U.S. patent application number 13/029900 was filed with the patent office on 2011-08-25 for methods for treating pain.
This patent application is currently assigned to The General Hospital Corporation. Invention is credited to Michael Costigan, Robert Griffin, Irmgard Tegeder, Clifford J. Woolf.
Application Number | 20110207753 13/029900 |
Document ID | / |
Family ID | 34619476 |
Filed Date | 2011-08-25 |
United States Patent
Application |
20110207753 |
Kind Code |
A1 |
Woolf; Clifford J. ; et
al. |
August 25, 2011 |
METHODS FOR TREATING PAIN
Abstract
The present invention features methods and compositions for
preventing, reducing, or treating a traumatic, metabolic or toxic
peripheral nerve lesion or pain including, for example, neuropathic
pain, inflammatory and nociceptive pain by administering to a
mammal in need thereof a compound that reduces the expression or
activity of BH4. According to this invention, this reduction may be
achieved by reducing the enzyme activity of any of the BH4
synthetic enzymes, such as GTP cyclohydrolase (GTPCH), sepiapterin
reductase (SPR), or dihydropteridine reductase (DHPR); by
antagonizing the cofactor function of BH4 on BH4-dependent enzymes;
or by blocking BH4 binding to membrane bound receptors. The
compounds of the invention may be administered alone or in
combination with a second therapeutic agent. The invention also
provides methods for diagnosing pain or a peripheral nerve lesion
in a mammal by measuring the levels of BH4 or its metabolites in
biological sample. Alternatively, pain or a peripheral nerve lesion
may be diagnosed by measuring the levels or activity of any one of
the BH4 synthetic enzymes in tissue samples of a mammal. Also
disclosed are screening methods that make use of BH4 or BH4
synthetic enzymes, BH4-dependent enzymes, and BH4-binding receptors
for the identification of novel therapeutics for the treatment,
prevention, or reduction of pain.
Inventors: |
Woolf; Clifford J.; (Newton,
MA) ; Costigan; Michael; (Somerville, MA) ;
Griffin; Robert; (Boston, MA) ; Tegeder; Irmgard;
(Frankfurt, DE) |
Assignee: |
The General Hospital
Corporation
Boston
MA
|
Family ID: |
34619476 |
Appl. No.: |
13/029900 |
Filed: |
February 17, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10987289 |
Nov 12, 2004 |
7906520 |
|
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13029900 |
|
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60520536 |
Nov 13, 2003 |
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Current U.S.
Class: |
514/263.1 ;
250/362; 435/7.1; 506/7; 514/262.1; 514/272; 514/282; 514/415;
514/653 |
Current CPC
Class: |
C07D 487/04 20130101;
C07D 239/48 20130101; C07D 473/18 20130101; A61K 31/4045 20130101;
A61P 25/04 20180101; A61K 31/165 20130101; A61P 25/00 20180101;
A61K 31/519 20130101; A61K 31/535 20130101; A61P 43/00 20180101;
A61K 31/54 20130101 |
Class at
Publication: |
514/263.1 ;
514/262.1; 514/272; 514/415; 514/653; 514/282; 435/7.1; 506/7;
250/362 |
International
Class: |
A61K 31/52 20060101
A61K031/52; A61K 31/519 20060101 A61K031/519; A61K 31/505 20060101
A61K031/505; A61K 31/405 20060101 A61K031/405; A61K 31/137 20060101
A61K031/137; A61K 31/485 20060101 A61K031/485; G01N 33/53 20060101
G01N033/53; C40B 30/00 20060101 C40B030/00; A61P 25/04 20060101
A61P025/04; A61P 25/00 20060101 A61P025/00; G01T 1/164 20060101
G01T001/164 |
Claims
1. A method of treating, reducing, or preventing pain or the
consequences or development of a peripheral nerve lesion in a
mammal, said method comprising administering to said mammal a
composition that reduces the tetrahydrobiopterin (BH4) biological
activity in an amount sufficient to treat, reduce, or prevent pain
or the exacerbation of a peripheral nerve lesion due to
overproduction of BH4.
2. The method of claim 1, wherein said mammal is a human.
3. The method of claim 1, wherein the pain is reduced by reducing
the BH4 levels in primary sensory neurons or dorsal horn
neurons.
4. The method of claim 3, wherein said primary sensory neurons are
in a dorsal root ganglion or a trigeminal ganglion.
5. The method of claim 4, wherein said dorsal horn neurons are in
the spinal cord or spinal nucleus of the trigeminal in the
brainstem.
6. The method of claim 1, wherein the reduction in BH4 biological
activity is the result of a reduction in BH4 synthesis or
recycling.
7. The method of claim 1, wherein said BH4 biological activity is
reduced by increasing the expression, GTPCH-binding, or activity of
GTP cyclohydrolase feedback regulatory protein (GFRP).
8. The method of claim 6, wherein said reduction in BH4 synthesis
is the result of a reduction in the level or biological activity of
at least one enzyme selected from the group consisting of
sepiapterin reductase (SPR), Pyruvoyltetrahydropterin Synthase
(PTPS), GTP cyclohydrolase (GTPCH), Pterin-4.alpha.-carbinolamine
dehydratase, and dihydropteridine reductase (DHPR).
9. The method of claim 8, wherein said reduction in BH4 synthesis
is the result of a reduction in the biological activity of at least
one enzyme selected from the group consisting of sepiapterin
reductase (SPR), GTP cyclohydrolase (GTPCH), and dihydropteridine
reductase (DHPR).
10. The method of claim 8, wherein the biological activity of at
least two of said enzymes is reduced.
11. The method of claim 10, wherein the biological activity of at
least three of said enzymes is reduced.
12. The method of claim 8, wherein said biological activity is
reduced by at least 10%.
13. The method of claim 12, wherein said biological activity is
reduced by at least 40%.
14. The method of claim 1, wherein said composition comprises
methotrexate.
15. The method of claim 1, wherein said composition comprises at
least one compound selected from the group consisting of 2,4
diamino 6-hydroxypyrimidine (DAHP), Tetrahydro-L-biopterin,
L-Sepiapterin, 7,8-dihydro-L-Biopterin,
6,7-dimethyltetrahydropterin hydrochloride, and 8-bromo-cGMP.
16. The method of claim 1, wherein said composition comprises at
least one compound selected from the group consisting of
N-acetyl-serotonin (NAS), N-Chloroacetylserotonin,
N-Methoxyacetylserotonin, and N-Chloroacetyldopamine.
17. The method of claim 1, wherein said composition comprises a
compound having the formula: ##STR00055## wherein R.sup.1 is H,
C.sub.1-6 alkyl, halo, NO.sub.2, CN, CO.sub.2R.sup.4,
CONR.sup.4R.sup.5, SO.sub.2R.sup.4, SO.sub.2NR.sup.4R.sup.5,
OR.sup.4, or NR.sup.4R.sup.5, wherein each of R.sup.4 and R.sup.5
is, independently, H, C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl,
C.sub.1-4 alkaryl, or C.sub.1-4 alkheteroaryl, R.sup.2 is H,
C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or
C.sub.1-4 alkheteroaryl, and R.sup.3 is H, C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, C.sub.1-4
alkheteroaryl, CO.sub.2R.sup.6, CONR.sup.7R.sup.8, SO.sub.2R.sup.6,
or SO.sub.2NR.sup.7R.sup.8, wherein R.sup.6 is C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl and each of R.sup.7 and R.sup.8 is, independently, H,
C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or
C.sub.1-4 alkheteroaryl; R.sup.3 is as above and R.sup.1 and
R.sup.2 together are represented by ##STR00056## wherein the N, O,
or S of the R.sup.1/R.sup.2 linkage forms a bond to the
pyrimidinone ring and each of R.sup.9, R.sup.10, R.sup.11,
R.sup.12, and R.sup.13 is, independently H, C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl; R.sup.3 is as above and R.sup.1 and R.sup.2 together
are represented by ##STR00057## wherein the N of the
R.sup.1/R.sup.2 linkage forms a bond to the pyrimidinone ring, each
of R.sup.12 and R.sup.13 is as above, and R.sup.14 is OR.sup.4,
halo, NO.sub.2, CN, CO.sub.2R.sup.7, CONR.sup.7R.sup.8,
SO.sub.2R.sup.7, SO.sub.2NR.sup.7R.sup.8, C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl, wherein each of R.sup.4, R.sup.7 and R.sup.8 is,
independently, H, C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.12 aryl,
C.sub.1-C.sub.4 alkaryl, heteroaryl, or C.sub.1-C.sub.4
alkheteroaryl; or R.sup.1 and R.sup.2 together are represented by
##STR00058## wherein the N of the R.sup.1/R.sup.2 linkage forms a
bond to the pyrimidinone ring, each of R.sup.9, R.sup.10, R.sup.11,
and R.sup.14 are as above, R.sup.3 does not exist, and a double
bond is formed between the carbon bearing R.sup.14 and the nitrogen
bearing R.sup.2.
18. The method of claim 17, wherein said composition comprises a
compound of formula (I), wherein R.sup.1 is H, C.sub.1-6 alkyl,
halo, NO.sub.2, CN, CO.sub.2R.sup.4, CONR.sup.4R.sup.5,
SO.sub.2R.sup.4, SO.sub.2NR.sup.4R.sup.5, OR.sup.4, or
NR.sup.4R.sup.5, wherein each of R.sup.4 and R.sup.5 is,
independently, H, C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl,
C.sub.1-4 alkaryl, or C.sub.1-4 alkheteroaryl, R.sup.2 is H,
C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or
C.sub.i-4 alkheteroaryl, and R.sup.3 is H, C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, C.sub.1-4
alkheteroaryl, CO.sub.2R.sup.6, CONR.sup.7R.sup.8, SO.sub.2R.sup.6,
or SO.sub.2NR.sup.7R.sup.8, wherein R.sup.6 is C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl and each of R.sup.7 and R.sup.8 is, independently, H,
C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or
C.sub.1-4 alkheteroaryl.
19. The method of claim 1, wherein said composition comprises a
compound of formula: ##STR00059## wherein R.sup.1 and R.sup.2
together are represented by ##STR00060## wherein the N, O, or S of
the R.sup.1/R.sup.2 linkage forms a bond to the pyrimidinone ring
and each of R.sup.9, R.sup.10, R.sup.11, R.sup.12, and R.sup.13 is,
independently H, C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl,
alkaryl, or C.sub.1-4 alkheteroaryl.
20. The method of claim 1, wherein said composition comprises a
compound of formula: ##STR00061## wherein R.sup.3 is as above and
R.sup.1 and R.sup.2 together are represented by ##STR00062##
wherein the N of the R.sup.1/R.sup.2 linkage forms a bond to the
pyrimidinone ring, each of R.sup.12 and R.sup.13 is as above, and
R.sup.14 is OR.sup.4, halo, NO.sub.2, CN, CO.sub.2R.sup.7,
CONR.sup.7R.sup.8, SO.sub.2R.sup.7, SO.sub.2NR.sup.7R.sup.8,
C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or
C.sub.1-4 alkheteroaryl, wherein each of R.sup.4, R.sup.7 and
R.sup.8 is, independently, H, C.sub.1-C.sub.6 alkyl, C.sub.6-12
C.sub.12 aryl, C.sub.1-C.sub.4 alkaryl, heteroaryl, or
C.sub.1-C.sub.1 alkheteroaryl.
21. The method of claim 1, wherein said composition comprises a
compound of formula: ##STR00063## wherein R.sup.1 and R.sup.2
together are represented by ##STR00064## wherein the N of the
R.sup.1/R.sup.2 linkage forms a bond to the pyrimidinone ring, each
of R.sup.9,R.sup.10, R.sup.11, R.sup.12, and R.sup.14 are as above,
R.sup.3 does not exist, and a double bond is formed between the
carbon bearing R.sup.14 and the nitrogen bearing R.sup.2.
22. The method of claim 1, wherein said composition comprises a
compound of formula: ##STR00065## wherein R.sup.1 and R.sup.2
together are represented by ##STR00066## wherein the N, O, or S of
the R.sup.1/R.sup.2 linkage forms a bond to the pyrimidinone ring
and each of R.sup.9, R.sup.10, R.sup.11, R.sup.12, and R.sup.13 is,
independently H, C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl,
C.sub.1-4 alkaryl, or C.sub.1-4 alkheteroaryl.
23. The method of claim 1, wherein said composition comprises a
compound of formula: ##STR00067## wherein R.sup.3 is as above and
R.sup.1 and R.sup.2 together are represented by ##STR00068##
wherein the N of the R.sup.1/R.sup.2 linkage forms a bond to the
pyrimidinone ring, each of R.sup.12 and R.sup.13 is as above, and
R.sup.14 is OR.sup.4, halo, NO.sub.2, CN, CO.sub.2R.sup.7,
CONR.sup.7R.sup.8, SO.sub.2R.sup.7, SO.sub.2NR.sup.7R.sup.8,
C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or
C.sub.1-4 alkheteroaryl, wherein each of R.sup.4, R.sup.7 and
R.sup.8 is, independently, H, C.sub.1-C.sub.6 alkyl,
C.sub.6-C.sub.12 aryl, C.sub.1-C.sub.4 alkaryl, heteroaryl, or
C.sub.1-C.sub.4 alkheteroaryl.
24. The method of claim 1, wherein said composition comprises a
compound of formula: ##STR00069## wherein R.sup.1 and R.sup.2
together are represented by ##STR00070## wherein the N of the
R.sup.1/R.sup.2 linkage forms a bond to the pyrimidinone ring, each
of R.sup.9, R.sup.10, R.sup.11, and R.sup.14 are as above, R.sup.3
does not exist, and a double bond is formed between the carbon
bearing R.sup.14 and the nitrogen bearing R.sup.2.
25. The method of claim 18, wherein said composition comprises a
compound selected from the group consisting of ##STR00071##
26. The method of claim 1, wherein said composition comprises a
compound having the formula: ##STR00072## wherein R.sup.15 is H,
C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or
C.sub.1-4 alkheteroaryl; and R.sup.16 is H, C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, C.sub.1-4
alkheteroaryl, CO.sub.2R.sup.17, CONR.sup.18R.sup.19,
SO.sub.2R.sup.17 or SO.sub.2NR.sup.18R.sup.19, wherein R.sup.17 is
C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or
C.sub.1-4 alkheteroaryl and each of R.sup.18 and R.sup.19 is,
independently, H, C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl,
C.sub.1-4 alkaryl, or C.sub.1-4 alkheteroaryl.
27. The method of claim 1, wherein said pain is acute pain.
28. The method of claim 1, wherein said pain is chronic pain.
29. The method of claim 1, wherein said pain is selected from the
group consisting of peripheral and central neuropathic pain,
inflammatory pain, functional pain nociceptive pain, and
headache.
30. The method of claim 1, further comprising a second therapeutic
agent.
31. The method of claim 30, wherein said second therapeutic agent
is an analgesic agent.
32. The method of claim 30, wherein said analgesic agent is a
non-steroidal anti-inflammatory agent (NSAIDs), opioid receptor
agonist, tricyclic antidepressant, SSRI, anticonvulsant, clonidine,
sodium or calcium channel blocker, potassium channel opener, 5-HT1D
receptor agonist.
33. The method of claim 32, wherein said second therapeutic agent
is an inhibitor of an enzyme selected from the group consisting of
nitric oxide synthase (NOS), tyrosine hydroxylase, tryptophan
hydroxylase I (non-neuronal TphI), tryptophan hydroxylase II
(neuronal Tph II), phenylalanine hydroxylase,
dopamine-.beta.-hydroxylase, N-methyltransferase, and ether lipid
oxidase.
34. The method of claim 30, wherein said therapeutic agent that
reduces the levels of tetrahydrobiopterin (BH4) and said second
therapeutic agent are administered within one hour of each
other.
35. The method of claim 30, wherein said therapeutic agent that
reduces the levels of tetrahydrobiopterin (BH4) and said second
therapeutic agent are administered simultaneously.
36. The method of claim 30, wherein said therapeutic agent that
reduces the levels of tetrahydrobiopterin (BH4) and said second
analgesia-inducing compound are administered in the same
pharmaceutical formulation.
37. A method of diagnosing pain or a peripheral nerve lesion in a
mammal, said method comprising detecting an increase in BH4, BH4
metabolite, BH4 precursor, or BH4 intermediate in a biological
sample from said mammal.
38. The method of claim 37, wherein said BH4 metabolite is pterin,
biopterin, 7,8 dihydropterin, 7,8-dihydroxanthopterin,
xanthopterin, isoxanthopterin, leucopterin, 7,8-dihydroneopterin,
or neopterin.
39. The method of claim 37, wherein said BH4 intermediate is
7,8-dihydroneopterin triphosphate, neopterin, or 6-pyruvoyl
tetrahydropterin.
40. The method of claim 37, wherein said biological sample is
selected from the group consisting of blood, serum, plasma, tissue
sample, urine, cerebrospinal fluid, synovial fluid, tissue exudate,
or tissue sample.
41. The method of claim 37, wherein said increase is at least a 20%
increase relative to control conditions.
42. A method of diagnosing pain or a peripheral nerve lesion in a
mammal, said method comprising detecting an increase in the
activity or level of a BH4 synthetic enzyme in primary sensory
neurons or dorsal horn neurons of said mammal.
43. The method of claim 42, wherein said BH4 synthetic enzyme is
selected from the group consisting of sepiapterin reductase (SPR),
Pyruvoyltetrahydropterin Synthase (PTPS), GTP cyclohydrolase
(GTPCH), Pterin-4.alpha.-carbinolamine dehydratase, and
dihydropteridine reductase (DHPR).
44. The method of claim 42, wherein said detecting is performed by
imaging techniques.
45. The method of claim 44, wherein said imaging is positron
emission tomography (PET).
46. The method of claim 42, wherein said increase is at least a 20%
increase relative to control conditions.
Description
[0001] This application claims benefit of the filing date of the
copending U.S. Provisional Application No. 60/520,536, filed Nov.
13, 2003, hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] In general, the invention features methods for diagnosing,
treating, reducing, or preventing pain.
BACKGROUND OF THE INVENTION
[0003] The sensation of pain is a common symptom that may be
indicative of an underlying disease or injury, or the expression of
an abnormal function within the nervous system. Pain is often the
primary incentive for which treatment is sought.
[0004] Pain can take a variety of forms depending on its origin.
Pain may be described as being peripheral neuropathic if the
initiating injury occurs as a result of a complete or partial
transection of a nerve or trauma to a nerve plexus. Alternatively,
pain is described as being central neuropathic following a lesion
to the central nervous system, such as a spinal cord injury or a
cerebrovascular accident. Inflammatory pain is a form of pain that
is caused by tissue injury or inflammation (e.g., in postoperative
pain or rheumatoid arthritis). Following a peripheral nerve injury,
symptoms are typically experienced in a chronic fashion, distal to
the site of injury and are characterized by hyperesthesia (enhanced
sensitivity to a natural stimulus), hyperalgesia (abnormal
sensitivity to a noxious stimulus), allodynia (widespread
tenderness, associated with hypersensitivity to normally innocuous
tactile stimuli), and/or spontaneous burning or shooting
lancinating pain. In inflammatory pain, symptoms are apparent, at
least initially, at the site of injury or inflamed tissues and
typically accompany arthritis-associated pain, musculo-skeletal
pain, and postoperative pain. Nociceptive pain is the pain
experienced in response to a noxious stimulus, such as a needle
prick or during trauma or surgery. Functional pain refers to
conditions in which there is no obvious peripheral pathology or
lesion to the nervous system. This particular form of pain is
generated by abnormal function of the nervous system and conditions
characterized by such pain include fibromyalgia, tension-type
headache, and irritable bowel syndrome. The different types of pain
may coexist or pain may be transformed from inflammatory to
neuropathic during the natural course of the disease, as in
post-herpetic neuralgia.
[0005] Although one approach for the treatment of pain is the
removal of the causative or etiological agent (disease modifying
therapy), the pain often outlasts the duration of the initiating
cause. Accordingly, symptomatic control is essential. In cases in
which the sensation of pain becomes unbearable, rapid and effective
analgesia is imperative (e.g., postoperative state, burns, trauma,
cancer, and sickle cell crisis). Currently, there exist a wide
variety of analgesic agents useful for the management of pain,
including for example non-steroidal analgesic agents (NSAIDs),
anticonvulsants, and opioid analgesics. Despite their efficacy, the
chronic use of such agents is often not recommended because of the
potential debilitating side effects, such as gastric irritation,
toxicity to the liver, respiratory depression, sedation,
psychotomimetic effects, constipation, nausea, tolerance,
dependence, and the risk of abuse. Also, these agents are sometimes
suboptimal, particularly for neuropathic and functional pain.
[0006] Thus, better therapeutic strategies are required for the
treatment and management of pain.
SUMMARY OF THE INVENTION
[0007] In general, the present invention features methods for the
diagnosis, treatment, reduction, and prevention of pain or of
endogenous mechanisms that further increase a traumatic, metabolic
or toxic peripheral nerve lesion in a mammal. According to this
invention, a mammal (e.g., a human) is administered a composition
(e.g., methotrexate) that reduces tetrahydrobiopterin (BH4)
biological activity such that pain is reduced, prevented, or
treated. Alternatively, pain may be reduced in the mammal being
treated by decreasing the levels or activity of any one of the
enzymes involved in the synthesis of BH4, i.e. BH4 synthetic
enzymes. In this regard, BH4 synthesis may be reduced by decreasing
the biological activity of at least one, two, three, or more than
three of the following enzymes: sepiapterin reductase (SPR),
Pyruvoyltetrahydropterin Synthase (PTPS), GTP cyclohydrolase
(GTPCH), Pterin-4.alpha.-carbinolamine dehydratase, and
dihydropteridine reductase (DHPR). Such activity may be reduced by
at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95%, 99%, or
even 100% relative to an untreated control. Alternatively, BH4
biological activity may be reduced by increasing the expression or
GTPCH-binding or other biological activity of GTP cyclohydrolase
feedback regulatory protein (GFRP). GFRP biological activity may be
increased by administering a BH4 or phenylalanine analog that
specifically binds to GFRP or a GTPCH:GFRP complex. Traumatic nerve
lesions include those caused by mechanical insults and compressive
injuries. Compressive injuries may be caused by external trauma or
injury, or from internal conditions and disease states, such as a
compression resulting from an infiltrative tumor. Metabolic nerve
lesions amenable to treatment using the methods of this invention
include, for example, diabetic peripheral neuropathies, heritable
neuropathies, or neuropathies caused by infectious agents such as
the human immunodeficiency virus (HIV). Toxic nerve lesions
include, for example, those cause by other therapeutic agents
(e.g., chemotherapeutics), or chemicals and environmental toxicants
(e.g., heavy metals and organic solvents).
[0008] According to this invention, a reduction in pain may result,
for example, from changes in the function of primary sensory
neurons or neurons within the dorsal horn of the spinal cord, in
the brainstem, or in the brain. Such changes may result, for
example, from a reduction in the synthesis of BH4, leading to a
reduction in the activity of various enzymes (e.g., nitric oxide
synthase) that utilize BH4 as a cofactor and to a reduction in the
activation of membrane-bound BH4-binding receptors following its
release from cells. BH4 action on BH4-binding receptors may be
inhibited or reduced by means of competitive or non-competitive
BH4-like receptor antagonists. BH4 action on enzymes which use BH4
as a cofactor may be inhibited by means of BH4-like competitive
antagonists. Such binding or activity may be reduced by at least
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95%, 99%, or even 100%
relative to an untreated control.
