Application of endogenous metabolite micromolecule GGOH in preparation of liver cancer drugs

Abstract

The invention discloses an application of an endogenous metabolite micromolecule GGOH in preparation of liver cancer drugs, the structural formula of GGOH is as shown in formula (I): GGOH can be spontaneously converted into GGPP after entering the liver, and the GGOH can specifically up-regulate gluconeogenesis in the liver and reduce glycolysis level, so that the glucose metabolism steady state of the liver is improved, and the occurrence and development of fatty liver diseases are inhibited. The invention discloses the application of the endogenous metabolite micromolecule GGOH in preparing the fatty liver disease medicine or treating the liver cancer for the first time, clarifies the key regulation effect of the GGOH / GGPP level in the occurrence and development of the fatty liver disease, opens up a new direction for treating the fatty liver disease related to metabolic disorder, and has important scientific significance and clinical application prospects.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of an endogenous metabolite, the small molecule GGOH, in the preparation of liver cancer drugs. Background Technology

[0002] Glucose and lipid metabolism disorders lead to non-alcoholic fatty liver disease (NAFLD). The regulation of glucose and lipid metabolic homeostasis is fundamental to maintaining bodily functions. In my country, due to changes in lifestyle and dietary structure in recent decades, the glucose and lipid metabolism patterns of some populations have changed, leading to a significant increase in the incidence of metabolic syndromes such as obesity and diabetes. Statistics show that the number of NAFLD patients in China is expected to exceed 300 million by 2030, and NAFLD has replaced viral hepatitis as the leading cause of chronic liver disease. The liver, as the central organ of metabolism, bears the brunt of this damage. Long-term glucose and lipid metabolism disorders trigger chronic liver diseases such as NAFLD and liver fibrosis. 10%-30% of NAFLD patients will progress to non-alcoholic steatohepatitis (NASH), of whom 25% will develop cirrhosis within 10 years, and 5%-15% will eventually develop liver cancer. Therefore, glucose and lipid metabolic homeostasis plays a crucial role in the occurrence, development, prevention, early diagnosis, and treatment of fatty liver disease. The pathogenesis of NAFLD is complex, involving multiple factors such as insulin resistance, lipid metabolism disorders, oxidative stress, and gut microbiota dysbiosis. Currently, there are limited clinical treatments with unsatisfactory efficacy, making it urgent to develop new therapeutic targets and drugs.

[0003] Current treatments for NAFLD primarily focus on lifestyle interventions, but these are effective in only 30% of patients. Existing clinical treatments mainly address symptoms and target single aspects of the NAFLD pathogenesis, but their efficacy is limited, and there is a lack of specific drugs targeting NASH. For example, insulin sensitizers such as metformin and pioglitazone can improve insulin resistance, but their effects on improving hepatic steatosis and inflammation are limited, and pioglitazone has side effects such as increasing the risk of heart failure. Hepatoprotective and anti-inflammatory drugs such as polyene phosphatidylcholine and silymarin can only partially improve liver function indicators and cannot reverse the process of liver fibrosis. Statins and fibrates can lower blood lipids and reduce hepatic lipid deposition, but they carry the risk of elevated liver enzymes and are only suitable for patients with concurrent hyperlipidemia. Some antioxidants, such as vitamin E and glutathione, are only effective for mild NASH inflammation, and their long-term safety is questionable. In recent years, some new drugs have begun to be used to treat NAFLD: obeticholic acid can regulate bile acid metabolism and has anti-inflammatory and anti-fibrotic effects, but clinical trials have shown that patients may experience adverse reactions such as itching and dyslipidemia; semaglutide, which has been widely used in the field of weight loss in recent years, is a GLP-1 analog that can improve insulin resistance, promote weight loss, and inhibit liver lipid synthesis. Clinical trials have shown that 48% of patients experienced NASH remission, but it requires long-term injections and is accompanied by gastrointestinal side effects. In summary, existing drugs for treating NAFLD have limitations such as limited efficacy of single-target drugs, significant side effects, poor patient compliance, and inability to reverse "lean NAFLD". Clinically, it is necessary to combine the latest pathological mechanisms and clinical needs to break through existing bottlenecks and fill the gap in drugs for the treatment of NASH.

[0004] GGOH (geranylgeraniol) is a naturally occurring straight-chain diterpenoid compound with the molecular formula C2. 20 H 24 O, an intermediate metabolite of the mevalonic acid (MVA) pathway, is widely found in everyday foods such as rice, fruits, vegetables, and annatto seeds. It has been listed as a "Generally Recognized As Safe" (GRAS) substance by regulatory agencies in many countries.

