Title of Invention

"A PHARMACEUTICAL COMPOSITION FOR TREATING GLIOBLASTOMA"

Abstract It is an object of the present invention to provide a novel therapeutic agent for glioblastoma. In accordance with the present invention, it was found that compounds having an antagonistic action against AMPA receptor are useful as therapeutic agents for glioblastoma, particularly primary glioblastoma de nova with a high malignancy level, and the object has been attained. (FIG). nil
Full Text DESCRIPTION
Technical Field
The present invention relates to a novel
pharmaceutical use of a compound having an antagonistic
action against AMPA receptor as a therapeutic agent for
glioblastoma.
Background of the Invention
Glioma is the general name of tumors originating
from ependymoblasts, which is the stem cell for glia, in
the course of the differentiation thereof into ependymal
cell, astroglia and oligodendroglia [Seikagaku Jiten
(Dictionary of Biochemistry), the third edition, Tokyo
Kagaku Dojin, Tokyo, 1998].
Glioblastoms multiforme (referred to as glioblastoma
hereinafter) highly invades the central nervous system and
is ranked at a higher malignancy level among gliomas.
Thus, glioblastoma is a typical one of malignant brain
tumors.
Additionally, the lethal rate thereof is very high
so that the onset thereof induces death in 9 to 12 months.
Even today with the progress in the various therapeutic
methods thereof, the 5-year survival from 1986 to 1990 is
8.0%, compared with the 5-year survival 20 years ago namely
the 5-year survival from 1969 to 1975, which was 11.9%.
Thus, almost no difference is observed (Neurology Progress,
43(3), 338-350, 1999). Accordingly, it is strongly desired
that an effective therapeutic method thereof should be
established.
Tumor cells of glioblastoma are the most
undifferentiated among brain tumors, so the tumor cells
have high potentials of migration and proliferation and are
highly invasive, leading to very poor prognosis.
Glioblastoma is classified into primary glioblastoma
de novo and secondary glioblastoma, depending on the
difference in the gene mechanism of the malignant
transformation of undifferentiated astrocyte or precursor
cells. Secondary glioblastoma occurs in a young generation
of 45 years old or younger. In 4 to 5 years on average,
secondary glioblastoma occurs from astrocytoma through
undifferentiated astrocytoma. Meanwhile, primary
glioblastoma de novo frequently occurs in an older
generation of the mean age of 55 years old. Generally,
primary glioblastoma de novo takes the form of fulminant
glioblastoma (referred to as de novo glioblastoma as well)
which occurs within 3 months from the state with no
clinical or pathological abnormalities [Pathology and
Genetics of the Nervous Systems. 29-39 (IARC Press, Lyon,
France, 2000)].
Glioblastoma migrates along myelinated nerve and
spreads widely in the central nerve. Therefore, surgical
treatment thereof cannot show any satisfactory therapeutic
effect (Neurol. Med. Chir. (Tokyo) 34, 91-94, 1994; Neurol.
Med. Chir. (Tokyo) 33, 425-458, 1993; Neuropathology 17,
186-188, 1997). Additionally, pharmaceutical agents with
an indication for glioblastoma are limited in Japan to for
example ranimustine and interferon. Additionally, the
efficacy thereof is insufficient.
a-amino-3-hydroxy-5-methyl-4-isoxazole propionic
acid (AMPA)-type glutamate receptors belong to ionotropic
glutamate receptors are responsible for rapid nerve
conduction of almost all excitatory synapses (Trends
Neurosci. 16, 359-365, 1993; Annu. Rev. Neurosci. 17, 31-
108, 1994; Prog. Neurobiol. 54, 581-618, 1998). AMPA
receptors are expressed in many glyacytes like those in
neurons (Trends Pharmacol. Sci. 21, 252-258, 2000). AMPA
receptors are composed of four subunits, namely GluRl
through GluR4. The Ca2+ permeability of an AMPA receptor
depends on the composition with the subunits thereof.
Specifically, an AMPA receptor with the 61uR2 subunit has
low Ca2+ permeability, while an AMPA receptor without GluR2
has high Ca2+ permeability. A higher content of the GluR2
subunit decreases the Ca2+ permeability more (Trends
Neurosci. 16, 359-365, 1993; Annu. Rev. Neurosci. 17, 31-
108, 1994; Prog. Neurobiol. 54, 581-618, 1998). Further,
the unique feature of GluR2 is described as follows. In case that one amino acid
residue in the second hydrophobk region (M2), which is originally arginine ( R),
is substituted with glutamlne (Q), the resulting homomer receptor composed of
the substituted GluR2 (Q) has high Ca2* permeability (Trends Neurosci. 16,359-
365, 1993, Annu. Rev. Neurosci. 17, 31-108, 1994). In other words, such
receptor at a higher content of the GluR2 subunft when the subunit is GkiR (Q)
type is highly permeable to Ca2+. When the subunit is GluR2 (R), however, the
resulting receptor does not shows any Ca2+ permeability.
Concerning glioma cells and glutamate, reports showed that the expression of
the GluR2 gene is low in C6-glioma (I Neurosci. Res. 46,164-178,1996) and
that 66% of glioblastoma culture cells respond to a glutamate receptor against
and depolarize (Eur. J. Neurosci. 10,2153-2162,1998).
The following reports have been issued about the inhibiting action of antagonists
against the ionotropic glutamate receptor family on glioma and the like.
1) Document WO 00/24395 discloses an invention relating to the method for
inhibiting the interaction between AMPA receptor complexes and
glutamate for cancer treatment, wherein specific examples of brain tumor
include medulbblastoma classified as a fetal tumor and human brain
astrocytoma as one glioma type. The Reference describes
the in vitro effect of an antagonist GYK152446 against AMPA
receptor on brain astrocytoma.
However, the reference does not disclose or suggests
the use of the antagonist against AMPA receptor for
treatment of glioblastoma with high malignancy to have
resistance to radiotherapy or chemotherapy.
2) Non-patent reference 1 by the inventor
described above includes a description that antagonists
against NMDA- and AMPA receptors are highly sensitive to
tumor cells derived from peripheral cells but poorly
sensitive to tumor cells derived from nerve and glyacites.
Additionally, the reference describes that these
antagonists are useful as therapeutic agents of peripheral
cancer. Thus, the effect of the invention on glioblastoma
cannot be anticipated on the basis of the in vitro effect
using the tumor cells with lower malignancy levels than
that of glioblastoma.
3) Non-patent reference 2 reports that MK-801
and memantin as antagonists against NMDA receptor exert a
