WO2021205341A1

WO2021205341A1 - 1-methylnicotinamide for the prevention/treatment of inflammatory airway diseases

Info

Publication number: WO2021205341A1
Application number: PCT/IB2021/052849
Authority: WO
Inventors: Konrad PALKA; Jerzy GĘBICKI; Marzena WIECZORKOWSKA
Original assignee: Pharmena S.A.
Priority date: 2020-04-07
Filing date: 2021-04-06
Publication date: 2021-10-14

Abstract

The object of the present invention is 1-methylnicotinamide (1-MNA) for use in a method of preventing or treating a disease associated with an inflammatory reaction in the airways or with a defective epithelial barrier in the airways, wherein the disease is selected from allergic diseases or diseases associated with an infection. Another object of the invention is a method of treatment of a disease associated with an inflammatory reaction in the airways in a subject or with a defective epithelial barrier in the airways in a subject, wherein the disease is selected from allergic diseases or diseases associated with an infection, wherein the subject is administered with 1-MNA or a pharmaceutically acceptable salt thereof.

Description

1-METHYLNICOTINAMIDE FOR THE PREVENTION/TREATMENT OF INFLAMMATORY AIRWAY DISEASES

TECHNICAL FIELD

The present invention relates to 1 -methylnicotinamide (1 -MNA) for use in a method of preventing or treating a disease associated with an inflammatory reaction in the airways, or with a defective epithelial barrier in the airways.

BACKGROUND ART

It is known that allergens and pathogens, such as bacteria or viruses, may activate inflammasomes, and such activation is also postulated for coronavirus Sars-CoV-2. The activated NLRP3 initiates a flurry of immune reactions that can result in deadly cytokine storms (httos ://www.evolutamente.it/covid-19-pneumonia-inflammasomes-the-melatonin- connection/).

According to independent studies (Shi Y. et al., COVID-19 infection: the perspectives on immune responses; Cell Death Differ. 2020, https://doi.Org/10.1038/s41418-020-0530-3) vitamin B3 (niacin or nicotinamide) is highly effective in preventing lung tissue inflammation and fibrosis. It is proposed to supply vitamin B3 food supplements to the COVID-19 patients. The authors recommends to use vitamin B3 as soon as coughing begins. It is also recommended that blocking IL-6, IL-1 and TNF-a may also be beneficial for COVID-19 patients.

Recently, it was also found that 1 -MNA levels in urine are down-regulated in infants with Respiratory syncytial virus (RSV) infection compared to healthy controls (T uri K.N. et al., Using urine metabolomics to understand the pathogenesis of infant respiratory syncytial virus (RSV) infection and its role in childhood wheezing; Metabolomics 2018, 14, 135).

1 -methylnicotinamide (1 -MNA) is a quaternary pyridinium compound. It is a metabolite of nicotinamide. 1-MNA can exist in various pharmaceutically acceptable salt forms, e.g., 1 -MNA chloride. It is a naturally occurring metabolite of nicotinic acid and nicotinamide, two main forms of the B3 vitamin (niacin). Numerous studies suggest that 1 -MNA affects the proper function of the vascular endothelium and cardiovascular system.

1 -MNA salts, such as 1 -MNA chloride are known, both alone and in combination with other ingredients, such as statins, for use in treatment of cardiovascular diseases and as vasoprotective agents (see e.g. W02007074406, W02005067927, W02006024545). In the prior art, 1 -MNA was shown to be useful in treatment of inter alia cardiovascular diseases, due to the fact that its positively charged molecules bind to the negatively charged glycosamionoglycans present on the vascular endothelium surface due to electrostatic interactions. This binding can result in manifestation of various endothelial effects, some of which can be positive from pharmacologic view point, for example release of NO and/or prostacyclin. 1-MNA hence increases NO bioavailability in the endothelium, as well as increasing FMD (Flow Mediated Dilation) and eNOS-mediated NO release from endothelial cells (Domagala T.B. et al. Nitric oxide production and endothelium-dependent vasorelaxation ameliorated by N1 -methylnicotinamide in human blood vessels; Flypertension. 2012 Apr;59(4):825-32). It may thus prevent endothelial cell dysfunction by increasing the bioavailability of nitric oxide. Further, this activity can result in the treatment or prevention of lipoprotein abnormalities (as discussed e.g. in W02007074406).

1 -MNA was indeed shown to reduce atherosclerotic plaque, inflammation and cholesterol content in the brachiocephalic artery in ApoE/LDLR2/2 animal model (Mateuszuk L, et. al. Antiatherosclerotic Effects of 1 -Methylnicotinamide in Apolipoprotein E/Low-Density Lipoprotein Receptor-Deficient Mice: A Comparison with Nicotinic Acid;, J Pharmacol Exp Ther. 2016, 356(2), 514.). Anti-atherosclerotic activity was associated with vascular endothelial stimulation, and increased concentrations of 6-keto-prostaglandin PGF 1a and nitrates/nitrites in aortic rings and inhibition of platelet activation (limiting the TXB 2 generation). It was additionally found that 1 -MNA raises adiponectin levels in blood (WO2018015862).

An anti-thrombotic activity has also been indicated, e.g. in W02007103450. This is associated with 1-MNA role as an activator of prostacyclin production, in particular the release of prostacyclin (PGI 2 ) mediated by cyclooxygenase-2 (COX-2) (Chlopicki S. et. al. 1 -Methylnicotinamide (MNA), a primary metabolite of nicotinamide, exerts anti-thrombotic activity mediated by a cyclooxygenase 2/ prostacyclin pathway;, Br. J. Pharmacol., 2007, 152, 230), since endogenous prostacyclin (PGI 2 ) is the strongest endogenous inhibitor of platelet aggregation. W02005067927 also discloses that compounds such as 1 -MNA release endogenous prostacyclin (PGI2) from vascular endothelium.

Additionally, an effect of decreasing C-reactive protein levels has also been shown (see WO2018015861)

Other known uses of 1 -MNA include activity against rheumatoid arthritis (see e.g. W02005067927, W02007103450), inflammatory bowel disease (see e.g. W02006024545), mental or neuropsychological disorders and Alzheimer’s disease, obesity and related disorders, as well as obesity-related cancers (W02007074406, W02008096203). EP1147086 discloses the use of 1 -MNA in the treatment of anti-inflammatory skin diseases and cosmetic treatment of the skin.

In humans, 1-MNA is mainly produced by the metabolism of dietary nicotinamide. It is metabolized to 1-methyl-2-pyridone-5-carboximide (2-PYR) and to 1 -methyl-4-pyridone-3- carboximide (4-PYR) by aldehyde oxidase (see Fig. 1).

1 -MNA can be obtained directly from foods (seaweed, green tea). Dietary intake is responsible for only a very small portion of the 1 -MNA total pool in human body. 1 -MNA can also be obtained from foods containing vitamin B3. In humans, close to 60% of niacin is metabolized into 1 -MNA.

During oxidation of 1-MNA by aldehyde oxidase, additional H2O2 formation occurs. The enzyme is present mainly in liver, lung, kidney and endocrine tissues (Moriwaki Y. et al., Widespread cellular distribution of aldehyde oxidase in human tissues found by immunohistochemistry staining; Histol. Histopathol., 2001 , 16, 745).

H2O2 is a diffusible small molecule that easily crosses biological membranes and can modulate the activity of different cellular proteins. Reactive oxygen species (ROS) have been linked with the activation of the NLRP3 inflammasome. Inflammasomes play a crucial role in infections. Inflammasome activation results in caspase-1 activation and secretion of the pro-inflammatory cytokines: interleukin (IL)-1 b and IL-18 (NLRP3 Inflammasome — A Key Player in Antiviral Responses; Zhao C. et al., Front. Immunol., 2020, 11 , 211). It has been recently shown that increase in H2O2 is specifically involved in the activation of the NLRP3 inflammasome. In fact, an early and transient increase in H2O2 is essential for the activation of innate immunity, avoiding the development of a systemic inflammatory response syndrome (Huet O. et al., Protective Effect of Inflammasome Activation by Hydrogen Peroxide in a Mouse Model of Septic Shock;, Crit. Care Med., 2017, 45, e184).

DISCLOSURE OF INVENTION

The object of the present invention is 1 -methylnicotinamide (1-MNA) or a pharmaceutically acceptable salt thereof for use in a method of preventing or treating a disease associated with an inflammatory reaction in the airways or with a defective epithelial barrier in the airways, wherein the disease is selected from allergic diseases or diseases associated with an infection.

In a preferred embodiment, the disease is associated with a viral infection in the airways.

In a preferred embodiment, the viral infection is an infection with Sars-CoV-2.

In another preferred embodiment, the viral infection is an infection with an influenza virus.

In another preferred embodiment, the disease is an allergic disease. In a preferred embodiment, the allergic disease is asthma.

In a preferred embodiment, 1-MNA or a pharmaceutically acceptable salt thereof is administered to prevent, alleviate or eliminate at least one of the following: coughing, throat ache, shortness of breath or difficulty breathing or dyspnoea, inflammation in the respiratory tract, e.g. lungs, fibrosis in lungs symptoms and/or symptoms related to muscles, such as myopathies, muscle fatigue, fatigue or muscle pains.

In a particularly preferred embodiment, 1-MNA or a pharmaceutically acceptable salt thereof is administered via an inhalation or intranasal route.

In a preferred embodiment, the disease is associated with a viral infection in the airways, and 1 -MNA or a pharmaceutically acceptable salt thereof is administered in order to prevent, alleviate or eliminate viral myositis, muscle pain, myopathy and/or fatigue.

In another preferred embodiment, the disease is associated with eosinophilia.

Another object of the invention is a method of treatment of a disease associated with an inflammatory reaction in the airways in a subject or with a defective epithelial barrier in the airways in a subject, wherein the disease is selected from allergic diseases or diseases associated with an infection, wherein the subject is administered with 1 -MNA or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the viral infection is an infection with Sars-CoV-2 or with an influenza virus.

In another preferred embodiment, the disease is an allergic disease.

In a preferred embodiment, the allergic disease is asthma.

In another preferred embodiment, the disease is associated with a viral infection in the airways, and 1 -MNA or a pharmaceutically acceptable salt thereof is administered in order to prevent, alleviate or eliminate viral myositis, muscle pain, myopathy and/or fatigue. In another preferred embodiment, the disease is associated with eosinophilia.

