Technical Field

The present invention relates to the ability of vinyldithiins, analogues and derivatives thereof to regulate sodium transport and fluid homeostasis and to treat of disorders with symptoms related to elevated sodium transport and difficulties associated with mucociliary clearance.

Background

Epithelial sodium channel (ENaC) and the basolateral Na ⁺ /K ⁺ -ATPase solute pump regulate the flow of sodium ions (Na ⁺ ) across the cell membrane and controls fluid homeostasis in multiple organs of the body, including the colon, kidney,

gastrointestinal tract and the lung. In the lung, the transport of sodium ions is specifically important for maintaining airway surface liquid (ASL) depth and viscosity, allowing optimal gas exchange and mucociliary clearance to reduce long term infection. Dysregulation of ion transport and related fluid balance has been reliably implicated in multiple diseases and conditions, including cystic fibrosis, hypertension, Liddle's disease, pseudoaldosteronism type ι and diabetes.

The movement of ions and water across epithelial cell membranes is tightly regulated by multiple selective ion channels, including apical located CI ^" and Na ⁺ channels and the basolateral K ⁺ and Na ⁺ transporters. Cystic fibrosis transmembrane conductance regulator (CFTR) is a c-AMP activated ATP-binding cassette (ABC) transporter channel which controls the passive diffusion of chloride ions (CI ) and other anions across the apical membrane down their electrochemical gradient. The flow of CI ^" out of the cell is passively followed by Na ⁺ . This movement of ions creates an osmotic gradient which promotes the movement of water across the epithelium and hydration of the ASL. Ion channels and transporters situated on the basolateral membrane, including Na ⁺ /K ⁺ - ATPase control the movement of ions from the interstitial space into the cell and vice versa.

Active transport of Na ⁺ from the ASL prevents hypersecretion into the airways and occurs via the apical located ENaC, down an electrochemical gradient into the cell. The electrochemical gradient is maintained by the co-ordinated function of basolateral Na ⁺ and K ⁺ channels and apical CI ^" channels. Dysregulation of ENaC and sodium

absorption is known to cause severe lung dysfunction, either due to excessive fluid build-up, or to dehydration of the airway surface liquid and increasingly viscous mucus which is difficult to remove.

Liddle's disease is caused by mutations in ENaC which alter the regulatory mechanisms controlling channel endocytosis and degradation, resulting in increased channel expression and activity. It is an autosomal dominant disorder which leads to increased Na ⁺ transport across the epithelium, severe high blood pressure and hypertension.

Less extreme alterations in ENaC function and Na ⁺ absorption are also linked to occurrences of high blood pressure, dry eye syndrome, hypertension and kidney related problems in people who do not have genetic mutations associated with specific diseases. The sensitivity of multiple systems and the potential severity of symptoms associated with ENaC dysregulation suggest that multiple mechanisms keep channel function under tight regulation.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene which affect the expression of functioning channels at the cell membrane and Ck movement. The alteration of Ck movement has an effect on the amount of Na ⁺ which passively flows into the ASL. The reduced function of CFTR has also been associated with an increased activity of ENaC and related Na ⁺ hyperabsorption. The combined dysregulation of Ck and Na ⁺ movement leads to increasingly dehydrated airways and viscous mucus that is more prone to harbouring long-term infections. Current treatments of cystic fibrosis focus on limiting and treating the lung damage caused by thick mucus and infection. Antibiotics are used to treat lung infections but are also frequently administered prophylactically. Mechanical devices and inhaled medications (such as dornase alpha) are used to alter and clear the thickened mucus. Ivacaftor (trade name Kalydeco) is a "potentiator" of CFTR and is used for the treatment of cystic fibrosis in people having one of several specific mutations in the CFTR protein.

Accordingly, there is a need for compositions affecting sodium current or fluid homeostasis, or compositions that have mucolytic properties.

