PHARMACEUTICAL COMPOSITION CONTAINING B-LAPACHONE AS ACTIVE INGREDIENT FOR PREVENTION OR TREATMENT OF CHOLESTATIC LIVER DISEASE

Abstract

The present invention relates to a pharmaceutical composition containing β-lapachone as an active ingredient for prevention or treatment of cholestatic liver disease, and can provide agents for effectively preventing and treating cholestatic liver disease.

Claims

1. A pharmaceutical composition for prevention or treatment of cholestatic liver disease, the pharmaceutical composition containing β-lapachone or a pharmaceutically acceptable salt thereof as an active ingredient.

2. The pharmaceutical composition of claim 1, wherein the cholestatic liver disease is at least one selected from the group consisting of primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), progressive familial intrahepatic cholestasis (PFIC), benign recurrent intrahepatic cholestasis, intrahepatic cholestasis of pregnancy (ICP), cholestasis caused by viral hepatitis, cholestasis caused by alcoholic hepatitis, drug-induced cholestasis, cholestasis during parenteral nutrition, cholestasis due to malignant tumor, post-liver transplantation cholestasis, infectious cholestasis, and Alagille syndrome (AS).

3. The pharmaceutical composition of claim 1, wherein the composition inhibits fibrosis and inflammation of cholangiocytes.

4. The pharmaceutical composition of claim 1, wherein the composition improves the level of at least one blood index selected from the group consisting of AST, ALT, ALP, and bilirubin in the blood.

5. The pharmaceutical composition of claim 3, wherein the inhibiting of fibrosis is inhibiting at least one selected from fibrosis factors consisting of collagen type I alpha 1 (Col1α1), collagen type IV alpha 1 (Col4α1), alpha-smooth muscle actin (α-SMA), fibronectin, transforming growth factor beta 1 (TGF-β1), collagen type I alpha 2 (Col1α2), and transforming growth factor beta 2 (TGF-β2).

6. The pharmaceutical composition of claim 3, wherein the inhibiting of inflammation is inhibiting at least one selected from inflammatory cytokine factors consisting of interleukin-1beta (IL-1β), interleukin-6 (IL-6), interleukin-18 (IL-18), interferon-γ (INF-γ), tumor necrosis factor-α (TNF-α), tumor necrosis factor-β (TNF-β), and monocyte chemoattractant protein-1 (MCP-1).

7. The pharmaceutical composition of claim 1, wherein the cholestatic liver disease is accompanied by inflammatory bowel disease.

8. The pharmaceutical composition of claim 7, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

9. The pharmaceutical composition of claim 7, wherein the composition inhibits fibrosis and inflammatory cytokines in colon tissues.

10. A health functional food for prevention or amelioration of cholestatic liver disease, the heath functional food comprising β-lapachone or a pharmaceutically acceptable salt thereof as an active ingredient.

11. The health functional food of claim 10, wherein the cholestatic liver disease is accompanied by inflammatory bowel disease.

12. The health functional food of claim 11, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

13. A method for prevention or treatment of cholestatic liver disease, comprising administering to a mammal in need thereof a therapeutically effective amount of a pharmaceutical composition containing β-lapachone or a pharmaceutically acceptable salt thereof as an active ingredient.

14. The method of claim 13, wherein the cholestatic liver disease is at least one selected from the group consisting of primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), progressive familial intrahepatic cholestasis (PFIC), benign recurrent intrahepatic cholestasis, intrahepatic cholestasis of pregnancy (ICP), cholestasis caused by viral hepatitis, cholestasis caused by alcoholic hepatitis, drug-induced cholestasis, cholestasis during parenteral nutrition, cholestasis due to malignant tumor, post-liver transplantation cholestasis, infectious cholestasis, and Alagille syndrome (AS).

15. The method of claim 13, wherein the composition inhibits fibrosis and inflammation of cholangiocytes.

16. The method of claim 13, wherein the composition improves the level of at least one blood index selected from the group consisting of AST, ALT, ALP, and bilirubin in the blood.

