U.S. patent application number 14/719232 was filed with the patent office on 2015-09-10 for melanocortin receptor-specific peptides.
This patent application is currently assigned to ASTRAZENECA AB. The applicant listed for this patent is AstraZeneca AB. Invention is credited to John H. Dodd, Yi-Qun Shi, Wei Yang.
Application Number | 20150252077 14/719232 |
Document ID | / |
Family ID | 43301172 |
Filed Date | 2015-09-10 |
United States Patent
Application |
20150252077 |
Kind Code |
A1 |
Dodd; John H. ; et
al. |
September 10, 2015 |
Melanocortin Receptor-Specific Peptides
Abstract
The invention relates to melanocortin receptor-specific cyclic
peptides of Formula (I) ##STR00001## or a pharmaceutically
acceptable salt thereof, where R.sub.1, R.sub.2, R.sub.3, R.sub.4a,
R.sub.4b, R.sub.4c, R.sub.5, x and y are as defined in the
specification. These compounds are particularly useful in the
treatments of energy homeostasis and metabolism related (e.g.
diabetes), food intake related and/or energy balance and body
weight related diseases, disorders and/or conditions, including
obesity, overweight and diseases, disorders and/or conditions
associated with obesity and/or overweight, such as type 2 diabetes
and metabolic syndrome.
Inventors: |
Dodd; John H.; (Cranbury,
NJ) ; Shi; Yi-Qun; (Cranbury, NJ) ; Yang;
Wei; (Cranbury, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AstraZeneca AB |
Sodertalje |
|
SE |
|
|
Assignee: |
ASTRAZENECA AB
Sodertalje
SE
|
Family ID: |
43301172 |
Appl. No.: |
14/719232 |
Filed: |
May 21, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13886864 |
May 3, 2013 |
9040663 |
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14719232 |
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12795469 |
Jun 7, 2010 |
8455617 |
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13886864 |
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61184932 |
Jun 8, 2009 |
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Current U.S.
Class: |
514/1.9 ;
514/10.7; 514/4.8; 514/6.7; 514/6.8; 514/6.9; 514/7.4 |
Current CPC
Class: |
A61P 9/12 20180101; A61P
1/04 20180101; A61K 38/00 20130101; A61P 9/00 20180101; A61P 3/10
20180101; A61P 19/02 20180101; C07K 7/06 20130101; A61P 5/50
20180101; A61P 11/00 20180101; A61P 43/00 20180101; A61P 7/00
20180101; C07K 7/56 20130101; A61P 3/04 20180101; A61P 25/00
20180101; A61P 25/20 20180101; A61P 3/00 20180101; A61P 9/10
20180101; A61P 5/00 20180101; A61P 3/08 20180101; A61P 3/06
20180101; A61P 1/16 20180101 |
International
Class: |
C07K 7/56 20060101
C07K007/56 |
Claims
1.-17. (canceled)
18. A method of treating a disease, disorder and/or condition
responsive to MC4 receptor activation in a mammal comprising
administering to mammal in need thereof a therapeutically effective
amount of a cyclic peptide of Formula (I) ##STR00188## or a
pharmaceutically acceptable salt thereof, wherein: R.sub.1 is
--NH--C(.dbd.O)-- or --C(.dbd.O)--NH--; R.sub.2 is --H or
--CH.sub.2--, wherein if R.sub.2 is --CH.sub.2--, then R.sub.2
forms with R.sub.3 a pyrrolidine ring, wherein the pyrrolidine ring
optionally substituted with --OH; R.sub.3 is --(CH.sub.2).sub.2--
if R.sub.2 is --CH.sub.2--, and otherwise R.sub.3 is selected from
the group consisting of: --(CH.sub.2).sub.1-4NH.sub.2, --CH.sub.3,
--CH(CH.sub.3).sub.2, --C(CH.sub.3).sub.3, --CH.sub.2OH,
--CH(OH)CH.sub.3, --(CH.sub.2).sub.1-2CONH.sub.2,
--CH.sub.2(imidazole), --(CH.sub.2).sub.1-2CO.sub.2H,
--CH.sub.2OBz, --CH(CH.sub.3)OBz, --CH.sub.2Ph(CONH.sub.2),
--(CH.sub.2).sub.2NHCOCH.sub.3,
--(CH.sub.2).sub.3NHC(.dbd.NH)NH.sub.2 and
--(CH.sub.2).sub.2SO.sub.2CH.sub.3; R.sub.4a, R.sub.4b and R.sub.4c
are each independently selected from the group consisting of --H,
--Cl, --F, --CN, --OH, --CF.sub.3, --CH.sub.3, --OCH.sub.3,
--NO.sub.2, --Ph and --NH.sub.2, with the proviso that at least one
of R.sub.4a, R.sub.4b and R.sub.4c is not hydrogen and that
R.sub.4a, R.sub.4b and R.sub.4c cannot all be --Cl, --CH.sub.3 or
--OCH.sub.3; R.sub.5 is --OH or --N(R.sub.6a)(R.sub.6b); R.sub.6a
and R.sub.6b are each H; x is 1 to 4; and y is 1 to 4.
19. The method according to claim 18, wherein the disease, disorder
and/or condition is diabetes, obesity and conditions associated
with obesity.
20. A method of reducing food intake, body weight and/or body
weight gain in a mammal comprising administering to a mammal a
therapeutically effective amount of a cyclic peptide of Formula (I)
##STR00189## or a pharmaceutically acceptable salt thereof,
wherein: R.sub.1 is --NH--C(.dbd.O)-- or --C(.dbd.O)--NH--; R.sub.2
is --H or --CH.sub.2--, wherein if R.sub.2 is --CH.sub.2--, then
R.sub.2 forms with R.sub.3 a pyrrolidine ring, wherein the
pyrrolidine ring optionally substituted with --OH; R.sub.3 is
--(CH.sub.2).sub.2-- if R.sub.2 is --CH.sub.2--, and otherwise
R.sub.3 is selected from the group consisting of:
--(CH.sub.2).sub.1-4NH.sub.2, --CH.sub.3, --CH(CH.sub.3).sub.2,
--C(CH.sub.3).sub.3, --CH.sub.2OH, --CH(OH)CH.sub.3,
--(CH.sub.2).sub.1-2CONH.sub.2, --CH.sub.2(imidazole),
--(CH.sub.2).sub.1-2CO.sub.2H, --CH.sub.2OBz, --CH(CH.sub.3)OBz,
--CH.sub.2Ph(CONH.sub.2), --(CH.sub.2).sub.2NHCOCH.sub.3,
--(CH.sub.2).sub.3NHC(.dbd.NH)NH.sub.2 and
--(CH.sub.2).sub.2SO.sub.2CH.sub.3; R.sub.4a, R.sub.4b and R.sub.4c
are each independently selected from the group consisting of --H,
--Cl, --F, --CN, --OH, --CF.sub.3, --CH.sub.3, --OCH.sub.3,
--NO.sub.2, --Ph and --NH.sub.2, with the proviso that at least one
of R.sub.4a, R.sub.4b and R.sub.4c is not hydrogen and that
R.sub.4a, R.sub.4b and R.sub.4c cannot all be --Cl, --CH.sub.3 or
--OCH.sub.3; R.sub.5 is --OH or --N(R.sub.6a)(R.sub.6b); R.sub.6a
and R.sub.6b are each H; x is 1 to 4; and y is 1 to 4.
21. The method according to claim 18 or claim 20, wherein the
cyclic peptide is of Formula (II) ##STR00190## or a
pharmaceutically acceptable salt thereof.
22. The method according to claim 18 or claim 20, wherein for the
cyclic peptide, R.sub.1 is C(.dbd.O)--NH; x is 2; and y is 3.
23. The method according to claim 18 or claim 20, wherein for the
cyclic peptide, R.sub.1 is NH--C(.dbd.O)--; x is 3; and y is 2.
24. The method according to claim 18 or claim 20, wherein for the
cyclic peptide, R.sub.2 is H or --CH.sub.2--, wherein if R.sub.2 is
--CH.sub.2--, then R.sub.2 forms with R.sub.3 a pyrrolidine ring,
wherein the pyrrolidine ring is optionally substituted with --OH;
and R.sub.3 is --(CH.sub.2).sub.2-- if R.sub.2 is --CH.sub.2--, and
otherwise R.sub.3 is selected from the group consisting of:
--(CH.sub.2).sub.1-4NH.sub.2, --CH.sub.3, --CH.sub.2OH,
--CH(OH)CH.sub.3, --(CH.sub.2).sub.1-2CONH.sub.2,
--(CH.sub.2).sub.2NHCOCH.sub.3,
--(CH.sub.2).sub.3NHC(.dbd.NH)NH.sub.2 and
--(CH.sub.2).sub.2SO.sub.2CH.sub.3.
25. The method according to claim 18 or claim 20, wherein for the
cyclic peptide, R.sub.2 is H; and R.sub.3 is selected from the
group consisting of: ##STR00191##
26. The method according to claim 18 or claim 20, wherein for the
cyclic peptide, R.sub.2 and R.sub.3 together form an unsubstituted
pyrrolidine ring.
27. The method according to claim 18 or claim 20, wherein for the
cyclic peptide, R.sub.4a is in the 4-position and is --C.ident.N;
and R.sub.4b and R.sub.4c are each --H.
28. The method according to claim 18 or claim 20, wherein for the
cyclic peptide, R.sub.4a is in the 4-position and is --F; and
R.sub.4b and R.sub.4c are each --H.
29. The method according to claim 18 or claim 20, wherein the
cyclic peptide is selected from the group consisting of:
TABLE-US-00014 (SEQ ID NO: 48)
Ac-Arg-cyclo(Glu-Asn-D-Phe(4-CN)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID NO:
51) Ac-Arg-cyclo(Glu-Gln-D-Phe(4-CN)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID
NO: 74) Ac-Arg-cyclo(Glu-Asn-D-Phe(4-F)-Arg-Trp-Orn)-NH.sub.2; (SEQ
ID NO: 90) Ac-Arg-cyclo(Glu-Asn-D-Phe(4-CN)-Arg-Trp-Orn)-OH; (SEQ
ID NO: 91) Ac-Arg-cyclo(Glu-Gln-D-Phe(4-CN)-Arg-Trp-Orn)-OH; (SEQ
ID NO: 97) Ac-Arg-cyclo(Glu-Gln-D-Phe(4-F)-Arg-Trp-Orn)-NH.sub.2;
(SEQ ID NO: 44)
Ac-Arg-cyclo(Glu-Pro-D-Phe(4-CN)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID NO:
72) Ac-Arg-cyclo(Glu-Pro-D-Phe(4-F)-Arg-Trp-Orn)-NH.sub.2; and (SEQ
ID NO: 107) Ac-Arg-cyclo(Glu-Pro-D-Phe(4-F)-Arg-Trp-Orn)-OH;
or a pharmaceutically acceptable salt of any of the foregoing.
30. The method according to claim 19, wherein the disease, disorder
and/or condition is selected from the group consisting of: insulin
resistance, impaired glucose tolerance, type 2 diabetes, metabolic
syndrome, dyslipidemia, hyperlipidemia, hypertension, heart
disorders, cardiovascular disorders, non-alcoholic fatty liver
disease, joint disorders, secondary osteoarthritis,
gastroesophageal reflux, sleep apnea, atherosclerosis, stroke,
macro- and micro-vascular diseases, steatosis and gall stones.
Description
TECHNICAL FIELD
[0001] The present invention relates to melanocortin
receptor-specific cyclic peptides which may be used in the
treatment of melanocortin receptor-mediated diseases, indications,
conditions and syndromes, in particular energy homeostasis and
metabolism related (e.g. diabetes), food intake related and/or
energy balance and body weight related diseases, disorders and/or
conditions, including obesity, overweight and diseases, disorders
and/or conditions associated with obesity and/or overweight, such
as type 2 diabetes and metabolic syndrome.
BACKGROUND
[0002] The following discussion refers to a number of publications
by author(s) and year of publication, and that due to recent
publication dates certain publications are not to be considered as
prior art vis-a-vis the present invention. Discussion of such
publications herein is given for more complete background and is
not to be construed as an admission that such publications are
prior art for patentability determination purposes.
[0003] A family of melanocortin receptor types and subtypes have
been identified, including melanocortin-1 receptors (MC1-R)
expressed on normal human melanocytes and melanoma cells,
melanocortin-2 receptors (MC2-R) for ACTH (adrenocorticotropin)
expressed in cells of the adrenal gland, melanocortin-3 and
melanocortin-4 receptors (MC3-R and MC4-R) expressed primarily in
cells in the hypothalamus, mid-brain and brainstem, and
melanocortin-5 receptors (MC5-R), expressed in a wide distribution
of peripheral tissues. MC1-R has been suggested to be associated
with hair and skin pigmentation and inflammation, MC2-R is believed
to mediate steroidogenesis, MC3-R has been suggested to be
associated with energy homeostasis, food intake, and inflammation,
MC4-R is believed to control feeding behavior, energy homeostasis,
and sexual function (e.g. erectile function), and MC5-R has been
suggested to be involved in the exocrine gland system.
[0004] Significant work has been done in determining the structure
of melanocortin receptors, including both the nucleic acid
sequences encoding for the receptors and the amino acid sequences
constituting the receptors. MC4-R is a G protein-coupled,
7-transmembrane receptor that is believed to be expressed primarily
in the brain.
[0005] MC4-R inactivation has been shown to result in obesity
(Hadley, 1999, Ann N Y Acad Sci., 885:1-21). Agouti-related protein
(AgRP) is an endogeneous compound that has been suggested to be a
MC antagonist or an inverse agonist on MC4-R. The
.alpha.-melanocyte stimulating hormone (.alpha.-MSH) is believed to
be the principle endogenous MC4-R agonist.
[0006] Also peripherally located MC4-R receptors have been
suggested to be involved in the control of energy homeostasis, and
the role of MC4-R signalling in the vagus nerve and its relevance
for treatment of obesity and diabetes is discussed by Gautron et
al, The Journal of Comparative Neurology, 518:6-24 (2010).
[0007] Peptides specific for MC4-R, and secondarily peptides
specific for MC3-R, are believed to be useful in regulation of
mammalian energy homeostasis, including use as agents for
attenuating food intake and body weight gain. MC4-R agonist
peptides are believed to be useful for treating sexual dysfunction,
including male erectile dysfunction, and for decreasing food intake
and body weight gain, such as for treatment of obesity. Such
peptides may also be employed for decreasing voluntary ethanol
consumption, treatment of drug addictions, and the like. MC4-R
agonist peptides, as well as MC3-R agonist peptides, may further be
employed for treatment of circulatory shock, ischemia, hemorrhagic
shock, inflammatory diseases and related diseases, indications,
conditions and syndromes. MC4-R antagonist peptides, by contrast,
are believed to be useful for weight gain aid, such as for use in
treatment of cachexia, sarcopenia, wasting syndrome or disease, and
anorexia. Such peptides may also be employed for treatment of
depression and related disorders. (Wikberg et al, Nature Reviews,
Drug Discovery, 7, 307, (2008); Adan et al, British J. Pharm., 149,
815-827 (2006); Nogueiras et al, J. Clin., Invest., 117(11):
3475-3488 (2007); Maaser et al, Ann. N.Y. Acad. Sci., 1072, 123-134
(2006); Giuliani et al, British J. Pharm., 150, 595-603 (2007);
Balbani, Expert Opin. Ther. Patents, 17(3), 287-297 (2007); and
Navarro et al, Alcohol. Clin. Exp. Res., 29(6), 949-957 (2005)).
Melanocortin receptor-specific peptides include cyclic .alpha.-MSH
analog peptides such as
Ac-Nle-cyclo(-Asp-His-D-Phe-Arg-Trp-Lys)-NH.sub.2 (SEQ ID NO:1)
(See U.S. Pat. Nos. 5,674,839 and 5,576,290) and
Ac-Nle-cyclo(-Asp-His-D-Phe-Arg-Trp-Lys)-OH (SEQ ID NO:2) (See U.S.
Pat. Nos. 6,579,968 and 6,794,489). These and other melanocortin
receptor-specific peptides generally contain the central
tetrapeptide sequence of native .alpha.-MSH,
His.sup.6-Phe.sup.7-Arg.sup.8-Trp.sup.9 (SEQ ID NO:3), or a mimetic
or variation thereof, including the substitution of D-Phe for
Phe.sup.7. Other peptides or peptide-like compounds asserted to be
specific for one or more melanocortin receptors are disclosed in
U.S. Pat. Nos. 5,731,408, 6,054,556, 6,350,430, 6,476,187,
6,600,015, 6,613,874, 6,693,165, 6,699,873, 6,887,846, 6,951,916,
7,008,925, and 7,176,279; in U.S. published patent application
Publication Nos. 2001/0056179, 2002/0143141, 2003/0064921,
2003/0105024, 2003/0212002, 2004/0023859, 2005/0130901,
2005/0187164, 2005/0239711, 2006/0105951, 2006/0111281,
2006/0293223, 2007/0027091, 2007/0105759, 2007/0123453,
2007/0244054, and 2008/0039387; and in international patent
applications nos. WO 98/27113, WO 99/21571, WO 00/05263, WO
99/54358, WO 00/35952, WO 00/58361, WO 01/30808, WO 01/52880, WO
01/74844, WO 01/85930, WO 01/90140, WO 02/18437, WO 02/26774, WO
03/006604, WO 2004/099246, WO 2004/046166, WO 2005/000338, WO
2005/000339, WO 2005/000877, WO 2005/030797, WO 2005/060985,
WO2006/048449, WO 2006/048450, WO 2006/048451, WO 2006/048452, WO
2006/097526, WO 2007/008684, WO 2007/008704, and WO 2007/009894.
Notwithstanding the intense scientific and pharmaceutical interest
in melanocortin receptor-specific peptides, evidenced by numerous
articles in the scientific literature and numerous patent
applications and issued patents (Nozawa et al, Expert Opin. Ther.
Patents 18(4):403-427 (2008); Bednarek et al, Expert Opin. Ther.
Patents 14(3):327-336 (2004); Todorovic et al, Peptides, 26,
2026-2036 (2005); and Ujjainwalla et al, Current Topics in Med.
Chem., 7, 1068-1084 (2007)), no melanocortin receptor-specific
peptide has been approved as a drug for any therapeutic indication.
Indeed, there are no reports of any melanocortin receptor-specific
peptide for any therapeutic indication having advanced past Phase
II clinical trials. There remains a significant and substantial
need for melanocortin receptor-specific peptides for use in
pharmaceutical applications. It is against this background that the
present invention was made.
SUMMARY OF THE INVENTION
[0008] It is therefore an object of the present invention to
provide novel compounds that are useful for the treatment of
diseases, disorders and/or conditions responsive to modulation,
including activation, of MC4-R and/or MC3-R, in particular
treatment of energy homeostasis and metabolism related (e.g.
diabetes), food intake related and/or energy balance and body
weight related diseases, disorders and/or conditions, including
obesity, overweight and diseases, disorders and/or conditions
associated with obesity and/or overweight, such as type 2 diabetes
and metabolic syndrome.
[0009] In one aspect, the present invention relates to a cyclic
peptide of the structural Formula (I):
##STR00002##
including all enantiomers, stereoisomers or diastereoisomers
thereof, or a pharmaceutically acceptable salt of any of the
foregoing,
[0010] wherein: [0011] R.sub.1 is --NH--C(.dbd.O)-- or
--C(.dbd.O)--NH--; [0012] R.sub.2 is --H or --CH.sub.2--, and if
R.sub.2 is --CH.sub.2-- forms with R.sub.3 a pyrrolidine ring, the
pyrrolidine ring optionally substituted with --OH; [0013] R.sub.3
is --(CH.sub.2).sub.2-- if R.sub.2 is --CH.sub.2--, and otherwise
R.sub.3 is selected from
##STR00003##
[0014] R.sub.4a, R.sub.4b and R.sub.4c are each independently
selected from hydrogen, halo, (C.sub.1-C.sub.10)alkyl-halo,
(C.sub.1-C.sub.10)alkyl-dihalo, (C.sub.1-C.sub.10)alkyl-trihalo,
(C.sub.1-C.sub.10)alkyl, (C.sub.1-C.sub.10)alkoxy,
(C.sub.1-C.sub.10)alkylthio, aryl, aryloxy, nitro, nitrile,
sulfonamide, amino, monosubstituted amino, disubstituted amino,
hydroxy, carboxy, or alkoxy-carbonyl, on the proviso that at least
one of R.sub.4a, R.sub.4b and R.sub.4c is not hydrogen; [0015]
R.sub.5 is --OH or --N(R.sub.6a)(R.sub.6b); [0016] R.sub.6a and
R.sub.6b are each independently H or a C.sub.1 to C.sub.4 linear,
branched or cyclic alkyl chain; [0017] R.sub.7 is --H or
--C(.dbd.O)--NH.sub.2; [0018] w is in each instance independently 0
to 5; [0019] x is 1 to 5; [0020] y is 1 to 5; and [0021] z is in
each instance independently 1 to 5.
[0022] In another aspect, the present invention relates to a cyclic
peptide of Formula (I) which is of formula (II):
##STR00004##
or a pharmaceutically acceptable salt thereof.
[0023] In another aspect, the present invention relates to a cyclic
peptide of Formula I, in particular of Formula (II), wherein
R.sub.1 is --C(.dbd.O)--NH--, x is 2 and y is 3.
[0024] In another aspect, the present invention relates to a cyclic
peptide of Formula (I), in particular of Formula (II), wherein
R.sub.1 is --NH--C(.dbd.O)--, x is 3 and y is 2.
[0025] In another aspect, the present invention relates to a cyclic
peptide of Formula (I), in particular of Formula (II), wherein
R.sub.2 is --H or --CH.sub.2--, and if R.sub.2 is --CH.sub.2--
forms with R.sub.3 a pyrrolidine ring, the pyrrolidine ring
optionally substituted with --OH; and
[0026] R.sub.3 is --(CH.sub.2).sub.2-- if R.sub.2 is --CH.sub.2--,
and otherwise R.sub.3 is
##STR00005##
[0027] wherein w is in each instance independently selected from 0
to 5, and
z is in each instance independently selected from 1 to 5.
[0028] In another aspect, the present invention relates to a cyclic
peptide of Formula I, in particular of Formula (II) or (III),
wherein R.sub.2 is H, R.sub.3 is selected from
##STR00006##
[0029] In another aspect, the present invention relates to a cyclic
peptide of Formula I, in particular of Formula (II), wherein
R.sub.2 is --CH.sub.2-- and R.sub.3 is --(CH.sub.2).sub.2--,
R.sub.2 and R.sub.3 together forming an unsubstituted pyrrolidine
ring.
[0030] In another aspect, the present invention relates to a cyclic
peptide of Formula I, in particular Formula (II), wherein at least
one of R.sub.4a, R.sub.4b and R.sub.4c is selected from
##STR00007##
[0031] In another aspect, the present invention relates to a cyclic
peptide of Formula I, in particular Formula (II) or (III), wherein
R.sub.4a is in the 4 position and is --C.ident.N and R.sub.4b and
R.sub.4c are each H.
[0032] In another aspect, the present invention relates to a cyclic
peptide of Formula (I), in particular Formula (II) or (III),
wherein R.sub.4a is in the 4 position and is --F and R.sub.4b and
R.sub.4c are each H.
[0033] The peptides according to the invention are ligands of one
or more of the melanocortin receptors, in particular ligands of the
MC4-R, more particularly agonists (including full and partial
agonists) of the MC4-R. The term "ligands" as used herein include
peptides binding to the active site as well as peptides binding to
one or more allosteric sites of any one of said receptors.
