U.S. patent number RE36,547 [Application Number 08/782,452] was granted by the patent office on 2000-02-01 for method of simultaneously enhancing analgesic potency and attenuating dependence liability caused by exogenous and endogenous opioid agonists.
This patent grant is currently assigned to Albert Einstein College of Medicine of Yeshiva University. Invention is credited to Stanley M. Crain, Ke-Fei Shen.
United States Patent |
RE36,547 |
Crain , et al. |
February 1, 2000 |
**Please see images for:
( Certificate of Correction ) ( Reexamination Certificate
) ** |
Method of simultaneously enhancing analgesic potency and
attenuating dependence liability caused by exogenous and endogenous
opioid agonists
Abstract
This invention relates to a method of selectively enhancing the
analgesic potency of morphine and other clinically used
bimodally-acting opioid agonists and simultaneously attenuating
development of physical dependence, tolerance and other undesirable
side effects caused by the chronic administration of said
bimodally-acting opioid agonists comprising the co-administration
of a bimodally-acting opioid agonist which activates both
inhibitory and excitatory opioid receptor-mediated functions of
neurons in the nociceptive (pain) pathways of the nervous system
and an opioid receptor antagonist which selectively inactivates
excitatory opioid receptor-mediated side effects. This invention
also relates to a method of using excitatory opioid receptor
antagonists alone to block the undesirable excitatory side effects
of endogenous bimodally-acting opioid agonists which may be
markedly elevated during chronic pain. This invention further
relates to a method of long-term treatment of previously detoxified
opiate, cocaine and alcohol addicts utilizing said excitatory
opioid receptor antagonists, either alone or in combination with
low-dose methadone, to prevent protracted physical dependence, and
to compositions comprising an excitatory opioid receptor antagonist
of the invention and a bimodally-acting opioid agonist.
Inventors: |
Crain; Stanley M. (Leonia,
NJ), Shen; Ke-Fei (Flushing, NY) |
Assignee: |
Albert Einstein College of Medicine
of Yeshiva University (Bronx, NY)
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Family
ID: |
27378386 |
Appl.
No.: |
08/782,452 |
Filed: |
January 13, 1996 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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097460 |
Jul 27, 1993 |
5472943 |
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947690 |
Sep 19, 1992 |
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Reissue of: |
276966 |
Jul 19, 1994 |
05512578 |
Apr 30, 1996 |
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Current U.S.
Class: |
514/282; 514/811;
514/812 |
Current CPC
Class: |
G01N
33/9486 (20130101); A61K 38/33 (20130101); A61K
31/485 (20130101); A61K 31/00 (20130101); G01N
33/94 (20130101); A61K 31/485 (20130101); A61K
2300/00 (20130101); A61K 38/33 (20130101); A61K
2300/00 (20130101); G01N 2500/10 (20130101) |
Current International
Class: |
A61K
31/485 (20060101); A61K 38/33 (20060101); A61K
31/00 (20060101); G01N 33/94 (20060101); A61K
031/14 () |
Field of
Search: |
;514/282,811,812 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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9406426 |
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Mar 1994 |
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WO |
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9503804 |
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Sep 1995 |
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WO |
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Other References
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.
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.
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.
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.
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.
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(1986)..
|
Primary Examiner: Reamer; James H.
Attorney, Agent or Firm: Amster, Rothstein &
Ebenstein
Government Interests
STATEMENT OF GOVERNMENT INTEREST
This invention was made with government support under NIDA research
grant number DA 02031. As such, the government has certain rights
in the invention.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a Continuation-In-Part of application Ser. No.
08/097,460 filed Jul. 27, 1993, entitled METHOD OF SIMULTANEOUSLY
ENHANCING ANALGESIC POTENCY AND ATTENUATING DEPENDENCE LIABILITY
CAUSED BY MORPHINE AND OTHER OPIOID AGONISTS, .[.currently
pending,.]. .Iadd.now issued as U.S. Pat. No. 5,472,943,
.Iaddend.which is a Continuation-In-Part of application Ser. No.
07/947,690 filed Sep. 19, 1992, entitled A METHOD OF IDENTIFICATION
OF NON-ADDICTIVE OPIOID ANALGESICS AND THE USE OF SAID ANALGESICS
FOR TREATMENT OF OPIOID ADDICTION, now abandoned.
Claims
We claim:
1. A method for selectively enhancing the analgesic potency of a
bimodally-acting opioid agonist and simultaneously attenuating
anti-analgesia, hyperalgesia, hyperexcitability, physical
dependence and/or tolerance effects associated with the
administration of said bimodally-acting opioid agonist, comprising
administering to a subject an analgesic or sub-analgesic amount of
said bimodally-acting opioid agonist and an amount of an excitatory
opioid receptor antagonist effective to enhance the analgesic
potency of said bimodally-acting opioid agonist and attenuate the
anti-analgesia, hyperalgesia, hyperexcitability, physical
dependence and/or tolerance effects of said bimodally-acting opioid
agonist.
2. The method of claim 1 wherein the excitatory opioid receptor
antagonist is selected from the group consisting of naltrexone,
naloxone, etorphine, diprenorphine, dihydroetorphine, and similarly
acting opioid alkaloids and opioid peptides.
3. The method of claim 1 wherein the bimodally-acting opioid
agonist is selected from the group consisting of morphine, codeine,
fentanyl analogs, pentazocine, buprenorphine, methadone,
enkephalins, dynorphins, endorphins and similarly acting opioid
alkaloids and opioid peptides.
4. The method of claim 1 wherein the amount of the excitatory
opioid receptor antagonist administered is at least 100-1000 fold
less than the amount of the bimodally-acting opioid agonist
administered.
5. The method of claim 2 wherein the excitatory opioid receptor
antagonist is naltrexone.
6. The method of claim 3 wherein the bimodally-acting opioid
agonist is morphine.
7. The method of claim 3 wherein the bimodally-acting opioid
agonist is codeine.
8. The method of claim 1 wherein the mode of administration is
selected from the group consisting of oral, sublingual,
intramuscular, subcutaneous and intravenous.
9. The method of claim 1 wherein the opioid receptor antagonist is
naltrexone, and is administered orally.
10. A method for treating a detoxified opiate, cocaine or alcohol
addict so as to prevent protracted dependence thereon comprising
administering to the detoxified addict over a long term an amount
of an excitatory opioid receptor antagonist which does not block
but instead enhances the analgesic effect of morphine and other
bimodally-acting opioid agonists.
11. The method of claim 10 wherein the antagonist is administered
in combination with a sub-analgesic amount of a long-lasting
bimodally-acting opioid agonist.
12. The method of claim 11 wherein the opioid agonist is
methadone.
13. The method of claim 10 wherein the excitatory opioid receptor
antagonist is selected from the group consisting of naloxone,
naltrexone, etorphine, dihydroetorphine, diprenorphine, and
similarly acting opioid alkaloids and opioid peptides.
14. The method of claim 13 wherein the excitatory opioid receptor
antagonist is naltrexone.
15. The method of claim 11 wherein the bimodally-acting opioid
agonist is methadone and the excitatory opioid receptor antagonist
is naltrexone.
16. A composition comprising an analgesic or sub-analgesic amount
of a bimodally-acting opioid agonist and an amount of an excitatory
opioid receptor antagonist effective to enhance the analgesic
potency of said bimodally-acting opioid agonist and attenuate the
anti-analgesia, hyperalgesia, hyperexcitability, physical
dependence and/or tolerance effects of said bimodally-acting opioid
agonist.
17. The composition of claim 16 wherein the excitatory opioid
receptor antagonist is selected from the group consisting of
naltrexone, naloxone, etorphine, diprenorphine, dihydroetorphine,
and similarly acting opioid alkaloids and opioid peptides.
