Use of terpenoids in the treatment or prevention of fibrotic diseases

Abstract

The present invention relates to a method for preventing or treating a fibrotic condition, comprising administering to a subject in need thereof an effective amount of a composition, wherein the composition comprises triterpenes extracted from Antrodia camphorate or Anisomeles indica.

Description

[Technical field]

[0001] Fibroproliferative diseases are a burden to many people and are typical pathological sequelae of many persistent inflammatory diseases such as pulmonary fibrosis, progressive kidney disease, liver cirrhosis, atherosclerosis and benign prostatic hyperplasia. [Background technology]

[0002] Fibroproliferative diseases are a growing problem for many people and are common pathological sequelae of many persistent inflammatory diseases such as pulmonary fibrosis, progressive kidney disease, liver cirrhosis, atherosclerosis and prostatic hyperplasia.

[0003] Impaired renal repair after acute kidney injury induces fibrosis, which can ultimately lead to chronic kidney disease. Kidney injury repairs tissue by activating multipotent progenitor cells. However, as injury persists, these multipotent progenitor cells become dysfunctional and induce fibrotic repair, resulting in renal fibrosis. The pathogenesis of renal fibrosis is progressive, ultimately leading to end-stage renal failure, a devastating disorder requiring dialysis or kidney transplantation.

[0004] Nonalcoholic fatty liver disease (NAFLD) is a representative chronic liver disease and a disease with high unmet medical needs. Nonalcoholic steatohepatitis (NASH), a progressive variant of NAFLD, can lead to fibrosis, cirrhosis, and hepatocellular carcinoma. NAFLD and NASH are diseases that have become the focus of attention of the medical community in general, especially due to the increasing epidemic of diabetes and obesity worldwide. In the clinical evaluation of patients with abnormal aminotransferase levels, nonalcoholic fatty liver and its spectrum should be considered, especially in the case of obesity and diabetes. The prognosis of simple NAFLD is generally benign, but the presence of fibrosis, hepatocyte ballooning, inflammation, and Mallory bodies poses a risk of progression to cirrhosis.

[0005] Autoimmune hepatitis (AIH) is a chronic liver disease without a clear etiology, but may be characterized by hepatocellular inflammation. Severe AIH may progress to cirrhosis, hepatocellular carcinoma, and death. Cirrhosis develops in 40% of treated autoimmune hepatitis patients, depending on the duration of observation. Antifibrotic therapies that complement the anti-inflammatory and immunosuppressive effects of current regimens are emerging, and these regimens are expected to redirect the therapeutic focus of autoimmune hepatitis toward preventing, stabilizing, and reversing liver fibrosis.

[0006] Atherosclerosis, one of the main causes of the development of cardiovascular disease, is associated with vascular fibrosis. Vascular fibrosis involves the accumulation of extracellular matrix (ECM) proteins, especially collagen and fibronectin, in the vascular media, contributing to structural remodeling and scar formation. A deficiency of elastin or excess of collagen in the vascular wall leads to vascular fibrosis and increased stiffness.

[0007] In benign prostatic hyperplasia, deposition of collagen fibers in the prostate gland replaces broken muscle fibers, resulting in stiffening and weakening of the muscle tissue and deposition of prostatic fluid within the gland ducts. Prostatic fibrosis plays a central role in the development of bladder outlet obstruction in older men.

[0008] Antrodia camphorata (AC), a medicinal fungus, is a well-known Chinese folk medicine that is known to have many biological activities, especially antitumor effects in cancer cells in vitro and in animal models in vivo. Due to its diverse bioactive compounds, it is considered as an effective alternative phytotherapeutic agent or adjuvant for cancer treatment and immune-related diseases. To date, a total of 225 compounds have been isolated, identified and structurally elucidated, including macromolecules (nucleic acids, proteins and polysaccharides), small molecules (benzenoids, lignans, benzoquinones and maleic / succinic acid derivatives), terpenoids (lanostane triterpenes, ergostane triterpenes, diterpenes, monoterpenes and steroids), nucleotides (nucleobases and nucleosides), fatty acids and fatty acid esters.

[0009] Accumulative in vitro and in vivo studies have revealed antidiabetic, antihyperlipidemic, antihypertensive, anti-inflammatory, antioxidant, antibacterial, cardiovascular disease preventive, immunomodulatory, hepatoprotective and neuroprotective effects. However, the efficacy of Antrodia camphorata and its components in the treatment of fibrosis has not been evaluated.

[0010] Anisomeles indica, commonly known as "Indian Catmint", is a source of medicamentously active compounds and has various pharmacological effects. The plant is traditionally used as an analgesic, anti-inflammatory agent, and for skin problems. Medicinally, it has been shown to have various pharmacological activities, including antioxidant, antibacterial, anti-HIV, anti-Helicobacter pylori, and anti-cancer activities. It is also used in chronic rheumatism. Further studies have revealed the presence of various phytochemical constituents, mainly triterpenes, β-sitosterol, stigmasterol, flavones, apigenin, and obatdiolide. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows the separation of anthocin K, dehydrosulfurenic acid / sulfurenic acid, versisponic acid D and dehydroebric acid from Antrodia camphorata extract.

[0012] [Diagram 2] Figure 2A-2C Protective effects of Antrodia camphorata extract and compounds against cisplatin-induced renal injury in AKI mice. To analyze the effects of Antrodia camphorata extract and compounds, mice were administered cisplatin daily for 7 days starting from 3 weeks after the first dose and sacrificed at the 4th week. Morphological changes of kidney (A). Blood urea nitrogen (BUN) levels (B). Serum creatinine (CRE) levels (C). Data are shown as mean ± SEM (n=5). ### indicates p<0.001 compared to the control group. **p<0.01 and ***p<0.001 compared to the cisplatin group.

[0013] [Diagram 3] Figure 3. Protective effects of Antrodia camphorata extract and compounds against cisplatin-induced kidney injury in AKI mice. To analyze the effects of Antrodia camphorata extract and compounds, mice were administered cisplatin daily for 7 days starting from 3 weeks after the first dose and sacrificed at the 4th week. Kidneys were stained with H&E. After cisplatin administration, kidneys from each group are prepared for histological evaluation. Representative tissue sections of kidneys were stained with H&E and magnification was ×400. Data are shown as mean ± SEM (n=5). ### indicates p<0.001 compared to control group. **p<0.01, ***p<0.001 compared to cisplatin group. Duct cell necrosis is indicated by arrows; bars indicate 50 μm.

