JP7118635B2 - Pharmaceutical composition - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act (1) Program Abstracts of the 35th Annual Meeting of the Japan Society of Bone and Metabolism, June 25, 2017 (2) ASBMR 2017 Annual Meeting Abstract Book, August 28, 2017 Day

The present invention relates to a pharmaceutical composition that is taken for improving renal function or preventing renal damage.

Inorganic phosphate (Pi) is a substrate for ATP synthesis and is taken up into cells by phosphate transporters on the cytoplasmic membrane. On the other hand, excess inorganic phosphate in the extracellular fluid is stressful for cells. For example, a high phosphorus state induces apoptosis and causes calcification in various parts of the body, which may cause arteriosclerosis, myocardial infarction, arthritis, and the like. When renal function declines, the excretion of phosphorus is not performed normally, resulting in an increase in blood phosphorus concentration. Therefore, hyperphosphatemia is known to be one of the prognostic factors for abnormal bone mineral metabolism associated with chronic kidney disease (CKD).

The present inventors produced rats overexpressing type III sodium-dependent Pi transporter (Pit-1) among phosphate transporters, and examined the effect on bone metabolism. In expression rats, an increase in serum inorganic phosphorus concentration and a decrease in bone density were observed (see Non-Patent Document 1). In addition, an increase in urinary protein was observed in this Pit-1 overexpressing rat (see Non-Patent Document 2). Furthermore, glomerular epithelial cells, especially podocytes, of Pit-1 overexpressing rats had increased phosphate uptake, decreased number of foot processes, and thickened glomerular basement membrane. (See Non-Patent Document 2.). These symptoms suggest that Pit-1 overexpressing animals are one of the pathological models of nephrotic syndrome.

On the other hand, bisphosphonates are used as therapeutic agents for osteoporosis as bone resorption inhibitors. However, in cases of advanced renal failure, excretion may be delayed, and therefore it is contraindicated or should be administered with caution (see Non-Patent Document 3).

Suzuki, et al., Journal of Bone and Mineral Metabolism, 2010, vol.28(2), p.139-48. Sekiguchi, et al., American Journal of Physiology-Renal Physiology, 2011, vol.300(4), F848-F856 DOI: 10.1152/ajprenal.00334.2010 Brand name: Actonel (registered trademark) tablets (2.5 mg), generic name: package insert for risedronate sodium tablets (revised May 2016 (22nd edition)), EA Pharma Co., Ltd. and Eisai Co., Ltd. Matheny, et al., Bone, 2013, vol.57(1), p.277-283. Osawa et al., American Journal of Clinical Pathology, 1966, vol.45(1), p.7-20.

An object of the present invention is to provide a pharmaceutical composition that is taken for improving renal function or preventing renal damage.

As a result of intensive studies to solve the above problems, the present inventors found that administration of a bisphosphonate improves the increase in urinary protein and the thickening of the glomerular basement membrane observed in Pit-1 overexpressing animals. I completed the present invention.

That is, embodiments of the present invention relate to the following [1] to [6].
[1] A medicament containing a bisphosphonate, a pharmacologically acceptable salt thereof, or a solvate thereof as an active ingredient and taken for improving renal function or preventing renal damage. composition.
[2] The pharmaceutical composition of [1] above, wherein the bisphosphonate is one or more selected from the group consisting of risedronic acid, alendronic acid, etidronic acid, minodronic acid, zoledronic acid, and pamidronic acid.
[3] The pharmaceutical composition of [1] or [2], which is taken to improve the function of the glomerular basement membrane.
[4] The pharmaceutical composition of [1] or [2], which is taken for the purpose of reducing the amount of urinary protein.
[5] The pharmaceutical composition of any one of [1] to [4], which is administered to a patient with nephrotic syndrome.
[6] The pharmaceutical composition according to any one of [1] to [4], which is administered to patients with chronic kidney disease.

The pharmaceutical composition according to the present invention can improve urinary protein increase and glomerular basement membrane thickening caused by high phosphorus loading, and can improve glomerular basement membrane function. Therefore, the pharmaceutical composition according to the present invention is excellent as an active ingredient of a drug for improving renal function or preventing renal damage.

