U.S. patent application number 10/926682 was filed with the patent office on 2005-01-20 for compositions and methods for treatment of neurological disorders.
Invention is credited to Papke, Roger L..
Application Number | 20050014779 10/926682 |
Document ID | / |
Family ID | 21891823 |
Filed Date | 2005-01-20 |
United States Patent
Application |
20050014779 |
Kind Code |
A1 |
Papke, Roger L. |
January 20, 2005 |
Compositions and methods for treatment of neurological
disorders
Abstract
The present invention concerns methods for treating or
preventing neurological disorders characterized by dysfunction of
nicotine acetylcholine receptors by co-administration of
metanicotine and at least one compound which exhibits antagonist
activity, or both agonistic and antagonist activity, toward one or
more nicotinic acetylcholine receptor subtypes. The subject
invention, in another aspect, pertains to pharmaceutical
compositions containing metanicotine and at least one compound
which exhibits antagonistic activity, or both agonistic and
antagonistic activity, toward one or more nicotinic acetylcholine
receptor subtypes.
Inventors: |
Papke, Roger L.;
(Gainesville, FL) |
Correspondence
Address: |
SALIWANCHIK LLOYD & SALIWANCHIK
A PROFESSIONAL ASSOCIATION
PO BOX 142950
GAINESVILLE
FL
32614-2950
US
|
Family ID: |
21891823 |
Appl. No.: |
10/926682 |
Filed: |
August 25, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10926682 |
Aug 25, 2004 |
|
|
|
10036988 |
Dec 31, 2001 |
|
|
|
Current U.S.
Class: |
514/295 ;
514/343; 514/408; 514/547 |
Current CPC
Class: |
A61K 31/465 20130101;
A61K 31/465 20130101; A61K 31/4545 20130101; A61K 31/4545 20130101;
A61K 31/22 20130101; A61K 2300/00 20130101; A61K 2300/00 20130101;
A61K 31/22 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/295 ;
514/408; 514/547; 514/343 |
International
Class: |
A61K 031/473; A61K
031/4439; A61K 031/225 |
Goverment Interests
[0002] The subject invention was made with government support under
a research project supported by National Institutes of Health Grant
No. PO1 AG10485. The government has certain rights in this
invention.
Claims
What is claimed is:
1. A method for treating a neurological condition characterized by
dysfunction of nicotinic acetylcholine receptors comprising
co-administering metanicotine, or a pharmaceutically acceptable
salt or analogue thereof, and at least one compound exhibiting
antagonistic activity, or both agonist and antagonist activity,
toward one or more nicotinic acetylcholine receptor subtypes, to a
patient in need of such treatment.
2. The method, according to claim 1, wherein the compound is
selected from the group consisting of acetylcholine; nicotine;
3-[2,4-dimethoxybenzylid- ene]-anabaseine;
2-methyl-3-(2-(S)-pyrrolidinyl methoxy)pyridine;
(S)-3-methyl-S-(1-methyl-2-pyrrolidinyl)isoxazole;
(R)-5-(2-azetidinyl-methoxy)-2-chloropyridine; altinicline;
(.+-.)-4-{[2-(1-methyl-2-pyrrolidinyl) ethyl]thio}phenol
hydrochloride; epibatadine; and mecamylamine, or a pharmaceutically
acceptable salt or analogue thereof.
3. The method, according to claim 1, wherein the metanicotine and
the compound are administered to the patient consecutively.
4. The method, according to claim 1, wherein the metanicotine and
the compound are administered to the patient simultaneously.
5. The method, according to claim 1, wherein the metanicotine and
the compound are administered to the patient simultaneously and in
the form of a pharmaceutical composition.
6. The method, according to claim 1, wherein the neurological
condition is selected from the group consisting of Alzheimer's
disease, Parkinson's disease, Huntington's chorea, tardive
dyskinesia, hyperkinesias, mania, attention deficit disorder,
attention deficit hyperactivity disorder, sleep-wake disorder,
chronic-fatigue syndrome, tremor, epilepsy, neuropathic pain,
addiction, anxiety, dyslexia, schizophrenia, obsessive-compulsive
disorder and Tourette's syndrome, or combinations thereof.
7. The method, according to claim 1, wherein the patient is
suffering from the neurological condition.
8. The method, according to claim 1, wherein the route of
administration is selected from the group consisting of
intravenous, oral, and intra-nasal.
9. The method, according to claim 1, wherein the metanicotine and
the compound administered to the patient do not cause an adverse
side effect in the patient which is normally associated with
administration of the compound alone, or wherein the metanicotine
and the compound administered to the patient cause an adverse side
effect in the patient which is normally associated with
administration of the compound alone, but of decreased
intensity.
10. The method, according to claim 1, wherein the metanicotine and
the compound are administered in amounts sufficient to penetrate
the blood-brain barrier.
11. A pharmaceutical composition comprising metanicotine, or a
pharmaceutically acceptable salt or analogue thereof, and at least
one compound exhibiting antagonistic activity, or both agonist and
antagonist activity, toward one or more nicotinic acetylcholine
receptor subtypes.
12. The pharmaceutical composition, according to claim 1, wherein
said compound is selected from the group consisting of wherein the
compound is selected from the group consisting of acetylcholine;
nicotine; 3-[2,4-dimethoxybenzylidene]-anabaseine;
2-methyl-3-(2-(S)-pyrrolidinyl methoxy)pyridine;
(S)-3-methyl-S-(1-methyl-2-pyrrolidinyl)isoxazole;
(R)-5-(2-azetidinyl-methoxy)-2-chloropyridine; altinicline;
(.+-.)-4-{[2-(1-methyl-2-pyrrolidinyl) ethyl]thio}phenol
hydrochloride; epibatadine; and mecamylamine, or a pharmaceutically
acceptable salt or analogue thereof.
13. A method for modulating the activity of a compound upon a
nicotinic acetylcholine receptor, wherein the compound has an
antagonist, or a mixed agonist/antagonist, nicotinic acetylcholine
receptor profile, said method comprising contacting the compound
with metanicotine, or a pharmaceutically acceptable salt or
analogue thereof.
14. The method, according to claim 15, wherein the metanicotine
diminishes the antagonist activity of the compound upon a nicotinic
acetylcholine receptor when the compound is contacted with the
nicotinic acetylcholine receptor.
15. The method, according to claim 14, wherein the compound has one
or more side effects that are reduced or eliminated after
contacting the compound with the metanicotine.
16. The method, according to claim 13, wherein the compound is
contacted with the metanicotine in vitro.
17. The method, according to claim 13, wherein the compound is
contacted with the metanicotine in vivo.
18. The method, according to claim 14, further comprising
contacting the compound with the nicotine acetylcholine receptor in
vitro.
19. The method, according to claim 14, further comprising
contacting the compound with the nicotine acetylcholine receptor in
vivo.
20. The method, according to claim 13, wherein the compound is
selected from the group consisting of acetylcholine; nicotine;
3-[2,4-dimethoxybenzylidene]-anabaseine;
2-methyl-3-(2-(S)-pyrrolidinyl methoxy)pyridine;
(S)-3-methyl-S-(1-methyl-2-pyrrolidinyl)isoxazole;
(R)-5-(2-azetidinyl-methoxy)-2-chloropyridine; altinicline;
(.+-.)-4-{[2-(1-methyl-2-pyrrolidinyl) ethyl]thio}phenol
hydrochloride; epibatadine; and mecamylamine, or a pharmaceutically
acceptable salt or analogue thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The present application is a continuation of U.S.
application Ser. No. 10/036,988, filed Dec. 31, 2001, which is
hereby incorporated by reference herein in its entirety, including
any figures, tables, nucleic acid sequences, amino acid sequences,
or drawings.
BACKGROUND OF THE INVENTION
[0003] The nicotinic acetylcholine receptors (nAChRs) are members
of a superfamily of ligand-gated ion channels that mediate fast
signal transmission at synapses. The ion channel is formed from the
assembly of a membrane protein oligomer (a pentamer) that binds the
neurotransmitter, acetylcholine, its natural ligand. The nAChR also
binds agonists, such as nicotine, and nicotinic antagonists, such
as mecamylamine. The binding of two molecules of acetylcholine or
nicotine to the alpha subunits of the receptor induces a
conformational change, stabilizing the receptor's open-state, which
allows the flux of ions (e.g., sodium, calcium, potassium) across
the cell membrane. An influx may cause membrane depolarization and
the activation of voltage-gated ion channels for sodium and
calcium, resulting in the exocytotic release of neurotransmitters
and hormones from vesicular stores. The overall conductance as well
as the relative conductances of various ions depend on the subunit
composition of the receptor (Lindstrom et al., Ann. NY Acad. Sci.
[1995] 757:100-116; McGehee and Role, Annu. Rev. Physiol. [1995]
57:521-546). Nicotinic receptors are found in both muscle and
neuronal tissues and are therefore broadly classified as either
muscle-type or neuronal nicotinic AChRs.
[0004] There are multiple types of nAChRs in the brain associated
with synaptic function, signal processing or cell survival. The
therapeutic targeting of nicotinic receptors in the brain requires
the identification of drugs that may be selective for their ability
to activate or inhibit a limited range of these receptor
subtypes.