18. The composition of claim 16 wherein the bimodally-acting opioid
agonist is selected from the group consisting of morphine, codeine,
fentanyl analogs, pentazocine, methadone, buprenorphine,
enkephalins, dynorphins, endorphins and similarly acting opioid
alkaloids and opioid peptides.
19. The method of claim 1 wherein the bimodally-acting opioid
agonist is morphine and the excitatory opioid receptor antagonist
is naltrexone.
20. The method of claim 3 wherein the bimodally-acting opioid
agonist is methadone.
21. The composition of claim 16 wherein the amount of the
excitatory opioid receptor antagonist is at least 100-1000 fold
less than the amount of the bimodally-acting opioid agonist.
22. The composition of claim 17 wherein the excitatory opioid
receptor antagonist is naltrexone.
23. The composition of claim 18 wherein the bimodally-acting opioid
agonist is morphine.
24. The composition of claim 18 wherein the bimodally-acting opioid
agonist is methadone.
25. The composition of claim 16 wherein the bimodally-acting opioid
agonist is morphine and the excitatory opioid receptor antagonist
is naltrexone.
26. A method for treating pain in a subject comprising
administering to said subject an analgesic or sub-analgesic amount
of a bimodally-acting opioid agonist and an amount of an excitatory
opioid receptor antagonist effective to enhance the analgesic
potency of said bimodally-acting opioid agonist and attenuate the
anti-analgesia, hyperalgesia, hyperexcitability, physical
dependence and/or tolerance effects of said bimodally-acting opioid
agonist.
27. The method of claim 26 wherein the bimodally-acting opioid
agonist is selected from the group consisting of morphine, codeine,
fentanyl analogs, pentazocine, methadone, buprenorphine,
enkephalins, dynorphins, endorphins and similarly acting opioid
alkaloids and opioid peptides.
28. The method of claim 26 wherein the excitatory opioid receptor
antagonist is selected from the group consisting of naltrexone,
naloxone, etorphine, diprenorphine and dihydroetorphine, and
similarly acting opioid alkaloids and opioid peptides.
29. The method of claim 26 wherein amount of the excitatory opioid
receptor antagonist administered is at least 100-1000 fold less
than the amount of the bimodally-acting opioid agonist
administered.
30. The method of claim 26 wherein the excitatory opioid receptor
antagonist is naltrexone.
31. The method of claim 26 wherein the bimodally-acting opioid
receptor agonist is morphine.
32. The method of claim 26 wherein the bimodally-acting opioid
agonist is morphine and the excitatory opioid receptor antagonist
is naltrexone.
Description
FIELD OF THE INVENTION
This invention relates to a method of enhancing the analgesic
(inhibitory) effects of bimodally-acting opioid agonists, including
morphine, codeine and other clinically used opioid analgesics,
while at the same time attenuating anti-analgesic effects, physical
dependence, tolerance, hyperexcitability, hyperalgesia, and other
undesirable (excitatory) side effects typically caused by chronic
use of bimodally-acting (excitatory and inhibitory) opioid
agonists. As used herein, the term "opioid" refers to compounds
which bind to specific opioid receptors and have agonist
(activation) or antagonist (inactivation) effects at these
receptors, such as opioid alkaloids, including the agonist morphine
and the antagonist naloxone, and opioid peptides, including
enkephalins, dynorphins and endorphins. As used herein, the term
"opiate" refers to drugs derived from opium or related analogs.
In the instant invention, a very low dose of a selective excitatory
opioid receptor antagonist is combined with a reduced dose of a
bimodally-acting opioid agonist so as to enhance the degree of
analgesia (inhibitory effects) and attenuate undesired side effects
(excitatory effects). Opioid analgesia results from activation (by
opioid agonists) of inhibitory opioid receptors on neurons in the
nociceptive (pain) pathways of the peripheral and central nervous
systems. The undesirable side effects, including anti-analgesic
actions, hyperexcitability and hyperalgesia, the development of
physical dependence, and some types of tolerance result from
sustained activation (by bimodally-acting opioid agonists) of
excitatory opioid receptors on neurons in the nociceptive (pain)
pathways of the peripheral and central nervous systems. In
addition, in the instant invention, long-term administration of
ultra-low doses of the excitatory opioid receptor antagonists of
the invention, either alone or in combination with low doses of
conventional bimodally-acting opioid agonists, provides effective
maintenance treatment of previously detoxified opiate, alcohol and
cocaine addicts.
BACKGROUND OF THE INVENTION
Morphine or other bimodally-acting opioid agonists are administered
to relieve severe pain due to the fact that they have analgesic
effects mediated by their activation of inhibitory opioid receptors
on nociceptive neurons (see North, Trends Neurosci., Vol. 9, pp.
114-117 (1986) and Crain and Shen, Trends Pharmacol. Sci., Vol. 11,
pp. 77-81 (1990)). However, bimodally-acting opioid agonists also
activate opioid excitatory receptors on nociceptive neurons, which
attenuates the analgesic potency of the opioids and results in the
development of physical dependence thereon and increased tolerance
thereto (see Shen and Crain, Brain Res., Vol. 597, pp. 74-83
(1992)), as well as hyperexcitability, hyperalgesia and other
undesirable (excitatory) side effects. As a result, a long-standing
need has existed to develop a method of both enhancing the
analgesic (inhibitory) effects of bimodally-acting opioid agonists
and limiting the undesirable (excitatory) side effects caused by
such opioid agonists.
The grandparent Patent Application for the instant invention, Ser.
No. 07/947,690, relates to a specific group of opioid agonists for
use as low/non-addictive analgesics and for the treatment of opioid
addiction. In the grandparent Application, it is stated that this
group of opioid agonists bind to and activate inhibitory but not
excitatory opioid receptors. In contrast, morphine and most other
opioid alkaloids and peptides elicit bimodal effects by binding to
and activating both excitatory and inhibitory opioid receptors.
To date, no method has been discovered or developed whereby two
opioid compounds are co-administered, one of which binds to and
acts as a selective agonist at inhibitory opioid receptors to cause
analgesia and the other of which binds to and acts as a selective
antagonist at excitatory opioid receptors so as to attenuate
undesirable side effects caused by the administration of
bimodally-acting opioid agonists while simultaneously enhancing the
analgesic effects of said bimodally-acting opioid agonists.
It is therefore an object of this invention to provide a method of
enhancing the analgesic potency of morphine and other
bimodally-acting opioid agonists by blocking their anti-analgesic
side effects.
It is a further object of this invention to provide a method of
attenuating physical dependence, tolerance, hyperexcitability,
hyperalgesia and other undesirable side effects caused by the
chronic administration of bimodally-acting opioid agonists.
It is another object of this invention to provide a method for
maintenance treatment of previously detoxified opiate, cocaine and
alcohol addicts utilizing ultra-low doses of an excitatory opioid
receptor antagonists, either alone or in combination with long-term
administration of low doses of methadone.
It is yet another object of this invention to provide a composition
which enhances the analgesic effects of bimodally-acting opioid
agonists while simultaneously attenuating undesirable side effects
caused by said opioid agonists, including physical dependence,
tolerance, hyperexcitability and hyperalgesia.
It is still a further object of this invention to provide a
composition which is useful for treatment of opiate, cocaine and
alcohol addicts.
