Pharmaceutical composition for preventing or treating nephritis

Abstract

The present invention provides a pharmaceutical composition that can be usefully used for the prevention or treatment of nephritis.

Description

[Technical Field]

[0001] The present invention provides a pharmaceutical composition for preventing or treating nephritis. [Background technology]

[0002] Diseases that cause inflammation in the kidneys are broadly referred to as nephritis or nephritis, and the causes of inflammation can be bacterial or immune-mediated. Depending on the location of inflammation, it can be divided into interstitial nephritis (TIN), acute / chronic pyelonephritis, and glomerulonephritis. It can also occur as a complication of other diseases, and depending on the cause, it can be divided into diabetic nephropathy, lupus nephritis, etc. This type of nephritis can lead to complications of chronic renal failure, and once it develops, it is difficult to restore kidney function, so early and appropriate treatment is necessary.

[0003] Interstitial nephritis is a disease in which inflammatory cells infiltrate the interstitium between renal tubules, resulting in decreased kidney function. Causes of interstitial nephritis include medications, systemic diseases such as lupus (SLE), and infections. 50% of patients with acute interstitial nephritis experience a severe decrease in urine output (oliguria), and 5% experience gross hematuria. Joint pain, fever, blood eosinophilia, and skin rash may also occur, although some patients may exhibit no symptoms. If the symptoms of interstitial nephritis appear and blood tests show elevated BUN and creatinine levels due to decreased kidney function, interstitial nephritis is diagnosed and steroids are used for treatment.

[0004] The glomerulus is a tissue made up of a cluster of capillaries that is the basic unit that filters blood in the kidney. Glomerulonephritis is a disease that causes symptoms and signs when an inflammatory reaction occurs in the glomerulus due to immune dysfunction. Glomerulonephritis has a wide range of causes, but it is primarily caused by immunological mechanisms. Glomerulonephritis is diagnosed through urine tests, blood tests, and kidney tissue tests, and immunosuppressants are the primary treatment.

[0005] Meanwhile, prolyl-tRNA synthetase (PRS) is a member of the aminoacyl-tRNA synthetase (ARS) family of enzymes that activates amino acids for protein synthesis. Specifically, ARS forms aminoacyl adenylate (AA-AMP) and then transfers the activated amino acid to the 3′ terminus of the corresponding tRNA (translational function). Because ARS plays a crucial role in protein synthesis, ARS inhibition suppresses the growth and development of all cells. Therefore, ARS is recognized as a promising target for antibiotics and therapeutic agents that inhibit cellular overexpression (Nature, 2013, 494:121-125).

[0006] PRS exists and functions in the form of EPRS (Glutamyl-Prolyl-tRNA Synthetase) in the multisynthetase complex (MSC). In particular, EPRS functions as a translational silencer that suppresses the production of VEGF A (vascular endothelial growth factor A), a key factor in angiogenesis, among various MSCs. It has also been reported to be closely associated with various solid cancers (Nat. Rev. Cancer, 2011, 11, 708-718).

[0007] Therefore, the present inventors have conducted extensive research into methods for preventing or treating nephritis, and as a result have found that a specific PRS inhibitor, described below, can be used to prevent or treat nephritis, thereby completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention provides a pharmaceutical composition that can be usefully used for the prevention or treatment of nephritis. [Means for solving the problem]

[0009] In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating nephritis, comprising a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof: <Chemical formula 1> JPEG2025529524000002.jpg25143

[0010] The compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof is a compound described in Korean Patent Registration No. 10-2084772, specifically, the substance described in Example 40 of the specification. It is known that the substance can be used as a PRS inhibitor for the prevention or treatment of fibrosis.

[0011] The present invention has confirmed that the substance can also be applied to the prevention or treatment of nephritis, and as can be confirmed from the examples and experimental examples described below, it can be usefully used in the prevention or treatment of nephritis.

[0012] Meanwhile, the compound represented by Chemical Formula 1 can be used in the form of a pharmaceutically acceptable salt, and useful salts include acid addition salts formed with a pharmaceutically acceptable free acid. The free acid can be an inorganic or organic acid. Examples of inorganic acids include hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid, while examples of organic acids include citric acid, acetic acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, succinic acid, 4-toluenesulfonic acid, glutamic acid, and aspartic acid. Preferably, the pharmaceutically acceptable salt of the compound represented by Chemical Formula 1 is a hydrochloride salt.

[0013] Furthermore, the compound represented by Chemical Formula 1 can be prepared in a crystalline or amorphous form, and when prepared in a crystalline form, it may be optionally hydrated or solvated. In the present invention, not only stoichiometric hydrates of the compound represented by Chemical Formula 1 but also compounds containing various amounts of water may be included. The solvates of the compound represented by Chemical Formula 1 include both stoichiometric and non-stoichiometric solvates.