[0005] Brain nicotinic receptor systems have long been associated
with addiction. Recently, it has been shown that nicotinic receptor
systems may be involved with Tourette's syndrome (Silver, A. A. et
al., J. Am. Acad. Child Adolesc. Psychiatry [1996] 35:1631-1636;
Sanberg, P. R. et al., Lancet [1998] 352:705-706) and schizophrenia
(Adler, L. E. et al., Biol. Psychiatry [1992] 32:607-616, Adler, L.
E. et al. Am. J. Psychiatry [1993] 150:1856-1861; Leonard, S. et
al., Soc. Neurosci. Abstr. [1993] 19:837; Freedman, R. et al.,
Harvard Rev. Psychiatry [1994] 2:179-192) and that nicotinic drugs
may also have applications as analgesics and for the treatment of
Alzheimer's disease (Williams, M. et al., Drug News Perspect.
[1994] 7:205-223; Arneric, S. P. et al., Psychopharmacology: The
Fourth Generation of Progress [1995], pp. 95-110). With these newly
defined therapeutic endpoints, the challenge becomes understanding
how best to target nicotinic drugs to the receptor systems of the
brain.
[0006] The pharmacology of neuronal nicotinic receptors, however,
is very complex. With a gene family that includes at least nine
different .alpha. subunits (designated .alpha.2-.alpha.10) that in
some cases may function as homooligomers (.alpha.7-.alpha.10) or
alternatively combine with different neuronal .beta. subunits
(.beta.2-.beta.4), there is a great potential for structural
diversity just on the level of the basic pentamer receptor subunit
combinations (Papke, R. L. Prog. Neurobiol. [1993] 41:509-531).
Multiple receptor subtypes are commonly found on single neurons,
and single tissues have multiple neuronal cell types that differ in
the function of their nicotinic receptors (Mulle, C. et al., J.
Neurosci. [1991] 11:2588-2597).
[0007] One approach for sorting out significant elements in this
complex system is to study cloned receptor subunits in defined
combinations. The co-expression of .alpha.4 and .beta.2 subunits
represents one receptor subunit combination of particular interest,
as the primary high-affinity nicotinic receptor of the brain is
composed of these subunits (Whiting, P. J. and J. M. Lindstrom,
Biochemistry [1986] 25:2082-2093; Flores, C. M. et al., Mol.
Pharmacol. [1992] 41:31-37). Receptors containing the .alpha.3
subunit are also likely to be found in the brain but predominate in
the peripheral nervous system (Halvorsen, S. W. and D. K. Berg, J.
Neurosci. [1990] 10:1711-1718). The properties of both brain and
ganglionic nicotinic ACh receptor (nAChR) can be modified by the
co-assembly with the nonessential .alpha.5 subunit (Conroy, W. G.
et al., Neuron. [1992] 9:679-691; Wang, F. et al., J. Biol. Chem.
[1996] 271:17656-17665; Gerzanich, V. et al., J. Pharmacol. Exp.
Ther. [1998] 286:311-320). Another important type of brain
nicotinic receptor subtype are those that bind .alpha.-bungarotoxin
with high affinity. These receptors correspond to the .alpha.7
subunit gene products, which form homomeric receptors with high
calcium permeability and fast desensitization to high
concentrations of agonist.
[0008] Furthermore, nicotinic receptor subunits exhibit
considerable promiscuity in their ability to coassemble to form
functional channels in various expression systems. Therefore, it is
possible that alternative subunit combinations may result under
certain conditions (e.g., tissue injury, chronic drug exposure). By
recombinant expression study with specific combinations of receptor
subunits, the relative efficacy and potency of available nicotinic
agonists and antagonists have been defined (Brioni et al., Adv.
Pharmacol. [1997] 37:153-214; Holladay et al., J. Med. Chem. [1997]
40:4169-4194; Lloyd and Williams, J. Pharmacol. [2000]
292:461-467). Therefore, there would seem an opportunity for
developing drugs that have greatly increased selectivity with
respect to receptor subtype specificity. Unfortunately, subtype
selective agonists and antagonists have been only slowly
forthcoming.
[0009] Many of the experimental new nicotinic agents being
considered for clinical development, including GTS-21, ABT-418,
ABT-089, and SIB-1553A (Meyer et al., Brain Res. [1997] 768:49-56;
Papke et al., Br. J. Pharmacol. [1997] 120:429-438; Sullivan et
al., J. Pharmacol. Exp. Ther. [1997] 283:235-246; Lloyd et al.,
Life Sci. [1998] 62:1601-1606), have very mixed profiles of agonist
and antagonist activity, meaning that each agent has both
excitatory and inhibitory effects on nicotinic receptors in the
brain. This mixed pharmacological profile is also observed in the
prototypic cholinergic ion channel agonist, nicotine.
Unfortunately, because of their mixed agonist/antagonist profiles,
the toxic side effects produced by these agents hinder their
development as therapeutic drugs.
[0010] Accordingly, considerable need exists for agents which
selectively target nicotine acetylcholine receptors and individual
receptor subtypes (i.e., receptor subunit combinations), and which
avoid the toxic side effects associated with the administration of
compounds that are mixed activators and inhibitors of nicotine
acetylcholine receptors.
BRIEF SUMMARY OF THE INVENTION
[0011] The subject invention arose out of the discovery that when
metanicotine (also known as TC-2403, RJR-2403, and
(E)-N-methyl-4-(3-pyridinyl)-3-butene-1-amine) is co-administered
with a compound that both activates and inhibits one or more types
of nicotine acetylcholine receptors (nAChRs), metanicotine will
protect nicotinic acetylcholine receptor (nAChR) function from the
inhibitory effects of the compound.
[0012] The subject invention concerns methods of treating a patient
suffering from a neurological condition characterized by the
dysfunction of nAChRs by the co-administration of a metanicotine,
or a pharmaceutically acceptable salt or analogue thereof, and a
compound having either: (1) an antagonist profile of action toward
one or more nAChR subtypes; or (2) a mixed agonist/antagonist
profile of action toward one or more nAChR subtypes. The
metanicotine and the nAChR antagonist or mixed nAChR
agonist/antagonist can be simultaneously administered or
consecutively administered. If administered simultaneously, the
metanicotine and the nAChR antagonist, or the metanicotine and the
mixed nAChR agonist/antagonist, can be administered as separate
compounds, or administered together as a pharmaceutical composition
of the subject invention. Therefore, in another aspect, the subject
invention also pertains to pharmaceutical compositions containing
metanicotine, or a pharmaceutically acceptable salt or analogue
thereof, and an nAChR antagonist or mixed nAChR agonist/antagonist.
The combination of nicotinic receptor ligands, incorporating
metanicotine and a mixed agonist/antagonist or metanicotine and an
nAChR antagonist, results in the reduction or elimination of
undesirable effects that would otherwise be associated with
administration of the mixed agonist/antagonist alone or the
antagonist alone. This innovative approach facilitates the
development of therapies for a number of neurological disorders,
with improved selectivity for nAChR subtypes. Thus, the methods of
the subject invention provide a therapeutic window for utilization
of such nAChR antagonists or mixed agonists/antagonists in the
treatment of neurological conditions where one previously did not
exist.
[0013] Preferably, the methods and compositions of the subject
invention are administered to treat a patient suffering from a
neurological disorder associated with dysfunction of one or more
subtypes of neuronal nAChR. Neurological disorders which can be
treated with pharmaceutical compositions of the present invention,
and in accordance with methods of the present invention, include,
but are not limited to, presenile dementia (early onset Alzheimer's
disease), senile dementia (dementia of the Alzheimer's type),
Parkinsonism including Parkinson's disease, Huntington's chorea,
tardive dyskinesia, hyperkinesias, mania, attention deficit
disorder, attention deficit hyperactivity disorder, sleep-wake
disorder, chronic-fatigue syndrome, tremor, epilepsy, neuropathic
pain, addiction (e.g., nicotine addiction), anxiety, dyslexia,
schizophrenia, obsessive-compulsive disorder, and Tourette's
syndrome.
[0014] Metanicotine is an effective activator of the
.alpha.4.beta.2 neuronal nAChR subtype, with activity comparable
with that of acetylcholine (ACh) (Bencherif, M. et al., J.
Pharmacol. Exp. Ther. [1996] 279:1413-1421; Lippiello, P. M. et
al., J. Pharmacol. Exp. Ther. [1996] 279:1422-1429). Furthermore,
metanicotine can be distinguished from nicotine and other mixed
agonists/antagonists by the relatively low level of residual
inhibition (or desensitization) that occurs after receptor
activation (Papke et al., J. Neurochem. [2000] 75(1):204-216).
Although metanicotine is particularly useful in the methods and
compositions of the present invention for its ability to reduce the
inhibitory effects of nAChR antagonists or mixed nAChR
agonists/antagonists, therapeutic effect can also be derived from
the nAChR agonistic activity exhibited by metanicotine itself.
