CN112823796A - Application of protein tyrosine phosphatase SHP2 inhibitor in preparation of medicine for treating osteoarthritis - Google Patents

Abstract

The invention discloses a kind of allosteric inhibitor SHP099 of SHP2 which can be used for the treatment of osteoarthritis. Aiming at the DMM model and the natural aging-induced mouse osteoarthritis disease model, SHP099 can effectively inhibit the knee joint cartilage defect in mice and promote the repair effect of cartilage. At the cellular level, SHP099 significantly reduced the levels of cartilage-degrading enzymes and promoted the expression of cartilage synthesis genes under the stimulation of IL-1β, thereby achieving dual regulation of cartilage synthesis and catabolism. In addition, SHP099 also has a concentration gradient to promote chondrocyte differentiation.

Description

Application of protein tyrosine phosphatase SHP2 inhibitor in preparation of medicine for treating osteoarthritis

One, the technical field

The invention belongs to the technical field of pharmacy.

Second, background Art

Osteoarthritis (OA) is the most common joint disease in the middle-aged and elderly, and is pathologically characterized by destruction of the cartilage structure, narrowing of the joint space, hardening of subchondral bone and formation of marginal osteophytes accompanied by inflammation of the synovium. The primary symptoms of early OA include transient joint movement disorders such as joint pain, morning stiffness and tenderness, which subsequently develop into joint swelling and joint deformity, which severely affect the quality of life of the patient. Statistically, the incidence rate is 50% in people over 60 years old, and 80% in 75 years old. To date, however, there is no recognized effective treatment strategy for altering the progression of osteoarthritis disease, and the existing treatment technologies are primarily directed to providing some medications for relief of pain symptoms in osteoarthritis patients. Currently, the first choice of drugs in the drug treatment of osteoarthritis remains non-steroidal anti-inflammatory drugs. Although gastrointestinal safety of specific COX-2 inhibitors such as celecoxib is superior to that of traditional nonsteroidal anti-inflammatory drugs, long-term studies indicate that long-term use of specific COX-2 inhibitors may increase the risk of cardiovascular events such as myocardial infarction and cardiovascular thrombosis, which are warned by the FDA.

SHP099 was developed in 2016 as an allosteric inhibitor of protein tyrosine phosphatase SHP2, and research on this compound was focused mainly on anti-tumor. The unique pharmacological action of the compound for treating bone diseases, particularly osteoarthritis is not reported so far. The invention mainly discovers that SHP099 has a remarkable improvement effect on a mouse osteoarthritis model.

SHP099 has the following structural formula:

third, the invention

In order to solve the problem that the existing clinical osteoarthritis treatment medicines are all used for diminishing inflammation and easing pain, no medicine for reversing the OA changing process exists, and the invention provides a protein tyrosine phosphatase SHP2 inhibitor SHP099 which can be used for treating osteoarthritis related diseases.

For the DMM surgery-induced osteoarthritis model in mice, intraperitoneal administration of SHP 09910 mg/kg significantly reduced the OARSI score. The compound has bidirectional regulation on chondrocytes, and can inhibit the expression of matrix metalloproteinases such as Mmp13 and Mmp3 for degrading cartilage matrixes on one hand, thereby inhibiting the degradation of the chondrocytes; on the other hand, the expression of cartilage synthesis markers such as Aggrecan and Col2a1 can be promoted so as to promote cartilage synthesis.

The results indicate that the SHP099 can improve symptoms related to osteoarthritis of mice, has obvious effects on chondrocytes in particular, and can be applied to the treatment of osteoarthritis.

Drawings

FIG. 1SHP099 inhibits DMM surgery-induced cartilage damage in mice.

FIG. 1-A shows the results of safranin fast green staining in a model of OA induced by surgery with DMM administered with SHP 099; FIG. 1-B shows the scoring results of OA articular cartilage damage in the OA model induced by DMM surgery administered SHP 099; FIG. 1-C are IHC and IF results of SHP099 administration to treat OA-associated marker expression in articular cartilage tissue in a DMM surgically-induced OA model; FIG. 1-D are statistical results of OA-associated marker expression in articular cartilage tissue in the DMM surgically-induced OA model treated with SHP099 administration. Results after treatment were expressed as mean ± standard deviation. *: p <0.05, x: p <0.01, x: p <0.001vs DMM (Student's-t test).