Summary According to a first aspect of the invention, a pharmaceutical composition is provided comprising a vinyldithiin or an analogue or derivative thereof, for use in therapy to reduce sodium ion uptake in the cell. In some embodiments, the reduction in sodium ion uptake in cells improves fluid homeostasis and/or improves mucociliary clearance.

In some embodiments, the activity of an epithelial sodium channel (ENaC) and/or a Na ⁺ /K ⁺ -ATPase is modulated to reduce sodium ion uptake in the cell.

In some embodiments, the modulation of the activity of the ENaC comprises modulating expression of one or more genes encoding one or more subunits of the ENaC. In some embodiments, the expression of genes encoding one or more of subunits of the ENaC is modulated by the inhibition of a mineralocorticoid receptor. In some embodiments the expression of genes encoding one or more of subunits of the ENaC is modulated by the inhibition of the glucocorticoid receptor. In some embodiments, the composition is for regulating the osmolarity of the periciliary fluid of the lung of a patient. In some embodiments, the osmolarity of the periciliary fluid of the patient is regulated by reducing the absorption of sodium ions into the cell. In some embodiments, the composition is for enhancing mucociliary clearance in the lung of a patient.

In some embodiments, the composition is for treating cystic fibrosis. In some embodiments, the composition is for treating one or more conditions selected from the group consisting of: hypertension, congestive heart failure, Liddle's syndrome, pseudoaldosteronism type 1, diabetes, dry eye syndrome, renal dysfunction, cirrhosis, hypokalemia, bronchitis, bronchiectasis, asthma, Chronic Pulmonary Obstructive Disorder (COPD), Sjorgen's syndrome, corticosteroid respiratory related diseases, alkalosis and Cushing Syndrome. In some embodiments, the composition further comprises one or more additional active agents.

In some embodiments, the one or more additional active agent is an antibiotic. In some embodiments, the antibiotic is a tobramycin, a ciprofloxacin and/or a combination thereof.

In some embodiments, the one or more additional active agent is an agent for thinning the mucus in the lungs. In some embodiments, the agent is selected from the group consisting of dornase alfa, saline, mannitol dry powder and mannitol solution.

In some embodiments, the one or more additional active agent is a potentiator of the CFTR channel, such as Ivacaftor or Lumacaftor.

In some embodiments, the one or more additional active agent opens an alternative chloride channel to CFTR, such as denufosol.

In some embodiments, the one or more additional active agent is a bronchodilator. In some embodiments, the one or more bronchodilator is selected from the group consisting of: beta-2 agonists, such as salbutamol, salmeterol, formoterol and vilanterol; anticholinergics, such as ipratropium, tiotropium, aclidinium and glycopyrronium; and theophylline.

In some embodiments, the one or more additional active agent is a corticosteroid. In some embodiments, the one or more corticosteroid is selected from the group consisting of: beclometasone, betamethasone, budesonide, flunisolide, fluticasone, mometasone and triamcinolone.

In some embodiments, the composition is for administration to the lung.

In some embodiments, the composition is a dry powder.

In some embodiments, the composition is a solution or suspension.

In some embodiments, a dose of the composition comprises from about o.oi mg to about 1500 mg vinyl dithiin per kg of subject body weight per day administered in one or more administrations. In some embodiments, the dose of the composition comprises from about o.i mg to about 50 mg per kg of subject body weight per day, administered in one or more administrations. According to a second aspect of the invention, a method of reducing sodium ion uptake in cells is provided, comprising administering a pharmaceutical composition comprising a vinyldithiin or an analogue or derivative thereof.

In some embodiments, the method comprises administering a composition as defined in embodiments of the first aspect of the invention.

Brief Description of the Drawings

Embodiments of the invention will now be described, by way of example only, with reference to the drawings in which:

Figure 1 shows data reflecting the change in sodium current following the application of 2-vinyl"4H-i,3-dithiin to a three-dimensional human epithelial cell model.

Figure 2 shows data reflecting the change in sodium channel activity following the application of 2-vinyl-4H-i,3-dithiin in terms of the average of delta Isc.

