U.S. patent application number 12/045265 was filed with the patent office on 2008-10-30 for methods and compositions for treating thalamocortical dysrhythmia.
Invention is credited to Rodolfo R. Llinas, Mutsuyuki Sugimori, Francisco Urbano.
Application Number | 20080268067 12/045265 |
Document ID | / |
Family ID | 39496063 |
Filed Date | 2008-10-30 |
United States Patent
Application |
20080268067 |
Kind Code |
A1 |
Llinas; Rodolfo R. ; et
al. |
October 30, 2008 |
Methods and Compositions for Treating Thalamocortical
Dysrhythmia
Abstract
This invention relates to methods of inhibiting a Ca.sub.v3
calcium channel in a cell using a C.sub.2-C.sub.10 alkyl alcohol,
or mixtures thereof. This invention further relates to methods of
treating a thalamocortical dysrhythmia disorder in a mammal and for
treating a neurological disorder in a mammal associated with the
thalamocortical dysrhythmia using a C.sub.2-C.sub.10 alkyl alcohol,
a lipophilic molecule with a partition coefficient substantially
similar to that of a C.sub.2-C.sub.10 alkyl alcohol, or mixtures
thereof, or a pharmaceutical composition comprising the same.
Inventors: |
Llinas; Rodolfo R.; (New
York, NY) ; Sugimori; Mutsuyuki; (New York, NY)
; Urbano; Francisco; (Brooklyn, NY) |
Correspondence
Address: |
WILMERHALE/BOSTON
60 STATE STREET
BOSTON
MA
02109
US
|
Family ID: |
39496063 |
Appl. No.: |
12/045265 |
Filed: |
March 10, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60906085 |
Mar 9, 2007 |
|
|
|
Current U.S.
Class: |
424/641 ;
424/676; 436/501; 514/108; 514/259.41; 514/271; 514/321; 514/327;
514/356; 514/400; 514/415; 514/647; 514/652; 514/655; 514/722;
514/724; 514/731; 514/743; 703/11 |
Current CPC
Class: |
A61P 25/08 20180101;
A61P 25/18 20180101; A61K 45/06 20130101; A61K 31/045 20130101;
A61P 25/28 20180101; A61P 25/06 20180101; A61P 25/10 20180101; A61K
31/045 20130101; A61P 25/16 20180101; A61P 9/12 20180101; A61P
25/24 20180101; A61P 25/00 20180101; A61P 25/02 20180101; A61K
2300/00 20130101 |
Class at
Publication: |
424/641 ;
514/724; 703/11; 436/501; 514/271; 424/676; 514/731; 514/400;
514/647; 514/722; 514/743; 514/108; 514/415; 514/321; 514/652;
514/259.41; 514/327; 514/356; 514/655 |
International
Class: |
A61K 33/30 20060101
A61K033/30; A61K 31/045 20060101 A61K031/045; G06G 7/58 20060101
G06G007/58; A61K 33/16 20060101 A61K033/16; A61P 25/08 20060101
A61P025/08; A61K 31/05 20060101 A61K031/05; A61K 31/135 20060101
A61K031/135; A61K 31/02 20060101 A61K031/02; A61K 31/4045 20060101
A61K031/4045; A61K 31/137 20060101 A61K031/137; A61K 31/381
20060101 A61K031/381; A61K 31/4422 20060101 A61K031/4422; A61K
31/4168 20060101 A61K031/4168; A61K 31/451 20060101 A61K031/451;
A61K 31/517 20060101 A61K031/517; A61K 31/138 20060101 A61K031/138;
A61K 31/4525 20060101 A61K031/4525; A61K 31/66 20060101 A61K031/66;
A61K 31/08 20060101 A61K031/08; A61K 31/4164 20060101 A61K031/4164;
A61P 25/28 20060101 A61P025/28; A61P 25/02 20060101 A61P025/02;
G01N 33/566 20060101 G01N033/566; A61K 31/515 20060101
A61K031/515 |
Claims
1. A method of reducing a low-threshold-activated calcium current
in a cell by having a Ca.sub.v3 plasmalemmal-bound calcium channel,
the method comprising superfusing the extracellular environment of
the cell with a C.sub.2-C.sub.10 alkyl alcohol, or mixtures
thereof, in an amount sufficient to modify the voltage-dependent
activation kinetics or single channel ionic conductance of the
Ca.sub.v3 calcium channel, thereby reducing the
low-threshold-activated calcium current in the cell.
2. The method of claim 1, wherein the cell is in a mammal in need
of inhibiting a Ca.sub.v3 calcium channel.
3. The method of claim 1, wherein the cell is a neuron, an
interneuron, a projection thalamic neuron, a reticular thalamic
neuron, a cortical interneuron, cortical pyramidal neuron, a basal
ganglion neuron, a hippocampus neuron, an amygdala neuron, a tectal
neuron, or a cerebellar neuron.
4. The method of claim 1, wherein the Ca.sub.v3 calcium channel is
selected from the group consisting of a Ca.sub.v3.1 calcium
channel, a Ca.sub.v3.2 calcium channel, a Ca.sub.v3.3 calcium
channel, and combinations thereof.
5. The method of claim 1, wherein the C.sub.2-C.sub.10 alkyl
alcohol is administered in a pharmaceutically acceptable excipient,
carrier, or diluent.
6. The method of claim 1, wherein the C.sub.2-C.sub.10 alkyl
alcohol is present in the extracellular environment at a
concentration of from about 10 .mu.M to about 100 .mu.M.
7. A method of inhibiting a Ca.sub.v3 plasmalemmal-bound calcium
channel in a cell, the method comprising administering to the
mammal a C.sub.2-C.sub.10 alkyl alcohol, or mixtures thereof, at a
dose of from about 0.001 mg/kg body weight to about 20 mg/kg body
weight of the mammal, the alcohol modifying the voltage dependent
activation kinetics or single channels ionic conductance of the
Ca.sub.v3 calcium channel, thereby inhibiting the Ca.sub.v3 calcium
channel.
8. The method of claim 7, wherein the alcohol is administered
orally.
9. The method of claim 7, wherein the alcohol is administered
parenterally.
10. The method of claim 7, wherein the C.sub.2-C.sub.10 alkyl
alcohol is 1-octanol.
11. The method of claim 7, wherein the C.sub.2-C.sub.10 alkyl
alcohol is 2-octanol.
12. The method of claim 7, wherein the mammal has a thalamocortical
dysrhythmia disorder.
13. A method of treating a neurological disorder in a mammal in
need thereof, the method comprising administering to the mammal a
therapeutically effective amount of a Ca.sub.v3 calcium channel
inhibitor, provided that the neurological disorder is not tremor or
Parkinson's tremor.
14. A method of treating a thalamocortical dysrhythmia disorder
that is not tremor or Parkinson's tremor in a mammal in need
thereof, the method comprising administrating to the mammal a
therapeutically effective amount of a Ca.sub.v3 calcium channel
inhibitor wherein the inhibitor binds to the Ca.sub.v3 calcium
channel, thereby reducing a low voltage-activated calcium current
in the cell of the mammal.
15. The method of claim 14, wherein the Ca.sub.v3 calcium channel
inhibitor is a C.sub.2-C.sub.10 alkyl alcohol, or mixtures
thereof.
16. The method of claim 14, wherein the Ca.sub.v3 calcium channel
inhibitor is a lipophilic molecule with a partition coefficient
substantially similar to that of a C.sub.2-C.sub.10 alkyl
alcohol.
17. The method of claim 14, wherein the Ca.sub.v3 calcium channel
inhibitor is a lipophilic molecule with a partition coefficient
substantially similar to that of an octanol.
18. The method of claim 14, wherein the Ca.sub.v3 calcium channel
inhibitor is a lipophilic molecule with a molecular weight less
than about 160.
19. The method of claim 14, wherein the thalamocortical dysrhythmia
disorder is petit mal epilepsy.
20. The method of claim 14, wherein the thalamocortical dysrhythmia
disorder is tinnitus.
21. The method of claim 14, wherein the thalamocortical dysrhythmia
disorder is neurogenic pain.
22. The method of claim 14, wherein the thalamocortical dysrhythmia
disorder is obsessive-compulsive disorder.
23. The method of claim 14, wherein the thalamocortical dysrhythmia
disorder is Tourettes disease.
24. The method of claim 14, wherein the thalamocortical dysrhythmia
disorder is chronic depression.
25. The method of claim 14, wherein the thalamocortical dysrhythmia
disorder is autism or Asperger's syndrome.
26. The method of claim 14, wherein the thalamocortical dysrhythmia
disorder is schizoaffective psychosis.
27. The method of claim 14, wherein the thalamocortical dysrhythmia
disorder is Parkinson's disorder.
28. The method of claim 14, wherein the thalamocortical dysrhythmia
disorder is Migraine.
29. The method of claim 14, wherein the thalamocortical dysrhythmia
disorder is Absence Epilepsy.
30. The method of claim 14, wherein the thalamocortical dysrhythmia
disorder is restless legs syndrome.
31. The method of claim 14, wherein the C.sub.2-C.sub.10 alkyl
alcohol is administered in a pharmaceutically acceptable excipient,
carrier, or diluent.
32. The method of claim 14, wherein the therapeutically effective
amount administered is from about 0.001 mg/kg body weight to about
20 mg/kg body weight of the mammal.
33. The method of claim 14, wherein the therapeutically effective
amount administered is from about 0.1 mg/kg body weight to about 10
mg/kg body weight of the mammal.
34. The method of claim 33, wherein the therapeutically effective
amount administered is from about 0.3 mg/kg body weight to about
3.0 mg/kg body weight of the mammal.
35. The method of claim 33, wherein the therapeutically effective
amount administered is about 1.0 mg/kg body weight of the
mammal.
36. The method of claim 14, wherein the alcohol is administered
orally.
37. The method of claim 14, wherein the alcohol is administered
parenterally.
38. The method of claim 14, wherein the C.sub.2-C.sub.10 alkyl
alcohol is selected from the group consisting of (a)
CH.sub.3--(CH.sub.2).sub.n--OH, wherein n is an integer from 1 to
9, (b) CH.sub.3--CH(OH)--(CH.sub.2).sub.m--CH.sub.3, wherein m is
an integer from 0 to 7, (c)
CH.sub.3--CH.sub.2--CH(OH)--(CH.sub.2).sub.p--CH.sub.3, wherein p
is an integer from 0 to 6, (d)
CH.sub.3--CH.sub.2--CH.sub.2--CH(OH)--(CH.sub.2).sub.q--CH.sub.3,
wherein q is an integer from 0 to 5, (e)
CH.sub.3--CH.sub.2--CH.sub.2--CH.sub.2--CH(OH)--(CH.sub.2).sub.w--CH.sub.-
3, wherein w is an integer from 0 to 4, and (f) mixtures
thereof.
39. The method of claim 14, wherein the C.sub.2-C.sub.10 alkyl
alcohol is 1-octanol.
40. The method of claim 14, wherein the C.sub.2-C.sub.10 alkyl
alcohol is 2-octanol.
41. A method for identifying an alkyl alcohol that modifies the
voltage-dependent activation kinetics or single channel ionic
conductance of a Ca.sub.v3 plasmalemmal-bound calcium channel
protein, the method comprising: (a) providing atomic coordinates of
the Ca.sub.v3 calcium channel protein; (b) subjecting an alkyl
alcohol to computational molecular docking; and (c) selecting the
alcohol that has the optimal virtual binding to the a Ca.sub.v3
calcium channel protein, the optimal virtual binding indicating
that the alcohol binds to the a Ca.sub.v3 calcium channel protein
or alters the properties of the lipid bi-layer at the channel's
voltage sensing site.
42. The method of claim 41, wherein the computational molecular
docking utilizes a computer screening method selected from the
group consisting of Virtual library screening (VLS), ICM, ICM-VLS,
DOCK, and FlexX.
43. A method of identifying a chemical entity which binds to a
Ca.sub.v3 calcium channel, the method comprising comparing a
structure model of the Ca.sub.v3 calcium channel with a structure
model for the chemical entity, and detecting (i) a binding surface
on the Ca.sub.v3 calcium channel for the chemical entity, or (ii)
an alteration of the properties of the lipid bi-layer at the
channel's voltage sensing site for the chemical entity.
44. The method of claim 43, wherein the structural model of the
Ca.sub.v3 calcium channel is derived from a structure-predicting
algorithm.
45. The method of claim 43, wherein the Ca.sub.v3 calcium channel
is selected from the group consisting of a Ca.sub.v3.1 calcium
channel, a Ca.sub.v3.2 calcium channel, a Ca.sub.v3.3 calcium
channel, and combinations thereof.
46. The method of claim 43, wherein the structure model of the
Ca.sub.v3 calcium channel is derived from atomic coordinates
determined by subjecting a crystal comprising a Ca.sub.v3 calcium
channel to X-ray diffraction measurements.
47. A method of identifying an inhibitor of a Ca.sub.v3 calcium
channel in a cell, the method comprising: contacting an isolated
Ca.sub.v3 plasmalemmal-bound calcium channel with a candidate
compound; and detecting the presence of a complex, or lack thereof,
between the Ca.sub.v3 calcium channel and the compound, the
candidate compound being an inhibitor if it forms a complex with
the Ca.sub.v3 calcium channel or alters the properties of the lipid
bi-layer at the channel's voltage sensing site.
