JPH11209324A - Antiosteoporotic agent and (r)-8-hydroxy-2,6-dimethyl-2-octenoic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To prepare an antiosteoporotic agent or an agent for inhibiting production of interleukin 6, hardly providing side effect, and capable of manifesting excellent osteoporosis-inhibiting activities and activities for inhibiting the production of the interleukin 6 by using an extract of Cistanchis herba as an active ingredient. SOLUTION: This antiosteoporotic agent contains (A) an extract of Cistanchis herba [e.g. the one obtained by finely cutting the Cistanchis herba, repeating the extraction of the cut Cistanchis herba with a solvent such as water, acetone and a lower alcohol for 2-10 times, and optionally subjecting the obtained extract to concentration, liquid-liquid distribution, adsorption chromatography or the like, and concretely the one containing (R)-8-hydroxy-2,6-dimethyl-2-octenoic acid of the formula] as an active ingredient. Preferably, the medicine is administered by forming the medicine into an orally administrable preparation such as a tablet, a granule and a solution, or a parenterally administrable preparation such as an injection, a suppository and a nebula, and the daily dose of the compound of the formula is 0.01-1,000 μg/kg-body weight, Concretely, the medicine is also useful for treatment or the like of chronic arthrorheumatism.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a medicament for bone diseases such as osteoporosis, and a novel (R) -8-hydroxy-2,6-dimethyl-2-octenoic acid or a pharmaceutically acceptable salt thereof. . Further, the present invention relates to an anti-osteoporosis agent and an interleukin-6 production inhibitor containing the active ingredient as an active ingredient.

[0002]

2. Description of the Related Art Osteoporosis is a disease in which the balance between bone resorption and bone formation is impaired due to metabolic disorders, endocrine disorders, and aging, resulting in a decrease in bone mass and bone porosity. Symptoms include severe back pain and bone fractures resulting from bone erosion, which can lead to bedridden and fatal elderly people. As osteoporosis therapeutic agents, drugs having a lumbar back pain relieving action, a bone resorption suppressing action, an osteogenesis promoting action are considered, and calcitonin, vitamin D, calcium preparations and the like are used. There is still no drug that is sufficiently satisfactory in this respect, and a therapeutic agent for osteoporosis that is highly effective and has few side effects is desired.

[0003] Interleukin 6 (IL-6) is produced from various cells such as monocytes, T cells, B cells, vascular endothelial cells, fibroblasts, and osteoblasts, and differentiates B cells into antibody-producing cells. Induction, induction of acute phase protein synthesis from hepatocytes,
Induction of differentiation of cranial nerve cells, induction of proliferation and differentiation of hematopoietic cells,
It is known to be a substance having a variety of physiological activities such as inducing osteoclast differentiation (Experimental Medicine, Vol. 7, 1
No. 1989). The relationship between IL-6 and various diseases includes, for example, autoimmune diseases in which autoantibody production such as chronic interstitial rheumatism and Castleman syndrome is considered to be involved in the cause or exacerbation of the disease (Euro
pean Journal of Immunology, 18, 1797, Experimental Medicine, 7, 50, 1989). Also, in the area of cancer, it has been reported that IL-6 is an autocrine growth factor for multiple myeloma (Nature, 332, 83, 1).
988) and cancer cachexia (Kakexia) substances (Endocrinology, 128, 265).
7, 1991). In the area of infectious disease, blood IL-6 concentration of sepsis patients is negatively correlated with survival rate (Jour
nal of Experimental Medicine, 169, 333,
1989, Blood, 74, 1704, 1989
Year)) have also been reported. In addition, IL-6 is an acute-phase protein of the inflammatory response, and is considered to be a cause of various symptoms due to inflammation such as fever, chills, and malaise, exhaustion after surgical operations such as organ resection, and delayed recovery.