FIG. 2SHP099 promotes repair of cartilage damage in mice under conditions of natural aging.

FIG. 2-A shows the safranin fast green staining results at the joint area 6 weeks after SHP099 administration to aged mice (20M); FIG. 2-B shows the thickness of articular cartilage 6 weeks after SHP099 administration to aged mice; FIG. 2-C is an IHC result of OA-associated marker expression in articular cartilage tissue of aged mice after SHP099 administration treatment; results are expressed as mean ± standard deviation; *: p <0.05, x: p <0.01, x: p <0.001vs PBS group (Student's-t test).

FIG. 3 at the cellular level, SHP099 inhibits cartilage degradation and promotes cartilage synthesis.

FIG. 3: A-F are respectively the mRNA level influence of SHP099 on genes Mmp3, Mmp13, Adamts5 and genes Sox9, Col2a1, Acan and the like related to cartilage degradation under the IL-1 beta induction condition. FIG. 3-G shows the effect of SHP099 on protein levels of MMP3, MMP13, SOX9, etc., under IL-1 β -inducing conditions. FIG. 3-H is the effect of SHP099 on chondrocyte survival. Results are expressed as mean ± standard deviation; *: p <0.05, x: p <0.01, x: p <0.001vs IL-1. beta. (Student's-t test).

FIG. 4SHP099 promotes the synthesis of cartilage explant ECM in OA patients.

FIG. 4A-B shows the results of safranine fast green staining of cartilage explants of OA patients under the action of SHP 099. Results are expressed as mean ± standard deviation: **: p <0.01, x: p <0.001vs 0 (Student's-t test).

Fig. 5SHP099 promotes chondrocyte differentiation.

FIG. 5-A shows the results of Alsinoblue staining of differentiation of primary chondrocytes by SHP 099; FIG. 5, B-D shows the effect of SHP099 on the mRNA levels of cartilage synthesis-related genes Sox9, Col2a1, and Acan in primary chondrocyte process. Results are expressed as mean ± standard deviation: **: p <0.01, x: p <0.001vs DM (Student's-t test).

Detailed Description

SHP099 was purchased from MCE.

Example 1: SHP099 inhibits DMM surgery-induced cartilage damage in mice

The technical method comprises the following steps: mouse osteoarthritis model induced by medial meniscal imbalance (DMM) surgery^[1]Immunohistochemical staining, immunofluorescence, safranin fast green staining

Wild C57BL/6 male mice are bred in SPF animal room at the age of 8-10 weeks, are fed with free water at 21 + -2 deg.C, and are fed alternately for 12h day and night. After anesthetizing the mice with avertin (350ul-400ul), the hair was shaved off from the knee joint of the right leg of the mice, the knee joint was exposed, and the medial meniscus-tibial ligament was cut with a surgical blade to free the medial meniscus. The next day, the mice were randomly divided into 3 groups, a normal group (Sham group), a model group (DMM group), and a SHP099(10mg/kg) group. The DMM + SHP099 group was administered intraperitoneally once a day at 100. mu.l each, and the normal group and the model group were administered with an equal amount of PBS. After 42 days of treatment, the experimental animals were euthanized, the right leg was fixed and dehydrated and embedded, and the cartilage injury was examined by safranine fast green staining for repair, and the tibial injuries were scored according to the Osteoarthritis cartilage pathology evaluation system OARSI Score system [1] established by the International Association for Osteoarthritis Research (OARSI).

Example 3: SHP099 promotes repair of cartilage damage in a model of natural aging.

The technical method comprises the following steps: model of natural aging, safranin fast green staining

Wild C57BL/6 male mice were raised to 20 months of age and grown under the same conditions. Mice were randomly divided into 2 groups, PBS group and SHP099(10mg/kg) group. After 42 days of drug administration treatment, the experimental animals are euthanized, the right leg is taken to be fixed, dehydrated, embedded and sliced, the cartilaginous injury repair situation is observed by safranine fast green staining, the tibial injury is scored,

example 4: at the cellular level, SHP099 inhibits cartilage degradation and promotes cartilage synthesis.