[0009] Furthermore, levels of tetrahydrobiopterin (BH4), its
precursors, or its metabolites may be measured in a biological
sample obtained from a mammal (e.g., plasma, tissue sample,
cerebrospinal fluid, synovial fluid, or tissue exudates) and, in
turn, serve as diagnostic tools and as biomarkers of pain or nerve
injury. Methods for measuring BH4 are described, for example, in
Powers et al. (1988) J Chromatogr 432:321-328; Blau et al. (1994)
Clin Chim Acta 226:159-169; Ponzone et al. (1994) Eur J Pediatr
153:616; Zorzi et al. (2002) Mol Genet Metab 75:174-177; and
Shiraki et al. (1994) Eur J Pediatr 153:616. Optionally, an
increase in levels or activities of any one of the BH4 synthetic
enzymes may also diagnose pain in a mammal.
[0010] The methods of this invention are therefore useful for the
diagnosis, treatment, reduction, or prevention of various forms of
pain, for example, nociceptive pain, inflammatory pain, functional
pain and neuropathic pain, all of which may be acute or chronic.
Thus, the mammal being treated may be diagnosed as having
peripheral diabetic neuropathy, compression neuropathy, post
herpetic neuralgia, trigeminal or glossopharyngeal neuralgia, post
traumatic or post surgical nerve damage, lumbar or cervical
radiculopathy, AIDS neuropathy, metabolic neuropathy, drug induced
neuropathy, complex regional pain syndrome, arachnoiditis, spinal
cord injury, bone or joint injury, tissue injury, psoriasis,
scleroderma, pruritis, cancer (e.g., prostate, colon, breast, skin,
hepatic, or kidney), cardiovascular disease (e.g., myocardial
infarction, angina, ischemic or thrombotic cardiovascular disease,
peripheral vascular occlusive disease, or peripheral arterial
occlusive disease), sickle cell anemia, migraine cluster or
tension-type headaches, inflammatory conditions of the skin,
muscle, or joints, fibromyalgia, irritable bowel syndrome, non
cardiac chest pain, cystitis, pancreatitis, or pelvic pain.
Alternatively, the pain for which treatment is being sought may be
the result of a traumatic injury, surgery, burn of the cutaneous
tissue (caused by a thermal, chemical, or radiation stimulus), or a
sunburn.
[0011] According to this invention, the mammal being treated may be
administered with a composition containing, for example, at least
one of the following compounds: methotrexate,
trimethoprim-sulfamethoxazole, 2,6 diamino hydroxypyrimidine
(DAHP), Tetrahydro-L-biopterin, L-Sepiapterin,
7,8-dihydro-L-Biopterin, 6,7-dimethyltetrahydropterin
hydrochloride, 8-bromo-cGMP, N-acetyl-serotonin (NAS),
N-Chloroacetylserotonin, N-Methoxyacetylserotonin, and
N-Chloroacetyldopamine.
[0012] Alternatively, the composition of the invention may contain
a compound having the formula:
##STR00001##
such that R.sup.1 is a H, C.sub.1-6 alkyl, halo, NO.sub.2, CN,
CO.sub.2R.sup.4, CONR.sup.4R.sup.5, SO.sub.2R.sup.4,
SO.sub.2NR.sup.4R.sup.5, OR.sup.4, or NR.sup.4R.sup.5. Each of
R.sup.4 and R.sup.5 may be, independently, a H, C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl; R.sup.2 may be a H, C.sub.1-6 alkyl, C.sub.6-12
aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4 alkheteroaryl;
and R.sup.3 may be a H, C.sub.1-6 alkyl, C.sub.6-12 aryl,
heteroaryl, C.sub.1-4 alkaryl, C.sub.1-4 alkheteroaryl,
CO.sub.2R.sup.6, CONR.sup.7R.sup.8, SO.sub.2R.sup.6, or
SO.sub.2NR.sup.7R.sup.8, where R.sup.6 is a C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl, and each of R.sup.7 and R.sup.8 is, independently, a
H, C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl,
or C.sub.1-4 alkheteroaryl.
[0013] Alternatively, R.sup.3 may be as above and R.sup.1 and
R.sup.2 together may be represented by
##STR00002##
where the N, O, or S of the R.sup.1/R.sup.2 linkage forms a bond to
the pyrimidinone ring and each of R.sup.9, R.sup.10, R.sup.11,
R.sup.12, and R.sup.13 is, independently, a H, C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl.
[0014] Optionally, R.sup.3 may be as above and R.sup.1 and R.sup.2
together may be represented by
##STR00003##
where the N of the R.sup.1/R.sup.2 linkage forms a bond to the
pyrimidinone ring, each of R.sup.12 or R.sup.13 is as above, and
R.sup.14 is a OR.sup.4, halo, NO.sub.2, CN, CO.sub.2R.sup.7,
CONR.sup.7R.sup.8, SO.sub.2R.sup.7, SO.sub.2NR.sup.7R.sup.8,
C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or
C.sub.1-4 alkheteroaryl, such that each of R.sup.7 and R.sup.8 is,
independently, a H, C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.12 aryl,
heteroaryl, C.sub.1-C.sub.4 alkaryl, or C.sub.1-C.sub.4
alkheteroaryl.
[0015] As another alternative, R.sup.1 and R.sup.2 together may be
represented by
##STR00004##
where the N of the R.sup.1/R.sup.2 linkage forms a bond to the
pyrimidinone ring, each of R.sup.9, R.sup.10, R.sup.11, and
R.sup.14 are as above, R.sup.3 does not exist, and a double bond is
formed between the carbon bearing the R.sup.14 and the nitrogen
bearing R.sup.2.
[0016] Optionally, the composition may have be a compound of
formula (I), such that R.sup.1 is a H, C.sub.1-6 alkyl, halo,
NO.sub.2, CN, CO.sub.2R.sup.4, CONR.sup.4R.sup.5, SO.sub.2R.sup.4,
SO.sub.2NR.sup.4R.sup.5, OR.sup.4, or NR.sup.4R.sup.5. Accordingly,
each of R.sup.4 and R.sup.5 may be, independently, a H, C.sub.1-6
alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl, R.sup.2 may be a H, C.sub.1-6 alkyl, C.sub.6-12
aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4 alkheteroaryl,
and R.sup.3 may be a H, C.sub.1-6 alkyl, C.sub.6-12 aryl,
heteroaryl, C.sub.1-4 alkaryl, C.sub.1-4 alkheteroaryl,
CO.sub.2R.sup.6, CONR.sup.7R.sup.8, SO.sub.2R.sup.6, or
SO.sub.2NR.sup.7R.sup.8, where R.sup.6 is a C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl and each of R.sup.7 and R.sup.8 may be,
independently, a H, C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl,
C.sub.1-4 alkaryl, or C.sub.1-4 alkheteroaryl.
[0017] If desired, the composition contains a compound of
formula:
##STR00005##
in which R.sup.1 and R.sup.2 together may be represented by
##STR00006##
such that the N, O, or S of the R.sup.1/R.sup.2 linkage forms a
bond to the pyrimidinone ring and each of R.sup.9, R.sup.10,
R.sup.11, R.sup.12, and R.sup.13 is, independently, a H, C.sub.1-6
alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl.
[0018] Alternatively, the composition contains a compound of
formula:
##STR00007##
in which R.sup.3 is as above and R.sup.1 and R.sup.2 together are
represented by
##STR00008##
where the N of the R.sup.1/R.sup.2 linkage forms a bond to the
pyrimidinone ring, each of R.sup.12 and R.sup.13 is as above, and
R.sup.14 is a OR.sup.4, halo, NO.sub.2, CN, CO.sub.2R.sup.7,
CONR.sup.7R.sup.8, SO.sub.2R.sup.7, SO.sub.2NR.sup.7R.sup.8,
C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or
C.sub.1-4 alkheteroaryl, where each of R.sup.4, R.sup.7 and R.sup.8
is, independently, a H, C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.12
aryl, C.sub.1-C.sub.4 alkaryl, heteroaryl, or C.sub.1-C.sub.4
alkheteroaryl.
[0019] Alternatively, the composition contains a compound of
formula:
##STR00009##
in which R.sup.1 and R.sup.2 together may be represented by
##STR00010##
where the N of the R.sup.1/R.sup.2 linkage forms a bond to the
pyrimidinone ring, each of R.sup.9, R.sup.10, R.sup.11, and
R.sup.14 are as above, R.sup.3 does not exist, and a double bond is
formed between the carbon bearing R.sup.14 and the nitrogen bearing
R.sup.2.
[0020] If the composition contains a compound of formula:
##STR00011##
R.sup.1 and R.sup.2 together may be represented by
##STR00012##
where the N, O, or S of the R.sup.1/R.sup.2 linkage forms a bond to
the pyrimidinone ring and each of R.sup.9, R.sup.10, R.sup.11,
R.sup.12, and R.sup.13 is, independently, a H, C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl.
[0021] Alternatively, the composition contains a compound of
formula:
##STR00013##
in which R.sup.3 is as above and R.sup.1 and R.sup.2 together are
represented by
##STR00014##
where the N of the R.sup.1/R.sup.2 linkage forms a bond to the
pyrimidinone ring, each of R.sup.12 and R.sup.13 is as above, and
R.sup.14 is a OR.sup.4, halo, NO.sub.2, CN, CO.sub.2R.sup.7,
CONR.sup.7R.sup.8, SO.sub.2R.sup.7, SO.sub.2NR.sup.7R.sup.8,
C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or
C.sub.1-4 alkheteroaryl, such that each of R.sup.7 and R.sup.8 is,
independently, H, C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.12 aryl,
C.sub.1-C.sub.4 alkaryl, heteroaryl, or C.sub.1-C.sub.4
alkheteroaryl.
[0022] Optionally, the composition may contain a compound of
formula:
##STR00015##
in which R.sup.1 and R.sup.2 together are represented by
##STR00016##
where the N of the R.sup.1/R.sup.2 linkage forms a bond to the
pyrimidinone ring, each of R.sup.9, R.sup.10, R.sup.11, and
R.sup.14 are as above, R.sup.3 does not exist, and a double bond is
formed between the carbon bearing R.sup.14 and the nitrogen bearing
R.sup.2.
[0023] Exemplary compounds that may be contained within the
composition of the invention include, for example:
##STR00017##
[0024] Furthermore, the composition of the invention may contain a
compound having the formula:
##STR00018##
in which R.sup.15 is a H, C.sub.1-6 alkyl, C.sub.6-12 aryl,
heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4 alkheteroaryl and
R.sup.16 is a H, C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl,
C.sub.1-4 alkaryl, C.sub.1-4 alkheteroaryl, CO.sub.2R.sup.17,
CONR.sup.18R.sup.19, SO.sub.2R.sup.17, or
SO.sub.2NR.sup.18R.sup.19. R.sup.17 may be a C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl and each of R.sup.18 and R.sup.19 may be,
independently, a H, C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl,
C.sub.1-4 alkaryl, or C.sub.1-4 alkheteroaryl.
[0025] If desired, a second therapeutic agent, such as an analgesic
agent, may be administered in combination with the composition of
the invention, including for example, a non-steroidal
anti-inflammatory agent (NSAIDs) (including acetaminophen,
non-COX-2 selective agents, or COX-2 selective inhibitory drugs),
opioid receptor agonist (e.g., morphine, codeine, hydrocodone,
hydromorphone, levorphanol, methadone, meperidine, butorphanol,
bupranorphine, nalbuphine, alfentanil, sufentanil, fentanyl,
tramadol, pentazocine, propoxyphene, or oxycodone), tricyclic
antidepressant (e.g., doxepin, amitriptyline, imipramine,
nortriptyline, desipramine, or venlafaxine), SSRIs (e.g.
paroxetine, sertraline, fluoxetine, or citalopram), anticonvulsants
(e.g., phenytoin, carbamazepine, oxcarbazepine, lamotrigine,
valproate, pregabalin, or gabapentin), voltage-gated sodium channel
blockers, membrane stabilizers, nerve blockers (e.g. lidocaine,
bupivicaine, prilocaine, or mexilitine), N-type calcium channel
blockers (e.g ziconitide), serotonin receptor agonists such as
5-HT1D receptor agonists (e.g. sumatriptam, zolmatriptan,
rizotriptan, naratriptan, almotriptan, or frovatriptan), steroids
(e.g. cortisone, hydrocrtisone, prednisolone, or
methylprednisolone). According to this invention, the second
therapeutic agent may or may not have a therapeutic effect (such as
analgesia) when administered as a single agent but results in such
an effect (or an additive or synergistic increase) when
administered in combination with the composition of the invention.
Other exemplary analgesic agents useful in the invention may
include, for example, acetaminophen, acetylsalicylic acid,
ibuprofen, naproxen, fenoprofen, indomethacin, ketorolac,
rofecoxib, celecoxib, valdecoxib, paracoxib, clonazepam, diazepam,
capsaicin, ketamine, clonidine, or baclofen.
[0026] Alternatively, the second therapeutic agent may be an
inhibitor of any enzyme which utilizes BH4 as a cofactor including
any of the following enzymes: all isoforms of nitric oxide synthase
(NOS) such as eNOS, iNOS, or nNOS; tyrosine hydroxylase; tryptophan
hydroxylase I (non-neuronal Tph1) and II (neuronal Tph2);
phenylalanine hydroxylase; dopamine-.beta.-hydroxylase;
N-methyltransferase; and ether lipid oxidase. The second
therapeutic agent may also include agents that inhibit direct
effects of BH4 independent of its co-enzyme function, such as
agents that interfere with BH4 binding to membrane bound
receptors.
[0027] The composition of the invention and the second therapeutic
agent may be administered together (as two separate formulations or
a single formulation) or separately (e.g., within one hour, two
hours, three hours, six hours, or twenty four hours of each
other).
[0028] In another aspect, the invention features a method for
diagnosing pain or a traumatic, metabolic or toxic peripheral nerve
lesion in a mammal by measuring the levels of BH4, BH4 precursors,
or intermediates (e.g., 7,8-dihydroneopterin triphosphate and
6-pyruvoyl tetrahydropterin), or BH4 metabolites (e.g., pterin,
bipterin, 7,8 dihydropterin, 7,8 dihydroxanthopterin, xanthopterin,
isoxanthopterin, leucopterin, or neopterin) in a biological sample
of a mammal (e.g., in the serum, plasma, urine, cerebrospinal
fluid, synovial fluid, tissue exudates, or tissue samples).
According to this aspect of the invention, levels of BH4, BH4
precursors, BH4 intermediates, or BH4 metabolites serve as
biomarkers of pain such that an increase in any of these molecules
diagnoses pain in the mammal. Alternatively, the clinical diagnosis
of pain may be supported in a mammal by measuring the level (e.g.,
mRNA or protein levels) or activity of any one of the BH4 synthetic
enzymes (e.g., sepiapeterin reductase (SPR),
Pyruvoyltetrahydropterin Synthase (PTPS), GTP cyclohydrolase
(GTPCH), Pterin-4.alpha.-carbinolamine dehydratase, and
dihydropteridine reductase (DHPR)) in the mammal. Similarly, the
pain diagnosis is supported by determining an increase in the
activity or level of such enzymes.
[0029] The present invention also provides a method for identifying
a candidate compound for treating, reducing, or preventing pain or
endogenous mechanisms that further increase a traumatic, metabolic
or toxic peripheral nerve lesion in a mammal. The method involves
the steps of: (a) contacting a cell synthesizing BH4 with a
candidate compound; and (b) measuring the BH4 level or activity
(e.g., ability to function as a co-factor or ability to bind
membrane-bound receptors). A compound that decreases the level or
activity of BH4 relative to the BH4 level or activity in a cell not
contacted with the compound is identified as a candidate compound
for treating, reducing, or preventing pain or a traumatic,
metabolic or toxic peripheral nerve lesion in a mammal.
Alternatively, the cell in step (a) may express any one of the BH4
synthetic enzymes (e.g., sepiapeterin reductase (SPR),
Pyruvoyltetrahydropterin Synthase (PTPS), GTP cyclohydrolase
(GTPCH), Pterin-4.alpha.-carbinolamine dehydratase, and
dihydropteridine reductase (DHPR)) and, optionally, step (b)
involves measuring the expression or biological activity of such
enzymes to assess the inhibitory activity of the candidate
compound. If desired, the BH4 synthetic enzyme may be a protein
fusion gene. In preferred embodiments, step (b) involves the
measurement of BH4 levels or activity, or alternatively, the
measurement of the mRNA or protein levels or enzyme activity of one
of the BH4 synthetic enzymes. Preferably, the cell is a mammalian
cell (e.g., a human or rodent cell).
[0030] In a related aspect, the invention provides an alternative
method for identifying a candidate compound for treating, reducing,
or preventing pain or a traumatic, metabolic or toxic peripheral
nerve lesion in a mammal. This method involves the steps of: (a)
identifying a pteridine or specific BH4 binding site on a protein
(e.g., a BH4-binding receptor or BH4-dependent enzyme); (b)
contacting such a BH4 binding protein (receptor or enzyme) with a
candidate compound; and (c) determining whether the candidate
compound binds to the BH4 site on the protein and inhibits BH4
binding or activity on the protein (for example, by itself binding
to the BH4 site). Compounds that inhibit BH4 activity or binding
are identified as candidate compounds for treating, reducing, or
preventing pain or a a traumatic, metabolic or toxic peripheral
nerve lesion. Optionally, this method may further involve
contacting the candidate compound with any one of the enzymes that
use BH4 as a cofactor (e.g., NOS) or membrane receptors that bind
BH4 and determining whether the candidate compound binds and/or
inhibits the activity of such an enzyme or receptor.
[0031] The invention also provides yet another method for
identifying a candidate compound for treating, reducing, or
preventing pain or a traumatic, metabolic or toxic a peripheral
nerve lesion in a mammal. This method involves the steps of: (a)
providing GTP cyclohydrolase (GTPCH), GTPCH Feedback Regulatory
Protein (GFRP), and a candidate compound; (b) measuring the binding
of the GTPCH and the GFRP; and (c) identifying a candidate compound
as useful for treating, reducing, or preventing pain or a
traumatic, metabolic or toxic peripheral nerve lesion, wherein the
binding of the GTPCH and GFRP is increased in the presence of the
candidate compound. In preferred embodiments, the GTPCH and GFRP
are human proteins. In this method, the candidate compound may
preferably bind to either GFRP or the GTPCH:GFRP complex.
[0032] In preferred embodiments, the method also tests the ability
of the candidate compound to reduce the expression of one or all of
the BH4 synthetic enzymes in a cell, for example, a mammalian cell
such as a rodent or human cell leading to a reduction in BH4
levels. Most preferably, the BH4 synthetic enzyme gene is human
sepiapterin reductase (SPR), Pyruvoyltetrahydropterin Synthase
(PTPS), GTP cyclohydrolase I (GTPCH I),
Pterin-4.alpha.-carbinolamine dehydratase, or dihydropteridine
reductase (DHPR).
[0033] The present invention further includes kits for carrying out
the methods of the invention. For example, the invention includes a
kit containing a composition that reduces the level and activity of
tetrahydrobiopterin (BH4) in an amount sufficient to treat, reduce,
or prevent pain or a traumatic, metabolic or toxic peripheral nerve
lesion as well as instructions for delivery of the composition to a
mammal for treating, reducing, or preventing pain or a traumatic,
metabolic or toxic peripheral nerve lesion.
[0034] The present invention further includes a diagnostic kit for
the measurement of BH4, its precursors and intermediates (e.g.,
7,8-dihydroneopterin triphosphate and 6-pyruvoyl tetrahydropterin),
or metabolites (e.g., pterin, biopterin, 7,8 dihydropterin, 7,8
dihydroxanthopterin, xanthopterin, isoxanthopterin, leucopterin,
and neopterin) from a biological sample of a mammal (e.g., serum,
plasma, urine, cerebrospinal fluid, synovial fluid, tissue
exudates, and tissue samples). For example, the invention includes
a kit containing an antibody that is specific to BH4 as well as
instructions for diagnosing pain in a mammal.
[0035] The invention also features methods for identifying a BH4
target protein. The first method consists of the steps of (a)
providing a sample and BH4; (b) contacting the sample with the BH4
under conditions that allow binding between the sample proteins and
the BH4; and (c) assessing the binding of the BH4 to a sample
protein by detecting the BH4, wherein a sample protein that binds
to BH4 is identified as a BH4 target protein. In one embodiment,
the assessing step (c) uses a detection method such as mass
spectrometry, surface plasmon resonance microscopy, or atomic force
microscopy. Biological samples which can be used in this method to
identify BH4 target proteins may come from any source. Preferred
samples are extracts prepared from mammalian nervous tissue such
as, for example, nervous tissue containing the dorsal horn or the
dorsal root ganglia. Preferably, samples contain membrane-bound
proteins.
[0036] A second method for identifying a BH4 target protein
contains the steps of (a) providing BH4 and an array, wherein the
array consists of a plurality of immobilized purified protein
species, wherein each of the protein species in the array is
spatially separated from each of the other protein species; (b)
contacting the array with the BH4 under conditions that allow
binding between the protein species and the BH4; and (c) assessing
the binding of the BH4 to the protein species, wherein a protein
species that binds to the BH4 is identified as a BH4 target
protein.
[0037] In preferred embodiments of either of the two foregoing
methods, the BH4 is detectably labeled. Useful detectable labels
include, for example, a radioisotope (e.g., tritium) or biotin. In
other useful embodiments, the assessing step (c) requires the use
of a BH4-specific antibody.
[0038] Finally, the invention features compositions for reducing
BH4 biological activity.
[0039] Compositions of the invention may have the formula:
##STR00019##
such that R.sup.1 is a H, C.sub.1-6 alkyl, halo, NO.sub.2, CN,
CO.sub.2R.sup.4, CONR.sup.4R.sup.5, SO.sub.2R.sup.4,
SO.sub.2NR.sup.4R.sup.5, OR.sup.4, or NR.sup.4R.sup.5. Each of
R.sup.4 and R.sup.5 may be, independently, a H, C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl; R.sup.2 may be a H, C.sub.1-6 alkyl, C.sub.6-12
aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4 alkheteroaryl;
and R.sup.3 may be a H, C.sub.1-6 alkyl, C.sub.6-12 aryl,
heteroaryl, C.sub.1-4 alkaryl, C.sub.1-4 alkheteroaryl,
CO.sub.2R.sup.6, CONR.sup.7R.sup.8, SO.sub.2R.sup.6, or
SO.sub.2NR.sup.7R.sup.8, where R.sup.6 is a C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl, and each of R.sup.7 and R.sup.8 is, independently, a
H, C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl,
or C.sub.1-4 alkheteroaryl.
[0040] Alternatively, R.sup.3 may be as above and R.sup.1 and
R.sup.2 together may be represented by
##STR00020##
where the N, O, or S of the R.sup.1/R.sup.2 linkage forms a bond to
the pyrimidinone ring and each of R.sup.9, R.sup.10, R.sup.11,
R.sup.12, and R.sup.13 is, independently, a H, C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl.
[0041] Optionally, R.sup.3 may be as above and R.sup.1 and R.sup.2
together may be represented by
##STR00021##
where the N of the R.sup.1/R.sup.2 linkage forms a bond to the
pyrimidinone ring, each of R.sup.12 or R.sup.13 is as above, and
R.sup.14 is a OR.sup.4, halo, NO.sub.2, CN, CO.sub.2R.sup.7,
CONR.sup.7R.sup.8, SO.sub.2R.sup.7, SO.sub.2NR.sup.7R.sup.8,
C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or
C.sub.1-4 alkheteroaryl, such that each of R.sup.7 and R.sup.8 is,
independently, a H, C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.12 aryl,
heteroaryl, C.sub.1-C.sub.4 alkaryl, or C.sub.1-C.sub.4
alkheteroaryl.
[0042] As another alternative, R.sup.1 and R.sup.2 together may be
represented by
##STR00022##
where the N of the R.sup.1/R.sup.2 linkage forms a bond to the
pyrimidinone ring, each of R.sup.9, R.sup.10, R.sup.11, and
R.sup.14 are as above, R.sup.3 does not exist, and a double bond is
formed between the carbon bearing the R.sup.14 and the nitrogen
bearing R.sup.2.
