CN113425723B - Application of Pim1 small-molecule inhibitor in preparation of product for preventing and treating ankylosing spondylitis - Google Patents
Application of Pim1 small-molecule inhibitor in preparation of product for preventing and treating ankylosing spondylitis Download PDFInfo
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Abstract
The invention belongs to the technical field of medical biology, and particularly relates to application of a Pim1 small molecular inhibitor in preparation of a product for preventing and treating ankylosing spondylitis. Compared with the IL-17A monoclonal antibody, the pim1 treats the AS by blocking Th17 differentiation, and is positioned upstream of the IL-17A monoclonal antibody in mechanism, so the invention can provide a new target and a new way for treating the AS and provides a new choice for AS patients, particularly patients with poor treatment effect of the IL-17A monoclonal antibody.
Description
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to an application of a Pim1 small-molecule inhibitor in preparation of a product for preventing and treating ankylosing spondylitis.
Background
Ankylosing Spondylitis (AS) is a common autoimmune disease. The incidence of AS is high, about 0.2-0.54% in China, and the proportion of AS is 2-3:1 in young and strong years. The pathological characteristics of AS are chronic progressive inflammation and pathological osteogenesis which affect the middle axial bone joint, the clinical manifestations are mainly inflammatory lumbago and backache and middle axial bone joint ankylosis deformity, in the chronic progress of the disease, the labor capacity of the patient is gradually reduced and even lost, and the life quality is seriously influenced. Because AS can not be completely cured, the method brings heavy economic burden to countries and individuals.
At present, the pathogenesis of AS is not yet establishedIt is fully elucidated, but more and more studies in recent years have shown that Th17 cells and their secreted IL-17A play an important driving role in the development of AS. Th17 is caused by peripheral blood
T cells develop, the differentiation level of the T cells is in positive correlation with the activity of AS diseases, and the secreted IL-17A participates in a plurality of processes of AS pathophysiology. IL-17A plays a promoting role in the early stage of AS ATD to mediate inflammation, and IL-17A also serves AS an amplifier of ATD to induce local interstitial cells to generate inflammatory factors to participate in the reaction process of inflammation. In bone tissue, IL-17A inhibits osteoblasts and chondrocytes from producing a matrix, induces osteoblasts to express RANKL, activates osteoclasts, and causes bone destruction. In addition, IL-17A plays a role in sensitizing the symptoms of low back pain of AS patients, and IL-17A enables the patients to be more sensitive to inflammatory stimuli by reducing the stimulation threshold of pain receptors, so that the patients feel stronger pain.
At present, the drugs for clinically treating AS mainly include nonsteroidal anti-inflammatory drugs (NSAIDs), tumor Necrosis Factor Inhibitors (TNFi), disease-relieving antirheumatic drugs (DMARDs), and the like. The NSAIDs can improve symptoms of low back pain, morning stiffness and joint pain of partial patients, but the treatment response of partial patients to the NSAIDs is poor and the side effect is obvious after long-term administration. DMARDs are mainly used to treat peripheral arthritis, but have poor therapeutic effect on spine disease-predominant AS. TNFa can block the activity of TNF and inhibit the progress of pathological changes, and is suitable for patients with symptoms outside the relevant segment and without the effect of NSAIDs treatment. Based on the important role and clinical evaluation of IL-17A in AS generation and development, the IL-17A monoclonal antibody secukinumab is approved for treating AS in China in 2020. Although Secukinumab has a certain clinical therapeutic effect, it still has a poor effect on some AS patients. In addition, secukinumab is expensive, and its use causes a large economic burden on most patients, and thus its universality is not high. Therefore, there is a need to explore new AS therapeutic targets and to find small molecule inhibitors that specifically inhibit the target in order to bring safe and effective new methods for the clinical treatment of AS.
