JP2010090094A - Oatp-r genetic expression boosting composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an OATP(Organic Anion Transporting Polypeptide)-R genetic expression boosting composition in which safety is higher and which has an OATP-R genetic expression boosting action, and to provide a prophylaxis/treatment agent or the like for renal dysfunction including the OATP-R genetic expression boosting composition. <P>SOLUTION: The OATP-R genetic expression boosting composition includes statin or a salt thereof which may be pharmacologically permitted. As the statin, cerivastatin, atorvastatin, fluvastatin, simvastatin, lovastatin, pitavastatin, pravastatin, mevastatin, rosuvastatin, and cilastatin can be suitably exemplified. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an OATP-R gene expression enhancing composition, an agent for preventing / treating renal dysfunction containing the OATP-R gene expression enhancing composition, and the like.

  The number of patients with renal dysfunction, such as patients with renal insufficiency, especially those undergoing renal dialysis, has been increasing year by year. Currently, 200,000 patients are undergoing maintenance penetration treatment, and an additional 30,000 patients every year. The above has led to the introduction of dialysis. Since dialysis therapy costs about 5 million yen per person per year, medical expenses of about 1 trillion yen are constantly required for 200,000 people. In recent years, the concept of chronic kidney disease (CKD) has been proposed, and recognition of the importance of its prevention and treatment is increasing. Since CKD is a common disease and the number of patients is enormous (approximately 20 million people), countermeasures against CKD are an urgent issue. Under these circumstances, if a method for preventing symptoms of renal dysfunction such as renal failure or CKD, or a treatment that alleviates such symptoms and delays the introduction of dialysis, the patient's QOL (quality of quality) Not only contributes to life), but can also greatly reduce medical costs and contribute greatly to society.

  Examples of therapeutic agents for renal diseases such as renal failure include those containing a single-chain hepatocyte growth factor as an active ingredient (see Patent Document 1), those containing a specific steroid (see Patent Document 2), colonies What uses a stimulating factor (CSF) as an active ingredient is known (refer patent document 3). However, the obtained therapeutic effect is not sufficient, and a better and more practical preventive and therapeutic agent for renal dysfunction has been demanded.

  Hypertension is a type of lifestyle-related disease, and the number of patients is also increasing. Hypertension is also a risk of onset of ischemic heart disease, stroke, renal failure, etc., so prevention and treatment are highly necessary. As a therapeutic agent for hypertension, in addition to a normal antihypertensive agent, an agent containing a coffee bean extract as an active ingredient (see Patent Document 4), or an ingredient containing an angiotensin II receptor antagonistic activity (patent) Document 5) is known. However, there has been a demand for better and more practical preventive and therapeutic agents for hypertension.

  There are a wide variety of transporters in the kidney, and they are involved in various transport of substances. In particular, the organic anion transporting polypeptide (OATP) family is a family of transporters that function as an evacuation / detoxification pump for removing organic anions, etc. that are no longer necessary from the living body. Is considered to be a transporter that plays an important role in the pharmacokinetics of endogenous substances and drugs due to its wide substrate recognition and diverse organ distribution. The present inventor isolated 15 or more organic anion transporters for the first time in the world, and bile acids, thyroid hormones, steroid hormones, prostaglandins and the like are not simply diffused but transported through the cell membrane by organic anion transporters. We have reported our knowledge for the first time in the world. Furthermore, the present inventor discovered OATP-R (also referred to as OATP4C1 or OATP-M1), an organic anion transporter expressed only in the kidney for the first time in the world (Non-Patent Document 1), and applied for a patent on the contents thereof. Has been registered in Japan (see Patent Document 6).

  Furthermore, the present inventors analyzed the 5 ′ upstream transcription region in detail in order to clarify the regulation of OATP-R expression. As a result, allyl hydrocarbon receptor (AHR), which is a nuclear receptor for dioxins, was analyzed. It was found that a xenobiotic response element (XRE) -like sequence known as a repetitive sequence to which is bound exists in the 5 ′ upstream transcription region of the OATP-R gene of human or rat (see Patent Document 7). Furthermore, the present inventors produced a cell in which a plasmid in which a reporter gene is arranged downstream of the 5 ′ upstream transcription region of human OATP-R was transfected into a Hela cell expressing AHR, and a reporter using the cell was used. The assay was performed. As a result, the transcription activity of the reporter gene is increased by 3-methylcholanthrene (3-MC) binding to AHR, and in the 5 ′ upstream transcription region described above, around −100 bases upstream from the transcription start point. In the case where the XRE-like region is present, it was found that the increase in transcriptional activity is particularly remarkable (see Patent Document 7). Moreover, since the transcription activity of the reporter gene is significantly reduced by removing or modifying the sequence of the XRE-like region in the 5 ′ upstream transcription region, the transcriptional control of the human OATP-R gene involves the XRE-like region. The possibility of being related was suggested (refer patent document 7). As described above, 3-MC has OATP-R gene expression enhancing activity, but 3-MC is a dioxin-like substance, and thus could not be administered to humans from the viewpoint of toxicity and the like.

