U.S. patent application number 12/937456 was filed with the patent office on 2011-11-10 for improving agent for neuropathic pain.
This patent application is currently assigned to SEIKAGAKU CORPORATION. Invention is credited to Shiro Imagama, Kenji Kadomatsu, Yukihiro Matsuyama, Akiomi Tanaka.
Application Number | 20110275137 12/937456 |
Document ID | / |
Family ID | 41199136 |
Filed Date | 2011-11-10 |
United States Patent
Application |
20110275137 |
Kind Code |
A1 |
Matsuyama; Yukihiro ; et
al. |
November 10, 2011 |
IMPROVING AGENT FOR NEUROPATHIC PAIN
Abstract
An object of the present invention is to provide a substance
which can be used as an active ingredient for improving neuropathic
pain having a novel mechanism of action different from those of
currently available agents and, therefore, provide an improving
agent for neuropathic pain which rarely interacts with currently
available agents and also does not have adverse reactions similar
to those of currently available agents. An improving agent for
neuropathic pain due to a hyperalgesic response of the present
invention as a means for resolution is characterized by comprising,
as an active ingredient, a lyase (an elimination enzyme) which has
an activity of degrading a chondroitin sulfate chain of a
chondroitin sulfate proteoglycan, and is typified by chondroitinase
ABC which selectively removes chondroitin sulfate and dermatan
sulfate of a proteoglycan.
Inventors: |
Matsuyama; Yukihiro; (Aichi,
JP) ; Kadomatsu; Kenji; (Aichi, JP) ; Imagama;
Shiro; (Aichi, JP) ; Tanaka; Akiomi; (Tokyo,
JP) |
Assignee: |
SEIKAGAKU CORPORATION
Tokyo
JP
NATIONAL UNIVERSITY CORPORATION NAGOYA UNIVERSITY,
Nagoya-shi
JP
|
Family ID: |
41199136 |
Appl. No.: |
12/937456 |
Filed: |
April 14, 2009 |
PCT Filed: |
April 14, 2009 |
PCT NO: |
PCT/JP2009/057503 |
371 Date: |
March 1, 2011 |
Current U.S.
Class: |
435/232 |
Current CPC
Class: |
A61K 38/51 20130101;
A61P 25/04 20180101; A61P 25/28 20180101; C12N 9/2402 20130101;
C12Y 302/01096 20130101; A61P 25/00 20180101; C07D 471/10 20130101;
A61P 25/02 20180101 |
Class at
Publication: |
435/232 |
International
Class: |
C12N 9/88 20060101
C12N009/88 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 14, 2008 |
JP |
2008-105183 |
Claims
1. An improving agent for neuropathic pain due to a hyperalgesic
response, comprising, as an active ingredient, a lyase having an
activity of degrading a chondroitin sulfate chain of a chondroitin
sulfate proteoglycan.
2. The improving agent according to claim 1, wherein the lyase is
chondroitinase ABC.
3. The improving agent according to claim 2, wherein the
neuropathic pain due to a hyperalgesic response is allodynia.
4. The improving agent according to claim 3, wherein the
neuropathic pain due to a hyperalgesic response is neuropathic pain
caused by a central nervous disorder.
5. The improving agent according to claim 4, wherein the central
nervous disorder is a spinal cord injury.
6. The improving agent according to claim 1, wherein the
neuropathic pain due to a hyperalgesic response is allodynia.
7. The improving agent according to claim 1, wherein the
neuropathic pain due to a hyperalgesic response is neuropathic pain
caused by a central nervous disorder.
8. The improving agent according to claim 2, wherein the
neuropathic pain due to a hyperalgesic response is neuropathic pain
caused by a central nervous disorder.
9. (canceled)
10. The improving agent according to claim 6, wherein the
neuropathic pain due to a hyperalgesic response is neuropathic pain
caused by a central nervous disorder.
11. The improving agent according to claim 7, wherein the central
nervous disorder is a spinal cord injury.
12. The improving agent according to claim 8, wherein the central
nervous disorder is a spinal cord injury.
13. The improving agent according to claim 10, wherein the central
nervous disorder is a spinal cord injury.
14. (canceled)
Description
TECHNICAL FIELD
[0001] The present invention relates to an improving agent for
neuropathic pain useful as a therapeutic agent for allodynia or the
like.
BACKGROUND ART
[0002] Neuropathic pain is a type of pain caused by damage to the
central nervous or the peripheral nervous and includes symptoms
such as a hyperalgesic response in which the threshold to
spontaneous pain or invasive stimulation is decreased, and
mechanical allodynia in which non-invasive mechanical stimulation
or tactile stimulation which does not usually cause pain is
mistakenly perceived as severe pain. Neuropathic pain, particularly
allodynia is characterized in that intractable burning pain or
stabbing pain lasts continuously for a long time and causes a
decrease in the effectiveness of rehabilitation due to pain or the
like, and is a pathological condition that significantly decreases
the QOL of patients. It can be said that there is almost no
satisfactory drug therapy for neuropathic pain and the development
of a drug that satisfies both efficacy and safety for neuropathic
pain has been demanded. However, the development of such a drug has
not progressed as it should have. One of the reasons for this is
considered to be that the mechanism of pathogenesis is not single
and several mechanisms are intricately involved in the onset of the
disease. The detailed pathogenic mechanisms of allodynia have not
been elucidated yet, however, involvement of abnormal neuronal
circuit formation due to an organic change and a functional change
in neurons in the pain transmission pathway including nociceptive
neurons has been proposed recently. Here, as the organic change, a
phenomenon in which A.beta. fibers terminating in laminae III-V of
the spinal cord dorsal horn elongate its sprout axon into lamina II
of the spinal cord dorsal horn which is the center of the pain
transmission called substantia gelatinosa after a nerve injury can
be exemplified. As the functional change, a phenomenon in which
A.beta. fibers presynaptically activate C fibers via interneurons,
and the like can be exemplified (Non-patent document 1). Further,
as a new factor for inducing abnormal neuronal circuit formation,
involvement of expression of a guidance molecule which guides nerve
fibers to a specific site and of release of a neuronal activator
such as BDGF (brain-derived growth factor) or the like which is an
allodynia enhancing factor accompanying the activation of spinal
microglia has been proposed (Non-patent document 2).
[0003] As pathological conditions exhibiting neuropathic pain,
diseases caused by central nervous disorders and peripheral nervous
disorders are known. Examples of the central nervous disease
include brain disorders, multiple sclerosis, and spinal cord
injuries, and examples of the peripheral nervous disease include
diabetes and herpes zoster.
[0004] In the current treatment of neuropathic pain, nonsteroidal
antiinflammatory drugs (NSAIDs), opioid analgesics, and analgesic
adjuvants (such as an NMDA receptor antagonist, an antidepressant,
an antiarrhythmic, an anticonvulsant, or an antispasmodic) are
used, however, neuropathic pain is resistant to therapy using
NSAIDs and opioid analgesics, and it is very difficult to control
with medication. Further, other than NSAIDs and opioid analgesics,
as the analgesic adjuvant, an antidepressant (such as a tricyclic
type, a tetracyclic type, an SSRI (a selective serotonin reuptake
inhibitor), or an SNRI (a selective serotonin-noradrenaline
reuptake inhibitor)), an anticonvulsant (such as carbamazepine), an
antispasmodic (baclofen), an antiarrhythmic (such as lidocaine), an
NMDA receptor antagonist (such as ketamine), a steroid (such as
betamethasone), a therapeutic agent for pain based on the
activation of a descending pain inhibitory system (Neurotropin, a
formulation containing an extract isolated from the inflamed skin
of rabbits inoculated with a vaccinia virus which is approved as a
therapeutic agent for postherpetic neuralgia and the like), or the
like is used (Non-patent document 3). However, the current
situation is that these medicinal agents for improving peripheral
pain have strong adverse reactions, and the medicinal agents for
central pain have a low efficacy, and therefore, the development of
a medicinal agent having a more stable and higher therapeutic
effectiveness has been demanded.
[0005] On the other hand, in the central nervous system, a
proteoglycan is considered to exert an activity of regulating
various neuronal functions such as an axon guidance function by
binding to various growth factors, cell adhesion factors, and so on
(Non-patent documents 4 and 5). In central nervous diseases such as
a spinal cord injury, it is known that a proteoglycan having
chondroitin sulfate side chain is a neuronal axonal regeneration
inhibitory factor which forms a glial scar in an injured area
thereof. It has been reported that chondroitinase ABC, which is an
enzyme capable of degrading chondroitin sulfate, dermatan sulfate
(also referred to as chondroitin sulfate B), and the like forming a
side chain of a proteoglycan, degrades a chondroitin sulfate side
chain (including a dermatan sulfate side chain, hereinafter, the
same shall apply) of a proteoglycan expressed in a glial scar in a
spinal cord injury rat model and has an activity of improving a
hind limb motor function (Non-patent document 8). It is considered
that the neuronal axonal regeneration inhibitory activity of a
chondroitin sulfate proteoglycan is exhibited mainly through an
activation of Rho kinase (Non-patent document 6). On the other
hand, it has been reported that ATP known as a pain substance
strongly activates spinal microglia and causes an increase in the
production of various mediators which promote abnormal neuronal
circuit formation, synaptic trafficking accompanied by cytoskeletal
reconstruction, or a release of a neurotransmitter and is involved
in neuropathic pain. ATP activates also Rac which is a member of
the Rho family (Non-patent document 7).
