Bacillus cereus inhalant and application thereof in treating pulmonary fibrosis

Abstract

The invention discloses a bacillus cereus inhalant and application of the bacillus cereus inhalant in treatment of pulmonary fibrosis. After the pulmonary administration of the bacillus cereus inhalant, more bacillus cereus exist in the lung, the bacillus cereus secretes collagenase, and the collagenase degrades collagen in fibrotic tissues, so that the pulmonary fibrosis resisting effect is achieved. After pulmonary fibrosis model animals inhale the bacillus cereus inhalant, the respiratory function and the athletic ability are greatly improved, the respiratory function and the athletic ability are remarkably different from those of non-administrated model animals in the aspects of lung tissue appearance, collagen deposition, the inflammatory factor level and the hydroxyproline level, and part of indexes are close to those of healthy animals.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to Bacillus cereus inhalation preparations and their use in the treatment of pulmonary fibrosis. Background Technology

[0002] Probiotics play a wide range of roles in regulating gut microbiota balance, enhancing immune function, and improving metabolic diseases. Studies have shown that probiotics not only alleviate gastrointestinal inflammation but also have immunomodulatory effects, demonstrating potential value in cardiovascular protection, antioxidation, and anti-tumor applications. Bacillus cereus (BC) is a probiotic whose oral formulation has been approved in China for the treatment of childhood diarrhea, immune modulation, and Helicobacter pylori infection.

[0003] Pulmonary fibrosis is the end-stage pathological manifestation of various lung diseases, characterized by abnormal alveolar structural repair, excessive proliferation of fibroblasts, and extracellular matrix deposition. Idiopathic pulmonary fibrosis (IPF), a common type, has an incompletely understood etiology, but dysregulation of trauma repair and inflammatory response are considered key driving factors. Current treatment options are limited, and there is an urgent need to develop novel therapies that can clear fibrotic tissue.

[0004] Lung-based drug delivery systems, with their local targeting, rapid onset of action, and high pulmonary bioavailability, have become a research hotspot in the treatment of respiratory diseases. Inhalers, including aerosols, liquids, and powders, deliver drug formulations into the lungs via oral inhalation and are widely used in respiratory diseases, characterized by rapid action, minimal systemic side effects, and high safety. Powder inhalers have seen rapid development recently, utilizing micronized drugs and combining them with inhaled excipients to enhance the efficiency of drug and carrier deposition in the lungs. Porous microspheres, with their large internal spatial structure, significantly reduce their aerodynamic particle size, preventing clearance by pulmonary macrophages and facilitating drug efficacy. Summary of the Invention

[0005] The inventors unexpectedly discovered that Bacillus cereus can treat pulmonary fibrosis.

[0006] This invention discloses a Bacillus cereus inhaler for its use in the treatment of pulmonary fibrosis.

[0007] There are no limitations on the preparation steps of the Bacillus cereus inhalant in this invention; as long as the corresponding Bacillus cereus inhalant is obtained, the requirements of this invention can be met. The dosage form of the Bacillus cereus inhalant is selected from inhaled liquid formulations and inhaled powder formulations, with inhaled powder formulations being preferred.

[0008] The Bacillus cereus content in the Bacillus cereus inhaler is selected from 0.1% to 100%, preferably from 2% to 50%, and more preferably from 5% to 20%.

[0009] The aerodynamic particle size of Bacillus cereus inhalers is 1–5 μm to ensure good lung deposition.

[0010] The Bacillus cereus in the Bacillus cereus inhaler is selected from free bacteria, coated bacteria, microencapsulated bacteria, and microsphere-borne bacteria, with microsphere-borne bacteria being preferred. Several states can coexist.

[0011] Free bacteria refer to isolated, unengineered Bacillus cereus, maintaining their original state. Coated bacteria refer to Bacillus cereus with one or more layers of coating on its surface through engineering treatment. Microencapsulated bacteria refer to Bacillus cereus encapsulated in microcapsules. Microsphere-borne bacteria refer to Bacillus cereus and microspheres mutually adsorbed or encapsulated within microspheres. Microsphere-borne bacteria are selected from solid microsphere-borne bacteria and porous microsphere-borne bacteria, with porous microsphere-borne bacteria being preferred. Solid microsphere-borne bacteria refer to Bacillus cereus embedded in microspheres formed of polymer materials. Porous microsphere-borne bacteria refer to Bacillus cereus in a free-form manner mutually adsorbed or encapsulated within microspheres.

