Hydroxyethyl cellulose gel composition containing bacteriophage

Abstract

The present invention provides a hydrogel comprising hydroxyethyl cellulose (HEC) and bacteriophage. The present invention provides a hydrogel or a lyophilized product of the hydrogel for use in a method for treating and / or preventing bacterial infection. The present invention provides a method for preparing a hydrogel comprising hydroxyethyl cellulose (HEC) and bacteriophage.

Description

Technical Field

[0001] The present invention relates to a hydrogel containing hydroxyethyl cellulose (HEC) and bacteriophage, and its lyophilized product, and the use of such a hydrogel or lyophilized product, particularly the use of such a hydrogel or lyophilized product in the treatment and prevention of bacterial infections.

[0002] As antibiotic resistance increases, bacterial infections have generally become a problem over the past few years. This usually affects all medical fields, but particularly when a larger surface of the patient's body comes into contact with foreign objects.

[0003] This basically applies, for example, when large wounds due to surgery, particularly skin surgery, are exposed to contaminated air, suture materials, or wound dressings. For example, surgically removing damaged skin or tissue from patients suffering from inverse acne or pressure ulcers / bedsore ulcers often leaves large wounds. These usually heal slowly and have a very high risk of repeated bacterial infections, and this risk is further increased because they need to heal in an open state after wound debridement. Furthermore, postoperative bloodstream infections caused by existing peripheral wounds such as diabetic chronic wounds can also cause serious problems.

[0004] Bacterial infection of vascular grafts has become a major burden in cardiovascular medicine, associated with increased morbidity and mortality. Various factors related to this medical field, such as patient frailty, biofilm formation, or immunosuppression, have an adverse effect on antibiotic treatment, thereby inhibiting the success of treatment. Therefore, additional treatment strategies are needed. The antibacterial properties of bacteriophages were discovered 100 years ago, but the focus on antibiotics in Western medicine since the mid-20th century has delayed the further development of bacteriophage therapy. As a result, much of the experience and knowledge gained up to that point regarding the mechanism of action, handling, clinical use, and limitations of bacteriophages was lost. However, the concurrent emergence of drug resistance and personalized medicine has created a need for additional means of treating bacterial infections.

[0005] This is particularly applicable to the treatment of bacterial infections in surgery. In particular, cardiovascular repairs in open-heart surgery and endovascular surgery both carry a risk of nosocomial infection, and prosthetic graft infection is generally a prolonged, acute disease that is often life-threatening in association with other complications. The morbidity rate of nosocomial infection in surgical patients ranges from 4% to 15% in patients requiring intensive care. Standard treatments of resection and autologous vascular reconstruction are very risky in immunocompromised patients, so alternative therapies are urgently needed. Furthermore, prosthetic graft infection is one of the most feared complications in cardiovascular surgery, associated not only with increased hospital costs but also with significant morbidity and mortality. The most common pathogens are Staphylococcus aureus, Staphylococcus epidermidis, other coagulase-negative staphylococci, enterobacterales, Escherichia coli, Pseudomonas, and corynebacteria. Bacteria often attach to prosthetic materials and, with the help of biofilms that inhibit phagocytosis and current standard treatments, protect themselves from the local immune response and antibiotics, enhancing their pathogenicity. Furthermore, systemic antibiotic therapy is often inadequate because effective saturation concentrations are rarely achieved in the inflamed tissue around the prosthetic vascular graft.

[0006] Bacteriophages (or simply "phages", from the Greek for "bacteria eater") are viruses that selectively infect bacterial cells and were first described in detail in 1917 by Felix Hubert d'Herelle in Canada. They are very stable in the environment and contribute significantly to regulating the amount of bacteria throughout the world. In principle, bacteriophages can be found wherever the corresponding bacteria are present, but they can only grow where a host is present. Bacteriophages are specific and, in most cases, affect only strains within one bacterial species and rarely cross species boundaries. When a lytic bacteriophage initiates the lytic cycle of virus propagation, the phage kills the corresponding bacteria by lysis, and when a bacterial host cell is infected, it then begins the process of replication, bacterial destruction, and release of new phage particles, which is controlled by enzymes and the interaction between the bacteria and phage genes. On the other hand, lysogenic phages initiate the lysogenic cycle, and the nucleic acid of the bacteriophage is either integrated into the genome of the host bacterium or forms a circular replicon in the cytoplasm of the bacterium. Here, the lytic cycle is initiated by external triggers such as ultraviolet light or temperature sensitivity.

[0007] Since bacteriophages are dynamic biological agents that grow within host bacteria, they impose various requirements on clinical research, and the regulatory approval process becomes complex because it is more suitable for passive and chemically defined drugs such as antibiotics. The therapeutic use of bacteriophages is common in countries of the former Soviet bloc, but lacks randomized placebo-controlled double-blind clinical trials that provide scientifically usable data to enable regulatory approval in the Western Hemisphere. There are increasing case reports of successful phage therapy for life-threatening infections. However, although some clinical studies have been conducted, most have been unable to provide clear evidence of the effectiveness of phage therapy, and one study evaluating phage cocktails against Escherichia coli and Pseudomonas aeruginosa (P. aeruginosa) infections in burns was terminated early due to insufficient effectiveness.

[0008] Only bacteriophage solutions are currently available, and due to adhesion and hydrodynamics, topical application is difficult. Even if bacteriophages are successfully delivered to the site of infection, especially when applied circularly to an infected anastomosis etc., they may flow into the surrounding tissues and onto the tissue. This usually does not pose a risk of local or systemic adverse reactions, but the effectiveness is reduced. For example, the preservation of bacteriophages on the surface of a prosthesis is not known. To ensure the interaction between bacteriophages and bacteria, it is essential to keep the bacteriophages at the application site for a sufficient time, and semi-solid and solid formulations are required.

[0009] In view of the above, it remains an object in the art, for example, to provide an antibacterial composition that can be applied during surgery.

[0010] This problem has been solved by the present invention which provides a hydrogel comprising hydroxyethyl cellulose (HEC) and bacteriophage in a first aspect.

[0011] The inventors have surprisingly discovered that a hydrogel containing hydroxyethyl cellulose (HEC) and bacteriophage exhibits excellent properties compared to other hydrogels. Specifically, among the most preferred gel forms tested, hydroxyethyl cellulose (HEC) was unexpectedly found to be the best gel former. In contrast to previous studies, the hydrogel containing hydroxyethyl cellulose (HEC) provides a solid gel and, at the same time, has been found to be easily applicable and spreadable without pain, for example, on the skin of a patient. In contrast to other gels tested, this hydrogel also showed a hardness that could be applied to the thickness of individual layers. In previous studies, the mesh size of the hydrogel containing HEC was too small and the HEC-based gel was considered not suitable for phage release applications because of insufficient release of infectious phages (see, for example, Chang et al., Int J Pharm. August 10, 2021; 605: 120850). In contrast, the inventors of the present invention have surprisingly been able to show in vitro and with an extended access range that the hydrogel of the present invention containing HEC not only exhibits the ability to retain phage but also can delay and continuously release phage. This was shown indirectly in the extended access range and animal experiments and directly in the lyophilized product experiments. Furthermore, it has been found that the phage maintains its lytic activity while being retained in the HEC-containing hydrogel according to the present invention.

[0012] The following medical uses relate to the success of treating patients who are not suitable for extensive surgical approaches, patients who did not show a favorable response to treatment according to general guidelines (e.g., patients who did not show a favorable response to the administration of conventional antibiotics due to antibiotic resistance), or patients with a bacterial infection in a difficult-to-access location (e.g., within an aortic stent, TEVAR stent graft, or biofilm).

[0013] From the viewpoints of tactile sensation, a good and uniform sense of protection on the skin, and ease of application, and regarding the desired layer thickness, the advantages of the hydrogel containing HEC were focused on in the following medical applications. In particular, the hydrogel of the present invention containing HEC showed convincing results from the viewpoint of phage release for preventing bacterial infection.

[0014] The success of the treatment was related to bacterial infection in the field of cardiovascular medicine. First, patients with a TEVAR stent graft were successfully treated by applying bacteriophages both outside and inside the blood vessels (Example 2).

[0015] Second, patients with a chronically infected and occluded aorta bilateral femoral artery Dacron (registered trademark) bypass also had successful treatment with bacteriophages (Example 3). In this example, the infected implant was clinically covered with a bacterial biofilm, but it could not be completely removed mechanically (manually). This shows a great advantage of the treatment of bacterial infection using the hydrogel according to the first aspect of the present invention. Since the hydrogel has the ability to localize in the biofilm in combination with the delayed release of bacteriophages, effective and continuous long-term treatment of the biofilm and the accompanying infectious diseases has become possible. This also further supports the option of systemic antibiotic combination therapy with the hydrogel of the present invention.

[0016] In the next step, the hydrogel containing HEC and bacteriophages was tested in the situation of skin surgery. Patients suffering from inverse acne showed an improvement in wound healing after surgery when treated with the hydrogel containing HEC and bacteriophages, as compared with the previous general wound treatment (Example 6). The above excellent results of the hydrogel containing HEC and bacteriophages in wound healing were supported by a bacterial infection mouse model (Example 7). Finally, it can be further shown that the lyophilized product of the hydrogel containing HEC and bacteriophages also shows the release and activity of the desired bacteriophages (Examples 4 and 5).

[0017] In summary, the inventors were able to disprove the prior art and show that hydrogels containing HEC are excellent tools that can retain phages, maintain their lytic activity, and release phages in a delayed manner.

[0018] The inventors can further disprove the general guidelines regarding implant removal and reimplantation. In contrast to the guidelines, the newly developed method using the hydrogel or lyophilizate according to the present invention can hold the infected implant in situ and enable minimally invasive surgery to enhance antibiotic sensitivity.

[0019] Also, in the field of biofilms, the hydrogel or lyophilizate according to the present invention is advantageous compared to previously applied methods. The hydrogel or lyophilizate according to the present invention adheres to the surface of the biofilm and enables the destruction of the biofilm by releasing phages in a delayed manner.

[0020] Compared to the treatment methods using the aqueous and individual phage solutions applied so far, the hydrogel or lyophilizate according to the present invention has shown that the wound healing in mouse experiments was significantly improved by 2.5 times. The treatment was successful not only on the skin but also in the body.

[0021] Furthermore, instead of individual treatment methods, the hydrogel or lyophilizate of the present invention provides a standard treatment using a standard phage cocktail for treating and / or preventing bacterial infections.

[0022] Finally, the hydrogel or lyophilizate of the present invention showed all of these advantages without showing any undesirable side effects, even when injected intravascularly.

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

[0024] In the context of the present invention (see Example 1), the inventor has discovered that HEC is a particularly suitable gelling agent for preparing gels with desired tactile and other properties. In particular, the hydrogels of the present invention can be steam sterilized, which is an important property for their intended use in the treatment of bacterial infections, particularly during surgery.

[0025] Furthermore, as shown in Example 2, the claimed composition can be shown to be particularly effective in the treatment of bacterial infections when applied during surgery.

[0026] In the context of the present invention, the terms "phage(s)" and "bacteriophage(s)" are used interchangeably.

[0027] In the context of the present invention, the following abbreviations are used.

[0028]

Table 1

[0029] As mentioned above, bacteriophages are well-known antibacterial agents. In the context of the present invention, any type of bacteriophage can be used. It is also possible to prepare a customized gel formulation that determines the type of bacteria to be treated and selects the corresponding bacteriophage.

[0030] In another embodiment of the present invention, a standardized bacteriophage composition that is active against a preselected type of bacteria is used. For example, the bacteriophages contained in the hydrogels of the present invention can be active against Staphylococcus aureus, Staphylococcus epidermidis, other coagulase-negative staphylococci, Enterobacteriaceae bacteria, Escherichia coli, Pseudomonas, and Corynebacterium. Such bacteriophages are known in the art as described above.