The technical method comprises the following steps: chondrocyte in vitro culture, qRT-PCR

After taking out chondrocytes of a wild C57 mouse within three days, digesting the chondrocytes for 30 minutes by using pancreatin, continuously digesting the chondrocytes for more than 2 hours by using 0.2% type II collagenase, collecting the cells in batches, adding DMEM/F12 containing 10% serum, and culturing the cells in a CO2 constant temperature incubator. In vitro culture to F1 passage followed by inoculation in 24-well plates. SHP099 is diluted into 10mmol of mother liquor with DMSO before use, subpackaged at 50ul per tube, stored at-20 deg.C, and freeze-thaw repeatedly is avoided, and other genes related to cartilage catabolism are detected. After SHP099 pre-treated cells for 2h, 10ng/ml IL-1 beta is added in vitro to stimulate chondrocytes to simulate inflammatory reaction of osteoarthritis, after 12h, RNA in the cells is extracted by Trizol harvest cells and is inverted into cDNA, and qRT-PCR is carried out to detect the expression of genes for degrading cartilage, such as Mmp13, Mmp3, Adamts5 and the like.

Example 5: SHP099 promotes the synthesis of chondrocyte extracellular matrix (ECM) in OA patients.

The technical method comprises the following steps: cartilage explant culture experiment, safranin fast green staining

Cutting a knee joint cartilage sample of an osteoarthritis patient subjected to joint replacement surgery into small blocks of about 1cm, and placing the small blocks in a 24-hole plate; after starvation for 24h, 1ml of DMEM/F12 culture medium containing 10% serum is added, the culture medium contains SHP099 with different concentrations, and after 7 days of culture, cartilage explants are fixed and decalcified and embedded; safranine fast green staining was performed to observe the synthesis of the chondrocyte extracellular matrix.

Example 6: SHP099 promotes chondrocyte differentiation

The technical method comprises the following steps: chondrocyte differentiation experiment, alcian blue staining, qRT-PCR

Mouse primary chondrocytes were cultured in a proliferation medium (DMEM/F12 containing 10% serum) and plated onto a 24-well plate after the cells were confluent, and the culture medium was changed to a cartilage differentiation medium (formula of cartilage differentiation medium: ITS1x, 10ng/ml TGF beta 3, dexamethasone 100nM, vitamin C50. mu.g/ml, proline 40. mu.g/ml, sodium pyruvate 1 mM). And SHP099 drugs with different concentration gradients are added under the condition of a differentiation medium, Alnew blue staining is carried out after 1.3.7 culture, RNA is extracted from cells after two days of differentiation culture and is inverted into cDNA, qRT-PCR is carried out, and the expression conditions of Sox9, Col2a1 and Acan in the cells are detected.

Analysis of SHP099 pharmacological experiment results

1) Effect of SHP099 on DMM-induced mouse osteoarthritis model

As shown in FIG. 1, OARSI scores were significantly higher in the normal group (Sham group) compared to the model group (DMM group); the OARSI score was significantly reduced after administration of SHP 09910 mg/kg.

As shown in fig. 1, compared with the normal group, the expression of cartilage degradation and hypertrophy-related proteins such as tibial MMP13, MMP3, and COLX in the model group is significantly increased, the expression of cartilage synthesis-related gene SOX9 is decreased, and the immunofluorescence and immunohistochemical staining results show that SHP099 can significantly inhibit the expression of cartilage degradation enzyme and hypertrophy of chondrocytes, and promote the expression of cartilage synthesis factor SOX 9.

2) Effect of SHP099 on model of cartilage degeneration in mice under conditions of Natural aging

As shown in FIG. 2, the treatment with SHP099 significantly promoted the increase in cartilage thickness compared to the aged group

3) Effect of SHP099 on IL-1 beta-induced mouse osteoarthritis model in vitro

As shown in FIG. 3, at the cellular level, SHP099 inhibited cartilage degradation and promoted cartilage synthesis

4) Effect of SHP099 on model of cartilage degeneration in mice under conditions of Natural aging

As shown in FIG. 3, the treatment with SHP099 significantly promoted the increase in cartilage thickness compared to the aged group

5) Effect of SHP099 on IL-1 beta-induced mouse osteoarthritis model in vitro

As shown in fig. 4, at the cellular level, SHP099 inhibited cartilage degradation and promoted cartilage synthesis SHP099 had an effect on a model of cartilage degeneration in mice under conditions of natural aging.

Claims (1)

1. the application of protein tyrosine phosphatase SHP2 inhibitor SHP099 in the preparation of medicine for the treatment of osteoarthritis, wherein the structural formula of SHP099 is as follows:

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