Multidrug Composition for the Treatment of Ankylosing Spondylitis

Abstract

This invention discloses a multi-drug composition for the treatment of ankylosing spondylitis. For the first time, this invention addresses the etiology of streptococcus-related diseases by proposing a synergistic treatment strategy combining a classic tumor necrosis factor antagonist (such as etanercept) with a narrow-spectrum antibiotic (benzathine penicillin). This approach breaks through the traditional treatment strategy that targets only the inflammatory pathway, providing a novel entry point for AS treatment.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, to multi-drug compositions for treating ankylosing spondylitis, and particularly to the application of antibiotics in improving the therapeutic effect of ankylosing spondylitis. Background Technology

[0002] Ankylosing spondylitis (AS) is a common chronic inflammatory disease of the spondyloarthritis family, often leading to spinal dysfunction and severely impacting patients' quality of life. Currently, the exact cause of this disease is not fully understood.

[0003] In clinical treatment, current methods mainly include nonsteroidal anti-inflammatory drugs (NSAIDs) and biological agents such as tumor necrosis factor antagonists. However, the efficacy of NSAIDs varies among individuals, while biological agents may face issues such as symptom recurrence after discontinuation, potential risks of long-term use, and gradual decline in efficacy in some patients. Therefore, there remains an unmet clinical need for the treatment of this disease, and there is an urgent need to explore more fundamental intervention strategies by starting with the pathogenesis of the disease.

[0004] In recent years, some studies have suggested a possible link between microbial infection and the pathogenesis of ankylosing spondylitis (AS). For example, reports have shown the presence of specific microbial components in samples from some patients; animal experiments have also observed that exposure to certain microbial components can induce radiographic changes similar to spondyloarthritis. At the cellular level, these microbial components can activate macrophages and polarize them towards a pro-inflammatory phenotype, thereby promoting the release of inflammatory factors. These inflammatory factors involve signaling pathways such as TNF-α and IL-17, which are the targets of some current clinical biologics. Although existing research reveals a possible link between microbial infection and the disease, no studies have confirmed that antibiotics can improve the treatment effect of AS, and antibiotics are not used or recommended for adjuvant therapy in routine clinical treatment. Summary of the Invention

[0005] This invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the purpose of this invention is to provide the application of antibiotics in improving the treatment efficacy of ankylosing spondylitis (AS). This invention, for the first time, proposes an intervention treatment from the perspective of streptococcal-related etiology, suggesting a synergistic treatment strategy combining a classic tumor necrosis factor antagonist (such as etanercept) with a narrow-spectrum antibiotic (benzathine penicillin). This breaks through the traditional treatment approach that only targets the inflammatory pathway, providing a completely new entry point for AS treatment.

[0006] A first aspect of the invention provides the use of the composition in the preparation of a medicament for treating or adjunctive treatment of ankylosing spondylitis.

[0007] In some embodiments of the present invention, the composition includes antibiotics and anti-inflammatory drugs.

[0008] In some embodiments of the present invention, the anti-inflammatory drug includes nonsteroidal anti-inflammatory drugs and tumor necrosis factor antagonists.

[0009] In some embodiments of the present invention, the anti-inflammatory drug includes, but is not limited to, etanercept.

[0010] In some embodiments of the present invention, the antibiotic is an antibiotic that has growth-inhibiting or killing effects on streptococci.

[0011] In some embodiments of the present invention, the antibiotic includes, but is not limited to, benzathine penicillin.

[0012] In some embodiments of the present invention, the dosage form of the composition includes, but is not limited to, tablets, capsules, granules, solutions, and injections.

[0013] In some embodiments of the present invention, the medicament further contains pharmaceutically acceptable excipients.

[0014] In some embodiments of the present invention, the pharmaceutically acceptable excipients are rationally selected based on factors such as the product form of the drug, its intended use, and the route of administration, and include, but are not limited to: buffers, coenzymes, enzyme protectants, metal ions, catalysts, defoamers, diluents (such as starch, dextrin, sucrose, lactose, mannitol, etc.), absorbents (such as calcium sulfate, dicalcium phosphate, etc.), wetting agents (such as ethanol), binders (such as hydroxypropyl methylcellulose, povidone, etc.), solvents, pH adjusters, antibacterial agents (such as sodium sulfite, sodium thiosulfate, etc.), isotonic adjusters (such as glucose, sodium chloride, etc.), chelating agents (such as disodium EDTA), etc.

[0015] In some embodiments of the present invention, the antibiotic and anti-inflammatory drug are administered alone or simultaneously during administration.

[0016] In some embodiments of the invention, the antibiotic and anti-inflammatory drug are administered separately during administration.

[0017] In some embodiments of the present invention, the dosage of the antibiotic is 200,000-300,000 U / mL / time.

[0018] In some embodiments of the present invention, the dosage of the antibiotic is 240,000 U / mL / time.

[0019] In some embodiments of the present invention, the dosage of the anti-inflammatory drug is 15-40 mg / time.

[0020] In some embodiments of the present invention, the dosage of the anti-inflammatory drug is 25 mg / time.

[0021] In some embodiments of the present invention, the administration frequency is 1-5 times / week.

[0022] In some embodiments of the present invention, the administration frequency is 1-2 times / week.

[0023] In some embodiments of the present invention, etanercept is administered twice a week.