48. A pharmaceutical composition comprising: a therapeutically
effective amount of a C.sub.2-C.sub.10 alkyl alcohol, or mixtures
thereof; and at least one other therapeutic agent selected from the
group consisting of: a barbiturate or barbituric acid derivative,
an anesthetic agent, a tinnitus treating agent, a selective
serotonin reuptake inhibitor, an antidepressant agent, a
neuroleptic, an antihypertensive agent, a calcium channel blocker,
and an ACE inhibitor, and combinations thereof.
49. The pharmaceutical composition of claim 48, wherein the
C.sub.2-C.sub.10 alkyl alcohol is 1-octanol.
50. The pharmaceutical composition of claim 48, wherein the
C.sub.2-C.sub.10 alkyl alcohol is 2-octanol.
51. The composition of claim 48, wherein the barbiturate or
barbituric acid derivative is selected from the group consiting of
sodium thiopental, pentobarbital, secobarbital, amobarbital,
butabarbital, barbital, phenobarbital, butalbital, cyclobarbital,
allobarbital, methylphenobarbital, secobarbital, vinylbital and
methohexital.
52. The composition of claim 48, wherein the anesthetic agent is
selected from the group consisting of propofol, etomidate,
isoflurane, halothane, and ketamine.
53. The composition of claim 48, wherein the tinnitus-treating
agent is selected from the group consisting of zinc
supplementation, acamprosate, etidronate or sodium fluoride,
lignocaine, carbemazepine, melatonin, sertraline and vitamin
combinations.
54. The composition of claim 48, wherein the selective serotonin
reuptake inhibitor is selected from the group consisting of
paroxetine, sertraline, fluoxetine and fluvoxamine.
55. The composition of claim 48, wherein the antidepressant agent
is selected from the group consisting of Harmaline, Iproniazid,
Isocarboxazid, Moclobemide, Nialamide, Pargyline, Phenelzine,
Toloxatone, Tranylcypromine, Brofaromine, Moclobemide, Amineptine,
Phenmetrazine, Vanoxerine, Bupropion, Atomoxetine, Maprotiline,
Reboxetine, Viloxazine, Duloxetine, Milnacipran, Nefazodone
Venlafaxine, Alaproclate, Citalopram, Escitalopram, Etoperidone,
Fluoxetine, Fluvoxamine, Paroxetine, Sertraline, Zimelidine,
Tianeptine, Amitriptyline, Amoxapine, Butriptyline, Clomipramine,
Dibenzepin, Dothiepin, Imipramine, Iprindole, Lofepramine,
Melitracen, Nortriptyline, Opipramol, Trimipramine, Maprotiline,
Mianserin, Nefazodone, and Trazodone.
56. The composition of claim 48, wherein the neuroleptic selected
from the group consisting of risperidone, ziprasidone, haloperidol,
pimozide and fluphenazine.
57. The composition of claim 48, wherein the antihypertensive agent
is selected from the group consisting of clonidine and
guanfacine.
58. The composition of claim 48, wherein the calcium channel
blocker is selected from the group consisting of amlodipine,
felodipine, nicardipine, nifedipine, nimodipine, nisoldipine,
nitrendipine, lacidipine, lercanidipine, verapamil, gallopamil,
diltiazem, mibefradil, and menthol.
59. The composition of claim 48, wherein the ACE inhibitor is
selected from the group consisting of enalapril, ramipril,
quinapril, perindopril, lisinopril, benazepril and fosinopril.
60. A method of treating a thalamocortical dysrhythmia disorder in
a mammal in need thereof, the method comprising administrating to
the mammal a therapeutically effective amount of a pharmaceutical
composition comprising of a C.sub.2-C.sub.10 alkyl alcohol, or
mixtures thereof; and at least one other therapeutic agent.
61. The method of claim 60, wherein the C.sub.2-C.sub.10 alkyl
alcohol is 1-octanol, 2-octanol or mixtures thereof, and wherein
the at least one other therapeutic agent is selected from the group
consisting of: anticonvulsant or antiepileptic agent, a barbiturate
or barbituric acid derivative, an anesthetic agent, a
tinnitus-treating agent, a selective serotonin reuptake inhibitor,
an antidepressant agent, a neuroleptic, an antihypertensive agent,
an antipsychotic agent, a calcium channel blocker, an ACE
inhibitor, and a beta-blocker.
62. A method of treating a neurological disorder in a mammal in
need thereof, the method comprising administering to the mammal a
therapeutically effective amount of a pharmaceutical composition
comprising of a C.sub.2-C.sub.10 alkyl alcohol, or mixtures
thereof, and at least one other therapeutic agent, wherein
neurological disorder is not tremor or Parkinson's tremor.
63. The method of claim 62, wherein the C.sub.2-C.sub.10 alkyl
alcohol is 1-octanol, 2-octanol or mixtures thereof, and wherein
the at least one other therapeutic agent is selected from the group
consisting of: anticonvulsant or antiepileptic agent, a barbiturate
or barbituric acid derivative, an anesthetic agent, a
tinnitus-treating agent, a selective serotonin reuptake inhibitor,
an antidepressant agent, a neuroleptic, an antihypertensive agent,
an antipsychotic agent, a calcium channel blocker, an ACE
inhibitor, and a beta-blocker.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn.119(e) to U.S. Provisional Patent Application No.
60/906,085 filed on Mar. 9, 2007, which is hereby incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to the medical sciences generally and
neurophysiology and neuropharmacology in particular. More
specifically, this invention relates to methods and compositions
for treating a neurological disorder in a mammal and for an
associated thalamocortical dysrhythmia using a C.sub.2-C.sub.10
alkyl alcohol, or a lipophilic molecule with a partition
coefficient substantially similar to that of a C.sub.2-C.sub.10
alkyl alcohol, or mixtures thereof.
BACKGROUND OF THE INVENTION
[0003] Neurological disorders that affect the central nervous
system, the peripheral nervous system, and the autonomic nervous
system strick millions of people worldwide. These varied disorders
include, but are not limited to, thalamocortical dysrhythma,
neurogenic pain, obsessive-compulsive disorder, depression, panic
disorder, Parkinson's disease, schizophrenia, rigidity, dystonia,
tinnitus, tremor, and epilepsy. In particular, these and other
neurological disorders occur when the coordinate, controlled
electrical activity at the cortical level of the brain becomes
disrupted, thereby leading to uncoordinated electrical activity and
abnormal neuronal oscillation.
[0004] In particular, thalamocortical dysrhythmia refers to a
neurological and/or psychiatric condition arising from the abnormal
rhythmicity in particular components of the thalamocortical circuit
(Llinas et al. (1999) Proc. Natl. Acad. Sci. USA, 96:15222-15227).
At the cellular level, the abnormal activity of the thalamic
neurons is caused by an increase of low frequency oscillatory
activity due to protracted activation of the T type calcium
channels because of direct modification of the channel properties
or more commonly, abnormal hyperpolarization of the thalamic
neurons due to excess inhibition or deafferentation. Such abnormal
activity is transmitted to the related cortical area to which the
given thalamic neurons are oscillating generating a recurrent
attractor that is maintained by the recursive nature of the
circuit. At the macrocellular level, the abnormal rhythmicity
interferes with the communication among and between different
regions of the brain, and thereby impairs the motor and cognitive
skills that are controlled by those regions of the cortex.
[0005] Spike output in neuronal cell types is affected by
low-voltage-activated Cav3-type calcium currents arising from the
Ca.sub.v channels. Low-voltage-activated (LVA) calcium currents
provide an important contribution to spike output patterns of
neurons. Cav3-type channels are recognized as key determinants of
LVA calcium-dependent responses, including low-threshold calcium
spikes (LTS), bistable behavior, rebound depolarizations and
augmentation of synaptic responses. Cav3-type channels are
important to cell and circuit functions that range from sensory and
pain transmission through thalamocortical sleep-wake cycles. The
three isoforms of the Cav3-type calcium channel, i.e., Ca.sub.v3.1,
Ca.sub.v3.2, and Ca.sub.v3.3, can differ in their voltage-dependent
and kinetic properties, demonstrating the potential to
differentially affect spike output. In thalamus, differences in the
distribution and kinetic properties of Cav3-type currents have been
shown to be capable of influencing the nature of oscillatory output
of principal cells and inhibitory intemeurons involved in the
sleep-wake cycle, suggesting a selective distribution or modulation
of Ca.sub.v3 channel isoforms over discrete regions of the cell
axis.
[0006] There are many drugs currently available for treating
neurological disorders. These include, but are not limited to,
anticonvulsants, antiepileptics, barbiturates, barbituric acid
derivatives, anesthetic agents, tinnitus-treating agents, selective
serotonin reuptake inhibitors, antidepressant agents, neuroleptic
agents, antihypertensive agents, antipsychotic agents, calcium
channel blockers, ACE inhibitors, and beta-blockers. However, many
of such drugs are limited in their effectiveness and by their
significant side effects. For example, some of these drugs are
known to cause lightheadedness, depression insomnia, weakness,
fatigue, hallucinations, side-effects which severely limit their
use in human population. In particular, beta blockers,
anticonvulsants, and benzodiazepines have been shown to be
partially beneficial to some patients. However, beta blockers can
cause changes in blood pressure and heart rate, and are
contraindicated in patients with heart block, asthma, and
congestive heart failure.
[0007] In addition, some anticonvulsants can cause acute nausea and
vomiting, fatigue, sleepiness, confusion and incoordination, while
others can cause memory and speech abnormalities, sedation,
incoordination, and metabolic dysfunction.
[0008] Physical trauma has been used to treat some neurological
disorders. For example, certain surgical procedures have been used
in the most severe cases of essential tremor, destroying a part of
the brain including, the globus pallidus pars interna (GPi) nucleus
in the basal ganglia or implanting electrodes into the same area of
the brain including connecting them to a pacemaker-like battery
that stimulates regions of the brain to diminish the tremor. Such
procedures carry a high risk of infection and bleeding or equipment
malfunction. Additionally, many existing treatments for some
neurological disorders are poorly effective in that they do not
stop the disorders completely, and in most instances, they do not
prevent or even delay the progression of the disease.
[0009] Thus, there is a need for more effective treatments of
neurological disorders that have reduced or no side-effects.
SUMMARY OF THE INVENTION
[0010] It has been determined that certain alkyl alcohols, or
mixtures thereof, can modify the voltage-dependent activation
kinetics or single channel ionic conductance of a Ca.sub.v3 calcium
channel in a mammalian cell. This finding has been exploited to
develop the present invention, which is directed to methods of
treating neurological disorders in a mammal associated with
thalamocortical dysrhythmia.
[0011] In one aspect, the invention relates to a method of reducing
a low-threshold-activated calcium current in a cell by having a
Ca.sub.v3 plasmalemmal-bound calcium channel. The method comprises
superfusing the extracellular environment of the cell with a
C.sub.2-C.sub.10 alkyl alcohol, or mixtures thereof, in an amount
sufficient to modify the voltage-dependent activation kinetics or
single channel ionic conductance of the Ca.sub.v3 calcium channel
such that the low-threshold-activated calcium current in the cell
is reduced. In some other embodiments, the C.sub.2-C.sub.10 alkyl
alcohol is present in the extracellular environment at a
concentration of from about 10 .mu.M to about 100 .mu.M. In certain
embodiments, the cell is in a mammal in need of inhibiting a
Ca.sub.v3 calcium channel.
[0012] In some other embodiments, the cell is a neuron, such as an
intemeuron, a projection thalamic neuron, a reticular thalamic
neuron, a cortical intemeuron, cortical pyramidal neuron, a basal
ganglion neuron, a hippocampus neuron, an amygdala neuron, a tectal
neuron, or a cerebellar neuron. In particular embodiments, the
Ca.sub.v3 calcium channel is selected from the group consisting of
a Ca.sub.v3.1 calcium channel, a Ca.sub.v3.2 calcium channel, a
Ca.sub.v3.3 calcium channel, and combinations thereof. In certain
embodiments, the C.sub.2-C.sub.10 alkyl alcohol is administered in
a pharmaceutically acceptable excipient, carrier, or diluent.
[0013] In another aspect, the invention relates to a method of
inhibiting a Ca.sub.v3 plasmalemmal-bound calcium channel in a cell
of a mammal in need thereof. The method comprises administering to
the mammal a C.sub.2-C.sub.10 alkyl alcohol, or mixtures thereof,
at a dose of from about 0.001 mg/kg body weight to about 20 mg/kg
body weight of the mammal, for example, from about 0.01 mg/kg to
about 1 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, from about
0.3 mg/kg to about 3 mg/kg. The alcohol modifies the
voltage-dependent activation kinetics or single channels ionic
conductance of the Ca.sub.v3 calcium channel, thereby inhibiting
the Ca.sub.v3 calcium channel. In one embodiment, the alcohol is
administered orally. In another embodiment, the alcohol is
administered parenterally. In particular embodiments, the
C.sub.2-C.sub.10 alkyl alcohol is 1-octanol or 2-octanol. In a
further embodiment, the mammal has a thalamocortical dysrhythmia
disorder, and in another embodiment, the mammal is a human.
