Pharmaceutical composition for preventing or treating adverse drug reactions by statin

Abstract

The present disclosure relates to a pharmaceutical composition for preventing or treating statin-induced adverse effects or a pharmaceutical composition for co-administration with statin, the pharmaceutical composition containing, as an active ingredient, at least one selected from the group consisting of an isoprenoid-based compound, zaragozic acid, terbinafine, and ketoconazole. The pharmaceutical composition according to the present disclosure may prevent and/or treat adverse statin effects that can be induced by statin, that is, can be induced at any time by oxisterols present at abnormal levels in the body. The pharmaceutical composition can not only treat but also prevent the adverse effects of various statin therapeutics whose use has recently increased rapidly, and thus it is expected that the pharmaceutical composition can be widely used for various diseases and the utilization thereof can further be increased.

Claims

1. A method for preventing or treating statin-induced adverse drug reactions, the method comprising administering to a subject in need thereof a pharmaceutically effective amount of an isoprenoid-based compound, wherein the isoprenoid-based compound is any one or more selected from the group consisting of farnesyl pyrophosphate, mevalonate, isopentenyl pyrophosphate, and geranylgeranyl pyrophosphate.

2. The method of claim 1, wherein the statin is any one selected from the group consisting of atorvastatin, rosuvastatin, simvastatin, pitavastatin, pravastatin, fluvastatin, lovastatin, cerivastatin, and mevastatin.

3. The method of claim 1, wherein the adverse drug reactions are caused by statin administration in a state in which oxysterols produced in vivo by lipopolysaccharides are abnormally increased compared to those in normal people.

4. The method of claim 3, wherein the adverse drug reactions are caused by damage to any one or more cells selected from the group consisting of kidney tubule cells, nerve cells, and pancreatic cells.

5. The method of claim 4, wherein a disease caused by the damage to kidney tubule cells is any one selected from the group consisting of acute renal failure, acute tubular necrosis injury, and ischemic reperfusion injury.

6. The method of claim 4, wherein a disease caused by the damage to nerve cells is any one selected from the group consisting of cognitive dysfunction, dementia, Parkinson's disease, Alzheimer's disease, Huntington's syndrome, stroke, and spinal nerve damage.

7. The method of claim 4, wherein a disease caused by the damage to pancreatic cells is diabetes.

8. The method of claim 3, wherein oxysterols produced in vivo by lipopolysaccharides are abnormally increased due to the inflammation.

9. The method of claim 1, further comprising administering a pharmaceutically effective amount of statin to the subject in need thereof.

10. The method of claim 9, further comprising administering an ezetimibe formulation, a niacin extended-release formulation, or an amlodipine formulation to the subject in need thereof.

11. The method of claim 1, wherein the adverse drug reactions are damage to kidney tubule cells, nerve cells or pancreatic cells caused by statin administration in a state in which oxysterols produced in vivo by lipopolysaccharides are abnormally increased compared to those in normal people.

12. The method of claim 11, wherein the oxysterols produced in vivo by lipopolysaccharides are abnormally increased due to inflammation.

Description

DESCRIPTION OF DRAWINGS

(1) FIGS. 1 and 2 show the results of analyzing the relationship between various types of statins and oxysterol according to an Example of the present disclosure.

(2) FIG. 3 shows the results of analyzing the relationship between various types of oxysterols and statin according to an Example of the present disclosure.

(3) FIGS. 4 to 6 show the results of evaluating the preventive and therapeutic effects of isoprenoid-based compounds on the adverse statin effects of co-administration of statin and oxysterol according to an Example of the present disclosure.

(4) FIG. 7 shows the results of analyzing the difference in adverse statin effects between tissues according to an Example of the present disclosure.

(5) FIG. 8 shows the results of analyzing the difference in adverse statin effects on pancreas according to an Example of the present disclosure.

(6) FIG. 9 shows the results of real-time polymerase chain reaction performed to analyze the mechanism of adverse effects of co-administration of statin and oxysterol according to an Example of the present disclosure.

(7) FIG. 10 shows the results of Western blotting performed to analyze the mechanism of adverse effects of co-administration of statin and oxysterol according to an Example of the present disclosure.