proliferation inhibition in the C6 and R62 glioma
transplanted rat models.
However, it is criticized that the previous research
on glioma using such animal models are criticized since
they do not represent real growth of tumor (non-patent
Reference 3)
4) Additionally, non-patent reference 4 as one
critical review of the non-patent reference 1 suggests the
possibility of the inhibition AMPA receptors and NMDA
receptors as a multiple therapy for glioma. However, the
authors are suspicious about whether or not the animal
models using C6 and R62 glioma used in the non-patent
reference 1 can reflect human tumor.
Based on those described above, these previous
techniques do not include any specific descriptions about
the possibility that antagonists against AMPA receptor will
become a therapeutic agent of the most malignant human
glioblastoma among various types of glioma. The effect on
C6 and RG2 glioma does not suggest any possible therapeutic
effect on human glioblastoma.
Thus, the invention is not described in any these
previous techniques, and it cannot be derived readily from
them.
[Patent Reference 1]
PCT International Publication pamphlet WO 00/24395
[Non-patent Reference 1]
Proceedings of the National Academy of Sciences of
United States of America 98 (11), 6372-6377, 2001
[Non-patent Reference 2]
Nature Medicine 7(9), 1010-1015, 2001
[Non-patent Reference 3]
Nature Medicine 6(4), 369-370, 2000
[Non-patent Reference 4]
Nature Medicine 7(9), 994-995, 2001
Disclosure of the Invention
It is an objective of the invention to provide a
therapeutic agent for glioblastoma, which has a novel
action mechanism.
The inventors made investigations so as to attain
the object. The inventors found that the GluR1 and/or
61uR4 subunit was expressed widely in glioblastoma cells,
particularly human primary glioblastoma cell and functioned
as a Ca2+-permeable AMPA receptor. Specifically, the
inventors found that the transformation of biological Ca2+-
permeable AMPA receptor into Ca2+-non-permeable AMPA
receptor by transfection of the GluR2(R) gene with
adenovirus vector inhibited migration and induced the
apoptosis of glioblastoma cells. In other words, the
inhibition of Ca2+ permeability by the presence of the
subunit GluR2(R) induces the cell death of glioblastoma
cells. Further, the inventors found that in contrast,
excess expression of Ca2+-permeable AMPA receptor promoted
not only the morphological change and growth of the tumor
cell but also the migration ability.
Still further, the inventor established an animal
model highly reflecting the pathological feature of human
glioblastoma and used this model for verifying the effect
in accordance with the invention. The animal model with a
transplanted human glioblastoma cell line CGNH-89 as
established by the inventor reflects the pathological
feature of human glioblastoma, where the cell vigorously
invades the inside of brain parenchyma and the subpial
brain to induce disseminated meningitis.
Accordingly, the inventors certified that a
compound, which has high antagonistic effect against AMPA
receptor inhibits tumor growth in an animal model
reflecting human glioblastoma, based on the findings that
the AMPA receptors of glioblastoma cell were composed of
the GluRl and/or GluR4 subunit not GluR2 and the high Ca2+
permeability of the AMPA receptor was responsible for the
marked growth and migration abilities of human glioblastoma
cell. Thus, the invention has been achieved.
Therefore, the present invention relates to a
therapeutic agent for glioblastoma, which comprises a
compound having an activity of inhibiting an AMPA receptor
as the active ingredient.
Preferably, the present invention relates to a
therapeutic agent for glioblastoma, wherein the compound
having an activity of inhibiting an AMPA receptor is [7-
(lH-imidazol-1-yl)-6-nitro-2,3-dioxo-3,4-dihydroquinoxalin-
1(2H)-yl]acetic acid or a salt thereof or a hydrate
thereof.
Additionally, the present invention relates to a
therapeutic agent for glioblastoma, wherein the compound
having an activity of inhibiting an AMPA receptor is 2,3-
dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline or a
salt thereof.
Still additionally, the present invention relates to
a therapeutic agent for glioblastoma, wherein the compound
having an activity of inhibiting an AMPA receptor is 2-[N-
(4-chlorophenyl)-N-methylamino]-4H-pyrido[3,2-e]-1,3-
thiazin-4-one or a salt thereof.
Additionally, the present invention relates to a
pharmaceutical composition for a therapeutic agent for
glioblastoma, which comprises a therapeutically effective
amount of a compound having an activity of inhibiting an
AMPA receptor and a pharmaceutically acceptable carrier;
the aforementioned pharmaceutical composition
wherein the compound having an activity of inhibiting an
AMPA receptor is preferably [7-(lH-imidazol-1-yl)-6-nitro-
2,3-dioxo-3,4-dihydroquinoxalin-l(2H)-yl]acetic acid or a
salt thereof or a hydrate thereof;
the aforementioned pharmaceutical composition
wherein the compound having an activity of inhibiting an
AMPA receptor is preferably 2,3-dihydroxy-6-nitro-7-
sulfamoyl-benzo(F)-quinoxaline or a salt thereof; and
the aforementioned pharmaceutical composition
wherein the compound having an activity of inhibiting an
AMPA receptor is preferably 2-[N-(4-chlorophenyl)-N-
methylamino]-4H-pyrido[3,2-e]-1,3-thiazin-4-one or a salt
there of.
Still more, the present invention relates to the use
of a compound having an activity of inhibiting an AMPA
receptor for the manufacture of a medicament for treating
glioblastoma comprising a clinically effective amount of
the compound;
the aforementioned use thereof wherein the compound
having an activity of inhibiting an AMPA receptor is
preferably [7-(lH-imidazol-1-yl)-6-nitro-2,3-dioxo-3,4-
dihydroquinoxalin-1(2H)-yl]acetic acid or a salt thereof or
a hydrate thereof;
the aforementioned use thereof wherein the compound
having an activity of inhibiting an AMPA receptor is
preferably 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-
quinoxaline or a salt thereof; and
the aforementioned use thereof wherein the compound
having an activity of inhibiting an AMPA receptor is
preferably 2-[N-(4-chlorophenyl)-N-methylamino]-4H-
pyrido[3,2-e]-1,3-thiazin-4-one or a salt thereof or a
hydrate thereof.
Furthermore, the present invention relates to a
method for treating glioblastoma comprising administering a
therapeutically effective amount of a compound having an
activity of inhibiting an AMPA receptor to a patient with

