CN107661501B - Pharmaceutical composition for treating ankylosing spondylitis, drug target and application thereof - Google Patents

Abstract

The invention discloses a pharmaceutical composition for treating ankylosing spondylitis, a pharmaceutical target and application thereof, wherein the pharmaceutical composition comprises a reagent for inhibiting or preventing CXCL5 expression or a CXCL5 antagonist. The invention can simultaneously relieve chronic inflammation and pathological osteogenesis of AS patients, has good effects of controlling the worsening progress of diseases and preventing the generation of deformity, and CXCL5 can be used AS a target point to be applied to the preparation or screening of the medicament for treating the ankylosing spondylitis.

Description

Pharmaceutical composition for treating ankylosing spondylitis, drug target and application thereof

Technical Field

The invention relates to the field of ankylosing spondylitis medicaments, in particular to a medicinal composition for treating ankylosing spondylitis, a medicinal target and application thereof.

Background

The pathogenesis of AS is not clear, and no radical treatment is available, and the treatment method can be roughly divided into three types, namely non-drug treatment, drug treatment and operation treatment, wherein the non-drug treatment mainly comprises education on disease knowledge of patients and families thereof, long-term psychosocial and rehabilitation treatment on the patients, persuading the patients to reasonably and insistently exercise physical exercises, keeping the chest in a straight posture in front of chest, abdomen and eyes when standing, giving necessary physical treatment to pain or inflammatory joints or soft tissues, recommending the smoking patients to quit smoking, preferably using non-steroidal anti-inflammatory drugs, other common drugs also comprise tumor necrosis factor (TNF- α) antagonists and disease-improving antirheumatic drugs (DMARDs), and for patients with late AS, pathological changes caused by the involvement of hip joints, narrowing, strengthening and straightening joints and treating vertebral deformity, adopting artificial joint replacement surgery, and for patients with limited spinal motion, and fixing points of vertebral bodies to correct vertebral column deformity, and usually correcting vertebral column deformity by 2-segment fixation.

Although the diagnosis and treatment of AS have been well developed in recent years, the exact cause of the disease is still not proven, and a radical treatment method is still not found. AS the disease progresses, patients with AS can develop pain in the waist, back, neck, hips, and joints, with severe cases even suffering from spinal deformities and joint stiffness.

In response to the shortcomings of the existing therapies, the present invention is directed to finding new targets for treating AS in an attempt to control the progression of disease progression and prevent the development of malformations.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a pharmaceutical composition and application thereof.

The invention also aims to provide application of CXCL5 as a target point in preparing or screening a medicament for treating ankylosing spondylitis.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a pharmaceutical composition comprising an agent that inhibits or prevents the expression of CXCL5, or a CXCL5 antagonist.

The CXCL5 is increased to cause inflammatory reaction in an AS patient body and cause pathological bone formation, and the inventor discovers that the CXCL5 antagonist can effectively relieve inflammation and spinal ossification conditions of an AS mouse animal model by carrying out tail vein injection on the AS mouse animal model.

As a preferred embodiment of the pharmaceutical composition of the present invention, the CXCL5 antagonist is a mouse CXCL5 monoclonal antibody.

As a preferred embodiment of the pharmaceutical composition of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

As a preferred embodiment of the pharmaceutical composition of the present invention, the dosage form of the pharmaceutical composition is an injection or a lyophilized preparation.

The invention also provides a reagent for inhibiting or preventing CXCL5 expression or an application of the CXCL5 antagonist in preparing a medicament for treating ankylosing spondylitis.

As a preferred embodiment of the use according to the invention, the use of the agent for inhibiting or preventing the expression of CXCL5, or of the CXCL5 antagonist, for the preparation of a medicament for the treatment of pathological osteogenesis and/or chronic inflammation in ankylosing spondylitis.

The invention also provides application of CXCL5 as a target point in preparation or screening of a medicament for treating ankylosing spondylitis.

The inventor finds that the CXCL5 is increased to cause inflammatory reaction in an AS patient body and cause pathological bone formation, and the inventor finds that the CXCL5 antagonist can effectively relieve inflammation and spinal ossification conditions of an AS mouse animal model by carrying out tail vein injection on the AS mouse animal model, thereby suggesting that the CXCL5 antagonist is a novel method for effectively treating AS, and the CXCL5 can be used AS a target point to prepare or screen a medicament for treating ankylosing spondylitis.

Compared with the prior art, the invention has the beneficial effects that:

compared with the existing AS treatment technology, the traditional Chinese medicine composition can simultaneously relieve chronic inflammation and pathological osteogenesis of AS patients, and has good effects on controlling the worsening progress of diseases and preventing deformity; the treatment method has no side effects such as abnormal death or infection of the mice and the like, and shows that the side effects are small; the CXCL5 can be used as a target point to be applied to the preparation or screening of the medicament for treating the ankylosing spondylitis.

