WO2016131459A2

WO2016131459A2 - Method for the inactivation of pathogens using electrically produced silver ions

Info

Publication number: WO2016131459A2
Application number: PCT/DE2016/200101
Authority: WO
Inventors: Paul Schaffrath
Original assignee: Paul Schaffrath
Priority date: 2015-02-19
Filing date: 2016-02-19
Publication date: 2016-08-25
Also published as: WO2016131459A3

Abstract

The present invention relates to a method for the inactivation of pathogens by electrically produced silver ions using a device, comprising a cathode and an anode with a first silver-containing surface, the method comprising the steps of: - bringing the first surface of the anode into contact with a composition which comprises at least a pathogen, and - applying a voltage to the cathode and anode. The invention further relates to a virus which is inactivated by bringing it into contact with electrically produced silver ions as well as to the use of a pathogen which is inactivated with the method according to the invention as a vaccine.

Description

METHOD FOR INACTIVATING DISEASES WITH ELECTRICALLY PRODUCED SILVERIONS

The present invention relates to a method for the inactivation of pathogens with electrically generated silver ions, in particular for vaccine production and in therapeutic use.

Background of the invention

The development of effective vaccines for the preventive control of viral and bacterial infectious diseases is a central component of medical development. In the production of vaccines, it is imperative to inactivate the pathogens in the vaccine.

Basically, a distinction is made here between live vaccines which contain viruses or bacteria in attenuated form and dead vaccines containing inactivated and killed viruses or bacteria. In principle, it can be stated that a so-called attenuated live vaccine is significantly more effective than a dead vaccine, since the pathogens in the live vaccine are still able to reproduce in a controlled, attenuated form and thus trigger a clear immune response. For example, in the production of live vaccines, viral mutants possessing antigens such as the wild-type virus but having reduced pathogenicity are used.

In contrast, dead vaccines are more easily tolerated and rule out the danger of a disease triggered by the still active pathogens. In particular, dead vaccines, for example in pregnancies, are indicated in order to exclude any risk for the still unborn child.

The prior art discloses various ways to inactivate microorganisms in vaccine recovery. Here are, for example chemical processes are used in which microorganisms are inactivated by means of formaldehyde, hydroxylamine or ß-propio-lactone. These processes have the disadvantage that the pathogens are so strongly changed in their structure by denaturation that the immuno-reaction of the body aimed at by the vaccination is achieved only to a limited extent or the vaccine protection even fails completely.

In addition, it is disadvantageous that longer exposure times of these substances are required for inactivation of the microorganisms. Thus, the commonly used method of virus inactivation in vaccine production by treating viruses with formalin requires a long incubation period of up to 15 days to ensure a sufficient reduction in viral activity.

An alternative option is to irradiate viruses. An example of this is the irradiation of viruses by means of UV-C radiation, as used in the disinfection of indoor air for use. This method is based on the cytotoxic and mutagenic effects of UV-C radiation. Also, this method has the disadvantage that the structure of the vaccine can be so badly damaged that it becomes unusable as a vaccine.

In addition, UV-C rays can penetrate limited in, for example, liquids, so there is a risk that the pathogens are not completely inactivated. In addition, it is especially problematic to distribute the radiation dose evenly enough to inactivate all pathogens, on the other hand also to limit so that no beyond the desired level destruction or denaturation takes place, which would reduce the Impfeigenschaften the substance. An example of a chemical inactivation of viruses is described in EP 1 942 932 B1, in which hydrogen peroxide is used for inactivation.

The inactivation of enveloped and non-enveloped viruses with UV light is described, for example, in US 2006/0270017 A1. Another irradiation method for inactivation is disclosed in DE 3 505 728 C2, wherein the treatment with laser beam pulses leads to the complete destruction of the nucleic acids contained in the samples. Against this background, it is an object of the present invention to provide a method for the inactivation of pathogens, which overcomes at least one of the aforementioned disadvantages.

Summary of the invention

The present invention is based inter alia on the discovery that electrically generated silver ions have an activity against pathogens such as viruses and bacteria. Thus it could be shown in experiments that contacting with a silver anode and simultaneously applying a micro-current not only leads to the killing of bacteria, but also of viruses.

Accordingly, the invention relates to a method of inactivating pathogens by electrically generated silver ions using a device comprising a cathode and an anode having a first silver-containing surface, the method comprising the steps of:

Contacting the first surface of the anode with a composition comprising at least one pathogen and

- Applying a voltage to the cathode and anode.

The method according to the invention can be used in particular for inactivating pathogens, such as bacteria or viruses, for vaccine production. Consequently, the method according to the invention is in particular an in vitro method.

In addition, the inventor was able to show that even very small streams, which are uncritical for humans, are sufficient to remove the pathogens, In particular, bacteria or viruses to completely inactivate, so that the method is also suitable for therapeutic use.

Sonnit also relates to electrically generated silver ions for use in the treatment of bacterial infections or viral infections, the treatment being by means of a device comprising a cathode and an anode, optionally via a voltage source, electronically conductively connected to the cathode having a first silver-containing surface, carried out and the method comprises the following steps:

Contacting the first surface of the anode with a tissue of a patient infested by the pathogen

Generating an ion or electron conducting connection between the tissue and the cathode, and

- Apply a voltage between the cathode and anode.

Furthermore, the subject of the invention is a virus inactivated by contacting with electrically generated silver ions. The invention also relates to the use of a pathogen inactivated with the in vitro method of the invention as a vaccine.

The invention also relates to a device for the in vitro inactivation of pathogens, comprising: a lower part (6) with an upper-side receptacle (2) which forms the cathode,

a silver-coated insert (7) which forms the anode, as a counterpart to the receptacle (2) in the lower part (6), and

a narrow uniformly formed gap space (1 1) between the insert (7) and the lower part (6) for receiving a liquid. Finally, the subject matter of the invention is a device for inactivating pathogens, comprising a device head and a device body, which can be covered in particular by a hand, and a cathode arranged on the surface of the device body and an anode arranged on the device head with an at least partially silver-containing surface wherein a voltage can be generated on the device between the cathode and the anode, and a current flows upon contact of the cathode and the anode with tissue of the same body, and wherein the device has a control circuit which ensures a preset current independent of the actual body resistance.

characters

Fig. 1 shows an embodiment of the device according to the invention in a flat shell-like design in a lateral view as a continuous design.

Fig. 2 shows an embodiment of the device according to the invention in a flat shell-like design in plan view as a continuous design.

Fig. 3 shows an embodiment of the device in a design with high side walls in a lateral view.

Fig. 4 shows an embodiment of the device in a design with high side walls in plan view.

Fig. 5 shows the experimental setup for the treatment of bacterial pathogens according to Example 1. The silver plates near the shell edge at the positions 12 °° - 2 °° - 4 °° - 6 °° - 8 °° - 10 °° o'clock.

FIG. 6 shows detailed photographs of the culture media with bacterial grass after exposure to the electrically generated silver ions and incubation of the bacteria. FIG. 6A shows shell 1, FIG. 6B shell 2, FIG. 6C shell 3, and FIG. 6D shell 4 from example 1. FIG. 7 shows detailed photos of the culture media with bacterial grass after the action of the electrically generated silver ions and incubation of the bacteria from Example 2.

8 shows detailed photos of the culture media with bacterial grass after the action of the electrically generated silver ions and incubation of the bacteria from Example 3. FIG. 9 shows a diagram of the experimental setup according to Example 9 for the inactivation of viruses in vitro.

FIG. 10 shows a circuit diagram of a control circuit which, in the therapeutic treatment device according to the invention, provides a constant current independent of body resistance.

DETAILED DESCRIPTION OF THE INVENTION According to the invention, the term "pathogenic agent" means a substance or organism which can cause health-damaging processes in other organisms In medicine, the property of pathogenicity is assigned to such pathogens. According to the invention, pathogens may be microorganisms and viruses. According to the invention, "microorganisms" comprise algae, bacteria, parasites, and fungi.

According to the invention, a "virus" is understood to mean an infectious particle that arises from a strain of genetic material (DNA or RNA) and a protein coat.

First, as nucleic acid (DNA or RNA) in the cells of the host. The nucleic acid contains the information for its replication and for Reproduction of the second virus form. The host cell replicates the nucleic acid.

