Patent Description:
In recent years, it has been revealed that curcumin has a pharmacological action such as a tumor formation inhibitory action, an anti-oxidant action, an anti-inflammatory action, a cholesterol-lowering action, an anti-allergic action, a brain disease preventive action, and a heart disease preventive and therapeutic action, and it has been studied to use the curcumin, for example, for food (for example, functional food), pharmaceuticals, or cosmetics.

However, it is known that curcumin is hardly soluble in water, and therefore, is hardly absorbed in the body when orally ingested as it is. In addition, it is indicated that most of the curcumin that has been slightly absorbed by oral ingestion is present in the blood as a curcumin conjugate conjugated with glucuronic acid and/or sulfuric acid, and free form of curcumin is present only slightly in the blood (Non Patent Literature <NUM>).

In general, it is well known that in a case where foreign substances are absorbed in the body, many of the foreign substances are inactivated by in vivo metabolism such as conjugation. In order to evaluate whether or not a curcumin conjugate has a pharmacological action, A. Pal, et al. investigated the cell growth inhibitory effect of a curcumin conjugate on KBM-<NUM>, Jurkat cells, U266 and A549 cell lines in vitro, and found that free curcumin has an effect of suppressing the growth of these cells (anti-cancer effect) , while indicating that there is no such an effect with the curcumin conjugate (curcumin monoglucuronide and curcumin diglucuronide) (Non Patent Literature <NUM>). In addition, when investigated the influence on the growth inhibitory effect of a curcumin conjugate on human hepatocellular carcinoma cell lines (HepG2 cells) and the gene expression, by oral administration, Shoji, et al. found the cell growth inhibitory effect with free curcumin, however, the effect was not found with the curcumin conjugate (curcumin monoglucuronide), and indicated that as to also the influence on the gene expression, the action was extremely low with the curcumin conjugate (curcumin monoglucuronide) as compared to that with free curcumin (Non Patent Literature <NUM>). As described above, it is considered that curcumin is also conjugated, as a result of which a pharmacological action possessed by the curcumin is inactivated.

For this reason, in order to sufficiently obtain the expected pharmacological action of curcumin, it is required to sufficiently increase the concentration of free curcumin in the blood. However, due to the reasons described above, it is extremely difficult to increase the concentration of free curcumin in the blood even if curcumin is ingested orally.

Therefore, it is conceivable to administer curcumin parenterally into the body, however, since curcumin is hardly soluble in water, curcumin itself cannot be administered parenterally. For this reason, for example, a method in which a poorly water-soluble compound such as curcumin is subjected to a treatment for increasing the solubility, and then the obtained preparation is administered parenterally has been proposed (Patent Literatures <NUM> and <NUM>). <NPL> discloses that glucuronidated curcumin may act as a prodrug that is specifically activated (deconjugated) by murine bone marrow cells to form curcumin, which can mediate anti - tumor cell effects within bone that limit the progression of osteolytic human BCa metastases in turmeric - treated mice.

However, in order to obtain a curcumin aqueous solution by inclusion of curcumin with cyclodextrin, the amount of cyclodextrin is required to be <NUM> to <NUM> times the amount of the curcumin (Patent Literature <NUM>), and the curcumin aqueous solution was not usable for parenteral administration.

Therefore, an obj ect of the present invention is to provide a curcumin preparation that is highly water soluble, can maintain the concentration of free curcumin in the blood sufficiently high by being administered parenterally, can effectively obtain a pharmacological action of curcumin, and is highly safe.

Therefore, the present inventors have conducted intensive studies in order to solve the problem described above, it was found that when a water-soluble substance conjugate of curcumin is administered parenterally, surprisingly, the concentration of free curcumin in the blood can be maintained at a high level, and as a result, a pharmacological action possessed by curcumin can be sufficiently obtained. In addition, the curcumin conjugate contained in the pharmaceutical composition for parenteral administration is an in vivo metabolite of curcumin, and therefore, has extremely high safety.

That is, the present invention is to provide the following.