[0034] Thus, the peptides of the invention can be used as a
medicament, in particular for the treatment of disorders, diseases,
or conditions responsive to modulation of the MC3-R and/or MC4-R,
and in particular disorders, diseases, or conditions responsive to
the activation of the MC4-R. More particularly, the peptides of the
invention are believed to attenuate food intake, body weight and/or
body weight gain and are therefore believed to be useful for
treatments of energy homeostasis and metabolism related (e.g.
diabetes), food intake related and/or energy balance and body
weight related diseases, disorders and/or conditions, including
obesity and overweight, and diseases, disorders and/or conditions
associated with obesity and/or overweight, such as type 2 diabetes
and metabolic syndrome, in a patient in need thereof. The patient
may be a human or non-human animal, in particular a human.
[0035] Peptides of the invention may have advantageous properties
compared to peptides of the prior art, in particular enhanced
potency and/or enhanced selectivity. These advantages may provide
for corresponding useful properties in practice. For example, when
used as pharmaceutical agents, peptides of the present invention
may be used at a lower daily clinical dose, may have longer
duration of action, and/or an improved side effect profile.
[0036] In another aspect of the invention, there is provided a
method of treating disorders, diseases, or conditions responsive to
modulation of the MC4-R and/or MC3-R, such as disorders, diseases,
or conditions responsive to activation of the MC4-R, in particular
energy homeostasis and metabolism related (e.g. diabetes), food
intake related and/or energy balance and body weight related
diseases, disorders and/or conditions, including obesity and
overweight, and diseases, disorders and/or conditions associated
with obesity and/or overweight, such as type 2 diabetes and
metabolic syndrome, by administering a therapeutically effective
amount of a peptide of the invention to a patient in need
thereof.
[0037] According to a further aspect of the invention, there is
provided a method of reducing food intake, body weight and/or body
weight gain by administering a pharmacologically effective amount
of a peptide of the invention to an individual, such as a human, in
need thereof.
[0038] According to a further aspect of the invention, there is
provided a method of preventing body weight regain after weight
loss by administering a pharmacologically effective amount of a
peptide of the invention to an individual, such as a human, in need
thereof.
[0039] In a further aspect, the invention provides the use of a
peptide of Formula I in the preparation of a medicament for
treatment of a disease, disorder and/or condition responsive to
activation of the MC4-R, in particular energy homeostasis and
metabolism related (e.g. diabetes), food intake related and/or
energy balance and body weight related diseases, disorders and/or
conditions, including obesity and overweight, and diseases,
disorders and/or conditions associated with obesity and/or
overweight, such as type 2 diabetes and metabolic syndrome.
[0040] In another aspect, the present invention provides a
melanocortin receptor-specific peptide-based pharmaceutical
composition for use in treatment of melanocortin receptor-mediated
diseases, indications, conditions and syndromes, in particular
diseases, disorders, conditions and/or syndromes responsive to
modulation of MC4-R, such as activation of MC4-R, comprising a
peptide of Formula I and a pharmaceutically acceptable carrier.
[0041] In another aspect, the present invention provides a
peptide-based melanocortin receptor-specific pharmaceutical
composition comprising a peptide of Formula I and a
pharmaceutically acceptable carrier, wherein the peptide is a
selective MC4-R ligand, for use in treatment of energy homeostasis
and metabolism related (e.g. diabetes), food intake related and/or
energy balance and body weight related diseases, disorders and/or
conditions, including obesity, overweight and diseases, disorders
and/or conditions associated with obesity and/or overweight, such
as type 2 diabetes and metabolic syndrome.
[0042] In another aspect, the present invention provides peptides
which are specific for MC4-R and which are partial or full agonists
at MC4-R. In particular, the present invention provides peptides
which are specific for MC4-R and which are partial agonists at
MC4-R.
[0043] In another aspect, the present invention provides a specific
MC4-R cyclic peptide that is effective over a significant dose
range.
[0044] Other aspects and novel features, and the further scope of
applicability of the present invention will be set forth in part in
the detailed description to follow, and in part will become
apparent to those skilled in the art upon examination of the
following, or may be learned by practice of the invention. The
aspects of the invention may be realized and attained by means of
the instrumentalities and combinations particularly pointed out in
the appended claims.
DETAILED DESCRIPTION OF THE INVENTION
1.0 Definitions
[0045] Before proceeding with the description of the invention,
certain terms are defined as set forth herein.
[0046] In the sequences given for the peptides according to the
present invention, the amino acid residues have their conventional
meaning as given in Chapter 2400 of the Manual of Patent Examining
Procedure, 8.sup.th Ed. Thus, "Ala" is alanine, "Asn" is
asparagine, "Asp" is aspartic acid, "Arg" is arginine, "Cys" is
cysteine, "Gly" is glycine, "Gln" is glutamine, "Glu" is glutamic
acid, "His" is histidine, "Ile" is isoleucine, "Leu" is leucine,
"Lys" is lysine, "Met" is methionine, "Phe" is phenylalanine, "Pro"
is proline, "Ser" is serine, "Thr" is Threonine, "Trp" is
tryptophan, "Tyr" is tyrosine, and "Val" is valine, and so on. It
is to be understood that "D" isomers are designated by a "D-"
before the three letter code or amino acid name, such that for
example D-Phe is D-phenylalanine Amino acid residues not
encompassed by the foregoing have the following definitions:
TABLE-US-00001 Side Chain or Abbreviation Common Name Amino Acid
Structure Cit citrulline ##STR00008## Dab diaminobutyric acid
##STR00009## Dab(Acetyl) 2-amino, 4-acetylamino- butyric acid
##STR00010## Dap diaminoproprionic acid ##STR00011## Hyp
hydroxyproline ##STR00012## Met(O) methionine sulfoxide
##STR00013## Met(O.sub.2) methionine sulfone ##STR00014## Nle
norleucine ##STR00015## Nva norvaline ##STR00016## Orn ornithine
##STR00017## Phe(2-CF.sub.3) 2-trifluoromethyl phenylalanine
##STR00018## Phe(2- C(.dbd.O)--NH.sub.2) 2-carbamoyl- phenylalanine
##STR00019## Phe(2-Me) 2-methyl phenylalanine ##STR00020##
Phe(2-CN) 2-cyano phenylalanine ##STR00021## Phe(2-Cl) 2-chloro
phenylalanine ##STR00022## Phe(2,4-diCl) 2,4-dichloro phenylalanine
##STR00023## Phe(2,4-diMe) 2,4-dimethyl phenylalanine ##STR00024##
Phe(2-F) 2-flouro phenylalanine ##STR00025## Phe(2-NO.sub.2)
2-nitro phenylalanine ##STR00026## Phe(3-CF.sub.3)
3-triflouromethyl phenylalanine ##STR00027## Phe(3-
C(.dbd.O)--NH.sub.2) 3-carbamoyl- phenylalanine ##STR00028##
Phe(3-CN) 3-cyano phenylalanine ##STR00029## Phe(3-Cl) 3-chloro
phenylalanine ##STR00030## Phe(3,4-diCl) 3,4-dichloro phenylalanine
##STR00031## Phe(3-F) 3-fluoro phenylalanine ##STR00032##
Phe(3,4,5-triF) 3,4,5-trifluoro phenylalanine ##STR00033##
Phe(3,4-diF) 3,4-difluoro phenylalanine ##STR00034## Phe(3,5-diF)
3,5-difluoro phenylalanine ##STR00035## Phe(3-Me) 3-methyl
phenylalanine ##STR00036## Phe(3-NO.sub.2) 3-nitro phenylalanine
##STR00037## Phe(3,4-diOMe) 3,4-dimethoxy phenylalanine
##STR00038## Phe(4- C(.dbd.O)--NH.sub.2) 4-carbamoyl- phenylalanine
##STR00039## Phe(4-Me) 4-methyl phenylalanine ##STR00040##
Phe(4-CF.sub.3) 4-trifluoromethyl phenylalanine ##STR00041##
Phe(4-CN) 4-cyano phenylalanine ##STR00042## Phe(4-Cl) 4-chloro
phenylalanine ##STR00043## Phe(4-F) 4-fluoro phenylalanine
##STR00044## Phe(4-NH.sub.2) 4-amino phenylalanine ##STR00045##
Phe(4-NO.sub.2) 4-nitro phenylalanine ##STR00046## Phe(4-Ph)
4-phenyl phenylalanine ##STR00047## Phe(4-OMe) 4-methoxy
phenylalanine ##STR00048## Phe(4-tBu) 4-tert butyl phenylalanine
##STR00049## Ser(Bzl) O-benzyl-serine ##STR00050## Thr(OBzl)
O-benzyl- threonine ##STR00051##
[0047] The term "acyl" includes a group R(C.dbd.O)--, where R is an
organic group, such as an alkyl, aryl, heteroaryl, carbocyclyl or
heterocyclyl. Thus, when reference is made herein to a substituted
acyl group, it means that said organic group (R) is substituted. An
example is the acetyl group CH.sub.3--C(.dbd.O)--, referred to
herein as "Ac". A peptide or aliphatic moiety is "acylated" when an
alkyl or substituted alkyl group as defined above is bonded through
one or more carbonyl {--(C.dbd.O)--} groups. A peptide is most
usually acylated at the N-terminus
[0048] The term "alkane" includes linear or branched saturated
hydrocarbons. Examples of linear alkane groups include methane,
ethane, propane, and the like. Examples of branched or substituted
alkane groups include methylbutane or dimethylbutane,
methylpentane, dimethylpentane or trimethylpentane, and the like.
In general, any alkyl group may be a substitutent of an alkane.
[0049] The term "alkene" includes unsaturated hydrocarbons that
contain one or more double carbon-carbon bonds. Examples of such
alkene groups include ethylene, propene, and the like.
[0050] The term "alkenyl" includes a linear monovalent hydrocarbon
radical of two to six carbon atoms or a branched monovalent
hydrocarbon radical of three to six carbon atoms containing at
least one double bond; examples thereof include ethenyl,
2-propenyl, and the like.
[0051] The term "alkyl" includes a straight or branched chain
saturated aliphatic hydrocarbon group. C.sub.1-10 alkyl means an
alkyl having from 1 to 10 carbon atoms. Non-limiting examples of
such alkyl radicals include methyl, ethyl, propyl, isopropyl,
butyl, sec-butyl, tertiary butyl, pentyl, isopentyl, hexyl,
isohexyl, and the like.
[0052] The term "alkyne" includes a linear monovalent hydrocarbon
radical of two to six carbon atoms or a branched monovalent
hydrocarbon radical of three to six carbon atoms containing at
least one triple bond; examples thereof include ethyne, propyne,
butyne, and the like.
[0053] The term "aryl" includes a monocyclic or bicyclic aromatic
hydrocarbon radical of 6 to 12 ring atoms, and optionally
substituted independently with one or more substituents selected
from alkyl, haloalkyl, cycloalkyl, alkoxy, alkythio, halo, nitro,
acyl, cyano, amino, monosubstituted amino, disubstituted amino,
hydroxy, carboxy, or alkoxy-carbonyl. The term "aryl" also includes
bicyclic aromatic ring systems wherein one ring is aromatic and one
ring is non-aromatic (including saturated or partially saturated
rings). In bicyclic aromatic ring systems, two or more ring carbons
are common to two adjoining rings (the rings are "fused rings").
Examples of an aryl group include phenyl, biphenyl, indanyl,
naphthyl, 1-naphthyl, and 2-naphthyl, derivatives thereof, and the
like.
[0054] The term "aralkyl" includes a radical --R.sup.aR.sup.b where
R.sup.a is an alkylene (a bivalent alkyl) group and R.sup.b is an
aryl group as defined above. Examples of aralkyl groups include
benzyl, phenylethyl, 3-(3-chlorophenyl)-2-methylpentyl, and the
like.
[0055] The term "aliphatic" includes compounds with hydrocarbon
chains, such as for example alkanes, alkenes, alkynes, and
derivatives thereof.
[0056] As used herein, the term "amide" includes compounds that
have a trivalent nitrogen attached to a carbonyl group, i.e.
--C(.dbd.O)--NH.sub.2 (i.e. primary amide), --C(.dbd.O)--NHR.sub.c
and --C(.dbd.O)--NR.sub.cR.sub.d, wherein each of R.sub.c and
R.sub.d independently represents an organic group. When reference
is made herein to a substituted amide group, it means that at least
one of said organic groups (R.sub.c and R.sub.d) is substituted.
Examples of amides include methylamide, ethylamide, propylamide,
and the like.
[0057] The term "amine" includes --NH.sub.2 (i.e. an amino group),
--NHR.sub.a and --NR.sub.aR.sub.b, wherein each of R.sub.a and
R.sub.b independently represents an organic group. When reference
is made herein to a substituted amine group, it means that at least
one of said organic groups (R.sub.a and R.sub.b) is
substituted.
[0058] The term "nitrile" includes the functional group
--C.ident.N.
[0059] The term "halogen" (or "halo") is intended to include the
halogen atoms fluorine, chlorine, bromine and iodine.
[0060] The term "alkyl-halo" includes an alkyl substituted with one
halogen atom, such as --CH.sub.2F. The term "alkyl-dihalo" includes
an alkyl substituted with two halogen atoms, such as --CHF.sub.2.
The term "alkyl-trihalo" includes an alkyl substituted with three
halogen atoms, such as --CF.sub.3.
[0061] The term "alkylthio" includes --S-alkyl wherein alkyl is as
defined above. Non-limiting examples of C.sub.1-C.sub.10 alkylthio
include methylthio, ethylthio, n-propylthio, iso-propylthio, and
n-butylthio.
[0062] The term "nitro" is intended to include --NO.sub.2.
[0063] The term "hydroxy" is intended to include --OH.
[0064] The term "alkoxy" includes --O-alkyl wherein alkyl is as
defined above. C.sub.1-C.sub.10 alkoxy includes an alkyl having
from 1 to 10 carbon atoms. Non-limiting examples of
C.sub.1-C.sub.10 alkoxy include methoxy, ethoxy, n-propyloxy,
iso-propyloxy, and 2-methyl-1-propyloxy.
[0065] The term "aryloxy" includes --O-aryl wherein aryl is as
defined above.
[0066] The term "alkoxycarbonyl" includes --C(.dbd.O)--O--R,
wherein R is an alkyl as defined above. Non-limiting examples of
C.sub.1-C.sub.10 alkoxycarbonyl include methoxycarbonyl,
ethoxycarbonyl, isopropoxy-carbonyl and isopentoxycarbonyl.
[0067] The term "carboxy" includes --C(.dbd.O)OH.
[0068] The term "oxo" includes .dbd.O.
[0069] The term "sulfonamide" includes a sulfonyl group connected
to an amine group, i.e. --S(.dbd.O).sub.2NH.sub.2,
--S(.dbd.O).sub.2NHR.sub.a, and --S(.dbd.O).sub.2NR.sub.aR.sub.b,
wherein each of R.sub.a and R.sub.b independently represents an
organic group. When reference is made herein to a substituted
sulfonamide group, it means that at least one of said organic
groups (R.sub.a and R.sub.b) is substituted.
[0070] The term "composition", as in pharmaceutical composition, is
intended to encompass a product comprising the active
ingredient(s), and the inert ingredient(s) that make up the
carrier, as well as any product which results, directly or
indirectly, from combination, complexation or aggregation of any
two or more of the ingredients, or from dissociation of one or more
of the ingredients, or from other types of reactions or
interactions of one or more of the ingredients. Accordingly, the
pharmaceutical compositions utilized in the present invention
encompass any composition made by admixing an active ingredient and
one or more pharmaceutically acceptable carriers.
[0071] By a melanocortin receptor "agonist" is meant an endogenous
substance, drug substance or compound, including a compound such as
the peptides of the present invention, which can interact with a
melanocortin receptor and initiate a pharmacological response,
including but not limited to adenyl cyclase activation,
characteristic of the melanocortin receptor.
[0072] By ".alpha.-MSH" is meant the peptide
Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH.sub.2
(SEQ ID NO:4) and analogs and homologs thereof, including without
limitation NDP-.alpha.-MSH.
[0073] By "NDP-.alpha.-MSH" is meant the peptide
Ac-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH.sub.2
(SEQ ID NO:5) and analogs and homologs thereof.
[0074] By "EC.sub.50" is meant the molar concentration of an
agonist, including a partial agonist, which produced 50% of the
maximum possible response for that agonist. By way of example, a
test compound which, at a concentration of 72 nM, produces 50% of
the maximum possible response for that compound as determined in a
cAMP assay in an MC4-R cell expression system has an EC.sub.50 of
72 nM. Unless otherwise specified, the molar concentration
associated with an EC.sub.50 determination is in nanomoles per
liter (nM).
[0075] By "Ki (nM)" is meant the equilibrium inhibitor dissociation
constant representing the molar concentration of a competing
compound that binds to half the binding sites of a receptor at
equilibrium in the absence of radioligand or other competitors. In
general, the numeric value of the Ki is inversely correlated to the
affinity of the compound for the receptor, such that if the Ki is
low, the affinity is high. Ki may be determined using the equation
of Cheng and Prusoff (Cheng Y., Prusoff W. H., Biochem. Pharmacol.
22: 3099-3108, 1973):
[0076] Ki may be expressed in terms of specific receptors (e.g.,
MC1-R, MC3-R, MC4-R or MC5-R) and specific ligands (e.g.
.alpha.-MSH or NDP-.alpha.-MSH).
[0077] By "inhibition" is meant the percent attenuation, or
decrease in receptor binding, in a competitive inhibition assay
compared to a known standard. Thus, by "inhibition at 1 .mu.M
(NDP-.alpha.-MSH)" is meant the percent decrease in binding of
NDP-.alpha.-MSH by addition of a determined amount of the compound
to be tested, such as 1 .mu.M of a test compound, such as under the
assay conditions hereafter described. By way of example, a test
compound that does not inhibit binding of NDP-.alpha.-MSH has a 0%
inhibition, and a test compound that completely inhibits binding of
NDP-.alpha.-MSH has a 100% inhibition. Typically, as described
hereafter, a radio assay is used for competitive inhibition
testing, such as with I.sup.125-labeled NDP-.alpha.-MSH, or a
lanthanide chelate fluorescent assay, such as with
Eu-NDP-.alpha.-MSH. However, other methods of testing competitive
inhibition are known, including use of label or tag systems other
than radioisotopes, and in general any method known in the art for
testing competitive inhibition may be employed in this invention.
It may thus be seen that "inhibition" is one measure to determine
whether a test compound attenuates binding of .alpha.-MSH to
melanocortin receptors.
[0078] By "binding affinity" is meant the ability of a compound or
drug to bind to its biological target, expressed herein as Ki
(nM).
[0079] By "intrinsic activity" is meant the maximal stimulation
(functional activity) achievable by a compound in a specified
melanocortin receptor expressing cell system, such as the maximal
stimulation of adenylyl cyclase. The maximal stimulation achieved
by .alpha.-MSH or NDP-.alpha.-MSH is designated as an intrinsic
activity of 1.0 (or 100%) and a compound capable of stimulating
half the maximal activity that of .alpha.-MSH or NDP-.alpha.-MSH is
designated as having an intrinsic activity of 0.5 (or 50%). A
compound of this invention that under assay conditions described
herein has an intrinsic activity of 0.7 (70%) or higher is
classified as an agonist, a compound with intrinsic activity
between 0.1 (10%) and 0.7 (70%) is classified as a partial agonist,
and a compound with intrinsic activity below 0.1 (10%) is
classified as inactive or having no intrinsic activity. In one
aspect, the cyclic peptides of the present invention may generally
be characterized as a partial agonist at MC4-R with respect to
.alpha.-MSH or NDP-.alpha.-MSH.
[0080] In general, "functional activity" is a measure of the
signaling of a receptor, or measure of a change in
receptor-associated signaling, such as a melanocortin receptor, and
in particular MC4-R or hMC4-R, upon activation by a compound.
Melanocortin receptors initiate signal transduction through
activation of heterotrimeric G proteins. In one aspect,
melanocortin receptors signal through G.alpha..sub.S, which
catalyzes production of cAMP by adenylyl cyclase. Thus
determination of stimulation of adenylyl cyclase, such as
determination of maximal stimulation of adenylyl cyclase, is one
measure of functional activity, and is the primary measure
exemplified herein. However, it is to be understood that
alternative measures of functional activity may be employed in the
practice of this invention, and are specifically contemplated and
included within the scope of this invention. Thus, in one example
intracellular free calcium may be measured, such as reported by and
using the methods disclosed in Mountjoy K. G. et al., Melanocortin
receptor-medicated mobilization of intracellular free calcium in
HEK293 cells. Physiol Genomics 5:11-19, 2001, or Kassack M. U. et
al., Functional screening of G protein-coupled receptors by
measuring intracellular calcium with a fluorescence microplate
reader. Biomol Screening 7:233-246, 2002. It is also possible to
measure activation by measurement of the production of inositol
triphosphate or diacylglycerol from phosphatidylinositol
4,5-biphosphate, such as by use of radioassays. Yet another measure
of functional activity is receptor internalization, resulting from
activation of regulatory pathways, such as using the methods
disclosed in Nickolls S. A. et al., Functional selectivity of
melanocortin 4 receptor peptide and nonpeptide agonists: evidence
for ligand specific conformational states. J Pharm Exper
Therapeutics 313:1281-1288, 2005. Yet another measure of functional
activity is the exchange, and exchange rate, of nucleotides
associated with activation of a G protein receptor, such as the
exchange of GDP (guanosine diphosphate) for GTP (guanosine
triphosphase) on the G protein a subunit, which may be measured by
any number of means, including a radioassay using guanosine
5'-(.gamma.-[.sup.35S]thio)-triphosphate, as disclosed in Manning
D. R., Measures of efficacy using G proteins as endpoints:
differential engagement of G proteins through single receptors. Mol
Pharmacol 62:451-452, 2002. Various gene-based assays have been
developed for measuring activation of G-coupled proteins, such as
those disclosed in Chen W. et al., A colorimetric assay from
measuring activation of Gs- and Gq-coupled signaling pathways. Anal
Biochem 226:349-354, 1995; Kent T. C. et al., Development of a
generic dual-reporter gene assay for screening G-protein-coupled
receptors. Biomol Screening, 5:437-446, 2005; or Kotarsky K. et
al., Improved receptor gene assays used to identify ligands acting
on orphan seven-transmembrane receptors. Pharmacology &
Toxicology 93:249-258, 2003. The colorimetric assay of Chen et al.
has been adapted for use in measuring melanocortin receptor
activation, as disclosed in Hruby V. J. et al., Cyclic lactam
.alpha.-melanocortin analogues of
Ac-Nle.sup.4-cyclo[Asp.sup.5,D-Phe.sup.7,Lys.sup.10]
.alpha.-melanocyte-stimulating hormone-(4-10)-NH.sub.2 with bulky
aromatic amino acids at position 7 shows high antagonist potency
and selectivity at specific melanocortin receptors. J Med Chem
38:3454-3461, 1995. In general, functional activity may be measured
by any method, including methods of determining activation and/or
signaling of a G-coupled receptor, and further including methods
which may be hereafter developed or reported. The terms "treat,"
"treating" and "treatment," as used herein, contemplate an action
that occurs while a patient is suffering from the specified
disease, disorder and/or condition, which reduces the severity of
the disease, disorder and/or condition. Moreover, the terms
"treat," "treating" and "treatment," as used herein are intended to
embrace therapeutic (curative), prophylactic (preventing),
controlling and palliative treatment of the indicated diseases,
disorders and/or conditions.
