Application of CXCR2 antagonist as new generation of selective Senlytics in skeletal muscle system / senile diseases

Abstract

The invention relates to an application of a CXCR2 antagonist as a new generation of selective Senlytics in a skeletal muscle system / senile diseases. The invention discloses a CXCR2 antagonist represented by AZD5069, which can be used for selectively eliminating harmful senescent cells (SCs) for expressing CXCR2, and an application of the CXCR2 antagonist in treatment of skeletal muscle system / senile diseases. Researches find that AZD5069 can inhibit expression of p53 (one of key genes of an aging signal channel) and Bcl-2 (key genes for antagonizing SCs apoptosis) through CXCR2 so as to selectively eliminate harmful SCs subgroups (CXCR2 + SCs, Detrimental SCs and dSCs) in callus, and Age-Associated Fracture Repair (AAFR) is promoted, so that the AZD5069 can be used for selectively eliminating harmful SCs subgroups (CXCR2 + SCs, Detrimental SCs and dSCs) in callus, and the AZD5069 can be used for selectively eliminating harmful SCs subgroups (CXCR2 + SCs, Detrimental SCs and dSCs) in callus. The invention provides a new choice for clinically researching and developing new drugs for skeletal muscle system / senile diseases.

Description

Technical Field

[0001] The present invention belongs to the field of new drug applications and the field of medical technology, and particularly relates to a CXCR2 antagonist represented by AZD5069, which can be used as a new generation of selective senolytics drugs and applied to musculoskeletal system / senile diseases. Background Art

[0002] Delayed fracture healing or nonunion has always been one of the great challenges faced by clinicians, with an incidence of about 5% to 10%. With the continuous improvement of the treatment and survival of patients with severe trauma, the incidence of nonunion has also increased significantly. However, as one of the many factors affecting fracture healing, the aging of patients is particularly important for fracture healing.

[0003] The occurrence of fractures in the elderly, especially in patients with osteoporosis, can lead to extremely high mortality and disability rates, causing patients to lose their ability to live independently, resulting in longer hospitalization time and high medical expenses, and placing a huge burden on the patient's family and the entire society's medical and health system.

[0004] Fracture healing is a unique process in which damaged bone structures are reconstructed to form new bone with similar structure and biomechanical properties to normal bone. Generally speaking, the fracture healing process can be divided into four stages: (1) inflammatory response phase; (2) recruitment and proliferation of mesenchymal progenitor cells (MPCs); (3) differentiation of stem cells into bone; and (4) bone remodeling. Therefore, fracture healing requires the recruitment of MPCs and their proliferation and differentiation into bone. However, with age, the proliferation capacity of MPCs is significantly reduced, resulting in the atrophy of the stem cell pool, which in turn leads to a sharp decline in fracture healing ability.

[0005] Senescent cells (SCs) are a newly discovered type of cells associated with age-related degenerative diseases. SCs increase in number with age and are associated with many age-related diseases. Khosla S et al. found that SCs are associated with age-related osteoporosis, and the use of senolytics or INK-ATTACDeletion of SCs in mouse models can treat osteoporosis. In vitro experiments also showed that SCs-related culture media can inhibit osteoblast differentiation and promote osteoclast differentiation. Liu C et al. proposed that in postmenopausal osteoporosis, estrogen deficiency-induced bone loss is due to the accumulation of SCs and the formation of a pro-inflammatory microenvironment caused by SASP secretion, and targeting SCs with dasatinib (Dasatinib, D) + quercetin (Quercetin, Q) can restore the osteogenic differentiation of MPCs and improve osteoporotic bone regeneration function. JeonOH et al. found that there are a large number of SCs in the articular cartilage of mice with primary osteoarthritis in the elderly or traumatic osteoarthritis caused by anterior cruciate ligament transection, and the process of osteoarthritis was significantly slowed down after SCs were deleted using senolytics or p16-3MR mice. Xu M et al. also found in the study of temporomandibular joint degeneration that the elimination of SCs can also improve the proliferation of subchondral bone.

