U.S. patent application number 10/735506 was filed with the patent office on 2004-07-08 for novel methods and compositions for alleviating pain.
This patent application is currently assigned to Allergan, Inc.. Invention is credited to Donello, John E., Gil, Daniel W..
Application Number | 20040132824 10/735506 |
Document ID | / |
Family ID | 29582080 |
Filed Date | 2004-07-08 |
United States Patent
Application |
20040132824 |
Kind Code |
A1 |
Gil, Daniel W. ; et
al. |
July 8, 2004 |
Novel methods and compositions for alleviating pain
Abstract
The present invention provides a method for the long-term relief
of chronic pain in a subject by activating in the subject an
analgesic .alpha.-adrenergic receptor in the absence of .alpha.-2A
receptor activation over a period of at least three days, such that
relief of chronic pain is maintained in the absence of continued
activation of said receptor. The analgesic .alpha.-adrenergic
receptor can be, for example, the .alpha.-2B receptor.
Inventors: |
Gil, Daniel W.; (Corona Del
Mar, CA) ; Donello, John E.; (Dana Point,
CA) |
Correspondence
Address: |
Cathryn Campbell
McDERMOTT, WILL & EMERY
4370 La Jolla Village Drive, 7th Floor
San Diego
CA
92122
US
|
Assignee: |
Allergan, Inc.
|
Family ID: |
29582080 |
Appl. No.: |
10/735506 |
Filed: |
December 11, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10735506 |
Dec 11, 2003 |
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10153154 |
May 21, 2002 |
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Current U.S.
Class: |
514/570 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 31/538 20130101; A61P 25/02 20180101; A61P 3/10 20180101; A61K
31/4178 20130101; A61P 19/02 20180101; A61K 31/4164 20130101; A61P
25/06 20180101; A61P 25/04 20180101; A61K 31/4168 20130101; A61K
31/498 20130101; A61P 29/00 20180101; A61P 29/02 20180101; A61P
43/00 20180101; Y02A 50/30 20180101; A61P 1/04 20180101; A61K
31/137 20130101; A61K 31/137 20130101; A61K 2300/00 20130101; A61K
31/4164 20130101; A61K 2300/00 20130101; A61K 31/4168 20130101;
A61K 2300/00 20130101; A61K 31/4178 20130101; A61K 2300/00
20130101; A61K 31/498 20130101; A61K 2300/00 20130101; A61K 31/538
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/570 |
International
Class: |
A61K 031/19 |
Claims
We claim:
1. A method of alleviating pain in a subject, comprising
administering to said subject a pharmaceutical composition
comprising an effective amount of an .alpha.-adrenergic agonist and
a pharmaceutical composition comprising an effective amount of a
selective .alpha.-2A antagonist.
2. The method of claim 1, wherein said pain is neuropathic
pain.
3. The method of claim 2, wherein said pain results from diabetic
neuropathy.
4. The method of claim 1, wherein said pain is visceral pain.
5. The method of claim 1, wherein said pain is post-operative
pain.
6. The method of claim 1, wherein said pain results from cancer or
cancer treatment.
7. The method of claim 1, wherein said pain is inflammatory
pain.
8. The method of claim 7, wherein said pain is arthritic pain.
9. The method of claim 7, wherein said pain is irritable bowel
syndrome pain.
10. The method of claim 1, wherein said pain is headache pain.
11. The method of claim 1, wherein said .alpha.-adrenergic agonist
is a pan-.alpha.-2 agonist.
12. The method of claim 11, wherein said pan-.alpha.-2 agonist is a
pan-.alpha.-1 pan-.alpha.-2 agonist.
13. The method of claim 1, wherein said .alpha.-adrenergic agonist
is a compound selected from the group consisting of clonidine,
brimonidine, tizanidine, dexemedetomidine, norepinephrine, a
compound represented by the formula 23[FORMULA 1], a compound
represented by the formula 24[FORMULA 2], and all pharmaceutically
acceptable salts, esters, amides, sterioisomers and racemic
mixtures thereof.
14. The method of claim 1, 11, 12 or 13, wherein said selective
.alpha.-2A antagonist is a 4-imidazole or a pharmaceutically
acceptable salt, ester, amide, sterioisomer or racemic mixture
thereof.
15. The method of claim 14, wherein said selective .alpha.-2A
antagonist is a compound represented by the formula 25[FORMULA 13]
or a pharmaceutically acceptable salt, ester, amide, sterioisomer
or racemic mixture thereof.
16. The method of claim 1, wherein said selective .alpha.-2A
antagonist is BRL 48962 or a pharmaceutically acceptable salt,
ester, amide, sterioisomer or racemic mixture thereof.
17. The method of claim 1, wherein said selective .alpha.-2A
antagonist is peripherally limited.
18. The method of claim 1, wherein said .alpha.-adrenergic agonist
and said selective .alpha.-2A antagonist each is administered
peripherally.
19. The method of claim 1 or claim 18, wherein said
.alpha.-adrenergic agonist is administered orally.
20. The method of claim 1 or claim 18, wherein said selective
.alpha.-2A antagonist is administered orally.
21. The method of claim 1 or claim 18, wherein said
.alpha.-adrenergic agonist is administered through a subcutaneous
minipump.
22. The method of claim 1 or claim 18, wherein said selective
.alpha.-2A antagonist is administered through a subcutaneous
minipump.
23. The method of claim 1, wherein said .alpha.-adrenergic agonist
and said selective .alpha.-2A antagonist each is administered
repeatedly or continuously over a period of at least three
days.
24. The method of claim 23, wherein pain alleviation continues in
the absence of significant .alpha.-adrenergic agonist levels in
said subject.
25. An analgesic composition, comprising an .alpha.-adrenergic
agonist with minimal .alpha.-2A agonist activity, said agonist
having the ability to produce peripheral analgesia without
concomitant sedation.
26. The analgesic composition of claim 25, wherein said peripheral
analgesia is sufficient to reduce pain by at least 50% without
concomitant sedation.
27. The analgesic composition of claim 26, wherein at least a
10-fold greater dose is required to produce a 20% reduction in
motor or muscular activity than the dose required to reduce pain by
at least 50%.
28. The analgesic composition of claim 27, wherein at least a
100-fold greater dose is required to produce a 20% reduction in
motor or muscular activity than the dose required to reduce pain by
at least 50%.
29. The analgesic composition of claim 28, wherein at least a
1000-fold greater dose is required to produce a 20% reduction in
motor or muscular activity than the dose required to reduce pain by
at least 50%.
30. The analgesic composition of claim 25 or claim 26, further
having a substantial absence of hypotensive effects.
31. The analgesic composition of claim 25 or claim 26, wherein said
agonist is not a thiourea or a derivative thereof.
32. The analgesic composition of claim 25 or claim 26, wherein said
agonist is not a thiourea or 4-imidazole or a derivative
thereof.
33. A method of alleviating pain in a subject, comprising
peripherally administering to said subject a pharmaceutical
composition comprising an effective amount of an .alpha.-adrenergic
agonist with minimal .alpha.-2A agonist activity, thereby producing
peripheral analgesia without concomitant sedation.
34. The method of claim 33, wherein said peripheral analgesia is
sufficient to reduce pain by at least 50% without concomitant
sedation.
35. The method of claim 33 or claim 34, wherein said peripheral
analgesia occurs in the substantial absence of hypotensive
effects.
36. The method of claim 33 or claim 34, wherein said
.alpha.-adrenergic agonist with minimal .alpha.-2A agonist activity
is not a thiourea or a derivative thereof.
37. The method of claim 33 or claim 34, wherein said
.alpha.-adrenergic agonist with minimal .alpha.-2A agonist activity
is not a thiourea or 4-imidazole or a derivative thereof.
38. The method of claim 33, wherein said pain is neuropathic
pain.
39. The method of claim 38, wherein said pain results from diabetic
neuropathy.
40. The method of claim 33, wherein said pain is visceral pain.
41. The method of claim 33, wherein said pain is post-operative
pain.
42. The method of claim 33, wherein said pain results from cancer
or cancer treatment.
43. The method of claim 33, wherein said pain is inflammatory
pain.
44. The method of claim 43, wherein said pain is arthritic
pain.
45. The method of claim 43, wherein said pain is irritable bowel
syndrome pain.
46. The method of claim 33, wherein said pain is headache pain.
47. The method of claim 33, wherein said .alpha.-adrenergic agonist
with minimal .alpha.-2A agonist activity is an .alpha.-2B agonist
with minimal .alpha.-2A agonist activity.
48. The method of claim 47, wherein said .alpha.-2B agonist with
minimal .alpha.-2A agonist activity is a thione.
49. The method of claim 48, wherein said .alpha.-2B agonist with
minimal .alpha.-2A agonist activity is a compound represented by
the formula 26[FORMULA 3] or a pharmaceutically acceptable salt,
ester, amide, sterioisomer or racemic mixture thereof.
50. The method of claim 49, wherein said .alpha.-2B agonist with
minimal .alpha.-2A agonist activity is the (-) enantiomer of a
compound represented by the formula 27[FORMULA 3] or a
pharmaceutically acceptable salt or ester thereof.
51. The method of claim 48, wherein said .alpha.-2B agonist with
minimal .alpha.-2A agonist activity is a compound represented by
the formula 28[FORMULA 11] or a pharmaceutically acceptable salt,
ester, amide, sterioisomer or racemic mixture thereof.
52. The method of claim 47, wherein said .alpha.-2B agonist with
minimal .alpha.-2A agonist activity is an imidazolone.
53. The method of claim 52, wherein said .alpha.-2B agonist with
minimal .alpha.-2A agonist activity is a compound represented by
the formula 29[FORMULA 4] or a pharmaceutically acceptable salt,
ester, amide, sterioisomer or racemic mixture thereof.
54. The method of claim 53, wherein said .alpha.-2B agonist with
minimal .alpha.-2A agonist activity is the (+) enantiomer of a
compound represented by the formula 30[FORMULA 4] or a
pharmaceutically acceptable salt or ester thereof.
55. The method of claim 47, wherein said .alpha.-2B agonist with
minimal .alpha.-2A agonist activity is a compound represented by a
formula selected from the group consisting of 31and all
pharmaceutically acceptable salts, esters, amides, sterioisomers
and racemic mixtures thereof.
56. The method of claim 33, wherein said .alpha.-adrenergic agonist
with minimal .alpha.-2A agonist activity is administered
orally.
57. The method of claim 33, wherein said .alpha.-adrenergic agonist
with minimal .alpha.-2A agonist activity is administered through a
subcutaneous minipump.
58. A method of screening for effective agents that produce
peripheral analgesia without concomitant sedation, comprising the
steps of: (a) contacting an .alpha.-2A receptor with an
.alpha.-adrenergic agonist having analgesic activity; and (b)
determining whether said agonist has .alpha.-2A agonist activity,
wherein the absence of .alpha.-2A agonist activity indicates that
said .alpha.-adrenergic agonist having analgesic activity is an
effective agent that produces peripheral analgesia without
concomitant sedation.
59. A method of screening for effective agents that produce
peripheral analgesia without concomitant sedation, comprising the
steps of: (a) contacting an .alpha.-2A receptor with an agent; (b)
determining whether said agent has .alpha.-2A agonist activity; (c)
contacting an .alpha.-2B receptor with said agent; and (d)
determining whether said agent has .alpha.-2B agonist activity,
wherein the absence of .alpha.-2A agonist activity and the presence
of .alpha.-2B agonist activity indicate that said agent is an
effective agent that produces peripheral analgesia without
concomitant sedation.
60. A method of screening for effective agents that produce
peripheral analgesia without concomitant sedation, comprising the
steps of: (a) peripherally administering an .alpha.-adrenergic
agonist to a control animal having at least wild type levels of
.alpha.-2A receptor activity; (b) assaying for analgesia in said
control animal; (c) peripherally administering to a corresponding
animal having reduced levels of .alpha.-2A receptor expression or
activity an amount of said .alpha.-adrenergic agonist similar or
greater than the amount administered to said control animal; and
(d) assaying for analgesia in said corresponding animal having
reduced levels of .alpha.-2A receptor expression or activity,
wherein the absence of analgesia in said control animal and the
presence of analgesia in said corresponding animal having reduced
levels of .alpha.-2A receptor expression or activity indicate that
said .alpha.-adrenergic agonist has excessive .alpha.-2A agonist
activity; and wherein the presence of analgesia in said control
animal and the presence of analgesia in said corresponding animal
having reduced levels of .alpha.-2A receptor expression or activity
indicate that said .alpha.-adrenergic agonist is an effective agent
that produces peripheral analgesia without concomitant
sedation.
61. The method of claim 60, wherein said control animal is wild
type at both .alpha.-2A receptor loci.
62. The method of claim 61, wherein said control animal is a wild
type animal.
63. The method of claim 62, wherein said wild type animal is a wild
type mouse.
64. The method of claim 60 or 63, wherein said corresponding animal
has a homozygous point mutation at the .alpha.-2A receptor
locus.
65. The method of claim 64, wherein said corresponding animal has a
point mutation within the .alpha.-2A receptor coding sequence.
66. The method of claim 65, wherein said point mutation occurs at a
residue analogous to Asp79.
67. The method of claim 66, wherein said point mutation is an Asp79
to Asn mutation.
68. The method of claim 60 or 63, wherein said corresponding animal
has a homozygous .alpha.-2A knockout mutation.
69. The method of claim 60 or 63, wherein, in steps (b) and (d),
analgesia is assayed following sulprostone sensitization.
70. The method of claim 60, further comprising: (e) peripherally
administering said .alpha.-adrenergic agonist to a corresponding
animal having reduced levels of .alpha.-2B receptor expression or
activity; and (f) assaying for analgesia in said corresponding
animal having reduced levels of .alpha.-2B receptor expression or
activity, wherein the absence of analgesia in said control animal
and the presence of analgesia in said corresponding animal having
reduced levels of .alpha.-2A receptor expression or activity
indicate that said .alpha.-adrenergic agonist has excessive
.alpha.-2A agonist activity; and wherein the presence of analgesia
in said control animal, the presence of analgesia in said
corresponding animal having reduced levels of .alpha.-2A receptor
expression or activity and the absence of analgesia in said
corresponding animal having reduced levels of .alpha.-2B receptor
expression or activity indicate that said .alpha.-adrenergic
agonist is an effective agent that produces peripheral analgesia
without concomitant sedation.
71. A method of screening for effective agents that produce
peripheral analgesia without concomitant sedation, comprising the
steps of: (a) peripherally administering an .alpha.-adrenergic
agonist to a control animal having at least wild type levels of
.alpha.-2B receptor activity; (b) assaying for analgesia in said
control animal; (c) peripherally administering said
.alpha.-adrenergic agonist to a corresponding animal having reduced
levels of .alpha.-2B receptor expression or activity; and (d)
assaying for analgesia in said corresponding animal having-reduced
levels of .alpha.-2B receptor expression or activity, wherein the
presence of analgesia in said control animal and the absence of
analgesia in said corresponding animal having reduced levels of
.alpha.-2B receptor expression or activity indicate that said
.alpha.-adrenergic agonist is an effective agent that produces
peripheral analgesia without concomitant sedation.
72. The method of claim 71, wherein said control animal is wild
type at both .alpha.-2B receptor loci.
73. The method of claim 72, wherein said control animal is a wild
type animal.
74. The method of claim 73, wherein said wild type animal is a wild
type mouse.
75. The method of claim 71, wherein said corresponding animal has a
heterozygous .alpha.-2B knockout mutation.
76. The method of claim 71, wherein said corresponding animal has a
homozygous .alpha.-2B knockout mutation.
77. The method of claim 71 or 74, wherein, in steps (b) and (d),
analgesia is assayed following sulprostone sensitization.
78. A method for the long-term relief of chronic pain in a subject,
comprising activating in said subject an analgesic
.alpha.-adrenergic receptor in the absence of .alpha.-2A receptor
activation over a period of at least three days, such that relief
of chronic pain is maintained in the absence of continued
activation of said receptor.
79. The method of claim 78, comprising administering to said
subject a pharmaceutical composition comprising an effective amount
of an .alpha.-adrenergic agonist with minimal .alpha.-2A
agonist-activity over a period of at least three days, such that
relief of chronic pain is maintained in the absence of significant
agonist levels in said subject.
80. The method of claim 78, comprising administering to said
subject a pharmaceutical composition comprising an effective amount
of an .alpha.-adrenergic agonist and a pharmaceutical composition
comprising an effective amount of a selective .alpha.-2A antagonist
over a period of at least three days, such that relief of chronic
pain is maintained in the absence of significant agonist levels in
said subject.
81. The method of claim 79 or 80, wherein relief of chronic pain is
maintained for at least three weeks in the absence of significant
agonist levels in said subject.
82. The method of claim 78, wherein said pain is neuropathic
pain.
83. The method of claim 82, wherein said pain results from diabetic
neuropathy.
84. The method of claim 78, wherein said pain is visceral pain.
85. The method of claim 78, wherein said pain is post-operative
pain.
86. The method of claim 78, wherein said pain results from cancer
or cancer treatment.
87. The method of claim 78, wherein said pain is inflammatory
pain.
88. The method of claim 87, wherein said pain is arthritic
pain.
89. The method of claim 87, wherein said pain is irritable bowel
syndrome pain.
90. The method of claim 78, wherein said pain is headache pain.
91. The method of claim 79, wherein said .alpha.-adrenergic agonist
with minimal .alpha.-2A agonist activity is an .alpha.-2B agonist
with minimal .alpha.-2A agonist activity.
92. The method of claim 91, wherein said .alpha.-2B agonist with
minimal .alpha.-2A agonist activity is a thione.
93. The method of claim 92, wherein said .alpha.-2B agonist with
minimal .alpha.-2A agonist activity is a compound represented by
the formula 32[FORMULA 3] or a pharmaceutically acceptable salt,
ester, amide, sterioisomer or racemic mixture thereof.
94. The method of claim 93, wherein said .alpha.-2B agonist with
minimal .alpha.-2A agonist activity is the (-) enantiomer of a
compound represented by the formula 33[FORMULA 3] or a
pharmaceutically acceptable salt or ester thereof.
95. The method of claim 92, wherein said .alpha.-2B agonist with
minimal .alpha.-2A agonist activity is a compound represented by
the formula 34[FORMULA 11] or a pharmaceutically acceptable salt,
ester, amide, sterioisomer or racemic mixture thereof.
96. The method of claim 91, wherein said .alpha.-2B agonist with
minimal .alpha.-2A agonist activity is an imidazolone.
97. The method of claim 96, wherein said .alpha.-2B agonist with
minimal .alpha.-2A agonist activity is a compound represented by
the formula 35[FORMULA 4] or a pharmaceutically acceptable salt,
ester, amide, sterioisomer or racemic mixture thereof.
98. The method of claim 97, wherein said .alpha.-2B agonist with
minimal .alpha.-2A agonist activity is the (+) enantiomer of a
compound represented by the formula 36[FORMULA 4] or a
pharmaceutically acceptable salt or ester thereof.
99. The method of claim 91, wherein said .alpha.-2B agonist with
minimal .alpha.-2A agonist activity is a compound represented by a
formula selected from the group consisting of 37and all
pharmaceutically acceptable salts, esters, amides, sterioisomers
and racemic mixtures thereof.
100. The method of claim 79, wherein said .alpha.-adrenergic
agonist with minimal .alpha.-2A agonist activity is administered
peripherally.
101. The method of claim 100, wherein said .alpha.-adrenergic
agonist with minimal .alpha.-2A agonist activity is administered
orally.