SUMMARY OF THE INVENTION
This invention is directed to a method of selectively enhancing the
analgesic potency of morphine and other conventional
bimodally-acting opioid agonists and simultaneously attenuating
undesirable side effects, including physical dependence, caused by
the chronic administration of said opioid agonists. Morphine and
other bimodally-acting (inhibitory/excitatory) opioid agonists bind
to and activate both inhibitory and excitatory opioid receptors on
nociceptive neurons which mediate pain. Activation of inhibitory
receptors by said agonists causes analgesia. Activation of
excitatory receptors by said agonists results in anti-analgesic
effects, hyperexcitability, hyperalgesia, as well as development of
physical dependence and tolerance and other undesirable side
effects. A series of antagonists which bind to excitatory opioid
receptors (e.g., diprenorphine, naltrexone and naloxone)
selectively block excitatory opioid receptor functions of
nociceptive types of DRG neurons at 1,000 to 10,000-fold lower
concentrations than are required to block inhibitory opioid
receptor functions in these neurons. The co-administration of a
bimodally-acting opioid agonist together with an ultra-low dose of
an opioid antagonist which binds to and inactivates excitatory, but
not inhibitory, opioid receptors results in the blocking of
excitatory anti-analgesic side effects of said opioid agonists on
these neurons, thereby resulting in enhanced analgesic potency.
This enhanced analgesic potency permits the use of lower doses of
morphine or other conventional opioid analgesics.
The preferred excitatory opioid receptor antagonists of the
invention include naltrexone and naloxone, in addition to
etorphine, dihydroetorphine, and diprenorphine which are disclosed
in parent U.S. patent application Ser. No. 08/097,460 and similarly
acting opioid alkaloids and opioid peptides. Prior hereto, clinical
uses of naloxone and naltrexone have been formulated to be
administered at much higher doses (e.g. 50 mg), which block
inhibitory opioid receptor functions mediating analgesia in
addition to blocking excitatory opioid receptors. These high doses
of antagonist are required as an antidote for acute opiate agonist
overdose (e.g., respiratory depression). However, in the instant
invention, long-term oral administration of ultra-low doses of
naltrexone (for example about 1 .mu.g) alone or in combination with
low doses of methadone (e.g. mg) prevents protracted physical
dependence which underlies resumption of drug abuse in previously
detoxified opiate, cocaine and alcohol addicts. This is in contrast
to clinical use of naltrexone prior hereto, wherein large (50 mg)
tablets (Trexan) are administered, which produce dysphoria and
other aversive side effects, and long-term treatment with high
doses of methadone which results in physical dependence on
methadone.
The opioid agonists of the invention include morphine or other
bimodally-acting (inhibitory/excitatory) opioid alkaloids or opioid
peptides that are in clinical use as analgesics, including codeine,
fentanyl analogs, pentazocine, buprenorphine, methadone and
endorphins.
Further, in chronic pain patients, the excitatory opioid receptor
antagonists of the invention are administered alone in ultra-low
doses to enhance the analgesic potency and decrease the dependence
liability of endogenous (as opposed to exogenous) opioid peptides,
including enkephalins, dynorphins and endorphins, so as to
facilitate physiologic mechanisms which normally regulate opioid
responsivity and nociceptive systems.
BRIEF DESCRIPTION OF THE DRAWINGS
The above brief description, as well as further objects and
features of the present invention, will be more fully understood by
reference to the following detailed description of the presently
preferred albeit illustrative, embodiments of the present invention
when taken in conjunction with the accompanying drawings
wherein:
FIG. 1 represents the structural formulae of the bimodally-acting
opioid agonist morphine and the preferred excitatory opioid
receptor antagonists of the invention, naltrexone and naloxone.
Naltrexone is the N-cyclopropylmethyl congener of naloxone;
FIG. 2 represents the direct inhibitory effect of etorphine on the
action potential duration (APD) of nociceptive types of sensory
neurons and the blocking effect of etorphine on the excitatory
response (APD prolongation) elicited by morphine. Acute application
of low (pM-nM) concentrations of etorphine to naive dorsal root
ganglion (DRG) neurons elicits dose-dependent, naloxone-reversible
inhibitory shortening of the APD. In contrast, morphine and other
bimodally-acting opioid agonists elicit excitatory APD prolongation
at these low concentrations which can be selectively blocked by
<pM levels of etorphine, resulting in unmasking of potent
inhibitory APD shortening by nM morphine;
FIG. 3 represents dose-response curves of different opioids,
showing that etorphine and dihydroetorphine elicit only inhibitory
dose-dependent shortening of the APD of DRG neurons at all
concentrations tested (fM-.mu.M). In contrast, dynorphin A (as well
as morphine and other bimodally-acting opioids) elicits
dose-dependent excitatory APD prolongation at low concentrations
(fM-nM) and requires much higher concentrations (about 0.1-1 .mu.M)
to shorten the APD, thereby resulting in-a bell-shaped
dose-response curve;
FIGS. 4A and 4B represent the selective blocking of excitatory
APD-prolonging effects elicited by morphine in DRG neurons by
co-administration of a low (pM) concentration of diprenorphine,
thereby unmasking potent dose-dependent inhibitory APD shortening
by low concentrations of morphine (comparable to the inhibitory
potency of etorphine). In contrast, co-treatment with a higher (nM)
concentration of DPN blocks both inhibitory as well as excitatory
opioid effects;
FIG. 5 represents similar selective blocking of excitatory
APD-prolonging effects elicited by morphine in DRG neurons when
co-administered with a low (pM) concentration of naltrexone,
thereby unmasking potent inhibitory APD shortening by low
concentrations of morphine. In contrast, a higher (.mu.M)
concentration of naltrexone blocks both inhibitory as well as
excitatory opioid effects; and
FIG. 6 represents the assay procedure used to demonstrate that
selective antagonists at excitatory opioid receptors prevent
development of tolerance/dependence during chronic co-treatment of
DRG neurons with morphine.
DETAILED DESCRIPTION OF THE INVENTION
This invention is directed to a method of selectively enhancing the
analgesic effect caused by the administration of a bimodally-acting
opioid agonist and simultaneously attenuating undesirable side
effects caused by the chronic administration of said
bimodally-acting opioid agonists. This is performed by
simultaneously inactivating excitatory opioid receptor-mediated
functions of neurons in the nociceptive (pain) pathways and
activating inhibitory opioid receptor-mediated mediated functions
of nociceptive neurons. Low doses of a bimodally-acting opioid
agonist and an excitatory opioid receptor antagonist are
co-administered. The bimodally-acting opioid agonist binds to
inhibitory receptors on nociceptive neurons so as to activate
inhibitory opioid receptor-mediated functions, including analgesia,
and concomitantly activates excitatory opioid receptors on
nociceptive neurons. The excitatory opioid receptor antagonist
binds to excitatory receptors on said neurons and thereby
inactivates excitatory opioid receptor-mediated functions,
including anti-analgesic effects, physical dependence and tolerance
to the opioid agonist, hyperexcitability and hyperalgesia.
Alternatively, the excitatory opioid receptor antagonists of the
invention can be used to pretreat patients prior to administering
bimodally-acting exogenous opioids thereto, or used alone to
enhance the analgesic potency and decrease the dependence liability
of endogenous opioid peptides including enkephalins, dynorphins and
endorphins, which are markedly unregulated in chronic pain
patients.
In addition, this invention is directed to the use of said
excitatory opioid receptor antagonists and opioid agonists for
maintenance treatment of previously detoxified opiate addicts.
Because addiction to cocaine and alcohol are also mediated by
specific opioid-sensitive brain cell networks (see Gardner, et al.
Substance Abuse 2 ed. pp. 70-99 (1992)), and because addiction to
cocaine and alcohol are mediated by specific opioid-sensitive brain
cell networks, the method of the invention for treating opiate
addicts can also be used for the treatment of cocaine or alcohol
addicts. Further, this invention is directed to a composition
comprising an excitatory opioid receptor antagonist and a
bimodally-acting opioid agonist.