[0081] As used herein, the term "pharmacologically effective
amount" (including "therapeutically effective amount") means an
amount of a peptide according to the invention that is sufficient
to induce a desired therapeutic or biological effect.
[0082] As used herein, the term "therapeutically effective amount"
means the amount of a peptide of the invention that will elicit a
biological or medical response in the mammal that is being treated
by a medical doctor or other clinician.
[0083] As used herein, the term "prophylactically effective" or
"preventive" means the amount of a peptide of the invention that
will prevent or inhibit affliction or mitigate affliction of a
mammal with a medical condition that a medical doctor or other
clinician is trying to prevent, inhibit, or mitigate before a
patient begins to suffer from the specified disease or
disorder.
[0084] The term "diabetes" includes Type 1 Diabetes, which is
insulin-dependent diabetes mellitus as diagnosed according to
criteria published in the Report of the Expert Committee on the
Diagnosis and Classification of Diabetes Mellitus (Diabetes Care,
Vol. 24, Supp. 1, January 2001) whereby fasting plasma glucose
level is greater than or equal to 126 milligrams per deciliter and
for which the primary cause is pancreatic beta cell destruction,
Type 2 Diabetes, which is non-insulin-dependent diabetes mellitus
as diagnosed according to criteria published in the Report of the
Expert Committee on the Diagnosis and Classification of Diabetes
Mellitus whereby fasting plasma glucose level is greater than or
equal to 126 milligrams per deciliter, and latent autoimmune
diabetes mellitus of adults (LADA).
[0085] The term "metabolic syndrome" refers to metabolic disorders,
particularly glucose and lipid regulatory disorders, including
insulin resistance and defective secretion of insulin by pancreatic
beta cells, and may further include conditions and states such as
abdominal obesity, dyslipidemia, hypertension, glucose intolerance
or a prothrombitic state, and which may further result in disorders
such as hyperlipidemia, obesity, diabetes, insulin resistance,
glucose intolerance, hyperglycemia, and hypertension.
2.0 Clinical Indications and Utility
[0086] The compositions and methods disclosed herein can be used
for both medical applications and animal husbandry or veterinary
applications. Typically, the methods are used in humans, but may
also be used in other mammals. The term "patient" is intended to
denote a mammalian individual, and is so used throughout the
specification and in the claims. The primary applications of the
present invention involve human patients, but the present invention
may be applied to laboratory, farm, zoo, wildlife, pet, sport or
other animals. Clinical indications and specific utilities include
the following:
[0087] 2.1 Obesity and Related Metabolic Syndrome.
[0088] Peptides of Formula (I), and in particular Formula (II) or
(III), have been found to be ligands of the MC4 receptor. In
particular, peptides of Formula (I) are believed to be useful in
treating diseases, disorders and/or conditions responsive to
modulation of the MC4-R function, more particularly activation of
the MC4-R, i.e. diseases, disorders and/or conditions which would
benefit from agonism (including full or partial agonism) at the
MC4-R, including energy homeostasis and metabolism related (such as
diabetes, in particular type 2 diabetes; dyslipidemia; fatty liver;
hypercholesterolemia; hypertriglyceridemia; hyperuricacidemia;
impaired glucose tolerance; impaired fasting glucos; insulin
resistance syndrome; and metabolic syndrome), food intake related
(such as hyperphagia; binge eating; bulimia; and compulsive eating)
and/or energy balance and body weight related diseases, disorders
and/or conditions, more particularly such diseases, disorders and
conditions characterized by excess body weight and/or excess food
intake.
[0089] Peptides of Formula (I), and in particular Formula (II) or
(III), are particularly believed to be useful for treatment of body
weight related diseases, disorders and/or conditions characterized
by excess body weight, including obesity and overweight (by
promotion of weight loss, maintenance of weight loss, and/or
prevention of weight gain, including medication-induced weight gain
or weight gain subsequent to cessation of smoking), and diseases,
disorders and/or conditions associated with obesity and/or
overweight, such as insulin resistance; impaired glucose tolerance;
type 2 diabetes; metabolic syndrome; dyslipidemia (including
hyperlipidemia); hypertension; heart disorders (e.g. coronary heart
disease, myocardial infarction); cardiovascular disorders;
non-alcoholic fatty liver disease (including non-alcoholic
steatohepatitis); joint disorders (including secondary
osteoarthritis); gastroesophageal reflux; sleep apnea;
atherosclerosis; stroke; macro and micro vascular diseases;
steatosis (e.g. in the liver); gall stones; and gallbladder
disorders.
[0090] Peptides of Formula (I), and in particular Formula (II) or
(III), are particularly believed to be useful for treatment of
obesity and type 2 diabetes, more specifically obesity.
[0091] It will be understood that there are medically accepted
definitions of obesity and overweight. A patient may be identified
by, for example, measuring body mass index (BMI), which is
calculated by dividing weight in kilograms by height in meters
squared, and comparing the result with the definitions. The
recommended classifications for BMI in humans, adopted by the
Expert Panel on the Identification, Evaluation and Treatment of
Overweight and Obesity in Adults, and endorsed by leading
organizations of health professionals, are as follows: underweight
<18.5 kg/m.sup.2, normal weight 18.5-24.9 kg/m.sup.2, overweight
25-29.9 kg/m.sub.2, obesity (class 1) 30-34.9 kg/m.sup.2, obesity
(class 2) 35-39.9 kg/m.sup.2, extreme obesity (class 3).gtoreq.40
kg/m.sup.2 (Practical Guide to the Identification, Evaluation, and
Treatment of Overweight and Obesity in Adults, The North American
Association for the Study of Obesity (NAASO) and the National
Heart, Lung and Blood Institute (NHLBI) 2000). Modifications of
this classification may be used for specific ethnic groups and for
children. Another alternative for assessing overweight and obesity
is by measuring waist circumference. There are several proposed
classifications and differences in the cutoffs based on ethnic
group. For instance, according to the classification from the
International Diabetes Federation, men having waist circumferences
above 94 cm (cut off for europids) and women having waist
circumferences above 80 cm (cut off for europids) are at higher
risk of diabetes, dyslipidemia, hypertension and cardiovascular
diseases because of excess abdominal fat. Another classification is
based on the recommendation from the Adult Treatment Panel III
where the recommended cutoffs are 102 cm for men and 88 cm for
women. However, the peptides of Formula (I) may also be used for
reduction of self-diagnosed overweight and for decreasing the risk
of becoming obese due to life style, genetic considerations,
heredity and/or other factors.
[0092] It is believed that peptides of Formula (I), and in
particular Formula (II) or (III), upon administration to an animal,
including man, will reduce food intake, body weight and/or body
weight gain in that animal.
[0093] Without being bound by any theory, it is believed that
peptides of Formula (I), and in particular Formula (II) or (III),
act by modulating appetite and/or satiety, increasing metabolic
rate, reducing intake of and/or craving for fat and/or
carbohydrates.
[0094] Without being bound by any theory, it is also believed that
peptides of Formula (I), and in particular Formula (II) or (III),
act by enhancing glucose tolerance and/or decreasing insulin
resistance. It is therefore believed that peptides of Formula (I)
can be useful also for treatment of type 2 diabetes in underweight
and normal weight individuals as well as in overweight and obese
individuals.
[0095] Peptides of the invention might also be useful for (i)
prevention of organ or tissue damage caused by hypoperfusion due to
vessel occlusion (e.g. caused by thrombosis), haemorrhage, trauma,
surgery, haemorrhagic shock, cardiogenic shock, toxic shock or
septic shock or (iii) treatment of male and female sexual
dysfunctions, such as male erectile dysfunction or female sexual
arousal dysfunction.
[0096] According to a further aspect of the invention, there is
provided a peptide of Formula (I), and in particular Formula (II)
or (III), as previously defined for use as a medicament.
[0097] In another aspect, the invention provides the use of a
peptide of Formula (I), and in particular Formula (II) or (III),
for treatment of diseases, disorders and/or conditions responsive
to modulation of the MC4-R, such as diseases, disorders and/or
conditions responsive to activation of the MC4-R, in particular
energy homeostasis and metabolism related (e.g. diabetes), food
intake related and/or energy balance and body weight related
diseases, disorders and/or conditions, including obesity,
overweight and diseases, disorders and/or conditions associated
with obesity and/or overweight, such as type 2 diabetes and
metabolic syndrome.
[0098] In a further aspect, the invention provides the use of a
peptide of Formula (I), and in particular Formula (II) or (III), in
the preparation of a medicament for treatment of diseases,
disorders and/or conditions responsive to modulation of the MC4-R,
such as activation of the MC4-R, in particular energy homeostasis
and metabolism related (e.g. diabetes), food intake related and/or
energy balance and body weight related diseases, disorders and/or
conditions, including obesity, overweight and diseases, disorders
and/or conditions associated with obesity and/or overweight, such
as type 2 diabetes and metabolic syndrome.
[0099] Peptides of the invention are advantageously more selective
(i.e. higher affinity and/or higher specificity) for MC4-R and/or
MC3-R than for MC1-R, MC2-R and MC5-R, in particular MC1-R. In
particular, peptides of the invention are advantageously more
selective for MC4-R than for any of MC3-R and MC1-R. Peptides of
the invention are suitably at least 3-fold, in particular 10-fold,
more particularly 30-fold, more selective for MC4-R than for any of
MC1-R, MC2-R, MC3-R and MC5-R. Some peptides of the invention are
even more than 100-fold, such as even about 150-fold, more
selective for MC4-R than MC1-R as determined in the receptor
binding assay described in 7.1. It is noted that the selectivity
profile may affect in vivo safety and side effects obtained upon
administration of the peptides.
[0100] Upon treatment of energy homeostasis and metabolism related,
food intake related and/or energy balance and body weight related
diseases, disorders and/or conditions, it is desirable to reduce or
eliminate unwanted side-effects that may result from MC4-R
activation, such as sexual side-effects, including penile erection,
and blood pressure effects.
[0101] Peptides of the invention are believed to be useful for
treatment of energy homeostasis and metabolism related (e.g.
diabetes), food intake related and/or energy balance and body
weight related diseases, disorders and/or conditions, including
obesity, overweight and diseases, disorders and/or conditions
associated with obesity and/or overweight, such as type 2 diabetes
and metabolic syndrome, without causing substantial adverse
cardiovascular effects, including a substantial increase in blood
pressure.
[0102] Peptides of the invention are believed to be useful for
treatment of energy homeostasis and metabolism related (e.g.
diabetes), food intake related and/or energy balance and body
weight related diseases, disorders and/or conditions, including
obesity, overweight and diseases, disorders and/or conditions
associated with obesity and/or overweight, such as type 2 diabetes
and metabolic syndrome, without causing substantial unwanted sexual
effects resulting from MC4-R activation, such as penile
erection.
[0103] It is believed that peptides of Formula I, and in particular
Formula (II) or (III), possess a satisfactory pharmacological
profile and promising biopharmaceutical properties, such as
toxicological profile, metabolism and pharmacokinetic properties,
solubility, and permeability. It will be understood that
determination of appropriate biopharmaceutical properties is within
the knowledge of a person skilled in the art.
3.0 Combination Therapy for Certain Indications
[0104] The peptides, compositions and methods of the present
invention may be used for treatment of any of the foregoing
diseases, indications, conditions or syndromes, or any disease,
indication, condition or syndrome which is melanocortin receptor
mediated, by administration in combination with one or more other
pharmaceutically active compounds. Such combination administration
may be by means of a single dosage form which includes both a
peptide of the present invention and one more other
pharmaceutically active compound, such single dosage form including
a tablet, capsule, spray, inhalation powder, injectable liquid or
the like. Alternatively, combination administration may be by means
of administration of two different dosage forms, with one dosage
form containing a peptide of the present invention, and the other
dosage form including another pharmaceutically active compound. In
this instance, the dosage forms may be the same or different.
Without meaning to limit combination therapies, the following
exemplifies certain combination therapies which may be
employed.
[0105] 3.1 Combination Therapy for Obesity and Related Metabolic
Syndrome.
[0106] One or more peptides of the invention may be combined with
one or more other pharmacologically active agent(s) that is (are)
useful in the treatment of various weight and feeding-related
disorders, such as obesity and/or overweight, in particular other
anti-obesity drugs that affect energy expenditure, glycolysis,
gluconeogenesis, glucogenolysis, lipolysis, lipogenesis, fat
absorption, fat storage, fat excretion, hunger and/or satiety
and/or craving mechanisms, appetite/motivation, food intake, or
gastrointestinal motility. Drugs that reduce energy intake include,
in part, various pharmacological agents, referred to as anorectic
drugs, which are used as adjuncts to behavioral therapy in weight
reduction programs.
[0107] Generally, a total dosage of the below-described obesity
control agents or medications, when used in combination with one or
more peptides of the present invention can range from 0.1 to 3,000
mg/day, preferably from about 1 to 1,000 mg/day and more preferably
from about 1 to 200 mg/day in single or 2-4 divided doses. The
exact dose, however, is determined by the attending clinician and
is dependent on such factors as the potency of the compound
administered, the age, weight, condition and response of the
patient.
[0108] One or more peptides of the invention may be combined with
one or more other pharmacologically active agent(s) that is (are)
useful in the treatment of diabetes, such as other anti-diabetic
drugs.
[0109] One or more peptides of the invention may in addition or
alternatively further be combined with one or more other
pharmacologically active agent(s) that is (are) useful in the
treatment of diseases, disorders and/or conditions associated with
obesity and/or overweight, such as insulin resistance; impaired
glucose tolerance; type 2 diabetes; metabolic syndrome;
dyslipidemia (including hyperlipidemia); hypertension; heart
disorders (e.g. coronary heart disease, myocardial infarction);
cardiovascular disorders; non-alcoholic fatty liver disease
(including non-alcoholic steatohepatitis); joint disorders
(including secondary osteoarthritis); gastroesophageal reflux;
sleep apnea; atherosclerosis; stroke; macro and micro vascular
diseases; steatosis (e.g. in the liver); gall stones; and
gallbladder disorders.
[0110] According to a further aspect of the invention there is
provided a combination treatment comprising the administration of a
pharmacologically effective amount of a peptide according to the
invention, or a pharmaceutically acceptable salt thereof,
optionally together with a pharmaceutically acceptable diluent or
carrier, with the simultaneous, sequential or separate
administration one or more of the following agents selected from:
[0111] insulin and insulin analogues; [0112] insulin secretagogues,
including sulphonylureas (e.g. glipizide) and prandial glucose
regulators (sometimes called "short-acting secretagogues"), such as
meglitinides (e.g. repaglinide and nateglinide); [0113] agents that
improve incretin action, for example dipeptidyl peptidase IV
(DPP-4) inhibitors (e.g. vildagliptin, saxagliptin, and
sitagliptin), and glucagon-like peptide-1 (GLP-1) agonists (e.g.
exenatide); [0114] insulin sensitising agents including peroxisome
proliferator activated receptor gamma (PPAR.gamma.) agonists, such
as thiazolidinediones (e.g. pioglitazone and rosiglitazone), and
agents with any combination of PPAR alpha, gamma and delta
activity; [0115] agents that modulate hepatic glucose balance, for
example biguanides (e.g. metformin), fructose 1,6-bisphosphatase
inhibitors, glycogen phopsphorylase inhibitors, glycogen synthase
kinase inhibitors, and glucokinase activators; [0116] agents
designed to reduce/slow the absorption of glucose from the
intestine, such as alpha-glucosidase inhibitors (e.g. miglitol and
acarbose); [0117] agents which antagonise the actions of or reduce
secretion of glucagon, such as amylin analogues (e.g. pramlintide);
[0118] agents that prevent the reabsorption of glucose by the
kidney, such as sodium-dependent glucose transporter 2 (SGLT-2)
inhibitors (e.g. dapagliflozin); [0119] agents designed to treat
the complications of prolonged hyperglycaemia, such as aldose
reductase inhibitors (e.g. epalrestat and ranirestat); and agents
used to treat complications related to micro-angiopathies; [0120]
anti-dyslipidemia agents, such as HMG-CoA reductase inhibitors
(statins, e.g. rosuvastatin) and other cholesterol-lowering agents;
PPAR.alpha. agonists (fibrates, e.g. gemfibrozil and fenofibrate);
bile acid sequestrants (e.g. cholestyramine); [0121] cholesterol
absorption inhibitors (e.g. plant sterols (i.e. phytosterols),
synthetic inhibitors); cholesteryl ester transfer protein (CETP)
inhibitors; inhibitors of the ileal bile acid transport system
(IBAT inhibitors); bile acid binding resins; nicotinic acid
(niacin) and analogues thereof; anti-oxidants, such as probucol;
and omega-3 fatty acids; [0122] antihypertensive agents, including
adrenergic receptor antagonists, such as beta blockers (e.g.
atenolol), alpha blockers (e.g. doxazosin), and mixed alpha/beta
blockers (e.g. labetalol); adrenergic receptor agonists, including
alpha-2 agonists (e.g. clonidine); angiotensin converting enzyme
(ACE) inhibitors (e.g. lisinopril), calcium channel blockers, such
as dihydropyridines (e.g. nifedipine), phenylalkylamines (e.g.
verapamil), and benzothiazepines (e.g. diltiazem); angiotensin II
receptor antagonists (e.g. candesartan); aldosterone receptor
antagonists (e.g. eplerenone); centrally acting adrenergic drugs,
such as central alpha agonists (e.g. clonidine); and diuretic
agents (e.g. furosemide); [0123] haemostasis modulators, including
antithrombotics, such as activators of fibrinolysis; thrombin
antagonists; factor VIIa inhibitors; anticoagulants, such as
vitamin K antagonists (e.g. warfarin), heparin and low molecular
weight analogues thereof, factor Xa inhibitors, and direct thrombin
inhibitors (e.g. argatroban); antiplatelet agents, such as
cyclooxygenase inhibitors (e.g. aspirin), adenosine diphosphate
(ADP) receptor inhibitors (e.g. clopidogrel), phosphodiesterase
inhibitors (e.g. cilostazol), glycoprotein IIB/IIA inhibitors (e.g.
tirofiban), and adenosine reuptake inhibitors (e.g. dipyridamole);
[0124] anti-obesity agents, such as appetite suppressant (e.g.
ephedrine), including noradrenergic agents (e.g. phentermine) and
serotonergic agents (e.g. sibutramine), pancreatic lipase
inhibitors (e.g. orlistat), microsomal transfer protein (MTP)
modulators, diacyl glycerolacyltransferase (DGAT) inhibitors, and
cannabinoid (CB1) receptor antagonists (e.g. rimonabant); [0125]
feeding behavior modifying agents, such as orexin receptor
modulators and melanin-concentrating hormone (MCH) modulators;
[0126] glucagon like peptide-1 (GLP-1) receptor modulators; [0127]
neuropeptideY (NPY)/NPY receptor modulators; [0128] pyruvate
dehydrogenase kinase (PDK) modulators; [0129] serotonin receptor
modulators; [0130] leptin/leptin receptor modulators; [0131]
ghrelin/ghrelin receptor modulators; or [0132] monoamine
transmission-modulating agents, such as selective serotonin
reuptake inhibitors (SSRI) (e.g. fluoxetine), noradrenaline
reuptake inhibitors (NARI), noradrenaline-serotonin reuptake
inhibitors (SNRI), triple monoamine reuptake blockers (e.g.
tesofensine), and monoamine oxidase inhibitors (MAOI) (e.g.
toloxatone and amiflamine), or a pharmaceutically acceptable salt,
solvate, solvate of such a salt or a prodrug thereof, optionally
together with a pharmaceutically acceptable carrier to a mammal,
such as man, in need of such therapeutic treatment.
[0133] According to an additional further aspect of the present
invention there is provided a combination treatment comprising the
administration of a pharmacologically effective amount of a
compound according to the invention, or a pharmaceutically
acceptable salt thereof, optionally together with a
pharmaceutically acceptable carrier, with the simultaneous,
sequential or separate administration of very low calorie diets
(VLCD) or low-calorie diets (LCD).
4.0 Methods of Making
[0134] In general, the peptides of the present invention may be
synthesized by solid-phase synthesis and purified according to
methods known in the art. Any of a number of well-known procedures
utilizing a variety of resins and reagents may be used to prepare
the peptides of the present invention.
[0135] The cyclic peptides of the present invention may be readily
synthesized by known conventional procedures for the formation of a
peptide linkage between amino acids. Such conventional procedures
include, for example, any solution phase procedure permitting a
condensation between the free alpha amino group of an amino acid or
residue thereof having its carboxyl group and other reactive groups
protected and the free primary carboxyl group of another amino acid
or residue thereof having its amino group or other reactive groups
protected. In a preferred conventional procedure, the cyclic
peptides of the present invention may be synthesized by solid-phase
synthesis and purified according to methods known in the art. Any
of a number of well-known procedures utilizing a variety of resins
and reagents may be used to prepare the peptides of the present
invention.
[0136] The process for synthesizing the cyclic peptides may be
carried out by a procedure whereby each amino acid in the desired
sequence is added one at a time in succession to another amino acid
or residue thereof or by a procedure whereby peptide fragments with
the desired amino acid sequence are first synthesized
conventionally and then condensed to provide the desired peptide.
The resulting peptide is then cyclized to yield a cyclic peptide of
the invention.
[0137] Solid phase peptide synthesis methods are well known and
practiced in the art. In such methods the synthesis of peptides of
the invention can be carried out by sequentially incorporating the
desired amino acid residues one at a time into the growing peptide
chain according to the general principles of solid phase methods.
These methods are disclosed in numerous references, including
Merrifield, R. B., Solid phase synthesis (Nobel lecture). Angew
Chem 24:799-810 (1985) and Barany et al., The Peptides, Analysis,
Synthesis and Biology, Vol. 2, Gross, E. and Meienhofer, J., Eds.
Academic Press 1-284 (1980).
[0138] In chemical syntheses of peptides, reactive side chain
groups of the various amino acid residues are protected with
suitable protecting groups, which prevent a chemical reaction from
occurring at that site until the protecting group is removed. Also
common is the protection of the alpha amino group of an amino acid
residue or fragment while that entity reacts at the carboxyl group,
followed by the selective removal of the alpha amino protecting
group to allow a subsequent reaction to take place at that site.
Specific protecting groups have been disclosed and are known in
solid phase synthesis methods and solution phase synthesis
methods.
[0139] Alpha amino groups may be protected by a suitable protecting
group, including a urethane-type protecting group, such as
benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl, such as
p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,
p-bromobenzyloxycarbonyl, p-biphenyl-isopropoxycarbonyl,
9-fluorenylmethoxycarbonyl (Fmoc) and p-methoxybenzyloxycarbonyl
(Moz) and aliphatic urethane-type protecting groups, such as
t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl,
isopropoxycarbonyl, and allyloxycarbonyl (Alloc). Fmoc are
preferred for alpha amino protection.
[0140] Guanidino groups may be protected by a suitable protecting
group, such as nitro, p-toluenesulfonyl (Tos), Z,
pentamethylchromanesulfonyl (Pmc), adamantyloxycarbonyl,
pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) and Boc. Pbf and Pmc
are preferred protecting groups for Arg.
[0141] The peptides of the invention described herein were prepared
using solid phase synthesis, such as by means of a Symphony
Multiplex Peptide Synthesizer (Rainin Instrument Company/Protein
Technologies Inc) automated peptide synthesizer, using programming
modules as provided by the manufacturer and following the protocols
set forth in the manufacturer's manual.
