JP2015164910A - Medicament for prevention or treatment of osteoporosis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an osteoporosis therapeutic agent that is inexpensive more than before.SOLUTION: A medicament for prevention or treatment of osteoporosis comprises an ultra fine bubble solution as an active ingredient.

Description

  The present invention relates to a pharmaceutical for preventing or treating osteoporosis containing ultrafine bubbles as an active ingredient.

  Osteoporosis is a disease state in which bone mass decreases due to estrogen reduction or aging due to menopause, and bone fragility is likely to cause fracture with a slight external force. In particular, fractures of the vertebral body and proximal femur are known not only to significantly reduce physical ability but also to increase the risk of death by about 6 to 8 times, which is a major problem in an aging society.

  As existing osteoporosis therapeutic agents, bisphosphonate preparations, raloxifene preparations, and the like are known, but all of them contain a specific pharmaceutical compound as an active ingredient, and the production costs correspondingly. On the other hand, the number of osteoporosis patients in Japan is said to be more than 12 million, and the number of patients is expected to increase further in the future as the aging society progresses. However, about 80% of them are currently untreated, and further spread of treatment is desired in the future. Therefore, there is a demand for a therapeutic agent for osteoporosis that is excellent in compliance with treatment and can be manufactured at a lower cost from the viewpoint of medical economics.

  Ultra fine bubbles are nano-sized (typically 1000 nm or less) microbubbles that exist in a solvent such as water, and have properties that are significantly different from normal bubbles. For example, it can exist in a solvent in a stable state for a long period of time, and the bubble surface is charged with a negative charge, so that it can be adsorbed to a positively charged substance. In recent years, the effect of ultra fine bubbles in various fields, for example, the effect of promoting the growth of plants, fish, mice, etc. (Non-patent Document 1), the bactericidal effect (Patent Document 1), the cleaning effect of contaminants (for example, Patent Document 2), etc. Has been reported. However, there is currently no knowledge about the therapeutic effect of ultrafine bubbles on osteoporosis.

JP 2013-180956 JP 2013-140096

PLOS ONE, June 2013, Volume 8, Issue 6, e65339

  An object of this invention is to provide a cheaper osteoporosis therapeutic agent.

  Ovariectomy can cause a decrease in blood estrogen and create a condition similar to postmenopausal osteoporosis. In addition, administration of steroid can create a state similar to steroidal osteoporosis. As a result of intensive studies, the present inventors have found that the bone mass significantly reduced by ovariectomy or steroid administration can be recovered by administration of the ultrafine bubble solution. As a result of further research based on this finding, the present invention was completed.

  That is, the present invention includes the following aspects.

  Item 1. A pharmaceutical for preventing or treating osteoporosis containing an ultrafine bubble solution as an active ingredient.

  Item 2. Item 2. The preventive or therapeutic drug according to Item 1, wherein the mode particle diameter of the ultrafine bubble is 1000 nm or less.

  Item 3. Item 3. The preventive or therapeutic drug according to Item 1 or 2, wherein the gas in the ultrafine bubble is at least one selected from the group consisting of air, oxygen, nitrogen, carbon dioxide, ozone, neon, and argon.

Item 4. Item 4. The preventive or therapeutic drug according to any one of Items 1 to 3, wherein the ultrafine bubble concentration in the ultrafine bubble solution is 1 × 10 ⁵ cells / mL or more.

  Item 5. Item 5. The preventive or therapeutic drug according to any one of Items 1 to 4, wherein the ultrafine bubble is an ultrafine bubble prepared by a gas-liquid mixed shear method.

  Ultra fine bubble solutions can be manufactured very inexpensively. For this reason, according to this invention, a cheaper therapeutic agent can be provided compared with the conventional osteoporosis therapeutic agent which uses a pharmaceutical compound as an active ingredient.

