Use of camptothecin glycoside A in preparation of drugs for resisting liver fibrosis or its complications

Abstract

The present application relates to the technical field of medicine, and particularly relates to application of Calanolide A in preparation of medicine for resisting fibrosis or complications thereof. The present application proves that Calanolide A has a strong effect of down-regulating gene expression of human liver stellate cells (LX-2) COL1A1 and COL3A1 in a molecular level experiment, so the compound has the potential to be developed into medicine for resisting liver fibrosis, and has higher safety and lower risk of side effects.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more particularly to the use of crotonin A in the preparation of drugs for the treatment of fibrosis or its complications. Background Technology

[0002] Liver fibrosis is a compensatory response secondary to liver damage and inflammation caused by various pathogenic factors during the tissue repair process. It is a common result and pathological process in the development of many chronic liver diseases. Its characteristic feature is an imbalance between the proliferation and degradation of the extracellular matrix, primarily collagen, leading to abnormal deposition of fibrous connective tissue within the liver. Liver fibrosis is a reversible disease. If the damaging factors are not removed over a long period, the fibrotic process will continue and eventually develop into cirrhosis. However, effective treatment at this stage can prevent liver fibrosis from progressing to fatal diseases such as liver cancer and liver failure.

[0003] Compared to fibrosis in other organs, liver fibrosis is more difficult to cure. On one hand, the causes of liver fibrosis (chronic viral hepatitis, alcoholic liver disease, etc.) are not only difficult to eliminate but also prone to recurrence, continuously damaging the liver. On the other hand, the specific cellular and molecular mechanisms leading to liver fibrosis differ from those in fibrosis in other organs. For example, cardiac fibrosis mainly involves the activation and proliferation of myocardial fibroblasts, while liver fibrosis primarily involves the activation of hepatic stellate cells. Therefore, drugs that have shown good efficacy in treating fibrosis in other organs are difficult to apply to the treatment of liver fibrosis.

[0004] Currently, there are few drugs approved for the treatment of liver fibrosis and related chronic liver diseases, and their safety profiles still need improvement. Ursodeoxycholic acid (UDCA) is approved for the treatment of primary biliary cirrhosis and primary sclerosing cholangitis, but some patients do not respond well to UDCA. Obeticholic acid (OCA) is approved for the treatment of primary cholangitis, but adverse reactions such as pruritus are quite significant. Although silymarin, a natural product, is widely used in the prevention and treatment of liver diseases, its poor water solubility, poor oral absorption, and low bioavailability greatly limit its application.

[0005] It is evident that while current treatments for liver fibrosis have varying degrees of efficacy, they all possess certain limitations. Chemically synthesized drugs and biological agents often exhibit significant side effects, while drugs based on natural products, although possessing advantages such as stable sources, diverse activities, and lower toxicity, also face challenges such as complex mechanisms of action and difficulties in quality control.

[0006] Brucea javanica (L.) Merr., the dried, ripe fruit of the Simaroubaceae plant, is harvested in autumn when the fruit is ripe. Impurities are removed, and the fruit is sun-dried. It is bitter and cold in nature, and slightly toxic. It can clear heat and detoxify, treat malaria, and stop dysentery; externally, it can corrode warts. It is commonly used for dysentery and malaria; externally, it can treat warts and corns. Although Brucea javanica is widely used in traditional Chinese medicine, there are currently no commercially available drugs using it as the main ingredient for treating liver fibrosis. Therefore, the development of drugs targeting Brucea javanica is of innovative significance. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide the use of crotonin A in the preparation of a medicament for the treatment of fibrosis or its complications.

[0008] This invention provides the use of crotonin A in the preparation of drugs for the prevention and treatment of liver fibrosis or its complications.

[0009] The molecular formula of crotonin A described in this invention is C 32 H 42 O 16 It has the molecular structure shown in formula (I). It is a triterpenoid glycoside compound isolated from the plant Brucea javanica, belonging to the Simaroubaceae family.

