SILVER NANOPARTICLES FOR USE IN THE TREATMENT OF RESPIRATORY INFECTIONS

Description

Technical field

[0001] The invention relates to an aqueous formulation containing silver nanoparticles for use as a drug. State of the art

[0002] According to a general definition, "nanoparticles" are particles with a size of less than or equal to 100 nm. The use of the prefix "nano" thus distinguishes them, according to the official definition in ISO TC 229, from particles in the sub-micrometer range (> 100 nm). Generally, it can be assumed that substances designated as nanomaterials possess altered chemical and physical properties. Nanometals exhibit different colors than the corresponding metals; for example, gold is red.

[0003] Furthermore, it is scientifically proven that nanoparticles of a substance possess an increased surface energy. The smaller the particles, the higher their surface energy. Consequently, nanoparticles are generally considered unstable, as their high surface energy allows them to readily react to form new compounds or larger, more stable aggregates. For example, with nanometals, this means that even particles of noble metals oxidize rapidly with atmospheric oxygen once their size reaches the nanometer range.

[0004] Technologically usable nanoparticles are obtained when their surfaces are chemically or physically protected and thus stabilized. Methods for stabilizing nanoparticles in dispersions are known from the prior art (see, e.g., WO 2008 / 017176 A1, WO 2009 / 072911 A1). Various processes exist for producing stabilized metallic nanoparticles. The nanoparticles can, for example, be supported on solid substrates. In addition, metal nanoparticles can be stabilized during production using polymers such as polyvinylpyrrolidone in the polyol process. Finally, metal nanoparticles can be produced by a PVD (Physical Vapor Deposition) process, in which the underlying metal is vaporized. Polymers or silicones are used to stabilize the nanoparticles produced in this way.

[0005] The antiviral activity of metal nanoparticles has been the subject of research for some time. An overview of this research area can be found in Maduray et al., ("Metal Nanoparticles: a Promising Treatment for Viral and Arboviral Infections" Biological Trace Element Research 199, 3159-3176 (2021), https: / / doi.org / 10.1007 / s12011-020-02414-2). Furthermore, the use of silver nanoparticles as an antiviral agent against SARS-CoV-2 was investigated, not as a drug, but for disinfection (Jeremiah et al. "Potent antiviral effect of silver nanoparticles on SARS-CoV-2" Biochemical and Biophysical Research Communications, Volume 533, Issue 1, 26 November 2020, pp. 195-200, doi: 10.1016 / j.bbrc.2020.09.018). The study demonstrated the antiviral efficacy of silver nanoparticles (AgNPs), which, however, is limited by the cytotoxic effects associated with the silver nanoparticles.

[0006] LV XIAONAN ET AL: "Inhibitory effect of silver nanomaterials on transmissible virus-induced host cell infections", BIOMATERIALS, ELSEVIER, AMSTERDAM, NL, Vol. 35, No. 13, 10 February 2014 (2014-02-10), pages 4195-4203, ISSN: 0142-9612, DOI: 10.1016 / J.BIOMATERIALS.2014.01.054 shows in vitro antiviral efficacy of silver nanoparticles against coronaviruses.

[0007] Zachar (“Formulations for COVID-19 Early Stage Treatment via Silver Nanoparticles Inhalation Delivery at Home and Hospital”, ScienceOpen Preprints, DOI: 10.14293 / S2199-1006.1.SOR-.PPHBJEO.v1) investigated the possibility of achieving an effective minimum inhibitory concentration (MIC) of silver nanoparticles at various target sites in the respiratory system to suppress both viral and bacterial respiratory infections. Applications include (i) controlling local COVID-19 outbreaks through early treatment at home and (ii) reducing the risk of ventilator-associated pneumonia (VAP) in a hospital intensive care unit.The aim was to propose a first-line intervention with the potential to suppress the spread of viral infection throughout the respiratory system, thereby allowing more time for an adequate immune response and reducing the risk of infection exacerbation and spread. Furthermore, the available credible evidence for human safety via inhalation is discussed to facilitate clinical trials. Based on previously published experimental data on the antiviral efficacy of colloidal silver, a modeling method and calculation are proposed to achieve the antiviral MIC of silver particles at various locations in the respiratory system by: (a) analyzing the required concentration as a function of nanoparticle size; and (b) calculating the required aerosol delivery properties.To calculate the required dose, he considers deposition losses and the inhalation time fraction of the normal breathing cycle. He evaluates the independent targeting of: (i) the tracheal-bronchial tree (mucus volume of approximately 1 cc) and (ii) the alveoli (total mucus volume of approximately 10 cc). No experimental evidence is provided. It is shown that the dosage is sensitive to the size of the silver nanoparticles, with 3 nm to 7 nm being the optimal size based on calculations. The effective antibacterial MIC is estimated at 10 µg / ml based on calculations, but for greater safety, 25 µg / ml is given as a reasonable target concentration to be achieved in the respiratory tract mucus. In particular, he claims that using colloidal silver with 5 nm particles, inhalation of standard 5 µm diameter aerosol droplets (e.g.,Using commercially available ultrasonic nebulizers, a sufficient MIC can be achieved with: (i) deposition of only 0.25 cm³ < a source concentration of 100 ppm (µg / ml) in the bronchial tree and (ii) deposition of 1 cm³ < a source concentration of 250 ppm (µg / ml) in the pulmonary alveoli. After taking deposition losses into account and considering that the active inhalation time is only about 1 / 3 of the respiratory cycle, he calculates that a practically effective MIC can be achieved with these aerosol doses: (a) for the upper respiratory tract and bronchial tree with 2 cc of a 100 µg / ml colloidal silver source, while (b) for the pulmonary alveoli, 6 cc of a 200 µg / ml colloidal silver source are used. This would be reduced by a factor of 3 if a breath-operated ultrasonic nebulizer is used.He concludes from the calculations that an effective MIC can be achieved in both the bronchial tree and the alveoli (although the specific aerosol delivery may differ). Since respiratory infections most commonly begin in the upper respiratory tract, he suggests that the presented method is best used early as a first-line treatment to suppress the progression of the infection.

[0008] Coronaviruses are widespread among mammals and birds. In humans, they primarily cause mild colds, but can sometimes also cause severe pneumonia. In early 2020, the new beta-coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus type 2) was identified as the cause of the disease COVID-19.

[0009] SARS-CoV-2 uses the enzyme ACE2 as a receptor to enter host cells. ACE2 levels are particularly high in the respiratory tract, which is why SARS-CoV-2 very frequently causes respiratory infections. Pneumonia can develop, usually in the second week of illness, and may progress to acute respiratory distress syndrome (ARDS) requiring mechanical ventilation, potentially necessitating extracorporeal membrane oxygenation (ECMO).

[0010] Only a proportion of COVID-19 cases are severe. Treatment focuses on providing optimal supportive measures according to the severity of the illness (e.g., oxygen therapy, fluid replacement, and, if necessary, antibiotics to treat bacterial co-infections) as well as treating any relevant underlying medical conditions.

[0011] Many different specific therapeutic approaches (directly antiviral, immunomodulatory) have been and are being investigated in studies during the SARS-CoV-2 pandemic. As an immunomodulatory drug, dexamethasone is used in patients with SARS-CoV-2 infection who require supplemental oxygen or mechanical ventilation.

[0012] As a direct-acting antiviral drug, remdesivir (Veklury®) received conditional marketing authorization from the EMA on July 3, 2020, for use in patients with pneumonia requiring supplemental oxygen (low- or high-flow oxygen therapy or non-invasive ventilation) in the early stages of the disease. Monoclonal antibodies also have direct antiviral effects. Several monoclonal antibodies are currently being investigated in clinical trials.

[0013] Since the SARS-CoV-2 pandemic continues to pose a health threat to all of humanity, there remains a need for medications to treat virus-based respiratory diseases, and in particular to treat COVID-19. Description of the invention

[0014] The invention, as characterized in the claims, is based on the objective of providing a drug for the treatment of virus-based diseases of the respiratory tract, in particular for the treatment of COVID-19.

[0015] This problem is solved according to the invention by the silver nanoparticle-containing formulation for use as a medicament in the prevention and / or treatment of microbial and / or virus-based diseases of the respiratory tract according to claim 1. Further advantageous details, aspects and embodiments of the present invention will become apparent from the dependent claims, the description, the examples and the figures.