17. The method of claim 15, wherein the inhibiting of fibrosis comprises inhibiting at least one selected from fibrosis factors consisting of collagen type I alpha 1 (Col1α1), collagen type IV alpha 1 (Col4α1), alpha-smooth muscle actin (α-SMA), fibronectin, transforming growth factor beta 1 (TGF-β1), collagen type I alpha 2 (Col1α2), and transforming growth factor beta 2 (TGF-β2).

18. The method of claim of 15, wherein the inhibiting of inflammation comprises inhibiting at least one selected from inflammatory cytokine factors consisting of interleukin-1beta (IL-1β), interleukin-6 (IL-6), interleukin-18 (IL-18), interferon-γ (INF-γ), tumor necrosis factor-α (TNF-α), tumor necrosis factor-β (TNF-β), and monocyte chemoattractant protein-1 (MCP-1).

19. The method of claim 13, wherein the cholestatic liver disease is accompanied by inflammatory bowel disease.

20. The method of claim 19, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0035] FIG. 1 shows images of the tissue surrounding the portal vein (PV) after the application or non-application of β-lapachone (BL) in DDC-induced cholestatic liver disease animal models.

[0036] FIG. 2 shows effects of β-lapachone (BL) on the collagen tissue surrounding the portal vein in DDC-induced cholestatic liver disease animal models (Panel A: images of the tissue surrounding the portal vein stained with Sirius Red and Masson's trichrome, Panel B: Graphs of quantification of images of the tissue stained with Sirius Red and Masson's trichrome).

[0037] FIG. 3 presents graphs showing effects of β-lapachone (BL) on expression of fibrosis-related genes in DDC-induced cholestatic liver disease mice (n=6).

[0038] FIG. 4 presents graphs showing effects of β-lapachone (BL) on blood indexes (ALT, AST, ALP, and bilirubin) in DDC-induced cholestatic liver disease mice (n=6).

[0039] FIG. 5 presents graphs showing effects of β-lapachone (BL) on expression of fibrosis-related genes in DDC-induced cholestatic liver disease mice (n=6).

[0040] FIG. 6 presents graphs showing effects of β-lapachone (BL) on expression of inflammation-related genes in DDC-induced cholestatic liver disease mice (n=6).

[0041] FIG. 7 presents graphs showing effects of β-lapachone (BL) on blood indexes (total bilirubin and ALP) in DDC-induced cholestatic liver disease animal models.

[0042] FIG. 8 presents graphs showing effects of β-lapachone (BL) on the blood indexes AST, ALT, and ALP in Poly I:C-induced cholestatic liver disease animal models.

[0043] FIG. 9 presents a graph showing effects of β-lapachone (BL) on the survival rate of inflammatory bowel disease mice in DSS-induced inflammatory bowel disease mice.

[0044] FIG. 10 presents graphs showing the effects of β-lapachone on the body weight change (A) and the colitis score (B) in DSS-induced inflammatory bowel disease mice (n=10).

[0045] FIG. 11 presents colon length comparison images (A) and a graph showing same (B) in DSS-induced inflammatory bowel disease mice.

[0046] FIG. 12 presents a graph showing effects of β-lapachone (BL) on inflammatory cytokines of the colon tissue in DSS-induced inflammatory bowel disease mice.

[0047] FIG. 13 presents a graph showing effects of β-lapachone (BL) on fibrosis-related mRNA expression in LX-2 hepatic stellate cell models.

[0048] FIG. 14 presents graphs showing effects of β-lapachone on expression of the inflammatory cytokine proteins IL-1p and TNF-α in the macrophage line Raw264.7 cell model.

[0049] FIG. 15 presents graphs showing effects of β-lapachone (BL) on expression of the inflammatory cytokines IL-1β and IL-18 proteins in peripheral blood mononuclear cells (PMBCs).

BEST MODE FOR CARRYING OUT THE INVENTION

[0050] Hereinafter, unless otherwise specified, β-lapachone is used as a concept encompassing β-lapachone itself and pharmaceutically acceptable salts thereof.

