GOLD-CONTAINING AGENTS FOR THE TREATMENT OF LUNG INFECTIONS

Abstract

The present application relates to pharmaceuticals for inhalation, containing aurothioglucose, and to an inhaler, preferably a powder inhaler, dosing inhaler or atomizer containing such pharmaceuticals. The application provides these pharmaceuticals for use in the prevention and therapy of pulmonary diseases, in particular infectious and inflammatory-infectious pulmonary diseases.

Claims

1. A medicament for inhalation, containing aurothioglucose.

2. A medicament for inhalation, containing aurothioglucose; and N-acetylcysteine.

3. A medicament for inhalation, containing aurothioglucose; a virostatic, selected from the group consisting of remdesivir, molnupiravir, favipiravir, ribavirin, lopinavir, umifenovir, nelfinavir and/or ritonavir, favipiravir, molnupiravir and/or ribavirin, in particular favipiravir; and N-acetylcysteine.

4. The medicament as claimed in claim 2, wherein the medicament contains aurothioglucose and N-acetylcysteine in a molar ratio of between 1:40 and 10:1 (aurothioglucose:N-acetylcysteine).

5. A medicament for inhalation, the medicament as claimed in claim 1, containing aurothioglucose; an active substance selected from hydroxychloroquine, chloroquine and/or ivermectin; and N-acetylcysteine.

6. The medicament as claimed claim 1, wherein the medicament contains a support material, comprising a carbohydrate, comprising lactose and/or mannose.

7. The medicament as claimed in claim 1, wherein the medicament is presented as a powder formulation, micronized.

8. The medicament as claimed in claim 1, wherein the medicament is presented as a solution or as an aerosol.

9. An inhaler, comprising at least one of a powder inhaler, dosing inhaler or nebulizer, containing a medicament as claimed in claim 1.

10. The medicament as claimed in claim 1, for use in the prevention or treatment of lung diseases.

11. The medicament for use as claimed in claim 10, wherein the lung disease is an infectious or mixed inflammatory and infectious lung disease.

12. The medicament for use as claimed in claim 10, wherein the lung disease is a lung infection, comprising at least one of a viral or mixed viral and bacterial lung infection.

13. The medicament for use as claimed in claim 10, wherein the lung disease is a disease which is caused by Coronaviridae which are included in the RNA virus group, in particular SARS-CoV-1, SARS-CoV-2 or MERS-CoV.

14. The medicament for use as claimed in claim 10, wherein the medicament is administered in a dose containing between 0.001 μmol and 450 μmol of gold.

15. The medicament for use as claimed in claim 10, wherein the medicament is administered in a dose containing between 0.1 μg and 1000 μg of gold per kg of body weight of the patient.

16. The medicament for use as claimed in claim 10, wherein the use is by means of inhalation, comprising at least one of liquid inhalation or powder inhalation.

17. The medicament for use as claimed in claim 10, wherein the use is by means of an inhaler comprising at least one of a powder inhaler, dosing inhaler or nebulizer.

Description

[0109] The present invention will now be illustrated with the aid of the following examples and figures; they are obviously not limited thereto.

[0110] FIG. 1. Anti-inflammatory effectiveness of auroacetylcysteine in a mouse model for acute allergic asthma: experimental setup.

[0111] FIG. 2. Anti-inflammatory effectiveness of auroacetylcysteine in a mouse model for acute allergic asthma: cell count in bronchoalveolar lavage for the investigation of inflammation of the respiratory tract. Graph: mean±SD, n=5 except for AAC group (n=4 (because of one death) * P<0.05 compared with placebo (support material), Kruskal-Wallis Test, Dunn's multiple comparison test.

[0112] FIG. 3. Anti-inflammatory effectiveness of auroacetylcysteine in a mouse model for acute allergic asthma: histological investigation of lung tissue.

[0113] FIG. 4. Anti-inflammatory effectiveness of auroacetylcysteine in a mouse model for acute allergic asthma: investigation of lung tissue with HE and LUNA staining.

