WO2004108123A1 - Preventive and/or remedy for kidney diseases - Google Patents

Abstract

It is intended to provide a drug which is efficacious for kidney diseases such as acute kidney failure. This object can be achieved by a preventive and/or a remedy for kidney diseases which contains as the active ingredient a heat shock protein inducer, its salt or a hydrate of the same. More specifically speaking, a preventive and/or a remedy for kidney diseases which contains as the active ingredient 6,10,14,18-tetramethyl-5,9,13,17-nanodecatetraen-2-one that is a prenyl ketone compound.

Description

 Specification

 Agent for preventing and / or treating kidney disease

 Technical field

 The present invention relates to the prevention and / or treatment of renal disease, and more particularly, to a preventive and / or therapeutic agent for acute renal failure.

 Background art

 [0002] Acute renal failure (hereinafter, simply referred to as "ARF") is a clinically significant problem, and the mortality rate of patients suffering from such a disorder is extremely high (for example, see Non-Patent Documents 1 to 3). ). The current treatment method is symptomatic treatment, and the mortality rate can hardly be improved at present (for example, see Non-Patent Documents 1 and 4). For example, acute tubular necrosis, one of the causes of acute renal failure, is still known as one of the most lethal diseases (40-60%). However, although treatment has been successful and there are cases where improvement healing is possible, permanent renal dysfunction often remains.

 [0003] This ARF is often frequently associated with multiple organ failure. In addition to the complexity of multi-organ disease, the presence of ARF has significantly increased mortality. Ischemic reperfusion induced by renal insufficiency (hereinafter sometimes simply referred to as “I / R”) is a common etiology of acute renal insufficiency (for example, see Non-Patent Document 5). I / R occurs when renal perfusion decreases during shock. I / R is also a serious complication in aortic or renal vascular surgery (eg, see Non-Patent Document 6). In addition, I / R disorders are unavoidable in kidney transplants and occur during organ recovery and storage. I / R dysfunction is highly associated with acute rejection episodes and subsequent graft dysfunction (see, eg, Non-Patent Documents 7 and 8). These problems are always observed in cadaveric kidney transplants that undergo warm or cold ischemia followed by reperfusion. The unsatisfactory results from such transplants hinder the expansion of the donor pool through the use of marginal donor organs (see, for example, Non-Patent Document 9). Therefore, efforts to reduce kidney damage exposed to I / R induced by injury are important.

[0004] The pathogenesis of I / R disorders is complex, but seizures due to oxygen free radicals (eg, Reference 10) and the subsequent inflammatory response are important factors to consider (eg, see Non-Patent Document 7). Numerous studies have shown that the antioxidant status of organs is important by the primary endogenous defense against free radical-induced damage. Various endogenous antioxidants including α-tocopherol, lazaroid, probucol and scavenger superoxide dismutase have been reported to prevent renal I / R disorders (eg, Non-Patent Document 1). Other studies have identified various inflammatory mediators, tumor necrosis factor α, cell-cell adhesion factor 1, Ρ-selectin, platelet activating factor, Gro a, macrophage inflammatory protein 2, MCP 1, midkine, and others that may be involved. Have been. In therapeutic prevention targeting these molecules, the renal I / R disorder was successfully attenuated in experimental animals (for example, see Non-Patent Documents 1 and 3).

 [0005] Despite many of the studies described above, no treatment has proven effective in humans. And much interest has been focused on inducing ischemic tolerance. This is a reasonable strategy, as it is based on the original defense mechanism. The concept of ischemic tolerance was established based on the premise that quasi-lethal heat shock or low levels of ischemic damage induced resistance to cells and tissues and then to exposure to heat or ischemic damage (For example, see Non-Patent Document 11). The mechanism of action of this resistance has not been fully elucidated. It is believed that force heat shock proteins (Hsps) play an important role in this process. Drugs that induce Hsp expression have been energetically studied. However, although several drugs that induce Hsp expression are known, some drugs are also known to be toxic or have adverse side effects (eg, Non-Patent Documents 12 to 14). reference). Thus, finding non-toxic compounds that can effectively induce Hsps in the kidney provides important therapeutic benefits.

[0006] By the way, geranylgeral acetone (hereinafter simply referred to as "GGA" t) is a kind of acyclic polyisoprene and has been used since 1984 for the treatment of gastritis and gastric ulcer without adverse reactions. It is an anti-ulcer agent that is used clinically. This GGA is known to induce overexpression of Hsp-familuin in the gastric mucosa and small intestine when administered orally to rats (for example, see Non-Patent Documents 15 and 16). Recently, GGA's organ protection effect Has been observed in the heart (for example, see Non-Patent Document 17) and the liver (for example, see Non-Patent Documents 18 and 19).

