U.S. patent application number 14/974249 was filed with the patent office on 2016-06-23 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 | 20160176930 14/974249 |
Document ID | / |
Family ID | 56128666 |
Filed Date | 2016-06-23 |
United States Patent
Application |
20160176930 |
Kind Code |
A1 |
ZADINA; James E. ; et
al. |
June 23, 2016 |
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 amino acid and (ii) a D-amino acid substitution in
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 |
United States Department of Veterans Affairs
The Administrators of the Tulane Educational Fund |
Washington
New Orleans |
DC
LA |
US
US |
|
|
Assignee: |
The Administrators of the Tulane
Educational Fund
New Orleans
LA
United States Department of Veterans Affairs
Washington
DC
|
Family ID: |
56128666 |
Appl. No.: |
14/974249 |
Filed: |
December 18, 2015 |
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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14974249 |
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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: |
514/18.4 ;
514/21.1; 530/317 |
Current CPC
Class: |
C07K 7/64 20130101; G01N
2333/726 20130101; G01N 2500/04 20130101; C07K 7/06 20130101; C07K
14/665 20130101; A61K 38/00 20130101; C07K 7/56 20130101; G01N
33/9486 20130101; G01N 2333/70571 20130101 |
International
Class: |
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 and Merit Award, Grant No. I01BX001489 from
the Department of Veteran Affairs, Grant No. DM090595 from the
Department of Defense, and Grant No. N00014-09-is 1-0648 from the
Office of Naval Research of the Department of Defense. The United
States government has certain rights in this invention.
Claims
1. A cyclic peptide of Formula I: TABLE-US-00004 (I)
H-Tyr-cyclo[X.sub.1-X.sub.2-X.sub.3-X.sub.4]-X.sub.5,
wherein X.sub.1 and X.sub.4 each independently is an acidic D-amino
acid or a basic D-amino acid; X.sub.2 and X.sub.3 each
independently is an aromatic amino acid; X.sub.5 is selected from
the group consisting of 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, 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 and D-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 and D-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.
7. The peptide of claim 1, wherein R is H and X.sub.5 is
Gly-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:
TABLE-US-00005 (SEQ ID NO: 3)
Tyr-cyclo[D-Lys-Trp-Phe-Glu]-Gly-NH.sub.2, (SEQ ID NO: 4)
Tyr-cyclo[D-Glu-Phe-Phe-Lys]-Gly-NH.sub.2 and (SEQ ID NO: 7)
Tyr-cyclo[D-Orn-Phe-p-Cl-Phe-Asp]-Val-NH.sub.2.
10. A cyclic peptide of Formula I: TABLE-US-00006 (I)
H-Tyr-cyclo[X.sub.1-X.sub.2-X.sub.3-X.sub.4]-X.sub.5,
wherein (i) 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; or (ii) X.sub.1 is selected from the
group consisting of D-Asp and D-Glu; and X.sub.4 is selected from
the group consisting of D-Lys, D-Orn, Lys, and Orn; 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 X.sub.3 is selected from the group consisting of Phe,
D-Phe, and p-Y-Phe, wherein Y is selected from the group consisting
of NO.sub.2, F, Cl, and Br X.sub.5 is selected from the group
consisting of 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, and
Val-NH.sub.2; 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 cyclic peptide of claim 10, wherein X.sub.5 is
Gly-NH.sub.2.
12. The cyclic peptide of claim 10, wherein X.sub.1 is selected
from the group consisting of D-Lys and D-Orn; X.sub.4 is selected
from the group consisting of D-Asp and D-Glu; and X.sub.5 is
Gly-NH.sub.2.
13. The cyclic peptide of claim 10, wherein the peptide has the
amino acid sequence of SEQ ID NO: 4.
14. A cyclic peptide of Formula I: TABLE-US-00007 (I)
H-Tyr-cyclo[X.sub.1-X.sub.2-X.sub.3-X.sub.4]-X.sub.5,
wherein X.sub.1 is an acidic or basic D-amino acid, and X.sub.4 is
a basic or acidic amino acid; X.sub.2 is Trp; X.sub.3 is selected
from the group consisting of Phe, D-Phe, and p-Y-Phe, wherein Y is
selected from the group consisting of NO.sub.2, F, Cl, and Br;
X.sub.5 is Gly-NH.sub.2; 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.
15. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and the peptide of claim 1.
16. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and the peptide of claim 10.
17. A pharmaceutical composition comprising a pharmaceutically
acceptable carrier and the peptide of claim 14.
18. A method of treating pain comprising administering to a subject
an analgesic amount of the peptide of claim 1.
19. A method of treating pain comprising administering to a subject
an analgesic amount of the peptide of claim 10.
20. A method of treating pain comprising administering to a subject
an analgesic amount of the peptide of claim 14.
21. A method for treating a drug dependence comprising
administering to a subject a therapeutically effective amount of
the peptide of claim 1.
22. A method for treating a drug dependence comprising
administering to a subject a therapeutically effective amount of
the peptide of claim 10.
23. A method for treating a drug dependence comprising
administering to a subject a therapeutically effective amount of
the peptide of claim 14.
24. A method for treating a patient with a history of substance
abuse comprising administering to a subject a therapeutically
effective amount of the peptide of claim 1.
25. A method for treating a patient with a history of substance
abuse comprising administering to a subject a therapeutically
effective amount of the peptide of claim 10.
26. A method for treating a patient with a history of substance
abuse comprising administering to a subject a therapeutically
effective amount of the peptide of claim 14.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 14/268,057, filed on May 2, 2014, which is a
continuation of U.S. application Ser. No. 13/477,423, filed on May
22, 2012, now U.S. Pat. No. 8,716,436, 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.
INCORPORATION OF SEQUENCE LISTING
[0003] 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.
FIELD OF THE INVENTION
[0004] 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.
BACKGROUND OF THE INVENTION
[0005] 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 opioid receptor
("MOR", Ref. 3,20,21), delta opioid receptor ("DOR", Ref 6,9), and
kappa ("KOR", Ref 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).
[0006] 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. 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] Metabolically stable analogs of the endomorphins, endogenous
opioids highly selective for the Mu opioid receptor (MOR) are
described herein. Compared to morphine, the pentapeptide and
hexapeptide compounds (EM analogs) of Formula I showed dramatically
improved analgesia-to-side-effect ratios. At doses providing equal
or greater antinociception compared to morphine in the rat, the
analogs showed reduced (a) respiratory depression, (b) impairment
of motor coordination, (c) tolerance and hyperalgesia, (d) glial
p38/CGRP/P2X7 receptor signaling, and (e) reward/abuse potential in
both conditioned place preference and self-administration tests.
Differential effects on glial activation indicate a mechanism for
the relative lack of side effects by the analogs compared to
morphine. The results indicate that EM analogs of Formula I provide
excellent pain relief mediated by selective MOR activation, but
with remarkably safer side effect profiles compared to opioids like
morphine.
[0009] The EM analogs compounds of Formula I, described below,
provide antinociceptive effects equal or greater than morphine with
less respiratory depression, motor impairment, tolerance, immune
reactivity, and reward/abuse liability, relative to morphine.
Opioids, and morphine in particular, induce proinflammatory glial
cell, CGRP, or P2X7 receptor activation, which leads to undesirable
tolerance to chronic opioid administration, and the need to
increase dosages for effective pain relief. The analogs of Formula
I do not induce proinflammatory glial cell, CGRP, or P2X7 receptor
activation, and thus avoid the tolerance to chronic administration
that is associated with opioids. Overall, the EM analogs of Formula
I represent a major advance for pain research by surprisingly
providing equally effective analgesia compared to morphine, with
absent or substantially reduced side effects.
[0010] 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 amino acid, (ii)
side-chain to side-chain cyclization, and (iii) in some
embodiments, a substitution in position 2. 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.
[0011] 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
and X.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.
[0012] 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, 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, (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.
[0013] 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.
[0014] 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.
[0015] 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).
[0016] 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.
[0017] 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.
[0018] 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).
[0019] 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).
[0020] 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.
[0021] 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.
[0022] 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 I to form a
compound-receptor complex, (ii) detecting the complex, and (iii)
quantifying the amount of complex formed.
[0023] Another aspect of the invention is directed to the use of a
peptide of Formula I to 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.