The pharmaceutical composition for parenteral administration according to the present invention, in which a specific water-soluble substance conjugate of curcumin is contained as an active component, can maintain the concentration of free curcumin in the blood at a high level for a long period of time by being administered intravenously, and as a result, a pharmacological action (for example, anti-tumor action) possessed by curcumin can beeffectively obtained. In addition, the pharmaceutical composition for parenteral administration is a water-soluble substance conjugate of curcumin, which contains an in vivo metabolite of curcumin as an active component, and therefore, has extremely high safety.

The pharmaceutical composition for parenteral, specifically intravenous administration according to the present invention contains a specific water-soluble substance conjugate of curcumin as an active component.

In the present invention, as the curcumin, chemically synthesized curcumin may be used, or curcumin distributed as turmeric pigment may be used. Examples of the turmeric pigment include powdered turmeric obtained by pulverizing a dried rhizome of curcuma longa LINNE, crude curcumin or oleoresin (turmeric oleoresin) obtained by extracting the powdered turmeric with an appropriate solvent (for example, ethanol, fat and oil, propylene glycol, hexane, or acetone), and purified curcumin.

In addition, curcumin includes both of keto and enol forms of curcumin, which are tautomers.

The water-soluble substance forming a conjugate with curcumin, is a water-soluble substance usually present in a living body, namely from glucuronic acid.

Herein, as the amino acid, an amino acid present in a living body, for example, an essential amino acid can be mentioned.

The binding molar ratio of the curcumin in a curcumin conjugate to the water-soluble substance is prefer ably curcumin : water-soluble substance = <NUM> : <NUM> to <NUM> : <NUM>, more preferably curcumin : water-soluble substance = <NUM> : <NUM> to <NUM> : <NUM>, and further more preferably curcumin : water-soluble substance = <NUM> : <NUM>.

The conjugated form (binding form) of the curcumin and the water-soluble substance is a form of the following formula (<NUM>). <CHM>
(in the formula, at least one of R<NUM> is a residue of glucuronic acid as the water-soluble substance, and R<NUM> is a hydrogen atom.

A water-soluble substance conjugate of curcumin can be produced by the method described in Non Patent Literature <NUM> or <NUM>.

The form of the pharmaceutical composition for parenteral administration containing a water-soluble substance conjugate of curcumin is not limited as long as it is administered intravenuously as a composition for injection (injection).

The content of the water-soluble substance of curcumin in the pharmaceutical composition for parenteral administration according to the present invention is not particularly limited, and is preferably <NUM> to <NUM>% by mass, more preferably <NUM> to <NUM>% by mass, and furthermore preferably <NUM> to <NUM>% by mass.

In the composition for parenteral administration according to the present invention, in addition to the active components described above, for example, water, a saline solution, a pH adjusting agent, sugars, an acid, an alkali, a buffer agent, an isotonizing agent, a stabilizer, an analgesic, and an antiseptic agent can be mixed.

Herein, examples of the sugars include monosaccharides, disaccharides, trisaccharides, polysaccharides, and a sugar alcohol. Examples of the acid and the alkali include a water-soluble inorganic acid, a water-soluble inorganic acid salt, a water-soluble organic acid, a water-soluble organic acid salt, an amino acid, and an amino acid salt.

In addition, the form of the composition for parenteral administration according to the present invention may be a form of a powder filler (crystal or freeze-dried product) to be dissolved at the time of use, or may be a form of an aqueous solution.

As described in Examples later, the composition for parenteral administration according to the present invention can maintain the concentration in blood of free curcumin having a pharmacological action at a high level over a long period of time by being administered intravenously. In addition, the composition for parenteral administration exhibits an excellent pharmacological action (for example, anti-tumor effect) bybeing administered intravenously, and therefore, is useful particularly as an anti-cancer agent, an anti-inflammatory agent, a cholesterol-lowering agent, an anti-allergic agent, a cognitive function improving agent, or a heart disease preventive and therapeutic agent, and in particular, is useful as an anti-cancer agent.

Next, the present invention will be described in more detail by way of Examples.