[0016] The present invention provides a formulation for use as a medicament in the prevention and / or treatment of microbial and / or virus-based diseases of the respiratory tract. The formulation comprises 0.02 wt% to 0.5 wt% silver nanoparticles with a mean particle size of 10 nm to 20 nm in water and at least two different stabilizers from the group of nonionic surfactants. The weight ratio of silver nanoparticles to the total stabilizer content is in the range of 10:2 to 10:50. The at least two different stabilizers are selected from the group consisting of polyoxyethylene monoalkyl esters, polyoxypropylene monoalkyl esters, polyoxyethylene dialkyl esters, polyoxypropylene dialkyl esters, polyoxyethylene trialkyl esters, polyoxypropylene trialkyl esters, and mixtures thereof.

[0017] In the present text, all statements of proportions in wt.% refer to the weight of the entire formulation as a 100% basis.

[0018] The water base typically used for the formulation is distilled water or a physiological solution, i.e., a 0.9% sodium chloride solution.

[0019] The formulation contains at least two stabilizers selected from the group consisting of polyoxyethylene monoalkyl esters, polyoxypropylene monoalkyl esters, polyoxyethylene dialkyl esters, polyoxypropylene dialkyl esters, polyoxyethylene trialkyl esters, and polyoxypropylene trialkyl esters. These stabilizers are surfactant compounds belonging to the group of nonionic surfactants, which are liquid at room temperature. Nonionic surfactants are surfactant chemicals that have uncharged polar and nonpolar regions within a single molecule. Furthermore, nonionic surfactants do not contain dissociated functional groups.

[0020] These stabilizers encapsulate the silver nanoparticles in the aqueous solution at varying concentrations, stabilizing them in the formulation before administration. They also ensure that the silver nanoparticles can be transported to their target site in the body, where they can exert their optimal effect. While not based on a scientific explanation, they also contribute to the long-lasting effectiveness of the main active ingredient, the silver nanoparticle, effectively ensuring a delayed release of the drug.

[0021] The silver nanoparticle formulation according to the invention, intended for use as a drug in the prevention and / or treatment of microbial and / or virus-based diseases, contains, in other words, dispersion-stabilized silver nanoparticles that cannot agglomerate into larger agglomerates because the stabilizers used are liquid in the temperature range of 0°C to 240°C. Many silver nanoparticle products are also known from the prior art that are offered as dry powders, but due to their tendency to agglomerate during transport and storage, they can only be dispersed in organic solvents, such as acrylic and epoxy resins, with a high input of mechanical energy, and even then only incompletely. These silver nanoparticle products are therefore unsuitable for use as a drug.

[0022] The at least two stabilizers in the formulation intended for use as a drug in the prevention and / or treatment of microbial and / or virus-based diseases according to the invention can be different stabilizers from one of the aforementioned classes of chemical compounds or stabilizers from different classes of compounds. For example, in a combination of three different stabilizers, three different polyoxyethylene monoalkyl esters can be used, but two different polyoxyethylene monoalkyl esters and one polyoxypropylene monoalkyl ester, or, for example, one polyoxypropylene dialkyl ester, one polyoxyethylene trialkyl ester, and one polyoxypropylene trialkyl ester can also be used. Any combination of these nonionic surfactants is possible.

[0023] Surprisingly, the above-mentioned aqueous formulation containing silver nanoparticles, which is currently only known from the prior art for the surface treatment of wood, in impregnating oils for air filters, as a textile dyeing solution, for promoting plant growth, for the production of antimicrobial surfaces, for the production of coatings for nonwoven / film laminates, for the production of coatings for decorative textiles, in varnishes and adhesives, in silicones, in thermoplastic polymers, in wood plastic composites (WPC), in polyolefin molded parts, in PVC and plastisols, and for the production of fibers, has been shown to have excellent effects when used as a drug in the prevention and / or treatment of microbial diseases and / or virus-based diseases of the respiratory tract.

[0024] According to a particularly preferred embodiment of the present invention, the silver nanoparticle-containing aqueous formulation is used in the treatment of virus-based respiratory tract diseases. The use of the formulation as a drug in the treatment of these diseases shows particularly positive effects with regard to the recovery time and also with regard to the completeness of recovery. It has also been shown that, presumably due to the stabilization present, such formulations can be safely administered over a wider concentration range, either intravenously or via inhalation, without exhibiting any toxic effects.

[0025] The proposed formulation is administered intravenously or by inhalation in humans. It can be used both curatively and preventively. Intravenous administration is preferred in severely ill patients; particularly good results have been demonstrated in terminal or near-terminal COVID-19 patients.

[0026] Clinical studies, which will be explained in more detail below, have shown that the administration of water-dispersed silver nanoparticles (AgNP) with a size particularly in the range of 10 nm to 20 nm into the lungs by nebulization using bronchodilators leads to faster healing, especially in mild cases of virus-based respiratory tract diseases.

[0027] In moderate to severe cases, systemic administration of silver nanoparticles can reduce mortality in patients with virus-based respiratory diseases, particularly COVID patients.

[0028] Due to the strong antiviral activity of silver, it is assumed that at least some of the viruses are killed directly via the respiratory epithelium. With systemic administration, it is likely that lethal viremia in the blood is reduced.

[0029] It is known that COVID-19 pneumonia involves a disruption of the immune system, leading to an overproduction of inflammatory cytokines. Since silver nanoparticles are able to reduce the production of inflammatory cytokines while simultaneously increasing the production of anti-inflammatory cytokines, this mechanism could potentially lead to lower mortality.

[0030] It can also be assumed that the Ag+ ions released from the AgNPs cause a change in the pH of the respiratory epithelium, leading to an environment hostile to the viruses. Experimental results indicate that there is a direct, low-pH-dependent fusion activation of coronaviruses during entry into host cells. Due to the resulting reduced viral load in the respiratory epithelium and other bodily fluids, the probability of transmission from infected individuals to healthy individuals is decreased. As is well known, coughing or sneezing with the expulsion of virus-laden droplets represents one of the main routes of infection.

[0031] It is known that Ag+ ions are released from the silver nanoparticles, which could inhibit the binding of the virus to the respiratory epithelium. Due to the long half-life of the silver nanoparticles, a further positive effect could be a reduced likelihood of secondary bacterial infections, which frequently occur in severe cases of COVID-19.

[0032] The formulation intended for use as a drug in the prevention and / or treatment of microbial diseases and / or virus-based diseases is particularly preferably a silver nanoparticle-containing, dispersed, aqueous formulation.

[0033] The aqueous formulation containing silver nanoparticles is particularly preferred for acute rhinitis, chronic rhinitis, paryngitis, herpangina, lateral angina, tonsillitis, laryngitis, tracheitis, acute bronchiolitis, chronic bronchiolitis, acute bronchitis, chronic bronchitis, aveolitis, pneumonia, acute sinusitis, chronic sinusitis, herpetic gingivastomatitis, herpetic aphthosis, nasal herpes, adenovirus pharyngoconjunctivitis, infectious mononucleosis, acute asthma, chronic asthma, COPD (chronic obstructive pulmonary disease), laryngeal diphtheria, otitis media, inflammation of the Eustachian tube and / or COVID-19.

[0034] Particularly positive effects with regard to the length of the recovery period as well as with regard to the completeness of recovery result when there is a combination of two or more virus-based diseases of the respiratory tract, possibly combined with bacterial secondary infections.

[0035] Increasingly positive effects are observed when the virus is a member of the families Picornaviridae, Orthomyxoviridae, Paramyxoviriae, Coronaviridae, Adenoviridae and / or Herpesviridae, a virus of the genus Rhinovirus, Enterovirus, Influenzavirus, Pneumovirus, Metapneumovirus, Betacoronavirus, Mastadenovirus, Simplexvirus and / or Varicellovirus, a virus of the species Rhinivirus, Cosackievirus-1, Cosackievirus-2, Echovirus, Poliovirus, Influenzavirus A, Influenzavirus B, Parainfluenzavirus, Respiratory Syncitual Virus (RSV), Human Metapneumovirus, Severe Acute Respiratory Syndrome-related Coronavirus (SARS-CoV), Human Adenovirus A, Human Adenovirus B, Human Adenovirus C, Human Adenovirus D, Human Adenovirus E, Human Adenovirus F. human herpes virus 4 (HHV-4), herpes simplex virus 1 (HSV-1),Herpes simplex virus 2 (HSV-2) and / or varicella zoster virus (VZV), and in particular a virus of the subtype severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0036] The aqueous formulation containing silver nanoparticles is particularly suitable for intravenous administration or administration via inhalation as an aerosol. In studies conducted to date, both of these dosage forms have proven to be easy to use and highly effective.