The present inventors have found that 1 -MNA has substantial effectiveness in reducing secretion of proinflammatory cytokines in human airway epithelial cells and endothelial cells stimulated towards inflammation. A decrease in overall reactive oxygen species (ROS) was also observed without an adverse effect on cell viability. Additionally, 1-MNA was shown to be capable of reducing caspase activation in human cells after inflammasome stimulation. Due to this, 1-MNA may have a potent anti-inflammatory effect in conditions of viral infection, comparable to steroids. 1 -MNA however is not associated with adverse effects commonly observed for steroid treatment. It may therefore be used instead steroid treatment or as a supplementary treatment, possibly reducing the necessary steroid dosage.

In addition, the present inventors have shown in an animal model of respiratory viral infection that 1-MNA may be administered by an inhalatory route (such as, but not limited to, intratracheal) and results in significant attenuation of BAL (bronchoalveolar lavage) cellular infiltrate, namely significantly lower BAL lymphocytes and a dose dependent decrease in BAL mononuclear cells and neutrophils. A decrease in cytokine levels and serum CRP was also observed. These in vivo results additionally support 1 -MNA usefulness in treatment of airway conditions associated with inflammation, such as infections (e.g. viral infections) or allergic diseases. In a particular example, the effectiveness in reducing inflammation and caspase activation in conditions of inflammasome activation, shows that 1-MNA may be beneficial in prevention, treatment or alleviation of symptoms of Sars-CoV-2 infection.

Without wishing to be bound by theory, the present inventors postulate that 1 -MNA serving as a substrate for aldehyde oxidase may trigger a mitohormetic reactive oxygen species signal through H2O2 and regulate inflammasomes activity, helping to avoid the development of a systemic inflammatory response syndrome. The effect is especially beneficial, as it was found by the present inventors that 1 -MNA decreases significantly the blood level of hsCRP and TNF-a, reducing overall inflammation. 1 -MNA may therefore reduce a deleterious increase in inflammatory response, including lung tissue inflammation and fibrosis, in response to contact with allergens or pathogens, e.g. in patients suffering from viral infections in the respiratory tract, such as but not limited to COVID-19 patients.

One example of a disease associated with lung fibrosis is idiopatic pulmonary fibrosis (IPF).

IPF is a serious chronic disease that affects the tissue surrounding the air sacs, or alveoli, in the lungs. It causes scar tissue growth inside the lungs, making it hard to breathe. The cause of IPF is unknown and since there is no treatment currently available, it is usually fatal. It typically affects people who are around 70 to 75 years old, and is rare in people under 50 Whenever a reference is made in the present description to lung fibrosis, it is also meant to include IPF. Without further wishing to be bound by any theory, 1 -MNA may also be useful in treating and/or preventing delirium, including but not limited to, intensive care unit delirium (ICU delirium), postoperative delirium (POD delirium) and delirium in Covid-19, since this condition is associated with inflammatory processes.

Moreover, an intact functional mucosal barrier is considered to be crucial for the maintenance of airway homeostasis as it protects the host immune system from exposure to allergens and noxious environmental triggers, as well as pathogens. The airway epithelium is the first site of contact with inhaled particles like allergens or pathogens, e.g. viral particles. The physical barrier against external environment, including inhaled pathogens, is formed by various junctional complexes connecting the epithelial cells to one another. A disturbed composition of interepithelial junctions will increase the accessibility of foreign particles to the submucosal region (Steelant B, et al. Restoring airway epithelial barrier dysfunction: a new therapeutic challenge in allergic airway disease. Rhinology. 2016 Sep;54(3):195-205). Impaired airway epithelial barrier function may be an important, key player in development of both allergic diseases and infections in airways. It should be noted that epithelial barrier defects are linked with chronicity and severity of airway inflammation. As a consequence, restoring barrier function might be a useful strategy in airway disease treatment.

The precise pathogenesis of barrier dysfunction in patients with mucosal inflammation and its implications on tissue inflammation and systemic absorption of exogenous particles are only partly understood. Opening of interepithelial junctions facilitates the entrance of allergens and other harmful particles in the mucosal and sub-mucosal region with the activation of the immune and inflammatory system as a consequence. Evidence for barrier dysfunctions underlying the pathology in upper and lower airways has only recently been obtained. The first evidence for barrier dysfunction in the lower airways was found in 2008. Patients with mild asthma had a disturbed expression of ZO-1 and b-catenin as shown by semi-quantitative immunofluorescence staining of bronchial biopsies (de Boer Wl. et al. Altered expression of epithelial junctional proteins in atopic asthma: possible role in inflammation. Can J Physiol Pharmacol 2008; 86:105-12). Additionally, it was shown that different junction-forming proteins (i.e. occludin and ZO-1) are involved in the pathology of allergic rhinitis compared to proteins (occludin and claudin-4) linked with the pathology of chronic rhinosinusitis, illustrating that the regulation of the function of proteins in the junctions can be linked with disease origin (Steelant et al. op. cit.). Additionally, TNF-a was shown as an important factor promoting epithelial barrier dysfunction (Hardyman MA, Wilkinson E, Martin E, Jayasekera NP, Blume C, Swindle EJ, Gozzard N, Holgate ST, Howarth PH, Davies DE, Collins JE. TNF-a-mediated bronchial barrier disruption and regulation by src-family kinase activation. J Allergy Clin Immunol. 2013 Sep;132(3):665-675.e8; Steelant et al. op. cit.). Restoration of barrier defects might reduce the excessive penetration of inhaled allergens and foreign particles (such as viruses) into the submucosal regions, ultimately resolving the activation of the immune system and the occurrence of symptoms. Available treatment options are currently mostly limited to corticosteroids as general anti-inflammatory agents. Other proposed treatments include epidermal growth factor and keratinocyte growth factor or targeting kinases in airways.

Shintani et al. Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates airway epithelial barrier integrity. Allergol Int. 2015 Sep;64 Suppl:S54-63, teaches that inhaled corticosteroids enhance airway epithelial barrier integrity. In particular, dexamethasone was shown to have this effect.

The present inventors propose that 1 -MNA may both regulate inflammasome activation by triggering a reactive oxygen species signal and enhance airway epithelial barrier integrity protecting against microbiological pathogens and allergic agents. These effects may be especially beneficial in prevention or treatment of viral infections, in particular infections associated with strong inflammatory response in the respiratory tract tissues, such as Sars- CoV-2 or influenza. Without wishing to be bound by theory, it is believed that corticosteroid effects in promoting epithelial barrier integrity in airways is at least in part effected by increasing expression of AOX1 (aldehyde oxidase) and NNMT (nicotinamide N-methyltransferase). Such increases for both enzymes were shown for dexamethasone. Both of these enzymes play a role in 1-MNA metabolism, with NNMT being involved in 1 -MNA synthesis and AOX1 catalyzing 1-MNA conversion to two pyridones (Fig. 1). The latter reaction produces H2O2 as described hereinabove, which mediates regulation of inflammasome activity. As indicated previously, 1 -MNA was shown to be down-regulated in ongoing infections. It is proposed that the presence of sufficient levels of 1 -MNA may be actually be an important factor in preventing and alleviating detrimental immune reactions in the airways, promoting epithelial barrier integrity in the respiratory tract and thus be useful in prevention and treatment of allergic diseases in the airways, e.g., asthma or respiratory diseases associated with infections, in particular viral infections, such as COVID-19, caused by Sars-CoV-2, or influenza.

As discussed above, it was hypothesized that proinflammatory factors, such as TNF-a may be crucial in epithelial barrier dysfunction and it may be a potential target in patients suffering from viral infections, such as COVID-19 or influenza.

General anti-inflammatory activity of 1 -MNA, e.g. when applied topically on skin has been shown previously (Gebicki J. et al. 1-Methylnicotinamide: a potent anti-inflammatory agent of vitamin origin; Pol. J. Pharmacol. 2003, 55, 109). Other works have shown that 1 -MNA protects against liver inflammatory damage when administered intravenously in mice (Jakubowski A. et al., 1-Methylnicotinamide protects against liver injury induced by concanavalin A via a prostacyclin-dependent mechanism: A possible involvement of IL-4 and TNF-a; Int. Immunopharmacol., 2016, 31 , 98).

The present inventors have shown however that 1 -MNA is capable of significantly reducing proinflammatory factors in blood and as such it is a good candidate to be used in prevention and/or treatment of allergic diseases in the airways, e.g. asthma or respiratory diseases, especially associated with infections, in particular viral infections, such as COVID-19, caused by Sars-CoV-2, or influenza. The present inventors were also able to show an anti inflammatory activity in an animal model of a respiratory viral infection when 1 -MNA was administered by inhalation (and therefore directly to the site of infection).

Furthermore, it was shown by the present inventors that 1 -MNA is capable of significantly reducing proinflammatory cytokines locally in the airways (e.g. as measured in BAL fluid). In particular, 1-MNA is capable of lowering the levels of factors associated with inflammasome activation (in particular IL-1 b, TGF-b).

Additionally, it was observed by the present inventors that 1 -MNA, even when administered via inhalation, is also capable of systemic activity and decreasing C-reactive protein levels in serum.

Furthermore, the present inventors have shown, in an animal model of pulmonary fibrosis, that 1 -MNA, administered by inhalation, was effective in lowering cell infiltration, and in particular eosinophil infiltration in airways (shown for bronchoalveolar lavage). In fact, 1 -MNA salt was more effective in lowering BAL eosinophils than nintedanib, which is an approved treatment for pulmonary fibrosis. The effect was visible as quickly as 3 days after the treatment was initiated and was even more pronounced after 21 days.

It is known in the art that eosinophils accumulate at sites of allergic inflammation, where they release a number of inflammatory mediators, such as radical oxygen species, cytokines, and lipid mediators. Eosinophils also produce TGF-b, which may contribute to airway fibrosis, being important to pulmonary fibrosis and other severe respiratory diseases. Therefore the strong reduction in eosinophils by 1-MNA is an encouraging characteristic as it suggests the likely benefit in numerous respiratory diseases, including but not limited to infections, such as viral infections. The effect is also important due to implications in treatment of allergic diseases associated with symptoms affecting the respiratory tract. In a particular example, a subtype of asthma is called “severe eosinophilic asthma” where patients demonstrate evidence of eosinophilia that often requires high maintenance doses of oral corticosteroids to maintain reasonable disease control. This is another indication for the inhaled use of 1 -MNA either as a separate inhaled therapy or in addition to inhaled corticosteroids in order to reduce the corticosteroid dose required for treatment.