[0004] Plants collectively referred to as Citrus japonicus include Orobanchaceae, hon-oniku, and oniku. Hongoniku (Cistanche salsa) is an all-parasitic plant that grows in China and infests shrubs. In addition, the Japanese plant Onik (Boschniakia glabra) is also used as a substitute for Hong Onik and is said to have the same effect (Hirokawa Medicinal Plant Encyclopedia, 1963, Hirokawa Shoten, Wakan Crude Drug, 1971, Hirokawa Shoten, etc.) ). However, there is no report at all that the extract of Cistanche salsa is effective for the treatment and prevention of osteoporosis.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a substance useful as a medicament having a therapeutic / preventive effect on osteoporosis and an interleukin-6 production inhibitory effect.

[0006]

Means for Solving the Problems As a result of intensive studies to solve the above problems, it has been found that extracts of Cistanche salsa are capable of suppressing osteoporosis in osteoporosis model mice. Furthermore, the present inventors have found that a novel compound contained in Cistanche salsa, represented by the following formula (1), is effective for the treatment of osteoporosis, and has been found to inhibit the production of interleukin 6, thereby completing the present invention.

[0007] That is, the present invention provides an anti-osteoporosis agent containing an extract of Cistanche salsa as an active ingredient.

Further, the present invention provides the following formula (1)

[0009]

Embedded image

An antiosteoporotic agent comprising as an active ingredient a novel compound (R) -8-hydroxy-2,6-dimethyl-2-octenoic acid or a pharmaceutically acceptable salt thereof represented by the formula (1): The indicated (R) -8-hydroxy-
Provided is an interleukin-6 production inhibitor comprising 2,6-dimethyl-2-octenoic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

[0011]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The raw materials used for obtaining the extract as an active ingredient of the present invention are collectively referred to as "Nikujuyo" as described above. Examples thereof include Hongoniku (Cistanchesalsa) and Onik ( Boschniakia glabra) and the like are considered to have similar effects (Hirokawa Medicinal Plant Encyclopedia, 1963, Hirokawa Shoten,
1971, Hirokawa Shoten, etc.). Therefore, these plants may be used alone or in combination. These plants are already known as crude drugs, and it is considered that there is no particular problem in safety.

The extract of Cistanche salsa as an active ingredient of the present invention is extracted from the above-mentioned raw materials of plants and the like using a solvent capable of effectively extracting the active ingredient, and if necessary, by partially purifying them. can get. The solvent used in this extraction is not particularly limited as long as it is a solvent that can effectively extract the active ingredient.For example, water, acetone, or lower alcohols such as methanol, ethanol, and propanol, or A mixed solution thereof, a chlorine-containing organic solvent such as chloroform, a lower fatty acid ester such as ethyl acetate, or the like can be used.

In extracting the active ingredient of the present invention, the method is not particularly limited as long as the method can effectively extract the active ingredient. The material may be subjected to extraction. Also,
The raw materials may be subjected to extraction as they are, but in order to enhance the extraction efficiency, these raw materials may be finely cut and then extracted with a solvent. Furthermore, the extraction efficiency can be increased by repeating the extraction about 2 to 10 times. The extraction is not particularly limited as long as the method can effectively extract the active ingredient, but the extraction can be performed for several hours to several months, at room temperature or at a high temperature, and sometimes at a low temperature. .

Further, in order to increase the concentration of the active ingredient in the extract, if necessary, the obtained extract is further subjected to means such as concentration, liquid-liquid distribution, adsorption chromatography, normal phase or reverse phase chromatography. It is also possible to attach.

In order to produce the anti-osteoporosis agent according to the present invention, the extract of Cistanche salsa as an active ingredient may be formulated as an active ingredient in combination with a known nontoxic pharmaceutical carrier according to a conventional method. The anti-osteoporosis agent according to the present invention can be administered in various dosage forms. For example, tablets, granules, powders, capsules and other solid preparations, solutions, suspensions, and emulsions can be used as oral preparations. And parenteral administration agents include injections, suppositories, sprays, transdermal absorbers, etc., and these preparations are prepared by conventional means in preparations. can do. Examples of the above non-toxic pharmaceutical carriers include glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, albumin,
Water, physiological saline and the like. Also, if necessary,
Conventional additives such as a stabilizer, a lubricant, a wetting agent, an emulsifier, and a binder can be appropriately added. In the therapeutic agent for osteoporosis or the interleukin-6 production inhibitor according to the present invention, the dose of the active ingredient is appropriately selected and determined according to the patient's age, body weight, symptoms, degree of disease, administration schedule, formulation, and the like. , For example, 1-500g per day
/ Kg body weight, and may be administered in several divided doses per day.