1 is a diagram showing the measurement results of body weight (g) of rats in each group in Example 1. FIG. 1 is a diagram showing the measurement results of blood albumin levels (g/dL) of rats in each group in Example 1. FIG. 1 is a diagram showing the results of measurement of serum creatinine levels (mg/dL) in rats of each group in Example 1. FIG. 1 is a diagram showing the results of measurement of serum calcium levels (mg/dL) in rats of each group in Example 1. FIG. 1 is a diagram showing the results of measuring serum phosphate levels (mg/dL) in rats of each group in Example 1. FIG. 1 is a diagram showing the measurement results of urinary calcium levels (mg/day) of rats in each group in Example 1. FIG. 1 is a diagram showing the measurement results of urinary phosphoric acid levels (mg/dL) in rats of each group in Example 1. FIG. 1 is a diagram showing the measurement results of TmP/GFR (mg/dL) of rats in each group in Example 1. FIG. 1 is a diagram showing the measurement results of urinary protein amounts (mg/day) of rats in each group in Example 1. FIG. 1 is a transmission electron micrograph of the kidney glomerular basement membrane of rats of each group in Example 1. FIG. 1 is a diagram showing measurement results of glomerular basement membrane thickness (nm) of rats of each group in Example 1. FIG. 1 is a diagram showing the measurement results of the thickness (nm) of the stratum densa of rats in each group in Example 1. FIG. In Example 2, results of measurement of anti-connexin43 antibody fluorescence intensity (× 1000 count) per glomerulus in images in which the glomerular basement membrane of each group of rats was co-fluorescently stained with anti-connexin43 antibody and anti-ZO-1 antibody. It is a figure showing. In Example 2, the results of measurement of the anti-desmin antibody fluorescence intensity (× 1000 count) per glomerulus in images obtained by co-fluorescently staining the glomerular basement membrane of the rats of each group with an anti-desmin antibody and an anti-laminin antibody are shown. It is a diagram. In Example 2, the results of measurement of the anti-nephrin antibody fluorescence intensity (× 1000 count) per glomerulus in images obtained by co-fluorescently staining the glomerular basement membrane of the rats of each group with an anti-nephrin antibody and an anti-laminin antibody are shown. It is a diagram. In Example 3, urinary microalbumin levels ( FIG. 10 is a diagram showing results of mean values of mg/g Cr).

Hereinafter, the present invention will be described in detail by showing embodiments. The pharmaceutical composition according to this embodiment contains a bisphosphonate or a pharmacologically acceptable salt thereof, or a solvate thereof as an active ingredient, and is taken for improving renal function or preventing renal damage. be. Bisphosphonates can improve renal function caused by high phosphorus load. Although the mechanism of action by which bisphosphonates improve renal function has not been elucidated in detail, bisphosphonate administration improves nephrosis (increase in urinary protein) caused by high phosphorus load, and also improves glomerular basement membrane thickening. . From this, it is considered that bisphosphonates improve the function of the glomerular basement membrane, and particularly have the effect of reducing the load on the glomerular basement membrane due to high phosphorus load. Bisphosphonates are widely used as bone resorption inhibitors, but their administration to patients with renal impairment should be avoided. Nonetheless, it is surprising that bisphosphonates themselves are effective in improving renal function.

Bisphosphonates that are active ingredients of the pharmaceutical composition according to this embodiment include risedronic acid, etidronic acid, alendronic acid, minodronic acid, zoledronic acid, pamidronic acid, tiludronic acid, monidronic acid, neridronic acid, olpadronic acid, and clodrone. acid, ibandronic acid and the like. The active ingredient of the pharmaceutical composition according to this embodiment is preferably risedronic acid, etidronic acid, alendronic acid, minodronic acid, zoledronic acid, or pamidronic acid. The active ingredient of the pharmaceutical composition according to this embodiment may contain one type of bisphosphonate, or may contain two or more types of bisphosphonates.