[0142] Solid phase synthesis is commenced from the C-terminal end
of the peptide by coupling a protected alpha amino acid to a
suitable resin. Such starting material is prepared by attaching an
alpha amino-protected amino acid by an amide linkage to a
4-(2',4'-dimethoxylphenyl-aminomethyl-phenoxy (Rink Amide) resin, a
4-(2',4'-dimethoxylphenyl-aminomethyl)-phenoxyacetamido-norleucyl-MBHA
resin, an aminoxanthen-3-yloxy-merifiel resin (Sieber Amide) resin,
or by an ester linkage to a p-benzyloxybenzyl alcohol (Wang) resin,
a 2-chlorotrityl chloride resin or by other means well known in the
art. Fmoc-Linker-BHA resin supports are commercially available and
generally used when feasible. The resins are carried through
repetitive cycles as necessary to add amino acids sequentially. The
alpha amino Fmoc protecting groups are removed under basic
conditions. Piperidine, piperazine, diethylamine, or morpholine
(20-40% v/v) in N,N-dimethylformamide (DMF) may be used for this
purpose.
[0143] Following removal of the alpha amino protecting group, the
subsequent protected amino acids are coupled stepwise in the
desired order to obtain an intermediate, protected peptide-resin.
The activating reagents used for coupling of the amino acids in the
solid phase synthesis of the peptides are well known in the art.
After the peptide is synthesized, if desired, the orthogonally
protected side chain protecting groups may be removed using methods
well known in the art for further derivatization of the
peptide.
[0144] Typically, orthogonal protecting groups are used as
appropriate. For example, the peptides of the invention contain
multiple amino acids with an amino group-containing side chain. In
one aspect, an Allyl-Alloc protection scheme is employed with the
amino acids forming a lactam bridge through their side chains, and
orthogonal protecting groups, cleavable under different reactive
conditions, use for other amino acids with amino group-containing
side chains. Thus, for example, Fmoc-Orn(Alloc)-OH and
Fmoc-Glu(OAll)-OH amino acids (Glu(OAll) refers to glutamic acid
5-allyl ester) can be employed for the positions forming a lactam
bridge upon cyclization, while other amino acids with amino
group-containing side chains have a different and orthogonal
protecting group, such as with Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Pbf)-OH,
Fmoc-Dab(Pbf)-OH or the like. Other protecting groups may be
similarly employed; by way of example and not limitation, Mtt/OPp
(4-methyltrityl/2-phenylisopropyl) can be employed with the side
chains forming a lactam bridge upon cyclization, with orthogonal
protecting groups being utilized for other positions that are not
cleavable using conditions suitable for cleavage of Mtt/OPp.
[0145] Reactive groups in a peptide can be selectively modified,
either during solid phase synthesis or after removal from the
resin. For example, peptides can be modified to obtain N-terminus
modifications, such as acetylation, while on resin, or may be
removed from the resin by use of a cleaving reagent and then
modified. Similarly, methods for modifying side chains of amino
acids are well known to those skilled in the art of peptide
synthesis. The choice of modifications made to reactive groups
present on the peptide will be determined, in part, by the
characteristics that are desired in the peptide.
[0146] In the peptides of the present invention, in one embodiment
the N-terminus group is modified by introduction of an N-acetyl
group. In one aspect, a method is employed wherein after removal of
the protecting group at the N-terminal, the resin-bound peptide is
reacted with acetic anhydride in DMF in the presence of an organic
base, such aspyridine. Other methods of N-terminus acetylation are
known in the art, including solution phase acetylation, and may be
employed.
[0147] The peptide can, in one embodiment, be cyclized prior to
cleavage from the peptide resin. For cyclization through reactive
side chain moieties, the desired side chains are deprotected, and
the peptide suspended in a suitable solvent and a cyclic coupling
agent added. Suitable solvents include, for example DMF,
dichloromethane (DCM) or 1-methyl-2-pyrrolidone (NMP). Suitable
cyclic coupling reagents include, for example,
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TBTU),
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU),
benzotriazole-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluoroph-
osphate (BOP),
benzotriazole-1-yl-oxy-tris(pyrrolidino)phosphoniumhexafluorophosphate
(PyBOP), 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TATU),
2-(2-oxo-1(2H)-pyridyl)-1,1,3,3-tetramethyluronium
tetrafluoroborate (TPTU) or
N,N'-dicyclohexylcarbodiimide/1-hydroxybenzotriazole (DCCI/HOBt).
Coupling is conventionally initiated by use of a suitable base,
such as N,N-diisopropylethylamine (DIPEA), sym-collidine or
N-methylmorpholine (NMM).
[0148] The cyclized peptides can then be cleaved from solid phase,
using any suitable reagent, such as ethylamine in DCM or various
combinations of agents, such as trifluoroacetic acid (TFA),
tri-isopropylsilane (TIS), dimethoxybenezene (DMB), water and the
like. The resulting crude peptide is dried and remaining amino acid
side chain protecting groups, if any, are cleaved using any
suitable reagent, such as TFA in the presence of water, TIS,
2-mercaptopethane (ME), and/or 1,2-ethanedithiol (EDT). The final
product is precipitated by adding cold ether and collected by
filtration. Final purification is by reverse phase high performance
liquid chromatography (RP-HPLC), using a suitable column, such as a
C.sub.18 column, or other methods of separation or purification,
such as methods based on the size or charge of the peptide, can
also be employed. Once purified, the peptide can be characterized
by any number of methods, such as high performance liquid
chromatography (HPLC), amino acid analysis, mass spectrometry, and
the like.
[0149] For peptides of the present invention which have a
C-terminus substituted amide derivative or N-alkyl group, synthesis
may proceed by solid phase synthesis commenced from the C-terminal
end of the peptide by coupling a protected alpha amino acid to a
suitable resin. Such methods for preparing substituted amide
derivatives on solid-phase have been described in the art. See, for
example, Barn D. R. et al., Synthesis of an array of amides by
aluminum chloride assisted cleavage on resin bound esters.
Tetrahedron Letters, 37:3213-3216 (1996); DeGrado W. F. and Kaiser
E. T., Solid-phase synthesis of protected peptides on a polymer
bound oxime: Preparation of segments comprising the sequences of a
cytotoxic 26-peptide analogue. J. Org. Chem., 47:3258-3261 (1982).
Such a starting material can be prepared by attaching an alpha
amino-protected amino acid by an ester linkage to a
p-benzyloxybenzyl alcohol (Wang) resin or an by amide linkage to a
4-(2',4'-dimethoxylphenyl-aminomethyl-phenoxy (Rink Amide) resin by
well known means. The peptide chain is grown with the desired
sequence of amino acids. Before cleavage, the peptide is cyclized
on the solid phase. Peptides employing a p-benzyloxybenzyl alcohol
(Wang) resin may be cleaved from resin by aluminum chloride in DCM,
and peptides employing a Rink Amide resin may be cleaved by mixture
of TFA, TIS and water.
[0150] While synthesis has been described primarily with reference
to solid phase Fmoc chemistry, it is to be understood that other
chemistries and synthetic methods may be employed to make the
cyclic peptides of the invention, such as by way of example and not
limitation, methods employing Boc chemistry, solution chemistry,
and other chemistries and synthetic methods.
[0151] 5.1 Salt Form of Cyclic Peptides of the Present
Invention.
[0152] It shall be understood that as used herein all references to
peptides according to the invention, including a specific chemical
formula or name, are intended to include all pharmaceutically
acceptable salts, solvates, hydrates, polymorphs, prodrugs,
metabolites, stereoisomers, and tautomeric isomers thereof.
[0153] Cyclic peptides of the present invention may be in the form
of any pharmaceutically acceptable salt. The term "pharmaceutically
acceptable salts" refers to salts prepared from pharmaceutically
acceptable non-toxic bases or acids including inorganic or organic
bases and inorganic or organic acids (see "Handbook of
Pharmaceutical Salts: Properties, Selection and Use", P. H. Stahl,
P. G. Wermuth, IUPAC, Wiley-VCH, 2002). Salts derived from
inorganic bases include aluminum, ammonium, calcium, copper,
ferric, ferrous, lithium, magnesium, manganic salts, manganous,
potassium, sodium, zinc, and the like. Particularly preferred are
the ammonium, calcium, lithium, magnesium, potassium, and sodium
salts. Salts derived from pharmaceutically acceptable organic
non-toxic bases include salts of primary, secondary, and tertiary
amines, substituted amines including naturally occurring
substituted amines, cyclic amines, and basic ion exchange resins,
such as arginine, betaine, caffeine, choline,
N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,
2-dimethylaminoethanol, ethanolamine, ethylenediamine,
N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine,
histidine, hydrabamine, isopropylamine, lysine, methylglucamine,
morpholine, piperazine, piperidine, polyamine resins, procaine,
purines, theobromine, triethylamine, trimethylamine,
tripropylamine, tromethamine, and the like. When cyclic peptide of
the present invention is basic, acid addition salts may be prepared
from pharmaceutically acceptable non-toxic acids, including
inorganic and organic acids. Such acids include acetic,
benzenesulfonic, benzoic, camphorsulfonic, carboxylic, citric,
ethanesulfonic, formic, fumaric, gluconic, glutamic, hydrobromic,
hydrochloric, isethionic, lactic, maleic, malic, mandelic,
methanesulfonic, malonic, mucic, nitric, pamoic, pantothenic,
phosphoric, propionic, succinic, sulfuric, tartaric,
p-toluenesulfonic acid, trifluoroacetic acid, and the like. Acid
addition salts of peptides of the present invention are prepared in
a suitable solvent for the peptide and an excess of an acid, such
as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic,
trifluoroacetic (TFA), citric, tartaric, maleic, succinic or
methanesulfonic acid. The acetate, ammonium acetate and TFA acid
salt forms are especially useful.
[0154] Where peptides of the present invention include an acidic
moiety, suitable pharmaceutically acceptable salts may include
alkali metal salts, such as sodium or potassium salts, or alkaline
earth metal salts, such as calcium or magnesium salts. It is also
to be understood that certain peptides of the Formula (I) can exist
in solvated forms, including solvates of the free peptide or
solvates of a salt of the compound, as well as unsolvated forms.
The term "solvate" is used herein to describe a molecular complex
comprising the compound of the invention and one or more
pharmaceutically acceptable solvent molecules, for example,
ethanol. The term "hydrate" is employed when said solvent is water.
It is to be understood that all polymorphs, including mixtures of
different polymorphs, are included within the scope of the claimed
peptides.
[0155] 5.2 Pharmaceutical Compositions.
[0156] The invention provides a pharmaceutical composition that
includes one or more cyclic peptides of the present invention and a
pharmaceutically acceptable carrier. When formulated with a
pharmaceutically acceptable carrier, the compound of the invention
may be present in the pharmaceutical composition in a concentration
from 0.1 to 99.5%, such as from 0.5 to 95%, by weight of the total
composition. The choice of carrier is within the knowledge of a
person skilled in the art and depends on, for instance, the mode of
administration, the dosage form, and the physical properties of the
active compound, such as solubility and stability. The term
"carrier" as used herein relates to a therapeutically inactive
ingredient. The dosage form may be a solid, semi-solid or liquid
system. The formulation may be an immediate and/or modified
release, including delayed-, sustained-, pulsed-, controlled-,
targeted and programmed release formulation.
[0157] The carrier may be a liquid formulation, and is preferably a
buffered, isotonic, aqueous solution. Pharmaceutically acceptable
carriers also include excipients, such as diluents, carriers and
the like, and additives, such as stabilizing agents, preservatives,
solubilizing agents, buffers and the like, as hereafter
described.
[0158] The cyclic peptide compositions of the present invention may
be formulated or compounded into pharmaceutical compositions that
include at least one cyclic peptide of the present invention
together with one or more pharmaceutically acceptable carriers,
including excipients, such as diluents, carriers and the like, and
additives, such as stabilizing agents, preservatives, solubilizing
agents, buffers and the like, as may be desired. Formulation
excipients may include polyvinylpyrrolidone, gelatin, hydroxy
propyl cellulose (HPC), acacia, polyethylene glycol, mannitol,
sodium chloride and sodium citrate. For injection or other liquid
administration formulations, water containing at least one or more
buffering constituents is preferred, and stabilizing agents,
preservatives and solubilizing agents may also be employed. For
solid administration formulations, any of a variety of thickening,
filler, bulking and carrier additives may be employed, such as
starches, sugars, cellulose derivatives, fatty acids and the like.
For topical administration formulations, any of a variety of
creams, ointments, gels, lotions and the like may be employed. For
most pharmaceutical formulations, non-active ingredients will
constitute the greater part, by weight or volume, of the
preparation. For pharmaceutical formulations, it is also
contemplated that any of a variety of measured-release,
slow-release or sustained-release formulations and additives may be
employed, so that the dosage may be formulated so as to provide
delivery of a peptide of the present invention over a period of
time.
[0159] In general, the actual quantity of cyclic peptides of the
present invention administered to a patient will vary between
fairly wide ranges depending on the mode of administration, the
formulation used, and the response desired.
[0160] In practical use, the cyclic peptides of the invention can
be combined as the active ingredient in an admixture with a
pharmaceutical carrier according to conventional pharmaceutical
compounding techniques. The carrier may take a wide variety of
forms depending on the form of preparation desired for
administration, for example, oral, parenteral (including
intravenous), urethral, vaginal, nasal, buccal, sublingual, or the
like. In preparing the compositions for oral dosage form, any of
the usual pharmaceutical media may be employed, such as, for
example, water, glycols, oils, alcohols, flavoring agents,
preservatives, coloring agents and the like in the case of oral
liquid preparations such as, for example, suspensions, elixirs and
solutions; or carriers such as starches, sugars, microcrystalline
cellulose, diluents, granulating agents, lubricants, binders,
disintegrating agents and the like in the case of oral solid
preparations such as, for example, powders, hard and soft capsules
and tablets.
[0161] Because of their ease of administration, tablets and
capsules represent an advantageous oral dosage unit form. If
desired, tablets may be coated by standard aqueous or non-aqueous
techniques. The amount of active peptide in such therapeutically
useful compositions is such that an effective dosage will be
obtained. In another dosage unit form, sublingual constructs may be
employed, such as sheets, wafers, tablets or the like.
[0162] The tablets, pills, capsules, and the like may also contain
a binder such as povidone, gum tragacanth, acacia, corn starch or
gelatin; diluents; fillers such as microcrystalline cellulose;
excipients such as dicalcium phosphate; a disintegrating agent such
as corn starch, potato starch or alginic acid; preservatives;
colorants; a lubricant such as magnesium stearate; and a sweetening
agent such as sucrose, lactose or saccharin. When a dosage unit
form is a capsule, it may contain, in addition to materials of the
above type, a liquid carrier such as fatty oil. Various other
materials may be utilized as coatings or to modify the physical
form of the dosage unit. For instance, tablets may be coated with
shellac, sugar or both. A syrup or elixir may contain, in addition
to the active ingredient, sucrose as a sweetening agent, methyl and
propylparabens as preservatives, a dye and a flavoring such as
cherry or orange flavor.
[0163] If formulated for oral delivery, the peptide is preferably
formulated and made such that it is encased in an enteric
protectant, more preferably such that it is not released until the
tablet or capsule has transited the stomach, and optionally has
further transited a portion of the small intestine. In the context
of this application it will be understood that the term enteric
coating or material refers to a coating or material that will pass
through the stomach essentially intact but will disintegrate after
passing through the stomach to release the active drug substance.
Materials that may be used includes cellulose acetate phthalate,
hydroxypropylmethyl-ethylcellulose succinate,
hydroxypropylmethylcellulose phthalate, polyvinyl acetate
phthalate, and methacrylic acid-methyl methacrylate copolymer. The
enteric coating employed promotes dissolution of the dosage form
primarily at a site outside the stomach, and may be selected such
that the enteric coating dissolves at a pH of approximately at
least 5.5, more preferable at a pH of from about 6.0 to about
8.0.
[0164] Any of a variety of permeation enhancers may be employed, to
increase uptake in the intestines upon dissolution of the enteric
coating. In one aspect, permeation enhancers increase either
paracellular or transcellular transport systems. Representative,
non-limiting examples of such permeation enhancers include calcium
chelators, bile salts (such as sodium cholate), and fatty acids. In
some embodiments, peptides or polypeptides that act as substrates
for intestinal proteases are further added. Cyclic peptides may
also be administered parenterally. Solutions or suspensions of
these active peptides may for instance be prepared in water mixed
with for instance hydroxy-propylcellulose. Dispersions can also be
prepared in glycerol, liquid polyethylene glycols and mixtures
thereof in oils. These preparations may optionally contain a
preservative to prevent the growth of microorganisms.
[0165] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases, the form must be sterile and must be
fluid to the extent that it may be administered by syringe. The
form must be stable under the conditions of manufacture and storage
and must be preserved against the contaminating action of
microorganisms such as bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, a polyol, for example glycerol, propylene glycol or liquid
polyethylene glycol, suitable mixtures thereof, and vegetable
oils.
[0166] The cyclic peptides of the present invention may be
therapeutically applied by means of nasal administration. By "nasal
administration" is meant any form of intranasal administration of
any of the cyclic peptides of the present invention. The peptides
may be in an aqueous solution, such as a solution including saline,
citrate or other common excipients or preservatives. The peptides
may also be in a dry or powder formulation.
[0167] If in an aqueous solution, the cyclic peptides may be
appropriately buffered by means of saline, acetate, phosphate,
citrate, acetate or other buffering agents, which may be at any
physiologically acceptable pH, such as from about pH 4 to about pH
7. A combination of buffering agents may also be employed, such as
phosphate buffered saline, a saline and acetate buffer, and the
like. In the case of saline, a 0.9% saline solution may be
employed. In the case of acetate, phosphate, citrate, and the like,
a 50 mM solution may be employed. In addition to buffering agents,
a suitable preservative may be employed, to prevent or limit
bacteria and other microbial growth. One such preservative that may
be employed is 0.05% benzalkonium chloride.
[0168] In an alternative embodiment, cyclic peptides of the present
invention may be administered directly into the lung.
Intrapulmonary administration may be performed by means of a
metered dose inhaler, a device allowing self-administration of a
metered bolus of a peptide of the present invention when actuated
by a patient during inspiration. Any of a variety of different
techniques may be used to make dry powder microparticles, including
but not limited to micro-milling, spray drying and a quick freeze
aerosol followed by lyophilization.
[0169] The cyclic peptides of the present invention may be
therapeutically administered by means of an injection of a
sustained release formulation. In general, any of a number of
injectable and bioerodible polymers may be employed in a sustained
release injectable formulation. Alternatively other sustained
release formulations may be employed, including formulations
permitting subcutaneous injection, which other formulations may
include one or more of nano/microspheres, liposomes, emulsions
(such as water-in-oil emulsions), gels, insoluble salts or
suspensions in oil The formulation may be such that an injection is
required on a daily, weekly, monthly or other periodic basis,
depending on the concentration and amount of cyclic peptide, the
sustained release rate of the materials employed, and other factors
known to those of skill in the art.
[0170] 5.3 Routes of Administration.
[0171] If a composition including one or more peptides of the
present invention is administered by injection, the injection may
be intravenous, subcutaneous, intramuscular, intraperitoneal or
other means known in the art.
[0172] The peptides of the present invention may be formulated by
any means known in the art, including but not limited to
formulation as tablets, capsules, caplets, suspensions, powders,
lyophilized preparations, suppositories, ocular drops, skin
patches, oral soluble formulations, sprays, aerosols and the like,
and may be mixed and formulated with buffers, binders, excipients,
stabilizers, anti-oxidants and other agents known in the art. In
general, any route of administration by which the peptides of
invention are introduced across an epidermal layer of cells may be
employed. Administration means may thus include administration
through mucous membranes, buccal administration, oral
administration, dermal administration, inhalation administration,
nasal administration, urethral administration, vaginal
administration, and the like.
[0173] 5.4 Therapeutically Effective Amount.
[0174] In general, the actual quantity of cyclic peptide of the
present invention administered to a patient will vary between
fairly wide ranges depending upon the mode of administration, the
patient (including weight, sex, health condition and diet), the
formulation used, and the response desired. The dosage for
treatment is administration, by any of the foregoing means or any
other means known in the art, of an amount sufficient to bring
about the desired therapeutic effect. The cyclic peptides of the
present invention are highly active. For example, the cyclic
peptide can be administered (as a single dose or in divided daily
doses) at about 0.1, 0.5, 1, 5, 50, 100, 500, 1000 or 5000 .mu.g/kg
body weight, depending on the specific peptide selected, the
desired therapeutic response, the route of administration, the
formulation and other factors known to those of skill in the
art.
6.0 Peptides of the Present Invention
[0175] In one aspect, the present invention relates to a cyclic
peptide of the structural Formula (I):
##STR00052##
including all enantiomers, stereoisomers or diastereoisomers
thereof, or a pharmaceutically acceptable salt of any of the
foregoing,
[0176] wherein:
R.sub.1 is --NH--C(.dbd.O)-- or --C(.dbd.O)--NH--; [0177] R.sub.2
is --H or --CH.sub.2--, and if R.sub.2 is --CH.sub.2-- forms with
R.sub.3 a pyrrolidine ring, the pyrrolidine ring optionally
substituted with --OH; [0178] R.sub.3 is --(CH.sub.2).sub.2-- if
R.sub.2 is --CH.sub.2--, and otherwise R.sub.3 is selected from
[0178] ##STR00053## [0179] R.sub.4a, R.sub.4b and R.sub.4c are each
independently selected from hydrogen, halo,
(C.sub.1-C.sub.10)alkyl-halo, (C.sub.1-C.sub.10)alkyl-dihalo,
(C.sub.1-C.sub.10)alkyl-trihalo, (C.sub.1-C.sub.10)alkyl,
(C.sub.1-C.sub.10)alkoxy, (C.sub.1-C.sub.10)alkylthio, aryl,
aryloxy, nitro, nitrile, sulfonamide, amino, monosubstituted amino,
disubstituted amino, hydroxy, carboxy, or alkoxy-carbonyl, on the
proviso that at least one of R.sub.4a, R.sub.4b and R.sub.4c is not
hydrogen; [0180] R.sub.5 is --OH or --N(R.sub.6a)(R.sub.6b); [0181]
R.sub.6a and R.sub.6b are each independently H or a C.sub.1 to
C.sub.4 linear, branched or cyclic alkyl chain; [0182] R.sub.7 is
--H or --C(.dbd.O)--NH.sub.2; [0183] w is in each instance
independently 0 to 5; [0184] x is 1 to 5; [0185] y is 1 to 5; and
[0186] z is in each instance independently 1 to 5.
[0187] In another aspect, the present invention relates to a cyclic
peptide of Formula (I) which is of formula (II):
##STR00054##
or a pharmaceutically acceptable salt thereof.
[0188] In another aspect, the present invention relates to a cyclic
peptide of Formula (I) which is of Formula (III):
##STR00055##
or a pharmaceutically acceptable salt thereof.
[0189] In one aspect, the invention provides a cyclic heptapeptide
which contains a core sequence derived from His-Phe-Arg-Trp within
the cyclic portion, and where the amino acid in the first position
is Arg and is outside the cyclic portion. Without being bound by
any theory, the Arg residue is believed to largely contribute to
the low activity at MC1-R.
[0190] Further, the core sequence in the peptides of Formula I
includes a substituted D-Phe. Without being bound by any theory, it
is believed that the intrinsic activity may be lowered while
substantially maintaining potency by using substituted D-Phe in
peptides in accordance with the invention. The peptide called Ref
Ex 1 (including unsubstituted D-Phe) in Table 1 can be compared
with one or more of Examples 1-5, 9-11, 17, 26 and 27. It may be
noted that Table 1 includes mean values.