- Second, as a virion, which is discharged from the host cells, and a spread to other hosts allows. The term "virus" used according to the invention therefore also encompasses the virion.

The present invention is based inter alia on the discovery that electrically generated silver ions can be used to inactivate viruses as well as bacteria. In addition, a device for the in vitro inactivation of such pathogens has been developed.

The antibacterial effect of silver ions, especially electrically generated silver ions, is known. A device for inactivating bacteria in the oral cavity has also been described in the prior art, see WO 2009/143827 A1. Such a device has at least one anode with a first silver-containing surface. The device further comprises at least one cathode.

According to the invention, an "anode" is understood to mean an electrode on which an oxidation reaction takes place, due to the silver contained in the surface of the anode, this oxidation reaction leads to the release of silver ions.

"Electrically generated silver ions" are therefore according to the invention ions which are released from an anode due to an oxidation reaction.

According to the invention, a "cathode" is understood to mean an electrode at which a reduction reaction takes place, in which electrons are supplied via the electrical conductor and emitted to the chemical reactants via the electrode. especially ion-conducting, composition instead. The invention thus provides a method of inactivating pathogens by electrically generated silver ions using a device comprising at least one cathode and at least one anode with a first silver-containing surface, the method comprising the following steps:

Contacting the first surface of the anode with a composition comprising at least one pathogen,

Applying a voltage between cathode and anode.

For the application of a voltage, the device optionally comprises a voltage source. The oxidation reaction at the anode and a concomitant measurable current is achieved when the composition comprising at least one pathogen, in addition to the contact with the anode is also directly or indirectly connected via an ion-conducting compound to the cathode.

The voltage may either be present before contacting the pathogen-containing composition with the anode and / or the cathode or may be applied only after contacting. The voltage can be generated for example via a voltage source in the device according to the invention. Alternatively, the potential difference between the cathode and the anode may be sufficient to result in contact with the composition for leakage of silver ions on the anode surface such as a measurable current between the anode and cathode.

According to one embodiment of the method according to the invention, this is an in vitro method. Such an in vitro method for inactivating pathogens can be used, for example, for the treatment of blood plasma, for the sterilization of culture media or for the production of vaccines.

As shown in the examples, the silver concentrations measurable in the tissue or in solution after carrying out the method according to the invention very low, in particular in the range of less than 10 pg per ml. The US Environmental Protection Agency (EPA) allows a silver intake of 5 pg silver / kg body weight per day, which corresponds to 350 pg at 70 kg body weight. The WHO recommends limiting maximum silver intake to 180 pg per day. The concentrations of silver ions achieved with the method according to the invention are thus well below the permissible limit values.

Consequently, the method according to the invention can also be used to treat substances which are subsequently supplied to humans, for example substances in pharmaceutical production. In addition, a treatment on humans is harmless to health.

According to one embodiment of the method according to the invention, it is a method for inactivating pathogens in vaccine production.

The pathogen can be a microorganism. The microorganism is in particular a bacterium or a fungus. The bacterium may be an eubacterium or an archaebacterium. Preference is given to eubacteria.

In a preferred embodiment, the pathogen is a virus. In addition to the nucleic acids (DNA or RNA) and the protein shell (capsid), viruses can additionally have a lipid bilayer interspersed with viral membrane proteins. This lipid bilayer forms a shell around the capsid. Such viruses are referred to as enveloped, viruses without such envelope as unwrapped. Viruses are classified by their host specificity, the presence of a lipid envelope, and the type of nucleic acid.

The viruses according to the invention are in particular human or animal pathogenic viruses. Human pathogenic viruses are viruses that cause diseases in humans, animal pathogenic viruses are viruses that cause diseases in animals. The viruses can be enveloped or non-enveloped viruses. According to the invention, the enveloped viruses are preferably selected from double-stranded DNA viruses, single (+) strand RNA viruses, single (-) strand RNA viruses. Double-stranded DNA viruses are preferably selected from viruses of the family Toxiviridae, Herpesviridae and Hepadnaviridae. Vested single (+) strand RNA viruses are preferably selected from viruses of the families Togaviridae, Flaviviridae, Coronaviridae and Retroviridae. Examples of enveloped single (-) strand RNA viruses are Arenaviridae, Bornaviridae, Bunyaviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae and Rhabdoviridae.

The major representatives of the non-enveloped viruses include double-stranded DNA viruses, single-stranded DNA viruses and double-stranded RNA viruses. According to the invention, the non-enveloped double-stranded DNA viruses include in particular the families of Adenoviridae, Polyomaviridae and Papillomaviridae. Important representatives of the unencapsulated single-stranded DNA viruses according to the invention are the viruses of the family Parvoviridae. Major members of the single (+) strand RNA viruses are viruses of the family Caliciviridae, Hepaviridae and Picornaviridae.

An exemplary representative of Poxviridae is Orthopoxvirus variola, the trigger of true smallpox. Important representatives of herpesviridae are herpes simplex virus 1 (HSV-1), the causative agent of herpes simplex, herpes labialis and herpes stomatis aphtosa, herpes simplex virus 2 (HSV-2) of herpes simplex and genital herpes Varicella zoster virus (VZV), trigger of chickenpox and shingles, human herpes virus (HHV-6), triggers of three-day fever, Epstein-Barr virus (EBV), also known as human herpes Virus 4 (HHV-4) is known and triggers the Pfeiffer's glandular fever as well as the Burkitt lymphoma.

Exemplary member of the family Hepadnaviridae is the hepatitis B virus (HBV). An important member of the Togaviridae family is Rubivirus (rubella virus), the causative agent of the rubella. The family of Flaviviridae includes the hepatitis C virus (HCV), the dengue virus that causes dengue fever, as well as the yellow fever virus, as well as the TBE virus, the trigger of the tick-borne encephalitis.

The family of Coronaviridae includes, for example, the SARS-associated coronavirus, which cause, for example, atypical pneumonia (SARS) and toroviruses (triggers of gastroenteritis).

The family of Retroviridae includes human T-lymphotropic virus 1 (HTLV-1), inducer of adult T-cell leukemia and human immunodeficiency viruses type I and II (HIV-I and HIV-II). To the family of the Filoviridae belongs in particular the Marburg virus, trigger of the Marburg fever, the Ebola virus.

The family of Orthomyxoviridae includes, in particular, various types of influenza viruses, such as influenza A and B viruses, which cause influenza. The Paramyxoviridae family includes, in particular, the human parainfluenza virus, the cause of colds, the measles virus and the mumps virus.

The family of the Rhabdoviridae includes in particular the rabies virus (RABV), triggers of rabies. The Adenoviridae family includes, in particular, human adenoviruses A - F, whose symptoms include colds, colds and diarrhea, and JC Polyoma Virus (JCV), which leads to progressive multifocal leukoencephalopathy (PML) in immunosuppressed patients.

The Papillomaviridae family includes in particular human papillomaviruses 16, 18 and 30, triggers of the cervix tumor. Representatives of the family Parvoviridae are in particular the adeno-associated viruses (AAV) 2, 3 and 5 as well as the parvovirus B19, which causes ring rubella. The family Reoviridae include in particular different types of rotaviruses, triggers gastroenteritis (diarrhea). The family of Caliciviridae include, in particular, human noroviruses that cause vomiting diarrhea (gastroenteritis). Important representative of Hepeviridae is the hepatitis E virus. The family of the Picornaviridae include in particular the polioviruses type 1 - 3, triggers of polio as well as the human enteroviruses and rhinoviruses that cause colds and the hepatitis A virus. In a preferred embodiment, the virus is an enveloped virus. Without wishing to be bound by this theory, it is assumed that the inactivating effect against pathogens, in particular the bactericidal or virucidal effect, is connected with an interaction of the free silver ions with the lipid structures of the bacterial cell membrane or the virus envelope. Therefore, the method of the invention may be particularly suitable for the inactivation of enveloped viruses.

In one embodiment, the pathogen is a virus of the family Herpesviridae. In particular, the virus is of the family Herpesviridae selected from Epstein-Barr virus, herpes simplex 1, murine cytomegalovirus and murine herpesvirus 4.

In one embodiment, the pathogen is an Epstein-Barr virus, particularly of type A or herpes simplex virus 1 (HSV-1).

According to one embodiment, the pathogen is a virus of the family Flaviviridae, in particular a Zika virus.

