MELANIN-CONCENTRATING HORMONE ANALOGS

ANALOGUES DE L'HORMONE CONCENTRATRICE DE LA MELANINE

English Abstract

The present invention features truncated MCH analogs active at the MCH
receptor. The truncated MCH analogs are optionally modified peptide
derivatives of mammalian MCH. The analogs can bind to the MCH receptor and,
preferably, bring about signal transduction. MCH analogs have a variety of
different uses including being used as a research tool and being used
therapeutically.

French Abstract

La présente invention concerne des analogues de l'hormone concentratrice de la mélanine (MCH) tronqués actifs au niveau du récepteur de MCH. Les analogues de MCH tronqués sont éventuellement des dérivés de peptide modifié de MCH mammifère. Ces analogues peuvent se lier au récepteur de MCH et, de préférence, créer une transduction de signal. Les analogues de MCH possèdent une variété d'utilisations, y compris, les utilisations thérapeutiques et en tant qu'outil de recherche.

Claims

WHAT IS CLAIMED IS:

1. An optionally substituted peptide having the structure:

Image

herein X1 is an optionally present amino acid that, if present, is either
alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine,
methionine,
glycine, serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine,
arginine,
histidine, aspartic acid, or glutamic acid, or a derivative thereof;
X2 is an optionally present amino acid that, if present, is either alanine,
valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine,
glycine,
serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine,
arginine, histidine,
aspartic acid, or glutamic acid, or a derivative thereof;
X3 is an optionally present amino acid that, if present, is either alanine,
valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine,
glycine,
serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine,
arginine, histidine,
aspartic acid or glutamic acid, or a derivative thereof;
X4 is an optionally present amino acid that, if present, is either alanine,
valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine,
glycine,
serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine,
arginine, histidine,
aspartic acid, glutamic acid, or norleucine, or a derivative thereof;
X5 is an optionally present amino acid that, if present, is either alanine,
valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine,
glycine,
serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine,
arginine, histidine,
aspartic acid or glutamic acid, or a derivative thereof;
X6 is an optionally present amino acid that, if present is either
arginine, alanine, leucine, glycine, lysine, proline, asparagine, serine,
histidine,
nitroarginine, norleucine, or des-amino-arginine, or a derivative thereof,
X7 is either cysteine, homocysteine, or penicillamine, or a derivative
thereof;
X8 is either methionine, norleucine, leucine, isoleucine, valine,
methioninesulfoxide, or methioninesulfone, or a derivative thereof;



26




X9 is either leucine, isoleucine, valine, alanine, methionine, or 5-
aminopentanoic acid, or a derivative thereof;
X10 is either glycine, alanine, leucine, norleucine, cyclohexylalanine,
5-aminopentanoic acid, asparagine, serine, sarcosine, isobutyric, or gamma-
aminobutyric acid, or a derivative thereof;
X11 is either arginine, lysine, citrulline, histidine, or nitroarginine, or a
derivative thereof;
X12 is either valine, leucine, isoleucine, alanine, or methionine, or a
derivative thereof;
X13 is either phenylalanine, tyrosine, D-( p-benzoylphenylalanine),
tryptophan, (1')- and (2')-naphthylalanine, cyclohexylalanine, or mono and
multi-
substituted phenylalanine wherein each substituent is independently selected
from the
group consisting of O-alkyl, alkyl, OH, NO2, NH2, F, I, and Br; or a
derivative
thereof;
X14 is either arginine, lysine, histidine, norarginine, or 5-
aminopentanoic acid or a derivative thereof;
X15 is either proline, alanine, valine, leucine, isoleucine, methionine,
sarcosine, or 5-aminopentanoic acid, or a derivative thereof;
X16 is either cysteine, homocysteine, or penicillamine, or a derivative
thereof;
X17 is an optionally present amino acid that, if present, is either
alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine,
methionine,
glycine, serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine,
arginine,
histidine, aspartic acid or glutamic acid, or a derivative thereof;
Z1 is an optionally present protecting group that, if present, is
covalently joined to the N-terminal amino group;
Z2 is an optionally present protecting group that, if present, is
covalently joined to the C-terminal carboxy group;
or a labeled derivative of said peptide;
or a pharmaceutically acceptable salt of said peptide or of said labeled
derivative.

2. The peptide of claim 1, wherein X1, X2, X3, X4, and X5 are
not present; X6 is arginine; and X17 is tyrosine or tryptophan.



27




3. The peptide of claim 1, wherein X1, X2, X3, X4, X5, and
X17are not present; and X6 is arginine.

4. The peptide of claim 3, wherein Z1 is -C(O)CH3 and Z2 is
-NH2.

5. The peptide of claim 1, wherein said peptide is either
SEQ. ID. NO. 7, SEQ. ID. NO. 8, SEQ. ID. NO. 9, SEQ. ID. NO. 10, or
a pharmaceutically acceptable salt thereof.

6. The peptide of claim 1, wherein said peptide is either
SEQ. ID. NO. 7, SEQ. ID. NO. 8, or a pharmaceutically acceptable salt thereof.

7. The peptide of claim 1, wherein X1, X2, X3, X4, X5 and X17
are not present;
X6 is either arginine, D-arginine, D-norleucine, D-proline, D-serine, or D-
asparagine;
X7 is cysteine;
X8 is either methionine, norleucine, or N-methyl norleucine;
X9 is leucine;
X10 is either glycine, alanine, leucine, norleucine, asparagine, serine, D-
norleucine,
D-proline, gamma-aminobutyric acid, or sarcosine;
X11 is arginine;
X12 is valine;
X13 is phenylalanine, (2')napthylalanine, p-fluoro-phenylalanine, tyrosine, or
cyclohexylalanine;
X14 is arginine;
X15 is either proline or sarcosine; and
X16 is either cysteine or D-cysteine.

8. The peptide of claim 1, wherein said peptide consists of a
sequence selected from the group consisting of: 7, 8, 10, 15, 24, 25, 27, 28,
30-49, 51,
52, 56, 57, 61, 62, 63, 65-67, 69-72, and 77.



28




9. A method of screening for a compound able to bind a MCH
receptor comprising the step of measuring the ability of said compound to
effect
binding of the peptide of any one of claims 1-8 to either said receptor, a
fragment of
said receptor comprising a MCH binding site, a polypeptide comprising said
fragment, or a derivative of said polypeptide.

10. The method of claim 9, wherein said method measures the
ability of said peptide to bind to said receptor or said fragment thereof.

11. The method of claim 10, wherein said peptide is radiolabeled.

12. The method of claim 9, wherein said peptide is a radiolabeled
derivative of SEQ. ID. NO. 8 or a pharmaceutically acceptable salt thereof.

13. A method for increasing weight in a subject comprising the step
of administering to said subject an effective amount of the peptide of any one
of
claims 1-8 to produce a weight increase.

14. A method for increasing appetite in a subject comprising the
step of administering to said subject an effective amount of the peptide of
any one of
claims 1-8 to produce an appetite increase.

15. A method for measuring the ability of a compound to decrease
weight or appetite in a subject comprising the steps of:

a) administering to said subject an effective amount of the peptide
of any one of claims 1-8 to produce a weight increase or appetite increase,
b) administering said compound to said subject, and
c) measuring the change in weight or appetite of said subject.

16. The method of claim 15, wherein said subject is either a rat or a
mouse.



29

Description

CA 02399509 2002-08-05
WO 01/57070 PCTNSO1/03293
TITLE OF THE INVENTION
MELANIN-CONCENTRATING HORMONE ANALOGS
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority to provisional application U.S.
Serial No. 60/179,967, filed February 3, 2000, which is hereby incorporated by
reference herein.
BACKGROUND OF THE INVENTION
y10 Neuropeptides present in the hypothalamus play a major role in
mediating the control of body weight. (Flier, et al., 1998. Cell, 92, 437-
440.)
Melanin-concentrating hormone (MCH) produced in mammals is a cyclic 19-amino
acid neuropeptide synthesized as part of a larger pre-prohormone precursor in
the
hypothalamus which also encodes neuropeptides NEI and NGE. (Nahon, et al.,
1990.
Mol. Efzdocrinol. 4, 632-637; Vaughan, et al., U.S. Patent No. 5,049,655; and
Vaughan;~ et al., 1989. EudocriiZOlogy 12~, 1660-1665.) MCH was first
identified in
salmon pituitary, and in fish ~MCH affects melanin aggregation thus affecting
skin
pigmentation. In trout and eels MCH has also been shown to be involved in
stress
induced or CRF-stimulated ACTH release. (Kawauchi, et al., 1983. Nature 305,
321-323.)
In humans two genes encoding MCH have been identified that are
expressed in the brain. (Breton, et al., 1993. Mol. Brain Res. 18, 297-310.)
In
mammals MCH has been localized primarily to neuronal cell bodies of the
hypothalamus which are implicated in the control of food intake, including
perikarya
of the lateral hypothalamus and zona inertia. (Knigge, et al., 1996. Peptides
17,
1063-1073.)
Pharmacological and genetic evidence suggest that the primary mode
of MCH action is to promote feeding (orexigenic). MCH mRNA is up regulated in
fasted mice and rats, in the oblob mouse and in mice with targeted disruption
in the
gene for neuropeptide Y (NPY). (Qu, et al., 1996. Nature 380, 243-247 and
Erickson, et al., 1996. Natacre 381, 415-418.) Injection of MCH centrally
(ICV)
stimulates food intake and MCH antagonizes the hypophagic effects seen with
a melanocyte stimulating hormone (ocMSH). (Qu, et al., 1996. Nata~re 380. 243-
247.)
MCH deficient mice are lean, hypophagic and have increased metabolic rate.
(Shimada, et al., 1998. Nature 396, 670-673.) The administration of MCH has
been
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indicated to useful for promoting eating, appetite or the gain or maintenance
of
weight. (Maratos-Flier, U.S. Patent No. 5,849,708.)
MCH action is not limited to modulation of food intake as effects on
the hypothalamic-pituitary-axis have been reported. (Nahon, 1994. Critical
Rev. in
Neurobiol. 8, 221-262.) MCH may be involved in the body response to stress as
MCH can modulate the stress-induced release of CRF from the hypothalamus and
ACTH from the pituitary. In addition, MCH neuronal systems may be involved in
reproductive or maternal function.
SUMMARY OF THE INVENTION
The present invention features truncated MCH analogs active at the
MCH receptor. The truncated MCH analogs are optionally modified peptide
derivatives of mammalian MCH. The analogs can bind to the MCH receptor and,
preferably, bring about signal transduction. MCH analogs have a variety of
different
uses including being used as a research tool and being used therapeutically.
Thus, a first aspect of the present invention describes a truncated MCH
analog. The truncated MCH analog is an optionally modified peptide having the
structure:
z1_ X1_X2_X3_X4_XS_X6-X7 X8_X9_X10_X11_X12_X13_X14_X15-X16_X17_z2
wherein Xl is an optionally present amino acid that, if present, is either
alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine,
methionine,
glycine, serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine,
arginine,
histidine, aspartic acid, or glutamic acid, or a derivative thereof;
X2 is an optionally present amino acid that, if present, is either alanine,
valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine,
glycine,
serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine,
arginine, histidine,
aspartic acid, or glutamic acid, or a derivative thereof;
X3 is an optionally present amino acid that, if present, is either alanine,
valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine,
glycine,
serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine,
arginine, histidine,
aspartic acid or glutamic acid, or a derivative thereof;