  On the other hand, a statin is a general term for drugs that lower the LDL cholesterol level in blood by inhibiting the action of HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase. As a more detailed mechanism of action, statins inhibit the action of HMG-CoA reductase, which is the rate-limiting enzyme of the mevalonate pathway, thereby reducing cholesterol biosynthesis in the liver, resulting in the maintenance of cholesterol homeostasis. Therefore, the expression of LDL receptor in the liver is increased, the uptake of LDL cholesterol from the blood to the liver is promoted, and the cholesterol in the blood is decreased. Various statins have been developed as therapeutic agents for hyperlipidemia so far (see Patent Document 8, Patent Document 9, etc.) and are currently widely used as therapeutic agents for hyperlipidemia. In addition, it has been reported that blood pressure decreases when pravastatin is administered to Dahl salt-sensitive rats (see Non-Patent Document 2). However, it has not been known that statins enhance the expression of OATP-R.

JP 2008-195628 A JP 2005-506321 A Table 2005/034986 JP 2006-083182 A Japanese Patent Laid-Open No. 06-312926 Japanese Patent No. 4008481 JP 2008-1000092 A Japanese Patent Laying-Open No. 2005-089300 Special table 2003-524582 PNAS March 9, 2004.vol.101, no.10, 3569-3574 Hypertens. Res., 2005, vol.28, no.12, 1009-1015

  An object of the present invention is to provide an OATP-R gene expression enhancing composition having higher safety and an OATP-R gene expression enhancing action, and prevention of renal dysfunction containing the OATP-R gene expression enhancing composition・ To provide therapeutic agents.

  In order to solve the above-mentioned problems, the present inventors screened drugs having an action of enhancing human OATP-R gene expression from various drugs using the assay system of Patent Document 7 described above. The present inventors have found that statins already in practical use and widely used as hyperlipidemia agents enhance the expression of human OATP-R gene. Furthermore, when the present inventors administered a statin to ACHN cells, which are human kidney-derived cells, the mRNA of the human OATP-R gene increased in the ACHN cells, and accordingly, the thyroid gland, which is an OATP-R substrate. We found that the uptake activity of hormones into cells increased. The present inventors have also found that statins enhance the expression of OATP-R gene in the kidney when statins are orally administered to rats. Furthermore, the present inventors have found that when a statin is continuously given to a rat in addition to a high salt diet, an increase in blood pressure is significantly suppressed as compared to a rat fed with a high salt diet alone. . In addition, renal function improvement obtained by orally administering statin to rats does not depend on changes in blood pressure or renal clearance of creatinine, but by enhancing OATP-R expression by statin administration, ADMA, etc. It was found that the excretion of substances accumulated during renal failure was promoted and kidney damage was reduced. Based on the above findings, the present inventors have completed the present invention.

  That is, the present invention relates to (1) an OATP-R gene expression enhancing composition containing statin or a pharmaceutically acceptable salt thereof, and (2) statin is cerivastatin, atorvastatin, fluvastatin (fluvastatin) ), Simvastatin, lovastatin, pitavastatin, pravastatin, mevastatin, rosuvastatin, and cilastatin, as described in (1) above OATP-R gene expression enhancing composition, (3) OATP-R gene expression enhancing composition according to (1) above, wherein the statin is cerivastatin or atorvastatin, and (4) OATP-R The gene is a human OATP-R gene or a rat A OATP-R gene for OATP-R gene expression enhancing composition according to any one of the above (1) to (3).

  The present invention also relates to (5) a preventive / therapeutic agent for renal dysfunction comprising an OATP-R gene expression enhancing composition containing statin or a pharmaceutically acceptable salt thereof, and (6) renal dysfunction is acute. Prevention of renal dysfunction according to (5) above selected from the group consisting of renal failure, chronic renal failure, acute nephritis, chronic nephritis, acute tubular necrosis, tubular interstitial disorder, chronic kidney disease and nephrotic syndrome It relates to a therapeutic agent.

  Furthermore, the present invention relates to (7) a method of using a statin or a pharmaceutically acceptable salt thereof in the production of a composition for enhancing OATP-R gene expression.

  Furthermore, the present invention relates to (8) a method of using a statin or a pharmaceutically acceptable salt thereof in the manufacture of a preventive / therapeutic agent for renal dysfunction.

  The OATP-R gene expression enhancing composition of the present invention can enhance the expression of the OATP-R gene that is specifically expressed in the kidney. OATP-R encoded by the OATP-R gene is known to have an activity to excrete renal failure substances such as bile acids, digoxin, ouabain, cAMP and the like (see Japanese Patent Application No. (See also No. 2008-264673), and by enhancing the expression of the OATP-R gene, the renal function is improved and the function of excluding renal failure substances from the body is promoted. It is thought that it is obtained.

  Further, by using the OATP-R gene expression enhancing composition (preventive / therapeutic agent for renal dysfunction) of the present invention, it becomes possible to excrete uremic substances that cannot be removed by dialysis. Furthermore, when the OATP-R gene expression enhancing composition of the present invention (prophylactic / therapeutic agent for renal dysfunction) is administered to a patient with renal dysfunction, the progression of renal dysfunction can be stopped or delayed, It is not necessary to perform complicated treatments such as dialysis, and the time for introducing complicated treatments such as dialysis can be delayed. If at least the progression of renal dysfunction can be delayed, then there will be time to choose effective treatments and methods for patients with renal dysfunction, and the optimal treatment for each patient with renal dysfunction will be selected. It becomes possible.