[0006] Further, it has been revealed that spinal microglia of the
spinal cord dorsal horn are activated by a nerve injury, and
stimulation of P2X4 receptors strongly expressed there causes
neuropathic pain, and it has been proposed that the Rho kinase
signal transduction pathway is involved as one of the pathways of
these activation cascades (Non-patent document 7).
[0007] The efficacy of a chondroitinase in the treatment of a motor
function for an injury of the central nervous system has been
supported not only by, for example, Non-patent document 8, but also
by Patent document 1 and the like. However, the efficacy as
described in these documents means or intends that a function (such
as a motor function) or a sensation which is lost by a nervous
disorder is restored or recovered by degrading a chondroitin
sulfate side chain of a proteoglycan and promoting the regeneration
of injured neuronal axons or the like, and there is no reported
case of an improving activity of a chondroitinase for pain caused
by a nervous disorder as far as the present inventors have known.
It is considered that it cannot be said that there is not at all an
association between a recovery effect on a motor function or the
like and an effect on neuropathic pain, however, at least there is
no technical common knowledge that if there is a recovery effect on
a motor function or the like, an improving effect on neuropathic
pain is exhibited or can be expected. In fact, Bradbury, E. J. et
al. confirmed a recovery efficacy of a chondroitinase on a motor
function in a spinal cord injury rat model, however, they have
reported that a recovery efficacy on a sensory function was not
obtained (Non-patent document 8). Further, in the study of abnormal
pain of human patients with a spinal cord injury (Non-patent
document 9), an association between the level of residual motor
ability and the presence or absence of abnormal pain such as
allodynia is not observed, and therefore, the recovery of a motor
function is not associated with the recovery of neuropathic
pain.
[0008] On the other hand, it is known that abnormal elongation of
primary afferent nerves adversely affect the function of
nociceptive neurons and can lead to abnormal hypersensitivity to
pain stimulations. Barritt, A. W. et al. studuied the possibility
in which the plasticity induced by a treatment with chondroitinase
ABC after a spinal cord injury (a lumbar spinal injury) has adverse
reactions. As a result of the study, they confirmed that the
elongation of primary afferent nerves was observed, but
connectivity of nociceptive neurons or development of mechanical
allodynia or thermal hyperalgesia was not observed by the treatment
(Non-patent document 10).
[0009] Accordingly, it has not been known so far that a
chondroitinase has an activity of improving neuropathic pain.
[0010] Patent document 1: JP-T-2005-526740 (WO 2003/074080) [0011]
Non-patent document 1: MINAMI Toshiaki, et al., "Plasticity of
pain", Seibutsu Butsuri, 41(1), 15-19 (2001) [0012] Non-patent
document 2: TSUDA Makoto, "A new mechanism of neuropathic pain
through ATP receptors in spinal microglia", Folia Pharmacologica
Japonica, 129, 349-353 (2007) [0013] Non-patent document 3: ISEKI
Masako et al., "Current clinical status of and future expectations
for pain alleviation agents", Folia Pharmacologica Japonica, 128,
326-329 (2006) [0014] Non-patent document 4: Crespo, D. et al.,
2007, How does chondroitinase promote functional recovery in the
damaged CNS?, Experimental Neurology 206(2), 159-71 [0015]
Non-patent document 5: Wit, J. D. et al., 2007, Semaphorins:
Receptor and Intracellular Signaling Mechanisms, Chapter 7,
Proteoglycans as Modulators of Axon Guidance Cue Function, 73-89
[0016] Non-patent document 6: Monnier, P. P. et al., 2003, The
Rho/ROCK pathway mediates neurite growth-inhibitory activity
associated with the chondroitin sulfate proteoglycans of the CNS
glial scar, Mol. Cell. Neurosci. 22, 319-330 [0017] Non-patent
document 7: Honda, S. et al., 2001, Extracellular ATP or ADP Induce
Chemotaxis of Cultured Microglia through Gi/o-Coupled P2Y
Receptors, J. Neurosci. 21(6), 1975-1982 [0018] Non-patent document
8: Bradbury, E. J. et al., 2002, Chondroitinase ABC promotes
functional recovery after spinal cord injury, Nature 416, 636-640
[0019] Non-patent document 9: Specified Non-profit Corporation,
JAPAN SPINAL CORD FOUNDATION (JSCF), Fact-finding report
publication about abnormal sharp pain with the SCI), September,
2004, http://www.jscf.org/jscf/SIRYOU/ssk07/ssk07-01.htm [0020]
Non-patent document 10: Barritt, A. W. et al., 2006, Chondroitinase
ABC promotes sprouting of intact and injured spinal systems after
spinal cord injury, Journal of Neuroscience 26(42), 10856-10867
DISCLOSURE OF THE INVENTION
Problems that the Invention is to Solve
[0021] An object of the present invention is to provide a substance
which can be used as an active ingredient of an improving agent for
neuropathic pain having a novel mechanism of action different from
those of currently available agents and, therefore, provide an
improving agent for neuropathic pain which rarely interacts with
currently available agents and also does not have adverse reactions
similar to those of currently available agents.
Means for Solving the Problems
[0022] The present inventors found that activation of Rho kinase is
a common intracellular transduction pathway involved in neuronal
axonal regeneration inhibition and pathological formation such as
development of neuropathic pain, and made a hypothesis, by
considering the above-mentioned biological activity of a
proteoglycan, that a proteoglycan having chondroitin sulfate side
chain induces continuous abnormal activation of neurons, glial
cells, or the like after a nervous disorder such as a nerve injury,
and also guides sensory nerves to the pain transmission pathway so
as to induce abnormal neuronal circuit formation, such that it is
involved in the development of neuropathic pain, particularly
allodynia.
[0023] Then, the present inventors conceived that if an enzyme
having an activity of degrading a chondroitin sulfate chain of a
chondroitin sulfate proteoglycan (hereinafter, also referred to as
CSPG) such as a chondroitinase is continuously administered to the
injured site of an individual starting from immediately after the
occurrence of a nervous disorder, for example, a spinal cord injury
to remove the chondroitin sulfate chain of the chondroitin sulfate
proteoglycan, the continuous abnormal activation of neurons or
non-neuronal cells such as glial cells caused by the proteoglycan
is canceled, and this canceling leads to the cancellation of the
neuronal axon guidance function of the proteoglycan and
consequently achieving a therapeutic effectiveness on neuropathic
pain. Based on the conception, the present inventors continuously
administered a lyase (an elimination enzyme, hereinafter, the same
shall apply) such as a chondroitinase having an activity of
degrading a chondroitin sulfate chain of a chondroitin sulfate
proteoglycan to an injured site in the intrathecal space of a
spinal cord injury rat model, thereby promoting the improvement of
a sensory nerve function, and therefore, they found for the first
time that the enzyme can improve neuropathic pain due to a
hyperalgesic response, particularly allodynia.
[0024] An improving agent for neuropathic pain due to a
hyperalgesic response according to the present invention
(hereinafter, also referred to as the improving agent of the
present invention) made on the basis of the above finding is
characterized by comprising, as an active ingredient, a lyase
having an activity of degrading a chondroitin sulfate chain of a
chondroitin sulfate proteoglycan as described in claim 1.
[0025] Further, the improving agent according to claim 2 is
characterized in that, in the improving agent according to claim 1,
the lyase is chondroitinase ABC.
[0026] Further, the improving agent according to claim 3 is
characterized in that, in the improving agent according to claim 1
or 2, the neuropathic pain due to a hyperalgesic response is
allodynia.
[0027] Further, the improving agent according to claim 4 is
characterized in that, in the improving agent according to any one
of claims 1 to 3, the neuropathic pain due to a hyperalgesic
response is neuropathic pain caused by a central nervous
disorder.
[0028] Further, the improving agent according to claim 5 is
characterized in that, in the improving agent according to claim 4,
the central nervous disorder is a spinal cord injury.
Effect of the Invention
[0029] The present invention is based on the fact that the present
inventors found that chondroitinase ABC which is one of the lyases
degrading chondroitin sulfate forming a side chain of a
proteoglycan into unsaturated disaccharides improves neuropathic
pain caused by a nervous disorder.
[0030] More specifically, based on an overall consideration made by
the present inventors with respect to various findings having been
reported so far as described above, a possibility that a
proteoglycan having a chondroitin sulfate side chain functions as
an aggravating factor for abnormal neuronal circuit formation in
the spinal cord dorsal horn after a nerve injury and a possibility
that an inhibitory effect on abnormal neuronal circuit formation is
obtained by degrading and removing the chondroitin sulfate chain of
the proteoglycan were assumed, and as a method for removing the
proteoglycan, chondroitinase ABC which can degrade chondroitin
sulfate was selected and the selected substance was continuously
administered intrathecally. As a result, it was found that the
substance has an improving activity on a sensory nerve function and
also has an improving effect on neuropathic pain due to a
hyperalgesic response, particularly allodynia. This finding was
found for the first time by the present inventors this time, and
based on this finding, the present invention provides a novel
improving agent for neuropathic pain through the inhibition of
abnormal neuronal circuit formation as a basic concept by a lyase
having an activity of degrading a chondroitin sulfate side chain of
a proteoglycan.