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X4 is an optionally present amino acid that, if present, is either alanine,
valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine,
glycine,
serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine,
arginine, histidine,
aspartic acid, glutamic acid, or norleucine, or a derivative thereof;
XS is an optionally present amino acid that, if present, is either alanine,
valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine,
glycine,
serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine,
arginine, histidine,
aspartic acid or glutamic acid, or a derivative thereof;
X6 an optionally present amino acid that, if present is either arginine,
alanine, leucine, glycine, lysine, proline, asparagine, serine, histidine,
nitroarginine,
norleucine, or des-amino-arginine, or a derivative thereof,
X~ is either cysteine, homocysteine, or penicillamine, or a derivative
thereof;
Xg is either methionine, norleucine, leucine, isoleucine, valine,
methioninesulfoxide, or methioninesulfone, or a derivative thereof;
X9 is either leucine, isoleucine, valine, alanine, methionine, or 5-
aminopentanoic acid, or a derivative thereof;
X10 is either glycine, alanine, leucine, norleucine, cyclohexylalanine,
5-aminopentanoic acid, asparagine, serine, sarcosine, isobutyric, or gamma-
aminobutyric acid, or a derivative thereof;
X11 is either arginine, lysine, citrulline, histidine, or nitroarginine, or a
derivative thereof;
X12 is either valine, leucine, isoleucine, alanine, or methionine, or a
derivative thereof;
X13 is either phenylalanine, tyrosine, D-( p-benzoylphenylalanine),
tryptophan, (1')- and (2')-naphthylalanine, cyclohexylalanine, or mono and
multi-
substituted phenylalanine wherein each substituent is independently selected
from the
group consisting of O-alkyl, alkyl, OH, N02, NH2, F, I, and Br; or a
derivative
thereof;
X14 is either arginine, lysine, histidine, norarginine, or 5-
aminopentanoic acid or a derivative thereof;
X15 is either proline, alanine, valine, leucine, isoleucine, methionine,
sarcosine, or 5-aminopentanoic acid, or a derivative thereof;
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X16 is either cysteine, homocysteine, or penicillamine, or a derivative
thereof;
X1~ is an optionally present amino acid that, if present, is either
alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine,
methionine,
glycine, serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine,
arginine,
histidine, aspartic acid or glutamic acid, or a derivative thereof;
Z1 is an optionally present protecting group that, if present, is
covalently joined to the N-terminal amino group;
Z2 is an optionally present protecting group that, if present, is
covalently joined to the C-terminal carboxy group;
or a labeled derivative of said peptide;
or a pharmaceutically acceptable salt of said peptide or of said labeled
derivative.
Unless otherwise stated, those amino acids with a chiral center are
provided in the L-enantiomer. Reference to "a derivative thereof ' refers to
the
corresponding D-amino acid, N-alkyl-amino acid and (3-amino acid.
Another aspect of the present invention describes a method of
screening for a compound able to bind a MCH receptor. The method comprises the
step of measuring the ability of the compound to effect binding of a truncated
MCH
analog to either the MCH receptor, a fragment of the receptor comprising a MCH
binding site, a polypeptide comprising such a fragment, or a derivative of the
polypeptide.
Another aspect of the present invention describes a method for
increasing weight in a subject. The method comprises the step of administering
to the
subject an effective amount of a truncated MCH analog to produce a weight
increase.
Another aspect of the present invention describes a method for
increasing appetite in a subject. The method comprises the step of
administering to
the subject an effective amount of a truncated MCH analog to produce an
appetite
increase.
Another aspect of the present invention describes a method for
measuring the ability of a compound to decrease weight or appetite in a
subject. The
method comprising the steps of:
a) administering to the subject an effective amount of a truncated
MCH analog to produce a weight increase or appetite increase,
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b) administering the compound to the subject, and
c) measuring the change in weight or appetite of the subject.
Other features and advantages of the present invention are apparent
from the additional descriptions provided herein including the different
examples.
The provided examples illustrate different components and methodology useful
in
practicing the present invention. The examples do not limit the claimed
invention.
Based on the present disclosure the skilled artisan can identify and employ
other
components and methodology useful for practicing the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 illustrates the results of an alanine scan where different amino
acid residues of human MCH were replaced with alanine. The binding assay was
performed by measuring inhibition of (12~I-tyrosine, phenylalaninel3)-MCH
binding
to cloned human MCH receptor (CHO clone). Cyclization sites (S-S) are
indicated by
"*"
DETAILED DESCRIPTION OF THE INVENTION
Truncated MCH analogs contain about 10 to about 17 groups that are
amino acids or amino acid derivatives. Using the present application as a
guide
truncated MCH analogs can be produced having significant MCH receptor
activity,
and in some cases having activity equal to or better than naturally occurnng
mammalian MCH. The smaller size of truncated MCH analogs offers advantages
over longer-length MCH such as ease of synthesis and/or increased solubility
in
physiological buffers.
The MCH receptor is a G-protein coupled receptor that appears to be
able to couple to Gi and Gq. Several references describe a receptor that is
indicated to
be a MCH receptor. (Chambers, et al., 1999. Nature 400, 261-265; Saito, et
al., 1999.
Nature 400, 265-269; Bachner, et al., 1999. FEBS Letters 457:522-524; and
Shimomura, et al., 1999. Biochemical aiad Biophysical Research Communications
261, 622-626. These references are not admitted to be prior art to the claimed
invention.)
The nucleic acid encoding for different variants of a MCH receptor is
provided for by SEQ. ID. NOS. 1-3. The encoded amino acid sequences of the
variants are provided by SEQ. ID. NOS. 4-6. The variants differ from each
other by
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the presence of additional amino acids at the N-terminal. One or more of these
variants may be a physiological MCH receptor.
Significant MCH activity is preferably at least about 50%, at least
about 75%, at least about 90%, or at least about 95%, the activity of
mammalian
MCH as determined by a binding assay or MCH receptor activity assay. Examples
of
such assays are provided below.
MCH analogs have a variety of different uses including being used as a
research tool and being used therapeutically. Research tool applications
generally
involve the use of a truncated MCH analog and the presence of a MCH receptor
or
fragment thereof. The MCH receptor can be present in different environments
such as
a mammalian subject, a whole cell, and membrane fragments. Examples of
research
tool applications of truncated MCH analogs include screening for compounds
active
at the MCH receptor, determining the presence of the MCH receptor in a sample
or
preparation, examining the role or effect of MCH, and examining the role or
effect of
MCH antagonists.
Truncated MCH analogs can be used to screen for both MCH agonists
and MCH antagonists. Screening for MCH agonists can be performed, for example,
by using a truncated MCH analog in a competition experiment with test
compounds.
Screening for MCH antagonists can be performed, for example, by using a
truncated
MCH analog to produce MCH receptor activity and then measuring the ability of
a
compound to alter MCH receptor activity.
Truncated MCH analogs can be administered to a subject. A "subject"
refers to a mammal including, for example, a human, a rat, a mouse, or a farm
animal.
Reference to subject does not necessarily indicate the presence of a disease
or
disorder. The term subject includes, for example, mammals being dosed with a
truncated MCH analog as part of an experiment, mammals being treated to help
alleviate a disease or disorder, and mammals being treated prophylactically to
retard
or prevent the onset of a disease or disorder.
MCH agonists can be used to achieve a beneficial effect in a subject.
For example, a MCH agonist can be used to facilitate a weight gain,
maintenance of
weight and/or an appetite increase. Such effects are particularly useful for a
patient
having a disease or disorder, or under going a treatment, accompanied by
weight loss.
Examples of diseases or disorders accompanied by weight loss include anorexia,
A)DS, wasting, cachexia, and frail elderly. Examples of treatments accompanied
by
weight loss include chemotherapy, radiation therapy, and dialysis.
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MCH antagonists can also be used to achieve a beneficial effect in a
patient. For example, a MCH antagonist can be used to facilitate weight loss,
appetite
decrease, weight maintenance, cancer (e.g., colon or breast) treatment, pain
reduction,
stress reduction and/or treatment of sexual dysfunction.
Truncated MCH Analo
A truncated MCH analog is an optionally modified peptide having the
structure:
z1_ X1_X2_X3_X4_XS_X6-X7 X8_X9_X10_X11_X12_X13_X14_X15-X16_X17_z2
wherein X1 is an optionally present amino acid that, if present, is either
alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine,
methionine,
glycine, serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine,
arginine,
histidine, aspartic acid, or glutamic acid, or a derivative thereof;
preferably, X1 if
present is aspartic acid or glutamic acid; more preferably, X1 if present is
aspartic
acid; and more preferably, X1 is not present;
X2 is an optionally present amino acid that, if present, is either alanine,
valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine,
glycine,
serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine,
arginine, histidine,
aspartic acid, or glutamic acid, or a derivative thereof; preferably, X2 if
present is
phenylalanine or tyrosine; more preferably, X2 if present is phenylalanine;
and more
preferably, X2 is not present;
X3 is an optionally present amino acid that, if present, is either alanine,
valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine,
glycine,
serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine,
arginine, histidine,
aspartic acid or glutamic acid, or a derivative thereof; preferably, X3 if
present is
aspartic acid or glutamic acid; more preferably, X3 if present is aspartic
acid; and
more preferably, X3 is not present;
X4 is an optionally present amino acid that, if present, is either alanine,
valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine,
glycine,
serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine,
arginine, histidine,
aspartic acid, glutamic acid, or norleucine, or a derivative thereof;
preferably, X4 if
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present is methionine, leucine, isoleucine, valine, alanine or norleucine;
more
preferably, X4 if present is methionine; and more preferably, X4 is not
present;
XS is an optionally present amino acid that, if present, is either alanine,
valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine,
glycine,
serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine,
arginine, histidine,
aspartic acid or glutamic acid, or a derivative thereof; preferably, XS if
present is
leucine, methionine, isoleucine, valine or alanine; more preferably, XS if
present is
leucine; and more preferably, XS is not present;
X6 is an optionally present amino acid that, if present is either
arginine, alanine, leucine, glycine, lysine, proline, asparagine, serine,
histidine,
nitroarginine, norleucine, or des-amino-arginine, or a derivative thereof;
preferably
X6 is not present or is either arginine, D-arginine, D-norleucine, D-proline,
D-serine,
or D-asparagine; more preferably X6 is arginine or D-arginine;
X~ is either cysteine, homocysteine, or penicillamine, or a derivative
thereof; preferably, X~ is cysteine;
Xg is either methionine, norleucine, leucine, isoleucine, valine,
methioninesulfoxide, or methioninesulfone, or a derivative thereof;
preferably, Xg is
methionine, norleucine, or N-methyl norleucine;
X9 is either leucine, isoleucine, valine, alanine, methionine, or 5-
aminopentanoic acid, or a derivative thereof; preferably, X9 is leucine;
X10 is either glycine, alanine, leucine, norleucine, cyclohexylalanine,
5-aminopentanoic acid, gamma-aminobutyric acid, asparagine, serine, sarcosine,
or
isobutyric or a derivative thereof; preferably, X10 is either glycine,
alanine, leucine,
norleucine, asparagine, serine, D-norleucine, D-proline, gamma-aminobutyric
acid, or
sarcosine; more preferably X10, is either glycine, leucine, norlecine,
asparagine, or
serene;
X11 is either arginine, lysine, citrulline, histidine, or nitroarginine, or a
derivative thereof; preferably, X11 is arginine;
X12 is either valine, leucine, isoleucine, alanine, or methionine, or a
derivative thereof; preferably, X12 is valine;
X13 is either phenylalanine, tyrosine, D-( p-benzoylphenylalanine),
tryptophan, ( 1' )- and (2' )-naphthylalanine, cyclohexylalanine, or mono and
multi-
substituted phenylalanine wherein each substituent is independently selected
from the
group consisting of O-alkyl, alkyl, OH, N02, NH2, F, I, and Br; or a
derivative