[0015] Mixed nAChR agonists/antagonists that can be utilized in the
subject invention include, but are not limited to, acetylcholine
(ACh), nicotine, GTS-21 (also known as
3-[2,4-dimethoxybenzylidene]-anabaseine and DMXB), ABT-089 (also
known as 2-methyl-3-(2-(S)-pyrrolidinyl methoxy)pyridine), ABT-418
(also known as (S)-3-methyl-S-(1-methyl-2-pyrr-
olidinyl)isoxazole), ABT-594 (also known as
(R)-5-(2-azetidinyl-methoxy)-2- -chloropyridine), SIB-1508Y (also
known as altinicline), SIB-1553A (also known as
(.+-.)-4-{[2-(1-methyl-2-pyrrolidinyl)ethyl]thio}phenol
hydrochloride), and epibatadine, or pharmaceutically acceptable
salts or analogues thereof having a mixed agonist/antagonist nAChR
profile. Those nAChR antagonists that can be utilized in the
subject invention include, but are not limited to,
mecamylamine.
[0016] The fact that metanicotine can protect nicotinic receptors
from the inhibitory after-effects of other potentially therapeutic
agonists is of great clinical significance. The subject invention
permits the tuning of the selectivity of specific compounds to
increase desired effects and diminish side effects. In this way,
co-administration of metanicotine with other compounds can provide
a means to tune a spectrum of effects to enhance receptor
subtype-selective activation, thereby producing a more positive
profile of effects. In one embodiment, metanicotine and/or the
mixed nAChR agonist/antagonist interact with one or more of the
nAChR .alpha.2-.alpha.10 and .beta.2-.beta.4 subunits. In another
embodiment, metanicotine and/or the mixed nAChR agonist/antagonist
interact with heteromeric nAChR subunit combinations of
.alpha.2-.alpha.6 and .beta.2-.beta.10, homomeric nAChR subunit
combinations of .alpha.7-.alpha.10, or both. In a further
embodiment, metanicotine and/or the nAChR antagonist interact with
one or more of the nAChR .alpha.2-.alpha.10 and .beta.2-.beta.4
subunits.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIGS. 1A and 1B show the effects of .beta.4 TM2 6' 10'
mutations on control ACh responses. Figure A shows control ACh
responses from an oocyte expressing wild-type .alpha.3.beta.4
receptors showing the stability of the responses recorded at 5
minute intervals. Figure B shows responses of oocytes expressing
.alpha.3.beta.4 (.beta.4 TM2 6' 10') receptors. As shown in the
upper trace, when ACh is applied at five minute intervals there is
a significant drop in the second response, and then control ACh
responses remain relatively stable. The lower trace shows that
control ACh responses show essentially full recovery between the
first and second applications with a 10 minute wash period.
[0018] FIGS. 2A and 2B show the concentration-response curves for
the effect of nicotine on .alpha.3.beta.4 and
.alpha.3.beta.4(6'F10'T) receptors. FIG. 2A shows the
concentration-response relationship for the activation of the peak
currents by nicotine. Data are expressed relative to ACh maximum
responses, see methods. FIG. 2B shows the concentration-response
relationship for the recovery of control ACh response amplitude,
measured five minutes after nicotine was applied at the indicated
concentrations. Each point represents the average normalized
response of at least four cells.
[0019] FIGS. 3A-3D show the concentration-response curves for the
effect of DMXB and TC-2403 on .alpha.3.beta.4 and
.alpha.3.beta.4(6'F10'T) receptors. FIG. 3A shows the
concentration-response relationship for the activation of the peak
currents by DMXB. Data are expressed relative to Ach maximum
responses, see methods. FIG. 3B shows the concentration-response
relationship for the recovery of control Ach response amplitude,
measured 5 minutes after DMXB was applied at the indicated
concentrations. FIG. 3C shows the concentration-response
relationship for the activation of the peak currents by TC-2403.
Data are expressed relative to Ach maximum responses, see methods.
FIG. 3D shows the concentration-response relationship for the
recovery of control ACh response amplitude, measured five minutes
after TC-2403 was applied at the indicated concentrations. Each
point represents the average normalized response of at least four
cells.
[0020] FIGS. 4A and 4B show the effect of hyperpolarization on the
inhibition measured after the application of agonists. FIG. 4A
shows oocytes expressing wild-type .alpha.3.beta.4 receptors which
were treated with 100 .mu.M DMXB at a holding potential of either
-50 or -100 mV. After a five minute wash at the test potential,
control applications of ACh were measured and expressed relative to
initial control responses obtained at the same potential. FIG. 4B
shows oocytes expressing .alpha.3.beta.4(6'F10'T) receptors which
were treated with the indicated agonists at a holding potential of
either -50 or -100 mV. After a five minute wash at the test
potential, control applications of ACh were measured and expressed
relative to initial control responses obtained at the same
potential. For this experiment, the initial ACh control
measurements were obtained ten minutes before the experimental
agonist application in order to minimize the residual inhibition
produced by the first ACh control. (* indicates p<0.05; **
indicates p<0.01; *** indicates p<0.001.
[0021] FIGS. 5A-5D show use-dependence of inhibition by DMXB. FIG.
5A shows raw data obtained from an oocyte expressing wild-type
.alpha.3.beta.4 receptors. The cell was first stimulated with 100
.mu.M ACh alone (left arrow) and then with 30 DMXB in combination
with 100 .mu.M ACh (thick gray trace). After a five minute wash,
the cell was re-tested with 100 .mu.M ACh (recovery, right arrow).
FIG. 5B shows data obtained from oocytes expressing wild-type
.alpha.3.beta.4 receptors treated with 30 or 100 .mu.M DMXB, alone
or in combination with 100 .mu.M ACh. After a five minute wash, the
cells were then re-evaluated for their response to control
applications of 100 .mu.M Ach. The data plotted represent average
residual ACh control responses of at least four oocytes. FIG. 5C
shows raw data obtained from an oocyte expressing wild-type
.alpha.3.beta.4(6'F10'T) receptors. The cell was first stimulated
with 100 .mu.M ACh alone (left arrow) and then with 30 DMXB in
combination with 100 .mu.M ACh (thick gray trace). After a five
minute wash, the cell was re-tested with 100 .mu.M Ach (recovery,
right arrow). FIG. 5D shows data obtained from oocytes expressing
.alpha.3.beta.4(6'F10'T) receptors treated with 30 .mu.M DMXB,
alone or in combination with 100 .mu.M ACh. After a five minute
wash, the cells were then re-evaluated for their response to
control applications of 100 .mu.M ACh. The data plotted represent
average residual ACh control responses of at least four
oocytes.
[0022] FIGS. 6A and 6B show protection of receptor function. FIG.
6A shows oocytes expressing wild-type .alpha.3.beta.4 receptors
which were treated with 100 .mu.M DMXB alone or in the presence of
20 .mu.M QX-314, 100 .mu.M tetracaine, or TC-2403. Control 100
.mu.M ACh responses were then measured after a five minute wash and
compared to the initial ACh control responses. Only the
co-application of 100 .mu.M TC-2403 was effective at decreasing the
residual inhibition measured after the application of 100 .mu.M
DMXB. In FIG. 6B, the bars on the right illustrate the results
obtained when oocytes expressing .alpha.3.beta.4(6'F10'T) receptors
were treated with 100 .mu.M DMXB alone or in co-application with
200 .mu.M QX-314, 100 .mu.M tetracaine, or 100 .mu.M TC-2403.
Control 100 .mu.M ACh responses were measured after a five minute
wash and compared to the initial ACh control responses. Only the
co-application of 100 .mu.M TC-2403 was effective at decreasing the
residual inhibition measured after the application of 100 .mu.M
DMXB. The bars on the left illustrate the results obtained when
oocytes expressing .alpha.3.beta.4(6'F10'T) receptors were treated
with 300 .mu.M nicotine alone or in co-application with 200 .mu.M
QX-314, 300 .mu.M tetracaine, or 100 .mu.M TC-2403. Note that
nicotine is an efficacious agonist for the mutant receptor, and we
have shown that the inhibitory effects of tetracaine on the mutant
receptor are decreased with high levels of agonist activation
(Papke, R. L. et al., Mol. Pharm. 60:1-10). Therefore, a somewhat
higher concentration of tetracaine was used in this nicotine
protection experiment. Control 100 .mu.M ACh responses were
measured after a five minute wash and compared to the initial ACh
control responses. Only the co-application of 100 .mu.M TC-2403 was
effective at decreasing the residual inhibition measured after the
application of 300 .mu.M nicotine Note that for both panels A and
B, cells were pre-equilibrated for 12 seconds with QX-314,
tetracaine, or TC-2403 before co-application of DMXB or nicotine
with the indicated agents.
BRIEF DESCRIPTION OF SEQUENCES
[0023] SEQ ID NO. 1 is the twenty amino acid residues of the second
transmembrane sequence of the nicotinic acetylcholine receptor
.alpha.3 subunit.
[0024] SEQ ID NO. 2 is the twenty amino acid residues of the second
transmembrane sequence of the nicotinic acetylcholine receptor
.beta.4 subunit.
[0025] SEQ ID NO. 3 is the twenty amino acid residues of the second
transmembrane sequence of the nicotinic acetylcholine receptor
.beta.1 subunit.
