U.S. patent application number 10/324268 was filed with the patent office on 2003-08-07 for antiviral agents.
This patent application is currently assigned to Takara Shuzo Co., Ltd.. Invention is credited to Chono, Hideto, Kato, Ikunoshin, Koyama, Nobuto, Sagawa, Hiroaki, Takesako, Kazutoh.
Application Number | 20030149114 10/324268 |
Document ID | / |
Family ID | 27303901 |
Filed Date | 2003-08-07 |
United States Patent
Application |
20030149114 |
Kind Code |
A1 |
Sagawa, Hiroaki ; et
al. |
August 7, 2003 |
Antiviral agents
Abstract
An antiviral agent which is characterized in containing at least
one compound selected from a group consisting of
4,5-dihydroxy-2-cyclopenten-- 1-one represented by the following
formula [I] and an optically active substance and a salt thereof as
an effective component. 1
Inventors: |
Sagawa, Hiroaki; (Otsu-shi,
JP) ; Koyama, Nobuto; (Otsu-shi, JP) ; Chono,
Hideto; (Otsu-shi, JP) ; Takesako, Kazutoh;
(Otsu-shi, JP) ; Kato, Ikunoshin; (Otsu-shi,
JP) |
Correspondence
Address: |
KENNEDY COVINGTON LOBDELL & HICKMAN, LLP
100 NORTH TRYON STREET
BANK OF AMERICA CORPORATE CTR. SUITE 4200
CHARLOTTE
NC
28202
US
|
Assignee: |
Takara Shuzo Co., Ltd.
Kyoto-shi
JP
|
Family ID: |
27303901 |
Appl. No.: |
10/324268 |
Filed: |
December 19, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10324268 |
Dec 19, 2002 |
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09367185 |
Aug 9, 1999 |
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6518317 |
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09367185 |
Aug 9, 1999 |
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PCT/JP98/00816 |
Feb 26, 1998 |
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Current U.S.
Class: |
514/690 ;
424/442 |
Current CPC
Class: |
A61P 31/18 20180101;
Y10S 514/894 20130101; C07C 323/59 20130101; C07C 323/58 20130101;
A23L 33/10 20160801; A61P 31/14 20180101; A23K 20/105 20160501;
A61K 31/122 20130101; A23L 2/52 20130101; A61P 31/12 20180101 |
Class at
Publication: |
514/690 ;
424/442 |
International
Class: |
A61K 031/12; A23K
001/165 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 17, 1997 |
JP |
82376/97 |
Jul 22, 1997 |
JP |
210193/97 |
Aug 8, 1997 |
JP |
225533/97 |
Claims
What is claimed is:
1. An antiviral agent which is characterized in containing at least
one compound selected from a group consisting of
4,5-dihydroxy-2-cyclopenten-- 1-one represented by the following
formula [I] and an optically active substance and a salt thereof as
an effective component. 5
2. An antiviral agent according to claim 1 in which the virus are
human AIDS virus and hepatitis C virus.
3. An antiviral agent according to claim 1 in which the antiviral
agent is antiviral agent for human being, antiviral agent for
non-human animals or antiviral agent for plants.
4. An antiviral agent according to claim 3 in which the antiviral
agent is an antiviral agent for domestic animals, domestic fowls,
fish or shrimps.
5. An antiviral food or beverage which is characterized in
containing at least one compound selected from a group consisting
of 4,5-dihydroxy-2-cyclopenten-1-one represented by the following
formula [I] and an optically active substance and a salt thereof as
an effective component. 6
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to pharmaceuticals, foods and
beverages which are useful to pathogenic organisms due to their
antiviral action.
PRIOR ART
[0002] In the antiviral action, there will be an action of inducing
the resisting ability to virus such as inhibition of infection of
virus to cells, inhibition of multiplication of virus in the
infected cells, etc., an action of selectively killing the cells
which are infected by virus, and an action of inactivating (i.e.,
eliminating the infecting ability of) the virus itself.
[0003] There is a neutralizing antibody as a substance which has an
action of inactivating the virus itself while there is vaccine as a
means for inducing such an antibody. However, although the
induction of the antibody by administration of vaccine is effective
in preventing the infection of virus, effective therapeutic methods
using antibody are rarely available at present. In addition, there
is no effective therapeutic method in which virus is directly
inactivated by pharmaceuticals.
[0004] With regard to action of inducing a resisting ability to
virus, there will be inhibition of replication of genome of virus,
inhibition of transcription of gene of virus, inhibition of
synthesis of protein of virus, inhibition of folding of protein of
virus, etc. In inducing such actions, there will be suppression of
activity or expression of transcription factor in the cell,
suppression of activity or expression of transcription factor
derived from virus, induction of heat shock proteins, etc. An
example of the substances which are capable of inducing such an
action is prostaglandin.
[0005] Examples of the agents which selectively kill the cells
which are infected by virus are acyclovir, ganciclovir, sorivudine,
etc. which have been used as drugs against herpes virus.
PROBLEMS TO BE SOLVED BY THE INVENTION
[0006] Against virus, it is more effective to cope with by means of
a synergistic action than by means of a single action. For example,
even when a substance which selectively kills the virus-infected
cells is administered, it is very difficult to completely eliminate
the virus because, until the infected cells are killed, new virus
is generated and other cells are infected by that. On the other
hand, even when a substance which has an action of inducing a
resisting ability to virus is administered, it is not possible to
eliminate the infected cells.
[0007] An object of the present invention is to develop the
compounds which have a function of inducing a resistance to virus
into cells and a function of selectively killing the virus-infected
cells and to offer pharmaceuticals such as antiviral agent, agent
for improving the hepatic functions, agents for inducing heat shock
proteins, agents for preventing the carcinogenesis by oncogene,
agents for preventing the chemical carcinogenesis, etc. and
antiviral foods or beverages wherein the above-mentioned compound
is contained.
MEANS TO SOLVE THE PROBLEM
[0008] The present inventors have conducted an intensive study for
achieving such an object and have found that, when cells are
treated with a compound which has a function of inducing a
resistance to virus and of selectively killing the cells infected
by virus, said virus-infected cells are selectively damaged and,
therefore, amount of the virus which is generated until death of
said cells decreases and that, since the cells which are not yet
infected by virus acquire a resistance to virus due to
administration of the compound, growth of the virus is suppressed
even if it is newly infected. In other words, it has been found
that the compound which has a function of inducing a resistance to
virus into cells and of selectively killing the cells infected by
virus is extremely effective for elimination of virus such as human
AIDS virus or hepatitis C virus.
[0009] The function of the compound used in the present invention
for inducing a resistance to virus into the cells can be measured
by treating the compound to the cells prior to infection of virus
followed by using inhibition of infection of virus to the cells,
inhibition of replication of genome of virus, inhibition of
transcription of gene of virus, inhibition of synthesis of protein
of virus, inhibition of folding of protein of virus, etc. as the
indexes or the yardsticks.
[0010] Further, the function of selectively killing the cells which
are infected by virus can be measured by comparing the survival
rate of the virus-infected cells with that of the uninfected
cells.
[0011] There is absolutely no limitation for the compound having a
function of inducing a resistance to virus into cells and also of
selectively killing the cells infected by virus so far as said
compound has both of those functions.
[0012] Now the present inventors have found
4,5-dihydroxy-2-cyclopenten-1-- one represented by the formula [I]
(hereinafter, just referred to as "the cyclopentenone") or an
optically active compound or a salt thereof as the compound having
a function of inducing a resistance to virus into cells and also of
selectively killing the cells infected by virus whereupon the
present invention has been achieved.
[0013] Outline of the present invention is that the first feature
of the present invention relates to an antiviral agent which is
characterized in containing at least one compound selected from a
group consisting of 4,5-dihydroxy-2-cyclopenten-1-one represented
by the following formula [I] and an optically active substance and
a salt thereof as an effective component. 2
[0014] The second feature of the present invention relates to
antiviral food or antiviral beverage which is characterized in
containing at least one compound selected from a group consisting
of 4,5-dihydroxy-2-cyclopen- ten-1-one represented by the formula
[I] and an optically active substance and a salt thereof as an
effective component.
[0015] In a preferred embodiment of the present invention, examples
of the virus are human AIDS virus and hepatitis C virus. Examples
of the antiviral agent are antiviral agent for human being,
antiviral agent for non-human animals (such as antiviral agent for
domestic animals, domestic fowls, fish or shrimps) and antiviral
agent for plants.
BRIEF EXPLANATION OF THE DRAWINGS
[0016] FIG. 1 is a graph showing the relation between the
concentration of the cyclopentenone and the survival rate when
CEM-SS cells are used;
[0017] FIG. 2 is a graph showing the relation between the
concentration of the cyclopentenone and the survival rate when H9
cells are used;
[0018] FIG. 3 is a graph showing the relation between the
concentration of the cyclopentenone and the survival rate when
CEM-3B cells are used;
[0019] FIG. 4 is a graph showing the relation between the
concentration of the cyclopentenone and the survival rate when
H9-3B cells are used;
[0020] FIG. 5 is a graph showing an inhibiting action of the
cyclopentenone against carcinogenicity.
[0021] FIG. 6 shows a CD of p-dimethylaminobenzoyl derivative of
(-)-cyclopentenone and a stereostructure of (-)-cyclopentenone.
[0022] FIG. 7 shows a CD of p-dimethylaminobenzoyl derivative of
(+)-cyclopentenone and a stereostructure of (+)-cyclopentenone.
EMBODIMENTS OF THE INVENTION
[0023] The cyclopentenone represented by the formula [I] used in
the present invention covers both isomers where the configurations
of hydroxyl groups at 4- and 5-positions are cis and trans. In the
present invention, any of cis-cyclopentenone, trans-cyclopentenone
and a mixture of cis- and trans-cyclopentenone may be used. It is
also possible to use optically active substances thereof.
[0024] cis-Cyclopentenone may be prepared by a chemical synthesis
[Helvetica Chimica Acta, volume 55, pages 2838-2844 (1972)].
trans-Cyclopentenone may be prepared either by a chemical synthesis
[Carbohydrate Res., volume 247, pages 217-222 (1993)] or by heating
uronic acid such as glucuronic acid, uronic acid derivative such as
glucuronolactone or a substance containing the same (refer to
PCT/JP97/03052). In the present invention, it is also possible to
use such a heated product or partially purified product or purified
product thereof.
