阅读说明：本技术 一种治疗骨髓炎的组合物及其应用 (Composition for treating osteomyelitis and application thereof ) 是由 余斌 张先荣 李凯群 陈昱辉 于 2021-08-26 设计创作，主要内容包括：本发明公开了一种治疗骨髓炎的组合物及其应用,包括抗生素和PD-1/PD-L1抑制剂,本发明发现PD-L1/PD-1中和抗体药物辅助抗生素治疗急慢性感染性骨髓炎,从激活自身固有免疫入手,应用PD-L1/PD-1中和抗体,通过部分解除急慢性感染性骨髓炎中固有免疫的抑制状态,恢复巨噬细胞杀菌能力,辅助抗生素达到更好的杀灭病原体的效果,减少感染复发,改善骨愈合。(The invention discloses a composition for treating osteomyelitis and application thereof, wherein the composition comprises antibiotic and PD-1/PD-L1 inhibitor, and the invention discovers that PD-L1/PD-1 neutralizing antibody drug assists antibiotic to treat acute and chronic infectious osteomyelitis, starts with activating innate immunity, and adopts PD-L1/PD-1 neutralizing antibody to partially relieve the suppression state of innate immunity in acute and chronic infectious osteomyelitis, recover the sterilization capability of macrophages, assist antibiotic to achieve better effect of killing pathogen, reduce infection recurrence and improve bone healing.)

1. A composition comprising an antibiotic and a PD-1/PD-L1 inhibitor.

2. The composition according to claim 1, wherein the mass ratio of the antibiotic to the PD-1/PD-L1 inhibitor is (2-3.3): 1.

3. Composition according to claim 1, characterized in that the antibiotic is an antibiotic against bacterial infections, preferably against staphylococcus aureus.

4. The composition of claim 1, wherein the antibiotic is at least one of β -lactams, aminoglycosides, quinolones, macrolides, glycopeptides, tetracyclines, sulfonamides, chloramphenicol, lincosamides, and oxazolidinones;

preferably, the beta-lactams comprise at least one of penicillins, cephalosporins and atypical beta-lactams;

preferably, the aminoglycoside is gentamicin.

5. The composition of claim 1, wherein the PD-1/PD-L1 inhibitor is selected from at least one of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-1 small molecule inhibitor, an anti-PD-L1 small molecule inhibitor, an anti-PD-1/PD-L1 interacting small molecule inhibitor, interfering RNA of PD-1, and interfering RNA of PD-L1.

6. The composition of claim 5, wherein the anti-PD-1 antibody is at least one of Nivolumab, Torpilimab, Keytruda, Tislelizumab, Pembrolizumab, Cemiplimimab, a biological analog thereof, a biological optimization thereof, and a biological equivalent thereof.

7. The composition of claim 5, wherein the anti-PD-L1 antibody is at least one of Atezolizumab, Durvalumab, Avelumab, KN035, CS1001, MSB2311, BGB-A333, KL-A167, SHR-1316, STI-A1014, a biological analog thereof, a biological optimal thereof, and a biological equivalent thereof.

8. The composition of claim 5, wherein the anti-PD-1 small molecule Inhibitor is at least one of PD-1Inhibitor1, PD-1Inhibitor2, a biological analog thereof, a biological optimization thereof, and a biological equivalent thereof; preferably, the anti-PD-L1 small molecule inhibitor is at least one of Lin 281632, Tomivosertib, a biological analog thereof, a biological optimization thereof, and a biological equivalent thereof; preferably, the anti-PD-1/PD-L1 interaction small molecule Inhibitor is at least one of BMS-1, BMS202, BMS-1001, BMS-1166, PD-1/PD-L1 Inhibitor 3, a biological analog thereof, a biological optimization thereof, and a biological equivalent thereof; preferably, the interfering RNA is siRNA and/or shRNA.