The inventors have discovered that certain compounds act as
excitatory opioid receptor antagonists, that is, they bind to and
inactivate excitatory opioid receptors on neurons in the
nociceptive pathways. The excitatory opioid receptor antagonists of
the invention are preferably selected from the group consisting of
naloxone, naltrexone, diprenorphine, etorphine and
dihydroetorphine. One of the excitatory opioid receptor antagonists
of the invention, naltrexone, can be administered orally at very
low doses. For example, naltrexone can be administered at a level
as low as 1 .mu.g and will have selective antagonist action at
excitatory, but not inhibitory, opioid receptors. All previous
clinical use of naltrexone, as well as naloxone, has been at much
higher (>mg) doses which results in antagonist actions at both
inhibitory as well as excitatory opioid receptors. In addition,
since the antagonists enhance the analgesic potency of the
agonists, the agonists become effective when administered at
markedly reduced doses which would otherwise be sub-analgesic.
The alkaloid opioid receptor antagonists of the invention
inactivate mu, delta, kappa and other subtypes of excitatory opioid
receptors. Etorphine and dihydroetorphine have very similar
chemical structures and are potent analgesics which selectively
activate inhibitory but not excitatory opioid receptors (see Shen
and Crain, Brain Res., Vol. 636, pp. 286-297 (1994)). Naltrexone,
naloxone (see FIG. 1) and diprenorphine have slightly different
chemical structures than etorphine and dihydroetorphine, which
results in their acting as general opioid receptor antagonists at
all types of inhibitory and excitatory opioid receptors (see Shen
and Crain, Brain Res., Vol. 491, pp. 227-242 (1989) and Brain Res.,
Vol. 636, (1994)). Nevertheless, at very low (pM) concentrations,
these compounds are all capable of selectively binding to and
acting as antagonists at excitatory, but not inhibitory, opioid
receptors on nociceptive DRG neurons.
The bimodally-acting opioid agonists of this invention preferably
include morphine, codeine, methadone, pentazocine buprenorphine,
fentanyl analogs, endorphins, and other opioid alkaloids and opioid
peptides. Typically, the opioid agonists of the invention are mu,
delta, kappa or epsilon opioid receptor agonists, and are capable
of binding to inhibitory opioid receptors on neurons in the pain
pathway. When these bimodally-acting agonists bind to inhibitory
opioid receptors, they thereby activate inhibitory opioid
receptor-mediated functions, including analgesia.
As discussed below, the inventors have discovered by studies of
nociceptive DRG neurons that certain compounds (the excitatory
opioid receptor antagonists of the invention), when used for
pretreatment or when co-administered with bimodally-acting opioid
agonists, are capable at very low dosages of enhancing the
analgesic effects of the bimodally-acting opioid agonists at least
100-1000 fold by inactivating excitatory anti-analgesic side
effects of said agonists. In addition, the excitatory opioid
receptor antagonists of the invention prevent development of opioid
tolerance and dependence which are mediated by sustained activation
of excitatory opioid receptor functions.
In addition, the excitatory opioid receptor antagonists of the
invention can be administered either alone or in conjunction with
low, sub-analgesic doses of inhibitory opioid receptor agonists for
long-term maintenance treatment of previously detoxified opiate,
cocaine and alcohol addicts to prevent protracted physical
dependence (see Goldberg, et al. (1969) and Crain, et al. (1992)),
which underlies resumption of drug abuse.
The long-term treatment of detoxified addicts with selective
antagonists blocks sustained activation of excitatory opioid
receptor functions by endogenous opioid peptides. These peptides
are present in the brain at concentrations that are well above the
markedly reduced threshold required to activate chronic
morphine-sensitized excitatory opioid receptors, thereby blocking
the cellular mechanism proposed to underlie protracted physical
dependence. Further, the excitatory opioid receptor antagonists can
be administered alone to chronic pain patients to enhance the
analgesic potency and decrease the dependence liability of
endogenous opioid peptides, including enkephalins, dynorphins and
endorphins which normally regulate nociceptive (pain) sensitivity
and which are elevated during chronic pain.
Ordinarily, most conventional bimodally-acting opioid agonists are
administered clinically in milligram dosages. By co-administering
bimodally-acting opioid agonists with the excitatory opioid
receptor antagonists of the invention, it is possible to achieve an
analgesic effect with 10-100 times lower doses of the
bimodally-acting opioid agonist than when said opioid agonist is
administered alone. This is because the excitatory opioid receptor
antagonists of the invention enhance the analgesic effects of the
bimodally-acting opioid agonists by attenuating the anti-analgesic
excitatory side effects of said opioid agonists. Hence,
bimodally-acting opioid agonists which are administered with the
excitatory opioid receptor antagonists of the invention are
administered in an amount 10-100 times less than the amount of that
bimodally-acting opioid agonist which has typically been
administered for analgesia.
According to the present invention, the dose of excitatory opioid
receptor antagonist to be administered is 100-1000 times less than
the dose of bimodally-acting opioid agonist to be administered, for
example, about 1 microgram of said antagonist together with
100-1000 micrograms of said agonist. These estimates of dosages are
based on studies of nociceptive DRG neurons in culture. The
excitatory opioid receptor antagonists, as well as the inhibitory
opioid agonists, can be administered orally, sublingually,
intramuscularly, subcutaneously or intravenously. Naltrexone is
particularly useful since it can be administered orally at 1 .mu.g
doses, has long-lasting action and has been safely used in
treatment of opiate addiction at 50 mg doses several times per week
for several years (see Greenstein et al., Subst. Abuse, 2d ed.
(1992) and Gonzales et al., Drugs, Vol. 35, pp. 192-213 (1988).
The co-administration of the opioid agonists and excitatory opioid
receptor antagonists of the invention simultaneously activates
inhibitory functions of nociceptive neurons mediating pain and
inactivates excitatory functions of the same or other nociceptive
neurons. In order to demonstrate this, electrophysiologic studies
on the effects of opioids on nociceptive types of mouse sensory DRG
neurons in tissue cultures were performed. It is shown below that
this bimodal modulation is mediated by activating putative
excitatory opioid receptors in addition to previously characterized
inhibitory opioid receptors on sensory neurons.
It is shown that at low pM-nM concentrations, nearly all
bimodally-acting opioids, including morphine, enkephalins,
dynorphins, endorphins and specific mu, delta and kappa opioid
agonists, elicit naloxone-reversible dose-dependent excitatory
effects manifested by prolongation of the calcium-dependent
component of the action potential duration (APD) of DRG neurons. In
contrast, the same opioids generally elicit inhibitory APD
shortening effects when applied at higher concentrations (0.1-1
.mu.M).
The excitatory opioid effects on sensory neurons have been shown to
be mediated by opioid receptors that are coupled via a
cholera-toxin-sensitive stimulatory GTP-binding protein, Gs, to
adenylate cyclase/cyclic AMP/protein kinase A-dependent ionic
conductances that prolong the APD (resembling, for example,
beta-adrenergic receptors). (See Crain and Shen, Trends Pharmacol.
Sci., Vol. 11, pp. 77-81 (1990)). On the other hand, inhibitory
opioid effects are mediated by opioid receptors that are coupled
via pertussis toxin-sensitive inhibitory G proteins: Gi to the
adenylate cyclase/cyclic AMP system and Go to ionic conductances
that shorten the APD (resembling, for example, alpha.sub.2
-adrenergic receptors). Shortening by opioids of the action
potential of primary sensory neurons has generally been considered
to be a useful model of their inhibition of calcium influx and
transmitter release at presynaptic terminals in the dorsal spinal
cord, thereby accounting for opioid-induced analgesia in vivo. (See
North, Trends Neurosci., Vol. 9, pp. 114-117 (1986) and Crain and
Shen, Trends Pharmacol. Sci., Vol. 11, pp. 77-81 (1990)).