DETAILED DISCLOSURE OF THE INVENTION
[0026] The present invention relates to methods for the prevention
or treatment of disorders, such as central nervous system
disorders, which are characterized by the dysfunction of nicotinic
acetylcholine receptors (nAChRs). In a preferred embodiment, the
methods of the present invention involve co-administration of
metanicotine, or a pharmaceutically acceptable salt or analogue
thereof, and at least one compound having a mixed
agonist/antagonist profile of action toward one or more nAChR
subtypes to a patient. The metanicotine and the mixed nAChR
agonist/antagonist can be simultaneously administered or
consecutively administered. If administered simultaneously, the
metanicotine and the mixed nAChR agonist/antagonist can be
administered as separate compounds, or administered together as a
pharmaceutical composition. Therefore, in another aspect, the
present invention includes a pharmaceutical composition containing
metanicotine, or a pharmaceutically acceptable salt or analogue
thereof, and at least one compound having a mixed
agonist/antagonist profile of action toward one or more nAChR
subtypes.
[0027] As used herein, the term "agonist" means those agents or
compounds that directly or indirectly interact with one or more
subtypes of nAChR and stimulate or facilitate activity of the
nAChR. These include both "direct agonists", i.e., those that bind
to the same site on the receptor as the natural ligand,
acetycholine, and "indirect agonists", i.e., those that bind to
alternative sites on the receptor. Agonist activity can include
stabilizing the receptor open-state, for example. As used herein,
the term "antagonist" means those agents or compounds that directly
or indirectly interact with one or more types of nAChR and inhibit
the activity of the nAChR. Nicotinic antagonists may block the
acetylcholine binding site, affect the agonist affinity state of
the receptor, may block the integral ion channel itself, or they
may bind to alternatives and so induce or stabilize nonconducting
states of the receptor.
[0028] As used herein, the term "mixed agonist/antagonist" and
"compounds exhibiting a mixed agonist/antagonist nAChR profile"
refer to compounds that act as both agonists and antagonists toward
at least one nAChR subtype. The agonist and antagonist activity can
occur at the same nAChR subunit or at different nAChR subunits.
Mixed agonists/antagonists include those compounds which, upon
exposure to an nAChR, initially increase receptor activation, but
will subsequently decrease receptor responsiveness
("agonist-induced residual inhibition"). Agonist-induced residual
inhibition includes classical desensitization produced by the
binding of agonist to the activation sites, or alternatively
decrease in subsequent evoked responses due to the effect of
agonist binding at sites other than those sites which promote
activation. These compounds may simply be described as "agonists"
or "partial agonists" in the scientific literature, and as
"inhibitory agonists" herein.
[0029] Mixed nAChR agonists/antagonists that can be utilized in the
subject invention include, but are not limited to, acetylcholine
(ACh), nicotine, GTS-21 (also known as
3-[2,4-dimethoxybenzylidene]-anabaseine and DMXB), ABT-089 (also
known as 2-methyl-3-(2-(S)-pyrrolidinyl methoxy)pyridine), ABT-418
(also known as (S)-3-methyl-S-(1-methyl-2-pyrr-
olidinyl)isoxazole), ABT-594 (also known as
(R)-5-(2-azetidinyl-methoxy)-2- -chloropyridine), SIB-1508Y (also
known as altinicline), SIB-1553A (also known as
(.+-.)-4-{[2-(1-methyl-2-pyrrolidinyl)ethyl]thio}phenol
hydrochloride), and epibatadine, or pharmaceutically acceptable
salts or analogues thereof having a mixed agonist/antagonist nAChR
profile. nAChR antagonistis that can be utilized in the subject
invention include, but are not limited to, mecamylamine (also known
as 3-methylamino-2,2,3-trime- thylnorcamphane). Mecamylamine and
other nAChR antagonists that can be utilized in the subject
invention are described in U.S. Pat. No. 6,034,079 (Sanberg et
al.), for example.
[0030] Receptor specificities for the above mixed nAChR
agonists/antagonists have been described in the scientific
literature (e.g., Papke R. L. et al., J. Neurochem. [2000]
75:204-216; de Fiebre et al., Mol. Pharmacol. [1995] 47:164-171;
Meyer et al., Brain Res. [1997] 768:49-56; Sullivan et al., J.
Pharm. Exp. Ther. [1997] 283:235-246; Papke et al., Br. J. Pharm.
[1997] 120:429-438; Donnelly-Roberts et al., J. Pharmacol. Exp.
Ther. [1998] 285:777-786; Cosford et al., J Med Chem [1996]
39:3235-3237; Cosford et al., Pharm Acta Helv [2000] 74:125-130;
Washburn et al., 27th Annual Meeting of the Society for
Neuroscience [1997] p. 477; Vernier et al., J Med Chem [1999]
42:1684-1686).
[0031] In one embodiment, metanicotine and/or the mixed nAChR
agonist/antagonist interact with one or more of the nAChR
.alpha.2-.alpha.9 and .beta.2-.beta.4 subunits. In another
embodiment, metanicotine and/or the mixed nAChR agonist/antagonist
interact with heteromeric nAChR subunit combinations of
.alpha.2-.alpha.6 and .beta.2-.beta.4, homomeric nAChR subunit
combinations of .alpha.7-.alpha.9, or both. In a further
embodiment, metanicotine and/or the nAChR antagonist interact with
one or more of the nAChR .alpha.2-.alpha.10 and .beta.2-.beta.4
subunits.
[0032] Neurological disorders that can be treated with the methods
and compositions of the subject invention include disorders
associated with dysfunction of nAChRs activity and particularly
those disorders characterized by dysfunction of nicotinic
cholinergic neurotransmission, including disorders involving
neuromodulation of neurotransmitter release. The nAChR dysfunction
can involve hyperactivity or hypoactivity of one or more nAChR
subtypes. nAChR hypoactivity can be caused, for example, by
dysfunction of existing receptors, or by deficits of binding sites
related to alterations of nAChR synthesis on the levels of (i)
transcription, (ii) translation and posttranslational
modifications, and (iii) receptor transport and turnover, including
membrane insertion.
[0033] The methods and pharmaceutical compositions of the present
invention are useful for treating those types of conditions and
disorders for which other types of nicotinic receptor ligands have
been proposed as therapeutics, for example. The methods and
pharmaceutical compositions of the present invention are useful for
the prevention and treatment of disorders, such as central nervous
system (CNS) disorders, that are characterized by an alteration in
normal nAChR function, e.g., alteration in normal neurotransmitter
release. Such neurological disorders can be drug induced; can be
attributed to genetic predisposition, infection or trauma; or can
be of unknown etiology. Such neurological disorders include
neuropsychiatric disorders, neurological diseases and mental
illnesses; and include neurodegenerative diseases, behavioral
disorders, cognitive disorders and cognitive affective
disorders.
[0034] Preferably, the methods and compositions of the subject
invention are administered to treat a patient suffering from a
neurological disorder associated with dysfunction of one or more
subtypes of neuronal nAChR. Neurological disorders which can be
treated with pharmaceutical compositions of the present invention,
and in accordance with methods of the present invention, include,
but are not limited to, presenile dementia (early onset Alzheimer's
disease), senile dementia (dementia of the Alzheimer's type),
Parkinsonism including Parkinson's disease, Huntington's chorea,
tardive dyskinesia, hyperkinesias, mania, attention deficit
disorder, attention deficit hyperactivity disorder, sleep-wake
disorders, chronic-fatigue syndrome, tremor, epilepsy, neuropathic
pain, addiction (e.g., nicotine addiction), anxiety, dyslexia,
schizophrenia, obsessive-compulsive disorder, and Tourette's
syndrome.
[0035] Other disorders associated with dysfunction of one or more
subtypes of neuronal nAChR which can be treated with the
pharmaceutical compositions of the present invention, and in
accordance with methods of the present invention, can be found
throughout the scientific literature (e.g., Benowitz N. L., Annu.
Rev. Pharmacol. Toxicol. [1996] 36:597-613; Vidal C., Mol. Chem.
Neuropathol. [1996] 28:3-11; Newhouse P. A. et al., Drugs Aging
[1997] 11:206-228; Adler L. E. et al., Schizophr. Bull. [1998]
24:189-202; Lloyd G. K. et al., Life Sci. [1998] 62:1601-1606;
Lloyd G. K. and Williams M., Brain Res. [2000] 768:49-56;
Mihailescu S. and Drucker-Colin R., Arch. Med. Res. [2000]
31:131-144; Newhouse P. A. and Kelton M., Pharm. Acta. Helv. [2000]
74:91-101; Rusted J. M., and Newhouse P. A., Behav. Brain Res.
[2000] 113:121-129; and Young J. M. et al. Clin. Ther. [2001]
23:532-565).
[0036] The methods and pharmaceutical compositions of the present
invention provide therapeutic benefit to individuals suffering from
such disorders and exhibiting clinical manifestations of such
disorders in that the compounds within those compositions,
particularly when employed in effective amounts, have the potential
to (i) exhibit nicotinic pharmacology and affect relevant nicotinic
receptor sites (e.g., act as a pharmacological agonist to activate
nicotinic receptors), and (ii) elicit neurotransmitter secretion,
and hence prevent and suppress the symptoms associated with those
diseases. In addition, the compounds have the potential to (i)
increase the number of nicotinic cholinergic receptors of the brain
of the patient, (ii) exhibit neuroprotective effects, and/or (iii)
when employed in effective amounts do not cause appreciable adverse
side effects (e.g., significant increases in blood pressure and
heart rate, significant negative effects upon the gastro-intestinal
tract, and significant effects upon skeletal muscle).