[0024] Six critical side effects of currently used opioids were
surprisingly reduced or absent with the compounds of Formula I:
abuse liability, respiratory depression, motor impairment,
tolerance, hyperalgesia, and glial activation. This extensive
profile of low side effects for peptides shown to cross the blood
brain barrier and provide prolonged antinociception is
unprecedented. The lack of respiratory depression and motor
impairment provides an improved safety profile. The lack of glial
activation by a mu agonist, where morphine induces a
proinflammatory response, is a novel finding that suggests both a
mechanism for the reduced side effects of the analogs and a
therapeutic profile with reduced inflammatory complications. The
associated reduction in tolerance indicates improved long-term
effects. The absence of CPP, and especially SA in the sensitive
long-access paradigm, indicates abuse is unlikely. Pain therapy
with opioids continues to present troubling questions for doctors
and patients who must weigh the risk of causing adverse side
effects with effectively alleviating pain. This struggle could be
significantly reduced by development of novel opioids with the
potent analgesia and low side-effect profile shown by the EM analog
compounds of Formula I.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] 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.
[0030] 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.
[0031] 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.
[0032] 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).
[0033] 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.
[0034] 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).
[0035] FIG. 11 shows EM analog structures and receptor selectivity.
(A) Chemical structures of cyclized, D-amino acid-containing
analogs: Tyr-c[D-Lys-Trp-Phe-Glu]-NH.sub.2 (SEQ ID NO: 1, ZH850,
Analog 1), Tyr-c[D-Glu-Phe-Phe-Lys]-NH.sub.2 (SEQ ID NO: 2, ZH831,
Analog 2), Tyr-c-[D-Lys-Trp-Phe-Asp]-NH.sub.2 (SEQ ID NO: 5, ZH809,
Analog 3) and Tyr-c[D-Lys-Trp-Phe-Glu]-Gly-NH.sub.2 (SEQ ID NO: 4,
ZH853, Analog 4). (B) In vitro opioid receptor selectivity of
binding and activation by analogs and reference compounds morphine,
DAMGO and EMs to cloned human mu, delta and kappa opioid receptors.
Values represent means of 2-4 independent assays in duplicate. (C)
In vivo selectivity illustrated by antagonism of analog-induced
antinociception by naloxone, but not by delta (NTI) or kappa (nBNI)
antagonists. AUC was calculated from antinociceptive % MPE scores.
To provide similar AUC values, Analogs 1 and 2 were given at 3.2
mg/kg while Analogs 3 and 4 were given at 5.6 and 1.8 mg/kg
respectively. Differences among treatments (F.sub.3,18=8.30,
F.sub.3,17=9.34, F.sub.3,16=17.33, F.sub.3,20=18.93, p<0.001 for
Analogs 1-4, respectively) were attributable to a highly
significant reduction of antinociception by naloxone
(***p<0.001), while NTI and nBNI treatment did not affect
antinociceptive scores (n=5-7).
[0036] FIG. 12 demonstrates Analog 4 stability and antinociceptive
effectiveness after peripheral administration. (A, B): Analog 4 was
incubated at 37.degree. C. in human (A) or rat (not shown) plasma
or physiological saline (B) and stability was analyzed at various
time points by HPLC. Means.+-.SEM of duplicate samples at each
point are plotted. Dashed lines represent the 50% degradation
point. Linear regressions were significant for plasma and saline
(F.sub.1,32 =216, F.sub.1,26=242, p<0.0001) with R.sup.2 of 0.87
and 0.86, respectively. (C-E): Dose-dependent TF antinociception
after i.v., s.c., and oral administration of Analog 4. Two-way
ANOVA showed a significant effect of dose (F.sub.3,276=252.4,
F.sub.4,310=76.00, F.sub.3,182=117.00) time (F.sub.12,276=17.7,
F.sub.10,310=20.4, F.sub.10,182=26.5) and interaction
(F.sub.36,276=4.3, F.sub.40,310=4.1, F.sub.30,182=4.8), all
p<0.0001 for i.v., s.c., and oral, respectively. (D) The MOR
antagonist .beta.-FNA blocked the effects of Analog 4 (3.2 mg/kg,
s.c. [F.sub.2,192=53.51, p<0.0001]). Time points at which Analog
4 produced significant differences from vehicle are shown at the
top of each graph (**,***,**** =p<0.01, 0.001, 0.0001 for
highest dose vs vehicle; +,++,+++,++++=p<0.01, 0.001, 0.0001 for
middle dose vs vehicle, #=p<0.05 for lowest dose vs. vehicle.
n=5-11).
[0037] FIG. 13 illustrates reduced impairment of respiration and
motor coordination with BBB penetration. (A) Time course of change
in minute ventilation (MV) after morphine (5.6, 10 mg/kg i.v.) or
EM Analog 4 at doses (3.2, 5.6 mg/kg i.v) producing greater
duration of antinociception. (B) Mean % inhibition over 20 min
showed significant differences after various drug doses
(F.sub.9,58=6.19, p<0.0001). (C) Duration of antinociception
varied among drug groups (F.sub.9,65=8.70, p<0.001) with Analog
4 producing almost twice the duration of morphine. Dashed lines
illustrate the range of morphine responses for reference. (D) For
motor coordination, morphine (squares), Analog 2 (diamonds) or
Analog 4 (triangles) were administered in cumulative doses
increasing every 20 min followed 15 min later by TF and rotorod
tests. % MPE values for antinociception (TF, filled symbols) and %
maximum possible inhibition (% MPI) values for rotorod inhibition
(open symbols) were determined. Differences in antinociception
(F.sub.3,327.sup.=348.9, F.sub.15,327=93.51, p<0.0001, for drug
and time, respectively) reflected significantly longer and greater
total antinociception for Analogs 2 and 4 relative to morphine, and
differences in motor impairment (F.sub.3,231=15.19,
F.sub.10,231=2.34, p<0.0001, 0.05, for drug and time,
respectively) reflected less total impairment. (E) Significantly
different motor impairment AUC/antinociceptive AUC ratios
(F.sub.3,21=4.43, p<0.05) were found and (F) dose-response
curves showed that, on a molar basis, Analogs 2 and 4 were
significantly more potent than morphine (ED.sub.50=3.5.+-.0.39,
3.3.+-.0.13, and 5.2.+-.0.41 .mu.mol for Analog 2, Analog 4, and
morphine respectively). (G) Antinociception after peripheral
administration of the analogs was reduced by central administration
of the opioid antagonist N1x-M (F.sub.11,43=23.01, p<0.0001)
suggesting blood-brain-barrier penetration. TF data were converted
to area under the curve (AUC). +, ++, +++,++++p<0.05, 0.01,
0.001, 0.0001 relative to vehicle; *,**, ***, ****p<0.05, 0.01,
0.001, 0.0001 relative to morphine, n=5-8.
[0038] FIG. 14 illustrates reduced tolerance, glial and CGRP
activation after EM analogs relative to morphine. (A) Tail-flick
dose-response curves were determined before (pre, open symbols) and
after (post, closed symbols) 7 days of intrathecal (i.t.) drug
delivery, and shifts in ED.sub.50 were determined as an index of
relative tolerance. The acute potencies of the analogs were 30-fold
greater than that of morphine before chronic infusions. The
ED.sub.50 of morphine shifted 37.7-fold while those of the analogs
shifted on average 13.5-fold reflecting significantly less
tolerance (F.sub.1,114=135, p<0.0001). (B) Photomicrographs of
representative samples of dorsal horn immunostaining for the
astrocytic marker GFAP, the microglial marker Iba1, and the MAPK
signaling kinase associated with microglial activation pp38 [20x;
insets: 63.times. (GFAP, Iba1) and 40.times. (pp38), Scale bar=100
.mu.m]. (C) Significant differences in immunostaining were observed
for all three markers (F.sub.5,25=3.61, p<0.05, F.sub.5,24=4.71,
p<0.01, F.sub.5,25=16.83, p<0.0001 for GFAP, Iba1 and pp38,
respectively). All three were activated by morphine, but none were
activated by the analogs. Values for all analogs were significantly
below those of morphine for Iba1 and pp38. (D) Representative
photomicrographs (Scale bar=50 .mu.m) of CGRP expression. (E)
Quantification showing increased CGRP immunostaining after chronic
morphine, but not analogs (F.sub.5,24=4.50, p<0.01). CGRP levels
after the analogs were significantly reduced compared to morphine.
