Hydroxyethylcellulose gel compositions comprising bacteriophages

Abstract

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

Description

[0001] The present invention relates to a hydrogel comprising hydroxyethylcellulose (HEC) and bacteriophages and a lyophilisate thereof, and uses of such hydrogel or lyophilisate, in particular, in the treatment and prevention of bacterial infections.

[0002] Bacterial infections in general have become an increasing problem over the past few years, with antibiotic resistance increasing. This generally affects all areas of medicine, especially when large surfaces of a subject's body come into contact with materials that are foreign to it.

[0003] This applies essentially to the case of larger wounds, such as those resulting from surgery, especially dermatological surgery, which are exposed to potentially contaminated air, suture material or dressings. For example, surgical excision of damaged skin or tissue in patients with inverse acne or bedsores / pressure ulcers often leaves larger wounds. These wounds are often slow to heal and carry a significant risk of recurrent bacterial infection, a risk that is even increased by the need for open treatment of the wound after wound debridement. In addition, blood-borne infections post-operatively and from pre-existing peripheral wounds, such as chronic diabetic wounds, can cause serious problems.

[0004] Bacterial infections of vascular grafts represent a major burden in cardiovascular medicine, associated with increased morbidity and mortality. Different factors associated with this medical field, such as patient frailty, biofilm formation or immunosuppression, can have a negative impact on antibiotic therapy and thereby inhibit treatment success. Therefore, further treatment strategies are needed. The antibacterial properties of bacteriophages were discovered 100 years ago, but since the middle of the twentieth century, the further development of phage therapy has been slowed down by the emphasis on antibiotics in Western medicine. Therefore, the experience and knowledge gained so far in terms of the mechanism of action, operation, clinical application and limitations of phages have been largely lost. However, the simultaneous emergence of antimicrobial resistance and personalized medicine has led to the need for further means to treat bacterial infections.

[0005] This is particularly applicable to the treatment of bacterial infections in surgical settings. Specifically, both open and endovascular surgical cardiovascular repairs have the risk of nosocomial infection, and prosthetic vascular graft infection is usually troublesome, associated with other complications, and often seriously life-threatening. In patients requiring intensive care treatment, the prevalence of nosocomial infection in surgical patients ranges from 4% to 15%. The standard treatment of resection and autologous vascular reconstruction may have an alarmingly high risk for damaged patients, so alternative treatments are urgently needed. In addition, prosthetic vascular graft infection is one of the most worrying complications in cardiovascular surgery and is associated with high morbidity, high mortality, and increased hospitalization costs. The most common pathogenic bacteria are Staphylococcus aureus, Staphylococcus epidermidis, other coagulase-negative staphylococci, Enterobacterales, Escherichia coli, Pseudomonas, and corynebacteria. Bacteria often increase their virulence by attaching themselves to prosthetic materials, thereby protecting themselves from local immune responses and antibiotics with the help of biofilms that inhibit phagocytosis and current standard therapies. Furthermore, systemic antibiotic therapy is often inadequate because effective saturating concentrations are rarely achieved in inflamed periprosthetic tissues.

[0006] Bacteriophages (or simply "phages"; from the Greek: "bacteria eaters") are viruses that selectively infect bacterial cells and were first described in 1917 by the Canadian Félix Hubert d'Hérelle. They are fairly stable in the environment and contribute significantly to the regulation of global bacterial mass. In principle, phages can be found anywhere their counterpart bacteria are present, but can only multiply where their host is. Phages are specific and almost always affect only strains within one bacterial species, rarely crossing species boundaries. Lytic phages initiate the lytic cycle of viral multiplication; phages kill their counterpart bacteria by lysis: once infected, the bacterial host cell begins the multiplication process, destroying the bacterium and releasing new phage particles; this process is controlled by enzymes and the interaction of bacterial and phage genes. On the other hand, lytic phages initiate the lytic cycle: the phage nucleic acid is integrated into the genome of the host bacterium or forms a circular replicon in the bacterial cytoplasm. The lytic cycle here is initiated by an external trigger, for example by UV light or heat.

[0007] Phages are dynamic biological agents that multiply within host bacteria, which imposes different requirements on clinical research and complicates the regulatory approval process from drugs that are more amenable to passive, chemically defined compositions, such as antibiotics. Although the therapeutic use of phages is common in Soviet bloc countries, there is a lack of randomized, placebo-controlled, double-blind studies that could provide scientifically viable data to gain regulatory approval in the Western hemisphere. An increasing number of case reports describe successful treatment of life-threatening infections with phage therapy. However, although some clinical studies have been conducted, most have failed to provide definitive evidence of the effectiveness of phage therapy: one study evaluating a cocktail of phages against Escherichia coli and Pseudomonas aeruginosa infections in burn wounds was terminated prematurely due to insufficient efficacy.

[0008] Currently only phage solutions are available, and topical administration is difficult due to adhesion and flow dynamics. Even if phages are successfully delivered to the site of infection, they may enter and reach surrounding tissues, especially in cases of circulatory administration, such as anastomotic infections. Although this does not generally present a risk of local or systemic adverse reactions, it will compromise efficacy. For example, the preservation of phages on prosthetic surfaces is unclear. Phages must remain at / in the site of administration for a sufficient time to ensure phage and bacterial interaction; this requires semisolid and solid formulations.

[0009] In view of the foregoing, it remains an object in the art to provide antibacterial compositions that can be administered, for example, during surgical procedures.

[0010] This object is addressed by the present invention, which in a first aspect provides a hydrogel comprising hydroxyethylcellulose (HEC) and bacteriophage.

[0011] The inventors surprisingly found that hydrogels containing hydroxyethylcellulose (HEC) and bacteriophages show excellent properties compared to other hydrogels in medical applications. In particular, it was unexpectedly found that hydroxyethylcellulose (HEC) was the best gel former in the most suitable gel form tested. In contrast to previous studies, it was found that hydrogels containing hydroxyethylcellulose (HEC) provided solid gels that can be easily and painlessly applied and spread on, for example, the skin of a patient at the same time. In contrast to other gels tested, it concomitantly showed a hardness that allowed the application of a single layer thickness. Previous studies have shown that the mesh size in hydrogels containing HEC is allegedly too small, and HEC-based gels are not suitable for phage release applications because they show poor release of infectious phages (see, e.g., Chang et al., Int JPharm. 2021Aug 10; 605: 120850). In contrast, the inventors of the present invention can surprisingly show in vitro and in expanded use that the hydrogels containing HEC of the present invention not only show the ability to retain phages, but also can continuously release phages in a delayed manner. This was demonstrated indirectly in scale-up and animal experiments, and directly in lyophilisate experiments. Furthermore, the bacteriophages were found to retain their lytic activity when stored in a hydrogel comprising HEC according to the invention.

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

[0013] The following medical applications focus on the superior hydrogels comprising HEC, taking into account the tactile sensation, i.e. a good, even, even protected feeling on the skin, as well as the properties of easy application, but also the desired layer thickness. In particular, the inventive hydrogels comprising HEC show convincing results in terms of bacteriophage release for the prevention of bacterial infections.

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

[0015] Second, patients with chronic infection and occlusion of the aorta-bifemoral arteries Patients with bypasses were also successfully treated with phage (Example 3). In this example, the infected implant was clinically evidently covered with a bacterial biofilm that could not be completely mechanically (manually) removed. This shows the significant advantage of using a hydrogel according to the first aspect of the invention to treat bacterial infections, due to its ability to localize at the biofilm and combined with delayed phage release, enabling effective and continuous long-term treatment of biofilms and infections associated therewith. This also further confirms the protocol of the hydrogel of the invention for systemic co-treatment with antibiotics.

[0016] In the next step, the hydrogel comprising HEC and bacteriophage was tested in the context of dermatological surgery. Patients with inverse acne after surgery showed improved wound healing when treated with a hydrogel comprising HEC and bacteriophage compared to conventional wound treatments to date (Example 6). The excellent results of the above-mentioned hydrogel comprising HEC and bacteriophage in terms of wound healing were confirmed in a bacterial infection mouse model (Example 7). Finally, it could be further shown that lyophilizates of hydrogels comprising HEC and bacteriophage also showed the desired phage release and activity (Examples 4 and 5).

[0017] In conclusion, the inventors have improved the prior art and could show that hydrogels comprising HEC are excellent tools capable of retaining bacteriophages, maintaining their lytic activity and releasing them with a delayed release.

[0018] The inventors could further improve conventional guidelines for implant removal and re-implantation. In contrast, the newly developed method using the hydrogel or lyophilisate according to the present invention allows for minimally invasive surgery, keeps infected implants in place and increases antibiotic sensitivity.

[0019] Also in the field of biofilms, the hydrogel or lyophilisate according to the invention has advantages over previously applied methods. The hydrogel or lyophilisate according to the invention allows the disruption of biofilms by tight adhesion to their surface and delays the release of bacteriophages.

[0020] The hydrogel or lyophilisate according to the invention showed a significantly improved wound healing of 2.5 times in mouse experiments compared to the treatment methods applied so far using aqueous solutions and phage solutions alone. The successful treatment was carried out in vivo as well as transdermally.

[0021] Furthermore, instead of individual therapies, the hydrogels or lyophilisates of the present invention provide standard therapy using a standard phage cocktail for the treatment and / or prevention of bacterial infections.

[0022] Finally, although the hydrogels or lyophilisates of the present invention show all these advantages, they do not show any adverse side effects, even when administered intravascularly.

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

[0024] In the context of the present invention (see Example 1), the inventors have found that HEC is a gel forming agent that is particularly suitable for preparing gels with desirable tactile and other properties. In particular, the hydrogels of the present invention can be steam sterilized, which is an important property for the intended use in treating bacterial infections, especially during surgery.

