JPH11292790A - Therapeutic agent for renal disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject therapeutic agent containing an expression inhibitor for a perchloric acid-soluble protein as an active ingredient, capable of promoting the proliferation of uriniferous tubuliform cells and useful for treating renal diseases. SOLUTION: This therapeutic agent containing an expression inhibitor for a perchloric acid-soluble protein(PSP) as an active ingredient is obtained. The expression inhibitor for the PSP is capable of inhibiting the expression of the PSP in uriniferous tubuliform cells and is preferably a PSP derived from a human kidney or its derivative. The PSP is a protein extracted with perchloric acid and having 14 kDa molecular weight and can be extracted and purified from a biological tissue. A substance in which a part of the amino acid sequence of the PSP is deleted or substituted with other amino acids, etc., is cited as the PSP derivative. The therapeutic agent is used together with a filler, an extender, etc., and can be converted into a pharmaceutical preparation composition. The active ingredient is contained in an amount of preferably 1-70 wt.% in the pharmaceutical preparation. The daily dose thereof administered is preferably 0.01 μg to 10 mg based on 1 kg body weight expressed in terms of the PSP.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a therapeutic agent for renal disease.

[0002]

2. Description of the Related Art The kidney is an organ that is responsible for maintaining body fluid homeostasis and excreting urea-based waste products in the body. Also, kidneys are tubules,
In particular, the proximal tubule functions as an endocrine metabolic regulatory system that produces vitamin D activation, hormones and enzymes such as erythropoietin and ACE converting enzyme.

[0003] Kidney diseases include glomerulopathy causing glomerular injury (typically, chronic glomerulonephritis) and renal tubular injury (typically, ischemic acute renal failure).
It is roughly divided into two. Among them, the latter interferes with the endocrine metabolic regulation system and is therefore closely related to the development of renal osteodystrophy, renal anemia, renal hypertension and the like. In any case, if the nephritis becomes chronic, its treatment becomes difficult,
Eventually, renal failure occurs, necessitating hemodialysis. Hemodialysis imposes a heavy burden on patients, and also raises the problem of side effects as the dialysis period becomes longer.

[0004] The predominant theory is that renal disease develops due to hemodynamic abnormalities associated with glomerular injury and the resulting hyperfiltration or perfusion of residual glomeruli.
However, it has been pointed out that as the research by molecular biology techniques in recent years progresses, tubular interstitial injury rather than glomerular injury is deeply involved in the development of renal disease (Kidney int. , 53, 1439 (1997)].

[0005] Unlike the glomeruli, the renal tubules are regenerable organs. In particular, it is known that renal regeneration is performed from the proximal tubules. Therefore, studies have been conducted on promoting the cell proliferation of the proximal tubule as a means for preventing or treating the progression of renal disease by elucidating the above mechanism.

[0006] As a drug for treating renal disease, there is no particularly effective drug except for steroid drugs used for acute nephritis and mild chronic nephritis. Therefore, in chronic nephritis which is not applied to drugs, it has been mainly performed to delay the progression to chronic renal failure due to dietary restrictions.

[0007] In recent years, as the relationship between the progression of renal disease and the renal tubule has been elucidated as described above, in order to obtain a novel therapeutic agent for renal disease, a search for a substance that promotes renal tubular cell proliferation has been made. Is being done. In particular, various cell growth factors in a living body are noted, such as EGF (epidermal growth factor), TG
F-α (transforming growth factor), HGF (hepatocyte growth factor), IGF-1 (insulin growth factor) and the like have been studied. However, none of the above has been established, and there are many negative reports. For example, it has been reported that the most studied IGF-1 is not effective in uremic patients with chronic renal failure [Pediatr.
hrol., 4, 654 (1990)) and reports that there were no significant differences in various indices even in patients with acute renal failure (Kidney int.,
45, 1731 (1994)]. As for EGF,
It is known that mesangial cells associated with glomerulonephritis can be proliferated or that cancer cells can be proliferated, and practical application is considered to be practically impossible.

[0008] As mentioned earlier, it is possible to greatly contribute to the treatment of renal disease if the cell regeneration of the damaged tubule can be promoted by promoting the cell proliferation of the tubule. It has been strongly desired in clinical practice that such drugs be developed and effective renal disease therapeutic agents be developed.