TABLE-US-00002 TABLE 1 0.1 ng/mL Doxycycline K.sub.i EC.sub.50
Intrinsic (nM) (nM) activity (%) Ref Ac-Arg-cyclo(Glu-Dab-D- 0.65
0.33 94 Ex 1 Phe-Arg-Trp-Orn)-NH.sub.2 Ref Ac-Arg-cyclo(Glu-Dab-D-
8 3.0 92 Ex 2 Phe-Arg-Trp-Orn)-OH
[0191] In a further embodiment of the invention, there are provided
peptides according to Formula (I), and in particular Formula (II)
or (III), wherein one or more of R.sub.4a, R.sub.4b and R.sub.4c
are each independently selected from hydrogen, halo,
(C.sub.1-C.sub.10)alkyl-halo, (C.sub.1-C.sub.10)alkyl-dihalo,
(C.sub.1-C.sub.10)alkyl-trihalo, (C.sub.1-C.sub.10)alkyl,
(C.sub.1-C.sub.10)alkoxy, phenyl, nitro, nitrile, amino or hydroxy,
on the proviso that at least one of R.sub.4a, R.sub.4b and R.sub.4c
is not hydrogen.
[0192] In a further embodiment of the invention, there are provided
peptides according to Formula (I), and in particular Formula (II)
or (III), wherein one or more of R.sub.4a, R.sub.4b and R.sub.4c
are each independently selected from hydrogen, halo,
(C.sub.1-C.sub.4)alkyl-halo, (C.sub.1-C.sub.4)alkyl-dihalo,
(C.sub.1-C.sub.4)alkyl-trihalo, (C.sub.1-C.sub.4)alkyl,
(C.sub.1-C.sub.4)alkoxy, phenyl, nitro, nitrile, amino or hydroxy,
on the proviso that at least one of R.sub.4a, R.sub.4b and R.sub.4c
is not hydrogen.
[0193] In a further embodiment of the invention, there are provided
peptides according to Formula (I), and in particular Formula (II)
or (III), wherein one or more of R.sub.4a, R.sub.4b and R.sub.4c
are each independently selected from [0194] fluoro, chloro,
##STR00056##
[0195] In a further embodiment of the invention, there are provided
peptides according to Formula (I), and in particular Formula (II)
or (III), wherein one or more of R.sub.4a, R.sub.4b and R.sub.4c
are each independently selected from hydrogen, halo,
(C.sub.1-C.sub.4)alkyl-halo, (C.sub.1-C.sub.4)alkyl-dihalo,
(C.sub.1-C.sub.4)alkyl-trihalo, (C.sub.1-C.sub.4)alkyl,
(C.sub.1-C.sub.4)alkoxy, nitro, nitrile, amino or hydroxy, on the
proviso that at least one of R.sub.4a, R.sub.4b and R.sub.4c is not
hydrogen.
[0196] In a further embodiment of the invention, there are provided
peptides according to Formula (I), and in particular Formula (II)
or (III), wherein one or more of R.sub.4a, R.sub.4b and R.sub.4c
are each independently selected from [0197] fluoro, chloro,
##STR00057##
[0198] In a further embodiment of the invention, there are provided
peptides according to Formula (I), and in particular Formula (II)
or (III), wherein R.sub.4a, is selected from [0199] fluoro,
chloro,
##STR00058##
[0199] and R.sub.4b and R.sub.4c are each H.
[0200] In a further embodiment of the invention, there are provided
peptides according to Formula (I), and in particular Formula (II)
or (III), wherein R.sub.4a is selected from --C.ident.N or --F, and
R.sub.4b and R.sub.4c are each H.
[0201] In a particular embodiment of the invention, there are
provided peptides according to Formula (I), and in particular
Formula (II) or (III), wherein R.sub.4a is in the 4 position and is
--C.ident.N and R.sub.4b and R.sub.4c are each H.
[0202] In another particular embodiment of the invention, there are
provided peptides according to Formula (I), and in particular
Formula (II) or (III), wherein R.sub.4a is in the 4 position and is
--F and R.sub.4b and R.sub.4c are each H.
[0203] The peptides encompassed within Formula (I) contain one or
more asymmetric elements such as stereogenic centers, stereogenic
axes and the like, so that the peptides encompassed within Formula
(I) can exist in different stereoisomeric forms. For both specific
and generically described peptides, including the peptides
encompassed within Formula (I), all forms of isomers at all chiral
or other isomeric centers, including enantiomers and diastereomers,
are intended to be covered herein. The peptides of the invention
each include multiple chiral centers, and may be used as a racemic
mixture or an enantiomerically enriched mixture, in addition to use
of the peptides of the invention in enantiopure preparations.
Typically, the peptides of the invention will be synthesized with
the use of chirally pure reagents, such as specified L- or D-amino
acids, using reagents, conditions and methods such that
enantiomeric purity is maintained, but it is possible and
contemplated that racemic mixtures may be made. Such racemic
mixtures may optionally be separated using well-known techniques
and an individual enantiomer may be used alone. In cases and under
specific conditions of temperature, solvents and pH wherein
peptides may exist in tautomeric forms, each tautomeric form is
contemplated as being included within this invention whether
existing in equilibrium or predominantly in one form. Thus a single
enantiomer of a peptide of Formula (I), which is an optically
active form, can be obtained by asymmetric synthesis, synthesis
from optically pure precursors, or by resolution of the
racemates.
[0204] The peptides of formulas (II) and (III) are specific
stereoisomeric forms of the peptides of Formula (I), but the
invention should not be construed as being limited to the
stereoisomeric forms encompassed by formulas (II) and (III).
[0205] The invention is further intended to include prodrugs of the
present peptides, which on administration undergo chemical
conversion by metabolic processes before becoming active
pharmacological peptides. In general, such prodrugs will be
functional derivatives of the present peptides, which are readily
convertible in vivo into a peptide of Formula (I). Prodrugs are any
covalently bonded compounds, which release the active parent
peptide drug of Formula (I) in vivo. Conventional procedures for
the selection and preparation of suitable prodrug derivatives are
described, for example, in "Design of Prodrugs", ed. H. Bundgaard,
Elsevier, 1985. Typical examples of prodrugs have biologically
labile protecting groups on a functional moiety, such as for
example by esterification of hydroxyl, carboxyl or amino functions.
Thus by way of example and not limitation, a prodrug includes
peptides of Formula (I) wherein an ester prodrug form is employed,
such as, for example, lower alkyl esters of the R group of Formula
(I), such as where R is --OH, which lower alkyl esters may include
from 1-8 carbons in an alkyl radical or aralkyl esters which have
6-12 carbons in an aralkyl radical. Broadly speaking, prodrugs
include compounds that can be oxidized, reduced, aminated,
deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed,
alkylated, dealkylated, acylated, deacylated, phosphorylated or
dephosphorylated to produce an active parent peptide drug of
Formula (I) in vivo.
[0206] Certain modifications of peptides of Formula I may be made
in order to enhance the half-life of the peptide (see G. Pasuta and
F. M. Veronese (2007) "Polymer-drug conjugation, recent
achievements and general strategies" Progress in Polymer Science 32
(8-9): 933-961).
[0207] The subject invention also includes peptides which are
identical to those recited in Formula (I), but for the fact that
one or more atoms depicted in Formula (I) are replaced by an atom
having an atomic mass or mass number different from the atomic mass
or mass number usually found in nature. Examples of isotopes that
can be incorporated into peptides of the invention include isotopes
of hydrogen, carbon, nitrogen and oxygen, such as .sup.2H, .sup.3H,
.sup.13C, .sup.14C, .sup.15N, .sup.18O and .sup.17O, respectively.
Peptides of the present invention and pharmaceutically acceptable
salts or solvates of said peptides which contain the aforementioned
isotopes and/or other isotopes of other atoms are within the scope
of this invention. Certain isotopically-labeled peptides of the
present invention, for example those into which radioactive
isotopes such as .sup.3H and .sup.14C are incorporated, may have
use in a variety of assays, such as in drug and/or substrate tissue
distribution assays. Substitution with heavier isotopes, such as
substitution of one or more hydrogen atoms with deuterium
(.sup.2H), can provide pharmacological advantages in some
instances, including increased metabolic stability. Isotopically
labeled peptides of Formula (I) can generally be prepared by
substituting an isotopically labeled reagent for a non-isotopically
labeled reagent.
[0208] In one embodiment of the peptides according to Formula (I),
and in particular Formula (II) or (III), R.sub.1 is
--C(.dbd.O)--NH--, x is 2 and y is 3.
[0209] In another embodiment of the peptides according to Formula
(I), and in particular Formula (II) or (III), R.sub.1 is
--C(.dbd.O)--NH--, x is 1 and y is 4.
[0210] In another embodiment of the peptides according to Formula
(I), and in particular Formula (II) or (III), R.sub.1 is
--NH--C(.dbd.O)--, x is 3 and y is 2.
[0211] In still another embodiment of the peptides according to the
Formula (I), and in particular Formula (II) or (III), R.sub.1 is
--NH--C(.dbd.O)--, x is 4 and y is 1.
[0212] In a particular embodiment of the peptides according to the
Formula (I), and in particular Formula (II) or (III), R.sub.1 is
--C(.dbd.O)--NH--, x is 2 and y is 3, or alternatively R.sub.1 is
--NH--C(.dbd.O)--, x is 3 and y is 2. More specifically, R.sub.1 is
--C(.dbd.O)--NH--, x is 2 and y is 3. It has surprisingly and
unexpectedly been found that peptides according to the invention
having a lactam bridge wherein the amide bond is positioned by
means of the side chain of Glu (at the N-terminus end of the cyclic
portion) and the side chain of Orn (at the C-terminus end of the
cyclic portion) generally provides improved potency as determined
by EC.sub.50, in particular in the herein described low density
hMC4-R system, compared to peptides wherein the amide bond is
positioned by means of the side chains of Asp and Lys, but which
are otherwise identical. This discovery is contrary to assertions
in the prior art that the location and direction of an amide bond
in the lactam bridge of melanocortin receptor specific peptides is
of little importance for activity and does not interact with
receptors. See, for example, Bednarek M A et al., Potent and
selective peptide agonists of .alpha.-melanotropin action at human
melanocortin receptor 4: their synthesis and biological evaluation
in vitro. Biochem. Biophys. Res. Comm. 286:641-645 (2001).
[0213] In an embodiment of the peptides according to Formula (I),
and in particular Formula (II) or (III), R.sub.2 is --H or
--CH.sub.2--, and if R.sub.2 is --CH.sub.2-- forms with R.sub.3 a
pyrrolidine ring, the pyrrolidine ring optionally substituted with
--OH;
[0214] R.sub.3 is --(CH.sub.2).sub.2-- if R.sub.2 is --CH.sub.2--,
and otherwise R.sub.3 is selected from
##STR00059##
[0215] wherein w is in each instance independently selected from 0
to 5, and
[0216] z is in each instance independently selected from 1 to
5.
[0217] In an embodiment of the peptides according to Formula (I),
and in particular Formula (II) or (III), R.sub.2 is --H or
--CH.sub.2--, and if R.sub.2 is --CH.sub.2-- forms with R.sub.3 a
pyrrolidine ring, the pyrrolidine ring optionally substituted with
--OH;
[0218] R.sub.3 is --(CH.sub.2).sub.2-- if R.sub.2 is --CH.sub.2--,
and otherwise R.sub.3 is selected from
##STR00060##
[0219] wherein w is in each instance independently selected from 0
to 5, and
[0220] z is in each instance independently selected from 1 to
5.
[0221] In an embodiment of the peptides according to Formula (I),
and in particular Formula (II) or (III), R.sub.2 is H.
[0222] In an embodiment of the peptides according to Formula (I),
and in particular Formula (II) or (III), R.sub.2 is H or
--CH.sub.2--, and if R.sub.2 is --CH.sub.2-- forms with R.sub.3 a
pyrrolidine ring, the pyrrolidine ring optionally substituted with
--OH; and
[0223] R.sub.3 is --(CH.sub.2).sub.2-- if R.sub.2 is --CH.sub.2--,
and otherwise R.sub.3 is selected from
##STR00061##
[0224] Without being bound by any theory, replacing His by a
non-aromatic amino acid, in particular a non-polar, small aliphatic
amino acid, such as Pro or Ala, or a polar uncharged or charged
amino acid, such as Lys, Asp, Gln and Asn, is believed to
contribute to attenuation of blood pressure effects. Thus, the His
position in the core sequence may for instance be replaced by Dab,
Dab(Acetyl), Ser, Met(O), Met(O.sub.2), Thr, Hyp, Gln, Pro, Ala,
Asn, Cit, Orn, Dap, Lys, or Arg, in particular Pro, Asn or Gln.
[0225] Thus, in a further embodiment of the invention, there are
provided peptides of Formula (I), in particular of Formula (II) or
(III), wherein R.sub.2 is --H or --CH.sub.2--, and if R.sub.2 is
--CH.sub.2-- forms with R.sub.3 a pyrrolidine ring, the pyrrolidine
ring optionally substituted with --OH; and R.sub.3 is
--(CH.sub.2).sub.2-- if R.sub.2 is --CH.sub.2--, and otherwise
R.sub.3 is selected from
##STR00062##
[0226] wherein w is in each instance independently selected from 0
to 5, and
[0227] z is in each instance independently selected from 1 to
5.
[0228] In a further embodiment of the invention, there are provided
peptides of Formula (I), in particular of Formula (II) or (III),
wherein R.sub.2 is --H or --CH.sub.2--, and if R.sub.2 is
--CH.sub.2-- forms with R.sub.3 a pyrrolidine ring, the pyrrolidine
ring optionally substituted with --OH; and R.sub.3 is
--(CH.sub.2).sub.2-- if R.sub.2 is --CH.sub.2--, and otherwise
R.sub.3 is selected from
##STR00063##
[0229] wherein w is in each instance independently selected from 0
to 5, and
[0230] z is in each instance independently selected from 1 to
5.
[0231] In a particular embodiment of the invention, there are
provided peptides of Formula (I), in particular of Formula (II) or
(III), wherein R.sub.2 is --H or --CH.sub.2--, and if R.sub.2 is
--CH.sub.2-- forms with R.sub.3 a pyrrolidine ring, the pyrrolidine
ring optionally substituted with --OH; and
[0232] R.sub.3 is --(CH.sub.2).sub.2-- if R.sub.2 is --CH.sub.2--,
and otherwise R.sub.3 is selected from
##STR00064##
[0233] wherein w is in each instance independently selected from 0
to 5, in particular 0 to 2, such as 0, and
[0234] z is in each instance independently selected from 1 to 5, in
particular 1 to 4.
[0235] In a further particular embodiment of the invention, there
are provided peptides of Formula (I), in particular of Formula (II)
or (III), wherein R.sub.2 is --H or --CH.sub.2--, and if R.sub.2 is
--CH.sub.2-- forms with R.sub.3 a pyrrolidine ring, the pyrrolidine
ring optionally substituted with --OH; and
[0236] R.sub.3 is --(CH.sub.2).sub.2-- if R.sub.2 is --CH.sub.2--,
and otherwise R.sub.3 is selected from
##STR00065##
[0237] wherein w is in each instance independently selected from 0
to 5, in particular 0 to 2, such as 0, and
[0238] z is in each instance independently selected from 1 to 5, in
particular 1 to 4.
[0239] In further embodiments of the peptides according to Formula
(I), and in particular Formula (II) or (III), R.sub.2 is H or
--CH.sub.2--, and if R.sub.2 is --CH.sub.2-- forms with R.sub.3 a
pyrrolidine ring, the pyrrolidine ring optionally substituted with
--OH; and
[0240] R.sub.3 is selected from
##STR00066##
[0241] In another embodiment of the invention, there are provided
peptides of Formula (I), in particular of Formula (II) or (III),
wherein R.sub.2 is H and R.sub.3 is selected from
##STR00067##
[0242] In another embodiment of the invention, there are provided
peptides of Formula (I), in particular of Formula (II) or (III),
wherein R.sub.2 is H and R.sub.3 is selected from
##STR00068##
[0243] In another embodiment of the invention, there are provided
peptides of Formula (I), and in particular Formula (II) or (III),
wherein R.sub.2 is --CH.sub.2-- and R.sub.3 is
--(CH.sub.2).sub.2--, R.sub.2 and R.sub.3 together forming an
unsubstituted pyrrolidine ring.
[0244] In still another embodiment of the invention, there are
provided peptides of Formula (I), and in particular Formula (II) or
(III), wherein R.sub.2 is --CH.sub.2-- and R.sub.3 is
--(CH.sub.2).sub.2--, R.sub.2 and R.sub.3 together forming an
pyrrolidine ring substituted with --OH. In a further embodiment of
the peptides according to Formula (I), and in particular Formula
(II) or (III), R.sub.5 is --NH.sub.2 or --OH.
[0245] In a specific embodiment of the peptides according to
Formula (I), and in particular Formula (II) or (III), R.sub.5 is
--NH.sub.2.
[0246] One or more of the above embodiments may be combined to
provide further specific embodiments of the peptides according to
the invention.
[0247] Thus, in a particular embodiment of the invention, there are
provided peptides according to Formula (I), in particular Formula
(II) or (III), wherein
[0248] R.sub.1 is --C(.dbd.O)--NH--, x is 2 and y is 3;
[0249] R.sub.2 is --H or --CH.sub.2--, and if R.sub.2 is
--CH.sub.2-- forms with R.sub.3 a pyrrolidine ring, the pyrrolidine
ring optionally substituted with --OH;
[0250] R.sub.3 is --(CH.sub.2).sub.2-- if R.sub.2 is --CH.sub.2--,
and otherwise R.sub.3 is selected from
##STR00069##
[0251] w is in each instance independently selected from 0 to
5;
[0252] z is in each instance independently selected from 1 to
5;
[0253] one or more of R.sub.4a, R.sub.4b and R.sub.4c are each
independently selected from [0254] fluoro, chloro,
##STR00070##
[0255] and
[0256] R.sub.5 is --NH.sub.2 or --OH.