[0037] The present invention also relates to a method for providing
prevention of a neurological condition or disorder to a patient
susceptible to such a condition or disorder. For example, the
method involves administering to a patient an amount of
metanicotine and an amount of a compound having either antagonist
nAChR activity, or mixed agonist/antagonist nAChR activity, wherein
the metanicotine and compound are, together, effective for
providing some degree of prevention of the progression of a
neurological disorder (i.e., provide protective effects),
amelioration of the symptoms of a neurological disorder, and
amelioration of the reoccurrence of a neurological disorder. The
methods of the present invention also involve administering an
effective amount of a pharmaceutical composition incorporating a
compound having either an nAChR antagonist activity profile or a
mixed agonist/antagonist nAChR activity profile, and metanicotine,
or a pharmaceutically acceptable salt or analogue thereof.
Alternatively, the method involves the co-administration of a
compound having an antagonist nAChR activity profile or a mixed
agonist/antagonist nAChR activity profile, and metanicotine, where
the compound is administered consecutively (before or after), or
simultaneous with, metanicotine. The compounds can optionally be
optically active, possessing substituent groups of a character such
that those compounds possess optical activity. Optically active
compounds can be employed as racemic mixtures or as
enantiomers.
[0038] As some of the compounds exhibiting antagonist nAChR
profiles or mixed agonist/antagonist nAChR profiles exhibit
analgesic effects, the methods of the subject invention also
pertain to methods for alleviating pain in a patient by
co-administering metanicotine and a compound exhibiting either
antagonist activity, or both agonist and antagonist activity,
toward one or more nAChRs, wherein the compound also exhibits
analgesic activity. The inhibitory effects of the compound on the
activity of one or more nAChR subtypes is reduced by
co-administration of metanicotine, thereby reducing any
side-effects associated with the inhibition, and increasing the
compound's therapeutic effectiveness.
[0039] The manner in which metanicotine and the nAChR antagonist,
or metanicotine and the mixed nAChR agonist/antagonist, can be
administered can vary. The compounds and compositions can be
administered by inhalation; in the form of an aerosol either
nasally or using delivery articles of the type set forth in U.S.
Pat. No. 4,922,901 to Brooks, et al. and 5,099,861 to Clearman et
al.; orally (e.g., in liquid form within a solvent such as an
aqueous liquid, or within a solid carrier); intravenously (e.g.,
within a saline solution); or transdermally (e.g., using a
transdermal patch). Exemplary methods for administering such
compounds and compositions will be apparent to the skilled artisan.
Certain methods suitable for administering compounds and
compositions useful according to the subject invention are set
forth in U.S. Pat. No. 4,965,074, to Leerson. The administration
can be intermittent, or at a gradual, continuous, constant or
controlled rate.
[0040] The compounds used in the subject invention, including
metanicotine, the nAChR antagonist, and the mixed nAChR
agonists/antagonists, can each be employed in a free base form or
in a salt form (e.g., as pharmaceutically acceptable salts).
Examples of suitable pharmaceutically acceptable salts include
inorganic acid addition salts such as hydrochloride, hydrobromide,
sulfate, phosphate, and nitrate; organic acid addition salts such
as acetate, galactarate, propionate, succinate, lactate, glycolate,
malate, tartrate, citrate, maleate, flimarate, methanesulfonate,
p-toluenesulfonate, and ascorbate; salts with acidic amino acid
such as aspartate and glutamate; alkali metal salts such as sodium
salt and potassium salt; alkaline earth metal salts such as
magnesium salt and calcium salt; ammonium salt; organic basic salts
such as trimethylamine salt, tritheylamine salt, pyridine salt,
picoline salt, dicyclohexylamine salt, and
N,N'-dibenzylethylenedia- mine salt; and salts with basic amino
acid such as lysine salt and arginine salt. The salts may be in
some cases hydrates or ethanol solvates.
[0041] As used herein, "analogues" of metanicotine include those
compounds which reduce or eliminate one or more of the inhibitory
effects of a nAChR antagonist or a mixed nAChR agonist/antagonist,
when the metanicotine analogue is co-administered with the nAChR
antagonist or the mixed nAChR agonist/antagonist. As used herein,
"analogues" of a particular nAChR antagonist include those
compounds which retain the ability to have an antagonistic effect
on one or more subtypes of nAChR and "analogues" of a particular
mixed nAChR agonist/antagonist include those compounds which retain
the ability to have both an agonistic effect and antagonistic
effect on one or more subtypes of nAChR.
[0042] The patient being administered the metanicotine and nAChR
antagonist, or metanicotine and mixed nAChR agonist/antagonist, can
be a human being, or other mammal.
[0043] The dose of metanicotine and nAChR antagonist, or
metanicotine and mixed nAChR agonist/antagonist, is that amount
effective to treat the neurological condition from which the
patient suffers or is susceptible to. As used herein, the terms
"effective amount", "effective dose", or "therapeutic amount" are
intended to mean that amount sufficient to pass across the
blood-brain barrier of the patient, to interact with relevant
receptor sites in the brain of the patient, and to elicit
neuropharmacological effects (e.g., elicit neurotransmitter
secretion, thus resulting in effective treatment of the disease or
condition). The effective dose can vary, depending upon factors
such as the condition of the patient, the severity of the symptoms
of the disorder, and the manner in which the metanicotine and mixed
nAChR agonist/antagonist is administered. Treatment of a
neurological condition involves a decrease of one or more symptoms
associated with the particular condition.
[0044] The metanicotine and nAChR antagonist, or metanicotine and
mixed nAChR agonist/antagonist, administered according to the
methods of the subject invention can traverse the blood-brain
barrier of the patient. As such, they have the ability to enter the
patient's central nervous system. The log P values of typical
compositions, which are useful in carrying out the present
invention are generally greater than about -0.5, often are greater
than about 0, and frequently are greater than about 0.5. The log P
values of such compositions are generally less than about 3, often
are less than about 2, and frequently are less than about 1. Log P
values provide a measure of the ability of a compound to pass
across a diffusion barrier, such as a biological membrane. See, for
example, Hansch, et al., J. Med. Chem. 11:1 (1968).
[0045] The pharmaceutical compositions of the subject invention
have the ability to modulate one or more nAChR functions, such as
secretion of a neurotransmitter or upregulation of one or more
types of nAChR. The compositions of the subject invention have the
ability to bind to, and cause activation of, nicotinic cholinergic
receptors of the brain of the patient. As such, such compositions
have the ability to act as nicotinic agonists. nAChR function that
may be modulated includes neurotransmitter release, such as
acetylcholine, dopamine, serotonin, and norepinephrine, from the
relevant neurons (Summers and Giacobini, J. Neurosci. Res. [1995]
20:683-689; Summers et al., Neurochem. Res. [1996]
21:1181-1186).
[0046] The metanicotine and nAChR antagonist, or metanicotine and
mixed nAChR agonist/antagonist, whether administered separately, or
as a pharmaceutical composition of the present invention, can
include various other components as additives or adjuncts.
Exemplary acceptable components or adjuncts which can be employed
in relevant circumstances include antioxidants, free radical
scavenging agents, peptides, growth factors, antibiotics,
bacteriostatic agents, immunosuppressives, anticoagulants,
buffering agents, anti-inflammatory agents, anti-pyretics, time
release binders, anesthetics, steroids, and corticosteroids. Such
components can provide additional therapeutic benefit, act to
affect the therapeutic action of the metanicotine and mixed nAChR
agonist/antagonist, or act towards preventing any potential side
effects which may be posed as a result of administration of the
metanicotine and mixed nAChR agonist/antagonist.
[0047] The metanicotine and nAChR antagonist, or metanicotine and
mixed nAChR agonist/antagonist, whether administered separately or
as a pharmaceutical composition of the subject invention, can be
formulated according to known methods for preparing
pharmaceutically useful compositions. Formulations are described in
a number of sources which are well known and readily available to
those skilled in the art. For example, Remington's Pharmaceutical
Science (Martin E W [1995] Easton Pa., Mack Publishing Company,
19.sup.th ed.) describes formulations which can be used in
connection with the subject invention. Formulations suitable for
parenteral administration include, for example, aqueous sterile
injection solutions, which may contain antioxidants, buffers,
bacteriostats, and solutes which render the formulation isotonic
with the blood of the intended recipient; and aqueous and
nonaqueous sterile suspensions which may include suspending agents
and thickening agents. The formulations may be presented in
unit-dose or multi-dose containers, for example sealed ampoules and
vials, and may be stored in a freeze dried (lyophilized) condition
requiring only the condition of the sterile liquid carrier, for
example, water for injections, prior to use. Extemporaneous
injection solutions and suspensions may be prepared from sterile
powder, granules, tablets, etc. It should be understood that in
addition to the ingredients particularly mentioned above, the
formulations of the subject invention can include other agents
conventional in the art having regard to the type of formulation in
question.
