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WO2019075865A1 - Applications d'hederagénine et de dérivé ou de sel d'hederagénine dans la préparation de médicaments pour le traitement de l'arthrose - Google Patents

Applications d'hederagénine et de dérivé ou de sel d'hederagénine dans la préparation de médicaments pour le traitement de l'arthrose Download PDF

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WO2019075865A1
WO2019075865A1 PCT/CN2017/113615 CN2017113615W WO2019075865A1 WO 2019075865 A1 WO2019075865 A1 WO 2019075865A1 CN 2017113615 W CN2017113615 W CN 2017113615W WO 2019075865 A1 WO2019075865 A1 WO 2019075865A1
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chondrocytes
heds
salt
joint
derivative
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Chinese (zh)
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马仁强
陆静云
马艺华
叶治营
周清
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Guangzhou Boji Medical Biotechnological Co Ltd
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Guangzhou Boji Medical Biotechnological Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the present invention relates to a novel use of an ivy saponin and a derivative thereof or a salt thereof, and particularly to the use of an ivy saponin and a derivative thereof or a salt thereof for the preparation of a medicament for treating osteoarthritis.
  • Arthritis refers to inflammatory lesions of the joint and its surrounding tissues caused by inflammation, infection, trauma or other factors, which are characterized by red, swollen, hot, painful and dysfunctional joints; can be classified into rheumatoid arthritis according to the cause , osteoarthritis, ankylosing spondylitis, reactive arthritis, gouty arthritis, the most common of which are rheumatoid arthritis and osteoarthritis.
  • Rheumatoid arthritis is an autoimmune disease of unknown etiology characterized by symmetrical, aggressive synovitis with the most involvement of proximal interphalangeal joints, metacarpophalangeal joints, wrist and toe joints. See; late appearance of muscle atrophy around the joints, joint rigidity, ulnar subluxation of the metacarpophalangeal joint, finger "swan neck” and “button flower” and other deformities. Laboratory tests may have elevated erythrocyte sedimentation rate, C-reactive protein and rheumatoid factor, but no specificity; anti-cyclic citrullinated peptide antibody, anti-keratin antibody positive specificity > 90%.
  • Osteoarthritis is the second most common cause of threats to human health. "The most immediate symptom is joint pain.”
  • OA is a chronic progressive osteoarthrosis, a chronic joint disease characterized by degeneration of bone and joint cartilage and peripheral bone hyperplasia. It is also known as degenerative arthritis. It is often found in the middle-aged and elderly population, often involving the whole body.
  • the joints with large weights such as the spine, hip joint and knee joint in the joint cause the joint to be stiff and deformed; the specific manifestations are: the distal interphalangeal joint and the proximal interphalangeal joint have a bony nodule, a square hand, and a "snake.
  • the type of hand, knee varus, knee valgus, etc. so that joint function is seriously affected; in the laboratory examination, erythrocyte sedimentation rate, C-reactive protein and rheumatoid factor are generally no abnormalities.
  • OA joint degenerative disease
  • OA is characterized by progressive degeneration of articular cartilage, chondrocyte apoptosis, subchondral bone reconstruction, inflammation, synovitis and osteophyte formation, causing pain in affected joints and limited activity.
  • ASA American College of Rheumatology
  • Primary OA is also called "idiopathic OA". The exact cause is unclear. It is considered to be caused by various factors such as genetics, environment and climate.
  • Secondary OA refers to secondary joint trauma, congenital or hereditary diseases, endocrine and metabolic diseases, inflammatory joint diseases, endemic joint diseases, and other osteoarthrosis.
  • Interleukin-1 Interleukin-1
  • TNF tumor necrosis factor
  • IL-6 interleukin-6
  • IL-1 ⁇ Interleukin-1
  • IL-17 tumor necrosis factor
  • IL-18 interleukin-6
  • LIF leukemia inhibitory factor
  • chemokines also play important roles in the pathophysiology of OA.
  • proinflammatory cytokines involved in OA IL-1 ⁇ and TNF are considered to be the most important cytokines, mainly produced by chondrocytes, monocytes, osteoblasts and synovial tissues; IL-1 ⁇ is mainly involved in cartilage destruction.
