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Title:
PHARMACEUTICAL COMPOSITIONS AND USES THEREOF
Document Type and Number:
WIPO Patent Application WO/2019/043375
Kind Code:
A1
Abstract:
The present invention relates to the use of ajoene, analogues and derivatives thereof to regulate sodium absorption and fluid homeostasis and to treat disorders with symptoms related to elevated sodium transport and difficulties associated with mucociliary clearance. The invention provides pharmaceutical compositions comprising ajoene or an analogue or derivative thereof, for reducing sodium ion uptake in cells. This may be achieved by modulating the activity of an epithelial sodium channel (ENa C) and/or a Na+/K+ - ATPase.

Inventors:
GRAZ CARL JORG MICHAEL (GB)
SYKES LUCY HELEN (GB)
Application Number:
PCT/GB2018/052432
Publication Date:
March 07, 2019
Filing Date:
August 29, 2018
Export Citation:
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Assignee:
NEEM BIOTECH LTD (GB)
International Classes:
A61K31/105; A61P11/00; A61P11/02
Foreign References:
Other References:
PATRICK KRUMM ET AL: "Thiol-reactive compounds from garlic inhibit the epithelial sodium channel (ENaC)", BIOORGANIC & MEDICINAL CHEMISTRY, PERGAMON, GB, vol. 20, no. 13, 10 May 2012 (2012-05-10), pages 3979 - 3984, XP028490911, ISSN: 0968-0896, [retrieved on 20120517], DOI: 10.1016/J.BMC.2012.05.021
T. H. JAKOBSEN ET AL: "Ajoene, a Sulfur-Rich Molecule from Garlic, Inhibits Genes Controlled by Quorum Sensing", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, vol. 56, no. 5, 6 February 2012 (2012-02-06), pages 2314 - 2325, XP055132367, ISSN: 0066-4804, DOI: 10.1128/AAC.05919-11
CIFELLI P ET AL: "An RCT of macerated garlic oil in patients with CF & chronic Pseudomonas aeruginosa", JOURNAL OF CYSTIC FIBROSIS, ELSEVIER, NL, vol. 7, 1 June 2008 (2008-06-01), pages S68, XP022714564, ISSN: 1569-1993, [retrieved on 20080601], DOI: 10.1016/S1569-1993(08)60261-1
Attorney, Agent or Firm:
GILL, Siân et al. (GB)
Download PDF:
Claims:
Claims

1. A pharmaceutical composition comprising ajoene or an analogue or derivative thereof, for use in therapy to reduce sodium ion uptake in cells.

2. A pharmaceutical composition for use as claimed in claim 1, wherein the reduction in sodium ion uptake in cells improves fluid homeostasis and/ or improves mucociliary clearance. 3. A pharmaceutical composition for use as claimed in either or the preceding claims, wherein the activity of an epithelial sodium channel (ENaC) and/or a Na+/K+ - ATPase solute pump is modulated to reduce sodium ion uptake in the cell.

4. A pharmaceutical composition for use as claimed in any one of the preceding claims, wherein the modulation of the activity of the ENaC comprises modulating expression of one or more genes encoding one or more subunits of the ENaC.

5. A pharmaceutical composition for use as claimed in claim 4, wherein the expression of genes encoding one or more of subunits of the ENaC is modulated by the inhibition of an estrogen receptor.

6. A pharmaceutical composition for use as claimed in any one of the preceding claims, wherein the modulation of the activity of the ENaC comprises altered phosphorylation of one or more subunits of the ENaC.

7. A pharmaceutical composition for use as claimed in claim 6, wherein the altered phosphorylation of one or more subunits of the ENaC is a result of the inhibition of the activity of PKC5. 8. A pharmaceutical composition for use as claimed in claim 7, wherein the altered phosphorylation of one or more subunits of the ENaC prevents intracellular trafficking of one or more subunits of the ENaC to the cell membrane.

9. A pharmaceutical composition for use as claimed in any one of the preceding claims, wherein the modulation of the activity of the ENaC comprises inhibiting protease cleavage of one or more subunits of the ENaC.

10. A pharmaceutical composition for use as claimed in claim 9, wherein the protease cleavage of one or more subunits of the ENaC is inhibited by inhibition of the activity of cathepsin B.