[0014] [Figure 4]Figure 4A-4E Antrodia camphorata extracts and compounds regulated (A) TNF-α, (B) IL-1β, (C) IL-6, (D) TGF-β, and (E) albumin in serum. Serum levels of TNF-α, IL-1β, IL-6, TGF-β, and albumin were measured by commercial ELISA kits. Data are shown as mean ± SEM (n=5). ### indicates p<0.001 compared to control samples. **p<0.01 and ***p<0.001 compared to cisplatin alone group.

[0015] [Diagram 5] Figure 5A-5B Effects of ARH005-EA (A) and ARH (B) on cisplatin-induced TWEAK, α-SMA, P53 and P21 signaling expression in the kidney. Protein levels of TWEAK, α-SMA, P53 and P21 protein expression in kidney homogenates were assessed by Western blot analysis after cisplatin challenge.

[0016] [Figure 6] FIG. 6 shows the course of the CCl4-induced fibrosis model.

[0017] [Figure 7] 7A-7C show (A) delta weight, (B) liver weight, and (C) liver / body weight ratio, respectively.

[0018] [Figure 8] 8A-8C show serum levels of (A) AST, (B) ALT, and (C) AST / ALT in rats after CCl4-induced liver injury, respectively.

[0019] [Figure 9] 9A-9E show (A) inflammation, (B) vacuolization, (C) necrosis, (D) fibrosis, and (E) total histological score of the liver.

[0020] [Figure 10]Figure 10. Representative histological sections of the liver stained with H&E.

[0021] [Figure 11] FIG. 11 shows the course of Con A (concanavalin A)-induced acute hepatitis model.

[0022] [Figure 12] Figures 12A-12C show the process of obatodiolide (AR100-DS1) on GOT, GPT and body weight. (A) Serum GOT and (B) serum GPT 24 hours after 15 mg / kg Con A challenge. (C) Body weight before and after 15 mg / kg Con A challenge. Data shown as mean ± SEM (n=9). *p<0.05 compared to Veh by t-test. Veh, vehicle; Dex, dexamethasone.

[0023] [Figure 13] Figure 13. Effect of obatodiolide (AR100-DS1) on liver injury. (A) Naive, (B) 15 mg / kg Con A (Veh), (C) 2019-0321-1 and (D) dexamethasone and (E) histopathological scores of necrosis. Data shown as mean ± SEM (n=9). ***p<0.001 compared to Veh by Student's t-test. Veh is vehicle, Dex is dexamethasone.

[0024] [Figure 14] FIG. 14 shows the course of a rabbit model of atherosclerosis.

[0025] [Figure 15] Figure 15 shows the initial and final mean body weights of rabbits.† and * indicate P<0.05 compared with the control and HF groups, respectively.

[0026] [Figure 16]16A-16C show the changes in AST, ALT, and BUN between W0 groups in each rabbit group. † and * indicate P<0.05 compared with the control group and HF group, respectively.

[0027] [Figure 17] 17A-17D show the changes in TG, TC, HDL-C, and LDL-C between W0 groups in each rabbit group. † and * indicate P<0.05 compared with the control group and the HF group, respectively.

[0028] [Figure 18] 18A-18C show the changes in AST, ALT, and BUN between W4 groups in each rabbit group. † and * indicate P<0.05 compared with the control group and the HF group, respectively.

[0029] [Figure 19] 19A-19D show the changes in TG, TC, HDL-C, and LDL-C between W4 groups in each rabbit group. † and * indicate P<0.05 compared with the control group and the HF group, respectively.

[0030] [Figure 20] 20A-20C show the changes in AST, ALT, and BUN between W8 groups in each rabbit group. † and * indicate P<0.05 compared with the control group and HF group, respectively.

[0031] [Figure 21] 21A-21D show the changes in TG, TC, HDL-C, and LDL-C between W8 groups in each rabbit group. † and * indicate P<0.05 compared with the control group and the HF group, respectively.

[0032] [Figure 22] 22A-22C show the changes in AST, ALT, and BUN between W12 groups in each rabbit group. † and * indicate P<0.05 compared with the control group and the HF group, respectively.

[0033] [Figure 23] 23A-23D show the changes in TG, TC, HDL-C, and LDL-C between W12 groups in each rabbit group. † and * indicate P<0.05 compared with the control group and the HF group, respectively.

[0034] [Figure 24] FIG. 24 shows histopathological examination of aortic fatty streak lesions in a hypercholesterolemic rabbit model after a 12-week study.

[0035] [Diagram 25] FIG. 25 shows HE staining of coronary artery sections after sacrifice in each rabbit group.

[0036] [Figure 26] Figure 26 shows HE staining of the coronary arteries after sacrifice in each rabbit group, N: neointimal layer; M: medial layer.

[0037] [Figure 27] Figure 27 shows the signs of vascular restenosis, expressed as the ratio of neointima area to media area (N / M ratio). N, neointima layer; M, media layer. *p<0.05, **p<0.01, ***p<0.001, respectively, compared with the HFD group.

[0038] [Figure 28] FIG. 28 shows histopathological examination of cardiac tissue in a hypercholesterolemic rabbit model after a 12-week study.

[0039] [Figure 29] FIG. 29 shows photographs of the liver appearance in a hypercholesterolemic rabbit model after a 12 week study.

[0040] [Diagram 30] FIG. 30 shows histopathological examination of liver tissue in a hypercholesterolemic rabbit model after a 12-week study.

[0041] [Diagram 31] FIG. 31 shows the body weight and lung weight of the animals.

[0042] [Diagram 32] FIG. 32 shows histopathological changes in the lungs during bleomycin-induced pulmonary fibrosis in mice.

[0043] [Diagram 33] FIG. 33 shows Masson's Trichrome staining of lungs in bleomycin-induced pulmonary fibrosis in mice.