[0114] FIG. 5. Anti-inflammatory effectiveness of auroacetylcysteine in a mouse model for acute allergic asthma: investigation of cell populations. Graph: mean±SD, n=5 except for AAC group (n=4 (because of one death) * P<0.05 compared with placebo (support material), Kruskal-Wallis Test, Dunn's multiple comparison test.

[0115] FIG. 6. Dose-effect curves for aurothioglucose (6A), aurothioglucose/N-acetylcysteine in a ratio of 1/2 (6B) and aurothiomalate (6C) as an inhibitor of SARS-CoV-2 PLpro.

[0116] FIG. 7. Influence of added N-acetylcysteine on the inhibition of SARS-CoV-2 PLpro by 1.0 μM aurothioglucose (n=2-3). AG=aurothioglucose; N=N-acetylcysteine. The ratios given are molar ratios.

[0117] FIG. 8. Activating influence of added N-acetylcysteine on the inhibition of the spike/ACE2 interaction by 20 μM aurothioglucose. N-acetylcysteine alone produced no inhibition of the spike/ACE2 interaction (97% of control at 100 μM). AG=aurothioglucose; N=N-acetylcysteine. The ratios given are molar ratios.

[0118] FIG. 9. Cytotoxicity of auranofin (AF), aurothiomalate (AM), aurothioglucose (AG), and the combinations of AM or AG with N-acetylcysteine (N) in a molar ratio of 1/2 (AM-N, AG-N, concentrations with respect to AM or AG), as well as auroacetylcysteine after 24 hours in CaLu-3 cells (n=3).

[0119] FIG. 10. Removal of zinc from PLpro by disulfiram, aurothioglucose (AG) and the mixture of aurothioglucose with N-acetylcysteine in a molar ratio of 1:2 (AG-N). (values given in comparison with untreated enzyme (PLpro)).

EXAMPLES

Example 1. Production of Single Doses for Inhalation

[0120] 1.a. Liquid ampoule: A solution of 1.5 mg of auranofin in 2.5 mL sodium chloride-containing water was introduced into a single dose container and introduced into a nebulizer for use.

[0121] 1.b. Dry ampoule: An ampoule was filled with 1.5 mg of aurothioglucose. Prior to use, 2.5 mL of water was injected and the solution was introduced into an inhaler. The prepared product had to be consumed within 3 hours.

Example 2. Production of Multiple Doses for Inhalation

[0122] 2.a. Powder spray: 30 mg of micronized aurothioglucose was suspended in 300 microlitres of ethanol. 30 mg of sorbitan trioleate was added and then added to a spray bottle with 15 g of propellant, with cooling; there were 300 spray applications of the product.

[0123] 2.b. Dry inhaler: 0.2 mg of micronized aurothioglucose was blended with 12 mg of lactose and prepared for powder inhalation with the strict exclusion of moisture. This was dependent on the type of inhaler and could, if necessary, involve compression into a disk.

Example 3. Assay of the Anti-Inflammatory Action of Inhaled Auroacetylcysteine in a Pre-Clinical Pilot Study in a Mouse Model for Acute Allergic Asthma by Means of Intranasal Application

