Hydroxyethyl cellulose gel composition containing bacteriophage

Abstract

This invention provides a hydrogel comprising hydroxyethyl cellulose (HEC) and bacteriophage.

Description

[0001] This invention relates to hydrogels containing hydroxyethyl cellulose (HEC) and bacteriophages, as well as lyophilized versions thereof, and the use of such hydrogels or lyophilized versions, particularly in the treatment and prevention of bacterial infections.

[0002] With increasing antibiotic resistance, bacterial infections have become a growing problem in recent years. This typically affects all areas of healthcare, especially when a subject's large body surface comes into contact with foreign materials.

[0003] This essentially applies to cases involving large wounds, such as those resulting from surgical procedures, particularly dermatological surgery, which expose the wounds to potentially contaminated air, suture materials, or dressings. For example, large wounds are often left after surgical removal of damaged skin or tissue from a patient with acne recurrent or pressure ulcers. These wounds typically heal slowly and carry a significant risk of recurrent bacterial infections, a risk that is further increased by the need for open treatment after wound debridement. Furthermore, postoperative infections and bloodstream infections originating from pre-existing surrounding wounds, such as chronic wounds from diabetes, can lead to serious problems.

[0004] Bacterial infections of vascular grafts represent a major burden in cardiovascular medicine, associated with increased morbidity and mortality. Various factors relevant to this field, such as patient debility, biofilm formation, or immunosuppression, negatively impact antibiotic therapy, thereby inhibiting treatment success rates. Therefore, further treatment strategies are needed. The antibacterial properties of bacteriophages were discovered over 100 years ago, but since the mid-20th century, the emphasis on antibiotics in Western medicine has slowed the further development of phage therapy. Consequently, much of the experience and knowledge gained to date regarding the mechanisms of action, manipulation, clinical applications, and limitations of phages has been lost. However, the simultaneous emergence of antimicrobial resistance and personalized medicine necessitates further approaches to treating bacterial infections.

[0005] This is particularly relevant for treating bacterial infections in the surgical setting. Specifically, both open and endovascular surgical repair of cardiovascular tissues carry a risk of hospital-acquired infections, and prosthetic graft infections are often troublesome, associated with other complications, and frequently life-threatening. The prevalence of hospital-acquired infections in surgical patients requiring intensive care ranges from 4% to 15%. Standard treatments such as resection and autologous vascular reconstruction can pose alarmingly high risks to affected patients, thus necessitating alternative treatments. Furthermore, prosthetic graft infections are among the most concerning complications of cardiovascular surgery and are associated with high morbidity, high mortality, and increased hospitalization costs. The most common pathogens are Staphylococcus aureus, Staphylococcus epidermidis, other coagulase-negative staphylococci, Enterobacterales, Escherichia coli, Pseudomonas, and Corynebacteria. Bacteria typically increase their virulence by attaching themselves to prosthetic materials, thereby protecting themselves from local immune responses and antibiotics with the help of biofilms that inhibit phagocytosis and current standard therapies. Furthermore, systemic antibiotic therapy is often inadequate because effective saturation concentrations are rarely achieved in the inflamed tissue surrounding the prosthesis.

[0006] Bacteriophages (or simply "phages"; derived from the Greek word for "bacterial devourer") are viruses that selectively infect bacterial cells and were first described in 1917 by Canadian Félix Hubert d'Hérelle. They are quite stable in the environment and make important contributions to regulating global bacterial quality. In principle, phages can be found anywhere their corresponding bacteria are present, but they can only reproduce in the presence of their host. Phages are specific and almost always affect strains within a single bacterial species, rarely crossing species boundaries. Lysogenic phages initiate the lysis cycle of viral replication; phages kill their corresponding bacteria by lysis: once infected, the bacterial host cell begins the replication process, destroying the bacteria and releasing new phage particles; this process is controlled by enzymes and the interaction of bacterial and phage genes. On the other hand, lytic phages initiate the lysis cycle: the phage nucleic acid is integrated into the host bacterial genome or forms a circular replicon in the bacterial cytoplasm. The lysis cycle here is initiated by an external trigger, such as ultraviolet light or heat.

[0007] Bacteriophages are dynamic biological agents that multiply within host bacteria, which presents different requirements for clinical research and complicates the regulatory approval process for drugs with more passive, defined chemical compositions, such as antibiotics. Although the therapeutic use of phages was common in the Soviet bloc countries, there was a lack of randomized, placebo-controlled, double-blind studies providing scientifically usable data for approval by regulatory authorities in the Western Hemisphere. A growing number of case reports describe the successful treatment of life-threatening infections with phage therapy. However, despite some clinical studies, most have failed to provide definitive evidence of the effectiveness of phage therapy: one study evaluating a phage mixture against Escherichia coli and Pseudomonas aeruginosa infections in burn wounds was terminated prematurely due to insufficient efficacy.

[0008] Currently, only phage solutions are available, and local application is challenging due to adhesion and flow dynamics. Even if phages are successfully delivered to the site of infection, they may enter and reach surrounding tissues, especially in cases of cyclic application, such as anastomotic infections. While this generally does not pose a risk of local or systemic adverse reactions, it will impair efficacy. For example, the preservation of phages on prosthetic surfaces is unclear. Phages must remain at / within the application site for a sufficient time to ensure phage-bacterial interaction; this necessitates both semi-solid and solid formulations.

[0009] In view of the above, providing antibacterial compositions remains a goal in the art, which can be applied, for example, during surgical procedures.

[0010] The present invention addresses this objective by providing a hydrogel comprising hydroxyethyl cellulose (HEC) and bacteriophages in a first aspect.

[0011] The inventors have surprisingly discovered that hydrogels containing hydroxyethyl cellulose (HEC) and phages exhibit superior properties compared to other hydrogels in medical applications. Specifically, hydroxyethyl cellulose (HEC) was unexpectedly found to be the best gel-forming agent among the most suitable gel forms tested. Contrary to previous studies, the hydrogel containing hydroxyethyl cellulose (HEC) provides a solid gel that can be easily and painlessly applied and spread onto, for example, a patient's skin. It also exhibits a stiffness that allows for the application of individual layer thicknesses, unlike other gels tested. Previous studies have shown that the pore size in HEC-containing hydrogels is allegedly too small, and that HEC-based gels are unsuitable for phage release applications because they exhibit poor release of infectious phages (see, for example, Chang et al., Int JPharm. 2021 Aug 10; 605:120850). In contrast, the inventors of this invention have surprisingly demonstrated, both in vitro and in expanded use, that the HEC-containing hydrogel of this invention not only exhibits the ability to retain phages but also enables the continuous release of phages in a delayed manner. This was indirectly demonstrated in expanded use and animal experiments, and directly demonstrated in lyophilized experiments. Furthermore, when stored in an HEC-containing hydrogel according to the invention, the phages were found to retain their lytic activity.

[0012] The following medical applications involve the successful treatment of patients who are not suitable for extensive surgical procedures, who do not respond to routine guideline treatments (such as the administration of routine antibiotics due to antibiotic resistance), or who have bacterial infections in hard-to-access locations (such as aortic stents, TEVARs, or biofilms).

[0013] Considering the tactile sensation—a pleasant, even, or even protective feeling on the skin—as well as ease of application and the required layer thickness, the following medical applications focus on superior hydrogels containing HEC. In particular, the hydrogels of this invention containing HEC have shown compelling results in phage release to prevent bacterial infection.

[0014] Successful treatment involves bacterial infections in the cardiovascular field. Firstly, patients with TEVAR were successfully treated using extravascular and intravascular phage applications (Example 2).

[0015] Secondly, patients with chronic infection and occlusion of the aorta and bilateral femoral arteries Patients who underwent bypass surgery were also successfully treated with phages (Example 3). In this example, the infected implant was clinically clearly covered by a bacterial biofilm that could not be completely removed mechanically (manually). This demonstrates the significant advantage of using the hydrogel according to the first aspect of the invention to treat bacterial infections, as it can be positioned at the biofilm and bind with delayed phage release, enabling effective and continuous long-term treatment of the biofilm and associated infection. This also further confirms the hydrogel of the invention for systemic co-treatment with antibiotics.

[0016] In the next step, the hydrogel containing HECs and phages was tested in a dermatological surgical setting. Patients with paradoxical acne post-surgery showed improved wound healing when treated with the hydrogel containing HECs and phages compared to conventional wound treatments to date (Example 6). A mouse model of bacterial infection confirmed the superior wound healing results of the hydrogel containing HECs and phages described above (Example 7). Finally, it can be further demonstrated that the lyophilized form of the hydrogel containing HECs and phages also exhibits the desired phage release and activity (Examples 4 and 5).

[0017] In summary, the inventors have improved upon the prior art and demonstrated that hydrogels containing HEC are excellent tools for retaining phages, maintaining their lytic activity, and delaying their release.

[0018] Inventors can further refine conventional guidelines for implant removal and reimplantation. Conversely, the novel development method using the hydrogel or lyophilized material according to the invention allows for minimally invasive surgery, keeping infected implants in situ and increasing antibiotic sensitivity.

[0019] Similarly, in the field of biofilms, the hydrogels or lyophilized products according to the invention offer advantages over previously applied methods. The hydrogels or lyophilized products according to the invention allow for the disruption of biofilms and the delay of phage release through tight adhesion to their surfaces.

[0020] Compared to treatments administered to date using aqueous solutions and phage solutions alone, the hydrogel or lyophilized product according to the invention showed a 2.5-fold significant improvement in wound healing in mouse experiments. Successful treatment was achieved both in vivo and percutaneously.

[0021] Furthermore, instead of individual therapies, the hydrogels or lyophilized products of the present invention provide a standard therapy for treating and / or preventing bacterial infections using standard phage mixtures.

[0022] Finally, while the hydrogels or lyophilized products of the present invention exhibit all these advantages, they have not shown any adverse side effects, even when administered intravascularly.

[0023] In a first aspect, the present invention provides a hydrogel comprising hydroxyethyl cellulose (HEC) and bacteriophage.

[0024] In the context of this invention (see Example 1), the inventors have found that HEC is a gelling agent, particularly suitable for preparing gels with desired tactile and other properties. In particular, the hydrogels of this invention are steam sterilizable, which is an important property for their intended use in treating bacterial infections, especially during surgical procedures.

[0025] Furthermore, as shown in Example 2, it can be demonstrated that the claimed composition is particularly effective in treating bacterial infections when applied during surgical procedures.

[0026] In the context of this invention, the terms "bacteriophage" and "bacterial phage" are used interchangeably.

[0027] In the context of this invention, the following abbreviations are used:

[0028] AMR antimicrobial resistance

[0029] CT computed tomography scan

[0030] EVAR (Endovascular Aortic Repair)

[0031] MRSA (Methicillin-resistant Staphylococcus aureus)

[0032] PET Positron Emission Tomography

[0033] SAVR (Surgical Aortic Valve Replacement)

[0034] TAVR (Transcatheter Aortic Valve Replacement)

[0035] TEVAR endovascular repair of the thoracic aorta

[0036] As mentioned above, bacteriophages are well-known antibacterial agents. In the context of this invention, any type of bacteriophage can be used. Customized gel formulations can also be prepared, wherein the type of bacteria to be treated is determined, and then the corresponding bacteriophage is selected.

[0037] In another embodiment of the invention, a standardized phage composition having activity against a pre-selected type of bacteria is used. For example, the phage contained in the hydrogel of the present invention may have activity against Staphylococcus aureus, Staphylococcus epidermidis, other coagulase-negative staphylococci, Enterobacteriaceae, Escherichia coli, Pseudomonas, and Corynebacterium. Such phages are known in the art, as described above.

[0038] In the context of this invention, lytic phages are preferred. This is advantageous because their antibacterial activity begins immediately upon encountering the corresponding bacteria. The phages contained in the hydrogel according to the first aspect can be thermally stable and / or cryogenically stable. When the phages contained in the hydrogel according to the first aspect are cryogenically stable, they are preferably stable even at low temperatures such as about -90°C to -20°C. In another preferred embodiment, the phages in the hydrogel of the present invention are thermally stable. In another preferred embodiment, the phages are cryogenically stable.