[0010]

[0011] The COL1A1 and / or COL3A1 genes are closely related to collagen production and are key regulatory factors in the process of liver fibrosis. This invention demonstrates that crotonin A has a strong downregulating effect on the expression of COL1A1 and / or COL3A1 genes in human hepatic stellate cells (LX-2) at the molecular level, thereby achieving an anti-liver fibrosis effect.

[0012] In this invention, the prevention and treatment include significantly downregulating the expression of COL1A1 and / or COL3A1 genes, thereby reducing extracellular matrix production. Furthermore, the COL1A1 and / or COL3A1 genes mentioned in this invention are the COL1A1 and / or COL3A1 genes found in hepatic stellate cells.

[0013] In this invention, the target of prevention and treatment is humans or mammals, such as bovines, equines, sheep, suidae, canines, felines, rodents, and primates. In this embodiment, human-derived hepatic stellate cells are used as the experimental subject to verify anti-liver fibrosis. Therefore, the target of prevention and treatment in this invention is preferably humans. That is, this invention provides the application of crotonin A in the preparation of drugs for preventing and treating liver fibrosis or its complications.

[0014] In this invention, the complications of liver fibrosis include, but are not limited to, small bile duct hyperplasia, functional liver failure, portal hypertension-induced bleeding, ascites, hepatic encephalopathy, hepatorenal syndrome, hepatopulmonary syndrome, and liver cancer.

[0015] In this embodiment of the invention, the effective dose for anti-liver fibrosis is 5-80 μM. For example, 5, 10, 20, 40, or 80 μM.

[0016] Furthermore, the present invention also provides a medicament for preventing and treating liver fibrosis or its complications, comprising crotonin A and pharmaceutically acceptable excipients.

[0017] The pharmaceutical compositions of the active ingredients of this invention can be prepared according to methods known in the art. For this purpose, the active pharmaceutical ingredients of this invention can be combined with one or more solid or liquid pharmaceutical excipients and / or adjuvants to formulate suitable administration or dosage forms for use as human or veterinary medicine. In this invention, the pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, lubricants, flavoring agents, colorants, or preservatives. The fillers may be selected from starch, powdered sugar, lactose, or microcrystalline cellulose; the binders may be selected from povidone, hydroxypropyl methylcellulose, or polyvinyl alcohol; the disintegrants are selected from sodium carboxymethyl starch, crospovidone, crospovidone sodium carboxymethyl cellulose, or low-substituted hydroxypropyl cellulose. The lubricants are selected from magnesium stearate, micronized silica gel, talc, and polyethylene glycol. The flavoring agents are selected from sweeteners (such as sucrose, aspartame, etc.), flavoring agents (such as fragrances, flavorings, etc.), or acidulants (such as citric acid, malic acid, etc.); the colorants include, but are not limited to, iron oxide red, iron oxide yellow, and carmine. The preservatives include, but are not limited to, parabens, benzoic acid and their salts.

[0018] The active ingredient of this invention or a combination of drugs containing it can be administered in unit dose form, and the route of administration can be enteric or non-enteric, such as oral, intravenous, intramuscular, intraperitoneal, nasal, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc., with oral administration being preferred.

[0019] The dosage form can be liquid, solid, or semi-solid. For example, tablets include, but are not limited to, regular tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets; capsules include, but are not limited to, hard capsules, soft capsules, and enteric-coated capsules; injections include, but are not limited to, aqueous injections, powder injections, and infusions. The drugs of this invention can also be formulated into other dosage forms commonly used in the art, such as solutions including, but not limited to, true solutions and colloidal solutions; emulsions including, but not limited to, o / w, w / o, and double emulsions; semi-solid dosage forms including, but not limited to, ointments, gels, pastes, etc.; suspensions, powders, suppositories, films, patches, aerosols, sprays, etc.

[0020] The active ingredients of this invention can be formulated into ordinary preparations, sustained-release preparations, controlled-release preparations, targeted preparations, and various microparticle delivery systems.

