PL247149B1 - The use of dimethyl sulfoxide in practice and treatment of infections caused by the SARS-CoV-2 virus and its variants and a pharmaceutical composition for use in the prevention and treatment of upper respiratory tract infection caused by the SARS-CoV 2 virus and its variants - Google Patents

Abstract

The invention relates to the use of dimethyl sulfoxide in the prevention and treatment of infections caused by SARS-CoV-2, preferably in the prevention of neurotropic consequences of infection with the SARS-CoV-2 virus or its variants. The invention also relates to the use of dimethyl sulfoxide for the production of preparations used in the prevention and treatment of infection with the SARS-CoV-2 virus or its variants, preferably for the production of liquid preparations in the form of an aqueous solution for the oral cavity and throat, for the nose and for nebulization, as well as for the production of preparations in semi-solid form for the nose, preferably in the form of a gel. Furthermore, the invention relates to a pharmaceutical composition comprising dimethyl sulfoxide (DMSO) as an active substance for use in the prevention and treatment of an upper respiratory tract infection caused by the SARS-CoV-2 virus or its variants.

Description

Description of the invention

The subject of the invention is the use of dimethyl sulfoxide in the prevention and treatment of infections caused by the SARS-CoV-2 virus and its variants, which may constitute an active substance for the production of preparations used in the prevention and treatment of infections with the SARS-CoV-2 virus. In addition, the subject of the invention is a pharmaceutical composition for use in the prevention and treatment of upper respiratory tract infection caused by the SARS-CoV-2 virus and its variants. In mammals, the nasopharynx plays a key role in exposure to pathogens transmitted in the form of microparticles suspended in the air or an artificially generated respiratory mixture. Neutral pH and a relatively large absorption surface and the lack of digestive enzymes and glands responsible for diluting the contents of the nasal cavity create special conditions for direct contact of pathogens with the epithelial cells of the nasopharyngeal mucosa. Natural physical barriers include a layer of mucus covering the mucous membrane, which includes microvilli (cilli), the movements of which enable continuous removal of mucus from the nasopharynx and elimination of adsorbed microparticles. Additionally, mucus provides a microenvironment for numerous nerve endings of the olfactory bulb and nerve endings responsible for taste perception. Penetration of pathogens through the mucous barrier allows direct access to nerve endings and to epithelial cells of the mucous membrane. Lymphatic cell deposits located under the epithelium of the mucous membrane create a complex system of cellular response to pathogens that have crossed the superficial mucous layer and are called NALT (nasal associated lymphoid tissue). Changes in the chemical composition of mucus or disorders in its production are an important factor facilitating the transmission of pathogens to epithelial cells and nerve endings. Viral infections transmitted by droplets involve the transmission of non-inactivated viruses through the mucus covering the epithelium, enabling infection of the epithelial cells of the mucous membrane and, in selected cases, the transfer of pathogens along nerve fibers to the cells of the central nervous system. Smell and/or taste disorders, including their complete loss and dysfunction of the central nervous system with a varying clinical spectrum, are a common consequence of viral infections with the portal of infection located in the nasopharynx, including those caused by the SARS-CoV2 virus and its variants. In SARS-CoV-2 infection, one of the first characteristic symptoms is the loss of the sense of smell and taste. SARS-CoV-2 infection is often accompanied by central symptoms, which often last much longer than the viral infection. These phenomena confirm the key role of the nasopharyngeal cavity, oral cavity, nasopharynx and throat in infection with the SARS-CoV-2 virus and its variants. Therefore, preventing infection in the nasal cavity, nasopharynx and pharynx, i.e. in the entry gates of pathogenic microorganisms into the body, is crucial in the process of counteracting the development of pathological processes. Literature data indicate that during SARS-CoV-2 infection (Severe acute respiratory syndrome Coronavirus 2), one of the key pathophysiological elements accompanying the development of Covid-19 is the activation of the aryl hydrocarbon receptor (AhR) (Liu et al., 2020; Giovannoni et al., 2021). Literature data indicate that AhR activation may occur either as a result of increased expression and activity of the IDO1 enzyme (indoleamine 2,3-dioxygenase 1) or the expression and activity of the TIPARP enzyme (TCDD-inducible poly [ADP-ribose] polymerase) (Grunewald et al., 2020; Turski et al., 2020). Activation of AhR by TIPARP may occur independently of the IDO-dependent activation pathway (Grunewald et al., 2020). Based on the above, it can be indicated that AhR, IDO1 and TIPARP are potential therapeutic targets important from the point of view of combating the spread of the Covid-19 epidemic caused by SARS-CoV-2. IDO1 activation occurs, among others, under the influence of interleukin IL-6. Activation of IDO1 leads to an increase in the conversion of tryptophan to kynurenine. This process occurs intracellularly, therefore the intensity of its course is determined not only by the activity of the IDO1 enzyme, but also by the amount of intracellularly available tryptophan. Tryptophan is transported across biological barriers by the large amino acid transporter LAT1 (L-type / large neutral amino acid transporter 1; SLC7A5). Due to the fact that AhR activation and the development of AhR-dependent pathophysiological processes can occur independently via different pathways, it was decided to find substances that act simultaneously on AhR, IDO and TiPARP, as well as on LAT1 and IL6, and cause a decrease in their expression and/or activity. The aim of the search was to find means to effectively counteract the spread of the Covid-19 epidemic, acting simultaneously on several key mechanisms activated by SARS-CoV-2. The search for substances with a given activity profile was carried out by exploring sets of gene expression profiles available from the Pharmaco Atlas tool of the BaseSpace Correlation program

