CN117482096A - Application of LF3 in preparation of arthritis treatment drugs - Google Patents
Application of LF3 in preparation of arthritis treatment drugs Download PDFInfo
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Classifications
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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
- A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
- A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
-
- 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
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- Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Immunology (AREA)
- Epidemiology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Rheumatology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physical Education & Sports Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses application of LF3 in preparation of a medicament for treating arthritis and a pharmaceutical composition containing LF3 and used for treating osteoarthritis, and belongs to the technical field of medicaments. The LF3 can obviously reduce the expression of MMP-13, ADAMTS5 and COX-2 genes and lighten the inhibition effect after IL-1 beta stimulation. The pharmaceutical composition comprises LF3 powder, DMSO, PEG300, tween-80 and physiological saline; wherein the molecular formula of LF3 is C 20 H 24 N 4 O 2 S 2 . The pharmaceutical composition can improve the inflammatory manifestation and cartilage destruction of knee osteoarthritis and reduce the formation of cartilage injury, thereby delaying the progress of the disease course of osteoarthritis, has no toxicity and has obvious treatment effect on osteoarthritis.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to application of LF3 in preparation of medicines for treating arthritis.
Background
Arthritis is a widely known inflammatory disease occurring in joints and surrounding tissues of the human body and can be divided into tens of kinds. More than 1 hundred million people suffering from arthritis in China are growing. Clinically, the symptoms of red, swelling, heat, pain, dysfunction and joint deformity of joints are that the serious symptoms cause joint disability and influence the life quality of patients. Clinically common arthritis mainly includes the following: rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, gouty arthritis, reactive arthritis, infectious arthritis, traumatic arthritis, psoriatic arthritis, and enteropathic arthritis.
Among them, osteoarthritis (OA), which is the most common type of arthritis, is one of the common age-related degenerative diseases. Most patients with OA are middle-aged and elderly people, and OA is prone to disability. In pathological manifestations, OA mainly undergoes changes such as articular cartilage degeneration, subchondral bone sclerosis, synovial hyperplasia inflammation, and marginal osteophyte formation; clinically, OA patients are mainly manifested as chronic pain, stiffness, swelling, deformity of the joints, so that joint movement limitation occurs later in the disease. Because the exact cause and pathogenesis of the OA are not completely clear, the treatment means of the OA are very limited, mainly aiming at relieving symptoms and recovering functions, no therapeutic drug which has obvious toxic and side effects and can greatly improve the OA is found at present. Therefore, the search for more effective drugs for the treatment of osteoarthritis is a major problem currently in urgent need.
LF3 is a 4-thiourea-benzenesulfonamide derivative, and researches show that LF3 serving as an inhibitor of beta-catenin/TCF 4 interaction can effectively reduce tumor growth and induce differentiation in a xenograft model of a colon cancer mouse, and does not cause cell death or interfere with cadherin-mediated intercellular adhesion. Based on this, experiments show that: LF3 can increase Na+ channel activity in mice HL-1 cardiomyocytes; LF3 can reduce hepatic fibrosis in mice; LF3 can prevent the development of pulmonary arterial hypertension (Pulmonary hypertension, PH) in rats. At present, no report on the aspect of treating arthritis by LF3 is known.
Disclosure of Invention
In order to overcome the defects of the prior art, one of the purposes of the invention is to provide the application of LF3 in preparing medicines for treating arthritis.
In order to solve the problems, the technical scheme adopted by the invention is as follows:
application of LF3 in preparation of arthritis treatment drugs, wherein the molecular formula of LF3 is C 20 H 24 N 4 O 2 S 2 。
As a preferred embodiment of the present invention, the arthritis is osteoarthritis.
It is another object of the present invention to provide a pharmaceutical composition for treating osteoarthritis, comprising LF3 powder, DMSO, PEG300, tween-80, and physiological saline; wherein the molecular formula of LF3 is C 20 H 24 N 4 O 2 S 2 。
As a preferred embodiment of the invention, the LF3 powder has a purity of 99.55% and is available from MedChemExpress.
As a preferred embodiment of the present invention, the pharmaceutical composition is in the form of an injection.
Further, the application method of the pharmaceutical composition is intraperitoneal injection.
As a preferred embodiment of the invention, the LF3 powder is dissolved in DMSO to form a clarified LF3 stock solution with LF3 concentration of more than or equal to 25.0mg/mL, and then the clarified LF3 stock solution is mixed with PEG300, tween-80 and normal saline to prepare the clarified pharmaceutical composition with concentration of more than or equal to 2.5 mg/mL.
