an NPY
mediated physiological response in a subject, e.g. to suppress
appetite, such analogs of PYY(22-36) having the formula: ##STR21##
wherein [0140] X is a chain of 0-5 amino acids, inclusive, where
the N-terminal amino acid is bonded to R.sup.1 and R.sup.2 by the
nitrogen of the amino group of the N-terminal amino acid; [0141] Y
is a chain of 0-4 amino acids, inclusive, where the C-terminal
amino acid has a carboxylamide group; which is independently bonded
to R.sup.3 and R.sup.4, e.g., ##STR22## [0142] R.sup.1 and R.sup.2
are each independently bonded to the amino group of the N-terminal
amino acid and selected from H, (C.sub.1-C.sub.12)alkyl (e.g.
methyl), (C.sub.6-C.sub.18)aryl (e.g. phenyl),
(C.sub.1-C.sub.12)acyl (formyl, acetyl, napthaleneacetyl, and
myristoyl), (C.sub.7-C.sub.18)aralkyl (e.g. benzyl), and
(C.sub.7-C.sub.18)alkaryl (e.g. p-methlyphenyl); [0143] R.sup.3 and
R.sup.4 are each independently bonded to the amide group of the
C-terminus amino acid, e.g., ##STR23## [0144] (where R denotes the
side chain group of the amino acid, e.g. R.dbd.H in Gly, etc.), and
selected from H, (C.sub.1-C.sub.12)alkyl (e.g. methyl),
(C.sub.6-C.sub.18)aryl (e.g. phenyl), (C.sub.1-C.sub.12)acyl
(formyl, acetyl, napthaleneacetyl, and myristoyl),
(C.sub.7-C.sub.18)aralkyl (e.g. benzyl), and
(C.sub.7-C.sub.18)alkaryl (e.g. p-methlyphenyl); [0145] A.sup.22 is
an aromatic amino acid, Ala, Aib, Anb, N-Me-Ala or is deleted;
[0146] A.sup.23 is Ser, Thr, Ala, Aib, N-Me-Ser, N-Me-Thr, N-Me-Ala
or is deleted; [0147] A.sup.24 is Leu, Ile, Nle, Val, Trp, Gly,
Nva, Aib, Anb, N-Me-Leu or is deleted; [0148] A.sup.25 is Arg, Lys,
homo-Arg, diethyl-homo-Arg, Lys-p.epsilon.-NH--R (where R is H, a
branched or straight chain (C.sub.1-C.sub.10) alkyl group, or an
aryl group), Orn or is deleted; [0149] A.sup.26 is Ala, His, Thr,
3-Me-His, 1-Me-His, .beta.-pyrazolylalaline, N-Me-His, Arg, Lys,
homo-Arg, diethyl-homo-Arg, Lys-.epsilon.-NH--R (where R is H, a
branched or straight chain (C.sub.1-C.sub.10) alkyl group, an aryl
group, or a pharmaceutically acceptable salt thereof), Orn or is
deleted; [0150] A.sup.27 is an aromatic amino acid; [0151] A.sup.28
is Leu, Ile, Nle, Val, Trp, Aib, Anb or N-Me-Leu; [0152] A.sup.29
is Asn, Ala, Gln, Gly, Trp or N-Me-Asn; [0153] A.sup.30 is Leu,
Ile, Nle, Nva, Fla, Val, Trp, Aib, Anb or N-Me-Leu; [0154] A.sup.31
is Val, Leu, Nle, Nva, Ile, Trp, Aib, Anb or N-Me-Val; and [0155]
A.sup.32 is Thr, Ser, D-Trp, N-Me-Ser or N-Me-Thr.
[0156] In preferred embodiments, Y is
A.sup.33-A.sup.34-A.sup.35-A.sup.36 wherein
[0157] A.sup.33 is Arg, Lys, homo-Arg, diethyl-homo-Arg,
Lys-.epsilon.-NH--R (where R is H, a branched or straight chain
(C.sub.1-C.sub.10) alkyl group, or an aryl group), Cys, or Orn
[0158] A.sup.34 is Cys, Gln, Asn, Ala, Gly, N-Me-Gin, Aib, or
Anb;
[0159] A.sup.35 is Arg, Lys, homo-Arg, diethyl-homo-Arg,
Lys-.epsilon.-NH--R (where R is H, a branched or straight chain
(C.sub.1-C.sub.10) alkyl group, or an aryl group), Cys, or Orn;
and
[0160] A.sup.36 is an aromatic amino acid, Cys, or a
pharmaceutically acceptable salt thereof.
[0161] Most preferably, the compound of formula (V) includes
N-.alpha.-Ac[Nle.sup.24,28, Trp.sup.30, Nva.sup.31,
.psi..sup.35/36]PYY(22-36)-NH.sub.2 (SEQ. ID. NO. 7), referred to
as B-48, wherein .psi. is --CH2-NH--.
[0162] Concerning formulas IV and V, reference to the
(C.sub.1-C.sub.12)acyl for R1 and R2 can include an aliphatic acyl
(e.g. CH.sub.3CO) or an aromatic acyl (e.g. C.sub.6H.sub.5CO).
[0163] Additionally, the formulas of I-V optionally include at
least one pseudopeptide bond between amino acids residues. By
"psuedopeptide bond" is meant that the carbon atom participating in
the bond between two residues is reduced from a carbonyl carbon to
a methylene carbon, i.e., CH2-NH; or less preferably that of CO--NH
is replaced with any of CH.sub.2--S, CH.sub.2--CH.sub.2,
CH.sub.2--O, or CH.sub.2--CO. A psuedopeptide peptide bond is
symbolized herein by ".psi.". Preferably, the psuedopeptide bonds
are located between one or more amino acid residues. In addition,
such psuedopeptide bond analogs can be used to form dimeric
analogs. A detailed discussion of psuedopeptide bonds is given in
Coy et al. (1998) Tetrahedon 44:835-841.
[0164] Accordingly, in another aspect and as already specifically
illustrated by certain of the preferred compounds, the invention
features dimers, trimers, etc. of compounds having the formula
(I-V). These dimers, trimers, etc. may be prepared, for example, by
dimerizing compounds of formula (I-V) with dicarboxylic acids
(e.g., succinic acid), cystine, or diaminodicarboxylic acid (e.g.,
2,6-diaminopimelic acid) as is known in the art. Resulting
compounds can include, for example, pentapeptide linear tandem or
parallel dimers.
[0165] In another aspect, the invention features a method of
controlling the food intake, i.e. appetite, of a subject comprising
administering to said subject the compound of formula I, II, or III
alone or in combination with formulas IV or V. These compounds also
can be conjugated to carriers, such carriers can include albumin,
such as cationized albumin (Endocrinology 126:977-984 (1990); J.
Pharmacol Exp. Therao. 268:791-796 (1994)), polyethylene glycol,
polyglutamic acid, polyleucine, polyisoleucine or polylysine, e.g.,
MAP.
[0166] In other preferred embodiments, a therapeutically effective
amount of a compound of formula I, II, or III alone, or in
combination with formulas IV or V, and a pharmaceutically
acceptable carrier substance, e.g., magnesium carbonate or lactose,
together form a therapeutic composition capable of suppressing an
NPY mediated physiological response. This composition can be in the
form of a pill, tablet, capsule, liquid, or sustained released
tablet for oral administration; or a liquid for nasal
administration as drops or spray; or a liquid for intravenous,
subcutaneous, parenteral, or intraperitoneal administration.