[0006] However, in recent years, the clinical trial results of senolytic drugs have been greatly biased and have led to the failure of clinical trials. Therefore, the study of SCs and senolytic drugs requires further mechanism research. We further found that SCs have heterogeneity, especially SCs from the same lineage have different cell functions. Saul D& Khosla S's research group and our research team simultaneously published articles and found that SCs aggregation occurs in the callus of early-middle stage fracture healing in young or old mice (1-10 days after fracture). The difference is that compared with the research method of our team, Khosla S et al. found in their study that using D+Q to eliminate SCs during the whole process of fracture healing can promote fracture healing in young mice. However, this study ignored the fact that long-term use of D itself can promote bone synthesis and increase bone mass. Based on the reason that SCs increase in the early-middle stage, our team only gave D+Q to fracture mice during this period, and found that D+Q can only promote fracture healing in old mice. Ren's study also found that knocking out the p16 gene only promotes fracture healing in 12-month-old mice. Campisi J et al. found that the elimination of SCs in 16-3MR mice would significantly delay wound closure, because the SCs accumulated in the damaged skin secrete platelet-derived growth factor-AA (PDGF-AA), which is necessary for skin healing. However, the study by Hardman MJ et al. found that the use of CXCR2 antagonists in mice with skin damage significantly promoted skin repair of wounds while reducing the number of SCs in the skin. Different research results of eliminating all SCs in tissues in fracture models of young and old mice confirmed that there were differences in the composition of SCs in the callus of young and old mice, and further suggested the possibility of different functional cell subpopulations of SCs. This team used single cell RNA sequencing (scRNA-seq) for the first time to conduct further cell subpopulation analysis of p16+p21+p18+ SCs in mouse callus. The study found that SCs can be further divided into three subpopulations: SC-1 subpopulation (CXCR2 + TGFβ1 + ), SC-2 subset (CXCR2 + TGFβ1 - ) and SC-3 subsets (CXCR2 - TGFβ1 +). TGFβ1 is one of the important components of SASP secreted by SCs. Studies have found that SCs in the callus of aged mice inhibit the proliferation of MPCs by secreting TGFβ1, thereby weakening the fracture healing ability. In addition, studies have found that SC-1 and SC-2 subpopulations can significantly inhibit the growth of CaMPCs, while SC-3 subpopulation can promote the growth of CaMPCs. Therefore, we define SC-1 and SC-2 subpopulations as harmful SCs subpopulations (CXCR2 + SCs, Detrimental SCs, dSCs). SC-3 subpopulation is a neutral SCs subpopulation (CXCR2 - TGFβ1 + SCs, Neutral SCs, nSCs). CXCR2 is widely expressed in various cells, such as neutrophils. The CXCR2 signaling pathway plays an important role in the migration of neutrophils from the bone marrow to the site of inflammation or injury.

[0007] AZD5069 is a CXCR2 antagonist. The molecular formula is C 18 H 22 F2N4O5S2, relative molecular weight is 476.52, chemical structure is as follows Figure 1 As shown. Clinical trials for its therapeutic effects on asthma, chronic obstructive pulmonary disease, pancreatic cancer and head and neck tumors are ongoing. AZD5069 can selectively eliminate dSCs and retain nSCs with potential beneficial effects. Therefore, compared with traditional senolytics (D+Q), it better promotes the growth and proliferation of CaMPCs, thereby promoting fracture healing in the elderly.

[0008] At present, there are no research reports on the effects of AZD5069-related products on the skeletal muscle system. Summary of the invention

[0009] The purpose of the present invention is to study the effects of AZD5069 on the skeletal muscle system / senile diseases and its related mechanisms of action, and to develop a new generation of selective senolytic drugs based on the CXCR2 target of dSCs.

[0010] To achieve the above objectives, the present invention adopts the following technical solutions.

[0011] 1. AZD5069 is a new generation of selective senolytics used in the treatment of geriatric diseases.

[0012] 2. Application of AZD5069 in musculoskeletal system / senile diseases.

[0013] 3. A drug for treating elderly musculoskeletal system diseases / senile diseases, wherein the active ingredient is AZD5069.

[0014] Preferably, the dose of AZD5069 is 100 mg / kg, three times / week (on alternate days).

[0015] Further preferably, the dose of AZD5069 is 100 mg / kg, once a day.

[0016] Beneficial effects of the present invention.

[0017] The present invention uses a CXCR2 antagonist AZD5069 for the treatment of elderly musculoskeletal system diseases / senile diseases for the first time. Specifically, AZD5069 is used for the first time to specifically eliminate SCs expressing CXCR2, rather than eliminating all SCs. Moreover, AZD5069 can effectively eliminate dSCs and promote fracture healing at a dose of 100 mg / kg. The present invention provides a new option for clinical research and development of new drugs for elderly musculoskeletal system diseases / senile diseases.

[0018] Compared with existing technologies, AZD5069 can selectively eliminate dSCs, reverse the adverse effects caused by them, and promote AAFR. AZD5069 has a good selective elimination effect on dSCs and retains the neutral SCs subpopulation (Neutral SCs, nSCs). Therefore, AZD5069 has a stronger therapeutic effect on senile fractures than traditional senolytics dasatinib (Dasatinib, D) + quercetin (Quercetin, Q). In addition, when D+Q is used to treat certain diseases (such as osteoarthritis), the instability of clinical experimental results caused by the elimination of all SCs including beneficial SCs will be effectively avoided. For example, our laboratory found that there was no obvious effect when D+Q was used to treat young fracture mice, while AZD5069, because it only eliminated dSCs, could significantly promote fracture healing ( Figure 5 D). BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the chemical structural formula of AZD5069.