[0039] The concentration of bacteriophages in a hydrogel can be any concentration that is effective in treating bacterial infections and can vary depending on the bacteriophages used and the expected bacterial concentration.

[0040] As a minimum value, for example, 10 can be used. 2 Phage concentration of pfu / ml.

[0041] In one implementation, the concentration of the bacteriophage is 10. 2 pfu / ml up to 10 8 pfu / ml, preferably 10 4 pfu / ml up to 10 8 pfu / ml or 10 5 pfu / ml up to 10 7 pfu / ml.

[0042] In another preferred embodiment, the concentration of bacteriophage in the hydrogel is 10. 2 pfu / ml up to 10 8 pfu / ml, preferably 10 2 pfu / ml up to 10 7 pfu / ml, more preferably 10 2 pfu / ml up to 10 6 pfu / ml, optimal value 10 2 pfu / ml up to 10 5.5 pfu / ml.

[0043] Before adding the phage to the composition of the present invention, it is preferable to sterilize the phage, more preferably by steam sterilization. During the sterilization process, the initial phage concentration may decrease accordingly. The reduction in the number of phages is pre-calculated so that the phage concentration of the final product is at least 10. 2 pfu / ml.

[0044] In another preferred embodiment, the bacteriophage is sterilized by aseptic filtration.

[0045] Bacteriophages can be added to the hydrogel of the present invention in solution form. For example, when preparing the hydrogel of the present invention, the phage solution can be 50%, and the HEC gel stock solution (optionally having other components, see below) can also be 50%.

[0046] Phage preparations can be produced in accordance with applicable GMP guidelines.

[0047] The hydrogel of the present invention is a hydrogel containing hydroxyethyl cellulose (HEC).

[0048] Hydroxyethyl cellulose (=HEC) has the following chemical formula

[0049]

[0050] R = H or CH2CH2OH

[0051] (source: https: / / apothekenwiki.com / wp-

[0052] content / uploads / 2017 / 12 / Hydroxyethylcellulose-Strukturformel.png )

[0053] HEC is known in the art. In the context of this invention, any suitable length of HEC can be used. In particular, n can vary between 300 and 15,000, more preferably between 500 and 10,000, and most preferably between 2,000 and 8,000.

[0054] In the context of this invention, HEC is used as a gelling agent because it can be demonstrated in this context that HEC produces a stable gel scaffold on an aqueous substrate. The gel matrix formed by HEC provides sufficient space to retain a defined concentration of phages, allowing for distribution that facilitates release. Furthermore, the viscosity of the final product can be easily adjusted by the concentration of the gelling agent, enabling variations in the HEC percentage to provide more possibilities for products with the same material composition. In addition, the pharmaceutical uses of HEC are well-known, and it has a low allergenic potential.

[0055] Among other gelling agents commonly used in the pharmaceutical industry, HEC is the only one that can be steam sterilized, as shown in the attached examples. This sterilization process increases the viscosity of the gel, and this "increase in viscosity" can therefore be considered when preparing the compositions of the present invention. Under the additional influence of body / skin temperature, the product can float at the application site by reabsorbing moisture from intervessel water. Through this process from different viscosity levels to aqueousity, bacteriophages are released, and their effects exceed:

[0056] Another reason for using hydroxyethyl cellulose is that the gelling agent has low sensitivity, resulting in a final product that is also insensitive to pH changes and electrolytes. HEC gel production, and equivalent phage HEC gel production, is easy, inexpensive, and rapid to achieve in both artificial manufacturing and industrial-scale processing. No special equipment or surfaces are required.

[0057] The hydrogel of the present invention preferably contains 3% to 10% HEC, more preferably 5% to 8% HEC or 6% to 7% HEC, and most preferably 6.5% HEC.

[0058] In a preferred embodiment, the hydrogel of the first aspect contains 3% to 10% HEC, preferably 5% to 9% HEC, more preferably 6% to 9% HEC, particularly preferably 6% to 7% HEC, and most preferably 6.5% HEC.

[0059] The hydrogel of this invention can be prepared by mixing the components and adding water to obtain the final concentration. In principle, the preparation of HEC hydrogels is known in the art.

[0060] Gel preparation is carried out manually or by technical processes and is known in the art. For example, the dissolving component can be dissolved in a first portion of water. Subsequently, HEC can be incorporated into the water under moderate stirring. Under further moderate stirring, at least the remaining required water can be added to the gel material. After the hydrogel is prepared, it can be sterilized, preferably by steam sterilization.

[0061] The phage can be added to the gel, for example, in the form of a phage solution. Mechanical force may be required to dissolve the phage into the gel matrix. This can be done by stirring the composition while adding the phage solution or by a dual-syringe technique: thus, one syringe may contain the phage solution, and a second syringe may provide the gel. Through a connector, the gel and the phage solution are bound together by first pressing the phage solution into the gel. This process can be repeated multiple times, and vice versa.

[0062] In a preferred embodiment, the hydrogel also contains CaCl2 and glycerol.

[0063] Calcium can be used to stabilize bacteriophages and thus maintain their activity. Calcium can be added to the composition in the form of readily soluble calcium chloride, but other calcium salts can also be used. This is achieved by dissolving CaCl2 in the composition, especially in the contained water, so that calcium is expressed as calcium ions (CaCl2). 2+ It can be utilized by bacteriophages in the form of ). Calcium chloride itself has only low chemical reactivity, does not affect the pH of the adjusted product, and has no relevant effect on the permeation process at the following concentrations (e.g., 10% to 33%).

[0064] The hydrogel of the present invention may contain 10% to 33% CaCl2, preferably 15% to 25% or 17% to 23%, and more preferably 20% to 22%, most preferably 21.5%.

[0065] Glycerin can be used to improve adhesion properties and delay product liquefaction under the influence of body temperature and fluids, as it acts as a lubricant, dispersant, and adhesion promoter. Glycerin is also hygroscopic, thus not only supporting systemic degradation and the release of bacteriophages onto the surface of the applied PhaTEC-drug, but also contributing to the residue-free degradation of the product. In the case of surgical sutures, for example, glycerin is incorporated to make the sutures a self-absorbable product.

[0066] The hydrogel of the present invention may contain 6% to 22% glycerol, preferably 8% to 18%, more preferably 10% to 16% or 12% to 15%, and most preferably 13.5% glycerol.

[0067] Glycerin can be added to the composition in the form of 85% glycerin.

[0068] In a preferred embodiment, if the hydrogel of the first aspect comprises CaCl2 and glycerol, the hydrogel may contain 10% to 33% CaCl2, preferably 15% to 25% or 17% to 23%, and more preferably 20% to 22%, most preferably 21.5%.

[0069] In another preferred embodiment, if the hydrogel of the first aspect comprises CaCl2 and glycerol, the hydrogel may contain 6% to 22% glycerol, preferably 8% to 18%, more preferably 10% to 16% or 12% to 15%, and most preferably 13.5% glycerol.

[0070] In another preferred embodiment, if the hydrogel of the first aspect comprises CaCl2 and glycerol, then the hydrogel may contain...

[0071] a) 10% to 33% CaCl2, preferably 15% to 25% CaCl2 or 17% to 23% CaCl2, more preferably 20% to 22% CaCl2, and most preferably 21.5% CaCl2; and additional

[0072] b) 6% to 22% glycerol, preferably 8% to 18% glycerol, more preferably 10% to 16% glycerol or 12% to 15% glycerol, and most preferably 13.5% glycerol.

[0073] In another preferred embodiment, the hydrogel of the present invention further contains a buffer. Buffers suitable in the context of the present invention include Tris-HCl (pH = 7.4), sodium chloride, and magnesium chloride.

[0074] The hydrogel of the present invention may also contain other salts, such as sodium chloride, magnesium chloride, calcium chloride and potassium chloride.

[0075] The hydrogel of the present invention preferably has a physiological pH, such as 7, 7.2, 7.4, 7.6, or 7.8. Using a buffer solution to ensure a physiological pH can also help reduce pH differences in infected inflammatory tissues.

[0076] In a particularly preferred embodiment, the hydrogel of the present invention comprises a buffer solution containing sodium chloride, magnesium chloride (x7H2O) and Tris-HCl, with a pH of 7.4.

[0077] In another preferred embodiment, the hydrogel according to the first aspect of the invention has a pH of 7.0 to 7.8, preferably 7.1 to 7.6, more preferably 7.2 to 7.5, and most preferably 7.4.

[0078] The HEC stock solution used to prepare the HEC hydrogel of the present invention may contain, for example (based on 100g): HEC (hydroxyethyl cellulose): 13g = 13%.

[0079] CaCl2 solution * 43g = 43%

[0080] 85% glycerin: 27g = 27%

[0081] Water: 17g = 17%

[0082] In a preferred embodiment, the hydrogel of the present invention comprises

[0083] 10 2 pfu / ml up to 10 8 pfu / ml phage,

[0084] 3% to 10% HEC

[0085] 10% to 33% CaCl2, and

[0086] 6% to 22% glycerin.

[0087] In another preferred embodiment, the hydrogel of the present invention comprises

[0088] 10 2 pfu / ml up to 10 8 pfu / ml phage,

[0089] 5% to 8% HEC

[0090] 10% to 33% CaCl2, and

[0091] 6% to 22% glycerin.

[0092] In a preferred embodiment of the present invention, the hydrogel of the present invention comprises

[0093] 10 4 pfu / ml up to 10 8 pfu / ml phage,

[0094] 3% to 10% HEC

[0095] 15% to 25% CaCl2, and

[0096] 8% to 18% glycerin.

[0097] In a more preferred embodiment of the present invention, the hydrogel of the present invention comprises

[0098] 10 5 pfu / ml up to 10 7 pfu / ml phage,

[0099] 6.5% HEC

[0100] 21.5% CaCl2, and

[0101] 13.5% glycerin.

[0102] In another preferred embodiment, the hydrogel according to the first aspect of the invention comprises 10 2 pfu / ml up to 10 8 pfu / ml phage,

[0103] 3% to 10% HEC

[0104] 10% to 33% CaCl2, and

[0105] 6% to 22% glycerin;

[0106] Preferred

[0107] 10 2 pfu / ml up to 10 7 pfu / ml phage,

[0108] 5% to 8% HEC

[0109] 10% to 33% CaCl2, and

[0110] 6% to 22% glycerin;

[0111] More

[0112] 10 2 pfu / ml up to 10 6 pfu / ml phage,

[0113] 3% to 10% HEC

[0114] 15% to 25% CaCl2, and

[0115] 8% to 18% glycerin;

[0116] or

[0117] 10 2 pfu / ml up to 10 5.5 pfu / ml phage,

[0118] 3% to 10% HEC

[0119] 15% to 25% CaCl2, and

[0120] 8% to 18% glycerin;

[0121] And the optimal choice

[0122] 10 2 pfu / ml up to 10 5.5 pfu / ml phage,

[0123] 6.5% HEC

[0124] 21.5% CaCl2, and

[0125] 13.5% glycerin.

[0126] The water used in the composition of the present invention may be water for injection (Aqua ad injectabilia).

[0127] In one exemplary embodiment, the hydrogel of the present invention is prepared using the following components.

[0128]

[0129]

[0130] In a preferred embodiment, the hydrogel of the present invention has a viscosity of 1,000 mPas to up to 100,000 mPas; preferably 10,000 mPas to 80,000 mPas, more preferably 40,000 mPas to 80,000 mPas or 40,000 mPas to 60,000 mPas.

[0131] In a preferred embodiment of the invention, the hydrogel is sterile. Preferably, the hydrogel has been steam sterilized.

[0132] The sterile state can be achieved by sterilizing the HEC hydrogel and the phage solution separately, or by sterilizing the hydrogel of the present invention, i.e., the hydrogel containing hydroxyethyl cellulose (HEC) and phage.

[0133] In a second aspect, the present invention relates to lyophilized hydrogels according to the first aspect.