[0037] Preferably, the aqueous formulation containing silver nanoparticles is combined with at least one other pharmaceutical active ingredient and / or at least one other non-pharmaceutical active ingredient. This significantly increases the range of applications for the formulation.

[0038] Particularly preferred is the at least one further pharmaceutical active ingredient selected from the group consisting of antivirals, sympathomimetics, proteins, peptides, nucleic acids, and immunosuppressants. Also preferred are embodiments in which the at least one further non-pharmaceutical active ingredient is selected from the group consisting of pharmaceutically acceptable inorganic or organic acids or bases, polymers, copolymers, block copolymers, monosaccharides, polysaccharides, ionic and non-ionic surfactants or lipids, as well as mixtures thereof, albumin, transferrin, and DNA repair proteins, such as kinase inhibitors.

[0039] Particularly preferred in the formulation are stabilizers in the form of a combination of non-ionic surfactants from two different classes of compounds: polyoxyethylene monoalkyl esters, polyoxypropylene monoalkyl esters, polyoxyethylene dialkyl esters, polyoxypropylene dialkyl esters, polyoxyethylene trialkyl esters and polyoxypropylene trialkyl esters.

[0040] According to a particularly preferred embodiment, the stabilizers are selected from the group consisting of polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan tristearate, polyoxyethylene glyceryl trioleate, polyoxyethylene glyceryl monolaurate, polyoxyethylene glyceryl monooleate, polyoxyethylene glyceryl monostearate, polyoxyethylene glyceryl monoricinoleate, castor oil, hydrogenated castor oil, soybean oil and mixtures thereof.

[0041] Stabilizers are often not known by their chemical names but by their respective trade names. Preferred stabilizers are Tween20TM, Tween40 ™< , Tween60 ™< , Tween8 ™< , Polysorbate ™< , Tagat TO ™< , Tagat TO V ™< , Tagat L2 ™< , Tagat S2 ™< , Tagat R40 ™< , Triton X 100 ™< , Hydrogenated Castoroil, PEG 20 glyceryl stearate, PEG 20 glyceryl laurate, PEG 40 castoroil, PEG 25 glyceryl trioleate, Newcol ™< , Montane ™< , Lonzest ™< , Liposorb ™< , Nonion ™< , Kuplur ™< , Ionet ™< , Kemotan ™< , Grillosan ™< , Ethylan ™< , Glycomul ™< , Emsorb ™< , Disponil ™< , Amisol ™< , Armotan ™< , Sorbax ™< , Sorbitan ™< , Span ™< and Tego Pearl ™< .

[0042] Particularly preferred are the stabilizers Tween20™<, also known as polysorbate 20 or polyoxyethylene(20) sorbitan monolaurate, and PEG 25 glyceryl trioleate, also known as PEG 25 trioleate or 1-[2,3-bis(2-oxopropoxy)propoxy]propan-2-one. A mixture of these two stabilizers is especially preferred, and the best results are obtained when the two stabilizers are present in a 1:1 ratio.

[0043] The above list of stabilizers is not complete, as different manufacturers market the same or similar products under different names, or new non-ionic surfactants of the above-mentioned compound classes may be synthesized in the future and also be used.

[0044] Preferably, the at least two stabilizers are present in the formulation in a ratio ranging from 1:1 to 2:1.

[0045] Since at least two non-ionic surfactants are used as stabilizers for the nanosilver, there is a quantitative relationship between the concentrations of the stabilizers and the silver. The formulation has a silver nanoparticle to stabilizer ratio in the range of 10:2 to 10:50, preferably 10:5 to 10:10. Preferably, the silver to stabilizer ratio is from 10:5 to 10:20, and particularly preferably from 10:6 to 10:10. The "silver to stabilizer ratio" refers to the "ratio of silver to the sum of all stabilizers present." Using the preferred ratios results in particularly stable dispersions of silver nanoparticles.

[0046] The silver nanoparticles preferably have a size distribution in which 90 wt% of the particles are smaller than 25 nm or smaller than 20 nm.

[0047] Silver nanoparticles are particularly preferred if they have a distribution such that 99 wt% are smaller than 20nm.

[0048] Alternatively or additionally, silver nanoparticles with a distribution are preferred in which 66 wt.% of the particles are smaller than 25 nm, preferably smaller than 20 nm, particularly preferably smaller than 18 nm, and / or in which 33 wt.% of the particles are smaller than 20 nm, preferably smaller than 15 nm, particularly preferably smaller than 10 nm.

[0049] The particle size distribution, along with the mean size (diameter), is preferably determined using electron microscopy: TEM gratings (400 mesh, coated with 8 nm carbon) are incubated for 20 s on a 10 ml drop of a silver nanoparticle suspension diluted 1:90 with nanopure water. The excess suspension is then filtered off, and the gratings are imaged. Images with a size of 1024 x 1024 pixels and a pixel size of 0.376 nm are processed using the image processing software IPS version 1.123 (Visometrics, Konstanz, Germany) to determine the particle size of the silver nanoparticles in the suspension. A total of at least 500 particles, preferably 500 to 1000 particles, are analyzed. These measurement results are presented as a histogram and density plot using the R software (http: / / www.r-project.org / r ...org) and at the same time the mean (mean particle size), the standard deviation and their robust counterparts (median and MAD) of the size measurement are determined.

[0050] According to the invention, the silver nanoparticles have a mean particle size of 10 nm to 20 nm, particularly preferably of 15 to 20 nm.

[0051] The formulation is liquid and preferably contains no other solid components that would restrict its further uses. This applies to the concentrate used as the starting formulation as well as to the formulation after dilution of the concentrate with water in a ratio of 1:100.

[0052] More than two stabilizers may be present in the formulation. In this case, the content of a first stabilizer is in the range of 30 wt.% to 90 wt.%, preferably between 40 wt.% and 60 wt.%, and particularly preferably between 45 wt.% and 55 wt.%. The remaining weight fraction up to 100% is divided among the other stabilizers used in combination, which in turn are distributed in proportions of 0 to 100 wt.%. The wt.% values ​​given here, therefore, refer to the total weight of stabilizers as a 100% basis, contrary to other specifications. Particularly preferably, the formulation contains one or more stabilizers selected from the group consisting of Tagate TO V ™<, Tween20 ™<, Tween80 ™<, PEG 25 glyceryl trioleate, and Tagate L2 ™<. Using these stabilizers, particularly stable and universally applicable dispersions are obtained that are effective as medicines.

[0053] According to a particularly preferred embodiment of the present invention, the formulation contains a mixture of PEG 25 glyceryl trioleate and Tween20™< as a stabilizer. Formulations in which the ratio of PEG 25 glyceryl trioleate to Tween20™< is in the range of 1:2 to 2:1 are particularly preferred, and formulations in which the ratio of PEG 25 glyceryl trioleate to Tween20™< is approximately 1:1 are especially preferred.

[0054] It is well known that the efficacy of medications in general depends extremely strongly on the dosage and thus on the concentration of the active ingredient in the administered medication. Too low a concentration of the active ingredient results in a deficient effect when treating patients with the medication. Too high a concentration of the active ingredient leads, in most cases, to poisoning of the patient. The studies conducted to date have determined that the proportion of 0.02 wt% to 0.5 wt% silver nanoparticles according to the invention in the aqueous formulation sufficiently fulfills these conditions.

[0055] According to the invention, the silver nanoparticles are contained in the formulation in a proportion of 0.02 wt.% to 0.5 wt.%, preferably in a proportion of 0.03 wt.% to 0.4 wt.%, particularly preferably in a proportion in the range of 0.04 wt.% to 0.3 wt.%, and most preferably in a proportion in the range of 0.05 wt.% to 0.2 wt.%. As the concentration range of silver nanoparticles narrows, the positive properties of the formulation used as a drug increase significantly. The proportion of 0.1 wt.% silver nanoparticles used in the studies has proven to be ideal.