In particular, the composition containing the pathogens may contain a plurality of pathogens. This can be a plurality of identical pathogens. Alternatively, the composition may also contain a plurality of different pathogens. Finally, it can be a different pathogens that are present in each case in a plurality.

According to one embodiment of the invention, the composition containing the pathogen, in particular a virus, is a liquid. This may be, for example, a virus serum, a solution of purified viruses or purified bacteria. In a virus serum According to the invention, it is a cell culture supernatant of a virus-infected cell culture containing the virus. The production of viruses such as bacteria or their purification is known in the art. A solution containing purified viruses or bacteria contains, in addition to the viruses or bacteria, only the solvent, in particular water and salts necessary for the stabilization of the pathogen. However, the solution is essentially free of other microorganisms, viruses or components thereof.

According to one embodiment of the invention, the composition containing pathogens is a virus serum.

According to one embodiment of the method according to the invention for the in vitro inactivation of pathogens, the device has a container into which the at least one cathode and at least one anode and the composition containing the pathogen is introduced. In particular, the composition is introduced into the container such that it is in contact with both the at least one cathode and the silver-containing surface of the at least one anode. It can be applied to the cathode and anode either already a voltage. Alternatively, the voltage is applied only when the composition containing the pathogen, in particular a liquid, has already been introduced into the container. According to one embodiment, the at least one cathode and at least one anode are arranged relative to one another such that the silver-containing surface and a surface of the cathode are at a constant distance from one another. Such a constant distance is advantageous, since a uniform current and hence a uniform charge carrier density can thus be ensured. This ensures a uniform inactivation effect over the entire sample.

According to one embodiment of the method according to the invention, the constant distance is in the range of 0.5 to 5 mm. Above 5 mm takes the Inactivation effect strongly. Thus, a complete inactivation of the pathogens in the composition at a distance above 5 mm can no longer be guaranteed. Although theoretically there is no minimum distance, for practical reasons the distance should not be less than 0.5 mm, otherwise the ingestible volume of the composition would be too low. Preferably, the distance between the surface of the cathode and surface and anode is in the range of 1 to 3 mm. From a distance of 1 mm, a reasonable volume of liquid can be guaranteed. Up to a distance of 3 mm, there is no measurable reduction in the inactivation effect. Particularly preferably, the distance of the cathode and anode in the range of 1, 5 to 2.5 mm.

According to a further embodiment of the method according to the invention, a voltage between cathode and anode is applied, which ensures a constant output current at the anode of at least 0.01 mA. Preferably, a voltage is applied which ensures a current of at least 0.05 mA. It could be shown that even a small leakage current - for example 0.05 mA - is sufficient to achieve complete deactivation of the pathogens. Particularly preferably, a voltage is applied to the cathode and anode, which ensures a constant output current at the anode of at least 0.1 mA. Depending on the device design, problems with current of less than 0.1 mA can cause it to remain constant.

The current of 0.1 mA corresponds to the value that can be achieved when two differently noble alloys come into contact and form a galvanic element. The charging-related currents are, because they are so small, absolutely harmless, but can lead to an unpleasant sensation (Patient Information DGZMK, German Society of Dentistry, Oral and Maxillofacial Surgery). Thus, a current can be generated without external power source.

According to one embodiment of the method according to the invention, a voltage is applied to the cathode and anode, which ensures a constant output current at the anode of at most 10 mA, preferably of at most 5 mA, more preferably of at most 1 mA. According to one embodiment, a voltage is applied to the cathode and anode which ensures a constant output current at the anode of at most 10 mA. According to one embodiment of the method according to the invention, the current density at the anode is at least 0.15 A / m ² , preferably at least 0.25 A / m ² , particularly preferably 0.28 A / m ² . A value of 0.28 A / m ² , as shown in the examples, is sufficient for complete inactivation of the viruses. It has been shown that the duration of the current impact is of particular importance for the degree of inactivation. The experiments presented in the examples indicate that, depending on the design of the device and volume of the composition to be treated, there is a certain minimum current density necessary to achieve an inactivating effect on viruses, bacteria or other pathogens. Above this minimum current density, a large improvement in inactivation efficiency is not necessarily achieved. In contrast, the inactivation efficiency is strongly dependent on the exposure time.

According to one embodiment of the method according to the invention, the pathogen-containing composition is for a duration of at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, preferably at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 90 minutes, at least 120 Kept under power for a few minutes.

The necessary exposure time of the electrically generated silver ions depends on the type of pathogen, on the current strength or applied voltage and on the type of application. It can be seen from the examples that the HSV-1 virus is completely inactivated after just five minutes at a current of 0.1 mA given the experimental setup. In one embodiment, the pathogen is a virus, the current strength is at least 0.1 mA, and the exposure time is at least five minutes. According to one embodiment if the pathogen is an HSV, preferably HSV-1, the current strength is at least 0.1 mA and the exposure time is at least five minutes. The exposure time is preferably at least 10 minutes, more preferably at least 20 minutes.

In a further embodiment, the pathogen is an HSV, in particular HSV-1, the current intensity is at least 0.05 mA and the exposure time is at least 15 minutes. The exposure time is preferably at least 30 minutes.

According to a further embodiment, the pathogen is an Epstein-Barr virus (EBV), in particular EBV type A, the current intensity is at least 0.1 mA and the exposure time is at least 15 minutes. The exposure time is preferably at least 30 minutes.

For bacteria, the preferred duration of use is at least 30 minutes for a current of at least 0.1 mA in direct contact. For inactivation of bacteria for which a penetration depth must be overcome, the minimum exposure time is preferably 60 minutes at a current of 0.1 mA.

To inactivate viruses, the virus titer in the suspension may be, for example, in the range of 10 ² to 10 ⁸ TCID50. TCID50 stands for Tissue Culture Infection Dose 50 and quantifies the amount of virus needed to kill 50% of the infected cells or produce a cytopathic effect in 50% of the inoculated tissue culture cells. Preferably, the virus titer is in the range of 10 ³ to 10 ⁷ , more preferably in the range of 10 ⁴ to 10 ⁶ TCDI50.

According to one embodiment of the method according to the invention, the in vitro inactivation of a pathogen-containing liquid is carried out with a device comprising the following components: a lower part (6) with an upper-side receptacle (2) which forms the cathode a silver-coated insert (7), which forms the anode, as a counterpart to the receptacle (2) in the lower part (6),

a narrow uniformly formed gap space (1 1) between the insert (7) and the lower part (6), wherein the liquid is filled into the gap space (7).

The device for inactivating pathogens comprises at least one cathode and an anode having a first silver-containing surface. According to one embodiment of the device, the first surface of the anode contains at least 50%, preferably at least 70%, particularly preferably at least 90% silver.

According to a preferred embodiment of the device according to the invention for inactivating pathogens, this is basically formed from an outer or lower part, which has a cup-shaped receptacle on the upper side 10, for example. In this recording, as a counterpart to the inclusion positively shaped insert, for example, a cup-shaped insert used, so that a narrow and uniformly formed gap space between this insert and the outer and lower part is formed. This gap serves to accommodate the liquid to be treated, which is treated by an input of silver ions.

With this device, with precise dosage of the parameters of the treatment, the pathogens can still be completely preserved and merely put out of action and inactivated. This is the desired result, for example, to treat a serum as a starting point for vaccine production, since the pathogens contained in the serum no longer represent a danger to the recipient and still takes place a reaction of the immune system to the remaining but disabled pathogens. Alternatively, the parameters can also be chosen so that complete destruction of the viruses is achieved.

The lower negative-shaped receiving part is in this design, the cathode. This needs no special coating and can, for example Stainless steel or similar exist. It is merely ensured that no undesired ion entry into the liquid takes place from this cathode as a receptacle, which could hinder the actual treatment. For this purpose, the cathode may for example also be coated with silver. Otherwise, no special coating is required in a basic design of the device.

The positively shaped insert forms the anode, at least partially containing silver on the surface. The anode is preferably coated with silver, in particular pure silver, or consists of pure silver. In the closed, inserted state, there is a completely uniform gap space with a distance of approximately 2 to 5 mm between the insert and the receptacle, as a result of which the liquid to be treated is distributed over a wide area in this gap space. This ensures that the treatment and the entry of silver ions over the entire surface into the liquid can be made evenly.