Disclosure of Invention
In order to overcome the defects of the prior art, in vivo and in vitro experiments show that the specific small molecule inhibitors of Pim1 such as AZD1208 and the like can effectively prevent and treat the ankylosing spondylitis by taking Pim1 as a new target point, and provide a new target and a new way for preventing and treating the ankylosing spondylitis.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention aims to provide application of a Pim1 small-molecule inhibitor in preparation of a product for preventing and treating ankylosing spondylitis, wherein the Pim1 small-molecule inhibitor is a biological functional molecule with the molecular weight of less than 1000 Da.
Preferably, the Pim1 small molecule inhibitor includes, but is not limited to, the Pim1 specific inhibitor AZD1208.
The molecular formula of AZD1208 is C 21 H 21 N 3 O 2 S, molecular weight 379.48, structure as follows:
th17 and IL-17A secreted by the Th17 have important functions in the generation and development of AS, and currently, monoclonal antibodies antagonizing IL-17A are clinically used for treating AS, but the treatment effect on partial patients is still poor.
Th17 is derived from
CD4+ T cells, the differentiation of which includes the steps of activation, proliferation and differentiation, are involved in a plurality of Ca2+ related pathways and the phosphorylation of a plurality of proteins at a molecular level, and Th17 and IL-17A secreted by the Th17 have important roles in the generation and development of AS. Pim1 is a phosphorylation kinase in cells, can bind and phosphorylate proteins, can regulate Ca < 2+ > channels in cells, has anti-apoptosis and proliferation promoting effects, and therefore can be a potential target for regulating Th17 differentiation.
Therefore, the invention starts from the source of IL-17A, explores a target point for inhibiting Th17 differentiation, blocks the secretion of IL-17A, starts with small molecular compounds, and explores a safe, effective and high-universality medicine for treating AS. The invention takes Pim1 AS a new AS treatment target, finds that a specific small molecule inhibitor AZD1208 of Pim1 can inhibit CD4+ T cells from differentiating to Th17, blocks the secretion of IL-17A at the source, has good curative effect on AS, shows good anti-inflammatory effect in-vivo and in-vitro experiments, and also shows the characteristic of low toxicity. The small molecular inhibitor Pim1 is used AS a target point to indicate that the AS can be effectively prevented and treated.
Preferably, the prevention and treatment of ankylosing spondylitis is inhibition of Th17 differentiation.
Preferably, the product for preventing and treating ankylosing spondylitis includes, but is not limited to, medicaments for preventing and treating ankylosing spondylitis and health-care functional foods for preventing and treating ankylosing spondylitis.
The second purpose of the invention is to provide a drug for preventing and treating ankylosing spondylitis, which takes Pim1 small-molecule inhibitor as a main active ingredient.
Preferably, the Pim1 small molecule inhibitor includes, but is not limited to, the Pim1 specific inhibitor AZD1208.
Preferably, in order to improve the application range of the medicine, the medicine also comprises a pharmaceutically acceptable carrier and/or an auxiliary material. The carrier and/or adjuvant comprises sour agent, toning agent, flavoring agent, sweetening agent, or their combination.
Preferably, the dosage form of the medicament includes, but is not limited to, injection, oral liquid, tablet, granule, capsule, and pill.
The third purpose of the invention is to provide a health-care functional food for preventing and treating ankylosing spondylitis, which takes a Pim1 small-molecule inhibitor as a main active ingredient.
Preferably, the Pim1 small molecule inhibitor includes, but is not limited to, the Pim1 specific inhibitor AZD1208.
Preferably, to increase the applicability of the food product, the food product further comprises a food acceptable carrier and/or auxiliary material. The carrier and/or adjuvant comprises diluent, excipient, filler, binder, humectant, disintegrating agent, absorption enhancer, sweetener, flavoring agent, or their combination.
Preferably, the food product is in the form of, but not limited to, a decoction, a beverage, a candy, an oral liquid, a capsule, a tablet, and a powder.