[0005] Sources and Biosynthesis: Plants and Foods: GGOH can be found in agricultural products such as rice, wheat, tomatoes, and carrots at concentrations of 0.1-1 mg / kg. -1 The highest content (2-3%) is found in the seed oil of the tropical annatto tree. Microbial synthesis: In recent years, through synthetic biology techniques, MVA-enhanced pathways have been constructed in *Saccharomyces cerevisiae* or *Escherichia coli*, achieving fermentation titers exceeding 6g / L in 5L tanks. -1 This provides a green alternative for large-scale production. After oral administration, GGOH is phosphorylated in the liver in two steps to the active form geraniylgeraniyl pyrophosphate GGPP, which participates in the geraniylgeraniylylation modification of proteins and regulates the membrane localization and signal transduction of small G proteins such as Ras, Rho, and Rap.

[0006] Since the 1990s, Japan has marketed it as an oral anti-ulcer drug, with no adverse reactions observed at a daily dose of 300mg. In 2017, it was included in the list of "Brain Health Functional Ingredients" and recommended for relieving mental fatigue. Based on its medicinal and edible properties, GGOH is being developed as a "metabolic reprogramming" functional ingredient, targeting populations including obese and diabetic patients who are intolerant to existing NAFLD drugs and require long-term regulation of glucose and lipid metabolism.

[0007] GGOH is an endogenous small molecule that plays a dual role as both a nutrient and a signaling agent. Through a GGPP-dependent protein modification network, it regulates energy metabolism, cell survival, and differentiation across tissues, providing a safe and efficient new approach for nutritional intervention and drug development for metabolic disorders.

[0008] In summary, there is currently a lack of small molecule compounds with GGOH as a precursor that can be used to regulate the balance of hepatic glucose and lipid metabolism in the preparation of liver cancer drugs. Summary of the Invention

[0009] To address the problems of the prior art, the purpose of this invention is to provide an application of the endogenous metabolite small molecule GGOH in the preparation of liver cancer drugs.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] In one aspect, this application provides the use of the endogenous metabolite small molecule GGOH in the preparation of drugs for fatty liver disease.

[0012] Secondly, this application provides the application of the endogenous metabolite small molecule GGOH in the preparation of liver cancer drugs.

[0013] Thirdly, this application provides a pharmaceutical composition for treating fatty liver disease or liver cancer.

[0014] Fourthly, this application provides a method for inhibiting the occurrence and development of fatty liver.

[0015] The first aspect of this application provides the use of the endogenous metabolite small molecule GGOH in the preparation of drugs for fatty liver disease, wherein the structural formula of GGOH is shown in formula (I):

[0016]

[0017] After entering the liver, GGOH is spontaneously converted into GGPP, which inhibits hepatocyte glycolysis, upregulates gluconeogenesis, and restores hepatic glucose and lipid metabolism homeostasis, thereby inhibiting the occurrence and development of fatty liver disease.

[0018] Furthermore, fatty liver disease includes any one of simple fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), NASH-associated liver fibrosis, or NASH-associated cirrhosis.

[0019] The second aspect of this application provides the application of the endogenous metabolite small molecule GGOH in the preparation of liver cancer drugs. After entering the liver, GGOH can be spontaneously converted into GGPP, which inhibits abnormal glycolysis in hepatocytes, upregulates gluconeogenesis, and reverses the Warburg effect, thereby inhibiting the proliferation of liver cancer cells and inducing their apoptosis.

[0020] Furthermore, liver cancer includes primary hepatocellular carcinoma (HCC), hepatocellular carcinoma combined with non-alcoholic steatohepatitis (NASH), or hepatocellular carcinoma driven by glucose and lipid metabolism disorders.

[0021] Furthermore, GGOH restores hepatic glucose homeostasis by reducing the activity of the key glycolytic enzyme PKM2 and activating the rate-limiting gluconeogenesis enzyme FBP1.

[0022] Furthermore, GGOH reduces the levels of triglycerides and cholesterol esters in hepatocytes by inhibiting the expression and activity of key enzymes in the lipid synthesis pathway, such as ACC, FASN, and SCD1, thereby alleviating hepatic steatosis.

[0023] A third aspect of this application provides a pharmaceutical composition for treating fatty liver disease or liver cancer, the pharmaceutical composition comprising a therapeutically effective amount of the compound GGOH of claim 1 or 3, and a pharmaceutically acceptable carrier or excipient.

[0024] Furthermore, the dosage form includes any one of oral formulations, injectable formulations, or targeted delivery formulations.