[0014] The invention also provides a method of treating a
thalamocortical dysrhythmia disorder that is not tremor or
Parkinson's tremor in a mammal in need thereof. The method
comprises administrating to the mammal a therapeutically effective
amount of a Ca.sub.v3 calcium channel inhibitor which binds to the
Ca.sub.v3 calcium channel, thereby reducing a low voltage-activated
calcium current in the cell of the mammal. In one embodiment, the
mammal is a human. In some embodiments, the inhibitor is a
C.sub.2-C.sub.10 alkyl alcohol, or mixtures thereof
[0015] In a further aspect, the invention provides a method of
treating a neurological disorder in a mammal in need thereof
provided that the neurological disorder is not tremor or
Parkinson's tremor. The method comprises administering to the
mammal a therapeutically effective amount of a Ca.sub.v3 calcium
channel inhibitor. In certain embodiments, the Ca.sub.v3 calcium
channel inhibitor is a C.sub.2-C.sub.10 alkyl alcohol, or mixtures
thereof. In other embodiments, the Ca.sub.v3 calcium channel
inhibitor is a lipophilic molecule with a partition coefficient
substantially similar to that of a C.sub.2-C.sub.10 alkyl alcohol.
In a particular embodiment, the Ca.sub.v3 calcium channel inhibitor
is a lipophilic molecule with a partition coefficient substantially
similar to that of an octanol. In another embodiment, the Ca.sub.v3
calcium channel inhibitor is a lipophilic molecule with a molecular
weight less than about 160.
[0016] In one embodiment, the neurological disorder is a
thalamocortical dysrhythmia disorder. In some embodiments, the
thalamocortical dysrhythmia disorder being treated is petit mal
epilepsy, tinnitus, neurogenic pain, Tourettes disease, chronic
depression, autism or Asperger's syndrome, schizoaffective
psychosis, Parkinson's disorder, Migraine, Absence Epilepsy, or
restless legs syndrome.
[0017] In certain embodiments, the therapeutically effective amount
of Ca.sub.v3 calcium channel inhibitor administered is from about
0.001 mg/kg body weight to about 20 mg/kg body weight of the
mammal, from about 0.01 mg/kg body weight to about 1 mg/kg body
weight of the mammal, from about 0.1 mg/kg body weight to about 10
mg/kg body weight of the mammal, from about 0.03 mg/kg body weight
to about 0.3 mg/kg body weight of the mammal, from about 0.3 mg/kg
body weight to about 3.0 mg/kg body weight of the mammal, or about
1.0 mg/kg body weight of the mammal.
[0018] In certain embodiments, the C.sub.2-C.sub.10 alkyl alcohol
is selected from the group consisting of (a)
CH.sub.3--(CH.sub.2).sub.n--OH, wherein n is an integer from 1 to
9, (b) CH.sub.3--CH(OH)--(CH.sub.2).sub.m--CH.sub.3, wherein m is
an integer from 0 to 7, (c)
CH.sub.3--CH.sub.2--CH(OH)--(CH.sub.2).sub.p--CH.sub.3, wherein p
is an integer from 0 to 6, (d)
CH.sub.3--CH.sub.2--CH.sub.2--CH(OH)--(CH.sub.2).sub.q--CH.sub.3,
wherein q is an integer from 0 to 5, (e)
CH.sub.3--CH.sub.2--CH.sub.2--CH.sub.2--CH(OH)--(CH.sub.2).sub.w--CH.sub.-
3, wherein w is an integer from 0 to 4, and (f) mixtures
thereof.
[0019] The invention also provides a use of a Ca.sub.v3 calcium
channel inhibitor which binds to the Ca.sub.v3 calcium channel,
thereby reducing a low voltage-activated calcium current in the
cell of the mammal, in the manufacture of a medicament for the
treatment of a neurological disorder in a mammal, provided that the
neurological disorder is not tremor or Parkinson's tremor.
[0020] The invention further provides a use of a Ca.sub.v3 calcium
channel inhibitor which binds to the Ca.sub.v3 calcium channel,
thereby reducing a low voltage-activated calcium current in the
cell of the mammal, in the manufacture of a medicament for the
treatment of a thalamocortical dysrhythmia disorder in a mammal,
provided that the thalamocortical dysrhythmia disorder is not
tremor or Parkinson's tremor.
[0021] The invention also provides a method for identifying an
alkyl alcohol that modifies the voltage-dependent activation
kinetics or single channel ionic conductance of a Ca.sub.v3
plasmalemmal-bound calcium channel protein. The method comprises
(a) providing atomic coordinates of the Ca.sub.v3 calcium channel
protein; (b) subjecting an alkyl alcohol to computational molecular
docking; and (c) selecting the alcohol that has the optimal virtual
binding to the a Ca.sub.v3 calcium channel protein. Optimal virtual
binding indicates that the alcohol binds to the a Ca.sub.v3 calcium
channel protein or alters the properties of the lipid bilayer at
the channel's voltage sensing site. In certain embodiments, the
optimal virtual binding is substantially similar to that as shown
in FIG. 8. In some embodiments, the computational molecular docking
utilizes a computer screening method selected from the group
consisting of Virtual library screening (VLS), ICM, ICM-VLS, DOCK,
and FlexX.
[0022] In another aspect, the invention provides a method of
identifying a chemical entity which binds to a Ca.sub.v3 calcium
channel. The method comprises comparing a structure model of the
Ca.sub.v3 calcium channel with a structure model for the chemical
entity, and detecting (i) a binding surface on the Ca.sub.v3
calcium channel for the chemical entity, or (ii) an alteration of
the properties of the lipid bi-layer at the channel's voltage
sensing site for the chemical entity. In certain embodiments, the
structural model of the Ca.sub.v3 calcium channel is derived from a
structure-predicting algorithm. In some other embodiments, the
Ca.sub.v3 calcium channel is selected from the group consisting of
a Ca.sub.v3.1 calcium channel, a Ca.sub.v3.2 calcium channel, a
Ca.sub.v3.3 calcium channel, and combinations thereof. In certain
embodiments, the structure model of the Ca.sub.v3 calcium channel
is derived from atomic coordinates determined by subjecting a
crystal comprising a Ca.sub.v3 calcium channel to X-ray diffraction
measurements.
[0023] In yet another aspect, the invention provides a method of
identifying an inhibitor of a Ca.sub.v3 calcium channel in a cell.
The method comprises contacting an isolated Ca.sub.v3
plasmalemmal-bound calcium channel with a candidate compound, and
detecting the presence of a complex, or lack thereof, between the
Ca.sub.v3 calcium channel and the compound. The candidate compound
is an inhibitor if it forms a complex with the Ca.sub.v3 calcium
channel or alters the properties of the lipid bilayer at the
channel's voltage sensing site.
[0024] In a further aspect, the invention provides a pharmaceutical
composition comprising: a therapeutically effective amount of a
C.sub.2-C.sub.10 alkyl alcohol, a lipophilic molecule with a
partition coefficient substantially similar to that of a
C.sub.2-C.sub.10 alkyl alcohol, or mixtures thereof; and at least
one other therapeutic agent.
[0025] In certain embodiments, the Ca.sub.v3 calcium channel
inhibitor is a C.sub.2-C.sub.10 alkyl alcohol, or mixtures thereof.
In some embodiments, the C.sub.2-C.sub.10 alkyl alcohol is
1-octanol or 2-octanol. In other embodiments, the Ca.sub.v3 calcium
channel inhibitor is a lipophilic molecule with a partition
coefficient substantially similar to that of a C.sub.2-C.sub.10
alkyl alcohol. In a particular embodiment, the Ca.sub.v3 calcium
channel inhibitor is a lipophilic molecule with a partition
coefficient substantially similar to that of an octanol. In another
embodiment, the Ca.sub.v3 calcium channel inhibitor is a lipophilic
molecule with a molecular weight less than about 160.
[0026] In certain embodiments, the at least one other therapeutic
agent is selected from the group consisting of an anticonvulsant or
antiepileptic agent, a barbiturate or barbituric acid derivative,
an anesthetic agent, a tinnitus-treating agent, a selective
serotonin reuptake inhibitor, an antidepressant agent, a
neuroleptic agent, an antihypertensive agent, an antipsychotic
agent, a calcium channel blocker, an ACE inhibitor, a beta-blocker,
and combinations thereof.
[0027] In yet another aspect, the invention provides a
pharmaceutical composition comprising: a therapeutically effective
amount of a C.sub.2-C.sub.10 alkyl alcohol, or mixtures thereof;
and at least one other therapeutic agent selected from the group
consisting of: a barbiturate or barbituric acid derivative, an
anesthetic agent, a tinnitus treating agent, a selective serotonin
reuptake inhibitor, an antidepressant agent, a neuroleptic, an
antihypertensive agent, a calcium channel blocker, and an ACE
inhibitor.
[0028] In certain embodiments, the anticonvulsant or antiepileptic
agent is selected from the group consisting of carbamazepine,
clobazam, clonazepam, ethosuximide, felbamate, fosphenytoin,
flurazepam, gabapentin, gabapentin, lamotrigine, levetiracetam,
oxcarbazepine, mephenytoin, phenobarbital, phenytoin, pregabalin,
primidone, sodium valproate, tiagabine, topiramate, valproate
semisodium, valproic acid, and vigabatrin, diazepam, lorazepam,
paraldehyde, pentobarbital, amiloride,
.alpha.-methyl-.alpha.-phenylsuccinimide, pentylenetetrazole and
tert-butyl-bicyclo[2.2.2] phosphorothionate.
[0029] In certain other embodiments, the barbiturate or barbituric
acid derivative is selected from the group consiting of sodium
thiopental, pentobarbital, secobarbital, amobarbital, butabarbital,
barbital, phenobarbital, butalbital, cyclobarbital, allobarbital,
methylphenobarbital, secobarbital, vinylbital and methohexital. In
certain embodiments, the anesthetic agent is selected from the
group consisting of propofol, etomidate, isoflurane, halothane, and
ketamine. In certain other embodiments, the tinnitus-treating agent
is selected from the group consisting of botulinum toxin,
propranolol, clonazepam, zinc supplementation, acamprosate,
etidronate or sodium fluoride, lignocaine, carbemazepine,
melatonin, sertraline and vitamin combinations. In certain
embodiments, the selective serotonin reuptake inhibitor is selected
from the group consisting of paroxetine, sertraline, fluoxetine and
fluvoxamine.
[0030] In some embodiments, the antidepressant agent is selected
from the group consisting of Harmaline, Iproniazid, Isocarboxazid,
Moclobemide, Nialamide, Pargyline, Phenelzine, Selegiline,
Toloxatone, Tranylcypromine, Brofaromine, Moclobemide, Amineptine,
Phenmetrazine, Vanoxerine, Bupropion, Atomoxetine, Maprotiline,
Reboxetine, Viloxazine, Duloxetine, Milnacipran, Nefazodone
Venlafaxine, Alaproclate, Citalopram, Escitalopram, Etoperidone,
Fluoxetine, Fluvoxamine, Paroxetine, Sertraline, Zimelidine,
Tianeptine, Amitriptyline, Amoxapine, Butriptyline, Clomipramine,
Desipramine, Dibenzepin, Dothiepin, Doxepin, Imipramine, Iprindole,
Lofepramine, Melitracen, Nortriptyline, Opipramol, Protriptyline,
Trimipramine, Maprotiline, Mianserin, Nefazodone, and
Trazodone.
[0031] In certain embodiments, the neuroleptic agent is selected
from the group consisting of risperidone, ziprasidone, haloperidol,
pimozide and fluphenazine. In certain other embodiments, the
antihypertensive agent is selected from the group consisting of
clonidine and guanfacine. In certain embodiments, the antipsychotic
agent is selected from the group consisting of clozapine,
risperidone, olanzapine, quetiapine, ziprasidone, aripiprazole, and
amisulpride, valproate semisodium or divalproex sodium, lithium
salts, risperidone, quetiapine and atomoxetine. In certain
embodiments, the calcium channel blocker is selected from the group
consisting of amlodipine, felodipine, nicardipine, nifedipine,
nimodipine, nisoldipine, nitrendipine, lacidipine, lercanidipine,
verapamil, gallopamil, diltiazem, mibefradil, and menthol. In
certain other embodiments, the ACE inhibitor is selected from the
group consisting of enalapril, ramipril, quinapril, perindopril,
lisinopril, benazepril and fosinopril. In particular embodiments,
the beta-blocker is selected from the group consisting of
dichloroisoprenaline, practolol, pronethaolol, alprenolol,
carteolol, levobunolol, mepindolol, metipranolol, nadolol,
oxprenolol, penbutolol, pindolol, propranolol, sotalol, timolol,
acebutolol atenolol, betaxolol, bisoprolol, esmolol, metoprolol,
nebivolol, carvedilol, celiprolol, labetalol and butoxamine.
[0032] In another aspect, the invention provides a method of
treating a neurological disorder in a mammal in need thereof,
comprises administering to the mammal a therapeutically effective
amount of the pharmaceutical composition described hereinabove.