[0024] In some embodiments of the present invention, the dosing frequency of benzathine penicillin is: once every 2 weeks.

[0025] In some embodiments of the present invention, the dosage and frequency of administration can be reasonably adjusted based on physician experience and treatment practice to achieve the corresponding therapeutic effect or purpose.

[0026] A second aspect of the invention provides the use of antibiotics as a therapeutic promoter for ankylosing spondylitis.

[0027] In some embodiments of the present invention, the antibiotic is an antibiotic that has growth-inhibiting or killing effects on streptococci.

[0028] In some embodiments of the present invention, the antibiotic includes, but is not limited to, benzathine penicillin.

[0029] In some embodiments of the present invention, the treatment of ankylosing spondylitis is mainly based on anti-inflammatory drugs, and the antibiotics are used to enhance the therapeutic effect or efficacy of the main treatment.

[0030] In some embodiments of the present invention, administering any dose of antibiotic alone does not have a therapeutic effect on AS.

[0031] A third aspect of the invention provides a pharmaceutical composition comprising an antibiotic and an anti-inflammatory drug.

[0032] In some embodiments of the invention, the antibiotic is as defined above.

[0033] In some embodiments of the invention, the anti-inflammatory drug is as defined above.

[0034] The beneficial effects of this invention are: 1. This invention provides an innovative treatment approach that addresses the shortcomings of existing treatment regimens. Current clinical treatments for ankylosing spondylitis (AS) primarily rely on single-drug antagonists targeting a single inflammatory molecular pathway (such as tumor necrosis factor antagonists). These treatments generally suffer from limited ASAS20 / ASAS40 remission rates, high disease rebound rates after discontinuation, and a tendency for efficacy to decline or fail with long-term use, and may also induce opportunistic infections. The method in this invention breaks through the traditional treatment approach that targets only inflammatory pathways. For the first time, it intervenes from the perspective of streptococcal-related etiology, proposing a synergistic treatment strategy that combines a classic tumor necrosis factor antagonist (such as etanercept) with a narrow-spectrum antibiotic (benzathine penicillin), providing a completely new entry point for AS treatment.

[0035] 2. This invention achieves a dual synergistic effect of etiology and inflammation blocking in the treatment of AS. The combined medication in this invention achieves a dual synergistic effect by eliminating potential sources of infection and reducing the risk of opportunistic infections caused by monotherapy with tumor necrosis factor antagonists. This overcomes the problems that benzathine penicillin alone is ineffective and that monotherapy with tumor necrosis factor antagonists has limited therapeutic effects.

[0036] 3. The combination regimen in this invention significantly improves clinical efficacy and safety. This invention demonstrates that the benzathine penicillin / etanercept combination regimen significantly improves treatment response rates compared to etanercept monotherapy. At 12-week follow-up, the ASAS20 response rate in the combination treatment group was approximately 67.8% (compared to 62.5% in the control group), and the ASAS40 response rate was approximately 46.4% (compared to 34.7% in the control group). Furthermore, subjects showed significant improvements in disease activity, physical function, and quality of life scores, and no serious adverse events occurred.

[0037] 4. The solution in this invention fills a current treatment gap. Currently, there are no published guidelines or indications to support the use of antibiotics for the treatment of ankylosing spondylitis. This invention is the first to apply narrow-spectrum antibiotics to the etiological intervention of AS and combine them with tumor necrosis factor antagonists, achieving a synergistic treatment upgrade from "symptom control" to "etiological intervention + inflammation blocking", providing an effective and safe new approach to improve the long-term prognosis of patients. Attached Figure Description

[0038] Figure 1 The image shows a representative fluorescence spectrum of peripheral blood mononuclear cells (PBMCs) from the subjects in Example 1. Green represents fluorescent probes carrying streptococcal 16S rRNA, and blue represents cell nuclei. 28 and 29 represent different subjects.

[0039] Figure 2The image shows a representative fluorescence spectrum of macrophages in the hip tissue of the subjects in Example 2. Green represents fluorescent probes carrying streptococcal 16S rRNA, and blue represents cell nuclei. 0229 and 0613 represent different subjects.

[0040] Figure 3 The results of the animal experiments in Example 3 are shown. A and B are images of the rabbit sacroiliac joints without streptococcal vesicle modeling; C and D are images of the rabbit sacroiliac joints 2 months after streptococcal vesicle modeling.

[0041] Figure 4 The diagram shows the results of validating the molecular mechanism at the cellular level in Example 4. A shows the results of Western blot detection of iNOS, TNF-α, and β-actin protein expression in macrophages of each group; B shows the results of immunofluorescence staining detection of the localization and expression of iNOS (red) and DAPI (blue) in macrophages of each group; C shows the relative expression of IL-1β mRNA in macrophages of each group using qRT-PCR; D shows the relative expression of TNF-α mRNA in macrophages of each group using qRT-PCR; and E shows the results of flow cytometry detection of CD86 in macrophages of each group. + F4 / 80 + The percentage of double-positive cells; F represents the CD86 count in each group measured by flow cytometry. + Cell percentage; G represents the iNOS integrated optical density of macrophages in each group, measured using immunofluorescence.