(8) FIG. 11 shows the results of H & E staining performed to analyze adverse statin effects in the presence of oxysterols in vivo according to an Example of the present disclosure.

(9) FIG. 12 shows the results of PAS staining performed to analyze adverse statin effects in the presence of oxysterols in vivo according to an Example of the present disclosure.

(10) FIG. 13 shows the results of NGAL immunohistochemistry performed to analyze adverse statin effects in the presence of oxysterols in vivo according to an Example of the present disclosure.

(11) FIG. 14 shows the results of NGAL immunohistochemistry performed to analyze adverse statin effects in the presence of oxysterols in vivo according to an Example of the present disclosure.

(12) FIG. 15 shows the results of NGAL immunohistochemistry performed to analyze adverse statin effects in the presence of oxysterols in vivo according to an Example of the present disclosure.

(13) FIG. 16 is a graph showing the results of quantifying the results of NGAL immunohistochemistry according to an Example of the present disclosure.

(14) FIGS. 17 and 18 show the results of H & E staining performed to evaluate the effects of an isoprenoid-based compound on the prevention and treatment of statin-induced adverse effects in vivo according to an Example of the present disclosure.

(15) FIGS. 19 and 20 show the results of PAS staining performed to evaluate the effects of an isoprenoid-based compound on the prevention and treatment of statin-induced adverse effects in vivo according to an Example of the present disclosure.

(16) FIGS. 21 and 22 show the results of NGAL immunohistochemistry performed to evaluate the effects of an isoprenoid-based compound on the prevention and treatment of statin-induced adverse effects in vivo according to an Example of the present disclosure.

(17) FIG. 23 is a graph showing the results of quantifying the results of NGAL immunohistochemistry according to an Example of the present disclosure.

BEST MODE

(18) In accordance with an embodiment of the present disclosure, there is provided a pharmaceutical composition for preventing or treating statin-induced adverse drug reactions, the pharmaceutical composition containing, as an active ingredient, at least one selected from the group consisting of an isoprenoid-based compound, zaragozic acid, terbinafine, and ketoconazole.

(19) In accordance with another embodiment of the present disclosure, there is provided a pharmaceutical composition for co-administration with statin, the pharmaceutical composition containing, as an active ingredient, at least one selected from the group consisting of an isoprenoid-based compound, zaragozic acid, terbinafine, and ketoconazole.

(20) In accordance with still another embodiment of the present disclosure, there is provided a method for preventing or treating statin-induced adverse drug reactions, the method including administering to a subject in need thereof a pharmaceutically effective amount of at least one selected from the group consisting of an isoprenoid-based compound, zaragozic acid, terbinafine, and ketoconazole.

MODE FOR INVENTION

(21) Hereinafter, the present disclosure will be described in more detail with reference to examples. It will be obvious to those skilled in the art that these examples are merely to illustrate the present disclosure in more detail and the scope of the present disclosure according to the gist of the present disclosure is not limited to these examples.

EXAMPLES

Example 1: Analysis of Relationship Between Statins and Oxysterols

(22) 1.1. Analysis of Relationship Between Various Types of Statins and Oxysterol

(23) To analyze the relationship between statins and oxysterols, the human kidney tubule cell line HK-2 (human kidney-2 cell line; ATCC CRL-2190™) was seeded into a 24-well plate at a density of 1×10.sup.5 per well. In addition, each well was treated with 5 μM of atorvastatin or 1 μM of fluvastatin, and 0 or 0.1 μg/mL of 25-hydroxycholesterol (25-HC), a kind of oxysterol, was added to each well. Subsequently, the cells were cultured in an incubator at 37° C. under 5% CO.sub.2 for 72 hours, and then the degree of cell death in each well was analyzed using an LDH assay kit (Takara, Cat #. MK401). The results are shown in FIGS. 1 and 2.

(24) As shown in FIGS. 1 and 2, it was confirmed that when the cells were treated with the statin or the oxysterol alone, no cell death was induced, but when the statin and the oxysterol were co-administered to the cells, rapid cell death was induced. From these results, it was confirmed that, regardless of the type of statin, significant cell death in the kidney tubule cell line was induced even when the oxysterol was present at a concentration of 0.1 μg/mL, which corresponds to a low concentration at which no toxicity occurs. This suggests that when oxysterol is present in vivo, protein prenylation may be inhibited regardless of the dose of statin, and hence adverse effects such as cell death may occur.