Non-Patent Document 1: BONVENTRE JV, WEINBERG JM: Recent advances in the pathophysiology of ischemic acute renal failure.J Am Soc Nephrol 14: 2199-2210, 2003

Non-patent document 2: SINGRI N, AHYA SN, LEVIN ML: Acute renalfailure. JAMA 289: 747-751, 2003

Non Patent Literature 3: DE VRIESE AS: Prevention and treatment of acute renal failure in sepsis.J Am Soc Nephrol 14: 792-805, 2003

Non-Patent Document 4: RONCO C, BELLOMO R: Prevention of acute renalfailure in the critically ill.Nephron Clin Pract 93: C13-C20, 2003

Non-Patent Document 5: PADANILAM BJ, LEWINGTON AJ: Molecularmechanisms of cell death and regeneration in acute ischemic renal injury.Curr Opin NephrolHypertens 8: 15-19, 1999

Non-Patent Document 6: MYERS BD, MORAN SM: Hemodynamically mediatedacute renal failure.N Engl J Med 314: 97-105, 1986

Non-Patent Document 7: HALLORAN PF, BATIUK TD, GOES NB, CAMPBELL

P: Strategiesto improve the immunologic management of organ transplants.

ClinicalTransplantation 9: 227—236, 1995

Non-Patent Document 8: TROPPMANN C, GILLINGHAM KJ, BENEDETTI E, et

ahDelayedgraft function, acute rejection, and outcome after cadaver

renaltransplantation. The multivariate analysis.Transplantation 59: 962--968, 1995 Non-Patent Document 9: KOOTSTRA G: The asystolic, or non-heartbeating, donor.

Transplantation 63: 917—921, 1997

Non-Patent Document 10: WEIGHT SC, BELL PR, NICHOLSON ML: Renal

ischaemia --- reperlusion injury. Br J Surg83: 162--170, 1996

Non-Patent Document 11: WEIGHT SC, BELL PR, NICHOLSON ML: Renal

ischaemia --- reperlusion injury. Br J Surg83: 162--170, 1996 Non-patent document 12: MORRIS SD, CUMMING DV, LATCHMAN DS, YELLON

DM: Specincinduction of the 70— kD heat stress proteins by the tyrosine

kinase inhibitor herbimycin— A protects rat neonatal cardiomyocytes. A new pharmacological route to stress protein expression? J Clin Invest 97: 706-712, 1996 Non-patent document 13: MAULIK N, ENGELMAN RM, WEI Z, et

al: Drug—inducedheat—shock preconditioning improves postischemicventricular recovery after cardiopulmonary bypass. lrculation 92: 11381—11388,1995

Non-Patent Document 14: SUN L, CHANG J, KIRCHHOFF SR, KNOWLTON AA: Activation of HSF and selective increase in heat-shock proteins by acute dexamethasone treatment.American Journal of PhysiologyHeart and Circulatory Physiology 278: H1091-H1097, 2000

Non-patent document 15: HIRAKAWA T, ROKUTAN K, NIKAWA T, KISHI

K: Geranylgeranylacetoneinduces heat shock proteins in cultured guinea pig gastric mucosal cells andrat gastric mucosa.Gastroenterology 111: 345-357, 1996

Non-Patent Document 16: TSURUMA T, YAGIHASHI A, KOIDE S, et al:

Geranylgeranylacetoneinduces heat shock protein— 73 in rat small intestine.

Transplant Proc 31: 572-573, 1999

Non-Patent Document 17: 0〇 IE T, TA AHASHIN, SAI AWA T, et ahSingleoral dose of geranylgeranylacetone induces heat—shockprotein 72 and renders protection against ischemia / reperfusion injury in ratheart.circulation 104: 1837—1843, 2001

Non-patent document 18: FUDABA Y, OHDAN H, TASHIRO H, et

al: Geranylgeranylacetone, a heat shock protein inducer, prevents primary graft nonfunctionin rat liver transplantation.Transplantation 72: 184—189, 2001

Non-Patent Document 19: ODA H, MIYAKE H, IWATA T, et

ahGeranylgeranylacetonesuppresses inflammatory responses and improves survival after massive hepatectomy in rats.Journal of Gastrointestinal Surgery 6: 464-72 discussion 47, 2002

Disclosure of the invention Problems the invention is trying to solve

 [0007] There is no effective preventive and / or therapeutic agent for renal diseases such as acute renal failure as described above, so that a powerful agent is eagerly desired.

[0008] In view of the above, an object of the present invention is to provide a drug effective for renal disease.

 Means for solving the problem

The present inventors have conducted intensive studies and found that a specific acyclic polyisoprene conjugate induces heat shock protein in the kidney and is effective for the prevention and / or treatment of kidney disease. With the knowledge, the present invention has been completed.

[0010] That is, according to the first aspect, the present invention provides a prophylactic and / or therapeutic agent for renal disease comprising a heat shock protein inducer or a salt thereof or a hydrate thereof as an active ingredient. provide.

 [0011] In a preferred embodiment, the preventive and / or therapeutic agent for renal disease or Z according to the present invention is the heat shock protein inducer prenyl ketone compound.