Figure 3 is a graph showing the evaluation of the IC50 of 2-vinyl-4H-i,3-dithiin based on results from modified Ussing chambers.

Detailed Description

The present invention relates to compositions comprising vinyldithiins or analogues or derivatives thereof, for reducing sodium ion uptake in cells. More specifically, the vinyldithiin or analogue or derivatives may modulate the activity of an epithelial sodium channel (ENaC) and/or the activity of Na ⁺ /K ⁺ -ATPase solute pump, to reduce sodium ion uptake in a cell. Vinyldithiins have previously been investigated as candidates for preventing

cardiovascular disease and as antioxidants. Divalent organosulfur compounds, such as vinyldithiins, are known to reduce oxidative stress via a radical process, which would be relevant to the protection of the cardiovascular system. Vinyldithiins from garlic have been shown to significantly lower the incidence of platelet aggregation. Reduction of platelet aggregation may result in reduction of myocardial infarction or ischemic stroke. Vinyldithiins can release small but biologically relevant amounts of hydrogen sulfide through a hydrolytic process and it is known that hydrogen sulphide has cardioprotective effects in cases of ischemia related to its effects as an endogenous messenger molecule, primarily via its reduction of the rate of cellular metabolism.

It has now been discovered that vinyldithiins are modulators of sodium channel activity and can therefore be used to reduce sodium ion uptake in a cell. It has also been discovered that vinyldithiins may be used as promoters of mucociliary transport. The administration of a vinyldithiin has been shown to have effects on signalling pathways that regulate ENaC and Na ⁺ /K ⁺ -ATPase solute pump. As a result, vinyldithiins may be used for the treatment of disorders associated with dysregulation of sodium transport and mucociliary clearance difficulties. Analogues and derivatives

Vinyldithiins include 3-vinyl-4H-i,2-dithiin and 2-vinyl-4H-i,3-dithiin. They are organosulfur phytochemicals formed in the breakdown of allicin from crushed garlic {Allium sativum). In some embodiments, the present invention relates to 2-vinyl-4H-i,3-dithiin.

As shown below, allicin (l) decomposes into: 2-propenesulfenic acid (2) which reverts to allicin; and thioacrolein (3) which gives rise to Diels-Alder dimers 3-vinyl-4H-i,2- dithiin (4) and 2-vinyl-4H-i,3-dithiin (5).

Vinyldithiins can be derived from allicin under specific conditions. Vinyldithiins for use in the present invention may be natural, synthetic or semi-synthetic. As used herein, vinyldithiin analogues and derivatives refer to compounds that are functionally equivalent to vinyldithiins, and, in some embodiments, functionally equivalent to 2-vinyl-4H-i,3-dithiin. Examples of vinyldithiin analogues and derivatives include:

A B where Ti, T2, T3, T4 and T5 independently from each other represent CH ₃ , CH2-R ¹ , CH— O— R ¹ , or C=0, with R ¹ is selected from a group consisting of a hydrogen, a straight-chain or branched-chain alkyl group having 1 to 12 carbon atoms, a cycloalkyl group having 3 to 10 carbon atoms and optionally comprising one or more ring heteroatoms, a substituted or unsubstituted aryl group with 6 to 12 ring atoms which can comprise one or more ring heteroatoms which may form a bicyclic compound, a substituted or unsubstituted C0-C12- aryl-Ci- C ₆ - alkyl group which can comprise one or more heteroatoms, a substituted or unsubstituted aryloxy group wherein aryl has 6 to 12 ring atoms and can comprise one or more ring heteroatoms, a (— C(O)— Ra) group, a (-C(S)-Ra) group, a (-C(O)-ORa) group, a (-(CH ₂ ) _n -CRa=CRaRb) group, or a (- C(O)— (CH ₂ )n— CRa=CRaRb) group wherein n is zero or an integer from 1 to 12 and Ra and Rb independently from each other are selected from a group consisting of hydrogen, a straight-chain or branched-chain alkyl group having 1 to 6 carbon atoms that is unsubstituted or substituted by hydroxy, a cycloalkyl group having 3 to 10 carbon atoms and optionally comprising one or more ring heteroatoms, and a substituted or unsubstituted aryl group with 6 to 12 ring atoms which can comprise one or more ring heteroatoms, and a substituted or unsubstituted C0-C12- aryl-Ci- C6-alkyl group which can comprise one or more heteroatoms.