The measurement result of the bone mass in Example 1 is shown. In FIG. 1, “OVX” indicates an ovariectomy group, “sham” indicates an ovariectomy sham operation group, “raw meal” indicates a physiological saline administration group, and “oxygen UFB” indicates oxygen ultrafine bubble-containing physiological saline. A water administration group is shown, and “nitrogen UFB” shows a physiological saline administration group containing nitrogen ultrafine bubbles. The measurement result of the amount of cancellous bone in Example 2 is shown. In FIG. 2, “steroid” indicates a steroid administration group, “sham” indicates a non-steroid administration group, “saline” indicates a physiological saline administration group, and “UFB” indicates an ultrafine bubble solution administration group. The same applies to FIGS. 3 and 4. The measurement result of the amount of cortical bone in Example 2 is shown. The measurement result of the osteoclast number of the femur in Example 2 is shown. The measurement result of the osteoclast number differentiated from the spleen cell in vitro in Example 3 is shown.

  The present invention relates to a drug for preventing or treating osteoporosis containing an ultrafine bubble solution as an active ingredient (hereinafter sometimes simply referred to as “medicament of the present invention”).

  Ultra fine bubbles are not particularly limited as long as they are nano-sized microbubbles. As the ultra fine bubble, a so-called “nano bubble” can also be used. The ultrafine bubble can be, for example, a microbubble having a mode particle size of 1000 nm or less, preferably 500 nm or less, more preferably 300 nm or less, still more preferably 200 nm or less, and even more preferably 50 to 150 nm.

  The gas in the ultra fine bubble is not particularly limited as long as it is a gas capable of forming the ultra fine bubble. Examples of such gas include air, oxygen, nitrogen, carbon dioxide, ozone, neon, and argon, and preferably air, oxygen, nitrogen, and the like. The medicament of the present invention is considered to exhibit osteoporosis preventive or therapeutic effects due to the properties of the ultra fine bubbles themselves, not the properties of the gases in the ultra fine bubbles.

  The ultra fine bubble solution (hereinafter sometimes simply referred to as “solution”) is not particularly limited as long as it is a solution containing ultra fine bubbles as a solute.

  The solvent of the solution is not particularly limited as long as it is a solvent that can generate and retain ultrafine bubbles. As such a solvent, for example, water (tap water, purified water, ion exchange water, pure water, ultrapure water, deionized water, distilled water, etc.) can be used. Further, a solvent obtained by adding a small amount of alcohol such as ethanol, glycerin or the like to water may be used as a solvent. A solvent may be used individually by 1 type and may be used in combination of 2 or more type.

The ultra fine bubble density | concentration in a solution is not specifically limited as long as the effect of this invention can be exhibited. The ultrafine bubble concentration is, for example, 1 × 10 ⁵ cells / mL or more, preferably 1 × 10 ⁶ cells / mL or more, more preferably 1 × 10 ⁷ cells / mL or more, and further preferably 1 × 10 ⁸ cells / mL or more. More preferably, it can be 1 × 10 ⁸ pieces / mL to 1 × 10 ⁹ pieces / mL.

  Solutes other than ultrafine bubbles are not particularly limited as long as ultrafine bubbles can be generated and maintained. Examples of such solutes include sodium chloride and buffering agents.

  Ultra fine bubbles can be prepared according to a known production method of nano-sized microbubbles. For example, it can be produced by a method such as a gas-liquid mixed shear method, a static mixer method, a venturi method, a cavitation method, a vapor condensation method, an ultrasonic method, a swirl flow method, a pressure dissolution method, or a fine hole method. Among these, from the viewpoint that the effects of the present invention can be more reliably exhibited, it can be preferably prepared by a gas-liquid mixed shear method.

  The osteoporosis to which the medicament of the present invention is applied may be either primary osteoporosis or secondary osteoporosis. Examples of primary osteoporosis include senile osteoporosis, postmenopausal osteoporosis, and the like according to the cause. Among these, from the viewpoint that the effects of the present invention can be more reliably exhibited, preferably postmenopausal osteoporosis is exemplified. . As secondary osteoporosis, lifestyle-related disease-related osteoporosis, steroidal osteoporosis and the like are listed according to the cause, and among these, steroidal osteoporosis is preferable from the viewpoint that the effects of the present invention can be more reliably exhibited. Can be mentioned. In addition, from the viewpoint of more surely exerting therapeutic and preventive effects on these diseases, the gas in the ultra fine bubble is more preferably air, oxygen, and the like, and still more preferably oxygen and the like. .