Therefore, an advantageous embodiment of the device is a cup-shaped receptacle with a correspondingly shaped insert. It is also a variety of other designs possible, with a formation of corners or blunt edges, for example, in the intermeshing box-shaped elements disadvantages has in particular as far as the cleaning of the device. Therefore, a cup-shaped receptacle with a correspondingly shaped insert is a preferred design.

The device is expediently not provided as a closed structural unit. Due to the oxidation reaction at the silver anode, prolonged use may cause the silver coating to discolor. This can mean a functionality restriction. In this case, the insert should be removable to clean the silver surface can. The device is expediently designed so that it is easy to clean and sterilize, since, after a treatment of, for example, a serum prior to the treatment of another serum, it must be ensured that no residues are left in the device which lead to contamination of the new one Could lead to a trial. In principle, the device can be designed, for example, for the sterilization of larger amounts of liquid advantageously as Durchlaufentkeimer. In a continuous degasser, the liquid to be treated is moved in a continuous stream at low DC and ionized by the apparatus. The movement of the liquid can be accomplished, for example, by gravity or by means of pumps.

Preferably, the device is operated by batch process. In this case, the device is supplied to a defined amount of liquid for ionization, which remains in the gap until the completion of the treatment and is removed again after the inactivation of the pathogen.

In this embodiment, no means for liquid transport such as a pump is required because the liquid is not moved through the device.

The illustration of Figure 1 shows the device according to the invention in a shell-like flat design, in which a side inlet 8 and a further arranged on the opposite side of drain 9 are provided, wherein the inlet is disposed above the level of the liquid to be treated in the device, which is shown by the dashed line 10.

The device is formed into one of the lower part 6, in which an insert 7 is inserted accurately. Between these components is the gap space 1 1, which serves to accommodate the liquid to be treated. It is relevant in this case that the gap space 1 1 has a continuous uniformly over the surface extending height, so that the distance between the anode surface 1 of the insert 7 and the cathode surface of the receptacle 2 in the lower part 6 is constant over the surface. This is of great importance, since the liquid located and treated in this gap should be ionized over the entire surface at a constant value, for which it is of central importance that the distance between the insert 7 acting as an anode and the receptacle 2 in the lower part 6 which acts as a cathode is constant, whereby the liquid is ionized at each point in the device equally by applying the weak direct current with silver. In the design illustrated in Figures 1 and 2, the device is closed on the upper side by a cover 3, for example, this cover 3 may be connected to the insert 7 and this defined in its position relative to the receptacle 2 in the lower part 6.

In the design shown in Figures 1 and 2 as Durchlaufentkeimer the arrows 4 indicate the flow direction of the liquid through the device, since here by means of a pump, the liquid to be treated is to be moved continuously through the device.

Figures 3 and 4 illustrate a design in which a liquid is filled and then treated, without a passage through the device should take place here. Depending on the pathogen to be inactivated, a defined residence time in the device is determined in order to achieve the desired degree of inactivation.

The design illustrated in FIGS. 3 and 4 also has a lower part 6, which has a receptacle 2 into which the insert 7 with its silver-coated anode surface 1 is inserted in such a way that a gap space 1 1 forming uniformly over the surface is formed. The constant The constant formation of the gap space 1 1 can be seen in particular by the synopsis of Figures 3 and 4.

Also in this embodiment of the device according to the invention is provided to form the insert 7 from the receptacle 2 of the lower part 6 removable. This is particularly relevant in order to be able to remove the insert 7, which is to be cleaned as a silver-coated component after use, from the device for this purpose. The therapeutic application of electrically generated silver ions has already been described, for example, in WO 2009/143827 A1. However, as shown in the examples, the inventor has now been able to demonstrate that in addition to a treatment of the tissue surface pathogens can be killed inside the tissue using the method. Unlike by Moisture contact resulting silver ions are electrically generated, ie released in a circuit at the positive pole, able to penetrate tissue several millimeters and thus to pass anatomical barriers, which are insurmountable obstacles for antibiotics. Accordingly, the invention further relates to electrically generated silver ions for use in the treatment of infections, wherein the treatment is carried out by means of a device comprising at least one cathode and at least one anode with a first silver-containing surface and the method comprises the following steps:

- Contacting the first surface of the anode with a

Tissue of a pathogen infested patient, generating an ionic or electron conducting connection between the tissue and the cathode and

Applying a voltage between cathode and anode.

The tissue to be treated can be, for example, the skin, a mucous membrane or the tissue of an open injury. The pathogen may be located on the body surface of the patient or in the body, for example in the bloodstream or tissue. The disease agent may also have entered an open injury.

According to the invention, the infection can be a viral infection, a fungal infection or a bacterial infection. A bacterial pathogen has preferably already penetrated into the tissue.

According to one embodiment of the electrically generated silver ions for use in the treatment, the pathogen is at most 10, preferably at most 5, more preferably at most 3 mm away from the surface of the tissue. In these penetration depths, a completely inactivating effect can be achieved when the current is well tolerated by the body.

According to one embodiment of the electrically generated silver ion treatment process, the bacterial pathogen belongs to a genus selected from the group consisting of Escherichia, Staphylococcus, Streptococcus, Pseudomonas, Enterococcus, Klebsiella, Providencia, Acinetobacter, and Borrelia.

With the therapy method according to the invention, both Gram-positive and Gram-negative bacteria can be killed, so that the treatment method is not limited to one of the bacterial groups.

In particular, the electrically generated silver ions are suitable for the treatment of bacteria which are either inaccessible to antibiotic treatment or resistant to such treatment. The antibiotic-resistant pathogens are also referred to as multi-resistant pathogens (MRE).

A well-known example of MRE is methicillin-resistant Staphylococcus aureus (MRSA). This has a mutation in the mecA gene expressing the penicillin binding protein PBP2a. This has the effect that the beta-lactam antibiotics such as penicillins, cephalosporins and carbapenems no longer dock on the surface of the pathogen and thus can not develop any activity. In addition, resistance to the fluoroquinolones, macrolides and clindamycin is often found. Cytotoxin PVL producing MRSA strains can cause recurrent deep skin and soft tissue infections, often without classical pus formation or recognizable portal of entry. Because these infections often occur outside the hospital, they are also referred to as community-aquired MRSA. These infections often affect children and young adults. In rare cases necrotizing pneumonia with high mortality occurs.

Another relevant group are Gram-negative multidrug-resistant bacteria (MRGN). According to recent resistance statistics, these pathogens show the highest growth rates among all MREs in recent years. These are mainly enterobacteria such as E. coli and Klebsiella spp. and nonfermenters such as Pseudomonas aeruginosa and Acinetobacter baumannii. The resistance is based on a variety of different resistance mechanisms, which usually affect several classes of antibiotics and their genetic code can be transmitted between different bacterial species. An example is the NDM 1 (New Delhi metallo-β-lactamase) gene and the NDM-1 strains derived therefrom.

This not only allows multidrug-resistant pathogens to be transferred from one patient to another, but also the actual resistance genes, which in turn are then incorporated by the bacteria belonging to the patient's normal flora. Under a non-targeted antibiotic therapy, these germs can then multiply unhindered and lead to infections (e.g., the urinary tract, wounds, etc.).

According to one embodiment of the method according to the invention for treating infections with electrically generated silver ions, the bacterium is selected from the group consisting of Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Pseudomonas aeruginosa, Providentia stuartii, Acinetobacter spec, Klebsiella oxytoca, Borrelia burgdorferi, Staphylococcus aureus , Streptococcus dysgalactiae, Streptococcus pyogen es, Enterococcus faecalis, Staphylococcus epidermis. It is preferably an MRSA strain. According to a further embodiment, the pathogen is a fungus. Preferably, the fungus selected from Candida albicans and Geotrichum.

The device for therapeutic inactivation of pathogens may be configured as in WO 2009/143827 A1, which is hereby incorporated by reference into this specification.

The device for the therapeutic inactivation of pathogens accordingly comprises a device head and a device body. The device body is in particular comprehensible by a human hand. The device body can thus also be referred to as the handle of the device. On the surface of the device body or the handle, a cathode is arranged. The device head has an anode with an at least partially silver-containing surface. The device also includes means for generating voltage. It acts For example, it is a battery. The battery may be rechargeable, for example. The voltage generating means may in particular be a voltage in the range of 5 to 20 V, preferably in the range of 10 to 14 V.

