DE102021000989A1 - Means for binding and displacing corona viruses on living cells and surfaces - Google Patents

Abstract

The invention relates to the use of formulations containing lactic acid bacteria for the neutralization of corona viruses (in particular SARS-CoV-2) on surfaces.

Description

field of invention

The invention relates to the use of formulations containing lactic acid bacteria for the neutralization of corona viruses (in particular SARS-CoV-2) on human and animal cells and surfaces by physical binding and displacement.

Prior Art and Background of the Invention

COVID-19 is a notifiable infectious disease that can occur as a result of infection with the novel coronavirus SARS-CoV-2 and that shows a broad but non-specific spectrum of symptoms. The virus is primarily absorbed via the respiratory tract and can then also be detected there. It was first described in Wuhan in December 2019. The virus particles, which are 60 to 160 nm in size, have a virus envelope in which several different types of membrane proteins are embedded. The characteristic appearance of the corona virus is due to the many club-shaped structures on the surface, the so-called spikes, that protrude outwards by about 20 nm. They consist of portions of the large glycosylated S protein (spike protein). These parts carry both a receptor binding domain with which the virus can dock to a cell and a subunit which, as a fusion protein, causes the viral envelope and cell membrane of host cells to fuse.

The COVID-19 pandemic first spread epidemically in China in January and February 2020 and became a worldwide pandemic with more than 102 million people infected (as of February 2, 2021) and more than 2.2 million fatalities (as of February 2021 ).

Infection with the new type of corona virus often occurs through droplet transmission, but also through inhaling virus-carrying aerosols. Staying unprotected in closed, poorly ventilated rooms is particularly risky. The Robert Koch Institute (RKI) does not rule out transmission through contaminated surfaces.

The virus has been detected in the secretions of the nose and throat, in sputum, stool, tears, blood, in aerosols and on surfaces. The main transmission route for SARS-CoV-2 is the respiratory intake of virus-containing liquid particles (breathing, coughing, speaking, sneezing).

Like other respiratory pathogens, the virus is believed to be spread primarily through virus-containing particles. These are released by infected people when they breathe, cough, sneeze, speak and sing and are then ingested by healthy people. The transition between aerosol and droplet infection is fluid. The WHO defines droplets as particles with a diameter of 5-10 µm. Particles that are smaller are called aerosols. Due to their size, droplets sink quickly to the ground). The smaller the particles, the longer they can remain in the air. The risk of aerosol transmission is increased in small, poorly ventilated spaces and activities that involve high levels of particulate emissions, such as loud speaking or singing.

After aerosols containing SARS-CoV2 have entered the airways, the spike protein of the virus, like the highly conserved spike proteins of other coronaviruses, preferentially binds to the angiotensin-converting enzyme 2 (ACE2) receptor on the surface of epithelial cells. As a transmembrane receptor, it enables the coronaviruses to penetrate, among other things, the epithelial cells of the airways and the parenchymal cells of the lungs. The viruses multiply there and are released after the cells have died and infect other cells.

As a prophylactic measure, in addition to wearing mouth and nose protection, frequent cleaning or disinfecting of hands and surfaces is recommended. Since the surfactants usually contained in soap and detergents destroy the fatty layer of corona viruses, these surfactants are sufficient in everyday life to largely rid hands and other surfaces of pathogens. The RKI does not recommend routine surface disinfection in domestic and public areas; disinfection that is necessary in individual cases should be carried out as a wipe and not as a spray disinfection, since the latter is less effective and the disinfectant can be inhaled. For this purpose, alcohol-based disinfection products are offered, the use of which is limited. On the one hand, alcohol dries out the skin, and on the other hand, vapors are produced when it is applied to large areas, which raises fire protection problems. An application in the nose, mouth or throat is not indicated.

The German Society for Hospital Hygiene recommends virucidal gargles and virucidal nasal sprays to "reduce the viral load at the portals of entry, since the probability of infection increases with exposure and the initial viral load has an influence on the severity of the infection".