[0134] Therefore, the term "lyophilized hydrogel" describes any product obtained by freeze-drying a hydrogel according to the first aspect of the invention. The term "lyophilization" describes any low-temperature dehydration process. Typically, the temperature is lowered at least until the water is frozen, and the pressure is reduced to cause the water to sublimate. Methods for freeze-drying are well known to those skilled in the art.

[0135] The starting point can be a hydrogel according to the first aspect of the invention. The gel is typically stored in a cool place, such as a refrigerator, at a temperature of up to 8°C, before the freeze-drying process begins.

[0136] At the start of the freeze-drying process, the hydrogel of the first aspect of the invention can be filled into (sterile) containers that typically already have the desired product shape. These containers can be cups, etc., made of foil designed for this purpose, for example using thermoforming techniques. The thickness of the resulting product can also be predetermined in this process step and is usually a result of the filling height of the container.

[0137] The duration and intensity of freezing and vacuum application must generally be adapted to the product size, i.e., the gel volume. The freeze dryer (equipment) used, for example, may also affect the process parameters to be applied due to different capacities. Therefore, the following process data will be considered as examples.

[0138] For example, in the first step, the hydrogel can be frozen at -20°C for 2 hours. In the second step, it can then be primarily dried at 2.5 mbar and -10°C for approximately 40 hours. The third step may include a secondary drying at >1 mbar and 10°C, which can be carried out for approximately 20 hours.

[0139] Generally, freezing processes are considered crucial for the production of freeze-dried products containing bacteriophages: they should be rapid and yield highly amorphous products. Rapid freezing methods typically also ensure that the lytic activity of the bacteriophages is preserved. Regardless of the volume to be frozen, the freezing time is typically <5 hours, more preferably <2 hours, and even more preferably <1.5 hours.

[0140] Further drying time depends primarily on the performance of the equipment used and typically has only a minor impact on the performance of the resulting product.

[0141] Preferably, the method for preparing the lyophilized hydrogel according to the first aspect of the present invention includes the following steps:

[0142] a) Provide a hydrogel according to the first aspect of the invention;

[0143] b) Freezing step a) at a temperature of at most -10°C, preferably -20°C, more preferably -40°C, and most preferably -60°C.

[0144] The hydrogel; time < 5 hours, preferably < 2 hours, more preferably < 1.5 hours and most preferably ≤ 1 hour;

[0145] c) The cryogel from step b) is subjected to primary drying at ≤2.5 mbar and ≤10°C for about 40 hours, preferably at 1.5 mbar and -30°C for about 40 hours;

[0146] d) Perform approximately two drying cycles at >1 mbar and 10°C for about 20 hours.

[0147] To maintain elasticity, the resulting freeze-dried product preferably has a residual water content of >2%.

[0148] Typically, the resulting lyophilized product may contain

[0149] 10 2 pfu / ml up to 10 8 pfu / ml phage,

[0150] 5% to 25% HEC

[0151] 24% to 80% CaCl2, and

[0152] 13% to 50% glycerin;

[0153] Preferably,

[0154] 10 2 pfu / ml up to 10 8 pfu / ml phage,

[0155] 10% to 20% HEC

[0156] 40% to 60% CaCl2, and

[0157] 20% to 40% glycerin;

[0158] More preferably,

[0159] 10 2 pfu / ml up to 10 8 pfu / ml phage,

[0160] 13% to 17% HEC

[0161] 45% to 55% CaCl2, and

[0162] 25% to 35% glycerin;

[0163] Most preferably,

[0164] 10 2 pfu / ml up to 10 8 pfu / ml phage,

[0165] 14% to 16% HEC

[0166] 51% to 53% CaCl2, and

[0167] 30% to 33% glycerin.

[0168] Examples of the composition of the hydrogel and the resulting lyophilized product:

[0169]

[0170] Preferably, the hydrogel according to the first aspect does not contain any preservative system. More preferably, the hydrogel according to the first aspect does not contain methylparaben, propylparaben, phenoxyethanol, and / or potassium sorbate.

[0171] Sterilization of freeze-dried hydrogels:

[0172] In medical applications, the purpose is typically to provide sterile products, especially when the product is designed for implantation, such as in the case of anastomosis and for packing wound cavities. However, the mandatory nature of sterility is often determined by the intended use of the product, making non-sterile products also plausible.

[0173] The aseptic state achieved through freeze-drying can be achieved using pre-sterilized phage-containing hydrogels in an aseptic freeze-drying process. Furthermore, based on experimental results with the gel, other types of sterilization, such as steam sterilization, are also possible. In particular, since the steam used in steam sterilization does not directly contact the product, no water / water vapor is introduced into the product. Other aspects, such as the support pressure set in the steam sterilization process, must generally be considered.

[0174] In practice, when applied to a subject's body, the lyophilized hydrogel typically decomposes according to the second aspect. First, the lyophilized hydrogel swells back to a gel (semi-solid) state under the influence of body temperature and water. In the second step, the gel from the first step is usually further diluted by body water and transforms into a liquid state. Finally, the diluted liquid hydrogel is physiologically degraded and excreted.

[0175] Compared to hydrogels containing HECs and phages, lyophilized products of the same hydrogel have demonstrated numerous advantages. For example, lyophilized products of phage-containing hydrogels exhibit a (strong) delay in phage release, which can be further determined by the gel composition and is highly desirable, for instance, in the treatment of infected wounds. Furthermore, lyophilized products of the same hydrogel are easier to cut and stretch, and offer better adjustability and adaptability compared to hydrogels containing HECs and phages. It is also highly elastic, thus not compromising physiological dynamics.

[0176] Furthermore, the shape and thickness of the product, as well as the phage cycle, can be easily determined through the manufacturing process (e.g., also depending on the amount of hydrogel freeze-dried, etc.). Additionally, during application, covering (if necessary with the aid of suture materials) and surface repair are possible, etc. Moreover, due to the sterilization potential of the gel, the freeze-dried product from aseptic production (=freeze-dried) is also sterile.

[0177] In a third aspect of the invention, the invention relates to the use of hydrogels according to the first aspect of the invention or lyophilized products according to the second aspect of the invention in methods of treating and / or preventing bacterial infections.

[0178] Clinical areas where infections can be treated using hydrogels according to the first aspect or lyophilized materials according to the second aspect include, for example, cardiac surgery (such as artificial hearts, drive systems, artificial heart valves), vascular surgery (such as vascular implantation, dialysis shunts), orthopedic surgery (especially accident orthopedic surgery) (such as the entire field of implantable prostheses, such as fully implantable prostheses for the knee and hip joints (TEP), dermatology (such as paradoxical acne), and emergency medical services (such as wound dressings).

[0179] Similarly, the present invention relates to a method for treating and / or preventing bacterial infections in a subject, wherein a hydrogel according to a first aspect of the invention or a lyophilized product according to a second aspect of the invention is applied to the subject.

[0180] In a preferred embodiment of the third aspect, a lyophilized version of the hydrogel of the first aspect or the hydrogel of the second aspect is applied during surgical procedures.

[0181] The subject can be any animal, but preferably, the subject is a mammal, and more preferably, a human.

[0182] The hydrogel according to the first aspect of the invention or the lyophilized product according to the second aspect of the invention can be used to treat and prevent bacterial infections. According to the invention, the term "treatment" means that pre-existing bacteria are neutralized by bacteriophages contained in the hydrogel, while the term "prevention" means that bacteria are not yet present when the hydrogel or lyophilized product is applied.

[0183] The hydrogel or lyophilized material can be applied to any accessible surface of the subject. This includes, but is not limited to, the subject's skin or any accessible mucous membranes. The hydrogel or lyophilized material of the present invention is particularly suitable for treating bacterial infections of wounds.

[0184] In a preferred embodiment, the hydrogel or lyophilized hydrogel used according to the third aspect is applied to surgical sites, implants, anastomoses, wounds, abrasions, incisions, punctures, incomplete skin and / or mucosal tissues, catheter entry points, sterile material coverings, wound and / or implant coverings, and / or suture materials.

[0185] Hydrogels can be applied in any possible manner. These methods include, but are not limited to, manual application, application via syringe, via needle, or via catheter. Similarly, lyophilized materials can also be applied in any possible manner. These methods include, but are not limited to, manual application, or application via tools such as tweezers. The dispensing of hydrogels or lyophilized materials is primarily done manually or with any auxiliary device to cover the site / area to be covered by the product.

[0186] In addition, hydrogels or lyophilized materials can be applied to any implant to be attached to or inserted into the subject or to an implant already inserted into the subject.

[0187] In a preferred embodiment, the hydrogel or lyophilized hydrogel used according to the third aspect is used for the treatment and / or prevention of bacterial infection of the implant.

[0188] Specifically, the use of hydrogels according to the first aspect or lyophilized materials according to the second aspect has proven particularly useful and promising in the field of implant infection. According to prescribed treatment guidelines, each infected material must be removed and replaced with autologous material, but this is not always feasible. In particular, when reoperation occurs in the cardiovascular field, subjects are generally unlikely to survive such procedures. Since hydrogels according to the first aspect or lyophilized materials according to the second aspect can be applied minimally invasively, their use significantly increases the likelihood of subject survival. Typically, implants are also not autologous replacements (such as artificial hearts, pacemakers, etc.). In such cases, it is highly advantageous that when treated with the hydrogels or lyophilized materials according to the invention, the implant can remain in situ, allowing for in-situ treatment of the infection.

[0189] Therefore, in another preferred embodiment of the invention, the hydrogel or lyophilized hydrogel for use according to the third aspect can be directly applied to the surface of an implant outside or inside the subject.

[0190] According to the present invention, the implant may be a joint prosthesis, a vascular prosthesis, a heart valve, a catheter, an artificial heart, a dental implant, and a minimally invasive transarterial heart valve (TAVI).

[0191] In a preferred embodiment of the third aspect, the implant is an in-situ prosthesis (preferably an articular prosthesis, more preferably a fully in-situ prosthesis for the knee or hip), a vascular prosthesis, a heart valve, a catheter, an artificial heart, a drive system, a dialysis shunt, a dental implant, and / or a minimally invasive transcatheter aortic valve implant (TAVI).

[0192] In another preferred embodiment, the hydrogel or lyophilized hydrogel used according to the third aspect is used for the treatment and / or prevention of prosthetic vascular graft infection.

[0193] Medical fields in which the hydrogels or lyophilized products of the present invention can be applied include, but are not limited to, surgery, orthopedic surgery, radiation medicine, nephrology, diabetology such as recurrent diabetic foot syndrome, dermatology, cardiology, wound care, nursing facilities / services, and ambulance services.

[0194] Possible indications for the hydrogels or lyophilized products of this invention include, for example, surgical sites, implants, anastomoses, wounds such as deep wounds or puncture wounds, abrasions, incisions, puncture wounds, incomplete skin and mucous membrane tissues, pressure ulcers, entry points for catheters such as urinary catheters / IV-catheters, coverings for sterile materials, wound and implant coverings, and suture materials. Therefore, the term "pressure ulcer" can describe any type of pressure ulcer.

[0195] In a preferred embodiment of the invention, the hydrogel or lyophilized product of the invention is applied during surgical procedures.

[0196] The present invention (see Examples 2 and 3) has demonstrated that the hydrogel of the present invention, when applied during surgical procedures, is particularly useful for the treatment and prevention of bacterial infections.

[0197] During surgical procedures, the hydrogels or lyophilized products of the present invention can be applied in all possible ways. For example, phage gels can be applied manually and dispensed in the same manner, primarily by a surgeon during surgery or by any other person, especially when applied externally to the body, such as to a wound or skin.

[0198] Furthermore, application and distribution at the desired site / area can be accomplished using all available aids, primarily syringes, needles, and catheters. Therefore, phage gel can be administered via minimally invasive intervention or into the body / wound cavity.

[0199] Due to its viscosity and the possibility of adapting the viscosity to the environment (e.g., during surgery), especially when using a dual-injector technique with hydrogels, and due to the robustness of the product provided by the combination of galenic formulations and phages, it can be used in all possible applications.