In a preferred embodiment, the vinyldithiin analogue or derivative is:

C D wherein X is a carbon or a heteroatom selected from a group consisting of an oxygen, a sulphur, an alkyl, an aryl, a substituted nitrogen and substituted phosphorus. The purity of the vinyldithiin analogues or derivatives herein may be in the range of, for example, about 50% to about 100%, or, for example, about 60% to about 80%, or, for example, about 70% to about 90%, or, for example, about 90% to about 100%.

Sodium transport

ENaC facilitates Na ⁺ absorption across the apical membrane of epithelia in the distal nephron, respiratory and reproductive tracts and exocrine glands, and so ENaC is primarily found in the epithelial cells of the kidney, lungs and colon. The Na ⁺ concentration affects extracellular fluid osmolality and consequently the movement of fluids, changing fluid volume and blood pressure. In the lung, ENaC is located along the entire length of the cilia and regulates the osmolarity of the periciliary fluid and its function is essential to maintain fluid volume at a depth necessary for the motility of the cilia and clearing of the mucosal surface.

Na ⁺ /K ⁺ -ATPase is an enzyme located in the basolateral membrane that pumps sodium ions and potassium ions across the membrane against their concentration gradient. It is an active solute pump that primarily functions to move 3 sodium ions out of the cell, for every 2 potassium ions it pumps in. This creates an electrochemical gradient within the cell and regulates the transport of sodium. Vinyldithiins have been shown to inhibit sodium current in a dose-dependent manner. Sodium absorption across the epithelium regulates fluid secretion onto the apical surface. Tightly regulated sodium flux is required to maintain an optimal level of airway hydration in the lung and allow mucociliary clearance without hypersecretion of fluid. It has also been demonstrated that vinyldithiins, analogues and derivatives modulate activity of the ENaC by modulating expression of one or more genes encoding one or more subunits of the ENaC on the cell membrane. It has been shown that vinyldithiins, analogues or derivatives thereof modulate the activity of the ENaC as a result of inhibiting mineralocorticoid receptors (MRs).

Aldosterone acts through MRs to increase the expression of individual subunits of ENaC, as well as related regulatory proteins, through direct binding of MRs to regulatory elements in gene promoter regions. Activation of MRs by aldosterone has been shown to increase the expression of serine/threomne-protem Idnase (SGKi) which inhibits ubiquitination by Nedd4-2 and degradation of ENaC from the channel membrane. Activation has further been found to increase the expression of ENaC subunits by antagonising the methylation of H3K79 of ocENaC. It has also been shown that vinyldithiins, analogues or derivatives thereof modulate the activity of the ENaC as a result of inhibiting the glucocorticoid receptor (GR). GR activates ENaC by increasing expression of subunits of ENaC through similar mechanisms as MR, and GILZ, which inhibits the activation of ERK1/2, the activation of Nedd4-2 and reduces the degradation of SGKi.

Vinyldithiins, analogues or derivatives have been shown to increase mucociliary clearance.

Vinyldithiins, analogues or derivatives thereof have been shown to increase cilia beat frequency.