9. Use of a composition according to any one of claims 1 to 8 in any one of the following (I) to (V):

(I) preparing a medicament for treating infectious diseases caused by staphylococcus aureus;

(II) preparing a medicament for treating osteomyelitis;

(III) preparing a medicament for reducing bacterial load in bone tissue;

(IV) preparing a medicament for reducing bone tissue lesions;

(V) preparing a medicament for enhancing macrophage function;

preferably, the osteomyelitis is caused by infection of staphylococcus aureus and other pathogenic bacteria with up-regulated expression of PD-L1/PD-1 in bone tissues of patients.

10. A medicament for treating osteomyelitis, which comprises the composition as claimed in any one of claims 1 to 8, and is preferably osteomyelitis infected by other pathogenic bacteria with up-regulated PD-L1/PD-1 expression in bone tissues of patients or osteomyelitis caused by Staphylococcus aureus infection.

Technical Field

The invention relates to the technical field of medicines, and particularly relates to a composition for treating osteomyelitis and application thereof.

Background

Infectious osteomyelitis is an inflammation of bones and bone marrow caused by pathogenic bacteria, and is characterized by a local severe inflammation and a progressive bone destruction of bones, and is often secondary to infection caused by open trauma of long bones or after internal fixation of fractures. The incidence of trauma and fractures due to traffic accidents, falls, etc. has continued to rise in recent years, and the number of patients with infectious osteomyelitis has increased year by year. Infectious osteomyelitis is always a clinical treatment problem of orthopedics at home and abroad, treatment schemes such as thorough debridement, systemic and local antibiotic application and the like which are conventionally adopted are generally limited in curative effect, infection is often delayed and not healed, the recurrence rate is high, osteonecrosis, delayed union of fracture and even nonunion are easily developed, and the like, so that a serious patient can cause limb shortness and deformity, great inconvenience is brought to life and work of the patient, and heavy economic burden is brought to families and society.

Common pathogenic bacteria for infectious osteomyelitis include staphylococcus aureus, staphylococcus epidermidis, pseudomonas aeruginosa, escherichia coli and the like. Since bone marrow tissue is an important immune organ and hematopoietic organ of the body, and contains various hematopoietic stem cells and progenitor cells, and various mature immune cells such as macrophages, neutrophils, T and B lymphocytes, etc., local infection of bone tissue will destroy the function of local immune cells of bone marrow. Research shows that the decrease of host innate immunity caused by various bacterial infections is one of the important reasons for the delayed infection. Therefore, the method helps to prevent infection and simultaneously assist host-directed therapy (host-directed therapy) to regulate host immune response so as to effectively defend pathogenic bacteria, is a new idea for treating various bacterial infections including acute and chronic infectious osteomyelitis, but no related medicine has the function of host-directed therapy in clinical practice at present.

Phagocytosis of pathogenic bacteria by macrophages and neutrophils in the infected local area is the first line of defense of the host innate immune system against infection. Among them, the bactericidal function of macrophages is crucial to the effectiveness of local tissues in combating infection. Research reports that some pathogenic bacteria such as Staphylococcus aureus (s. aureus) can escape from killing of macrophages and colonize the macrophages for a long time, the macrophages are used as main reservoirs in cells after infection of the s. aureus and other pathogenic bacteria, and the change of antibacterial capacity of the macrophages can be one of sources causing recurrent outbreak of infectious osteomyelitis. Therefore, under the condition of innate immunity inhibition, how to recover the antibacterial capacity of macrophages to realize 'host-oriented therapy' has important significance for clinical treatment of acute and chronic infectious osteomyelitis.

The prior art has the following disadvantages: in the case of osteomyelitis, the current treatment regimen is surgical removal of infected tissue and strict antibiotic treatment to minimize bacterial load. However, treatment methods often do not completely eliminate pathogens and re-infection often occurs due to increased tolerance of biofilm-embedded bacteria to most antibiotics, a decrease in host innate immunity caused by s. Ultimately, this may lead to unhealed bone defects, stiffness of the affected joint and even infection of limb amputations.

Disclosure of Invention

The invention aims to provide a composition for treating osteomyelitis, which can restore the antibacterial capacity of macrophages to realize 'host-oriented therapy'.

