WO2019029526A1 - 二甲双胍在制备预防和治疗骨关节炎以及缓解骨关节炎引起的疼痛症状相关药物中的应用 - Google Patents

二甲双胍在制备预防和治疗骨关节炎以及缓解骨关节炎引起的疼痛症状相关药物中的应用 Download PDF

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WO2019029526A1
WO2019029526A1 PCT/CN2018/099180 CN2018099180W WO2019029526A1 WO 2019029526 A1 WO2019029526 A1 WO 2019029526A1 CN 2018099180 W CN2018099180 W CN 2018099180W WO 2019029526 A1 WO2019029526 A1 WO 2019029526A1
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metformin
group
osteoarthritis
intervention
chondrocyte
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PCT/CN2018/099180
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French (fr)
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雷光华
曾超
魏捷
崔洋
丁翔
李辉
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中南大学湘雅医院
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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
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • 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
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • 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 metformin, in particular to the use of metformin in the preparation of a medicament for the prevention and treatment of osteoarthritis and for alleviating pain symptoms caused by osteoarthritis.
  • Osteoarthritis is a joint degenerative disease characterized by articular cartilage degeneration, subchondral bone sclerosis and osteophyte formation. It is mainly characterized by post-activity joint pain, activity limitation and joint deformation. Clinical manifestations, often involving weight-bearing joints.
  • OA Osteoarthritis
  • the disability rate of OA ranks second among all diseases, second only to cardiovascular disease.
  • the Chinese epidemiological survey the prevalence of OA in people over 65 years old is more than 50%. About 80% of OA patients have a certain degree of exercise restriction, and 25% of OA patients have significant influence on daily life.
  • the cost of treating OA can be as high as 150 billion yuan. With the global aging population and the increase in obesity, the prevalence of OA will increase further.
  • OA patients are mostly middle-aged and elderly, often accompanied by other systemic diseases, such as digestive system and cardiovascular diseases, and OA's first-line treatment drugs (symptoms relief) such as Non-steroidal antiinflammatory drugs (Non-steroidal antiinflammatory drugs,
  • NSAIDs Non-steroidal antiinflammatory drugs
  • the use of NSAIDs is likely to cause an increase in the risk of gastrointestinal side effects and cardiovascular events. Therefore, it is urgent to explore safe and effective treatments for patients with early and mid-stage OA.
  • metformin As one of the most widely used drugs for the treatment of type 2 diabetes worldwide, metformin has been recommended as a first-line drug due to its established efficacy, good safety and low cost. It has been used for more than 60 years. history. Metformin can reduce the production of glycogen, reduce the absorption of sugar in the small intestine, and increase the uptake and utilization of peripheral sugars, increase the sensitivity of insulin, thereby improving blood glucose tolerance and reducing basic and postprandial blood glucose. Unlike other types of hypoglycemic drugs, metformin does not generally cause hypoglycemia in patients with type 2 diabetes or normal subjects. In addition to type 2 diabetes, metformin has been shown to be useful in the treatment of other metabolic diseases such as the metabolic syndrome.
  • metformin can reduce the risk of various malignant tumors, such as breast cancer, colon cancer and prostate cancer, that is, metformin may have super-intended use, but whether metformin has anti-cancer Efficacy is still a huge controversy in academia.
  • AMPK adenosine monophosphate-activated protein kinase
  • metformin as an AMPK agonist, may delay the occurrence and development of OA by inhibiting inflammatory response and chondrocyte apoptosis.
  • metformin can regulate the metabolism of microbial folate and methionine to delay aging, and OA is a common aging disease.
  • metabolic diseases such as diabetes may be a risk factor for the development of OA, which may increase the risk of OA, and metformin, as a first-line drug for type 2 diabetes, is very effective in treating diabetes and reducing hyperglycemia. .
  • metformin can activate AMPK, and AMPK may play an important role in the development of OA; at the macro level, metformin has been found to have anti-aging effects, and OA is a common aging disease; metformin It is a classic first-line drug for the treatment of type 2 diabetes, and diabetes is considered a possible risk factor for OA. However, it has not been mentioned in previous patents and publications that metformin can be used to protect chondrocytes and cartilage, and no studies have found that metformin can be used for the prevention and treatment of OA and alleviating the pain symptoms caused by it.
  • metformin in the first embodiment of the present invention, it has been shown by epidemiological investigation that metformin can significantly reduce the risk of osteoarthritis, and that metformin can also delay the progression of osteoarthritis.
  • metformin can further inhibit cartilage matrix degradation and degeneration by activating AMPK, thereby further exerting a cartilage protective function.
  • metformin treatment not only reduces the degree of cartilage damage in the mouse OA model, but also is effective in reducing cartilage matrix degradation, reducing cartilage surface wear and delaying cartilage degeneration, and Relieve the pain symptoms caused by osteoarthritis.
  • the present invention provides the use of metformin for the preparation of a medicament for the prevention and treatment of osteoarthritis.
  • the present invention also provides the use of metformin in the preparation of a medicament for alleviating the symptoms of pain caused by osteoarthritis.
  • the present invention also provides a pharmaceutical composition for preventing and treating osteoarthritis and alleviating pain symptoms of osteoarthritis, wherein the pharmaceutical composition contains a salt of metformin or metformin (a medically acceptable salt such as metformin hydrochloride) ). And a pharmaceutically acceptable diluent or carrier.
  • a salt of metformin or metformin a medically acceptable salt such as metformin hydrochloride
  • the pharmaceutical composition further contains a molecule for enhancing the extracellular matrix, and the weight ratio of the salt of metformin or metformin to the molecule which enhances the extracellular matrix is 1:0.2 to 1:1.
  • the molecule that enhances the extracellular matrix is collagen or glycosaminoglycan.
  • the pharmaceutical composition further comprises a pharmacologically active substance and/or a biologically active substance, and the weight ratio of the salt of metformin or metformin to the pharmacologically active substance and/or the biologically active substance is 1:0.2 to 1: 1.
  • the pharmacologically active substance and/or biologically active substance is a growth factor, a hormone and/or a vitamin.
  • the pharmaceutical composition further comprises an excipient, and the pharmaceutical composition is formulated into any dosage form.
  • the weight ratio of the metformin or metformin salt to the excipient is from 1:10 to 1:100.
  • a human or animal can be treated with an effective amount of metformin or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the same.
  • the metformin of the present invention or a pharmaceutically acceptable salt thereof is formulated into a dosage form suitable for oral and intra-articular injection, and is used for the prevention and treatment of osteoarthritis and the relief of pain symptoms of osteoarthritis. It is effective in reducing the degradation of cartilage matrix, reducing the degree of cartilage surface wear and delaying cartilage degeneration, can delay the occurrence and development of osteoarthritis, and at the same time, it can alleviate the pain symptoms caused by osteoarthritis, and achieve "species and cures" The role.
  • the metformin of the present invention or a pharmaceutically acceptable salt thereof and the other one or more pharmaceutically acceptable carriers/excipients may be prepared as a pharmaceutical composition for preventing and treating osteoarthritis, preferably for intra-articular injection Forms such as solutions and gels.
  • the pharmaceutical form may also consist of the form in which some or all of the components are in dry form (e.g., lyophilized), which are reconstituted with an aqueous solution or other suitable vehicle prior to use.
  • the formulation can be prepared by methods known in the art using known excipients such as binders, disintegrants, fillers, stabilizers, diluents and colorants. They also include delayed release or sustained release formulations prepared using suitable polymers known in the pharmaceutical arts.
  • the pharmaceutical composition is in the form of an oral dosage form or a joint cavity injection.
  • water is preferred as the solvent.
  • metformin or a pharmaceutically acceptable salt thereof with other molecules useful for enhancing the extracellular matrix, such as collagen or glycosaminoglycans, or with other pharmacological and/or biologically active substances such as growth. Combination of factors, hormones and/or vitamins.
  • the present invention is effective for OA
  • the invention can alleviate the pain symptoms caused by OA and achieve the effect of "speculating the symptoms and treating the symptoms";
  • the invention opens up a new use of the traditional hypoglycemic drug metformin, which is safe for metformin, and is not suitable for normal people.