[0033] The invention also provides a method of treating a
thalamocortical dysrhythmia disorder in a mammal in need thereof,
comprises administrating to the mammal a therapeutically effective
amount of the pharmaceutical composition described hereinabove.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0034] The foregoing and other objects of the present invention,
the various features thereof, as well as the invention itself may
be more fully understood from the following description, when read
together with the accompanying drawings in which:
[0035] FIG. 1A is a schematic representation of recording from an
experiment showing the effect of 1-octanol on Ca.sub.v3 calcium
current at different voltage levels using a voltage clamp
technique.
[0036] FIG. 1B is a schematic representation of recording showing
the effect of 1-octanol on the peak of the fast component
(T-currents) but not the slow component when both low and high
voltage activated calcium currents are open in a cell.
[0037] FIG. 2A is a schematic representation of recording from an
experiment showing the effect of 2-octanol on Ca.sub.v3 calcium
current at different voltage levels using a voltage clamp
technique.
[0038] FIG. 2B a schematic representation of recording of an
experiment showing the effect of 2-octanol on the current-voltage
(i.e., I-V curve) relationship for the same thalamic neuron as
described in FIG. 2A, but for a wider range of holding
potentials.
[0039] FIG. 3A is a schematic representation of recording from an
experiment showing the effect of 1-octanol on thalamic low
frequency oscillations using a current clamp technique (a pair of
low threshold spikes firing at 2 Hz generated after the rebound of
a short hyperpolarizing pulse is applied).
[0040] FIG. 3B is a schematic representation of recording from an
experiment showing the corresponding rebound of a short
hyperpolarizing pulse that is applied in FIG. 3A.
[0041] FIG. 4A is a schematic representation of recording from an
experiment showing the effect of 1-octanol on low-threshold calcium
current using a current clamp technique.
[0042] FIG. 4B is a schematic representation of recording from an
experiment showing the corresponding ramps of current that is
applied in FIG. 4A.
[0043] FIG. 5A is schematic representation of a recording from an
experiment showing that low threshold spikes can be activated
following TTX removal from the external solution using a current
clamp technique.
[0044] FIG. 5B is a schematic representation of more detailed
recording from an experiment showing that low threshold spikes can
be activated following TTX removal from the external solution using
a current clamp technique.
[0045] FIG. 6A are schematic representations of recordings from an
experiment showing the effect of tetrodotoxin ("TTX") on
low-threshold calcium current using a current clamp technique, in
both wild type (top) and alpha1A-KO calcium channel knockout
(bottom) mice.
[0046] FIG. 6B are schematic representations of recordings from an
experiment showing the effect of TTX plus 1-octanol on
low-threshold calcium current using a current clamp technique, in
both wild type (top) and alpha1A-KO calcium channel knockout
(bottom) mice.
[0047] FIG. 7A is a graphic representation of an experiment showing
the normal frequency profiles presented in wild type mice.
[0048] FIG. 7B is a graphic representation of an experiment showing
the normal frequency profiles presented in alpha-1A knockout
mice.
[0049] FIG. 7C is a graphic representation of an experiment
comparing the frequency profiles presented in both wild-type and
alpha1A-knockout mice.
[0050] FIG. 7D is a graphic representation of an experiment
describing the effect of a 1-octanol injection on the frequency
profiles presented in wild-type mice.
[0051] FIG. 8 is a schematic representation of a three-dimension
molecular model of a specific binding between 1-octanol and the
subunit of a Ca.sub.v3 calcium channel shown in one low energy
conformation.
DETAILED DESCRIPTION OF THE INVENTION
[0052] All references cited herein are incorporated herein by
reference in their entirety and for all purposes to the same extent
as if each individual publication or patent or patent application
was specifically and individually indicated to be incorporated by
reference in its entirety for all purposes. To the extent
publications and patents or patent applications incorporated by
reference contradict the disclosure contained in the specification,
the specification is intended to supercede and/or take precedence
over any such contradictory material.
[0053] The major theoretical framework of motor and cognitive
functions hypothesizes that motor and cognitive functions arise
from coordinate electrical activity at the cortical level of the
brain. Coordinate electrical activity refers to controlled
electrical discharges within the brain at both cellular and
macrocellular levels. Controlled electrical discharges facilitate
communication within and among different regions of the outer layer
of the brain (i.e., the cortex), and thereby coordinate the brain's
ability to give rise to both motor and cognitive functions.
[0054] At the cellular level, neurons within the brain interact and
communicate through electrical signals that are sent between
neurons. Neurons transmit electrical signals via an electrochemical
process, whereby an exchange of ions occurs across the neuron's
membrane, thereby causing an electrical discharge. When a neuron is
in its rest state, the neuron accumulates and maintains a negative
charge within its membrane, thereby polarizing a negative potential
(typically -70 mV) between the inside and outside of the neuron.
The neuron discharges when a stimulus event increases the membrane
potential beyond a certain threshold value (typically -55 mV),
thereby triggering an exchange of ions across the neuron's membrane
and depolarizing the neuron. The depolarization and exchange of
ions causes a positive discharge, also known as a "spike" or
"impulse," that peaks at a net positive potential (typically +30
mV). This positive discharge is sent from the neuron through its
axon(s) to the dendrites of recipient neurons, which receive, and
subsequently respond to, the electrical signal. After the positive
discharge, the transmitting neuron returns to its resting state,
thereby completing the discharge cycle.
[0055] A calcium channel is a structure that spans cell membrane
lipid bilayer and allows calcium ions to move passively across the
membrane according to the calcium electrochemical gradient between
the interior of the cell (where calcium concentration is lower) and
the extra-cellular fluid (where calcium concentration is higher).
These channels are highly specific to calcium ions, although
specificity with respect to other divalent cations is never
absolute. Importantly, calcium channels can be activated by changes
in the electrical field across the membrane and are therefore said
to be "voltage-gated."
[0056] Both central and peripheral neurons possess multiple types
of voltage-gated calcium channels that often are differentiated on
the basis of the membrane potentials at which they are activated.
Low-voltage activated calcium current, also called Ca.sub.v3-type
current, has been observed in a variety of cell types including
neurons (White et al. (1989) Proc. Natl. Acad. Sci. 86: 6802-6806);
Cribbs et al. (2001) Cir. Res. 88:403). Ca.sub.v3-type current
appears to play a key role in the regulation of neuronal bursting
and low-amplitude voltage oscillations (Llinas et al. (2006) J.
Neurophysiol, 95: 3297-3308).
[0057] At the macrocellular level, different regions of the brain
are responsible for different cognitive and motor functions.
Different layers of the cortex control different motor skills as
well as distinctive cognitive skills including speech, hearing,
sight, touch, smell and thought. The cortex itself has six main
cellular levels of neurons (termed levels I-VI) that communicate
intracellularly via electrical impulses. Thus, normal cognitive and
motor functions are the product of coordinate electrical activity
that occurs at the cortical level.
[0058] Also at the macrocellular level, the thalamus resides within
the center of the brain and acts as a "communications hub" between
different regions of the brain, including the cortex. The normal
electrical activity of the thalamus is also coordinated, in that
the thalamus fires electrical impulses at specific intervals and in
a controlled fashion. A complete network of neurons exist between
the thalamus and the cortex, thereby creating corticothalamic
pathways that facilitate communication and interaction between the
thalamus and the cortex.
[0059] The thalamus, itself, is divided into regions that include
the sensory thalamus and the reticular nucleus. The sensory
thalamus is stimulated by signals from other sensory inputs from
the body and communicates those inputs to the cortex. The reticular
nucleus surrounds the sensory thalamus and acts to suppress the
sensory thalamus from transmitting signals at certain times, such
as sleep, when the cortex is to be desensitized from communication
with the rest of the body. Thus, the reticular nucleus suppresses
the electrical activity and discharge of the sensory thalamus.
[0060] The thalamus and the cortex are connected through both
specific and nonspecific corticothalamic pathways. Specific
pathways refer to pathways between the thalamus and particular
sensory or motor input regions of the cortex, typically connecting
at layer IV of the cortex. Nonspecific pathways refer to pathways
between the thalamus and non-sensory and non-motor input regions of
the cortex, typically connecting at layers I, IV and/or V of the
cortex. Afferent corticothalamic pathways communicate signals from
the thalamus to the cortex, whereas efferent corticothalamic
pathways communicate signals from the cortex to the thalamus,
thereby closing the communication loop between the cortex and the
thalamus.
[0061] Coordinate electrical activity is characterized by normal
neuronal oscillation (i.e., normal frequencies of electrical
oscillation by neurons and neuronic regions), wherein neurons and
neuronic regions of the brain discharge electrical impulses at
particular frequencies, thereby causing electrical oscillation. At
the cellular level, inhibitors and neuronal inputs properly control
the chemical release of neurons and thereby facilitate normal
electrical discharges by the neurons. At the macrocellular level,
the interaction and communication between properly discharging
neurons causes normal, coordinate electrical activity characterized
by electrical oscillation at different frequencies between and
among particular regions of the brain.
[0062] Neuronal oscillation generally occurs in a plurality of
distinct frequency bands. These frequency bands include the theta
(.theta.) band, which includes low frequency oscillations in the
4-8 Hz range that are most commonly associated with the four-phase
sleep cycle of human beings. Another significant frequency band is
the gamma (.gamma.) band, which includes high frequency
oscillations in the 20-50 Hz range that are associated with
sensorimotor and cognitive functions. Individuals experience
specific types and amounts of .theta. and .gamma. band activity
based on factors including their mental activity level and physical
state. For instance, a person who is asleep will typically
experience the four-phase theta-band oscillation cycle associated
with sleep, whereas a person who is awake and active will
experience gamma-band oscillation at the cortical level to perform
cognitive and motor functions.
[0063] The present invention relates, in part, to a method of
reducing a low-threshold-activated calcium current in a cell by
having a Ca.sub.v3 plasmalemmal-bound calcium channel. In this
method, the extracellular environment of the cell is superfused
with a C.sub.2-C.sub.10 alkyl alcohol, or mixtures thereof, in an
amount sufficient to modify the voltage-dependent activation
kinetics or single channel ionic conductance of the Ca.sub.v3
calcium channel such that the low-threshold-activated calcium
current in the cell is reduced. As used herein, the term
"superfused" refers adding to the fluid that surrounds the
tissue.
[0064] The term "C.sub.2-C.sub.10 alkyl alcohol" as used herein
refers to one or more hydroxyl groups (--OH), attached to a
C.sub.2-C.sub.10 alkyl group at any available carbon position. The
term "C.sub.2-C.sub.10 alkyl" refers to a linear or branched,
saturated hydrocarbon having from 2 to 10 carbon atoms.
Representative C.sub.2-C.sub.10 alkyl groups include, but are not
limited to, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl,
nonyl, decanyl, and their respective isomers. Thus, representative
C.sub.2-C.sub.10 alkyl alcohols include, but not limited to,
ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol,
nonanol, deanol and their respective isomers.
[0065] The C.sub.2-C.sub.10 alkyl alcohol can be a straight chain
alkyl alcohol such as: CH.sub.3--(CH.sub.2).sub.n--OH, wherein n is
an integer from 1 to 9,
CH.sub.3-CH(OH)--(CH.sub.2).sub.m--CH.sub.3, wherein m is an
integer from 0 to 7;
CH.sub.3--CH.sub.2--CH(OH)--(CH.sub.2).sub.p--CH.sub.3, wherein p
is an integer from 0 to 6;
CH.sub.3--CH.sub.2--CH.sub.2--CH(OH)--(CH.sub.2).sub.q--CH.sub.3,
wherein q is an integer from 0 to 5;
CH.sub.3--CH.sub.2--CH.sub.2--CH.sub.2--CH(OH)--(CH.sub.2).sub.w--CH.sub.-
3, wherein w is an integer from 0 to 4; and mixtures of any two or
more of the above alkyl alcohols.
[0066] Alternatively, the C.sub.2-C.sub.10 alkyl alcohol can be a
branched chain alkyl alcohol such as:
(CH.sub.3).sub.2CH--(CH.sub.2).sub.n--OH, wherein n is an integer
from 0 to 6;
CH.sub.3--CH(OH)--(CH.sub.2).sub.m--CH(CH.sub.3).sub.2, wherein m
is an integer from 0 to 5;
CH.sub.3--CH.sub.2--CH(OH)--(CH.sub.2).sub.p--CH(CH.sub.3).sub.2,
wherein p is an integer from 0 to 4;
CH.sub.3--CH.sub.2--CH.sub.2--CH(OH)--(CH.sub.2).sub.q--CH(CH.sub.3).sub.-
2, wherein q is an integer from 0 to 3;
CH.sub.3--CH.sub.2--CH.sub.2--CH.sub.2--CH(OH)--(CH.sub.2).sub.w--CH(CH.s-
ub.3).sub.2, wherein w is an integer from 0 to 2; and mixtures of
any two or more of the above alkyl alcohols.
[0067] The C.sub.2-C.sub.10 alkyl alcohol can be a primary, a
secondary or a tertiary alcohol. In particular, the
C.sub.2-C.sub.10 alkyl alcohol can be 1-octanol or 2-octanol. The
C.sub.2-C.sub.10 alkyl alcohol can be present in the extracellular
environment at a concentration of from about 10 .mu.M to about 100
.mu.M.
