COMPATIBLE SOLUTES FOR PREVENTING OR TREATING SARS-COV-2 INFECTIONS

Abstract

The present invention relates to the use of organic and highly water-soluble compatible solutes or a solute mixture, preferably in the form of an inhalable, oropharyngeally, nasally and intravenously administrable composition, in the prevention or treatment of diseases caused by ss(+)RNA viruses of the Coronavriridae family, preferably of those diseases caused by SARS-CoV-1, SARS-CoV-2, MERS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-NL63 and/or HCoV-229E. Particularly suitable solutes in the meaning of the invention are ectoine and its derivatives, Glycoin, mannosylglycerate (Firoin) and mannosylglyceramide (Firoin-A), which, due to their strong water-binding capacity, reduce the binding of the viruses to the receptors of the host cell in the transitional epithelium, e.g. eye, in the internal epithelium, e.g. lung, and in the endothelium and thus reduce or prevent the multiplication of the viruses. According to the invention, prevention is enabled by a reduced infectious sputum and breath, and treatment and rehabilitation of the affected tissues is enabled by the membrane protective properties of the compatible solutes according to the invention.

Claims

1. A method of preventing or treating a disease caused by a ss(+)RNA virus of the Coronavriridae family, said method comprising administering to a patient in need thereof a composition comprising at least one compatible solute or solute mixture, wherein the at least one compatible solute or solute mixture is selected from organic and highly water soluble compounds.

2. The method according to claim 1, wherein the at least one solute has a water-binding capacity of greater than or equal to 7 mol/mol H.sub.2O/solute.

3. The method according to claim 1, wherein the at least one solute is selected from glyceryl glucoside (Glycoin), glycine betaine, mannosylglycerate (Firoin), mannosylglyceramide (Firoin-A), ectoine and its derivatives of formula I and/or II and the physiologically compatible salts, amides and esters of the aforementioned compounds, wherein in formula I and in formula II ##STR00002## R1=H or alkyl, R2=H, COOH, COO-alkyl or CO—NH—R5, R3 and R4 are each independently H or OH, R5=H, alkyl, an amino acid residue, dipeptide residue or tripeptide residue n=1, 2 or 3, Alkyl=an alkyl radical with C1-C4 carbon atoms.

4. The method according to claim 1, wherein the disease is caused by an ss(+)RNA virus of the genus Betacoronavirus and/or Alphacoronavirus.

5. The method according to claim 1, to wherein the disease is caused by an ss(+)RNA virus selected from SARS-CoV-1, SARS-CoV-2, MERS-CoV, HCoV-HKU1, HCoV-OC43, HCoV-NL63 and/or HCoV-229E.

6. The method according to claim 1, wherein the disease comprises infection and/or inflammation of the transitional epithelial tissues and/or internal epithelial tissues.

7. The method according to claim 1, wherein the disease comprises infection and/or inflammation of the endothelium.

8. The method according to claim 1, wherein the ss(+)RNA virus interacts with at least one membrane-bound protein or component thereof on human cells and uses this protein or component thereof as a receptor for binding to the cell.

9. The method according to claim 1, wherein the ss(+)RNA virus interacts with a receptor selected from angiotensin converting enzyme 2 (ACE2), aminopeptidase N (APN) and/or dipeptidyl peptidase 4 (DPP4).

10. The method according to claim 1, wherein the at least one compatible solute reduces or prevents the unfolding and/or opening of the viral protein suitable for binding to the human receptor of the ss(+)RNA virus.

11. The method according to claim 1, wherein multiplication of the ss(+)RNA virus is reduced or prevented.

12. The method according to claim 1, wherein the at least one compatible solute shields the membrane of transitional epithelia, the internal epithelial tissues, and/or the endothelium.

13. The method according to claim 1, wherein the at least one solute is administered in combination with anti-viral compounds, anti-inflammatory compounds, interleukin blockers, anti-inflammatory anti-cytokines, inhibitors of viral receptors and/or vaccines.