[0025] Furthermore, as shown in Example 2, it could be shown that the claimed composition is particularly effective in treating bacterial infections when administered during surgery.

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

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

[0028] AMR Antimicrobial resistance

[0029] CT Computerized tomography

[0030] EVAR Endovascular aortic repair

[0031] MRSA Methicillin-resistant Staphylococcus aureus

[0032] PET Positron Emission Tomography

[0033] SAVR Surgical Aortic Valve Replacement

[0034] TAVR Transcatheter Aortic Valve Replacement

[0035] TEVAR Thoracic endovascular aortic repair

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

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

[0038] In the context of the present invention, it is preferred to use lytic bacteriophages. This has the advantage that when the bacteriophage encounters the corresponding bacteria, its antibacterial action starts immediately. The bacteriophage contained in the hydrogel according to the first aspect may be thermostable and / or cryostable. When the bacteriophage contained in the hydrogel according to the first aspect are cryostable, they are preferably stable even at low temperatures such as about -90°C to -20°C. In another preferred embodiment, the bacteriophage in the hydrogel of the present invention is thermostable. In another preferred embodiment, the bacteriophage is cryostable.

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

[0040] As a minimum value, for example, 10 2 The phage concentration was expressed as pfu / ml.

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

[0042] In another preferred embodiment, the concentration of phage in the hydrogel 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.

[0043] Before adding the bacteriophage to the composition of the present invention, the bacteriophage is preferably sterilized, more preferably steam sterilized. During the sterilization process, the initial bacteriophage concentration may decrease accordingly. The reduction in the number of bacteriophages is calculated in advance so that the bacteriophage concentration of the final product is at least 10 2 pfu / ml.

[0044] In another preferred embodiment, the phage are sterilized by sterile filtration.

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

[0046] Phage preparations can be produced according to applicable GMP guidelines.

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

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

[0049]

[0050] R=H or CH2CH2OH

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

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

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

[0054] In the context of the present invention, HEC is used as a gel forming agent because it can be demonstrated in the context of the present invention that HEC produces a stable gel scaffold on an aqueous substrate. The gel matrix formed by HEC provides enough space to retain a defined concentration of bacteriophage, allowing its distribution to facilitate release. In addition, the viscosity of the final product can be easily adjusted by the concentration of the gel forming agent, so that the variation of the HEC percentage provides more possibilities for products with the same material composition. In addition, the pharmaceutical use of HEC is well known and has a low allergen potential.

[0055] Among other gel formers commonly used in the pharmaceutical industry, HEC is the only one that can be steam sterilized, as shown in the accompanying examples. This sterilization process increases the viscosity of the gel, so this "viscosity increase" can be taken into account when preparing the composition of the present invention. With the additional influence of body / skin temperature, the product can float at the application site by reabsorption of water from the interstitial body water by the product. Through this process from different viscosity levels to watery, the phages are released, and as they continue to spread in smaller doses at the application site, their effects exceed:

[0056] Another reason for using hydroxyethylcellulose is that the gel former has low sensitivity, and therefore the final product also has low sensitivity to pH changes and electrolytes. HEC gel production and equally bacteriophage HEC gel production is easy, cheap and fast to achieve in both manual manufacturing and industrial scale processing. No special equipment or surfaces are required.

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

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

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

[0060] Gel manufacturing is performed manually or by a technical process and is known in the art. For example, the soluble components can be dissolved in a first portion of water. Subsequently, under moderate stirring, the HEC can be incorporated into the water. Under further moderate stirring, at least the remaining required water can be added to the gel material. After the hydrogel is prepared, the hydrogel can be sterilized, preferably steam sterilized.

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

[0062] In a preferred embodiment, the hydrogel further comprises CaCl2 and glycerol.

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

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

[0065] Glycerol can be used to improve adhesive properties and delay product liquefaction under the influence of body temperature and body fluids, as it acts as a lubricant, spreading agent and adhesion promoter. Glycerol is also hygroscopic and therefore not only supports systemic degradation and release of bacteriophages onto the surface of the applied PhaTEC-drug, but also contributes to residue-free degradation of the product. For example, in the case of surgical sutures, the inclusion of glycerol in the surgical suture makes it a self-absorbable product.

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

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

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

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

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

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

[0072] b) 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.

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

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

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

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

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

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

[0079] CaCl2 solution * : 43g=43%

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

[0081] Water: 17g = 17%

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

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

[0084] 3% to 10% HEC,

[0085] 10% to 33% CaCl2, and

[0086] 6% to 22% glycerol.

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

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

[0089] 5% to 8% HEC,

[0090] 10% to 33% CaCl2, and

[0091] 6% to 22% glycerol.

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

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

[0094] 3% to 10% HEC,

[0095] 15% to 25% CaCl2, and

[0096] 8% to 18% glycerol.

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

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

[0099] 6.5% HEC,

[0100] 21.5% CaCl2, and

[0101] 13.5% Glycerol.

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

[0103] 3% to 10% HEC,

[0104] 10% to 33% CaCl2, and

[0105] 6% to 22% glycerol;

[0106] Best

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

[0108] 5% to 8% HEC,

[0109] 10% to 33% CaCl2, and

[0110] 6% to 22% glycerol;

[0111] More preferred

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

[0113] 3% to 10% HEC,

[0114] 15% to 25% CaCl2, and

[0115] 8% to 18% glycerol;

[0116] or

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

[0118] 3% to 10% HEC,

[0119] 15% to 25% CaCl2, and

[0120] 8% to 18% glycerol;

[0121] And the best choice

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

[0123] 6.5% HEC,

[0124] 21.5% CaCl2, and

[0125] 13.5% Glycerol.

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

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

[0128]

[0129]

[0130] In a preferred embodiment, the hydrogel of the invention has a viscosity of 1000 mPas up to 100,000 mPas; preferably 10,000 mPas to 80,000 mPas, more preferably 40,000 mPas to 80,000 mPas or 40,000 mPas to 60,000 mPas.

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

[0132] Sterility can be achieved by sterilizing the HEC hydrogel and the bacteriophage solution separately, or by sterilizing the hydrogel of the present invention, ie, the hydrogel comprising hydroxyethylcellulose (HEC) and bacteriophage.

[0133] In a second aspect, the present invention relates to a lyophilisate of the hydrogel according to the first aspect.

[0134] Thus, the term "lyophilisate of a hydrogel" describes any product obtained by freeze drying a hydrogel according to the first aspect of the invention. The term "freeze drying" describes any low temperature dehydration process. Typically, the temperature is reduced at least until freezing of the water is achieved, and the pressure is reduced to cause sublimation of the water. Methods for freeze drying are well known to those skilled in the art.

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

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

[0137] The duration of freezing and application of the vacuum as well as its intensity must generally be adapted to the product size, i.e. the gel volume. The freeze dryer (apparatus) used may also influence the process parameters to be applied, e.g. due to different capacities. Therefore, the following process data are to be regarded as examples.

[0138] For example, in a first step, the hydrogel may be frozen at a temperature of -20°C for 2 hours. In a second step, primary drying may then be performed at 2.5 mbar and -10°C for approximately 40 hours. The third step may include secondary drying at >1 mbar and 10°C and may be performed for approximately 20 hours.

[0139] In general, the freezing process can be considered to be crucial for the production of a phage-containing freeze-dried product: it should be rapid and a highly amorphous product should be obtained. Rapid freezing methods generally also ensure that the lytic activity of the phage is maintained. Regardless of the volume to be frozen, the freezing process time is generally a freezing time of <5 hours, more preferably a freezing time of <2 hours, and more preferably a freezing time of <1.5 hours.

[0140] Further drying times depend largely on the capabilities of the equipment used and generally have only a minor effect on the properties of the resulting product.

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

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

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

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

[0145] c) subjecting the frozen hydrogel of step b) to primary drying at ≤ 2.5 mbar and ≤ 10°C for about 40 hours, preferably at 1.5 mbar and -30°C for about 40 hours;

[0146] d) Approximately secondary drying is carried out at >1 mbar and 10° C. for about 20 hours.

[0147] In order to maintain the elastic state, the resulting lyophilisate preferably has a residual water content of >2%.

[0148] Typically, the resulting lyophilisate may contain

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

[0150] 5% to 25% HEC,

[0151] 24% to 80% CaCl2, and

[0152] 13% to 50% glycerol;

[0153] Preferably,

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

[0155] 10% to 20% HEC,

[0156] 40% to 60% CaCl2, and

[0157] 20% to 40% glycerol;

[0158] More preferably,

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

[0160] 13% to 17% HEC,

[0161] 45% to 55% CaCl2, and

[0162] 25% to 35% glycerol;

[0163] Most preferably,

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

[0165] 14% to 16% HEC,

[0166] 51% to 53% CaCl2, and

[0167] 30% to 33% glycerin.

[0168] Examples of compositions of hydrogels and resulting lyophilisates:

[0169]

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

[0171] Sterilization of freeze-dried hydrogels:

[0172] In the case of medical applications, the aim is usually to provide a sterile product, especially when the product is designed for implantation in the body, such as in the case of anastomosis and for packing of wound cavities. However, the mandatory feature of sterility is usually determined by the intended use of the product, so that non-sterile products can also be justified.

[0173] Freeze-dried sterility can be achieved by using a pre-sterilized phage-containing hydrogel in an aseptic freeze-drying process. In addition, based on the experimental results with the gel, other types of sterilization, such as steam sterilization, are also possible, especially since the steam from the steam sterilization does not directly come into contact with the product and therefore no water / water vapor is introduced into the product. Other aspects must usually be considered, such as the support pressure set in the steam sterilization process.