(25) 1.2. Analysis of Relationship Between Various Types of Oxysterols and Statin

(26) In order to examine whether the same results appear even in the presence of various types of oxysteols, an experiment was performed in the same manner as Example 1.1. As statin, 5 μM of atorvastatin was used, and as oxysterols, 0.1 μg/mL of 25-hydroxycholesterol, 7-ketocholesterol, cholesterol 5,6-epoxide, 24-hydroxycholesterol and 27-hydroxycholesterol were used. The results are shown in FIG. 3.

(27) As shown in FIG. 3, it was confirmed that, in the cell line to which the oxysterols were administered alone, no cell death occurred, but in all the experimental groups to which the statin and each of the oxysterols were co-administered, cell death was induced. From these results, it was confirmed that, regardless of the type of oxysterol, significant cell death was induced in the kidney tubule cell line when the statin was present even at a low concentration at which no toxicity occurs. This suggests that statin-induced adverse effects may occur in the presence of oxysterols in vivo.

(28) From the above results, it was confirmed that strong cytotoxicity to the kidney tubule cells was induced by co-administration of various types of oxystreols and statins. Through this, it was confirmed that at a low concentration of the oxysterol or a low concentration of the statin, no cell toxicity was induced, but when the oxysterol and the statin were co-administered, strong cytotoxicity to the kidney tubule cells could be induced even at low concentrations of the oxysterol and the statin. In addition, it could be confirmed that when the oxysterol is present at high concentration in vivo, administration of the statin for treatment can cause adverse effects such as acute renal failure (acute kidney injury).

Example 2: Evaluation of Effect of Isoprenoid-Based Compounds on Reduction of Adverse Effects

(29) In order to examine whether isoprenoid-based compounds can reduce adverse effects which are induced by co-administration of oxysterol and statin, the cell line was treated with oxysterol (0.1 μg/mL of 25-hydroxycholestyerol) and statin (5 μM of atorvastatin) in the same manner as Example 1.1, and treated with 10 μM of farnesyl pyrophosphate, 200 μM of mevalonate or 10 μM of geranylgeranyl pyrophosphate, which is an isoprenoid-based compound. In addition, the cells were treated with 10 μg/mL of 25-hydroxycholesterol and 1 μM of zaragozic acid. The results are shown in FIGS. 4 to 6.

(30) As shown in FIGS. 4 to 6, it was confirmed that cell death induced by co-administration of the oxysterol and the statin was inhibited by the isoprenoid-based compound. In addition, it was confirmed that even when the cells were treated with zaragozic acid, the adverse effects were reduced to some degrees, and thus the viability of the cells increased. From these results, it can be confirmed that, regarding adverse effects induced by statin administration, the isoprenoid-based compound or the zaragozic acid allows protein phenylation to be induced by the mevalonate pathway, and when the isoprenoid-based compound or the zaragozic acid is co-administered with statin, it can prevent adverse effects, which can be induced by statin administration when the in vivo concentrations of oxysterols are outside normal levels, or reduce the extent of the adverse effects. Furthermore, it could be confirmed that the adverse effects that occurred could also be treated.

Example 3: Analysis of Relationship Between Adverse Effects of Co-Administration of Statin with Oxysterol and Tissue

(31) In order to examine whether the adverse effects of co-administration of statin with oxysterol show different results depending on tissues in vivo, in addition to the kidney tubule cell line HK-2, primary renal tubule cells (ATCC® PCS-400-010™), renal mesangial cells (ScienCell Cat. # #4200), primary hepatocytes (cultured after isolation from C57BL6/J mice), and pancreatic Ins-le cells (ThermoFisher Scientific) were treated with oxysterol (0.1 μg/mL of 25-hydroxycholesterol) and statin in the same manner as Example 1.1, and the degree of cell death was analyzed. Primary neurons were obtained by euthanizing the mother mouse and then collecting brain tissue from the fetal head in the womb. After removing the arachnoid and pia mater from the obtained brain tissue, the hippocampus was isolated and transferred to DM media (dissection media; 490 mL of HBSS, 5 mL of HEPES, and 5 mL of penicillin/streptomycin), and then the cerebral cortex was isolated therefrom. Generally, the cerebral cortex was isolated from two mouse fetuses, and the hippocampus was used in an amount isolated from all the fetuses isolated from one mother mouse. Each isolated hippocampus or cerebral cortex was placed in a 15-mL tube, and washed and disrupted with trypsin and DM media, and the cells were collected. For image acquisition, the collected cells were seeded in PM media (plating media; 485 mL of Neurobasal media, 10 mL of B-27 supplement, and 5 mL of 100 X glutamax) at a concentration of 1×10.sup.5 to 5×10.sup.5 cells/mL, and for experimental use, the cells was seeded in PM media at a concentration of 5×10.sup.5 to 1×10.sup.6 cells/mL. Then, the cells were cultured for 2 weeks and used in the experiment. The results are shown in FIGS. 7 and 8.