With the means for generating voltage, a voltage can be generated between the cathode and the anode. Upon application of the voltage between cathode and anode and a contact of the cathode and the anode with tissue of the same body, a current flows. In this case, both the cathode must be in direct or conductive contact with a tissue of the body as well as the anode with another location of the same tissue or with another tissue.

The problem is that the resistance (body resistance) formed by the body varies. It is given as an average of 1 to 3 kQ (IEC Report 60479), but can vary considerably depending on the skin moisture and varies from person to person.

On the one hand, the body resistance varies in proportion to the distance between the contact surfaces of the anode and the cathode on the same body. On the other hand, the human body due to different nature different body resistances. This would result in different amounts for the current at a given voltage. For example, in an application in the mouth, in which the anode touches the oral mucosa and the cathode, the inner surface of the hand would give a different body resistance in a first person and thus a different current.

However, in order to ensure a pre-adjustable constant current and thus in particular a constant leakage current at the silver anode, the device according to the invention for therapeutic inactivation of pathogens on a control circuit, which ensures a preset independent of the body resistance current. The control circuit used according to the invention in the device preferably has a field-effect transistor (FE transistor). For example, the FE transistor may be a depletion type. Together with the FE transistor, a first resistor may be connected in series.

FIG. 10 shows a circuit diagram for a particularly simple embodiment of a control circuit for an output current independent of the body resistance. The circuit is based on a field effect transistor Q1, which has the usual connections Drain D, Source S and Gate G. The field effect transistor used is a depletion type and is therefore in a conducting state without the presence of a control voltage at the gate G. With increasing control voltage at the gate, the drain-source path is increasingly high impedance. Between the gate G and the source S, a resistor R1 is arranged, which adjusts the current output by the circuit. Between the drain D and the connection point between gate G and resistor R1, a series circuit of a resistor R2, a light emitting diode and a reverse-biased Zener diode (Zener diode) D1 is connected. At the connection point between resistor R2 and drain D is the connection pole KG, at the connection point between resistor R1, gate G and Zener diode D1, the connection pole KBF is formed. The terminal KBF is connected to the negative terminal of a battery with an output voltage of 12V. The positive pole of the battery is connected to the silver plate (anode). The connection pole KG is connected to the stainless steel handle (cathode). The connection between silver anode and stainless steel handle is closed by contact with different surfaces of a patient's body. Thus, the skin resistance of the patient's body determines the voltage applied between the terminal poles KG and KBF.

During operation of the circuit, the output current is kept constant. When the gate-source voltage exceeds 2 V, no current flows. The operating point of the circuit is UA = 1, 5 V. The control range of the circuit is between UA and the output voltage of the battery, ie between 1, 5 V and 12 V. The value of the resistor R1 can be the output of the circuit constant current to adjust. With the resistor R1 of 6.2 kΩ used in Fig. 10, the current is 50 μΑ. The at This circuit maximum permissible body resistance, which is adjustable to an output current of 0.1 mA, is (12 V - 2V) / 0.1 mA = 10V / 0.1 mA = 100 kQ. Since typical body resistances are around 10 kΩ, the circuit can meet the required control range in typical situations.

The elements resistance R2, light emitting diode and Zener diode D1 serve as the battery test. If the resistance between silver plate and stainless steel handle becomes very low, for example due to a short circuit, a voltage close to the battery voltage of 12 V is present between the connecting poles KG and KBF. As a result, the voltage across the series circuit of resistor R2, light emitting diode and Zener diode D1 is so large that the breakdown voltage of the Zener diode D1 is exceeded. The current branch becomes conductive and a current limited by the resistor R2 can flow, which causes the LED to light up. With a deliberately caused short circuit can thus be controlled battery.

In the present case, it has been shown for the first time that viruses can be inactivated by contacting them with electrically generated silver ions. Thus, another aspect of the invention is a virus inactivated by contacting with electrically generated silver ions.

Viruses according to the invention have been described in detail above. In particular, the virus according to the invention is an enveloped virus. In one embodiment, it is an enveloped single-stranded (+) virus.

Preferably, it is a virus from the family of herpes Viridae. In particular, it is a virus selected from Epstein-Barr virus, herpes simplex 1, murine cytomegalovirus and murine herpesvirus 4.

With the electrically generated silver ions, different levels of inactivation are possible. From a pure attenuation over a complete one Inactivation until destruction of the virus structure. Based on the descriptions in the application, the person skilled in the art can determine the necessary parameters for the respective type of virus for inactivation. In particular, it can vary the duration of action, the current intensity and the distance between the electrodes. In particular, depending on whether he wants to achieve complete inactivation or even destruction of the virus. The level of inactivation may also depend on sample specific parameters - concentration of virus, solvent, etc. The person skilled in the art can, for a particular type of virus, determine the parameters to be set for the desired level of inactivation on the device by simple trial and error, and then apply them generally to this type of virus. Under certain circumstances, the sample-specific parameters must be taken into account.

For the functional characterization, several methods known in the art are available to the person skilled in the art, for example the immunostaining of viral proteins, plaque count and virus propagation assays. In addition, the treated viruses can be tested for their quality by means of virus binding assays, Western blots against various viral proteins and transmission electron microscopy.

The immunogenic properties of the treated viruses can be tested, for example, with T-cell assays and the immunization of mice. The inventively inactivated viruses are inactivated in particular by a method as described above.

For use as a vaccine, it is essential that the immunogenic properties be maintained. Thus, the virus is in particular a virus whose immunogenic properties substantially correspond to the properties of the untreated virus. This is understood according to the invention that the silver-treated viruses have an immune response of at least 50% of at least 60%, of at least 70% of 80%, of at least 90% of at least 95% in comparison with the trigger untreated virus. The method of determination is the T-cell assay.

In addition to viruses, other pathogens such as bacteria come as vaccines in questions. Examples include Clostridium tetani, trigger of tetanus, Salmonella typhi, trigger of typhoid fever (typhus) or pneumococci, cause of pneumonia.

The present invention thus further relates to the use of a pathogen inactivated by the method of the invention as a vaccine.

Examples

The present invention will be described in detail by the following non-limiting examples.

Example 1 - Treatment of multidrug-resistant staphylococci with electrically generated silver ions

1.1 Experiment description

For the test series the device "SilVi" is used. With this device, silver ions can be generated electrically. The handle contains a power source and control electronics to achieve a constant current. The handle also has a partially metal surface as a cathode, which comes into contact with the palm during gripping. The anode, a pure silver plate on the device head, is placed on the diseased tissue. This closes the circuit and silver ions are released to the affected region. The electronics ensure that the current remains constant at the set value regardless of the body resistance. The silver anode is always connected to the handle via a cable.

Four stainless steel bowls were used, freshly filled by the laboratory with a DEV medium from the company Oxoid. The culture media of two dishes was coated with a 0.5 McFarland solution of one MRSA patient strain (hereinafter abbreviated to PAT), the other two with an MRSA ATTC reference strain. Six SilVi instruments were positioned on each of the four cups so that the device heads with the silver plate were located on the surface of the medium.

In order to make the test setup visually clear, the silver plates were located near the edge of the bowl at the positions 12 °° - 2 °° - 4 °° - 6 °° - 8 °° - 10 °° o'clock. This is shown in FIG. 5. These are referred to below as measuring points 1 to 6. The handles (minus poles) were connected via a cable with the associated steel shell. In order to make the / nv / trcA / request as realistic as possible, a resistance of 10 kQ was interposed to simulate the electrical body resistance that was missing in the experiment.

For the experiment, a higher value was chosen to ensure that the achieved effect is achieved even with above-average body resistance and thus poorer electrical conductivity.

The devices were calibrated to currents of 0.4 mA, 0.2 mA and 0.1 mA. Action times of 15 to 120 minutes were tested.

As a negative control, another device was additionally placed in dish 1 so that the device head with the silver plate came to rest centrally in the middle of the dish. However, its handle was not cabled to the shell, so that no current could flow.

This should be ruled out that an antimicrobial effect already takes place by the pressure of the device head or by the contact of the silver surfaces without electricity. After the reaction time, the devices were removed from the dishes and the dishes incubated for 12 hours in the incubator at 36 ° C.