[0051] The causes of cholestasis are very diverse, such as various drug side effects, infections, tumors, bile duct tumors, cysts, bile duct stone, stenosis, and physical pressure on the bile ducts, and examples of cholestatic liver disease according to causes include primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), progressive familial intrahepatic cholestasis (PFIC), benign recurrent intrahepatic cholestasis, intrahepatic cholestasis of pregnancy (ICP), cholestasis caused by viral hepatitis, cholestasis caused by alcoholic hepatitis, drug-induced cholestasis, cholestasis during parenteral nutrition, cholestasis due to malignant tumor, post-liver transplantation cholestasis, infectious cholestasis, and Alagille syndrome (AS).

[0052] Hereinafter, preferable exemplary embodiments of the present disclosure will be described in detail. However, the present disclosure is not limited to the exemplary embodiments described herein and can be embodied in many different forms. Rather, these exemplary embodiments are provided so that the present disclosure will be thorough and complete and will fully convey the scope of the disclosure to those skilled in the art.

Example 1: Construction of Cholestatic Liver Disease Mouse Animal Models

[0053] 3,5-Diethoxycarbonyl-1,4-dihydrocollidine (DDC) inhibits the activity of ferrochelatase, which inserts Fe into protoporphyrin IX to generate heme, to induce the accumulation of protoporphyrin in the liver, and the accumulation and crystallization in the bile ducts due to the characteristics of hydrophobic protoporphyrin that can be excreted out of the liver only through the bile causes inflammation and fibrosis inside and outside the bile ducts. Since cholestasis, inflammation, and fibrosis in the liver tissue are induced by the above mechanism, DDC diet-induced animal models are one of the most used rodent cholestatic liver disease animal models.

[0054] DDC-induced cholestatic liver disease mouse animal models were prepared according to the method of Elisa Pose et al. by using 8-week-old C57BL/6 male mice (Samtako, Korea). Briefly, cholestatic liver disease was induced by feeding a standard rodent diet supplemented with 0.1% (w/w) DDC for 7 or 14 days. The 12-hour light and dark cycle was maintained and animals were allowed to free access to water. The standard rodent diet included medium wheat, wheat, corn, and corn gluten flour, and soybean oil (14% protein). All animal related procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Korea Research Institute of Bioscience and Biotechnology (KRIBB), KRIBB-AEC-20165).

Example 2: Preventive Effect of β-Lapachone on Lesions in Cholestatic Liver Disease Mouse Animal Models

[0055] 2.1. Histology Analysis of Cholestatic Liver Disease Mouse Animal Models

[0056] The long-term supply of DDC results in porphyrin plugs in the ducts to damage the biliary epithelium, thereby inducing duct obstruction. A toxic accumulation of bile in the bile ducts leads to cholangiocyte activation and ductular reaction proliferation, and is positive for Sirius Red staining and Masson's trichrome staining.

[0057] The tissue staining of the cholestatic liver disease animal models was measured through H&E staining (basic tissue staining), Sirius Red staining (collagen staining), and Masson's trichrome staining (collagen, cytoplasm, muscle fibers, etc.), and the tissue staining was measured by a skilled researcher.

[0058] After the mice were fed a diet containing 0.1% DDC for 14 days, tissue staining was performed for inflammation, fibrosis, and adhesion molecules around the portal vein, and the results are shown in FIG. 1.

[0059] To investigate the preventive effect of β-Lapachone (BL) on cholestatic liver disease, the mice were fed a diet with 0 (DDC-vehicle), 40 mg/kg (DDC-BL40) and 80 mg/kg (DDC-BL80) 3 days before DDC treatment (total 17 days) and, after 3 days, were fed a diet with DDC (total 14 days). After 14 days of DDC treatment, the mice were sacrificed and the liver was harvested including the portal vein (PV) and bile ducts of the mice, and the surrounding lesions were confirmed by H&E, and are shown in FIG. 1.