  The medicament of the present invention can be the ultrafine bubble solution itself, or can contain components other than the ultrafine bubble solution (hereinafter sometimes simply referred to as “additive”). The additive is not particularly limited as long as it is a pharmaceutically acceptable ingredient. For example, a base, a carrier, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, Examples include disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, and chelating agents. When the medicament of the present invention contains an additive, the medicament of the present invention can be produced by using the additive according to a conventional method corresponding to the dosage form.

  The medicament of the present invention can be in any dosage form such as a liquid, emulsion, injection, suspension, capsule, etc., preferably a liquid, emulsion, injection, etc., more preferably It can be an injection.

  The administration target of the medicament of the present invention is an osteoporosis patient or a subject who may develop osteoporosis. A subject that may develop osteoporosis is a subject that has factors (for example, menopause, aging, lifestyle-related diseases, steroid administration, etc.) that cause osteoporosis. The medicament of the present invention is also suitable as a prophylactic medicament because it does not exert an adverse effect such as excessive increase in bone mass even if administered before the onset of osteoporosis.

  The administration route of the medicament of the present invention may be oral administration or parenteral administration. Specific examples of parenteral administration include intraperitoneal administration, subcutaneous administration, intravenous administration, transarterial administration, intradermal administration, and intramuscular administration.

  The dosage form and effective dosage of the medicament of the present invention depend on the administration subject, administration route, dosage form, patient condition, doctor's judgment, etc., and are not limited. For adults, 50 to 800 ml (more preferably 200 to 400 mL) can be administered per conversion in terms of ultrafine bubble solution. In addition, as an administration form, it is preferable to administer once every 1 to 5 days (preferably 2 days).

  The medicament of the present invention may be used in combination with other preventive or therapeutic agents for osteoporosis. Examples of other preventive or therapeutic agents include alendronate, risedronate, raloxifene hydrochloride, etidronate, active vitamin D3 preparation, calcitonin preparation, vitamin K2 preparation, female hormone preparation, calcium preparation and the like. Other preventive or therapeutic drugs may be used alone or in combination of two or more. However, from the viewpoint that it can be produced at a lower cost, the medicament of the present invention preferably does not contain these existing pharmaceutical compounds.

  The ultra fine bubble solution is also useful as an agent for inhibiting differentiation into osteoclasts.

  EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

Example 1. Analysis of therapeutic effect of post-menopausal osteoporosis with ultrafine bubble solution The ultrafine bubble solution was administered to mice that had been ovariectomized to force osteoporosis, and the bone mass was measured. Specifically, it was performed as follows.

<1-1. Creation of test mouse>
Eight week old female C57Bl / 6 mice (Oriental Yeast Co., Ltd.) were placed prone under anesthesia. The caudal site (surgical site) about 2 cm from the final rib was shaved with a clipper so as not to damage the skin. The shaved part was wiped off with gauze and disinfected with 70% alcohol. The surgical site was cut about 1 cm laterally from the outside about 5 mm from the midline, and the subcutaneous tissue was peeled off. Further, the peritoneum was incised by about 1 cm so as not to damage the abdominal organs, and the ovaries were confirmed. In the ovariectomy group (OVX group), the ovaries were pulled out, the oviducts and blood vessels were ligated with sutures, and the ovaries were excised with a scalpel. After confirming that no ovary was left behind, the resected end was disinfected with 70% alcohol and delivered completely into the abdominal cavity. The contralateral ovary was similarly removed. In the oophorectomy sham operation group (Sham group), the ovary was pulled out and confirmed, and then it was completely delivered into the abdominal cavity again. The peritoneum and skin were sutured with 1-2 needles. The suture was disinfected with 70% alcohol.

<1-2. Preparation of Ultra Fine Bubble Solution>
Using a nanobubble generator (trade name: BUVITAS, manufactured by Ligaric, Inc.), an ultrafine bubble-containing physiological saline in which the gas in the bubble is oxygen was produced by a gas-liquid mixed shear method (containing oxygen ultrafine bubbles) Saline). Similarly, a physiological saline containing nitrogen ultrafine bubbles was also produced. These ultrafine bubble-containing physiological saline had an ultrafine bubble mode particle size of 105 nm and an ultrafine bubble concentration of 3.43 × 10 ⁸ / mL.