[0042] Figure 5The diagram shows the results of validating the molecular mechanism at the cellular level in Example 4. A is a schematic diagram of the experimental procedure for co-culturing macrophage conditioned medium and BMSCs; B shows the results of ELISA detection of PGD2 secretion levels in macrophage supernatants of each group; C shows the results of ELISA detection of TNF-α secretion levels in macrophage supernatants of each group; D shows the results of ELISA detection of IL-17A secretion levels in macrophage supernatants of each group; E shows the results of ELISA detection of IL-6 secretion levels in macrophage supernatants of each group; F shows the results of Western blot detection of PPARγ, FABP4, and β-actin protein expression in BMSCs of each group; G shows the results of Western blot gray-scale quantification of the relative expression of PPARγ / β-actin in BMSCs of each group; H shows the results of Western blot gray-scale quantification of the relative expression of FABP4 / β-actin in BMSCs of each group; I shows the results of Oil Red O staining absorbance quantification of the adipogenic capacity of BMSCs of each group; J shows the results of qRT-PCR detection of PPARγ in BMSCs of each group. The results of relative mRNA expression; K represents the results of relative C / EBPα mRNA expression in BMSCs of each group detected by qRT-PCR; L represents the results of lipid droplet (orange-red) formation in BMSCs of each group observed by Oil Red O staining.

[0043] Figure 6 A graph comparing the ASAS20 response rates of the two groups of subjects at week 12 of treatment in different experimental groups.

[0044] Figure 7 A graph comparing the ASAS40 response rates of the two groups of subjects at week 12 of treatment in different experimental groups.

[0045] Figure 8 A comparison of the improvement rates of BASDAI scores between the two groups at week 12 of treatment in different experimental groups.

[0046] Figure 9 Electron micrographs of the extracted Streptococcus agalactiae exosomes and their particle size distribution. Detailed Implementation

[0047] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0048] Example 1 In this embodiment, peripheral blood was extracted from 30 AS patients and 28 normal controls, and mononuclear cells were isolated for in situ fluorescence hybridization detection of streptococcal 16S rRNA (FISH).

[0049] The specific detection method is as follows: the collected mononuclear cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes, washed with PBS, and the cell concentration was adjusted to 1×10⁻⁶. 6 Cells / mL. Add 10 μL of cell suspension to a poly-L-lysine-coated slide and bake at 56°C for 30 minutes. Permeate the slide with 0.1% Triton X-100 for 10 minutes, then wash with PBS. Fix the slide with 1% paraformaldehyde at room temperature for 10 minutes, then wash with PBS. Dehydrate sequentially with 70%, 80%, 90%, and 100% ethanol for 5 minutes each, then dry at room temperature. Add 20-40 μL of prehybridization solution to the sample and incubate at 37°C for 30 minutes. Add 20-40 μL of hybridization reaction solution (probe + hybridization solution) to the sample to be tested and incubate at 73°C for 5-8 minutes. Quickly transfer to 37°C for overnight hybridization (16-20 h). Wash with preheated wash solutions I, II, III, and IV at 42°C. Stain with 20-40 μL of DAPI for 10 minutes, wash with PBS, and mount with anti-fluorescence quenching mounting medium. Take a laser confocal image.

[0050] The probes used are: Streptococcus agalactiae probe sequence: 5'-GTAAACACCAAACMTCAGCG-3' (SEQ ID NO: 1).

[0051] The prehybridization solution, hybridization solution, washing solution and mounting medium used were all from the RNA-FISH (short chain) Kit (purchased from BersinBio).

[0052] The results are as follows Figure 1 As shown.

[0053] It can be found that streptococcal 16S rRNA is present in the PBMCs of AS patients, indicating that streptococci are present in their peripheral blood.

[0054] Example 2 In this embodiment, perihip tissue was collected from patients who underwent total hip arthroplasty, including 14 patients with avascular necrosis of the femoral head and 15 patients with normal femoral head necrosis. The obtained perihip tissue was subjected to in situ fluorescent hybridization (FISH) detection of streptococcal 16S rRNA.

[0055] The specific detection method is as follows: the collected perihip tissue is prepared into paraffin sections. The paraffin sections are placed in xylene for 15 minutes; then rehydrated sequentially with 100%, 90%, 80%, and 70% ethanol, and washed with PBS; the sections are permeated with 0.1% Triton X-100 for 10 minutes and then washed with PBS. Fix the slides with 1% paraformaldehyde at room temperature for 10 minutes, wash with PBS; dehydrate in a gradient of 70%, 80%, 90%, and 100% ethanol for 5 minutes each, and dry at room temperature; add 20-40 μL of prehybridization solution to the sample and incubate at 37°C for 30 minutes; add 20-40 μL of hybridization reaction solution (probe + hybridization solution) to the sample to be tested, incubate at 73°C for 5-8 minutes, and quickly transfer to 37°C for overnight hybridization (16-20 h); wash with washing buffers I, II, III, and IV preheated to 42°C; stain with 20-40 μL of DAPI for 10 minutes, wash with PBS, and mount with anti-fluorescence quenching mounting medium; take laser confocal images.

[0056] The agalactia streptococcus probe used is the same as in the above-described embodiments.

[0057] The results are as follows Figure 2 As shown.

[0058] It was found that streptococcal 16S rRNA deposition was prevalent in the perihip tissues of AS patients, mainly located in the nucleus region of macrophages within the tissue. This suggests that there is a high probability of deposition of streptococcal nucleic acid, protein, and other components, or ectopic colonization without active bacterial structures, in the perihip tissues of AS patients.