Mucociliary clearance

As discussed above, epithelial sodium channels regulate the fluid secretion onto the apical surface that in the lung directly controls airway surface liquid height and the rate of mucociliary clearance. Subjects with pulmonary disorders, including cystic fibrosis, have a drastically reduced rate of mucociliary clearance, leading to the establishment of chronic bacterial infections that are difficult to clear. Persistent lung infections are the leading cause of death in people with cystic fibrosis, with 80-90% of patients developing respiratory failure due to bacterial infection and airway inflammation. Vinyldithiin, such as 2-vinyl-4H-i,3-dithiin, was tested on a 3D epithelial cell model, derived from bronchial cells from patients with cystic fibrosis (MucilAir™-CF, from Epithelix), to establish effects on the rate of mucociliary transport. It was demonstrated that vinyldithiin increases the rate of mucociliary clearance in a patient-derived 3D epithelial cell model 24 hours after administration, compared to no treatment controls. Vinyldithiin increases the ciliary beat frequency in the same 3D cell model. Disease applications

The effects of vinyldithiins on ion transport, fluid homeostasis and mucociliary clearance indicate a novel benefit of vinyldithiins and analogues and derivatives thereof for treatment of disorders related to dysregulated ion transport and related mucociliary clearance deficits.

The activity of vinyldithiins and analogues and derivatives thereof indicates that these compounds may be used in the treatment of a number of diseases, disorders and conditions which are directly or indirectly associated with local or systemic imbalance in Na ⁺ and water homeostasis, and/or with increased ENaC activity.

In cystic fibrosis and related disorders, there is an elevated Na ⁺ current across the epithelium as a result of the malfunctioning CFTR protein, with increased absorption of Na ⁺ leading to dehydration of the apical membrane. Vinyldithiins, analogues and derivatives thereof are effective in reducing the physiological symptoms of cystic fibrosis that are caused by dysregulated ion transport, for example, bronchiectasis.

Increased or enhanced ENaC function leads to several forms of hypertension, including but not limited to salt-sensitive hypertension, obesity-related hypertension as well as Liddle's syndrome, pseudoaldosteronism Type 1, and diabetes. These may therefore also be treated by administration of vinyldithiins, analogues and derivatives thereof.

Vinyldithiins, analogues and derivatives thereof may also be useful in treating congestive heart failure, renal dysfunction, cirrhosis, hypokalemia, bronchitis, bronchiectasis, asthma, Chronic Pulmonary Obstructive Disorder (COPD), Sjorgen's syndrome, dry eye syndrome and corticosteroid respiratory related diseases such as chronic bronchitis and emphysema, alkalosis and Cushing Syndrome. Formulations & Administration

The pharmaceutical composition may take the form, for example, of solid preparations including tablets, capsules, dragees, lozenges, granules, powders, pellets and cachets; and liquid preparations including suspensions, sprays, emulsions and solutions.

In some embodiments of the invention, the pharmaceutical composition comprising a vinyldithiin or an analogue or derivative thereof is intended for local administration. In some embodiments, the local administration is to the lung. This allows the vinyldithiin, analogue or derivative to be directly administered to the lung epithelia where the ENaC is located.

In some embodiments, the composition may be suitable for administration by inhalation. In some embodiments, the composition is a dry powder. Such

compositions may be administered using a dry powder inhaler. In other embodiments, the composition is a solution or suspension. Such compositions may be administered using a pressurised metered dose inhaler or the like.

In some embodiments of the invention, the pharmaceutical composition comprising a vinyldithiin or an analogue or derivative thereof is intended for systemic

administration. In some embodiments, the systemic administration is, for example, suitable for oral, nasal, suppository, intravenous or intradermal application.

In some embodiments, a therapeutically effective dose of the composition comprises from about o.oi mg to about 1500 mg vinyldithiin per kg of subject body weight per day, administered in one or more doses over the course of a day (24 hours). In some embodiments, which may be preferred, the therapeutically effective dose of the composition comprises from about 0.1 mg to about 50 mg vinyldithiin per kg of subject body weight per day, administered in one or more doses over the course of a day (24 hours). The vinyldithiin may be 2-vinyl-4H-i,3-dithiin.

