Use of cryptotanshinone in preparation of drug for treating non-alcoholic fatty liver disease

Abstract

Disclosed is use of cryptotanshinone or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating non-alcoholic fatty liver diseases, including simple fatty liver and non-alcoholic steatohepatitis. Cryptotanshinone can significantly reduce the contents of cholesterol, triglyceride, and low-density lipoprotein in mice with a non-alcoholic fatty liver disease, decrease the levels of liver injury indicators ALT and AST, and significantly ameliorate blood glucose level and insulin resistance.

Description

Application of cryptotanshinone in preparing medicine for treating non-alcoholic fatty liver disease Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and in particular relates to an application of cryptotanshinone in the preparation of a medicine for treating non-alcoholic fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a metabolic disorder, a serious chronic metabolic disease, and one of the most common causes of chronic liver disease worldwide. NAFLD has become the leading chronic liver disease in my country. NAFLD is a multi-system metabolic disease that not only affects liver structure and function, but is also closely associated with the high incidence of metabolic syndrome, type 2 diabetes, arteriosclerotic cardiovascular disease, and colorectal cancer. Over the past 20 years, the proportion of NAFLD in chronic liver disease has increased from 47% to 75%, and it is expected to become the main cause of cirrhosis and primary liver cancer in the next 10 years. However, there is currently no recognized clinical treatment for NAFLD. Therefore, the use of traditional Chinese medicine to treat NAFLD has attracted much attention. It is of great significance to deeply understand the pathogenic mechanism of NAFLD and seek effective therapeutic targets.

[0003] Hyperlipidemia refers to high blood lipid levels, which can directly lead to several serious health-threatening diseases, such as atherosclerosis, coronary heart disease, and pancreatitis. Currently, statins are the most commonly used medications for treating hyperlipidemia. However, statins are prone to causing adverse reactions, including liver damage, blood sugar fluctuations, and muscle pain. Fibrates, also used to treat hyperlipidemia, are also prone to gastrointestinal side effects.

[0004] Patent application CN112294828A discloses the use of cryptotanshinone as an inflammation inhibitor. Cryptotanshinone acts as an inflammasome inhibitor, inhibiting ASC speck formation, subsequent caspase-1p20 activation, and IL-1β secretion. Cryptotanshinone is a potential treatment for diseases associated with abnormal inflammasome activation.

[0005] Cryptotanshinone (CTS), a major chemical component of the traditional Chinese medicine Danshen (Salvia miltiorrhiza), has been shown to have a long half-life and possess antibacterial, anti-inflammatory, and antioxidant properties, while also promoting fat metabolism. However, the effectiveness of CTS in treating NAFLD remains unclear. There are no reports of cryptotanshinone's therapeutic effects on nonalcoholic fatty liver disease (NAFLD) and hyperlipidemia.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide an application of cryptotanshinone in the preparation of a medicine for treating non-alcoholic fatty liver disease.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] The first aspect of the present invention provides a use of cryptotanshinone or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating non-alcoholic fatty liver disease.

[0010] The molecular formula of the cryptotanshinone (CTS) is C 19 H 20 O3, the structural formula is as follows:

[0011] The non-alcoholic fatty liver disease includes simple fatty liver and non-alcoholic steatohepatitis.

[0012] The pharmaceutically acceptable salt is an acid addition salt formed by cryptotanshinone and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

[0013] The medicine for treating non-alcoholic fatty liver disease also includes pharmaceutically acceptable excipients.

[0014] Non-alcoholic hepatic steatosis, also known as simple fatty liver, is an early manifestation of NAFLD. Macrovesicular or predominantly macrovesicular steatosis affects more than 5% of hepatocytes. Non-alcoholic steatohepatitis (NASH) is an inflammatory subtype of NAFLD, characterized by hepatic steatosis, evidence of hepatocellular damage (ballooning), and inflammation, with or without liver fibrosis. Over time, NASH may progress to cirrhosis, end-stage liver disease, or the need for liver transplantation. 20% of patients with simple fatty liver will develop NASH, and 20% of patients with NASH will develop cirrhosis. The widely accepted pathogenesis is based on the "multiple hit" theory, which includes multiple factors such as lipotoxicity, mitochondrial dysfunction, endoplasmic reticulum stress, adipose tissue dysfunction, inflammatory cytokines, immune system dysregulation, and enteric endotoxins. Western medicine treatments for NAFLD primarily focus on controlling obesity, diabetes, and lowering blood lipids. For example, pioglitazone and metformin are used to treat diabetic NAFLD. In addition, vitamin E can significantly improve liver steatosis and inflammatory damage. However, there is currently no recognized clinical treatment for NAFLD. Simple fatty liver is an early manifestation of fatty liver, with excessive fat deposition in the liver as the main pathological manifestation. The liver can return to normal after the cause is removed. When liver cell damage occurs, it progresses to non-alcoholic steatohepatitis, a severe stage of non-alcoholic fatty liver disease, which can further develop into cirrhosis or even liver cancer. Treatment emphasizes eliminating the cause and protecting the liver and reducing enzymes. Currently, there is a lack of specific drugs.