The active ingredient of the pharmaceutical composition according to this embodiment may be a pharmacologically acceptable salt of a bisphosphonate, or a solvate of a bisphosphonate or a pharmacologically acceptable salt thereof. good. Examples of pharmacologically acceptable salts of bisphosphonates include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as magnesium salts and calcium salts; aluminum salts; trimethylamine salts, triethylamine salts, and dicyclohexylamine. salts, dibenzylamine salts, phenethylbenzylamine salts, procaine salts, morpholine salts, pyridine salts, piperidine salts, N-ethylpiperidine salts and other amine salts; ammonium salts; lysine salts, arginine salts and other basic amino acid salts; is mentioned. Examples of the solvent constituting the solvate of the bisphosphonate or its salt include water and alcohols such as ethanol. The active ingredient of the pharmaceutical composition according to the present embodiment is preferably an alkali metal salt of a bisphosphonate, a hydrate of an alkali metal salt of a bisphosphonate, or a hydrate of a bisphosphonate. Hydrates or hydrates of bisphosphonates are more preferred. Hereinafter, bisphosphonates, pharmaceutically acceptable salts of bisphosphonates, and solvates of bisphosphonates or pharmaceutically acceptable salts thereof may be collectively referred to simply as "bisphosphonates."

Among them, the active ingredient of the pharmaceutical composition according to this embodiment includes risedronate sodium hydrate, alendronate sodium hydrate, etidronate disodium, minodronic acid hydrate, zoledronic acid hydrate, or Disodium pamidronate hydrate is preferred. A commercially available bisphosphonate drug can also be used as the active ingredient of the pharmaceutical composition according to this embodiment.

Bisphosphonates are effective in improving renal function or preventing renal damage. Therefore, the pharmaceutical composition according to the present embodiment is effective as a therapeutic or prophylactic agent for chronic kidney disease, and can also be used as a therapeutic or prophylactic agent for various diseases in which deterioration of renal function causes onset or exacerbation. It is valid. Diseases in which decreased renal function causes onset or exacerbation include, for example, abnormal bone mineral metabolism associated with chronic kidney disease (CKD-MBD), diabetic nephropathy, nephrotic syndrome, chronic nephritis, IgA nephropathy, nephrosclerosis. disease, etc.

When the pharmaceutical composition according to this embodiment is administered to animals including humans, the dosage form may be either oral administration or parenteral administration. , for example, solid formulations such as powders, granules, capsules, tablets, chewable formulations; liquid formulations such as solutions and syrups; - Examples include pulmonary sprays. The pharmaceutical composition according to this embodiment can be formulated into medicaments of these dosage forms by ordinary methods.

From the viewpoint of reducing the burden on patients, the pharmaceutical composition according to this embodiment is preferably orally administered to the subject. A preferred dosage form for oral administration is a tablet. On the other hand, for subjects to whom oral administration is difficult, the pharmaceutical composition according to the present embodiment can be administered intravenously or arterially as an infusion. In addition, the pharmaceutical composition according to the present embodiment may contain various appropriate pharmaceutically acceptable additives as necessary for formulation. Such additives include, for example, excipients, binders, lubricants, solvents, disintegrants, solubilizers, suspending agents, emulsifiers, tonicity agents, stabilizers, soothing agents, preservatives , antioxidants, flavoring agents, coloring agents, and the like.

Examples of excipients include sugars such as lactose, glucose, and D-mannitol, starches, organic excipients such as celluloses such as crystalline cellulose; inorganic excipients such as calcium carbonate, kaolin, and titanium oxide; is mentioned. Binders include pregelatinized starch, gelatin, gum arabic, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, D-mannitol, trehalose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol and the like. Lubricants include fatty acid salts such as stearic acid and stearates, talc, and silicates. Examples of the solvent include purified water, physiological saline, and the like. Examples of disintegrants include low-substituted hydroxypropyl cellulose, chemically modified cellulose and starches. Examples of solubilizing agents include polyethylene glycol, propylene glycol, trehalose, benzyl benzoate, ethanol, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate and the like. Suspending or emulsifying agents include sodium lauryl sulfate, gum arabic, gelatin, lecithin, glyceryl monostearate, polyvinyl alcohol, polyvinylpyrrolidone, celluloses such as sodium carboxymethylcellulose, polysorbates, polyoxyethylene hydrogenated castor oil, and the like. mentioned. Tonicity agents include sodium chloride, potassium chloride, sugars, glycerin, urea and the like. Stabilizers include polyethylene glycol, sodium dextran sulfate, and other amino acids. Examples of pain relievers include glucose, calcium gluconate, procaine hydrochloride and the like. Preservatives include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like. Antioxidants include sulfites, ascorbic acid, and the like. Flavoring agents include: Sweeteners, flavoring agents and the like commonly used in the pharmaceutical and food fields are included. Coloring agents include those commonly used in the pharmaceutical and food fields.