[0012] When Bacillus cereus in an inhaler exists in a coated state, the coating process can be selected from fluidized bed coating, extrusion coating, gel coating, spray drying coating, freeze drying coating, and layer-by-layer coating. Fluidized bed coating involves spraying and drying a Bacillus cereus suspension onto a carrier using a fluidized bed coating machine. The carrier is selected from albumin, dextrin, lactose, and mannitol. Extrusion coating involves extruding a hydrocolloid solution of Bacillus cereus through a nozzle to generate droplets, which are then freely dripped into a gel solution. The droplets solidify through gelation or by forming a film on the surface, thus producing porous hydrogel beads. The gelling agent in the gel solution is selected from alginate, chitosan, and poloxamer. Spray drying coating involves spraying a suspension containing Bacillus cereus through a spray dryer nozzle into a drying chamber, where it rapidly dries upon contact with hot air, forming dry particles. The freeze-drying coating process involves placing a suspension containing Bacillus cereus into a freeze dryer, first freezing it to convert water into ice, then removing the ice through sublimation, and finally removing the unfrozen water through desorption to obtain dried Bacillus cereus particles. The layer-by-layer coating process involves using chemical electrostatic deposition to alternately adsorb positively and negatively charged materials onto the surface of Bacillus cereus, forming multiple layers of coating.

[0013] When Bacillus cereus in an inhaler exists in a microcapsule-loaded state, the microcapsule preparation process can be selected from emulsification, extrusion, spray drying, droplet drying, spray cooling, and in-situ polymerization. The emulsification method involves mixing a Bacillus cereus suspension with a wall material solution, then dispersing the mixture into an oil phase containing an emulsifier for further emulsification. Continued stirring leads to microcapsule formation. Finally, the aqueous phase is removed, and the microcapsules are obtained by centrifugation, washing, and drying. The extrusion method involves mixing Bacillus cereus with a wall material to prepare a water-soluble polysaccharide solution. The polysaccharide mixture is extruded through a syringe needle, forming free-falling droplets that enter a coagulating solution to obtain microcapsules. The spray drying method involves spraying a liquid dispersion of Bacillus cereus into a drying chamber, where it comes into contact with hot air, rapidly evaporating the solvent to form dried microcapsules. The droplet method prepares microcapsules containing Bacillus cereus through a chemical reaction between sodium alginate and calcium chloride solutions. Specifically, a suspension of Bacillus cereus is directly mixed with a sodium alginate solution of a certain concentration, and then dripped uniformly into a calcium chloride solution to induce a gelation reaction and form microcapsules. The spray cooling method involves mixing a suspension of Bacillus cereus with a wall material and spraying the mixture through a nozzle into a cooling medium, causing the droplets to rapidly cool and solidify to form microcapsules. The in-situ polymerization method involves suspending Bacillus cereus in a solution containing monomers and an initiator, initiating a polymerization reaction under specific conditions, causing the monomers to polymerize on or around the bacteria to form microcapsule walls, thus encapsulating the bacteria.

[0014] The microspheres are made from chitosan, polycaprolactone, polylactic acid, and polylactic-glycolic acid copolymer, with polylactic-glycolic acid copolymer being the preferred material. Polylactic-glycolic acid copolymer (PLGA) is a synthetically produced biodegradable material commonly used in sustained-release drug delivery systems. Chitosan is a natural polymer material with a positive charge, and microspheres can be formed by adding ionic crosslinking agents such as sodium tripolyphosphate.

[0015] The microspheres were prepared by emulsification or precipitation.

[0016] When using the emulsification method to prepare microspheres, the following steps can be taken:

[0017] (1) Dissolve biodegradable polymer materials in an organic solvent to obtain an oil phase;

[0018] (2) The gelatin was heated and dissolved in an aqueous solution containing a stabilizer to obtain an aqueous phase;

[0019] (3) Under high-speed stirring or ultrasonic conditions, the aqueous phase is slowly added dropwise to the oil phase to form a W / O emulsion;

[0020] (4) Transfer the emulsion to a large amount of water and stir continuously to allow the organic solvent to evaporate and solidify the microspheres to precipitate.