Other diseases and pathological states which are associated with eosinophilia and thus can be treated or prevented or alleviated with 1 -MNA use include: eosinophilic angiocentric fibrosis, eosinophilic cellulitis, eosinophilic colitis, eosinophilic endocarditis, eosinophilic enteritis, eosinophilic esophagitis, eosinophilic fasciitis, eosinophilic gastroenteritis, eosinophilic gastroenterocolitis.

Furthermore, many diseases or disorders associated with an inflammatory reaction in the airways or with a defective epithelial barrier in the airways, in particular diseases or disorders associated with infections or the respiratory tract, in particular viral infections, are additionally associated with symptoms related to muscles, such as myopathies, muscle fatigue, fatigue and/or muscle pains. For example, COVID-19 is often associated with myopathy and muscle fatigue (Chen Tao, Wu Di, Chen Huilong, Yan Weiming, Yang Danlei, Chen Guang et al. Clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study BMJ 2020; 368 :m109). A study on a population of 201 COVID-19 patients in China, showed that over 30% of the subjects exhibited fatigue as one of the symptoms (with over 50% of ICU patients having this symptom) (Chang, De & Lin, Minggui & Wei, Lai & Xie, Lixin & Zhu, Guangfa & Dela Cruz, Charles & Sharma, Lokesh. (2020). Epidemiologic and Clinical Characteristics of Novel Coronavirus Infections Involving 13 Patients Outside Wuhan, China. JAMA. 323. 10.1001 /jama.2020.1623). A similar set of symptoms is associated with other viruses causing acute infections in the airways, such as influenza.

The present inventors note that 1-MNA is additionally involved in signaling in muscles. Low levels of this molecule may play a part in myopathies, muscle fatigue and the like (with decreased levels correlating with viral infection as discussed above). On the other hand, endogenous 1 -MNA levels may be increased in correlation with physical exercise and/or caloric restriction (see e.g. Strom, K., Morales-Alamo, D., Ottosson, F. et al. N1- methylnicotinamide is a signaling molecule produced in skeletal muscle coordinating energy metabolism. Sci Rep 8, 3016 (2018)).

The present inventors have unexpectedly found that 1-MNA is additionally capable of increasing plasma levels of lipoprotein lipase, to an extent which has been previously described following moderate physical exercise, with the effect observed as quickly as two hours after a single administration. Lipoprotein lipase is an enzyme expressed inter alia in muscle tissue and essential for controlling plasma triglyceride catabolism, HDL cholesterol, and other metabolic risk factors. Myalgia and myositis, such as viral myositis (a state of viral induced inflammation in muscles, being the main cause of the muscle-related symptoms as listed above) is often associated with changes in numerous muscle-expressed enzymes. The present inventors propose that 1-MNA may additionally help alleviating other symptoms associated with diseases or disorders associated with an inflammatory reaction in the airways or with a defective epithelial barrier in the airways, in particular diseases or disorders associated with infections or the respiratory tract, in particular viral infections, such as e.g. myopathies, muscle fatigue, fatigue and/or muscle pains.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

In this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), as well as the terms "one or more," and "at least one" can be used interchangeably herein.

As used herein the terms "treat," "treatment," or "treatment of" refers to (i) reducing the potential for a disease or disorder (e.g., an allergic disease, an example of an allergic disease being asthma; a disease associated with infections in the respiratory tract, in particular viral infections, in particular COVID-19, or influenza), (ii) reducing the occurrence of a disease or disorder, (iii) reducing the severity of a disease or disorder, preferably, to an extent that the subject suffers less or no longer suffers discomfort and/or altered function due to it, (iv) reducing an indication or marker of a disease or disorder such as reducing the blood or serum TNF-a level, or (v) a combination thereof.

As shown by the present application, 1 -MNA may also effectively prevent development of inflammation or reduce the severity of inflammation when administered before exposure to a pro-inflammatory factor. 1 -MNA is capable of preventing pathological changes associated with inflammasome activation, such as cellular infiltration and fibrotic lesions in lungs.

The terms "subject," "individual" or "patient" as used herein refer to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy of a disease or disorder (e.g., an allergic disease, such as e.g. asthma, a disease associated with infections in the respiratory tract, in particular viral infections, in particular COVID-19 or influenza) is desired. As used herein, the terms "subject," "individual" or "patient" include any human or nonhuman animal. The term "nonhuman animal" includes all vertebrates, e.g., mammals and non-mammals, such as mice, nonhuman primates, sheep, dogs, cats, horses, cows, bears, chickens, amphibians, reptiles, etc. In preferred embodiments, a subject is a human. The term "administration" or "administering" of a drug or a medication, as used herein, includes delivering, applying, or giving the therapy or drug to a subject including self-administering by the subject.

1 -MNA is administered to a subject in need thereof in a form of a therapeutically acceptable salt. The term "pharmaceutically acceptable salt" refers to a salt of an acidic or basic group of a base compound that is generally safe, non-toxic, neither biologically nor otherwise undesirable, and useful for either or both veterinary use and/or human pharmaceutical use. The disclosure herein discloses 1 -MNA, which is capable of forming a wide variety of salts with anions of various inorganic and organic acids.

As used herein, the term "1 -methylnicotinamide," also known as 1 -MNA, is a quaternary pyridinium compound the cation of which has the structural formula of

Non-limiting pharmaceutically acceptable salts of 1 -MNA include sulfate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, amino acid salts, salts of mono-, di- and tricarboxylic acids, e.g., acetate, benzoate, salicylate, hydroxyacetate, lactate, maleate, malonate, malate, tartrate, bitartrate, isonicotinate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, pamoate (i.e., 1 ,1'-methylene-bis-(2-hydroxy-3-naphthoate)), oxalate and citrate salts. In some embodiments, a pharmaceutically acceptable salt includes chloride, benzoate, salicylate, acetate, citrate, and lactate salt of 1 -MNA. In preferred embodiments, a salt of 1 -MNA is 1 MNA chloride, also known as TRIA 662. 1 -MNA chloride is commercially available (e.g., Sigma, Cayman Chemical).

Pharmaceutically acceptable 1 -MNA salts can be prepared from nicotinamide by methods known to persons skilled in the art. Salts with halogen anion can be prepared from nicotinamide by direct methylation with methyl halogenide as known in the art, e.g., as described in AT 131118, GB348345, US3614408, and US4115390. Salts with a non-halogen anion can be prepared by substitution of a halogen anion to another anion, for example by treatment with a salt of the another anion, such as for example sodium or silver salt of the another anion. As an illustration, lactate and acetate can be prepared by the treatment of a halogenide, preferably chloride, with silver lactate or acetate, respectively. Salicylate can be prepared by the treatment of a halogenide, preferably chloride, with sodium salicylate.

Oral, topical or intravenous administration routes of pharmaceutically acceptable 1 -MNA salts are known in the art. However, in order to treat or prevent allergic diseases in the respiratory tract and/or diseases and conditions associated with infections, in particular viral infections, such as with Sars-CoV-2, it may be unexpectedly beneficial to administer 1-MNA salts through an inhalation route, nasal route or in particular through nebulization. In this way, the active substance administered may be delivered directly to the airways, wherein it may act on regulating inflammatory reactions and promoting epithelial barrier integrity. Such administration may be particularly useful in preventing allergic and/or excessive inflammatory reactions, in particular in lung tissues, preventing fibrosis and/or alleviating symptoms. These administration routes enable local delivery and confer a potentially higher concentration of the active substance in the target organ, in this case pulmonary drug concentrations, and lower systemic concentrations. Inhalation is typically associated with high pulmonary efficacy and minimal systemic side effects. The lung, as a target, represents an organ with a complex structure and multiple pulmonary-specific pharmacokinetic processes, and hence there are processes that are specific to the pulmonary environment and the inhalation route, making pulmonary pharmacokinetics generally distinct and much more complex than those of drugs administered via other routes. Therefore it is never clear whether an orally administered substance can be provided effectively into the respiratory tract and whether it will have local efficacy and to what extent.

It is preferred that the pharmaceutically acceptable 1 -MNA salt of the present invention is provided in form of particles or liquid droplets, preferably within a range of about 0.5-5 pm in diameter, since particles or droplets within this range have the highest probability of being deposited in the lungs.

A nasal aerosol or inhalation composition can be prepared according to techniques well known in the art of pharmaceutical formulation. Certain technologies suitable for administration by inhalation employ liposomes and lipid complexes which may provide a prolonged therapeutic effect of the drug in the lung. For administration by inhalation, the compound for use according to the present invention is thus conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. Therefore, according to the present invention pharmaceutically acceptable 1-MNA salt is administered in a therapeutically effective amount, by any suitable administration route, however in particular the route of administration is inhalation, intranasal, or similar, e.g. via a nebulizer.

1 -methylnicotinamide (1 -MNA) for use according to the present invention is for use in a method of preventing or treating a disease associated with an inflammatory reaction in the airways or with a defective epithelial barrier in the airways, wherein the disease is selected from allergic diseases or diseases associated with an infection. An allergic disease, within the meaning of the present invention, is a disease, disorder or condition caused by hypersensitivity of the immune system (typically against substances in the environment which are normally harmless), associated with symptoms in the respiratory system, such as coughing, shortness of breath or dyspnoea and/or inflammation in the airways, e.g. in lungs. An example of an allergic disease is asthma. A disease associated with an infection, within the meaning of the present invention, is a disease, disorder or condition associated with an infection within the respiratory system, such as a bacterial or a viral infection. Such a disease is usually associated with symptoms from the respiratory tract, such as coughing, shortness of breath or dyspnoea, throat ache and/or inflammation in the airways, e.g. in lungs. A disease associated with an infection is in particular a disease associated with a viral infection, the virus preferably being one associated with an acute infection of the respiratory system, such as an adenovirus, a respiratory syncytial virus, a rhinovirus, a coronavirus, an influenza virus, a parainfluenza virus. The virus may in particular be Sars-CoV-2 (causing COVID-19) or an influenza virus.

1 -methylnicotinamide (1-MNA) salt for use according to the present invention may be used for treatment of an ongoing disease associated with an inflammatory reaction in the airways or with a defective epithelial barrier in the airways, wherein the disease is selected from allergic diseases or diseases associated with an infection. This may mean that 1 -MNA salt may be administered to a subject that is already showing symptoms in the respiratory tract (such as, but not limited to, coughing, throat ache, shortness of breath or difficulty breathing, or dyspnoea, inflammation in the respiratory tract, e.g. lungs; additionally symptoms may include myopathies, muscle fatigue, fatigue and/or muscle pains; fever may or may not be present), with an aim of inter alia alleviating one or more of the symptoms.