[0124] The anti-inflammatory effectiveness of auroacetylcysteine (AAC) was tested in 2 doses (10 mg/kg and 100 mg/kg) in a mouse model for acute allergic asthma with 5 mice each time, compared with dexamethasone (1 mg/kg). There were 5 mice in each of 4 groups: AAC/dexamethasone/placebo (vehicle)/untreated in triplicate per group in a setup for intranasal application in an aerosol chamber (FIG. 1). The evaluation was carried out as follows: inflammation of the respiratory tract was determined by bronchoalveolar lavage (BAL). To this end, the cell count was determined in the bronchoalveolar lavage (FIG. 2). There was a significant difference compared with the placebo. The reduction of the total cell count was comparable with dexamethasone. The inflammation of the lung tissue was determined by means of a histological investigation (FIG. 3), HE and LUNA staining (FIG. 4); mucous production was determined by means of PAS staining. Compared with dexamethasone, however, different cell populations were influenced. Primarily eosinophilic and neutrophilic granulocytes as well as lymphocytes were reduced by AAC. Macrophages were increased compared with dexamethasone. (FIG. 5). In addition, the serum-specific Ag-IgG1 was measured (ELISA). The following results were obtained: auroacetylcysteine reduced the inflammatory parameters both peribronchially as well as in the parenchyma in a concentration of 10 mg/kg. The anti-inflammatory effect in the tissue was comparable with that of dexamethasone. The treatment of mice with acute exacerbation of allergic asthma with 10 mg/kg auroacetylcysteine over 5 days reduced the total number of inflammatory cells in the bronchial secretions, the extent of the inflammation in the respiratory tract, the number of eosinophils and neutrophiles in the respiratory tract, infiltrates of inflammatory cells in the lung parenchyma.

Example 4. Production of Auroacetylcysteine

[0125] 5 g of tetrachloroauric acid was mixed with 5 mL of water and cooled with ice. 3.18 g of 2,2′-thiodiethanol was added dropwise over 45 minutes, with vigorous stirring. Addition was complete when the solution was colourless and free from precipitate. 1.75 g of N-acetylcysteine in 27 mL of water was added slowly to this solution, with the formation of a white precipitate. This suspension was stirred for 1 hour and filtered through a vacuum filter. The precipitate was washed with 30 mL of water to which 1 drop of 2 N hydrochloric acid had been added, and then dried overnight. Auroacetylcysteine was obtained in a quantitative yield.

Example 5. Inhibition of SARS-CoV-2 Protease PLpro by Gold Compounds

[0126] The inhibition of the SARS-CoV-2 protease Papain-Like Protease (PLpro) was determined as follows: the test substances were dissolved in water as stock solutions and diluted one hundred-fold with HEPES buffer (50 mM HEPES, pH 7.5, 0.1 mg/mL foetal calf serum, 0.1% Triton-X-100), so that micromolar concentrations were obtained. Volumes of 50 μL of a 200 nM solution of SARS-CoV-2 PLpro in HEPES buffer or pure HEPES buffer (negative control) were pipetted into the wells of a black 96-well microtitre plate. 50 μL of the solutions of the test substances or pure HEPES buffer were added to each well (positive control) and the resulting solutions were mixed and incubated for one hour at 37° C. Next, a volume of 100 μL of a 100 μM solution of the substrate Z-Arg-Leu-Arg-Gly-Gly-AMC was added to all of the samples, mixed thoroughly, and the fluorescence emission was recorded over 10 minutes every 30 seconds (λ.sub.ex=355 nm, λ.sub.em=460 nm, 37° C., Victor™ X4 Perkin Elmer 2030 microplate reader). The increase in the fluorescence emission followed a linear trend (r.sup.2>0.97) and the enzyme activities in the individual samples were determined as the slopes thereof. The percentage calculation of the enzyme activity was given with respect to the untreated control (positive control). The results for the negative controls were used in order to confirm the absence of false positive results, for example by reaction of the test substance with the substrate. The IC.sub.50 values were considered to be those concentrations at which the test substance inhibited the enzyme activity by 50% compared with the positive control.

[0127] Results: the following IC.sub.50 values were determined from the dose-effect curves (FIG. 6):

[0128] Aurothioglucose: 7.03 μM (+/−2.31 μM)

[0129] Aurothioglucose/N-acetylcysteine in a molar ratio of 1/2:9.55 μM (+/−1.61 μM) (with respect to the quantity of aurothioglucose)

[0130] Aurothiomalate: 0.60 μM (+/−0.25 μM)

[0131] The addition of more equivalents of N-acetylcysteine to aurothioglucose did not substantially change the inhibition of the PLpro (FIG. 7).