[0031] According to the present invention, an improving agent for
neuropathic pain due to a hyperalgesic response and a therapeutic
agent for allodynia, each containing, as an active ingredient, a
lyase such as a chondroitinase having an activity of degrading a
chondroitin sulfate chain of a chondroitin sulfate proteoglycan,
for example, chondroitinase ABC which is a lyase degrading
chondroitin sulfate forming a side chain of a proteoglycan into
unsaturated disaccharides are provided. The improving agent for
neuropathic pain of the present invention has a novel mechanism of
action different from those of opioid analgesics and analgesic
adjuvants currently used for the treatment of pain. Therefore,
according to the present invention, an unprecedented novel method
for treating pain which rarely interacts with currently available
agents and also does not have adverse reactions similar to those of
currently available agents can be provided.
[0032] Further, according to the present invention, a method for
preventing or improving abnormal neuronal circuit formation after a
nervous disorder by enzymatically degrading a chondroitin sulfate
proteoglycan which abnormally increases in the injured site or
impairing the function thereof can be provided.
[0033] Further, according to the present invention, a method for
improving neuropathic pain including allodynia can be provided by,
in an injured site, for example, inhibiting activation of Rho
kinase, degrading a chondroitin sulfate side chain or a dermatan
sulfate side chain of a proteoglycan which guides the pathway of
elongation of neuronal axons, or impairing the function of a
proteoglycan.
[0034] Further, according to the present invention, a method for
restoring or recovering a function (such as a motor function) or
the like which is lost by a nervous disorder as well as improving
neuropathic pain due to a hyperalgesic response including allodynia
by continuously administering a low dosage of a lyase having an
activity of degrading a chondroitin sulfate chain of a chondroitin
sulfate proteoglycan, particularly a chondroitin sulfate degrading
enzyme such as chondroitinase ABC to the injured site (for example,
in the subarachnoid cavity (in the intrathecal space)) for a given
period of time after the occurrence of the nervous disorder can be
provided.
[0035] Specifically, according to the present invention, an agent
for the application to a nervous disorder for continuously
administering a lyase having an activity of degrading a chondroitin
sulfate chain of a chondroitin sulfate proteoglycan at a dose of 6
mU to 15000 mU per human adult per day to an injured site of the
nervous disorder, characterized by containing the enzyme as an
active ingredient, and a method for continuously administering the
agent for a period from immediately after to 3 days after receiving
an injury up to 8 weeks can be provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 A graph showing an improving effect of chondroitinase
ABC on mechanical allodynia in Example 1 (response to pressure
stimulation: touch test).
[0037] FIG. 2 A graph showing an improving effect of chondroitinase
ABC on thermal allodynia in Example 1 (response to thermal
stimulation: tail flick test).
[0038] FIG. 3 A graph showing a recovery activity of chondroitinase
ABC on a sensory nerve in Example 1 (by somatosensory evoked
potential (SEP) measurement).
[0039] FIG. 4 A graph showing the results of evaluation of a hind
limb motor nerve function (BBB test) using chondroitinase ABC in
Reference example 1.
[0040] FIG. 5 A graph showing the results of evaluation of a hind
limb motor nerve function (grid test) using chondroitinase ABC in
Reference example 1.
[0041] FIG. 6 A graph showing the results of evaluation of a hind
limb motor nerve function (footprint test) using chondroitinase ABC
in Reference example 1.
[0042] FIG. 7 A graph showing a recovery activity of chondroitinase
ABC on a motor nerve in Reference example 1 (by motor nerve evoked
potential (MEP) measurement).
BEST MODE FOR CARRYING OUT THE INVENTION
[0043] The improving agent of the present invention is
characterized by comprising, as an active ingredient, a lyase
having an activity of degrading a chondroitin sulfate chain of a
chondroitin sulfate proteoglycan.
[0044] The enzyme which can be used as the active ingredient of the
improving agent of the present invention (hereinafter, also
referred to as the enzyme of the present invention) is not
particularly limited as long as it is a lyase having an activity of
degrading a chondroitin sulfate chain of a chondroitin sulfate
proteoglycan which can be present in the nervous system (such as
Neurocan, Phosphacan, RPTP, Brevican, NG2, Versican, Aggrican,
Decorin, Biglycan, or CD44) by an elimination reaction, however,
generally, a lyase called a chondroitinase (a chondroitin sulfate
degrading enzyme) is preferred.
[0045] The chondroitinase is a lyase which degrades chondroitin
sulfate into unsaturated disaccharides in an eliminating manner,
and a chondroitinase derived mainly from a microorganism is
preferred as the enzyme to be used as the active ingredient of the
improving agent of the present invention. As the chondroitinase,
the following chondroitinase ABC is particularly preferred.
[0046] Chondroitinase ABC (EC 4.2.2.20 or EC 4.2.2.4) is an enzyme
that mainly produces unsaturated disaccharides having a
.DELTA.4-hexuronic acid residue at the nonreducing terminal by
cleaving an N-acetylhexosaminide linkage in a glycosaminoglycan
including chondroitin sulfate through an elimination reaction, and
is an enzyme that strongly catalyzes the degradation of chondroitin
sulfate A derived from mammalian cartilage (having a high
chondroitin-4-sulfate content), chondroitin sulfate C derived from
shark cartilage (having a high chondroitin-6-sulfate content), and
chondroitin sulfate B derived from mammalian skin (dermatan
sulfate), and weakly catalyzes the degradation of hyaluronic acid,
and degrades also chondroitin sulfate A, dermatan sulfate, and
chondroitin sulfate C in a chondroitin sulfate side chain of a
chondroitin sulfate proteoglycan (Yamagata, T. et al., J. Biol.
Chem., 243: 1523-1535 (1968), Hamai A. et al., J. Biol. Chem. 272:
9123-9130 (1997)). As a reagent for research for removing a
mucopolysaccharide from an animal tissue or for identifying a
mucopolysaccharide in a tissue, this enzyme is commercially
available as a reagent from Seikagaku Biobusiness Corporation and
the commercial products of the enzyme produced by bacteria such as
Proteus vulgaris have a code No. 100330 (Chondroitinase ABC
(Proteus vulgaris)) and a code No. 100332 (Chondroitinase ABC
Protease Free (Proteus vulgaris)). Other than these, an enzyme
purified to a high purity (which is a single protein and does not
contain endotoxins, nucleic acids, contaminant proteins, etc.) and
having an extremely high specific activity is more preferred
because a clinical trial using it has been performed as a
therapeutic agent for intervertebral disc herniation and the
activity thereof on the living body has been sufficiently
elucidated. In particular, chondroitinase ABC having a high purity
and a high specific activity described in Japanese Patent No.
3980657, U.S. Pat. Nos. 6,184,023, 5,773,277, and 5,763,205 is most
preferred. Incidentally, the above-mentioned chondroitinase ABC is
the same enzyme as the following enzymes with other names.
[0047] Chondroitinase ABC Type 1
[0048] Chondroitin ABC endolyase 1
[0049] Chondroitin ABC lyase I
[0050] Chondroitin sulfate endolyase
[0051] Chondroitin ABC eliminase
[0052] Further, a gene of a substance having the above-mentioned
chondroitinase ABC activity has been cloned, and the amino acid
sequence of the protein thereof and also the nucleotide sequence of
the DNA encoding the protein have been identified (U.S. Pat. No.
5,578,480, JP-T-2007-520447 (WO2004/103299)). As a typical amino
acid sequence, an amino acid sequence represented by Sequence
Listing No. 1 described in U.S. Pat. No. 5,578,480 can be
exemplified. This amino acid sequence comprises 1021 amino acid
residues, wherein amino acids 1 to 24 form a signal sequence and a
mature protein comprises 997 amino acid residues (amino acids 25 to
1021). Further, as another typical amino acid sequence, an amino
acid sequence represented by Sequence Listing No. 2 described in
the amino acid sequence database (UniProtKB/Swiss-Prot: Entry name
CABC_PROVU, Primary accession number P59807, Protein name
Chondroitin ABC endolyase 1 [Precursor]) can be exemplified. This
amino acid sequence also comprises 1021 amino acid residues,
however, it is different from the amino acid sequence represented
by Sequence Listing No. 1 in two amino acids (amino acid 694:
Q.fwdarw.E, amino acid 738: D.fwdarw.N). Further, the amino acid
sequence described in SEQ ID NO: 1 of U.S. Patent Application
Publication No. 2006/0233782 is that of a mature protein, and
therefore comprises 997 amino acid residues, however, it is
different from the amino acid sequence ranging from amino acids 25
to 1021 of the amino acid sequence represented by Sequence Listing
No. 1 in four amino acids (amino acid 154: A.fwdarw.T, amino acid
295: I.fwdarw.T, amino acid 694: Q.fwdarw.E, amino acid 738:
D.fwdarw.N). That is, even an enzyme having an amino acid sequence
with a substitution of at least four amino acids (for example,
amino acids 154, 295, 694, and 738) on the basis of the amino acid
sequence represented by Sequence Listing No. 1 can be regarded as
an enzyme having a comparable chondroitinase ABC activity.