In some embodiments, the composition comprises vinyldithiin, such as 2-vinyl-4H-i,3- dithiin, in a concentration of from about 0.01 to about 500 μΜ vinyldithiin, or from about 0.05 to about 100 μΜ vinyldithiin, or from about 0.05 to about 200 μΜ vinyldithiin. In some embodiments, which may be preferred, the composition comprises vinyldithiin in a concentration of from about 0.50 μΜ to about 200 μΜ vinyldithiin.

The pharmaceutical compositions may include pharmaceutically acceptable carriers and excipients such as diluents, adjuvants, vehicles, fillers, binders, disintegrating agents, wetting agents, emulsifying agents, suspending agents, perfuming agents, buffers, dispersants, thickeners, solubilising agents, lubricating agents and dispersing agents. Use of such carriers and excipients are commonly known in the field of the art, for example, but not limited to, colloidal silicon dioxide, cellulose, magnesium stearate, titanium dioxide, Sepineo™ P600, cyclodextrin and the like.

In some embodiments of the invention, in addition to a vinyldithiin or an analogue or derivative thereof, the compositions further comprise one or more active agents. The additional active agent may have the effect of modulating the activity of ENaC, for example amiloride. Alternatively or in addition, the further active agent may modulate the activity of another epithelial ion channel, such as a Ck channel, for example CFTR. As such, the additional active agent may be Ivacaftor or Lumicaftor, which potentiates CFTR, or it may open an alternative chloride channel, such as denufosol.

In some embodiments, the one or more additional active agent is an agent for thinning the mucus in the lungs. For example, the agent may be selected from the group consisting of dornase alfa, saline, mannitol dry powder and mannitol solution. In some embodiments, the saline solution is a hypertonic, a hypotonic or an isotonic saline. In some embodiments, the saline solution has a concentration range of from about 0.1% to about 12%.

In some embodiments, the one or more additional active agent is an antibiotic. Classes of antibiotics include aminoglycosides, ansamycins, carbacephem, carbapenems, cephalosporins, glycopeptides, lincosamides, macrolides, monobactams, penicillins, polypeptides, quinolones, sulfonamides and tetracyclines.

For example, the antibiotic may be selected from the group consisting of: Ampicillin, Bacampicillin, Carbenicillin indanyl, Mezlocillin, Piperacillin, Ticarcillin, Amoxicillin- clavulanic acid, Ampicillin-sulbactam, Benzylpenicillin, Cloxacillin, Dicloxacillin, Methicillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin Tazobactam, Ticarcillin Clavulanic acid, Nafcillin, Cefadroxil, Cefazolin, Cephalexin, Cephalothin, Cephapirin, Cephradine, Cefaclor, Cefamandole, Cefonicid, Cefotetan, Cefoxitin, Cefprozil,

Cefmetazole, Cefuroxime, Loracarbef, Cefdinir, Ceftibuten, Cefoperazone, Cefixime, Cefotaxime, Cefpodoxime proxetil, Ceftazidime, Ceftizoxime, Ceftriaxone, Cefepime, Azithromycin, Clarithromycin, Clindamycin, Dirithromycin, Erythromycin,

Lincomycin, Troleandomycin, Cinoxacin, Ciprofloxacin, Enoxacin, Gatifloxacin, Grepafloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, Oxolinic acid, Gemifloxacin, Perfloxacin, Imipenem Cilastatin, Meropenem, Aztreonam, Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Streptomycin, Tobramycin, Paromomycin, Teicoplanin, Vancomycin, Demeclocycline, Doxycycline, Methacycline, Minocycline,

Oxytetracycline, Tetracycline, Chlortetracycline, Mafenide, Silver Sulfadiazine, Sulfacetamide, Sulfadiazine, Sulfamethoxazole, Sulfasalazine, Sulfisoxazole,

Trimethoprim-Sulfamethoxazole, Sulfamethizole, Rifampin, Rifabutin, Rifapentine, Linezolid, Quinupristin Dalfopristin, Bacitracin, Chloramphenicol, Fosfomycin, Isoniazid, Methenamine, Metronidazole, Mupirocin, Nitrofurantoin, Nitrofurazone, Novobiocin, Polymyxin, Spectinomycin, Trimethoprim, Colistin, Cycloserine,

Capreomycin, Ethionamide, Pyrazinamide, Para-aminosalicylic acid, Fluoroquinolone and Erythromycin ethylsuccinate.

