U.S. patent application number 15/704730 was filed with the patent office on 2018-08-09 for mu opioid receptor agonist analogs of the endomorphins.
This patent application is currently assigned to The Administrators of the Tulane Educational Fund. The applicant listed for this patent is The Administrators of the Tulane Educational Fund, United States Department of Veterans Affairs. Invention is credited to Laszlo HACKLER, James E. ZADINA.
Application Number | 20180222940 15/704730 |
Document ID | / |
Family ID | 45441820 |
Filed Date | 2018-08-09 |
United States Patent
Application |
20180222940 |
Kind Code |
A1 |
ZADINA; James E. ; et
al. |
August 9, 2018 |
MU OPIOID RECEPTOR AGONIST ANALOGS OF THE ENDOMORPHINS
Abstract
The invention relates to cyclic peptide agonists that bind to
the mu (morphine) opioid receptor and their use in the treatment of
acute and/or chronic pain. Embodiments of the invention are
directed to cyclic pentapeptide and hexapeptide analogs of
endomorphin that have (i) a carboxy-terminal extension with an
amidated hydrophilic amino acid and (ii) a substitution in amino
acid position 2. These peptide analogs exhibit decreased tolerance
relative to morphine, increased solubility compared to similar
tetrapeptide analogs, while maintaining favorable or improved
therapeutic ratios of analgesia to side effects.
Inventors: |
ZADINA; James E.; (Metairie,
LA) ; HACKLER; Laszlo; (Metairie, LA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Administrators of the Tulane Educational Fund
United States Department of Veterans Affairs |
New Orleans
Washington |
LA
DC |
US
US |
|
|
Assignee: |
The Administrators of the Tulane
Educational Fund
New Orleans
LA
United States Department of Veterans Affairs
Washington
DC
|
Family ID: |
45441820 |
Appl. No.: |
15/704730 |
Filed: |
September 14, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14268057 |
May 2, 2014 |
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15704730 |
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13477423 |
May 22, 2012 |
8716436 |
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14268057 |
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PCT/US2011/043306 |
Jul 8, 2011 |
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13477423 |
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61363039 |
Jul 9, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2333/70571
20130101; A61K 38/12 20130101; A61P 1/12 20180101; A61P 25/30
20180101; C07K 5/126 20130101; C07K 7/06 20130101; A61P 25/36
20180101; G01N 2500/04 20130101; A61K 38/00 20130101; A61K 45/06
20130101; G01N 2333/726 20130101; G01N 33/9486 20130101; A61P 25/04
20180101; A61P 1/04 20180101; A61P 1/00 20180101; C07K 14/665
20130101; C07K 7/64 20130101 |
International
Class: |
C07K 7/06 20060101
C07K007/06; C07K 14/665 20060101 C07K014/665; A61K 38/12 20060101
A61K038/12; A61K 45/06 20060101 A61K045/06; C07K 5/12 20060101
C07K005/12; G01N 33/94 20060101 G01N033/94; C07K 7/64 20060101
C07K007/64 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] A portion of the work described herein was supported by a
Senior Career Research Scientist Award and Competitive Merit Review
Program funding grant from the Department of Veteran Affairs to
James E. Zadina. The United States government has certain rights in
this invention.
Claims
1. A cyclic peptide of Formula I:
H-Tyr-cyclo[X.sub.1--X.sub.2--X.sub.3--X.sub.4]--X.sub.5 (I),
wherein X.sub.1 and X.sub.4 each independently is an acidic amino
acid or a basic amino acid; X.sub.2 and X.sub.3 each independently
is an aromatic amino acid; X.sub.5 is NHR, Ala-NHR, Arg-NHR,
Asn-NHR, Asp-NHR, Cys-NHR, Glu-NHR, Gln-NHR, Gly-NHR, His-NHR,
Ile-NHR, Leu-NHR, Met-NHR, Orn-NHR, Phe-NHR, Pro-NHR, Ser-NHR,
Thr-NHR, Trp-NHR, Tyr-NHR, or Val-NHR, wherein R is H or an alkyl
group; and there is an amide bond between an amino group and a
carboxylic acid group on side chains of amino acids X.sub.1 and
X.sub.4; with the proviso that when X.sub.1 is an acidic amino
acid, then X.sub.4 is a basic amino acid; and when X.sub.1 is a
basic amino acid, then X.sub.4 is an acidic amino acid.
2. The peptide of claim 1, wherein: (i) X.sub.1 is selected from
the group consisting of D-Lys, D-Orn, Lys, and Orn; and X.sub.4 is
selected from the group consisting of D-Asp, D-Glu, Asp, and Glu;
or (ii) X.sub.1 is selected from the group consisting of D-Asp,
D-Glu, Asp, and Glu; and X.sub.4 is selected from the group
consisting of D-Lys, D-Orn, Lys, and Orn.
3. The peptide of claim 1, wherein: X.sub.2 is selected from the
group consisting of Trp, Phe, and N-alkyl-Phe, wherein the alkyl
group of N-alkyl-Phe comprises 1 to about 6 carbon atoms; and
X.sub.3 is selected from the group consisting of Phe, D-Phe, and
p-Y-Phe, wherein Y is NO.sub.2, F, Cl, or Br.
4. The peptide of claim 3, wherein X.sub.2 is N-methyl-Phe.
5. The peptide of claim 3, wherein X.sub.3 is p-Cl-Phe.
6. The peptide of claim 1, wherein R is H and X.sub.5 is
NH.sub.2.
7. The peptide of claim 1, wherein R is H and X.sub.5 is
Ala-NH.sub.2, Arg-NH.sub.2, Asn-NH.sub.2, Asp-NH.sub.2,
Cys-NH.sub.2, Glu-NH.sub.2, Gln-NH.sub.2, Gly-NH.sub.2,
His-NH.sub.2, Leu-NH.sub.2, Met-NH.sub.2, Orn-NH.sub.2,
Phe-NH.sub.2, Pro-NH.sub.2, Ser-NH.sub.2, Thr-NH.sub.2,
Trp-NH.sub.2, Tyr-NH.sub.2, or Val-NH.sub.2.
8. The peptide of claim 1, wherein the alkyl group is a methyl,
ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl,
hexyl, isohexyl, heptyl, or isoheptyl group.
9. The peptide of claim 1, selected from the group consisting of:
Tyr-cyclo[D-Lys-Trp-Phe-Glu]-NH.sub.2 (SEQ ID NO:1),
Tyr-cyclo[D-Glu-Phe-Phe-Lys]-NH.sub.2 (SEQ ID NO:2),
Tyr-cyclo[D-Lys-Trp-Phe-Glu]-Gly-NH.sub.2 (SEQ ID NO:3),
Tyr-cyclo[D-Glu-Phe-Phe-Lys]-Gly-NH.sub.2 (SEQ ID NO:4),
Tyr-cyclo[D-Lys-Trp-Phe-Asp]-NH.sub.2 (SEQ ID NO:5),
Tyr-cyclo[D-Glu-N-Me-Phe-Phe-Lys]-NH.sub.2 (SEQ ID NO:6), and
Tyr-cyclo[D-Orn-Phe-p-Cl-Phe-Asp]-Val-NH.sub.2 (SEQ ID NO:7).
10. A cyclic peptide of Formula I:
H-Tyr-cyclo[X.sub.1--X.sub.2--X.sub.3--X.sub.41--X.sub.5 (I),
wherein X.sub.1 is a basic D-amino acid and X.sub.4 is an acidic
amino acid; X.sub.2 is Trp and X.sub.3 is an aromatic amino acid;
and X.sub.5 is NHR, wherein R is H or an alkyl group; and there is
an amide bond between an amino group and a carboxylic acid group on
side chains of amino acids X.sub.1 and X.sub.4.
11. The peptide of claim 10, wherein: X.sub.1 is selected from the
group consisting of D-Lys and D-Orn; and X.sub.4 is selected from
the group consisting of D-Asp, D-Glu, Asp, and Glu.
12. The peptide of claim 10, wherein: X.sub.3 is selected from the
group consisting of Phe, D-Phe, and p-Y-Phe, wherein Y is NO.sub.2,
F, Cl, or Br.
13. The peptide of claim 10, wherein X.sub.3 is p-Cl-Phe.
14. The peptide of claim 10, wherein R is H.
15. The peptide of claim 10, wherein R is an alkyl group selected
from methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl,
isopentyl, hexyl, isohexyl, heptyl, and isoheptyl group.
16. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and the peptide of claim 1.
17. A method of treating pain comprising administering to a subject
an analgesic amount of the peptide of claim 1.
18. The method of claim 17, wherein the pain is selected from the
group consisting of pain from a gastrointestinal disorder, chronic
pain, and neuropathic pain.
19. A method for treating a drug dependence comprising
administering to a subject a therapeutically effective amount of
the peptide of claim 1.
20. A method of activating a mu-opioid receptor comprising
contacting the mu-opioid receptor with the peptide of claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S Application Serial
No. 14/268,057, filed on May 2, 2014, which is a divisional of U.S
Application Serial No. 13/477,423, filed on May 22, 2012, now U.S.
Pat. No. 8,716,436, issued May 6, 2014, which is a
continuation-in-part of PCT/US2011/43306, filed on Jul. 8, 2011,
which claims the benefit of U.S. Provisional Application Ser. No.