The technical scheme adopted by the invention is as follows:

in a first aspect of the invention, a composition is provided comprising an antibiotic and a PD-1/PD-L1 inhibitor.

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

In some preferred embodiments of the invention, the antibiotic and the PD-1/PD-L1 inhibitor are present in a 2:1 mass ratio.

In some embodiments of the invention, the antibiotic is an antibiotic against a bacterial infection.

In some preferred embodiments of the invention, the antibiotic is an antibiotic against staphylococcus aureus.

In some embodiments of the invention, the antibiotic is at least one of a β -lactam, an aminoglycoside, a quinolone, a macrolide, a glycopeptide, a tetracycline, a sulfonamide, chloramphenicol, a lincosamide, and an oxazolidinone.

In some preferred embodiments of the present invention, the beta-lactams comprise at least one of penicillins, cephalosporins, and atypical beta-lactams.

In some preferred embodiments of the invention, the aminoglycoside is gentamicin.

In some embodiments of the invention, the PD-1/PD-L1 inhibitor is selected from at least one of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-1 small molecule inhibitor, an anti-PD-L1 small molecule inhibitor, an anti-PD-1/PD-L1 interacting small molecule inhibitor, an interfering RNA of PD-1, and an interfering RNA of PD-L1.

In some embodiments of the invention, the anti-PD-1 antibody is at least one of Nivolumab, toriplalimab, Keytruda, Tislelizumab, Pembrolizumab, cemipimab, a biological analog thereof, a biological optimization thereof, and a biological equivalent thereof.

In some embodiments of the 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, a biological analog thereof, a biological optimal thereof, and a biological equivalent thereof.

In some embodiments of the invention, the anti-PD-1 small molecule Inhibitor is at least one of PD-1Inhibitor1, PD-1Inhibitor2, a biological analog thereof, a biological optimization thereof, and a biological equivalent thereof.

In some embodiments of the invention, the anti-PD-L1 small molecule inhibitor is at least one of Lin 281632, Tomivosertib, a biological analog thereof, a biological optimization thereof, and a biological equivalent thereof.

In some embodiments of the invention, the anti-PD-1/PD-L1 interaction small molecule Inhibitor is at least one of BMS-1, BMS202, BMS-1001, BMS-1166, PD-1/PD-L1 Inhibitor 3, a biological analog thereof, a biological optimization thereof, and a biological equivalent thereof.

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

In a second aspect of the present invention, there is provided a use of the composition of the first aspect of the present invention in any one of the following (I) to (V):

(I) preparing a medicament for treating infectious diseases caused by staphylococcus aureus;

(II) preparing a medicament for treating osteomyelitis;

(III) preparing a medicament for reducing bacterial load in bone tissue;

(IV) preparing a medicament for reducing bone tissue lesions;

(V) preparing the medicine for enhancing the function of the macrophage.

In some embodiments of the invention, the infectious disease caused by staphylococcus aureus is specifically an infectious disease of bone and surrounding soft tissue caused by staphylococcus aureus.

In some embodiments of the invention, the osteomyelitis is an infection of osteomyelitis caused by infection with staphylococcus aureus or other pathogenic bacteria with up-regulated PD-L1/PD-1 expression in bone tissue of patients.

In a third aspect of the present invention, there is provided a medicament for the treatment of osteomyelitis comprising a composition according to the first aspect of the present invention.

In some embodiments of the invention the osteomyelitis is osteomyelitis infected with Staphylococcus aureus or other pathogenic bacteria with up-regulated PD-L1/PD-1 expression in the bone tissue of patients.

In some embodiments of the invention, the medicament further comprises an adjuvant or carrier.

In some embodiments of the invention, the pharmaceutical is in a dosage form of: injection, powder, capsule, tablet, ointment, suppository, aerosol, oral preparation, pill, drop, sustained release tablet, suspension, granule, buccal preparation, granule, drop, pellet, powder, solution, cream, patch, lozenge or any form combination thereof.