 [0012] In a preferred embodiment of the preventive and / or therapeutic agent for renal disease according to the present invention, the prenyl ketone compound is

 [0013] [Formula 1]

6, 10, 14, 18-tetramethyl-5,9,13,17-nanodecatetraen-2-one represented by

In a preferred embodiment, the preventive and / or therapeutic agent for renal disease or Z or the therapeutic agent according to the present invention, Kidney disease is acute renal failure.

 In a preferred embodiment of the agent for preventing and / or treating renal disease according to the present invention, the heat shock protein is heat shock protein 70.

[0016] Further, according to a second aspect, the present invention provides a method for preventing and / or treating renal disease, comprising a step of administering a heat shock protein inducer or a salt or hydrate thereof. .

 [0017] In a preferred embodiment of the method for preventing and / or treating renal disease according to the present invention, the above-mentioned heat shock protein inducer is a prenyl ketone compound.

 [0018] In a preferred embodiment of the method for preventing and / or treating renal disease according to the present invention, the prenyl ketone compound is

[0019] [Formula 1]

6, 10, 14, 18-tetramethyl-5,9,13,17-nanodecatetraen-2-one represented by

 [0020] In a preferred embodiment of the method for preventing and / or treating renal disease according to the present invention, the renal disease is acute renal failure.

In a preferred embodiment of the method for preventing and / or treating kidney disease according to the present invention, the heat shock protein is heat shock protein 70.

[0022] Further, according to the third aspect, the present invention provides a method for preventing renal disease and containing a heat shock protein inducer or a salt thereof or a hydrate thereof as an active ingredient. Provides the use of a heat shock protein inducer or a salt thereof or a hydrate thereof for producing a therapeutic agent.

 [0023] In a preferred embodiment of the use according to the present invention, the heat shock protein inducer is a prenyl ketone compound.

 [0024] In a preferred embodiment of the use according to the present invention, the prenyl ketone compound is represented by the following formula:

6, 10, 14, 18-tetramethyl-5,9,13,17-nanodecatetraen-2-one represented by

 [0026] In a preferred embodiment of the use according to the present invention, the renal disease is acute renal failure.

[0027] In a preferred embodiment of the use according to the present invention, the heat shock protein is heat shock protein 70.

 The invention's effect

 According to the present invention, the compound according to the present invention, 6,10,14,18-tetramethyl-5,9,13,17-nanodecatetraen-2-one, is a heat shock protein in the kidney It induces 70 and is effective for the prevention and / or treatment of acute renal failure. BEST MODE FOR CARRYING OUT THE INVENTION

The following embodiment is an exemplification for describing the present invention, and is not intended to limit the present invention to only this embodiment. The present invention may take various forms without departing from the gist thereof. It can be implemented in a state.

 The present invention provides a nontoxic prophylactic and / or therapeutic agent that is effective for treating renal diseases, particularly renal diseases such as acute renal failure. Hereinafter, the present invention will be described in detail.

 In the present invention, the compound which is an active ingredient is a prenyl ketone compound represented by the formula (I).

 The compound name of the compound suitably used in the present invention is 6,10,14,18-tetramethyl-5,9,13,17-nanodecatetraen-2-one (alias: geranylgeralacetone; general name: teprenone) , Hereinafter simply referred to as “GGA”). The GGA used in the present invention has a double bond in four places in its structure, and there are a total of eight types of geometric isomers. It may be one isomer or a mixture of two or more. Among these, preferred compounds include (5E, 9E, 13E) -6,10,14,18-tetramethyl-5,9,13,17-nonadecatetraen-2-one and (5Z,

 9E, 13E) —6, 10, 14, 18—tetramethyl-5, 9, 13, 17—nonadecatetraen-2-one.

 [0032] GGA used in the present invention is a known compound and can be obtained as a reagent or an industrial raw material. GGA can be synthesized according to the method disclosed in, for example, JP-A-53-145922.

GGA, which is an active ingredient of the preventive and / or therapeutic agent for renal diseases according to the present invention, is preferably a salt capable of forming a salt. (E.g., hydrofluoride, hydrochloride, hydrobromide, hydroiodide, etc.), inorganic acid (e.g., sulfate, nitrate, perchlorate, phosphate, carbonate, bicarbonate) Salts, etc.) Organic sorbate (eg, acetate, trifluoroacetate, oxalate, maleate, tartrate, fumarate, citrate, etc.), organic sulfonate (eg, methane snolefonic acid) Salts, trifluoromethanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, camphorsulfonate, etc.), amino acid salts (eg, aspartate, glutamate, etc.), quaternary amine salts , Alkali metal salts (eg, sodium salt, potassium salt, etc.), alkaline earth metal salts (eg, magnesium salt, calcium salt, etc.), etc. can give.

 [0034] GGA or a salt thereof as an active ingredient used in the present invention may be an anhydride or may form a hydrate. GGA or a salt thereof may have a crystal polymorph, but is not limited thereto, and any one of the crystal forms may be a single crystal or a mixture of crystal forms. Further, metabolites generated by decomposing GGA or a salt thereof used in the present invention in a living body are also included in the claims of the present invention.