14. The method according to claim 1, wherein the at least one solute is administered to a patient who comes from a region with high fine dust pollution, belongs to a professional group with high fine dust pollution, has a previous vascular disease and/or a chronic respiratory disease.

15. The method according to claim 1, wherein the at least one compatible solute or solute mixture is present in the composition in an amount of greater than or equal to 0.0001% by weight to less than or equal to 70% by weight based on a total content of the composition.

16. The method according to claim 15, wherein the composition is in i) a solid form including powder, granules, capsules, lozenges and effervescent tablets, ii) a liquid form including solution, injection, infusion and suspension, and/or iii) as a mixture including spray, aerosols and inhalants.

17. The method according to claim 15, wherein the composition is in liquid form for solution for infusion or in liquid form suitable for use with a nebulizer and/or respirator.

18. The method according to claim 15, wherein an infusion is administered in combination with an inhalant.

19. A kit comprising at least one ready-to-use composition according to claim 15 for inhalation, and a device for administering the composition.

20. The method according to claim 15, wherein a device for the controlled delivery of the composition is used, wherein the device is suitable for generating aerosols of the composition, allows the composition to be inhaled via the mouth and/or nose, ensures a metered delivery of a defined spray burst of a liquid or dry composition, ensures a metered delivery of a liquid composition into the eyes, and/or allows the composition to be sprayed in the oral cavity, throat and/or nasal cavity

21. A method of identifying a compatible solute according to claim 1, comprising the steps of providing a cell line which has membrane-bound surface proteins as potentially viral receptors, contacting the cells with a compound which is potentially a compatible solute according to claim 1, adding a viral receptor binding domain comprising a measurable signal, incubation of approach of the cells contacted with the potentially compatible solute and the viral receptor binding domain comprising a measurable signal, recording the signal measurable on the cell and determination of a reduced binding between the viral receptor binding domain and the human membrane-bound surface protein.

Description

DESCRIPTION OF THE FIGURES

[0116] FIG. 1 Gating strategy to exclude the dead cells from the analysis. A) FL1 shows the green fluorescence resulting from the detection of cell-bound SARS-Cov-2 S1. Dead cells were counterstained with propidium iodide. Dead cell staining is visible by a shift in the FL2 axis. Thus, cells in gate R2 are live cells. B) The cells in gate R2 were plotted as a histogram and the mean fluorescence intensity of these cells was determined.

[0117] FIG. 2 Proof of the binding of the Cov2 S1 protein to A549 cells using flow cytometry. The mean fluorescence intensity of living cells is shown.

[0118] FIG. 3A Proof of the effect of ectoine compared to NADA on the binding of the Cov2 S1 protein to A549 cells. Cov2 S1 protein binding to A549 cells were measured using immunofluorescence and subsequent flow cytometry. The mean fluorescence intensity of living cells is shown. Measurement series n=2 for 1.25% and 2.5%, n=1 for 5%; the mean values from both measurements are shown.

[0119] FIG. 3B Depiction of the influence of ectoine on the binding of the Cov2 S1 protein to A549 cells (as in FIG. 3A), with the concentration range being extended to 0.25% to 7.5% ectoine. The mean values from both measurements are shown.

[0120] FIG. 4A Depiction shows the results as a relative increase in fluorescence. The increase in fluorescence of A549 cells due to the binding of the Cov2 S1 protein to the cell surface was calculated by dividing the mean fluorescence intensity of the protein-stimulated cells by the intensity of the non-protein-stimulated cells.

[0121] FIG. 4B The data of all experiments were combined, the background fluorescence eliminated and plotted against the concentration of the Cov2 S1 protein. The inhibitory effect of ectoine is significant even when the standard deviation is taken into account. In comparison, cells preincubated with NADA are comparable to untreated cells

[0122] FIG. 5 This figure shows all possible formulations and applications of the solutes according to the invention, or compositions containing at least one solute or solute mixture, preferably ectoine and/or its derivatives, summarized. Only the infusion solution is not shown in this figure.