[0025] For example, when D-glucuronic acid is used as a uronic acid
and its 1% solution is heated at 121.degree. C. for four hours, the
cyclopentenone is produced in the heat-treated substance. The
cyclopentenone in this heat-treated substance is extracted with a
solvent and the extract is concentrated. Then, this concentrated
extract is separated by means of a silica gel column
chromatography, the eluted cyclopentenone fraction is concentrated,
the cyclopentenone is extracted with chloroform from the
concentrate and the extract of the concentrate is subjected to a
normal phase column chromatography whereupon the cyclopentenone in
the heat-treated substance is isolated.
[0026] Physical property of the cyclopentenone will be given as
hereunder. Incidentally, a mass spectrometric analysis of the
cyclopentenone was conducted using a mass spectrometer DX302
(manufactured by Nippon Denshi). Further, measurement of an NMR
using heavy chloroform as a solvent was conducted by JNM-A 500
(manufactured by Nippon Denshi). Specific rotation was measured by
a DIP-370 polarimeter (manufactured by Nippon Bunko); ultraviolet
absorption spectrum was measured by a UV-2500 spectrophotometer
(manufactured by Shimadzu); and infrared absorption spectrum (IR)
was measured by an FTIR-8000 infrared spectrophotometer
(manufactured by Shimadzu).
[0027] MS m/z 115 [M+H].sup.+
[0028] .sup.1H--NMR (CDCl.sub.3): .delta.4.20 (1H, d, J=2.4 Hz,
5-H), 4.83 (1H,m, 4-H), 6.30 (1H, dd, J=1.2, 6.1 Hz, 2-H), 7.48
(1H, dd, J=2.1, 6.1 Hz, 3-H).
[0029] Incidentally, the chemical shift value of the .sup.1H-NMR
was given on a basis that the chemical shift value of CHCl.sub.3
was 7.26 ppm.
[0030] Optical rotation: [.alpha.].sub.D.sup.20 0.degree. (c 1.3,
water)
[0031] UV: .lambda..sub.max 215 nm (water)
[0032] IR (KBr method): absorptions were noted at 3400, 1715, 1630,
1115, 1060, 1025 cm.sup.-1.
[0033] When the isolated cyclopentenone is subjected to an optical
resolution, (-)-4,5-dihydroxy-2-cyclopenten-1-one, and
(+)-4,5-dihydroxy-2-cyclopenten-1-one are obtained. It goes without
saying that the cyclopentenone obtained by a synthetic method can
be subjected to an optical resolution as well.
[0034] For example, the cyclopentenone is dissolved in ethanol. To
this ethanolic solution is further added hexane/ethanol (94/6) to
prepare a cyclopentenone solution. The cyclopentenone can be
optically resolved when this sample solution is subjected to an
HPLC using, for example, a Chiral Pack AS (manufactured by Daicel
Chemical Industries) under such a condition that the column
temperature was 40.degree. C. and the mobile phase was
hexane/ethanol (94/6).
[0035] Optical rotation of the optically resolved
(-)-trans-4,5-dihydroxy-- 2-cyclopenten-1-one [hereinafter,
referred to as (-)-cyclopentenone] is [.alpha.].sub.D.sup.20
-105.degree. (c 0.30, ethanol) while that of the optically resolved
(+)-trans-4,5-dihydroxy-2-cyclopenten-1-one [hereinafter, referred
to as (+)-cyclopentenone] is [.alpha.].sub.D.sup.20 +104.degree. (c
0.53, ethanol). Incidentally, the optical rotation was measured by
the above-mentioned polarimeter of the type DIP-370 (manufactured
by Nippon Bunko).
[0036] After that, each of (-)-cyclopentenone and
(+)-cyclopentenone was subjected to structural analysis by means of
mass analysis and nuclear magnetic resonance (NMR), measurement of
UV absorption spectrum and measurement of infrared absorption
spectrum by the method mentioned already. As a result, both
optically active substances showed the same result as that of the
cyclopentenone before the optical resolution.
[0037] Each of the optically resolved (-)-cyclopentenone and
(+)-cyclopentenone was converted to a p-dimethylaminobenzoyl
derivative, the circular dichroism spectrum (CD) was measured using
a circular dichroism dispersimeter of type J-720 (manufactured by
Nippon Bunko) and the result was applied to a dibenzoate chirality
rule [J. Am. Chem. Soc., volume 91, pages 3989-3991 (1969)] to
determine the configuration.
[0038] CD of p-dimethylaminobenzoyl derivative of
(-)-cyclopentanone and stereostructure of (-)-cyclopentenone are
shown in FIG. 6. In the drawing, the ordinate indicates molar
circular dichroism while the abscissa indicates wave length (nm).
Incidentally, the above stereostructure is given hereunder as the
formula [II] 3
[0039] CD of p-dimethylaminobenzoyl derivative of
(+)-cyclopentanone and stereostructure of (+)-cyclopentenone are
shown in FIG. 7. In the drawing, the ordinate indicates molar
circular dichroism while-the abscissa indicates wave length (nm).
Incidentally, the above stereostructure is given hereunder as the
formula [III] 4
[0040] As shown in FIG. 6, FIG. 7, formula [II] and formula [III],
the (-) -cyclopentenone is (-)-(4R,5S)
-trans-4,5-dihydroxy-2-cyclopenten-1-one while the
(+)-cyclopentenone is (+)-(4S,5R)-trans-4,5-dihydroxy-2-cyclope-
nten-1-one.
[0041] The above-mentioned cyclopentenones or an optically active
substance thereof may be manufactured by any method, i.e. they may
be manufactured by a method disclosed in this specification or by
means of chemical synthesis; and trans- and cis-cyclopentenone or a
mixture thereof may be used in the present invention as well.
Naturally, an optically active substance of cyclopentenones
obtained by a chemical synthetic method is also included in an
optically active substance disclosed in the present invention.
[0042] Examples of the salt of the cyclopentenone or optically
active substance thereof are pharmaceutically acceptable salts and
they may be prepared by known converting methods.
[0043] The compound which is used in the present invention having a
function of inducing a resistance to virus into cells and also
having a function of selectively killing the cells infected by
virus such as, for example, the cyclopentenone, an optically active
substance or a salt thereof has an antiviral action and it is now
possible to prepare an antiviral agent using at least one compound
selected from the above as an effective component.
[0044] That is, the compound having a function of inducing a
resistance to virus into cells and also having a function of
selectively killing the cells infected by virus is used as an
effective component and is made into a pharmaceutical preparation
by compounding with known pharmaceutical carriers, it is now
possible to prepare an antiviral agent. Generally, at least one of
the compound selected from the compound having a function of
inducing a resistance to virus into cells and also having a
function of selectively killing the cells infected by virus such
as, for example, the cyclopentenone, an optically active substance
or a salt thereof is compounded with a pharmaceutically acceptable
liquid or solid carrier and, if necessary, solvent, dispersing
agent, emulsifier, buffer, stabilizer, filler, binder,
disintegrating agent, lubricant, etc. are added thereto to give an
antiviral agent which may be in solid such as tablets, granules,
diluted powders, powders, capsules, etc. or in liquid such as
solutions, suspensions, emulsions, etc. Further, this may be in a
dry preparation which can he made into liquid by adding an
appropriate carrier before use.
[0045] The pharmaceutical carrier may be selected depending upon
the above-mentioned mode of the administration and form of the
preparation. In the case of oral preparations, starch, lactose,
sugar, mannitol, carboxymethyl cellulose, corn starch, inorganic
salts, etc. may be used. In the manufacture of oral preparations,
binders, disintegrating agents, surface-active agents, lubricants,
fluidity promoters, taste-correctives, coloring agents, flavors,
etc. may be further compounded therewith.
[0046] On the other hand, in the case of parenteral preparations,
they may be prepared by common methods where at least one of the
compound selected from the compound having a function of inducing a
resistance to virus into cells and also having a function of
selectively killing the cells infected by virus such as, for
example, the cyclopentenone, an optically active substance or a
salt thereof which is an effective component of the present
invention is dissolved or suspended in a diluent such as distilled
water for injection, physiological saline solution, aqueous
solution of glucose, vegetable oil for injection, sesame oil,
peanut oil, soybean oil, corn oil, propylene glycol, polyethylene
glycol, etc. followed, if necessary, by adding bactericides,
stabilizers, isotonic agents, analgesics, etc. thereto.
[0047] The antiviral agent of the present invention is administered
by an appropriate route depending upon the form of the preparation.
There is no particular limitation for the method of administration
as well and it may be administered by means of oral use, external
use and injection. Injection preparations are administered, for
example, intravenously, intramuscularly, subcutaneously,
intracutaneously, etc. while preparations for external use include
suppositories, etc.
[0048] The dose as the antiviral agent is not particularly
specified but may be appropriately determined depending upon the
dosage form, administration method, purpose of the use and age,
body weight, conditions, etc. of the patient. Usually, however, the
amount of at least one of the compound selected from the compound
having a function of inducing a resistance to virus into cells and
also having a function of selectively killing the cells infected by
virus such as, for example, the cyclopentenone, an optically active
substance or a salt thereof contained in the preparation for an
adult is 0.1 .mu.g-20 mg/kg per day. As a matter of course, the
dose may vary depending upon various factors and, therefore, the
dose less than the above-mentioned one may be sufficient in some
cases while, in other cases, the dose more than the above may be
necessary. The agent of the present invention may be administered
orally as it is and, further, the agent may be taken daily after
adding to common food and/or beverage as well. Furthermore, the
compound having a function of inducing a resistance to virus into
cells and also having a function of selectively killing the cells
infected by virus such as, for example, the cyclopentenone, an
optically active substance or a salt thereof may be used as a
material for the antiviral agent, antiviral food or beverage.
[0049] The compound having a function of inducing a resistance to
virus into cells and also having a function of selectively killing
the cells infected by virus such as, for example, the
cyclopentenone, an optically active substance or a salt thereof
used the present invention has antiviral activity against DNA
virus, RNA virus, retrovirus and viroid.
[0050] Accordingly, it may be used as antiviral agent for human
being, antiviral agent for non-human animals such as that effective
to viral diseases (e.g. for domestic animals, domestic fowls and
cultured animals such as fish and shrimp), antiviral agent for
plants such as that for viral diseases of agricultural and
horticultural products (e.g. flowers and vegetables) and antiviral
agent for useful animate things.