[0056] According to a further preferred embodiment, the silver nanoparticle-containing, dispersed, aqueous formulation contains ammonium nitrate in a proportion of 0.025 wt.% to 2.0 wt.%, preferably 0.05 wt.% to 1.0 wt.%, particularly preferably around 0.075 wt.% per 0.1 wt.% silver nanoparticles.

[0057] Regarding particle size, reference is again made to the definition formulated at the beginning, according to which the silver nanoparticles have a particle size of less than 100 nm. In the formulation, the silver nanoparticles are present in a particle size of 10 nm to 20 nm, particularly preferably 15 nm to 20 nm. The morphology of the silver nanoparticles can exhibit shapes such as triangles, cubes, spheres, rods, or platelets.

[0058] According to a further, particularly preferred embodiment, the silver nanoparticles have a size distribution in which 99 wt.% of the silver nanoparticles are smaller than 30 nm, preferably smaller than 20 nm. Embodiments in which 90 wt.% of the silver nanoparticles are smaller than 20 nm are preferred. Furthermore, embodiments in which 66 wt.% of the silver nanoparticles are smaller than 18 nm are preferred. Finally, embodiments in which 33 wt.% of the silver nanoparticles are smaller than 15 nm, preferably smaller than 10 nm, are also preferred.

[0059] The formulation intended for use as a drug in the prevention and / or treatment of microbial and / or virus-based diseases can be prepared as follows: a) Providing an aqueous concentrate containing silver nanoparticles, wherein the concentrate comprises 1 wt% to 60 wt% silver nanoparticles with a mean particle size of 10 nm to 20 nm and at least two stabilizers from the group of nonionic surfactants, wherein the ratio of silver nanoparticles to stabilizer is in the range of 10:2 to 10:50, wherein the at least two stabilizers are selected from the group consisting of polyoxyethylene monoalkyl esters, polyoxypropylene monoalkyl esters, polyoxyethylene dialkyl esters, polyoxypropylene dialkyl esters, polyoxyethylene trialkyl esters, polyoxypropylene trialkyl esters and mixtures thereof. b) Diluting the concentrate provided in step a) with water in a ratio of 1:100 vol%.

[0060] A process for producing a dispersed aqueous concentrate containing silver nanoparticles, wherein the concentrate comprises 1 wt.% to 60 wt.% silver nanoparticles and at least two stabilizers from the group of non-ionic surfactants, wherein the ratio of silver nanoparticles to stabilizer is in the range of 10:2 to 10:50, wherein the at least two stabilizers are selected from the group consisting of polyoxyethylene monoalkyl esters, polyoxypropylene monoalkyl esters, polyoxyethylene dialkyl esters, polyoxypropylene dialkyl esters, polyoxyethylene trialkyl esters, polyoxypropylene trialkyl esters and mixtures thereof, are described in detail in EP 2 515 660 B1.

[0061] Reference is hereby explicitly made to EP 2 515 660 B1 and to the content of the document with regard to the production of a silver nanoparticle-containing, dispersed, aqueous starting formulation prior to dilution with water in a ratio of 1:100, i.e., with regard to the production of a concentrate with 1 wt.% to 60 wt.% silver nanoparticles with a mean particle size of 10 nm to 20 nm and at least two stabilizers from the group of non-ionic surfactants, wherein the ratio of silver nanoparticles to stabilizer is in the range of 10:2 to 10:50, wherein the at least two stabilizers are selected from the group consisting of polyoxyethylene monoalkyl esters, polyoxypropylene monoalkyl esters, polyoxyethylene dialkyl esters, polyoxypropylene dialkyl esters, polyoxyethylene trialkyl esters, polyoxypropylene trialkyl esters and mixtures thereof.

[0062] A process for preparing the above-described silver nanoparticle-containing, dispersed, aqueous starting formulation prior to its dilution with water in a ratio of 1:100 comprises the following steps: Providing a silver salt, providing at least two stabilizers selected from the group consisting of polyoxyethylene monoalkyl esters, polyoxypropylene monoalkyl esters, polyoxyethylene dialkyl esters, polyoxypropylene dialkyl esters, polyoxyethylene trialkyl esters, polyoxypropylene trialkyl esters and mixtures thereof, providing a reducing agent, providing the solvent water, preparing a solution of silver salt, stabilizer and reducing agent, adding a base to the solution, wherein the addition of the base is carried out continuously over a period of 5 h to 48 h such that the pH of the formulation is between 0 and 6.

[0063] This manufacturing process yields a starting formulation with a very narrow distribution of the particle sizes of the nanoparticles.

[0064] Preferably, the base is added continuously over a period of 9 to 30 hours. This results in a starting formulation with a particularly narrow distribution of the nanoparticle sizes.

[0065] The described manufacturing process is a reductive chemical process. Accordingly, the silver particles are produced from their salts through chemical reduction. Generally, any chemical or physical reducing agent can be used to produce the silver nanoparticles. Physical reducing agents include temperature increases or irradiation with light. The use of a chemical reducing agent is advantageous because it allows for conversion rates of 100 percent and very high reaction rates.

[0066] It has been shown that in the presence of at least two stabilizers from the group consisting of polyoxyethylene monoalkyl esters, polyoxypropylene monoalkyl esters, polyoxyethylene dialkyl esters, polyoxypropylene dialkyl esters, polyoxyethylene trialkyl esters, and polyoxypropylene trialkyl esters, very strong reducing agents can be used, leading to an increased reaction rate without the simultaneous risk of forming a larger proportion of large, undesirable silver particles. Among the chemical reducing agents, those are preferred that do not generate reaction byproducts remaining in the reaction mixture, such as the corresponding oxidized form of the respective reducing agent, which would impair the quality of the silver nanoparticle-containing, dispersed, aqueous starting formulation.Therefore, a reducing agent is preferably used which reacts with the silver ions of the silver salt to form elemental silver and otherwise predominantly gaseous reaction products.

[0067] Reducing agents that can leave the reaction solution in their oxidized form as a gas, such as hydrazine hydrate, are particularly preferred. Of course, the silver nanoparticles can also be obtained with any other reducing agent.

[0068] Any silver salt can be used to prepare the starting formulation. For practical reasons, the choice of silver salt depends on the solvent used, as different salts have different solubility properties. The silver salt with the best solubility in the given solvent is always preferred. InThe following examples describe some solvents and the respective suitable silver salts. However, the manufacturing process described above is not limited to the silver salt / solvent pairings mentioned.

[0069] The reductive production of silver can be formulated as a pair of redox equations. The first half-equation is the reduction equation, according to which the silver cation is reduced from the silver salt to elemental silver. The second half-equation describes the corresponding oxidative process for the oxidation of the reducing agent to the conjugate oxidation product, which ideally leaves the reaction solution in a gaseous state. All reducing agents have in common that one proton is produced for each electron transferred. This proton contributes to a significant drop in the pH of the entire reaction solution. This decrease in pH is responsible for the undesirable cessation of the reaction. Therefore, an alkali must be added to neutralize the protons that are released and slow down the overall reaction.

[0070] The type of alkali, the concentration of the alkali, and the speed at which the alkali is added are crucial for the distribution of the particle sizes of the nanoparticles in the manufactured formulation.

[0071] Ammonia, potassium bicarbonate, or sodium hydroxide are preferably used as the base. Using these bases results in particularly stable dispersions with a narrow distribution of nanoparticle sizes.

[0072] The amount of alkali added must be measured so that a pH-neutral dispersion is obtained after the reaction is complete. The pH value will then be between pH 5 and pH 9.

[0073] Bases, or proton acceptors, are defined by their pKb values. The pKb value is the negative decadic logarithm of the proton concentration at equilibrium and is therefore a measure of the strength of the base.

[0074] Suitable bases for the preparation of the starting formulation have a pKb value in the range of -2 to 10.5, preferably 1.5 to 9.1, particularly preferably in the range of 3.5 to 7.5.

