U.S. patent application number 10/284889 was filed with the patent office on 2003-05-08 for method for the suppression of visceral pain by regulating t type calcium channel.
Invention is credited to Kim, Chan-Ki, Kim, Dae-Soo, Shin, Hee-Sup.
Application Number | 20030086980 10/284889 |
Document ID | / |
Family ID | 19715647 |
Filed Date | 2003-05-08 |
United States Patent
Application |
20030086980 |
Kind Code |
A1 |
Shin, Hee-Sup ; et
al. |
May 8, 2003 |
Method for the suppression of visceral pain by regulating T type
calcium channel
Abstract
The disclosure concerns a method for the suppression of visceral
pain by regulating the T-type calcium channel; a visceral pain
inhibitor that includes a T-type calcium channel inhibitor as an
effective ingredient; and a method of screening a visceral pain
inhibitor by investigating the suppression activity of T-type
calcium channels. Particularly, the present invention relates to a
method for the suppression of visceral pain by regulating an alpha
1G T-type calcium channel in the central nervous system and alpha
1H and alpha 1I T-type calcium channels in the peripheral nervous
system; a visceral pain inhibitor that includes a T-type calcium
channel inhibitor as an effective ingredient; and a method of
screening a visceral pain inhibitor by investigating the
suppression activity of T-type calcium channels. The method of the
present invention can be effectively used to suppress visceral pain
by regulating T-type calcium channel in a precise mechanism without
any side effects.
Inventors: |
Shin, Hee-Sup; (Uiwang-si,
KR) ; Kim, Dae-Soo; (Seoul, KR) ; Kim,
Chan-Ki; (Seoul, KR) |
Correspondence
Address: |
Michael N. Mercanti
Roberts and Mercanti, L.L.P.
Suite 203
105 Lock Street
Newark
NJ
07103
US
|
Family ID: |
19715647 |
Appl. No.: |
10/284889 |
Filed: |
October 31, 2002 |
Current U.S.
Class: |
424/646 ;
435/7.1; 514/1 |
Current CPC
Class: |
G01N 33/6872 20130101;
A61P 25/04 20180101; G01N 2500/04 20130101; A61P 1/00 20180101;
A61K 31/4184 20130101; A61P 43/00 20180101; A61K 31/00 20130101;
A61P 29/00 20180101; A61K 33/00 20130101; A61P 25/02 20180101; A61P
25/00 20180101 |
Class at
Publication: |
424/646 ;
435/7.1; 514/1 |
International
Class: |
A61K 033/26; A61K
031/00; G01N 033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 2, 2001 |
KR |
2001/68180 |
Claims
What is claimed is:
1. A method for the suppression of visceral pain by regulating the
T-type calcium channel.
2. The method as set forth in claim 1, wherein the T-type calcium
channel is selected from a group consisting of alpha 1G, alpha 1H
and alpha 1I T-type calcium channels.
3. The method as set forth in claim 1, wherein the suppression of
visceral pain is achieved by activating the function of alpha 1G
T-type calcium channel in the central nervous system.
4. The method as set forth in claim 1, wherein the suppression of
visceral pain is achieved by inhibiting the function of alpha 1H
and alpha 1I T-type calcium channels in the peripheral nervous
system.
5. A visceral pain inhibitor containing the T-type calcium channel
inhibitor as an effective ingredient.
6. The visceral pain inhibitor as set forth in claim 5, wherein the
T-type calcium channel is alpha 1H or alpha 1I T-type calcium
channel.
7. The visceral pain inhibitor as set forth in claim 5, wherein the
T-type calcium channel inhibitor is selected from a group
consisting of mibefradil and Ni.sup.2+.
8. A screening method of the visceral pain inhibitor by
investigating the suppression activity of the T-type calcium
channel.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method for the
suppression of visceral pain by regulating the T-type calcium
channel; a visceral pain inhibitor that includes a T-type calcium
channel inhibitor as an effective ingredient; and a method of
screening a visceral pain inhibitor by investigating the
suppression activity of T-type calcium channels. Particularly, the
present invention relates to a method for the suppression of
visceral pain by regulating an alpha 1G T-type calcium channel in
the central nervous system and alpha 1H and alpha 1I T-type calcium
channels in the peripheral nervous system; a visceral pain
inhibitor that includes a T-type calcium channel inhibitor as an
effective ingredient; and a method of screening a visceral pain
inhibitor by investigating the suppression activity of T-type
calcium channels.
