HK40081414B - Provision of bacteriophages in various dosage forms and bacteriophage application device - Google Patents

Description

The invention relates to an intracorporeal bacteriophage delivery in the form of a gel comprising bacteriophages for use in a method for the treatment or prophylaxis of bacterial infections, wherein the gel is sterile and is applied intracorporeally during surgery.

As bacteriophages, also called BPH or phages for short, different groups of viruses are referred to that are specialized on bacteria as host cells, i.e., which specifically infect bacteria. Thus, the host, for example, a mammal and especially a human, is not infected. An infection of bacteria or other pathogenic microorganisms by virulent phages leads to the lytic cycle and ultimately to lysis and destruction of the bacteria. Endotoxins are bacterial toxins that, unlike exotoxins, are not secreted by living bacteria, but are released only through autolysis.

Phages have found a wide range of applications in medicine, veterinary medicine, biology, agricultural sciences, food processing, especially in the field of genetic engineering. For example, phages are used in medicine due to their host specificity for identifying bacterial pathogens. The use of phages for the treatment of bacterial infections was discovered by Felix d'Herelle long before the discovery of penicillin and antibiotics. However, phage therapy was later considered impractical with the introduction of chemotherapy using antibiotics and gradually fell into oblivion. Due to the increasing occurrence of multiple antibiotic resistances, research is currently being conducted again on the application of bacteriophages as an alternative to antibiotics in human medicine.

A series of publications address this state of the art and deal with the general status of phage therapies and the regional statuses of medical approvals for drug products based on phages.

From the printed publication US 2004/0063 189 A1, a method for identifying and combating Bacillus anthracis on surfaces using phages is known.

Furthermore, from the publication Qadir et al. "Phage therapy: progress in pharmacokinetics," Braz. J. Pharm. Sci. 2018; 54(1): e17093, pp. 1-9, information is available on the general status of phage therapies and the regional statuses of medical approvals for drugs based on phages.

From the printed publication WO 2006/047870 A1, bacteriophage compositions and methods for their preparation are known.

Bacteriophages can be obtained from nature. For this purpose, water samples, blood samples, swabs, human or animal secretions, or other samples are taken and spread on nutrient plates. By incubating these plates (36°C - 37°C for 24 hours), existing bacteriophages can be detected through lysis plaques. By detecting the existing prokaryotes, an initial assessment can be made regarding which bacterium the found phage is lytically active against.

For purification, the plaque is punched out from the bacterial lawn and incubated in a snap-cap vessel with liquid nutrient medium for at least 10 minutes. The liquid nutrient medium is then removed, sterilized by filtration, and applied onto a previously inoculated nutrient agar plate with the corresponding bacterium, and then incubated again for 24 hours at 36°C–37°C. A plaque is punched out again and processed as described. This cycle should be repeated at least five times to ensure that the isolated phages are only clones of a single phage.

To generate larger quantities of phage clones of a phage, the sterilized phage solution from the snap-cap container is applied onto an inoculated plate after at least fivefold purification and incubated again as described. Then, an extraction buffer is added to the plate, which is moved over the agar for 30 minutes using a shaking apparatus. The extraction buffer is then removed and sterilized by filtration, resulting in a sterile phage solution.

A bacteriophage solution can be stabilized by adding stabilizers, such as CaCl₂, and adjusted to a physiological pH using substances like HCl, CH₃COOH, or CH₃COO⁻. The further addition of preservatives may be indicated if the primary packaging does not protect against microbial contamination (e.g., in non-sterile products). Preservatives such as potassium sorbate are used in this case.

Antimicrobial resistance is becoming a serious problem for the healthcare system worldwide. For decades, insufficient research has been conducted on the development of fundamentally new antibiotics, so only a few preparations have reached the market. Since then, the pressure has greatly increased to implement new effective concepts for reducing infections caused by problematic pathogens. The urgency has been recognized from the political side, and extensive funding programs have been launched both nationally and internationally. A key pillar of many publicly funded measures is the research and development of therapeutics whose effects are based on new mechanisms and/or minimize the formation of resistance.

In medical fields, infection of a foreign body is associated with an increased rate of complications and mortality; in this regard, current and prospective antibiotic therapy is exhausted.

The urgent need for antibiotic alternatives is the demand for the invention on this side.

Problems arise due to the low stability of phages in the body, as they are eliminated by phagocytic cells as foreign bodies within a relatively short time.