[0174] In practice, the lyophilisate of the hydrogel according to the second aspect will generally decompose when applied to the body of a subject. First, the lyophilisate of the hydrogel swells back to a gel (= semi-solid) state under the action of body temperature and water. In a second step, the gel of the first step is generally further diluted by body water and converted into a liquid state. Finally, the diluted liquid hydrogel is physiologically degraded and excreted.

[0175] Compared to a hydrogel comprising HEC and bacteriophage, a freeze-dried product of the same hydrogel has been shown to have many advantages. For example, a freeze-dried product of a hydrogel containing bacteriophages shows a (strong) delay in the release of the bacteriophages, which can be further determined by the gel composition and is very desirable in the case of, for example, treating infected wounds. In addition, a freeze-dried product of the same hydrogel is easier to cut and stretch, as well as having better adjustability and adaptability, than a hydrogel comprising HEC and bacteriophages. It is also very elastic and therefore does not compromise physiological dynamic structures.

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

[0177] In a third aspect of the present invention, the present invention relates to a hydrogel according to the first aspect of the present invention or a lyophilisate according to the second aspect of the present invention for use in a method for treating and / or preventing bacterial infections.

[0178] Clinical areas where infections frequently occur and which can be treated using the hydrogel according to the first aspect or the lyophilisate according to the second aspect are, for example, cardiac surgery (such as artificial hearts, transmission systems, artificial heart valves), vascular surgery (such as vascular implants, dialysis shunts), orthopedics (especially accident orthopedics) (such as the complete field of endoprostheses, e.g. total endoprostheses (TEPs) for the knee and hip joints), dermatology (such as acne inversa) and emergency medical services (such as wound dressings).

[0179] Likewise, the present invention relates to a method for treating and / or preventing a bacterial infection in a subject, wherein the hydrogel according to the first aspect of the invention or the lyophilisate according to the second aspect of the invention is administered to the subject.

[0180] In a preferred embodiment of the third aspect, the hydrogel of the first aspect or the lyophilisate of the hydrogel of the second aspect is administered during surgery.

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

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

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

[0184] In a preferred embodiment, the hydrogel for use according to the third aspect or the lyophilizate of the hydrogel is applied to a surgical site, an implant, an anastomosis, a wound, an abrasion, an incision, a puncture, an incomplete skin tissue and / or mucosal tissue, an entry point for a catheter, a covering of sterile material, a wound and / or implant covering, and / or a suture material.

[0185] The hydrogel can be applied by any possible means. These include, but are not limited to, manual application, application by a syringe, by a needle, by a catheter. Similarly, the lyophilisate can also be applied by any possible means. These include, but are not limited to, manual application, or application by tools such as tweezers. The distribution of the hydrogel or lyophilisate is mainly done by hand or any auxiliary device to cover the site / area to be covered by the product.

[0186] Furthermore, the hydrogel or lyophilisate may be applied to any implant to be attached to or inserted into a subject or to an implant already inserted into a subject.

[0187] In a preferred embodiment, the hydrogel for use or the lyophilisate of the hydrogel according to the third aspect is used for treating and / or preventing bacterial infection of implants.

[0188] Specifically, it has been demonstrated that the treatment using the hydrogel according to the first aspect or the lyophilized product according to the second aspect is particularly useful and promising in the field of implant infection. According to the prescribed treatment guidelines, each infected material must be removed and replaced with autologous material, but this is not always feasible. In particular, when reoperation occurs in the cardiovascular field, it is usually unlikely that the subject will survive in such an operation. Since the hydrogel according to the first aspect or the lyophilized product according to the second aspect can be applied in a minimally invasive manner, their use significantly increases the possibility of the subject's survival. Typically, implants are also unable to be replaced by themselves (such as artificial hearts, pacemakers, etc.). In such cases, it is very advantageous that, when treated with the hydrogel or lyophilized product according to the present invention, the implant can be left in place and infection is allowed to be treated in situ.

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

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

[0191] In a preferred embodiment of the third aspect, the implant is an endoprosthesis (preferably a joint prosthesis, more preferably a total knee or hip endoprosthesis), a vascular prosthesis, a heart valve, a catheter, an artificial heart, a transmission system, a dialysis shunt, a dental implant and / or a minimally invasive heart valve transcatheter aortic valve implant (TAVI).

[0192] In another preferred embodiment, the hydrogel for use or the lyophilisate of the hydrogel according to the third aspect is used for treating and / or preventing infections in prosthetic vascular grafts.

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

[0194] Possible indications for the hydrogel or lyophilisate of the invention are, for example, surgical sites, implants, anastomoses, wounds such as deep wounds or wound holes, abrasions, incisions, puncture wounds, incomplete skin tissue and mucosal tissue, bedsores / pressure ulcers, entry points for catheters such as urinary catheters / IV-catheters, coverings of sterile materials, wound and implant coverings, and suture materials. Thus, the term "bedsores / pressure ulcers" may describe any type of bedsores.

[0195] In a preferred embodiment of the invention, the hydrogel or lyophilisate according to the invention is administered during surgery.

[0196] The present invention (see Examples 2 and 3) has shown that the hydrogels of the present invention are particularly useful for treating and preventing bacterial infections when administered during surgical procedures.

[0197] During surgery, the hydrogel or lyophilisate of the invention can be applied by all possible means. For example, the phage gel can be applied manually and distributed in the same manner, mainly by the surgeon at the time of surgery or by any other person, especially when applied externally to the body, such as to a wound or skin.

[0198] Furthermore, application and distribution at / on the desired site / area can be accomplished by all auxiliary devices (mainly syringes, needles and catheters). Thus, the phage gel can be administered under minimally invasive interventions or in body / wound cavities.

[0199] Due to the viscosity and the possibility to adapt the viscosity to the environment present (such as during surgery), especially when using the double syringe technique in the case of hydrogels, and due to the robustness of the product provided by the galenic and phage combination, all possible applications can be used.

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

[0201] In a particularly preferred embodiment of the present invention, as described above, the hydrogel or lyophilisate of the present invention is used to treat and / or prevent bacterial infection of implants. In this embodiment of the present invention, the hydrogel or lyophilisate of the present invention can be directly applied to implants in vitro or in vivo, for example during surgery.

[0202] In another preferred embodiment, the hydrogel for use or the lyophilisate of the hydrogel according to the third aspect is used for treating and / or preventing bacterial infections caused by dermatological surgery, preferably bacterial infections caused by dermatological surgery associated with acne vulgaris treatment or bedsores / pressure ulcers.

[0203] Generally, the hydrogel for use according to the third aspect or the lyophilizate of the hydrogel can be used to treat and / or prevent bacterial infections caused by any type of skin surgery. This means that during any type of skin surgery, a wound is formed, which may be difficult to heal without any bacterial infection problems, especially due to the size or location of the wound, and the hydrogel according to the first aspect or the lyophilizate according to the second aspect can be applied to treat and / or prevent bacterial infections to support wound healing.

[0204] Examples of such wounds are well known to those skilled in the art, such as wounds resulting from skin biopsy, electrosurgery, cautery, flaps, skin grafts, Mohs microscopically controlled excision, cryotherapy (e.g., with liquid nitrogen), skin lesion excision, liposuction, vitiligo surgery, acne vulgaris treatment, or bedsores / pressure ulcers.

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

[0206] a) providing HEC in the form of a solid substance,

[0207] b) dissolving the solid HEC of step a) in a liquid component comprising water and bacteriophage, and

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

[0209] All aspects relating to the first, second and third aspects of the invention, particularly as they relate to the characteristics of the hydrogel, hydrogel formulation and bacteriophage, also apply to the fourth aspect of the invention.

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

[0211] Preferably, in the method for treating and / or preventing bacterial infections, the hydrogel or a lyophilisate of the hydrogel is administered during surgery.

[0212] Also preferably, in a method for treating and / or preventing bacterial infection, the hydrogel or a lyophilisate of the hydrogel is applied to a surgical site, an implant, an anastomosis, a wound, an abrasion, an incision, a puncture, an incomplete skin tissue and / or a mucosal tissue, such as an entry point for a catheter, a covering of a sterile material, a wound and / or an implant covering, and / or a suture material.

[0213] More preferably, the hydrogel or the lyophilisate of the hydrogel is used for treating and / or preventing bacterial infection of an implant in a method for treating and / or preventing bacterial infection.

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

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

[0216] In particular, when the present invention relates to a method for treating and / or preventing bacterial infection, the hydrogel or the lyophilisate of the hydrogel is used for treating and / or preventing infection of a prosthetic vascular graft.

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

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

[0219] The invention is further illustrated by the accompanying examples and drawings, which are intended to explain rather than limit the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0220] Figure 1 :

[0221] The sodium hyaluronate gel was deposited on the filter and then stored for 24 hours at 37° C. The gel was too hard to spread well on the filter paper and showed clumping as the gel hardened.

[0222] Figure 2 :

[0223] Sodium Hyaluronate Gel on Skin: The gel appeared to harden even on the skin. Due to the hard state of the gel, the application and spreading attempt on the arm shown was painful.

[0224] Figure 3 :

[0225] Carbomer gel on skin: The gel showed highly sticky properties. It was difficult to spread on the skin. Moreover, due to the stickiness, application and spreading were painful. The envisaged gel layer was not applicable.

[0226] Figure 4 :

[0227] Carbomer gel: To show the stickiness of carbomer gel, a small amount of gel was taken between two fingers. Then the fingers were spread out. As a result, the gel stuck to the fingers and became thin in the middle. Therefore, this already shows that the application is too difficult and the gel is too sticky.

[0228] Figure 5 :

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

[0230] Figure 6 :

[0231] HEC gel on the skin: Application was tested on the skin. The application was very easy. In addition, the envisaged gel layer could be applied.

