A mixture for the preparation of freeze-dried tablets with antibacterial effect against Staphylococcus aureus and Pseudomonas aeruginosa, containing bacteriophages and lytic enzymes with optimal stability and efficiency of the active ingredients

Abstract

The solid formulation (tablet) prepared from a specific liquid mixture containing bacteriophages and endolysins active against the important pathogens Staphylococcus aureus and Pseudomonas aeruginosa as active ingredients is therapeutically applicable and dose-stable for the treatment of infections caused by these antibiotic-resistant pathogens. It also allows the bacteriophages and their enzymes to be maintained and stored at relatively high temperatures and other conditions that are unfavorable for these active ingredients.

Description

[Technical Field]

[0001] The present invention relates to a mixture for preparing a lyophilized single-dose dosage form (tablet) containing a bacterial virus (bacteriophage, or phage for short) in the form of a phage lysate or purified product in combination with its antibacterial enzyme (endolysin) as an active ingredient. The formulation of this dosage form provides a balanced composition of the individual components, ensuring appropriate technical parameters for the dosage / administration form, while simultaneously maintaining maximum and long-term stability of the active ingredients (phage and endolysin). Therefore, this dosage form can be applied in clinical settings to treat bacterial infections caused by pathogenic strains of Staphylococcus aureus and Pseudomonas aeruginosa. Specifically, these are two phages that act specifically against Staphylococcus aureus and Pseudomonas aeruginosa bacteria, as well as an endolysin with a unique amino acid sequence, LysMB10. [Background technology]

[0002] The World Health Organization (WHO) has identified bacterial resistance to antibiotics as one of the greatest threats to human health in the 21st century, not only because of increased mortality but also because of the significant burden it places on healthcare systems (URL1). The most dangerous antibiotic-resistant pathogens include Staphylococcus aureus and Pseudomonas aeruginosa. Staphylococcus aureus is a typical example of the bacterium responsible for many serious infections, including osteomyelitis, sepsis, and wound and skin infections. Similarly, Pseudomonas aeruginosa (P. aeruginosa) causes a wide range of diseases, including wound infections and pneumonia (particularly dangerous in patients with cystic fibrosis). Both of these pathogens are opportunistic, meaning they primarily pose a risk to immunocompromised patients and often hospital environments (Barer 2018). Therefore, resistance in these pathogens complicates healthcare delivery and limits the potential for antibiotic treatment of infections, leading to the need to explore alternatives, such as phage therapy (Abedon et al. 2011).

[0003] Bacteriophages (or phages for short) are bacterial viruses that lyse bacteria. Phage therapy exploits this property to treat bacterial infections. While this approach has been studied since the early 20th century, it lost ground in the Western world after the discovery and widespread use of antibiotics (though some research has continued in former Soviet republics, such as Georgia). Only in the fields of biotechnology and molecular biology has interest in phages continued (Abedon et al. 2011). The discovery of antibiotics is the primary cause of this decline. However, phage therapy offers many advantages, particularly a reduced risk of side effects due to minimal disruption to the patient's normal flora (compared to antibiotics) and the ability of phages to adapt to bacterial defense mechanisms (LOC-Carrillo and Abedon 2011). Initially, limited knowledge of phage biology obscured its therapeutic applications. Modern advances in molecular biology, whole-genome sequencing, genomics, and proteomics have provided a thorough understanding of phage biology and its interactions with host bacteria. Due to our expanding knowledge of bacteriophages and the rise of antibiotic resistance, interest in phage therapy has grown worldwide (Abdelrahman 2021). Similarly, interest has grown in the antibacterial applications of phage enzymes (called endolysins) that can lyse host cells. These enzymes cleave the peptidoglycan in the cell wall during the final stage of the phage life cycle, killing the bacterial cell. In the case of Gram-positive bacteria such as Staphylococcus aureus, endolysins can also be applied from the outside of the bacterial cell (Abdelrahman 2021). In all of these biotechnological treatment protocols for bacterial infections, maintaining the stability of antibacterial activity over time as long as possible while simultaneously maintaining a consistent dosage is crucial. Both of these properties are crucial for the practical application of phages and their endolysins (Abdelrahman 2021).