REFERENCES

[0123] Costa et al. da Costa M S, Santos H, Galinski E A. An overview of the role and diversity of compatible solutes in Bacteria and Archaea. Adv Biochem Eng Biotechnol. 1998; 61:117-53. [0124] Dong et al. Dong, J.; Xu, X.; Liang, Y.; Head, R.; Bennett, L. Inhibition of angiotensin converting enzyme (ACE) activity by polyphenols from tea (Camellia sinensis) and links to processing method. Food Funct. 2011, 2, 310-319. [0125] Fang Li Structure, Function, and Evolution of Coronavirus Spike Proteins. Annu. Rev. Virol. 2016. 3:237-61. doi: 10.1146/annurev-virology-110615-042301 [0126] Hahn et al. Marc Benjamin Hahn, Frank Uhlig, Tihomir Solomun, Jens Smiatek and Heinz Sturmad Combined influence of ectoine and salt: spectroscopic and numerical evidence for compensating effects on aqueous solutions Phys. Chem. Chem. Phys., 2016, 18, 28398 [0127] Hoffmann et al. Markus Hoffmann, Hannah Kleine-Weber, Simon Schroeder, . . . , Marcel A. Müller, Christian Drosten, Stefan Pöhlmann SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor Cell 181, 271-280 Apr. 16, 2020 Elsevier Inc. https://doi.org/10.1016/j.cell.2020.02.052 [0128] Roychoudhury et al. Roychoudhury A, Haussinger D, Oesterhelt F. Effect of the compatible solute ectoine on the stability of the membrane proteins. Protein Pept Lett. 2012 August; 19(8):791-4. [0129] Tay et al. Matthew Zirui Tay, Chek Meng Poh, Laurent Rénia, Paul A. MacAry and Lisa F. P. Ng The trinity of COVID-19: immunity, inflammation and intervention. Nature Reviews, Immunology doi: 10.1038/s41577-020-0311-8 [0130] Tortoricia M. Alejandra Tortoricia David Veeslera, Structural insights into coronavirus entry. Advances in Virus Research, Volume 105. 2019. doi.org/10.1016/bs.aivir.2019.08.002 [0131] Uhal et al. Bruce D. Uhal, MyTrang Dang, Vinh Dang, Roger Llatos, Esteban Cano, Amal Abdul-Hafez, Jonathan Markey, Christopher C. Piasecki and Maria Molina-Molina Cell cycle dependence of ACE-2 explains downregulation in idiopathic pulmonary fibrosis Eur Respir J 2013; 42: 198-210 doi: 10.1183/09031936.00015612 [0132] Wang et al. Bolin Wang, Ruobao Li, Zhong Lu, Yan Huang Does comorbidity increase the risk of patients with COVID-19: evidence from meta-analysis. AGING 2020, Vol. 12, No. 7 [0133] Wu et al. Xiao Wu M S, Rachel C. Nethery PhD, M. Benjamin Sabath M A, Danielle Braun PhD, Francesca Dominici PhD. Exposure to air pollution and COVID-19 mortality in the United States preprint doi: 10.1101/2020.04.05.20054502 [0134] Zhao et al. Qianwen Zhao, Meng, Rahul Kumar, Yinlian Wu, Jiaofeng Huang, Ningfang Lian, Yunlei Deng, Su Lin. The impact of COPD and smoking history on the severity of Covid-19: A systemic review and meta-analysis. Accepted article

[0135] The following examples show the efficacy of selected compatible solutes to demonstrate the feasibility of the invention, but without limiting the invention to them.