The dosage of the pharmaceutical composition according to the present embodiment varies depending on the subject's renal function condition, age, body weight, pathology of tissues other than the kidney, administration method, administration interval, etc. It can be appropriately set in consideration of dosage. For example, in the case of oral administration, 0.01 to 50 mg, preferably 0.1 to 10 mg, particularly preferably 0.5 to 5 mg of bisphosphonate is administered as a daily dose per adult once or in several doses. be able to. In addition, 1 to 100 mg, preferably 5 to 100 mg, particularly preferably 10 to 100 mg of bisphosphonate can be administered per adult once or several times a week to a year. In the case of parenteral administration, for example, 0.0001 to 10 mg, preferably 0.001 to 10 mg, particularly preferably 0.01 to 0.01 to 10 mg of bisphosphonate per adult is divided into once or several times per week to year. 5 mg can be injected intravenously or continuously intravenously for up to 24 hours.

EXAMPLES Next, the present embodiment will be described in more detail with reference to Examples, etc., but the present invention is not limited to these.

[Example 1]
In Pit-1 overexpressing animals, phosphate overload damages renal podocytes and thickens the glomerular basement membrane, ultimately leading to nephrotic syndrome.
Therefore, we examined whether the administration of bisphosphonates, which have a competitive effect on phosphorus, improved urinary protein levels and glomerular basement membrane thickening in Pit-1 overexpressing rats, one of the pathological models of nephrotic syndrome. did. Sodium risedronate was used as the bisphosphonate. In addition, Pit-1 overexpressing rats prepared by the method described in Non-Patent Document 1 were used.

<Administration of risedronate sodium>
5-week-old Pit-1 overexpressing rats and control rats (Wister strain rats), CC group: control rat group (n = 4) not administered with risedronate sodium, CR group: risedronic acid Control rat group administered sodium (n = 5), TG-C group: Pit-1 overexpressing rat group (n = 3) not administered risedronate sodium, TG-R group: administered risedronate sodium They were divided into a total of 4 groups, a Pit-1 overexpressing rat group (n=3). According to the method of Matheny et al. (see Non-Patent Document 4), the CR group and the TG-R group were subcutaneously injected with 5 μg/kg body weight of risedronate sodium twice a week. The CC group and the TG-C group were subcutaneously injected twice a week with an equal volume of physiological saline at the same timing as the CR group and the TG-R group.

<Collection and component analysis of blood and urine, and measurement of body weight>
Blood and urine collection and body weight measurements were performed at 5 weeks of age (before administration of risedronate sodium), 8 weeks of age, and 12 weeks of age. Blood was collected from the tail vein. After fasting for 12 hours, urine was collected for 24 hours.
Blood albumin level (g/dL), serum creatinine level (mg/dL), serum calcium level (mg/dL), serum phosphate level (mg/dL), urinary calcium level (mg/day), urine Phosphate level (mg/dL), renal proximal tubular phosphate reabsorption threshold (TmP/GFR) (mg/dL), and urinary protein level (mg/day) were measured by conventional methods.