[0059] Example 3 In this embodiment, an animal experiment was used to detect whether streptococcal infection was one of the causes of AS.

[0060] The specific method is as follows: Select healthy male New Zealand White rabbits (weighing 2.5–3.0 kg), raise them under SPF conditions, with an ambient temperature of 22 ± 2 °C, relative humidity of 50-60%, 12-hour light-dark cycle, and free access to food and water.

[0061] Take commercially available Streptococcus agalactiae ( Streptococcus agalactiae BNCC 353769 and Lactobacillus rhamnosus ( Lactobacillus rhamnosusBNCC 136673, after resuscitation and culture, *Streptococcus agalactiae* was inoculated into brain-heart infusion (BHI) broth and cultured (37°C for 18-24 h). The activated strain was then transferred to fresh culture medium for secondary culture under the same conditions. The bacterial culture supernatant was collected for the isolation of extracellular vesicles (EVs). The obtained bacterial culture supernatant was centrifuged sequentially at 3000×g for 10 min and then at 10000×g for 30 min to remove cells and debris. It was then filtered through a 0.22 µm top-mounted filter. EVs (precipitate) were collected by ultracentrifugation (100000×g for 70 min at 4°C). The precipitate was resuspended in PBS to obtain exosomes derived from *Lactobacillus rhamnosus* (LB EVs) or *Streptococcus agalactiae* (Saga EVs) (electron microscopy images and particle size distribution are shown in the figure). Figure 9 (As shown).

[0062] Exosomes derived from *Lactobacillus rhamnosus* (LB EVs) or *Streptococcus agalactiae* (Saga EVs) were isolated, purified, and resuspended in sterile PBS. Experimental animals were randomly divided into three groups: a PBS control group, an LB EVs group, and a Saga EVs group. All groups received the medication via slow intravenous injection from the ear vein, at a dose calculated based on body weight (100 μg / kg), with a single injection volume controlled between 0.5 and 1.0 mL. The PBS control group received an equal volume of sterile PBS. The animals' general condition and body weight changes were observed daily after administration.

[0063] Three months after injection, animals underwent magnetic resonance imaging (MRI). Appropriate anesthesia was administered before imaging, and the animals were placed in a supine position and secured within a small animal coil. A 3.0 T whole-body MRI scanner was used to acquire T1-weighted and T2-weighted images or STIR sequences of the target sites. Scanning parameters were kept consistent across groups to ensure image comparability.

[0064] MRI images were evaluated under blinded conditions by two researchers with radiology experience, analyzing indicators such as changes in inflammatory signals, bone marrow edema, or structural changes. Animals were euthanized at the experimental endpoint, and relevant tissues were collected for subsequent histological and molecular biological analysis.

[0065] It was found that continuous injection of streptococcal vesicles into the marginal ear vein of New Zealand white rabbits successfully induced sacroiliac joint intramedullary edema and well-defined fat deposition similar to ankylosing spondylitis (AS) on day 30 post-injection (red arrows). Figure 3Symptoms include peripheral joint swelling, which can be observed 60 days after injection. Specifically, in images of rabbit sacroiliac joints never treated with streptococcal vesicle modeling, the sacroiliac joint surface is intact, with no internal fat deposition. However, in images of rabbit sacroiliac joints treated with streptococcal vesicle modeling for 2 months, significant intramedullary fat deposition, subchondral bone sclerosis, and serrated changes on the articular surface are clearly visible. This suggests that, at the animal experimental level, streptococcal infection may be one of the pathogenic factors of AS.

[0066] Example 4 In this embodiment, the mechanism by which streptococcal infection leads to AS is further explained based on cell experiments.

[0067] The specific experimental method is as follows: (1) Culture *Streptococcus agalactiae* and *Lactobacillus rhamnosus* according to the method described in the above examples, collect the bacterial cultures of *Streptococcus agalactiae* and *Lactobacillus rhamnosus*, centrifuge at 8000 × g for 20 minutes at 4°C, collect the supernatant, filter through a 0.22 μm filter membrane, and then precipitate the vesicles by ultracentrifugation at 150000 × g for 70 minutes (4°C). Resuspend the obtained vesicles in sterile PBS, aliquot and store at -80°C.

[0068] (2) Mouse RAW264.7 macrophages were seeded in DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and cultured routinely at 37°C in a 5% CO2 incubator. Before the experiment, the cells were sputtered at 5 × 10⁻⁶ cells / year. 5 Cells were seeded at a rate of 100 cells / well in 6-well plates and allowed to adhere overnight. The following day, cells were treated with either Streptococcus agalactiae-derived vesicles or Lactobacillus rhamnosus vesicles for 24 hours; the control group received only an equal volume of PBS. After treatment, cells and culture supernatant were collected for subsequent analysis.

[0069] Simultaneously, rat bone marrow mesenchymal stem cells (BMSCs) were processed. Specifically, BMSCs were isolated from the lavage fluid of the femoral and tibia bone marrow cavities of 4-6 week old SD rats using standard methods and cultured in α-MEM medium containing 10% FBS and 1% penicillin-streptomycin. Cells from passages 3-5 were used for subsequent experiments. The cultured BMSCs were then subjected to a 2×10⁻⁶ ppm... 4 Cells were seeded per well in 24-well plates. After adhesion, the medium was replaced with conditioned medium containing the supernatant of RAW264.7 culture. The culture was treated for 24 h, and then adipogenic differentiation was assessed (including Oil Red O staining, qRT-PCR and Western blot detection).