the disease; such method wherein the compound having an activity of inhibiting
an AMPA receptor is preferably [7- (lH-imidazol-l-yl)-6-nitro-2, 3-dioxo-3, 4-
dihydroquinoxalin-1 (2H)-yl] acetic acid or a salt thereof or a hydrate thereof;
the aforementioned method wherein the compound having an activity of
inhibiting an AMPA receptor is preferably 2,3-dihydroxy-6-nitro-7-sulfamoyl-
benzo (F)-quinoxaline or a salt thereof; and
the aforementioned method wherein the compound having an activity of
inhibiting an AMPA receptor is preferably 2-[N-(4-chlorophenyl)-N-methylamino]-
4H-pyrido [3,2-e]-l, 3-thiazin-one or a salt thereof.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWING
Fig. 1 shows a graph of tumor volumes in animate administered intraperitoneally
with control (PBS) and 100 mg/kg compound A in a repeated manner for 14 days
from the next day of tumor transplantation.
Best Mode for Carrying out the Invention
The present invention is now described in details as follows.
Glioblastoma means primary glioblastoma de novo and secondary glioblastoma,
preferably primary glioblastoma de novo.
The therapeutic agent for glioblastoma means having
an effect of inhibiting growth of glioblastoma, an effect
of prolonging the survival period, or an effect of raising
the survival rate.
The effect of inhibiting growth of glioblastoma
means an effect of reducing or not increasing the volume of
glioblastoma when the compound for use in accordance with
the present invention is administered to an animal,
preferably a human patient with the onset of glioblastoma,
compared with the volume thereof when no such compound is
administered.
The effect of prolonging the survival period means
an effect of prolonging the survival period when the
compound for use in accordance with the present invention
is administered to an animal, preferably a human patient
with the onset of glioblastoma, compared with the mean
survival period when none of various therapeutic treatments
indicated for the disease or no treatment is done.
Preferably, the effect means an effect of significantly
prolonging the survival period.
The effect of raising the survival rate means an
effect of raising the survival rate during a period of one
month to 12 months, a period of one year to 5 years or a
period of 5 years or longer when the compound for use in
accordance with the present invention is administered to an
animal, preferably a human patient with the onset of