Figure 1 shows the measurement results of body weight (g), Figure 2 shows the measurement results of blood albumin level (g/dL), Figure 3 shows the measurement results of serum creatinine level (mg/dL), and serum calcium level (mg /dL), serum phosphate (mg/dL) in FIG. 5, urinary calcium (mg/day) in FIG. (mg/dL) is shown in FIG. 7, TmP/GFR (mg/dL) is shown in FIG. 8, and urinary protein amount (mg/day) is shown in FIG. In the figure, the columns with "-" for "Pit-1TG" and "-" for "Risedronate" are the results of group C-C, the columns for "-" for "Pit-1TG" and "+" for "Risedronate" is the result of the CR group, "Pit-1TG" is "+", "Risedronate" is "-" column is the result of the TG-C group, "Pit-1TG" is "+", "Risedronate" Columns with "+" indicate the results of the TG-R group, respectively. All measured values are mean±SE (standard error), and “*” means “p<0.05”. As a result, no differences between groups were observed in body weight gain rate, serum calcium level, serum phosphorus level and albumin level between the 8th and 12th weeks of age. On the other hand, the amount of urinary protein at 12 weeks of age was clear in the TG-C group (42.2 ± 27.0 mg/day) compared to the CC group (7.1 ± 3.6 mg/day). (p=0.037). The decrease in urinary protein excretion in the TG-R group was not statistically significant compared to the TG-C group, but the significant difference from the CC group disappeared.

<Observation of glomerular basement membrane>
Kidney tissue was collected from 8-week-old rats housed under the same conditions. Similar to the method described in Non-Patent Document 2, glomerular basement membranes of the collected kidneys were observed with a transmission electron microscope (product name: JEM-1010, manufactured by JEOL).
The thickness of the glomerular basement membrane was measured from just below the cell membrane of the epithelial foot process to just below the endothelial cells according to the method of Osawa et al. (see Non-Patent Document 5).

Transmission electron micrographs of kidney glomerular basement membrane collected from each group of rats are shown in FIG. 10, the measurement results of glomerular basement membrane thickness are shown in FIG. are shown in FIG. 12, respectively. In the figure, the columns with "-" for "Pit-1TG" and "-" for "Risedronate" are the results of group C-C, the columns for "-" for "Pit-1TG" and "+" for "Risedronate" is the result of the CR group, "Pit-1TG" is "+", "Risedronate" is "-" column is the result of the TG-C group, "Pit-1TG" is "+", "Risedronate" Columns with "+" indicate the results of the TG-R group, respectively. All measured values are mean±SE, and "*" means "p<0.05". As shown in Figure 11, the thickness of the glomerular basement membrane was 206.1 ± 75.1 mm in the TG-R group and 221.1 ± 88.4 mm in the TG-C group (p = 0.016). . That is, as shown in FIG. 10, thickening of the glomerular basement membrane was observed in the 8-week-old TG-C group, but the degree of thickening was mild in the TG-R group compared to the TG-C group. there were.

These results suggest that bisphosphonates such as risedronic acid can ameliorate both urinary protein increase and glomerular basement membrane thickening in Pit-1 overexpressing rats. It has been shown that it may be possible to attenuate Moreover, in Pit-1 overexpressing rats, serum phosphate level was not affected by risedronic acid administration, suggesting that the effect of bisphosphonates attenuating high phosphorus load is not due to inhibition of bone metabolism, but directly to podocytes. It was suggested that it is a working effect.

Even if the serum phosphorus level is normal at the early stage of bone mineral metabolism disorder, the secretion of PTH (parathormone), FGF23 (fibroblast growth factor 23) and the like is enhanced in response to a slight phosphorus load. These lead to a decrease in bone density and a decrease in active vitamin D, and are one of the factors that exacerbate abnormalities in bone mineral metabolism. The glomerular basement membrane has been reassessed for its importance as a renal protein filtration barrier, and therapeutic interventions that preserve its physiological function may slow the progression of bone mineral metabolism disorders.

[Example 2]
In the same manner as in Example 1, 5-week-old Pit-1-overexpressing rats and control rats (Wister strain rats) were administered with risedronate sodium, and the renal glomerular basement membrane at 12-week-old. The lesion status was assessed by immunostaining. Administration of risedronate sodium and physiological saline was carried out in the same manner as in Example 1.