11. A pharmaceutical composition for use as claimed in any one of the preceding claims, wherein the modulation of the activity of the ENaC comprises increasing levels of glutathione. 12. A pharmaceutical composition for use as claimed in any one of the preceding claims, wherein the modulation of the activity of the ENaC comprises increasing paraoxonase activity.

13. A pharmaceutical composition for use as claimed in any one of the preceding claims, for regulating the osmolarity of the periciliary fluid of the lung of a patient.

14. A pharmaceutical composition for use as claimed in claim 13, wherein the osmolarity of the periciliary fluid of the patient is regulated by reducing the absorption of sodium ions into the cell.

15. A pharmaceutical composition for use as claimed in any one of the preceding claims, for enhancing mucociliary clearance in the lung of a patient.

16. A pharmaceutical composition for use as claimed in any one of the preceding claims, for treating cystic fibrosis.

17. A pharmaceutical composition for use as claimed in any one of claims 1 to 15, 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), alkalosis, Cushing syndrome and Sjorgen's syndrome.

18. A pharmaceutical composition for use as claimed in any one of the preceding claims, further comprising one or more additional active agents.

19. A pharmaceutical composition for use as claimed in claim 18, wherein the one or more additional active agent is an antibiotic.

20. A pharmaceutical composition for use as claimed in claim 19, wherein the antibiotic is a tobramycin, a ciprofloxacin or a combination thereof.

21. A pharmaceutical composition for use as claimed in claim 18, wherein the one or more additional active agent is an agent for thinning the mucus in the lungs. 22. A pharmaceutical composition for use as claimed in claim 21, wherein the agent for thinning mucus in the lungs is selected from the group consisting of dornase alfa, saline, mannitol dry powder and mannitol solution.

23. A pharmaceutical composition for use as claimed in claim 18, wherein the one or more additional active agent is a potentiator of the CFTR channel, such as Ivacaftor or Lumacaftor.

24. A pharmaceutical composition for use as claimed in claim 18, wherein the one or more additional active agent opens an alternative chloride channel to CFTR, such as denufosol.

25. A pharmaceutical composition for use as claimed in claim 18, wherein the one or more additional active agent is a bronchodilator. 26. A pharmaceutical composition for use as claimed in claim 25, wherein 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. 27. A pharmaceutical composition for use as claimed in claim 18, wherein the one or more additional active agent is a corticosteroid.

28. A pharmaceutical composition for use as claimed in claim 27, wherein the one or more corticosteroid is selected from the group consisting of: beclometasone, betamethasone, budesonide, flunisolide, fluticasone, mometasone and triamcinolone.

29. A pharmaceutical composition for use as claimed in any one of the preceding claims, wherein the composition is for administration to the lung.

30. A pharmaceutical composition for use as claimed in claim 29, wherein the composition is a dry powder.

31. A pharmaceutical composition for use as claimed in claim 29, wherein the composition is a solution or suspension. 32. A pharmaceutical composition for use as claimed in any one of the preceding claims, wherein a dose of the composition comprises from about 0.01 mg to about 100 mg ajoene per kg of subject body weight per day, administered in one or more administrations. 33· A method of reducing sodium ion uptake in cells by administering a pharmaceutical composition comprising ajoene or an analogue or derivative thereof.

34. A method as claimed in claim 33, wherein the composition is as claimed in any one of claims 2 to 32.

Description:
Pharmaceutical Compositions and Uses Thereof

Technical Field

The present invention relates to the ability of ajoene, analogues and derivatives thereof to regulate sodium transport and fluid homeostasis and to treat 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 transport of sodium ions (Na + ) across the cell membrane and control 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 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 ajoene or an analogue or derivative thereof, for use in therapy to reduce sodium ion uptake in cells. 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 an estrogen receptor.

In some embodiments, the modulation of the activity of the ENaC comprises altered phosphorylation of one or more subunits of the ENaC. In some embodiments, the altered phosphorylation of one or more subunits of the ENaC is a result of the inhibition of the activity of PKC5.

In some embodiments, the altered phosphorylation of one or more subunits of the ENaC prevents intracellular trafficking of one or more subunits of the ENaC to the cell membrane.

In some embodiments, the modulation of the activity of the ENaC comprises inhibiting protease cleavage of one or more subunits of the ENaC. In some embodiments, the protease cleavage of one or more subunits of the ENaC is inhibited by inhibition of the activity of cathepsin B.

