Patent Application
20110230447
The present invention relates to an osteogenesis promoter or a pharmaceutical composition having [4-(methylthio)phenylthio] methanebisphosphonic acid, which is one type of bisphosphonic acid, or a pharmaceutically acceptable salt thereof as an active ingredient; and a method for promoting osteogenesis that is constituted from administering the osteogenesis promoter or the pharmaceutical composition to a subject requiring treatment.
Bisphosphonic acid (BP, BPs) has a strong bone resorption inhibitory action resulting from inhibition of the function of osteoclasts, and to date, it has been widely used as a medical agent for various diseases with increased bone resorption such as osteoporosis, hypercalcemia, Paget disease, and neoplastic bone destruction.
Bisphosphonic acid is broadly categorized into, based on a chemical structure of side chains, (1) a compound group having an alkyl side chain, (2) a compound group having a halogen side chain, (3) a compound group having an aminoalkyl side chain, and (4) a compound group having a cyclic side chain. Various phai inacological properties of bisphosphonic acid such as its bone resorption inhibitory activity and the action mechanism thereof are known to vary significantly according to differences in the structure of these side chains.
For example, while a bisphosphonate compound (N-BPs: pamidronate, alendronate, risedronate, incadronate, zoledronate, etc.) having a nitrogen atom in the side chain inhibits a farnesyl pyrophosphate synthetase and a geranylgeranyl pyrophosphate synthetase in the mevalonate pathway and blocks prenylation of a low molecular G protein, a bisphosphonate compound (non N-BPs: etidronate, elodronate, tiludronate, etc.) not having the nitrogen atom in the side chain is known to have a high structural similarity with pyrophosphoric acid and foim an ATP (adenosine triphosphate) analog. Moreover, N-BPs, compared to non N-BPs, is known to have a bone resorption inhibitory activity of 100-10,000 times or more.
Application of [4-(methylthio) phenylthio] methane, which is one type of the bisphosphonate compounds not having a nitrogen atom in a side chain, to rheumatoid arthritis and an anti-inflammatory action are described in non-patent documents 1 to 4, and an improvement effect of bone metabolism disorders, an inhibitory effect of interleukin-1 production and action, and an antioxidative effect are described in the patent document 1 and non-patent document 5. Furthermore, it is known that this compound can be used as a medical agent for periodontal disease (patent document 4 and non-patent documents 6 and 7). However, the osteogenesis promoting action of the above compound was conventionally not known.
Moreover, the effect of bisphosphonic acid derivatives is variable, and it was well-known in the field of the technical field that when different bisphosphonic acids are used, the opposite effects may be caused and that even if the same bisphosphonic acid is used, according to the concentration, a varying biological reaction is caused (non-patent document 8).
On the other hand, as a substance having the osteogenesis promoting action, to date, BMP, FGF, IGF, and statins are well-known. However, for these substances, there was a problem in that because they are protein factors that promote osteogenesis and at the same time promote bone resorption (BMP) and that induce inflammation to application sites (BMP, statins), the manufacturing cost was high, antigenicity was a problem (BMP, FGF, IGF), affinity for a bone was low, special DDS (Drug Delivery System) was required (BMP, FGF, IGF, statins), etc.
The present invention is intended to solve the problems described above. That is, the primary object of the present invention is to offer a compound that can be produced through a chemical synthesis at a low cost, that has an excellent osteogenesis promoting activity, that has high affinity for bones, and that can be applied without requiring any special DDS; and a method for promoting osteogenesis by administering the compound and for applying to the formation or regeneration of a bone.
The inventor of the present invention, carried out intensive studies in order to solve the above problems, and newly discovered that [4-(methylthio)phenylthio] methanebisphosphonic acid has an osteogenesis promoting action, which was not known to date, and completed the present invention.
That is, the present invention relates to aspects described below.
[Aspect 1] An osteogenesis promoter having [4-(methylthio)phenylthio] methanebisphosphonic acid or a pharmaceutically acceptable salt thereof as an active ingredient.
[Aspect 2] An osteogenesis promoter having a sodium salt of [4-(methylthio)phenylthio] methahebisphosphonic acid as an active ingredient.
[Aspect 3] An osteogenesis promoter having [4-(methylthio)phenylthio] methanebisphosphonic acid disodium salt as an active ingredient.
[Aspect 4] An osteogenesis promoter according to any one of Aspects 1 to 3 that is constituted with the active ingredients being adsorbed to a sustained-releasing agent that is constituted from calcium phosphate.
