U.S. patent application number 10/380522 was filed with the patent office on 2003-09-18 for remedies for amyotrophic lateral sclerosis.
Invention is credited to Funakoshi, Hiroshi, Nakamura, Toshikazu, Sun, Woong.
Application Number | 20030176347 10/380522 |
Document ID | / |
Family ID | 28042097 |
Filed Date | 2003-09-18 |
United States Patent
Application |
20030176347 |
Kind Code |
A1 |
Nakamura, Toshikazu ; et
al. |
September 18, 2003 |
Remedies for amyotrophic lateral sclerosis
Abstract
The invention presents a therapeutic agent (and progress
suppressant) for amyotrophic lateral sclerosis (ALS) containing HGF
and/or HGF gene as active ingredient. HGF has an effect of
improving the motor function of ALS and life span through two
actions, that is, direct neuronutrient factor activity on
motoneurons, and indirect improving action of glutamate
cytotoxicity on motoneurons by maintaining the level of glutamate
transporter in astrocytes. Hence, HGF and/or HGF gene can be used
as an effective therapeutic agent not known in the past.
Inventors: |
Nakamura, Toshikazu; (Osaka,
JP) ; Funakoshi, Hiroshi; (Osaka, JP) ; Sun,
Woong; (Seoul, KR) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Family ID: |
28042097 |
Appl. No.: |
10/380522 |
Filed: |
May 14, 2003 |
PCT Filed: |
February 28, 2001 |
PCT NO: |
PCT/JP01/01539 |
Current U.S.
Class: |
514/9.5 ;
514/44R |
Current CPC
Class: |
A61K 38/1833 20130101;
A61K 48/005 20130101; A61K 48/00 20130101 |
Class at
Publication: |
514/12 ;
514/44 |
International
Class: |
A61K 038/18; A61K
048/00 |
Claims
1. A remedy for amyotrophic lateral sclerosis (ALS), containing HGF
and/or HGF gene as active ingredient.
2. A progress suppressant for ALS, containing HGF and/or HGF gene
as active ingredient.
3. An induction suppressant of caspase-1 in motoneurons, containing
HGF and/or HGF gene as active ingredient.
4. A phosphorylation promoter of Akt in spinal cord, containing HGF
and/or HGF gene as active ingredient.
5. A decrease suppressant of glial-specific glutamate transporter
(EAAT2/GLT-1) in astrocytes, containing HGF and/or HGF gene as
active ingredient.
Description
TECHNICAL FIELD
[0001] The present invention relates to a remedy for amyotrophic
lateral sclerosis (ALS). More particular, the invention relates to
a remedy for ALS containing HGF (hepatocyte growth factor) and/or
HGF genes as active ingredients.
BACKGROUND ART
[0002] Amyotrophic lateral sclerosis (ALS) is a severe
neurodegenerative disease characterized by progressive loss of
motoneurons and degeneration of motor axons, which results in motor
dysfunction and shortening of life span. About 15% of ALS patients
are patients with familial ALS (FALS), and about 15% to 25% of FALS
patients carry mutations in the gene encoding Cu.sup.2+/Zn.sup.2+
superoxide dismutase (SOD1). Transgenic mice with high levels of
mutant SOD1 protein and activity develop diseases similar to both
familial and sporadic ALS, and such muted SOD1 overexpressing
transgenic mice are used as a model for ALS. In the invention, too,
G93A mice (Science, 264, 1772-1775, 1994) were used as muted SOD1
(G93A) overexpressing transgenic mice.
[0003] Since degeneration of motoneurons is thought to be a first
sign of the disease, many approaches had been focused to directly
support the survival of motoneurons. However, these attempts were
not satisfactory.
[0004] On the other hand, Brujin et al. reported that prominent
SOD1 containing inclusions in astrocytes appear prior to clinical
signs and increase markedly in abundance during disease
progression, implicating astrocytes as primary targets for
mutant-SOD1 mediated damage in mutant SOD1 (G85R) overexpressing
transgenic mice (Neuron, 18, 327-338, 1997). Therefore, a
bifunctional substance that can restore the function of astrocytes
and directly promote motoneurons from each cell death would be more
ideal for the treatment of ALS. Such substance, however, has not
been known yet.
[0005] Hepatocyte growth factor (HGF) was first identified as a
potent mitogen for mature hepatocytes, and its gene was cloned in
1989 (Biochem. Biophys. Res. Commun., 122, 1450-1459, 1984; Nature,
342, 440-443, 1989). Although HGF was discovered as a hepatotrophic
factor, recent extensive studies on expression and functional
analysis including knockout/in mice strategies revealed HGF as a
new neurotrophic factor (Ciba. Found. Symp., 212, 198-211, 1997;
Nat. Neurosci., 2, 213-217, 1999). HGF shows neurotrophic
activities on hippocampus, cerebral cortex, midbrain dopaminergic,
cerebellum granular, sensory and motoneurons and sympathetic
neuroblasts (Ciba. Found. Symp., 212, 198-211, 1997; Brain Res.
Mol. Brain Res., 32, 197-210, 1995). Especially, HGF is shown to be
one of the most potent survival promoting factors for motoneurons
comparable with glial cell line derived neurotrophic factors (GDNF)
in vitro (Neuron, 17, 1157-1172, 1996). Neurotrophic effects of HGF
on embryonic spinal motor neurons during development and on adult
motor neurons after axotomy of hypoglossal nerve are shown in vivo
(J. Neurosci., 20, 326-337, 2000; Eur. J. Neurosci., 11, 4139-4144,
1999). However, nothing has been reported about the role of HGF in
ALS, or clinical effect of expression of HGF on ALS, and therefore
it has been known whether HGF would be a remedy for ALS or not.
[0006] In this background, the present inventors investigated into
possibility of using HGF as a remedy for ALS in order to present a
novel remedy for ALS.
[0007] The inventors first studied the role of HGF in ALS by using
G93A transgenic mice as a model for ALS. As a result, it is found
that c-Met/HGF receptor-like immunoreactivity (c-Met-IR) is
localized in motoneurons of G93A transgenic mice similarly to that
of wildtype littermates. Quantification of the levels of the
expression of c-met and HGF mRNA in the ventral horn of spinal cord
(where motoneurons locate) revealed that they were progressively
increased during the progression of ALS in G93A transgenic mice.
These results suggested the roles of HGF on ALS motoneurons.
[0008] Using neuron-specific enolase promoter (NSE), the inventors
generated transgenic mice overexpressing rat HGF in a
neuron-specific manner, and explored the effect of HGF on ALS. As a
result, it was discovered for the first time that HGF is effective
to attenuate motoneuronal death and suppress axonal degeneration of
motoneurons. It was also found that HGF exerts a neuroprotective
effect not only on motoneurons but also on DRG sensory neurons
against ALS related neurotoxicity. The neuroprotective effect of
HGF was also indicated from the delayed loss of muscle weight in
ALS.
[0009] The inventors further studied if the progress of ALS can be
actually suppressed (delayed) by HGF or not by using the same
transgenic mice. To a great surprise, start of paralysis was
delayed, the life span was extended, and motor functions were
improved by an extremely small expression of HGF (about 2 times of
wildtype mouse and G93A mouse).
[0010] On the basis of these findings, it was discovered for the
first time that the HGF has a therapeutic effect on ALS.
[0011] The inventors further investigated into the mechanism of
suppressing progress of ALS by HGF, and revealed that HGF brings
about the ALS improving effects by at least three new mechanisms as
explained below.