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thereof; preferably, X13 is phenylalanine, (2')napthylalanine, p-fluoro-
phenylalanine,
tyrosine, or cyclohexylalanine;
X14 is either arginine, lysine, histidine or norarginine, or 5-
aminopentanoic acid, or a derivative thereof; preferably, X14 is arginine;
X15 is either proline, alanine, valine, leucine, isoleucine, methionine,
sarcosine, or 5-aminopentanoic acid, or a derivative thereof; preferably, X15
is proline
or sarcosine;
X16 is either cysteine, homocysteine, or penicillamine, or a derivative
thereof; preferably, X16 is cysteine or D-cysteine;
X1~ is an optionally present amino acid that, if present, is either
alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine,
methionine,
glycine, serine, threonine, tyrosine, cysteine, asparagine, glutamine, lysine,
arginine,
histidine, aspartic acid or glutamic acid, or a derivative thereof;
preferably, X1~ if
present is tyrosine or tryptophan; more preferably X1~ is not present;
Z1 is an optionally present protecting group that, if present, is
covalently joined to the N-terminal amino group;
Z2 is an optionally present protecting group that, if present, is
covalently joined to the C-terminal carboxy group;
or a labeled derivative of said peptide;
or a pharmaceutically acceptable salt of said peptide or of said labeled
derivative.
The present invention is meant to comprehend diastereomers as well as
their racemic and resolved enantiomerically pure forms. Truncated MCH analogs
can
contain D-amino acids, L-amino acids or a combination thereof. Preferably,
amino
acids present in a truncated MCH analog are the L-enantiomer.
In different embodiments, MCH analogs contain a preferred (or more
preferred) group at one or more different locations. More preferred
embodiments
contain preferred (or more preferred) groups in each of the different
locations.
A protecting group covalently joined to the N-terminal amino group
reduces the reactivity of the amino terminus under ira vivo conditions. Amino
protecting groups include optionally substituted -C1_lo alkyl, optionally
substituted
-Ca-to alkenyl, optionally substituted aryl, -C1_~ alkyl optionally
substituted aryl,
-C(O)-(CHZ)1_~-COOH, -C(O)- C1_6 alkyl, -C(O)-optionally substituted aryl,
9


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-C(O)-O-C1_~ alkyl, or -C(O)-O-optionally substituted aryl. Preferably, the
amino
terminus protecting group is acetyl, propyl, succinyl, benzyl,
benzvloxycarbonyl or t-
butyloxycarbonyl.
A protecting group covalently joined to the C-terminal carboxy group
reduces the reactivity of the carboxy terminus under in vivo conditions. The
carboxy
terminus protecting group is preferably attached to the a-carbonyl group of
the last
amino acid. Carboxy terminus protecting groups include amide, methylamide, and
ethylamide.
"Alkyl" refers to carbon atoms joined by carbon-carbon single bonds.
The alkyl hydrocarbon group may be straight-chain or contain one or more
branches
or cyclic groups. Preferably, the alkyl group is 1 to 4 carbons in length.
Examples of
alkyl include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, and t-
butyl. Alkyl
substituents are selected from the group consisting of halogen (preferably -F
or -Cl)
-OH, -CN, -SH, -NHZ, -NO2, -C1_2 alkyl substituted with 1 to 6 halogens
(preferably
-F or -Cl, more preferably -F), -CF;, -OCH3, or -OCF3.
"Alkenyl" refers to a hydrocarbon group containing one or more
carbon-carbon double bonds. The alkenyl hydrocarbon group may be straight-
chain or
contain one or more branches or cyclic groups. Preferably, the alkenyl group
is 2 to 4
carbons in length. Alkenyl substituents are selected from the group consisting
of
halogen (preferably -F or -Cl), -OH, -CN, -SH, -NHS, -NO2, -C1_~ alkyl
substituted
with I to 5 halogens (preferably -F or -Cl, more preferably -F), -CF;, -OCH3,
or
-OCF3.
"Aryl" refers to an optionally substituted aromatic group with at least
one ring having a conjugated pi- electron system, containing up to two
conjugated or fused ring systems. Aryl includes carbocyclic aryl. heterocyclic
aryl
and biaryl groups. Preferably, the aryl is a 5 or 6 membered ring, more
preferably
benzyl. Aryl substituents are selected from the group consisting of -C,_4
alkyl,
-C~_4 alkoxy, halogen (preferably -F or -Cl), -OH, -CN, -SH, -NHS, -NO~, -CI_2
alkyl
substituted with 1 to 5 halogens (preferably -F or -Cl, more preferably -F), -
CF3, or
-OCF;.
A labeled derivative indicates the alteration of a substituent with a
detectable label. Examples of detectable labels include luminescent,
enzymatic, and
radioactive labels. A preferred radiolabel is l2sI. Both the type of label and
the
position of the label can effect MCH activity. Labels should be selected so as
not to
substantially alter the activity of the truncated MCH analog at the MCH
receptor. The


CA 02399509 2002-08-05
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effect of a particular label on MCH activity can be determined using assays
measuring
MCH activity and/or binding.
In naturally occurring full length MCH, alteration of the tyrosine at
position 13 by labeling with lzsl substantially effects MCH activity. (Drozdz,
et al.,
1995. FEES letters 359, 199-202.) lz'I labeled analogs of full length
mammalian
MCH having substantial activity can be produced, for example, by replacing the
tyrosine at position 13 with a different group, then replacing valine at
position 19 with
tyrosine, and labeling the tyrosine. Examples of such analogs include
[lz'I][Phel3,
Tryl9]-MCH and (D-(p-benzoylphenylalanine)13, tyrosinel9)-MCH. (Drozdz, et
al.,
FEBS letters 359, 199-202, 1995; and Drozdz, et al., J. Peptide Sci. 5, 234-
242,
1999.)
In preferred embodiments the optionally modified peptide has the
structure:
Zl_ X6-X7 X8_X9_X10_X11_X12_X13_X14_X15-X16_X17_z2
wherein the different groups, and preferred groups, are as described
above.
In different embodiments the truncated MCH analog is a peptide of
SEQ. ID. NOS. 7, 8, 9, or 10, a labeled derivative of said peptide or a
pharmaceutically acceptable salt of said peptide or of said labeled
derivative. SEQ.
)D. NOS. 7-12 are made up of L-amino acids and have the following sequences
("*"
indicates cyclization (S-S)):
* *
SEQ. >D. NO. 7: Ac-Arg-Cys-Met-Leu-Gly-Arg-Val-Tyr-Arg-Pro-Cys-amide;
SEQ. ID. NO. 8: Ac-Arg-Cys-Met-Leu-Gly-Arg-Val-Phe-Arg-Pro-Cys-Tyr-amide;
*
SEQ. ID. NO. 9: Ac-Cys-Met-Leu-Gly-Arg-Val-Tyr-Arg-Pro-Cys-amide;
SEQ. >D. NO. 10:
* x
Asp-Phe-Asp-Met-Leu-Arg-Cys-Met-Leu-Gly-Arg-Val-Tyr-Arg-Pro-Cys-amide;
11


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i'VO 01/57070 PCT/USO1/03293
* *
SEQ. )17. NO. 12: Ac-Cys-Met-Leu-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Gln-Val;
SEQ. >D. NO. 13:
*
Asp-Phe-Asp-Nle-Leu-Arg-Cys-Nle-Leu-Gly-Arg-V al-Tyr-Arg-Pro-Cys-Trp-Gln-
Val;
SEQ. >D. NO. 14:
* *
Asp-Phe-Ala-Met-Leu-Arg-Cys-Met-Leu-Gly-Arg-Val-Phe-Arg-Pro-Cys-Trp-Gln-
Tyr.
In additional embodiments the peptide has a sequence selected from
the group consisting of SEQ. >D. NOs. 7, 8, 10, 15, 24, 25, 27, 28, 30-49, 51,
52, 56,
57, 61, 62, 63, 65-67, 69-72, and 77, is a labeled derivative of said peptide
or a
pharmaceutically acceptable salt of said peptide or of said labeled
derivative.
Preferred sequences are those with an IC50 less than 0.3 nM, preferably less
than 0.1
nM; and/or those having a % activation greater than about 90%, preferably
greater
than 100%. Examples of preferred sequences are provided in Example 4, Tables 1-
7.
Truncated MCH analogs can be produced using techniques well known
in the art. For example, a polypeptide region of a truncated MCH analog can be
chemically or biochemically synthesized and, if desired modified to produce a
blocked N-terminus and/or blocked C-terminus. Techniques for chemical
synthesis of
polypeptides are well known in the art. (See e.g., Vincent, in Peptide and
Protein
Drug Delivery, New York, N.Y., Dekker, 1990.) Examples of techniques for
biochemical synthesis involving the introduction of a nucleic acid into a cell
and
expression of nucleic acids are provided in Ausubel, Current Protocols in
Molecular-
Biology, John Wiley, 1987-1998, and Sambrook, et al., in Molecular Cloning, A
Laboratory Manual, 2°d Edition, Cold Spring Harbor Laboratory
Press, 1989.
MCH Receptor Binding Assay
Assays measuring the ability of a compound to bind a MCH receptor
employ a MCH receptor, a fragment of the receptor comprising a MCH binding
site, a
12


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polypeptide comprising such a fragment, or a derivative of the polypeptide.
Preferably, the assay uses the MCH receptor or a fragment thereof.
A polypeptide comprising a MCH receptor fragment that binds MCH
can also contain one or more polypeptide regions not found in a MCH receptor.
A
S derivative of such a polypeptide comprises a MCH receptor fragment that
binds MCH
along with one or more non-peptide components.
The MCH receptor amino acid sequence involved in MCH binding can
be readily identified using labeled MCH or truncated MCH analogs and different
receptor fragments. Different strategies can be employed to select fragments
to be
tested to narrow down the binding region. Examples of such strategies include
testing
consecutive fragments about 15 amino acids in length starting at the N-
terminus, and
testing longer length fragments. If longer length fragments are tested, a
fragment
binding MCH can be subdivided to further locate the MCH binding region.
Fragments used for binding studies can be generated using recombinant nucleic
acid
techniques.
Binding assays can be performed using individual compounds or
preparations containing different numbers of compounds. A preparation
containing
different numbers of compounds having the ability to bind to the MCH receptor
can
be divided into smaller groups of compounds that can be tested to identify the
compounds) binding to the MCH receptor. In an embodiment of the present
invention a test preparation containing at least 10 compounds is used in a
binding
assay.
Binding assays can be performed using recombinantly produced MCH
receptor polypeptides present in different environments. Such environments
include,
for example, cell extracts and purified cell extracts containing the MCH
receptor
polypeptide expressed from recombinant nucleic acid or naturally occurring
nucleic
acid; and also include, for example, the use of a purified MCH receptor
polypeptide
produced by recombinant means or from naturally occurnng nucleic acid which is
introduced into a different environment.
Screening for MCH Receptor Active Compounds
Screening for MCH active compounds is facilitated using a
recombinantly expressed MCH receptor. Using recombinantly expressed MCH
receptor polypeptides offers several advantages such as the ability to express
the
receptor in a defined cell system so that response to MCH receptor active
compounds
13