In some embodiments, the modulation of the activity of the ENaC comprises increasing levels of glutathione. In some embodiments, the modulation of the activity of the ENaC comprises increasing paraoxonase activity. 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 l, diabetes, dry eye syndrome, renal dysfunction, cirrhosis, hypokalemia, bronchitis, bronchiectasis, asthma, Chronic Pulmonary Obstructive Disorder (COPD), alkalosis, Cushing syndrome and Sjorgen's 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 CF R 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; or 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.oimg to about loomg ajoene 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 ajoene 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 ion channel activity following the application of ajoene to a three-dimensional human epithelial cell model.

Figure 2 is a graph showing the evaluation of the IC50 of ajoene based on results from modified Ussing chambers.

Figure 3 shows the effect of ajoene on mucociliary clearance after 4 hours exposure on a 3D human epithelial cell model created cells from a subject with cystic fibrosis. Detailed Description

The present invention relates to compositions comprising ajoene or analogues or derivatives thereof, for reducing sodium ion uptake in cells. More specifically, the ajoene or analogues or derivatives may modulate the activity of an epithelial sodium channel (ENaC) and/or the activity of a Na + /K + - ATPase, to reduce sodium ion uptake in a cell.

Ajoene has previously been shown to have anti-hypertensive, antioxidant, anti- thrombotic, anti-infective and anti-inflammatory properties and has also been found to act as a quorum sensing inhibitor against biofilm forming bacteria such as

Pseudomonas aeruginosa.

It has now been discovered that ajoene is a modulator of sodium channel activity and can therefore be used to reduce sodium ion uptake in a cell. It has also been discovered that ajoene may be used as a promoter of mucociliary transport. The administration of ajoene has been shown to have effects on signalling pathways and protease inhibition. As a result, ajoene may be used for the treatment of disorders associated with dysregulation of sodium transport and mucociliary clearance difficulties.

Analogues and derivatives

References made to ajoene herein refer to mixtures of the isomers of ajoene, as well as purified isomers, including E-ajoene and Z- ajoene. Ajoene is organosulfur compound found in plants of allium genus, for example in garlic {Allium sativum) or onion {Allium cepa L.), containing sulfoxide and disulfide functional groups. It is formed of up to four isomers, with either an E- or Z- central alkene and can differ in the chirality of the sulfoxide.

Ajoene can be derived from thermal degradation of allicin under specific conditions. Suitable ajoene for use in the present invention may be natural, synthetic or semisynthetic and obtained via methods described for example in WO2010/100487 and WO2016/083781. In some embodiments of the invention, the ajoene for use may be synthetic or semi-synthetic.

As used herein, ajoene analogues and derivatives refer to compounds that are functionally equivalent to ajoene. For example, ajoene analogues and derivatives may contain the sulfoxide and/ or the vinyl disulfide core whilst including different terminal end-groups. In some embodiments that maybe preferred, ajoene analogues and derivatives may contain both the sulfoxide and the vinyl disulfide core whilst including different terminal end-groups.

Examples of ajoene analogues and derivatives are as indicated below:

Ti and T2 independently of each other represent the diradicals **CH 3 **CH 2 ,

**CH— O— R 1 , or **C=0, wherein 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 carbohydrate having 5 to 12 carbon atoms, a substituted or

unsubstituted aryl group with 6 to 12 ring atoms which can comprise one or more ring heteroatoms, a substituted or unsubstituted C6-Ci 2 -aryl-Ci-C6-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 Z, 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 C6-Ci 2 -aryl- Ci-C6-alkyl group which can comprise one or more heteroatoms.

Vinyl disulfide B may be substituted for a trisulphide with an allyl group present.

The purity of the ajoene, analogues or derivatives used in the compositions may, for example, be in the range of from about 50% to about 100%, or from about 60% to about 80%, or from about 70% to about 90%, or from 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 osmolarity 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 peri ciliary 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.

