A composition for treating osteomyelitis and use thereof

Abstract

The application discloses a composition for treating osteomyelitis and application thereof, and comprises an antibiotic and a PD-1 / PD-L1 inhibitor. It is found that a PD-L1 / PD-1 neutralizing antibody drug can assist antibiotic treatment of acute and chronic infectious osteomyelitis, and the application starts from activating the self innate immunity, applies the PD-L1 / PD-1 neutralizing antibody, removes the inhibition state of the innate immunity in the acute and chronic infectious osteomyelitis, restores the bactericidal capacity of macrophages, and assists the antibiotic to achieve a better effect of killing pathogens, reduces infection recurrence, and improves bone healing.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a composition for treating osteomyelitis and its application. Background Technology

[0002] Infectious osteomyelitis is an inflammation of the bone and bone marrow caused by pathogens, characterized by severe local inflammation and progressive bone destruction. It often occurs secondary to infections following open trauma to long bones or internal fixation surgery. In recent years, due to the continuously increasing incidence of trauma and fractures caused by traffic accidents and falls, the number of patients with infectious osteomyelitis has been growing year by year. Infectious osteomyelitis has always been a challenging problem in orthopedic clinics both domestically and internationally. Conventional treatments such as thorough debridement and the use of systemic and local antibiotics usually have limited efficacy. Infections are often persistent and have a high recurrence rate, easily progressing to osteonecrosis, delayed fracture healing, or even nonunion. In severe cases, it can lead to limb shortening and deformity, causing great inconvenience to patients' lives and work, and imposing a heavy economic burden on families and society.

[0003] Common pathogens causing infectious osteomyelitis include Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Escherichia coli. Since bone marrow is an important immune and hematopoietic organ, containing various hematopoietic stem cells and progenitor cells, as well as mature immune cells such as macrophages, neutrophils, T and B lymphocytes, local infection of bone tissue inevitably disrupts the function of local immune cells in the bone marrow. Studies have shown that the decline in host innate immunity caused by various bacterial infections is one of the important reasons for the protracted nature of infections. Therefore, while fighting infection, adjuvant "host-directed therapy" to regulate the host immune response to effectively defend against pathogens is a new approach to treating various bacterial infections, including acute and chronic infectious osteomyelitis. However, currently, there are no drugs in clinical practice with "host-directed therapy" capabilities.

[0004] Macrophages and neutrophils form the first line of defense against infection in the host's innate immune system by phagocytizing pathogens at the site of infection. The strength of macrophages' bactericidal function is crucial for the effectiveness of local tissues in combating infection. Studies have reported that some pathogens, such as Staphylococcus aureus (S. aureus), can evade macrophage killing and colonize macrophages long-term. As the main intracellular reservoir for pathogens like S. aureus after infection, changes in the antibacterial capacity of macrophages may be one of the sources of recurrent infectious osteomyelitis. Therefore, restoring the antibacterial capacity of macrophages to achieve "host-directed therapy" under conditions of innate immune suppression is of great significance for the clinical treatment of acute and chronic infectious osteomyelitis.

[0005] The limitations of existing technologies: For osteomyelitis, current treatment options involve surgical removal of infected tissue and rigorous antibiotic therapy to minimize the bacterial load. However, due to the increased resistance of bacteria embedded in biofilms to most antibiotics, the decreased host immunity caused by *S. aureus* infection, and the presence of dormant bacteria within biofilms, bone niches, and / or host cells, treatments often fail to completely eradicate the pathogen, frequently leading to reinfection. Ultimately, this can result in non-healing bone defects, stiffness in affected joints, and even amputation of the infected limb. Summary of the Invention

[0006] The purpose of this invention is to provide a composition for treating osteomyelitis that can restore the antibacterial ability of macrophages to achieve "host-directed therapy".

[0007] The technical solution adopted in this invention is:

[0008] In a first aspect, the present invention provides a composition comprising an antibiotic and a PD-1 / PD-L1 inhibitor.

[0009] In some embodiments of the present invention, the mass ratio of the antibiotic to the PD-1 / PD-L1 inhibitor is (2 to 3.3):1.

[0010] In some preferred embodiments of the present invention, the mass ratio of the antibiotic to the PD-1 / PD-L1 inhibitor is 2:1.

[0011] In some embodiments of the present invention, the antibiotic is an antibiotic against bacterial infection.

[0012] In some preferred embodiments of the present invention, the antibiotic is an antibiotic against Staphylococcus aureus.