[0075] In addition to the base strength, the addition rate to the reaction solution for preparing the formulation is also crucial. If the addition is too rapid, the particle size spectrum shifts towards larger particles, which in extreme cases are in the micrometer range. Conversely, if the addition is too slow, yields greater than 90% will not be achieved, as the nanosilver already formed catalytically degrades reducing agents, leaving no further reaction partners available for generating silver nanoparticles.

[0076] Experiments have shown that, for a batch size of 50 kg, the addition rate of the lye should be in the range of 9 to 30 hours to achieve the high quality of silver nanoparticles according to the invention. For correspondingly smaller batch sizes, the lye addition time decreases analogously. The addition time cannot be extended indefinitely, as the catalytic degradation of the reducing agent by already formed nanosilver significantly impairs the overall yield after 48 hours at the latest.

[0077] The lye is added at a rate that ensures the pH of the dispersion remains between 0 and 6. A higher pH leads to an excessively rapid reaction and thus uncontrolled particle growth. A pH that is too low causes the reaction to stop, preventing the formation of nanosilver.

[0078] Particularly when producing a nanoparticle-containing mixture by adding inorganic salts, the dispersion obtained by adding a base to the solution of silver salt, stabilizers, and reducing agent can be separated into two chemical phases, 1 and 2. Phase 1 contains the silver nanoparticles in the liquid stabilizer mixture. The solvent, the inorganic salts, and the byproduct ammonium nitrate are located in Phase 2, which is above Phase 1. The two phases can then be easily separated by decanting off the upper Phase 2. What remains is Phase 1, which consists only of the silver nanoparticles and the liquid stabilizers.

[0079] The silver nanoparticles, which are present in the form of a dispersion and typically have particle sizes of 1 nm to 20 nm and are chemically stabilized, are separated in this way from the by-product ammonium nitrate contained in the dispersion and the solvent used, in particular water.

[0080] Particularly good results are achieved when the solvent is water and the inorganic salts are water-soluble. In this case, the dispersion obtained by adding a base to the solution of silver salt, stabilizer, and reducing agent is separated into two chemical phases, 1 and 2, by the addition of water-soluble inorganic salts. Phase 1 contains the silver nanoparticles in the liquid stabilizer mixture. The solvent, water, the water-soluble salts, and the byproduct ammonium nitrate are located in Phase 2, which is above Phase 1. The two phases can be separated by decanting off the upper aqueous Phase 2. What remains is Phase 1, which consists only of the silver nanoparticles and the liquid stabilizers.

[0081] To characterize suitable salts in more detail, their cationic and anionic components must be considered separately. Salts generally consist of at least one cation and at least one anion. An undesirable interaction of cations with the stabilized silver nanoparticle-containing formulation is not expected, since the silver, and in particular the silver preferably used, exists either as uncharged silver or as a positively charged cation.

[0082] Since water is used as the solvent, the selection of suitable cation / anion combinations in the form of appropriate salts is based on the idea that phase separation is caused by the stress on water's hydration capacity by charged ions. Hydration capacity is expressed as the ability to form hydrogen bonds. It is also the reason for the stability of the dispersion in the water medium. Thus, there is a certain competition between the stabilizers coating the silver nanoparticles and the dissolved reaction byproducts, such as ammonium nitrate.

[0083] The stabilizers used are nonionic macromolecules that interact with water molecules only through weak dipole-dipole interactions. The addition of salts aims to counteract the stabilizing influence of hydrogen bonds on the overall dispersion by introducing electrically charged ions that interact much more strongly with the water dipoles. Generally, the degree of interaction with water depends on the ionic radius and charge, such that the interaction increases with decreasing ionic radii and increasing ionic charge. The solubility of the salts in water is also important when selecting suitable salts. As the solubility of the salts in water increases, so does the formation of interaction forces with the water molecules.

[0084] The selection of anions is further limited by the need to avoid unwanted interactions with existing silver cations. This excludes all anions that form sparingly soluble compounds with the silver used, and especially with silver itself, such as halides, chalcogenides, and their oxygen compounds.

[0085] The inorganic salt therefore particularly preferably comprises at least one cation from period 3 or period 4 of the periodic table of elements and at least one element from group 5 of the periodic table of elements as a component of the anion, wherein the inorganic salt particularly preferably comprises nitrogen as a component of the anion. The phase formed after the addition of the inorganic salt is particularly preferably decanted from the silver nanoparticle-containing mixture, which consists essentially of silver nanoparticles and stabilizers. Brief description of the drawings

[0086] Preferred embodiments of the invention are described below with reference to the drawings, which serve only for illustration and are not to be interpreted restrictively.

[0087] The drawings show: Fig. 1 UVVis spectrum of a 5,000-fold diluted aqueous solution of a silver nanoparticle-containing, dispersed aqueous concentrate of a nanosilver dispersion with 10.0 wt% silver; Fig. 2 Measured particle sizes and calculated curve of the scanning electron microscope (SEM) data sets; Fig. 3 Transmission electron microscope (TEM) image of the silver nanoparticles; Fig. 4 TEM of a 5,000-fold diluted aqueous solution of a silver nanoparticle-containing, dispersed aqueous concentrate. Description of preferred embodiments Example 1: Formulation of nanosilver with hydrazine hydrate, ammonia, Tagat TO V ™< and Tween20 ™<

[0088] 7,000 g of silver nitrate, 1,760 g of Tagat TO V ™< (non-ionic polyoxyethylene-25 glycerol trioleate), 1,760 g of Tween20 ™< (polyoxyethylene(20) sorbitan monolaurate), and 512 g of hydrazine hydrate are dissolved in 28,439 g of deionized water. The solution is stirred for 3 hours. Then, 5,000 g of 14% ammonia solution are added continuously over a period of 24 hours. The reaction is complete after the addition and yields a dispersion with a silver content of 10.0 wt%. The particle size and distribution are determined using a UV-Vis spectrum ( Figure 1 ) determined. The result is a 10 percent nanosilver dispersion with a nanosilver particle size of 1-20 nm.

[0089] Methods for determining particle sizes, such as those described above using TEM, are complex for nanomaterials. Therefore, an indirect method was developed that simply determines the extinction of the plasmon resonance of the silver nanoparticles (in water), and this method correlates very well with the results of TEM measurements for these systems. This is a simple and rapid measurement that determines the absorption of the silver nanoparticles at a wavelength of 350–700 nm.

[0090] The wavelength of maximum absorption corresponds to the nano-silver particle size (415 nm, for example, corresponds to a mean diameter of 17 nm, determined as described above using TEM). The peak height correlates with the amount of nano-silver and the number of nano-silver particles in the dispersion.

[0091] The absorption spectrum is measured on a 5,000-fold diluted aqueous solution of a dispersed aqueous concentrate of silver nanoparticles containing 10.0 wt% silver nanoparticles, thus containing 20 ppm of nanosilver, and is clear and deep yellow in color. The UV-Vis spectrum is recorded in the wavelength range from 750 nm to 350 nm. The measured absorption values ​​show a peak with a maximum at 410 nm - 420 nm and a peak width at half maximum of approximately 80 nm.

[0092] The dispersing properties of the resulting 10 percent dispersion are excellent in both polar and non-polar solvents; that is, without any further chemical effort (dispersing aids) or mechanical effort (ultrasound, Ultraturax, etc.), an absolutely clear solution is obtained, which only has a coloration caused by the plasmon effect of the silver.