BACKGROUND OF THE INVENTION
[0002] Visceral pain is usually caused by excessive dilation of
internal organs, necrosis of these cells or intensive contraction
or acute relaxation of internal organs. When there is a tumor,
infection or congestion in internal organs, slight mechanical
stimulus, acidic or basic solution might cause severe pain.
Visceral pain caused especially by tumors cannot be easily
suppressed even with an excessive amount of morphine, so
neuro-surgical operations such as partial myelotomy of the spinal
cord are frequently used (Gybels, Pain Headache, 1989, 11:1-402).
However, the bilateral cordotomies or commissural myelotomies of
spinothalamic tract have many side effects. Relatively, the midline
myelotomy that severs the upper middle part of the T10 spinal cord
is known as an effective remedy (Nauta, J. Neurosurg., 1997,
86:538-542).
[0003] The above result proves that the visceral pain signal is
delivered to the brain through the spinal cord, which supports the
fact that the visceral pain signal is delivered through a different
channel from other pains. According to a MRI test carried out on
a-visceral-pain-induced monkey, it can be known that visceral pain
induces the activation of thalamus (Willis, Proc. Natl. Acad. Sci.
USA., 1999, 96:7675-79) The result of the test, after all, tells
that the visceral pain is delivered from the pain sensory cells in
the end of the internal organs through the spinal cord to thalamus.
Particularly, thalamus is known as an important sensory processing
organ since it delivers the stimulus to the cerebral cortex
(McCormick, Curr. Opin. Neurobiol., 1994, 4:550-556).
[0004] The calcium in nerve cells plays an important role in
delivering signals between nerve cells. Calcium has many different
delivery paths, however, when delivering peripheral stimuli, the
voltage-activated calcium channel is crucial. The voltage-activated
calcium channel can be categorized into the high voltage-activated
calcium channel (HVA) that is activated at a higher voltage than
the resting membrane potential and the low voltage-activated
calcium channel (LVA) that is activated at a lower voltage. The HVA
calcium channel can be subdivided into L, P/Q, N or R-type
depending on the pharmacological property of the current, and the
LVA calcium channel is differentiated as T-type (Tsien, Trends
Neurosci., 1988, 11:431-438).
[0005] The HVA calcium channel is evenly expressed from the
peripheral sensory cells to the central nervous system, and is well
known to play an important role in transmission of the sense of
pain and reflection. The inhibitors against these channels are
already commercially available as various anodynes (Schaible, Prog.
Brain Res., 2000, 129:173-190). However, it is not yet clearly
understood how the LVA calcium channel that generates the T-type
calcium current can regulate pain. The reason why the T-type
calcium current is categorized as one of the functions of the LVA
calcium channel is that when the excitability of nerve cells
lowers, the calcium current are generated so that the excitability
increases again (Llinas, J. Physiol (Lond), 1981, 315:549-567;
McCormick, Neuroscience, 1990, 39:103-113). Thus, the nerve cells
excited by the T-type calcium channel have the property of burst
firings and induce a type of excitability different from tonic
firings (Llinas, J. Physiol (Lond), 1981, 315:549-567). The channel
protein of the T-type calcium channel is encoded by three different
genes, which are referred to as alpha1G, alpha1H and alpha1I
respectively (Perez-Reyes, Nature, 1998, 391:896-900). It is known
that the alpha1G and alpha1H T-type calcium channels are expressed
in the back of the spinal cord, and that the alpha1G is expressed
in thalamocortical relay neurons (Talley, J. Neurosci., 1999,
19:1895-1911), and that is identical with the delivery path of the
visceral pain. Recently, it has been proved in an experiment using
a T-type calcium current inhibitor, mibefradil, that the function
of the T-type calcium current in the peripheral nerves is related
to hyperalgesic reaction against thermo-stimuli or mechanical
stimuli by reducing agents (Todorovic, Neuron, 2001, 31:75-85),
however, it has not yet been found which T-type calcium channel is
related. Mibefradil (RO40-5967) was initially known for lowering
blood pressure (Clozel, Cardiovasc Drugs Ther., 1990, 4:731-736;
Hefti, Arzneimittelforschung, 1990, 40:417-421), and was reported
to have a suppression effect (Viana, Cell Calcium, 1997,
22:299-311). Recently, it has been reported that Mibefradil has the
most selective suppression effect on T-type calcium channels.
[0006] Thus, the present inventors have studied about visceral pain
with alpha1G-/- transgenic mice and found that the alpha1G-/-
transgenic mice show hyperalgesia to visceral pain caused by acetic
acid. In wild-type mice, visceral pain caused by acetic acid could
be alleviated by administration of mibefradil at the periphery but
enhanced when mibefradil is injected in the brain. The present
invention has been accomplished by confirming that visceral pain
can be modulated by controlling the T-type calcium channel.
