U.S. patent application number 16/814519 was filed with the patent office on 2020-12-03 for charged ion channel blockers and methods for use.
This patent application is currently assigned to President and Fellows of Harvard College. The applicant listed for this patent is Children's Medical Center Corporation, President and Fellows of Harvard College. Invention is credited to Bruce P. Bean, Sooyeon Jo, Jinbo Lee, David P. Roberson, Sebastien Talbot, Clifford J. Woolf.
Application Number | 20200377459 16/814519 |
Document ID | / |
Family ID | 1000005030594 |
Filed Date | 2020-12-03 |
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United States Patent
Application |
20200377459 |
Kind Code |
A1 |
Bean; Bruce P. ; et
al. |
December 3, 2020 |
CHARGED ION CHANNEL BLOCKERS AND METHODS FOR USE
Abstract
The invention provides compounds, compositions, methods, and
kits for the treatment of pain, itch, and neurogenic
inflammation.
Inventors: |
Bean; Bruce P.; (Brookline,
MA) ; Woolf; Clifford J.; (Newton, MA) ; Lee;
Jinbo; (Andover, MA) ; Jo; Sooyeon;
(Auburndale, MA) ; Roberson; David P.; (Cambridge,
MA) ; Talbot; Sebastien; (Montreal, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
President and Fellows of Harvard College
Children's Medical Center Corporation |
Cambridge
Boston |
MA
MA |
US
US |
|
|
Assignee: |
President and Fellows of Harvard
College
Cambridge
MA
Children's Medical Center Corporation
Boston
MA
|
Family ID: |
1000005030594 |
Appl. No.: |
16/814519 |
Filed: |
March 10, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15749885 |
Feb 2, 2018 |
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PCT/US2016/045354 |
Aug 3, 2016 |
|
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16814519 |
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62200205 |
Aug 3, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 211/34 20130101;
A61K 45/06 20130101; C07D 207/16 20130101; C07D 207/08 20130101;
C07C 237/04 20130101; C07D 211/14 20130101 |
International
Class: |
C07D 211/34 20060101
C07D211/34; A61K 45/06 20060101 A61K045/06; C07C 237/04 20060101
C07C237/04; C07D 207/08 20060101 C07D207/08; C07D 207/16 20060101
C07D207/16; C07D 211/14 20060101 C07D211/14 |
Claims
1. A quaternary amine compound having formula (I) ##STR00125##
wherein R.sup.1F and R.sup.1G together complete a heterocyclic ring
having at least one nitrogen atom; each of R.sup.1A and R.sup.1B is
independently selected from H, halogen, C.sub.1-4 alkyl, C.sub.2-4
alkenyl, C.sub.2-4 alkynyl, OR.sup.1I, NR.sup.1JR.sup.1K,
NR.sup.1LC(O)R.sup.1M, S(O)R.sup.1N, SO.sub.2R.sup.1OR.sup.1P,
SO.sub.2NR.sup.1QR.sup.1R, SO.sub.3R.sup.1S, CO.sub.2R.sup.1T,
C(O)R.sup.1U, and C(O)NR.sup.1VR.sup.1W; each of R.sup.1I,
R.sup.1J, R.sup.1K, R.sup.1L, R.sup.1M, R.sup.1N, R.sup.1O,
R.sup.1P, R.sup.1Q, R.sup.1R, R.sup.1S, R.sup.1T, R.sup.1U,
R.sup.1V and R.sup.1W is, independently selected from H, C.sub.1-4
alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, and C.sub.2-4
heteroalkyl; R.sup.1C is H; X.sup.1 is --NH(C)O--; each of R.sup.1D
and R.sup.1E is independently selected from H, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, and C.sub.2-4 heteroalkyl,
wherein R.sup.1D and R.sup.1E are independently optionally
substituted with halogen, C.sub.3-8 cycloalkyl, aryl, or
heteroaryl; or R.sup.1D and R.sup.1E together form a 3-6-membered
heterocyclic or heteroalkyl ring; R.sup.1H is selected from
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, and
C.sub.2-4 heteroalkyl, wherein R.sup.1H is substituted with
halogen, C.sub.3-8 cycloalkyl, aryl, or heteroaryl.
2. (canceled)
3. The compound of claim 1, wherein each of R.sup.1A and R.sup.1B
is independently selected from H, halogen, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, and NR.sup.1JR.sup.1K; and
each of R.sup.1J and R.sup.1K is independently selected from H,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, and
C.sub.2-4 heteroalkyl.
4. The compound of claim 1, wherein R.sup.1D is C.sub.1-4 alkyl
optionally substituted with halogen, C.sub.3-8 cycloalkyl, aryl, or
heteroaryl.
5. The compound of claim 1, wherein R.sup.1E is H or C.sub.1-4
alkyl optionally substituted with halogen, C.sub.3-8 cycloalkyl,
aryl, or heteroaryl.
6. The compound of claim 1, wherein R.sup.1H is C.sub.1-4 alkyl
optionally substituted with halogen, C.sub.3-8 cycloalkyl, aryl, or
heteroaryl.
7-21. (canceled)
22. A composition comprising the quaternary amine compound of claim
1 and a pharmaceutically acceptable excipient.
23. The composition of claim 21, wherein said composition is
formulated for oral, intravenous, intramuscular, rectal, cutaneous,
subcutaneous, topical, transdermal, sublingual, nasal, inhalation,
vaginal, intrathecal, epidural, or ocular administration.
24. A method for treating pain, itch, or a neurogenic inflammatory
disorder in a patient, said method comprising administering to said
patient a composition comprising the quaternary amine compound of
claim 1, wherein said compound inhibits one or more voltage-gated
ion channels present in nociceptors and/or pruriceptors when
applied to the internal face of said channels but does not
substantially inhibit said channels when applied to the external
face of said channels, and wherein said compound is capable of
entering nociceptors or pruriceptors through a channel-forming
receptor when said receptor is activated and inhibiting said one or
more voltage-gated ion channels present in said nociceptors.
25. The method of claim 24, wherein said channel-forming receptor
is a transient receptor potential ion channel (TRP channel-forming
receptor)
26. The method of claim 25, wherein said TRP channel-forming
receptor is activated by an exogenous or endogenous agonist.
27. The method of claim 25, wherein said TRP channel-forming
receptor is TRPA1 or TRPV1.
28. The method of claim 27, wherein said compound is capable of
entering nociceptors or pruriceptors through said TRPA1 or TRPV1
receptor when said receptor is activated.
29. The method of claim 24, wherein said compound inhibits
voltage-gated sodium channels.
30. The method of claim 24, wherein said pain is selected from the
group consisting of neuropathic pain, inflammatory pain,
nociceptive pain, pain due to infections, and procedural pain.
31. The method of claim 24, wherein said neurogenic inflammatory
disorder is selected from the group consisting of allergic
inflammation, asthma, chronic cough, conjunctivitis, rhinitis,
psoriasis, inflammatory bowel disease, a*-interstitial cystitis,
and atopic dermatitis.
32. (canceled)
33. The compound of claim 1, wherein R.sup.1H is C.sub.1-4 alkyl
optionally substituted with aryl.
34. The compound of claim 1, wherein R.sup.1H is C.sub.1-4 alkyl
optionally substituted with heteroaryl.
35. The compound of claim 1, wherein R.sup.1D is hydrogen.
36. The compound of claim 1, wherein R.sup.1E is hydrogen.
37. The compound of claim 1, wherein R.sup.1A is methyl and
R.sup.1B is methyl.
Description
RELATED APPLICATIONS
[0001] This application is a national stage filing under 35 U.S.C.
.sctn. 371 of international PCT application, PCT/US2016/045354,
filed Aug. 3, 2016, which claims priority under 35 U.S.C. .sctn.
119(e) to U.S. Provisional Application, U.S. Ser. No. 62/200,205,
filed on Aug. 3, 2015 each of which is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] The invention features compositions and methods for
selective inhibition of pain- and itch sensing neurons (nociceptors
and pruriceptors) and treatment of neurogenic inflammation by
targeting nociceptors with drug molecules of small molecule weight,
while minimizing effects on non-pain-sensing neurons or other types
of cells. According to the method of the invention, small,
hydrophilic drug molecules gain access to the intracellular
compartment of pain-sensing neurons via entry through large pore
receptor/ion channels that are present in pain- and itch-sensing
neurons but to a lesser extent or not at all in other types of
neurons or in other types of tissue.
[0003] Local anesthetics such as lidocaine and articaine act by
inhibiting voltage-dependent sodium channels in neurons. These
anesthetics block sodium channels and thereby the excitability of
all neurons (an excitable cells in the cardiovascular system), not
just pain-sensing neurons (nociceptors). Thus, while the goal of
topical or regional anesthesia is to block transmission of signals
in nociceptors to prevent pain, administration of local anesthetics
also produces unwanted or deleterious effects such as general
numbness from block of low threshold pressure and touch receptors,
motor deficits from block of motor axons and other complications
from block of autonomic fibers. Local anesthetics are relatively
hydrophobic molecules that gain access to their blocking site on
the sodium channel by diffusing into or through the cell membrane.
Charged derivatives of these compounds (such as QX-314, a
quaternary nitrogen derivative of lidocaine), which are not
membrane-permeant, have no effect on neuronal sodium channels when
applied to the external surface of the nerve membrane but can block
sodium channels if somehow introduced inside the cell, for example
by diffusion from a micropipette used for whole-cell
electrophysiological recording from isolated neurons. Pain- and
itch-sensing neurons differ from other types of neurons in
expressing (in most cases) the TRPV1 receptor/channel, activated by
painful heat or by capsaicin, the pungent ingredient in chili
pepper. Other types of receptors selectively expressed in various
types of pain-sensing and itch-sensing (pruriceptor) neurons
include but are not limited to TRPA1, and P2X(2/3) receptors.
[0004] Neuropathic, inflammatory, and nociceptive pain differ in
their etiology, pathophysiology, diagnosis, and treatment.
Nociceptive pain occurs in response to the activation of a specific
subset of high threshold peripheral sensory neurons, the
nociceptors by intense or noxious stimuli. It is generally acute,
self-limiting and serves a protective biological function by acting
as a warning of potential or on-going tissue damage. It is
typically well-localized. Examples of nociceptive pain include but
are not limited to traumatic or surgical pain, labor pain, sprains,
bone fractures, burns, bumps, bruises, injections, dental
procedures, skin biopsies, and obstructions.
[0005] Inflammatory pain is pain that occurs in the presence of
tissue damage or inflammation including postoperative,
post-traumatic pain, arthritic (rheumatoid or osteoarthritis) pain
and pain associated with damage to joints, muscle, and tendons as
in axial low back pain, severe nociceptive pain may transition to
inflammatory pain if there is associated tissue injury.
[0006] Neuropathic pain is a common type of chronic, non-malignant
pain, which is the result of an injury or malfunction in the
peripheral or central nervous system and serves no protective
biological function. It is estimated to affect more than 1.6
million people in the U.S. population. Neuropathic pain has many
different etiologies, and may occur, for example, due to trauma,
surgery, herniation of an intervertebral disk, spinal cord injury,
diabetes, infection with herpes zoster (shingles), HIV/AIDS,
late-stage cancer, amputation (including mastectomy), carpal tunnel
syndrome, chronic alcohol use, exposure to radiation, and as an
unintended side-effect of neurotoxic treatment agents, such as
certain anti-HIV and chemotherapeutic drugs.
[0007] Neuropathic pain is frequently described as "burning,"
"electric," "tingling," or "shooting" in nature. It is often
characterized by chronic dynamic allodynia (defined as pain
resulting from a moving stimulus that does not ordinarily elicit a
painful response, such as light touch) and hyperalgesia (defined as
an increased sensitivity to a normally painful stimulus), and may
persist for months or years beyond the apparent healing of any
damaged tissues.
[0008] Pain may occur in patients with cancer, which may be due to
multiple causes; inflammation, compression, invasion, metastatic
spread into bone or other tissues.
[0009] There are some conditions where pain occurs in the absence
of a noxious stimulus, tissue damage or a lesion to the nervous
system, called dysfunctional pain and these include but are not
limited to fibromyalgia, tension type headache, and irritable bowel
disorders.
[0010] Migraine is a headache associated with the activation of
sensory fibers innervating the meninges of the brain.
[0011] Itch (pruritus) is a dermatological condition that may be
localized and generalized and can be associated with skin lesions
(rash, atopic eczema, wheals). Itch accompanies many conditions
including but not limited to stress, anxiety, UV radiation from the
sun, metabolic and endocrine disorders (e.g., liver or kidney
disease, hyperthyroidism), cancers (e.g., lymphoma), reactions to
drugs or food, parasitic and fungal infections, allergic reactions,
diseases of the blood (e.g., polycythemia vera), and dermatological
conditions. Itch is mediated by a subset of small diameter primary
sensory neurons, the pruriceptor, that share many features of
nociceptor neurons, including but not limited to expression of
TRPV1 channels. Certain itch mediators--such as eicosanoids,
histamine, bradykinin, ATP, and various neurotrophins have
endovanilloid functions. Topical capsaicin suppresses
histamine-induced itch. Pruriceptors like nociceptors are therefore
a suitable target for this method of delivering ion channel
blockers.
[0012] Neurogenic inflammation is a mode of inflammation mediated
by the efferent (motor) functions of sensory neurons, in which
pro-inflammatory mediator molecules released in the periphery by
pain-sensing neurons (nociceptors) both activate a variety of
inflammatory pathways in immune cells and also act on the vascular
system to alter blood flow and capillary permeability.
[0013] Neurogenic inflammation contributes to the peripheral
inflammation elicited by tissue injury, autoimmune disease,
infection, allergy, exposure to irritants in a variety of tissues,
and is thought to play an important role in the pathogenesis of
numerous disorders (e.g. migraine, arthritis, rhinitis, gastritis,
colitis, cystitis, and sunburn). One way to reduce neurogenic
inflammation is to block excitability in nociceptors, thereby
preventing the activation of nociceptor peripheral terminals and
the release of pro-inflammatory chemicals.
[0014] Despite the development of a variety of therapies for pain,
itch, and neurogenic inflammation, there is a need for additional
agents.
SUMMARY OF THE INVENTION
[0015] In a first aspect, the invention features a quaternary amine
compound having formula (I)
##STR00001##
[0016] R.sup.1F and R.sup.1G together complete a heterocyclic ring
having at least one nitrogen atom; and wherein
[0017] each of R.sup.1A, R.sup.1B, and R.sup.1C is independently
selected from H, halogen, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, OR.sup.1I, NR.sup.1JR.sup.1K,
NR.sup.1LC(O)R.sup.1M, S(O)R.sup.1N, SO.sub.2R.sup.1OR.sup.1P,
SO.sub.2NR.sup.1OR.sup.1R, SO.sub.3R.sup.1S, CO.sub.2R.sup.1T,
C(O)R.sup.1U, and C(O)NR.sup.1VR.sup.1W; and each of R.sup.1I,
R.sup.1J, R.sup.1K, R.sup.1L, R.sup.1M, R.sup.1N, R.sup.1O,
R.sup.1P, R.sup.1Q, R.sup.1R, R.sup.1S, R.sup.1T, R.sup.1U,
R.sup.1V, and R.sup.1W is, independently, selected from H,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, and
C.sub.2-4 heteroalkyl; wherein
[0018] X.sup.1 is selected from --CR.sup.1XR.sup.1Y,
--NR.sup.1ZC(O)--, --OC(O)--, --SC(O)--, --C(O)NR.sup.1AA--,
--CO.sub.2--, and --OC(S)--; and each of R.sup.1X, R.sup.1Y,
R.sup.1Z, and R.sup.1AA is independently selected from H, C.sub.1-4
alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, and C.sub.2-4
heteroalkyl; wherein
[0019] each of R.sup.1D and R.sup.1E is independently selected from
H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-4
heteroalkyl, optionally substituted with halogen, C.sub.3-8 cyclic
alkyl, aryl, or heteroaryl, and C.sub.3-6 cycloalkyl or R.sup.1D
and R.sup.1E together form a 3-6-membered heterocyclic or
heteroalkyl ring; and wherein
[0020] R.sup.1H is selected from C.sub.1-4 alkyl, C.sub.2-4
alkenyl, C.sub.2-4 alkynyl, C.sub.2-4 heteroalkyl, optionally
substituted with halogen, C.sub.3-8 cyclic alkyl, aryl, or
heteroaryl, and C.sub.3-6 cycloalkyl.
[0021] In some embodiments, X.sup.1 is --NHC(O)--. In some
embodiments, each of R.sup.1A and R.sup.1B is independently
selected from H, halogen, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, and NR.sup.1JR.sup.1K; and each of R.sup.1J and
R.sup.1K is independently selected from H, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, and C.sub.2-4 heteroalkyl; or
wherein at least one R.sup.1C is present. In certain other
embodiments, R.sup.1D is C.sub.1-4 alkyl optionally substituted
with halogen, C.sub.3-8 cyclic alkyl, aryl, or heteroaryl, R.sup.1E
is H and C.sub.1-4 alkyl optionally substituted with halogen,
C.sub.3-8 cyclic alkyl, aryl, or heteroaryl, or R.sup.1H is
C.sub.1-4 alkyl optionally substituted with halogen, C.sub.3-8
cyclic alkyl, aryl, or heteroaryl.
[0022] In some embodiments, the compound is a compound in Table 1.
In some embodiments, the compound is:
##STR00002##
[0023] In a second aspect, the invention features a quaternary
amine compound having formula (II)
##STR00003##
[0024] m is 0 or 1; wherein
[0025] each of R.sup.2A, R.sup.2B, and R.sup.2C, is independently
selected from H, halogen, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, CF.sub.3, OR.sup.2H, NR.sup.2IR.sup.2J,
NR.sup.2KC(O)R.sup.2L, S(O)R.sup.2M, SO.sub.2R.sup.2NR.sup.2O,
SO.sub.2NR.sup.2PR.sup.2O, SO.sub.3R.sup.2R, CO.sub.2R.sup.2S,
C(O)R.sup.2T, and C(O)NR.sup.2UR.sup.2V; and each of R.sup.2H,
R.sup.2I, R.sup.2J, R.sup.2K, R.sup.2L, R.sup.2M, R.sup.2N,
R.sup.2O, R.sup.2P, R.sup.2Q, R.sup.2R, R.sup.2S, R.sup.2T,
R.sup.2U, and R.sup.2V is, independently, selected from H,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, and
C.sub.2-4 heteroalkyl; wherein
[0026] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9 and each R.sup.2F is,
independently, selected from halogen, C.sub.1-4 alkyl, C.sub.2-4
alkenyl, C.sub.2-4 alkynyl, CF.sub.3, OR.sup.2H, NR.sup.2I,
R.sup.2J, NR.sup.2KC(O)R.sup.2L, S(O)R.sup.2M,
SO.sub.2R.sup.2NR.sup.2O, SO.sub.2NR.sup.2PR.sup.2Q,
SO.sub.3R.sup.2R, CO.sub.2R.sup.2S, C(O)R.sup.2T, and
C(O)NR.sup.2UR.sup.2V; and each of R.sup.2H, R.sup.2I, R.sup.2J,
R.sup.2K, R.sup.2L, R.sup.2M, R.sup.2N, R.sup.2O, R.sup.2P,
R.sup.2Q, R.sup.2R, R.sup.2S, R.sup.2T, R.sup.2U, and R.sup.2V is,
independently, selected from H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, and C.sub.2-4 heteroalkyl; and wherein
[0027] each of R.sup.2D and R.sup.2E is, independently, selected
from C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
C.sub.2-4 heteroalkyl, optionally substituted with halogen, cyclic
alkyl, aryl, or heteroaryl, and C.sub.3-6 cycloalkyl In some
embodiments, each of R.sup.2D and R.sup.2E is C.sub.1-4 alkyl that
is optionally substituted with halogen, cyclic alkyl, aryl, or
heteroaryl. In another embodiment, each of R.sup.2A, R.sup.2B, and
R.sup.2C is independently selected from H, halogen, C.sub.1-4
alkyl, and CF.sub.3; or wherein at least one R.sup.2C is present;
or wherein at least one R.sup.2F is present. In certain
embodiments, the compound is a compound in Table 2. In some
embodiments, the compound is
##STR00004##
[0028] In a third aspect, the invention features a quaternary amine
compound having general formula (III)
##STR00005##
wherein
[0029] n is 0, 1, 2, or 3; wherein
[0030] each of R.sup.3A, R.sup.3B, and R.sup.3C is independently
selected from H, halogen, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, OR.sup.3I, NR.sup.3JR.sup.3K,
NR.sup.3LC(O)R.sup.3M, S(O)R.sup.3N, SO.sub.2R.sup.3OR.sup.3P,
SO.sub.2NR.sup.3OR.sup.3R, SO.sub.3R.sup.3S, CO.sub.2R.sup.3T,
C(O)R.sup.3U, and C(O)NR.sup.3VR.sup.3W; and each of R.sup.3I,
R.sup.3J, R.sup.3K, R.sup.3L, R.sup.3M, R.sup.3N, R.sup.3O,
R.sup.3P, R.sup.3Q, R.sup.3R, R.sup.3S, R.sup.3T, R.sup.3U,
R.sup.3V, and R.sup.3W is independently selected from H, C.sub.1-4
alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, and C.sub.2-4
heteroalkyl; wherein
[0031] X.sup.3 is selected from --NHC(O)--, and --C(O)NH;
wherein
[0032] each of R.sup.3D and R.sup.3E can, independently, be
selected from H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4
alkynyl, C.sub.2-4 heteroalkyl, optionally substituted with
halogen, cyclic alkyl, aryl, or heteroaryl, and C.sub.3-6
cycloalkyl, or R.sup.1D and R.sup.1E together can form a
3-6-membered heterocyclic or heteroalkyl ring; and wherein
[0033] each of R.sup.3F, R.sup.3G, and R.sup.3H is independently
selected from C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4
alkynyl, C.sub.2-4 heteroalkyl, optionally substituted with
halogen, cyclic alkyl, aryl, or heteroaryl, and C.sub.3-6
cycloalkyl
[0034] In particular embodiments, X.sup.3 is --NHC(O)--. In other
embodiments, n is 0 or 1. In some embodiments, each of R.sup.3A,
R.sup.3B, and R.sup.30 is independently selected from H, C.sub.1-4
alkyl, and NR.sup.3JR.sup.3K; and each of R.sup.3J and R.sup.3K is
independently selected from H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, and C.sub.2-4 heteroalkyl. In yet another
embodiment, each of R.sup.3E, R.sup.3F, and R.sup.3G is
independently selected from C.sub.1-4 alkyl optionally substituted
with halogen, cyclic alkyl, aryl, or heteroaryl, and C.sub.3-6
cycloalkyl. In particular embodiments, the compound is any one of
Compound Nos. 21-24 in Table 3. In yet another embodiment, the
compound is
##STR00006##
[0035] In a fourth aspect, the invention also features a
composition including the quaternary amine compound of any one of
the compounds in Tables 1-3 or a compound of formulas I through III
and a pharmaceutically acceptable excipient. The composition can be
formulated for oral, intravenous, intramuscular, rectal, cutaneous,
subcutaneous, topical, transdermal, sublingual, nasal, inhalation,
vaginal, intrathecal, epidural, or ocular administration.