Alternatively or additionally, the 1 -MNA salt for use according to the present invention may be used for prevention of a disease associated with an inflammatory reaction in the airways or with a defective epithelial barrier in the airways, wherein the disease is selected from allergic diseases or diseases associated with an infection. Therefore, 1 -MNA salt may be administered, as described herein, to a subject being at a risk of developing a disease associated with an inflammatory reaction in the airways or with a defective epithelial barrier in the airways, wherein the subject is not yet showing any or all of the symptoms as described herein above. 1-MNA salt may be administered as described herein, by any of the herein described administration routes, in particular by inhalation and/or orally, as a preventive measure, e.g. also but not only for longer time periods, in order to prevent the subject from developing symptoms or to render the symptoms less severe if they do appear or shorten the time period of symptoms etc. In one embodiment, 1 -MNA is administered in a form of a dietary supplement, in order to prevent onset, development or worsening of a disease associated with an inflammatory reaction in the airways or with a defective epithelial barrier in the airways, in particular to prevent onset, development or worsening of any or all of symptoms as described above.

Alternatively or additionally, the 1 -MNA salt for use according to the present invention may be used for prevention of a disease associated with eosinophilia. Such a disease may be caused by or associated with infection in airways (e.g. viral infection). Alternatively or additionally, it may be an allergic disease, such as, but not limited to eosinophilic asthma and/or a disease selected from the group of : eosinophilic angiocentric fibrosis, eosinophilic cellulitis, eosinophilic colitis, eosinophilic endocarditis, eosinophilic enteritis, eosinophilic esophagitis, eosinophilic fasciitis, eosinophilic gastroenteritis, eosinophilic gastroenterocolitis.

The term "therapeutically effective amount" as used herein refers to an amount of a drug effective to "treat" a disease or disorder in a subject and/or to prevent or reduce the risk, potential, possibility or occurrence of a disease or disorder (e.g. an allergic disease, a disease associated with infections in the respiratory tract, in particular viral infections, in particular COVID-19). A "therapeutically effective amount" includes an amount of a drug or a therapeutic agent that provides some improvement or benefit to a subject having or at risk of having a disease or disorder (e.g. an allergic disease, such as e.g. asthma, a disease associated with infections in the respiratory tract, in particular viral infections, in particular COVID-19 or influenza). Thus, a "therapeutically effective" amount is an amount that reduces the risk, potential, possibility or occurrence of a disease or disorder or provides some alleviation, mitigation, and/or decrease in at least one clinical symptom of a disease or disorder (e.g. an allergic disease, such as e.g. asthma, a disease associated with infections in the respiratory tract, in particular viral infections, in particular COVID-19 or influenza).

The therapeutic effective amount of the pharmaceutically acceptable 1-MNA salt for the treatment may depend on factors including the blood or serum levels of proinflammatory factors, severity of infection in the subject before the treatment, the presence or absence of various conditions (e.g., presence or absence or severity of cardiovascular disorders and/or allergic diseases or reactions, such as asthma), age and gender of the subject and can be adjusted by a person of ordinary skill in the art (e.g., a doctor). The expected therapeutic effect involves at least one selected from the following: a decrease in one or more proinflammatory factors, e.g. TNF-a, IL-1 b, IFN-A1, IL-6, IP-10 and/or KC (keratinocyte-derived cytokine), a decrease in overall lung inflammation, a decrease in caspase activity, a decrease in inflammasome activity, a decrease in or elimination of pulmonary fibrosis, an enhancement in epithelial barrier integrity in airways, a decrease in immunological cellular infiltrate in airways, e.g. lymphocytes mononuclear cells and/or neutrophils, and/or a decreased or eliminated eosinophilia, and/or an alleviation of at least one of the symptoms associated with an airway infection, such as viral infection, the symptoms including coughing, throat ache and/or shortness of breath or difficulty breathing or dyspnoea etc and/or symptoms related to muscles, such as myopathies, muscle fatigue, fatigue or muscle pains.

In some embodiments, the therapeutically effective amount of a pharmaceutically acceptable 1 -MNA salt is from about 50 to 1000 mg per day. It is noted that if 1 -MNA salt for use according to the present invention is administered by inhalation, the dose may preferably be lower than oral dose would be. Therefore, in a preferred embodiment, the therapeutically effective amount of a pharmaceutically acceptable 1 -MNA salt is from about 15 to 100 mg per day or from about 50 to 1000 mg per day, in particular when administered by inhalation. In another preferred embodiment, the therapeutically effective amount of a pharmaceutically acceptable 1 -MNA salt is from about 50 to 500 mg per day, or 50 to 200 mg per day, or 50 to 100 mg per day, in particular when administered by inhalation. In other embodiments, the therapeutically effective amount of a pharmaceutically acceptable 1 -MNA salt is from about 500 to about 1000 mg, from about 1000 to about 8000 mg, from about 1000 mg to about 7000 mg, from about 1000 mg to about 6000 mg, from about 1000 mg to about 5000 mg, from about 1000 mg to about 4000 mg, from about 1000 mg to about 3000 mg, from about 1000 mg to about 2000 mg, from about 2000 mg to about 8000 mg, from about 2000 mg to about 7000 mg, from about 2000 mg to about 6000 mg, from about 2000 mg to about 5000 mg, from about 2000 mg to about 4000 mg, from about 2000 mg to about 3000 mg, from about 3000 mg to about 8000 mg, from about 3000 mg to about 7000 mg, from about 3000 mg to about 6000 mg, from about 3000 mg to about 5000 mg, from about 3000 mg to about 4000 mg, from about 4000 mg to about 8000 mg, from about 4000 mg to about 7000 mg, from about 4000 mg to about 6000 mg, from about 4000 to about 5000 mg, from about 5000 mg to about 8000 mg, from about 5000 mg to about 7000 mg, from about 5000 mg to about 6000 mg, from about 6000 mg to about 8000 mg, from about 6000 mg to about 7000 mg, from about or 7000 mg to about 8000 mg, per day.

In some embodiments, the therapeutically effective a pharmaceutically acceptable 1 -MNA salt is about 50 mg, about 100 mg, about 200 mg, about 500 mg, 1000 mg, about 2000 mg, about 3000 mg, about 4000, about 5000 mg, about 6000 mg, about 7000 mg, or about 8000 mg, per day.

In some embodiments, the therapeutically effective amount of a pharmaceutically acceptable 1 -MNA salt is administered once a day, twice a day, or three times a day or more. A therapeutically effective amount can be administered in one dose or divided into multiple doses, as long as the dose is sufficiently high that the subject benefits from the dose or treatment. In a preferred embodiment, 1 -MNA salt is administered twice a day.

In an embodiment, 1 -MNA salt is administered by inhalation or intranasally, preferably twice a day. An exemplary dose may be from about 50 to 1000 mg per day. In a particular but non limiting example, the 1 -MNA salt may be administered in an amount corresponding to 18.75 - 75 mg/kg of body weight, twice a day.

The 1-MNA salt may be administered by only one administration route (preferably, by inhalation, intranasally or similar, so that the 1 -MNA salt is delivered directly to the airways, in particular to lungs). However, in another preferred embodiment, the therapeutically effective amount of a pharmaceutically acceptable 1 -MNA salt may be administered via more than one administration route, e.g. both by inhalation and orally. The two ways of administration may be effected simultaneously, sequentially or separately. For example, but without limitation, administration by inhalation may be provided twice a day and oral administration may be provided once a day, with the time of oral administration being directly before or after administration by inhalation or at a separate time point entirely.

When the pharmaceutically effective amount of a pharmaceutically acceptable 1 -MNA salt is administered orally, e.g., comprised in a pharmaceutically acceptable oral dosage form, such oral dosage forms can also comprise a suitable amount of one or more pharmaceutically acceptable excipients, including a diluent, suspending agent, solubilizer, binder, disintegrant, preservative, coloring agent, lubricant, and the like. The pharmaceutical excipients can be a liquid, such as water or oil, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. The pharmaceutical excipient can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. In one embodiment, the pharmaceutically acceptable excipient is sterile when administered to a human subject. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. The pharmaceutical compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Examples of pharmaceutically acceptable carriers and excipients that can be used to formulate oral dosage forms are known in the art, e.g., described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986).

For inhalation or intranasal or similar routes providing 1 -MNA salts directly to the airways, the active substance may be present in a composition, the composition also comprising a suitable amount of one or more pharmaceutically acceptable excipients, including a diluent, suspending agent, solubilizer, binder, preservative, coloring agent, lubricant, and the like. The composition may be in a liquid form or a solid form (e.g. powder). Pharmaceutically acceptable excipients that are volatile or non-volatile may be included. Volatile excipients, when heated, are concurrently volatilized, aerosolized and inhaled with the drug. Classes of such excipients are known in the art and include, without limitation, gaseous, liquid and solid solvents. The following is a list of exemplary carriers within the classes: water; terpenes, such as menthol; alcohols, such as ethanol, propylene glycol, glycerol and other similar alcohols.

1 -MNA salts may also optionally be administered in conjunction with other active ingredients either sequentially or simultaneously. The other active ingredients may be for example anti inflammatory compounds, e.g. corticosteroids, such as prednisone, dexamethasone, fluticasone propionate.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 shows primary 1 -MNA metabolic pathways.

Fig. 2 summarizes the results for blood levels of proinflammatory factors in subjects receiving either 1-MNA chloride or placebo.

Fig. 3 shows the result for plasma lipoprotein lipase (LPL) activity in subjects receiving either 1 -MNA chloride or placebo.

Fig. 4 shows secreted cytokine levels for HBEC3-KT cells stimulated with a TLR3 receptor agonist (poly(l:C)) and treated with 1 -MNA. The measured cytokines are IL-1 b (4A), IFN-A1 (4B), IFN-A2/3 (4C), IFN-b (4D), TNF (4E), GM-CSF (4F).

Fig. 5 shows secreted cytokine levels for HUVEC/Tert2 cells stimulated with a TLR3 receptor agonist (poly(l:C)) and treated with 1 -MNA. The measured cytokines are IL-6 (5A), IL-8 (5B), IP-10 (5C). ^* P<0.05, ^** P<0.01.

Fig. 6 shows relative differences in reactive oxygen species (ROS) after 1 -MNA treatment, measured by DCFDA fluorescence in HBEC3-KT cell line. Fig. 6A and 6B show the results in cells stimulated for 6 and 24h respectively with 10 pg/mL poly(l:C) and Fig. 6C and 6D show the results in cells stimulated for 6 and 24h respectively with 30 pg/mL poly(l:C). ^* P<0.05, ^** P<0.01 , ^**** P<0.001.