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WO 01/57070 PCT/USO1/03293
can more readily be differentiated from responses to other receptors. For
example, the
MCH receptor can be expressed in a cell line such as HEK 293, COS 7, and CHO
not
normally expressing the receptor by an expression vector, wherein the same
cell line
without the expression vector can act as a control.
Screening for MCH receptor active compounds is facilitated through
the use of a truncated MCH analog in the assay. The use of a truncated MCH
analog
in a screening assay provides for MCH receptor activity. The effect of test
compounds on such activity can be measured to identify, for example,
allosteric
modulators and antagonists. Additionally, such assays can be used to identify
agonists.
MCH receptor activity can be measured using different techniques
such as detecting a change in the intracellular conformation of the MCH
receptor, Gi
or Gq activity, and/or intracellular messengers. Gi activity can be measured
using
techniques well known in the art such as a melonaphore assay, assays measuring
cAMP production, inhibition of cAMP accumulation, and binding of 35S-GTP.
cAMP can be measured using different techniques such as radioimmunoassay and
indirectly by cAMP responsive gene reporter proteins.
Gq activity can be measured using techniques such as those measuring
intracellular Ca2+. Examples of techniques well known in the art that can be
employed to measure Ca2+ include the use of dyes such as Fura-2 and the use of
Ca2+-bioluminescent sensitive reporter proteins such as aequorin. An example
of a
cell line employing aequorin to measure G-protein activity is HEK293/aeql7.
(Button, et a1.,1993. Cell Calcium 14, 663-671, and Feighner, et al., 1999.
Science
284, 2184-2188, both of which are hereby incorporated by reference herein.)
Chimeric receptors containing a MCH binding region functionally
coupled to a G protein can also be used to measure MCH receptor activity. A
chimeric MCH receptor contains an N-terminal extracellular domain; a
transmembrane domain made up of transmembrane regions, extracellular loop
regions, and intracellular loop regions; and an intracellular carboxv
terminus.
Techniques for producing chimeric receptors and measuring G protein coupled
responses are provided for in, for example, International Application Number
WO
97/05252, and U.S. Patent Number 5,264,565, both of which are hereby
incorporated
by reference herein.
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Weight or Appetite Alteration
Truncated MCH analogs can be used in methods to increase or
maintain weight and/or appetite in a subject. Such methods can be used, for
example,
as part of an experimental protocol examining the effects of MCH antagonists,
to
achieve a beneficial effect in a subject and/or to further examine the
physiological
effects of MCH.
Experimental protocols examining the effects of MCH antagonists can
be performed, for example, by using a sufficient amount of a truncated MCH
analog
to produce a weight or appetite increase in a subject and then examining the
effect of
a test compound. Changes in weight and appetite can be measured using
techniques
well known in the art.
Increasing weight or appetite can be useful for maintaining weight or
producing a weight or appetite gain in an under weight subject, or in a
patient having
a disease or undergoing treatment that effects weight or appetite. In
addition, for
example, farm animals such as pigs, cows and chickens can be treated to gain
weight.
Under weight subjects include those having a body weight about 10%
or less, 20% or less, or 30% or less, than the lower end of a "normal" weight
range or
Body Mass Index ("BMI"). "Normal" weight ranges are well known in the art and
take into account factors such as a patient age, height, and body type.
BMI measures your height/weight ratio. It is determined by calculating
weight in kilograms divided by the square of height in meters. The BMI
"normal"
range is 19-22.
Administration
Truncated MCH analogs can be formulated and administered to a
subject using the guidance provided herein along with techniques well known in
the
art. The preferred route of administration ensures that an effective amount of
compound reaches the target. Guidelines for pharmaceutical administration in
general
are provided in, for example, Remiizgton's Pharmaceutical Sciences 18'''
Edition, Ed.
Gennaro, Mack Publishing, 1990, and Modern Pharmaceutics 2nd Edition, Eds.
Banker and Rhodes, Marcel Dekker, Inc., 1990, both of which are hereby
incorporated
by reference herein.
Truncated MCH analogs can be prepared as acidic or basic salts.
Pharmaceutically acceptable salts (in the form of water- or oil-soluble or
dispersible
products) include conventional non-toxic salts or the quaternary ammonium
salts that


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
are formed, e.g., from inorganic or organic acids or bases. Examples of such
salts
include acid addition salts such as acetate, adipate, alginate, aspartate,
benzoate,
benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,
fumarate,
glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate,
hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate,
maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,
pamoate,
pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate,
succinate,
tartrate, thiocyanate, tosylate, and undecanoate; and base salts such as
ammonium
salts, alkali metal salts such as sodium and potassium salts, alkaline earth
metal salts
such as calcium and magnesium salts, salts with organic bases such as
dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such
as
arginine and lysine.
Truncated MCH analogs can be administered using different routes
including oral, nasal, by injection, transdermal, and transmucosally. Active
ingredients to be administered orally as a suspension can be prepared
according to
techniques well known in the art of pharmaceutical formulation and may contain
microerystalline cellulose for imparting bulk, alginic acid or sodium alginate
as a
suspending agent, methylcellulose as a viscosity enhancer, and
sweeteners/flavoring
agents. As immediate release tablets, these compositions may contain
microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate
and
lactose and/or other excipients, binders, extenders, disintegrants, diluents
and
lubricants.
Truncated MCH analogs may also be administered in intravenous (both
bolus and infusion), intraperitoneal, subcutaneous, topical with or without
occlusion,
or intramuscular form. When administered by injection, the injectable solution
or
suspension may be formulated using suitable non-toxic, parenterally-acceptable
diluents or solvents, such as Ringer's solution or isotonic sodium chloride
solution, or
suitable dispersing or wetting and suspending agents, such as sterile, bland,
fixed oils,
including synthetic mono- or diglycerides, and fatty acids, including oleic
acid.
Suitable dosing regimens are preferably determined taking into factors
well known in the art including type of subject being dosed; age, weight, sex
and
medical condition of the subject; the route of administration; the renal and
hepatic
function of the subject; the desired effect; and the particular compound
employed.
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Optimal precision in achieving concentrations of drug within the range
that yields efficacy without toxicity requires a regimen based on the kinetics
of the
drug's availability to target sites. This involves a consideration of the
distribution,
equilibrium, and elimination of a drug. The daily dose for a subject is
expected to be
between 0.01 and 1,000 mg per subject per day.
Truncated MCH analogs can be provided in kit. Such a kit typically
contains an active compound in dosage forms for administration. A dosage form
contains a sufficient amount of active compound such that a weight or appetite
increase can be obtained when administered to a subject during regular
intervals, such
as 1 to 6 times a day, during the course of 1 or more days. Preferably, a kit
contains
instructions indicating the use of the dosage form for weight or appetite
increase and
the amount of dosage form to be taken over a specified time period.
EXAMPLES
Examples are provided below to further illustrate different features of
the present invention. The examples also illustrate useful methodology for
practicing
the invention. These examples do not limit the claimed invention.
Example 1: Synthesis of MCH Analog-s
MCH analogs were produced using the procedures described below
and varying the stepwise addition of amino acid groups. Other procedures for
producing and modifying peptides are well known in the art.
Elongation of peptidyl chains on 4-(2',4'-dimethoxyphenyl-Fmoc-
aminomethyl)-phenoxy resin and the acetylation of the N-terminal amino groups
of
the peptides was performed on a 431A ABI peptide synthesizer. Manufacture-
supplied protocols were applied for coupling of the hydroxybenzotriazole
esters of
amino acids in N-methylpyrrolidone (NMP). The fluorenylmethyloxycarbonyl
(Fmoc) group was used as a semipermanent alpha-amino protecting group. whereas
the side chains protecting groups were: tert-butyl for aspartic acid and
tyrosine,
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for arginine, and
trityl for
cysteine.
Peptides were cleaved from the resin with TFA containing ~ % of
anisole. After 2 hours at room temperature the resin was filtered, washed with
TFA
and the combined filtrates were evaporated to dryness in vacuo. The residue
was
17


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triturated with ether, the precipitate which formed was filtered off. washed
with ether,
and dried.
Crude peptides were dissolved in 5 % acetic acid in water, and the pH
of the solutions were adjusted to ca. 8.2 with diluted ammonium hydroxide. The
reaction mixtures were stirred vigorously while 0.05 % solution of potassium
ferricyanide (K3Fe(CN)6) in water was added dropwise till the reaction mixture
remained yellow for about 5 minutes. After an additional 20 minutes oxidation
was
terminated with ca. 1 ml of acetic acid and the reaction mixtures were
lyophilized.
Crude lyophilized peptides were analyzed by analartical reverse-phase
high-pressure liquid chromatography (RP HPLC) on a C18 Vydac column attached
to
a Waters 600E system with automatic Wisp 712 injector and 991 Photodiode Array
detector. A standard gradient system of 0-100% buffer B in 30 minutes was used
for
analysis: buffer A was 0.1 % trifluoroacetic acid in water and buffer B was
0.1 %
trifluoroacetic acid in acetonitrile. HPLC profiles were recorded at 210 nm
and 280
nm. Preparative separations were performed on a Waters Delta Prep 4000 system
with a semipreparative C18 RP Waters column. The above-described solvent
system
of water and acetonitrile, in a gradient of 20-80 % buffer B in 60 minutes,
was used
for separation. The chromatographically homogenous compounds were analyzed by
electrospray mass spectrometry.
Example 2: Aequorin Bioluminescence Functional Assay
The aequorin bioluminescence assay is a reliable test for measuring the
activity of G protein-coupled receptors that couple through the Ga protein
subunit
family consisting of Gq and G11 and leads to the activation of phospholipase
C,
mobilization of intracellular calcium and activation of protein kinase C.
Measurement of MCH receptor activity in the aequorin-expressing
stable reporter cell line 293-AEQ17 (Button et al., Cell Calcium 14:663-671,
1993)
was performed using a Luminoskan RT luminometer (Labsystems Inc.,
Gaithersburg,
MD). 293-AEQ17 cells (8 x 105 cells plated 18 hours before transfection in a
T75
flask) were transfected with 22 ~g of human MCH receptor plasmid using 264 ~g
lipofectamine. The open reading frame cDNA (SEQ. ID. NO. 1 ) encoding the
human
MCH receptor inserted in the mammalian expression vector pcDNA-3 (Invitrogen,
Carlsbad, CA) was used for expression studies. Following approximately 40
hours of
expression the apo-aequorin in the cells was charged for 4 hours with
coelenterazine
(10 p,M) under reducing conditions (300 ~M reduced glutathione) in ECB buffer
(140
18


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WO 01/57070 PCT/USO1/03293
mM NaCI, 20 mM KCI, 20 mM HEPES-NaOH [pH=7.4], 5 mM glucose, 1 mM
MgCl2, 1 mM CaCl2, 0.1 mg/ml bovine serum albumin).
The cells were harvested, washed once in ECB medium and
resuspended to X00,000 cells/ml. 100 ~l of cell suspension (corresponding to
Sx104
cells) was then injected into the test plate containing MCH or MCH analogs,
and the
integrated light emission was recorded over 30 seconds, in 0.5 second units.
20 ~uL of
lysis buffer (0.190 final Triton X-100 concentration) was then injected and
the
integrated light emission recorded over 10 seconds, in 0.5 second units. The
"fractional response" values for each well were calculated by taking the ratio
of the
integrated response to the initial challenge to the total integrated
luminescence
including the Triton X-100 lysis response.
Example 3: Radiolabeled MCH-R Binding Assay
Activity of truncated MCH analogs was assayed by measuring the
ability of the analog to inhibit binding of [lzsl]-human MCH (Phel3, Tyri9
substituted) to membranes prepared from cells stably expressing the human MCH
receptor. Human MCH (Phe~3, Tyrl~ substituted) used in the assay was
radiolabeled
with lzsI at l9Tyr to a specific activity of 2000 Ci/mmol (NEN Life Science
Products,
Boston, MA).
Cell membranes were prepared on ice. Each T-75 flask was rinsed
twice with 10 ml of Enzyme-free Cell Dissociation Buffer (Specialty Media,
Lavallette, NJ), and the cell monolayer was detached in an additional 10 ml of
Enzyme-free Cell Dissociation Buffer by incubation at room temperature for 10
minutes. Dissociated cells were centrifuged (500 x g for 10 minutes at
4°C),
resuspended in ~ ml homogenization buffer (10 mM Tris-HCI, pH 7.4, 0.01 mM
Pefabloc, 10 p.M phosphoramidon, 40 p.g/ml bacitracin) and then homogenized
using
a glass homogenizer (10-15 strokes). The homogenate was centrifuged for 10
minutes
(1,000 x g at 4°C). The resulting supernatant was then centrifuged at
38,700 x g for
15 minutes at 4°C. Pelleted membranes were resuspended (passed through
25 gauge
needle 5 times), snap-frozen on liquid nitrogen, and stored at -80°C
until use.
Binding was performed in a 96-well filter assay or Scintillation
Proximity Assay (SPA)-based format using cell membranes from a stable CHO or
HEK-293 cell line expressing the MCH receptor. For the filter assay, reactions
were
performed at 20°C for 1 hour in a total volume of 0.2 ml containing:
0.05 ml of
membrane suspension (~3 ~.g protein), 0.02 ml of ['25I]-human MCH (Phel3,
Tyr'~
19