Engine (Kupershmidt et al., 2010). After data mining, it was unexpectedly found that only dimethyl sulfoxide causes a decrease in the expression of all 5 selected molecular targets, i.e. AhR, IDO, TIPARP, LAT1 and IL-6. Dimethyl sulfoxide is known to have antiviral activity. In vitro studies conducted on human foreskin fibrblasts (HFF) cell lines and Vero cell line showed that dimethyl sulfoxide at concentrations of 0.7-2% reduces the infection capacity of the herpes simplex virus (HSV). The mechanism of action was indicated as (a) a decrease in the infection capacity of the virion, (b) inhibition of viral DNA replication, (c) a decrease in the level of transcription of HSV genes (Aguilar et al., 2002). In vitro studies conducted on MDCK (Madin-Darby canine kidney) and L cell lines have shown that dimethyl sulfoxide, depending on the concentration, stimulates or inhibits the virulence of viruses. Dimethyl sulfoxide at a concentration of up to 4% increases the virulence of the FVP (Influenza virus strain A/fowl plague/Rostock/34; H7N1), NDV (Newcastle disease virus, strain Italy) and SFV (Semliki Forest virus, strain Osterrieth) viruses, and at higher concentrations of up to 10% inhibits the virulence of these viruses (Scholtissek and Muller, 1988). There is no data on the effect of dimethyl sulfoxide on the SARS-CoV-2 virus. From the publication of Ali Hosseinzadeh et al.: “Application of nasal spray containing dimethyl sulfoxide (DSMO) and ethanol during the COVID-19 pandemic may protect healthcare workers: A randomized controlled trials”; medRxiv 2021.07.06.21259749 it is known that the use of dimethyl sulfoxide (DSMO) and ethanol in aerosol may protect healthcare workers during the COVID-19 pandemic.

Unexpectedly, it turned out that dimethyl sulfoxide can be used in the prevention and treatment of the consequences of SARS-CoV-2 virus infection.

The invention solves the problem of the new use of dimethyl sulfoxide and compositions with dimethyl sulfoxide in the prevention and treatment of the consequences of SARS-CoV-2 virus infection. In addition, the invention solves the problem of using dimethyl sulfoxide for the production of preparations for the prevention and treatment of the consequences of SARS-CoV-2 virus infection, especially used for the oral cavity, including the throat and nose. Additionally, it has been shown that dimethyl sulfoxide reduces the expression of the amino acid transporter LAT1 and the expression of the proinflammatory cytokine IL-6.

The essence of the invention is the use of dimethyl sulfoxide in the prevention and treatment of infections caused by SARS-CoV-2 and its variants, in particular in the prevention of the occurrence of neurotropic consequences of infection with the SARS-CoV-2 virus and its variants.