[0044] [Diagram 34] FIG. 34 shows the effect of A. camphorata extracts and compounds on hydroxyproline content in bleomycin-induced lung injury in mice.

[0045] [Diagram 35] 35A-35D show that A. camphorata extracts and compounds modulated (A) TNF-α, (B) IL-1β, (C) IL-6 (D) and TGF-β (E) in BALF.

[0046] [Diagram 36] FIG. 36 shows the modulatory effects of A. camphorata extracts and compounds on pulmonary MPO activity in BLM-induced mice.

[0047] [Figure 37] FIG. 37 shows that HepG2 cells were treated with different concentrations of oleic acid (OA) to induce steatosis.

[0048] [Figure 38] 38A-38B show 24-h OA-induced steatosis in HepG2 cells.

[0049] [Figure 39] FIG. 39 shows the cytotoxicity study of compounds (3.7 μg / mL, 11.1 μg / mL, 33.3 μg / mL, 100 μg / mL and 200 μg / mL) in HepG2 cells.

[0050] [Diagram 40] FIG. 40 shows the anti-steatotic test of compounds in HepG2 cells.

[0051] Detailed Description of the Invention For the convenience of explaining the present invention, the central ideas expressed in the above summary of the invention will be explained by concrete examples. Various items in the embodiments are shown in ratios, dimensions, deformation amounts or displacements suitable for illustration, and do not mean the ratios of the actual elements as described above.

[0052] The term "terpene" refers to a large and diverse group of organic compounds whose basic structure follows a general principle: a 2-methylbutane residue (usually, but not precisely, an isoprene unit, (C5) n The carbon skeleton of terpenes is made up of C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21, C22, C30, C41, C52, C63, C74, C85, C96, C97, C98, C99, C101, C112, C123, C134, C146, C152, C165, C176, C186, C197, C198, C199, C201, C213, C224, C236, C248, C256, C368, C370, C371, C372, C373, C374, C375, C481, C492, C 10 ), Sesqui-(C 15 ), Ji-(C 20 ), sester-(C 25 ), Tri-(C 30 ) and tetraterpenes (C 40 ) are distinguished. [ka]

[0053] The terms "subject", "individual", "host" and "patient" are used interchangeably herein and refer to living animals, including humans and non-human animals. A subject may be, for example, an organism having immune cells capable of responding to antigenic stimulation and transmitting stimulatory and inhibitory signals via cell surface receptor binding. A subject may be a mammal. Human or non-human mammals include, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats and mice. The term "subject" does not exclude individuals who are completely normal or normal in all respects with respect to a disease.

[0054] The term "treatment" refers to a therapeutic or prophylactic measure that may be administered to a subject having a medical disorder or a subject who may ultimately acquire the disorder in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or a recurring disorder, or to prolong the survival of the subject beyond that which would be expected in the absence of such treatment.

[0055] By "therapeutically effective amount" is meant an amount of the subject compound that may elicit a desired response, e.g., a biological or medical response in a tissue, system, animal or human, as desired by a researcher, veterinarian, physician, or other clinician.

[0056] Measurement of biochemical parameters: Serum creatinine and serum urea are assessed using colorimetric kits according to the manufacturer's instructions. The kit for the former marker is purchased from (HUMAN Diagnostics Worldwide, Magdeburg, Germany) and a chemistry analyzer (Roche Diagnostics, Cobas Mira Plus, Rotkreuz, Switzerland).

[0057] Renal histopathology: The anterior portion of the left hepatic lobe from each mouse was fixed in 10% formaldehyde phosphate buffer, embedded in paraffin, cut into 5 μm sections, stained with hematoxylin and eosin (H&E), and examined histologically under a light microscope (Nikon, ECLIPSE, TS100, Tokyo, Japan). Images were taken with a digital camera (NIS- Elements D 2.30, SP4, Build 387) at a magnification of 400x.

[0058] TNF-α, IL-6, and IL-1β cytokines in serum: Serum concentrations of proinflammatory cytokines (i.e., tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β) will be assessed with the relevant enzyme-linked immunosorbent assay (ELISA) kits (Biosource International Inc., Sunnyvale, CA, USA) according to the manufacturer's instructions.

[0059] Western blot analysis of kidney tissue: Liver tissue is homogenized at 4°C using a lysis buffer consisting of 0.6% NP-40, 150 mM NaCl, 10 mM HEPES (pH 7.9), 1 mM EDTA, and 0.5 mM PMSF. The homogenized samples are centrifuged at 3000 revolutions per minute (rpm) for 10 min at 4°C to obtain the supernatant. Equal total cellular protein amounts in the supernatant are measured using a protein standard of bovine serum albumin (BSA). Proteins (50 μg) were resolved by denaturing 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using standard methods, followed by transfer to a PVDF membrane (Immobilon, Millipore, Bedford, MA, USA) for electroblotting and blocking with 10% nonfat milk. The membranes were incubated with appropriate dilutions of specific primary antibodies at 4°C, washed three times with TBS / Tween (TBST) buffer, and then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at 37°C (overnight). The membranes were washed three times before probing for immunoreactive proteins with enhanced chemiluminescence (ECL) reagents (Thermo Scientific, Hudson, NH, USA). Band intensities of scanned membranes were quantified and presented as relative intensities compared to controls using Image J software (NIH, Bethesda, MD, USA).

[0060] Statistical analysis: Data from animal studies are presented as mean and standard error of the mean (±SEM). Student's t-test was used to examine differences between multiple or two groups. Statistical significance is indicated as *p<0.05, **p<0.01 and ***p<0.001.

[0061] Example 1: Preparation of Antrodia camphorata extract

[0062] 100 g of the fruiting body of Antrodia camphorata is refluxed in methanol for 6 hours, and the extract is collected and dried to obtain a total of 15 g of methanol extract of Antrodia camphorata.