Ajoene has 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 ajoene modulates activity of the ENaC by modulating expression of one or more genes encoding one or more subunits of the ENaC on the cell membrane. For example, the expression of genes encoding one or more of subunits of the ENaC is modulated by the inhibition of an estrogen receptor by ajoene. There are two main types of classic estrogen receptor, alpha (ERa) and beta (ΕΡνβ), and an additional G-protein coupled receptor which has been found to bind estrogen and mediate non-genomic effects. ΕΡνβ is more highly expressed in the lung than ERa. There are estrogen receptor response elements in the promoter region of all three ENaC subunits, and stimulation of alveolar cells with estrogen and related hormones has been found to increase the expression of alpha and gamma ENaC subunits and to result in channel activation. It has also been demonstrated that ajoene modulates the activity of the ENaC as a result of altered phosphorylation of one or more subunits of the ENaC. In some

embodiments, this altered phosphorylation is a result of the activation of protein kinase D. PKC5 is a serine threonine protein kinase with a role in cell signalling. It has been implicated in the signalling cascade induced by aldosterone which promotes the intracellular trafficking of ENaC to the cell membrane. Aldosterone stimulated localisation of ENaC subunits via activation of protein kinase D was found to be blocked by a specific PKC5 inhibitor. PKC5 has also been found to inhibit basolateral potassium channels KCNQi:KCNE3 by direct association with channel subunits, as well as phosphorylation of AMP-dependent kinase.

Inhibition of these channels leads to increased Na + absorption through ENaC in colonic epithelium. This mechanism has also been found to specifically mediate the activation of ENaC by estrogen. PKC5 further mediates mucin secretion by the phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS). A dominant-negative PKC5 construct transfected into human bronchial epithelial 1 cells inhibited PMA- induced mucin hypersecretion and phosphorylation of MARCKS, whereas wild-type construct increased PKC5 and mucin secretion, resulting in more viscous mucus.

It has been shown that ajoene dose dependency inhibits the activity of PKC5. This in turn will prevent the intracellular trafficking of ENaC to the cell membrane, thereby reducing the sodium uptake into the cell via ENaC. Ajoene, analogues or derivatives thereof may also modulate the activity of the ENaC as a result of inhibiting Cathepsin B. Cathepsin B is an acid protease that is present on the apical membrane of epithelia and is secreted into the airway surface liquid. This protease has been shown to cleave the a and Y subunits of ENaC, resulting in increased activation of the channel and Na + hyper- absorption. Unlike prostasin, which is inactive below pH 7.0, Cathepsin B has been found to be more active in acidic conditions. Activation of Cathepsin B was found to correlate with increased abundance of ENaC on the cell membrane and reduced airway surface liquid. Inhibition of Cathepsin B reduced the activation of ENaC. Patients with CF have been found to have a more acidic lung environment, suggesting that there may be an over activation of Cathepsin B contributing to the hyper-activation of ENaC.

Ajoene, analogues or derivatives thereof may also modulate the activity of the ENaC as a result of increasing levels of glutathione. ENaC activity is regulated by oxidative stress. Glutathione is the predominant antioxidant in the lung and changes to the glutathione redox potential have been found to impact ENaC activity. Application of oxidised glutathione (GSSG) inhibited the open probability of ENaC, through S-glutathionylation of ENaC. Glutathione (GSH) is excreted, along with CI " and HCOy, by CFTR channels and has antioxidant and mucolytic properties. Patients with CF have been found to have reduced extracellular GSH and reduced GSH:GSSG ratio. Ajoene has been found to return liver GSH content back to normal in a model of morphine-dependence in mice. It has also been found to be an inhibitor of human glutathione reductase and predicted to inhibit glutathione S- transferase and also induce glutamate cysteine ligase, which in turn increases GSH. The potential of hydrogen sulfide donors for the inhibition of ENaC has been investigated and have been suggested to mediate the vasoactive properties of some garlic derived compounds, including ajoene.

Ajoene, analogues or derivatives thereof may also modulate the activity of the ENaC as a result of increasing paraoxonase activity.

As disclosed by Shil et al in The FASEB Journal (April 2016) vol. 30 no. 1 Supplement 1223.9, paraoxonase-2 (PON-2) is a membrane-bound lactonase with unique antioxidative and anti-atherosclerotic properties, and thus has been associated with cardiovascular diseases. PON-2 was been shown to have an inhibitory effect on ENaC, and it was concluded that PON-2 interacts with ENaC and modulates channel expression.

It has been found that ajoene increases paraoxonase activity, which in turn reduces ENaC activity.

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.

