Title of Invention

STABILIZED COMPOSITIONS OF AQUEOUS REDUCED COENZYME Q SOLUTION

Abstract The present invention provides a reduced coenzyme Q-containing solution capable of being retained, more stably 5against oxidation, by using a reduced coenzyme Q which is hydrophobic and susceptible to oxidation and thus unstable. The aqueous solution containing a reduced coenzyme Q obtained in the present invention is a solution containing a reduced coenzyme Q and an antioxidant such as vitamin C lOand/or a chelating agent such as ethylenediaminetetraacetic acid.
Full Text 1

DESCRIPTION
AN AQUEOUS SOLUTION CONTAINING A REDUCED COENZYME Q

TECHNICAL FIELD
The present invention relates to a solution containing a reduced coenzyme Q as a constituent and, more particularly, to an aqueous solution containing an lOantioxidant and/or a chelating agent added thereto for maintaining a reduced coenzyme Q stable against oxidation.
BACKGROUND ART Coenzymes Q are essential constituents widely
15distributed in living bodies, from bacteria to mammals and are known as mitochondrial electron transfer system constituents in cells of living bodies. Through repeated oxidation and reduction in mitochondria, coenzymes Q perform their function as transmitter components in the
20electron transfer system and, further, reduced coenzymes Q are known to have antioxidant activity. In humans, coenzyme Q10, whose coenzyme Q side chain comprises 10 repeating structures, is the main component and, generally, about 40 to 90% thereof occurs in reduced form in living
25bodies. The physiological activities of coenzymes. Q may be energy production activation through mitochondrial activation, cardiac function activation, cell' membrane stabilizing effect, and cell protecting effect through antioxidant activity.
30 Coenzymes Q are known to be useful in various
application fields. For example, oxidized coenzyme Q10 is used as a remedy for congestive heart failure owing to its effects on the heart. Besides such medical uses, they are orally used as nutrients or nutritional supplements, like
35vitamins. However, coenzymes Q are highly liposoluble and

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hardly soluble in water and, therefore, only oral preparations and dermal preparations are known as their practical uses.
In recent years, various reports have been published 5about the aggravation of diseases due to increases in oxidative stress in blood. Typical examples are arteriosclerosis, complications of diabetes and the like diseases. These diseases are caused and/or aggravated by denaturation of lipids and the like due to various
lOoxidative stresses occurring in blood. For counteracting such effects of oxidative stresses, antioxidant activity promotion by administration of an antioxidant is effective. Vitamin E is a compound representative of the liposoluble antioxidant substances considered to be more effective in
15inhibiting lipid peroxidation and is in wide use in disease prevention and so on.
Recently, it has been reported that the coexistence of reduced coenzyme Q10 is important for vitamin E to properly perform its antioxidant activity (Bowry et al.,
201993, J. American Chemical Society, 115, 6029-6044), and the importance of coenzymes Q as liposoluble antioxidant substances is becoming clear.
Coenzymes Q have themselves strong antioxidant activity and, therefore, the antioxidant activity in blood can be effectively enhanced by sending a sufficient amount of reduced coenzymes Q in solubilized form into blood. The enhanced antioxidant activity in blood is considered to be useful widely in preventing vascular lesions during ischemia-reperfusion, preventing restenosis in arteriosclerosis, preventing vascular lesions following cerebral infarction, preventing arteriosclerosis, preventing complications of diabetes, and preventing a number of other diseases from being aggravated supposedly by active oxygen species. .Furthermore, by sending it into the living body in a new delivery form, namely by drip, it