Another preferred form for administration includes a biodegradable
sustained-release composition for intramuscular administration to a
subject in need of the composition. Preferably, the composition
includes a lipophilic salt and is suitable for administration in
the form of an oil emulsion or dispersion to a subject in need of
the composition.
[0167] In yet another aspect, the invention features methods for
controlling an NPY mediated physiological response in a subject;
such methods involve administering a compound of formula I, II, or
III alone, or in combination with formulas IV or V, to a subject in
a dosage effective to control blood pressure, cardiovascular
response, libido, circadian rhythm, hyperlipidimia, chronic
pancreatitis, and nonalcoholic fatty liver disease including
nonalcoholic steatohepatitis.
[0168] The NPY Y.sub.4 receptor agonists including pancreatic
polypeptide (PP), analogs thereof, and peptide fragments of PP,
e.g. PP(32-36), and analogs thereof such as those of compounds
(I-III), either alone or in combination with NPY Y.sub.2 receptor
agonists including peptide YY (PYY) (3-36), analogs thereof, and to
peptide fragments of PYY(3-36), e.g. PYY(22-36) and PYY(25-36), and
analogs thereof such as those of compounds (IV-V), also can be
useful in treating any number of illnesses that involve eating
disorders, cardiovascular function, alterations in sexual function,
as well as disorders of sleep and circadian rhythms (see, e.g.,
Harrison's Principles of Internal Medicine, McGraw-Hill Inc., New
York, 12th ed.).
[0169] Other features and advantages of the invention will be
apparent to one skilled in the art.
[0170] The symbol A1, A2, A3, and the like; and Tyr, Lys or the
like, as found in a peptide sequence herein stands for an amino
acid residue, e.g., --N--CH(R)--CO-- when it is at the N-terminus,
or --NH--CH(R)--CO-- when it is at any other position, where R
denotes the side chain (or identifying group) of an amino acid or
its residue. For example, R is CH.sub.2COOH for Asp, R is --H for
Gly, R is --CH.sub.2OH for Ser, R is --CH.sub.3 for Ala and R is
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2NH.sub.2 for Lys.
[0171] As set forth above and for convenience in describing this
invention, the conventional and certain unconventional
abbreviations for the various amino acids are used. They are
familiar to those skilled in the art; but for clarity are listed
below. All peptide sequences mentioned herein are written according
to the usual convention whereby the N terminal amino acid is on the
left and the C-terminal amino acid is on the right. A short line
between two amino acid residues indicates a peptide bond.
[0172] Abbreviations (common):
[0173] Asp=D=Aspartic Acid
[0174] Ala=A=Alanine
[0175] Arg=R=Arginine
[0176] Asn =N=Asparagine
[0177] Cys =C=Cysteine
[0178] Gly=G=Glycine
[0179] Glu=E=Glutamic Acid
[0180] Gin=Q=Glutamine
[0181] His =H=Histidine
[0182] Ile=I=Isoleucine
[0183] Leu=L=Leucine
[0184] Lys=K=Lysine
[0185] Met=M=Methionine
[0186] Phe =F=Phenylalanine
[0187] Pro=P=Proline
[0188] Ser=S=Serine
[0189] Thr=T=Threonine
[0190] Trp=W=Tryptophan
[0191] Tyr=Y=Tyrosine
[0192] Val=V=Valine.
[0193] Abbreviations (uncommon):
[0194] Aoc=8-aminooctanoic acid
[0195] Orn=Ornithine
[0196] Nal=2-napthylalanine
[0197] Thi=2-thienylalanine
[0198] Pcp=4-chlorophenylalanine
[0199] Bth=3-benzothienyalanine
[0200] Bip=4,4'-biphenylalanine
[0201] Tic=tetrahydroisoquinoline-3-carboxylic acid
[0202] Aib=aminoisobutyric acid
[0203] Anb=alpha-aminonormalbutyric acid
[0204] Ac.sub.6c=1-aminocyclohexanecarboxylic acid
[0205] D-Pal=beta-(3-pyridyl)alanine;
[0206] Tcc=tetrahydrocarbolenecarboxylic acid
[0207] Abu=.alpha.-aminonormalbutyric acid
[0208] hArg(Pr).sub.2=N,N'-guanidino-(dipropyl)-homoarginine
[0209] Tic-OH=1,2,3,4 tetrahydroisoquinoline-7-hydroxy-3-carboxylic
acid
[0210] Dip=3,3-diphenylalanine
[0211] 2-Nal=3(2-naphthylalanine)
[0212] Tfp=Paratrifluoromethyl phenylalanine
[0213] Fla=3-(9-Fluorenyl)alanine
[0214] Flg=9-Fluorenylglycine
[0215] Cit=Citruline
[0216] Adp=2,5-diaminoadipic acid
[0217] Pim=2,6-diaminopimelicacid
[0218] Sub=2,7-diaminosuberic acid
[0219] Nle=Norleucine
[0220] Nva=Norvaline
[0221] Thz=4-Thiazolylalanine
[0222] Dpr=Dap=2,3-diaminopropionic acid
[0223] Pyr=Pyroglutamic acid
[0224] Tip=1,2,3,4-tetrahydronorhamman-3-carboxylic acid
[0225] Pen=Penicillamine.
[0226] Other Abbreviations:
[0227] Fmoc =N-(9-fluorenyl)methoxycarbonyl.
[0228] The terms "C2-C4-alkenyl" and "C2-C6-alkenyl" as used herein
refer to a 2 to 4 to 6 straight- or branched-chain of carbon atoms
which contains a carbon-carbon double bond, such as allyl,
propenyl, butanol, isoprenyl and the like.
[0229] The terms "C1-C18-alkyl" as used herein refer to straight or
branched chain alkyl radicals having from 1 to 18 carbon atoms
including, but not limited to, methyl, ethyl, propyl, isopropyl,
butyl, isobutyl, sec-butyl, pentyl, neopentyl hexyl, and the
like.
[0230] The term "C6-C18-aryl" as used herein refers to phenyl or to
a "bicyclic carbocyclic" group or "bicyclic carbocycle" having two
fused carbocyclic rings, each ring having 5, 6 or 7 carbon atoms,
and each ring being fully saturated, partially saturated or
aromatic. Bicyclic carbocyclic groups include, but are not limited
to, naphthyl, tetrahydronaphthyl, decalin, indanyl, indenyl and the
like.
[0231] The term "C7-C18-arylalkyl" as used herein refers to an aryl
group appended to a C1-C4-alkyl radical including, but not limited
to, benzyl, phenethyl, naphthylmethyl and the like.
[0232] The term "bicyclic heterocycle" as used herein refers to a
group having two fused rings, one or both of which are heterocyclic
rings as defined herein. When both rings are not heterocyclic, the
other ring is carbocyclic and is saturated, partially saturated or
aromatic, preferably a benzene ring. Bicyclic heterocyclic groups
can be unsubstituted or monosubstituted or disubstituted with
substituents independently selected from hydroxy, halo, oxo
(.dbd.O), amino, C1-C4-alkylamino, di-(C1-C4)-alkylamino,
C1-C4-alkoxy, thio-C1-C4-alkoxy, carboxy, C1-C4-alkoxycarbonyl,
C1-C4-alkyl, C3-C8-cycloalkyl, --OSO.sub.3H and halo-C1-C4-alkyl.