[0257] Some specific peptides of the invention are one or more of
the following:
TABLE-US-00003 (SEQ ID NO: 6)
Ac-Arg-cyclo(Glu-Dab-D-Phe(2-Cl)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID NO:
7) Ac-Arg-cyclo(Glu-Dab-D-Phe(3-Cl)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID
NO: 8) Ac-Arg-cyclo(Glu-Dab-D-Phe(4-Cl)-Arg-Trp-Orn)-NH.sub.2; (SEQ
ID NO: 9) Ac-Arg-cyclo(Glu-Dab-D-Phe(2,4-diCl)-Arg-Trp-Orn)-
NH.sub.2; (SEQ ID NO: 10)
Ac-Arg-cyclo(Glu-Dab-D-Phe(3,4-diCl)-Arg-Trp-Orn)- NH.sub.2; (SEQ
ID NO: 11) Ac-Arg-cyclo(Glu-Dab-D-Phe(3-CN)-Arg-Trp-Orn)-NH.sub.2;
(SEQ ID NO: 12)
Ac-Arg-cyclo(Glu-Dab-D-Phe(4-CN)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID NO:
13) Ac-Arg-cyclo(Glu-Dab-D-Tyr-Arg-Trp-Orn)-NH.sub.2; (SEQ ID NO:
14) Ac-Arg-cyclo(Glu-Dab-D-Phe(2-F)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID
NO: 15) Ac-Arg-cyclo(Glu-Dab-D-Phe(3-F)-Arg-Trp-Orn)-NH.sub.2; (SEQ
ID NO: 16) Ac-Arg-cyclo(Glu-Dab-D-Phe(4-F)-Arg-Trp-Orn)-NH.sub.2;
(SEQ ID NO: 17)
Ac-Arg-cyclo(Glu-Dab-D-Phe(2-CN)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID NO:
18) Ac-Arg-cyclo(Glu-Dab-D-Phe(2-CF.sub.3)-Arg-Trp-Orn)-NH.sub.2;
(SEQ ID NO: 19)
Ac-Arg-cyclo(Glu-Dab-D-Phe(3-CF.sub.3)-Arg-Trp-Orn)-NH.sub.2; (SEQ
ID NO: 20) Ac-Arg-cyclo(Glu-Dab-D-Phe(4-Me)-Arg-Trp-Orn)-NH.sub.2;
(SEQ ID NO: 21)
Ac-Arg-cyclo(Glu-Dab-D-Phe(4-OMe)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID
NO: 22) Ac-Arg-cyclo(Glu-Dab-D-Phe(3,5-diF)-Arg-Trp-Orn)- NH.sub.2;
(SEQ ID NO: 23)
Ac-Arg-cyclo(Glu-Dab-D-Phe(2-Me)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID NO:
24) Ac-Arg-cyclo(Glu-Dab-D-Phe(3-Me)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID
NO: 25)
Ac-Arg-cyclo(Glu-Dab-D-Phe(4-CF.sub.3)-Arg-Trp-Orn)-NH.sub.2; (SEQ
ID NO: 26) Ac-Arg-cyclo(Glu-Dab-D-Phe(2,4-diMe)-Arg-Trp-Orn)-
NH.sub.2; (SEQ ID NO: 27)
Ac-Arg-cyclo(Glu-Dab-D-Phe(2-NO.sub.2)-Arg-Trp-Orn-NH.sub.2; (SEQ
ID NO: 28) Ac-Arg-cyclo(Glu-Dab-D-Phe(3-NO.sub.2)-Arg-Trp-Orn)-
NH.sub.2; (SEQ ID NO: 29)
Ac-Arg-cyclo(Glu-Dab-D-Phe(4-NO.sub.2)-Arg-Trp-Orn)- NH.sub.2; (SEQ
ID NO: 30) Ac-Arg-cyclo(Glu-Dab-D-Phe(3,4-diOMe)-Arg-Trp-
Orn)-NH.sub.2; (SEQ ID NO: 31)
Ac-Arg-cyclo(Glu-Dab-D-Phe(3,4-diF)-Arg-Trp-Orn)- NH.sub.2; (SEQ ID
NO: 32) Ac-Arg-cyclo(Glu-Dab-D-Phe(3,4,5-triF)-Arg-Trp-
Orn)-NH.sub.2; (SEQ ID NO: 33)
Ac-Arg-cyclo(Glu-Dab-D-Phe(4-NH.sub.2)-Arg-Trp-Orn)-NH.sub.2; (SEQ
ID NO: 35) Ac-Arg-cyclo(Glu-Dab-D-Phe(4-tBu)-Arg-Trp-Orn)-NH.sub.2;
(SEQ ID NO: 36)
Ac-Arg-cyclo(Glu-Ser-D-Phe(2-Cl)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID NO:
37) Ac-Arg-cyclo(Glu-Thr-D-Phe(2-Cl)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID
NO: 38) Ac-Arg-cyclo(Glu-Hyp-D-Phe(2-Cl)-Arg-Trp-Orn)-NH.sub.2;
(SEQ ID NO: 39)
Ac-Arg-cyclo(Glu-Gln-D-Phe(2-Cl)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID NO:
40) Ac-Arg-cyclo(Glu-Ser-D-Phe(3-Cl)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID
NO: 41) Ac-Arg-cyclo(Glu-Thr-D-Phe(3-Cl)-Arg-Trp-Orn)-NH.sub.2;
(SEQ ID NO: 42)
Ac-Arg-cyclo(Glu-Hyp-D-Phe(3-Cl)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID NO:
43) Ac-Arg-cyclo(Glu-Gln-D-Phe(3-Cl)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID
NO: 44) Ac-Arg-cyclo(Glu-Pro-D-Phe(4-CN)-Arg-Trp-Orn)-NH.sub.2;
(SEQ ID NO: 45)
Ac-Arg-cyclo(Glu-Hyp-D-Phe(4-CN)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID NO:
46) Ac-Arg-cyclo(Glu-Ala-D-Phe(4-CN)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID
NO: 48) Ac-Arg-cyclo(Glu-Asn-D-Phe(4-CN)-Arg-Trp-Orn)-NH.sub.2;
(SEQ ID NO: 49)
Ac-Arg-cyclo(Glu-Thr-D-Phe(4-CN)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID NO:
50) Ac-Arg-cyclo(Glu-Ser-D-Phe(4-CN)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID
NO: 51) Ac-Arg-cyclo(Glu-Gln-D-Phe(4-CN)-Arg-Trp-Orn)-NH.sub.2;
(SEQ ID NO: 54) Ac-Arg-cyclo(Glu-Orn-D-Phe(2-C1)-Arg-Trp-Orn)-OH;
(SEQ ID NO: 60) Ac-Arg-cyclo(Glu-Orn-D-Phe(3-C1)-Arg-Trp-Orn)-OH;
(SEQ ID NO: 61) Ac-Arg-cyclo(Glu-Dab(Acetyl)-D-Phe(2-Cl)-Arg-Trp-
Orn)-NH.sub.2; (SEQ ID NO: 63)
Ac-Arg-cyclo(Glu-Ser-D-Phe(2-F)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID NO:
64) Ac-Arg-cyclo(Glu-Gln-D-Phe(2-F)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID
NO: 65) Ac-Arg-cyclo(Glu-Hyp-D-Phe(4-F)-Arg-Trp-Orn)-NH.sub.2; (SEQ
ID NO: 66) Ac-Arg-cyclo(Glu-Dap-D-Phe(3-Cl)-Arg-Trp-Orn)-NH.sub.2;
(SEQ ID NO: 67)
Ac-Arg-cyclo(Glu-Dap-D-Phe(2-Cl)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID NO:
69) Ac-Arg-cyclo(Glu-Hyp-D-Phe(2-F)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID
NO: 70) Ac-Arg-cyclo(Glu-Ser-D-Phe(4-F)-Arg-Trp-Orn)-NH.sub.2; (SEQ
ID NO: 71) Ac-Arg-cyclo(Glu-Pro-D-Phe(2-F)-Arg-Trp-Orn)-NH.sub.2;
(SEQ ID NO: 72)
Ac-Arg-cyclo(Glu-Pro-D-Phe(4-F)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID NO:
73) Ac-Arg-cyclo(Glu-Met(O.sub.2)-D-Phe(4-F)-Arg-Trp-Orn)-
NH.sub.2; (SEQ ID NO: 74)
Ac-Arg-cyclo(Glu-Asn-D-Phe(4-F)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID NO:
78) Ac-Arg-cyclo(Glu-Asn-D-Phe(2-Me)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID
NO: 81) Ac-Arg-cyclo(Glu-Asn-D-Phe(3,5-diF)-Arg-Trp-Orn)- NH.sub.2;
(SEQ ID NO: 89)
Ac-Arg-cyclo(Glu-Asn-D-Phe(2-Cl)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID NO:
90) Ac-Arg-cyclo(Glu-Asn-D-Phe(4-CN)-Arg-Trp-Orn)-OH (SEQ ID NO:
91) Ac-Arg-cyclo(Glu-Gln-D-Phe(4-CN)-Arg-Trp-Orn)-OH; (SEQ ID NO:
92) Ac-Arg-cyclo(Glu-Dab-D-Phe(3-0Me)-Arg-Trp-Orn)- NH.sub.2; (SEQ
ID NO: 93) Ac-Arg-cyclo(Glu-Pro-D-Phe(2-Cl)-Arg-Trp-Orn)-NH.sub.2;
(SEQ ID NO: 97)
Ac-Arg-cyclo(Glu-Gln-D-Phe(4-F)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID NO:
121) Ac-Arg-cyclo(Glu-Asn-D-Phe(2-CF.sub.3)-Arg-Trp-Orn)- NH.sub.2;
(SEQ ID NO: 100) Ac-Arg-cyclo(Glu-Asn-D-Phe(3,4,5-triF)-Arg-Trp-
Orn)-NH.sub.2; (SEQ ID NO: 102)
Ac-Arg-cyclo(Glu-Gln-D-Phe(3,4,5-triF)-Arg-Trp- Orn)-NH.sub.2; (SEQ
ID NO: 104) Ac-Arg-cyclo(Glu-Arg-D-Phe(3-Cl)-Arg-Trp-Orn)-NH.sub.2;
(SEQ ID NO: 105)
Ac-Arg-cyclo(Glu-Lys-D-Phe(3-Cl)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID NO:
106) Ac-Arg-cyclo(Glu-Orn-D-Phe(3-Cl)-Arg-Trp-Orn)-NH.sub.2 (SEQ ID
NO: 107) Ac-Arg-cyclo(Glu-Pro-D-Phe(4-F)-Arg-Trp-Orn)-OH; (SEQ ID
NO: 111) Ac-Arg-cyclo(Glu-Asp-D-Phe(4-CN)-Arg-Trp-Orn)-NH.sub.2;
(SEQ ID NO: 112) Ac-Arg-cyclo(Glu-Asp-D-Phe(4-CN)-Arg-Trp-Orn)-OH;
(SEQ ID NO: 113)
Ac-Arg-cyclo(Glu-Glu-D-Phe(4-CN)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID NO:
114) Ac-Arg-cyclo(Glu-Glu-D-Phe(4-CN)-Arg-Trp-Orn)-OH; (SEQ ID NO:
115) Ac-Arg-cyclo(Glu-Gln-D-Phe(2-Me)-Arg-Trp-Orn)-NH.sub.2;
(SEQ ID NO: 119)
Ac-Arg-cyclo(Glu-Gln-D-Phe(2-CF.sub.3)-Arg-Trp-Orn)-NH.sub.2;
or a pharmaceutically acceptable salt of any of the foregoing.
[0258] Particularly, the invention relates to:
TABLE-US-00004 (SEQ ID NO: 48)
Ac-Arg-cyclo(Glu-Asn-D-Phe(4-CN)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID NO:
74) Ac-Arg-cyclo(Glu-Asn-D-Phe(4-F)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID
NO: 90) Ac-Arg-cyclo(Glu-Asn-D-Phe(4-CN)-Arg-Trp-Orn)-OH,
or a pharmaceutically acceptable salt of any of the foregoing.
[0259] Particularly, the invention also relates to:
TABLE-US-00005 (SEQ ID NO: 97)
Ac-Arg-cyclo(Glu-Gln-D-Phe(4-F)-Arg-Trp-Orn)-NH.sub.2, (SEQ ID NO:
51) Ac-Arg-cyclo(Glu-Gln-D-Phe(4-CN)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID
NO: 91) Ac-Arg-cyclo(Glu-Gln-D-Phe(4-CN)-Arg-Trp-Orn)-OH;
or a pharmaceutically acceptable salt of any of the foregoing.
[0260] Particularly, the invention also relates to:
TABLE-US-00006 (SEQ ID NO: 107)
Ac-Arg-cyclo(Glu-Pro-D-Phe(4-F)-Arg-Trp-Orn)-OH; (SEQ ID NO: 44)
Ac-Arg-cyclo(Glu-Pro-D-Phe(4-CN)-Arg-Trp-Orn)-NH.sub.2; (SEQ ID NO:
72) Ac-Arg-cyclo(Glu-Pro-D-Phe(4-F)-Arg-Trp-Orn)-NH.sub.2,
or a pharmaceutically acceptable salt of any of the foregoing.
[0261] Some further specific peptides of the invention are:
TABLE-US-00007 (SEQ ID NO: 75)
Ac-Arg-cyclo(Orn-Asn-D-Phe(4-F)-Arg-Trp-Glu)-NH.sub.2; (SEQ ID NO:
76) Ac-Arg-cyclo(Orn-Asn-D-Phe(2-F)-Arg-Trp-Glu)-NH.sub.2; (SEQ ID
NO: 77) Ac-Arg-cyclo(Orn-Asn-D-Phe(3-Me)-Arg-Trp-Glu)-NH.sub.2;
(SEQ ID NO: 79)
Ac-Arg-cyclo(Orn-Asn-D-Phe(2-Me)-Arg-Trp-Glu)-NH.sub.2; (SEQ ID NO:
80) Ac-Arg-cyclo(Orn-Asn-D-Phe(3,4-diF)-Arg-Trp-Glu)- NH.sub.2;
(SEQ ID NO: 82) Ac-Arg-cyclo(Orn-Asn-D-Phe(3,5-diF)-Arg-Trp-Glu)-
NH.sub.2; (SEQ ID NO: 96)
Ac-Arg-cyclo(Orn-Asn-D-Phe(4-CN)-Arg-Trp-Glu)-NH.sub.2; (SEQ ID NO:
99) Ac-Arg-cyclo(Orn-Gln-D-Phe(4-CN)-Arg-Trp-Glu)-NH.sub.2; (SEQ ID
NO: 101) Ac-Arg-cyclo(Orn-Asn-D-Phe(3,4,5-triF)-Arg-Trp-
Glu)-NH.sub.2; (SEQ ID NO: 103)
Ac-Arg-cyclo(Orn-Gln-D-Phe(3,4,5-triF)-Arg-Trp- Glu)-NH.sub.2; (SEQ
ID NO: 110) Ac-Arg-cyclo(Orn-Pro-D-Phe(2-F)-Arg-Trp-Glu)-NH.sub.2;
(SEQ ID NO: 116)
Ac-Arg-cyclo(Orn-Gln-D-Phe(2-Me)-Arg-Trp-Glu)-NH.sub.2; (SEQ ID NO:
117) Ac-Arg-cyclo(Orn-Gln-D-Phe(2-Cl)-Arg-Trp-Glu)-NH.sub.2; (SEQ
ID NO: 118) Ac-Arg-cyclo(Orn-Asn-D-Phe(2-Cl)-Arg-Trp-Glu)-NH.sub.2;
(SEQ ID NO: 120)
Ac-Arg-cyclo(Orn-Gln-D-Phe(2-CF.sub.3)-Arg-Trp-Glu)-NH.sub.2; (SEQ
ID NO: 122)
Ac-Arg-cyclo(Orn-Asn-D-Phe(2-CF.sub.3)-Arg-Trp-Glu)-NH.sub.2;
or a pharmaceutically acceptable salt of any of the foregoing.
[0262] 6.1 Specific Peptides.
[0263] Peptides of the following structures were synthesized by the
general methods described above, and except where indicated MC1-R
Ki and MC4-R Ki values for each peptide were determined in
competitive binding assays using [I.sup.125]-NDP-.alpha.-MSH as
described in 7.1 below. All peptides were prepared in the TFA acid
salt form, except for the peptides of Examples 43, 46 and 67, which
were prepared in the acetate salt form.
[0264] The syntheses of some specific peptides of the invention are
illustrated below. These peptides were prepared using solid phase
peptide synthesis by means of a Symphony Multiplex Peptide
Synthesizer (Rainin Instrument Company/Protein Technologies Inc)
automated peptide synthesizer.
Step 1: Coupling of Orn
[0265] The Sierber resin 9-Fmoc-Aminoxanthen-3-yloxy-polystyrene
resin (0.39 mol/g, ChemPep Inc., #151902) was swelled in 3.times.5
mL of DMF for 10 min. Thereafter, Fmoc was deprotected using
2.times.5 mL of 20% piperidine in DMF for 10 min. The resin was
then washed in 6.times.5 mL DMF for 30 sec. 5 mL of 200 mM
Fmoc-Orn(Alloc)-OH in DMF and 5 mL 200 mM HBTU containing 400 mM
NMM in DMF was added and after 30 min the resin was washed with
3.times.5 mL DMF for 30 sec.
Step 2: Coupling of Next 6 Amino Acids (AA)
[0266] The resin from step 1 was first swelled in 3.times.5 mL of
DMF for 30 sec, Fmoc was deprotected using 2.times.5 mL of 20%
piperidine in DMF for 10 min and then washed with 6.times.5 mL DMF
for 30 sec. 5 mL of 200 mM Fmoc-AA-OH solution and 5 mL 200 mM HBTU
containing 400 mM NMM in DMF was added and after 30 min the resin
was washed with 3.times.5 mL DMF for 30 sec.
[0267] This step was repeated for each amino acid (AA).
Step 3: Acetylation
[0268] Fmoc was deprotected using 2.times.5 mL of 20% piperidine in
DMF for 10 min and the resin was then washed with 3.times.5 mL DMF
for 30 sec. Thereafter, 5 mL of 50% Ac.sub.2O/DMF solution was
added and after 30 min the peptide resin was washed with 3.times.5
mL DMF for 30 sec and 6.times.5 mL DCM for 30 sec.
Step 4: Allyl/Alloc Deprotection
[0269] The peptide resin (0.6 mmol) was mixed with phenylsilane
(Oakwood Chemical, #S13600) (20 eq.) in 20 mL of DCM and bubbled
with nitrogen for 5 min.
[0270] Tetrakis(triphenylphosphine)-palladium(0) (Strem Chemicals,
Inc., #46-2150) (0.2 eq.) was added and the mixture was agitated
with nitrogen for 1 hour. The procedure was repeated one time for 1
hour and an additional time for 30 min with fresh reagents. The
treated peptide resin was then washed with DCM x 3 and DMF x 3.
Step 5: Lactam Formation
[0271] The lactam ring was formed on the peptide resin using TBTU
(2 eq.) and ethyldiisopropylamine (DIEA) (4 eq.) in 20 mL DMF for 1
hour. A second coupling may be needed if a positive Kaiser
Ninhydrin test is observed.
Step 6: Peptide Cleavage
[0272] The peptide resin (0.6 mmol) was mixed with 20 mL of 5%
sodium diethyldithio-carbamate trihydrate (NaCS.sub.2NEt.sub.2,
Aldrich, #228680) in DMF for 20 min and then washed with
DMF.times.3, DCM.times.3 and diethyl ether.times.2.
[0273] The resin (0.6 mmol) was then stirred in a 25 mL of
TFA/TIS/H2O (90:5.0:5.0 v/v/v) for 2.5 hours. The resin was
filtered. The filtrate was concentrated to about 10 mL in volume
and about 140 mL of cold diethyl ether (pre-cooled to about
0.degree. C.) was added.
[0274] The mixture was vortexed, and then placed in the
refrigerator (about -4.degree. C.) for 1 h, centrifuged for 5 min
at 2800 rpm, and the ether layer was decanted.
[0275] The peptide was washed with 90 mL of cold diethyl ether
(pre-cooled to about 0.degree. C.), vortexed, centrifuged for 5 min
at 2800 rpm, and ether layer decanted.
[0276] The resulting solid was dissolved in 50% AcOH/H.sub.2O and
stored at room temperature overnight.
[0277] The crude peptide solution was concentrated to afford solid
crude peptide for HPLC purification.
[0278] After HPLC purification, the peptide TFA salt was converted
to peptide acetate salt using ion exchange (.times.100 eq.). The
anion exchange resin used was Dowex SBR LC NG, OH-form (Supelco,
Cat#14036-U).
Example 46
TABLE-US-00008 [0279] (SEQ ID NO: 51)
Ac-Arg-cyclo(Glu-Gln-D-Phe(4-CN)-Arg-Trp-Orn)-NH.sub.2
[0280] The procedure described above was followed in the
preparation of the title peptide.
[0281] The amino acids added in step 2 were, in the order of being
coupled, Fmoc-Trp(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-D-Phe(4-CN)--OH,
Fmoc-Gln(Trt)-OH, Fmoc-Glu(OAll)-OH, and Fmoc-Arg(Pbf)-OH.
[0282] The resulting peptide was purified by HPLC (column: Atlantis
dC18 OBD.TM. 19.times.100 mm (5.mu.., Waters part #186001367) using
10% MeOH/H.sub.2O containing 0.1% TFA (solvent A) and 90%
MeOH/H.sub.2O containing 0.1% TFA (solvent B). A gradient of 0%-5%
of solvent B for 5 min and 5%-35% of solvent B for 30 min was
used.
[0283] The peptide yield was 10%.
Example 67
TABLE-US-00009 [0284] (SEQ ID NO: 72)
Ac-Arg-cyclo(Glu-Pro-D-Phe(4-F)-Arg-Trp-Orn)-NH.sub.2
[0285] The procedure described above was followed in the
preparation of the title peptide except for that 15 mL of 5% sodium
diethyldithio-carbamate trihydrate in DMF, 16 mL of
TFA/TIS/H.sub.2O and 90 mL+60 mL of diethyl ether were used in step
6. Moreover, the filtrate was concentrated to 5 mL.
[0286] The amino acids added in step 2 were, in the order of being
coupled, Fmoc-Trp(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-D-Phe(4-F)--OH,
Fmoc-Pro-OH, Fmoc-Glu(OAll)-OH, and Fmoc-Arg(Pbf)-OH.
[0287] The resulting peptide was purified by HPLC (column: Atlantis
dC18 OBD.TM. 19.times.100 mm (5.mu.., Waters part #186001367) using
10% MeOH/H.sub.2O containing 0.1% TFA (solvent A) and 90%
MeOH/H.sub.2O containing 0.1% TFA (solvent B). A gradient of 5%-10%
of solvent B for 5 min and 10%-40% of solvent B for 30 min was
used.
[0288] The peptide yield was 16%.