The present invention is based on the object of revealing bacteriophage preparations, also known as bacteriophage depots, which can be applied using technical devices, and to show corresponding application devices for these, in order to apply the corresponding bacteriophage preparations in a time- and location-controlled manner, thereby providing a preventive broad-spectrum antibiotic without side effects that can be easily administered.

In particular, the aim is to apply bacteriophages in different aggregate states and galenic compositions as biologically active agents for infection prophylaxis or infection therapy to foreign bodies and to homologous as well as xenogenic tissue.

This task or tasks is solved by the inventive intracorporeal bacteriophage delivery in the form of a gel comprising bacteriophages for use in a method for the treatment or prevention of bacterial infections, wherein the gel is sterile and is applied intracorporeally during an operation.

A two-syringe bacteriophage delivery system is characterized by the fact that a first syringe is prepared with a bacteriophage solution and a second syringe is prepared with a gel, wherein these two syringes can be connected to each other, particularly via a connector, allowing the gel to be mixed with the bacteriophage solution, which then is present in one syringe for application. In this case, the gels and also the bacteriophage solutions can be adapted accordingly to the specific requirements. In particular, different gels and/or different bacteriophage solutions can be stored separately and mixed together as needed to form an individual bacteriophage solution-gel combination.

The following are detailed descriptions of the aforementioned explanations of the invention: A bacteriophage depot or phage delivery or depot is a unit in which at least one bacteriophage or phage is provided as a phage application, which maintains stability in a time-defined manner after being introduced into a body.

It was particularly recognized that the bacteriophage is suitable both for the treatment and for the prevention of bacterial diseases or inflammations or fungal infections, especially due to the very low associated side effects. Due to the mechanism of action, which involves the multiplication of active bacteriophages only upon infection of the host by a corresponding bacterium or fungus, it is possible to administer prophylactically small amounts of bacteriophages and endotoxins that do not harm the host organism, but which can multiply rapidly when bacteria or fungi to be combated appear. In general, high doses of bacteriophages can be administered in acute infections, since they selectively lyse the pathogenic microorganisms without harming the host organism, for example, a mammal.

The following examples illustrate technical implementations of bacteriophage application for use according to the claimed invention. The objective of the presented bacteriophage application is the introduction of therapeutically active bacteriophages for the prevention and/or treatment and/or reduction of a bacterial infection. However, this is not about the therapeutic procedure itself, but rather about the necessary products/applications required for this purpose.

Intracorporeal Application: Sterile Bacteriophage Gel (release-modulated)

The sterile bacteriophage gel can be applied anywhere intracorporeally. The preparation is carried out from the corresponding bacteriophage solution: a gel is produced using a gelling agent (e.g., HPMC, ...) without including the water content that will later be provided by the bacteriophage solution, and then sterilized. Adhesive substances can be added to improve adhesion to different materials (PTFE, ceramics, Dacron, titanium, ...). The bacteriophage solution is sterilized by filtration under sterile conditions and stored sterile in a syringe. The sterilized gel is also stored sterile in a syringe. For better storage, the two components are mixed by a two-syringe technique just before application by an operating team.

The properties of the resulting gel, and thus also the bacteriophage release rate from it, are defined by the amount of gelling agent used.

For this reason, different gel bases should be kept in syringes. All bacteriophage solutions, which differ in their composition of bacteriophages, can be combined with all gel bases using the two-syringe technique.

An operator can therefore decide during the operation which viscosity/release profile and which bacteriophage composition to apply. Less viscous gels release their contents quickly, while highly viscous bacteriophage gels release them over a longer period of time. The individual syringes (gel base and bacteriophage solution) are each sterile packaged and are provided sterile by the non-sterile OR staff upon request. The sterile OR staff mixes the components using the two-syringe technique over a specified period of time. The bacteriophage gel is now ready for application.

Further production variants: bacteriophage solutions are mixed with the first part of the gel by means of machine production and then sterilized by filtration. The modulation of this base gel is carried out by adding the gelling agent in a subsequent production step under sterile conditions. Machine-assisted, sterile filling of bacteriophage solution and base gel. The base gel is then sterilized in the final container. The combination is flexible, similar to the manually described initial procedure above.