[0021] (5) Collect the microspheres by centrifugation and wash them with water to remove residual solvent and impurities.

[0022] In step (1) above, the biodegradable polymer material is selected from polylactic acid-glycolic acid copolymer and polylactic acid, and the organic solvent is selected from dichloromethane, ethyl acetate, isopropyl ether, and chloroform. In step (1) above, the stabilizer is selected from polyvinyl alcohol, poloxamer, methylcellulose, and soluble polysaccharides.

[0023] When using the precipitation method to prepare microspheres, the following steps can be taken:

[0024] (1) Dissolve biodegradable polymer materials in organic solvents to obtain an oil phase;

[0025] (2) Under stirring conditions, the oil phase is slowly added to the non-solvent to precipitate microspheres;

[0026] (3) Collect the microspheres by centrifugation and wash them with water to remove residual solvent and impurities.

[0027] A porogen is added during the microsphere preparation process, and the resulting microspheres are then subjected to a porogen removal step to obtain porous microspheres. Adjusting the amount of porogen yields porous microspheres with different porosities. The porogen is selected from ammonium bicarbonate, ammonium acetate, gelatin, and soluble polysaccharides. The porogen removal step is selected from heating, freeze-drying, and dissolution.

[0028] Microspheres were co-incubated with a suspension containing Bacillus cereus. After the microspheres were adsorbed onto the surface of Bacillus cereus or encapsulated by Bacillus cereus, they were dried using a suitable method to obtain Bacillus cereus microspheres. Bacillus cereus inhalation agents were then screened to obtain the inhalation agents.

[0029] When using gelatin as a pore-forming agent to prepare porous microspheres, the following steps can be taken:

[0030] (1) Take an appropriate amount of polylactic acid-glycolic acid copolymer and dissolve it in dichloromethane as the oil phase;

[0031] (2) Dissolve an appropriate amount of gelatin in water to form the internal aqueous phase;

[0032] (3) The aqueous phase is slowly added to the oil phase under ultrasound to obtain a W / O primary emulsion;

[0033] (4) Dissolve an appropriate amount of polyvinyl alcohol in water as the external aqueous phase;

[0034] (5) Add the initial emulsion to the external aqueous phase and shear at high speed to obtain a W / O / W double emulsion. Continue stirring to remove dichloromethane and solidify the emulsion droplets to obtain solid microspheres.

[0035] (6) Add solid microspheres to water and stir continuously at 40-55℃ to remove internal gelatin. Collect the precipitate by centrifugation, wash and dry to obtain porous microspheres.

[0036] The preparation of Bacillus cereus inhalant can be carried out using the following steps:

[0037] (1) Preparation of porous microspheres;

[0038] (2) Add Bacillus cereus to the porous microsphere suspension, stir for an appropriate time to prepare Bacillus cereus porous microspheres, sieve to obtain microspheres of suitable particle size, and dry to obtain Bacillus cereus inhalant.

[0039] Porous microspheres can be adsorbed onto the surface of Bacillus cereus. Through their porous structure, they achieve a lower aerodynamic particle size, thereby obtaining a greater lung deposition efficiency.

[0040] Fibronectin (FN) has the property of binding to flagellin proteins on the surface of bacteria. Engineered Bacillus cereus is obtained by attaching fibronectin to its surface, and further prepared into a Bacillus cereus inhaler. Upon inhalation into the lungs, the surface-modified fibronectin prevents the rapid clearance of Bacillus cereus by pulmonary macrophages, thereby prolonging the residence time of Bacillus cereus in the lungs and enhancing drug efficacy.

[0041] The lung deposition rate of Bacillus cereus inhalers can be determined using a new generation impactor (NGI), and the fine particle fraction (FPF) should be greater than 10%.