[0060] As shown in FIG. 1, the DDC treatment group (DDC-vehicle) as a control group showed increased lesions (indicated by arrows) in the portal vein (PV) and bile ducts of the mice compared with the normal group (standard diet, SD). It was also identified that the DDC-induced cholestatic liver disease mice (DDC-BL40 and DDC-BL80) treated with β-lapachone (BL) showed significant lesion reductions (indicated by arrows) around the portal vein (PV) and bile ducts compared with the control group (DDC-vehicle) treated with a vehicle. It was also identified that the lesion reduction was significant in the group treated with 80 mg/kg (BL80) β-lapachone (BL) compared with the group treated with 40 mg/kg (BL40), indicating that β-lapachone (BL) reduced the lesions of the DDC-treated portal vein and bile ducts depending on the concentration thereof.

[0061] These results indicate that the β-lapachone treatment has a preventive effect, such as a reduction in lesions of the portal vein and bile ducts in the cholestatic liver disease occurrence environment.

[0062] 2.2. Effect of β-Lapachone on Collagen Amount in Cholestatic Liver Disease Mouse Animal Models

[0063] The liver harvested in Example 2.1 was investigated for tissues or connective tissues around cells through Sirius Red and Masson's trichrome staining, which are shown in FIG. 2A.

[0064] As shown in FIG. 2A, the Sirius Red and Masson's trichrome staining results confirmed that the formation of the connective tissues was increased by DDC and the β-lapachone treatment inhibited such fibrosis progression. FIG. 2B is a quantification of stained portions of FIG. 2A in digital images.

[0065] It can be seen from the results that the β-lapachone treatment had a preventive effect on fibrosis due to the increase in connective tissue in the cholestatic liver disease occurrence environment.

[0066] Statistical analysis was performed on the results obtained from independent experiments (means±SEM) throughout the entire examples by two-tailed Student's t-test. The differences were considered significant when p<0.05. As for survival comparison experiments, the results were plotted and analyzed according to Kaplan-Meier survival analysis or product-limit method, with a log-rank (Mantel-Cox) test (Prism, version 5.0, GraphPad Software).

[0067] 2.3. Identification of Effect of β-Lapachone on (mRNA) Transcriptional Levels of Fibrosis-Associated Genes in Cholestatic Liver Disease Mouse Animal Models

[0068] In the cholestatic liver disease mouse models, the bile ducts were damaged and fibrosis was induced, by DDC, and accordingly, the transcriptional levels of collagen type I alpha 1 (Col1α1), collagen type IV alpha 1 (Col4α1), alpha-smooth muscle actin (α-SMA), fibronectin, transforming growth factor beta 1 (TGF-β1), collagen type I alpha 2 (Col1α2), and transforming growth factor beta 2 (TGF-β2), which are important factors in the development of fibrosis, were confirmed to be increased.

[0069] The expression of the factors in the liver tissue obtained from the cholestatic liver disease mouse animal models of Example 2.1 was performed by a real-time polymerase chain reaction was performed according to the following procedure. Total RNA from liver samples was extracted using Tri-RNA Reagent (Favrogen BIOTECH CORP, Nong-Ke Rd, Taiwan) according to the procedure presented by Favrogen, and was reverse transcribed into cDNA by using PrimeScript™ RT reagent Kit with gDNA Eraser (TAKARA Korea Biomedical Inc, Seoul, 08506, Korea). Quantitative PCR was performed using TB Green™ Premix Ex Tag™ II (Tli RNaseH Plus), ROX plus (TAKARA Korea Biomedical Inc, Seoul, 08506, Korea) and QuantStudio 5 Real-Time PCR Instrument (Thermo Fisher Scientific, Waltham, Mass., USA). The primer sequences used are shown in Table 1 below.

TABLE-US-00001 TABLE 1 Primer Gene name Category Sequence Col1a1 Forward 5′-CCTGAGTCAGCAGATTGAGAACA-3′ Reverse 5′-CCAGTACTCTCCGCTCTTCCA-3′ Col1a2 Forward 5′-TTCTGCAGGGTTCCAACGAT-3′ Reverse 5′-TGTCTTGCCCCATTCATTTG-3′ Fibronectin Forward 5′-AGGCAGAAAACAGGTCTCGATT-3′ Reverse 5′-CAGAATGCTCGGCGTGATG-3′ α-Sma Forward 5′-CACGGCATCATCACCAACTG-3′ Reverse 5′-GGCCACACGAAGCTCGTTAT-3′ TGFβ1 Forward 5′-GCAGTGGCTGAACCAAGGA-3′ Reverse 5′-AGAGCAGTGAGCGCTGAATC-3′ TGFβ2 Forward 5′-CAGCGCTACATCGATAGCAA-3′ Reverse 5′-CCTCGAGCTCTTCGCTTTTA-3′