[0051] Examples of DNA virus infecting the animals are pox virus,
herpes virus, adenovirus, hepatitis B virus, papilloma virus,
polyoma virus, Epstein-Barr virus and baculovirus while an example
of DNA virus infecting the plants is cauliflower mosaic virus.
Examples of RNA virus infecting the animals are rotavirus, rubella
virus, Japanese encephalitis virus, dengue virus, Newcastle disease
virus, measles virus, mumpus virus, distemper virus, influenza
virus, vesicular stomatitis virus, human poliomyelitis virus,
hepatitis A virus and hepatitis C virus while examples of RNA virus
infecting the plants are tobacco mosaic virus, wheat dwarf virus,
rice stripe virus and tobacco ringspot virus. Examples of
retrovirus are adult T cell leukemia virus and human acquired
immunodeficiency syndrome virus and an example of virois is potato
spindle tuber viroid.
[0052] The cyclopentenone, an optically active substance thereof or
a salt thereof is effective for therapy and prevention of viral
diseases of non-human mammals and birds such as chicken and turkey
and cold-blooded animals such as fish and such a compound has an
antiviral activity to the following non-human viruses. They are
sciruid herpesvirus of type 1, cavlid herpesvirus of type 1,
lagomorph herpesvirus of type 1, phasianid herpesvirus of type 1,
phasianid herpesvirus of type 2, turkey herpesvirus of type 1,
anatid herpesvirus of type 1, catfish herpesvirus of type 1, equid
herpesvirus of type 3, bovid herpesvirus of type 1, bovid
herpesvirus of type 3, bovid herpesvirus of type 4, porcine
herpesvirus of type 1, porcine herpesvirus of type 2, murid
herpesvirus of type 1, cebid herpesvirus of type 1, cebid
herpesvirus of type 2, tupalid herpesvirus of type 1, canine
herpesvirus of type 1, feline herpesvirus of type 1, equid
herpesvirus of type 1 and equid herpesvirus of type 2.
[0053] Viral diseases of birds such as Marek disease can be
prevented and/or cured by the compound used in the present
invention by the method known in veterinary or breeding such as
that the antiviral agent of the present invention is injected to
birds or added to feed or drinking water. Further, when the
compound used in the present invention is directly added to pool,
water tank, holding tank, or water, seawater, etc. in a breeding
area or is mixed with the feed, the following diseases can be
similarly prevented and/or cured. They are viral diseases of fish
living in a narrow sector such as pool, water tank, holding tank or
breeding area infected with herpesvirus such as petite catfish
virus, herpesvirus solomons and nerka virus and their examples are
infectious necrotizing disease of hematopoietic organs, infectious
diseases of herpesvirus or infectious necrotizing disease of
pancreas of fish of salmon family, viral hemorrhagic septicemia of
rainbow trout, spring viremia of carps, lymphocystis of various
fish, viral necrotizing disease of erythrocytes of sea fish and
anadromous fish, rhabdoviral disease of flatfish and the like,
viral necrotizing disease of pancreas and liver of fry of
yellowtail and the like, snout ulcer of torafugu (a kind of
glovefish), etc. Incidentally, the precise regulation in
administering the compound used in the present invention and the
antiviral agent of the present invention is naturally dependent
upon the necessity for each animals to be treated, type of the
treatment and judgement of the breeder.
[0054] The non-human animals to which the antiviral agent of the
present invention is administered are able to maintain their health
whereby the improvement in survival rate, growing rate, spawning
rate, etc. is significant.
[0055] The compound having a function of inducing a resistance to
virus into cells and also having a function of selectively killing
the cells infected by virus such as, for example, the
cyclopentenone, an optically active substance or a salt thereof
used the present invention inhibits the synthesis of those viral
proteins and inhibits the synthesis of virus genome as well and,
accordingly, it exhibits a powerful antiviral action. In addition,
it selectively kills the cells infected by those viruses.
[0056] For example, even in the patients suffering from human
immunodeficiency virus (hereinafter, abbreviated as HIV), all of
the CD4-positive cells are not infected by HIV but only a part of
them are infected by it. The antiviral agent of the present
invention inhibits the production of HIV in those infected cells,
at the same time, selectively kills the infected cells, and induces
the resisting ability to virus to the uninfected cells whereby it
is possible to remove the HIV from the cells.
[0057] The cyclopentenone, an optically active substance or a salt
thereof has an ability of improving the hepatic function and an
induction activity of the heat shock protein besides the
above-mentioned antiviral activity. An agent for improving the
hepatic function and an agent for inducing the heat shock protein
containing at least one compound selected from the cyclopentenone,
an optically active substance or a salt thereof can be made into a
pharmaceutical preparation by the same manner as in the case of the
above-mentioned antiviral agent and can be administered by the same
manner as in the case of the antiviral agent.
[0058] The dose as the agent for improving the hepatic function and
for inducing the heat shock protein is not particularly specified
but may be appropriately determined depending upon the dosage form,
administration method, purpose of the use and age, body weight,
conditions, etc. of the patient. Usually, however, the amount of at
least one of the compound selected from the cyclopentenone, an
optically active substance or a salt thereof contained in the
preparation for an adult is 0.1 .mu.g-20 mg/kg per day. As a matter
of course, the dose may vary depending upon various factors and,
therefore, the dose less than the above-mentioned one may be
sufficient in some cases while, in other cases, the dose more than
the above may be necessary. The agent of the present invention may
be administered orally as it is and, further, the agent maybe taken
daily after adding to common food and/or beverage as well. Further,
at least one of the compound selected from the cyclopentenone, an
optically active substance or a salt thereof may be used as a
material for the food or beverage for improving the hepatic
function or for inducing the heat shock protein.
[0059] When the cyclopentenone, an optically active substance or a
salt thereof is taken, disorder in hepatic function is improved and
GOT and GPT values become normal.
[0060] Moreover, the cyclopentenone, an optically active substance
or a salt thereof has an induction activity of heat shock protein
such as heat shook protein 70 kDa (HSP70), etc. and has an
antiviral activity to RNA virus and DNA virus such as hepatitis
virus, AIDS virus, influenza virus, vesicular stomatitis virus and
herpesvirus. Heat shock protein participates in cancer immunity and
has biodefense activity. When the cyclopentenone, an optically
active substance or a salt thereof is taken, viral diseases such as
cold by influenza can be prevented and cured.
[0061] Incidentally, heat shock protein is a general name for the
protein whose synthesis is induced when cell or individual is
subjected to a sudden temperature change which is higher than
normal temperature to an extent of around 5-10.degree. C. and it
widely exists in prokaryotes and high eukaryotes. Examples of known
heat shock protein are HSP90, HSP70, ubiquitin and HSP26. Among
them, HSP70 is a kind of molecular chaperone and is bonded to
protein where folding is not completed or is incompletely done to
assist the formation of stereostructure. Amino acid sequence of the
heat shock protein has been well conserved durina the course of
evolution and HSP70 is identical with DnaK protein of Escherichia
coli. In human being, there are about ten HSP70 genes and some of
them are expressed constitutionally while other are induced by
various stimulations. Besides the heat shock, synthesis of heat
shock protein is induced by various chemical substances and by
cellular damage such as oxidation.
[0062] C. Amici, et al. reported [Journal of Virology, volume 68,
pages 6890-6899 (1994)] that, when animal cells infected with
Sendai virus are incubated in the presence of prostaglandin A.sub.1
having .alpha., .beta.-unsaturated carbonyl group, synthesis of
HSP70 and HSP90 is induced and that, during the synthesis of HSP70
is induced, synthesis of virus protein is inhibited. Further, A.
Rossi, et al. reported [The Journal of Biological Chemistry, volume
271, pages 32192-32196 (1996)] that, like in the case of
prostaglandin A.sub.1, 2-cyclopenten-1-one induces the synthesis of
HSP70 and inhibits the synthesis of vesicular stomatitis virus
protein.
[0063] An ability of the cyclopentenone used in the present
invention for inducing HSP70 is noted at 10 .mu.M and becomes
maximum at 20-30 .mu.M and this can be said to be a very high
inducing ability as compared with the fact that a concentration of
several hundred .mu.M is required for 2-cyclopenten-1-one for
inducing the HSP70. This ability is equivalent to the ability
inducing the HSP70 by prostaglandin A.sub.1 and, since the
molecular weight of the cyclopentenone is not more than one-third
of that of prostaglandin A.sub.1, the cyclopentenone has a higher
inducing ability than prostaglandin A.sub.1 when compared in terms
of concentration by weight.
[0064] Since the cyclopentenone, an optically active substance
thereof or a salt thereof used in the present invention has such a
high inducing ability to heat shock protein, it has antiviral
activity to DNA virus, RNA virus, retrovirus and viroid. Examples
of such virus and viroid are those which were mentioned
hereinabove.
[0065] In addition, the cyclopentenone, an optically active
substance thereof or a salt thereof has an inhibition activity of
the growth of cancer cells which are transformed by cancer gene and
has an activity of preventing the carcinogenesis due to cancer
gene.
[0066] For example, papilloma virus is a DNA virus belonging to
family Papovaviridae and genus Papillomavirus and, with respect to
human papilloma virus (HPV), HPV of type 16 which is a cause of
cervical cancer has been known for example.
[0067] The cyclopentenone, an optically active substance thereof or
a salt thereof has an inhibition activity to the growth of cells
which are cancerated by cancer gene E7 of an HPV16 type. Thus, an
inhibiting agent to the growth of cancer cells which are cancerated
by virus can be offered by the use of at least one compound
selected from the cyclopentenone, an optically active substance
thereof or a salt thereof as an effective component whereby
canceration by cancer gene can be prevented.
[0068] Incidentally, the cyclopentenone, an optically active
substance thereof or a salt thereof has an inhibition activity to
carcinogenesis in two steps as an initiator and a promoter and it
is now possible to offer an inhibiting agent to chemical
canceration containing at least one compound selected from the
cyclopentenone, an optically active substance thereof or a salt
thereof as an effective component.
[0069] Accordingly, it is possible to offer food or beverage for
prevention of carcinogenesis containing at lest one compound
selected from the cyclopentenone, an optically active substance
thereof or a salt thereof.
[0070] An agent for preventing the carcinogenesis by oncogene or an
agent for suppressing the chemical carcinogenesis containing at
least one compound selected from the cyclopentenone and an
optically active substance and a salt thereof can be made into a
preparation and administered by a method similar to the antiviral
agent.