61/363,039, filed on Jul. 9, 2010, each of which is incorporated
herein by reference in its entirety.
FIELD OF THE INVENTION AND INCORPORATION OF SEQUENCE LISTING
[0003] The present invention relates to peptide agonists that bind
to the mu (morphine) opioid receptor and their use in the treatment
of acute and chronic pain. The biological sequence information in
this application is included in an ASCII text file having the file
name "TU386CIPSEQ.txt", created on Aug. 24, 2012, and having a file
size of 3,011 bytes, which is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0004] Activation of the mu opioid receptor is among the most
effective means of alleviating a wide range of pain conditions. Of
the recently cloned opioid receptors e.g., mu (3,20,21), delta
(6,9), and kappa (12-14), the vast majority of clinically used
opioids act at the mu receptor. As illustrated in genetically
altered "knock-out" mice, the absence of the mu receptor eliminates
the analgesic effects of morphine (8), illustrating its central
role in opioid-induced pain relief. The unique effectiveness of mu
agonists can be attributed to several factors, including their
presence in numerous regions of the nervous system that regulate
pain processing and activation of multiple mechanisms that limit
pain transmission (e.g., inhibiting release of excitatory
transmitters from the peripheral nervous system and decreasing
cellular excitability in the central nervous system).
[0005] Limitations on the use of opioids result from negative side
effects, including abuse liability, respiratory depression, and
cognitive and motor impairment. Major efforts to develop compounds
that maintain analgesic properties while reducing the negative side
effects have met with limited success. This is evident from the
recent epidemic of prescription drug abuse. Numerous attempts at
targeting alternative mechanisms of pain relief to avoid these side
effects have generally been met with similar problems, i.e., a
profile of adverse effects that are different from opioids, but
often sufficiently serious to warrant removal from the market
(e.g., COX inhibitors) or lack of approval to enter the market
(e.g., TRP receptor antagonists). Over 100 million patients
annually in the United States experience acute or chronic pain and
frequently do not achieve adequate relief from existing drugs due
to limited efficacy or excessive side effects.
[0006] Elderly patients tend to show greater sensitivity to severe
pain and recent guidelines of the American Geriatric Society
suggest greater use of opioids and reduction of non-steroidal
anti-inflammatory drugs (NSAIDs) (10). Impairment of motor and
cognitive function can be more debilitating in the elderly than in
younger patients, particularly due to increased risk of fractures
(7). Opioids with reduced motor and cognitive impairment are
therefore a growing unmet need.
[0007] Natural endogenous peptides from bovine and human brain that
are highly selective for the mu opioid receptor relative to the
delta or kappa receptor have been described (23 and U.S. Pat. No.
6,303,578 which is incorporated herein by reference in its
entirety). These peptides are potent analgesics and have shown
promise of reduced abuse liability (22) and respiratory depression
(4,5), as measured in rodent studies. The limited metabolic
stability of the natural peptides led to the development of
cyclized, D-amino acid-containing tetrapeptide analogs of the
endomorphins (U.S. Pat. No. 5,885,958 which is incorporated herein
by reference in its entirety) of sufficient metabolic stability to
produce potent analgesia in rodents after peripheral
administration. A lead compound from this group reportedly was
3-fold more potent than morphine in alleviating neuropathic pain
and showed reduced rewarding properties in animal models that are
correlated with abuse potential. While these results are promising,
the development of additional compounds showing equal or better
properties is desirable. The instant invention addresses this need
by providing peptide analogs having unexpectedly better solubility
and side-effect profiles than the previously described
materials.
SUMMARY OF THE INVENTION
[0008] An embodiment of the instant invention is directed to
pentapeptide and hexapeptide analogs of endomorphins that differ
from the previously described tetrapeptide analogs by having (i) a
carboxy-terminal extension with an amidated hydrophilic amino acid,
(ii) a substitution in amino acid position 2; or (iii) a
combination of (i) and (ii). The pentapeptide and hexapeptide
analogs of the present invention exhibit increased solubility
relative to the tetrapeptides while maintaining favorable
therapeutic ratios of analgesia-to-side effects.
[0009] The compounds of the present invention are cyclic peptides
that act as mu opioid receptor agonists with high affinity. These
compounds provide relief of acute pain, chronic pain, or both, and
comprise or consist of compounds of Formula I:
(I) H-Tyr-cyclo[X.sub.1--X.sub.2--X.sub.3--X.sub.4]--X.sub.5.
X.sub.1 and X.sub.4 each independently is an acidic amino acid
(i.e., an amino acid comprising a carboxylic acid-substituted
side-chain) or a basic amino acid (i.e., an amino acid comprising
an amino-substituted side-chain), with the proviso that if X.sub.1
is an acidic amino acid (e.g., D-Asp or D-Glu), then X.sub.4 is a
basic amino acid (e.g., Lys, Orn, Dpr, or Dab), and vice versa.
Preferably, X.sub.1 is D-Asp, D-Glu, D-Lys, D-Orn, D-Dpr or D-Dab;
while X.sub.4 preferably is Asp, Glu, Lys, Orn, Dpr or Dab. X.sub.2
andX.sub.3 each independently is an aromatic amino acid (i.e., an
amino acid comprising an aromatic group in the side chain thereof).
For example, X.sub.2 preferably is Trp, Phe, or N-alkyl-Phe, where
the alkyl group preferably comprises 1 to about 6 carbon atoms,
i.e., a (C.sub.1 to C.sub.6) alkyl group. X.sub.3 preferably is
Phe, D-Phe, or p-Y-Phe where Y is NO.sub.2, F, Cl, or Br. X.sub.5
is selected from the group consisting of --NHR, Ala-NHR, Arg-NHR,
Asn-NHR, Asp-NHR, Cys-NHR, Glu-NHR, Gln-NHR, Gly-NHR, His-NHR,
Ile-NHR, Leu-NHR, Met-NHR, Orn-NHR, Phe-NHR, Pro-NHR, Ser-NHR,
Thr-NHR, Trp-NHR, Tyr-NHR, and Val-NHR; where R is H or an alkyl
group (e.g. a (C.sub.1 to C.sub.10) alkyl group such as methyl,
ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, isopentyl,
hexyl, isohexyl, heptyl, or isoheptyl). The peptide of Formula I is
cyclic (shown as "c[X.sub.1--X.sub.2--X.sub.3--X.sub.4]" or
"cyclo[X.sub.1--X.sub.2--X.sub.3--X.sub.4]" in the formulas
described herein) by virtue of an amide linkage between the
carboxylic acid and amino substituents of the side chains of amino
acid residues X.sub.1 and X.sub.4. For example, the linkage can be
an amide bond formed between the side chain amino group of the
D-Lys, D-Orn, D-Dpr, D-Dab, Lys, Orn, Dpr, or Dab with the side
chain carboxyl group of D-Asp, D-Glu, Asp, or Glu.
[0010] In one embodiment of the invention directed to a peptide of
Formula I, X.sub.5 is NHR, R is H, and X.sub.5 can be --NH.sub.2
(i.e., the peptide is an amidated pentapeptide), or Ala-NH.sub.2,
Arg-NH.sub.2, Asn-NH.sub.2, Asp-NH.sub.2, Cys-NH.sub.2,
Glu-NH.sub.2, Gln-NH.sub.2, Gly-NH.sub.2, Ile-NH.sub.2,
Leu--NH.sub.2, Met-NH.sub.2, Orn-NH.sub.2, Phe-NH.sub.2,
Pro-NH.sub.2, Ser-NH.sub.2, Thr-NH.sub.2, Trp-NH.sub.2,
Tyr-NH.sub.2, or Val-NH.sub.2, (i.e., the peptide is an amidated
hexapeptide). In one particular embodiment, X.sub.5 is NH.sub.2. In
other particular embodiments, X.sub.5 is Ala-NH.sub.2,
Arg-NH.sub.2, Asn-NH.sub.2, Asp-NH.sub.2, Cys-NH.sub.2,
Glu-NH.sub.2, Gln-NH.sub.2, Gly-NH.sub.2, His-NH.sub.2,
Ile-NH.sub.2, Leu-NH.sub.2, Met-NH.sub.2, Orn-NH.sub.2,
Phe-NH.sub.2, Pro-NH.sub.2, Ser-NH.sub.2, Thr-NH.sub.2,
Trp-NH.sub.2, Tyr-NH.sub.2, or Val-NH.sub.2.
[0011] Another embodiment of the invention is directed to a peptide
of Formula I, wherein X.sub.1 is D-Asp, D-Glu, D-Lys, or D-Orn; and
X.sub.4 is Asp, Glu, Lys, or Orn.
[0012] Another embodiment of the invention is directed to a
compound of Formula I, wherein X.sub.5 is NHR and R is a (C.sub.1
to C.sub.10) alkyl.
[0013] Another embodiment of the invention is directed to a peptide
of Formula I, wherein the aromatic amino acid of X.sub.2 is Trp,
Phe, or N-alkyl-Phe, and the alkyl group of N-alkyl-Phe is a
(C.sub.1 to C.sub.6) alkyl. In one particular embodiment, X.sub.2
is N-methyl-Phe (N-Me-Phe).