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WO 01/57070 PCT/USO1/03293
substituted; 30 pM), 0.01 ml of competitor and 0.12 ml of binding buffer (50
mM
Tris-HC1, pH 7.4, 10 mM MgCl2, 2 mM EDTA, 200 ~g/ml bacitracin, 1 ~,M
phosphoramidon).
Bound radioligand was separated by rapid vacuum filtration (Packard
Filtermate 96-well cell harvester) through GF/C filters pretreated for 1 hour
with 1 %
polyethylenimine. After application of the membrane suspension to the filter,
the
filters were washed 3 times with 3 ml each of ice-cold 50 mM Tris-HCI, pH 7.4,
10
mM MgCl2, 2 mM EDTA, 0.04 % Tween 20 and the bound radioactivity on the
filters was quantitated by scintillation counting (TopCount device). Specific
binding
(>80 % of total) is defined as the difference between total binding and non-
specific
binding conducted in the presence of 100 nM unlabeled human MCH.
For the SPA-based assay, WGA-PVT beads (NEN Life Sciences
Products) were resuspended in Dulbecco's PBS with calcium and magnesium (500
mg beads in 4 ml PBS). For each 96-well assay plate, 0.18 ml of beads was pre-
coated with MCH receptor by mixing with 0.2 ml MCH receptor CHO cell
membranes (~ 0.2-4 mg protein) and 1.5 ml SPA assay buffer (50 mM Tris-HCI, pH
7.4, 10 mM MgCl2, 2 mM EDTA, 0.1 % BSA, 12 % glycerol). The suspension was
mixed gently for 20 minutes, 12.3 ml of assay buffer and protease inhibitors
were
added (final concentration given): 2 ~g/ml leupeptin, 10 qM phosphoramidon, 40
~g/ml bacitracin, 5 ~.g/ml aprotinin, 0.1 mM Pefabloc.
Coated beads were kept on ice until use. For each well, 0.145 ml of
beads were added to Optiplate assay plates (Packard 6005190), followed by
0.002-
0.004 ml of competitor and 0.05 ml of [l2sl]-human MCH (Phel', Tyrl~
substituted;
pM). Binding reactions were allowed to proceed at room temperature for 3
hours.
25 Quantitation was performed by scintillation counting (TopCount device).
Example 4: MCH Activity
The activity of different MCH analogs was measured using the
procedures described in Examples 2 and 3 above. Tables 1-7 illustrate the
activity of
30 different truncated MCH analogs and mammalian MCH (SEQ. ID. NO. 11). Figure
1
illustrates the results of replacing different amino acids of mammalian MCH
with
alanine. Based on the guidance provided herein, additional MCH analogs active
at the
MCH receptor can be obtained.
20


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TABLE 1
SEQ. ID. Binding AssayECSp (nM) % Activation at 10
NO. IC50 (nM) ~M


11 0.3 36 100


7 0.12 18 123 ;


8 0.16 36 123


9 1.6 300 74


0.3 99 j


12 6.4 492 3 I


13 1.5 65.9


14 0.5 62.2


ICSp was determined using a SPA based assay.
ECSp (nM) and % Activation at 10 ~M were determined using aequorin functional
assays.
Table 2 illustrates the affect of different D-amino acids.
TABLE 2
Ac-Arg6-Cys~-Met$-Leu9-G
1y' -Arg"-Val'
2-Tyr'
3-Arg'
4-Pro'
5-Cys'
6-N H2


Activit


SEQ. ID. Compound ICSp ECSp Activation
NO. (nM) (nM) %


11 0.3 30.9 100


7 0.5 20 99


D-ArQ 0.46 45 86


16 D-C s 7.78 909 34


17 D-Met >1000 Inactive


18 D-Leu 1520 Inactive


19 D-ArQ >1000 Inactive


D-Val 381 Inactive


21 D-T r >1000 Inactive


22 D-Ar 368 Inactive


23 D-Pro 584 Inactive


24 D-Cys 0.8 133 76


Table 3 illustrates the effect of different N-methyl-amino acids.
21


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
TABLE 3
Ac-Args-Cys~-M
et$-Le
ug-G lye
-Arg' 1-Val
12-Tyre
3-Arg'
4-P ro
~ 5-Cys16-N
H2


Activit


SEQ. ID. Compound IC50 EC50 Activation
NO. (nM) (nM)


11 0.3 30.9 100


7 1.4 20 99


25 N-Me-Nle 0.16 20 110


26 N-Me-Leu 1070 > 10000 3
@ 1


27 Sar 2.3 140 95


28 N-Me-Ars 43 10 110


29 N-Me-Ara 643 >1000


30 Sar ' 0.36 25 113 ,
j


Table 4 illustrates the affect of different alterations to position 6 of the
SEQ. >D. NO. 7 MCH analog.
TABLE 4
Xs-Cys~-Met$-Leu9-Glyi-Argi
1_Va112-Tyrl3-Argl4-Pro1'-Cysl6-NH2


Activit


SEQ. ID. Position IC50 ECSp Activation
NO. 6 (~) (nM)
modification


11 0.3 30.9 100


7 1.4 20 99


31 Ac-Ala 27 114 135


32 Ac-Nle 40 117 107


33 Ac-Pro 3.4 59 133


34 Ac-Asn 2.6 150 96


35 Ac-Ser 4.5 207 120


36 Ac-Glu 19 935 113


37 H 12 809 120


38 Ac 1.6 144 82


39 Ars 0.13 14 106


40 4 NH,-Ar~0.48 38.5 49


41 Ac-D-Ara 0.46 45 86


42 Ac-D-Nle 1.2 110 97


43 Ac-D-Pro 0.82 60 96


44 Ac-D-Asn 3 340 94


45 Ac-D-Ser 2.3 170 93


46 Ac-D-Glu 8 ~ 820-


22


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
Table 5 illustrates the affect of different alterations to position 10 of
the SEQ. >D. NO. 7 MCH analog.
TART.F 5
Ac-Arg6-Cys~-Met$-Leu9-1
-Arg11-Va112-Tyre
3-Arg t
4-P rot
5-Cys~ 6-N
H2


Activit


SEQ. ID. Position 10 IC50 ECSp Activation
NO. modification ~nMl (~) %


11 0.3 30.9 100


7 0.5 20 99


47 Ala 0.59 31 104


48 Leu 0.06 23 106


49 Nle 0.04 15 106


50 Pro 700 519 4


51 Asn 0.23 23 106


52 Ser 0.32 65 104


53 L s 110 4500 25


54 Glu 190 > 10000 12


55 D-Leu 16 750 23


56 D-Nle 2.4 215 33


57 D-Pro 1.2 190 90


58 D-Glu 40% @ > 10000
1


59 D-L s >1000 >10000


60 -Ala 390 >1000 3.2


61 -Abu 2.1 30.6 101


Table 6 illustrates the affect of different alterations to position 13 of
the SEQ. >D. NO. 7 MCH analog.
23


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
TABLE 6
Ac-Arg6-Cys~-Met$-Leu9-G
1y1 -Arg"-Val~
2-Tyrl3-Argl4-ProlS-Cys16-N
H2


Activit


SEQ. ID. Position 13 ICSp ECSp Activation
NO. modification


11 0.3 30.9 100


7 1.4 20 99


62 Phe 1 46 96


63 T 3.8 890 83


64 His 13.1 3400 66


65 (2~Na1 0.15 54 105


66 Phe( F) 0.6 108 98


67 Phe( NH2) I 3.2 610 88


68 Phe( COON) > 1000 > 10000


I 69 Cha 0.09 122 93


Table 7 illustrates the affect of some alteration combinations and some
alterations to position 8 of the SEQ. >I7. NO. 7 MCH analog.
TABLE 7
Ac-Args-Cys~-Met$-Leu9-G
lye -Arg'
1-Val12-Tyr13-Arg~
4-P ro15-Cys16-N
H2


Activit


SEQ. ID. NO. Compound ICSp ECSp Activation
(~) (nM) %


11 0.3 30.9 100


7 1.4 20 99


70 Ava ' 3.7 587 82


71 D-Ar ,Ava' 3.7 1080 72


72 Ava ' 6.2 406 75


73 D-Ar ,Ava ' ' 19.5 1300 28


74 D-Pro ,Ava '' 700 1530 3


75 DAr ,Ava '' 250 >10000 3


76 Ava ' , Ava ' ' S0 > 10000 3


77 Nle 0.5 44 105


78 DAr~ ,D-Nle 25 72 ( 4


24


CA 02399509 2002-08-05
WO 01/57070 PCT/USOi/03293
Other embodiments are within the following claims. While several
embodiments have been shown and described, various modifications may be made
without departing from the spirit and scope of the present invention.


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
SEQUENCE LISTING
<110> Merck & Co., Inc.
<120> MELANIN-CONCENTRATING HORMONE ANALOGS
<130> 20590Y PCT
<150> 60/179,967
<151> 2000-02-03
<160> 78
<170> FastSEQ for Windows Version 4.0
<210> 1
<211> 1062
<212> DNA
<213> Human
<400>
1


atggacctggaagcctcgctgctgcccactggtcccaacgccagcaacacctctgatggc 60


cccgataacctcacttcggcaggatcacctcctcgcacggggagcatctcctacatcaac 120


atcatcatgccttcggtgttcggcaccatctgcctcctgggcatcatcgggaactccacg 180


gtcatcttcgcggtcgtgaagaagtccaagctgcactggtgcaacaacgtccccgacatc 240


ttcatcatcaacctctcggtagtagatctcctctttctcctgggcatgcccttcatgatc 300


caccagctcatgggcaatggggtgtggcactttggggagaccatgtgcaccctcatcacg 360


gccatggatgccaatagtcagttcaccagcacctacatcctgaccgccatggccattgac 420


cgctacctggccactgtccaccccatctcttccacgaagttccggaagccctctgtggcc 480


accctggtgatctgcctcctgtgggccctctccttcatcagcatcacccctgtgtggctg 540


tatgccagactcatccccttcccaggaggtgcagtgggctgcggcatacgcctgcccaac 600


ccagacactgacctctactggttcaccctgtaccagtttttcctggcctttgccctgcct 660


tttgtggtcatcacagccgcatacgtgaggatcctgcagcgcatgacgtcctcagtggcc 720


cccgcctcccagcgcagcatccggctgcggacaaagagggtgacccgcacagccatcgcc 780


atctgtctggtcttctttgtgtgctgggcaccctactatgtgctacagctgacccagttg 840


tccatcagccgcccgaccctcacctttgtctacttatacaatgcggccatcagcttgggc 900


tatgccaacagctgcctcaacccctttgtgtacatcgtgctctgtgagacgttccgcaaa 960


cgcttggtcctgtcggtgaagcctgcagcccaggggcagcttcgcgctgtcagcaacgct 1020


cagacggctgacgaggagaggacagaaagcaaaggcacctga 1062


<210>
2


<211>
1254


<212>
DNA


<213>
Human


<400>
2


atgaagaagggagtggggagggcagttgggcttggaggcggcagcggctgccaggctacg 60


gaggaagacccccttcccaactgcggggcttgcgctccgggacaaggtggcaggcgctgg 120


aggctgccgcagcctgcgtgggtggaggggagctcagctcggttgtgggagcaggcgacc 180


ggcactggctggatggacctggaagcctcgctgctgcccactggtcccaacgccagcaac 240


acctctgatggccccgataacctcacttcggcaggatcacctcctcgcacggggagcatc 300


tcctacatcaacatcatcatgccttcggtgttcggcaccatctgcctcctgggcatcatc 360


gggaactccacggtcatcttcgcggtcgtgaagaagtccaagctgcactggtgcaacaac 420


gtccccgacatcttcatcatcaacctctcggtagtagatctcctctttctcctgggcatg 480


cccttcatgatccaccagctcatgggcaatggggtgtggcactttggggagaccatgtgc 540


accctcatcacggccatggatgccaatagtcagttcaccagcacctacatcctgaccgcc 600


atggccattgaccgctacctggccactgtccaccccatctcttccacgaagttccggaag 660


ccctctgtggccaccctggtgatctgcctcctgtgggccctctccttcatcagcatcacc 720


cctgtgtggctgtatgccagactcatccccttcccaggaggtgcagtgggctgcggcata 780


cgcctgcccaacccagacactgacctctactggttcaccctgtaccagtttttcctggcc 840


-1-


CA 02399509 2002-08-05
WO 01/5'7070 PCT/USO1/03293
tttgccctgccttttgtggtcatcacagccgcatacgtgaggatcctgcagcgcatgacg 900


