JP2005314381A - Prophylactic/therapeutic/ameliorating agent for proliferative nephropathy - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a medicament useful for a therapeutic agent, and the like, for proliferative nephropathy, especially nephritis (diabetic nephritis or hypertensive nephritis). <P>SOLUTION: This invention relates to the medicament for prophylactic/therapeutic/ameliorating agent for proliferative nephropathy, comprising a decoy type nucleic acid medicine which inhibits coupling of a transcription factor to a coupling point, and is selected from decoy of NF-κB, decoy of STAT-6, decoy of AP-1, decoy of Ets and decoy of E2F. The medicament is suitable for prophylaxis/therapy/amelioration of nephritis (diabetic nephritis or hypertensive nephritis), glomerulosclerosis or albuminuria. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a preventive / treating / ameliorating agent for proliferative renal diseases such as nephropathy, glomerulosclerosis or albuminuria, comprising a decoy nucleic acid drug as an active ingredient.

  Currently, there are approximately 7 million diabetic patients in Japan, and an estimated 20 million potential diabetic reserve groups. There are various causes of diabetes, which is regarded as a lifestyle-related disease, and many factors such as diet, gene, endocrine, and inflammation are involved, and it is formed on a complex interrelationship.

  Diabetes is a chronic disease, and its complications include microangiopathy, nephropathy, retinopathy, neurosis, etc., and there is a great physical and mental burden as an individual patient. The social disease that Japanese society has from now to the future has progressed. According to a recent survey by the Japanese Society for Dialysis Therapy, the number of diabetic patients is about 35% among new dialysis patients.

  Among the complications, diabetic nephropathy is associated with dilation of the glomerular basement membrane, proliferation of mesangium cells and fibrosis represented by Kimmelstiel-Wilson nodules, and these pathological changes are diabetic. It is a direct exacerbation factor for nephropathy.

  The stages of diabetic nephropathy can be classified from the first stage to the fifth stage, and treatment methods according to the respective stages are known as follows.

  Stage 1 (early nephropathy): A period in which glomeruli are considered to have high blood pressure, but no renal disorder that can be detected by current general testing methods is observed. Strict blood sugar control is effective.

  Second stage (early nephropathy): The time when slight proteinuria (microalbuminuria) is observed in a precise urinalysis. Systemic blood pressure begins to rise. Strict blood glucose control and blood pressure management are effective (blood pressure is about 130/85 mmHg). Angiotensin converting enzyme inhibitor is useful.

  Third stage (apparent nephropathy): Period (first period) when persistent proteinuria (abnormality is revealed by a simple proteinuria test) is observed. As it further progresses, glomerular function (waste waste excretion function) decreases (late stage). It is easy to accompany high blood pressure. In addition to strict blood glucose control and hypertension treatment (blood pressure is about 130/85 mmHg), it is necessary to limit the amount of salt and protein in the diet (about 0.8 grams of dietary protein per kilogram of standard body weight). Angiotensin converting enzyme inhibitor is useful. At this time, it will be difficult to completely prevent the progression of nephropathy.

  4th stage (renal failure stage): Period when abnormalities are observed in general blood tests due to impaired renal function. Urinary protein increases and blood protein decreases (called nephrotic syndrome) in many cases. Antihypertensive therapy (about 140-150 / 90-95mmHg), salt restriction, low protein diet are required. Extreme exercise is not possible. It is also necessary to prepare for the introduction of dialysis therapy.

  5th period (dialysis therapy period): The period when renal function was abolished and introduced into chronic hemodialysis therapy.

Diabetic nephropathy is thought to be caused by changes in lifestyle habits, and is also the leading cause of dialysis in patients with renal failure (2000). It has been demanded.
Gene Ther. 2000 Aug; 7 (15): 1326-32. Exp Nephrol. 2001; 9 (3): 181-90 Genes Immun. 2001 0ct; 2 (6): 304-8.

  Non-Patent Document 1 discloses that a synthetic oligonucleotide (ODN), which is a decoy compound of NF-κB, is used for the treatment of nephritis caused by inflammatory changes in tissues. There is no disclosure about.

  It is an object of the present invention to provide an agent for preventing / treating / ameliorating proliferative kidney disease caused by proliferative changes.

  The present inventors used Otsuka-Long-Evance-Tokushima-Fatty (OLETF) rats, which are known as type 2 diabetes model animals and are known as the only model capable of causing lesions very similar to those of human diabetic nephropathy. The present inventors have found that gene expression of various growth factors and receptors can be suppressed by transfecting decoy ODN into the kidney of an animal that has already entered the renal lesion formation stage.