EXAMPLES

Example 1: Inhibition of Binding of the SARS-CoV-2 Spike S1 Protein to A549 Cells by Compatible Solutes

[0136] Material

[0137] Ectoine: (4S)-2-Methyl-1,4,5,6-tetrahydropyrimidine-4-carbon acid, CAS Nr. 96702-03-3

[0138] NADA: Nγ-Acetyl-L-2,4-diaminobutyric acid, CAS Nr. 1190-46-1

[0139] Hydroxyectoine: CAS-Nr. 165542-15-4

[0140] Glycoin: CAS-Nr. 22160-26-5

[0141] Mannosylglycerate: CAS-Nr. 164324-35-0

[0142] Glycine-Betaine: CAS Nr. 107-43-7

[0143] L-Prolin: CAS-Nr. 147-85-3

[0144] SARS-CoV-2 Spike S1 protein: trenzyme life science services, Cat. No. P2020-001, Lot No.: 11PO

[0145] Method

[0146] Angiotensin-converting enzyme 2 (ACE2)-expressing A549 cells from the American Type Culture Collection (ATCC) were used (Uhal et al. 2013). The adherent cells were routinely cultured in T-75 flasks in DMEM with 10% FCS containing penicillin and streptomycin as antibiotics in a CO.sub.2 incubator (5% CO.sub.2, 37° C.). When the bottom of the flask was 2/3 covered with cells, they were detached with accutase.

[0147] To perform the assay, the A549 cells were cultured to confluence in DMEM containing 10% FCS, and confluent culture was continued for another four days during which time the cell culture medium was replaced.

[0148] For the inhibition experiment, the cells were detached with accutase. Each 105 cells were transferred to a sample tube and pretreated with different concentrations of the compatible solute (Table 2) to give a final volume of 100 μl.

[0149] After 30 min incubation, the HIS-tagged SARS-Cov2-S1 protein is added to each tube and incubated for an additional 60 min on a tumble shaker at 450 rpm and room temperature.

TABLE-US-00004 TABLE 3 compatible Solute + CoV2 S1 Concentration [%] Amount of CoV2 compatible Solute S1 [μg] 7.5 5 2 1 0 5 5 2 1 0 2.5 5 2 1 0 1.25 5 2 1 0 0.75 5 2 1 0 0.25 5 2 1 0 0 5 2 1 0

[0150] The evaluation was carried out via immunofluorescence using flow cytometry. Binding of the SARS-CoV-2 Spike 51 protein is analyzed by indirect immunofluorescence using a CyFlow SL (Sysmex GmbH) flow cytometer. The cells were first centrifuged at 300×g, 5 min and then resuspended in 100 μl PBS with 1.5% fetal calf serum and 10 mM sodium azide. The cells were then challenged for 30 minutes with 5 μg/ml mouse antibody directed to the HIS tag (Biolegend). After washing (PBS with 1.5% fetal calf serum and 10 mM sodium azide), the cells were labeled with the secondary antibody (20 μg/ml, rabbit anti-mouse IgG (H+L), Alexafluor 488, Invitrogen) incubated for 20 minutes. The cells were then washed once and resuspended in 1 ml PBS without azide/FCS. Shortly before the measurement, 10 μl of a 0.5 mg/ml propidium iodide solution were added. Thereafter, the cells were immediately filtered through a 50 μm cell strainer and then immediately measured. All steps are performed at 4° C. in the dark.

[0151] The fluorescence of the A549 cells is then analyzed by flow cytometry. For this purpose, a region is placed on the live cells (the dead cells can be excluded from the analysis since they absorb the red propidium iodide dye) and the geometric mean relative fluorescence (MFI) of the cells in this region is determined as in FIG. 1 shown.

Example 2: Proof of the Binding of the SARS-Cov2-S1 Protein as a Function of the Concentration in A549 Cells

[0152] A549 cells are grown to confluency in DMEM with 10% FCS. Thereafter, the cells were supplied with fresh cell culture medium every day. Subsequently, the cells were stimulated with different concentrations of the SARS-Cov2-S1 protein without an extremolyte, as described in example 1. The analysis was performed by cytometry.