102. The method of claim 100, wherein said .alpha.-adrenergic
agonist with minimal .alpha.-2A agonist activity is administered
through a subcutaneous minipump.
103. The method of claim 80, wherein said .alpha.-adrenergic
agonist is a pan-.alpha.-2 agonist.
104. The method of claim 103, wherein said pan-.alpha.-2 agonist is
a pan-.alpha.-1 pan-.alpha.-2 agonist.
105. The method of claim 80, wherein said .alpha.-adrenergic
agonist is a compound selected from the group consisting of
clonidine, brimonidine, tizanidine, dexemedetomidine,
norepinephrine, a compound represented by the formula 38[FORMULA
1], a compound represented by the formula 39[FORMULA 2], and all
pharmaceutically acceptable salts, esters, amides, sterioisomers
and racemic mixtures thereof.
106. The method of claim 80, 103, 104 or 105, wherein said
selective .alpha.-2A antagonist is a 4-imidazole or a
pharmaceutically acceptable salt, ester, amide, sterioisomer or
racemic mixture thereof.
107. The method of claim 106, wherein said selective .alpha.-2A
antagonist is a compound represented by the formula 40[FORMULA 13]
or a pharmaceutically acceptable salt, ester, amide, sterioisomer
or racemic mixture thereof.
108. The method of claim 80, 103, 104 or 105, wherein said
selective .alpha.-2A antagonist is BRL 48962 or a pharmaceutically
acceptable salt, ester, amide, sterioisomer or racemic mixture
thereof.
109. The method of claim 80, 103, 104 or 105, wherein said
selective .alpha.-2A antagonist is peripherally limited.
110. The method of claim 80, wherein said .alpha.-adrenergic
agonist and said selective .alpha.-2A antagonist each is
administered peripherally.
111. The method of claim 80 or claim 110, wherein said
.alpha.-adrenergic agonist is administered orally.
112. The method of claim 80 or claim 110, wherein said .alpha.-2A
antagonist is administered orally.
113. The method of claim 80 or claim 110, wherein said
.alpha.-adrenergic agonist is administered through a subcutaneous
minipump.
114. The method of claim 80 or claim 110, wherein said selective
.alpha.-2A antagonist is administered through a subcutaneous
minipump.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates generally to the treatment of pain
and the long-term reversal of chronic pain and, in particular, to
.alpha.-adrenergic agonists, and selective antagonists of the
.alpha.-2A adrenergic receptor.
[0003] 2. Background Information
[0004] Clinical pain encompasses nociceptive and neuropathic pain.
Each type of pain is characterized by hypersensitivity at the site
of damage and in adjacent normal tissue. While nociceptive pain
usually is limited in duration and responds well to available
opioid therapy, neuropathic pain can persist long after the
initiating event has healed, as is evident, for example, in the
"ghost pain" that often follows amputation. Chronic pain syndromes
such as chronic neuropathic pain are triggered by any of a variety
of insults, including surgery, compression injury or trauma,
infectious agent, toxic drug, inflammatory disorder, or a metabolic
disease such as diabetes or ischemia.
[0005] Unfortunately, chronic pain such as chronic neuropathic pain
generally is resistant to available drug therapy. Furthermore,
current therapies have serious side-effects such as cognitive
changes, sedation, nausea and, in the case of narcotic drugs,
addiction. Many patients suffering from neuropathic and other
chronic pain are elderly or have medical conditions that limit
their tolerance to the side-effects associated with available
analgesic therapy. The inadequacy of current therapy in relieving
neuropathic pain without producing intolerable side-effects often
is manifest in the depression and suicidal tendency of chronic pain
sufferers.
[0006] .alpha.-2 adrenergic agonists, which are devoid of
respiratory depressant effects and addictive potential, are being
developed as alternatives to current analgesics. Such drugs are
useful analgesic agents when administered spinally. However,
undesirable pharmacological properties of .alpha.-adrenergic
agonists, specifically sedation and hypotension, limit the utility
of these drugs when administered orally or by other peripheral
routes. Thus, there is a need for effective analgesic agents that
can be administered by oral or other peripheral routes and that
lack undesirable side-effects such as sedation and hypotension. The
present invention satisfies this need and provides related
advantages as well.
[0007] The present invention also provides new therapy for chronic
pain sufferers, who, until now, have faced a lifetime of daily
medication to control their pain. Unfortunately, available
treatments for chronic neuropathic pain, such as tricyclic
antidepressants, anti-seizure drugs and local anesthetic
injections, only alleviate symptoms temporarily and to varying
degrees. No available treatment reverses the sensitized pain state
or cures pain such as neuropathic pain. Effective drugs that can be
administered, for example, once or several times a month and that
maintain analgesic activity for several weeks or months, are
presently not available. Thus, there is a need for novel methods of
providing long-term relief from chronic pain. The present invention
satisfies this need and also provides related advantages.
SUMMARY OF THE INVENTION
[0008] The present invention provides a method of alleviating pain
in a subject by administering to the subject a pharmaceutical
composition containing an effective amount of an .alpha.-adrenergic
agonist and a pharmaceutical composition containing an effective
amount of a selective .alpha.-2A antagonist. The methods of the
invention are useful for alleviating a variety of types of pain
including, but not limited to, neuropathic pain such as the pain
resulting from diabetic neuropathy; visceral pain; post-operative
pain; pain resulting from cancer or cancer treatment; inflammatory
pain resulting, for example, from arthritis or irritable bowel
syndrome; headache pain and muscle pain.
[0009] A variety of .alpha.-adrenergic agonists are useful in the
invention including pan-.alpha.-2 agonists and pan-.alpha.-1
pan-.alpha.-2 agonists. .alpha.-adrenergic agonists useful in
alleviating pain according to a method of the invention include,
without limitation, clonidine, brimonidine, tizanidine,
dexemedetomidine, norepinephrine, Compound 1 and Compound 2, and
all pharmaceutically acceptable salts, esters, amides,
sterioisomers and racemic mixtures thereof.
[0010] A variety of selective .alpha.-2A antagonists also are
useful in the invention. Such selective .alpha.-2A antagonists
include, without limitation, 4-imidazoles such as Compound 13 and
pharmaceutically acceptable salts, esters, amides, sterioisomers
and racemic mixtures thereof, and BRL 48962 or pharmaceutically
acceptable salts, esters, amides, sterioisomers and racemic
mixtures thereof. In one embodiment, the invention is practiced
with a peripherally limited selective .alpha.-2A antagonist.
[0011] Various routes of administration can be useful for
alleviating pain according to a method of the invention. In one
embodiment, both the .alpha.-adrenergic agonist and selective
.alpha.-2A antagonist are administered peripherally. In other
embodiments, the .alpha.-adrenergic agonist is administered orally
or through a subcutaneous minipump. In further embodiments, the
.alpha.-adrenergic agonist is administered orally or through a
subcutaneous minipump, and the selective .alpha.-2A antagonist is
administered by any peripheral route. In yet other embodiments, the
selective .alpha.-2A antagonist is administered orally or through a
subcutaneous minipump. If desired, the selective .alpha.-2A
antagonist can be administered orally or through a subcutaneous
minipump, and the .alpha.-adrenergic agonist can be administered by
any peripheral route such as orally or through a subcutaneous
minipump.
[0012] In one embodiment, the invention provides a method of
alleviating pain in a subject by administering to the subject a
pharmaceutical composition containing an effective amount of an
.alpha.-adrenergic agonist and a pharmaceutical composition
containing an effective amount of a selective .alpha.-2A
antagonist, where the .alpha.-adrenergic agonist and the selective
.alpha.-2A antagonist each is administered repeatedly or
continuously over a period of at least three days. In such a
method, pain alleviation can continue, for example, in the absence
of significant .alpha.-adrenergic agonist levels in the
subject.
[0013] The present invention further provides an analgesic
composition that contains an .alpha.-adrenergic agonist with
minimal .alpha.-2A agonist activity having the ability to produce
peripheral analgesia without concomitant sedation. In one
embodiment, the analgesic composition produces peripheral analgesia
without concomitant sedation and in the substantial absence of
hypotensive effects. In another embodiment, the invention provides
an analgesic composition that produces peripheral analgesia
sufficient to reduce pain by at least 50% without concomitant
sedation. In further embodiments, at least a 10-fold, 100-fold or
1000-fold greater dose of the analgesic composition is required to
produce a 20% reduction in motor or muscular activity than the dose
of the analgesic composition that reduces pain by at least 50%. In
a further embodiment, the invention provides an analgesic
composition that produces peripheral analgesia sufficient to reduce
pain by at least 50% without concomitant sedation and in the
substantial absence of hypotensive effects. In another embodiment,
the invention provides an analgesic composition that contains an
.alpha.-adrenergic agonist with minimal .alpha.-2A agonist activity
having the ability to produce peripheral analgesia without
concomitant sedation, where the agonist is not a thiourea or
derivative thereof. In a further embodiment, the invention provides
an analgesic composition that contains an .alpha.-adrenergic
agonist with minimal .alpha.-2A agonist activity having the ability
to produce peripheral analgesia without concomitant sedation, where
the agonist is not a thiourea or 4-imidazole or derivative
thereof.
[0014] Further provided by the invention is a method of alleviating
pain in a subject by peripherally administering to the subject a
pharmaceutical composition containing an effective amount of an
.alpha.-adrenergic agonist with minimal .alpha.-2A agonist
activity, thereby producing peripheral analgesia without
concomitant sedation. Such peripheral analgesia can be sufficient
to reduce pain, for example, by at least 50% without concomitant
sedation. In another embodiment, the peripheral analgesia occurs in
the substantial absence of hypotensive effects. In one embodiment,
the method is practiced using an .alpha.-adrenergic agonist with
minimal .alpha.-2A agonist activity which is not a thiourea or
derivative thereof. In another embodiment, the method is practiced
using an .alpha.-adrenergic agonist with minimal .alpha.-2A agonist
activity which is not a thiourea or 4-imidazole or derivative
thereof. Pain of various types and etiologies can be alleviated
according to a method of the invention. As non-limiting examples,
the methods of the invention can be useful in alleviating
neuropathic pain, such as the pain resulting from diabetic
neuropathy; visceral pain; post-operative pain; pain resulting from
cancer or cancer treatment; inflammatory pain such as arthritic
pain or irritable bowel syndrome pain; headache pain and muscle
pain.
[0015] A variety of .alpha.-adrenergic agonists with minimal
.alpha.-2A agonist activity can be useful in the methods of the
invention. In one embodiment, the .alpha.-adrenergic agonist with
minimal .alpha.-2A agonist activity is an .alpha.-2B agonist with
minimal .alpha.-2A agonist activity. Such an agonist can be, for
example, a thione such as Compound 3 or Compound 11 or a
pharmaceutically acceptable salt, ester, amide, sterioisomer or
racemic mixture thereof. In one embodiment, a method of the
invention is practiced with an .alpha.-2B agonist with minimal
.alpha.-2A agonist activity which is the (-) enantiomer of Compound
3 or a pharmaceutically acceptable salt or ester thereof.
[0016] .alpha.-2B agonists with minimal .alpha.-2A agonist activity
useful in the invention further include, but are not limited to,
imidazolones. A useful imidazolone .alpha.-2B agonist with minimal
.alpha.-2A agonist activity can be, for example, Compound 4 or a
pharmaceutically acceptable salt, ester, amide, sterioisomer or
racemic mixture thereof. In one embodiment, the .alpha.-2B agonist
with minimal .alpha.-2A agonist activity is the (+) enantiomer of
Compound 4 or a pharmaceutically acceptable salt, ester or amide
thereof. In additional embodiments, a method of the invention is
practiced using one of the following .alpha.-2B agonists with
minimal .alpha.-2A agonist activity: Compound 5, Compound 6,
Compound 7, Compound 8, Compound 9 or Compound 14, or a
pharmaceutically acceptable salt, ester, amide, sterioisomer or
racemic mixture thereof. An .alpha.-adrenergic agonist with minimal
.alpha.-2A agonist activity can be peripherally administered by any
of a variety of routes including, without limitation, oral
administration and administration via subcutaneous minipump.
[0017] The present invention also provides a method of screening
for effective agents that produce peripheral analgesia without
concomitant sedation by contacting an .alpha.-2A receptor with an
.alpha.-adrenergic agonist having analgesic activity; and
determining whether the agonist has .alpha.-2A agonist activity,
where the absence of .alpha.-2A agonist activity indicates that the
.alpha.-adrenergic agonist having analgesic activity is an
effective agent that produces peripheral analgesia without
concomitant sedation.
[0018] Further provided herein is a method of screening for
effective agents that produce peripheral analgesia without
concomitant sedation by contacting an .alpha.-2A receptor with an
agent; determining whether the agent has .alpha.-2A agonist
activity; contacting an .alpha.-2B receptor with the agent; and
determining whether the agent has .alpha.-2B agonist activity,
where the absence of .alpha.-2A agonist activity and the presence
of .alpha.-2B agonist activity indicate that the agent is an
effective agent that produces peripheral analgesia without
concomitant sedation.
[0019] The invention also provides a method of screening for
effective agents that produce peripheral analgesia without
concomitant sedation by peripherally administering an
.alpha.-adrenergic agonist to a control animal having at least wild
type levels of .alpha.-2A receptor activity; assaying for analgesia
in the control animal; peripherally administering to a
corresponding animal having reduced levels of .alpha.-2A receptor
expression or activity an amount of the .alpha.-adrenergic agonist
similar or greater than the amount administered to the control
animal; and assaying for analgesia in the corresponding animal
having reduced levels of .alpha.-2A receptor expression or
activity, where the absence of analgesia in the control animal and
the presence of analgesia in the corresponding animal having
reduced levels of .alpha.-2A receptor expression or activity
indicate that the .alpha.-adrenergic agonist has excessive
.alpha.-2A agonist activity, and where the presence of analgesia in
the control animal and the presence of analgesia in the
corresponding animal having reduced levels of .alpha.-2A receptor
expression or activity indicate that the .alpha.-adrenergic agonist
is an effective agent that produces peripheral analgesia without
concomitant sedation. In such a method of the invention, the
control animal can be, for example, wild type at both .alpha.-2A
receptor loci. In one embodiment, the control animal is a wild type
animal such as a wild type mouse. A variety of corresponding
animals are useful in a screening method of the invention. In one
embodiment, the invention is practiced with a corresponding animal
having a homozygous point mutation at the .alpha.-2A receptor
locus. In another embodiment, the invention is practiced with a
corresponding animal having a point mutation within the .alpha.-2A
receptor coding sequence. Such a point mutation can occur, for
example, at a residue analogous to Asp79 and can be, for example,
an Asp79 to Asn mutation. In a further embodiment, the invention is
practiced with a corresponding animal having a homozygous
.alpha.-2A knockout mutation. It is understood that a variety of
methodologies can be used to assay for analgesia in the methods of
the invention, including, but not limited to, assaying for
analgesia following sulprostone sensitization.
[0020] If desired, a method of the invention for screening for
effective agents that produce peripheral analgesia without
concomitant sedation can be practiced by (a) peripherally
administering an .alpha.-adrenergic agonist to a control animal
having at least wild type levels of .alpha.-2A and .alpha.-2B
receptor activity; (b) assaying for analgesia in the control
animal; (c) peripherally administering to a corresponding animal
having reduced levels of .alpha.-2A receptor expression or activity
an amount of the .alpha.-adrenergic agonist similar or greater than
the amount administered to the control animal; (d) assaying for
analgesia in the corresponding animal having reduced levels of
.alpha.-2A receptor expression or activity; (e) peripherally
administering the .alpha.-adrenergic agonist to a corresponding
animal having reduced levels of .alpha.-2B receptor expression or
activity; and (f) assaying for analgesia in the corresponding
animal having reduced levels of .alpha.-2B receptor expression or
activity, where the absence of analgesia in the control animal and
the presence of analgesia in the corresponding animal having
reduced levels of .alpha.-2A receptor expression or activity
indicate that the .alpha.-adrenergic agonist has excessive
.alpha.-2A agonist activity, and where the presence of analgesia in
the control animal, the presence of analgesia in said corresponding
animal having reduced levels of .alpha.-2A receptor expression or
activity, and the absence of analgesia in the corresponding animal
having reduced levels of .alpha.-2B receptor expression or activity
indicate that the .alpha.-adrenergic agonist is an effective agent
that produces peripheral analgesia without concomitant
sedation.
[0021] The present invention additionally provides a method of
screening for effective agents that produce peripheral analgesia
without concomitant sedation by peripherally administering an
.alpha.-adrenergic agonist to a control animal having at least wild
type levels of .alpha.-2B receptor activity; assaying for analgesia
in the control animal; peripherally administering the
.alpha.-adrenergic agonist to a corresponding animal having reduced
levels of .alpha.-2B receptor expression or activity; and assaying
for analgesia in the corresponding animal having reduced levels of
.alpha.-2B receptor expression or activity, where the presence of
analgesia in the control animal and the absence of analgesia in the
corresponding animal having reduced levels of .alpha.-2B receptor
expression or activity indicate that the .alpha.-adrenergic agonist
is an effective agent that produces peripheral analgesia without
concomitant sedation.
[0022] Such a method of the invention can be practiced with a
variety of control animals, for example, a control animal which is
wild type at both .alpha.-2B receptor loci. In one embodiment, the
control animal is a wild type animal. In a further embodiment, the
control animal is a wild type mouse. Similarly, a variety of
corresponding animals are useful in the screening methods of the
invention, including corresponding animals which have a
heterozygous .alpha.-2B knockout mutation or a homozygous
.alpha.-2B knockout mutation. Analgesia can be assayed using any of
a variety of methodologies. In one embodiment, analgesia is assayed
following sulprostone sensitization.
[0023] The present invention further provides a method for the
long-term relief of chronic pain in a subject. The method is
practiced by activating in the subject an analgesic
.alpha.-adrenergic receptor in the absence of .alpha.-2A receptor
activation over a period of at least three days, such that relief
of chronic pain is maintained in the absence of continued receptor
activation. In one embodiment, a method of the invention is
practiced by administering to the subject a pharmaceutical
composition containing an effective amount of an .alpha.-adrenergic
agonist with minimal .alpha.-2A agonist activity over a period of
at least three days, such that relief of chronic pain is maintained
in the absence of significant agonist levels in the subject. Relief
of chronic pain can be maintained, for example, for at least three
weeks in the absence of significant agonist levels in the subject.
It is understood that the methods of the invention can be used for
the long-term relief of any type of chronic pain. As non-limiting
examples, such a method can be used for the long-term relief of
neuropathic pain; visceral pain; post-operative pain; pain
resulting from cancer or cancer treatment; or inflammatory
pain.
[0024] Long-term chronic pain relief can be achieved according to a
method of the invention with any of a variety of .alpha.-adrenergic
agonists with minimal .alpha.-2A agonist activity. Long-term
chronic pain relief can be achieved, for example, using an
.alpha.-2B agonist with minimal .alpha.-2A agonist activity.
Exemplary .alpha.-2B agonists with minimal .alpha.-2A agonist
activity include, without limitation, thiones such as Compound 3 or
Compound 11, or pharmaceutically acceptable salts, esters, amides,
sterioisomers or racemic mixtures thereof. In one embodiment, such
a thione .alpha.-2B agonist with minimal .alpha.-2A agonist
activity is the (-) enantiomer of Compound 3, or a pharmaceutically
acceptable salt or ester thereof. Exemplary .alpha.-2B agonists
with minimal .alpha.-2A agonist activity further include, without
limitation, imidazolones such as Compound 4 or a pharmaceutically
acceptable salt, ester, amide, sterioisomer or racemic mixture
thereof. In one embodiment, such a imidazolone .alpha.-2B agonist
with minimal .alpha.-2A agonist activity is the (+) enantiomer of
Compound 4, or a pharmaceutically acceptable salt or ester thereof.