The novel substance (R) -8-hydroxy-2,6-dimethyl-2-octenoic acid of the present invention can be obtained from extracts of Cistanche salsa extract obtained by the above-mentioned method, as shown in Examples. Furthermore, isolation can be achieved by repeating purification until the single substance is obtained by means such as concentration, liquid-liquid distribution, adsorption chromatography, normal phase or reverse phase chromatography. The obtained single substance is represented by the above formula (1) because of its physicochemical properties and the like.
2,6-dimethyl-2-octenoic acid (optical purity 100% e
e) was decided. The corresponding (S) -form has been reported in the literature (Phytochemistry, 22, 255-258, 1983).
This (R) -isomer is a novel compound that has not been published in the literature.

Further, the novel substance (R) -8 according to the present invention
-Hydroxy-2,6-dimethyl-2-octenoic acid is
For example, as shown in the Examples, commercially available (+)-(R) -citronellol can be used as a starting material to chemically synthesize according to the method described in the above-mentioned literature (see Synthesis Examples).

In the present invention, examples of the salt include a sodium salt, a potassium salt, a lithium salt, a calcium salt, an ammonium salt, and a salt with ethanolamine.

The (R) -8-hydroxy-2,6 of the present invention
In order to use dimethyl-2-octenoic acid or a pharmaceutically acceptable salt thereof as an anti-osteoporosis agent or an interleukin-6 production inhibitor, (R)-obtained as described above.
8-Hydroxy-2,6-dimethyl-2-octenoic acid or a pharmaceutically acceptable salt thereof is used as an active ingredient, and may be formulated in accordance with a conventional method in combination with a known nontoxic pharmaceutical carrier. The anti-osteoporosis agent and the interleukin 6 production inhibitor according to the present invention can be administered in various dosage forms. For example, as oral administration agents, solid agents such as tablets, granules, powders, and capsules, solutions Preparations, suspensions, liquid preparations such as emulsions, lyophilized preparations, and the like. Parenteral administration preparations include, in addition to injection preparations, suppositories, sprays, transdermal absorbents, and the like. It can be prepared by conventional means on the formulation. Examples of the non-toxic carrier for pharmaceuticals include, for example, glucose, lactose, sucrose, starch, mannitol,
Examples include dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, albumin, water, and physiological saline.
If necessary, conventional additives such as a stabilizer, a lubricant, a wetting agent, an emulsifier, and a binder can be appropriately added. In the therapeutic agent for osteoporosis or the interleukin-6 production inhibitor according to the present invention, the dose of the active ingredient depends on the patient's age, body weight, symptoms, degree of disease, administration schedule,
It is appropriately selected and determined depending on the formulation form, for example,
The dose is about 0.01 to 1000 μg / kg body weight per day, and the administration may be carried out several times a day.

[0020]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which should not be construed as limiting the invention thereto.

Example 1 (Preparation of Cistanche salsa extract) Cistanche salsa (Uchida Wanhan Yakuhin Co., Ltd., lot number 2226)
29, Hongonik, Cistanche salsa) (100 g) was extracted with 1 L of ethanol, and the extract was distilled off under reduced pressure to obtain about 6 g of a brown oily substance.

Example 2 ((R) -8-hydroxy-2,
Purification of 6-dimethyl-2-octenoic acid Nikujuyou (Uchida Wako Pharmaceutical Co., Ltd., lot number 2324)
19, Hongonik, Cistanche salsa) was extracted with 40 L of ethanol, and the extract was distilled off under reduced pressure to obtain 80.1 g of an oily substance. Add 1 L of water to this oily substance.
And extracted 4 times with 1 L of chloroform. The chloroform fraction was concentrated to dryness to obtain 6.8 g of an oily substance.