[0014] Another embodiment of the invention is directed to a peptide
of Formula I, wherein the aromatic amino acid residue of either
X.sub.2 or X.sub.3 is Phe, D-Phe, Trp, D-Trp, D-Tyr, N-alkyl-Phe,
and the alkyl group of N-alkyl-Phe is (C.sub.1 to C.sub.10) alkyl
or p-Y-Phe, wherein Y is NO.sub.2, F, Cl, or Br.
[0015] Another embodiment of the invention is directed to a peptide
of Formula I, wherein the aromatic amino acid of X.sub.3 is Phe,
D-Phe, or p-Y-Phe, wherein Y is NO.sub.2, F, Cl, or Br. In one
particular embodiment, X.sub.3 is p-Cl-Phe.
[0016] Another embodiment of the invention is directed to a peptide
of Formula I selected from the group consisting of
Tyr-c[D-Lys-Trp-Phe-Glu]-NH.sub.2 (SEQ ID NO:1);
Tyr-c[D-Glu-Phe-Phe-Lys]-NH.sub.2 (SEQ ID NO:2);
Tyr-c[D-Lys-Trp-Phe-Glu]-Gly-NH.sub.2 (SEQ ID NO:3);
Tyr-c[D-Glu-Phe-Phe-Lys]-Gly-NH.sub.2 (SEQ ID NO:4);
Tyr-c[D-Lys-Trp-Phe-Asp]-NH.sub.2 (SEQ ID NO:5);
Tyr-c[D-Glu-N-Me-Phe-Phe-Lys]-NH.sub.2 (SEQ ID NO:6); and
Tyr-c[D-Orn-Phe-p-Cl-Phe-Asp]-Val-NH.sub.2 (SEQ ID NO:7).
[0017] Another aspect of the invention is directed to a
pharmaceutical composition comprising a peptide of Formula I and a
pharmaceutically acceptable carrier (e.g., a diluent or
excipient).
[0018] Yet another aspect of the invention is directed to the use
of a peptide of Formula I in a method of treating a patient having
a condition that responds to an opioid, or a condition for which
opioid treatment is standard in the art. Such a method comprises or
consists of administering to the patient an effective amount of a
peptide of Formula I of the invention. Particular embodiments of
this method can be followed for the purpose of providing at least
one effect selected from (i) analgesia (pain relief), (ii) relief
from a gastrointestinal disorder such as diarrhea, (iii) therapy
for an opioid drug dependence, and (iv) treatment of any condition
for which an opioid is indicated. In some embodiments the peptides
of Formula I can be used to treat acute or chronic pain. Uses for
the peptides of Formula I also include, but are not be limited to,
use as antimigraine agents, immunomodulatory agents,
immunosuppressive agents or antiarthritic agents. Certain
embodiments of the methods of the present invention, such as
treatment of pain or opioid drug dependence, are directed to
patients having a history of opioid substance abuse. In certain
embodiments of the present methods, the peptide is administered
parenterally (e.g., intravenous). This invention also relates to a
peptide of Formula I for use in one of said methods of
treatment.
[0019] Another aspect of the invention is directed to a method of
activating or regulating a mu-opioid receptor by contacting the
mu-opioid receptor with a compound of the invention, as well as the
use of the peptide of Formula I in such a treatment.
[0020] Another aspect of the invention is directed to a method of
measuring the quantity of a mu opioid receptor in a sample using a
peptide of Formula I. This method can comprise or consist of the
following steps: (i) contacting a sample suspected of containing a
mu opioid receptor with a peptide of Formula Ito form a
compound-receptor complex, (ii) detecting the complex, and (iii)
quantifying the amount of complex formed.
[0021] Another aspect of the invention is directed to the use of a
peptide of Formula Ito perform a competitive assay method of
detecting the presence of a molecule that binds to a mu opioid
receptor. This method can comprise or consist of the following
steps: (i) contacting a sample suspected of containing a molecule
that binds to a mu opioid receptor with a mu opioid receptor and a
peptide of Formula I, wherein the compound and receptor form a
compound-receptor complex; (ii) measuring the amount of the complex
formed in step (i); and (iii) comparing the amount of complex
measured in step (ii) with the amount of a complex formed between
the mu opioid receptor and the peptide in the absence of said
sample.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 shows Tyr-c[D-Lys-Trp-Phe-Glu]-NH.sub.2 (SEQ ID
NO:1), which is described as "Compound 1" in the following
disclosure. The structural and basic molecular formulae, as well as
the molecular weight (MW), are shown for Compound 1.
[0023] FIG. 2 shows Tyr-c[D-Glu-Phe-Phe-Lys]-NH.sub.2 (SEQ ID
NO:2), which is described as "Compound 2" in the following
disclosure. The structural and basic molecular formulae, as well as
the molecular weight (MW), are shown for Compound 2.
[0024] FIG. 3 shows Tyr-c[D-Glu-Phe-Phe-Lys]-Gly-NH.sub.2 (SEQ ID
NO:4), which is described as "Compound 4" in the following
disclosure. The structural and basic molecular formulae, as well as
the molecular weight (MW), are shown for Compound 4.
[0025] FIG. 4 shows opioid receptor binding activity for Compound
1. (A) mu receptor binding of "Compound 1" (triangles) or DAMGO
(squares). (B) Antagonist activity of Compound 1 against binding of
SNC80 to delta receptor.
[0026] FIG. 5 shows effects of compounds on antinociception and
respiration. (A) Effects of Compounds 1 and 2 on antinociception as
compared with morphine. **=p<0.01. (B) Effects of Compounds 1
and 2 on respiratory minute volume (MV) over a 20-minute period as
compared to morphine. *p<0.05. ***p<0.001.
[0027] FIG. 6 shows the effects of Compound 2 on antinociception
and motor impairment. (A) The effects of Compound 2 (filled
triangles) and morphine sulfate (MS, filled squares) on
antinociception were measured by the tail flick (TF) test. Also,
the effects of Compound 2 (open triangles) and morphine sulfate
(open squares) on motor behavior were measured. (*=p<0.05). (B)
The bar graph shows the ratio of the area under the curve (AUC) for
percent motor impairment relative to the AUC for percent
antinociception. This ratio is significantly greater (*p<0.05)
for morphine than for Compound 2, consistent with greater motor
impairment relative to analgesia for morphine.
[0028] FIG. 7 shows the effects of compounds on drug abuse
liability. (A) The effects of Compound 1 (filled triangles),
morphine (filled squares), and vehicle (filled circles) on
antinociception were measured by the tail flick (TF) test.
*p<0.05. (B) The cumulative doses of either morphine or Compound
1 that were shown to produce maximal antinociception as shown in
(A) were tested for the ability to induce conditioned place
preference (CPP). ***p<0.01.
[0029] FIG. 8 shows the duration and relative potency of compounds
in reversing chronic pain induced by nerve injury (neuropathic
pain). (A) The decrease in paw pressure required for withdrawal
after nerve injury surgery was reversed by morphine and Compounds
1, 2, and 5 (squares, down triangles, diamonds, and up triangles,
respectively). Times at which the reversal was significantly above
vehicle (p<0.05 to 0.001) are shown in bars at the top. Scores
for Compound 1 were also significantly above those of morphine from
155 to 215 min (dashed bar). Compound 5 showed similar reversal (80
min) relative to morphine, and Compounds 1 and 2 showed
significantly longer reversal (120 and 260 min, respectively)
relative to morphine. (B) Dose-response curves show that all three
analogs are significantly more potent than morphine, as determined
by the dose required to fully (100%) reverse hyperalgesia
(pre-surgical minus post-surgical pressure).
[0030] FIG. 9 shows the extent of tolerance produced by intrathecal
delivery of morphine or Compound 2 for 1 week via an osmotic
minipump. Cumulative dose-response curves (four increasing
quarter-log doses) were used and responses expressed as % maximum
possible effect (% MPE) in a tail-flick test were determined before
and after implantation of a minipump. The shift in ED.sub.50 after
Compound 2 (about 8.5-fold) was significantly less than that after
morphine (64 fold), consistent with reduced induction of tolerance
by the analog. Similar results were observed with Compounds 1 and
5.
[0031] FIG. 10 shows activation of glia after 1 week of treatment
with morphine but not analogs. Integrated density of GFAP (A) and
pp38 (B) staining in morphine-treated, but not analog-treated rats
is significantly increased relative to those given vehicle. In
addition, the density of staining after morphine is significantly
greater than that after analogs (*, **, ***=p<0.05, 0.01,
0.001,respectively; n=5-7).
DETAILED DESCRIPTION OF THE INVENTION
[0032] Peptides of Formula I, which are cyclic pentapeptide and
hexapeptide analogs of endomorphin-1 (Tyr-Pro-Trp-Phe-NH.sub.2, SEQ
ID NO:8) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH.sub.2, SEQ ID NO:9)
were prepared. In each case, the cyclic portion of the peptide is
formed from amino acid residues 2 through 4, while the Tyr residue
(residue 1) is attached to residue 2 as a branch. Non-limiting
examples of peptides with the composition of Formula I include
Compounds 1-7 below, wherein the side chains of amino acid residues
2 (X.sub.1) and 5 (X.sub.4) in the sequence are linked by an amide
bond between the side-chains thereof. The formulae of Compounds 1,
2, 3, 4, 5, 6, and 7 are shown in Table 1.