  • Figure 1 MMP-13 mRNA RQ results, 1) C, chondrocytes routine culture control group; 2) C + IL-1, OA chondrocyte control group; 3) C + IL-1 + 1 mM, OA chondrocytes Metformin (1 mM) was intervened in group A; 4) C+IL-1+10 mM, OA chondrocyte metformin (10 mM) was intervened in group B; 5) C+IL-1+20 mM, OA chondrocyte metformin (20 mM) was intervened in group C; RQ, relative expression; IL-1, interleukin-1; *, p ⁇ 0.05.
  • Figure 2 Western blotting to detect the expression of type II collagen and internal reference protein after metformin intervention. From left to right, the lanes were: 1) C, chondrocytes routinely cultured; 2) C+IL-1, OA chondrocyte control group; 3) C+IL-1+1 mM, OA chondrocyte metformin (1 mM) Intervention group A; 4) C+IL-1+10 mM, OA chondrocyte metformin (10 mM) intervention group B; 5) C+IL-1+20 mM, OA chondrocyte metformin (20 mM) intervention group C; IL-1 , interleukin-1; COL2A1, type II collagen; Tublin, tubulin.
  • Figure 3 Western blotting detection of MMP-13 and internal reference protein expression after metformin intervention. From left to right, the lanes were: 1) C, chondrocytes routinely cultured; 2) C+IL-1, OA chondrocyte control group; 3) C+IL-1+1 mM, OA chondrocyte metformin (1 mM) Intervention group A; 4) C+IL-1+10 mM, OA chondrocyte metformin (10 mM) intervention group B; 5) C+IL-1+20 mM, OA chondrocyte metformin (20 mM) intervention group C; IL-1 , interleukin-1; MMP-13, matrix metal degrading enzyme-13; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
  • Figure 4 MMP-13 mRNA RQ results, 1) C+IL-1, OA chondrocyte control group; 2) C+IL-1+metformin, OA chondrocyte metformin intervention group; 3) C+IL-1 +metformin+Dorsomorphin, OA chondrocyte metformin (10 mM) intervention + AMPK block group; 4) C+IL-1+metformin+DMSO, OA chondrocyte metformin (10 mM) intervention +AMPK vehicle control group; RQ, relative expression IL-1, interleukin-1, DMSO, dimethyl sulfoxide; *, p ⁇ 0.05.
  • Figure 5 Western blotting was performed to detect the expression of type II collagen and p-AMPK protein after 24 hours of intervention with metformin and AMPK blockers. From left to right, the lanes were: 1) C+IL-1, OA chondrocyte control group; 2) C+IL-1+metformin, OA chondrocyte metformin (10 mM) intervention group; 3) C+IL-1 +metformin+Dorsomorphin, OA chondrocyte metformin intervention (10 mM) + AMPK blocking group; 4) C+IL-1+metformin+DMSO, OA chondrocyte metformin intervention (10 mM)+AMPK vehicle control group; IL-1, leukocyte Interleukin-1; COL2A1, type II collagen; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
  • IL-1 leukocyte Interleukin-1
  • COL2A1 type II collagen
  • GAPDH glyceraldehyde
  • Figure 6 Western blotting was performed to detect the expression of type II collagen and p-AMPK protein after 48 hours of intervention with metformin and AMPK blockers. From left to right, the lanes were: 1) C+IL-1, OA chondrocyte control group; 2) C+IL-1+metformin, OA chondrocyte metformin (10 mM) intervention group; 3) C+IL-1 +metformin+Dorsomorphin, OA chondrocyte metformin intervention (10 mM) + AMPK blocking group; 4) C+IL-1+metformin+DMSO, OA chondrocyte metformin intervention (10 mM)+AMPK vehicle control group; IL-1, leukocyte Interleukin-1; COL2A1, type II collagen; Tublin, tubulin.
  • Figure 7 Western blotting was performed to detect the expression of MMP-13 and internal reference protein after 48 hours of intervention with metformin and AMPK blockers. From left to right, the lanes were: 1) C+IL-1, OA chondrocyte control group; 2) C+IL-1+Metformin, OA chondrocyte metformin intervention group; 3) C+IL-1+Metformin+ Dorsomorphin, OA chondrocyte metformin intervention + AMPK block group; 4) C+IL-1+Metformin+DMSO, OA chondrocyte metformin intervention + AMPK vehicle control group; IL-1, interleukin-1; MMP-13, Matrix metal degrading enzyme-13; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
  • FIG. 8 Scanning electron microscopy results of knee joint cartilage in blank group mice. It can be seen that the cartilage surface is smooth and complete (the left is 35 times the general view, the right is 500 times the microscopic)
  • FIG 9 Scanning electron microscopy results of knee joint cartilage in sham-operated mice. It can be seen that the cartilage surface is smooth, see a small range of mild degeneration (left is 35 times the general view, right is 500 times the microscopic)
  • FIG 10 Scanning electron microscopy results of knee joint cartilage in saline-administered mice. Visible cartilage extensive range of cracks, exfoliation, subchondral bone exposure (left is 35 times the general view, right is 500 times microscopic)
  • FIG. 11 Scanning electron microscopy results of knee joint cartilage in mice with metformin. It can be seen that the cartilage has cracks and exfoliation, but the range is small and the degree is light. The subchondral bone is not exposed (the left is 35 times the gross view, the right is 500 times the microscopic)
  • FIG 12 Scanning electron microscopy results of knee joint articular cartilage in saline. Visible cartilage extensive range of cracks, exfoliation, subchondral bone exposure (left is 35 times the general view, right is 500 times microscopic)
  • FIG 13 Scanning electron microscopy results of knee joint articular cartilage injected into the knee joint of metformin. It can be seen that the cartilage has cracks and exfoliation, but the extent is small, the degree is light, and the subchondral bone is exposed less (the left is 35 times the general view, the right is 500 times the microscopic)
  • Figure 14 Scanning electron microscopy scores of knee joint cartilage in each group of mice. Among them, 1) C, blank group; 2) Sham, sham operation group; 3) NS-IG, saline gavage group; 4) Metformin-IG, metformin gavage group; 5) NS-IA, saline knee joint Intraluminal injection group; 6) Metformin-IA, metformin knee joint injection group.
  • NS saline
  • IG intragastric administration
  • IA intra-articular injection
  • Metformin metformin *, p ⁇ 0.05.
  • Fig. 15 Effect of metformin on the threshold of mechanical contraction of OA mice. *, P ⁇ 0.05 (compared with saline gavage group and metformin gavage group)
  • Figure 16 Effect of intra-articular injection of metformin on the mechanical threshold of OA mice. *, P ⁇ 0.05 (compared with saline intra-articular injection group and metformin knee joint injection group)
  • Figure 17 Effect of metformin on the weight difference of the hind limbs of OA mice. *, P ⁇ 0.05 (compared with saline gavage group and metformin gavage group)
  • Fig. 18 Effect of intra-articular injection of metformin on the weight difference of the hind limbs of OA mice. *, P ⁇ 0.05 (compared with saline intra-articular injection group and metformin knee joint injection group)
  • Example 1 The epidemiology of Example 1 demonstrates that metformin reduces the risk of developing osteoarthritis and delays its progression.
  • the study was conducted in a prospective cohort study of 4,796 subjects with osteoarthritis or a high risk risk profile for osteoarthritis.
  • the cohort has collected 8 years of follow-up data (annual follow-up), including basic demographic data, clinical features, and evaluation results of knee imaging studies.
  • the correlation was selected from those with osteoarthritis in the baseline population with a KL rating of 2 or 3, and subjects with one follow-up record were included in the study.
  • MIF Medication Inventory Form
  • the results of the imaging examination of all subjects were based on the evaluation results of the database imaging review center.
  • the follow-up records for each year were compared with baseline, and the KL level of the knee joint of the study subject was changed from 0 or 1 to 2 and above during the follow-up period.
  • patients with OA had a KL level of knee joint during the follow-up period compared with baseline during the 8-year follow-up period (level 2 changed to grade 3 or grade 4, 3 The level becomes level 4) It can be judged as the progress of OA.
  • Quantitative data were statistically described using mean and standard deviation, and qualitative data were statistically described using frequency and percentage.
  • the difference of quantitative data was statistically analyzed by t test (normal distribution data) or nonparametric test ( skewed distribution data), and the difference of qualitative data was statistically analyzed by chi-square test.
  • the correlation between metformin and OA progression risk was analyzed by Generalized Estimated Equation (GEE). All data were statistically analyzed by knee joint, and the relative risk (RR) and 95% were calculated.
  • the confidence interval (95% confidence interval, 95% CI) can be used to evaluate the correlation between the two.