As the test animals, <NUM>-week old SD rats (male, with a body weight of <NUM> to <NUM>, CHARLES RIVER LABORATORIES JAPAN, INC. ) were used.

The intravenous administration was performed as follows: a predetermined amount of the curcumin monoglucuronide prepared in Production Example <NUM> was dissolved into water for injection so as tobe <NUM>/kg; and the obtained preparation was administered intravenously to each of the test animals (n = <NUM>) fasted for <NUM> hours or more before the administration via an indwelling catheter in the femoral artery of each of the test animals. In this regard, the catheter was indwelled in the femoral artery under isoflurane anesthesia on the day of administration and blood collection.

Blood was collected via a catheter from the femoral artery of each of test animals by using a heparin-treated syringe under the restraint condition in a Ballman cage without anesthesia. In this regard, around <NUM> of blood was collected after each lapse of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> minutes from the start of intravenous administration, and the collected blood was centrifuged (at <NUM>, <NUM> rpm, for <NUM> minutes, at <NUM>) to obtain the plasma.

The concentration of each of the curcumin conjugate and free curcumin in plasma was determined by the following methods.

Into <NUM>µL of the collected plasma, <NUM>µL of a <NUM> acetate buffer solution (pH <NUM>) and <NUM>µL of a β-glucuronidase solution (around <NUM>, <NUM> units/mL) or distilled water were added, and the resultant mixture was kept at <NUM> for <NUM> hour, and then into the resultant mixture, <NUM>µL of <NUM>% (v/v) methanol containing <NUM> ng/mL of mepronil, which was an internal standard liquid, was added. Next, into the mixture, <NUM> of chloroform was added, and the resultant mixture was stirred for <NUM> minute with a vortex mixer, then the mixture was treated for <NUM> minutes by using an ultrasonic generator, and the treated mixture was fractionated into a chloroform layer and a water layer by centrifugation (<NUM>,<NUM>×g, for <NUM> minutes, at room temperature). Further, this fractionation process was repeated twice. After that, the chloroform layer was collected, and dried and solidified by distilling off the solvent with a vacuum centrifugal concentrator, and to the dried and solidif iedmatter, <NUM>µL of <NUM>% (v/v) methanol was added, and then the resultant mixture was centrifuged (<NUM>,<NUM>×g, for <NUM> minutes, at room temperature) to collect a supernatant liquid.

In this regard, in the following test, a supernatant liquid prepared by using a β-glucuronidase solution was used as an enzyme-treated sample, and a supernatant liquidprepared by using distilled water in place of the β-glucuronidase solution was used as a non-enzyme-treated sample.

By analyzing <NUM>µL of the enzyme-treated or non-enzyme-treated sample prepared in the above-described "a. Pretreatment" using LC-MS/MS (manufactured by Shimadzu Corporation) , the concentration of each of the curcumin conjugate and free curcumin in plasma was determined.

That is, by measuring the concentration of the curcumin contained in the enzyme-treated sample that had been obtained by the treatment with β-glucuronidase, the total concentration of curcumin in plasma was measured.

On the other hand, by measuring the concentration of the curcumin in the non-enzyme-treated sample that had not been treated with β-glucuronidase, the concentration of free curcumin in plasma was measured.

Further, by subtracting the concentration of free curcumin from the total concentration of curcumin, the concentration of the curcumin conjugates contained in plasma was calculated.

In this regard, the LC-MS/MS analysis conditions were set to be a LC column of Atlantis T3 (<NUM> × <NUM>, <NUM>, manufactured by Waters), a column temperature of <NUM>, a flow rate of <NUM>/min, and a mobile phase of A of a <NUM>% formic acid aqueous solution and B of <NUM>% formic acid/acetonitrile, and the gradient elution was performed under the following conditions (Table <NUM>). In addition, the MS analysis conditions were set to be an ionization mode of Electron Spray thermo ionization (ESI), Positive, a measurement mode of Multiple Reaction Monitoring (MRM), curcumin of <NUM> -> <NUM> (m/z), and mepronil of <NUM> → <NUM> (m/z), and the evaluation was performed.