 As the prophylactic and / or therapeutic agent according to the present invention, GGA may be used as it is, or a known pharmaceutically acceptable carrier or the like, for example, an excipient (specifically, , Lactose, sucrose, starch, mannitol, etc.), binders (specifically, starch arsenide, gum arabic, dextrin cellulose, polybutylpyrrolidone, etc.), lubricants (specifically, stearin Raw materials for pharmaceutical preparations, such as disintegrants, coloring agents, flavoring agents, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, etc. It may be formulated by a commonly used method by blending the components used as above. If necessary, components such as a blood flow promoter, a bactericide, an anti-inflammatory agent, a cell activator, a vitamin, an amino acid, a humectant, and a keratolytic agent may be added.

 [0036] The formulation of the prophylactic and / or therapeutic agent according to the present invention includes tablets, powders, fine granules, granules, coated tablets, capsules, syrups, troches, inhalants, suppositories. Preparations, injections, freeze-dried preparations, ointments, ophthalmic ointments, eye drops, nasal drops, ear drops, poultices, lotions and the like.

 [0037] In the present invention, the dosage form of GGA is not particularly limited, but is preferably orally administered. GGA is available under the trade name "Selvex" (made by Eisai Co., Ltd.).

 [0038] The GGA-containing prophylactic and / or therapeutic agent according to the present invention is useful for preventing and / or treating renal diseases in mammals (eg, humans, mice, rats, guinea pigs, egrets, dogs, dogs, monkeys, etc.). And particularly useful for treating and / or preventing human kidney disease.

[0039] The dose of the prophylactic and / or therapeutic agent containing GGA according to the present invention is appropriately selected depending on conditions such as symptoms, age, and body weight. One 1000 mg, more preferably 100-500 mg.

[0040] The disease for which the GGA-conjugate according to the present invention is effective is a renal disease, and acute renal failure, acute tubular necrosis, chronic renal failure, tubulointerstitial disorder, acute nephritis, chronic nephritis, nephrosis, Although renal dysfunction is mentioned, it is particularly effective for acute renal failure.

[0041] The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited thereto. Various changes and modifications can be made by those skilled in the art based on the description of the present invention, and these changes and modifications are also included in the present invention.

Example

 (Laboratory animals)

 Male Spraugue-Dawley rats purchased from Chubu Kagaku Kagaku Co., Ltd. (Nagoya II, Japan) were used and fed adequately. All handling of experimental animals complied with the “Nagoya University School of Medicine Animal Experiment Guidelines”.

 (Experimental sample)

 GGA (trade name "Celvex", manufactured by Eisai Co., Ltd.) was prepared as an emulsion using a 5% gum arabic solution and 0.008% tocopherol. The same dose of solvent was used as placebo. In the drawings, placebo may be displayed as a vehicle.

 (Statistical analysis)

 All figures are expressed as mean SEM values. Continuous changes in heat shock protein 70 (hereinafter simply referred to as “Hsp70”) were analyzed by two-way ANOVA (mutual analysis of squared deviation) followed by Tukey's test. The relative density of Hsp, blood urea nitrogen and serum creatine levels, and the number of TUNEL and ED-1-positive cells were determined by Student t-test. The evaluation of tubular injury and tubular column formation was compared by the Mann-Whitnes U test. A significant difference was defined as a P value of 0.05 or less.

Analysis of Hsp70 expression in vivo

 To determine the optimal dose to induce Hsp70 in the kidney,

GGA was administered to rats as the experimental samples described above (0,200, kg / kg body weight).

400, 800 mg of GGA). 24 hours after GGA and vehicle administration, pen injection by peritoneal injection They were killed by anesthesia with tobarbital (50 mg I kg). Immediately after, the kidney was excised and analyzed by the stamp lot method. FIG. 1 shows the in vivo (A) and in vitro (B) experimental procedures used in the present invention.

 [0044] To examine the time course of Hsp70 expression, kidneys were collected 12, 24, and 48 hours after a single administration of GGA (400 mg / kg). In addition, prior to collecting the kidney 48 hours later, the expression of Hsp70 was examined immediately after administration of GGA (400 mg I kg) and also after two oral administrations of 24 hours after administration. Some kidneys were subjected to Western blot analysis and other kidneys were frozen using OCT® conjugate (Miles, Elkhart, IN USA) for immunostaining. The GGA administration group and the placebo group each used 6 rats.

 [0045] FIG. 2 shows the GGA expression relative to Hsp70 expression in rats 24 hours after oral administration of GGA.

 FIG. 9 shows the results of the effect of the dose. From the results shown in Fig. 2 (A), 400

 It can be seen that the expression of Hsp70 was significantly increased in rats to which mg I kg of GGA was administered, and that the expression of Hsp70 was not significantly increased even when 800 mg / kg of GGA was administered. From these results, the optimal dose of GGA for inducing Hsp70 in rat kidney was 400 mg I kg, and it was decided to use this dose in the following experiments.