As a preferred embodiment of the present invention, the DMSO is present in the pharmaceutical composition in a volume ratio of 5 to 10%.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, LF3 powder is dissolved in DMSO to form LF3 stock solution, and experiments prove that the LF3 powder can obviously reduce the expression of MMP-13, ADAMTS5 and COX-2 genes (P < 0.05) and lighten the inhibition effect after IL-1 beta stimulation (P < 0.05); LF3 stock solution is mixed with PEG300, tween-80 and normal saline to prepare a pharmaceutical composition, and the pharmaceutical composition is absorbed in physiological environment to play a pharmacological role, and experiments prove that the pharmaceutical composition can improve the inflammatory manifestation of knee osteoarthritis and cartilage destruction, reduce the formation of cartilage injury, improve the calcification condition of cartilage, relatively increase the area of cartilage tissue, reduce cartilage degradation, increase cartilage thickness and decrease arranged cartilage cells, and reduce cartilage destruction, so that the pharmaceutical composition provided by the invention has remarkable treatment effect on osteoarthritis.
In addition, in order to reduce the toxicity of DMSO to animals, LF3 stock solution, PEG300, tween-80 and physiological saline are mixed according to a specific proportion, so that the final concentration of DMSO is not more than 10%, and the pharmaceutical composition has no toxicity. Meanwhile, because the DMSO has extremely strong bactericidal power, the prepared pharmaceutical composition is a sterile solution, and in actual production, the aseptic production can be realized by only maintaining the operation environment and instrument sterility.
Drawings
FIG. 1 is a graph showing the results of the measurement of chondrocyte CCK-8 according to example 2 of the present invention;
FIG. 2 is a graph showing qRT-PCR results of related genes of interest in various chondrocytes according to example 2 of the present invention;
FIG. 3 is a graph showing the results of Western Blot detection of major target proteins within various groups of chondrocytes according to example 2 of the present invention;
FIG. 4 is a Micro-CT image of a mouse knee joint in example 3 of the present invention;
FIG. 5 is a graph showing the results of O-fast green staining and OARSI scoring of mouse knee safranin in example 3 of the present invention;
FIG. 6 is a graph showing the results of immunofluorescence and immunohistochemistry of knee joint in mice in example 3 of the present invention.
Detailed Description
The invention is described in further detail below with reference to the drawings and the detailed description.
The pharmaceutical composition for treating osteoarthritis provided by the invention comprises LF3 powder (with the purity of 99.55%, purchased from MedChemexpress company), DMSO, PEG300, tween-80 and physiological saline; wherein the molecular formula of LF3 is C 20 H 24 N 4 O 2 S 2 The molecular weight is 416.56, and the molecular structural formula is shown as follows:
the preparation method of the pharmaceutical composition comprises the following steps:
s1, adding DMSO into LF3 powder, and dissolving to obtain LF3 stock solution with LF3 concentration more than or equal to 25.0 mg/mL;
s2, adding the LF3 stock solution into the PEG300, uniformly mixing, then adding Tween-80, and uniformly mixing; finally adding physiological saline and uniformly mixing to obtain the clear pharmaceutical composition with concentration of more than or equal to 2.5 mg/mL. Wherein, the volume ratio of DMSO in the pharmaceutical composition is 5-10%.
Taking 1mL of working solution as an example, taking 100 mu L of 25.0mg/mL of clarified DMSO stock solution, adding the stock solution into 400 mu L of PEG300, and uniformly mixing; adding 50 mu L of Tween-80 into the system, and uniformly mixing; then, 450. Mu.L of physiological saline was further added to fix the volume to 1mL.
The above medicinal composition can be prepared at present and used in the same day. If precipitation and precipitation occur during the preparation process, the solution can be assisted by heating and/or ultrasonic treatment to clarify the solution.
Example 1: preparation method of pharmaceutical composition for treating osteoarthritis
The present embodiment provides a method for preparing a pharmaceutical composition for treating osteoarthritis, comprising the steps of:
s1, taking 100mg of LF3 powder, adding 4.0mL of DMSO and dissolving to obtain 25.0mg/mL of LF3 stock solution;
s2, sequentially mixing LF3 stock solution with the mass ratio of 10%, 40% of PEG300, 5% of Tween-80 and 45% of physiological saline: taking 1mL of the pharmaceutical composition as an example, 100 mu L of 25.0mg/mL of clarified LF3 stock solution is added into 400 mu L of PEG300, and the mixture is uniformly mixed; adding 50 mu LTwen-80 into the system, and uniformly mixing; then, 450. Mu.L of physiological saline was further added to fix the volume to 1mL.