(F,G) Upregulation of P2X7 receptors in microglial cells. (F)
Representative photomicrographs (Scale bar=20 .mu.m); and (G)
quantification showing chronic morphine upregulated P2X7 receptors
(t.sub.7=2.50, p<0.05), OX-42-labeled microglial cells
(F.sub.2,16=3.96, p<0.05), and especially microglial cells
containing P2X7 receptors (merge) (F.sub.2,12=15.61, p<0.001).
By contrast, Analog 4 did not alter OX-42 levels or microglial
co-labeling with P2X7 receptors. +, ++, ++++=p<0.05, 0.01,
0.0001 significantly different from vehicle; *,**,*** =p<0.05,
0.01, 0.001, compared to morphine. Tolerance: n=6-12; IHC: n=5-6
rats, 4-6 sections per rat.
[0039] FIG. 15 demonstrates that chronic morphine, but not Analog
4, induces thermal hyperalgesia. Tail flick latencies were measured
on day 1 (baseline, BL) and after 7 day infusion (D7) of morphine
or Analog 4. Heat settings for baseline latencies were set to 10
sec to allow assessment of increased thermal sensitivity with a
cutoff of 20 sec. Latencies were significantly reduced after 7 day
infusion of morphine, but not Analog 4. [+=p<0.05 relative to
BL, n=12 (morphine), 8 (Analog 4)].
[0040] FIG. 16 illustrates tests for abuse liability. (A) Doses of
morphine and EM analogs providing equal antinociception 20 min
after injection. (B) Conditioned place behavior at
equi-antinociceptive doses showed morphine produced a significant
increase in time spent in the drug-paired box (CPP), while rats
given EM analogs were not significantly different from controls
(F.sub.5, 51=3.25, p<0.05). (C) CPP for morphine occurred in a
classic inverted-U dose-response fashion, whereas analogs did not
produce CPP (F.sub.6,57=3.97, p<0.01). (D) Self-administration:
Rats elevated lever pressings when required to obtain a
sub-antinociceptive dose of morphine (F.sub.1, 18=20.33,
p<0.001), but not for infusions ("inf") of the analogs, and only
Analog 2 showed a small but significant increase on the active
"drug lever" vs. inactive lever on the final trial (F.sub.5,
272=36.11, p<0.0001). (E) Active (filled bars) and inactive
(open bars) lever pressings averaged across sessions 5-7 (FR3-5)
from Panel D. Active lever pressings for morphine, but not analogs,
were significantly greater than the inactive lever and vehicle
(F.sub.11, 78=10.16, p<0.0001). (F) A variable dose experiment
showed that as the available dose was lowered, rats worked harder
to obtain morphine, but not analog infusions (F.sub.4,
240=32.05,p<0.0001). (G) Lever pressings for antinociceptive
doses of morphine were significantly greater than the inactive
lever (morphine 1 mg/kg/inf: F.sub.1, 55=18.33, p<0.0001; 3
mg/kg/inf: F.sub.1, 49=21.66, p<0.0001), while these doses of
Analog 4 did not produce self-administrations. (H) Number of
infusions and intake per 12 h shows that Analog 4 was not
self-administered at any dose compared to morphine. (I) Active
lever pressings show rats increased workload effort for morphine
infusions at a range of doses (F.sub.6, 42=5.395, p<0.001) while
no escalation was made for Analog 4 (F.sub.3, 25=2.05, p=0.1321).
+,++,+++p<0.05, 0.01, 0.001 relative to vehicle; *,**,***
p<0.05, 0.01, 0.001 relative to morphine; #, ##p<0.05, 0.01
relative to the inactive lever. n=8 rats/group for CPP; n=5-10
rats/group SA.
[0041] FIG. 17 shows that EM analogs provide potent and prolonged
relief of neuropathic pain induced by the spared nerve injury (SNI)
model in the rat. As demonstrated in Panel A, prior to SNI surgery
("pre-surgery"), an average pressure of about 182 g applied to the
hindpaw with a Randall-Selitto device was required to elicit a paw
withdrawal response. At 7 to 10 days post-surgery, the animals
showed hyperalgesia, indicated by a reduction in the average
pressure (to about 74 g) required to elicit withdrawal. Drugs were
administered as intrathecal cumulative doses chosen to produce full
alleviation of the hyperalgesia. Pressure tolerance (pain
alleviation) was significantly greater than vehicle until 135 min
after injection for morphine, and 235 min or more for the analogs
(p<0.05-0.0001). In addition, scores for analog 2 were
significantly above those of morphine at 135 min, from 135-235 min
for Analog 1, and from 95-235 min for Analog 4 (bars above time
points, p<0.05-0.0001). `ns vs veh`=time at which morphine
scores were no longer significantly different than vehicle.
Dose-response curves (Panel B) showed that all 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 (pre-surgical minus
post-surgical pressure). While potency differences among the
analogs were not significant (p>0.05), the analogs reversed
mechanical hypersensitivity at doses about 80-fold lower than
morphine (average of 0.02 vs 1.61 .mu.g for morphine, p<0.0001),
n=6-11.
[0042] FIG. 18 illustrates the antinociceptive effects of several
hexapeptides of the Formula I in which the L-amino acid (AA) in
X.sub.5 is varied. Antinociception was measured in the tail flick
test following subcutaneous injection to mice. All compounds were
compared at 3.2 and 5.6 mg/kg; results from 9 peptides with
X.sub.2=Trp are shown, along with the listed amino acid substituted
in position 6 (X.sub.5). The Gly-containing peptide (Analog 4, SEQ
ID NO: 3) provided potent antinociception after peripheral
injection. Panel A shows the time course of antinociception induced
by Analog 4, expressed as maximum possible effect (% MPE, as
described for FIG. 6). The 5.6 mg/kg dose maintained
antinociception >50% MPE for 80 min. The area under the curve
(AUC) for the 3.2 and 5.6 mg/kg doses are plotted in Panel B along
with those of the other analogs. Two peptides (dark bars) produced
potent antinociception similar to the Analog 4, i.e., a peptide
with substitution of a basic AA (Arg) in position 6, and a peptide
with substitution of 2,6-dimethyl-L-tyrosine (DMT) in place of Tyr
in position one (DMT.sup.1-Gly). Significant reduction of potency
was observed by substitution of Tyr, Met, and Pro in position 6
(white bars), relative to Analog 4, although the compounds were
still active, while Leu, Gln, and Ser showed intermediate potency
(grey bars). Some preference for smaller moieties within a class is
indicated by the greater potency of Ser vs Tyr for hydroxyl AAs,
and for Gly vs Leu for aliphatic AAs. Positive charge appears
preferable to negative charge as reflected in the greater potency
of Arg vs Gln. Panel C: In addition to Trp.sup.3 hexapeptides, a
Phe.sup.3 Gly.sup.6 hexapeptide was compared to the reference
Trp.sup.3-Gly.sup.6 peptide (SEQ ID NO: 3). For this comparison,
HCl salt forms of the peptides were tested while those in A and B
were acetate salts. The analogs with an HCl (C) salt form provided
a greater area under the curve relative to acetate salt (A,B), and
the Phe.sup.3 analog was fully effective (C); *,*** =p<0.05,
0.001 main effect of drug relative to Gly.sup.6 analog, n=5. The
results indicate that both Tyr and DMT are effective in position 1,
preferential amino acids in position 6 are Gly and Arg, and that to
a lesser extent, several additional amino acids in position 6 can
induce antinociception.
DETAILED DESCRIPTION OF THE INVENTION
[0043] 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 Comound 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)
[0044] 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.
[0045] Compounds 1 (FIG. 1), 2 (FIG. 2), 5 and 6 are examples of
cyclic pentapeptides, and Compounds 3, 4 (FIG. 3) and 7 are
examples of cyclic hexapeptides of the instant invention.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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
.alpha.-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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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.