  • Model 1 calculates the RR value without covariate correction
  • Model 2 gender, age, body mass index as covariates into the model to correct the RR value
  • model 3 Gender, age, body mass index, exercise volume, education level, degree of knee pain, history of knee joint injury, and KL grade at baseline were used as covariates to adjust RR values in the model. Covariates were measured at baseline. value. All statistical analyses were performed using SAS 9.4 software. The difference was statistically significant at P ⁇ 0.05, and the tests were bilateral tests.
  • the RR value of knee OA progression in the metformin group was 0.34 (95% CI: 0.22 to 0.52, P ⁇ 0.001); adjusted age, gender, BMI, joint injury history, knee pain level, education level, exercise volume, and knee KL level Afterwards, the RR value of the OA progression in the metformin group compared with the metformin group was 0.33 (95% CI: 0.21 to 0.51, P ⁇ 0.001).
  • Model 2 Correcting age, gender, BMI
  • Model 3 Based on Model 2, baseline knee injury history, knee pain level, education level, exercise volume, and knee KL level were corrected.
  • Model 2 Correcting age, gender, BMI
  • Model 3 Based on Model 2, baseline knee injury history, knee pain level, education level, exercise volume, and knee KL level were corrected.
  • Example 2 Cell Level Verification Metformin inhibits OA chondrocyte matrix degradation by activating AMPK
  • Metformin was purchased from Sigma and dissolved in Phosphate Buffer solution (PBS).
  • the AMPK blocker compound C was purchased from Sigma.
  • Interleukin-1 ⁇ (IL-1 ⁇ ) was purchased from R&D Systems.
  • IL-1 ⁇ Interleukin-1 ⁇
  • pAMPK Thr-172 site phosphorylated AMPK
  • AMPK ⁇ AMPK ⁇
  • MMP-13 matrix metalloproteinase 13
  • Col2a1 type II collagen
  • Neonatal mouse chondrocytes were obtained from hip, knee and ankle cartilage of 4-day newborn mice, and primary mouse chondrocytes were cultured. Newborn mice were purchased from Changsha Hunan Slack Jingda Experimental Animal Co., Ltd.
  • the first generation of neonatal mouse knee chondrocytes were cultured in DMEM-F12 medium containing fetal bovine serum (10%) and double antibody (penicillin 50 U/ml, streptomycin 50 U/ml), and placed in CO 2 culture.
  • the cells were cultured in a box (at a constant temperature of 37 ° C, 5% CO 2 , 95% air, 100% humidity), and then incubated with DMEM/F12 containing 5% FBS for 24 hours to synchronize the cells in the non-proliferative phase and the inactive phase.
  • the cells are grown to 70%-80%, the culture is removed and the intervention is performed as follows:
  • Chondrocytes routine culture control group 10% FBS in DMEM/F12 complete medium
  • OA chondrocyte control group 10% FBS DMEM/F12 complete medium was cultured, and 5 ng/ml IL-1 ⁇ was added for intervention;
  • OA chondrocyte metformin intervention group A 10% FBS DMEM/F12 complete medium culture, 5 ng / ml of IL-1 ⁇ was added for intervention, and 1 mM metformin was added for intervention;
  • OA chondrocyte metformin intervention group B 10% FBS DMEM/F12 complete medium culture, 5 ng / ml IL-1 ⁇ was added for intervention, and 10 mM metformin was added for intervention;
  • OA chondrocyte metformin intervention group C 10% FBS DMEM/F12 complete medium was cultured, 5 ng/ml IL-1 ⁇ was added for intervention, and 20 mM metformin was added for intervention.
  • OA chondrocyte control group 10% FBS DMEM/F12 complete medium was cultured, and 5 ng/ml IL-1 ⁇ was added for intervention;
  • OA chondrocyte metformin intervention group A 10% FBS DMEM/F12 complete medium culture, 5 ng / ml IL-1 ⁇ was added for intervention, and 10 mM metformin was added for intervention;
  • OA chondrocyte metformin intervention + AMPK block group 10% FBS DMEM/F12 complete medium was cultured, 5 ng/ml IL-1 ⁇ was added for intervention, and 10 mM metformin and 10 uM Dorsomorphin (AMPK blocker) were added. Intervention;
  • OA chondrocyte metformin intervention + vehicle control group 10% FBS in DMEM/F12 complete medium, 5 ng/ml IL-1 ⁇ was added for intervention, and 10 mM metformin and DMSO (Dorsomorphin vehicle control) were added for intervention. .
  • chondrocyte mRNA and protein were extracted after intervention of the corresponding group of chondrocytes for 24 hours, the expression of MMP-13 mRNA in chondrocytes was detected by qRT-PCR, and the chondrocytes were detected by Western Blot. Expression of type II collagen and MMP-13.
  • the RQ values of MMP-13 mRNA in group B and group C of metformin cells in OA chondrocytes were 0.788 ⁇ 0.048 and 0.711 ⁇ 0.048, respectively, which were lower than OA chondrocytes. In the control group, the difference was statistically significant (p ⁇ 0.001; p ⁇ 0.001).
  • metformin can inhibit the degradation and degeneration of chondrocyte matrix by activating AMPK, and further play a role in cartilage protection.
  • Example 3 verified that metformin can alleviate the degeneration of knee joint cartilage in mice and alleviate the pain symptoms of osteoarthritis in mice.
  • Metformin was dissolved in physiological saline to prepare a 10 -1 M metformin solution, which was stored at -20 ° C until use.
  • An experimental OA model was constructed by inducing OA formation in the right knee joint of the right knee joint in general anesthesia.
  • mice were randomly divided into the following 6 groups:
  • Sham operation group sham operation
  • DMM was used to construct a mouse knee OA model 3 days after the start of normal saline gavage (10ml / kg), once a day for 8 weeks;
  • Metformin group Metformin (200mg/kg) was administered 3 days after DMM to construct mouse knee OA model, once a day for 8 weeks.
  • mice were sacrificed 8 weeks after operation, and the right knee joint specimen was taken.
  • the femur and tibia were carefully separated under a stereo microscope and fixed in 2.5% glutaraldehyde solution for 24 hours.
  • the specimens were processed according to the SEM standard. Observed and photographed under a scanning electron microscope. The degree of wear of the knee cartilage surface of each group of mice was evaluated according to the scanning electron microscopy scoring system.
  • the rating rules are:
  • the cartilage surface is relatively smooth, but visible cartilage fragments are gathered together;
  • the surface of the cartilage is rough, and irregular pieces of cartilage are scattered.
  • the mechanical pain threshold was determined using a von Frey pain tester:
  • Stimulus bending force is 1,1.4,2,4,6,8,10and 15g, from small to large;
  • mice appear to be lifted, lame or evasive, the behavior is considered as a positive reaction, otherwise it is a negative reaction;
  • the difference in weight of the hind limbs was measured with a hindlimb weight gauge:
  • the threshold was greater than the threshold of the reflex foot reflex in the saline-administered group, and the difference was statistically significant (P ⁇ 0.001).
  • the difference in weight of the hind limbs of the mice was statistically significant (P ⁇ 0.001).
  • the difference in the weight of the hind limbs of the mice injected with saline in the intra-articular injection group was lower than that of the mice injected into the intra-articular injection group of metformin. The difference was statistically significant. Significance (P ⁇ 0.001)
  • metformin treatment can not only reduce the degree of cartilage damage in mouse OA model, but also reduce cartilage matrix degradation, reduce cartilage surface wear and delay cartilage degeneration, and relieve pain symptoms caused by osteoarthritis. .
  • mice showed any adverse toxicity at doses up to 200 mg/kg (oral) and 0.1 mmol/kg (knee joint injection). The mice weighed in each group. There was no significant difference between them.