[0068] As used herein, "about" means a numeric value having a range
of .+-.10% around the cited value. For example, a range of "about
10 .mu.M to about 100 .mu.M" "includes the range" "about 9 .mu.M to
about 110 .mu.M," the range "about 9 .mu.M to about 90 .mu.M" "the
range" "about 11 .mu.M to about 110 .mu.M," as well as "11 .mu.M to
about 90 .mu.M," and all ranges in between.
[0069] The cell being treated is a neuron, e.g., an interneuron, a
projection thalamic neuron, a reticular thalamic neuron, a cortical
interneuron, cortical pyramidal neuron, a basal ganglion neuron, a
hippocampus neuron, an amygdala neuron, a tectal neuron, or a
cerebellar neuron.
[0070] Spike output in neuronal cell types is affected by
low-voltage-activated Ca.sub.v3-type calcium currents arising from
Ca.sub.v3 channels. There are three isoforms of the Ca.sub.v3-type
calcium channel, i.e., Ca.sub.v3.1, Ca.sub.v3.2, and Ca.sub.v3.3,
and they can differ in their voltage-dependent and kinetic
properties, showing the potential to differentially affect spike
output. In thalamus, differences in the distribution and kinetic
properties of Ca.sub.v3-type currents have been shown capable of
influencing the nature of oscillatory output of principal cells and
inhibitory interneurons involved in the sleep-wake cycle,
suggesting a selective distribution or modulation of Ca.sub.v3
channel isoforms over discrete regions of the cell axis. (see,
e.g., McKay et al. (2006) Eur. J. Neurosci. 24:2581-2594.).
[0071] The C.sub.2-C.sub.10 alkyl alcohol can be administered in a
pharmaceutically acceptable excipient, carrier, or diluent.
[0072] The phrase "pharmaceutically-acceptable excipient, carrier,
or diluent" as used herein means a pharmaceutically-acceptable
material, composition or vehicle, such as a liquid or solid filler,
diluent, excipient, solvent or encapsulating material, involved in
carrying or transporting the subject pharmaceutical agent from one
organ, or portion of the body, to another organ, or portion of the
body. Each carrier must be "acceptable" in the sense of being
compatible with the other ingredients of the formulation and not
injurious to the patient. Some examples of materials which can
serve as pharmaceutically-acceptable carriers include: sugars, such
as lactose, glucose and sucrose; starches, such as corn starch and
potato starch; cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa
butter and suppository waxes; oils, such as peanut oil, cottonseed
oil, safflower oil, sesame oil, olive oil, corn oil and soybean
oil; glycols, such as propylene glycol; polyols, such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters, such as ethyl
oleate and ethyl laurate; agar; buffering agents, such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;
isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer
solutions; and other non-toxic compatible substances employed in
pharmaceutical formulations. Wetting agents, emulsifiers and
lubricants, such as sodium lauryl sulfate, magnesium stearate, and
polyethylene oxide-polypropylene oxide copolymer as well as
coloring agents, release agents, coating agents, sweetening,
flavoring and perfuming agents, preservatives and antioxidants can
also be present in the compositions.
[0073] The present invention also provides a method of inhibiting a
Ca.sub.v3 plasmalemmal-bound calcium channel in a cell of a mammal
in need thereof. The method comprises administering to the mammal a
C.sub.2-C.sub.10 alkyl alcohol, or mixtures thereof, at a dose of
from about 0.001 mg/kg body weight to about 20 mg/kg body weight of
the mammal, for example, from about 0.01 mg/kg body weight to about
1 mg/kg body weight of the mammal, from about 0.1 mg/kg body weight
to about 10 mg/kg body weight of the mammal, from about 0.03 mg/kg
body weight to about 0.3 mg/kg body weight of the mammal, from
about 0.3 mg/kg body weight to about 3.0 mg/kg body weight of the
mammal, or about 1.0 mg/kg body weight of the mammal, the alcohol
modifying the voltage dependent activation kinetics or single
channels ionic conductance of the Ca.sub.v3 calcium channel, and
thereby inhibiting the Ca.sub.v3 calcium channel.
[0074] The alcohol can be administered to the mammal orally or
parenterally as described below.
[0075] In another aspect, the invention provides a method of
treating a thalamocortical dysrhythmia disorder in a mammal in need
thereof, the method comprising administrating to the mammal a
therapeutically effective amount of a Ca.sub.v3 calcium channel
inhibitor that binds to the Ca.sub.v3 calcium channel, thereby
reducing a low voltage-activated calcium current in a cell of the
mammal.
[0076] A thalamocortical dysrhythmia disorder refers to a
neurological and/or psychiatric condition arising from the abnormal
rhythmicity in particular components of the thalamocortical circuit
(Llinas et al. (1999) Proc. Natl. Acad. Sci. USA, 96:15222-15227).
Thalamocortical dysrhythmia occurs when the coordinate, controlled
electrical activity at the cortical level of the brain becomes
disrupted, thereby leading to uncoordinated electrical activity and
abnormal neuronal oscillation. At the macrocellular level, the
abnormal rhythmicity interferes with the communication among and
between different regions of the brain, and thereby impairs the
motor and cognitive skills that are controlled by those regions of
the cortex.
[0077] Aspects of the present invention provide a method of
treating a neurological disorder in a mammal in need thereof,
comprising administering to the mammal a therapeutically effective
amount of a pharmaceutical composition as described
hereinabove.
[0078] Other aspects of the present invention provide a method of
treating a thalamocortical dysrhythmia disorder in a mammal in need
thereof, comprising administrating to the mammal a therapeutically
effective amount of a pharmaceutical composition as described
hereinabove.
[0079] Non-limiting examples of the neurological disorder
associated with thalamocortical dysrhythmia include: Petit Mal
Epilepsy (Coulter et al. (1990) Br. J. Pharmacol. 100:800-806;
Perez-Reyes et al. (2003) Physiol. Rev. 83:117-161; Kim et al.
(2001) Neuron, 31: 35-45; Song et al. (2001) Soc. Neurosci. Abstr.
27: 14; Volkmann et al. (1996) Neurol., 46:1359-1370); Parkinson's
Disease (Moran et al. (2004) Soc. Neurosci. Abst. 30:676.12;
Jeanmonod et al. (1996) Brain, 119: 363-375; Llinas et al. (2005)
Trends Neurosci., 28:325-333); Tinnitus Llinas et al. (2005) Trends
Neurosci., 28:325-333); Jeanmonod et al. (2001) Thalams &
Related Systems 1:71-79); neurological pain or Central Pain
(Schwartzman et al. (2001) Arch. Neurol. 58:1547-1550; Schulman et
al. (2005) Thalamus & Related Systems 3(1): 33-39);
Obsessive-Compulsive Disorder (Jeanmonod et al. (2003) Thalamus
& Related Systems 2:103-113); Tourettes Disease (Moran et al.
(2004) Soc. Neurosci. Abst. 30:676.12); Chronic Depression
(Schulman et al. (2001) Soc. Neurosci. Abst. 27); Autism (Sinton et
al. (1989) Plugers Arch., 414:31-36); Schizoaffective Psychosis
(Moran et al. (2004) Soc. Neurosci. Abst. 30:676.12); Migraine;
Absence Epilepsy; and restless legs syndrome, among others.
[0080] Aspects of the invention also provide a method of treating a
neurological disorder that is not tremor or Parkinson's tremor in a
mammal in need thereof. In this method, a therapeutically effective
amount of a Ca.sub.v3 calcium channel inhibitor is provided to the
mammal.
[0081] In the therapeutic methods described above, the Ca.sub.v3
calcium channel inhibitor is a C.sub.2-C.sub.10 alkyl alcohol, or
mixtures thereof. In addition, the Ca.sub.v3 calcium channel
inhibitor can be a lipophilic molecule with a partition coefficient
substantially similar to that of a C.sub.2-C.sub.10 alkyl alcohol.
The Ca.sub.v3 calcium channel inhibitor can also be a lipophilic
molecule with a partition coefficient substantially similar to that
of an octanol. In certain cases, the Ca.sub.v3 calcium channel
inhibitor can be a lipophilic molecule with a molecular weight less
than about 160. The lipophilic molecule is a fat soluble molecule
that can penetrate a plasmalemmal cell membrane that can interact
specifically with the Ca.sub.v3 type calcium channel. As used
herein, "substantially similar" means a value having a range of at
least .+-.5% around the targeted value.
[0082] The lipophilic molecule typically has a molecular weight
less than about 160. A partition coefficient is a measure of
differential solubility of a compound in two solvents. The
logarithmic ratio of the concentrations of the solute in the
solvent is called log P (sometimes LogP). Typically, the partition
coefficient is based on the solvents octanol and water. The
octanol-water partition coefficient is a measure of the
hydrophobicity and hydrophilicity of a substance. A classical
method of log P determination is the shake-flask method, which
consists of mixing a known amount of solute in a known volume of
octanol and water, then measuring the distribution of the solute in
each solvent. The most common method of measuring the distribution
of the solute is by UV/VIS spectroscopy. A faster method of log P
determination makes use of high-performance liquid chromatography.
The log P of a solute can be determined by correlating its
retention time with similar compounds with known log P values.
[0083] For all of these therapeutic methods, the therapeutically
effective amount of the alcohol administered can be from about
0.001 mg/kg body weight to about 20 mg/kg body weight of the
mammal, for example, from about 0.01 mg/kg body weight to about 1
mg/kg body weight of the mammal, from about 0.1 mg/kg body weight
to about 10 mg/kg body weight of the mammal, from about 0.03 mg/kg
body weight to about 0.3 mg/kg body weight of the mammal, from
about 0.3 mg/kg body weight to about 3.0 mg/kg body weight of the
mammal, or about 1.0 mg/kg body weight of the mammal.
[0084] The invention also provides a pharmaceutical composition for
treating a neurological disorder in a mammal in need thereof. The
composition comprises a therapeutically effective amount of
C.sub.2-C.sub.10 alkyl alcohol, or mixtures thereof, and at least
one other therapeutic agent. The composition also comprises a
therapeutically effective amount of a lipophilic molecule with a
partition coefficient substantially similar to that of a
C.sub.2-C.sub.10 alkyl alcohol, or mixtures thereof, and at least
one other therapeutic agent.
[0085] The at least one other therapeutic agent is also
administered in an amount effective in reducing the symptoms on the
disease or disorder, whether used additively or synergistically in
combination with the alcohol. The at least one other therapeutic
agent can be administered separately from the pharmaceutical
compositions described herein, or it can be administered
simultaneously and/or successively with the pharmaceutical
compositions described herein.
[0086] Effective amounts of the at least one other therapeutic
agent are known to those skilled in the art. However, it is within
the skilled artisan's purview to determine the at least one other
therapeutic agent's optimal effective amount range. In some cases,
the patient in need of treatment is being treated with one or more
other therapeutic agents. In some other cases, the patient in need
of treatment is being treated with at least two other therapeutic
agents.
[0087] The at least one other therapeutic agent can be, but not
limited to, an anticonvulsant or antiepileptic agent, a barbiturate
or barbituric acid derivative, an anesthetic agent, a
tinnitus-treating agent, a selective serotonin reuptake inhibitor,
an antidepressant agent, a neuroleptic agent, an antihypertensive
agent, an antipsychotic agent, a calcium channel blocker, an ACE
inhibitor, and a beta-blocker.
[0088] The anticonvulsant or antiepileptic agent can suppress the
rapid and excessive firing of neurons that start a seizure, or can
prevent the spread of the seizure within the brain and offer
protection against possible excitotoxic effects that may result in
brain damage. Many useful anticonvulsant or antiepileptic agents
block sodium channels, calcium channels, AMPA receptors, or NMDA
receptors. Some useful anticonvulsant or antiepileptic agents
inhibit the metabolism of GABA or increase its release.
[0089] A useful anticonvulsant or antiepileptic agent includes, but
is not limited to, carbamazepine, clobazam, clonazepam,
ethosuximide, felbamate, fosphenytoin, flurazepam, gabapentin,
lamotrigine, levetiracetam, oxcarbazepine, mephenytoin,
phenobarbital, phenytoin, pregabalin, primidone, sodium valproate,
tiagabine, topiramate, valproate semisodium, valproic acid, and
vigabatrin, diazepam and lorazepam, paraldehyde, and pentobarbital.
A useful barbiturate or barbituric acid derivative includes, but is
not limited to, sodium thiopental, pentobarbital, secobarbital,
amobarbital, butabarbital, barbital, phenobarbital, butalbital,
cyclobarbital, allobarbital, methylphenobarbital, secobarbital,
vinylbital, and methohexital.
[0090] Useful anesthetic agents include, but are not limited to,
propofol, etomidate, isoflurane, halothane, and ketamine. A useful
tinnitus-treating agent includes, but is not limited to, botulinum
toxin, propranolol and clonazepam, zinc supplementation,
acamprosate, etidronate or sodium fluoride, lignocaine,
carbemazepine, melatonin, sertraline, and vitamin combinations.