  That is, the present invention provides, as a means for solving the problem, the prevention / proliferation of proliferative kidney disease comprising, as an active ingredient, a decoy nucleic acid drug (hereinafter referred to as “decoy”) that inhibits the binding of a transcriptional regulator to a binding site. It provides treatment / amelioration agents.

  The “decoy” or “decoy compound” used in the present invention is a site on a chromosome to which NF-κB, STAT-6, AP-1, Ets, E2F, etc. bind, or NF-κB, STAT-6, AP-1 , Binds to a site on the chromosome to which other transcriptional regulatory factors of genes controlled by Ets and E2F, etc. bind (target binding site), NF-κB, STAT-6, AP-1, Ets and E2F, etc. It refers to compounds that antagonize binding to these target binding sites. Exemplary decoys or decoy compounds are nucleic acids and analogs thereof.

  The prophylactic / therapeutic / ameliorating agent for proliferative kidney disease caused by proliferative change according to the present invention is effective in the treatment of proliferative renal disease caused by proliferative change.

  The prophylactic / therapeutic / ameliorating agent for proliferative kidney disease of the present invention (hereinafter referred to as “drug for proliferative renal disease”) uses a decoy that inhibits the binding of a transcriptional regulatory factor to the binding site as an active ingredient, and if necessary And a pharmaceutically acceptable carrier.

  As the decoy, those disclosed in WO02 / 066070 and US2002-0052333 A1 can be used, and among them, at least one selected from the following decoys is preferable.

NF-κB decoy (SEQ ID NO: 2) (see WO02 / 066070)
STAT-6 decoy (SEQ ID NO: 4) (see WO02 / 066070)
AP-1 decoy (SEQ ID NO: 5) (see WO02 / 066070)
Ets decoy (SEQ ID NO: 6) (see WO02 / 066070)
E2F decoy (SEQ ID NO: 7 and SEQ ID NO: 8 are shifted to form a double strand) (see US2002-0052333 A1)
As the decoy, an oligonucleotide containing a complement of the above-mentioned decoy, a variant thereof, and a compound containing these in the molecule can also be used. The oligonucleotide may be DNA or RNA, or may contain a nucleic acid modification and / or pseudo-nucleic acid in the oligonucleotide.

  Oligonucleotides include oligonucleotides with thiophosphate diester bonds (S-oligos) in which the oxygen atom of the phosphodiester bond is replaced with sulfur atoms, and oligonucleotides in which the phosphodiester bond is replaced with an uncharged methyl phosphate group Those modified so as to be less susceptible to degradation in vivo.

  Oligonucleotides, variants thereof or compounds containing these in the molecule may be single-stranded or double-stranded, and may be linear or circular. A variant is a nucleic acid to which a part of the above sequence is mutated, substituted, inserted, or deleted, and to which NF-κB or the like or other transcriptional regulatory factors of genes controlled by NF-κB or the like bind. It means a nucleic acid that has the ability to specifically antagonize a binding site.

  As the decoy, an NF-κB decoy is particularly preferable. NF-κB is a transcription factor involved in inflammation / immunity, cell proliferation, and canceration. When NF-κB responds to a signal from the outside of the cell, its inhibitor molecule I-κB is degraded and released. The released I-κB moves from the cytoplasm to the nucleus and binds to various DNA recognition sites, thereby interleukin (IL) -1, IL-6, IL-8 involved in cell proliferation and inflammation. It acts as an important mediator for the expression of cytokines such as GM-CSF, TNF, chemokines, interferons and various adhesion factors (E-selectin, VCAM-1, ICAM-1). Synthetic oligonucleotide (ODN), a decoy compound of NF-κB, has a synthetic double-stranded DNA with high affinity for NF-κB and inhibits the binding of nuclear factors to the promoter region of the target gene. To prevent gene transactivation.

  The NF-κB decoy preferably includes SEQ ID NO: 1 in the base sequence, particularly a double-stranded nucleic acid (NF-κB decoy oligonucleotide) consisting of SEQ ID NO: 2 (SEQ1) and SEQ ID NO: 3 (SEQ2) (US6, 262, 033).

  Decoy can be manufactured by the method described in WO02 / 066070.

  The agent for proliferative kidney disease of the present invention can be made into a dosage form according to the administration method using decoy alone or, if necessary, a pharmaceutically acceptable carrier and decoy. For example, WO02 / 066070 The administration method (oral administration or parenteral administration) and dosage form described in 1).

  Examples of dosage forms for oral administration include tablets, pills, dragees, capsules, solutions, syrups and the like.

  Examples of parenteral administration methods include topical administration, dermal application administration, intraarterial administration, intramuscular administration, subcutaneous administration, intravenous administration, nasal administration, and the like, and a liquid preparation including an injection is preferable.