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Abstract

本发明公开了二甲双胍在制备预防和治疗骨关节炎以及缓解骨关节炎引起的疼痛症状相关药物中的应用。本发明对OA疗效确切；开拓了传统降血糖药物二甲双胍的新用途，二甲双胍安全，对正常人没有明显降血糖作用，不会引起低血糖；费用低，耐受性好，易被患者接受。

Description

二甲双胍在制备预防和治疗骨关节炎以及缓解骨关节炎引起的疼痛症状相关药物中的应用 技术领域
本发明涉及一种二甲双胍的新用途，具体是二甲双胍在制备预防和治疗骨关节炎以及缓解骨关节炎引起的疼痛症状相关药物中的应用。
背景技术
骨关节炎(Osteoarthritis,OA)是一种以关节软骨退变、软骨下骨硬化和骨赘形成为主要特点的关节退行性疾病，并以活动后关节疼痛、活动受限和关节变形为其主要临床表现，常累及负重关节。美国50岁以上人群中，OA的致残率位居所有疾病中的第二位，仅次于心血管疾病。中国流行病学调查研究显示，65岁以上人群中OA的患病率超过50％，约80％的OA患者存在一定程度的运动受限，25％的OA患者日常生活受到明显影响，而每年用于治疗OA的费用或高达1500亿元。随着全球老龄化人口以及肥胖人口的增加，OA的患病率还将进一步升高。
目前，国内外尚无明确有效且安全的可延缓OA病情进展的药物。最新的国际OA权威指南明确提出，许多一直以来被广泛用于治疗OA的药物，如透明质酸、氨基葡萄糖、硫酸软骨素和双醋瑞因等，由于最新的高质量的循证医学证据的出现，其有效性在世界范围内引起了广泛的争议，甚至被认定为疗效不确定或不推荐使用(McAlindon T E等，Osteoarthritis and Cartilage，2014，22(3):363‐388)。针对早中期OA的治疗往往只能起到缓解疼痛和改善功能的作用。由于OA患者多为中老年人，常伴有其它系统疾病，如消化系统以及心血管系统疾病，而OA的一线治疗药物(症状缓解)如非甾体类抗炎药(Non‐steroidal antiinflammatory drugs，NSAIDs)的使用易引起胃肠道副作用和心血管事件风险的增加，因此，亟待探索出针对早中期OA患者的安全有效的治疗药物。
二甲双胍作为全世界范围内用于治疗2型糖尿病最广泛的药物之一，由于其确定的疗效、良好的安全性和较低的花费而被推荐为一线药物，距今已有60余年的临床应用历史。二甲双胍可减少肝糖的产生，降低小肠对糖的吸收，并且还可增加外周糖的摄取和利用，提高胰岛素的敏感性，从而提高血糖耐受性，降低基础和餐后血糖。与其他类型降血糖药物不同，二甲双胍单独应用时一般不引起2型糖尿病病人或正常人产生低血糖。除了2型糖尿病，二甲双胍已被证实还可用于治疗其他代谢性疾病，例如代谢综合征。此外，近年来涌现出了大量的观察性研究，发现二甲双胍可降低多种恶性肿瘤的发病风险，例如乳腺 癌、结肠癌和前列腺癌等，即二甲双胍可能具有超说明书用途，但二甲双胍是否具有抗癌功效目前在学术界仍存巨大争议。
既往研究表明OA软骨细胞及软骨组织中腺苷一磷酸激活的蛋白激酶(AMP‐activated protein kinase，AMPK)的表达较正常软骨细胞及软骨组织降低，提示AMPK在OA的发病过程中可能发挥重要作用。包括阿司匹林在内的多种抗炎药已被证实可以通过激活AMPK发挥抗炎作用，AMPK被激活后还可抑制软骨细胞凋亡，因此AMPK可作为治疗OA的潜在靶点。据此可以推测，二甲双胍作为一种AMPK激动剂，可能通过抑制炎症反应和软骨细胞凋亡从而延缓OA的发生和发展。同时，已有研究通过线虫模型发现，二甲双胍可调节微生物叶酸和蛋氨酸代谢从而具有延缓衰老的功效，而OA是一种常见的衰老性疾病，随着年龄的增加，OA的患病率不断增加。此外，既往研究提到糖尿病等代谢性疾病可能为OA发生发展的危险因素，即可能升高OA发病的风险，而二甲双胍作为2型糖尿病的一线药物，治疗糖尿病、降低高血糖的功效已十分明确。
在分子水平，二甲双胍可通过激活AMPK，而AMPK在OA的发生发展中可能起到了重要作用；在宏观水平，二甲双胍已被发现具有抗衰老的功效，而OA是一种常见的衰老性疾病；二甲双胍是一种经典的用于治疗2型糖尿病的一线药物，而糖尿病被认为是OA的可能的危险因素。但是，既往的专利和出版物中均未提到二甲双胍可用于保护软骨细胞和软骨，更无研究发现二甲双胍可用于OA的预防和治疗并缓解其引起的疼痛症状。
发明内容
本发明的第一个实施例中，已通过流行病学调查研究表明二甲双胍可显著降低骨关节炎的发病风险，并且，二甲双胍还可延缓骨关节炎的进展。
本发明的第二个实施例中，已通过细胞实验表明二甲双胍可通过激活AMPK从而抑制软骨细胞基质降解和退变，进一步发挥软骨保护功能。
本发明的另一个实施例中，已表明二甲双胍治疗不但可减轻小鼠OA模型的软骨损伤程度，其在减少软骨基质降解、降低软骨表面磨损程度和延缓软骨退变中是有效的，同时还可缓解骨关节炎引起的疼痛症状。
因此，本发明提供了二甲双胍在制备预防和治疗骨关节炎药物中的应用。
本发明还提供了二甲双胍在制备缓解骨关节炎引起疼痛症状的药物中的应用。
本发明还提供了一种预防和治疗骨关节炎以及缓解骨关节炎疼痛症状的药物组合物，所述药物组合物中含有二甲双胍或二甲双胍的盐(医学上可以接受的盐，如二甲双胍盐酸盐)。以及一种药学上可接受的稀释剂或载体。
优选地，所述药物组合物中还含有用于增强细胞外基质的分子，所述二甲双胍或二甲双胍的盐与增强细胞外基质的分子的重量比为1:0.2至1:1。所述增强细胞外基质的分子为胶原蛋白或糖胺聚糖。
优选地，所述药物组合物中还含有药理学活性物质和/或生物活性物质，所述二甲双胍或二甲双胍的盐与药理学活性物质和/或生物活性物质的重量比为1:0.2至1:1。药理学活性物质和/或生物活性物质为生长因子、激素和/或维生素。
优选地，所述药物组合物中还含有赋形剂，将药物组合物做成任何剂型，。所述所述二甲双胍或二甲双胍的盐与赋形剂的重量比为1:10至1:100。
利用本发明，可以用有效数量的二甲双胍或其药学上可接受的盐或含有它们中存在的药物组合物治疗人或动物。
下面对本发明作进一步说明：
本发明所述二甲双胍或其药学上可接受的盐，配制成适用于口服和关节腔注射的剂型，应用于骨关节炎的预防和治疗以及骨关节炎疼痛症状的缓解。其在减少软骨基质降解、降低软骨表面磨损程度和延缓软骨退变中是有效的，可延缓骨关节炎的发生发展，同时，它还可缓解骨关节炎引起的疼痛症状，达到“标本兼治”的作用。可将本发明所述二甲双胍或其药学上可接受的盐与其他一种或多种可药用的载体/赋形剂制备成预防和治疗骨关节炎的药物组合物，优选适于关节内注射的形式诸如溶液剂和凝胶剂。药用形式还可由以下的形式构成，其中一些或所有组分是干燥形式(如冻干)，在使用前用水溶液或其他适合的溶媒将其重新配制。
所述制剂能使用已知的赋形剂诸如粘合剂、崩解剂、填充剂、稳定剂、稀释剂和着色剂通过本领域所公知的方法而制备。它们还包括用制药技术中已知的适合的聚合物制备的延迟释放或缓释制剂。所述药物组合物的剂型为口服剂型或关节腔注射剂。
对于制备适于注射的液体形式而言，优选水作为溶剂。
如果需要的话，有可能将二甲双胍或其药学上可接受的盐与其他可用于增强细胞外基质的分子，如胶原蛋白或糖胺聚糖联合，或者与其他药理学和/或生物活性物质如生长因子、激素和/或维生素联合。
本发明具有如下优点：
1)本发明对OA疗效确切；
2)本发明可缓解OA引起的疼痛症状，达到“标本兼治”的作用；
3)本发明开拓了传统降血糖药物二甲双胍的新用途，二甲双胍安全，对正常人没有
明显降血糖作用，不会引起低血糖；
4)费用低，耐受性好，易被患者接受；
5)除外二甲双胍传统口服剂型，本发明开发了关节腔注射剂型，且对OA疗效确切。
附图说明
图1 MMP-13 mRNA RQ值结果，其中，1)C,软骨细胞常规培养对照组；2)C+IL-1,OA软骨细胞对照组；3)C+IL-1+1mM,OA软骨细胞二甲双胍(1mM)干预A组；4)C+IL-1+10mM,OA软骨细胞二甲双胍(10mM)干预B组；5)C+IL-1+20mM,OA软骨细胞二甲双胍(20mM)干预C组；RQ,相对表达量；IL-1,白细胞介素-1；*，p＜0.05.