[0015] The second aspect of the present invention provides a use of cryptotanshinone or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating hyperlipidemia.

[0016] The pharmaceutically acceptable salt is an acid addition salt formed by cryptotanshinone and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

[0017] The drug for treating hyperlipidemia also includes pharmaceutically acceptable excipients.

[0018] Hyperlipidemia refers to a disease that can seriously endanger human health due to disorders in indicators such as cholesterol, triglycerides, high-density lipoprotein, and low-density lipoprotein. It is often closely related to genetics, medication history, and unhealthy lifestyle habits. Hyperlipidemia can induce a variety of diseases such as hypertension, diabetes, coronary heart disease, and fatty liver. For example, elevated serum triglycerides can reduce insulin sensitivity; while increased insulin resistance will also increase serum triglyceride levels. Currently, the most commonly used drug for the treatment of hyperlipidemia is statins. However, statins are prone to causing adverse reactions such as liver damage, blood sugar fluctuations, and muscle pain. Fibrates used to treat hyperlipidemia are also prone to gastrointestinal reactions.

[0019] Non-alcoholic fatty liver disease (NAFLD) is often associated with metabolic disorders such as overweight / obesity, hyperlipidemia, and diabetes, leading to its term "metabolism-related fatty liver disease." Hyperlipidemia, also known as dyslipidemia, typically refers to elevated plasma triglycerides (TG) and total cholesterol (TC), and can also include elevated low-density lipoprotein (LDL) and decreased high-density lipoprotein (HDL). Currently, there is a lack of multifunctional medications that can effectively reduce weight, lower blood lipids, and control blood sugar.

[0020] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0021] The present invention found that cryptotanshinone can reduce liver lipid droplet density and liver TC and TG levels in mice with non-alcoholic fatty liver disease, lower liver damage indicators ALT and AST, reduce mouse body weight and liver weight, and improve blood sugar levels and insulin resistance. In summary, the traditional Chinese medicine monomer cryptotanshinone can effectively improve non-alcoholic fatty liver disease and hyperlipidemia.

[0022] The present invention discovered through a high-fat-fed non-alcoholic fatty liver disease mouse model that cryptotanshinone has a significant anti-non-alcoholic fatty liver effect, and cryptotanshinone significantly reduces the body weight and liver weight of mice with non-alcoholic fatty liver disease; histologically, cryptotanshinone significantly improves fatty degeneration of liver tissue and liver fat droplet density; cryptotanshinone significantly reduces the cholesterol, triglyceride, and low-density lipoprotein levels in mice with non-alcoholic fatty liver disease, and reduces the liver damage indicators ALT and AST; cryptotanshinone significantly improves the blood sugar level and insulin resistance of mice with non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG1 is a schematic diagram of body weight changes in mice with non-alcoholic fatty liver disease.

[0024] FIG2 is a schematic diagram showing changes in food intake in mice with non-alcoholic fatty liver disease after drug administration.

[0025] FIG3 is a statistical diagram of liver weight in mice with non-alcoholic fatty liver disease.

[0026] FIG4 is a statistical diagram of changes in TC and TG in serum and liver of mice with non-alcoholic fatty liver disease.

[0027] FIG5 is a statistical diagram of changes in serum HDL-c, FFA, and LDL-c in mice with non-alcoholic fatty liver disease.

[0028] FIG6 is a schematic diagram of HE staining results of liver damage and Oil Red O staining of liver lipid droplet accumulation in mice with non-alcoholic fatty liver disease.

[0029] FIG7 is a statistical diagram of the detection of serum ALT, AST, and ALP in mice with non-alcoholic fatty liver disease.

[0030] FIG8 is a schematic diagram showing changes in glucose tolerance and insulin tolerance in mice with non-alcoholic fatty liver disease. DETAILED DESCRIPTION

[0031] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. Those skilled in the art should understand that the following specific description is illustrative rather than restrictive and should not be used to limit the scope of protection of the present invention.