[0070] The DDC-induced cholestatic liver disease mice also treated with β-lapachone were investigated for the transcriptional levels of collagen type I alpha 1 (Col1α1), collagen type IV alpha 1 (Col4α1), alpha-smooth muscle actin (α-SMA), fibronectin, transforming growth factor beta 1 (TGF-β1), and transforming growth factor beta 2 (TGF-β2), which are fibrosis-related genes associated with the progress of cholestatic liver disease, and compared with the vehicle-treated mice as the control group in view of expression levels, and the results are shown in FIG. 3. As shown in FIGS. 3A to 3F, similar to the results of the reductions in portal vein lesions and fibrosis, the transcriptional levels of Col1α1, Col4α1, α-SMA, fibronectin, and TGF-β1 were significantly reduced in the DCC-induced cholestatic liver disease mice (DDC-BL40 and DDC-BL80) treated with β-lapachone (BL) compared with the DCC-induced cholestatic liver disease mice (DDC-vehicle) treated with a vehicle. These results in the DDC-induced cholestatic liver disease mice indicate that the reductions in portal vein lesions and fibrosis were made at the level of fibrosis-related gene expression, and pathological changes during the occurrence of cholestatic liver disease were inhibited by the pre-treatment with β-lapachone (BL).

Example 3: Identification of Effect of β-Lapachone in Cholestatic Liver Disease Mouse Animal Models

[0071] To investigate the effect of β-lapachone (BL) on the occurrence of cholestatic liver disease, both 1% DDC and β-lapachone were administered to mice to characterize animal models.

[0072] 3.1. Identification of Effect on Blood Indexes (ALT, AST, ALP, and Bilirubin)

[0073] After 8-week-old C57BL/6 male mice (Samtako, Korea) were divided into each group (=6), the mice were fed a diet with normal (vehicle), control (DDC), DDC-+β-lapachone 20 mg/kg, DDC+β-lapachone 40 mg/kg, DDC+β-lapachone 80 mg/kg, DDC+β-lapachone 100 mg/kg, and positive control DDC+ursodeoxycholic acid 100 mg/kg, and DDC+obeticholic acid 30 mg/kg (total 7 days). After 7 days of treatment, the mice were sacrificed and then the blood was collected from the heart. Thereafter, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and bilirubin, which are blood indexes, were analyzed, and shown in FIG. 4.

[0074] As shown in FIG. 4, the DDC treatment significantly increased the levels of ALT, AST, ALP, and bilirubin in the blood, and β-lapachone (BL) inhibited ALT, AST, ALP, and bilirubin, increased by the DDC treatment, depending on the concentration thereof, and showed excellent effects compared with ursodeoxycholic acid (UDCA) and obeticholic acid (OCA).

[0075] 3.2. Identification of Effect on Transcriptional Levels of Fibrosis-Related Genes

[0076] After the cholestatic liver disease mouse animal models in Example 3.1 were sacrificed and the liver tissue was harvested therefrom, the mRNA levels of fibrosis-related genes were measured by a method similar to the method in Example 2.3.

[0077] In the cholestatic liver disease mouse models, the bile ducts were damaged and fibrosis was induced, by DDC, and accordingly, the transcriptional levels of collagen type I alpha 1 (Col1α1), transforming growth factor beta 1 (TGF-β1), and collagen type I alpha 2 (Col1α2), which are important factors in the development of fibrosis, were investigated, and the results are shown in FIG. 5.

[0078] As shown in FIG. 5, DDC significantly increased the transcriptional levels of Col1α1, TGF-β1, and Col1α2, which are genes associated with fibrosis, and β-lapachone (BL) inhibited the transcriptional levels of Col1α1, TGF-β1, and Col1α2 depending on the concentration thereof.