  • IL-1 ⁇ and TNF can simultaneously induce the production of a large number of inflammatory and degrading factors.
  • High levels of IL-1 ⁇ and TNF expression in synovial fluid, synovial tissue, subchondral bone and cartilage in patients with OA have demonstrated that IL-1 ⁇ and TNF reduce major cell extracellular activity by inhibiting chondrocyte protein anabolic activity. Synthesis of matrix components.
  • IL-1 ⁇ and TNF stimulate chondrocytes to release a variety of proteolytic enzymes, including matrix metalloproteinases (MMPs); play an important role in cartilage destruction, thereby enabling alternative anti-cytokine therapy, especially for targeted therapy of MMPs, Will become the main means of treatment of OA.
  • MMPs matrix metalloproteinases
  • the current goal of OA treatment is to relieve pain, prevent and delay the progression of the disease, protect joint function, and improve quality of life.
  • the guidelines for the diagnosis and treatment of osteoarthritis suggest that the treatment plan should be individualized, taking into account the patient's risk factors, the location of the affected joints, joint structure changes, inflammation, pain, accompanying disease and other specific conditions and conditions.
  • the principle of treatment should be based on non-drug therapy combined with drug therapy, if necessary, surgical treatment.
  • Non-pharmacological treatment plays an important role in the treatment of OA, including patient education, exercise, life guidance and physical therapy; drug treatment is mainly divided into drugs for controlling symptoms, drugs for improving disease and chondroprotective agents; for medical treatment Patients with no significant curative effect, severe lesions, and significant joint function disorders may consider surgical treatment to correct for deformities and improve joint function.
  • the main route of surgical treatment is through arthroscopic surgery and open surgery.
  • Oral drugs include acetaminophen, non-steroidal anti-inflammatory drugs (NSAIDs), and opioids.
  • NSAIDs non-steroidal anti-inflammatory drugs
  • Synovitis of OA is not a major factor in the early stage of the disease. Therefore, mild analgesics can be used as a preferred drug such as acetaminophen for short-term use.
  • the main adverse reactions are gastrointestinal symptoms and hepatotoxicity.
  • NSAIDs have both analgesic and anti-inflammatory effects. They are the most commonly used drugs for controlling OA symptoms. They mainly reduce prostaglandin synthesis by inhibiting cyclooxygenase activity, and reduce pain and swelling caused by joint inflammation and improve joints.
  • Injectables are glucocorticoids, hyaluronic acid and NSAIDs.
  • Intra-articular injection of long-acting glucocorticoids can relieve pain and reduce exudation. The effect lasts for several weeks to several months, but should not be repeated in the same joint. The injection interval should not be shorter than 4-6. Months.
  • Non-drug therapy and simple analgesics can not be used in the treatment of knee joint OA with intra-articular injection of hyaluronic acid (glass acid), which is effective in relieving joint pain, increasing joint mobility and protecting cartilage. For several months; has a good effect on mild to moderate OA.
  • Knee intra-articular injection once a week, 4 to 6 weeks for a course of treatment the frequency of injection can be adjusted according to the patient's symptoms.
  • Intramuscular injection of NSAIDs has a fast onset and gastrointestinal reactions are not obvious.
  • Topical applications include NSAIDs and capsaicin.
  • Topical topical NSAIDs can reduce joint pain and have less adverse reactions; capsaicin emulsion can consume P substance from local sensory nerve endings, which can relieve joint pain and tenderness.
  • the slow acting drug (DMOAD) and chondroprotective agent for treating osteoarthritis generally have a slower onset of action, and it takes several weeks to be effective; it has the effects of reducing the activity of matrix metalloproteinase and collagenase, and is effective against inflammation and pain. It can protect articular cartilage and delay the development of OA.
  • the commonly used drugs such as glucosamine, chondroitin sulfate and diacerein may have certain effects; however, it has a long onset time, high cost and time cost, and has certain restrictions on its promotion. .
  • Glucosamine is a natural amino monosaccharide, which is an essential component for the synthesis of proteoglycans in human articular cartilage matrix; it can improve the metabolism of articular cartilage, improve the repair ability of articular cartilage, protect damaged articular cartilage, and relieve pain of OA. Symptoms, improve joint function, delay the pathological process and disease progression of OA, and thus have both symptomatic regulation and structural regulation effects; need to continue taking more than 8 weeks, and more stable after 1 year of use.