[0014] On the other hand, examples of the nephritis include tubulo-interstitial nephritis, glomerulonephritis, acute pyelonephritis, and chronic pyelonephritis.

[0015] As used herein, the term "prevention" refers to any action that inhibits or delays the onset, spread, and recurrence of the disease by administering the composition of the present invention, and "treatment" refers to any action that improves or favorably alters the symptoms of the disease by administering the composition of the present invention. Specifically, the pharmaceutical composition of the present invention has the effect of improving renal function. In addition, the pharmaceutical composition of the present invention has the effect of reducing inflammatory cells (CD4+, CD8+, or gdT) in kidney tissue.

[0016] The pharmaceutical compositions of the present invention can be formulated into oral or parenteral dosage forms according to standard pharmaceutical practice. These dosage forms may contain, in addition to the active ingredient, additives such as pharmaceutically acceptable carriers, adjuvants, or diluents.

[0017] Suitable carriers include, but are not limited to, physiological saline, polyethylene glycol, ethanol, vegetable oil, and isopropyl myristate, and diluents include, but are not limited to, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and / or glycine. The compound of the present invention can also be dissolved in oil, propylene glycol, or other solvents commonly used in the preparation of injection solutions. Furthermore, for topical application, the compound of the present invention can be formulated into ointments or creams.

[0018] The pharmaceutical dosage forms of the compounds of the present invention may be in the form of their pharmaceutically acceptable salts or solvates, and may be used alone or in combination with other pharmaceutically active compounds, or in suitable combinations. For example, the pharmaceutical composition of the present invention may additionally contain other active ingredients used for the prevention or treatment of nephritis. When the pharmaceutical composition of the present invention additionally contains other active ingredients, the weight ratio of the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof to the other active ingredients is preferably 1:0.1 to 1:10.

[0019] The compounds of the present invention can be formulated into injections by dissolving, suspending, or emulsifying them in aqueous solvents such as common saline, 5% dextrose, or non-aqueous solvents such as synthetic fatty acid glycerides, higher fatty acid esters, or propylene glycol. The dosage forms of the present invention can contain conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, and preservatives.

[0020] The pharmaceutical composition according to the present invention may be administered orally or parenterally. Depending on the administration method, the composition according to the present invention may contain 0.001 to 99% by weight, preferably 0.01 to 60% by weight, of the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[0021] The pharmaceutical compositions of the present invention can be administered to mammals, including rodents, mice, livestock, and humans, by a variety of routes, including oral, rectal, or intravenous, intramuscular, subcutaneous, intrauterine, intradural, or intracerebroventricular injection, although any route of administration is contemplated. [Effects of the Invention]

[0022] The pharmaceutical composition according to the present invention can be usefully used for the prevention or treatment of nephritis. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 is a diagrammatic view showing the process of separating blood from a normal person using a centrifuge in Experimental Example 1 of the present invention. [Figure 2] FIG. 1 shows the results of the inhibition of cell proliferation in CD4+ and CD8+ T cells by the compounds of the present invention in Experimental Example 1 of the present invention. [Figure 3] FIG. 1 shows the results of IL-17 cytokine suppression in gdT cells by the compounds of the present invention in Experimental Example 2 of the present invention. [Figure 4] FIG. 1 shows the results of measuring blood BUN and creatinine, which are primary indicators of renal function after treatment with the compound of the present invention, in a mouse model of renal failure treated with adenine diet in Experimental Example 3 of the present invention. [Figure 5] FIG. 1 shows the results of measuring the degree of fibrosis by immunochemical staining of mouse kidney tissues after treatment with the compound of the present invention in a mouse model of renal failure treated with dietary adenine in Experimental Example 3 of the present invention. [Figure 6] FIG. 1 shows the results of fibrosis factor inhibition after treating a rat kidney cell line with the compound of the present invention in Experimental Example 3 of the present invention. [Figure 7] FIG. 1 shows the results of fibrosis factor inhibition after treating human primary kidney cells with the compound of the present invention in Experimental Example 4 of the present invention. [Figure 8]FIG. 1 shows the results of measuring blood BUN and creatinine, which are primary indicators of renal function after treatment with the compound of the present invention, in a mouse model of preventing renal failure by feeding adenine in Experimental Example 5 of the present invention. [Figure 9] FIG. 1 shows the results of measuring the level of collagen accumulation in kidney tissues in a mouse model of preventing renal failure induced by dietary adenine treatment with the compound of the present invention in Experimental Example 5 of the present invention. [Figure 10] FIG. 1 shows the results of confirming the effects on damaged glomerular tissue after treating an NTN model with the compound of the present invention in Experimental Example 6 of the present invention. [Figure 11] FIG. 1 shows the results of immunochemical staining of mouse kidney tissue to confirm the effects on damaged glomerular tissue after treatment of an NTN model with the compound of the present invention in Experimental Example 6 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention will be described in more detail with reference to the following examples, but the following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0025] Example The following compound was prepared in the same manner as in Example 40 of Korean Patent Registration No. 10-2084772, and is hereinafter referred to as "PRSi" or "Example." JPEG2025529524000003.jpg25154 1 H NMR (500 MHz, MeOD): δ 9.67 (s, 1H). 8.02 (d, 1H), 7.82 (d, 1H), 4.62 (m, 2H), 3.60 (m, 1H), 3.28 (m, 1H), 2.99 (m, 2H), 2.25 (m, 2H), 2.08 (m, 2H), 1.99 (m, 1H), 1.78 (m, 2H), 1.54 (m, 1H)