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becomes possible to provide patients with a serious illness or a brain disease, who are incapable of oral intake, with coenzymes Q. It is thus expected that solubilization of coenzymes Q will bring about a number of merits. 5 As is well known, coenzymes Q can occur in both the oxidized form and reduced form, and a number of investigations have so far been made about the method of solubilizing oxidized coenzyme Q10 (ubidecarenone or ubiquinone).
10 As for the solubilization of oxidized coenzyme Q10, various methods have been reported, for example coating with liposomes, suspension using a surfactant or an oil/fat, and the like (Japanese Kokai Publication Hei-05-186340, Japanese Kokai Publication Hei-07-69874, Japanese
15Kohyo Publication 2000-510841). However, few examples have been put to practice use. One of the reasons is that while it is necessary for oxidized coenzyme Q10 to be converted to the reduced form by the action of a reductase or the like in order to perform its antioxidant activity, no
20reductase is present in blood and, therefore, no
antioxidant activity against oxidative stresses in blood can be expected upon administration by injection or the like.
On the other hand, reduced coenzyme Q10 itself has
25antioxidant activity, hence it is a substance; much expected to be of great utility in the prevention/treatment of such diseases as mentioned above. However, it has. not been put into practical use because of its drawback that it is susceptible to oxidation and thus unstable. Although a
30search report describing the preparation of liposome-coated reduced coenzyme Q10 for the purpose of studying oxidoreductases and so forth is available (Kishi et al., 1999, BioFactors, 10, 131-138), the liposomes used were prepared extemporaneously in each experiment. Any method
35of stably solubilizing reduced coenzymes Q has not been

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known at all.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide 5an aqueous solution improved in oxidation stability of reduced coenzymes Q.
The present inventors made investigations in an attempt to accomplish the above object and, as a result, found out an aqueous solution composition suitable for lOincreasing the stability of reduced coenzymes Q, which has led to completion of the present invention.
Thus, the present invention provides an.aqueous solution containing a reduced coenzyme 'Q, which comprises an antioxidant and/or a chelating agent in the same aqueous 15solution.
DETAILED DESCRIPTION OF THE INVENTION The aqueous solution of the invention is an aqueous solution containing a reduced coenzyme Q 20represented by the following formula (1):

in the formula, n represents an integer of 1 to 12:
I which comprises an antioxidant and/or a chelating agent in the same aqueous solution.
The coenzyme Q includes the species represented by 35the following formula (1):


5

in the formula, n represents an integer of 1 to 12:
and the species represented by the following formula (2):

25in the formula, n represents an integer of 1 to 12. The formula (1) represents the reduced form of a coenzyme Q and the formula (2) represents the oxidized form of a coenzyme
Q.
In the practice of the invention, the coenzyme Q to 30be used may be any of those in which the number (n in the formulas) of repeating units in the side chain is 1 to 12, as represented by the above formulas (1) and (2). However, the one having 10 repeating units in the side' chain, namely coenzyme Q10, can be used most adequately. 35 In the practice of the invention, an oxidized

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coenzyme Q may coexist with the reduced coenzyme Q to be used. In that case, the content of the reduced coenzyme Q in the whole amount of coenzymes Q is preferably not less than 20% by weight, more preferably not less than 40% by 5weight, most preferably not less than 60% by weight.
The method of preparing the reduced coenzyme Q is not particularly restricted but, for example, the method comprising producing a coenzyme Q by any of the methods known in the art,' for example by synthesis, fermentation,
lOextraction from a natural source, or the like method and then concentrating the reduced coenzyme Q fraction in the eluate by chromatography can be employed. In this case, it is also possible to add a conventional reducing agent, such as sodium borohydride or sodium dithionite (hydrosulfite
15sodium), to the coenzyme Q if necessary and, after
reduction of the oxidized coenzyme Q contained in the above coenzyme Q to a reduced coenzyme Q in the conventional manner, subject the reduction product to chromatography for concentration. It can also be obtained by the method
20comprising reacting a commercially available high-purity coenzyme Q with such a reducing agent as mentioned above. The method of preparing the aqueous solution of the invention is not particularly restricted but, for example, the aqueous solution can be obtained (1) by coating a
25reduced coenzyme Q and an appropriate antioxidant and/or an appropriate chelating agent with an appropriate liposome base to give liposomes, (2) by adding an aqueous solution of an appropriate antioxidant and/or an appropriate chelating agent to reduced coenzyme Q-containing liposomes,
30or (3) by solubilizing or emulsifying a reduced coenzyme Q and an appropriate antioxidant and/or an appropriate chelating agent by means of an appropriate surfactant.
The aqueous solution of the invention is not particularly restricted but may be any one containing a
35reduced coenzyme Q and an antioxidant and/or a chelating