(32) As shown in FIGS. 7 and 8, it was confirmed that, in the renal mesangial cells and the primary hepatocytes, co-administration of the statin and the oxysterol showed no cytotoxicity, whereas, in the primary renal tubule cells and the pancreatic Ins-le cells, significant cell death was induced by co-administration of the statin and the oxysterol. In addition, it was confirmed that when each of the statin or the oxysterol was administered alone, no cell death was observed even in primary nerve cells, but when the statin and the oxysterol were co-administered to the primary nerve cells, cell death significantly increased. From the above results, it was confirmed that cytotoxicity caused by co-administration of the oxysterol and the statin occurred in a cell-specific manner. This suggests that adverse effects caused by administration of statin in a state in which the concentration of oxysterols in vivo is high occur in a tissue-specific manner. In particular, it can be confirmed that the adverse effects cause damage to kidney tubule cells, nerve cells and pancreatic cells.

Example 4: Analysis of Mechanism of Adverse Effects Caused by Co-Administration of Statin and Oxysterol

(33) In order to examine the causes of adverse effects induced by co-administration of statin and oxysterol, the HK-2 cell line was seeded into a 24-well plate at a density of 1×10.sup.5 cells per well. Then, each well was treated with 5 μM of atorvastatin or 1 μM of fluvastatin, and 0.1 μg/mL of 25-hydroxycholesterol was added to each well. Next, the cells were cultured in an incubator at 37° C. under 5% CO.sub.2 for 24 hours, and then mRNA was extracted from the cells using an RNA isolation kit (Qiagen). In addition, protein was extracted using a ReadyPrep Protein Extraction kit (Bio-rad). Using the extracted mRNA as a template, cDNA was synthesized using a Prime Script RT-PCR kit (Takara). Using the synthesized cDNA, the expression level of HMGCR (3-hydroxy-3-methylglutaryl CoA reductase; HMG-CoA reductase) gene was quantified by real-time polymerase chain reaction. The primer sequences used for the real-time polymerase chain reaction are shown in Table 1 below, and the results of the quantification are shown in FIG. 9. In addition, the extracted protein was analyzed by Western blotting using anti-cleaved SREBP2 (sterol regulatory element binding protein 2) antibody (Abcam), and the results of the analysis are shown in FIG. 10.

(34) TABLE-US-00001 TABLE Gene Primer sequence HMG-CoA Forward: 5′-CAGGATGCAGCACAGAATGT-3′ reductase Reverse: 5′-CTTTGCATGCTCCTTGAACA-3′ GAPDH Forward: 5′-GCACAGTCAAGGCCGAGAAT-3′ Reverse: 5′-GCCTTCTCCATGGTGGTGAA-3′

(35) As shown in FIG. 9, it was confirmed that when the cells were treated with a low concentration of the statin, the expression of HMGCR increased, but when the statin and the oxysterol were co-administered to the cells, the expression of HMGCR decreased. From these results, it could be confirmed that when a low concentration of the statin was administered to the cells, the expression of HMGCR was increased by positive feedback, but in the presence of the oxysterol, the positive feedback was inhibited.

(36) In addition, as shown in FIG. 10, it was confirmed that when the cells were treated with a low concentration of the statin, activated SREBP2 increased, but in the presence of the oxysterol, the increase in SREBP2 was inhibited.