glioblastoma, compared with the survival rate when none of
various therapeutic treatments indicated for the disease or
no treatment is done. Preferably, the effect means an
effect of significantly raising the survival rate.
The compound usable in accordance with the present
invention is a compound inhibiting the AMPA-type glutamate
receptor.
The compound with an activity inhibiting the AMPA
receptor includes a compound binding to the ligand binding
site of the AMPA receptor in a competitive or non-
competitive manner to inhibit the binding between the AMPA
receptor and glutamate; or a compound not binding to the
binding site of the AMPA receptor but binding to the
allosteric regulation site of the AMPA receptor, thus,
block the glutamate neurotransmission.
Preferably, the compound is 2,3-dihydroxy-6-nitro-7-
sulfamoyl-benzo(F)-quinoxaline (NBQX), [7-(lH-imidazol-1-
yl)-6-nitro-2,3-dioxo-3,4-dihydroquinoxalin-l(2H)-yl]acetic
acid (zonanpanel) disclosed as an antagonist against AMPA
receptor in WO 96/10023, or a salt thereof, or 2-[N-(4-
chlorophenyl)-N-methylamino]-4H-pyrido[3,2-e]-1,3-thiazin-
4-one (referred to as compound B hereinbelow) or a salt
thereof.
[7-(lH-Imidazol-1-yl)-6-nitro-2,3-dioxo-3,4-
dihydroquinoxalin-1(2H)-yl]acetic acid forms a salt with an
acid or a base. Preferably, -the salt is a pharmaceutically
acceptable salt.
The salt with an acid includes acid addition salts
with inorganic acids such as mineral acids including
hydrochloric acid, hydrobromic acid, hydroiodic acid,
sulfuric acid, nitric acid, and phosphoric acid, and
organic acids such as formic acid, acetic acid, propionic
acid, oxalic acid, malonic acid, succinic acid, fumaric
acid, maleic acid, lactic acid, malic acid, citric acid,
tartaric acid, carbonic acid, picric acid, methanesulfonic
acid, ethanesulfonic acid, and glutamate. The salt with a
base includes for example salts with inorganic bases such
as sodium, potassium, magnesium, calcium and aluminium,
organic bases such as methylamine, ethylamine, and
ethanolamine, or salts with basic amino acids such as
arginine and ornithine, and ammonium salt. Furthermore,
the compound may form hydrates, solvates with ethanol and
the like, crystal polymorphism.
Preferably, the compound is [7-(lH-imidazol-1-yl)-6-
nitro-2,3-dioxo-3,4-dihydroquinoxalin-l(2H)-yl]acetic
acid*monohydrate (compound A).
2-[N-(4-Chlorophenyl)-N-methylamino]-4H-pyrido[3,2-
e]-1,3-thiazin-4-one (compound B) may form a salt with an
acid. The salt with an acid includes for example inorganic
acids such as hydrochloric acid, hydrobromic acid,
hydroiodic acid, sulfuric acid, nitric acid, and phosphoric