[0009]

Means for Solving the Problems The present inventors have previously discovered a protein having a protein synthesis inhibitory activity extracted by perchloric acid from a liver cytoplasmic fraction and named it a perchloric acid soluble protein (PSP). [J. Biol. Chem., 270, 300
60 (1995)]. In addition, they found that a protein having almost the same amino acid sequence was localized not only in the liver but also in the proximal tubule of the kidney [Japan Society of Nutrition and Food Science (1997)]. Therefore,
Furthermore, as a result of further research on this PSP, it was found that the expression of PSP was high immediately before the proliferation of proximal tubule cells entered the stationary phase, and if the expression of PSP was suppressed, the cells could be proliferated. And reached the knowledge. Also, PS
Surprisingly, when P itself is applied to proximal tubule cells, an unexpected phenomenon occurs in which intracellular PSP expression is suppressed, and concomitantly promotes tubular cell proliferation as described above. , Found that renal disease can be treated,
The present invention has been completed.

[0010] That is, the present invention provides a therapeutic agent for renal disease comprising a PSP expression inhibitor as an active ingredient. The present invention also provides a therapeutic agent for renal disease comprising PSP or a derivative thereof as an active ingredient.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The therapeutic agent for renal disease of the present invention comprises PSP
Although an expression-suppressing substance is contained as an active ingredient, a typical example of the substance is PSP itself.

PSP is a protein having a molecular weight of about 14 kDa extracted by perchloric acid and can be easily isolated and purified by a known method. Since PSP extracted from the liver and those extracted from the kidney have almost the same primary structure, any of them can be used as the active ingredient of the present invention. It is preferably used. In addition, PSP has a highly retained primary structure in most organisms.
Although SP can be used, it is preferable to use human-derived PSP from the viewpoint of efficacy and safety.

[0013] PSP is described, for example, in the literature [J. Biol. Chem.,
270, 30060 (1995)], and can be extracted and purified from living tissues. That is, a mitochondrial supernatant obtained by homogenizing the liver or kidney can be subjected to perchloric acid extraction and trichloroacetic acid extraction, and further purified by a conventional protein purification method such as a CM-Sephadex column. Alternatively, the mitochondrial supernatant can be purified by using a gel filtration and an ion exchange chromatography column. As another method, there can be mentioned a method obtained by culturing primary culture cells or cell lines that produce PSP and purifying them from the culture. Furthermore, using known genetic engineering techniques,
The gene encoding PSP may be inserted into an appropriate vector, inserted into an appropriate host, transformed, and obtained from a culture of this transformant. In this case, for example, the human kidney PSP gene is known as the above gene [Eur. J. Biochem., 242 (2), 339 (1996)], and various plasmid vectors and bovine papilloma virus DNA are used as the vector. And host cells include Escherichia coli, Bacillus subtilis, yeast, Chinese hamster ovary cells, mouse C127 cells and the like.

The derivatives of PSP used in the present invention include, for example, those in which a part of the amino acid sequence of PSP is deleted or substituted with another amino acid, or in which another amino acid sequence is partially inserted or added, Those to which sugar chains and the like are bonded are exemplified.

[0015] The therapeutic agent for renal disease of the present invention comprises, as an active ingredient, at least one substance that inhibits PSP expression in cells, such as PSP or a derivative thereof, which is used together with a suitable pharmaceutical carrier. Practically used as a form of a typical pharmaceutical preparation composition. As the pharmaceutical carrier, a filler, a bulking agent, a binder, and a filler generally used, depending on the use form of the pharmaceutical,
Examples thereof include diluents or excipients such as humectants, disintegrants, surfactants, and lubricants. These are appropriately selected and used depending on the dosage form of the preparation.

As the administration form of the pharmaceutical preparation, various forms can be selected according to the purpose of treatment, and typical examples thereof include injections (solutions, suspensions, etc.). For injections, the active ingredient is dissolved in a suitable solvent (eg, sterile water, buffer,
(Physiological saline), sterilized by filtration through a filter or the like, and then filled into a sterile container. In the above injection, diluents such as ethanol, macrogol, propylene glycol, polyoxyethylene sorbitan fatty acid ester, isotonic agents such as glucose, glycerin, mannitol, sorbitol, and solubilizers, stabilizers and the like are appropriately used. They can be added and blended. The injection may be lyophilized and redissolved at the time of use for use. Other administration forms include oral preparations such as tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules and the like.

The amount of the active ingredient to be contained in the pharmaceutical preparation of the present invention is not particularly limited and may be appropriately selected from a wide range. Good.

The dose of the above-mentioned pharmaceutical preparation is appropriately selected depending on its usage, age, sex, degree of disease and the like of the patient.
When PSP is used as an active ingredient, it is usually preferable that the PSP be used in an amount of about 0.01 μg to 10 mg per kg of body weight per day, and the preparation can be administered once to several times a day.

[0019]

Next, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

Test Example The following test shows that intracellular P in renal proximal tubule
The effect of suppressing SP expression and the effect of promoting cell proliferation were examined.