After dissolving this oily substance in 90 mL of chloroform-methanol (2: 1), silica gel powder 13.4 was obtained.
g was added and mixed well with stirring, and the solvent was removed under reduced pressure. This silica gel powder was charged into a silica gel column (φ4 × 39 cm) previously filled with hexane. Hexane
The fraction eluted with 6.7 L of a mixed solvent of acetone (90:10) was removed. Then, hexane-acetone (80:
Elution was carried out with the mixed solvent of 20), and 500 mL was fractionated. Fraction No. 6-No. In (8), (R) -8-hydroxy-2,6-dimethyl-2-octenoic acid was eluted. These fractions were collected and dried under reduced pressure to obtain 89 mg of an oily substance.

This oily substance was dissolved in 1 mL of chloroform-methanol (2: 1) to prepare a sample for high performance liquid column chromatography performed under the following conditions (column size: φ20 × 300 mm, carrier: TSKgel ODS).
-80TS (manufactured by Tosoh Corporation), solvent composition; 40% methanol, 60% water, flow rate: 20 mL / min, temperature: 23
° C, detection wavelength: 210 nm, device: Tosoh HLC-8
070). A fraction having a retention time of 48 to 58 minutes was collected, the solvent was distilled off under reduced pressure, and the residue was extracted with ethyl acetate and concentrated to dryness to give 8.5 mg of (R)-as a colorless oily substance.
8-Hydroxy-2,6-dimethyl-2-octenoic acid was obtained. High performance liquid column chromatography, ¹ H-NM
As a result of instrumental analysis of R and the like, it was confirmed that the purity was 99% or more. The obtained (R) -8-hydroxy-2,6-
The physicochemical properties of dimethyl-2-octenoic acid are as follows. (A) Mass spectrometry value: EI mass spectrum m / z 16
_{8 (M-H 2 O)} , SIMS m / z 187 (M +
H), 209 (M + Na) (b) High-resolution mass spectrometry: HREI mass spectrum
Found m / z 168.1151 (M-H 2 O), ( calculated as C ₁₀ H ₁₆ O ₂₎ theory m / z 168.1149 (c) Specific rotation: [α] _D ²² +5.6 ° (C = 0.4
(25, chloroform) (d) Ultraviolet absorption spectrum: methanol λmax
nm (?) 215 (9890). (E) Infrared absorption spectrum: NaCl (Neat)
3440, 2930, 1688, 1642, 1281,
1057 cm ^-1 (f) ¹ H-NMR spectrum: (400 MHz, CD
Cl ₃ ) δ 0.92 (3H, d, J = 6.5 Hz),
1.30 (1H, m), 1.41 (1H, m), 1.4
6 (1H, m), 1.61 (1H, m), 1.62 (1
H, m), 1.83 (3H, s), 2.21 (2H,
m), 3.70 (2H, m), 6.88 (1H, t, J
= 7.1 Hz) (g) ¹³ C-NMR spectrum: (100 MHz, CD
Cl ₃ ) δ 173.1, 144.9, 127.1, 6
0.9, 39.6, 35.6, 29.3, 26.4, 1
9.3, 12.0

Synthesis Example 1 (Synthesis of (R)-and (S) -8-hydroxy-2,6-dimethyl-2-octenoic acid) A known method (Phytochemistry, vol. 22, p. 255-258, 1983)
(R) -8-hydroxy-2,6-dimethyl-2-octenal (170 mg, 1.0 mmol) synthesized from commercially available (+)-(R) -citronellol according to tert-butyl alcohol (20 mL). Dissolved in 2-methyl-2-butene
(5 mL) was added. Sodium chlorite (833 mg,
9.2 mmol) and sodium dihydrogen phosphate (833 mg, 6.
9 mmol) in water (8.3 mL) was added dropwise over 30 minutes, and the mixture was further stirred at room temperature for 3 hours. After the reaction solution was concentrated under reduced pressure, water was further added for ether extraction, and the aqueous layer was acidified with dilute hydrochloric acid, and then the ether extraction was repeated. The obtained organic layers were combined, washed with water and dried over sodium sulfate. The residue obtained by evaporating the solvent under reduced pressure is subjected to silica gel column chromatography (3% to 1%).
(R) -8-hydroxy-2,6-dimethyl-2.
-Octenoic acid (160 mg, 86%) was obtained as a colorless oil. The spectrum data of this product other than the specific rotation was completely consistent with (R) -8-hydroxy-2,6-dimethyl-2-octenoic acid obtained from nature. [α] _D ²⁰ +
7.9 ゜ (c 0.455, CHCl ₃ )