Fig. 7 shows relative differences in reactive oxygen species (ROS) after 1 -MNA treatment, measured by DCFDA fluorescence in HUVEC/Tert2 cell line. Fig. 7A and 7B show the results in cells stimulated for 6 and 24h respectively with 10 pg/mL poly(l:C) and Fig. 7C and 7D show the results in cells stimulated for 6 and 24h respectively with 30 pg/mL poly(l:C). ^* P<0.05, ^** P<0.01 , ^**** P<0.001.

Fig. 8 demonstrates cell viability of HBEC3-KT cells treated with 1-MNA. Fig. 8A and 8B show the results in cells stimulated for 6 and 24h respectively with 10 pg/mL poly(l:C) and Fig. 8C and 8D show the results in cells stimulated for 6 and 24h respectively with 30 pg/mL poly(l:C).

Fig. 9 demonstrates cell viability of HUVEC/Tert2 cells treated with 1-MNA. Fig. 9A and 9B show the results in cells stimulated for 6 and 24h respectively with 10 pg/mL poly(l:C) and Fig. 9C and 9D show the results in cells stimulated for 6 and 24h respectively with 30 pg/mL poly(l:C).

Fig. 10 shows caspase-1 activity, as measured by Caspase Glo-1 protocol (Promega) in HUVEC/Tert2 cells stimulated with LPS or LPS and nigericin and treated with 1 -MNA. ^* P<0.05, ^** P<0.01.

Fig. 11 shows IL-1 b levels in HUVEC/Tert2 cells stimulated with LPS or LPS and nigericin and treated with 1 -MNA. ^* P<0.05, ^** P<0.01.

Fig. 12 demonstrates the effect of 1-MNA administered by the intratracheal route in a model of H1 N1 induced pulmonary inflammation model on BAL neutrophil (12A), eosinophil (12B), mononuclear cell (12C) and lymphocyte counts (12D). TRIA-662 indicates 1-MNA chloride. Displayed as mean ± s.e.m. +++ p<0.001 when H1N1/Vehicle was compared to the Saline/Vehicle control group; ^* p<0.05, ^** p<0.01 and ^*** p<0.001 when compared to the H1 N1/Vehicle control group.

Fig. 13 shows the effect of 1-MNA administered by the intratracheal route in a model of H1 N1 induced pulmonary inflammation model on BAL keratinocyte-derived cytokine (KC; pg/mL). TRIA-662 indicates 1-MNA chloride. Displayed as mean ± s.e.m. ++ p<0.01 when H1 N1 /Vehicle was compared to the Saline/Vehicle control group; ^* p< 0.05 and ^*** p<0.001 when compared to the H1 N1 /Vehicle control group.

Fig. 14 shows the influence of 1-MNA administered by the intratracheal route in a model of H1 N1 induced pulmonary inflammation model on serum CRP levels. Displayed as mean ± s.e.m. +++ p<0.001 when H1 N1/Vehicle was compared to the Saline/Vehicle control group; ^* p<0.05 and ^*** p<0.001 when compared to the H1 N1/Vehicle control group.

Fig. 15 demonstrates the effect of 1-MNA administered by the intratracheal route in rat model of bleomycin induced pulmonary fibrosis on BAL total cell (15A), neutrophil (15B), mononuclear cell (15C), lymphocyte (15D) and eosinophil (15E) counts, on day 3. TRIA indicates 1 -MNA chloride. Data expressed as mean ± S.E.M.

Fig. 16 demonstrates the effect of 1-MNA administered by the intratracheal route in rat model of bleomycin induced pulmonary fibrosis on BAL total cell (16A), neutrophil (16B), mononuclear cell (16C), lymphocyte (16D) and eosinophil (16E) counts, on day 21. TRIA indicates 1 -MNA chloride. Data expressed as mean ± S.E.M.

Fig. 17 shows lung parameters after 21 days of study with 1 -MNA administered by the intratracheal route in rat model of bleomycin induced pulmonary fibrosis; resistance (17A), elastance (17B), compliance (17C), FVC (17D), FEV (17E) and PEF (17F). TRIA indicates 1 -MNA chloride. Data expressed as mean ± S.E.M.

Fig. 18 shows cytokine levels after 3 days of study with 1 -MNA administered by the intratracheal route in rat model of bleomycin induced pulmonary fibrosis; 11-1 b (18A), MCP-1 (18B), MIP-1a (18C). TRIA-662 indicates 1-MNA chloride. Data expressed as mean ± S.E.M.

Fig. 19 shows determined values after 21 days of study with 1 -MNA administered by the intratracheal route in rat model of bleomycin induced pulmonary fibrosis, forTGF-b (19A), CRP (19B). TRIA-662 indicates 1-MNA chloride. Data expressed as mean ± S.E.M.

EXAMPLES

Example 1. 1-MNA significantly decreases TNF-a levels.

The study was a randomized, double-blind, placebo-controlled, forced dose-escalation, multi center study conducted in Canada (22 Sites) among patients with dyslipidemia. One of the objectives was the measurement of inflammatory markers: hs-CRP, TNFa.

Diagnosis and Main Inclusion Criteria:

Males and females, at least 18 years of age and £ 80 years of age Compliant to the dietary-controlled 6 to 8-week lead-in period

Mean serum LDL-C at levels for which lipid-modifying drug therapy was not indicated according to investigator judgment under ATP III guidelines,

Mean serum TG 200 mg/dL (2.26 mmol/L) but £ 500 mg/dL (5.65 mmol/L). Number of Subjects and Disposition:

Planned to enter approximately 200 subjects in the baseline lead-in period to randomize approximately 64 subjects in the double-blind period (3:1 ratio).

164 subjects entered the baseline period, 71 subjects randomized:

Placebo = 22

1 -MNA chloride = 49

In a diet-controlled lead-in period (6-8 weeks), the subjects were administered 1000 mg of placebo thrice daily. After the lead-in period, the treatment group received 500 mg x2 of 1 -MNA chloride thrice daily for two weeks, and then 1000 mg x2 thrice daily for 12 weeks. The placebo groups was administered placebo in exactly the same regime. The treatment phase was followed by a 30-day follow-up observation phase.

The analysis of proinflammatory factors in blood showed that 1 -MNA decreased the blood level of high sensitivity C-reactive protein (hsCRP) by 17% (vs placebo), and reduced the blood level of tumour necrosis factor TNF-a by 14% (vs placebo). In patients with the highest initial level of TNF-a, 1-MNA reduced its level by 29% (vs placebo). The results are summarized on

Fig. 2.

These results demonstrate that 1 -MNA is capable of significantly lowering TNF-a levels which supports its proposed function in not only regulating inflammation but also promoting epithelial barrier integrity.

Example 2. 1-MNA significantly increases plasma activity of lipoprotein lipase.

A short-term effect of 1 -MNA chloride administration was investigated in a human study. Subjects were administered an oral single dose of ether a placebo (n=4) or 90 mg of 1 -MNA chloride (n=8). The subjects were not administered heparin. Blood samples were collected form subjects, at the beginning of the study, before treatment and 2 and 5 h after treatment. Plasma for LPL measurement was collected in EDTA containing tubes and stored in -70°C until analysis. Non-heparin stimulated lipoprotein lipase activity in plasma was measured in the samples, and values before administration, as well as 2 hours and 5 hours after administration were compared.

It was observed that 90 mg 1-MNA produced (2 hrs after treatment) a statistically significant (p<0.014) increase of non-heparin stimulated lipoprotein lipase activity to an extent which has been previously described following moderate physical exercise (Fig. 3) Example 3. 1-MNA in treatment of an acute airway infection; administration by inhalation

A person was showing symptoms of an acute airway infection in the form of cough, dyspnoea and fever during a trip to Spain (by the end of February 2020). Immediately after return (which was one day after the onset of the symptoms), the person started inhalation treatment with 1 - MNA, which lasted for 7 days. 1 g of 1 -MNA chloride was dissolved in 50 ml. of water. The active substance was administered by nebulisation with 5 ml. of the above solution used for one dose (thus, a unit dose was 100 mg/inhalation). Inhalation with such a unit dose was carried out twice daily. The subject additionally received a daily oral dose of 250 mg of 1-MNA chloride. After the treatment, a clear alleviation of symptoms was observed.

The infection was suspected to be caused by Sars-CoV-2 virus, due to the symptoms and a time period and circumstances of onset (symptoms occurred shortly after the subject was travelling on a train in Spain in February 2020, while a man with severe dry cough was seated behind for the duration of the train ride).

Example 4. Activity of 1-MNA chloride on human lung epithelial and endothelial cells undergoing TLR3 receptor stimulation (a viral infection model).

Assays were performed on two human cell lines, FIBEC3-KT (normal human bronchial epithelial cells immortalized with CDK4 and hTERT) and FIUVEC/Tert2 (hTERT immortalized human umbilical vein endothelial cell line). Both cell lines were cultured in 37°C in atmosphere with 5% CO2. FIBEC3-KT (ATCC) were cultured in PneumaCult-Ex Medium (StemCell Technologies). FIUVEC-Tert2 (ATCC) were cultured in EGM-2 Endothelial Cell Growth Medium (Lonza).

One day before the experiment, the cells were seeded, in amount of 2 x 10⁴ on 96-well plates in 100 pi of respective medium and cultured overnight. The cells were then treated. The specific treatment and control groups were as follows:

- untreated cells (no stimulation with poly(l:C), no test treatment) (control);

- cells stimulated with 10 pg/mL poly(l:C), no test treatment (control);

- cells stimulated with 30 pg/mL poly(l:C), no test treatment (control);

- cells without stimulation, treated with 1 mM 1 -MNA chloride (control);

- cells without stimulation, treated with 10 mM 1 -MNA chloride (control);

- cells stimulated with 10 pg/mL poly(l:C) and treated with 1 mM 1 -MNA chloride;

- cells stimulated with 10 pg/mL poly(l:C) and treated with 10 mM 1 -MNA chloride; - cells stimulated with 30 pg/mL poly(l:C) and treated with 1 mM 1 -MNA chloride;

- cells stimulated with 30 pg/mL poly(l:C) and treated with 10 mM 1 -MNA chloride;

- cells stimulated with 10 pg/mL poly(l:C) and treated with 10 mM nicotinamide (NA) (control);

- cells stimulated with 30 pg/mL poly(l:C) and treated with 10 mM nicotinamide (NA) (control);

- cells stimulated with 10 pg/mL poly(l:C) and treated with 1 mM dexamethasone (dex) (control);

- cells stimulated with 30 pg/mL poly(l:C) and treated with 1 mM dexamethasone (dex) (control).