Compared with the prior art, the invention has the beneficial effects that:
the invention uses a specific small molecule inhibitor AZD1208 of Pim1, proves the important function of Pim1 in Th17 differentiation through in vivo and in vitro experiments, proves that Pim1 can be used AS a target for blocking Th17 differentiation and IL-17A secretion, and verifies the safety, effectiveness and feasibility of using AZD1208 to treat AS. Meanwhile, compared with the IL-17A monoclonal antibody, the pim1 treats the AS by blocking Th17 differentiation, and is positioned at the upstream of the IL-17A in mechanism, so the invention can provide a new target and a new path for treating the AS and provides a new choice for AS patients, particularly patients with poor treatment effect of the IL-17A monoclonal antibody.
Drawings
FIG. 1 is a flow chart of an experiment of example 1;
FIG. 2 shows Pim1 expression during the differentiation of CD4+ T cells into Th 17;
FIG. 3 is a graph of differentiation of CD4+ T cells to Th17 following treatment with AZD1208 inhibitor;
figure 4 is a graph of the toxic effect of AZD1208 on CD4+ T cells;
FIG. 5 shows the arthritis scores of CIA mice treated with the pam 1-specific inhibitor AZD1208 after injection;
FIG. 6 shows changes in liver and kidney function indices of CIA mice treated with the pim 1-specific inhibitor AZD1208.
Detailed Description
The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the respective embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.
Example 1 Pim1 Small molecule inhibitor has preventive and therapeutic effects on ankylosing spondylitis by inhibiting Th17 differentiation
The experimental flow is shown in fig. 1, and the specific process is as follows:
1. in vitro experiments
(1) Inhibition of Pim1 Effect on Th17 differentiation
CD4+ T cells were sorted from human peripheral blood mononuclear cells (from healthy blood donor volunteers) using human CD4 magnetic beads (purchased from America, whirlwind, cat # 130-045-101) as follows:
adding peripheral blood gently onto Ficoll separating medium at 1:1 volume ratio, centrifuging at 400g rotation speed (acceleration 1, deceleration 0) for 35min, carefully sucking up leucocyte layer with Pasteur pipette after centrifuging, washing with PBS twice, centrifuging to remove supernatant, suspending cells with 1640 medium containing 10% FBS, counting, and suspending cells per 10 6 2uL of magnetic beads and 8uLbuffer (PBS 100mL +0.5% BSA (0.5 g) +2mM EDTA (0.654 g)) were added to each cell, adjusted to pH 7.2 with NaOH, filtered to sterilize, mixed well, and incubated at 4 ℃ for 15 minutes in the dark. Adding an appropriate amount of buffer, centrifuging for 10min at 300g, discarding the supernatant, and mixing with 500. Mu.L of buffer/10 8 And resuspending the cells. Placing the separation column on a magnetic frame, moistening with buffer, injecting cells into the separation column, washing with buffer for 3 times, removing the separation column from the magnetic frame, adding 1mL buffer, washing out cells in the separation column, centrifuging for 5min at 1500 rpm, discarding supernatant, resuspending with 1mL 1640 medium containing 10% FBS, counting, and performing filtration with a filter medium of 0.6 × 10 6 cell/Kong Chongban.
Under the condition of Th17 polarization (anti-CD 3 ug/mL, anti-CD28 ug/mL, IL-23 ng/mL, IL-6 20ng/mL and TGF-beta 5 ng/mL), CD4+ T cells are induced to be differentiated to Th17, meanwhile, a pim1 specific small molecule inhibitor AZD1208 20uM or DMSO-treated cells with the same volume are given, and after 3 days of culture, the proportion of Th17 differentiation is detected by flow cytometry by taking CD4 and IL-17A as markers.