TABLE-US-00001 TABLE 1 Compound H-Tyr- X.sub.1- X.sub.2- X.sub.3-
X.sub.4- X.sub.5 SEQ ID NO: 1 Tyr- c[D-Lys Trp Phe Glu] NH.sub.2
(SEQ ID NO: 1) 2 Tyr- c[D-Glu Phe Phe Lys] NH.sub.2 (SEQ ID NO: 2)
3 Tyr- c[D-Lys Trp Phe Glu] Gly-NH.sub.2 (SEQ ID NO: 3) 4 Tyr-
c[D-Glu Phe Phe Lys] Gly-NH.sub.2 (SEQ ID NO: 4) 5 Tyr- c[D-Lys Trp
Phe Asp] NH.sub.2 (SEQ ID NO: 5) 6 Tyr- c[D-Glu N--Me-Phe Phe Lys]
NH.sub.2 (SEQ ID NO: 6) 7 Tyr- c[D-Orn Phe p-Cl-Phe Asp]
Val-NH.sub.2 (SEQ ID NO: 7)
[0033] In some embodiments, the peptides of Formula I includes
peptides with an N-alkylated phenylalanine in position 3 (X.sub.2).
Alkyl groups suitable in the peptides of the present invention
include (C.sub.1 to C.sub.10) alkyl groups, preferably (C.sub.1 to
C.sub.6) alkyl groups (e.g., methyl or ethyl). Compound 6
illustrates a cyclic analog whose linear primary amino acid
sequence contains an N-methylated phenylalanine in position 3.
Other peptides of this invention include compounds wherein the
amino acid at position 4 (X.sub.3) is p-Y-phenylalanine, wherein Y
is NO.sub.2, F, Cl or Br, in order to enhance receptor binding and
potency. An exemplary peptide (Compound 7), whose linear primary
amino acid sequence is provided in SEQ ID NO:7, has a
p-chlorophenylalanine (p-Cl-Phe) in position 4.
[0034] Compounds 1 (FIG. 1), 2 (FIGS. 2), 5 and 6 are examples of
cyclic pentapeptides, and Compounds 3, 4 (FIGS. 3) and 7 are
examples of cyclic hexapeptides of the instant invention.
[0035] For reference, the abbreviations for amino acids described
herein include alanine (Ala), arginine (Arg), asparagine (Asn),
aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid
(Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine
(Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline
(Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine
(Tyr), valine (Val), ornithine (Orn), naphthylalanine (Nal),
2,3-diaminopropionic acid (Dpr), and 2,4-diaminobutyric acid (Dab).
The L- or D-enantiomeric forms of these and other amino acids can
be included in the peptides of Formula I. Other amino acids, or
derivatives or unnatural forms thereof such as those listed in the
2009/2010 Aldrich Handbook of Fine Chemicals (incorporated herein
by reference in its entirety, particularly those sections therein
listing amino acid derivatives and unnatural amino acids) can be
used in preparing compounds of the invention.
[0036] In Formula I, X.sub.1 can be, for example, D-Asp, D-Glu,
D-Lys, D-Orn, D-Dpr or D-Dab, and X.sub.4 can be, for example, Asp,
Glu, Lys, Orn, Dpr or Dab. In general, an amino acid or derivative
thereof can be used as X.sub.1 or X.sub.4 if it contains either an
amino group or a carboxyl group in its side chain. In some
embodiments, the amino acid used for X.sub.1 can be a
D-enantiomeric form of such amino acid.
[0037] X.sub.2 and X.sub.3 in Formula I are aromatic amino acids.
Examples of such amino acids are unsubstituted or substituted
aromatic amino acids selected from the group consisting of
phenylalanine, heteroarylalanine, naphthylalanine (Nal),
homophenylalanine, histidine, tryptophan, tyrosine, arylglycine,
heteroarylglycine, thyroxine, aryl-beta-alanine, and
heteroaryl-beta-alanine. Examples of substituted versions of these
aromatic amino acids are disclosed in U.S. Pat. No. 7,629,319,
which is herein incorporated by reference in its entirety. As used
herein, "aromatic amino acid" refers to an a-amino acid comprising
an aromatic group (including aromatic hydrocarbon and aromatic
heterocyclic groups) in the side-chain thereof.
[0038] In some embodiments, X.sub.2 in Formula I can be
N-alkyl-Phe, where the alkyl group comprises 1 to about 6 carbon
atoms. Alternatively, the alkyl group can comprise about 1, 2, 3,
4, 5, 6, 7, 8, 9, or 10 carbons, for example. The alkyl group can
be a methyl (i.e., X.sub.2 is N-Me-Phe), ethyl, propyl, isopropyl,
butyl, isobutyl, pentyl, isopentyl, hexyl, isohexyl, heptyl, or
isoheptyl group, or any other branched form thereof, for example.
By definition, the alkyl group of N-alkyl-Phe is linked to the
a-amino group of phenylalanine. This alpha amino group is involved
in an amide bond with the X.sub.1 residue in certain peptides of
the invention; therefore, the alpha amino group of X.sub.2 (when
N-alkyl-Phe) as it exists in such peptides is a tertiary amide.
[0039] In some embodiments X.sub.3 in Formula I is para-Y-Phe
(p-Y-Phe), where Y is NO.sub.2, F, Cl, or Br, for example. For
example, X.sub.3 can be p-Cl-Phe. Alternatively, the NO.sub.2, F,
Cl, or Br groups can be linked in the ortho or meta positions of
the phenyl ring of Phe. Any aromatic amino acid incorporated in the
compounds of the invention such as at X.sub.2 or X.sub.3 can have
the above groups linked thereto in the ortho, meta, or para
positions.
Solubility.
[0040] The solubility of the peptides of Formula I (e.g., in saline
or physiologic buffer) typically is enhanced relative to the prior
art tetrapeptide analogs of the endomorphins. Addition of a
hydrophilic amino acid and amidated C-terminus to the relatively
hydrophobic tetrapeptide sequences Tyr-cyclo[D-Lys-Trp-Phe] (SEQ ID
NO:10) and Tyr-cyclo[D-Lys-Phe-Phe] (SEQ ID NO:11), resulted in an
unexpectedly high improvement in solubility while maintaining or
improving functionality. For example, Compound 1 was soluble in
water, saline and 20% PEG/saline at about 43, 21 and 90 mg/mL,
respectively, compared to less than about 2 mg/mL for the
previously described compounds. While increases in solubility are
associated with improved pharmaceutical delivery properties, higher
solubility is also often associated with reduced functional
activity (e.g., receptor binding) that may depend on lipophilicity.
Surprisingly however, as described in examples below, the
functional properties of the compounds of the invention are not
diminished, and indeed are generally improved.
Methods of Preparation of the Peptides of Formula I.
[0041] The peptides of Formula I can be prepared by conventional
solution phase (2) or solid phase (18) methods with the use of
proper protecting groups and coupling agents; references 2 and 20
are herein incorporated by reference in their entirety. Such
methods generally utilize various protecting groups on the various
amino acid residues of the peptides. A suitable deprotection method
is employed to remove specified or all of the protecting groups,
including splitting off the resin if solid phase synthesis is
applied. The peptides can be synthesized, for example, as described
below.
[0042] Peptides of Formula I were synthesized on Rink Amide resin
via Fmoc chemistry. A t-butyl group was used for Tyr, Glu, Asp side
chain protection and Boc was used for Lys, Orn and Trp side chain
protection. All materials were obtained from EMD Biosciences, Inc
(San Diego, Calif.). The peptide was assembled on Rink Amide resin
by repetitive removal of the Fmoc protecting group and coupling of
protected amino acid. HBTU
(O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate;
CAS # 94790-37-1) and HOBT (N-hydroxybenzotriazole; CAS #
2592-95-2) were used as coupling reagents in N,N-dimethylformamide
(DMF) and diisopropylethylamine (DIPEA) was used as a base. The
resin was treated with an aqueous cocktail of trifluoroacetic acid
and triisopropylsilane (TFA/TIS/H.sub.2O cocktail) for cleavage and
removal of the side chain protecting groups. Crude peptide was
precipitated with diethyl ether and collected by filtration.
[0043] Cyclization of the linear
Fmoc-Tyr-c[X.sub.1--X.sub.2--X.sub.3--X.sub.4]--X.sub.5 precursors:
About 1 mmol of peptide was dissolved in about 1000 mL DMF and
about 2 mmol DIPEA was added to the solution, followed by a
solution of HBTU (about 1.1 mmol) and HOBT (about 1.1 mmol) in
about 100 mL DMF. The reaction mixture was stirred at room
temperature overnight. Solvent was removed in vacuo. The resulting
solid residue was washed with 5% citric acid, saturated NaCl,
saturated NaHCO.sub.3, and water. The final solid was washed with
diethyl ether and dried under high vacuum.
[0044] Preparation of
Tyr-c[X.sub.1--X.sub.2--X.sub.3--X.sub.4]--X.sub.5 peptides. The
solids obtained above were dissolved in 20% piperidine/DMF. The
mixture was stirred at room temperature for about 1 hour. Solvent
was removed in vacuo. Residues were dissolved in 10% aqueous
acetonitrile (MeCN/H.sub.2O) and lyophilized.