[0031] In the context of the present invention, it is preferred to use lytic bacteriophages. This has the advantage that the antibacterial effect of the bacteriophage is initiated immediately upon encountering the corresponding bacteria. The bacteriophages contained in the hydrogel according to the first aspect can be heat-stable and / or freeze-thaw stable. When the bacteriophages contained in the hydrogel according to the first aspect are freeze-thaw stable, they are preferably stable even at low temperatures, for example, at about minus 90 °C to minus 20 °C. In another preferred embodiment, the bacteriophages in the hydrogel of the present invention are heat-stable. In another preferred embodiment, the bacteriophages are freeze-thaw stable.

[0032] The concentration of phages in the hydrogel may be any concentration effective for the treatment of bacterial infections and may vary depending on the bacteriophage used and the expected bacterial concentration.

[0033] For example, a phage concentration of at least 10 2 pfu / ml may be used.

[0034] In one embodiment, the concentration of bacteriophages is between 10 2 pfu / ml and 10 8 pfu / ml, preferably between 10 4 pfu / ml and 10 8 pfu / ml, or between 10 5 pfu / ml and 10 7 pfu / ml.

[0035] Also, in a preferred embodiment, the concentration of bacteriophages in the hydrogel is between 10 2 pfu / ml and 10 8 pfu / ml, preferably between 10 2 pfu / ml and 10 7 pfu / ml, more preferably between 10 2 pfu / ml and 10 6 pfu / ml, most preferably between 10 2 pfu / ml and 10 5.5 pfu / ml.

[0036] Before adding the phage to the composition of the present invention, the bacteriophage is preferably sterilized, more preferably steam sterilized. The initial phage concentration may decrease accordingly during the sterilization process. This decrease in the phage amount is pre-calculated so that the final product has a phage concentration of at least 10 2 pfu / ml.

[0037] In another preferred embodiment, the bacteriophage is sterilized by sterile filtration.

[0038] The bacteriophage may be added to the hydrogel of the present invention in a solution state. For example, when preparing the hydrogel of the present invention, the bacteriophage solution may be 50%, and the HEC gel stock solution (optionally containing other components, see below) may also be 50%.

[0039] The bacteriophage preparation can be manufactured according to the corresponding GMP guidelines.

[0040] The hydrogel of the present invention is a hydroxyethylcellulose (HEC)-containing hydrogel.

[0041] Hydroxyethylcellulose (=HEC) has the following chemical formula.

[0042]

Chemical formula

[0043] (Source: https: / / apothekenwiki.com / wp-content / uploads / 2017 / 12 / Hydroxyethylcellulose-Strukturformel.png)

[0044] HEC is known in the art. In the context of the present invention, HEC of any suitable length 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.

[0045] In the context of the present invention, HEC is used as a gelling agent because it can be shown in the context of the present invention that HEC provides a stable gel scaffold based on water. The gel matrix formed by HEC provides sufficient space to hold phages at a defined concentration in a distribution that promotes release. Furthermore, since the viscosity of the final product can be easily adjusted by the concentration of the gelling agent, a change in the HEC percentage expands the possibilities of further products even with the same material composition. Furthermore, HEC is well-known in pharmaceutical applications and has low allergenicity.

[0046] Among other gelling agents commonly used in the pharmaceutical industry, as shown in the attached examples, HEC is the only gelling agent that can be steam sterilized. Since this sterilization process increases the viscosity of the gel, this "viscosity increase" can be taken into account when preparing the composition of the present invention. Under the further influence of body temperature / skin temperature, the product moisture is reabsorbed from the interstitial body moisture, so the product may float at the application site. Phages are released through this process of becoming aqueous through various viscosity levels and continue to spread at the application site, so they act with a small dose.

[0047] Another reason for using hydroxyethyl cellulose is that the gelling agent is less sensitive, and thus the final product is less sensitive to pH changes and electrolytes. The production of HEC gels and likewise the production of phage HEC gels are easy, inexpensive, and rapid for both manual production and industrial-scale handling. No special equipment or surfaces are required.

[0048] The hydrogel of the present invention preferably contains 3-10% of HEC, more preferably 5-8% of HEC, or 6-7% of HEC, and most preferably 6.5% of HEC.

[0049] In a preferred embodiment, the hydrogel of the first aspect contains 3% to 10% of HEC, preferably 5-9% of HEC, more preferably 6-9% of HEC, particularly preferably 6% to 7% of HEC, and most preferably 6.5% of HEC.

[0050] The hydrogel of the present invention can be prepared by mixing the individual components and adding water to obtain the final concentration. In principle, the preparation of HEC hydrogels is known in the art.

[0051] The production of the gel can be carried out manually or by a technical process, which is known in the art. For example, the dissolved components may be dissolved in the first portion of water. Then, while stirring moderately, HEC may be mixed with water. Further, while stirring moderately, at least the remaining required water may be added to the gel mass. After preparing the hydrogel, the hydrogel may be sterilized, preferably by steam sterilization.

[0052] The bacteriophage may be added to the gel, for example, in the form of a bacteriophage solution. Mechanical force may be required to dissolve the phage in the gel matrix. This may be done by stirring the composition while adding the bacteriophage solution, or by the two-syringe-technique, i.e., the first syringe contains the bacteriophage solution and the second syringe contains the gel. By first pushing the phage solution into the gel, the gel and the bacteriophage solution can be combined via a connector. This process may be repeated several times in the opposite direction.

[0053] In a preferred embodiment, the hydrogel further contains CaCl2 and glycerol.

[0054] Calcium can help stabilize and maintain the activity of bacteriophages. Calcium may be added to the composition in the form of calcium chloride, which is easily soluble, but other calcium salts may also be used. By dissolving CaCl2 in the composition, especially the water contained therein, calcium becomes available to the phage as calcium ions (Ca 2+ 2+). Calcium chloride itself has only low chemical reactivity, does not affect the adjusted pH of the product, and at the amounts described below (for example, concentrations between 10% and 33%), has no effect on the osmosis process.

[0055] The hydrogel of the present invention may contain 10 to 33%, preferably 15 to 25%, or 17 to 23%, more preferably 20 to 22%, and most preferably 21.5% of CaCl2.

[0056] Glycerol can serve as a lubricant, spreading agent, and adhesion promoter, so it can be used to enhance adhesion properties and delay the liquefaction of the product under the influence of body temperature and body fluids. In addition, since glycerol is hygroscopic, it not only supports the systematic degradation and phage release on the surface of the applied PhaTEC formulation, but also contributes to the degradation without product residues. By containing glycerol, as in the case of suture threads, it becomes a self-absorbable product.

[0057] The hydrogel of the present invention contains 6 to 22%, preferably 8 to 18%, more preferably 10 to 16%, or 12 to 15% of glycerol, and most preferably 13.5% of glycerol.

[0058] Glycerol may be added to the composition in the form of 85% glycerol.

[0059] In a preferred embodiment, when the hydrogel of the first aspect contains CaCl2 and glycerol, the hydrogel may contain 10% to 33%, preferably 15% to 25%, or 17% to 23%, more preferably 20% to 22%, and most preferably 21.5% of CaCl2.

[0060] In a more preferred embodiment, when the hydrogel of the first aspect contains CaCl2 and glycerol, the hydrogel may contain 6% to 22%, preferably 8% to 18%, more preferably 10% to 16%, or 12% to 15% glycerol, and most preferably may contain 13.5% glycerol. Also, in a preferred embodiment, when the hydrogel of the first aspect contains CaCl2 and glycerol, the hydrogel a) may contain 10% to 33% CaCl2, preferably 15% to 25% CaCl2 or 17% to 23% CaCl2, more preferably 20% to 22% CaCl2, most preferably 21.5% CaCl2, and further, b) may contain 6% to 22% glycerol, preferably 8% to 18% glycerol, more preferably 10% to 16% glycerol, or 12% to 15% glycerol, most preferably 13.5% glycerol.

[0061] In a more preferred embodiment, the hydrogel of the present invention further contains a buffer solution. Suitable buffer solutions in the context of the present invention include Tris-HCl (pH = 7.4), sodium chloride, and magnesium chloride.

[0062] The hydrogel of the present invention may further contain other salts such as sodium chloride, magnesium chloride, calcium chloride, and potassium chloride.

[0063] The hydrogel of the present invention can preferably have a physiological pH, for example, it can have a pH of 7, 7.2, 7.4, 7.6, or 7.8. Using a buffer solution to ensure physiological pH may help reduce the pH difference in infected inflamed tissues in some cases.

[0064] In a particularly preferred embodiment, the hydrogel of the present invention contains a buffer solution containing sodium chloride, magnesium chloride (×7H2O), and Tris HCl (pH = 7.4).

[0065] In another preferred embodiment, the pH of the hydrogel according to the first aspect of the present invention is from 7.0 to 7.8, preferably from 7.1 to 7.6, more preferably from 7.2 to 7.5, and most preferably, the pH is 7.4.

[0066] The HEC stock solution for preparing the HEC hydrogel of the present invention may, for example, contain the following (based on 100 g). HEC (hydroxyethyl cellulose): 13 g = 13% CaCl2 solution * : 43 g = 43% Glycerol 85%: 27 g = 27% Water: 17 g = 17%

[0067] In a preferred embodiment, the hydrogel of the present invention 10 2 pfu / ml to 10 8 pfu / ml of bacteriophage, 3 to 10% of HEC, 10 to 33% of CaCl2, and 6 to 22% of glycerol is included.

[0068] In a more preferred embodiment, the hydrogel of the present invention 10 2 pfu / ml to 10 8 pfu / ml of bacteriophage, 5 to 8% of HEC, 10 to 33% of CaCl2, and 6 to 22% of glycerol is included.

[0069] In a preferred embodiment of the present invention, the hydrogel of the present invention 10 4 pfu / ml to 10 8 pfu / ml of bacteriophage, 3 to 10% of HEC, 15 to 25% of CaCl2, and 8 to 18% of glycerol is included.

[0070] In a more preferred embodiment of the present invention, the hydrogel of the present invention 10 5 pfu / ml to 10 7 pfu / ml of bacteriophage, 6.5% of HEC, 21.5% of CaCl2, and 13.5% of glycerol are included.

[0071] In another preferred embodiment, the hydrogel according to the first aspect of the present invention 10 2 pfu / ml to 10 8 pfu / ml of bacteriophage, 3 to 10% of HEC, 10 to 33% of CaCl2, and 6 to 22% of glycerol are included, preferably 10 2 pfu / ml to 10 7 pfu / ml of bacteriophage, 5 to 8% of HEC, 10 to 33% of CaCl2, and 6 to 22% of glycerol are included, more preferably 10 2 pfu / ml to 10 6 pfu / ml of bacteriophage, 3 to 10% of HEC, 15 to 25% of CaCl2, and 8 to 18% of glycerol are included, or or 10 2 pfu / ml to 10 5.5 pfu / ml of bacteriophage, 3 to 10% of HEC, 15 to 25% of CaCl2, and 8 to 18% of glycerol are included, most preferably 10 2 pfu / ml to 10 5.5 bacteriophage of pfu / ml, 6.5% HEC, 21.5% CaCl2, and 13.5% glycerol are included.

[0072] The water used in the composition of the present invention may be water for injection (Aqua ad injectabilia).

[0073] In an exemplary embodiment, the hydrogel of the present invention is prepared using the following components.

[0074] [Table 2]

[0075] In a preferred embodiment, the hydrogel of the present invention has a viscosity of 1000 mPas to 100,000 mPas, preferably 10,000 to 80,000 mPas, more preferably 40,000 to 80,000 mPas, or 40,000 to 60,000 mPas.