acid, and organic acids such as formic acid, acetic acid,
propionic acid, oxalic acid, malonic acid, succinic acid,
fumaric acid, maleic acid, lactic acid, malic acid, citric
acid, tartaric acid, carbonic acid, picric acid,
methanesulfonic acid, ethanesulfonic acid, and acidic amino
acids such as aspartate and glutamate. Preferably, the
compound is in the free form.
Furthermore, compound B or a salt thereof may
sometimes be isolated in the form of various solvates such
as hydrate and ethanol solvate or in the form of substances
in polymorphic crystal thereof. The compound B encompasses
such various hydrates, solvates and substances in
polymorphic crystal thereof.
Other than the compounds described above,
antagonists against AMPA receptor include those described
below in the table.
The compounds described above can be produced with
reference to the synthetic methods described in the
references or using general synthetic methods. Otherwise,
these compounds may be available from companies having
produced, marketing, and having developed these compounds
and the like.
The therapeutic agent of the present invention may
further be used in combination with other pharmaceutical
agents. For example, the antagonists against AMPA receptor
may be administered singly or in combination with other
antitumor drugs or other growth inhibitors or other
pharmaceutical agents or nutritious agents.
Numerous antitumor drugs have been commercially
available or under way of development so far. These can be
selected as pharmaceutical agents to be concurrently used
in combination for treating glioblastoma. Such antitumor
drugs include for example antibiotic drugs, alkylating
agents, metabolic antagonists, immunological agents and
interferon-type agents. Specifically, the antitumor drugs
are for example interferon-beta (immunoenhancer
interferon), nimustine hydrochloride (alkylating agent),
ranimustine (alkylating agent), etoposide (alkaloid),
carboplastin and cisplatin (platinum formulation) and
temozolomide (alkylating agent).
A pharmaceutical formulation containing the compound
for use in accordance with the present invention or one
type or two types or more of the salt thereof as the active
ingredients are prepared, using carriers, excipients and
other additives for general use in formulation.
The carriers and excipients for formulation may be
solid or liquid, and include for example lactose, magnesium
stearate, starch, talc, gelatin, agar, pectin, gum arabic,
olive oil, sesame oil, cacao butter, and ethylene glycol
and other routine such ones.
Administration thereof may satisfactorily be either
oral administration via tablets, pills, capsules, granules,
powders, liquids and the like or parenteral administration
via injections via intravenous injections, intramuscular
injections and the like, suppositories and transcutaneous
agents.
The dose is appropriately determined, depending on
each case, in terms of the symptom, age, sex and the like
of a subject to be dosed. Generally, the daily dose of the
compound of the present invention is about 100 to 2,000 mg
per adult per day, preferably about 900 mg per adult per
day. 100 to 2,000 mg per adult per day by dividing the
daily dose into one or two to four doses per day. In case
of intravenous administration or sustained intravenous
administration, the administration may satisfactorily be
administered for one hour to 24 hours per day.
As described above, the dose can be determined by
taking into consideration various conditions. A dose less
than the range described above may also be used in case
that the dose is effective.
The compound for use in accordance with the present
invention is mainly administered by parenteral
administration, specifically including subcutaneous
administration, intramuscular administration, intravenous
administration, transcutaneous administration, intrahecal
administration, epidural administration, intrajoint
administration and local administration, or may also be
administered in various dosage forms, for example by oral
administration if possible.
The injections for parenteral administration include
for example sterile, aqueous or non-aqueous solutions,
suspensions and emulsions. The aqueous solutions and
suspensions include for example distilled water for
injections and physiological saline. The non-aqueous
solutions and suspensions include for example propylene
glycol, polyethylene glycol, vegetable oils such as olive
oil, alcohols such as ethanol, and Polysorbate 80 (under
trade name). Such composition may contain auxiliary agents
such as preservatives, moistening agents, emulsifying
agents, dispersing agents, stabilizers (for example,
lactose) and dissolution auxiliary agents (for example,
meglumine). These are sterilized by filtering through
bacteria-retaining filters, blending sterilizing agents, or