(37) From the above results, it could be confirmed that when the cells were treated with a low concentration of the statin, the adverse effects of the statin were reduced by positive feedback, but in the presence of the oxysterol, the positive feedback was inhibited, and hence damage to the cells was induced, and finally, adverse effects on various tissues were induced.

Example 5: Evaluation of Adverse Effects of Co-Administration of Statin and Oxysterol In Vivo

(38) In order to examine whether co-administration of statin and oxysterol also induces adverse effects in vivo, 100 mg of statin (atorvastatin or fluvastatin) was added to 1 kg of feed so that the statin could be administered at a dose of about 20 mg to 30 mg per kg of mouse body weight. The mixture was fed to 8-week-old C57BL6/J mice for 14 days. After 14 days, lipopolysaccharide (LPS, SIGMA) was administered intraperitoneally to mice at a lipopolysaccharide dose of 12 mg per kg of mouse body weight so that the concentration of oxysterol in the body reached 5 ng/mL to 5 μg/mL, thereby producing oxysterol in the mouse body (see “J Lipid Res (2009) 50:2258-2264; PNAS (2009) 106: 16764-16769”). 72 hours after administration of the lipopolysaccharide, the mice were euthanized, and then kidney tissue was extracted by performing cardiac perfusion with 4% paraformaldehyde in DPBS (Dulbecco's phosphate-buffered saline). The extracted kidney tissue was subjected to H & E (Hematoxylin & Eosin) staining, PAS (periodic acid-Schiff) staining and immunohistochemistry of neutrophil gelatinase-associated lipocalin (NGAL) that is an early diagnostic marker of acute renal failure, thereby determining whether acute kidney damage and acute renal failure would occur. The results are shown in FIGS. 11 to 16.

(39) FIG. 11 shows the results of performing H & E staining. In FIG. 11, the blue arrow indicates the site where a cast was formed, and the blue arrowhead indicates the site where tubule cell vacuolization was induced. As shown in FIG. 11, it was confirmed that when the generation of oxysterol was induced in the experimental group to which the statin was administered, acute renal failure was induced.

(40) FIG. 12 shows the results of performing PAS staining. In FIG. 12, the red arrow indicates the site where a cast was formed. As shown in FIG. 12, it was confirmed that when the generation of oxysterol was induced in the experimental group to which the statin was administered, acute renal failure was induced.

(41) FIGS. 13 to 15 show the results of performing NGAL immunohistochemistry. In FIGS. 13 to 15, red represents phalloidin indicating the tubule structure, green represents NGAL, and blue represents DAPI indicating the cell nucleus. In addition, FIG. 16 is a graph showing the results of quantifying NGAL staining. As shown in FIGS. 13 to 16, it could be confirmed that only when the generation of oxysterol was induced in the experimental group to which the statin was administered, the expression of NGAL, an early diagnostic marker of acute renal failure, significantly increased.

(42) From the above results, it could be confirmed that statin-induced adverse effects were not induced by administration of a low concentration of the statin in vivo or by the oxysterol alone, but administration of a low concentration of the statin in the presence of the oxysterol could induce rapid damage to the cells, thus inducing adverse effects such as acute renal failure.

Example 6: Evaluation of Effect of Isoprenoid-Based Compound on Reduction of Adverse Effects In Vivo

(43) In order to examine whether administration of an isoprenoid-based compound reduces statin-induced adverse effects even in vivo, 100 mg of statin (fluvastatin) was added to 1 kg of feed, and the mixture was fed to 8-week-old C57BL6/J mice for 14 days. Subsequently, mevalonate was administered intraperitoneally to the mice at a dose of 30 mg per kg of mouse body weight for 4 consecutive days, and then lipopolysaccharide (SIGMA) was administered intraperitoneally to mice at a lipopolysaccharide dose of 12 mg per kg of mouse body weight, thereby producing oxysterol in the mouse body. 72 hours after administration of the lipopolysaccharide, the mice were euthanized, and then kidney tissue was extracted by performing cardiac perfusion with 4% paraformaldehyde in DPBS (Dulbecco's phosphate-buffered saline). The extracted kidney tissue was subjected to H & E (Hematoxylin & Eosin) staining, PAS (periodic acid-Schiff) staining and immunohistochemistry of neutrophil gelatinase-associated lipocalin (NGAL) that is an early diagnostic marker of acute renal failure, thereby determining whether acute kidney damage and acute renal failure would occur. In addition, the concentrations of total cholesterol, high-density lipoprotein (HDL) and low-density lipoprotein (LDL) were measured. The results are shown in FIGS. 17 to 23.