[0076] In a preferred embodiment of the present invention, the hydrogel of the present invention is sterilized. Preferably, the hydrogel is steam sterilized.

[0077] Sterilization can be achieved by sterilizing the HEC hydrogel and the bacteriophage solution separately, or by sterilizing the hydrogel of the present invention, i.e., the hydrogel containing hydroxyethyl cellulose (HEC) and bacteriophage.

[0078] In a second aspect, the present invention relates to a lyophilized product of the hydrogel according to the first aspect.

[0079] Accordingly, the term "lyophilizate of the hydrogel" refers to any product obtained by freeze-drying the hydrogel according to the first aspect of the present invention. The term "freeze-dry" refers to any low-temperature dehydration process. Usually, the temperature is lowered until at least the freezing of water is achieved, and the pressure is lowered to sublime the water. The method of freeze-drying is well known to those skilled in the art.

[0080] The starting point may be a hydrogel according to the first aspect of the present invention. Before starting the freeze-drying process, the gel is usually stored at a temperature of up to 8 °C in a cool place such as a refrigerator until the start of the freeze-drying process.

[0081] At the start of the freeze-drying process, the hydrogel of the first aspect of the present invention may be filled into (sterile) containers that usually already have the desired product shape. These containers can be, inter alia, cups made of foil designed for this purpose, for example using thermoforming techniques. The thickness of the resulting product may also be predetermined in this process step, but usually it is the result of the filling height of the container.

[0082] The freezing, and the duration and intensity of the applied vacuum generally have to be adapted to the size of the product, i.e., the volume of the gel. The freeze-dryer (device) used may also influence the process parameters applied, for example due to differences in capacity. Therefore, the following process data should be considered as examples.

[0083] For example, in the first step, the hydrogel may be frozen at a temperature of -20 °C for 2 hours. In the second step, the primary drying may then be carried out at 2.5 mbar and -10 °C for about 40 hours. The third step may include a secondary drying at above 1 mbar and 10 °C, which may be carried out over about 20 hours.

[0084] Generally, in the production of freeze-dried products containing phages, the freezing process can be considered very important. This process should be fast, and a highly amorphous product should be obtained. Also, the fast freezing process ensures the overall maintenance of the lytic activity of bacteriophages. Regardless of the amount to be frozen, the freezing treatment time is usually less than 5 hours of freezing time, more preferably less than 2 hours of freezing time, and even more preferably less than 1.5 hours of freezing time.

[0085] The further drying time mainly depends on the performance of the equipment used and usually has very little impact on the properties of the resulting product.

[0086] Preferably, the method for preparing a freeze-dried product of a hydrogel according to the first aspect of the present invention comprises a) providing a hydrogel according to the first aspect of the present invention; b) freezing the hydrogel of step a) at a temperature of up to -10°C, preferably -20°C, more preferably -40°C, and most preferably -60°C for less than 5 hours, preferably less than 2 hours, more preferably less than 1.5 hours, and most preferably 1 hour or less; c) performing primary drying of the frozen hydrogel of step b) at 2.5 mbar or less and 10°C or less for about 40 hours, preferably at 1.5 mbar and -30°C for about 40 hours; d) performing secondary drying at above 1 mbar and 10°C for about 20 hours and including.

[0087] To maintain the elastic state, the obtained freeze-dried product preferably has a residual moisture content of more than 2%.

[0088] Generally, the obtained freeze-dried product 10 2 pfu / ml to 10 8 pfu / ml of bacteriophage, 5 - 25% of HEC, 24 - 80% of CaCl2, and 13 - 50% of glycerol may include, preferably, 10 2 pfu / ml to 10 8 pfu / ml of bacteriophage, 10 to 20% of HEC, 40 to 60% of CaCl2, and 20 to 40% of glycerol may include, more preferably, 10 2 pfu / ml to 10 8 pfu / ml of bacteriophage, 13 to 17% of HEC, 45 to 55% of CaCl2, and 25 to 35% of glycerol may include, most preferably, 10 2 pfu / ml to 10 8 pfu / ml of bacteriophage, 14 to 16% of HEC, 51 to 53% of CaCl2, and 30 to 33% of glycerol may be included. Examples of the composition of the hydrogel and the obtained lyophilized product:

[0089]

Table 3

[0090] 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.

[0091] Sterilization of the lyophilized hydrogel: In the context of medical use, the overall aim is to provide sterile products, which is particularly applicable when the product is designed for implantation into the body, for example in the case of anastomosis and packing of wound cavities. However, since the essential property of sterility generally depends on the intended use of the product, non-sterile products can also be appropriate.

[0092] Sterilization of the freeze-dried product can be achieved by a sterilizing freeze-drying process using the aforementioned sterilized bacteriophage-containing hydrogel. Furthermore, according to the experimental results of the gels described, other types of sterilization such as steam sterilization are also possible. In particular, since the steam for steam sterilization does not come into direct contact with the product, water / water vapor is not introduced into the product. Further aspects such as the support pressure set in the steam sterilization process should usually be considered.

[0093] In practice, when applied to the patient's body, the lyophilized product of the hydrogel according to the second aspect usually decomposes. First, the lyophilized product of the hydrogel expands due to body temperature and body moisture and returns to the gel (= semi-solid) state. In the second step, the gel from the first step is generally further diluted by body moisture and becomes a liquid state. Finally, the diluted liquid hydrogel is physiologically decomposed and excreted.

[0094] It has been found that the lyophilized product of the same hydrogel has many advantages compared to the hydrogel containing HEC and bacteriophage. For example, the lyophilized product of the bacteriophage-containing hydrogel has been shown to release the bacteriophage very slowly, which can be further determined by the gel composition and is highly desirable, for example, for the treatment of infected wounds. Furthermore, compared to the hydrogel containing HEC and bacteriophage, the lyophilized product of the same hydrogel is much easier to cut and stretch, and is also better regulated and adaptable. Moreover, since it is very elastic, it does not damage the physiologically dynamic structure.

[0095] Furthermore, the shape and thickness of the product, and thus the duration of the phage, can be easily determined by the manufacturing process (and also depends on, for example, the amount of lyophilized hydrogel). Furthermore, during application, coating and surface repair are possible, especially (using a suture material if necessary). Furthermore, since the gel can be sterilized, the lyophilized product (= freeze-dried) is also in a sterilized state by sterilized production.

[0096] In a third aspect of the invention, the invention relates to a hydrogel according to the first aspect of the invention, or a lyophilized product according to the second aspect of the invention, for use in a method of treating and / or preventing a bacterial infection. Typical clinical fields where infections frequently occur and can be treated using the hydrogel according to the first aspect, or the lyophilized product according to the second aspect, include, for example, cardiac surgery (e.g., artificial heart, drive line, artificial heart valve), vascular surgery (e.g., vascular implant, dialysis shunt), orthopedic surgery (especially orthopedics for traffic accidents) (e.g., internal prosthesis, e.g., artificial knee joint and artificial hip joint (TEP)), dermatology (e.g., inverse acne), and emergency medical services (e.g., wound dressing).

[0097] Similarly, the invention relates to a method of treating and / or preventing a bacterial infection in a patient, the method comprising applying to the patient a hydrogel according to the first aspect of the invention, or a lyophilized product according to the second aspect of the invention.

[0098] In a preferred embodiment of the third aspect, the hydrogel of the first aspect, or the lyophilized product of the hydrogel of the second aspect, is applied during surgery.

[0099] The patient may be any animal, but preferably the patient is a mammal, more preferably a human.

[0100] The hydrogel according to the first aspect of the present invention, or the lyophilized product according to the second aspect of the present invention, can be used for treating and / or preventing bacterial infections. According to the present invention, the term "treatment" means that existing bacteria are neutralized by bacteriophages contained in the hydrogel, and the term "prevention" means that when the hydrogel or the lyophilized product is applied, no bacteria are present yet.

[0101] The hydrogel or the lyophilized product can be applied to any accessible surface of the patient. Any accessible surface of the patient includes, but is not limited to, the patient's skin or any accessible mucous membrane. The hydrogel or the lyophilized product of the present invention is particularly suitable for treating bacterial infections in wounds.

[0102] In a preferred embodiment, the hydrogel or the lyophilized product of the hydrogel for use according to the third aspect is applied to a surgical site, an implant, an anastomosis, a wound, an abrasion, an incision, a puncture wound, wounded skin tissue and / or mucous membrane tissue, the entrance of a catheter, the coating of a sterile material, the coating material of a wound and / or an implant, and / or a suture material.

[0103] The hydrogel can be applied by any possible means. These include, but are not limited to, manual application, application by a syringe, a needle, or a catheter. Similarly, the lyophilized product can also be applied by any possible means. These include, but are not limited to, manual application, application by tools such as forceps. The distribution of the hydrogel or the lyophilized product is mainly carried out manually or by any auxiliary device to cover the site / area to be covered by the product.

[0104] Furthermore, the hydrogel or the lyophilized product may be applied to any implant to be attached or inserted into the patient's body, or any implant already inserted into the patient.

[0105] In a preferred embodiment, a hydrogel or a lyophilized product of a hydrogel for use according to the third aspect is used for the treatment and / or prevention of bacterial infection of an implant.

[0106] Specifically, it has been found that the treatment using a hydrogel according to the first aspect or a lyophilized product according to the second aspect is particularly useful and promising in the field of implant infections. According to the established treatment guidelines, each infected material must be removed and replaced with autologous material, which is not always possible. In particular, when reoperation is performed in the field of the cardiovascular system, it is almost impossible for a patient to survive such an operation. Since the hydrogel according to the first aspect or the lyophilized product according to the second aspect can be applied minimally invasively, the use thereof significantly increases the survival rate of the patient. In very many cases, implants (e.g., artificial hearts, pacemakers, etc.) cannot be autotransplanted either. In such cases, it is very advantageous that the implant treated with the hydrogel or lyophilized product according to the present invention can be retained in situ and the infection can be treated in situ.

[0107] Therefore, in a more preferred embodiment of the present invention, a hydrogel or a lyophilized product of a hydrogel for use according to the third aspect can be directly applied to the surface of an implant either outside or inside the patient's body.

[0108] According to the present invention, the implant may be an artificial joint, an artificial blood vessel, a heart valve, a catheter, an artificial heart, a dental implant, and a transcatheter aortic valve implantation (TAVI) implant for a minimally invasive heart valve.

[0109] In a preferred embodiment of the third aspect, the implant is an internal prosthesis (preferably an artificial joint, more preferably an artificial knee joint or an artificial hip joint), an artificial blood vessel, a heart valve, a catheter, an artificial heart, a drive line, a dialysis shunt, a dental implant, and / or a transcatheter aortic valve implantation (TAVI) implant for a minimally invasive heart valve.

[0110] In a further preferred embodiment, the hydrogel or the lyophilized product of the hydrogel for use according to the third aspect is used for the treatment and / or prevention of artificial blood vessel graft infection.

[0111] Medical fields to which the hydrogel or the lyophilized product of the present invention can be applied include, but are not limited to, surgery, orthopedics, radiology, nephrology, for example, diabetic patients with severely infected diabetic foot lesions, dermatology, cardiology, wound care, nursing facilities / services, and emergency services.

[0112] Possible indications for the hydrogel or the lyophilized product of the present invention include, for example, surgical sites, implants, anastomoses, wounds, for example, deep wounds or cavities, abrasions, incisions, puncture wounds, damaged skin tissue and mucosal tissue, pressure ulcers / bedsore ulcers, catheters, for example, the entrances of urethral catheters / IV catheters, coating of sterile materials, coating materials for wounds and implants, and suture materials. The term "pressure ulcer / bedsore ulcer" may thereby represent any kind of bed sore.