[0032] Example 1

[0033] 1. Experimental Materials

[0034] 1.1 Experimental animals and Chinese medicinal materials: Male C57BL / 6 mice, weighing 20-25 g, 6 weeks old, were purchased from JESTJET Co., Ltd. Cryptotanshinone was purchased from Stendra.

[0035] 1.2 Establishment of a Nonalcoholic Fatty Liver Disease Mouse Model: Mice were fed a high-fat diet (HFD), with fat providing 60% of the total calories. Long-term (>2 months) HFD feeding naturally develops hepatic steatosis in mice. After 7 days of adaptive feeding, mice were randomly assigned to a normal diet (NCD) plus 0.5% sodium hydroxymethylcellulose group (n=10; once daily, 200 μl each time); a high-fat diet (HFD) plus 0.5% sodium hydroxymethylcellulose group (n=10; once daily, 200 μl each time); a high-fat diet plus low-dose cryptotanshinone (20 mg / kg / d) (HFD+CTS-L) group (n=10; once daily, 200 μl each time); and a high-fat diet plus high-dose cryptotanshinone (60 mg / kg / d) (HFD+CTS-H) group (n=10; once daily, 200 μl each time). After 16 weeks of HFD feeding to induce obesity, mice were continued on the HFD diet and given oral medication for 8 weeks. The weight of food was recorded once a week to measure food intake, and the weight changes of mice were recorded once a week. Cryptotanshinone was dissolved using 0.5% sodium hydroxymethylcellulose (CMC-Na), and 0.5% CMC-Na was gavaged into the NCD group and HFD group. Before sampling, mice were fasted overnight but not watered. On the day of sampling, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital 50 mg / kg, 50 μL / 10 g, and weighed and recorded. After the mice were fully anesthetized, blood was collected from the eyeballs of the mice, and the whole blood was collected, placed at room temperature for half an hour, centrifuged at 3000 rpm for 15 minutes, and the collected serum was stored at -80°C. After blood collection, the mice were killed by dislocating the neck, and the chest wall was cut open layer by layer at the midline. The left liver lobe was divided into two equal pieces, each piece was fixed in 4% paraformaldehyde, and the remaining liver was placed in an EP tube with liquid nitrogen for freezing and storage.

[0036] 1.3 Measurement of food intake and body weight: The weight of food was recorded once a week to measure food intake, and the weight changes of mice were detected and recorded once a week.

[0037] 1.4 Tissue H&E staining and Oil Red staining:

[0038] (1) H&E staining: Dewax the paraffin sections and process the sections according to the following steps: xylene immersion for 30 min, 100% alcohol for 10 min, 95% alcohol for 10 min, ddH2O for 10 min, hematoxylin for 10 min, rinse with running water for 5 min, eosin for 90 s, rinse with running water for 10 min, 95% alcohol for 20 s, 100% alcohol for 20 s, xylene for 20 s, and seal with neutral gum.

[0039] (2) Oil red staining: Fixation of fresh frozen sections: Rewarm the frozen sections and dry them, fix them in the fixative for 15 minutes, wash them with tap water and air dry. Immerse the sections in the oil red staining solution for 8-10 minutes (cover and protect from light). Background differentiation: Take out the sections, let them stand for 3 seconds, then immerse them in two cylinders of 60% isopropyl alcohol for differentiation, 3 seconds and 5 seconds respectively. Immerse the sections in two cylinders of pure water for 10 seconds each. Hematoxylin staining: Take out the sections, let them stand for 3 seconds, then immerse them in hematoxylin for 3-5 minutes, and then rinse them in three cylinders of pure water for 5 seconds, 10 seconds and 30 seconds respectively. Differentiate them in differentiation solution (with 60% alcohol as solvent) for 2-8 seconds, wash them in two cylinders of distilled water for 10 seconds each, and blue them in the blueing solution for 1 second. Gently immerse the sections in two cylinders of tap water for 5 seconds and 10 seconds each, and examine the staining effect under a microscope. Sealing: Sealing with glycerol gelatin sealing medium.

[0040] 1.5 Biochemical index detection: Hitachi fully automatic biochemical analyzer was used to measure blood lipids and liver function related indicators in mouse serum.