TABLE-US-00010 MC4- MC1- R Ki R Ki No. Structure Amino Acid
Sequence (nM) (nM) 1 ##STR00071## Ac-Arg-cyclo(Glu-Dab-
D-Phe(2-Cl)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO: 6) 3 1 2
##STR00072## Ac-Arg-cyclo(Glu-Dab- D-Phe(3-Cl)-Arg-Trp-
Orn)-NH.sub.2 (SEQ ID NO: 7) 0.75 1 3 ##STR00073##
Ac-Arg-cyclo(Glu-Dab- D-Phe(4-Cl)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID
NO: 8) 0.08 0.3 4 ##STR00074## Ac-Arg-cyclo(Glu-Dab-
D-Phe(2,4-diCl)-Arg- Trp-Orn)-NH.sub.2 (SEQ ID NO: 9) 0.055 0.5 5
##STR00075## Ac-Arg-cyclo(Glu-Dab- D-Phe(3,4-diCl)-Arg-
Trp-Orn)-NH.sub.2 (SEQ ID NO: 10) 0.055 0.2 6 ##STR00076##
Ac-Arg-cyclo(Glu-Dab- D-Phe(3-CN)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID
NO: 11) 33 43 7 ##STR00077## Ac-Arg-cyclo(Glu-Dab-
D-Phe(4-CN)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO: 12) 0.833 5 8
##STR00078## Ac-Arg-cyclo(Glu-Dab- D-Tyr-Arg-Trp-Orn)- NH.sub.2
(SEQ ID NO: 13) 75 15 9 ##STR00079## Ac-Arg-cyclo(Glu-Dab-
D-Phe(2-F)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO: 14) 0.7 0.8 10
##STR00080## Ac-Arg-cyclo(Glu-Dab- D-Phe(3-F)-Arg-Trp-
Orn)-NH.sub.2 (SEQ ID NO: 15 2 2 11 ##STR00081##
Ac-Arg-cyclo(Glu-Dab- D-Phe(4-F)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO:
16) 0.25 0.5 12 ##STR00082## Ac-Arg-cyclo(Glu-Dab-
D-Phe(2-CN)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO: 17) 45 35 13
##STR00083## Ac-Arg-cyclo(Glu-Dab- D-Phe(2-CF.sub.3)-Arg-Trp-
Orn)-NH.sub.2 (SEQ ID NO: 18) 0.6 3 14 ##STR00084##
Ac-Arg-cyclo(Glu-Dab- D-Phe(3-CF.sub.3)-Arg-Trp- Orn)-NH.sub.2 (SEQ
ID NO: 19) 0.95 9 15 ##STR00085## Ac-Arg-cyclo(Glu-Dab-
D-Phe(4-Me)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO: 20) 0.145 0.75 16
##STR00086## Ac-Arg-cyclo(Glu-Dab- D-Phe(4-OMe)-Arg-
Trp-Orn)-NH.sub.2 (SEQ ID NO: 21) 0.25 0.15 17 ##STR00087##
Ac-Arg-cyclo(Glu-Dab- D-Phe(3,5-diF)-Arg- Trp-Orn)-NH.sub.2 (SEQ ID
NO: 22) 2 -- 18 ##STR00088## Ac-Arg-cyclo(Glu-Dab-
D-Phe(2-Me)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO: 23) 0.5 -- 19
##STR00089## Ac-Arg-cyclo(Glu-Dab- D-Phe(3-Me)-Arg-Trp-
Orn)-NH.sub.2 (SEQ ID NO: 24) 0.4 -- 20 ##STR00090##
Ac-Arg-cyclo(Glu-Dab- D-Phe(4-CF.sub.3)-Arg-Trp- Orn)-NH.sub.2 (SEQ
ID NO: 25) 0.06 -- 21 ##STR00091## Ac-Arg-cyclo(Glu-Dab-
D-Phe(2,4-diMe)-Arg- Trp-Orn)-NH.sub.2 (SEQ ID NO: 26) 0.085 -- 22
##STR00092## Ac-Arg-cyclo(Glu-Dab- D-Phe(2-NO.sub.2)-Arg-Trp-
Orn-NH.sub.2 (SEQ ID NO: 27) 11 -- 23 ##STR00093##
Ac-Arg-cyclo(Glu-Dab- D-Phe(3-NO.sub.2)-Arg-Trp- Orn)-NH.sub.2 (SEQ
ID NO: 28) 0.7 -- 24 ##STR00094## Ac-Arg-cyclo(Glu-Dab-
D-Phe(4-NO.sub.2)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO: 29) 0.45 -- 25
##STR00095## Ac-Arg-cyclo(Glu-Dab- D-Phe(3,4-diOMe)-Arg-
Trp-Orn)-NH.sub.2 (SEQ ID NO: 30) 35 -- 26 ##STR00096##
Ac-Arg-cyclo(Glu-Dab- D-Phe(3,4-diF)-Arg- Trp-Orn)-NH.sub.2 (SEQ ID
NO: 31) 0.3 -- 27 ##STR00097## Ac-Arg-cyclo(Glu-Dab-
D-Phe(3,4,5-triF)-Arg- Trp-Orn)-NH.sub.2 (SEQ ID NO: 32) 0.667 --
28 ##STR00098## Ac-Arg-cyclo(Glu-Dab- D-Phe(4-NH.sub.2)-Arg-Trp-
Orn)-NH.sub.2 (SEQ ID NO: 33) 48 -- 29 ##STR00099##
Ac-Arg-cyclo(Glu-Dab- D-Phe(4-Ph)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID
NO: 34) 0.5 -- 30 ##STR00100## Ac-Arg-cyclo(Glu-Dab-
D-Phe(4-tBu)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO: 35) 4 -- 31
##STR00101## Ac-Arg-cyclo(Glu-Ser- D-Phe(2-Cl)-Arg-Trp-
Orn)-NH.sub.2 (SEQ ID NO: 36) 8 300 32 ##STR00102##
Ac-Arg-cyclo(Glu-Thr- D-Phe(2-Cl)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID
NO: 37) 11 540 33 ##STR00103## Ac-Arg-cyclo(Glu-Hyp-
D-Phe(2-Cl)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO: 38) 1 143 34
##STR00104## Ac-Arg-cyclo(Glu-Gln- D-Phe(2-Cl)-Arg-Trp-
Orn)-NH.sub.2 (SEQ ID NO: 39) 7 128 35 ##STR00105##
Ac-Arg-cyclo(Glu-Ser- D-Phe(3-Cl)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID
NO: 40) 10 810 36 ##STR00106## Ac-Arg-cyclo(Glu-Thr-
D-Phe(3-Cl)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO: 41) 6 380 37
##STR00107## Ac-Arg-cyclo(Glu-Hyp- D-Phe(3-Cl)-Arg-Trp-
Orn)-NH.sub.2 (SEQ ID NO: 42) 2 430 38 ##STR00108##
Ac-Arg-cyclo(Glu-Gln- D-Phe(3-Cl)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID
NO: 43) 3 290 39 ##STR00109## Ac-Arg-cyclo(Glu-Pro-
D-Phe(4-CN)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO: 44) 4 1550 40
##STR00110## Ac-Arg-cyclo(Glu-Hyp- D-Phe(4-CN)-Arg-Trp-
Orn)-NH.sub.2 (SEQ ID NO: 45) 2 880 41 ##STR00111##
Ac-Arg-cyclo(Glu-Ala- D-Phe(4-CN)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID
NO: 46) 3 4300 42 ##STR00112## Ac-Arg-cyclo(Glu-Val-
D-Phe(4-CN)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO: 47) 6 420 43
##STR00113## Ac-Arg-cyclo(Glu-Asn- D-Phe(4-CN)-Arg-Trp-
Orn)-NH.sub.2 (SEQ ID NO: 48) 5 870 44 ##STR00114##
Ac-Arg-cyclo(Glu-Thr- D-Phe(4-CN)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID
NO: 49) 18 1625 45 ##STR00115## Ac-Arg-cyclo(Glu-Ser-
D-Phe(4-CN)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO: 50) 10 10000 46
##STR00116## Ac-Arg-cyclo(Glu-Gln- D-Phe(4-CN)-Arg-Trp-
Orn)-NH.sub.2 (SEQ ID NO: 51) 4 425 47 ##STR00117##
Ac-Arg-cyclo(Glu- Ser(Bzl)-D-Phe(2-Cl)- Arg-Trp-Orn)-NH.sub.2 (SEQ
ID NO: 52) 0.3 25 48 ##STR00118## Ac-Arg-cyclo(Glu-
Phe(3-C(.dbd.O)--NH.sub.2)-D- Phe(2-Cl)-D-Phe-Arg-
Trp-Orn)-NH.sub.2 (SEQ ID NO: 53) 15 545 49 ##STR00119##
Ac-Arg-cyclo(Glu-Orn- D-Phe(2-Cl)-Arg-Trp- Orn)-OH (SEQ ID NO: 54)
8 8 50 ##STR00120## Ac-D-Arg-cyclo(Orn- His-D-Phe(2-Cl)-Arg-
Trp-Glu)-NH.sub.2 (SEQ ID NO: 55) 1 2 51 ##STR00121##
Ac-D-Arg-cyclo(Orn- His-D-Phe(3-Cl)-Arg- Trp-Glu)-NH.sub.2 (SEQ ID
NO: 56) 0.6 3 52 ##STR00122## Ac-D-Arg-cyclo(Orn-
His-D-Phe(4-CN)-Arg- Trp-Glu)-NH.sub.2 (SEQ ID NO: 57) 0.6 6 53
##STR00123## Ac-D-Arg-cyclo(Orn- His-D-Phe(2-F)-Arg-
Trp-Glu)-NH.sub.2 (SEQ ID NO: 58) 0.95 0.75 54 ##STR00124##
Ac-D-Arg-cyclo(Orn- His-D-Phe(3,4-diF)-Arg- Trp-Glu)-NH.sub.2 (SEQ
ID NO: 59) 0.25 0.7 55 ##STR00125## Ac-Arg-cyclo(Glu-Orn-
D-Phe(3-Cl)-Arg-Trp- Orn)-OH (SEQ ID NO: 60) 20 23 56 ##STR00126##
Ac-Arg-cyclo(Glu- Dab(Acetyl)-D-Phe(2- Cl)-Arg-Trp-Orn)-NH.sub.2
(SEQ ID NO: 61) 11 30 57 ##STR00127## Ac-D-Arg-cyclo(Orn-
His-D-Phe(4-F)-Arg- Trp-Glu)-NH.sub.2 (SEQ ID NO: 62) 0.65 1 58
##STR00128## Ac-Arg-cyclo(Glu-Ser- D-Phe(2-F)-Arg-Trp-
Orn)-NH.sub.2 (SEQ ID NO: 63) 15 138 59 ##STR00129##
Ac-Arg-cyclo(Glu-Gln- D-Phe(2-F)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO:
64) 7 58 60 ##STR00130## Ac-Arg-cyclo(Glu-Hyp- D-Phe(4-F)-Arg-Trp-
Orn)-NH.sub.2 (SEQ ID NO: 65) 1 89 61 ##STR00131##
Ac-Arg-cyclo(Glu-Dap- D-Phe(3-Cl)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID
NO: 66) 4 25 62 ##STR00132## Ac-Arg-cyclo(Glu-Dap-
D-Phe(2-Cl)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO: 67) 9 28 63
##STR00133## Ac-Arg-cyclo(Glu- Thr(Bzl)-D-Phe(2-Cl)-
Arg-Trp-Orn)-NH.sub.2 (SEQ ID NO: 68) 0.35 12 64 ##STR00134##
Ac-Arg-cyclo(Glu-Hyp- D-Phe(2-F)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO:
69) 3 53 65 ##STR00135## Ac-Arg-cyclo(Glu-Ser- D-Phe(4-F)-Arg-Trp-
Orn)-NH.sub.2 (SEQ ID NO: 70) 5 43 66 ##STR00136##
Ac-Arg-cyclo(Glu-Pro- D-Phe(2-F)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO:
71) 6 95 67 ##STR00137## Ac-Arg-cyclo(Glu-Pro- D-Phe(4-F)-Arg-Trp-
Orn)-NH.sub.2 (SEQ ID NO: 72) 5 130 68 ##STR00138##
Ac-Arg-cyclo(Glu- Met(O.sub.2)-D-Phe(4-F)- Arg-Trp-Orn)-NH.sub.2
(SEQ ID NO: 73) 2 33 69 ##STR00139## Ac-Arg-cyclo(Glu-Asn-
D-Phe(4-F)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO: 74) 3 150 70
##STR00140## Ac-Arg-cyclo(Orn-Asn- D-Phe(4-F)-Arg-Trp-
Glu)-NH.sub.2 (SEQ ID NO: 75) 3 170 71 ##STR00141##
Ac-Arg-cyclo(Orn-Asn- D-Phe(2-F)-Arg-Trp- Glu)-NH.sub.2 (SEQ ID NO:
76) 9 335 72 ##STR00142## Ac-Arg-cyclo(Orn-Asn-
D-Phe(3-Me)-Arg-Trp- Glu)-NH.sub.2 (SEQ ID NO: 77) 4 2300 73
##STR00143## Ac-Arg-cyclo(Glu-Asn- D-Phe(2-Me)-Arg-Trp-
Orn)-NH.sub.2 (SEQ ID NO: 78) 4 160 74 ##STR00144##
Ac-Arg-cyclo(Orn-Asn- D-Phe(2-Me)-Arg-Trp- Glu)-NH.sub.2 (SEQ ID
NO: 79) 5 405 75 ##STR00145## Ac-Arg-cyclo(Orn-Asn-
D-Phe(3,4-diF)-Arg- Trp-Glu)-NH.sub.2 (SEQ ID NO: 80) 3 190 76
##STR00146## Ac-Arg-cyclo(Glu-Asn- D-Phe(3,5-diF)-Arg-
Trp-Orn)-NH.sub.2 (SEQ ID NO: 81) 20 260 77 ##STR00147##
Ac-Arg-cyclo(Orn-Asn- D-Phe(3,5-diF)-Arg- Trp-Glu)-NH.sub.2 (SEQ ID
NO: 82) 8 190 78 ##STR00148## Ac-D-Arg-cyclo(Asp-
Gln-D-Phe(3,4-diF)- Arg-Trp-Lys)-NH.sub.2 (SEQ ID NO: 83) 8 515 79
##STR00149## Ac-D-Arg-cyclo(Asp- Gln-D-Phe(2-Cl)-Arg-
Trp-Lys)-NH.sub.2 (SEQ ID NO: 84) 10 395 80 ##STR00150##
Ac-D-Arg-cyclo(Asp- Gln-D-Phe(3-Cl)-Arg- Trp-Lys)-NH.sub.2 (SEQ ID
NO: 85) 7 1750 81 ##STR00151## Ac-D-Arg-cyclo(Asp-
Gln-D-Phe(4-CN)-Arg- Trp-Lys)-NH.sub.2 (SEQ ID NO: 86) 13 10000 82
##STR00152## Ac-D-Arg-cyclo(Asp- Gln-D-Phe(2-F)-Arg-
Trp-Lys)-NH.sub.2 (SEQ ID NO: 87) 10 220 83 ##STR00153##
Ac-D-Arg-cyclo(Asp- Gln-D-Phe(4-F)-Arg- Trp-Lys)-NH.sub.2 (SEQ ID
NO: 88) 6 260 84 ##STR00154## Ac-Arg-cyclo(Glu-Asn-
D-Phe(2-Cl)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO: 89) 7 245 85
##STR00155## Ac-Arg-cyclo(Glu-Asn- D-Phe(4-CN)-Arg-Trp- Orn)-OH
(SEQ ID NO: 90) 64 10000 86 ##STR00156## Ac-Arg-cyclo(Glu-Gln-
D-Phe(4-CN)-Arg-Trp- Orn)-OH (SEQ ID NO: 91) 40 3968 87
##STR00157## Ac-Arg-cyclo(Glu-Dab- D-Phe(3-OMe)-Arg-
Trp-Orn)-NH.sub.2 (SEQ ID NO: 92) 3 -- 88 ##STR00158##
Ac-Arg-cyclo(Glu-Pro- D-Phe(2-Cl)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID
NO: 93) 5 -- 89 ##STR00159## Ac-Arg-cyclo(Asp-Asn-
D-Phe(4-CN)-Arg-Trp- Lys)-NH.sub.2 (SEQ ID NO: 94) 48 -- 90
##STR00160## Ac-Arg-cyclo(Asp-Gln- D-Phe(4-CN)-Arg-Trp-
Lys)-NH.sub.2 (SEQ ID NO: 95) 5 -- 91 ##STR00161##
Ac-Arg-cyclo(Orn-Asn- D-Phe(4-CN)-Arg-Trp- Glu)-NH.sub.2 (SEQ ID
NO: 96) 4 -- 92 ##STR00162## Ac-Arg-cyclo(Glu-Gln-
D-Phe(4-F)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO: 97) 2 -- 93
##STR00163## Ac-Arg-cyclo(Lys-Gln- D-Phe(4-CN)-Arg-Trp-
Asp)-NH.sub.2 (SEQ ID NO: 98) 1 -- 94 ##STR00164##
Ac-Arg-cyclo(Orn-Gln- D-Phe(4-CN)-Arg-Trp- Glu)-NH.sub.2 (SEQ ID
NO: 99) 0.9 -- 95 ##STR00165## Ac-Arg-cyclo(Glu-Asn-
D-Phe(3,4,5-triF)-Arg- Trp-Orn)-NH.sub.2 (SEQ ID NO: 100) 3 -- 96
##STR00166## Ac-Arg-cyclo(Orn-Asn- D-Phe(3,4,5-triF)-Arg-
Trp-Glu)-NH.sub.2 (SEQ ID NO: 101) 2 -- 97 ##STR00167##
Ac-Arg-cyclo(Glu-Gln- D-Phe(3,4,5-triF)-Arg- Trp-Orn)-NH.sub.2 (SEQ
ID NO: 102) 0.75 -- 98 ##STR00168## Ac-Arg-cyclo(Orn-Gln-
D-Phe(3,4,5-triF)-Arg- Trp-Glu)-NH.sub.2 (SEQ ID NO: 103) 0.75 --
99 ##STR00169## Ac-Arg-cyclo(Glu-Arg- D-Phe(3-Cl)-Arg-Trp-
Orn)-NH.sub.2 (SEQ ID NO: 104) 0.5 -- 100 ##STR00170##
Ac-Arg-cyclo(Glu-Lys- D-Phe(3-Cl)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID
NO: 105) 4 -- 101 ##STR00171## Ac-Arg-cyclo(Glu-Orn-
D-Phe(3-Cl)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO: 106) 1 -- 102
##STR00172## Ac-Arg-cyclo(Glu-Pro- D-Phe(4-F)-Arg-Trp- Orn)-OH (SEQ
ID NO: 107) 30 730 103 ##STR00173## Ac-Arg-cyclo(Asp-Pro-
D-Phe(4-F)-Arg-Trp- Lys)-NH.sub.2 (SEQ ID NO: 108) 4 -- 104
##STR00174## Ac-Arg-cyclo(Asp-Pro- D-Phe(2-F)-Arg-Trp-
Lys)-NH.sub.2 (SEQ ID NO: 109) 78 -- 105 ##STR00175##
Ac-Arg-cyclo(Orn-Pro- D-Phe(2-F)-Arg-Trp- Glu)-NH.sub.2 (SEQ ID NO:
110) 7 -- 106 ##STR00176## Ac-Arg-cyclo(Glu-Asp-
D-Phe(4-CN)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO: 111) 647 10000 107
##STR00177## Ac-Arg-cyclo(Glu-Asp- D-Phe(4-CN)-Arg-Trp- Orn)-OH
(SEQ ID NO: 112) 6463 10000 108 ##STR00178## Ac-Arg-cyclo(Glu-Glu-
D-Phe(4-CN)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO: 113) 128 10000 109
##STR00179## Ac-Arg-cyclo(Glu-Glu- D-Phe(4-CN)-Arg-Trp- Orn)-OH
(SEQ ID NO: 114) 1090 10000 110 ##STR00180## Ac-Arg-cyclo(Glu-Gln-
D-Phe(2-Me)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO: 115) 5 105 111
##STR00181## Ac-Arg-cyclo(Orn-Gln- D-Phe(2-Me)-Arg-Trp-
Glu)-NH.sub.2 (SEQ ID NO: 116) 6 145 112 ##STR00182##
Ac-Arg-cyclo(Orn-Gln- D-Phe(2-Cl)-Arg-Trp- Glu)-NH.sub.2 (SEQ ID
NO: 117) 4 83 113 ##STR00183## Ac-Arg-cyclo(Orn-Asn-
D-Phe(2-Cl)-Arg-Trp- Glu)-NH.sub.2 (SEQ ID NO: 118) 7 255 114
##STR00184## Ac-Arg-cyclo(Glu-Gln- D-Phe(2-CF.sub.3)-Arg-Trp-
Orn)-NH.sub.2 (SEQ ID NO: 119) 8 93 115 ##STR00185##
Ac-Arg-cyclo(Orn-Gln- D-Phe(2-CF.sub.3)-Arg-Trp- Glu)-NH.sub.2 (SEQ
ID NO: 120) 4 235 116 ##STR00186## Ac-Arg-cyclo(Glu-Asn-
D-Phe(2-CF.sub.3)-Arg-Trp- Orn)-NH.sub.2 (SEQ ID NO: 121) 7 380 117
##STR00187## Ac-Arg-cyclo(Orn-Asn- D-Phe(2-CF.sub.3)-Arg-Trp-
Glu)-NH.sub.2 (SEQ ID NO: 122) 5 540
7.0 Tests and Assays Employed in Evaluation of the Peptides of the
Present Invention
[0289] The melanocortin receptor-specific peptides of this
invention may be tested by a variety of assay systems and animal
models to determine binding, functional status and efficacy.
[0290] 7.1 Competitive Inhibition Assay Using
[I.sup.125]-NDP-.alpha.-MSH.
[0291] A competitive inhibition binding assay was performed for
exemplified peptides according to the invention using membrane
homogenates prepared from HEK-293 cells that express recombinant
hMC4-R, hMC3-R, or hMC5-R, and from B-16 mouse melanoma cells
(containing endogenous MC1-R). In some instances, HEK-293 cells
that express recombinant hMC1-R were employed. Assays were
performed in 96 well GF/B Millipore multiscreen filtration plates
(MAFB NOB10) pre-coated with 0.5% bovine serum albumin (Fraction
V). Membrane homogenates were incubated with 0.2 nM (for hMC4-R)
0.4 nM (for MC3-R and MC5-R) or 0.1 nM (for mouse B16 MC1-R or
hMC1-R) [I.sup.125]-NDP-.alpha.-MSH (Perkin Elmer) and increasing
concentrations of test peptides of the present invention in buffer
containing 25 mM HEPES buffer (pH 7.5) with 100 mM NaCl, 2 mM
CaCl.sub.2, 2 mM MgCl.sub.2, 0.3 mM 1,10-phenanthroline, and 0.2%
bovine serum albumin. After incubation for 60 minutes at 37.degree.
C., the assay mixture was filtered and the membranes washed three
times with ice-cold buffer. Filters were dried and counted in a
gamma counter for bound radioactivity. Non-specific binding was
measured by inhibition of binding of [I.sup.125]-NDP-.alpha.-MSH in
the presence of 1 .mu.M NDP-.alpha.-MSH. Maximal specific binding
(100%) was defined as the difference in radioactivity (cpm) bound
to cell membranes in the absence and presence of 1 .mu.M
NDP-.alpha.-MSH. Radioactivity (cpm) obtained in the presence of
test peptides was normalized with respect to 100% specific binding
to determine the percent inhibition of [I.sup.125]-NDP-.alpha.-MSH
binding. Each assay was conducted in triplicate and the actual mean
values are described, with results less than 0% reported as 0%. Ki
values for test peptides of the present invention were determined
using Graph-Pad Prism.RTM. curve-fitting software. Results from
this assay are presented herein (see 6.1). For some peptides, a
mean value of more than two values is presented.
[0292] 7.2 Assay for Agonist Activity.
[0293] Accumulation of intracellular cAMP was examined as a measure
of the ability of peptides of the present invention to elicit a
functional response in HEK-293 cells that express MC4-R. Confluent
HEK-293 cells that express recombinant hMC4-R were detached from
culture plates by incubation in enzyme-free cell dissociation
buffer. Dispersed cells were suspended in Earle's Balanced Salt
Solution containing 10 mM HEPES (pH 7.5), 1 mM MgCl.sub.2, 1 mM
glutamine, 0.5% albumin and 0.3 mM 3-isobutyl-1-methyl-xanthine
(IBMX), a phosphodiesterase inhibitor. The cells were plated in
96-well plates at a density of 0.5.times.10.sup.5 cells per well
and pre-incubated for 10 minutes. Cells were exposed for 15 minutes
at 37.degree. C. to peptides of the present invention dissolved in
DMSO (final DMSO concentration of 1%) at a concentration range of
0.05-5000 nM in a total assay volume of 200 .mu.L. NDP-.alpha.-MSH
was used as the reference agonist. cAMP levels were determined by
an HTRF.RTM. cAMP cell-based assay system from Cisbio Bioassays
utilizing cryptate-labeled anti-cAMP and d2-labeled cAMP, with
plates read on a Perkin-Elmer Victor plate reader at 665 and 620
nM. Data analysis was performed by nonlinear regression analysis
with Graph-Pad Prism.RTM. software. The maximum efficacies of test
peptides of the present invention were compared to that achieved by
the reference melanocortin agonist NDP-.alpha.-MSH, used as full
agonist benchmark in this context.
[0294] 7.3 High and Low Density hMC4-R Functional Assay.
[0295] A HEK293 cell line transfected with human MC4-R (Palatin
Technologies, US, with license from the University of Michigan) was
used. The human MC4-R was introduced to HEK293 by using the
T-REx.TM. System, Invitrogen. The T-REx.TM. System employs a
tetracycline-regulated mammalian expression system that uses
regulatory elements from the E. coli Tn10-encoded tetracycline
(Tet) resistance operon. By use of the T-REx.TM. System, expression
of the gene of interest, the human MC4-R gene, is repressed in the
absence of tetracycline or doxycycline and induced in the presence
of tetracycline or doxycycline (see T-REx.TM. System Manual,
published by Invitrogen).
[0296] HEK293-T-REx-MC4-R cells were cultured in DMEM (Gibco
11965), supplemented with L-Glutamine (Gibco 25030), 10% fetal
bovine serum (FBS), 200 .mu.g/mL Zeocin (Invitrogen 46-0072) and 6
.mu.g/mL Blasticidin (Invitrogen 46-1120) in 5% CO.sub.2 and 95%
humidity at 37.degree. C. T-150 flasks of cells at 75% confluence
were incubated with two concentrations of doxycycline (0.1 ng/mL to
provide a low density hMC4-R system and 10 ng/mL to provide a high
density hMC4-R system) in 5% CO.sub.2 at 37.degree. C. for 16-18
hours to induce MC4-R expression. On the day of the assay, the
cells were washed with PBS (Gibco 14190) and harvested using cell
dissociation buffer (Gibco 13150-016), then centrifuged and
resuspended in Hanks' Balanced Salt Solution (+Ca, +Mg) (Gibco
14025), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES) (pH 7.4) (Sigma H0887), 1 mM L-Glutamine (Gibco 25030), 1
mg/mL bovine serum albumin (BSA) (Sigma A3311) and 0.3 mM
3-isobutyl-1-methyl-xanthine (IBMX). Cells were then counted and
volume was adjusted to 2.5.times.10.sup.5 cells per ml.
[0297] The cells were then dispensed into 96-well plates (BD
353916) in 198 .mu.L (about 5.times.10.sup.4) cells/well and
incubated for 10 minutes at 37.degree. C. The compound to be tested
was diluted with DMSO to a final concentration of 1 mM. Serial
dilution was prepared in polypropylene removable 12-well library
tube strips (VWR cat#83009-682). 120 .mu.L of the 1 mM compound
stock was pipetted in the second column on the plate. Using the
Janus liquid handler the compound was serially diluted 1:10 (25
.mu.L compound+225 .mu.L DMSO) to a total of 9 concentrations
(representing final assay concentrations ranging from 10.sup.-5 to
10.sup.13 M).
[0298] 2 .mu.L of the standard, [Nle.sup.4,
D-Phe.sup.7]-alpha-Melanocyte Stimulating Hormone
(NDP-.alpha.-MSH), or compound was added to the 96-well plate using
the Janus robotic system. All assay samples were run in duplicate
(i.e. each sample was in two low dox and two high dox plates,
respectively). The plates were gently shaken and incubated for 15
minutes at 37.degree. C. The reaction was stopped by adding 15
.mu.L of lysis buffer per well and the plates were shakened for 30
minutes at room temperature.