The invention has the beneficial effects that:

the invention provides a pharmaceutical composition, which comprises a PD-1/PD-L1 inhibitor and an antibiotic, and finds that a PD-L1/PD-1 neutralizing antibody drug assists the antibiotic to treat acute and chronic infectious osteomyelitis, starts with activating the innate immunity, and uses a PD-L1/PD-1 neutralizing antibody to partially relieve the suppressed state of the innate immunity in the acute and chronic infectious osteomyelitis, recover the sterilization capability of macrophages, and assist the antibiotic to achieve a better effect of killing pathogens, reduce infection relapse, improve bone healing and achieve a better treatment effect.

Drawings

FIG. 1 is a graph showing the effect of a neutralizing antibody against PD-1in a model of infectious osteomyelitis of S.aureus in mice on the treatment of acute and chronic infectious osteomyelitis with the aid of antibiotics. Wherein FIG. 1A is a representative diagram of the detection of bone tissue and embedded nail bacteria coated plate; FIG. 1B is a graph of the quantification of bacteria attached to the surface of femoral tissue; FIG. 1C is a quantitative graph of bacteria attached to the surface of the implantable nail.

FIG. 2 is a graph showing the effect of PD-1 neutralizing antibody in adjuvant antibiotic treatment on acute and chronic infectious osteomyelitis in a mouse model with S.aureus infectious osteomyelitis by immunofluorescence assay. Wherein FIG. 2A is a representation of S.aureus in mouse bone marrow; fig. 2B is a graph of s.

FIG. 3 shows H & E staining of bone tissue in the gentamicin alone treatment group and in the combined PD-1 neutralizing antibody treatment group. Wherein FIG. 3A is an H & E staining plot, yellow arrows show large range abscess foci, blue arrows show localized abscess foci; fig. 3B is the bone tissue infection lesion score.

FIG. 4 shows H & E staining of bone tissue in the gentamicin alone treatment group and in combination with the neutralizing antibody PD-L1 treatment group. Wherein FIG. 4A is an H & E staining plot, yellow arrows show large range abscess foci, blue arrows show localized abscess foci; fig. 4B is the bone tissue infection lesion score.

FIG. 5 shows the infection of femur by gentamicin alone and by PD-1 neutralizing antibody. Wherein FIG. 5A is a representative view of Micro-CT scanning; FIG. 5B is a statistical plot of bone density of infected femurs; FIG. 5C is a bone mass score histogram for an infected femur; FIG. 5D is a statistical plot of the number of trabeculae of infected femurs; FIG. 5E is a statistical plot of trabecular thickness of the left femur; fig. 5F is a trabecular gap statistical plot of infected femurs.

FIG. 6 shows the infection of femur by the gentamicin alone treatment group and the combined PD-L1 neutralizing antibody treatment group. Wherein FIG. 6A is a representative view of Micro-CT scanning; FIG. 6B is a statistical plot of bone density of infected femurs; FIG. 6C is a bone mass score histogram for an infected femur; FIG. 6D is a statistical plot of the number of trabeculae from infected femurs; FIG. 6E is a statistical plot of trabecular thickness of the left femur; fig. 6F is a trabecular gap statistical plot of infected femurs.

FIG. 7 shows the dilution factor of the coated plate.

Fig. 8 shows phagocytosis of s. aureus coated plates by macrophages alone and in combination with PD-L1/PD-1 neutralizing antibody. Wherein FIG. 8A is a representative drawing of a coated sheet; FIG. 8B is a calibration chart of the coated plate.

Figure 9 is a graph of the blocking of macrophage mitophagy by s. Wherein FIG. 9A is a representative view; fig. 9B is a quantification chart.

Represents p < 0.05; represents p < 0.01; represents p < 0.001; p is significance.

Isotype: gentamicin + isotype control antibody treatment group.

PD-1 Ab: gentamicin + PD-1 antibody treatment group.