SUMMARY OF THE INVENTION
[0007] It is an object of the present invention to provide a method
for the suppression of visceral pain by regulating the T-type
calcium channel.
[0008] It is another object of the present invention to provide a
visceral pain inhibitor that includes a T-type calcium channel
inhibitor as an effective ingredient.
[0009] It is a further object of the present invention to provide a
method of screening a visceral pain inhibitor by investigating the
suppression activity of T-type calcium channels.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is a graph showing the responses of alpha 1G-/- mice
and normal mice against mechanical stimuli such as paw withdrawal
and tail flick test,
[0011] .circle-solid.: normal mice .smallcircle.: alpha 1G-/-
mice
[0012] FIG. 2 is a graph showing the responses of alpha 1G-/- mice
and normal mice against thermostimulus Hyperalgesia that is caused
by a thermostimulus by radiation or by inflammation,
[0013] .box-solid.: normal mice .quadrature.: alpha 1G-/- mice
[0014] FIG. 3 is a graph showing the responses of alpha 1G-/- mice
and normal mice against pain in internal organs that is caused by
acetic acid,
[0015] .circle-solid.: normal mice .smallcircle.: alpha 1G-/-
mice
[0016] FIG. 4 is a graph showing the suppression degree in
accordance with the concentration of a T-type calcium channel
inhibitor, mibefradil.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0017] The present invention provides a method for the suppression
of visceral pain by regulating the T-type calcium channel.
[0018] The present invention also provides a visceral pain
inhibitor that includes a T-type calcium channel inhibitor as an
effective ingredient.
[0019] The present invention also provides a method of screening a
visceral pain inhibitor by investigating the suppression activity
of T-type calcium channels.
[0020] Hereinafter, the present invention is described in
detail.
[0021] The present invention provides a method for the suppression
of visceral pain by regulating the T-type calcium channel.
[0022] The present invention provides a method for the suppression
of visceral pain either by activating alpha 1G T-type calcium
channel function in the brain or by suppressing alpha 1H and alpha
1I T-type calcium channel function in the peripheral nervous
system.
[0023] The T-type calcium channel is categorized into alpha 1G,
alpha 1H and alpha 1I depending on the organization unit of
small-pore forming, and in the present invention, the inventors
carried out an experiment related to pain by using the alpha 1G-/-
transgenic mice so as to suppress the function of the alpha 1G
protein, one of the component of alpha 1G T-type calcium channel.
It has been known that mechanical stimuli, which are acute pains,
are controlled by spinal reflex, and the responsiveness of the
mechanical stimuli is proportionate with the intensity of the pains
received by the peripheral organs. The alpha 1G-/- mice do not have
much difference from the normal mice in the paw withdrawal and tail
flick test (see FIG. 1). In addition, the result of the thermal
pain response analysis using radiant heat in which spinal reflex
and supraspinal mechanism is involved shows that the alpha 1G-/-
mice does not have much difference from the normal mice, either in
thermo-stimulus hyperalgesia caused by inflammation reaction or
thermo-stimulus by infrared radiation (see FIG. 2). As mentioned
above, deducing from the fact that the alpha 1G-/- mice reacts
normally to thermal or mechanical stimuli, it can be known that the
loss of the alpha 1G T-type calcium channel do not affect the
development of the peripheral sensory organs, that is, the nerves
that are involved in spinal reflection and inflammation
reaction.
[0024] From the observation of the response of the internal pains
induced by acetic acid, the alpha 1G-/- mice show serious
hyperalgesia to the visceral pain induced by acetic acid (see FIG.
3). It shows that the alpha 1G T-type calcium channel is
selectively involved in the control of visceral pain. In order to
find out whether the alpha 1G T-type calcium channel that is
involved in visceral pain control functions in the peripheral
sensory organs or in the part where the spine is connected to the
brain and thalamus, the present inventors injected mibefradil, a
T-type inhibitor, in the same spot of acetic acid injection. The
result was that there was analgesia to the visceral pain caused by
the acetic acid (see FIG. 4). Mibefradil in this case suppresses
the T-type calcium channel only in the peripheral nervous system
since it cannot pass through the brain barrier inside the body.
[0025] From the result, it can be deduced that the pain suppression
of the alpha 1G T-type calcium channel only functions in the
central nervous system, not in the peripheral nervous system. It
can also be known that the other types of the T-type calcium
channels, alpha 1H and alpha 1I calcium channels increase pain in
the peripheral nervous system.