[0043] Optionally, the composition may have be a compound of
formula (I), such that R.sup.1 is a H, C.sub.1-6 alkyl, halo,
NO.sub.2, CN, CO.sub.2R .sup.4, CONR.sup.4R.sup.5, SO.sub.2R.sup.4,
SO.sub.2NR.sup.4R.sup.5, OR.sup.4, or NR.sup.4R.sup.5. Accordingly,
each of R.sup.4 and R.sup.5 may be, independently, a H, C.sub.1-6
alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl, R.sup.2 may be a H, C.sub.1-6 alkyl, C.sub.6-12
aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4 alkheteroaryl,
and R.sup.3 may be a H, C.sub.1-6 alkyl, C.sub.6-12 aryl,
heteroaryl, alkaryl, C.sub.1-4 alkheteroaryl, CO.sub.2R.sup.6,
CONR.sup.7R.sup.8, SO.sub.2R.sup.6, or SO.sub.2NR.sup.7R.sup.8,
where R.sup.6 is a C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl,
C.sub.1-4 alkaryl, or C.sub.1-4 alkheteroaryl and each of R.sup.7
and R.sup.8 may be, independently, a H, C.sub.1-6 alkyl, C.sub.6-12
aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl.
[0044] If desired, the composition contains a compound of
formula:
##STR00023##
in which R.sup.1 and R.sup.2 together may be represented by
##STR00024##
such that the N, O, or S of the R.sup.1/R.sup.2 linkage forms a
bond to the pyrimidinone ring and each of R.sup.9, R.sup.10,
R.sup.11, R.sup.12, and R.sup.13 is, independently, a H, C.sub.1-6
alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl.
[0045] Alternatively, the composition contains a compound of
formula:
##STR00025##
in which R.sup.3 is as above and R.sup.1 and R.sup.2 together are
represented by
##STR00026##
where the N of the R.sup.1/R.sup.2 linkage forms a bond to the
pyrimidinone ring, each of R.sup.12 and R.sup.13 is as above, and
R.sup.14 is a OR.sup.4, halo, NO.sub.2, CN, CO.sub.2R.sup.7,
CONR.sup.7R.sup.8, SO.sub.2R.sup.7, SO.sub.2NR.sup.7R.sup.8,
C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or
C.sub.1-4 alkheteroaryl, where each of R.sup.4, R.sup.7 and R.sup.8
is, independently, a H, C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.12
aryl, C.sub.1-C.sub.4 alkaryl, heteroaryl, or C.sub.1-C.sub.4
alkheteroaryl.
[0046] Alternatively, the composition contains a compound of
formula:
##STR00027##
in which R.sup.1 and R.sup.2 together may be represented by
##STR00028##
where the N of the R.sup.1/R.sup.2 linkage forms a bond to the
pyrimidinone ring, each of R.sup.9, R.sup.10, R.sup.11, and
R.sup.14 are as above, R.sup.3 does not exist, and a double bond is
formed between the carbon bearing R.sup.14 and the nitrogen bearing
R.sup.2.
[0047] If the composition contains a compound of formula:
##STR00029##
R.sup.1 and R.sup.2 together may be represented by
##STR00030##
where the N, O, or S of the R.sup.1/R.sup.2 linkage forms a bond to
the pyrimidinone ring and each of R.sup.9, R.sup.10, R.sup.11,
R.sup.12, and R.sup.13 is, independently, a H, C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl.
[0048] Alternatively, the composition contains a compound of
formula:
##STR00031##
in which R.sup.3 is as above and R.sup.1 and R.sup.2 together are
represented by
##STR00032##
where the N of the R.sup.1/R.sup.2 linkage forms a bond to the
pyrimidinone ring, each of R.sup.12 and R.sup.13 is as above, and
R.sup.14 is a OR.sup.4, halo, NO.sub.2, CN, CO.sub.2R.sup.7,
CONR.sup.7R.sup.8, SO.sub.2R.sup.7, SO.sub.2NR.sup.7R.sup.8,
C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or
C.sub.1-4 alkheteroaryl, such that each of R.sup.7 and R.sup.8 is,
independently, H, C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.12 aryl,
C.sub.1-C.sub.4 alkaryl, heteroaryl, or C.sub.1-C.sub.4
alkheteroaryl.
[0049] Optionally, the composition may contain a compound of
formula:
##STR00033##
in which R.sup.1 and R.sup.2 together are represented by
##STR00034##
where the N of the R.sup.1/R.sup.2 linkage forms a bond to the
pyrimidinone ring, each of R.sup.9, R.sup.10, R.sup.11, and
R.sup.14 are as above, R.sup.3 does not exist, and a double bond is
formed between the carbon bearing R.sup.14 and the nitrogen bearing
R.sup.2.
[0050] Exemplary compounds that may be contained within the
composition of the invention include, for example:
##STR00035##
[0051] Furthermore, the composition of the invention may contain a
compound having the formula:
##STR00036##
in which R.sup.15 is a H, C.sub.1-6 alkyl, C.sub.6-12 aryl,
heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4 alkheteroaryl and
R.sup.16 is a H, C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl,
C.sub.1-4 alkaryl, C.sub.1-4 alkheteroaryl, CO.sub.2R.sup.17,
CONR.sup.18R.sup.19, SO.sub.2R.sup.17, or
SO.sub.2NR.sup.18R.sup.19. R.sup.17 may be a C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl and each of R.sup.18 and R.sup.19 may be,
independently, a H, C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl,
C.sub.1-4 alkaryl, or C.sub.1-4 alkheteroaryl.
[0052] The compositions of the invention may be combined with any
second therapeutic suitable for use in the above-described methods.
The compositions alone or in combination with any second
therapeutic may be present in a therapeutic composition in
association with a pharmaceutically acceptable carrier or
excipient.
[0053] By "BH4 synthetic enzyme fusion gene" is meant a promoter
and/or all or part of a coding region of a BH4 synthetic enzyme
(e.g., sepiapeterin reductase (SPR), Pyruvoyltetrahydropterin
Synthase (PTPS), GTP cyclohydrolase (GTPCH),
Pterin-4.alpha.-carbinolamine dehydratase, and dihydropteridine
reductase (DHPR)) operably linked to a second, heterologous nucleic
acid sequence. In preferred embodiments, the second, heterologous
nucleic acid sequence is a reporter gene, that is, a gene whose
expression may be assayed; exemplary reporter genes include,
without limitation, those genes encoding glucuronidase (GUS),
luciferase, chloramphenicol transacetylase (CAT), green fluorescent
protein (GFP), alkaline phosphatase, and (3-galactosidase.
[0054] By a "candidate compound" is meant a chemical, be it
naturally-occurring or artificially-derived. Candidate compounds
include, for example, peptides, polypeptides, synthetic organic
molecules, naturally occurring organic molecules, nucleic acids,
peptide nucleic acids, and components thereof.
[0055] By "dominant negative protein" is meant any polypeptide
having at least 50%, 70%, 80%, 90%, 95%, or even 99% sequence
identity to 10, 20, 35, 50, 100, 150, or more than 150 amino acids
of the wild type protein to which the dominant negative protein
corresponds. In addition to inactivating mutations, dominant
negative proteins may consist of deletions or truncations of a
wild-type molecule. For example, a dominant negative BH4 synthetic
enzyme may be a truncated BH4 synthetic enzyme mutant that has a
deletion such that it no longer functions to produce BH4 or its
intermediates (e.g., 7,8-dihydroneopterin triphosphate and
6-pyruvoyl tetrahydropterin) including, for example, a GTPCH that
has lost its catalytic activity, thereby disrupting the BH4
synthetic pathway.
[0056] By "opioid receptor agonist" is meant any naturally
occurring, semi-synthetic, or synthetic compound that binds to the
mu, kappa, or delta opioid receptor subtypes and mimics the
function of opioids at these receptors. The opioid receptor agonist
may be a peptide or a non-peptide compound. Preferably, opioid
receptor agonists have a K.sub.d for at least one opioid receptor
subtype of <1 .mu.M, more preferably <100 nM, most preferably
<10 nM, or even <1 nM. Opioid receptor agonists include
generally, for example, members from the phenanthrene, phenyl
heptylamine, phenylpiperidine, morphinan, and benzomorphan chemical
families. Opioid receptor agonists include, for example, morphine,
hydormorphone, oxymorphone, codeine, oxycodone, hydrocodone,
methadone, meperidine, levorphanol, nalbuphine, sufentanil,
alfentanil, buprenorphine, pentazocine, propoxyphene and
butorphanol.
[0057] By "an effective amount" is meant an amount of a compound,
alone or as part of a combination according to the invention,
required to prevent, reduce, or eliminate the sensation of pain.
The effective amount of active compound(s) used to practice the
present invention for therapeutic treatment of pain varies
depending upon the manner of administration, the age, and body
weight, of the subject as well as the underlying pathology that is
causing the pain. Ultimately, the attending physician or
veterinarian will decide the appropriate amount and dosage regimen.
Such amount is referred to as an "effective" amount.
[0058] By "pain" is meant all types of pain including, for example,
peripheral and central neuropathic pain, functional pain,
inflammatory pain or nociceptive pain, whether acute or chronic.
Exemplary pain conditions include post-operative or post-traumatic
pain, chronic lower back pain, pain of rheumatoid arthritis,
osteoarthritis, fibromyalgia, cluster headaches, post-herpetic
neuralgia, phantom limb pain, central stroke pain, dental pain,
opioid-resistant pain, visceral pain, bone injury pain, labor pain,
pain resulting from burns including sunburns, post-partum pain,
migraine, tension type headache, angina pain, and genitourinary
tract-related pain (e.g., cystitis).
[0059] By "reduce the tetrahydrobiopterin (BH4) biological
activity" is meant to reduce a functional outcome associated with a
biological activity attributed to BH4. Generally, the reduction of
BH4 biological activity may be the result of, for example, a
reduction in the amount (level) of the BH4 molecules and may be
affected by reducing/inhibiting de novo BH4 synthesis,
increasing/accelerating BH4 catabolism, or a combination of the
two.
[0060] Alternatively, the biological activity of BH4 may be reduced
by inhibiting the effect of BH4 at a target molecule. For example,
a competitive BH4 inhibitor that binds to a BH4 binding site on an
effector protein (e.g, an enzyme) will reduce the biological
activity. Non-competitive BH4 inhibitors are also included in this
definition. Likewise, a BH4 binding molecule which effectively
sequesters BH4 and prevents it from binding to an effector molecule
also has the effect of reducing BH4 biological activity. Such
reduction may be, for example, a decrease of least 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100%, relative
to control conditions. More specifically, BH4 level or activity may
be decreased, for example, by reducing the enzyme activity of
enzymes involved in the BH4 synthesis pathway, such as GTP
cyclohydrolase (GTPCH), Sepiapterin Reductase (SPR), and
Dihydropteridine reductase (DHPR). Preferably, such enzyme activity
is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
100%, or even greater than 100%, relative to a control. GTPCH may,
for example, be inhibited by increasing the levels or binding
activity of GTPCH Feedback Regulatory Protein (GFRP).
[0061] By "treating, reducing, or preventing pain" is meant
preventing, reducing, or eliminating the sensation of pain in a
subject before, during, or after it has occurred. As compared with
an equivalent untreated control, such reduction or degree of
prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%,
or 100% as measured by any standard technique known in the art. To
treat pain, according to the methods of this invention, the
treatment does not necessarily provide therapy for the underlying
pathology that is causing the painful sensation. Treatment of pain
can be purely symptomatic.
[0062] By "diagnosing pain" is meant detecting pain caused by any
stimulus in a mammal including damage to peripheral nerves. For
example, pain may be diagnosed by detecting a surrogate marker that
is associated or correlated with the sensation of pain. According
to the invention, pain or a traumatic, metabolic or toxic
peripheral nerve lesion is diagnosed by measuring and detecting an
increase in the levels of BH4, BH4 intermediates, BH4 precursors,
or BH4 metabolites in a biological sample from the mammal (e.g.,
serum, plasma, urine, cerebrospinal fluid, synovial fluid, tissue
exudates, or tissue samples). Pain is diagnosed in a mammal if such
levels are increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 80%,
90%, 95%, 100, or more than 100% above a control. Alternatively,
the levels or activity of any one of BH4 synthetic enzymes are
measured and pain is diagnosed if an increase in the level or
activity of a BH4 enzyme is detected. Desirably, such increase is
at least 10%, 20%, 30%, 40%, 50%, 60%, 80%, 90%, 95%, 100%, or more
than 100% above control conditions.
[0063] By "specific for" as used herein in reference to an antibody
is meant an increased affinity of an antibody for a particular
protein or antigen, relative to an equal amount of any other
protein or antigen. For example, an antibody (e.g., a human
monoclonal antibody) that is specific for BH4 desirably has an
affinity for BH4 that is least 2-fold, 5-fold, 10-fold, 30-fold, or
100-fold greater than for an equal amount of any other antigen,
including related antigens. Binding of an antibody to another
protein or antigen may be determined as described herein, and by
any number of standard methods in the art, e.g., Western analysis,
ELISA, or co-immunoprecipitation.
[0064] By "substantially identical," when referring to a protein or
polypeptide, is meant a protein or polypeptide exhibiting at least
75%, but preferably 85%, more preferably 90%, most preferably 95%,
or even 99% identity to a reference amino acid sequence. For
proteins or polypeptides, the length of comparison sequences will
generally be at least 20 amino acids, preferably at least 30 amino
acids, more preferably at least 40 amino acids, and most preferably
50 amino acids or the full length protein or polypeptide. Nucleic
acids that encode such "substantially identical" proteins or
polypeptides constitute an example of "substantially identical"
nucleic acids; it is recognized that the nucleic acids include any
sequence, due to the degeneracy of the genetic code, that encodes
those proteins or polypeptides. In addition, a "substantially
identical" nucleic acid sequence also includes a polynucleotide
that hybridizes to a reference nucleic acid molecule under high
stringency conditions.
[0065] By "high stringency conditions" is meant any set of
conditions that are characterized by high temperature and low ionic
strength and allow hybridization comparable with those resulting
from the use of a DNA probe of at least 40 nucleotides in length,
in a buffer containing 0.5 M NaHPO.sub.4, pH 7.2, 7% SDS, 1 mM
EDTA, and 1% BSA (Fraction V), at a temperature of 65 C, or a
buffer containing 48% formamide, 4.8.times.SSC, 0.2 M Tris-Cl, pH
7.6, 1.times. Denhardt's solution, 10% dextran sulfate, and 0.1%
SDS, at a temperature of 42 C. Other conditions for high stringency
hybridization, such as for PCR, Northern, Southern, or in situ
hybridization, DNA sequencing, etc., are well known by those
skilled in the art of molecular biology. See, e.g., F. Ausubel et
al., Current Protocols in Molecular Biology, John Wiley & Sons,
New York, N.Y., 1998, hereby incorporated by reference.
[0066] As used herein, the terms "alkyl" and the prefix "alk-" are
inclusive of both straight chain and branched chain saturated or
unsaturated groups, and of cyclic groups, including cycloalkyl and
cycloalkenyl groups. Unless otherwise specified, acyclic alkyl
groups contain 1 to 6 carbons. Cyclic groups can be monocyclic or
polycyclic and preferably have 3 to 8 ring carbon atoms. Exemplary
cyclic groups include cyclopropyl, cyclopentyl, cyclohexyl, and
adamantyl groups. The alkyl group may be substituted or
unsubstituted. Exemplary substituents include alkoxy, aryloxy,
sulfhydryl, alkylthio, arylthio, halogen, hydroxyl, fluoroalkyl,
perfluoralkyl, amino, aminoalkyl, disubstituted amino, quaternary
amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups.
[0067] By "aryl" is meant a carbocyclic aromatic ring or ring
system. Unless otherwise specified, aryl groups contain 6 to 18
carbons. Examples of aryl groups include phenyl, naphthyl,
biphenyl, fluorenyl, and indenyl groups.
[0068] By "heteroaryl" is meant an aromatic ring or ring system
that contains at least one ring hetero-atom (e.g., O, S, Se. N, and
P). Unless otherwise specified, heteroaryl groups contain 1 to 9
carbons. Heteroaryl groups include furanyl, thienyl, pyrrolyl,
imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl,
isothiazolyl, triazolyl, tetrazolyl, oxadiazolyl, oxatriazolyl,
pyridyl, pyridazyl, pyrimidyl, pyrazyl, triazyl, benzofuranyl,
isobenzofuranyl, benzothienyl, indole, indazolyl, indolizinyl,
benzisoxazolyl, quinolinyl, isoquinolinyl, cinnolinyl,
quinazolinyl, naphtyridinyl, phthalazinyl, phenanthrolinyl,
purinyl, and carbazolyl groups.
[0069] By "heterocycle" is meant a non-aromatic ring or ring system
that contains at least one ring heteroatom (e.g., O, S, Se, N, and
P). Unless otherwise specified, heterocyclic groups contain 2 to 9
carbons. Heterocyclic groups include, for example, dihydropyrrolyl,
tetrahydropyrrolyl, piperazinyl, pyranyl, dihydropyranyl,
tetrahydropyranyl, dihydrofuranyl, tetrahydrofuranyl,
dihydrothiophene, tetrahydrothiophene, and morpholinyl groups.
[0070] Aryl, heteroaryl, or heterocyclic groups may be
unsubstituted or substituted by one or more substituents including
for example a C.sub.1-6 alkyl, hydroxy, halo, nitro, C.sub.1-6
alkoxy, C.sub.1-6alkylthio, trifluoromethyl, C.sub.1-6 acyl,
arylcarbonyl, heteroarylcarbonyl, nitrile, C.sub.1-6
alkoxycarbonyl, alkaryl (in which the alkyl group has 1 to 6 carbon
atoms), and alkheteroaryl (in which the alkyl group has 1 to 6
carbon atoms).
[0071] By "alkoxy" is meant a chemical substituent of the formula
--OR, where R is an alkyl group. By "aryloxy" is meant a chemical
substituent of the formula --OR , where R is an aryl group. By
"alkaryl" is meant a chemical substituent of the formula --RR ,
where R is an alkyl group and R is an aryl group. By "alkheteraryl"
is meant a chemical substituent of the formula -RR , where R is an
alkyl group and R is a heteroaryl group.
[0072] By "halide" or "halogen" or "halo" is meant bromine,
chlorine, iodine, or fluorine.
[0073] Overall, the present invention provides significant
advantages over standard therapies for the diagnosis, treatment,
and prevention of pain. Based on our results, the administration of
a therapeutic agent (e.g., methotrexate) that reduces the level or
activity of BH4 attenuates pain in part by interfering with the
activity of enzymes that utilize BH4 as a co-factor or
membrane-bound receptors that bind to BH4 and modulate neuronal
excitability or transmitter release. The present invention further
allows the diagnosis of pain in a mammal by measuring and detecting
an increase in the levels of BH4, BH4 intermediates, BH4
metabolites, or BH4 precursors, or alternatively, by measuring and
detecting an increase in the activity or levels of any one the BH4
synthetic enzymes. In addition, the candidate compound screening
methods provided by the present invention allow for the
identification of novel therapeutics that modify the injury process
and mitigate the symptoms by reducing the synthesis or action of
BH4.
BRIEF DESCRIPTION OF THE DRAWINGS
[0074] FIG. 1A is a graph representing the microarray expression
profile analysis of twenty-three genes, including Dihydropteridine
reductase (DHPR), in the dorsal root ganglia (DRG) during
development and adulthood, before and following peripheral nerve
injury.
[0075] FIG. 1B represents a Northern slot blot analysis confirming
mRNA regulation of DHPR in the dorsal root ganglia during
development and in the adult, before and following peripheral nerve
injury.
[0076] FIG. 2A is a schematic diagram outlining the
Tetrahydrobiopterin (BH4) synthesis pathway.
[0077] FIG. 2B is a schematic diagram showing the catabolism of
tetrahydrobiopterin.
[0078] FIG. 3 is a table showing the change in expression levels of
members of the BH4 synthetic pathway before and following
peripheral nerve injury detected by microarrays.
[0079] FIG. 4A is a picture of a Northern blot analysis showing
mRNA expression of GTP cyclohydrolase (GTPCH) in the dorsal root
ganglia (DRG) before (naive) and following peripheral nerve
injury.
[0080] FIG. 4B is a series of photomicrographs showing non-isotopic
in situ localization of GTPCH mRNA in the DRG before (naive) and
following peripheral nerve injury.
[0081] FIG. 4C is a series of photomicrograph of DRGs showing
increased GTPCH mRNA in neurons 3 days and 3 weeks after a
peripheral nerve injury as detected by isotopic in situ
hybridization.
[0082] FIGS. 4D-4F are a series of graphs representing GTPCH, DHPR,
and sepiapterin reductase (SPR) mRNA levels as measured by Northern
Blot analysis in the DRG over a period of 14 days following
peripheral nerve injury (*p<0.05; **p<0.01;
***p<0001).
[0083] FIGS. 5A and 5B represent a series of graphs showing protein
levels and enzyme activity of GTPCH before and following peripheral
nerve injury (*p<0.05; **p<0.01; ***p<0001).
[0084] FIG. 5C is a bar graph showing BH4 levels in the DRG before
and following peripheral nerve injury.
[0085] FIG. 6A are a series of graphs showing the ratio of
experimental to naive intensity readings from triplicate Affymetrix
microarrays over time, of the various members of the BH4 synthetic
pathway in three different neuropathic pain models (spared nerve
injury (SNI), chronic constriction injury (CCI) and the spinal
nerve ligation (SNL) models. FIG. 6A shows the expression profile
in the DRG. Changes in the expression of BH4 synthetic enzymes in
the dorsal horn are shown in FIG. 6B. The enzymes are present both
in the DRG and dorsal horn.
[0086] FIGS. 7A and 7B are a series of graphs showing the ratio of
experimental to naive intensity readings from triplicate Affymetrix
microarrays over time of the various members of the BH4 synthetic
pathway in the DRG (7A) and dorsal horn (7B) after peripheral
inflammation produced by intraplantar complete Freund's
adjuvant.
[0087] FIGS. 8A-8D represent a series of graphs showing the effect
of DAHP on nociceptive behavior in the SNI model of neuropathic
pain. Figures on the left show the threshold to mechanical stimuli
applied with von Frey hairs (von Frey threshold). Figures on the
right show the duration of paw licking, shaking and lifting
following acetone application to the paw as a measure for cold
allodynia. In FIG. 8A, rats were treated with DAHP (180 mg/kg/d
i.p.) for three days starting three days after surgery (early
treatment). Nociceptive behavior was assessed daily before and
after drug injection. In FIG. 8B, the treatment period was extended
to five days (180 mg/kg/d i.p.) starting three days after surgery
and pain behavior was assessed daily before and after drug
injection and once every second day for an additional week after
stopping the daily drug injections. In FIG. 8C, treatment with DAHP
(180 mg/kg/d i.p. for 5 days) was started 17 days after surgery
i.e. in the chronic phase without any treatment in the early phase.
Nociceptive behavior was assessed once daily 3h after drug
injection. In FIG. 8D, animals received a continuous spinal
infusion of DAHP (6 mg/kg/d) for 14 days. The infusion was started
directly after surgery. Nociceptive behavior was assessed once
daily.
[0088] FIGS. 9A and 9B are two graphs showing the effect of DAHP on
nociceptive behavior in the CCI model of neuropathic pain. FIG. 9A
shows the threshold to mechanical stimuli applied with von Frey
hairs (von Frey threshold). FIG. 9B shows the duration of paw
licking, shaking and lifting following acetone application to the
paw as a measure for cold allodynia. DAHP treatment (180 mg/kg/d
i.p. for 5 days) was started 3 days after surgery and nociceptive
behavior was assessed daily before and after drug injection and
once every second day after stopping daily drug injections.
[0089] FIG. 10 shows the effect of DAHP on the flinching behavior
in the Formalin test. DAHP (180 mg/kg) was injected i.p. one hour
before injection of formalin into the hindpaw. Flinches were
counted for one hour starting right after formalin injection.