[0071] In the manufacture of the antiviral food or antiviral
beverage of the present invention, it is possible to use a compound
which has a function of inducing a resistance to virus into the
cells and also a function of selectively killing the virus-infected
cells such as the cyclopentenone, an optically active substance or
a salt thereof. It is also possible to use a heat-treated product
of uronic acid containing the cyclopentenone or a partially
purified or a purified cyclopentenone obtained from said
heat-treated substance.
[0072] Further, in the manufacture of action-expressing food or
action-expressing beverage having an action of improving the
hepatic function, inducing the heat shock proteins, preventing the
carcinogenesis, etc., it is also possible to use the
cyclopentenone, an optically active substance or a salt thereof or
a heat-treated product of uronic acid containing the cyclopentenone
or a partially purified or a purified cyclopentenone obtained from
said heat-treated substance.
[0073] Thus, food or beverage which manufactured by diluting and/or
adding the cyclopentenone, an optically active substance thereof or
a salt thereof, or a material selected from the
cyclopentenone-containing heat treated product, partially purified
cyclopentenone and purified cyclopentenone from the heat treated
product is covered by the antiviral food or beverage of the present
invention.
[0074] There is no particular limitation for the method of
manufacturing the antiviral food or beverage of the present
invention but cooking, processing and commonly-used manufacturing
methods for food or beverage may be applied provided that an
effective amount of at least one compound selected from the
compound having a function of inducing a resistance to virus into
cells and also having a function of selectively killing the cells
infected by virus such as, for example, the cyclopentenone, an
optically active substance or a salt thereof is contained in the
resulting food or beverage.
[0075] There is no particular limitation for the shape of the
antiviral food or beverage of the present invention so far as one
compound selected from the compound having a function of inducing a
resistance to virus into cells and also having a function of
selectively killing the cells infected by virus such as, for
example, the cyclopentenone, an optically active substance or a
salt thereof is contained therein, added thereto and/or diluted
therein. Thus, the shape includes the ones which can be orally
taken such as tablets, granules, capsules, gel and sol.
[0076] There is no particular limitation for the shape of the food
or beverage having an action of improving the hepatic function,
inducing the heat shock proteins, preventing the carcinogenesis so
far as one compound selected from the cyclopentenone, an optically
active substance or a salt thereof having an action of improving
the hepatic function, inducing the heat shock proteins, preventing
the carcinogenesis is contained therein, added thereto and/or
diluted therein. Thus, the shape includes the ones which can be
orally taken such as tablets, granules, capsules, gel and sol.
[0077] The food or beverage of the present invention contains the
cyclopentenone, an optically active substance or a salt thereof
having the physiological activities and, due to various
physiological activities of said compound such as antiviral
activity, activity of improving the hepatic function, inducing the
heat shock proteins, preventing the carcinogenesis, etc., it is a
healthy food or beverage having viral diseases-preventing and
-treating effects, hepatic function improving effect,
carcinogenesis-preventing effect, etc. and, further, it is food or
beverage which is useful for maintaining the homeostasis of living
body.
[0078] No toxicity was observed in the compound used in the present
invention even when the dose which is effective to achieve those
physiological activities is administered. In the case of oral
administration for example, no dead case was observed in rats by a
single oral administration of 100 mg/kg of any of the
cyclopentenone, an optically active substance or a salt
thereof.
[0079] To sum up, the pharmaceutical agent of the present invention
can be used as a therapeutic or a preventive agent for viral
diseases, hepatic diseases, cancer, etc. and is particularly useful
for the therapy of AIDS induced by HIV and for improvement of said
syndrome.
EXAMPLES
[0080] The present invention will be further illustrated by way of
the following examples although the present invention is never
limited to those examples. Incidentally, "%" used in the examples
stands for "% by weight".
Referential Example 1
[0081] D-Glucuroic acid (G 5269; manufactured by Sigma) (10 g) was
dissolved in 1 liter of water, heated at 121.degree. C. for four
hours and concentrated in vacuo until about 10 ml. This was mixed
with 40 ml of an upper layer of a 3:2:2 mixture of butyl acetate,
acetic acid and water and centrifuged and the resulting supernatant
liquid was concentrated in vacuo until about 10 ml.
[0082] The above extract was applied to silica gel (BW-300SP;
2.times.28 cm; manufactured by Fuji Silycia) for a column
chromatography and separated using an upper layer of a 3:2:2
mixture of butyl acetate, acetic acid and water as an eluate at the
flow rate of about 5 ml/minute under a pressure of 0.2
kg/cm.sup.2using a compressor. Fractionation was conducted to make
a volume of one fraction 10 ml and a part of each fraction was
analyzed by a thin layer chromatography whereupon cyclopentenone of
a high purity was contained in 61st to 80th fractions. Those
fractions were collected, concentrated in vacuo, extracted with 40
ml of chloroform and the extract was concentrated in vacuo to
afford 100 mg of cyclopentenone.
[0083] The fraction was separated by means of a normal phase HPLC
using a Palpack type S column (manufactured by Takara Shuzo) and,
when a detection was conducted by an ultraviolet absorption of 215
nm, the purity was found to be 98%.
[0084] The above cyclopentenone (113.9 mg) was dissolved in 2.85 ml
of ethanol. To this ethanolic solution was added 3.85 ml of
hexane/ethanol (94/6) to prepare a cyclopentenone solution (17
mg/ml). This solution was filtered through a filter of 0.5 .mu.m to
prepare a sample solution for an optical resolution HPLC.
[0085] This sample solution was applied to an optical resolution
HPLC, each of the fractions of the (-)-cyclopentenone in the
earlier peak and the (+)-cyclopentenone in the later peak was
collected and evaporated to dryness in vacuo to give 43.2 mg of the
(-)-cyclopentenone and 43.0 mg of the (+)-cyclopentenone.
[0086] Conditions for Optical Resolution HPLC.
[0087] Columns: Chiral Pack AS (manufactured by Daicel) 2.0
cm.times.25.0 cm
[0088] Column temperature: 40.degree. C.
[0089] Mobile phase: hexane/ethanol (94/6)
[0090] Flow rate: 14.0 ml/minute
[0091] Detection: UV 210 nm
[0092] Amount of the charged sample: 150 .mu.l (2.55 mg)
[0093] Each of the (-)-cyclopentenone and (+)-cyclopentenone
obtained herein contains about 1% of enantiomer and, therefore,
they were subjected to an optical resolution under the
above-mentioned conditions again. As a result, 19.7 mg of the
(-)-cyclopentenone containing no enantiomer was obtained from 30.0
mg of the (-)-cyclopentenone of the earlier peak while, from 37.4
mg of the (+)-cyclopentenone of the later peak, 27.7 mg of the
(+)-cyclopentenone containing no enantiomer was obtained.
Incidentally, the eluting times in optical resolution HPLC of the
(-)-cyclopentenone and (+)-cyclopentenone were 33 minutes and 40
minutes, respectively.
Example 1
[0094] (1) An RPMI 1640 medium (5 ml) containing 10% of fetal
bovine serum which contained 2.times.10.sup.5 cells/ml of human
promyelocytic leukemia cells HL-60 (ATCC CCL-240) was placed in
each well of a six-well plate, incubated at 37.degree. C. for 24
hours in the presence of 5% of CO.sub.2, then the cyclopentenone
described in the referential example 1 was added thereto to make
its final concentration 0, 10, 20, 30, 40, 50, or 100 .mu.M and the
incubation was further continued for eight hours more.
[0095] After completion of the incubation, cell numbers were
counted and the cells were recovered by centrifugation and washed
with phosphate-buffered saline (PBS) to prepare
cyclopentenone-treated cells. In the meanwhile, cells which were
heated at 45.degree. C. for ten minutes followed by subjecting to
the same incubation were prepared as well.
[0096] The cells treated as such were subjected to an
SDS-polyacrylamid gel electrophoresis (SDS-PAGE) by a method
mentioned in "Molecular Cloning" [Cold Spring Harbor Laboratory
Press,(1989)]. The treated cells were suspended in an SDS-PAGE
sample buffer to make the concentration 2.5.times.10.sup.6
cells/ml, the resulting cell suspension was treated at 100.degree.
C. for ten minutes and each 5 .mu.l thereof was applied to two
sheets of SDS-PAGE gels (5% stacking gel; 10% separation gel) to
conduct an electrophoresis. One of the gels was Coomassie stained
while another gel was subjected to a blotting to a polyvinylidene
difluoride transfer membrane (Immobilon.TM., manufactured by
Millipore, catalog no. IPVH000-10). The membrane was subjected to a
blocking at 4.degree. C. for one night with Block Ace (manufactured
by Dainippon Pharmaceutical; catalog no. UK-B25).
[0097] The blocked membrane was made to react with monoclonal
antibody HSP 72/73 (Ab-1) (manufactured by Oncogene Research
Products, catalog no. HSP01) which would specifically react with
heat-induced heat shock protein of 70 kDa and washed with TBS
containing 0.05% of Tween 20 followed by further washing with TBS.
After that, it was made to react with peroxidase-compounded
secondary antibody HRP-Rabbit Anti-Mouse IgG (H+L) (manufactured by
Zymed Laboratories, catalog no. 61-6520) and washed by the same
manner as in the above operation. The membranes which were treated
with primary and secondary antibodies as such were made to react
with Renaissance.TM. (a chemiluminor reagent manufactured by Dupont
NEN, catalog no. NEL-100) and photosensitized with an X-ray film to
confirm the induction of heat shock protein of 70 kDa.
[0098] The result was that, by addition of cyclopentenone (20 to 30
.mu.M), induction of heat shock protein of 70 kDa which is almost
same as heat-treated at 45.degree. C. for 10 minutes was confirmed.
Intensity of the induction is shown in Table 1. In Table 1, "+"
indicates degree of intensity of induction and the more the numbers
of "+", the more the intensity of induction. Incidentally, "-"
means that no induction was noted and ".+-." means the induction
was slight.
1 TABLE 1 Induction of Heat Shock Treated Cells Proteins Heated at
45.degree. C. for 10 minutes +++ 0 .mu.M of the cyclopentenone - 10
.mu.M of the cyclopentenone + 20 .mu.M of the cyclopentenone +++ 30
.mu.M of the cyclopentenone +++ 40 .mu.M of the cyclopentenone ++
50 .mu.M of the cyclopentenone + 100 .mu.M of the cyclopentenone
.+-.