[0113] In a preferred embodiment of the present invention, the hydrogel or the lyophilized product of the present invention is applied during surgery.

[0114] In the present invention, it has been shown that the hydrogel of the present invention is particularly useful for the treatment and prevention of bacterial infections when applied during surgery (see Examples 2 and 3).

[0115] During surgery, the hydrogel or the lyophilized product of the present invention can be applied by any possible means. For example, the bacteriophage gel may be manually applied mainly by a surgeon during surgery or, particularly when applied outside the body such as to a wound or the skin, by any other person, and distributed in a similar manner.

[0116] Furthermore, the application and distribution to the desired site / area can be carried out by any auxiliary device, often a syringe, a needle, and a catheter. Therefore, the bacteriophage gel can be applied by minimally invasive intervention or applied into the body / cavity.

[0117] It has a high viscosity and can be adjusted according to the situation, such as during surgery, especially when using the two-syringe method in the case of a hydrogel. It can be adjusted according to the robustness of the product obtained by combining a galenos preparation and a bacteriophage, so it can be used for all possible applications.

[0118] In a preferred embodiment, the hydrogel or lyophilized product thereof for use according to the third aspect is applied manually and / or with one or more auxiliary devices, preferably one or more syringes, needles and / or catheters, to the surface of the patient's body or the surface of the implant.

[0119] In a particularly preferred embodiment of the present invention, as already described above, the hydrogel or lyophilized product of the present invention is used for the treatment and / or prevention of bacterial infections of implants. In this embodiment of the present invention, the hydrogel or lyophilized product of the present invention can be applied directly to the implant, either extracorporeally or intracorporeally, for example during surgery.

[0120] In a further preferred embodiment, the hydrogel or lyophilized product thereof for use according to the third aspect is used for the treatment and / or prevention of bacterial infections caused by skin surgery, preferably bacterial infections caused by skin surgery related to inverse acne treatment or decubitus ulcers / pressure ulcers.

[0121] Generally, the hydrogel or lyophilized product thereof for use according to the third aspect can be used for the treatment and / or prevention of bacterial infections caused by any type of skin surgery. This means that, no matter what type of skin surgery a wound is formed during, especially when it may be difficult to heal without problems of bacterial infection due to its size or location, the hydrogel according to the first aspect or the lyophilized product according to the second aspect can be applied to assist wound healing and treat and / or prevent bacterial infections.

[0122] Examples of such wounds are well known to those skilled in the art and include, for example, skin biopsies, electro-surgery, cauterization, skin flaps, skin grafts, Mohs micrographic surgery, cryotherapy (e.g., using liquid nitrogen), excision of skin lesions, liposuction, vitiligo surgery, treatment of inverse acne, or wounds resulting from pressure sores / pressure ulcers.

[0123] In a fourth aspect, the invention is a method for preparing a hydrogel comprising hydroxyethyl cellulose (HEC) and bacteriophage, comprising: a) providing the HEC in a solid state; b) dissolving the solid HEC of step a) in a liquid component comprising water and bacteriophage; c) optionally adding water until a desired viscosity is reached. The method relates to a method comprising the steps of:

[0124] All aspects related to the first, second, and third aspects of the invention also apply to this fourth aspect of the invention, particularly when related to the characteristics of hydrogels, hydrogel formulations, and bacteriophages.

[0125] Finally, the invention also relates to a method for treating and / or preventing bacterial infections, comprising the use of a hydrogel according to the first aspect or the use of a lyophilizate according to the second aspect.

[0126] Preferably, in the method for treating and / or preventing bacterial infections, the hydrogel or the lyophilizate of the hydrogel is applied during surgery.

[0127] Also preferably, in the method for treating and / or preventing bacterial infections, the hydrogel or the lyophilizate of the hydrogel is applied to the surgical site, implant, anastomosis, wound, abrasion, incision, puncture wound, damaged skin tissue and / or mucosal tissue, for example, the catheter entry, the coating of sterile materials, the coating material of the wound and / or implant, and / or the suture material.

[0128] More preferably, in a method for treating and / or preventing bacterial infection, the hydrogel or the lyophilized product of the hydrogel is used to treat and / or prevent bacterial infection of the implant.

[0129] Most preferably, in a method for treating and / or preventing bacterial infection, in a method of using a hydrogel or a lyophilized product of a hydrogel for treating and / or preventing bacterial infection of an implant, the hydrogel or the lyophilized product of the hydrogel is directly applied to the surface of the implant either outside or inside the patient's body.

[0130] In particular, in a method for treating and / or preventing bacterial infection, the implant may be an artificial joint, an artificial blood vessel, a heart valve, a catheter, an artificial heart, a dental implant, and / or a transcatheter aortic valve implantation (TAVI) implant for minimally invasive heart valve.

[0131] In particular, when the present invention relates to a method for treating and / or preventing bacterial infection, a hydrogel or a lyophilized product of a hydrogel is used to treat and / or prevent infection of an artificial blood vessel graft.

[0132] Preferably, when the present invention relates to a method for treating and / or preventing bacterial infection, the hydrogel or the lyophilized product of the hydrogel is used for the treatment and / or prevention of bacterial infection caused by skin surgery, preferably bacterial infection caused by skin surgery related to inverse acne treatment or decubitus ulcer / pressure ulcer.

[0133] Also preferably, when the present invention relates to a method for treating and / or preventing bacterial infection, the hydrogel or the lyophilized product of the hydrogel is applied manually and / or with one or more auxiliary devices, preferably one or more syringes, needles and / or catheters, to the surface of the patient's body or the surface of the implant.

[0134] The present invention will be further described by the accompanying examples and drawings, which are intended to illustrate the present invention and not to limit the present invention.

Brief Description of the Drawings

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Example

[0136] Example 1 Discovery of a phage-containing gel galenos preparation suitable for clinical use: Laboratory trajectory Abstract In vitro laboratory tests were conducted to discover a suitable base gel for PhaTEC's phage gel product. The list of gel former candidates included hydroxyethyl cellulose (= HEC), sodium hyaluronate, poloxamer, carbomer, and silicon dioxide (= SiO2 [high dispersion]). The tests were carried out according to the spreadability, tactile and sensory properties on filter paper and skin, as well as the gel viscosity and behavior after 24 hours at 37 °C. As bacteriophages, a cocktail of various bacteriophages (SniPha 360) available from "Phage24" in Austria was used. The required amount is placed on the gel that will be the base of the subsequent PhaTEC product. All tests were carried out aseptically in the laboratory of Pohl-Boskamp GmbH & Co. KG, taking into account GLP (Good Laboratory Practice).

[0137] Regarding sterilization, in our experiments, various methods are shown in which the HEC gel and the phage cocktail result in a sterile product.

[0138] Furthermore, both the HEC gel and the phage can be steam sterilized. Thus, the PhaTEC product containing the gel and the phage can be sterilized in the final container, enabling easy and cost-effective industrial production.

[0139] 1. Background 1. Stability and Spreadability The PhaTEC product is used for application in vivo and in vitro. In particular, this product is applied to mucosal and skin areas, surgical sites such as wounds and anastomoses. For this purpose, appropriate spreadability (to cover the application area) is required, along with the stability of the product to create / apply, for example, the thickness of individual layers and keep them at the application site. Furthermore, the gel should be spreadable so that it can be easily applied to sensitive areas such as new wounds without pain. Additionally, especially when applied externally, coolness is desired to suppress the inflammatory reaction and provide a pleasant tactile sensation in the application area.

[0140] 2. Sterilizability Since we are pursuing products intended for both in vitro and in vivo applications, the products, and thus the initial components such as the base gel, must provide the possibility of sterilization.

[0141] 3. Appropriate spreading around the application site The viscosity of the gel should decrease upon introduction of skin temperature or body temperature and body fluids so that the phages contained therein are released over a certain period of time. As a result, the release of the therapeutic agent is prolonged, but it does not affect the body due to dilution and degradation of the product by the physiological excretion process.

[0142] 4. Feel on the skin For all medical and pharmaceutical requirements, patient approval is essential. Since phages do not cause a physical reaction and have no side effects, the gel felt by the patient, especially when applied to the skin / in vitro, should be comfortable on the skin and / or mucous membranes.

[0143] 2. Gel manufacturing First, all gels were prepared with a 13% content of gel-forming agent. Carbomer, sodium hyaluronate, and HEC gels were the easiest to prepare.

[0144] From the five formulations, only three solid gels were obtained, namely HEC gel, carbomer gel, and sodium hyaluronate gel. Silicon dioxide formed a viscous liquid at the applied concentration but did not form a solid gel and was not completely dissolved. Poloxamer showed islands of unbonded gels and furthermore, much water was not absorbed (Figure 1).

[0145] 3. Tactile / sensory skin test Method Various gel formations were tested on the skin surface of the same subject. Sensory feel was observed within the range of skin reactions.

[0146] Sodium hyaluronate (Natriumhyaluronat EP) gel was too hard and could not spread well on the filter paper. Figure 1 On the skin, especially in areas of hairy skin such as the arm used in the test, it was in a hard state, so application and spreading were painful. Figure 2 Carbomer (Carbomer EP: Carboprol 71G) gel had high adhesiveness, so it was difficult to spread on filter paper and almost impossible to spread on the skin. Furthermore, on the skin, especially in areas of hairy skin such as the arm used in the test, due to its adhesiveness, application and spreading were painful. Figures 3 and 4 HEC (Natrosol 250 HX) gel was felt to be the most comfortable. It could be easily spread on filter paper and the skin, and at the same time had a hardness that allowed it to be applied to the thickness of individual layers. Figures 5 and 6

[0147] Results The feel of these gels was very different. These gels showed very different properties. SiO2 (highly dispersed silicon dioxide, Aeroperl 300 Pharma) and poloxamer (Polaxamer 407) gels were not tested on filter paper and / or skin because the SiO2 gel was in a liquid state and the poloxamer gel formed islands. The carbomer gel was found to have high adhesiveness and was difficult to apply due to its flow characteristics. The adhesiveness of the carbomer gel was very unpleasant when applied to the skin and was painful in hairy areas such as the arm (in the test). The cooling effect was felt almost immediately. The gel did not stay at the application site, especially on the skin, and immediately flowed around in an undirected and uncontrolled manner. It was not possible to apply it to an appropriate layer thickness.

[0148] Sodium hyaluronate gel was painless but too hard to apply easily. It could hardly spread on filter paper and the skin, and especially in hairy areas such as the arm (in the test), it was painful and difficult to spread due to its hardness.

[0149] The HEC gel was hard yet easy to apply without pain and stayed at the desired application site. Here, it was not only possible to demonstrate good spreadability on filter paper and skin, but also the feel on the skin was comfortable. The application to the skin was easy and painless. The distribution of the gel could be done individually with respect to the desired layer thickness. After some time, a cooling effect occurred depending on the thickness of the applied layer. Furthermore, the applied gel layer gave a feel like that of a protective layer such as a plaster.

[0150] 4. Behavior of the Gelatinous Galenos Formulation under Dry Conditions The heat exposure at 37 °C was carried out using a drying oven. To test the desired cooling effect in the case of topical application, gels (10 ml each) were placed in the center of filter paper and marked at the boundaries. The gels were stored at 37 °C for 24 hours to demonstrate the cooling effect due to drying of the gels, and thus evaporation cooling.

[0151] Method The tactile / sensory test after 24 hours at 37 °C was carried out using the gels placed in a covered cup in a drying cabinet. Figures 7, 8, 9

[0152] Results Appropriate protection against dehydration was provided by the primary packaging. Under physiological conditions, the product is liquefied by interstitial moisture and physiologically decomposed.