[0045] Purification of the crude lyophilized peptides was performed
with reverse phase high performance liquid chromatography
(RP-HPLC). The HPLC system GOLD 32 KARAT (Beckman) consisting of
the programmable solvent module 126 and the diode array detector
module 168 was used in the purification and the purity control of
the peptides. Reverse phase HPLC was performed using a gradient
made from two solvents: (A) 0.1% TFA in water and (B) 0.1% TFA in
acetonitrile. For preparative runs, a VYDAC 218TP510 column
(250.times.10 mm; Alltech Associates, Inc.) was used with a
gradient of 5-20% solvent B in solvent A over a period of 10 min,
20-25% B over a period of 30 minutes, 25-80% B over a period of 1
minute and isocratic elution over 9 minutes at a flow rate of about
4 mL/min, absorptions being measured at both 214 and 280 nm. The
same gradient was used for analytical runs on a VYDAC 218TP54
column (250.times.4.6 mm) at a flow rate of about 1 mL/min.
Pharmaceutical Preparations.
[0046] The instant invention also provides pharmaceutical
preparations which contain a pharmaceutically effective amount of
the peptides in a pharmaceutically acceptable carrier (e.g., a
diluent, complexing agent, additive, excipient, adjuvant and the
like). The peptide can be present for example in a salt form, a
micro-crystal form, a nano-crystal form, a co-crystal form, a
nanoparticle form, a mirocparticle form, or an amphiphilic form.
The carrier can be an organic or inorganic carrier that is suitable
for external, enteral or parenteral applications. The peptides of
the present invention can be compounded, for example, with the
usual non-toxic, pharmaceutically acceptable carriers for tablets,
pellets, capsules, liposomes, suppositories, intranasal sprays,
solutions, emulsions, suspensions, aerosols, targeted chemical
delivery systems (15), and any other form suitable for use.
Non-limiting examples of carriers that can be used include water,
glucose, lactose, gum acacia, gelatin, mannitol, starch paste,
magnesium trisilicate, talc, corn starch, keratin, colloidal
silica, potato starch, urea and other carriers suitable for use in
manufacturing preparations, in solid, semisolid, liquid or aerosol
form. In addition auxiliary, stabilizing, thickening and coloring
agents and perfumes can be used.
The present invention also provides pharmaceutical compositions
useful for treating pain and related conditions, as described
herein. The pharmaceutical compositions comprise at least one
peptide of Formula I in combination with a pharmaceutically
acceptable carrier, vehicle, or diluent, such as an aqueous buffer
at a physiologically acceptable pH (e.g., pH 7 to 8.5), a
polymer-based nanoparticle vehicle, a liposome, and the like. The
pharmaceutical compositions can be delivered in any suitable dosage
form, such as a liquid, gel, solid, cream, or paste dosage form. In
one embodiment, the compositions can be adapted to give sustained
release of the peptide.
[0047] In some embodiments, the pharmaceutical compositions
include, but are not limited to, those forms suitable for oral,
rectal, nasal, topical, (including buccal and sublingual),
transdermal, vaginal, parenteral (including intramuscular,
subcutaneous, and intravenous), spinal (epidural, intrathecal), and
central (intracerebroventricular) administration. The compositions
can, where appropriate, be conveniently provided in discrete dosage
units. The pharmaceutical compositions of the invention can be
prepared by any of the methods well known in the pharmaceutical
arts. Some preferred modes of administration include intravenous
(iv), topical, subcutaneous, oral and spinal.
[0048] Pharmaceutical formulations suitable for oral administration
include capsules, cachets, or tablets, each containing a
predetermined amount of one or more of the peptides, as a powder or
granules. In another embodiment, the oral composition is a
solution, a suspension, or an emulsion. Alternatively, the peptides
can be provided as a bolus, electuary, or paste. Tablets and
capsules for oral administration can contain conventional
excipients such as binding agents, fillers, lubricants,
disintegrants, colorants, flavoring agents, preservatives, or
wetting agents. The tablets can be coated according to methods well
known in the art, if desired. Oral liquid preparations include, for
example, aqueous or oily suspensions, solutions, emulsions, syrups,
or elixirs. Alternatively, the compositions can be provided as a
dry product for constitution with water or another suitable vehicle
before use. Such liquid preparations can contain conventional
additives such as suspending agents, emulsifying agents,
non-aqueous vehicles (which may include edible oils),
preservatives, and the like. The additives, excipients, and the
like typically will be included in the compositions for oral
administration within a range of concentrations suitable for their
intended use or function in the composition, and which are well
known in the pharmaceutical formulation art. The peptides of the
present invention will be included in the compositions within a
therapeutically useful and effective concentration range, as
determined by routine methods that are well known in the medical
and pharmaceutical arts. For example, a typical composition can
include one or more of the peptides at a concentration in the range
of at least about 0.01 nanomolar to about 1 molar, preferably at
least about 1 nanomolar to about 100 millimolar.
[0049] Pharmaceutical compositions for parenteral, spinal, or
central administration (e.g. by bolus injection or continuous
infusion) or injection into amniotic fluid can be provided in unit
dose form in ampoules, pre-filled syringes, small volume infusion,
or in multi-dose containers, and preferably include an added
preservative. The compositions for parenteral administration can be
suspensions, solutions, or emulsions, and can contain excipients
such as suspending agents, stabilizing agents, and dispersing
agents. Alternatively, the peptides can be provided in powder form,
obtained by aseptic isolation of sterile solid or by lyophilization
from solution, for constitution with a suitable vehicle, e.g.
sterile, pyrogen-free water, before use. The additives, excipients,
and the like typically will be included in the compositions for
parenteral administration within a range of concentrations suitable
for their intended use or function in the composition, and which
are well known in the pharmaceutical formulation art. The peptides
of the present invention will be included in the compositions
within a therapeutically useful and effective concentration range,
as determined by routine methods that are well known in the medical
and pharmaceutical arts. For example, a typical composition can
include one or more of the peptides at a concentration in the range
of at least about 0.01 nanomolar to about 100 millimolar,
preferably at least about 1 nanomolar to about 10 millimolar.
[0050] Pharmaceutical compositions for topical administration of
the peptides to the epidermis (mucosal or cutaneous surfaces) can
be formulated as ointments, creams, lotions, gels, or as a
transdermal patch. Such transdermal patches can contain penetration
enhancers such as linalool, carvacrol, thymol, citral, menthol,
t-anethole, and the like. Ointments and creams can, for example,
include an aqueous or oily base with the addition of suitable
thickening agents, gelling agents, colorants, and the like. Lotions
and creams can include an aqueous or oily base and typically also
contain one or more emulsifying agents, stabilizing agents,
dispersing agents, suspending agents, thickening agents, coloring
agents, and the like. Gels preferably include an aqueous carrier
base and include a gelling agent such as cross-linked polyacrylic
acid polymer, a derivatized polysaccharide (e.g., carboxymethyl
cellulose), and the like. The additives, excipients, and the like
typically will be included in the compositions for topical
administration to the epidermis within a range of concentrations
suitable for their intended use or function in the composition, and
which are well known in the pharmaceutical formulation art. The
peptides of the present invention will be included in the
compositions within a therapeutically useful and effective
concentration range, as determined by routine methods that are well
known in the medical and pharmaceutical arts. For example, a
typical composition can include one or more of the peptides at a
concentration in the range of at least about 0.01 nanomolar to
about 1 molar, preferably at least about 1 nanomolar to about 100
millimolar.
[0051] Pharmaceutical compositions suitable for topical
administration in the mouth (e.g., buccal or sublingual
administration) include lozenges comprising the peptide in a
flavored base, such as sucrose, acacia, or tragacanth; pastilles
comprising the peptide in an inert base such as gelatin and
glycerin or sucrose and acacia; and mouthwashes comprising the
active ingredient in a suitable liquid carrier. The pharmaceutical
compositions for topical administration in the mouth can include
penetration enhancing agents, if desired. The additives,
excipients, and the like typically will be included in the
compositions of topical oral administration within a range of
concentrations suitable for their intended use or function in the
composition, and which are well known in the pharmaceutical
formulation art. The peptides of the present invention will be
included in the compositions within a therapeutically useful and
effective concentration range, as determined by routine methods
that are well known in the medical and pharmaceutical arts. For
example, a typical composition can include one or more of the
peptides at a concentration in the range of at least about 0.01
nanomolar to about 1 molar, preferably at least about 1 nanomolar
to about 100 millimolar.
[0052] A pharmaceutical composition suitable for rectal
administration comprises a peptide of the present invention in
combination with a solid or semisolid (e.g., cream or paste)
carrier or vehicle. For example, such rectal compositions can be
provided as unit dose suppositories. Suitable carriers or vehicles
include cocoa butter and other materials commonly used in the art.
The additives, excipients, and the like typically will be included
in the compositions of rectal administration within a range of
concentrations suitable for their intended use or function in the
composition, and which are well known in the pharmaceutical
formulation art. The peptides of the present invention will be
included in the compositions within a therapeutically useful and
effective concentration range, as determined by routine methods
that are well known in the medical and pharmaceutical arts. For
example, a typical composition can include one or more of the
peptides at a concentration in the range of at least about 0.01
nanomolar to about 1 molar, preferably at least about 1 nanomolar
to about 100 millimolar.