1.2 Results

1.2. 1 evaluation dish 1 PA T

All 6 measuring points with electricity were 100% bacteria-free. Also bacteria-free was a 2.5 millimeter wide yard around the contact surfaces of the silver plates, clearly recognizable, as the silver plates had left an edgy imprint on the edge in the nutrient medium. The remaining surface of the medium was completely covered with a bacterial lawn, also the central negative control measuring point, where the silver plate was without electricity for 120 minutes. Table 1: Shell 1 PAT

FIG. 6A shows a detail photograph of the shell 1 after incubation for 12 h at 36 ° C., in which the impressions of the electrodes are clearly visible. The central impression of the negative control without electricity is completely overgrown. This suggests that the bactericidal effect of silver-containing wound dressings or dressings in the action of electrically generated silver ions is significantly lower.

1.2.2 Evaluation of shell 2 PA T Table 2: Shell 2 PAT

Position Current [mA] Duration [min] Result

1 0,4 60 100% bacteria-free

2 0.2 60 100% bacteria-free

3 0.2 30 bacteria by about 90%

reduced

4 0.1 45 bacteria by about 90%

reduced

5 0.1 30 bacteria by about 90%

reduced

6 0.1 15 Chance of growth The culture medium of the shell 2 after carrying out the experiment is shown in FIG. 6B. It showed a complete elimination of bacteria at the measuring points on 1 and 2 with surrounding courtyard of 2.5 millimeters. At the other measuring points there was weak bacterial growth, about 90% less than at the untreated remaining area. For electrodes 3, 4 and 5, a surrounding edge area of about 2 mm was completely free of growth.

1.2.3 Evaluation of shell 3 MRSA A TTC reference strain

Test parameters are shown in Table 3.

Table 3: Shell 3 MRSA ATTC reference strain

The culture medium of the shell 3 after carrying out the experiment is shown in FIG. 6C. The result was a complete elimination of all bacteria at all measuring points including surrounding courtyard of 2.5 mm.

1.2.4 Evaluation of shell 4 MRSA A TTC reference strain

The test parameters are shown in Table 4.

Table 4: Shell 4 MRSA ATTC reference strain

Position Current [mA] Duration [min] Result

1 0,4 60 100% bacteria-free

2 0.2 60 100% bacteria-free 3 0.2 30 100% bacteria-free

4 0, 1 45 100% bacteria-free

5 0, 1 30 100% bacteria-free

6 0, 1 15 100% bacteria-free

The culture medium of the shell 4 is shown in FIG. 6D. The positions 1 - 4 were completely bacteria-free including surrounding courtyard of 2.5 millimeters. In position 5 tiny bacterial colonies were visible (about 99% reduction), the surrounding courtyard of 2.5 millimeters was free. At position, the surrounding yard of 2.5 millimeters was also free.

Example 2 - Treatment of various other bacterial strains with electrically generated silver ions

In this experiment, the effect of electrically generated silver ions on different bacterial species was tested at constant experimental parameters.

Eight stainless steel trays were filled with a DEV medium from the company Oxoid. The dishes were divided into four quadrants and coated with 4 different 0.5 McFarland bacterial solutions each. In each quadrant, a "SilVi" device was then positioned.

The negative control, a device head of pure silver without power, was centrally located in shell 8 (third quadrant). Trays 1 and 2 were fed with Staphylococcus aureus, Escherichia coli, Streptococcus dysgalactiae equisimilis, Streptococcus pyogenes, Pseudomonas aeruginosa, Enterococcus faecalis and Staphylococcus epidermidis. On trays 3 to 8 a total of 24 different MRSA patient strains were streaked. After one hour of exposure (0.1 mA current), an incubation in the incubator at 37 ° C. for 18 hours followed.

The results are summarized in Table 5. All 32 experimental batches were completely bacteria-free, as shown graphically in FIG. Table 5: Overview of the bacteria used and test results

Cup Quadrant Name Result

1 1 Staphylococcus aureus no growth

1 2 Escherichia coli no growth

1 3 Streptococcus dysgalactiae no growth

1 4 Streptococcus pyogenes no growth

2 1 Pseudomonas aeruginosa no growth

2 2 Enterococcus faecalis no growth

2 3 Pseudomonas aeruginosa no growth

2 4 Staphylococcus epidermidis no growth

3 1 MRSA no growth

3 2 MRSA no growth

3 3 MRSA no growth

3 4 MRSA no growth

4 1 MRSA no growth

4 2 MRSA no growth

4 3 MRSA no growth

4 4 MRSA no growth

5 1 MRSA no growth

5 2 MRSA no growth

5 3 MRSA no growth

5 4 MRSA no growth

6 1 MRSA no growth

6 2 MRSA no growth

6 3 MRSA no growth

6 4 MRSA no growth

7 1 MRSA no growth

7 2 MRSA no growth

7 3 MRSA no growth

7 4 MRSA no growth

8 1 MRSA no growth

8 2 MRSA no growth

8 3 MRSA no growth

8 4 MRSA no growth Example 3

As in the previous experiments with the gram-positive pathogens, stainless steel trays filled with a DEV medium (Oxoid) were used.

The dishes were divided into quadrants and coated with 0.5 McFarland solutions of various pathogens. The assignment of the quadrants is described below.

Bowl 1

1) Klebsiella pneumoniae

2) Escherichia coli

3) Enterobacter cloacae

4) Escherichia coli

Bowl 2

5) Pseudomonas aeruginosa

6) Klebsiella pneumoniae

7) Providentia stuartii

8) Acinetobacter spec.

Bowl 3

9) Klebsiella oxYtoca

10) Escherichia coli

1 1) Escherichia coli

12) Escherichia coli shell 4

13) Candida albicans

14) Candida albicans

15) Candida albicans

16) Geotrichum Bowl 5

17) Escherichia coli

18) Klebsiella pneumoniae

19) Klebsiella pneumoniae

No. 1 -9 was MRGN.

The devices were positioned so that the silver plate of each device was in the center of each test area. The exposure time was 1 hour. All devices were calibrated to the current of 0.1 mA. The handles were connected by cable to the stainless steel plates, and in each case an intermediate 10 kΩ resistor was used as a substitute for the missing body resistance in the experimental setup.

After one hour, the circuits were interrupted, the devices removed and the samples incubated. As a negative control, a device without current flow was positioned at the lower edge of the measuring field 1 in the dish 1, at the border to measuring field 2.

The evaluation after 24 hours of incubation gave the results shown in FIG. 8: All tested bacteria were 100% killed in the region of the contact area of the silver electrodes. On the measuring field 16, no growth of the sprout fungus Geotrichum was recognizable over the entire surface. It is thought that the soil is unsuitable for Geotrichum.

The surrounding inhibitory sites were different, the most extensive (about 5 mm) in Candida albicans. There was no antimicrobial effect in the negative control area. Example 4 - Treatment of borrelia in culture with electrically generated silver ions

A low passage of the B. afzelii strain PKo was used, which could originally be isolated from the skin of a patient. The strain was taken 10 days before the start of the experiment in 2 x 7 ml MKP medium and grown at 33 ° C in 2 hermetically sealed 8 ml glass tube. Before starting the test, the Borrelia were examined for dark field microscopy for appearance and motility, the cultures were mixed and adjusted to about 10 ⁶ Borrelia / ml.

The experiments took place because of the high oxygen sensitivity of Borrelia with oxygen occlusion. Petri dishes with a diameter of 34 mm and a height of 10 mm were used (Fischer Laboratory GmbH, Frankfurt am Main), which were filled 5 mm high with the Borrelia-containing culture medium. The Borrelia concentration was 10 ⁶ / ml. To allow a DC to act on the solution in the desired constant current, the Petri dishes were prepared as follows:

At the bottom of each Petri dish was a minus pole a pure silver electrode. 5 mm above the bottom of the tray was positioned a round pure silver plate with a diameter of 25 mm and a thickness of 0.6 mm, which was fixed at the edge of the petri dish by means of a holding structure made of sterilisable plastic and silver wire.

To check the current parameters, all circuits were checked by means of a multimeter before and after the test. In addition, each circuit was equipped with control LEDs in order to be able to check the uniform current flow during the experiment. All Petri dishes including attached electrodes were sterilized and sealed before the experiments. In order to exclude any damage to the nnikroaerophilen Borrelien by oxygen contact, all experiments were carried out under exclusion of oxygen in a glass vessel (dessicator) with Gas Paks (Gas Generating Kit, OXOID). The experimental conditions of the individual samples are given in Table 6 below. The following test conditions were selected:

Table 6: Test Conditions of Samples 3 to 11

Two negative controls (Samples 1 and 2) were used to ensure that the antibacterial effect was not due to nonspecific effects but to the combination of silver ions with microcurrent.