[0021] In some embodiments, the dosage form of the drug is a tablet, and the pharmaceutically acceptable excipients include hydroxypropyl methylcellulose, talc, lactose, magnesium stearate, and ethanol; in this embodiment, the tablet contains 0.11% crotonoside A by mass.

[0022] In other embodiments, the dosage form of the drug is a capsule, and the pharmaceutically acceptable excipients include microcrystalline cellulose, starch, sodium metabisulfite, magnesium stearate, and ethanol; in this embodiment, the mass fraction of crotonoside A in the capsule is 0.45%.

[0023] In other embodiments, the dosage form of the drug is granules, and the pharmaceutically acceptable excipients include starch, sodium metabisulfite, magnesium stearate, and ethanol; in this embodiment, the mass fraction of crotonoside A in the granules is 0.54%.

[0024] In other embodiments, the dosage form of the drug is an oral liquid, and the pharmaceutically acceptable excipients include sucrose, sodium bisulfite, methylparaben, sodium bicarbonate, and water; in this embodiment, the concentration of crotonin A in the oral liquid is 0.025 g / L.

[0025] In other embodiments, the drug is in the form of an injection, and the pharmaceutically acceptable excipients include vitamin C, sodium chloride, sodium bicarbonate, and water. In this embodiment, the concentration of crotonin A in the injection is 0.030 g / L.

[0026] Furthermore, the medicament described in this invention may also include other therapeutic agents for liver fibrosis.

[0027] Other treatment agents for liver fibrosis include ursodeoxycholic acid, obeticholic acid, and / or silymarin.

[0028] This invention provides the application of the triterpenoid glycoside compound crocin A in the preparation of anti-hepatic fibrosis drugs. The naturally derived compound crocin A has a strong effect on downregulating the expression of COL1A1 and COL3A1 genes in human hepatic stellate cells (LX-2) in molecular-level experiments. Therefore, this compound has the potential to be developed into an anti-hepatic fibrosis drug with higher safety and lower risk of side effects. Attached Figure Description

[0029] Figure 1 The compound crocin A significantly downregulated the expression of COL1A1 and COL3A1 in the concentration range of 5-80 μM. Detailed Implementation

[0030] This invention provides the application of crotonin A in the preparation of drugs for treating fibrosis or its complications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0031] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0032] Example 1

[0033] I. Using qPCR technology, the effects of monomeric compounds on the expression of COL1A1 and COL3A1 genes were discovered. The specific experimental procedure is as follows (using LX-2 cells as an example):

[0034] 1. LX-2 cells were seeded at 200,000 cells / well in 12-well plates. After 24 hours of cell adhesion and growth, a monomeric compound with a final concentration of 5-80 μM was added. A solvent control group was set up and the cells were treated for 24 hours.

[0035] 2. Use a kit to extract RNA and reverse it into cDNA.

[0036] 3. qPCR reaction, program: 95℃, 3min; (95℃, 3s; 60℃, 30s) 40 cycles.

[0037] The primer sequences are as follows:

[0038] Gene name Forward primer Reverse primer COL1A1 GAGGGCCAAGACGAAGACATC CAGATCACGTCATCGCACAAC COL3A1 CTTCTCTCCAGCCGAGCTTC GTAGTCTCACAGCCTTGCGT

[0039] 4. Data Processing: Using the gene expression level of the blank control group as a reference, the change in gene expression was described by calculating the ratio of gene expression levels between the drug-treated group and the blank control group. A ratio greater than 1 indicates upregulation of gene expression; a ratio less than 1 indicates downregulation of gene expression. A t-test was used to compare whether the difference in gene expression levels between the blank control group and the drug-treated group was significant. P < 0.05 was considered significant (*), and P < 0.01 was considered highly significant (**).

[0040] II. The effects of different concentrations of the compound crotonin A on the expression of COL1A1 and COL3A1 genes, the specific process is as follows:

[0041] 1. The compound crocin A was prepared as a 50 mM DMSO stock solution, and its effects on the expression of COL1A1 and COL3A1 genes were tested at 80, 40, 20, 10 and 5 μM, respectively.