[0093] Figure 4shows a transmission electron micrograph (TEM) of the dilute dispersion from Example 1. The one in the Figure 4 The visible dark areas correspond to the nano-silver particles, which have a particle size of 1 - 30 nm. Example 2: Formulation of nanosilver with hydrazine sulfate, potassium bicarbonate, Tagat TO V ™< and Tween80TM

[0094] 7,000 g of silver nitrate, 2,360 g of Tagat TO VTM, 1,160 g of Tween80 (polyoxyethylene(80) sorbitan monolaurate), and 1,331 g of hydrazine sulfate are dissolved in 27,620 g of deionized water. The solution is stirred for 3 hours. Then, 5,000 g of potassium bicarbonate solution (1,900 g KHCO3) is added continuously over a period of 30 hours. The reaction is complete after the addition and yields a dispersion with a silver content of 10.0 wt%. The particle size and distribution are determined by a UV-Vis spectrum (analogous to [reference to previous method]). Figure 1) determined. The result is a 10 percent nanosilver dispersion with a nanosilver particle size of 1-30 nm. Example 3: Formulation of nanosilver with glucose, sodium hydroxide, Tagat L2™< (PEG-20 glyceryl laurate) and Tween20™<

[0095] 7,000 g of silver nitrate, 2,360 g of Tagat L2, 1,160 g of Tween20, and 3,708 g of glucose are dissolved in 25,243 g of deionized water. The solution is stirred for 3 hours. Then, 5,000 g of sodium hydroxide solution (760 g NaOH) is added continuously over a period of 30 hours. The reaction is complete after the addition of the other components and yields a dispersion with a silver content of 10.0 wt%. The particle size and distribution are determined using a UV-Vis spectrum. The result is a 10% nanosilver dispersion with a nanosilver particle size of 1–30 nm. Example 4:Further processing of the dispersion from Example 1 to produce a liquid, water- and salt-free formulation with silver nanoparticles

[0096] The aqueous dispersion containing silver nanoparticles obtained in Example 1 is contaminated with the byproduct ammonium nitrate. The silver nanoparticles have particle sizes of 1–20 nm and are chemically stabilized. The silver content is 25 wt%. The water content is 366 g and the ammonium nitrate content is 185 g. 1000 g of this dispersion is placed in a beaker and heated to 45°C with stirring. The separation of the dispersion into two phases is initiated by the addition of 78 g of potassium nitrate. After the addition and complete dissolution of the potassium nitrate, the heating is removed and the stirrer is switched off. The separation of the phases can be observed after cooling. The upper, clear, aqueous phase 1 is completely decanted. The remaining phase 2 has a dark brown color with syrupy flow properties and a weight of 449 g.

[0097] An analysis of aqueous phase 1 reveals a salt content of 263 g and a water content of 366 g.

[0098] The analysis of the silver-containing phase 2 yields the following data: a) Analysis of the total silver content by ashing at 800°C yields 250 g of silver. Based on the total formulation, this corresponds to a silver content of 56 wt%. b) The analysis of the particle size distribution of the obtained silver is in Figure 1 The determination is performed here by measuring the UV-Vis absorption spectrum in the wavelength range of 700 nm to 350 nm. The peak maximum at 415 nm corresponds to a mean particle size of 15 nm. The peak half-width of a maximum of 80 nm is a measure of the narrow particle size distribution. The correlation with the SEM (Scanning Electron Microscope) and TEM (Transmission Electron Microscope) analyses, as described in Figure 2 and Figure 3The representation allows the specification of the particle size distribution of D99 < 20 nm (99% of the particle diameters are smaller than 20 nm), as well as guideline values ​​D33% < 7 nm and D66 < 17 nm, and as an average value of approximately 15 nm. Example 5: Pilot studies on the use of a silver nanoparticle-containing, dispersed, aqueous formulation as a drug for the treatment of COVID-19

[0099] Two Phase 2 clinical trials (Study A and Study B) were conducted as pilot studies. All study participants suffered from COVID-19 pneumonia, clinically diagnosed as moderate to severe. Such moderate to severe COVID-19 pneumonia represents a serious, life-threatening illness for which no reliably effective treatment is currently available.

[0100] The silver nanoparticle-containing, dispersed, aqueous formulation used in the pilot studies was based on a concentrate (purity of silver nanoparticles: 99.99%) with the following composition: substance Percentage [wt.%) Water 74,5 AgNP 10,0 Ammonium nitrate 7,5 Polysorbate 20 4,0 PEG 25 Trioleate 4,0

[0101] The silver nanoparticles AgNP used have the following particle size distribution: D99: 20 nm; D66: approx. 17 nm; D33: approx. 7 nm. The particle size distribution was determined using the following method: Transmission Electron Microscopy (TEM) as described above.

[0102] Polysorbate-20 (CAS number 9005-64-5) is equivalent to the above-mentioned Tween 20 ™< , is a non-ionic surfactant and has an HLB value of 16.7.

[0103] PEG 25 Trioleate is Peg-25 glyceryl trioleate (CAS 68958-64-5).

[0104] To prepare the silver nanoparticle-containing, dispersed, aqueous formulation used in the pilot studies, the above concentrate was diluted with distilled water in a ratio of 1:100. The resulting formulation exhibits the following properties: State of matter: fluid Color: brownish Odor: odorless pH value at 20°C: 8,0 Density: 1.1 g / cm3 Boiling point: 100°C AgNP concentration: 0.1 wt.%

[0105] The average size of the silver nanoparticles is around 17 nm (TEM measurements), D99 < 20 nm. The nanoparticles have a spherical shape and are readily soluble in water. Dosage calculation inhalation

[0106] The NOAEL (No Observed Adverse Effect Level) of AgNP for the Sprague-Dawley rat is 100 µg / m³ [Ji JH, Jung JH, Kim SS, Yoon JU, Park JD, Choi BS, Chung YH, Kwon IH, Jeong J, Han BS, Shin JH. Twenty-eight-day inhalation toxicity study of silver nanoparticles in Sprague-Dawley rats. Inhalation toxicology. 2007 Jan 1;19(10):857-71]. This corresponds to a value of 59 µg / m³ in humans.

[0107] The exposure time planned in the pilot studies is 8 hours per day, the exposure duration is 13 weeks, and the exposure frequency is 65 / 365. From this data, the average permissible dose can be calculated using the following formula (Exposure Assessment Tools by Routes - Inhalation): ADD = C air × Inhalationsrate × Expositionszeit × Expositionshäufigkeit × Expositionsdauer / Körpergewicht × Mittelungszeit Tage = 0.059 × 32.4 × 8 × 0.178 × 0.25 / 60 × 0.25 = 0 , 003 mg / kg = 3 μg / kg

[0108] For an exposure time of 8 hours per day and an exposure frequency of 65 days, the permissible total dose for 13 weeks can be calculated as follows: 3 μ g / kg × 65 = 195 μ g / kg Systemic intravenous administration

[0109] The systemic dose of AgNP for the human model can be calculated according to Nair AB, Jacob S. "A simple practice guide for dose conversion between animals and human" Journal of basic and clinical pharmacy. 2016 Mar;7(2):27.

[0110] The following applies to the human equivalent dose (HED): HED mg / kg = Tier NOAEL mg / kg × Gewicht Tier kg / Gewicht Mensch kg 1 − 0 , 67

[0111] Taking into account (see: Kim YS, Song MY, Park JD, Song KS, Ryu HR, Chung YH, Chang HK, Lee JH, Oh KH, Kelman BJ, Hwang IK. Subchronic oral toxicity of silver nanoparticles. Particle and fibre toxicology. 2010; 7(1):1-1) Reference body weight mouse = 0.02 kg Reference body weight human = 60 kg NOAEL - 4 mg / kg The resulting HED value is 0.3 mg / kg. Research design and methodology Case selection

[0112] Study A included 10 patients as the case group and 10 patients as the control group. All patients suffered from COVID-19 pneumonia, clinically diagnosed as moderate or severe. All patients were found to be infected with the delta variant of SARS-CoV-2. All patients were properly informed and gave their consent.

[0113] Study B included 10 patients as the case group and 10 patients as the control group. All patients suffered from COVID-19 pneumonia, clinically diagnosed as moderate or severe. All patients were found to be infected with the Omicron variant of SARS-CoV-2. All patients were properly informed and gave their consent.

[0114] In total, 20 patients were included in the case group and 20 patients in the control group.

[0115] The severity of the illness was assessed using the Early Warning Sign (EWS) score based on the Indian system, with an EWS score of 5 or higher indicating severe illness. The EWS score takes into account age, blood pressure, pulse, respiratory rate, and oxygen saturation. The definition provided in "National Early Warning Score (NEWS), Standardising the assessment of acute-illness severity in the NHS, Report of a working party July 2012, Royal College of Physicians" can serve as a basis for this assessment.