[0036] In a fifth aspect, the invention features a method for
treating pain, itch, or a neurogenic inflammatory disorder in a
patient, the method including administering to the patient a
composition including the quaternary amine compound of any one of
the compounds in Tables 1-3 or a compound of formulas I through
III, wherein the compound inhibits one or more voltage-gated ion
channels present in nociceptors and/or pruriceptors when applied to
the internal face of the channels but does not substantially
inhibit the channels when applied to the external face of the
channels, and wherein the compound is capable of entering
nociceptors or pruriceptors through a channel-forming receptor when
the receptor is activated and inhibiting the one or more
voltage-gated ion channels present in the nociceptors.
[0037] In certain embodiments, the channel-forming receptor is a
transient receptor potential ion channel (TRP channel-forming
receptor). In other embodiments, the TRP channel-forming receptor
is activated by an exogenous or endogenous agonist. In yet other
embodiments, the TRP channel-forming receptor is TRPA1 or TRPV1. In
particular embodiments, the compound is capable of entering
nociceptors or pruriceptors through the TRPA1 or TRPV1 receptor
when the receptor is activated. In yet other embodiments, the
compound inhibits voltage-gated sodium channels. In yet another
embodiment, the pain is selected from the group consisting of
neuropathic pain, inflammatory pain, nociceptive pain, pain due to
infections, and procedural pain, or wherein the neurogenic
inflammatory disorder is selected from the group consisting of
allergic inflammation, asthma, chronic cough, conjunctivitis,
rhinitis, psoriasis, inflammatory bowel disease, and interstitial
cystitis, atopic dermatitis. In particular embodiments, the
compositions of the invention include a quaternary amine compound
selected from the group consisting of:
##STR00007##
Definitions
[0038] By "biologically active" is meant that a molecule, including
biological molecules, such as nucleic acids, peptides,
polypeptides, and proteins, exerts a physical or chemical activity
on itself or other molecule. For example, a "biologically active"
molecule may possess, e.g., enzymatic activity, protein binding
activity (e.g., antibody interactions), or cytotoxic activities
(e.g., anti-cancer properties). Biologically active agents that can
be used in the methods and kits described herein include, without
limitation, an antibody or antibody fragment, an antibiotic, a
polynucleotide, a polypeptide, a protein, an anti-cancer agent, a
growth factor, and a vaccine.
[0039] By "inflammation" is meant any types of inflammation, such
those caused by the immune system (immune-mediated inflammation)
and by the nervous system (neurogenic inflammation), and any
symptom of inflammation, including redness, heat, swelling, pain,
and/or loss of function.
[0040] By "neurogenic inflammation" is meant any type of
inflammation mediated or contributed to by neurons (e.g.
nociceptors) or any other component of the central or peripheral
nervous system.
[0041] The term "pain" is used herein in the broadest sense and
refers to all types of pain, including acute and chronic pain, such
as nociceptive pain, e.g. somatic pain and visceral pain;
inflammatory pain, dysfunctional pain, idiopathic pain, neuropathic
pain, e.g., centrally generated pain and peripherally generated
pain, migraine, and cancer pain.
[0042] The term "nociceptive pain" is used to include all pain
caused by noxious stimuli that threaten to or actually injure body
tissues, including, without limitation, by a cut, bruise, bone
fracture, crush injury, burn, and the like. Pain receptors for
tissue injury (nociceptors) are located mostly in the skin,
musculoskeletal system, or internal organs.
[0043] The term "somatic pain" is used to refer to pain arising
from bone, joint, muscle, skin, or connective tissue. This type of
pain is typically well localized.
[0044] The term "visceral pain" is used herein to refer to pain
arising from visceral organs, such as the respiratory,
gastrointestinal tract and pancreas, the urinary tract and
reproductive organs. Visceral pain includes pain caused by tumor
involvement of the organ capsule. Another type of visceral pain,
which is typically caused by obstruction of hollow viscus, is
characterized by intermittent cramping and poorly localized pain.
Visceral pain may be associated with inflammation as in cystitis or
reflux esophagitis.
[0045] The term inflammatory pain includes pain associates with
active inflammation that may be caused by trauma, surgery,
infection and autoimmune diseases.
[0046] The term "neuropathic pain" is used herein to refer to pain
originating from abnormal processing of sensory input by the
peripheral or central nervous system consequent on a lesion to
these systems.
[0047] The term "procedural pain" refers to pain arising from a
medical, dental or surgical procedure wherein the procedure is
usually planned or associated with acute trauma.
[0048] The term "itch" is used herein in the broadest sense and
refers to all types of itching and stinging sensations localized
and generalized, acute intermittent and persistent. The itch may be
idiopathic, allergic, metabolic, infectious, drug-induced, due to
liver, kidney disease, or cancer. "Pruritus" is severe itching.
[0049] By "patient" is meant any animal. In one embodiment, the
patient is a human. Other animals that can be treated using the
methods, compositions, and kits of the invention include but are
not limited to non-human primates (e.g., monkeys, gorillas,
chimpanzees), domesticated animals (e.g., horses, pigs, goats,
rabbits, sheep, cattle, llamas), and companion animals (e.g.,
guinea pigs, rats, mice, lizards, snakes, dogs, cats, fish,
hamsters, and birds).
[0050] Compounds useful in the invention include but are not
limited to those described herein in any of their pharmaceutically
acceptable forms, including isomers such as diastereomers and
enantiomers, salts, esters, amides, thioesters, solvates, and
polymorphs thereof, as well as racemic mixtures and pure isomers of
the compounds described herein.
[0051] By "low molecular weight" is meant less than about 500
Daltons.
[0052] The term "pharmaceutically acceptable salt" represents those
salts which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of humans and lower
animals without undue toxicity, irritation, allergic response and
the like, and are commensurate with a reasonable benefit/risk
ratio. Pharmaceutically acceptable salts are well known in the art.
The salts can be prepared in situ during the final isolation and
purification of the compounds of the invention, or separately by
reacting the free base function with a suitable organic acid.
Representative acid addition salts include but are not limited to
acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate,
benzoate, bisulfate, borate, butyrate, camphorate,
camphorsulfonate, citrate, cyclopentanepropionate, digluconate,
dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate,
glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,
hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, isethionate,
lactobionate, lactate, laurate, lauryl sulfate, malate, maleate,
malonate, mesylate, methanesulfonate, 2-naphthalenesulfonate,
nicotinate, nitrate, oleate, oxalate, palmitate, pamoate,
pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,
pivalate, propionate, stearate, succinate, sulfate, tartrate,
thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the
like. Representative alkali or alkaline earth metal salts include
but are not limited to sodium, lithium, potassium, calcium,
magnesium, and the like, as well as nontoxic ammonium, quaternary
ammonium, and amine cations, including, but not limited to
ammonium, tetramethylammonium, tetraethylammonium, methylamine,
dimethylamine, trimethylamine, triethylamine, ethylamine, and the
like.
[0053] In the generic descriptions of compounds of this invention,
the number of atoms of a particular type in a substituent group is
generally given as a range, e.g., an alkyl group containing from 1
to 4 carbon atoms or C.sub.1-4 alkyl. Reference to such a range is
intended to include specific references to groups having each of
the integer number of atoms within the specified range. For
example, an alkyl group from 1 to 4 carbon atoms includes each of
C.sub.1, C.sub.2, C.sub.3, and C.sub.4. A C.sub.1-12 heteroalkyl,
for example, includes from 1 to 12 carbon atoms in addition to one
or more heteroatoms. Other numbers of atoms and other types of
atoms may be indicated in a similar manner.
[0054] As used herein, the terms "alkyl" and the prefix "alk-" are
inclusive of both straight chain and branched chain groups and of
cyclic groups, i.e., cycloalkyl. Cyclic groups can be monocyclic or
polycyclic and preferably have from 3 to 6 ring carbon atoms,
inclusive. Exemplary cyclic groups include cyclopropyl, cyclobutyl,
cyclopentyl, and cyclohexyl groups.
[0055] By "C.sub.1-4 alkyl" is meant a branched or unbranched
hydrocarbon group having from 1 to 4 carbon atoms. A C.sub.1-4
alkyl group may be substituted or unsubstituted. Exemplary
substituents include alkoxy, aryloxy, sulfhydryl, alkylthio,
arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl, amino,
aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl,
carboxyalkyl, and carboxyl groups. C.sub.1-4 alkyls include,
without limitation, methyl, ethyl, n-propyl, isopropyl,
cyclopropyl, cyclopropylmethyl, n-butyl, iso-butyl, sec-butyl,
tert-butyl, and cyclobutyl.
[0056] By "C.sub.2-4 alkenyl" is meant a branched or unbranched
hydrocarbon group containing one or more double bonds and having
from 2 to 4 carbon atoms. A C.sub.2-4 alkenyl may optionally
include monocyclic or polycyclic rings, in which each ring
desirably has from three to six members. The C.sub.2-4 alkenyl
group may be substituted or unsubstituted. Exemplary substituents
include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide,
hydroxyl, fluoroalkyl, perfluoralkyl, amino, aminoalkyl,
disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl,
and carboxyl groups. C.sub.2-4 alkenyls include, without
limitation, vinyl, allyl, 2-cyclopropyl-1-ethenyl, 1-propenyl,
1-butenyl, 2-butenyl, 3-butenyl, 2-methyl-1-propenyl, and
2-methyl-2-propenyl.
[0057] By "C.sub.2-4 alkynyl" is meant a branched or unbranched
hydrocarbon group containing one or more triple bonds and having
from 2 to 4 carbon atoms. A C.sub.2-4 alkynyl may optionally
include monocyclic, bicyclic, or tricyclic rings, in which each
ring desirably has five or six members. The C.sub.2-4 alkynyl group
may be substituted or unsubstituted. Exemplary substituents include
alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxy,
fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino,
quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups.
C.sub.2-4 alkynyls include, without limitation, ethynyl,
1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and 3-butynyl.
[0058] By "C.sub.2-6 heterocyclyl" is meant a stable 5- to
7-membered monocyclic or 7- to 14-membered bicyclic heterocyclic
ring which is saturated partially unsaturated or unsaturated
(aromatic), and which consists of 2 to 6 carbon atoms and 1, 2, 3
or 4 heteroatoms independently selected from N, O, and S and
including any bicyclic group in which any of the above-defined
heterocyclic rings is fused to a benzene ring. The heterocyclyl
group may be substituted or unsubstituted. Exemplary substituents
include alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide,
hydroxy, fluoroalkyl, perfluoralkyl, amino, aminoalkyl,
disubstituted amino, quaternary amino, hydroxyalkyl, carboxyalkyl,
and carboxyl groups. The nitrogen and sulfur heteroatoms may
optionally be oxidized. The heterocyclic ring may be covalently
attached via any heteroatom or carbon atom which results in a
stable structure, e.g., an imidazolinyl ring may be linked at
either of the ring-carbon atom positions or at the nitrogen atom. A
nitrogen atom in the heterocycle may optionally be quaternized.
Preferably when the total number of S and O atoms in the
heterocycle exceeds 1, then these heteroatoms are not adjacent to
one another. Heterocycles include, without limitation, 1H-indazole,
2-pyrrolidonyl, 2H,6H-1,5,2-dithiazinyl, 2H-pyrrolyl, 3H-indolyl,
4-piperidonyl, 4aH-carbazole, 4H-quinolizinyl,
6H-1,2,5-thiadiazinyl, acridinyl, azocinyl, benzimidazolyl,
benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,
benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,
benzisothiazolyl, benzimidazalonyl, carbazolyl, 4aH-carbazolyl,
b-carbolinyl, chromanyl, chromenyl, cinnolinyl,
decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,
dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl,
imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl,
indolinyl, indolizinyl, indolyl, isobenzofuranyl, isochromanyl,
isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,
isothiazolyl, isoxazolyl, morpholinyl, naphthyridinyl,
octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,
1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,
oxazolidinyl, oxazolyl, oxazolidinylperimidinyl, phenanthridinyl,
phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl,
phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl,
piperidinyl, pteridinyl, piperidonyl, 4-piperidonyl, pteridinyl,
purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl,
pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,
pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl,
pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,
quinuclidinyl, carbolinyl, tetrahydrofuranyl,
tetrahydroisoquinolinyl, tetrahydroquinolinyl,
6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,
1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl,
thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl,
thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl,
1,2,5-triazolyl, 1,3,4-triazolyl, xanthenyl. Preferred 5 to 10
membered heterocycles include, but are not limited to, pyridinyl,
pyrimidinyl, triazinyl, furanyl, thienyl, thiazolyl, pyrrolyl,
pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, tetrazolyl,
benzofuranyl, benzothiofuranyl, indolyl, benzimidazolyl,
1H-indazolyl, oxazolidinyl, isoxazolidinyl, benzotriazolyl,
benzisoxazolyl, oxindolyl, benzoxazolinyl, quinolinyl, and
isoquinolinyl. Preferred 5 to 6 membered heterocycles include,
without limitation, pyridinyl, pyrimidinyl, triazinyl, furanyl,
thienyl, thiazolyl, pyrrolyl, piperazinyl, piperidinyl, pyrazolyl,
imidazolyl, oxazolyl, isoxazolyl, and tetrazolyl.
[0059] By "C.sub.6-12 aryl" is meant an aromatic group having a
ring system comprised of carbon atoms with conjugated .pi.
electrons (e.g., phenyl). The aryl group has from 6 to 12 carbon
atoms. Aryl groups may optionally include monocyclic, bicyclic, or
tricyclic rings, in which each ring desirably has five or six
members. The aryl group may be substituted or unsubstituted.
Exemplary substituents include alkyl, hydroxy, alkoxy, aryloxy,
sulfhydryl, alkylthio, arylthio, halide, fluoroalkyl, carboxyl,
hydroxyalkyl, carboxyalkyl, amino, aminoalkyl, monosubstituted
amino, disubstituted amino, and quaternary amino groups.
[0060] By "C.sub.7-14 alkaryl" is meant an alkyl substituted by an
aryl group (e.g., benzyl, phenethyl, or 3,4-dichlorophenethyl)
having from 7 to 14 carbon atoms.
[0061] By "C.sub.3-10 alkheterocyclyl" is meant an alkyl
substituted heterocyclic group having from 3 to 10 carbon atoms in
addition to one or more heteroatoms (e.g., 3-furanylmethyl,
2-furanylmethyl, 3-tetrahydrofuranylmethyl, or
2-tetrahydrofuranylmethyl).
[0062] By "C.sub.1-7 heteroalkyl" is meant a branched or unbranched
alkyl, alkenyl, or alkynyl group having from 1 to 7 carbon atoms in
addition to 1, 2, 3 or 4 heteroatoms independently selected from
the group consisting of N, O, S, and P. Heteroalkyls include,
without limitation, tertiary amines, secondary amines, ethers,
thioethers, amides, thioamides, carbamates, thiocarbamates,
hydrazones, imines, phosphodiesters, phosphoramidates,
sulfonamides, and disulfides. A heteroalkyl may optionally include
monocyclic, bicyclic, or tricyclic rings, in which each ring
desirably has three to six members. The heteroalkyl group may be
substituted or unsubstituted. Exemplary substituents include
alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxyl,
fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino,
quaternary amino, hydroxyalkyl, hydroxyalkyl, carboxyalkyl, and
carboxyl groups. Examples of C.sub.1-7 heteroalkyls include,
without limitation, methoxymethyl and ethoxyethyl.
[0063] By "halide" is meant bromine, chlorine, iodine, or
fluorine.
[0064] By "fluoroalkyl" is meant an alkyl group that is substituted
with a fluorine atom.
[0065] By "perfluoroalkyl" is meant an alkyl group consisting of
only carbon and fluorine atoms.
[0066] By "carboxyalkyl" is meant a chemical moiety with the
formula --(R)--COOH, wherein R is selected from C.sub.1-7 alkyl,
C.sub.2-7 alkenyl, C.sub.2-7 alkynyl, C.sub.2-6 heterocyclyl,
C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl, or
C.sub.1-7 heteroalkyl.
[0067] By "hydroxyalkyl" is meant a chemical moiety with the
formula --(R)--OH, wherein R is selected from C.sub.1-7 alkyl,
C.sub.2-7 alkenyl, C.sub.2-7 alkynyl, C.sub.2-6 heterocyclyl,
C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl, or
C.sub.1-7 heteroalkyl.
[0068] By "alkoxy" is meant a chemical substituent of the formula
--OR, wherein R is selected from C.sub.1-7 alkyl, C.sub.2-7
alkenyl, C.sub.2-7 alkynyl, C.sub.2-6 heterocyclyl, C.sub.6-12
aryl, C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl, or C.sub.1-7
heteroalkyl.
[0069] By "aryloxy" is meant a chemical substituent of the formula
--OR, wherein R is a C.sub.6-12 aryl group.
[0070] By "alkylthio" is meant a chemical substituent of the
formula --SR, wherein R is selected from C.sub.1-7 alkyl, C.sub.2-7
alkenyl, C.sub.2-7 alkynyl, C.sub.2-6 heterocyclyl, C.sub.6-12
aryl, C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl, or C.sub.1-7
heteroalkyl.
[0071] By "arylthio" is meant a chemical substituent of the formula
--SR, wherein R is a C.sub.6-12 aryl group.
[0072] By "quaternary amino" is meant a chemical substituent of the
formula --(R)--N(R')(R'')(R''')+, wherein R, R', R'', and R''' are
each independently an alkyl, alkenyl, alkynyl, or aryl group. R may
be an alkyl group linking the quaternary amino nitrogen atom, as a
substituent, to another moiety. The nitrogen atom, N, is covalently
attached to four carbon atoms of alkyl, heteroalkyl, heteroaryl,
and/or aryl groups, resulting in a positive charge at the nitrogen
atom.
[0073] By "charged moiety" is meant a moiety which gains a proton
at physiological pH thereby becoming positively charged (e.g.,
ammonium, guanidinium, or amidinium) or a moiety that includes a
net formal positive charge without protonation (e.g., quaternary
ammonium). The charged moiety may be either permanently charged or
transiently charged.
[0074] As used herein, the term "parent" refers to a channel
blocking compound which can be modified by quaternization or
guanylation of an amine nitrogen atom present in the parent
compound. The quaternized and guanylated compounds are derivatives
of the parent compound. The guanidyl derivatives described herein
are presented in their uncharged base form. These compounds can be
administered either as a salt (i.e., an acid addition salt) or in
their uncharged base form, which undergoes protonation in situ to
form a charged moiety.
[0075] By "therapeutically effective amount" means an amount
sufficient to produce a desired result, for example, the reduction
or elimination of pain, itch, or neurogenic inflammation in a
patient (e.g., a human) suffering from a condition, disease, or
illness that is caused wholly or in part by neurogenic inflammation
(e.g. asthma, arthritis, colitis, contact dermatitis, diabetes,
eczema, cystitis, gastritis, migraine headache, psoriasis,
rhinitis, rosacea, or sunburn).