[0091] Useful selective serotonin reuptake inhibitors include, but
are not limited to, paroxetine, sertraline, fluoxetine, and
fluvoxamine as well as an antidepressant such as clomipramine. The
at least one other active agent may also include gabapentin,
lamotrigine, olanzapine and risperidone.
[0092] Useful neuroleptic agents include, but are not limited to,
risperidone, ziprasidone, haloperidol, pimozide and fluphenazine.
Useful antihypertensive agents include, but not limited to,
clonidine and guanfacine, atomoxetine (a non-stimulant drug
approved for the treatment of attention-deficit hyperactivity
disorder). A useful antidepressant agent includes, but is not
limited to, clomipramine. A useful atypical antipsychotic agent
includes, but is not limited to, clozapine, risperidone,
olanzapine, quetiapine, ziprasidone, aripiprazole, and amisulpride,
or a typical antipsychotic agent such as chlorpromazine and
haloperidol. A useful schizoaffective disorder-treating agent
includes, but is not limited to, valproate semisodium or divalproex
sodium, Lithium salts, Risperidone, and quetiapine.
[0093] The calcium channel blockers are a class of drugs with
effects on many excitable cells of the body, like the muscle of the
heart, smooth muscles of the vessels or neuron cells. Many calcium
channel blockers work by blocking L-type voltage gated calcium
channels in the heart, blood vessels, or neuron cells in a brain.
This prevents calcium levels from increasing as much in the cells
when stimulated, leading to less contraction.
[0094] Useful calcium channel blockers include, but are not limited
to, amlodipine, felodipine, nicardipine, nifedipine, nimodipine,
nisoldipine, nitrendipine, lacidipine, lercanidipine, verapamil,
gallopamil, diltiazem, mibefradil, and menthol. The at least one
other active agent mentioned above may also include other classes
of pharmaceutical agents that have overlapping effects as calcium
channel blockers such as ACE inhibitors, beta-blockers, and
nitrates. ACE inhibitors include enalapril, ramipril, quinapril,
perindopril, lisinopril, benazepril, and fosinopril. Beta-blockers
include, but not limited to, dichloroisoprenaline, practolol,
pronethaolol, alprenolol, carteolol, levobunolol, mepindolol,
metipranolol, nadolol, oxprenolol, penbutolol, pindolol,
propranolol, sotalol, timolol, acebutolol atenolol, betaxolol,
bisoprolol, esmolol, metoprolol, nebivolol, carvedilol, celiprolol,
labetalol, and butoxamine.
[0095] The at least one other therapeutic agent mentioned above may
alternatively be Parkinson's disorder-treating agent such as, but
not limited to, levodopa and its derivatives, carbidopa,
benserazide, bromocriptine, pergolide, pramipexole, ropinirole,
cabergoline, apomorphine, lisuride, selegiline and rasagiline.
These agents are commercially available.
[0096] Alternatively, or additionally, the at least one other
active agent mentioned above is amiloride (an epithelial sodium
channel blocker), .alpha.-methyl-.alpha.-phenylsuccinimide,
pentylenetetrazole, tert-butyl-bicyclo[2.2.2]phosphorothionate.
These agents are commercially available.
[0097] Formulations of the present invention include those suitable
for oral, nasal, topical (including buccal and sublingual), rectal,
vaginal and/or parenteral administration. The formulations may
conveniently be presented in unit dosage form and may be prepared
by any methods well known in the art of pharmacy. The amount of
active ingredient which can be combined with a carrier material to
produce a single dosage form will vary depending upon the mammal
being treated and the particular mode of administration. The amount
of active ingredient, which can be combined with a carrier material
to produce a single dosage form will generally be that amount of
the compound which produces a therapeutic effect. Generally, out of
100%, this amount will range, for example, from about 1% to about
99% of active ingredient, from about 5% to about 70%, from about
10% to about 30%.
[0098] Therapeutic compositions or formulations of the invention
suitable for oral administration may be in the form of capsules,
cachets, pills, tablets, lozenges (using a flavored basis, usually
sucrose and acacia or tragacanth), powders, granules, or as a
solution or a suspension in an aqueous or non-aqueous liquid, or as
an oil-in-water or water-in-oil liquid emulsion, or as an elixir or
syrup, or as pastilles (using an inert base, such as gelatin and
glycerin, or sucrose and acacia) and/or as mouth washes and the
like, each containing a predetermined amount of a compound of the
present invention as an active ingredient. A compound of the
present invention may also be administered as a bolus, electuary or
paste.
[0099] In solid dosage forms of the invention for oral
administration (capsules, tablets, pills, dragees, powders,
granules and the like), the alcohol or inhibitor according to the
invention is mixed with one or more pharmaceutically-acceptable
carriers, such as sodium citrate or dicalcium phosphate, and/or any
of the following: fillers or extenders, such as starches, lactose,
sucrose, glucose, mannitol, and/or silicic acid; binders, such as,
for example, carboxymethylcellulose, alginates, gelatin, polyvinyl
pyrrolidone, sucrose and/or acacia; humectants, such as glycerol;
disintegrating agents, such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, sodium
carbonate, and sodium starch glycolate; solution retarding agents,
such as paraffin; absorption accelerators, such as quaternary
ammonium compounds; wetting agents, such as, for example, cetyl
alcohol, glycerol monostearate, and polyethylene
oxide-polypropylene oxide copolymer; absorbents, such as kaolin and
bentonite clay; lubricants, such a talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, and mixtures thereof, and coloring agents. In the case of
capsules, tablets and pills, the pharmaceutical compositions may
also comprise buffering agents. Solid compositions of a similar
type may also be employed as fillers in soft and hard-filled
gelatin capsules using such excipients as lactose or milk sugars,
as well as high molecular weight polyethylene glycols and the
like.
[0100] Liquid dosage forms for oral administration of the compounds
of the invention include pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active ingredient, the liquid dosage forms may
contain inert diluents commonly used in the art, such as, for
example, water or other solvents, solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof. Additionally,
cyclodextrins, e.g., hydroxypropyl-.beta.-cyclodextrin, may be used
to solubilize compounds.
[0101] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents. Suspensions, in addition to the alcohols or
inhibitors according to the invention, may contain suspending
agents as, for example, ethoxylated isostearyl alcohols,
polyoxyethylene sorbitol and sorbitan esters, microcrystalline
cellulose, aluminum metahydroxide, bentonite, agar--agar and
tragacanth, and mixtures thereof.
[0102] Formulations of the pharmaceutical compositions of the
invention for rectal or vaginal administration may be presented as
a suppository, which may be prepared by mixing one or more alcohols
or inhibitors according to the invention, with one or more suitable
nonirritating excipients or carriers comprising, for example, cocoa
butter, polyethylene glycol, a suppository wax or a salicylate, and
which is solid at room temperature, but liquid at body temperature
and, therefore, will melt in the rectum or vaginal cavity and
release the active pharmaceutical agents of the invention.
Formulations of the present invention which are suitable for
vaginal administration also include pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing such carriers
as are known in the art to be appropriate.
[0103] Dosage forms for the topical or transdermal administration
of an alcohol or other inhibitor according to the invention include
powders, sprays, ointments, pastes, creams, lotions, gels,
solutions, patches and inhalants. The active compound may be mixed
under sterile conditions with a pharmaceutically-acceptable
carrier, and with any preservatives, buffers, or propellants which
may be required.
[0104] The ointments, pastes, creams and gels may contain, in
addition to an alcohol or other inhibitor according to the
invention, excipients, such as animal and vegetable fats, oils,
waxes, paraffins, starch, tragacanth, cellulose derivatives,
polyethylene glycols, silicones, bentonites, silicic acid, talc and
zinc oxide, or mixtures thereof.
[0105] Powders and sprays can contain, in addition to a compound of
this invention, excipients such as lactose, talc, silicic acid,
aluminum hydroxide, calcium silicates and polyamide powder, or
mixtures of these substances. Sprays can additionally contain
customary propellants, such as chlorofluorohydrocarbons and
volatile unsubstituted hydrocarbons, such as butane and
propane.
[0106] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention.
[0107] Pharmaceutical compositions of this invention suitable for
parenteral administration comprise one or more alcohols or
inhibitors according to the invention in combination with one or
more pharmaceutically-acceptable sterile isotonic aqueous or
nonaqueous solutions, dispersions, suspensions or emulsions, or
sterile powders which may be reconstituted into sterile injectable
solutions or dispersions just prior to use, which may contain
antioxidants, buffers, bacteriostats, solutes which render the
formulation isotonic with the blood of the intended recipient or
suspending or thickening agents.
[0108] In some cases, in order to prolong the effect of the alcohol
or inhibitor according to the invention, it is desirable to slow
the absorption of the alcohol or inhibitor from subcutaneous or
intramuscular injection. This may be accomplished by the use of a
liquid suspension of crystalline or amorphous material having poor
water solubility. The rate of absorption of the drug then depends
upon its rate of dissolution, which, in turn, may depend upon
crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally-administered composition is
accomplished by dissolving or suspending the alcohol or inhibitor
in an oil vehicle. One strategy for depot injections includes the
use of polyethylene oxide-polypropylene oxide copolymers wherein
the vehicle is fluid at room temperature and solidifies at body
temperature.
[0109] The pharmaceutical compounds of this invention may be
administered alone or in combination with other pharmaceutical
agents, or with other anti-thalamocortical dysrhythmia drugs or
medicaments, as well as in combination with a pharmaceutically
acceptable carrier or dilutent as described above.
[0110] The amount of pharmacological agent in the oral unit dosage
form, with as a single or multiple dosage, is an amount that is
effective for treating a neurological disorder. As one of skill in
the art will recognize, the precise dose to be employed will depend
on a variety of factors, examples of which include the condition
itself, the seriousness of the condition being treated, the
particular composition used, as well as various physical factors
related to the individual being treated. In vitro or in vivo assays
can optionally be employed to help identify optimal dosage
ranges.
[0111] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration. In some cases, these kits are
designed for daily administration over the specified term or cycle
of administration, in some cases for the number of prescribed
administrations per day, and organized so as to indicate a single
formulation or combination of formulations to be taken on each day
of the regimen or cycle. In some cases, the kit shall have a
calendar or days-of-the-week designation directing the
administration of the appropriate compositions on the appropriate
day or time.
[0112] The present invention further provides a method for
identifying an alkyl alcohol that modifies the voltage-dependent
activation kinetics or single channel ionic conductance of a
Ca.sub.v3 plasmalemmal-bound calcium channel protein. In this
method, atomic coordinates of the Ca.sub.v3 calcium channel protein
are provided. An alkyl alcohol is then subjected to computational
molecular docking. The alcohol that has the optimal virtual binding
to the a Ca.sub.v3 calcium channel protein is then selected. The
optimal virtual binding indicates that the alcohol binds to the a
Ca.sub.v3 calcium channel protein or alters the properties of the
lipid bi-layer at the channel's voltage sensing site.
[0113] Computational molecular docking can utilize a computer
screening method such as Virtual library screening (VLS) (ICM-VLS,
the proposed methodology (software) for this work, represents the
state-of-the-art in flexible ligand docking). Optimal virtual
binding may be substantially similar to that as shown in FIG.
8.
[0114] Computational molecular docking is a research technique for
predicting whether one molecule will bind to another, usually a
protein. Protein-ligand docking is performed by modelling the
interaction between protein and ligand: if the geometry of the pair
is complementary and involves favorable biochemical interactions,
the ligand will potentially bind the protein in vitro or in vivo.
Where the activity of a small molecule ligand (e.g., octanol) is
known and desired and the structure of the target protein (e,g.,
Ca.sub.v3-type calcium channel) is accessible, computational
molecular docking may identify the binding site on the protein, map
the pharmacophore space and afford rational improvement of the
binding of the said selected ligand to the target protein to
eliminate negative biological properties while retaining it
therapeutic activity.
[0115] In the instant invention, the computational molecular
docking of a molecule such as octanol to the Cav3-type calcium
channel is specifically addressed to identify optimized ligands to
binding a Ca.sub.v3 calcium channel and thereby reducing the
low-threshold-activated calcium current in a cell. Docking is
utilized in the field of drug design (most drugs are small
molecules), and using a computational approach allows researchers
to quickly screen large databases of potential drugs. Useful
docking algorithms for studying the interaction of octanol with
Cav3-type calcium channels are flexible ligand docking programs
that have performed well in VLS, a procedure whereby thousands of
molecules are docked in a short time to the protein in order to
detect high-affinity binding agents that may be screened to
pharmaceutical activity. See, e.g., Schapira et al., (2000) Proc.
Natl. Acad. Sci. U.S.A, 97:1008-1013; Schapira et al., (2003) Proc.
Natl. Acad. Sci. U.S.A, 100:7354-7359); Wang et al., (2003) Semin.
Oncol., 30:133-142; Degterev et al., (2001) Nat. Cell Biol.,
3:173-182; Gruneberg et al., (2002) J. Med. Chem., 45:3588-3602.
ICM-VLS, the proposed methodology (software) for this work,
represents the state-of-the-art in flexible ligand docking.
[0116] The structure of the Cav3-type calcium channel is not known.