[0116] In the specific context of COVID-19, the following protocol can be used in particular for determining the EWS: National Early Warning Score (NEWS) 2, Standardising the assessment of acute-illness severity in the NHS Additional implementation guidance, Update: March 2020, Royal College of Physicians (https: / / www.rcplondon.ac.uk / file / 20716 / download) Study type: Open-label, randomized controlled trial. Study location: Tertiary care COVID hospital. Inclusion criteria

[0117] RT-PCR-confirmed COVID-19 patients of both sexes, aged over 18 years, with an EWS score of 5 or higher and clinically diagnosed moderate to severe pneumonia were included. Patients had to declare their willingness to participate in the study. Exclusion criteria

[0118] Patients with an EWS score below 5 and patients who did not express a willingness to participate in the study. Description of the treatment

[0119] First, the human equivalent dose was calculated from the NOAEL (No Observed Adverse Effect Level) value. The result of the calculation explained above is 0.3 mg / kg. For a 60 kg patient, the total dose is 18 mg.

[0120] At the start of the study, 1% of the total dose, i.e., 1.8 mg, was administered for 3 days, corresponding to 5.4 mg of water-soluble AgNP. Patients were selected from their daily patient status sheets according to the inclusion and exclusion criteria described above. Informed consent was obtained either from the patients themselves or from their relatives after counseling on the risks and benefits. A total of 20 patients were selected and randomly assigned to a treatment group and a control group using block randomization.

[0121] The patients in the case group received 1.8 ml of the 1:100 diluted concentrate (AgNP concentration: 0.1 wt%) in 500 ml saline solution and were infused over 1 hour on three consecutive days while observing all aseptic precautions. In The control group did not receive AgNP. Both the patients in the case group and the control group received standard treatment with oxygen, antibiotics, corticosteroids, low-molecular-weight heparin, and medications for comorbidities such as insulin for diabetes and antihypertensives for high blood pressure.

[0122] All patients were clinically monitored every 6 hours for level of consciousness, pulse, blood pressure, oxygen saturation, respiratory rate, urine output, and infusion site. The results and observations were recorded on a patient status sheet every 12 hours. Routine blood tests were performed on the fourth day after the first dose. Patients who showed improvement and were hemodynamically stable were discharged according to guidelines. Patients were followed for 10 days after the infusion. Statistical methodology

[0123] The original variables were compared in the total sample using a t-test for continuous variables and a chi-square test (followed by a Wilcoxon test) for nominal variables. Significance (alpha) was set at p < 0.05.

[0124] The outcome variable (favorable outcome or discharge as opposed to unfavorable outcome or death) was compared between the medication status (case group vs. control group) using the chi-square test (with exact Fisher corrections) and the Wilcoxon test. Results of Study A: Delta variant of SARS-CoV-2

[0125] Of the 10 people in the control group, one person was excluded from the analysis during the follow-up period because the person was discharged against medical advice and withdrew their consent. Table 1A: Case group with age, EWS score at baseline, comorbidities and outcome Serial No. Initial value EWS Age [years] Gender Comorbidities Current status Duration of hospital stay [days] 1 15 49 female Coronary artery disease with global ischemia deceased - 2 12 71 masculine Type 2 diabetes mellitus deceased - 3 7 52 female Type 2 diabetes mellitus dismissed 5 4 9 60 female High blood pressure dismissed 5 5 10 84 masculine no comorbidity dismissed 5 6 8 30 masculine no comorbidity dismissed 5 7 8 72 masculine Type 2 diabetes mellitus dismissed 7 8 11 62 masculine High blood pressure dismissed 9 9 10 79 masculine High blood pressure dismissed 10 10 13 62 masculine High blood pressure, type 2 diabetes mellitus deceased - Table 1B: Control group with age, EWS score at baseline, comorbidities and outcome Serial No. Initial value EWS Age [years] Gender Comorbidities Current status Duration of hospital stay [days] 1 12 71 masculine Type 2 diabetes mellitus removed from follow-up due to discharge at self-risk and withdrawal of consent - 2 8 58 masculine no comorbidity deceased - 3 12 42 masculine High blood pressure, type 2 diabetes mellitus deceased - 4 8 68 masculine High blood pressure dismissed 12 5 13 74 masculine Parkinson deceased - 6 12 70 female High blood pressure deceased - 7 11 40 masculine High blood pressure deceased - 8 7 84 masculine no comorbidity deceased - 9 8 65 masculine Type 2 diabetes mellitus deceased - 10 8 76 masculine High blood pressure deceased -

[0126] A comparison of Tables 1A and 1B shows a trend towards more favorable disease courses (discharge after clinical resolution) in the adjuvant AgNP group (case group) compared to the control group with usual treatment.

[0127] The mortality rate was 80% in the control group and 30% in the case group. The average length of hospital stay was 12 days in the control group and 7 ± 2 days in the case group. Results of Study B: Omicron variant of SARS-CoV-2

[0128] Table 2A: Case group with age, EWS score at baseline, comorbidities and outcome Serial No. Initial value EWS Age [years] Gender Comorbidities Current status Duration of hospital stay [days] 1 13 70 masculine Type 2 diabetes mellitus, high blood pressure deceased - 2 9 75 masculine Type 2 diabetes mellitus, high blood pressure dismissed 7 3 8 76 masculine High blood pressure dismissed 8 4 9 66 masculine Type 2 diabetes mellitus, hypertension, CKD deceased - 5 11 80 masculine Type 2 diabetes mellitus dismissed 7 6 10 83 masculine Type 2 diabetes mellitus, high blood pressure dismissed 6 7 12 70 masculine Type 2 diabetes, hypertension, COPD, CAD deceased - 8 10 93 masculine Type 2 diabetes mellitus, high blood pressure deceased - 9 10 85 female Type 2 diabetes, high blood pressure, COPD dismissed 7 10 7 66 female Type 2 diabetes mellitus, high blood pressure dismissed 12 Table 2B: Control group with age, EWS score at baseline, comorbidities and outcome Serial No. Initial value EWS Age [years] Gender Comorbidities Current status Duration of hospital stay [days] 1 10 68 female Type 2 diabetes mellitus, high blood pressure deceased - 2 8 77 masculine Type 2 diabetes mellitus dismissed 10 3 7 75 masculine Type 2 diabetes mellitus, high blood pressure dismissed 10 4 8 78 female Type 2 diabetes mellitus deceased - 5 8 86 masculine Type 2 diabetes mellitus, high blood pressure deceased - 6 7 81 female Type 2 diabetes mellitus deceased - 7 9 65 female Type 2 diabetes mellitus dismissed 18 8 10 85 masculine Type 2 diabetes mellitus, DVT deceased - 9 5 56 masculine Type 2 diabetes mellitus, hypertension, CKD deceased - 10 12 75 masculine Type 2 diabetes mellitus, high blood pressure deceased -

[0129] A comparison of Tables 2A and 2B shows a trend towards more favorable disease courses (discharge after clinical resolution) in the adjuvant AgNP group (case group) compared to the control group with usual treatment.

[0130] The mortality rate was 70% in the control group and 40% in the case group. The average length of hospital stay was 13 ± 4 days in the control group and 8 ± 2 days in the case group. Table 3: Comparison of continuous output variables between the groups (case group [+]; control group [-]) Demographic data statistics Case group control group In total N 20 20 40 Gender [N[%] Masculine 15 (75%) 15 (75%) 30 (75%) Female 5 (25%) 5 (25%) 10 (25%) Age [years] Mean (Standard deviation) 69,3 (14,5) 69,7 (12,8) 69,5 (13,5) median 70.5 72.5 71 Min 30 40 30 Max 93 86 93 Masculine 10,3 (1,7) 9,1 (2,4) EWS Female 9,6 (3,3) 9,2 (1,9) In total 10,1 (2,1) 9,2 (2,2)

[0131] The values ​​in Table 3 show that the individuals in the case group and the control group had comparable gender, age, and EWS severity on average. Table 4: Comparison of nominal output variables status P (Wilcoxon test) Case group control group Gender masculine 15 (75%) 15 (75%) 1.000 female 5 (25%) 5 (25%) Comorbidities Yes 19 (95,0%) 18 (90%) 0,5483 No 1 (5,0%) 2 (10%) EWS upon enrollment in study 10,1 (2,1) 9,2 (2,2) 0,1911

[0132] The values ​​in Table 4 show that there were no statistically significant differences between the case and control groups with regard to the patients' ages. Furthermore, the baseline variables of the case and control groups are statistically compared. Table 5: Comparison of disease progression Result p cheap unfavorable Case group Study A 7 (70,0%) 3 (30,0%) Control group Study A 1 (11,1%) 8 (88,9%) Case group Study B 6 (60,0%) 4 (40,0%) Control group Study B 3 (30,0)% 7 (70,0%) 0,0881 Case group Total 13 (65,0%) 7 (35,0%) Control group total 4 (20,0%) 15 (75,0%)

[0133] A trend towards a more favorable outcome was observed in the case group compared to the control group (p = 0.0881). This means that an unfavorable disease course, including patient death, was more likely in the control group than in the case group. Compatibility of the formulation

[0134] One patient in the case group (Patient No. 1) died within 12 hours of first receiving the formulation. The patient had global cardiac ischemia. It is unclear whether this was due to COVID-19 itself. The effect of AgNP on cardiac ischemia is the subject of further investigation. None of the other patients in the case group experienced an acute deterioration in their clinical status, including vital signs. There is no association between the death of Patient No. 1 and treatment with the formulation.