[0200] In a preferred embodiment, the hydrogel or lyophilized hydrogel used in accordance with the third aspect is applied manually and / or using one or more auxiliary devices, preferably one or more syringes, needles and / or catheters, to the surface of the subject's body or the surface of the implant.

[0201] In a particularly preferred embodiment of the invention, as described above, the hydrogel or lyophilized material of the present invention is used to treat and / or prevent bacterial infections of implants. In this embodiment of the invention, the hydrogel or lyophilized material of the present invention can be applied directly to the implant, either in vitro or in vivo, for example, during surgical procedures.

[0202] In another preferred embodiment, the hydrogel or lyophilized hydrogel used according to the third aspect is for the treatment and / or prevention of bacterial infections caused by dermatological surgery, preferably bacterial infections caused by dermatological surgery related to the treatment of atypical acne or pressure ulcers.

[0203] Typically, hydrogels or lyophilized hydrogels used according to the third aspect can be used to treat and / or prevent bacterial infections resulting from any type of dermatological surgery. This means that wounds are formed during any type of dermatological surgery, and especially due to the size or location of the wound, they may be difficult to heal without any bacterial infection issues. Hydrogels according to the first aspect or lyophilized hydrogels according to the second aspect can be applied to treat and / or prevent bacterial infection to support wound healing.

[0204] Examples of such wounds are well known to those skilled in the art, such as wounds resulting from skin biopsy, electrosurgery, cauterization, flaps, skin grafts, Moss microsurgical controlled excision, cryotherapy (e.g., with liquid nitrogen), excision of skin lesions, liposuction, vitiligo surgery, treatment of paradoxical acne, or pressure ulcers.

[0205] In a fourth aspect, the present invention relates to a method for preparing a hydrogel comprising hydroxyethyl cellulose (HEC) and bacteriophage, the method comprising the following steps:

[0206] a) Provide HEC in solid form,

[0207] b) Dissolve the solid HEC from step a) in a liquid component containing water and bacteriophage, and

[0208] c) Optionally add water until the desired viscosity is achieved.

[0209] All aspects relating to the first, second, and third aspects of the invention, especially when they relate to the characteristics of hydrogels, hydrogel formulations, and bacteriophages, are also applicable to the fourth aspect of the invention.

[0210] Finally, the present invention also relates to a method for treating and / or preventing bacterial infections, the method comprising using a hydrogel according to the first aspect or a lyophilized product according to the second aspect.

[0211] Preferably, in methods of treating and / or preventing bacterial infections, the hydrogel or a lyophilized form of the hydrogel is applied during surgical procedures.

[0212] Preferably, in methods of treating and / or preventing bacterial infections, the hydrogel or a lyophilized hydrogel is applied to surgical sites, implants, anastomoses, wounds, abrasions, incisions, punctures, incomplete skin tissue and / or mucosal tissue, such as catheter entry points, sterile material coverings, wound and / or implant coverings, and / or suture materials.

[0213] More preferably, in methods of treating and / or preventing bacterial infection, the hydrogel or a lyophilized form of the hydrogel is used to treat and / or prevent bacterial infection of the implant.

[0214] Most preferably, in the method of treating and / or preventing bacterial infection, wherein the hydrogel or lyophilized hydrogel is used to treat and / or prevent bacterial infection of the implant, the hydrogel or lyophilized hydrogel is applied directly to the surface of the implant outside the subject or inside the subject.

[0215] In particular, in methods of treating and / or preventing bacterial infections, the implant may be a joint prosthesis, a vascular prosthesis, a heart valve, a catheter, an artificial heart, a dental implant, and / or a minimally invasive transcatheter aortic valve implant (TAVI).

[0216] In particular, when the present invention relates to methods for treating and / or preventing bacterial infections, the hydrogel or lyophilized hydrogel is used for treating and / or preventing prosthetic vascular graft infections.

[0217] Preferably, when the present invention relates to a method for treating and / or preventing bacterial infections, the hydrogel or a lyophilized form of the hydrogel is used to treat and / or prevent bacterial infections caused by dermatological surgery, preferably bacterial infections caused by dermatological surgery associated with treatment of atypical acne or pressure ulcers.

[0218] More preferably, when the invention relates to a method for treating and / or preventing bacterial infections, the hydrogel or a lyophilized hydrogel is applied manually and / or using one or more auxiliary devices, preferably one or more syringes, needles and / or catheters, to the surface of the subject's body or the surface of an implant.

[0219] The present invention is further illustrated by the accompanying embodiments and drawings, which are intended to explain rather than limit the invention. Attached Figure Description

[0220] Figure 1 :

[0221] Sodium hyaluronate gel was placed on a filter and then stored at 37°C for 24 hours. The gel was too stiff to spread well on the filter paper and exhibited agglomeration due to its hardening.

[0222] Figure 2 :

[0223] Sodium hyaluronate gel on the skin: The gel even hardens on the skin. Due to the gel's hardness, attempts to apply and spread it on the arm shown were painful.

[0224] Figure 3 :

[0225] Carbomer gel on skin: This gel exhibits highly adhesive properties. It is difficult to spread on the skin. Furthermore, due to its stickiness, application and spreading are painful. The intended gel layer is not suitable.

[0226] Figure 4 :

[0227] Carbomer Gel: To demonstrate the viscosity of carbomer gel, a small amount was taken between two fingers. The fingers were then spread apart. The gel stuck to the fingers and thinned in the middle. This indicates that it is too difficult to apply and the gel is too sticky.

[0228] Figure 5 :

[0229] HEC gel on the filter before storage at 37°C for 24 hours: The gel exhibits solid properties and good spreading ability / simultaneous application possibility.

[0230] Figure 6 :

[0231] HEC gel on skin: Application tested on skin. Application is very easy. Furthermore, the envisioned gel layer can also be applied.

[0232] Figure 7 :

[0233] HEC gel after 24 hours at 37°C: The gel has dried and shrunk under storage conditions. The post-storage solidity characteristics, without any packaging to protect the gel from water evaporation, demonstrate the necessity of adequate primary packaging. Furthermore, the gel retains its shape as it was in storage, indicating continued coverage of the application area.

[0234] Figure 8 :

[0235] Poluxamer gel after 24 hours at 37°C: The gel has dried out under storage conditions. The post-storage solids characteristics, without any protective packaging against water evaporation, highlight the necessity of adequate primary packaging. The gel did not retain its storage shape, showing changes in application area coverage and a shift towards a cubic shape.

[0236] Figure 9 :

[0237] Sodium hyaluronate gel after 24 hours at 37°C: The gel had dried and shrank under storage conditions. The post-storage solid characteristics, without any packaging to protect the gel from water evaporation, highlight the necessity of adequate primary packaging. Furthermore, the gel did not retain its storage shape, indicating a change in application area coverage. Viscosity increased with moisture loss.

[0238] Figure 10 :

[0239] HEC gel after aseptic manufacturing: direct loading method, European Pharmacopoeia (Ph.Eur.) 2.6.1, showed no signs of bacterial contamination after prolonged testing to increase the likelihood of detection.

[0240] Figure 11 :

[0241] Plaques were still detectable after steam sterilization of bacteriophages: on a standardized bacterial bioload, the bacteriophages had diffused after steam sterilization. After a period of incubation, the plaques indicated that the bacteriophages survived the steam sterilization process and remained active.

[0242] Figure 12 :

[0243] Aseptic preparation of HEC gel: Figure 12 The results show the sterility of the phage-gel combination.

[0244] Figure 13 :

[0245] The steam-sterilized gel: direct loading method, European Pharmacopoeia 2.6.1, showed no signs of bacterial contamination after prolonged testing to increase the likelihood of detection.

[0246] Figure 14 :

[0247] Preoperative PET-CT. Representative slide from a preoperative [F18]fluorodeoxyglucose PET-CT scan. Increased levels of tracer accumulation around the stent graft in the thoracic aorta are a sign of active inflammation.

[0248] Figure 15 :

[0249] Intraoperative image from the first treatment step: The descending aorta has been mechanically debrided from the infected tissue surrounding the aorta.

[0250] Figure 16 :

[0251] Intraoperative image from the first treatment step: two endo-sponges are placed around the aorta.

[0252] Figure 17 :

[0253] Intraoperative image from the first treatment step: Finally, before closing the thoracic cavity, another vacuum sponge is applied for ventral coverage of the aorta (C).

[0254] Figure 18 :

[0255] The ready-to-use aortic stent graft system was manually covered with phage gel.

[0256] Figure 19 :

[0257] Lateral X-ray after stent implantation shows that the infected stent graft is completely covered by the new stent graft.

[0258] Figure 20 :

[0259] Three months after the implantation of a phage gel-coated stent, a PET-CT scan showed no signs of infection in or around the thoracic aorta.

[0260] Figure 21 :

[0261] PET-CT showed an infected aortic-bifemoral artery graft with infection of the right femoral artery tissue.

[0262] Figure 22 :

[0263] Recording of surgical procedures and application of phage gel.

[0264] Figure 23 :

[0265] Three months after the intervention, a PET-CT scan showed no signs of infection around the aorta or both femoral arteries.

[0266] Figure 24 :

[0267] An exemplary image of a freeze-dried hydrogel.

[0268] Figure 25 :

[0269] Lyophilized products were prepared from hydrogels containing bacteriophages.

[0270] Figure 26 :

[0271] Four types of lyophilized hydrogels were placed in the center of an agar plate containing Staphylococcus aureus.

[0272] Figure 27 :

[0273] Bacterial-free (circled) plaques on the culture medium.

[0274] Figure 28 :

[0275] Experimental setup.

[0276] Figure 29 :

[0277] Data on phage release from products B1, B2, C1, and C2 over time in Example 5: the x-axis represents phage (BPh) concentration in [pfu / ml], and the y-axis represents time in [minutes].

[0278] Figure 30 :

[0279] Hurley stage II / III in the left armpit (left image) and right buttock (right image).

[0280] Figure 31 :

[0281] Surgical treatment of the left axilla involved the application of a hydrogel containing HEC and a phage. First, the left axilla was given local anesthesia (upper left), followed by surgical removal of the affected tissue (left axilla surgical incision, upper right), application of the phage and carrier (lower left), and then suturing of the axilla skin (lower right).

[0282] Figure 32 :

[0283] The condition of the armpit after 2 weeks (left) and 10 weeks (right).

[0284] Figure 33 :

[0285] The condition of the buttocks after 2 weeks (top) and 10 weeks (bottom).

[0286] Figure 34 :

[0287] Wound seize decreases over time, where the x-axis represents time and the y-axis indicates wound seize.

[0288] Example 1

[0289] Discovery of suitable phages containing gel-gallen formulations for clinical use: Laboratory bench

[0290] summary

[0291] In vitro laboratory tests were conducted to identify a suitable base gel for PhaTEC phage gel products. The list of gelling agents included hydroxyethyl cellulose (HEC), sodium hyaluronate, poloxamer, carbomer, and silica (SiO2 [highly dispersed]). Tests were performed based on spreadability on filter paper and skin, tactile and sensory properties, gel consistency, and behavior after 24 hours at 37°C. For the phages, a mixture of different phages (SniPha 360), available from Phage24 in Austria, was used. These requirements were for the gel that would become the subsequent base for PhaTEC products. All tests were conducted under sterile conditions in the Pohl-Boskamp GmbH & Co. KG laboratory, following GLP (Good Laboratory Practice).

[0292] Regarding sterilization, our experiments demonstrate multiple methods for producing sterile products of HEC gel and phage mixtures.

[0293] Furthermore, both the HEC gel and the phage can be steam sterilized. Therefore, PhaTEC products containing both gel and phage can be sterilized in the final container, enabling simple and cost-effective industrial production. 1. Background Technology

[0295] 1. Stability and spreadability

[0296] PhaTEC products are intended for both internal and external application. Specifically, the product will be applied to mucosal and skin areas, wounds, and surgical sites such as anastomoses. For this purpose, appropriate spreadability (to cover the application area) is required, along with product stability, for example, to create / apply a single layer thickness and remain at the application site. Furthermore, the gel should be spreadable so that it can be easily and painlessly applied to sensitive areas, such as fresh wounds. Additionally, cooling is necessary, especially in the case of external application, to counteract inflammation and provide a comfortable sensation at the application site.