TABLE-US-00005 TABLE 4 MFI-values of FIG. 2. The corresponding coefficient of variation in percent (CV %) of the fluorescence intensity of the cells is also given. MFI CV % Unstained A549 1.17 71.39 A549 control staining 1.84 87.62 A549 + 1μCov2 S1 2.29 65.24 A549 + 2μCov2 S1 2.90 70.55 A549 + 5μCov2 S1 4.23 88.75

[0153] In a comparison of unstained (“unstained A549”) and stained A549 control cells (“A549 control staining”), a slight background staining is measured. Control stained A549 cells are cells that were not stimulated with Cov2 S1 protein but treated with a Fitc-labeled antibody for staining. Taking these controls into account, the signal of the bound spike protein was already detectable after stimulating the cells with 1 μg Cov2 S1. Increased protein binding to the A549 cells was detectable by using higher amounts of the protein.

[0154] Thus, by means of this assay, a concentration-dependent binding of a binding domain, such as Cov2 S1, to ACE-expressing cells, such as A549, can be detected and a substance can be examined for its effect of inhibiting the binding of Cov2 S1 to A549 cells.

[0155] The assay can be carried out in a modified form using the cell lines mentioned below. Any cell line which expresses angiotensin-converting enzyme 2 (ACE2), aminopeptidase N (APN) and/or dipeptidyl peptidase 4 (DPP4) is a suitable cell line for the purposes of the assay according to the invention. The detailed properties of the cell lines listed below can be looked up, inter alia, in the protein atlas: https://www.proteinatlas.org/ENSG0000013Q234-ACE2/cell and are known to the person skilled in the art.

TABLE-US-00006 TABLE 5 ACE-Receptor expressing cell lines Name ACE2 Origin Provider (Availability examples) COS7 + Kidney Creative biogene CSC-RO0290, (monkey) LGCStandards ATCC ® CRL-1651 ™ Hek293 + human Creative biogene CSC-RO0292/ kidney LgcStandards ATCC ® CRL-1573 ™ Hek293T + human Creative biogene CSC-RO0641 kidney LgcStandards ATCC ® CRL-11268 ™ Hep2G + Liver LgcStandards ATCC ® HB-8065 ™ HUVEC + Endothelium LgcStandards ATCC ® CRL-1730 ™ RPMI-8226 + Peripheral LgcStandards ATCC ® CCL-155 ™ blood

[0156] A suitable antibody which detects the expression of the receptor on the cell surface is preferably included in the assay. Cell lines that show low mRNA expression for ACE are cultivated in DMEM with at least 10% FCS. Hep 2G cells show high expression of the RNA for ACE. HUVEC cells express ACE2 and are cultured in Vascular Cell Basal Medium using the Endothelial Cell Growth Kit (ATCC). As a control, MRC5 cells that do not express the ACE receptor are included. In addition, Hoffmann et al. 2020 demonstrated that the cells are not infected by SARS CoV2. Therefore, the MRC5 cells represent an excellent control for the specificity of the binding assay used. MRC5 cells are cultured in DMEM with 10% FCS.

[0157] The HUVEC cell line, which is already used for ACE inhibition tests (Don et al.), is particularly suitable for providing evidence of a potential Covid-19 endotheliitis as currently discussed and the positive effect of compatible solutes according to the invention, such as the ectoine.

[0158] In the binding experiment, the aforementioned cell lines are incubated with the CoV2 S1 concentrations shown in Table 6.

Example 3: Influence of Ectoine on the Binding of the SARS-Cov2-S1 Protein to A549 Cells

[0159]

TABLE-US-00007 TABLE 6 compatible Solute + CoV2 S1 Concentration [%] Amount of compatible Solute CoV2 S1 [μg] 20 5 2 1 0 15 5 2 1 0 10 5 2 1 0 5 5 2 1 0 2.5 5 2 1 0 1.25 5 2 1 0 0 5 2 1 0

[0160] In order to investigate whether ectoine has an influence on the binding of the Cov2 S1 protein to A549 cells, detached cells were examined with different concentrations of a solute according to the invention, such as ectoine. For this purpose, the cells were pre-incubated with the respective solute and then treated with different concentrations of the Cov2 S1 protein (see example 1 and 2).