Examples of bicyclic heterocycles include indole, 5-hydroxyindole,
quinoline, isoquinoline, tetrahydroisoquinoline, quinoxaline,
benzimidazole, benzofuran, and the like.
[0233] The term "cyclo-C3-C10-alkyl" as used herein refers to an
aliphatic monocyclic of 3 to 10 or bicyclic group having 6 to 10
carbon atoms including, but not limited to, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, adamantyl, and the
like.
[0234] The term "halo" or "halogen" as used herein refers to
chloro, bromo, iodo or fluoro.
[0235] The term "halo-C1-C4-alkyl" as used herein refers to a lower
alkyl radical in which one to three hydrogen atoms have been
replaced by a halogen including, but not limited to, chloromethyl,
2-fluoroethyl, trifluoromethyl and the like.
[0236] The term "monocyclic heterocyclic group" or "monocyclic
heterocycle" as used herein refers to any 3- or 4-membered ring
containing a heteroatom selected from oxygen, nitrogen and sulfur,
or a 5- or 6-membered ring containing carbon atoms and one, two or
three nitrogen atoms; one nitrogen and one sulfur atom; or one
nitrogen and one oxygen atom; wherein the 5-membered ring has 0-2
double bonds and the 6-membered ring has 0-3 double bonds; wherein
the nitrogen and sulfur heteroatoms may optionally be oxidized; and
wherein the nitrogen heteroatom may optionally be quaternized.
Heterocycles include, but are not limited to, pyridyl, imidazolyl,
furyl, thienyl, pyrazinyl, pyrrolyl, pyrimidyl and the like.
Heterocyclics may be unsubstituted or mono- or disubstituted with
substituents independently selected from hydroxy, halo, oxo
(.dbd.O), amino, C1-C4-alkylamino, (C1-C4)-2-alkylamino,
C1-C4-alkoxy, thio-C1-C4-alkoxy, carboxy, C1-C4-alkoxycarbonyl,
C1-C4-alkyl, C3-C8-cycloalkyl, --OSO.sub.3H and
halo-C1-C4-alkyl.
[0237] The term "peptide bond" as used herein refers to the
chemical bond between carbon and nitrogen in the bivalent group
CONH that unites amino acid residues in a peptide.
[0238] In addition, the above compounds may contain two or more
asymmetric carbon atoms and thus can exist as pure diastereomers,
mixtures of diastereomers, diastereomeric racemates or mixtures of
diastereomeric racemates. As such, the present invention includes
within its scope all of the isomeric forms. In keeping with
standard peptide nomenclature, J. Biol. Chem., 1969, 243:3557-59,
abbreviations for amino acid residues are used herein.
[0239] It is noted that all amino acid residue sequences are
represented herein by formulae whose left to right orientation is
in the conventional direction of amino-terminus to
carboxy-terminus.
Administration
[0240] The amount of active ingredient that may be combined with
the carrier materials to produce a single dosage form will vary
depending upon the host treated, the particular treatment and the
particular mode of administration.
[0241] It will be understood, however, that the specific dose level
for any particular patient will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, sex, diet, time of administration,
rate of excretion, drug combination, and the severity of the
particular disease undergoing therapy.
[0242] However, generally speaking the following guidelines will
suffice. When an NPY Y.sub.4 receptor agonist, such as those of
compounds (I-III), is either used alone or in combination with an
NPY Y.sub.2 receptor agonist, such as those of compounds (IV-V), as
an agonist(s) of NPY in a human subject, the total daily dose
administered in single or divided doses may be in amounts, for
example, from 0.001 to 1000 mg a day and more usually 1 to 1000 mg.
Dosage unit compositions may contain such amounts of submultiples
thereof to make up the daily dose.
[0243] The compounds useful in the present inventive method may be
administered by any suitable means. One skilled in the art will
appreciate that many suitable methods of administering the
compounds to an animal in the context of the present invention, in
particular a human, are available, and, although more than one
route may be used to administer a particular compounds, a
particular route of administration may provide a more immediate and
more effective reaction than another route.
[0244] The compositions according to the present invention may be
formulated for administration by any suitable route such as the
oral, rectal, nasal, topical (dermal) or parenteral administration
route. Thus, the composition may be in the form of tablets,
capsules, suspensions, emulsions, solutions, injectables,
suppositories, sprays, aerosols and in other suitable form.
[0245] Formulations for oral use include tablets that contain the
active ingredient in admixture with non-toxic pharmaceutically
acceptable excipients. These excipients may be, for example, inert
diluents, such as calcium carbonate, sodium chloride, lactose,
calcium phosphate or sodium phosphate; granulating and
disintegrating agents, for example, potato starch or alginic acid;
binding agents, for example, starch, gelatin or acacia; and
lubricating agents, for example, magnesium stearate, stearic acid
or talc. Other pharmaceutically acceptable excipients can be
colorants, flavoring agents, plasticizers, humectants etc. The
tablets may be uncoated or they may be coated by known techniques,
optionally to delay disintegration and absorption in the
gastrointestinal tract and thereby provide a sustained action over
a longer period. For example, a time delay material such as
glyceryl monostearate or glyceryl distearate may be employed.
[0246] Formulations for oral use may also be presented as chewing
tablets, or as hard gelatin capsules wherein the active ingredient
is mixed with an inert solid diluent, for example, calcium
carbonate, calcium phosphate or kaolin, or as soft gelatin capsules
wherein the active ingredient is mixed with water or an oil medium,
for example, peanut oil, liquid paraffin, or olive oil.
[0247] Powders, dispersible powders or granules suitable for
preparation of an aqueous suspension by addition of water are also
convenient dosage forms of the present invention. Formulation as a
suspension provide the active ingredient in admixture with a
dispersing or wetting agent, suspending agent and one or more
preservatives. Suitable dispersing or wetting agents are, for
example, naturally-occurring phosphatides, as e.g. lecithin, or
condensation products of ethylene oxide with e.g. a fatty acid, a
long chain aliphatic alcohol or a partial ester derived from fatty
acids and a hexitol or a hexitol anhydrides, for example,
polyoxyethylene stearate, polyoxyethylene sorbitol monooleate,
polyoxyethylene sorbitan monooleate etc. Suitable suspending agents
are, for example, sodium carboxymethylcellulose, methylcellulose,
sodium alginate etc.
[0248] The present agents can also be administered in the form of
liposomes. As is known in the art, liposomes are generally derived
from phospholipids or other lipid substances. Liposomes are formed
by mono- or multi-lamellar hydrated liquid crystals that are
dispersed in an aqueous medium. Any non-toxic, physiologically
acceptable and metabolizable lipid capable of forming liposomes can
be used. The present compositions in liposome form can contain, in
addition to the peptides of the present invention, stabilizers,
preservatives, excipients, and the like. The preferred lipids are
the phospholipids and the phosphatidyl cholines (lecithins), both
natural and synthetic.
[0249] Methods to form liposomes are known in the art. See, for
example, Prescott, Ed., Methods in Cell Biology, Volume XIV,
Academic Press, New York, N.Y. (1976), p. 33 et seq.
[0250] The pharmaceutical formulation may also be administered
parenterally (intravenous, intramuscular, subcutaneous or the like)
in dosage forms or formulations containing conventional, non-toxic
pharmaceutically acceptable carriers and adjuvants. The formulation
and preparation of such compositions is well-known to those skilled
in the art of pharmaceutical formulation.