[0076] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0077] FIG. 1A shows use-dependent block of Na.sub.v1.7 sodium
channels by 100 .mu.M QX-314 (middle trace) or the sodium channel
blocker compound, BW 8186 (Compound 6) (bottom trace) applied
intracellularly in whole-cell patch clamp recordings, with currents
through sodium channels elicited by depolarizing voltage pulses
applied at increasing frequencies, with rest periods in between to
assay recovery from use-dependence. Top trace shows the lack of
significant block when the same protocol was applied in the absence
of drug. FIG. 1B shows that compound 6, applied externally (middle
trace) has very little effect (comparable to the absence of drug)
when tested with the same protocol.
[0078] FIGS. 2A-2D show reversal of CFA-induced thermal
hyperalgesia by charged sodium channel blockers. FIGS. 2A and 2C
show time course (0, 1, 6, and 24 hours) of changes in thermal
nociceptive sensitivity measured as response latency (seconds) to a
constant radiant heat source applied to the plantar surface of hind
paw in mice co-treated with 20 .mu.L (injected in the left hindpaw)
of CFA (50% emulsion) and saline, and when the CFA was co-applied
with QX-314 (1%, hollow square), N-ethyl etidocaine (Compound 21,
1%; dark square), or ACS8180-3B (Compound 3, 1%; solid triangle).
FIGS. 2B and 2D show the nociceptive heat pain response time before
CFA treatment and after treatment with the different compounds 1
hour post CFA injection. The data are means.+-.SEM of 8-16 mice per
group. The statistical comparison with pre-CFA (*) and with
CFA+Vehicle (+) is indicated by +P<0.05; ++P<0.01 and +++,***
P<0.001.
[0079] FIGS. 3A-3B are data showing reversal of paw
incision-induced thermal hyperalgesia by charged sodium channel
blockers. FIG. 3A shows thermal nociceptive threshold observed
between the contra and ipsilateral paw of rats that received (1
hour prior to testing) an acute intraplantar injection (50 .mu.l)
of saline, QX-314 (0.5%), or N-ethyl etidocaine (Compound 21)
(0.5%). FIG. 3B shows thermal nociceptive threshold observed in
rats that underwent surgical incision to their left hindpaw
following injection of saline, QX-314, or N-ethyl etidocaine
(Compound 21) compared to their contralateral paw. The data are
means.+-.SEM of 8 rats per group. The statistical comparison with
the contralateral paw (*) is indicated by ***P<0.001.
[0080] FIGS. 4A-4C are results showing that N-ethyl-etidocaine
(Compound 21) does not induce neurotoxicity. Representative picture
of ATF3 (dark shading, FIGS. 4A-4C) expression in mice dorsal root
ganglion slice exposed 8 weeks earlier to an acute hindpaw
injection of CFA+N-ethyl-etidocaine (Compound 21) (1%, 20
.mu.l).
[0081] FIG. 5A-5E are results showing that lung sensory neuron
silencing with N-ethyl-etidocaine (Compound 21) reduces allergic
airway inflammation. OVA-exposed mice (day 21) develop increases in
BALF total (FIG. 5A), and CD45+ cells (FIG. 5B), including
eosinophil (FIG. 5C), macrophage (FIG. 5D) and T-cell (FIG. 5E)
counts. In comparison to vehicle treatment, the silencing of
sensory neurons using aerosolized QX-314 (100 .mu.M, hollow square)
or N-ethyl-etidocaine (Compound 21) (100 .mu.M, dark square)
decreased these levels. N-ethyl-etidocaine (Compound 21) shows a
tendency toward a greater decrease in comparison to QX-314. Data
expressed as mean S.E.M; Two-tailed unpaired Student's t-test
(n=8-16 animals/group; 1-2 cohorts).
DETAILED DESCRIPTION OF THE INVENTION
[0082] We have identified new quaternary ammonium compounds that
are capable of passing through open TRP channel-forming receptors
that are expressed on nociceptors and/or pruriceptors but not on
motor neurons that are more potent than QX-314 as ion channel
blockers when applied inside cells. Because they are positively
charged, the ion channel blockers of the present invention are not
membrane-permeant and thus cannot enter cells that do not express
TRP channel-forming receptors. Since TRP channel-forming receptors
are often more active in tissue conditions associated with pain
(such as inflammation) due to release of endogenous ligands or
activation by thermal stimuli, the ion channel blockers of the
invention can be used alone to selectively target activated
nociceptors in order to effectively treat (e.g., eliminate or
alleviate) pain, itch, or neurogenic inflammation. The ion channel
blockers of the invention can also be used in combination with one
or more exogenous TRP channel-forming receptor agonists to
selectively target nociceptors in order to effectively treat (e.g.,
eliminate or alleviate) pain, itch, or neurogenic inflammation.
[0083] Voltage-dependent ion channels in pain-sensing neurons are
currently of great interest in developing drugs to treat pain.
Blocking voltage-dependent sodium channels in pain-sensing neurons
can block pain signals by interrupting initiation and transmission
of the action potential, and blocking calcium channels can prevent
neurotransmission of the pain signal to the second order neuron in
the spinal cord. Moreover, blocking voltage-dependent sodium
channels in nociceptors can reduce or eliminate neurogenic
inflammation by preventing activation of nociceptor peripheral
terminals and the release thereof pro-inflammatory chemicals.
[0084] Heretofore, a limitation in designing small organic
molecules that block sodium channels or calcium channels is that
they must be active when applied externally to the target cell. The
vast majority of such externally-applied molecules are hydrophobic
and can pass through membranes. Because of this, they will enter
all cells and thus have no selectivity for affecting only
nociceptors.
[0085] Some inhibitors, such as the cationic lidocaine derivative
QX-314, are membrane-impermeant and are only effective when present
inside the nociceptor cell, and thus must pass through through the
cell membrane via a channel or receptor, such as a transient
receptor potential ion channel (TRP channels, e.g., TRPAV1, TRPA1,
and P2X(2/3)), in order to produce an effect. Under normal
circumstances, most TRP channels in nociceptors are not active but
require a noxious thermal, mechanical, or chemical stimulus to
activate them. For example, TRP channels in nociceptors can be
activated by an exogenous TRP ligand (i.e. TRP agonist) such as
capsaicin, which opens the TRPV1 channel. Thus, one approach to
selectively targeting nociceptors is to co-administer the
membrane-impermeant ion channel inhibitor with an exogenous TRP
ligand that permits passage of the inhibitor through the TRP
channel into the cell. In addition to capsaicin, the exogenous TRP
ligand can also be another capsaicinoid, mustard oil, or lidocaine.
In another example, TRP channels may be active in response to
exogenous irritant activators such as inhaled acrolein from smoke
or chemical warfare agents such as tear gas.
[0086] Under certain circumstances, TRP channels can be activated
in the absence of exogenous TRP agonists/ligands by endogenous
inflammatory activators that are generated by tissue damage,
infection, autoimmunity, atopy, ischemia, hypoxia, cellular stress,
immune cell activation, immune mediator production, and oxidative
stress. Under such conditions, endogenous molecules (e.g., protons,
lipids, and reactive oxygen species) can activate TRP channels
expressed on nociceptors, allowing membrane-impermeant,
voltage-gated ion channel blockers to gain access to the inside of
the nociceptor through the endogenously-activated TRP channels.
Endogenous inflammatory activators of TRP channels include, for
example, prostaglandins, nitric oxide (NO), peroxide (H.sub.202),
cysteine-reactive inflammatory mediators like 4-hydroxynonenal,
endogenous alkenyl aldehydes, endocannabinoids, and immune
mediators (e.g., interleukin 1 (IL-1), nerve growth factor (NGF),
and bradykinin, whose receptors are coupled to TRP channels).
[0087] The invention is described in more detail below.
Charged Ion Channel Blockers
[0088] Compounds that can be used in the compositions, kits, and
methods of the invention include compounds of formulas (I).
##STR00008##
[0089] In formula (I), R.sup.1F and R.sup.1G together complete a
heterocyclic ring having at least one nitrogen atom. In preferred
embodiments, the heterocyclic ring is a 6-membered ring or a
5-membered ring. In addition, each of R.sup.1A, R.sup.1B, and
R.sup.1C can, independently, be selected from H, halogen, C.sub.1-4
alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, OR, NR.sup.1JR.sup.1K,
NR.sup.1LC(O)R.sup.1M, S(O)R.sup.1N, SO.sub.2R.sup.1OR.sup.1P,
SO.sub.2NR.sup.1QR.sup.1R, SO.sub.3R.sup.1S, CO.sub.2R.sup.1T,
C(O)R.sup.1U, and C(O)NR.sup.1VR.sup.1W and each of R.sup.1I,
R.sup.1J, R.sup.1K, R.sup.1L, R.sup.1M, R.sup.1N, R.sup.1O,
R.sup.1P, R.sup.1Q, R.sup.1R, R.sup.1S, R.sup.1T, R.sup.1U,
R.sup.1V, and R.sup.1W can, independently, be selected from H,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, and
C.sub.2-4 heteroalkyl. In preferred embodiments, the compounds of
the invention have at least one independent R.sup.1C. X.sup.1 can
be selected from --CR.sup.1XR.sup.1Y--, --NR.sup.1ZC(O)--,
--OC(O)--, --SC(O)--, --C(O)NR.sup.1AA--, --CO.sub.2--, and
--OC(S)--. In a preferred embodiment, X.sup.1 is --NHC(O)--. Each
of R.sup.1X, R.sup.1Y, R.sup.1Z, and R.sup.1AA can, independently,
be selected from H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4
alkynyl, and C.sub.2-4 heteroalkyl. Each of R.sup.1D and R.sup.1E
can, independently, be selected from H, C.sub.1-4 alkyl, C.sub.2-4
alkenyl, C.sub.2-4 alkynyl, C.sub.2-4 heteroalkyl, optionally
substituted with halogen, cyclic alkyl, aryl, or heteroaryl, and
C.sub.3-6cycloalkyl, or R.sup.1D and R.sup.1E together can form a
3-6-membered ring (cyclic alkyl or heterocyclic). R.sup.1H can be
selected from C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4
alkynyl, C.sub.2-4 heteroalkyl, optionally substituted with
halogen, cyclic alkyl, aryl, or heteroaryl, and C.sub.3-6
cycloalkyl. Exemplary compounds of formula (I) include those listed
in Table 1. These compounds can be prepared using methods analogous
to those described in Examples 1-6.
TABLE-US-00001 TABLE 1 Com- pound Molecular % Inhibition No.
Molecular Structure Weight at 100 .mu.M 1 ##STR00009## 261.39 40 2
##STR00010## 275.42 42 3 ##STR00011## 289.44 78 4 ##STR00012##
275.42 43 5 ##STR00013## 289.44 34 6 ##STR00014## 303.47 83 7
##STR00015## 275.42 TBD 8 ##STR00016## 289.23 TBD 9 ##STR00017##
275.42 TBD 10 ##STR00018## 289.44 TBD 11 ##STR00019## 303.47 TBD 12
##STR00020## 289.44 TBD
[0090] Compounds that can be used in the compositions, kits, and
methods of the invention include compounds of formulas (II).
##STR00021##
[0091] In formula (II), m can be 0 or 1, each of R.sup.2A,
R.sup.2B, and R.sup.2C can, independently, be selected from H,
halogen, C.sub.1-4 alkyl, C.sub.2-4alkenyl, C.sub.2-4alkynyl,
CF.sub.3, OR.sup.2H, NR.sup.2IR.sup.2J, NR.sup.2KC(O)R.sup.2L,
S(O)R.sup.2M, SO.sub.2R.sup.2NR.sup.2O, SO.sub.2NR.sup.2PR.sup.2Q,
SO.sub.3R.sup.2R, CO.sub.2R.sup.2S, C(O)R.sup.2T, and
C(O)NR.sup.2UR.sup.2V; and each of R.sup.2H, R.sup.2I, R.sup.2J,
R.sup.2K, R.sup.2L, R.sup.2MR.sup.2N, R.sup.2O, R.sup.2P, R.sup.2Q,
R.sup.2R, R.sup.2S, R.sup.2T, R.sup.2U, and R.sup.2V can,
independently, be selected from H, C.sub.1-4alkyl,
C.sub.2-4alkenyl, C.sub.2-4 alkynyl, and C.sub.2-4 heteroalkyl. n
can be 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9, and each R.sup.2F can,
independently be selected from halogen, C.sub.1-4 alkyl, C.sub.2-4
alkenyl, C.sub.2-4 alkynyl, CF.sub.3, OR.sup.2H, NR.sup.2IR.sup.2J,
NR.sup.2K(O)R.sup.2L, S(O)R.sup.2M, SO.sub.2R.sup.2NR.sup.2O,
SO.sub.2NR.sup.2PR.sup.2Q, SO.sub.3R.sup.2R, CO.sub.2R.sup.2S,
PC(O)R.sup.2T, and C(O)NR.sup.2UR.sup.2V; and each of R.sup.2H,
R.sup.2I, R.sup.2J, R.sup.2K, R.sup.2L, R.sup.2M, R.sup.2N,
R.sup.2R, R.sup.2S, R.sup.2T, R.sup.2U, and R.sup.2V can,
independently, be selected from H, C.sub.1-4 alkyl, C.sub.2-4
alkenyl, C.sub.2-4 alkynyl, and C.sub.2-4 heteroalkyl. In preferred
embodiments, the compounds of the invention have at least one
independent R.sup.2C. In another embodiment, compounds of the
invention have at least one R.sup.2F and up to nine R.sup.2F.
R.sup.2D and R.sup.2E can be selected from C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-4 heteroalkyl,
optionally substituted with halogen, cyclic alkyl, aryl, or
heteroaryl, and C.sub.3-6 cycloalkyl. Exemplary compounds of
formula (II) include those listed in Table 2. These compounds can
be prepared using methods analogous to those described in Examples
7-10.
TABLE-US-00002 TABLE 2 Com- pound Molecular % Inhibition No.
Molecular Structure Weight at 100 .mu.M 13 ##STR00022## 247.36 33
14 ##STR00023## 275.42 62 15 ##STR00024## 289.44 40 16 ##STR00025##
279.38 70 17 ##STR00026## 261.39 TBD 18 ##STR00027## 279.38 TBD 19
##STR00028## 315.36 TBD 20 ##STR00029## 369.33 TBD
[0092] Compounds that can be used in the compositions, kits, and
methods of the invention include compounds of formulas (III).
##STR00030##
[0093] In formula (III), n can be 0, 1, 2, or 3, each of R.sup.3A,
R.sup.3B, and R.sup.3C can, independently, be selected from H,
halogen, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
OR.sup.3I, NR.sup.3JR.sup.3K, NR.sup.3LC(O)R.sup.3M, S(O)R.sup.3N,
SO.sub.2R.sup.3OR.sup.3P, SO.sub.2NR.sup.3QR.sup.3R,
SO.sub.3R.sup.3S, CO.sub.2R.sup.3T, C(O)R.sup.3U, and
C(O)NR.sup.3VR.sup.3W; and each of R.sup.3I, R.sup.3J, R.sup.3K,
R.sup.3L, R.sup.3M, R.sup.3N, R.sup.3O, R.sup.3P, R.sup.3Q,
R.sup.3R, R.sup.3S, R.sup.3T, R.sup.3U, R.sup.3V, and R.sup.3W can,
independently, be selected from H, C.sub.1-4 alkyl, C.sub.2-4
alkenyl, C.sub.2-4 alkynyl, and C.sub.2-4 heteroalkyl. X.sup.3 can
be selected from --NHC(O)--, and --C(O)NH. Each of R.sup.3D and
R.sup.3E can, independently, be selected from H, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-4 heteroalkyl,
optionally substituted with halogen, cyclic alkyl, aryl, or
heteroaryl, and C.sub.3-6cycloalkyl, or R.sup.3D and R.sup.3E
together can form a 3-6-membered ring (cyclic alkyl or
heterocyclic). Each of R.sup.3F, R.sup.3G, and R.sup.3H can be
selected from C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4
alkynyl, C.sub.2-4 heteroalkyl, optionally substituted with
halogen, cyclic alkyl, aryl, or heteroaryl, and
C.sub.3-6cycloalkyl. Exemplary compounds of formula (III) include
those listed in Table 3. These compounds can be prepared using
methods analogous to those described in Examples 11-14.
TABLE-US-00003 TABLE 3 Compound Molecular % Inhibition No.
Molecular Structure Weight at 100 .mu.M 21 ##STR00031## 305.49 97
22 ##STR00032## 250.37 0 23 ##STR00033## 278.42 56 24 ##STR00034##
277.43 15 25 ##STR00035## 277.43 3 26 ##STR00036## 264.39 TBD 27
##STR00037## 292.45 TBD
Synthesis The synthesis of charge-modified ion channel blockers may
involve the selective protection and deprotection of alcohols,
amines, ketones, sulfhydryls or carboxyl functional groups of the
parent ion channel blocker, the linker, the bulky group, and/or the
charged group. For example, commonly used protecting groups for
amines include carbamates, such as tert-butyl, benzyl,
2,2,2-trichloroethyl, 2-trimethylsilylethyl, 9-fluorenylmethyl,
allyl, and m-nitrophenyl. Other commonly used protecting groups for
amines include amides, such as formamides, acetamides,
trifluoroacetamides, sulfonamides, trifluoromethanesulfonyl amides,
trimethylsilylethanesulfonamides, and tert-butylsulfonyl amides.
Examples of commonly used protecting groups for carboxyls include
esters, such as methyl, ethyl, tert-butyl, 9-fluorenylmethyl,
2-(trimethylsilyl)ethoxy methyl, benzyl, diphenylmethyl,
O-nitrobenzyl, ortho-esters, and halo-esters. Examples of commonly
used protecting groups for alcohols include ethers, such as methyl,
methoxymethyl, methoxyethoxymethyl, methylthiomethyl,
benzyloxymethyl, tetrahydropyranyl, ethoxyethyl, benzyl,
2-napthylmethyl, O-nitrobenzyl, P-nitrobenzyl, P-methoxybenzyl,
9-phenylxanthyl, trityl (including methoxy-trityls), and silyl
ethers. Examples of commonly used protecting groups for sulfhydryls
include many of the same protecting groups used for hydroxyls. In
addition, sulfhydryls can be protected in a reduced form (e.g., as
disulfides) or an oxidized form (e.g., as sulfonic acids, sulfonic
esters, or sulfonic amides). Protecting groups can be chosen such
that selective conditions (e.g., acidic conditions, basic
conditions, catalysis by a nucleophile, catalysis by a Lewis acid,
or hydrogenation) are required to remove each, exclusive of other
protecting groups in a molecule. The conditions required for the
addition of protecting groups to amine, alcohol, sulfhydryl, and
carboxyl functionalities and the conditions required for their
removal are provided in detail in T. W. Green and P. G. M. Wuts,
Protective Groups in Organic Synthesis (2.sup.nd Ed.), John Wiley
& Sons, 1991 and P. J. Kocienski, Protecting Groups, Georg
Thieme Verlag, 1994.
[0094] Charge-modified ion channel blockers can be prepared using
techniques familiar to those skilled in the art. The modifications
can be made, for example, by alkylation of the parent ion channel
blocker using the techniques described by J. March, Advanced
Organic Chemistry: Reactions, Mechanisms and Structure, John Wiley
& Sons, Inc., 1992, page 617. The conversion of amino groups to
guanidine groups can be accomplished using standard synthetic
protocols. For example, Mosher has described a general method for
preparing mono-substituted guanidines by reaction of
aminoiminomethanesulfonic acid with amines (Kim et al., Tetrahedron
Lett. 29:3183 (1988)). A more convenient method for guanylation of
primary and secondary amines was developed by Bernatowicz employing
1H-pyrazole-1-carboxamidine hydrochloride;
1-H-pyrazole-1-(N,N'-bis(tert-butoxycarbonyl)carboxamidine; or
1-H-pyrazole-1-(N,N'-bis(benzyloxycarbonyl)carboxamidine. These
reagents react with amines to give mono-substituted guanidines (see
Bernatowicz et al., J. Org. Chem. 57:2497 (1992); and Bernatowicz
et al., Tetrahedron Lett. 34:3389 (1993)). In addition, Thioureas
and S-alkyl-isothioureas have been shown to be useful intermediates
in the syntheses of substituted guanidines (Poss et al.,
Tetrahedron Lett. 33:5933 (1992)).
[0095] Charge-modified ion channel blockers can be prepared by
alkylation of an amine nitrogen in the parent compound as shown in
Scheme 1.
##STR00038##
[0096] Alternatively, charge-modified ion channel blockers can be
prepared by introduction of a guanidine group. The parent compound
can be reacted with a cynamide, e.g., methylcyanamide or
pyrazole-1-carboxamidine derivatives. Alternatively, the parent
compound can be reacted with cyanogens bromide followed by reaction
with methylchloroaluminum amide as shown in Scheme 2. Reagents such
as 2-(methylthio)-2-imidazoline can also be used to prepare
suitably functionalized derivatives (Scheme 3).
##STR00039##
##STR00040##
Any ion channel blocker containing an amine nitrogen atom can be
modified as shown in Schemes 1-5. Exemplary synthetic schemes for
particular charged ion channel blockers of the present invention
are further detailed in Examples 1-14.