[0004] Each bacteriophage differs in the range of bacteria it can infect and subsequently lyse. Phages can be polyvalent, acting against a wide range of bacteria, or highly specialized against specific pathogenic microorganisms. This is similar to endolysins, which generally act against multiple bacterial species within the same genus, but their activity can vary (Abdelrahman 2021). Combining different phages (cocktails) may broaden the spectrum of antimicrobial activity. Furthermore, the isolation of specific natural mutants may demonstrate antimicrobial activity against other resistant bacteria. The bacteriophage referred to here refers to a specific phage isolated as a phage strain active against resistant bacteria. In this way, phages broaden the spectrum of microbial activity not only against antibiotic-resistant bacteria but also against other, phage-insensitive bacteria.

[0005] While the use of phages and endolysins in liquid form is common, the preparation of dosage / drug / storage forms remains a major scientific challenge. Available literature indicates that phages have been processed into liquid, semi-solid, and solid forms (Jault et al., 2019, Brown et al., 2018, Khanal et al., 2021). Liquid preparation is less time-consuming and less expensive, but it is more susceptible to microbial contamination and presents greater challenges in storage rooms where relatively high temperatures can compromise the stability of antimicrobial activity (Malik et al. 2017). Furthermore, unlike semi-solid and solid drug forms, liquids have limitations in certain dosage forms. While the main advantage of semi-solid forms is ease of administration, they suffer from similar challenges. In both cases, preservatives and antioxidants can be used to maintain microbiological quality, but these can negatively impact antimicrobial efficacy and product quality (Subils et al., 2012). Solid formulations generally pose a lower risk of microbial contamination and therefore do not require the use of preservatives. Furthermore, they have a high stability of antibacterial activity, precise dosage, and low storage requirements. The main drawbacks are the more complex optimization and higher formulation costs (Komarek 2006). Proven methods for processing phages into solid formulations include spray drying and freeze drying (Malik et al. 2017).

[0006] In spray drying, a solution of solid material is exposed to high-temperature drying gas in a drying chamber, forming powder particles that are then expelled from the drying chamber. This process is primarily applied to the production of inhalation powders (Hoe et al. 2014). This method has been tested multiple times with phages, achieving good stability of phage titer (Malik et al. 2017). However, as mentioned above, spray drying produces nothing but powder particles, which make dosing and storage more complicated and require additional processing, such as compression, which can negatively affect phage efficiency, primarily due to temperature changes (Khanal et al., 2021).

[0007] Lyophilization (freeze-drying) is commonly used to process bacteriophages into solid form. This process involves freezing the sample, followed by primary drying with ice sublimation, and finally secondary drying, where residual water is evaporated at high temperatures (Malik et al. 2017). Phages in the lyophilizate can remain effective for months, or even years (Clark 1962). However, this stability is affected by many factors, including the conditions of the lyophilization process, the excipients used (mainly bulking agents and cryoprotectants), and the storage and thawing of the resulting lyophilizate (Malik et al. 2017). Furthermore, in contrast to chemicals, the stability and efficiency of phages vary from strain to strain, requiring experimental optimization of the lyophilization process and the optimal composition of the medium suitable for maintaining maximum activity of a particular phage. Similar principles apply to enzymes.

[0008] It should be emphasized that phages are typically freeze-dried in sealed vials to produce so-called lyophilized cakes. However, this method is more complicated in terms of administration and dosage; for example, each cake is stored separately in a vial, occupying more space. Furthermore, the cakes must be dissolved before handling, reducing the benefits of freeze-drying. A solution to this problem is to prepare single-dose lyophilizates by transferring a mixture of filler and cryoprotectant, phage lysate (or purified product, as the case may be), and phage enzyme solution into a metal mold before freeze-drying begins. This achieves high stability of their antibacterial effects. Furthermore, the manufactured single-dose formulations can be stored in a single primary package, reducing storage space requirements and simplifying and accurately administering the single doses.