  • Chondroitin sulfate competitively inhibits the activity of degrading enzymes, reduces the destruction of cartilage matrix and synovial fluid components; improves the blood circulation of synovial membrane and subchondral bone by reducing the formation of fibrin thrombus; can effectively alleviate the symptoms of OA and reduce Pain, improve joint function, and reduce the amount of NSAIDs or other analgesics.
  • Diacerein is an interleukin (IL)-1 inhibitor that inhibits cartilage degradation, promotes cartilage synthesis, and inhibits synovial inflammation. It can not only effectively improve the symptoms of osteoarthritis, relieve pain, improve joint function, and has a follow-up effect; it can also delay the progression of OA, and has a structural regulation. This drug does not inhibit the synthesis of prostaglandins. Generally take no less than 3 months.
  • IL-1 interleukin-1 inhibitor that inhibits cartilage degradation, promotes cartilage synthesis, and inhibits synovial inflammation. It can not only effectively improve the symptoms of osteoarthritis, relieve pain, improve joint function, and has a follow-up effect; it can also delay the progression of OA, and has a structural regulation. This drug does not inhibit the synthesis of prostaglandins. Generally take no less than 3 months.
  • Ivy saponin is a common aglycone in Chinese medicinal materials, mainly found in sequelae, pre-existing seeds, Mutong, honeysuckle and so on.
  • studies on ivy saponins and derivatives thereof or salts thereof have focused on anti-diabetic and anti-depressant, and the use of ivy saponins and derivatives thereof or salts thereof for the preparation of a medicament for treating osteoarthritis No reports have been reported yet.
  • the primary object of the present invention is to provide an application of the Ivy saponin represented by the following formula (I) and a derivative thereof or a salt thereof for the preparation of a medicament for treating osteoarthritis;
  • R 3 is H, Na, K or NH 3 .
  • R 1 and R 2 are the same and represent H or —CO(CH 2 ) 2 COOH.
  • R 3 is H.
  • R 1 and R 2 represent -CO(CH 2 ) n COOH
  • the carboxyl group thereof may further react with a base to form one or more salts, preferably, the ivy sapogenin represented by the general formula (I) and its derivative.
  • the salt of the substance is a sodium salt or a potassium salt.
  • the ivy saponin can be extracted from a Chinese medicinal material by a known preparation method or directly obtained from the market.
  • the ivy saponin derivative can be obtained from the saponin by an acylation reaction, an esterification reaction or a transesterification reaction, and the derivative can be further reacted with a base to obtain a salt thereof.
  • Another object of the present invention is to provide a medicament for treating osteoarthritis comprising the ivy saponin having the formula (I) and a derivative thereof or a salt thereof and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field, including, but not limited to, a specific classification, including: one or more of an excipient such as starch or water; Or one or more of starch, lactose, dextrin, sucrose, microcrystalline cellulose or mannitol; binders such as pregelatinized starch, dextrin, cellulose derivatives, alginates, gelatin Or one or more of polyvinylpyrrolidone or polyethylene glycol; a disintegrating agent such as sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, microcrystalline cellulose, sodium carboxymethyl cellulose or One or more of vidoketone and the like; a lubricant such as magnesium stearate, micronized silica gel, talc, hydrogenated vegetable oil, polyethylene glycol, sodium lauryl sulfate or glycerin or the like a plurality of; a wetting agent such as one or more of water or
  • the ivy saponin having the formula (I) and a derivative thereof or a salt thereof according to the present invention can also be used in combination in the form of a composition, for example, the ivy saponin of the present invention and a derivative thereof can be used. Or a salt thereof is added to a pharmaceutical composition suitable for administration to a subject.
  • the composition, particularly the pharmaceutical composition may take a wide variety of forms including, for example, one or more of a liquid, semi-solid, and solid dosage form; wherein the pharmaceutical composition comprises a therapeutically effective amount of formula (I)
  • the illustrated ivy sapogenin and its derivatives or salts thereof are the active ingredients, together with one or more pharmaceutically acceptable carriers.