[0012] (1) Induction suppressing action of caspase-1 on
motoneurons
[0013] At middle stage of ALS, caspase-1 is thought to play an
important role in the progress of the disease, because caspase-1 is
shown to be activated and/or induced in motoneurons of transgenic
mice overexpressing mutated SOD1 (Proc. Natl. Acad. Sci. U.S.A.,
95, 15763-15768, 1998; Science, 288, 335-339, 2000), and
introduction of dominant negative inhibitor for caspase-1 in G93A
mice was successfully delayed in mortality for about 2 weeks
Nature, 388, 31, 1997; J. Exp. Med., 185, 933-940, 1997).
Accordingly, the inventors examined whether HGF can modify the
induction of caspase-1, and disclosed that HGF has an effect of
suppressing induction of caspase-1 in ALS motoneurons.
[0014] (2) Phosphorylation of Akt in spinal cord
[0015] Phosphorylation of Akt is suggested to be involved in the
survival promoting activity of HGF in cerebral cortex neurons and
kidney epithelial cells, and HGF is shown to induce Bcl-xl
expression and block massive apoptosis in the liver in fluminant
hepatitis models (Biochem. Biophys. Res. Commun., 244, 683-690,
1998; Hepatology, 30, 151-159, 1999). Accordingly, the present
inventors explored if phosphorylation of Akt would be induced by
expression of HGF, and recognized phosphorylation of Akt
specifically in the spinal cord of ALS.
[0016] (3) Suppression of down-regulation of glial-specific
glutamate transporter (EAAT2/GLT1) in reactive astrocytes
[0017] It has been indicated that glutamate-mediated excitotoxicity
contributes to motor neuron degeneration of ALS by reduction of
glutamate clearance. Consistently with this hypothesis, it is
reported that in the spinal cord and motor cortex of patients with
sporadic ALS, glutamate transport activity is decreased remarkably
(N. Engl. J. Med., 326, 1464-1468, 1992), and immunoreactivity for
glial-specific glutamate transporter (EAAT2/GLT-1), which locates
in astrocytes and is thought to be a major transporter to suppress
glutamatergic neurotoxicity, selectively disappears (Ann. Neurol.,
38, 73-84, 1995). The reduction of EAAT2 in G85R type ALS model
transgenic mice (Neuron, 18, 327-338, 1997), and SOD1 mutants (A4V
and I113T)-linked inactivation of a glial glutamate transporter in
ALS (Nat. Neurosci., 2, 427-433, 1999) are also reported. The
inventors previously studied the role of HGF on EAAT2 because
glutamatergic neurotoxicity was thought to play a part in
motoneuron degeneration in ALS. As a result, it was found that HGF
functions to suppress down-regulation of EAAT2 in ALS and maintain
a functional astrocytes in endstage of ALS.
[0018] It was thus disclosed that HGF improves ALS by at least
three mechanisms, that is, induction suppressing action of
caspase-1 on motoneurons, phosphorylation of Akt, and suppression
of down-regulation of glial-specific glutamate transporter
(EAAT2/GLT1) in reactive astrocytes.
[0019] Thus, HGF has an effect of improving the motor function of
ALS and life span through two actions, that is, direct
neuronutrient factor activity on motoneurons, and indirect
improving action of glutamate cytotoxicity on motoneurons by
maintaining the level of glutamate transporter in astrocytes. Such
bifunctional growth factor has not bee known so far, and HGF is a
first example. Such functions of HGF suggest possibility
therapeutic usefulness of HGF (gene or protein) in ALS and related
motoneuron diseases.
SUMMARY OF THE INVENTION
[0020] The invention is devised on the basis of the above findings.
That is, the invention presents:
[0021] (1) Remedy for ALS, containing HGF and/or HGF gene as active
ingredient.
[0022] (2) Progress suppressant for ALS, containing HGF and/or HGF
gene as active ingredient.
[0023] (3) Induction suppressant of caspase-1 in motoneurons,
containing HGF and/or HGF gene as active ingredient.
[0024] (4) Phosphorylation promoter of Akt in spinal cord,
containing HGF and/or HGF gene as active ingredient.
[0025] (5) Decrease suppressant of glial-specific glutamate
transporter (EAAT2/GLT-1) in astrocytes, containing HGF and/or HGF
gene as active ingredient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIG. 1 is a microphotograph showing results of
immunohistological analysis of expression of c-Met (C-Met-IR) in
motoneurons in G93A transgenic mice (G93A) and wildtype littermates
(wildtype). In the figure, 2 mo denotes 2 months of age, and 8 mo
is 8 months of age.
[0027] FIG. 2 is a graph showing results of quantification of HGF
and c-met RNA in spinal cord by competitive RT-PCR, in progress
stages of disease of G98A mice, in comparison with wildtype mice of
same age. The axis of abscissas denotes the age, and the axis of
ordinates represents the c-met RNA level (left) or HGF RNA level
(right). In the figure, the arrowhead indicates the band of c-met
or HGF, and the bar shows the band of competitor.
[0028] FIG. 3 is an electrophoretic photograph showing results of
expression of exogenous HGF in each tissue of HGF transgenic mice
prepared by using neuron-specific enolase promoter, analyzed by
RNase protection assay. In the figure, the arrow indicates the band
corresponding to the exogenous HGF, and the bar shows the band
corresponding to the endogenous HGF. Also in the figure, Br is the
brain, Lu is the lung, Ht is the heart, Li is the liver, Kd is the
kidney, Sp is the spleen, St is the stomach, Sc is the spinal cord,
Ts is the testis, Ms is the muscle, In is the intestine, and Sk is
the skin.
[0029] FIG. 4 is a graph showing the level of plasma HGF in
2-month-old wildtype and HGF transgenic mouse littermates (n=6). It
was measured by means of enzyme-linked immunosorbent assay
(ELISA).
[0030] FIG. 5 is a graph showing ELISA analysis results of HGF
contents in the whole spinal cord in wildtype and HGF transgenic
mouse littermates at various growth stages after birth. In the
figure, P2 is 2 days after birth, P14 is 14 days, 1 mo is 1 month
of age, and 6 mo is 6 months of age.
[0031] FIG. 6 is a microphotograph showing histological analysis
results of number and morphology of motoneurons of ventral horn of
lumbar spinal cord. In the figure, wildtype is wildtype mice, HGF
is HGF single transgenic mice, G93A is G93A transgenic mice (ALS
endstage), and G93A/HGF is double transgenic mice (ALS
endstage).
[0032] FIG. 7 is a graph showing quantitative comparison of number
of motoneurons in the lumbar spinal cord in transgenic mice. In the
figure, .largecircle. is wildtype, .circle-solid. is HGF single
transgenic mice, .DELTA. is G93A transgenic mice, and
.tangle-solidup. is double transgenic mice.
[0033] FIG. 8 is a graph showing the wet weight of gastrocnemius
muscle of transgenic mice. In the figure, .largecircle. is
wildtype, .circle-solid. is HGF single transgenic mice, .DELTA. is
G93A transgenic mice, and .tangle-solidup. is double transgenic
mice.
[0034] FIG. 9 is a microphotograph of segment (1 .mu.m) of ventral
root (V, upper panel) and dorsal root (D, lower panel) of
8-month-old transgenic mice.
[0035] FIG. 10 is a graph showing ELISA analysis results of HGF
level in the spinal cord at the age of 2, 6 and 8 months in
transgenic mice.