  Further, the OATP-R gene expression enhancing composition of the present invention has an action of suppressing an increase in blood pressure in addition to an action of improving renal function.

  The OATP-R gene expression enhancing composition of the present invention is not particularly limited as long as it contains a statin or a pharmaceutically acceptable salt thereof (hereinafter also referred to as “statin”). And the like, as long as they are statins having activity to enhance OATP-R gene expression, and are not particularly limited. For example, cerivastatin, atorvastatin, fluvastatin (fluvastatin), simvastatin, lovastatin ), Pitavastatin, pravastatin, mevastatin, rosuvastatin and cilastatin, or derivatives thereof, among them, cerivastatin, atorvastatin, or derivatives thereof More suitable examples Rukoto can. Two or more of these statins can be used in combination. When used in the human body, cerivastatin, atorvastatin, fluvastatin, simvastatin, lovastatin, pitavastatin, pravastatin, mevastatin, which is already marketed as a therapeutic agent for hyperlipidemia and has been confirmed to be safe from the viewpoint of toxicity , Statins such as rosuvastatin and silastatin can be particularly preferably used.

  The above-mentioned “derivatives” of statins are substances that change the position of substituents of statins while maintaining the basic skeleton of statins, or replace one molecule of a statin with another molecule while maintaining the basic structure of the statin This means a substance obtained by adding a specific substituent to a statin while maintaining the basic skeleton of the statin, a substance obtained by removing a specific substituent from the statin while maintaining the basic skeleton of the statin, and the like. The basic skeleton of the statin includes, for example, a skeleton common to any two or more structural formulas of the respective statin structural formulas shown in FIG. Among the statin derivatives, statin lactones and esters can be particularly preferably exemplified.

  Although the details of the mechanism of action by which statins and the like enhance OATP-R gene expression are unclear, it is considered that the basic skeleton of statin produces some action and enhances OATP-R gene expression.

  The above-mentioned “statin having activity to enhance OATP-R gene expression” is not used when co-cultured with human or rat kidney cell lines capable of expressing OATP-R gene or administered to human or rat. Compared with the case of presence or non-administration, it refers to a statin or the like in which the expression of OATP-R in cells capable of expressing OATP-R gene such as kidney cells is increased at the mRNA level and / or protein level. More specifically, in the case of humans, in the human OATP-R gene expression assay using human renal cancer cell lines in Examples 1 and 2 described later and the luciferase assay in Example 7 described later, control (statin non-administration or In the case of control (non-statin administration) in the intracellular uptake assay of statins etc. in which the mRNA expression and transcriptional activity of the human OATP-R gene are increased or the thyroid hormone in Examples 3 and 4 described later than in the case of DMSO administration) In the case of the rat, more specifically, in the in vivo OATP-R gene expression assay using the rat of Example 5 described later, the control (water ) MR of rat OATP-R gene Including statins such as A expression is increased.

  The degree of increase in the mRNA expression of the OATP-R gene is not particularly limited, but is preferably 5% or more, more preferably 10% or more, and even more preferably 15% or more as a percentage compared to the control. A certain case can be preferably exemplified, and the degree of increase in the amount of thyroid hormone uptake is not particularly limited, but is preferably at least%, more preferably 10%, as compared to the control. As mentioned above, the case where it is 15% or more more preferably can be illustrated suitably. The OATP-R gene is not particularly limited as long as it is an OATP-R gene having an XRE-like sequence upstream of the OATP-R coding region, and the human OATP-R gene, rat OATP-R gene Can be particularly preferably exemplified.

  “Pharmaceutically acceptable salts” include non-toxic alkali metal salts, alkaline earth metal salts, ammonium salts and the like such as sodium, potassium, lithium, calcium, magnesium, barium and ammonium. In addition, the above salts include non-toxic acid addition salts obtained by reacting statins with appropriate organic or inorganic acids. Representative non-toxic acid addition salts include, for example, hydrochloride, hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, Borate, benzoate, lactate, phosphate, p-toluenesulfonate (tosylate), citrate, maleate, fumarate, succinate, tartrate, sulfonate, glycolate , Maleate, ascorbate, benzenesulfonate, napsilate and the like. Such salts can be prepared by methods well known in the art. Among these salts, nontoxic alkali metal salts and alkaline earth metal salts can be preferably exemplified, and sodium salts and calcium salts can be particularly suitably exemplified.

  As the above-mentioned various statins, those commercially available as hyperlipidemic agents can be used, and Japanese Patent Publication No. 2-46031, US Pat. No. 4,231,938, US Pat. No. 346,227, US Pat. No. 5,354,772, US Pat. No. 5,273,995, US Pat. No. 5,177,080, US Pat. No. 3,983,140, It can also be appropriately produced by combining known reactions described in US Pat. No. 5,260,440, International Patent Application WO 00/42024, US Pat. No. 5,753,675 and the like.