[0063] Example 2: Preparation of active ingredients: Anthocin K, dehydrosulfurenic acid / sulfurenic acid, versisponic acid D and dehydroebric acid

[0064] The methanol extract of Antrodia camphorata was further separated by silica column chromatography using n-hexane / ethyl acetate / methanol as eluent to obtain the following fractions (see Figure 1): ARH101-DS1 (RS-Anthosin K), ARH101-DS2 (dehydrosulfurenic acid / sulfurenic acid), ARH101-DS3 (versiponic acid D) and ARH101-DS4 (dehydroevulinic acid). [ka]

[0065] Example 3: Preparation of AR003 extract

[0066] 100 grams of Antrodia camphorata (petri dish culture) is refluxed with methanol for 6 hours, and the extract is collected and dried under reduced pressure to obtain 15 grams of Antrodia camphorate ARH003 extract.

[0067] Example 4: Preparation of AR003-E extract

[0068] 200 g of Antrodia camphorata (petri dish culture) is refluxed with ethanol for 6 hours, and the extract is collected and dried to obtain a total of 18 g of ethanol extract of AR003-E Antrodia camphorata.

[0069] Example 5: Preparation of AR004 extract

[0070] 100 g of Antrodia camphorata (wood culture) is refluxed with methanol for 6 hours, and the extract is collected and dried under reduced pressure to obtain Antrodia camphorate ARH004 extract.

[0071] Example 6: Preparation of AR005-EA extract

[0072] 100 g of Antrodia camphorata (solid culture) is refluxed with ethyl acetate for 6 hours, and the extract is collected and dried to obtain a total of 12 g of EA extract of Antrodia camphorata.

[0073] Example 7: Preparation of Anisomeles indica Extract

[0074] The Anisomeles indica extract is prepared by the following process: (1) An ethanol extract of Anisomeles indica is taken, added to a chromatographic column packed with silica, and gradient elution with the eluents "n-hexane / ethyl acetate", "hexane / ethyl acetate / methanol" and "methanol" is performed to obtain an Anisomeles indica isolate. (2) The Anisomeles indica isolate is separated using a chromatographic column packed with silica, and gradient elution with the eluents "dichloromethane", "dichloromethane / methanol" and "methanol" is performed to obtain a separated concentrate. (3) The separated concentrate is recrystallized with the solvent "n-hexane / ethyl acetate" to obtain Anisomeles indica microcrystals.

[0075] Example 8: Preparation of the active ingredient: Obatodiolide (AR100-DS1)

[0076] 200 g of the ethanol extract of Anisomeles indica was taken and added to a silica-filled chromatography column (10 x 15 cm). Gradient elution was performed with 1200 ml of each eluent: n-hexane / ethyl acetate (ratios of 10 / 1, 5 / 1, 3 / 1, 1 / 1), hexane / ethyl acetate / methanol (ratios of 6 / 4 / 1, 3 / 2 / 1), and methanol, to obtain 140 g of the initial isolated solution.

[0077] 140 g of the first isolated solution is separated using a silica-filled chromatographic column (10 x 15 cm), and a gradient elution is performed with 1000 ml of each eluent: "dichloromethane", "dichloromethane / methanol (10 / 1, 5 / 1, 7 / 3 ratio)" and "methanol" to obtain a separated concentrated substance. The separated concentrated substance is further recrystallized using a solvent: "n-hexane / ethyl acetate" to obtain a crystal. The crystal is identified as a diterpenoid compound with the chemical structure of obatodiolide by nuclear magnetic resonance spectroscopy (H1-NMR). The crystal is compared with a standard product of obatodiolide by high performance liquid chromatography (HPLC) analysis, and confirmed to be an obatodiolide compound. [ka]

[0078] Metabolites from Obatodiolide (AR100-DS1): +O, +Cysteine: m / z: 466, M2, M3, M4 + Glutathione: m / z: 636, M6, M7 +O:m / z:345, M8, M9

[0079] [Table 1-1] [Table 1-2] [Table 1-3]

[0080] Example 9: Mouse model of cisplatin-induced kidney injury

[0081] Male C57BL / 6 mice aged 7–8 weeks were obtained from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan). Animals are housed in Plexiglas cages with a 12-h dark-light cycle at a constant temperature of 22 ± 1 °C and relative humidity of 55 ± 5% for at least 2 weeks prior to the experiment. Animals are provided with food and water ad libitum. All experimental procedures were performed in accordance with the guidelines of the Institutional Animal Ethics Committee, and the protocols have been approved by the Committee for the Management and Supervision of Animal Experiments.

[0082] Renal fibrosis is induced by multiple injections of low doses of cisplatin. Cisplatin (5 mg / kg / injection; P4394, Sigma-Aldrich, St Louis, MO) is injected intraperitoneally three times at weeks 0, 1, and 3. Mice are sacrificed 6 weeks after the first injection of cisplatin (n=6). To analyze the effects of the samples, mice are injected intraperitoneally every day for 7 days starting 4 weeks after the first injection of cisplatin and sacrificed at 4 weeks (n=6).

[0083] Example 10: Antrodia camphorata extracts and compounds attenuated renal dysfunction and histopathological changes in cisplatin-induced mice.

[0084] Morphological changes in the kidney are shown in Figure 2A. CRE and BUN are characteristic of renal function. Figures 2B and 2C show that cisplatin injection at three 10 mg / kg CP doses (weeks 0, 1, and 3) highly elevated serum CRE and BUN levels (p<0.001) compared to the control group, indicating the occurrence of nephrotoxicity in cisplatin-treated mice. Treatment with ARH005-EA and ARH003-E at a dose of 1000 mg / kg and compounds (AR101-DS4 and AR100-DS1) exerted significant renal protective effects in a dose-dependent manner compared to the cisplatin-stimulated group, as demonstrated by the normalization of CRE and BUN (p<0.001).

[0085] Example 11: Antrodia camphorata extracts and compounds reduce renal dysfunction and damage caused by multiple cisplatin treatments.

[0086] Histopathological changes were analyzed to determine whether Antrodia camphorata extract and compound affected renal failure in cisplatin-stimulated mice. The kidney tissue of the control group was completely normal, characterized by clear tubular and glomerular structures with clear and normal nuclei. The kidneys of cisplatin-stimulated mice had severe renal damage, inducing tubular epithelial damage, inflammatory cell infiltration, tubular cell swelling, intratubular cast formation, and tubular dilation. However, treatment with Antrodia camphorata extract and compound (AR100-DS1) at a dose of 1000 mg / kg significantly improved the necrosis and inflammatory infiltrating cells in the kidney tissue (see Figure 3).