As shown in fig. 2 and fig. 3, it was found that the expression of Pim1 was up-regulated during the differentiation of CD4+ T cells into Th17, but the proportion of Th17 differentiation was significantly reduced after inhibiting Pim1 activity using AZD 1208;
(2) Toxicity of AZD1208 inhibitors on CD4+ T cells
The toxicity of the drug on the cells was detected using CCK-8 enzyme-labeled assay. The specific detection is as follows: to a 96-well plate, 100uL Th17 polarization-cultured CD4+ T cells (about 5000 cells) were added, and AZD1208 or an equal volume of DMSO was added, respectively, while a blank was set by adding a culture solution containing no cells, and 5 duplicate wells were set for each group. To each well, 10uL of CCK-8 solution was added, incubated for 2 hours, and absorbance was measured at 450nm using a microplate reader. In the detection range, the absorbance at 450nm is approximately proportional to the cell activity.
As shown in fig. 4, it was found that inhibition of Pim1 using AZD1208 was less toxic to CD4+ T cells.
2. In vivo experiments
(1) Inhibition of the therapeutic effect of Pim1 on CIA
As an arthritis-inducing subject, 6-8 week-old DBA/1 female mice (purchased from Jiangsu Jiejiao Kangbiotech Co., ltd.) were used, CFA (Freund's complete adjuvant, 1 mg/mL) and type II collagen (2 mL/mL) were completely emulsified on ice, transferred to a 1mL syringe, centrifuged, and then 100uL was intradermally injected into the groin of each mouse. After 21 days, the mice were boosted (Freund's incomplete adjuvant IFA + type II collagen), collagen-induced arthritis models (CIA) were established, and 5 CIA mice per group were divided into experimental and control groups. AZD1208 was dissolved in DMSO to make a 60mg/mL solution and diluted with methylcellulose to a 3mg/mL suspension, and the CIA was treated by gavage with AZD1208 (control: equivalent ratio of methylcellulose to DMSO) at a dose of 1 dose per day at 30mg/kg body weight for 21 consecutive doses, with an arthritis score every 3 days.
As shown in fig. 5, after 21 days of continuous administration, AZD1208, a specific small molecule inhibitor of Pim1, was found to significantly reduce the arthritis score in CIA mice.
(2) Toxic effects of AZD1208 inhibitors in vivo
After the above CIA mice were continuously administered for 21 days, the mice were sacrificed by dislocation of the spine on day 21, and peripheral blood was collected to detect liver and kidney function indices [ serum aspartate Aminotransferase (AST) by MDH method, alanine Aminotransferase (ALT) by lactate dehydrogenase method, serum creatinine (Scr) by picric acid method, and serum urea nitrogen (BUN) by urease-glutamate dehydrogenase method ]. As shown in figure 6, it was found that inhibition of Pim1 activity in vivo using AZD1208 did not cause severe impairment of liver and kidney function.
The experiments show that the Pim1 specific small molecule inhibitor AZD1208 can inhibit the differentiation of CD4+ T cells to Th17, block the secretion of IL-17A at the source, has a good curative effect on Ankylosing Spondylitis (AS), shows a good anti-inflammatory effect in vivo and in vitro experiments, and also shows the characteristic of low toxicity. Therefore, pim1 can be used AS a new target point for AS treatment.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.
Claims (4)
- The application of the Pim1 small molecular inhibitor in preparing a product for preventing and treating ankylosing spondylitis is characterized in that the Pim1 small molecular inhibitor is a Pim1 specific inhibitor AZD1208, and the prevention and treatment of the ankylosing spondylitis is to inhibit Th17 differentiation.
- 2. The use according to claim 1, wherein the product for the prevention and treatment of ankylosing spondylitis is a medicament for the prevention and treatment of ankylosing spondylitis.
- 3. The use of claim 2, wherein the medicament for preventing and treating ankylosing spondylitis further comprises a pharmaceutically acceptable carrier and/or adjuvant.
- 4. The use of claim 2, wherein the medicament is in the form of injection, oral liquid, tablet, granule, capsule, pill.
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| Title |
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| "AZD1208, a potent and selective pan-Pim kinase inhibitor, demonstrates efficacy in preclinical models of acute myeloid leukemia";Erika K Keeton，et al;《Blood》;20140206;第123卷(第6期);第905-913页 * |
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