  Among the “statins or pharmaceutically acceptable salts thereof” used in the OATP-R gene expression enhancing composition of the present invention, cerivastatin sodium, cerivastatin lactone, atorvastatin calcium, fluvastatin sodium, simvastatin, lovastatin, pitavastatin Calcium, pravastatin sodium, mevastatin, rosuvastatin calcium and cilastatin sodium can be exemplified.

  The OATP-R gene expression enhancing composition of the present invention is not particularly limited as long as it contains the above-mentioned statin and the like, but as an optional component, other components that enhance the OATP-R gene and pharmaceutically acceptable A carrier may further be contained. Pharmaceutically acceptable carriers include, for example, excipients, binders, solvents, solubilizers, suspending agents, emulsifiers, isotonic agents, buffers, stabilizers, soothing agents, antiseptics, antiseptics, and the like. Optional components such as an oxidizing agent and a coloring agent can be blended.

  The dosage form of the OATP-R gene expression enhancing composition of the present invention is not particularly limited, and may be the above-described statin or the like, or a solid preparation such as a powder, granule or tablet. It may be a liquid agent such as a solution, an emulsion, or a suspension. In addition, examples of the method for administering the OATP-R gene expression enhancing composition of the present invention include oral administration and intravenous administration. However, since administration is simple, oral administration can be preferably exemplified. .

  The dose of the OATP-R gene expression enhancing composition of the present invention is not particularly limited as long as the activity of enhancing OATP-R gene expression is obtained, and the dose can be appropriately adjusted according to the administration subject. The administration target is not particularly limited as long as the activity of enhancing OATP-R gene expression is obtained, but mammals can be exemplified, among which humans and rats can be preferably exemplified, and humans are particularly preferred. It can be illustrated.

  The OATP-R gene expression enhancing composition of the present invention can also be used as an agent for preventing or treating renal function. OATP-R is known to have an activity to excrete renal failure substances such as bile acids, digoxin, ouabain, cAMP and the like, and improves renal function by enhancing the expression of OATP-R gene. This is because it is considered that the function of discharging renal failure substances to the outside of the body is promoted, and the preventive effect and therapeutic effect of renal dysfunction are obtained. In fact, Example 8 described later demonstrates that the OATP-R gene expression enhancing composition of the present invention significantly increases ADMA clearance, which is one of the indicators of renal function.

  The renal dysfunction is not particularly limited as long as it is a functional disorder related to urine formation or excretion in the kidney. For example, acute renal failure, chronic renal failure, acute nephritis, chronic nephritis, acute tubular necrosis, urine Preferred examples include tubulointerstitial disorder, chronic kidney disease and nephrotic syndrome.

  In addition to the OATP-R gene expression enhancing composition of the present invention, the above-mentioned renal function preventive / therapeutic agent of the present invention may further contain another preventive / therapeutic agent for renal function as an optional component. .

  In addition, the OATP-R gene expression enhancing composition of the present invention has an action of suppressing an increase in blood pressure in addition to an action of improving renal function (see the assay of Example 6 described later). Preferably, it can also be used effectively for the prevention and treatment of hypertension associated with renal dysfunction. In order to obtain an inhibitory effect on blood pressure increase, it is necessary to administer more statin than simply obtaining an OATP-R gene expression enhancing effect, but the appropriate dose is appropriately adjusted according to the administration subject. Etc. can be confirmed.

  The present invention also includes a method for using a statin or a pharmaceutically acceptable salt thereof in the production of an OATP-R gene expression enhancing composition, and a statin or a pharmaceutically acceptable salt thereof in the production of a preventive / therapeutic agent for renal dysfunction. A method of using the obtained salt and a method for preventing and treating renal dysfunction characterized by administering a statin or a pharmaceutically acceptable salt thereof to a mammal (particularly human or rat) are also included. The contents of the words in these uses and methods and the preferred embodiments thereof are as described above.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

[Human OATP-R gene expression assay using ACHN cells, a human renal cancer cell line 1]
Similar to 3-MC, which is known to enhance mRNA expression of the human OATP-R (hOATP-R) gene, in order to confirm whether statin enhances mRNA expression of the human OATP-R gene, Quantitative RT-PCR was performed on the human OATP-R gene using ACHN cells, a renal cancer cell line. Specifically, the following method was used.

The ACHN cell line, which is a human renal cancer cell line, was provided by the Medical Cell Resource Center, Institute of Aging Medicine, Tohoku University. This ACHN cell is a cell capable of expressing the OATP-R gene. The ACHN cells were dispensed into a 24-well plate at 2.4 × 10 ⁵ cells per well, and a specific medium (RPMI1640 medium containing FBS, penicillin, and streptomycin, final concentrations of FBS 10%, penicillin 100 U / ml, respectively) In a medium added to 100 μg / ml of streptomycin), the cells were cultured for 48 hours under conditions of 37 ° C. and 5% CO ₂ . After confirming that the ACHN cells were 70-80% confluent, the medium was replaced with serum-free medium OPTI-MEM1, and after culturing for 4 hours, the same amount of serum-free medium in 24 wells was used. Specific media was added into each well. As this specific medium, a sample medium obtained by adding pravastatin (Prava) as a sample to 20% FBS / RPMI1640 medium at a final concentration of 100 μM was used. Further, in place of the sample medium, 20% FBS / RPMI 1640 medium (negative control medium) with no sample added (control) or 20% FBS / with added dimethyl sulfoxide (DMSO) as a sample at a final concentration of 0.1% by mass. RPMI1640 medium (negative control medium) and 20% FBS / RPMI1640 medium (positive control) supplemented with 3-MC (3MC) as a sample at a final concentration of 5 μM were used.