When the pharmaceutical composition is used, the low-dose group and the high-dose group are respectively injected with the drugs by the abdominal cavity according to the dosages of 1mg/kg and 10 mg/kg.
Example 2: in vitro detection of proliferation-toxicity of mouse chondrocyte CCK-8 cells and drug intervention experiment after stimulation by IL-1 beta
The procedure for CCK-8 cell proliferation-toxicity assay was as follows:
s1, using F2 generation chondrocytes, adjusting the concentration of cell suspension, and adding 100 mu L of the cell suspension into each hole of a 96-well plate to adjust the density of cells to be detected to 5000/hole.
S2: cells were allowed to adhere to the walls of the incubator at 37℃for 24 hours, 5 wells were provided for each group, one plate was treated according to LF3 (0, 20, 40, 80, 120. Mu.M), and the other plate was treated with IL-1β8ng/ml simultaneously with LF3 (treatment time of the drug in the following experiment).
S3: after the treatment was completed 10ul of CCK-8 was added to each well, taking care not to generate air bubbles, and incubated at 37 ℃ for 2 hours in the dark.
S4: after the incubation time is over, the absorbance can be read at a wavelength of 450nm using a fully automatic microplate reader. A control group (cell viability 100%) was defined and the cell activities of the remaining groups were calculated according to the following formula to determine whether LF3 was toxic to cells. Cell viability= [ interference group λ (450 nm) -blank group λ (450 nm) ]/[ control group λ (450 nm) -blank group λ (450 nm) ]x100%.
The procedure for the cell intervention test was as follows:
s1, 5mg of LF3 powder is taken, 1.2003mL of DMSO is added to dissolve the LF3 powder into an LF3 stock solution with the concentration of 10mM, and the LF3 stock solution is used for cell intervention experiments.
S2, culturing cells by using a six-hole plate and an F12 complete culture medium, and randomly dividing the cartilage cells of the mice into 4 groups for test: normal, IL-1β (8 ng/ml), low dose intervention (20 uM at LF3 intervention), high dose intervention (80 uM at LF3 intervention). The medicine is stimulated for 24 hours, then total RNA and protein of the cartilage cells are extracted, and real-time fluorescence quantitative PCR (RT-qPCR) and Western immunoblotting (Western blot) experiments are respectively carried out, and the results are shown in figures 2 and 3.
As shown in fig. 1, the LF3 drug intervention concentration was not significantly toxic to chondrocytes in the 120uM range, thus demonstrating that the pharmaceutical composition of the present invention was not significantly cytotoxic.
FIG. 2 shows that MMP-13, ADAMTS5, COX-2 gene expression levels were significantly increased in the IL-1β group compared to the normal control group; the LF 3-intervention group significantly reduced MMP-13, ADAMTS5, COX-2 gene expression (P < 0.05) compared to the model group. For chondroprotective cytokines such as COL2, agg and SOX-9, the inhibition effect following IL-1β stimulation was reduced following LF3 intervention (P < 0.05).
FIG. 3 shows that LF3 intervention group has increased intracellular Collagen II and Aggrecan protein expression (P < 0.05) compared with IL-1 beta group; MMP13 and ADAMTS5 protein expression levels were significantly reduced (P < 0.05).
Example 3: knee osteoarthritis mouse model test
1. Experimental animals, modeling and administration
40 male C57 mice were purchased from Changsha Biotechnology Co., ltd, 10 weeks old. All mice are raised in animal houses matched with the national medical college life and scientific research center in the right river, the environment accords with the clean-level experimental animal management standard, the corncob padding is replaced every 2-3 days, and special maintenance feed and required clean drinking water for the mice are regularly supplemented. It was randomly divided into 4 groups, respectively: normal, modular, low dose, high dose. The model and high-low dose treatment groups were modeled for knee arthritis using DMM (free right lower limb medial meniscus, cut medial collateral ligament) and the drug of example 1 was injected intraperitoneally 2 weeks after DMM: (1) low dose treatment group: dose 1mg/kg (2) high dose treatment group: the injection of 10mg/kg into the abdominal cavity of the mice was performed under conventional sterilization to avoid infection, and normal group and model group were injected with physiological saline for 6 weeks 1 time a day. Samples of articular cartilage were taken 6 weeks after administration, and care was taken to preserve normal structures such as knee articular cartilage for pathological morphological observation.