[0153] 5. Sterilization 5.1. Sterile Manufacturing 5.1.1. Gel Method The gel was prepared in a sterile state in laboratory-scale tests. For this purpose, pre-sterilized equipment and benches (laminar flow) were used. The prepared HEC gel was tested according to the direct filling method according to the European Pharmacopoeia 2.6.1 (Figure 5).

[0154] Results There was no bacterial growth until the 9th day when the extended test ended, 24 hours later. No microbial growth was observed throughout the entire period. Figure 10 5.1.2. Phage The clear goal of PhaTEC is to make phage products industrially manufacturable in a standardized and easy way. The main aspect of simple and industrial manufacturing is to sterilize the product in the final container. Two possibilities of gel sterilization were described above and demonstrated by corresponding tests. Phages can be sterilized by sterile filtration as previously tested and described.

[0155] Method Phages were sterile filtered and did not show bacterial growth in different media after 24 hours, 48 hours, and 72 hours at 37°C. The cocktail showed a positive reaction to Escherichia coli cultured on agar and a sample of phage cocktail containing about 10 7 pfu / ml was steam sterilized, and tested with steam-sterilized BPG on the same bacterial strain with corresponding fresh agar.

[0156] Results Plaques of about 6×10^2 pfu / ml were detectable. This leads to the result that the steam-sterilized bacteriophage from the phage cocktail still has lytic activity against the corresponding bacterial strain. Figure 11 As a conclusion, it was demonstrated that phages can be steam sterilized.

[0157] 5.2. Steam Sterilization 5.2.1. Gel Method Gels were prepared in a normal clean laboratory environment in addition to sterile manufacturing and steam sterilized in the final container. Figure 12 Results The steam-sterilized HEC gel did not show bacterial growth throughout the entire period when tested, as described in "Sterile Preparation". Figure 13

[0158] 6. Summary Among the most suitable gelling agents tested, hydroxyethyl cellulose is the best gelling agent, and the characteristics of tactile sensation, good and uniform protective feeling on the skin, ease of application, and the desired layer thickness are only a part of the aspects leading to this result.

[0159] Regarding sterilization, various methods leading to sterilized products of HEC gel and phage cocktails are shown. All individual tests led to the conclusion that all the above methods and procedures are suitable for sterilizing PhaTEC products.

[0160] Furthermore, both HEC gel and phage can be steam sterilized. Therefore, PhaTEC products containing gel and phage can be sterilized in the final container, enabling easy and cost-effective industrial production.

[0161] Example 2 Success of treatment of infected TEVAR stent grafts by application of bacteriophage both extravascularly and intravascularly Abstract 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, alternative solutions to inappropriate antibiotic treatment and extensive surgery are urgently needed.

[0162] Case A 67-year-old female was admitted due to an infected stent graft in the thoracic aorta. Local infection was confirmed by PET-CT imaging. Due to co-existing diseases, surgical resection of the stent graft could not be performed. Therefore, as a last resort, a three-stage approach of local bacteriophage therapy was carried out. First, left thoracotomy was performed to excise the para-aortic tissue, the bacteriophage suspension was applied around the aorta, and an irrigation-vacuum-system was installed. After repeatedly instilling the bacteriophage suspension for 3 days, as the second stage, the negative pressure sponge was removed and a bacteriophage-containing gel was locally applied around the aorta. In the third stage, the bacteriophage-containing gel was applied to the thoracic stent graft and then endovascularly injected into the infected stent. After 28 days, the patient was discharged with normalized infection parameters. PET-CT imaging at 3 months after the intervention showed no signs of infection within or around the thoracic aorta.

[0163] Conclusion This case demonstrates the successful treatment of an infected intravascular stent graft by applying bacteriophages both extravascularly and intravascularly as a novel approach using a stent graft coated with bacteriophages. This success was only possible with the hydrogel containing HEC and bacteriophages according to the first aspect of the present invention, which can localize the phages in situ at the infection site and release the bacteriophages continuously in a delayed manner.

[0164] Keywords: Staphylococcus aureus sepsis, graft infection, phage therapy, antibiotic resistance Background In vascular surgery, infection of vascular grafts is a serious complication. In particular, infection of endovascular aortic stent grafts is associated with high mortality and morbidity rates of up to 75%. These endovascular procedures are often performed on elderly patients with multiple co-morbidities who are not candidates for open aortic repair, which often involves laparotomy. Therefore, the need for excision of infected stent grafts and in-situ reconstruction with autologous tissue or non-anatomic replacement is associated with postoperative early morbidity and mortality rates exceeding 20%. Even if treatment is successful, the reinfection rate can be up to 20%.

[0165] Bacteria embedded in the tissue around the artificial blood vessel form a biofilm adhering to the surface, and thus have a maximum 1000-fold resistance to antibiotics. Conversely, even targeted antibiotic treatment can only suppress stent graft infection and is not an option for radical treatment. To reduce the morbidity and mortality associated with inevitable surgical treatment, a less invasive alternative approach is urgently needed. In this context, bacteriophages and their lytic activity are promising treatment options.

[0166] Case In August 2020, a 67-year-old female patient was admitted due to deterioration of the general condition and chest pain on breathing. She had a prominent cough when taking a deep breath, no sputum, and a fever up to 38.6°C. The possibility of infection by Sars-CoV-2 was excluded. The patient's white blood cell count was 16.7×10 9 / l, and the serum C-reactive protein value was 199.6 mg / l. Secondary findings included the condition after thoracic and abdominal stent graft implantation (COOK stent 34 / 152 mm) following Stanford type B aortic dissection in February 2009. Additionally, the patient suffered from Osler's disease requiring treatment with prednisone, the condition after pulmonary embolism, arterial hypertension, an atrophied left kidney, and diverticulosis of the sigmoid colon.

[0167] Chest X-ray examination showed no evidence of pneumonia. Antibiotic therapy with ampicillin / sulbactam and roxithromycin was initiated. After Staphylococcus aureus was detected in the blood culture, the antibiotic therapy was switched to flucloxacillin, and 5 days later, it was switched to cefuroxime due to allergic skin eczema.

[0168] The possibility of endocarditis was excluded. The patient showed progression of known leukocytoclastic vasculitis that had developed on both legs, arms, intercostal spaces of the chest, hands, and soles of the feet, but there was no involvement of the kidneys. The skin symptoms improved with intensive prednisolone therapy. The progression of vasculitis was considered a reaction to systemic infection. The antibiotic treatment was switched to meropenem and cefazolin.

[0169] Chest and abdominal computed tomography scans did not reveal infectious foci. To rule out the possibility of the aortic stent graft as an infectious focus, 18F-fluorodeoxyglucose PET-CT was performed.

[0170] As a result, increased metabolic activity throughout the proximal aortic stent was visualized pathologically, starting from the level of the center of the aortic arch and extending to the level of the eighth thoracic vertebra, which was indicative of stent infection in the symptomatic stage. Furthermore, inflammatory swelling of the mediastinal soft tissue and left pleural effusion were seen. Figure 14

[0171] Due to the poor condition of the patient and several co-existing diseases, surgical resection of the infected stent graft and anatomical reconstruction with autologous tissue could not be performed. The patient himself desired an alternative to indefinite systemic antibacterial treatment. Therefore, in accordance with Article 37 of the Helsinki Declaration and the local ethics committee (A 2021-0132), an experimental approach using local administration of bacteriophages was planned as a last resort.

[0172] Bacteriophage therapy As a radical treatment strategy, a three-step approach was implemented for both extravascular and intravascular administration of SnifA360 (Phage24.com, Austria). SnifA360 is a commercial cocktail of lytic bacteriophages against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pyogenes, Proteus vulgaris, and Proteus mirabilis.

[0173] After explaining the risks and benefits of the experimental procedure, the patient consented to the treatment. First, the extravascular treatment was performed by left thoracotomy. It was found that the visceral pleura was adherent to the aorta. After collecting local swab specimens for microbiological analysis, debridement and jet lavage were performed. Then, 20 ml of SnifA360 diluted with 100 ml of 0.9% NaCl was applied dropwise to the infected para-aortic tissue. Next, two Endo-SPONGE® (B.Braun, Melsungen, Germany) were placed on the lesser and greater curvatures of the aortic arch and the proximal descending aorta, respectively, followed by the placement of a V.A.C.® GRANFOAM™ dressing sponge (18 × 12.5 × 3.2 cm, KCI Medizinprodukte GmbH, Wiesbaden, Germany) (Figure 2C). The Endo-SPONGE was connected to a V.A.C. VERAFLO® treatment system (KCI Medizinprodukte GmbH). To avoid contact of the sponge with the lung, a Suprasorb® CNP drainage film (25 × 20 cm, Lohman-Rauscher GmbH&Co., Neuwied, Germany) was placed on the sponge. After placing the chest tube, the chest wall was closed and the patient was admitted to the intermediate intensive care unit.

[0174] Therefore, once a day, the intrapleural fluid was drained via a V.A.C. VERAFLO (trademark) therapy system 2 hours before bacteriophage therapy. Subsequently, the sponge was rinsed three times with 500 ml of 0.9% NaCl each time and drained. Then, the endosponge was rinsed with 20 ml of Snifa 360 diluted with 100 ml of 0.9% NaCl. Both the endosponge drainage and the chest tube were fixed until the next day. Figures 15, 16, 17

[0175] As a second stage, the patient underwent thoracotomy again 3 days later. After removing all the negative pressure sponges, the aorta and the surrounding tissues were coated with 40 ml of Snifa 360 mixed with 15.8% hydroxyethyl cellulose gel. The chest wall was closed and the patient was admitted to the intermediate intensive care unit.

[0176] The third stage was performed 3 days later. Therefore, two sterile Relay NBS PLUS stent grafts (Vascutek Terumo - Bolton Medical, Vascutek Germay GmbH, Hamburg, Germany) coated with a mixture of 40 ml of Snifa 360 and 15.8% hydroxyethyl cellulose gel were released. Then, the grafts coated with bacteriophage externally were reassembled and placed intravascularly. This was performed via the left common femoral artery. After an angiographic overview, the two grafts were placed with the infected stent grafts slightly overlapping. Figures 18, 19

[0177] All the invasive procedures were performed successfully without any observed side effects. After the endovascular treatment, the patient was transferred to the general ward and recovered immediately. The infection parameters decreased, the prescription of antibiotics was discontinued, and the patient recovered. The symptoms of vasculitis also disappeared. After extending the collection of physical therapy in the hospital for 4 weeks, the patient was discharged and transferred to follow - up rehabilitation therapy in good general condition.

[0178] In the PET-CT scan performed 3 months after bacteriophage therapy, no signs of infection were detected within or around the thoracic aorta. The patient further recovered, and no infection parameters were detected. Figure 20

[0179] Discussion This case demonstrates the successful treatment of an infected endovascular stent graft with local bacteriophage therapy. To our knowledge, this is the first time that bacteriophages have been infiltrated into a stent graft and locally administered intravascularly.

[0180] Bacteriophages are known as powerful antibacterial therapeutic agents due to their lytic activity. Compared with other antibacterial treatment strategies such as local rifampicin treatment, bacteriophages have no cytotoxic effect on vascular cells 1 . Furthermore, they have the advantage of acting on both multidrug-resistant bacteria and biofilm-forming bacteria. Recently, a case series of 8 patients with infections of vascular grafts, surgical wounds, or implanted medical devices further demonstrated the feasibility of using various bacteriophages with lytic activity to successfully treat bacterial infections 2 . The use of bacteriophages has successfully treated infections of vascular implants, but bacteriophage therapy is not yet common and is not an officially recommended option for infectious diseases in the Western Hemisphere.