[0232] Figure 7 :

[0233] HEC gel after 24 hours at 37°C: The gel has shrunk under storage conditions. In the absence of any packaging to protect the gel from water evaporation, the solid character after storage shows the necessity of adequate primary packaging. In addition, the gel retains the shape it was in when stored, showing continuous coverage of the application area.

[0234] Figure 8 :

[0235] Poloxamer gel after 24 hours at 37°C: The gel has shrunk under storage conditions. In the absence of any packaging to protect the gel from water evaporation, the solid characteristics after storage show the necessity of adequate primary packaging. The gel does not maintain the shape it had when stored, showing changes in the coverage of the application area and a transition to a cubic shape.

[0236] Fig. 9 :

[0237] Sodium hyaluronate gel after 24 hours at 37°C: The gel has shrunk under storage conditions. In the absence of any packaging to protect the gel from water evaporation, the solid characteristics after storage show the necessity of adequate primary packaging. In addition, the gel does not maintain the shape it had when stored, showing changes in coverage of the application area. Viscosity increases with the loss of water.

[0238] Fig.10 :

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

[0240] Fig.11 :

[0241] Plaques can still be detected after steam sterilization of bacteriophages: On a standardized bacterial bioburden, bacteriophages have spread after steam sterilization. After a period of incubation, plaques indicate that the bacteriophages survived the steam sterilization process and are still active.

[0242] Fig.12 :

[0243] Sterile preparation of HEC gel: Fig.12 Sterility results were shown for the combination of phage and gel.

[0244] Fig.13 :

[0245] Gel after steam sterilization: Direct loading method, Ph. Eur. 2.6.1, showed no signs of bacterial contamination after prolonged testing to increase the probability of detection.

[0246] Fig.14 :

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

[0248] Fig.15 :

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

[0250] Fig.16 :

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

[0252] Fig.17 :

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

[0254] Fig.18 :

[0255] The aortic stent graft system was released off the shelf and the surface was manually coated with phage gel.

[0256] Fig.19 :

[0257] Lateral X-ray after stent implantation, showing complete coverage of the infected stent graft by the new stent graft.

[0258] Fig. 20 :

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

[0260] Fig.21 :

[0261] PET-CT showed an infected aorto-bifemoral vascular graft with right femoral artery tissue infection.

[0262] Fig. 22 :

[0263] Documentation of surgical procedures and administration of phage gel.

[0264] Fig.23 :

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

[0266] Fig.24 :

[0267] Exemplary pictures of freeze-dried hydrogels.

[0268] Fig.25 :

[0269] Lyophilisates were prepared from the hydrogels containing bacteriophages.

[0270] Fig.26 :

[0271] Four freeze-dried hydrogels were placed on the center of an agar plate containing Staphylococcus aureus.

[0272] Fig. 27 :

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

[0274] Fig.28 :

[0275] Experimental setup.

[0276] Fig.29 :

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

[0278] Fig.30 :

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

[0280] Fig.31 :

[0281] Surgical treatment of the left axilla included the application of a hydrogel containing HEC and bacteriophage. First, the left axilla was given local anesthesia (upper left), followed by surgical excision of the affected tissue (surgical incision of the left axilla, upper right), application of bacteriophage and vector (lower left), and subsequent suturing of the axilla skin (lower right).

[0282] Fig.32 :

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

[0284] Fig.33 :

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

[0286] Fig.34 :

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

[0288] Example 1

[0289] Discovering suitable phage-containing gel-galenic formulations for clinical use: a laboratory testbed

[0290] summary

[0291] In vitro laboratory tests were performed to find a suitable base gel for the PhaTEC phage gel product. The list of gel formers includes hydroxyethylcellulose (=HEC), sodium hyaluronate, poloxamer, carbomer and silicon dioxide (=SiO2 [highly dispersed]). The tests were carried out based on spreadability on filter paper and on the skin, tactile and sensory properties as well as gel consistency and behavior after 24 hours at 37°C. For the phages, we used a mixture of different phages (SniPha 360), which can be purchased from the Austrian company "Phage24". These requirements are for the gel that will become the subsequent basis for the PhaTEC products. All tests were carried out under sterile conditions in the laboratories of Pohl-Boskamp GmbH & Co. KG and in compliance with GLP (Good Laboratory Practice).

[0292] Regarding sterilization, in our experiments, various methods were demonstrated to produce a sterile product of the HEC gel and phage mixture.

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

[0294] 1. Stability and spreadability

[0295] PhaTEC products are used for in vivo and in vitro administration. Among them, the product will be applied to mucosal and skin areas, wounds and surgical sites such as anastomoses. For this reason, appropriate spreadability (to cover the application area) is required while having product stability, for example, to produce / apply a single layer thickness and stay at the application site. In addition, the gel should be spreadable so that it can be easily and painlessly applied to sensitive areas, such as fresh wounds. In addition, cooling is necessary, especially in the case of in vitro administration, in order to counteract the inflammatory response and provide a comfortable feeling on the application area.

[0296] 2. Possibility of sterilization

[0297] Since a product intended for in vitro as well as in vivo administration is sought, the product and therefore the initial components such as the base gel must offer the possibility of sterility.

[0298] 3. Spread fully around the application site

[0299] The viscosity of the gel should be reduced by the introduction of skin or body temperature and fluids so that the contained phages are then released within a defined period of time. The product degrades due to dilution and physiological excretion processes, which results in a prolonged release of the therapeutic agent, thus not causing an impact on the body.

[0300] 4. Feeling on the skin

[0301] As with all medical and pharmaceutical requirements, patient acceptance is crucial. Since bacteriophages do not cause any physical reaction and have no side effects, the gel that the patient feels, especially when applied to the skin / external body, must be comfortable on the skin and / or mucous membranes.

[0302] 2. Gel Fabrication

[0303] Initially, all gels were prepared with 13% gel former. Carbomer, sodium hyaluronate, and HEC gels were the easiest to prepare.

[0304] From the five preparations, only three solid gels were obtained:

[0305] HEC gel, carbomer gel, and sodium hyaluronate gel. Silica formed a viscous liquid at the applied concentration, but not a solid gel, and did not completely dissolve. Poloxamer showed gel islands that were not connected to each other; in addition, it did not absorb much water ( Figure 1 ).

[0306] 3. Tactile / sensory skin test

[0307] method:

[0308] The different gel formations were tested on the skin surface of the same tester. In terms of the observed skin reaction, it was felt to be sensitive.

[0309] Sodium hyaluronate (Sodium Hyaluronate EP) gel was too stiff to spread well on the filter paper. Figure 1 .

[0310] Because of this hard state, application and spreading on the skin (especially hairy skin areas such as the arm used in the test) can be painful. Figure 2 .

[0311] Carbomer (Carbomer EP; Carboprol 71G) gel is highly sticky and therefore difficult to spread on filter paper and almost impossible to spread on the skin. In addition, due to this stickiness, application and spreading on the skin (especially hairy skin areas, such as the arm used in the test) can cause pain: Figure 3 , Figure 4 .

[0312] The HEC (Natrosol 250HX) gel was the most comfortable to use: it spread easily on the filter paper and on the skin, while having a hardness that allowed application of a single layer thickness: Figure 5 , Figure 6 .

[0313] result:

[0314] It has been shown that the sensation of these gels is very different: These gels show very different properties: While SiO2 and poloxamer gels were not tested on filter paper and / or skin due to the liquid state of SiO2 gels (hochdisperses Siliciumdioxid; Aeroperl 300 Pharma) and due to the island formation of poloxamer gels (Poloxamer 407), carbomer gels were found to be highly adhesive and difficult to apply due to their free-flowing properties. The stickiness of carbomer gels is very uncomfortable when applied to the skin and causes pain in hairy areas such as (in the test) the arms. The cooling effect is almost immediately apparent. The gels, especially on the skin, do not stay at the application site, but immediately flow into the surrounding area in an undirected and uncontrolled manner. It is not possible to apply an appropriate layer thickness.

[0315] Sodium hyaluronate gel is too hard for painless, simple application: it can hardly be spread on filter paper and on the skin, especially in hairy areas such as (in the test) the arm, because its hardness causes pain and is difficult to spread.

[0316] Although hard, the HEC gel is easy and painless to apply and stays on the application site as desired: here, it not only shows good spreadability on the filter paper and on the skin; the feeling on the skin is also pleasant. Application on the skin is simple and painless. The gel distribution can be carried out individually according to the desired layer thickness. Due to the applied layer thickness, the cooling effect occurs after a while. In addition, the applied gel layer creates the feeling of a protective layer, just like a plaster or the like.

[0317] 4. Behavior of Gel-Galenic Formulations under Dry Conditions

[0318] Heat exposure at 37°C was performed with the aid of a drying cabinet. To test the cooling effect required when applied externally, the gels (10 ml each) were placed in the center of a filter paper and the borders were marked. The gels were stored at 37°C for 24 hours to demonstrate the drying of the gel and the cooling effect due to evaporative cooling.

[0319] method:

[0320] The tactile / sensory test after 24 hours at 37°C was performed in a dry cabinet with the gel in a cup with a lid. Figure 7 , Figure 8 , Fig. 9 .

[0321] result:

[0322] The primary packaging provides adequate protection against dehydration. Under physiological conditions, the product is liquefied by interstitial water and physiological degradation occurs.

[0323] 5. Sterility

[0324] 5.1. Aseptic production

[0325] 5.1.1. Gel

[0326] method:

[0327] The gel was prepared aseptically in a laboratory-scale test. For this purpose, pre-sterilized equipment and workbench (laminar air flow) were used. The prepared HEC gel was tested according to the direct loading method of European Pharmacopoeia 2.6.1. Figure 5 ).

[0328] result:

[0329] There was no bacterial growth after 24 hours until day 9 (when the extended test ended). There was no significant microbial growth over the entire period. Fig.10 .