[Aspect 5] An osteogenesis promoter according to Aspect 4, wherein calcium phosphate is selected from a group that is constituted from α-tricalcium phosphate, β-tricalcium phosphate, octa-calcium phosphate, and hydroxyapatite.
[Aspect 6] A pharmaceutical composition including an osteogenesis promoter according to any one of Aspects 1-5 and having an osteogenesis promoting action.
[Aspect 7] A pharmaceutical composition according to Aspect 6 that is in the form of an aqueous solution.
[Aspect 8] A pharmaceutical composition according to Aspect 7 having the form of injection.
[Aspect 9] A method for promoting osteogenesis that is constituted from administering the osteogenesis promoter according to any one of Aspects 1 to 5 or the pharmaceutical composition according to any one of Aspects 6 to 8.
[Aspect 10] A method for promoting osteogenesis that is constituted from locally administering to a site the osteogenesis promoter according to any one of Aspects 1 to 5 or the pharmaceutical composition according to any one of Aspects 6 to 8.
[Aspect 11] A method for promoting osteogenesis for a site that is constituted from locally administering to the site the osteogenesis promoter according to any one of Aspects 1 to 5 or the pharmaceutical composition according to any one of Aspect 6 to 8.
[Aspect 12] A method according to Aspect 11 that is constituted from locally administering to an alveolar bone site.
By causing [4-(methylthio)phenylthio] methanebisphosphonic acid, which is one type of bisphosphonic acid, or the pharmaceutically acceptable salt thereof to act, osteogenesis promotion was observed in a surrounding site of administration in the living body, in osteoblast-like cell lines, expression of alkaline phosphatase (ALP) activity and/or osteogenesis related genes was increased significantly, and it was possible to induce, in the bone organ culture system, promotion of collagen synthesis, promotion of osteogenesis, and/or promotion of production of the bone matrix, and increase of the bone mass, significantly.
The present invention relates to an osteogenesis promoter or a pharmaceutical composition having [4-(methylthio)phenylthio] methanebisphosphonic acid, which is one type of bisphosphonic acid, or a pharmaceutically acceptable salt thereof as an active ingredient; and a method for promoting osteogenesis that is constituted from administering the compound to a subject requiring treatment.
In the present invention, an “osteogenesis promoting action” refers to at least one of an alkaline phosphatase (ALP) activity that is known as the index for differentiating osteoblast precursor cells to osteoblasts in the osteogenesis promotion in the living body and in the culture system of the osteoblast-like cell lines; and/or an activity to significantly increase the expression of osteogenesis-related genes of type-I collagen, osteocalcin, bone sialoprotein, etc., known as other index for promoting osteogenesis; an activity, in the bone organ culture system such as the skull cap, that induces promotion of collagen synthesis, promotion of osteogenesis, and/or promotion of production of the bone matrix, and increases of the bone mass; and an activity that may be considered to be practically the same or is correlated physiologically or pharmacologically to these activities.
[4-(methylthio)phenylthio] methanebisphosphonic acid or a pharmaceutically acceptable salt thereof that is contained as an active ingredient in the osteogenesis promoter or the pharmaceutically acceptable salt thereof of the present invention, for example, can be manufactured with any known method to those skilled in the art, as described in the patent documents 1 to 3.
As a salt that may be a pharmaceutically acceptable for [4-(methylthio)phenylthio] methanebisphosphonic acid, any known salt to those skilled in the art, for example, sodium salt, potassium salt, etc., can be mentioned. As a specific example, [4-(methylthio)phenylthio] methanebisphosphonic acid (abbreviation: “MPMBP”) showing the structural formula in
The osteogenesis promoter of the present invention is useful as a pharmaceutical composition having various drug formulations such as foams of oral administration such as a tablet, a capsule, a powdered drug, a granule, and a pill, and such as forms of non-oral administration such as an injection, a syrup, an ointment, a buccal tablet, a suppository, an oral cleaning agent, and a topical drug, and the pharmaceutical composition shows the osteogenesis promoting action.
To the pharmaceutical composition of the present invention, by taking into consideration of the above drug formulations, aside from the active ingredients of the present invention, a carrier, an excipient, a bonding agent, a lubricant, a disintegrator, a sustained-releasing agent, a buffer, a coating agent, a coloring agent, etc., which may be pharmaceutically acceptable to those skilled in the art, can be included as appropriate. The osteogenesis promoter or the pharmaceutical composition of the present invention can be formulated easily with any known manufacturing method to those skilled in the art.