[0297] 2. Sterilization possibility

[0298] Because of the pursuit of products intended for both in vitro and in vivo application, the product and therefore its initial components, such as the base gel, must offer the possibility of a sterile state.

[0299] 3. Spread it thoroughly around the application area.

[0300] The viscosity of the gel should be reduced by introduction through the skin or body temperature and fluids to allow for the subsequent release of the contained phages within a defined timeframe. The product degrades due to dilution and physiological excretion, resulting in a prolonged release of the therapeutic agent, thus having no adverse effects on the body.

[0301] 4. Sensation on the skin

[0302] Patient acceptance is crucial for all medical and pharmaceutical needs. Since phages do not cause any physical reactions and have no side effects, the gel, especially when applied to the skin / external application, must be comfortable on the skin and / or mucous membranes.

[0303] 2. Gel manufacturing

[0304] Initially, all gels were prepared using a 13% gelling agent. Carbomer, sodium hyaluronate, and HEC gels were the easiest to prepare.

[0305] Only three solid gels were obtained from the five formulations:

[0306] HEC gel, carbomer gel, and sodium hyaluronate gel. Silica forms a viscous liquid at the applied concentration, but not a solid gel, and does not completely dissolve. Poloxamer exhibits gel islands that are not connected to each other; furthermore, it does not absorb much water. Figure 1 ).

[0307] 3. Tactile / Sensory Skin Test

[0308] method:

[0309] Different gel formations were tested on the same test subject's skin. Sensitive skin reactions were observed.

[0310] Sodium hyaluronate (Sodium hyaluronate EP) gel is too stiff to spread well on filter paper. Figure 1 .

[0311] Because of its hardness, applying and spreading it on the skin (especially in hairy areas, such as the arm used in the test) can cause pain. Figure 2 .

[0312] Carbomer (Carbomer EP; Carboprol 71G) gel is highly adhesive, making it difficult to spread on filter paper and virtually impossible to spread on skin. Furthermore, due to this stickiness, application and spreading on skin (especially in hairy areas, such as the arm used in the test) causes pain. Figure 3 , Figure 4 .

[0313] HEC (Natrosol 250HX) gel offers the most comfortable feel: it spreads easily on filter paper and skin, while also having the firmness to allow for the application of individual layer thicknesses. Figure 5 , Figure 6 .

[0314] result:

[0315] It has been demonstrated that these gels feel very different: they exhibit very different properties. Carbomer gel was found to be highly adhesive and difficult to apply due to its free-flowing nature, as SiO2 gel (highly dispersed silica; Aeroperl 300 Pharma) is liquid and poloxamer gel (poloxamer 407) does not exhibit island-like formation, even when tested on filter paper and / or skin. The stickiness of carbomer gel is very uncomfortable when applied to the skin and causes pain in hairy areas such as the arm (in the test). The cooling effect is almost immediate. The gel, especially on the skin, does not remain at the application site but immediately and uncontrollably flows into the surrounding area. An appropriate layer thickness cannot be applied.

[0316] Sodium hyaluronate gel is too hard for painless, easy application: it is almost impossible to spread on filter paper and skin, especially in hairy areas such as (in the test) the arm, where its hardness causes pain and makes it difficult to spread.

[0317] Although HEC gel is firm, it is still easy and painless to apply and can remain on the application site as needed: it exhibits good spreadability not only on filter paper and skin, but also feels comfortable on the skin. Application to the skin is simple and painless. Gel dispensing can be done individually according to the desired layer thickness. Due to the applied layer thickness, a cooling effect occurs after a period of time. Furthermore, the applied gel layer creates a protective layer sensation, much like a plaster or similar ointment.

[0318] 4. Behavior of gel-gallen formulations under dry conditions

[0319] Heat exposure at 37°C was performed using a drying oven. To test the required cooling effect when applied externally, each 10 ml of gel was placed in the center of filter paper and the boundaries were marked. The gels were then stored at 37°C for 24 hours to demonstrate the drying of the gels and the cooling effect due to evaporative cooling.

[0320] method:

[0321] The tactile / sensory test was performed in a dry oven with a covered cup of gel after 24 hours at 37°C. Figure 7 , Figure 8 , Figure 9 .

[0322] result:

[0323] Primary packaging provides sufficient protection against dehydration. Under physiological conditions, the product is liquefied by interstitial water and undergoes physiological degradation.

[0324] 5. Aseptic state

[0325] 5.1. Aseptic Production

[0326] 5.1.1. Gel

[0327] method:

[0328] The gels were prepared aseptically in laboratory-scale testing. For this purpose, pre-sterilized equipment and workbenches (laminar flow) were used. The HEC gels were prepared according to the direct loading method as described in European Pharmacopoeia 2.6.1. Figure 5 ).

[0329] result:

[0330] No bacterial growth was observed 24 hours later until day 9 (when the extended test ended). No significant microbial growth was observed throughout the period. Figure 10 .

[0331] 5.1.2. Bacteriophage

[0332] PhaTEC's explicit objective is to enable the industrial, standardized, and easy production of its phage products. A key aspect of simple industrial production is the sterilization of the product in the final container. Two possibilities for gel sterilization have been described above and confirmed through corresponding tests. Phages can also be sterilized via aseptic filtration, as tested and described above.

[0333] method:

[0334] The bacteriophages were sterile filtered and no bacteria grew on different culture media after 24, 48, and 72 hours at 37°C. The mixture tested positive on E. coli cultures on agar, containing approximately 10... 7 The phage mixture samples were steam-sterilized and tested on fresh, appropriate agar using steam-sterilized BPG.

[0335] result:

[0336] Plaques were detected at approximately 6 × 10^2 PFU / ml. As a result, steam-sterilized phages from our phage mixture still exhibited lytic activity against the corresponding bacterial strains. Figure 11 .

[0337] In conclusion, it has been proven that steam sterilization of bacteriophages is possible.

[0338] 5.2. Steam sterilization

[0339] 5.2.1. Gel

[0340] method:

[0341] In addition to aseptic manufacturing, the gel is also prepared in a clean laboratory environment and steam-sterilized in the final container. Figure 12 .

[0342] result:

[0343] When tested as described in "Sterile Preparations", the steam-sterilized HEC gel showed no microbial growth throughout the entire time. Figure 13 .

[0344] 6. Summary

[0345] Of the most suitable gelling agents tested, hydroxyethyl cellulose was the best: tactile sensation, i.e., a good, even, or even protective feeling on the skin, as well as its ease of application and the required layer thickness, are just some of the factors that contribute to this result.

[0346] Regarding sterilization, there are multiple methods that demonstrate the production of sterile products containing a mixture of HEC gel and bacteriophage. All individual tests conclude that all the methods and procedures described above are suitable for sterilizing PhaTEC products.

[0347] Furthermore, both the HEC gel and the phage can be steam sterilized. Therefore, PhaTEC products containing both gel and phage can be sterilized in the final container, enabling simple and cost-effective industrial production.

[0348] Example 2

[0349] The use of extravascular and intravascular phages successfully treated infected TEVAR.

[0350] summary

[0351] Objective: Graft infection is a serious complication in vascular surgery. Surgical resection of infected aortic stent grafts is associated with high mortality and morbidity. Therefore, alternatives to inadequate antibiotic treatment and major surgery are urgently needed.

[0352] Case

[0353] A 67-year-old woman was admitted to the hospital due to infection of a thoracic aortic stent graft. PET-CT imaging confirmed localized infection. Surgical resection of the stent graft was not feasible due to comorbidities. Therefore, a three-step approach using localized phage therapy was adopted as a last resort. First, the para-aortic tissue was debrided via a left thoracotomy, a phage suspension was applied around the aorta, and a vacuum irrigation system was installed. As a second step, after three days of repeated alternating infusions of the phage suspension, the vacuum sponge was removed, and a phage-containing gel was locally applied around the aorta. In the third step, the phage-containing gel was applied to the thoracic stent graft, which was then placed intravascularly into the infected stent. The patient was discharged 28 days later with normal infection parameters. Three months after intervention, PET-CT imaging showed no signs of infection in or around the thoracic aorta.

[0354] in conclusion:

[0355] This case demonstrates that infected intravascular stent grafts can be successfully treated by applying phages to both extravascular and intravascular sites (as a novel approach) using phage-coated stent grafts. This success is likely solely due to the hydrogel comprising HEC and phage according to the first aspect of the invention, which allows for in-situ localization of the phage at the infection site and continuous, delayed release of the phage.

[0356] Keywords:

[0357] Staphylococcal sepsis, graft infection, phage therapy, antibiotic resistance Background Technology

[0358] In vascular surgery, graft infection is a serious complication. In particular, infection of endovascular aortic stent grafts is associated with a morbidity and mortality rate as high as 75%. Because these endovascular surgeries are often performed on elderly patients with multiple comorbidities who are unsuitable for open aortic repair, the removal of infected stent grafts and in situ reconstruction with autologous tissue or extraanatomical replacement is necessary, which is associated with early postoperative morbidity and mortality rates exceeding 20%. Even with successful treatment, the reinfection rate is as high as 20%.

[0359] Bacteria embedded in the tissue surrounding the prosthesis form a surface-adhering biofilm, thus exhibiting resistance to antibiotics up to 1000 times greater. Conversely, even targeted antibiotic therapy can only suppress stent graft infection, not provide a cure.

[0360] To reduce the morbidity and mortality rates associated with unavoidable surgical procedures, alternative minimally invasive methods are urgently needed. In this context, bacteriophages and their lytic activity represent a promising treatment option.

[0361] Case

[0362] In August 2020, a 67-year-old female patient was admitted to the hospital due to worsening general condition and chest pain. She presented with a pronounced cough upon deep inspiration, no sputum, and a fever reaching 38.6°C. SARS-CoV-2 infection was ruled out. The patient's white blood cell count was 16.7 × 10⁻⁶. 9 The patient's serum C-reactive protein level was 199.6 mg / L. Secondary findings included the patient's condition following a thoracic and abdominal stent graft implantation (COOK stent 34 / 152 mm) after a type B aortic dissection at Stanford University in February 2009. Additionally, the patient suffered from Osler disease requiring prednisolone treatment, post-pulmonary embolism, arterial hypertension, left renal atrophy, and sigmoid diverticulosis.

[0363] Chest X-ray showed no signs of pneumonia. Antibiotic treatment was initiated with ampicillin / sulbactam and roxithromycin. After Staphylococcus aureus was detected in blood cultures, the antibiotic regimen was switched to flucloxacillin, and after five days, it was switched to cefuroxime due to allergic dermatitis.

[0364] Endocarditis was ruled out. The patient presented with a known progression of leukocytoclastic vasculitis involving both legs, arms, cleavage, hands, and soles of the feet, but not the kidneys. Treatment with prednisolone improved the skin symptoms. The progression of vasculitis was considered a response to a systemic infection. Antibiotic treatment was switched to meropenem and cefazolin.

[0365] Computed tomography scans of the chest and abdomen did not show any signs of infection. To rule out the possibility of the aortic stent graft being an infected site, a fluorodeoxyglucose PET-CT scan was performed.

[0366] The results showed increased pathological metabolic activity throughout the proximal aortic stent, starting at the level of the mid-aortic arch and extending to the level of the eighth thoracic vertebra, indicating stent infection. Additionally, inflammatory mediastinal soft tissue swelling and left pleural effusion were observed. Figure 14 .

[0367] Due to the severity of the patient's condition and several comorbidities, surgical resection and autologous anatomical reconstruction of the infected stent graft were not feasible. The patient desired an alternative to the uncertain systemic antibacterial therapy. Therefore, in accordance with Article 37 of the Declaration of Helsinki and the requirements of the local ethics committee (A 2021-0132), an experimental approach using local phage application was planned as a last resort.