In the same manner as described above, commercially available (-)-(S)
-(S) -8-hydroxy-2,6 from citronellol
-Dimethyl-2-octenoic acid was synthesized. [α] _D ²⁰ -5.7
゜ (c 0.525, CHCl ₃ ) (optical purity 65% ee)

Reference Example 1 (Confirmation of stereochemical structure of (R) -8-hydroxy-2,6-dimethyl-2-octenoic acid obtained from Cistanche salsa) (R)-obtained by the above chemical synthesis method 8-hydroxy-2,6-dimethyl-2-octenoic acid (10.3 mg, 0.0
To a solution of 553 mmol) in ether (1.4 mL) at room temperature was added an ether solution of diazomethane until the starting material disappeared. After quenching excess diazomethane with a small amount of acetic acid, the reaction solution was washed with a saturated sodium hydrogen carbonate solution and saturated saline, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane: ethyl acetate = 2: 1) to give methyl (R) -8-hydroxy-2,6-dimethyl-2-octenoate.
(8.7 mg, 79%) was obtained as a colorless oil.

¹ H-NMR (200 MHz) (CDCl ₃ ): δ 0.86 (3H,
d, J = 6.3Hz), 1.38 (5H, m), 1.77 (3H, d, J = 1.2 Hz),
2.12 (2H, dt, J = 7.4, 7.4Hz), 3.62 (6H, m), 6.68
(1H, dt, J = 1.2, 7.4Hz).

The obtained (R) -8-hydroxy-2,
Methyl 6-dimethyl-2-octenoate (2.2 mg, 0.011
pyridine (8.0 μL, 0.10 mmol) and benzoyl chloride (4.0 mmol) at 0 ° C.
μL, 0.034 mmol) and stirred at room temperature for 4 hours. A saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture under ice cooling, and ether extraction was performed. The organic layer was washed with saturated saline, and the solvent was distilled off under reduced pressure. The resulting residue was purified by silica gel thin-layer chromatography (chloroform) to give (R) -8-benzoyloxy-2,6- Methyl dimethyl-2-octenoate (4.5 mg, 69%) was obtained as a colorless oil.

¹ H-NMR (200 MHz) (CDCl ₃ ): δ1.00 (3H,
d, J = 6.2Hz), 1.50 (5H, m), 1.84 (3H, d, J = 1.2 Hz),
2.22 (2H, dt, J = 7.4, 7.4Hz), 3.72 (3H, s), 4.37
(2H, dt, J = 1.5, 7.3Hz), 6.76 (1H, dt, J = 1.2, 7.4H
z), 7.50 (3H, m), 8.06 (2H, td, J = 1.5, 6.9Hz).

By the same method as described above, (R) -8-hydroxy-2,6-dimethyl-2-octenoic acid obtained from nature and (S) -8-hydroxy-2,6-chemically synthesized are obtained. Dimethyl-2-octenoic acid is also 8-
Benzoyloxy-2,6-dimethyl-2-octenoate was introduced into a column, and these were subjected to HPLC using an analytical optical isomer separation column (DAICEL CHIRALCEL OD, column size φ
0.46 × 25 cm, hexane: isopropyl alcohol =
9: 1, 0.5 mL / min). As a result, the synthetic (R) -form and those derived from (R) -8-hydroxy-2,6-dimethyl-2-octenoic acid obtained from nature showed a single peak with a retention time of 15.4 minutes. From this result, it was found that (R) -8-hydroxy-2,6-dimethyl-2-octenoic acid obtained from nature had an optical purity of 100% ee. On the other hand, the sample of the synthesized (S) -isomer showed a major peak at a retention time of 12.6 minutes and a minor peak at 15.4 minutes, indicating that the optical purity was about 65% ee. Then, the (S) -isomer sample (98.8 mg) contaminated with the (R) -isomer was collected using a preparative column (DAICEL CHIRALCELO).
D, column size φ2 × 25 cm, hexane: isopropyl alcohol = 9: 1, 6 mL / min) and optically pure (S) -8-benzoyloxy-2,6-dimethyl-2-octenoic acid 73.6 mg of methyl, (R) -8-benzoyloxy-
15.2 mg of methyl 2,6-dimethyl-2-octenoate was obtained.