[0251] For parenteral use, the pharmaceutical compositions
according to the invention may comprise the thermogenic compounds
in the form of a sterile injection. To prepare such a composition,
the compounds are dissolved or suspended in a parenterally
acceptable liquid vehicle. Among acceptable vehicles and solvents
that may be employed are water, water adjusted to a suitable pH by
addition of an appropriate amount of hydrochloric acid, sodium
hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution
and isotonic sodium chloride solution. The aqueous formulation may
also contain one or more preservatives, for example, methyl, ethyl
or n-propyl p-hydroxybenzoate.
[0252] For the rectal application, suitable dosage forms for a
composition according to the present invention include
suppositories (emulsion or suspension type), and rectal gelatin
capsules (solutions or suspensions). In a typical suppository
formulation, the compounds are combined with an appropriate
pharmaceutically acceptable suppository base such as cocoa butter,
esterified fatty acids, glycerinated gelatin, and various
water-soluble or dispersible bases like polyethylene glycols and
polyoxyethylene sorbitan fatty acid esters. Various additives like,
for example, enhancers or surfactants, may be incorporated.
[0253] For the nasal application, typical dosage forms for a
composition according to the present invention include nasal sprays
and aerosols for inhalation. In a typically nasal formulation, the
active ingredients are dissolved or dispersed in a suitable
vehicle. The pharmaceutically acceptable vehicles and excipients
and optionally other pharmaceutically acceptable materials present
in the composition such as diluents, enhances, flavoring agents,
preservatives, etc., are all selected in accordance with
conventional pharmaceutical practice in a manner understood by the
persons skilled in the art of formulating pharmaceuticals.
[0254] The pharmaceutical compositions according to the invention
may also be administered topically on the skin for percutaneous
absorption in dosage forms or formulations containing
conventionally non-toxic pharmaceutically acceptable carriers and
excipients including microspheres and liposomes. The formulations
include creams, ointments, lotions, liniments, gels, hydrogels,
solutions, suspensions, pastes, plasters and other kinds of
transdermal drug delivery systems. The pharmaceutically acceptable
carriers or excipients may include emulsifying agents,
antioxidants, buffering agents, preservatives, humectants,
penetration enhancers, chelating agents, gel-forming agents,
ointment bases, perfumes and skin protective agents.
[0255] Examples of emulsifying agents are naturally occurring gums,
e.g., gum acacia or gum tragacanth, naturally occurring
phosphatides, e.g., soybean lecithin and sorbitan monooleate
derivatives.
[0256] Examples of antioxidants are butylated hydroxy anisole
(BHA), ascorbic acid and derivatives thereof, tocopherol and
derivatives thereof and cysteine.
[0257] Examples of preservatives are parabens and benzalkonium
chloride.
[0258] Examples of humectants are glycerin, propylene glycol,
sorbitol and urea.
[0259] Examples of penetration enhancers are propylene glycol,
DMSO, triethanoiamine, N,N-dimethylacetamide,
N,N-dimethylformamide, 2-pyrrolidone and derivatives thereof,
tetrahydrofurfuryl alcohol and AZONE.RTM..
[0260] Examples of chelating agents are sodium EDTA, citric acid
and phosporic acid.
[0261] Examples of gel forming agents are Carbopol, cellulose
derivatives, bentonit, alginates, gelatin and PVP.
[0262] Examples of ointment bases are beeswax, paraffin, cetyl
palmitate, vegetable oil, sorbitan esters of fatty acids (Span),
polyethyleneglycols, and condensation products between sorbitan
esters of fatty acids and ethylene oxide, e.g., polyoxyethylene
sorbitan monooleate (Tween).
[0263] The formulation and preparation of the above-mentioned
compositions is well-known to those skilled in the art of
pharmaceutical formulation. Specific formulation can be found in
"Remington's Pharmaceutical Sciences" incorporated herein by
reference.
[0264] In one aspect the present invention relates to a method for
treatment of overweight or obesity in individuals, in particular in
humans or for reducing the adipose tissue mass/lean mass body mass
ratio of an individual, in particular a human or a domestic
animal.
[0265] In the present context the term "overweight" is used as an
indication of a body with a weight exceeding the "desirable
weight", whereas the term "obesity" is used when the body weight is
20% or more above the "desirable weight". Desirable weights for
humans are given by the Council on Scientific Affairs defining the
desirable weights for humans according to Metropolitan Height and
Weight Tables as the midpoint of the range of the medium-frame
individuals.
[0266] In another aspect, the present invention relates to a method
for the treatment of diseases that are complications to overweight
or obesity. These diseases or conditions include, for example,
diabetes mellitus type II, hypercholesterolemia,
hypertriglyceridaemia and hypertension.
[0267] In another aspect, the present invention also relates to a
method of reducing adipose tissue mass/lean body mass ratio or
treating overweight or obesity or complications thereof by means of
subjecting the individuals to a diet regimen. The diet regimen into
which the individuals may be subjected in connection with the
administration of compositions of the present invention may include
a low carbohydrate, a low fat and a low energy regimen, e.g., a
diet of from 800-2500 kcal/day.
Veterinary Use
[0268] The NPY Y.sub.4 receptor agonists including pancreatic
polypeptide (PP), analogs thereof, and peptide fragments of PP,
e.g. PP(32-36), and analogs thereof such as those of compounds
(I-III), either alone or in combination with NPY Y.sub.2 receptor
agonists including peptide YY (PYY) (3-36), analogs thereof, and to
peptide fragments of PYY(3-36), e.g. PYY(22-36) and PYY(25-36), and
analogs thereof such as those of compounds (IV-V), can also be
administered to domestic animals in order to improve the
performance of the animal (daily weight gain and feed utilization)
or to improve carcass quality or both. Carcass quality is generally
improved when the fat tissue mass/lean mass body mass ratio is
decreased, i.e., when the body content of meat is increased e.g.,
at the expense of the body content of fat.
[0269] The improvements in performance and carcass quality are
suggested to be caused by a reduced fat accretion and/or by an
increased skeletal muscle accretion. In growing animals, the amount
of lipid present is suggested to be governed by the relative rates
of lipolysis and lipogenesis. Stimulation of lipolysis and/or
inhibition of lipogenesis in fat tissue may lead to a reduced fat
accretion. In vivo and in vitro studies with both pigs and
ruminants may indicate that certain beta agonists stimulate
lipolysis and inhibit lipogenesis in fat tissue leading to a
reduced fat accretion.
[0270] Administration to an animal of the compositions according to
the present invention may be useful in order to increase the lean
body mass at the expense of body fat, particularly in domestic
animals like pigs, hogs, cattle, sheep and poultry. The composition
may be given in admixture with the feed in a suitable dose
corresponding to the size of the animal.
Peptide Synthesis
[0271] The peptides of the present invention can be synthesized by
any suitable method, such as by exclusively solid-phase techniques,
by partial solid-phase techniques, by fragment condensation or by
classical solution addition. A detailed description of certain of
these methods is contained in "The Peptides, Vol. 1", Gross and
Meinenhofer, Eds., Academic Press, New York, 1979. Coupling methods
employed include the carbodiimide method
(1,3-dicyclohexylcarbodiimide [DCC],
1-(3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride [EDCI])
with the option of racemization preventing additives
(1-hydroxybenzotriazole [HOBT]), the mixed anhydride method, the
azide method, the acid chloride method, the symmetrical anhydride
method, the use of bis(2-oxo-3-oxazolidinyl)phosphinic chloride
(BOP--Cl), and the active ester method (N-hydroxysuccinimide
esters, 4-nitrophenol esters, 2,4,5-trichlorophenol esters, and the
like). The synthesis of the PYY analogs of U.S. Pat. Nos.