Each variant was done in three technical and three biological replicates. Treatment was commenced with 1-MNA chloride in concentration of 1 or 10 mM in deionized water or nicotinamide (NA, Sigma, cat. N3376-100G, 10 mM) or a potent steroid dexamethasone (dex; Sun-Farm, 1 mM in deionized water) as positive control, and incubated for 30 minutes. Next, an agonist of the TLR3 receptor - poly(l:C) (polyinosinic:polycytidylic acid, Sigma, cat. P9582- 5MG; stock 10 mg/ml_ in water, RNase and DNase free; work solution 1 mg/ml_ in PBS) was added in concentration of 10 or 30 pg/mL. The cells were incubated further for 24 hours and frozen in -80°C for further analysis.

Treated and control cells were analyzed for secreted cytokine levels, viability and reactive oxygen species production. Cytokines were assessed using LEGENDplex™ Human Anti-Virus Response Panel (11-plex) with V-bottom Plate (Biolegend) and evaluated with FACS Cantoll (BD) flow cytometer. Statistical significance for differences between groups were evaluated using Anova with a post-hoc Tukey test.

For ROS analysis, the cells were seeded and incubated as indicated above. Treatment groups were also identical to described above. Each variant was done in three technical and three biological replicates. ROS were evaluated using DCFDA/H2DCFDA - Cellular ROS Assay Kit (Abeam). Briefly, 1 -MNA, dexamethasone and nicotinamide were added as above and cells were incubated for 30 minutes. Next, the medium was discarded and the cells were washed with 100 mI 1x Buffer. 100 mI of DCFDA Solution and Poly(l:C) (in concentrations as indicated above) were then added. The cells were incubated for 1 h in 37°C in the dark. Then, DCFDA Solution was removed and 100 mI IxBuffer with 5% FBS and Poly(l:C) in the appropriate concentration were added, while positive control was treated with 100 mI TBHP 100 mM. Next, the cells were incubated in 37°C and 5% CO2 and ROS levels were determined by fluorescence measurement every hour for 6 hours; Ex/Em=485/535. Another measurement was also done 24 h after treatment with stimulants. The results are shown as a ratio of absolute values for fluorescence intensity for a sample in relation respective control (cells without stimulants). Statistical significance for differences between groups were evaluated using Anova with a post-hoc Tukey test.

For general cytotoxicity, the cells were seede in the amoun of 9x 10³ on 96-well plates in 100 pi of respective medium and cultured overnight. Then, the cells were treated with stimulants as described above in treatment groups as indicated above.

General cytotoxicity was evaluated in all wells with CellTox Green Cytotoxicity Assay (Promega), utilizing CellTox Green dye, which does not penetrate through cell membranes of viable cells. Wells with positive control were additionally prepared, and these cells were treated with Lysis Solution for 30 min before first measurement. For assessment, 20 mI of CellTox Green Reagent (20 mI of CellTox Green Dye in 2 ml of Assay Buffer) was added to each well. After 15 min of incubation in darkness and room temperature, the cells were kept in 37°C, 5% CO2 and fluorescence was measured at Ex/Em = 485/520 nm, 6h and 24 h after treatment. Results are shown as percent in relation to fluorescence intensity of unstimulated cells and positive control (lysed cells). Statistical significance for differences between groups were evaluated using Anova with a post-hoc Tukey test.

Results

Results for cytokine levels in HBEC3-KT cell line for IL-1 b, IFN-A1, IFN-A2/3, IFN-b, TNF, GM-CSF are shown on Fig. 4. As shown on Fig. 4, 1-MNA caused a decrease in pro- inflammatory cytokines (IL-1 b, IFN- A1 and TNF) in a dose dependent manner, with the 10 mM dose reverting these inflammation mediators to base levels comparable to unstimulated cells. It should be noted that all three cytokines are involved in strong inflammatory responses associated with local response against viral infection.

Fig. 5 shows results in HUVEC/Tert2 cell line for the levels of IL-6, IL-8 and IP-10 (cxcMO). The levels of IL-1/?, IFN-21 , IFN-22/3, IFN-a2, IL-10, TNF, GM-CSF were below measurement threshold. 1 -MNA was shown to be capable of significantly decreasing proinflammatory cytokine levels in endothelial cells.

Therefore, 1 -MNA was shown to be capable of inhibiting different types of cytokines, in particular, cytokines normally secreted in response to viral ligands (dsRNA). The analyzed cytokines are often elevated in states of viral infection and their higher levels correlate with more sever disease progression (such as IP-10 in SARS; see e.g. Yang XY, Yao GH, Xu J, Zhong NS: The possible mechanism of lung injury induced by severe acute respiratory syndrome coronavirus spike glycoprotein. Zhonghua Jie Fie Fie Flu Xi Za Zhi 2006, 29(9):587- 590). Fig. 6 and 7 show results for ROS levels, as measured by DCFDA fluorescence in both cells lines in the groups as defined above. Respective cell viabilities are shown on Fig. 8 and 9 respectively. Stimulation with poly(l:C) causes an increase in cell-damaging ROS levels, but 1 -MNA is demonstrated to have alleviating effect in a dose-dependent manner, which was significant in cells after 24h exposure to the TLR3 receptor agonist. The effect obtained with 1 -MNA was comparable to dexamethasone and improved in comparison with the corresponding concentration of nicotinamide. There was no significant influence on cell viability by any evaluated treatment (Fig. 8 and 9).

The above indicates that 1 -MNA may have a potent anti-inflammatory effect in conditions of viral infection, comparable to steroids. 1 -MNA however is not associated with adverse effects commonly observed for steroid treatment. As an immunomodulator, this compound may be especially useful in alleviating states when a massive infection causes production of high amounts of cytokines and inducing tissue damage.

Example 5. Activity of 1-MNA chloride on human endothelial cells undergoing NLRP3 inflammasome stimulation.

Inflammasomes are large intracellular multiprotein complexes playing a crucial role in innate immunity. They detect and respond to a large range of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), such as uric acid crystals. Inflammasomes contain a member of the NOD-like receptor (NLR) family, such as NLRP3 and IPAF, by which they are defined. The NLR protein recruits the inflammasome- adaptor protein ASC, which in turn interacts with caspase-1 leading to its activation. Once activated, caspase-1 promotes maturation of proinflammatory cytokines, such as interleukin (IL)-1 b and IL-18. Inflammasome activation is crucial for host defense against pathogens (such as viruses).

This study utilized the HUVEC/Tert2 human cell line, as described in Example 4 above. The cells were seeded at 5 x 10⁴ per well on a 96-well plate in 100 pi of EGM2 medium and incubated overnight. The cells were then treated with 1 -MNA chloride (1 mM or 10 mM in deionized water), 1 pg/mL lipopolysaccharide (LPS; from Escherichia coli 0111 :B4, Sigma, in deionized water; stock 1 mg/mL in deionized water) from bacterial cell walls (a proinflammatory TLR4 receptor agonist) and/or nigericin (nigericin being a known NLRP3 Inflammasome inducer) (nigericin sodium salt, TOCRIS, cat. 4312/10, stock 6.7 mM in pure ethanol).

The treatment groups were:

- 1-MNA chloride, 1 mM, for 0.5 h;

- 1 -MNA chloride, 10 mM, for 0.5 h; - LPS 1 pg/mL for 2.5 h, then nigericin 1 mM for 2 h;

- 1 -MNA chloride, 1 mM, for 0.5 h, then LPS 1 pg/mL for 2.5 h, then nigericin 1 mM for 2 h;

- 1 -MNA chloride, 10 mM, for 0.5 h, then LPS 1 mg/mL for 2.5 h, then nigericin 1 mM for 2 h;

- LPS 1 mg/mL for 2.5 h, then 1 -MNA chloride 1 mM, for 0.5 h, then nigericin 1 mM for 2 h;

- LPS 1 mg/mL for 2.5 h, then 1-MNA chloride 10 mM, for 0.5 h, then nigericin 1 mM for 2 h.

Each variant was done in two technical replicates and three biological replicates.

After the incubation times as above, 50 mI of supernatants were collected and frozen in -80°C for further analysis.

Next, the cells were assayed for IL-1 b secretion levels and caspase-1 activation. IL-1 b Human Uncoated ELISA kit (Thermofisher) was utilized for IL-1 b, according to the manufacturer’s instructions.

Caspase-1 activation was determined with Caspase-Glo 1 Inflammasome Assay kit (Promega). The assay utilizes a caspase-1 substrate, which is activated in the presence of the active enzyme. Briefly, the lyophilized Z-WEHD Substrate was suspended in Caspase-Glo 1 Buffer. Such prepared buffer was aliquoted and stored in -20°C. Caspase-Glo 1 Reagent was prepared on the same day by adding MG-132 inhibitor. Half of the Caspase-Glo 1 Reagent volume was transferred to a new vessel and Ac-YVAD-CHO was added obtaining the Caspase-Glo 1 YVAD-CHO Reagent. 50 mI of Caspase-Glo 1 Reagent was added to each well, and the wells were spontaneously secreted caspase-1 was to be inhibited were treated with 50 mI of Caspase-Glo 1 YVAD-CHO Reagent. The cells were incubated for 1 h in room temperature and luminescence was measured using Multiskan Go (ThermoScientific) plate reader.

Results

The results are shown on Fig. 10 (caspase-1) and Fig. 11 (IL-1 b levels). Stimulation with LPS and nigericin did not induce a significant increase in caspase-1 . However, 1-MNA was shown to decrease constitutive caspase-1 activity.

IL-1 b secretion was elevated after stimulation with LPS and nigericin. The increase was inhibited by 1 -MNA in both concentrations, when added after LPS and before nigericin. This indicates involvement in a mechanism associated with cytokine proteolysis and/or secretion.

The obtain results indicate that 1 -MNA may have a pleiotropic immunomodulatory effect which can be beneficial towards reducing inflammation, induced by infection, such as viral infection and inflammasome activation, which is a component of numerous pathological states, in particular in the respiratory system, e.g. in lungs.

Example 6. 1-MNA effect in in a murine H1N1 Induced Pulmonary Inflammation Model.

The purpose of this study was to evaluate the efficacy of 1 -MNA in a murine H1 N1 pulmonary inflammation model.