[0026] As described above, the T-type calcium channel functions
wholly differently in the peripheral nervous system and in the
central nervous system concerning visceral pain control, and the
T-type calcium channel is activated when the resting membrane
potential lowers, thus suppressing the membrane potential from
being low, which increase the activity of the visceral pain sensory
cells in the peripheral organs. This is supported by the fact that
the T-type calcium channel functions against hyperalgesia to
mechanical or thermal stimuli induced by reducing agents. As for
visceral pain, the dilation of intestinal cells or outflow of
reduced substrates from cells caused by necrosis is brought along,
so it is highly likely that hyperalgesia is already included in the
normal algesia.
[0027] The present invention also provides a visceral pain
inhibitor that includes a T-type calcium channel inhibitor as an
effective ingredient.
[0028] When a visceral pain inhibitor that includes a T-type
calcium channel inhibitor as an effective ingredient is injected
into a body, the inhibitor reacts with alpha 1H and alpha 1I T-type
calcium channel, which will eventually suppress visceral pain by
suppressing the functions of the above-mentioned alpha 1H and alpha
1I T-type calcium channel.
[0029] In the present invention, the T-type calcium channel
inhibitor is selected from a group consisting of mibefradil and
Ni.sup.2+.
[0030] Moreover, the present invention provides a method of
screening a visceral pain inhibitor by investigating the
suppression activation of the T-type calcium channel.
[0031] In the present invention, the suppression activity of the
T-type calcium channel of chemical materials or natural materials
is investigated; the materials that have suppression activity to
the T-type calcium channel are selected; and among the selected
materials, the material that has an analegesic effect only on the
visceral pain induced by acetic acid, et al. is found by carrying
out experiments related pain with alpha 1G-/- mice and normal
mice.
[0032] In accordance with the present invention, the T-type calcium
channel inhibitor has a suppression effect on visceral pain in a
precise mechanism without any side effects, therefore, the selected
material from these T-type calcium channel inhibitors can be used
as a visceral pain inhibitor.
EXAMPLES
[0033] Practical and presently preferred embodiments of the present
invention are illustrative as shown in the following Examples.
[0034] However, it will be appreciated that those skilled in the
art, on consideration of this disclosure, may make modifications
and improvements within the spirit and scope of the present
invention.
Example 1
Generation and Management of Alpha 1G-/- Transgenic Mice
[0035] <1-1> Generation of Alpha 1G-/- Transgenic Mice
[0036] The present inventors have produced transgenic mice that
have the genotype of alpha 1G-/- by using an embryo that has the
genotype of alpha 1G+/- (International Depository Authority: Korea
Institute of Bioscience and Biotechnology Gene Bank, Accession No:
KCTC 10086 BP). Particularly, the heterozygote transgenic mice
having alpha 1G-/- genotype was produced by transplanting the
embryo having alpha 1G+/- genotype to a surrogate mother, and the
homozygote transgenic mice having alpha 1G-/- genotype was produced
by crossbreeding male and female of the above heterozygote
transgenic mice.
[0037] <1-2> Management of Animal
[0038] All animals were allowed to access freely to food and water
under the environment of controlled temperature and humidity, and
they were bred under the condition in which the daytime starts at 8
o'clock in a 12 hour cycle of daytime and nighttime. All male and
female F2 mice were used in the experiments when they are 8-15 week
old.
Example 2
Analysis of the Response to Mechanical Stimuli
[0039] In order to observe the response of the alpha 1G-/- mice to
mechanical stimuli, the present inventors carried out a paw
withdrawal test and a tail flick test.
[0040] <2-1> Paw Withdrawal Test
[0041] Paw withdrawal test was based on that described by Mogil et
al (Mogil et al., J. Neurosci., 1999, 19:RC25). Particularly, the
alpha 1G-/- mice were placed individually on a fine mesh metal
floor and allowed to acclimate for at least 2 hr. The mechanical
threshold was measured using calibrated von Frey filaments
(Stoelting) and was defined as the bending force, in grams, at
which the mice withdraws its paw. The filament was applied from
underneath the floor, through the mesh, to the plantar surface of
the paw for each limb. The response score was assessed as the total
numbers of paw withdrawals in 10 consecutive trials for each
filament and the average value of the response was used in the
analysis.
[0042] <2-2> Tail Flick Test
[0043] The local pressure required to elicit tail flick was
determined using von Frey filaments. The alpha 1G-/- mice were
habituated in the mice restrainer 30 minutes every day for 2 weeks.
The bending force of each monofilament was applied locally to the
tail resting on a table. Only flicking of the pressed tail was
defined as a nociceptive response. The response score was assessed
as the average of the total tail flicking number in 10 consecutive
trials with an interval of 10 min between each filament
application.