(Other Reference Information)
KEGG GENES
ype: YP00824
[0053] hypothetical protein [EC:4.2.2.20 4.2.2.21]; K08961
chondroitin-sulfate-ABC endolyase; K08962 chondroitin-sulfate-ABC
exolyase; [Sanger: YP00824] [NCBI-GI: 16121135] [NCBI-Gene ID:
1173663] [UniProt: Q0WIL0] ypk: y3211 ypk: y3211 chondroitin lyase
[EC:4.2.2.20 4.2.2.21]; K08961 chondroitin-sulfate-ABC endolyase;
K08962 chondroitin-sulfate-ABC exolyase; [NCBI-GI: 22127087]
[NCBI-Gene ID: 1148158] [UniProt: Q8CZX2] ypa: YPA.sub.--0445 ypa:
YPA.sub.--0445 hypothetical protein [EC:4.2.2.20 4.2.2.21]; K08961
chondroitin-sulfate-ABC endolyase; K08962 chondroitin-sulfate-ABC
exolyase; [JGI: YPA0445] [NCBI-GI: 108806442] [NCBI-Gene ID:
4121007] [UniProt: Q1CAV9] ypn: YPN.sub.--3027
ypn: YPN.sub.--3027
[0054] chondroitin-sulfate-ABC endolyase/chondroitin-sulfate-ABC
exolyase [EC:4.2.2.20 4.2.2.21]; K08961 chondroitin-sulfate-ABC
endolyase; K08962 chondroitin-sulfate-ABC exolyase; [NCBI-GI:
108813187] [NCBI-Gene ID: 4125950] [UniProt: Q1CF76] yps: YPTB3073
yps: YPTB3073 possible chondroitin ABC lyase [EC:4.2.2.20
4.2.2.21]; K08961 chondroitin-sulfate-ABC endolyase; K08962
chondroitin-sulfate-ABC exolyase; [NCBI-GI: 51597387][NCBI-Gene ID:
2954593] [UniProt: Q667C0] Entrez Gene GENE ID 2827933
http://www.ncbi.nlm.nih.gov/sites/entrez?db=gene&cmd=searc
h&term=chondroitinase+ABC
[0055] Further, a plurality of mutated proteins of chondroitinase
ABC (including fragmented proteins having the enzymatic activity)
have also been produced, and these enzymes produced by genetic
recombination are also included in the present invention as long as
they can function as an active ingredient of the improving agent of
the present invention (see, for example, JP-T-2007-516229 (WO
2004/110359), JP-T-2007-532094 (WO 2004/110360), JP-T-2007-520447
(WO 2004/103299), WO 2005/087920, etc.).
[0056] Further, chondroitinase ABC (Type 2) which is different from
the above-mentioned chondroitinase ABC (Type 1) has also been
isolated and purified from Proteus vulgaris and then subjected to
cloning, and these enzymes are also included in the present
invention as long as they can function as an active ingredient of
the improving agent of the present invention (see, Hamai A. et al.,
J. Biol. Chem. 272: 9123-9130 (1997), U.S. Pat. Nos. 5,741,692 and
5,498,536, etc.).
[0057] Incidentally, chondroitinase ABC which is purified to a high
purity and has an extremely high specific activity described in
Japanese Patent No. 3980657, U.S. Pat. Nos. 6,184,023, 5,773,277,
and 5,763,205 is an enzyme which does not include the
above-mentioned Type 2, and is most preferred as an active
ingredient of the improving agent of the present invention. That
is, the improving agent of the present invention is a
pharmaceutical product aiming at the administration to a human body
or the like, and therefore, it is preferred that chondroitinase ABC
which is used as the active ingredient has a pharmaceutically
acceptable grade and impurities derived from a microorganism that
produces this enzyme such as endotoxins, nucleic acids, and
proteases are removed as much as possible. It is preferred that the
amounts of endotoxins, nucleic acids, and proteases are not higher
than the detection limits by a common analytical method, and for
example, the amount of endotoxins is preferably 5.0 pg/100 U or
less when measurement is performed using Toxicolor (registered
trademark) system manufactured by Seikagaku Corporation. Further,
the amount of nucleic acids is preferably not higher than the
detection limit when measurement is performed by a threshold method
(DNA analyzer: Threshold (manufactured by Molecular Device
Corporation)). The amount of proteases is preferably 0.1% or less
of the total protein when measurement is performed using
FITC-casein as a substrate.
[0058] The origin of chondroitinase ABC is not limited to the
above-mentioned Proteus vulgaris, and chondroitinase ABC may be
derived from other bacterium belonging to the genus Proteus, and
moreover, it may be derived from a bacterium belonging to the genus
Bacteroides such as Bacteroides stercoris (see Hong SW. et al., Eur
J. Biochem. 2002 June; 269(12): 2934-40).
[0059] The enzyme which is the active ingredient of the improving
agent of the present invention, that is, a lyase having an activity
of degrading CSPG can effectively degrade and remove a chondroitin
sulfate side chain and a dermatan sulfate side chain of a
proteoglycan expressed in an injured site of a spinal cord injury
or can impair the function thereof by directly administering the
lyase to the injured site. As a target molecule, Neurocan,
Phosphacan, RPTP, Brevican, NG2, Versican, Aggrican, Decorin,
Biglycan, CD44, and the like, which are each a proteoglycan having
a chondroitin sulfate and/or dermatan sulfate side chain expressed
in astrocytes, oligodendrocytes, microglia, neurons, and the like,
which constitute central nerve tissues, can be exemplified. As is
presumed from the fact that there are a lot of molecular species,
the activity of such a proteoglycan having a chondroitin sulfate
side chain and/or a dermatan sulfate side chain on
neurophysiological functions including a central nerve regeneration
phenomenon is very versatile, and much of which is still unknown.
However, from the past studies, it has been proposed that there are
two main activities: one activity is exhibited as a neuronal axonal
regeneration inhibitory molecule; and the other activity is
exhibited as a neuronal axon guidance molecule. The former is the
activity of inhibiting nerve regeneration, and the latter is the
activity of promoting nerve regeneration. The present inventors
found that there is a possibility that CSPG may induce and promote
abnormal neuronal circuit formation. Since the abnormal neuronal
circuit formation after a nerve injury leads to the occurrence of
neuropathic pain such as allodynia occurring after a spinal cord
injury, it was found that by degrading CSPG which abnormally
increases in an injured site after a nervous disorder with the
enzyme of the present invention which is the active ingredient of
the improving agent of the present invention, that is, a lyase
having an activity of degrading CSPG, it is possible to prevent or
treat the occurrence of neuropathic pain.
[0060] The active ingredient of the improving agent of the present
invention is formulated into a parenteral preparation by a common
procedure in the same manner as a conventional parenteral enzyme
preparation, is parenterally (intravenously, subcutaneously,
intrathecally (into the intrathecal space), intradermally,
intramuscularly, internodally, percutaneously, or the like)
administered to a mammal (such as human, non-human primate, rat,
mouse, rabbit, cattle, horse, pig, dog, or cat) through an
administration route appropriate to a target disease, and is used
for treating or preventing the target disease of the animal.
[0061] Examples of the parenteral preparation include liquid
preparations (such as solution preparations, suspensions, and
ophthalmic preparations), solid preparations (such as lyophilized
preparations, powder preparations, granule preparations,
microcapsules, liposomes, and lipospheres), and semi-solid
preparations (such as ointments). For example, in the case where
the medicinal agent of the present invention is produced as a
liquid preparation, the liquid preparation can be produced by
dissolving or dispersing the enzyme of the present invention of a
pharmaceutically acceptable grade in water for injection or a
solution obtained by appropriately adding the water, a
pharmaceutically acceptable additive or carrier such as a isotonic
agent (such as sodium chloride, potassium chloride, glycerin,
mannitol, sorbitol, boric acid, borax, glucose, or propylene
glycol), a buffer agent (such as a phosphoric acid buffer, an
acetic acid buffer, a boric acid buffer, a carbonic acid buffer, a
citric acid buffer, a Tris buffer, a glutamic acid buffer, or an
.epsilon.-aminocaproic acid buffer), a preservative (such as a
p-hydroxybenzoic acid ester, chlorobutanol, benzyl alcohol,
benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric
acid, or borax), a nonionic surfactant (such as a polyoxyethylene
sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil,
a sucrose fatty acid ester, or polyoxyethylene polyoxypropylene
glycol), a thickener (such as polyvinyl alcohol), a stabilizing and
activity retaining agent (such as serum albumin, gelatin, sucrose
(saccharose), polyethylene glycol, a dextran, lactose, maltose,
mannitol, xylitol, sorbitol, inositol, sodium edetate, sodium
citrate, or ascorbic acid), a pH adjusting agent (such as
hydrochloric acid, sodium hydroxide, phosphoric acid, or acetic
acid), a solubilizing agent, an antioxidant, a substance effective
for preventing adsorption onto a container, or the like. Further,
the medicinal agent of the present invention can be formulated into
a solid preparation for dissolution before use by drying such a
liquid preparation through a drying method (such as lyophilization)
which does not affect the pharmacological function such as an
activity of the enzyme of the present invention. As a specific
example, a liquid preparation can be prepared by dissolving or
suspending the enzyme of the present invention of a
pharmaceutically acceptable grade in physiological saline, water
for injection, an isotonic solution, an oily solution, or the
like.