  After adding these specific media and culturing for 24 hours, the cultured cells were added to 10 ml of TRIZOL solution (manufactured by Invitrogen) and homogenized for 1 minute at the maximum speed (100,000 rpm) of a Polytron crusher. Two times the amount of chloroform solution was added to the homogenized solution, stirred, and then centrifuged, and the aqueous phase portion was transferred to another tube. Two times the amount of isopropyl alcohol was added to this aqueous phase, followed by cooling and centrifugation at 15000 rpm to extract RNA. Using the extracted RNA as a template, cDNA synthesis was performed using a SuperScriptIII RTS first strand cDNA synthesis kit (Invitrogen). Quantitative RT-PCR was performed using TaqMan Probe (Applied Biosystems) to quantify the mRNA expression level of the human OATP-R gene in the cultured cells.

  Each sample was quantified at N = 5, and the average value of mRNA expression level was calculated for each sample. The average mRNA expression level in the case of water was taken as 1, and the average mRNA expression level of each sample was corrected to calculate the relative mRNA amount. The results of the above quantitative RT-PCR are shown in the left panel of FIG. As can be seen from the results in the left panel of FIG. 1, when pravastatin was administered, the relative mRNA amount of the human OATP-R gene was significantly and significantly increased compared to when water or DMSO as a control was administered. It was. Moreover, the amount of human OATP-R gene mRNA (relative expression level with respect to GAPDH) when pravastatin was administered was significantly increased compared to 3-MC. From these results, it was shown that pravastatin increases mRNA expression of human OATP-R gene in kidney cells.

[Human OATP-R gene expression assay using ACHN cells, a human renal cancer cell line 2]
Instead of adding pravastatin or the like as a sample, quantitative RT-PCR was performed in the same manner as in Example 1 except that fluvastatin (Fluva) was added at 10 μM, 30 μM, or 100 μM (each final concentration). . The result is shown in the right panel of FIG. When fluvastatin was administered, the mRNA level of human OATP-R gene (relative expression level relative to GAPDH) was significantly increased at any concentration compared to the control. From these results, it was shown that fluvastatin also increased mRNA expression of human OATP-R gene in kidney cells.

[Intracellular uptake assay of thyroid hormone 1]
To confirm whether statins not only increase mRNA expression of human OATP-R gene but also increase expression of human OATP-R and enhance the function of OATP-R in cells, T3 intracellular uptake assay was performed using thyroid hormone (triiodothyronine: T3) as a substrate and ACHN cells. Specifically, the following method was used.

First, triiodothyronine ( ¹²⁵ I-T3) (made by Amersham) having radioactive iodine ( ¹²⁵ I) as iodine and ACHN cells were prepared. After culturing ACHN cells in 20% FBS / RPMI1640 medium for 48 hours, ¹²⁵ I-T3 was added to the medium, and 10 μM (final concentration) fluvastatin was added as a sample. After incubating the ACHN cells at 37 ° C. for 24 hours after the addition of fluvastatin, the ACHN cells were separated, the protein in the details was dissolved in 0.1N NaOH, and a part of the protein was quantified by the BCA method. The ^{125 I-T3} content in the resulting protein (N = 5), the radiation is measured by measuring, the average value was calculated. Moreover, the average value of ¹²⁵ I-T3 amount was calculated by the same method except that no fluvastatin was added. In both cases, the mean value (fmol) of ¹²⁵ I-T3 content per mg of isolated protein is shown in the left panel of FIG. As can be seen from the results in the left panel of FIG. 2, when fluvastatin was administered, the uptake amount of ¹²⁵ I-T3 was significantly and significantly increased as compared with the case of administering water as a control. . From these results, it was shown that fluvastatin also increased the expression of human OATP-R in kidney cells and enhanced the function of OATP-R in the cells to improve the transport ability.

[Thyroid hormone uptake assay 2]
A thyroid hormone uptake assay was performed in the same manner as in Example 3 except that the incubation time was changed from 24 hours to 48 hours. In this modified assay method, instead of adding 10 μM (final concentration) of fluvastatin as a sample, 100 μM, 300 μM or 500 μM (final final concentration) of pravastatin (Prava, respectively) was added, and thyroid hormone ( ¹²⁵ I -Intracellular uptake assay of T3) was performed. These results are shown in the right panel of FIG. 2 in terms of the average value (fmol) of ¹²⁵ I-T3 content per mg of isolated protein for each sample. As can be seen from the right panel of FIG. 2, when pravastatin was administered, the uptake of ¹²⁵ I-T3 was significantly increased at any concentration compared to the control, but the concentration of pravastatin was particularly 100 μM. In the case, the increase in the intake was particularly excellent. In addition, even when 10 μM fluvastatin was administered, the increase in the uptake of ¹²⁵ I-T3 was particularly excellent. From these results, it was shown that pravastatin also increases the expression of human OATP-R in kidney cells and enhances the function of OATP-R in the cells to improve the transport ability.