[0330] 5.1.2. Bacteriophage

[0331] The explicit aim of PhaTEC is to make its phage products industrially, standardized and easily produced. A major aspect of simple industrial production is the sterilization of the product in the final container. The two possibilities of gel sterilization are described above and have been confirmed by corresponding tests. Phage can be sterilized by sterile filtration, as tested and described above.

[0332] method:

[0333] The phages were sterile filtered and no bacterial growth was observed on different media after 24, 48 and 72 hours at 37°C. The mixture tested positive on E. coli bacterial culture on agar and contained approximately 10 7 Samples of the phage cocktail at pfu / ml were steam sterilized and tested against the same bacterial strains on fresh corresponding agar using steam sterilized BPG.

[0334] result:

[0335] Plaques can be detected, about 6×10^2 PFU / ml. As a result, the steam-sterilized phages from our phage cocktail still have lytic activity against the corresponding bacterial strains. Fig.11 .

[0336] In conclusion, it has been demonstrated that steam sterilization of bacteriophages is possible.

[0337] 5.2. Steam sterilization

[0338] 5.2.1. Gel

[0339] method:

[0340] In addition to aseptic manufacturing, the gel is prepared in a normal laboratory clean environment and steam sterilized in the final container. Fig.12 .

[0341] result:

[0342] When tested as described for the "Sterile Preparation", the steam sterilized HEC gel showed no microbial growth at all times. Fig.13 .

[0343] 6. Summary

[0344] Among the most suitable gel formers tested, hydroxyethylcellulose was the best gel former: the tactile sensation, ie the good, even and even protected feeling on the skin, and the easy application properties, but also the desired layer thickness, were only some of the aspects that led to this result.

[0345] Regarding sterilization, there are various methods shown to produce a sterile product of HEC gel and phage mixture. All individual tests have led to the conclusion that all methods and procedures given above are suitable for rendering the PhaTEC product sterile.

[0346] Furthermore, both HEC gel and bacteriophages can be steam sterilized. Therefore, PhaTEC products containing gel and bacteriophages can be sterilized in the final container, enabling simple and cost-effective industrial production.

[0347] Example 2

[0348] Infected TEVAR was successfully treated using extravascular phage and intravascular phage application

[0349] summary

[0350] Objective: Graft infection is a serious complication during vascular surgery. Surgical resection of infected aortic stent grafts is associated with high mortality and morbidity. Therefore, alternatives to inadequate antibiotic therapy and major surgical intervention are urgently needed.

[0351] Case

[0352] A 67-year-old woman was admitted with a thoracic aortic stent graft infection. Local infection was confirmed by PET-CT imaging. Surgical removal of the stent graft was not feasible due to comorbidities. Therefore, a three-step approach for local phage therapy was adopted as a last resort. First, para-aortic tissue was debrided by left thoracotomy, phage suspension was applied around the aorta, and an irrigation vacuum system was installed. After three days of repeated alternating instillations of phage suspension, as a second step, the vacuum sponge was removed and a phage-containing gel was applied topically around the aorta. In the third step, the phage-containing gel was applied to the thoracic stent graft, which was then placed intravascularly into the infected stent. After 28 days, the patient was discharged with normal infection parameters. Three months after the intervention, PET-CT imaging showed no signs of infection in or around the thoracic aorta.

[0353] in conclusion:

[0354] This case demonstrates that infected intravascular stent grafts can be successfully treated by administering phages to both extravascular sites and (as a novel approach) intravascular sites using phage-coated stent grafts. This success is only possible due to the hydrogel comprising HEC and phages according to the first aspect of the present invention, which allows for in situ localization of phages at the site of infection and continuous release of phages in a delayed manner.

[0355] Keywords:

[0356] Staphylococcus aureus sepsis, transplant infection, phage therapy, antibiotic resistance Background Art

[0358] Infection of vascular grafts is a serious complication in vascular surgery. In particular, infection of endovascular aortic stent grafts is associated with high morbidity and mortality rates of up to 75%. Because these endovascular procedures are often performed in elderly patients with multiple comorbidities who are not suitable for open aortic repair, the infected stent graft needs to be removed and reconstructed in situ with autologous tissue or replaced extra-anatomically, which is associated with early postoperative morbidity and mortality rates exceeding 20%. Even when treatment is successful, reinfection rates are as high as 20%.

[0359] Bacteria embedded in periprosthetic tissue form surface-adherent biofilms and are therefore up to 1,000-fold more resistant to antibiotics. Vice versa, even targeted antibiotic therapy can only suppress stent graft infection, rather than being a curative treatment option.

[0360] To reduce the morbidity and mortality of unavoidable surgical treatment, alternative minimally invasive approaches are urgently needed. In this context, bacteriophages and their bacteriolytic activity are a promising therapeutic option.

[0361] Case

[0362] In August 2020, a 67-year-old female patient was admitted to the hospital due to worsening of her general condition and chest respiratory pain. The patient had a significant cough with no sputum during deep inspiration and a fever of up to 38.6°C. Sars-CoV-2 infection was ruled out. 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 thoracoabdominal stent graft implantation (COOK stent 34 / 152 mm) after Stanford type B aortic dissection in February 2009. In addition, the patient had Osler disease requiring prednisolone treatment, a condition after pulmonary embolism, arterial hypertension, left renal atrophy, and sigmoid colon diverticulosis.

[0363] Chest X-ray showed no signs of pneumonia. Antibiotic therapy with ampicillin / sulbactam and roxithromycin was initiated. Antibiotic therapy was changed to flucloxacillin after Staphylococcus aureus was detected in blood cultures and, five days later, to cefuroxime due to allergic skin eczema.

[0364] Endocarditis was ruled out. The patient had progression of a known leukocytoclastic vasculitis involving the legs, arms, cleavage, hands, and soles of the feet, but not the kidneys. Skin symptoms improved with prednisolone therapy. The progression of the vasculitis was thought to be a response to a systemic infection. Antibiotic therapy was changed to meropenem and cefazolin.

[0365] Computed tomography of the chest and abdomen showed no infection. To exclude the possibility of the aortic stent graft as a lesion of infection, fluorodeoxyglucose PET-CT was performed.

[0366] Results showed that increased pathological metabolic activity was observed throughout the proximal aortic stent, starting at the level of the mid-aortic arch and extending to the level of the eighth thoracic vertebra, a sign of stent infection. Inflammatory mediastinal soft tissue swelling and left pleural effusion were also observed. Fig.14 .

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

[0368] Phage therapy

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

[0370] After explaining the risks and benefits of the experimental procedure, the patient agreed to the treatment. First, extravascular treatment was performed through a left thoracotomy. The visceral pleura was found to be adherent to the aorta. After obtaining a local swab sample for microbiological analysis, debridement and jet irrigation were performed. Then, 20 ml of SniPha 360 (diluted in 100 ml of 0.9% NaCl) was dripped onto the infected para-aortic tissue. Then, two endogenous sponges (Endo- B. Braun, Melsungen, Germany) were placed on the lesser and greater curvatures of the aortic arch and the proximal descending aorta, and then GRANFOAM TM Dressing sponge (18 × 12.5 × 3.2 cm, KCI Medizinprodukte GmbH, Wiesbaden, Germany) Figure 2 C) Connect the internal sponge to the VACVERAFLO TM Treatment system (KCI Medizinprodukte GmbH). To avoid contact between the sponge and the Lunge, A CNP drainage membrane (25 x 20 cm, Lohman-Rauscher GmbH & Co., Neuwied, Germany) was placed on top of the sponge. After placement of the chest tube the chest cavity was closed and the patient was transferred to the intermediate care unit.

[0371] There, once a day, the intrathoracic fluid is removed by VACVERAFLO two hours before phage therapy. TM The treatment system was withdrawn, and the sponge was subsequently flushed and drained three times with 500 ml of 0.9% NaCl each. The internal sponge was then flushed with 20 ml of SniPha 360 diluted in 100 ml of 0.9% NaCl. Both the internal sponge drain and the chest tube were clamped until the next day. Fig.15 , Fig.16 , Fig.17 .

[0372] As a second step, the patient underwent another thoracotomy three days later. After all vacuum sponges were removed, the aorta and surrounding tissues were covered with 40 ml of SniPha 360 mixed with 15.8% hydroxyethylcellulose gel. The thorax was closed and the patient was transferred to the intermediate care unit.

[0373] The third step was performed after another three days. Therefore, two sterile RELAY NBS PLUS stent grafts (Vascutek Terumo-BoltonMedical, Vascutek Germay GmbH, Hamburg, Germany) coated with a mixture of 40 ml SniPha 360 and 15.8% hydroxyethylcellulose gel were released. Then, the grafts coated with phage on the outside were reassembled for intravascular placement. This was performed through the left common femoral artery. After overview angiography, the two grafts were placed with a small overlap with the infected stent graft. Fig.18 , Fig.19 .

[0374] All invasive procedures were uneventful and no side effects were observed. After endovascular treatment, the patient was transferred to the general ward and recovered quickly. The infection parameters decreased, antibiotic medication was stopped, and the patient recovered. The signs of vasculitis also disappeared. After four weeks of long physical therapy activities in the hospital, the patient was discharged in general good condition and continued rehabilitation.

[0375] A PET-CT scan performed three months after phage therapy showed no detectable signs of infection in or around the thoracic aorta. The patient further recovered with no detectable infection parameters. Fig. 20 .

[0376] discuss

[0377] This case demonstrates that infected intravascular stent grafts can be successfully treated with local phage therapy. To our knowledge, this is the first time that stent grafts have been impregnated with phages for local intravascular administration.