[0181] In this case, due to the patient's health status and significant comorbidities, the patient could not undergo surgical resection of the infected stent graft and anatomical reconstruction of the thoracic aorta. By applying bacteriophages to both the vascular lumen and the perigraft tissue, the treatment of graft infection caused by Staphylococcus aureus was successful. The intravascular administration of bacteriophages using a releasable stent graft coated with bacteriophages and reassembled under sterile conditions before insertion into the patient was an important issue for this approach. Therefore, by directly administering phages intravascularly to the infected site, the concentration, contact time, and invasion of bacteriophages into the infected tissue could be maximized.

[0182] Multiple surgical steps were performed, including two thoracotomies and administration into an endovascular stent graft, and the pain caused by each treatment was significantly less compared to conventional surgical treatments. The extension of the postoperative period in the hospital was because the patient's condition was already weak at the beginning, and a comprehensive collection of physical therapy was required.

[0183] The patient's physical condition improved significantly over time, and no signs of infection were seen in the follow-up PET-CT scan three months later, so bacteriophage therapy is considered to have been successful. However, in order to ensure the success of permanent treatment, the patient's follow-up is being continuously carried out.

[0184] In summary, this case report demonstrates that bacteriophage therapy can be an option for radical treatment for patients who are not suitable for extensive surgical approaches. It is particularly important that this success was only possible with the hydrogel containing HEC and bacteriophage according to the first aspect of the present invention, which can localize the phage in situ at the infection site and can release the bacteriophage continuously in a delayed manner.

[0185] Example 3 Success of bacteriophage treatment for a chronically infected and occluded Dacron® bypass of the bilateral femoral arteries of the aorta.

[0186] Objective Graft infection is a serious and dreaded complication in vascular surgery. Surgical resection of an infected aortic stent graft is associated with high mortality and morbidity. Therefore, alternatives to inappropriate antibiotic treatment and extensive surgery are essential.

[0187] Case A 66-year-old patient was admitted due to infection of a chronic occlusive bilateral femoral artery Dacron® prosthetic vascular bypass. After several attempts at femoral surgical revascularization in the past medical history, a poor tissue condition with chronic wound infection and graft exposure occurred on both femoral artery sides. Local infection was confirmed by PET-CT imaging examination. Due to the patient's co-existing diseases, the only justification for the prosthetic vascular bypass was that it was a medical indication and reasonable. Furthermore, we intended to treat bilateral femoral wound healing disorders. Bacteriophage was contemplated as an alternative treatment option for intraoperative and postoperative treatment of graft-related soft tissue infection. After laparotomy was reopened, the infected aortic prosthetic vascular was removed and the aorta was sutured. The bacteriophage suspension was instilled into Tabotamb-Snow® and then placed retroperitoneally. After interrupting the femoral anastomosis, using the same approach, fleeces impregnated with bacteriophage were placed bilaterally on the femoral sides. The wound was joined and closed without further drainage. After a 10-day hospital stay, the patient was able to be discharged with a self-perceived healthy state, a non-irritating wound state, and without systemic inflammatory parameters. In the PET-CT imaging examination at the 3rd month after the intervention, no signs of infection were shown around the aorta or around the bilateral femoral regions.

[0188] Summary This case demonstrates the supportive antibacterial effect of bacteriophage in septic aortic surgery in high-risk patients and the success of secondary closure of chronically infected femoral wounds. In particular, it demonstrates the excellent effectiveness of the hydrogel containing HEC and bacteriophage according to the first aspect of the present invention, which can localize the phage in situ at the infection site and can continuously release the bacteriophage in a delayed manner.

[0189] Background In vascular surgery, infection of vascular grafts is considered a serious complication. In particular, infection of aortic grafts is associated with a high mortality and morbidity rate of up to 75%. Since these procedures are often performed on patients with multiple co-existing diseases, the need for explantation of the infected graft and the ongoing battle with associated abdominal infections are related to a postoperative early morbidity and mortality rate exceeding 20%. Despite early achievement of treatment success, the general reinfection rate can reach up to 20% of all cases. This is mainly due to bacterial colonies lurking in the tissues around the artificial blood vessel, which form a biofilm adhering to the surface and thus have up to 1000-fold resistance to antibiotic administration. Even targeted antibiotic action suitable for antibacterial susceptibility testing can only suppress graft infection and is not an option for radical treatment. The most common pathogens associated with graft infection are Staphylococcus aureus, Staphylococcus epidermidis, and other coagulase-negative staphylococci, Enterobacterales bacteria, Pseudomonas aeruginosa, and Corynebacterium [3]. These bacteria constantly enhance their specific pathogenicity by adhering to the prosthetic material and avoid the local immune response by forming a biofilm that prevents phagocytosis. Furthermore, systemic antibiotic therapy is often insufficient because effective saturation concentrations are lacking in the inflamed tissues around the artificial blood vessel. To reduce the morbidity and mortality associated with surgical treatment, which is often inevitable, a less invasive approach for appropriately treating the infection of the surrounding tissues is urgently needed. In this context, bacteriophages and their lytic activity are promising treatment options.

[0190] Case In November 2020, a 66-year-old male patient was referred to the emergency department by his family doctor with clinical symptoms of acute abdomen. Examination revealed peritonitis in the lower abdomen and scattered tenderness throughout the abdomen. The possibility of infection by SARS-CoV-2 was excluded. Further examination showed that the body temperature had risen to 39.2°C, and blood tests revealed a white blood cell count of 9.4×109 / l and a serum C-reactive protein level of 90.2 mg / l. Chest X-ray examination showed no evidence of pneumonia. The possibility of endocarditis was excluded. Antibiotic therapy with ampicillin / sulbactam was initiated at the usual intravenous dosage. Blood culture was positive for methicillin-sensitive Staphylococcus aureus. Secondary findings included a state of widespread arteriosclerotic occlusive disease. Since multiple vascular surgeries were required for both legs, it was finally revealed that the patient had undergone a right thigh amputation 12 months ago, and on the left side, a chronically occluded polytetrafluoroethylene (PTFE) Stockmann bypass remained in situ.

[0191] After several attempts at femoral surgical revascularization in the past medical history, a poor tissue condition with exposure of the graft material due to chronic wound infection had occurred in both femoral arteries. The presence of Staphylococcus aureus and Escherichia coli was revealed by wound swab, indicating a polymicrobial infection. The peripheral blood flow of the lower limbs was compensated. First, an abdominal CT scan was performed, and then aorto-bifemoral artery grafts that were occluded and infected were suspected. Subsequently, the PET-CT scan performed showed a visibly increased metabolic activity in the graft area, so it was diagnosed that the chronically occluded and infected aorto-bifemoral artery prosthetic bypass, followed by bilateral thigh infections, had caused skin wound healing disorder. Figure 21

[0192] Due to the patient's co-existing diseases, overall, effective treatment was intended by explanting the prosthetic vascular bypass and shortening the operation and anesthesia time as much as possible. Furthermore, a septic abdominal lavage program was planned to be postponed, and primary abdominal closure was intended. In this case, the use of bacteriophages was considered a reasonable alternative treatment option for the long-term treatment of local inflammation in the abdomen and thighs during and after the operation. The patient himself preferred an alternative solution compared to indefinite systemic antibacterial treatment. Therefore, in accordance with Article 37 of the Helsinki Declaration, in cooperation with the local ethics committee (A 2021-0208), an experimental approach using local administration of bacteriophages as a last resort was intended.

[0193] Bacteriophage therapy As a radical treatment strategy, Sniffa 360 (Phage24.com, Austria) was applied intraperitoneally and outside the abdominal wall. Sniffa 360 is a commercially available bacteriophage cocktail of lytic bacteriophages against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pyogenes, Proteus vulgaris, and Proteus mirabilis. After outlining both the potential risks and benefits of the experimental procedure, the patient consented to the treatment. When laparotomy was resumed, turbid fluid appeared in the abdomen. After the initial lavage, the retroperitoneum was opened and preparations were made to fix the proximal aorta. The Dacron® prosthetic vascular grafts in both femoral arteries of the aorta were encased in a biofilm and embedded in putrid fluid. The infected aortic prosthetic vascular graft was removed, and then the aorta was sutured. The prosthetic vascular graft was retrieved from the thigh after joining the legs of the prosthetic vascular graft. The bacteriophage suspension was instilled into Tabotamp®-Snow® and placed in the retroperitoneum around the infected site. The retroperitoneum and abdomen were primarily closed, and no further drainage was performed. After removing the femoral anastomosis, the wound was excised and joined, and washed with a sharp curette. In the same manner, fleece impregnated with bacteriophages was placed bilaterally on the thigh side, and the wound was closed again without further drainage.

[0194] The operation time was 52 minutes, and there was no significant blood loss. Subsequently, the patient was transferred to the intensive care unit and was able to be extubated without the need for catecholamines. After a 10-day hospital stay, the patient was able to be discharged with a subjective sense of health, a non-stimulating wound condition, and normal findings of blood inflammatory values. At the 3-month follow-up PET-CT imaging examination after the intervention, no signs of infection surrounding the aorta or both thigh regions were shown.

[0195] Discussion This case demonstrates the success of the treatment of a chronically infected and occluded aorta-bifemoral Dacron (registered trademark) bypass by local administration of bacteriophage. In particular, it was demonstrated that this success was only possible with the hydrogel containing HEC and bacteriophage according to the first aspect of the present invention, which can localize the phage in situ at the infection site and can release the bacteriophage continuously in a delayed manner.

[0196] Approximately 50 - 65% of prosthetic infections are thought to be the result of intraoperative bacterial contamination [3, 4]. Although the classification is arbitrary, early (up to 30 days postoperatively) and late infections are generally distinguished [3, 4]. Early prosthetic infections are thought to be the result of intraoperative contamination, and late infections are often thought to be the result of hematogenous bacterial spread, but the in-depth evidence regarding this is limited. Late infections usually result from insufficient tissue integration of the prosthetic graft bed. Common pathogens are Staphylococcus, Enterobacteriaceae, and Corynebacterium [3, 4]. Bacteriophage (or simply "phage", Greek for "bacteria eater") is a virus that selectively infects bacterial cells and was first described in detail in 1917 by Felix Hubert d'Herelle in Canada.

[0197] Currently, bacteriophages are known as powerful antibacterial therapeutic agents due to their lytic activity. When exposed to an inflammatory environment, they are very stable and greatly contribute to the regulation of the bacterial population across the globe. Bacteriophages can only grow where their host is present. Bacteriophages are highly specific and thus mainly affect strains within one bacterial species and rarely cross species boundaries [5].

[0198] In the first (lytic) cycle of virus growth, the phage kills the corresponding bacteria by lysis. Once the bacterial host cell is infected, it then initiates the processes of replication, bacterial destruction, and release of new phage particles, which are controlled by enzymes and the interaction between bacteria and phage genes. In the second (lysogenic) cycle, the nucleic acid of the bacteriophage is integrated into the genome of the host bacterium or forms a circular replicon in the cytoplasm of the bacterium. Compared with other antibacterial treatment strategies such as topical rifampicin treatment, no cytotoxic effect is found in vascular cells in the case of bacteriophages.

[0199] Furthermore, they are effective against multi-drug resistant bacteria and biofilm-forming bacteria. Recently, a case series of eight patients with infections of vascular grafts, surgical wounds, or implanted medical devices further demonstrated the feasibility of using various bacteriophages with lytic activity to successfully treat bacterial infections. The use of bacteriophages led to the successful treatment of vascular implant infections, but bacteriophage therapy is still not common and is not an officially recommended option for infectious diseases in the Western Hemisphere. Therefore, by directly administering phages to the abdominal back and intraperitoneally at the infection site, the concentration of bacteriophages, contact time, and invasion of the surrounding tissue of the infected graft could be maximized. We were able to perform a reliable treatment regarding short operation time, complete closure of the skin / wound, and exclusion of drainage. In our cases, no bacteriophage-related clinical adverse events were found. At the 3-month follow-up PET-CT scan, no signs of infection were seen. Bacteriophage therapy is considered to have been successful.