[0087] Example 12: Antrodia camphorata extracts and compounds counteract cisplatin-induced changes in pro-inflammatory cytokines and albumin.

[0088] Evaluation of pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and TGF-β levels in serum is performed by ELISA. Cisplatin-treated kidney injury mice showed significantly increased NO, TNF-α, IL-1β and IL-6 levels in serum compared to the control group (Figure 4A-4E, respectively). Treatment with Antrodia camphorata extract (AR005-EA) and compound (AR100-DS1) at a dose of 1000 mg / kg significantly improved necrosis and inflammatory infiltrating cells in kidney tissue processing, and improved NO, TNF-α, IL-1β and IL-6 production after cisplatin challenge.

[0089] Example 13: Inhibition of Cisplatin-induced Kidney Injury TWEAK, α-SMA, P53 and P21 Protein Expression

[0090] We investigated whether pretreatment with Antrodia camphorata extract (ARH005-EA) and compound (AR100-DS1) inhibited cisplatin-induced TWEAK, α-SMA, P53 and P21 protein expression. The results revealed that treatment with ARH005-EA and ARH inhibited the expression of TWEAK, α-SMA, P53 and P21 proteins in kidney tissues after cisplatin challenge (Figures 5A and 5B).

[0091] Example 14: CCl4-induced chronic liver fibrosis in rats

[0092] As shown in Figure 6, 8-week-old male SD rats are administered 0.4 mg / kg of CCl4 twice a week for 8 weeks. Blood samples are taken at weeks 0, 2, 4, 6 and 8. The animals are sacrificed at the end of 8 weeks for histopathological examination. Figures 7A, 7B, and 7C show the delta weight, liver weight, and liver / body weight ratio, respectively. The liver weight of the naive group is not significantly different from that of the vehicle group. However, the liver / body weight ratio of the naive group is significantly smaller than that of the vehicle group. The liver weight and liver / body weight ratio of the 50 mg / kg AR100-DS1 group are significantly larger compared to the vehicle and naive groups.

[0093] Example 15: Serum Liver Enzyme Profiles

[0094] The levels of clinical biochemistry, such as aminotransferase (AST) and alanine aminotransferase (ALT), are evaluated to determine the enzyme activities in the liver of the control and experimental groups (shown in Figures 8A-8C). The levels of AST, ALT and the AST / ALT ratio of the naive group showed no significant changes during the experiment. The serum AST and ALT levels of animals in each test group increased significantly as the experiment progressed. However, a smaller increase in AST and ALT could be observed at W6 and W8 in the 50 mg / kg AR100-DS1 group compared to the vehicle group.

[0095] Example 16: Liver histological evaluation

[0096] Eight weeks after induction with CCl4, the vehicle group suffered significant liver damage, including elevated AST and ALT, decreased AST / ALT ratio, inflammation, fibrosis, vacuolization and necrosis. As shown in Figures 9A-9E and 10, the livers of the 50 mg / kg AR100-DS1 group had a smooth surface without atrophy and hardening, and the liver weight and liver / body weight ratio were significantly larger than those of the vehicle and naive groups. Overall, these results indicate that AR100-DS1 may partially repair CCl4-induced liver injury.

[0097] Example 17: Effect of obatodiolide (AR100-DS1) on Con A (concanavalin A)-induced acute hepatitis in BALB / c mice

[0098] Intravenous injection of concanavalin A (Con A) is a widely used strategy to study T cell-mediated hepatitis. Con A is a lectin that can activate CD4+ T cells, produce cytokines, and cause hepatocyte damage. Dexamethasone (Dex) is a long-acting synthetic corticosteroid that has been used as an anti-inflammatory and immunosuppressant drug. To evaluate the effects of obatdiolide (AR100-DS1) on serum glutamic pyruvic transaminase (GOT), glutamic oxaloacetic transaminase (GPT), circulating cytokines, and liver histopathology in Con A-induced acute hepatitis in BALB / c mice.

[0099] Con A and Dex were purchased from Sigma Aldrich (USA). TM Immunoassay kits were purchased from Corning Inc. GOP and GPT Fuji Dri-Chem slides were purchased from Winning Medical Inc. (Taiwan).

[0100] Male BALB / c mice (7–9 weeks old) were purchased from BioLASCO Taiwan Co., Ltd. or the National Laboratory Animal Center (NLAC, Taiwan). Animals were housed five per cage and provided with food and water ad libitum during the experiment. Room temperature was kept at 23 ± 2 °C with alternating 12-h light-dark cycles. Animals were allowed to acclimate for 1 week before the experiment to minimize the effects of stress. All experimental protocols involving animals and their care were approved by ITRI's Institutional Animal Care and Use Committee (IACUC) (ITRI-IACUC-2018-041 and ITRI-IACUC-2018-050; accredited by AAALAC) and conducted in accordance with the regulations of the Taiwan Agriculture Council.

[0101] Con A was dissolved in pyrogen-free saline at a concentration of 3 mg / mL and intravenously injected at 15 or 20 mg / kg body weight to induce hepatitis. Obatodiolide (AR100-DS1) and Dex were orally administered 30 min before, 4 h, and 8 h after Con A administration. Blood and liver tissues were collected 24 h after Con A treatment (Figure 11). Serum was stored at 80°C until analysis.

[0102] To assess the level of hepatocellular injury after Con A treatment, serum GPT and GOT levels are measured using Fuji Dri-Chem slides (Fuji, Japan). Sera from the same group are pooled for cytokine assays. Cytokine levels are measured using ProcartaPlex TM Measured by immunoassay kit according to manufacturer's instructions. Data are presented as mean ± SEM. T-test is used to analyze the differences between drug and vehicle treated groups. Differences are considered statistically significant when p-value is less than 0.05. Obatodiolide (AR100-DS1) at 50 mg / kg significantly reduced the GPT value elevated by Con A (109 ± 25 U / L vs 368 ± 107 U / L, p < 0.05) and slightly improved the elevation of GOT (261 ± 45 U / L vs 410 ± 56 U / L) (Figure 12).