[0009] There has been limited research on single-dose solid formulations containing phages and their enzymes that allow for precise phage dosage and enhanced efficacy stability (Khanal et al., 2021). This patent focuses on the methodology for preparing lyophilized single-dose formulations containing bacteriophages and their enzymes that facilitate the administration of phages and their enzymes in the treatment of infections caused by pathogenic strains of Pseudomonas aeruginosa and Staphylococcus aureus.

[0010] The above problems regarding the stabilization and activity spectrum of the active ingredient during the manufacturing process of the lyophilized formulation are solved by the unique composition of the tablet mix containing specific antimicrobial components (phages and endolysins). [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Abedon, Stephen T., Sarah J. Kuhl, Bob G. Blasdel, and Elizabeth Martin Kutter. 2011. Phage Treatment of Human Infections. Bacteriophage 1 (2): 66-85. https: / / doi.org / 10.4161 / bact.1.2.15845.

[0012] [Non-patent document 2] Abdelrahman, F.; Easwaran, M.; Daramola, OI; Ragab, S.; Lynch, S.; Oduselu, TJ; Khan, FM; Ayobami, A.; Adnan, F.; Torrents, E.; et al. 2021. Phage-Encoded Endolysins. Antibiotics 10 (124): https: / / doi.org / 10.3390 / antibiotics10020124

[0013] [Non-Patent Document 3] Anany, H., Chen, W., Pelton, R., & Griffiths, M. W. (2011). Biocontrol of Listeria monocytogenes and Escherichia coli O157:H7 in meat by using phages immobilized on modified cellulose membranes. Applied and Environmental Microbiology, 77(18), 6379-6387. https: / / doi.org / 10.1128 / AEM.05493-11

[0014] [Non-Patent Document 4] Barer a Irving. Medical Microbiology, 19th Edition: A Guide to Microbial Infections: Pathogenesis, Immunity, Laboratory Investigation and Control. 19. United Kingdom: Elsevier, 2018. ISBN 978-0-7020-7200-0.

[0015] [Non-Patent Document 5] Brown, T. L., Petrovski, S., Chan, H. T., Angove, M. J., & Tucci, J. (2018). Semi-solid and solid dosage forms for the delivery of phage therapy to epithelia. Pharmaceuticals, 11(1), 1-12. https: / / doi.org / 10.3390 / ph11010026

[0016] ​​​​Carlson, K. 2005. Working with bacteriophages: Common techniques and methodological approaches In: Kutter, E., Sulakvelidze, A, (ed.) Bacteriophages: Biology and Application, p. 437-4 CRC Press, Boca Raton, FL, USA

[0017]

Direct Environment 7

[0018]

Outdoor Track 8

[0019]

Outdoor Tools9

[0020] [Non-Patent Document 10] Hoe, Susan, James W. Ivey, Mohammed A. Boraey, Abouzar Shamsaddini-Shahrbabak, Emadeddin Javaheri, Sadaf Matinkhoo, Warren H. Finlay, and Reinhard Vehring. 2014. Use of a Fundamental Approach to Spray-Drying Formulation Design to Facilitate the Development of Multi-Component Dry Powder Aerosols for Respiratory Drug Delivery. Pharmaceutical Research 31 (2): 449-65. https: / / doi.org / 10.1007 / s11095-013-1174-5.