Drawings

FIG. 1 is a graph showing the results of measuring the amount of CXCL5 in the serum of patients and healthy individuals who have AS (HD means healthy individuals and AS means AS patients);

FIG. 2 is a graph of the effect of CXCL5 on TNF- α secretion by monocytes macrophages;

FIG. 3 is a alizarin red staining diagram;

FIG. 4 shows the results of quantification of alizarin red staining;

FIG. 5 is a graph showing quantitative measurement of ALP activity;

FIG. 6 is a flow chart of the experiment of example 2.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples. It will be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The test methods used in the following examples are all conventional methods unless otherwise specified; materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

The inventor researches to find that the CXCL5 level is obviously increased compared with that of normal people, and the CXCL5 is increased to cause inflammatory reaction in AS patients and cause pathological bone formation.

(1) Detection of CXCL5 levels in serum of AS patients

Respectively extracting serum of AS patients and serum of health people of the same period, detecting the content of CXCL5 in the serum according to the kit instructions, wherein the result is shown in figure 1, and the result in figure 1 shows that the content of CXCL5 in the serum of AS patients is 1620 +/-76.25 pg/mL; the content of CXCL5 in the serum of healthy people is 351.7 +/-19.78 pg/mL, p is less than 0.05, and the level of CXCL5 in the serum of AS patients is obviously higher than that of normal people.

(2) Effect of CXCL5 on TNF- α secretion by monocytes

Separating Peripheral Blood Mononuclear Cells (PBMC) from human peripheral blood by density gradient centrifugation, and magnetically separating with CD14Bead sorting to CD14⁺Monocytes, CD14⁺Monocyte is expressed in 2X 10⁵One of the cells was inoculated into a 24-well plate, the culture medium was RPMI-1640 containing 10% serum, CXCL5 was added to the culture medium at a concentration of 0, 1, 5, 10 and 20ng/mL, and after 5 days of culture, the supernatant was collected and tested for the level of TNF- α secretion by ELISA, the results are shown in FIG. 2.

As shown in figure 2, compared with a control group, CXCL5 can promote mononuclear macrophages to secrete TNF- α, and the fact that CXCL5 is increased to cause inflammatory reaction in AS patients is shown.

(3) Effect of CXCL5 on osteogenic differentiation of Mesenchymal Stem Cells (MSC)

Separating and purifying MSC from bone marrow of healthy volunteers by density gradient centrifugation-wall pasting method, and purifying MSC at 5 × 10⁴Planting the cells/hole into a 12-hole plate, respectively adding 0, 1, 5, 10 and 20ng/mL of CXCL5 into a culture medium of the mesenchymal stem cells, wherein the culture medium is DMEM containing 10% serum, and the formula of an osteogenesis inducing solution is as follows: 100IU/mL penicillin, 100IU/mL streptomycin, 0.1mM dexamethasone, 10mM glycerol phosphate, 50mM vitamin C. ALP activity was measured by induction up to day 10 and alizarin red staining was performed on day 14. Fig. 3 is a alizarin red staining graph, fig. 4 is a alizarin staining quantitative result graph, and fig. 5 is an ALP activity quantitative determination result graph.

The osteogenic differentiation enhancement of Mesenchymal Stem Cells (MSC) of AS patients is one of the important mechanisms of pathological osteogenesis of AS. Alizarin staining and ALP activity experimental results show that CXCL5 can promote MSC osteogenesis.

In conclusion, the increase of the serum CXCL5 of AS patients leads to the increase of the secretion of TNF- α by mononuclear macrophages, can cause in vivo inflammatory reaction, and simultaneously, the CXCL5 can promote MSC bone formation, thereby causing pathological bone formation.

Example 2

The inventor discovers that the CXCL5 antagonist can effectively relieve inflammation and spinal ossification conditions of an AS mouse animal model by tail vein injection of the CXCL5 antagonist to the AS mouse animal model, and the CXCL5 antagonist is a medicine for effectively treating AS. The specific flow is shown in fig. 6:

(1) AS mouse animal model construction

Female BALB/c mice 24 weeks old were selected and administered with intraperitoneal injection of Proteoglycan (Proteoglycan) (1 injection for each of weeks 0, 3 and 6, 3 injections, 100. mu.g each).

(2) Group administration

The AS mouse animal model was constructed and divided into three groups, including a control group, a TNF- α antagonist group and a CXCL5 antagonist group, and starting at week 10, the three groups were each administered with 0.1mg of tail vein injection of saline, TNF- α antagonist and CXCL5 antagonist, once every 4 weeks, and 6 total injections were administered up to week 30. the CXCL5 antagonist used in this study was mouse CXCL5 monoclonal antibody from NOVUS, cat # NBP 2-22026.