[0036] FIG. 11 is a graph showing comparative results of paralysis
start in G93A transgenic mice (.DELTA., n=15) and G93A/HGF double
transgenic mice (.tangle-solidup., n=16).
[0037] FIG. 12 is a graph showing comparative results of death
start in G93A transgenic mice (.DELTA., n=15) and G93A/HGF double
transgenic mice (.tangle-solidup., n=16).
[0038] FIG. 13 is a graph showing results of measurement of hind
limb stretch reflex in wildtype (.largecircle., n=14), HGF single
transgenic mice (.circle-solid., n=15), G93A transgenic mice
(.DELTA., n=15), and double transgenic mice (.tangle-solidup.,
n=16).
[0039] FIG. 14 is a graph showing results of measurement of holding
time on rotor rod in wildtype (.largecircle., n=14), HGF single
transgenic mice (.circle-solid., n=15), G93A transgenic mice
(.DELTA., n=15), and double transgenic mice (.tangle-solidup.,
n=16).
[0040] FIG. 15 is a graph showing results of measurement of stride
in wildtype (.largecircle., n=14), HGF single transgenic mice
(.circle-solid., n=15), G93A transgenic mice (.DELTA., n=15), and
double transgenic mice (.tangle-solidup., n=16).
[0041] FIG. 16 is a microphotograph showing immunostaining analysis
results of aggregation of mutant SOD1 in the lumbar spinal cord in
G93A and G93A/HGF littermates in progressive stages of disease. In
the figure, 4 mo is 4 months of age and 8 mo is 8 months of
age.
[0042] FIG. 17 is an electrophoretic photograph showing
immunoblotting analysis results of amount of mutant SOD1 in the
lumbar spinal cord of mice of wildtype (W), HGF single (H), G93A
(G), and double transgenic (G/H). In the figure, 2 mo is 2 months
of age, 6 mo is 6 months of age and 8 mo is 8 months of age.
[0043] FIG. 18 is an electrophoretic photograph showing
immunostaining results of phosphorylated Akt (p-Akt), Akt, Bcl-xL,
and Bcl-2 in the lumbar spinal cord of 8-month-old mice of wildtype
(W), HGF single (H), G93A (G), and double transgenic (G/H).
[0044] FIG. 19 is a microscopic photograph of GFAP
immunohistological analysis results of ventral horn of lumbar
spinal cord of 8-month-old mice of four types.
[0045] FIG. 20 is a quantitative graph immunohistological analysis
by GFAP of FIG. 19. In the figure, .largecircle. is wildtype,
.circle-solid. is HGF single transgenic, .DELTA. is G93A
transgenic, and .tangle-solidup. is double transgenic (n=3 in each
group).
[0046] FIG. 21 is an electrophoretic photograph showing
immunoblotting results of EAAT2, GFAP, and c-Met in the spinal cord
of four types of mice at the age of 2, 6,and 8 months. In the
figure, W is wildtype, H is HGF single transgenic, G is G93A
transgenic, and G/H is double transgenic
[0047] FIG. 22 is a graph showing the relative level of EAAT2 after
standardization (EAAT2 level is divided by GFAP level in order to
standardize) (in each group, n=4 mice).
[0048] FIG. 23 is an electrophoretic photograph showing
immunoblotting of EAAT2 and c-Met in astrocytes of primary culture
after HGF treatment.
[0049] FIG. 24 is a quantitative graph showing results of
immunoblotting of FIG. 23 (in each group, n=2).
DETAILED DESCRIPTION OF PREFERRED EMBODIMENT
[0050] HGF used in the invention is a known substance, and its base
sequence and amino acid sequence are disclosed, for example, in
Nature, 342, 440 (1989), Japanese Patent No. 2777678, Biochem.
Biophys. Res. Commun., 163, 967 (1989), Biochem. Biophys. Res.
Commun.,172, 321 (1990), etc. This HGF may be manufactured in any
method as far as it is refined to be a pharmaceutical level.
Commercial products may be also used (for example, Code No. HGF-101
of Toyobo).
[0051] For example, HGF may be manufactured by cultivating primary
culture cells or established cells producing HGF, and obtaining HGF
by separating and purifying from the culture supernatant. Or, by a
gene engineering technique, a gene encoding HGF is incorporated in
a proper vector, and it is inserted into a proper host to
transform, and a desired recombinant HGF can be obtained from the
culture supernatant of this transformant (for example, see Nature,
342, 440,1989; Japanese Laid-open Patent No. 5-111383, Biochem.
Biophys. Res. Commun., 163, 967,1989, etc.). The host cell is not
particularly limited, and various host cells hitherto used in the
gene engineering techniques can be used, such as Escherichia coli,
yeast or animal cells. Animal cells include CHO cell, COS cell, and
mouse C127 cell (all available from ATCC, etc.). Thus obtained HGF
may have a structure analogous to natural type HGF as far as it has
an action substantially same as natural type HGF. That is, the
category of HGF of the invention includes protein having action as
HGF among 1) protein encoded by DNA for hybridizing with cDNA of
HGF in stringent conditions, and 2) protein of amino acid sequence
having one or plural (preferably several) amino acids replaced,
deleted and/or added in the amino acid sequence of HGF. Herein, the
DNA for encoding the protein of 1) or 2) can be obtained easily,
for example, by site-specific mutagenesis induction method, PCR
method, or ordinary hybridization method, and specifically it can
be executed by reference to the textbook "Molecular Cloning 2nd
Ed.," Cold Spring Harbor Laboratory Press (1989), or the like.
[0052] The invention has disclosed for the first time that HGF is
useful as remedy for ALS, specifically as progress suppressant of
ALS. HGF has an effect of improving the motor function of ALS and
life span through two actions, that is, direct neuronutrient factor
activity on motoneurons, and indirect improving action of glutamate
cytotoxicity on motoneurons by maintaining the level of glutamate
transporter in astrocytes. Such bifunctional growth factor has not
been known in the past, and HGF is a first example. Having these
two actions, HGF is capable of retarding and suppressing the
progress of ALS and related motoneuron diseases.
[0053] As the mechanism of action of HGF on ALS, HGF is considered
to have ALS improving effects by exhibiting at least the following
three actions.
[0054] (1) Induction suppressing action of caspase-1 on
motoneurons
[0055] (2) Phosphorylation of Akt in spinal cord
[0056] (3) Suppression of down-regulation of glial-specific
glutamate transporter (EAAT2/GLT1) in reactive astrocytes
[0057] Endogenous growth factor having such action has not been
known hitherto, and HGF is a first growth factor showing these
actions. By exhibiting these actions, HGF can retard or suppress
the progress of ALS and related motoneuron diseases.
[0058] Motoneuron diseases relating to ALS include progressive
spinal muscular atrophy, progressive bulbar paralysis, primary
lateral sclerosis, infantile or juvenile muscular atrophy,
Fazio-Londe syndrome, Charcot-Marie-Tooth disease, and others, but
are not limited to these diseases.
[0059] The remedy of the invention may be prepared in various
pharmaceutical formulas (such as liquid and solid), but generally
it is manufactured as an injection of active ingredient of HGF only
or together with a usual carrier. The injection can be prepared in
an ordinary method, and, for example, HGF is dissolved in a proper
solvent (for example, sterilized water, buffer solution,
physiological saline), and filtered and sterilized by a filter, and
poured into an aseptic container. The content of HGF in the
injection is usually adjusted to be 0.0002 to 0.2 w/v %, preferably
about 0.001 to 0.2 w/v %.