Exogenous TRP Channel-Forming Receptor Agonists
[0097] TRPV1 agonists that can be employed in the methods and kits
of the invention include but are not limited to any that activates
TRPV1 receptors on nociceptors and allows for entry of at least one
inhibitor of voltage-gated ion channels. A suitable TRPV1 agonist
is capsaicin or another capsaicinoids, which are members of the
vanilloid family of molecules. Naturally occurring capsaicinoids
are capsaicin itself, dihydrocapsaicin, nordihydrocapsaicin,
homodihydrocapsaicin, homocapsaicin, and nonivamide. Other suitable
capsaicinoids and capsaicinoid analogs and derivatives for use in
the compositions and methods of the present invention include
naturally occurring and synthetic capsaicin derivatives and analogs
including, e.g., vanilloids (e.g., N-vanillyl-alkanedienamides,
N-vanillyl-alkanedienyls, and N-vanillyl-cis-monounsaturated
alkenamides), capsiate, dihydrocapsiate, nordihydrocapsiate and
other capsinoids, capsiconiate, dihydrocapsiconiate and other
coniferyl esters, capsiconinoid, resiniferatoxin, tinyatoxin,
civamide, N-phenylmethylalkenamide capsaicin derivatives, olvanil,
N-[(4-(2-aminoethoxy)-3-methoxyphenyl)methyl]-9Z-octa-decanamide,
N-oleyl-homovanillamide, triprenyl phenols (e.g., scutigeral),
gingerols, piperines, shogaols, guaiacol, eugenol, zingerone,
nuvanil, NE-19550, NE-21610, and NE-28345. Additional
capsaicinoids, their structures, and methods of their manufacture
are described in U.S. Pat. Nos. 7,446,226 and 7,429,673, which are
hereby incorporated by reference.
[0098] Additional suitable TRPV1 agonists include but are not
limited to eugenol, arvanil (N-arachidonoylvanillamine),
anandamide, 2-aminoethoxydiphenyl borate (2APB), AM404,
resiniferatoxin, phorbol 12-phenylacetate 13-acetate
20-homovanillate (PPAHV), olvanil (NE 19550), OLDA
(N-oleoyldopamine), N-arachidonyldopamine (NADA),
6'-iodoresiniferatoxin (6'-IRTX), C18 N-acylethanolamines,
lipoxygenase derivatives such as 12-hydroperoxyeicosatetraenoic
acid, inhibitor cysteine knot (ICK) peptides (vanillotoxins),
piperine, MSK195
(N-[2-(3,4-dimethylbenzyl)-3-(pivaloyloxy)propyl]-2-[4-(2-aminoethoxy)-3--
methoxyphenyl]acetamide), JYL79
(N-[2-(3,4-dimethylbenzyl)-3-(pivaloyloxy)propyl]-N'-(4-hydroxy-3-methoxy-
benzyl)thiourea), hydroxy-alpha-sanshool, 2-aminoethoxydiphenyl
borate, 10-shogaol, oleylgingerol, oleylshogaol, and SU200
(N-(4-tert-butylbenzyl)-N'-(4-hydroxy-3-methoxybenzyl)thiourea).
Still other TRPV1 agonists include amylocaine, articaine,
benzocaine, bupivacaine, carbocaine, carticaine, chloroprocaine,
cyclomethycaine, dibucaine (cinchocaine), dimethocaine (larocaine),
etidocaine, hexylcaine, levobupivacaine, lidocaine, mepivacaine,
meprylcaine (oracaine), metabutoxycaine, piperocaine, prilocaine,
procaine (novacaine), proparacaine, propoxycaine, risocaine,
ropivacaine, tetracaine (amethocaine), and trimecaine.
[0099] TRP1A agonists that can be employed in the methods,
compositions, and kits of the invention include any that activates
TRP1A receptors on nociceptors or pruriceptors and allows for entry
of at least one inhibitor of voltage-gated ion channels. Suitable
TRP1A agonists include but are not limited to cinnamaldehyde,
allyl-isothiocynanate (mustard oil), diallyl disulfide, icilin,
cinnamon oil, wintergreen oil, clove oil, acrolein,
hydroxy-alpha-sanshool, 2-aminoethoxydiphenyl borate,
4-hydroxynonenal, methyl p-hydroxybenzoate, and
3'-carbamoylbiphenyl-3-yl cyclohexylcarbamate (URB597).
[0100] P2X agonists that can be employed in the methods,
compositions, and kits of the invention include any that activates
P2X receptors on nociceptors or pruriceptors and allows for entry
of at least one inhibitor of voltage-gated ion channels. Suitable
P2X agonists include but are not limited to 2-methylthio-ATP, 2'
and 3'-O-(4-benzoylbenzoyl)-ATP, and
ATP5'-O-(3-thiotriphosphate).
Additional Agents
[0101] If desired, one or more additional biologically active
agents typically used to treat neurogenic inflammation may be used
in combination with a composition of the invention described
herein. The biologically active agents include, but are not limited
to, acetaminophen, NSAIDs, glucocorticoids, narcotics (e.g.
opioids), tricyclic antidepressants, amine transporter inhibitors,
anticonvulsants, antiproliferative agents, and immune modulators.
The biologically active agents can be administered prior to,
concurrent with, or following administration of a composition of
the invention, using any formulation, dosing, or administration
known in the art that is therapeutically effective.
[0102] Non-steroidal anti-inflammatory drugs (NSAIDs) that can be
administered to a patient (e.g., a human) suffering from neurogenic
inflammation in combination with a composition of the invention
include, but are not limited to, acetylsalicylic acid, amoxiprin,
benorylate, benorilate, choline magnesium salicylate, diflunisal,
ethenzamide, faislamine, methyl salicylate, magnesium salicylate,
salicyl salicylate, salicylamide, diclofenac, aceclofenac,
acemethacin, alclofenac, bromfenac, etodolac, indometacin,
nabumetone, oxametacin, proglumetacin, sulindac, tolmetin,
ibuprofen, alminoprofen, benoxaprofen, carprofen, dexibuprofen,
dexketoprofen, fenbufen, fenoprofen, flunoxaprofen, flurbiprofen,
ibuproxam, indoprofen, ketoprofen, ketorolac, loxoprofen, naproxen,
oxaprozin, pirprofen, suprofen, tiaprofenic acid, mefenamic acid,
flufenamic acid, meclofenamic acid, tolfenamic acid,
phenylbutazone, ampyrone, azapropazone, clofezone, kebuzone,
metamizole, mofebutazone, oxyphenbutazone, phenazone,
sulfinpyrazone, piroxicam, droxicam, lornoxicam, meloxicam,
tenoxicam, and the COX-2 inhibitors celecoxib, etoricoxib,
lumiracoxib, parecoxib, rofecoxib, valdecoxib, and pharmaceutically
acceptable salts thereof.
[0103] Glucocorticoids that can be administered to a patient (e.g.,
a human) suffering from neurogenic inflammation in combination with
a composition of the invention include, but are not limited to,
hydrocortisone, cortisone acetate, prednisone, prednisolone,
methylprednisolone, dexamethasone, betamethasone, triamcinolone,
beclometasone, fludrocortisones acetate, deoxycorticosterone
acetate, aldosterone, and pharmaceutically acceptable salts
thereof.
[0104] Narcotics that can be administered to a patient (e.g., a
human) suffering from neurogenic inflammation in combination with a
composition of the invention include, but are not limited, to
tramadol, hydrocodone, oxycodone, morphine, and pharmaceutically
acceptable salts thereof.
[0105] Antiproliferative and immune modulatory agents that can be
administered to a patient (e.g., a human) suffering from neurogenic
inflammation in combination with a composition of the invention
include, but are not limited to, alkylating agents, platinum
agents, antimetabolites, topoisomerase inhibitors, dihydrofolate
reductase inhibitors, antitumor antibiotics, antimitotic agents,
aromatase inhibitors, thymidylate synthase inhibitors, DNA
antagonists, farnesyltransferase inhibitors, pump inhibitors,
histone acetyltransferase inhibitors, metalloproteinase inhibitors,
ribonucleoside reductase inhibitors, TNF-alpha agonists, TNF-alpha
antagonists or scavengers, interleukin 1 (IL-1) antagonists or
scavengers, endothelin A receptor antagonists, retinoic acid
receptor agonists, hormonal agents, antihormonal agents,
photodynamic agents, and tyrosine kinase inhibitors.
Formulation of Compositions
[0106] The administration of the compounds of the invention may be
by any suitable means that results in the reduction of perceived
pain sensation at the target region. The compounds of the invention
may be contained in any appropriate amount in any suitable carrier
substance, and are generally present in amounts totaling 1-95% by
weight of the total weight of the composition. The composition may
be provided in a dosage form that is suitable for oral, parenteral
(e.g., intravenous, intramuscular), rectal, cutaneous,
subcutaneous, topical, transdermal, sublingual, nasal, vaginal,
intrathecal, epidural, or ocular administration, or by injection,
inhalation, or direct contact with the nasal or oral mucosa.
[0107] Thus, the composition 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, implants, sprays, or aerosols. The
compositions may be formulated according to conventional
pharmaceutical practice (see, e.g., Remington: The Science and
Practice of Pharmacy, 20th edition, 2000, ed. A. R. Gennaro,
Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia
of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,
1988-1999, Marcel Dekker, New York).
[0108] Each compound may be formulated in a variety of ways that
are known in the art. For example, the first and second agents may
be formulated together or separately. Desirably, the first and
second agents are formulated together for the simultaneous or near
simultaneous administration of the agents.
[0109] The individually or separately formulated agents can be
packaged together as a kit. Non-limiting examples include but are
not limited to kits that contain, e.g., two pills, a pill and a
powder, a suppository and a liquid in a vial, two topical creams,
etc. The kit can include optional components that aid in the
administration of the unit dose to patients, such as vials for
reconstituting powder forms, syringes for injection, customized IV
delivery systems, inhalers, etc. Additionally, the unit dose kit
can contain instructions for preparation and administration of the
compositions.
[0110] The kit may be manufactured as a single use unit dose for
one patient, multiple uses for a particular patient (at a constant
dose or in which the individual compounds may vary in potency as
therapy progresses); or the kit may contain multiple doses suitable
for administration to multiple patients ("bulk packaging"). The kit
components may be assembled in cartons, blister packs, bottles,
tubes, and the like.
Controlled Release Formulations
[0111] Each compound of the invention, alone or in combination with
one or more of the biologically active agents as described herein,
can be formulated for controlled release (e.g., sustained or
measured) administration, as described in U.S. Patent Application
Publication Nos. 2003/0152637 and 2005/0025765, each incorporated
herein by reference. For example, a compound of the invention,
alone or in combination with one or more of the biologically active
agents as described herein, can be incorporated into a capsule or
tablet that is administered to the site of inflammation.
[0112] Any pharmaceutically acceptable vehicle or formulation
suitable for local infiltration or injection into a site to be
treated (e.g., a painful surgical incision, wound, or joint), that
is able to provide a sustained release of compound of the
invention, alone or in combination with one or more of the
biologically active agents as described herein, may be employed to
provide for prolonged elimination or alleviation of inflammation,
as needed. Slow release formulations known in the art include
specially coated pellets, polymer formulations or matrices for
surgical insertion or as sustained release microparticles, e.g.,
microspheres or microcapsules, for implantation, insertion,
infusion or injection, wherein the slow release of the active
medicament is brought about through sustained or controlled
diffusion out of the matrix and/or selective breakdown of the
coating of the preparation or selective breakdown of a polymer
matrix. Other formulations or vehicles for sustained or immediate
delivery of an agent to a preferred localized site in a patient
include, e.g., suspensions, emulsions, gels, liposomes and any
other suitable art known delivery vehicle or formulation acceptable
for subcutaneous or intramuscular administration.
[0113] A wide variety of biocompatible materials may be utilized as
a controlled release carrier to provide the controlled release of a
compound of the invention, alone or in combination with one or more
biologically active agents, as described herein. Any
pharmaceutically acceptable biocompatible polymer known to those
skilled in the art may be utilized. It is preferred that the
biocompatible controlled release material degrade in vivo within
about one year, preferably within about 3 months, more preferably
within about two months. More preferably, the controlled release
material will degrade significantly within one to three months,
with at least 50% of the material degrading into non-toxic
residues, which are removed by the body, and 100% of the compound
of the invention being released within a time period within about
two weeks, preferably within about 2 days to about 7 days. A
degradable controlled release material should preferably degrade by
hydrolysis, either by surface erosion or bulk erosion, so that
release is not only sustained but also provides desirable release
rates. However, the pharmacokinetic release profile of these
formulations may be first order, zero order, bi- or multi-phasic,
to provide the desired reversible local anesthetic effect over the
desired time period.
[0114] Suitable biocompatible polymers can be utilized as the
controlled release material. The polymeric material may comprise
biocompatible, biodegradable polymers, and in certain preferred
embodiments is preferably a copolymer of lactic and glycolic acid.
Preferred controlled release materials which are useful in the
formulations of the invention include the polyanhydrides,
polyesters, co-polymers of lactic acid and glycolic acid
(preferably wherein the weight ratio of lactic acid to glycolic
acid is no more than 4:1 i.e., 80% or less lactic acid to 20% or
more glycolic acid by weight)) and polyorthoesters containing a
catalyst or degradation enhancing compound, for example, containing
at least 1% by weight anhydride catalyst such as maleic anhydride.
Examples of polyesters include polylactic acid, polyglycolic acid
and polylactic acid-polyglycolic acid copolymers. Other useful
polymers include protein polymers such as collagen, gelatin, fibrin
and fibrinogen and polysaccharides such as hyaluronic acid.
[0115] The polymeric material may be prepared by any method known
to those skilled in the art. For example, where the polymeric
material is comprised of a copolymer of lactic and glycolic acid,
this copolymer may be prepared by the procedure set forth in U.S.
Pat. No. 4,293,539, incorporated herein by reference.
Alternatively, copolymers of lactic and glycolic acid may be
prepared by any other procedure known to those skilled in the art.
Other useful polymers include polylactides, polyglycolides,
polyanhydrides, polyorthoesters, polycaprolactones,
polyphosphazenes, polyphosphoesters, polysaccharides, proteinaceous
polymers, soluble derivatives of polysaccharides, soluble
derivatives of proteinaceous polymers, polypeptides, polyesters,
and polyorthoesters or mixtures or blends of any of these.
Pharmaceutically acceptable polyanhydrides which are useful in the
present invention have a water-labile anhydride linkage. The rate
of drug release can be controlled by the particular polyanhydride
polymer utilized and its molecular weight. The polysaccharides may
be poly-1,4-glucans, e.g., starch glycogen, amylose, amylopectin,
and mixtures thereof. The biodegradable hydrophilic or hydrophobic
polymer may be a water-soluble derivative of a poly-1,4-glucan,
including hydrolyzed amylopectin, hydroxyalkyl derivatives of
hydrolyzed amylopectin such as hydroxyethyl starch (HES),
hydroxyethyl amylose, dialdehyde starch, and the like. The
polyanhydride polymer may be branched or linear. Examples of
polymers which are useful in the present invention include (in
addition to homopolymers and copolymers of poly(lactic acid) and/or
poly(glycolic acid)) poly[bis(p-carboxyphenoxy) propane
anhydride](PCPP), poly[bis(p-carboxy)methane anhydride] (PCPM),
polyanhydrides of oligomerized unsaturated aliphatic acids,
polyanhydride polymers prepared from amino acids which are modified
to include an additional carboxylic acid, aromatic polyanhydride
compositions, and co-polymers of polyanhydrides with other
substances, such as fatty acid terminated polyanhydrides, e.g.,
polyanhydrides polymerized from monomers of dimers and/or trimers
of unsaturated fatty acids or unsaturated aliphatic acids.
Polyanhydrides may be prepared in accordance with the methods set
forth in U.S. Pat. No. 4,757,128, incorporated herein by reference.
Polyorthoester polymers may be prepared, e.g., as set forth in U.S.
Pat. No. 4,070,347, incorporated herein by reference.
Polyphosphoesters may be prepared and used as set forth in U.S.
Pat. Nos. 6,008,318, 6,153,212, 5,952,451, 6,051,576, 6,103,255,
5,176,907 and 5,194,581, each of which is incorporated herein by
reference.
[0116] Proteinaceous polymers may also be used. Proteinaceous
polymers and their soluble derivatives include gelation
biodegradable synthetic polypeptides, elastin, alkylated collagen,
alkylated elastin, and the like. Biodegradable synthetic
polypeptides include poly-(N-hydroxyalkyl)-L-asparagine,
poly-(N-hydroxyalkyl)-L-glutamine, copolymers of
N-hydroxyalkyl-L-asparagine and N-hydroxyalkyl-L-glutamine with
other amino acids. Suggested amino acids include L-alanine,
L-lysine, L-phenylalanine, L-valine, L-tyrosine, and the like.
[0117] In additional embodiments, the controlled release material,
which in effect acts as a carrier for a compound of the invention,
alone or in combination with one or more biologically active agents
as described herein, can further include a bioadhesive polymer such
as pectins (polygalacturonic acid), mucopolysaccharides (hyaluronic
acid, mucin) or non-toxic lectins or the polymer itself may be
bioadhesive, e.g., polyanhydride or polysaccharides such as
chitosan.
[0118] In embodiments where the biodegradable polymer comprises a
gel, one such useful polymer is a thermally gelling polymer, e.g.,
polyethylene oxide, polypropylene oxide (PEO-PPO) block copolymer
such as Pluronic.TM. F127 from BASF Wyandotte. In such cases, the
local anesthetic formulation may be injected via syringe as a
free-flowing liquid, which gels rapidly above 30.degree. C. (e.g.,
when injected into a patient). The gel system then releases a
steady dose of a compound of the invention, alone or in combination
with one or more biologically active agents as described herein, at
the site of administration.
Solid Dosage Forms for Oral Use
[0119] 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.
[0120] Two or more compounds may be mixed together in a tablet,
capsule, or other vehicle, or may be partitioned. In one example,
the first compound is contained on the inside of the tablet, and
the second compound is on the outside, such that a substantial
portion of the second compound is released prior to the release of
the first compound.
[0121] Formulations for oral use may also be provided 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, granulates, and pellets 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.
[0122] 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
methylcellulose, carnauba wax and stearyl alcohol, carbopol 934,
silicone, glyceryl tristearate, methyl acrylate-methyl
methacrylate, polyvinyl chloride, polyethylene, and/or halogenated
fluorocarbon.
[0123] The liquid forms in which the compounds and compositions of
the present invention can be incorporated for administration orally
include aqueous solutions, suitably flavored syrups, aqueous or oil
suspensions, and flavored emulsions with edible oils such as
cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as
elixirs and similar pharmaceutical vehicles.
[0124] Generally, when administered to a human, the oral dosage of
any of the compounds of the combination of the invention will
depend on the nature of the compound, and can readily be determined
by one skilled in the art. Typically, such dosage is normally about
0.001 mg to 2000 mg per day, desirably about 1 mg to 1000 mg per
day, and more desirably about 5 mg to 500 mg per day. Dosages up to
200 mg per day may be necessary.
[0125] Administration of each drug in a combination therapy, as
described herein, can, independently, be one to four times daily
for one day to one year, and may even be for the life of the
patient. Chronic, long-term administration will be indicated in
many cases.
Parenteral Formulations
[0126] Formulations suitable for parenteral administration (e.g.,
by injection), include aqueous or non-aqueous, isotonic,
pyrogen-free, sterile liquids (e.g., solutions, suspensions), in
which the compound is dissolved, suspended, or otherwise provided
(e.g., in a liposome or other microparticulate). Such liquids may
additional contain other pharmaceutically acceptable ingredients,
such as anti-oxidants, buffers, preservatives, stabilisers,
bacteriostats, suspending agents, thickening agents, and solutes
which render the formulation isotonic with the blood (or other
relevant bodily fluid) of the intended recipient. Examples of
excipients include, for example, water, alcohols, polyols,
glycerol, vegetable oils, and the like. Examples of suitable
isotonic carriers for use in such formulations include Sodium
Chloride Injection, Ringer's Solution, or Lactated Ringer's
Injection. Typically, the concentration of the compound in the
liquid is from about 1 ng/ml to about 10 .mu.g/ml, for example from
about 10 ng/ml to about 1 .mu.g/ml. The formulations may be
presented in unit-dose or multi-dose sealed containers, for
example, ampoules and vials, and may be stored in a freeze-dried
(lyophilised) condition requiring only the addition of the sterile
liquid carrier, for example water for injections, immediately prior
to use. Extemporaneous injection solutions and suspensions may be
prepared from sterile powders, granules, and tablets.
Topical Formulations
[0127] A composition of the invention, alone or in combination with
one or more of the biologically active agents described herein, can
also be adapted for topical use with a topical vehicle containing
from between 0.0001% and 25% (w/w) or more of active
ingredient(s).
[0128] In a preferred combination, the active ingredients are
preferably each from between 0.0001% to 10% (w/w), more preferably
from between 0.0005% to 4% (w/w) active agent. The cream can be
applied one to four times daily, or as needed. Performing the
methods described herein, the topical vehicle containing the
composition of the invention, or a combination therapy containing a
composition of the invention is preferably applied to the site of
inflammation on the patient. For example, a cream may be applied to
the hands of a patient suffering from arthritic fingers.