[0042] 2. The experimental steps and result interpretation are the same as in "Using qPCR technology to discover the effect of monomeric compounds on the expression of COL1A1 and COL3A1 genes 1-4".

[0043] 3. Results of compound PCR experiments

[0044] Table 1. PCR results of compound crotonin A at different concentrations.

[0045] Compound concentration (μM) COL1A1 COL3A1 Control group (DMSO) 1.00 1.00 5 0.17 0.10 10 0.20 0.08 20 0.16 0.07 40 0.13 0.08 80 0.13 0.08

[0046] like Figure 1 As shown, the results indicate that the compound crotonin A significantly downregulated the expression of COL1A1 and COL3A1 in the concentration range of 5-80 μM.

[0047] Comparative Example 1: Effects of different concentrations of the positive compound silymarin on the expression of COL1A1 and COL3A1 genes

[0048] The specific process is as follows:

[0049] 1. Silymarin, a positive compound, was prepared as a 50 mM DMSO stock solution, and its effects on the expression of COL1A1 and COL3A1 genes were tested at 80 and 5 μM, respectively.

[0050] 2. The experimental steps and result interpretation are the same as in "Using qPCR technology to discover the effect of monomeric compounds on the expression of COL1A1 and COL3A1 genes 1-4".

[0051] 3. PCR results of different concentrations of the positive compound silymarin

[0052] compound COL1A1 COL3A1 Control group (DMSO) 1.00 1.00 Silymarin 80μM 0.83 0.52 silymarin 5μM 1.12 0.78

[0053] The results showed that the compound crocin A was superior to the positive control drug silymarin in downregulating the expression levels of COL1A1 and COL3A1 genes.

[0054] Comparative Example 2: Effects of different monomeric compounds at the same concentration on the expression of COL1A1 and COL3A1 genes

[0055] The specific process is as follows:

[0056] 1. The monomeric compounds luteolin, oleuropein, and linoleic acid were prepared into a 50 mM DMSO stock solution, and the effects on the expression of COL1A1 and COL3A1 genes at 5 μM were tested.

[0057] 2. The experimental steps and result interpretation are the same as in "Using qPCR technology to discover the effect of monomeric compounds on the expression of COL1A1 and COL3A1 genes 1-4".

[0058] 3. PCR results of different compounds in Brucea javanica at a concentration of 5 μM

[0059] compound COL1A1 COL3A1 Control group (DMSO) 1.00 1.00 Brucine A 5μM 0.17 0.10 Luteolin 5μM 0.55 0.53 5 μM oleuropein 0.85 0.76 Linoleic acid 5μM 1.07 0.94

[0060] The results showed that at a concentration of 5 μM, the compound crotonin A was superior to other compounds in Brucea javanica, such as luteolin, oleuropein, and linoleic acid, in downregulating the expression levels of COL1A1 and COL3A1 genes.

[0061] Example 2

[0062] This embodiment provides a tablet using crocin A as the active pharmaceutical ingredient, and its components are as follows:

[0063] crocin A 20.0mg Hydroxypropyl methylcellulose 18g talcum powder 0.4g lactose 0.2g magnesium stearate 0.2g Anhydrous ethanol Appropriate amount

[0064] Make 100 pieces.

[0065] Take crocin A, hydroxypropyl methylcellulose, talc, lactose, and magnesium stearate, mix them evenly, add anhydrous ethanol to make a soft mass (the amount of anhydrous ethanol added is such that the soft mass can be clumped when squeezed and dispersed when pressed), pass it through a 24-mesh sieve to make granules, dry it, add magnesium stearate, mix it evenly, and compress it into tablets.

[0066] Example 3

[0067] This embodiment provides a capsule formulation using crocin A as the active pharmaceutical ingredient, the components of which are as follows:

[0068]

[0069]

[0070] Make 100 pills.