[0053] According to one embodiment, pharmaceutical compositions of
the present invention suitable for vaginal administration are
provided as pessaries, tampons, creams, gels, pastes, foams, or
sprays containing a peptide of the invention in combination with
carriers as are known in the art. Alternatively, compositions
suitable for vaginal administration can be delivered in a liquid or
solid dosage form. The additives, excipients, and the like
typically will be included in the compositions of vaginal
administration within a range of concentrations suitable for their
intended use or function in the composition, and which are well
known in the pharmaceutical formulation art. The peptides of the
present invention will be included in the compositions within a
therapeutically useful and effective concentration range, as
determined by routine methods that are well known in the medical
and pharmaceutical arts. For example, a typical composition can
include one or more of the peptides at a concentration in the range
of at least about 0.01 nanomolar to about 1 molar, preferably at
least about 1 nanomolar to about 100 millimolar.
[0054] Pharmaceutical compositions suitable for intra-nasal
administration are also encompassed by the present invention. Such
intra-nasal compositions comprise a peptide of the invention in a
vehicle and suitable administration device to deliver a liquid
spray, dispersible powder, or drops. Drops may be formulated with
an aqueous or non-aqueous base also comprising one or more
dispersing agents, solubilizing agents, or suspending agents.
Liquid sprays are conveniently delivered from a pressurized pack,
an insufflator, a nebulizer, or other convenient means of
delivering an aerosol comprising the peptide. Pressurized packs
comprise a suitable propellant such as dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide,
or other suitable gas as is well known in the art. Aerosol dosages
can be controlled by providing a valve to deliver a metered amount
of the peptide. Alternatively, pharmaceutical compositions for
administration by inhalation or insufflation can be provided in the
form of a dry powder composition, for example, a powder mix of the
peptide and a suitable powder base such as lactose or starch. Such
powder composition can be provided in unit dosage form, for
example, in capsules, cartridges, gelatin packs, or blister packs,
from which the powder can be administered with the aid of an
inhalator or insufflator. The additives, excipients, and the like
typically will be included in the compositions of intra-nasal
administration within a range of concentrations suitable for their
intended use or function in the composition, and which are well
known in the pharmaceutical formulation art. The peptides of the
present invention will be included in the compositions within a
therapeutically useful and effective concentration range, as
determined by routine methods that are well known in the medical
and pharmaceutical arts. For example, a typical composition can
include one or more of the peptides at a concentration in the range
of at least about 0.01 nanomolar to about 1 molar, preferably at
least about 1 nanomolar to about 100 millimolar.
[0055] Optionally, the pharmaceutical compositions of the present
invention can include one or more other therapeutic agent, e.g., as
a combination therapy. The additional therapeutic agent will be
included in the compositions within a therapeutically useful and
effective concentration range, as determined by routine methods
that are well known in the medical and pharmaceutical arts. The
concentration of any particular additional therapeutic agent may be
in the same range as is typical for use of that agent as a
monotherapy, or the concentration may be lower than a typical
monotherapy concentration if there is a synergy when combined with
a peptide of the present invention.
[0056] In another aspect, the present invention provides for the
use of the peptides of Formula I for treatment of pain, treatment
of discomfort associated with gastrointestinal disorders, and
treatment of drug dependence. Methods for providing analgesia
(alleviating or reducing pain), relief from gastrointestinal
disorders such as diarrhea, and therapy for drug dependence in
patients, such as mammals, including humans, comprise administering
to a patient suffering from one of the aforementioned conditions an
effective amount of a peptide of Formula I. Diarrhea may be caused
by a number of sources, such as infectious disease, cholera, or an
effect or side-effect of various drugs or therapies, including
those used for cancer therapy. Preferably, the peptide is
administered parenterally or enterally. The dosage of the effective
amount of the peptides can vary depending upon the age and
condition of each individual patient to be treated. However,
suitable unit dosages typically range from about 0.01 to about 100
mg. For example, a unit dose can be in the range of about 0.2 mg to
about 50 mg. Such a unit dose can be administered more than once a
day, e.g., two or three times a day.
[0057] All of the embodiments of the peptides of Formula I can be
in the "isolated" state. For example, an "isolated" peptide is one
that has been completely or partially purified. In some instances,
the isolated compound will be part of a greater composition, buffer
system or reagent mix. In other circumstances, the isolated peptide
may be purified to homogeneity. A composition may comprise the
peptide or compound at a level of at least about 50, 80, 90, or 95%
(on a molar basis or weight basis) of all the other species that
are also present therein. Mixtures of the peptides of Formula I may
be used in practicing methods provided by the invention.
[0058] Additional embodiments of the current invention are directed
towards methods of using the peptides of Formula I disclosed herein
in medicinal formulations or as therapeutic agents, for example.
These methods may involve the use of a single peptide, or multiple
peptides in combination (i.e., a mixture). Accordingly, certain
embodiments of the invention are drawn to medicaments comprising
the peptides of Formula I, and methods of manufacturing such
medicaments.
[0059] As used herein, the terms "reducing," "inhibiting,"
"blocking," "preventing", alleviating," or "relieving" when
referring to a compound (e.g., a peptide), mean that the compound
brings down the occurrence, severity, size, volume, or associated
symptoms of a condition, event, or activity by at least about 7.5%,
10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 100% compared to how the
condition, event, or activity would normally exist without
application of the compound or a composition comprising the
compound. The terms "increasing," "elevating," "enhancing,"
"upregulating","improving," or "activating" when referring to a
compound mean that the compound increases the occurrence or
activity of a condition, event, or activity by at least about 7.5%,
10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 27.5%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 150%, 200%, 250%,
300%, 400%, 500%, 750%, or 1000% compared to how the condition,
event, or activity would normally exist without application of the
compound or a composition comprising the compound.
[0060] The following examples are included to demonstrate certain
aspects of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples,
which represent techniques known to function well in practicing the
invention, can be considered to constitute preferred modes for its
practice. However, those of skill in the art should, in light of
the present disclosure, appreciate that many changes can be made in
the specific disclosed embodiments and still obtain a like or
similar result without departing from the spirit and scope of the
invention. The examples are provided for illustration purposes only
and are not intended to be limiting.
EXAMPLE 1
Binding and Activation of Human Opioid Receptors.
[0061] The peptides of Formula I showed surprisingly high affinity
(subnanomolar) for the human mu opioid receptor with selective
binding relative to the delta and kappa opioid receptors. The
compounds were tested in standard binding assays using
.sup.3H-DAMGO (tritiated [D-Ala.sup.e, N-Me-Phe.sup.4,
Gly-ol]-enkephalin; CAS #78123-71-4), .sup.3H-DPDPE (CAS#
88373-73-3), and .sup.3H-U69593 (CAS#96744-75-1) to label mu, delta
and kappa receptors, respectively, in membranes from CHO cells
expressing human cloned receptors. As shown in Table 2,
endomorphin-1 (EM1, SEQ ID NO:8) and endomorphin -2 (EM2, SEQ ID
NO:9) are the most selective endogenous mu agonists previously
reported. Analogs based on these natural opioids show greater
affinity for the mu receptor, albeit with less selectivity.
Tetrapeptide endomorphin analogs described earlier (U.S. Pat. No.
5,885,958; ckl, Tyr-c[D-Lys-Trp-Phe] (SEQ ID NO:10); ck2,
Tyr-c[D-Lys-Phe-Phe] (SEQ ID NO:11)) showed the highest affinity of
the compounds tested. Peptides of Formula I, which include a
hydrophilic amino acid and amidated carboxy-terminus (Compounds 1,
2, 5) retained high affinity binding, but increased selectivity for
the mu receptor.
TABLE-US-00002 TABLE 2 Compound binding to opioid receptors.
K.sub.i (nM) Selectivity Mu Delta Kappa Delta/Mu Kappa/Mu Morphine
0.92 242 56 264 61 DAMGO 0.78 589 334 754 429 EM1 2.07 1215
>10000 587 >5000 EM2 1.32 5704 >10000 4328 >5000 ck1
0.32 28 35 90 111 ck2 0.36 3 12 9 33 Compound 1 0.49 132 128 267
260 Compound 2 0.73 69 71 94 98 Compound 5 0.43 140 29 328 67
[0062] Receptor Activation: GTP.gamma.S Functional Assay.
Functional activation of the three opioid receptors was tested in
standard assays in which the non-hydrolysable GTP analog,
.sup.35S-GTP.gamma.S, was used to quantify activation of cloned
human opioid receptors expressed in cell membranes. FIG. 4A shows
that Compound 1 is a full efficacy agonist with significantly
greater potency than the reference compound, DAMGO. FIG. 4B shows
that Compound 1 exhibits unexpected full efficacy as a delta
antagonist; i.e., it is able to inhibit the delta activation
produced by an ED.sub.80 dose of the reference delta agonist, SNC80
(CAS # 156727-74-1). Table 3 shows that all agonists tested are
potent activators of the mu receptor, with EC.sub.50 (median
effective concentration) values at low-nanomolar to sub-nanomolar
concentrations. All compounds were found to be full efficacy
(>90%) agonists at the mu receptor. The endomorphins and the
compounds of Formula I of the invention show remarkable selectivity
for receptor activation, with delta activation below 50% at
concentrations up to 10 .mu.M, reflecting selectivity >100000.