  • the medicament comprising the ivy saponin represented by the general formula (I) and a derivative thereof or a salt thereof can be prepared into various dosage forms by a conventional production method well known in the art, for example, by mixing the active ingredient with one or more carriers. And then make it into the desired dosage form.
  • the pharmaceutical dosage form includes a tablet, a capsule, a granule, a suspension, an emulsion, a solution, a syrup or an injection, and is taken orally or by injection (including one of intravenous injection, intravenous drip, intramuscular injection or subcutaneous injection). Treatment or scientific research of osteoarthritis and its directly related diseases by one or more routes of administration.
  • the above pharmaceutical or pharmaceutical composition can be administered by various routes, and in the form of use, the preferred form is an oral preparation (such as a tablet, a coated tablet, a capsule, a solution or a suspension), and a parenteral administration form.
  • an oral preparation such as a tablet, a coated tablet, a capsule, a solution or a suspension
  • a parenteral administration form e.g., an injection, an ointment or a patch
  • a tablet or a capsule e.g., a tablet or a capsule.
  • the test results prove that the Ivy saponin represented by the general formula (I) and its derivatives or salts thereof have a breakthrough in the treatment mechanism of the osteoarthritis, which not only promotes cartilage Cell proliferation significantly inhibits H 2 O 2 -induced chondrocyte apoptosis; and promotes chondrocyte secretion of matrix metalloproteinase inhibitor (TIMP-1), inhibits chondrocyte secretion of matrix metalloproteinases (MMPs), IL-6 It can also inhibit the inflammatory response induced by MIA, inhibit the expression of IL-1 ⁇ , TNF- ⁇ in the serum of osteoarthritis rats and the degradation of proteoglycan and type II collagen in cartilage. Rats given a good therapeutic effect of 1 ⁇ 10mg / kg.
  • the effective dosage of the ivy saponin having the formula (I) and a derivative thereof or a salt thereof according to the present invention is 5 to 100 mg/time, and the dosage is 1-3 times a day;
  • the dosage is usually determined by the age and weight of the patient or user as well as the condition of the body or the condition of the patient.
  • the invention has the following beneficial effects:
  • the ivy saponin represented by the general formula (I) and a derivative thereof or a salt thereof can promote the proliferation of primary chondrocytes and can significantly inhibit the chondrocyte apoptosis induced by H 2 O 2 . It inhibits the secretion of MMPs and IL-6 by chondrocytes and promotes the secretion of TIMP-1 by chondrocytes.
  • the administration of the Ivy saponin and its derivatives or its salts in vivo can inhibit the inflammatory response induced by MIA and inhibit IL-1 ⁇ in serum.
  • TNF- ⁇ expression and inhibition of matrix metalloproteinase MMPs, promote the expression of TIMP-1, indicating that the Ivy saponin and its derivatives or their salts have protective knee articular cartilage, improve osteoarthritis, and have the exact treatment of osteoarthritis The effect can improve the quality of life of patients with osteoarthritis.
  • the present invention has found through pharmacological studies the pharmacology of the Ivy saponin represented by the general formula (I) and derivatives thereof or salts thereof
  • the activity breaks through the clinical simple anti-inflammatory and analgesic, improves joint cavity lubrication, or cartilage protection.
  • cartilage damage repair promotes chondrocyte proliferation, inhibits degradation of proteoglycan and type II collagen in cartilage, and is superior to positive drug Diacerein
  • anti-cartilage damage inhibittion of chondrocytes secreting MMPS, promoting TIMP-1 secretion, inhibiting secretion of IL-6 by chondrocytes
  • good anti-inflammatory effects reducing serum IL-1 ⁇ , TNF- ⁇ levels
  • triple action on osteoarthritis may form a breakthrough therapy, truly play a part-time treatment, become a clinical first-line medication.
  • Figure 1A 10 times magnification of the primary chondrocyte morphology photograph isolated and cultured for 24 h in Example 1;
  • Figure 1B 10 times magnification of the primary chondrocyte morphology photograph of the isolated culture of Example 1 for 72 h;
  • Figure 1C Photograph of passaged chondrocytes of passage (passage of passage 3) of Example 1 of 10 times magnification;
  • Figure 2 Photograph of HEDS for gross knee observation of knee joints in rats with osteoarthritis induced by sodium iodoacetate.