[0103] Liver tissues are fixed in 10% phosphate-buffered formaldehyde, embedded in paraffin, and stained with hematoxylin-eosin (H&E) to confirm histopathology. Histopathology is examined under a microscope by veterinary pathologists at BioLASCO Taiwan Co., Ltd. All microscopic lesions are graded from 0 to 4 according to the severity grading system criteria as follows: 0=none, 1=individual cell necrosis, 2=≦30% lobular necrosis, 3=≦60% lobular necrosis, 4=>60% lobular necrosis. In histopathological analysis, obatodiolide (AR100-DS1) improved hepatic necrosis (score 0.2±0.2 vs 1.4±0.2, p<0.05) (Figure 13). The results indicate that obatodiolide (AR100-DS1) reduced serum GOP and GPT and attenuated Con A-induced hepatic necrosis.

[0104] Example 18: Evaluation of the preventive effects of Antrodia camphorata extract and AR101-DS2 on atherosclerosis and liver fibrosis

[0105] Experimental model

[0106] Male New Zealand white rabbits weighing 2-3 kg are individually caged and kept in a temperature- and humidity-controlled room. The light / dark cycle is 12 hours each. After several days of acclimation, the animals are sequentially assigned to six feeding groups: standard rabbit chow, standard rabbit chow containing 0.5% cholesterol, standard rabbit chow containing both 0.5% cholesterol and 10 mg / kg lovastatin, standard rabbit chow containing both 0.5% cholesterol and 1% ARH003, standard rabbit chow containing both 0.5% cholesterol and 1% ARH004, and standard rabbit chow containing both 0.5% cholesterol and 10 mg / kg AR101-DS2. Groups other than the standard rabbit chow group were fed standard rabbit chow containing 0.5% cholesterol for 4 weeks (see Figures 14-15). The daily feeding amount for each rabbit is 50 g / kg body weight / day. The chow is administered for 8 weeks after the animals have become accustomed to the new environment. At the beginning and end of the 12-week study, rabbits are anesthetized with an intramuscular injection of Zoletil 50 (1 mL / kg) (Virbac, France) and blood samples are taken. Finally, after sacrificing the rabbits, the aorta (from the aortic arch to the iliac artery bifurcation) and the whole liver are harvested for further histopathological analysis.

[0107] Male New Zealand White rabbits (n=30) weighing 2-3 kg were divided into the following groups: (ND) standard rabbit chow, n = 5; (HF) standard rabbit chow containing 0.5% cholesterol, n = 6; (L) standard rabbit chow containing both 0.5% cholesterol and 10 mg / kg lovastatin, n = 4; (AR003) standard rabbit chow containing both 0.5% cholesterol and 1% ARH003, n=5; (AR004) standard rabbit chow containing both 0.5% cholesterol and 1% ARH004, n=5; (AR101-DS2) Standard rabbit chow containing both 0.5% cholesterol and 10 mg / kg AR101-DS2, n=5; daily feeding amount of each rabbit is 50 g / kg body weight per day.

[0108] blood chemistry analysis

[0109] Animals are fasted overnight prior to blood collection. Blood is collected from the rabbit marginal ear vein into BD Vacutainer EDTA blood collection tubes. Plasma is separated by centrifugation at 3,000 rpm for 10 minutes at 4°C. Figures 16-23 show measurements of changes in blood chemistry parameters including serum levels of low density lipoprotein (LDL), cholesterol (Chol), triglycerides (TG), glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT).

[0110] Aortic Fatty Streak Staining

[0111] The aorta is opened longitudinally to expose the intimal surface and gently flushed with saline (see Figures 24-26). The aorta is incubated in 2% (w / v) Sudan IV and flushed for 1 min with several concentrations of ethanol (100%, 90%, 80%, 70%, and 60%), followed by rinsing with pure water. The photographs shown in Figure 28 were acquired using a digital camera (Nikon D80, Japan) and quantified with an Alpha Imager 2200 documentation system (Alpha Innotech, USA). The progression of aortic fatty streak lesions is shown as the percentage of stained area relative to the total area (Figure 27).

[0112] method 1. Hydration of cells or tissues. i. Use microscope slides containing frozen or rehydrated tissue sections (see step 12 for sectioning paraffin-embedded tissues) fixed in alcohol- or aldehyde-based fixatives (Fischer et al. 2008). ii. Immerse the slide in H2O for 30 seconds with manual agitation. Rinsing with water is important; the hematoxylin will precipitate along with the salts and buffers. Staining can be performed after immunohistochemistry or hybridization with a non-fluorescent detection system. 2. Immerse the slides in a Coplin jar containing Mayer's hematoxylin and agitate for 30 seconds. 3. Rinse the slide with HO for 1 minute. Estimate the staining intensity at this point and repeat steps 2 and 3 if necessary. 4. Stain the slides with 1% Eosin Y solution for 10-30 seconds with agitation. 5. Dehydrate sections in two changes of 95% alcohol and two changes of 100% alcohol for 30 s each. Some colorimetric substrates are soluble in alcohol. 6. Extract the alcohol with two changes of xylene. If staining on plastic slides or plastic culture dishes is used, do not use xylene or a xylene-based mounting medium as it will dissolve the plastic. 7. Add 1-2 drops of mounting medium and cover with a coverslip. If alcohol is not available, use glycerol or other aqueous mounting medium to mount the coverslip.

[0113] reagent Cells or tissues of interest on a microscope slide (see step 1.i) Eosin Y (1% aqueous solution; EM Diagnostic Systems) Ethanol (95%, 100%) Instead of ethanol, methanol or FlexAlcohol (Richard-Allan Scientific) can be used (see step 5). Hematoxylin, Mayer (Sigma) Mayer's hematoxylin is the easiest to use and is compatible with most colorimetric substrates. Mounting medium (Canada Balsam, Sigma C1795) If alcohol is not available, use glycerol or other aqueous mounting media (see step 7).