Example 6. Inhibition of the Interaction of the SARS-CoV-2 Spike Protein with the ACE2-Receptor

[0132] The influence of the test substances on the spike/ACE2 interaction can be determined by ELISA. To this end, a 96-well plate was coated with the receptor binding domains of the spike protein and stored overnight at 4° C. The wells of the microtitre plate were emptied, replaced with a blocking solution for 2 hours, washed and emptied. The test substances and controls were added to the mixture with the ACE2 receptor and incubated for one hour at 37° C. The wells were washed. Streptavidin-horseradish peroxidase conjugate was added and incubation was carried out for one hour at room temperature. After washing again, 3,3′,5,5′-tetramethylbenzidine was added to a solution. After 5 minutes at room temperature, the absorption was determined at 450 nm (Perkin Elmer Victor X4 microplate reader). The activity remaining after the inhibitor addition was calculated as a percentage with respect to the untreated control.

[0133] Result:

[0134] 20 μM aurothioglucose resulted in an inhibition of the spike/ACE2 interaction (FIG. 8). The inhibition could be substantially increased by adding N-acetylcysteine. A molar ratio of aurothioglucose to N-acetylcysteine of 1:2 proved to be particularly effective.

Example 7. Cytotoxicity in CaLu-3 Cells

[0135] In order to determine the cytotoxicity, CaLu-3 cells were cultured in 96-well microtitre plates. The cell culture medium was replaced with fresh medium which contained the gold compounds in concentrations of 25, 50 or 100 μM and incubated for 24 hours at 37° C./5% CO.sub.2. Next, the remaining cells were photometrically determined using crystal violet stain (Victor X4 microplate reader). The quantity of cells in the treated samples was calculated as a percentage with respect to an untreated control.

[0136] The results are shown in FIG. 9. In the experiment, auranofin exhibited toxicity in CaLu-3 cells (<20% of the untreated control at 25 μM). Aurothiomalate, aurothioglucose, the combinations of aurothiomalate or aurothioglucose with N-acetylcysteine in a molar ratio of 1:2, as well as auroacetylcysteine exhibited no relevant cytotoxicity in concentrations of up to 100 μM.

Example 8. Removal of Zinc from PLpro

[0137] In addition to a cysteine in the catalytic centre of the enzyme, the protease Plpro contains further cysteines in a zinc binding domain which stabilise the structure and function of the enzyme. The removal of the bound zinc constitutes an interesting mechanism for the action of inhibitors for PLpro.

[0138] In order to establish whether the inhibitors are Zn-removing agents, the presence of the Zn.sup.2+ cation in solution was determined as follows. The inhibitor compounds were prepared as stock solutions in DMSO, as stock solutions in water or DMSO and diluted one hundred-fold with HEPES buffer (50 μM HEPES, pH 7.5) to 100 μM concentrations. Volumes of 50 μL of SARSCoV-2 PLpro (Elabscience) in HEPES buffer or blank HEPES buffer (control for false positive results) were placed in the wells of a black 96-well microtitre plate (Nunclon, Nunc). Volumes of 50 μL of the inhibitor solutions or 1% DMSO in HEPES buffer (control) were added. The resulting solutions (500 nM SARS-CoV-2 PLpro, 0.5% DMSO, 50 μM test compound or blank HEPES buffer) were mixed. A volume of 100 μL with 2.0 μM of the zinc-specific fluorophore FluoZin™-3 (Invitrogen/LifeTechnologies) was added to all of the wells. The resulting solutions were mixed and the fluorescence emission was determined after 10 min, every 10 min over a period of 90 minutes, at 37° C. (λ.sub.exc=485 nm, λ.sub.em=535 nm); Victor X4 microtitre plate reader). The relative fluorescence was calculated by dividing the absolute fluorescence emission of the well which contained the inhibitor by the absolute fluorescence of the corresponding well which contained the enzyme but no inhibitor (control). Wells which contained the inhibitor but no enzyme were used to check for false positive results. None of the tested compounds exhibited false positive results.