图2 Western blotting检测二甲双胍干预后II型胶原和内参蛋白表达。各泳道从左到右依次为：1)C,软骨细胞常规培养对照组；2)C+IL-1,OA软骨细胞对照组；3)C+IL-1+1mM,OA软骨细胞二甲双胍(1mM)干预A组；4)C+IL-1+10mM,OA软骨细胞二甲双胍(10mM)干预B组；5)C+IL-1+20mM,OA软骨细胞二甲双胍(20mM)干预C组；IL-1,白细胞介素-1；COL2A1，II型胶原；Tublin，微管蛋白。
图3 Western blotting检测二甲双胍干预后MMP-13和内参蛋白表达。各泳道从左到右依次为：1)C,软骨细胞常规培养对照组；2)C+IL-1,OA软骨细胞对照组；3)C+IL-1+1mM,OA软骨细胞二甲双胍(1mM)干预A组；4)C+IL-1+10mM,OA软骨细胞二甲双胍(10mM)干预B组；5)C+IL-1+20mM,OA软骨细胞二甲双胍(20mM)干预C组；IL-1,白细胞介素-1；MMP-13,基质金属降解酶-13；GAPDH，甘油醛-3-磷酸脱氢酶。
图4 MMP-13 mRNA RQ值结果，其中，1)C+IL-1,OA软骨细胞对照组；2)C+IL-1+metformin,OA软骨细胞二甲双胍干预组；3)C+IL-1+metformin+Dorsomorphin,OA软骨细胞二甲双胍(10mM)干预+AMPK阻断组；4)C+IL-1+metformin+DMSO,OA软骨细胞二甲双胍(10mM)干预+AMPK溶媒对照组；RQ,相对表达量；IL-1,白细胞介素-1，DMSO,二甲基亚砜；*，p＜0.05.
图5 Western blotting检测二甲双胍和AMPK阻断剂干预24小时后II型胶原和p-AMPK蛋白表达。各泳道从左到右依次为：1)C+IL-1,OA软骨细胞对照组；2)C+IL-1+metformin,OA软骨细胞二甲双胍(10mM)干预组；3)C+IL-1+metformin+Dorsomorphin,OA软骨细胞 二甲双胍干预(10mM)+AMPK阻断组；4)C+IL-1+metformin+DMSO,OA软骨细胞二甲双胍干预(10mM)+AMPK溶媒对照组；IL-1,白细胞介素-1；COL2A1，II型胶原；GAPDH，甘油醛-3-磷酸脱氢酶。
图6 Western blotting检测二甲双胍和AMPK阻断剂干预48小时后II型胶原和p-AMPK蛋白表达。各泳道从左到右依次为：1)C+IL-1,OA软骨细胞对照组；2)C+IL-1+metformin,OA软骨细胞二甲双胍(10mM)干预组；3)C+IL-1+metformin+Dorsomorphin,OA软骨细胞二甲双胍干预(10mM)+AMPK阻断组；4)C+IL-1+metformin+DMSO,OA软骨细胞二甲双胍干预(10mM)+AMPK溶媒对照组；IL-1,白细胞介素-1；COL2A1，II型胶原；Tublin，微管蛋白。
图7 Western blotting检测二甲双胍和AMPK阻断剂干预48小时后MMP-13和内参蛋白表达。各泳道从左到右依次为：1)C+IL-1,OA软骨细胞对照组；2)C+IL-1+Metformin,OA软骨细胞二甲双胍干预组；3)C+IL-1+Metformin+Dorsomorphin,OA软骨细胞二甲双胍干预+AMPK阻断组；4)C+IL-1+Metformin+DMSO,OA软骨细胞二甲双胍干预+AMPK溶媒对照组；IL-1,白细胞介素-1；MMP-13,基质金属降解酶-13；GAPDH，甘油醛-3-磷酸脱氢酶。
图8空白组小鼠膝关节软骨扫描电镜结果。可见软骨表面光滑、完整(左为35倍大体观，右为500倍微观)
图9假手术组小鼠膝关节软骨扫描电镜结果。可见软骨表面较光滑，见小范围轻度退变(左为35倍大体观，右为500倍微观)
图10生理盐水灌胃组小鼠膝关节软骨扫描电镜结果。可见软骨大范围裂纹、剥脱、软骨下骨暴露(左为35倍大体观，右为500倍微观)
图11二甲双胍灌胃组小鼠膝关节软骨扫描电镜结果。可见软骨存在裂纹、剥脱，但范围较小，程度较轻，基本未暴露软骨下骨(左为35倍大体观，右为500倍微观)
图12生理盐水膝关节腔内注射膝关节软骨扫描电镜结果。可见软骨大范围裂纹、剥脱、 软骨下骨暴露(左为35倍大体观，右为500倍微观)
图13二甲双胍膝关节腔内注射膝关节软骨扫描电镜结果。可见软骨存在裂纹、剥脱，但范围较小，程度较轻，软骨下骨暴露较少(左为35倍大体观，右为500倍微观)
图14各组小鼠膝关节软骨扫描电镜评分。其中，1)C,空白组；2)Sham,假手术组；3)NS-IG,生理盐水灌胃组；4)Metformin-IG,二甲双胍灌胃组；5)NS-IA,生理盐水膝关节腔内注射组；6)Metformin-IA,二甲双胍膝关节腔内注射组。NS,生理盐水；IG,灌胃；IA,关节腔内注射；Metformin二甲双胍；*，p＜0.05.
图15二甲双胍灌胃对OA小鼠机械缩足反射阈值的影响。*，P＜0.05(生理盐水灌胃组和二甲双胍灌胃组比较)
图16二甲双胍膝关节腔内注射对OA小鼠机械缩足反射阈值的影响。*，P＜0.05(生理盐水膝关节腔内注射组和二甲双胍膝关节腔内注射组比较)
图17二甲双胍灌胃对OA小鼠双后肢负重差值的影响。*，P＜0.05(生理盐水灌胃组和二甲双胍灌胃组比较)
图18二甲双胍膝关节腔内注射对OA小鼠双后肢负重差值的影响。*，P＜0.05(生理盐水膝关节腔内注射组和二甲双胍膝关节腔内注射组比较)
具体实施方式
实施例1流行病学证实二甲双胍可降低骨关节炎的发病风险，并延缓其进展。
1.研究方法
1.1研究对象
本研究的研究对象来源于一项前瞻性队列研究，共包含4796名患有骨关节炎或具有骨关节炎高危风险特征的研究对象。该队列已收集8年的随访数据(每年随访一次)，包括研究对象的基本人口学资料、临床特征和膝关节影像学检查的评价结果等。本研究中评价二甲双胍服用情况与OA发生风险的相关性选取的是基线人群中未患骨关节炎的人群(Kellgren and Lawrence(KL)＝0或KL＝1)；评价二甲双胍服用情况与OA进展风险的相关 性选取的是基线人群中患有骨关节炎且KL等级评分为2级或3级的人群，只要有一次随访记录的研究对象即可纳入到本研究中。
1.2二甲双胍服药情况的确定
所有研究对象的服药情况均依据“药物库存系统”(Medication Inventory Form，MIF)中过去30天内医生开具处方药的记录。MIF详细记录了每个研究对象在过去30天内被开具处方药物的商品名、剂型、药物成分名、用药史、用药频率等信息。服用二甲双胍定义为：OA发生或进展人群中，在OA发生和进展以前(基线-上一次随访这段时间)若有服药记录则记录为服用二甲双胍；OA未发生或未进展人群中，基线-随访终止前有服药记录则记录为服用二甲双胍。其他记录为未服用二甲双胍。
1.3骨关节炎发生和进展的确定
所有研究对象的影像学检查的评定结果采用的是该数据库影像学评阅中心的评价结果。在8年的随访期间内，每一年的随访记录与基线时相比，在随访期间研究对象的膝关节的KL等级由0级或1级变为2级及以上等级可判定为OA的发生；基线时为OA患者，在8年的随访期间内，每一年的随访记录与基线时相比，在随访期间内膝关节的KL等级升高(2级变为3级或4级，3级变为4级)可判定为OA的进展。
1.4统计学分析
定量资料采用均数和标准差进行统计描述，定性资料采用频数和百分比进行统计描述。定量资料的差异采用t检验(正态分布资料)或非参数检验(偏态分布资料)进行统计分析，定性资料的差异采用卡方检验进行统计分析。二甲双胍与OA进展风险的相关性采用广义估计方程(Generalized Estimated Equation,GEE)进行分析，所有数据均按膝关节进行统计分析，由其计算出的相对危险度(risk ratio,RR)和95％可信区间(95％confidence interval,95％CI)可评价二者之间的相关性。采用三个不同的模型来估计RR值及其95％CI：模型1，计算未经协变量校正的RR值；模型2，性别、年龄、体重指数作为协变量放入模型中校正RR值；模型3，性别、年龄、体重指数、运动量、教育程度、膝关节疼痛程度、膝关节是否有受伤史和基线时的KL分级作为协变量放入模型中校正RR值，协变量均采用基线时的测量值。所有统计分析均采用SAS 9.4软件进行，P<0.05时可认为差异具有统计学意义，检验均为双侧检验。
2.结果