Exemplary .alpha.-2B agonists with minimal .alpha.-2A agonist
activity also include, without limitation, compounds such as
Compound 5, Compound 6, Compound 7, Compound 8 and Compound 9, and
pharmaceutically acceptable salts, esters, amides, sterioisomers
and racemic mixtures thereof. An .alpha.-adrenergic agonist with
minimal .alpha.-2A agonist activity can be administered by any of a
variety of routes including, but not limited to, all routes of
peripheral administration, for example, oral administration or
administration via subcutaneous minipump.
[0025] In a further embodiment, a method of the invention is
practiced by administering to the subject a pharmaceutical
composition containing an effective amount of an .alpha.-adrenergic
agonist and a pharmaceutical composition containing an effective
amount of a selective .alpha.-2A antagonist over a period of at
least three days, such that relief of chronic pain is maintained in
the absence of significant agonist levels in the subject. Chronic
pain relief can be maintained, for example, for at least three
weeks in the absence of significant agonist levels in the subject.
A variety of .alpha.-adrenergic agonists are useful in the
invention including clonidine, brimonidine, tizanidine,
dexemedetomidine, norepinephrine and other pan-.alpha.-2 agonists
and pan-.alpha.-1 pan-.alpha.-2 agonists as well as Compound 1 or
Compound 2, and pharmaceutically acceptable salts, esters, amides,
sterioisomers and racemic mixtures thereof. Similarly, a variety of
selective .alpha.-2A antagonists are useful in long-term relief of
chronic pain including, without limitation, Compound 13 and
pharmaceutically acceptable salts, esters, amides, sterioisomers
and racemic mixtures thereof, and peripherally limited selective
.alpha.-2A antagonists.
[0026] Various routes of administration can be useful for
delivering pharmaceutical compositions for the long-term relief of
chronic pain. Such routes of administration encompass, but are not
limited to, peripheral administration, for example, oral
administration or administration via subcutaneous minipump. In one
embodiment, the .alpha.-adrenergic agonist and selective .alpha.-2A
antagonist both are administered peripherally. In other
embodiments, the .alpha.-adrenergic agonist is administered orally
or through a subcutaneous minipump, and the selective .alpha.-2A
antagonist is administered by any peripheral route. In still
further embodiments, the selective .alpha.-2A antagonist is
administered orally or through a subcutaneous minipump and the
.alpha.-adrenergic agonist is administered by a peripheral route,
including but not limited to, orally or via subcutaneous
minipump.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 shows the results obtained with intrathecal (1 .mu.g)
or intraperitoneal (30 .mu.g/kg) injection of Compound 1 into
sulprostone-sensitized wild type or .alpha.-2A knockout animals.
Pain sensitivity to light touch was scored as described below. (A)
Intrathecal injection into wild type mice. (B) Intrathecal
injection into .alpha.-2A knockout mice. (C) Intraperitoneal
injection into wild type mice. (D) Intraperitoneal injection into
.alpha.-2A knockout mice. Asterisks indicate a significant result
with a p value <0.05.
[0028] FIG. 2 shows-the results obtained with intraperitoneal
injection of Compound 2 into sulprostone-sensitized wild type and
.alpha.-2A knockout mice. (A) Intraperitoneal injection of 100
.mu.g/kg Compound 2 into wild type mice. (B) Intraperitoneal
injection of 100 .mu.g/kg Compound 2 into .alpha.-2A knockout mice.
Asterisks indicate a significant result with a p value
<0.05.
[0029] FIG. 3 shows that a peripheral .alpha.-2 receptor mediates
analgesia in .alpha.-2A knockout mice. Para-amino clonidine (PAC)
at 100 .mu.g/kg, alone or in combination with rauwolscine (Rau) at
0.3 .mu.g/kg, was administered to sulprostone-sensitized .alpha.-2A
knockout mice by intraperitoneal injection. Asterisks indicate a
significant result with a p value <0.05.
[0030] FIGS. 4A and 4B show that .alpha.-adrenergic agonists can be
effective peripheral analgesic agents in wild type animals when
.alpha.-2A receptor activation is blocked. Percentage allodynia
reversal is shown for each set of animals. (A) Chung model rats
were intraperitoneally administered 30 .mu.g/kg clonidine, 0.3
.mu.g/kg Compound 13, or both. (B) Chung model rats were
intraperitoneally administered 100 .mu.g/kg Compound 2, 0.3
.mu.g/kg Compound 13, or both. Asterisks indicate a significant
result with a p value <0.05.
[0031] FIGS. 4C and 4D compare the sedative and peripheral
analgesic profiles of clonidine and Compound 3. (C) Total activity
counts and percentage of allodynia reversal for intraperitoneally
administered clonidine. (D) Total activity counts and percentage of
allodynia reversal for intraperitoneally administered Compound 3.
The total number of activity counts for vehicle-treated animals was
approximately 2500. Asterisks indicate a significant result with a
p value <0.05.
[0032] FIG. 5 shows that analgesic activity of an
.alpha.-adrenergic agonist with minimal .alpha.-2A agonist activity
continues in the absence of plasma drug levels. (A) Chung model
rats were administered Compound 8, Compound 9, brimonidine,
para-amino-clonidine or vehicle for seven days via osmotic minipump
at the indicated concentrations. The percentage allodynia reversal
was measured over the course of 15 days from the time of osmotic
minipump implantation. The results obtained with Compounds 8 and 9
were significant with a p value of <0.05. (B) Chung model rats
were administered 0.1 mg/hr/kg drug Compound 8 for seven days via
osmotic minipump. The plasma concentration (ng/ml) of Compound 8
and the percentage of allodynia reversal (% MPE) at the same time
point were measured on the indicated days following pump
implantation.
[0033] FIG. 6 shows that reversal of allodynia does not require
ongoing receptor activation after several days dosing with Compound
8. The percentage of allodynia reversal in Chung model rats is
indicated at various days after initiation of Compound 8 drug
treatment. (A) Treatment with 0.1 mg/hr/kg Compound 8 via osmotic
minipump for seven days. On days 3 and 4, measurements were made
prior to and 0.30 minutes following a 0.3 .mu.g/kg intraperitoneal
dose of rauwolscine (R). (B) Treatment with 0.3 mg/kg Compound 8 by
oral dosing three times a day for three days. Measurements were
made after or prior to the first dose of the day as indicated.
Measurements continued for 11 days after dosing was completed.
Asterisks indicate a significant result with a p value
<0.05.
[0034] FIG. 7A shows that pan-.alpha.-2 agonists can produce
long-term pain relief when combined with a selective .alpha.-2A
antagonist. Drugs were administered for five days via osmotic
minipump at the following doses: brimonidine (42 .mu.g/kg/hr);
Compound 1 (0.1 mg/kg/hr); and Compound 13 (8 .mu.g/kg/hr). Each
compound and vehicle was administered alone; brimonidine and
Compound 1 also were administered together with Compound 13. The
percentage allodynia reversal was determined at various days from
the start of dosing. The results obtained after day 5 with the
combination of brimonidine and Compound 13 or Compound 1 and
Compound 13 were significant with a p value <0.05. FIG. 7B shows
that Compound 8 produces long-term pain relief of cold allodynia in
the Bennett model. Animals were treated for four days with 0.1
mg/hr/kg Compound 8 or saline by osmotic minipump, which was
removed on day 4. Paw withdrawal duration over a five minute period
(in seconds) is shown on the indicated days following initiation of
drug treatment.
[0035] FIG. 8 shows that Compound 8 produces long-term pain relief
in a rat model of irritable bowel syndrome. Rats were treated for
seven days by osmotic minipump; the abdominal withdrawal reflex to
a series of colorectal distensions (CRDs) was measured by
electromyography prior to, during, and subsequent to treatment. (A)
Normal rats treated with 50% DMSO vehicle. (B) Normal rats treated
with Compound 8 at 0.1 mg/hr/kg. (C) Sensitized rats treated with
50% DMSO vehicle. (D) Sensitized rats treated with Compound 8 at
0.1 mg/hr/kg. CRD, colorectal distension
[0036] FIG. 9 shows the analgesia obtained with
sulprostone-sensitized .alpha.-2B knockout mice. Wild type (+/+);
heterozygous (+/-) or homozygous (-/-) .alpha.-2B knockout mice
were treated with intrathecal vehicle (DMSO), intrathecal
sulprostone, sulprostone with intraperitoneal clonidine, or
sulprostone with intraperitoneal Compound 3. The total pain score
in six mice was determined. Asterisks indicate a significant result
with a p value <0.05.
[0037] FIG. 10 shows the peripheral analgesic effects of Compound
3, Compound 11 and Compound 4 at various oral doses of drug in
Chung model rats. (A) A single oral dose of 10 .mu.g/kg, 30
.mu.g/kg, 100 .mu.g/kg or 300 .mu.g/kg Compound 3. (B) A single
oral dose of 30 .mu.g/kg, 100 .mu.g/kg, 300 .mu.g/kg or 1000
.mu.g/kg Compound 11. (C) A single oral dose of 30 .mu.g/kg, 100
.mu.g/kg, 300 .mu.g/kg or 1000 .mu.g/kg Compound 4. Asterisks
indicate a significant result with a p value <0.05.
[0038] FIG. 11 shows long-term reversal of allodynia in Chung model
rats following 5 days of treatment by osmotic minipump with 0.1
mg/kg/hour Compound 3, Compound 4 or Compound 11. The results
obtained for all three compounds were significant, with a p value
<0.05.
[0039] FIG. 12 shows differential analgesic activity of enantiomers
of Compounds 3 and 4. (A) Percentage allodynia reversal obtained
with enantiomers and parent racemate of Compound 3 at the indicated
intraperitoneal doses. (B) Percentage allodynia reversal obtained
with enantiomers and parent racemate of Compound 4 at the indicated
intraperitoneal doses. The results obtained were significant, with
a p value <0.05, where an analgesic effect was observed.
DETAILED DESCRIPTION OF THE INVENTION
[0040] Adrenergic receptors mediate physiological responses to the
catecholamines, norephinephrine and epinephrine, and are members of
the superfamily of G protein-coupled receptors having seven
transmembrane domains. These receptors, which are divided
pharmacologically into .alpha.-1, .alpha.-2 and .alpha.-adrenergic
receptor types, are involved in diverse physiological functions
including functions of the cardiovascular and central nervous
systems. The .alpha.-adrenergic receptors mediate most excitatory
functions: .alpha.-1 adrenergic receptors generally mediate
responses in the effector organ, while .alpha.-2 adrenergic
receptors are located postsynaptically as well as presynaptically,
where they regulate release of neurotransmitters. Agonists of
.alpha.-2 adrenergic receptors currently are used clinically in the
treatment of hypertension, glaucoma, spasticity, and
attention-deficit disorder, in the suppression of opiate
withdrawal, and as adjuncts to general anesthesia.
[0041] The .alpha.-2 adrenergic receptors presently are classified
into three subtypes based on their pharmacological and molecular
characterization: .alpha.-2A/D (.alpha.-2A in human and .alpha.-2D
in rat); .alpha.-2B; and .alpha.-2C (Bylund et al., Pharmacol. Rev.
46:121-136 (1994); and Hein and Kobilka, Neuropharmacol. 34:357-366
(1995)). The .alpha.-2A and .alpha.-2B subtypes can regulate
arterial contraction in some vascular beds, and the .alpha.-2A and
.alpha.-2C subtypes mediate feedback inhibition of norepinephrine
release from sympathetic nerve endings. The .alpha.-2A subtype also
mediates many of the central effects of .alpha.-2 adrenergic
agonists (Calzada and Artiano, Pharmacol. Res. 44: 195-208 (2001);
Hein et al., Ann. NY Acad. Science 881:265-271 (1999); and Karger
(Ed.), .alpha.-Adrenoreceptors: Molecular Biology, Biochemistry and
Pharmacology (1991)).
[0042] Although non-selective .alpha.-adrenergic agonists having
.alpha.-2A agonist activity, such as clonidine and dexmeditomidine,
have been used for the treatment of various types of pain, such
drugs must be administered spinally to achieve analgesia that is
separable from sedation. The central analgesic effect of such
pan-agonists is mediated by the .alpha.-2A receptor expressed in
the dorsal horn within the spinal column. The present invention is
based on the surprising discovery that, in contrast to the
pro-analgesic function of the .alpha.-2A receptor in the spinal
column, a peripheral .alpha.-2A receptor mediates pain. The
invention further is based on identification of a peripherally
expressed adrenergic receptor, the .alpha.-2B receptor, which, when
activated, can produce peripheral analgesia.
[0043] As disclosed herein, .alpha.-adrenergic agonists with
.alpha.-2A activity such as the pan-agonists clonidine or
brimonidine produce no significant analgesia separable from
sedation upon peripheral administration to wild type animals.
However, analgesia separable from sedation was observed in
.alpha.-2A knockout mice treated peripherally with these drugs.
Furthermore, analgesic activity separable from sedation also was
observed in .alpha.-2A knockout mice following peripheral
administration of .alpha.-adrenergic agonists that do not readily
cross the blood-brain barrier, such as Compound 1 or
para-amino-clonidine (PAC; see Example IIB and FIG. 1D). This
peripheral analgesic activity was not observed in wild type animals
(FIG. 1C), indicating that a novel analgesic activity of
.alpha.-adrenergic agonists is unmasked by preventing activation of
a peripheral .alpha.-2A receptor.
[0044] Furthermore, as shown herein in Example III, Chung rats, a
well-accepted model of peripheral neuropathy, were peripherally
administered an .alpha.-2 pan-agonist such as clonidine together
with the selective .alpha.-2A antagonist, Compound 13, shown in
Table 1. In contrast to the results obtained when clonidine was
peripherally administered alone, peripheral co-administration of
clonidine with the selective .alpha.-2A antagonist produced an
analgesic effect separable from sedation, confirming that blockade
of the .alpha.-2A receptor can reveal a peripheral analgesic
activity of .alpha.-adrenergic agonists in genetically unaltered
animals (see FIG. 4A).
1TABLE 1 Structures of Various alpha-Adrenergic Agonists and
Antagonists COMPOUND 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11
11 12 12 13 13 14 14
[0045] As further disclosed herein in Example III, diverse
structural classes of .alpha.-adrenergic agonists with minimal
.alpha.-2A activity, such as Compounds 3, 4, 5, 6, 7 and 14,
produced analgesia without concomitant sedation when administered
by intraperitoneal injection to Chung model rats (see Table 2
below). These results demonstrate that the analgesic activity of
.alpha.-adrenergic agonists with minimal .alpha.-2A agonist
activity is distinct from the previously described analgesic
activity of .alpha.-adrenergic agents and further indicate that
.alpha.-adrenergic agonists characterized by having minimal
.alpha.-2A agonist activity can be useful peripheral analgesic
drugs.
[0046] The results disclosed herein in Example IV further
demonstrate that .alpha.-adrenergic agonists with minimal
.alpha.-2A agonist activity can produce long-term pain relief
lasting for at least six weeks following several days of drug
administration. As disclosed herein, Chung model animals were
treated for three to seven days using a subcutaneous osmotic
minipump with Compound 8, Compound 9, Compound 3 or Compound 4,
which are structurally diverse .alpha.-adrenergic agonists with
minimal .alpha.-2A agonist activity. Pain relief was observed
during the period of drug treatment; for example, Compound 8
alleviated allodynia 90-100%, and Compound 9 alleviated allodynia
60-80%. Furthermore, the analgesic effects of each of the agonists
tested continued for over a month after treatment was concluded,
and this long-term pain relief was not a property of a variety of
other analgesic agents tested.
[0047] As further disclosed herein, sampling of plasma
concentrations of Compound 8 at various time points revealed that
very low drug levels were present by day 10 following initiation of
drug treatment, and no plasma drug was detectable by day 14 (see
FIG. 5B), although pain was largely alleviated at this time. In
addition, the .alpha.-2 antagonist, rauwolscine, was assayed for
the ability to inhibit the analgesic activity of Compound 8 at
various time points following several days of dosing by oral gavage
or osmotic minipump. Notably, rauwolscine inhibited the analgesic
activity of Compound 8 on the third day of treatment but not on the
fourth day (see FIG. 6). In sum, these results indicate that the
duration of analgesic activity extends beyond the time drug remains
in the blood and that, following extended dosing,
.alpha.-adrenergic agonists with minimal .alpha.-2A agonist
activity can provide prolonged pain relief even in the absence of
continued receptor activation or plasma drug levels.
[0048] Additional results disclosed herein show that
.alpha.-adrenergic agonists with minimal .alpha.-2A agonist
activity also produce long-term pain relief in the Bennett partial
sciatic nerve ligation model and in an animal model of irritable
bowel syndrome (see Example IV, FIG. 7B and FIG. 8), demonstrating
that the observed long-term analgesic effects are not specific to
neuropathic pain. These results indicate that an .alpha.-adrenergic
agonist with minimal .alpha.-2A agonist activity can be used to
treat a variety of types of acute and chronic pain including, but
not limited to, neuropathic pain, visceral pain, inflammatory pain,
post-surgical pain and cancer pain.
[0049] Based on these findings, the present invention provides a
method of alleviating pain in a subject by agonizing a peripheral
.alpha.-adrenergic receptor other than the .alpha.-2A receptor. In
one embodiment, the invention provides a method of alleviating pain
in a subject by agonizing a peripheral .alpha.-2B receptor. The
present invention also provides a method of alleviating pain in a
subject by administering to the subject a pharmaceutical
composition containing an effective amount of an .alpha.-adrenergic
agonist and a pharmaceutical composition containing an effective
amount of a selective .alpha.-2A antagonist. Such methods are
useful for alleviating a variety of types of pain including, but
not limited to, neuropathic pain such as the pain resulting from
diabetic neuropathy; visceral pain; post-operative pain; pain
resulting from cancer or cancer treatment; inflammatory pain
resulting, for examples from arthritis or irritable bowel syndrome;
headache pain and muscle pain.
[0050] A variety of .alpha.-adrenergic agonists are useful in the
invention including pan-.alpha.-2 agonists and pan-.alpha.-1
pan-.alpha.-2 agonists as well as .alpha.-2B agonists.
.alpha.-adrenergic agonists useful in alleviating pain according to
a method of the invention encompass, without limitation, clonidine,
brimonidine, tizanidine, dexemedetomidine, norepinephrine, Compound
1 and Compound 2, and all pharmaceutically acceptable salts,
esters, amides, sterioisomers and racemic mixtures thereof.
[0051] A variety of selective .alpha.-2A antagonists also are
useful in the invention. Such selective .alpha.-2A antagonists
include, without limitation, Compound 13 and pharmaceutically
acceptable salts, esters, amides, sterioisomers and racemic
mixtures thereof, and BRL 48962 and BRL 44408 and pharmaceutically
acceptable salts, esters, amides, sterioisomers and racemic
mixtures thereof (Young et al., Eur. J. Pharmacol. 168:381-386
(1989)). In one embodiment, the invention is practiced with a
peripherally limited selective .alpha.-2A antagonist.