  An injection can be obtained by dissolving or suspending a decoy in a physiologically compatible buffer such as Hanks' solution, Ringer's solution, or buffered physiological saline.

  When the injection is made into an aqueous injection suspension, a substance that stabilizes the viscosity of the suspension, for example, sodium carboxymethylcellulose, sorbitol, or dextran can be contained. The injection can be an oily injection suspension containing a fatty acid such as sesame oil, a synthetic fatty acid ester such as ethyl oleate or triglyceride, and a liposome.

  When the agent for proliferative kidney disease of the present invention is used as an injection, it is desirable from the viewpoint of enhancing the therapeutic effect and the like to combine the injection of the agent and ultrasonic irradiation.

  Specifically, a mixture of NF-κB, etc. and contrast agent Optison (trade name; contrast agent manufactured by Mallinkrodt, USA) is administered to the circulating blood and circulates only to the target kidney. In order to introduce the blood from the blood side, the kidney is irradiated with ultrasonic waves (for example, using the ultrasonic gene transfer device described in the Examples), and decoy ODN is specifically transferred into mesangial cells which are renal glomerular support cells. Apply the method to effect. In addition, ultrasonic irradiation to the kidney may be performed directly on the kidney after laparotomy or may be performed on the abdomen from outside the body without performing laparotomy.

The ultrasonic irradiation conditions are 1 to 10 W / cm ² , preferably ² to 5 W / cm ² , and 30 seconds to 30 minutes, preferably 5 to 25 when using the ultrasonic gene introduction apparatus described in the Examples. Minutes are fine. At this time, the duty ratio can be 10 to 100%, preferably 50 to 100%.

Injection and ultrasound irradiation
(1) A method of starting ultrasonic irradiation after injection,
(2) A method of simultaneously performing ultrasonic irradiation and injection,
(3) A method of performing injection while irradiating with ultrasonic waves,
Any one of these methods can be applied, but in the present invention, the methods (1) and (2) are particularly preferred from the viewpoint of enhancing the therapeutic effect and the like.

  The dose and frequency of administration of the drug for proliferative kidney disease of the present invention vary depending on the administration method, treatment period, age, body weight, etc., but in the case of injection, it is usually 1 mg to 1 mol in terms of the amount of decoy which is an active ingredient. Administer 1 g / day once a day or divided into multiple doses.

  The agent for proliferative kidney disease of the present invention is suitable as a prophylactic agent, therapeutic agent and ameliorating agent for nephropathy (for example, diabetic nephropathy or hypertensive nephropathy), glomerulosclerosis or albuminuria.

Example 1 [Introduction of FITC-labeled NF-κB decoy into kidney by perfusion method]
Rats used two female Wistar (retired) 1-year-olds. After laparotomy, the renal artery of the left kidney was isolated, cannulated by surflow into the renal artery, and perfused with heparin solution, followed by FITC-labeled NF-κB decoy 200 μg, Optison (trade name) (10%) and physiological The drug of the present invention mixed with saline (total 500 μl) was injected.

Sonication was started immediately after drug injection. The ultrasonic irradiation conditions were continuous (100%) with an ultrasonic gene transfer apparatus (Sonitoron 1000, Nepagene, Chiba), 1 W / cm ² , and 10 minutes.

  Kidney tissues were collected immediately after ultrasound irradiation, 4 hours later, and 4 days later. The kidney tissue cross-section was embedded in a compound, frozen, and nuclear stained with DAPI (4 ', 6-diamidino-2-phenylindole). And observed under a fluorescence microscope. At the end of the experiment, neither kidney had infarction.

  The fluorescence of FITC is strong on the right side compared to the part not exposed to ultrasound (left side of the photo). Ultrasound-irradiated kidney was observed only on the half surface, and there was almost no fluorescence on the non-irradiated back half surface (FIG. 1).

  Fluorescence was not observed in arterioles in the renal tissue, but was partially observed in tubular epithelial cells, but was mainly observed in glomerular cells (FIG. 2).

After 4 hours, although fluorescence remained in the glomeruli, it decreased from immediately after irradiation. On the other hand, the tubule was dominant, and it was distributed granularly in the epithelial cells (FIG. 3).

  Fluorescence still remained in the glomerulus even after 4 days, and was observed in the tubular epithelial cells as granules. In the contralateral kidney, almost no fluorescence was observed immediately, 4 hours, and 4 days later.

Example 2 [Introduction of FITC-labeled NF-κB decoy into kidney by intravenous injection method]
In order to examine the effects of body weight and kidney size, mice and rats were used to examine the efficiency of FITC-labeled NF-κB decoy introduction into the kidney.