[0090] FIGS. 11A-11E are a series of graphs showing the effect of
DHAP on thermal hyperalgesia in the CFA model which is a model for
inflammatory pain. FIG. 11A shows the paw withdrawal latency in
response to radiant heat (Hargreaves test) of the CFA treated,
inflamed paw. On the left end of the graph, two doses of DAHP (180
mg/kg i.p.) were injected, the first 30 minutes before injection of
CFA into the hindpaw, the second 4 hours after CFA (indicated with
arrows D1 and D2). On the right, a single dose (dotted arrow D1) of
DAHP (180 mg/kg i.p.) was injected 24 h after induction of paw
inflammation without any treatment during the first 24 h following
CFA injection. FIG. 11B shows the respective paw withdrawal
latencies of the non-inflamed contralateral paw. In FIGS. 11C-11D,
the effects of systemically administered DAHP (180 mg/kg i.p.
single dose; 11C) are compared with the effects of a single
intrathecal dose (1 mg/kg i.t.; 11D) which was administered via a
lumbar spinal catheter. In both cases, treatment was started 24
hours after CFA injection into the hindpaw. Because of slightly
different baseline levels (11C compared with 11D) effects of DAHP
after i.p. and i.t. treatment are additionally presented as
percentage change of paw withdrawal latency (FIG. 11E) to allow for
a direct comparison of both routes of administration.
[0091] FIG. 12 is a bar graph showing the increase of the paw
weight of the inflamed paw compared with the contralateral paw. Paw
weight was determined 48 hours following CFA injection. The
increase of the paw weight is a measure for the inflammatory paw
edema and therefore allows assessment of paw inflammation.
[0092] FIGS. 13A and 13B are a series of graphs showing the effects
of DAHP on the response to tactile (13A) and heat stimuli (13B) in
naive rats. A single dose of DAHP (180 mg/kg i.p.) or vehicle was
injected at time "zero".
[0093] FIGS. 14A-14C represent a series of graphs showing
pharmacokinetic features of DAHP and the
pharmacokinetic-pharmacodynamic relationship. FIG. 14A shows the
time course of DAHP plasma concentrations following i.p. injection
of a single dose of 180 mg/kg at time "zero". In addition,
concentrations of DAHP in cerebrospinal fluid (CSF) were determined
at an early and late time point. In FIG. 14B, plasma concentrations
were fitted according to a one-compartment PK model with first
order input and first order elimination. PK parameters are
presented where Cmax is the maximum concentration, tmax the time of
the maximum concentration, k01 is the absorption rate constant,
t1/2abs. represents the absorption half-life, k10 is the
elimination rate constant, t1/2 e1 is the elimination half-life,
and C1 is the clearance. In FIG. 14C, pooled plasma concentration
and effect data of the CFA model were used to assess the PK/PD
relationship employing a standard sigmoidal Emax model.
[0094] FIGS. 15A and 15B show the effect of the sepiapterin
reductase inhibitor, N-acetyl-serotonin (NAS) on thermal
hyperalgesia in the CFA model. The paw withdrawal latency (PWL) to
radiant heat (Hargreaves test) is a measure for the heat
sensitivity. NAS treatment (single dose of 50 mg/kg i.p.) was
started 24 hours after CFA injection into the hindpaw. Data are
presented as percentage change of the PWL compared with the PWL of
the non-inflamed contralateral paw. FIG. 15B shows the increase of
the paw weight of the inflamed paw compared with the contralateral
paw. Paw weight was determined 48 hours following CFA injection.
The increase of the paw weight is a measure for the inflammatory
paw edema.
[0095] FIGS. 16A-16D show the effects of systemic (16A) and
intrathecal (16B) treatment with methotrexate (MTX) on the
nociceptive behavior in the SNI model of neuropathic pain. In FIGS.
16A, and 16B the left panel shows the threshold to mechanical
stimuli applied with von Frey hairs (von Frey threshold). The right
panel shows the duration of paw licking, shaking and lifting
following acetone application to the paw as a measure for cold
allodynia. In FIG. 16A MTX (0.2 mg/kg/d) was injected i.p. once
daily starting 5 days after SNI surgery. Nociceptive behavior was
assessed once daily three hours after drug injection. In FIG. 16B,
MTX was administered as continuous intrathecal infusion (0.1
mg/kg/d for 14 days) starting right after SNI surgery. Nociceptive
behavior was assessed once daily. FIGS. 16C and 16D show the body
weight gain of animals during systemic (16C) and intrathecal (16D)
MTX treatment. Determination of the weight gain was used to assess
general well-being and potential toxic effects of MTX.
[0096] FIG. 17 is a table showing various sepiapterin reductase
inhibitors (Smith et al., (1992) J Biol Chem 267: 5599-5607).
[0097] FIG. 18 shows the chemical structures of BH4, guanine, and
DAHP
[0098] FIG. 19 is a table representing potential GTPCH
inhibitors.
[0099] FIG. 20 shows the structure of GTPCH and the binding of its
substrate to the catalytic center of the enzyme. Hydrogen bonds
between aminoacids of GTPCH and the substrate GTP are shown as
dotted lines.
[0100] FIG. 21 shows the interaction of GTPCH-I with the feedback
regulatory protein (GFRP) and binding site of phenylalanine in the
interface of both proteins. The binding site of BH4 and
GFRP-dependent GTPCH-I inhibitors is thought to be similar to that
of phenylalanine.
[0101] FIGS. 22A and 22B are a series of graphs demonstrating that
the anti-nociceptive effect of DAHP follows a dose-response
relationship for mechanical stimuli (FIG. 22A) and thermal stimuli
(FIG. 22B) in the SNI model of nerve injury. In this graph, closed
triangles represent vehicle, i.p.; open triangles represent DAHP at
90 mg/kg/day, i.p.; open squares represent DAHP at 180 mg/kg/day,
i.p.; and open circles represent DAHP at 270 mg/kg/day, i.p.
[0102] FIGS. 23A and 23B are a series of graphs demonstrating the
pro-nociceptive effects of BH4. FIG. 23A demonstrates that
intrathecal administration of BH4 reduces the paw withdrawal
latency to a thermal (heat) stimulus in naive rats. FIG. 23B
demonstrates the pro-nociceptive effects of BH4 in animals having a
pre-existing thermal hypersensitivity (ipsilateral) compared to
controls (contralateral). Hypersensitivity was induced using the
CFA model of paw inflammation. In these experiments, BH4 was
injected at time "zero" after measurement of baseline paw
withdrawal latency.
[0103] FIG. 24A is a series of photo micrographs showing cFos
immunoreactivity in ipsilateral dorsal horn neurons two hours after
formalin injection in DAHP and vehicle-treated rats. FIG. 24B is a
bar graph quantifying the number of cFos immunoreactive cell bodies
observed under each condition.
[0104] FIG. 25 is a bar graph showing the number of apoptotic cell
profiles, using TUNEL staining, observed in the L4/L5 dorsal horn 7
days after SNI surgery of animals treated with either 180 mg/kg/day
DAHP or vehicle control. Apoptotic neurons were detected by in situ
TUNEL labeling and counted by a blinded observer.
[0105] FIG. 26A is a series of graphs demonstrating that there is
no significant difference between wild-type and nNOS knockout mice,
using the SNI model either with or without treatment using DAHP, in
response to mechanical or thermal (cold) stimuli. In this figure,
closed circles represent nNOS knockout mice with DAHP; open
circles, nNOS knock out mice with vehicle; closed triangles, wild
type mice with DAHP; and open triangles, wild type mice with
vehicle. FIG. 26B is a series of line graphs demonstrating that
L-NAME, a NOS inhibitor, does not enhance the anti-nociceptive
effects of DAHP to mechanical or thermal (cold) stimuli in the SNI
model. In this figure, closed triangles represent L-NAME
administration; open circles, L-NAME+DAHP administration; closed
circles, DAHP administration; and open triangles, vehicle
administration. L-NAME was administered at 25 mg/kg, i.p., single
dose, and DAHP was administered at 120 mg/kg, i.p., single dose.
The antinociceptive efficacy of this high L-NAME dose is weaker
(about 50%) than that of a moderate dose of DAHP.
[0106] FIG. 27A is a series of photomicrographs of in situ
hybridization of GTPCH-I in the ipsilateral (lesioned) and
contralateral (control) dorsal root ganglia 3 days (top panel) and
14 days (bottom panel) after SNI surgery. FIG. 27B is a series of
photomicrographs of GTPCH-I in situ hybridization 14 days after SNI
surgery and treatment with either DAHP or vehicle control. These
data demonstrate that DAHP does not affect GTPCH-1 expression in
the SNI model.
[0107] FIG. 28A is a series of photomicrographs demonstrating that
GTPCH-1 mRNA is not normally expressed in the spinal cord but,
three days after SNI surgery, isolated GTPCH-1-expressing motor
neurons could be observed. FIG. 28B is a series of photomicrographs
demonstrating that GFRP is expressed in isolated DRG neurons, but
that the expression pattern does not change three days after SNI
surgery.
[0108] FIG. 29 is a series of photomicrographs showing, by in situ
hybridization, the expression of GTPCH-I (left column) and, by
immunofluorescence, the expression of NF200, Griffonia
simplicifolia isolectin B4 (IB4), CGRP, and nNOS (center column).
The number of cells co-expressing GTPCH-I and each of the
identified proteins is expressed as a percentage of cells
expressing GTPCH-I (right column).
[0109] FIG. 30 is a series of bar graphs quantifying the levels of
the BH4 metabolites biopterin and neopterin in the DH, DRG, and ScN
following SNI surgery with and without DAHP treatment, compared to
control.
[0110] FIG. 31 are photomicrographs showing the co-localization of
GTPCH-I mRNA, using in situ hybridization, and ATF-3 protein
localization, using immunohistochemistry, following the SNI
neuronal injury model. The arrows indicate cells that express GTPCH
mRNA expression and also nuclear staining for ATF-3. Overall,
80-90% of GTPCH expressing neurons were ATF-3 positive
demonstrating that GTPCH-1 upregulation occurs mostly in injured
neurons.
DETAILED DESCRIPTION
[0111] In general, the present invention features methods and
compounds for treating, reducing, or preventing pain or a
traumatic, metabolic or toxic peripheral nerve lesion by
administering to a mammal a therapeutically effective amount of a
composition that reduces the level or activity of
tetrahydrobiopterin (BH4). The invention also provides methods for
diagnosing pain or a traumatic, metabolic or toxic peripheral nerve
lesion in a mammal by detecting an increase in the levels of BH4,
BH4 intermediates, BH4 precursors, or BH4 metabolites in a
biological sample obtained from a mammal (e.g., serum, plasma,
urine, cerebrospinal fluid, synovial fluid, tissue exudates, or
tissue samples). Alternatively, pain may be diagnosed by detecting
an increase in the activity or levels of any one of the BH4
synthetic enzymes. In yet another general embodiment, the invention
provides methods for identifying novel therapeutics for pain based
on their ability to decrease the level or activity of BH4 or its
synthetic enzymes or its ability to interfere with binding of BH4
to a BH4 receptor or BH4-dependent enzyme.
[0112] The invention stems from our discovery that inhibiting the
synthesis of BH4 by interfering with the biological activity of any
of the enzymes involved in the synthesis of BH4 (e.g., GTP
cyclohydrolase (GTPCH), Sepiapterin Reductase (SPR), and
Dihydropteridine reductase (DHPR), see FIG. 2A) results in
prevention, treatment, and reduction in pain, i.e. analgesia. To
this end, we show that the administration of 2,4 diamino
6-hydroxypyrimidine (DAHP), an inhibitor of GTPCH; Methotrexate
(MTX), an inhibitor of DHPR; or N-acetyl-serotonin (NAS), an
inhibitor of SPR, results in profound analgesia in various rat
models of acute post-injury pain hypersensitivity, peripheral
neuropathic and inflammatory pain, and without an effect on basal
pain sensitivity, sedative effect, or gross disruption of motor
function. This reduction in pain is mediated, at least in part, by
the interference with or by a reduction in the activity of various
enzymes for which BH4 is a key co-factor. Such enzymes include, for
example, nitric oxide synthases and catecholamine synthetic
enzymes. In addition, the reduction in the levels of BH4 may also
reduce its release from cells in the nervous system and its direct
actions, for example on neurotransmitter release or ion channel
activity which may be mediated through BH4 binding to membrane
bound receptors that modify neuronal function.
[0113] The methods of this invention are therefore useful for the
diagnosis, treatment, reduction, or prevention of various forms of
clinical pain, namely inflammatory pain, functional pain and
neuropathic pain, whether acute or chronic. Exemplary conditions
that may be associated with pain include, for example, soft tissue,
joint, bone inflammation and/or damage (e.g., acute trauma,
osteoarthritis, or rheumatoid arthritis), myofascial pain syndromes
(fibromylagia), headaches (including cluster headache, migraine and
tension type headache), myocardial infarction, angina, ischemic
cardiovascular disease, post-stroke pain, sickle cell anemia,
peripheral vascular occlusive disease, cancer, inflammatory
conditions of the skin or joints, diabetic neuropathy, and acute
tissue damage from surgery or traumatic injury (e.g., burns,
lacerations, or fractures). The present invention is also useful
for the treatment, reduction, or prevention of musculo-skeletal
pain (after trauma, infections, and exercise), neuropathic pain
caused by spinal cord injury, tumors, compression, inflammation,
dental pain, episiotomy pain, deep and visceral pain (e.g., heart
pain, bladder pain, or pelvic organ pain), muscle pain, eye pain,
orofacial pain (e.g., odontalgia, trigeminal neuralgia,
glossopharyngeal neuralgia), abdominal pain, gynecological pain
(e.g., dysmenorrhea and labor pain), pain associated with nerve and
root damage due to trauma, compression, inflammation, toxic
chemicals, metabolic disorders, hereditary conditions, infections,
vasculitis and autoimmune diseases, central nervous system pain,
such as pain due to spinal cord or brain stem damage,
cerebrovascular accidents, tumors, infections, demyelinating
diseases including multiple sclerosis, low back pain, sciatica, and
post-operative pain. Conditions that are amenable to treatment
according to the present invention are described in detail, for
example, in U.S. Ser. No. 10/348,381 as well as U.S. Pat. Nos.
6,593,331 and 6,593,331, all of which are hereby incorporated by
reference.
[0114] Briefly, we conducted a cluster analysis of changes in
expression of developmentally- and peripheral nerve
injury-regulated genes in the dorsal root ganglion using
high-density oligonucleotide microarrays. Interestingly, we found
that a particular cluster of genes was characterized by high levels
of expression during development, down-regulation during adulthood,
and induction following nerve injury. Such exemplary genes include
DHPR (a member of the BH4 synthetic pathway).
[0115] A triplicate analysis of lumbar DRG microarrays three days
post-axotomy (sciatic nerve transection) further revealed that
three of the approximately 200 genes that were up-regulated were
members of the BH4 synthetic pathway (e.g., GTPCH, SPR and DHPR).
Their induction under such conditions was validated by Northern
blot analysis, Northern slot blots, in situ hybridization, and
Western blot analysis. We further confirmed that three of the four
members of the BH4 synthetic pathway were up-regulated in primary
sensory neurons after peripheral nerve injury in at least three
models of peripheral neuropathic pain and that this was concomitant
with an increase in BH4 levels in the dorsal root ganglion.
Furthermore, using DAHP (a GTPCH inhibitor) as well as various
other inhibitors of BH4 synthetic enzymes (e.g., NAS and
methotrexate), we showed that inhibition of the BH4 synthetic
pathway induces analgesia in numerous neuropathic pain models as
well as in inflammatory pain and a post-injury pain
hypersensitivity model. The analgesic action could be demonstrated
after systemic delivery and intrathecal delivery. The latter
indicates an action on the nervous system including the DRG and
spinal cord. The degree of analgesia matched, or in the case of the
spared nerve injury peripheral neuropathic pain model, was far
greater than that achieved by any of the conventionally used
analgesics, including morphine, gabapentin, carbamazepine,
amytryptiline, and rofecoxib.
Therapeutic Agents
Inhibitors of the BH4 Pathway
[0116] BH4 is enzymatically synthesized de novo from guanosine
5'-triphosphate (GTP) via two intermediates, 7,8-dihydroneopterin
triphosphate and 6-pyruvoyl tetrahydropterin (see FIG. 2A).
According to the present invention, the administration of any agent
that inhibits or modulates the biological activity of at least one,
two, three, or more than three of any of the BH4 synthetic enzymes
shown in FIG. 2A to reduce BH4 synthesis induces analgesia. Such
enzymes include, for example, sepiapeterin reductase (SPR),
Pyruvoyltetrahydropterin Synthase (PTPS), GTP cyclohydrolase
(GTPCH), Pterin-4.alpha.-carbinolamine dehydratase, and
dihydropteridine reductase (DHPR). The analgesic effect that
results from the reduction in BH4 levels is caused, at least in
part, by a reduction in the biological activity of enzymes for
which BH4 is a co-factor and of membrane-bound receptors to which
BH4 binds.
[0117] In this regard, BH4 is an essential cofactor of several
enzymes (e.g., the hydroxylases of the three aromatic amino acids
phenylalanine, tyrosine, and tryptophan;
[0118] ether lipid oxidase; and the three nitric oxide synthase
(NOS) isoenzymes, eNOS, iNOS, and nNOS) and therefore plays a key
role in a number of biological processes, including
neurotransmitter formation and signaling in pain pathways. Indeed,
a number of enzymes that are regulated by BH4 have previously been
involved in pain and include for example, NOS (Meller et al. (1992)
Neuroscience 50:7-10; Minami et al., (1995) Neurosci. Lett.
201:239-242; Yamaguchi and Naito (1996) Can. J. Anaesth.
43:975-981; Aley et al., (1998) J. Neurosci. 18:7008-7014; Handy
and Moore (1998) Neuropharmacology 37:37-43; Levy and Zochodne
(1998) Eur. J. Neurosci. 10:1846-1855; Guhring et al. (2000) J.
Neurosci. 20:6714-6720; Levy et al. (2000) Eur J Neurosci
12:2323-2332, tyrosine hydroxylase (Ma and Bisby (1999) Neurosci
Lett 275:117-120; Lindqvist et al. (2000) Muscle Nerve
23:1214-1218) and tryptophan hydroxylase.
[0119] According to the present invention, an inhibitor of the BH4
pathway is any agent having the ability to reduce the production or
the activity of BH4 by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, or 100% relative to an untreated control cell as
determined by any standard method in the art, including those
described herein. Alternatively, the inhibitor may treat, prevent,
or reduce pain when administered to a mammal by at least 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% relative to an untreated
control. Such reduction or prevention in pain may be measured by
any technique known in the art such as those described herein.
Exemplary compounds that may be used according to this invention
include DAHP, an inhibitor of GTPCH; MTX, an inhibitor of DHPR;
NAS, an inhibitor of SPR; Tetrahydro-L-biopterin, L-Sepiapterin,
7,8-dihydro-L-Biopterin, 6,7-dimethyltetrahydropterin
hydrochloride, N-acetyl-serotonin (NAS), N-Chloroacetylserotonin,
N-Methoxyacetylserotonin, N-Chloroacetyldopamine, as well as any of
the compounds identified by any of the screening methods of the
invention. Other agents that may be used are described further
below.
[0120] Optionally, the BH4 inhibitor may be a small molecule
antagonist or an antisense to any of the BH4 synthetic enzymes. RNA
interference (RNAi) may also be used to target the BH4 synthetic
pathway as it provides a powerful method of gene silencing in
eukaryotic cells including mammalian cells such as the primary
sensory neurons of the present invention. The basic technique of
RNAi involves introducing sequence-specific double-stranded RNA
into neurons in order to generate a nonheritable, epigenetic
knockout of gene function that phenocopies a null mutation in the
targeted gene. RNA interference has previously been described
(O'Neil N J, et al., Am J Pharmacogenomics (2001): 45-53).
[0121] Alternatively, the analgesic agent may be a dominant
negative protein or a nucleic acid encoding a dominant negative
protein that interferes with the biological activity of BH4 or any
of the BH4 synthetic enzymes. A dominant negative protein is any
amino acid molecule having a sequence that has at least 50%, 70%,
80%, 90%, 95%, or even 99% sequence identity to at least 10, 20,
35, 50, 100, or more than 150 amino acids of the wild type protein
to which the dominant negative protein corresponds. For example, a
dominant-negative BH4 synthetic enzyme may have mutation such that
it no longer able to produce BH4.
[0122] According to this invention, the dominant negative protein
may be administered as an expression vector. The expression vector
may be a non-viral vector or a viral vector (e.g., retrovirus,
recombinant adeno-associated virus, or a recombinant adenoviral
vector). Alternatively, the dominant negative protein may be
directly administered as a recombinant protein to dorsal root
ganglia or the spinal cord using, for example, microinjection
techniques.
Inhibitors of GTP Cyclohydrolase I (GTPCH)
[0123] The rate-limiting enzyme in BH4 de novo biosynthesis is GTP
cyclohydrolase I (GTPCH), which converts GTP to
7,8-dihydroneopterin triphosphate (see FIG. 2A). Over time, the
accumulation of BH4 causes a feedback inhibition of GTPCH, in a
reaction mediated by the GTPCH Feedback Regulatory Protein (GFRP).
In the presence of phenylalanine, GFRP induces a feed-forward
activation of GTPCH activity by enhancing GTP binding (Maita et
al., (2002) Proc. Natl. Acad. Sci. U.S.A. 99: 1212-1217). However,
in the presence of BH4, GFRP induces feedback inhibition of GTPCH
enzyme activity (Yoneyama and Hatakeyama, (1998) J. Biol. Chem.
273:20102:20108); Yoneyama and Hatakeyama, (2001) Protein Sci. 10:
981-878) such that BH4 production is auto-inhibited.
[0124] Accordingly, any agent that inhibits GTPCH activity can
efficiently reduce the production of BH4 and in turn, induce
analgesia. GTPCH inhibitors may compete with the substrate GTP for
binding to the catalytic center of the enzyme (FIG. 20).
Alternatively, GTPCH inhibitors (for example BH4 analogs) may
inhibit BH4 production by binding to the GTPCH/GFRP complex (FIG.
21) and thereby mimic the feedback inhibition of BH4. Although GFRP
mRNA is abundant in brainstem neurons, it remains undetectable in
dopamine neurons of the midbrain and in norepinephrine neurons of
the locus coeruleus (Kapatos et al., (1999) J. Neurochem. 72:
669-675). Thus, the GFRP-dependency of GTPCH inhibitors may confer
specificity and simultaneously avoid dopamine-related side
effects.
[0125] Here, we have shown, for example, that the specific GTPCH
inhibitor, 2,4-Diamino-6-hydroxypyrimidine (DAHP), induces
analgesia in various neuropathic pain and inflammatory models (see
FIGS. 8-13). DAHP-mediated inhibition of GTPCH is mediated by
GFRP-dependent (at low concentrations) and GFRP-independent (at
higher concentrations) mechanisms. Accordingly, DAHP mimics BH4 in
its indirect mechanism of GTPCH inhibition at low concentrations.
At higher concentrations DAHP competes with the physiological
GTPCH-substrate, guanosine-triphosphate (GTP) for binding to the
catalytic site (Xie et al., (1998) J. Biol. Chem.
273:21091-21098).
[0126] Biochemical and crystallographic studies on the interaction
of GTPCH with GTP reveal that hydrogen bonds are formed between
highly conserved amino acids found within the active site of GTPCH
and the pyrimidine portion of guanine i.e. the nitrogen atoms at
position 1, 2 and 3 and the oxygen at position 6, respectively (see
FIG. 20) (Rebelo et al., (2003) J. Mol. Biol. 326: 503-516). This
particular portion of the guanine structure exactly matches the
pyrimidine structure of DAHP as well as a portion of the pteridine
structure of tetrahydrobiopterin (BH4), which is the end product of
the enzymatic cascade.