The essence of the invention is also a pharmaceutical composition containing dimethyl sulfoxide (DMSO) as an active substance for use in the prevention and treatment of upper respiratory tract infection caused by the SARS-CoV-2 virus and its variants, where DMSO is present in an amount of 20% to 50% by volume. The composition is preferably in the form of an aqueous solution for the throat. The composition is preferably in the form of an aqueous solution for nebulization. The composition is preferably in the form of an aqueous solution for the nose. The composition is preferably in the form of a nasal spray. The composition is preferably in a semi-solid form, preferably in the form of a nasal gel.

The advantage of the invention is the new use of dimethyl sulfoxide in the prevention and treatment of infections caused by SARS-CoV-2 and its variants and compositions with dimethyl sulfoxide in the prevention and treatment of the consequences of SARS-CoV-2 virus infection. Dimethyl sulfoxide can be used to produce preparations for the prevention and treatment of the consequences of SARS-CoV-2 virus infection, especially those used for the oral cavity, including the throat and nose. In connection with the waves of infections caused by the SARSCoV-2 virus and a small range of medicinal products, it was found that dimethyl sulfoxide can be an effective agent in the prevention and treatment of SARS-CoV-2 and its variants and the consequences of infections caused by the SARS-CoV-2 virus and its variants.

The results of studies confirming the use of dimethyl sulfoxide in the prevention and treatment of the consequences of SARS-CoV-2 virus infection are presented in the examples below.

Example 1

Based on the conducted research, the issue of simultaneous activation of AhR, IDO, TPARP in SARS-CoV-2 was discussed.

The database of microarray experiments and RNAseq of the Genevestigator package (Hruz et al., 2008) was used for data mining concerning the effect of SARS-CoV-2 infection on the expression level of genes encoding AhR, IDO and TiPARP proteins. The Perturbations tool was used to extract the results of experiments concerning changes in the expression level of AhR, IDO and TiPARP genes in response to SARS-CoV-2 infection (experimental sample) compared to uninfected cells or cells subjected to mock infection (control sample). Gene expression level for the experimental and control samples

PL 247149 Β1 was expressed as the logarithm to the base of 2 (Iog2) of the number of transcripts per million increased by one (TPM + 1). The difference in the expression levels thus expressed for the experimental sample in relation to the control sample (Iog2ratio) was taken as the value describing the change in the expression level of the studied genes.

The Genevestigator database identified 38 RNAseq experiments investigating the effect of SARS-CoV-2 infection on gene expression levels compared to controls. The control was uninfected or mock infected. The studies were conducted in the following research models:

• Calu-3 cells • A549 cells • Cardiomyocytes • Alveolar cell type 2 organoids • CaCo-2 cells • Colon organoids • Lung organoids • Fetal stomach organoids • Stomach organoids • NCI-H1299 cells • NHBE cells • Choroid plexus organoids • Cornea • Pancreatic endocrine-like cells • Primary respiratory epithelial cells • Sclera • Corneal limbus • Cardiac fibroblasts • Liver organoids • WTC-11 pluripotent stem cells • Endothelial cells

Among these experiments, a statistically significant (p-value < 0.01) increase in the expression level (upregulation) of the TIPARP gene was observed in 13 cases, and a statistically significant decrease in the expression level (downregulation) in 1 case. For AhR, 8 experiments were observed with a statistically significant increase in the expression level, with no experiments with a decrease in the expression level. For IDO, 7 experiments were observed with an increase in the expression level, with no experiments with a decrease in the expression level. Based on the available data, the effect of infection of lung epithelial cells of the Calu-3 line with SARS-CoV-2 virus on the change in the expression of the AHR, IDO1 and TIPARP genes over time was determined. The extracted data indicate that SARS-CoV-2 leads to a time-dependent increase in the expression level of the AHR, IDO1 and TIPARP genes, which encode AhR, IDO and TiPARP, respectively. The results are presented in Table 1.

Table 1. Effect of SARS-CoV-2 virus infection on AhR, IDO and TiPARP gene expression.

Expression Time after infection 12 hrs 24 hrs AhR 1.24 0.46 IDO 3.65 5.29 TIPARP 3.24 2.39

Results are expressed as log2ratio mean.