[0378] Bacteriophages are considered effective antibacterial treatments due to their lytic activity. In contrast to other antibacterial treatment strategies such as topical rifampicin treatment, phages have no cytotoxic effects on vascular cells. 1 In turn, they have the advantage of being able to act both against multidrug-resistant bacteria and against bacteria organized in biofilms. A recent case series of eight patients with infections of vascular grafts, surgical wounds, or implanted medical devices further demonstrated the feasibility of using different phages with lytic activity to successfully treat bacterial infections. 2 Although phages have been used to successfully treat vascular implant infections, phage therapy remains uncommon and there are no officially recommended protocols for the infection in the Western Hemisphere.

[0379] In this case, the patient’s medical condition and severe comorbidities made him unsuitable for surgical resection of the infected stent graft and reconstruction of the thoracic aorta. Graft infection with Staphylococcus aureus was successfully treated by administering phages into the vessel lumen and onto the surrounding coagulation tissue. The intravascular administration of phages using a releasable stent graft coated with phages and reassembled under sterile conditions before insertion into the patient is an important issue of this approach. The intravascular administration of phages directly to the site of infection ensures maximum concentration, contact time, and penetration of phages into the infected tissue.

[0380] Despite the multiple surgical steps, including two thoracotomies and endovascular stent graft administration, the corresponding pain caused by each procedure was significantly reduced compared with traditional surgical treatment. The prolonged postoperative hospital stay was due to the patient's inherently weak physical condition and the need for extensive physical therapy activities.

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

[0382] In conclusion, this case report demonstrates that phage therapy may be a curative treatment option for patients who are not suitable for major surgical approaches. Of particular importance, this success was only possible due to the hydrogel comprising HEC and phage according to the first aspect of the present invention, which allows for in situ localization of phage at the site of infection and continuous release of phage in a delayed manner.

[0383] Example 3

[0384] Successful treatment of chronically infected and occluded aorta-bifemoral arteries with bacteriophages bypass

[0385] Purpose:

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

[0387] Case:

[0388] A 66-year-old patient presented with chronic occlusion of the aorta-bifemoral arteries The patient was admitted to our hospital due to prosthetic bypass. After various transfemoral surgical recanalization attempts in the past medical history, there was an unfavorable tissue condition on both sides of the femoral artery with chronic wound infection and graft exposure. Local infection was confirmed by PET-CT imaging. Due to the patient's comorbidities, there was no medical indication and reasonable explanation other than the sole explanation of prosthetic bypass. In addition, we aimed to treat bilateral femoral artery wound healing disorder. Bacteriophages are considered as an alternative treatment option for intraoperative and postoperative management of graft-related soft tissue infection. After repeat laparotomy, the infected aortic prosthesis was resected and the aorta was sutured. The phage suspension was instilled into the The wound was mobilized and closed without further drainage. After 10 days of hospitalization, the patient was discharged with a subjective feeling of well-being, non-irritating wound condition, and no systemic inflammatory parameters. Three months after the intervention, PET-CT imaging showed no signs of infection around the aorta or both femoral artery territories.

[0389] Summarize:

[0390] This case demonstrates the supportive antibacterial effect of phages in suppurative aortic surgery and in the successful secondary closure of chronic infected femoral artery wounds in high-risk patients. Most importantly, it demonstrates the excellent efficiency of the hydrogel comprising HEC and phages according to the first aspect of the present invention, which allows the in situ localization of phages at the site of infection and the continuous release of phages in a delayed manner. Background Art

[0392] Infection of vascular grafts is considered a serious complication during vascular surgery. Infection of aortic grafts in particular is associated with high morbidity and mortality rates of up to 75%. Since these procedures are often performed in patients with multiple comorbidities, the need to remove infected grafts and to fight extensively with associated abdominal infections is associated with early postoperative morbidity and mortality rates of even more than 20%. Despite initial successful treatment, overall reinfection rates can be as high as 20% of cases. This is mainly due to bacterial colonies becoming embedded in the periprosthetic tissue and then forming surface-adherent biofilms that are therefore up to 1000-fold resistant to antibiotic administration. Even targeted antimicrobial agents suitable for antimicrobial susceptibility testing can only inhibit graft infection but cannot be a curative treatment option. The most common pathogenic bacteria associated with graft inflammation are Staphylococcus aureus, Staphylococcus epidermidis and other coagulase-negative staphylococci, Enterobacteriaceae, Pseudomonas aeruginosa, and Corynebacterium [3]. These bacteria regularly enhance their specific virulence by attaching to the prosthetic material and thus avoid local immune responses by forming biofilms that hinder phagocytosis. Furthermore, systemic antibiotic therapy is often inadequate due to the lack of effective saturation concentrations within inflamed periprosthetic tissues. Minimally invasive approaches to adequately treat periprosthetic tissue infections are urgently needed to reduce the morbidity and mortality associated with surgical interventions, which are often unavoidable. In this setting, bacteriophages and their bacteriolytic activity represent a promising therapeutic option.

[0393] Case

[0394] In November 2020, a 66-year-old male patient was referred to the emergency ward by his general practitioner with clinical symptoms of an acute abdomen. Examination revealed widespread tenderness in all abdominal subregions with peritonitis in the lower abdomen. SARS-CoV-2 infection was ruled out. Further examinations revealed an elevated temperature of 39.2°C, blood tests revealed a white blood cell count of 9.4 × 109 cells / l, and an elevated serum C-reactive protein of 90.2 mg / l. Chest X-ray showed no signs of pneumonia. Endocarditis was ruled out. Calculated antibiotic therapy with ampicillin / sulbactam was initiated at conventional doses intravenously. Blood cultures were positive for methicillin-susceptible Staphylococcus aureus. Secondary findings included the presence of widespread arterial occlusive disease. Due to the need for multiple vascular surgeries in both legs, the patient ultimately underwent right thigh amputation 12 months ago; a long-term occluded polytetrafluoroethylene (PTFE) Stockmann bypass remained in situ in the left thigh.

[0395] After various attempts at surgical recanalization via the femoral artery in the past medical history, there was an unfavorable tissue condition in both femoral arteries with chronic wound infection leading to exposure of the graft material. Wound swabs revealed the presence of Staphylococcus aureus and Escherichia coli, indicating a polymicrobial infection. Peripheral blood flow to the lower extremities was compensated. Initially, an abdominal CT scan was performed, and then it was assumed that the aorto-bifemoral graft was occluded and infected. A subsequent PET-CT scan showed a marked increase in metabolic activity in the graft area, which led to the diagnosis of a chronically occluded and infected aorto-bifemoral prosthetic bypass, followed by infection of the bifemoral arteries, leading to impaired skin wound healing. Fig.21 .

[0396] Due to the patient's comorbidities, we generally want to shorten the operation and anesthesia time as much as possible and perform effective treatment by removing the prosthetic bypass. In addition, we plan to abandon the planned irrigation of the suppurative abdomen and intend to perform primary closure of the abdomen. For the long-term treatment of local inflammation in the abdomen and femoral artery area during and after surgery, the use of bacteriophages was considered a feasible alternative treatment option in this case. The patient himself preferred an alternative solution compared to unlimited and lasting systemic antibacterial treatment. Therefore, according to Article 37 of the Declaration of Helsinki and in agreement with the local ethics committee (A 2021-0208), it was hoped to use an experimental method of local phage application as a last resort.

[0397] Phage therapy

[0398] As a curative treatment strategy, in vivo and in vitro abdominal administration of SniPha 360 (Phage24, Austria) was performed. SniPha 360 is a commercially available phage cocktail against lytic phages of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pyogenes, Proteus vulgaris, and Proteus mirabilis. After an overview of the potential risks and benefits of the experimental procedure, the patient consented to the treatment. When repeat laparotomy was performed, turbid fluid was present in the abdomen. After initial flushing, the retroperitoneum was opened and the proximal aorta was prepared for clamping. Aorta-bifemoral artery The prosthesis showed biofilm shells and was embedded in rancid fluid. The infected aortic prosthesis was removed and the aorta was sutured. The prosthesis was removed via the femoral artery after mobilization of the leg with the prosthesis. The phage suspension was instilled into It was then placed in the retroperitoneum around the infection. The retroperitoneum and abdomen were closed primary without further drainage. After the femoral anastomosis was removed, the wound was debrided, mobilized, and irrigated with a sharp spoon. The phage-soaked fabric was then placed on both sides of the femoral artery side according to the same principle, and the wound was closed again without further drainage.

[0399] The operation time was 52 minutes, with no significant blood loss. Subsequently, the patient could be transferred to the intensive care unit and extubated without the need for catecholamines. After 10 days of hospitalization, the patient was discharged with a subjective feeling of well-being, non-irritating wound conditions, and normal inflammatory values ​​in the blood. Three months after the intervention, PET-CT imaging showed no signs of infection around the aorta or both femoral artery territories.

[0400] discuss

[0401] This case demonstrates the successful treatment of a chronically infected occluded aorto-bifemoral artery by local bacteriophage application It was shown in particular that this success was only possible due to the hydrogel comprising HEC and bacteriophages according to the first aspect of the invention, which allows for in situ localization of the bacteriophages at the site of infection and for the continuous release of the bacteriophages in a delayed manner.

[0402] It is thought that approximately 50% to 65% of prosthesis infections are the result of bacterial contamination during surgery [3,4]. Infections are generally divided into early (within 30 days after surgery) and late infections, although this classification is arbitrary [3,4]. Early prosthesis infections are generally thought to be the result of intraoperative contamination and late infections the result of hematogenous bacterial transmission, but strong evidence for this is limited. Late infections are usually caused by inadequate tissue integration of the prosthesis into the graft bed. Common pathogens are Staphylococci, Enterobacteriaceae, and Corynebacteria [3,4]. Bacteriophages (or simply "phages"; Greek: "bacteria eaters") are viruses that selectively infect bacterial cells and were first described by Canadian Félix Hubert d'Hérelle in 1917.