[0079] 3.3. Identification of Effect on Transcriptional Levels of Inflammation-Related Genes

[0080] In the cholestatic liver disease, the inflammation in the portal vein and damage to the bile ducts in the liver occur chronically, resulting in cholestasis and liver fibrosis. The mRNA levels of inflammatory cytokine genes in the liver tissue of the cholestatic liver disease mouse animal models obtained by the same method as in Example 3.1 were measured, and shown in FIG. 6. The primer sequences used for gene expression analysis are shown in Table 2 below.

TABLE-US-00002 TABLE 2 Gene Primer name Category Sequence TNFα Forward 5′-CTGAGGTCAATCTGCCCAAGTAC-3′ Reverse 5′-CTTCACAGAGCAATGACTCCAAAG-3′ IL-1β Forward 5′-GAAAGCTCTCCACCTCAATGG-3′ Reverse 5′-AGGCCACAGGTATTTTGTCGT-3′

[0081] As shown in FIG. 6, DDC significantly increased the transcriptional levels of TNFα and IL-1β, which are inflammation-related genes, and β-lapachone (BL) inhibited the transcriptional levels of TNFα and IL-1β.

Example 4: Identification of Treatment Effect of β-Lapachone in Cholestatic Liver Disease-Induced Mouse Animal Models

[0082] The treatment effect of β-lapachone in cholestatic liver disease-induced mouse animals were investigated. After 8-week-old C57BL/6 male mice (Samtako, Korea) were divided into each group (=6), each group excluding a control group was fed a diet containing 0.1% DDC for 3 days. Thereafter, the mice were fed a diet with DDC control (vehicle), DDC-+β-lapachone 20 mg/kg, DDC-+β-lapachone 40 mg/kg, DDC-+β-lapachone 80 mg/kg, and positive control DDC+obeticholic acid 30 mg/kg for 4 days (total 7 days). After 7 days of feeding, the mice were sacrificed and then the blood was collected from the heart. Thereafter, the levels of bilirubin and ALP, which are blood indexes, were analyzed, and shown in FIG. 7.

[0083] As shown in FIG. 7, the DDC treatment significantly increased the levels of bilirubin and ALP in the blood, and β-lapachone inhibited bilirubin and ALP, increased by the DDC treatment, depending on the concentration thereof. In addition, β-lapachone showed an excellent effect compared with ursodeoxycholic acid (UDCA) and obeticholic acid (OCA).

Example 5: Identification of Treatment Effect of β-Lapachone in Primary Biliary Cirrhosis (PBC)-Induced Mouse Animal Models

[0084] Of cholestatic liver diseases, primary biliary cirrhosis (PBC) may be introduced to a similar state by using a viral RNA mimic and the Toll-like receptor polyinosinic-polycytidylic acid (Poly I:C).

[0085] After 8-week-old C57BL/6 male mice (Samtako, Korea) were divided into each group (=6), each experimental group excluding a normal group was intraperitoneally administered 5 mg/kg Poly I:C twice a week for 8 weeks. Thereafter, the mice were fed a diet with control (vehicle), β-lapachone 40 mg/kg, β-lapachone 80 mg/kg, and positive control ursodeoxycholic acid 100 mg/kg and obeticholic acid 30 mg/kg for 8 weeks. On the 16th week of treatment, the experiment was ended, and the mice were sacrificed and then the blood was collected from the heart. Thereafter, the levels of the blood indexes AST, ALT, and ALP were analyzed, and shown in FIG. 8.

[0086] As shown in FIG. 8, the levels of AST, ALT, and ALP in the blood were increased by Poly I:C, and β-lapachone (BL) inhibited AST, ALT, and ALP, increased by Poly I:C. UDCA among the controls inhibited AST, ALT, and ALP increased by DDC, but β-lapachone (BL) showed an excellent effect compared with the comparative substances ursodeoxycholic acid (UDCA) and obeticholic acid (OCA).

Example 6: Identification of Treatment Effect of β-Lapachone in Inflammatory Bowel Disease-Induced Mouse Animal Models

[0087] Approximately 80% of cholestatic liver disease patients suffer from inflammatory bowel disease (IBD), and thus the present inventors identified the effect of β-lapachone (BL) in inflammatory bowel disease mouse animal models.