Reference Example 2 (Production of optically pure (S) -8-hydroxy-2,6-dimethyl-2-octenoic acid) Optically pure (S) -8 obtained in Reference Example 1 -Methyl benzoyloxy-2,6-dimethyl-2-octenoate
(72.3 mg, 0.24 mmol) was dissolved in methanol (2.0 mL), 1N sodium hydroxide (1.0 mL, 1.0 mmol) was added, and the mixture was stirred at room temperature for 18.5 hours. After dilution with water, ether extraction was performed. The aqueous layer was further adjusted to acidic pH with 1N hydrochloric acid, and then ether extraction was repeated. The organic layers were combined, washed with water and saturated saline, dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The resulting residue was subjected to silica gel column chromatography (hexane: ethyl acetate = 2: 1 to 1: 1). Purification allows optically pure (S) -8-hydroxy-
2,6-dimethyl-2-octenoic acid (42.4 mg, 96%)
Was obtained as a colorless oil. [α] _D ²⁰ -8.3 ゜ (c 0.505, CHCl ₃ )

Test Example 1 (Effect of Cistanche Tubulosa Extract on Osteoporosis) When the ovaries of mice are excised, osteoporosis develops and the etiology and pathology are similar to human postmenopausal osteoporosis. Used as a model animal. Therefore, the following test was conducted to examine the effect of Cistanche salsa extract on osteoporosis. 4 weeks old
The ovaries of ddY mice (females) were excised under ketamine hydrochloride anesthesia. The day after ovariectomy, the Cistanche salsa extract obtained by the method of Example 1 was orally administered. Cistanche salsa extract is suspended in a 10% cremophor (Sigma) aqueous solution, and 16 mg
/0.2 ml / mouse / day was orally administered 5 times a week for 4 consecutive weeks. Three mice were used in each group. Four weeks later, after measuring the body weight, the mice were sacrificed and the dry weight of the femur was measured. Table 1 shows the mean ± standard error of the femur dry weight / body weight of each group.

[0034]

[Table 1] Table 1. Dry femur weight per body weight 群 group Femur dry weight / body weight (mg / G) ────────────────────────────────── 1. Sham operation control group 1.41 ± 0.02 2. Ovariectomized control group 1.32 ± 0.04 3. Cistanche salsa extract administration group 1.44 ± 0.03 ──────────────────────────────────

From these results, it was found that the extract of Cistanche salsa was effective in treating osteoporosis.

Test Example 2 ((R) -8-hydroxy-2,
Osteoporosis inhibitory effect of 6-dimethyl-2-octenoic acid) (R) -8-hydroxy- obtained from Cistanche salsa
The following test was conducted to examine the effect of 2,6-dimethyl-2-octenoic acid on osteoporosis. 4 weeks old ddY
The ovaries of the mice (females) were removed under ketamine hydrochloride anesthesia. From the day after ovariectomy, 0.32, 1.6, and 8 μg of (R) -8-hydroxy-2,6-dimethyl-2-octenoic acid ((R) -HDOA) purified from Cistanche salsa by the method of Example 2 was used.
The dose was intraperitoneally administered at a dose of / kg / day five times a week for four consecutive weeks. The compound (R) -8-hydroxy-2,6-dimethyl-2-octenoic acid was dissolved in a 10% aqueous solution of DMSO, and 0.2 ml / day was intraperitoneally administered to each mouse. The group was also intraperitoneally administered with a 10% DMSO aqueous solution at 0.2 ml / day per mouse. Five mice were used in each group. Four weeks later, after measuring the body weight, the mice were sacrificed and the dry weight of the femur was measured. The femoral strength was measured by a three-point bending test using a bone hardness tester TK-252C (manufactured by Muromachi Kikai Co., Ltd.). Table 2 shows the femur dry weight / body weight of each group, Table 3 shows the bone load as an index of bone strength (failure load), and Table 4 shows the average value ± standard error of the bone yield energy. . The test for the significant difference between each group and the ovariectomized control group was performed by Student's t-test. The group without any description is not significantly different from the ovariectomized control group (p ≧ 0.05).