[0127] The structural homology between DAHP, guanine, and BH4 (see
FIG. 18) indicates that it is the pyrimidine portion in these
molecules that is likely to be crucial for binding to GTPCH.
Analysis of various pyrimidine-molecules with modifications at
these sites reveals that alterations at position 2, 4 and 6 are
associated with a reduction or loss of GTPCH inhibitory activity
(Yoneyama et al. (2001) Arch. Biochem. Biophys. 388:67-73) (see
FIG. 19). On the other hand, guanine itself (i.e. part of the
physiological substrate) and guanine analogs with modifications at
position 7 or 8 inhibit GTPCH with a higher potency (approximately
10 fold higher than DAHP) (Yoneyama et al., supra) (FIG. 19). Thus,
drugs containing a 5-membered ring like guanine (thus also sharing
higher similarity with BH4) have an increased inhibitory effect.
Interestingly, BH4 is about 10 times more potent than guanine
analogs (Yoneyama et al., supra) (FIG. 19) and 100 times more
potent than DAHP, therefore suggesting that the side chain attached
to C-6 of BH4 confers further potency and/or specificity. This is
supported by the finding that an 6S-BH4 enantiomer and an analog of
BH4 with no side chain at C-6 are more than one order of magnitude
less effective than BH4 (Harada et al. (1993) Science
260:1507-1510). Accordingly, the potency of DAHP can be increased,
for example, by increasing its structural similarity to BH4 while
retaining substitutions or modifications that prevent its use as a
cofactor for BH4-dependent enzymes (e.g., tyrosine, phenylalanine,
and tryptophan hydroxylases; glycerol ether monooxygenases; and
nitric oxide synthases (which utilize various N-alkyl and
N-aryl-hydroxyguanidines such as
N-(4-Chlorophenyl)N'-hydroxyguanidine as substrates for the
production of nitric oxide (Moali et al. (2001) Chem. Res. Toxicol.
14:202-210; Renodon-Corniere et al., (1999) Biochemistry 38:
4663-8)).
[0128] In light of the above, the mammal being treated according to
the present invention may be administered with a composition
containing a compound having the formula:
##STR00037##
such that R.sup.1 is a H, C.sub.1-6 alkyl, halo, NO.sub.2, CN,
CO.sub.2R.sup.4, CONR.sup.4R.sup.5, SO.sub.2R.sup.4,
SO.sub.2NR.sup.4R.sup.5, OR.sup.4, or NR.sup.4R.sup.5. Each of
R.sup.4 and R.sup.5 may be, independently, a H, C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl; R.sup.2 may be a H, C.sub.1-6 alkyl, C.sub.6-12
aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4 alkheteroaryl;
and R.sup.3 may be a H, C.sub.1-6 alkyl, C.sub.6-12 aryl,
heteroaryl, C.sub.1-4 alkaryl, C.sub.1-4 alkheteroaryl,
CO.sub.2R.sup.6, CONR.sup.7R.sup.8, SO.sub.2R.sup.6, or
SO.sub.2NR.sup.7R.sup.8, where R.sup.6 is a C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl, and each of R.sup.7 and R.sup.8 is, independently, a
H, C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl,
or C.sub.1-4 alkheteroaryl.
[0129] Alternatively, R.sup.3 may be as above and R.sup.1 and
R.sup.2 together may be represented by
##STR00038##
where the N, O, or S of the R.sup.1/R.sup.2 linkage forms a bond to
the pyrimidinone ring and each of R.sup.9, R.sup.10, R.sup.11,
R.sup.12, and R.sup.13 is, independently, a H, C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl.
[0130] Optionally, R.sup.3 may be as above and R.sup.1 and R.sup.2
together may be represented by
##STR00039##
where the N of the R.sup.1/R.sup.2 linkage forms a bond to the
pyrimidinone ring, each of R.sup.12 or R.sup.13 is as above, and
R.sup.14 is a OR.sup.4, halo, NO.sub.2, CN, CO.sub.2R.sup.7,
CONR.sup.7R.sup.8, SO.sub.2R.sup.7, SO.sub.2NR.sup.7R.sup.8,
C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or
C.sub.1-4 alkheteroaryl, such that each of R.sup.7 and R.sup.8 is,
independently, a H, C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.12 aryl,
heteroaryl, C.sub.1-C.sub.1 alkaryl, or C.sub.1-C.sub.1
alkheteroaryl.
[0131] As another alternative, R.sup.1 and R.sup.2 together may be
represented by
##STR00040##
where the N of the R.sup.1/R.sup.2 linkage forms a bond to the
pyrimidinone ring, each of R.sup.9, R.sup.10, R.sup.11, and
R.sup.14 are as above, R.sup.3 does not exist, and a double bond is
formed between the carbon bearing the R.sup.14 and the nitrogen
bearing R.sup.2.
[0132] Optionally, the composition may have be a compound of
formula (I), such that R.sup.1 is a H, C.sub.1-6 alkyl, halo,
NO.sub.2, CN, CO.sub.2R.sup.4, CONR.sup.4R.sup.5, SO.sub.2R.sup.4,
SO.sub.2NR.sup.4R.sup.5, OR.sup.4, or NR.sup.4R.sup.5. Accordingly,
each of R.sup.4 and R.sup.5 may be, independently, a H, C.sub.1-6
alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl, R.sup.2 may be a H, C.sub.1-6 alkyl, C.sub.6-12
aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4 alkheteroaryl,
and R.sup.3 may be a H, C.sub.1-6 alkyl, C.sub.6-12 aryl,
heteroaryl, C.sub.1-4 alkaryl, C.sub.1-4 alkheteroaryl,
CO.sub.2R.sup.6, CONR.sup.7R.sup.8, SO.sub.2R.sup.6, or
SO.sub.2NR.sup.7R.sup.8, where R.sup.6 is a C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl and each of R.sup.7 and R.sup.8 may be,
independently, a H, C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl,
C.sub.1-4 alkaryl, or C.sub.1-4 alkheteroaryl.
[0133] If desired, the composition contains a compound of
formula:
##STR00041##
in which R.sup.1 and R.sup.2 together may be represented by
##STR00042##
such that the N, O, or S of the R.sup.1/R.sup.2 linkage forms a
bond to the pyrimidinone ring and each of R.sup.9, R.sup.10,
R.sup.11, R.sup.12, and R.sup.13 is, independently, a H, C.sub.1-6
alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl.
[0134] Alternatively, the composition contains a compound of
formula:
##STR00043##
in which R.sup.3 is as above and R.sup.1 and R.sup.2 together are
represented by
##STR00044##
where the N of the R.sup.1/R.sup.2 linkage forms a bond to the
pyrimidinone ring, each of R.sup.12 and R.sup.13 is as above, and
R.sup.14 is a OR.sup.4, halo, NO.sub.2, CN, CO.sub.2R.sup.7,
CONR.sup.7R.sup.8, SO.sub.2R.sup.7, SO.sub.2NR.sup.7R.sup.8,
C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or
C.sub.1-4 alkheteroaryl, where each of R.sup.4, R.sup.7 and R.sup.8
is, independently, a H, C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.12
aryl, C.sub.1-C.sub.4 alkaryl, heteroaryl, or C.sub.1-C.sub.4
alkheteroaryl.
[0135] Alternatively, the composition contains a compound of
formula:
##STR00045##
in which R.sup.1 and R.sup.2 together may be represented by
##STR00046##
where the N of the R.sup.1/R.sup.2 linkage forms a bond to the
pyrimidinone ring, each of R.sup.9, R.sup.10, R.sup.11, and
R.sup.14 are as above, R.sup.3 does not exist, and a double bond is
formed between the carbon bearing R.sup.14 and the nitrogen bearing
R.sup.2.
[0136] If the composition contains a compound of formula:
##STR00047##
R.sup.1 and R.sup.2 together may be represented by
##STR00048##
where the N, O, or S of the R.sup.1/R.sup.2 linkage forms a bond to
the pyrimidinone ring and each of R.sup.9, R.sup.10, R.sup.11,
R.sup.12, and R.sup.13 is, independently, a H, C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl.
[0137] Alternatively, the composition contains a compound of
formula:
##STR00049##
in which R.sup.3 is as above and R.sup.1 and R.sup.2 together are
represented by
##STR00050##
where the N of the R.sup.1/R.sup.2 linkage forms a bond to the
pyrimidinone ring, each of R.sup.12 and R.sup.13 is as above, and
R.sup.14 is a OR.sup.4, halo, NO.sub.2, CN, CO.sub.2R.sup.7,
CONR.sup.7R.sup.8, SO.sub.2R.sup.7, SO.sub.2NR.sup.7R.sup.8,
C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or
C.sub.1-4 alkheteroaryl, such that each of R.sup.7 and R.sup.8 is,
independently, H, C.sub.1-C.sub.6 alkyl, C.sub.6-C.sub.12 aryl,
C.sub.1-C.sub.4 alkaryl, heteroaryl, or C.sub.1-C.sub.4
alkheteroaryl.
[0138] Optionally, the composition may contain a compound of
formula:
##STR00051##
in which R.sup.1 and R.sup.2 together are represented by
##STR00052##
where the N of the R.sup.1/R.sup.2 linkage forms a bond to the
pyrimidinone ring, each of R.sup.9, R.sup.10, R.sup.11, and
R.sup.14 are as above, R.sup.3 does not exist, and a double bond is
formed between the carbon bearing R.sup.14 and the nitrogen bearing
R.sup.2.
[0139] Exemplary compounds that may be contained within the
composition of the invention include, for example:
##STR00053##
These compounds are shown in FIG. 19.
[0140] Furthermore, the composition of the invention may contain a
compound having the formula:
##STR00054##
in which R.sup.15 is a H, C.sub.1-6 alkyl, C.sub.6-12 aryl,
heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4 alkheteroaryl and
R.sup.16 is a H, C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl,
C.sub.1-4 alkaryl, C.sub.1-4 alkheteroaryl, CO.sub.2R.sup.17,
CONR.sup.18R.sup.19, SO.sub.2R.sup.17, or
SO.sub.2NR.sup.18R.sup.19. R.sup.17 may be a C.sub.1-6 alkyl,
C.sub.6-12 aryl, heteroaryl, C.sub.1-4 alkaryl, or C.sub.1-4
alkheteroaryl and each of R.sup.18 and R.sup.19 may be,
independently, a H, C.sub.1-6 alkyl, C.sub.6-12 aryl, heteroaryl,
C.sub.1-4 alkaryl, or C.sub.1-4 alkheteroaryl.
[0141] Further exemplary compounds include Tetrahydro-L-biopterin
(BH4.2HCl, a reduced pterin that is a noncompetitive inhibitor of
GTP cyclohydrolase I with a Ki of 15.7 .mu.M); L-Sepiapterin (a
reduced pterin that is 12 times more potent than oxidized pterins
as a GTP cyclohydrolase I inhibitor and has an IC50 of 12.7.+-.1.8
.mu.M); 7,8-dihydro-L-Biopterin (BH2, a metabolic end product of
GTP cyclohydrolase I in vitro, which functions as a noncompetitive
inhibitor of GTP cyclohydrolase I (Ki of 14.4 .mu.M) and is
approximately 12 times more potent as an inhibitor than oxidized
pterins, folates, and aminopterins); and
6,7-dimethyltetrahydropterin hydrochloride (a noncompetitive
inhibitor of GTP cyclohydrolase I (IC50 of 76 to 112 .mu.M)). As
discussed above, the inhibition of GTPCH may also be accomplished
using inhibitors that act on GTPCH in a GFRP-independent GTP
competitive fashion (e.g., guanine derivatives as shown in FIG. 19
and as described above).
[0142] Activators of GTPCH Feedback Regulatory Protein (GFRP)
[0143] GFRP is the endogenous inhibitor of BH4 synthesis by GTPCH.
The binding of GFRP to GTPCH is, itself, a BH4-dependent event,
making GFRP a negative feedback regulator of BH4 production. It has
recently been discovered that DAHP, a molecule that is structurally
similar to BH4, binds to GFRP and promotes GTPCH inhibition
(Kolinsky et al., J. Biol. Chem., Manuscript M405370200, Jul. 29,
2004). Thus, molecules that mimic the actions of BH4 and/or DAHP at
GFRP by enhancing the binding of GFRP to GTPCH and inhibiting the
production of BH4 are useful in the methods of this invention.
Alternatively, molecules that facilitate the binding of GFRP and
GTPCH, but not through a binding at the BH4 site on GFRP are also
useful. Such molecules include, for example, bi-functional
antibodies or cross-linking agents.
[0144] Inhibitors of Sepiapterin Reductase
[0145] Sepiapterin Reductase (SPR) functions as the final synthetic
enzyme in the BH4 pathway. Here, we show that NAS (50 mg/kg i.p.,
an inhibitor of SPR, produces analgesia in a model of inflammatory
pain (FIGS. 15A and 15B). Other sepiapterin reductase inhibitors
with increased potency include N-Chloroacetylserotonin,
N-Methoxyacetylserotonin, and N-Chloroacetyldopamine.
[0146] Inhibitors of Dihydropteridine Reductase (DHPR)
[0147] The BH4 salvage arm, which involves DHPR, allows a recycling
of oxidized BH4 without de novo synthesis. The inhibition of DHPR,
which is upregulated in DRGs and the spinal cord following nerve
injury, induces analgesia by inhibiting the recycling of BH4 from
BH2. For example, the administration of methotrexate (MTX, also
known as amethopterin hydrate (0.1 mg/kg/d as continuous
intrathecal infusion or 0.2 mg/kg i.p. once daily) results in pain
reduction without directly affecting BH4 de novo synthesis (see
FIGS. 16A-16F). MTX is an inhibitor of dihydrofolate reductase and
has been approved for use in humans as an immunosuppressant. It is
typically used at low dosages for the treatment of rheumatoid
arthritis (Weinstein et al. (1985) Am J Med 79: 331-7, Williams et
al. (1985) Arthritis Rheum;28: 721-30, Weinblatt et al. (1985) N
Engl J Med 312: 818-22, Hoffmeister et al. (1983) Am J Med 75:
69-73, Giannini et al. (1992) N Engl J Med 326: 1043-9). While
systemically administered MTX primarily reaches peripheral targets
and DRGs, MTX delivered spinally targets the spinal cord and DRGs.
Systemically administered MTX (at low doses) does not penetrate the
blood brain barrier because it is a substrate for ATP-binding
cassette (ABC) transporters (probenicid-sensitive multi-drug
resistance protein (MRP) 1-3). Consequently, systemic treatment
with MTX may require the co-administration with probenicid or other
inhibitors of MRPs or organic anion transporters to ensure that MTX
reaches the spinal cord and brain.
[0148] If desired, analgesia may be induced by simultaneously
blocking both parts of the BH4 pathway, namely the biosynthesis and
the salvage pathways, to achieve an additive effect. For example,
the reduction in pain may be significantly enhanced by blocking BH4
synthesis by simultaneously inhibiting GTPCH using DAHP and DHPR
using methotrexate (or other DHPR inhibitors that achieve higher
concentration in the central nervous system).
[0149] Inhibitors of Pterin-4.alpha.-carbinolamine dehydratase
(PCD)
[0150] Quinoid dihydropterin products are strong inhibitors of the
Pterin-4.alpha.-carbinolamine dehydratase (PCD) (having KI's of
about one half of their respective Km's) and may therefore be used
according to the present invention. (Rebrin et al. (1995)
Biochemistry 34: 5801-10).
Second Therapeutic Agents
[0151] The composition of the present invention may be administered
either alone or in combination with a second therapeutic agent,
such as an analgesic agent used in the treatment of nociception,
inflammatory, functional or neuropathic pain. According to this
invention, the second therapeutic agent may or may not produce a
therapeutic effect when administered on its own, but results in
such an effect (e.g., pain reduction) when administered with the
composition of the invention.
[0152] Exemplary analgesic agents include, nonsteroidal
anti-inflammatory agents (NSAIDs) (e.g. rofexocib, celecoxib,
valdecoxib, paracoxib, salicylic acid, acetominophen, diclofenac,
piroxican indomethacin, ibuprofen, and naproxen), opioid analgesics
(e.g., propoxyphene, meperidine, hydromorphone, hydrocodone,
oxycodone, morphine, codeine, and tramodol), NMDA antagonist
analgesics (e.g., 2-piperdino-1 alkanol derivatives, ketamine,
dextormethorphan, eliprodil, or ifenprodil), anesthetic agents
(e.g., nitrous oxide, halothane, fluothane) local anesthetics
(lidocaine, etidocaine, ropivacaine, chloroprocaine, sarapin, and
bupivacaine), benzodiazepines (diazepam, chlordiazepoxide,
alprazolam, and lorazepam), capsaicin, tricyclic antidepressants
(e.g., amitriptyline, perphanazine, protriptyline, tranylcypromine,
imipramine, desimipramine, and clomipramine), skeletal muscle
relaxant analgesics (flexeril, carisoprodol, robaxisal, norgesic,
and dantrium), migraine therapeutic agents (e.g., elitriptan,
sumatriptan, rizatriptan, zolmitriptan, and naratriptan),
anticonvulsants (e.g., phenytoin, lamotrigine, pregabalin,
carbamazepine, oxcarbazepine, topiramate, valproic acid, and
gabapentin), baclofen, clonidine, mexilitene, diphenyl-hydramine,
hydroxysine, caffeine, prednisone, methylprednisone, decadron,
paroxetine, sertraline, fluoxetine, tramodol, ziconotide,
levodopa.
[0153] If desired, the mammal being treated may be administered
with more than one agent that inhibits the production of BH4.
Optionally, the composition of the invention may contain more than
one such inhibitor. Alternatively, the mammal may further be
administered with specific inhibitors of enzymes that function
downstream of BH4, in addition to the composition of the invention.
Such inhibitors are described below.
BH4-depedent Enzymes
[0154] BH4 is an essential cofactor of several enzymes, i.e. the
hydroxylases of the three aromatic amino acids phenylalanine,
tyrosine, and tryptophan; ether lipid oxidase; and of the three
nitric oxide synthase (NOS) isoenzymes. Thus, BH4 plays a key role
in a number of biological processes including neurotransmitter
formation and signaling pathways.
[0155] Nitric oxide (NO) is released from nociceptive neurons
following NMDA receptor stimulation and diffuses back to the
presynaptic neuron where it causes further glutamate release by
stimulating the guanylyl cyclase/cGMP/cGMP dependent kinase
pathway. Furthermore, NO modulates excitability of the postsynaptic
neuron. Inhibitors of NO synthase, such as L-NAME
(N.sup.G-Nitro-L-arginine-methyl ester), reduce inflammatory
hyperalgesia and neuropathic allodynia in various models and
accordingly, may be administered with or admixed in the composition
of the invention. Given that drugs, which block the activity of all
NOS enzymes, are generally more effective than specific inhibitors
of either neuronal NOS (nNOS) or inducible NOS (iNOS), both enzymes
are most likely involved in pain. nNOS is constitutively expressed
in neurons and upregulated after peripheral nociceptive
stimulation. iNOS is upregulated in the spinal cord after
peripheral nociceptive stimulation particularly in glial cells and
produces much higher NO levels than nNOS. Although endothelial
nitric oxide synthase (eNOS) is primarily expressed in endothelial
cells, studies in knockout mice have shown that this enzyme also
contributes to pain modulation. All of these enzymes are dependent
on BH4, employing it as a cofactor. Inhibitors of the NOS pathway
that may be used to induce analgesia include inhibitors of NOS-1
(nNOS) such as N-Methyl-L-arginine (M 7033), N-Nitro-L-arginine (N
5501), 7-Nitroindazole (N 7778),
1-(2-Trifluoromethylphenyl)imidazole (T 7313), L-Thiocitrulline,
S-Methyl-L-thiocitrulline (M 5171); inhibitors of NOS-2 (iNOS),
such as Aminoguanidine (A 8835, A 7009), S-Benzylisothiourea (B
9138), 1-(2-Trifluoromethylphenyl)imidazole (T 7313),
L-N6-(1-Iminoethyl)lysine (I 8021), and 1400W (W 4262); and
inhibitors of NOS-3 (eNOS) include N-Methyl-L-arginine (M 7033),
N-Nitro-L-arginine (N 5501), N-Iminoethyl-L-ornithine (I 8768), and
7-Nitroindazole (N 7778).
[0156] Tyrosine hydroxylase catalyses the first step in
cathecholamine synthesis, i.e., the production of dopamine from the
amino acid tyrosine in a reaction that requires the presence of
BH4. Tryptophan hydroxylase is the key enzyme in serotonin
synthesis. Noradrenaline and serotonin act as neurotransmitters in
descending inhibitory neurons arising from the locus coeruleus and
nucleus raphe magnus, respectively. Reduction of co-factor
availability for these enzymes may therefore result in an increase
of pain. To overcome this potential disadvantage, the composition
of the present invention may be administered in combination with a
5HT receptor agonist and/or a centrally acting alpha receptor
agonist such as clonidine. Various 5HT-1 agonists have
antinociceptive effects when administered alone such as the 5-HT-1
agonists m-trifluoromethylphenyl-piperazine (TFMPP) and
7-trifluoromethyl-4(4-methyl-1-piperazinyl)-pyrrolo(1,2-1a)quinoxaline
(CGS 12066B) (Alhaider et al., (1993) J Pharmacol Exp Ther.
265:378-85) or the 5HT-2A agonist, FR143166 (Ochi et al. (2002) Eur
J Pharmacol. 452:319-24). Alternatively, a potential decrease of
serotonin and/or noradrenaline synthesis may be outweighed by
combination treatment with a reuptake inhibitor such as a tricyclic
antidepressant (e.g. amitriptyline) or a selective serotonin
reuptake inhibitor (e.g. paroxetine, see above).
Direct Effects of BH4
[0157] In addition to its co-factor function, the R enantiomer of
BH4 (6R-BH4) exhibits various direct effects on neurons when
delivered via microdialysis to certain regions of the brain. For
example, 6R-BH4 increases the release of dopamine in the striatum.
Given that these effects persist in the presence of a tyrosine
hydroxylase inhibitor (alpha-methyl-p-tyrosine), it is probably not
caused by an increase of dopamine synthesis (Koshimura et al.
(1995) J Neurochem. 65: 827-30). 6R-BH4 also increases the release
of other neurotransmitters such as acetylcholine in the hippocampus
(Ohue et al. (1991) Neurosci Lett. 128:93-6) and glutamate and
serotonin in the striatum and frontal cortex (Mataga et al. (1991)
Brain Res. 551: 64-71). The neurotransmitter releasing effects of
BH4 are inhibited with a calcium channel antagonist (Koshimura et
al. (1995) J Neurochem. 65:827-30). The addition of 6R-BH4 to
microdialysis perfusion fluid also increases calcium currents in
the motor nucleus of the vagus in rats whereas the addition of
L-DOPA or the nitric oxide donor Sin-1 has no effect, therefore
suggesting that these effects are independent of tyrosine
hydroxylase or NOS activity, respectively (Shiraki T et al., (1996)
Biochem Biophys Res Commun. 221: 181-5). The S-enantiomer of BH4
(6S-BH4) or the precursor sepiapterin has no effect on
neurotransmitter release. 6R-BH4 may therefore act through a
specific "BH4-receptor." However, such a membrane bound
extracellular binding site for BH4 has not yet been
identified/characterized.
Screening for Potential Novel BH4 Binding Sites
[0158] The results described herein suggest the existence of a
novel membrane bound or intracellular BH4 binding molecule that
functions as a BH4 target protein. Although the characteristics of
BH4 as a coenzyme are well described in the literature its binding
to other proteins and its transport mechanisms have not been
investigated. Novel proteomic approaches have been developed that
allow for a high throughput screening of binding sites of small
molecules such as BH4. Large-scale protein chips i.e.,
two-dimensional displays of individual proteins have been
constructed by immobilizing large numbers of purified proteins on
microplates. They are used, for example, to assay protein-protein
interactions, drug-target or enzymes-substrate interactions.