[In vivo OATP-R gene expression assay]
In order to confirm whether or not the OATP-R gene expression enhancing action of statin can be obtained in vivo, an in vivo OATP-R gene expression assay was performed using rats. Specifically, the following method was used.

  Ten SD rats were divided into a water (control) administration group (5 animals) and a pravastatin (Prava) administration group (5 animals). In the pravastatin administration group, pravastatin was orally administered at 200 mg / kg / day for 7 days using water as a solvent. The control group received the same amount of water as that administered to the pravastatin administration group. After 7 days of administration, each SD rat was dissected and the kidney was removed. 1 g of the extracted kidney was added to 10 ml of TRIZOL solution (manufactured by Invitrogen) and homogenized for 1 minute at the maximum speed (100,000 rpm) of a polytron crusher. Two times the amount of chloroform solution was added to the homogenized solution, stirred, and then centrifuged, and the aqueous phase portion was transferred to another tube. Two times the amount of isopropyl alcohol was added to this aqueous phase, followed by cooling and centrifugation at 15000 rpm to extract RNA. Using the extracted RNA as a template, cDNA synthesis was performed using a SuperScriptIII RTS first strand cDNA synthesis kit (Invitrogen). Quantitative RT-PCR was performed using TaqMan Probe (Applied Biosystems) in the same manner as described in Example 1, and mRNA expression level of OATP-R gene in rat kidney cells (relative expression level with respect to GAPDH) Was quantified for each sample group at N = 5, and the average value of mRNA expression level was calculated for each sample. The mRNA expression level of rat OATP-R gene when using water as a control was set to 1, and the average value of the mRNA expression level of each sample was corrected to calculate the relative mRNA level. These results are shown in the right panel of FIG. As can be seen from the right panel of FIG. 3, it was shown that pravastatin's OATP-R gene expression enhancing activity can also be obtained in vivo. The mRNA expression level of the cyp1a1 gene having an XRE-like sequence similar to that of the rat OATP-R gene and human OATP-R gene was quantified by quantitative RT-PCR by the same method as described in Example 1. As a result, it was shown that mRNA expression of Cyp1a1 gene in rat kidney cells was also enhanced by pravastatin (FIG. 3 left panel). CYP1a1 is an enzyme that oxidizes a low-molecular-weight organic compound and works with other induced detoxification enzymes to control a so-called detoxification reaction. Therefore, statins such as pravastatin are considered not only to prevent and treat renal dysfunction, but also to improve renal detoxification.

[Assay for inhibiting blood pressure elevation of statin in vivo]
In order to confirm whether statin has a blood pressure increase inhibitory effect, an in vivo blood pressure increase inhibitory effect confirmation assay was performed using rats. Specifically, the following method was used.

  15 dahl salt-sensitive rats were divided into a low salt diet (LS) (control) administration group (5 mice), a high salt diet (HS) administration group (5 mice), a high salt diet and pravastatin (HS + statin) administration group ( 5). In the HS + statin administration group, instead of drinking water, a 1 mg / ml pravastatin aqueous solution was given at about 60-80 ml / day per rat. The blood pressure of each group of rats before the assay (0 W) was measured with a tail cuff, and it was confirmed that there was no blood pressure difference between the groups. In addition, when blood pressure fluctuated due to examination and treatment, measurement was resumed after confirming that blood pressure returned to the baseline. Moreover, as a telemetry system, the product made by Data Sciences International (DSI) is used, respectively. Telemetry transmitter: TA11PA-C40, Telemetry receiver: RPC-1, Atmospheric pressure calibration device: APR-1, Data conversion matrix: DEM Data acquisition / analysis software: DataquestARTSilver Version2.2 was used. Thus, the systolic blood pressure of each group of rats was measured every week from the start of the assay up to 4 weeks later. The transition of the mean blood pressure of each group at each time is shown in FIG. As can be seen from the results in FIG. 4, the mean blood pressure in the systolic period increased in the HS administration group every week as compared with the LS administration group. However, in the HS + statin administration group, the mean blood pressure has changed almost in the same way as in LS, and after 3 weeks (3W) and 4 weeks (4W) from the start of the assay, the mean blood pressure increased compared to the HS administration group. It was significantly suppressed.

[Luciferase assay using HEK293 cells]
As described in Example 2 2) of Patent Document 7 above, a reporter plasmid incorporating a luciferase gene downstream of the transcriptional active region of the human OATP-R gene is used as HEK239, a human fetal kidney-derived cell line. Cells were transfected. The effect of various statins on the transcriptional activity of the human OATP-R gene was examined by conducting a luciferase assay using these cells. FIG. 5 shows the result of correcting the expression level of luciferase when using DMSO as a control as 1. As can be seen from the results in FIG. 5, simvastatin ([1] in FIG. 5), lovastatin ([2] in FIG. 5), fluvastatin sodium ([3] in FIG. 5), cerivastatin sodium ([4] in FIG. 5) ), Cerivastatin lactone ([5] in FIG. 5), pitavastatin calcium ([6] in FIG. 5), pravastatin sodium ([7] in FIG. 5), mevastatin ([8] in FIG. 5), atorvastatin calcium (FIG. 5) [9]), rosuvastatin calcium ([10] in FIG. 5), and silastatin sodium ([11] in FIG. 5) all increase the transcriptional activity of the human OATP-R gene compared to DMSO as a control. It was shown that Among them, fluvastatin sodium, cerivastatin lactone, and atorvastatin calcium were particularly excellent in increasing the transcription activity of human OATP-R.