[0070] The specific steps of qRT-PCR were as follows: RAW264.7 cells and BMSCs from each group were treated, and total RNA was extracted using a commercially available kit (EZBioscience EZ-press RNA Purification Kit) according to the manufacturer's instructions. RNA concentration and purity were measured using a NanoDrop spectrophotometer. 1 μg of the extracted total RNA was used to reverse transcribe cDNA using a commercially available kit (PrimeScript™ RT Reagent Kit with gDNA Eraser) to eliminate genomic DNA contamination. Real-time quantitative PCR was performed using TB Green™ Premix Ex Taq™ II on a QuantStudio™ 5 Real-Time PCR System. Primers were designed targeting mouse TNF-α, IL-6, Nos2 (encoding iNOS), Pparg, Cebpa, and the internal control genes Gapdh and β-actin.

[0071] The specific primer information is as follows: Gapdh F: 5'-CATGGCCTTCCGTGTTCCTA-3' (SEQ ID NO: 2); Gapdh R: 5'-GCGGCACGTCAGATCCA-3' (SEQ ID NO: 3).

[0072] β-actin F: 5'-GCAGGAGTACGATGAGTCCG-3' (SEQ ID NO: 4); β-actin R: 5'-ACGCAGCTCAGTAACAGTCC-3' (SEQ ID NO: 5).

[0073] TNF-α F: 5'-CCCAATCTGTGTCCTTCTAACT-3' (SEQ ID NO: 6); TNF-α R: 5'-CAGCGTCTCGTGTGTTTCT-3' (SEQ ID NO: 7).

[0074] IL-6 F: 5'-TAGTCCTTCCTACCCCAATTTCC-3' (SEQ ID NO: 8); IL-6 R: 5'-TTGGTCCTTAGCCACTCCTTC-3' (SEQ ID NO: 9).

[0075] iNOS F: 5'-TCACCTTCGAGGGCAGCCGA-3' (SEQ ID NO: 10); iNOS R: 5'-TCCGTGGCAAAGCGAGCCAG-3' (SEQ ID NO: 11).

[0076] Pparg F: 5'-CCTTTACCACGGTTGATTTCTC-3' (SEQ ID NO: 12); Pparg R: 5'-GGCTCTACTTTGATCGCACTTT-3' (SEQ ID NO: 13).

[0077] Cebpa F: 5'-CAAGAACAGCAACGAGTACCG-3' (SEQ ID NO: 14); Cebpa R: 5'-GTCACTGGTCAACTCCAGCAC-3' (SEQ ID NO: 15).

[0078] The amplification program was as follows: 95℃ pre-denaturation for 30 seconds; followed by 95℃ for 5 seconds, 60℃ for 30 seconds, repeated 40 times. Three replicates were set up for each sample. Relative mRNA expression levels were calculated using a 2-1 ratio. -ΔΔCt The method is calculated and standardized using the intrinsic parameter Gapdh.

[0079] The specific steps of Western blotting were as follows: RAW264.7 cells and BMSCs were collected separately after treatment. Total protein was extracted using RIPA lysis buffer containing protease inhibitors, and quantified using the BCA method. 30 μg of the extracted total protein was subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. iNOS, TNF-α, PPARγ, and FABP4 were detected, with β-actin as the internal control. The primary antibodies used were anti-iNOS antibody (dilution 1:2000, purchased from Proteintech), anti-TNF-α antibody (dilution 1:4000, purchased from Proteintech), anti-PPARγ antibody (dilution 1:4000, purchased from Proteintech), anti-FABP4 antibody (dilution 1:2000, purchased from Proteintech), and anti-β-actin antibody (dilution 1:20000, purchased from Proteintech). After incubation with primary antibody, HRP-labeled secondary antibody (dilution 1:2000, purchased from Beyotime) was added and incubated at room temperature for 1 hour. ECL color development was performed, ChemiDoc MP imaging was used, and grayscale quantification was performed using ImageJ.

[0080] The specific steps for immunofluorescence staining were as follows: RAW264.7 cells were seeded in a confocal dish, treated with the vesicles as described in the above examples, fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 5% BSA for 1 hour. Primary antibody (the above-mentioned anti-iNOS antibody, diluted 1:200) was added, and the cells were incubated overnight at 4°C. The next day, secondary antibody (Alexa Fluor® 594, diluted 1:500) was added, and the nuclei were counterstained with DAPI. Observation was performed using a confocal microscope.

[0081] The specific steps for ELISA detection are as follows: The concentrations of TNF-α, IL-6, and IL-17A in the supernatant of RAW264.7 cells treated with vesicles for 24 hours were detected using commercially available ELISA kits (Mouse TNF-α ELISA kit, purchased from Elabscience; Mouse IL-6 ELISA kit, purchased from Elabscience; Mouse IL-17A ELISA kit, purchased from Aifang Biotechnology). The specific detection methods were all performed according to the instructions for use. The absorbance was read at 450 nm using an ELISA reader, and the concentrations (pg / mL) of TNF-α, IL-6, and IL-17A were calculated based on the standard curve.