5,604,203, and 6,046,167 to Balasubramaniam is fully disclosed
therein, which is hereby incorporated by reference herein, and
further is generally discussed below.
[0272] The techniques of exclusively solid-phase synthesis are set
forth in the textbook "Solid-Phase Peptide Synthesis" Stewart &
Young, Freeman & Co., San Francisco, 1969, and are exemplified
by the disclosure of U.S. Pat. No. 4,105,603, issued Aug. 8, 1978
to Vale et al. The fragment condensation method of synthesis is
exemplified in U.S. Pat. No. 3,972,859 (Aug. 3, 1976). Other
available syntheses are exemplified by U.S. Pat. No. 3,842,067
(Oct. 15, 1974) and U.S. Pat. No. 3,862,925 (Jan. 28, 1975). All of
the above references are incorporated herein by reference.
[0273] Synthesis by the use of recombinant DNA techniques, for
purposes of this application, should be understood to include the
suitable employment of a structural gene coding for peptides, or
peptide fragments, to transform a microorganism, using an
expression vector including a promoter and operator together with
such structural gene, and causing such transformed microorganism to
express the peptide or such a synthetic peptide fragment. A
non-human animal may also be used to produce the peptide by
gene-farming using such a structural gene in the microinjection of
embryos as described in U.S. Pat. No. 4,870,009 issued Sep. 26,
1989, incorporated herein by reference.
[0274] When the peptides are not prepared using recombinant DNA
technology, they are preferably prepared using solid phase
synthesis, such as that described by Merrifield, J. Am. Chem. Soc.,
85, p 2149 (1964), although other equivalent chemical syntheses
known in the art can also be used as previously mentioned.
Solid-phase synthesis is commenced from the C-terminus of the
peptide by coupling a protected alpha-amino acid to a suitable
resin as generally set forth in U.S. Pat. No. 4,244,946 issued Jan.
21, 1981 to Rivier et al., the disclosure of which is incorporated
herein by reference. A starting material for analogs of the present
invention can, for example, be prepared by attaching alpha-amino-
and side-chain-protected Tyr to a BHA resin.
[0275] The compounds of the invention may be prepared by stepwise
coupling of the amino acids or by coupling together fragments of
dipeptide length or greater. Thus, the free carboxylic acid moiety
from one amino acid or peptide fragment is activated and allowed to
condense with the free nitrogen group of the second amino acid or
peptide fragment. The coupling reactions are conducted in solvents
such as methylene chloride (CH.sub.2Cl.sub.2), tetrahydrofuran
(THF), dimethylformamide (DMF) or other such solvents.
[0276] During the coupling process, the non-participating
carboxylic acids or amines on the reacting set of amino acids or
peptide fragments are protected by a protecting group that can be
selectively removed at a later time if desired. A detailed
description of these groups and their selection and chemistry is
contained in "The Peptides, Vol. 3", Gross and Meinenhofer, Eds.,
Academic Press, New York, 1981, incorporated herein in its entirety
by reference. Thus, useful protective groups for the amino group
are benzyloxycarbonyl(Cbz), t-butyloxycarbonyl(t-BOC),
2,2,2-trichloroethoxycarbonyl(Troc), t-amyloxycarbonyl,
4-methoxybenzyloxycarbonyl, 2-(trichlorosilyl)ethoxycarbonyl,
9-fluorenylmethoxycarbonyl(Fmoc), phthaloyl, acetyl (Ac), formyl,
trifluoroacetyl, and the like.
[0277] Examples of useful protective groups for the carboxylic acid
include esters, such as methyl, ethyl, benzyl, t-butyl,
2,2,2-trichloroethyl, allyl, 4-nitrobenzyl, and the like. Removal
of these protecting groups may be accomplished selectively by
employing various acid or base catalyzed hydrolytic,
hydrogenolytic, thermal or dissolving metal conditions.
[0278] Generally, peptides will be synthesized by stepwise solid
phase methodology developed by using an automated Applied Biosystem
Model 430A peptide synthesizer. Tertiary butyloxy-carbonyl (Boc)
amino acids with benzyl or halobenzyl based side chain protecting
groups (Asp & Glu with Obzl or OcHex; Ser & Thr with Bzl:
Cys with pMeBzl; Tyr with 2BrZ; Lys with 2ClZ; Arg with Tos; His
with Bom; Trp with CHO) will be used in conjunction with
phenylacetamidomethyl (PAM) resin. In the case of the synthesis of
peptide amides, benzyldrylamine (BHA) or
paramethylbenzylhydrylamine (MBHA) will be used instead of PAM
resin.
[0279] Boc-aminoacid-PAM-resin, using
Boc-aminoacyloxy-methyl-phenylacetic acid and aminomethyl resin, is
available commercially. The Boc-aminoacid-PAM-resin thus prepared
eliminates the possibility of chain termination by
tri-fluoroacetylation. Attachment to BHA or MBHA resin will be
performed by way of preformed symmetrical anhydride.
[0280] Coupling and deprotection functions are generally carried
out automatically by the instrument. The standard program provided
by the manufacturers are modified to incorporate a double coupling
procedure, first in DMF and then in CH.sub.2Cl.sub.2. Altering the
polarity of the solvents improves the coupling. All amino acids,
except Asn, Gln and Arg, will generally be coupled as preformed
symmetrical anhydrides. Asn, Gln and Arg are double coupled as
preformed 1-hydroxy-benzotriazole esters to avoid side reactions.
Resin samples taken during these reactions may be assayed by
quantitative procedure to determine the degree of coupling. Other
standard cleaving reagents and conditions for removal of specific
alpha-amino protecting groups may be used as described in Schroder
& Lubke, "The Peptides" Vol. 1, pp 72-75 (Academic Press 1965),
incorporated herein in its entirety by reference.
[0281] In the case of coupling unusual amino acids, suitable
conditions (solubility, coupling times) will be first developed
before using in automated mode. In some cases these couplings will
be carried out manually (e.g.: pseudopeptides, N-Me-amino acids).
Pseudopeptide bonds will be incorporated by the methods described
earlier. The t-Boc-amino acid aldehyde will be obtained by reducing
N-methoxy-N-methylamide derivatives of Boc-amino acids with
LiAlH.sub.4. The aldehyde obtained will be reacted immediately with
the a-amino group of the peptide attached to the resin in DMF
containing 1.0% HOAC in the presence of an equivalent amount of
NaBH.sub.3CN. At the end of the reaction, the presence of secondary
amine is tested for with ninhydrin (wine-red color). The secondary
amine formed will then be blocked by reacting with 2 equivalents of
Z(2-Cl)OSU, 2 equivalents of HOBT, and 4 equivalents of
diisopropylethyamine until ninhydrin gives a yellow color. This way
the formation of branched peptide is prevented. Coupling of
sterically hindered amino acids (e.g.: N-Me-amino acids,
C.alpha.MeLeu, Aib) will be effected by a HOAT or HATU which has
been shown to be superior to BOP/HOBT.