[0062] Further, the medicinal agent of the present invention can
also be formulated into a sustained-release preparation using a
hydrogel such as Duraseal (trademark) I (available from Confluent
Surgical, Inc.), Duraseal (trademark) II (available from Confluent
Surgical, Inc.), or Spray Gel. Duraseal (trademark, collagen based
on a gel foam).
[0063] The medicinal agent as prepared as described above is
administered through an administration method appropriate to the
target disease, severity of the symptoms, subject in need of
administration, and the like, and improvement (cure or prevention
of occurrence) of neuropathic pain can be achieved. For example, as
one example of the administration method, a method of topically
administering the medicinal agent to an injured site of a nervous
disorder or a periphery thereof can be exemplified. More
specifically, a method of continuously administering the medicinal
agent, for example, in the subarachnoid cavity (in the intrathecal
space) by an osmotic pump using a microtube can be exemplified.
[0064] The dosage and duration of administration of the medicinal
agent prepared using the improving agent of the present invention
should be appropriately determined by a health care professional
such as a medical doctor according to the conditions including the
target disease, animal species in need of administration, age and
body weight, severity of the symptoms, and health conditions, and
are not particularly limited. That is, the dosage and duration of
administration of the medicinal agent may be effective dosage and
duration for, for example, inhibiting activation of Rho kinase, or
degrading a chondroitin sulfate side chain and a dermatan sulfate
side chain of a proteoglycan which guides the pathway of elongation
of neuronal axons (also referred to as regeneration of neuronal
axons) or impairing the function of a proteoglycan in an injured
site.
[0065] In a spinal cord injury model, an intrathecal dosage per day
of the improving agent of the present invention to a rat (body
weight: 0.3 kg, amount of cerebrospinal fluid: 0.3 mL) is, in terms
of chondroitinase ABC expressed in enzyme unit, from 0.03 mU to 30
mU, preferably from 0.3 mU to 10 mU, most preferably about 3 mU.
When this dosage is converted into a dosage for a human adult (body
weight: 60 kg, amount of cerebrospinal fluid: 150 mL) with the body
weight and the amount of cerebrospinal fluid of the rat taken as
0.3 kg and 0.3 mL, respectively, the dosage for a human adult is
about 200 times as much as that for the rat on the basis of the
body weight, and about 500 times as much as that for the rat on the
basis of the amount of cerebrospinal fluid in consideration of
direct administration thereof into the dura mater. Accordingly, the
dosage for a human adult (average body weight: 60 kg, average
amount of cerebrospinal fluid: 150 mL) is as follows.
[0066] In the case of conversion on the basis of the body weight,
the intrathecal dosage per day is from 6 mU to 6000 mU, preferably
from 60 mU to 2000 mU, most preferably about 600 mU.
[0067] Further, in the case of conversion on the basis of the
amount of cerebrospinal fluid, the intrathecal dosage per day is
from 15 mU to 15000 mU, preferably from 150 mU to 5000 mU, most
preferably about 1500 mU.
[0068] Therefore, in overall consideration of the above-mentioned
conversion methods, the intrathecal dosage per day is from 6 mU to
15000 mU, preferably from 60 mU to 5000 mU, most preferably from
about 600 mU to 1500 mU.
[0069] The duration of administration should be determined by a
health care professional such as a medical doctor in consideration
of the expression level of a chondroitin sulfate proteoglycan in an
injured site and a possibility of occurrence of adverse reactions
or the like and according to the symptoms, however, as a guide, in
the case of continuous administration, it is up to about 8 weeks,
preferably up to about 4 weeks, more preferably up to about 2 weeks
after receiving an injury. The administration start time is
preferably immediately after to about 3 days after receiving an
injury.
[0070] Incidentally, in the case where chondroitinase ABC is used
as the active ingredient, a preferred specific activity thereof is
not less than about 300 U/mg protein, and therefore, the
above-mentioned intrathecal dosage per day corresponds in terms of
the weight of the enzyme protein as follows: 6 mU corresponds to
about 0.02 .mu.g or less, 15000 mU corresponds to about 50 .mu.g or
less, 60 mU corresponds to about 0.2 .mu.g or less, 5000 mU
corresponds to about 17 .mu.g or less, and 600 mU corresponds to
about 2 .mu.g or less. Incidentally, 1 unit (1 U) of the enzyme
amount (enzyme titer) of chondroitinase ABC is defined as an enzyme
amount with which 1 .mu.mol of unsaturated disaccharides is
produced per minute when chondroitin sulfate C derived from shark
cartilage is used as a substrate and reacted with the enzyme at pH
8.0 and 37.degree. C. Incidentally, the dosage of "a lyase having
an activity of degrading a chondroitin sulfate chain of a
chondroitin sulfate proteoglycan" (the enzyme of the present
invention) can also be determined by using the enzyme titer of each
enzyme for the chondroitin sulfate proteoglycan which is a
substrate as an index, and the range thereof is from 6 mU to 15000
mU in the same manner as chondroitinase ABC.
[0071] Incidentally, the amount or concentration of the enzyme per
unit dosage form of the medicinal agent prepared using the
improving agent of the present invention is determined according to
the above-mentioned dosage. However, it is not necessary that the
preparation have the amount or concentration of the enzyme when it
is administered, and for example, immediately before or at the time
of administration, the preparation may be diluted with a diluent to
an effective and safe concentration. Accordingly, the present
invention also provides a kit containing such a diluent and the
above-mentioned medicinal agent of the present invention in
combination.
[0072] The improving agent of the present invention is used for
improving, curing, or preventing neuropathic pain due to a
hyperalgesic response caused by an acute disorder or a chronic
disorder in the central nervous system (CNS) including the spinal
cord and brain or the peripheral nervous system, particularly
allodynia and sensory nerve dysfunction. Examples of the disease
causing neuropathic pain to which the improving agent of the
present invention is effectively applied include central nervous
disorders, specifically CNS injuries and diseases including spinal
cord injuries, traumatic brain injuries, and the like can be
exemplified as typical examples. However, the improving agent of
the present invention can be effectively applied particularly to
spinal cord injuries. The spinal cord injuries include diseases and
traumatic injuries such as neuronal crush injuries caused by a
traffic accident, a fall accident, a contused wound, a gunshot
wound, and another injury. By administering the improving agent of
the present invention, clinical improvement can be obtained in
neuropathic pain due to a hyperalgesic response accompanying such a
disease, particularly allodynia and sensory nerve dysfunction.
[0073] It is also possible to perform the treatment of neuropathic
pain by using the medicinal agent prepared using the improving
agent of the present invention in combination with another
medicinal agent known to have an improving effect on neuropathic
pain. Examples of such a medicinal agent include, as illustrated
below, NSAIDs, opioids, and analgesic adjuvants. Examples of the
opioid analgesic include morphine, fentanyl, oxycodone, codeine
phosphate, pethidine, buprenorphine, tramadol, pentazocine, and
butorphanol. Further, examples of the analgesic adjuvant include
antidepressants such as a tricyclic type (such as amitriptyline or
nortriptyline), a tetracyclic type (such as Tetramide), an SSRI
(such as paroxetine or fluvoxamine), and an SNRI (milnacipran);
anticonvulsants such as carbamazepine, lamotrigine, zonisamide,
valproic acid, and clonazepam; antispasmodics such as baclofen;
antiarrhythmics such as lidocaine and mexiletine; NMDA receptor
antagonists such as ketamine, dextromethorphan, amantadine, and
ifenprodil; and formulations containing an extract isolated from
the inflamed skin of rabbits inoculated with a vaccinia virus such
as Neurotropin. Further, gabapentin (an anticonvulsant) and
pregabalin which are each a calcium channel blocker (bonding to an
.alpha.2 .delta. subunit) and have been receiving public attention
recently can also be exemplified. It is also possible to use the
present inventive medicinal agent in combination with a medicinal
agent having an antiinflammatory activity, for example, a steroid
agent such as methylprednisolone sodium succinate, dexamethasone,
or betamethasone; or fasudil which is an Rho kinase inhibitor.
EXAMPLES
[0074] Hereinafter, the present invention will be described in
detail with reference to Examples, however, the present invention
should not be interpreted by limiting to the following
description.
Example 1
Improving Activity of Chondroitinase ABC on Allodynia and Sensory
Nerve Dysfunction Such as Hyperalgesia or the Like after Spinal
Cord Injury
(Experimental Method)
[0075] As animals, S.D. rats (Nippon SLC Co., Ltd., female, 9 weeks
of age) were used. Under anesthesia with a cocktail of ether and
ketamine, the ninth and the twelfth thoracic vertebrae were
laminectomized, and a spinal cord injury on the ninth thoracic
vertebra was induced by crushing using IH-0400 Impactor. After the
injury, small incision was done to the dura mater of the twelfth
thoracic vertebra and a microtube was inserted into the
subarachnoid cavity under a microscope. The microtube was connected
to an osmotic pump (Alzet Osmotic Pump) in which a test sample was
previously filled. The tube and the pump were fixed onto the
interspinal ligament and the muscle. Thereafter, the wound of the
muscle layer and the skin was closed. After the operation, the rat
was orally given an antibiotic for two weeks. The manipulative
urination was performed once a day until micturition reflex was
recovered. The test sample was continuously administered
intrathecally for 14 days starting from immediately after the
injury (administration rate: 12 .mu.L/24 hours; total dose: 168
.mu.L/14 days). As the test sample, the following two types were
used.