SARS-CoV-2 infection causes an increase in the expression of all 3 tested genes after 12 hours, the highest for IDO (Iog2ratio = 3.65). The increase in the expression level for TIPARP expressed as Iog2ratio was 3.24, and for AhR 1.24. After 24 hours from infection, the increased level of expression of these genes is still maintained; the highest for IDO (Iog2ratio = 5.29), lower for TIPARP (Iog2ratio = 2.39) and

PL 247149 Β1 small for AhR (Iog2ratio = 0.46). Based on the above, it was found that in SARS-CoV2 infection there is an increase in the expression of genes encoding AhR, IDO and TiPARP. The dynamics of changes are noteworthy, especially after 12 hours, which indicates that prophylactic, appropriately early use of drugs acting on 3 genes encoding AhR, IDO and TiPARP may be more effective than treatment of an advanced disease.

Example 2

Substances reducing the expression of 3 genes encoding AhR, IDOI, TIPARP

Data mining in the field of pharmacology and gene expression was performed based on the BaseSpace Correlation Engine database (Kupershmidt et al., 2010) and the Pharmaco Atlas tool available in this program. The threshold values adopted in the program to identify chemical compounds that affect the expression of AhR (gene: AHR), IDO (gene: IDO1) and TIPARP (gene: TIPARP) in a statistically significant way were as follows: (a) maximum p value: 0.05; (b) minimum value of absolute fold change in gene expression: 1.2. Based on the adopted threshold values for each of the studied genes, a list of chemical substances that significantly affect its expression was obtained. Compounds that significantly increased the expression level of the studied genes were rejected from the lists. Only compounds that statistically significantly decreased the expression of each of the genes were retained. The following substances were identified: (a) 190 substances reducing AhR expression, (b) 108 substances reducing IDO expression, (c) 191 substances reducing TIPARP expression. Among the identified substances, only two substances were found that inhibit the expression of all three tested genes, which are indicated in the figure, where in Fig. 1 the Venn diagram presents the identified substances that statistically significantly reduce the level of expression of the AHR, IDO1 and TIPARP genes. The identified substances that simultaneously have a negative effect on the level of AHR, IDO1 and TIPARP are dimethyl sulfoxide and SB-203580 (C21H16FN3OS; CAS 152121-47-6).

Example 3

Substances reducing the expression of 5 genes: AhR, IDO, TIPARP, LAT1, IL-6

Data mining was performed as described in Example 2. As a result of the studies, only one substance was identified that reduces the expression of all five studied genes. It is dimethyl sulfoxide. It turned out that the substance SB-203580 causes an increase in LAT1 expression (impact strength 29; number of studies 3) and an increase in IL-6 expression (impact strength 3; number of studies 3). Detailed parameters of the effect of dimethyl sulfoxide are placed in Table 2.

Table 2. Substances causing a decrease in the expression of five genes: AHR, IDO, TIPARP, LAT1 and IL-6

Substance Relative measure of the strength of the impact (0- 100) Direction of influence Number of studies Gen Dimethyl sulfoxide 28 decrease in expression level 3 AHR 18 decrease in expression level 1 IDO1 16 decrease in expression level 2 TIPARP 26 decrease in expression level 5 LAT1 31 decrease in expression level 3 IL-6

Dimethyl sulfoxide was found to have the unique property of simultaneously reducing the expression of 5 genes: AHR, IDO, TIPARP, LAT1 and IL-6.

Example 4

Mechanism of action of dimethyl sulfoxide

Dimethyl sulfoxide (C2H6OS; CAS 67-68-5) is an organic compound from the sulfoxide group. Due to its polarity, it is miscible with water and is used as a solvent. In medicine, it is

PL 247149 Β1 applied locally to the bladder in cystitis. In veterinary medicine, it is used externally to lubricate areas after mechanical injuries to relieve pain. Dimethyl sulfoxide is used as a solvent for drugs used externally and systemically. It facilitates the transport of substances into the tissues and is used as an ingredient in cosmetics. In addition, dimethyl sulfoxide is used to store cells and tissues in deep freezing due to its cryoprotective effect (https://www.drugs.com/vet/domoso-solution.html).