[0037]

[Table 2] Table 2. Dry femur weight per body weight 群 group Femur dry weight / body weight (mg / G) ─────────────────────────────────── 1. Sham operation control group 1.43 ± 0.03 (p <0.005) 2. Ovariectomized control group 1.21 ± 0.03 (R) -HDOA administration group (0.32 μg / kg / day) 1.28 ± 0.03 4. (R) -HDOA administration group (1.6 μg / kg / day) 1.34 ± 0.03 (p <0.05) (R) -HDOA administration group (8 μg / kg / day) 1.36 ± 0.03 (p <0.01) ──────────────────────────── ──────

[0038]

[Table 3] Table 3. Bone breaking force 群 group Bone breaking force (N) ──────── ──────────────────────── 1. Sham operation control group 14.4 ± 0.8 (p <0.05) Ovariectomized control group 12.7 ± 0.2 (R) -HDOA administration group (0.32 μg / kg / day) 13.1 ± 0.7 (R) -HDOA administration group (1.6 μg / kg / day) 14.1 ± 0.2 (p <0.005) (R) -HDOA administration group (8 μg / kg / day) 13.5 ± 0.5 ────────────────────────────────

[0039]

[Table 4] Table 4. Bone breaking energy ─────────────────────────────── group Bone breaking energy (mJ) ───────── ────────────────────── 1. Sham operation control group 3.35 ± 0.26 2. Ovariectomized control group 2.90 ± 0.22 3. (R) -HDOA administration group (0.32 μg / kg / day) 3.46 ± 0.31 4. (R) -HDOA administration group (1.6 μg / kg / day) 3.46 ± 0.05 (p <0.05) (R) -HDOA administration group (8 μg / kg / day) 3.38 ± 0.20 ────────────────────────────────

From these results, the compound (R) of the present invention was obtained.
-8-Hydroxy-2,6-dimethyl-2-octenoic acid has been found to be effective in treating osteoporosis.

Test Example 3 (Effect of (S)-and (R) -8-hydroxy-2,6-dimethyl-2-octenoic acid on osteoporosis) (S)-and (R) -8-hydroxy-2, The following test was conducted to examine the effect of 6-dimethyl-2-octenoic acid on osteoporosis. The ovary of a 4-week-old ddY mouse (female) was excised under ketamine hydrochloride anesthesia. Ovariectomy
(R) -8-Hydroxy-2,6-dimethyl-2 obtained by the method described in Synthesis Example 2 and Reference Example 2 from the day after arrival.
-Octenoic acid (optical purity 100% ee) and (S) -8-
Hydroxy-2,6-dimethyl-2-octenoic acid (optical purity 100% ee) was administered at a dose of 8, 16, or 32 μg / kg / day for 5 weeks.
Intraperitoneally for four consecutive weeks. 10% test compound
Dissolve in DMSO aqueous solution and intraperitoneally administer 0.2 ml / day per mouse, 10% in sham control group and ovariectomy control group
DMSO aqueous solution was intraperitoneally administered at 0.2 ml / day per mouse. Five mice were used in each group. Four weeks later, after measuring the body weight, the mice were sacrificed and the dry weight of the femur was measured. Tables 5 and 6 show the mean ± standard error of femur dry weight / body weight of each group. Table 5 shows (R) -8-hydroxy-2,6-dimethyl-2-octenoic acid ((R) -HDOA), and Table 6 shows (S) -8-
Hydroxy-2,6-dimethyl-2-octenoic acid ((S)-
HDOA) test results. The test for the significant difference between each group and the ovariectomized control group was performed by Student's t-test. Unless otherwise noted, there was no significant difference from the ovariectomized control group (p
≧ 0.05).