[0048] All patents, patent applications, and publications referred
to or cited herein are incorporated by reference in their entirety
to the extent they are not inconsistent with the explicit teachings
of this specification.
Materials and Methods
[0049] cDNA clones. Rat cDNA clones for the neuronal receptors
(Heinemann et al., Proceedings of NATO Conference on Mechanism of
Action of Nicotinic Acetylcholine Receptor [1986]) were used. The
sequences of the TM2 domains of the relevant subunits are shown
below. Adopting the terminology proposed by Miller et al. (Miller,
C. Neuron [1988] 2:1195-1205), the 20 residues in the proposed
second transmembrane sequence are identified as 1' through 20'.
1 intracellular MEMBRANE SPANNING II extracellular ALPHA3
ValThrLeuCysIleSerValLeuLeuSerLeuThrValPheL-
euLeuValIleThrGluThrIleProSerThr (SEQ ID NO. 1) BETA4
MetThrLeuCysIleSerValLeuLeuAlaLeuThrPhePheLeuLeuLeuIleSerLysIleValProProT-
hr (SEQ ID NO. 2) BETA1 MetGlyLeuSerIlePheAlaLeuLeuThrLeuT-
hrValPheLeuLeuLeuLeuAlaAspLysValProGluThr (SEQ ID NO. 3) 1' 6' 10'
20'
[0050] Construction of site-directed mutants. Site-directed
mutagenesis was conducted with QUICKCHANGE kits (STRATAGENE,
LaJolla, Calif.). In brief, two complimentary oligonucleotides were
synthesized which contain the desired mutation flanked by 10-15
bases of unmodified nucleotide sequence. Using a thermal cycler,
Pfu DNA polymerase extended the sequence around the whole vector,
generating a plasmid with staggered nicks. Each cycle built only
off the parent strands, therefore there was no amplification of
misincorporations. After 12-16 cycles, the product was treated with
Dpn I, which digested the methylated parent DNA into numerous small
pieces. The product was then transformed into E. coli cells, which
repaired the nicks. Mutations were confirmed by DNA sequencing.
[0051] Preparation of RNA. After linearization and purification of
cloned cDNAs, RNA transcripts were prepared in vitro using the
appropriate mMessage mMachine kit from AMBION Inc. (Austin,
Tex.).
[0052] Expression in Xenopus oocytes. Mature (>9 cm) female
Xenopus laevis African toads (NASCO, Ft. Atkinson, Wis.) were used
as a source of oocytes. Prior to surgery, frogs were anesthetized
by placing the animal in a 1.5 g/L solution of MS222
(3-aminobenzoic acid ethyl ester). Eggs were removed from an
incision made in the abdomen. In order to remove the follicular
cell layer, harvested oocytes were treated with collagenase (1.25
mg/ml) from Worthington Biochemical Corporation (Freehold, N.J.)
for 2 hours at room temperature in calcium-free Barth's solution
(88 mM NaCl, 10 mM HEPES pH 7.6, 0.33 mM MgSO.sub.4, 0.1 mg/ml
gentamicin sulfate). Subsequently, stage 5 oocytes were isolated
and injected with 50 nl (5-20 ng) each of a mixture of the
appropriate subunit cRNAs following harvest. Recordings were made 1
to 7 days after injection depending on the cRNAs being tested.
[0053] Chemicals. DMXB (GTS-21) was supplied by Taiho
Pharmaceuticals (Japan). QX-314, tetracaine, (-)-Nicotine, and all
other chemicals for electrophysiology were obtained from Sigma
Chemical Co. (St. Louis Mo.). Fresh acetylcholine stock solutions
were made daily in Ringer's solution and diluted.
[0054] Electrophysiology. Oocyte recordings were made with a Warner
Instruments (Hamden, Conn.) OC-725C oocyte amplifier and RC-8
recording chamber interfaced to either a Macintosh or Gateway
personal computer. Data were acquired using Labview software
(NATIONAL INSTRUMENTS) or pClamp8 (AXON INSTRUMENTS) and filtered
at a rate of 6 Hz. Oocytes were placed in a Warner recording
chamber with a total volume of about 0.6 ml and perfused at room
temperature with frog Ringer's solution (115 mM NaCl, 2.5 mM KCl,
10 mM HEPES pH 7.3, 1.8 mM CaCl.sub.2) containing 1 .mu.M atropine
to inhibit potential muscarinic responses. A Mariotte flask filled
with Ringer's solution was used to maintain a constant hydrostatic
pressure for drug deliveries and washes. Drugs were diluted in
perfusion solution and loaded into a 2 ml loop at the terminus of
the perfusion line. A bypass of the drug-loading loop allowed bath
solution to flow continuously while the drug loop was loaded, and
then drug application was synchronized with data acquisition by
using a 2-way electronic valve. The rate of bath solution exchange
and all drug applications was 6 ml/min. Current electrodes were
filled with a solution containing 250 mM CsCl, 250 mM CsF and 100
mM EGTA and had resistances of 0.5-2 M.OMEGA.. Voltage electrodes
were filled with 3 M KCl and had resistances of 1-3 M.OMEGA..
[0055] Experimental protocols and data analysis. Current responses
to drug application were studied under two-electrode voltage clamp
at a holding potential of -50 mV unless otherwise noted. Holding
currents immediately prior to agonist application were subtracted
from measurements of the peak response to agonist. All ACh and
other drug applications were separated by wash periods of 5 minutes
unless otherwise noted. At the start of recording, all oocytes
received two initial control applications of 100 .mu.M ACh. While
there was frequently a rundown between the first and second
responses to 100 .mu.M ACh, it was determined in a series of
control experiments, that for both the wild-type and mutant
receptors, ACh responses were essentially stable after the second
100 .mu.M ACh application (see FIG. 1). Once ACh responses
stabilized, responses to experimental drug applications were
obtained in alternation with further 100 .mu.M control ACh
applications. In order to correct for the variability in the level
of channel expression in each oocyte, all drug application
responses were normalized to the respective ACh control response
obtained 5 minutes previous to the experimental drug
application.
[0056] In order to measure residual inhibitory effects of
experimental drug applications, and to otherwise confirm the
continued stability of the ACh control responses in a given cell,
comparisons were made between the responses to 100 .mu.M ACh
obtained 5 minutes before an experimental drug application (C1) and
the 100 .mu.M ACh response obtained after a further 5 minute wash
(C2). The ratio of C2/C1 is referred to as the recovery response
(FIGS. 2B, 3B and 3D). Measurements of C2/C1 that were <0.75
were taken to indicate that the experimental drug application
produced some form of residual inhibition. An oocyte was not used
for further experimental drug applications when the measurements of
C2/C1 became less than 0.75.
[0057] Means and standard errors (SEM) were calculated from the
normalized responses of at least four oocytes for each experimental
concentration. For concentration-response relations (FIGS. 2 &
3), data were plotted using Kaleidagraph 3.0.2 (ABELBECK SOFTWARE;
Reading, Pa.), and curves were generated from the Hill equation. 1
Response = I max [ agonist ] n [ agonist ] n + ( EC50 ) n ( Formula
I )
[0058] In Formula I, I.sub.max denotes the maximal response for a
particular agonist/subunit combination, and n represents the Hill
coefficient. I.sub.max, n, and the EC50 were all unconstrained for
the fitting procedures. In order to show the relative efficacy of
each experimental agonist compared to ACh, for all the
concentration response curves, the data were initially normalized
to the 100 .mu.M ACh responses obtained in the same cells and then
scaled by the ratio of 100 .mu.M ACh control responses to the
maximal ACh responses determined in separate experiments (not
shown). For both the wild-type and mutant receptors, maximal
responses to ACh were obtained with 1 mM ACh. The responses of
wild-type and mutant receptors to 1 mM ACh were respectively,
2.9.+-.0.2 (n=4) and 1.7.+-.0.13 (n=4) times larger than the 100
.mu.M ACh control responses in the same cells.
[0059] As noted above, measurements of agonist-induced residual
inhibition were made based on changes in the response to control
100 .mu.M ACh applications. The values for the C2/C1 ratios were
plotted as functions of the experimental agonist concentrations
applied between the C2 and C1 control responses. The data were then
fit to the Hill equation with n constrained to equal -1 for the
calculation of IC50 values. Determination of significant
differences between experimental and control groups (FIGS. 4A-4B
and 5A-5D) was made by t-test (unpaired two-tailed).
[0060] For experiments assessing voltage-dependence of inhibition,
oocytes were voltage-clamped at the indicated holding potential for
both control applications of ACh alone and test applications of
experimental agonists and/or antagonists. After a 5 minute wash
period, cells were given another control ACh application at the
indicated potential so that residual inhibition could be
evaluated.
[0061] Following are examples which illustrate procedures for
practicing the invention. These examples should not be construed as
limiting. All percentages are by weight and all solvent mixture
proportions are by volume unless otherwise noted.