[0026] Experimental Example 1: Inhibitory effect on CD4+ and CD8+ T cell proliferation Since the adenine-fed animal model confirmed that CD4+, CD8+, and gdTcell (IL-17) were the main factors in the renal failure model, Experimental Examples 1 and 2 of the present invention confirmed the effects of the compounds of the present invention on these main factors.

[0027] After collecting 10 ml of blood from a normal subject, Ficoll density gradient medium (Cytiva 17144003) and PBS were added as shown in Figure 1, and the immune cell layer (PBMC) was separated using a centrifuge.

[0028] PBMCs were transferred to a new tube using a pipette. After adding RBC lysis buffer and centrifuging, CD8 or CD4 cells were isolated using CD8 microbeads (Miltenyl 130-045-201) or CD4 microbeads (Miltenyl 130-096-533). CFSE (-(and-6)-Carboxyfluorescein Diacetate, Succinimidyl Ester) labeling solution (Invitrogen C1157) was added to the isolated cells. Compounds of the examples were dissolved in DMSO to a 5 mM stock concentration, and then treated with the compounds at 1.25 μM, 2.5 μM, 5 μM, and 10 μM. Five days later, the level of cell proliferation was measured by flow cytometry based on the amount of CFSE staining. The results are shown in Figure 2.

[0029] As shown in FIG. 2, it was confirmed that cell proliferation was inhibited when CD4+ and CD8+ T cells were treated with the compounds of the examples.

[0030] Experimental Example 2: Inhibition of IL-17 factor in gdT cells Ten milliliters of normal blood was collected, Ficoll density gradient medium (Cytiva, 17144003) and PBS were added, and the immune cell layer (PBMC) was separated by centrifugation. The PBMCs were transferred to a new tube using a pipette. RBC lysis buffer was added to the PBMCs and centrifuged. The cells were then cultured with 10 ng / ml of human IL-2 and IL-15 protein (cytokine) (for IL-17A induction). Example compounds were dissolved in DMSO to make 5 mM stocks and treated with 10 μM of the example compounds. On days 3 and 5 of cytokine treatment, 10 ng / ml of human IL-2 and IL-15 protein (cytokine) were added, centrifuged, and the supernatant was collected. IL-17A expression levels were determined in the collected supernatant using an IL-17A ELISA kit (R&D Systems, DY371). The results are shown in Figure 3.

[0031] As shown in Figure 3, when gdT cells isolated from the blood of a normal individual were treated with the compounds of the examples, it was confirmed that IL-17 cytokine, a major factor in gdT cells, was suppressed.

[0032] Experimental Example 3: Renal function improvement and anti-fibrotic effect - Adenine dietary treatment of mouse renal failure model As described in the literature (Ali et al. J Pharmacol Toxicol Methods 2013; Jia et al. BMC Nephrol 2013; Tanaka et al. Am J Pathol 2009), C57BL / 6 male mice were fed a diet containing adenine. Two weeks after the start of adenine intake, the mice were orally administered 5 mg / kg, 10 mg / kg, and 20 mg / kg of the example compound daily for two weeks (Figure 4). The negative control group (vehicle) was administered saline. Blood BUN and creatinine, primary indicators of renal function, were measured. The results are shown in Figure 4.

[0033] As shown in FIG. 4, it was confirmed that oral administration (once a day) of the compound of the present invention significantly inhibited the deterioration of renal function in a dose-dependent manner.

[0034] The mouse kidney tissues were also subjected to immunochemical staining to measure the degree of fibrosis, and the results are shown in FIG.

[0035] As shown in FIG. 5, it was confirmed that oral administration of the compound of the present invention significantly inhibited excessive accumulation of collagen in kidney tissue in a dose-dependent manner in a mouse model of renal failure induced by dietary adenine.

