KR100745125B1 - Drug delivery systems loading a bacteriophage for curing infection by a bacteria and method for manufacturing the same - Google Patents

Abstract

The present invention relates to the use of bacteriophage as an infectious agent in order to improve the treatment limited to antibiotics mainly used for the treatment of bacterial infections. In particular, bacteriophages are formulated using a drug delivery system and used for bacterial infections.

Bacteriophage, Salmonellosis, Drug Delivery System, Bacterial Infection, Antibiotics    

Description

Drug delivery systems loading a bacteriophage for curing infection by a bacteria and method for manufacturing the same

1 is a schematic configuration diagram showing an embodiment of a chitosan-gelatin film preparation encapsulated bacteriophage of the present invention,

Figure 2 is a process chart for a temporary example of the manufacturing method of chitosan-gelatin film preparation containing the bacteriophage of the present invention,

Figure 3 is a process diagram for an embodiment of a method for producing PLA-PEG microparticles encapsulated bacteriophage of the present invention,

Figure 4 is a scanning micrograph showing the form of the PLA-PEG microparticles prepared in the present invention.

The treatment of infectious diseases with bacteriophages was first presented by Herler in 1917. Bacteriophages are characterized by lysogenic or lytic life cycles that destroy bacteria. Therefore, bacterial infections were expected to be effectively treated with bacteriophage and phage therapy was introduced. Phage, however, was not as effective in killing bacteria in vivo as the effect of killing bacteria in vitro. With the development of antibiotics in the 1940's, bacteriophages began to be excluded as a means of treating infections, and research on this was going downhill.

Recently, many side effects have occurred due to the abuse of antibiotics and unnecessary prescription. In particular, antibiotics are extracted from living organisms and frequently used as substances that inhibit or kill the growth of other microorganisms, and the bacteria change themselves to a new shape, making them resistant to death. More seriously, antibiotic development is not keeping pace with the emergence of resistant bacteria. The seriousness of the antibiotic-resistant so-called 'superbacteria' is emerging as a big problem. As a result, new natural antibiotics, vaccine development, and new methods of treatment are required.

Several countries, including Eastern Europe and India, have continued to work on the treatment of infectious diseases with bacteriophages and bacterial lysates. Phage therapy has been used extensively since World War II, and as a result, the treatment of bacteriophage infections is extremely effective. Indeed, phagetherapy, which has been performed in the last 14 years for 1473 patients with purulent infections in various tissues and organs, has shown high efficiency. However, in spite of the therapeutic effect, there are problems in practical use due to some problems of bacteriophage. For example, there is a problem that the administered bacteriophage is easily inactivated in the spleen, or the antibody to phage is generated in the body when repeatedly administered. Recently, attempts have been made to overcome various problems using bacteriophage peptides and to use phage as an infection treatment.

The present invention relates to the use of a drug delivery system to effectively use the bacteriophage as an infection treatment while solving the problems presented above.

The present invention intends to formulate a bacteriophage effective drug delivery system for the treatment of bacterial infections, and to provide for the manufacture of a drug delivery system for bacteriophage therapy for the treatment of infectious diseases, and in particular in the film or capsule formulation while surviving phages. It aims to do it.

In order to achieve the above object, in the present invention, the purified bacteriophage (a) was encapsulated in a film formulation in which gechitosan and gelatin were mixed, and (b) poly lactic acid (PLA) and polyethylene glycol (PEG) were used as capsules. The present invention relates to a bacteriophage transport film formulation and a capsule formulation and a method for producing the same by encapsulating bacteriophages in mixed microparticles and measuring survival. In the film preparation used in the present invention, polysaccharides such as alginic acid, curdlan, pullulan, pectin, skuleroglucan, dextran, starch, whey, soy protein, natural protein, and the like may be used. Polysaccharides or Poly lactide-co-glycolide (PLGA), gelatin, liposomes and the like can be used.

Bacteriophage is easily inactivated in gastric acid when administered orally, and is easily inactivated by the spleen when administered as blood through veins. Thus, if the bacteriophage can be protected from gastric acid and the circulation time in the blood can be increased, the therapeutic effect can be significantly increased.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Film Preparation of Chitosan Packed with Bacteriophage and Gelatin

In the present invention, the bacteriophage (P22) specific to Salmonella typhimurium strain was encapsulated in a film preparation and a capsule preparation. First, Salmonella typhimurium culture and phage (P22) growth were incubated using Lurina-Burtani (LB) agar medium, and 0.2 ml of the culture medium and 200 µl of the solution shown in Table 1 were added to a new test tube, and 5 µl of phage (P22) was added. Incubated at 37 ℃ conditions for 18 hours. To isolate the grown phage (P22), 0.1 ml of chloroform was added to a test tube incubated, stirred vigorously for about 1 minute, and centrifuged at 5,000 rpm for 10 minutes to filter and sterilize the supernatant to obtain bacteriophage (P22). All experiments were used by repeat culture propagation by the method described above. Bacteriophage (P22) obtained by growth was 4.4 × 10 ¹² PFU / ml.