 [0046] Figs. 2 (B) and (C) show the histology of the tubules obtained according to the present invention. Fig. 2 (B) shows the results after placebo administration, and Fig. 1 shows a histology of GGA after administration of GGA. It can be seen that Hsp70 expression was actually observed in tubular epithelial cells in the normal rat kidney. FIG. 2 (D) is a diagram showing the results of analysis of the expression of other heat shock proteins (eg, Hsp90, Hsp60, Hsp32) 24 hours after GGA administration and placebo administration to which a vehicle as a control was administered. From the results shown in FIG. 2 (D), it can be seen that no significant difference was observed in the expression of the other heat shock proteins between the vehicle administration and the GGA administration.

 FIG. 3 is a graph showing the time course of Hsp70 expression after administration of 400 mg / kg GGA to rats. From the results shown in the upper panel of FIG. 3, after a single oral administration of 400 mg I kg of GGA, Hsp70 expression increased after 12 hours, peaked after 24 hours, and decreased after 48 hours. On the other hand, after the administration of the vehicle, Hsp70 expression was not observed in a time-dependent manner.

[0048] Furthermore, two oral doses of GGA (400 mg I kg) (0 hour and 24 hours), and 48 hours later, Hsp70 expression was studied. As a result, the peak levels of Hsp70 expression were comparable to those induced 24 hours after a single dose, indicating that high levels of Hsp70 expression were maintained even after 48 hours of two oral doses. Suggests that

 [0049] Experimental protocol for I / R research

Rats in the GGA group (n = 9) received GGA (400 mg / kg) orally 24 hours before nephrectomy and 1 hour immediately before resection. Control rats ( _n = 9) received the same volume of vehicle. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg I kg body weight) and body temperature was maintained at the temperature of the electrical pad (37 ° C). Both kidneys were revealed by midline incision and bilateral renal arteries were clipped for 30 minutes with a 25 mm microaneurysm clamp to induce ischemia. After removal of the clamp, the muscle and skin were sutured separately. Rats were killed 24 hours after I / R injury and blood and kidney samples were collected for study.

 [0050] Histology and immunohistochemistry

A portion of the kidney was fixed with 4% paraformaldehyde, immersed in noraffin, and cut into small pieces of 4 μm ² . These pieces were stained with periodate Schiff reagent (PAS).

 [0051] On the other hand, the other part of the kidney was frozen using the OCT conjugate. A small piece of 2 μm thickness was fixed in acetone. According to the method described in the literature (SHIMIZU H, MARUYAMA

 ¾, YUZAWA et al: Anti-monocyte chemoattractant protein-1 gene therapy at tenuates renal injury induced by protein-overload proteinuria. J Am Soc Nephrol 14: 1496-1505, 2003), and immunostaining was performed. The primary antibodies used in this experiment were monoclonal antibodies against Hsp70 (Stress Gen Biotechnologies Corp., Victoria, B. and Canada), and ED-1 (anti-rat macrophage / monosite, Serotec, Raleigh, The secondary antibody was a goat anti-mouse IgG antibody fluorescein conjugate (Zymed laboratories, San Francisco, CA).

 [0052] Morphological analysis of histology and immunohistochemistry

J Am Soc Nephrol 14: 1496-1505, 2003), and evaluated using a semi-quantitative method with a slightly modified method. In other words, the severity of tubular columnar formation and tubular injury (dilation and degeneration) was scored according to the criteria described below, with 200 fields of 16 fields of non-overlapping renal medulla. 0 points: normal, 1 point: less than 30% of the relevant area, 2 points: 30-70% of the relevant area, 3 points: more than 70% of the relevant area. Stromal macrophage infiltration was evaluated by calculating the number of ED-1 positive cells by increasing the 16 non-overlapping fields of the extramedullary zone of the kidney by 400 times. Morphological analysis was performed by two observers using a Zeiss microscope (Oberkochen Germany), hiding each other.

FIG. 4 shows a histology reflecting the results of PAS staining performed in the present invention. PAS staining of kidney fragments from vehicle-treated rats revealed that the lesions were mainly in the extramedullary zone of the renal medulla, and that GGA treatment reduced the damage (Figure 4). (A) —See (D)).

 [0054] Next, the severity of tubular injury, including tubular dilatation and degeneration (vacuum alteration, loss of brush border, shedding of tubular epithelial cells, condensation of tubular cell nuclei), and reduction of tubular formation An evaluation was performed. FIGS. 4 (E)-(F) show the results of morphological analysis of histology in the present invention when vehicle and GGA were administered. Pretreatment with GGA resulted in tubule injury (1.7 vs. 0.1 vs. 0.9 vs. 0.2, P <0.01) and tubular formation (1.7 vs. 0.1 vs. 0.9 vs. 0.9).

 In both cases (0.2, P and 0.01), significant good results were obtained (see FIGS. 4 (E) and (F)). Also, the results shown in FIG. 4G indicated that the number of macrophages detected by ED-1 staining was significantly suppressed by GGA administration (15.3 individuals 0.9 vs 7.5 individuals 0.7, P and 0.01).