[0129] The compositions can be formulated using any
dermatologically acceptable carrier. Exemplary carriers include a
solid carrier, such as alumina, clay, microcrystalline cellulose,
silica, or talc; and/or a liquid carrier, such as an alcohol, a
glycol, or a water-alcohol/glycol blend. The therapeutic agents may
also be administered in liposomal formulations that allow
therapeutic agents to enter the skin. Such liposomal formulations
are described in U.S. Pat. Nos. 5,169,637; 5,000,958; 5,049,388;
4,975,282; 5,194,266; 5,023,087; 5,688,525; 5,874,104; 5,409,704;
5,552,155; 5,356,633; 5,032,582; 4,994,213; 8,822,537, and PCT
Publication No. WO 96/40061. Examples of other appropriate vehicles
are described in U.S. Pat. Nos. 4,877,805, 8,822,537, and EP
Publication No. 0586106A1. Suitable vehicles of the invention may
also include mineral oil, petrolatum, polydecene, stearic acid,
isopropyl myristate, polyoxyl 40 stearate, stearyl alcohol, or
vegetable oil.
[0130] The composition can further include a skin penetrating
enhancer, such as those described in "Percutaneous Penetration
enhancers", (eds. Smith E W and Maibach H I. CRC Press 1995).
Exemplary skin penetrating enhancers include alkyl
(N,N-disubstituted amino alkanoate) esters, such as dodecyl 2-(N,N
dimethylamino) propionate (DDAIP), which is described in patent
U.S. Pat. Nos. 6,083,996 and 6,118,020, which are both incorporated
herein by reference; a water-dispersible acid polymer, such as a
polyacrylic acid polymer, a carbomer (e.g., Carbopol.TM. or
Carbopol 940P.TM., available from B. F. Goodrich Company (Akron,
Ohio)), copolymers of polyacrylic acid (e.g., Pemulen.TM. from B.
F. Goodrich Company or Polycarbophil.TM. from A. H. Robbins,
Richmond, Va.; a polysaccharide gum, such as agar gum, alginate,
carrageenan gum, ghatti gum, karaya gum, kadaya gum, rhamsan gum,
xanthan gum, and galactomannan gum (e.g., guar gum, carob gum, and
locust bean gum), as well as other gums known in the art (see for
instance, Industrial Gums: Polysaccharides & Their Derivatives,
Whistler R. L., BeMiller J. N. (eds.), 3rd Ed. Academic Press
(1992) and Davidson, R. L., Handbook of Water-Soluble Gums &
Resins, McGraw-Hill, Inc., N.Y. (1980)); or combinations
thereof.
[0131] Other suitable polymeric skin penetrating enhancers are
cellulose derivatives, such as ethyl cellulose, methyl cellulose,
hydroxypropyl cellulose. Additionally, known transdermal
penetrating enhancers can also be added, if desired. Illustrative
are dimethyl sulfoxide (DMSO) and dimethyl acetamide (DMA),
2-pyrrolidone, N,N-diethyl-m-toluamide (DEET),
1-dodecylazacycloheptane-2-one (Azone.TM., a registered trademark
of Nelson Research), N,N-dimethylformamide, N-methyl-2-pyrrolidone,
calcium thioglycolate and other enhancers such as dioxolanes,
cyclic ketones, and their derivatives and so on.
[0132] Also illustrative are a group of biodegradable absorption
enhancers which are alkyl N,N-2-(disubstituted amino) alkanoates as
described in U.S. Pat. Nos. 4,980,378 and 5,082,866, which are both
incorporated herein by reference, including: tetradecyl
(N,N-dimethylamino) acetate, dodecyl (N,N-dimethylamino) acetate,
decyl (N,N-dimethylamino) acetate, octyl (N,N-dimethylamino)
acetate, and dodecyl (N,N-diethylamino) acetate.
[0133] Particularly preferred skin penetrating enhancers include
isopropyl myristate; isopropyl palmitate; dimethyl sulfoxide; decyl
methyl sulfoxide; dimethylalanine amide of a medium chain fatty
acid; dodecyl 2-(N,N-dimethylamino) propionate or salts thereof,
such as its organic (e.g., hydrochloric, hydrobromic, sulfuric,
phosphoric, and nitric acid addition salts) and inorganic salts
(e.g., acetic, benzoic, salicylic, glycolic, succinic, nicotinic,
tartaric, maleic, malic, pamoic, methanesulfonic,
cyclohexanesulfamic, picric, and lactic acid addition salts), as
described in U.S. Pat. No. 6,118,020; and alkyl
2-(N,N-disubstituted amino)-alkanoates, as described in U.S. Pat.
Nos. 4,980,378 and 5,082,866.
[0134] The skin penetrating enhancer in this composition by weight
would be in the range of 0.5% to 10% (w/w). The most preferred
range would be between 1.0% and 5% (w/w). In another embodiment,
the skin penetrating enhancer comprises between 0.5%-1%, 1%-2%,
2%-3%, 3%-4%, or 4%-5%, (w/w) of the composition.
[0135] The compositions can be provided in any useful form. For
example, the compositions of the invention may be formulated as
solutions, emulsions (including microemulsions), suspensions,
creams, foams, lotions, gels, powders, or other typical solid,
semi-solid, or liquid compositions (e.g., topical sprays) used for
application to the skin or other tissues where the compositions may
be used. Such compositions may contain other ingredients typically
used in such products, such as colorants, fragrances, thickeners
(e.g., xanthan gum, a fatty acid, a fatty acid salt or ester, a
fatty alcohol, a modified cellulose, a modified mineral material,
Krisgel 100.TM., or a synthetic polymer), antimicrobials, solvents,
surfactants, detergents, gelling agents, antioxidants, fillers,
dyestuffs, viscosity-controlling agents, preservatives, humectants,
emollients (e.g., natural or synthetic oils, hydrocarbon oils,
waxes, or silicones), hydration agents, chelating agents,
demulcents, solubilizing excipients, adjuvants, dispersants, skin
penetrating enhancers, plasticizing agents, preservatives,
stabilizers, demulsifiers, wetting agents, sunscreens, emulsifiers,
moisturizers, astringents, deodorants, and optionally including
anesthetics, anti-itch actives, botanical extracts, conditioning
agents, darkening or lightening agents, glitter, humectants, mica,
minerals, polyphenols, silicones or derivatives thereof, sunblocks,
vitamins, and phytomedicinals.
[0136] The compositions can also include other like ingredients to
provide additional benefits and improve the feel and/or appearance
of the topical formulation. Specific classes of additives commonly
use in these formulations include: isopropyl myristate, sorbic acid
NF powder, polyethylene glycol, phosphatidylcholine (including
mixtures of phosphatidylcholine, such as phospholipon G), Krisgel
100.TM. distilled water, sodium hydroxide, decyl methyl sulfoxide
(as a skin penetrating enhancer), menthol crystals, lavender oil,
butylated hydroxytoluene, ethyl diglycol reagent, and 95% percent
(190 proof) ethanol.
Formulations for Ophthalmic Administration
[0137] The compounds of the invention can also be formulated with
an ophthalmically acceptable carrier in sufficient concentration so
as to deliver an effective amount of the active compound or
compounds to the optic nerve site of the eye. Preferably, the
ophthalmic, therapeutic solutions contain one or more of the active
compounds in a concentration range of approximately 0.0001% to
approximately 1% (weight by volume) and more preferably
approximately 0.0005% to approximately 0.1% (weight by volume).
[0138] An ophthalmically acceptable carrier does not cause
significant irritation to the eye and does not abrogate the
pharmacological activity and properties of the charged sodium
channel blockers. Ophthalmically acceptable carriers are generally
sterile, essentially free of foreign particles, and generally have
a pH in the range of 5-8. Preferably, the pH is as close to the pH
of tear fluid (7.4) as possible. Ophthalmically acceptable carriers
are, for example, sterile isotonic solutions such as isotonic
sodium chloride or boric acid solutions. Such carriers are
typically aqueous solutions contain sodium chloride or boric acid.
Also useful are phosphate buffered saline (PBS) solutions.
[0139] Various preservatives may be used in the ophthalmic
preparation. Preferred preservatives include, but are not limited
to, benzalkonium potassium, chlorobutanol, thimerosal,
phenylmercuric acetate, and phenylmercuric nitrate. Likewise,
various preferred vehicles may be used in such ophthalmic
preparation. These vehicles include, but are not limited to,
polyvinyl alcohol, povidone, hydroxypropyl methyl cellulose,
poloxamers, carboxymethyl cellulose and hydroxyethyl cellulose.
[0140] Tonicity adjustors may be added as needed or convenient.
They include, but are not limited to, salts, particularly sodium
chloride, potassium chloride, etc., mannitol and glycerin, or any
other suitable ophthalmically acceptable tonicity adjustor.
[0141] Various buffers and means for adjusting pH may be used so
long as the resulting preparation is ophthalmically acceptable.
Accordingly, buffers include but are not limited to, acetate
buffers, citrate buffers, phosphate buffers, and borate buffers.
Acids or bases may be used to adjust the pH of these formulations
as needed. Ophthalmically acceptable antioxidants can also be
include. Antioxidants include but are not limited to sodium
metabisulfite, sodium thiosulfate, acetylcysteine, butylated
hydroxyanisole, and butylated hydroxytoluene.
Formulations for Nasal and Inhalation Administration
[0142] The pharmaceutical compositions of the invention can be
formulated for nasal or intranasal administration. Formulations
suitable for nasal administration, when the carrier is a solid,
include a coarse powder having a particle size, for example, in the
range of approximately 20 to 500 microns which is administered by
rapid inhalation through the nasal passage. When the carrier is a
liquid, for example, a nasal spray or as nasal drops, one or more
of the formulations can be admixed in an aqueous or oily solution,
and inhaled or sprayed into the nasal passage.
[0143] For administration by inhalation, the active ingredient can
be conveniently delivered in the form of an aerosol spray
presentation from pressurized packs or a nebulizer, with the use of
a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide
or other suitable gas. In the case of a pressurized aerosol the
dosage unit can be determined by providing a valve to deliver a
metered amount, Capsules and cartridges of, for example, gelatin
for use in an inhaler or insufflator can be formulated containing a
powder mix of the compound and a suitable powder base such as
lactose or starch.
Indications
[0144] The methods, compositions, and kits of the invention can be
used to treat pain or itch associated with any of a number of
conditions, including back and neck pain, cancer pain,
gynecological and labor pain, fibromyalgia, arthritis and other
rheumatological pains, orthopedic pains, post herpetic neuralgia
and other neuropathic pains, sickle cell crises, interstitial
cystitis, urethritis and other urological pains, dental pain,
headaches, postoperative pain, and procedural pain (i.e., pain
associated with injections, draining an abcess, surgery, dental
procedures, opthalmic procedures, ophthalmic irritation,
conjuctivitis (e.g., allergic conjunctivitis), eye redness, dry
eye, arthroscopies and use of other medical instrumentation,
cosmetic surgical procedures, dermatological procedures, setting
fractures, biopsies, and the like).
[0145] Since a subclass of nociceptors mediate itch sensation the
methods, compositions, and kits of the invention can also be used
to treat itch in patients with conditions like dermatitis,
infections, parasites, insect bites, pregnancy, metabolic
disorders, liver or renal failure, drug reactions, allergic
reactions, eczema, and cancer.
[0146] The methods, compositions, and kits of the invention can
also be used for the treatment of pyrexia (fever), hyperpyrexia,
malignant hyperthermia, or a condition characterized by elevated
body temperature.
[0147] The methods, compositions, and kits of the invention can
also be used to treat neurogenic inflammation and neurogenic
inflammatory disorders. Inflammation is a complex set of responses
to harmful stimuli that results in localized redness, swelling, and
pain. Inflammation can be innate or adaptive, the latter driven by
antigens and is mediated by immune cells (immune-mediated
inflammation). Neurogenic inflammation results from the efferent
functions of pain-sensing neurons (nociceptors), wherein
neuropeptides and other chemicals that are pro-inflammatory
mediators are released from the peripheral terminals of the
nociceptors when they are activated. This release process is
mediated by calcium influx and exocytosis of peptide containing
vesicles, and the pro-inflammatory neuropeptides include substance
P, neurokinin A and B (collectively known as tachykinins),
calcitonin gene-related peptide (CGRP), and vasoactive intestinal
polypeptide (VIP).
[0148] The release of peripheral terminal chemicals stimulate a
variety of inflammatory responses. First, the release of substance
P can result in an increase in capillary permeability such that
plasma proteins leak from the intravascular compartment into the
extracellular space (plasma extravasation), causing edema. This can
be detected as a wheal (a firm, elevated swelling of the skin)
which is one component of a triad of inflammatory responses-wheal,
red spot, and flare-known as the Lewis triple response. Second, the
release of CGRP causes vasodilation, leading to increased blood
flow. This can be detected as a flare, which is another component
of the Lewis triple response.
[0149] Substance P also has a pro-inflammatory action on immune
cells (e.g. macrophages, T-cells, mast cells, and dendritic cells)
via their neurokinin-1 (NK1) receptor. This effect has been
documented in allergic rhinitis, gastitis, and colitis, and
represents an interface between the neurogenic and immune-mediated
components of inflammation. Substance P released from one
nociceptor may also act on NK1 receptors on neighboring nociceptors
to sensitize or activate them, causing a spread of activation and
afferent/efferent function. These efferent functions of nociceptors
can be triggered by: 1) Direct activation of a nociceptor terminal
by a peripheral adequate stimulus applied to the terminal (e.g. a
pinch); 2) Indirect antidromic activation of a non-stimulated
nociceptor terminal by the axon reflex, wherein action potential
input from one terminal of a nociceptor, upon reaching a converging
axonal branch point in the periphery, results in an action
potential traveling from the branch point down to the peripheral
terminal of a non-stimulated terminal; and 3) Activation as a
result of activity in nociceptor central terminals in the CNS
traveling to the periphery (e.g., primary afferent depolarization
of central terminals produced by GABA can be sufficient to initiate
action potentials traveling the "wrong way").
[0150] Genomic analysis of lung resident ILC2 cells has revealed
expression of receptors for several neuropeptides released by
sensory neurons, including SP, CGRP and VIP, providing an
opportunity for nociceptors to directly communicate with these
cells. In particular, VIP is found to be expressed in NaV1.8+
nodose ganglion neurons, including lung afferents in OVA-exposed
mice. Cultured nodose ganglion neurons stimulated with capsaicin or
IL5 also released VIP while BALF from OVA-exposed mice contained
elevated VIP compared to vehicle-challenged mice (Talbot et al.,
Neuron 2015, in press). These data indicate that VIP is released in
the inflamed lung and can be blocked by silencing neurons with
charged sodium channel blockers of the present invention. In
addition, when CD4+ T cells cultured under T.sub.H2 skewing
conditions were exposed to recombinant mouse VIP, the transcript
levels of IL-13 and IL-5 increased, suggesting that VIP contributes
to the competence of T.sub.H2 cells to transcribe these type II
regulatory cytokines.
[0151] Immune mediator release from immune cells can also activate
nociceptors. Mast cells are found close to primary nociceptive
neurons and contribute to nociceptor sensitization in a number of
contexts. Injection of the secretagogue compound 48/80 promotes
degranulation of mast cells in the dura and leads to excitation of
meningeal nociceptors. Mast cell degranulation also contributes to
the rapid onset of nerve growth factor-induced thermal
hyperalgesia. Macrophages contribute to nociceptor sensitization by
releasing several soluble mediators. Expression of the chemokine
macrophage inflammatory protein-1.alpha. (MIP-1.alpha.) and its
receptors CCR1 and CCR5 is increased in macrophages and Schwann
cells after partial ligation of the sciatic nerve and contributes
to the development of neuropathic pain. Lymphocytes contribute to
the sensitization of peripheral nociceptors. T cells infiltrate the
sciatic nerve and dorsal root ganglion (DRG) after nerve injury.
Hyperalgesia and allodynia induced by nerve injury are markedly
attenuated or abrogated in rodents lacking T cells and the
immunosuppressant rapamycin attenuates neuropathic pain in rats,
partly owing to an effect on T cells. Among the subsets of T cells,
type 1 and 2 helper T cells (T.sub.H1 and T.sub.H2 cells) have been
shown to have different roles in neuropathic pain. T.sub.H1 cells
facilitate neuropathic pain behavior by releasing proinflammatory
cytokines (IL-2 and interferon-.gamma. (IFN.gamma.)), whereas
T.sub.H2 cells inhibit it by releasing anti-inflammatory cytokines
(IL-4, IL-10 and IL-13). The complement system also has a role in
inflammatory hyperalgesia and neuropathic pain. C5a, an
anaphylatoxin, is an important effector of the complement cascade
and upon binding to C5aR1 receptors on neutrophils it becomes a
potent neutrophil attractant (Ren & Dubner, Nat. Med.
16:1267-1276 (2010)).
[0152] Bacterial infections have been shown to directly activate
nociceptors, and that the immune response mediated through TLR2,
MyD88, T cells, B cells, and neutrophils and monocytes is not
necessary for Staphylococcus aureus-induced pain in mice (Chiu et
al., Nature 501:52-57 (2013)). Mechanical and thermal hyperalgesia
in mice is correlated with live bacterial load rather than tissue
swelling or immune activation. Bacteria induce calcium flux and
action potentials in nociceptor neurons, in part via bacterial
N-formylated peptides and the pore-forming toxin
.alpha.-haemolysin, through distinct mechanisms. Specific ablation
of Nav1.8-lineage neurons, which include nociceptors, abrogated
pain during bacterial infection, but concurrently increased local
immune infiltration and lymphadenopathy of the draining lymph node.
Thus, bacterial pathogens produce pain by directly activating
sensory neurons that modulate inflammation, an unsuspected role for
the nervous system in host-pathogen interactions. Data from Talbot
et al., Neuron 2015, in press have also suggested that nociceptors
are activated during exposure to allergens in sensitized
animals.
[0153] In certain disorders, neurogenic inflammation contributes to
the peripheral inflammation elicited by tissue injury, autoimmune
disease, infection, and exposure to irritants in soft tissue, skin,
the respiratory system, joints, the urogenital and GI tract, the
liver, and the brain. Neurogenic inflammatory disorders include
asthma, rhinitis, conjunctivitis, arthritis, colitis, contact
dermatitis, diabetes, eczema, cystitis, gastritis, migraine
headache, psoriasis, rhinitis, rosacea, and sunburn. pancreatitis,
chronic cough, chronic rhinosinusistis, traumatic brain injury,
polymicrobial sepsis, tendinopathies chronic urticaria, rheumatic
disease, acute lung injury, exposure to irritants, inhalation of
irritants, pollutants, or chemical warfare agents, as described
herein.
Assessment of Pain, Itch, and Neurogenic Inflammation
[0154] In order to measure the efficacy of any of the methods,
compositions, or kits of the invention, a measurement index may be
used. Indices that are useful in the methods, compositions, and
kits of the invention for the measurement of pain associated with
musculoskeletal, immunoinflammatory and neuropathic disorders
include a visual analog scale (VAS), a Likert scale, categorical
pain scales, descriptors, the Lequesne index, the WOMAC index, and
the AUSCAN index, each of which is well known in the art. Such
indices may be used to measure pain, itch, function, stiffness, or
other variables.
[0155] A visual analog scale (VAS) provides a measure of a
one-dimensional quantity. A VAS generally utilizes a representation
of distance, such as a picture of a line with hash marks drawn at
regular distance intervals, e.g., ten 1-cm intervals. For example,
a patient can be asked to rank a sensation of pain or itch by
choosing the spot on the line that best corresponds to the
sensation of pain or itch, where one end of the line corresponds to
"no pain" (score of 0 cm) or "no itch" and the other end of the
line corresponds to "unbearable pain" or "unbearable itch" (score
of 10 cm). This procedure provides a simple and rapid approach to
obtaining quantitative information about how the patient is
experiencing pain or itch. VAS scales and their use are described,
e.g., in U.S. Pat. Nos. 6,709,406 and 6,432,937.
[0156] A Likert scale similarly provides a measure of a
one-dimensional quantity. Generally, a Likert scale has discrete
integer values ranging from a low value (e.g., 0, meaning no pain)
to a high value (e.g., 7, meaning extreme pain). A patient
experiencing pain is asked to choose a number between the low value
and the high value to represent the degree of pain experienced.
Likert scales and their use are described, e.g., in U.S. Pat. Nos.
6,623,040 and 6,766,319.
[0157] The Lequesne index and the Western Ontario and McMaster
Universities (WOMAC) osteoarthritis index assess pain, function,
and stiffness in the knee and hip of OA patients using
self-administered questionnaires. Both knee and hip are encompassed
by the WOMAC, whereas there is one Lequesne questionnaire for the
knee and a separate one for the hip. These questionnaires are
useful because they contain more information content in comparison
with VAS or Likert. Both the WOMAC index and the Lequesne index
questionnaires have been extensively validated in OA, including in
surgical settings (e.g., knee and hip arthroplasty). Their metric
characteristics do not differ significantly.