Similarly, the delayed repolarization associated with the observed
opioid-induced prolongation of action potential has been
interpreted as evidence of excitatory effects of opioids on
nociceptive types of sensory neurons (see Shen and Crain, J.
Neurosci., (1994, in press)) that may result in enhanced calcium
influx and transmitter release at presynaptic terminals. This could
account for some types of hyperalgesia and hyperexcitatory states
elicited by opioids in vivo (see Crain and Shen, Trends Pharmacol.
Sci., Vol. 11, pp. 77-81 (1990); Shen and Crain, Brain Res., Vol.
491, pp. 227-242 (1989); and Shen and Crain, J. Neurosci.
(1994).
Chronic treatment of DRG neurons with typical bimodally-acting
(excitatory/inhibitory) opioids (e.g., 1 .mu.M D-ala.sup.2
-D-leu.sup.5 enkephalin (DADLE) or morphine for 1 week) results in
tolerance to the usual inhibitory APD-shortening effects of high
concentrations of these opioids and supersensitivity to the
excitatory APD-prolonging effects of these opioid agonists, as well
as the opioid antagonist, naloxone (see Crain and Shen, Brain Res.,
Vol. 575, pp. 13-24 (1992) and Shen and Crain, Brain Res., Vol.
597, pp. 74-83 (1992)). It has been suggested that the latter
electrophysiologic effects and related biochemical adaptations are
cellular manifestations of physical dependence that may underlie
some aspects of opiate addiction (see Shen and Crain, Brain Res.,
Vol. 597, pp. 74-83 (1992) and Terwilliger et al., Brain Res., Vol.
548, pp. 100-110 (1991)).
In contrast to bimodally-acting opioids, it has been discovered by
the inventors that the opioid alkaloids etorphine (see Bentley and
Hardy, Proc. Chem. Soc., pp. 220 (1963) and Blane et al., Brit. J.
Pharmacol. Chemother., Vol. 30, pp. 11-22 (1967)) and
dihydroetorphine (see Bentley and Hardy, J. Amer. Chem. Soc., Vol.
89, pp. 3281-3286 (1967)) uniquely elicit dose-dependent,
naloxone-reversible inhibitory effects on sensory neurons in
DRG-spinal cord explants, even at concentrations as low as 1 pM,
and show no excitatory effects at lower concentrations (see Shen
and Crain, Brain Res.,, Vol. 636, pp. 286-297 (1994)). In addition,
these potent inhibitory opioid receptor agonists also display
unexpected antagonist effects at excitatory opioid receptors on DRG
neurons. Acute pretreatment of DRG neurons with etorphine or
dihydroetorphine, at low concentrations (<pM) which do not alter
the APD, block the excitatory APD-prolonging effects of morphine
and other bimodally-acting opioids and unmask inhibitory
APD-shortening effects which normally require much higher
concentrations. The potent inhibitory effect of etorphine and
dihydroetorphine may be due to their selective activation of
inhibitory opioid receptor-mediated functions while simultaneously
inactivating excitatory opioid receptor-mediated functions in
sensory neurons. In contrast, bimodally-acting opioids activate
excitatory as well as inhibitory opioid receptors on DRG neurons,
thereby decreasing the net inhibitory effectiveness of these
agonists, resembling the attenuation of the inhibitory potency of
systemic morphine by the "anti-analgesic" (excitatory) effect of
dynorphin A release in spinal cord in mice (see Fujimoto et al.,
Neuropharmacol., Vol. 29, pp. 609-617, (1990)).
The inventors have discovered that at ultra-low (pM)
concentrations, naloxone and naltrexone act as selective
antagonists at excitatory opioid receptors on DRG neurons, thereby
unmasking potent inhibitory effects of bimodally-acting opioid
agonists. At nM concentrations, naloxone blocks both inhibitory APD
shortening in DRG neurons by .mu.M opioid agonists as well as
excitatory APD prolongation by pM-nM opioids. Systematic tests with
lower concentrations of naloxone have revealed that pM naloxone
acts selectively as an antagonist at excitatory opioid receptors.
In DRG neurons where fM-nM morphine elicited dose-dependent
excitatory APD prolongation, subsequent tests on the same neurons
in the presence of 1 pM naloxone showed a complete block of opioid
excitatory effects, and in some of the cells inhibitory APD
shortening was evoked at these low (fM-nM) morphine concentrations.
Similar unmasking of potent inhibitory effects of low
concentrations of morphine was obtained in another series of DRG
neurons tested with fM-nM morphine in the presence of pM
naltrexone, whereas higher concentrations of naltrexone (nM-.mu.M)
blocked both inhibitory as well as excitatory opioid effects (see
FIG. 5).
The selective antagonist action of ultra-low dose naloxone at
excitatory opioid receptors is consonant with in vivo data where
0.1 fg of naloxone (i.t.) enhanced a type of behavioral
(tail-flick) analgesia in mice shown to be mediated by an
endogenous dynorphin A-(1-17) anti-analgesic system, whereas 100 fg
of naloxone (i.t.) was required to significantly reduce analgesia
mediated by direct i.t. injection of morphine or k opioid agonists
(see Fujimoto et al., J. Pharm. Exp. Ther., Vol. 251, pp. 1045-1052
(1989)).
Co-administration of low (pM) concentrations of etorphine during
chronic treatment of DRG neurons with .mu.M levels of morphine is
effective in preventing development of the opioid excitatory
supersensitivity and tolerance that generally occurs after
sustained exposure to bimodally-acting opioids. Acute application
of 1 fM dynorphin A(1-13) or 10 nM naloxone to DRG neurons
chronically exposed to 3 .mu.M morphine together with 1 pM
etorphine (for greater than 1 week) did not evoke the usual
excitatory APD prolongation observed in chronic morphine-treated
cells, even when tested up to 6 hours after return to BSS.
Furthermore, there was little or no evidence of tolerance to the
inhibitory APD-shortening effects of .mu.M morphine.
If etorphine was acting simply as an agonist at inhibitory opioid
receptors, it might be predicted that the addition of 1 pM
etorphine together with a 10.sup.6 -fold higher concentration of
morphine would have a negligible effect on chronic morphine-treated
DRG neurons or would augment development of cellular signs of
dependence. However, the results obtained are accounted for by the
potent antagonist action of etorphine at excitatory opioid
receptors during chronic morphine treatment, thereby preventing
development of opioid excitatory supersensitivity and tolerance,
just as occurs during chronic opioid treatment of DRG neurons in
the presence of cholera toxin-B sub-unit (see Shen et al., Brain
Res., Vol. 575, pp. 13-24 (1992)), which selectively interferes
with GM.sub.1 ganglioside regulation of excitatory opioid receptor
functions (see Shen et al., Brain Res., Vol. 531, pp. 1-7 (1990)
and Shen et al., Brain Res., Vol. 559, pp. 130-138 (1991)).
Similarly, co-administration of ultra-low (pM) concentrations of
naloxone or naltrexone during chronic treatment of DRG neurons with
.mu.M levels of morphine was effective in preventing development of
the opioid excitatory supersensitivity and tolerance that generally
occurs after sustained exposure to bimodally-acting opioids. Acute
application of fM dynorphin A-(1-13) or fM morphine, as well as 1
nM naloxone to DRG neurons chronically exposed to 1 .mu.M morphine
together with 1 pM naloxone or naltrexone (for 1-10 weeks) did not
evoke the usual excitatory APD prolongation observed in chronic
morphine-treated cells (see Crain et al., (1992) and Shen et al.,
(1992)) tested after washout with BSS. Furthermore, there was no
evidence of tolerance to the usual inhibitory effects of .mu.M
opioids.