  • Example 1 In vitro experiment of therapeutic effect of ivy saponin represented by the general formula (I) and derivatives thereof or salts thereof on osteoarthritis
  • Hydrogen peroxide H 2 O 2
  • source Merck, Germany
  • fetal bovine serum FBS fetal bovine serum FBS
  • DMEM high glucose medium
  • trypsin Trypsin 1:250
  • HEPES solution all source: American life technologies company GIBCO
  • thiazole blue (MTT) source: Amresco, USA
  • dimethyl sulfoxide (DMSO) source: Nanjing Shengxing Biotechnology Co., Ltd.
  • type II collagenase source: Sigma, USA
  • toluidine blue dye solution trypan blue staining Liquid, source: Beijing Suo Laibao Technology Co., Ltd.
  • Positive control drug diacerein
  • source Shanghai Jingchun Biochemical Technology Co., Ltd.
  • toluidine blue staining is used as a method for identifying chondrocytes.
  • the cells cultured in the laser confocal dish were washed three times with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. Add 1% toluidine blue stain and incubate for 2 h at 37 °C. The cells were observed under an inverted fluorescence microscope and photographed.
  • the cells cultured in the laser confocal dish were removed and washed three times with PBS. 4% paraformaldehyde was fixed at room temperature for 30 min, and PBST (PBS + 0.1% Triton X 100) was permeabilized for 5 min. After 5% BSA was blocked at room temperature for 1 h, type II collagen antibody was added and incubated overnight at 4 °C. The anti-rabbit secondary antibody labeled with fluorescein isothiocyanate (FITC) was added the next day and incubated for 2 h at room temperature in the dark. Add 10 ⁇ g/L DAPI and incubate for 5 min at room temperature in the dark. Finally, anti-fluorescence quenching and sealing solution was added, and the cells were observed under an inverted fluorescence microscope and photographed.
  • FITC fluorescein isothiocyanate
  • Chondrocytes in the logarithmic growth phase were trypsinized and seeded in 96-well plates at 5000/well cells. Each well was inoculated with 100 ⁇ l and placed in a CO 2 incubator for static culture. 6 duplicate wells per group. After the cells were attached, the culture solution was discarded, and compound HED and HEDS (concentration: 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 ⁇ M) were added to each well, and the cell viability was measured by MTT assay after 3 days of incubation. The light absorption values of the respective wells were measured on a microplate reader at 570 nm and 650 nm, and the results were recorded.
  • Chondrocytes in the logarithmic growth phase were trypsinized and seeded in 96-well plates at 5000/well cells.
  • the experimental procedure was the same as in 2.2.3, and the positive control drug diacerein (concentration: 1 ⁇ M) was added, and after 7 days of culture, the cell viability was detected by MTT assay.
  • the light absorption values of the respective wells were measured on a microplate reader at 570 nm and 650 nm, and the results were recorded.
  • the cell growth curve was plotted with the concentration as the abscissa and the absorbance as the ordinate.
  • Chondrocytes in the logarithmic growth phase were trypsinized and seeded in 96-well plates at 5000/well cells. Each well was inoculated with 100 ⁇ l and placed in a CO 2 incubator for static culture. 6 duplicate wells per group. After culture for 24 hours, cells were attached to the well, and H 2 O 2 was added to each well (concentration: 0.1, 0.2, 0.4, 0.8 mM), and incubation was continued for 30 min, 1 h, 2 h, 4 h. The cell viability was measured by MTT method, and the light absorption values of each well were measured on a dual-wavelength of 570 nm and 650 nm by a microplate reader, and the results were recorded.
  • Chondrocytes in the logarithmic growth phase were trypsinized and seeded in a 60 x 15 mm dish at 2x10 5 cells/dish. The cells were placed in a CO 2 incubator for static growth. On the next day, the compounds HED, HEDS (0.01, 0.1, 1 ⁇ M) and the positive drug diacerein (1 ⁇ M) were administered separately. After 7 days of incubation, 0.8 mM H 2 O 2 was administered to each group for 4 hours. The adherent cells were washed once with PBS, and an appropriate amount of trypsin was added to digest the cells. For specific steps, refer to the instructions of the Biyuntian Biotechnology Annexin V-FITC Apoptosis Kit.