[0060] In pharmaceutical manufacture, preferably, a stabilizer is
added, and examples of the stabilizer include albumin, globulin,
gelatin, mannitol, glucose, dextran, and ethylene glycol. Further,
the preparation of the invention may include additives necessary
for manufacture, such as vehicle, dissolution aid, antioxidant,
analgesic, and isotonic agent. In a form of liquid preparation, it
is preferable to store it under frozen conditions or after the
removal of water by a process such as freeze-drying. The freeze-dry
preparation is used by adding distilled water for injection and
redissolving.
[0061] The preparation of the invention is administered through a
proper route depending on the dosage form. The route of
administration is not particularly limited as far as HGF acts
properly to suppress progress of ALS. General routes are
subcutaneous administration, intradermal administration,
intravenous administration, intra-aortic administration, muscular
administration, local injection, intraventricular administration,
and intraspinal administration. Further, it may be administered
through a device embedded in the body, and specifically it may be
administered to the diseased site continuously and gradually by
using a osmotic pump or the like, or a sustained-release agent (for
example, mini pellet preparation) may be buried near the diseased
site.
[0062] In particular, the intraspinal administration is a preferred
route because the preparation of the invention can reach the
motoneurons in the spinal cord directly.
[0063] The dose is adjusted properly depending on the symptom, age,
body weight and other conditions of patients, and usually the dose
of HGF is 0.001 mg to 1000 mg, preferably 0.01 mg to 100 mg, which
is divided in one to several portions a day.
[0064] As described in Examples below, it was proved that ALS was
improved by expression of a very small amount of HGF. That is, by
the HGF expression level of about 2 times of the HGF expression
amount in the spinal cord of wildtype mice and G93A transgenic
mice, the motor function was improved sufficiently at 6 months in
the period of first sign of symptoms of ALS, and the life was
expanded by 1 month in mice. Therefore, for patients with ALS, the
HGF preparation of the invention is preferred to be administered at
a dose of more than 2 times before HGF administration at least in
the HGF expression level in the spinal cord. The invention also
presents a remedy for ALS at a dose of more than 2 times before HGF
administration, as the HGF expression level in the spinal cord.
[0065] Recently, gene therapies using HGF gene have been reported
(see Circulation, 96, No. 3459, 1997; Nature Medicine, 5, 226-230,
1999; Circulation, 100, No. 1672, 1999; Gene Therapy, 7, 417-427,
2000, etc.), and this method is established technically. The
invention includes not only the administration of HGF protein as
mentioned above, but also a gene remedy for ALS by transducing HGF.
The gene therapy of HGF is explained below.
[0066] The HGF gene used in the invention refers to the gene
capable of expressing HGF (HGF protein). More specifically, cDNA of
HGF mentioned in Nature, 342, 440 (1989), Japanese Patent No.
2777678, Biochem. Biophys. Res. Commun., 163, 967 (1989), Biochem.
Biophys. Res. Commun.,172, 321 (1990), etc. is incorporated in a
proper expressing vector (non-viral vector, viral vector). The base
sequence of the cDNA encoding HGF is mentioned in the above
documents, and is also registered in the database of Genbank and
others. On the basis of such sequence information, a proper DNA
portion is used as primary of PCR, and, for example, by RT-PCR
reaction on the m-RNA derived from liver or leukocytes, cDNA of HGF
can be cloned. Such cloning operations can be done easily by those
skilled in the art by referring to the textbook such as "Molecular
Cloning 2nd Ed.," Cold Spring Harbor Laboratory Press (1989).
[0067] Further, the HGF gene of the invention is not limited to
those mentioned above, and as far as the expressed protein
substantially as the same action as HGF, it can be used as the HGF
gene of the invention. That is, among 1) DNA for hybridizing in
stringent conditions with the above-mentioned cDNA, and 2) DNA for
encoding protein of amino acid sequence having one or plural
(preferably several) amino acids replaced, deleted and/or added in
the amino acid sequence of the protein encoded by the cDNA, those
capable of protein having the action as HGF are included in the
category of the HGF gene of the invention. Herein,. the DNA of 1)
or 2) can be obtained easily, for example, by site-specific
mutagenesis induction method, PCR method, or ordinary hybridization
method, and specifically it can be executed by reference to the
textbook such as "Molecular Cloning" mentioned above.
[0068] The HGF gene of the invention can be applied in the same
diseases as the HGF protein. The HGF gene and HGF protein can be
used independently or in combination.
[0069] The gene transducing method, transducing format and
transducing amount used in the gene therapy of the invention are
described below.
[0070] When the gene remedy mainly composed of gene as active
ingredient is administered to patients, modes of administration are
roughly classified into a method of using non-viral vector and a
method of using viral vector, and the preparation and
administration are specifically described in the textbooks
(Supplementary Experimental Medicine, Fundamental technology of
gene therapy, Yodosha, 1996; Supplementary Experimental Medicine,
Gene transduction and expression analysis experiment, Yodosha,
1997; Gene therapy development research handbook edited by Japan
Society of Gene Therapy, NTS, 1999). These methods are specifically
described below.
[0071] A. Using non-viral vector
[0072] Using a recombination expression vector having an intended
gene integrated in a usual gene expression vector, the intended
gene can be transduced into the cell or tissue in the following
technique.
[0073] The gene transducing method into a cell includes lipofection
method, phosphoric acid-calcium coprecipitation method,
DEAE-dextran method, and DNA direct injection method using micro
glass tube.
[0074] The gene transducing method into a tissue includes gene
transduction method by internal type liposome, gene transduction
method by electrostatic type liposome, HVJ-liposome method,
modified HVJ-liposome method (HVJ-AVE liposome method), receptor
interstitial gene transduction method, method of transcribing DNA
molecule into cell together with carrier (metal particle) by
particle gun, naked-DNA direct transduction method, and method of
transduction by positive charge polymer, and a recombination
expression vector can be integrated into the cell.
[0075] In particular, the HVJ-liposome is prepared by sealing DNA
into liposome composed of lipid double membrane, and fusing this
liposome and inactivated hemagglutinating virus of Japan (HVJ). As
compared with the conventional liposome method, the HVJ-liposome
method is very high in the fusion activity with cell membrane, and
it is a preferred form of transduction. The preparing method of
HVJ-liposome is taught in the publications (Supplementary
Experimental Medicine, Fundamental technology of gene therapy,
Yodosha, 1996; Supplementary Experimental Medicine, Gene
transduction and expression analysis experiment, Yodosha, 1997; J.
Clin. Invest. 93, 1458-1464,1994; Am. J. Physiol. 271, R1212-1220,
1996), which should be referred to. Preferably, HVJ should be
obtained from Z strain (available via ATCC), but basically other
HVJ strains (for example, ATCC VR-907 or ATCC VR-105) may be also
used.
[0076] The direct transduction method of naked-DNA is the easiest
method of all methods mentioned above, and is hence a preferred
method of transduction.
[0077] The expression vector used herein may be any expression
vector as far as the intended gene can be expressed in the body,
and recommended expression vectors include pCAGGS (Gene 108,
193-200, 1991), PBK-CMV, pcDNA3.1, pZeoSV (Invidrogen Co.,
Stratagene Co.).