Table 3. Commercial products containing dimethyl sulfoxide approved for use as a drug

Name/ manufacturer Character Application References Dimethyl Sulfoxide Irrigation USP / Sandoz Canada Incorporated 95% solution for bladder flushing http://ftp.uspbpep.eom/v 29240/usp29nf24s0_m26 680.html Domoso / Zoetis, USA 90% solution for external use on animal skin as a spray for acute post-traumatic pain https ://w w w .drugs .c om/v et/domoso-solution.html Kemsol / Axxess Pharma Inc. 70% solution for external use https://go.drugbank.com/drugs/DB01093 Rimso-50 / Mylan Institutional LLC, Rockford, IL 61103, Ireland 50% solution for bladder irrigation in interstitial cystitis https://www.rxlist.com/ri mso-50- drug .htm#indication p

Dimethyl sulfoxide is officially approved by the US Food and Drug Administration (FDA) for the treatment of pain in the course of interstitial cystitis (Table; Rawls et al. Neurourol Urodyn. 2017). It is believed to have a beneficial effect on limb pain caused by trauma and therefore drugs are produced for use in veterinary medicine (Table 3). Dimethyl sulfoxide is applied externally to the skin to alleviate the toxic effects of extravasation of anticancer drugs during their intravenous administration. There are many scientific studies indicating the beneficial effects of dimethyl sulfoxide in various disease states, but many of them are not confirmed by other studies or the results of studies are insufficient to draw clear conclusions. The molecular mechanism of action of dimethyl sulfoxide is not known. The most frequently indicated are antioxidant, anti-inflammatory and immunomodulatory effects (Huang et al. Immunobiology. 2020). The adverse effects of dimethyl sulfoxide are assessed differently, which seems to depend on the dose or concentration used and the duration of use. It is considered that when dimethyl sulfoxide is used in moderate doses, the adverse effects are moderate and transient (Kollerup Madsen et al. 2018).

Example 5

Use of dimethyl sulfoxide in the treatment and prevention of infections caused by Covid-19 Use of aqueous solution of dimethyl sulfoxide in patients with symptoms of SARS-CoV-2. Case 1

An aqueous solution of dimethyl sulfoxide was used in Patient 1 (a 29-year-old woman) who was previously healthy and had not taken any medications. Family history was negative. After the patient was found to have a complete loss of smell and a partial loss of taste, a PCR test was performed (the next day), which confirmed infection with the SARS-CoV-2 virus. In the following days, other symptoms occurred: significant weakness of the body that made everyday functioning difficult, severe pain in the muscles of the whole body and the spine, especially in the lumbar-sacral section. During the course of the disease, no increase in body temperature or decrease in SpO2 was noted. After 3 days from the first symptoms - on the first day after the PCR test, Patient 1 started using an aqueous solution of dimethyl sulfoxide at a concentration of 50% (based on distilled water), applying it intranasally 3-4 times a day by lubricating the walls of the nasal cavity. Apart from that, Patient 1 did not use any other medications. After

PL 247149 Β1 first applications Patient 1 felt a partial return of smell and taste until the symptoms completely disappeared after 7 days. The remaining reported symptoms, i.e. significant weakness, lasted for about 20 days. Patient 1 did not use any other drugs in the course of the disease.

Case 2

A 50% aqueous solution of dimethyl sulfoxide (based on distilled water) was used in Patient 2 (female, age 20) with no other diagnosed diseases and no previous medication. Systemic symptoms: body temperature 37.7°C, normal well-being, slight headaches, troublesome cough. In addition, loss of smell and taste was noted. After 24 hours, a PCR test was performed, which confirmed infection with the SARS-CoV-2 virus. Three days after the test, dimethyl sulfoxide administration was started. The dimethyl sulfoxide solution was administered bilaterally into the nasal cavity in drops of 1-2 drops. The procedure was performed 3-4 times a day for 5 consecutive days. On the 2nd day of dimethyl sulfoxide use, Patient 2 noted improvement consisting in the gradual return of the sense of smell and taste. In addition, the patient did not report any adverse effects apart from skin irritation around the nostrils. The cough persisted for 5-7 days. No other symptoms of illness.