Salt forms can be, e.g., salts of inorganic acids such as
hydrochloride salts, phosphate salts, sulfate salts, bisulfate
salts, hemisulfate salts, and the like; or salts of organic acids,
such as acetate salts, aspartate salts, citrate salts, fumarate
salts, maleate salts, malate salts, lactate salts, hippurate salts,
tartrate salts, gluconate salts, succinate salts, and the like. The
carrier can be an organic or inorganic carrier, or a combination
thereof, which 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.
[0058] In another aspect, pharmaceutical compositions useful for
treating pain and related conditions utilizing the compounds of
Formula I are 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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
[0073] 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.2, 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 (EMI, 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; ck1, Tyr-c[D-Lys-Trp-Phe] (SEQ ID NO:10); ck2,
Tyr-c[D-Lys-Phe-Phe] (SEQ ID NO:11)) are also shown. Peptides of
Formula I, which include an 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
[0074] Receptor Activation: GTP.gamma.S Functional Assay.
[0075] 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. 4, Panel A
shows that Compound 1 is a full efficacy agonist with significantly
greater potency than the reference compound, DAMGO. FIG. 4, Panel B
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 Delta EC.sub.50 (nM) Selectivity 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
[0076] Receptor Activation: Beta-Arrestin Recruitment.
[0077] 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.
[0078] 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
[0079] 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.
[0080] FIG. 5, Panel A 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/kg 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. 5, Panel B). 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
[0081] 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
[0082] FIG. 6, Panel A 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).
[0083] 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. 6, Panel A). Despite this greater
antinociception, the motor impairment was significantly less than
that of morphine (FIG. 6, Panel B, * 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
[0084] 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
[0085] 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(dot)samhsa(dot)gov/2k9/painRelievers/nonmedicalTrends(dot)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.
[0086] When the cumulative doses of either morphine or Compound 1
that were shown to produce maximal antinociception (FIG. 7, Panel
A) were tested for the ability to induce CPP (FIG. 7, Panel B),
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. 7, Panel
A) 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
[0087] 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.
8, Panel A, 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.
8, Panel B) 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 (pre-surgical 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
[0088] 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.
[0089] 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 (19). 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
20.times.greater than morphine and shifts less than 20 fold. These
results show that EM analogs cause unexpected and significantly
less tolerance than morphine.
[0090] 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.
Example 7
Evaluation of a Hexapeptide Analog of Formula I Relative to
Pentapeptides
[0091] Chemicals:
[0092] A series of pentapeptide and hexapeptide compounds of
Formula I, i.e., Compounds 1 (SEQ ID NO: 1), 2 (SEQ ID NO; 2), 5
(SEQ ID NO: 5), and 4 (SEQ ID NO: 4) described above (referred to
hereinafter in this Example as Analog 1, Analog 2, Analog 3, and
Analog 4, respectively) were synthesized by standard solid phase
methods at 1 mMol on a Rink amide resin via
fluorenylmethyloxycarbonyl (Fmoc) chemistry with purity (>95%)
and sequence identity confirmed by HPLC and MS. Analogs selected
for full characterization (FIG. 11, Panel A) were synthesized at 2
g scale. Morphine sulfate and beta-funaltrexamine (.beta.-FNA) were
supplied by NIDA, naloxone and naloxone methiodide (Nlx-M) were
obtained from Sigma (St. Louis, Mo.), naltrindole (NTI) and
nor-binaltorphimine (nBNI) from Tocris (Ellisville, Mo.), and
Tyr-D-Ala.sup.2-N-MePhe.sup.4-Gly-ol (DAMGO) from Bachem (King of
Prussia, Pa.).
[0093] Receptor binding assays were conducted using cloned human
receptors in CHO-K1 cell membranes and the following ligands for
mu, delta, and kappa opioid receptors (MOR, DOR KOR), respectively:
.sup.3H-DAMGO, .sup.3H-DPDPE and .sup.3H-U69593. Membranes,
radioligands at their Kd concentrations, and varying concentrations
of test compounds were incubated in 50 mM Tris pH 7.4, 5 mM
MgCl.sub.2, 10 .mu.g/mL saponin, for 60 min at 25.degree. C.,
filtered over GF/B filters and counted in MICROSCINT 20
scintillation cocktail (Packard).
[0094] Activation of receptors was determined in GTP.gamma.S
assays. CHO-K1 cell membranes, as described above, were mixed with
GDP (1 .mu.M for MOR, 10 .mu.M for DOR and KOR) for 15 min on ice
followed by incubation with .sup.355-GTP.gamma.S (0.1 nM) and
scintillation proximity assay (SPA) beads in 20 mM HEPES (pH 7.4)
containing 10 mM saponin, MgCl.sub.2 (1 mM for MOR and DOR, 30 mM
for KOR) and 100 mM NaCl. After shaking for 2 min and
centrifugation (2000 rpm, 10 min), samples were incubated for 1
hour (hr) and counted. Percent efficacy was calculated relative to
activation by reference compounds DAMGO, SNC80, and U-50488 for mu,
delta and kappa receptors. All test compounds showed >95%
efficacy at MOR. DOR antagonism was assessed by inhibition of the
activation produced by SNC80 at its EC.sub.80.
[0095] Stability in 37.degree. C. Plasma and Saline.
[0096] To test analog degradation by human and rat plasma enzymes
in vitro, pooled normal human plasma was obtained from Innovative
Research (Novi Mich., cat.#IPLA-N) and rat aortic blood was drawn
in a 10 ml syringe rinsed with heparin, incubated at room
temperature 1 hr and centrifuged (2000 g.times.15', 4.degree. C.).
Analogs were incubated at 200 .mu.g/ml in fresh plasma 37.degree.
C. Aliquots (75 .mu.L=7.5 .mu.g) of the mixture were withdrawn at
various times and immediately added to 75 .mu.L ice cold 0.1M HCl,
and centrifuged at 30,000 g, 20 min, 4.degree. C. Aliquots (100
.mu.L, about 5 .mu.g) of the supernatant were frozen pending HPLC
analysis. Samples were diluted with 100 .mu.L 0.1% TFA in water and
analyzed on a Beckman System GOLD HPLC with a VYDAC 218TP54 C18
column using eluents comprising 0.1% TFA in water/acetonitrile
(AcN) mixtures. The samples were run at 1 mL/min in gradients of
5-20% AcN/10 min, 20-25% AcN/30 min, 25-60% AcN/1 min, 60% AcN/9
min, and 60-80% AcN/1 min with an absorbance detector and
absorbance was plotted as a function of elution time. Intact
peptide was calculated from area under the absorbance curve at the
appropriate retention time relative to a standard curve generated
with the same conditions. Degradation was calculated by linear
regression of nlog A/A.sub.0 at 280 nm absorbance. To test analog
stability as an injectable solution at physiological temperature,
Analog 4 was sterile-filtered and incubated at 37.degree. C. in
sterile saline (10 .mu.g/100 .mu.L). Aliquots were removed at
various times and analyzed by HPLC. Confirmation of peptide
MW/integrity was conducted on an Applied Biosystems VOYAGER-DE PRO
mass spectrometer.
[0097] Animals and Surgery:
[0098] Male CD-1 mice (22-25 g) and Sprague-Dawley rats (250-400 g,
Charles River, Wilmington, Mass.) were housed in a 12-h light/dark
cycle. All experiments were approved by the Tulane Institutional
Animal Care and Use Committee and conducted according to the NIH
Guide for the Care and Use of Laboratory Animals. All efforts were
made to minimize animal suffering, and to reduce the number of
animals used. No alternatives to in vivo techniques are available.
Drug injections to rats were given as described previously through
indwelling jugular vein (i.v.) (55) or intrathecal (i.t.) catheters
(60). Mice received subcutaneous (s.c.) injections at the nape of
the neck or oral administration by gavage.
[0099] Antinociception was determined in a standard tail flick (TF)
test wherein the latency to withdraw the tail from a heat source
was automatically measured (IITC, Woodland Hills, Calif.). Baseline
latencies were 3-4 sec with a cutoff time of 9 sec to prevent
tissue damage. Percent Maximum Possible Effect (% MPE) was
determined as [(latency-baseline latency)/(9-baseline
latency)]*100. Equi-antinociceptive asymptotic doses of morphine
and analogs producing .gtoreq.95 MPE were used in the CPP test.