tcctcagtggcccccgcctcccagcgcagcatccggctgcggacaaagagggtgacccgc 960


acagccatcgccatctgtctggtcttctttgtgtgctgggcaccctactatgtgctacag "020


ctgacccagttgtccatcagccgcccgaccctcacctttgtctacttatacaatgcggcc .080


atcagcttgggctatgccaacagctgcctcaacccctttgtgtacatcgtgctctgtgag 1140


acgttccgcaaacgcttggtcctgtcggtgaagcctgcagcccaggggcagcttcgcgct "200


gtcagcaacgctcagacggctgacgaggagaggacagaaagcaaaggcacctga "-254


<210>
3


<211>
1269


<212>
DNA


<213>
Human


<400>
3


atgtcagtgggagccatgaagaagggagtggggagggcagttgggcttggaggcggcagc 60


ggctgccaggctacggaggaagacccccttcccaactgcggggcttgcgctccgggacaa 120


ggtggcaggcgctggaggctgccgcagcctgcgtgggtggaggggagctcagctcggttg 180


tgggagcaggcgaccggcactggctggatggacctggaagcctcgctgctgcccactggt 240


cccaacgccagcaacacctctgatggccccgataacctcacttcggcaggatcacctcct 300


cgcacggggagcatctcctacatcaacatcatcatgccttcggtgttcggcaccatctgc 360


ctcctgggcatcatcgggaactccacggtcatcttcgcggtcgtgaagaagtccaagctg 420


cactggtgcaacaacgtccccgacatcttcatcatcaacctctcggtagtagatctcctc 480


tttctcctgggcatgcccttcatgatccaccagctcatgggcaatggggtgtggcacttt 540


ggggagaccatgtgcaccctcatcacggccatggatgccaatagtcagttcaccagcacc 600


tacatcctgaccgccatggccattgaccgctacctggccactgtccaccccatctcttcc 660


acgaagttccggaagccctctgtggccaccctggtgatctgcctcctgtgggccctctcc 720


ttcatcagcatcacccctgtgtggctgtatgccagactcatccccttcccaggaggtgca 780


gtgggctgcggcatacgcctgcccaacccagacactgacctctactggttcaccctgtac 840


cagtttttcctggcctttgccctgccttttgtggtcatcacagccgcatacgtgaggatc 900


ctgcagcgcatgacgtcctcagtggcccccgcctcccagcgcagcatccggctgcggaca 960


aagagggtgacccgcacagccatcgccatctgtctggtcttctttgtgtgctgggcaccc 1020


tactatgtgctacagctgacccagttgtccatcagccgcccgaccctcacctttgtctac 1080


ttatacaatgcggccatcagcttgggctatgccaacagctgcctcaacccctttgtgtac 1140


atcgtgctctgtgagacgttccgcaaacgcttggtcctgtcggtgaagcctgcagcccag 1200


gggcagcttcgcgctgtcagcaacgctcagacggctgacgaggagaggacagaaagcaaa 1260


ggcacctga
1269


<210>
4


<211>
353


<212>
PRT


<213>
Human


<400> 4
Met Asp Leu Glu Ala Ser Leu Leu Pro Thr Gly Pro Asn Ala Ser Asn
1 5 10 15
Thr Ser Asp Gly Pro Asp Asn Leu Thr Ser Ala Gly Ser Pro Pro Arg
20 25 30
Thr Gly Ser Ile Ser Tyr Ile Asn Ile Ile Met Pro Ser Val Phe Gly
35 40 45
Thr Ile Cys Leu Leu Gly Ile Ile Gly Asn Ser Thr Val Ile Phe Ala
50 55 60
Val Val Lys Lys Ser Lys Leu His Trp Cys Asn Asn Val Pro Asp Ile
65 70 75 80
Phe Ile Ile Asn Leu Ser Val Val Asp Leu Leu Phe Leu Leu Gly Met
85 90 95
Pro Phe Met Ile His Gln Leu Met Gly Asn Gly Val Trp His Phe Gly
100 105 110
Glu Thr Met Cys Thr Leu Ile Thr Ala Met Asp Ala Asn Ser Gln Phe
115 120 125
Thr Ser Thr Tyr Ile Leu Thr Ala Met Ala Ile Asp Arg Tyr Leu Ala
130 135 140
-2-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
Thr Val His Pro Ile Ser Ser Thr Lys Phe Arg Lys Pro Ser Val Ala
145 150 155 160
Thr Leu Val Ile Cys Leu Leu Trp Ala Leu Ser Phe Ile Ser Ile Thr
165 170 175
Pro Val Trp Leu Tyr Ala Arg Leu Ile Pro Phe Pro Gly Gly Ala Val
180 185 190
Gly Cys Gly Ile Arg Leu Pro Asn Pro Asp Thr Asp Leu Tyr Trp Phe
195 200 205
Thr Leu Tyr Gln Phe Phe Leu Ala Phe Ala Leu Pro Phe Val Val Ile
210 215 220
Thr Ala Ala Tyr Val Arg Ile Leu Gln Arg Met Thr Ser Ser Val Ala
225 230 235 240
Pro Ala Ser Gln Arg Ser Ile Arg Leu Arg Thr Lys Arg Val Thr Arg
245 250 255
Thr Ala Ile Ala Ile Cys Leu Val Phe Phe Val Cys Trp Ala Pro Tyr
260 265 270
Tyr Val Leu Gln Leu Thr Gln Leu Ser Ile Ser Arg Pro Thr Leu Thr
275 280 285
Phe Val Tyr Leu Tyr Asn Ala Ala Ile Ser Leu Gly Tyr Ala Asn Ser
290 295 300
Cys Leu Asn Pro Phe Val Tyr Ile Val Leu Cys Glu Thr Phe Arg Lys
305 310 315 320
Arg Leu Val Leu Ser Val Lys Pro Ala Ala Gln Gly Gln Leu Arg Ala
325 330 335
Val Ser Asn Ala Gln Thr Ala Asp Glu Glu Arg Thr Glu Ser Lys Gly
340 345 350
Thr
<210> 5
<211> 417
<212> PRT
<213> Human
<400> 5
Met Lys Lys Gly Val Gly Arg Ala Val Gly Leu Gly Gly Gly Ser Gly
1 5 10 15
Cys Gln Ala Thr Glu Glu Asp Pro Leu Pro Asn Cys Gly Ala Cys Ala
20 25 30
Pro Gly Gln Gly Gly Arg Arg Trp Arg Leu Pro Gln Pro Ala Trp Val
35 40 45
Glu Gly Ser Ser Ala Arg Leu Trp Glu Gln Ala Thr Gly Thr Gly Trp
50 55 60
Met Asp Leu Glu Ala Ser Leu Leu Pro Thr Gly Pro Asn Ala Ser Asn
65 70 75 80
Thr Ser Asp Gly Pro Asp Asn Leu Thr Ser Ala Gly Ser Pro Pro Arg
85 90 95
Thr Gly Ser Ile Ser Tyr Ile Asn Ile Ile Met Pro Ser Val Phe Gly
100 105 110
Thr Ile Cys Leu Leu Gly Ile Ile Gly Asn Ser Thr Val Ile Phe Ala
115 120 125
Val Val Lys Lys Ser Lys Leu His Trp Cys Asn Asn Val Pro Asp Ile
130 135 140
Phe Ile Ile Asn Leu Ser Val Val Asp Leu Leu Phe Leu Leu Gly Met
145 150 155 160
Pro Phe Met Ile His Gln Leu Met Gly Asn Gly Val Trp His Phe Gly
165 170 175
Glu Thr Met Cys Thr Leu Ile Thr Ala Met Asp Ala Asn Ser Gln Phe
180 185 190
Thr Ser Thr Tyr Ile Leu Thr Ala Met Ala Ile Asp Arg Tyr Leu Ala
195 200 205
-3-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
Thr Val His Pro Ile Ser Ser Thr Lys Phe Arg Lys Pro Ser Val Ala
210 215 220
Thr Leu Val Ile Cys Leu Leu Trp Ala Leu Ser Phe Ile Ser Ile Thr
225 230 235 240
Pro Val Trp Leu Tyr Ala Arg Leu Ile Pro Phe Pro Gly Gly Ala Val
245 250 255
Gly Cys Gly Ile Arg Leu Pro Asn Pro Asp Thr Asp Leu Tyr Trp Phe
260 265 270
Thr Leu Tyr Gln Phe Phe Leu Ala Phe Ala Leu Pro Phe Val Val Ile
275 280 285
Thr Ala Ala Tyr Val Arg Ile Leu Gln Arg Met Ti-ir Ser Ser Val Ala
290 295 300
Pro Ala Ser Gln Arg Ser Ile Arg Leu Arg Thr Lys Arg Val Thr Arg
305 310 315 320
Thr Ala Ile Ala Ile Cys Leu Val Phe Phe Val Cys Trp Ala Pro Tyr
325 330 335
Tyr Val Leu Gln Leu Thr Gln Leu Ser Ile Ser Arg Pro Thr Leu Thr
340 345 350
Phe Val Tyr Leu Tyr Asn Ala Ala Ile Ser Leu Gly Tyr Ala Asn Ser
355 360 365
Cys Leu Asn Pro Phe Val Tyr Ile Val Leu Cys Glu Thr Phe Arg Lys
370 375 380
Arg Leu Val Leu Ser Val Lys Pro Ala Ala Gln Gly Gln Leu Arg Ala
385 390 395 400
Val Ser Asn Ala Gln Thr Ala Asp Glu Glu Arg Thr Glu Ser Lys Gly
405 410 415
Thr
<210> 6
<211> 422
<212> PRT
<213> Human
<400> 6
Met Ser Val Gly Ala Met Lys Lys Gly Val Gly Arg Ala Val Gly Leu
1 5 10 15
Gly Gly Gly Ser Gly Cys Gln Ala Thr Glu Glu Asp Pro Leu Pro Asn
20 25 30
Cys Gly Ala Cys Ala Pro Gly Gln Gly Gly Arg Arg Trp Arg Leu Pro
35 40 45
Gln Pro Ala Trp Val Glu Gly Ser Ser Ala Arg Leu Trp Glu Gln Ala
50 55 60
Thr Gly Thr Gly Trp Met Asp Leu Glu Ala Ser Leu Leu Pro Thr Gly
65 70 75 80
Pro Asn Ala Ser Asn Thr Ser Asp Gly Pro Asp Asn Leu Thr Ser Ala
85 90 95
Gly Ser Pro Pro Arg Thr Gly Ser Ile Ser Tyr Ile Asn Ile Ile Met
100 105 110
Pro Ser Val Phe Gly Thr Ile Cys Leu Leu Gly Ile Ile Gly Asn Ser
115 120 125
Thr Val Ile Phe Ala Val Val Lys Lys Ser Lys Leu His Trp Cys Asn
130 135 140
Asn Val Pro Asp Ile Phe Ile Ile Asn Leu Ser Val Val Asp Leu Leu
145 150 155 160
Phe Leu Leu Gly Met Pro Phe Met Ile His Gln Leu Met Gly Asn Gly
165 170 175
Val Trp His Phe Gly Glu Thr Met Cys Thr Leu Ile Thr Ala Met Asp
180 185 190
Ala Asn Ser Gln Phe Thr Ser Thr Tyr Ile Leu Thr Ala Met Ala Ile
195 200 205
-4-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
Asp Arg Tyr Leu Ala Thr Val His Pro Ile Ser Ser Thr Lys Phe Arg
210 215 220
Lys Pro Ser Val Ala Thr Leu Val Ile Cys Leu Leu Trp Ala Leu Ser
225 230 235 240
Phe Ile Ser Ile Thr Pro Val Trp Leu Tyr Ala Arg Leu Ile Pro Phe
245 250 255
Pro Gly Gly Ala Val Gly Cys Gly Ile Arg Leu Pro Asn Pro Asp Thr
260 265 270
Asp Leu Tyr Trp Phe Thr Leu Tyr Gln Phe Phe Leu Ala Phe Ala Leu
275 280 285
Pro Phe Val Val Ile Thr Ala Ala Tyr Val Arg Ile Leu Gln Arg Met
290 295 300
Thr Ser Ser Val Ala Pro Ala Ser Gln Arg Ser Ile Arg Leu Arg Thr
305 310 315 320
Lys Arg Val Thr Arg Thr Ala Ile Ala Ile Cys Leu Val Phe Phe Val
325 330 335
Cys Trp Ala Pro Tyr Tyr Val Leu Gln Leu Thr Gln Leu Ser Ile Ser
340 345 350
Arg Pro Thr Leu Thr Phe Val Tyr Leu Tyr Asn Ala Ala Ile Ser Leu
355 360 365
Gly Tyr Ala Asn Ser Cys Leu Asn Pro Phe Val Tyr Ile Val Leu Cys
370 375 380
Glu Thr Phe Arg Lys Arg Leu Val Leu Ser Val Lys Pro Ala Ala Gln
385 390 395 400
Gly Gln Leu Arg Ala Val Ser Asn Ala Gln Thr Ala Asp Glu Glu Arg
405 410 415
Thr Glu Ser Lys Gly Thr
420
<210> 7
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<221> ACETYLATION
<222> (1)...(1)
<221> AMIDATION
<222> (11)...(11)
<221> DISULFID
<222> (2)...(11)
<223> MCH Analog
<400> 7
Arg Cys Met Leu Gly Arg Val Tyr Arg Pro Cys
1 5 10
<210> 8
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<221> ACETYLATION
<222> (1)...(1)
<221> AMIDATION
<222> (12)...(12)
-5-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
<221> DISULFID
<222> (2)...(11)
<223> MCH Analog
<400> 8
Arg Cys Met Leu Gly Arg Val Phe Arg Pro Cys Tyr
1 5 10
<210> 9
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<221> ACETYLATION
<222> (1)...(1)
<221> AMIDATION
<222> (10)...(10)
<221> DISULFID
<222> (1)...(10)
<223> MCH Analog
<400> 9
Cys Met Leu Gly Arg Val Tyr Arg Pro Cys
1 5 10
<210> 10
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<221> AMIDATION
<222> (16)...(16)
<221> DISULFID
<222> (7)...(16)
<223> MCH Analog
<400> 10
Asp Phe Asp Met Leu Arg Cys Met Leu Gly Arg Val Tyr Arg Pro Cys
1 5 10 15
<210> 11
<211> 19
<212> PRT
<213> Human
<220>
<221> DISULFID
<222> (7)...(16)
<400> 11
Asp Phe Asp Met Leu Arg Cys Met Leu Gly Arg Val Tyr Arg Pro Cys
1 5 10 15
Trp Gln Val
-6-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
<210> 12
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> MCH Analog
<221> DISULFID
<222> (1)...(10)
<221> ACETYLATION
<222> (1)...(1)
<400> 12
Cys Met Leu Gly Arg Val Tyr Arg Pro Cys Trp Gln Val
1 5 10
<210> 13
<211> 19
<212> PRT
<213> Artificial Sequence
<220>
<223> MCH Analog
<221> DISULFID
<222> (7)...(16)
<221> MOD_RES
<222> (4) ..(4)
<223> Xaa = Norleucine
<221> MOD_RES
<222> (8) ..(8)
<223> Xaa = Norleucine
<400> 13
Asp Phe Asp Xaa Leu Arg Cys Xaa Leu Gly Arg Val Tyr Arg Pro Cys
1 5 10 15
Trp Gln Val
<210> 14
<211> 19
<212> PRT
<213> Artificial Sequence
<220>
<223> MCH Analog
<221> DISULFID
<222> (7)...(16)
<400> 14
Asp Phe Ala Met Leu Arg Cys Met Leu Gly Arg Val Phe Arg Pro Cys
1 5 10 15
Trp Gln Tyr
<210> 15