Chronic co-treatment of nociceptive types of DRG neurons with
morphine together with ultra-low (pM) concentrations of naltrexone
or naloxone can therefore prevent the cellular manifestations of
tolerance and dependence that generally occur in chronic
morphine-treated DRG neurons. This data for naltrexone and naloxone
on chronic morphine-treated nociceptive DRG neurons provides
evidence that the formulation of opioid analgesic preparations
comprising ultra-low doses of these excitatory opioid receptor
antagonists and morphine (or codeine) will result in enhanced
analgesic potency and low dependence liability.
The unmasking by pM naloxone or naltrexone of potent inhibitory
(APD-shortening) effects of low pM-nM concentrations of morphine in
DRG neurons accounts for the paradoxical enhancement by low-dose
naloxone of: (1) morphine analgesia in humans (see Gillman et al.,
Intern. J. Neurosci., Vol. 48, pp. 321-324 (1989); Gillman et al.,
J. Nuerol. Sciences, Vol. 49, pp. 41-49 (1981); and South African
J. Science Vol. 83, pp. 560-563 (1987); (2) buprenorphine analgesia
in humans and animals (see Pederson et al., Brit. J. Anaesth., Vol.
57, pp. 1045-1046 (1985); Schmidt et al., Anesthesia, Vol. 40, pp.
583-586 (1985); and Bergman et al., Arch. Int. Pharmacodyn., Vol.
291, pp. 229-237 (1988)); and (3) pentazocine analgesia in humans
(see Levine et al., J Clin, Invest., Vol. 82, pp. 1574-1577
(1988).
EXAMPLE 1
The effects of etorphine and dihydroetorphine on nociceptive types
of DRG neurons in culture are described in Example 1. Etorphine and
dihydroetorphine are the first compounds determined by the
inventors by electrophysiologic analyses on DRG neurons to have
specific antagonist action on excitatory opioid receptor functions
when applied at ultra-low (pM) concentrations. This is in contrast
to their well-known agonist action at inhibitory opioid receptors
when applied at higher concentrations.
Etorphine and Dihydroetorphine Act as Potent Selective Antagonists
at Excitatory Opioid Receptors on DRG Neurons Thereby Enhancing
Inhibitory Effects of Bimodally-Acting Opioid Agonists
Methods (Used in This and Following Examples): The experiments
described herein were carried out on dorsal root ganglion (DRG)
neurons in organotypic explants of spinal cord with attached DRGs
from 13-day-old fetal mice after 3 to 5 weeks of maturation in
culture. The DRG-cord explants were grown on collagen-coated
coverslips in Maximow depression-slide chambers. The culture medium
consisted of 65% Eagle's minimal essential medium, 25% fetal bovine
serum, 10% chick embryo extract. 2 mM glutamine and 0.6% glucose.
During the first week in vitro the medium was supplemented with
nerve growth factor (NGF-7S) at a concentration of about 0.5
.mu.g/ml, to enhance survival and growth of the fetal mouse DRG
neurons.
In order to perform electrophysiologic procedures, the culture
coverslip was transferred to a recording chamber containing about 1
ml of Hanks' balanced salt solution (BSS). The bath solution was
supplemented with 4 mM Ca.sup.2+ and 5 mM Ba.sup.2+ (i.e.,
Ca,Ba/BSS) to provide a prominent baseline response for
pharmacological tests. Intracellular recordings were obtained from
DRG perikarya selected at random within the ganglion. The
micropipettes were filled with 3M KCl (having a resistance of about
60-100 megohms) and were connected via a chloridized silver wire to
a neutralized input capacity preamplifier (Axoclamp 2A) for
current-clamp recording. After impalement of a DRG neuron, brief (2
msec) depolarizing current pulses were applied via the recording
electrode to evoke action potentials at a frequency of 0.1 Hz.
Recordings of the action potentials were stored on a floppy disc
using the P-clamp program (Axon Instruments) in a microcomputer
(IBM AT-compatible).
Drugs were applied by bath perfusion with a manually operated,
push-pull syringe system at a rate of 2-3 ml/min. Perfusion of test
agents was begun after the action potential and the resting
potential of the neuron reached a stable condition during >4
minute pretest periods in control Ca, Ba/BSS. Opioid-mediated
changes in the APD were considered significant if the APD
alteration was >10% of the control value for the same cell and
was maintained for the entire test period of 5 minutes. The APD was
measured as the time between the peak of the APD and the inflection
point on the repolarizing phase. The following drugs were used in
this and the following Examples: etorphine, diprenorphine and
morphine (gifts from Dr. Eric Simon); dihydroetorphine (gift from
Dr. B.-Y. Qin, China and United Biomedical, Inc.); naloxone (Endo
Labs); naltrexone, DADLE, dynorphin and other opioid peptides
(Sigma).
Opioid alkaloids and peptides were generally prepared as 1 mM
solutions in H.sub.2 O and then carefully diluted with BSS to the
desired concentrations, systematically discarding pipette tips
after each successive 1-10 or 1-100 dilution step to ensure
accuracy of extremely low (fM-pM) concentrations.
Results: Intracellular recordings were made from small- and
medium-size DRG neuron perikarya (about 10-30 .mu.m in diameter)
which generate relatively long APDs (greater than 3 msec in Ca/Ba
BSS) and which show characteristic responsiveness to opioid
agonists and other properties of primary afferent nociceptive
neurons as occur in vivo. Acute application of selective inhibitory
opioid receptor agonists, e.g., etorphine, to these DRG neurons
shortens the APD in 80-90% of the cells tested, whereas low
concentrations of bimodally-acting (excitatory/inhibitory) opioids,
e.g., morphine, dynorphin, enkephalins, prolong the APD in these
same cells. Relatively small numbers of large DRG neurons (about
30-50 .mu.m in diameter) survive in DRG-cord explants (about
10-20%) and show much shorter APDs (about 1-2 msec in Ca/Ba BSS),
with no clear-cut inflection or "hump" on the falling phase of the
spike. The APD of these large DRG neurons is not altered by
exogenous opioids.
The opioid responsiveness of DRG neurons was analyzed by measuring
the opioid-induced alterations in the APD of DRG perikarya. A total
of 64 DRG neurons (from 23 DRG-cord explants) were studied for
sensitivity to progressive increases in the concentration of
etorphine (n=30) or dihydroetorphine (n=38). Etorphine rapidly and
dose-dependently shortened the APD in progressively larger
fractions of DRG cells at concentrations from 1 fM (30% of cells;
n=26) to 1 .mu.M (80% of cells; n=16) (see FIGS. 2 and 3).
FIG. 2 shows that acute application of low (pM-nM) concentrations
of etorphine to naive DRG neurons elicits dose-dependent,
naloxone-reversible inhibitory shortening of the action potential
duration (APD). In contrast, dynorphin (and many other
bimodally-acting opioid agonists, e.g., morphine, DADLE) elicit
excitatory APD prolongation at these low concentrations (see FIG.
3), which can be selectively blocked by <pM levels of etorphine,
as well as by diprenorphine or naltrexone (see FIGS. 4 and 5). FIG.