本研究共纳入研究对象4378人，包括1805名男性和2573名女性，平均年龄为61.1±9.7岁，共有7475侧膝关节纳入评价。其中，3870侧基线时为非OA的膝关节用于研究 服用二甲双胍与OA发生风险的相关性，3605侧基线时为OA患病的膝关节用于研究服用二甲双胍与OA进展风险的相关性。OA发生以前(基线-上一次随访这段时间)服用二甲双胍组共包括251侧膝关节，未服用二甲双胍组共包括3619侧膝关节。OA进展以前(基线-上一次随访这段时间)服用二甲双胍组共包括314侧膝关节，未服用二甲双胍组共包括3291侧膝关节。纳入人群的基本信息请见表1(基线时为非OA膝关节)和表2(基线时为OA患病膝关节)。
广义估计方程的分析结果显示，未经校正时服用二甲双胍组相比未服用二甲双胍组膝关节OA发生的RR值为0.52(95％CI:0.28至0.97，P＝0.041)；校正年龄、性别、BMI后服用二甲双胍组相比未服用二甲双胍组膝关节OA发生的RR值为0.34(95％CI:0.18至0.65，P＝0.001)；校正年龄、性别、BMI、关节受伤史，膝关节疼痛程度，教育程度，运动量和膝关节KL等级后，服用二甲双胍组相比未服用二甲双胍组膝关节OA发生的RR值为0.30(95％CI:0.15至0.60，P<0.001)，以上结果提示服用二甲双胍的人群其OA发生的风险均低于未服用二甲双胍的人群。(表3)。未经校正时服用二甲双胍组相比未服用二甲双胍组膝关节OA进展的RR值为0.38(95％CI:0.25至0.58，P<0.001)；校正年龄、性别、BMI后服用二甲双胍组相比未服用二甲双胍组膝关节OA进展的RR值为0.34(95％CI:0.22至0.52，P<0.001)；校正年龄、性别、BMI、关节受伤史，膝关节疼痛程度，教育程度，运动量和膝关节KL等级后，服用二甲双胍组相比未服用二甲双胍组膝关节OA进展的RR值为0.33(95％CI:0.21至0.51，P<0.001)。以上结果提示服用二甲双胍的人群其OA发生和进展的风险均低于未服用二甲双胍的人群。以上结果提示服用二甲双胍的人群其OA进展的风险均低于未服用二甲双胍的人群。(表4)。
该结果表明患有骨关节炎的人群服用二甲双胍可能对骨关节炎的发生和进展均具有保护作用。
表1 纳入非OA膝关节人群基线时的基本信息(n＝3870)
Figure PCTCN2018099180-appb-000001
Figure PCTCN2018099180-appb-000002
n，number；PASE，Physical Activity Scale for the Elderly；KL，Kellgren and Lawrence
表2 纳入OA患病膝关节人群基线时的基本信息(n＝3605)
Figure PCTCN2018099180-appb-000003
Figure PCTCN2018099180-appb-000004
n，number；PASE，Physical Activity Scale for the Elderly；KL，Kellgren and Lawrence
表3 服用二甲双胍与OA发生风险的相关性
Figure PCTCN2018099180-appb-000005
模型1：未校正
模型2：校正年龄、性别、BMI；
模型3：在模型2的基础上，校正基线时膝关节受伤史，膝关节疼痛程度，教育程度，运动量和膝关节KL等级。
表4 服用二甲双胍与OA进展风险的相关性
Figure PCTCN2018099180-appb-000006
模型1：未校正
模型2：校正年龄、性别、BMI；
模型3：在模型2的基础上，校正基线时膝关节受伤史，膝关节疼痛程度，教育程度，运动量和膝关节KL等级。
实施例2细胞水平验证二甲双胍可通过激活AMPK抑制OA软骨细胞基质降解
1实验材料和方法
1.1试剂
二甲双胍购置于Sigma公司，溶解于磷酸盐缓冲液(Phosphate Buffer solution，PBS)。AMPK阻滞剂compound C购置于Sigma公司。白细胞介素1β(interleukin-1β，IL-1β)购置于R&D Systems公司。Thr-172位点磷酸化AMPK(pAMPK)、AMPKα、基质金属蛋白酶13(MMP-13)和II型胶原(Col2a1)一抗购置于Abcam和Cell Signaling公司。辣根过 氧化物酶偶联的二抗购置于Cell Signaling公司。
1.2实验细胞
本研究应用新生小鼠膝关节软骨细胞。新生小鼠软骨细胞取自4天新生小鼠的髋关节、膝关节和踝关节软骨，并培养原代小鼠软骨细胞，新生小鼠购置于长沙湖南斯莱克景达实验动物有限公司。
1.3细胞干预
取第1代新生小鼠膝关节软骨细胞培养于含有胎牛血清(10％)和双抗(青霉素50U/ml、链霉素50U/ml)的DMEM-F12培养基中，置于CO 2培养箱内(37℃恒温，5％CO 2，95％空气、100％湿度)培养，先予以含5％FBS的DMEM/F12孵育24h，使细胞同步化出现在非增殖期和非活动期。待细胞长至70％-80％时吸去培基，按以下分组进行干预：
1)检测二甲双胍对软骨细胞MMP-13和II型胶原表达影响，将体外培养的第1代小鼠软骨细胞随机分为以下各组予以干预：
a.软骨细胞常规培养对照组：予以10％FBS的DMEM/F12完全培养基培养；
b.OA软骨细胞对照组：予以10％FBS的DMEM/F12完全培养基培养，加入5ng/ml的IL-1β进行干预；
c.OA软骨细胞二甲双胍干预A组：予以10％FBS的DMEM/F12完全培养基培养，加入5ng/ml的IL-1β进行干预，同时加入1mM二甲双胍进行干预；
d.OA软骨细胞二甲双胍干预B组：予以10％FBS的DMEM/F12完全培养基培养，加入5ng/ml的IL-1β进行干预，同时加入10mM二甲双胍进行干预；
e.OA软骨细胞二甲双胍干预C组：予以10％FBS的DMEM/F12完全培养基培养，加入5ng/ml的IL-1β进行干预，同时加入20mM二甲双胍进行干预。
2)验证AMPK在二甲双胍调控MMP-13和II型胶原表达中的作用，将体外培养的第1代小鼠软骨细胞随机分为以下各组予以干预：
a.OA软骨细胞对照组：予以10％FBS的DMEM/F12完全培养基培养，加入5ng/ml的IL-1β进行干预；
b.OA软骨细胞二甲双胍干预A组：予以10％FBS的DMEM/F12完全培养基培养，加入5ng/ml的IL-1β进行干预，同时加入10mM二甲双胍进行干预；
c.OA软骨细胞二甲双胍干预+AMPK阻断组：予以10％FBS的DMEM/F12完全培养基培养，加入5ng/ml的IL-1β进行干预，同时加入10mM二甲双胍和10uM Dorsomorphin(AMPK的阻滞剂)进行干预；
d.OA软骨细胞二甲双胍干预+溶媒对照组：予以10％FBS的DMEM/F12完全培养基培养，加入5ng/ml的IL-1β进行干预，同时加入10mM二甲双胍和DMSO(Dorsomorphin 的溶媒对照)进行干预。
1.4结果指标检测
1)为检测二甲双胍对软骨细胞MMP-13和II型胶原表达影响，干预相应分组软骨细胞24小时后提取软骨细胞mRNA、蛋白，qRT-PCR检测软骨细胞MMP-13mRNA的表达，Western Blot检测软骨细胞II型胶原和MMP-13的表达。
2)为验证AMPK在二甲双胍调控MMP-13和II型胶原表达中的作用，干预相应分组软骨细胞24、48小时后提取软骨细胞mRNA、蛋白，qRT-PCR检测软骨细胞MMP-13mRNA的表达，Western Blot检测软骨细胞MMP-13、II型胶原和AMPKαThr172位点磷酸化的AMPK(p-AMPK)的表达。
2实验结果
2.1二甲双胍对软骨细胞II型胶原和MMP‐13表达影响
1)qRT-PCR检测各组小鼠软骨细胞干预后MMP-13的mRNA的RQ值如表5、图1所示，统计结果显示：相对于OA软骨细胞对照组，软骨细胞常规培养组MMP-13的mRNA的RQ值为0.038±0.017，低于OA软骨细胞对照组，差异具有统计学意义(p＜0.001)；OA软骨细胞二甲双胍干预A组MMP-13的mRNA的RQ值为0.993±0.041，与OA软骨细胞对照组差异无统计学意义(p＝0.811)，OA软骨细胞二甲双胍干预B组、C组MMP-13的mRNA的RQ值分别为0.788±0.048、0.711±0.048，低于OA软骨细胞对照组，差异具有统计学意义(p＜0.001；p＜0.001)。
表5 MMP-13 mRNA RQ值结果
Figure PCTCN2018099180-appb-000007
RQ,相对表达量；SD,标准差.