[0052] Various routes of administration can be useful for
alleviating pain according to a method of the invention. In one
embodiment, both the .alpha.-adrenergic agonist and selective
.alpha.-2A antagonist are administered peripherally. In other
embodiments, the .alpha.-adrenergic agonist is administered orally
or through a subcutaneous minipump. In further embodiments, the
.alpha.-adrenergic agonist is administered orally or through a
subcutaneous minipump, and the selective .alpha.-2A antagonist is
administered by any peripheral route. In yet other embodiments, the
selective .alpha.-2A antagonist is administered orally or through a
subcutaneous minipump. If desired, the selective .alpha.-2A
antagonist can be administered orally or through a subcutaneous
minipump while the .alpha.-adrenergic agonist is administered
peripherally, for example, orally or via subcutaneous minipump.
[0053] The invention also provides a method of alleviating pain in
a subject by administering to the subject a pharmaceutical
composition containing an effective amount of an .alpha.-adrenergic
agonist and a pharmaceutical composition containing an effective
amount of a selective .alpha.-2A antagonist, where the
.alpha.-adrenergic agonist and the selective .alpha.-2A antagonist
each is administered repeatedly or continuously over a period of at
least three days. In such a method, pain alleviation can continue,
for example, in the absence of significant .alpha.-adrenergic
agonist levels in the subject.
[0054] The present invention provides methods that rely on
administration of one or more pharmaceutical compositions to a
subject. As used herein, the term "subject" means any animal
capable of experiencing pain, for example, a human or other mammal
such as a primate, horse, cow, dog or cat.
[0055] The methods of the invention are used to treat both acute
and chronic pain, and, as non-limiting examples, pain which is
neuropathic, visceral or inflammatory in origin. In particular
embodiments, the methods of the invention are used to treat
neuropathic pain; visceral pain; post-operative pain; pain
resulting from cancer or cancer treatment; and inflammatory
pain.
[0056] Both acute and chronic pain can be treated by the methods of
the invention, and the term "pain" encompasses both acute and
chronic pain. As used herein, the term "acute pain" means
immediate, generally high threshold, pain brought about by injury
such as a cut, crush, burn, or by chemical stimulation such as that
experienced upon exposure to capsaicin, the active ingredient in
chili peppers. The term "chronic pain," as used herein, means pain
other than acute pain and includes, without limitation, neuropathic
pain, visceral pain, inflammatory pain, headache pain, muscle pain
and referred pain. It is understood that chronic pain is of
relatively long duration, for example, several years and can be
continuous or intermittent.
[0057] In one embodiment, the methods of the invention are used to
treat "neuropathic pain," which, as used herein, is a term that
means pain resulting from injury to a nerve. Neuropathic pain is
distinguished from nociceptive pain, which is the pain caused by
acute tissue injury involving small cutaneous nerves or small
nerves in muscle or connective tissue. In contrast to neuropathic
pain, pain involving a nociceptive mechanism usually is limited in
duration to the period of tissue repair and generally is alleviated
by available analgesic agents or opioids (Myers, Regional
Anesthesia 20:173-184 (1995)).
[0058] Neuropathic pain typically is long-lasting or chronic and
can develop days or months following an initial acute tissue
injury. Neuropathic pain can involve persistent, spontaneous pain
as well as allodynia, which is a painful response to a stimulus
that normally is not painful, or hyperalgesia, an accentuated
response to a painful stimulus that usually is trivial, such as a
pin prick. Neuropathic pain generally is resistant to opioid
therapy (Myers, supra, 1995).
[0059] The methods of the invention are useful in alleviating
neuropathic pain resulting from, without limitation, a trauma,
injury or disease of peripheral nerve, dorsal root ganglia, spinal
cord, brainstem, thalamus or cortex. Examples of neuropathic pain
which can be treated by the methods of the invention include
neuralgia such as post-herpetic neuralgia, deafferentation pain and
diabetic neuropathy. It is understood that the methods of the
invention are useful in alleviating neuropathic pain regardless of
the etiology of the pain. As examples, the methods of the invention
can be used to alleviate neuropathic pain resulting from a
peripheral nerve disorder such as neuroma; from nerve compression;
from nerve crush or stretch or incomplete nerve transsection; or
from a mononeuropathy or polyneuropathy. As further examples, the
methods of the invention are useful in alleviating neuropathic pain
resulting from a disorder such as dorsal root ganglion compression;
inflammation of the spinal cord; contusion, tumor or hemisection of
the spinal cord; and tumors or trauma of the brainstem, thalamus or
cortex.
[0060] As indicated above, the methods of the invention can
alleviate neuropathic pain resulting from a mononeuropathy or
polyneuropathy. A neuropathy is a functional disturbance or
pathological change in the peripheral nervous system and is
characterized clinically by sensory or motor neuron abnormalities.
The term mononeuropathy indicates that a single peripheral nerve is
affected, while the term polyneuropathy indicates that several
peripheral nerves are affected. The etiology of a neuropathy can be
known or unknown. Known etiologies include complications of a
disease or toxic state such as diabetes, which is the most common
metabolic disorder causing neuropathy, or irradiation, ischemia or
vasculitis. Polyneuropathies that can be treated by a method of the
invention can result, without limitation, from post-polio syndrome,
diabetes, alcohol, amyloid, toxins, HIV, hypothyroidism, uremia,
vitamin deficiencies, chemotherapy, ddC or Fabry's disease. It is
understood that the methods of the invention can be used to
alleviate pain of these or other neuropathies of known or unknown
etiology.
[0061] As additional non-limiting examples, the methods of the
invention can be used to treat chronic gastrointestinal
inflammations including Crohn's disease, ulcerative colitis,
gastritis, irritable bowel disease; and chronic visceral pain such
as pain caused by cancer or attendant to the treatment of cancer,
for example, attendant to chemotherapy or radiation therapy.
Similarly, the methods of the invention can be used to treat
chronic inflammatory pain resulting, for example, from arthritis
such as rheumatoid arthritis, gouty arthritis, or osteoarthritis;
spondylitis; or autoimmune diseases such as lupus erythematosus.
The methods of the invention further can be used to treat headache
pain; muscle pain; and the pain associated with substance abuse or
withdrawal and other types of pain of known or unknown
etiology.
[0062] Several of the methods of the invention rely, in part, on an
".alpha.-adrenergic agonist," which, as used herein, is a term
which means a compound having greater than 25% efficacy relative to
brimonidine at one or more .alpha.-2 adrenergic receptors or having
greater than 25% efficacy relative to phenylephrine at one or more
.alpha.-1 adrenergic receptors. Such a compound can be selective
for one or more .alpha.-adrenergic receptors, or can be
non-selective. Thus, the term .alpha.-adrenergic agonist
encompasses, without limitation, "pan-.alpha.-1 pan-.alpha.-2
agonists" such as norepinephrine, which have agonist activity at
all .alpha.-1 and .alpha.-2 receptors; pan-.alpha.-2 agonists;
.alpha.-2 selective agonists; .alpha.-2B agonists; and agonists
that are specific for a single .alpha.-adrenergic receptor. In
particular embodiments, a method of the invention utilizes an
.alpha.-adrenergic agonist having greater than 30%, 40%, 50%, 60%,
70%, 80%, 90%, 100% or 200% efficacy relative to brimonidine at one
or more .alpha.-2 adrenergic receptors. In additional embodiments,
a method of the invention utilizes an .alpha.-adrenergic agonist
having greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 200%
efficacy relative to phenylephrine at one or more .alpha.-1
adrenergic receptors. In another embodiment, the invention relies
on an .alpha.-adrenergic agonist lacking significant .alpha.-2A
antagonist activity.
[0063] Efficacy, also known as intrinsic activity, is a measure of
maximal receptor activation achieved by a compound and can be
determined using any accepted assay of .alpha.-adrenergic receptor
activation, such as a cAMP or Receptor Selection and Amplification
Technology (RSAT) assay described hereinbelow. Efficacy is
represented as a ratio or percentage of the maximal effect of the
drug to the maximal effect of a standard agonist for each receptor
subtype. Brimonidine (UK14304) generally is used as the standard
agonist for the .alpha.-2A, .alpha.-2B and .alpha.-2C receptors and
is used as the standard herein where efficacy of an .alpha.-2
receptor is defined. Phenylephrine is an accepted standard agonist
for the .alpha.-1A, .alpha.-1B and .alpha.-1D receptors and is used
herein as the standard where efficacy of an .alpha.-1 receptor is
defined (Messier et al., supra, 1995; Conklin et al., supra,
1993).
[0064] As disclosed herein, .alpha.-2B agonists can be useful in
alleviating pain or for the long-term relief of chronic pain. The
term ".alpha.-2B agonist," as used herein, means a compound having
greater than 25% efficacy relative to brimonidine at the .alpha.-2B
adrenergic receptor. It is understood that this term encompasses
agonists that are either selective or non-selective for the
.alpha.-2B receptor as compared to other .alpha.-adrenergic
receptors. Thus, the term ".alpha.-2B agonist" encompasses
pan-.alpha.-2 agonists and pan-.alpha.-1 pan-.alpha.-2 agonists as
well as agonists that are selective or specific for the .alpha.-2B
receptor, as described further below. In particular embodiments, a
method of the invention utilizes an .alpha.-2B agonist having
greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 200%
efficacy relative to brimonidine at the .alpha.-2B adrenergic
receptor. Exemplary .alpha.-2B agonists include clonidine,
brimonidine, Compounds 1 and 2, and Compounds 3 through 12 and 14,
and all pharmaceutically acceptable salts, esters, amides,
sterioisomers and racemic mixtures of these compounds. In a further
embodiment, a method of the invention relies on an .alpha.-2B
agonist lacking significant .alpha.-2A antagonist activity.
[0065] The term "pan-.alpha.-2 agonist," as used herein, means a
compound having greater than 25% efficacy relative to brimonidine
at each of the .alpha.-2A, .alpha.-2B and .alpha.-2C adrenergic
receptors and encompasses pan-.alpha.-1 pan-.alpha.-2 agonists. A
variety of pan-.alpha.-2 agonists are known in the art and include
clonidine, brimonidine, tizanidine, dexemedetomidine and
norepinephrine. A pan-.alpha.-2 agonist has, at a minimum, greater
than 25% efficacy relative to brimonidine at each of the
.alpha.-2A, .alpha.-2B and .alpha.-2C receptors; in particular
embodiments, a method of the invention is practiced with a
pan-.alpha.-2 agonist having greater than 30%, 40%, 50%, 60%, 70%,
80%, 90%, 100% or 200% efficacy relative to brimonidine at the
.alpha.-2A, .alpha.-2B and .alpha.-2C adrenergic receptors. It is
understood that the efficacy of a pan-.alpha.-2 agonist can be
different at the various .alpha.-2 receptors; as an example, a
pan-.alpha.-2 agonist can have greater than 25% efficacy at the
.alpha.-2A receptor, greater than 80% efficacy at the .alpha.-2B
receptor and greater than 40% efficacy at the .alpha.-2C
receptor.
[0066] The term "pan-.alpha.-1 pan-.alpha.-2 agonist," as used
herein, means a compound having greater than 25% efficacy relative
to phenylephrine at all .alpha.-1 receptors and having greater than
25% efficacy relative to brimonidine at all .alpha.-2 adrenergic
receptors. In particular embodiments, a method of the invention
relies on a pan-.alpha.-1 pan-.alpha.-2 agonist having greater than
30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 200% efficacy at all
.alpha.-1 and .alpha.-2 receptors relative to phenylephrine or
brimonidine, respectively.
[0067] As disclosed herein, a selective .alpha.-2A antagonist is
administered in conjunction with an .alpha.-adrenergic agonist to
alleviate pain or for the long-term relief of chronic pain. As used
herein, the term "selective .alpha.-2A antagonist" means a compound
having (1) an efficacy of less than 25% relative to brimonidine at
.alpha.-2A; (2) a Ki of less than 100 nM at .alpha.-2A; and further
having (3) at least a 10-fold greater Ki at .alpha.-2B than at
.alpha.-2A or an efficacy of greater than 25% relative to
brimonidine at .alpha.-2B. From this definition, it is clear to the
skilled person that non-selective antagonists such as rauwolscine
are not included within the scope of this term. Exemplary selective
.alpha.-2A antagonists are provided herein as Compound 13, BRL
48962 and BRL 44408. Pharmaceutically acceptable salts, esters,
amides, sterioisomers and racemic mixtures of these compounds also
are useful in the invention. A selective .alpha.-2A antagonist can
be a peripherally limited compound. Such a compound does not
readily cross the blood-brain barrier and, thus, upon peripheral
administration, is excluded from the central nervous system.
[0068] In addition to .alpha.-2A antagonist activity, a "selective
.alpha.-2A antagonist" also can have agonist or antagonist activity
at one or more adrenergic or other receptors, provided that the
compound satisfies the three criteria set forth above. As an
example, a compound having .alpha.-2C antagonist activity,
characterized by a Ki at .alpha.-2C of less than 100 nM and an
efficacy at .alpha.-2C of less than 25% relative to brimonidine,
and further having (1) an efficacy of less than 25% relative to
brimonidine at .alpha.-2A, a Ki of less than 100 nM at .alpha.-2A,
and (3) at least a 10-fold greater Ki at .alpha.-2B than at
.alpha.-2A is encompassed by the term "selective .alpha.-2A
antagonist" as defined herein. Similarly, compounds exhibiting
.alpha.-2B or other agonist activity in addition to .alpha.-2A
antagonist activity also are encompassed by the term "selective
.alpha.-2A antagonist." As an example, Compound 13 exhibits
.alpha.-2B agonist activity characterized by about 40% efficacy at
.alpha.-2B relative to brimonidine, thus satisfying criteria (3),
and further has (1) an efficacy of 5% relative to brimonidine at
.alpha.-2A and (2) a Ki of about 0.08 nM at .alpha.-2A and,
therefore, falls within the definition of the term "selective
.alpha.-2A antagonist" as used herein. In particular embodiments, a
method of the invention is practiced with a selective .alpha.-2A
antagonist having a Ki of less than 80 nM, 60 nM, 40 nM, 20 nM, 10
nM, 1 nM or 0.1 nM. In further embodiments, a method of the
invention is practiced with a selective .alpha.-2A antagonist
having at least a 20-fold, 30-fold, 40-fold, 50-fold, 100-fold,
200-fold, 500-fold or 1000-fold greater Ki at .alpha.-2B than at
.alpha.-2A. In still further embodiments, a method of the invention
is practiced with a selective .alpha.-2A antagonist having an
efficacy of greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or
200% relative to brimonidine at .alpha.-2B.
[0069] In one embodiment, the invention relies on a selective
.alpha.-2A antagonist with minimal .alpha.-2B antagonist activity.
As used herein, the term "selective .alpha.-2A antagonist with
minimal .alpha.-2B antagonist activity" means a selective
.alpha.-2A antagonist, as defined hereinabove, further having a Ki
at .alpha.-2B greater than 100 nM. As non-limiting examples, such
an antagonist can have a Ki at .alpha.-2B of greater than 200 nM,
300 nM, 400 nM, 500 nM, 1000 nM, 2000 nM, 3000 nM, 4000 nM or 5000
nM.
[0070] A specific .alpha.-2A antagonist also can be used to prevent
.alpha.-2A receptor activation in a method of the invention. As
used herein, the term "specific .alpha.-2A antagonist" means a
compound having (1) an efficacy of less than 25% relative to
brimonidine at .alpha.-2A; (2) a Ki of less than 100 nM at
.alpha.-2A; and further having (3) at each of the
.alpha.-adrenergic receptors other than .alpha.-2A either at least
a 10-fold greater Ki than at .alpha.-2A or an efficacy of greater
than 25% relative to brimonidine or phenylephrine.
[0071] The present invention also provides compositions and methods
which rely on an .alpha.-adrenergic agonist with minimal .alpha.-2A
agonist activity. In particular, the present invention provides an
analgesic composition that contains an .alpha.-adrenergic agonist
with minimal .alpha.-2A agonist activity having the ability to
produce peripheral analgesia without concomitant sedation. In one
embodiment, the analgesic composition produces peripheral analgesia
without concomitant sedation and in the substantial absence of
hypotensive effects. In another embodiment, the invention provides
an analgesic composition that produces peripheral analgesia
sufficient to reduce pain by at least 50% without concomitant
sedation. In further embodiments, at least a 10-fold, 100-fold or
1000-fold greater dose of the analgesic composition is required to
produce a 20% reduction in motor or muscular activity than the dose
of the analgesic composition required to reduce pain by at least
50%. In yet a further embodiment, the invention provides an
analgesic composition that produces peripheral analgesia sufficient
to reduce pain by at least 50% without concomitant sedation and in
the substantial absence of hypotensive effects. In another
embodiment, the invention provides an analgesic composition that
contains an .alpha.-adrenergic agonist with minimal .alpha.-2A
agonist activity having the ability to produce peripheral analgesia
without concomitant sedation, where the agonist is not a thiourea
or derivative thereof. In a further embodiment, the invention
provides an analgesic composition that contains an
.alpha.-adrenergic agonist with minimal .alpha.-2A agonist activity
having the ability to produce peripheral analgesia without
concomitant sedation, where the agonist is not a thiourea or
4-imidazole or derivative thereof.
[0072] The invention also provides a method of alleviating pain in
a subject by peripherally administering to the subject a
pharmaceutical composition containing an effective amount of an
.alpha.-adrenergic agonist with minimal .alpha.-2A agonist
activity, thereby producing peripheral analgesia without
concomitant sedation. Such peripheral analgesia can be sufficient
to reduce pain, for example, by at least 50% without concomitant
sedation. In another embodiment, the peripheral analgesia occurs in
the substantial absence of hypotensive effects. In a further
embodiment, the method is practiced using an .alpha.-adrenergic
agonist with minimal .alpha.-2A agonist activity which is not a
thiourea or derivative thereof. And, in yet a further embodiment,
the method is practiced using an .alpha.-adrenergic agonist with
minimal .alpha.-2A agonist activity which is not a thiourea or
4-imidazole or derivative thereof. Pain of various types and
etiologies can be alleviated according to a method of the
invention. As non-limiting examples, the methods of the invention
can be useful in alleviating neuropathic pain, such as the pain
resulting from diabetic neuropathy; visceral pain; post-operative
pain; pain resulting from cancer or cancer treatment; inflammatory
pain such as arthritic pain or irritable bowel syndrome pain;
headache pain and muscle pain.
[0073] A variety of .alpha.-adrenergic agonists with minimal
.alpha.-2A agonist activity can be useful in the methods of the
invention. In one embodiment, the .alpha.-adrenergic agonist with
minimal .alpha.-2A agonist activity is an .alpha.-2B agonist with
minimal .alpha.-2A agonist activity. Such an agonist can be, for
example, a thione such as Compound 3, or Compound 11 or a
pharmaceutically acceptable salt, ester, amide, sterioisomer or
racemic mixture thereof. In one embodiment, a method of the
invention is practiced with an .alpha.-2B agonist with minimal
.alpha.-2A agonist activity which is the (-) enantiomer of Compound
3 or a pharmaceutically acceptable salt or ester thereof.