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agent. When it is prepared using liposomes, as mentioned above under (1) or (2), it occurs as an aqueous solution with the liposomes being dispersed in the aqueous solution When it is prepared using a surfactant, as mentioned above 5under (3) , it occurs as an aqueous solution with a reduced coenzyme Q being solubilized or emulsified therein.
The antioxidant that can be used in the practice of the present invention is not particularly restricted but includes, for example, citric acid, citric acid
lOderivatives, vitamin C, vitamin C derivatives, probucol, licopene, vitamin A, carotenoids, vitamin B, vitamin B derivatives, flavonoids, polyphenols, glutathione, selenium, sodium thiosulfate, vitamin E, vitamin E derivatives, superoxide dismutase (SOD), glutathione
15peroxidase, glutathione-S-transferase, glutathione
reductase, catalase, ascorbate peroxidase and- mixtures of these.
Among them, citric acid, citric acid derivatives, vitamin C, vitamin C derivatives, glutathione and sodium
20thiosulfate are preferred, vitamin C, citric acid and the like are more preferred, and vitamin C is still more preferred.
The chelating agent is not particularly restricted but includes, for example, ethylenediaminetetraacetic acid
25and salts thereof, ethylenediaminediacetic acid and salts thereof, hydroxyiminodiacetic acid and salts thereof, hydroxyethylethylenediaminetetraacetic acid and salts thereof, diethylenetriaminepentaacetic acid and salts thereof, nitrilotriacetic acid and salts thereof,
30triethylenetetraminehexaacetic acid and salts thereof, dicarboxymethylglutamic acid tetrasodium salt, dihydroxymethylglycine, 1,3-propanediaminetetraacetic acid and salts thereof, 1,3-diamino-2-hydroxypropanetetraacetic acid and salts thereof, sodium gluconate,
35hydroxyethylidenediphosphonic acid, alkylenephosphonic

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acids, phosphonobutanetricarboxylic acid and mixtures of
these.
Among these, ethylenediaminetetraacetic acid and salts thereof, hydroxyethylethylenediaminetetraacetic acid 5and salts thereof, diethylenetriaminepentaacetic acid and salts thereof, sodium gluconate and
hydroxyethylidenediphosphonic acid are preferred, and
ethylenediaminetetraacetic acid and salts thereof are more
preferred.
10 In cases where liposomes are used in the aqueous . , solution of the invention, the liposome includes, for example, phospholipids such as refined soy lecithin and phosphatidylcholine, glicolipids such as digalactosylglyceride, and the like. From the viewpoint of
15the stabilization of reduced coenzyrnes Q against oxidation, refined soy lecithin, phosphatidylcholine and, the like are preferred.
When a surfactant is used in the aqueous solution of the, invention, the surfactant is not particularly
20restricted but includes, for example, carboxylate salt type
anionic surfactants and the like. Preferred from the
viewpoint of the oxidation stability of reduced coenzymes Q
are polysorbate 80, polyoxyethylene hydrogenated castor oil
and the like.
25 The reduced coenzyme Q concentration in the aqueous solution of the invention is not particularly. restricted but, from the viewpoint of oxidation stability,, solubility in aqueous solution, and/or the like, the proportion of the reduced coenzyme weight to the volume of the aqueous
30solution is preferably not lower than 0.001% (w/v) but not higher than 5% (w/v), more preferably not lower than 0.05% (w/v) but not higher than 1% (w/v). The antibxidant concentration is not particularly restricted, either. From the efficacy viewpoint, however, it is preferably not lower
35than 0.01% (w/v) but not higher than 50% (w/v), more