<1-3. Administration of Ultra Fine Bubble Solution>
For the ovariectomized group (OVX group) and the ovariectomized sham-operated group (Sham group), physiological saline, oxygen ultrafine bubble-containing physiological saline, or nitrogen ultrafine bubble-containing physiological saline, from 2 days after surgery It was intraperitoneally administered (200 μl / mouse / times) three times a week (at intervals of 2 days). During this period, normal food for mice (produced by Oriental Yeast Co., Ltd.) and water were freely ingested and kept in a breeding room maintained at constant temperature and humidity. On day 42 after the operation, the mice in each group were sacrificed under anesthesia.

<1-4. Bone mass measurement>
The femurs of both legs of each group of mice were removed and fixed with 10% formalin. The fixed femur was imaged using an X-ray micro CT apparatus (R_mCT, Rigaku, Tokyo, Japan). The shooting conditions were as follows: the number of pixels: 512 pixels × 512 pixels × 512 slice, pixel size: 10 μm, tube voltage: 90 kV, tube current: 200 μA. The femoral measurement site was a secondary cancellous bone region of 0.5 mm to 1.2 mm in the proximal direction from the growth plate cartilage at the distal end of the femur, excluding the growth cartilage site. About this measurement site | part, the cancellous bone volume ratio (BV / TV) was measured using the bone mass analysis software (TRI / 3D-BON, Ratoc System Engineering CO., LTD, Tokyo, Japan).

<1-5. Result>
The measurement results of each mouse are shown in Table 1 below. In Table 1, “OVX” indicates an ovariectomy group, “sham” indicates an ovariectomy sham operation group, “raw meal” indicates a physiological saline administration group, and “oxygen UFB” indicates oxygen ultrafine bubble-containing physiological saline. A water administration group is shown, and “nitrogen UFB” shows a physiological saline administration group containing nitrogen ultrafine bubbles.

  FIG. 1 shows the results of Table 1 shown in a box diagram. The notation of each group corresponds to Table 1. In FIG. 1, the tip of the ridge extending upward from the column indicates the 90th percentile value, the top of the column indicates the 75th percentile, the horizontal line in the column indicates the 50th percentile (median), and the bottom of the column indicates 25 It shows the percentile value, and the tip of the ridge extending downward from the column shows the 10th percentile value.

  From the comparison of sham and OVX, it was confirmed that the bone mass was greatly reduced by ovariectomy (Mann-Whitney U-test / P <0.01). The comparison between OVX raw diet and OVX oxygen UFB or OVX nitrogen UFB showed that the administration of the ultrafine bubble solution increased the amount of bone that had been reduced by ovariectomy (Mann-Whitney U-test /P<0.05). This increase phenomenon occurred in both the oxygen UFB group and the nitrogen UFB group, suggesting that it occurred regardless of the type of gas in the bubble. From the above, it was shown that the ultrafine bubble solution has a therapeutic effect on postmenopausal osteoporosis.

  On the other hand, there was no significant difference in bone mass between sham raw food and sham oxygen UFB or sham nitrogen UFB. This suggests that the ultrafine bubble solution does not affect the bone mass even if administered before the onset of osteoporosis.

Example 2 Analysis of therapeutic effect of steroidal osteoporosis by ultrafine bubble solution Ultrafine bubble solution was administered to mice that were forced to osteoporosis by administering steroid, and the bone mass and the number of osteoclasts were measured. Specifically, it was performed as follows.

<2-1. Creation of test mouse>
According to a conventional method, 5 mg of prednisolone pellets for animal experiments was embedded in the subcutaneous part of a 6-month-old female C57Bl / 6 mouse (steroid group). On the other hand, as the Sham group, a group was prepared which was treated in the same manner as the steroid administration group except that the prednisolone pellets were not buried.

<2-2. Ultra Fine Bubble Solution>
The oxygen ultrafine bubble-containing physiological saline prepared in 1-2 of Example 1 was used as an ultrafine bubble solution in the following experiment.