[0078] B. Using viral vector
[0079] As the viral vector, recombinant adeno virus, retro virus,
and other viral vectors are used. More specifically, by using
detoxicated retro virus, adeno virus, adeno companion virus, herpes
virus, vaccinia virus, pox virus, polio virus, Sindbis virus,
hemagglutinating virus of Japan, SV40, human immunodeficiency virus
(HIV), other DNA virus, or RNA virus, an intended gene is
transduced, and the cell is infected with the recombinant virus,
and the gene can be transduced into the cell.
[0080] Of these viral vectors, the infection efficiency of adeno
virus is known to be very high as compared with other viral
vectors, and from this viewpoint it is preferred to use the adeno
virus vector system.
[0081] The method of administration of the gene remedy of the
invention into patients includes both in-vivo method of introducing
the gene remedy directly into the body, and ex-vivo method of
taking out a certain cell from the body, transducing the gene
remedy into the cell, and putting back the cell into the body
(Nikkei Science, 20-45, April 1994; Monthly Pharmacology, 36 (1),
23-48, 1994; Experimental Medicine Extra, 12 (15), 1994; Gene
Remedy Development Research Handbook ed. by Japan Society of Gene
Therapy, NTS, 1999). In this invention, the in-vivo method is
preferred.
[0082] The route of administration to patient is not particularly
specified as far as HGF expressed in the body can exhibit its
action to suppress progression of ALS. General routes are
subcutaneous administration, intradermal administration,
intravenous administration, intra-aortic administration, muscular
administration, local injection, intraventricular administration,
and intraspinal administration. Further, it may be administered
through a device embedded in the body, and specifically it may be
administered to the diseased site continuously and gradually by
using a osmotic pump or the like, or a sustained-release agent (for
example, mini pellet preparation) may be buried near the diseased
site.
[0083] Above all, the intraspinal administration is a preferred
route of administration because the preparation of the invention
can directly reach the motoneurons in the spinal cord.
[0084] The dosage form includes various pharmaceutical preparations
suited to the route of administration (such as liquid). For
example, in the case of an injection preparation containing the
gene as the active ingredient, the injection can be prepared by a
conventional method, and, for example, it is dissolved in a proper
solvent (buffer solution such as PBS, physiological saline,
sterilized water, etc.), filtered and sterilized by a filter as
required, and poured into an aseptic container. A carrier or other
known matter may be added to the injection as required. In the case
of liposome such as HVJ-liposome, liposome preparations can be
manufactured in the dosage form of suspension, frozen matter, or
frozen matter concentrated by centrifugal separation.
[0085] The content of the DNA in the preparation may be properly
adjusted properly depending on the disease to be treated, age, body
weight and other conditions of patients, and usually the dose of
DNA of the invention is 0.0001 to 100 mg, preferably 0.001 to 10
mg, which is preferred to be administered once in every several
days to every several months.
[0086] Same as in the case of HGF protein mentioned above, the
invention also presents a remedy for ALS to be administered at a
dose so that the expression level of HGF in the spinal cord may be
more than 2 times before administration of HGF gene.
EXAMPLES
[0087] The invention is more specifically described below while
presenting Examples, but it must be noted that the invention is not
limited to these Examples alone.
[0088] Material and method
[0089] (1) Transgenic mice
[0090] Transgenic G93A mice ((SOD1-G93A)1Gurd1) were purchased from
Jackson Laboratory (Science, 264, 1772-1775, 1994). For the
maintenance, male G93A mice were mated with female C57B6 mice, and
genotyped by PCR and dot-blot hybridization.
[0091] Full-length rat HGF cDNA (Proc. Natl. Acad. Sci. U.S.A. 87,
3200-3204, 1990) tagged with KT3 epitope was amplified by PCR and
inserted into the downstream of neuron-specific enolase promoter
(Neuron, 18, 231-241, 1997) in pNSE-Ex vector (kindly provided by
Dr. Doherty). For the subcloning, Not I site was generated in the
pNSE-Ex vector by addition of linker. Full-base sequence of plasmid
was confirmed. HGF-KT3 showed similar potency to human recombinant
HGF in scattering assay of MDCK cell and in vitro survival assays
of primary hippocampal neuronal culture. Transgenic mice were
generated and analyzed for transgene integration as previously
described (J. Cell. Biol., 128, 185-199, 1995; Proc. Natl. Acad.
Sci. U.S.A., 95, 5269-5274, 1998) with slight modification.
Briefly, transgenic cassette was excised from the vector by Sal I
digestion and the DNA was injected into the embryo of C57B6 strain
that has matched genetic background with G93A transgenic mice.
Integration of transgene was examined by PCR and dot-blot using
SV40 poly(A) sequence in the transgene construct. Expression of
exogenous HGF was accessed by RNase protection assay, using probes
covering 3' end of HGF-KT3 cDNA and 5' region of SV40 poly(A)
signal sequence.
[0092] Progeny of NSE-HGF transgenic mice were crossed with G93A
transgenic mice. The pups from one dam were housed in the same cage
until onset of the phenotypes. After the first sign of onset,
animals were separately kept. Food and water were supplied in the
bottom for free access. When animal could not stand its body within
30 seconds, the time was used as the time of death.
[0093] (2) Quantification of HGF and c-met RNA in the ventral
horn
[0094] Quantitative competitive RT-PCR for the ventral horn of
spinal cord was performed as previously reported (Brain Res. Brain
Res. Protoc., 5, 190-197, 2000).
[0095] (3) RNase protection assay
[0096] RNase protection assay was preformed with an RPA II
ribonuclease protection assay kit (Ambion, Austin, Tex.) as
described previously (J. Cell. Biol., 123, 455-465, 1993; Science,
268, 1495-1499, 1995). To prepare an anti-sense cRNA probe specific
for HGF mRNA, a 326-bp fragment encompassing 3' end part of poly(A)
sequence of HGF-KT-3-polyA was inserted into pGEM-T vector. The
plasmid was linearized and transcribed in the presence of
.alpha.-[.sup.32P]CTP and prepared cRNA probe was used for the
hybridization. Exogenous HGF RNA gives 326 bp protected band and
endogenous HGF RNA gives shorter protected band (251 bp) because of
the lacking of KT-3 and poly(A) sequence.
[0097] (4) Behavioral tests
[0098] Behavioral tests were executed with 15 animals of each
genotype from 6 families. Extension of hind limb is normally
observed when a mouse is suspended in the air by its tail. Mice
with motor neuron disease commonly show the retraction of hind
limb. The score corresponds to the number of extending hind limbs.
For a rotor rod test, mice were placed on the rod rotating at a
speed of 20 rpm. The duration of each mouse remaining on the rod
was measured. If animal could remain on the rod for 4 minutes, the
test was finished and scored as 4 minutes. Footprint was collected
by letting the mouse walk on a straight way after dipping its hind
paw in the black ink. Stride was measured within the area showing
regular walking.
[0099] (5) Histological analyses
[0100] For the counting of the number of motoneurons, spinal cord
was fixed by series of ethanol and serially section (14 .mu.m) from
L5 to L4 after embedding into the paraffin. Number of motoneurons
(within L4-L5) on the ventral horn was counted from 20 sections on
every seventh section. The number was counted on densely stained
neurons by crystal violet with clear nucleolus in the defined area
of ventral horn.