2)Western blotting检测各组小鼠软骨细胞干预后蛋白的表达结果表明：IL-1干预后小鼠软骨细胞II型胶原蛋白表达降低、MMP-13蛋白表达升高，表明体外OA建模成功。随着二甲双胍干预浓度的提高，各组小鼠软骨细胞II型胶原表达逐渐升高，MMP-13表达降低(图2、3)。
2.2 AMPK在二甲双胍调控II型胶原和MMP-13表达中的作用
1)qRT-PCR检测各组小鼠软骨细胞二甲双胍(10mM)干预后48小时MMP-13的mRNA的RQ值如表6、图4所示，统计结果显示：相对于OA软骨细胞对照组，OA软骨细胞二甲双胍干预组和OA软骨细胞二甲双胍干预+AMPK溶媒对照组MMP-13的mRNA的RQ值分别为0.767±0.078、0.752±0.103，低于OA软骨细胞对照组，但差异不具有统计学意义(p＝0.623；p＝0.602)；OA软骨细胞二甲双胍干预+AMPK阻断组MMP-13的mRNA的RQ值为3.214±1.110，高于OA软骨细胞对照组(p＜0.001)、OA软骨细胞二甲双胍干预组(p＜0.001)和OA软骨细胞二甲双胍干预+AMPK溶媒对照组(p＜0.001)，差异具有统计学意义。
表6 MMP-13 mRNA RQ值结果
Figure PCTCN2018099180-appb-000008
RQ,相对表达量；SD,标准差.
2)Western blotting检测各组小鼠软骨细胞干预后24小时蛋白的表达结果表明：OA软骨细胞二甲双胍(10mM)干预组较OA软骨细胞对照组II型胶原表达升高，AMPKαThr172位点磷酸化的AMPK(p-AMPK)表达升高，OA软骨细胞二甲双胍干预(10mM)+AMPK阻断组在加入Dorsomorphin后p-AMPK表达下降，但II型胶原表达仍升高；OA软骨细胞二甲双胍干预+AMPK溶媒对照组II型胶原和p-AMPK蛋白表达与OA软骨细胞二甲双胍干预组无明显差别(图5)。
3)Western blotting检测各组小鼠软骨细胞干预后48小时蛋白的表达结果表明：OA软骨细胞二甲双胍(10mM)干预组较OA软骨细胞对照组II型胶原表达升高，后MMP-13表达降低，p-AMPK表达升高；OA软骨细胞二甲双胍干预(10mM)+AMPK阻断组在加入Dorsomorphin后II型胶原表达下降，MMP-13表达升高，p-AMPK无明显差异；OA软骨细胞二甲双胍干预+AMPK溶媒对照组II型胶原、MMP-13和p-AMPK蛋白表达与OA软骨细胞二甲双胍干预组无明显差别(图6、7)。
以上结果表明二甲双胍可通过激活AMPK从而抑制软骨细胞基质降解和退变，进一步发挥软骨保护功能。
实施例3动物实验验证二甲双胍可减轻小鼠膝关节软骨退变、缓解小鼠骨关节炎疼痛症 状。
1实验材料和方法
1.1实验动物
实验选用8周龄雄性C57BL/6J小鼠96只，所有动物饲养于中南大学实验动物学部，以SPF级标准饲料饲养，自由进食、饮水。
1.2实验药物
将二甲双胍溶于生理盐水，制成10 -1M二甲双胍溶液，-20℃保存备用。
1.3实验方法
1.3.1手术建模
对全身麻醉小鼠右膝关节行内侧半月板失稳术从而诱发OA形成，构建实验OA模型。
1.3.2分组及干预方法
将小鼠随机分为以下6组：
1)空白对照组：无特殊处理；
2)假手术组：予以假手术处理；
3)生理盐水灌胃组：行DMM术构建小鼠膝OA模型后3天开始予以生理盐水灌胃(10ml/kg)，每天一次，连续8周；
4)二甲双胍灌胃组：行DMM术构建小鼠膝OA模型后3天开始予以二甲双胍灌胃(200mg/kg)，每天一次，连续8周；
5)膝关节腔内注射生理盐水组：行DMM术构建小鼠膝OA模型后3天开始予以关节腔注射生理盐水(1ml/kg)，每周两次，连续8周；
6)膝关节腔内注射二甲双胍组：行DMM术构建小鼠膝OA模型后3天开始予以关节腔注射二甲双胍(0.1mmol/kg)，每周两次，连续8周。
1.3.3评价方法
1.3.3.1扫描电镜评估软骨表面磨损程度及退变程度
术后8周处死小鼠，取右侧膝关节标本，于体视显微镜下小心分离股骨和胫骨，分别置于2.5％戊二醛溶液中固定24小时，按扫描电镜检测标准进行标本处理后置于扫描电子显微镜下观察、拍照分析。按照扫描电镜评分系统对各组小鼠膝关节软骨表面的磨损程度进行评价。
评分细则为：
3分：软骨表面光滑，无明显裂痕；
2分：软骨表面相对光滑，但可见聚集在一起的软骨碎片；
1分：软骨表面粗糙，可见聚集在一起的软骨碎片；
0分：软骨表面粗糙，可见不规则的散在软骨碎片。
1.3.3.2小鼠疼痛评估
各组小鼠手术建模当天和建模后每隔3-4天评价疼痛并作前后比较，持续8周。采用von Frey测痛仪及双足平衡法测痛仪评估小鼠疼痛相关指标。
1)机械缩足反射阈值
用von Frey测痛仪测定机械痛觉阈值：
a.将小鼠置于干净的底部为铁丝网空格有机玻璃箱内，适应环境15-30min(以小鼠在笼中觅食、打闹等活动停止为准)；
b.采用Von Frey纤维毛针刺激小鼠左(造模侧)后肢足底中部；
c.刺激弯曲力为1,1.4,2,4,6,8,10and 15g，由小到大；
d.每次持续3s，如果小鼠出现抬足、舔足或躲避等行为视为阳性反应，否则为阴性反应；
e.若小鼠不出现上述反应，则换大一级力度的毛针；如出现阳性反应则给予相邻小一级力度的刺激；
f.能引起小鼠缩足反射的最小刺激记录为机械缩足反射阈值；
g.连续测定5次，每次间隔3min，取其平均值。
2)双后肢负重差值
用后肢负重测量仪测量双后肢负重差值：
a.将小鼠放入后肢负重测量仪中，待其安静，使其双后肢分别置于不同感应器上；
b.保持3s后，由每一侧的后肢产生的负重(g)将分别显示在测量仪上；
c.重复测量3次，取平均值；
d.计算双侧后肢负重差值。
2实验结果
2.1扫描电镜评价二甲双胍灌胃和膝关节腔内注射后小鼠膝关节软骨退变情况
1)电镜结果显示空白干预组小鼠膝关节软骨表面光滑、完整，无表面磨损、裂隙形成(见图8)。假手术组小鼠膝关节软骨表面较为光滑，可见小范围轻度退变(见图9)。生理盐水灌胃组小鼠膝关节软骨表面可见软骨大范围裂纹、剥脱、软骨下骨暴露(见图10)。二甲双胍灌胃组可见软骨存在裂纹、剥脱，但范围相比生理盐水灌胃组较小，程度较轻， 基本未暴露软骨下骨(见图11)。生理盐水膝关节腔内注射组小鼠膝关节软骨表面可见软骨大范围裂纹、剥脱、软骨下骨暴露(图12)。二甲双胍膝关节腔内注射组可见软骨存在裂纹、剥脱，但范围相比生理盐水膝关节腔内注射组较小，程度较轻，软骨下骨暴露少见(图13)。