PD-L1 Ab: gentamicin + PD-L1 antibody treatment group.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

Example 1

A mouse internal plant S.aureus infectious osteomyelitis model was prepared by starting the administration of gentamicin (4mg/ml,0.1ml, intraperitoneal injection, once a day) therapy to mice on the first day after surgery, starting the administration of PD-1 neutralizing antibody (Anti-PD-1Ab, # BE0146, Bio X Cell, NH, USA) (200. mu.g/mouse, once a day 3) or PD-L1 neutralizing antibody (Anti-PD-L1 Ab, # 0101, Bio X Cell, NH, USA) (200. mu.g/mouse, once a day 3) to mice treated with PD-1 or PD-L1 neutralizing antibody in combination with PD-1 neutralizing antibody (Anti-PD-1Ab, # BE0146, Bio X Cell), meanwhile, mice in the non-treated group were injected with Isotype control antibody (# BE0083, Bio X Cell, NH, USA) (200. mu.g/mouse, once every 3 days), and the postoperative bone tissue specimens were collected on the 14 th day after the operation. Removing soft tissue attached around the bone tissue, taking out the internal implanted nail, weighing and grinding the bone tissue, adding PBS solution according to the proportion of 10 mu L/mg, diluting the stock solution to 2000 times in a gradient manner, and taking 200 mu L for plating. The taken out internal implantation nail is placed in 1ml PBS solution for ultrasonic treatment to dissociate bacteria attached to the surface of the internal implantation nail, and the stock solution and the diluted 10 times bacterium solution are respectively taken for plate coating detection. For samples subjected to histomorphometric detection, bone specimens were fixed with 4% PFA for 24 hours, decalcified, and then paraffin or cryo-embedded sections were used for immunohistochemical staining or tissue immunofluorescence detection. For the samples subjected to the micct scan, the bone specimens were fixed with 4% PFA for 24 hours and then scanned using a Micro-CT scanner. Bone density (BMD), bone volume/tissue volume (BV/TV), trabecular number (tb.n), trabecular thickness (tb.th) and trabecular separation (tb.sp) were measured.

The results show that: as can be derived from fig. 1 and 2, treatment with PD-L1/PD-1 neutralizing antibody significantly reduced the local bacterial load in the femur compared to gentamicin alone for treatment of plant-infectious osteomyelitis in s. As can be seen from fig. 3 and 4, the treatment with PD-L1/PD-1 neutralizing antibody significantly improved bone tissue lesions compared to the treatment of plant infectious osteomyelitis in s. As can be seen from fig. 5 and 6, the combination of PD-L1/PD-1 neutralizing antibody treatment significantly improved cancellous bone mass loss following femoral infection compared to gentamicin alone for treatment of plant-infectious osteomyelitis in s.

Example 2

Mouse macrophage line Raw264.7 was cultured at 1X 10⁵CFU/cm²S.aureus was density-seeded in 6-well plates, cells were infected at MOI of 10 (ratio of bacterial CFU number to cell number) and treated with neutralizing antibody PD-1 (10. mu.g/mL) or PD-L1 (10. mu.g/mL), and controls were treated with equal volumes of isotype control antibody. After 1 hour incubation with 20. mu.g/mL lysozyme lysostaphin and 50. mu.g/mL gentamicin at 37 ℃ cleared of extracellular bacteria. To be sterileAfter the cells are rinsed twice by PBS solution, the cells are lysed, the cell lysate is diluted in a gradient way by 1 x, 10 x, 100 x, 500 x, 1000 x and 2000 x, the specific distribution is shown in figure 7, agar plates are coated for culture, and the bacterial load in each cell is calculated to reflect the phagocytosis. To investigate the effect of neutralizing antibodies to PD-1 or PD-L1 on the bactericidal capacity of cells, cells were collected after further culturing in growth medium and treatment with neutralizing antibodies to PD-1 or PD-L1 for 12 hours, rinsed, lysed, cell lysates were diluted in gradient, plated on agar plates, bacterial clones were counted and the bactericidal rate was calculated.

The results show that: from fig. 8 and 9, it can be concluded that the PD-L1/PD-1 neutralizing antibody does not affect macrophage phagocytic function, but significantly enhances macrophage bactericidal ability.

The present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.