Despite all the research in the field, there are still no means that effectively neutralize corona viruses and preventively prevent the infection in this way.

It is known that certain lactic acid bacterial species or strains of the intestine or vaginal tract can act against certain types of viruses such as HIV, HPV, hepatitis and other viruses, or that they can prevent the colonization of mucous membranes with these viruses , see Refs. [1]-[6].

Description of the invention

It has now been found that corona viruses (e.g. SARS-CoV2) can be prevented from infecting animal and human cells by lactic acid bacteria. In addition, it has been shown that lactic acid bacteria are also suitable for highly effectively neutralizing corona viruses on other surfaces, such as wood, metal, glass, ceramic or plastic surfaces.

Particularly suitable for this are commensal lactic acid bacteria of the genus Lactobacillus with the species Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus ruminis, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus brevis or Lactobacillus rhamnosus. It is also interesting for the proposed application that lactic acid bacteria that have been treated with heat or radiation, i.e. lactic acid bacteria that are no longer capable of dividing, also cause a neutralization of corona viruses.

However, the exact mechanisms of action have not been clarified, here various active (through released effectors), passive-immunological or passively inhibiting processes (e.g. virus binding to the bacterial surface or steric hindrance of the viruses to binding to epithelial cells, thereby strengthening the barrier function the mucous membrane/skin) is suspected. Due to the use of killed lactic acid bacteria and the absence of immune cells in the model studied, the observations made can only be explained by a physical binding of coronaviruses to the cell wall of lactic acid bacteria or a blockade of the virus binding to the human and animal cells, and are therefore a complete one surprising novel discovery. In principle, the invention thus allows the development of disinfectants, cleaning agents, medical products and foodstuffs, but in combination with pharmaceutical active ingredients also of drugs against or in the case of coronavirus infections with physical principles of action.

The possibility of using lactic acid bacteria that are no longer alive to neutralize corona viruses also allows the development of storage-stable neutralization products.

Furthermore, it was found that certain cell wall components and extracts from the described lactic acid bacteria can also neutralize corona viruses. These are cell wall components and extracts containing the S-layer proteins of the lactic acid bacteria. These proteins, their production and properties are described in the literature [7]-[11], so that further discussions are not necessary at this point.

The compositions according to the invention can be provided as a solution (in particular as an aqueous or alcoholic solution). Depending on the exact area of application, they can be used in dosage forms known from the pharmaceutical sector (e.g. tablets, syrup, granules, powder, capsules, effervescent tablets, aerosol spray, powder spray, hydrogel) or the food sector (e.g. lozenges, hard caramel, syrup, granules, powder, capsules, effervescent tablets, chewing gum) are offered. Further, you can also use in the form of cleaning products, such as soap, syndet, foam, stick, paste, emulsion, gel, hydrogel, mask, shampoo, liquid soap, deodorant, detergent, dishwashing liquid, detergent, cleaning cloth, disinfectant, air purifier, water filter or as Coating of masks, bandages, plasters or wound dressings are formulated.

When used in the veterinary or human medical sector, the formulations mentioned from the pharmaceutical or food sector are used in or on the eyes, ears, nose, mouth, pharynx, throat, bronchi, lungs, skin, hair, scalp, mucous membranes or on wounds . The formulations from the cosmetics sector are mainly used on the skin (including the scalp and hair). The formulations from the (household) cleaning sector are used in particular on surfaces made of wood, metal, plastic, ceramics, paint or glass.

The solvents, carriers, emulsifiers, detergents, stabilizers, fragrances and auxiliaries customary in cosmetic or pharmaceutical preparations and household cleaners can be used for the formulation. For use in the mouth/nose and throat and bronchi/lungs, the compositions can also be sprayed or nebulized. This can be done in the form of pump sprays, but also with powder nebulizers. Appropriate spraying or nebulizing devices are from the pharmaceutical industry known in the field (e.g. for nasal sprays or asthma preparations), so that further explanations are not necessary at this point.