[0074] .alpha.-2B agonists with minimal .alpha.-2A agonist activity
useful in the invention further include, but are not limited to,
imidazolones. A useful imidazolone .alpha.-2B agonist with minimal
.alpha.-2A agonist activity can be, for example, Compound 4 or a
pharmaceutically acceptable salt, ester, amide, sterioisomer or
racemic mixture thereof. In one embodiment, the .alpha.-2B agonist
with minimal .alpha.-2A agonist activity is the (+) enantiomer of
Compound 4 or a pharmaceutically acceptable salt, ester or amide
thereof. In additional embodiments, a method of the invention is
practiced using one of the following .alpha.-2B agonists with
minimal .alpha.-2A agonist activity: Compound 5, Compound 6,
Compound 7, Compound 8, Compound 9, Compound 14 or a
pharmaceutically acceptable salt, ester, amide, sterioisomer or
racemic mixture thereof. An .alpha.-adrenergic agonist with minimal
.alpha.-2A agonist activity can be peripherally administered by any
of a variety of routes including, without limitation, oral
administration and administration via subcutaneous minipump.
[0075] As used herein, the term "peripheral analgesia" means a
reduction in pain obtained following peripheral administration. As
discussed further below, peripheral administration means
introduction of an agent into a subject outside of the central
nervous system and encompasses any route of administration other
than direct administration to the spine or brain. The peripheral
analgesia can be sufficient to reduce pain, for example, by at
least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.
[0076] The compositions of the invention produce peripheral
analgesia without concomitant sedation. Sedation, as used herein,
is a term that means a reduction in motor or muscular activity. The
term "without concomitant sedation," as used herein, means that
relatively little reduction in motor or muscular activity
accompanies peripheral analgesia at one or more doses of drug. In
particular, a drug produces "peripheral analgesia without
concomitant sedation" if, upon peripheral administration, the dose
required to produce a 20% reduction in motor or muscular activity
is at least 3-fold greater than the dose required to reduce pain by
at least 50%. In particular embodiments, the dose required to
produce a 20% reduction in motor or muscular activity is at least
4-fold greater than, 5-fold greater than, 6-fold greater than,
7-fold greater than, 8-fold greater than, 9-fold greater than,
10-fold greater than, 25-fold greater than, 50-fold greater than,
100-fold greater than, 200-fold greater than, 500-fold greater
than, 1000-fold greater than, 2000-fold greater than, or 5000-fold
greater than the dose required to reduce pain by at least 50%.
Methods of determining the extent of pain reduction and the extent
of sedation following peripheral administration are well known in
the art and are described hereinbelow.
[0077] As used herein, the term ".alpha.-adrenergic agonist with
minimal .alpha.-2A agonist activity" means an .alpha.-adrenergic
agonist, as defined above, further characterized by (1) having less
than 25% efficacy relative to brimonidine at the .alpha.-2A
adrenergic receptor and (2) the ability to produce peripheral
analgesia without concomitant sedation in genetically unaltered
animals. It is understood that efficacy is measured using any
standard assay of agonist activity such as a cAMP or RSAT assay
described hereinbelow. Compounds 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12
as well as pharmaceutically acceptable salts, esters, amides,
sterioisomers and racemic mixtures thereof are provided herein as
exemplary .alpha.-adrenergic agonists with minimal .alpha.-2A
agonist activity; such compounds belong to diverse structural
classes as shown in Table 1 above. .alpha.-adrenergic agonists with
minimal .alpha.-2A agonist activity useful in the invention
encompass .alpha.-adrenergic agonists with activity at one or more
.alpha.-1 receptors, .alpha.-2B/C agonists with minimal .alpha.-2A
agonist activity, specific .alpha.-2B agonists with minimal
.alpha.-2A activity and specific .alpha.-2C agonists with minimal
.alpha.-2A activity, as described further below.
[0078] .alpha.-adrenergic agonists with minimal .alpha.-2A agonist
activity can readily be identified by screening .alpha.-adrenergic
agonists, for example, by screening those exhibiting less than 25%
efficacy at .alpha.-2A for functional activity in various in vitro
or in vivo assays. In particular, such agonists can be assayed for
peripheral analgesic activity without concomitant sedation in both
wild type and .alpha.-2A knockout mice, for example, using the
well-accepted model of sulprostone sensitized pain. Agonists with
more than minimal .alpha.-2A agonist activity can be eliminated as
those compounds which fail to produce peripheral analgesia without
concomitant sedation in wild type sulprostone sensitized animals,
although peripheral analgesia without concomitant sedation is
observed in .alpha.-2A knockout mice, as described further below.
In particular embodiments, a method of the invention is practiced
using an .alpha.-adrenergic agonist with minimal .alpha.-2A agonist
activity which produces, without concomitant sedation, peripheral
analgesia sufficient to reduce pain by at least 50%, or by at least
60%, 70%, 80%, 90% or 100% in genetically unaltered animals.
[0079] As used herein, the term ".alpha.-2B agonist with minimal
.alpha.-2A agonist activity" means a compound characterized by
having (1) greater than 25% efficacy relative to brimonidine at the
.alpha.-2B receptor; (2) a potency of less than 1000 nM at
.alpha.-2B or at least 100-fold greater potency at .alpha.-2B than
at .alpha.-2A; (3) less than 25% efficacy relative to brimonidine
at the .alpha.-2A receptor; and (4) the ability to produce
peripheral analgesia without concomitant sedation in genetically
unaltered animals. Provided herein as exemplary .alpha.-2B agonists
with minimal .alpha.-2A agonist activity are Compounds 3, 4, 5, 6,
7, 8, 9, 10, 11, 12 and 14 and pharmaceutically acceptable salts,
esters, amides, sterioisomers and racemic mixtures of these
compounds; as exemplified herein, these compounds are
characterized, in part, by the ability to produce peripheral
analgesia without concomitant sedation in genetically unaltered
animals. In particular embodiments, an .alpha.-2B agonist with
minimal .alpha.-2A agonist activity useful in the invention has an
efficacy of greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or
200% relative to brimonidine at .alpha.-2B. In additional
embodiments, an .alpha.-2B agonist with minimal .alpha.-2A agonist
activity has less than 20%, 15%, 10%, 5%, 2% or 1% efficacy
relative to brimonidine at .alpha.-2A. In further embodiments, an
.alpha.-2B agonist with minimal .alpha.-2A agonist activity has a
potency of less than 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400
nM, 300 nM, 200 nM, 100 nM, 50 nM, 10 nM or 1 nM at .alpha.-2B. In
still further embodiments, an .alpha.-2B agonist with minimal
.alpha.-2A agonist activity has at least 200-fold, 300-fold,
400-fold, 500-fold, 1000-fold or 10,000-fold greater potency at
.alpha.-2B than at .alpha.-2A. In additional embodiments, the
methods of the invention rely on an .alpha.-2B agonist with minimal
.alpha.-2A agonist activity having the ability to produce, without
concomitant sedation, peripheral analgesia sufficient to reduce
pain by at least 50% or by at least 60%, 70%, 80%, 90% or 100% in
genetically unaltered animals.
[0080] The term ".alpha.-2B/C agonist with minimal .alpha.-2A
agonist activity" as used herein, means a compound characterized by
having (1) greater than 25% efficacy relative to brimonidine at the
.alpha.-2B or .alpha.-2C receptor or both; (2) a potency of less
than 1000 nM at the .alpha.-2B or .alpha.-2C receptor or both, or
at least 100-fold greater potency relative to the .alpha.-2A
receptor at the .alpha.-2B or .alpha.-2C receptor or both; (3) less
than 25% efficacy relative to brimonidine at the .alpha.-2A
receptor; and (4) the ability to produce peripheral analgesia
without concomitant sedation in genetically unaltered animals.
[0081] The term "specific .alpha.-2B agonist with minimal
.alpha.-2A activity," as used herein, means a compound
characterized by having (1) greater than 25% efficacy relative to
brimonidine at .alpha.-2B; (2) a potency of less than 1000 nM at
.alpha.-2B or at least 100-fold greater potency at .alpha.-2B than
.alpha.-2A; (3) less than 25% efficacy or 50-fold less potency
relative to brimonidine at .alpha.-2A and .alpha.-2C; (4) less than
25% efficacy or 50-fold less potency relative to phenylephrine at
all .alpha.-1 receptors; and (5) the ability to produce peripheral
analgesia without concomitant sedation in genetically unaltered
animals.
[0082] Similarly, the term "specific .alpha.-2C agonist with
minimal .alpha.-2A activity," as used herein, means a compound
characterized by having (1) greater than 25% efficacy relative to
brimonidine at .alpha.-2C; (2) a potency of less than 1000 nM at
.alpha.-2C or at least 100-fold greater potency at .alpha.-2C than
.alpha.-2A; (3) less than 25% efficacy or 50-fold less potency
relative to brimonidine at .alpha.-2A and .alpha.-2B; (4) less than
25% efficacy or 50-fold less potency relative to phenylephrine at
all .alpha.-1 receptors; and (5) the ability to produce peripheral
analgesia without concomitant sedation in genetically unaltered
animals.
[0083] Agonist and antagonist activity, including selectivity and
specificity, can be characterized using any of a variety of routine
assays, including, without limitation, Receptor Selection and
Amplification Technology (RSAT) assays (Messier et al., Pharmacol.
Toxicol. 76:308-11 (1995); Conklin et al., Nature 363:274-6
(1993)); cyclic AMP assays (Shimizu et al., J. Neurochem.
16:1609-1619 (1969)); and cytosensor microphysiometry assays (Neve
et al., J. Biol. Chem. 267:25748-25753 (1992)). Such assays
generally are performed using cells that naturally express only a
single .alpha.-adrenergic receptor subtype, or using transfected
cells expressing a single recombinant .alpha.-adrenergic receptor
subtype. The adrenergic receptor can be a human receptor or homolog
thereof having a similar pharmacology. As disclosed herein, RSAT
assays were performed using cells transiently transfected with
human .alpha.-2A (c10 gene); rat .alpha.-2B (RNG gene); human
.alpha.-2C (c4 gene); bovine .alpha.-1A; hamster .alpha.-1B; and
rat .alpha.-1D.
[0084] The RSAT assay measures receptor-mediated loss of contact
inhibition resulting in selective proliferation of
receptor-containing cells in a mixed population of confluent cells.
The increase in cell number is assessed with an appropriate
detectable marker gene such as .beta.-galactosidase, if desired, in
a high throughput or ultra high throughput assay format. Receptors
that activate the G protein, Gq, elicit the proliferative response.
.alpha.-adrenergic receptors, which normally couple to Gi, activate
the RSAT response when coexpressed with a hybrid Gq protein
containing a Gi receptor recognition domain, designated Gq/i5.
[0085] As an example, an RSAT assay can be performed essentially as
follows. NIH-3T3 cells are plated at a density of 2.times.10.sup.6
cells in 15 cm dishes and maintained in Dulbecco's modified Eagle's
medium supplemented with 10% calf serum. One day later, cells are
cotransfected by calcium phosphate precipitation with mammalian
expression plasmids encoding p-SV-.beta.-galactosidase (5-10
.mu.g), receptor (1-2 .mu.g) and G protein (1-2 .mu.g). Carrier
DNA, for example 40 .mu.g salmon sperm DNA, also can be included to
increase transfection efficiency. Fresh media is added on the
following day; one to two days later, cells are harvested and
frozen in 50 assay aliquots. Transfected cells are thawed, and 100
.mu.l of cells added to 100 .mu.l aliquots of compound to be
tested, with various concentrations assayed in triplicate, for
example, in 96-well plates. Incubation continues for 72 to 96 hours
at 37.degree.. After washing with phosphate-buffered saline,
.beta.-galactosidase activity is determined by adding 200 .mu.l of
chromogenic substrate (3.5 mM
O-nitrophenyl-.beta.-D-galactopyranoside/0.5% NP-40 in phosphate
buffered saline), incubating overnight at 300, and measuring
optical density at 420 nm. The absorbancy is a measure of enzyme
activity, which depends on cell number and reflects
receptor-mediated cell proliferation. The EC.sub.50 and maximal
effect (efficacy) of each drug at each receptor is determined.
[0086] The present invention further provides a method for the
long-term relief of chronic pain in a subject. The method is
practiced by activating in the subject an analgesic
.alpha.-adrenergic receptor in the absence of .alpha.-2A receptor
activation over a period of at least three days, such that relief
of chronic pain is maintained in the absence of continued receptor
activation. In one embodiment, a method of the invention is
practiced by administering to the subject a pharmaceutical
composition containing an effective amount of an .alpha.-adrenergic
agonist with minimal .alpha.-2A agonist activity over a period of
at least three days, such that relief of chronic pain is maintained
in the absence of significant agonist levels in the subject. Relief
of chronic pain can be maintained, for example, for at least three
weeks in the absence of significant agonist levels in the subject.
It is understood that the methods of the invention can be used for
the long-term relief of any type of chronic pain. As non-limiting
examples, such methods can be used for the long-term relief of
neuropathic pain; visceral pain; post-operative pain; pain
resulting from cancer or cancer treatment; and inflammatory
pain.
[0087] Long-term chronic pain relief can be achieved according to a
method of the invention with any of a variety of .alpha.-adrenergic
agonists with minimal .alpha.-2A agonist activity. Long-term
chronic pain relief can be achieved, for example, using an
.alpha.-2B agonist with minimal .alpha.-2A agonist activity.
Exemplary .alpha.-2B agonists with minimal .alpha.-2A agonist
activity include, without limitation, thiones such as Compound 3
and Compound 11, and pharmaceutically acceptable salts, esters,
amides, sterioisomers and racemic mixtures thereof. In one
embodiment, such a thione .alpha.-2B agonist with minimal
.alpha.-2A agonist activity is the (-) enantiomer of Compound 3, or
a pharmaceutically acceptable salt or ester thereof. Exemplary
.alpha.-2B agonists with minimal .alpha.-2A agonist activity
further include, without limitation, imidazolones such as Compound
4 or a pharmaceutically acceptable salt, ester, amide, sterioisomer
or racemic mixture thereof. In one embodiment, such a imidazolone
.alpha.-2B agonist with minimal .alpha.-2A agonist activity is the
(+) enantiomer of Compound 4, or a pharmaceutically acceptable salt
or ester thereof. Exemplary .alpha.-2B agonists with minimal
.alpha.-2A agonist activity also include, without limitation,
Compound 5, Compound 6, Compound 7, Compound 8 and Compound 9, and
pharmaceutically acceptable salts, esters, amides, sterioisomers
and racemic mixtures thereof. In the methods of the invention, an
.alpha.-adrenergic agonist with minimal .alpha.-2A agonist activity
can be administered by any of a variety of routes including, but
not limited to, a route of peripheral administration such as oral
administration or administration via subcutaneous minipump.
[0088] The present invention further provides a method for the
long-term relief of chronic pain in a subject by administering to
the subject a pharmaceutical composition containing an effective
amount of an .alpha.-adrenergic agonist and a pharmaceutical
composition containing an effective amount of a selective
.alpha.-2A antagonist over a period of at least three days, such
that relief of chronic pain is maintained in the absence of
significant agonist levels in the subject. Chronic pain relief can
be maintained, for example, for at least three weeks in the absence
of significant agonist levels in the subject. A variety of
.alpha.-adrenergic agonists are useful in the invention including
clonidine, brimonidine, tizanidine, dexemedetomidine,
norepinephrine and other pan-.alpha.-2 agonists and pan-.alpha.-1
pan-.alpha.-2 agonists as well as Compound 1 or Compound 2, and
pharmaceutically acceptable salts, esters, amides, sterioisomers
and racemic mixtures thereof. Similarly, a variety of selective
.alpha.-2A antagonists are useful in long-term relief of chronic
pain including, without limitation, Compound 13 and
pharmaceutically acceptable salts, esters, amides, sterioisomers
and racemic mixtures thereof. It is understood that various routes
of administration are useful for delivering pharmaceutical
compositions for the long-term relief of chronic pain as discussed
further below. Such routes of administration encompass, but are not
limited to, peripheral administration, for example, oral
administration or administration via subcutaneous minipump.
[0089] The methods of the invention provide long-term relief of
chronic pain. As used herein," the term "long-term relief" means a
significant reduction in pain that lasts for at least ten days
following last administration of the pharmaceutical composition. In
particular embodiments, the relief lasts for at least fourteen
days, at least 21 days, at least 28 days, at least 60 days, or at
least 90 days following last administration of the pharmaceutical
composition. In further embodiments, the invention provides a
method for the long-term relief of chronic pain in which the pain
is reduced by at least 50% for at least ten days, at least fourteen
days, at least 21 days, at least 28 days, at least 60 days, or at
least 90 days following last administration of the pharmaceutical
composition. In additional embodiments, the invention provides a
method for the long-term relief of chronic pain in which the pain
is reduced by at least 80% for at least ten days, at least fourteen
days, at least 21 days, at least 28 days, at least 60 days, or at
least 90 days following last administration of the pharmaceutical
composition. In other embodiments, the invention provides a method
for the long-term relief of chronic pain in which the pain is
reduced by at least 90% for at least ten days, at least fourteen
days, at least 21 days, at least 28 days, at least 60 days, or at
least 90 days following last administration of the pharmaceutical
composition.
[0090] Methods for the long-term relief of chronic pain are
practiced by administering an .alpha.-adrenergic agonist with
minimal .alpha.-2A activity over a period of at least three days.
The agonist can be administered by repeated dosing or continuous
dosing over a period of at least three days, for example, over
three days, four days, five days, six days, seven days, eight days,
nine days or ten days. As non-limiting examples, the
.alpha.-adrenergic agonist with minimal .alpha.-2A activity can be
administered three times a day for three days, or three times a day
for four days, for example, orally three times a day for three
days, or orally three times a day for four days. As further
examples, the .alpha.-adrenergic agonist with minimal .alpha.-2A
activity can be administered continuously, for example,
intravenously, via implanted infusion minipump or using an extended
release formulation for three days, four days, five days, six days
or seven days.
[0091] It is understood that slow-release formulations can be
useful in the methods of the invention for the long-term relief of
chronic pain. It is further understood that, where repeated
administration is used, the frequency of administration depends, in
part, on the half-life of the agonist. If desired, a method of the
invention can be practiced by administering a single dose, or just
two or three doses, of an agonist with a long half-life, for
example, a half-life of at least 24 hours, 36 hours, 48 hours, or
72 hours.
[0092] It is understood that different means of drug delivery can
be combined in a method of the invention. As an example, continuous
intravenous administration on the first day can be combined with
repeated oral dosing on the second and third days to activate an
analgesic .alpha.-adrenergic receptor in the absence of .alpha.-2A
receptor activation over a period of at least three days, such that
relief of chronic pain is maintained in the absence of significant
agonist levels in said subject. It is understood that the frequency
and duration of dosing will be dependent, in part, on the relief
desired and the half-life of the agonist, and that a variety of
routes of administration are useful in the methods of the
invention, as detailed further hereinbelow.
[0093] Also encompassed by the invention are pharmaceutically
acceptable salts, esters and amides derived from the formula
representing the specified agonist or antagonist. Suitable
pharmaceutically acceptable salts of the agonists and antagonists
useful in the invention include, without limitation, acid addition
salts, which can be formed, for example, by mixing a solution of
the agonist or antagonist with a solution of an appropriate acid
such as hydrochloric acid, sulfuric acid, fumaric acid, maleic
acid, succinic acid, acetic acid, benzoic acid, citric acid,
tartaric acid, carbonic acid or phosphoric acid. Where an agonist
or antagonist carries an acidic moiety, suitable pharmaceutically
acceptable salts thereof can include alkali salts such as sodium or
potassium salts; alkaline earth salts such as calcium or magnesium
salts; and salts formed with suitable organic ligands, for example,
quaternary ammonium salts. Representative pharmaceutically
acceptable salts include, yet are not limited to, acetate,
benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate,
borate, bromide, calcium edetate, camsylate, carbonate, chloride,
clavulanate, citrate, dihydrochloride, edetate, edisylate,
estolate, esylate, fumarate, gluceptate, gluconate, glutamate,
glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide,
hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate,
lactobionate, laurate, malate, maleate, mandelate, mesylate,
methylbromide, methylnitrate, methylsulfate, mucate, napsylate,
nitrate, N-methylglucamine ammonium salt, oleate, pamoate
(embonate), palmitate, pantothenate, phosphate/diphosphate,
polygalacturonate, salicylate, stearate, sulfate, subacetate,
succinate, tannate, tartrate, teoclate, tosylate, triethiodide and
valerate.