Further, the calibration curve used for quantifying curcumin was prepared with the measurement under the conditions similar to those described above by using various kinds of standard solutions (curcumin concentration of <NUM> to <NUM> ng/mL) , which had been prepared by adding <NUM>µL of a <NUM>% ethanol solution containing <NUM> ng/mL of mepronil into <NUM>µL of a <NUM>% (v/v) methanol solution (curcumin standard solution) containing the curcumin in each amount of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> ng/mL.

Concentration of each of the curcumin conjugates and free curcumin in plasma in a case where the curcumin conjugates had been administered intravenously was examined (<FIG>).

The concentration of curcumin conjugates in plasma was <NUM> ± <NUM> ng/mL after the lapse of <NUM> minute from the intravenous administration, <NUM> ± <NUM> ng/mL after the lapse of <NUM> minutes from the intravenous administration, <NUM> ± <NUM> ng/mL after the lapse of <NUM> minutes from the intravenous administration, <NUM> ± <NUM> ng/mL after the lapse of <NUM> minutes from the intravenous administration, <NUM> ± <NUM> ng/mL after the lapse of <NUM> minutes from the intravenous administration, <NUM> ± <NUM> ng/mL after the lapse of <NUM> minutes from the intravenous administration, <NUM> ± <NUM> ng/mL after the lapse of <NUM> hour from the intravenous administration, <NUM> ± <NUM> ng/mL after the lapse of <NUM> hours from the intravenous administration, <NUM> ± <NUM> ng/mL after the lapse of <NUM> hours from the intravenous administration, and <NUM> ± <NUM> ng/mL after the lapse of <NUM> hours from the administration, and was reduced with the passage of time. In addition, at this time, AUC <NUM>-<NUM> (ng·h/mL) was <NUM> ± <NUM> ng/mL, and Cmax (ng/mL) was <NUM> ± <NUM> ng/mL.

On the other hand, it was found that the concentration of free curcumin in plasma was not observed before the intravenous administration of curcumin conjugates, however, was <NUM> ± <NUM> ng/mL after the lapse of <NUM> minute from the intravenous administration of curcumin conjugates, and the free curcumin were present at a high concentration in plasma. In addition, it was found that the concentration of free curcumin in plasma was <NUM> ± <NUM> ng/mL after the lapse of <NUM> minutes from the intravenous administration, <NUM> ± <NUM> ng/mL after the lapse of <NUM> minutes from the intravenous administration, <NUM> ± <NUM> ng/mL after the lapse of <NUM> minutes from the intravenous administration, <NUM> ± <NUM> ng/mL after the lapse of <NUM> minutes from the intravenous administration, <NUM> ± <NUM> ng/mL after the lapse of <NUM> minutes from the intravenous administration, <NUM> ± <NUM> ng/mL after the lapse of <NUM> hour from the intravenous administration, <NUM> ± <NUM> ng/mL after the lapse of <NUM> hours from the intravenous administration, <NUM> ± <NUM> ng/mL after the lapse of <NUM> hours from the intravenous administration, and <NUM> ± <NUM> ng/mL after the lapse of <NUM> hours from the intravenous administration, and the free curcumin was maintained at a high concentration in plasma for a long period of time. In addition, at this time, AUC <NUM>-<NUM> (ng·h/mL) was <NUM> ± <NUM> ng/mL, and Cmax (ng/mL) was <NUM> ± <NUM> ng/mL.

As described above, it was found that by administering curcumin conjugates parenterally, the concentration of free curcumin having a pharmacological action in blood can be maintained at a high level over a long period of time.

As the test animals, <NUM>-week old BALB/cAnNcr j-nu/nu (homo) mice (female, with a body weight of around <NUM> to <NUM>, CHARLES RIVER LABORATORIES JAPAN, INC. ), which had been purchased and acclimated to the environment for around <NUM> days, was used.