[0042] Bacillus cereus inhalation formulation exhibits high stability and excellent pulmonary delivery efficiency, making it suitable for treating pulmonary fibrosis. Animal efficacy studies have demonstrated that the Bacillus cereus inhalation formulation disclosed in this invention results in a significant amount of Bacillus cereus in the lungs after pulmonary administration. These Bacillus cereus secrete collagenase, which degrades collagen in fibrotic tissue, thereby exerting an anti-pulmonary fibrosis effect. Attached Figure Description

[0043] Figure 1 Scanning electron microscope images of porous microspheres

[0044] Figure 2 Transmission electron microscopy images, including Bacillus cereus (BC), fibronectin-engineered Bacillus cereus (FN-BC), and Bacillus cereus inhaler (GM-FN-BC).

[0045] Figure 3 Aerodynamic particle size distribution of Bacillus cereus inhalant. ***p<0.001

[0046] Figure 4 Mouse body weight changes

[0047] Figure 5 Mouse movement trajectory in open field experiment

[0048] Figure 6 Masson staining of mouse lung tissue appearance and tissue sections

[0049] Figure 7 IL-1β levels in mouse lung tissue. *p<0.05

[0050] Figure 8 HYP levels in mouse lung tissue. *p<0.05 Detailed Implementation

[0051] Example 1. Porous microspheres

[0052] 0.75g of gelatin was added to water to a final volume of 10g and dissolved in a 60°C water bath to form the inner aqueous phase. 1.7g of polyvinyl alcohol was dissolved in water at 60°C to form the outer aqueous phase. 0.75g of polylactic acid-glycolic acid copolymer was added to dichloromethane to a final volume of 15g and sonicated to dissolve, forming the oil phase. The inner aqueous phase was added dropwise to the oil phase in an ice bath and sonicated to obtain a milky white W / O primary emulsion. The primary emulsion was added dropwise to the outer aqueous phase and sheared at high speed to obtain a milky white W / O / W secondary emulsion. The mixture was stirred at low speed overnight to remove dichloromethane, and the emulsion droplets solidified to form solid microspheres. The solid microsphere suspension was transferred to 400ml of water and stirred at low speed at 45°C for 3 hours to remove gelatin, forming porous microspheres. The microspheres were centrifuged at high speed, and the precipitate was resuspended in 45°C water, washed, and dried to obtain powdered porous microspheres.

[0053] Take an appropriate amount of porous microspheres, attach them to conductive adhesive, sputter-coated with gold, and place them under a scanning electron microscope to observe the morphology of the microspheres. (Scanning electron microscope image of porous microspheres) Figure 1 The results show that the porous microspheres have a small particle size (<10μm) and a visible porous structure on the surface, so their aerodynamic particle size is smaller and can meet the inhalation requirements.

[0054] Example 2. Bacillus cereus inhalant

[0055] Take 100 mg of the porous microspheres from Example 1, suspend them in 4 ml of physiological saline, add 1 ml of Bacillus cereus suspension, shake on a shaker at 37°C and low speed for 2 hours, freeze dry, and pass through a 180-mesh sieve to prepare Bacillus cereus inhalation agent.

[0056] Example 3. Fibronectin-engineered Bacillus cereus

[0057] Take 1 ml of 0.2 mg / ml fibronectin solution, add 4 ml of physiological saline, mix well, add 5 ml of Bacillus cereus suspension, mix well, and shake on a shaker at 37℃ and low speed for 30 minutes to obtain fibronectin-engineered Bacillus cereus.

[0058] Dilute Bacillus cereus and fibronectin-engineered Bacillus cereus, place a small amount on a copper grid, add 5 μL of 2.5% phosphotungstic acid solution for staining for 30 seconds, remove excess staining solution, air dry, and observe under a transmission electron microscope. Figure 2 Transmission electron microscopy (TEM) images of different samples are shown, including Bacillus cereus (BC) and fibronectin-engineered Bacillus cereus (FN-BC). The images show that Bacillus cereus has a higher protein content on its surface after engineering.

[0059] Example 4. Bacillus cereus inhalation agent

[0060] Take 100 mg of the porous microspheres from Example 1, suspend them in 4 ml of physiological saline, add 1 ml of fibronectin-engineered Bacillus cereus suspension from Example 3, shake on a shaker at 37°C and low speed for 2 hours, freeze dry, and pass through a 180-mesh sieve to prepare Bacillus cereus inhalation agent.