Case 3

An aqueous solution of dimethyl sulfoxide was used prophylactically in Patient 3 (female, age 62), previously without other active diseases, body temperature 36.7°C, without symptoms of disease. Patient 3 was in contact with Covid-19 patients with comments on the place of work (hospital). Dimethyl sulfoxide was dissolved in distilled water in a volume ratio of 2:8 (20% solution). The dimethyl sulfoxide solution was administered bilaterally into the nasal cavity, thoroughly wetting the walls of the nasal cavity. The procedure was performed twice a day on the day when Patient 3 was in direct contact with Covid-19 patients. Patient 3 used general principles of antiviral prophylaxis and protective measures, such as a mask and a face shield. Patient 3 used dimethyl sulfoxide for 6 months on the days of contact with Covid-19 patients with a frequency of about once a week. During the period of use of dimethyl sulfoxide, Patient 3 did not experience any symptoms of SARS-CoV-2 infection, in particular loss of smell or taste. Patient 3 did not report any adverse effects. He did not take any other medications.

Example 6

Composition containing dimethyl sulfoxide for application to the nasal cavity in the form of a gel

Gel ingredients

Phase Ingredient CAS No. Contents And Dimethyl sulfoxide 67-68-5 50% Glycerol anhydrous 56-81-5 3% Propylene glycol 57-55-6 2% Carbopol Ultrcz 10 195739-91-4 0.5% Water 7732-18-5 35.65% B Polyvinylpyrrolidone k90 9003-39-8 5% Panthenol 81-13-0 1% C Triethanolamine 102-71-6 0.75% D Twccn 20 9005-64-5 1% Peppermint oil 8006-90-4 0.1% Preservative mixture Phenoxyethanol + Ethylhexylglycerin 122-99-6 and 70445-33-9 1%

The gel composition was obtained as follows:

PL 247149 Β1

In a vessel, the components of phase A from the table were mixed using a top mixer equipped with a dissolver-type mixer. The components of phase B were added and mixed. Then triethanolamine (C) was added and mixed until a uniformly thickened gel was obtained. Then the components of phase D were added and the whole was mixed. Then the obtained composition was evaluated:

The preparation is in the form of a gel, it is colorless, transparent, and homogeneous.

The study was carried out using a vibration viscometer, electronic pH meter, oscillation densitometer, pH = 7.4, density = 1.094 g/ml, kinematic viscosity = 4,510 mPas. The basic properties of the preparation characteristic for intranasal administration were obtained.

For the theological evaluation, the study was performed in a Searle-type rotational rheometer.

A test was conducted in three stages:

initial mixing, increasing shear rate mixing from 0.1/s to 300/s, decreasing shear rate mixing from 300/s to 0.1/s.

The drawing in Fig. 2 shows a graph of shear stress as a function of shear rate.

Figure 3 shows a graph of viscosity as a function of shear rate.

Flow parameters according to the Herschel-Bulkley Model:

To = 24,262 Pa; b = 78, 557; p = 0.45884

The gel was shown to exhibit pseudoplastic flow, low yield stress and low viscosity.

Then, the spreadability was assessed:

The test was performed using a manual extensometer with 200 g weights and a 1 mm scale.

Fig. 4 shows the gel spreading surface as a function of the applied pressure (N).

The spread of the preparation has been shown to be high.

The force of squeezing out of the container was then assessed.

The test was carried out using a texturometer (bending test) by measuring the force over a distance of 50 mm (squeezing from the tube by the rollers) or 10 mm (squeezing from the tube in the three-point bending test) or 20 mm (squeezing from the container) at a traverse speed of 1 cm/min.

Parameters of pharmaceutical packaging squeeze tests

Test Type Force (N) Energy (J) Maximum Mean 20 ml tube squeezing - 3-point bending test 18.5072 13.5757 0.13572 Squeezing from a 20 ml tube - through the rollers 12.2059 9.31304 0.46553 Squeezing from a 50 ml container 22.4117 20.2672 0.40528

The squeezing force from the packaging was assessed to be low, the lowest for the tube, and the force distribution was even.

An assessment of slippage was also made.

The test was performed with a texturometer (tensile test) using Plexiglas plates measuring 2.5x2.5 cm, measuring the force over a distance of 40 mm at a traverse speed of 1 cm/min.