[0025] The fourth aspect of this application provides a method for inhibiting the occurrence and development of fatty liver disease, wherein a therapeutically effective amount of the compound GGOH is administered to a patient to restore hepatic glucose and lipid metabolism homeostasis and inhibit disease progression.

[0026] Beneficial effects: This invention discloses for the first time the application of an endogenous metabolite small molecule GGOH in the preparation of drugs for fatty liver disease or the treatment of liver cancer, and elucidates the key regulatory role of GGOH / GGPP levels in the occurrence and development of fatty liver disease. It opens up a new direction for the treatment of fatty liver disease related to metabolic disorders and has important scientific significance and clinical application prospects.

[0027] Compared with the prior art, the present invention has the following advantages:

[0028] (1) Safe and highly biocompatible: This invention reveals for the first time the effect of compound GGOH in treating fatty liver disease by regulating the balance of glucose and lipid metabolism in the liver. GGOH and its spontaneous conversion products are small molecules of endogenous metabolites in the human body, which improves the safety and biocompatibility of the drug.

[0029] (2) Regulation of metabolic balance: GGPP activates FBP1, coordinates the balance of glucose metabolism in the liver, reduces abnormal glycolysis in liver tissue, and inhibits the occurrence and development of fatty liver disease at the molecular level, providing a new strategy for the treatment of fatty liver disease related to metabolic disorders.

[0030] (3) Clinical translation potential: significant efficacy: In the mouse model of fatty liver disease, GGOH supplementation significantly inhibited the occurrence and development of fatty liver disease, precisely targeting the core pathological chain of glucose and lipid metabolism disorders in the development of NAFL-NASH, covering the entire course of the disease from simple hepatic steatosis to fibrosis, and is especially suitable for special populations who are intolerant to existing drugs, filling the gap in clinical precision intervention.

[0031] (4) Research and development advantages: GGOH / GGPP are small molecules of endogenous metabolites and also important nutrient molecules in food (such as soy products). They have the advantage of being both food and medicine, which can significantly shorten the drug development cycle and cost. At the same time, combined with multi-level experimental verification (molecular, cellular, animal and clinical data), it lays a solid foundation for subsequent clinical trials. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the experimental examples of this application, the drawings used in the description of the experimental examples or prior art will be briefly introduced below. Obviously, the drawings described below are only some experimental examples of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0033] Abbreviations in the attached diagram Figure 1 This is a pathway diagram for mevalonic acid.

[0034] Figure 2 The GGPPS and GGPP levels of this invention were downregulated in patients with fatty liver disease, mouse models, and cell models.

[0035] Figure 3 The GGOH / GGPP treatment of the present invention significantly inhibits lipid accumulation in liver cells.

[0036] Figure 4 The GGOH / GGPP treatment of the present invention significantly inhibited the occurrence and development of fatty liver disease in mice.

[0037] Figure 5This is a graph showing the changes in mice before and after GGOH intervention treatment according to the present invention. Figure 5 A is a schematic diagram of the GGOH intervention for treating liver cancer (HCC) mice according to the present invention. Figure 5 B represents the overall changes in mouse liver tumors before and after GGOH intervention treatment according to this invention. Figure 5 C represents the size and number of liver tumors in mice before and after GGOH intervention treatment according to this invention. Detailed Implementation

[0038] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0039] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0040] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0041] In this invention, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0042] The first aspect of this application provides the application of the endogenous metabolite small molecule GGOH in the preparation of drugs for fatty liver disease. The structural formula of GGOH is shown in formula (I):

[0043]

[0044] After entering the liver, GGOH is spontaneously converted into GGPP, which inhibits hepatocyte glycolysis, upregulates gluconeogenesis, and restores hepatic glucose and lipid metabolism homeostasis, thereby inhibiting the occurrence and development of fatty liver disease.

[0045] In some embodiments, fatty liver disease includes any one of simple fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), NASH-associated liver fibrosis, or NASH-associated cirrhosis.

[0046] The second aspect of this application provides the application of the endogenous metabolite small molecule GGOH in the preparation of liver cancer drugs. After entering the liver, GGOH can be spontaneously converted into GGPP, which inhibits abnormal glycolysis of hepatocytes, upregulates gluconeogenesis, and reverses the Warburg effect, thereby inhibiting the proliferation of liver cancer cells and inducing their apoptosis.

[0047] In some embodiments, liver cancer includes primary hepatocellular carcinoma (HCC), hepatocellular carcinoma combined with non-alcoholic steatohepatitis (NASH), or hepatocellular carcinoma driven by glucose and lipid metabolism disorders.

[0048] In some embodiments, GGOH restores hepatic glucose homeostasis by reducing the activity of the key glycolytic enzyme PKM2 and activating the rate-limiting gluconeogenesis enzyme FBP1.