 [0055] To study the contribution of apoptosis to this process, a TUNEL assay described below was performed. FIG. 5 is a diagram showing the results of TUNEL ATSSEI in the vehicle treatment and GGA treatment of the extramedullary zone of rats. In the vehicle-treated rats, TUNEL-positive cells were localized mainly in the extramedullary zone of the kidney (see FIG. 5 (A)). On the other hand, as is clear from the results shown in FIGS. 5 (B) and (C), the number of TUNEL-positive cells in the extramedullary zone of the kidney was significantly reduced by GGA treatment (40.2 ± 4.9 vs. 18.2 ± 2.6). , P <0.01)

Measurement of blood urea nitrogen (BUN) and serum creatine After the end of the in vivo experiment, blood was collected from the aorta and serum was collected. BUN level and serum creatine (hereinafter referred to as " _s -Cr") level were measured using Diaauto UN or Diaauto Clare Kit (Daiya Shiyaku, Tokyo, Japan).

 [0056] Isolation and culture of rat proximal tubular cells

Rat proximal tubular epithelial cells (hereinafter simply referred to as “R-TECs”) were isolated from kidneys obtained from Spraugue-Dawley rats, and SHIMIZU H, MARUYAMA S, YUZAWA Y, et al: Anti- monocyte chemoattractant protein-1 gene therapy attenuates renal injury induced by protein-overload proteinuria.J Am Soc Nephrol 14: 149b-1505, 2003 with a minor modification to 10% fetal serum (FBS) and hormones. The cells were cultured in the contained K1 medium (224.25 ml Ham's F12, 226.25 ml DEME, 12.5 ml HEPES). R-TECs showed strong alkaline phosphatase reactivity and were confirmed by antibodies to cytokeratin (Labsystems, Helsinki, Finland), a marker for proximal tubules. After reaching confluence (1 × 10 ⁷ 110-cm dish), R-TECs were washed with FBS.

 FIG. 6 is a graph showing the effects of vehicle treatment and GGA treatment on BUN and s-Cr levels in rats suffering from renal ischemic reperfusion. The results shown in Figure 6 indicate that rats suffering from renal ischemic reperfusion have significantly elevated levels of BUN and s-Cr. The increase was significantly suppressed by pretreatment with GGA (BUN: 56.0, 10.8 vs. 22.2 ± 2.2, P <0.01, s-Cr: 1.1 ± 0.2 vs. 0.6 ± 0.1, P <0.05).

 [0058] In vitro experimental design

First, the induction of Hsp70 by GGA was examined in R-TECs. GGA and vehicle are IKEYAMA S, KUSUMOTO K, MIYAKE H, et al: A non-toxic heat shock protein 70 inducer, geranylgeranylacetone, suppresses apoptosis of cultured rat hepatocytes caused by hydrogen peroxide and ethanol.J Hepatol 35: 53 , 2001. Confluent primary cultures of R-TECs were grown at a continuous concentration of GGA (0, 0.1, 1, 10, 100 / zM) or in Hanks's solution without phenol red, which was supplemented with 0.1% FBS. After incubation in a vehicle at 37 ° C for 2 hours, Hsp70 expression was examined by Western blot. After administration of 10 M GGA or vehicle, the time course of Hsp70 expression was also examined. Next, the cytoprotective effect of GGA was examined. By GGA (10 μΜ) or vehicle

R-TECs were pre-treated for 2 hours, followed by a further 2 hour incubation with 0.5 mM hydrogen peroxide. After incubation, cell death was analyzed by TUNNEL assay.

 [0060] Western blot analysis

 SHIMIZU H, MARUYAMA S, YUZAWA Y, et al: Anti-monocyte chemoattractant protein-1 gene therapy attenuates renal injury induced oy protein-overload proteinuria.J Am Soc Nephrol 14: 1496-1505, 2003 Western blot analysis was performed as described. That is, whole kidney preparations or cultured cells were homogenized using RIPA buffer. 50 mg of the protein was separated by electrophoresis on 10% SDS-PAGE and electrophoretically transferred to a PVDF membrane (ImmobilonPsq; Millipore, Bedford, USA). After the electrophoresis, the cells were blocked with non-fat milk, and the PVDF membrane was incubated with the antibody. The primary antibodies used were antibodies against Hsp70, Hsp90, Hsp60 and Hsp32 (Stress Gen Biotechnologies Corp., Victoria, B.C., Canada). Secondary antibodies were goat F (ab ') anti-mouse Ig's, HRP conjugate, goat F (ab') anti-mouse Ig's, HRP conjugate (Biosource international, Camarillo, USA). Proteins were detected using an ECL kit (Amersham, Little Chalfont, UK) and quantified using NIH image software.