[0042]

[Table 5] Table 5. Activity of (R) -8-hydroxy-2,6-dimethyl-2-octenoic acid ((R) -HDOA) (femoral dry weight / body weight) ──────────────────── group Femur dry weight / body weight (mg / g) ────────────────── ──────────────── 1. Sham operation control group 1.54 ± 0.04 (p <0.01) Ovariectomized control group 1.34 ± 0.02 3. (R) -HDOA administration group (8 μg / kg / day) 1.32 ± 0.02 (R) -HDOA administration group (16 μg / kg / day) 1.42 ± 0.01 (p <0.05) (R) -HDOA administration group (32 μg / kg / day) 1.43 ± 0.01 (p <0.05) ──────────────────────────── ──────

[0043]

[Table 6] Table 6. (S) -8-Hydroxy-2,6-dimethyl-2-octenoic acid ((S) -HDOA) activity (femur dry weight / body weight) ──────────────────── group Femur dry weight / body weight (mg / g) ────────────────── ──────────────── 1. Sham operation control group 1.60 ± 0.06 (p <0.01) 2. Ovariectomized control group 1.39 ± 0.01 (S) -HDOA administration group (8 μg / kg / day) 1.31 ± 0.03 (S) -HDOA administration group (16 μg / kg / day) 1.35 ± 0.06 (S) -HDOA administration group (32 μg / kg / day) 1.34 ± 0.03 ───────────────────────────────── ──

From these results, (R) -8-hydroxy-2,6-dimethyl-2-octenoic acid is effective for the treatment of osteoporosis, while the optical isomer (S) -8-hydroxy-2, 6-Dimethyl-2-octenoic acid had no remarkable effect, indicating that the therapeutic effect on osteoporosis was (R) -body specific.

Test Example 4 Interleukin 6 (IL-6) production inhibitory activity test 1 × 10 ⁴ / ml mouse preosteoblast MC3T3E1 (medium; alpha MEM containing 10% fetal bovine serum) was placed in a 96-well multiplate. After the addition, the cells were cultured for 3 days in a CO ₂ incubator according to a conventional method. After confirming that the cells have become confluent, parathyroid hormone (PTH) 20 ng / ml or
(R) -8-hydroxy-2,6 according to PTH and dilution sequence
-Replaced with the above medium containing both dimethyl-2-octenoic acid. After further culturing for one day, the concentration of IL-6 produced in the culture supernatant was quantified by a bioassay. I
L-6 was quantified using a mouse hybridoma MH60 that proliferates in an IL-6-dependent manner according to a conventional method. This is 50% of the amount of IL-6 produced by PTH stimulation.
The concentration of the inhibitor required for% inhibition was defined as the IC ₅₀ value. (R) -8-hydroxy-2,6-dimethyl-2-
The IC ₅₀ value of the octenoic acid IL-6 production inhibitory activity is 27
μg / ml. From this result, it was found that (R) -8-hydroxy-2,6-dimethyl-2-octenoic acid had an activity of inhibiting interleukin-6 production.

[0046]

According to the present invention, the prevention and treatment of osteoporosis and the prevention and treatment of various diseases, such as autoimmune diseases, cachexia, and inflammation, which cause the onset or exacerbation of interleukin 6 can be performed.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Sachiko Kojima 10-3 Komoncho, Hachioji City, Tokyo

Claims (4)

[Claims] 1. An anti-osteoporosis agent comprising an extract of Cistanche salsa as an active ingredient. 2. The following formula (1): (R) -8-hydroxy-2,6-dimethyl-2-octenoic acid represented by 3. An anti-osteoporosis agent comprising, as an active ingredient, (R) -8-hydroxy-2,6-dimethyl-2-octenoic acid represented by the formula (1) or a pharmaceutically acceptable salt thereof. 4. Inhibition of interleukin-6 production comprising (R) -8-hydroxy-2,6-dimethyl-2-octenoic acid represented by the above formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. Agent.