[Assays on mechanism of action of statin for improving renal function]
A series of assays shown in FIG. 6 were performed in order to confirm whether or not the action of improving statin function by statins depends on changes in blood pressure. Specifically, the following method was used.

(1) Effect of Pravastatin Administration on Rat Blood Pressure First, 10 Wistar rats were prepared, and 5 rats were divided into a pravastatin (Prava) administration group and a control administration group. From one week before the 5/6 nephrectomy (5 / 6Nx) operation described below (−1 W), the pravastatin administration group was given a 0.1 mg / ml pravastatin solution at about 30 to 40 ml / day per rat, The control group received the same amount of water. Each operation was performed according to the schedule shown in FIG. The nephrectomy operation and blood pressure measurement of Wistar rats were performed as follows.

  Each Wistar rat was given a sleeping pill (43 mg / kg ketamine and 2.9 mg / kg xylazine) intraperitoneally, then laparotomized from the midline, and the right renal artery, right renal vein, and ureter into 4-0 silk thread. Ligated and the right kidney was removed. The branches of the left renal artery were ligated with 7-0 silk thread so that the remaining blood flow was 1/3, so that only 1/6 kidney remained in total with respect to the total kidney volume before laparotomy. In this way, 5/6 nephrectomy (5/6 nephrectomy) rats (hereinafter also referred to as “nephrectomy rats”) were prepared. In addition, in order to measure the blood pressure of each of these nephrectomized rats as needed, a telemetry main body was placed in the abdominal cavity of the rat, and the sensor portion was inserted and fixed from the left femoral movement. After 5/6 nephrectomy, each rat was individually housed in a cage, and blood pressure was measured for 10 seconds every 3 minutes by telemetry for 24 hours. In the pravastatin (Prava) administration group and the control administration group, blood pressure was measured with a tail cuff before performing nephrectomy, and it was confirmed that there was no difference in blood pressure between the two groups. In addition, when blood pressure fluctuated due to examination and treatment, measurement was resumed after confirming that blood pressure returned to the baseline. Moreover, as a telemetry system, the product made by Data Sciences International (DSI) is used, respectively. Telemetry transmitter: TA11PA-C40, Telemetry receiver: RPC-1, Atmospheric pressure calibration device: APR-1, Data conversion matrix: DEM Data acquisition / analysis software: DataquestARTSilver Version2.2 was used.

  As described above, the systolic blood pressure of the pravastatin (Prava) administration group and the control administration group was measured from the day after the nephrectomy to the 26th day, and the average blood pressure of each day was averaged within the same group. Was calculated. The result is shown in FIG. As can be seen from FIG. 7, no difference in systolic blood pressure was observed between the pravastatin administration group and the control administration group. In the assay of Example 6 described above, suppression of blood pressure increase was observed in the high salt diet + pravastatin administration group as compared to the high salt diet administration group, but in the experiment of Example 8 (1), There was no difference in systolic blood pressure between the pravastatin (Prava) administration group and the control administration group. This is thought to be due to the fact that the dose of pravastatin in the experiment of Example 8 (1) is considerably lower (less than 1/10) than that in the assay of Example 6.

(2) Creatinine Renal Clearance Assay Plasma and urine creatinine concentrations and urine volume in both groups of nephrectomized rats prepared and bred according to the schedule of FIG. 6 described above are the assay of Example 8 (4) described later. Using the same method, the renal clearance of creatinine (ml / min / kg) was calculated. The result is shown in FIG. As can be seen from FIG. 8, there was no significant difference in the renal clearance of creatinine between the pravastatin administration group and the control administration group.

(3) In vivo OATP-R gene expression assay Regarding the renal tissue of each rat excised by nephrectomy at 0 W of Example 8 (1) (ie, when one week has elapsed since the start of pravastatin administration) Quantitative RT-PCR was performed in the same manner as in Example 5, and the mRNA expression level of OATP-R gene in rat kidney cells (relative expression level relative to GAPDH) was measured for each rat in both groups. The value which averaged the mRNA expression level of OATP-R gene of each rat within the same group was computed. The result is shown in FIG. As can be seen from the results in FIG. 9, the mRNA expression level of the OATP-R gene was significantly increased in the rats in the pravastatin administration group as compared with the control administration group.

(4) In Vivo Asymmetric Dimethylarginine Measurement Assay Plasma and urine asymmetric dimethylarginine (ADMA) levels were measured in both groups of nephrectomized rats prepared and bred according to the schedule of FIG. ADMA is known as a renal failure substance, and is known to have an action of suppressing synthesis of nitric oxide and an action of increasing blood pressure.