[0101] Antibodies specific for the c-Met (Santa Cruz, 1:100),
antibodies specific for the HGF (Tokushu Meneki, 1:1000),
antibodies specific for the human SOD (Sigma, 1:200), antibodies
specific for the GFAP (Sigma, 1:1000), and antibodies specific for
the caspase-1 (Santa Cruz, 1:500) were applied to the sections for
1 hour to 4 hours at room temperature or overnight at 4.degree. C.
after blocking with 5% goat serum and mouse IgG blocking reagent
for 1 hour (M.O.M. kit, Vector Science). After washing,
biotinylated or fluorescence labeled secondary antibody was applied
and incubated for 15 minutes. For the fluorescence immunostaining,
sections were then observed under the fluorescence microscope after
counting staining with Hoechst 33342. Fluorescence image was
captured and digitized by CCD camera (Hamamatsu), and the
fluorescence level was measured using Adobe PhotoShop. To visualize
the signal recognized by biotinylated secondary antibody, ABC
solution was applied for 10 minutes and developed in the DAB
solution. The specificity of each staining was tested as previously
reported (Sun et al., 1999).
[0102] L5 root was dissected and fixed with 4%
paraformaldehyde/0.25% glutaldehyde for overnight, and after
postfix with osmium tetraoxide for 2 hours on ice, roots were
dehydrated and embedded in Epon812. Embedded roots were sectioned
(1 .mu.m) and stained with toluidine blue. Morphology of samples
was examined under the light microscope.
[0103] (6) Cell culture
[0104] Primary astrocytes were cultured as described previously
(Brain Res. Mol. Brain Res., 41, 259-268, 1996) with slight
modification. Cerebral cortex of postnatal day 2 G93A mice or
wildtype littermates was dissected and made into small pieces.
Tissue blocks were dissociated by incubation with 0.25% trypsin and
100 .mu.g/ml DNase I for 12 minutes in 37.degree. C. water bath
with shaking. The tissues were incubated by aspirating with
pipette, and dissociated cells were plated in DF (high glucose
DMEM/HamF12, 50:50) medium (GibCO BRL) with 10% fetal calf serum
onto the poly-L-ornithin-coated dishes at indicated densities.
Seven days after the plating when cells became confluent, cells
were washed two times with PBS, and treated with indicated dose of
recombinant HGF. Purity of the astrocytes was usually more than 95%
after 7 days of culture, as examined by morphology or NSE/GFAP
double staining.
[0105] (7) Western blotting
[0106] Lumbar spinal cord lysate was prepared by RIPA buffer. Fifty
micrograms of lysates was electrophoresed on a 10%
SDS-polyacrylamide gel (SDS-PAGE) and transferred to PVDF membrane.
The membrane was incubated with 5% non-fat skim milk in PBS
overnight at 4.degree. C. and anti-EAAT2 antibody (1:2,000,
Chemicon) was applied for 2 hours. After washing, the membrane was
incubated with HRP-conjugated anti-guinea pig IgG (1:3,000,
Chemicon), and an ECL chemiluminescence reaction was performed
(Amersham, Buckinghamshire, UK). The membrane was stripped and
re-probed with anti-GFAP (1:3,000 Sigma), human SOD (1:500, Sigma),
and anti-c-Met (1:400, Santa Cruz) antibody. Fifty micrograms of
lysates was electrophoresed on a 12% SDS-PAGE and applied for
phospho-Akt (Cell Signaling Tech.), Akt (Cell Signaling Tech.),
Bcl-xL/s (Santa Cruz), and Bcl-2 (Santa Cruz) antibodies as
described above. Band intensities were measured by Fluorochem image
analyzer (IS-8000).
[0107] (8) Determination of HGF concentration
[0108] HGF in tissues or plasma was measured by means of
enzyme-linked immunosorbent assay (ELISA) using anti-rat HGF
polyclonal antibody (Tokushu Meneki, Tokyo, Japan). The rat HGF
ELISA system specifically detects rat and mouse HGF with similar
affinity.
[0109] (9) Statistical analysis
[0110] Statistical comparisons were determined by Student's t-test.
For survival tests, statistical significance (p <0.05) was
evaluated by log rank test.
Example 1
Expression and regulation of HGF and c-Met in G93A transgenic
mice
[0111] In motoneurons of G93A transgenic mice, it was studied by
immunohistological analysis to observe presence or absence of
c-Met/HGF receptor-like immunoreactivity (c-Met-IR). As a result,
same as in motoneurons of wildtype littermates, localization of
c-Met-IR was discovered in motoneurons of 2-month-old G93A mice
(FIG. 1). At 8 months when G93A mice were at endstage, c-Met-IR was
observed in many cells in the ventral horn, and the wildtype
littermates of same age indicated a same level of c-Met-IR as those
of 2 months of age (FIG. 1).
[0112] Quantification of HGF and c-met mRNA in the ventral horn of
spinal cord in the progressive stage of G93A transgenic mice was
determined by the competitive RT-PCR, and compared with that of
wildtype mice of the same age. As a result, the expression level of
c-met and HGF mRNA in the ventral horn of the spinal cord in which
motoneurons localize is known to increase progressively in the
process of ALS in G93A transgenic mice (FIG. 2). These results
suggested the role of HGF in ALS motoneurons. Specifically,
c-Met-IR was expressed not only in the remaining motoneurons, but
also in the peripheral astrocytes, and the role of HGF in other
cells than motoneurons was suggested. It was hence taught that HGF
may be one of the endogenous factors for retarding the progress of
the disease.
Example 2
Preparation and characterization of transgenic mice excessively
expressing HGF neuron-specifically
[0113] To study the effect of HGF in ALS, transgenic mice
expressing rat HGF neuron-specifically were prepared by using
neuron-specific enolase promoter (NSE). From eight independent
lines of HGF transgenic mice, one line expressing the exogenous HGF
specifically at relatively low level specifically in the nervous
system was selected. FIG. 3, FIG. 4 and FIG. 5 show the
characteristics of the HGF transgenic mouse lines. By RNase
protection assay, exogenous HGF was known to be expressed in the
brain and spinal cord specifically among 12 tissues being tested
(arrow in FIG. 3). Among tissues of HGF transgenic mice, the
endogenous and whole HGF levels in the plasma were not different
from the levels in the wildtype littermates (FIG. 3 and FIG. 4). On
the other hand, the level of HGF protein increased along with the
growth of mice in the spinal cord, only after termination of
principal differentiation of the spinal cord nerves after birth
(FIG. 5).
[0114] Apparently, HGF transgenic mice could not be distinguished
from the wildtype transgenic mice unless the genotype was
determined. That is, no difference was noted in the size, body
weight, morphology, behavior and changes in motor neuron system
including many motoneurons and astrocytes, and weight of muscle at
any stage of growth ever investigated. This fact proves the
successful introduction of HGF specifically to the nervous system
without effects on the growth (see FIG. 6 to FIG. 10, FIG. 19 and
FIG. 20).
Example 3
Investigation of effect of HGF on ALS (1)
[0115] To study the role of HGF on ALS, heterozygotes (+/-) of HGF
transgenic mice were mated with heterozygotes (+/-) of G93A mice,
and transgenic mice having HGF introduced directly into ALS neurons
were prepared. By this mating, four different groups of mice were
created, that is, wildtype (W), transgenic to HGF only (HGF),
transgenic to G93A only (G93A), and double transgenic to both G93A
and HGF (G93A/HGF).
[0116] First of all, it was histologically analyzed to see if
expression of HGF would trigger neuroprotective action on
motoneurons. Results are shown in FIG. 6 and FIG. 7. Paraffin
segments (14 .mu.m) of the ventral horn of lumbar spinal cord at
endstage stained by crystal violet were evidently decreased in the
number of motoneurons of G93A mice (G93A), and the remaining
motoneurons were atrophied (FIG. 6). On the other hand, double
transgenic littermates (G93A/HGF) maintained spinal cord
motoneurons having many healthy forms evidently as compared with
G93A mice (FIG. 6). Wildtype and HGF single transgenic mice
presented a same number of healthy motoneurons (FIG. 6).