[0099] Same results were obtained in the case of the
(-)-cyclopentenone and the (+)-cyclopentenone as well.
Example 2
[0100] (1) HeLa cells (ATCC CCL-2) were incubated in a
Dulbecco-modified Eagle's medium (DMEM; manufactured by Nissuisha)
containing 10% fetal bovine serum in a 10 cm plate at 37.degree. C.
in the presence of 5% carbon dioxide gas until a 80%. confluence
was resulted, then the cyclopentenone was added thereto so that its
final concentration was made 0, 5, 10, 20 or 40 .mu.M and
incubation was continued for additional six hours under the
above-mentioned conditions. The medium was discarded, 1 ml of 10%
trichloroacetic acid was added to each of the wells and the cells
were recovered by a scraper.
[0101] The cells prepared as such were subjected to an SDS-PAGE and
a blotting according to a method of Example 1 to detect the
expression of 70 kd heat shock proteins.
[0102] As a result, induction of the 70 kd heat shock proteins was
noted in the sections to which the cyclopentenone of from 5 .mu.M
to 40 .mu.M was added. The result is given in Table 2.
Incidentally, in Table 2, the sign "+" shows the potency of signals
of the 70 kd heat shock proteins observed in the blotting and the
more the numbers of +, the more potent the signals. The sign ".+-."
means that the signal was very weak.
2 TABLE 2 Amount of 70 kd of Heat Treated Cells Shock Proteins 0
.mu.M of the cyclopentenone .+-. 5 .mu.M of the cyclopentenone + 10
.mu.M of the cyclopentenone ++ 20 .mu.M of the cyclopentenone +++
40 .mu.M of the cyclopentenone +++
[0103] (2) HeLa cells were incubated in a DMEM containing 10% fetal
bovine serum in a 10 cm plate at 37.degree. C. in the presence of
5% carbon dioxide gas until a 80% confluence was resulted, then the
cyclopentenone was added thereto so that its final concentration
was made 0, 5, 10, 20 or 40 .mu.M and incubation was continued for
additional six hours under the above-mentioned conditions. After
that, the cells were washed with the DMEM containing 5% of fetal
bovine serum, then the DMEM containing 5% of fetal bovine serum
which contained Ad5 dlX [an adenovirus; Saito, et al.; Journal of
Virology, volume 54, pages 711-719 (1985)] was added to the cells
so that the cells were infected therewith and incubation was
conducted for 20 hours. Incidentally, the multiplicity of
infections (m.o.i.) was adjusted to 50. The medium was discarded, 1
ml of 10% trichloroacetic acid was added to each of the wells and
the cells were recovered by a scraper.
[0104] Then SDS-PAGE and blotting of the treated cells obtained
hereinabove were conducted by the method of Example 1 to detect the
expression of hexon proteins of the adenovirus. Incidentally,
anti-adenovirus hexon antibody (AB 1056; manufactured by Chemicon
International Inc.) was used as the primary antibody.
[0105] In the section to which 10 .mu.M or higher cyclopentenone
was added, the amount of the hexon protein apparently decreased as
compared with the control to which none of the cyclopentenone was
added. In the sections to which 20 .mu.M or lower amount of the
cyclopentenone was added, growth of the cells similar to that in
the section to which none of the cyclopentenone was added was
observed.
[0106] (3) Viral DNA was extracted according to a method mentioned
in "Protocols for Experiments of Virus" (pages 24-25; published by
Medical View) from the HeLa cells which were treated with the
cyclopentenone and infected by adenovirus followed by incubation by
the same manner as in Example 2-(2)
[0107] Thus, cells infected by virus were washed with a saline
solution buffered by phosphoric acid, suspended in 1 ml of aqueous
solution of 0.6% sodium laurylsulfate (SDS) and 10 mM EDTA and 3.0
ml of 5M aqueous solution of sodium chloride was added thereto. The
mixture was allowed to stand at 0.degree. C. for one hour and
centrifuged and 3 ml of ethanol was added to the resulting
supernatant liquid followed by mixing. The precipitate obtained by
centrifugal separation was dissolved in 0.2 ml of TE buffer (10 mM
Tris-HCl of pH 8.0 and 1 mM EDTA), then 2 .mu.l of 10% SDS and 4
.mu.l of 10 mg/ml proteinase K (manufactured by Takara Shuzo) were
added thereto and the mixture was kept at 37.degree. C. for one
hours. This was extracted with a mixture of equal amounts of phenol
and chloroform twice, 20 .mu.l of 3M sodium acetate and 400 .mu.l
of ethanol were added to the aqueous layer, the mixture was
centrifuged and the precipitate was dissolved in 50 .mu.l of the TE
buffer to give a DNA solution. The DNA solution (10 .mu.l) was
digested with 10 units of EcoT22I (manufactured by Takara Shuzo)
and 1 .mu.l of 10 mg/ml ribonuclease A and then subjected to an
agarose gel electrophoresis to determine the amount of viral DNA.
The result was that there was an apparent decrease in the amount of
viral DNA in the sections to which 5 .mu.M or more of the
cyclopentenone was added as compared with the control where none of
the cyclopentenone was added.
[0108] (4) After HeLa cells were incubated by the same manner as in
Example 2-(2), DMEM containing 5% of fetal bovine serum which
contained adenovirus of type 5 (Adenoid 75; ATCC VR-5) was added to
the cells without addition of the cyclopentenone whereby the cells
were infected by that. Incidentally, multiplicity of infections
(m.o.i.) was adjusted to 50. After that, the cyclopentenone was
added thereto to make its final concentration 0, 5, 10, 20 or 40
.mu.M and incubation was conducted for 20 hours. After the
incubation, detection of hexon proteins of adenovirus was conducted
by the same manner as in Example 2-(2). The result was that there
was an apparent decrease in the amount of hexon proteins in the
sections to which 10 .mu.M or more of the cyclopentenone was added
as compared with the control to which none of the cyclopentenone
was added. Incidentally, growth of the cells were in the sections
to which 20 .mu.M or more of the cyclopentenone was added was as
same as that in the sections to which none of the cyclopentenone
was added.
[0109] (5) HeLa cells were incubated by the same manner as in
Example 2-(2) and treated with the cyclopentenone. After that, the
cells were infected by the adenovirus type 5 (Adenoid 75; ATCC
VR-5) by the same manner as in Example 2-(4) and incubated in a
medium to which the cyclopentenone was added. After the incubation,
detection of the hexon proteins of adenovirus was conducted by the
same manner as in Example 2-(2). The result was that there was an
apparent decrease in the amount of the hexon proteins in the
sections to which 10 .mu.M or more of the cyclopentenone was added
as compared with the control to which none of the cyclopentenone
was added. Incidentally, growth of the cells was noted in the
sections to which 20 .mu.M or less of the cyclopentenone was added
as same as in the section to which none of the cyclopentenone was
added.
[0110] (6) Amount of the viral DNA was measured by the same manner
as in Example 2-(3) from the HeLa cells which were treated with the
cyclopentenone infected by the adenovirus type 5 (Adenoid 75; ATCC
VR-5) followed by incubating by the same manner as in Example
2-(5). The result was that there was an apparent decrease in the
amount of the viral DNA in the sections to which 5 .mu.M or more of
the cyclopentenone was added as compared with the control to which
none of the cyclopentenone was added.
[0111] From the results of the above-mentioned Examples
2-(2).about.(6), it was apparent that administration of the
cyclopentenone before, after or before and after the infection
exhibited antiviral activity to adenovirus. Same results were
obtained in the case of the (-)-cyclopentenone and the
(+)-cyclopentenone as well.
Example 3
[0112] (1) Recombined retrovirus vector BAG having
.beta.-galactosidase gene and neomycin-resistant gene as reporter
genes as mentioned in Proceedings of the National Academy of
Sciences of the U. S. A., volume 84, pages 156-160 (1987) was
digested by a restriction enzyme BamHI to conduct a self-ligation
whereupon DOL wherefrom .beta.-galactosidase gene was eliminated
was constructed.
[0113] (2) DOL vector plasmid mentioned in Example 3-(1) was
transformed to E. coli HB 101, incubated in an L-broth medium,
plasmid was extracted from the collected cells and the DOL plasmid
was purified by means of a cesium chloride density-gradient
ultracentrifugation.
[0114] The purified DOL plasmid (10 .mu.g) was introduced into
retrovirus packaging cell .PSI. CRIP [Proceedings of the National
Academy of Sciences of the U.S.A., volume 85, pages 6460-6464
(1988)] using a cationic liposome (Trans 2T LT-1; manufactured by
Takara Shuzo).
[0115] The cells after the introduction were selected for two weeks
at 37.degree. C. under the condition of 5% CO.sub.2 in a 10% calf
serum-containing Dulbecco-modified Eagle's medium containing 0.4
mg/ml of G418 (Gibco), 20 colonies selected thereby were cloned and
promulgated in a plate having a diameter of 100 mm, the medium was
exchanged under the semiconfluent condition, the supernatant liquid
was recovered after 24 hours and filtered through a filer of 0.45
.mu.m (Milex HV; manufactured by Millipore) to give a supernatant
liquid of the virus. In the meanwhile, the cells were scraped off
with trypsin and preserved in liquid nitrogen.
[0116] Titer of the supernatant liquid of the virus obtained from
each of the colonies was determined by the method mentioned in the
following Example 3-(3) and the clone wherefrom the virus solution
of the highest titer was obtained was established as a recombinant
retrovirus producer cell .PSI. CRIP/DOL. The titer of the
supernatant liquid of the virus obtained from the producer cells at
the time of the establishment was 1.times.10.sup.6 colony forming
units (cfu)/ml. The producer cells established as such were
maintained in a 10% calf serum-containing Dulbecco-modified Eagle's
medium containing 0.2 mg/ml of G418.
[0117] (3) NIH3T3 cells (ATCC CRL-1658) were used for the
measurement of the titer of the virus. The NIH3T3 cells incubated
in a Dulbecco-modified Eagle's medium containing 10% calf serum
were transplanted at the rate of 50,000 cells/well of a six-well
plate (Iwaki Glass) and, on the next day, they were infected to
NIH3T3 cells for three hours using 1 ml of diluted virus solution
containing 8 .mu.g/ml of Polybrene (Sigma) For diluting the virus,
a Dulbecco-modified Eagle's medium containing 10% of calf serum was
used. After completion of the infection, more 2 ml of a
Dulbecco-modified Eagle's medium containing 10% of calf serum was
added for diluting the Polybrene. As from the next day, exchange
with a 10% calf serum-containing Dulbecco-modified Eagle's medium
containing 0.4 mg/ml of G418 was conducted. Selection was conducted
for two weeks by exchanging the medium as above every three to four
days whereupon colonies were formed. The resulting colonies were
stained by a conventional manner using a Giemsa staining liquid
(Gibco) to count. The value obtained by multiplying the counted
colony numbers by degree of the dilution was defined as cfu and
used as the titer of the virus.