[0021] [Non-Patent Document

[11] ] Jault, P., Leclerc, T., Jennes, S., Pirnay, J. P., Que, Y. A., Resch, G., Rousseau, A. F., Ravat, F., Carsin, H., Le Floch, R., Schaal, J. V., Soler, C., Fevre, C., Arnaud, I., Bretaudeau, L., & Gabard, J. (2019). Efficacy and tolerability of a cocktail of bacteriophages to treat burn wounds infected by Pseudomonas aeruginosa (PhagoBurn): a randomised, controlled, double-blind phase 1 / 2 trial. The Lancet Infectious Diseases, 19(1), 35-45. https: / / doi.org / 10.1016 / S1473-3099(18)30482-1

[0022] [Non-Patent Document 12] Khanal, D., Chang, R. Y. K., Hick, C., Morales, S., & Chan, H.-K. (2021). Enteric-coated bacteriophage tablets for oral administration against gastrointestinal infections. International Journal of Pharmaceutics, 609(July), 121206. https: / / doi.org / 10.1016 / j.ijpharm.2021.121206

[0023] [Non-Patent Document 13] Komarek, Pavel and Miloslava Rabiskova. Technology today: galenika. 3., preprac. a dopl. vyd. Prague: Ancient, c2006. ISBN 80-726-2423-7.

[0024]

Outdoor Tools 14

[0025]

Outdoor Tools 15

[0026]

Outdoor Content 16

[0027] [Non-Patent Document 17] Malik, Danish J., Ilya J. Sokolov, Gurinder K. Vinner, Francesco Mancuso, Salvatore Cinquerrui, Goran T. Vladisavljevic, Martha R.J. Clokie, Natalie J. Garton, Andrew G.F. Stapley, and Anna Kirpichnikova. 2017. Formulation, Stabilisation and Encapsulation of Bacteriophage for Phage Therapy. Advances in Colloid and Interface Science 249 (May): 100-133. https: / / doi.org / 10.1016 / j.cis.2017.05.014.

[0028] [Non-Patent Document 18] Mathias, J. R., Dodd, M. E., Walters, K. B., Yoo, S. K., Erik, A., & Huttenlocher, A. (2010). Phage-Bacterium War on Polymeric Surfaces-Anchored Eliminate Micobial Infections. Biomacromolecules, 33(11), 1212-1217. https: / / doi.org / 10.1021 / bm400290u.Phage-Bacterium

[0029] Non-Patent Document 19 Merabishvili M. Production of bacteriophages using bacterial suspension cultures for phage-therapy. In: Meyer HP, Schmidhalter DR, editors. Industrial scale suspension culture of living cells. 2014. Wiley VCH Verlag, Weinheim, Germany; 2014. pp. 537-543.

[0030] Non-Patent Document 20 Meyer HP, Schmidhalter DR, editors. Industrial scale suspension culture of living cells. 2014. WileyVCH Verlag, Weinheim, Germany; 2014. pp. 537-543.

[0031] Non-Patent Document 21 Subils, T., Aquili, V., Ebner, G., & Balague, C. (2012). Effect of preservatives on Shiga toxigenic phages and Shiga toxin of Escherichia coli O157:H7. Journal of Food Protection, 75(5), 959-965. https: / / doi.org / 10.4315 / 0362-028X.JFP-11-332

[0032] [Non-Patent Document 22] URL 1: Antimicrobial resistance, 17. 11. 2021,

[0033] [Non-Patent Document 23] https: / / www.who.int / news-room / fact-sheets / detail / antimicrobial-resistance Summary of the Invention

[0034] The present invention provides a mixture for preparing a freeze-dried tablet, the mixture having an antibacterial activity of 10% against Staphylococcus aureus and Pseudomonas aeruginosa. 7 ~10 11 The mixture contains a phage lysate / purified product of a bacteriophage working at a titer of PFU / ml, 0.01-0.3 mg / ml of endolysin LysMB10 produced in Escherichia coli and having the sequence of SEQ ID NO: 1, and also 5-50 g of bulking agent and 4-13 g of cryoprotectant per 100 ml of the mixture.

[0035] To prepare tablets, the mixture is filled into molds with cavities having the shape of the intended tablet and then freeze-dried.