(44) FIG. 17 shows the results of performing H & E staining, and FIGS. 19 and 20 show the results of performing PAS staining. In the figures, the azure arrow indicates the site where a cast was formed, and the blue arrowhead indicates the site where tubule cell vacuolization was induced. FIGS. 21 and 22 show the results of performing NGAL immunohistochemistry. In the figures, red represents phalloidin indicating the tubule structure, green represents NGAL, and blue represents DAPI indicating the cell nucleus. In addition, FIG. 23 is a graph showing the results of quantifying NGAL staining.

(45) As shown in FIGS. 17 to 20, it was confirmed that in the experimental mouse group in which the oxysterol was formed by administering only the lipopolysaccharide without administering the statin, cast formation and tubule cell vacuolization were induced, and in the experimental mouse group in which the oxysterol was formed after administering the statin, cast formation and tubule cell vacuolization were further induced. In addition, from the results of administering mevalonate (MVA), it was confirmed that the mevalonate (MVA) showed no therapeutic effect on kidney damage caused by oxysterol formation, but exhibited a significant effect in the experimental group to which the statin and the oxysterol were co-administered.

(46) As shown in FIGS. 21 to 23, it was confirmed that when generation of the oxysterol was induced without administration of the statin, the expression of NGAL, an early diagnostic marker of acute renal failure, increased, but when generation of the oxysterol was induced after administration of the statin, the expression of NGAL significantly increased. In addition, it was confirmed that when mevalonate (MVA) was administered to the experimental group in which generation of the oxysterol was induced without administration of the statin, the mevalonate (MVA) showed no significant therapeutic effect, but when the mevalonate (MVA) was administered to the experimental group in which generation of the oxysterol was induced after administration of the statin, the expression of NGAL significantly decreased.

(47) In addition, it was confirmed that in the mice to which the statin was administered alone, the concentrations of total cholesterol and low-density lipoprotein decreased compared to those in the control group to which the statin was not administered, and in the group to which the statin and the oxysterol were co-administered, the concentrations of total cholesterol and low-density lipoprotein decreased, but damage to kidney tissue was observed. Furthermore, it was confirmed that in the experimental group to which the statin, the oxysterol and the mevalonate (MVA) were all administered, the concentrations of total cholesterol and low-density lipoprotein decreased, but kidney tissue was not damaged. From the above results, it could be confirmed that the isoprenoid-based compound effectively reduced the adverse effects of the statin while maintaining the therapeutic effect of the statin.

(48) Through the above results, it could be confirmed that the adverse effects (cell damage in the kidney, pancreas, nerve, etc.) of a low concentration of the statin, that is, the statin at a defined daily dose usable for treatment, significantly increased in the presence of the oxysterol in vivo, and the isoprenoid-based compound or zaragozic acid could effectively prevent and treat the adverse effects of the statin. In addition, it could be confirmed that in cell damage caused by the oxysterol alone, the isoprenoid-based compound showed no preventive or therapeutic effect, but in adverse statin effects in the presence of the oxysterol, the isoprenoid-based compound significantly reduced cell damage. Therefore, it could be confirmed that the isoprenoid-based compound or zaragozic acid of the present disclosure may be effectively used to prevent adverse effects that may be caused by administration of a low concentration of statin, or to treat the adverse effects that occurred.

(49) Although the present disclosure has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only of a preferred embodiment thereof, and the scope of the present disclosure is limited thereto. Thus, the substantial scope of the present disclosure will be defined by the appended claims and equivalents thereof.

INDUSTRIAL APPLICABILITY

(50) The pharmaceutical composition, the pharmaceutical composition for co-administration with statin and the method of treating by administering the same according to the present disclosure may prevent and/or treat adverse statin effects that can be induced by statin, that is, can be induced at any time by oxisterols present at abnormal levels in the body. They can not only treat but also prevent the adverse effects of statin therapeutics whose use has recently increased rapidly, and thus it is expected that they can be widely used for various diseases and the utilization thereof can further be increased.