[0041] 1.6 Insulin tolerance test: Insulin was diluted with normal saline and injected at a concentration of 0.2 U / kg. Blood glucose was measured at 0, 15, 30, 60, and 90 min. The animals were fasted for 4 hours in the morning and allowed to drink water as normal. In the afternoon, the animals were weighed and numbered. Blood glucose was measured before insulin injection. The insulin injection dose was calculated based on body weight. Blood glucose was measured at 0, 15, 30, 60, and 90 min. At the end of the experiment, each cage was replenished with feed.

[0042] 1.7 Glucose tolerance test: Prepare a 10% glucose solution in normal saline at a dose of 1g / kg body weight. Fast the animals at 5:00 PM the day before, but not water. Blood glucose was measured 16 hours before injection at 9:00 AM the following morning. Glucose was injected intraperitoneally at 0.01ml per gram. Blood glucose was measured at intervals of 0, 15, 30, 60, 90, and 120 minutes. After the experiment, each cage was replenished with feed.

[0043] 2. Experimental results:

[0044] 2.1 Effects of Cryptotanshinone on Body Weight and Food Intake The results are shown in Figures 1 and 2. Figure 1 is a schematic diagram of weight changes in mice with non-alcoholic fatty liver disease. Figure 2 is a schematic diagram of food intake changes in mice with non-alcoholic fatty liver disease after drug administration. As shown in Figure 1, compared with the NCD control group, the HFD model group significantly increased body weight, while food intake was not affected. However, compared with the HFD model group, the HFD+CTS-L and HFD+CTS-H mice significantly decreased body weight. These results indicate that cryptotanshinone can reduce the weight gain induced by a high-fat diet in mice.

[0045] 2.2 Effect of Cryptotanshinone on Liver Weight in Mice with Non-alcoholic Fatty Liver Disease The results are shown in Figure 3, which is a statistical diagram of liver weight in mice with non-alcoholic fatty liver disease. Figure 3 shows, from left to right, liver weight, liver weight / body weight ratio, and fat / body weight ratio. The results showed that compared with the normal group, the liver weight, liver weight / body weight ratio, and fat / body weight ratio of mice in the HFD group were significantly increased. Compared with the HFD group, the liver weight, liver weight / body weight ratio, and fat / body weight ratio of mice in the HFD+CTS-L and HFD+CTS-H groups were significantly decreased, indicating that cryptotanshinone improved HFD-induced lipid accumulation and weight gain.

[0046] In summary, cryptotanshinone treatment can significantly reduce the liver weight of HFD mice, indicating that it has the effect of treating non-alcoholic fatty liver disease.

[0047] 2.3 Effects of Cryptotanshinone on Serum and Liver Triglycerides, Total Cholesterol, High-Density Lipoprotein (HDL), and Low-Density Lipoprotein in Mice with Non-Alcoholic Fatty Liver Disease The results are shown in Figures 4 and 5. Figure 4 is a statistical diagram of changes in serum TC and TG in the liver of mice with non-alcoholic fatty liver disease. Figure 5 is a statistical diagram of changes in serum HDL-c, FFA, and LDL-c in mice with non-alcoholic fatty liver disease.

[0048] Figure 4 shows, from left to right, the schematic diagrams of serum total cholesterol (TC) levels, liver TC levels, serum triglyceride (TG) levels, and liver TG levels. Compared with the control group, the HFD group showed significantly increased TC and TG levels in the serum and liver, suggesting abnormal lipid metabolism in the HFD group. Compared with the HFD group, the HFD+CTS-L and HFD+CTS-H groups showed significantly decreased TC and TG levels in the serum and liver, indicating that cryptotanshinone can improve HFD-induced lipid metabolism disorders.

[0049] Figure 5 shows, from left to right, the levels of serum low-density lipoprotein (LDL-c), serum free fatty acids (FFA), and serum high-density lipoprotein (HDL-c). Compared with the control group, the serum LDL-c, FFA, and HDL-c levels of mice in the HFD group were significantly increased, indicating that the HFD group had abnormal lipid metabolism. Compared with the HFD group, the serum LDL-c, FFA, and HDL-c levels of mice in the HFD+CTS-L and HFD+CTS-H groups were significantly decreased, indicating that cryptotanshinone can improve abnormal lipid metabolism in mice. It is worth mentioning that cryptotanshinone significantly reduced the LDL-c levels of mice induced by a high-fat diet. Elevated LDL-c is the main key factor in the pathogenesis of hyperlipidemia. Therefore, controlling LDL-c levels is clinically regarded as the primary goal of hyperlipidemia treatment to reduce the morbidity and mortality of cardiovascular disease.