  • the chondrocytes in the logarithmic growth phase were trypsinized and inoculated into a 60 x 15 mm dish at 2 x 10 5 cells/dish in the same manner as in 2.2.6.
  • the small dish was taken out from the incubator, washed with PBS, and then lysed by lysing, and centrifuged at 12,000 rpm for 10 min. The supernatant was taken, diluted 10 times with the sample diluent, and analyzed.
  • the specific steps refer to the ELISA kit instructions of Wuhan Boster Bioengineering Co., Ltd.
  • the chondrocytes in the logarithmic growth phase were trypsinized and inoculated into a 60 x 15 mm dish at 2 x 10 5 cells/dish in the same manner as in 2.2.6. Remove the small dish from the incubator, wash it with PBS and add Trizol lysate to fully lyse. 1st Strand cDNA Synthesis Kit instructions.
  • the staining results showed that the nucleus was dark blue, the cytoplasm was light blue, and the extracellular matrix was light blue.
  • the glycosaminoglycan sulfate in the cells was positive, indicating that the normal phenotypic protein expression of chondrocytes was active.
  • the staining results showed that the cytoplasm of the cells showed green fluorescence, and all of them were positive results.
  • the nucleus was stimulated by DAPI to induce blue fluorescence. Almost 100% of cells express type II collagen, suggesting that chondrocytes are highly pure.
  • the compound HED was 0.01-10 ⁇ M, and the compound HEDS was not cytotoxic to chondrocytes in the concentration range of 0.01-100 ⁇ M.
  • n 6, * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, compared with the control group
  • n 6, * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, compared with the control group
  • Compounds HED and HEDS can promote the proliferation of primary chondrocytes.
  • the cell proliferation curve can be obtained according to the MTT results.
  • the optimal rate of increase of HED in the concentration of 0.01 ⁇ M is 312.43% within seven days.
  • the optimal rate of increase of HEDS in the seven days at 0.01 ⁇ M was 368.04%.
  • the compounds HED and HEDS were better than the positive control drug diacerein in the concentration of 0.01 ⁇ M.
  • Table 3-6 The results are shown in Table 3-6.
  • n 5, * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, compared with the control group
  • n 5, * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, compared with the control group
  • n 5, * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, compared with the control group
  • n 5, * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, compared with the control group
  • n 6, * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, compared with the control group
  • n 3, ### P ⁇ 0.001 compared with the negative control group; ** P ⁇ 0.01, *** P ⁇ 0.001, compared with the model group.
  • n 3, ### P ⁇ 0.001 compared with the negative control group; ** P ⁇ 0.01, *** P ⁇ 0.001, compared with the model group.
  • n 3, ### P ⁇ 0.001 compared with the negative control group; * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, compared with the model group.
  • n 3, ### P ⁇ 0.001 compared with the negative control group; * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, compared with the model group.
  • chondrocytes can synthesize a large amount of substances such as type II collagen and proteoglycan (PG). Therefore, type II collagen and proteoglycan (PG) are characteristic secretions of cartilage, which is the identification of chondrocytes. The essential. The extracted cells were highly purified chondrocytes by toluidine blue staining and type II collagen immunofluorescence staining.
  • the toxicity and proliferation of the cytotoxic cells by the MTT assay were examined by MTT assay.
  • the results showed that the compound HED was 0.01-10 ⁇ M, HEDS was not cytotoxic to chondrocytes in the concentration range of 0.01-100 ⁇ M, and the compound promoted the proliferation of primary chondrocytes in a safe concentration range.
  • H 2 O 2 can induce chondrocyte apoptosis and articular cartilage degradation. Therefore, H 2 O 2 was used to establish a model of apoptosis induced by oxidative stress in articular chondrocytes, and MTT method was used to screen out appropriate H. 2 O 2 concentration and time of action, and then differentiated cells in each stage of death by Annexin V-FITC/PI double staining. The results showed that HED and HEDS can significantly inhibit H 2 O 2 induced chondrocytes in the concentration range of 0.01-1 ⁇ M. Apoptosis.