(1) Chondroitinase ABC (0.05 U/200 .mu.L, Seikagaku
Corporation)
[0076] (2) Physiological saline (200 .mu.L, control)
[0077] Incidentally, as the above-mentioned chondroitinase ABC, a
solution prepared by dissolving a lyophilized product obtained by
formulating a high-purity chondroitinase ABC (which shows a single
band in gel electrophoresis (SDS-PAGE) and has a specific activity
of 380 U/mg, and from which nucleic acids (DNAs) and proteases have
been removed) derived from Proteus vulgaris described in Japanese
Patent No. 3980657 and U.S. Pat. No. 6,184,023 into a preparation
by the method described in U.S. Pat. No. 6,007,810 (a method using
polyethylene glycol and saccharose) in physiological saline was
used.
Experimental Results Part 1: Improving Effect on Mechanical
Allodynia (Response to Pressure Stimulation: Touch Test)
[0078] Before the injury and once a week from one week after the
injury to 10 weeks thereafter, each rat was placed in a small box
and allowed to stand still for 15 minutes. When the rat became
quiet, a stimulation was applied to the plantar surface of the hind
limbs from the downside using von Frey filaments (several kinds of
filaments of different stimulation intensities). By using an
up-down method in which application of a strong stimulation and a
weak stimulation was repeated to determine a correct stimulation
threshold value (Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung,
J. M., Yaksh, T. L., Quantitative assessment of tactile allodynia
in the rat paw, J. Neurosci. Methods, 53, 55-63 (1994)), the
pressure stimulation of the filament by which the rat felt pain and
moved the hind limb was measured for each of the right and left
hind limbs, and an average value was calculated and shown (unit:
gram). The results of the cases where the two kinds of test samples
were administered, respectively, are shown in FIG. 1. As is
apparent from FIG. 1, in the group with the administration of
physiological saline, the pressure stimulation of the filament
showed a high value from one week to two weeks after the injury as
compared with that before the injury, and an insensitive response
to the stimulation applied to the hind limbs was observed. However,
as from four weeks after the injury, the pressure stimulation of
the filament showed a low value as compared with that before the
injury and, after six weeks from the injury, a hypersensitive
symptom in which a response was resulted even by a slight
stimulation to the hind limb was observed. On the other hand, in
the group with the administration of chondroitinase ABC, although a
similar tendency to that in the case of the group with the
administration of physiological saline was observed up to six weeks
after the injury, however, after seven weeks from the injury, a
hypersensitive response was gradually improved. After 10 weeks from
the injury, the pressure stimulation of the filament showed a
comparable level to that before the injury, and therefore, a
remarkable improving (treating) effect on mechanical allodynia was
observed in the administration group such that the sensory nerve
function returned to a normal level.
Experimental Results Part 2: Evaluation of Sensory Nerve Function
(Response to Thermal Stimulation: Tail Flick Test)
[0079] Before the injury and once a week from one week after the
injury to 10 weeks thereafter, the tail of each rat was immersed in
a bath of 55.degree. C. and the time until the rat flicks the tail
(response time) was measured (unit: second). The measurement was
performed three times with an interval of 15 minutes and an average
value was calculated and shown. The results of the cases where the
two kinds of test samples were administered, respectively, are
shown in FIG. 2. As is apparent from FIG. 2, in the group with the
administration of physiological saline, the response time was
reduced as the day went on after the injury as compared with that
before the injury and a hypersensitive response to thermal
stimulation was observed. On the other hand, in the group with the
administration of chondroitinase ABC, although a similar tendency
to that in the case of the group with the administration of
physiological saline was observed in an early stage after the
injury, however, the hypersensitive response to thermal stimulation
was improved as from five weeks after the injury and, after eight
weeks from the injury, the response time was stable and showed an
almost comparable level to that before the injury, and therefore, a
remarkable recovery (an improving (treating) effect on thermal
allodynia) was observed in the administration group such that the
sensory nerve function returned to a normal level.
Experimental Results Part 3: Somatosensory Evoked Potential
[0080] A somatosensory evoked potential (SEP) was recorded on day
56 after the operation. Under anesthesia with ketamine
hydrochloride, the wound made at the time of inducing the injury
was opened and the dura mater on the rostral and caudal sides in
the injured site of the spinal cord injury was exposed, and a
stimulation electrode and a measurement electrode were placed on
the dura mater on the caudal side and on the rostral side,
respectively. Then, a stimulation was applied for a given period of
time from the downside, and a potential transmitted to the upside
was measured. A ground wire was placed between respective
electrodes. The time from the stimulation to the first wave
(latency) and the time from the stimulation to the second wave
showing a clear wave peak (peak latency: PL) were measured (unit:
millisecond (ms)). For both waves, 100 measurements were summed and
averaged. The results of measured PL are shown in FIG. 3. As is
apparent from FIG. 3, the group with the administration of
chondroitinase ABC had a significant recovery activity on sensory
nerve also from an electrophysiological standpoint.
Reference Example 1
Improving activity of chondroitinase ABC on Motor Dysfunction after
Spinal Cord Injury
(Experimental Method)
[0081] An experiment was performed in the same manner as in Example
1.
Reference Experimental Results Part 1: Evaluation of Hind Limb
Motor Nerve Function (BBB Test)
[0082] One day after the injury and once a week from one week after
the injury to 10 weeks thereafter, the hind limb motor nerve
function was evaluated using a Basso-Beattie-Bresnahan (BBB) scale
in accordance with a method of Basso et al. (Basso, D. M., Beattie,
M. S. & Bresnahan, J. C., Graded histological and locomotor
outcomes after spinal cord contusion using the NYU weight-drop
device versus transection, Exp. Neurol., 139, 244-256 (1996)), and
the results were expressed as BBB scores. The test was performed in
a blind manner by two examiners and the score was expressed as an
average value of the results of two examiners. As for the
statistical analysis, intergroup comparisons for the respective
evaluation time points were performed using a Wilcoxon rank sum
test and determination was made with a P value of less than 5%
considered significant. The results of the cases where the two
kinds of test samples were administered, respectively, are shown in
FIG. 4. As is apparent from FIG. 4, in the group with the
administration of chondroitinase ABC, a significant recovery of the
hind limb motor nerve function was observed as compared with the
group with the administration of physiological saline.
Reference Experimental Results Part 2: Evaluation of Hind Limb
Motor Nerve Function (Grid Test)
[0083] Once a week from one week after the injury to 10 weeks
thereafter, each rat was placed on a net (grid) of 2 cm.times.2 cm
and allowed to walk thereon for three minutes, and the number of
times that the rat could grip the grid with the hind limb and the
total number of steps were measured. The ratio of the number of
times that the rat could grip the grid to the total number of steps
was defined as a % grip value and an average value was calculated
for each of the right and left hind limbs and shown. As for the
statistical analysis, intergroup comparisons for the respective
evaluation time points were performed using a t-test and
determination was made with a P value of less than 5% considered
significant. The results of the cases where two kinds of test
samples were administered, respectively, are shown in FIG. 5. As is
apparent from FIG. 5, in the group with the administration of
chondroitinase ABC, a significant recovery of the hind limb motor
nerve function was observed as compared with the group with the
administration of physiological saline.
Reference Experimental Results Part 3: Evaluation of Hind Limb
Motor Nerve Function (Footprint Test)
[0084] One day after the injury and once a week from one week after
the injury to 10 weeks thereafter, ink was applied to the hind
limbs of each rat and the rat was allowed to walk on a sheet of
paper with a size of 50 cm, whereby footprints of the hind limbs
were obtained. The stride length was measured for each of the right
and left hind limbs from the footprints of the hind limbs on the
paper, and an average value was calculated and shown (unit: mm). As
for the statistical analysis, intergroup comparisons for the
respective evaluation time points were performed using a t-test and
determination was made with a P value of less than 5% considered
significant. The results of the cases where the two kinds of test
samples were administered, respectively, are shown in FIG. 6. As is
apparent from FIG. 6, in the group with the administration of
chondroitinase ABC, a significant recovery of the hind limb motor
nerve function was observed as compared with the group with the
administration of physiological saline.
Reference Experimental Results Part 4: Motor Nerve Evoked
Potential
[0085] A motor nerve evoked potential (MEP) was recorded on day 56
after the operation. Under anesthesia with ketamine hydrochloride,
the wound made at the time of inducing the injury was opened and
the dura mater on the rostral side in the injured site of the
spinal cord injury was exposed, and a stimulation electrode was
placed on the dura mater on the rostral side. As recording
electrodes for both hind limbs, two needle electrodes were placed
on each of the right and left gastrocnemius muscles at an equal
interval. Then, a stimulation was applied for a given period of
time, and a potential transmitted to each of the right and left
hind limbs was measured. A ground wire was placed between
respective electrodes. The time from the stimulation until a wave
form was recorded (latency: L) and the time from the appearance of
the wave form to the disappearance thereof (duration) were measured
(unit: millisecond (ms)). For both waves, 50 measurements were
summed and averaged for each results of the right and left hind
limbs. The results of measured L are shown in FIG. 7. As is
apparent from FIG. 7, the group with the administration of
chondroitinase ABC had a significant recovery activity on motor
nerve also from an electrophysiological standpoint.