[0088] 6.1. Effects on Survival Rate, Body Weight, Colitis Score

[0089] Dextran sodium sulfate (DSS)-induced acute colitis mouse models were constructed using 8-week-old C57BL/6 female mice (Samtako, Korea).

[0090] After 8-week-old C57BL/6 female mice (Samtako, Korea) were divided into each group (=10), each experimental group was fed a standard rodent diet supplemented with 3% (w/w) DSS in drinking water for the first 5 days to induce acute colitis and orally administered control (vehicle), β-lapachone 40 mg/kg, and β-lapachone 80 mg/kg for 14 days.

[0091] All animal-related procedures were discussed and approved by the Institutional Animal Care and Use Committee of the Hanyang University. The survival rate over time is shown in FIG. 9, and the change rate in body weight of mice in each experimental group was measured and shown in FIG. 10A. The fecal occult bleeding per rectum, total bleeding loss, and fecal concentration were measured daily. As for the colitis score of each experimental group, the body weight reduction, bloody feces, firmness of feces, and the like were measured by two skilled researchers unaware of the treatment group, and convert to score as shown in Table 3, and the sum thereof was calculated and shown in FIG. 10B.

TABLE-US-00003 TABLE 3 Body weight Score reduction Feces condition Blood feces 0 No body weight Well-formed granular form No bleeding reduction 1  1-5% reduction 2  5-10% reduction Paste form that is not Partial bleeding attached to the anus or semi- formed granular form 3 10-20% reduction 4 20% or more Liquid form that is attached Overall bleeding reduction to the anus

[0092] As shown in FIG. 9, 70% died in the DSS-induced IBD mice, and 50% died in the β-lapachone 40 mg/kg experimental group, and only 20% died in the β-lapachone 80 mg/kg experimental group on the 14th day of the experiment. As shown in FIG. 10A, the body weight of the DSS-induced IBD mice was reduced, and reduced to 75% relative to baseline on Day 8 of the initiation of the experiment. In the groups treated with β-lapachone (BL), the body weight reduction by DSS was inhibited, and especially, the β-lapachone 80 mg/kg experimental group was recovered up to 90% of the initial body weight. As shown in FIG. 10B, the colitis score of the DSS-induced IBD mice was reduced depending on the treatment concentration of β-lapachone (BL).

[0093] 6.2. Effects on Colon Length Recovery and Inflammation Inhibition

[0094] By the same method as in Example 6.1, experimental groups were divided into a DSS untreated group (Control), a DSS treated group, and a DSS-+β-lapachone 80 mg/kg treated group, respectively, and treated. After the end of the experiment, the mice were sacrificed to remove the colon, and the accurate colon length was measured using a Vernier caliper, and is shown FIGS. 11A and 11B. In addition, the mRNA levels of genes of interleukin-1beta (IL-1β), interleukin-6 (IL-6), interleukin-18 (IL-18), interferon-γ (INF-γ), tumor necrosis factor-α (TNF-α), tumor necrosis factor-β (TNF-β), and monocyte chemoattractant protein-1 (MCP-1), which were inflammatory cytokine factors in the colon tissue, were analyzed by a real-time polymerase chain reaction through a conventional experiment procedure, and are shown in FIG. 12.

[0095] As shown in FIGS. 11A and 11B, the colon length of the mice was significantly shortened by DSS, and was recovered by the β-lapachone (BL) treatment.

[0096] The inhibition of inflammation may include an inhibition of inflammatory cytokines and/or an inhibition of cytokine gene expression. In this regard, as shown in FIG. 12, the mRNA levels of IL-1β, IL-6, IL-18, INF-γ, TNF-α, TNF-β, and MCP-1, which are inflammatory factors in the colon tissues of mice, were significantly increased, and each of the inflammatory cytokines, increased by DSS, was significantly reduced by the β-lapachone treatment.