[0299] Agonist stimulation of the MC4-R activates adenylate
cyclase, which is an enzyme that catalyses the formation
3',5'-cyclic adenosine monophosphate (cAMP) from adenosine
triphosphate (ATP). Thus, agonist stimulation of the MC4-R
increases the levels of cAMP. cAMP-levels were measured with the
cAMP dynamic 2 HTRF kit (CisBio cat#62AM4PEC; see manual published
by CisBio). cAMP levels were normalized against plate controls (1%
DMSO for 0%, 400 nM NDP-.alpha.-MSH for 100%) and a calibration
curve ranging from 712 nM to 0.04 nM cAMP (essentially as described
in the CisBio HTRF kit). The plates were incubated on a shaker at
room temperature for 1 hour and read on the Perkin-Elmer Victor
plate reader at 665 and 620 nm. Fluorescence ratios were then
calculated as described in the CisBio HTRF kit, with GraphPad Prism
software used to plot the change in fluorescence percent values
versus cAMP concentration using the variable slope dose response
curve and, based on calculated cAMP concentrations, to determine
EC.sub.50 values and percent activation. Results from this assay
are presented herein (see 8.0).
[0300] 7.4 Food Intake and Body Weight Change.
[0301] Change in food intake and body weight was evaluated for
selected peptides administered by subcutaneous injection route.
Male Sprague-Dawley rats were obtained from Hilltop Lab Animals,
Inc. (Scottdale, Pa.). Animals were individually housed in
conventional polystyrene hanging cages and maintained on a
controlled 12 hour on/off light cycle. Water and pelleted food
(ProLab RMH 2500, W.F. Fisher & Son Inc.) was provided ad
libitum. The rats were dosed subcutaneously with vehicle (3.2%
mannitol/50 mM Tris buffer) or selected peptides (1.0 mg/kg). The
changes in food intake for the 4 and 24 hour periods after dosing
and the changes in body weight for the 24 hour period after dosing
were determined. The changes in body weight and food intake for the
48 hour and 72 hour periods after dosing was also measured to
determine reversal of changes in body weight and food intake
effects back to baseline levels (not shown).
[0302] Food intake (FI, 0-4 h and 0-24 h) and body weight (BW, 0-24
h) results from studies with 1.0 mg/kg subcutaneously of Examples
43, 46 and 67 and corresponding vehicle controls run in parallel
are shown in Table 2 below, expressed as means.+-.SEM (n=8-10).
TABLE-US-00011 TABLE 2 Dose BW change Experiment Treatment (mg/kg,
s.c.) n FI 0-4 h (g) FI 0-24 h (g) 0-24 h (g) 1 Vehicle 10 10.53
.+-. 0.78 27.75 .+-. 1.00 1.49 .+-. 0.18 1 Ref Ex 2 1.0 10 4.41
.+-. 0.48 20.84 .+-. 1.01 -0.38 .+-. 0.46 1 Ex 43 1.0 10 6.85 .+-.
0.55 24.44 .+-. 0.85 0.45 .+-. 0.35 2 Vehicle 8 7.09 .+-. 0.39
27.46 .+-. 1.16 1.96 .+-. 0.55 2 Ref Ex 2 1.0 8 2.80 .+-. 0.27
21.81 .+-. 1.04 0.85 .+-. 0.45 2 Ex 46 1.0 8 3.71 .+-. 0.68 20.56
.+-. 1.14 -0.29 .+-. 0.55 3 Vehicle 8 7.76 .+-. 0.53 25.71 .+-.
0.25 2.25 .+-. 0.50 3 Ref Ex 2 1.0 8 3.17 .+-. 0.42 19.98 .+-. 0.60
-0.27 .+-. 0.48 3 Ex 67 1.0 8 4.20 .+-. 0.66 21.51 .+-. 1.27 -0.05
.+-. 0.39
[0303] 7.5 Induction of Penile Erection.
[0304] The ability of peptides of the present invention to induce
penile erection (PE) in male rats was evaluated with selected
peptides. Male Sprague-Dawley rats weighing 250-300 g were kept on
a 12 hour on/off light cycle with food and water ad libitum. All
behavioral studies were performed between 9 a.m. and 4 p.m. Groups
of 6-8 rats were administered peptides at a variety of doses via
the subcutaneous injection route. Immediately after treatment, rats
were placed into individual polystyrene cages (27 cm long, 16 cm
wide, and 25 cm high) for behavioral observation, typically by
remote video monitoring (High-speed Digital Video Recording System
EVS-DSX-16000DVD-H with CCD cameras, Epic Systems Inc., St. Louis,
Mo., USA) followed by blinded off-line scoring of video recordings.
Rats were observed for one hour, and the number of yawns, grooming
bouts and PEs were recorded in 10-minute bins.
[0305] PE results from studies with 1.0-3.0 mg/kg subcutaneously of
Examples 43, 46 and 67, along with Ref Ex 2, are shown in Table 3
below, expressed as total number of PE/rat, mean.+-.SEM (n=7-11)
and number of rats/group showing at least one erection over the
study period (% responders). Corresponding vehicle controls (3.2%
mannitol/50 mM Tris buffer) were run in parallel.
TABLE-US-00012 TABLE 3 Dose Total number of Treatment (mg/kg, s.c.)
n PE/rat 0-1 h % Responders Vehicle 11 1.0 .+-. 0.27 64 Ref Ex 2
3.0 7 6.0 .+-. 0.44 100 Ex 43 1.0 7 1.7 .+-. 0.36 29 3.0 7 6.0 .+-.
0.76 100 Ex 46 1.0 7 4.9 .+-. 1.1 100 3.0 7 4.4 .+-. 1.1 86 Ex 67
1.0 7 2.3 .+-. 0.42 86 3.0 7 3.3 .+-. 1.0 100
8.0 High and Low Density hMC4-R Functional Assay Results
[0306] Exemplified peptides were tested using the high and low
density hMC4-R functional assay as described in Section 7.3 above,
with the results as shown in Table 4 below. Table 4 includes both
single point values and mean values.
TABLE-US-00013 TABLE 4 0.1 ng/mL Doxycycline 10 ng/mL Doxycycline
Intrinsic Intrinsic Compound EC.sub.50 (nM) Activity (%) EC.sub.50
(nM) Activity (%) Example 1 0.34 91 0.045 96 Example 2 0.56 36
0.095 99 Example 3 0.07 28 0.16 100 Example 4 -- 11 0.245 100
Example 5 -- 5 0.38 92 Example 6 37 29 2 100 Example 7 0.3 48 0.135
100 Example 8 48 71 0.55 87 Example 9 0.09 91 0.035 89 Example 10
0.45 77 0.17 94 Example 11 0.065 83 0.06 90 Example 12 34 64 0.5 97
Example 13 0.125 85 0.06 96 Example 14 8 16 0.4 93 Example 15 0.165
16 0.2 90 Example 16 0.15 28 0.16 97 Example 17 0.95 64 0.12 95
Example 18 0.14 83 0.085 97 Example 19 0.42 35 0.22 97 Example 20
-- 0.5 0.75 53 Example 21 -- 8 0.5 97 Example 22 3 78 0.2 93
Example 23 2 17 0.55 95 Example 24 0.7 15 0.45 99 Example 25 75 23
2 94 Example 26 0.125 74 0.12 90 Example 27 0.7 37 0.33 97 Example
28 29 35 0.6 94 Example 29 3 14 0.8 100 Example 30 -- 2 25 9
Example 31 7 33 0.313 98 Example 32 18 34 0.543 96 Example 33 1 41
0.153 98 Example 34 1 79 0.137 97 Example 35 -- 6 1 84 Example 36
-- 8 1 89 Example 37 -- 6 0.55 90 Example 38 2 31 0.258 97 Example
39 -- 4 1 79 Example 40 -- 8 0.5 93 Example 41 -- 5 0.8 84 Example
42 -- 9 0.5 84 Example 43 1 43 0.177 100 Example 44 11 22 0.617 99
Example 45 -- 12 0.45 87 Example 46 0.666 40 0.173 97 Example 47
0.31 36 0.373 100 Example 48 -- 6 4 93 Example 49 2 74 0.235 93
Example 50 0.21 94 0.085 94 Example 51 0.64 22 0.4 95 Example 52
0.325 31 0.2 99 Example 53 0.13 86 0.13 90 Example 54 0.075 56
0.075 92 Example 55 10 28 0.8 98 Example 56 5 41 0.3 98 Example 57
0.25 89 0.095 100 Example 58 5 67 0.25 100 Example 59 0.6 90 0.1 98
Example 60 0.45 62 0.14 95 Example 61 -- 6 0.95 89 Example 62 16 23
0.47 84 Example 63 0.37 30 0.157 93 Example 64 1 57 0.08 90 Example
65 1 58 0.085 93 Example 66 5 32 0.265 88 Example 67 1 38 0.14 99
Example 68 0.85 55 0.075 91 Example 69 0.25 76 0.095 92 Example 70
0.4 64 0.1 91 Example 71 1 86 0.085 91 Example 72 3 11 0.35 84
Example 73 0.587 86 0.068 102 Example 74 0.943 68 0.11 100 Example
75 0.65 43 0.125 87 Example 76 3 53 0.135 85 Example 77 3 34 0.155
80 Example 78 2 48 0.2 89 Example 79 4 48 0.2 85 Example 80 6 13
0.65 86 Example 81 4 23 0.45 85 Example 82 2 79 0.095 95 Example 83
0.75 78 0.075 94 Example 84 0.633 78 0.09 97 Example 85 15 36 0.707
94 Example 86 9 36 0.483 96 Example 87 1 64 0.065 100 Example 88 3
15 0.35 89 Example 89 6 17 87 Example 90 4 31 0.283 96 Example 91 1
18 0.265 96 Example 92 0.1 81 0.04 89 Example 93 0.3 44 0.17 97
Example 94 0.24 32 0.09 92 Example 95 1 42 0.125 100 Example 96 2
24 0.235 100 Example 97 0.8 49 0.1 100 Example 98 0.45 47 0.14 100
Example 99 0.7 32 0.14 94 Example 100 6 27 0.27 93 Example 101 0.95
36 0.14 93 Example 102 18 30 0.725 97 Example 103 1 32 0.13 100
Example 104 191 16 10 100 Example 105 3 35 0.15 94 Example 106 3
113 77 Example 107 4 64 Example 108 3 9 59 Example 109 2 269 69
Example 110 0.6 84 0.06 105 Example 111 0.575 81 0.053 104 Example
112 0.39 83 0.07 101 Example 113 0.72 80 0.065 100 Example 114 1 73
0.185 117 Example 115 0.8 54 0.17 108 Example 116 0.68 77 0.095 104
Example 117 1 50 0.18 112
[0307] It is believed that peptides of the invention exhibiting an
intrinsic activity of equal to or greater than about 0.1 (10%),
such as greater than 0.2 (20%) or greater than about 0.3 (30%) or
greater than about 0.4 (40%) or greater than about 0.5 (50%) or
greater than about 0.6 (60%) %) or greater than about 0.7 (70%), or
greater than about 0.8 (80%), or greater than about 0.9 (90%), or
equal to or greater than about 1.0 (100%), in the above described
high density hMC4-R system (i.e. HEK293TRexMC4R cells treated with
10 ng/ml doxycycline), based on maximal stimulation of adenylyl
cyclase achievable by the compound in the same high density hMC4-R
system where the maximal stimulation achieved by .alpha.-MSH or
NDP-.alpha.-MSH is designated as an intrinsic activity of 1.0
(100%), provide agonist (full or partial) activity on the MC4
receptor.
[0308] A majority of the peptides of the Examples which were tested
in the above described high density hMC4-R system (i.e.
HEK293TRexMC4R cells treated with 10 ng/ml doxycycline) gave in
said assay EC.sub.50 values for cAMP production of less than 0.05
.mu.M, such as less than 0.01 .mu.M, and in particular less than
0.001 .mu.M.
[0309] As seen from Table 4, peptides of the invention may lack
intrinsic activity in the low density hMC4-R system and be full
agonists in the high density hMC4-R system.
[0310] As further seen from Table 4, peptides of the invention may
be partial agonists in the above described low density hMC4-R
system and full agonists in the above described high density hMC4-R
system.
[0311] As further seen from Table 4, peptides of the invention may
be full agonists in both the low density hMC4-R system and in the
high density hMC4-R system.
[0312] Without being bound by any theory, it is believed that
peptides that either lack intrinsic activity or are partial
agonists in the above described low density hMC4-R system (i.e.
peptides having intrinsic activity of from 0 to 70% in the low
density hMC4-R system) and are partial or full agonists in the
above described high density hMC4-R system (i.e. peptides having
intrinsic activity of equal to or above 10%, or above 70%, in the
high density hMC4-R system) will provide no or a reduced level of
sexual effects resulting from MC4 receptor activation, e.g. penile
erection, compared to peptides being full agonists in both the low
and the high density hMC4-R systems. As explained above, these
sexual effects are considered unwanted side-effects upon treatment
of energy homeostasis and metabolism related, food intake related
and/or energy balance and body weight related diseases, disorders
and/or conditions.
[0313] It is thus believed that peptides according to the invention
possess promising MC4 receptor affinity, efficacy and potency to be
useful for treatment of diseases, disorders and/or conditions
responsive to activation of the MC4 receptor, in particular energy
homeostasis and metabolism related (e.g. diabetes), food intake
related and/or energy balance and body weight related diseases,
disorders and/or conditions, including obesity, overweight and
diseases, disorders and/or conditions associated with obesity
and/or overweight, such as type 2 diabetes and metabolic
syndrome.
[0314] Although the invention has been described in detail with
particular reference to these preferred embodiments, other
embodiments can achieve the same results. Variations and
modifications of the present invention will be obvious to those
skilled in the art and it is intended to cover all such
modifications and equivalents.
Sequence CWU 1
1
12217PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 1Xaa Asp His Xaa Arg Trp Lys 1 5
27PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 2Xaa Asp His Xaa Arg Trp Lys 1 5
34PRTArtificial SequenceSynthetic melanocortin receptor binding
core sequence derived from alpha-MSH 3His Phe Arg Trp 1
413PRTArtificial SequenceHuman alpha-MSH 4Ser Tyr Ser Met Glu His
Phe Arg Trp Gly Lys Pro Val 1 5 10 513PRTArtificial
SequenceSynthetic melanocortin binding sequence derived from human
alpha-MSH 5Ser Tyr Ser Xaa Glu His Xaa Arg Trp Gly Lys Pro Val 1 5
10 67PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 6Arg Glu Xaa Xaa Arg Trp Xaa 1 5
77PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 7Arg Glu Xaa Xaa Arg Trp Xaa 1 5
87PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 8Arg Glu Xaa Xaa Arg Trp Xaa 1 5
97PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 9Arg Glu Xaa Xaa Arg Trp Xaa 1 5
107PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 10Arg Glu Xaa Xaa Arg Trp Xaa 1 5
117PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 11Arg Glu Xaa Xaa Arg Trp Xaa 1 5
127PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 12Arg Glu Xaa Xaa Arg Trp Xaa 1 5
137PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 13Arg Glu Xaa Xaa Arg Trp Xaa 1 5
147PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 14Arg Glu Xaa Xaa Arg Trp Xaa 1 5
157PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 15Arg Glu Xaa Xaa Arg Trp Xaa 1 5
167PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 16Arg Glu Xaa Xaa Arg Trp Xaa 1 5
177PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 17Arg Glu Xaa Xaa Arg Trp Xaa 1 5
187PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 18Arg Glu Xaa Xaa Arg Trp Xaa 1 5
197PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 19Arg Glu Xaa Xaa Arg Trp Xaa 1 5
207PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 20Arg Glu Xaa Xaa Arg Trp Xaa 1 5
217PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 21Arg Glu Xaa Xaa Arg Trp Xaa 1 5
227PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 22Arg Glu Xaa Xaa Arg Trp Xaa 1 5
237PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 23Arg Glu Xaa Xaa Arg Trp Xaa 1 5
247PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 24Arg Glu Xaa Xaa Arg Trp Xaa 1 5
257PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 25Arg Glu Xaa Xaa Arg Trp Xaa 1 5
267PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 26Arg Glu Xaa Xaa Arg Trp Xaa 1 5
277PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 27Arg Glu Xaa Xaa Arg Trp Xaa 1 5
287PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 28Arg Glu Xaa Xaa Arg Trp Xaa 1 5
297PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 29Arg Glu Xaa Xaa Arg Trp Xaa 1 5
307PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 30Arg Glu Xaa Xaa Arg Trp Xaa 1 5
317PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 31Arg Glu Xaa Xaa Arg Trp Xaa 1 5
327PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 32Arg Glu Xaa Xaa Arg Trp Xaa 1 5
337PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 33Arg Glu Xaa Xaa Arg Trp Xaa 1 5
347PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 34Arg Glu Xaa Xaa Arg Trp Xaa 1 5
357PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 35Arg Glu Xaa Xaa Arg Trp Xaa 1 5
367PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 36Arg Glu Ser Xaa Arg Trp Xaa 1 5
377PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 37Arg Glu Thr Xaa Arg Trp Xaa 1 5
387PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 38Arg Glu Xaa Xaa Arg Trp Xaa 1 5
397PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 39Arg Glu Gln Xaa Arg Trp Xaa 1 5
407PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 40Arg Glu Ser Xaa Arg Trp Xaa 1 5
417PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 41Arg Glu Thr Xaa Arg Trp Xaa 1 5
427PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 42Arg Glu Xaa Xaa Arg Trp Xaa 1 5
437PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 43Arg Glu Gln Xaa Arg Trp Xaa 1 5
447PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 44Arg Glu Pro Xaa Arg Trp Xaa 1 5
457PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 45Arg Glu Xaa Xaa Arg Trp Xaa 1 5
467PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 46Arg Glu Ala Xaa Arg Trp Xaa 1 5
477PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 47Arg Glu Val Xaa Arg Trp Xaa 1 5
487PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 48Arg Glu Asn Xaa Arg Trp Xaa 1 5
497PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 49Arg Glu Thr Xaa Arg Trp Xaa 1 5
507PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 50Arg Glu Ser Xaa Arg Trp Xaa 1 5
517PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 51Arg Glu Gln Xaa Arg Trp Xaa 1 5
527PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 52Arg Glu Xaa Xaa Arg Trp Xaa 1 5
538PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 53Arg Glu Xaa Xaa Xaa Arg Trp Xaa
1 5 547PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 54Arg Glu Xaa Xaa Arg Trp Xaa 1 5
557PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 55Xaa Xaa His Xaa Arg Trp Glu 1 5
567PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 56Xaa Xaa His Xaa Arg Trp Glu 1 5
577PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 57Xaa Xaa His Xaa Arg Trp Glu 1 5
587PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 58Xaa Xaa His Xaa Arg Trp Glu 1 5
597PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 59Xaa Xaa His Xaa Arg Trp Glu 1 5
607PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 60Arg Glu Xaa Xaa Arg Trp Xaa 1 5
617PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 61Arg Glu Xaa Xaa Arg Trp Xaa 1 5
627PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 62Xaa Xaa His Xaa Arg Trp Glu 1 5
637PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 63Arg Glu Ser Xaa Arg Trp Xaa 1 5
647PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 64Arg Glu Gln Xaa Arg Trp Xaa 1 5
657PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 65Arg Glu Xaa Xaa Arg Trp Xaa 1 5
667PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 66Arg Glu Xaa Xaa Arg Trp Xaa 1 5
677PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 67Arg Glu Xaa Xaa Arg Trp Xaa 1 5
687PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 68Arg Glu Xaa Xaa Arg Trp Xaa 1 5
697PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 69Arg Glu Xaa Xaa Arg Trp Xaa 1 5
707PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 70Arg Glu Ser Xaa Arg Trp Xaa 1 5
717PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 71Arg Glu Pro Xaa Arg Trp Xaa 1 5
727PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 72Arg Glu Pro Xaa Arg Trp Xaa 1 5
737PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 73Arg Glu Xaa Xaa Arg Trp Xaa 1 5
747PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 74Arg Glu Asn Xaa Arg Trp Xaa 1 5
757PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 75Arg Xaa Asn Xaa Arg Trp Glu 1 5
767PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 76Arg Xaa Asn Xaa Arg Trp Glu 1 5
777PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 77Arg Xaa Asn Xaa Arg Trp Glu 1 5
787PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 78Arg Glu Asn Xaa Arg Trp Xaa 1 5
797PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 79Arg Xaa Asn Xaa Arg Trp Glu 1 5
807PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 80Arg Xaa Asn Xaa Arg Trp Glu 1 5
817PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 81Arg Glu Asn Xaa Arg Trp Xaa 1 5
827PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 82Arg Xaa Asn Xaa Arg Trp Glu 1 5
837PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 83Xaa Asp Gln Xaa Arg Trp Lys 1 5
847PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 84Xaa Asp Gln Xaa Arg Trp Lys 1 5
857PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 85Xaa Asp Gln Xaa Arg Trp Lys 1 5
867PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 86Xaa Asp Gln Xaa Arg Trp Lys 1 5
877PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 87Xaa Asp Gln Xaa Arg Trp Lys 1 5
887PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 88Xaa Asp Gln Xaa Arg Trp Lys 1 5
897PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 89Arg Glu Asn Xaa Arg Trp Xaa 1 5
907PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 90Arg Glu Asn Xaa Arg Trp Xaa 1 5
917PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 91Arg Glu Gln Xaa Arg Trp Xaa 1 5
927PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 92Arg Glu Xaa Xaa Arg Trp Xaa 1 5
937PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 93Arg Glu Pro Xaa Arg Trp Xaa 1 5
947PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 94Arg Asp Asn Xaa Arg Trp Lys 1 5
957PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 95Arg Asp Gln Xaa Arg Trp Lys 1 5
967PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 96Arg Xaa Asn Xaa Arg Trp Glu 1 5
977PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 97Arg Glu Gln Xaa Arg Trp Xaa 1 5
987PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 98Arg Lys Gln Xaa Arg Trp Asp 1 5
997PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 99Arg Xaa Gln Xaa Arg Trp Glu 1 5
1007PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 100Arg Glu Asn Xaa Arg Trp Xaa 1 5
1017PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 101Arg Xaa Asn Xaa Arg Trp Glu 1 5
1027PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 102Arg Glu Gln Xaa Arg Trp Xaa 1 5
1037PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 103Arg Xaa Gln Xaa Arg Trp Glu 1 5
1047PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 104Arg Glu Arg Xaa Arg Trp Xaa 1 5
1057PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 105Arg Glu Lys Xaa Arg Trp Xaa 1 5
1067PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 106Arg Glu Xaa Xaa Arg Trp Xaa 1 5
1077PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 107Arg Glu Pro Xaa Arg Trp Xaa 1 5
1087PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 108Arg Asp Pro Xaa Arg Trp Lys 1 5
1097PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 109Arg Asp Pro Xaa Arg Trp Lys 1 5
1107PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 110Arg Xaa Pro Xaa Arg Trp Glu 1 5
1117PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 111Arg Glu Asp Xaa Arg Trp Xaa 1
5
1127PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 112Arg Glu Asp Xaa Arg Trp Xaa 1 5
1137PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 113Arg Glu Glu Xaa Arg Trp Xaa 1 5
1147PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 114Arg Glu Glu Xaa Arg Trp Xaa 1 5
1157PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 115Arg Glu Gln Xaa Arg Trp Xaa 1 5
1167PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 116Arg Xaa Gln Xaa Arg Trp Glu 1 5
1177PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 117Arg Xaa Gln Xaa Arg Trp Glu 1 5
1187PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 118Arg Xaa Asn Xaa Arg Trp Glu 1 5
1197PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 119Arg Glu Gln Xaa Arg Trp Xaa 1 5
1207PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 120Arg Xaa Gln Xaa Arg Trp Glu 1 5
1217PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 121Arg Glu Asn Xaa Arg Trp Xaa 1 5
1227PRTArtificial SequenceSynthetic melanocortin binding peptide
derived from human NDP-alpha-MSH 122Arg Xaa Asn Xaa Arg Trp Glu 1
5
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