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analogs


<221>DISULFID


<222>(2)...(11)


<221>ACETYLATION


<222>(1)...(1)


<221>MOD_RES


<222>(1)...(1)


<223>Xaa = D-Arginine


<221>AMIDATION


<222>(11)...(11)


<400>15


Xaa Tyr Arg Pro
Cys Cys
Met
Leu
Gly
Arg
Val


1 5 10


<210>16


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>DISULFID


<222>(2)...(11)


<221>MOD_RES


<222>(2)...(2)


<223>Xaa = D-Cysteine


<221>ACETYLATION


<222>(1)...(1)


<221>AMIDATION


<222>(11)...(11)


<400>16


Arg Tyr Arg Pro
Xaa Cys
Met
Leu
Gly
Arg
Val


1 5 10


<210>17


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>DISULFID


<222>(2)...(11)


<221>MOD
RES


<222>_
(3) ..(3)


_g_


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
<223> Xaa = D-Methionine
<221>ACETYLATION


<222>(1)...(1)


<221>AMIDATION


<222>(11)...(11)


<400>17


Arg Tyr Arg Pro
Cys Cys
Xaa
Leu
Gly
Arg
Val


1 5 10


<210>18


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>DISULFID


<222>(2)...(11)


<221>MOD
RES


<222>_
(4) .. (4)


<223>Xaa = D-Leucine


<221>ACETYLATION


<222>(1)...(1)


<221>AMIDATION


<222>(11)...(11)


<400>18


Arg Tyr Arg Pro
Cys Cys
Met
Xaa
Gly
Arg
Val


1 5 10


<210>19


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>DISULFID


<222>(2)...(11)


<221>RES
MOD


<222>_
(6) ..(6)


<223>Xaa = D-Arginine


<221>ACETYLATION


<222>(1)...(1)


<221>AMIDATION


<222>(11)...(11)


<400>19


Arg Tyr Arg Pro
Cys Cys
Met
Leu
Gly
Xaa
Val


1 5 10


-9-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
<210> 20
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> MCH Analog
<221> DISULFID
<222> (2)...(11)
<221> MOD_RES
<222> (7) ..(7)
<223> Xaa = D-Valine
<221> ACETYLATION
<222> (1)...(1)
<221> AMIDATION
<222> (11)...(11)
<400> 20
Arg Cys Met Leu Gly Arg Xaa Tyr Arg Pro Cys
1 5 10
<210> 21
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> MCH Analog
<221> DISULFID
<222> (2)...(11)
<221> MOD_RES
<222> (8) ..(8)
<223> Xaa = D-Tyrosine
<221> ACETYLATION
<222> (1)...(1)
<221> AMIDATION
<222> (11)...(11)
<400> 21
Arg Cys Met Leu Gly Arg Val Xaa Arg Pro Cys
1 5 10
<210> 22
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> MCH Analog
<221> ACETYLATION
<222> (1)...(1)
<221> MOD RES
-10-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
<222>(9) . . . (9)


<223>Xaa = D-Arginine


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>22


Arg Tyr Xaa Pro
Cys Cys
Met
Leu
Gly
Arg
Val


1 5 10


<210>23


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1) . . . (1)


<221>MOD_RES


<222>(10)...(10)


<223>Xaa = D-Proline


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>23


Arg Tyr Arg Xaa
Cys Cys
Met
Leu
Gly
Arg
Val


1 5 10


<210>24


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>MOD_RES


<222>(11)...(11)


<223>Xaa = D-Cysteine


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>24


Arg Tyr Arg Pro
Cys Xaa
Met
Leu
Gly
Arg
Val


1 5 10


-11-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
<210>25


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>MOD
RES


<222>_
(3) ..(3)


<223>Xaa = N-Me-Norleucine


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>25


Arg Tyr Arg Pro
Cys Cys
Xaa
Leu
Gly
Arg
Val


1 5 10


<210>26


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>RES
MOD


<222>_
(4) . . (4)


<223>Xaa = N-Me-Leucine


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>26


Arg Tyr Arg Pro
Cys Cys
Met
Xaa
Gly
Arg
Val


1 5 10


<210>27


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


-12-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
<221>MOD
RES


<222>_
(5) ..(5)


<223>Xaa = MeGly


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>27


Arg Tyr Arg Pro
Cys Cys
Met
Leu
Xaa
Arg
Val


1 5 10


<210>28


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>MOD
RES


<222>_
(6) ..(6)


<223>Xaa = N-Me-Arginine


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>28


Arg Tyr Arg Pro
Cys Cys
Met
Leu
Gly
Xaa
Val


1 5 10


<210>29


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>MOD
RES


<222>_
(9) ..(9)


<223>Xaa = N-Me-Arginine


<221>DISUI~FID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400> 29
-13-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
Arg Tyr Xaa Pro
Cys Cys
Met
Leu
Gly
Arg
Val


1 5 10


<210>30


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>MOD_RES


<222>(10)...(10)


<223>Xaa = MeGly


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>30


Arg Tyr Arg Xaa
Cys Cys
Met
Leu
Gly
Arg
Val


1 5 10


<210>31


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>31


Ala Tyr Arg Pro
Cys Cys
Met
Leu
Gly
Arg
Val


1 5 10


<210>32


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>MOD
RES


<222>_
(1) ..(1)


<223>Xaa = Norleucine


<221> DISULFID
14-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
<222> (2)...(11)
<221>AMIDATION


<222>(11)...(11)


<221>ACETYLATION


<222>(1)...(1)


<400>32


Xaa Tyr Arg Pro
Cys Cys
Met
Leu
Gly
Arg
Val


1 5 10


<210>33


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>33


Pro Tyr Arg Pro
Cys Cys
Met
Leu
Gly
Arg
Val


1 5 10


<210>34


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>34


Asn Tyr Arg Pro
Cys Cys
Met
Leu
Gly
Arg
Val


1 5 10


<210>35


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


-15-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
<221>ACETYLATION


<222>(1)...(1)


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>35


Ser Tyr Arg Pro
Cys Cys
Met
Leu
Gly
Arg
Val


1 5 10


<210>36


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>36


Glu Tyr Arg Pro
Cys Cys
Met
Leu
Gly
Arg
Val


1 5 10


<210>37


<211>10


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>AMIDATION


<222>(10)...(10)


<221>DISULFID


<222>(1)...(10)


<400>37


Cys Arg Pro Cys
Met
Leu
Gly
Arg
Val
Tyr


1 5 10


<210>38


<211>10


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


- 16-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
<221> DISULFID
<222> (1)...(10)
<221> AMIDATION
<222> (10)...(10)
<400> 38
Cys Met Leu Gly Arg Val Tyr Arg Pro Cys
1 5 10
<210> 39
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> MCH Analog
<221> AMIDATION
<222> (11)...(11)
<221> DISULFID
<222> (2)...(11)
<400> 39
Arg Cys Met Leu Gly Arg Val Tyr Arg Pro Cys
1 5 10
<210> 40
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> MCH Analog
<221> MOD_RES
<222> (1) ..(1)
<223> Xaa = Des-Amino-Arginine
<221> DISULFID
<222> (2)...(11)
<221> AMIDATION
<222> (11)...(11)
<400> 40
Xaa Cys Met Leu Gly Arg Val Tyr Arg Pro Cys
1 5 10
<210> 41
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> MCH Analog
<221> MOD_RES
<222> (1) ..(1)
<223> Xaa = D-Arginine
- 17-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<221>ACETYLATION


<222>(1)...(1)


<400>41


Xaa Tyr Arg Pro
Cys Cys
Met
Leu
Gly
Arg
Val


1 5 10


<210>42


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>MOD
RES


<222>_
(1) ..(1)