[0158] The AUSCAN (Australian-Canadian hand arthritis) index
employs a valid, reliable, and responsive patient self-reported
questionnaire. In one instance, this questionnaire contains 15
questions within three dimensions (Pain, 5 questions; Stiffness, 1
question; and Physical function, 9 questions). An AUSCAN index may
utilize, e.g., a Likert or a VAS scale.
[0159] Indices that are useful in the methods, compositions, and
kits of the invention for the measurement of pain include the Pain
Descriptor Scale (PDS), the Visual Analog Scale (VAS), the Verbal
Descriptor Scales (VDS), the Numeric Pain Intensity Scale (NPIS),
the Neuropathic Pain Scale (NPS), the Neuropathic Pain Symptom
Inventory (NPSI), the Present Pain Inventory (PPI), the Geriatric
Pain Measure (GPM), the McGill Pain Questionnaire (MPQ), mean pain
intensity (Descriptor Differential Scale), numeric pain scale (NPS)
global evaluation score (GES) the Short-Form McGill Pain
Questionnaire, the Minnesota Multiphasic Personality Inventory, the
Pain Profile and Multidimensional Pain Inventory, the Child Heath
Questionnaire, and the Child Assessment Questionnaire.
[0160] Itch can be measured by subjective measures (VAS, Lickert,
descriptors). Another approach is to measure scratch which is an
objective correlate of itch using a vibration transducer or
movement-sensitive meters.
[0161] The following examples are intended to illustrate the
invention, and is not intended to limit it.
Examples
Experimental Methods
In Vitro Electrophysiology
[0162] Whole-cell recordings were made of currents carried by
voltage-activated channels in HEK293 cells stably expressing human
Nav1.7 channels. Recordings were made using patch pipettes with
resistances of 2-3.5 MQ when filled with internal solution,
consisting of 61 mM CsF, 61 mM CsCl, 9 mM NaCl, 1.8 mM MgCl.sub.2,
9 mM EGTA, 14 mM creatine phosphate (tris salt), 4 mM MgATP, and
0.3 mM GTP (tris salt), 9 mM HEPES, pH adjusted to 7.2 with CsOH.
The shank of the electrode was wrapped with Parafilm in order to
reduce capacitance and allow optimal series resistance compensation
without oscillation. Seals were obtained and the whole-cell
configuration established with cells in Tyrode's solution (155 mM
NaCl, 3.5 mM KCl, 1.5 mM CaCl.sub.2), 1 mM MgCl.sub.2, 10 mM HEPES,
10 mM glucose, pH adjusted to 7.4 with NaOH) with 10 mM TEACl. To
ensure complete dialysis with pipette solution, recordings began
after 5 to 10 min after establishment of the whole-cell
configuration.
[0163] Currents were recorded at room temperature (21-23.degree.
C.) with an Axopatch 200 amplifier and filtered at 5 kHz with a
low-pass Bessel filter. The amplifier was tuned for partial
compensation of series resistance (typically 70-80% of a total
series resistance of 4-10 MO). Currents were digitized using a
Digidata 1322A data acquisition interface controlled by pClamp9.2
software (Axon Instruments) and analyzed using programs written in
Igor Pro 4.0 (Wavemetrics, Lake Oswego, Oreg.). Currents were
corrected for linear capacitative and leak currents determined
using 5 mV hyperpolarizations delivered from the resting potential
(usually -80 or -100 mV) and then appropriately scaled and
subtracted.
[0164] Sodium currents were evoked by 30-msec depolarizations from
-100 mV to -20 mV. To assay use-dependent block, pulses were
delivered at series of increasing rates: 0.05 Hz for 1-min, 0.33 Hz
for 1-min, 1 Hz for 1-min, 3 Hz for 1-min, 5 Hz for 30 seconds, 10
Hz for 30 seconds, with 1 minute rest between each series of
pulses. After the series of pulses to induce use-dependent block,
the time course of recovery was followed using pulses delivered at
01. Hz (2-min) and 0.05 Hz (1-min).
Animals
[0165] All procedures were approved by the Institutional Animal
Care and Use Committees. Mice and rats were housed in standard
environmental conditions (12-h light/dark cycle; 23.degree. C.;
food and water ad libitum) at facilities accredited by the
Association for Assessment and Accreditation of Laboratory Animal
Care. 8-week old male BALB/c (stock number: 001026) and C57BL/6J
(stock number 000664) mice were purchased from Jackson Laboratory
and respectively used in allergic airway inflammation (FIG. 5) and
pain hypersensitivity model (FIGS. 2A-2D). 6-week old male
Sprague-Dawley (stock number: 400) rats, 150.+-.25 grams, were
purchased from Charles River laboratory and used to assess pain
hypersensitivity (FIG. 3).
Immunofluorescence
[0166] Upon harvesting, the dorsal root ganglia were fixed
overnight in 4% para-formaldehyde, washed in PBS and cryoprotected
by sequential sucrose immersion (PBS 10-30% sucrose, Overnight).
Ganglia were mounted in O.C.T. (Tissue-tek), and serially cut in 20
.mu.m coronal sections with a cryostat. The sections were
thaw-mounted on Fisherbrand superfrost microscopy slides and kept
at -80.degree. C. On the day of the experiment, sections were
thawed at room temperature for 10 min. Sections were washed in PBS
for 5 min, blocked for 1 h at room temperature (PBS, 0.1% Triton
X-100, 5% BSA) and exposed to the primary antibodies (Overnight,
4.degree. C.), namely rabbit anti-mouse ATF3 (Sigma, # HPA001562).
Sections were then washed three times in PBS (5 min), exposed to
the secondary antibodies and DAPI (1:2000. Sigma, # D9542) (2 h,
dark), washed, coverslipped with Vectashield (Vector Labs) and
observed under confocal microscope (Leica, LSM-710).
Ovalbumin Sensitization and Airway Challenge
[0167] Allergic airway inflammation was studied in an ovalbumin
(OVA) based model. On day 0 and 7, mice were sensitized by a 200
.mu.l i.p. injections of a solution containing 1 mg/ml ovalbumin
(Sigma-Aldrich) and 5 mg/ml aluminum hydroxide (Sigma-Aldrich,
Boston, Ma). On day 14-17 (10:00 am) mice were exposed to 6% OVA
aerosol for 25 min to induce airways allergic inflammation. Drugs
were nebulized on day 18 (10:00 am) and inflammation (BALF cell
content)/airway hyper-responsiveness were assessed on day 21 (10:00
am).
Bronchoalveolar Lavage (BAL)
[0168] On day 21, mice were anesthetized following an
intraperitoneal injection of urethane (dose) and a 20 G sterile
catheter inserted longitudinally into the trachea. 2 ml of ice cold
PBS containing protease inhibitors (Roche) was injected into the
lung, harvested and stored on ice. BAL fluid underwent a 400 g
centrifugation (15 min; 4.degree. C.), the supernatant was
discarded and cells re-suspended in 200 .mu.l.
Airway Inflammatory and Differential Cell Count
[0169] Bronchoalveolar lavage fluid (BALF) cells were re-suspended
in FACS buffer (PBS, 2% FCS, EDTA), and incubated with Fc block
(0.5 mg/ml, 10 min; BD Biosciences). Cells were then stained with
monoclonal antibodies (FITC anti-mouse CD45, BD Biosciences, cat
no: 553079, PE anti-mouse Syglec-F, BD Biosciences, cat no: 552126;
APC anti-mouse GR-1, eBiosciences, cat no: 17-5931-81; PE-Cy7
anti-mouse CD3e, cat no: 25-0031-81; PerCP anti-mouse F4/80,
BioLegend, cat no: 123125; PE anti-mouse, BD Bioscience, cat no:
552126; 45 min, 4.degree. C. on ice) before data acquisition on a
FACS Canto II (BD Biosciences). A leukocyte differential count was
determined during flow cytometry analysis of cells expressing the
common leukocyte antigen CD45 (BD Pharmigen; cat no: 553079).
Specific cell populations were identified as follows: macrophages
as F4/80Hi-Ly6gNeg, eosinopohils as F4/80Int-Ly6gLo-SiglecFHi,
neutrophils as F4/80Lo-Ly6gHi-SiglecFNeg, and lymphocytes as
F4/80Neg-Ly6gNeg-CD3Pos. Total BAL cell counts were performed using
a standard hemocytometer, with absolute cell numbers calculated as
total BAL cell number multiplied by the percentage of cell
subpopulation as determined by FACS.
Thermal Hypersensitivity
[0170] The compounds were injected in the plantar surface of each
animal's right hind paw in 10 .mu.l (mice) or 50 .mu.l (rats)
volume of 1:1 emulsion of Complete Freund's Adjuvant (CFA). Thermal
hyperalgesia was examined by measuring the latency to withdrawal of
the hind paws from a focused beam of radiant heat applied to the
plantar surface using a Ugo Basile Plantar Test (Hargreaves)
apparatus. For 3 consecutive days, 8 weeks old Sprague Dawley male
rat will be habituated for 60 min on the Hargreaves apparatus. On
the 4th day, their thermal nociceptive threshold was evaluated. 1 h
later, the rats were lightly anesthetized under isoflurane (3%
induction, 2% maintenance) and received an intraplantar injection
of CFA in the presence or not of the test compounds. Impact of
treatment was analyzed 1 h prior and 1, 3, 6 and 24 h
post-treatment. Three trials were performed on each paw, by
alternating the starting paw, with an interval of 5 minutes. A
positive pain reaction was defined as sudden paw withdrawal,
flinching, and/or paw licking. With each reading the apparatus was
set with a cut-off time of 32 s.
Example 1. Synthesis of
1-(1-(2,6-dimethylphenylamino)-1-oxopropan-2-yl)-1-methylpyrrolidinium
Chloride (Compound 1)
Synthetic Scheme
##STR00041##
[0171] Step 1: Preparation of 2
##STR00042##
[0173] The solution of 1 (5.0 g, 32.86 mmol, 1.0 eq) in 30 mL
SOCl.sub.2 was refluxed at 8.degree. C. for 2 h. After competition,
the reaction mixture was directly concentrated in vacuum to give a
residue without further purification (7.8 g, y=120%).
Step 2: Preparation of 3
##STR00043##
[0175] To a solution of 2, 6-dimethylaniline (4.6 g, 38.2 mmol, 1.0
eq) in DCM (10 mL) and TEA (4.6 g, 45.8 mmol, 1.2 eq) was added 2
(7.8 g, 45.8 mmol, 1.2 eq) in DCM (20 mL) slowly at ice bath. Then
the reaction mixture was warmed to R.T. for 2 h. After competition,
the reaction mixture was adjusted to pH=5-6 with 2N HCl, extracted
with EA (150 mL.times.2). The combined organic phases was washed
with brine (100 mL), dried over Na.sub.2SO.sub.4, filtered and
concentrated in vacuum to give a residue. The residue was purified
by column chromatography to give the desired product (4.8 g, 50%
yield) as gray solid.
Step 3: Preparation of 4
##STR00044##
[0177] To a solution of 3 (2 g, 7.68 mmol, 1.0 eq) in Toluene (15
mL, c=0.5) was added pyrrolidine (1.17 g, 16.5 mmol, 2.0 eq). The
reaction mixture was refluxed at 110.degree. C. for 30 min. After
competition, the reaction mixture was directly concentrated in
vacuum to give a residue. The residue was purified by column
chromatography to give the desired product (1.8 g, 95% yield) as
yellow oil.
Step 4: Preparation of 5
##STR00045##
[0179] To a solution of 4 (500 mg, 2.03 mmol, 1.0 eq) in DCM (20
mL, c=0.1) was added MeI (721 mg, 5.08 mmol, 2.5 eq) at R.T.
overnight. The reaction mixture was directly concentrated in vacuum
to give a residue. The residue was washed with EA to give desired
product (233 mg, 30% yield).
Step 5: Preparation of Compound 1
##STR00046##
[0181] To a solution of 5 (233 mg, 0.602 mmol, 1.0 eq) in H.sub.2O
(1.6 mL, c=0.37) was added AgCl (172 mg, 2.0 eq). The reaction
mixture was heated at 60.degree. C. overnight. After competition,
the reaction mixture was filtered. The filtrate was lyophilized to
give desired product. (62 mg, 35% yield) as white solid. .sup.1H
NMR (300 MHz, DMSO): .delta.7.18-7.09 (m, 3H), 4.31 (q, J=7.2 Hz,
1H), 3.65-3.57 (m, 4H), 3.16 (s, 3H), 2.15-2.25 (m, 4H), 2.08 (s,
6H), 1.74 (d, J=6.9 Hz, 3H) ppm. HPLC purity: 100% at 220 nm; Mass:
m/z=261.5 (M+1, ESI+).
Example 2. Synthesis of
1-(1-(2,6-dimethylphenylamino)-2-methyl-1-oxopropan-2-yl)-1-methylpyrroli-
dinium Chloride (Compound 2)
Synthetic Scheme
##STR00047##
[0182] Step 1: Preparation of 2
##STR00048##
[0184] The solution of 1 (10.0 g, 59.88 mmol, 1.0 eq) in 60 mL
SOCl.sub.2 was refluxed at 80.degree. C. for 2 h. After
competition, the reaction mixture was directly concentrated in
vacuum to give a residue without further purification (15.0 g,
y=132%).
Step 2: Preparation of 3
##STR00049##
[0186] To a solution of 2, 6-dimethylaniline (8.16 g, 67.38 mmol,
1.0 eq) in DCM (50 mL) and TEA (8.18 g, 80.86 mmol, 1.2 eq) was
added 2 (15.0 g, 80.86 mmol, 1.2 eq) in DCM (50 mL) slowly at ice
bath. Then the reaction mixture was warmed to R.T. for 2 h. After
competition, the reaction mixture was adjusted to pH=5-6 with 2N
HCl, extracted with EA (200 mL.times.2). The combined organic
phases was washed with brine (150 mL), dried over Na.sub.2SO.sub.4,
filtered and concentrated in vacuum to give a residue. The residue
was purified by column chromatography to give the desired product
(14.8 g, 82% yield) as solid.
Step 3: Preparation of 4
##STR00050##
[0188] To a solution of 3 (1.7 g, 6.32 mmol, 1.0 eq) in THE (32 mL,
c=0.2) was added pyrrolidine (539 mg, 7.58 mmol, 1.2 eq) and NaH
(303 mg, 12.64 mmol, 2.0 eq) in THE (32 mL, c=0.2). The reaction
mixture was stirred at R.T. for 30 min. After competition, 30 mL
H.sub.2O was added slowly, extracted with EA (50 mL.times.2). The
combined organic phases was washed with brine (30 mL), dried over
Na.sub.2SO.sub.4, filtered and concentrated in vacuum to give a
residue. The residue was purified by column chromatography to give
the desired product (680 g, 68% yield) as white solid.
Step 4: Preparation of 5
##STR00051##
[0190] To a solution of 4 (150 mg, 0.577 mmol, 1.0 eq) in MeCN (5.7
mL, c=0.1) was added MeI (245 mg, 1.73 mmol, 3.0 eq). The reaction
mixture was refluxed overnight. After competition, the reaction
mixture was directly concentrated in vacuum to give a residue. The
residue was washed with EA to give desired product (170 mg, 73%
yield) as white solid
Step 5: Preparation of 6
##STR00052##
[0192] To a solution of 5 (170 mg, 0.423 mmol, 1.0 eq) in H.sub.2O
(1.4 mL, c=0.3) was added AgCl (121 mg, 2.0 eq). The reaction
mixture was heated at 60.degree. C. overnight. After competition,
the reaction mixture was filtered. The filtrate was lyophilized to
give desired product. (82 mg, 63% yield) as white solid. .sup.1H
NMR (300 MHz, DMSO): .delta.7.15-7.09 (m, 3H), 3.95-3.85 (m, 2H),
3.53-3.49 (m, 2H), 3.11 (s, 3H), 2.11-2.10 (m, 4H), 2.08 (s, 6H),
1.85 (s, 6H) ppm. HPLC purity: 95.6% at 220 nm; Mass: m/z=261.5
(M+1, ESI+).
Example 3. Synthesis of 1-(1-(2,
6-dimethylphenylamino)-1-oxobutan-2-yl)-1-ethylpyrrolidinium
Chloride (Compound 3)
Synthetic Scheme
##STR00053##
[0193] Step 1: Preparation of 2
##STR00054##
[0195] The solution of 1 (10.0 g, 60.2 mmol, 1.0 eq) in 60 mL
SOCl.sub.2 was refluxed at 80.degree. C. for 2 h. After
competition, the reaction mixture was directly concentrated in
vacuum to give a residue without further purification (15.0 g,
y=132%).
Step 2: Preparation of 3
##STR00055##
[0197] To a solution of 2, 6-dimethylaniline (8.2 g, 67.97 mmol,
1.0 eq) in DCM (200 mL) and TEA (8.25 g, 81.56 mmol, 1.2 eq) was
added 2 (15.0 g, 80.86 mmol, 1.2 eq) in DCM (50 mL) slowly at ice
bath. Then the reaction mixture was warmed to R.T. for 2 h. After
competition, the reaction mixture was adjusted pH 5-6 with 2N HCl,
extracted with EA (200 mL.times.2). The combined organic phases was
washed with brine (150 mL), dried over Na.sub.2SO.sub.4, filtered
and concentrated in vacuum to give a residue. The residue was
purified by column chromatography to give the desired product (3.0
g, 17% yield) as solid.
Step 3: Preparation of 4
##STR00056##
[0199] To a solution of 3 (3.0 g, 11.15 mmol, 1.0 eq) in Toluene
(22 mL, c=0.5) was added pyrrolidine (1.66 g, 23.42 mmol, 1.2 eq).
The reaction mixture was refluxed at 110.degree. C. for 3 h. After
competition, the reaction mixture was directly concentrated in
vacuum to give a residue. The residue was purified by column
chromatography to give the desired product (1.2 g, 41% yield) as
white solid.
Step 4: Preparation of 5
##STR00057##
[0201] To a solution of 4 (500 mg, 1.921 mmol, 1.0 eq) in MeCN (20
mL, c=0.1) was added EtI (749 mg, 4.802 mmol, 2.5 eq). The reaction
mixture was refluxed overnight. After competition, the reaction
mixture was directly concentrated in vacuum to give a residue. The
residue was washed with EA to give desired product (423 mg, 50%
yield) as white solid.
Step 5: Preparation of 6
##STR00058##
[0203] To a solution of 5 (423 mg, 1.016 mmol, 1.0 eq) in H.sub.2O
(4 mL, c=0.3) was added AgCl (291 mg, 2.033 mmol, 2.0 eq). The
reaction mixture was heated at 60.degree. C. overnight. After
competition, the reaction mixture was filtered. The filtrate was
lyophilized to give desired product. (83 mg, 63% yield) as white
solid. .sup.1H NMR (300 MHz, DMSO): .delta.10.30 (br, 1H),
7.26-7.19 (m, 3H), 4.23 (q, J=6.15, 1H), 3.95-3.80 (d, J=3.12, 2H),
3.70-3.55 (m, 4H), 3.22 (s, 6H), 2.15-2.11 (m, 6H),
1.47.about.1.44(d, J=4.23, 3H), 1.22-1.19 (d, J=4.40, 3H) ppm. HPLC
purity: 98% at 220 nm; Mass: m/z=289.5 (M+1, ESI+).
Example 4. Synthesis of
1-(1-(2,6-dimethylphenylamino)-1-oxopropan-2-yl)-1-methylpiperidinium
Chloride (Compound 4)
Synthetic Scheme
##STR00059##
[0204] Step 1: Preparation of 3
##STR00060##
[0206] To a solution of 1 (0.5 g, 1.95 mmol, 1.0 eq) in Toluene (10
ml, c=0.2) was added 2 (0.35 g, 4.1 mmol, 2.1 eq). After addition,
the mixture was heated to reflux. After completion, the suspension
was filtered and the filtrate was concentrated under reduce
pressure. The residue was purified by column chromatography to give
the desired product (0.4 g, yield=78.9%) as a solid.
Step 2: Preparation of 4
##STR00061##
[0208] To a solution of 3 (162 mg, 0.62 mmol, 1.0 eq) in MeCN (6
mL, c=0.1) was added MeI (220 mg, 2.5 eq). After addition, the
reaction mixture was heated to reflux for 5 h. After completion,
the reaction solution was concentrated under reduce pressure to
give the product (184 mg, yield=73.8%, HPLC: 93%) as a solid.
Step 3: Preparation of 5
##STR00062##
[0210] To a solution of 4 (122.7 mg, 0.3 mmol, 1.0 eq) in deionized
water (2 ml, c=0.15) was added AgCl (86 mg, 2.0 eq). After
addition, the reaction mixture was stirred at 60.degree. C.
overnight. After completion, the suspension was filtered and the
filtrate was used lyophilization to give the product (89.6 mg,
yield=96.1%) as a solid. HPLC purity: 95% at 220 nm; Mass: M+:
M-35.5=275.5; 2M-35.5=585.8; M-: M=310.5; M+35.5=345.5. .sup.1H NMR
(500 MHz, D.sub.2O): .delta. 7.1400-7.1981 (m; 3H), 4.4345 (s; 1H),
3.6625 (s; 1H), 3.5410 (s; 1H), 3.4610 (s; 2H), 3.2348 (s; 3H),
2.1289 (s; 6H), 1.9253 (d, J=28.3 Hz, 4H), 1.7403 (t, 4H), 1.6225
(s; 1H). ppm.