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preferably not lower than 0.05% (w/v) but not higher than 10% (w/v), relative to the whole amount of the aqueous solution. The chelating agent concentration is not particularly restricted, either. From the efficacy 5viewpoint, however, it is preferably not lower than 0.001% (w/v) but not higher than 10% (w/v), more preferably not lower than 0.005% (w/v) but not higher than 5% (w/v), relative to the whole amount of the aqueous solution. The antioxidant concentration and chelating agent concentration
lOmentioned above are applicable not only in the case where these are respectively used singly but also.in the case of combined use thereof.
The pH of the aqueous solution of the invention is not particularly restricted but may vary according to the
15intended use thereof. From the viewpoint of the stability of coenzymes Q, however, the pH is preferably not lower than 1.0 but not higher than 8.0, more preferably not lower than 2.0 but not higher than 7.6.
To the aqueous solution to be prepared in the above
20manner, there may be added one or more other
pharmaceutically acceptable preparation materials each at an appropriate addition level in the conventional manner. The preparation materials are not limited but there may be mentioned, for example, emulsifiers, isotonizing agents,
25buffers, solubilizing agents, correctives for. smell, preservatives, stabilizers and the like. Furthermore, a further active ingredient, for example a drug, and/or a nutritional supplement, may be added with the. intended use. The method of storing the aqueous solution
30composition according to the invention is not- particularly restricted but includes cold storage (e.g. at -80°C to 4°C), anaerobic storage in a tightly closed container, and storage under protection from light., and the like.
The aqueous solution of the invention which can be
35prepared in the above manner can preserve reduced coenzymes

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10
Q more stably from oxidation.
The reduced coenzyme Q-containing aqueous solution according to the invention can be used in a wide range, including medical, cosmetic, food, horticultural, and dairy
5uses, and the' like. As specific preparation forms, there may be mentioned injectable solutions, infusion solutions, liquid preparations, ophthalmic solutions, solutions for oral administration, lotions, hair tonics, milky lotions, sprays, aerosols, health drinks, liquid fertilizers,
lOpreserving solutions and so on. In the field of medicine, it can further be used as a preserving solution on the occasion of organ transplantation. Furthermore, the use as
¦ animal feeds and so on may also be mentioned. In addition, it can be used, as an antioxidant solution, in storing
15meat,. fish and other perishable foods.
BEST MODE FOR CARRYING OUT THE INVENTION The following examples illustrate the present invention in further detail. These examples are, however, 20by no means limitative of the scope of the invention.
(Example 1) Oxidation stability of liposome-coated reduced coenzyme Q10 as given by an antioxidant
For evaluating the oxidation stability of reduced
25coenzyme Qlo in liposomes, yolk lecithin (lecithin, product of Wako Pure Chemical Industries), which has poor oxidation stability, was selected as the liposome base, and the evaluation of substances capable of increasing the oxidation stability of reduced coenzyme Q10 (content 0.005%
30w/v) was performed under the storage condition of 40°C.
Reduced coenzyme Qi0-containing liposomes were prepared by the following method. Thus, a powder of reduced coenzyme Qi0 was dissolved in ethanol to give a 0.1 mg/ml solution. Similarly, a lecithin solution (3.2 mg/ml)
35in ethanol was prepared. Both solutions (1 ml each) were

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mixed together. To the resulting mixture was added 2 ml of chloroform and, then, the solvent was removed under reduced pressure. After complete elimination of the solvents, 2 ml of a 50 mM HEPES buffer solution (pH 7.4) was added, and 5the lipid film was dispersed using a mixer to give a milk-white suspension. This suspension was subjected to sonication in a nitrogen atmosphere at 4°C for 30 minutes for the formation of liposomes, followed by 20 minutes of centrifugation at 3,000 rpm to remove giant molecules as a
lOsedirnent. The fraction remaining unsettled after
centrifugation was used as reduced coenzyme Q10-containing liposome solution.
Liposomes containing an antioxidant or a chelating agent (0.05% w/v) were prepared as follows. In the case of
15vitamin E, a 10 mg/ml ethanol solution was prepared and added to a mixed solution containing reduced coenzyme Q10 and lecithin prior to the addition of chloroform in the above-mentioned process for preparing reduced coenzyme Q10-containing liposomes and, thereafter, liposomes were
20prepared in the same manner as mentioned above. In the case of other water-soluble antioxidants or chelating agents, each antioxidant or chelating agent was dissolved in the above-mentioned HEPES buffer solution to a concentration of 0.05% (w/v) and, thereafter, ¦ liposomes
25were prepared in the same manner.
In this manner, liposomes containing various antioxidant (vitamin E acetate, sodium thiosulfate, citric acid, vitamin C, vitamin E (a-tocopherol)) were prepared and evaluated for oxidation stability during storage at
3040°C in the ambient atmosphere. The results were expressed in terms of residual reduced coenzyme Q10 percentage.
For determining the residual reduced coenzyme Q10 percentage, 1 ml of hexane was first added to 0.05 ml of each liposome solution and, after 30 seconds of stirring,
35the mixture was separated into a hexane layer and an