[0020] Figure 2 : (A) t-SNE plot of single-cell RNA sequencing of mesenchymal cells (CD45-CD31-Ter119-) in callus; (B) t-SNE plot of cell subpopulations after further subdivision of SCs expressing p16; (C) Cell number statistics of three SCs cell subpopulations; (D) Labeling of each SCs subpopulation with different cell markers; (E) Tgfb1 and Cxcr2 define three cell subpopulations, namely group 1, Tgfb1+Cxcr2+; group 2, Tgfb1-Cxcr2+; and group 3, Tgfb1+Cxcr2-.

[0021] Figure 3 :(A) CaMPCs were cultured in vitro using p16TdTomato reporter mice and stimulated with hydrogen peroxide to generate SCs. (B) Total SCs positive for red fluorescence were further differentiated by TGFb1 and CXCR2 and sorted into three groups of SC subpopulations using flow cytometry. (C) The three cell subpopulations were cultured separately and conditioned medium was obtained. The conditioned medium was used to treat CaMPCs and its effect on the growth of CaMPCs was detected.

[0022] Figure 4 Flow cytometry was used to detect the number of dSCs and nSCs in the skeleton of young and old fracture mice on day 10.

[0023] Figure 5 :(A) The numbers of total SCs, dSCs and nSCs in the callus were detected on the 10th day after fracture in aged mice given AZD5069 or D+Q every other day;(B) The number of neutrophils in the callus;(C) The RNA expression levels of p53 and Bcl-2 in the callus;(D) The biomechanical test results on the 28th day after fracture;(EF) The numbers of total SCs, dSCs and nSCs in the callus of CXCR2 knockout mice 10 days after fracture and the biomechanical test results on the 28th day. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited to the following embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its content.

[0025] The AZD5069 of the present invention is a CXCR2 antagonist with a molecular formula of C 18 H 22 F2N4O5S2, relative molecular weight is 476.52. Chemical structure is as follows Figure 1 As shown, the English name is: N-(2-((2,3-difluorobenzyl)thio)-6-(((2R,3S)-3,4-dihydroxybutan-2-yl)oxy)pyrimidin-4-yl)azetidine-1-sulfonamide.

[0026] Example 1

[0027] AZD5069 selectively eliminates dSCs in callus and promotes AAFR.

[0028] Experimental procedures.

[0029] (1) A bilateral tibial fracture model was established in mice aged 4 and 20 months. The experimental group was intraperitoneally injected with AZD5069 at a dose of 100 mg / kg / d, and the positive control group was given senolytics dasatinib (D, 5 mg / kg) and quercetin (Q, 50 mg / kg). The callus cells of mice aged 4 and 20 months were compared, and the effects of selective elimination of dSCs on fracture healing were studied in cell and animal models using histochemistry, cell biology, immunoprecipitation and immunofluorescence techniques.

[0030] The experimental results show that dSCs will over-secrete p53 and Bcl-2, synergistically regulate the post-translational modification of PDGFRβ, inhibit the PDGFRβ signaling pathway, and thus lead to weakened osteogenesis and decreased fracture healing ability. By using AZD5069, dSCs can be selectively eliminated and the adverse effects brought by them can be reversed, promoting AAFR (see Figure 2 However, AZD5069 had no significant effect on the recruitment of nSCs into the callus (see Figure 2 B). The results of the study showed that AZD5069 selectively eliminated dSCs (CXCR2+) and retained nSCs (CXCR2-TGF1+) with potential beneficial effects. Compared with traditional senolytics (D+Q), AZD5069 has a higher ability to promote fracture healing and improve bone strength (see Figure 2 D). Moreover, the number of osteoblasts in the callus of mice treated with AZD5069 increased significantly, and CT results showed that the area of ​​woven bone and callus increased (see Figure 3 for details).

Claims

1. CXCR2 antagonists represented by AZD5069 are a new generation of selective Senolytics, which have the ability to selectively eliminate harmful SCs subpopulations.

2. Application of CXCR2 antagonists represented by AZD5069 in musculoskeletal system / senile diseases.

3. A senolytic drug for selectively eliminating SCs subpopulations or a drug for skeletal muscle system / senile diseases, characterized in that Its active ingredient is a CXCR2 antagonist represented by AZD5069.

4. A senolytic drug for selectively eliminating SCs subpopulations or a drug for skeletal muscle system / senile diseases, characterized in that Its active ingredient contains AZD5069.

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