Sections of the renal glomerular basement membrane collected from 12-week-old rats were co-fluorescently stained with anti-connexin43 antibody and anti-ZO-1 antibody, co-fluorescently stained with anti-desmin antibody and anti-laminin antibody, or with anti-nephrin antibody. Co-fluorescent staining of anti-laminin antibody was performed. Connexin43, desmin and nephrin are constituents of glomerular epithelial cells. ZO-1 is a molecule that constitutes a tight junction, and laminin is a molecule that constitutes the basement membrane of the extracellular matrix.

Quantification results by immunostaining of various extracellular matrices (connexin43, laminin, nephrin) are shown. Anti-connexin43 antibody fluorescence intensity (× 1000 count) per glomerulus measured from images of co-fluorescent staining of anti-connexin43 antibody and anti-ZO-1 antibody in FIG. 13, anti-desmin antibody and anti-laminin antibody in FIG. The anti-desmin antibody fluorescence intensity (× 1000 count) per glomerulus measured from the co-fluorescent staining image of the glomerulus measured from the anti-nephrin antibody and anti-laminin antibody co-fluorescent staining image in FIG. The anti-nephrin antibody fluorescence intensity (×1000 count) per piece is shown. In the figure, the columns with "-" for "Pit-1TG" and "-" for "Risedronate" are the results of group C-C, the columns for "-" for "Pit-1TG" and "+" for "Risedronate" is the result of the CR group, "Pit-1TG" is "+", "Risedronate" is "-" column is the result of the TG-C group, "Pit-1TG" is "+", "Risedronate" Columns with "+" indicate the results of the TG-R group, respectively. As a result, as shown in FIGS. 13 and 14, no particular difference was observed in the CR group compared to the CC group, but in the TG-C group, the expression of connexin43 and desmin in the glomerular basement membrane was enhanced, and this change was shown to be suppressed by administration of Risedronate. Moreover, as shown in FIG. 15, no inter-group difference was observed in the fluorescence intensity of the anti-nephrin antibody. That is, in the TG-C group, glomerular epithelial cell injury was the strongest, and although epithelial cell injury was observed in the TG-R group, the degree of epithelial cell injury was lessened than in the TG-C group. No epithelial cell injury was observed in the CC and CR groups.

[Example 3]
Among type 2 diabetes patients, administration of bisphosphonates to 21 cases of urinary microalbumin-positive patients other than renal transplant cases and diabetic nephropathy stage 4 and 5 patients, and effects on urinary microalbumin levels examined.

Specifically, risedronate sodium in 9 cases (7 females, 2 males), alendronate sodium hydrate in 7 cases (7 females), and minodronic acid hydrate in 5 cases (5 males) was administered. The dosing period ranged from 1 week to 18 months.

Figure 16 shows the mean ± SE of urinary microalbumin levels (mg/g Cr) before administration of bisphosphonate (in the figure, "before administration") and after administration (in the figure, "after administration") in all 21 cases. Show the results. The average urinary microalbumin level in these 21 cases was 52.7 mg/g Cr before administration of the bisphosphonate, whereas it was 39.1 mg/g Cr after administration of the bisphosphonate, which was clearly reduced. (p=0.016). More specifically, in 16 out of 21 cases, a decrease in urinary microalbumin levels was observed due to bisphosphonate administration. The rate of decrease in urinary microalbumin levels in these 16 cases (100-[urine microalbumin levels after administration]/[urine microalbumin levels before administration]×100) (%) was 0.7 to 77.2%. , the average value of 16 cases was 37.1%.

The pharmaceutical composition according to the present invention can reduce the amount of urinary protein and improve the thickening of the glomerular basement membrane. Therefore, the pharmaceutical composition according to the present invention is particularly effective as a drug for improving renal function and preventing renal damage.

Claims (2)

Pharmaceutical use characterized in that it contains risedronic acid or a pharmacologically acceptable salt thereof, or a solvate thereof as an active ingredient, and is taken for the reduction of urinary protein increased by high phosphorus loading. Composition. It contains risedronic acid or a pharmacologically acceptable salt thereof, or a solvate thereof as an active ingredient, and is taken to improve thickening of the renal glomerular basement membrane due to high phosphorus load. A pharmaceutical composition.

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