Doses of morphine and Analog 4 were tested at 0.25 log lower than
the maximal antinociceptive dose (producing 60-80% MPE), and 0.25
log higher than the maximal antinociceptive dose. Antinociception
after a 20 min respiratory test was scored as duration of
analgesia, defined as time that MPE was greater than 50%. Bolus
injections were used except for the rotorod (i.v.) and tolerance
(i.t.) tests, where cumulative dosing was used (44) with minor
modifications. Doses were increased in 0.25 log increments with
injections every 20 min, followed 15 minutes later by TF and
rotorod tests, or a TF test alone in the tolerance experiment.
[0100] Blood-Brain Barrier Penetration:
[0101] Intracerebroventricular (icy) administration of the opioid
antagonist naloxone-methiodide (Nlx-M) was used to test the central
effects of peripherally injected analogs. Rats were placed in a
stereotaxic apparatus under isoflurane/oxygen anesthetic (4-5%
induction, and 1.5-2.5% for maintenance). Infusions of Nlx-M (10
.mu.g/5 .mu.L, icy) or vehicle (5 .mu.L, icy) were made to the
right lateral ventricle (1.5 mm lateral, 0.7 mm posterior, and 3.5
mm ventral to the bregma) using a 5 .mu.L syringe. The 10 .mu.g icy
dose of Nlx-M was chosen based on reports (24,33) showing the
effectiveness of this icy dose in antagonizing systemic morphine.
Injection was made over 1 min and the syringe was held in place for
1 additional min to ensure adequate diffusion. About 20 min after
the icy injection, the analogs were injected (i.v.) and TF
latencies were measured 15, 30, 45, 60, 90, 120, 180, 240, 300, and
360 mins after injection.
[0102] Respiratory depression was measured in unanesthetized
free-moving rats in a whole body plethysmography system (Buxco,
Wilmington, N.C.) as previously described (29). Rats were given
saline (1 mL/kg) through an i.v. jugular PE-50 catheter connected
via swivel spring leash to a syringe outside the chamber. Minute
ventilation (MV, tidal volume.times.respiratory rate) was
determined for 20 min (vehicle baseline), then morphine or an EM
analog was injected. Change in MV (% vehicle baseline) was
determined over 20 min, the period of maximal effect. A TF test was
then conducted at 20 min intervals until antinociceptive scores
fell below 50% MPE, defined as the duration of antinociception.
Duration of antinociception, an index of total antinociception, was
used to assess antinociception relative to respiratory depression
for morphine and analogs.
[0103] Motor coordination was tested on a ROTOMEX-5 rotorod
apparatus (Columbus Instruments, Columbus, Ohio). Cumulative doses
were given as described in the antinociception section to produce
>90% MPE on the TF test. Only rats remaining on the rotorod for
180 seconds during training were tested, allowing determination of
% Maximum Possible Inhibition (% MPI) of motor coordination as
[100-(latency to fall/180.times.100)]. Antinociceptive ED.sub.50
values were calculated by nonlinear regression. An index of motor
impairment relative to antinociception was calculated from the area
under the curve (AUC) for MPI/AUC for MPE.
[0104] Tolerance was assessed by determining ED.sub.50s before and
after i.t. drug infusions for 7 days. Cumulative dosing with
quarter-log increases every 20 min were followed by a tail-flick
test 15 min after injection. Immediately after the test in naive
rats, osmotic minipumps (ALZET model 2001, Durect Corp, Cupertino,
Calif.) filled with vehicle, morphine, or analog and primed in 0.9%
saline at 37.degree. C. for 16 h, were implanted s.c. and connected
to a PE-8 (0.008 inch I.D.) i.t. catheter. The pumps delivered
8.times. the ED.sub.50/hr (2 .mu.g/hr for morphine, 0.056
.mu.g-0.075 .mu.g/hr for analog) for 7 days (53). A second
dose-response curve was generated on day 7. ED.sub.50 values are
presented along with the shift in ED.sub.50 that provides an index
of relative tolerance.
[0105] Hyperalgesia relative to morphine was determined at baseline
and on day 7 with separate TF scores in which the heat intensity
was set to evoke a baseline response >10 sec (cutoff 20 sec) to
detect decreased latencies (increased sensitivity).
[0106] Immunohistochemistry: Animals from the tolerance experiment
were deeply anesthetized with ketamine/xylazine (85/10 mg/kg) and
perfused transcardially with 0.1M PBS followed by 4%
paraformaldehyde. Spinal cords were post-fixed overnight at
4.degree. C., cryoprotected in 30% sucrose/0.1 M PBS for 48 hr, and
sectioned on a freezing microtome at 40 .mu.m. After 2 washes in
PBS and blocking with 5% normal goat serum/0.3% Triton X-100,
sections were incubated in primary antibody; GFAP (1:1000, ab7779,
Abcam, Cambridge, Mass.), Iba1 (1:1000, #019-19741, Wako, Richmond,
Va.), pp38 (1:100, #4511, Cell Signaling Technology, Danvers,
Mass.), OX-42 (1:100, #CBL1512, Millipore, Temecula, Calif.), or
CGRP (1:1000, T-4032, Peninsula Labs, San Carlos, Calif.) for 24 h
at 4.degree. C. on a slow rocker. The tissue was then washed twice,
re-blocked, incubated in donkey anti-rabbit secondary antibody
conjugated to ALEXA 488 dye (1:500 for GFAP, Iba1, CGRP, and 1:200
for pp38, #A21206 Invitrogen, Eugene, Oreg.) or ALEXA 594 dye
(1:500, #A21203, Invitrogen) for 2 hours (hrs) at room temperature
(RT), washed, and slide-mounted with PROLONG GOLD antifade reagent
(Life Technologies, Grand Island, N.Y.). GFAP- and
Ibal-immunoreactivities in lamina I-V of dorsal horn segments L4-L5
were quantified on a NIKON microscope with a HAMMAMATSU camera and
NIH IMAGEJ software. Images containing lamina I-II of the spinal
dorsal horn were analyzed for CGRP and OX-42 integrated density
using IMAGEJ software (50). A blinded observer determined
integrated density by thresholding the images using the default
IMAGEJ algorithm to reduce background and include positively
stained cells. Integrated density in the Region of Interest (ROI)
is equal to the product of area and mean gray value. The mean gray
value represents the sum of the intensity values/number of pixels
for all pixels above the threshold in the ROI. This method controls
for differences in background between slices and subjects. For
quantification of pp38, an observer blinded to treatment manually
counted punctate immunoreactive cells. For co-labeling experiments,
primary antibody against P2X7 receptors (P2X7R; 1:100, #APR-008,
Alamone Labs, Jerusalem, Israel) was incubated with OX-42
overnight. Tissue was washed and re-blocked as described above, and
finally incubated with appropriate secondary antibodies ALEXA 488
dye (1:500) and ALEXA 594 dye (1:500) before washing and mounting.
Quantification of P2X7R and OX-42 co-labeling was performed using
NIKON projection images constructed from 1 .mu.m thick image stacks
from lamina I-II. The number of OX-42 positive cells and
P2X7R/OX-42 co-labeled cells were counted to determine percent
co-labeling (27). A total of 5-6 rats per group and 4-6 slices/rat
were quantified for all experiments. Representative confocal images
were generated on a LEICA SP2 AOBS microscope.
[0107] Conditioned Place Preference (CPP):
[0108] Animals were allowed to freely explore an apparatus with a
smaller "start box" and two larger distinct compartments (gray vs.
black and white stripes of equal luminance; TSE Systems,
Chesterfield, Mo.). Mean time in each compartment for 2 morning and
2 afternoon sessions of 20 min each was determined as baseline. For
the next three days, the animals were given an i.v. injection of
drug and immediately confined to one compartment for 30 min, and
vehicle was given in the other. The time at which the drug or
vehicle was given (a.m. or p.m.) was counterbalanced, as was the
compartment in which the drug was given (preferred or
non-preferred, as determined during the baseline test). The
unbiased apparatus and design allows for detection of both drug
preference and aversion. After three days of conditioning, the
animals were allowed free access to all compartments in the
drug-free state for 20 min in the morning and afternoon. The mean
change in time spent in the drug-paired compartment was determined,
and a significant increase on the post-conditioning test relative
to that on the pre-conditioning test was interpreted as a CPP,
reflective of rewarding properties and potential abuse liability.