[0118] (4) The recombinant retrovirus producer cells .PSI. CRIP/DOL
mentioned in Example 3-(2) were transplanted to a six-well plate
and, when the semiconfluent state was resulted, exchanging with 1.5
ml of a 10% calf serum-containing Dulbecco-modified Eagle's medium
containing 0.about.20 .mu.M of the cyclopentenone was conducted.
After 24 hours, the supernatant liquid was recovered. Titer of the
virus in the recovered supernatant liquid were measured by the
method mentioned in Example 2-(3) and the influence of the
recombinant retrovirus producer cells on the productivity of virus
by addition of the cyclopentenone was investigated.
[0119] The titer of the virus solution obtained from the control
experimental section to which none of the cyclopentenone was added
was 9.5.times.10.sup.4 cfu/ml while those of the virus solutions in
the presence of 0.1, 0.5, 1.0, 2.0, 5.0, 10 and 20 .mu.M of the
cyclopentenone were 8.3.times.10.sup.4, 6.4.times.10.sup.4,
6.1.times.10.sup.4, 3.8.times.10.sup.4, 5.6.times.10.sup.4,
5.1.times.10.sup.4 and 4.1.times.10.sup.4 cfu/ml, respectively
whereupon it was ascertained that the titer of virus solutions
obtained from the producer cells decreases by addition of the
cyclopentenone. Thus, the an action of the cyclopentenone for
suppressing the virus productivity of the recombinant retrovirus
producer cells was confirmed.
[0120] (5) Control plasmid pcD2-Y expressing the G418-resistant
genes [Mol. Cell. Biol., volume 7, pages 2745-2752 (1987)] and
plasmid pcD2-16E7 expressing both HPV16-type E7 and G418-resistant
genes [Jpn. J. Cancer Res., volume 82, pages 1340-1343 (1881)] were
transformed to E. coli HB 101, incubated in an L-broth medium and
the plasmid was extracted from the collected cells and purified by
means of a cesium chloride density-gradient ultracentrifugation to
give vector plasmid for introduction of genes.
[0121] NIH3T3 cells were incubated in a Dulbecco-modified Eagle's
medium containing 10% of calf serum at 37.degree. C. under the
condition of 5% of CO.sub.2.
[0122] The purified plasmid (10 .mu.g) was introduced into the
NIH3T3 cells using a cationic liposome (TransIT LT-1; manufactured
by Takara Shuzo), the cells were selected for two weeks in a 10%
calf serum-containing Dulbecco-modified Eagle's medium containing
0.4 mg/ml of G418 (Gibco) under the condition of 5% of CO.sub.2 and
the resulting colonies were cloned, cultivated in a tissue culture
plate of a 100 mm diameter and preserved in liquid nitrogen
successively.
[0123] As a result thereof, each nine strains of NIH 3T3 cells into
which control vectors were introduced and NIH 3T3 cells which were
tumorgenically transformed by HPV 16 type E7 were established.
[0124] The cell strains into which the control vectors were
introduced were named NIH3T3/Y-1, NIH3T3/Y-2, NIH3T3/Y-3,
NIH3T3/Y-4, NIH3T3/Y-5, NIH3T3/Y-6, NIH3T3/Y-7, NIH3T3/Y-8 and
NIH3T3/Y-9.
[0125] The cell strains into which E7 was introduced were named
NIH3T3/E7-1, NIH3T3/E7-2, NIH3T3/E7-3, NIH3T3/E7-4, NIH3T3/E7-5,
NIH3T3/E7-6, NIH3T3/E7-7, NIH3T3/E7-8 and NIH3T3/E7-9.
[0126] (6) NIH3T3 cells, the cell strains into which the control
vectors were introduced and the cell strains into which E7 was
introduced were cultivated to an extent of 50-70% confluence in a
100-mm tissue culture plate using a Dulbecco-modified Eagle's
medium containing 10% of calf serum and washed with PBS and the
cells were scraped off with 0.25% trypsin-EDTA solution and
suspended in 5 ml of a Dulbecco-modified Eagle's medium containing
10% of calf serum.
[0127] A part of the suspension was taken out and cell density
thereof was calculated using a blood counter of a Newbauer type.
Based upon the resulting data, the suspension was diluted with a
Dulbecco-modified Eagle's medium containing 10% of calf serum and
sowed on a tissue culture plate having a diameter of 60 mm to make
the concentration 200 cells/plate and incubation was started in 3
ml of the medium. After 24 hours from the initiation of the
incubation, the cyclopentenone was added thereto to an extent of 5
.mu.M. After more 24 hours, the medium was exchanged with a fresh
one and the cyclopentenone was added to an extent of 5 .mu.M.
[0128] After that, the medium was exchanged and the cyclopentenone
was added to an extent of 5 .mu.M every two to three days. As a
control experimental section, a plate to which none of the
cyclopentenone was added was prepared and the medium was exchanged
in the same manner as above. Each incubation was conducted in three
runs. After incubating for nine days, fixation with methanol was
conducted and the colonies were stained with a Giemsa solution
(Gibco)
[0129] Incidentally, evaluation was conducted using NIH3T3,
NIH3T3/Y-1 and NIH3T3/E7-2.
[0130] Results of counting the stained colonies are given in Table
3. The cells into which E7 was introduced showed high sensitivity
to the cyclopentenone as compared with the control cells. Thus, the
cyclopentenone selectively acted to the cells transformed by
oncogenes.
3TABLE 3 Cells Numbers (average .+-. SD) of the Colonies Used
Control Cyclopentenone-Treated Cells NIH3T3 91.7 .+-. 11.9 85.3
.+-. 4.0 NIH3T3/Y-1 83.3 .+-. 8.4 71.3 .+-. 2.3 NIH3T3/E7-2 67.3
.+-. 3.2 22.3 .+-. 3.5
[0131] Similar results were obtained when other cell strains of
Example 3-(5) were used. In addition, the (-)-cyclopentenone and
the (+)-cyclopentenone gave the similar results as well.
Example 4
[0132] (1) To the MDCK cells (preserved at the Prefectural Public
Hygiene Laboratory, Osaka Prefecture) incubated in a 24-well
microplate using an Eagle's MEM containing 10% fetal bovine serum
in the presence of 5% carbon dioxide gas until monolayers were
obtained was added the cyclopentenone to make its final
concentration 0, 5, 10, 20 or 40 .mu.M and the incubation was
continued for six hours more under the above-mentioned
conditions.
[0133] After that, the cells were washed with PBS, infected by
influenza virus A/PR/8/34 strain (preserved at the Prefectural
Public Hygiene Laboratory, Osaka Prefecture) and incubated at
37.degree. C. for 30 minutes. Incidentally, the multiplicity of
infections (m.o.i.) was adjusted to 0.01. After incubation, the
cells were washed with PBS and incubated in an Eagle's MEM
containing 10 .mu.g/ml of trypsin.
[0134] The supernatant liquid of the infected cells was collected
on 0, 1, 2 and 3 day(s) thereafter and the titer of the virus was
determined by a PAP method using a focus counting method [J. Clin.
Microbiol., volume 28, pages 1308-1313 (1990)].
[0135] The result was that, in the sections to which 10 .mu.M or
more of the cyclopentenone was added, the titer of the virus
apparently lowered as compared with the control to which none of
the cyclopentenone was added. The result is given in Table 4. In
addition, the cells were not eliminated but adhered in each of the
sections to which the cyclopentenone was added.
4TABLE 4 Days Concentration of the Cyclopentenone (.mu.M) after 0 5
10 20 40 Infection pfu/ml pfu/ml pfu/ml pfu/ml pfu/ml 0 <1.0
.times. 10.sup.2 <1.0 .times. 10.sup.2 <1.0 .times. 10.sup.2
<1.0 .times. 10.sup.2 <1.0 .times. 10.sup.2 1 3.6 .times.
10.sup.5 4.0 .times. 10.sup.5 2.0 .times. 10.sup.5 2.2 .times.
10.sup.3 4.0 .times. 10.sup.2 2 1.0 .times. 10.sup.6 8.0 .times.
10.sup.5 7.2 .times. 10.sup.5 2.6 .times. 10.sup.5 1.9 .times.
10.sup.5 3 1.5 .times. 10.sup.5 9.6 .times. 10.sup.4 2.4 .times.
10.sup.5 3.8 .times. 10.sup.5 5.6 .times. 10.sup.5
[0136] (2) According to the same operations as in Example 4-(1)
influenza virus was added to the MDCK cells incubated to monolayers
in the absence of the cyclopentenone and said cells were infected
by said virus by the same manner as in Example 4-(1) and incubated
in an Eagle's MEM containing 10 .mu.g/ml of trypsin to which the
cyclopentenone was added to make its final concentration 0, 5, 10,
20 or 40 .mu.M. After that, the titer of the virus was determined
by the same manner as in Example 4-(1). The result was that, in the
sections to which 10 .mu.M or more of the cyclopentenone was added,
the titer of the virus apparently lowered as compared with the,
control to which none of the cyclopentenone was added. The result
is given in Table 5. In addition, the cells were not eliminated but
adhered in each of the sections to which the cyclopentenone was
added.
5TABLE 5 Days Concentration of the Cyclopentenone (.mu.M) after 0 5
10 20 40 Infection pfu/ml pfu/ml pfu/ml pfu/ml pfu/ml 0 <1.0
.times. 10.sup.2 <1.0 .times. 10.sup.2 <1.0 .times. 10.sup.2
<1.0 .times. 10.sup.2 <1.0 .times. 10.sup.2 1 4.2 .times.
10.sup.6 2.4 .times. 10.sup.5 1.9 .times. 10.sup.5 1.2 .times.