Table 1. Cultures for Transducing Bacteriophage (P22)

Culture Volume (v / v) LB liquid medium 20 ml 50 × VB * 400 μl 20% (w / w) glucose 200 μl

^* VB composition: Distilled water _{67 ml, MgSO 4 · 7H 2} O 1 g, citric acid · 2H 2 O 10 g, K 2 HPO 4 50 g, NaNH 4 HPO 4 · 4H 2 O 17.5 g.

1 is a schematic configuration diagram showing a chitosan-gelatin film preparation of the present invention. Figure 2 is a process chart for a temporary example of the chitosan-gelatin film production method of the present invention. The film was prepared by dissolving 4% (w / v) chitosan (average molecular weight 80,000) in an aqueous solution containing 10% (w / v) gelatin and 2% (v / v) acetic acid. 500 μl of bacteriophage (P22) was added to the mixed solution, stirred well, and cast on a plate to dry for 24 hours to prepare a chitosan-gelatin mixed film including phage. In the examples, experimental results were obtained without using an enteric coated coating material.

The prepared film was physically completely dissolved in a phosphate buffered saline (pH 7.4) solution and centrifuged to measure the activity of the bacteriophage (P22) encapsulated in the film. The result was that the number of formed plaques was 4.6 × 10 ⁷ PFU / ml. Survived and existed. The survival rate of bacteriophage in chitosan-gelatin mixed film with time is shown in Table 2. After the passage of time, the number of surviving phages in the film was not changed. At 6 hours, 5.4 × 10 ⁷ PFU / ml of bacteriophage (P22) survived and remained in the film.

Table 2. Survival of Bacteriophage (P22) Enclosed in Chitosan-Gelatin Mixed Films

time Survival Phage (PFU / ml) 0 4.6 × 10 ⁷ 0.5 4.8 × 10 ⁷ One 5.6 × 10 ⁷ 2 3.5 × 10 ⁷ 4 5.8 × 10 ⁷ 6 5.4 × 10 ⁷

Table 3 shows the results of the release of bacteriophage (P22) from the chitosan-gelatin mixed film. Release experiment of phage (P22) from the prepared mixed film was immersed in phosphate buffered saline (pH 7.4) and released while swelling at 37 ℃ for 10 hours. The discharged solution was collected at regular time intervals, and the supernatant was collected by centrifugation at 5,000 rpm for 10 minutes, and the released phage (P22) was measured for phage activity using a soft agar medium prepared in advance. After 4 hours, the chitosan-gelatin mixed film can be biodegraded through swelling and hydrolysis. The film formulation releases the phage (P22) over about 4 hours when attached to the oral mucosa or skin. It is expected to be effective treatment. In particular, chitosan has excellent mucoadhesive properties, and therefore, it is thought that chitosan is effective in inhibiting and killing the proliferation of bacteria attached to the mucosa due to its adhesion to the intestinal mucosa after oral administration. In addition, as shown in FIG. 3, the film prepared above is an enteric formulation such as eudragit, cellulose derivative-based cellulose acetate phthalates, hydroxypropylethyl cellulose, polyvinylacetate phthalates, and the like. By coating with scleroglucan, pectin, dextran and the like, it is useful as an agent capable of releasing bacteriophage slowly in the mucosa by preventing oral administration or relatively rapid degradation in the oral mucosa and protecting from gastric acid.

Table 3. Release of Bacteriophage (P22) from Chitosan-Gelatin Mixed Films

Release time Total Released Phage (PFU / ml) 10 minutes 1.4 × 107 30 minutes 2.0 × 107 1 hours 2.9 × 107 2 hours 3.3 × 107 3 hours 3.7 × 107 4 hours 4.0 × 107

Example 2 Preparation of PLA-PEG Microparticles Including Bacteriophage (P22)

First, since it is very important to investigate the stability of the bacteriophage (P22) for the organic solvent used in the preparation of microparticles, the survival rate in ethanol, dichloromethane, isopropyl alcohol, isooctane and the like was measured.

Table 4 shows the survival rate of the bacteriophage (P22) in the organic solvent. Bacteriophage was relatively stable in various organic solvents and relatively stable in ethanol. It was confirmed that the relative stability in the dichloromethane used for the production of PLA-PEG microparticles, it can be seen that it can be used to encapsulate the bacteriophage.