[0094] The functional groups of agonists and antagonists useful in
the invention can be modified to enhance the pharmacological
utility of the compound. Such modifications are well within the
knowledge of the skilled chemist and include, without limitation,
esters, amides, ethers, N-oxides, and pro-drugs of the indicated
agonist or antagonist. Examples of modifications that can enhance
the activity of an agonist or antagonist include, for example,
esterification such as the formation of C.sub.1 to C.sub.6 alkyl
esters, preferably C.sub.1 to C.sub.4 alkyl esters, wherein the
alkyl group is a straight or branched chain. Other acceptable
esters include, for example, C.sub.5 to C.sub.7 cycloalkyl esters
and arylalkyl esters such as benzyl esters. Such esters can be
prepared from the compounds described herein using conventional
methods well known in the art of organic chemistry.
[0095] Other pharmaceutically acceptable modifications include the
formation of amides. Useful amide modifications include, for
example, those derived from ammonia; primary C.sub.1 to C.sub.6
dialkyl amines, where the alkyl groups are straight or branched
chain; and arylamines having various substitutions. In the case of
secondary amines, the amine also can be in the form of a 5 or 6
membered ring. Methods for preparing these and other amides are
well known in the art.
[0096] It is understood that, where an agonist or antagonist useful
in the invention has at least one chiral center, the agonist or
antagonist can exist as chemically distinct enantiomers. In
addition, where an agonist or antagonist has two or more chiral
centers, the compound exists as diastereomers. All such isomers and
mixtures thereof are encompassed within the scope of the indicated
agonist or antagonist. Similarly, where an agonist or antagonist
possesses a structural arrangement that permits the structure to
exist as tautomers, such tautomers are encompassed within the scope
of the indicated agonist or antagonist. Furthermore, in crystalline
form, an agonist or antagonist may exist as polymorphs; in the
presence of a solvent, an agonist may form a solvate, for example,
with water or a common organic solvent. Such polymorphs, hydrates
and other solvates also are encompassed within the scope of the
indicated agonist or antagonist as defined herein.
[0097] An agonist or antagonist useful in the invention generally
is administered in a pharmaceutical composition. If desired, the
composition may, in some cases, be administered in conjunction with
one or more other therapeutic or analgesic substances, in the same
or different pharmaceutical compositions and by the same or
different routes of administration. As one example, an an
.alpha.-adrenergic agonist with minimal .alpha.-2A agonist activity
can be administered together with an analgesic agent such as
gabapentin. As another example, an .alpha.-adrenergic agonist with
minimal .alpha.-2A agonist activity can be administered together
with one or more cancer chemotherapeutic agents in an intravenous
cocktail.
[0098] A pharmaceutical composition useful in the invention
includes the active agonist or antagonist and further can include,
if desired an excipient such as a pharmaceutically acceptable
carrier or a diluent, which is any carrier or diluent that has
substantially no long term or permanent detrimental effect when
administered to a subject. Such an excipient generally is mixed
with active compound, or permitted to dilute or enclose the active
compound. A carrier can be a solid, semi-solid, or liquid agent
that acts as an excipient or vehicle for the active compound.
Examples of pharmaceutically acceptable carriers and diluents
include, without limitation, water, such as distilled or deionized
water; saline; and other aqueous media. It is understood that the
active ingredients can be soluble or can be delivered as a
suspension in the desired carrier or diluent.
[0099] A pharmaceutical composition further can include, if
desired, one or more agents such as emulsifying agents, wetting
agents, sweetening or flavoring agents, tonicity adjusters,
preservatives, buffers or anti-oxidants. Tonicity adjustors useful
in a pharmaceutical composition include salts such as sodium
chloride, potassium chloride, mannitol or glycerin and other
pharmaceutically acceptable tonicity adjustors. Preservatives
useful in the pharmaceutical compositions of the invention include,
without limitation, benzalkonium chloride, chlorobutanol,
thimerosal, phenylmercuric acetate, and phenylmercuric nitrate.
Various buffers and means for adjusting pH can be used to prepare a
pharmaceutical composition, including, but not limited to, acetate
buffers, citrate buffers, phosphate buffers and borate buffers.
Similarly, anti-oxidants useful in the pharmaceutical compositions
of the invention are well known in the art and include, for
example, sodium metabisulfite, sodium thiosulfate, acetylcysteine,
butylated hydroxyanisole and butylated hydroxytoluene. It is
understood that these and other substances known in the art of
pharmacology can be included in a pharmaceutical composition useful
in the invention.
[0100] The agonists and antagonists of the invention are
administered in effective amounts. Such an effective amount
generally is the minimum dose necessary to achieve the desired
therapeutic effect, which can be, for example, that amount roughly
necessary to reduce the discomfort caused by the pain to tolerable
levels. Such a dose generally is in the range of 0.1-1000 mg/day
and can be, for example, in the range of 0.1-500 mg/day, 0.5-500
mg/day, 0.5-100 mg/day, 0.5-50 mg/day, 0.5-20 mg/day, 0.5-10 mg/day
or 0.5-5 mg/day, with the actual amount to be administered
determined by a physician taking into account the relevant
circumstances including the severity of the pain, the age and
weight of the patient, the patient's general physical condition,
the cause of pain and the route of administration. Suppositories
and extended release formulations can be useful in the invention
and include, for example, dermal patches, formulations for deposit
on or under the skin and formulations for intramuscular
injection.
[0101] A pharmaceutical composition useful in the methods of the
invention can be administered to a subject by a variety of means
depending, for example, on the type of pain to be treated, the
agonist or antagonist to be included in the composition, and the
history, risk factors and symptoms of the subject. Routes of
administration suitable for the methods of the invention include
both systemic and local administration. As non-limiting examples, a
pharmaceutical composition useful for alleviating pain or for the
long-term relief of chronic pain can be administered orally or by
subcutaneous pump; by dermal patch; by intravenous, subcutaneous or
intramuscular injection; by topical drops, creams, gels or
ointments; as an implanted or injected extended release
formulation; by subcutaneous minipump or other implanted device; by
intrathecal pump or injection; or by epidural injection.
[0102] In particular embodiments, a method of the invention is
practiced by peripheral administration of a pharmaceutical
composition containing an agonist, a pharmaceutical composition
containing an antagonist, or both. As an example, one or both of a
pharmaceutical composition containing an .alpha.-adrenergic agonist
and a pharmaceutical composition containing a selective .alpha.-2A
antagonist can be administered peripherally. As used herein, the
term "peripheral administration" or "administered peripherally"
means introducing an agent into a subject outside of the central
nervous system. Peripheral administration encompasses any route of
administration other than direct administration to the spine or
brain. As such, it is clear that intrathecal and epidural
administration as well as cranial injection or implantation are not
within the scope of the term "peripheral administration" or
"administered peripherally." It further is clear that some
analgesic agents can cross the blood-brain barrier and, thus,
become distributed throughout the central and peripheral nervous
systems following peripheral administration.
[0103] Peripheral administration can be local or systemic. Local
administration results in significantly more of a pharmaceutical
composition being delivered to and about the site of local
administration than to regions distal to the site of
administration. Systemic administration results in delivery of a
pharmaceutical composition to essentially the entire peripheral
nervous system of the subject and may also result in delivery to
the central nervous system depending on the properties of the
composition.
[0104] Routes of peripheral administration useful in the methods of
the invention encompass, without limitation, oral administration,
topical administration, intravenous or other injection, and
implanted minipumps or other extended release devices or
formulations. A pharmaceutical composition useful in the invention
can be peripherally administered, for example, orally in any
acceptable form such as in a tablet, liquid, capsule, powder, or
the like; by intravenous, intraperitoneal, intramuscular,
subcutaneous or parenteral injection; by transdermal diffusion or
electrophoresis; topically in any acceptable form such as in drops,
creams, gels or ointments; and by minipump or other implanted
extended release device or formulation.
[0105] In some embodiments, the invention is practiced by
administering both a pharmaceutical composition containing an
effective amount of an .alpha.-adrenergic agonist and a
pharmaceutical composition containing an effective amount of a
selective .alpha.-2A antagonist to a subject. In such "combination"
therapy, it is understood that the agonist and antagonist can be
delivered independently or simultaneously, in the same or different
pharmaceutical compositions, and by the same or different routes of
administration. As an example, both agonist and antagonist can be
administered orally, with the agonist given twice daily and the
antagonist given once daily. As another example, the agonist can be
administered epidurally while the antagonist is administered
orally. As a further example, the agonist and antagonist can be
administered together in an intravenous "cocktail."
[0106] The present invention also provides a method of screening
for effective agents that produce peripheral analgesia without
concomitant sedation by contacting an .alpha.-2A receptor with an
.alpha.-adrenergic agonist having analgesic activity; and
determining whether the agonist has .alpha.-2A agonist activity,
where the absence of .alpha.-2A agonist activity indicates that the
.alpha.-adrenergic agonist having analgesic activity is an
effective agent that produces peripheral analgesia without
concomitant sedation.
[0107] Further provided herein is a method of screening for
effective agents that produce peripheral analgesia without
concomitant sedation by contacting an .alpha.-2A receptor with an
agent; determining whether the agent has .alpha.-2A agonist
activity; contacting an .alpha.-2B receptor with the agent; and
determining whether the agent has .alpha.-2B agonist activity,
where the absence of .alpha.-2A agonist activity and the presence
of .alpha.-2B agonist activity indicate that the agent is an
effective agent that produces peripheral analgesia without
concomitant sedation.
[0108] The invention also provides a method of screening for
effective agents that produce peripheral analgesia without
concomitant sedation by peripherally administering an
.alpha.-adrenergic agonist to a control animal having at least wild
type levels of .alpha.-2A receptor activity; assaying for analgesia
in the control animal; peripherally administering to a
corresponding animal having reduced levels of .alpha.-2A receptor
expression or activity an amount of the .alpha.-adrenergic agonist
similar or greater than the amount administered to the control
animal; and assaying for analgesia in the corresponding animal
having reduced levels of .alpha.-2A receptor expression or
activity, where the absence of analgesia in the control animal and
the presence of analgesia in the corresponding animal having
reduced levels of .alpha.-2A receptor expression or activity
indicate that the .alpha.-adrenergic agonist has excessive
.alpha.-2A agonist activity, and where the presence of analgesia in
the control animal and the presence of analgesia in said
corresponding animal having reduced levels of .alpha.-2A receptor
expression or activity indicate that the .alpha.-adrenergic agonist
is an effective agent that produces peripheral analgesia without
concomitant sedation. In such a method of the invention, the
control animal can be, for example, wild type at both .alpha.-2A
receptor loci. In one embodiment, the control animal is a wild type
animal such as a wild type mouse. A variety of corresponding
animals are useful in a screening method of the invention. In one
embodiment, the invention is practiced with a corresponding animal
having a homozygous point mutation at the .alpha.-2A receptor
locus. In another embodiment, the invention is practiced with a
corresponding animal having a point mutation within the .alpha.-2A
receptor coding sequence. Such a point mutation can occur, for
example, at a residue analogous to Asp79 and can be, for example,
an Asp79 to Asn mutation. In a further embodiment, the invention is
practiced with a corresponding animal having a homozygous
.alpha.-2A knockout mutation. It is understood that a variety of
methodologies can be used to assay for analgesia in the methods of
the invention, including, but not limited to, assaying for
analgesia following sulprostone sensitization.
[0109] If desired, a method of the invention for screening for
effective agents that produce peripheral analgesia without
concomitant sedation can be practiced by (a) peripherally
administering an .alpha.-adrenergic agonist to a control animal
having at least wild type levels of .alpha.-2A and .alpha.-2B
receptor activity; (b) assaying for analgesia in the control
animal; (d) peripherally administering to a corresponding animal
having reduced levels of .alpha.-2A receptor expression or activity
an amount of the .alpha.-adrenergic agonist similar or greater than
the amount administered to the control animal; (d) assaying for
analgesia in the corresponding animal having reduced levels of
.alpha.-2A receptor expression or activity; (e) peripherally
administering the .alpha.-adrenergic agonist to a corresponding
animal having reduced levels of .alpha.-2B receptor expression or
activity; and (f) assaying for analgesia in the corresponding
animal having reduced levels of .alpha.-2B receptor expression or
activity, where the absence of analgesia in the control animal and
the presence of analgesia in the corresponding animal having
reduced levels of .alpha.-2A receptor expression or activity
indicate that the .alpha.-adrenergic agonist has excessive
.alpha.-2A agonist activity, and where the presence of analgesia in
the control animal, the presence of analgesia in said corresponding
animal having reduced levels of .alpha.-2A receptor expression or
activity, and the absence of analgesia in the corresponding animal
having reduced levels of .alpha.-2B receptor expression or activity
indicate that the .alpha.-adrenergic agonist is an effective agent
that produces peripheral analgesia without concomitant
sedation.
[0110] The present invention additionally provides a method of
screening for effective agents that produce peripheral analgesia
without concomitant sedation by peripherally administering an
.alpha.-adrenergic agonist to a control animal having at least wild
type levels of .alpha.-2B receptor activity; assaying for analgesia
in the control animal; peripherally administering the
.alpha.-adrenergic agonist to a corresponding animal having reduced
levels of .alpha.-2B receptor expression or activity; and assaying
for analgesia in the corresponding animal having reduced levels of
.alpha.-2B receptor expression or activity, where the presence of
analgesia in the control animal and the absence of analgesia in the
corresponding animal having reduced levels of .alpha.-2B receptor
expression or activity indicate that the .alpha.-adrenergic agonist
is an effective agent that produces peripheral analgesia without
concomitant sedation.
[0111] Such a method of the invention can be practiced with a
variety of control animals, for example, a control animal which is
wild type at both .alpha.-2B receptor loci. In one embodiment, the
control animal is a wild type animal. In a further embodiment, the
control animal is a wild type mouse. Similarly, a variety of
corresponding animals are useful in the screening methods of the
invention, including corresponding animals which have a
heterozygous .alpha.-2B knockout mutation or a homozygous
.alpha.-2B knockout mutation. Analgesia can be assayed using any of
a variety of methodologies. In one embodiment, analgesia is assayed
following sulprostone sensitization.
[0112] The term "control animal," as used herein, means any animal
capable of experiencing pain. A control animal can, for example,
express wild type levels of endogenous .alpha.-2A receptor or can
express an .alpha.-2A receptor transgene in addition to endogenous
.alpha.-2A. Where a control animal is "wild type" at both
.alpha.-2A receptor loci, the animal has .alpha.-2A receptor
sequences that are naturally occurring and expressed at levels
normally found in nature. Similarly, where a control animal is
"wild type" at both .alpha.-2B receptor loci, the animal has
.alpha.-2B receptor sequences that are naturally occurring and
expressed at levels normally found in nature.
[0113] As used herein, the term "wild type animal" means an animal
such as a mouse or rat that is unaltered through molecular
genetics. A "wild type animal" can be a wild animal found in nature
or can be an inbred animal or other laboratory strain with a well
characterized genetic system.
[0114] The term "corresponding animal," as used herein, means an
animal of the same species as the control animal, which has reduced
levels of .alpha.-2A receptor expression or activity or which has
reduced levels of .alpha.-2B receptor expression or activity. A
corresponding animal generally is genetically identical to the
control animal except at one or more .alpha.-2A receptor loci or at
one or more .alpha.-2B receptor loci. As non-limiting examples, a
corresponding animal can entirely lack one or both .alpha.-2A genes
or one or both .alpha.-2B genes, have a deletion, insertion or
point mutation in the. .alpha.-2A or .alpha.-0.2B receptor coding
sequence, a deletion, insertion or point mutation in the 5' or 3'
regulatory sequence that reduces or eliminates .alpha.-2A or
.alpha.-2B expression, or express wild type levels of .alpha.-2A
receptor or .alpha.-2B receptor with reduced activity, such as a
murine .alpha.-2A receptor having an Asp79 to Asn mutation, which
abolishes about 80% of receptor activity. Thus, in one embodiment,
a screening method of the invention is practiced with a
corresponding animal having reduced levels of .alpha.-2A receptor
expression or activity due to a homozygous point mutation at a
residue analogous to Asp79; such an "analogous" residue is that
asparagine residue occurring in the same relative position in a
homolog of the murine .alpha.-2A receptor. .alpha.-2A and
.alpha.-2B knockout mouse strains as well as strains bearing
homozygous point mutations resulting in reduced levels of
.alpha.-2A receptor expression or activity are well known in the
art or can be prepared by standard methods (Hein et al., Ann. NY
Acad. Science 881:265-271 (1999); and Kable et al., J. Pharm.
Exper. Therapeutics 293:1-7 (2000)).
[0115] A screening method of the invention relies, in part, on
peripherally administering an .alpha.-adrenergic agonist to a
control animal and to a corresponding animal with reduced levels of
.alpha.-2A receptor expression or activity or a corresponding
animal with reduced levels of .alpha.-2B receptor expression or
activity, or both corresponding animals. It is understood that
administration to the control animal and administration to the one
or more corresponding animals can be performed simultaneously or in
any order. Similarly, it is understood that the two or more steps
in which analgesia is assayed can be performed simultaneously or in
any order. Assays for analgesic activity can be performed using any
established or reproducible pain model, including, but not limited
to, those exemplified herein. It is understood that analgesic
activity in the control and corresponding animals is assayed under
the same or similar conditions.
[0116] The following examples are intended to illustrate but not
limit the present invention.
EXAMPLE I
Preparation of .alpha.-Adrenergic Agonist and Antagonist
[0117] This example describes synthesis of various
.alpha.-adrenergic agonist, including .alpha.-adrenergic agonist
with minimal .alpha.-2A agonist activity such as .alpha.-2B agonist
with minimal .alpha.-2A agonist activity, and synthesis of a
selective .alpha.-2A antagonist. 15161718192021
[0118] 2-Bromo-5-fluorobenzyl bromide (3.0 g, 11 mmol, 1.0 equiv)
was dissolved in dimethyl sulfoxide (100 mL) at ambient
temperature. Sodium azide (2.8 g, 44 mmol, 4.0 equiv) was added to
the solution and the reaction mixture heated at reflux for one day.
The solution was cooled, quenched with water, and then extracted
with ethyl acetate. The organic extracts were combined, washed with
brine, dried over sodium sulfate and concentrated in vacuo.
Purification by column chromatography (10:1 hexanes/ethyl acetate)
afforded the azide (0.85 g, 34%). The azide (0.85 g, 3.7 mmol, 1.0
equiv) was dissolved in tetrahydrofuran (25 mL) and water (5.0 mL).