<NUM> × <NUM><NUM> or <NUM> × <NUM><NUM> colorectal cancer cells derived from adult human male colon HCT116 (ATCC No. CCL-<NUM>) were transplanted subcutaneously into the flank of each of the test animals by using a <NUM> injection needle.

Into each of the mice (n = <NUM> or <NUM>) inoculated with the cancer cells (HCT116), after each lapse of <NUM>, <NUM>, <NUM>, <NUM> and <NUM> days from the start of inoculation or after each lapse of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> days from the start of inoculation, <NUM> to <NUM>µL of the injection prepared by dissolving the curcumin monoglucuronide that had been prepared in the above-described Production Example <NUM> into water for injection (prepared so that curcumin monoglucuronide was administered in an amount of <NUM> or <NUM> per kg body weight) was administered intravenously by using an injection needle, and further the tumor size of each of the mice was examined with the passage of days. In this regard, in the control group, mice to which water for injection excluding curcumin monoglucuronide had been administered intravenously were used.

For the tumor size, after each lapse of <NUM>, <NUM>, <NUM>, <NUM> and <NUM> days from the start of administration or after each lapse of <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> days from the start of administration, the length, width and height of the tumor were measured by using a vernier caliper, and the tumor size was calculated by the following equation.

For the mice (n = <NUM>) inoculated with the <NUM> × <NUM><NUM> cancer cells (HCT116), the tumor after the lapse of <NUM> hours from the intravenous administration of curcumin monoglucuronide (<NUM>/kg) after the lapse of <NUM> days from the start of inoculation was removed from each of the mice, the removed tumor was rinsed with a saline solution, and then immediately frozen in liquid nitrogen, and the frozen tumor was stored in an ultra-low temperature tank at -<NUM>.

The frozen and stored tumor was thawed while cooling in ice, an adequate amount of a saline solution was added to the thawed tumor, and the thus obtained tumor was homogenized with a glass homogenizer, the concentration of each of the curcumin conjugates and free curcumin in the pulverized tumor obtained as described above were measured to examine the concentration of each of the curcumin conjugates and free curcumin contained in the tumor tissue.

The anti-tumor effect was examined in a case where <NUM> of curcumin conjugates (curcumin monoglucuronide) per kg body weight of a mouse was administered parenterally (<FIG>).

In a case where curcumin conjugates (curcumin monoglucuronide) were administered parenterally (<NUM>/kg - mouse), the tumor size was <NUM> ± <NUM><NUM> after the lapse of <NUM> day from the start of administration, <NUM> ± <NUM><NUM> after the lapse of <NUM> days from the start of administration, <NUM> ± <NUM><NUM> after the lapse of <NUM> days from the start of administration, <NUM> ± <NUM><NUM> after the lapse of <NUM> days from the start of administration, and <NUM> ± <NUM><NUM> after the lapse of <NUM> days from the start of administration.

On the other hand, in a case of the control group, the tumor size was <NUM> ± <NUM><NUM> after the lapse of <NUM> day from the start of administration, <NUM> ± <NUM><NUM> after the lapse of <NUM> days from the start of administration, <NUM> ± <NUM><NUM> after the lapse of <NUM> days from the start of administration, <NUM> ± <NUM><NUM> after the lapse of <NUM> days from the start of administration, and <NUM> ± <NUM><NUM> after the lapse of <NUM> days from the start of administration, and showed larger values at any time as compared to those in the case where the curcumin conjugates (curcumin monoglucuronide) had been administered parenterally.

In addition, when the biochemical test using the serum obtained by the blood collection after the lapse of <NUM> days from the start of administration was referred to Oriental Yeast Co. (biochemical test set (liver and biliary diseases set), <NUM> items), no abnormality was found in the test values.

In a case where curcumin conjugates (curcumin monoglucuronide) were administered parenterally (<NUM>/kg - mouse), the tumor size was <NUM> ± <NUM><NUM> after the lapse of <NUM> day from the start of administration, <NUM> ± <NUM><NUM> after the lapse of <NUM> days from the start of administration, <NUM> ± <NUM><NUM> after the lapse of <NUM> days from the start of administration, <NUM> ± <NUM><NUM> after the lapse of <NUM> days from the start of administration, <NUM> ± <NUM><NUM> after the lapse of <NUM> days from the start of administration, <NUM> ± <NUM><NUM> after the lapse of <NUM> days from the start of administration, and <NUM> ± <NUM><NUM> after the lapse of <NUM> days from the start of administration.