[0114] Cryosectioning of liver tissue

[0115] Rabbit liver tissue (shown in Figure 29) is perfused with saline and fixed in 10% (v / v) formalin neutralizing solution (JT Baker, Inc., USA) for 24 hours. The tissue is then embedded in Tissue Tek OCT Compound (#4583; Sakura Finetek Inc., USA). The embedded tissue is cut into 10 μm thick sections and stained with Sudan IV and hematoxylin (Merck, USA). Briefly, the sections are washed in pure water for 1 min to remove the OCT compound, washed in 50% (v / v) ethanol for 30 s, and then stained with 2% (w / v) Sudan IV for 1 h. After further washing in 50% (v / v) ethanol and pure water for 2 min, the sections are counterstained with hematoxylin. The photographs shown in Figure 30 were taken using a microscope equipped with a 10x objective lens and quantified with an Alpha Imager 2200 documentation system (Alpha Innotech, USA). The progression of fatty liver is shown as the percentage of the area of ​​oil droplets relative to the total liver tissue (cells).

[0116] [Table 2] TIFF2024524499000009.tif122160

[0117] Example 19: Protective Effects of Antrodia camphorata Extracts and Compounds Against Bleomycin-Induced Pulmonary Fibrosis in Mice

[0118] Animals and treatments

[0119] Specific pathogen-free ICR mice (male, weighing 18–22 g) were purchased from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan). They were housed in Plexiglas cages at a temperature of 22 ± 1°C, relative humidity of 55 ± 5%, and a 12-h light-dark cycle for at least 2 weeks prior to the experiment. Animals were provided with food and water. All experimental procedures were performed in accordance with the guidelines of the Institutional Animal Ethics Committee, and the protocol was approved by the Institutional Animal Care and Use Oversight Committee.

[0120] BLM-induced PF in mice

[0121] Mice were divided into 5 groups with 5 mice per group according to body weight: control, BLM, BLM+DEX (7.5mg / kg), BLM+ACH dose group (50mg / kg), and BLM+ACM dose group (25mg / kg), BLM+ACH dose group (50mg / kg), and BLM+ACM dose group (25mg / kg), BLM+AH dose group (50mg / kg), and BLM+AM dose group (25mg / kg), BLM+BH dose group (50mg / kg), and BLM+BM dose group (25mg / kg), BLM+CH dose group (50mg / kg), and BLM+CM dose group (25mg / kg), BLM+DH dose group (50mg / kg), and BLM+DM dose group (25mg / kg). BLM+EH dose group (50mg / kg), and BLM+EM dose group (25mg / kg) BLM. Pulmonary fibrosis (PF) was established in mice by a single intratracheal administration of 7.5mg / kg body weight BLM. Different doses of samples were administered intragastrically daily for 21 days after BLM injury, and DEX was used as a positive control. The control and model groups were administered with an equal volume of vehicle (0.9% NaCl) using the same schedule and route of administration.

[0122] Mouse body weights were recorded daily. Mice were sacrificed on day 21 with chloral hydrate hydrochloride anesthesia. Blood was collected for ELISA analysis, and whole lungs were removed and weighed. The right lung was fixed in 10% formalin, dehydrated, and embedded in paraffin. The left lung was used to measure hydroxyproline. Lung index was calculated using lung weight / body weight × 100%.

[0123] Experimental design

[0124] Male C57BL / 6 mice were randomly assigned to the following eight groups (n=6): 1. Group I: control; 2. Group II: Mice that received a single intraperitoneal injection of BLM (7.5 mg / kg BW). 3. Group III: Single dose (ACH, 0.5g / kg) 4. IV group: single dose (ACM, 1.0 g / kg) 5. Group V: purified AR101-DS1 (50 mg / kg) 6. Group VI: purified AR101-DS1 (25 mg / kg) 7. Group VII: purified AR101-DS2 (50 mg / kg) 8. Group VIII: purified AR101-DS2 (25 mg / kg) 7. Group VII: purified AR101-DS4 (50 mg / kg) 8. Group VIII: purified AR101-DS4 (25 mg / kg) 7. Group VII: purified AR100-DS1 (50 mg / kg) 8. Group VIII: purified AR100-DS1 (25 mg / kg) 7. Group VII: purified ARH013-RA1 (50 mg / kg) 8. Group VIII: purified ARH013-RA1 (25 mg / kg)

[0125] Sampling of BALF

[0126] Under anesthesia, BALF was collected four times through the tracheal cannula with 0.7 mL of saline. Approximately 2.5 mL (90%) of BAL fluid (BALF) was collected from each mouse examined. BALF supernatant was stored at -80°C until use.

[0127] Lung histopathology

[0128] The anterior portion of the right lung of each mouse was fixed in 10% formaldehyde phosphate buffer, embedded in paraffin, cut into 5 μm sections, processed with hematoxylin and eosin (H&E) staining, and subjected to histological examination under a light microscope (Nikon, ECLIPSE, TS100, Tokyo, Japan). Images were captured at 400x original magnification using a digital camera (NIS-Elements D 2.30, SP4, Build 387).

[0129] Hydroxyproline assay

[0130] The content of hydroxyproline was analyzed in lung tissue according to the instructions of the Hydroxyproline Assay Kit (Biosource International Inc., Sunnyvale, CA, USA). The lung tissues of the mice were ground and homogenized with 1 ml of 6 mol / L potassium chloride solution, hydrolyzed at 95 °C for 5 h, and the pH value was adjusted to 6.0-6.8. According to the instructions, the corresponding reagents were added to the reaction system, mixed well, and incubated at 60 °C for 15 min. After cooling, the mixture was centrifuged at 3500 rpm for 10 min, and the supernatant was collected. The absorbance values ​​of the supernatants from the samples were measured at 550 nm by a spectrophotometer, and the hydroxyproline content of each group was calculated.

[0131] TNF-α, IL-6 and IL-1β cytokines in serum

[0132] Serum concentrations of proinflammatory cytokines (i.e., tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β) were assessed with the relevant enzyme-linked immunosorbent assay (ELISA) kits (Biosource International Inc., Sunnyvale, CA, USA) according to the manufacturer's instructions.

[0133] Myeloperoxidase (MPO) assay

[0134] Pulmonary MPO activity was a reliable indicator for estimating inflammatory cell infiltration in the lungs. Lung tissues were homogenized and MPO levels were detected using a kit according to the manufacturer's instructions.