[0050] In summary, 8 weeks of cryptotanshinone treatment can significantly reduce the serum TC, TG, LDL-c, HDL-c and FFA levels of mice, and also improve the liver TC and TG levels, thereby reversing HFD diet-induced hyperlipidemia in mice.

[0051] 2.4 The results of the effect of cryptotanshinone on liver morphology in mice with non-alcoholic fatty liver disease are shown in Figure 6, which shows the results of HE staining of liver damage in mice with non-alcoholic fatty liver disease and a schematic diagram of Oil Red O staining of liver lipid droplet accumulation. As can be seen from the figure, compared with the normal control group, the HFD model group mice showed obvious liver steatosis, lobular inflammation, hepatocyte ballooning, and lipid droplet accumulation; compared with the HFD model group, the liver steatosis, lobular inflammation, hepatocyte ballooning, and lipid droplet accumulation in the HFD+CTS-L and HFD+CTS-H groups were significantly reduced. The results show that the cryptotanshinone of the present invention can improve liver steatosis and damage in mice caused by a high-fat diet.

[0052] 2.6 The effects of cryptotanshinone on liver function in mice with non-alcoholic fatty liver disease are shown in Figure 7. Figure 7 shows, from left to right, the statistical diagram of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels in mice with non-alcoholic fatty liver disease. As can be seen from the figure, compared with the normal control group, the ALT, AST, and ALP levels of mice in the HFD model group were significantly increased, indicating that the HFD model group mice had liver damage. Compared with the HFD model group, the ALT and AST levels of mice in the HFD+CTS-L and HFD+CTS-H groups were significantly decreased, indicating that cryptotanshinone has a hepatoprotective effect and can be used as a drug for the treatment of non-alcoholic fatty liver disease.

[0053] 2.7 Effects of Cryptotanshinone on the Glucose Tolerance Test (GTT) and Insulin Resistance Test (ITT) in Mice with Non-alcoholic Fatty Liver Disease The results are shown in FIG8 , which is a schematic diagram of changes in glucose tolerance and insulin tolerance in mice with non-alcoholic fatty liver disease.

[0054] Figure 8 shows, from left to right and top to bottom, the diagrams for the glucose tolerance test (OGTT), the area under the glucose tolerance curve (AUC), the insulin tolerance test (ITT), and the AUC. These results demonstrate that HFD-fed mice exhibited significantly higher OGTT and ITT values ​​at all time periods than mice fed a normal diet, demonstrating significant insulin resistance in the intraperitoneal glucose tolerance test. Cryptotanshinone administration improved glucose tolerance and insulin sensitivity in mice.

[0055] In summary, cryptotanshinone is a drug that can treat non-alcoholic fatty liver disease and hyperlipidemia.

[0056] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with this patent can make slight changes or modifications to equivalent embodiments using the above technical content without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the solution of the present invention.

Claims

1. A use of cryptotanshinone or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating hyperlipidemia.

2. The use of cryptotanshinone or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for treating hyperlipidemia, characterized in that: The pharmaceutically acceptable salt is an acid addition salt formed by cryptotanshinone and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

3. The use of cryptotanshinone or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for treating hyperlipidemia, characterized in that: The structural formula of the cryptotanshinone is as follows:

4. The use of cryptotanshinone or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for treating hyperlipidemia, characterized in that: The drug for treating hyperlipidemia also includes pharmaceutically acceptable excipients.

5. Use of cryptotanshinone or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating non-alcoholic fatty liver disease.

6. Use of cryptotanshinone or a pharmaceutically acceptable salt thereof according to claim 5 in the preparation of a medicament for treating non-alcoholic fatty liver disease, characterized in that: The structural formula of the cryptotanshinone is as follows:

7. Use of cryptotanshinone or a pharmaceutically acceptable salt thereof according to claim 5 in the preparation of a medicament for treating non-alcoholic fatty liver disease, characterized in that: The non-alcoholic fatty liver disease includes simple fatty liver and non-alcoholic fatty liver hepatitis.

8. Use of cryptotanshinone or a pharmaceutically acceptable salt thereof according to claim 5 in the preparation of a medicament for treating non-alcoholic fatty liver disease, characterized in that: The pharmaceutically acceptable salt is an acid addition salt formed by cryptotanshinone and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

9. Use of cryptotanshinone or a pharmaceutically acceptable salt thereof according to claim 5 in the preparation of a medicament for treating non-alcoholic fatty liver disease, characterized in that: The medicine for treating non-alcoholic fatty liver disease also includes pharmaceutically acceptable excipients.

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