[0061] The Bacillus cereus inhalant (GM-FN-BC) was diluted, and a small amount was placed on a copper grid. 5 μL of 2.5% phosphotungstic acid solution was added for staining for 30 seconds. Excess staining solution was aspirated, and the sample was air-dried and observed under a transmission electron microscope. A transmission electron microscope image of the Bacillus cereus inhalant is shown below. Figure 2 It can be seen that a large number of microspheres are adsorbed on the surface of Bacillus cereus.

[0062] Experimental Example 1. Determination of Lung Deposition Efficiency of Bacillus cereus Inhalant

[0063] Materials: Bacillus cereus lyophilized powder (BC); Bacillus cereus inhalant (GM-FN-BC) prepared according to Example 4.

[0064] Methods: The new generation drug impactor (NGI) is one of the standard devices for evaluating the pulmonary distribution of inhaled formulations. It consists of an artificial larynx, a pre-separator, and an 8-stage impactor collection disc. At an airflow rate of 60 ml / min, the cutoff particle sizes for stages 1 (S1), 2 (S2), and 3 (S3) were 11.7 μm, 4.46 μm, and 2.82 μm, respectively. Fluorescently labeled Bacillus cereus was used to detect the distribution of Bacillus cereus in each stage. The best pulmonary deposition was observed when the aerodynamic particle size of the inhaled formulation was in the range of 1–5 μm.

[0065] Results: Bacillus cereus inhalant was mainly distributed in grades 2 and 3, with a small amount deposited in grade 4. Bacillus cereus lyophilized powder was mainly found in the throat and grade 1, with a small amount in grades 2 and 3. Figure 3Fluorescence intensity analysis showed that the fluorescence intensity of the Bacillus cereus inhaler in the larynx and at grade 1 was significantly lower than that of the Bacillus cereus lyophilized powder (p < 0.001); however, the fluorescence intensity of the Bacillus cereus inhaler at grades 2 and 3 was significantly higher than that of the Bacillus cereus lyophilized powder (p < 0.001). Therefore, the Bacillus cereus inhaler has better lung deposition efficiency than the Bacillus cereus lyophilized powder.

[0066] Experimental Example 5. Therapeutic effect of Bacillus cereus inhalation on pulmonary fibrosis in mice.

[0067] Materials: Bacillus cereus lyophilized powder (BC), diluted to a certain concentration with physiological saline before use; Bacillus cereus inhaler (GM-FN-BC) prepared according to Example 4, diluted to a certain concentration with physiological saline before use; blank porous microspheres (BM) prepared according to Example 1, diluted to a certain concentration with physiological saline before use; bleomycin solution.

[0068] Methods: Healthy mice were injected with 50 μL of bleomycin solution via a pulmonary injection. A pulmonary fibrosis mouse model was established after 14 days, characterized by weight loss and lethargy. Animal grouping: 14 days after bleomycin pulmonary administration, the weight of the pulmonary fibrosis model mice was measured. Mice weighing 19–25 g were selected and grouped as follows: Healthy group (no pulmonary bleomycin administration), n=6; Pulmonary fibrosis model group, n=6; Blank porous microsphere group, n=6; Bacillus cereus group, n=6; Bacillus cereus inhalation group, n=6. The healthy and model groups were injected intratracheally with 20 μL of physiological saline, while the other groups were injected intratracheally with 20 μL of the corresponding material. Weight was recorded daily. Administration was repeated every other day for four consecutive days. The mice's condition was observed after administration, and weight changes were recorded. Open field tests were conducted on days 7 and 14 to assess locomotor behavior. Fourteen days later, the mice were dissected, and the upper lobe of the right lung was removed for lung tissue appearance and Masson staining observation. The lower lobe of the right lung was used to detect the content of hydroxyproline (HYP) and interleukin IL-1β using an ELISA kit.

[0069] Results: Healthy mice were more active, had shiny fur, stable breathing, and gradually increased in weight. Figure 4 Mice in other groups initially exhibited rapid breathing, but respiratory function improved in the BC and GM-FN-BC groups as the drug was administered and time progressed. Furthermore, the body weight of the GM-FN-BC group remained stable and was the highest among all groups except the healthy group.