10.sup.5 <1.0 .times. 10.sup.2 2 1.6 .times. 10.sup.6 1.5
.times. 10.sup.6 3.4 .times. 10.sup.5 1.0 .times. 10.sup.6 <1.0
.times. 10.sup.2 3 4.8 .times. 10.sup.5 1.9 .times. 10.sup.5 1.7
.times. 10.sup.5 7.2 .times. 10.sup.5 <1.0 .times. 10.sup.2
[0137] (3) According to the same operations as in Example 4-(1),
the MDCK cells which were incubated to monolayers and treated with
the cyclopentenone of the final concentration of 0, 20 or 40 .mu.M
for six hours and were infected by influenza virus by the same
manner as in Example 4-(1) and incubation was continued in an
Eagle's MEM containing 10 .mu.g/ml of trypsin in which the
cyclopentenone of the same concentration as same as before the
infection. After that, the titer of the virus was determined by the
same manner as in Example 4-(1). The result was that, in the
sections to which 20 .mu.M or more of the cyclopentenone was added,
the titer of the virus apparently lowered as compared with the
control to which none of the cyclopentenone was added. The result
is given in Table 6. In addition, the cells were not eliminated but
adhered in each of the sections to which the cyclopentenone was
added.
6TABLE 6 Days Concentration of the Cyclopentenone (.mu.M) after 0
20 40 Infection pfu/ml pfu/ml pfu/ml 0 <1.0 .times. 10.sup.2
<1.0 .times. 10.sup.2 <1.0 .times. 10.sup.2 1 4.6 .times.
10.sup.5 9.8 .times. 10.sup.4 <1.0 .times. 10.sup.2 2 6.6
.times. 10.sup.5 1.6 .times. 10.sup.5 <1.0 .times. 10.sup.2 3
1.0 .times. 10.sup.5 1.3 .times. 10.sup.5 <1.0 .times.
10.sup.2
[0138] (4) The same experiment as in Example 4-(1) was conducted
where the multiplicity of infections (m.o.i.) was adjusted to
0.001. The result was that, in the sections to which 10 .mu.M or
more of the cyclopentenone was added, the titer of the virus
apparently lowered as compared with the control to which none of
the cyclopentenone was added. The result is given in Table 7. In
addition, the cells were not eliminated but adhered in each of the
sections to which the cyclopentenone was added.
7TABLE 7 Days Concentration of the Cyclopentenone (.mu.M) after 0 5
10 20 40 Infection pfu/ml pfu/ml pfu/ml pfu/ml pfu/ml 0 <1.0
.times. 10.sup.2 <1.0 .times. 10.sup.2 <1.0 .times. 10.sup.2
<1.0 .times. 10.sup.2 <1.0 .times. 10.sup.2 1 <1.0 .times.
10.sup.2 <1.0 .times. 10.sup.2 <1.0 .times. 10.sup.2 <1.0
.times. 10.sup.2 <1.0 .times. 10.sup.2 2 3.6 .times. 10.sup.5
5.2 .times. 10.sup.5 6.2 .times. 10.sup.5 5.0 .times. 10.sup.5 4.4
.times. 10.sup.3 3 3.8 .times. 10.sup.4 4.0 .times. 10.sup.4 8.6
.times. 10.sup.4 1.1 .times. 10.sup.5 8.0 .times. 10.sup.3
[0139] (5) The same experiment as in Example 4-(2) was conducted
where the multiplicity of infections (m.o.i.) was adjusted to
0.001. The result was that, in the sections to which 10 .mu.M or
more of the cyclopentenone was added, the titer of the virus
apparently lowered as compared with the control to which none of
the cyclopentenone was added. The result is given in Table 8. In
addition, the cells were not eliminated but adhered in each of the
sections to which the cyclopentenone was added.
8TABLE 8 Days Concentration of the Cyclopentenone (.mu.M) after 0 5
10 20 40 Infection pfu/ml pfu/ml pfu/ml pfu/ml pfu/ml 0 <1.0
.times. 10.sup.2 <1.0 .times. 10.sup.2 <1.0 .times. 10.sup.2
<1.0 .times. 10.sup.2 <1.0 .times. 10.sup.2 1 6.0 .times.
10.sup.2 <1.0 .times. 10.sup.2 <1.0 .times. 10.sup.2 <1.0
.times. 10.sup.2 <1.0 .times. 10.sup.2 2 3.6 .times. 10.sup.5
8.8 .times. 10.sup.4 2.0 .times. 10.sup.5 1.8 .times. 10.sup.4
<1.0 .times. 10.sup.2 3 3.8 .times. 10.sup.4 2.6 .times.
10.sup.4 1.8 .times. 10.sup.4 1.2 .times. 10.sup.4 <1.0 .times.
10.sup.2
[0140] (6) The same experiment as in Example 4-(3) was conducted
where the multiplicity of infections (m.o.i.) was adjusted to
0.001. The result was that, in the sections to which 10 .mu.M or
more of the cyclopentenone was added, the titer of the virus
apparently lowered as compared with the control to which none of
the cyclopentenone was added. The result is given in Table 9. In
addition, the cells were not eliminated but adhered in each of the
sections to which the cyclopentenone was added.
9TABLE 9 Days Concentration of the Cyclopentenone (.mu.M) after 0 5
10 20 40 Infection pfu/ml pfu/ml pfu/ml pfu/ml pfu/ml 0 <1.0
.times. 10.sup.2 <1.0 .times. 10.sup.2 <1.0 .times. 10.sup.2
<1.0 .times. 10.sup.2 <1.0 .times. 10.sup.2 1 6.0 .times.
10.sup.3 <1.0 .times. 10.sup.2 <1.0 .times. 10.sup.2 <1.0
.times. 10.sup.2 <1.0 .times. 10.sup.2 2 6.2 .times. 10.sup.5
4.4 .times. 10.sup.5 4.8 .times. 10.sup.5 3.2 .times. 10.sup.4
<1.0 .times. 10.sup.2 3 3.6 .times. 10.sup.4 5.6 .times.
10.sup.4 2.8 .times. 10.sup.4 2.8 .times. 10.sup.4 <1.0 .times.
10.sup.2
[0141] From the results of the above-mentioned Examples
4-(1).about.(6), it was apparent that the cyclopentenone exhibited
antiviral activity to influenza virus. In addition, the (-)
-cyclopentenone and the (+)-cyclopentenone gave the similar results
as well.
Example 5
[0142] (1) Action of the Cyclopentenone to Human T Cells.
[0143] The cyclopentenone (0.5-5 .mu.M) was added to
2.times.10.sup.5 cells/ml of the CEM-SS cells (ATCC CCL-119) or to
H9 cells (ATCC HTB-176) and incubated for three days and the
numbers of living cells and of dead cells were counted whereupon
the survival rates of the cells were calculated.
[0144] The result was that, in both cells, there was no significant
decrease in the survival rate of the cells by addition of the
cyclopentenone. The result is given in FIG. 1 and in FIG. 2. Thus,
FIG. 1 and FIG. 2 are the graphs showing the relation between the
concentration of the cyclopentenone added and the survival rate of
the cells in which abscissa is the concentration (.mu.M) of the
cyclopentenone added while ordinate is the survival rate (%) of the
cells after incubating for three days. FIG. 1 is a result when
CEM-SS cells were used while FIG. 2 is a result when H9 cells were
used.
[0145] (2) Action of the Cyclopentenone to HIV-Infected T
Cells.
[0146] The cyclopentenone (1-5 .mu.M) was added to CEM-SS cells
infected by HIV-1.sub.IIIB (abbreviated as CEM-3B) or to H9 cells
infected by HIV-1.sub.IIIB (abbreviated as H9-3B) and incubated for
three days. In both cells, 90% or more of the cells were infected
by HIV-1.sub.IIIB. Numbers of living and dead cells were counted
and the survival rate of the cells was calculated therefrom.
[0147] The result was that, in both cells, the survival rate of the
cells significantly decreased by addition of 3 .mu.M of the
cyclopentenone and, in the case of addition of 5 .mu.M of the
cyclopentenone, the survival rate of the cells further decreased.
Thus, as compared with Example 5-(1), the cyclopentenone showed an
anti-HIV action. The result is shown in FIG. 3 and in FIG. 4. Thus,
FIG. 3 and FIG. 4 are the graphs showing the relation between the
concentration of the cyclopentenone added and the survival rate of
the cells in which abscissa is the concentration (.mu.M) of the
cyclopentenone added while ordinate is the survival rate (%) of the
cells after incubating for three days. FIG. 3 is the result when
CEM-3B were used while FIG. 4 is the result when H9-3B were
used.
Example 6
[0148] Concentration of the p24 antigen contained in the
supernatant liquid of the culture after incubation for three days
in the case of Example 5-(2) was measured. The result was that the
concentration of p24 decreased corresponding to the concentration
of the cyclopentenone added thereto whereupon the anti-HIV action
was noted. The result is shown in Table 10. In Table 10, the
figures in parentheses are the ratio of each of the supernatant
liquid of the cell cultures (when none of the cyclopentenone was
added) to the concentration of p24 expressed in terms of %.
10TABLE 10 Concentration of Concentration (ng/ml) of p24 in the
Cyclopentenone Supernatant Liquid of the cell cultures (.mu.M)
CEM-3B H9-3B 0 280 (100%) 210 (100%) 1 232 (83%) 203 (97%) 3 176
(63%) 157 (75%) 5 175 (63%) 148 (70%)
Example 7
[0149] Vero cells (ATCC CCL-81) was suspended in an Eagle's MEM
containing 10% of fetal bovine serum until the cell concentration
became 5.times.10.sup.4 cells/100 .mu.l, the suspension was placed
in a 96-well microtiter plate to such an extent that 100 .mu.l of
the cell suspension was poured into each well and incubated
overnight at 37.degree. C. in the presence of 5% carbon dioxide gas
and the Vero cells in a state of monolayers were prepared.
[0150] An Eagle's MEM medium to which the cyclopentenone was added
to make its final concentration 0, 5, 10, 20 or 40 .mu.M was added
to the cells and incubation was conducted at 37.degree. C. for
seven hours in the presence of 5% of carbon dioxide gas.
[0151] After completion of the incubation, the medium was removed,
washing with PBS was conducted twice, then Japanese encephalitis
virus (JEV Ja0Ar-363-70 strain) was inoculated to an extent of
4.9.times.10.sup.2 pfu/ml, incubation was conducted at 37.degree.
C. for 30 hours in the presence of 5% of carbon dioxide gas, the
cells were fixed by ethanol and a focus counting was conducted by
means of a focus counting by a PAP method [Arch. Virol., volume 86,
pages 129-135 (1985)].