[0161] In this “proof-of-concept” experiment with n=1, a lower fluorescence of the Cov2 S1 protein bound to A549 cells was measured even in the presence of 2.5% ectoine (data not shown). This indicates reduced binding of Cov2 S1 protein to A549 cells in the presence of ectoine. No effect on the binding between Cov2 S1 and the A549 cells was found for NADA compared to ectoine. Both the experimental mixture [1.25/2.5% NADA+5 μg Cov2 S1+A549] and [5 μg Cov2 S1+A549] show the same mean fluorescence (cf. FIGS. 2 and 3). Thus, NADA does not appear to affect the binding of the virus to the host cell.

[0162] To verify the experiment, the concentrations of 1.25% and 2.5% were repeated and 5% of the respective solute was also examined. The results are shown in table 7.

TABLE-US-00008 TABLE 7 MFI-values of FIG. 4. The corresponding coefficient of variation in percent (CV %) of the fluorescence intensity of the cells is also given. MFI CV % 5% Ectoin Without Cov2 S1 2.23 181.07 1μCov2 S1 2.07 88.77 2μCov2 S1 2.17 134.70 5μCov2 S1 2.43 123.09 2.5% Ectoin Without Cov2 S1 2.03 131.63 1μCov2 S1 2.43 142.38 2μCov2 S1 3.15 174.07 5μCov2 S1 2.61 159.93 1.25% Ectoin Without Cov2 S1 2.17 117.24 1μCov2 S1 3.09 151.13 2μCov2 S1 2.21 152.85 5μCov2 S1 3.25 144.79 5% γ-NADA Without Cov2 S1 1.77 102.48 1μCov2 S1 2.52 113.55 2μCov2 S1 2.68 124.69 5μCov2 S1 3.51 121.95 2.5% γ-NADA Without Cov2 S1 1.62 101.84 1μCov2 S1 2.35 129.73 2μCov2 S1 2.99 144.33 5μCov2 S1 4.26 130.39 1.25% γ-NADA Without Cov2 S1 1.61 83.86 1μCov2 S1 2.33 126.42 2μCov2 S1 3.06 142.59 5μCov2 S1 4.06 154.67

[0163] With 5% ectoine, a complete inhibition of the binding of the Cov2 S1 protein to A549 cells could be shown. In comparison, no inhibition could be achieved with NADA. Here the mean fluorescence in the presence of 1.25%, 2.5% or 5% NADA increases comparably to the mean fluorescence of the test set without a compatible solute (cf. FIG. 2). The experiment with NADA and ectoine was repeated with the concentrations listed in Table 3. It was confirmed that NADA had no effect on Cov2 S1 protein binding to A549 cells (data not shown), whereas ectoine repeatedly had a significant effect (FIG. 3b). All tests carried out were summarized in order to determine the significance of the effect. The background fluorescence of the A549 cells was subtracted and the specific values plotted against the concentration of Cov2 S1 protein binding (FIG. 4b). It was confirmed that ectoine has a significant effect on A549 cells and inhibits the binding of the Cov2 S1 protein. Significance calculation:

TABLE-US-00009 Mann Whitney test P value 0.0286 Exact or approximate P value? Exact P value summary * Significantly different (P < 0.05)? Yes One- or two-tailed P value? Two-tailed Sum of ranks in column Ectoin 10.26 %, NADA 5%, Mann-Whitney U 0 Difference between medians Median of column Ectoin 5% 0.3050, n = 4 Median of column NADA 5%,  1.820, n = 4 Difference: Actual 1.515 Difference: Hodges-Lehmann 1.505

Example 4: Influence of Glycoin and Ectoine on the Binding of the SARS-Cov2-S1-Proteins

[0164] Experiments analogous to Example 3 are carried out with the cell lines HUVEC, HEK293, RPMI-8226 and Hep G2 (Table 5). Ectoine and glycoin in different concentrations (Table 3) are tested as compatible solutes.