[0082] The specific steps for Oil Red O staining are as follows: After treatment, BMSCs are fixed with 4% PFA for 15 minutes, pretreated with 60% isopropanol for 5 minutes, stained with 0.5% Oil Red O working solution (isopropanol:water volume ratio 3:2) for 30 minutes, differentiated with 60% isopropanol, and counterstained with hematoxylin. Five fields of view (×200) are randomly selected and photographed under an inverted microscope, and the lipid droplet area ratio is quantified using ImageJ software.

[0083] The results are as follows Figure 4 and 5 As shown.

[0084] In the above experiment on macrophage polarization induced by Streptococcus agalactiae-derived vesicles, qRT-PCR results showed that after treatment with Streptococcus vesicles, the mRNA expression levels of TNF-α, iNOS, and IL-6 in macrophages were upregulated compared with the control group (without Streptococcus vesicle treatment) (all P < 0.05), exhibiting typical M1 pro-inflammatory transcriptional characteristics. Lactobacillus rhamnosus vesicles did not cause significant changes in the expression of these genes.

[0085] At the protein level, immunofluorescence staining showed that after treatment with streptococcal vesicles, iNOS signaling in macrophages was significantly enhanced, while the control group and LB Exo group maintained a weak signal.

[0086] Western blot results further confirmed this phenomenon. It was found that treatment with Streptococcus vesicles significantly increased the protein expression of iNOS and TNF-α, while Lactobacillus rhamnosus vesicles did not produce a detectable effect.

[0087] The corresponding grayscale analysis also showed that streptococcal vesicle treatment significantly enhanced the level of M1 marker protein.

[0088] Furthermore, based on the co-culture results, it was found that macrophage supernatant pretreated with streptococcal vesicles significantly promoted adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) through paracrine effects. ELISA results further confirmed a significant increase in the secretion levels of key pro-inflammatory cytokines TNF-α, IL-17A, and IL-6 in the supernatant. Moreover, after treating BMSCs with this supernatant, Western blot and gray-scale quantification results showed a significant upregulation of the expression of key adipogenic proteins PPARγ and FABP4. Simultaneously, qRT-PCR also confirmed increased mRNA levels of adipogenic transcription factors PPARγ and C / EBPα.

[0089] Functionally, based on Oil Red O staining quantification and microscopic observation results, it can be found that the formation of lipid droplets in BMSCs in the supernatant group treated with Streptococcus vesicles was significantly greater than that in the control group and the supernatant group treated with Lactobacillus rhamnosus vesicles (LB Exo).

[0090] These results collectively confirm that streptococcal vesicle-induced M1 macrophages can drive BMSCs to differentiate into adipocytes by secreting specific inflammatory factors, suggesting that the M1-dominated inflammatory microenvironment plays a promoting role in the formation of pathological fat deposition in the medulla oblongata of AS patients.

[0091] This demonstrates that *Streptococcus agalactiae* vesicles significantly induce macrophage polarization towards the M1 pro-inflammatory phenotype at the phenotypic, gene expression, and protein levels, while *Lactobacillus rhamnosus* vesicles, as a negative control, do not exhibit similar effects. This confirms that in cell experiments, macrophages phagocytose *Streptococcus agalactiae* vesicles, subsequently undergoing M1 polarization and secreting cytokines such as TNF-α, IL-6, and iNOS, inducing tissue inflammation. Simultaneously, paracrine IL-17 leads to increased levels of IL-6, TNF-α, and IL-7 in the supernatant of macrophages stimulated by *Streptococcus agalactiae* vesicles, further activating the MAPK pathway and initiating the AP-1 / NF-κB-mediated transcriptional program to induce COX-2 expression. COX-2, as the rate-limiting enzyme in prostaglandin synthesis, and its product PGD2 and downstream metabolites can activate PPARγ, thereby promoting adipocyte differentiation and inducing adipocyte differentiation in bone marrow mesenchymal stem cells. This explains, at the cell experimental level, the molecular mechanism behind the classic intramedullary inflammatory edema and intramedullary fat deposition in the sacroiliac joint of AS patients.

[0092] Example 5 In this embodiment, the feasibility and efficacy of antibiotics in adjunctive treatment of AS were tested based on clinical trials.

[0093] In this embodiment, the relevant research has been approved by the Ethics Committee of Nanfang Hospital, Southern Medical University. The ethics review approval number of Nanfang Hospital, Southern Medical University is NFEC-202410-K15-01, and the ethics review approval number of Yulin First Hospital, Shaanxi Province is NFEC-202410-K15-01. The clinical research registration number has been registered and filed on the Resman platform, the Chinese Clinical Trial Registry, and ClinicalTrials.gov as required.

[0094] In this study, 340 patients with ankylosing spondylitis (AS) were included and randomly assigned to either etanercept (ETN) maintenance therapy or benzathine penicillin / etanercept (BPG / ETN) combination therapy. The safety and feasibility of the BPG / ETN combination therapy were verified through 6, 12, and 24 weeks of clinical follow-up and imaging monitoring. Furthermore, the efficacy and safety of the BPG / ETN combination therapy versus ETN monotherapy in reducing disease activity, improving clinical symptoms and functional scores, enhancing quality of life, and improving imaging findings were compared.

[0095] In this embodiment, the grouping and drug administration details for each group are as follows: During the experimental period (from week 0 to week 24), the frequency and method of administration of benzathine penicillin or placebo in each group were intramuscular injections every 2 weeks.