KOH (0.20 g, 3.6 mmol, 0.97 equiv) was added to the solution
followed by triphenylphosphine (1.1 g, 4.4 mmol, 1.2 equiv). The
reaction mixture was stirred overnight at ambient temperature. The
reaction was quenched with hydrochloric acid (conc.) and extracted
with ethyl acetate. The aqueous layers were combined and made basic
with sodium hydroxide (pellet) until pH reached .about.14. The
aqueous layer was extracted with ethyl acetate, the organic layers
combined, washed with brine, dried over sodium sulfate and
concentrated in vacuo to afford the crude amine (0.35 g, 46%) as an
orange oil. 22
[0119] 2-Bromo-5-fluorobenzyl amine (0.35 g, 1.7 mmol, 1.0 equiv)
was dissolved in tetrahydrofuran (20 mL) and stirred overnight at
ambient temperature. The solvent was removed in vacuo and water (30
mL) was used to dissolve the concentrate. The solution was heated
at reflux overnight and then cooled to ambient temperature. Sodium
hydroxide (pellet) was added to the solution until the pH was
.about.14. The solution was extracted with ethyl acetate, the
organic extracts combined, washed with brine and dried over sodium
sulfate. The solution was concentrated in vacuo. The concentrate
was purified by column chromatography (30:1 chloroform/methanol
sat'd with ammonia). The product was isolated as a white solid. H
NMR (300 MHz, CDCl3 w/TMS): 57.46-7.50 (m, 1H), 7.16-7.19 (m, 1H),
6.83-6.90 (m, 1H), 4.43 (s, 2H), 4.31 (t, 2H, J=8.5 Hz), 3.79 (t,
2H, J=8.8 Hz
Example II
Peripheral Treatment of pain in .alpha.2-a Receptor Knock-Out Mice
Using .alpha.-Adrenergic Agonists
[0120] This example demonstrates that .alpha.-adrenergic agonists
are effective analgesic agents when administered peripherally in
the absence of .alpha.-2A receptor activation.
[0121] A. Peripherally Administered .alpha.-Adrenergic Agonists are
Effective Analgesic Agents in .alpha.-2A Knockout Mice
[0122] Non-specific .alpha.-adrenergic agonists were assayed in
.alpha.-2A receptor deficient ("knockout") mice (Hein et al.,
supra, 1999) using a mouse model of sulprostone sensitized pain in
which allodynia is evoked by intrathecal administration of a
selective prostaglandin E2 receptor agonist to conscious mice
essentially as described in Minami et al., Pain 57:217-223 (1994).
In this model, the pain response to stroking the flank with a paint
brush is scored eight times over a 35 minute period starting 15
minutes following spinal administration of sulprostone and
intrathecal or intraperitoneal administration of drug or control
vehicle. Sulprostone elicits a "pain" score of 12-13 on a 16-point
scale.
[0123] Consistent with previous clinical data, intrathecal
injection of clonidine, brimonidine (UK14304) or the charged
.alpha.-adrenergic agonist Compound 1 produced significant
analgesia separable from sedation in wild type mice. Furthermore,
intrathecal injection of clonidine, brimonidine or Compound 1 into
.alpha.-2A knockout mice did not produce analgesia, consistent with
the therapeutic target of spinally administered .alpha.-adrenergic
agonists being an .alpha.-2A receptor expressed in the dorsal horn
of the spinal cord. FIGS. 1A and 1B show the results obtained with
intrathecal administration of 1 .mu.g Compound 1 in wild type and
.alpha.-2A knockout mice. These results indicate that centrally
mediated analgesia by non-selective .alpha.-adrenergic agonists
requires .alpha.-2A receptor.
[0124] In contrast to the results obtained with intrathecal
injection, intraperitoneal injection of 30 .mu.g/kg Compound 1 as
well as clonidine or brimonidine failed to produce analgesia
separable from sedation in wild type mice. However, in .alpha.-2A
knockout mice, peripheral (intraperitoneal) treatment with
clonidine, UK14304 or Compound 1 resulted in significant analgesia
without sedation. See FIGS. 1C and 1D, which show that, upon
peripheral administration, Compound 1 only produced analgesia in
.alpha.-2A knockout mice but not in wild type animals. These
results indicate that .alpha.-adrenergic agonists can produce
analgesia without significant sedation when administered
peripherally in the absence of .alpha.-2A receptor activation.
[0125] Wild type and .alpha.-2A knockout mice also were treated
with 100 .mu.g/kg of the .alpha.-adrenergic agonist, Compound 2,
which, unlike clonidine, brimonidine, Compound 1 and PAC, has very
little .alpha.-1 activity and only weakly activates the .alpha.-2A
receptor with a relative efficacy of 40% relative to brimonidine in
in vitro assays. Like clonidine, Compound 2 readily crosses the
blood-brain barrier. In wild type mice, Compound 2 was not
analgesic when administered intraperitoneally, but was fully
analgesic in .alpha.-2A knockout mice when administered by the same
route (see FIG. 2). These results indicate that a variety of
.alpha.-adrenergic agonists with differing .alpha.-1 and .alpha.-2
receptor activity profiles and bioavailablility can be effective
peripheral analgesic agents when .alpha.-2A receptor activation is
prevented.
[0126] As disclosed above, activation of an .alpha.-2A receptor by
.alpha.-adrenergic agonists masks the peripheral analgesic activity
of these molecules. To test whether the masking effect was
dependent on a spinal or peripherally localized .alpha.-2A
receptor, mice were injected intrathecally or intraperitoneally
with Compound 1, a highly charged .alpha.-adrenergic agonist that
does not readily cross the blood-brain barrier. As indicated above,
intrathecal, but not intraperitoneal, injection produced
significant analgesia in wild type mice while the converse was true
in .alpha.-2A knockout mice: intraperitoneal, but not intrathecal,
injection resulted in analgesia. Similarly, another highly charged
.alpha.-adrenergic agonist that does not readily cross the
blood-brain barrier, para-amino clonidine (PAC), also was analgesic
at 100 .mu.g/kg in .alpha.-2A knockout mice when administered
peripherally. These results indicate that a novel analgesic
activity of non-selective .alpha.-adrenergic agonists such as
pan-agonists is unmasked by preventing activation of a peripheral
.alpha.-2A receptor.
[0127] Assays for allodynia in wild type and .alpha.-2A knockout
mice were performed essentially according to the method of Yaksh
and Harty, J. Pharmacology Exp. Ther. 244: 501-507 (1998). In
brief, mice were divided into groups of 5-6 animals. Control mice
were administered 5 ul DMSO while treated animals were injected
with 5 ul DMSO containing various doses of the indicated agent.
After intrathecal injection, each mouse was placed in an individual
13.times.8.5.times.13 cm Plexiglass enclosure with wood chips on
the floor for observation. Allodynia was assessed once every five
minutes over a 50 minute time period, with response recorded eight
times in the 15 to 50 minute time frame. Allodynia was assessed by
light stroking of the flank of the mice with a small paintbrush and
ranked as follows: 0, no response; 1, mild squeaking with attempts
to move away from the stroking probe; and 2, vigorous squeaking
evoked by the stroking probe, biting at the probe and strong
efforts to escape. The eight scores for each animal were added
together, and the mean for the group determined to give an average
total score.
[0128] B. A Peripheral .alpha.-2 Receptor Mediates Analgesia in
.alpha.-2A Knockout Mice
[0129] The non-selective .alpha.-2 antagonist, rauwolscine, was
assayed for its ability to affect peripheral analgesia produced by
para-amino-clonidine (PAC) in .alpha.-2A knockout mice.
Sulprostone-sensitized .alpha.-2A knockout mice were treated with
PAC, delivered at 100 .mu.g/kg by intraperitoneal injection. As
described above, PAC induced significant analgesia in the
.alpha.-2A knockout mice. The analgesic effect of PAC was blocked,
however, by intraperitoneal injection of rauwolscine (0.3 .mu.g/kg)
as shown in FIG. 3. These results indicate that a peripheral
.alpha.-2 receptor mediates analgesia in .alpha.-2A knockout
mice.
[0130] Sedative effects were analyzed by assessing exploratory
behavior in a darkened chamber as follows. Mice were weighed, and
test compound administered by intrathecal injection in a volume of
5 .mu.l or by intraperitoneal injection in 1 ml/kg volume at the
indicated dose. At predetermined time points corresponding with
analgesia measurements 5 to 30 minutes following the injection, the
animal's activity was determined automatically by placing the mouse
in a digicom analyzer chamber (Omnitech Electronic; Columbus,
Ohio). The digicom analyzer chamber contains photocell beams
criss-crossing the box that are broken as the animal moves around;
the chamber was modified for mice by raising the level of the
floor. Computer analysis of total animal movement proceeded over a
5-minute time period. Any given animal was used at most twice for
this protocol, as learned behavior can affect the data. All animals
received at least two weeks rest between studies.
EXAMPLE III
Relief of Pain by Peripheral Administration of .alpha.-Adrenergic
Agonists in Genetically Unaltered Animals
[0131] This example demonstrates that .alpha.-adrenergic agonists
can be peripherally administered to produce a significant analgesic
effect with less than a 20% reduction in motor or muscular activity
in animals having wild type .alpha.-adrenergic receptors.
[0132] A. Selective .alpha.-2A Antagonists can be Used in
Combination with Peripherally Administered .alpha.-Adrenergic
Agonists to Produce Analgesia
[0133] The Chung rat nerve ligation model is a well accepted model
of peripheral neuropathic pain. In the Chung model, partial
ligation of left spinal nerves L-5 and L-6 produces a long-lasting
hypersensitivity to light touch on the affected left foot. The
hypersensitivity is similar to pain experienced by humans with the
neuropathic condition of causalgia (Kim and Chung, Pain 50:355-363
(1992)).
[0134] When administered by intrathecal injection to Chung rats,
the pan-.alpha.-2 agonist clonidine produced significant analgesia
separable from sedation, while intraperitoneal administration only
produced unremarkable analgesia in the absence of sedative effects.
Compounds that do not readily cross the blood-brain barrier,
Compound 1 and PAC, also were assayed for activity in the Chung rat
model. As expected, neither compound was significantly analgesic at
non-sedating doses when administered peripherally.
[0135] Coadministration of clonidine with the selective .alpha.-2A
antagonist, Compound 13, shifted the analgesic dose response of
intraperitoneally administered clonidine to the left; when combined
with 0.3 .mu.g/kg Compound 13, only 30 .mu.g/kg was required to
give dramatic allodynia reversal (see FIG. 4A). This concentration
of clonidine reduces activity but was not nearly as sedating as the
100 .mu.g/kg dose that reverses allodynia in the absence of
Compound 13. Similarly, Compound 2 also was not analgesic when
given alone by peripheral administration at 100 .mu.g/kg to Chung
rats, yet produced significant analgesia when co-administered with
0.3 .mu.g/kg .alpha.-2A antagonist Compound 13 as shown in FIG. 4B;
no significant sedative effects were observed at this
concentration. Taken together, these results indicate that
selective .alpha.-2A antagonists can be administered together with
.alpha.-adrenergic agonists to unmask a novel, .alpha.-2A
receptor-independent, peripheral analgesic property of
.alpha.-adrenergic agonists.
[0136] .alpha.-2B/C selective agonists with some .alpha.-2A agonist
activity were unable to produce significant analgesia separable
from sedation when administered peripherally to Chung rats. As
disclosed above, the thione Compound 2 has slight .alpha.-2A
agonist activity, yet did not have analgesic activity when
administered peripherally in the absence of an .alpha.-2A
antagonist. This result indicates that peripheral analgesic
activity of .alpha.-adrenergic agonists is dependent upon an
extremely low level of .alpha.-2A receptor activation.
[0137] Chung rat surgeries of about 20 minutes duration were
performed essentially as follows. Male Sprague-Dawley rats weighing
100 to 120 grams were anesthetized with isofluorane. After shaving
and preparing the surgical site with betadine, an incision was made
from thoracic vertebra XIII down toward the sacrum, and muscle
separated from spinal vertebra (left side) at the L4-S2 levels. The
L6 vertebra were located, and the transverse process carefully
removed with a small rongeur to visually identify the L4-L6 spinal
nerves. L5 and L6 spinal nerves were isolated and tightly ligated
with 6-0 silk thread; the wound was sutured after complete
hemostasis was confirmed. A small amount of antibiotic ointment was
applied to the incised area before transferring the animals to a
plastic recovery cage under a regulated heat-temperature lamp.
Animals were not treated with any topical or local anesthetics
post-operatively.
[0138] Assessment of pain in Chung model rats was performed by
applying a light tactile stimulus (Von Frey hair) to the affected
surgical paw as follows. A 50% pain threshold was established by
applying the Von Frey hair in an up-and-down manner to the plantar
surface of the surgical paw with just enough force to bend them. A
positive response was recorded if the paw was withdrawn sharply.
The 50% paw withdrawal threshold was determined using the method of
Dixon et al., Ann. Rev. Pharmacol. Toxicol. 20:441-462 (1980). The
post-drug threshold was compared to the pre-drug threshold, and the
precent reversal of tactile sensitivity calculated based on a
normal threshold of 15.1 grams. The results were expressed as %
allodynia reversal, reflecting the percentage reversal of pain
threshold relative to a normal rat (100%).
[0139] Male Sprague-Dawley rats that were not subject to surgery
were used for assessment of sedative effects. Rats were weighed and
compound administered by intravenous or intraperitoneal injection
or given orally at the indicated dose. At a predetermined time
point 5 to 30 minutes following injection, the animal's activity
was determined automatically by placing the rat in a digicom
analyzer chamber (Omnitech Electronic). Total activity was analyzed
as described above.
[0140] B. Diverse Structural Classes of Peripherally Administered
.alpha.-2B/C-Selective Compounds with Minimal .alpha.-2A Activity
Relieve Pain
[0141] .alpha.-adrenergic agonists of diverse structural classes
with minimal .alpha.-2A agonist activity were administered
peripherally and assayed for the ability to relieve pain in the
Chung rat model. Table 2 shows the results obtained with the thione
Compound 3, the imidazolone Compound 4, the thiazole Compound 5,
the oxazole Compound 6, the thiourea Compound 7, and the
4-imidazole Compound 14. Each of these compounds, while
structurally diverse, are .alpha.2-B/C selective .alpha.-adrenergic
agonists having minimal .alpha.-2A agonist activity. As an example,
Compound 14 is an .alpha.-2B selective agonist having minimal
.alpha.-2A agonist activity yet having significant .alpha.-1
agonist activity.
[0142] As shown in Table 2, peripheral administration of each of
the diverse compounds produced analgesic activity at a dose that
did not reveal significant sedative effects. Conversely, diverse
.alpha.-adrenergic agonists having .alpha.-2A activity were assayed
and did not produce analgesia separable from sedation when
administered by intraperitoneal injection. These results
corroborate that diverse structural classes of .alpha.-adrenergic
agonists having minimal .alpha.-2A activity are characterized by
the ability to produce peripherally mediated analgesia without
concomitant sedation.
2TABLE 2 Peak Allodynia reversal in Chung rat model Peak allodynia
Sedative reversal effect COMPOUND (dose) (1 mg/kg) COMPOUND 3 81%
+/- 10% NS* (300 .mu.g/kg) COMPOUND 4 80% +/- 6.5% NS* (100
.mu.g/kg) COMPOUND 5 77% +/- 8.3% NS* ( 30 .mu.g/kg) COMPOUND 6 92%
+/- 5.2% NS* (100 .mu.g/kg) COMPOUND 7 60% +/- 8.0% NS* ( 3
.mu.g/kg) COMPOUND 14 77% +/- 6.2% NS* (100 .mu.g/kg) *No
significant effect.
[0143] A full comparison of the sedative and analgesic dose
response curves for clonidine and Compound 3 was performed using a
single intraperitoneal dose from 20 .mu.g/kg to 100 .mu.g/kg for
clonidine and 1 to 1000 .mu.g/kg for Compound 3. The percentage of
allodynia reversal and the reduction in total activity were
determined as described above. As shown in FIG. 4C, clonidine
sedation occurred at lower doses than the doses that produced
analgesia. In particular, clonidine was extremely sedating at the
100 .mu.g/kg dose that produced significant analgesia. These
results demonstrate that the analgesia resulting from peripheral
dosing with clonidine is not separable from sedation. In contrast,
the results shown in FIG. 4D demonstrate that Compound 3 produced
significant analgesia without producing sedation. Specifically,
sedation did not occur at doses 100-fold greater than those
producing a robust reversal of allodynia. These results demonstrate
that an .alpha.-2B adrenergic agonist with minimal .alpha.-2A
agonist activity can produce a 80% allodynia reversal without
concomitant sedation.
[0144] In sum, these results demonstrate that non-selective
.alpha.-adrenergic agonists with unremarkable analgesic activity
following systemic dosing can be turned into very effective agents
that produce peripheral analgesia without concomitant sedation when
combined with an agent that prevents activation of the .alpha.-2A
receptor. These results also demonstrate that the analgesic action
of selective .alpha.-2 agonists having only minimal .alpha.-2A
receptor agonist activity is distinct from the analgesic action of
.alpha.-adrenergic agents previously described.
EXAMPLE IV
Long-Term Relief of Chronic Pain
[0145] This example demonstrates that .alpha.-adrenergic agonists
with minimal .alpha.-2A activity can mediate a long-term reversal
of the chronic pain phenotype.
[0146] A. Long-Term Pain Relief in Chung Model Rats
[0147] Structurally distinct .alpha.-adrenergic agonists were
assayed for the ability to produce prolonged relief of chronic pain
in Chung model rats following extended dosing. In particular, Chung
model animals were dosed for seven days using a subcutaneous
osmotic minipump with vehicle control or 0.1 mg/kg/hour of the
following .alpha.-adrenergic agonist with minimal .alpha.2-A
activity: Compound 8, Compound 9, Compound 3 or Compound 4. Pain
relief was observed during the period of drug treatment; for
example, Compound 8 alleviated the allodynia 90-100%, and Compound
9 alleviated the allodynia 60-80%, as shown in FIG. 5A. Notably,
the analgesic effects of these compounds as well as Compound 3 and
Compound 4 continued for over a month after treatment was
concluded. Treated animals exhibited behavioral signs of being
cured, differing from untreated or vehicle-treated rats in that
they no longer guarded the surgical paw and placed this paw flat on
the bottom of their cage.
[0148] Prolonged pain relief also was observed following three days
of oral dosing. Chung model rats were administered three doses of
0.3 mg/kg Compound 8 by oral gavage between 8 a.m. and 6 p.m. for
three consecutive days. A 70-80% reversal of allodynia was
achieved, and allodynia did not increase during a more than three
week period of follow-up testing. These results indicate that, in
contrast to the relatively short duration of pain relief obtained
following a single intraperitoneal or oral dose of an .alpha.-2B/C
agonist such as Compound 8, a prolonged analgesic effect results
from repeated dosing with .alpha.-2B/C selective agonists with
minimal .alpha.2-A agonist activity.
[0149] Chung surgeries were performed, and pain assessed as
described above. Drugs administered via osmotic minipump were
delivered as follows. ALZET (Cupertino, Calif.) minipumps (Model
#1007D) were implanted subcutaneously on the rats' back between the
shoulder blades. Pumps were filled with test solution prior to
surgery, which was performed with aseptic procedures and sterilized
surgical instruments. After anesthetization with isoflorane and
midline incision between the shoulder blades through the muscle, a
subcutaneous pocket was exposed. The minipump was inserted into the
exposed pocket, before stapling the wounds closed and allowing
animals to recover under a warming light.
[0150] ALZET osmotic minipumps (model 1007D) hold a total volume of
100 .mu.l and delivered drug at the set rat of 0.5 .mu.l per hour
the indicated time period. Drugs were administered at the indicated
rate, typically 100 .mu.g/hr/kg, dissolved in 50% DMSO, with the
concentration of drug in the pump varied depending on animal
weight. Pumps were removed seven days following pump insertion.