  • Example 2 In vivo experiment of the therapeutic effect of ivy saponin and its derivative or its salt represented by the general formula (I) on osteoarthritis
  • mice 60 male Sprague-Dawley rats aged 8 weeks, weighing 200 ⁇ 50g, intra-articular injection of 2mg/mL iodine 50 ⁇ l of a physiological saline solution of sodium acetate caused a rat osteoarthritis model.
  • SD rats were randomly divided into 4 groups: normal control group (Con group) 10, model group (MIA group) 10, HEDS group (MIA+HEDS 0.1 mg/kg, A1 group; MIA+HEDS 1 mg/kg, A2) Group; MIA + HEDS 10 mg / kg, A3 group) 10 each.
  • MIA + Diacerein 10 mg / kg, D1 group The Con group and the MIA group were given normal saline every day.
  • the rats in the A1, A2, A3 and D1 groups were given HEDS (0.1, 1 or 10 mg/kg) and diacerein (10 mg/kg) per day. .
  • HEDS 0.1, 1 or 10 mg/kg
  • diacerein 10 mg/kg
  • Blood was collected from the abdominal aorta of each group, and the levels of IL-1 ⁇ and TNF- ⁇ were detected in the serum of rats.
  • the specific steps refer to the ELISA kit instructions of Wuhan Boster Bioengineering Co., Ltd.
  • Rats in each group were sacrificed on schedule, and the right knee joint was excised.
  • the articular cartilage tissue was quickly scraped off with a surgical blade, placed in a pre-frozen mortar, and 1 ml of Trizol lysate was added. After fully grinding, it was allowed to stand for 5 minutes. Centrifugation at 12,000 rpm and 4 ° C. Refer to the specific steps. 1st Strand cDNA Synthesis Kit instructions.
  • the degree of swelling of the joint was expressed by the diameter of the knee joint of the rat.
  • the diameter of the knee joint was significantly increased after MIA modeling compared with the normal group.
  • the 28 days after HEDS administration can alleviate joint swelling in rats, and has certain therapeutic effects on MIA-induced knee arthritis in rats. The results are shown in Figure 2 and Table 14.
  • n 6, # P ⁇ 0.05 , comparing ## P ⁇ 0.01, ### P ⁇ 0.001 and the negative control group; * P ⁇ 0.05, ** P ⁇ 0.01, comparing *** P ⁇ 0.001, with model group
  • the results of Q-PCR showed that the expression levels of type II collagen, proteoglycan and TIMP-1 in the MIA group were significantly decreased, and the expressions of MMP-2, MMP-9 and MMP-13 were significantly increased, and 1 mg/d after administration.
  • Kg, 10mg / kg dose of HEDS can increase the expression of type II collagen and proteoglycan and TIMP-1, reduce the expression of MMP-2, MMP-9, MMP-13, the effect is better than the positive drug diacerein, see the results Table 16.
  • n 3, # P ⁇ 0.05 , comparing ## P ⁇ 0.01, ### P ⁇ 0.001 and the negative control group; * P ⁇ 0.05, ** P ⁇ 0.01, comparing *** P ⁇ 0.001, with model group
  • an animal model of osteoarthritis was established using sodium iodoacetate (MIA).
  • MIA sodium iodoacetate
  • the MIA model has the advantages of simple and easy injection method and short cycle, which is suitable for small animal experiments, thus significantly reducing the requirements for drug dosage in drug screening.
  • the experimental results show that HED and HEDS have a certain therapeutic effect on experimental OA in rats. Mainly to inhibit joint swelling in experimental OA rats, degradation of extracellular matrix of chondrocytes, secretion of inflammatory factors IL-1 ⁇ and TNF- ⁇ in serum.
  • HEDS can promote the proliferation of chondrocytes, inhibit the damage of chondrocytes and the production of MMPs, thereby reducing the damage and degradation of articular cartilage.
  • the compounds also have a certain therapeutic effect on experimental OA.