Generally they require an expression library, cloned into E. coli,
yeast, or other similar expression systems from which the expressed
proteins are then purified, and immobilized. Any suitable protein
purification method known in the art may be used including, for
example, a His-tag. Cell free protein transcription/translation
such as ribosome display is an alternative for synthesis of
proteins. Phage or yeast display libraries may also be used.
[0159] Binding of BH4 may be detected directly by labeling BH4 for
example with tritium what has been described previously (Werner et
al., Biochem. J. 604: 189-193, 1994) or with biotin which may be
coupled to the primary amino group of BH4. Incorporation of tritium
may be achieved in a cell based system with over-expression of the
synthetic enzymes and [.sup.3H]-labeled GTP as the substrate
(Werner et al., 1994). BH4 can also be chemically labeled with
tritium using the commercially available 6R-BH4 hydrochloride as
template. As an alternative to BH4 labeling, its binding to novel
targets might also be detected by using a labeled capturing
molecule e.g., a peptide containing the BH4 binding site of one of
the enzymes that bind BH4 as cofactor or a BH4 antibody. Label-free
detection methods, including mass spectrometry, surface plasmon
resonance and atomic force microscopy are also available and avoid
alteration of the ligand. In addition to large scale protein arrays
two-dimensional gel electrophoresis of tissue or cell protein
extracts of the dorsal horn and DRGs can be used to screen for
novel binding sites. The gel is then blotted onto PVDF membranes
and exposed to labeled BH4. Positive protein spots can be analyzed
by mass fingerprinting using matrix-assisted laser desorption
ionization time of flight mass spectrometry (MALDI-TOF MS).
Diagnosis of Pain or a Peripheral Nerve Lesion Using the BH4
Pathway
[0160] According to this invention, pain or a traumatic, metabolic
or toxic peripheral nerve lesion may be diagnosed in a mammal by
measuring the levels of BH4, BH4 intermediates, BH4 precursors, or
BH4 metabolites in a biological sample obtained from a mammal
(e.g., serum, plasma, urine, cerebrospinal fluid, synovial fluid,
tissue exudate, or tissue sample). Pain or a peripheral nerve
lesion is diagnosed if an increase in such levels relative to a
control (at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,
100%, or more than 100% relative to control) is detected using any
standard method known in the art. Alternatively, pain or a
peripheral nerve lesion may be diagnosed if an increase in the
levels or activity of any of the BH4 synthetic enzymes is detected.
Desirably, such increase is at least 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 95%, 100%, or more than 100% relative to control
conditions. According to this invention, the BH4 pathway serves as
a biomarker of nerve injury and pain.
[0161] The present invention further provides a kit for diagnosing
pain or a traumatic, metabolic or toxic peripheral nerve lesion in
mammal involving the measurement of BH4, its precursors and
intermediates (e.g., 7,8-dihydroneopterin triphosphate neopterin
and 6-pyruvoyl tetrahydropterin) or metabolites (e.g., pterin,
biopterin, 7,8 dihydropterin, 7, 8 dihydroxanthopterin,
xanthopterin, isoxanthopterin, or leucopterin) in a biological
sample, such as serum, plasma, urine, cerebrospinal fluid, synovial
fluid, tissue exudates, and tissue samples. For example, the
invention may include an antibody specific for any one of the above
compounds (e.g., biopterin or neopterin) and instructions to
diagnose pain in a mammal. In this regard, serum may be isolated
from a mammal in which a condition associated with the symptom of
pain is tested or a traumatic, metabolic or toxic peripheral nerve
lesion is suspected, and subjected to an ELISA or RIA assay using
an antibody of the invention. Pain is diagnosed in the mammal if
the serum level of BH4 in the mammal's serum (as detected by the
antibody) is increased relative to the BH4 levels in a control
serum sample. The diagnosis of pain, a subtype of pain, or a
peripheral nerve lesion is in a mammal if the levels of the
compound being measured in the biological sample obtained from the
mammal is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 100%, or more than 100% relative to the level of the same
compound in a control biological sample.
Pain Models
[0162] Various models test the sensitivity of normal animals to
intense or noxious stimuli (physiological or nociceptive pain).
These tests include responses to thermal, mechanical, or chemical
stimuli.
[0163] Thermal stimuli usually involve the application of hot
stimuli (typically varying between 42 -55.degree. C.) including,
for example: radiant heat to the tail (the tail flick test),
radiant heat to the plantar surface of the hindpaw (the Hargreaves
test), the hotplate test, and immersion of the hindpaw or tail into
hot water. In such models, the end points include latency to a
painful response, the duration of the response, vocalization, and
licking of the paw. Immersion in cold water, acetone evaporation,
or cold plate tests may also be used to test cold pain
responsiveness.
[0164] Tests involving mechanical stimuli typically measure the
threshold for eliciting a withdrawal reflex of the hindpaw to
graded strength monofilament von Frey hairs (the outcome measure
being the force of the filament required to elicit a reflex) or to
a sustained pressure stimulus to a paw (e.g., the Ugo Basile
analgesiometer). The duration of a response to a standard pinprick
may also be measured.
[0165] When using a chemical stimulus, the response to the
application or injection of a chemical irritant (e.g., capsaicin,
mustard oil, bradykinin, ATP, formalin, acetic acid) to the skin,
muscle joints or internal organs (e.g., bladder or peritoneum) is
measured. The outcome measures include vocalization, licking of the
paw, writhing, or spontaneous flexion.
[0166] In addition, various tests assess pain sensitization by
measuring changes in the excitability of the peripheral or central
components of the pain neural pathway. In this regard, peripheral
sensitization (i.e. changes in the threshold and responsiveness of
high threshold nociceptors) can be induced by repeated heat stimuli
as well as the application or injection of sensitizing chemicals
(e.g. prostaglandins, bradykinin, histamine, serotonin, capsaicin,
mustard oil). The outcome measures are thermal and mechanical
sensitivity in the area of application/stimulation using the
techniques described above in behaving animals, or alternatively,
electrophysiological measurements of single sensory fiber receptive
field properties either in vivo or using isolated skin nerve
preparations. The electrophysiological, neurochemical or cell
biological properties of sensory neurons can also be used to study
these parameters indirectly (e.g., recordings from isolated sensory
neurons (e.g., dorsal root ganglion neurons in culture), activation
of signal transduction pathways by sensitizing stimuli (e.g.,
protein kinase C or A), or measurements of receptor or ion channel
phosphorylation). Central sensitization (i.e. changes in the
excitability of neurons in the central nervous system induced by
activity in peripheral pain fibers) can be induced by noxious
stimuli (e.g., heat), chemical stimuli (i.e. injection or
application of chemical irritants such as capsaicin, mustard oil,
or formalin), or electrical activation of sensory fibers. The
outcome measures may be behavioral (i.e. thermal and mechanical
responsiveness outside of the area of application, that is the area
of secondary hyperalgesia, or tactile allodynia (pain responses to
normally innocuous tactile stimuli)), electrophysiological (i.e.
receptive field properties of single central neurons),
neurochemical (i.e. activation of signal transduction pathways in
central neurons (e.g., ERK, p38, CREB, immediate early genes such
as c-fos, kinases, PKC, PKA, or src) or phosphorylation of
receptors or ion channels such as NMDA or AMPA receptors).
Functional imaging techniques may also be used to assess changes in
the patterns of activation.
[0167] Various pain tests have also been developed to measure the
effect of peripheral inflammation on pain sensitivity (Stein et
al., Pharmacol. Biochem. Behay. (1988) 31: 445-451; Woolf et al.,
Neurosci. (1994) 62: 327-331). The inflammation may be produced by
injection of an irritant (e.g., complete Freund's adjuvant,
carrageenan, turpentine, and croton oil) into the skin,
subcutaneously, into a muscle, into a joint, or into a visceral
organ. Alternatively, the generation of a controlled UV light burn
and ischemia or the administration of cytokines or inflammatory
mediators such as lipopolysaccharide (LPS) or nerve growth factor
(NGF) can also mimic the effects of inflammation. Following the
induction of inflammation, the outcome measures may include changes
in behavior (e.g., thermal and mechanical sensitivity (as discussed
above), weight bearing, visceral hypersensitivity (e.g., inflation
of balloons in bladder or bowel), spontaneous locomotor activity,
or performance in more complex behaviors such as place preference
tasks), in electrophysiology (e.g., in vivo and in vitro recordings
from primary sensory neurons and central neurons with particular
attention to changes in receptive field properties, excitability,
or synaptic input), in neurochemistry (e.g., changes in the
expression and distribution of transmitters, neuropeptides, and
proteins in primary sensory and central neurons, activation of
signal transduction cascades, expression of transcription factors,
and phosphorylation of proteins in neurons), and imaging techniques
to detect changes in neural activity.
[0168] Additionally, various tests assess peripheral neuropathic
pain using lesions of the peripheral nervous system. One such
example is the "axotomy pain model," for example, which involves
the complete transection of a peripheral nerve and in which one or
a plurality of peripheral nerve fibers is severed, either by
traumatic injury or experimental or surgical manipulation (Watson,
J. Physiol. (1973) 231:41). Other similar tests include the SNL
test which involves the ligation of a spinal segmental nerve (Kim
and Chung Pain (1992) 50: 355), the Seltzer model involving partial
nerve injury (Seltzer, Pain (1990) 43: 205-18), the spared nerve
injury (SNI) model (Decosterd and Woolf, Pain (2000) 87:149),
chronic constriction injury (CCI) model (Bennett (1993) Muscle
Nerve 16: 1040), tests involving toxic neuropathies such as
diabetes (streptozocin model), pyridoxine neuropathy, taxol,
vincristine, and other antineoplastic agent-induced neuropathies,
tests involving ischaemia to a nerve, peripheral neuritis models
(e.g., CFA applied peri-neurally), models of post-herpetic
neuralgia using HSV infection, and compression models. In all of
the above tests, outcome measures may be assessed, for example,
according to behavior (e.g., thermal and mechanical sensitivity as
above, weight bearing, spontaneous activity, or performance in more
complex behaviors such as place preference tasks),
electrophysiology (e.g., in vivo and in vitro primary sensory
neurons and central neurons with particular attention to changes in
membrane excitability, spontaneous activity, receptive field
properties, and synaptic input), neurochemistry (e.g., expression
and distribution of transmitters, neuropeptides and proteins in
primary sensory and central neurons, activation of signal
transduction cascades, expression of transcription factors, and
phosphorylation of proteins in neurons), and imaging techniques to
detect changes in neural activity. Furthermore, several pain tests
that mimic central neuropathic pain involve lesions of the central
nervous system including, for example, spinal cord injury (e.g.,
mechanical, compressive, ischemic, infective, or chemical). In
these particular tests, outcome measures are the same as those used
for peripheral neuropathic pain.
[0169] Various features of pain are shared between the above
models. In this respect, physiological pain is characterized by a
high threshold to mechanical and thermal stimuli and rapid
transient responses to such stimuli. Inflammatory and neuropathic
pain are characterized by displays of behavior indicating either
spontaneous pain (measured by spontaneous flexion, vocalization,
biting, or even self mutilation), abnormal hypersensitivity to
normally innocuous stimuli (allodynia), and an exaggerated response
to noxious stimuli (hyperalgesia).
Measurement of Pain
[0170] Thermal and mechanical threshold sensitivity may be measured
quantitatively, for example, in .degree. C., force in grams or
Newtons, or alternatively, as a measure of time to respond. For
thermal pain thresholds, the temperature of a hot stimulus
>40.degree. C. or a cold stimulus (<15 .degree. C.) that
elicits a flexion withdrawal response is typically measured. For
mechanical thresholds the force of a punctate mechanical stimulus
(<100 g) that elicits a flexion withdrawal response is measured.
The reduction in the latency of response to the stimulus, that is
the length of time the animal takes to respond, can also be
measured (typically <10 seconds). The actual values depend on
the nature of the test and the area of the body stimulated. One way
of testing an increase in pain sensitivity is to repeatedly apply a
stimulus close to threshold levels and look for an increase in the
proportion of positive responses to this fixed stimulus. For pain
responsiveness, the measurement is the duration or magnitude of a
response such as the amount of time an animal holds its limb in a
flexed position after a pinprick or a hot or cold stimulus.
Screening Assays
[0171] The present invention provides screening methods to identify
compounds that can inhibit the production or action of BH4. Useful
compounds include any agent that can inhibit the biological
activity or reduce the cellular level of BH4 or at least one or
more than one of any one of the enzymes shown in FIG. 2A. Such
enzymes include, for example, GTP cyclohydrolase (GTPCH),
Pyruvoyltetrahydropterin (PTPS), Sepiapterin Reductase (SPR), and
Dihydropteridine Reductase (DHPR). As discussed above, we have
shown that DAHP, NAS, and methotrexate are useful to treat, reduce,
or prevent pain. Using such agents as lead compounds, for example,
the present screening methods allow the identification of novel,
specific inhibitors of the BH4 synthetic pathway that function to
induce analgesia. The method of screening may involve
high-throughput techniques.
[0172] A number of methods are available for carrying out such
screening assays. According to one approach, candidate compounds
are added at varying concentrations to the culture medium of cells
expressing one or more of the BH4 synthetic enzymes. Gene
expression of the BH4 synthetic enzymes is then measured, for
example, by standard Northern blot analysis (Ausubel et al.,
supra), using any appropriate fragment prepared from the nucleic
acid molecule of the BH4 synthetic enzyme as a hybridization probe
or by real time PCR with appropriate primers. The level of gene
expression in the presence of the candidate compound is compared to
the level measured in a control culture lacking the candidate
molecule. If desired, the effect of candidate compounds may, in the
alternative, be measured at the level of BH4 production using the
same general approach and standard immunological techniques, such
as Western blotting, immunoprecipitation, or immunoassay with an
antibody specific to the BH4 synthetic enzyme or for BH4 (or its
intermediaries or metabolites). For example, immunoassays may be
used to detect or monitor the level of BH4 or GTPCH. Polyclonal or
monoclonal antibodies which are capable of binding to BH4, BH4
precursors, or BH4 metabolites may be used in any standard
immunoassay format (e.g., ELISA or RIA assay) to measure the levels
of BH4 or its precursors or metabolites. BH4 or its precursors or
metabolites can also be measured using mass spectroscopy, high
performance liquid chromatography, spectrophotometric or
fluorometric techniques, or combinations thereof. Total biopterin
(BH1, BH2, and BH4) content may further be measured as described
further below.
[0173] Alternatively, the screening methods of the invention may be
used to identify candidate compounds that decrease the biological
activity of BH4 by decreasing its binding to BH4-dependent enzymes
or BH4-binding receptors, or alternatively, that decrease the
activity or levels of any of the BH4 synthetic enzymes. For
example, a candidate compound may be tested for its ability to
decrease GTPCH activity in cells that naturally express the enzyme,
after transfection with cDNA for the enzyme, or in cell-free
solutions containing the enzyme, as described further below. The
effect of a candidate compound on the binding or activation of a
BH4-dependent enzyme (such as NOS) or a BH4-binding receptor (or
analogs) can be tested by radioactive and non-radiaoctive binding
assays, competition assays, enzyme activity assays, receptor
signaling assays.
[0174] As a specific example, mammalian cells (e.g., rodent cells)
that express a nucleic acid encoding a BH4 synthetic enzyme are
cultured in the presence of a candidate compound (e.g., a peptide,
polypeptide, synthetic organic molecule, naturally occurring
organic molecule, nucleic acid molecule, or component thereof).
Cells may either endogenously express the BH4 synthetic enzyme or
may alternatively be genetically engineered by any standard
technique known in the art (e.g., transfection and viral infection)
to overexpress the BH4 synthetic enzyme. The expression level of
the BH4 synthetic enzyme is measured in these cells by means of
e.g., Western blot analysis and subsequently compared to the level
of expression of the same protein in control cells that have not
been contacted by the candidate compound. A compound which promotes
a decrease in the level of BH4 or intermediary as a result of
reducing its synthesis by reducing the level or activity of one of
its synthetic enzymes is considered useful in the invention. Given
its ability to decrease the level or activity of BH4, such a
molecule may be used, for example, as an analgesic therapeutic
agent to treat, reduce, or prevent pain.
[0175] The activity of any of the BH4 synthetic enzymes may be
measured by the rate at which they consume substrate, e.g., GTP or
produce product, e.g., BH4 (see Werner et al. (1996) J Chromatogr B
Appl 684:51-58). Radiometric assays are based on the consumption of
labeled substrate. For example, GTPCH activity may be assessed by
measuring the release of labeled formic acid originating from a
labeled hydrogen atom of GTP and separation of formic acid from GTP
by charcoal (Viveros et al. (1981) Science 213: 349). HPLC-based
methods however, are superior to the radioactive method in that
HPLC allows determination of the product. For measuring GTPCH
activity, the tissue or cell homogenate containing GTPCH is
incubated with excess GTP (substrate) in the presence of EDTA to
ensure that the product 7,8 dihydropterin triphosphate is not
further metabolized by the downstream PTPS which requires Mg.sup.2+
to operate. The reaction is stopped by the addition of HCl and
iodine. This also results in oxidation of the labile
7,8-dihydroneopterin triphosphate to the more stable neopterin
triphosphate. Neopterin triphosphate may be analyzed directly by
ion-pair HPLC and fluorescence detection. Alternatively, the
mixture is treated with NaOH and alkaline phosphatase to yield
neopterin which can be analyzed using reversed-phase HPLC with
fluorescence detection, immunoassay or direct fluorescence in case
of "pure" samples (such as in vitro kinase assay or CSF).
[0176] Sepiapterin reductase activity is commonly assayed using
sepiapterin as artificial substrate and measuring levels of total
biopterin after oxidation of BH4 and BH2 to biopterin.
[0177] For determination of PTPS activity, the substrate 7,8
dihydroneopterin triphosphate is typically freshly prepared with
purified GTPCH. The incubation mixture also typically contains
purified sepiapterin reductase, so that PTPS activity may be
evaluated by measuring biopterin levels after oxidation of BH4 and
BH2.
[0178] Given its ability to decrease the levels of BH4 or the
levels or activity of a BH4 synthetic enzyme, such a molecule may
be used, for example, as an analgesic therapeutic agent to treat,
reduce, or prevent pain.
[0179] Alternatively, or in addition, candidate compounds may be
screened for those which specifically bind to or inhibit a BH4
synthetic enzyme, BH4-dependent enzyme, or BH4-binding receptor.
The efficacy of such a candidate compound is dependent upon its
ability to interact with BH4, a BH4 synthetic enzyme or a
BH4-binding enzyme or receptor. Such an interaction can be readily
assayed using any number of standard binding techniques and
functional assays (e.g., those described in Ausubel et al., supra).
For example, a candidate compound may be tested in vitro for
interaction and binding with BH4 and its ability to modulate pain
may be assayed by any standard assays (e.g., those described
herein).
[0180] In one particular example, a candidate compound that binds
to any of the BH4 synthetic enzymes may be identified using a
chromatography-based technique. For example, a recombinant BH4
synthetic enzyme protein may be purified by standard techniques
from cells engineered to express the BH4 synthetic enzymes (e.g.,
those described above) and may be immobilized on a column.
Alternatively, BH4 may be immobilized on a column. A solution of
candidate compounds is then passed through the column, and a
compound specific for either BH4 or one of the BH4 synthetic
enzymes is identified on the basis of its ability to bind to BH4 or
a BH4 synthetic enzyme and be immobilized on the column. To isolate
the compound, the column is washed to remove non-specifically bound
molecules, and the compound of interest is then released from the
column and collected. Compounds isolated by this method (or any
other appropriate method) may, if desired, be further purified
(e.g., by high performance liquid chromatography).
[0181] Screening for new inhibitors and optimization of lead
compounds may be assessed, for example, by assessing GTPCH activity
as described above. For screening of multiple substances, a 96
well-based enzyme assay may be used where purified recombinant
GTPCH is incubated together with substrate and the potential
inhibitor followed by oxidation and measurement of neopterin with a
fluorescence ELISA reader. Neopterin shows intense fluorescence and
can be directly measured.
[0182] Assays may also be based on BH4 measurement. BH4 shows no
intense fluorescence, because the rings of the molecule are not in
the fully oxidized, aromatic state. To circumvent this, a
differential oxidization method in which dihydrobiopterin and BH4
are measured following their oxidation to biopterin may be used,
with a limit of detection of 0.3 .mu.mol for biopterin with
fluorescence (Fukushima and Nixon, Anal. Biochem. (1980) 102:
176-188). Assays for measuring the activity of GTPCH or levels of
biopterin are described, for example, by Kaneko et al., Brain Res.
Brain Res. Protoc. (2001) 8:25-31; Ota et al., J. Neurochem. (1996)
67: 2540-2548; Brautigam et al., Physiol. Chem. (1982) 363:
341-343; Curtius et al., Eur. J. Biochem. (1985) 148: 413-419; Stea
et al., J. Chromatogr. (1979) 168: 385-393; Werner et al., J.
Chromatogr. (1996) 684: 51-58; Werner et al., Methods Enzymol.
(1997) 281: 53-61; Nagatsu et al., Anal. Biochem. (1981) 110:
182-189; and Geller et al., Biochem Biophys Res Commun (2000) 276:
633-41.
[0183] In addition, these candidate compounds may be tested for
their ability to function as analgesic agents (e.g., as described
herein). Compounds isolated by this approach may also be used, for
example, as therapeutics to treat, reduce, or prevent pain.
Compounds which are identified as binding to BH4, any of the BH4
synthetic enzymes, BH4 dependent enzymes, or BH4-binding receptors
with an affinity constant less than or equal to 10 mM are
considered particularly useful in the invention.
[0184] Ultimately, the analgesic efficacy of any of the candidate
compounds identified by the present screening methods may be tested
using any of the pain models described above.
[0185] Potential analgesics include organic molecules, peptides,
peptide mimetics, polypeptides, and antibodies that bind to a
nucleic acid sequence or polypeptide that encodes any of the BH4
synthetic enzymes or BH4 dependent enzymes or BH4 binding receptors
and thereby inhibit or extinguish their activity. Potential
analgesics also include small molecules that bind to and occupy the
binding site of such polypeptides thereby preventing binding to
cellular binding molecules, such that normal biological activity is
prevented. Other potential analgesics include antisense
molecules.
[0186] In addition to BH4, any of the BH4 synthetic enzymes may be
used upon expression, as a target for the screening of candidate
compounds. Furthermore, each of the compounds provided herein
(e.g., DAHP, NAS, methotrexate, BH4, guanine, or any of the
compounds found in FIG. 19) may also be used as lead compounds in
the discovery and development of analgesic compounds.
[0187] Test Compounds and Extracts
[0188] In general, compounds capable of inducing analgesia are
identified from large libraries of both natural products or
synthetic (or semi-synthetic) extracts or chemical libraries
according to methods known in the art. Those skilled in the field
of drug discovery and development will understand that the precise
source of test extracts or compounds is not critical to the
screening procedure(s) of the invention. Accordingly, virtually any
number of chemical extracts or compounds can be screened using the
methods described herein. Examples of such extracts or compounds
include, but are not limited to, plant-, fungal-, prokaryotic- or
animal-based extracts, fermentation broths, and synthetic
compounds, as well as modification of existing compounds. Numerous
methods are also available for generating random or directed
synthesis (e.g., semi-synthesis or total synthesis) of any number
of chemical compounds, including, but not limited to, saccharide-,
lipid-, peptide-, and nucleic acid-based compounds. Synthetic
compound libraries are commercially available from Brandon
Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee,
Wis.). Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant, and animal extracts are commercially
available from a number of sources, including Biotics (Sussex, UK),
Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft.
Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). In
addition, natural and synthetically produced libraries are
produced, if desired, according to methods known in the art, e.g.,
by standard extraction and fractionation methods. Furthermore, if
desired, any library or compound is readily modified using standard
chemical, physical, or biochemical methods.
[0189] In addition, those skilled in the art of drug discovery and
development readily understand that methods for dereplication
(e.g., taxonomic dereplication, biological dereplication, and
chemical dereplication, or any combination thereof) or the
elimination of replicates or repeats of materials already known for
their analgesic activity should be employed whenever possible.
[0190] When a crude extract is found to have an analgesic activity,
or a binding activity, further fractionation of the positive lead
extract is necessary to isolate chemical constituents responsible
for the observed effect. Thus, the goal of the extraction,
fractionation, and purification process is the careful
characterization and identification of a chemical entity within the
crude extract having analgesic activity. Methods of fractionation
and purification of such heterogenous extracts are known in the
art. If desired, compounds shown to be useful agents for the
treatment of pain are chemically modified according to methods
known in the art.
Pharmaceutical Therapeutics
[0191] The invention provides a simple means for identifying
compounds (including peptides, small molecule inhibitors, and
mimetics) capable of treating, reducing, or preventing pain.
Accordingly, a chemical entity discovered to have medicinal value
using the methods described herein are useful as either drugs or as
information for structural modification of existing analgesic
compounds, e.g., by rational drug design.
[0192] For therapeutic uses, the compositions or agents identified
using the methods disclosed herein may be administered
systemically, for example, formulated in a
pharmaceutically-acceptable buffer such as physiological saline.
Treatment may be accomplished directly, e.g., by treating the
animal with compounds that treat, reduce, or prevent pain by
interfering with the production of BH4 (by interfering with the
biological activity of any of the BH4 synthetic enzymes) or by
interfering directly with the biological activity of BH4 by
blocking activation of enzymes that use it as a cofactor or
receptors that bind to BH4. Preferable routes of administration
include, for example, subcutaneous, intravenous, intraperitoneally,
intramuscular, or intradermal injections, which provide continuous,
sustained levels of the drug in the patient. Treatment of human
patients or other animals will be carried out using a
therapeutically effective amount of an analgesic in a
physiologically-acceptable carrier. Suitable carriers and their
formulation are described, for example, in Remington's
Pharmaceutical Sciences by E. W. Martin. The amount of the
analgesic to be administered varies depending upon the manner of
administration, the age and body weight of the patient, and with
the type of disease and extensiveness of the disease. Generally,
amounts will be in the range of those used for other agents used in
the treatment of pain, although in certain instances lower amounts
will be needed because of the increased specificity of the
compound. A compound is administered at a dosage that inhibits
pain. For example, for systemic administration a compound is
administered typically in the range of 0.1 ng-10 g/kg body
weight.
[0193] The results of the invention are now described in more
detail in the following examples. These examples are provided to
illustrate the invention and should not be construed as
limiting.
EXAMPLE 1
Induction of Synthetic Enzymes of the BH4 Pathway by Peripheral
Nerve Injury
[0194] Transection of the peripheral axons of primary sensory
neurons results in profound alterations in their metabolism,
regenerative capacity, survival, excitability, transmitter
function, and sensitivity to diverse extrinsic and intrinsic
signals. These changes are mediated by transcriptional alterations
triggered both by the loss of trophic support from peripheral
target organs and by novel signals generated at the injury site.
These transcriptional changes lead to adaptive responses, such as
the capacity to survive the injury and re-grow the injured axon, as
well as maladaptive responses that can result in a change in
sensation, including the generation of neuropathic pain.
[0195] High-density rat oligonucleotide microarrays have been used
to detect changes in gene expression in the dorsal root ganglion
(DRG) following sciatic nerve transection (axotomy). The DRG
represents a dense collection of cell bodies of one general class
of neurons, the primary sensory neuron. The lesion has a uniform
impact on the cells, and the existence of a large pool of genes
with known regulation allows for quality controls for changes
identified by the microarrays.
[0196] Affymetrix rat U34A oligonucleotide arrays were used to
screen for changes in gene expression in DRG neurons during
maturation of the DRG in the embryo and after a sciatic nerve
lesion. In the first study we found that the expression profile of
several genes, including dihydropteridine reductase (DHPR), was
characterized by high levels of expression early during
development, a down-regulation during adulthood, and re-expression
following peripheral nerve injury (FIGS. 1A and 1B). A more
detailed study was then performed looking at alterations in gene
expression three days following a peripheral nerve (sciatic)
transection (axotomy, Ax) by comparing expression levels with
non-injured DRGs (naive, N) as described previously by Costigan et
al., (BMC Neuroscience (2002) 3:16), hereby incorporated by
reference. Nine biologically independent array hybridizations were
performed (six naive and three after axotomy). DRG tissue (L4 and
L5 from the left or ipsilateral side to the injury) from five male
Sprague-Dawley rats was pooled for each RNA population. Each RNA
sample was labeled separately and hybridized to a separate array.
Two comparisons were made using two sets of triplicate microarrays:
naive versus naive and naive versus axotomy. .sub.Genes were
defined as detected if they received a present or marginal call in
at least one of the arrays within each comparison. Since each
individual sample was pooled from five male Sprague-Dawley animals
of a similar age and from a single supplier (Charles River),
biological variation is likely to be minimal.
[0197] In addition to DHPR, this analysis further revealed that two
other members of the tetrahydrobiopeterin synthesis pathway (see
FIG. 2A) were also significantly upregulated by peripheral nerve
injury: GTP cyclohydrolase (GTPCH) and sepiapterin reductase (SPR)
(See FIG. 3).
EXAMPLE 2
Validation of Microarray Analysis
[0198] The induction of BH4 synthetic enzymes by axotomy was next
confirmed by various methods, such as Northern blot analysis,
Northern slot blot analysis, in situ hybridization, and Western
blot analysis. A sample from each group was prepared from
independent L4 and L5 DRG RNA samples extracted from different
groups of animals than those used for the arrays. FIG. 1B
represents a Northern slot blot analysis showing the expression
profile of DHPR during embryonic development and in the adult,
before and after axotomy and thereby confirming our microarray
data. Northern Blot analysis to detect GTPCH mRNA levels in naive
DRG and 3 days following injury clearly show the marked induction
of two transcripts of 3 kb and 1.2 kb (see FIG. 4A). FIG. 3
summarizes the degree of the induction of BH4 synthetic enzymes in
DRG following axotomy. In situ analysis further confirmed the
induction of GTPCH mRNA in neurons of the DRG three days post
peripheral nerve injury (FIG. 4B). FIGS. 4D-4F represent triplicate
Northern blot analysis demonstrating that the induction of GTPCH,
DHPR, and SPR mRNA transcript levels in the DRG following axotomy
is sustained for at least 2 weeks. We further show that the
increase in GTPCH mRNA levels following nerve injury is associated
with an increase in protein levels (FIG. 5A) and enzyme activity
(FIG. 5B). FIG. 5A represents a Western Blot analysis of GTPCH
protein levels in naive DRG and 1, 3, 7, and 14 days post-axotomy
showing a marked and sustained increase in GTPCH levels following
axotomy. GTPCH activity levels in the DRG are markedly higher at
seven days post axotomy relative to control (FIG. 5B). The amount
of BH4 in the DRG is also increased (FIG. 5C) seven days post
axotomy relative to control.
EXAMPLE 3
Changes in BH4 Synthetic Enzymes in Neuropathic and Inflammatory
Pain Models
[0199] Triplicate Affymetrix microarrays were used to establish the
time course of changes of expression of the BH4 synthetic pathway
members (GTPCH, SPR and DHPR as well as 6-pyruvoyl tetrahydropterin
synthase and the feedback regulatory protein, GTPCH feed-back
regulatory protein) in the DRG and in the dorsal horn of the spinal
cord in three independent peripheral neuropathic pain models and
after peripheral inflammation. GTPCH I, SPR, PTPS and DHPR were all
upregulated by a substantial degree and for prolonged periods in
the DRG in all three peripheral neuropathic pain models (FIGS.
6A-6J). The mRNA for all these enzymes was detectable in the dorsal
horn (i.e. are constitutively expressed) but showed minimal
alterations in expression in the pain models. In both the DRG and
dorsal horn, peripheral inflammation also did not produce marked
changes in the constitutive basal level of expression of the BH4
synthetic enzymes (FIG. 7A-I).
EXAMPLE 4
Effects of an Inhibitor of GTPCH on Neuropathic Pain
[0200] Based on these findings, we hypothesized that the BH4
pathway may have a role in the biological response to peripheral
nerve injury including activation of cell survival responses,
changes in excitability, alterations in transmitter function, and
change in growth status. In particular, we hypothesized that the
pathway may have a role in the generation of pain after peripheral
nerve injury (peripheral neuropathic pain) for example by
increasing NOS activity as a result of the increase in BH4
levels.
[0201] To assess whether the BH4 synthetic pathway is involved in
neuropathic pain, we examined if DAHP could elicit analgesia in
various pain models, such as the spared nerve injury (SNI) model
(FIGS. 8A-8H), the chronic constriction injury (CCI) model (FIGS.
9A and 9B), the formalin assay (FIG. 10), and the CFA model (FIGS.
11A-E).
[0202] Using the SNI model, we show that treatment with DAHP (180
mg/kg/day injected intraperitoneally) following surgery produced a
reduction in mechanical sensitivity (von Frey threshold) and cold
pain (cold allodynia by the application of acetone to the paw)
relative to rats injected with vehicle, whether treatment was
initiated at an early time point (e.g., three days post-surgery,
see FIGS. 8A-8D) or at a later time point (e.g., seventeen days
post-surgery, see FIGS. 8E-8F). Thus, treatment with DAHP could
produce analgesia even once neuropathic pain was established. DAHP
(6 mg/kg/day i.t.) also reduced mechanical and cold allodynia when
administered as a continuous intrathecal infusion through a lumbar
spinal catheter. The efficacy was comparable with intraperitoneal
treatment. The analgesic effects of DAHP were further confirmed in
the CCI model (180 mg/kg/d i.p.; FIGS. 9A and 9B). DAHP (single
i.p. dose of 180 mg/kg) also reduced the flinching behavior in the
formalin assay (FIG. 10).
[0203] The results of FIG. 8 demonstrating the analgesic effects of
DAHP in an SNI model were extended through the use of increasing
doses of DAHP. FIGS. 22A and 22B demonstrate that a dose-dependent
relationship exists between the amount of DAHP administered and the
nociceptive response to mechanical (von Frey test) or thermal (cold
allodynia) stimuli. This dose-effect relationship was linear in the
dose range tested. Up to the highest dose of 270 mg/kg/d, no
obvious neurological adverse effects were observed over 14 days of
treatment. These results further support the pharmacological effect
of BH4 pathway inhibitors.
[0204] Moreover, as expected, intrathecal administration of BH4 was
pro-nociceptive. FIG. 23A demonstrates that intrathecal
administration of BH4 onto the lumbar spinal cord through a
chronically implanted catheter reduces the paw withdrawal latency
to a thermal stimulus (Hargreaves model) in naive rats, indicating
an increased hypersensitivity to heat. Likewise, in rats with
pre-existing heat hypersensitivity, intrathecal BH4 administration
induced heat hypersensitivity in the ipsilateral, but not
contralateral, paw in a CFA-induced model of paw inflammation.
[0205] We next injected complete Freund's adjuvant (CFA) into the
right paw of rats to elicit paw inflammation. We show using the CFA
pain model that DAHP (180 mg/kg i.p.) reduced thermal hyperalgesia,
whether treatment was initiated 30 min before (FIG. 11A, left side)
or 24 h (FIGS. 11A, right side and 11C) after the CFA injection.
DAHP had no effect on the paw withdrawal latency of the
non-inflamed contralateral paw (FIG. 11B), indicating that DAHP had
no general obvious inhibitory effect on sensory and motor
functions. DAHP (1 mg/kg i.t.) also reduced thermal hyperalgesia
when it was delivered to the lumbar spinal cord by intrathecal
injection through a lumbar spinal catheter (FIG. 11D). Direct
comparison of intraperitoneal and intrathecal DAHP treatment
revealed that effects are similar with both routes of drug
administration (FIG. 11E) The fact that there was no difference in
the analgesic effect in rats treated with DAHP intrathecally and
intraperitoneally infers that DAHP is effective at the level of the
spinal cord and DRG.
[0206] We next measured the effect of DAHP on inflammatory paw
edema. As shown in FIG. 12, measuring the paw weight in the CFA
injected paw and the non-injected control paw showed no difference
in the degree of paw inflammation between DAHP treated and control
animals. Thus, because the administration of DAHP has no obvious
effect on inflammation (no anti-inflammatory action), our results
suggest that DAHP's analgesic effect is primarily a result of
changes in sensory processing in the nervous system
[0207] Using the von Frey and the Hargreaves thermal pain test, we
further show that the injection of DAHP in non-injured animals did
not result in a difference in motor activity. Based on these
results, and the absence of any detectable changes in locomotion
(FIGS. 13A-13B) the possibility that DAHP at the doses used has an
effect on general sensory and motor activity seems unlikely. In
addition there was not detectable change in the general level of
activity with no obvious signs of sedation.
[0208] We next performed pharmacokinetic studies to inspect the
levels of DAHP in plasma and CSF (see FIGS. 14A and 14B) and show
that the plasma concentration rapidly increased after i.p.
injection followed by a rapid distribution in the cerebrospinal
fluid. Furthermore, we confirmed that the increase in plasma DAHP
concentration over time correlated with the behavioral effect in
rats in response to DAHP treatment in the CFA model (FIG. 14C).
EXAMPLE 5
Effects of Other BH4 Inhibitors on Inflammatory or Neuropathic
Pain
[0209] We next evaluated the effect of inhibiting the BH4 synthetic
enzyme Sepiapterin reductase by administering N-acetyl-serotonin
(NAS). Similarly to DAHP, we show that NAS (50 mg/kg i.p.) resulted
in a reduction of thermal hyperalgesia in the CFA model (see FIG.
15A). NAS treatment also had no effect on the CFA-induced
inflammatory paw edema (FIG. 15B).
[0210] Similarly, we show that the administration of methotrexate,
an inhibitor of DHPR, could result in a reduction in pain in the
SNI model in response to mechanical and cold allodynia, in the
absence of detectable acute toxicity. MTX was administered at low
dose systemically (see FIGS. 16A and 16B) or by continuous lumbar
spinal delivery using an osmotic pump (0.1 mg/kg/d ay)(see FIGS.
16C and 16D). Toxicity was measured as body weight change over time
(see FIGS. 16E and 16F).
[0211] Overall, our results demonstrate that the inhibition of BH4
synthesis by administering DAHP, NAS, or MTX for example, results
in analgesia in response to thermal, mechanical, and chemical
stimuli. Based on these results, inhibiting the synthesis of BH4,
by reducing the biological activity of BH4 synthetic enzymes for
example, induces analgesia, and therefore, may be used to treat,
prevent, or reduce pain in a mammal in need thereof.
EXAMPLE 6
Molecular and Cellular Effects of DAHP on Neuropathic Pain
[0212] Measurement of c-FOS expression in dorsal horn neurons was
used as an objective indication of pain intensity. FIG. 24
demonstrates that c-FOS immunoreactivity is elevated in ipsilateral
dorsal horn neurons of animals two hours after receiving formalin
injection into the hindpaw. A significantly reduced elevation in
c-FOS levels were observed in animals also administered
intraperitoneal DAHP (p<0.05). This indicates that DAHP acts in
the BH4 metabolic pathway upstream of immediate early gene c-Fos
induction and reduces activation of neurons in the spinal cord.
[0213] Apoptosis of dorsal horn neurons contributes to the
development of neuropathic pain following a nerve injury. To
further investigate the cellular role of BH4 in neuropathic pain,
apoptotic profiles of L4/L5 dorsal horn neurons were evaluated
using TUNEL staining. FIG. 25 demonstrates that intraperitoneal
DAHP administration protects dorsal horn neurons from apoptosis in
the SNI model.
[0214] As discussed above, BH4 is an essential co-factor for nNOS
and iNOS isozymes which have been shown to contribute to pain
signaling in the nervous system. We have found that the
anti-nociceptive effects of DAHP do not differ between nNOS
knockout mice and wild-type mice (FIG. 26A), demonstrating the nNOS
is not essential for the production of the analgesic effects of
DAHP. We have also found that, DAHP induces a stronger
antinociceptive effect than a high systemic dose of L-NAME, a
non-specific NOS inhibitor, and that L-NAME does not further
increase the efficacy of DAHP when injected together (FIG. 26B).
Thus, the anti-nociceptive effects of DAHP cannot be attributed
merely to an inhibition of NO production and the pro-nociceptive
action of BH4 is not mediated solely through a NOS-dependent
mechanism. Taken together, these data suggest that the
pro-nociceptive effects of BH4 are likely mediated through a novel
target molecule.
EXAMPLE 7
Localization of GTPCH-I
[0215] We have characterized the localization of GTPCH-I.
Upregulation of this enzyme occurs in large and small to medium
sized DRG neurons after peripheral axonal injury (SNI model)
compared to unlesioned animals (FIGS. 27A and 27B). Upregulation of
GTPCH-I is not, however, observed in the CFA-induced paw
inflammation model. Forty to fifty percent of neurons that
upregulate GTPCH-I are also immunoreactive for neurofilament 200
(NF200) which is a marker for neurons having myelinated axons (FIG.
29). Thirty to forty percent of GTPCH-I mRNA positive neurons are
also immunoreactive for calcitonin gene related peptide (CGRP; FIG.
29). CGRP labels small to medium sized neuropeptide positive
sensory neurons, most of which are nociceptors. GTPCH-I expressing
neurons do not express nNOS and are not labeled with Griffonia
simplicifolia isolectin B4 (IB4; FIG. 29). IB4 labels small
unmyelinated GDNF (glial cell derived neurotrophic factor)
responsive neurons. GTPCH-I-expressing neurons show
immunoreactivity for ATF-3 which indicates that the upregulation
mainly occurs in injured neurons (FIG. 31).
[0216] The GTPCH-I transcript is not detectable in the dorsal horn
of the spinal cord in either control or SNI-lesioned animals.
Isolated injured motor neurons show GTPCH-I mRNA when their
peripheral axons are transected by a peripheral nerve injury (FIG.
28A). GTPCH-I feedback regulatory protein (GFRP) mRNA can be
detected in isolated DRG neurons and its expression does not change
after nerve injury (FIG. 28B).
EXAMPLE 8
Upregulation of BH4 Metabolites is Detected in Lesioned Dorsal Root
Ganglia
[0217] We measured the levels of biopterin and neopterin, stable
metabolites of BH4, in the DRG of animals following SNI lesion.
Neopterin is a stable metabolite of BH4 found following BH4
recycling. The presence of neopterin may be an index of new BH4
synthesis, whereas biopterin is indicative of BH4 recycling and
reuse, but not necessarily new synthesis. FIG. 30 demonstrates
elevated biopterin levels in the ipsilateral doral horn (DH), the
DRG, and the ScN compared to the contralateral side. Biopterin
increases were reversed in the DH and the ScN, but not the DRG,
with the administration of DAHP. Neopterin levels, by contrast,
were elevated only in the DRG and ScN, but not the DH. These
increases were reversed by DAHP administration. Together, these
data demonstrate the usefulness of measuring stable BH4 metabolites
as objective indicators of pain. Further, these data demonstrate
that DAHP inhibits BH4 biosynthesis in vivo and inhibition of the
BH4 biosynthetic pathway is a useful mechanism for inducing
analgesia.
[0218] The above experiments were performed using the following
materials and methods.
Materials and Methods
Surgical Procedures
[0219] All procedures were performed in accordance with
Massachusetts General Hospital animal care regulations. Adult male
Sprague Dawley rats (200-300 g) were anesthetized with halothane.
For the sciatic nerve transection (axotomy), the left sciatic nerve
was exposed at the mid thigh level, ligated with 3/0 silk, and
sectioned distally. The wound was sutured in two layers, and the
animals were allowed to recover. For SNI the tibial and peroneal
branch of the sciatic nerve were ligated and transected whereas the
sural nerve was spared. For CCI, the sciatic nerve was loosely
ligated with dexon 4/0 (three ligatures) and for the spinal
ligation model the L5 spinal segmental nerve was ligated.
Tissue and RNA Preparation
[0220] Animals were terminally anesthetized with CO.sub.2, the L4
and L5 DRGs rapidly removed, and stored at -80.degree. C. Total RNA
was extracted from homogenized DRG samples using acid phenol
extraction (TRIzol reagent, Gibco-BRL). RNA concentration was
evaluated by A.sub.260 measurement and quality assessed by
electrophoresis on a 1.5% agarose gel. Each RNA sample used for
hybridization of each array was extracted from rat L4 and L5 DRGs
(10 ganglia pooled from 5 animals, per sample).
Microarray Analysis
[0221] Affymetrix rat genome U34A oligonucleotide microarrays,
representing 8799 known transcripts and expressed sequence tags
(ESTs), were used according to the manufacturers instructions
(Santa Clara, Calif. http://www.affymetrix.com). Transcript
abundance is estimated by analysis of signal intensity of the probe
set for each transcript and comparison with mismatch controls. The
arrays are hybridized with biotin-labeled cRNA, prepared as per
standard Affymetrix protocol. Briefly, total RNA (8 .mu.g) from
DRGs was reverse transcribed using an oligo-dT primer coupled to a
T7 RNA polymerase binding site. Double-stranded cDNA was made and
biotinylated-cRNA synthesized using T7 polymerase. The cRNA was
hybridized for 16 hours to an array, followed by binding with a
streptavidin-conjugated fluorescent marker, and then incubated with
a polyclonal anti-streptavidin antibody coupled to phycoerythrin as
an amplification step. Following washing, the chips were scanned
with a Hewlett-Packard GeneArray laser scanner and data analyzed
using GeneChip software. External standards were included to
control for hybridization efficiency and sensitivity.
[0222] Hybridization levels for each species of mRNA detected on
the arrays are expressed by intensity (signal) and as present (P),
marginal (M) or absent (A) calls, calculated by Affymetrix software
(MAS 5.0, .alpha.1=0.04 .alpha.2=0.06). To normalize the array data
standard Affymetrix protocols were employed, each array was scaled
to a target signal of 2500 across all probe sets (MAS 5.0).
[0223] The arrays were grouped for comparison of a triplicate set
of naive data with a triplicate post-axotomy set. A probe set was
determined undetected if it received an A call in all of the six
arrays involved in the comparison. Detected were Present or
Marginal by MAS5.0 in at least one array for each analysis. Mean
signal and standard deviation were calculated for each detected
probe set. The p-value for rejecting the null hypothesis that the
mean signals were equal between the two triplicate sets was
calculated using an unpaired, two-tailed t-test for independent
samples with unequal variance (Satterthwaite's method).
Fold-differences between the mean signals (A and B) in the two
triplicate sets were calculated as max (A, B)/min(A, B) with down
regulation relative to naive expressed as negative.
cDNA Probe production
[0224] To generate specific probes for Northern blot hybridization
experiments, primers based on the rat accession number provided by
Affymetrix were designed, primer pairs were chosen using the
Primer3 software http://www-genome.wi.mit.edu/ from the 1000 most
3' nucleotides within each accession sequence. PCR was performed on
cDNA reverse transcribed from total RNA, extracted from lumbar
DRGs, using poly-dT as a primer to obtain cDNA fragments (141 to
596 bp). These fragments were subsequently cloned into the PCRII
vector (TA cloning Kit, Invitrogen) and the identity of each was
confirmed by sequencing in both directions. These cDNAs were
gel-purified and used to produce .sup.32P-labeled cDNA probes
(Prime-It kit, Stratagene).