In some embodiments, the one or more additional active agent is a bronchodilator. For example, the bronchodilator may be selected from the group consisting of: beta-2 agonists, such as salbutamol, salmeterol, formoterol and vilanterol; anticholinergics, such as ipratropium, tiotropium, aclidinium and glycopyrronium; or theophylline.

In some embodiments, the one or more additional active agent is a corticosteroid. For example, the corticosteroid may be selected from the group consisting of:

beclometasone, betamethasone, budesonide, flunisolide, fluticasone, mometasone, and triamcinolone.

The invention will now be described in detail by way of reference only to the following non-limiting examples. Experimental The experiments described below were carried out using 2-vinyl-4H-i,3-dithiin having more than 95% purity.

1. Extraction of 2-vinyl-4ff-i,3-dithiin

Crude product containing ajoene and vinyl dithiin was obtained using the method of

Thereafter, in order to extract 2-vinyl-4H-i,3-dithiin from the crude product, 20 ml water was added to approximately 2 g of the crude product, which was then transferred into a separation funnel. Hexane was used to then extract the organic extract. The collected organic extract was then dried over MgS0 ₄ followed by evaporation and then further purified by reverse phase automated flash chromatography.

The reverse phase automated flash chromatography conditions for isolating 2-vinyl- 4H-i,3-dithiin were:

The purified fraction of 2-vinyl-4H-i,3-dithiin will start eluting at +/-6.5 minutes and stop around +/-14 minutes. The purified fractions were collected and the organic phase (Acetonitrile) was evaporated at mild (100 bar, 33°C) conditions. The aqueous phase (water) was extracted with diethyl ether, where the collected organic extract was then dried over MgS0 ₄ followed by evaporation of the solvent under ambient conditions (350 bar, 24°C) to obtain pure product of 2-vinyl-4H-i,3-dithiin.

2. Dose dependent inhibition of Na ⁺ channel activity with vinyl dithiin

Methods

2-Vinyl"4H-i,3-dithiin was tested on a 3D epithelial cell model (MucilAir™, from Epithelix) to establish effects on Na ⁺ channel activity.

MucilAir™ is a fully differentiated three-dimensional in vitro cell model of the human airway epithelia. Epithelial cells were freshly isolated from nose and bronchi biopsies from a healthy donor, and then seeded onto a semi-porous membrane (Costar

Transwell, pore size 0.4 μπι). After approximately 45 days of culture at air-liquid interface, the epithelia were fully differentiated, both morphologically and functionally. Ussing chamber measurements were performed on the MucilAir™ in quadruplicate, at ten different concentrations of 2-vinyl-4H-i,3-dithiin (from 19.6 μg/ml up to 195.2 μg/ml) using sequential addition. Amiloride was used as a positive control. The change in sodium current was recorded following each addition of 2-vinyl-4H-i,3- dithiin.

Results

Sequential addition of 2-vinyl-4H-i,3-dithiin to the Ussing chamber solution resulted in a delayed dose-dependent reduction in sodium current. The average of delta Isc (increase in short-circuit current) shown in Figure 2 was calculated from Ussing chamber recording traces from 6 inserts (shown in Figure 1) with sequential application of 2-vinyl-4H-i,3-dithiin across 100 minutes, reflecting change in sodium ion channel activity following drug treatment. The graph in Figure 3 shows the evaluation of the IC50 of 2-vinyl-4H-i,3-dithiin based on results from modified Ussing chambers. IC50 was calculated as being 108.3 ± 4·ΐ μδ/ητΐ.