[0096] The BPG / ETN combination therapy group: 1.2 million units of benzathine penicillin were added to 5 mL of normal saline to obtain benzathine penicillin injection. During weeks 0-24, etanercept was administered subcutaneously twice weekly, with benzathine penicillin injection administered intramuscularly once every 2 weeks.

[0097] Control group: 25 mg etanercept was administered subcutaneously twice weekly from week 0 to 12, while a placebo (vitamin C powder dissolved in 5 mL of normal saline) was administered intramuscularly every 2 weeks. After week 13, the label was removed, and the patient was switched to intramuscular benzathine penicillin injection every 2 weeks, while the subcutaneous injection of 25 mg etanercept was maintained twice weekly.

[0098] Safety and study compliance were assessed in each subject at weeks 1, 2, and 3 after the first treatment, and medication instructions were given simultaneously. Safety assessments and survival follow-ups were conducted at weeks 6, 12, and 24. Assessment indicators included disease activity, physical function, quality of life, and sacroiliac joint involvement, and adverse events were recorded.

[0099] In this embodiment, the primary endpoint was the ASAS20 response rate at week 12 after medication. ASAS20 was defined as: at least three of the following indicators showed a 20% improvement, or an improvement of at least 10 units (VAS score), and none of the four indicators showing a 20% improvement showed a worsening compared to baseline.

[0100] 1) The patient's overall VAS score (Patient's-Global score); 2) VAS scores for nocturnal back pain and overall back pain assessed by the patient; 3) BASFI (Bath Ankylosing Spondylitis Functional index) score; 4) Inflammatory response: The average VAS scores of the last two items related to morning stiffness in BASDAI (the average of the subjects’ self-reported severity of morning stiffness (0-10, 0 = no symptoms, 10 = severe) and duration of morning stiffness (0-10, 0 = no morning stiffness, 10 = morning stiffness for 2 hours or more).

[0101] Similarly, ASAS40 corresponds to a 40% improvement.

[0102] In this embodiment, secondary endpoints include: 1) ASAS40 remission rate at each follow-up point; and 2) BASDAI score at each follow-up point.

[0103] The specific evaluation methods for each score are as follows: (1) The assessment was based on the methods outlined in the 2009 International Society for Assessment of Spondyloarthritis (ASAS) consensus (refer to Sieper J, Rudwaleit M, Baraliakos X, et al. The Assessment of Spondyloarthritis international Society (ASAS) handbook: a guide to assess spondyloarthritis. Ann Rheum Dis 2009;68 Suppl 2:ii1-44.), which included: using the Patient's Global Assessment and the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) score (refer to Garrett S, Jenkinson T, Kennedy LG, et al. A new approach to defining disease status in ankylosing spondylitis: the Bath Ankylosing Spondylitis Disease Activity Index. J Rheumatol Joint pain and disease activity were assessed using the Bath Ankylosing Spondylitis Functional Index (BASFI), SQOL-AS score (refer to Bodur H, Ataman S, Rezvani A, et al. Quality of life and related variables in patients with ankylosing spondylitis. Qual Life Res 2011;20(4):543-9.), Short Form Health Survey (SF-36), BASMI score, MASES score, BAS-G score, and Work Efficiency and Activity Impairment Questionnaire for Specific Health Problems (WPAI-SHP).

[0104] (2) Combined with imaging assessment. This includes: Based on pelvic radiographs, in this embodiment, all subjects underwent pelvic radiographs, and the bilateral sacroiliac joints were scored according to the modified New York criteria (refer to van der Linden S, Valkenburg HA, Cats A. Evaluation of diagnostic criteria for ankylosing spondylitis. Aproposal for modification of the New York criteria. Arthritis Rheum 1984;27(4):361-8.), scored by two trained radiology experts who were not familiar with the subjects' characteristics.

[0105] Based on MRI assessment of the sacroiliac joint, in this embodiment, all subjects underwent MRI examination at SIJ. The MRI examination of the sacroiliac joint primarily used the Spine and Joint Research Association of Canada score (SPARCC, see Maksymowych WP, Inman RD, Salonen D, et al. Spondyloarthritis researchConsortium of Canada magnetic resonance imaging index for assessment of sacroiliac joint inflammation in ankylosing spondylitis. Arthritis Rheum2005;53(5):703-9.) and the Spine and Joint Research Association of Canada sacroiliac joint structural score (SPARCCSSS, see Maksymowych WP, Wichuk S, Chiowchanwisawakit P, et al. Development and preliminary validation of the spondyloarthritis research consortium of Canada magnetic resonance imaging sacroiliac joint structural score. J Rheumatol2015;42(1):79-86.) to precisely quantify the inflammatory involvement of the sacroiliac joint. All MRI examinations were performed using two sequences: 3.0T spin-echo T-weighted sequence (T1-weighted spinecho, TIWSE) and short-tlinversion recovery sequence (STIR). For the TIWSE sequence, five consecutive planar images were selected, and for the STIR sequence, six consecutive planar images were selected. After concealing subject information and examination time points, the acquired images were randomly assigned to two researchers certified with the SPARCC Certificate of Scoring Proficiency. The SPARCC score was used to score sacroiliac joint bone marrow edema (0-72 points) on the STIR images. The SPARCCSSS score was used to score sacroiliac joint bone erosion (0-40 points), joint filling (0-20 points), fatty metaplasia (0-40 points), and joint ankylosis (0-20 points) on the TIWSE images. The specific scoring criteria were as follows: Sacroiliac joint MRI inflammation scoring: SPARCC assesses bone marrow edema. In each STIR sequence image, each sacroiliac joint is divided into 4 quadrants: superior ilium, inferior ilium, superior sacrum, and inferior sacrum. If a high signal intensity shadow is present in any quadrant, the corresponding sacroiliac joint quadrant is scored 1 point; otherwise, it is scored 0 points. If at least one of the high signal intensity shadows has a relatively strong signal intensity (comparable to the signal intensity of blood vessels around the joint), the corresponding sacroiliac joint score is increased by 1 point. If at least one of the high signal intensity shadows has a width >1 cm from the articular surface, the corresponding sacroiliac joint score is increased by 1 point. The maximum total score in each image is 8 (4 quadrants bilaterally) + 2 (bilateral strong signal) + 2 (bilateral wide signal) = 12 points. The 6 images selected from the STIR sequence total 72 points. A SPARCC score >2 indicates the presence of inflammation in the sacroiliac joint.