[0403] Bacteriophages are currently considered to be effective antibacterial therapeutics due to their lytic activity. They are quite stable when exposed to inflammatory environments and make an important contribution to regulating global bacterial mass. Bacteriophages can only reproduce where their host is located. They are highly specific and therefore mainly affect strains within one bacterial species, rarely crossing species boundaries [5].

[0404] In the first (lytic) cycle of viral reproduction, phages kill their bacterial counterparts by lysis: once infected, the bacterial host cell begins a multiplication process that destroys the bacteria and releases new phage particles; this process is controlled by enzymes and the interaction of bacterial and phage genes. In the second (lytic) cycle, phage nucleic acids are integrated into the genome of the host bacteria or form circular replicons in the bacterial cytoplasm. In contrast to other antibacterial treatment strategies such as topical rifampicin therapy, no cytotoxic effects of phages on vascular cells were found.

[0405] Furthermore, they are effective against multidrug-resistant bacteria as well as bacteria in biofilm tissues. Recently, a case series of eight patients with infections of vascular grafts, surgical wounds, or implanted medical devices further demonstrated the feasibility of successfully treating bacterial infections with different phages with lytic activity. Although phages have been used to successfully treat vascular implant infections, phage therapy remains uncommon and there is no officially recommended protocol for infections in the Western hemisphere. Direct administration of phages to the infection site in the retroperitoneum and intraperitoneum ensures maximum concentration, contact time, and penetration of phages into the infected peri-graft tissue. We were able to perform surgery within a short time, with definitive treatment in terms of complete skin / wound closure and without the need for any drainage. No phage-related clinical adverse events were detected in our cases. A three-month follow-up PET-CT scan showed no signs of infection. Phage therapy can be considered successful.

[0406] We realized the use of bacteriophages as an alternative treatment option to antibacterial local therapy for the long-term management of local inflammation in the abdominal and femoral artery regions during and after surgery. However, patients should be followed up continuously to ensure lasting treatment success. In conclusion, this case report suggests that phage therapy may be a curative treatment option for patients with bacterial graft and perigraft infections who are not suitable for major surgical approaches.

[0407] Example 4

[0408] Testing the activity of phage in lyophilized samples

[0409] method

[0410] To test the activity of phages in freeze-dried hydrogels, five phage gels in the form of freeze-dried products were prepared (composition: HEC: 13 g = 13%, CaCl2 solution: 43 g = 43%, 85% glycerol: 27 g = 27%, water: 17 g = 17%) (see Fig.25 ). The freeze-dried hydrogel was then placed in the center of an agar plate containing Staphylococcus aureus (see Fig.26 ) and incubated at 36°C for 24 h.

[0411] result

[0412] The freeze-dried hydrogel was confirmed to degrade through the gel state and then dilute (here: mainly by water from the culture medium). The activity of the phage still exists, which can be recognized by the corresponding bacteria-free (circled) plaques on the culture medium (see Fig. 27 ).

[0413] Example 5

[0414] Testing phage release from freeze-dried hydrogels

[0415] method

[0416] Preparation of lyophilisates

[0417] Hydrogels B and C used for freeze-drying had the following compositions:

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

[0419] CaCl2 solution: 43 g = 43%

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

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

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

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

[0424]

[0425] *BPh-Buffer>5×10 11 PFU / ml

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

[0427] Among products B1, B2, C1 and C2, C1 and C2 are the most solid formulations due to their gel former content (double amount of HEC), while 2 variants of B and C (B2 and C2) contain double concentration of BPh.

[0428] Release Test

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

[0430] During the next 2 hours, samples were drawn by extracting 1 ml every 15 minutes, thus without changing the sample volume. If necessary, the samples were diluted. Subsequently, the samples were distributed on fixed S. aureus plates and incubated at 37°C for 24 hours. In total, 2 runs were performed for each gel variant (n=2).

[0431] Release test results

[0432] In summary, the results showed a strong delayed release of phage from freeze-dried hydrogels (see Fig.29 ).

[0433] In addition, the curves show that the higher ratio of gel forming agent (C1 and C2) shows a stronger delayed release compared with the lower ratio of gel forming agent (B1 and B2). In addition, the lower phage concentration in the starting gel (B1, C1) also shows a lower phage release per unit time compared with the higher phage concentration (pfu / ml) in the starting gel (B2, C2).

[0434] Example 6

[0435] Clinical comparison of bacteriophage cocktails combined with galenic vectors in the treatment of acne inversa with gold standard

[0436] summary:

[0437] Acne inversa is a chronic skin disease. Painful inflammation occurs in the area of ​​hair follicles. As a result, abscesses, fistulas and nodular scars may form. Inflammation occurs mainly in the armpits and groin, anus and genital areas.

[0438] In this treatment attempt, the findings in the left axilla were treated with a hydrogel containing HEC and phages, in accordance with Article 37 of the Declaration of Helsinki, while clinically similar findings on the right buttock were treated with conventional open wound therapy.

[0439] Phage therapy has clear clinical advantages in both the short and long term.

[0440] introduce :

[0441] The patient was referred by her family physician with suspected acne inverse in multiple locations, especially the left axilla and right buttock (Hurley stage II / III). She had undergone surgery in the axilla in 1997 and on the posterior thigh in 2011 for acne inverse.

[0442] The condition has continued to deteriorate since last year, while on medical treatment with adalimumab (Hyrimoz) 40 mg and cefpodoxime 200 mg, as well as topical fusidic acid (Fucidine). The patient has severe pain, can hardly sit up, has pain with rotation of the right arm, and has limited mobility. Intermittent fevers are mentioned. Associated diseases are arterial hypertension, a factor V Leiden mutation, Zn pulmonary embolism (1996, 1998, 2003), and nicotine abuse.

[0443] On admission, the patient was found to have tender inflammatory nodules in the axillae, groin, and especially in the right gluteal region, as well as abscesses, fistulas, and scarring, equivalent to Hurley stage II / III (see Fig.31 ). Laboratory blood test revealed leukocytosis, with WBC count of 21×10 9 The CRP was 30 mg / l (3 mg / dl). No wound swab was collected.

[0444] method:

[0445] The patient was admitted for surgical care in conjunction with a planned phage injection as an attempt at healing in accordance with Article 37 of the Declaration of Helsinki. Local anesthesia was performed by subcutaneous injection of 20 ml of 1% Scandincain in the area of ​​the surgical axillary incision (see Fig.31 , upper left). After the infected soft tissue was excised en bloc through two separate incisions, 20 ml of a viscous phage galenic preparation (see Fig.31 , upper right and lower left), the formulation contains The company's phage mixture SniPha360 is against Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris and Proteus mirabilis. The mixture has a phage activity of at least 1×10 5 The phage concentration was 13 g = 13% (galenic composition: HEC: 13 g = 13%, CaCl2 solution: 43 g = 43%, 85% glycerol: 27 g = 27%, water: 17 g = 17%). The skin was then mobilized and primary closure was performed without further drainage (see Fig.31 , lower right). During hospitalization, only dry dressings were changed. Sutures were removed after 12 days of OR post-treatment.

[0446] In contrast, general anesthesia was required due to persistent pain under local anesthesia. In this case, open excision and open wound treatment was chosen as the gold standard treatment for acne inversa on the right buttocks. The VAC pump used during the operation was checked regularly and could be removed after 5 days. The wound was then rinsed daily and was clean and non-irritating before discharge. Further procedures were daily cleaning and irrigation of the wound with Octenisept solution and the use of wet dressings until the wound was completely scabby.

[0447] result:

[0448] The patient was referred to the outpatient department for clinical follow-up at 2 and 10 weeks after the surgical intervention. The right axillary wound remained clean and dry, with a 1 mm large, visibly oozing hole in the middle of the larger incisional wound (see Fig.32 , left), the wound closed without irritation 10 weeks after surgery (see Fig.32 , right). At the first wound examination 2 weeks later, the buttock wound was granulated but covered with fibrin (see Fig.33 , above). With continued wound healing, a scarred granulation wound appeared 10 weeks after surgery (see Fig.33 ,Down).

[0449] discuss:

[0450] Acne inversa is a painful, chronic inflammatory skin disorder that primarily affects the axillae, groin, pubic area, and anus. The disease typically begins as inflammation of the hair roots and then spreads in the form of recurrent inflammation and abscesses. An estimated 1% of the population is affected. Large numbers of bacteria, particularly Staphylococcus aureus, can be found in the purulent lesions of acne inversa / hidradenitis. The disease is often diagnosed too late, at an advanced stage. Those affected often withdraw from the disease due to fear and shame, which can disrupt their professional lives, personal and social relationships, and lead to depression.

[0451] Depending on the severity, it is classified as Hurley grade I to III, and those affected will present with recurrent purulent wounds. Individual inflammatory nodules or abscesses are often present in the surrounding healthy tissue, which is often seen in patients classified as Hurley grade I. In grade II, progressive, well-defined inflammation with scarring and fistula formation is described. In grade III, there are numerous, interconnected abscesses and fistulas.

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

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

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

[0455] For postoperative wound care of Hurley grade II to III, the guidelines recommend that the wound not be closed but left to heal “open”. Appropriate wound dressings and, if necessary, negative pressure wound therapy (NPWT) are used to cover the “open” wound.

[0456] This example impressively demonstrates the efficacy of phage application in direct comparison with conventional treatment methods. Early hospitalization and postoperative course were unremarkable in the axilla treated with phage, whereas in the buttocks wound area, a complex, prolonged and less aesthetic healing process could be documented.