Example 7: Anti-Fibrotic Effect of β-Lapachone at Cell Level

[0097] 7.1. Effect of β-Lapachone on Fibronectin and α-SMA Activity in LX-2 Hepatic Stellate Cell Line Models

[0098] Considering that inflammation and fibrosis of bile ducts cause cirrhosis in cholestatic liver disease, experiments were performed on the LX-2 hepatic stellate cell line model by using the method of Takaaki Higashi et al. (2017). The LX-2 cell line was subcultured in DMEM containing 10% FBS, 2 mM glutamine, 100 U penicillin, and 0.1 mg/ml streptomycin. The LX-2 cell line was added into a 6-well plate at a density of 16×10.sup.4 cells/well, and cultured in DMEM containing 1 ng/ml TGF-β1, 1 ng/ml TGF-β1+0.5 μM β-lapachone or 1 ng/ml TGF-β1+1 μM β-lapachone for 24 hours. Thereafter, the protein expression levels of the fibrosis-related genes fibronectin and α-SMA of the obtained cells were measured by protein immunoblotting according to a conventional method known in the art.

[0099] As shown in FIG. 13, when the control group LX-2 cell line was treated with TGF-β1, the protein expression levels of fibronectin and α-smooth muscle actin (α-SMA) associated with fibrosis were increased compared with the control group, but in the case of the administration of β-lapachone, the levels of fibronectin and α-SMA increased by TGF-β1 were reduced depending on the concentration of β-lapachone.

[0100] 7.2. Effect of β-Lapachone on Inflammatory Cytokine Protein Expression in Macrophage Line Raw264.7 Cell Model

[0101] The macrophage line Raw264.7 (ATCC TIB-71, Manassas, Va., USA) was subcultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin.

[0102] For inflammation induction, RAW264.7 cells were suspended in DMEM containing 2% FBS, inoculated in a 12-well plate to a cell number of 4×10.sup.5/ml, and incubated in 5% CO.sub.2 incubator at 37° C. for 24 hours. With replacement with fresh media, the cells were treated with appropriate concentrations of a corresponding substance, LPS (100 ng/ml), and 0.2, 0.5, 1, and 2 μM β-lapachone, cultured for 24 hours, treated with ATP (2.5 mM), and then cultured for additional 30 minutes, and the supernatant was collected. The release of cytokines (IL-1β and TNF-α) of RaW264.7 macrophages was quantified using each cell-free supernatant-ELISA set (Invitrogen, Billerica, Mass., USA) by Invitrogen according to the manufacture's instruction, and is shown in FIG. 14.

[0103] As shown in FIG. 14, when the Raw264.7 cell line was treated with LPS and ATP, the expression of inflammation-related proteins, IL-1β and TNF-α, was increased compared with the control group, but in cases of the administration of β-lapachone (BL), the expression levels of IL-1β and TNF-α protein, increased by LPS and ATP, was reduced depending on the concentration of β-lapachone.

[0104] 7.3. Effect of β-Lapachone on Secretion of Inflammatory Cytokines 1L-1β and IL-18 in Peripheral Blood Mononuclear Cells (PMBCs)

[0105] Considering that inflammatory cytokines mediate the pathogenic mechanism of cholestatic liver disease, the peripheral blood mononuclear cell model was used by a modified method of Boyum et al. (1968). Peripheral blood mononuclear cells isolated from healthy 8-week-old C57BL/6 male mice (Samtako, Korea) were maintained by primary culture in RPMI-1640 medium containing 10% FBS, 2 mM glutamine, 100 U penicillin, and 0.1 mg/ml streptomycin. Stable peripheral blood mononuclear cells (PBMCs) were treated with LPS at 200 ng/mL and ATP and β-lapachone at 0, 0.5, 1, 2 μM in the medium and, after 4 hours, the expression levels of IL-1β and IL-18 were investigated, and the results are shown in FIG. 15.

[0106] As shown in 15, when the Peripheral blood mononuclear cells were treated with LPS and ATP, the expression levels of inflammation-related proteins, IL-1β and IL-18, were increased compared with the control group, but in cases of the administration of β-lapachone (BL), the expression levels of IL-1β and IL-18, increased by LPS and ATP, were reduced depending on the concentration of β-lapachone (BL).