Claims

1. Formulation for use as a medicament in the prevention and / or treatment of microbial and / or viral diseases of the respiratory tract, wherein the formulation comprises, in water, 0.02% by weight to 0.5% by weight of silver nanoparticles with an average particle size of 10 nm to 20 nm and at least two different stabilisers from the group of non-ionic surfactants, wherein the weight ratio of silver nanoparticles to the sum of the stabilisers is in the range of 10:2 to 10:50, and wherein the at least two different stabilisers are selected from polyoxyethylene monoalkyl acid esters, polyoxypropylene monoalkyl acid esters, polyoxyethylene dialkyl acid esters, polyoxypropylene dialkyl acid esters, polyoxyethylene trialkyl acid esters, polyoxypropylene trialkyl acid esters and mixtures thereof.

2. Formulation for use as a medicament according to claim 1, characterised in that the virus-based disease is acute rhinitis, chronic rhinitis, pharyngitis, herpangina, lateral angina, tonsillitis, laryngitis, tracheitis, acute bronchiolitis, chronic bronchiolitis, acute bronchitis, chronic bronchitis, alveolitis, pneumonia, acute sinusitis, chronic sinusitis, herpetic gingivostomatitis, herpetic aphthous stomatitis, herpes nasalis, adenoviral pharyngoconjunctivitis, infectious mononucleosis, acute asthma, chronic asthma, COPD (chronic obstructive pulmonary disease), laryngeal diphtheria, otitis media, inflammation of the Eustachian tube and / or COVID-19, or that it involves a combination of two or more virus-based respiratory tract diseases, possibly in combination with at least one or more secondary bacterial infections and / or that the microbial disease is a pneumococcal, staphylococcal, enterobacterial (such as, in particular, Klebsiella or Escherichia coli), Pseudomonas aeruginosa, Haemophilus influenzae and / or Mycoplasma pneumoniae infection, possibly in combination with at least one or more secondary viral infections.

3. Formulation for use as a medicament according to any one of claims 1 or 2, characterised in that the virus is a virus of the family Picornaviridae, Orthomyxoviridae, Paramyxoviridae, Coronaviridae, Adenoviridae and / or Herpesviridae or that the virus is a virus of the genus Rhinovirus, Enterovirus, Influenzavirus, Pneumovirus, Metapneumovirus, Betacoronavirus, Mastadenovirus, Simplexvirus and / or Varizellovirus, preferably a virus of the species Rhinovirus, Cosackievirus-1, Cosackievirus-2, Echovirus, Poliovirus, Influenza virus A, influenza virus B, parainfluenza virus, respiratory syncytial virus (RSV), human metapneumovirus, severe acute respiratory syndrome-related coronavirus (SARS-CoV), human adenovirus A, human adenovirus B, human adenovirus C, human adenovirus D, human adenovirus E, human adenovirus F, human herpesvirus 4 (HHV-4), herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2) and / or varicella-zoster virus (VZV).

4. Formulation for use as a medicament according to any one of claims 1 to 3, characterised in that the virus is a virus of the subspecies severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), wherein the formulation can be used for preventive and / or curative treatment, preferably for the treatment of patients with an EWS score of at least 3, in particular of at least 4.

5. Formulation for use as a medicament according to any one of claims 1 to 4, characterised in that the formulation is suitable for intravenous administration, preferably for continuous administration or for bolus administration or for administration within 5-120 minutes per day, or for administration by inhalation as an aerosol.

6. Formulation for use as a medicament according to any one of claims 1 to 5, characterised in that the silver nanoparticles have a size distribution such that 99% by weight of the silver nanoparticles are smaller than 30 nm, preferably smaller than 20 nm; in which 90% by weight of the silver nanoparticles are smaller than 20 nm; and / or in which 66% by weight of the silver nanoparticles are smaller than 18 nm; and / or in which 33% by weight of the silver nanoparticles are smaller than 15 nm, preferably smaller than 10 nm.

7. Formulation for use as a medicament according to any one of claims 1 to 6, characterised in that the silver nanoparticles are present in a mean particle size of 15 nm to 20 nm.

8. Formulation for use as a medicament according to any one of claims 1 to 7, characterised in that the silver nanoparticles are present in the formulation in an amount of 0.03% by weight to 0.4% by weight, preferably in an amount in the range of 0.04% by weight to 0.3% by weight, particularly preferably in an amount in the range of 0.05% by weight to 0.2% by weight.

9. Formulation for use as a medicament according to any one of claims 1 to 8, characterised in that the stabilisers are selected from polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan tristearate, polyoxyethylene glyceryl trioleate, polyoxyethylene glyceryl monolaurate, polyoxyethylene glyceryl monooleate, polyoxyethylene glyceryl monostearate, polyoxyethylene glyceryl monoricinoleate and mixtures thereof.

10. Formulation for use as a medicament according to any one of claims 1 to 9, characterised in that at least two different stabilisers are present in a weight ratio in the range of 1:1 to 2:1, and / or that the weight ratio of silver nanoparticles to the sum of the stabilisers lies in the range from 2:1 to 1:1.

11. Formulation for use as a medicament according to any one of claims 1 to 10, characterised in that two different stabilisers are present, wherein the first of the stabilisers is selected as a polyoxyethylene monoalkyl acid ester, wherein the number of polyoxyethylene units is preferably in the range of 15 to 90, in particular in the range of 20 to 40, wherein the stabiliser is preferably selected as polyoxyethylene sorbitan-monolaurate, wherein the number of polyoxyethylene units lies in the range of 20 to 40, preferably at 20 or 21, and is particularly preferably selected as polyoxyethylene(20)-sorbitan-monolaurate, and wherein the second of the stabilisers is selected as a polyoxyethylene trialkyl acid ester, wherein the number of polyoxyethylene units is preferably in the range of 15 to 60, particularly in the range of 20 to 40, wherein the second stabiliser is preferably selected as polyoxyethylene glyceryl trioleate, wherein the number of polyoxyethylene units is in the range of 20 to 40, preferably 30, and is particularly preferably selected as polyoxyethylene (25) glyceryl trioleate, wherein the weight ratio of the first to the second stabiliser is preferably in the range of 2:1 to 1:2, preferably in the range of 1:1.

12. Formulation for use as a medicament according to any one of claims 1 to 11, characterised in that the formulation is present in combination with at least one further active pharmaceutical ingredient and / or at least one further non-pharmaceutical ingredient, wherein the at least one further active pharmaceutical ingredient is preferably selected from antivirals, sympathomimetics, proteins, peptides, nucleic acids and immunosuppressive agents, and wherein the at least one further non-pharmaceutical active ingredient is selected from pharmaceutically acceptable inorganic or organic acids or bases, polymers, copolymers, block copolymers, monosaccharides, polysaccharides, ionic and non-ionic surfactants or lipids, as well as mixtures thereof, albumin, transferrin and DNA repair proteins, such as kinase inhibitors.

13. Formulation for use as a medicament according to any one of claims 1 to 12, characterised in that the formulation contains ammonium nitrate in a proportion of 0.025% by weight to 2.0% by weight, preferably 0.05% by weight to 1.0% by weight, more preferably around 0.075% by weight, per 0.1% by weight of silver nanoparticles.