[0036] The tablet base composition is optimized to promote phage and enzyme stabilization and prevent a decrease in their concentration and activity, ensuring optimal efficacy. Furthermore, the combination of endolysin LysMB10 (produced by E. coli CCM9354) with bacteriophages DSM34648 (a phage active against Pseudomonas aeruginosa) and DSM34647 (a phage active against Staphylococcus aureus) significantly expands the range of commonly used bacteriophages and endolysins active against Staphylococcus aureus and Pseudomonas aeruginosa. Phages DSM34648 and DSM34647 were newly isolated from the environment or prepared by advanced laboratory evolution, and their lytic ranges are unique. Endolysin LysMB10 is a previously undescribed antibacterial enzyme active against a broad range of staphylococcal strains.

[0037] In the present invention, a single-dose solid formulation (freeze-dried tablet) containing a phage lysate and an endolysin is prepared using a specific freeze-drying process to create a mixture with an optimized composition to ensure the stability and high activity of the antibacterial ingredients. The specified mixture of the active antibacterial substances (phage lysate and endolysin), filler (fish gelatin, maltodextrin, or polyvinylpyrrolidone (PVP)), and cryoprotectant (mannitol) is filled into a special metal mold (Czech Utility Model No. CZ31295U1) and then freeze-dried under specified conditions. This procedure improves the stability of the active ingredients, especially at high temperatures, allowing for easier storage. The resulting product has improved microbiological quality, allowing it to be used for the treatment of bacterial infections caused by Staphylococcus aureus and Pseudomonas aeruginosa. The specific phage contained in the tablet has a specific solubility range that dissolves pathogenic strains isolated from hospital environments, including those isolated from hospitals, and acts synergistically with the uncharacterized endolysin LysMB10 (which has a unique amino acid sequence included in the attached sequence listing as SEQ ID NO:1).

[0038] Sequence 1: Primary sequence of endolysin (SEQ ID NO: 1)

[0039] Sequence 1: Amino acid sequence of endolysin LysMB10 METLKDAENYIKKAIGKGIDFDGQYGYQCADLSVDYMYYITDKKVRMWGNAKDLINNDFKGLATVYKNTPSFLAKKGDVFVMGANRGGYGHTGIVTSATLNSITVIEQNWLGGGATFSEVTTKRTHPY DTEMWFIRPKFAKSKTKETAKKVAKASAKKVTPKKSWSFKVGGEPIITRIGKPSLKATSGGSVKPNQKMTFNKLVKSEGYEWGKLTNYKGQTEYVPIRPLKQKGYWGVLKWNSSSVDKLAAALEHHHHHH [Brief explanation of the drawings]

[0040] [Figure 1] Figure 1: Detailed scanning electron microscope image of the surface of a dosage form prepared from a specific antimicrobial mixture.

[0041] [Figure 2] Figure 2: Detailed scanning electron microscope image of the surface of a dosage form prepared from a specific antimicrobial mixture.

[0042] [Figure 3] Figure 3: Detail of the top of a dosage form prepared from a specific antimicrobial mixture

[0043] [Figure 4] Figure 4: Detail of the bottom of a dosage form prepared from a specific antimicrobial mixture

[0044] [Figure 5] Figure 5: Detailed aspects of dosage forms prepared from specific antimicrobial mixtures

[0045] [Figure 6] Figure 6: Primary packaging sample DETAILED DESCRIPTION OF THE INVENTION

[0046] At least 1x10 8A mixture of phage lysate with a titer of at least PFU / ml and endolysin at concentrations (0.01-0.3 mg / ml) can be processed into various solid dosage forms (tablets). In this process, the active ingredient is placed in a special mold with a filler and cryoprotectant to produce a separate solid dosage form that is then frozen in the mold. The resulting lyophilized product maintains its tablet structure and, with appropriate packaging, can be stored at 4°C for at least three months, protected from light and air humidity. These dosage forms are manufactured with a phage titer of at least 10, which is the generally accepted minimum for therapeutic use (Merabishvili 2014). 6 ~10 7 It is necessary to have a phage titer that achieves PFU / ml and the activity of the endolysin purified by an appropriate method.