[0013] In some embodiments of the present invention, the antibiotic is at least one selected from β-lactams, aminoglycosides, quinolones, macrolides, glycopeptides, tetracyclines, sulfonamides, chloramphenicol, lincosamides, and oxazolidinones.

[0014] In some preferred embodiments of the present invention, the β-lactams include at least one of penicillins, cephalosporins, and atypical β-lactams.

[0015] In some preferred embodiments of the present invention, the aminoglycoside is gentamicin.

[0016] In some embodiments of the present invention, the PD-1 / PD-L1 inhibitor is selected from at least one of anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-1 small molecule inhibitor, anti-PD-L1 small molecule inhibitor, anti-PD-1 / PD-L1 interaction small molecule inhibitor, PD-1 interfering RNA, and PD-L1 interfering RNA.

[0017] In some embodiments of the present invention, the anti-PD-1 antibody is at least one of Nivolumab, Toripalimab, Keytruda, Tislelizumab, Pembrolizumab, Cemiplimab, its biosimilars, its biooptimizers, and its bioequivalents.

[0018] In some embodiments of the present invention, the anti-PD-L1 antibody is at least one of Atezolizumab, Durvalumab, Avelumab, KN035, CS1001, MSB2311, BGB-A333, KL-A167, SHR-1316, STI-A1014, their biosimilars, their biooptimizers, and their bioequivalents.

[0019] In some embodiments of the present invention, the anti-PD-1 small molecule inhibitor is at least one of PD-1Inhibitor 1, PD-1Inhibitor 2, its biosimilar, its biooptimizer, and its bioequivalence.

[0020] In some embodiments of the present invention, the anti-PD-L1 small molecule inhibitor is at least one of Lin28 1632, Tomivosertib, its biosimilars, its biooptimizers, and its bioequivalents.

[0021] In some embodiments of the present invention, the small molecule inhibitor against PD-1 / PD-L1 interaction is at least one of BMS-1, BMS202, BMS-1001, BMS-1166, PD-1 / PD-L1 Inhibitor 3, its biosimilars, its biooptimizers, and its bioequivalents.

[0022] In some embodiments of the present invention, the interfering RNA is siRNA and / or shRNA.

[0023] A second aspect of the present invention provides the use of the composition described in the first aspect of the present invention in any one of the following (I) to (V):

[0024] (I) To prepare drugs for treating infectious diseases caused by Staphylococcus aureus;

[0025] (II) Preparation of drugs for treating osteomyelitis;

[0026] (III) Preparation of drugs to reduce bacterial load in bone tissue;

[0027] (IV) Preparation of drugs to reduce bone tissue lesions;

[0028] (V) Prepare drugs that enhance macrophage function.

[0029] In some embodiments of the present invention, the infectious diseases caused by Staphylococcus aureus are specifically infectious diseases of bone and surrounding soft tissues caused by Staphylococcus aureus.

[0030] In some embodiments of the present invention, the osteomyelitis is osteomyelitis caused by Staphylococcus aureus infection or osteomyelitis caused by other pathogens that upregulate the expression of PD-L1 / PD-1 in the patient's bone tissue.

[0031] A third aspect of the present invention provides a medicament for treating osteomyelitis, comprising the composition described in the first aspect of the present invention.

[0032] In some embodiments of the present invention, osteomyelitis is osteomyelitis caused by Staphylococcus aureus infection or osteomyelitis caused by other pathogens that upregulate the expression of PD-L1 / PD-1 in the patient's bone tissue.

[0033] In some embodiments of the present invention, the drug further comprises excipients or carriers.

[0034] In some embodiments of the present invention, the dosage form of the drug is: injection, powder, capsule, tablet, ointment, suppository, aerosol, oral, pill, drop, sustained-release tablet, suspension, granule, lozenge, powder, drop, pill, powder, solution, cream, patch, lozenge, or any combination thereof.

[0035] The beneficial effects of this invention are:

[0036] This invention provides a pharmaceutical composition comprising a PD-1 / PD-L1 inhibitor and an antibiotic. This invention discovers that PD-L1 / PD-1 neutralizing antibody drugs, used as adjunctive antibiotics in the treatment of acute and chronic infectious osteomyelitis, activate the body's innate immunity. By applying PD-L1 / PD-1 neutralizing antibodies, the suppressed state of innate immunity in acute and chronic infectious osteomyelitis is partially relieved, restoring the bactericidal ability of macrophages. This assists antibiotics in achieving better pathogen-killing effects, reducing infection recurrence, improving bone healing, and achieving better therapeutic results. Attached Figure Description

[0037] Figure 1This image shows the effect of PD-1 neutralizing antibody as an adjunct to antibiotic treatment for acute and chronic infectious osteomyelitis in a mouse model of plant-derived S. aureus infectious osteomyelitis. Figure 1 A is a representative image of bacterial smear detection in bone tissue and implants; Figure 1 B is a quantitative diagram of bacteria attached to the surface of the femoral tissue; Figure 1 C is a quantitative diagram of bacteria attached to the surface of the implanted nail.