2. Micro-CT detection
The right lower limb bone tissue of the mice, which have been fixed and washed with the materials, was placed into a newly prepared 50ml centrifuge tube, and at least 3 knee joint tissues were randomly selected from each group for Micro-CT (Perkin Elmer Co., ltd.) scanning. And (3) installing the machine assembly, wherein the scanning range is the whole knee joint tissue, and performing three-dimensional reconstruction operation on the result after the scanning is finished. The results are shown in FIG. 4.
The Micro-CT image of the knee joint of the mouse in fig. 4 shows that, compared with the normal control group, the subchondral bone resorption, the bone vesicle, the bone defect and the formation of a large amount of osteophytes of the knee joint of the OA building module are significantly reduced, and the observation results show that the pharmaceutical composition of the embodiment 1 of the present invention can effectively reduce cartilage destruction, reduce cartilage injury and avoid excessive formation of osteophytes, so that the course of osteoarthritis is relieved.
3. Histomorphology detection
S1, decalcifying the specimen by using the prepared EDTA decalcification solution, dehydrating and transparentizing after 3 weeks, and embedding and tabletting.
S2, after selecting tablets under a mirror, putting the tablets into a 65 ℃ oven for 1.5 hours, and then dewaxing and hydrating.
S3, using a modified safranin-O-fast green cartilage staining kit (Soy Bao Co.) for staining: (1) the freshly prepared Weibert dye is subjected to liquid dyeing for 5 minutes and water washing; (2) the acidic differentiation liquid is differentiated for 3s, the differentiation is carried out one slice by one slice in sequence, and distilled water is used for washing for 10 minutes; (3) the solid green dyeing liquid is dyed for 5 minutes, and the slice is quickly washed for 10-15 seconds by weak acid solution so as to remove residual solid green and air-dried; (4) the safranine dyeing liquid is dyed for 10 to 15 minutes, and dehydrated according to the ethanol of 95 percent for 2 to 3 seconds and the absolute ethanol for 2 to 3 seconds; (5) the xylene is transparent and the optical resin seals. And after the sealing, the glass can be placed under an inverted microscope for observation and photographing. The results are shown in FIG. 5.
The results of the solid green staining of knee safranin O and the OARSI scoring in FIG. 5 show that the cartilage tissue on the surface layer of the knee joint of the mice in the normal control group is smooth and complete, no obvious fibrosis change is seen on the cartilage surface, and the size and the morphology of the cartilage cells in the shallow and deep layers are normal and the distribution arrangement is uniform. The pathological changes of the OA model set basically accord with the changes of human OA, the knee joint cartilage is seriously degenerated, the cartilage surface is uneven, the fibrosis is obvious, a large number of cracks reach subchondral bone deeply, synovitis is obvious, the arrangement of cartilage cells is disordered, and the like. Compared with the OA modeling group, the degeneration of knee joint cartilage of the low-dose drug group is reduced, only a small amount of fibrosis is seen on the surface cartilage of the high-dose drug group, the cell number is slightly less than that of the normal group, the thickness of a cartilage layer is close to that of the normal group, and the stained cartilage matrix is obviously increased. Therefore, the pharmaceutical composition can effectively relieve knee joint inflammation of mice, reduce the damage of cartilage attached to joint surfaces and delay cartilage degeneration of osteoarthritis.
4. Tissue immunofluorescence, immunohistochemical staining
(1) Tissue immunofluorescence
S1, placing a conventional tissue slice (4 μm) in a water bath at 60 ℃ in a citric acid buffer solution overnight, and repairing the tissue antigen.
S2, taking out the slide after the night, washing the slide with PBS for 1 time, then roughly throwing away the water on the slide, placing the slide in a wet box (the specimen faces upwards), and making tissue slices surround the tissue on the slide by an immunohistochemical pen without fixing the tissue slices, and then sealing the tissue slices for 30 minutes by using sealing liquid prepared from serum, triton and PBS.
S3, washing with PBS for 3 times, each time for 5 minutes; the primary antibody is diluted according to the proportion of the instruction book, and is dripped on the tissue in the circle and placed at 4 ℃ overnight.
S4, taking out the wet box from the refrigerator at the temperature of 4 ℃ after overnight, and placing the wet box at the room temperature for 30 minutes, wherein the wet box is washed with PBS for 3 times for 5 minutes each time; according to the protein selection 594/488 fluorescence secondary antibody, incubated for 1 hour at room temperature in the dark, PBS (containing 1%Tween 20) was washed 3 times for 5 minutes each.
S5, dripping DAPI on the tissue surface in the ring, clamping the cover glass by using forceps, tightly attaching the cover glass, taking care of not generating bubbles, and placing the cover glass in an inverted fluorescence microscope for observation and photographing after the operation is finished. The results are shown in FIG. 6.