Compounds 1 and 3, however, showed full efficacy delta antagonism;
Compound 1 exhibited this antagonism at a relatively low
concentration.
TABLE-US-00003 TABLE 3 Opioid receptor activation by compounds.
Agonist EC.sub.50 (nM) Selectivity Delta Antagonist mu delta kappa
delta/mu kappa/mu IC 50 efficacy MS.sup.a 3.90 1245 2404 319 616
DAMGO 1.98 3641 13094 1839 6613 ck1 0.21 138 469.51 658 2236 ck2
0.15 7 206.11 44 1374 EM1 1.82 >100000 >100000 >50000
>50000 4287 100 EM2 8.44 >100000 >100000 >10000
>10000 30000 88 Comp. 1 0.15 >100000 963.79 >500000 6425
105 93 Comp. 2 0.99 >100000 12114.00 >100000 12236 2750 51
Comp. 5 0.22 >100000 740.34 >400000 3365 557 100
.sup.amorphine sulfate
[0063] Receptor Activation: Beta-Arrestin Recruitment.
Beta-arrestin is an intracellular protein that is recruited to the
mu opioid receptor following activation by agonists. It has been
shown to activate intracellular signaling pathways that in many
cases are independent of well-known G-protein mediated pathways. It
has recently been shown that beta-arrestin knockout mice exhibit
altered responses to morphine, including increased analgesia and
decreased side effects such as tolerance, respiratory depression,
and constipation (16). These results indicate that the analgesic
and side-effects of morphine are separable by manipulation of cell
signaling processes. These findings also provide support for the
recent concept known variously as "functional selectivity", "biased
agonism","agonist directed signaling" and other descriptions.
According to this concept, agonists capable of producing a
different cascade of signaling at a given receptor could produce a
different profile of desired and undesired effects relative to
other agonists for that receptor. Three of the analogs of this
invention were tested and showed patterns of beta-arrestin
recruitment (ranging from high potency with low efficacy to
moderate potency with significant efficacy) that were different
from each other and from morphine. Together with the differential
analgesic/side-effect profiles relative to morphine described in
previous examples, the beta arrestin results suggest that these
compounds exhibit "functional selectivity", favoring analgesia over
adverse side-effects.
[0064] Beyond the value of high mu agonist selectivity (i.e.,
exclusion of potential side-effects resulting from activation of
multiple receptors), delta antagonism is expected to attenuate
opioid-induced tolerance, dependence, and reward. As first shown in
1991 (1) and supported in numerous studies since, delta antagonists
can reduce morphine-induced tolerance and dependence, while
maintaining or enhancing analgesia. Recent studies (11) have also
shown reduced rewarding properties of mu agonist/delta antagonists
as reflected in the conditioned place preference (CPP) test
described below. The activity of the peptides of Formula I (e.g.,
Compound 1) as mu agonists/delta antagonists as well as at mu/delta
receptor dimers indicate that the peptides will produce effective
analgesia with reduced tolerance, dependence, and reward (18).
EXAMPLE 2
Providing Analgesia of Greater Duration, but with Reduced
Respiratory Depression, Relative to Morphine After Intravenous
Administration.
[0065] Respiratory depression is a major safety issue in the use of
opioids. An opioid providing analgesia as effective as that
produced by morphine, but with less respiratory depression, would
be a major advance for the safe use of opioid analgesics.
Effectiveness after systemic administration, such as intravenous
(i.v.) injection, is unusual for peptide-based compounds, and would
be critical for the clinical utility thereof. Two peptides
(Compounds 1 and 2) were tested for their effects on respiration
(minute ventilation) and duration of antinociception relative to
morphine. Rats with indwelling jugular catheters were placed in a
BUXCO whole body plethysmograph apparatus for determining multiple
respiratory parameters. For 20 minutes following i.v. injection of
vehicle (saline), baseline minute ventilation was determined.
Animals were then injected with morphine or test compound and
changes from baseline were determined for 20 minutes, the period of
maximal inhibition of minute ventilation by all compounds. The
standard tail-flick (TF) test was used to determine
antinociception. A baseline test was conducted before placing the
animal in the BUXCO chamber, at the end of the 20-minute
respiratory test, and at every 20 minutes thereafter until the TF
latency returned to below 2x baseline TF. Baseline latencies were
3-4 seconds and a cut-off time ("maximal antinociception") was set
at 9 seconds to avoid tissue damage.
[0066] FIG. 5A shows that 10 mg/kg doses of Compounds 1 and 2
produced significantly longer antinociception than all other
treatments (**=p<0.01) and 5.6 mg/kg doses produced
antinociception similar to the 10 mg dose of morphine. Despite the
greater antinociceptive effect of Compounds 1 and 2, significantly
(*p<0.05) less inhibition of respiration was observed in both
doses of Compound 1 and in the 5.6 mg/kg dose of Compound 2 (FIG.
5B). These results indicate an unexpected and clearly safer
therapeutic profile for the peptides of Formula I over the current
standard opioid analgesic.
EXAMPLE 3
Providing Analgesia of Greater Duration than Morphine with Reduced
Impairment of Neuromotor Coordination and Cognitive Function.
[0067] Neuromotor and cognitive impairment are characteristics of
opioids that are of particular importance in two populations, i.e.,
military combat troops, where escape from immediate danger can
require unimpaired motor and cognitive skills, and the elderly,
where these impairments can exacerbate compromised function
including impaired balance, which can lead to increased risk of
fractures.
EXAMPLE 3a
Neuromotor Coordination
[0068] FIG. 6A illustrates that Compound 2 produces significantly
greater antinociception, but significantly reduced motor
impairment, relative to morphine (MS). Both compounds were
administered by cumulative intravenous (i.v.) doses in rats.
Increasing quarter-log doses were given every 20 minutes, and a
tail flick (TF) test (a test of latency to remove the tail from a
hot light beam) followed by a rotorod test were conducted about 15
minutes after each injection. Escalating doses were given until
each animal showed greater than 90% maximum possible effect (% MPE)
on the TF test, determined as: [(latency to TF minus baseline
latency)/(9 sec maximum (cut off) time to avoid tissue damage)
minus baseline)].times.100. The animal was then placed on a rod
that rotated at speeds escalating to 13 revolutions per minute
(RPM) over 3 minutes, and the latency to fall from the rod was
determined. Only animals that consistently remained on the rod for
the full 180 seconds during training in the drug-naive state were
tested. % Maximum Possible Inhibition (% MPI) of motor coordination
was determined as 100-(latency to fall/180.times.100).
[0069] The two compounds showed similar onset to maximal
antinociception, but Compound 2 produced significantly longer
antinociception, as reflected by TF latencies significantly
(*=p<0.05) longer than those of the morphine group at 135 and
155 minutes (FIG. 6A). Despite this greater antinociception, the
motor impairment was significantly less than that of morphine (FIG.
6B, * p<0.05). The impairment of motor behavior by morphine was
significantly above that of vehicle controls (* p<0.05) while
that of Compound 2 was not.
EXAMPLE 3b
Cognitive Impairment
[0070] A widely used standard test of cognitive function is the
Morris Water Maze (MWM). During training, rats learn to find a
hidden escape platform based on spatial memory. Average latency to
the platform, as well as average distance from the platform (a
measure unaffected by swim speed), decrease as the task is acquired
and provide indices of spatial memory. After 4 days of training, an
injection of morphine produced impairment of spatial memory, as
reflected by a significant increase in the latency to, and average
distance from, the platform. By contrast, Compound 2, at doses that
provide equal or greater antinociception than morphine, did not
produce significant impairment. These results indicate an
unexpected and superior therapeutic profile of the peptides of
Formula I with regard to cognitive function relative to the current
standard opioid analgesic.
EXAMPLE 4
Providing Analgesia of Greater Duration, but Reduced Reward,
Relative to Morphine
[0071] Opioids remain the standard treatment for relief of severe
pain, but diversion of pain medications for non-pain use has become
a serious national problem (see U.S. Department of Health and Human
Services Substance Abuse and Mental Health Services Administration,
found at world wide website
oas.samhsa.gov/2k9/painRelievers/nonmedicalTrends.pdf).
Considerable efforts in academia and industry have focused on
"tamper-proof" versions of opioid medications, but there has been
little success in developing opioids that provide highly effective
analgesia with minimal abuse potential. The conditioned place
preference (CPP) paradigm is a widely accepted model for
demonstrating rewarding properties of drugs, and all major classes
of abused drugs produce CPP, including opioids such as morphine and
heroin. Briefly, animals are first allowed, on Day 1, to freely
explore a 3-compartment apparatus consisting of a small "start box"
and two larger compartments that are perceptually distinct (gray
vs. black and white stripes in this example). For the next three
days, the animals are given an i.v. injection of drug and confined
to one compartment, and vehicle is given in the other. The time at
which the drug or vehicle is given (a.m. or p.m.) is
counterbalanced, as is the compartment in which the drug is given
(preferred or non-preferred, as determined during the baseline
test). This unbiased design allows for detection of both drug
preference and drug aversion. After three days of conditioning
(Days 2, 3 and 4), the animal is allowed free access to all
compartments on Day 5 in the drug-free state and the change in
absolute time and proportion of time spent in the drug-paired
compartment are determined. A significant increase in the time or
proportion of time spent in the drug-paired compartment on the
post-conditioning test day relative to that on the pre-conditioning
baseline test is interpreted as a conditioned place preference,
reflective of rewarding properties and potential abuse
liability.