<223>Xaa = D-Norleucine


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<221>ACETYLATION


<222>(1)...(1)


<400>42


Xaa Tyr Arg Pro
Cys Cys
Met
Leu
Gly
Arg
Val


1 5 10


<210>43


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>MOD
RES


<222>_
(1) ..(1)


<223>Xaa = D-Proline


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<221>ACETYLATION


<222>(1)...(1)


<400> 43
-18-


CA 02399509 2002-08-05
WO 01/57070 PCT/US01/03293
Xaa Cys Met Leu Gly Arg Val Tyr Arg Pro Cys
1 5 10
<210> 44
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> MCH Analog
<221> MOD_RES
<222> (1) ..(1)
<223> Xaa = D-Asparagine
<221> DISULFID
<222> (2)...(11)
<221> AMIDATION
<222> (11)...(11)
<221> ACETYLATION
<222> (1)...(1)
<400> 44
Xaa Cys Met Leu Gly Arg Val Tyr Arg Pro Cys
1 5 10
<210> 45
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> MCH Analog
<221> MOD_RES
<222> (1) ..(1)
<223> Xaa = D-Serine
<221> DISULFID
<222> (2)...(11)
<221> AMIDATION
<222> (11)...(11)
<221> ACETYLATION
<222> (1)...(1)
<400> 45
Xaa Cys Met Leu Gly Arg Val Tyr Arg Pro Cys
1 5 10
<210> 46
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> MCH Analog
<221> MOD RES
-19-


CA 02399509 2002-08-05
WO 01/57070 PCT/US01/03293
<222>(1)...(1)


<223>Xaa = D-Glutamic Acid


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<221>ACETYLATION


<222>(1)...(1)


<400>46


Xaa Pro
Cys Cys
Met
Leu
Gly
Arg
Val
Tyr
Arg


1 5 10


<210>47


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>AMIDATION


<222>(11)...(11)


<221>DISULFID


<222>(2)...(11)


<221>ACETYLATION


<222>(1)...(1)


<400>47


Arg Pro
Cys Cys
Met
Leu
Ala
Arg
Val
Tyr
Arg


1 5 10


<210>48


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>AMIDATION


<222>(11)...(11)


<221>DISULFID


<222>(2)...(11)


<400>48


Arg s Met Leu Leu Arg Val Pro
Cy Tyr Arg Cys


1 5 10


<210>49


<211>11


<212>PRT


<213>Artificial Sequence


-20-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>RES
MOD


<222>_
(5) ..(5)


<223>Xaa = Norleucine


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>49


Arg Pro
Cys Cys
Met
Leu
Xaa
Arg
Val
Tyr
Arg


1 5 10


<210>50


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>AMIDATION


<222>(11)...(11)


<221>DISULFID


<222>(2)...(11)


<400>50


Arg Pro
Cys Cys
Met
Leu
Pro
Arg
Val
Tyr
Arg


1 5 10


<210>51


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>AMLDATION


<222>(11)...(11)


<221>DISULFID


<222>(2)...(11)


<400>51


Arg Pro
Cys Cys
Met
Leu
Asn
Arg
Val
Tyr
Arg


1 5 10


-21-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
<210>52


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>AMIDATION


<222>(11)...(11)


<221>DISULFID


<222>(2)...(11)


<400>52


Arg Tyr Arg Pro
Cys Cys
Met
Leu
Ser
Arg
Val


1 5 10


<210>53


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>AMIDATION


<222>(11)...(11)


<221>DISULFID


<222>(2)...(11)


<400>53


Arg Tyr Arg Pro
Cys Cys
Met
Leu
Lys
Arg
Val


1 5 10


<210>54


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>AMIDATION


<222>(11)...(11)


<221>DISULFID


<222>(2)...(11)


<400> 54
-22-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
Arg Cys Met Leu Glu Arg Val Tyr Arg Pro Cys
1 5 10
<210> 55
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> MCH Analog
<221> ACETYLATION
<222> (1)...(1)
<221> MOD_RES
<222> (5) ..(5)
<223> Xaa = D-Leucine
<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>55


Arg Tyr Arg Pro
Cys Cys
Met
Leu
Xaa
Arg
Val


1 5 10


<210>56


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>MOD
RES


<222>_
(5) ..(5)


<223>Xaa = D-Norleucine


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>56


Arg Tyr Arg Pro
Cys Cys
Met
Leu
Xaa
Arg
Val


1 5 10


<210>57


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221> ACETYLATION
-23-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
<222> (1)...(1)
<221>RES
MOD


<222>_
(5) ..(5)


<223>Xaa = D-Proline


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>57


Arg Tyr Arg Pro
Cys Cys
Met
Leu
Xaa
Arg
Val


1 5 10


<210>58


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>MOD
RES


<222>_
(5) ..(5)


<223>Xaa = D-Glutamic
Acid


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>58


Arg Tyr Arg Pro
Cys Cys
Met
Leu
Xaa
Arg
Val


1 5 10


<210>59


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>MOD
RES


<222>_
(5) ..(5)


<223>Xaa = D-Lysine


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


-24-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
<400>59


Arg ys Met Leu Xaa Arg Val Pro
C Tyr Arg Cys


1 5 10


<210>60


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>MOD
RES


<222>_
(5) ..(5)


<223>Xaa = bAla


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>60


Arg Pro
Cys Cys
Met
Leu
Xaa
Arg
Val
Tyr
Arg


1 5 10


<210>61


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>MOD
RES


<222>_
(5) ..(5)


<223>Xaa = Gamma-Aminobutyric
Acid


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>61


Arg Pro
Cys Cys
Met
Leu
Xaa
Arg
Val
Tyr
Arg


1 5 10


<210>62


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


-25-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
<221>ACETYLATION


<222>(1)...(1)


<221>AMIDATION


<222>(11)...(11)


<221>DISULFID


<222>(2)...(11)


<400>62


Arg Phe Arg Pro
Cys Cys
Met
Leu
Gly
Arg
Val


1 5 10


<210>63


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>AMIDATION


<222>(11)...(11)


<221>DISULFID


<222>(2)...(11)


<400>63


Arg Trp Arg Pro
Cys Cys
Met
Leu
Gly
Arg
Val


1 5 10


<210>64


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>AMIDATION


<222>(11)...(11)


<221>DISULFID


<222>(2)...(11)


<400>64


Arg His Arg Pro
Cys Cys
Met
Leu
Gly
Arg
Val


1 5 10


<210>65


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


-26-


CA 02399509 2002-08-05
WO 01/57070 PCT/US01/03293
<221>ACETYLATION


<222>(1)...(1)


<221>MOD
RES


<222>_
(8) ..(8)


<223>Xaa = (2')Naphthylalanine


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>65


Arg Pro
Cys Cys
Met
Leu
Gly
Arg
Val
Xaa
Arg


1 5 10


<210>66


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>MOD
RES


<222>_
(8) ..(8)


<223>Xaa = P-Fluoro-Phenylalanine


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>66


Arg Pro
Cys Cys
Met
Leu
Gly
Arg
Val
Xaa
Arg


1 5 10


<210>67


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>MOD
RES


<222>_
(8) ..(8)


<223>Xaa = P-Amino-Phenylalanine


<221>DISULFID


<222>(2)...(11)


<221> AMIDATION
-27-


CA 02399509 2002-08-05
WO 01/57070 PCT/US01103293
<222> (11)...(11)
<400> 67
Arg Cys Met Leu Gly Arg Val Xaa Arg Pro Cys
1 5 10
<210> 68
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> MCH Analog
<221> ACETYLATION
<222> (1)...(1)
<221> MOD_RES
<222> (8) ..(8)
<223> Xaa = P-Carboxy-Phenylalanine
<221> DISULFID
<222> (2)...(11)
<221> AMIDATION
<222> (11)...(11)
<400> 68
Arg Cys Met Leu Gly Arg Val Xaa Arg Pro Cys
1 5 10
<210> 69
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> MCH Analog
<221> ACETYLATION
<222> (1)...(1)
<221> MOD_RES
<222> (8) . . (8)
<223> Xaa - Cyclohexylalanine
<221> DISULFID
<222> (2)...(11)
<221> AMIDATION
<222> (11)...(11)
<400> 69
Arg Cys Met Leu Gly Arg Val Xaa Arg Pro Cys
1 5 10
<210> 70
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
-28-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
<223> MCH Analog
<221>ACETYLATION


<222>(1)...(1)


<221>MOD
RES


<222>_
(4) ..(5)


<223>Xaa = 5-Aminopentanoic
Acid


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>70


Arg Pro
Cys Cys
Met
Xaa
Xaa
Arg
Val
Tyr
Arg


1 5 10


<210>71


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>MOD
RES


<222>_
(1) ..(1)


<223>Xaa = D-Arginine


<221>MOD
RES


<222>_
(4) ..(5)


<223>Xaa = 5-Aminopentanoic
Acid


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>71


Xaa Pro
Cys Cys
Met
Xaa
Xaa
Arg
Val
Tyr
Arg


1 5 10


<210>72


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>MOD
RES


<222>_
(9) ..(10)


<223>Xaa = 5-Aminopentanoic
Acid


-29-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>72


Arg Tyr Xaa Xaa
Cys Cys
Met
Leu
Gly
Arg
Val


1 5 10


<210>73


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>MOD
RES


<222>_
(1) ..(1)


<223>Xaa = D-Arginine


<221>MOD
RES


<222>_
(9) ..(10)


<223>Xaa = 5-AminopentanoicAcid


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>73


Xaa Tyr Xaa Xaa
Cys Cys
Met
Leu
Gly
Arg
Val


1 5 10


<210>74


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>MOD
RES


<222>_
(5) ..(5)


<223>Xaa = D-Proline


<221>MOD
RES


<222>_
(9) ..(10)


<223>Xaa = 5-AminopentanoicAcid


<221>DISULFID


<222>(2)...(11)


-30-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
<221> AMIDATION
<222> (11)...(11)
<400> 74
Arg Cys Met Leu Xaa Arg Val Tyr Xaa Xaa Cys
1 5 10
<210> 75
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> MCH Analog
<221> ACETYLATION
<222> (1)...(1)
<221> MOD_RES
<222> (1) ..(1)
<223> Xaa = Des-Amino-Arginine
<221> MOD_RES
<222> (9) ..(10)
<223> Xaa = 5-Aminopentanoic Acid
<221> DISULFID
<222> (2)...(11)
<221> AMIDATION
<222> (11)...(11)
<400> 75
Xaa Cys Met Leu Gly Arg Val Tyr Xaa Xaa Cys
1 5 10
<210> 76
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> MCH Analog
<221> ACETYLATION
<222> (1)...(1)
<221> MOD_RES
<222> (4) ..(5)
<223> Xaa = 5-Aminopentanoic Acid
<221> MOD_RES
<222> (9) ..(10)
<223> Xaa = 5-Aminopentanoic Acid
<221> DISULFID
<222> (2)...(11)
<221> AMIDATION
<222> (11)...(11)
<400> 76
-31-


CA 02399509 2002-08-05
WO 01/57070 PCT/USO1/03293
Arg Xaa
Cys Cys
Met
Xaa
Xaa
Arg
Val
Tyr
Xaa


1 5 10


<210>77


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>MOD
RES


<222>_
(3) ..(3)


<223>Xaa = Norleucine


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>77


Arg Pro
Cys Cys
Xaa
Leu
Gly
Arg
Val
Tyr
Arg


1 5 10


<210>78


<211>11


<212>PRT


<213>Artificial Sequence


<220>


<223>MCH Analog


<221>ACETYLATION


<222>(1)...(1)


<221>MOD
RES


<222>_
(1) ..(1)


<223>Xaa = Des-Amino-Arginine


<221>MOD
RES


<222>_
(5) ..(5)


<223>Xaa = D-Norleucine


<221>DISULFID


<222>(2)...(11)


<221>AMIDATION


<222>(11)...(11)


<400>78


Xaa Pro
Cys Cys
Met
Leu
Xaa
Arg
Val
Tyr
Arg


1 5 10


-32-