On the other hand, in a case of the control group, the tumor size was <NUM> ± <NUM><NUM> after the lapse of <NUM> day from the start of administration, <NUM> ± <NUM><NUM> after the lapse of <NUM> days from the start of administration, <NUM> ± <NUM><NUM> after the lapse of <NUM> days from the start of administration, <NUM> ± <NUM><NUM> after the lapse of <NUM> days from the start of administration, <NUM> ± <NUM><NUM> after the lapse of <NUM> days from the start of administration, <NUM> ± <NUM><NUM> after the lapse of <NUM> days from the start of administration, and <NUM> ± <NUM><NUM> after the lapse of <NUM> days from the start of administration, and showed larger values at any time as compared to those in the case where the curcumin conjugates (curcumin monoglucuronide) had been administered parenterally.

In addition, when the concentration of each of the curcumin conjugates and free curcumin contained in the tumor tissue was examined, the concentration of the curcumin conjugates in the tumor tissue was <NUM> ± <NUM> ng/g, and the concentration of the free curcumin in the tumor tissue was <NUM> ± <NUM> ng/g.

From these results, it was found that the curcumin conjugates exerted the effect of significantly suppressing the tumor growth by being administered parenterally. Further, it was observed that free curcumin having a pharmacological action was present in the tumor at a high concentration, and the tumor growth inhibitory effect was presumed to be due to the free curcumin.

Intravenous administration of a single dose of curcumin monoglucuronide was performed to each of the mice at each dose of <NUM>, <NUM>, <NUM> and <NUM>/kg, and the toxicity thereof was evaluated. In this regard, the number of mice in each group was set to be n = <NUM>.

As the test animals, <NUM>-week old Slc:ICR mice (male, with a body weight of <NUM> to <NUM>, Japan SLC, Inc. ), which had been purchased and acclimated to the environment for around <NUM> days, was used.

<NUM> to <NUM>µL of the solution prepared by dissolving the curcumin monoglucuronide prepared in the above-described Production Example <NUM> into a saline solution (in cases of <NUM>, <NUM> and <NUM>/kg) or water for injection (in a case of <NUM>/kg) (solution being prepared so that curcuminmonoglucuronide was administered in an amount of <NUM>, <NUM>, <NUM> or <NUM> per kg body weight) was administered intravenously to each of the mice (n=<NUM>) by using an injection needle.

Observation of the state of each of the mice was performed by recording the type, extent, expression time, recovery time, and death (death detection) time of all of the toxic signs observed macroscopically.

Observation was performed at a time immediately after administration, at a time after each lapse of <NUM> minutes, <NUM>, <NUM>, <NUM> and <NUM> hours from the start of administration, and once a day from the day following the date of administration (up to after the lapse of <NUM> days from the start of administration). In this regard, in the <NUM>-mg/kg administration group, acute symptoms were observed immediately after administration, and therefore, observation was frequently performed up to after the lapse of <NUM> hour from the start of administration.

The body weight was measured by using an electronic balance (UW4200S, Shimadzu Corporation) immediately before administration, and after each lapse of <NUM>, <NUM>, <NUM>, <NUM> and <NUM> days from the administration.

Blood biochemical test was performed by the following method. That is, mice were fasted for <NUM> hours or more after the end of the mouse observation period (after the lapse of <NUM> days from the start of administration), the fasted mice were subjected to laparotomy under anesthesia by intraperitoneal administration of pentobarbital sodium, and the blood was collected from the abdominal aorta (the amount of the blood was <NUM> or more). Next, the collected blood was anti-coagulated with heparin sodium, and then the anti-coagulated blood was centrifuged (<NUM>, <NUM> rpm, for <NUM> minutes) to obtain the plasma, and transaminase (AST and ALT) contained in the plasma was measured.