irradiation. Alternatively, these may be produced once
into a sterile solid composition and then dissolved or
suspended in sterile water or sterile solvents for
injections, prior to use.
As the solid composition for oral administration,
tablets, powders, granules and the like are used. In such
solid composition, one or more active substances are mixed
with at least one inert excipient, for example lactose,
mannitol, glucose, hydroxypropyl cellulose,
microcrystalline cellulose, starch, polyvinyl pyrrolidone,
metasilicic acid and magnesium aluminate. According to
general methods, the composition may contain inert
additives other than diluents, lubricants for example
magnesium stearate, disintegrators for example fibrin
calcium glucorate, stabilizers such as lactose and
dissolution auxiliary agents such as glutamate or aspartic
acid. On a needed basis, the tablets or pills may
satisfactorily be coated with sugar coating or with films
of stomach-soluble or gut-soluble substances such as
sucrose, gelatin, hydroxypropyl cellulose and
hydroxypropylmethyl cellulose phthalate.
The liquid composition for oral administration
includes for example pharmaceutically acceptable emulsions,
liquids, suspensions, syrups and elixirs and contains inert
diluents for general use, for example distilled water and
ethanol. The composition may contain auxiliary agents such
as moistening agents and suspending agents, sweetening
agents, flavoring agents, aromatic agents and
preservatives, other than the inert diluents.
[Examples]
The present invention is now described in detail in
the following Examples. However, the invention should not
be limited to those in the following Examples.
The therapeutic effect of the present invention was
confirmed by the following experimental method.
Experimental Example 1
Growith inhibition of glioblastoma in vitro
Human glioblastoma cell (CGNH-89 cell line) was used
at this experiment. These cells were randomly divided in
the following three groups: a group of a glutamate-free
medium (no glutamate group; control group); a group of
the glutamate-f ree medium added 100 mM glutamate (100 mM
glutamate group); a group of the glutamate-free medium
added 100 mM glutamate and 20 mM NBQX (100 mM glutamate +
20 mM NBQX group) . The cells were incubated for 48 hours.
The CGNH-89 cell was cultured in DMEM (Dulbecco"s modified
Eagle"s medium) supplemented with 10% dialyzed fetal calf
serum.
CGNH-89 cell line
CGNH-89 cell line was established through resection
from the tumor at the right front lobe of a 56-year old
lady according to the Explant method by Nicolas et al.
(Science 196, 60-63, 1977). The cell line shows activities
of glia cell fibrous acidic protein (GFAP), vimentin, A2B5,
O4 and myelin basic protein (J. Neuropathol. Exp. Neurol.,
57653-663, 1998) . Additionally, the mRNAs of GluRl through
GluR4 were analyzed with antibodies against the AMPA
receptor subunits GluRl, GluR2, GluR3 and GluR4 and RT-PCR.
Consequently, the cell line expresses GluRl and GluR4 but
not expresses GluR2 and GluR3. As described above, the
cell line has the same profile as that of the cell of
primary glioblastoma de novo.
Furthermore, the cell line is provided by the
inventor and is available on request to? the inventor.
The anti-tumor action was evaluated by calculating
the apoptosis score by TUNEL method (terminal
deoxynucleotide transferase (TdT)-mediated dUTP nick end
labeling) 48 hours after culturing. Additionally,
proliferation of the human glioblastoma cell was evaluated,
using the antibody Ki67.
The results are shown as mean ± standard deviation
and statistically analyzed by the Student"s t-test.
Significance level was set at p Test Results
In the no glutamate group, apoptosis was induced in
18% of the human glioblastoma cells within the 48-hour
incubation period. Additionally, almost no proliferation
was observed. In contrast, the addition of 100 mM
glutamate to the culture medium decreased the apoptosis
down to 3%, while the proliferative activity increased to
18.5%. Meanwhile, NBQX as an antagonist against AMPA
receptor inhibited the action of suppressing apoptosis as
induced by glutamate, so that NBQX induced the apoptosis.
Additionally, NBQX suppressed the glutamate-induced
proliferative activity (Table 1).
TUNEL-SCORE:
The number of TUNEL positive cell meaning a cell
with DNA fragmented by apoptosis in the microscopic field
of vision divided by the number of total cells represented
as PI (propidium iodine) positive nucleus in the same field
of vision is shown as the TUNEL SCORE.
Staining index with Ki-67:
The number of Ki-67 positive cells in the
microscopic field of vision as divided by the number of
total cells represented as PI (propidium iodine) positive
nucleus in the same field of vision is shown as the
staining index.