Slip test parameters of the preparation

Value Mean Standard deviation N Average static coefficient 0.03694 0.00051 3 Average dynamic coefficient 0.02861 0.00188 3 Average slip force 0.05613 N 0.00369 3 Average maximum slip force 0.07245 N 0.001 3

PL 247149 Β1

The sliding force of 1 g of the preparation was assessed to be low and the static component was low.

Example 7

Composition comprising dimethyl sulfoxide in the form of a nasal spray

Spray ingredients:

Ingredient CAS No. Contents Dimethyl sulfoxide 67-68-5 20.00% Hydroxypropyl methylcellulose 9004-65-3 0.1% Benzalkonium chloride 8001-54-5 0.01% Sodium edetate dihydrate 6381-92-6 0.05% Sodium hydrogen phosphate heptahydrate 7782-85-6 0.20% Sodium dihydrogen phosphate monohydrate 10049-21-5 0.64% Water 7732-18-5 79.00%

The composition was obtained as follows.

In a vessel, phosphates and sodium edetate were dissolved in water using an overhead mixer equipped with a paddle mixer. The liquid was stirred until the solids were dissolved. Hydroxypropyl methylcellulose, benzalkonium chloride and dimethyl sulfoxide were then added and mixed. The liquid was transferred to 30 ml spray bottles. The product was then assessed, where visual inspection showed that the composition was a colorless, transparent and homogeneous liquid.

The resulting fluid has the following basic parameters:

The test was carried out using a vibration viscometer, electronic pH meter, oscillating densitometer pH = 6.0, density = 1.03 g/ml, and kinematic viscosity = 2.57 mPas.

It was assessed that the basic properties of the preparation typical of a nasal spray were achieved.

Example 8

Composition containing dimethyl sulfoxide in the form of a nasal rinse

Liquid ingredients:

Ingredient CAS No. Contents Dimcty lo sulfo oxide 67-68-5 20.00% Benzalkonium chloride 8001-54-5 0.01% Water 7732-18-5 79.99%

The composition was obtained in the following way: the ingredients were mixed in a suitable stainless steel container and stirred vigorously until a clear solution was obtained. Then the mixture was filtered using only polyethylene tubes, a clarifying filter with a pore size of 0.45 µm. The liquid was transferred to a sterile container, constituting an auxiliary container. Plastic bottles with a nasal applicator with a capacity of 25 ml were filled using the blow-fill-seal technology, but no autoclaving was used. Then the composition was visually assessed, on the basis of which it was found to be a colorless, transparent and homogeneous liquid. The composition has basic parameters: The test was carried out using a vibrating viscometer, an electronic pH meter, an oscillating densitometer pH = 7, density = 1.02 g/ml, kinematic viscosity = 1.2 mPas.

It was assessed that the basic properties of the preparation typical of a nasal rinse fluid were obtained.

Example 9

Composition comprising dimethyl sulfoxide in the form of a mouthwash

PL 247149 Β1

Liquid ingredients:

Ingredient CAS No. Contents Dimethyl sulfoxide 67-68-5 20.00% Sodium lauroyl sarcosinate + water (Medialan® LD) 137-16-6 and 7732-18-5 0.50% Twccn 20 9005-64-5 0.50% Peppermint oil 8006-90-4 0.10% Sodium benzoate 532-32-1 0.05% Glycerol anhydrous 56-81-5 10.00% Indigotine 860-22-0 0.01% Water 7732-18-5 68.85%

The composition was obtained as follows:

Sodium benzoate was dissolved in a mixture of water and glycerol in a vessel and stirred using an overhead stirrer equipped with a paddle stirrer. The liquid was stirred until the sodium benzoate was dissolved. Then Medialan LD, Tween 20 and indigotine were added and mixed. Finally, peppermint oil was added dropwise to the mixture and the whole was mixed. The liquid was transferred to a 500 ml plastic bottle with a screw cap. Then, the composition was visually assessed, on the basis of which it was found to be a bluish, transparent and homogeneous liquid.

The composition has basic parameters:

The study was carried out using a vibration viscometer, electronic pH meter, oscillation densitometer, pH = 7.4; density = 1.05 g/ml kinematic viscosity = 3.80 mPas.