[0457] The successful use of phage mixtures as part of the phagocytic profile without prior sensitivity testing can be attributed to the use of different phages. The use of multiple phages increases the sensitivity rate and also targets mixed infections. Subcutaneous administration of standardized phage solutions combined with carriers increases local residence time and local phage concentrations in the initial stages of infection treatment. In addition to the phage solution, a gel galenic preparation is used as an application medium in order to prevent the phage solution from flowing out of the targeted area quickly. In this regard, the gel galenic preparation acts like a reservoir for phages. The application of liquid solutions is considered to be less efficient because the phage concentration cannot be optimally maintained locally.

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

[0459] Summarize:

[0460] In the present case report, the administration of a mixture of bacteriophages together with a galenically defined carrier, which together form a hydrogel according to the invention, was superior to conventional treatment of type II / III acne inversa according to Hurley.

[0461] Example 7

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

[0463] introduce

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

[0465] Therefore, the reduction in wound adhesion is an indirect indicator of the effectiveness of the corresponding treatment. Therefore, the time and the trend of the rate of wound reduction over time is an indirect but reliable scientific readout for the mouse model used.

[0466] describe:

[0467] In the mouse model, 5 groups of 22 mice were tested. On the first day, all mice had been infected subcutaneously with the same amount of S. aureus. Therefore, under ketamine / xylazine anesthesia, 100 μL of bacterial suspension containing the appropriate number of colony forming units (CFU) was injected subcutaneously into the shaved area.

[0468] On day 3, all mice showed wounds treated with:

[0469] I HEC in the composition na 0.5ml / mouse II Phage solution <![CDATA[10 5 pfu / ml]]> 0.5ml / mouse III Phage solution + HEC in the composition is 1:1 <![CDATA[10 5 pfu / ml]]> 1.0ml / mouse IV Phage solution + HEC in the composition is 1:2 <![CDATA[10 2.5 pfu / ml]]> 1.0ml / mouse V Untreated na

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

[0471] Therefore, the phage solution used was SniPha 360 phage, ie phage solution Sanubiom.

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

[0473] In the following days, the corresponding wound adhesions (based on the length and width of each lesion) were measured, the averages were calculated and the trends were analyzed. For the ratio evaluation, the slope of the phage solution group was used as the comparison group, so that the slope ratio = 1. The test results were continued until day 10,

[0474] Because the immune system then overlaps with the healing effects of the corresponding products.

[0475] result:

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

[0477]

[0478] This was particularly evident in Group IV, which had the best overall slope and a 2.5-fold better trend slope compared to Group II. Despite this, it maintained only half the phage concentration compared to Groups II and III. Thus, Group I was 1.5-fold better and Group II was about 2-fold better in terms of the trend toward reduced wound adhesion. Group II was comparable to Group V, which was untreated.

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

[0480] Many products are used for wound treatment, but there is no HEC gel in the composition. This test shows that the HEC in the composition can not only fully carry the phage at the application site, but also adhere to it and release the phage in a corresponding delayed manner. In addition, it is also conducive to wound healing: in the composition, the single HEC gel (Group I) without phage reduces the wound area faster than the phage solution, and its reduction trend is 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 hydrophilic gel. In addition, the adhesion properties of HEC in the composition described herein are also obvious: due to the tissue-friendly composition, the wounds moistened by the hydrogel heal better than the wounds of the mice in the phage solution group, which is also attributed to the physical shielding to the outside world.

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

[0482] Compared to group II (phage solution), groups III and IV showed 2 to 2.5 times the wound reduction rate of group II. Of particular note is group IV, which carried only half the phage concentration compared to groups II and III. The properties of HEC in the composition are most evident here. Due to the properties already explained, wound healing is accelerated. The sustained release of phage increases the interaction between phage and bacteria.

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

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

[0485] References for Example 2:

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

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

[0488] References for Example 3:

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

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

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

[0492] References for Example 7:

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

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

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

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

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

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

Claims

1. A hydrogel comprising hydroxyethyl cellulose (HEC) and bacteriophage.

2. The hydrogel according to claim 1, further comprising CaCl2 and glycerol.

3. The hydrogel according to claim 1, wherein the hydrogel comprises 10% to 33% CaCl2, preferably 15% to 25% or 17% to 23%, and more preferably 20% to 22%, most preferably 21.5%.

4. The hydrogel according to any one of claims 2 or 3, wherein the hydrogel comprises 6% to 22% glycerol, preferably 8% to 18%, more preferably 10% to 16% or 12% to 15%, most preferably 13.5% glycerol.

5. The hydrogel according to any one of the preceding claims, wherein the hydrogel comprises 3% to 10% HEC, preferably 5% to 9% HEC, more preferably 6% to 9% HEC, especially preferably 6% to 7% HEC, and most preferably 6.5% HEC.

6. The hydrogel according to any one of the preceding claims, wherein 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, and most preferably 10 2 pfu / ml to 10 5.5 pfu / ml.

7. A hydrogel according to any one of the preceding claims, comprising 10 2 pfu / ml to 10 8 pfu / ml phage, 3% to 10% HEC, 10% to 33% CaCl2, and 6% to 22% glycerol; Preferably include 10 2 pfu / ml to 10 8 pfu / ml phage, 5% to 8% HEC, 10% to 33% CaCl2, and 6% to 22% glycerol; or 10 4 pfu / ml to 10 8 pfu / ml phage, 3% to 10% HEC, 15% to 25% CaCl2, and 8% to 18% glycerol; More preferably include 10 5 pfu / ml to 10 7 pfu / ml phage, 6.5% HEC, 21.5% CaCl2, and 13.5% Glycerol.

8. A hydrogel according to any one of the preceding claims, comprising 10 2 pfu / ml to 10 8 pfu / ml phage, 3% to 10% HEC, 10% to 33% CaCl2, and 6% to 22% glycerol; Preferably include 10 2 pfu / ml to 10 7 pfu / ml phage, 5% to 8% HEC, 10% to 33% CaCl2, and 6% to 22% glycerol; More preferably include 10 2 pfu / ml to 10 6 pfu / ml phage, 3% to 10% HEC, 15% to 25% CaCl2, and 8% to 18% glycerol; or 10 2 pfu / ml to 10 5.5 pfu / ml phage, 3% to 10% HEC, 15% to 25% CaCl2, and 8% to 18% glycerol; and most preferably comprises 10 2 pfu / ml to 10 5.5 pfu / ml phage, 6.5% HEC, 21.5% CaCl2, and 13.5% Glycerol.

9. The hydrogel according to any one of the preceding claims, further comprising a buffer.

10. The hydrogel according to any one of the preceding claims, wherein the hydrogel has a pH of 7.0 to 7.8, preferably 7.1 to 7.6, more preferably 7.2 to 7.5, most preferably 7.

4.

11. The hydrogel according to any one of the preceding claims, wherein the viscosity of the hydrogel is from 1000 mPas up to 100,000 mPas, preferably from 10,000 mPas to 80,000 mPas, more preferably from 40,000 mPas to 80,000 mPas, and most preferably from 40,000 mPas to 60,000 mPas.

12. The hydrogel according to any one of the preceding claims, wherein the hydrogel is sterile, preferably wherein the hydrogel has been steam sterilized.

13. A lyophilisate of the hydrogel according to any one of the preceding claims.

14. The hydrogel or lyophilisate of the hydrogel according to any one of the preceding claims for use in a method for the treatment and / or prevention of bacterial infections.

15. The hydrogel or the lyophilisate of the hydrogel for use according to claim 14, wherein the hydrogel or the lyophilisate of the hydrogel is administered during a surgical procedure.

16. The hydrogel or the lyophilizate of the hydrogel for use according to any one of claims 14 to 15, wherein the hydrogel or the lyophilizate of the hydrogel is applied to a surgical site, an implant, an anastomosis, a wound, an abrasion, an incision, a puncture, an incomplete skin tissue and / or mucosal tissue, an entry point for a catheter, a covering of sterile material, a wound and / or implant covering, and / or a suture material.

17. The hydrogel or the lyophilizate of the hydrogel for use according to any one of claims 14 to 16, wherein the hydrogel or the lyophilizate of the hydrogel is used for treating and / or preventing bacterial infection of implants.

18. The hydrogel or the lyophilisate of the hydrogel for use according to claim 17, wherein the hydrogel or the lyophilisate of the hydrogel is directly applied to the surface of the implant outside the body of a subject or inside the body of a subject.

19. The hydrogel or lyophilizate of the hydrogel for use according to any one of claims 17 or 18, wherein the implant is an endoprosthesis (preferably a joint prosthesis, more preferably a total endoprosthesis of a knee or hip joint), a vascular prosthesis, a heart valve, a catheter, an artificial heart, a transmission system, a dialysis shunt, a dental implant and / or a minimally invasive heart valve transcatheter aortic valve implant (TAVI).

20. The hydrogel or the lyophilizate of the hydrogel for use according to any one of claims 14 to 19, wherein the hydrogel or the lyophilizate of the hydrogel is used for treating and / or preventing infections in prosthetic vascular grafts.

21. The hydrogel or lyophilizate of a hydrogel for use according to any one of claims 15 to 17, wherein the hydrogel or the lyophilizate of a hydrogel is used for treating and / or preventing bacterial infections caused by dermatological surgery, preferably bacterial infections caused by dermatological surgery associated with the treatment of inverse acne or bedsores / pressure ulcers.

22. The hydrogel or lyophilizate of a hydrogel for use according to any one of claims 14 to 21, wherein the hydrogel or the lyophilizate of a hydrogel is applied to the surface of a subject's body or to the surface of an implant manually and / or using one or more auxiliary devices, preferably one or more syringes, needles and / or catheters.

23. A method for preparing a hydrogel comprising hydroxyethyl cellulose (HEC) and bacteriophage, the method comprising the following steps a) providing HEC in the form of a solid substance, b) dissolving the solid HEC of step a) in a liquid component comprising water and bacteriophage, as well as c) Optionally add water until the desired viscosity is reached.

Images