* p ** p Based on the results, it was confirmed that the AMPA
receptor antagonist inhibits the suppression of the
apoptosis by glutamate and inhibits glutamate-induced
proliferation of glioblastoma in vitro.
Experimental Example 2
Growth inhibition on the glioblastoma (human glioblastoma)
in vivo
Nude mice (5-6 weeks old) were used at this
experiment. 107 cells of CGNH-89 used in Example 1 were
transplanted subcutaneously in the nude mice. On the next
day of transplantation, the mice were divided randomly in
two groups; a group (n = 12) to be administered with 100
mg/kg compound A and a group (n = 12) to be administered
with PBS (phosphate buffered saline) . The drug was
administered repeatedly intraperitoneally for 14 days
starting on the next day of transplantation. Up to day 8
after the completion of the administration, the mice were
observed. The tumor size was measured every two days with
a vernier micrometer, to calculate the volume according to
the formula (length x width2) x 1/2.
The results are shown in mean ± standard deviation
and statistically analyzed by the Student"s t-test. The
significance level was set at p Test results
Compound A as an antagonist against AMPA receptor
expressed a significant tumor growth inhibition on days 11
and 13 after the onset of administration and on days 2, 4,
6 and 8 after the completion of administration (Fig. 1).
Based on the results, it was confirmed that the
antagonist against AMPA receptor showed an excellent tumor
growth inhibition even in vivo on the glioblastoma cell
with the pathological feature of human glioblastoma.
Experimental Example 3
Growth inhibition on glioblastoma (human glioblastoma) in
the orthotopic xenograft model
Using an orthotopic xenograft model, in which CGNH-
89 is transplanted in the brain parenchyma of mouse or rat,
the anti-tumor effect of an antagonist against AMPA
receptor is evaluated. The dose of compound A is 50 mg/kg
or 10 mg/kg. On the next day of the transplantation of the
tumor, intraperitoneal administration of compound A
started for 14 days. On day 21, a specimen is prepared for
pathological analysis.
Example (Production of freeze-dried formulation)
33.3 g of meglumine was dissolved in 400 ml of
distilled water for injections, to which 10 g of compound A
was added for dissolution with stirring. 1,400 ml of
distilled water for injections was added to the resulting
solution. Additionally, 40 g of mannitol was dissolved in
the solution, to which distilled water for injections was
added to 2,000 ml. The resulting solution was filtered
aseptically by general method. 15 ml of the resulting
filtrate was charged in a 30-ml vial and freeze-dried by
general method, to prepare a freeze-dried formulation of
the compound for use in accordance with the present
invention.
Industrial Applicability
In accordance with the present invention, compounds
with an antagonistic action against AMPA receptor, which is
preferably zonanpanel or a salt or hydrate thereof or NBQX,
are useful as therapeutic agents of glioblastoma,
particularly primary glioblastoma de novo with a high
malignancy level.
WE CLAIM:
1. A pharmaceutical composition for the treatment of glioblastoma containing
10-500 mg of 2-[N-(4-chlorophenyl)-N-methylamino]-4H-pyrido[3,2-e]-1,3-
thiazin-4-one as an active ingredient in combination with a pharmaceutically
acceptable carrier, excipient and other additive, wherein the ratio of an active
ingredient to a pharmaceutically acceptable carrier, excipient and other
additive is 1:20 to 1:1.
2. A pharmaceutical composition for the treatment of glioblastoma containing
10-500 mg of 7-Acetyl-5-(4-aminophenyl)-8 (R ) -methyl-8, 9-dihydro-7H-1,3-
dioxolo[4,5-h][2,3]benzodiazepine (Talampanel) as an active ingredient in
combination with a pharmaceutically acceptable carrier, excipient and other
additive, wherein the ratio of an active ingredient to a pharmaceutically
acceptable carrier, excipient and other additive is 1:20 to 1:1.
3. A process for preparing a pharmaceutical composition for treating
glioblastoma which comprises mixing 2-[N-(4-chlorophenyl)-N-
methylamino]-4H-pyrido[3-2-e]-1.3-thiazin-4-one with a pharmaceutically
acceptable carrier, excipient and other additive in the ratio of 1:20 to 1:1.
4. The process for preparing a pharmaceutical composition for treating
glioblastoma which comprises mixing 7-Acetyl-5-(4-aminophenyl)-8(R)-
methyl-8,9-dihydro-7H-1,3-dioxolo[4,5-h][2,3]benzodiazepine
(Talampanel) with a pharmaceutically acceptable carrier, excipient and
other additive in the ratio of 1:20 to 1:1.
A pharmaceutical composition for the treatment of glioblastoma comprising 2-[N-
(4-chlorophenyl)-N-methylamino]-4H-pyrido[3,2-e]-1,3-thiazin-4-one or 7-Acetyl-
5-(4-aminophenyl)-8(R)-methyl-8,9-dihydro-7H-1,3:dioxolo[4,5-
h][2,3]benzodiazepine(Talampanel).