2)各组小鼠膝关节软骨扫描电镜评分结果见表7、图14。生理盐水灌胃组和生理盐水膝关节腔内注射组扫描电镜评分明显低于空白组和假手术组，差异具有统计学意义，证明OA手术建模成功。二甲双胍灌胃组扫描电镜评分高于生理盐水灌胃组，差异接近统计学意义(p＝0.069)。二甲双胍膝关节腔内注射组扫描电镜评分明显高于生理盐水膝关节腔内注射组，差异具有统计学意义(p＜0.001)。
表7 二甲双胍灌胃和膝关节腔内注射后小鼠膝关节软骨扫描电镜评分
Figure PCTCN2018099180-appb-000009
N，样本量
2.2二甲双胍灌胃和膝关节腔内注射后小鼠疼痛症状缓解情况
1)各组小鼠建模当天及建模后每3-4天测量机械缩足反射阈值，各组小鼠建模后不同时间机械缩足反射阈值的平均值见表8。
采用单因素方差分析比较建模当天至建模后56天各组小鼠机械缩足反射阈值单天测量的结果，统计结果表明：建模后14天(P＝0.005)、25天(P＝0.002)、28天(P＝0.034)、39天(P＜0.001)、42天(P＝0.002)、46天(P＝0.001)、49天(P＝0.005)、53天(P＜0.001)、56天(P＝0.001)二甲双胍灌胃组小鼠的机械缩足反射阈值高于生理盐水灌胃组，差异有统计学意义(图15)；建模后21天(P＝0.008)、32天(P＝0.005)、39天(P＜0.001)、46天(P＝0.003)、49天(P＝0.008)、53天(P＝0.022)、56天(P＝0.028)二甲双胍膝关节腔内注射组小鼠的机械缩足反射阈值高于生理盐水膝关节腔内注射组，差异有统计学意义(图16)。
采用重复测量资料方差分析检验比较建模当天至建模后56天各组小鼠机械缩足反射阈值的整体差别，统计结果表明：空白对照组小鼠缩足反射阈值大于生理盐水灌胃组(P＜0.001)、二甲双胍灌胃组(P＜0.001)、生理盐水膝关节腔内注射组(P＜0.001)、二甲双胍膝 关节腔内注射组(P＜0.001)小鼠的缩足反射阈值；空白对照组小鼠与假手术组小鼠缩足反射阈值差异无统计学意义(P＝0.214)；假手术组小鼠缩足反射阈值大于生理盐水灌胃组(P＜0.001)、二甲双胍灌胃组(P＝0.004)、生理盐水膝关节腔内注射组(P＝0.003)、二甲双胍膝关节腔内注射组(P＝0.004)小鼠的缩足反射阈值；二甲双胍灌胃组小鼠的缩足反射阈值大于生理盐水灌胃组小鼠的缩足反射阈值，差异具有统计学意义(P＜0.001)
表8 二甲双胍灌胃和膝关节腔内注射对OA小鼠机械缩足反射阈值的影响
Figure PCTCN2018099180-appb-000010
N，样本量
2)各组小鼠建模当天及建模后每3-4天测量双后肢负重差值，各组小鼠建模当天及建模后不同时间双后肢负重差值的平均值见表9。
采用单因素方差分析比较建模当天至建模后56天各组小鼠双后肢负重差值单天测量的结果，统计结果表明：建模后18天(P＝0.001)、25天(P＝0.022)、28天(P＜0.001)、35天(P＝0.005)、39天(P＝0.030)、42天(P＝0.003)、46天(P＝0.009)、49天(P＝0.024)、53天(P＝0.008)、56天(P＝0.009)二甲双胍灌胃组小鼠的后肢负重差值小于生理盐水灌胃组，差异有统计学意义(图17)；建模后21天(P＝0.005)、42天(P＜0.001)、46天(P＝0.021)、49天(P＝0.008)、53天(P＝0.009)、56天(P＜0.001)二甲双胍膝关节腔内注射组小鼠的后肢负重差值小于生理盐水膝关节腔内注射组，差异有统计学意义(图18)。
采用重复测量资料方差分析检验比较建模当天至建模后56天各组鼠双后肢负重差值的整体差别，统计结果表明：空白对照组小鼠双后肢负重差值小于假手术组(P＝0.003)、生理盐水灌胃组(P＜0.001)、二甲双胍灌胃组(P＜0.001)、生理盐水膝关节腔内注射组(P＜0.001)、二甲双胍膝关节腔内注射组(P＜0.001)小鼠的双后肢负重差值；假手术组小鼠双后肢负重差值大于生理盐水灌胃组(P＜0.001)、二甲双胍灌胃组(P＜0.001)、生理盐水膝关节腔内注射组(P＜0.001)、二甲双胍膝关节腔内注射组(P＜0.001)小鼠的双后肢负重差值，差异具有统计学意义；二甲双胍灌胃组小鼠的双后肢负重差值小于生理盐水灌胃组小鼠的双后肢负重差值，差异具有统计学意义(P＜0.001)；生理盐水膝关节腔内注射组小鼠双后肢负重差值小于二甲双胍膝关节腔内注射组小鼠，差异具有统计学意义(P＜0.001)。
表2-5 二甲双胍灌胃和膝关节腔内注射对OA小鼠双后肢负重差值的影响
Figure PCTCN2018099180-appb-000011
Figure PCTCN2018099180-appb-000012
N，样本量
以上结果表明二甲双胍治疗不但可减轻小鼠OA模型的软骨损伤程度，其在减少软骨基质降解、降低软骨表面磨损程度和延缓软骨退变中是有效的，同时还可缓解骨关节炎引起的疼痛症状。
另外，在小鼠体内试验的本药物，在最高达200mg/kg(口服)和0.1mmol/kg(膝关节腔注射)剂量下，均为显示出任何不利的毒性现象，小鼠体重在各组间无明显差异。

Claims (8)

  1. 二甲双胍在制备预防和治疗骨关节炎以及缓解骨关节炎引起的疼痛症状相关药物中的应用。
  2. 一种预防和治疗骨关节炎以及缓解骨关节炎引起的疼痛症状相关的药物组合物，其特征在于，所述药物组合物中含有二甲双胍或二甲双胍的盐，如二甲双胍盐酸盐。
  3. 如权利要求2所述的药物组合物，其特征在于，所述药物组合物中还含有用于增强细胞外基质的分子，所述二甲双胍或二甲双胍的盐与增强细胞外基质的分子的重量比为1:0.2至1:1。
  4. 如权利要求3所述的药物组合物，其特征在于，所述增强细胞外基质的分子为胶原蛋白或糖胺聚糖。
  5. 如权利要求2所述的药物组合物，其特征在于，所述药物组合物中还含有药理学活性物质和/或生物活性物质，所述二甲双胍或二甲双胍的盐与药理学活性物质和/或生物活性物质的重量比为1:0.2至1:1。
  6. 如权利要求5所述的药物组合物，其特征在于，药理学活性物质和/或生物活性物质为生长因子、激素和/或维生素。
  7. 如权利要求2所述的药物组合物，其特征在于，所述药物组合物中还含有赋形剂，所述所述二甲双胍或二甲双胍的盐与赋形剂的重量比为1:10至1:100。
  8. 如权利要求2至7任一项所述的药物组合物，其特征在于，所述药物组合物的剂型为口服剂型或注射剂。
PCT/CN2018/099180 2017-08-08 2018-08-07 二甲双胍在制备预防和治疗骨关节炎以及缓解骨关节炎引起的疼痛症状相关药物中的应用 WO2019029526A1 (zh)

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