Example 5. Synthesis of
1-(1-(2,6-dimethylphenylamino)-2-methyl-1-oxopropan-2-yl)-1-methylpiperid-
inium Chloride (Compound 5)
Synthetic Scheme
##STR00063##
[0211] Step 1: Preparation of 2
##STR00064##
[0213] To a mixture of 1 (10.0 g, 59.88 mmol) was added SOCl.sub.2
(60 ml, c=1.0). The mixture was heated to reflux. After completion,
the reaction mixture was concentrated under reduce pressure to give
the desired product (14.8 g, yield=128.8%) as a yellow oil.
Step 2: Preparation of 4
##STR00065##
[0215] To a mixture of 3 (6.0 g, 50 mmol, 1.0 eq) in DCM (100 ml,
c=0.5) was added TEA (10.1 g, 100 mmol, 2 eq). Then the solution
was added 2 (14.3 g, 77.1 mmol, 1.5 eq) in DCM (50 ml, c=1). The
reaction mixture was stirred at RT. over night. Then the mixture
was added water (60 ml) to stratify. The organic phases was washed
with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated
under reduce pressure. The residue was purified by column
chromatography and the cross sample was washed with n-hexane.
Combine the solid to give the product (5.7 g, yield=42.2%,
HPLC:99.5%).
Step 3: Preparation of 6
##STR00066##
[0217] To a solution of 4 (5.3 g, 19.6 mmol, 1.0 eq) and
K.sub.2CO.sub.3 (2.7 g, 19.6 mmol, 1.0 eq) in MeCN (98 ml, c=0.2)
was added 5 (2.5 g, 29.4 mmol, 1.5 eq). After addition, the mixture
was heated to reflux. After completion, the suspension was filtered
and the filtrate was concentrated under reduce pressure. The
residue was purified by column chromatography to give the desired
product (1.7 g, yield=31.6%, HPLC: 96.4%) as a solid.
Step 4: Preparation of 7
##STR00067##
[0219] 6 (1.6 g, 5.8 mmol, 1.0 eq) and MeCN (30 mL, c=0.2) was
added in sealed tube. To this solution, MeI (5 mL, 14.0 eq) was
added. After addition, the reaction mixture was stirred at
90.degree. C. for 32 h. After completion, the reaction solution was
concentrated under reduce pressure. The residue was purified by
column chromatography and washed with EA (5 ml.times.2) to give the
product (557 mg, yield=23%, HPLC: 99.6%) as a solid.
Step 4: Preparation of 8
##STR00068##
[0221] To a solution of 7 (200 mg, 0.48 mmol, 1.0 eq) in deionized
water (4 ml, c=0.12) was added AgCl (137.8 mg, 2.0 eq). After
addition, the reaction mixture was stirred at RT for 6 h. After
completion, the suspension was filtered and the filtrate was used
lyophilization to give the product (152 mg, yield=97%) as a solid.
HPLC purity: 100% at 220 nm; Mass: M+:M-35.5=289.5; 2M-35.5=613.8;
.sup.1H NMR (500 MHz, D.sub.2O): .delta. 7.0842-7.1578 (m, 3H),
3.5323 (d, J=7.85 Hz, 4H), 3.1250 (s, 3H), 2.0620 (s, 6H), 1.9314
(m, 2H), 1.7323-1.8119 (m, 9H), 1.32771.3543 (m, 2H) ppm.
Example 6. Synthesis of 1-(1-(2,
6-dimethylphenylamino)-1-oxobutan-2-yl)-1-ethylpiperidinium
(Compound 6)
Synthetic Scheme
##STR00069##
[0222] Step 1: Preparation of 2
##STR00070##
[0224] To a mixture of 1 (10.0 g, 59.88 mmol) was added SOCl.sub.2
(60 mL, c=1.0). The mixture was heated to reflux. After completion,
the reaction mixture was concentrated under reduce pressure to give
the desired product (9.2 g, yield=82.8%) as a yellow oil.
Step 2: Preparation of 4
##STR00071##
[0226] To a mixture of 3 (5.0 g, 41.3 mmol, 1.0 eq) in DCM (100 ml,
c=0.5) was added pyridine (4.9 g, 61.95 mmol, 1.5 eq). Then the
solution was added 2 (9.2 g, 49.59 mmol, 1.2 eq) in DCM (40 ml,
c=1.2). The reaction mixture was stirred at room temperature
overnight. Then the solution was added water (50 ml) to stratify.
The organic phase was washed with brine, dried over
Na.sub.2SO.sub.4, filtered and concentrated under reduce pressure.
The residue was washed with n-hexane. Combine the solid to give the
product (7.8 g, yield=70%, HPLC: 98.6%).
Step 3: Preparation of 6
##STR00072##
[0228] To a solution of NaH (0.35 g, 14.8 mmol, 2.0 eq) in THE (37
mL, c=0.4) was added 5 (0.75 g, 8.8 mmol, 1.2 eq). Then the
solution was added 4 (2.0 g, 7.4 mmol, 1.0 eq) in THE (20 mL,
c=0.37). After addition, the mixture was stirred at room
temperature overnight. After completion, the suspension was added
water (20 mL) and EA (50 mL) to stratify. The organic phases were
washed with water (50 mL.times.2). Then the organic phase was
adjusted pH to 2, extracted with EA (40 mL.times.2). The water
phases were adjusted pH to 9, then extracted with EA (40.times.2).
The combined organic phases was washed with brine, dried over
Na.sub.2SO.sub.4, filtered and concentrated under reduce pressure.
The residue was washed with n-hexane to give the desired product
(0.48 g, yield=24%, HPLC: 99.3%) as a solid.
Step 4: Preparation of 7
##STR00073##
[0230] 6 (0.48 g, 1.75 mmol, 1.0 eq) and MeCN (9 mL, c=0.2) was
added in sealed tube. To this solution, EtI (2 mL, 14.0 eq) was
added. After addition, the reaction mixture was stirred at
90.degree. C. for 10 h. After completion, the reaction solution was
concentrated under reduce pressure. The residue was purified by
column chromatography to give the product (470 mg, yield=62.6%,
HPLC: 99%) as a solid.
Step 4: Preparation of 8
##STR00074##
[0232] To a solution of 7 (200 mg, 0.465 mmol, 1.0 eq) in deionized
water (3 ml, c=0.15) was added AgCl (133 mg, 0.93 mmol, 2.0 eq).
After addition, the reaction mixture was stirred at room
temperature overnight. After completion, the suspension was
filtered and the filtrate was used lyophilization to give the
product (141 mg, yield=89.8%) as a solid. HPLC purity: at 220 nm;
Mass: M+1=339.4. .sup.1H NMR (300 MHz, D.sub.2O): .delta. 7.117 (m,
3H), 4.056 (dd, J=8.1 Hz, 1H), 3.712-3.808 (m, 1H), 3.656 (m,
J=13.2 Hz, 2H), 3.510-3.582 (m, 1H), 3.344 (m, 2H), 2.117 (s, 6H),
1.984-2.070 (m, 2H), 1.818 (m, 4H), 1.660 (m, 1H), 1.455 (m, 1H),
1.278 (t, J=7.2 Hz, 3H), 1.107 (t, 3H) ppm.
Example 7. Synthesis of
2-(2,6-dimethylphenylcarbamoyl)-1,1-dimethylpyrrolidinium Chloride
(Compound 13)
Synthetic Scheme
##STR00075##
[0233] Step 1: Preparation of 2
##STR00076##
[0235] To a mixture of 1 (10 g, 86.9 mmol, 1 eq) in DCM (189 mL,
c=0.46) was added TEA (35.2 g, 347.6 mmol, 4 eq) and then dropwise
(Boc).sub.2O (12.57 g, 99.03 mmol, 1.2 eq) at 0.degree. C. After
addition, the reaction mixture was warmed slowly to room
temperature and stirred for 3 h. The reaction mixture was directed
concentrated in vacuum to get a residue without further
purification (18 g, 98.36% yield) as white oil.
Step 2: Preparation of 3
##STR00077##
[0237] To a mixture of 2 (9 g, 42.6 mmol, 1 eq) in THE (106.5 ml,
c=0.4) was added ClCO.sub.2Et (5 g, 46.86 mmol, 1.1 eq) and then
dropwise 2,6-dimethylaniline (5.16 g, 42.6 mmol, 1 eq) at 0.degree.
C. The reaction mixture was stirred at 60.degree. C. refluxed
overnight. After completion, the reaction mixture was turned back
to room temperature. Then the mixture was filtered, the filtrate
was concentrated in vacuum. The crude product was purified by
column chromatography to afford pure product (10.4 g, 74%
yield).
Step 3: Preparation of 4
##STR00078##
[0239] To a solution of 3 (3.4 g, 10.68 mmol, 1 eq) in MeOH (53 mL)
was added 4N HCl/MeOH (5.34 mL, 21.36 mmol, 2 eq) at 0.degree. C.
After addition, the reaction mixture was stirred at room
temperature for 3 h. After completion, the reaction mixture was
concentrated in vacuum. The oil was washed with EA and then
filtered to afford a white solid (2.02 g, 74.6% yield).
Step 4: Preparation of 5
##STR00079##
[0241] To a solution of 4 (600 mg, 2.35 mmol, 1 eq) in MeCN (6.35
mL, c=0.37) was added K.sub.2CO.sub.3 (0.83 g, 5.99 mmol, 2.55 eq)
and MeI (333 mg, 235 mol, 1 eq) then string at room temperature
overnight. The mixture was concentrated in vacuum. The crude
product was pre-purified by column chromatography to afford pure
product (262 mg, 43.16% yield).
Step 5: Preparation of 6
##STR00080##
[0243] To a solution of 5 (91 mg, 0.43 mmol, 1 eq) in MeCN (4.3 mL,
c=0.1) was added MeI (152 mg, 1.075 mmol, 2.5 eq). After addition,
the reaction mixture was stirred at room temperature for 2 h. After
completion, the reaction mixture was concentrated in vacuum to
afford a white solid (140 mg, 87.5% yield).
Step 5: Preparation of 6
##STR00081##
[0245] To a solution of 6 (140 mg, 0.288 mmol, 1 eq) in water (0.78
mL, c=0.37) was added AgCl (81 mg, 0.576 mmol, 2 eq). After
addition, the reaction mixture was stirred at 60.degree. C.
overnight. After completion, the reaction mixture was filtered and
lyophilization at 0.degree. C. to give the desired product (92 mg,
80% yield) as a white solid. .sup.1H NMR (300 MHz, D.sub.2O):
.delta. 7.155-7.098 (m, 3H), 4.500-4.446 (t, J=8.1 Hz, 1H),
3.807-3.768 (m, 1H), 3.621-3.582 (m, 1H), 3.265 (s, 3H), 3.182 (s,
3H), 2.799-5.512 (m, 3H), 2.465-2.437 (m, 1H), 2.294-2.242 (m, 1H),
2.094 (s, 6H) ppm. HPLC purity: 99.07% at 220 nm; Mass: m/z=247.5
(M, ESI+).
Example 8. Synthesis of
2-(2,6-dimethylphenylcarbamoyl)-1,1-diethylpyrrolidinium Chloride
(Compound 14)
Synthetic Scheme
##STR00082##
[0246] Step 1: Preparation of 2
##STR00083##
[0248] To a mixture of 1 (10 g, 86.9 mmol, 1 eq) in DCM (189 mL,
c=0.46) was added TEA (35.2 g, 347.6 mmol, 4 eq) and then dropwise
(Boc).sub.2O (12.57 g, 99.03 mmol, 1.2 eq) at 0.degree. C. After
addition, the reaction mixture was warmed slowly to room
temperature and stirred for 3 h. The reaction mixture was directed
concentrated in vacuum to get a residue without further
purification (18 g, 98.36% yield) as white oil.
Step 2: Preparation of 3
##STR00084##
[0250] To a mixture of 2 (9 g, 42.6 mmol, 1 eq) in THF (106.5 ml,
c=0.4) was added ClCO.sub.2Et (5 g, 46.86 mmol, 1.1 eq) and then
dropwise 2,6-dimethylaniline (5.16 g, 42.6 mmol, 1 eq) at 0.degree.
C. The reaction mixture was stirred at 60.degree. C. and refluxed
overnight. After completion, the reaction mixture was turned back
to room temperature. Then the mixture was filtered, the filtrate
was concentrated in vacuum. The crude product was purified by
column chromatography to afford pure product (10.4 g, 74%
yield).
Step 3: Preparation of 4
##STR00085##
[0252] To a solution of 3 (3.4 g, 10.68 mmol, 1 eq) in MeOH (53 mL)
was added 4N HCl/MeOH (5.34 mL, 21.36 mmol, 2 eq) at 0.degree. C.
After addition, the reaction mixture was stirred at room
temperature for 3 h. After completion, the reaction mixture was
concentrated in vacuum. The oil was washed with EA and then
filtered to afford a white solid 2.02 g (74.6% yield).
Step 4: Preparation of 5
##STR00086##
[0254] To a solution of 4 (1.5 g, 5.9 mmol, 1 eq) in MeCN (16 mL,
c=0.37) was added K.sub.2CO.sub.3 (2 g, 5.99 mmol, 2.55 eq) and EtI
(1.84 g, 11.8 mmol, 2 eq) then string at 60.degree. C. overnight.
The second day, the mixture was concentrated in vacuum. The crude
product was purified by column chromatography to afford pure
product (262 mg, 43.16% yield).
Step 5: Preparation of 6
##STR00087##
[0256] To a solution of 6 (230 mg, 0.57 mmol, 1 eq) in water (0.78
mL, c=0.37) was added AgCl (163 mg, 1.14 mmol, 2 eq). After
addition, the reaction mixture was stirred at 60.degree. C.
overnight. After completion, the reaction mixture was filtered and
lyophilization at 0.degree. C. to give the desired product (190 mg,
80% yield) as a white solid. .sup.1H NMR (300 MHz, D.sub.2O):
.delta. 7.162-7.090 (m, 3H), 4.529-4.511 (m, 1H), 3.710-3.358 (m,
6H), 2.682-2.534 (m, 1H), 2.382-2.183 (m, 3H), 2.089 (s, 6H),
1.361-1.273 (m, 6H) ppm. HPLC purity: 96.5% at 220 nm; Mass:
m/z=275.5 (M, ESI+).
Example 9. Synthesis of
2-(2,6-dimethylphenylcarbamoyl)-1,1-diethylpiperidinium Chloride
(Compound 15)
Synthetic Scheme
##STR00088##
[0257] Step 1: Preparation of 2
##STR00089##
[0259] To a solution of 1 (10 g, 43.6 mmol, 1 eq) in THF (109 ml,
c=0.4) was added ClCO.sub.2Et (5.2 g, 47.9 mmol, 1.1 eq) and then
dropwise 2,6-dimethylaniline (5.8 g, 47.9 mmol, 1 eq) at 0.degree.
C. The reaction mixture was stirred at 60.degree. C. refluxed
overnight. After completion, the reaction mixture was turned back
to room temperature. Then the mixture was filtered, the filtrate
was concentrated in vacuum. The crude product was purified by
column chromatography to afford pure product (7.71 g, 53%
yield).
Step 3: Preparation of 3
##STR00090##
[0261] To a solution of 2 (7.71 g, 23.2 mmol, 1 eq) in MeOH (116 mL
c=0.2) was added 4N HCl/MeOH (11.6 mL, 46.4 mmol, 2 eq) at
0.degree. C. After addition, the reaction mixture was stirred at
room temperature for 3 h. After completion, the reaction mixture
was concentrated in vacuum. The oil was washed with EA and then
filtered to afford a white solid 5.1 g (81.86% yield).
Step 4: Preparation of 4
##STR00091##
[0263] To a solution of 3 (2.5 g, 9.3 mmol, 1 eq) in MeCN (25 mL,
c=0.37) was added K.sub.2CO.sub.3 (3.3 g, 23.7 mmol, 2.55 eq) and
EtI (1.45 g, 9.3 mmol, 1 eq) then string at room temperature
overnight. The mixture was concentrated in vacuum. The crude
product was pre-purified by column chromatography to afford pure
product (2 g, 80% yield).
Step 5: Preparation of 5
##STR00092##
[0265] To a solution of 4 (0.5 g, 1.92 mmol, 1 eq) in MeCN (25 mL,
c=0.37) was added, and EtI (749 mg, 4.8 mmol, 2.5 eq) then string
at 60.degree. C. overnight. The mixture was concentrated in vacuum.
The crude product was washed with PE to afford pure product (200
mg, 38.75% yield).
Step 4: Preparation of 6
##STR00093##
[0267] To a solution of 6 (217 mg, 0.52 mmol, 1 eq) in water (1.4
mL, c=0.37) was added AgCl (149 mg, 1.04 mmol, 2 eq). After
addition, the reaction mixture was stirred at 60.degree. C.
overnight. After completion, the reaction mixture was filtered and
lyophilized at 0.degree. C. to give the desired product (110 mg,
65% yield) as a white solid. .sup.1H NMR (300 MHz, D.sub.2O):
.delta. 7.127.about.7.107 (m, 3H), 4.292 (t, 1H), 3.896-3.765 (m,
1H), 3.733-3.412 (m, 4H), 3.365-3.254 (d, 1H), 2.457-2.310 (m, 1H),
2.283-2.231 (m, 1H), 2.083 (s, 6H), 1.875-1.526 (m, 4H), 1.290 (t,
J=4.5, J=3, 6H) ppm. HPLC purity: 96.4% at 220 nm; Mass: m/z=289.5
(M, ESI+).
Example 10. Synthesis of 2-(4-fluoro-2,
6-dimethylphenylcarbamoyl)-1, 1-dimethylpiperidinium Chloride
(Compound 16)
Synthetic Scheme
##STR00094##
[0268] Step 1: Preparation of 5-fluoro-1,
3-dimethyl-2-nitrobenzene
##STR00095##
[0270] 1 (6 g, 48.3 mmol, 1 eq) was cooled to -10.degree. C. and
nitric acid (9 g, 143.78 mmol, 3 eq) was added to it dropwise
during 20 min. The mixture was stirred at -15.degree. C. for 1 h,
then allowed to reach RT carefully and kept for 3 hr with stirring.
The mixture was poured into ice to give a yellow precipitate,
filtered and the filter cake dissolved with DCM. The organic phase
washed by brine, dried on Na.sub.2SO.sub.4, concentrated to give
the desired product 2 (6.8 g, 83% yield) as a white powder.
Step 2: Preparation of 4-fluoro-2, 6-dimethylaniline
##STR00096##
[0272] To a solution of 2 (16 g, 94.65 mmol, 1 eq) in MeOH (236 mL)
was added Pd/C (1.6 g, 10% w/w) and a couple of drops of Conc.HCl
under H.sub.2. The mixture was stirred for 5 hr at RT, then
filtered and the filtrate was concentrated. The residue was poured
into ice, adjusted pH to 10 with 2N NaOH, extracted with EA. The
organic phase was washed with brine (150 mL) and dried over
anhydrous Na.sub.2SO.sub.4 and filtered. The residue after rotary
evaporation was purified by column chromatography to give the
desired product 3 (5 g, 38.4% yield).
Step 3: Preparation of N-(4-fluoro-2,
6-dimethylphenyl)-1-methylpiperidine-2-carboxamide
##STR00097##
[0274] To a solution of 1-methylpiperidine-2-carboxylic acid
hydrochloride (2.27 g, 12.65 mmol, 1.1 eq) in DCM (46 mL, c=0.25)
was added TEA (5.12 g, 50.6 mmol, 4.4 eq) under nitrogen and
stirred at RT for 30 min. The mixture was cooled to 0.degree. C. at
an ice-bath, then ClCO.sub.2Et was added dropwise slowly during a
period of 20 min. To the mixture above was added 3 (1.6 g, 11.5
mmol, 1.0 eq) in DCM (2 mL) dropwise, stirred for overnight. The
mixture after rotary evaporation was purified by column
chromatography to give the desired product 4 (0.5 g, 16%
yield).
Step 4: Preparation of 2-(4-fluoro-2, 6-dimethylphenylcarbamoyl)-1,
1-dimethylpiperidinium Iodide
##STR00098##
[0276] To a solution of 4 (0.5 g, 1.89 mmol, 1.0 eq) in MeCN (35.8
mL) was added MeI (1.025 g, 7.225 mmol, 2.5 eq), stirred at
90.degree. C. in a 75 mL of sealed tube with stirring for 2 h.
After completion, removed the solvent to give the desired product 5
(760 g, 99% yield).
Step 5: Preparation of 2-(4-fluoro-2, 6-dimethylphenylcarbamoyl)-1,
1-dimethylpiperidinium Chloride
##STR00099##
[0278] To a solution of 5 (0.3 g, 0.739 mmol, 1.0 eq) in deionized
water (2.5 mL) was added AgCl (212 mg, 1.48 mmol, 2.0 eq), stirred
at 60.degree. C. with stirring for 30 min. After completion, the
reaction was filtered, and the filtrate was lyophilized to give 6
(160 g, 73% yield). .sup.1H NMR (300 MHz,
D.sub.2O-d.sub.6):.delta.6.90 (d, J=9.6 Hz, 2H), 4.21-4.11 (m, 1H),
3.69-3.60 (m, 1H), 3.48-3.35 (m, 1H), 3.27 (d, J=11.1 Hz, 6H),
2.30-2.18 (m, 2H), 2.17-2.00 (m, 6H), 1.95-1.55 (m, 5H) ppm.