Equi-antinociceptive asymptotic doses of morphine and analogs
producing >95% MPE were used in the CPP test. Doses of morphine
and Analog 4 were also tested at 0.25 log lower (producing 60-80%
MPE), and 0.25 log higher than the maximal antinociceptive
dose.
[0109] Self-administration (SA) tests were conducted in SA chambers
(MED Associates, St. Albans, Vt.) containing an inactive lever and
an active lever that regulated a computer-controlled infusion pump
outside each chamber. Infusions delivered through TYGON tubing in a
balanced arm swivel and metal spring leash allowed the animal free
movement and protected the infusion line. The protocol involved 7
sessions of 12-hour access to saline or drug during the dark cycle
(55). At the start of each 12-hour session, all rats received one
priming injection equivalent to the same dose available during the
session. The initial requirement of 1 active lever press per
infusion (fixed ratio 1, FR1) was escalated on days 3, 5 and 7 to
FR2, 3 and 5, respectively. When the FR requirement was completed,
an infusion occurred along with a 10 sec time out period when the
stimulus lamp turned off and no additional infusions were possible.
Pressings on the active lever that resulted in an infusion or
occurred during the 10-sec time out period were analyzed and
compared to inactive lever responding. Active lever pressings/12 h,
number of infusions/12 h, and intake (mg/kg/12 h) were analyzed
from data averaged from the FR3-5 sessions to compare SA at high FR
workload requirements to obtain infusion. In 12-hour variable dose
studies, SA sessions were conducted for 12 hours per day using a
descending dose paradigm in which 0.75, 0.3, 0.1, and 0
mg/kg/infusion of morphine or analogs were available on days 1-2,
3-4, 5-7, and 8-10, respectively.
[0110] Alleviation of chronic pain by the analogs was tested in the
spared nerve injury (SNI) model. At the site of the trifurcation of
the left sciatic nerve, the common peroneal and tibial branches
were tightly ligated and transected, leaving the sural branch
intact. Pain sensitivity was assessed by applying pressure to the
lateral edge of the hindpaw with a Randall-Selitto device. A
baseline measure was taken before surgery and at 7 to 10 days
postsurgery. Drugs were then administered intrathecally in
cumulative doses chosen to produce full alleviation of the
hyperalgesia. Duration of pain alleviation (>vehicle) was
assessed at 20 min intervals.
[0111] Data analysis: Data were analyzed by analysis of variance
(ANOVA) followed, when appropriate, by Bonferroni, Newman-Kuels, or
Dunnett post-tests using GRAPHPAD PRISM software (GraphPad, San
Diego, Calif.). Cumulative antinociceptive data from the tolerance
study was analyzed by non-linear regression to generate ED.sub.50
values. Drug tolerance ED.sub.50 values were determined after acute
and chronic administration. Immunohistochemical analysis of cell
counts, integrated density, or co-labeling was conducted by blinded
observers. Drugs were coded during in vivo experiments and tested
by blinded observers. All data is presented as the mean.+-.S.E.
with 95% confidence limits. Differences were considered
statistically significant when p<0.05.
Results
[0112] EM Analogs Bind Selectively with High Affinity and Efficacy
at Mu Opioid Receptors.
[0113] Analog structures are shown in FIG. 11, Panel A. Binding
assays with cloned human opioid receptors showed that all four
analogs had selectivity and subnanomolar affinity for MOR (FIG. 11,
Panel B). Analog 4 showed the highest selectivity. In
.sup.355-GTP.gamma.S assays the analogs showed full agonism at MOR,
had greater potency than morphine, DAMGO and EMs, and remarkable
selectivity for MOR activation (>100000 and >3000-fold higher
concentrations were required for delta or kappa activation).
Interestingly, Analogs 1, 3 and 4 showed high efficacy delta
antagonism at sub-micromolar concentrations. In vivo selectivity
was also demonstrated: FIG. 11, Panel C shows that antinociceptive
effects of all four analogs were significantly blocked by naloxone
(1 mg/kg), but not by antagonists for DOR (NTI) or KOR (nor-BNI) at
doses (1 mg/kg) known to antagonize selective agonists for those
receptors after i.v. injection in the rat (26,30).
[0114] EM Analogs Show High Solubility and Stability.
[0115] The EM analogs showed favorable solubility (40, 20, 20 and
50 mg/mL in water, 15, 15, 12, and 20 mg/mL in saline, and 90, 70,
50, and 50 mg/mL in 20% PEG400/saline for Analogs 1-4,
respectively), and stable plasma half-life in vitro. While the
parent endomorphins were metabolized rapidly in rat plasma with a
half-life of 5 min, Analogs 1-4 showed half-lives of 14, 46, 14,
and 81 hr. The longer value for Analog 4 relative to Analog 1
indicates that the glycine extension surprisingly enhances
stability. Analog 4 also was tested in human plasma and showed a
half-life of about 150 hours (FIG. 12, Panel A). Stability in
saline at 37.degree. C. was >1 year (FIG. 12. Panel B),
indicating a highly favorable "shelf life" and usefulness for
long-term infusion.
[0116] Em Analogs Produce Potent, Long Lasting and Mu-Selective
Antinociception after Peripheral Administration.
[0117] The stability of the analogs translated to effectiveness
after peripheral administration as shown by potent and long lasting
antinociception after intravenous (i.v.), subcutaneous (s.c.), and
oral administration. FIG. 12, Panels C-E illustrate these results
for Analog 4. At doses producing maximal % MPE, the durations of
antinociception for i.v. (FIG. 12, Panel C) s.c., (FIG. 12, Panel
D) and oral (FIG. 12, Panel E) administration were 240, 80, and 100
min, respectively. The duration of antinociception after i.v.
injection in rat for morphine and all analogs is shown in FIG. 13,
Panel C. FIG. 12, Panel D shows that the mu-selective antagonist
.beta.-FNA eliminated antinociception induced by s.c.
administration of Analog 4 in mouse, consistent with the
MOR-selective receptor binding data.
[0118] Respiratory Depression is Reduced or Absent after EM Analogs
at Doses Producing Equal or Greater Duration of Antinociception
Relative to Morphine.
[0119] Changes in MV over the 20 minute period of maximal drug
effect are illustrated for morphine and Analog 4 in FIG. 13, Panel
A, and average changes for all compounds in FIG. 13, Panel B.
Duration of antinociception determined from TF tests conducted at
20 min intervals thereafter are shown in FIG. 13, Panel C. Morphine
produced significant inhibition of MV at 5.6 and 10 mg/kg (FIG. 13,
Panel A,B) along with about 2-3 hr of antinociception (FIG. 13,
Panel C). By contrast, after EM analogs, respiratory inhibition was
below, and duration of antinociception (2-6 h) was at or above,
that produced by morphine (dashed lines). The rank order for
duration of antinociception was Analog
4>1>3>2>morphine. Analog 4 showed the clearest
separation with no respiratory depression in the presence of
significantly longer antinociception induced by lower doses. The
results indicate that the EM analogs are clearly more effective and
safer analgesics than morphine.
[0120] Motor Coordination is Impaired by Morphine but not EM
Analogs.
[0121] Impairment of motor coordination relative to antinociception
was tested in rotorod and TF tests. Analogs 2 and 4 produced
significantly longer and greater total antinociception than
morphine (FIG. 13, Panel D), were significantly more potent (FIG.
13, Panel F), and did not produce significant motor impairment
(FIG. 13, Panel D,E). Morphine showed significantly more impairment
than vehicle and the analogs (FIG. 13, Panel D,E). Analog 4
provided the greatest duration of antinociception and the least
motor impairment. Vehicle animals remained on the rotorod for the 3
min maximum (no inhibition) and vehicle TF values (not shown for
clarity) were below 10% MPE at all time-points, indicating that the
testing sequence did not produce stress- or rotorod-induced
antinociception. Differences in motor impairment ratio (FIG. 13,
Panel E) were significant with morphine (40.6+14.2) scores
significantly greater than those of vehicle (0.0), Analog 2
(8.0+8.0) and Analog 4 (4.7+2.0), indicating a 5-fold and 8.6-fold
better therapeutic ratio for Analogs 2 and 4 relative to
morphine.