[0047] For preparation, use crude phage lysate or at least 1 x 10 7Purified solutions with titers on the order of PFU / ml can be used. The phage lysate is purified and transferred to SM buffer (SM buffer composition: 100 mM NaCl, 8 mM MgSO4.7H2O, 50 mM Tris-Cl (pH 7.5) in 1000 ml of distilled water). This buffer is used for phage storage and is also suitable for lyophilization because its pH remains constant during lyophilization. Phage purification can be performed using commonly used protein purification methods, such as ultracentrifugation, tangential flow filtration, chromatography, or repurification using a centrifuge flask equipped with a filter adapter. These bacteriophages were isolated from strains that were not susceptible to other bacteriophages tested. The two bacteriophages mentioned above, DSM34648 and DSM34647, which are patent-deposited in the German DSMZ collection, possess a unique range that potentially extends their applicability to therapeutic phages of other pathogenic clinical isolates. These sequences are set out in the attached sequence listing as SEQ ID NO:2 (phage DSM34647) and SEQ ID NO:3 (phage DSM34648). Endolysin LysMB10, produced by Escherichia coli and deposited in the CCM collection at Brno, is a novel, previously unidentified protein with demonstrated antibacterial activity against Staphylococcus aureus. This enzyme complements the antibacterial action of bacteriophages and can be used against S. aureus without bacteriophages. The sequence of endolysin LysMB10 (SEQ ID NO:1) is provided separately.

[0048] This product is manufactured using phages from the MB Pharma Collection that infect Pseudomonas aeruginosa and Staphylococcus aureus, as well as a lytic enzyme that acts against Staphylococcus aureus. The bacteriophages and the E. coli strains that produce LysMB10 have been deposited in the German DSMZ collection under the Budapest Treaty and have also been patent-deposited with the CCM (for E. coli) under numbers CCM9354, DSM34648, and DSM34647. Their basic characteristics are shown in Tables 1 and 2 below.

[0049] [Table 1]

[0050] Table 1: Summary of phage characteristics [Table 2]

[0051] Table 2: Summary of the characteristics of endolysin LysMB10

[0052] The antibacterial efficacy of specific formulations of phages and endolysins is tested by the drop method on a double-layer agar medium (Garbe et al. 2010) or by the decrease in OD in liquid culture (as in the case of endolysins). For bacteriophages, one formulation is dissolved in 50 ml of sterile distilled water at room temperature for 3-5 minutes at 150 rpm before testing. The resulting solution is diluted and the dilution is divided by dropwise addition. Measurements are performed on multiple formulations to confirm the uniformity of their efficacy and, at the same time, the stability of the antibacterial effect of the active ingredient over time, preferably after at least 3 months.

[0053] The effect of endolysin was measured by measuring the turbidity (OD 600 The bacterial culture is washed twice with buffer and its specific gravity is measured by the decrease in OD. 600 = 0.5. Then, transfer to a cuvette, add one tablet, and measure the decrease in OD. [Example]

[0054] The composition of the lyophilized tablets and their preparation are set forth in Examples 1-3, each of which features a cryoprotectant-filler mixture suitable for stabilizing bacteriophages and endolysins. The proportions of each component are further detailed in Table 3. [Table 3] [Example]

[0055] 43.75 g of maltodextrin and 5.00 g of mannitol are mixed in a mixture of at least 1 x 10 9 The excipients are dissolved in 90 ml of the phage lysate / purified mixture at a concentration of 100 PFU / ml with continuous stirring at room temperature for 30 minutes. After dissolution of the excipients, the pH of the mixture is adjusted to 7.5 using hydrochloric acid and sodium hydroxide solutions. 10 ml of endolysin solution at a concentration of 0.3 mg / ml is added to the mixture. The resulting mixture is filtered through a 0.45 μm PES filter and then incubated at room temperature at a rotation speed of 150 rpm for at least 20 minutes and a maximum of 30 minutes. The amount of mixture is determined according to the number of dosage forms required; one dosage form corresponds to 0.5-1 ml of the mixture; in other words, 100-200 dosage forms are prepared from 100 ml of the mixture.