Ajoene 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 ajoene reduces the viscosity of mucus on the apical surface of human bronchial epithelial cells and increases the rate of mucociliary transport.

Patients with pulmonary disorders, including cystic fibrosis, have been found to have more viscous mucus and hypersecretion of mucins compared to healthy controls. The effect of ajoene at 2.5 and 5 μg/ml in the videos captured for mucociliary clearance analysis indicates a reduction in mucus viscosity and an increased rate of mucociliary transport.

Disease applications

In conjunction with the already well-established anti-inflammatory and antioxidant properties of ajoene, the effects of ajoene on ion transport, fluid homeostasis and mucus viscosity indicate a novel benefit of ajoene for treatment of disorders related to dysregulated ion transport and related mucociliary clearance deficits.

The activity of ajoene 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. Ajoene, analogues and derivatives thereof are effective in treating the underlying cause of cystic fibrosis and 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 l, and diabetes. These may therefore also be treated by administration of ajoene, analogues and derivatives thereof.

Ajoene additionally has therapeutic application as a selective estrogen receptor modulator (SERM) and for the treatment of related disorders, including but not limited to cystic fibrosis, dry eye syndrome and renal dysfunction. Ajoene may also be useful in treating congestive heart failure, cirrhosis, hypokalemia, bronchitis, bronchiectasis, asthma, Chronic Pulmonary Obstructive Disorder (COPD), Sjorgen's syndrome, 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 ajoene or an analogue or derivative thereof is intended for local administration. In some embodiments, the local administration is to the lung. This allows the ajoene, 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 ajoene 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.oimg to about loomg ajoene 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, the composition comprises ajoene in a concentration of from about 1 to about 500 μΜ ajoene, or from about 1 to about 100 μΜ ajoene, or from about 1 to about 200 μΜ ajoene.

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 ajoene 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 Cl ~ 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 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; and 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 ajoene having more than 95% purity.

The ajoene was produced by methods disclosed in WO2016/083781 to obtain pure ajoene of at least 15% to 30%. Thereafter, said ajoene undergoes two additional chromatography steps to obtain more than 95% pure ajoene.

1. Dose Dependent Inhibition of Na + Channel Activity with Ajoene

Methods

Ajoene 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 ajoene (from 2.36 μg/ml up to 21.24 ug/ml) using sequential addition. Amiloride was used as a positive control. The change in sodium current was recorded following each addition of ajoene.

Results

Sequential addition of ajoene to the Ussing chamber solution resulted in a delayed dose-dependent reduction in sodium current, as shown in Figure 1, with an IC50 of 12.69 ig/nd, as shown in Figure 2.

As shown in Figure 1, the average of delta Isc (increase in short-circuit current) was calculated from Ussing chamber recording traces from 6 inserts with sequential application of ajoene across 100 minutes, reflecting change in sodium ion channel activity following drug treatment. The graph in Figure 2 shows the evaluation of the IC50 of ajoene based on results from modified Ussing chambers. IC50 was calculated as being 12.69 ± 0.68 μg/ml.

Conclusions

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

2. Ajoene Increases Mucociliary Clearance

Methods

Ajoene 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. Ajoene had no negative effect on the viability of the epithelium at concentrations tested, as measured by TEER and a lactate dehydrogenase assay. Evidence of cytotoxicity was observed at doses 20x the highest test concentration. Inserts were placed into culture medium containing 2.5 or 5 μg/ml ajoene. 30 μιη Microbeads, suspended in 30 μΐ of NaCl with the same concentration of ajoene, 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 Axiovert 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

Ajoene at concentrations of 2.5 and 5 μg/ml significantly increased the rate of mucociliary clearance in the patient-derived 3D epithelial cell model by 42.5% and 43.7% respectively, compared to 0.9% NaCl control (p<o.ooi) (see Figure 3).

Figure 3 shows the measurement of MCC by nanoparticle velocity after 4 hours exposure. Means values are presented (+/- SEM, n=3). Student's t-test: ***p<o.ooi

Conclusions

The results indicate that the administration of ajoene 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 ajoene. In contrast, prior to the administration of ajoene there was very little mucociliary clearance observed.

It is hypothesised that the change in mucociliary clearance following the administration of ajoene 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) maybe practiced and provide for novel uses for ajoene, 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.