[0122] Cognitive Function is Impaired by Morphine, but not Analog
4.
[0123] Tests of cognitive function (spatial memory) in the Morris
Water Maze (MWM) revealed that Analog 4 shows a highly favorable
profile relative to morphine. 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, Analog 4 did not produce
significant impairment, even at doses that provided 2-3 times
greater duration of antinociception than morphine. 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.
[0124] EM analogs access and activate CNS receptors. FIG. 13, Panel
G shows that i.c.v. low-dose (10 .mu.g) injection of the
blood-brain barrier impermeable antagonist naloxone methiodide
(Nlx-M) significantly inhibited i.v. analog-induced antinociception
(p<0.05). These results indicate that full antinociceptive
effects require activation of CNS opioid receptors and that the
relative lack of respiratory and motor impairment by the analogs is
not due to lack of CNS entry.
[0125] EM Analogs Produce Less Tolerance and Hyperalgesia than
Morphine.
[0126] Tolerance and glial activation were tested in a
well-characterized model (53,19). Cumulative intrathecal (i.t.)
dosing of naive rats showed that EM analogs were initially 30-fold
(60-fold on a molar basis) more potent than morphine (FIG. 14,
Panel A). After 7 days of infusion, the ED.sub.50 of morphine
shifted 38-fold. By contrast, EM analogs, tested at doses matched
for antinociception, surprisingly produced a shift of only 14-fold,
demonstrating substantially less tolerance. Analog 4 was selected
for comparison to morphine for hyperalgesia. FIG. 15 shows that
7-day treatment with morphine, but not EM Analog 4, induced
hyperalgesia.
[0127] Activation of Glial Cells by Morphine, but not EM
Analogs.
[0128] Glial activation was assessed using markers for astrocytes
(GFAP), microglia (Iba-1 and OX-42), and the activated microglial
MAP kinase phospho-p38 (pp38) (FIG. 14, Panels B,C). Expression of
all 3 markers was significantly increased after morphine, but not
after any of the analogs. Expression of Iba-1 and pp38 after
analogs was significantly below that of morphine.
[0129] Upregulation of CGRP by Morphine, but not EM Analogs.
[0130] CGRP induction has been implicated in glial activation and
tolerance (56). FIG. 14, Panels D,E show CGRP staining was strongly
upregulated in the dorsal horn of the spinal cord by chronic
infusion of morphine, but not by any of the EM analogs.
[0131] Upregulation of P2X7 Receptors by Morphine, but not EM
Analog 4.
[0132] Because upregulation of microglial P2X7R has been implicated
in tolerance (27,62) changes in this marker after morphine and
Analog 4 administration were tested. Morphine, but not Analog 4,
upregulated P2X7 receptors in microglial cells (FIG. 14, Panels
F,G). Activated microglia contained high levels of P2X7 receptor
expression, confirmed by increased microglial OX-42 that strongly
co-labeled with P2X7 receptors. The high proportion of
co-localization of upregulated P2X7 and activated microglia after
morphine (21% vs 5% after Analog 4 or vehicle) suggests increased
ATP-P2X7-neurotrophin-cytokine mediated effects of morphine, but
not Analog 4. In summary, EM analogs produced more potent acute
antinociception and, after chronic administration at equi-effective
doses, induced substantially less tolerance and did not promote
glial reactivity, CGRP upregulation or P2X7 induction compared to
morphine.
[0133] Abuse liability was tested in two complementary paradigms,
CPP and SA. CPP was compared using equally asymptotic
antinociceptive doses that produced full antinociception (>95%
MPE) 20 min after injection (FIG. 16, Panel A), corresponding to
the period of confinement to the conditioning side. These doses
were determined as 3.2 mg/kg i.v. for morphine, 5.6, 3.2, 5.6, and
3.2 mg/kg for EM Analogs 1, 2, 3, and 4, respectively. FIG. 16,
Panels B and C show that after 3 days of conditioning, morphine,
but not the analogs, produced a significant increase in the time
spent on the drug-paired side, a place preference. None of the
compounds showed place aversion (decreased time on the drug-paired
side). Morphine and EM Analog 4 were also tested at 0.25 log
higher, and 0.25 log lower doses, but only morphine produced CPP.
(FIG. 16, Panel C).
[0134] While CPP has an advantage of testing for reward
associations in the drug-free state, SA more directly models drug
consumption. Rats were allowed to bar press for i.v. morphine, EM
analogs or vehicle for 12 h/day for 7 days with an escalating fixed
ratio schedule. As the effort required to receive the drug
increased from 1 to 5 presses per infusion (FIG. 16, Panel D), rats
significantly escalated bar pressings for morphine, but not for the
analogs (0.75 mg/kg/infusion). FIG. 16, Panel E compares active
drug lever presses to an inactive lever during high-effort sessions
with FR requirements set at 3-5 (e.g. 3-5 presses required for 1
infusion). Morphine produced significantly higher active lever
pressings than vehicle or the inactive lever, while pressings for
all analogs were significantly reduced by comparison. As doses were
gradually lowered (0.75, 0.3, and 0.1 mg/kg/infusion) across
sessions, rats self-administering morphine increased responding,
while rats given access to EM analogs did not escalate lever
pressings (FIG. 16, Panel F). At comparably antinociceptive
infusion doses (FIG. 16, Panels G-I) of morphine and EM Analog 4,
the same pattern emerged; pressings for morphine (1 and 3
mg/kg/infusion) were significantly elevated over the inactive
lever, whereas none of the doses of EM Analog 4 (1 and 3
mg/kg/infusion) were self-administered (FIG. 16, Panels G-I). Thus,
in CPP and SA models, the EM analogs showed significantly reduced
reward properties relative to morphine, consistent with low abuse
liability.
[0135] Support for the concept that Analog 4 could also be useful
for treating subjects with a history of abuse was demonstrated in a
drug discrimination paradigm. Rats were trained to discriminate
morphine injections from vehicle to receive food pellets. When
challenged with Analog 4, rats responded on the morphine lever,
indicating they could discriminate the EM analog as more similar to
morphine than vehicle. In combination with the lack of
self-administrations by Analog 4, these data convey a compelling
case for a compound that effectively substitutes for morphine, but
does not produce reward, indicating strong potential for opioid
addiction pharmacotherapy.
[0136] EM analogs provide potent and prolonged alleviation of
chronic pain. As shown in FIG. 17, Panel A, Analogs 1-3, and
especially Analog 4, provide unexpectedly potent and long-lasting
relief of neuropathic pain induced by the spared nerve injury (SNI)
model in the rat. Alleviation of mechanical hypersensitivity
(scores significantly greater than those after vehicle) lasted just
over 2 hours (about 135 min) after morphine and 4 hours or more
(>235 min) after administration of the EM analogs. An increase,
relative to morphine, in duration of relief was significant at 135
min for Analog 2, at 100 min for Analog 1 (135-155 min), and at
least about 140 min (95-235+ min) for Analog 4. Although potency
differences among the analogs were not significant, the analogs
were on average 80-fold more potent than morphine (p<0.0001). On
a molar basis, this represents an average of 180-fold greater
potency. The analogs, especially Analog 4, also produced more
potent and longer duration of relief than morphine 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.
[0137] As a result of the success of Analog 4 in the multiple tests
described here, several additional hexapeptides of Formula I and a
variant with 2,6-dimethyltyrosine (DMT) in place of the Tyr residue
at position 1 were synthesized and tested for antinociception in
the tail flick test following subcutaneous injection to mice. FIG.
18 illustrates that both Tyr and DMT were effective in position 1,
preferential amino acids in position 6 were Gly (Analog 4) and Arg.
Several additional amino acids in position 6 induced
antinociception to a lesser extent compared to Gly and Arg.
DISCUSSION
[0138] The EM analogs tested here provide potent antinociception
(FIG. 12, Panels C-E, FIG. 13, Panel D, FIG. 14, Panel A, and FIG.