[0368] Phage therapy

[0369] As a curative treatment strategy, a three-step approach was employed for the extravascular and intravascular application of SniPha 360 (Phage24, Austria). SniPha 360 is a commercially available mixture of lytic phages against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pyogenes, Proteus vulgaris, and Proteus mirabilis.

[0370] After the risks and benefits of the experimental procedure were explained, the patient consented to treatment. First, extravascular treatment was performed via a left thoracotomy. Adhesions between the visceral pleura and the aorta were found. After obtaining a local swab sample for microbiological analysis, debridement and spray irrigation were performed. Then, 20 ml of SniPha 360 (diluted in 100 ml of 0.9% NaCl) was instilled onto the infected para-aortic tissue. Next, two embedded sponges (Endo- B. Braun (Melsungen, Germany) placed them on the lesser and greater curvatures of the aortic arch and on the proximal descending aorta, respectively, followed by placement GRANFOAM TM Dressing sponge (18×12.5×3.2cm, KCI Medizinprodukte GmbH, Wiesbaden, Germany) Figure 2 C). Attach the built-in foam to VACVERAFLO TM Treatment system (KCI Medizinprodukte GmbH). To avoid contact between the sponge and the Lunge, A CNP drainage membrane (25×20cm, Lohman-Rauscher GmbH&Co., Germany) was placed on a sponge. After the chest tube was placed, the chest cavity was closed, and the patient was transferred to a ward requiring intermediate care.

[0371] There, once daily, intrathoracic fluid was drained through VACVERAFLO two hours before phage therapy. TM The treatment system was removed, and the sponge was then rinsed and drained three times with 500 ml of 0.9% NaCl. The internal sponge was then rinsed with 20 ml of SniPha 360 diluted in 100 ml of 0.9% NaCl. Both the internal sponge drainage tube and the chest tube were clamped until the next day. Figure 15 , Figure 16 , Figure 17 .

[0372] As a second step, the patient underwent a second thoracotomy three days later. After removing all vacuum sponges, the aorta and surrounding tissues were covered with 40 ml of SniPha 360 mixed with 15.8% hydroxyethyl cellulose gel. The chest cavity was closed, and the patient was transferred to a intensive care unit.

[0373] The third step was performed three days later. Two sterile RELAY NBS PLUS stent grafts (Vascutek Terumo-Bolton Medical, Vascutek Germay GmbH, Hamburg, Germany) coated with a mixture of 40 ml SniPha 360 and 15.8% hydroxyethyl cellulose gel were then released. The externally coated phage grafts were then reassembled for intravascular placement. This was performed via the left common femoral artery. After an overview angiography, the two grafts were placed with minimal overlap with the infected stent graft. Figure 18 , Figure 19 .

[0374] All invasive procedures went smoothly, and no side effects were observed. Following endovascular treatment, the patient was transferred to a general ward and recovered quickly. Infection parameters decreased, antibiotic treatment was discontinued, and the patient recovered. Signs of vasculitis also disappeared. After four weeks of intensive physical therapy in the hospital, the patient was discharged in good overall condition and began follow-up rehabilitation.

[0375] A PET-CT scan performed three months after phage therapy showed no signs of infection in or around the thoracic aorta. With no detectable infection parameters, the patient continued to recover. Figure 20 .

[0376] discuss

[0377] This case demonstrates that localized phage therapy can successfully treat infected intravascular stent grafts. To our knowledge, this is the first time that phage-impregnated stent grafts have been used for localized intravascular application.

[0378] Bacteriophages are considered effective antibacterial treatments due to their lytic activity. Compared to other antibacterial treatment strategies such as topical rifampicine, bacteriophages do not exhibit cytotoxic effects on vascular cells. 1 Conversely, their advantage lies in their ability to act on both multidrug-resistant bacteria and bacteria in biofilm tissues. Recently, a case series of eight patients with infections from vascular grafts, surgical wounds, or implanted medical devices further demonstrated the feasibility of successfully treating bacterial infections using various phages with lytic activity. 2 Although phages have been used to successfully treat vascular implant infections, phage therapy remains uncommon, and there is no officially recommended infection regimen in the Western Hemisphere.

[0379] In this case, the patient's physical condition and severe comorbidities made them unsuitable for surgical resection of the infected stent graft and thoracic aortic anatomical reconstruction. Staphylococcus aureus graft infection was successfully treated by administering bacteriophages into the vascular lumen and surrounding coagulated tissue. A key challenge of this approach is the intravascular administration of bacteriophages using releasable stent grafts coated with bacteriophages, followed by aseptic reassembly before insertion into the patient. Direct intravascular application of bacteriophages to the site of infection ensures maximum concentration, contact time, and penetration of the bacteriophages into the infected tissue.

[0380] Despite the multiple surgical steps, including two thoracotomies and endovascular stent graft placement, the associated pain associated with each procedure was significantly reduced compared to conventional surgical treatment. The prolonged postoperative hospital stay was due to the patient's pre-existing weakened condition and the need for extensive physical therapy.

[0381] Since the patient's condition improved significantly over time, and a three-month follow-up PET-CT scan showed no signs of infection, the phage therapy can be considered successful. However, continued follow-up is necessary to ensure lasting treatment success.

[0382] In summary, this case report demonstrates that phage therapy may be a curative treatment option for patients unsuitable for major surgical procedures. Of particular importance is that this success is likely solely due to the hydrogel comprising HEC and phage according to the first aspect of the invention, which allows for in-situ localization of the phage at the site of infection and for the continuous release of the phage in a delayed manner.

[0383] Example 3

[0384] Successful treatment of chronic infection and occlusion of the aorta and bifemoral arteries using bacteriophages bypass

[0385] Purpose:

[0386] Graft infection is a serious and devastating complication of vascular surgery. Surgical resection of infected aortic grafts is associated with high mortality and morbidity. Therefore, inadequate antibiotic treatment and alternatives to major surgery are essential.

[0387] Case:

[0388] A 66-year-old patient was diagnosed with chronic occlusion of the aorta and bilateral femoral arteries. The patient was admitted via prosthesis bypass. Following various transfemoral surgical recanalization attempts in the past medical history, unfavorable tissue conditions were observed bilaterally to the femoral arteries, accompanied by chronic wound infection and graft exposure. Local infection was confirmed by PET-CT imaging. Due to the patient's comorbidities, there was no medical indication or reasonable explanation other than prosthesis bypass. Furthermore, we aimed to treat bilateral femoral artery wound healing disorders. Phage therapy was considered an alternative treatment option for intraoperative and postoperative management of graft-associated soft tissue infections. After a repeat laparotomy, the infected aortic prosthesis was removed, and the aorta was sutured. A phage suspension was infused into… The phage-impregnated fabric was then placed retroperitoneally. After the femoral artery anastomosis was completed, the same principle was used to place phage-impregnated fabric on both sides of the femoral artery. The wound was mobilized and closed without further drainage. The patient was discharged after 10 days of hospitalization, subjectively feeling well, with no irritating wound condition and no systemic inflammatory parameters. Three months after the intervention, PET-CT imaging showed no signs of infection around the aorta or both femoral artery regions.

[0389] Summarize:

[0390] This case demonstrates the supportive antibacterial effect of bacteriophages in successful secondary closure of chronically infected femoral artery wounds in high-risk patients undergoing suppurative aortic surgery. Most importantly, it proves the superior efficiency of hydrogels containing HECs and bacteriophages according to the first aspect of the invention, allowing for in-situ localization of the bacteriophages at the site of infection and continuous release of the bacteriophages in a delayed manner. Background Technology

[0392] In vascular surgery, infection of vascular grafts is considered a serious complication. In particular, infection of aortic grafts is associated with a high morbidity and mortality rate of up to 75%. As these procedures are often performed on patients with multiple comorbidities, the removal of infected grafts and the extensive struggle with associated abdominal infections are required, which are associated with early postoperative morbidity and mortality rates of up to 20%. Despite initial successful treatment, the overall reinfection rate can be as high as 20% of cases. This is mainly because bacterial colonies are embedded in the tissue surrounding the prosthesis and then form a biofilm that adheres to the surface, thus exhibiting resistance to antibiotics up to 1,000 times. Even targeted antimicrobial agents suitable for antimicrobial susceptibility testing can only suppress graft infection, but cannot be a curative treatment. The most common pathogens associated with graft inflammation are Staphylococcus aureus, Staphylococcus epidermidis and other coagulase-negative staphylococci, Enterobacteriaceae, Pseudomonas aeruginosa and Corynebacterium[3]. These bacteria periodically enhance their specific virulence by attaching to the prosthetic material and thus avoid local immune responses by forming a biofilm that hinders phagocytosis. Furthermore, systemic antibiotic therapy is often inadequate due to the lack of effective saturation concentrations within the tissue surrounding the inflamed prosthesis. To reduce morbidity and mortality associated with often unavoidable surgical procedures, minimally invasive methods for adequately treating surrounding tissue infections are urgently needed. In this context, bacteriophages and their lytic activity represent a promising therapeutic approach.

[0393] Case

[0394] In November 2020, a 66-year-old male patient was referred to the emergency department by his general practitioner with clinical symptoms of acute abdomen. Examination revealed pervasive tenderness in all abdominal regions, accompanied by lower abdominal peritonitis. SARS-CoV-2 infection was ruled out. Further examination revealed a fever of 39.2°C, a white blood cell count of 9.4 × 10⁹ / L, and elevated serum C-reactive protein of 90.2 mg / L. Chest X-ray showed no signs of pneumonia. Endocarditis was ruled out. Calculated antibiotic treatment with ampicillin / sulbactam was initiated at the standard dose intravenously. Blood cultures were positive for methicillin-sensitive Staphylococcus aureus. Secondary findings included a pre-existing condition of pervasive arterial occlusive disease. Due to the need for multiple vascular surgeries in both legs, the patient had ultimately undergone a right thigh amputation 12 months prior; a long-term obstructive polytetrafluoroethylene (PTFE) Stockmann bypass remained in situ in the left thigh.

[0395] Following a history of various transfemoral surgical recanalization attempts, an unfavorable tissue condition was observed in both femoral arteries, accompanied by chronic wound infection leading to graft material exposure. Wound swabs revealed the presence of Staphylococcus aureus and Escherichia coli, indicating a multimicrobial infection. Peripheral blood flow in the lower extremities was compensated. Initially, an abdominal CT scan was performed, presuming aortofemoral graft occlusion and infection. Subsequent PET-CT scans revealed significantly increased metabolic activity in the graft area, leading to a diagnosis of chronically occluded and infected aortofemoral prosthesis bypass grafts, subsequently resulting in bilateral femoral artery infection and impaired skin wound healing. Figure 21 .

[0396] Due to the patient's comorbidities, we generally aim to minimize surgical and anesthesia time and achieve effective treatment through prosthetic bypass removal. Furthermore, we plan to forgo the planned irrigation of the purulent abdomen and intend to perform primary closure of the abdomen. Phage administration was considered a viable alternative in this case for long-term intraoperative and postoperative treatment of local inflammation in the abdominal and femoral artery regions. The patient preferred an alternative solution compared to indefinite systemic antibacterial therapy. Therefore, in accordance with Article 37 of the Declaration of Helsinki and in accordance with the local ethics committee (A 2021-0208), we wish to use an experimental approach involving local phage application as a last resort.

[0397] Phage therapy

[0398] As a curative treatment strategy, SniPha 360 (Phage24, Austria) was administered both in vivo and in vitro to the abdomen. SniPha 360 is a commercially available mixture of lytic phages against *Escherichia coli*, *Staphylococcus aureus*, *Pseudomonas aeruginosa*, *Streptococcus pyogenes*, *Proteus vulgaris*, and *Proteus mirabilis*. After outlining the potential risks and benefits of the experimental procedure, the patient consented to treatment. During the repeat laparotomy, turbid fluid was present in the abdomen. After initial irrigation, the retroperitoneal space was opened, and preparation was made to clamp the proximal aorta. Aorta-bifemoral artery The prosthesis exhibited a biofilm shell and was embedded in putrid liquid. The infected aortic prosthesis was removed, and the aorta was then sutured. After moving the leg with the prosthesis, it was removed via the femoral artery. A phage suspension was infused into... It was then placed in the retroperitoneal space surrounding the infection. The retroperitoneal space and abdomen were closed primarily without further drainage. After removing the femoral artery anastomosis, the wound was debrided, mobilized, and irrigated with a sharp instrument. Then, following the same principle, phage-impregnated fabric was placed bilaterally on the femoral artery side, and the wound was closed again without further drainage.