Table 4. Measurement of Stability of Bacteriophage (P22) in Various Organic Solvents

                                              (Unit: PFU / ml)

time Organic solvent ethanol Dichloromethane Isopropyl Alcohol Isooctane 0 4.4 × 1012 4.4 × 1012 4.4 × 1012 4.4 × 1012 1 hours 9.6 × 109 1.8 × 109 2.7 × 109 2.0 × 109 2 hours 9.6 × 109 1.8 × 109 2.4 × 109 1.5 × 109 4 hours 9.6 × 109 0.4 × 109 1.9 × 109 1.2 × 109 8 hours 5.6 × 109 0.2 × 109 1.6 × 109 1.0 × 109 12 hours 4.0 × 109 0.1 × 109 1.5 × 109 0.8 × 109

Figure 3 is a process chart for an embodiment of the PLA-PEG microparticles manufacturing method. Microparticles were prepared by some modification of the solvent evaporation method. PLA and PEG were mixed in various ratios (4: 1, 4: 2, 4: 4, weight ratio), and each ratio of PLA-PEG was dissolved in dichloromethane, and then stirred by adding 1 ml of a bacteriophage (P22) solution. The mixture was slowly mixed with 40 ml of poly vinyl alcohol (PVA, 5% (w / v)). The solvent was evaporated with continuous stirring at room temperature, centrifuged at 10,000 rpm for 5 minutes, washed 2-3 times and lyophilized.

Figure 4 is a scanning micrograph showing the form of the PLA-PEG microparticles prepared in the present invention. As a result of observing the prepared particles, all of them were spherical, and the particles were smooth and relatively uniform. As the ratio of PEG increased, the diameter of the particles was found to be smaller and more uniform.

Table 5 shows the results of measuring the size of the PLA-PEG microparticles prepared in the present invention. The particle size was 4.98 ± 0.84 μm in the PLA: PEG ratio of 4: 1, while the average particle size of 4: 4 in the ratio of 4: 4 was 3.45 ± 0.25 μm.

Table 5. Size of PLA-PEG Microparticles Encapsulated Bacteriophage (P22)

PLA: PEG (weight ratio) Particle size (μm) 4: 4 4.98 ± 0.84 4: 2 4.18 ± 0.68 4: 1 3.45 ± 0.25

PLA-PEG microparticles thus prepared were put in a tube containing phosphate buffered saline (pH 7.4) and HCl buffer (pH 1.2) to perform a release experiment at 37 ℃. Emissions were taken at regular intervals and centrifuged at 5,000 rpm for 10 minutes. The centrifuged phage (P22) was measured by the titration method using a pre-made soft agar medium.

Table 6 shows the results of the release trend of bacteriophage (P22) from the PLA-PEG microparticles prepared in the present invention. There was no difference in the release trend with increasing PEG ratio. Bacteriophage (P22) was released from the microparticles with a PLA: PEG ratio of 4: 1 at 10 minutes, and 2.8 × 10 ⁹ PFU / ml was released at 10 minutes, while microparticles with a ratio of 4: 4 had 3.1 × 10 ⁹ PFU / ml of phage ( P22) was released.

Therefore, it is possible to prepare the film and capsules encapsulated with bacteria and phage and to increase the therapeutic effect by inducing slow release.

Table 6. Bacteriophage (P22) Release from Embedded PLA-PEG Microparticles

                                                     (Unit: PFU / ml)

time PLA: PEG ratio 4: 4 4: 2 4: 1 10 minutes 3.1 × 109 3.3 × 109 2.8 × 109 1 hours 2.5 × 109 3.2 × 109 3.4 × 109 2 hours 2.3 × 109 2.9 × 109 3.1 × 109 4 hours 2.3 × 109 3.3 × 109 3.5 × 109

The method for formulating bacteriophage proposed in the present invention can be enclosed in a delivery system while the bacteriophage is alive, and this delivery system is expected to overcome the problems in the treatment of infection using phage. In addition to the advantages of the drug delivery system in general, it is not necessary to administer large amounts of phage, can increase mucoadhesiveness after oral administration, and prevent inactivation of phage from gastric acid. In addition, the phage administered into the blood can be absorbed into the spleen and inactivated, and the circulation time in the blood can be increased, and the frequent release of phage in the body can be reduced through slow release. Therefore, the development of such drug delivery system including bacteriophage is thought to be effective to treat bacterial infection.

Claims (6)

A formulation for treating Salmonella sp. Infection comprising bacteriophage P22 enclosed in a film matrix consisting of chitosan and gelatin. The formulation of claim 1 further comprising an enteric coating coating on the surface of the film matrix. A formulation for treating Salmonella sp. Infection comprising bacteriophage P22 encapsulated in particles consisting of polylactic acid (PLA) and polyethylene glycol (PEG). ⑴ mix chitosan and gelatin aqueous solution, 박 Bacteriophage P22 solution was mixed with the mixture obtained in step ,, (B) molding and drying the mixed solution obtained in step :: A method of preparing a preparation for treating Salmonella infection according to claim 1, comprising the step. 5. The method of claim 4, further comprising coating the product obtained in step (iii) with an enteric coating and then drying. (B) dissolving polylactic acid and polyethylene glycol in an organic solvent selected from the group consisting of ethanol, dichloromethane, isopropyl alcohol and isooctane; Mix the bacteriophage P22 solution with the solution obtained in step ⑵, Emulsify the mixture obtained in step iii and evaporate the solvent: To separate and lyophilize particles from the product obtained in step ⑶: Method for producing a preparation for treating Salmonella infection according to claim 3, comprising the step.

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