After the end of the observation period (after the lapse of <NUM> days from the start of administration), autopsy was performed as follows: blood was collected from each of the mice under anesthesia with pentobarbital sodium; and then the mice were sacrificed by exsanguination by cutting the abdominal aorta and the caudal vena; each of the sacrificed mice was dissected according to a pathological technique; and the cranium, the intrathoracic organs, the intraperitoneal organs, and the tissues thereof were observed macroscopically.

With regard to the state of a mouse in each administration group, in the <NUM>-mg/kg administration group, no abnormality was found in the states of all of the mice (n = <NUM>) throughout the observation period.

In the <NUM>-mg/kg administration group, piloerection was observed in <NUM> case (n = <NUM>) after the lapse of <NUM> hour from the start of administration, however, the mouse in this case was recovered later.

In the <NUM>-mg/kg administration group, reduction of locomotor activity was observed in all of the mice (n = <NUM>) from immediately after administration to after the lapse of <NUM> hours from the administration, loose stools were observed in all of the cases (n = <NUM>) from after the lapse of <NUM> minutes to <NUM> hour from the start of administration, and piloerection was observed in <NUM> cases (n = <NUM>) from after the lapse of <NUM> minutes to <NUM> hours from the start of administration, however, all of the symptoms were recovered later.

In the <NUM>-mg/kg administration group, prone position and reduction of locomotor activity were observed in all of the mice (n = <NUM>) from immediately after administration to after the lapse of <NUM> minutes from the start of administration. In addition, bradypnea was observed in <NUM> cases (n = <NUM>) immediately after administration and in <NUM> case (n = <NUM>) after the lapse of <NUM> minutes from the start of administration. Further, dyspnea was observed in <NUM> cases (n = <NUM>) within <NUM> minutes from the start of administration. Furthermore, paralytic gait was observed in <NUM> cases (n = <NUM>) within <NUM> minutes from the start of administration and in <NUM> case (n = <NUM>) from after the lapse of <NUM> minutes to <NUM> hour from the start of administration. Moreover, death was observed in <NUM> cases (n = <NUM>) within <NUM> minutes from the start of administration and in <NUM> case (n = <NUM>) from after the lapse of <NUM> minutes to <NUM> hour from the start of administration.

From these results, it was found to be safe at a single dose of <NUM>/kg or less, and found that <NUM>% lethal dose (LD<NUM>) was between <NUM>/kg and <NUM>/kg.

Changes in the body weight of a mouse in each group are shown in <FIG>. In the <NUM>-mg/kg administration group, all of the animals showed a steady weight gain up to after the lapse of <NUM> days from the start of administration. Slight weight loss was observed in <NUM> case (n = <NUM>) after the lapse of <NUM> day from the start of administration in the <NUM>-mg/kg administration group and in <NUM> cases (n = <NUM>) after the lapse of <NUM> day from the start of administration in the <NUM>-mg/kg administration group, however, all of the animals showed a steady weight gain up to after the lapse of <NUM> days from the start of administration.

For the blood biochemical test of a mouse in each group (<NUM>-, <NUM>- and <NUM>-mg/kg administration groups) , theAST levels in the <NUM>-, <NUM>- and <NUM>-mg/kg administration groups were <NUM> ± <NUM>, <NUM> ± <NUM> and <NUM> ± <NUM> IU/L, respectively, and the ALT levels in the <NUM>-, <NUM>- and <NUM>-mg/kg administration groups were <NUM> ± <NUM>, <NUM> ± <NUM> and <NUM> ± <NUM> IU/L, respectively, and which showed normal levels, respectively. In this regard, the test was not conducted in the <NUM>-mg/kg administration group because all of the mice died.

When autopsy was performed in each group (<NUM>-, <NUM>- and <NUM>-mg/kg administration groups) , no abnormality was found in all of the administration groups. In this regard, the autopsy was not performed in the <NUM>-mg/kg administration group because all of the mice died.