2A record 1 shows the action potential (AP) generated by a DRG
neuron in balanced salt solution containing 5 mM Ca.sup.2+ and 5 mM
Ba.sup.2+ (BSS). AP response in this record (and in all records
below) is evoked by a brief (2 msec) intracellular depolarizing
current pulse. FIG. 2A records 2-5 show that APD is not altered by
bath perfusion with 1 fM etorphine (Et) but is progressively
shortened in 1 pM, 1 nM and 1 .mu.M concentrations (5 minute test
periods). FIG. 2A record 6 shows that APD returns to control value
after transfer to BSS (9 minute test). FIG. 2B records 1 and 2 show
that APD of another DRG neuron is shortened by application of 1 nM
etorphine (2 minute test). FIG. 2B record 3 shows that APD returns
to control value after transfer to 10 nM naloxone (NLX). FIG. 2B
records 4 and 5 show that APD is no longer shortened by 1 nM or
even 1 .mu.M etorphine when co-perfused with 10 nM naloxone (5
minute test periods).
FIG. 2C records 1 and 2 show that APD of another DRG neuron is
prolonged by application of 3 nM morphine. FIG. 2C record 3 shows
that APD returns to control value by 5 minutes after washout FIG.
2C record 4 shows that application of 1 pM etorphine does not alter
the APD. FIG. 2C record 5 shows that APD is no longer prolonged by
3 nM morphine when co-perfused with 1 pM etorphine and instead is
markedly shortened to a degree which would require a much higher
morphine concentration in the absence of etorphine. Similar results
were obtained by pretreatment with 1 pM diprenorphine (see FIG. 4),
with 1 pM naltrexone (FIG. 5) or 1 pM naloxone. Records in this and
subsequent Figures are from DRG neurons in organotypic DRG-spinal
cord explants maintained for 3-4 weeks in culture.
FIG. 3 shows dose-response curves demonstrating that etorphine (Et)
(.quadrature.) and dihydroetorphine (DHE) (.diamond.) elicit only
inhibitory dose-dependent shortening of the APD of DRG neurons at
all concentrations tested (fM-.mu.M). In contrast, dynorphin A
(1-13) (Dyn) (X) (as well as morphine and other bimodally-acting
opioids) elicits dose-dependent excitatory APD prolongation at low
concentrations (fM-nM) and generally requires much higher
concentrations (about 0.1-1 .mu.M) to shorten the APD, thereby
resulting in a bell-shaped dose-response curve. Data were obtained
from 11 neurons for the etorphine tests, 13 for the DHE tests and
35 for the dynorphin tests; 5, 8 and 9 neurons were tested (as in
FIG. 2) with all four concentrations of etorphine, DHE and
dynorphin, respectively (from fM to .mu.M). For sequential
dose-response data on the same neuron, the lowest concentrations
(e.g., 1 fM) were applied first.
Dihydroetorphine was even more effective (n=38; FIG. 3). Naloxone
(10 nM) prevented the etorphine- and dihydroetorphine-induced APD
shortening which was previously elicited in the same cells (n=12.
FIG. 2B). These potent inhibitory effects of etorphine and
dihydroetorphine on DRG neurons at low concentrations are in sharp
contrast to the excitatory APD-prolonging effects observed in
similar tests with morphine and a wide variety of mu, delta and
kappa opioids. None of the DRG neurons tested with different
concentrations of etorphine or dihydroetorphine showed prominent
APD prolongation.
The absence of excitatory APD-prolonging effects of etorphine and
dihydroetorphine on DRG neurons could be due to low binding
affinity of these opioid agonists to excitatory opioid receptors.
Alternatively, these opioids might bind strongly to excitatory
receptors, but fail to activate them, thereby functioning as
antagonists. In order to distinguish between these two modes of
action, DRG neurons were pretreated with etorphine at low
concentrations (fM-pM) that evoked little or no alteration of the
APD. Subsequent addition of nM concentrations of morphine. DAGO,
DADLE or dynorphin to etorphine-treated cells no longer evoked the
usual APD prolongation observed in the same cells prior to exposure
to etorphine (n=11; see FIG. 2C). This etorphine-induced blockade
of opioid excitatory effects on DRG neurons was often effective for
periods up to 0.5-2 hours after washout (n=4).
These results demonstrate that etorphine, which has been considered
to be a "universal" agonist at mu, delta and kappa opioid receptors
(see Magnan et al., Naunyn-Schmiedeberg's Arch. Pharmacol., Vol.
319, pp. 197-205 (1982)), has potent antagonist actions at mu,
delta and kappa excitatory opioid receptors on DRG neurons, in
addition to its well-known agonist effects at inhibitory opioid
receptors. Pretreatment with dihydroetorphine (fM-pM) showed
similar antagonist action at excitatory opioid receptor mediating
nM opioid-induced APD prolongation (n=2). Furthermore, after
selective blockade of opioid excitatory APD-prolonging effects by
pretreating DRG neurons with low concentrations of etorphine
(fM-pM), which showed little or no alteration of the APD, fM-nM
levels of bimodally-acting opioids now showed potent inhibitory
APD-shortening effects (5 out of 9 cells) (see FIG. 2C and FIG. 4).
This is presumably due to unmasking of inhibitory opioid
receptor-mediated functions in these cells after selective blockade
of their excitatory opioid receptor functions by etorphine.
EXAMPLE 2
Diprenorphine, Naloxone and Naltrexone, at Low Concentrations, Show
Potent Selective Antagonist Action at Excitatory Opioid
Receptors
Drug tests: Mouse DRG-cord explants, grown for >3 weeks as
described in Example 1, were tested with the opioid antagonists,
diprenorphine, naltrexone and naloxone. Electrophysiological
recordings were made as in Example 1.
Results: The opioid receptor antagonists naloxone and diprenorphine
were previously shown to block, at nM concentrations, both
inhibitory APD shortening of DRG neurons by .mu.M opioid agonists
as well as excitatory APD prolongation by nM opioids. Tests at
lower concentrations have revealed that pM diprenorphine, as well
as pM naloxone or naltrexone, act selectively as antagonists at mu,
delta and kappa excitatory opioid receptors, comparable to the
antagonist effects of pM etorphine and dihydroetorphine. In the
presence of pM diprenorphine, morphine (n=7) and DAGO (n=7) no
longer elicited APD prolongation at low (pM-nM) concentrations (see
FIG. 4A). Instead, they showed progressive dose-dependent APD
shortening throughout the entire range of concentrations from fM to
.mu.M (see FIG. 4B), comparable to the dose-response curves for
etorphine and dihydroetorphine (see FIG. 3 and FIG. 2C). This
unmasking of inhibitory opioid receptor-mediated APD-shortening
effects by pM diprenorphine occurred even in the presence of
10.sup.6 -fold higher concentrations of morphine (see FIG. 4A.
records 11 vs. 5).
FIG. 4 shows that excitatory APD-prolonging effects elicited by
morphine in DRG neurons are selectively blocked by
co-administration of a low (pM) concentration of diprenorphine,
thereby unmasking potent dose-dependent inhibitory APD shortening
by low concentrations of morphine. FIG. 4A records 1-4 show that
APD of a DRG neuron is progressively prolonged by sequential bath
perfusions with 3 fM, 3 pM and 3 nM morphine (Mor). FIG. 4A record
5 shows that APD of this cell is only slightly shortened after
increasing morphine concentration to 3 .mu.M. FIG. 4A records 6 and
7 show that after transfer to BSS, the APD is slightly shortened
during pretreatment for 17 minutes with 1 pM diprenorphine (DPN).
FIG. 4A records 8-11 show that after the APD reached a stable value
in DPN, sequential applications of 3 fM, 3 pM, 3 nM and 3 .mu.M Mor
progressively shorten the APD, in contrast to the marked APD
prolongation evoked by these same concentrations of Mor in the
absence of DPN (see also FIG. 2C). FIG. 4B dose-response curves
demonstrate similar unmasking by 1 pM DPN of potent dose-dependent
inhibitory APD shortening by morphine (.quadrature.) in a group of
DRG neurons (n=7), all of which showed only excitatory APD
prolongation responses when tested prior to introduction of DPN
(X). Note that the inhibitory potency of morphine in the presence
of pM DPN becomes comparable to that of etorphine and
dihydroetorphine (see FIG. 3). In contrast, pretreatment with a
higher (nM) concentration of DPN blocks both inhibitory as well as
excitatory effects of morphine (.circle-solid.).