[0049] In some embodiments, GGOH reduces the content of triglycerides and cholesterol esters in hepatocytes by inhibiting the expression and activity of key enzymes in the lipid synthesis pathway, ACC, FASN and SCD1, thereby alleviating hepatic steatosis.

[0050] A third aspect of this application provides a pharmaceutical composition for treating fatty liver disease or liver cancer, the pharmaceutical composition comprising a therapeutically effective amount of the compound GGOH, and a pharmaceutically acceptable carrier or excipient.

[0051] In some embodiments, the dosage form includes any one of an oral formulation, an injection, or a targeted delivery formulation.

[0052] The fourth aspect of this application provides a method for inhibiting the occurrence and development of fatty liver disease, which involves administering a therapeutically effective amount of the compound GGOH to a patient to restore hepatic glucose and lipid metabolism homeostasis and inhibit disease progression.

[0053] Example 1

[0054] The present invention relates to the application of an endogenous metabolite, small molecule GGOH, in the preparation of a drug for fatty liver disease. The structural formula of GGOH is shown in formula (I):

[0055]

[0056] After entering the liver, GGOH is spontaneously converted into GGPP, which inhibits hepatocyte glycolysis, upregulates gluconeogenesis, and restores hepatic glucose and lipid metabolism homeostasis, thereby inhibiting the occurrence and development of fatty liver disease.

[0057] Fatty liver disease includes any one of simple fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), NASH-associated liver fibrosis, or NASH-associated cirrhosis.

[0058] Example 2

[0059] The present invention relates to the application of an endogenous metabolite, small molecule GGOH, in the preparation of liver cancer drugs. After entering the liver, GGOH can be spontaneously converted into GGPP, which inhibits abnormal glycolysis in hepatocytes, upregulates gluconeogenesis, and reverses the Warburg effect, thereby inhibiting the proliferation of liver cancer cells and inducing their apoptosis.

[0060] Liver cancer includes primary hepatocellular carcinoma (HCC), hepatocellular carcinoma combined with non-alcoholic steatohepatitis (NASH), or hepatocellular carcinoma driven by glucose and lipid metabolism disorders.

[0061] GGOH restores hepatic glucose homeostasis by reducing the activity of PKM2, a key enzyme in glycolysis, and activating FBP1, the rate-limiting enzyme in gluconeogenesis.

[0062] GGOH reduces hepatic steatosis by inhibiting the expression and activity of key lipid synthesis enzymes ACC, FASN, and SCD1, thereby lowering the levels of triglycerides and cholesterol esters in hepatocytes.

[0063] Example 3

[0064] The present invention provides a pharmaceutical composition for treating fatty liver disease or liver cancer, the pharmaceutical composition comprising a therapeutically effective amount of the compound GGOH, and a pharmaceutically acceptable carrier or excipient.

[0065] Example 4

[0066] The difference between Example 4 and Example 3 is as follows:

[0067] The dosage form is an oral preparation.

[0068] Example 5

[0069] The difference between Example 5 and Example 3 is as follows:

[0070] The dosage form is injection.

[0071] Example 6

[0072] The difference between Example 6 and Example 3 is as follows:

[0073] The dosage form is a targeted delivery formulation.

[0074] Example 7

[0075] The present invention discloses a method for inhibiting the occurrence and development of fatty liver disease by administering a therapeutically effective amount of the compound GGOH to the patient in order to restore hepatic glucose and lipid metabolism homeostasis and inhibit disease progression.

[0076] Experimental Example 1

[0077] GGOH inhibits lipid accumulation in liver cells.

[0078] This invention first constructs a NAFLD cell model using the THLE-2 hepatocyte line induced by gradient concentrations of PA / OA. It was found that as the PA / OA concentration increased, the level of GGPPS1 protein in the cells first increased and then decreased. Figure 2 A). Subsequently, the induced cells were treated with GGOH, and after staining the lipid droplets with Bodipy, it was found that the size and number of lipid droplets in the cells were significantly reduced after GGOH treatment. Figure 2 B).

[0079] Experimental Example 2

[0080] GGOH inhibits the development and progression of fatty liver disease in mice.

[0081] This invention first investigated the changes of GGPP synthase GGPPS1 in mouse NAFLD. This invention induced the NAFLD phenotype in mice using three dietary modalities: high-fat, methionine-restricted and choline-deficient diet (HFMRCD), high-fructose high-fat diet (HCHFD), and Western diet (WD). Figure 3 A). It was found that GGPPS1 levels showed an increasing trend in the early stages of the disease, followed by a decreasing trend in the later stages. Figure 3 B represents the changes in GGPPS1 protein levels in the livers of the three mouse models. Figure 3 C represents the change in GGPPS1 mRNA levels in the liver of HCHFD-induced NAFLD model mice. Figure 3 D represents the immunohistochemical results of GGPPS1 in the liver of HCHFD-induced NAFLD model mice.