[0200] To treat local inflammation of the abdomen and thighs over the long term during and after surgery, we recognized that the use of bacteriophages is an alternative option in antibacterial local treatment. However, in order to ensure the success of permanent treatment, the patient follow-up is being continuously conducted. In summary, this case report demonstrates that bacteriophage treatment can be an option for radical treatment for patients with graft and perigraft bacterial infections who are not suitable for extensive surgical approaches.

[0201] Example 4 Test of the bacteriophage activity of the lyophilizate Method To test the activity of bacteriophages in lyophilized hydrogels, five lyophilizates derived from phage gels (composition: HEC: 13 g = 13%, CaCl₂ solution: 43 g = 43%, glycerol 85%: 27 g = 27%, water: 17 g = 17%) were prepared (see Figure 25). Subsequently, the lyophilized hydrogel was placed at the center of an agar plate inoculated with Staphylococcus aureus (see Figure 26) and incubated at 36 °C for 24 hours.

[0202] Results The freeze-dried hydrogel was shown to be degraded through the gel state and then diluted (here mainly by water from the medium). The activity of the phages remained and could be recognized by the plaques (circled parts) on the medium that did not contain the corresponding bacteria (see Figure 27).

[0203] Example 5 Test of phage release from the lyophilized hydrogel Method Preparation of lyophilized product The hydrogels B and C used for lyophilization have the following compositions. HEC: B = 7 g = 7% / C 14 g = 14% CaCl₂ solution: 43 g = 43% Glycerol 85%: 27 g = 27% Water: B = 10 g = 23% / C = 16 g = 16%

[0204] These hydrogels containing bacteriophage were prepared as sterile gels, and lyophilization was also carried out in a sterile environment. The following lyophilized products were prepared and tested as follows. 7 g of B1 gel + BPh buffer * 3 ml + 4 ml of water for injection →> 15 × 10 11 PFU / ml 7 g of B2 gel + BPh buffer * 6 ml + 1 ml of water for injection →> 30 × 10 11 PFU / ml 7 g of C1 gel + BPh buffer * 3 ml + 4 ml of water for injection →> 15 × 10 11 PFU / ml 7 g of C2 gel + BPh buffer * 6 ml + 1 ml of water for injection →> 30 × 10 11 PFU / ml * BPh buffer > 5 × 10 11 PFU / ml BPh buffer: Tris buffer containing sodium chloride, magnesium chloride (×7H2O), and Tris-HCl, pH = 7.4

[0205] Among products B1, B2, C1, and C2, C1 and C2 are the firmest formulations due to the content of the gelling agent (twice the amount of HEC), and variations 2 of B and C (B2 and C2) contain twice the concentration of BPh.

[0206] Release test First, the lyophilized products B1, B2, C1, and C2 were each released into 100 ml of sterile physiological NaCl solution (0.9%) over 2 hours. Therefore, an NaCl solution sterilized by pre-sterile filtration was used. Additional utensils such as bottles and magnetic stirrers (stirring fish) were sterilized by steam sterilization in advance.

[0207] Furthermore, samples were taken by extracting 1 ml every 15 minutes over a further 2 hours without replacing the sample volume. Samples were diluted as necessary. Subsequently, the samples were distributed onto the set Staphylococcus aureus plates and incubated at 37 °C for 24 hours. In total, this was performed twice per gel variation (n = 2).

[0208] Results of release test In summary, the results showed that phages from the freeze-dried hydrogels were released very slowly (see Figure 29).

[0209] Furthermore, this curve shows that the higher the proportion of the gelling agent (C1 and C2), the stronger the sustained release compared to the case where the proportion of the gelling agent is low (B1 and B2). Furthermore, it was shown that when the phage concentration (pfu / ml) of the starting gel (B1, C1) was low, the phage release amount per hour was also low compared to the case where the phage concentration of the starting gel was high (B2, C2).

[0210] Example 6 Treatment of inverse acne using a bacteriophage cocktail with a galenos carrier in a clinical comparison with a gold standard Abstract Inverse acne is a chronic skin disease. Inflammation with pain occurs in the area of the hair follicles. As a result, there may be abscesses, fistulas, and nodular scars. The inflammation mainly occurs in the axillary region, as well as in the groin, anus, and genital area.

[0211] In this treatment attempt, in accordance with the treatment attempt under Article 37 of the Helsinki Declaration, a hydrogel containing HEC and bacteriophages was applied to the findings in the left axilla, and clinically similar findings on the right buttock were treated by open wound treatment as before.

[0212] There are obvious clinical advantages regarding bacteriophage therapy both in the short term and in the long term.

[0213] Introduction The patient was referred by his general practitioner because inverse acne was suspected at several sites, especially in the left axilla and right buttock (Hurley classification II / III). The patient had already undergone axillary surgery in 1997 and posterior thigh surgery due to inverse acne in 2011.

[0214] Since last year, under drug therapy with adalimumab (Hyrimoz) 40 mg, cefpodoxime 200 mg, and topical fusidic acid, continuous deterioration was observed. The patient felt severe pain, could hardly sit, rotation of the right arm was painful and movement was restricted. Intermittent fever was mentioned. Associated diseases are arterial hypertension, factor V Leiden mutation, pulmonary embolism due to Z.n. in 1996, 1998, 2003, and nicotine abuse.

[0215] Findings on admission (axilla, groin, and especially the right buttock) showed inflamed nodules with tenderness, as well as abscesses, fistulas, and scarring, corresponding to Hurley classification II / III (see Fig. 31). In the clinical blood test, the WBC was as high as 21×109 / l and the CRP was 30 mg / l (3 mg / dl), showing leukocytosis. No wound swab was taken.

[0216] Method As an attempt at cure according to Article 37 of the Helsinki Declaration, hospitalization was started for surgical treatment together with planned bacteriophage injection. 20 ml of 1% Scandicain was administered subcutaneously to the surgical access area in the axilla for local anesthesia (see upper left of Fig. 31). After en bloc resection of the infected soft tissue through two accesses, 20 ml of a viscous bacteriophage galenos preparation was injected subcutaneously onto the wound surface (see upper right and lower left of Fig. 31). This preparation had a phage concentration of at least 1×10 5It is a phage cocktail of Phage24 (trademark) company against Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, and Proteus mirabilis, including Snifa 360 (Composition of the Galenos preparation: HEC: 13 g = 13%, CaCl2 solution: 43 g = 43%, glycerol 85%: 27 g = 27%, water: 17 g = 17%). Then the skin was joined and primary closure was performed without further drainage (see the lower right of Figure 31). During the hospitalization treatment process, only the dry wound dressing was replaced. Twelve days after the surgery, the sutures were removed.

[0217] In contrast, general anesthesia had to be performed because pain persisted in local anesthesia. In this case, as the gold standard treatment for inverse acne on the right buttock, open resection and open wound treatment were selected. The intraoperative adaptive VAC pump was regularly inspected and could be removed after 5 days. Thereafter, the wound was washed with a shower daily and was clean and non-irritating at the time of discharge. As a further measure, the wound was washed daily with Octenisept solution until complete granulation occurred, the wound was perfused, and a moist wound dressing was used.

[0218] Results The patient visited the hospital 2 weeks and 10 weeks after the surgical intervention and received clinical follow-up. The right axillary wound remained continuously clean and dry, and there was a clearly oozing pore with a size of 1 mm in the center of the larger accessed wound (see the left of Figure 32), and it closed without irritation 10 weeks after the surgery (see the right of Figure 32). In the first wound examination 2 weeks later, the wound on the buttock had formed granulation but was covered with fibrin (see the upper part of Figure 33). Continuing the wound treatment, a scarred granulation wound was formed 10 weeks after the surgery (see the lower part of Figure 33).

[0219] Discussion Inverse acne is a painful, chronic inflammatory skin disease that mainly occurs in the axillae, groin, genitals, and anus. This disease often begins with inflammation of the hair follicles and then spreads in the form of recurrent inflammation and abscesses. It affects an estimated 1% of the population. A large number of bacteria are found in inverse acne / hidradenitis suppurativa lesions, particularly Staphylococcus aureus. This disease is often diagnosed at a rather advanced stage, quite late. Sufferers often withdraw due to fear and shame, which can disrupt their professional careers, personal and social relationships, and may lead to depression.

[0220] Depending on the severity, it is classified into Hurley stages I to III, and sufferers endure recurrent pus and festering wounds. In patients classified as Hurley stage I, individual inflammatory nodules or abscesses are usually seen around healthy tissue. In stage II, progressive and obvious inflammation with scarring and fistula formation is observed. In stage III, extensive interconnected abscesses and fistula tracts are seen. Hurley stage I: Local or systemic administration of antibiotics, surgical excision of individual lesions and affected areas as necessary Hurley stage II: Systemic administration of antibiotics, administration of antibodies, surgical excision of individual injuries, removal of the superficial tissue layer using a laser as necessary Hurley stage III: Systemic antibiotic administration, administration of antibodies, radical surgical excision of the affected tissue For wound treatment after surgery in Hurley stages II to III, according to the guideline therapy, it is recommended to let the wound "heal openly" rather than suturing it. A suitable wound dressing and, if necessary, negative pressure wound therapy (NPWT) are used to cover the "open" wound.

[0221] This example impressively demonstrated the efficacy of bacteriophage application in direct comparison with conventional treatment methods. Regarding the axilla treated with bacteriophage, neither the initial hospital course nor the postoperative course was remarkable, but in the area of the buttock wound, a complex, time-consuming, and cosmetically poor healing process was recorded.

[0222] The successful use of bacteriophage cocktails without prior susceptibility testing as part of a phagogram is thought to be due to the use of a variety of phages. Using multiple bacteriophages improved sensitivity and targeted mixed infections as well. Subcutaneous administration of a standardized bacteriophage solution with a carrier increased local residence time and local bacteriophage concentration at the initial stage of infection treatment. To avoid the bacteriophage solution flowing off immediately from the target site, a gel-like galenos preparation was used as a coating medium in addition to the bacteriophage solution. In this regard, the described gel-like galenos preparation functions like a reservoir for phages. The application of liquid solutions is considered to be less effective because the concentration of bacteriophages cannot be optimally maintained locally.

[0223] The application of bacteriophages in the form of a hydrogel according to the present invention is a patient-friendly, inexpensive, side-effect-free, rapid treatment approach and has proven to give excellent results in this trial. From the perspective of medical economics, there are clearly different healing processes for the application of bacteriophages, showing advantages.

[0224] Summary In this case report, the application of a bacteriophage cocktail with a galenos-defined carrier that together forms the hydrogel according to the present invention is superior to the conventional treatment of inverse acne of grades II / III according to the Hurley classification.

[0225] Example 7 Comparison of wound healing in mice with wounds caused by subcutaneous bacterial infection treated with various forms of bacteriophage-containing hydrogels First Subcutaneous injection of Staphylococcus aureus into healthy mice generally causes bacterial infection and leads to open wounds. Therefore, since bacterial infection causes and expands skin destruction, reduction of bacterial load is essential for wound healing. In particular, usually, the less bacteria there are, the faster the wound closes. In the process of infection, in the healing of these wounds supported by the treatment with the hydrogel according to the present invention, when the healing is completed, the wound size shrinks and the wound is completely closed.

[0226] Therefore, the reduction of wound size serves as an indirect indicator of the effect of each treatment. The tendency of how fast the wound shrinks over time, which is related to time, is certain scientific information that is indirect but certain for the mouse model used.