  The mice were 3 WBB6F1 + / + males, weighing about 25 g, and 2 months old. After laparotomy of the mouse under anesthesia, the left kidney was exposed, and the pharmaceutical agent of the present invention mixed with FITC-NF-κB decoy 400 μg / 100 μ and an ultrasonic contrast agent Optison (trade name) 100 μl just before ultrasonic irradiation was intravenously injected. .

Sonication was started immediately after drug injection. The conditions of ultrasonic irradiation to the kidney were 2 W / cm ² with an ultrasonic gene transfer apparatus, duty ratios of 50% and 100%, irradiation time of 5 minutes and 10 minutes.

  In the case of mice, when the irradiation time was kept constant (10 minutes) and the duty ratio was decreased, the renal cell introduction rate decreased. Only in the place where the ultrasonic irradiation probe was applied, the fluorescence was localized and the depth remained in the cortex. Fluorescence was also observed in the glomeruli and tubules (FIG. 4).

  When the duty ratio was decreased, the intracellular introduction rate was also decreased. Conversely, bleeding spots were less than continuous irradiation. When looking at the kidney on the other side that was not irradiated with ultrasonic waves, fluorescence was observed in the glomerular cells, but fluorescence was observed in the tubular epithelial cells.

On the other hand, in rats (2 male Wistars, 8 weeks old, body weight of about 300 g), FITC-NF-κB decoy 600 μg and Optison (trade name) (300 μl) were mixed immediately before administration and intravenously injected, with a duty ratio of 50 %, 100%, 2 W / cm ² , irradiation time of 5 minutes, 10 minutes, and 20 minutes were performed, and since the diameter of the probe was about 5 mm, the entire kidney was irradiated while moving the probe slowly.

Under the optimum conditions in mice (2 W / cm ² , duty ratio 50%, irradiation time 10 minutes), the introduction efficiency of rats was clearly poor. The gene transfer efficiency almost the same as that of the mouse was 2 W / cm ² , the duty ratio was 50%, and the irradiation time was 20 minutes (FIGS. 5 and 6).

  After the introduction of FITC-NF-κB decoy, fluorescence is specifically observed in the glomeruli, and the fluorescence distribution is present in cells (mesangial cells) in connective tissue, not in the epithelium or endothelium ( Figures 5 and 6).

  Fluorescence in the tubular epithelial cells was observed in a granular form immediately after irradiation (FIG. 7A), but gradually decreased with time, and almost disappeared on the sixth day (FIG. 7B).

Example 3 [Treatment and gene expression by NF-κB decoy in the kidney of diabetes model animals]
OLETF rats (two), male, 20 months old (average body weight 625 g), which are human type 2 diabetes model animals, were used. After anesthesia, the right kidney was exposed by making a longitudinal incision about 5 cm from the midline. The intestine was carefully brought to one side, and a mixture of 15 mg of NF-κB decoy / 1.5 ml of physiological saline and 500 μl of Optison (trade name) was administered under anesthesia. The ultrasonic irradiation conditions were a duty ratio (50%), 2 W / cm ² , 20 minutes, and the entire right kidney was irradiated immediately after injection (Table 1). Since the diameter of the probe was small at about 5 mm, the entire irradiation was performed while moving the probe slowly.

  After 20 minutes of ultrasonic irradiation, the kidneys were slightly swollen and the color was dark brown, but the color gradually returned. The kidney was returned to its original location, the incision was sutured, and then kept normally for 1 month. One month later, the left and right kidneys were collected under deep anesthesia, and after separating the renal cortex, the cortical tissue was immediately frozen in liquid nitrogen and stored until genetic analysis.

[Examination of histological changes]
Both glomerulosclerosis and stromal disorder, which represent renal histological damage, were clearly progressed in OLETF rats compared to the control group, but the right kidney treated with ultrasound after NF-κB decoy administration and the left of the control There was no significant difference between the kidneys in both rats. The urinary cylinders seen when glomerular and tubule function declines were observed in the OLTF rats, although many PAS-positive unstructured urinary cylinders (*) were found in the tubular lumen (Fig. 8C), but only in the control group. Only a few were observed (FIG. 8A = left kidney, FIG. 8B = right kidney). However, sonic cast right kidney (FIG. 8D) in OLETF rats had a marked decrease in urinary column formation compared to control left kidney (FIG. 8C).