Animal Model

Female mouse (BALB/c), accepted by regulatory agencies. Weight range 18-20 g, approximately 7-8 weeks of age, obtained from Charles River (UK) Ltd. The animals were allowed to acclimatize for at least 5 days before treatment. They were randomized into study groups using random tables so that the group mean weights were approximately equal. Mice were kept in groups up to five with food and water ad libitum.

Route of Administration

Mice were dosed intranasally with H1N1 (strain: PR8; in PBS, 8.0 x 10² pfu/mL, 40 pfu/50 pl_). Vehicle (water for injection) and 1 -MNA chloride were administered intratracheally (1 -MNA was dissolved in water for injection to produce concentrations of 15 mg/ml_, 30 mg/ml_ or 60 mg/ml_). Ethyl (3/?,4/?,5S)-4-acetamido-5-amino-3-pentan-3-yioxycydohexene-1-carboxylate (COMP1) was administered by the oral route (dissolved in water for injection in the amount of 3 mg/ml_).

Table 1. Treatment Groups and Doses

1-MNA, COMP1 and vehicle were administered twice daily from 2h prior to virus administration on Day 0 through and including the morning of Day 5. 1 -MNA and vehicle were administered at a fixed volume at 25 pL/animal. Therefore, dose level and formulation concentration were calculated based on a 20 g mouse. ^** COMP1 was administered orally via oral gavage at a dose volume of 10 mL/kg.

⁺ H1 N1 was administered at a fixed dose volume of 50 mI_ per animal on Day 0.

⁺⁺ PK animals were not challenged with virus and were culled 2 h after the final administration of test item. After completion of the study protocol, the animals were sacrificed (and blood samples were taken) and bronchoalveolar lavages were collected through a tracheal cannula by washing the airways 3 times with 0.3 ml. phosphate buffer saline. BAL fluid was pooled and kept on ice. The BAL fluid samples were centrifuged at 800 x g for 10 min at 4°C and supernatant was frozen in -80°C prior to cytokine analysis. Cell pellet was resuspended in 0.5 mL PBS. Cell counts were performed using XT-2000iV (Sysmex UK Ltd.). Each sample was vortexed for 5 seconds before analysis. A total and differential cell count (neutrophils, eosinophils, mononuclear cells (includes monocytes and macrophages) and lymphocytes) were reported as number of cells per animal. Fluid supernatants were analyzed for cytokine levels (IL-6, TFN- a, IFN-g, IP-10 and KC). Serum CRP levels were evaluated in blood samples collected from animals using Mouse C-Reactive Protein ELISA kit.

Following completion of BAL fluid collection procedure, lungs were excised and weighed. The whole lung was infused in 10% neutral buffered formalin for a minimum of 24 hours an tissues were processed to paraffin blocks. 4-5 pm sections were stained with haematoxylin and eosin. Light microscope analysis included semi-quantitative assessment (grading) of inflammatory cell infiltrate and tissue inflammation.

Results

Fig. 12 shows an effect of 1-MNA administered by the intratracheal route in this pulmonary inflammation model on BAL (bronchoalveolar lavage) neutrophil, lymphocyte, mononuclear cell and eosinophil counts. TRIA-662 indicates 1-MNA, the doses administered are shown on the figure. Detailed results on Day 5 are also shown in Table 2 below.

Table 2. Effect of 1-MNA and COMP1 in a Murine Model of H1N1 Inflammation on BAL Total and Differential Cell Counts (Day 5)

Values rounded s.e.m. Standard error of the mean.

+++ p<0.001 when H1 N1/Vehicle was compared to the Saline/Vehicle control group; ^* p< 0.05, ^** p<0.01 and ^*** p<0.001 when compared to the H1 N1 /Vehicle control group. Fig. 13 shows determined KC levels in bronchoalveolar lavage of the above-described model (TRIA-662 indicates 1-MNA chloride). Detailed results are provided in Table 3 below.

Table 3. Effect of 1-MNA and COMP1 in a Murine Model of H1N1 Inflammation on BAL Cytokine Levels

Values rounded s.e.m. Standard error of the mean. +++ p<0.001 when H1 N1/Vehicle was compared to the Saline/Vehicle control group; ^* p<0.05, ^** p<0.01 and ^*** p<0.001 when compared to the H1 N1 /Vehicle control group.

BLQ Below limit of quantification (<1.52 pg/mL (IP-10, TNFa, IL-6, KC, IFN-y).

ALQ Above limit of quantification ((<3330 pg/mL (IP-10, TNFa) (<10000 pg/mL (IP-10, TNFa, IL-6, KC, IFN-y)).

Data extrapolated from BLQ and ALQ values where possible, when more than 66% of values are BLQ or ALQ, the mean is BLQ or ALQ.

Fig. 14 shows the effects of 1-MNA administered by the intratracheal route in the model of H1 N1 induced pulmonary inflammation on serum CRP levels. TRIA-662 indicates 1-MNA chloride. Detailed results are also provided in Table 4 below.

Table 4. Effect of 1-MNA and COMP1 in a Murine Model of H1N1 induced pulmonary inflammation on serum CRP Levels

Values rounded s.e.m. Standard error of the mean. +++ p<0.001 when H1 N1/Vehicle was compared to the Saline/Vehicle control group; ^* p< 0.05 and ^*** p<0.001 when compared to the H1 N1/Vehicle control group.

To summarize, twice daily intratracheal administration of 1-MNA (18.75, 37.5 and 75 mg/kg) resulted in a statistically significant attenuation of BAL cellular infiltrate, namely significantly lower BAL lymphocytes and a dose dependent decrease in BAL mononuclear cells and neutrophils. Twice daily intratracheal administration of 1-MNA (18.75, 37.5 and 75 mg/kg) result in a significantly lower BAL KC when compared to the H1 N1 vehicle control at the 75 mg/kg dose level. Twice daily intratracheal administration of 1 -MNA resulted in lower serum CRP which reached statistical significance (17.3% reduction, p<0.05) at 75 mg/kg when compared to the H1 N1 vehicle control group.

1 -MNA is capable of reducing pathological changes in lungs, caused by H1 N1 challenge. Occurrence of lesions, such as diffuse alveolar damage, hyaline membrane formation, mucosal/submucosal mononuclear cell infiltration, mucosal infiltration of eosinophils, microthrombi, multifocal desquamation of the epithelium or even interstitial fibrosis, is reduced in 1-MNA treated animals.

Example 7. Efficacy of 1-MNA in a rat model of bleomycin induced pulmonary fibrosis.

The purpose of this study is to evaluate the efficacy of TRIA-662 in a rat model of bleomycin induced pulmonary fibrosis.

Animal Model

Male Rat (Sprague Dawley, CRLCD) accepted by regulatory agencies were obtained from Charles River (UK Ltd. The animals were 7-8 weeks old and were within the range of 220-250 g at the beginning of the study. They were randomized into study groups using random tables so that the group mean weights were approximately equal. Rats were kept in groups up to five with food and water ad libitum.

Methods

Rats were administered with either vehicle (water for injection) or 1 -MNA chloride (dissolved in water; stock 500 mg/mL) intratracheally to mimic the clinical route of administration, twice daily, until and including the PM dose on the day prior to the scheduled termination day (Day 3 or Day 20), 2 h prior to intratracheal dosing with bleomycin sulfate from Streptomyces verticillus (Sigma-Aldrich; 0.9% w/v saline; 3 I.U./mL), to illicit a fibrotic response in the lungs. Animals were weighed on the morning of dosing. Animals greater than or equal to 250 g were be administered 0.75 I.U. of bleomycin sulfate (the dose by bodyweight of a 250 g animal) or vehicle (dose volume: 250 pL/animal). Animals weighing less than 250 g on day of dosing were dosed by bodyweight with either bleomycin sulfate or vehicle (dose volume: 1 mL/kg). For animals in Group 7 Fluticasone Propionate (Sigma; 0.9% w/v saline; stock 2 mg/mL) was dosed once daily until the day prior to the scheduled termination day (Day 3 or Day 20). A dose volume of 0.5 mL/kg was be administered via the intratracheal route under recoverable gaseous anesthesia (isoflurane/oxygen mix). Nintedanib ethanosulfate (kinase inhibitor) (in water for injection stock 12 mg/ml_ salt, 10 mg/ml_ free base) and dexamethasone (Sigma; 1 % HPMC/0.5% Tween80 in purified water; stock 0.3 mg/ml_) were administered by the oral route (oral gavage) to mimic the clinical route of administration. Nintedanib 10 mL/kg was administered in Group 6, once daily until and including the PM dose on the day prior to the scheduled termination day (Day 3 or Day 20). Dexamethasone 10 mL/kg was administered in Group 8, once daily until and including the PM dose on the day prior to the scheduled termination day (Day 3 or Day 20). First oral doses were on Day 0, approximately 2h before bleomycin dosage.

Protocol details are summarized in Table 5 below.

Table 5. Treatment groups and protocol. TRIA-662 indicate 1-MNA. BID = b.i.d. - twice per day; QD = q.d. - once per day.

^*Vehicle, 1 -MNA, and fluticasone Propionate were administered intratracheally at a dose volume of 0.5 ml/kg. Animals in Group 5 were dosed at a dose volume of 0.33 mL/kg from the Day 1 PM dose onwards. Animals in Group 7 were dosed at a dose volume of 0.25 mL/kg from Day 7 onwards

^** Nintedanib and Dexamethasone were administered orally at a dose volume of 10 mL/kg.

+ Dose and concentration expressed as mg of active pharmaceutical ingredient

++ Dose volume was 1 mL/kg for intratracheal challenge for animals weighing under 250 g on the day of bleomycin dosing, for animals 250 g or greater a fixed volume of 0.25 mL (0.75 I.U) was administered.

On Day 3, 8 animals from each group were sacrificed by an overdose of pentobarbitone administered by the intraperitoneal route and bronchoalveolar lavages were collected through a tracheal cannula, by washing the airways 3 times with 3 mL of phosphate buffer saline. First lavage aliquot was separated and kept on ice (Tube A). The latter two BAL fluid aliquots were pooled and kept on ice (Tube B). The BAL fluid samples were centrifuged at 800 g for 10 min in 4°C. Supernatant from Tube A was retained and kept in -80°C for cytokine analysis. Cell pellets in both A and B were resuspended in 5 mL PBS and kept on ice for cell count. A total and differential cell count of the BAL cells was performed using the XT-2000iV (Sysmex UK Ltd). The samples were vortexed for approximately 5 seconds and analyzed. A total and differential cell count (neutrophils, eosinophils, mononuclear cells (includes monocytes and macrophages) and lymphocytes) were reported as number of cells per animal. Fluid supernatants were analyzed for cytokine levels (MIP-1a, I L-1 b, IL-6, IL-17A, MCP-1 , and TNF- a).