As sample urine, urine accumulated in a metabolic cage at the time shown in FIG. 6 was used for each rat. As the plasma for the sample, plasma separated from blood collected at the time shown in FIG. 6 was used for each rat. Urine and plasma samples, internal standard was stirred at adjusted methanol solution, further stirred by adding CHCl _3, then centrifuged 5 min at 4600g, ultrafiltration filter the supernatant (Fraction Centrifugation at 4 ° C. and 9100 g for 2 hours. Centrifugal concentration of the obtained filtrate was asked to measure the concentration of ADMA in each sample by liquid chromatography using SRL. It was. Then, the ADMA concentration in plasma was calculated as an average value (nmol / ml) of all the rats for each rat, and an average value was calculated within the same group. For ADMA in urine, the amount of ADMA excretion per day (nmol / day) of the rat at each time was first calculated from the measured ADMA concentration, and the average value was calculated for all times. Calculated, and a value obtained by further averaging within the same group was calculated. In addition, based on the urinary ADMA concentration, the ADMA concentration in plasma, and the urine volume per minute, ADMA renal clearance (ADMA concentration in urine × urine volume per minute / ADMA concentration in plasma; ml / Min) was calculated. These results are shown in FIG. The plasma ADMA concentration did not change much between the pravastatin-administered group and the control-administered group (see the left panel of FIG. 10), but the amount of ADMA in the urine (middle panel of FIG. 10) and renal clearance of ADMA were A significant increase was confirmed in the administration group compared to the control group.

(5) DDAH1 and DDAH2 renal mRNA expression assay In addition, the reason why the ADMA concentration in the plasma of the nephrectomy TG (+) rat group in Example 8 (4) is low is not the enhancement of ADMA degradation. In order to confirm, the expression of DDAH1 and DDAH2 which are ADMA degrading enzymes in vivo in the kidney was examined by quantitative PCR. The result is shown in FIG. As a result, DDAH1 and DDAH2 mRNA expression (relative expression level with respect to GAPDH) in the nephrectomy TG (+) rat group was not different from that in the nephrectomy TG (−) rat group.

(6) As described above, AMDA excretion from the kidney by statins does not depend on changes in blood pressure or changes in renal clearance of creatinine, and statin enhances OATP-R expression to increase AMDA from the kidneys. It was found that excretion was increased.

Both the left panel and the right panel show the results of a human OATP-R gene expression assay using ACHN cells, which are human renal cancer cell lines. Both the left panel and the right panel are diagrams showing the results of an intracellular thyroid hormone uptake assay. FIG. 3 right panel: shows the results of in vivo rat OATP-R gene expression assay. FIG. 3 left panel: shows the results of an in vivo rat CYP1a1 gene expression assay. It is a figure which shows the result of the blood pressure increase inhibitory effect confirmation assay of a statin in vivo. It is a figure which shows the result of the luciferase assay using HEK293 cell. It is a figure which shows the outline | summary of a series of assays for confirming whether the renal function improvement effect by a statin is dependent on the change of a blood pressure, etc. It is a figure which shows the blood pressure of a systole in the pravastatin (Prava) administration group rat and the control administration group rat. The horizontal axis of the graph shows the number of days elapsed since nephrectomy, and the vertical axis shows blood pressure (mmHg). It is a figure which shows the result of the renal clearance assay of a creatinine in a pravastatin (Prava) administration group rat and a control administration group rat. It is a figure which shows the result of the OATP-R gene expression assay in vivo. Left panel: It is a figure which shows the ADMA density | concentration in plasma in a pravastatin administration group and a control administration group. Middle panel: A diagram showing the amount of ADMA in urine in the pravastatin administration group and the control administration group. Right panel: ADMA renal clearance in the pravastatin administration group and the control administration group. It is a figure which shows the structural formula of various statins. It is a figure which shows the expression of DDAH1 and DDAH2 in the kidney of a nephrectomy TG (+) rat group and a nephrectomy TG (-) rat group.

Claims (8)

An OATP-R gene expression enhancing composition containing a statin or a pharmaceutically acceptable salt thereof. Statins include cerivastatin, atorvastatin, fluvastatin, simvastatin, lovastatin, pitastatin, pitavastatin, pravastatin, mevastatin, rosuvastatin and rosuvastatin The composition for enhancing OATP-R gene expression according to claim 1 selected from the group consisting of (cilastatin). The composition for enhancing OATP-R gene expression according to claim 1, wherein the statin is cerivastatin or atorvastatin. The OATP-R gene expression enhancing composition according to any one of claims 1 to 3, wherein the OATP-R gene is a human OATP-R gene or a rat OATP-R gene. A prophylactic / therapeutic agent for renal dysfunction comprising an OATP-R gene expression enhancing composition comprising a statin or a pharmaceutically acceptable salt thereof. The kidney according to claim 5, wherein the renal dysfunction is selected from the group consisting of acute renal failure, chronic renal failure, acute nephritis, chronic nephritis, acute tubular necrosis, tubular interstitial disorder, chronic kidney disease and nephrotic syndrome. Preventive / therapeutic agent for dysfunction. A method of using a statin or a pharmaceutically acceptable salt thereof in the production of an OATP-R gene expression enhancing composition. A method of using a statin or a pharmaceutically acceptable salt thereof in the manufacture of a preventive / therapeutic agent for renal dysfunction.