The formulations according to the invention can be produced in a customary manner. In the following examples, individual embodiments of the invention, namely the compositions and the production of the formulation, are explained in more detail.

examples

The invention is explained further by the examples below, without these being intended to have a limiting effect.

Example 1:

The lactic acid bacteria strains are used alive or killed by heat or gamma radiation and dried. For example, the lactic acid bacteria strain Lactobacillus gasseri DSM 25908 is freeze-dried after fermentation with protective substances, and the lyophilizate is treated by gamma irradiation.

Another example is a preparation in which the Lactobacillus crispatus strain DSM 25988 ferments after pre-cultivation from working ampoules in bioreactors under exclusion of air and heating in aqueous yeast extract/glucose solution and at the end of the fermentation step after pH adjustment for 20 to 60 minutes between 50 and 80°C, then the cells are centrifuged off and lyophilized with a protective agent.

The lactobacillus-containing active ingredients produced in this way are used, for example, as a component of disinfectants or household cleaners, or as a cosmetic product, food, medical product or pharmaceutical product in dosage forms that are effective locally on mucous membranes, the skin or wounds, such as powders, granules, syrups, gels, pastes , sprays, nebulizable dosage forms, inhalable dosage forms, foams, sticks, masks, bandages, plasters, rinsing solutions, lozenges, buco-adhesive films, coatings, sweets, dairy products or similar dosage forms, and preferably several times a day prophylactically against infection with the SARS CoV2 virus or other corona viruses are used, thus avoiding infection, e.g. of the respiratory mucous membranes, the skin or wounds.

Example 2:

With exemplary application of L. crispatus (DSM 25988), L. gasseri (DSM 25908) or L. reuteri (DSM 17648) as a powder in pH-adjusted (pH 7.4), serum-free medium in concentrations of 0.2, 0.5 , 1.0, 2.0, 5.0, 10.0, 20.0 and 44.0 mg/ml in cultures of Vero cells at a density of 5 × 10 ⁴ cells and in the presence of 20000 RLU of a rVSV-SARS-CoV-2 mutant (D614G) was shown after 24 hours of incubation at 37°C, a 50% reduction (mean inhibitory concentration) in the viral load in the Vero cells by means of fluorescence compared to negative controls (IC ₅₀ ) at concentrations of the active ingredients of 7.4, 2.9 and 5.9 mg/mL.

Vero cells are an established cell line derived from normal green monkey kidney cells and represent a standard model for testing antivirals.

At the same time, the concentration at which 50% of the Vero cells die due to cytotoxicity (CC ₅₀ ) of the 3 active ingredients was 44.2, 5.4 and 9.6 mg/mL, for example.

This results in an exemplary efficacy window for all 3 preparations in which the inhibition of the infection of the Vero cells with a SARS-CoV2 mutant can be effectively suppressed with mild cytotoxicity at the same time. This finding is surprising and completely new. The cytotoxicity of the active ingredients in clinical use can also be classified as significantly lower due to the protective mechanisms of the mucous membranes through mucus and saliva, or on the skin through the horny layer, which means that the therapeutic window in practical use should be larger.

Various mechanisms can be used for the observed effect. Vero cells are known to produce abundant fibronectin and collagen IV on the cell wall surface.

On the one hand, the lactic acid bacteria themselves bind to certain surface structures such as fibronectin in the natural target tissues, such as epithelial cells of the vaginal, gastrointestinal or respiratory tract, to extracellular matrix proteins, mucus components, saliva components, to connective tissue cells in wounds or to Keratinocytes of the skin, and thus prevent binding, absorption and proliferation of corona viruses on or in these target structures by crowding out and blocking binding sites. On the other hand, corona viruses can Glycoproteins, such as RGD motifs, may themselves be bound to adhesins of the lactic acid bacteria, and thus be prevented from infecting other structures.