As negative controls served

- A equipped with silver electrodes Petri dish with Borrelia culture without current flow (sample 1) and

- a Petri dish with platinum electrodes instead of those made of silver with a current of 0.2mA. Both negative control experiments were carried out over a period of 30 minutes. All samples were monitored after dark field microscopic examination, transferred to a 2 ml plastic tube (Eppendorf) and fed with new medium. Further microscopic controls with appropriate evaluation were carried out after 1 day and, associated with medium change, then once a week for 6 weeks.

Results

In both negative control groups, samples 1 and 2, normal growth with many, well-mobile Borrelia was detectable for all dark-field microscopic controls over 6 weeks. The concentration of Borrelia at the final check was 5 x 10 ⁷ / ml.

In the samples 3 to 1 1 (verum group) on the day after the treatment still mobile Borrelia were visible dark-field microscopically. The count showed no significant difference compared to the negative controls.

After one week, the mobility in samples 3 to 1 1 was greatly reduced and after two weeks the Borrelia bacteria showed no movement. Only immovable structures were seen, which decreased slowly in the weekly controls. After six weeks, no Borrelia could be seen.

Example 5 - Action of electrically generated silver ions on tissue

As a test material for the in vitro experiments were used preparations of pig oral mucosa and pig jawbone. The preparations used were 1 mm thick and had a size of about 1 x 1 cm. Several identical SilVi prototypes (see experiment 1) were used which were calibrated to a constant direct current of 0.3 mA. The current constancy was checked by measurements immediately before and after the experiments. For the experiments, the tissue samples were placed on a steel plate. Were The treatment devices were positioned so that the silver anode was in contact with the surface of the tissue sample.

The cathodes were each connected to the steel plate via an intermediate 10 kΩ resistor, which simulates the electrical resistance of the human body in the experimental set-up.

A total of eight tissue samples were measured, including two negative controls, two silver ion limit testing samples and four silver ion penetration depth probes when used by SilVi.

5.1 Determination of the silver content of untreated samples:

Tissue samples 1 (porcine oral mucosa) and 2 (porcine bone) were checked for their silver content as negative controls untreated to exclude any pre-existing contamination of the samples.

The complete samples were analyzed for their silver content by atomic absorption spectroscopy analysis. Methodology: Ph. Eur. 6.012.02.23. 00 (AAS graphite tube after acid digestion)

The silver content of both samples was below the detection threshold (<0.025 pg). Therefore, contamination of the tissues with silver prior to testing can be excluded.

5.2 Silver content after application of silver ions under microcurrent

Samples 3 and 4 (both porcine oral mucosa) were treated with SilVi for 30 and 60 minutes, respectively, as described in the experimental set-up. The complete samples were analyzed for their silver content by atomic absorption spectroscopy analysis.

Methodology. Ph. Eur. 6.012.02.23. 00 (AAS graphite tube after acid digestion) Tissue sample 3 (pig oral mucosa, 30 min): 0.298 pg Tissue sample 4 (porcine oral mucosa, 60 min): 0.625 pg

5.3 Checking the penetration depth in mucous membrane

In this experiment, it was to be determined whether mucosal tissue is a barrier to the penetration of silver ions into deeper tissue layers.

For this purpose, three approximately 1 mm thick and 10 mm x 10 mm large porcine mucosal layers were positioned one above the other. The bottom layer lay on the steel plate (negative pole), on the top lay the silver device head on (plus pole). Only the silver content of the middle (sample 5) and lower layer (sample 6) was measured.

The silver content of the upper layer was not measured, since the aim of the experiment was to verify the ability to penetrate into deeper tissue layers.

The test duration was 30 minutes at a current of 0.3 mA.

Sample 5 (middle layer, porcine oral mucosa): 0.527 pg

Sample 6 (lower layer, porcine oral mucosa): 0.291 pg

Methodology. Ph. Eur. 6.012.02.23. 00 (AAS graphite tube after acid digestion)

The measured values of the treated samples show that the silver ions are able to penetrate several millimeters of mucous membrane. The concentrations were below 1 pg, well below the permissible limits.

Verification of the ability to penetrate bone tissue with tissue samples 7 and 8.

In this experiment, it was to be determined whether bone tissue is a barrier to the penetration of silver ions into deeper tissue layers. To A bone preparation was positioned between two mucosal preparations, each about 1 mm thick and about 10 mm x 10 mm in size.

The lowest layer lay on the steel cathode, on the uppermost was the head of the device with the silver anode. The test duration was 30 minutes at a current of 0.3 mA.

5.4 Verification of penetration into mixed tissue

only the silver content of the middle (sample 7, bone) and lower layer (sample 8, mucosa) was measured. The upper layer was discarded. Methodology: Ph. Eur. 6.0 12.02.23. 00 (AAS graphite tube after acid digestion)

Measured silver concentrations

Sample 7 (middle layer, porcine jawbone): 0.324 pg

Sample 8 (lower layer, porcine oral mucosa): 0.633 pg

The measured values of the treated samples demonstrate the ability of silver ions to penetrate bone tissue as well as mucosal tissue. These measurements were well below the permissible limits.

Finally, it should be expressly understood that the above-described embodiments of the device according to the invention are only for the purpose of discussion of the claimed teaching, but not limit these to the embodiments.

Example 6 - Inactivation of viruses with electrically generated silver ions

For inactivation, the apparatus shown in FIG. 9 is used. This includes six sample chambers. The chambers have an elliptical cross section with a length in the longitudinal direction of 27 mm and in the transverse direction of 20 mm. The depth of the chamber is 2 mm. The chambers have been optimized to accommodate SilVi equipment. The silver anode of the SilVi device is also elliptical with a length in the longitudinal direction of 25 mm and in the transverse direction of 18 mm. The silver anode head of the SilVi device closes the Chamber like a lid. The opposite side of the SilVi device is formed by a soft brass plate coated with silver.

The inactivation of four members of the herpes family was tested:

1) Epstein Barr virus (EBV)

2) herpes simplex virus (HSV I)

3) murine cytomegalovirus (mCMV)

4) murine herpesvirus 4 (mHV 4)

The viruses were produced using the human embryonic virus line HEK-293 in Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich). The medium additionally contained 10% heat-inactivated fetal bovine serum (HIFBS, Sigma-Aldrich), 1% L-glutamine and 1% penicillin-streptomycin (Gibco).

Virus preparation proceeded as follows: HEK-293 cells were grown to confluency in 70 cm ² bottles. Then the culture medium was removed and a virus / medium inoculum added to the cells and allowed to grow for several hours at 37 ° C to allow the cells to ingest the virus. Subsequently, the cells were incubated for a further three days at 37 ° C until the cells have a cytopathic effect. To obtain the virus culture, the cells were then filtered off.

These supernatants with infectious herpesviruses were first examined for their virus titer by plaque count. In addition, the ability of EBV to transform primary B cells was measured.

Subsequently, the supernatant was filled in the chambers described above. The samples in the different chambers were then treated with different currents and over different time periods. These are summarized in Table 7. Table 7. Parameters tested for in vitro virus inactivation

After treatment with electrically generated silver ions, virus titer was again determined as a measure of the infectious potential of the treated viruses by means of plaque count and the ability to transform primary B cells.

Finally, it was tested to what extent the treatment of viruses with silver ions influences their immunogenic properties. For this purpose, a T-cell assay was carried out on the one hand.

T cell clones directed against EBV proteins and B cells are firstly incubated with untreated or silver ion-treated viruses and the results are compared. Second, humanized mice were infected with silver ion-treated viruses. Six weeks later, T cells were isolated from the spleen and tested for their ability to recognize EBV proteins. Furthermore, mice were inoculated with silver ion-treated viruses and treated with wild-type virus six weeks later. For a further six weeks, these mice were examined histologically and by qPCR to detect a chronic viral infection. The results are summarized below.

Example 7 - Inactivation of HSV-1 by electrically generated silver ions

This example demonstrates that inactivation of HSV-1 by electrically generated silver ions is possible.