[Examination of gene expression in the renal cortex]
The collected RNA in the renal cortex is amplified as cDNA by RT-PCR, and the left kidney sample is used as a control (cy5), the right kidney is used as a treatment sample (cy3), and a DNA chip (Atlas Glass Rat 1.0 Microarray, 1081 gene, CLONTECH , CA, USA). Among all 1081 genes, 49 genes related to growth factors and growth factor receptors are included. Nine kinds of hose-keeping genes (Glyceraldehyde-3-phosphate dehydrogenase, alpha-tubulin, Ornitine decarboxylase, myosin IB, beta actin, Ribosomal protein S29, polyubiquitin, phospholipase A2, hypoxanthine-guanine The average value of phosphoribosyltransferase was corrected. Finally, 1062 genes could be examined among 1081 genes. The frequency distribution of the examined genes is shown (FIG. 9).

  As is clear from FIG. 9, the expression distribution of all genes is almost normal, but the center of the whole is shifted slightly from 1 to the left. If the rejection area at both ends is about 10%, the decreased gene has an expression ratio of 0.75 or less, the decreased gene has an expression ratio of 0.75 to less than 0.8, and the increased gene has an expression ratio of 1.2. Table 2 shows that genes with an expression ratio of 1.5 or more and other genes (0.8 or more and less than 1.5) have no variation.

  As is clear from Table 2, there were very few genes with large fluctuations as a whole. Table 3 shows the rate of change of genes related to growth factors, growth factor receptors, and blood pressure among the genes that changed.

  As is clear from Table 3, the most decreased was renin, and the PDGF and VEGF receptor genes related to other growth factors and cytokines were decreased. TGFβ, α and TNF showed a decreasing trend. On the contrary, the increase was not found in the growth factor / cytokine, but the increase was observed in cytochrome P450 expressed in the tubule, and the most increased was neulexin 3.

Fluorescence micrograph (100 times) showing FITC distribution in the kidney irradiated with ultrasound after administration by perfusion of FITC-NF-κB decoy from rat renal artery. Fluorescence micrograph (100 times) showing FITC distribution in tubules under the same conditions as in FIG. Fluorescence micrograph (100 times) showing FITC distribution in renal tubules 4 hours after administration of FITC-NF-κB decoy under the same conditions as in FIG. Fluorescence micrograph (100 ×) showing FITC distribution in renal tissue sonicated after systemic administration of FITC-NF-κB decoy to mice. Fluorescence micrograph (100 ×) showing FITC distribution in renal tissue sonicated after systemic administration by intravenous injection of FITC-NF-κB decoy to rats. Fluorescence micrograph (100 ×) showing FITC distribution in renal tissue sonicated after systemic administration by intravenous injection of FITC-NF-κB decoy to rats. Fluorescence micrograph (100 times) showing FITC distribution in tubular epithelial cells sonicated after systemic administration by intravenous injection of FITC-NF-κB decoy under the same conditions as in FIGS. Fluorescence micrograph (100 times) showing renal tissue images (PAS staining) one month after ultrasonic irradiation of the right kidney after systemic administration of NF-κB decoy by intravenous injection to rats. The figure which shows a gene expression frequency.

Claims (8)

  A prophylactic, therapeutic, or ameliorating agent for proliferative kidney disease, comprising as an active ingredient a decoy nucleic acid drug that inhibits the binding of a transcriptional regulator to the binding site.   The prophylactic kidney disease prevention according to claim 1, wherein the decoy nucleic acid drug is at least one selected from decoy nucleic acid drugs of NF-κB, STAT-6, AP-1, Ets, and E2F.・ Treatment / improvement agent.   The prophylactic / therapeutic / improving agent for proliferative kidney disease according to claim 1 or 2, wherein the decoy nucleic acid drug is a decoy nucleic acid drug of NF-κB.   The prophylactic / therapeutic / ameliorating agent for proliferative kidney disease according to any one of claims 1 to 3, wherein the NF-κB decoy nucleic acid medicine comprises SEQ ID NO: 1 in the base sequence.   The prevention / treatment / improvement of proliferative kidney disease according to claim 1, wherein the NF-κB decoy nucleic acid pharmaceutical is a double-stranded nucleic acid consisting of SEQ ID NO: 2 and SEQ ID NO: 3. Agent.   The proliferative renal disease preventive / treating / ameliorating agent according to any one of claims 1 to 5, wherein the proliferative renal disease is nephropathy, glomerulosclerosis or albuminuria.   The prophylactic / therapeutic / ameliorating agent for proliferative kidney disease according to claim 6, wherein the nephropathy is diabetic nephropathy or hypertensive nephropathy.   The prophylactic / therapeutic / ameliorating agent for proliferative kidney disease according to any one of claims 1 to 7, wherein the dosage form is a liquid preparation containing an injection.