For example, examples of appropriate carriers include lactose, starch, sucrose, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, and croscarmellose sodium. Or, appropriate bonding agents include starch, gelatin, or glucose, anhydrous lactose, free-flow lactose, β-lactose, natural sugar such as a corn sweetener, gum Arabic, guar gum, a natural or synthetic gum such as tragacanth or sodium alginate, carboxymethyl-cellulose, polyethylene glycol, wax, etc. Moreover, lubricants used as the dosage for of these include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium chloride, etc. Furthermore, in order to orally administer in the liquid form such as an elixir, syrup, and a form composition, oral drug components can be combined with pharmaceutically acceptable non-toxic oral non-active carriers, such as ethanol, glycerol, and water. Moreover, to the pharmaceutical carriers that are constituted from soluble polymers such as polyvinylpyrrolidone, pyran polymer, polyhydroxypropylmethacrylamide, the active ingredients may be combined.
As a preferred example of the osteogenesis promoter or the pharmaceutical composition of the present invention, a drug formulation that, in the state in which the active ingredients are adsorbed to the sustained-releasing agent that is constituted from calcium phosphate such as α-tricalcium phosphate, β-tricalcium phosphate, octa-calcium phosphate, and hydroxyapatite, is included in an aqueous solution in which the osmotic pressure and pH are adjusted to the physiological range, can be mentioned. Moreover, it is preferable that the osmotic pressure and pH of this aqueous solution are adjusted to the physiological range.
The content and the dose of the active ingredients that are contained in the osteogenesis promoter or the pharmaceutical composition of the present invention can be selected by those skilled in the art appropriately, according to an administration plan, an oral drug of specified bisphosphonic acid compound that is selected, the age of recipient patients, size, gender and physical condition, type and the degree of seriousness of disorders to be treated, and other relevant medical and physical factors. Moreover, the appropriate amount can be determined based on a typical experiment of an animal model and on a clinical test on human. Generally, the appropriate amount of the active ingredients is selected such that significant osteogenesis promoting effects are obtained.
Therefore, although the dose, administration intervals, etc., can be determined appropriately according to an administration subject, administration route, symptoms, etc., with respect to the amount of the active ingredients, it is approximately 0.1 mg to 5 g, preferably approximately 1 mg to 2 g, and by dividing it into one to several times daily, appropriate duration, for example, for 1 day to 30 days, oral or non-oral administration is performed. For example, an injection can be locally administered to treatment sites. Moreover, although the type, sites, etc., of bones to which formation is promoted according to the present invention are not limited, as the treatment sites or the surrounding sites, for example, an alveolar hone, a peri-implant bone, a site after removal of bone cyst, etc., can be mentioned.
Although the present invention is described in detail below with reference to Examples, these Examples are simply for explanation purposes only, and they are intended to provide as references of the specific Examples. Although these examples are intended to provide explanations of specific detailed Examples of the present invention, they are not intended to limit or restrict the scope of the invention disclosed in the present specification. In the present invention, it should be understood that various Examples are possible based on the idea of the present invention. Moreover, for cases in which a commercially available reagent or kit is used, the protocols attached to those, drugs attached, etc., are used.