(2) Tissue immunohistochemistry
S1, placing a conventional tissue slice (4 μm) in a water bath at 60 ℃ in a citric acid buffer solution overnight, and repairing the tissue antigen.
S2, adding a proper amount of endogenous peroxidase blocking agent (10 min at room temperature) after overnight, and flushing with PBS for 3 times each for 3 minutes.
S3, dropwise adding a proper amount of primary antibody (Aggrecan, MMP 13) according to the tissue size, standing at 4 ℃ overnight, and then washing with PBS for 3 times, each time for 3 minutes.
S4, dropwise adding a reaction enhancement solution (20 minutes at 37 ℃), and washing with PBS for 3 times, each time for 3 minutes.
S5, dripping the enhanced enzyme-labeled goat anti-mouse/rabbit IgG polymer (20 minutes at 37 ℃) and washing 3 times by PBS for 3 minutes each time.
S6, dripping DAB color development liquid for 8 minutes at room temperature, and then flushing with distilled water; incubation of hematoxylin staining solution for 3 minutes, washing with distilled water, and returning blue with 1% ammonia water; and (5) photographing and observing the sealing sheet after airing. The results are shown in FIG. 6.
The immunofluorescence and histochemical results of the mouse knee joint sections of FIG. 6 show that the Col2A1 and Aggrecan expression levels of the OA modeling module are significantly reduced (P < 0.05) and MMP13 and ADAMTS5 expression levels are significantly increased (P < 0.05) compared with the Sham group; compared with the OA modeling group, the Col2A1 and Aggrecan expression levels of the high-dose drug group are obviously increased (P < 0.05), and the MMP13 and ADAMTS5 expression levels are obviously reduced (P < 0.05).
Example 3 relevant experimental procedures were performed using the DMM surgery induced mouse OA model, as can be seen: (1) Compared with a modeling module, the Micro-CT and safranin-O-solid green results show that the whole cartilage surface of the drug treatment group has better flatness, reduced cartilage loss, reduced joint synovial inflammation, subchondral bone sclerosis, osteophyte formation and the like; (2) Immunofluorescence and histochemical results show that after intraperitoneal injection of the pharmaceutical composition containing LF3, the content of COL2A1 and Agg in knee joint of the pharmaceutical treatment group is higher than that of OA modeling, and the expression of MMP13 and ADAMTS5 in mice after OA modeling is also remarkably reduced. In general, from the in vivo experimental point of view, it is proved that the pharmaceutical composition containing LF3 can effectively reduce joint inflammation and damage of knee joint cartilage of mice, and further plays a role in delaying the progress of osteoarthritis of mice.
The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, but any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention are intended to be within the scope of the present invention as claimed.
Claims (8)
- The application of LF3 in preparing a medicament for treating arthritis is characterized in that: the molecular formula of the LF3 is C 20 H 24 N 4 O 2 S 2 。
- 2. The use according to claim 1, characterized in that: the arthritis is osteoarthritis.
- 3. A pharmaceutical composition for the treatment of osteoarthritis, characterized in that: comprises LF3 powder, DMSO, PEG300, tween-80 and physiological saline; wherein the molecular formula of LF3 is C 20 H 24 N 4 O 2 S 2 。
- 4. A pharmaceutical composition for the treatment of osteoarthritis as claimed in claim 3, wherein: the dosage form of the pharmaceutical composition is injection.
- 5. The pharmaceutical composition for treating osteoarthritis as claimed in claim 4, wherein: the application method of the pharmaceutical composition is intraperitoneal injection.
- 6. A pharmaceutical composition for the treatment of osteoarthritis as claimed in claim 3, wherein: the LF3 powder is dissolved in DMSO to form a clarified LF3 stock solution with LF3 concentration more than or equal to 25.0mg/mL, and the clarified LF3 stock solution is mixed with PEG300, tween-80 and normal saline to prepare the clarified pharmaceutical composition.
- 7. The pharmaceutical composition for treating osteoarthritis as claimed in claim 6, wherein: the volume ratio of the DMSO in the pharmaceutical composition is 5-10%.
- 8. A method of preparing a pharmaceutical composition for the treatment of osteoarthritis according to any one of claims 3 to 7, characterized in that: the method comprises the following steps:s1, adding DMSO into LF3 powder, and dissolving to obtain LF3 stock solution with LF3 concentration more than or equal to 25.0 mg/mL;s2, adding the LF3 stock solution into the PEG300, uniformly mixing, then adding Tween-80, and uniformly mixing; and finally adding physiological saline and uniformly mixing to obtain the compound.
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