(3) Detection of inflammation and pathological osteogenesis conditions in mice

Mice in three groups are sacrificed by adopting a cervical dislocation method from the 14 th week, once every 4 weeks to the 34 th week for 6 times, peripheral blood serum of the mice is separated after the sacrifice, the levels of CRP, TNF- α, IL17 and IL23 of the mice are detected, the 3 rd to 5 th lumbar vertebra segments of the spinal column of the mice are taken and sequentially fixed, decalcified, embedded in paraffin and sliced, and the pathological bone formation condition of the spinal column of the mice is observed by HE staining and Masson staining, wherein the pathological bone formation scoring standard comprises the following steps of 0 score of normal, 1 score of slight hyperplasia of the edge of the vertebral body, 2 score of obvious osteophyte formation of the edge of the vertebral body, 3 score of bone bridge formation of the edge of one side vertebral body, and 4 score of bone bridge formation and interbody fusion of the edge of the vertebral body.

(4) Results

Compared with a control group, the TNF- α antagonist group and the CXCL5 antagonist group can inhibit the in-vivo inflammation level of an AS mouse animal model (CRP, TNF- α, IL17 and IL23 are all obviously reduced), the CXCL5 antagonist group has stronger inflammation inhibition capability than the TNF- α antagonist group, compared with the control group, the CXCL5 antagonist group can obviously inhibit the pathological bone formation of the spine of the AS mouse animal model, the TNF- α antagonist group does not influence the pathological bone formation progress, and the side effects of abnormal death or infection of mice and the like are not seen in the three groups.

TABLE 1C reactive protein (CRP) levels (ng/mL)

Time of day	Control group	TNF- α antagonist group	CXCL5 antagonist group
				14 weeks	23.1±1.3	22.1±3.2	18.2±1.2
For 18 weeks	24.2±1.8	20.5±2.9	15.5±1.0
				22 weeks	25.3±2.2	19.2±1.0	13.9±0.9
For 26 weeks	20.9±0.9	16.7±0.8	8.6±0.5
				30 weeks	21.5±2.5	17.2±1.4	7.2±0.2
34 weeks	26.7±1.8	11.4±1.9	5.7±0.3

TABLE 2 TNF- α levels (pg/mL)

Time of day	Control group	TNF- α antagonist group	CXCL5 antagonist group
				14 weeks	323.6±34.6	333.6±25.1	335.1±23.7
For 18 weeks	403.4±44.1	308.1±24.9	303.2±45.1
				22 weeks	394.7±34.9	299.1±17.8	291.6±15.4
For 26 weeks	401.6±22.7	253.3±27.1	220.3±22.2
				30 weeks	386.1±39.1	165.7±12.4	155.2±18.9
34 weeks	397.8±24.1	110.4±31.9	101.1±8.0

TABLE 3 Interleukin 17 levels (pg/mL)

TABLE 4 Interleukin 23 levels (pg/mL)

Time of day	Control group	TNF- α antagonist group	CXCL5 antagonist group
				14 weeks	111.4±11.5	121.2±15.2	121.1±9.9
For 18 weeks	136.1±15.1	111.7±14.1	91.2±16.8
				22 weeks	105.0±10.1	112.1±11.0	88.3±5.9
For 26 weeks	109.7±7.8	117.2±17.3	57.9±5.1
				30 weeks	116.4±15.7	94.2±11.1	35.6±6.1
34 weeks	145.1±19.5	112.6±16.2	34.7±6.9

TABLE 5 spinal pathological osteogenesis score

Time of day	Control group	TNF- α antagonist group	CXCL5 antagonist group
				14 weeks	4.5±0.5	4.4±1.2	4.2±1.9
For 18 weeks	4.8±1.2	4.8±2.2	3.3±1.0
				22 weeks	5.1±1.1	4.9±1.0	2.3±1.1
For 26 weeks	5.8±2.1	5.7±2.2	2.0±0.8
				30 weeks	7.4±1.7	7.1±4.2	1.2±0.3
34 weeks	7.8±3.3	7.7±3.1	0.8±0.2

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims (2)

1. Use of a CXCL5 antagonist for the preparation of a medicament for the treatment of pathological osteogenesis in ankylosing spondylitis and chronic inflammation in ankylosing spondylitis.
2. The application of CXCL5 as a target spot in preparing or screening medicaments for treating the pathological osteogenesis of ankylosing spondylitis and the chronic inflammation of ankylosing spondylitis.

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