EXAMPLE 1
Beta Subunit TM2 Mutations Promote Agonist-Induced Residual
Inhibition by ACh and Nicotine
[0062] As previously reported for receptors containing chimeric
.beta.4 subunits (Webster, J. C. et al., Br. J. of Pharmacol.
[1999] 127:1337-1348), receptors containing the .beta.4 6' and 10'
point mutations showed decreased responses to repeated applications
of ACh and nicotine, suggesting that these agonists produce some
form of residual inhibition, as shown in FIGS. 1A and 1B. While the
inhibition produced by nicotine persisted for up to an hour (not
shown), inhibition produced by ACh was more reversible, with
essentially full recovery after 7-8 minutes of wash.
[0063] The concentration-response function for nicotine's
activation and inhibition of wild-type and mutant receptors is
shown in FIGS. 2A and 2B. The presence of the 6' and 10' mutations
in the .beta.4 subunit appeared to increase the efficacy of
nicotine compared to ACh and to substantially increase the
agonist-induced residual inhibition measured after a 5 minute wash.
However, the interpretation of the efficacy data in FIG. 2A is
complicated by the fact that the measurement is based on comparison
to ACh-evoked currents. Noncompetitive inhibitory or desensitizing
effects limit the apparent efficacy of nicotine in wild-type
.alpha.3-containing receptors (Papke, R. L. et al., J. Neurochem.
[2000] 75:204-216), and the effect of the 6'/10' mutations seems to
be primarily on the rate of recovery from nicotine-induced
inhibition. It may be the case that the 6'/10' mutations are having
the effect of producing more agonist-induced residual inhibition
during an ACh-evoked response. If this is the case, then much of
the apparent increase in nicotine's relative efficacy may be due to
a decrease in ACh's absolute efficacy.
EXAMPLE 2
TM2 Mutations Differentially Regulate the Activation and Inhibition
of Subtype-Selective Agonists
[0064] As shown in FIGS. 2A and 2B, the 6'/10' mutations appear to
influence both activation and agonist-induced residual inhibition,
raising the question of whether these effects are likely to
represent multiple consequences of these agonists binding to a
single site on the receptor (i.e. the activation binding site), or
alternatively represent effects from binding to multiple sites on
the receptors. In order to test this, the effects of other agonists
on the wild-type and mutant receptors were investigated.
Specifically, two subtype-selective agents were used that
previously have been reported to be only weak partial agonists on
wild-type .alpha.3.beta.4 receptors, DMXB and TC-2403
(metanicotine). DMXB is an .alpha.7-selective partial agonist
(Meyer, E. M. et al., Brain Res. [1997] 768:49-56) that can produce
agonist-induced residual inhibition of wild-type receptors in the
absence of strong activation. As shown in FIG. 3A, the 6'/10'
.beta.4 mutations did not cause DMXB to appear as a more
efficacious agonist than for the wild-type receptor, but they did
cause DMXB to produce more inhibition after it was applied at 100
or 300 M (p<0.001), as shown in FIG. 3B. In contrast, the
greatest effect of the 6'/10' .beta.4 mutations on the activity of
the .alpha.4.beta.2-selective agonist TC-2403 was in measurement of
apparent efficacy, as shown FIG. 3C, since there was no significant
increase in residual inhibition at concentrations less than 1 mM,
as shown in FIG. 3D. Even after the application of 1 mM TC-2403,
residual inhibition was minimal; responses were still 70.+-.6% of
the pre-application control values.
2TABLE I Curve fits for Hill equations Agonist Receptor Max
response* n EC.sub.50 (.mu.M) Figure reference Nicotine
.alpha.3.beta.4 0.88 .+-. 0.04 1.2 .+-. 0.2 96 .+-. 16 2A
.alpha.3.beta.4 (6'F10'T) 0.20 .+-. 0.05 0.8 .+-. 0.3 30 .+-. 18 2B
TC-2403 .alpha.3.beta.4 0.09 .+-. 0.01 2.1 .+-. 0.2 347 .+-. 12 3C
.alpha.3.beta.4(6'F10'T) 0.38 .+-. 0.01 2.1 .+-. 0.2 111 .+-. 5 3C
ACh.sup..dagger. .alpha.3.beta.4 1.0 2.1 .+-. 0.6 68 .+-. 12
.alpha.3.beta.4 (6'F10'T) 1.0 0.8 .+-. 0.1 72 .+-. 17 *relative to
ACh Maximum .sup..dagger.data taken from Papke et al., 2001
[0065]
3TABLE II IC.sub.50 values Holding FIGURE Drug Receptor potential
IC.sub.50 (.mu.M) reference Nicotine .alpha.3.beta.4 -50 >1000
2B .alpha.3.beta.4 (6'F10'T) -50 29 .+-. 5 2B DMXB .alpha.3.beta.4
-50 121 .+-. 48 3B .alpha.3.beta.4 (6'F10'T) -50 23 .+-. 17 3B
EXAMPLE 3
The Residual Inhibition Produced by Agonists is Voltage
Dependent
[0066] The voltage dependence of the residual inhibition of
wild-type .alpha.3.beta.4 and .alpha.3.beta.4(6'F10'T) receptors
produced by DMXB was evaluated, as well as the enhanced inhibition
of .alpha.3.beta.4(6'F10'T) mutant receptors by nicotine and ACh to
see if the inhibition had properties that would be consistent with
open channel blockade. Cells were held at either -50 mV or -100 mV
and tested for their response to control concentrations of ACh.
After a 10 min wash, test agonists (ACh, DMXB or nicotine) were
applied at the concentrations indicated. Cells were then washed for
5 min and tested again for their response to a control ACh
application. Cells were held at the indicated holding potential
throughout the entire procedure. As shown in FIGS. 4A and 4B, the
residual inhibition of both wild-type and mutant receptors was
enhanced if the cells were held at a hyperpolarized potential. This
would be consistent with inhibition associated with binding to a
channel-associated site (e.g. open channel block).
EXAMPLE 4
DMXB-Induced Inhibition of Mutant Receptors is Use-Dependent
[0067] As shown in FIG. 5A, the inhibition of wild-type
.alpha.3.beta.4 receptors by DMXB is not enhanced when the weak
partial agonist DMXB is co-applied with the strong agonist ACh.
This is in contrast with the previous observation that DMXB-induced
inhibition of .alpha.4.beta.2 receptors is enhanced when the drug
is co-applied with ACh (de Fiebre, C. M. et al. Mol. Pharmacol.
[1995] 47:164-171). Interestingly, this property is apparently
altered in the receptors containing the .beta.4(6'F10'T) subunit,
such that the co-application of ACh with 30 .mu.M DMXB produced a
large increase in the residual inhibition, as shown in FIG. 5B.
While the results presented in FIGS. 5A-5D suggest a clear
difference in the inhibition of wild-type and mutant receptors by
DMXB, interpretation of the data should also include consideration
that as a weak partial agonist, DMXB effectively prevents ACh from
being effective during the co-applications. That is, since DMXB is
apparently capable of both competitive interactions with ACh at the
activation site, and a use-dependent binding to a channel
associated site, when ACh is co-applied with DMXB, ACh cannot
promote the use-dependent effects because DMXB is blocking
activation. Specifically, the responses of both wild-type and
mutant receptors to 100 .mu.M ACh were decreased a similar amount
when the ACh was co-applied with 30 .mu.M DMXB. The responses of
.alpha.3.beta.4 z.alpha.3.beta.4(6'F10'T) receptors were reduced to
13.+-.2%. When 100 PM DMXB was co-applied to wild-type receptors
with 100 .mu.M ACh, the activation was only 4.+-.0.2% the
activation produced by ACh alone. It may be the case that, for the
wild-type receptors, DMXB may have a similar affinity for both
activation sites and inhibitory sites so that competitive
inhibition masks use-dependent effects. If in the mutant receptors,
there is increase only in affinity for the inhibitory site,
compared to wild-type, then use-dependent inhibition would become
more apparent.
EXAMPLE 5
Protection of (6'F10'T) Receptors from Residual Inhibition Produced
by Mixed Agonists/Antagonists
[0068] Since QX-314 (also known as
2-(triethylamino)-N-(2,6-dimethylphenyl- )-acetamide) and
tetracaine both produce a readily reversible voltage-dependent
inhibition of .alpha.3.beta.4 receptors, it was determined whether,
by pre-treating the receptors with a high concentration of these
agents so as to saturate their binding sites, the receptors'
function could be protected from the relatively long-lived
inhibition produced by the application of 100 .mu.M DMXB. As shown
in FIG. 6A, neither of these agents provided any protection from
the DMXB-induced inhibition. However, when cells were pretreated
with TC-2403 (metanicotine), there was a significant (p<0.05)
reduction in the residual inhibition produced by DMXB, as shown in
FIG. 6A. These same agents were evaluated for their ability to
protect .alpha.3.beta.4(6'F10'T) receptors from the residual
inhibition produced by either 100 .mu.M DMXB or 300 .mu.M nicotine.
As shown in FIG. 6B, significant protection of receptor function
was provided by TC-2403 and not by the local anesthetics (p<0.01
for DMXB inhibition and p<0.05 for nicotine inhibition).