[0056] The mixture is filled into sterile aluminum molds and cooled to -80° C. During filling, the molds are maintained at a temperature of -60 to -80° C. Then, freeze-drying is carried out as follows: 1. Freeze the sample at -30°C for 20 minutes. 2. Primary drying is carried out at a pressure of 19.99 Pa (150 mTorr) and a temperature of -30°C for 960 minutes (i.e., 16 hours). 3. Secondary drying is carried out at a temperature gradient of 0.1°C / min from -30°C to 20°C at a pressure of 19.99 Pa (150 mTorr). After removal from the freeze-drying device and molds, the resulting dosage forms (tablets) are placed in primary packaging and stored under optimized conditions at 4°C (or room temperature). [Example]

[0057] Fish gelatin can also be used as a bulking agent. 12 g of mannitol and 12 g of fish gelatin are heated to approximately 70°C in 50 ml of water with continuous stirring. The mixture is then heated to 50°C, after which the phage lysate / purification product (Table 3) is added to a total volume of 90 ml. The phage concentration should be at least 1 x 10 9The resulting mixture is stirred at room temperature for 30 minutes. After dissolving the excipients, the pH of the mixture is adjusted to 7.5 using a solution of hydrochloric acid and sodium hydroxide. After adjusting the pH of the mixture, 10 ml of endolysin solution is added to achieve a final endolysin concentration of 0.02 mg / ml. The final mixture is filtered through a 0.45 μm PES filter and subsequently held at room temperature for at least 20 minutes at 150 rpm and a maximum of 30 minutes. The amount of mixture is determined based on the number of dosage forms required; one dosage form corresponds to 0.5-1 ml of the mixture, so 100-200 dosage forms can be prepared from 100 ml of the mixture.

[0058] The mixture is filled into sterile aluminum molds and cooled to −80° C. During filling, the molds are maintained at a temperature of −60 to −80° C. Then, freeze-drying is carried out according to a routine procedure. 1. Freeze the sample at -30°C for 20 minutes. 2. Primary drying is carried out at a pressure of 19.99 Pa (150 mTorr) and a temperature of -30°C for 960 minutes (i.e., 16 hours). 3. Perform secondary drying at a pressure of 150 mTorr with a temperature gradient of 0.1°C / min from -30°C to 20°C. After removal from the freeze-drying apparatus and molds, the resulting dosage forms (tablets) are transferred to primary packaging and optimized for storage at 4°C (or room temperature). [Example]

[0059] Polyvinylpyrrolidone K90 (PVP90) can be used as a bulking agent. 7.5 g of PVP90 and 5.00 g of mannitol can be mixed in a volume of at least 1 x 10 9The phage lysate / purified product mixture is dissolved at a concentration of 0.02 mg / ml in 90 ml of the mixture with continuous stirring at room temperature for 30 minutes. After dissolving the excipients, the pH of the mixture is adjusted to 7.5 using hydrochloric acid and sodium hydroxide solution. After adjusting the pH of the mixture, 10 ml of endolysin solution is added to achieve a final endolysin concentration of 0.02 mg / ml. The final mixture is filtered through a 0.45 μm PES filter and then kept at room temperature for at least 20 minutes at 150 rpm and a maximum of 30 minutes. The amount of mixture is determined based on the number of dosage forms required; one dosage form corresponds to 0.5-1 ml of the mixture, so 100-200 dosage forms can be prepared from 100 ml of the mixture.