[0106] Sacroiliac joint MRI structural injury scoring: SPARCCSSS is used to assess sacroiliac joint structural injury. When assessing bone erosion and fatty infiltration, the quadrant calculation method for the sacroiliac joint is the same as SPARCCSSS. When assessing fat filling and joint ankylosis, each sacroiliac joint in each T1WSE sequence image is divided into upper and lower quadrants. The scoring criteria for each category of injury are as follows: Bone erosion: Subchondral bone defect of the sacrum or ilium, covered by low signal intensity, 1 point per quadrant; Fatty infiltration: Uniform high signal intensity shadow with a width >1 cm appearing from the articular surface of the subchondral bone, 1 point per quadrant; Fat filling: High signal intensity shadow appearing at the site of bone erosion or surrounding articular surfaces, 1 point per quadrant; Joint ankylosis: Bridge-shaped high signal intensity shadow forming between the sacrum and ilium, 1 point per quadrant. In each image, bone erosion and fatty infiltration each scored a maximum of 8 points, while fat filling and joint ankylosis each scored a maximum of 4 points. In the five images of the TIW sequence, the total scores for bone erosion and fatty infiltration were 0-40 points, while the total scores for fat filling and joint ankylosis were 0-20 points.

[0107] The results are as follows Figure 6-8 As shown.

[0108] It can be observed that, based on the primary endpoint and assessed according to the above criteria, the results show that the ASAS20 response rate at week 12 in the BPG / ETN combination therapy group was significantly better than that in the ETN monotherapy maintenance therapy group. Based on the secondary endpoint, the results show that the ASAS40 response rate at week 12 in the BPG / ETN combination therapy group was higher than that in the ETN monotherapy group, demonstrating a deeper trend toward clinical remission. Figure 7 The BASDAI score of the BPG / ETN combination therapy group decreased significantly from baseline compared to the ETN monotherapy group. Figure 8 This further supports the synergistic effect of the combined regimen in controlling AS inflammation and clinical symptoms.

[0109] In conclusion, it can be seen that the combination therapy of BPG / ETN is more beneficial to the treatment of AS than ETN alone.

[0110] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of a composition in the preparation of a medicament for treating or assisting in treating ankylosing spondylitis; wherein The composition comprises: an antibiotic and an anti-inflammatory agent.

2. Use according to claim 1, characterized in that, The anti-inflammatory agent comprises a non-steroidal anti-inflammatory agent and a tumor necrosis factor antagonist; Preferably, the anti-inflammatory agent comprises etanercept.

3. Use according to claim 1, characterized in that, The antibiotic is an antibiotic having a growth inhibitory or killing effect on streptococcus; Preferably, the antibiotic comprises benzathine penicillin.

4. Use according to claim 1, characterized in that, The dosage form of the composition comprises: a tablet, a capsule, a granule, a solution and an injection.

5. The use according to claim 1, characterized in that, The medicament further comprises a pharmaceutically acceptable adjuvant; Preferably, the pharmaceutically acceptable adjuvant comprises at least one of: a buffer, a metal ion, a catalyst, an antifoaming agent, a diluent, an absorbent, a wetting agent, a binder, a solvent, a pH adjusting agent, an antibacterial agent, an isotonicity adjusting agent and a chelating agent.

6. Use according to claim 1, characterized in that, The antibiotic and the anti-inflammatory agent are administered separately or simultaneously. Optionally, the antibiotic is administered at a dosage of 200-300 thousand U / mL / time; and the anti-inflammatory agent is administered at a dosage of 15-40 mg / time.

7. Use according to claim 6, characterized in that, The administration frequency is 1-5 times / week; Preferably, 1-2 times / week.

8. Use of an antibiotic as an ankylosing spondylitis treatment promoter; Preferably, the antibiotic is an antibiotic having a growth inhibitory or killing effect on streptococcus; Preferably, the antibiotic comprises benzathine penicillin.

9. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises an antibiotic and an anti-inflammatory agent; Preferably, the antibiotic is an antibiotic having a growth inhibitory or killing effect on streptococcus; Preferably, the antibiotic comprises benzathine penicillin.

10. The pharmaceutical composition of claim 9, wherein, The anti-inflammatory agent comprises a non-steroidal anti-inflammatory agent and a tumor necrosis factor antagonist; Preferably, the anti-inflammatory agent comprises etanercept.

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