Conclusions

The results indicate that the administration of vinyldithiin to epithelial cells from healthy subjects leads to a change in the Na ⁺ channel activity. This change in channel activity is due to vinyldithiin inhibiting the activity of ENaC present in the MucilAir™ cells. The data indicates a dose-dependent effect of vinyldithiin. 3. Dithiin increases mucociliary clearance

Methods

2-Vinyl-4H-i,3-dithiin was tested on a 3D epithelial cell model in an air-liquid interface, derived from bronchial cells from patients with cystic fibrosis (MucilAir™- CF, from Epithelix), to establish effects on the rate of mucociliary transport.

MucilAir™-CF (AF5o8 homozygous) were cultured for a minimum of 45 days as described above. Each Transwell cell culture insert was washed apically with

MucilAir™ culture medium 3 days before the experiment.

This washing step removes accumulated mucus and minimizes the risks of interference. Trans-Epithelial Electrical Resistance (TEER) was measured before and after exposure to compounds to verify that all the selected inserts satisfy quality control, with appropriate formation of tight junctions and barrier function.

2-Vinyl-4H-i,3-dithiin had no negative effect on the viability of the epithelium at concentrations tested.

Inserts were placed into culture medium containing 80 or 160 μg/ml 2-vinyl-4H-i,3- dithiin. 30 μπι Microbeads, suspended in 30 μΐ of NaCl with the same concentration of 2-vinyl"4H-i,3-dithiin, were applied on the apical surface of MucilAir™-CF. Three inserts were used per condition and compared to a 0.9% NaCl vehicle control. The epithelia were incubated at 37°C; 5% C0 ₂ ; 100% humidity. Mucociliary clearance analysis was evaluated at 4 hours using a high-speed acquisition camera (Sony) connected to an Axi overt 200M microscope (Zeiss). 30 second movies (3

movies/insert) showing the movement of the Microbeads were taken and analysed using the imaging software Image Pro Plus (Mediacy). The movement of the beads was tracked and the velocity of each particle was calculated in order to determine the average speed of mucociliary clearance.

Results

2-Vinyl-4H-i,3-dithiin at concentrations of 80 and 160 μg/ml increased the rate of mucociliary clearance in the patient-derived 3D epithelial cell model in a dose- dependent manner. 2-Vinyl-4H-i,3-dithiin increases the ciliary beat frequency in the same 3D cell model. Conclusions

The results indicate that the administration of vinyldithiin to epithelial cells from a subject suffering from cystic fibrosis leads to a change in mucociliary clearance. There was a marked increase in mucociliary clearance following the administration of vinyldithiin. In contrast, prior to the administration of vinyldithiin there was very little mucociliary clearance observed.

It is hypothesised that the change in mucociliary clearance following the administration of vinyldithiin was due a reduction in the viscosity of mucus on the apical surface of the human bronchial epithelial cells. It is hypothesised that the viscosity of the mucus is affected by the change in Na ⁺ flow which increases the extracellular ion concentration and therefore triggers movement of water out of the cells. This increases the ASL volume and depth, and reduces the viscosity of the mucus. In order to address various issues and advance the art, the entirety of this disclosure shows by way of illustration various embodiments in which the claimed invention(s) may be practiced and provide for novel uses for vinyldithiins, analogues and derivatives thereof in therapy. The advantages and features of the disclosure are of a

representative sample of embodiments only, and are not exhaustive and/or exclusive. They are presented only to assist in understanding and teach the claimed features. It is to be understood that advantages, embodiments, examples, functions, features, structures, and/or other aspects of the disclosure are not to be considered limitations on the disclosure as defined by the claims or limitations on equivalents to the claims, and that other embodiments may be utilised and modifications may be made without departing from the scope and/ or spirit of the disclosure. Various embodiments may suitably comprise, consist of, or consist essentially of, various combinations of the disclosed elements, components, features, parts, steps, means, etc. In addition, the disclosure includes other inventions not presently claimed, but which may be claimed in future.