[0061] The effect of GGA was studied using R-TECs, which were cultured renal epithelial cells. FIG. 7 shows the results of Hsp70 expression induced by treatment with various concentrations of vehicle and GGA in R-TECs used in the present invention. From the results of the Western plot analysis shown in FIG. 7, it was found that the GGA force of 10 / zM Hsp70 expression was significantly increased. On the other hand, from the results shown in FIGS. 7 (A) and (C), the result of the 10 M GGA treatment was different from the result observed after incubation with 100 M GGA. In addition, as shown in the results of Figs. 7 (B) and (D), from the time course analysis of Hsp70 expression, GGA treatment at 10 / zM temporarily increased the protein level of Hsp70 expression in R-TECs. It was demonstrated that the protein level peaked after 1 to 2 hours, and then rapidly decreased after 8 hours. As is evident from the results shown in Fig. 7 (E), other It was found that the expression did not affect the expression of heat shock proteins, specifically Hsp90, Hsp60 and Hsp32.

 [0062] TUNEL Atsusey

 Using the TUNEL method (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling), stain with a paraffin-immersed kidney fragment (4 m thick) by staining with an in situ apoptosis detection kit (Takara, Shiga, Japan) The number of apoptotic cells was determined for R-TECs fixed to 4% paraformaldehyde. The number of apoptotic cells in the extramedullary zone of the renal medulla was expressed in a total of 1000 tubular cells by increasing the 16 non-overlapping fields by 400 times and counting them.

 [0063] To study tubule damage due to oxidative stress, R-TECs were cultured in a medium containing 0.5 mM hydrogen peroxide. FIG. 8 is a diagram comparing the number of positive cells obtained by the TUNEL method performed in the present invention. The arrows in the figure indicate positive cells. From the results shown in FIG. 8, the number of TUNEL-positive cells was significant for vehicle-treated R-TECs. On the other hand, pretreatment with GGA significantly reduced the number of TUNEL-positive cells (66.3 ± 10.4 vs 7.0 ± 3.3, P <0.01).

 Industrial applicability

According to the present invention, heat shock protein is induced in the kidney by administering GGA, and is effective for the prevention and / or treatment of kidney diseases, particularly acute renal failure, and contains GGA as an active ingredient And a prophylactic and / or therapeutic agent for renal disease.

 BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 shows the in vivo (A) and in vitro (B) experimental procedures used in the present invention.

 (A) shows the experimental procedure of rats that were orally administered GGA (400 mg I kg) and vehicle 24 hours and 1 hour before surgery. Both kidneys were in an ischemic state for 30 minutes and died 24 hours later. (B) R-TECs were treated with GGA (10 M) and vehicle for 2 hours and then incubated with 0.5 mM hydrogen peroxide for 2 hours. After the incubation, the cells were fixed, and TUNEL assay was performed.

FIG. 2 is a graph showing the results of the effect of GGA dose on Hsp70 expression in rats 24 hours after oral administration of GGA. (A) 24 hours after oral GGA administration FIG. 2 shows the results of Western blot analysis of the dose-dependent expression of Hsp70. Hsp70 expression was not further increased at a dose of 800 mg / kg of significantly increased power in rats fed GGA (400 mg I kg). The P value for the vehicle-treated rats was 0.05 or less (P 0.05) ₀ (B and C) indicates the results of the immunohistochemical study, and the normal rat kidney (B) Substantial levels of Hsp70 expression were observed in tubular epithelial cells. Oral administration of GGA increased the level of Hsp70 expression, especially in renal tubular cells in the extramedullary zone of the kidney (see (C)). (D) shows the results of examining the expression of other Hsp proteins by Western blotting. At 24 hours after administration of GGA (400 mg I kg), expression of Hsp90, Hsp60 and Hsp32 was not induced in the kidney. Open columns are the results of vehicle administration and shaded columns are the results of GGA administration.

[Fig. 3] Fig. 3 is a graph showing the time course of Hsp70 expression after administration of 400 mg I kg of GGA to rats. The levels of Hsp70 expression were investigated at 12, 24, and 48 hours after a single oral GGA (400 mg / kg) dose. Hsp70 expression increased by 12 hours, peaked after 24 hours, and decreased after 48 hours. No time-dependent change in the level of Hsp70 expression in the vehicle-treated group was observed. Also, after two oral doses of GGA (400 mg / kg) (0 h and 24 h), the level of Hsp70 expression at 48 h was determined. The peak level of Hsp70 expression was comparable to the peak level induced 24 hours after a single oral dose. The open column shows the results of vehicle administration, and the shaded column shows the results of GGA administration. The P value for vehicle-treated rats was less than 0.05 (P <0.05).