FIG. 5 shows that excitatory APD-prolonging effects elicited by
morphine in DRG neurons (.smallcircle.) are also selectively
blocked by co-administration of a low (pM) concentration of
naltrexone (NTX), thereby unmasking potent dose-dependent
inhibitory APD shortening by low concentrations or morphine (X). In
contrast, pretreatment with a higher (.mu.M) concentration of NTX
blocks both inhibitory as well as excitatory effects of morphine
(.quadrature.) (similar blockade occurs with 1 nM NTX). These
dose-response curves are based on data from 18 neurons, all of
which showed only excitatory APD prolongation responses when tested
prior to introduction of NTX. The inhibitory potency of morphine in
the presence of pM NTX becomes comparable to that of etorphine and
dihydroetorphine (see FIG. 3).
EXAMPLE 3
Chronic Co-treatment of DRG Neurons with Morphine and
Ultra-low-dose Naloxone or Naltrexone Prevents Development of
Opioid Excitatory Supersensitivity ("Dependence") and Tolerance
Co-administration of ultra-low (pM) concentrations of naloxone or
naltrexone during chronic treatment of DRG neurons with .mu.M
levels of morphine was effective in preventing development of
opioid excitatory supersensitivity and tolerance which generally
occurs after sustained exposure to bimodally-acting opioids. Acute
application of fM dynorphin A-(1-13) or fM morphine (n=21), as well
as 1 nM naloxone (n=11), to DRG neurons chronically exposed to 1
.mu.M morphine together with 1 pM naloxone or naloxone or
naltrexone (for 1-10 weeks) did not evoke the usual excitatory APD
prolongation observed in chronic morphine-treated cells tested
after washout with BSS (see FIG. 6). Furthermore, there was no
evidence of tolerance to the usual inhibitory effects of .mu.M
opioids (n=6) (FIG. 6).
These results are consonant with previous data that blockade of
sustained opioid excitatory effects by cholera toxin-B sub-unit
during chronic morphine treatment of DRG neurons prevents
development of tolerance and dependence. (See Shen and Crain, Brain
Res., Vol. 597, pp. 74-83 (1992)). This toxin sub-unit selectively
interferes with GMl ganglioside regulation of excitatory opioid
receptor functions (see Shen and Crain, Brain Res., Vol. 531, pp.
1-7 (1990) and Shen et al., Brain Res., Vol. 559, pp. 130-138
(1991)).
Similarly, in the presence of pM etorphine, chronic .mu.M
morphine-treated DRG neurons did not develop signs of tolerance or
dependence. FIG. 6 outlines the assay procedure used for testing
the effectiveness of these and other antagonists at excitatory
opioid receptors in preventing development of tolerance/dependence
during chronic co-treatment of DRG neurons with morphine.
Excitatory Opioid Receptor Antagonists Enhance Analgesic Potency
and Reduce Dependence Liability and Other Side Effects of Morphine
or Other Conventional Opioid Analgesics When Administered in
Combination
Electrophysiological studies on DRG neurons in culture indicated
that pretreatment with low fM-pM concentrations of naltrexone,
naloxone, diprenorphine, etorphine or dihydroetorphine is
remarkably effective in blocking excitatory APD-prolonging effects
of morphine or other bimodally-acting opioid agonists by selective
antagonist actions at mu, delta and kappa excitatory opioid
receptors on these cells. In the presence of these selective
excitatory opioid receptor antagonists, morphine and other
clinically used bimodally-acting opioid agonists showed markedly
increased potency in evoking the inhibitory effects on the action
potential of sensory neurons which are generally considered to
underlie opioid analgesic action in vivo.
These bimodally-acting opioid agonists became effective in
shortening, instead of prolonging, the APD at pM-nM (i.e.,
10.sup.-12 -10.sup.-9 M) concentrations, whereas 0.1-1 .mu.M (i.e.,
10.sup.-7 -10.sup.-6 M) levels were generally required to shorten
the APD (FIGS. 4B and 5). Selective blockade of the excitatory side
effects of these bimodally-acting opioid agonists eliminates the
attenuation of their inhibitory effectiveness that would otherwise
occur. Hence, according to this invention, the combined use of a
relatively low dose of one of these selective excitatory opioid
receptor antagonists, together with morphine or other
bimodally-acting mu, delta or kappa opioid agonists, will markedly
enhance the analgesic potency of said opioid agonist, and render
said opioid agonist comparable in potency to etorphine or
dihydroetorphine, which, when used alone at higher doses, are
>1000 times more potent than morphine in eliciting
analgesia.
Co-administration of one of these excitatory opioid receptor
antagonists at low (pM) concentration (10.sup.-12 M) during chronic
treatment of sensory neurons with 10.sup.-6 M morphine or other
bimodally-acting opioid agonists (>1 week in culture) prevented
development of the opioid excitatory supersensitivity, including
naloxone-precipitated APD-prolongation, as well as the tolerance to
opioid inhibitory effects that generally occurs after chronic
opioid exposure. This experimental paradigm was previously utilized
by the inventors on sensory neurons in culture to demonstrate that
co-administration of 10.sup.-7 M cholera toxin-B sub-unit, which
binds selectively to GMl ganglioside and thereby blocks excitatory
GMl-regulated opioid receptor-mediated effects, but not opioid
inhibitory effects (see Shen and Crain, Brain Res., Vol. 531, pp.
1-7 (1990)), during chronic opioid treatment prevents development
of these plastic changes in neuronal sensitivity that are
considered to be cellular manifestations related to opioid
dependence/addiction and tolerance in vivo (see Shen and Crain,
Brain Res., Vol. 597, pp. 74-83 (1992)).
Hence, according to this invention, the sustained use of a
relatively low clinical dose of one of these selective excitatory
opioid receptor antagonists, e.g., about 1 microgram of naltrexone,
naloxone, etorphine, dihydroetorphine or diprenorphine, in
combination with 100-1000 micrograms of morphine or other
conventional bimodally-acting opioid analgesics will result in
analgesia comparable to that elicited by said analgesics when
administered alone in >10 milligram doses and will attenuate or
even prevent development of tolerance, physical dependence and
other undesirable excitatory side effects generally associated with
said analgesics. Furthermore, administration of .mu.g doses of
these excitatory opioid receptor antagonists alone will enhance the
analgesic effects of endogenous opioid peptides and thereby
decrease chronic pain.
Treatment of Detoxified Opiate Addicts
Long-term maintenance treatment of previously detoxified opiate,
cocaine and alcohol addicts to prevent protracted dependence is
carried out by long-term oral administration of ultra-low doses
(about 1 .mu.g) of naltrexone. Ultra-low dose naltrexone
selectively blocks resumption of the sustained activation of
excitatory opioid receptor functions that are required for the
development of protracted opioid dependence as well as
opioid-mediated cocaine and alcohol dependence without inducing
dysphoria or other adverse side effects caused by high-dose
naltrexone blockade of inhibitory opioid receptor functions.
Alternatively, ultra-low dose (about 1 .mu.g) naltrexone can be
administered long-term in combination with low-dose methadone to
provide effective treatment for addiction.
Although the invention herein has been described with reference to
particular embodiments, it is to be understood that these
embodiments are merely illustrative of various aspects of the
invention. Thus, it is to be understood that numerous modifications
may be made in the illustrative embodiments and other arrangements
may be devised without departing from the spirit and scope of the
invention.
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