[0227] Explanation In the mouse model, five groups of mice, each containing 22 mice, were tested. On the first day, all mice were subcutaneously infected with the same amount of Staphylococcus aureus. Therefore, 100 μL of a bacterial suspension containing an appropriate number of colony-forming units (CFU) was subcutaneously injected into the shaved area under ketamine / xylazine anesthesia.

[0228] On the third day, when all mice showed wounds, the following treatments were performed.

[0229]

Table 4

[0230] Here, the phage solution used was Snifa 360 phage - phage solution, Sanubiom.

[0231] The formulations of Groups III and IV were prepared as follows: An appropriate amount of HEC in the described composition was placed in the first Omnifix Luer lock syringe, and the second Omnifix syringe was filled with the phage solution. The two syringes were connected with an Omnifix adapter, and the phage solution was pushed into the carrier. The formulation was thoroughly mixed by pushing the solution back and forth at least 10 times between the two syringes through the connector.

[0232] The next day, each wound size was measured (based on the length and width of each lesion), and the average value and trend were examined. In the ratio evaluation, the slope of the phage solution group was used as the comparison group, and the slope ratio was set to 1. The test results were considered until the 10th day, but after that, the immune system overlapped with the healing effect of each product, so it was not considered.

[0233] Results The results showed the superiority of the combination of gel and phage.

[0234]

Table 5

[0235] This is particularly prominent in Group IV, with the overall slope being the highest compared to Group II, and the slope of the trend being 2.5 times higher. However, compared to Groups II and III, Group IV has only half the phage concentration. Therefore, in terms of the tendency for the wound size to shrink, Group I is 1.5 times better and Group II is about 2 times better. Group II is equivalent to Group V that was left untreated.

[0236] The aforementioned trend indicates that the combination of phage and gel is superior to the non-treatment group and the phage solution group (Group II).

[0237] Many products are used for wound treatment, but the HEC gel in the described composition is not used. Tests have shown that the HEC in the described composition can properly retain phages at the application site, adhere adhesively, and release phages with an appropriate delay. Furthermore, it is also beneficial for wound healing. In the group with only the HEC gel in the described composition without phages (Group I), the wound area in the trend decreased at a rate 1.5 times that of the phage solution. This effect can be explained, on the one hand, by the properties of the contained water and the hydrophilic gel. Furthermore, the adhesive properties of the HEC in the composition described herein are also evident. Due to the tissue-friendly composition, wounds moistened with the hydrogel healed better than the wounds of the mice in the phage solution group, which is also due to the physical shielding to the outside.

[0238] In Group II, when the phage solution was applied to the wound, it immediately flowed off the wound. Even when coated with an appropriate wound dressing, this could not be avoided, or the possibility of the interaction between phages and bacteria could not be increased. This is clearly shown by the equivalent values of the phage solution group (Group II) and the non-treatment group (Group V).

[0239] Groups III and IV showed a wound reduction rate 2 to 2.5 times that of Group II (phage solution). Of particular note is Group IV, which has only half the phage concentration compared to Groups II and III. The properties of the HEC of the described composition are most obvious here. Due to the properties already described, wound healing was promoted. The continuous release of phages increased the interaction between phages and bacteria.

[0240] The phage solution (Group II) and Group III were carried out with the same amount of phage solution, but here only the difference regarding the carrier became clear. Group III was more than twice as excellent ( = faster) regarding wound healing. Furthermore, it was shown that the delayed release was enhanced as the proportion of the carrier increased. Group IV had a phage concentration of only 10 2.5 pfu / ml and showed the fastest wound healing rate among the trends.

[0241] As a result, in the HEC in this described composition, the success of treatment was shown only at a phage concentration of 10 5 pfu / ml (Group III), which was two times superior to the phage solution (Group II).

[0242] References for Example 2: 1. Szilagyi DE, Smith RF, Elliott JP, Vrandecic MP (1972) Infection in arterial reconstruction with synthetic grafts. Ann Surg 176: 321-333. 2. Zuhlke HV, Harnoss BM, Lorenz EP (1994) Postoperative Infektionen in der Gefaβchirurgie. In: Septische Gefaβchirurgie Blackwell Wiss Verlag. References for Example 3: [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. [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 Cardiovascular Imaging 2019;12:722-9. [5] Baddour LM, Wilson WR, Bayer AS, FowlerVG, Tleyjeh IM, Rybak MJ, et al. Infective Endocarditis in Adults: Diagnosis,Antimicrobial Therapy, and Management of Complications: A Scientific Statementfor Healthcare Professionals From the American Heart Association. Circulation2015;132:1435-86. References for Example 7: 6-F. Altamirano, J. Barr - Phage Therapy in the Postantibiotic Era - Clin Microbiol Rev. 2019 Jan16;32(2):e00066-18. doi: 10.1128 / CMR.00066-18. 7-D. Malik, I. Sokolov, G. Vinner, F.oMancuso, A Kirpichnikova - Formulation, stabilisation and encapsulation of bacteriophage for phage therapy. Adv Colloid Interface Sci. 2017Nov;249:100-133. doi: 10.1016 / j.cis.2017.05.014. Epub 2017 May 14. 8-L. Kasman, J. Norris - Overcoming the PhageReplication Threshold: a Mathematical Model with Implications for PhageTherapy - Journal of Virology 2002 Vol. 76, No. 11,doi.org / 10.1128 / jvi.76.11.5557-5564.2002 9 - E. Morello, L. Debarbieux - Pulmonary Bacteriophage Therapy on Pseudomonas aeruginosa Cystic Fibrosis Strains: First Steps Towards Treatment and Prevention - 2011 plos on collection psychiology, doi.org / 10.1371 / journal.pone.0016963 10 - H. madavi, S. padmanabhan - Therapeutic Potential of Staphylococcal Bacteriophages 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 11 - D. Rhoads, A. Sulakvelidze - Bacteriophage therapy of venous leg ulcers in 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 hydroxyethyl cellulose (HEC) and bacteriophage.

2. CaCl ２ The hydrogel according to claim 1, further comprising glycerol.

3. CaCl ２ The hydrogel according to claim 1, comprising 10% to 33%, preferably 15% to 25%, or 17% to 23%, more preferably 20% to 22%, and most preferably 21.5%.

4. The hydrogel according to claim 2 or 3, comprising 6% to 22% glycerol, preferably 8% to 18%, more preferably 10% to 16%, or 12% to 15%, most preferably 13.5%.

5. The hydrogel according to any one of claims 1 to 4, comprising 3% to 10% HEC, preferably 5 to 9% HEC, more preferably 6 to 9% HEC, particularly preferably 6% to 7% HEC, most preferably 6.5% HEC.

6. The concentration of the bacteriophage is 10 ２ pfu / ml to 10 ８ pfu / ml, preferably 10 ２ pfu / ml to 10 ７ pfu / ml, more preferably 10 ２ pfu / ml to 10 ６ pfu / ml, most preferably 10 ２ pfu / ml to 10 ５．５ pfu / ml, and the hydrogel according to any one of claims 1 to 5

7. 10 ２ pfu / ml to 10 ８ pfu / ml of bacteriophage, 3 to 10% HEC, 10 to 33% CaCl ２ , and 6 to 22% glycerol comprising, preferably 10 ２ pfu / ml to 10 ８ bacteriophage of pfu / ml, 5 to 8% HEC, 10 to 33% CaCl ２ , and 6 to 22% glycerol comprising, or or 10 ４ pfu / ml to 10 ８ a bacteriophage of pfu / ml, 3 to 10% HEC, 15 to 25% CaCl ２ , and 8 to 18% glycerol comprising, more preferably 10 ５ pfu / ml to 10 ７ bacteriophage of pfu / ml, 6.5% HEC, 21.5% CaCl ２ , and 13.5% glycerol The hydrogel according to any one of claims 1 to 6, comprising.

8. 10 ２ pfu / ml to 10 ８ bacteriophage of pfu / ml, 3 to 10% HEC, 10 to 33% CaCl ２ , and 6 to 22% glycerol comprising, preferably 10 ２ pfu / ml to 10 ７ bacteriophage of pfu / ml, 5 to 8% HEC, 10 to 33% CaCl ２ and 6 to 22% glycerol comprising, more preferably 10 ２ pfu / ml to 10 ６ bacteriophage of pfu / ml, 3 to 10% HEC, 15 to 25% CaCl ２ and 8 to 18% glycerol comprising, or or 10 ２ pfu / ml to 10 ５．５ a bacteriophage of pfu / ml, 3 to 10% HEC, 15 to 25% CaCl ２ , and 8 to 18% glycerol comprising, most preferably 10 ２ pfu / ml to 10 ５．５ bacteriophage of pfu / ml, 6.5% HEC, 21.5% CaCl ２ , and 13.5% glycerol The hydrogel according to any one of claims 1 to 7, comprising.

9. The hydrogel according to any one of claims 1 to 8, further comprising a buffer solution.

10. The hydrogel according to any one of claims 1 to 9, having a pH of 7.0 to 7.8, preferably 7.1 to 7.6, more preferably 7.2 to 7.5, most preferably a pH of 7.

4.

11. The hydrogel according to any one of claims 1 to 10, having a viscosity of 1000 mPas to 100,000 mPas, preferably 10,000 to 80,000 mPas, more preferably 40,000 to 80,000 mPas, most preferably 40,000 to 60,000 mPas.

12. The hydrogel according to any one of claims 1 to 11, which is sterilized, preferably steam sterilized.

13. A lyophilized product of the hydrogel according to any one of claims 1 to 12.

14. The hydrogel or lyophilized product of the hydrogel according to any one of claims 1 to 13 for use in the treatment and / or prevention of bacterial infections.

15. The hydrogel or lyophilized product of the hydrogel according to claim 14 for use during surgery.

16. The hydrogel or lyophilized product of the hydrogel according to claim 14 or 15 for application to a surgical site, implant, anastomosis, wound, abrasion, incision, puncture wound, damaged skin tissue and / or mucosal tissue, catheter entry, coating of sterile materials, coating material for wounds and / or implants, and / or suture materials.

17. The hydrogel or lyophilized product of the hydrogel according to any one of claims 14 to 16 for use in the treatment and / or prevention of bacterial infection of implants.

18. The hydrogel or lyophilized product of the hydrogel according to claim 17 for direct application to the surface of the implant, either outside or inside the patient.

19. The hydrogel or lyophilized product of the hydrogel according to claim 17 or 18, wherein the implant is an internal prosthesis (preferably an artificial joint, more preferably an artificial knee joint or artificial hip joint), artificial blood vessel, heart valve, catheter, artificial heart, drive line, dialysis shunt, dental implant, and / or a transcatheter aortic valve implantation (TAVI) implant for minimally invasive heart valve.

20. The hydrogel or lyophilized product of the hydrogel according to any one of claims 14 to 19 for use in the treatment and / or prevention of infection of artificial blood vessel grafts.

21. The hydrogel or lyophilized product of the hydrogel according to any one of claims 15 to 17 for use in the treatment and / or prevention of bacterial infections caused by skin surgery, preferably bacterial infections caused by skin surgery related to inverse acne treatment or decubitus ulcers / pressure ulcers.

22. The hydrogel or lyophilized product of the hydrogel according to any one of claims 14 to 21 for use in the treatment and / or prevention of bacterial infections, applied manually and / or with one or more auxiliary devices, preferably one or more syringes, needles and / or catheters, to the surface of the patient's body or the surface of the implant.

23. A method for preparing a hydrogel comprising hydroxyethyl cellulose (HEC) and bacteriophage, comprising: a) preparing HEC in a solid state; b) dissolving the solid HEC of step a) in a liquid component comprising water and bacteriophage; c) optionally adding water until a desired viscosity is reached and including the method.

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