[0071] After mixing crotonin A with starch, microcrystalline cellulose, and sodium metabisulfite, anhydrous ethanol is added to make a soft mass (the amount of anhydrous ethanol added is such that the soft mass can be clumped when squeezed but crumbles when pressed). The mass is then passed through a 24-mesh sieve to make granules, dried, and magnesium stearate is added. The mixture is then filled into capsules.

[0072] Example 4

[0073] This embodiment provides a granule formulation using crocin A as the active pharmaceutical ingredient, with the following components:

[0074] crocin A 35.0mg starch 6g Sodium metabisulfite 0.2g magnesium stearate 0.2g Anhydrous ethanol Appropriate amount

[0075] Make 100 bags.

[0076] After mixing crotonin A with starch and sodium bisulfite, anhydrous ethanol is added to make a soft mass (the amount of anhydrous ethanol added is such that the soft mass can be clumped when squeezed but crumbles when pressed). The mass is then passed through a 24-mesh sieve to make granules, dried, and magnesium stearate is added. The mixture is then mixed and packaged.

[0077] Example 5

[0078] This embodiment discloses an oral liquid using the compound crotonin A as a raw material, the components of which are as follows:

[0079] crocin A 25.0mg sucrose 2.0g Sodium bisulfite 0.2g Methylparaben 0.2g Sodium bicarbonate 0.1mL Water for Injection 1000mL

[0080] Prepare 100 vials. After mixing the above components, dispense them using standard oral liquid preparation methods.

[0081] Example 6

[0082] This embodiment provides an injection using crotonin A as the active pharmaceutical ingredient, the components of which are as follows:

[0083] crocin A 30.0mg Vitamin C 0.2g Sodium chloride 6.0g Sodium bicarbonate 0.1mL Water for Injection 1000mL

[0084] Prepare 100 vials. After mixing the above components, 100 vials can be obtained using conventional injection preparation methods.

[0085] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of crotonin A in the preparation of drugs for the prevention and treatment of liver fibrosis or its complications.

2. The application according to claim 1, characterized in that, The prevention and treatment include suppressing the levels of the COL1A1 and / or COL3A1 genes.

3. The application according to claim 1, characterized in that, The COL1A1 and / or COL3A1 genes are COL1A1 and / or COL3A1 genes in hepatic stellate cells.

4. The application according to claim 1, characterized in that, The target of the prevention and control measures is humans or other mammals.

5. The application according to claim 4, characterized in that, The mammals mentioned include bovines, equines, sheep, pigs, canines, felines, rodents, and primates.

6. The application according to any one of claims 1 to 5, characterized in that, The dosage for prevention and treatment is 5-80 μM.

7. Drugs for the prevention and treatment of liver fibrosis or its complications, including crotonin A and pharmaceutically acceptable excipients.

8. The medicament according to claim 7, characterized in that, The dosage form is tablets, and the pharmaceutically acceptable excipients include hydroxypropyl methylcellulose, talc, lactose, magnesium stearate, and ethanol; Its dosage form is capsules, and the pharmaceutically acceptable excipients include microcrystalline cellulose, starch, sodium metabisulfite, magnesium stearate and ethanol; Its dosage form is granules, and the pharmaceutically acceptable excipients include starch, sodium metabisulfite, magnesium stearate and ethanol; Its dosage form is an oral liquid, and the pharmaceutically acceptable excipients include sucrose, sodium bisulfite, methylparaben, sodium bicarbonate and water; Its dosage form is an injection, and the pharmaceutically acceptable excipients include vitamin C, sodium chloride, sodium bicarbonate, and water.

9. The medicament according to claim 8, characterized in that, The tablet contains 0.11% crotonin A by mass. The mass fraction of crotonin A in the capsule is 0.45%. The mass fraction of crotonin A in the granules is 0.54%. The concentration of crotonin A in the oral liquid is 0.025 g / L; The concentration of crotonin A in the injection is 0.030 g / L.

10. The medicament according to any one of claims 7 to 9, characterized in that, It also includes other treatments for liver fibrosis. Other treatment agents for liver fibrosis include ursodeoxycholic acid, obeticholic acid, and / or silymarin.

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