[0399] The procedure took 52 minutes with no significant blood loss. The patient was then transferred to the intensive care unit and extubated without the need for catecholamines. Ten days after the intervention, the patient was discharged in good condition, with no irritating wounds and normal blood inflammation levels. Three months post-intervention, PET-CT imaging showed no signs of infection around the aorta or both femoral arteries.

[0400] discuss

[0401] This case demonstrates the successful treatment of chronic inflammatory occlusion of the aorta and bilateral femoral arteries via local phage application. Bypass. In particular, this success is likely due solely to the hydrogel containing HEC and phage according to the first aspect of the invention, which allows the phage to be localized in situ at the site of infection and to be released continuously in a delayed manner.

[0402] It is generally believed that approximately 50% to 65% of prosthesis infections result from bacterial contamination during surgery [3,4]. Infections are typically classified as early (within 30 days post-surgery) and late infection, although this classification is arbitrary [3,4]. Early prosthesis infections are often considered to be the result of intraoperative contamination, while late infection is attributed to bloodborne bacterial transmission, but strong evidence for this is limited. Late infection is usually caused by inadequate tissue integration of the prosthesis into the transplant bed. Common pathogens are Staphylococcus, Enterobacteriaceae, and Corynebacterium [3,4]. Bacteriophages (or simply "phages"; Greek for "bacterial devourer") are viruses that selectively infect bacterial cells and were first described in 1917 by the Canadian Félix Hubert d'Hérelle.

[0403] Currently, bacteriophages are considered effective antibacterial therapies due to their lytic activity. They are quite stable when exposed to inflammatory environments and make important contributions to regulating global bacterial quality. Bacteriophages can only reproduce in the place where their host is located. They are highly specific and therefore mainly affect strains within a single bacterial species, rarely crossing species boundaries [5].

[0404] In the first (lysis) cycle of viral replication, bacteriophages kill their corresponding bacteria through lysis: once infected, the bacterial host cell begins the replication process, destroying the bacteria and releasing new phage particles; this process is controlled by enzymes and the interaction of bacterial and phage genes. In the second (lysis) cycle, the phage nucleic acid is integrated into the host bacterial genome or forms a circular replicon in the bacterial cytoplasm. No cytotoxic effects of phages on vascular cells were observed compared to other antibacterial treatment strategies such as topical rifampin treatment.

[0405] Furthermore, they are effective against multidrug-resistant bacteria and bacteria in biofilm tissues. A recent case series of eight patients with infections of vascular grafts, surgical wounds, or implanted medical devices further demonstrates the feasibility of successfully treating bacterial infections using various phages with lytic activity. Although phages have been used to successfully treat vascular implant infections, phage therapy remains uncommon, and there are no officially recommended infection protocols in the Western Hemisphere. Direct application of phages to the infection site in the retroperitoneal space and intraperitoneum ensures maximum phage concentration, contact time, and penetration into the tissue surrounding the infected graft. We were able to perform surgery quickly, achieving definitive treatment with complete skin / wound closure without any drainage. No phage-related adverse clinical events were detected in our cases. Three-month PET-CT follow-up scans showed no signs of infection. The phage therapy can be considered successful.

[0406] To address localized inflammation in the abdominal and femoral artery regions during and long-term postoperative treatment, we recognized the potential of phage therapy as an alternative to topical antibacterial treatment. However, continued patient follow-up is crucial to ensure sustained treatment success. In conclusion, this case report suggests that phage therapy may be a curative treatment option for patients with bacterial graft-versus-graft infections who are unsuitable for major surgical procedures.

[0407] Example 4

[0408] Testing the activity of bacteriophages in lyophilized products

[0409] method

[0410] To test the activity of phages in lyophilized hydrogels, five lyophilized phage gels were prepared (composition: HEC: 13g = 13%, CaCl2 solution: 43g = 43%, 85% glycerol: 27g = 27%, water: 17g = 17%) (see [reference]). Figure 25 Subsequently, the lyophilized hydrogel was placed in the center of an agar plate containing Staphylococcus aureus (see [link]). Figure 26 ), and incubate at 36℃ for 24 hours.

[0411] result

[0412] The freeze-dried hydrogel was shown to degrade in the gel state, followed by dilution (primarily through water from the culture medium). Phage activity remained, which could be identified by the corresponding bacteria-free (circled) phage plaques on the culture medium (see [link to article]). Figure 27 ).

[0413] Example 5

[0414] Testing phage release in lyophilized hydrogels

[0415] method

[0416] Preparation of freeze-dried products

[0417] Hydrogels B and C used for lyophilization have the following composition:

[0418] HEC: B=7g=7% / C 14g=14%

[0419] CaCl2 solution: 43g = 43%

[0420] 85% glycerin: 27 g = 27%

[0421] Water: B = 10 g = 23% / C = 16 g = 16%

[0422] These phage-containing hydrogels were prepared as sterile gels and also lyophilized in a sterile environment.

[0423] The following lyophilized products were prepared and tested as described below:

[0424]

[0425] *BPh-Buffer > 5 × 10 11 PFU / ml

[0426] Bph-Buffer: Tris buffer containing sodium chloride, magnesium chloride (x 7 H2O) and Tris-HCl, pH=7.4.

[0427] Among products B1, B2, C1 and C2, C1 and C2 are the most solid formulations due to their gelling agent content (double the amount of HEC), while the two variants of B and C (B2 and C2) contain double the concentration of BPh.

[0428] Release test

[0429] First, freeze-dried products B1, B2, C1, and C2 were each released into 100 ml of saline solution, i.e., sterile physiological saline solution (0.9%), for 2 hours. Therefore, the saline solution used was pre-sterilized by aseptic filtration. Further equipment, such as bottles and stir bar, was pre-sterilized by steam sterilization.

[0430] Over the next 2 hours, samples were drawn at a rate of 1 ml every 15 minutes, without changing the sample volume. Samples were diluted if necessary. Subsequently, samples were allocated onto fixed Staphylococcus aureus plates and incubated at 37°C for 24 hours. In summary, each gel variant was run twice (n=2).

[0431] Release test results

[0432] In summary, the results indicate that phages derived from freeze-dried hydrogels exhibit a strong delayed release (see [link to study]). Figure 29 ).

[0433] Furthermore, the curves show that higher proportions of gel-forming agents (C1 and C2) exhibit a stronger delayed release compared to lower proportions of gel-forming agents (B1 and B2). Additionally, lower phage concentrations in the starting gels (B1 and C1) also showed lower phage release per unit time compared to higher phage concentrations (pfu / ml) in the starting gels (B2 and C2).

[0434] Example 6

[0435] A clinical comparison of phage mixtures with Galen vectors for the treatment of atypical acne with gold standard.

[0436] summary:

[0437] Acne paradoxica is a chronic skin condition. Painful inflammation occurs in the hair follicle areas. This can result in abscesses, fistulas, and nodular scarring. Inflammation primarily occurs in the armpits and groin, anal and genital areas.

[0438] In this treatment attempt, in accordance with Article 37 of the Declaration of Helsinki, a hydrogel containing HEC and phage was applied to treat the finding in the left axilla, while a clinically similar finding on the right buttock was treated with a routine open wound.

[0439] In both the short and long term, phage therapy has significant clinical advantages.

[0440] introduce :

[0441] The patient was referred by their family doctor with suspected paradoxical acne in multiple locations, particularly the left armpit and right buttock (Hurley stage II / III). The patient had undergone axillary surgery in 1997 and posterior thigh surgery in 2011 for paradoxical acne.

[0442] Since last year, despite treatment with adalimumab (Hyrimoz) 40mg, cefpodoxime 200mg, and topical fusidic acid (Fucidine), the patient's condition has continued to worsen. The patient experiences severe pain, is almost unable to sit up, experiences pain and limited mobility in the right arm when rotating it. Intermittent fever has been reported. Related medical conditions include hypertension, a factor V Leiden mutation, Zn pulmonary embolism (1996, 1998, 2003), and nicotine abuse.

[0443] Upon admission, the patient presented with inflammatory nodules tender to pressure in the armpit, groin, and especially the right buttock area, along with abscesses, fistulas, and scarring, equivalent to Hurley stage II / III (see [link to relevant documentation]). Figure 31 Laboratory blood tests revealed an elevated white blood cell count, with a WBC count of 21 × 10⁻⁶. 9 The patient's blood count was 1 / L, and CRP was 30 mg / L (3 mg / dL). No wound swabs were collected.

[0444] method:

[0445] According to Article 37 of the Declaration of Helsinki, the patient was admitted for surgical care in conjunction with a planned phage injection as an attempt to heal. Local anesthesia was administered by subcutaneous injection of 20 ml of 1% Scandincain into the area of ​​the surgical axillary incision (see [link to relevant documentation]). Figure 31 (Top left). After the infected soft tissue was removed en bloc through two separate incisions, 20 ml of a viscous phage galenine preparation was injected subcutaneously into the wound surface (see [link]). Figure 31 (Top right and bottom left), the formulation contains ingredients from Phage The company's phage mixture SniPha360, which is resistant to Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, and Proteus mirabilis, has a concentration of at least 1 × 10⁻⁶. 5 The phage concentration (composition of galenine preparation: HEC: 13g = 13%, CaCl2 solution: 43g = 43%, 85% glycerol: 27g = 27%, water: 17g = 17%) was then mobilized into the skin and primary closure was performed without further drainage (see [link to original text]). Figure 31 (Lower right). During hospitalization, only dry dressings were changed. Sutures were removed 12 days after OR treatment.

[0446] Conversely, due to the persistent pain associated with local anesthesia, general anesthesia is required. In this case, open excision and open wound treatment were chosen as the gold standard for treating paradoxical acne on the right buttock. The VAC pump used during the procedure was checked regularly and removed after 5 days. The wound was then rinsed daily until it was clean and non-irritating before discharge. Further procedures included daily cleaning and rinsing of the wound with Octenesept solution and applying wet dressings until the wound was completely scabbed over.

[0447] result:

[0448] The patient was transferred to the outpatient department for clinical follow-up at 2 and 10 weeks after surgical intervention. The right axillary wound remained clean and dry, with a 1 mm puncture hole in the center of the larger incision (see [link to original text]). Figure 32 (Left), the wound closed without irritation 10 weeks after surgery (see...) Figure 32 (Right). At the initial wound examination two weeks later, the buttock wound appeared as granulation tissue but was covered with fibrin (see [link]). Figure 33 (See above). With continued wound treatment, a granulation-like scar developed 10 weeks post-surgery (see above). Figure 33 ,Down).

[0449] discuss:

[0450] Acne paradoxically is a painful, chronic inflammatory skin condition that primarily affects the armpits, groin, pubic region, and anus. The disease typically begins with inflammation of the hair follicles and then spreads as recurrent inflammation and abscesses. It is estimated that about 1% of the population is affected. A large number of bacteria, particularly Staphylococcus aureus, can be found in the purulent lesions of acne paradoxically / hidradenitis suppurativa. The disease is often diagnosed at an advanced stage. Those affected often withdraw due to fear and shame, which can disrupt their careers, personal and social relationships, and lead to depression.

[0451] Based on severity, Hurley grades I through III are defined, with affected patients exhibiting recurrent purulent wounds. Individual inflammatory nodules or abscesses are often present in the surrounding healthy tissue, which is common in patients classified as Hurley grade I. Grade II is characterized by progressive, well-defined inflammation with scarring and fistula formation. Grade III is characterized by numerous, interconnected abscesses and fistulas.

[0452] • Hurley I: Local or systemic administration of antibiotics, and surgical excision of individual wounds / areas if necessary.