On Day 21 remaining animals were anaesthetized using a mix of 100 mg/mL ketamine, 1 mg/mL medetomine and water for injection with a final ration of 2.4:1 :9 dosed at 4 mL/kg via the intraperitoneal route. Once a surgical plane of anesthesia was reached, animals were tracheotomized and transferred to a plethysmograph and a forced maneuvers lung function procedure was performed using eSpira Forced Maneuvers system for the rat (EMMS). Approximately 3 maneuvers were performed, and values obtained for each animal for FEV100, (Forced Expiratory Volume in 100 msecs), FVC (Forced Vital Capacity) and PEF (Peak Expiratory Flow). FVC (forced vital capacity) is the maximum amount of air that can be exhaled when blowing out as fast as possible. FEV (forced expiratory volume) is the volume of air that can forcibly be blown out in first 100 msecs, after full inspiration. PEF (peak expiratory flow) is the maximal flow exhaled from the lungs when blowing out at a steady rate. The values for each animal were averaged and group means derived.

Following the forced maneuvers procedure, animals were placed on a rodent ventilator. Various parameters were measured using the FlexiVent system, such as resistance, compliance, and elastance.

The animals were terminated, at which time blood samples were collected. Levels of C-reactive protein in serum were measured with ELISA. BAL lavages were collected and processed as described above. Additionally, TGF-b was determined in BAL fluid.

Following completion of BAL fluid collection procedure, lungs were excised and weighed. The right lung lobes were dissected away. The left lung was infused in 10% neutral buffered formalin for a minimum of 24 hours an tissues were processed to paraffin blocks. 4-5 pm sections were stained with haematoxylin and eosin or masons trichrome (enabling differentiation of cells from connective tissue deposits). Light microscope analysis included semi-quantitative assessment (grading) of inflammatory cell infiltrate and tissue inflammation. The right lung was snap frozen in liquid nitrogen prior to lung hydroxyproline assessment. For measurement, the frozen samples were homogenized in distilled water at a ratio of 1 ml_: 1 g of lung tissue. Following homogenization, samples were hydrolyzed and analyzed for levels of hydroxyproline using a commercially available hydroxyproline assay kit from Sigma Aldrich cat. no. MAK008. The concentration of hydroxyproline in each sample was derived from a standard curve using the formula below:

C = S_a /Sv (pg/ pL) where,

S_a = Amount of hydroxyproline in unknown sample (pg) from standard curve S_v = Sample volume (pL) added into the wells C = Concentration of hydroxyproline in samples

Lung hydroxyproline levels are expressed in pg per whole lung. Individual values were averaged to derive mean values for each treatment group.

Results

Fig. 15 shows the obtained cell counts in BAL (bronchoalveolar lavage) after 3 days. It should be noted that all treatments, not just intratracheal administered treatments, showed elevated total white blood cell, neutrophil, monocyte, and lymphocyte counts relative to placebo.

While bleomycin alone was associated with significant elevation in eosinophils in the bronchoalveolar fluid, all treatments showed reduced eosinophil counts, with more pronounced effects from the 1-MNA treatments, fluticasone, and dexamethasone. The lower doses of 1 -MNA seem to be more effective in lowering BAL eosinophils than nintedanib, which is currently approved for the treatment of pulmonary fibrosis. 1-MNA lowered eosinophils similarly to fluticasone and dexamethasone, despite not being a corticosteroid.

Eosinophils accumulate at sites of allergic inflammation, where they release a number of inflammatory mediators, such as radical oxygen species, cytokines, and lipid mediators. Eosinophils also produce TGF-b, which may contribute to airway fibrosis, having substantial influence on pulmonary fibrosis and other severe respiratory diseases. Therefore the strong reduction in eosinophils by 1-MNA is an encouraging characteristic as it demonstrates the likely benefit of this compound in respiratory diseases.

Fig. 16 shows the obtained cell counts in BAL (bronchoalveolar lavage) after 21 days. The effect of 1-MNA is even more pronounced, in particular for eosinophils (see Fig. 16E).

Fig. 17 summarizes the measured lung function parameters after 21 days. 1-MNA is shown to have a particularly visible effect on compliance, with the dose 75 mg/kg providing values not only improved in comparison to bleomycin without treatment but also comparable to vehicle treated (non-fibrotic) lung.

Fig. 18 shows the determined cytokine levels in BAL after 3 days of treatment. 1-MNA treatment even after only 3 days caused a marked decrease in proinflammatory cytokines and in particular reduced MIP-1a (with a stronger effect than dexamethasone) and IL-1 b. In the utilized model of lung fibrosis, bleomycin induces NLRP3 inflammasome activation via HIF-1a, which in turn induces IL-1 b (as visible on Fig. 18A; bleomycin only displays a large increase in this cytokine). 11-1 b is then effectuating an increase in TGF-b, which is one of the key signaling molecules associated with fibrotic alterations in lungs (see e.g. Alyaseer et al. The Role of NLRP3 Inflammasome Activation in the Epithelial to Mesenchymal Transition Process During the Fibrosis. Frontiers in Immunology 11/2020, 883). The capability of 1-MNA to decrease IL-1 b are therefore indicative of usefulness of the compound to interrupt this pathway and to counteract lung fibrosis. This is further supported by results obtained after 21 days (see Fig. 19), which shows that 1-MNA treatment also reduces TGF-b levels (Fig. 19A). An effect on C- reactive protein is also visible, indicating a persisting anti-inflammatory activity. These results are consistent with an observed decrease in neutrophil and eosinophil count (Fig. 16), as these two cell types are observed in larger numbers after inflammasome activation (

1 -MNA is indeed capable of reducing lung fibrosis. Fibrosis can be defined as an accumulation of fibrous connective tissue, in particular extracellular matrix (ECM) components such as type I collagen and fibronectin. This process is the end result of chronic inflammatory processes, such as induced via inflammasome activation. Bleomycin challenge induces fibrotic lesions which are evident at day 21 post injury and there is a difference in degree of fibrosis when comparing the bleomycin-only treated organs with bleomycin with 1-MNA. 1-MNA treatment not only provides significant anti-inflammatory activity already after 3 days of treatment (and at a level comparable or better then nintedanib), but is also associated with a smaller degree of fibrotic lesions after 21 days.

Claims

1. 1-methylnicotinamide (1-MNA) or a pharmaceutically acceptable salt thereof for use in a method of preventing or treating a disease associated with an inflammatory reaction in the airways or with a defective epithelial barrier in the airways, wherein the disease is selected from allergic diseases or diseases associated with an infection.

2. 1-methylnicotinamide (1-MNA) or a pharmaceutically acceptable salt thereof for use according to claim 1 , wherein the disease is associated with a viral infection in the airways.

3. 1-methylnicotinamide (1-MNA) or a pharmaceutically acceptable salt thereof for use according to claim 2, wherein the viral infection is an infection with Sars-CoV-2.

4. 1-methylnicotinamide (1-MNA) or a pharmaceutically acceptable salt thereof for use according to claim 2, wherein the viral infection is an infection with an influenza virus.

5. 1-methylnicotinamide (1-MNA) or a pharmaceutically acceptable salt thereof for use according to claim 1 , wherein the disease is an allergic disease.

6. 1-methylnicotinamide (1-MNA) or a pharmaceutically acceptable salt thereof for use according to claim 5, wherein the allergic disease is asthma.

7. 1-methylnicotinamide (1-MNA) or a pharmaceutically acceptable salt thereof for use according to any of the preceding claims, wherein 1 -MNA or a pharmaceutically acceptable salt thereof is administered to prevent, alleviate or eliminate at least one of the following: coughing, throat ache, shortness of breath or difficulty breathing or dyspnoea, inflammation in the respiratory tract, especially lungs, fibrosis in lungs symptoms and/or symptoms related to muscles, especially myopathies, muscle fatigue, fatigue or muscle pains, and delirium, especially intensive care unit delirium (ICU delirium), postoperative delirium (POD delirium) and delirium in Covid-19.

8. 1-methylnicotinamide (1-MNA) or a pharmaceutically acceptable salt thereof for use according to any of the preceding claims, wherein 1-MNA or a pharmaceutically acceptable salt thereof is administered via an inhalation or intranasal route.

9. 1-methylnicotinamide (1-MNA) or a pharmaceutically acceptable salt thereof for use according to any of the preceding claims, wherein the disease is associated with a viral infection in the airways, and 1-MNA or a pharmaceutically acceptable salt thereof is administered in order to prevent, alleviate or eliminate viral myositis, muscle pain, myopathy and/or fatigue.

10. 1-methylnicotinamide (1-MNA) or a pharmaceutically acceptable salt thereof for use according to any of the preceding claims, wherein the disease is associated with eosinophilia.

11. A method of treatment of a disease associated with an inflammatory reaction in the airways in a subject or with a defective epithelial barrier in the airways in a subject, wherein the disease is selected from allergic diseases or diseases associated with an infection, wherein the subject is administered with 1 -MNA or a pharmaceutically acceptable salt thereof.

12. The method of claim 11 , wherein the disease is associated with a viral infection in the airways.

13. The method of claim 12, wherein the viral infection is an infection with Sars-CoV-2 or with an influenza virus.

14. The method of claim 11 , wherein wherein the disease is an allergic disease.

15. The method of claim 14, wherein the allergic disease is asthma.

16. The method of claim 11 , wherein 1 -MNA or a pharmaceutically acceptable salt thereof is administered to prevent, alleviate or eliminate at least one of the following: coughing, throat ache, shortness of breath or difficulty breathing or dyspnoea, inflammation in the respiratory tract, especially lungs, fibrosis in lungs symptoms and/or symptoms related to muscles, especially myopathies, muscle fatigue, fatigue or muscle pains, and delirium, especially intensive care unit delirium (ICU delirium), postoperative delirium (POD delirium) and delirium in Covid-19.

17. The method of claim 11 , wherein 1 -MNA or a pharmaceutically acceptable salt thereof is administered via an inhalation or intranasal route.

18. The method of claim 11 , wherein the disease is associated with a viral infection in the airways, and 1-MNA or a pharmaceutically acceptable salt thereof is administered in order to prevent, alleviate or eliminate viral myositis, muscle pain, myopathy and/or fatigue.

19. The method of claim 11 , wherein the disease is associated with eosinophilia.