The following cells and culture solutions were used for the test. (1) PSP is described in the literature [J. Biol. Che.
m., 270, 30060 (1995)], and used was purified from rat kidney and further dialyzed against phosphate buffered saline. (2) Rat renal cortical epithelial cells NRK-
52E (70% or more of renal proximal tubule cells): manufactured by Dainippon Pharmaceutical. (3) Dulbecco's modified Eagle culture (DME
M): manufactured by INC Biomedicals. (4) Fetal calf serum (FCS): manufactured by Boehringer Mannheim.
(5) NEAA solution: manufactured by ICN Biomedicals.
(6) Phosphate buffer: manufactured by Nissui (Ca, Mg-free Dulbecco's phosphate buffer). (7) Penicillin-streptomycin solution: manufactured by ICN Biomedicals. (8) Trypsin-EDTA solution: manufactured by ICN Biomedicals.

That is, first, NRK-52E cells were
The cells were suspended in M + NEAA medium supplemented with 5% immobilized FCS, 100 units / ml penicillin and 100 μg / ml streptomycin, and the cell count was determined on a collagen-coated 12-well plate (manufactured by Becton Dickinson). Seed so as to have a density of 10 × 10 ⁴ cells / well, and further added PSP to a concentration of 10 ⁻⁷ M,
Culture was started at 37 ° C. in an environment of 5% carbon dioxide and 95% oxygen. In addition, what added the same amount of phosphate buffer instead of PSP was used as a control.

On days 4 and 8 of culture, trypsin-E
The cells were detached using a DTA solution, suspended in a phosphate buffer, a part thereof was taken out, and the number of cells was counted. The remaining cells were first homogenized under ice-cooling at 800 rpm for 15 strokes, and further homogenized at 4 ° C. for 30 minutes.
After centrifugation at 2,000 rpm, perchloric acid was added to the supernatant to a concentration of 5%, and the mixture was centrifuged at 4 ° C. for 10 minutes at 12,000 rpm, and trichloroacetic acid was added to the resulting supernatant at a concentration of 20%.
And centrifuged at 12,000 rpm for 30 minutes at 4 ° C. to obtain a precipitate containing PSP. The PSP was detected by a Western blotting method as usual, and measured by a densitometer.

FIG. 1 shows the intracellular PSP amount (mean value) on days 4 and 8 of the culture, and FIG. 2 shows the number of cells (mean ± standard deviation).
Shown in

As a result, it was found that in the cells to which PSP was added, the amount of PSP in tubular cells was decreased, and the number of cells was significantly increased.

Formulation Example 1 In 100 ml of physiological saline, the same P as used in the test example was used.
After dissolving 1 mg of SP, 1 g of mannitol and 10 mg of polysorbate 80, sterile-filtrating, filling each 1 ml into a vial, freeze-drying and sealing to obtain a freeze-dried preparation.

Formulation Example 2 100 ml of 0.02 M phosphate buffer (containing 0.15 M sodium chloride and 0.01% polysorbate 80)
In it, 1 mg of PSP and 100 mg of human serum albumin same as those used in the test example were dissolved and sterile-filtered, and then 1 ml
Each was filled in a vial, lyophilized, and then sealed to obtain a lyophilized preparation.

Formulation Example 3 In 100 ml of distilled water for injection, 1 mg of PSP, 1 g of sorbitol, 2 g of glycine and 10 mg of polysorbate 80, which are the same as those used in the test example, are dissolved, and sterile-filtered.
Each ml was filled into a vial, lyophilized, and then sealed to obtain a lyophilized preparation.

[0029]

According to the present invention, it is possible to promote renal tubular cell proliferation and to treat various renal diseases.

[Brief description of the drawings]

FIG. 1 is a graph showing the effect of PSP administration on suppressing the expression of PSP in tubular cells.

FIG. 2 is a graph showing the effect of PSP administration on promoting the proliferation of tubular cells.

Claims (5)

[Claims] A remedy for renal diseases comprising a perchloric acid-soluble protein expression inhibitor as an active ingredient. 2. The perchloric acid-soluble protein expression inhibitor,
The therapeutic agent for renal disease according to claim 1, which suppresses the expression of perchloric acid-soluble protein in tubular cells. 3. The perchloric acid-soluble protein expression suppressing substance,
The therapeutic agent for renal disease according to claim 1 or 2, which is a perchloric acid-soluble protein or a derivative thereof. 4. The therapeutic agent for renal disease according to claim 3, wherein the perchloric acid-soluble protein or its derivative is derived from kidney. 5. The therapeutic agent for renal disease according to claim 3, wherein the perchloric acid-soluble protein or its derivative is derived from human kidney.