12
aqueous layer by centrifugation {3,000 rpm, 1 minute). Then, the hexane layer was recovered and evaporated to dryness in a nitrogen atmosphere, the residue was dissolved in 0.2 ml of ethanol, and the solution was subjected to 5assaying by HPLC. The HPLC was carried out under per se known analytical conditions using an electrochemical detector, and oxidized coenzyme Q10 and reduced coenzyme Q10 were respectively quantitated. The residual reduced coenzyme Q10 percentage was calculated as a percentage
lOrelative to the amount of reduced coenzyme Q10 on the occasion of preparing liposomes.
As a result, the antioxidant-free liposomes underwent oxidation in such a manner that the residual percentage lowered to 10% or below during 1 to 2 days of storage,
15whereas the residual percentage was 63% after 2 days of storage in the case of vitamin E acetate-containing liposomes, 76% after 3 days of storage in the case of sodium thiosulfate-containing liposomes, 54% after 8 days of storage in the case of citric acid-containing liposomes,
20or 68% after 15 days of storage in the case of vitamin C-containing liposomes. However, the addition of vitamin E (a-tocopherol) failed to produce any remarkable effect.
It was thus found that some antioxidants are strongly effective and others are mildly effective in stabilizing
25reduced coenzyme Q10 in liposomes. More specifically,
vitamin C was the most effective, followed by citric acid, sodium thiosulfate, vitamin E acetate, and vitamin E in that order.
30(Example 2) Oxidation stability of liposome-coated reduced coenzyme Q10 as given by a chelating agent
The effect of a chelating agent on the residual reduced coenzyme Q10 percentage was evaluated in the same manner as in Example 1. Liposomes containing 0.05% (w/v)
35of ethylenediaminetetraacetic acid were prepared in the

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same manner as in Example 1 and stored in the air at 40°C for evaluating the stabilizing effect of the chelating agent.
The reduced coenzyme Q10 in chelating agent-free 51iposomes was oxidized to a residual percentage of 10% or below during 1 to 2 days of storage, whereas the ethylenediaminetetraacetic acid-containing liposomes showed a residual percentage of 76% after 43 days of storage.
It is known in the art that the chelating agent lOethylenediaminetetraacetic acid is more or less effective in protecting substances susceptible to oxidation. However, the finding obtained in this example that it is comparable or superior in such protective effect to
antioxidants was guite unexpected. 15
(Example 3) Effect of an antioxidant on a surfactant
solution - (1)
To a 0.1% aqueous polysorbate 80 solution containing
0.005% (w/v) of reduced coenzyme Q10 was added vitamin C, 20as an antioxidant, to a concentration of 0.05% (w/v), the
solution was stored at 40°C, and the effect of the
antioxidant was evaluated.
The solution without addition of the antioxidant gave
a residual percentage of 27% after 6 days of storage. On 25the contrary, 88% of the reduced form was retained in the
vitamin C-containing solution even after 6 days of storage.
(Example 4) Effect of an antioxidant on a surfactant solution - (2)
30 To a solution containing 0.1% of polyoxyethylene hydrogenated castor oil (HCO-60; product of Nikko Chemicals) and 0.005% (w/v) of reduced coenzyme Q10 was added vitamin C or citric acid, as an antioxidant, to a concentration of 0.05% (w/v), the solution was stored at
35400C, and the effect of the antioxidant was evaluated.

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The solution without addition of the antioxidant gave a residual percentage of 17% after 7 days of storage. On the contrary, 73% of the reduced form was retained in the vitamin C-containing solution even after 14 days of 5storage, and 61% of the reduced form was maintained in the citric acid-containing solution even after 71 days of storage.
(Example 5) Effect of a chelating agent on a surfactant
lOsolution - (1)
The effect of a chelating agent was evaluated in the same manner as in Example 3 except that
ethylenediaminetetraacetic acid was used as the chelating agent in lieu of the antioxidant.
15 The solution without addition of the chelating agent gave a residual percentage of 27% after 6 days of storage, whereas 80% of the reduced form was retained in the ethylenediaminetetraacetic acid-containing solution even after 78 days of storage, indicating the same potent
20protective effect as in the case of liposomes..
(Example 6) Effect of a chelating agent on a surfactant solution - (2)
The effect of a chelating agent was evaluated in the
25same manner as in Example 4 except that
ethylenediaminetetraacetic acid was used as the chelating agent in lieu of the antioxidant.
The solution without addition of the chelating agent gave a residual percentage of 17% after 7 days of storage,
30whereas 74% of the reduced form was retained in the
ethylenediaminetetraacetic acid-containing solution even after 167 days of storage. The result obtained that reduced coenzyme Q10 can be maintained at high residual percentages under such high temperature conditions as 40°C
35over a period as long as nearly half a year was quite