[0082] Given that this invention discovered the relationship between the level of the GGPP synthase GGPPS1 and the progression of NAFLD in the liver of mice, this invention subsequently investigated the role of GGOH in the process of NAFLD in mice. The results showed that after 6 weeks of induction with a high-fat, methionine-restricted, and choline-deficient diet (HFMRCD), wild-type mice exhibited a significant NASH phenotype in their livers, while two weeks after intraperitoneal injection of GGOH, it significantly suppressed liver fibrosis and inflammation-related genes and phenotypes in mice. Figure 4 A is a schematic diagram of GGOH treatment in NAFLD mice. Figure 4 B represents the change in liver weight ratio in mice before and after GGOH treatment. Figure 4 C represents the appearance characteristics of mouse livers before and after GGOH treatment. Figure 4 D represents the changes in the levels of GGPPS1 and fibrin α-SMA proteins in mouse liver before and after GGOH treatment. Figure 4 E represents the morphological characteristics of mouse liver cells before and after GGOH treatment, including cell size, degree of fibrosis, and lipid droplet accumulation.

[0083] Experimental Example 3

[0084] GGOH inhibits the development and progression of liver cancer in mice.

[0085] This invention further investigated the role of GGOH in the development of primary hepatocellular carcinoma in mice. The results showed that after 30 weeks of induction with a high-fat diet and chemical drugs, wild-type mice exhibited a clear primary hepatocellular carcinoma phenotype in their livers. However, intraperitoneal injection of GGOH during modeling significantly inhibited the development and progression of primary hepatocellular carcinoma in mice. Figure 5 A is a schematic diagram of GGOH intervention in the treatment of hepatocellular carcinoma (HCC) mice. Figure 5 B represents the overall changes in mouse liver tumors before and after GGOH intervention. Figure 5 C represents the size and number of liver tumors in mice before and after GGOH intervention.

[0086] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above-described experimental examples. The experimental examples and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. The application of a small molecule endogenous metabolite, GGOH, in the preparation of drugs for fatty liver disease, characterized in that: The structural formula of the GGOH is shown in formula (I): After entering the liver, GGOH is spontaneously converted into GGPP, which inhibits hepatocyte glycolysis, upregulates gluconeogenesis, and restores hepatic glucose and lipid metabolism homeostasis, thereby inhibiting the occurrence and development of fatty liver disease.

2. The application according to claim 1, characterized in that: The fatty liver disease includes any one of simple fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), NASH-associated liver fibrosis, or NASH-associated cirrhosis.

3. The use of the endogenous metabolite small molecule GGOH as described in claim 1 in the preparation of liver cancer drugs, characterized in that: After entering the liver, GGOH can be spontaneously converted into GGPP, which inhibits abnormal glycolysis in hepatocytes, upregulates gluconeogenesis, and reverses the Warburg effect, thereby inhibiting the proliferation of liver cancer cells and inducing their apoptosis.

4. The application according to claim 3, characterized in that: The liver cancers mentioned include primary hepatocellular carcinoma (HCC), hepatocellular carcinoma combined with non-alcoholic steatohepatitis (NASH), or hepatocellular carcinoma driven by glucose and lipid metabolism disorders.

5. The application according to claim 3, characterized in that: The GGOH restores hepatic glucose homeostasis by reducing the activity of the key glycolytic enzyme PKM2 and activating the rate-limiting gluconeogenesis enzyme FBP1.

6. The application according to claim 3, characterized in that: The GGOH reduces hepatic steatosis by inhibiting the expression and activity of key lipid synthesis enzymes ACC, FASN, and SCD1, thereby lowering the levels of triglycerides and cholesterol esters in hepatocytes.

7. A pharmaceutical composition for treating fatty liver disease or liver cancer, characterized in that: The pharmaceutical composition comprises a therapeutically effective amount of the compound GGOH of claim 1 or 3, and a pharmaceutically acceptable carrier or excipient.

8. The pharmaceutical composition according to claim 7, characterized in that: The dosage form includes any one of oral formulations, injectable formulations, or targeted delivery formulations.

9. A method for inhibiting the occurrence and development of fatty liver disease, characterized in that: Administering a therapeutically effective amount of the compound GGOH as described in claim 1 or 3 to a patient to restore hepatic glucose and lipid metabolism homeostasis and inhibit disease progression.

Images