[0069] The mutations characterized in the beta subunit TM2 domain
were first identified because they determined the sensitivity of
neuronal receptor subtypes to selective noncompetitive inhibitors
classified as ganglionic blockers based on their preferential
inhibition of receptors containing neuronal-type beta subunits
(i.e. .beta.2 or .beta.4). It was noted that in addition to having
a reduced sensitivity to the classic ganglionic blocker
mecamylamine, compared to wild-type, receptors with the
.beta.4(6'F10'T) subunit also showed increased residual inhibition
after exposure to nicotine (Webster, J. C. et al., Br. J. of Pharm.
[1999] 127:1337-1348). Therefore, in order to determine to what
extent the effects of these mutations might be generalized to other
agonists, the analysis of .alpha.3.beta.4(6'F10'T) receptors was
expanded to look at the effects of these mutations on the activity
of the two selective partial agonists DMXB and TC-2403. Competition
experiments were conducted in order to determine whether the
increased agonist-induced residual inhibition observed was most
consistent with enhanced desensitization, channel block by agonist
(steric inhibition), or alternatively some form of allosteric
inhibition.
[0070] A strict classical definition of desensitization requires
that the inhibition follows from binding to the very same sites
where the agonists bind to promote channel activation. On the other
hand, channel block or "steric" inhibition would be distinguished
as the binding of the agonist molecules to a site (or sites) within
the conduction pathway, such that the presence of the agonist
prevents current flow. In the case of allosteric inhibition, the
agonists would be assumed to be binding to a class of sites that
selectively stabilized the closed or desensitized states without
directly blocking the channel.
[0071] The results obtained with the two selective agonists DMXB
and TC-2403 suggest that the effects of the 6'/10' mutations on
apparent efficacy and inhibition may not be interdependent since
DMXB manifested only increased inhibition and TC-2403 manifested
primarily an increase in relative efficacy. One possibility might
be that the mutations are affecting the coupling efficiency between
agonist binding and channel gating for some agonists (i.e. nicotine
and TC-2403), but at the same time improving a binding site within
the channel for a steric inhibition (i.e. open-channel block) by
other agonists (i.e. all but TC-2403). Alternatively, the mutations
may simply promote more rapid desensitization (although, again
TC-2403 would be the exception).
[0072] However, defining desensitization as an inactivation process
promoted by the binding (or retention of) agonist at the activation
binding site, it would seem unlikely that the inactivation of
.alpha.3.beta.4(6'F10'T) receptors by DMXB would represent
desensitization since it is promoted by the binding of ACh to the
activation site. This apparent use-dependence would also suggest
that DMXB, nicotine, and ACh may have their enhanced inhibitory
effects by binding to sites within the ion channel such as those
associated with open channel block. Such a mechanism would also be
consistent with the observed voltage dependence for inhibition of
both the wild-type and mutant receptors.
[0073] The local anesthetic QX-314 has been characterized as an
open channel blocker of various nAChR subtypes (Neher, E. and J. H.
Steinbach J Physiol [1978] 277:135-176; Pascual, J. M. and A.
Karlin J. Gen. Physiol. [1998] 112(5):611-621; Horn, R. et al.,
Science [1980] 210(4466):205-207; Francis, M. M. et al., Biophys.
J. [1998] 74(5):2306-2317; Wilson, G. and A. Karlin Proc. Natl.
Acad. Sci. USA [2001] 98(3):1241-1248) (3, 12-15). Tetracaine has
been shown to have both competitive and noncompetitive effects on
nAChR function. When functioning as a noncompetitive antagonist,
tetracaine appears to have comparable affinity for receptors in the
resting and open states (Middleton, R. E. et al., Mol. Pharmacol.
[1999] 56(2):290-299; Takayama, H. et al., J. Pharmacol. Exp. Ther.
[1989] 251(3):1083-1089; Papke, R. L. and R. E. Oswald J. Gen.
Physiol. [1989] 93:785-811; Blanton, M. P. et al., J. Biol. Chem.
[2000] 275(5):3469-3478; Gallagher, M. J. and J. B. Cohen Mol.
Pharmacol. [1999] 56(2):300-307). Therefore, these two agents
should serve as effective probes for channel-associated sites and
in fact may distinguish between sites associated with different
forms of channel blockade. The fact that the residual inhibition
produced by DMXB and nicotine was unperturbed by the binding of
either QX-314 or tetracaine would argue against the idea that these
agonists produce that inhibition by binding to the same
channel-associated site recognized by the local anesthetics.
[0074] Furthermore, the observation that protection from inhibition
was provided by a drug which lacks intrinsic inhibitory activity
suggests that the binding site protected by TC-2403 is unlikely to
be a site within the conduction pathway (i.e. an open channel block
site), or TC-2403 itself would have had inhibitory effects. An
alternative interpretation therefore is that the agonists are
working at a proposed allosteric regulatory site (Arias, H. R. J.
Neurosci. Res. [1996] 44(2):97-105; Rozental, R. et al., J.
Pharmacol Exp. Ther. [1989] 249(1):123-130; Yost, C. S. and B. A.
Dodson Cell Mol. Neurobiol. [1993] 13(2):159-172; Min, C. K. and G.
A. Weiland Brain Res. [1992] 586(2):348-351), the accessibility of
which is regulated by gating.
[0075] Since TC-2403 can protect receptor function from the
long-term inhibition by mixed agonists/antagonists, TC-2403 may
bind to an allosteric site also recognized by other agonists but
not promote the inhibition of function through that binding. As
shown in FIGS. 3A-3D, TC-2403 is an effective activator of
.alpha.3.beta.4(6'F10'T) receptors, but unlike ACh, TC-2403 acts to
prevent rather than promote DMXB-induced inhibition. This would
indicate that the protective effects of TC-2403 are not associated
with its binding to the activation sites (where it behaves like
ACh) but rather to different sites where the other agonists promote
inhibition but where TC-2403 does not. This would be most
consistent with an allosteric binding site, since as noted above,
it seems unlikely that TC-2430 would bind to a site within the
conduction pathway and not inhibit function. However, since the
effects of the inhibitory agonists (i.e. ACh, DMXB and nicotine) do
appear to be voltage dependent, if they are associated with binding
to an allosteric site, then it may be the case that the
conformation or accessibility of such a site is influenced by
gating and/or membrane voltage.
[0076] It appears that a specific sequence in the TM2 domain can
regulate both the sensitivity of specific nicotinic receptor
subtypes to channel blocking agents and effects at sites outside of
the ion channel conduction pathway, presumably by affecting
gating-dependent conformational changes in the receptor. This
effect of beta subunit TM2 sequence on gating-dependent
conformational changes is consistent with our previous report on
the regulation of voltage-independent use-dependent inhibition by
BTMPS (Francis, M. M. et al., Biophys. J. [1998] 74(5):2306-2317).
While the 6'/10' mutation decreases the inhibitory effects of BTMPS
at sites outside the ion channel, these mutations increase
inhibition by selected agonists and shift the affinity of
tetracaine away from a channel associated site and toward the
activation binding site.
EXAMPLE 6
Evaluation of Compounds for Agonist, Antagonist, or Mixed
Agonist/Antagonist nAChR Activity and Nicotinic Specificity
[0077] Binding of the agonist, antagonist, or mixed nAChR
agonists/antagonists utilized in the methods and compositions of
the present invention to relevant receptor sites can be determined
in accordance with the techniques described in U.S. Pat. No.
5,597,919 to Dull et al. Neurotransmitter release can be measured
using techniques similar to those previous published (Bencherif et
al., J PET [1996] 279:1413-1421). The determination of the
interaction of the compounds with muscle receptors can be carried
out in accordance with the techniques described in U.S. Pat. No.
5,597,919 to Dull et al. For example, the maximal activation for
individual compounds can be determined as a percentage of the
maximal activation induced by (S)-(-)-nicotine. Preferable
compounds have the capability to activate human CNS receptors
without activating muscle-type nicotinic acetylcholine receptors.
Therefore, at certain levels, preferred compounds show CNS effects
to a significant degree but do not show undesirable muscle effects
to any significant degree. The determination of the interaction of
the compounds with ganglionic receptors can be carried out in
accordance with the techniques described in U.S. Pat. No. 5,597,919
to Dull et al. The measurement and characterization of nAChR
inhibition exhibited by a given compound applied alone, and when
co-applied with metanicotine, can be determined using the methods
described herein (e.g., Examples 1-5).
Sequence CWU 1
1
3 1 25 PRT Rattus norvegicus 1 Val Thr Leu Cys Ile Ser Val Leu Leu
Ser Leu Thr Val Phe Leu Leu 1 5 10 15 Val Ile Thr Glu Thr Ile Pro
Ser Thr 20 25 2 25 PRT Rattus norvegicus 2 Met Thr Leu Cys Ile Ser
Val Leu Leu Ala Leu Thr Phe Phe Leu Leu 1 5 10 15 Leu Ile Ser Lys
Ile Val Pro Pro Thr 20 25 3 25 PRT Rattus norvegicus 3 Met Gly Leu
Ser Ile Phe Ala Leu Leu Thr Leu Thr Val Phe Leu Leu 1 5 10 15 Leu
Leu Ala Asp Lys Val Pro Glu Thr 20 25
* * * * *