[0044] As a result, the alpha 1G-/- mice do not have any difference
from the normal mice in responding to thermal or mechanical stimuli
using von Frey (FIG. 1)
Example 3
Responses to Radiant Heat and Hot Plate
[0045] The present inventors examined the thermal pain response by
using radiant heat assay (Hargreaves test) wherein the mechanism of
spinal reflex and supraspine is involved.
[0046] <3-1> Paw Withdrawal Test
[0047] The present inventors measured hind-paw withdrawal latency
by Hargreaves' method (Hargreaves et al., Pain, 1988, 32:77-88)
using an Ugo Basile plantar test apparatus (Stoelting). Mice were
placed in a Plexiglas box on an elevated glass plate and acclimated
for 2 hr before testing. The tests were performed at low (20) and
high (40) intensities. Response was defined as withdrawal of a paw
when head turning and paw licking were observed. The time was
defined as the paw withdrawal latency. Five to ten minutes were
allowed between each trial on both hind-paws and 4 to 5 trials were
averaged for each mice.
[0048] <3-2> Hot Plate Test
[0049] Thermal pain response was assessed using the hotplate test
(Mogil et al., J. Neurosci., 1999, 19:RC25). For the hot-plate
test, the mice was habituated for 2 days in a transparent testing
box (14.times.14.times.20 cm) with a metal bottom. The mice was
then placed on the box pre-heated to the desired temperature in a
thermo-regulated water bath, and the time was recorded to the first
hind-paw licking or jumping response (cut-off time, 60 s).
[0050] As a result, the alpha 1G-/- mice did not have much
difference from the normal mice in the response to thermal
hyperalgesia caused by inflammation reaction or thermostimuli by
infrared radiation (FIG. 2). Thus, it was confirmed that the loss
of the alpha 1G T-type calcium channel did not affect the
development of the peripheral sensory organs, that is, the nerves
that are involved in spinal reflection and inflammation
reaction.
Example 4
Analysis of Visceral Pain Induced by Acetic Acid (Writhing
Test)
[0051] The present inventors injected 0.6% acetic acid into the
peritoneal cavity of mice to examine the acetic acid-induced
visceral pain response. The visceral pain is elicited secondarily
to a delayed inflammatory response and induced abdominal stretching
and writhing behavior (Gyires and Torma, Arch Int. Pharmacodyn.
Ther., 1984, 267:131-140). Mice were placed individually in a
transparent home cage (24.times.18.times.12 cm) and allowed to
acclimate for at least 60 min. Then, 0.6% acetic acid (5.0 mg/kg)
was injected into the peritoneum, after which the mice was returned
to the testing chamber. The number of abdominal stretches or
writhing motions was counted for 20 min. All mice were used only
once in this experiment.
[0052] As a result, the alpha 1G-/- mice showed severe hyperalgesia
to the visceral pain induced by acetic acid (FIG. 3), which means
that the alpha 1G T-type calcium channel is involved selectively in
controlling the sense of visceral pain.
Example 5
Analysis of Visceral Pain Induced by Mibefradil
[0053] In order to find where the alpha 1G T-type calcium channel
functions in relation to controlling visceral pain, whether the
peripheral nerves or the part where the spine was connected to
thalamus and brain, the T-type inhibitor, mibefradil, was injected
into peritoneum, the same spot where the acetic acid (the visceral
pain inducer for the normal mice) was injected.
[0054] Particularly, in order to find out how mibefradil, the
T-type calcium channel inhibitor functions, mibefreail was
dissolved in 0.9% of NaCl at the concentration of 5 mg/ml. The
degree of writhing was measured in a visceral pain inducing
experiment 20 minutes after the injection of the said mibefradil at
the concentration of 1, 10 and 30 mg/kg respectively in peritoneum
of the mice.
[0055] As a result, the mibefradil induced analgesia to the
visceral pain caused by acetic acid in the normal mice (FIG.
4).
INDUSTRIAL APPLICABILITY
[0056] As shown above, a method of the present invention can be
effectively used to suppress visceral pain by regulating T-type
calcium channel in a precise mechanism without any side
effects.
[0057] Those skilled in the art will appreciate that the concepts
and specific embodiments disclosed in the foregoing description may
be readily utilized as a basis for modifying or designing other
embodiments for carrying out the same purposes of the present
invention. Those skilled in the art will also appreciate that such
equivalent embodiments do not depart from the spirit and scope of
the invention as set forth in the appended claims.
* * * * *