[0070] Open field experiments revealed that mice in the model group and BM treatment group were lethargic and had very little activity, while the BC group still had relatively little activity on day 7, but its activity level increased significantly on day 14. The GM-FN-BC group showed high activity levels on both day 7 and day 14. Figure 5 ).

[0071] Lung tissue dissection revealed that the lungs of healthy mice appeared reddish, while the model group, BM group, and BC group showed hemorrhages and white nodules. The lung tissue of the GM-FN-BC group was smooth and without hemorrhage. Figure 6 Masson staining of lung tissue, which involves staining collagen, was used to observe the degree of fibrosis. Results showed that the collagen staining area in the GM-FN-BC group was significantly reduced compared to the model group, approaching that of the healthy group. The BC group also showed some improvement, while the remaining groups all exhibited a relatively large number of stained areas. Figure 6 ).

[0072] IL-1β is an important inflammatory factor. Inflammation levels significantly increase after tissue fibrosis. IL-1β measurement results showed that the IL-1β level in the GM-FN-BC group was significantly lower than that in the model group and the BM group, and the BC group also showed some improvement. Figure 7 ).

[0073] Hydroxyproline is an important amino acid that constitutes collagen and is also an important indicator of the degree of fibrosis. HYP measurement results showed that the HYP level in the GM-FN-BC group was significantly lower than that in the model group, and there was no difference compared to the healthy group; the BC group also showed some improvement. Figure 8 ).

[0074] The above results demonstrate that Bacillus cereus inhaler has a good anti-pulmonary fibrosis effect. Its mechanism may be that it enters the pulmonary fibrosis area, secretes collagenase, degrades collagen in the fibrosis area, breaks down fibrotic tissue, and plays an immunomodulatory role, ultimately reversing pulmonary fibrosis.

Claims

1. A Bacillus cereus inhalant, characterized in that... Used to treat pulmonary fibrosis.

2. The Bacillus cereus inhalant as described in claim 1, wherein the dosage form is selected from inhaled liquid formulations and inhaled powder formulations.

3. The Bacillus cereus inhalant as described in claim 1, wherein the Bacillus cereus content is selected from 0.1% to 100%, preferably from 2% to 50%, and more preferably from 5% to 20%.

4. The Bacillus cereus inhalant as described in claim 1, wherein the aerodynamic particle size is 1–5 μm.

5. The Bacillus cereus inhalant as described in claim 1, wherein the Bacillus cereus exists in a state selected from free bacteria, coated bacteria, microencapsulated bacteria, and microsphere-borne bacteria, preferably microsphere-borne bacteria, and several states may coexist.

6. The Bacillus cereus inhalant as described in claim 5, wherein the microspheres in the microsphere-carrying bacteria are porous microspheres.

7. The Bacillus cereus inhalant as described in claim 6, wherein the porous microspheres and Bacillus cereus adsorb each other.

8. The Bacillus cereus inhalant as described in claim 6, wherein the porous microspheres are prepared by the following steps: (1) Take an appropriate amount of polylactic acid-glycolic acid copolymer and dissolve it in dichloromethane as the oil phase; (2) Dissolve an appropriate amount of gelatin in water to form the internal aqueous phase; (3) The aqueous phase is slowly added to the oil phase under ultrasound to obtain a W / O primary emulsion; (4) Dissolve an appropriate amount of polyvinyl alcohol in water as the external aqueous phase; (5) Add the initial emulsion to the external aqueous phase and shear at high speed to obtain a W / O / W double emulsion. Continue stirring to remove dichloromethane and solidify the emulsion droplets to obtain solid microspheres. (6) Add solid microspheres to water and stir continuously at 40-55℃ to remove internal gelatin. Collect the precipitate by centrifugation, wash and dry to obtain porous microspheres.

9. The Bacillus cereus inhalant as described in claims 1 to 4, prepared by the following steps: (1) Preparation of porous microspheres; (2) Add Bacillus cereus to the porous microsphere suspension, stir for an appropriate time to prepare Bacillus cereus porous microspheres, sieve to obtain microspheres of suitable particle size, and dry to obtain Bacillus cereus inhalant.

10. The Bacillus cereus inhalant as described in claim 1, wherein fibronectin is attached to the surface of Bacillus cereus before the preparation of the Bacillus cereus inhalant.

Images