[0038] Figure 2 This image shows the effect of PD-1 neutralizing antibodies as adjuvant antibiotics in treating acute and chronic infectious osteomyelitis in a mouse model of plant-derived S. aureus-induced osteomyelitis, as detected by immunofluorescence assay. Figure 2 A is a representative image of S. aureus in mouse bone marrow; Figure 2 B is a quantitative map of S. aureus per unit area of ​​bone marrow.

[0039] Figure 3 The image shows the H&E staining of bone tissue in the gentamicin-only treatment group and the group treated with a combination of PD-1 neutralizing antibody. Figure 3 A is an H&E staining image, with yellow arrows indicating large-scale abscesses and blue arrows indicating localized abscesses; Figure 3 B represents the bone tissue infection lesion score.

[0040] Figure 4 The images show the H&E staining of bone tissue in the gentamicin-only treatment group and the group treated with a combination of gentamicin and PD-L1 neutralizing antibody. Figure 4 A is an H&E staining image, with yellow arrows indicating large-scale abscesses and blue arrows indicating localized abscesses; Figure 4 B represents the bone tissue infection lesion score.

[0041] Figure 5 The study compared femoral bone infection in the gentamicin-only treatment group and the group treated with a combination of PD-1 neutralizing antibody. Figure 5 A is a representative image from a Micro-CT scan; Figure 5 B is a bone mineral density graph of the infected femur; Figure 5 C is a statistical graph of bone volume fraction in the infected femur; Figure 5 D is a statistical chart of the number of trabeculae in the infected femur; Figure 5 E is a statistical diagram of the trabecular thickness of the left femur; Figure 5 F is a statistical diagram of the trabecular gaps in the infected femur.

[0042] Figure 6 The study compared femoral bone infection in the gentamicin-only treatment group and the group treated with a combination of PD-L1 neutralizing antibody. Figure 6 A is a representative image from a Micro-CT scan; Figure 6 B is a bone mineral density graph of the infected femur; Figure 6C is a statistical graph of bone volume fraction in the infected femur; Figure 6 D is a statistical chart of the number of trabeculae in the infected femur; Figure 6 E is a statistical diagram of the trabecular thickness of the left femur; Figure 6 F is a statistical diagram of the trabecular gaps in the infected femur.

[0043] Figure 7 This refers to the dilution factor for the coating plate.

[0044] Figure 8 The images show the effects of macrophages phagocytizing S. aureus alone versus phagocytosis by macrophages combined with PD-L1 / PD-1 neutralizing antibodies on S. aureus. Figure 8 A is a representative drawing of the coating plate; Figure 8 B is the quantitative chart for coating.

[0045] Figure 9 The study investigated the effect of PD-1 neutralizing antibody on the blockade of S. aureus infection-induced mitochondrial autophagy in macrophages. Figure 9 A is the representative diagram; Figure 9 B is a quantitative graph.

[0046] * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; p represents significance.

[0047] Isotype: Gentamicin + isotype control antibody treatment group.

[0048] PD-1Ab: Gentamicin + PD-1 antibody treatment group.

[0049] PD-L1 Ab: Gentamicin + PD-L1 antibody treatment group. Detailed Implementation