[0453] ●Hurley II: Systemic administration of antibiotics, administration of antibodies, surgical excision of individual wounds, and, if necessary, removal of superficial tissue layers using laser.

[0454] ●Hurley III: Systemic administration of antibiotics, administration of antibodies, and complete surgical removal of the affected tissue.

[0455] For postoperative wound care of Hurley grade II to III, guidelines recommend against suturing the wound and instead allow it to heal "openly." Appropriate wound dressings, and if necessary, negative pressure wound therapy (NPWT), should be used to cover the "open" wound.

[0456] This example impressively demonstrates the efficacy of phage application compared directly to conventional treatments. No significant abnormalities were observed in early hospitalized patients or in the axillary region treated with phage in terms of postoperative course, while a complex, lengthy, and aesthetically unappealing healing process was recorded in the gluteal wound area.

[0457] The successful use of phage mixtures as part of a phagocytic profile without prior susceptibility testing can be attributed to the use of different phages. Using multiple phages improves susceptibility and also targets mixed infections. Subcutaneous administration of standardized phage solutions bound to a carrier increases local residence time and local phage concentration during the initial phase of infection treatment. In addition to the phage solution, a gel-gallen formulation is used as the application medium to prevent rapid outflow of the phage solution from the target area. In this respect, the gel-gallen formulation acts as a reservoir for the phage. Application of liquid solutions is considered less efficient because phage concentrations cannot be optimally maintained locally.

[0458] The application of phages in the hydrogel form according to the present invention has proven to be a patient-friendly, inexpensive, side-effect-free, and rapid treatment method, achieving excellent results in current treatment trials. From a medical-economic perspective, the advantages of phage application can be seen due to the significantly different healing process.

[0459] Summarize:

[0460] In this case report, the application of a phage mixture with a carrier defined by the Galen formulation (which together form the hydrogel of the present invention) was superior to conventional treatment for type II / III paradoxical acne according to Hurley.

[0461] Example 7

[0462] Comparison of wound healing caused by subcutaneous bacterial infection in mice treated with various forms of phage-containing hydrogels

[0463] introduce

[0464] Subcutaneous injection of Staphylococcus aureus in healthy mice typically leads to bacterial infection, resulting in open wounds. Therefore, reducing the bacterial load is crucial for wound healing, as bacterial infection can cause and exacerbate skin damage. In particular, wounds generally close more quickly when fewer bacteria are present. During infection, healing of these wounds supported by treatment with the hydrogel according to the invention results in reduced wound adhesions until complete healing and full wound closure.

[0465] Therefore, reduced wound adhesions are an indirect indicator of the corresponding treatment effect. Thus, time and the trend of wound reduction over time are indirect but reliable scientific readings for the mouse model used.

[0466] describe:

[0467] In the mouse model, five groups of mice, each with 22 mice, were tested. On day one, all mice were subcutaneously infected with the same amount of Staphylococcus aureus. Therefore, under ketamine / toluidine anesthesia, 100 μL of a bacterial suspension containing an appropriate number of colony-forming units (CFU) was subcutaneously injected into the shaved area.

[0468] On day 3, all mice showed improvement in wounds treated with the following substance:

[0469] I HEC in the composition na 0.5 ml / mouse II bacteriophage solution <![CDATA[10 5 pfu / ml]]> 0.5 ml / mouse III The ratio of phage solution to HEC in the composition is 1:1. <![CDATA[10 5 pfu / ml]]> 1.0 ml / mouse IV The ratio of phage solution to HEC in the composition is 1:2. <![CDATA[10 2.5 pfu / ml]]> 1.0 ml / mouse V Untreated na

[0470] (HEC composition: HEC (hydroxyethyl cellulose): 13g = 13%, CaCl2 solution*: 43g = 43%, 85% glycerol: 27g = 27%, water: 17g = 17%)

[0471] Therefore, the phage solution used is SniPha 360 phage, i.e., phage solution Sanubiom.

[0472] The formulations for Groups III and IV were prepared as follows: A sufficient volume of HEC from the composition was placed in a first Omnifix Luer lock syringe, while a second Omnifix syringe was filled with the phage solution. Both syringes were connected to the Omnifix adapter, and the phage solution was pushed into the carrier. The formulation was thoroughly mixed by pushing the solution back and forth between the two syringes through the connector at least ten times.

[0473] Over the next few days, corresponding wound adhesions were measured (based on the length and width of each lesion), averages were calculated, and trends were analyzed. For ratio assessment, the slope of the phage solution group was used as the comparison group, such that the slope ratio = 1. Testing results were maintained until day 10.

[0474] This is because the subsequent immune system overlaps with the healing effects of the corresponding products.

[0475] result:

[0476] The results demonstrate the superiority of the gel-phage combination:

[0477]

[0478] This was particularly evident in group IV, which exhibited the best overall slope and a 2.5-fold better trend slope compared to group II. Nevertheless, it maintained only half the phage concentration compared to groups II and III. Therefore, in terms of the trend toward reduced wound adhesions, group I was 1.5 times better, and group II was approximately 2 times better. Group II was comparable to the untreated group V.

[0479] The aforementioned trends indicate that the phage-gel combination is superior to the untreated group and the phage solution group (Group II).

[0480] Many products are used for wound treatment, but the composition described herein does not contain HEC gel. This test showed that the HEC in the composition not only adequately carries the phage at the application site but also adheres to it and releases the phage in a correspondingly delayed manner. Furthermore, it promotes wound healing: in the composition, the HEC gel alone (Group I) without the phage reduced the wound area faster than the phage solution, with a reduction trend 1.5 times greater. This effect can be explained, in part, by the water content and the properties of the hydrophilic gel. Moreover, the adhesive properties of HEC in the composition described herein are also evident: wounds moistened with hydrogel healed better in mice than those in the phage solution group due to the tissue-friendly composition, which is also attributed to the physical shielding against external factors.

[0481] In Group II, when the phage solution was applied to the wound, it rapidly leaked out. Even covering the wound with appropriate wound coverings could not prevent this or increase the likelihood of phage-bacteria interaction. This is clearly demonstrated by the comparable values ​​between the phage solution group (Group II) and the untreated group (Group V).

[0482] Compared to group II (phage solution), groups III and IV showed a wound reduction rate of 2 to 2.5 times that of group II. Group IV is particularly noteworthy, carrying only half the phage concentration compared to groups II and III. The properties of HEC in the composition are most evident here. Wound healing is accelerated due to the explained properties. Sustained phage release increases phage-bacteria interaction.

[0483] Phage solutions (Group II) and Group III were prepared with the same amount of phage solution, where differences related only to the vector became significant: Group III was more than twice as good (=fast) in wound healing. Furthermore, the results showed that the greater the proportion of vector, the stronger the delayed release effect: Group IV showed the fastest wound healing rate, with a phage concentration of only 10... 2.5 pfu / ml.

[0484] The results showed that, using the HEC described in the composition, only when the phage concentration was 10... 5 Treatment success was only demonstrated at pfu / ml (Group III), which is twice that of phage solution (Group II).

[0485] References for Example 2:

[0486] 1.Szilagyi DE, Smith RF, Elliott JP, Vrandecic MP (1972) Infection inarterial reconstruction with synthetic grafts. Ann Surg 176:321-333.

[0487] 2.Zühlke HV, Harnoss BM, Lorenz EP (1994) Postoperative Infektionen inder In:Septische Blackwell Wiss Verlag.

[0488] References for Example 3:

[0489] [3] Kirklin JK, Pagani FD, Kormos RL, Stevenson LW, Blume ED, Myers SL, et al. Eighth annual INTERMACS report: Special focus on framing the impact of adverse events. The Journal of Heart and Lung Transplantation 2017;36:1080–6.

[0490] [4] Kim J, Feller ED, Chen W, Liang Y, Dilsizian V. FDG PET / CT for Early Detection and Localization of Left Ventricular Assist Device Infection: Impact on Patient Management and Outcome. JACC Cardiovasc Imaging 2019; 12: 722–9.

[0491] [5] Baddour LM, Wilson WR, Bayer AS, Fowler VG, Tleyjeh IM, Rybak MJ, et al. Infective Endocarditis in Adults: Diagnosis, Antimicrobial Therapy, and Management of Complications: A Scientific Statement for Healthcare Professionals From the American Heart Association. Circulation 2015; 132: 1435–86.

[0492] References for Example 7:

[0493] 6 - F. Altamirano, J. Barr - Phage Therapy in the Postantibiotic Era - Clin Microbiol Rev. 2019 Jan 16; 32(2): e00066 - 18. doi: 10.1128 / CMR.00066 - 18.

[0494] 7 - D. Malik, I. Sokolov, G. Vinner, F. o Mancuso, A Kirpichnikova - Formulation, stabilisation and encapsulation of bacteriophage for phage therapy. Adv Colloid Interface Sci. 2017 Nov; 249: 100 - 133. doi: 10.1016 / j.cis.2017.05.014. Epub 2017 May 14.

[0495] 8-L.Kasman,J.Norris-Overcoming the Phage Replication Threshold:aMathematical Model with Implications for Phage Therapy-Journal ofVirology2002Vol.76,No.11, doi.org / 10.1128 / jvi.76.11.5557-5564.2002

[0496] 9–E.Morello,L.Debarbieux-Pulmonary Bacteriophage Therapy onPseudomonas aeruginosa Cystic Fibrosis Strains:First Steps Towards Treatmentand Prevention–2011plos on collection psychiology,doi.org / 10.1371 / journal.pone.0016963

[0497] 10–H.madavi,S.padmanabhan-Therapeutic Potential ofStaphylococcalBacteriophages for Nasal Decolonization of Staphylococcus aureus in Mice-Advances in Microbiology Vol.3 No.1(2013),Article ID:29179,9 pages DOI:10.4236 / aim.2013.31008

[0498] 11–D.Rhoads,A.Sulakvelidze-Bacteriophage therapy of venous leg ulcersin humans:results of a phase I safety trial– Journal of wound care 2013,Vol.18,No.6,doi.org / 10.12968 / jowc.2009.18.6.42801

Claims

1. A hydrogel comprising 3% to 10% hydroxyethyl cellulose (HEC), bacteriophage, 10% to 33% CaCl2, and 6% to 22% glycerol, wherein the concentration of the bacteriophage is 10%. 2 pfu / ml up to 10 8 pfu / ml.

2. The hydrogel of claim 1, wherein the hydrogel contains 15% to 25% CaCl2.

3. The hydrogel of claim 1, wherein the hydrogel contains 17% to 23% CaCl2.

4. The hydrogel of claim 1, wherein the hydrogel contains 20% to 22% CaCl2.

5. The hydrogel of claim 1, wherein the hydrogel contains 21.5% CaCl2.

6. The hydrogel of claim 1, wherein the hydrogel contains 5% to 8% HEC.

7. The hydrogel of claim 1, wherein the hydrogel contains 6% to 7% HEC.

8. The hydrogel of claim 1, wherein the hydrogel contains 6.5% HEC.

9. The hydrogel of claim 1, wherein the hydrogel contains 8% to 18% glycerol.

10. The hydrogel of claim 1, wherein the hydrogel contains 10% to 16% glycerol.

11. The hydrogel of claim 1, wherein the hydrogel contains 12% to 15% glycerol.

12. The hydrogel of claim 1, wherein the hydrogel comprises 13.5% glycerol.

13. The hydrogel of claim 1, wherein the hydrogel further comprises a buffer.

14. The hydrogel of claim 1, wherein the viscosity of the hydrogel is from 1,000 mPas to up to 100,000 mPas.

15. The hydrogel of claim 1, wherein the viscosity of the hydrogel is from 10,000 mPas to 80,000 mPas.

16. The hydrogel of claim 1, wherein the hydrogel is sterile.

17. The hydrogel of claim 1, wherein the hydrogel has been steam sterilized.

18. Use of the hydrogel according to any one of the preceding claims in the preparation of a medicament for treating or preventing bacterial infections.

19. The use according to claim 18, wherein the hydrogel is applied during surgical procedures.

20. The use according to claim 19, wherein the hydrogel is used for treating and / or preventing bacterial infection of the implant.

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