Regular local application of the active ingredients as components of cosmetics, food, medical devices or pharmaceuticals can reduce the risk of colonization of target tissues with corona viruses and prevent active infection, disease and virus spread. In disinfectants and cleaners, the active ingredients can reduce the viral load on surfaces by absorbing corona viruses on lactic acid bacteria after the lactic acid bacteria/virus complexes have been removed.

Example 3:

The lyophilizates from Example 1 are formulated as throat lozenges or chewing gum, each having a strength of 20 mg Lactobacillus crispatus DSM 25988 or 50 mg Lactobacillus reuteri DSM 17648 per dose. With an average amount of saliva of 10 ml released when sucking a candy, for example, a concentration in the saliva of around 2 mg/mL or 5 mg/mL is achieved with these doses. This means that with throat lozenges of the strength mentioned, a mild inhibition of infection with coronaviruses can be achieved, but with chewing gum, based on the preclinical data, a clear inhibition.

According to the invention, due to the better effect-to-toxicity ratio of Lactobacillus crispatus DSM 25988 compared to Lactobacillus reuteri DSM 17648, Lactobacillus crispatus DSM 25988 pastilles with an active ingredient quantity in the range of approx. 50 mg to 200 mg, or a product with approx. 20 to 40 mg Lactobacillus gasseri DSM 25908 per dose best suited for an anti-coronavirus product.

Example 4:

The lyophilizates from Example 1 are formulated as a liquid or semisolid aqueous or alcoholic solution in a concentration of 20-200 mg/mL and filled into pump spray bottles customary in pharmaceuticals. The solution is characterized in that it contains solvents, preferably water, physiological saline, phosphate buffer or ethanol, stabilizers such as sodium edetate, benzalkonium chloride, propylene glycol, sorbic acid or sodium benzoate, and dispersing ingredients such as polysorbates, e.g. polysorbate 80, moisturizing ingredients such as aloe vera, contains hyaluronic acid, glycerin, panthenol or dexpanthenol, as well as flavors, plant extracts or essential oils such as thyme extract, sage extract, eugenol, menthol, chamomile oil, clove oil, eucalyptus oil, aniseed oil, sage oil or peppermint oil, and sweeteners such as sodium cyclamate, sorbitol, maltitol, xylitol, Steviol glycosides or erythritol and thickeners such as ectoine, xanthan, cellulose, pectin, or methyl cellulose. The solution can be sprayed into the mouth and throat area or into the nose and can have a prophylactic effect by neutralizing any corona viruses that may have entered. Preferably, 0.05 to 0.5 ml of the solution is given every 2-6 hours during the waking phase.

Example 5:

The lyophilizates from Example 1 are formulated as an aqueous or alcoholic solution in a concentration of 20-100 mg/l and filled into commercially available spray bottles. The solution can be sprayed onto common surfaces used in households and businesses (wood, plastic, glass, metal) and wiped off with a cloth after 30 s - 5 minutes. On surfaces contaminated with coronaviruses, there is a 60-80% reduction in surface load.

Example 5:

The lyophilizates from Example 1 are formulated in powder form and filled into powder nebulizers customary in pharmaceuticals. The powder can be sprayed into the mouth and throat for inhalation purposes while inhaling, reaches the respiratory tract, in particular the bronchi, through the nebulization, and can have a prophylactic effect there by binding any corona viruses that may enter. Usually 250-1000 µg of the powder preparation are nebulized in one spray. The dose range is between 0.1 and 20 mg/kg body weight. Preferably 0.1 to 10 mg/kg body weight of the powder preparation is given 2-3 times a day during the waking phase.