Preparation Example 1
[0086] A solution preparation containing the above-mentioned
high-purity chondroitinase ABC at a concentration of 0.05 U/200
.mu.L was prepared by dissolving the chondroitinase ABC in
physiological saline.
INDUSTRIAL APPLICABILITY
[0087] The present invention has industrial applicability in the
point that an improving agent for neuropathic pain caused by a
nervous disorder, particularly a therapeutic agent for allodynia
and sensory dysfunction, containing, as an active ingredient, a
lyase which has an activity of degrading a chondroitin sulfate
chain of a chondroitin sulfate proteoglycan, and is typified by
chondroitinase ABC which selectively removes a chondroitin sulfate
side chain and a dermatan sulfate side chain forming side chains of
a proteoglycan can be provided.
Sequence CWU 1
1
211021PRTProteus vulgaris 1Met Pro Ile Phe Arg Phe Thr Ala Leu Ala
Met Thr Leu Gly Leu Leu1 5 10 15Ser Ala Pro Tyr Asn Ala Met Ala Ala
Thr Ser Asn Pro Ala Phe Asp 20 25 30Pro Lys Asn Leu Met Gln Ser Glu
Ile Tyr His Phe Ala Gln Asn Asn 35 40 45Pro Leu Ala Asp Phe Ser Ser
Asp Lys Asn Ser Ile Leu Thr Leu Ser 50 55 60Asp Lys Arg Ser Ile Met
Gly Asn Gln Ser Leu Leu Trp Lys Trp Lys65 70 75 80Gly Gly Ser Ser
Phe Thr Leu His Lys Lys Leu Ile Val Pro Thr Asp 85 90 95Lys Glu Ala
Ser Lys Ala Trp Gly Arg Ser Ser Thr Pro Val Phe Ser 100 105 110Phe
Trp Leu Tyr Asn Glu Lys Pro Ile Asp Gly Tyr Leu Thr Ile Asp 115 120
125Phe Gly Glu Lys Leu Ile Ser Thr Ser Glu Ala Gln Ala Gly Phe Lys
130 135 140Val Lys Leu Asp Phe Thr Gly Trp Arg Ala Val Gly Val Ser
Leu Asn145 150 155 160Asn Asp Leu Glu Asn Arg Glu Met Thr Leu Asn
Ala Thr Asn Thr Ser 165 170 175Ser Asp Gly Thr Gln Asp Ser Ile Gly
Arg Ser Leu Gly Ala Lys Val 180 185 190Asp Ser Ile Arg Phe Lys Ala
Pro Ser Asn Val Ser Gln Gly Glu Ile 195 200 205Tyr Ile Asp Arg Ile
Met Phe Ser Val Asp Asp Ala Arg Tyr Gln Trp 210 215 220Ser Asp Tyr
Gln Val Lys Thr Arg Leu Ser Glu Pro Glu Ile Gln Phe225 230 235
240His Asn Val Lys Pro Gln Leu Pro Val Thr Pro Glu Asn Leu Ala Ala
245 250 255Ile Asp Leu Ile Arg Gln Arg Leu Ile Asn Glu Phe Val Gly
Gly Glu 260 265 270Lys Glu Thr Asn Leu Ala Leu Glu Glu Asn Ile Ser
Lys Leu Lys Ser 275 280 285Asp Phe Asp Ala Leu Asn Ile His Thr Leu
Ala Asn Gly Gly Thr Gln 290 295 300Gly Arg His Leu Ile Thr Asp Lys
Gln Ile Ile Ile Tyr Gln Pro Glu305 310 315 320Asn Leu Asn Ser Gln
Asp Lys Gln Leu Phe Asp Asn Tyr Val Ile Leu 325 330 335Gly Asn Tyr
Thr Thr Leu Met Phe Asn Ile Ser Arg Ala Tyr Val Leu 340 345 350Glu
Lys Asp Pro Thr Gln Lys Ala Gln Leu Lys Gln Met Tyr Leu Leu 355 360
365Met Thr Lys His Leu Leu Asp Gln Gly Phe Val Lys Gly Ser Ala Leu
370 375 380Val Thr Thr His His Trp Gly Tyr Ser Ser Arg Trp Trp Tyr
Ile Ser385 390 395 400Thr Leu Leu Met Ser Asp Ala Leu Lys Glu Ala
Asn Leu Gln Thr Gln 405 410 415Val Tyr Asp Ser Leu Leu Trp Tyr Ser
Arg Glu Phe Lys Ser Ser Phe 420 425 430Asp Met Lys Val Ser Ala Asp
Ser Ser Asp Leu Asp Tyr Phe Asn Thr 435 440 445Leu Ser Arg Gln His
Leu Ala Leu Leu Leu Leu Glu Pro Asp Asp Gln 450 455 460Lys Arg Ile
Asn Leu Val Asn Thr Phe Ser His Tyr Ile Thr Gly Ala465 470 475
480Leu Thr Gln Val Pro Pro Gly Gly Lys Asp Gly Leu Arg Pro Asp Gly
485 490 495Thr Ala Trp Arg His Glu Gly Asn Tyr Pro Gly Tyr Ser Phe
Pro Ala 500 505 510Phe Lys Asn Ala Ser Gln Leu Ile Tyr Leu Leu Arg
Asp Thr Pro Phe 515 520 525Ser Val Gly Glu Ser Gly Trp Asn Asn Leu
Lys Lys Ala Met Val Ser 530 535 540Ala Trp Ile Tyr Ser Asn Pro Glu
Val Gly Leu Pro Leu Ala Gly Arg545 550 555 560His Pro Phe Asn Ser
Pro Ser Leu Lys Ser Val Ala Gln Gly Tyr Tyr 565 570 575Trp Leu Ala
Met Ser Ala Lys Ser Ser Pro Asp Lys Thr Leu Ala Ser 580 585 590Ile
Tyr Leu Ala Ile Ser Asp Lys Thr Gln Asn Glu Ser Thr Ala Ile 595 600
605Phe Gly Glu Thr Ile Thr Pro Ala Ser Leu Pro Gln Gly Phe Tyr Ala
610 615 620Phe Asn Gly Gly Ala Phe Gly Ile His Arg Trp Gln Asp Lys
Met Val625 630 635 640Thr Leu Lys Ala Tyr Asn Thr Asn Val Trp Ser
Ser Glu Ile Tyr Asn 645 650 655Lys Asp Asn Arg Tyr Gly Arg Tyr Gln
Ser His Gly Val Ala Gln Ile 660 665 670Val Ser Asn Gly Ser Gln Leu
Ser Gln Gly Tyr Gln Gln Glu Gly Trp 675 680 685Asp Trp Asn Arg Met
Gln Gly Ala Thr Thr Ile His Leu Pro Leu Lys 690 695 700Asp Leu Asp
Ser Pro Lys Pro His Thr Leu Met Gln Arg Gly Glu Arg705 710 715
720Gly Phe Ser Gly Thr Ser Ser Leu Glu Gly Gln Tyr Gly Met Met Ala
725 730 735Phe Asp Leu Ile Tyr Pro Ala Asn Leu Glu Arg Phe Asp Pro
Asn Phe 740 745 750Thr Ala Lys Lys Ser Val Leu Ala Ala Asp Asn His
Leu Ile Phe Ile 755 760 765Gly Ser Asn Ile Asn Ser Ser Asp Lys Asn
Lys Asn Val Glu Thr Thr 770 775 780Leu Phe Gln His Ala Ile Thr Pro
Thr Leu Asn Thr Leu Trp Ile Asn785 790 795 800Gly Gln Lys Ile Glu
Asn Met Pro Tyr Gln Thr Thr Leu Gln Gln Gly 805 810 815Asp Trp Leu
Ile Asp Ser Asn Gly Asn Gly Tyr Leu Ile Thr Gln Ala 820 825 830Glu
Lys Val Asn Val Ser Arg Gln His Gln Val Ser Ala Glu Asn Lys 835 840
845Asn Arg Gln Pro Thr Glu Gly Asn Phe Ser Ser Ala Trp Ile Asp His
850 855 860Ser Thr Arg Pro Lys Asp Ala Ser Tyr Glu Tyr Met Val Phe
Leu Asp865 870 875 880Ala Thr Pro Glu Lys Met Gly Glu Met Ala Gln
Lys Phe Arg Glu Asn 885 890 895Asn Gly Leu Tyr Gln Val Leu Arg Lys
Asp Lys Asp Val His Ile Ile 900 905 910Leu Asp Lys Leu Ser Asn Val
Thr Gly Tyr Ala Phe Tyr Gln Pro Ala 915 920 925Ser Ile Glu Asp Lys
Trp Ile Lys Lys Val Asn Lys Pro Ala Ile Val 930 935 940Met Thr His