[0152] The result was that, in the sections to which 40 .mu.M of
the cyclopentenone was added, the numbers of the focus apparently
decreased as compared with the control in which none of the
cyclopentenone was added. The result is given in Table 11.
Incidentally, the cells were not eliminated but adhered in each of
the sections to which the cyclopentenone was added.
11 TABLE 11 Cyclopentenone Concentration (.mu.M) pfu/ml 0 3.0
.times. 10.sup.7 10 1.6 .times. 10.sup.7 20 2.1 .times. 10.sup.7 40
4.9 .times. 10.sup.6
[0153] (2) Vero cells which were incubated in a 24-well microplate
using an Eagle's MEM containing 10% of fetal bovine serum in the
presence of 5% of carbon dioxide gas at 37.degree. C. until the
monolayers were resulted were washed with PBS, infected by
4.times.10.sup.2 pfu/ml of Japanese encephalitis virus (JEV
Ja0Ar-363-70 strain) and incubated at 37.degree. C. for 90
minutes.
[0154] After the incubation, the cells were washed with PBS and
incubated in an MEM to which the cyclopentenone was added to make
its final concentration 0, 5, 10, 20 or 40 .mu.M.
[0155] The supernatant liquid of the infected cells was collected
after 0, 1, 2 and 3 day(s) and the titer of the virus were
determined means of the focus counting by a PAP method [J. Clin.
Microbiol., volume 28, pages 1308-1313 (1990)].
[0156] The result was that, in the sections to which 10 .mu.M or
more of the cyclopentenone was added, the numbers of the focus
apparently decreased as compared with the control in which none of
the cyclopentenone was added. The result is given in Table 12.
Incidentally, the cells were not eliminated but adhered in each of
the sections to which the cyclopentenone was added.
12 TABLE 12 Concentration of the Days after Inoculation of Virus
Cyclopentenone 1 2 3 (.mu.M) pfu/ml pfu/ml pfu/ml 0 6.0 .times.
10.sup.3 5.6 .times. 10.sup.6 1.1 .times. 10.sup.7 10 6.0 .times.
10.sup.3 2.4 .times. 10.sup.6 4.4 .times. 10.sup.6 20 6.4 .times.
10.sup.3 1.9 .times. 10.sup.6 3.0 .times. 10.sup.6 40 0 0 0
[0157] From the above result for Example 7-(1) and (2), it is
apparent that the cyclopentenone exhibits an antiviral activity to
the Japanese encephalitis virus. Incidentally, the Japanese
encephalitis virus belongs to a species of the same type as the
hepatitis C virus does and, under the present circumstances where
incubation of the hepatitis C in vitro has not been established
yet, the Japanese encephalitis virus is used as a model of the
hepatitis C virus. Consequently, the cyclopentenone is effective as
a therapeutic agent for the hepatitis C as well.
[0158] In the meanwhile, the same result was obtained for the
(-)-cyclopentenone and the (+)-cyclopentenone as well.
Example 8
[0159] When a female who was diagnosed to be hepatitis C five year
ago and showed no improvements in hepatic functions where both GOT
and GPT were around 150 in spite of a treatment with interferon and
Minofagen Strong took the beverage which was prepared according to
Example 13 at the dose of 50 ml (containing 2 mg of the
cyclopentenone) for two months, both GOT and GPT were improved to
80. When she took it for additional one month, both GOT and GPT
became 30 whereupon a significant improvement in hepatic function
was noted.
Example 9
[0160] Hair on the back of mice of ICR strain (purchased from
Nippon SLC; seven weeks age; female) was shaved and DMBA
(dimethylbenzanthracene) as an initiator in a form of a solution in
acetone was applied thereto at the dose of 50 .mu.g/mouse. After
one week, TPA (12-o-tetrasecanoylphorbo- l 13-acetate) as a
promoter in a form of a solution in acetone at the dose of 1
.mu.g/mouse was applied twice a week to the site where the
initiator was applied until the completion of the test while 80%
ethanolic solution of the cyclopentenone or 80% ethanol (control)
was applied one hour before each application of TPA whereupon the
anti-carcinogenic action to carcinogenesis caused by a two-stage
carcinogenesis on skin was observed for 20 weeks.
[0161] The control group (a group applied with a vehicle only)
showed a carcinogenic rate of 100% (12 mice out of 12) within 15
weeks while the cyclopentenone strongly suppressed the
carcinogenesis and the carcinogenic rate of the mice administered
with 2.5 mg within 15 weeks was 8.3% (1 mouse out of 12) and that
within and after 19 weeks was 25% (3 mice out of 12). The result is
given in FIG. 5. Thus, FIG. 5 is a graph showing the
anti-carcinogenic action of the cyclopentenone in which ordinate is
a carcinogenic rate while abscissa is time (weeks). In the graph,
open triangles, black triangles and open circles stand for a group
treated with 2.5 mg of the cyclopentenone per mouse (12 mice in
total), a group treated with 0.8 mg of the cyclopentenone per mouse
(11 mice in total) and a control group (12 mice in total),
respectively.
[0162] Incidentally, in the anti-inflammatory test of TPA in
conchae of mice, the cyclopentenone showed no anti-inflammatory
activity by application of 2.5 mg (per mouse) to concha of the
mouse.
[0163] To sum up, the cyclopentenone showed an anti-promoter action
in a two-stage chemical carcinogenesis. Heated product of
glucuronic acid containing the cyclopentenone, the
(-)-cyclopentenone and the (+)-cyclopentenone showed the same
result as well.
Example 10
Injection Preparations
[0164] (1) Cyclopentenone was added to a physiological saline
solution (as listed in the Japanese Pharmacopoeia) in a
concentration of 1% to prepare an injection preparation.
[0165] (2) (-)-cyclopentenone and glycyrrhizic acid were added to a
physiological saline solution (the same as above) in concentrations
of 0.5% and 0.1%, respectively, to prepare an injection
preparation.
Example 11
Tablets
[0166] (1) A tablet containing 10 mg of cyclopentenone and an
appropriate amount of microcrystalline cellulose was prepared and
coated with sugar to manufacture a tablet preparation.
[0167] (2) A tablet containing 0.1 mg of (+)-cyclopentenone, 10 mg
of dipotassium glycyrrhizinate and an appropriate amount of
microcrystalline cellulose was prepared and coated with sugar to
manufacture a tablet preparation.
Example 12
Ointment
[0168]
13 cyclopentenone 1 g Absorption ointment (as listed in the 99 g
Japanese Pharmacopoeia)
[0169] First, cyclopentenone was well kneaded with a small amount
of absorption ointment and then the residual absorption ointment
was gradually added thereto and kneaded therewith until homogeneity
was resulted to prepare an ointment preparation.
[0170] This ointment was applied to the affected part for four to
five times a day.
Example 13
[0171] (1) Pectin (Pomosin Pectin LM-13CG; manufactured by
Hercules) (5 kg) was added to 100 liters of tap water and the
mixture was heated from the liquid temperature of 28.degree. C. to
120.degree. C. by means of blowing steam thereinto during 35
minutes, kept at 120.degree. C. for five hours with stirring and
cooled to prepare 135 liters of cooled mixture. To this were added
1.35 kg of Celite #545 (manufactured by Celite) and 1.35 kg of
Silica #600-S (manufactured by Chuo Silica) as filter aids and
filtration was conducted using a compact filter (6-inch filter
paper in 16 stages; ADVANTEC #327) precoated with 0.1 kg of Celite
#545 and 0.1 kg of Silica #600-S. The resulting filtrate was
subjected to a continuous instant heating treatment (at 98.degree.
C. for 60 seconds) using a plate heater (manufactured by Nichihan
Seisakusho) followed by cooling to prepare 150 liters of
heat-treated pectin solution containing the cyclopentenone.
[0172] Said heat-treated pectin solution containing the
cyclopentenone had pH of about 3.5, acidity of 6.2 ml and sugar
degree of 5.8 Brix %. Incidentally, pH was measured by a pH meter,
acidity was expressed in terms of the amount (ml) of 0.1N NaOH used
for neutralizing to pH 7.0 and sugar degree was measured by a Brix
saccharometer.
[0173] (2) Beverage was prepared according to the following
formulation.
14 Fructose-Glucose-Liquid Sugar 5.00% Sugar 4.00% Acidic agent
1.20% Perfumes 0.30% Cyclopentenone-containing material 0.5% Pure
water balance Total 100.00%
[0174] The heat-treated pectin solution containing the
cyclopentenone mentioned in Example 13-(1) was used as the
cyclopentenone-containing material and its amount calculated on a
solid basis was added. This beverage (100 ml) contains 4 mg of the
cyclopentenone.
MERIT OF THE INVENTION
[0175] The present invention offers an antiviral agent containing a
compound having a function of inducing a resistance to virus to
cells and a function of selectively killing the virus-infected
cells such as the cyclopentenone, an optically active substance or
a salt thereof as an effective component. The antiviral agent of
the present invention selectively kills the virus-infected cells
and gives a resistance to virus to normal cells which are not
infected by virus and, as a result of synergistic action thereof,
it is an antiviral agent which is extremely useful for the therapy
of intractable viral diseases such as AIDS and hepatitis C and also
for the improvement in symptoms thereof. In addition, the present
invention offer a pharmaceutical agent containing the
cyclopentenone, an optically active substance or a salt thereof
which exhibits various physiological activities such as an action
of improving the hepatic function, an action of inducing the heat
shock proteins, an action of preventing the viral carcinogenesis
and an anti-promoter action. Said pharmaceutical agent affords a
drug which is useful for maintaining the homeostasis of living
body, particularly maintaining the health of stomach and
intestine.
[0176] The present invention further offers an antiviral food and
antiviral beverage containing a compound having a function of
inducing a resistance to virus to cells and a function of
selectively killing the virus-infected cells such as the
cyclopentenone, an optically active substance or a salt thereof as
an effective component. Such food and beverage are useful as food
and beverage for improving the symptoms of various diseases caused
by virus. In addition, the present invention offers food and
beverage containing the cyclopentenone, an optically active
substance or a salt thereof which exhibits physiological activity
such as an action of improving the hepatic function, an action of
inducing the heat shock proteins, an action of preventing the viral
carcinogenesis and an anti-promoter action and said food and
beverage are useful for maintaining the homeostasis of living body,
particularly maintaining the health of stomach and intestine.
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