Example 5: Influence of Glycoin, Mannosylglycerate, Hydroxyectoine and Ectoine on the Binding of the SARS-Cov2-S1 Protein

[0165] Experiments analogous to example 3 are carried out with the cell lines HUVEC, HEK293, RPMI-8226 and Hep G2 (Table 5), the Cells are not pre-incubated with ectoine, but ectoine or glycoin and Cov2 S1 protein are administered at the same time. Ectoine, hydroxyectoine, mannosylglycerate and glycoin are tested as solutes in various concentrations (Table 3).

[0166] As a further approach, the Cov2 S1 protein is pre-incubated with ectoine or glycine and then the cells are incubated with the pre-incubated mixture [Cov2 S1 protein+ectoine] in different concentrations according to Table 3. The pre-incubation [Cov2 S1 protein+ectoine] takes place for 5 min, 10 min and 30 min each at room temperature. The analysis then takes place analogously to example 3. The experiment is carried out with the cell lines HUVEC, HEK293, RPMI-8226 and Hep G2 (Table 5).

Example 6: Calculation of the Increase in Relative Fluorescence

[0167] To make the experiments comparable, the increase in fluorescence of A549 cells due to the binding of the Cov2 S1 protein to the cell surface was calculated by dividing the mean fluorescence intensity of the cells by the mean fluorescence intensity of the background staining. To determine background staining, cells were stained with an antibody in the absence of Cov2 S1. The results are shown in FIG. 5.

[0168] It can be seen that in the absence of a compatible solute, a concentration-dependent increase in mean fluorescence (fold fluorescence) is detected upon addition of the Cov2 S1 protein to A549 cells. While ectoine already shows a positive effect at 1.25% and inhibits the binding of the Cov2 S1 protein to A549 cells, no significant effect can be demonstrated for NADA at the same concentration. The effect of ectoine increases with increasing concentration and achieves complete inhibition of binding at 5% ectoine. In comparison, little inhibition of binding is shown with 5% NADA.

Example 7: Atomic Force Spectroscopy to Determine the Effect of Compatible Solutes on the Stability of Membranes

[0169] Based on the method of Roychoudhury et al. a method is carried out using atomic force microscopy to detect a bond between a viral membrane-bound protein, in particular peplomer, and a human membrane-bound surface protein. The procedure is based on the following steps.

[0170] The binding domain to be tested or the entire protein is bound to a surface. The surface can be a membrane and even a whole cell that expresses the receptor such as ACE2, APN and/or DPP4. At least two different approaches are tested, one approach contains the human viral receptor with a compatible solute (ectoine 1 M) in a buffer (300 mM KCl and 20 mM Tris at pH 7.8) and one approach contains the human viral receptor in the buffer without solute. In a next step, the tip of the atomically guideable arm (AFM tip) of the device (AFM device from Asylum Research, Olympus OMCL TR400 silicon nitride canilever with a spring constant of 20 pN/nm) is provided with a viral receptor binding domain, preferably S1, or the whole protein, preferably of SARS-CoV-2. The protein has an HIS tag. This tip is then approached to the bound sample (viral receptor with/without solute) and gradually brought into contact. While approaching as well as during moving away from the bound protein, the Newton force is versus the distance between the potential binding partners—here ACE2 and S1—and recorded as a force curve (retraction speed of 400 nm/s).

[0171] The measured power of the experimental approaches with solute compared to without solute allow conclusions to be drawn about molecular interactions between a membrane-bound protein, such as ACE2, and the potential binding partner, such as the SARS-CoV-2 protein, and statements about the shielding effect of the solute on membrane-bound proteins, as already described for ectoine in Roychoudhury et al. The method is suitable for testing all compatible solutes.