[0117] Using six independent animals in each group of mice, the
number of motoneurons in lumbar spinal cord was quantitatively
compared. As a result, at the age of 6 months, G93A mice began to
lose motoneurons in the lumbar spinal cord gradually, and at the
age of 8 months, only 40% of motoneurons were left over in G93A
mice, as compared with motoneurons of wildtype or HGF single
transgenic littermates (FIG. 7). By contrast, double transgenic
littermates were evidently improved in the number of motoneurons as
compared with G93A transgenic mice (FIG. 7).
[0118] The survival promoting activity of HGF on motoneurons has
been proved to be different between the lumbar level and cervical
level in chicken embryo, and it was tested to see if introduction
of HGF would decrease death of motoneurons on the cervical level.
As a result, in G93A mice, 55% of cervical motoneurons remained at
the age of 8 months. It is a feature of the mice in this model that
the death of motoneurons at the cervical level is smaller than at
the lumbar level. In the double transgenic mice, at the cervical
level, evidently, a greater number of motoneurons remained
(87.8.+-.2.4%), which suggests that HGF is effective on both lumbar
and cervical motoneurons.
[0119] Using at least three independent animals in each group of
mice, the effect of HGF on axon degeneration was studied by
histological analysis. Degeneration of axon of large-diameter the
ventral root was observed in G93A mice at the age of 8 months (FIG.
9), while the axon degeneration of the ventral root was moderate in
double transgenic littermates, and the ventral root was hardly
changed. In the dorsal root, a medium degeneration was noted in
G93A mice, but it was visually normal in double transgenic
littermates (FIG. 9) These facts suggest that HGF effectively
prevents degeneration of both ventral root and dorsal root.
Further, in endstage double transgenic mice, if the ventral root
was poor, the dorsal root was healthy. Therefore, HGF was known to
have a neuroprotective effect not only on motoneurons but also on
DRG sensory nerves relating to the ALS related neurotoxicity.
Moreover, the neuroprotective effect of HGF was also suggested from
the retarding effect of decrease in the gastrocnemius muscle (FIG.
8).
Example 4
Investigation of effect of HGF on ALS (2)
[0120] Using the same four types of mice, it was tested to see if
introduction of HGF would improve the start of paralysis, life
span, and motility of ALS.
[0121] Start of paralysis was observed at the average age of
243.8.+-.4.7 days (average .+-. standard deviation, median
=242.+-.14 days) in heterozygote (+/-) G93A mice, and the start was
evidently later in double transgenic mice (G93A/HGF), beginning at
the age of 271.9.+-.5.6 days (median 282.5.+-.9.5 days, p =0.004)
(FIG. 11).
[0122] Start of death in G93A mice was the average number of
survival of 259.5.+-.5.0 days (median =259.+-.11 days). In double
transgenic mice, the life was extended to 286.8.+-.6.5 days, and
the average life was extended by 27.3 days (median =294.+-.14.5
days, p =0.003, FIG. 12).
[0123] Motility was tested by reflex of hind limb extension,
holding time of rotor rod, and stride measurement. HGF single
transgenic mice were similar in motility to wildtype littermates.
G93A mice were decreased in hind limb extension from the age of 5
months, rotor rod capacity from 6 months, and stride progress from
7 months. On the other hand, double transgenic mice decrease very
slowly in all functional parameters as compared with G93A mice
(FIG. 13 to FIG. 15).
[0124] FIG. 10 shows HGF protein levels of spinal cord in four
groups in the progressive stages of ALS. HGF level was about 2
times higher at 2 months and 6 months in HGF transgenic mice and
G93A/HGF littermates, which suggests that a small addition of HGF
sufficiently improves the motor functions in 6 months, and extends
the life span for 1 month. However, insufficient production of HGF
in G93A/HGF double transgenic mice at 8 months, that is, same
levels of HGF in HGF, G93A, and G93A/HGF at the endstage suggested
possibility of reflection of imperfect improvement of ALS, and
expression of HGF a higher level is expected.
[0125] Thus, expression of HGF gene on ALS neurons suggested an
evident improvement of life span and motor function in ALS.
Example 5
Investigation of action mechanism of HGF on ALS (1)
[0126] In order to study the mechanism of possibility of decrease
of motoneuron deaths in double transgenic mice, decrease of motor
and sensory axon degeneration, and slow decrease of motor function,
that is, to see if the transduced HGF would change the initial
event of ALS, change the endstage event, or improve the speed of
progress of disease without having effect on any specific event,
one of the earliest signs of the disease was tested. Aggregation of
mutant SOD1 is reported as an earliest event in animal models, and
aggregation of SOD1 is reported in patients with familial ALS.
[0127] The total volume of mutant SOD1 in spinal cord extract was
investigated by immunoblotting by using an antibody specific to
human SOD1 antibody, that is, capable of recognizing only the
mutant human SOD1, not the endogenous mouse SOD1. Three independent
animals were tested in each group. In both G93A and double
transgenic mice, mutant SOD1 was detected from the age of 2 months.
The total volume of mutant SOD1 increased in both G93A and double
transgenic mice in a similar time course. The total volume of
mutant SOD1 in G93A mice was not different from that of HGF
littermates (FIG. 17).
[0128] Aggregation of SOD1 was investigated. Aggregation of mutant
SOD1 in spinal cord was detected prominently at the ventral horn in
initial stage (at the age of about 4 months) in G93A mice, and the
volume of aggregation evidently increased at the age of 8 months in
the endstage of animals (FIG. 16). Aggregation of mutant SOD1 in
double transgenic mice corresponded to aggregation in G93A
littermates at the age of 4 months (FIG. 16), and specific
immunoresponse was not detected in wildtype or HGF single
transgenic littermates. Aggregation of mutant SOD1 in double
transgenic mice was evidently increased at 8 months (FIG. 16), and
the volume seemed to be slightly less as compared with G93A
littermates, but the difference was very small. These results
suggested that HGF would not improve the origin of neurotoxicity
and initial events of the disease in this model.
[0129] Caspase-1 is believed to play an important role in progress
of ALS (Nature, 388, 31, 1997; J. Exp. Med., 185, 933-940, 1997).
Therefore, to see if HGF would improve induction of caspase-1 or
not, a double-labeled immunohistochemical method was attempted by
using anti-caspase-1 antibody, and anti-tubulin III antibody
staining mature neurons.
[0130] Caspase-1 immunoresponse (caspase-1-IR) was below the limit
of detection in both wildtype and HGF littermates at any moment of
test. In G93A mice, caspase-1-IR was specifically induced in large
tubulin III immunostained cells at 6 months, and co-localized. It
means induction of caspase-1 in motoneurons. Level of caspase-1 in
G93A decreased at 8 months, and only a faint capase-1-IR was
detected. In double transgenic mice, at 6 months of age, a very
weak caspase-1-IR was indicated as compared with G93A mice in large
tubulin III immunoresponse cells, which infers that HGF would
decrease the level of induction of caspase-1 in motoneurons in the
midstage of ALS.