A total of 9 Japanese male rabbits with the average weight of 2.2 kg were used and they were divided into 3 groups of 3 rabbits each. Under the periosteum of the palatal alveolar bone between the teeth of the maxillary first molar and the maxillary second molar on the left side (experiment side) of the domestic rabbits in each group, 50 μl of a 0.1, 1.0, or 10 mM MPMBP solution was injected every 4 days for 6 times (all performed under anesthesia using Nembutal). To the same sites on the opposite side (the right side), 50 μl of a physiological saline solution (0.9% NaCl solution) was injected in the same manner, and was set to be a control. On the 4th day after the final injection, the animals were euthanized with excessive administration of Nembutal, and the maxillary bone was removed and fixed with a 10% neutral formalin solution (pH 7.4). With the alveolar bone in the vicinity of drug administration sites and the palatine bone as the region of interest, 3-dimensional analysis was performed using a pQCT (peripheral Quantitative Computerized Tomography;
Using the mouse skull-derived osteoblast-like cell line MC3T3-E1 cells (RIKEN Bio Resource Center, CELL BANK: RCB 1126), with respect to the alkaline phosphatase (ALP) activity, known as one index of osteogenesis, and also known as the index of differentiation of osteoblast precursor cells to osteoblasts, what kind of effects MPMBP has was studied. After MC3T3-E1 cells were cultured in a α-MEM (10% bovine serum supplementation) and after the confluent was reached, under the presence or absence of MPMBP (1, 10, and 100 μM), for 6, 10, or 20 days, they were cultured in the culture plate of 48 wells. After completion of culturing, a cell homogenate was prepared, and using the method by Lowry et al (J Biochem 1954; 207: 19-37), the ALP activity (per well) was measured. The results were such that MPMBP increased the ALP activity in a dose-dependent manner and with time (
Using the bone organ culture system, what kind of effects MPMBP has on production of the bone matrix was studied. From 3-6 day old mice, calvariae were collected aseptically. These were cultured using the Stern and Krieger method (Calcif Tissue hit 1983; 35: 172-17) with DMEM (10% bovine serum supplementation, no supplementation of heparin, supplementation or no supplementation of LPS 10 μg/ml) for 48 hours. After completion of culturing, with the method described by Suzuki, Takeyama et al (J Histochem Cytochem 2005; 53: 1525-1537), a double staining of the type I collagen and alkaline phosphatase (
The upper panels are Nomarski differential interference contrast images of the bone surface. The middle panels are images of the bone surface after the double staining of the type I collagen and alkaline phosphatase was performed, and the lower panels are images of the bone section after the double staining of the type I collagen and alkaline phosphatase was performed, respectively. Using rhodamine for the type I collagen, red color could be observed, and using a substrate (ELF-97) that fluoresces when dephosphorylated for alkaline phosphatase (stain osteoblasts), the enzyme activity could be observed in green color. By adding MPMBP into a culture fluid, regardless of the presence or absence of LPS (10 μg/ml) (lipopolysaccharide: one of the bone resorption promoting factors), compared to the control bone, production of the bone matrix (type I collagen: sites in red) significantly increased. For osteoblasts, under the presence of MPMBP, production of the collagen fibers increased; therefore, they were covered with the collagen fibers that they themselves produced, and no clear observation could be made, as was the case with the control bone. Moreover, based on the observation of the Nomarski differential interference contrast images, under the presence of MPMBP, it can be observed that the bone resorption was inhibited resulting from LPS and the size of the resorption cavity that was formed by LPS was significantly small, compared to the control bone.
As above, zoledronate works such that production of the bone matrix is inhibited, and in contrast, it was clear that MPMBP acts such that it increases production of the bone matrix. Moreover, the degree is significantly higher, compared to the effect of alendronate, which is the only one in which the osteogenesis promoting action is reported.
As an experiment to clarify the mechanism of the osteogenesis promoting action of MPMBP, using the osteoblast-based cell line MC3T3-E1 cells, the effect on expression of osteogenesis-related genes was studied using the real-time RT-PCR method. Moreover, the effect was compared with the action of zoledronate.
In the experiment, the osteoblast-based cell line MC3T3-E1 cells that reached the confluent after cultured with α-MEM (10% bovine serum supplementation) were used. For 3 days, they were cultured (used a dish with the diameter of 60 mm) under the presence of MPMBP (1, 10, or 100 μM) or zoledronate (0.1, 0.5 or 2.00 μM). After completion of culturing, cells were scraped and using Trizol, RNA was extracted. From 5 μg of RNA, cDNA was synthesized, specific primers were set with respect to alkaline phosphatase, the type I-α collagen, osteocalcin, and bone sialoprotein genes, PCR amplification was performed in the thermal cycler, and the amount of expression of the above genes was measured.
The results were such that for MPMBP, at the concentration of 100 μM, expression of the above genes known to be the index for promotion of osteogenesis was increased significantly in all genes (
As above, for all four Examples, it was newly confirmed that MPMBP has the promoting effects with respect to osteogenesis.
The osteogenesis promoter having [4-(methylthio)phenylthio] methanebisphosphonic acid or the pharmaceutically acceptable salt thereof as an active ingredient can be widely applied to the repair, reconstruction, filling, etc., of defective sites after removal of sockets, cysts, and tumors; alveolar bone defective sites absorbed by periodontal disease; alveolar clefts resulting from congenital anomaly; fractured sites; peri-implant bones, etc.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2008-225484 | Sep 2008 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2009/003758 | 8/5/2009 | WO | 00 | 5/16/2011 |