[0072] When the cumulative doses of either morphine or Compound 1
that were shown to produce maximal antinociception (FIG. 7A) were
tested for the ability to induce CPP (FIG. 7B), morphine produced a
significant (***p<0.01) increase in the time spent on the drug
side, while Compound 1 did not, even though significantly
(*p<0.05) greater antinociception (FIG. 7A) was observed with
Compound 1 from about 140 to 180 minutes after its injection.
Compounds 2 and 5 also showed no significant CPP at doses producing
antinociception equal to those of morphine that produced CPP. In a
complementary paradigm in which rats were provided access to
morphine or EM analogs for self-administration, access to morphine,
but not analogs, resulted in significant self-administration. These
findings are consistent with less abuse liability for the novel
analogs relative to morphine.
EXAMPLE 5
Alleviation of Chronic Pain.
[0073] Chronic pain affects a large proportion of the population.
One form of chronic pain, neuropathic pain, is particularly
difficult to treat. FIG. 8 shows that Compounds 1, 2 and 5 provide
unexpectedly potent relief of neuropathic pain induced by the
spared nerve injury (SNI) model in the rat. As demonstrated in FIG.
8A, prior to SNI surgery ("pre-surgery"), an average pressure of
about 177 g applied to the hindpaw with a Randall-Selitto device
was required to elicit a paw withdrawal response. About 7 to 10
days post-surgery, the animals showed hyperalgesia, indicated by a
reduction in the average pressure (to about 70 g) required to
elicit withdrawal. Drugs were administered as intrathecal
cumulative doses chosen to produce full alleviation of the
hyperalgesia. Times at which the reversal was significantly
(p<0.05 to 0.001) above vehicle are shown in bars at the top.
Compound 5 showed similar reversal times (about 80 min), and
Compounds 1 and 2 showed significantly longer reversal times (about
120 and 260 min, respectively) relative to morphine (about 80 min).
Scores for Compound 1 were also significantly above those of
morphine from 155-215 min (dashed bar). Dose-response curves (FIG.
8B) showed that all three analogs are significantly more potent
than morphine, as determined by the dose required to fully (100%)
reverse the hyperalgesia, i.e., return to the pre-surgical baseline
response (presurgical minus post surgical pressure). Compounds 1,
2, and 5 reversed mechanical hypersensitivity at doses about
80-fold to 100-fold lower than morphine (about 0.01 to 0.014 .mu.g
compared to about 1.14 .mu.g for morphine). On a molar basis, this
represents about 180 to 240 fold greater potency than morphine
against neuropathic pain. Similar results were observed after other
forms of chronic pain including post-incisional (post-operative)
and inflammatory pain induced by Complete Freund's Adjuvant (CFA).
The foregoing examples are illustrative, but not exhaustive, as to
the types of acute or chronic pain for which the peptides of
Formula I are effective.
EXAMPLE 6
Reduced Tolerance and Glial Activation Relative to Morphine
[0074] A major limiting factor for the usefulness of opioid
medications is tolerance, which requires increasing doses to
maintain an analgesic effect. Reduction of the potential for
tolerance would be a very important advantage for a novel
analgesic. In addition, several recent studies have shown that
repeated opioid exposure sometimes leads to "paradoxical"
opioid-induced pain. Increased responsiveness to normally noxious
stimuli (hyperalgesia) or normally non-noxious stimuli such as
touch (allodynia) have been reported. Explanations for the
tolerance and opioid induced hypersensitivity include the
possibility that activation of glia, a reflection of an
inflammatory response, results in an increased release of
substances that activate or sensitize neuronal transmission of
nociceptive signals. Specifically, enhanced release of
"pronociceptive" cytokines and chemokines are thought to mediate
the enhanced pain sensitivity sometimes observed after chronic
exposure to opioids. In addition, several studies have linked this
phenomenon to opioid tolerance based on the concept that increasing
doses of opioids are required to overcome the increased
pronociceptive effects of the released compounds. Described below
are the unexpected findings that: (1) Compounds 1, 2 and 5 produce
significantly less tolerance relative than morphine, and (2) that
in direct comparison to morphine, and in contrast to morphine and
most clinically used opioids, the analogs do not induce an
inflammatory glial activation response after chronic
administration. In addition to their potential value for reduced
escalation of doses required during chronic administration, the
analogs of Formula I could be ideal for opioid rotation and for a
wide range of situations where ongoing inflammatory conditions may
be exacerbated by treatment with morphine. This approach would also
be superior to use of an anti-inflammatory agent as an adjuvant to
opioid treatment.
[0075] Compounds 1, 2 and 5 all showed greater potency, reduced
tolerance and reduced glial activation relative to morphine. For
simplicity, only Compound 2 is shown in comparison to morphine in
FIG. 9. The experiment was designed to model clinical use of
opioids by titrating to full antinociception in each subject, and
maintaining steady blood levels, in this case through use of
osmotic minipumps. Doses producing matched initial antinociception
were determined for morphine and analog by intrathecal injection of
the cumulative dosing paradigm described above for the rotorod and
neuropathic pain models. Doses were increased until each rat
achieved full antinociception (100% MPE). The ED.sub.50 for all
compounds in opioid naive animals was determined and Compound 2 was
found to be over 20-fold more potent (p<0.001) than morphine
(ED.sub.50=0.01 .mu.g.+-.0.001 compared to 0.253 .mu.g.+-.0.05 for
morphine, n=5-7). This translates on a molar basis to about 40-fold
greater potency for the analog. Immediately after the first test,
ALZET osmotic minipumps (Durect Corp, Cupertino, Calif.) were
implanted subcutaneously and connected to the intrathecal catheter.
The primed pumps delivered morphine or analog at 2.mu.g/hr or 0.056
.mu.g/hr for about 7 days, respectively. The 2 .mu.g/hr morphine
dose was chosen based on previous studies in which this dose was
shown to produce glial activation in the dorsal horn in a similar
paradigm (Tawfik et al., 2005). The dose of analog was chosen using
a similar ratio to the ED.sub.50 (about 7.times. to 8.times.). A
second cumulative dose-response curve was generated on Day 7 after
minipump implantation to determine the shift in ED.sub.50 as an
index of relative tolerance. As shown in FIG. 9, the ED.sub.50 of
morphine shifted to 16.+-.3.3 .mu.g (over 60-fold) while that of
compound 2 shifted only about 8.5 fold to about 0.11.+-.0.02 .mu.g.
Compounds 1 and 5 showed similar results with potencies over 20x
greater than morphine and shifts less than 20 fold. These results
show that EM analogs cause unexpected and significantly less
tolerance than morphine.
[0076] As shown in FIG. 10, morphine produced significant glial
activation, but for all 3 analogs, activation was not significantly
different from vehicle and was significantly less than morphine,
establishing differential glial effects for morphine compared to EM
analogs (Compounds, 1, 2, and 5). Rats used in the above tolerance
experiment were perfused after the final behavioral test and
analyzed for glial activation as indicated by (A) GFAP staining for
astroglia and (B) phospho-p38, a signaling pathway activated in
microglia by morphine. Five sections from each of 5-7 animals/group
were analyzed for integrated density of staining with the IMAGE J
program. Morphine, but none of the analogs, showed significantly
greater induction than vehicle. Values for all analogs were
significantly below those of morphine (*,**,***=p<0.05,
0.01,0.001, respectively, compared to indicated groups). These data
provide evidence that, at doses producing equal or greater
antinociception, the analogs produce unexpectedly less glial
activation and this is associated with reduced tolerance.
[0077] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0078] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
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Sequence CWU 1
1
1115PRTArtificial SequenceCyclic endomorphin analog; residues 2-5
form a cyclic structure 1Tyr Xaa Trp Phe Glu1 5 25PRTArtificial
SequenceCyclic endomorphin analog; residues 2-5 form a cyclic
structure 2Tyr Xaa Phe Phe Lys1 5 36PRTArtificial SequenceCyclic
endomorphin analog; residues 2-5 form a cyclic structure 3Tyr Xaa
Trp Phe Glu Gly1 5 46PRTArtificial SequenceCyclic endomorphin
analog; residues 2-5 form a cyclic structure 4Tyr Xaa Phe Phe Lys
Gly1 5 55PRTArtificial SequenceCyclic endomorphin analog; residues
2-5 form a cyclic structure 5Tyr Xaa Trp Phe Asp1 5 65PRTArtificial
SequenceCyclic endomorphin analog; residues 2-5 form a cyclic
structure 6Tyr Xaa Phe Xaa Lys1 5 76PRTArtificial SequenceCyclic
endomorphin analog; residues 2-5 form a cyclic structure 7Tyr Xaa
Phe Xaa Asp Val1 5 84PRTHomo sapiens 8Tyr Pro Trp Phe1 94PRTHomo
sapiens 9Tyr Pro Phe Phe1 104PRTArtificial SequenceCyclic
tetrapeptide; residues 2-4 form a cyclic structure 10Tyr Xaa Trp
Phe1 114PRTArtificial SequenceCyclic tetrapeptide; residues 2-4
form a cyclic structure 11Tyr Xaa Phe Phe1
* * * * *