It was assessed that the basic properties of the preparation characteristic of a fluid for oral and throat therapy were achieved.

Example 10

Composition comprising dimethyl sulfoxide in the form of a nebulizer fluid

Liquid ingredients:

Ingredient CAS No. Contents Dimethyl sulfoxide 67-68-5 20.00% Water 7732-18-5 80.00%

The composition was obtained as follows: The ingredients were mixed in a suitable stainless steel container and stirred vigorously until a clear solution was obtained. Then the mixture was filtered using only polyethylene tubes, a pre-filter with a 0.22 µm sterilizing membrane. The liquid was transferred to a sterilized container, constituting an auxiliary container. Under aseptic conditions, plastic ampoules of 5 or 10 ml were filled using blowfill-seal technology. It was not autoclaved. Then, the composition was visually assessed, on the basis of which it was found to be a colorless, transparent and homogeneous liquid.

The composition has basic parameters:

The study was carried out using a vibration viscometer, electronic pH meter, oscillation densitometer, pH = 7; density = 1.02 g/ml, kinematic viscosity = 1.2 mPas,

It was assessed that the basic properties of the preparation characteristic of a nebulization fluid were obtained.

As shown in the above examples, in SARS-CoV-2 infection there is an increase in the expression level (upregulation) of 3 molecular markers, i.e. genes encoding AhR, IDO and TiPARP. Moreover, unexpectedly, it was shown that downregulation (downregulation) of 3 molecular markers, i.e. expression of genes encoding AhR, IDO and TiPARP occurs under the influence of dimethyl sulfoxide.

Additionally, dimethyl sulfoxide has been shown to reduce the expression of the amino acid transporter LAT1 and the cytokine IL-6.

The mechanism of action of dimethyl sulfoxide causing downregulation of the expression of 5 genes encoding AhR, IDO, TIPARP, LAT1 and IL-6 is unknown.

The studies conducted have shown that people infected with the SARS-CoV-2 virus who were intranasally administered dimethyl sulfoxide in an aqueous solution experienced a rapid recovery of the perception of smell and taste and did not experience systemic symptoms of Covid-19 or adverse reactions associated with intranasal administration of dimethyl sulfoxide.

In the light of the presented studies, it was demonstrated that it is possible to produce a gel for intranasal use, a nasal spray, liquids for rinsing the nasal cavity, mouth and throat, and a liquid for nebulisation containing dimethyl sulfoxide.

Due to the privileged importance of the nasal cavity, oral cavity, nasopharynx and pharynx as the first gateway to infection with microorganisms transmitted by droplets, including the SARS-CoV-2 virus, the application of dimethyl sulfoxide to the mucous membrane of these anatomical structures causes the cells of the mucous membrane of these areas to change their genotype and phenotype, making them less susceptible to the virulent action of microorganisms.

The results obtained in the presented examples confirm the effectiveness of using dimethyl sulfoxide to prevent and treat SARS-CoV-2 infection.

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Claims (9)

1. Dimethyl sulfoxide for use in the prevention and treatment of infections caused by the SARS-CoV-2 virus and its variants, in particular in the prevention of the occurrence of neurotropic consequences of infection with the SARS-CoV-2 virus and its variants. PL 247149 Β1 2. Dimethyl sulfoxide for use according to claim 1 in the form of a liquid, aqueous solution for the oral cavity and throat, for the nose and for nebulization. 3. Dimethyl sulfoxide for use according to claim 1 in a semi-solid nasal form, preferably in the form of a gel. 4. A pharmaceutical composition for use in the prevention and treatment of upper respiratory tract infection caused by the SARS-CoV-2 virus and its variants, comprising dimethyl sulfoxide as an active substance in an amount of 20% to 50% by volume. 5. The composition for use according to claim 4 in the form of an aqueous solution for the throat. 6. The composition for use according to claim 4 in the form of an aqueous solution for nebulization. 7. The composition for use according to claim 4 in the form of an aqueous nasal solution. 8. The composition for use according to claim 4 in the form of a nasal spray. 9. The composition for use according to claim 4 in the form of a semi-solid, preferably a nasal gel.