However, the crystallographic structure of the related K.sub.vAP
and K.sub.v1.2 voltage gated K+ channels has been elucidated (Long
et al. (2005) Science, 309:897-903; Jiang et al. (2003) Nature,
423:33-41). ICM docking and VLS has been successfully used to map
the pharmacophore space of homology models. Accordingly, homology
models of the H and G forms of the Cav3-type calcium channels are
constructed and octanol is molecularly docked by computational
computer modeling to these homology models. A model of the P-type
calcium channel may serve as a negative control.
[0117] A three-dimensional model of the complex of octanol with
Cav3-type calcium channels emerges from this study. The chemical
groups of octanol facing the solvent and not buried in contact with
the protein are candidate sites for synthetic alteration to improve
the drug-like properties of octanol.
[0118] FIG. 8 shows a molecular modeling diagram in one low energy
conformation which demonstrates an affinity binding between
1-octanol and a homology model of a subunit of the Ca.sub.v3
calcium channel. In further detail, a homology model of a subunit
of the Ca.sub.v3 calcium channel was constructed, and 1-octanol was
docked to it and shown to have a preference for non-specific
docking to the lipid phase. One low energy conformation (the one
shown in FIG. 8) indicates affinity for the surface between S2 and
S5 near the voltage gated S4.
[0119] The invention also provides a method of identifying a
chemical entity which binds to a Ca.sub.v3 calcium channel. In this
method, a structure model of the Ca.sub.v3 calcium channel is
compared with a structure model for the chemical entity. A binding
surface on the Ca.sub.v3 calcium channel for the chemical entity is
then detected.
[0120] The invention includes the use of the structural
co-ordinates obtainable by subjecting a crystal comprising
Ca.sub.v3 calcium channel to X-ray diffraction measurements and
deducing the structural co-ordinates from the diffraction
measurements, to identify, screen, characterize, design or modify a
chemical entity. The invention further includes a chemical entity
identified by such a method of the invention, wherein the chemical
entity reduces the Ca.sub.v3 calcium channel-mediated
low-threshold-activated calcium current of a Ca.sub.v3-channel
expressing cell.
[0121] The Ca.sub.v3 calcium channel is identified as a therapeutic
target of alkyl alcohols. The structure of the Ca.sub.v3 channel is
also identified, and therefore allowing for identification of the
amino acid residues involved in binding of alkyl alcohols, such as
octanol as well as other inhibitors, to Ca.sub.v3 channels.
[0122] The identification of the interaction and the structures
allows for the characterization or identification of chemical
entities which can bind, and in particular, which can reduce
Ca.sub.v3-mediated low-threshold activated calcium current in a
Ca.sub.v3 expression to the Ca.sub.v3 channel. A number of
different types of inhibitors can be identified as discussed in
further detail below.
[0123] The structure of crystallised Ca.sub.v3 channels is
envisaged. Typically, the structural coordinates used are
obtainable by subjecting a crystal comprising a Ca.sub.v3 calcium
channel protein, or a fragment thereof, to X-ray diffraction
measurements and deducing the structural co-ordinates from the
diffraction measurements, to identify, screen, characterize, design
or modify a chemical entity. The structural co-ordinates indicate
the positions of individual atoms within the crystal and give an
indication of the space available for adjusting the position of
individual atoms when designing a chemical entity.
[0124] The crystal subjected to X-ray diffraction methods comprises
a Ca.sub.v3 channel protein or a fragment thereof. The Ca.sub.v3
protein may be from any source but is most usefully a human
Ca.sub.v3 protein. The Ca.sub.v3 calcium channel may be a modified
form. For example, the Ca.sub.v3 channel protein may be modified by
insertion, deletion, n-terminal or C-terminal addition, or
substitution of amino acid by another amino acid. Amino acid
substitutions may be conservative substitutions. Typically, when
crystallised, a Ca.sub.v3 mutant will adopt a similar 3-dimension
structure to that adopted by the wild-type channel protein.
[0125] References to Ca.sub.v3 channel protein herein refer to
Ca.sub.v3 and homologs thereof. Amino acid residues are defined
with reference to the position of the Ca.sub.v3 polypeptide. The
relevant amino acid residues of homologues of Ca.sub.v3 are the
equivalent amino acid residues, base on, for example, the best
alignment of the homologue to a human or mouse Ca.sub.v3.
[0126] A Ca.sub.v3 polypeptide may be isolated by any suitable
means for use in crystallization studies. For example, a Ca.sub.v3
polypeptide may be purified using biochemical means from a suitable
source. Typically, however, it is convenient to over-express
Ca.sub.v3 in cells and purify Ca.sub.v3 from those cells. Thus, a
polynucleotide encoding a Ca.sub.v3 may be used in the construction
of a vector. The Ca.sub.v3 may be crystallized according to any
method known to those skilled in the art. X-ray diffraction may be
carried according to any suitable method. The data collected from
X-ray diffraction experiments may be processed to deduce the
structural co-ordinates of Ca.sub.v3 calcium channel using any
suitable method.
[0127] The invention provides the use of structural co-ordinates to
identify, characterize, design or screen a chemical entity. The
chemical entity may be one which binds to Ca.sub.v3, and/or which
acts as an inhibitor of Ca.sub.v3-mediated low-threshold activated
calcium current a cell. Alternatively, the chemical entity may be a
modified Ca.sub.v3 polypeptide to alter the activity of the
Ca.sub.v3 calcium channel.
[0128] A chemical entity which binds to or inhibits a Ca.sub.v3
calcium channel is any chemical entity capable of forming an
association with a Ca.sub.v3 polypeptide. The binding or inhibition
may be non-specific, for example, such an entity may also bind to
or inhibit other calcium channels. More usefully, an agent may be
designed or identified which specifically binds to or inhibits the
Ca.sub.v3 calcium channel. An agent may be designed or identified
which is a specific inhibitor of Ca.sub.v3, but not other proteins
generally or calcium channels in particular.
[0129] The structural co-ordinates of Ca.sub.v3 allow with skill in
the art to predict which amino acids are important in binding an
alkyl alcohol and reducing low-threshold activate calcium current.
The substrate binding site may be shown as a 2-dimensional
representation or a 3-dimensional representation produced by
physical models or displayed on a computer screen. Such
representations can be used to design, identify or screen chemical
entities which bind to or inhibit or are predicted to bind to or
inhibit the Ca.sub.v3 calcium channel. Such representations can
also be used to identify modifications of Ca.sub.v3 that alter its
activity characteristics.
[0130] The representations of the structures may be used in other
ways. For example, the representations of the Ca.sub.v3 polypeptide
may be used to model constraints by the putative introduction of
covalent bonds between the atoms which come close together when
Ca.sub.v3 functions. Representation of the Ca.sub.v3 calcium
channel may be used to predict which residues of Ca.sub.v3 are
likely to be involved in steric hindrance. Such residues may be
modified, replaced or deleted to decrease esoteric hindrance in
order to increase avidity of the peptide for its substrates.
[0131] In general, it will be most useful to process the structural
co-ordinates obtainable according to the invention in
computer-based methods in order to identify or design chemical
entities with the desired molecular structure or to identify
chemical entities whose structure is complementary to all or part
of another chemical entity interest. Thus, chemical entities which
are structurally compatible with a Ca.sub.v3 polypeptide may be
identified or designed.
[0132] Such computer-based methods fall into two broad classes:
database methods, and de novo designed methods. In database
methods, the chemical entity of interest is compared to all
chemical entities present in a database of chemical structures and
chemical identities whose structure is in some way similar to the
compound of interest identified. The structures in the database are
based either on experimental data, generated by NMR or X-ray
crystallography, or models of 3 dimensional structures based on 2
dimensional data. In de novo design methods, models of chemical
entities, for example, such as those which might bind to a
Ca.sub.v3 target, are generated by a computer program using
information derived from known structures and/or theoretical
rules.
[0133] Similarly, the Ca.sub.v3 structural coordinates may be used
to screen for the expected activity of chemical entities selected,
designed or shown to be modulators such as inhibitors of
low-threshold-activated calcium current. For example, the compounds
may be screened to assess the likelihood of a Ca.sub.v3 binding
agent additionally reducing a low-threshold-activated calcium
current in a cell. Such screening methods may be useful in
identifying agents which selectively reduce Ca.sub.v3-mediated
low-threshold-activated calcium current, but not other calcium
channels generally or other activities of the Ca.sub.v3 calcium
channel, in particular.
[0134] Chemical entities designed or selected according to the
methods of the invention may be tested and optimized using
computational or experimental evaluation. Experimental methods to
assay for the Ca.sub.v3-mediated low-threshold-activated calcium
current are described in more detail below.
[0135] Based on the structure of the Ca.sub.v3 polypeptide, a
number of different types of inhibitors can be identified.
[0136] The following examples illustrate the representative modes
of making and practicing the present invention, but are not meant
to limit the scope of the invention since alternative methods may
be utilized to obtain similar results.
[0137] Alternatively, an alteration of the properties of the lipid
bi-layer at the channel's voltage sensing site for the chemical
entity is detected. Detecting a binding surface or detecting the
alteration of the properties of the lipid bi-layer is indicative of
the presence of a chemical entity which binds to the Ca.sub.v3
calcium channel.
[0138] In other aspects, the present invention provides a method of
identifying an inhibitor of a Ca.sub.v3 calcium channel in a cell.
In this method, an isolated Ca.sub.v3 plasmalemmal-bound calcium
channel is contacted with a candidate compound, and the presence of
a complex, or lack thereof, between the Ca.sub.v3 calcium channel
and the compound is then detected. The candidate compound is an
inhibitor if it forms a complex with the Ca.sub.v3 calcium channel
or alters the properties of the lipid bi-layer at the channel's
voltage sensing site. The inhibitor can be any type of molecule or
chemical entity that can bind to a Ca.sub.v3 channel and stop it
from functioning as a Ca.sub.v3 calcium channel, e.g., which can
reduce Ca.sub.v3-mediated low-threshold activated calcium current
in a Ca.sub.v3 expression to the Ca.sub.v3 channel.
[0139] The image is oriented with the extracellular mileu at the
top and the cytosol at the bottom. The voltage gated paddle
containing S4 is at the bottom right. The protein chain is
presented as a continuous amino acid sequence with an N- to
C-terminus.
[0140] Compounds used as pharmaceuticals are usually selective for
their intended target or the targets involved in producing the
desired effect. A lack of selectivity can lead to toxic side
effects that render particular compounds unsuitable for use in
human or animal therapy. One approach to identifying compounds that
are selective for the intended target is to undertake structural,
mechanistic and other analyses on the intended agents and to use
the information gained to aid in the preparation of selective
compounds, or more selective compounds (relative to those
previously known), for use as pharmaceuticals for use in humans or
animals. The description describes structural and other studies on
the Ca.sub.v3 calcium channels that enable the design of selective
inhibitors of Ca.sub.v3.1, Ca.sub.v3.2, and Ca.sub.v3.3 on a
closely related calcium channels.
[0141] The structural model of the Ca.sub.v3 calcium channel
structural factors or structural coordinates determined by
projecting the Ca.sub.v3 amino acid sequence onto the structure of
a known channel protein.
[0142] The invention will be more fully understood by reference to
the following examples. They should not, however, be construed as
limiting the scope of the invention.
EXAMPLES
Example 1
Preparation of Brain Slices Containing Ventrobasal Thalamic
Nucleus
[0143] Long-Evans rats (two to four weeks old, 40-50 g; either sex;
Taconic Farms) and Harley guinea pigs (four to eight weeks old,
50-300 g) were anesthetized with pentobarbital (Nembutal, 120 mg/Kg
i.p.), decapitated and the bone and dura mater covering the
cortical surface were carefully peeled away. The rostral part of
the brain was glued onto microslicer stage of a vibroslicer (Leica
Microsistemas, Bannockburn, Ill., USA) containing a low
Na.sup.+/high sucrose artificial cerebrospinal fluid ("ACSF")
solution (248 mM sucrose, 26 mM NaHCO.sub.3, 1.25 mM
NaH.sub.2PO.sub.4, 5 mM KCl, 3 mM MgSO.sub.4, 0.5 mM CaCl.sub.2, 10
mM (+)-sodium-L-ascorbate, 3 mM sodium pyruvate and 10 mM glucose,
aerated with 95% O.sub.2/5% CO.sub.2 to a final pH of 7.4). 180
.mu.m to 240 .mu.m transverse slices were obtained containing both
thalamic and cortical areas. After cut, slices were allowed to
recover in an incubation chamber at 37.degree. C. for at least 30
min. containing continuously oxygenated combination of half-half
low Na.sup.+/high sucrose-normal ACSF (124 mM NaCl, 5 mM KCl, 1.25
mM KH.sub.2PO.sub.4, 26 mM NaHCO.sub.3, 1.2 mM MgCl.sub.2, 2.4 mM
CaCl.sub.2, and 10 mM glucose, pH 7.4).