Example 6:

The lyophilisates from example 1 are formulated as a hydrogel as an aqueous solution in a concentration of 20-200 mg/l. The hydrogel is applied into the nasal cavity using an applicator or cotton swab. The hydrogel is characterized in that it contains pantothenol, in particular dexpanthenol, and at least one humectant, which is water-soluble or water-dispersible natural or synthetic polymers that form gels or viscous solutions in aqueous systems, polyhydric alcohols, mono-, di- and/or Includes polysaccharides. Polymers are preferably used. Suitable polymers are, for example, collagen derivatives (animal and vegetable), polyalkylene glycols, in particular polyethylene glycols, polyglycerols, alginates, carageen, pectins, tragacanth, gums, in particular gum arabic, cellulose derivatives, in particular cellulose ethers and/or cellulose esters, polyvinyl alcohol, polyvinylpyrrolidone and derivatives thereof and/or dextran. Particularly preferred cellulose derivatives for the purposes of the present invention include hydroxyethyl cellulose and/or methylhydroxypropyl cellulose. Preferably, 500 mg of the hydrogel is applied to each nostril every 4-6 hours during the waking phase.

literature

1. [1] A. Lopez-Santamarina et al., "Probiotic Effects against Virus Infections: New Weapons for an Old War," 2021.
2. [2] Lehtoranta, L, Latvala, S, and Lehtinen, MJ, "Role of Probiotics in Stimulating the Immune System in Viral Respiratory Tract Infections: A Narrative Review."
3. [3] Kou YR, Zhang Y, Yang JX, Villena J, Ar JO, and Kitazawa H. The Modulation of Mucosal Antiviral Immunity by Immunobiotics: Could They Offer Any Benefit in the SARS-CoV-2 Pandemic? “Front. physiol. | www.frontiersin.org, vol. 1, p. 699, 2020.
4. [4] Guadalupe Martinez M, Prado Acosta M, Candurra NA, and Ruzal SM, "S-layer proteins of Lactobacillus acidophilus inhibits JUNV infection," 2012.
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7. [7] Ulla Hynönen, Airi Palva, "Lactobacillus surface layer proteins: structure, function and applications", Appl Microbiol Biotechnol (2013) 97:5225-5243
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9. [9] Jenni Antikainen et al., "Domains in the S-layer protein CbsA of Lactobacillus crispatus involved in adherence to collagens, laminin and lipoteichoic acids and in self-assembly", Molecular Microbiology (2002) (2), 381-394
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Claims (10)

Use of lactic acid bacteria to prevent contamination of human and animal cells with coronaviruses. Use of lactic acid bacteria to bind and neutralize corona viruses on surfaces. Use of lactic acid bacteria according to claim 1 or 2 , characterized in that lactic acid bacteria belonging to the genus Lactobacillus with the species Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus ruminis, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus brevis or Lactobacillus rhamnosus are used. Use of lactic acid bacteria according to claim 1 , 2 or 3 , characterized in that lactic acid bacteria of the strain L. crispatus with the deposit number DSM 25988, the strain L. gasseri with the deposit number DSM 25908 and/or the strain L. reuteri with the deposit number DSM 17648 are used. Use of lactic acid bacteria according to one of Claims 1 until 4 , characterized in that the lactic acid bacteria are used in live form or in a form which has been killed by heat, UV radiation or gamma radiation. Use of lactic acid bacteria according to one of Claims 1 until 5 , characterized in that the lactic acid bacteria are used in the form of cell wall components or an extract of their surface proteins. Use of lactic acid bacteria according to claim 5 or 6 , characterized in that the lactic acid bacteria, their cell wall components or extracts are used in lyophilized form. Use of lactic acid bacteria according to one of Claims 1 until 7 , characterized in that the lactic acid bacteria as a solution, be formulated as a cream, a lotion, a gel, a coating or a nebulizable powder. Use of lactic acid bacteria according to one of Claims 1 until 8th for neutralizing corona viruses on surfaces made of wood, metal, plastic, ceramics, paintwork or glass. Use of lactic acid bacteria according to one of Claims 1 until 8th for the production of preparations for the neutralization of corona viruses on animal or human skin, mucous membranes or wounds.