[0282] For the final cleavage, the N-.alpha.-Boc group and the
Nin-CHO will be first removed with 50% TFA/CH.sub.2Cl.sub.2 and 20%
piperidine-DMF from the protected peptide resin before detaching
the target peptide using HF containing p. cresol (5%). If Cys and
Met are present, p. thicresol (2.5%) will also be added to the HF
reaction mixture. If problems are encountered during the standard
HF method, then the "low/high" HF procedure will be used.
[0283] The materials are then purified. After initial fractionation
on Sephadex G-25, the peptide material will be subjected to
reversed phase high performance liquid chromatography (RPLC) on
C.sub.18 Vydac columns. However, peptides may be first subjected to
ion exchange chromatography before RPLC, depending upon the
heterogeneity of the crude peptide. The homogeneity of the purified
product may be confirmed by analytical RPLC using two different
solvent systems, amino acid analysis, complete sequencing, and mass
spectral analysis.
[0284] For analysis, the peptide resins are hydrolyxed using 12N
HCl/HOAc/phenol (2:1:1) for 24 hours at 110.degree. C. The free
peptides are hydrolyzed for 24 hours in 6N HCl containing 0.1%
phenol or 4N methane sulfonic acid at 110.degree. C. and are
quantified on a Waters Pico Tag system. Peptide hormones and
fragments are then subjected to complete sequencing on an automated
gas phase sequencer (Applied Biosystem, Model 470A).
[0285] For the production of a compound of the invention where any
one or several of the constituent amino acids bear an N-alkyl
group, specifically methyl, the corresponding N-alkyl amino acid
can be prepared via the method described by Benoiton (Can. J.
Chem., 1977, 55:906) or Shuman ("Peptides: Proceedings of the 7th
American Peptide Symposium", D. Rich, E. Gross, Eds., Pierce
Chemical Co., Rockford, Ill. 1981, p 617), wherein the t-BOC- or
Cbz-protected amino acid is treated with a base in the presence of
a chelating agent such as a crown ether and then quenched with
methyl iodide. An alternative method described by Freidinger (J.
Org. Chem., 1983, 48:77), in which triethylsilane reduction of the
oxazolidinone of an amino acid directly produces the N-methyl
derivative may also be utilized.
[0286] The reduced carbonyl amide bond surrogates can be prepared
in a manner similar to that described by Martinez (J. Med. Chem.
1987, 30:1366). The N-alpha-t-BOC protected amino acid (with
appropriate protection of side chain functional groups) is
converted to the 3,5-dimethylpyrazolide, which is then reduced with
lithium aluminum hydride. The resulting aldehyde is then allowed to
condense with an amino acid or peptide bearing a free amino
terminus. Reduction of the Schiff base that is formed as a result
of the condensation is accomplished using sodium cyanoborohydride
to yield the desired compound having a reduced amide bond.
[0287] Functionalization of the epsilon-amino group of the lysine
(Lys) or homologous (e.g., Orn) residue is achieved via activation
of the acid fragment as the active ester (N-hydroxysuccinimide,
2,4,5-trichlorophenol, etc.) or, if no other free carboxylic acid
function is present on the peptide, coupling using any of the
methods mentioned above is applicable. In addition, the
functionalization of the epsilon-amino group may be accomplished by
reaction with various alkyl and aryl isocyanates, as well as alkyl
and aryl isothiocyanates.
[0288] The sulfuric acid esterification of the phenolic residues
may be conducted using a variety of known reagents such as the
pyridine-sulfuric anhydride or the pyridine-sulfur trioxide
complex. Use of pyridinium acetyl sulfate as described by Penke and
Rivier ("Proceedings of the 8th American Peptide Symposium", V.
Hruby, D. Rich, Eds., Pierce Chemical Company, Rockford, Ill.;
1983; p. 119), may also be applied to prepare the sulfuric acid
ester derivative of the peptides.
[0289] The compounds of the present invention can be used in the
form of salts derived from inorganic or organic acids. These salts
include but are not limited to the following: acetate, adipate,
alginate, citrate, aspartate, benzoate, benzenesulfonate,
bisulfate, butyrate, camphorate, camphorsulfonate, digluconate,
cyclopentanepropionate, dodecylsulfate, ethanesulfonate,
glucoheptonate, glycerophosphate, hemisulfate, heptonate,
hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide,
2-hydroxy-ethanesulfonate, lactate, maleate, methanesulfonate,
nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate,
persulfate, 3-phenylpropionate, picrate, pivalate, propionate,
succinate, tartrate, thiocyanate, tosylate, and undecanoate. Also,
the basic nitrogen-containing groups can be quaternized with such
agents as loweralkyl halides, such as methyl, ethyl, propyl, and
butyl chloride, bromides, and iodides; dialkyl sulfates like
dimethyl, diethyl, dibutyl, and diamyl sulfates, long chain halides
such as decyl, lauryl, myristyl and stearyl chlorides, bromides and
iodides, aralkyl halides like benzyl and phenethyl bromides, and
others. Water or oil-soluble or dispersible products are thereby
obtained.
[0290] The pharmaceutically acceptable salts of the present
invention can be synthesized which contain a basic or acidic moiety
by conventional chemical methods. Generally, the salts are prepared
by reacting the free base or acid with stoichiometric amounts or
with an excess of the desired salt forming inorganic or organic
acid or base in a suitable solvent or various combinations of
solvents.
[0291] Examples of preferred salts are those with therapeutically
acceptable organic acids, e.g., acetic, lactic, maleic, citirc,
malic, ascorbic, succinic, benzoic, or pamoic acid, as wells as
polymeric acids and salts with inorganic acids such as the
hydrohalic acids, e.g., hydrocholoric and sulfuric acids.
[0292] In addition, pseudopeptide bonds may, if desired, may be
introduced at various positions, e.g., between amino acid residues
A1-A2 or between residues A2-A3. Optically pure Boc-AA-CHO can be
obtained in good yields and coupled directly to the --NH2 group of
the peptide resin by published methods (Sasaki et al., Peptides
8:119-121, 1987; Fehrentz et al., Synthesis pp. 676-678, 1983). The
secondary amine in the pseudopeptide bond is capped with Z(2-Cl).
This is introduced by reacting the peptide resin with Z(2-Cl)--OSU
(2 equiv.), HOBT (2 equiv.) and DIEA (4 equiv.) for 10-60 min. The
red wine color of ninhydrin with secondary amine turns yellow at
the end of capping.
[0293] Exemplary compounds of the present invention include:
##STR24##
[0294] These compounds may be combined with NPY Y.sub.2 receptor
agonists including PYY(3-36), analogs thereof, and peptide
fragments of PYY(3-36), e.g. PYY(22-36) and PYY(25-36), and analogs
thereof such as those of formulas (IV-V).
[0295] Other analogs of the invention can be prepared as above and
tested for their biological activity effectiveness as antagonists
or agonists using the methods described below and those commonly
known in the art.