<2-3. Administration of Ultra Fine Bubble Solution>
For the steroid administration group (steroid group) and non-steroid administration group (Sham group), physiological saline or ultrafine bubble solution is administered intraperitoneally (200 μl / mouse /) three times a week (every 2 days) from the day after administration. Times). During this time, food and water were freely ingested and kept in a breeding room kept at constant temperature and humidity. After 8 weeks, each group of mice was sacrificed under anesthesia.

<2-4. Bone mass measurement>
According to the same method as in Example 1-4, cancellous bone mass volume ratio (BV / TV) and cortical bone mass volume ratio (Cv / Av) were measured.

<2-5. Measurement of the number of osteoclasts in the femur>
The femur was stained with TRAP according to a standard method, and the number of osteoclasts (/ mm ² ) was counted.

<2-6. Result>
Fig. 2 shows the measurement results of cancellous bone volume ratio (BV / TV), Fig. 3 shows the measurement results of cortical bone volume ratio (Cv / Av), and the measurement results of osteoclast number (/ mm ² ) 4 shows. 2 to 4, “steroid” represents a steroid administration group, “sham” represents a non-steroid administration group, “saline” represents a physiological saline administration group, and “UFB” represents an ultrafine bubble solution administration group. Show. 2 and 3, the tip of the ridge extending upward from the column indicates the 90th percentile value, the top of the column indicates the 75th percentile value, the horizontal line in the column indicates the 50th percentile value (median value), The lower end of the indicates the 25th percentile, and the tip of the ridge extending downward from the column indicates the 10th percentile.

  2 and 3, it was confirmed from the comparison between sham + raw food and steroid + raw food that bone mass was decreased by administration of steroid (Mann-Whitney U-test / P = 0.003 (FIG. 2), P = 0.02 (Figure 3)). The comparison of steroid + raw food and steroid + UFB showed that the bone volume decreased by steroid administration increased by administration of the ultrafine bubble solution (Mann-Whitney U-test / P = 0.003). (Figure 2), P = 0.027 (Figure 3)). From the above, it was shown that the ultrafine bubble solution has a therapeutic effect on steroidal osteoporosis.

  In FIG. 4, it was confirmed that the number of osteoclasts was significantly increased by administration of steroids by comparing sham + raw food and steroid + raw food (Mann-Whitney U-test / P <0.05). And the comparison of steroid + saline and steroid + UFB showed that the number of osteoclasts increased by steroid administration decreased by the administration of ultrafine bubble solution (Mann-Whitney U-test / P <0.05).

Example 3 FIG. Osteoclast-differentiated mouse spleen cells in ultrafine bubble solution in the presence of 10 ng / ml M-CSF and 50 ng / ml RANKL, 48-well plate (for TRAP staining), 12-well plate (for RNA) The cells were cultured in (1 × 10 ⁵ cells / ml, 500 μl / well (for TRAP), 1000_μl / well (for RNA)). The culture solution was prepared by using water alone (0%), a solution obtained by mixing water and half of the ultrafine bubble solution (50%), or an ultrafine bubble solution (100%) as a solvent. Five days after the start of culture, the cells in the well were stained with TRAP according to a standard method, and the number of osteoclasts (/ well) was counted. The results are shown in FIG.

  FIG. 5 shows that the ultrafine bubble solution suppresses osteoclast differentiation.

Claims (5)

A pharmaceutical for preventing or treating osteoporosis containing an ultrafine bubble solution as an active ingredient. 2. The preventive or therapeutic drug according to claim 1, wherein the mode particle diameter of the ultrafine bubble is 1000 nm or less. The preventive or therapeutic drug according to claim 1 or 2, wherein the gas in the ultrafine bubble is at least one selected from the group consisting of air, oxygen, nitrogen, carbon dioxide, ozone, neon, and argon. The pharmaceutical for prevention or treatment according to any one of claims 1 to 3, wherein the concentration of ultrafine bubbles in the ultrafine bubble solution is 1 x 10 ⁵ / mL or more. The preventive or therapeutic pharmaceutical according to any one of claims 1 to 4, wherein the ultra fine bubble is an ultra fine bubble prepared by a gas-liquid mixed shearing method.