Arg Gln Lys Asp Thr Leu Ile Val Ser Ala Val Thr Pro945 950 955
960Asp Leu Asn Met Thr Arg Gln Lys Ala Ala Thr Pro Val Thr Ile Asn
965 970 975Val Thr Ile Asn Gly Lys Trp Gln Ser Ala Asp Lys Asn Ser
Glu Val 980 985 990Lys Tyr Gln Val Ser Gly Asp Asn Thr Glu Leu Thr
Phe Thr Ser Tyr 995 1000 1005Phe Gly Ile Pro Gln Glu Ile Lys Leu
Ser Pro Leu Pro 1010 1015 102021021PRTProteus vulgaris 2Met Pro Ile
Phe Arg Phe Thr Ala Leu Ala Met Thr Leu Gly Leu Leu1 5 10 15Ser Ala
Pro Tyr Asn Ala Met Ala Ala Thr Ser Asn Pro Ala Phe Asp 20 25 30Pro
Lys Asn Leu Met Gln Ser Glu Ile Tyr His Phe Ala Gln Asn Asn 35 40
45Pro Leu Ala Asp Phe Ser Ser Asp Lys Asn Ser Ile Leu Thr Leu Ser
50 55 60Asp Lys Arg Ser Ile Met Gly Asn Gln Ser Leu Leu Trp Lys Trp
Lys65 70 75 80Gly Gly Ser Ser Phe Thr Leu His Lys Lys Leu Ile Val
Pro Thr Asp 85 90 95Lys Glu Ala Ser Lys Ala Trp Gly Arg Ser Ser Thr
Pro Val Phe Ser 100 105 110Phe Trp Leu Tyr Asn Glu Lys Pro Ile Asp
Gly Tyr Leu Thr Ile Asp 115 120 125Phe Gly Glu Lys Leu Ile Ser Thr
Ser Glu Ala Gln Ala Gly Phe Lys 130 135 140Val Lys Leu Asp Phe Thr
Gly Trp Arg Ala Val Gly Val Ser Leu Asn145 150 155 160Asn Asp Leu
Glu Asn Arg Glu Met Thr Leu Asn Ala Thr Asn Thr Ser 165 170 175Ser
Asp Gly Thr Gln Asp Ser Ile Gly Arg Ser Leu Gly Ala Lys Val 180 185
190Asp Ser Ile Arg Phe Lys Ala Pro Ser Asn Val Ser Gln Gly Glu Ile
195 200 205Tyr Ile Asp Arg Ile Met Phe Ser Val Asp Asp Ala Arg Tyr
Gln Trp 210 215 220Ser Asp Tyr Gln Val Lys Thr Arg Leu Ser Glu Pro
Glu Ile Gln Phe225 230 235 240His Asn Val Lys Pro Gln Leu Pro Val
Thr Pro Glu Asn Leu Ala Ala 245 250 255Ile Asp Leu Ile Arg Gln Arg
Leu Ile Asn Glu Phe Val Gly Gly Glu 260 265 270Lys Glu Thr Asn Leu
Ala Leu Glu Glu Asn Ile Ser Lys Leu Lys Ser 275 280 285Asp Phe Asp
Ala Leu Asn Ile His Thr Leu Ala Asn Gly Gly Thr Gln 290 295 300Gly
Arg His Leu Ile Thr Asp Lys Gln Ile Ile Ile Tyr Gln Pro Glu305 310
315 320Asn Leu Asn Ser Gln Asp Lys Gln Leu Phe Asp Asn Tyr Val Ile
Leu 325 330 335Gly Asn Tyr Thr Thr Leu Met Phe Asn Ile Ser Arg Ala
Tyr Val Leu 340 345 350Glu Lys Asp Pro Thr Gln Lys Ala Gln Leu Lys
Gln Met Tyr Leu Leu 355 360 365Met Thr Lys His Leu Leu Asp Gln Gly
Phe Val Lys Gly Ser Ala Leu 370 375 380Val Thr Thr His His Trp Gly
Tyr Ser Ser Arg Trp Trp Tyr Ile Ser385 390 395 400Thr Leu Leu Met
Ser Asp Ala Leu Lys Glu Ala Asn Leu Gln Thr Gln 405 410 415Val Tyr
Asp Ser Leu Leu Trp Tyr Ser Arg Glu Phe Lys Ser Ser Phe 420 425
430Asp Met Lys Val Ser Ala Asp Ser Ser Asp Leu Asp Tyr Phe Asn Thr
435 440 445Leu Ser Arg Gln His Leu Ala Leu Leu Leu Leu Glu Pro Asp
Asp Gln 450 455 460Lys Arg Ile Asn Leu Val Asn Thr Phe Ser His Tyr
Ile Thr Gly Ala465 470 475 480Leu Thr Gln Val Pro Pro Gly Gly Lys
Asp Gly Leu Arg Pro Asp Gly 485 490 495Thr Ala Trp Arg His Glu Gly
Asn Tyr Pro Gly Tyr Ser Phe Pro Ala 500 505 510Phe Lys Asn Ala Ser
Gln Leu Ile Tyr Leu Leu Arg Asp Thr Pro Phe 515 520 525Ser Val Gly
Glu Ser Gly Trp Asn Asn Leu Lys Lys Ala Met Val Ser 530 535 540Ala
Trp Ile Tyr Ser Asn Pro Glu Val Gly Leu Pro Leu Ala Gly Arg545 550
555 560His Pro Phe Asn Ser Pro Ser Leu Lys Ser Val Ala Gln Gly Tyr
Tyr 565 570 575Trp Leu Ala Met Ser Ala Lys Ser Ser Pro Asp Lys Thr
Leu Ala Ser 580 585 590Ile Tyr Leu Ala Ile Ser Asp Lys Thr Gln Asn
Glu Ser Thr Ala Ile 595 600 605Phe Gly Glu Thr Ile Thr Pro Ala Ser
Leu Pro Gln Gly Phe Tyr Ala 610 615 620Phe Asn Gly Gly Ala Phe Gly
Ile His Arg Trp Gln Asp Lys Met Val625 630 635 640Thr Leu Lys Ala
Tyr Asn Thr Asn Val Trp Ser Ser Glu Ile Tyr Asn 645 650 655Lys Asp
Asn Arg Tyr Gly Arg Tyr Gln Ser His Gly Val Ala Gln Ile 660 665
670Val Ser Asn Gly Ser Gln Leu Ser Gln Gly Tyr Gln Gln Glu Gly Trp
675 680 685Asp Trp Asn Arg Met Glu Gly Ala Thr Thr Ile His Leu Pro
Leu Lys 690 695 700Asp Leu Asp Ser Pro Lys Pro His Thr Leu Met Gln
Arg Gly Glu Arg705 710 715 720Gly Phe Ser Gly Thr Ser Ser Leu Glu
Gly Gln Tyr Gly Met Met Ala 725 730 735Phe Asn Leu Ile Tyr Pro Ala
Asn Leu Glu Arg Phe Asp Pro Asn Phe 740 745 750Thr Ala Lys Lys Ser
Val Leu Ala Ala Asp Asn His Leu Ile Phe Ile 755 760 765Gly Ser Asn
Ile Asn Ser Ser Asp Lys Asn Lys Asn Val Glu Thr Thr 770 775 780Leu
Phe Gln His Ala Ile Thr Pro Thr Leu Asn Thr Leu Trp Ile Asn785 790
795 800Gly Gln Lys Ile Glu Asn Met Pro Tyr Gln Thr Thr Leu Gln Gln
Gly 805 810 815Asp Trp Leu Ile Asp Ser Asn Gly Asn Gly Tyr Leu Ile
Thr Gln Ala 820 825 830Glu Lys Val Asn Val Ser Arg Gln His Gln Val
Ser Ala Glu Asn Lys 835 840 845Asn Arg Gln Pro Thr Glu Gly Asn Phe
Ser Ser Ala Trp Ile Asp His 850 855 860Ser Thr Arg Pro Lys Asp Ala
Ser Tyr Glu Tyr Met Val Phe Leu Asp865 870 875 880Ala Thr Pro Glu
Lys Met Gly Glu Met Ala Gln Lys Phe Arg Glu Asn 885 890 895Asn Gly
Leu Tyr Gln Val Leu Arg Lys Asp Lys Asp Val His Ile Ile 900 905
910Leu Asp Lys Leu Ser Asn Val Thr Gly Tyr Ala Phe Tyr Gln Pro Ala
915 920 925Ser Ile Glu Asp Lys Trp Ile Lys Lys Val Asn Lys Pro Ala
Ile Val 930 935 940Met Thr His Arg Gln Lys Asp Thr Leu Ile Val Ser
Ala Val Thr Pro945 950 955 960Asp Leu Asn Met Thr Arg Gln Lys Ala
Ala Thr Pro Val Thr Ile Asn 965 970 975Val Thr Ile Asn Gly Lys Trp
Gln Ser Ala Asp Lys Asn Ser Glu Val 980 985 990Lys Tyr Gln Val Ser
Gly Asp Asn Thr Glu Leu Thr Phe Thr Ser Tyr 995 1000 1005Phe Gly
Ile Pro Gln Glu Ile Lys Leu Ser Pro Leu Pro 1010 1015 1020
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References