[0135] Histopathological analysis

[0136] The right lung was embedded in paraffin wax, fixed in 10% formalin, and sectioned, and sections were stained with hematoxylin and eosin (H&E) or Masson's trichrome staining.

[0137] statistical analysis

[0138] Data from animal studies were presented as the mean and standard error of the mean (±SEM). Differences between multiple groups or between two groups were examined using Student's t-test. Statistical significance was indicated as *p<0.05, **p<0.01, and ***p<0.001.

[0139] At the end of the entire experiment, the body weight and lung weight of the animals were recorded. Compared with the control animals, the body weight change of the animals administered bleomycin (BLM) was significantly reduced. Compared with the other experimental groups, the lung index [(lung weight / body weight) × 100] showed a significant increase in the bleomycin-administered animals (Table 1 and Figure 31). The lung indexes of ACH, BH, and DH were significantly reduced.

[0140] TIFF2024524499000010.tif230161

[0141] Example 20: Antrodia camphorata extracts and compounds attenuated pulmonary dysfunction and histopathological changes in BLM-induced mice.

[0142] To investigate the therapeutic effects of A. camphorata extracts and compounds, histopathological changes in the lungs of mice were evaluated. Inflammatory infiltration and tissue structure integrity were observed by H&E staining (Figure 32). The degree of fibrosis in lung tissue was measured by Masson staining (Figure 33). In the control group, several histological findings were observed, such as thin alveolar walls, intact alveolar structure, normal alveolar septa, and less infiltration of inflammatory cells in the pulmonary mesenchyme. After 21 days of BLM administration, alveolar edema, significant increase in septal width, and increased inflammatory cell infiltration were observed. Administration of A. camphorata extracts and compounds improved inflammatory infiltration and damaged structure of lung tissue compared to the BLM group.

[0143] Masson staining showed that lung tissue and septa were extensively stained blue 21 days after BLM administration, suggesting that pulmonary fibrosis in the BLM group was more severe than that in the normal group. After treatment with A. camphorata extracts and compounds, the blue areas decreased and the degree of fibrosis was alleviated. After 21 days of BLM modeling, the scores of alveolitis and fibrosis were significantly reduced after therapy with A. camphorata extracts and compounds. The above results suggest that A. camphorata extracts and compounds can reduce the degree of lung inflammation and fibrosis in pulmonary fibrosis mice.

[0144] Example 21: Pulmonary fibrosis markers

[0145] Hydroxyproline content is an important indicator of collagen deposition in lung tissue. To quantify the degree of pulmonary fibrosis, the hydroxyproline content in lung tissue was measured in each group and is shown in Figure 34. BLM obviously increased HP content compared with the control group (p<0.001). Antrodia camphorata extract (1.0 g / kg) and AH, BH, and DH significantly reduced the recovery of lung HP (p<0.001).

[0146] Example 22: Antrodia camphorata extracts and compounds counteract bleomycin-induced changes in pro-inflammatory cytokines.

[0147] Pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and TGF-β levels in serum were assessed by ELISA. BLM-treated kidney injury mice had significantly increased NO, TNF-α, IL-1β and IL-6 levels in serum compared to the control group (Figures 35A-34E). Treatment with A. camphorata extract and compounds (BH and DH) at a dose of 1.0 g / kg significantly improved necrosis and inflammatory infiltrating cells in lung tissue processing improved TNF-α, IL-1β, IL-6 and TGF-β production after BLM challenge (p<0.001).

[0148] Example 23: Effects of A. camphorata extracts and compounds on pulmonary MPO activity

[0149] As shown in Figure 36, a significant increase in MPO levels was observed in response to BLM challenge compared to the control group (p<0.01). Meanwhile, administration of A. camphorata extracts AH, BH, and DH together with Dex obviously suppressed MPO activity compared to the BLM group (p<0.001), and showed a stronger effect than A. camphorata extract and compound groups (p<0.05) (Figure 36).

[0150] Example 24. Effect of a combination of A. camphorata extract and obatodiolide on oleic acid-induced steatosis in HepG2 cells

[0151] Oleic acid (OA) induces steatosis in HepG2 cells, which can be considered as an in vitro model of human fatty liver disease. Steatosis was induced by treating HepG2 cells with various concentrations of oleic acid (OA). Intracellular lipids were stained with Oil Red O (ORO) and quantified by colorimetric assay. 0.5 mM OA was selected for further testing (Figures 37 and 38). HepG2 cells were treated with test compounds (3.7 μg / mL, 11.1 μg / mL, 33.3 μg / mL, 100 μg / mL and 200 μg / mL) and incubated for 48 hours (Figure 39). HepG2 cells were seeded in 24-well plates and deprived of FBS for 24 hours. These cells were then treated with various concentrations of test compounds for 6 hours and then induced with 0.5 mM OA for 24 hours. The cells were gently washed with PBS, fixed with paraformaldehyde at RT, and then stained with Oil Red (ORO) solution for 30 min at room temperature. ORO staining was extracted with pure isopropanol and optical density was detected at 510 nm (Figure 40). AR101-DS4, AR100-DS1 and AR100-DS1+AR101-DS2 show inhibition against OA-induced steatosis in HepG2 cells at non-cytotoxic concentrations.

Claims

Claim 1 A composition for preventing or treating fibrotic conditions, the composition comprising a combination of obatoclide and at least one 4-fused ring triterpenoid extracted from Antrodia camphorate. Claim 2 The at least one 4-fused ring triterpenoid is 【Chemical 1】 selected from the group consisting of, the composition according to claim 1. Claim 3 The composition according to claim 1 or 2, wherein the fibrotic condition is liver fibrosis, kidney fibrosis, vascular fibrosis, pulmonary fibrosis or benign prostatic hyperplasia. Claim 4 The composition according to claim 1 or 2, which further reduces renal insufficiency and kidney injury. Claim 5 A composition for reducing non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), or liver inflammation, vacuolization and necrosis, the composition comprising a combination of obatoclide and at least one 4-fused ring triterpenoid extracted from Antrodia camphorate.

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