[0060] The mixture is filled into molds and cooled to -80° C. During filling, the molds are maintained at a temperature of -60 to -80° C. Then, freeze-drying is carried out according to a routine procedure. 1. Freeze the sample at -30°C for 20 minutes. 2. Primary drying is carried out at a pressure of 19.99 Pa (150 mTorr) and a temperature of -30°C for 960 minutes (i.e., 16 hours). 3. Perform secondary drying at a pressure of 150 mTorr with a temperature gradient of 0.1°C / min from -30°C to 20°C. After removal from the freeze-drying equipment and molds, the resulting dosage forms (tablets) are transferred to primary packaging and optimized for storage at 4°C (or room temperature). [Example]

[0061] 43.75 g of maltodextrin and 5.00 g of mannitol were added to 90 ml of the phage lysate / purification mixture to obtain at least 1 x 10 9The mixture is dissolved at a concentration of 0.02 mg / ml with continuous stirring at room temperature for 30 minutes. After dissolution of the excipients, the pH of the mixture is adjusted to 7.5 using hydrochloric acid and sodium hydroxide solutions. After adjusting the pH of the mixture, 10 ml of endolysin solution is added to achieve a final endolysin concentration of 0.02 mg / ml. The final mixture is filtered through a 0.45 μm PES filter and then held at room temperature for at least 20 minutes at 150 rpm and a maximum of 30 minutes. The amount of mixture is determined based on the number of dosage forms required; one dosage form corresponds to 0.5-1 ml of the mixture, so 100-200 dosage forms can be prepared from 100 ml of the mixture.

[0062] The mixture is filled into molds and cooled to -80° C. During filling, the molds are maintained at a temperature of -60 to -80° C. Then, freeze-drying is carried out according to a routine procedure. 1. Freeze the sample at -30°C for 20 minutes. 2. Primary drying is carried out at a pressure of 19.99 Pa (150 mTorr) and a temperature of -30°C for 960 minutes (i.e., 16 hours). 3. Perform secondary drying at a pressure of 150 mTorr with a temperature gradient of 0.1°C / min from -30°C to 20°C. After removal from the freeze-drying apparatus and molds, the resulting dosage forms (tablets) are placed in primary packaging and optimized for storage at 4°C (or room temperature). [Industrial Applicability]

[0063] Lyophilized single-dose dosage forms (tablets) containing phage lysates / purified products and endolysins prepared from the mixture can be applied in phage therapy against infections caused by a wide range of strains of the pathogenic bacteria Staphylococcus aureus and Pseudomonas aeruginosa, which are known to be sensitive to these active ingredients.

[0064] Reference to deposited biological material The bacteriophage and the E. coli strain producing LysMB10 have been deposited in accordance with the Budapest Treaty with the German DSMZ collection or (in the case of E. coli) as patent deposits with the CCM under numbers CCM9354, DSM34648 and DSM34647.

Claims

1. Contains bacteriophage and lytic enzymes with optimized stability and efficiency of the active ingredients, ７ ~10 １１ A mixture for preparing freeze-dried tablets having antibacterial effect against Staphylococcus aureus and Pseudomonas aeruginosa, comprising a phage lysate / purified product of a bacteriophage acting on Staphylococcus aureus and Pseudomonas aeruginosa at a titer of PFU / ml, 0.01 to 0.3 mg / ml of an endolysin produced by Escherichia coli and having the sequence of SEQ ID NO: 1, and further comprising 5 to 50 g of a bulking agent and 4 to 13 g of a cryoprotectant per 100 ml of the mixture.

2. 2. The mixture according to claim 1, characterized in that the bacteriophage is at least one phage selected from the group comprising phage DSM 34647 and phage DSM 34648.

3. 3. The mixture according to claim 1 or 2, characterized in that the filler is selected from the group comprising maltodextrin, fish-derived gelatin and polyvinylpyrrolidone.

4. 4. The mixture according to claim 1, wherein the cryoprotectant is mannitol.

5. Phage titer is 10 ７ ~10 １１ 1. An antibacterial mixture for use in the treatment of bacterial infections caused by the pathogenic strains Staphylococcus aureus and Pseudomonas aeruginosa in humans and veterinary fields, comprising PFU / ml of specific bacteriophages DSM 34647 and / or DSM 34648, 0.01 to 0.3 mg / ml of endolysin of SEQ ID NO: 1, and also 5 to 50 g of bulking agent and 4 to 13 g of cryoprotectant per 100 ml of mixture.

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