15 15
unexpected.
INDUSTRIAL APPLICABILITY
In accordance with the invention, liquid preparations 5containing reduced coenzymes Q, which are highly useful as antioxidant substances or nutritional supplement components, can be provided more stably.

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WE CLAIM:
1. An aqueous solution containing a reduced coenzyme Q represented by the following formula (1):

in the formula, n represents an integer of 1 to 12:
which comprises a chelating agent such as herein described in the same aqueous solution,
wherein the proportion of the reduced coenzyme weight to the volume of the aqueous solution is not lower than 0.001 w/v % but not higher than 5 w/v %, and
the concentration of chelating agent is not lower than 0.001 w/v % but not higher than 10 w/v % relative to the whole amount of the aqueous solution.
2. An aqueous solution containing a reduced coenzyme Q represented by the following formula (1):


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in the formula, n represents an integer of 1 to 12:
which comprises at least one antioxidant selected from the group consisting of citric acid, a citric acid derivative such as herein described and sodium thiosulfate, in the same aqueous solution,
wherein the proportion of the reduced coenzyme weight to the volume of the aqueous solution is not lower than 0.001 w/v % but not higher than 5 w/v %, and
the concentration of antioxidant is not lower than 0.01 w/v % but not higher than 50 w/v % relative to the whole amount of the aqueous solution.
3. An aqueous solution as claimed in Claim 1 or 2, wherein the reduced coenzyme Q is reduced coenzyme Q10 represented by the following formula (2)

4. An aqueous solution as claimed in any of claims 1 to 3, wherein the chelating agent is at least one agent selected from the group consisting of ethyl en ediaminetetracetic acid, salts of ethylenediaminetetracetic acid, hydroxyethylethylenediaminetetraacetic acid, salts of hydroxyethylethylenediamine-tetraacetic acid, diethylenetriaminepentaacetic acid, salts of diethylenetriaminepentaacetic acid, sodium gluconate or hydroxyethylidenediphosphonic acid.
The present invention provides a reduced coenzyme Q-containing solution capable of being retained, more stably 5against oxidation, by using a reduced coenzyme Q which is hydrophobic and susceptible to oxidation and thus unstable.
The aqueous solution containing a reduced coenzyme Q obtained in the present invention is a solution containing a reduced coenzyme Q and an antioxidant such as vitamin C lOand/or a chelating agent such as ethylenediaminetetraacetic acid.


Documents:


Patent Number 212685
Indian Patent Application Number 00397/KOLNP/2004
PG Journal Number 50/2007
Publication Date 14-Dec-2007
Grant Date 12-Dec-2007
Date of Filing 25-Mar-2004
Name of Patentee KANEKA CORPORATION
Applicant Address 2-4, NAKANOSHIA3-CHOME KITA-KU, OSAKA-SHI OSAKA-530-8288
Inventors:
# Inventor's Name Inventor's Address
1 FUJII KENJI 5-11 HANYAMANAKAODAI
2 HOSOE KAZUNORI 8-17, NISHIHATA 3-CHOME TAKASAGO-SHI JAPAN.
3 HIDAKATAKAYOSHI 21-8 HONTAMON 2-CHOME TARUMI-KU, KOBEHI JAPAN.
4 KAWABE TAIZO 7-203, SHIMONODA 1 CHOME SHIKAMA KU HIMEJI SHI JAPAN.
PCT International Classification Number C07C
PCT International Application Number PCT/JP02/10516
PCT International Filing date 2002-10-10
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 2001-312181 2001-10-10 Japan