From these results, it was found to be safe at a single dose of up to <NUM>/kg without finding any abnormalities.

It was examined whether or not curcumin diglucuronide (manufactured by SynInnova Laboratories Inc. ) was deconjugated with β-glucuronidase. In this regard, as a control, curcumin monoglucuronide was used for the examination.

As the curcumin monoglucuronide, curcumin monoglucuronide prepared in the above-described Production Example <NUM> was used. In addition, as the curcumin diglucuronide, commercially available curcumin diglucuronide (manufactured by SynInnova Laboratories Inc. ) was used.

Into <NUM>µL of a <NUM> acetate buffer solution (pH <NUM>) and <NUM>µL of a β-glucuronidase solution (around <NUM>, <NUM> units/mL) , <NUM>µL of curcumin monoglucuronide (<NUM>µg/mL, n = <NUM>) or <NUM>µL of curcumin diglucuronide (<NUM>µg/mL, n = <NUM>) was added, and the resultant mixture was kept at <NUM> for <NUM> hour, and into this mixture, <NUM>µL of <NUM>% (v/v) methanol containing <NUM> ng/mL of mepronil, which was an internal standard liquid, was added. Next, into the resultant mixture, <NUM> of chloroform was added, and the mixture was stirred for <NUM> minute with a vortex mixer, and then the stirred mixture was treated for <NUM> minutes by using an ultrasonic generator, and the treated mixture was fractionated into a chloroform layer and a water layer by centrifugation (<NUM>,<NUM>×g, for <NUM> minutes, at room temperature). Further, this fractionation process was repeated twice. After that, the chloroform layer was collected, and dried and solidified by distilling off the solvent with a vacuum centrifugal concentrator, and to the dried and solidifiedmatter, <NUM>µL of <NUM>% (v/v) methanol was added, and then the resultant mixture was centrifuged (<NUM>,<NUM>×g, for <NUM> minutes, at room temperature) to collect a supernatant liquid, and the collected supernatant liquid was taken as an enzyme-treated sample.

In <NUM>µL of the enzyme-treated sample prepared in the above (<NUM>), free curcumin was measured by the method described in "b. Measurement method" in (<NUM>) in Test Example <NUM>.

In this regard, the calibration curve used for quantifying curcumin was prepared with the measurement under the conditions similar to those described above by using a supernatant liquid (curcumin concentration of <NUM> to <NUM> ng/mL), which had been prepared as follows : into <NUM>µL of a <NUM> acetate buffer solution (pH <NUM>) and <NUM>µL of a β-glucuronidase solution (around <NUM>, <NUM> units/mL), <NUM>µL of a <NUM>% (v/v) methanol solution (curcumin standard solution) containing the curcumin in each amount of <NUM>, <NUM>, <NUM> and <NUM> ng/mL and <NUM>µL of a <NUM>% ethanol solution containing <NUM> ng/mL of mepronil were added; and the resultant mixture was subjected to the treatment by chloroform and the solvent removal by distillation with a vacuum centrifugal concentrator in a similar manner as in the above (<NUM>) ; then into this mixture, <NUM>µL of <NUM>% (v/v) methanol was added; and then the resultant mixture was centrifuged (<NUM>,<NUM>×g, for <NUM> minutes, at room temperature) to obtain the supernatant liquid.

The concentration of the curcumin contained in the enzyme-treated sample, which had been obtained by enzymatically treating curcumin monoglucuronide with β-glucuronidase, was <NUM> ± <NUM> ng/mL.

On the other hand, the concentration of the curcumin contained in the enzyme-treated sample, which had been obtained by treating curcumin diglucuronide with β-glucuronidase, was <NUM> ± <NUM> ng/mL.

Claim 1:
A pharmaceutical composition comprising curcumin monoglucuronide of the following formula (f):
<CHM>
as an active component, for use as a medicament selected from an anti-cancer agent, an anti-inflammatory agent, a cholesterol-lowering agent, an anti-allergic agent, a cognitive function improving agent, and a heart disease preventive and therapeutic agent, wherein the medicament is administered intravenously.