15, Panel A) with reduction or absence of six major side effects of
currently used opioids: abuse liability, respiratory depression,
impairment of motor coordination, tolerance, hyperalgesia, and
glial activation. While all analogs proved superior to morphine
against the side effects tested, some differences indicate that
they may provide new tools for understanding analgesia vs.
side-effects and their separation for clinical application. First,
Analog 2 (SEQ ID NO: 2) (an EM2 analog) showed low motor impairment
relative to that produced by morphine and two analogs of EM1, i.e.,
Analog 1 (SEQ ID NO: 1) and Analog 3 (SEQ ID NO: 5). The latter
analogs, however, showed preferable profiles in respiratory and
abuse liability tests. Analog 4 (SEQ ID NO: 4), based on the EM1
structure, showed that remarkably low motor impairment, as well as
favorable respiratory and abuse liability profiles, could be
achieved in a single molecule. Analog 4 proved unexpectedly
superior in tests of respiratory depression, motor impairment (FIG.
13), and abuse liability (FIG. 14), while producing the most potent
antinociceptive effects (FIG. 13, Panels C and D). All four analogs
produced far less tolerance than morphine (FIG. 14, Panel A) and
did not induce glial cell (FIG. 14, Panel B) or CGRP (FIG. 14,
Panel D) activation. While morphine upregulated microglial P2X7
receptors, Analog 4 did not (FIG. 14, Panel F). Compared to
currently available opioids, the prolonged antinociception and lack
of adverse effects from the analogs shown here is
unprecedented.
[0139] Analogs of Endomorphins.
[0140] The parent compounds of these analogs, the endomorphins
(EMs) (23), showed early promise for a profile of potent analgesia
with reduced side effects (29,22). The linear native peptides,
however, are metabolically labile. While "constant renewal" methods
such as viral vector-based delivery of these peptides have provided
prolonged pain relief (47,59), structural modifications are
required for more stable drug-like compounds. Numerous analogs of
EMs have been developed with the goal of optimizing drug-like
properties and avoiding side-effects (37,40,41,42,52). The approach
described herein has focused on cyclization combined with D-amino
acid substitution in a cyclic pentapeptide or hexapeptide side
chain-to-side chain structure. This strategy permits amidation or
extension of the C-terminus, and provides greater solubility and
improved pharmacological profiles relative to linear peptide or
side chain to C-terminus cyclic structures that were described
previously. The resulting compounds of Formula I show high
solubility, stability and favorable bioavailability as suggested by
activity after peripheral (including oral) administration (FIG. 12)
and penetration of the blood-brain barrier, as shown by central
antagonism of antinociception produced by peripheral administration
of the analogs (FIG. 13, Panel G). These advantageous drug
characteristics are accompanied by the favorable spectrum of
antinociception/side effect profiles described above.
[0141] Opioid-induced respiratory depression can be deadly, but the
analogs described herein (especially Analogs 1 and 4) did not
impair respiration even at equi- or greater-antinociceptive doses
compared to morphine. Analog 4 showed no significant respiratory
depression at doses producing significantly longer antinociception
than morphine. Analogs 2 and 4 also did not produce impairment of
motor coordination, while rats given morphine were significantly
impaired at equi-antinociceptive doses. Older adults who take
opioids for pain have an increased risk for falls and accidents.
Opioids such as those shown here, that alleviate pain without
producing motor deficits, would be particularly useful for this
population.
[0142] The analogs described herein produced significantly less
tolerance than morphine even though the antinociceptive effects
were matched. Numerous mechanisms have been postulated for opioid
tolerance. Some mechanisms of relevance to the current study
include effects of agonist efficacy, mu agonist/delta antagonist
properties, and glial activation. Stevens et al. (53) showed that
sufentanyl produced less tolerance than morphine at comparably
effective doses. They suggested that high efficacy agonists have a
lower receptor reserve requirement for maintaining antinociception,
and therefore show less tolerance. The analogs described herein are
consistent with this hypothesis, since they are high efficacy
agonists and show reduced tolerance. Combining a delta (DOR)
antagonist with a MOR agonist reduces tolerance (1). This profile
for Analogs 1, 3 and 4 (FIG. 11, Panel B) could contribute to the
relative lack of tolerance. However, since the delta antagonist
effect of the analogs is of high efficacy but low potency, further
studies would be required to establish the relative potency ratio
of the MOR agonism/DOR antagonism required to affect tolerance. In
addition, Analog 2 showed reduced tolerance despite a lack of DOR
antagonism, indicating the likely involvement of multiple
mechanisms and the importance of in vivo tests for assessing this
complex process.
[0143] A potentially important contributing mechanism to the
reduced tolerance produced by the analogs described herein is
differential glial activation. Many studies show that morphine
activates glia through a variety of mechanisms, resulting in
production of proinflammatory cytokines [IL-1, IL-6, IL-18,
TNF.alpha. (27,39,49)] and PG, ATP, NO, and ROS (57), which are
also "pronociceptive" and contribute to morphine tolerance and
hyperalgesia (28,39,49,51,19,57,58). Phosphorylation of microglial
p38 (pp38), which induces many of these pronociceptive molecules
(38), plays a key role in tolerance paradigms (28,34,38,56).
[0144] While morphine-induced glial activation is well-established,
the mechanism is the subject of considerable debate. Three leading
hypotheses are (1) activation of a non-opioid site on microglial
TLR4/1VID-2 receptors (38,58), (2) direct action at MOR on
microglia that stimulates purinergic (P2X) receptor signaling
(31,35,62), and (3) the release of proinflammatory CGRP that
activates glial-neuron inflammatory crosstalk (56). All three of
these proposed mechanisms ultimately converge onto the p38 pathway
to induce the release of pronociceptive molecules, so a crucial
finding here is that chronic exposure to the analogs did not change
pp38 levels while morphine significantly upregulated pp38. In
addition, EM Analog 4 unexpectedly did not change CGRP levels or
P2X7 receptor (P2X7R) expression, in contrast to morphine which
robustly increased the "activated" microglia phenotype that
strongly co-labeled with P2X7Rs. This is consistent with studies
showing direct CGRP (56) and P2X7R (27,62) involvement in the
development of tolerance to morphine. Hence, the reduced tolerance
and hyperalgesia after EM analogs may be partly due to less glial
reactivity. The present study is the first to our knowledge in
which MOR-selective agonists did not induce glial, pp38, CGRP or
P2X7R activation, while morphine, at the same antinociceptive
effectiveness, produced significant activation.
[0145] Morphine-induced glial reactivity raises a host of issues
concerning exacerbation of conditions associated with inflammation,
including post-operative-induced (34), nerve injury-induced
(34,43,49) and inflammation-induced (43) pain, traumatic brain
injury (45), neurodegenerative diseases (32), and advancing age
(25). Pain therapy in these conditions would be best served by
opioids shown here that do not exacerbate inflammatory responses.
The discovery of opioid induced proinflammatory responses has led
to efforts to develop anti-inflammatory medications as adjuncts to
opioid treatment to enhance analgesia and reduce tolerance. The
compounds of Formula I provide a clearly preferable approach, in
that immune modulators would not be required.
[0146] In addition to tolerance, differential glial activation may
contribute to the differences between the analogs and morphine in
other side effects. The microglial cell inhibitor minocycline
blocked the respiratory depressive effects of morphine at similar
doses and time frame used here (36), but see (63). Several studies
have linked glial reactivity to morphine-induced reward behaviors.
Morphine-induced CPP was associated with increased expression of
Iba1 and pp38 in the nucleus accumbens (NAc) (61). Systemic (36)
and intra-accumbens (61) minocycline blocked morphine-induced CPP,
and intra-NAc injection of media from cultured astrocytes
potentiated CPP for morphine (46).
[0147] Although numerous EM analogs have been developed, abuse
liability effects have not been reported previously. Because of the
importance of this side-effect, the analog compounds described
herein were tested in both conditioned place preference (CPP) and
self-administration (SA) paradigms. Tested at a range of
physiologically relevant doses, Analog 4 did not produce CPP or
aversive behaviors. Morphine promoted reward effects under
long-term access SA conditions, the most sensitive SA paradigm
(55), while the analogs did not. A recent review of rat
self-administration studies showed that they were concordant with
at least one of two human clinical indicators of abuse liability in
64 of 71 (90.1%) drug cases (48). Based on this correlation, the
likelihood of abuse in humans is low for the analogs. Circumventing
abuse liability is a major advance, since most available opioids
produce rewarding effects in CPP and/or SA models (48,54).
[0148] 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.
[0149] 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
* * * * *