[0131] Phosphorylation Akt is reported to be related to survival
promotion activity of HGF in cerebral cortical neurons and renal
epithelial cells, and HGF is known to induce Bcl-xL expression and
block massive apoptosis in the liver in fluminant hepatitis models
(Biochem. Biophys. Res. Commun., 244, 683-690, 1998; Hepatology,
30, 151-159, 1999). Accordingly, to explore the mechanism of HGF
for decreasing apoptosis, phosphorylation of Akt and adjustment of
Bcl-2 family gene were investigated.
[0132] By Western blotting, the amount of Akt was not changed in
any littermate, and, by contrast, Akt was evidently phosphorylated
specifically in the spinal cord of G93A/HGF mice at 8 months (FIG.
18). A considerably low level of Akt phosphorylation in the spinal
cord of HGF mice seems to suggest the lower level of c-Met in HGF
mice as compared with the level in double transgenic mice (FIG.
21). These results support the survival promotion activity of HGF
partially through the activation of Akt.
[0133] Bcl-xL and Bcl-2 protein were not induced in the spinal cord
at 8 months of age (FIG. 18).
[0134] As disclosed from these experiments, it was indicated that
HGF retarded the progress of ALS by preventing caspase-1 induction
in motoneurons and phosphorylation of Akt in the spinal cord.
Example 6
Investigation of action mechanism of HGF on ALS (2)
[0135] It has been indicated that glutamate-mediated excitotoxicity
contributes to motoneuron degeneration of ALS by reduction of
glutamate clearance. Consistently with this hypothesis, it is
reported that in the spinal cord and motor cortex of patients with
sporadic ALS, glutamate transport activity is decreased remarkably
(N. Engl. J. Med., 326, 1464-1468, 1992), and immunoreactivity for
glial-specific glutamate transporter (EAAT2/GLT-1), which locates
in astrocytes and is thought to be a major transporter to suppress
glutamatergic neurotoxicity, selectively disappears (Ann. Neurol.,
38, 73-84, 1995). The reduction of EAAT2 in G85R type ALS model
transgenic mice (Neuron, 18, 327-338, 1997), and SOD1 mutants (A4V
and I113T)-linked inactivation of a glial glutamate transporter in
ALS (Nat. Neurosci., 2, 427-433, 1999) are also reported. The role
of HGF on EAAT2 was studied because glutamatergic neurotoxicity was
thought to play a part in motoneuron degeneration in ALS.
[0136] First, it was explored if c-Met-IR would be localized in
reactive astrocytes or not, and if HGF would change the
pathogenesis of astrocytes and level of glutamate transporter in
reactive astrocytes. By double staining of spinal cord using
anti-GFAP and anti-c-Met antibody, c-Met-IR was found to be
localized in the remaining large neurons and GFAP positive reactive
astrocytes. Not being contradictory to immunohistochemical results,
levels of c-Met particularly increased at 8 months in G93A mice and
double transgenic mice (FIG. 21, lower panel), which suggested that
not only motoneurons but also astrocytes would be targets of HGF in
ALS at 8 months (endstage).
[0137] In wildtype or HGF single transgenic mice, astrocytes were
mainly white matter and localized near the central tubule, and very
slightly localized in the ventral horn (FIG. 19). In G93A mice,
reactive astrocytes progressively increased in the ventral horn at
6 months when motoneuron death was not observed in G93A mice (FIG.
19). By contrast, in double transgenic littermates, the number of
reactive astrocytes in the ventral horn was extremely small (FIG.
19). Quantification of reactivity intensity of GFAP-IR disclosed
small immunoresponse activities at the ventral horn of about 40%
and 60%, as compared with those of G93A mice at 6 and 8 months,
respectively (FIG. 20).
[0138] As a result of immunoblotting of EAAT2, GFAP and c-Met in
spiral cord in mice at 2, 6 and 8 months of age, induction of GFAP
was noted from the age of 6 months in both G93A mice and G93A/HGF
mice, a specific EAAT2 down-regulation was observed in G93A mice at
8 months, and a specific up-regulation of c-Met was recorded in
G93A and G93A/HGF mice at 8 months. In G93A/HGF mice, a total level
of EAAT2 was maintained at 8 months.
[0139] The level of EAAT2 in G93A mice at 8 months of age was
markedly decreased by 40%, as compared with that of wildtype or HGF
single transgenic littermates (FIG. 22). By contrast, in double
transgenic mice, as compared with wildtype or HGF single transgenic
littermates, a higher level of EAAT2 (140%) was detected in the
lumbar spinal cord (FIG. 22). To evaluate the level of EAAT2 in
individual astrocytes, the level of EAAT2 was divided by the level
of GFAP. Only 11% of EAAT2 were left over in G93A mice, while 63%
of EAAT2 was left over in double transgenic mice (FIG. 22).
Although increase of astrocytes began at 6 months, decrease of
EAAT2 in astrocytes and induction of c-Met occurred parallel only
at the endstage, which seems to be a noteworthy record.
[0140] As a result of investigation of action of HGF on primary
culture of astrocytes, it was known that HGF treatment caused to
increase the level of EAAT2 in primary culture of astrocytes in
both G93A mice and wildtype littermates (FIG. 23 and FIG. 24). It
means that prevention of decrease of EAAT2 in astrocytes directly
owes to activity of HGF in astrocytes.
[0141] It was thus disclosed that double transgenic mice would
maintain functional astrocytes at the endstage, while G93A mice
would excessively produce nonfunctional astrocytes from the
viewpoint of glutamate clearance.
Example 7
Progress suppressing effect of ALS by HGF administration and HGF
gene transduction
[0142] In G93A mice as model of ALS, from the age of 6 months when
a first sign of ALS is recognized, HGF protein preparation is
administered to a proper site such as spinal cord at appropriate
times and dose on consecutive days. In a control group, a
preparation not containing HGF protein is administered. To evaluate
the dose and number of administrations, the dose and the number of
times are changed in groups. Further, in the HGF group and control
group, progress suppressing effects of ALS are evaluated as
mentioned in Examples 3 and 4. Possibility of usefulness of HGF as
remedy for ALS will be confirmed when the ALS progress suppressing
effect will be recognized in the HGF group.
[0143] By conducting a similar experiment by using HGF gene instead
of HGF, usefulness of HGF gene as remedy for ALS will be
confirmed.
[0144] Pharmaceutical preparation 1
[0145] A solution containing 1 mg of HGF, 1 g of mannitol and 10 mg
of polysorbate 80 in 100 ml of physiological saline was aseptically
prepared, and dispensed in 1 ml vials, and was freeze-dried and
sealed, and freeze-dry preparations were obtained.
[0146] Pharmaceutical preparation 2
[0147] An aqueous solution containing 1 mg of HGF and 100 mg of
human serum albumin in 100 ml of 0.02M phosphate buffer (containing
0.15 M NaCl and 0.01% polysorbate 80, pH 7.4) was aseptically
prepared, and dispensed in 1 ml vials, and was freeze-dried and
sealed, and freeze-dry preparations were obtained.
[0148] Industrial Applicability
[0149] The invention presents a therapeutic agent (and progress
suppressant) for ALS containing HGF and/or HGF gene as active
ingredient. HGF has an effect of improving the motor function of
ALS and life span through two actions, that is, direct
neuronutrient factor activity on motoneurons, and indirect
improving action of glutamate cytotoxicity on motoneurons by
maintaining the level of glutamate transporter in astrocytes. Such
bifunctional growth factor has not bee known so far, and therefore
HGF and/or HGF gene can be used as an effective therapeutic agent
not known in the past.
* * * * *