FIG. 4 shows a histology reflecting the results of PAS staining performed in the present invention. Small pieces ((A) and (C)) from vehicle-treated rats showed tubular columnar formation, tubular dilatation and tubular degeneration (vacuum vesicles, missing brush borders, shedding of skin cells on tubules, Condensation of tubular nuclei) mainly occurred in the extramedullary zone of the kidney. Only renal strength ((B) and (D)) and moderate tubule damage in GGA-treated rats were observed. Pretreatment with GGA attenuated all of the above changes (see (B) and (D)). (A) and (B) are low-magnification (100 ×) photographs of a kidney piece stained with PAS, and (C) and (D) are results of high magnification (400 ×) of a kidney piece stained with PAS. It is a photograph of (fold). Tubular injury and tubular cast formation To evaluate, the kidney pieces were analyzed using the semi-quantitative evaluation method. GGA treatment resulted in significantly better scores (see (E) and (F)). The number of ED-1 positive cells (macrophages) was significantly suppressed by GGA administration (see (G)).

FIG. 5 is a diagram showing the results of TUNEL Atssey in the vehicle treatment and GGA treatment of the extramedullary zone of the renal kidney of rats. The arrows in FIG. 5 indicate TUNEL-positive cells. In vehicle-treated rats, TUNEL-positive cells are localized mainly in the extramedullary zone of the kidney (see (A)). The number of apoptotic cells was hardly observed in rats administered with GGA (see (B)). The number of TUNEL-positive cells was expressed for a total of 1000 tubular cells (see (C)). GGA significantly suppressed tubular apoptotic cell death (P <0.01).

 FIG. 6 is a graph showing the effects of vehicle treatment and GGA treatment on BUN and s-Cr levels in rats suffering from renal ischemic reperfusion. Rats suffering from renal I / R showed significant increases in BUN and serum back creatine levels. The pretreatment with GGA significantly suppressed the increase.

 FIG. 7 shows the results of Hsp70 expression induced by treatment with various concentrations of vehicle and GGA in R-TECs used in the present invention. Western blot analysis shows that 10 / zM GGA significantly increases Hsp70 expression. The results differed from those observed after incubation with 100 / zM GGA (see FIGS. 7 (A) and (C)). From the time course of Hsp70 expression, with 10 / zM GGA, the protein level of Hsp70 in R-TECs temporarily increased, peaked in 1 to 2 hours, and then rapidly after 8 hours. It decreased (see FIGS. 7 (B) and 7 (D)). The expression of other Hsps (Hsp90, Hsp60 and Hsp32) was not affected by GGA administration (see FIG. 7 (E)).

 FIG. 8 is a diagram comparing the number of positive cells obtained by the TUNEL method performed in the present invention. R-TECs were incubated in a medium containing 0.5 mM hydrogen peroxide. The number of TUNEL-positive cells was significantly observed in vehicle-treated R-TECs (see (A)). GGA pretreatment significantly reduced the number of TUNEL-positive cells (see (B)). The difference was statistically significant (see (C), P-0.01).

Claims

The scope of the claims

 [1] A prophylactic and / or therapeutic agent for renal disease, comprising a heat shock protein inducer or a salt thereof or a hydrate thereof as an active ingredient.

 [2] The preventive and / or therapeutic agent for renal disease according to claim 1, wherein the heat shock protein inducer is a prenyl ketone compound.

 [3] The prenyl ketone compound is

 [Formula 1]

3. The preventive and / or therapeutic agent for renal disease according to claim 2, which is 6, 10, 14, 18-tetramethyl-5, 9, 13, 17-nanodecatetraen-2-one represented by the following formula:

4. The preventive and / or therapeutic agent for renal disease according to any one of claims 1 to 3, wherein the renal disease is acute renal failure.

[5] The preventive and / or therapeutic agent for renal disease according to any one of claims 1 to 4, wherein the heat shock protein is heat shock protein 70.

[6] A method for preventing and treating Z, or a method for treating renal disease, comprising administering a heat shock protein inducer, a salt thereof, or a hydrate thereof.

[7] The method for preventing and / or treating renal disease according to claim 6, wherein the heat shock protein inducer is a prenyl ketone compound.

[8] The prenyl ketone compound,

[Formula 1]

8. The method for preventing and / or treating a renal disease according to claim 7, wherein the method is 6, 10, 14, 18-tetramethyl-5, 9, 13, 17-nanodecatetraen-2-one represented by the following formula:

[9] The method for preventing and / or treating renal disease according to any one of claims 6 to 8, wherein the renal disease is acute renal failure.

[10] The method for preventing and / or treating renal disease according to any one of claims 6 to 9, wherein the heat shock protein is heat shock protein 70.

[11] A heat shock protein inducer or a salt thereof for producing a prophylactic and / or therapeutic agent for renal disease or a remedy containing a heat shock protein inducer or a salt thereof or a hydrate thereof as an active ingredient. Or use of their hydrates.

[12] The use according to claim 11, wherein the heat shock protein inducer is a prenyl ketone compound.

[13] The prenyl ketone compound,

[Formula 1]

The use according to claim 12, which is 6,10,14,18-tetramethyl-5,9,13,17-nanodecatetraen-2-one represented by the formula:

[14] The use according to any one of claims 11 to 13, wherein the renal disease is acute renal failure.

 [15] The use according to any one of claims 11 to 14, which is the heat shock protein card shock protein 70.