Functional Assays
Animals. Cell Lines and Cultures, And Reagents
[0296] Any suitable in vivo or in vitro system may be utilized to
assay and test the effectiveness of the compounds of the invention,
and combinations thereof as discussed above. Such assays may employ
in vivo methods for evaluating physiological responses, e.g., blood
pressure, renovascular function, feeding behavior, or circadian
rhythm, or in vivo biochemical systems evaluating receptor binding
in a suitable cell line, e.g., SK-N-MC (ATCC HBT 10) or SK-N-BE(2)
(Barnes et al. In Vitro, 17: 619 631, 1981); or in isolated cells,
e.g., cells isolated from the spleen, kidney, heart or brain. A
number of in vivo and in vitro biochemical systems known to those
skilled in the art are available for testing antagonists to
hypothalamic NPY receptors, e.g. the Y-1, Y-2, and Y-3 receptor
categories. Described below are assay methods which can be utilized
with cell lines such as SK-N-MC and SK-N-BE2 or isolated cardiac
membranes which possess the high-affinity hypothalamic NPY receptor
sites. Other systems are also known for evaluating NPY antagonists
to the hypothalamic NPY receptor, e.g. VSM cells (Sheikh et al.,
Am. J. Physiol. 260: G250 G257, 1991) and HEL cells (Motulsky et
al. Amer. J. Physiol. 255: E880-E885, 1988); Y-2 receptor, e.g.,
kidney (Sheikh et al., Am. J. Physiol 26:F978-F984), spleen
(Lunberg et al., Eur. J. Pharmal. 145:21-29, 1988), dorsal root
ganglion (Bleakman et al., Br. J. Pharmal. 103:1781-1789, 1991) and
hippocampal cells (Sheikh et al., J. Biol. Chem. 265:8304 8310,
1990); and Y-3 receptors, e.g., in cardiac ventricular membranes
(Balasubramaniam et al., Peptides 11: 545-550, 1990), chromaffin
cells, rat gastric mucosa (Michel, M. C., Trends in Pharmol. Sci.
12: 389-394, 1991) and brain stem.
In Vitro Biochemical Assays
[0297] The ability of the compounds of the invention, and
combinations thereof as discussed above, to act as antagonists of
NPY can be demonstrated by any number of methods known in the art.
For example, the compounds can be shown to compete with iodinated
neuropeptide Y for receptors using the methods described by
Lundberg et al. (Eur. J. Pharmol. 145: 21-29, 1988); Gordon et al.
(J. Neurochemistry 55:506-513, 1990); Walker et al. (Mol.
Pharmacol. 34:779 792, 1988); Balasubramaniam et al. (Peptides
10:1283-1286, 1989).
[0298] In one example demonstrating antagonists to hypothalamic NPY
receptors, rat hypothalamus was isolated and the membranes were
prepared for binding and adenylate cyclase studies according to
standard methods (Unden et al. 1984. Eur. J. Biochem 145: 525-530;
Westlind-Danielsson et al., Neurosci. Lett. 74: 237-242 (1987)).
Displacement studies are performed in a total volume of 0.25 ml 20
mM HEPES buffer, pH 7.4, containing 1% bovine serum albumin, 0.1%
bacitracin, 300 .mu.m PMSF and 5 KIU/ml aprotinin. In a standard
assay, 100 .mu.g of membrane/tube is incubated in a shaking water
bath at 24.degree. C. for 45 min with [.sup.125I-Tyr]-NPY (20,000
CPM) as described by Balasubramaniam et al, (Peptides 11: 545-550,
1990), in the presence of increasing concentrations of NPY (10
.mu.OsM). At the end of incubation, 1.0 ml of iced cold buffer is
added, centrifuged at 10,000.times.g for 10 min, and the
supernatant removed by aspiration. The tube containing the pellet
is counted for bound radioactivity in a micromedic gamma
counter.
[0299] An example of assaying adenylate cyclase activity of
hypothalamic and cerebral cortex membranes is now described.
[0300] Adenlyate cyclase activity of the hypothalamic and cerebral
cortex membranes is determined by incubating 50 .mu.g of membranes
in a total volume of 0.20 ml Tris-HC.mu.30 mM pH 7.4 buffer
containing 150 mM NaCl, 8.25 mM MgCl2, 0.75 mM EGTA, 1.5
theophylline, 20 .mu.g/ml aprotinin, 100 .mu.g/ml bacitracin, 1
mg/ml bovine serume albumin, 1 mM ATP, 20 mM creatine phosphate, 1
mg/ml phosphocreatine kinase, 10 .mu.M isopreternol, 10 .mu.M GTP,
and various concentrations of peptides (0-10 .mu.M). After
incubating the mixture at 35.degree. C. for 15 min in a shaking
water bath, the reaction is arrested by the addition of 100 .mu.M
EDTA and boiling for 3 min. cAMP is extracted and quantitated by
radioimmunoassay. All the points in the binding and adenlyate
cyclase are the means of at least three parallel experiments
performed in duplicate.
In Vivo Assays
[0301] Any suitable in vivo model system can be used to evaluate
the properties of the compounds of the invention, and combinations
thereof as discussed above. Such models, without limitation,
include those used to evaluate feeding and memory behavior (Flood
et al., Peptides 10:963-966), and vasoconstriction and hypertension
(Balasubramaniam et al. Biochemm et Biophys Acta 997: 176-188,
(1989)).
[0302] Thus, in one example, feeding studies can be performed using
Spraque Dawley rats (350-450 g) with paraventricular hypothalmic
cannulae to investigate effects of PP, NPY, and/or PYY analogs
(Chance et al., Peptides 10: 1283, 1286 (1989)).
[0303] The following Examples set forth preferred methods for
synthesizing the analogs of PP and of fragments thereof, such as
the pentapeptide analogs of formulas (I-III) of the present
invention, as well as the analogs of PYY and of fragments thereof,
such as those of formulas (IV-V), using the solid-phase technique
which generally is in accordance with the procedure set forth in
U.S. Pat. No. 4,415,558 to Vale, et al., issued Nov. 15, 1983, the
disclosure of which is incorporated herein by reference.
EXAMPLES
Example I
[0304] Analogs of PP(32-36) and, more specifically, the
pentapeptide parallel dimer, ##STR25## which includes the
tetra-methylene spacer, was synthesized generally by standard
stepwise t-Boc-solid phase methods and purified by reversed phase
chromatography as is known in the art (J. Med. Chem. 2000, 49,
3420-3427).
[0305] More specifically, to obtain the parallel dimer, B-74,
N-.alpha.-Boc-D/L-diamino-dicarboxylic acids (0.5 equiv) was
coupled manually to the N-terminus of the protected pentapeptide
peptide resin in the presence of equivalent quantities of DIC,
HOBT, and DIEA. Free-peptide amides then were obtained by standard
HF cleavage of the protected peptide-MBHA resin in the presence of
about 5% p-cresol and about 2.5% thiocresol and purified by
reversed phase chromatography.
Example II
[0306] To study the feeding patterns of animals treated with B-74,
seven to eight week old C57BL/6 male mice (Harlan Laboratories,
Indianapolis, Ind.) were individually housed in cages in a
temperature controlled room (25.degree. C.) under 12-hour
light/dark cycles. Mice had free access to water and standard chow
and were acclimatized to daily ip saline injections. After one week
of acclimatizing, mice were fasted for 18 hours before the
experiment, and saline (0.1 ml), hPP (10 mmol/mice), or B-74 (100
nmol/mice) in saline (0.1 ml) were injected intraperitoneally. Food
was provided ten (10) minutes later, and the 2 and 4 hour food
intake was monitored. Statistical significance was determined by
ANOVA, with individual means being compared post-hoc by Tukey's
corrected t-test.