Example 11. Synthesis of
2-(4-aminophenylamino)-N,N,N-triethyl-2-oxoethanaminium (Compound
22)
Synthetic Scheme
##STR00100##
[0279] Step 1: Preparation of 3
##STR00101##
[0281] To a mixture of 2 (240 mL, 7.2 eq) was added 1 (30 g, 1.0
eq) in batches. The mixture was stirred at 30.degree. C. overnight.
After completion, the suspension was filtered and the filter cake
washed with EA (30 mL.times.2), 150 mL EA was added to the filter
cake and stirred for 30 min at 30.degree. C. Then the suspension
was filtered and the filter cake washed with EA (30 mL.times.2),
the filtrate was concentrated under reduce pressure. The residue
was added to 90 mL Acetonitrile/acetone (1:2) and stirred at RT.
over night. The suspension was filtered and the filter cake was
dried under reduce pressure to give the product (9.7 g,
yield=23.3%).
Step 2: Preparation of 5
##STR00102##
[0283] To a mixture of 3 (1.0 g, 7.6 mmol, 1.0 eq) and TEA (1.5 g,
15.2 mmol, 2.0 eq) in THE (19 ml, c=0.4) was added ClCO.sub.2Et
(0.9 g, 8.36 mmol, 1.1 eq). Then the solution was added 4 (1.15 g,
8.36 mmol, 1.1 eq). The reaction mixture was stirred at reflux
overnight. Then the suspension was filtered and the filtrate was
concentrated under reduce pressure. The residue was dissolved with
EA and washed with acid-base to give the product (0.35 g,
yield=20%). .sup.1HNMR (300 MHz DMSO): .delta. 10.2268 (s, 1H),
8.2159 (d, J=9.18 Hz, 2H), 7.9377 (d, J=9.15 Hz, 2H), 3.2379 (s,
2H), 2.5458-2.6470 (dd, 4H), 1.0139 (t, 6H).
Step 3: Preparation of 6
##STR00103##
[0285] To a mixture of 5 (300 mg, 1.19 mmol, 1.0 eq) in MeCN (12
ml, c=0.1) was added EtI (651.5 mg, 4.18 mmol, 3.5 eq). The
reaction mixture was heated to reflux overnight. After completion,
the reaction solution was concentrated under reduce pressure to
give the product (200 mg, yield=41%, HPLC: 98%).
Step 4: Preparation of 7
##STR00104##
[0287] To a solution of 5 (200 mg, 0.49 mmol, 1.0 eq) in deionized
water (4 mL, c=0.12) was added AgCl (140.8 mg, 0.98 mmol, 2.0 eq).
Then the solution was heated to 60.degree. C. and stirred
overnight. After completion, the suspension was filtered and the
filtrate was used to next step.
Step 5: Preparation of 8
##STR00105##
[0289] To the filtrate of step 3 was added Pd/C (30 mg, m/m=0.2) 2
drops of 2N HCl. The solution was displaced with H.sub.2. Then the
solution was stirred at 35.degree. C. overnight. After completion,
the suspension was filtered was lyophilized and the residue was
washed with EA to give the product (92.4 mg, yield=66%). HPLC
purity: 98.8% at 220 nm, 99% at 254 nm; Mass:M+: M-35.5=250.5;
M-:M=285.5; M+35.5=321.4. .sup.1H NMR (300 MHz, D.sub.2O): .delta.
7.4742 (d, J=8.7 Hz, 2H), 7.2281 (d, J=8.79 Hz, 2H), 4.1406 (s,
2H), 3.5985 (dd, J=7.2 Hz, 6H), 1.3526 (t, J=7.2 Hz, 9H). ppm.
Example 12. Synthesis of 2-(4-amino-2, 6-dimethylphenylamino)-N, N,
N-triethyl-2-oxoethanaminium Chloride (Compound 23)
Synthetic Scheme
##STR00106##
[0290] Step 1: Preparation of N-(2,
6-dimethylphenyl)-4-methylbenzenesulfonamide
##STR00107##
[0292] To a solution of 1 (15 g, 123.8 mmol, 1 eq) in Pyridine (334
mL, c=0.37) was added TsCl (28 g, 148.54 mmol, 1.2 eq), heated to
reflux at 115.degree. C. for 4 hours. The reaction mixture was
cooled to RT, removed the solvent, adjusted pH to 6 with 2N HCl,
washed with water, extracted with EA, washed by brine, concentrated
and the residue recrystallized from hot ethanol to give the desired
product 2 (19.8 g, 56% yield) as a white powder.
Step 2: Preparation of N-(2,
6-dimethyl-4-nitrophenyl)-4-methylbenzenesulfonamide
##STR00108##
[0294] To a suspension of 2 (19.8 g, 71.9 mmol, 1 eq) in AcOH:
H.sub.2O (150 MI: 100 mL) was added NaNO.sub.2 under nitrogen with
stirring, con.HNO.sub.3 (9 g, 194 mmol, 2 eq) was added dropwise
during a period of 10 min, refluxed at 110.degree. C. for 5 hr.
After completion, the reaction was diluted with H.sub.2O (150 mL),
extracted with EA (200 mL), adjusted pH to 8 with 1N NaOH. The
organic phase was washed with brine (150 mL) and dried over
anhydrous Na.sub.2SO.sub.4. The filtrate was evaporated in vacuum
to give 3 (16 g, 70% yield) as a powder.
Step 3: Preparation of 2, 6-dimethyl-4-nitroaniline
##STR00109##
[0296] A solution of 3 (23 g, 72 mmol, 1.0 eq) in
con.H.sub.2SO.sub.4 (232 mL, c=0.3) was heated at 60.degree. C. for
1 h with stirring. After completion, the reaction was poured into
ice (1000 mL) and adjusted pH=8 with 20% NaOH.extracted with EA
(200 mL). The organic phase was washed with brine (15 mL) and dried
over anhydrous Na.sub.2SO.sub.4 and filtered. The residue after
rotary evaporation was purified by column chromatography to give
the desired product 4 (8 g, 66% yield).
Step 4: Preparation of 2-chloro-N-(2, 6-dimethyl-4-nitrophenyl)
Acetamide
##STR00110##
[0298] To a mixture of 4 (4 g, 24 mmol, 1.0 eq) in DCM (30 mL,
c=0.8) was added pyridine (2.28 g, 28.8 mmol, 1.2 eq),
2-chloroacetyl chloride (3.25 g, 28.8 mmol, 1.2 eq) dropwise at
0.degree. C., stirred at RT for overnight with stirring. After
completion, the reaction was filtered, and the cake was poured into
water, adjusted pH to 4 with 2N HC, filtered and the cake dried to
give the desired product 5 (4.4 g, 76% yield).
Step 5: Preparation of 2-(diethylamino)-N-(2,
6-dimethyl-4-nitrophenyl) Acetamide
##STR00111##
[0300] A mixture of 5 (4 g, 16.48 mmol, 1.0 eq) in diethylamine
(46.7 mL, 639 mmol, 38.8 eq) was heated to reflux at 55.degree. C.
for 5 hr, removed solvent, poured into water, extracted with EA
(100 mL.times.3). The organic phase was washed with brine (15 mL)
and dried over anhydrous Na.sub.2SO.sub.4 and filtered. The residue
after rotary evaporation was purified by column chromatography to
give the desired product 6 (2.17 g, 47% yield).
Step 6: Preparation of 2-(2, 6-dimethyl-4-nitrophenylamino)-N, N,
N-triethyl-2-oxoethanaminium Iodide
##STR00112##
[0302] To a solution of 6 (1 g, 3.58 mmol, 1.0 eq) in MeCN (35.8
mL) was added EtI (1.4 g, 8.95 mmol, 2.5 eq), stirred at 90.degree.
C. in a 75 mL of sealed tube with stirring for 3 day. After
completion, removed the solvent to give the desired product 7 (1.48
g, 95% yield).
Step 7: 2-(2, 6-dimethyl-4-nitrophenylamino)-N, N,
N-triethyl-2-oxoethanaminium Chloride
##STR00113##
[0304] To a solution of 7 (0.5 g, 1.15 mmol, 1.0 eq) in deionized
water (2 mL) was added AgCl (329 mg, 2.3 mmol, 2.0 eq), stirred at
60.degree. C. with stirring for overnight. After completion, the
reaction was filtered to give the filtrate 8.
Step 8: Preparation of N-(4-amino-2,
6-dimethylphenyl)-2-(diethylamino) Acetamide
##STR00114##
[0306] To a mixture of 8 above was added Pd/C (39.5 mg, 10% w/w),
the suspension was degassed under vacuum and purged with H.sub.2
several times, stirred for 6 hr at RT. After completion, the
reaction was filtered, and the cake was washed with EA (1.5 mL),
filtrated to give the desired product 9 (250 mg, 74% yield) as a
white powder. .sup.1H NMR (300 MHz, D.sub.2O-d.sub.6): .delta.7.05
(s, 2H), 4.22 (s, 2H), 3.53-3.50 (m, 6H), 2.11 (s, 6H), 1.30-1.25
(m, 9H) ppm.
Example 13. Synthesis of
3-(2,6-dimethylphenylamino)-N,N,N-triethyl-3-oxopropan-1-aminium
Chloride (Compound 24)
Synthetic Scheme
##STR00115##
[0307] Step 1: Preparation of 2
##STR00116##
[0309] To a mixture of 1 (10 g, 82.52 mmol, 1 eq) in DCM (50 mL,
c=1.6) was added TEA (10.02 g, 99.03 mmol, 1.2 eq) and was drop
wised 3-chloropropanoyl chloride (12.57 g, 99.03 mmol, 1.2 eq) in
DCM (50 mL, c=1.6) at 0.degree. C. After addition, the reaction
mixture was warmed slowly to room temperature and stirred for 2 h.
After completion, the reaction solution was adjusted to pH=3-4 with
1N HCl and extracted with DCM (2.times.50 mL). The combined organic
phases was adjusted to pH=7-8 with saturated NaHCO.sub.3. The
combined organic phases was washed with brine, dried over
Na.sub.2SO.sub.4, filtered and concentrated in vacuum at 25.degree.
C. The residue was purified by column chromatography to give the
desired product (12.46 g, 71.3% yield, included 46% N-(2,
6-dimethylphenyl) acryl amide) as a white solid.
Step 2: Preparation of 3
##STR00117##
[0311] To 2 (8 g, 37.79 mmol, 1 eq) was added diethylamine (107.24
g, 1466.29 mmol, 38.8 eq). The reaction mixture was stirred at
55.degree. C. overnight. After completion, the reaction mixture was
concentrated in vacuum. And then the residue diluted with water
(150 mL) and extracted with EA (3.times.100 mL). The combined
organic phases was washed with brine, dried over Na.sub.2SO.sub.4,
filtered and concentrated in vacuum. The residue was purified by
column chromatography to give the desired product (5.75 g, 61%
yield) as yellow oil.
Step 3: Preparation of 4
##STR00118##
[0313] To a solution of 3 (3 g, 12.08 mmol, 1 eq) in ACN (121 MI,
c=0.1) was added EtI (5.65 g, 36.24 mmol, 3 eq). After addition,
the reaction mixture was stirred at 85.degree. C. overnight. After
completion, the reaction mixture was concentrated in vacuum. The
residue was purified by column chromatography to give the desired
product (3.6 g, 74% yield) as a white solid.
Step 4: Preparation of 5
##STR00119##
[0315] To a solution of 4 (300 mg, 0.74 mmol, 1 eq) in water (1.2
mL, c=0.6) was added fresh AgCl (212 mg, 1.48 mmol, 2 eq) at
6-7.degree. C. After addition, the reaction mixture was stirred at
6-7.degree. C. for 4 min. After completion, the reaction mixture
was filtered and lyophilized at 0.degree. C. to give the desired
product (133 mg, 57% yield) as a white solid. .sup.1H NMR (300 MHz,
D.sub.2O): .delta. 7.063-7.128 (m, 3H), 3.495 (t, J=7.8 Hz, J=8.1
Hz, 2H), 3.220-3.293 (m, 4H), 2.909 (t, J=7.8 Hz, J=8.1 Hz, 2H),
2.100 (d, J=22.5 Hz, 6H), 1.212 (t, J=7.2 Hz, 6H) ppm. HPLC purity:
99.7% at 220 nm.
Example 14. Synthesis of
2-(4-aminobenzamido)-N,N,N-triethylethanaminium Chloride
Hydrochloride (Compound 26)
Synthetic Scheme
##STR00120##
[0316] Step 1: Preparation of 3
##STR00121##
[0318] To a mixture of 1 (1.6 g, 9.46 mmol, 1.1 eq) and TEA (1.7 g,
17.2 mmol, 2 eq) in THE (25 mL, c=0.37) was added ClCO.sub.2Et (1.0
g, 9.46 mmol, 1.1 eq) slowly. Then 2 (1.0 g, 8.6 mmol, 1 eq) was
added to the mixture. The mixture was stirred at RT. for 5 h. After
completion, the suspension was filtered and the filtrate was
concentrated under reduce pressure. The residue was purified by
column chromatography and washed with NaOH to give the product (1.0
g, yield=43.8%, HPLC: 99.8%).
Step 2: Preparation of 4
##STR00122##
[0320] To a mixture of 3 (200 mg, 0.75 mmol, 1.0 eq) in MeCN (7.5
ml, c=0.1) was added EtI (293 mg, 1.88 mmol, 2.5 eq). The reaction
mixture was heated to reflux overnight. After completion, the
reaction solution was concentrated under reduce pressure to give
the product (313.8 mg, yield=99.3%, LCMS: 100%).
Step 3: Preparation of 5
##STR00123##
[0322] To a solution of 4 (195.7 mg, 0.46 mmol, 1.0 eq) in
deionized water (4 mL, c=0.1) was added AgCl (133 mg, 0.93 mmol,
2.0 eq). Then the solution was heated to 60.degree. C. and stirred
overnight. After completion, the suspension was filtered and the
filtrate was used to next step.
Step 4: Preparation of 6
##STR00124##
[0324] To the filtrate of step 3 was added MeOH (4 mL), Pd/C (15
mg, m/m=0.1), 2 drops of 2N HCl. The solution was displaced with
H.sub.2. Then the solution was stirred at 35.degree. C. overnight.
After completion, the suspension was filtered and the 4N HCl/MeOH
(0.5 mL) was added to the solution and stirred for 30 min. Then the
solution was lyophilized and the residue was washed with EA to give
the product (55 mg, yield=36%). HPLC purity:95.9% at 220 nm; 96.7%
at 254 nm; Mass: M+:M-35.5=264.5, M-:M+35.5=334.5; .sup.1H NMR (300
MHz, D.sub.2O): .delta. 7.8252 (d, J=8.3 Hz, 2H), 7.4115 (d, J=8.4
Hz, 2H), 3.7562 (t, J=6.6 Hz, 2H), 3.3613 (m, 9H), 1.2785 (t, J=7.5
Hz, 10H) ppm.
Example 15. Compound 6 has Greater Efficacy than QX-314 in
Inhibiting Sodium Channels when Applied Inside Cells
[0325] FIG. 1A shows the time course of peak sodium current as a
function of time for cells dialyzed with either 100 micromolar
QX-314 or 100 micromolar BW8186 (Compound 6) and stimulated with a
series of 30-msec depolarizations to -20 mV from a holding
potential of -100 mV. To induce use-dependent block, the
depolarizations were delivered by series of increasing rates: 0.05
Hz for 1-min, 0.33 Hz for 1-min, 1 Hz for 1-min, 3 Hz for 1-min, 5
Hz for 30 seconds, 10 Hz for 30 seconds, with 1 minute rest between
each series of pulses. After the series of pulses to induce
use-dependent block, the time course of recovery was followed using
pulses delivered at 01. Hz (2-min) and 0.05 Hz (1-min). Peak sodium
current was plotted as a function of experimental time (1-min per
division on the time axis).
[0326] As can be seen in FIG. 1A, QX-314 and Compound 6 both show
strong use dependent accumulation of block at stimulation
frequencies from 1 to 10 Hz, with only partial recovery during
1-minute rest intervals. QX-314 produced use-dependent inhibition
of sodium current to about 20% of the initial value, followed by
partial recovery to about 40% of the initial value after 3 minutes
of slow stimulation. Compound 6 produced more profound
use-dependent block, to about 3% of the initial sodium current, and
recovered very little during 3 minutes of slow stimulation, to
about 5% of the initial value. Thus, compared with QX-314, Compound
6 shows more accumulation of use-dependent block and also
strikingly less recovery from block during periods of no
stimulation or a long period of slow stimulation (0.05 Hz) after
development of block. This indicates that Compound 6 is trapped in
the cell and may result in a prolonged analgesic effect in
vivo.
[0327] FIG. 1B shows that Compound 6 is far more effective when
applied inside the cell (presented inside the recording pipette
during whole cell recording so that the cell dialyzed) than when it
is applied outside the cell. This supports the idea that, like
QX-314, Compound 6 should have only weak effects on neuronal
activity unless it can enter neurons through activated TRPV1,
TRPA1, or other large-pore channels, present selectively in neurons
mediating pain and itch.
Example 16. Compound 21 was as Effective as QX-314 in Blocking Paw
Incision-Induced Thermal Hyperalgesia
[0328] The effects of N-ethyl etidocaine ("NEE", Compound 21)
compared to QX-314 were tested in a mouse and rat models of thermal
hyperalgesia. Inflammation-related hyperalgesia evoked by
intraplantar injection of CFA in mice is reversed by Compound 21
(NEE) or by QX-314 (FIGS. 2A-2D). In rats (FIG. 3A), no differences
in thermal nociceptive threshold were observed between the
contralateral (untreated) paw and the hind paw that received an
acute intraplantar injection (50 .mu.l) of saline, QX-314 (0.5%) or
N-ethyl etidocaine (Compound 21) (0.5%). One week later the rats
underwent surgical incision to their left hindpaw. One hour later
the rats received acute intradermal injections of compound 21 or
QX-314 directly into the surgical wound. When compared to their
untreated contralateral paw, the animals that received the saline
treatment showed a significant thermal hyperalgesia at 1 and 3 hour
post-treatment (FIG. 3B). This effect was absent in animals treated
with QX-314 or Compound 21, suggesting that Compound 21 is as
effective as QX-314 in decreasing pain sensitivity in conditions of
thermal hyperalgesia.
Example 17. Compounds 3, 6, and 21 have Effects on Thermal
Nociceptive Response Latency when Injected Alone
[0329] Some data in the literature suggests that QX-314, when
injected perineurally, can induce neurotoxicity 8 weeks
post-treatment. To evaluate if such toxicity occurred with NEE
(Compound 21), mice were injected with a combination of CFA+NEE
(1%) 8 weeks prior to DRG extraction. DRGs extracted from these
mice were stained for ATF3, a transcription factor specifically
increased in injured neurons. No ATF3 expression was observed in
the CFA+NEE (1%) treated mice (FIG. 4B and FIG. 4C). To confirm the
validity of the staining, a DRG slice from an ATF3-GFP reporter
mice that underwent a sciatic nerve injury was labeled in parallel
and co-localization of the ATF3 antibody (dark shading) was
observed with the ATF3 reporter staining (light shading) (FIG.
4A).
[0330] It is known that QX-314 reverses allergic airway
inflammation by interrupting the interplay between the pain neuron
and the immune system. To determine if NEE (Compound 21) works by
the same mechanism, the effect of NEE (Compound 21) on airway
inflammation was then assessed. Aerosolized NEE (Compound 21) (100
uM, 6%, 20 min, day 18) blocked bronchoalveolar lavage fluid immune
cell influx (day 21), specifically CD45+cells, including
eosinophils, macrophages, and T cells, in a murine model of
allergic airway inflammation induced by Ovalbumin (FIGS.
5A-5E).
[0331] Of the five new compounds that show an improved in vitro
NaV1.7 use-dependent block over QX-314, ACS-3B (Compound 3) and
N-ethyl-etidocaine (Compound 21) showed potent blockade of
CFA-induced thermal hyperalgesia without long lasting analgesia in
naive animals. Further, N-ethyl-etidocaine (Compound 21) potently
reversed lung allergic airway inflammation.
Other Embodiments
[0332] Various modifications and variations of the described method
and system of the invention will be apparent to those skilled in
the art without departing from the scope and spirit of the
invention. Although the invention has been described in connection
with specific desired embodiments, it should be understood that the
invention as claimed should not be unduly limited to such specific
embodiments. Indeed, various modifications of the described modes
for carrying out the invention that are obvious to those skilled in
the fields of medicine, immunology, pharmacology, endocrinology, or
related fields are intended to be within the scope of the
invention.
[0333] All publications mentioned in this specification are herein
incorporated by reference to the same extent as if each independent
publication was specifically and individually incorporated by
reference.
* * * * *