Documents:

01330-kolnp-2004-abstract.pdf

01330-kolnp-2004-claims.pdf

01330-kolnp-2004-correspondence.pdf

01330-kolnp-2004-description (complete).pdf

01330-kolnp-2004-drawings.pdf

01330-kolnp-2004-form 1.pdf

01330-kolnp-2004-form 13.pdf

01330-kolnp-2004-form 18.pdf

01330-kolnp-2004-form 2.pdf

01330-kolnp-2004-form 26.pdf

01330-kolnp-2004-form 3.pdf

01330-kolnp-2004-form 5.pdf

01330-kolnp-2004-letter patent.pdf

01330-kolnp-2004-reply first examination report.pdf


Patent Number 216859
Indian Patent Application Number 01330/KOLNP/2004
PG Journal Number 12/2008
Publication Date 21-Mar-2008
Grant Date 19-Mar-2008
Date of Filing 10-Sep-2004
Name of Patentee ASTELLAS PHARMA INC.
Applicant Address 3-11, NIHONBASHI-HONCHO 2-CHOME CHUO-KU TOKYO, JAPAN.
Inventors:
# Inventor's Name Inventor's Address
1 ISHIUCHI SHOGO 13-7, FURUICHI -CHO 1-CHOME, MAEBASHI-SHI-SHI, GUNMA, 371-0844 JPAN.
PCT International Classification Number A61K45/00
PCT International Application Number PCT/JP03/03867
PCT International Filing date 2003-03-27
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 2002-094313 2002-03-29 Japan