[0172] The atomic force spectroscopy is carried out with the following combinations as an example

[0173] Ectoine—Spike S1 protein

[0174] Hydroxyectoine—Spike S1 protein

[0175] Glycoin—Spike S1 protein

[0176] Mannosylglycerat—Spike S1 protein

Example 8: Examples of Formulations of the Solute According to the Invention A-1) Solute-Containing Inhalant Solution 13%

[0177] The aim of an inhalant within the meaning of the invention is to wet the lung surface as well and as extensively as possible with a thin layer of ectoine hydrate. A theoretically maximally and completely wetted lung is calculated using the data from Hahn et al. and Roychoudhury et al.

[0178] The area of a hydrated ectoine: 3.5e10.sup.−10 m*3.5e10.sup.−10 m*3.14=3.85e10.sup.−19 m.sup.2 (circle formula). Distance ectoine to a water molecule=0.35 nm

[0179] Assuming that this is equal to the radius and the surface area of the lungs=100-150 m.sup.2, it is calculated that 2.6e10.sup.20 ectoine molecules are required to achieve a monolayer hydration shell for a complete occupancy of the area. For two hydration shells, 5.2e10.sup.20 ectoine molecules would be required

[0180] It should be noted that the above calculation applies to a rather small lung and the area can be up to 1.5 times larger. The assumed radius of hydrated ectoine at 0.35 nm is the closest possible state. A less dense state (r=0.8 nm) would require less ectoine. Therefore, the present calculation is only to be regarded as indicative and may need to be adjusted to the size and/or age of the patient in individual cases.

[0181] Molecular weight ectoine: 142.16 g/mol

[0182] 1 mol=6.022e10.sup.23 particles

[0183] Amount of ectoine=5.2e10.sup.20/(6.022e10.sup.23×mol-1)=0.864e10.sup.−3 mol=0.123 g ectoine

[0184] With a 13% solution (130 g/I) that would be 0.00094 l or 0.94 mL

[0185] A dose of between 1-1.5 mL of an inhalant solution containing 13% solute would therefore be required to effectively arrive in the lungs so that ectoine is theoretically distributed homogeneously in the lungs. This determined concentration is based on the assumption of a single application

[0186] A-2) Solute Inhalant Solution Containing 3.9%

[0187] Starting from an inhalant solution containing 3.9% solute, a three-time application, e.g. distributed throughout the day, necessary to achieve a dose comparable to the 13% solution.

[0188] A) Infusion Solution 4%

[0189] An isotonic NaCl solution of 0.9% has an osmolar activity of 286 mOsmol/kg H.sub.2O. A 2% ectoine solution has an osmolarity of 147 mOsmol/kg H.sub.2O. An isotonic ectoine solution thus has a concentration of 3.89%.

[0190] An isotonic infusion solution is preferred so as not to irritate or damage the tissue. Therefore, an isotonic ectoine infusion solution contains 3.89% ectoine. Another combination is an ectoine infusion solution in combination with a NaCl solution suitable for infusion. Such a product contains a small amount of NaCl in combination with an adapted ectoine solution, resulting in an isotonic ectoine-NaCl infusion solution.

[0191] With the other solutes within the meaning of the invention, corresponding infusion solutions can be calculated according to their osmolarity.

TABLE-US-00010 Solute Solute [%] NaCl [%] Ectoine 3.0 3:5 3.8 4 0.9++ Hydroxyectoine * * * * 0.9++ Betaine * * * * 0.9++ Glycoin * * * * 0.9++ *To be determined according to the osmolarity or ++ to be adapted to a physiologically compatible solution

[0192] In an i.v. pharmacokinetics study, an amount of ectoine of 100 mg/kg was used in the rat. Oral toxicity data indicate a NOAL of 2000 mg/kg body weight/day. Orally taken ectoine (1000 mg/kg) resulted in a plasma concentration of 99 μg/ml in rats.