[0151] B. Analgesic Effects Extend Beyond the Time Drug Persists in
the Plasma
[0152] To determine whether the persistent reversal of allodynia
was due to the continued presence of drug in the animals, rats were
treated for seven days using a subcutaneous osmotic minipump with
0.1 mg/kg/hour Compound 8. Plasma concentrations of Compound 8 were
sampled on days 3, 6, 8, 10 and 14 following pump insertion and
determined by liquid chromatography-mass spectrometry/mass
spectrometry (LC-MS/MS). As shown in FIG. 5, minimal drug levels
were detected on day 10, and no drug remained in the plasma by day
14. These results indicate that pain relief can be achieved
following extended dosing, even in the absence of plasma drug
levels.
[0153] C. Analgesia does not Require Receptor Activation Following
Extended Dosing
[0154] Chung model rats treated with Compound 8 for seven days by
osmotic minipump or for three days by oral gavage consistently
exhibited prolonged relief from allodynia. The .alpha.-2
antagonist, rauwolscine, was assayed for the ability to inhibit
this anti-allodynic action at various time points. Notably, as
shown in FIG. 6A, rauwolscine at 0.3 .mu.g/kg i.p. inhibited the
analgesic activity of Compound 8 when injected on the third day of
treatment but not on the fourth day.
[0155] Similarly, oral dosing was performed three times a day with
0.3 mg/kg Compound 8 for three days. The morning assessment of
analgesia was performed approximately 14 hours after the last dose.
Chung model rats exhibited complete tactile allodynia on days 2 and
3, when assayed prior to the first Compound 8 dose of the morning.
However, as shown in FIG. 6B, on the morning of day 4, allodynia
was dramatically reduced and it did not reappear during the time
frame of testing.
[0156] These results indicate that, following several days of
dosing with an .alpha.-adrenergic agonist with minimal .alpha.-2A
activity, drug is no longer required for continued analgesic
activity. These results further indicate that receptor activation
sufficient for a prolonged analgesic effect can be continuous or
intermittent.
[0157] D. Long-Term Pain Relief is Not a General Property of
Analgesic Agents
[0158] A variety of drugs that relieve acute pain were assayed for
long-term pain alleviation in Chung rats. In particular, the
anti-convulsant, gabapentin (3 mg/kg, oral, TID, three days); the
anti-depressant, amitriptyline (0.1 mg/kg/hour, infusion minipump,
seven days); and two non-selective .alpha.-adrenergic agonists with
.alpha.-2A activity, brimonidine (0.04 mg/kg/hour, infusion
minipump, seven days) and Compound 1 (0.1-mg/kg/hr, infusion
minipump, seven days), were administered to Chung model rats at
doses that acutely alleviated tactile allodynia in this model. In
all cases, the allodynia returned completely prior to or following
cessation of treatment.
[0159] However, as shown in FIG. 7A, when brimonidine or Compound 1
was co-administered with the .alpha.-2A selective antagonist,
Compound 13 at 0.2 mg/kg/day, greater analgesia was observed during
the dosing period, and this analgesic effect continued after
completion of drug administration. These results indicate that an
.alpha.-adrenergic agonist with minimal .alpha.-2A agonist activity
or a non-selective .alpha.-adrenergic agonist administered in
conjunction with a selective .alpha.-2A antagonist can produce a
long-lasting analgesic effect after extended dosing that cannot be
achieved by similar compounds having .alpha.-2A agonist
activity.
[0160] E. Long-Term Pain Relief in the Bennett Partial Sciatic
Nerve Ligation Model
[0161] The .alpha.-2B/C selective agonist Compound 8 was tested in
a second rat nerve injury model of neuropathic pain, the Bennett
partial sciatic nerve ligation model. This rat model produces a
peripheral mononeuropathy with disorders of pain sensation similar
to those seen in man (Bennett and Xie, Pain 33:87-107 (1988)). In
the Bennett model, nerve injury is created by loosely tying
constrictive ligatures around the sciatic nerve, causing
degeneration of nerve distal to the constriction. Allodynia and
hyperalgesia are produced by the constriction injury in addition to
spontaneous pain (Bennett and Xie, supra, 1988). In particular,
cold allodynia, the sensation of pain from cold stimuli, is one
manifestation of altered pain sensation: Bennett model animals
frequently lift the paw of the surgical limb off the cold surface,
in contrast to control animals.
[0162] Compound 8 was administered by osmotic minipump over a
period of four days. As shown in FIG. 7B, cold allodynia was
completely alleviated both during the four day treatment period and
for the more than three week testing period following pump removal.
These results indicate that .alpha.-2 adrenergic agonists with
minimal .alpha.-2A agonist activity such as Compound 8 have
analgesic properties applicable to different types of neuropathic
pain.
[0163] Bennett surgeries of about 20 minute duration were performed
as follows. Male Sprague-Dawley rats. (approximately 250-300 grams)
were anesthetized by isoflurane/oxygen inhalation. After preparing
the surgical site by shaving and application of betadine, an
incision was made slightly to the left of midline over the pelvic
girdle. Slightly caudal and ventral to the left hip joint, a faint
separation of muscle groups was visualized, and a small
(approximately 10 to 25 mm) incision made just below the separation
of the muscle groups. Muscle was bluntly separated until the
sciatic nerve was visible parallel to the length of the femur. A 7
to 10 mm length of sciatic nerve was cleared carefully from
underlying tissue, before loosely tying four ligatures (6/0, silk)
around the sciatic nerve with approximately 1 mm spacing between
ligatures. Ligatures were tied so that the diameter of the nerve
was slightly constricted and blood flow retarded but not arrested.
Excess suture material was trimmed, the muscle groups approximated,
and the skin incision closed with wound clips, which were removed
10-14 days post surgery. Animals were not administered any
post-operative topical or local anesthetic.
[0164] Chronic drug administration was achieved via an osmotic
minipump, which was implanted subcutaneously on the back of the rat
one week after the Bennett surgery as described above. Assessment
of pain response in Bennett animals was performed seven days after
surgery as follows. To assess response to thermal stimuli, rats
were placed under a clear plastic chamber (18 cm.times.29
cm.times.12.5 cm) on a chilled (0-4.degree. C.) metal floor, which
is not noxious to a normal animal, and the time that the surgical
paw was raised off of the cold floor recorded over a 5 minute
period. On the day of experiment, test drug was administered (IP or
PO in 1 ml/kg volume in a dose ranging from 1 to 1000 ug/kg)
without anesthetization. In some cases, animals were used for
subsequent experiments over a 3 month period after receiving at
least three days rest in between studies.
[0165] F. Long-Term Pain Relief in a Model of Irritable Bowel
Syndrome
[0166] This example demonstrates that an .alpha.-2 adrenergic
agonist with minimal .alpha.-2 activity such as Compound 8
alleviates pain in a rat model of chronic visceral
hypersensitivity.
[0167] A well-accepted model of chronic visceral hypersensitivity
is described in Al-Chaer et al., Gastroenterology 119:1276-85
(2000). In brief, colons of neonatal male Sprague Dawley rats were
sensitized by repeated inflation of a colonic balloon on postnatal
days 8 to 21, with colonic balloons inflated to pressures ranging
from 20-80 mm Hg. Three months later, the abdominal withdrawal
reflex (AWR) response of the adult rats to graded colorectal
distension (CRD) was quantified by electromyography (EMG) recording
from the abdominal wall muscle. Sensitized rats exhibited both
allodynic pain and hyperalgesia, showing an exaggerated response to
normally nonpainful levels of balloon inflation (20 mm Hg) as well
as to painful stimuli (40-80 mm Hg), which are visceral pain
symptoms similar to those in human patients with irritable bowel
syndrome. Unsensitized animals exhibited very little response to
balloon inflation of 20 mm Hg and a mild response up to an EMG
intensity unit of 1, with CRD up to 80 mm Hg. Each level of CRD was
administered for 20 seconds every four minutes and repeated for a
total of five times.
[0168] Groups of 8-10 normal and sensitized adult Sprague Dawley
rats (approximately 3 months old) were implanted with subcutaneous
minipumps to deliver either 50% DMSO or Compound 8 in 50% DMSO at a
dose of 100 ug/kg/hr over a period of 7 days. The abdominal
withdrawal reflex to a graded series of colorectal distensions (20,
40, 60, 80 mm Hg) was measured by abdominal EMG recording prior to
pump implantation, on days 2 and 5 of the subcutaneous infusion,
and 2 days, 1 week, 2 weeks, and 4 weeks after the pumps were
removed.
[0169] As shown in FIGS. 8A and 8B, the EMG response following
treatment of non-sensitized control rats with 50% DMSO vehicle
(FIG. 8A) or Compound 8 (FIG. 8B) did not change significantly from
pre-treatment levels; EMG intensity ranged from approximately 0 to
about 1.5. In contrast, the EMG response in the sensitized rats was
much greater, ranging approximately from intensities of 1 to 3.5
(see FIGS. 8C and 8D); this increased pain response in the
sensitized rats was not reduced following treatment with vehicle as
shown in FIG. 8C. In contrast, during and following the treatment
with Compound 8, the increased EMG response in the sensitized rats
was completely alleviated. As shown in FIG. 8D, pain was reduced to
0 to 1.5, which is the level seen in non-sensitized rats.
Furthermore, colorectal allodynia and hyperalgesia did not return
during the time period tested, which was 4 weeks following
cessation of drug treatment.
[0170] These results demonstrate that an .alpha.-2 adrenergic
agonist with minimal .alpha.2-A activity can be used for the
long-term relief of colorectal pain such as irritable bowel
syndrome pain. These results further indicate that the observed
analgesic effects are not specific to peripheral neuropathic pain
and that an .alpha.-2 adrenergic agonist with minimal .alpha.2-A
activity or an .alpha.-2 adrenergic agonist administered in
conjunction with a selective .alpha.2-A antagonist can be used to
treat a variety of types of acute and chronic pain such as
neuropathic, visceral, inflammatory, post-surgical and cancer
pain.
EXAMPLE V
Peripheral .alpha.-Adrenergic Agonist Analgesic Activity is
Mediated by the .alpha.-2B Receptor
[0171] This example demonstrates that peripheral .alpha.-adrenergic
agonist analgesic activity can be mediated by activation of the
.alpha.-2B receptor.
[0172] In wild type mice, an intraperitoneal dose of clonidine (500
ug/kg) alleviated sulprostone-induced tactile allodynia but
produced concomitant sedation. In contrast, intraperitoneal
Compound 3 (100 ug/kg), an .alpha.-adrenergic agonist with minimal
.alpha.-2A agonist activity, alleviated sulprostone-induced tactile
allodynia without concomitant sedation. The analgesic effect of
these doses of clonidine and Compound 3 was determined in
.alpha.-2B heterozygous (-/+) and homozygous (-/-) knockout mice
using the smallest Von Frey hair at 1.65 grams of force. At this
force, the smallest Von Frey hair does not evoke a pain response in
untreated .alpha.-2B knockout mice nor in their wild type
litter-mates. Allodynia was assessed and ranked as described
above.
[0173] As shown in FIG. 9, clonidine alleviated the analgesia in
both heterozygous and homozygous .alpha.-2B knockout strains, with
no difference from its effect in wild type mice; again, the
analgesia was accompanied by sedation. In contrast, Compound 3 was
not analgesic in either the heterozygous or homozygous .alpha.-2B
knockout mice (FIG. 9). Similar results were obtained with other
compounds. In particular, like clonidine, the .alpha.-adrenergic
pan-agonist, brimonidine, was analgesic in .alpha.-2B knockout
mice, while Compound 8, an .alpha.-adrenergic agonist with minimal
.alpha.-2A agonist activity, failed to show analgesic activity in
heterozygous or homozygous .alpha.-2B knockout mice.
[0174] These results demonstrate that the mechanism of analgesia of
.alpha.-adrenergic agonists with minimal .alpha.-2A agonist
activity is distinct from the mechanism of analgesia of
.alpha.-adrenergic pan-agonists. These results further indicate
that peripheral .alpha.-adrenergic analgesic activity is mediated
by activation of the .alpha.-2B receptor.
EXAMPLE VI
Characterization of .alpha.-2B and .alpha.-2B/C Selective
.alpha.-Adrenergic Agonists
[0175] This example describes the receptor subtype selectivity,
oral analgesic activity and absence of sedative and cardiovascular
side effects associated with Compounds 3, 11 and 4.
[0176] A. Receptor Subtype Selectivity Profiles of Compounds 3, 11
and 4
[0177] As shown in Table 3, each of Compounds 3, 11 and 4 are
selective for .alpha.-2 adrenergic receptors, with little or no
activity at .alpha.-1 receptors. Furthermore, each of these
compounds is a .alpha.-2B/C selective compound exhibiting no
detectable activity at the .alpha.-2A receptor in the in vitro RSAT
assay. Compound 4 was selective for the .alpha.-2B receptor,
exhibiting more than 10-fold greater activity at the .alpha.-2B
receptor than the .alpha.-2C receptor. Compound 3 was specific for
the .alpha.-2B receptor as it was characterized by about 100-fold
greater activity at the .alpha.-2B receptor than the .alpha.-2C
receptor.
3TABLE 3 .alpha.-Adrenergic receptor selectivity profile RSAT
activity (nM)/ % efficacy Compound/class 2A 2B 2C 1A 1B 1D Compound
3 NA 24 >2000 >2000 NA >2000 thione/racemate (90%)
Compound 11 NA 42 247 1713 NA >2000 thione (80%) (0.3) Compound
4 NA 15 202 NA NA >2000 imidazolone (100%) (0.5) NA = not active
(EC.sub.50 .gtoreq. 10,000)
[0178] B. Oral Analgesic Activity of Compounds 3, 11 and 4
[0179] Various concentrations of Compounds 3, 11, and 4 were
administered orally to Chung model rats as described above. As
shown in FIG. 10A, 30 .mu.g/kg oral Compound 3 resulted in 70-100%
allodynia reversal. The analgesic effect was seen quickly, in less
than 20 minutes. Moreover, when administered as a single oral dose,
the effect was transient with analgesia essentially gone by 2 hours
following administration. FIG. 10B shows that oral Compound 11 also
alleviated pain in the Chung rat model. A dose of 0.1 mg/kg was
sufficient to reduce the allodynia by about 60-90%. Again, the
analgesic effect was gone by 2 hours post-administration following
a single oral dose. As shown in FIG. 10C, Compound 4 exhibited
linear dose-responsiveness in alleviating pain: a dose of 30
.mu.g/kg was sufficient for an analgesic effect, and, at 0.3 mg/kg
Compound 4, about 60-80% of the allodynia was reversed. At 1 mg/kg,
the allodynia was reversed to a greater extent, and the pain relief
was of longer duration. However, essentially no pain relief was
observed after four hours, even at the highest dose (FIG. 10C).
These results corroborate that .alpha.-adrenergic agonists with
minimal .alpha.-2A activity can act as analgesic agents when
administered orally.
[0180] C. Long-Term Pain Relief with Compounds 3, 11 and 4
[0181] Compounds 3, 11, and 4 were administered for five days via
osmotic minipump. Drug administration was discontinued on day 5, at
which time the minipumps were removed. Allodynia was assayed over a
period of about a month. As shown in FIG. 11, about 80% allodynia
reversal was achieved by each of the three compounds. Furthermore,
the analgesic effect of Compounds 11, and 4 was maintained at
essentially the same level over the entire four week test period.
These results indicate that Compounds 3, 11 and 4 are effective
analgesic agents for long-term pain relief and further corroborate
that, following extended dosing, .alpha.-adrenergic agonists with
minimal .alpha.-2A activity can be used to treat chronic pain.
[0182] D. Side Effect Profiles of Compounds 3, 11 and 4
[0183] Compounds 3, 11 and 4 were administered intraperitoneally at
1 mg/kg, which is higher than the dose required to produce peak
allodynia reversal (see Table 2). Sedative effects were assayed as
above. In addition, these compounds were assayed for cardiovascular
side effects in monkeys at 0.5 mg/kg intravenous administration, or
3 mg/kg orally, and Compound 3 was assayed for cardiovascular
effects in rats. The lack of .alpha.-2 antagonist activity of a 3
mg/kg dose was assessed by testing reversal of the sedative effects
of clonidine coadministered intraperitoneally at a 0.1 mg/kg dose
("sedation reversal").
[0184] As shown in Table 4, no significant sedative or
cardiovascular side effects were observed for Compound 3, Compound
11 or Compound 4 at the indicated doses, which were higher than the
doses required to achieve 60-90% allodynia reversal.
4TABLE 4 Sedative and Cardiovascular side effects Rat Sedation
Monkey Rat sedation reversal cardiovascular cardiovascular Compound
(i.p.) (i.p.) (BP, HR) (BP, HR) Compound NS* NS NS 0.5 mg/kg iv NS
3 mg/kg i/a 3 1 mg/kg 3 mg/kg Compound NS NS NS 0.5 mg/kg iv -- 11
1 mg/kg 3 mg/kg NS 3 mg/kg po Compound NS NS NS 3 mg/kg po -- 4 1
mg/kg 3 mg/kg *No significant effect
[0185] Cardiovascular effects were assayed in approximately six
cynomolgus monkeys weighing roughly 4 kg with the indicated dose
and compound administered by intravenous or intraperitoneal
administration. Monkeys were weighed, and the appropriate
concentration of a 0.1 ml/kg intravenous dosing solution or a 1.0
ml/kg intraperitoneal dosing solution injected. Intravenous
injections were via the cephalic arm vein. Blood pressure and heart
rate measurements were made prior to and at 0.5, 1, 2, 4 and 6
hours after drug administration with a BP 100S automated
sphygmomanometer (Nippon Colin; Japan). Cardiovascular effects were
determined in rats as described in Altman et al., Mol. Pharm.
56:154-161 (1999).
[0186] These results indicate that the analgesic effects of
.alpha.-adrenergic agonists with minimal .alpha.-2A activity can be
achieved without significant sedative or cardiovascular side
effects.
EXAMPLE VII
Analgesic Activity of Enantiomers of Compounds 3 and 4
[0187] This example demonstrates that enantiomers of the
.alpha.-adrenergic agonists with minimal .alpha.-2A agonist
activity can exhibit differential analgesic activity.
[0188] Enantiomers of Compound 3 and Compound 4 were prepared as
described above in Example I and designated Compound 10 and
Compound 12, respectively. Both enantiomers and the parent racemate
were assayed for analgesic activity following intraperitoneal
administration to Chung model rats. As shown in FIG. 12A, allodynia
reversal was obtained with the (-) enantiomer of Compound 3
(designated Compound 10) but not with the (+) enantiomer. The (-)
enantiomer was as effective as parent racemic mixture in relieving
pain.
[0189] Similarly, FIG. 12B shows the percentage of allodynia
reversal obtained in Chung model rats given various concentrations
of each enantiomer and parent agonist Compound 4 by intraperitoneal
administration. As shown in the figure, the (+) enantiomer
(designated Compound 12) was at least as effective as the parent
compound in reducing allodynia at all doses tested.
[0190] These results demonstrate that enantiomers of .alpha.-2B/2C
selective agonists can exhibit differential analgesic activity and
further indicate that the (-) enantiomer of Compound 3 and the (+)
enantiomer of Compound 4 are effective analgesic agents.
[0191] All journal article, reference and patent citations provided
above, in parentheses or otherwise, whether previously stated or
not, are incorporated herein by reference in their entirety.
[0192] Although the invention has been described with reference to
the examples provided above, it should be understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
claims.
* * * * *