[0225] Northern Blot Analysis
[0226] Total RNA was size separated by electrophoresis on a 1.5%
agarose/formaldehyde gel (10 .mu.g of total RNA per lane) and
transferred to a Hybond N+ nylon membrane. Membranes were
hybridized with labeled-probes (see above) in ExpressHyb (Clontech)
overnight at 65.degree. C., washed, and exposed to X-ray film with
an intensifying screen at -80.degree. C.
Slot Blots
[0227] Total RNA (1.25 .mu.g) was directly transferred to Hybond N+
nylon membrane under vacuum using a Hoefer PR648 slot blot
apparatus (Amersham Pharmacia Biotech). Levels of hybridization
were quantified using the 24450 phosphorimager system (Molecular
Dynamics, Sunnyvale Calif.). The blots were probed for cyclophilin
as a loading control. Loading levels between samples on each blot
were normalized using the cyclophilin levels from the control
blot.
Isotopic in Situ Hybridization
[0228] DRGs were rapidly removed, embedded in OCT (Tissue Tek), and
frozen. Sections were cut serially at 6 .mu.m. Isotopic-in situ
hybridization was carried out using forty-eight base pair
oligonucleotide probes, designed to have 50% G-C content and be
complementary to the mRNAs whose accession numbers were provided by
Affymetrix. Probes were 3'-end labeled with .sup.35S or
.sup.33P-dATP using a terminal transferase reaction and
hybridization carried out. Autoradiograms were generated by dipping
slides in NTB2 nuclear track emulsion and storing in the dark at
4.degree. C. Sections were exposed for 1-8 weeks (depending on the
abundance of transcript), developed, fixed, and viewed under
darkfield using a fiber-optic darkfield stage adapter (MVI).
Controls to confirm specificity of oligonucleotide probes included
hybridization of sections with labeled probe with a 1,000-fold
excess of cold probe or labeled probe with a 1,000-fold excess of
another, dissimilar cold probe of the same length and similar G-C
content.
Assays for GTPCH Activity and Biopterin Concentration
[0229] Screening for new inhibitors and optimization of lead
compounds may be assessed, for example, by assessing GTPCH
activity. In this regard, its inhibition by various chemicals may
be determined by incubating the enzyme with GTP and measuring the
level of biopterin or neopterin production using fluorometric,
radiolabel, immunoassay, spectrophotometry, and HPLC
techniques.
[0230] Using the preferred method, tissue neopterin and biopterin
levels were determined with a liquid chromatography tandem mass
spectrometry method. After acidic pH oxidation with iodine
according to Fukushima and Nixon (Methods Enzymol. 66: 429-436,
1980) tissues were extracted by solid phase extraction employing
Oasis MCX extraction cartridges and concentrations of total
biopterin, neopterin and the internal standard rhamnopterin were
determined by liquid chromatography coupled to tandem mass
spectrometry. HPLC analysis was done under gradient conditions
using a Nucleosil C8 column. MS/MS analyses were performed on an
API 4000 Q TRAP triple quadrupole mass spectrometer with a Turbo
Ion Spray source. Precursor-to-product ion transitions of m/z 236
192 for biopterin, m/z 252 192 for neopterin, m/z 265 192 for
rhamnopterin were used for the MRM. Concentrations of the
calibration standards, quality controls and samples were evaluated
by Analyst software 1.4 (Applied Biosystems). Linearity of the
calibration curve was proven from 0.1-50 ng/ml. The coefficient of
correlation for all measured sequences was at least 0.99. The
intra-day and inter-day variability was <10%.
[0231] Alternative methods to determine BH4 employ radioenzymatic
assays that require the production of separate and individual
antibodies specific for each pterin species and/or oxidation state.
Separation of pteridines is accomplished by chromatographic
techniques and HPLC. HPLC with fluorescence detection enables rapid
and sensitive determination of many biologically occurring pterins
(including biopterin and pterin) in the picomole range in a single
chromatographic run.
[0232] Tissue homogenates are centrifuged and the resulting
supernatant is used for both enzyme and protein assays.
[0233] GTP cyclohydrolase I activity is assayed as described by
Duch et al. (Mol. Cell Endocrinol. (1986) 47: 209-16) with the
following modifications. The reaction mixture (500 ml) contained
0.1 M Tris-Cl (pH 7.8), 0.3 M KCl, 2.5 mM EDTA, 10% glycerol, 1 mM
GTP, and the enzyme. The reaction was carried out at 37.degree. C.
for one hour in the dark and was terminated by the addition of 50
ml of iodine solution (1.0% 12, 2.0% KI in 1.0 N HCl). After
keeping the mixture at room temperature for one hour, excess iodine
is reduced by addition of 50 ml of 2.0% ascorbate. The mixture was
supplemented with 50 ml of 1.0 N NaOH and then incubated with 3.0
units (100 ml) of alkaline phosphatase at 37.degree. C. for one
hour. The reaction was stopped by the addition of 100 ml of 1.0 N
acetic acid. After centrifugation, the supernatant was applied to a
Whatman Partisil 10 ODS column (4.6.times.3.times.250 mm) connected
to a Cosmosil 10 C18 column (4.6.times.3.times.50 mm). Neopterin
was eluted isocratically with a solvent of 50 mM sodium acetate
buffer (pH 5.0) containing 0.1 mM EDTA and 5% methanol at a flow
rate of 0.8 ml/min. The column temperature was maintained at
25.degree. C. The eluate was monitored with a fluoromonitor
(excitation, 350 nm; emission 440 nm). Protein concentration was
determined using a dye-binding assay kit (Bio-Rad) using
immunoglobulin-G as a standard.
Cellular Biopterin Content
[0234] Total biopterin (BH1, BH2, and BH4) was measured in tissue
lysates after acidic oxidation of reduced forms of biopterin with
iodines. Following centrifugation at 12,000 rpm (three times for
five minutes each), cell lysates are treated with 1% I.sub.2
containing 2% KI in 1N HCl for one hour at 37.degree. C. in the
dark. Samples were then centrifuged at 12,000 rpm (three times for
five minutes each) and the supernatants treated with ascorbate (0.1
M) to remove residual I.sub.2. Extracts were then neutralized with
1 N NaOH followed by 200 mM Tris-Cl (pH 7.8). Biopterin was
quantitated by C18 reverse HPLC using an online fluorescence
detector.
[0235] The determination of cellular biopterin content is described
in detail in the following references, Harada et al., Science
(1993) 260: 1507-10, Kapatos et al., J. Neurochem. (1999) 72:
669-75, Maita et al., Proc. Natl. Acad. Sci. USA (2002) 99: 1212-7;
Moali et al., Chem Res Toxicol (2001)14: 202-10, Rebelo et al., J.
Mol. Biol. (2003) 326: 503-16; Renodon-Comiere et al., Biochemistry
(1999) 38: 4663-8; Xie et al., J. Biol. Chem. (1998) 273: 21091-8;
Yoneyama et al., J. Biol. Chem. (1998) 273: 20102-8; Yoneyama et
al., Protein Sci. (2001) 10: 871-8; Yoneyama et al. Arch. Biochem.
Biophys. (2001) 388: 67-73, all of which are hereby incorporated by
reference.
Dosages
[0236] The dosage of individual components or therapeutic
combinations of the present invention can be readily determined by
those skilled in the art of pain management. For example, the dose
of an analgesic administered according to the present invention
will be the same or less than that which is practiced in the
art.
Formulation of Pharmaceutical Compositions
[0237] The administration of any compound of this invention may be
by any suitable means that results in a concentration of the
compound that is effective for the treatment of pain. The
compound(s) may be contained in any appropriate amount in any
suitable carrier substance, and is generally present in an amount
of 1-95% by weight of the total weight of the composition. The
composition may be provided in a dosage form that is suitable for
the oral, parenteral (e.g., intravenous, intramuscular, or
subcutaneous injection), rectal, or transdermal (topical)
administration route. Thus, the composition(s) may be in the form
of, e.g., tablets, capsules, pills, powders, granulates,
suspensions, emulsions, solutions, gels including hydrogels,
pastes, ointments, creams, plasters, drenches, osmotic delivery
devices, suppositories, enemas, injectables, or implants. The
pharmaceutical compositions may be formulated according to
conventional pharmaceutical practice (see, e.g., Remington: The
Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro,
Lippincott Williams & Wilkins, 2000 and Encyclopedia of
Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,
1988-1999, Marcel Dekker, New York).
[0238] Pharmaceutical compositions according to the invention may
be formulated to release the active compound (drug) substantially
immediately upon administration or at any predetermined time or
time period after administration. The latter types of compositions
are generally known as controlled release formulations, which
include (i) formulations that create a substantially constant
concentration of the drug within the body over an extended period
of time; (ii) formulations that after a predetermined lag time
create a substantially constant concentration of the drug within
the body over an extended period of time; (iii) formulations that
sustain drug action during a predetermined time period by
maintaining a relatively, constant, effective drug level in the
body with concomitant minimization of undesirable side effects
associated with fluctuations in the plasma level of the active drug
substance (sawtooth kinetic pattern); (iv) formulations that
localize drug action by, e.g., spatial placement of a controlled
release composition adjacent to or in the diseased tissue or organ;
and (v) formulations that target drug action by using carriers or
chemical derivatives to deliver the drug to a particular target
cell type.
[0239] Administration of compounds in the form of a controlled
release formulation is especially preferred in cases in which the
compound, either alone or in combination, has (i) a narrow
therapeutic index (i.e., the difference between the plasma
concentration leading to harmful side effects or toxic reactions
and the plasma concentration leading to a therapeutic effect is
small; in general, the therapeutic index, TI, is defined as the
ratio of median lethal dose (LD50) to median effective dose
(ED50)); (ii) a narrow absorption window in the gastro-intestinal
tract; or (iii) a very short biological half-life so that frequent
dosing during a day is required in order to sustain the plasma
level at a therapeutic level.
[0240] Any of a number of strategies can be pursued in order to
obtain controlled release in which the rate of release outweighs
the rate of metabolism of the compound in question. In one example,
controlled release is obtained by appropriate selection of various
formulation parameters and ingredients, including, e.g., various
types of controlled release compositions and coatings. Thus, the
drug is formulated with appropriate excipients into a
pharmaceutical composition that, upon administration, releases the
drug in a controlled manner. Examples include single or multiple
unit tablet or capsule compositions, oil solutions, suspensions,
emulsions, microcapsules, microspheres, nanoparticles, patches, and
liposomes.
Solid Dosage Forms for Oral Use
[0241] Formulations for oral use include tablets containing the
active ingredient(s) in a mixture with non-toxic pharmaceutically
acceptable excipients. These excipients may be, for example, inert
diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol,
microcrystalline cellulose, starches including potato starch,
calcium carbonate, sodium chloride, lactose, calcium phosphate,
calcium sulfate, or sodium phosphate); granulating and
disintegrating agents (e.g., cellulose derivatives including
microcrystalline cellulose, starches including potato starch,
croscarmellose sodium, alginates, or alginic acid); binding agents
(e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium
alginate, gelatin, starch, pregelatinized starch, microcrystalline
cellulose, magnesium aluminum silicate, carboxymethylcellulose
sodium, methylcellulose, hydroxypropyl methylcellulose,
ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and
lubricating agents, glidants, and antiadhesives (e.g., magnesium
stearate, zinc stearate, stearic acid, silicas, hydrogenated
vegetable oils, or talc). Other pharmaceutically acceptable
excipients can be colorants, flavoring agents, plasticizers,
humectants, buffering agents, and the like.
[0242] The tablets may be uncoated or they may be coated by known
techniques, optionally to delay disintegration and absorption in
the gastrointestinal tract and thereby providing a sustained action
over a longer period. The coating may be adapted to release the
active drug substance in a predetermined pattern (e.g., in order to
achieve a controlled release formulation) or it may be adapted not
to release the active drug substance until after passage of the
stomach (enteric coating). The coating may be a sugar coating, a
film coating (e.g., based on hydroxypropyl methylcellulose,
methylcellulose, methyl hydroxyethylcellulose,
hydroxypropylcellulose, carboxymethylcellulose, acrylate
copolymers, polyethylene glycols and/or polyvinylpyrrolidone), or
an enteric coating (e.g., based on methacrylic acid copolymer,
cellulose acetate phthalate, hydroxypropyl methylcellulose
phthalate, hydroxypropyl methylcellulose acetate succinate,
polyvinyl acetate phthalate, shellac, and/or ethylcellulose).
Furthermore, a time delay material such as, e.g., glyceryl
monostearate or glyceryl distearate may be employed.
[0243] The solid tablet compositions may include a coating adapted
to protect the composition from unwanted chemical changes, (e.g.,
chemical degradation prior to the release of the active drug
substance). The coating may be applied on the solid dosage form in
a similar manner as that described in Encyclopedia of
Pharmaceutical Technology, supra.
[0244] If more than one drug is administered simultaneously, the
drugs may be mixed together in the tablet, or may be partitioned.
In one example, a first drug is contained on the inside of the
tablet, and a second drug is on the outside, such that a
substantial portion of the second drug is released prior to the
release of the first drug.
[0245] Formulations for oral use may also be presented as chewable
tablets, or as hard gelatin capsules wherein the active ingredient
is mixed with an inert solid diluent (e.g., potato starch, lactose,
microcrystalline cellulose, calcium carbonate, calcium phosphate or
kaolin), or as soft gelatin capsules wherein the active ingredient
is mixed with water or an oil medium, for example, peanut oil,
liquid paraffin, or olive oil. Powders and granulates may be
prepared using the ingredients mentioned above under tablets and
capsules in a conventional manner using, e.g., a mixer, a fluid bed
apparatus or a spray drying equipment.
Controlled Release Oral Dosage Forms
[0246] Controlled release compositions for oral use may, e.g., be
constructed to release the active drug by controlling the
dissolution and/or the diffusion of the active drug substance.
[0247] Dissolution or diffusion controlled release can be achieved
by appropriate coating of a tablet, capsule, pellet, or granulate
formulation of compounds, or by incorporating the compound into an
appropriate matrix. A controlled release coating may include one or
more of the coating substances mentioned above and/or, e.g.,
shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl
alcohol, glyceryl monostearate, glyceryl distearate, glycerol
palmitostearate, ethylcellulose, acrylic resins, dl-polylactic
acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl
acetate, vinyl pyrrolidone, polyethylene, polymethacrylate,
methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels,
1,3 butylene glycol, ethylene glycol methacrylate, and/or
polyethylene glycols. In a controlled release matrix formulation,
the matrix material may also include, e.g., hydrated
metylcellulose, carnauba wax and stearyl alcohol, carbopol 934,
silicone, glyceryl tristearate, methyl acrylate-methyl
methacrylate, polyvinyl chloride, polyethylene, and/or halogenated
fluorocarbon.
[0248] A controlled release composition containing one or more of
the compounds of the claimed combinations may also be in the form
of a buoyant tablet or capsule (i.e., a tablet or capsule that,
upon oral administration, floats on top of the gastric content for
a certain period of time). A buoyant tablet formulation of the
compound(s) can be prepared by granulating a mixture of the drug(s)
with excipients and 20-75% w/w of hydrocolloids, such as
hydroxyethylcellulose, hydroxypropylcellulose, or
hydroxypropylmethylcellulose. The obtained granules can then be
compressed into tablets. On contact with the gastric juice, the
tablet forms a substantially water-impermeable gel barrier around
its surface. This gel barrier takes part in maintaining a density
of less than one, thereby allowing the tablet to remain buoyant in
the gastric juice.
Liquids for Oral Administration
[0249] Powders, dispersible powders, or granules suitable for
preparation of an aqueous suspension by addition of water are
convenient dosage forms for oral administration. Formulation as a
suspension provides the active ingredient in a mixture with a
dispersing or wetting agent, suspending agent, and one or more
preservatives. Suitable dispersing or wetting agents are, for
example, naturally-occurring phosphatides (e.g., lecithin or
condensation products of ethylene oxide with a fatty acid, a long
chain aliphatic alcohol, or a partial ester derived from fatty
acids) and a hexitol or a hexitol anhydride (e.g., polyoxyethylene
stearate, polyoxyethylene sorbitol monooleate, polyoxyethylene
sorbitan monooleate, and the like). Suitable suspending agents are,
for example, sodium carboxymethylcellulose, methylcellulose, sodium
alginate, and the like.
Parenteral Compositions
[0250] The compound(s) may also be administered parenterally by
injection, infusion, or implantation (intravenous, intramuscular,
subcutaneous, or the like) in dosage forms, formulations, or via
suitable delivery devices or implants containing conventional,
non-toxic pharmaceutically acceptable carriers and adjuvants. The
formulation and preparation of such compositions are well known to
those skilled in the art of pharmaceutical formulation.
Formulations can be found in Remington: The Science and Practice of
Pharmacy, supra.
[0251] Compositions for parenteral use may be provided in unit
dosage forms (e.g., in single-dose ampoules), or in vials
containing several doses and in which a suitable preservative may
be added (see below). The composition may be in form of a solution,
a suspension, an emulsion, an infusion device, or a delivery device
for implantation, or it may be presented as a dry powder to be
reconstituted with water or another suitable vehicle before use.
Apart from the active drug(s), the composition may include suitable
parenterally acceptable carriers and/or excipients. The active
drug(s) may be incorporated into microspheres, microcapsules,
nanoparticles, liposomes, or the like for controlled release.
Furthermore, the composition may include suspending, solubilizing,
stabilizing, pH-adjusting agents, and/or dispersing agents.
[0252] As indicated above, the pharmaceutical compositions
according to the invention may be in the form suitable for sterile
injection. To prepare such a composition, the suitable active
drug(s) are dissolved or suspended in a parenterally acceptable
liquid vehicle. Among acceptable vehicles and solvents that may be
employed are water, water adjusted to a suitable pH by addition of
an appropriate amount of hydrochloric acid, sodium hydroxide or a
suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic
sodium chloride solution. The aqueous formulation may also contain
one or more preservatives (e.g., methyl, ethyl or n-propyl
p-hydroxybenzoate). In cases where one of the compounds is only
sparingly or slightly soluble in water, a dissolution enhancing or
solubilizing agent can be added, or the solvent may include 10-60%
w/w of propylene glycol or the like.
Controlled Release Parenteral Compositions
[0253] Controlled release parenteral compositions may be in form of
aqueous suspensions, microspheres, microcapsules, magnetic
microspheres, oil solutions, oil suspensions, or emulsions.
Alternatively, the active drug(s) may be incorporated in
biocompatible carriers, liposomes, nanoparticles, implants, or
infusion devices. Materials for use in the preparation of
microspheres and/or microcapsules are, e.g.,
biodegradable/bioerodible polymers such as polygalactin,
poly-(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L-glutamnine)
and, poly(lactic acid). Biocompatible carriers that may be used
when formulating a controlled release parenteral formulation are
carbohydrates (e.g., dextrans), proteins (e.g., albumin),
lipoproteins, or antibodies. Materials for use in implants can be
non-biodegradable (e.g., polydimethyl siloxane) or biodegradable
(e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid)
or poly(ortho esters)).
Rectal Compositions
[0254] For rectal application, suitable dosage forms for a
composition include suppositories (emulsion or suspension type),
and rectal gelatin capsules (solutions or suspensions). In a
typical suppository formulation, the active drug(s) are combined
with an appropriate pharmaceutically acceptable suppository base
such as cocoa butter, esterified fatty acids, glycerinated gelatin,
and various water-soluble or dispersible bases like polyethylene
glycols and polvoxyethylene sorbitan fatty acid esters. Various
additives, enhancers, or surfactants may be incorporated.
Percutaneous and Topical Compositions
[0255] The pharmaceutical compositions may also be administered
topically on the skin for percutaneous (transdermal) absorption in
dosage forms or formulations containing conventionally non-toxic
pharmaceutical acceptable carriers and excipients including
microspheres and liposomes. The formulations include creams,
ointments, lotions, liniments, gels, hydrogels, solutions,
suspensions, sticks, sprays, pastes, plasters, and other kinds of
transdermal drug delivery systems. The pharmaceutically acceptable
carriers or excipients may include emulsifying agents,
antioxidants, buffering agents, preservatives, humectants,
penetration enhancers, chelating agents, gel-forming agents,
ointment bases, perfumes, and skin protective agents.
[0256] Examples of emulsifying agents are naturally occurring gums
(e.g., gum acacia or gum tragacanth) and naturally occurring
phosphatides (e.g., soybean lecithin and sorbitan monooleate
derivatives). Examples of antioxidants are butylated hydroxy
anisole (BHA), ascorbic acid and derivatives thereof, tocopherol
and derivatives thereof, butylated hydroxy anisole, and cysteine.
Examples of preservatives are parabens, such as methyl or propyl
p-hydroxybenzoate, and benzalkonium chloride. Examples of
humectants are glycerin, propylene glycol, sorbitol, and urea.
Examples of penetration enhancers are propylene glycol, DMSO,
triethanolamine, N,N-dimethylacetamide, N,N-dimethylformamide,
2-pyrrolidone and derivatives thereof, tetrahydrofurfuryl alcohol,
and AZONE.TM.. Examples of chelating agents are sodium EDTA, citric
acid, and phosphoric acid. Examples of gel forming agents are
CARBOPOL.TM., cellulose derivatives, bentonite, alginates, gelatin
and polyvinylpyrrolidone. Examples of ointment bases are beeswax,
paraffin, cetyl palmitate, vegetable oils, sorbitan esters of fatty
acids (Span), polyethylene glycols, and condensation products
between sorbitan esters of fatty acids and ethylene oxide (e.g.,
polyoxyethylene sorbitan monooleate (TWEEN.TM.)).
[0257] The pharmaceutical compositions described above may be
applied by means of special drug delivery devices such as dressings
or alternatively plasters, pads, sponges, strips, or other forms of
suitable flexible material.
Controlled Release Percutaneous and Topical Compositions
[0258] There are several approaches for providing rate control over
the release and transdermal permeation of a drug, including:
membrane-moderated systems, adhesive diffusion-controlled systems,
matrix dispersion-type systems, and microreservoir systems. A
controlled release percutaneous and/or topical composition may be
obtained by using a suitable mixture of the above-mentioned
approaches.
[0259] In a membrane-moderated system, the active drug is present
in a reservoir which is totally encapsulated in a shallow
compartment molded from a drug-impermeable laminate, such as a
metallic plastic laminate, and a rate-controlling polymeric
membrane such as a microporous or a non-porous polymeric membrane
(e.g., ethylene-vinyl acetate copolymer). The active compound is
only released through the rate-controlling polymeric membrane. In
the drug reservoir, the active drug substance may either be
dispersed in a solid polymer matrix or suspended in a viscous
liquid medium such as silicone fluid. On the external surface of
the polymeric membrane, a thin layer of an adhesive polymer is
applied to achieve an intimate contact of the transdermal system
with the skin surface. The adhesive polymer is preferably a
hypoallergenic polymer that is compatible with the active drug.
[0260] In an adhesive diffusion-controlled system, a reservoir of
the active drug is formed by directly dispersing the active drug in
an adhesive polymer and then spreading the adhesive containing the
active drug onto a flat sheet of substantially drug-impermeable
metallic plastic backing to form a thin drug reservoir layer.
[0261] A matrix dispersion-type system is characterized in that a
reservoir of the active drug substance is formed by substantially
homogeneously dispersing the active drug substance in a hydrophilic
or lipophilic polymer matrix and then molding the drug-containing
polymer into a disc with a substantially well-defined surface area
and thickness. The adhesive polymer is spread along the
circumference to form a strip of adhesive around the disc.
[0262] In a microreservoir system, the reservoir of the active
substance is formed by first suspending the drug solids in an
aqueous solution of water-soluble polymer, and then dispersing the
drug suspension in a lipophilic polymer to form a plurality of
microscopic spheres of drug reservoirs.
Other Embodiments
[0263] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. Although
the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding,
it will be readily apparent to those of ordinary skill in the art
in light of the teachings of this invention that certain changes
and modifications may be made thereto without departing from the
spirit or scope of the appended claims.
[0264] Other embodiments are within the claims.
* * * * *
References