[0139] As can be seen in FIG. 10, aurothioglucose as well as the mixture of aurothioglucose with acetylcysteine resulted in removal of zinc from the PLpro which was comparable with the reference compound disulfiram. These results are in agreement with the inhibition of the enzyme activity of PLpro by aurothioglucose and are further confirmation of its relevance.

Example 9. Inhibition of the SARS-CoV-2 Protease 3CLpro by Gold Compounds

[0140] The inhibition of the SARS-CoV-2 protease 3CLpro was determined as follows: the test substances were dissolved in water as stock solutions and diluted one hundred-fold with HEPES buffer (50 mM HEPES, pH 7.5, 0.1 mg/mL foetal calf serum, 0.1% Triton-X-100), so that micromolar concentrations were obtained. Volumes of 50 μL of a 300 nM solution of SARS-CoV-2 3CL protease (Mpro) MBP-tag in HEPES buffer or pure HEPES buffer (negative control) were pipetted into the wells of a black 96-well microtitre plate. 50 μL of the test substance solutions or pure HEPES buffer (positive control) was respectively added thereto and the resulting solutions were mixed and incubated for one hour at 37° C. Next, a volume of 100 μL of a 50 μM solution of the substrate DABCYL-Lys-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-Met-Glu-EDANS trifluoroacetate was added to all of the samples, mixed thoroughly, and the fluorescence emission was recorded every 3 minutes over 75 minutes (λ.sub.ex=60 nm, λ.sub.em=460 nm, 37° C., Victor™ X4 Perkin Elmer 2030 microtitre plate reader). The evaluation was carried out in an analogous manner to the method with PLpro.

[0141] Results: the gold compounds are good inhibitors for SARS-CoV-2 3CLpro. The following IC.sub.50 values were determined: auranofin: 11.69 μM (±0.40 μM); aurothioglucose: 8.25 μM (±0.04 μM); aurothiomalate: 22.89 μM (±0.72 μM).

Example 10. Inhibition of Infectivity of Bovine Coronavirus (BCoV) in Cell Structure

[0142] As a surrogate for SARS-CoV-2, upon which experiments can only be carried out in biosafety level 3 (BSL3) laboratories, bovine coronavirus (BCoV), a virus which is related to SARS-CoV-2, which is also classified in the Betacoronavirus genus, was used because these experiments could be carried out in the lower biosafety level 2, BSL2. The receiving cell cultures used were Madin Darby Bovine Kidney (MDBK) cells.

[0143] After incubating different concentrations of aurothioglucose with 100 Tissue Culture Infectious Dose 50 (TCID50) of bovine coronavirus at 37° C. for one hour, the aurothioglucose-bovine coronavirus suspension was inoculated onto the MDBK cells receiving this virus and incubation was carried out for a further 12-48 hours at 37° C. and under a 5% CO.sub.2 atmosphere. At an aurothioglucose concentration of 256 μM, a 10-fold reduction in the viral load was observed.

[0144] Adding 64 μM favipiravir at the time of inoculating the aurothioglucose-bovine coronavirus suspension onto the cell cultures also resulted in a reduction in the viral load formed.

Example 11. Therapeutic Treatment

[0145] In order to prepare a preparation A, 3 mg of micronized aurothioglucose are blended with 9 mg of lactose and placed in a multi-dose powder inhaler. Four Covid-19 patients with the onset of symptoms less than 48 hours prior to the start of treatment are treated by administration of 2 spray applications each of 200 μg of the powder formulation over a period of 10 days, 2×daily.

[0146] In order to prepare a combination preparation B, liquid ampoules are produced, each with 0.1 mg of aurothioglucose and 0.4 mg of N-acetylcysteine dissolved in 2.5 mL of water. Four artificially ventilated Covid-19 patients are treated with one liquid ampoule per day by means of a nebulizer over a period of 10 days.

REFERENCES

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