Example 2
Whole-Cell Patch Recording Methods
[0144] Patch recordings were performed at 35.degree. C. in a
chamber constantly perfused with normal ACSF solution containing
carbachol (50 .mu.M-100 .mu.M, a cholinergic agonist, both
nicotinic and muscarinic), tetrodotoxin (100 nM-500 nM, a
voltage-dependent sodium channel blocker) and an equivalent
proportion of the vehicle Tween-80 used to dissolve octanol (Llinas
et al. (1982) Nature 297:406-408.). Patch electrodes were made from
borosilicate glass and had resistances of 3 to 8 M.OMEGA. when
filled with either high potassium intracellular solution (130 mM
KMeSO.sub.3, 10 mM NaCl, 10 mM HEPES, 1 mM EGTA, 4 mM Mg-ATP, 0.4
mM Na-GTP, 2 mM MgCl.sub.2, 10 mM sucrose and 10 mM
phosphocreatine, pH 7.3, 290 mOsm); or either a high cesium/QX314
intracellular solution (120 mM CsMeSO.sub.3, 8 mM NaCl, 10 mM
HEPES, 5 mM EGTA, 10 mM TEA-Cl (triethylamine chloride), 4 mM
Mg-ATP, 0.5 mM GTP, 7 mM phosphocreatine, pH 7.3 (29 mOsm)). High
potassium solution was used when octanol-dependent effects were
studied on both basic membrane properties as well as on the pattern
of action potentials of thalamic neurons. High Cs/QX314 was used to
quantify the blocking effect of octanol on the voltage-sensitive
calcium currents.
[0145] Neurons were recorded using an Axopatch 700B amplifier (Axon
Instruments, Molecular Devices, Sunnyvale, Calif., USA) in
combination with the PCLAMP 10.0 software (Axon Instruments,
Molecular Devices, Sunnyvale, Calif., USA). Data were filtered at 5
kHz, digitalized, and stored on a computer to further analysis
off-line. Access resistance (8-20M.OMEGA.) was continuously
monitored during experiments.
Example 3
A. Inhibition of Ca.sub.v3 Plasmalemmal-Bound Calcium Channel With
1-Octanol
[0146] FIGS. 1A and 1B show the patch recording of individual
thalamic neurons from the ventrobasal nucleus obtained from the
rodents' brain slices prepared according to Example 1. The thalamic
nucleus processes the somatosensory information from rodents'
whiskers. The effect of 10 .mu.M octanol on calcium channels at two
different voltage levels was shown.
[0147] Using a holding level where only low voltage activated
calcium currents can be open (-30 mV), octanol blocks peak fast
activated Ca.sub.v3.1 calcium current (ICa) amplitude by about 30%
(FIG. 1A). When both low and high voltage activated calcium
currents are open, octanol blocks the peak of the fast component
(Ca.sub.v3.1) leaving unchanged the slow component that remains
open at the end of the depolarized pulse (-10 mV)( FIG. 1B).
Therefore, 1-octanol is shown to specifically block low threshold
activated Ca.sub.v3-type currents from Ventrobasal thalamic
neurons.
B. Inhibition of Ca.sub.v3 Plasmalemmal-Bound Calcium Channel with
2-Octanol
[0148] FIGS. 2A and 2B show the blocking effect of clinical
concentrations of 2-octanol directly on the low threshold activated
calcium currents using a high Cs/QX314 intracellular solution in a
voltage-clamp mode, as described in Example 2.
[0149] When a concentration of 100 .mu.M 2-octanol is applied, the
low threshold activated Ca.sub.v3 calcium currents are completely
abolished. FIG. 2A shows four representative Ca.sub.v3 calcium
current activated after depolarizing the thalamic neuron from a
holding potential of -70 mV to -60 mV, -30 mV, -20 mV and -10 mV
using square pulses (FIG. 2A, top). In this case, a progressive
reduction of the Ca.sub.v3 calcium currents peak is observed for
increasing 2-octanol concentrations. FIG. 2B shows the
current-voltage (i.e., I-V curve) relationship for the same
thalamic neuron described in FIG. 2A, but for a wider range of
holding potentials. Similarly, Ca.sub.v3 peak currents are
partially reduced at 10 M 2-octanol while almost abolished at 100
.mu.M 2-octranol.
C. Inhibition of Thalamic Low Frequency Oscillations with
2-Octanol
[0150] FIGS. 3A and 3B show the effect of clinical levels of
2-octanol on thalamic cell intrinsic oscillatory activity using
intracellular recording from thalamic cells in vitro.
[0151] Octanol selectively blocks thalamic low frequency
oscillations. This finding is in keeping with the results
illustrated in FIGS. 1 and 2, as such oscillations are mediated by
low threshold activated calcium conductance (Jahnsen et al., J.
Physiol. (London) 346:205-226). FIG. 3A (black lines) shows a pair
of low threshold spikes firing at 2 Hz generated after the rebound
of a short hyperpolarizing pulse is applied (FIG. 3B). Once the
activation paradigm is established, five min. of perfusion with 10
.mu.M 2-octanol results in a complete abolishment of the
oscillatory activity, which is partially reversible after 40 min.
of washout to remove the 2-octanol.
D. Inhibition of Ca.sub.v3 Plasmalemmal-Bound Calcium Channel with
1-Octanol
[0152] FIGS. 4A and 4B are traces showing the effect of 1-octanol
on low-threshold calcium spikes using a current clamp technique, as
described in Example 2.
[0153] The results show that the effect of 1-octanol on the low
threshold spikes is concentration-dependent. The application of
1-octanol increments membrane resistance (i.e., a greater change in
membrane potential after the injection of the same amplitude
current pulse) after blocking Cav3 calcium currents (as shown in
FIGS. 1 and 2). FIGS. 4A and 4B also show that 1-octanol at the
concentration of 50 .mu.M (dotted lines) further decreases the peak
of low threshold spikes activated using ramps of current instead of
square pulses.
E. Activation of Low Threshold Spikes in Thalamic Neurons
[0154] FIGS. 5A and 5B demonstrate that low threshold spikes can be
activated following TTX removal from the external solution using a
current clamp technique. After TTX removal from the external normal
ACSF solution (as described in Example 2), low threshold spikes can
be activated in the presence of fast action potential mediated by
voltage-sensitive sodium channels. In control conditions and after
a long hyperpolarizing pulse, thalamic neurons respond with a low
threshold spike on top of which several action potentials can be
observed (FIGS. 5A and 5B). Under these conditions, after
2-octanol's block of low threshold spike, the increment in input
resistance notably increased the frequency of action potentials of
the thalamic neurons (FIGS. 5A and 5B).
F. Inhibition of Thalamic High-Frequency Subthreshold Oscillations
with 1-Octanol with TTX Comparison with TTX Plus 1-Octanol
[0155] The effect of (1) tetrodotoxin ("TTX") and (2) TTX and
1-octanol on the high-frequency subthreshold oscillation previously
described in the Ventrobasal thalamic neurons (Pedroarena &
Llinas (1997) Proc. Natl. Acad. Sci. U S A. 94(2): 724-728) was
tested. Using patch configuration and high-K.sup.+ intracellular
solution, in the presence of TTX (2 .mu.M), thalamic neurons from
wild-type mouse respond with a low threshold spike after
hyperpolarizing square pulses (FIG. 6A, wildtype, up left black
lines) while depolarizing square pulses result in a clear 40 Hz
oscillation (FIG. 6A, wildtype, up left, inset). Following the
application of 50 .mu.M octanol, 40 Hz oscillations are observed
while the rebound low threshold spike is abolished (FIG. 6,
wildtype, up right, inset, red lines). When the alpha-1A subunit of
the P/Q-type calcium channels is absent due to a homozygous
knock-out mutation (FIG. 6, alpha1A-KO), a clear low threshold
spike is present in the thalamic neurons (also blocked by octanol,
see FIG. 6B, alpha1A-KO, bottom right) while no high-frequency 40
Hz oscillations are present (FIGS. 6A and 6B, alpha1A-KO, bottom
left and right insets) either before or after octanol
application.
[0156] The examples show that applications of clinical
concentrations of octanol at micromolar level administration result
in a reversible membrane conductance modification that can
significantly reversibly block the low threshold activated
Ca.sub.v3 calcium currents of the neurons present in the thalamic
Ventrobasal nucleus. These results in an electrical activation
change where thalamic neurons fail to generate low frequencies,
either spontaneously or by being evoked. This effect is further
demonstrated by increasing the concentration of octanol to 50 mM
and determining the resulting effects on the low threshold spikes
generated by a slow change in membrane potential using a ramp of
current in the presence of TTX.
[0157] This selective block of Ca.sub.v3 calcium currents allows
continued activity of these neurons at higher frequencies. Indeed,
octanol does not appear to affect the high frequency oscillations
in the thalamic neurons. Accordingly, the specific repression of
low frequency oscillations with octanol provides an appropriate
pharmacological tool to reduce the low frequency oscillations
observed in patients with thalamocortical dysrhythmias while
leaving untouched the mechanisms by which high-frequency
oscillations are generated in the thalamocortical system.
Example 4
The Effect of Octanol on Frequency Profiles Presented in Wild Type
and Alpha-1A Knockout Mice
[0158] Alpha1A knockout mice, which have an enhanced Ca.sub.v3
calcium channel activity, develop progressive neurological symptoms
resembling Thalamo-cortical Dysrhythmia characterized specifically
by ataxia and dystonia, before dying, around four weeks after birth
(Jun et al. (1999) Proc. Natl. Acad. Sci. U S A.
96(26):15245-50).
[0159] FIG. 7A shows an EEG recording during 30 seconds from a wild
type mouse where a characteristic low amplitude EEG recording can
be observed. When a Fast Fourier transform analysis is performed in
two segments of 1 second each (FIG. 7A, segments a and b) from the
same wild type trace, a wide range of frequencies is observed.
Importantly, the presence of high frequencies in the range of gamma
band is observed (FIG. 7A, red arrows). In contrast, an alpha-1A
knockout mouse presents a totally different EEG recoding. As shown
in FIG. 7B, the presence of high amplitude low frequency is
characteristic. Furthermore, the frequency analysis of these
alpha-1A animals shows no high frequencies presented when
one-second interval is used (FIG. 7B, segments c and d).
[0160] As in the FIG. 7C the wild type (another way to say control)
and the mutant mouse recordings are compared. The increase activity
produced by the increase of Cav3 channel results in a large
increase in low frequencies (Thalamo-cortical Dysrhythmia [TCD]). A
more detail plot of frequencies show that the lower frequencies
(theta 2-8 Hz) are increased and the higher frequencies are absent
(red bars in the plot) while the control displays the normal 10 Hz
(alpha rhythm) and 40 Hz (gamma rhythm). It is expected that
Octanol given to the mutant mice will reduce the amplitude of the
theta rhythm, demonstrating is use as a possible TCD pharmaceutical
compound.
[0161] The administration of 1-octanol (Sinton et al., (1989)
Pflugers Archiv 414: 31-36) in wild types versus knockout mice
allows the quantification of the effects of such a drug in the high
and low frequencies in the EEG. Since knockout mice only present
low frequencies, a dose-response curve of 1-octanol can be easily
prepared. Also, an augmentation in the ratio of high/low
frequencies is expected for the wild types treated with
1-octanol.
[0162] As shown in FIG. 7D, following octanol administration (i.p.,
0.2 mg/kg) the second line of EEG activity and a faster sweep
inset. Analysis of the frequency components plotted in blue bars
show a decrease in the low frequencies (0-15 Hz) and an increase in
hi frequencies (30-45 Hz) consistent with the fact that Cav3
channels are responsible for low frequencies and their block
produces an increase in high frequency.
Example 5
Methods for Identifying an Alkyl Alcohol that Binds to a Ca.sub.v3
Plasmalemmal-Bound Calcium Channel Using Computational Molecular
Docking
[0163] A homology model of a single subunit of the Ca.sub.v3
calcium channel is made as described above, and 1-Octanol is docked
to it using computational methods. The results in FIG. 8 show a
preference for non-specific docking to the lipid phase. One low
energy conformation indicates affinity for the surface between S2
and S5 near the voltage gated S4.
[0164] The image is oriented with the extracellular milieu at the
top and the cytosol at the bottom. The voltage gated paddle
containing S4 is in white at the bottom right. The protein chain is
colored in a gradient from N- to C-terminus with dark blue being
the N-terminus and dark red being the C-terminus. FIG. 8 shows an
image of the model of the tetramer of the Ca.sub.v3 calcium
channel. The yellow balls at the bottom are the location of the ion
channel and correspondingly the domain in which they are located is
the transmembrane domain of the channel. The low energy
conformation of octanol is shown in green near the channel in the
voltage gate area of the tetramer.
Equivalents
[0165] All numbers expressing quantities of ingredients, reaction
conditions, analytical results and so forth used in the
specification and claims are to be understood as being modified in
all instances by the term "about." Accordingly, unless indicated to
the contrary, the numerical parameters set forth in the
specification and attached claims are approximations that may vary
depending upon the desired properties sought to be obtained by the
present invention. At the very least, and not as an attempt to
limit the application of the doctrine of equivalents to the scope
of the claims, each numerical parameter should be construed in
light of the number of significant digits and ordinary rounding
approaches.
[0166] Modifications and variations of this invention can be made
without departing from its spirit and scope, as will be apparent to
those skilled in the art. The specific embodiments described herein
are offered by way of example only and are not meant to be limiting
in any way. It is intended that the specification and examples be
considered as exemplary only, with a true scope and spirit of the
invention being indicated by the following claims.
* * * * *