[0307] The results are shown in FIG. 1. Specifically,
intraperitoneal (ip) injections of PP (10 nmol/mice) and B-74 (100
nmol/mice) inhibited the 2 h and 4 h food intake to a greater
extent than the control.
Example III
[0308] The PYY (22-36) analog, N-.alpha.-Ac[Nle.sup.24,28,
Trp.sup.30, Nva.sup.31, .psi..sup.35/36]PYY(22-36)-NH.sub.2 (SEQ.
ID. NO. 7), referred to as B-48, wherein .psi. is --CH2-NH--, was
similarly synthesized by standard stepwise t-Boc-solid phase
methods and purified by reversed phase chromatography as is known
in the art according, and as referred to in Example I.
[0309] To further study the feeding patterns of animals treated
with B-48, experiments were performed in seven to eight week old
C57BL/6 male mice (Harlan Laboratories, Indianapolis, Ind.) after
acclimatization of mice for one week. More specifically, in
preliminary studies, overnight (18 h) fasted animals were injected
(ip) with 0.1 ml of saline or saline (0.1 ml) containing various
concentrations of B-48 (0.025, 0.050, 0.100 or 0.150 .mu.mol/mice)
(n=8-10 per group). The mice were provided with a known quantity of
chow 10 min later. The food intake was monitored over 1-24 h. The
results (not shown) revealed that a minimum dose of 0.050
.mu.mol/mice of B-48 was required to exhibit significant inhibitory
effects on food intake.
Example IV
[0310] To further compare the inhibitory effects of injecting (ip)
0.050 .mu.mol of B-48 or B-74 alone or together (B-48+B-74) on the
food intake by fasted mice, additional experiments were
conducted.
[0311] Accordingly, to study the feeding patterns of animals
treated with B-48 or B-74 alone or together (B-48+B-74), seven to
eight week old C57BL/6 male mice (Harlan Laboratories,
Indianapolis, Ind.) were individually housed in cages in a
temperature controlled room (25.degree. C.) under 12-hour
light/dark cycles. The mice had free access to water and standard
chow. After acclimatizing for one week, mice were fasted 18 hours
before the experiment. The fasted animals were injected (ip) with
0.1 ml of saline or saline (0.1 ml) containing B-48 (0.050
.mu.mol/mice) or B-74 (0.050 .mu.mol/mice) alone or together,
B-48+B-74 (0.100 .mu.mol/mice, i.e. 0.050 .mu.mol/mice per each
compound), (n=8-10 per group). The mice were provided with a known
quantity of chow 10 minutes later. The food intake was monitored
over 1-24 h. Statistical significance was determined by ANOVA, with
individual means being compared post-hoc by Tukey's corrected
t-test.
[0312] The results are shown in FIGS. 2 and 3. Specifically,
treatment with B-74 or B-48 alone inhibited food intake to a
greater extent than the control over the 24 h time period, while
the combination treatment (B-48+B-74) inhibited food intake to a
greater extent than either B-48 or B-74 taken alone. Table I below
utilizes the data from FIGS. 2 and 3 to further illustrate the
percentage inhibition of food intake by B-74 (0.05 .mu.mol), B-48
(0.050 .mu.mol), and B-74 (0.05 .mu.mol)+B-48 (0.050 .mu.mol) in
the fasted mice at the 2 h, 4 h, 8 h, and 24 h marks.
TABLE-US-00003 TABLE I Percentage of Inhibition of Food Intake
Compared to Saline Hours after Injection B-74 vs. (B-48 + B-74)
B-48 vs. (B-48 + B-74) 2 35 vs. 85 39 vs. 72 4 26 vs. 63 30 vs. 54
8 14 vs. 44 16 vs. 30 24 7 vs. 24 7 vs. 20
[0313] Accordingly, as shown in FIGS. 2 and 3, and Table I,
combined treatment of B-48+B-74 caused greater inhibition of food
intake at all time points as compared to individual treatment with
either B-74 or B-48, or the control.
[0314] While the present invention has been illustrated by the
description of the various embodiments thereof, and while the
embodiments have been described in considerable detail, it is not
intended to restrict or in any way limit the scope of the appended
claims to such detail. Additional advantages and modifications will
readily appeared to those skilled in the art. The invention in its
broader aspects is therefore not limited to the specific details,
representative apparatus and methods and illustrative examples
shown and described. Accordingly, departures may be made from such
details without departing from the scope of Applicants' general
inventive concept. Various features of the invention are emphasized
in the claims that follow.
Sequence CWU 1
1
10 1 36 PRT human neuropeptide Y MOD_RES (36)..(36) AMIDATION 1 Tyr
Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp 1 5 10
15 Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr
20 25 30 Arg Gln Arg Tyr 35 2 36 PRT porcine peptide YY 2 Tyr Pro
Ala Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu 1 5 10 15
Leu Ser Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr 20
25 30 Arg Gln Arg Tyr 35 3 36 PRT human peptide YY 3 Tyr Pro Ile
Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu 1 5 10 15 Leu
Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr 20 25
30 Arg Gln Arg Tyr 35 4 12 PRT Artificial synthesized by man
MOD_RES (1)..(1) ACETYLATION MOD_RES (12)..(12) AMIDATION 4 Arg His
Tyr Leu Asn Trp Val Thr Arg Gln Arg Tyr 1 5 10 5 12 PRT Artificial
synthesized by man misc_feature Reduced peptide bond between 11 and
12 (-CH2-NH-) MOD_RES (1)..(1) ACETYLATION MOD_RES (12)..(12)
AMIDATION 5 Arg His Trp Leu Asn Leu Val Thr Arg Gln Arg Tyr 1 5 10
6 12 PRT Artificial synthesized by man misc_feature Reduced peptide
bond between 11 and 12 (-CH2- NH-) MOD_RES (1)..(1) ACETYLATION
MOD_RES (12)..(12) AMIDATION 6 Arg His Tyr Leu Asn Trp Val Thr Arg
Gln Arg Tyr 1 5 10 7 15 PRT artificial synthesized by man
MISC_FEATURE reduced peptide bond between 14 and 15 (-CH2- NH-)
MOD_RES (1)..(1) ACETYLATION MOD_RES (3)..(3) Nle MOD_RES (7)..(7)
Nle MOD_RES (10)..(10) Nva MOD_RES (15)..(15) AMIDATION 7 Ala Ser
Xaa Arg His Tyr Xaa Asn Trp Xaa Thr Arg Gln Arg Tyr 1 5 10 15 8 36
PRT Artificial human pancreatic peptide (PP) 8 Ala Pro Leu Glu Pro
Val Tyr Pro Gly Asp Asn Ala Thr Pro Glu Gln 1 5 10 15 Met Ala Gln
Tyr Ala Ala Asp Leu Arg Arg Tyr Ile Asn Met Leu Thr 20 25 30 Arg
Pro Arg Tyr 35 9 10 PRT Artificial synthesized by man MOD_RES
(10)..(10) AMIDATION 9 Tyr Arg Leu Arg Tyr Tyr Arg Leu Arg Tyr 1 5
10 10 15 PRT Artificial synthesized by man MOD_RES (15)..(15)
AMIDATION 10 Tyr Arg Leu Arg Tyr Tyr Arg Leu Arg Tyr Tyr Arg Leu
Arg Tyr 1 5 10 15
* * * * *