  • the above-mentioned raw materials and auxiliary materials are respectively passed through a 100 mesh sieve, and the sieved HED, sodium carboxymethyl starch, starch, and microcrystalline cellulose are weighed according to the above-mentioned prescription amount, and uniformly mixed, and the soft material is made into an appropriate amount with 50% medicinal ethanol.
  • the wet granules of 20 mesh sieves were dried at 55 ° C for 4 hours, granulated with a 24-mesh sieve, uniformly mixed with magnesium stearate, and compressed, and 932 pieces were prepared.
  • the above-mentioned raw materials and auxiliary materials are respectively passed through a 100 mesh sieve, and the sifted HEDS, microcrystalline cellulose, lactose and hypromellose are weighed according to the above-mentioned prescription amount, uniformly mixed, and made into a soft material with 60% medicinal ethanol.
  • the wet granules of 20 mesh sieves were dried at 55 ° C for 4 hours, granulated with a 24-mesh sieve, uniformly mixed with magnesium stearate, and tableted to obtain 948 tablets.
  • the above-mentioned raw materials and auxiliary materials are respectively passed through an 80 mesh sieve, and the sieved HED, sodium carboxymethyl starch, and microcrystalline cellulose are weighed according to the above-mentioned prescription amount, and uniformly mixed, and 50% of medicinal ethanol is used to make soft materials, and 20 meshes are obtained.
  • the wet granules were sieved, dried at 55 ° C for 4 hours, granulated with a 24-mesh sieve, uniformly mixed with magnesium stearate, and filled with capsules to prepare 951 capsules.
  • the above-mentioned raw materials and auxiliary materials are respectively passed through a 80 mesh sieve, and the sifted HEDS, dextrin and sucrose are weighed according to the above-mentioned prescription amount, uniformly mixed, and wetted with purified water to form a soft material, and passed through a 12-mesh sieve to prepare wet granules, 55 ° C The mixture was dried for 4 hours, and the whole granules were dispensed by a composite aluminum film to obtain 964 bags.

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  • Orthopedic Medicine & Surgery (AREA)
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Abstract

La présente invention concerne des applications d'hederagénine représentée par la formule générale (I) et un dérivé ou un sel d'hederagénine dans la préparation de médicaments pour le traitement de l'arthrose. Dans la formule générale (I), R1 et R2 sont identiques ou différents et représentent indépendamment l'un de l'autre H ou -CO(CH2)nCOOH, n=1-3 ; et R3 est H, Na, K ou NH3.
PCT/CN2017/113615 2017-10-19 2017-11-29 Applications d'hederagénine et de dérivé ou de sel d'hederagénine dans la préparation de médicaments pour le traitement de l'arthrose Ceased WO2019075865A1 (fr)

Applications Claiming Priority (2)

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CN201710979238.7A CN107550917B (zh) 2017-10-19 2017-10-19 常春藤皂苷元及其衍生物或其盐在制备治疗骨性关节炎药物中的应用
CN201710979238.7 2017-10-19

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WO2019075865A1 true WO2019075865A1 (fr) 2019-04-25

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1385158A (zh) * 2002-05-20 2002-12-18 吉林天药科技股份有限公司 常青藤皂苷元在制备抗高血脂症药物中的应用
WO2003099305A1 (fr) * 2002-05-27 2003-12-04 Eun-Bang Lee Composition contenant un extrait soluble d'acetate d'ethyle produit a partir de kalopanax pictus nakai, et derives de kalopanaxsaponin a isoles dudit extrait pour prevenir ou traiter une maladie inflammatoire ou rhumatismale

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100546999C (zh) * 2007-08-13 2009-10-07 中国科学院新疆理化技术研究所 常春藤皂苷元及其制备方法和用途

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1385158A (zh) * 2002-05-20 2002-12-18 吉林天药科技股份有限公司 常青藤皂苷元在制备抗高血脂症药物中的应用
WO2003099305A1 (fr) * 2002-05-27 2003-12-04 Eun-Bang Lee Composition contenant un extrait soluble d'acetate d'ethyle produit a partir de kalopanax pictus nakai, et derives de kalopanaxsaponin a isoles dudit extrait pour prevenir ou traiter une maladie inflammatoire ou rhumatismale

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CN107550917B (zh) 2020-04-24

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