[0050] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0051] Example 1

[0052] A mouse model of endophytic osteomyelitis caused by *S. aureus* was established. Mice were treated with gentamicin (4 mg / ml, 0.1 ml, intraperitoneal injection, once daily) starting on the first day post-surgery. Mice in the combined PD-1 or PD-L1 neutralizing antibody treatment group were given either PD-1 neutralizing antibody (Anti-PD-1Ab, #BE0146, Bio X Cell, NH, USA) (200 μg / mouse, every 3 days) or PD-L1 neutralizing antibody (Anti-PD-L1 Ab, #BE0101, Bio X Cell, NH, USA) (200 μg / mouse, every 3 days) starting on the 5th day post-surgery. Meanwhile, untreated mice were injected with isotype control antibody (Isotype control antibody, #BE0083, Bio X Cell, NH, USA) (200 μg / mouse, every 3 days). Bone tissue samples were collected from the operated side on the 14th day post-surgery. The soft tissue surrounding the bone was removed, and the implanted screws were taken out. The bone tissue was weighed, crushed, and added to PBS solution at a ratio of 10 μL / mg. The stock solution was serially diluted to 2000 times, and 200 μl was plated. The removed implanted screws were placed in 1 ml of PBS solution and sonicated to release bacteria attached to their surface. The stock solution and the 10-fold diluted bacterial solution were plated separately for detection. For samples undergoing histomorphological examination, bone specimens were fixed with 4% PFA for 24 hours, decalcified, and then paraffin-embedded or frozen for sectioning for immunohistochemical staining or tissue immunofluorescence detection. For samples undergoing microCT scanning, bone specimens were fixed with 4% PFA for 24 hours and then scanned using a micro-CT scanner. Bone mineral density (BMD), bone volume / tissue volume (BV / TV), trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th), and trabecular bone separation (Tb.Sp) were measured.

[0053] The results show that: Figure 1 and Figure 2 It can be concluded that, compared with gentamicin alone in treating vegetative osteomyelitis in mice (S. aureus), combined PD-L1 / PD-1 neutralizing antibody therapy can significantly reduce the local bacterial load in the femur. Figure 3 and Figure 4 It can be concluded that, compared with gentamicin alone in treating vegetative osteomyelitis in mice (S. aureus), combined treatment with PD-L1 / PD-1 neutralizing antibodies significantly improved bone tissue lesions. Figure 5 and Figure 6 It can be concluded that, compared with gentamicin alone in treating vegetative osteomyelitis in mice (S. aureus), combined treatment with PD-L1 / PD-1 neutralizing antibody can significantly improve bone loss in cancellous bone after femoral infection.

[0054] Example 2

[0055] Mouse macrophage cell line Raw264.7 was cultured at 1×10⁻⁶ cells / mL. 5 CFU / cm 2 Six-well plates were densely seeded with *S. aureus* at an MOI of 10 (bacterial CFU to cell number ratio) and treated with neutralizing antibodies PD-1 (10 μg / mL) or PD-L1 (10 μg / mL). The control group was treated with an equal volume of isotype control antibody. One hour later, cells were incubated at 37°C with 20 μg / mL lysostaphin and 50 μg / mL gentamicin to eliminate extracellular bacteria. Cells were lysed after washing twice with sterile PBS, and the cell lysis buffer was serially diluted at 1×, 10×, 100×, 500×, 1000×, and 2000×. Specific distribution details are shown in [link to relevant documentation]. Figure 7 Cells were cultured on agar plates, and the bacterial load in each cell was calculated to reflect the phagocytic amount. To investigate the effect of PD-1 or PD-L1 neutralizing antibodies on the bactericidal ability of cells, cells were cultured in growth medium and treated with PD-1 or PD-L1 neutralizing antibodies for 12 hours. Cells were then collected, washed, lysed, and the cell lysates were serially diluted, cultured on agar plates, and bacterial clones were counted and the bactericidal rate was calculated.

[0056] The results show that: Figure 8 and Figure 9 It can be concluded that PD-L1 / PD-1 neutralizing antibodies do not affect the phagocytic function of macrophages, but can significantly enhance the bactericidal ability of macrophages.

[0057] The above detailed embodiments have provided a comprehensive description of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. The use of a composition in the preparation of a medicament for treating osteomyelitis caused by Staphylococcus aureus infection, characterized in that, The composition consists of gentamicin and PD. 1 / PD Composed of L1 inhibitors; including gentamicin and PD 1 / PD The mass ratio of L1 inhibitors is (2–3.3):1; the PD 1 / PD L1 inhibitors are selected from anti-PD 1. Antibody, anti-PD L1 antibody.

2. The application according to claim 1, characterized in that, The anti-PD 1. The antibody is at least one of Nivolumab, Toripalimab, Keytruda, Tislelizumab, Pembrolizumab, or Cemiplimab.

3. The application according to claim 1, characterized in that, The anti-PD L1 antibodies include Atezolizumab, Durvalumab, Avelumab, KN035, CS1001, MSB2311, and BGB. A333, KL A167, SHR 1316 or STI At least one of A1014.

4. The application according to claim 1, characterized in that, Use of the composition according to claim 1 in any one of (I) to (III): (I) Preparation of drugs to reduce bacterial load in bone tissue; (II) Preparation of drugs to reduce bone tissue lesions; (III) Prepare drugs that enhance macrophage function.

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