[0036] The mouse kidney tissue was homogenized using a tissue homogenizer (Qiagen's TissueLyser II), centrifuged, and the supernatant was collected. Cells were isolated from the supernatant by centrifugation and cultured. The cultured cells were treated with 5 ng / ml TGFβ and 2.5 μM, 5 μM, and 10 μM of the example compounds, respectively. After 48 hours, the cells were harvested and analyzed by Western blot for quantitative analysis of fibrotic factor proteins. The results are shown in Figure 6.

[0037] As shown in Figure 6, when rat kidney cell line (NRK-49F cell line) was treated with the compound of the present invention, it was confirmed that fibrotic factors (collagen, fibronectin, αSMA) were significantly inhibited.

[0038] Experimental Example 4: Inhibitory effect of fibrosis factors Human kidney tissue (preserved after biopsy) was placed in digestion buffer, minced with scissors, and then disintegrated by incubating on a shaker for 90 minutes. The disintegrated tissue was filtered through a 70 μm filter cell strainer, and the remaining tissue was filtered again using a 40 μm filter cell strainer. The filtered disintegrated tissue solution was centrifuged to separate and culture cells. The cultured cells were treated with 5 ng / ml TGFβ and 1.25 μM, 2.5 μM, or 5 μM of the example compounds, respectively. After 48 hours, the cells were harvested and analyzed by Western blot for quantitative analysis of fibrotic factor proteins. The results are shown in Figure 7.

[0039] As shown in FIG. 7, it was confirmed that the compounds of the examples were treated in human primary kidney cells and resulted in significant inhibition of fibrotic factors (collagen, fibronectin, αSMA).

[0040] Experimental Example 5: Renal function improvement and anti-fibrotic effect - Adenine dietary administration in a mouse model of renal failure prevention As described in the literature (Ali et al. J Pharmacol Toxicol Methods 2013; Jia et al. BMC Nephrol 2013; Tanaka et al. Am J Pathol 2009), C57BL / 6 male mice were fed a diet containing adenine. Simultaneously with the initiation of adenine administration, the example compounds were orally administered at 5 mg / kg, 10 mg / kg, and 20 mg / kg daily for four weeks (Figure 8). A negative control group (vehicle) was administered saline. Blood BUN and creatinine, primary indicators of renal function, were measured. The results are shown in Figure 8 below.

[0041] As shown in Figure 8, normal mice were fed an adenine diet for 4 weeks and simultaneously orally administered the compound of the present invention (once daily) for 4 weeks. As a result, it was confirmed that the deterioration of renal function was significantly suppressed in a dose-dependent manner. In other words, the compound demonstrated a preventive effect by suppressing kidney damage caused by the adenine diet.

[0042] The mouse kidney tissues were also subjected to immunochemical staining to measure the degree of fibrosis. The results are shown in Figure 9. As shown in Figure 9, it was confirmed that the excessive accumulation of collagen in kidney tissues was significantly inhibited in a dose-dependent manner by simultaneous oral administration of the compound of the present invention in a mouse model of renal failure induced by adenine diet.

[0043] Experimental Example 6: Renal function improvement and anti-fibrotic efficacy - NTN (Nephrotoxic serum-induced Nephritis) model C57BL / 6 male mice were immunized with 200 μg of sheep IgG and Freund's complement adjuvant. Five days later, 10 μg of sheep nephrotoxic serum was injected to induce disease. Starting from the day of disease induction, the example compounds were orally administered at doses of 5 mg / kg, 10 mg / kg, and 20 mg / kg for seven days. A negative control group received vehicle saline. Renal function (uPCR, BUN) was evaluated on day 8. The results are shown in Figure 10.

[0044] The NTN model is a glomerulonephritis model that induces inflammation in the glomeruli, resulting in proteinuria. As shown in Figure 10, this inflammation occurred specifically in the glomeruli (left vehicle in Figure 10), with minimal damage to the renal tubules (right vehicle in Figure 10). Oral administration of the example compounds for one week significantly reduced proteinuria, demonstrating their effectiveness on damaged glomerular tissue (PRSi 5 mg / kg in the left side of Figure 10, and Figure 11).

Claims

1. A pharmaceutical composition for preventing or treating nephritis, comprising a compound represented by the following chemical formula 1 or a pharmaceutically acceptable salt thereof: <Chemical formula 1>

2. The nephritis is tubulointerstitial nephritis, glomerulonephritis, acute pyelonephritis, or chronic pyelonephritis; The pharmaceutical composition of claim 1.

3. The pharmaceutically acceptable salt is a hydrochloride salt. The pharmaceutical composition of claim 1.

4. The pharmaceutical composition has an effect of improving renal function. The pharmaceutical composition of claim 1.

5. The pharmaceutical composition has the effect of reducing inflammatory cells CD4+, CD8+ or gdT in kidney tissue. The pharmaceutical composition of claim 1.

Images