For this purpose, a starting solution of the HSV-1 suspension with an infection titre of 1 × 10 ⁵ × ³⁸ (TCID50) was used. For the inactivation experiments, the starting solution was diluted 1:10. For inactivation, the apparatus shown in FIG. 9 and described in Example 6 was used. The virus suspension was treated with 0, 1 mA or 0.05 mA current for 2, 5 or 15 minutes. Subsequently, the respective virus suspensions were frozen at -80 ° C.

For the HSV-1 infection experiments, human embryonic lung fibroblasts were used. The trypsinized lung fibroblasts were cultured in Eagle's Minimal Essential Medium which additionally contained non-essential amino acids, 2 mM L-glutamine, 25 mM HEPES, 10% fetal calf serum and 1 μg / ml ciprofloxacin hydrochloride. The cells were then incubated as monolayer cell cultures in cell culture tubes (Nunclon® Tube) at 37 ° C and 1% CO2 for approximately 48 hours. After incubation, the culture medium was changed and added to re-thawed virus suspension to be tested. After adding the virus suspension to the lung fibroblasts, they were incubated for 7 days at 37 ° C and 1% CO2. Subsequently, the vitality of the cells was evaluated by light microscopy (see Table 8). Batches # 1 and # 5 were included as negative controls to ensure that the antiviral effect was not due to nonspecific effects or only by the combination of microcurrent and silver ions. Approach No. 6 served as a positive control, here the untreated virus starting solution was used.

Table 8: Inactivation of HSV-1 as a function of current and time

* Titer about 1x10 ⁴

* ^* Titer approx. 1x10 ⁵

When the cells died after 5 days, i. virus titer was cytotoxic, no virus inactivation occurred, at 100% viral inactivation cell growth was normal.

In both negative controls (batch no. 1 and no. 5) normal cell growth was observed. In the positive control (batch no. 6), as expected, all cells had died after 5 days.

In runs no. 2, 7 and 8, no complete inactivation of the viruses took place and the cells died after 5 days. In runs 3, 4 and 9, the viruses were so damaged that no infection of the cells occurred and normal cell growth took place. The results show that the combination of microcurrent and silver ions reliably inactivates HSV-1. It is enough to have a current of 0, 1 mA and a contact time of 5 minutes to inactivate the virus. Example 8 - Inactivation of EBV type A by electrically generated silver ions

This experiment was performed to investigate the inactivation of Epstein-Barr virus (EBV) type A by means of electrically generated silver ions. For this purpose, 2 ml each PBS buffered virus suspension with a TCID50 of 1 x 10 ⁶ each in a stainless steel dish were added. Subsequently, the silver electrode was immersed in the suspension and the circuit closed. The application times and the current intensities were varied (see Table 9). The thus treated EBV were then added to a B-lymphocyte suspension and then incubated. The activity of the viruses was determined by the absence of transformation of B lymphocytes. For this purpose, the transformed and non-transformed B lymphocytes were microscopically determined and counted in one batch. The percentage of untransformed B lymphocytes to the total amount of B lymphocytes is thus a measure of inactivation. The results are summarized in Table 9.

Table 9: Inactivation of EBV as a function of current and time

Experimental setup Amperage Application time Inactivated viruses

1 stainless steel bowl + SilVi 0.05 mA 5 min 5%

2 stainless steel bowl + SilVi 0.05 mA 10 min 30%

3 stainless steel bowl + SilVi 0.05 mA 15 min 30% 4 stainless steel bowl + SilVi 0.1 mA 1 min 5%

5 stainless steel bowl + SilVi 0.1 mA 2 min 90%

6 stainless steel bowl + SilVi 0.1 mA 5 min 90%

7 stainless steel bowl + SilVi 0 mA 15 min 10%

8 0 mA untreated / 0%

negative control

The EBV inactivation tests also show a dependency of the virus inactivation on the current intensity and the duration of action of the electrically generated silver ions.

It can be seen that up to 30% of viruses could be inactivated after 10 and 15 minutes, respectively, when using the SilVi device with a current of 0.05 mA. By contrast, the increase in the current intensity to 0.1 mA already resulted in an inactivation rate of 90% after 2 minutes. In summary, the results show that electrically generated silver ions already at 0.1 mA and a short application time lead to a reliable inactivation of the EBV.

LIST OF REFERENCE NUMBERS

1 anode surface

2 recording

3 lids

4 flow direction of the liquid

6 lower part

7 use

8 lateral inlet

9 process

11 gap space

Claims

A method of inactivating pathogens by electrically generated silver ions using a device comprising at least one

Cathode and one, at least one anode having a first silver-containing surface, said method comprising the steps of:

- Apply a voltage between the cathode and anode.

2. The method according to claim 1, characterized in that the at least one pathogen is a microorganism, in particular a bacterium or a fungus.

3. The method according to claim 1, characterized in that the at least one pathogen is a virus.

4. The method according to claim 3, characterized in that the virus is a clogged virus, in particular a culled virus is selected from the

Group consisting of Epstein-Barr virus (EBV), herpes simplex virus (HSV-1), murine cytomegalovirus (mCMV) and murine herpesvirus 4.

5. The method according to any one of claims 1 to 4, characterized in that the constant distance in the range of 0.5 to 6 mm, preferably in the range of 1 to 3 mm, more preferably in the range of 1, 5 to 2.5 mm.

6. The method according to any one of claims 1 to 5, characterized in that a voltage is applied to the cathode and anode, which ensures a constant output current at the anode of at least 0.05 mA, preferably of at least 0.1 mA.

7. The method according to any one of claims 1 to 6, characterized in that the voltage for a period of at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 30 minutes, preferably at least 60 minutes.

8. The method according to any one of claims 1 to 7, characterized in that the device comprises the following components:

a lower part (6) with an upper-side receptacle (2), which forms the cathode,

a silver-coated insert (7) which forms the anode, as a counterpart to the receptacle (2) in the lower part (6),

a narrow uniformly formed gap space (1 1) between the insert (7) and the lower part (6),

the composition containing the pathogens is a liquid and the liquid is filled into the gap space (7).

9. Electrically generated silver ions for use in the therapeutic treatment of infections, wherein the treatment is carried out by means of a device comprising a cathode and an anode optionally electronically conductively connected to the cathode having a first silver-containing surface with the cathode and the method the following steps include:

Contacting the first surface of the anode with a tissue of a patient afflicted by the pathogen,

Generating an ion or electron conductive connection between the tissue and the cathode and

Applying a voltage between cathode and anode.

Electrically generated silver ions for use according to claim 9, characterized in that the tissue is the skin, a mucous membrane or the tissue of an open injury.

1 1. Electrically generated silver ions for use according to claim 9 or 10, characterized in that the pathogen at a distance of at most 15 mm, preferably at most 10, more preferably at most 5 and in particular at most 3 mm away from the surface of the fabric.

12. Electrically generated silver ions for use according to any one of claims 9 to 1 1, characterized in that the pathogen is a bacterium.

13. Electrically generated silver ions for use according to any one of claims 9 to 1 1, characterized in that the pathogen is a virus, in particular an enveloped virus.

14. Electrically produced silver ions for use according to any one of claims 9 to 13, characterized in that a voltage at cathode and anode is applied, which ensures a constant output current at the anode of at least 0.05 mA, preferably of at least 0.1 mA ,

15. Electrically produced silver ions for use according to any one of claims 9 to 14, characterized in that the voltage is maintained for a period of at least 30 minutes, preferably at least 60 minutes.

16. Virus inactivated by contacting with electrically generated silver ions.

17. Virus inactivated by a method according to any one of claims 1 to 9.

18. Use of a pathogen inactivated by a method according to any one of claims 1 to 9, as a vaccine.

19. A device for inactivating pathogens, comprising:

a lower part (6) with an upper-side receptacle (2), which forms the cathode,

a narrow uniformly formed gap space (1 1) between the insert (7) and the lower part (6) for receiving a liquid.

20. A device for inactivating pathogens, comprising a device head and a device body, which is particularly encompassed by a hand, and arranged on the surface of the device body cathode and an anode arranged on the anode head with an at least partially silver-containing surface, wherein the device between the

Cathode and the anode, a voltage can be generated and in contact of the cathode and the anode with tissue of the same body, a current flows, characterized in that the device has a control circuit which ensures a preset independent of the actual body resistance current.