Further possibilities for sterile bacteriophage gels: A. The bacteriophages are uniformly embedded in a hydrogel matrix. The release from the hydrogel is determined by the proportion of hydrogel-forming substance relative to the water content. The product does not interact with skin or mucous membrane cells, is inert towards the human organism, and can be used intracorporeally; possible hydrogel-forming agents are carbomers, cellulose ethers, gelatin, alginates, betonid, and highly dispersed silicon dioxide. B. The phages are embedded in a lipophilic gel matrix. The release from the hydrogel is determined by the proportion of lipophilic gel-forming agent relative to the water content. In addition to the antibacterial mechanism, the product is strongly emollient and supports the natural wound healing process as a complement to antibacterial therapy/prevention. Possible lipophilic gel-forming agents are highly dispersed silicon dioxide, aluminum soaps, zinc soaps, particularly those with an appropriate lipophilic base, such as mineral oil or liquid triglycerides. C. The bacteriophages are embedded in an amphiphilic gel matrix. The release from the hydrogel is determined by the proportion of amphiphilic gel-forming agent relative to the water content.

All implants can also be introduced minimally invasively via a syringe. A percutaneous application, for example, into an abscess, is therefore possible.

The following examples illustrate technical implementations of the bacteriophage application for use according to the claimed invention. The objective of the described bacteriophage application is the introduction of therapeutically active bacteriophages for the prevention and/or reduction of a bacterial infection.

Implementation examples of the invention are described in detail in the figure description based on the accompanying drawing, and further other embodiments are also shown below, which are not illustrated figuratively. These descriptions are intended to explain the invention and should not be interpreted as limiting: It shows FIG. 1 a schematic representation of an embodiment of an intracorporeal bacteriophage application;

In Fig. 1, a schematic representation of an embodiment of an intracorporeal bacteriophage application is shown, wherein a sterile bacteriophage gel is produced from a bacteriophage solution and can be applied intracorporeally everywhere via bacteriophage application devices.

During production, a gel is made using a gelling agent, such as HPMC or similar, without the water content of the later bacteriophage solution, and then sterilized. Adhesive substances can be added during this process, which improve adhesion to different materials (PTFE, ceramic, Dacron, titanium, zinc oxide...).

The bacteriophage solution is sterile-filtered under sterile conditions and, for example, stored sterile in a syringe. The sterilized gel is also kept sterile under sterile conditions, for example, in a syringe. For better storage, the two components are mixed using a two-syringe technique just immediately before application.

The properties of the resulting gel, and thus also the bacteriophage release rate from it, are defined by the amount of gelling agent used.

For this reason, different gel bases are stored, for example, in syringes. All bacteriophage solutions, which differ in their composition of bacteriophages, can be combined with all gel bases using the two-syringe technique.

Other possibilities for supply include a stepwise approach, for example, where bacteriophage solutions are mixed with the first part of the gel by means of machine production and then sterilized by filtration. The modulation of this base gel can then be carried out in a subsequent production step under sterile conditions by adding the gelling agent appropriately. Furthermore, it is possible to perform the machine-assisted, sterile filling of the BPH solution and the base gel, and subsequently sterilize the base gel in the final container. The combination can then take place flexibly.

Other possibilities to provide sterile bacteriophage gels include embedding the phages homogeneously within a hydrogel matrix. The release from the hydrogel is determined by the proportion of hydrogel-forming agent relative to the water content. The product does not interact with skin or mucous membrane cells, is inert towards the human organism, and can be used intracorporeally, or the phages can be embedded in a lipophilic gel matrix. The release from the hydrogel is determined by the proportion of lipophilic gel-forming agent relative to the water content. The product is strongly emollient beyond its antibacterial mechanism and supports the natural wound healing process as a complement to antibacterial therapy/prevention. The phages can be embedded in an amphiphilic gel matrix. The release from the hydrogel is determined by the proportion of amphiphilic gel-forming agent relative to the water content.

All implants can also be introduced minimally invasively via the syringe. It is now for the first time possible to apply them percutaneously, for example into an abscess, in order to treat it.

The viscosity, release rate, and BPH composition can be set immediately before application. Less viscous gels release quickly, while highly viscous BPH gels release over a longer period of time.

A particularly notable application example of targeted phage application is prosthetics.

Prosthetic infections still represent one of the most severe complications in reconstructive surgery today. Prosthetic infections, especially when, for example, the aorta is involved, often have a fatal course; this particular surgical field not only appears highly complex but also particularly prone to complications due to the large number of plastic prostheses used.

Claims (1)

1. A gel comprising bacteriophages for use in a method for the treatment or prophylaxis of bacterial infections, wherein the gel is sterile and is applied intracorporeally during an operation.