WO2022167778A1

WO2022167778A1 - Ebselen as adenosine receptor modulator

Info

Publication number: WO2022167778A1
Application number: PCT/GB2022/000014
Authority: WO
Inventors: Vijay Kumar PABBATHI; Sriharsha KANTAMNENI
Original assignee: Haiku Therapeutics Ltd
Priority date: 2021-02-02
Filing date: 2022-02-01
Publication date: 2022-08-11

Abstract

Ebselen, 2-Phenyl-1,2-benzisoselenazol-3(2H)-one, (I) is a modulator of the adenosine A2A receptor and is indicated for use in the treatment or prevention of a disease or condition mediated by the adenosine A2A receptor.

Description

EBSELEN AS ADENOSINE RECEPTOR MODULATOR

The present invention relates to modulation of the adenosine A2A receptor, and in particular to the compound Ebselen as a modulator of the adenosine A2A receptor.

Background to the Invention

Adenosine receptors belong to the superfamily of G protein-coupled receptors (GPCRs). GPCRs constitute one of the primary signal transduction systems in eukaryotic cells and target for numerous clinically used medicines. An overview of their characteristic structural features, molecular interactions, cellular functions, and their roles in pain, cancer, and neurodegenerative, inflammatory, and autoimmune diseases, is given by Borea et. al., in Physiol Rev 98: 1591-1625, 2018, May 30, 2018; doi:10.1152/physrev.00049.2017.

The adenosine A2A receptor is one of four adenosine receptors and, through coupling to G-proteins, stimulates adenylate cyclase (AC) and increases cyclic adenosine monophosphate (cAMP) levels. They play a role in many diseases including cardiovascular diseases, autoimmune diseases, neurodegenerative and psychiatric diseases, cancers, and inflammatory diseases. A2A receptor modulators (agonists and antagonists) have been indicated in the treatment of a variety of diseases and conditions, such as described below.

Bleeding Disorders

Bleeding disorders include haemophilia, von Willebrand disease, coagulation disorders, platelet disorders and clotting factor deficiencies. They affect the way the body controls blood clotting. Treatment generally involves medicines or clotting factor replacement therapy.

US 2014/0271606 discloses methods for treating bleeding disorders, in particular, haemophilia, with A2A receptor antagonists. Administration of a A2A receptor antagonist was shown to reduce blood loss in subjects with Haemophilia A and Haemophilia B, demonstrating pro-haemostatic properties of A2A receptor antagonists. This document further discloses methods including administration of an A2A receptor antagonist with one for more of Factor VIII, Factor IX, and Factor XI.

Bleeding disorders and obstetric complications are discussed by Gernsheimer, in ‘Congenital and acquired bleeding disorders in pregnancy’, Hematology Am Soc Hematol Educ Program (2016) 2016 (1): 232-235, with particular reference to von Willebrand disease, coagulation factor deficiencies, and inherited disorders of platelet number or function.

Endometriosis and diseases of the reproductive system

Endometriosis is a chronic inflammatory condition in which tissue similar to the lining of the womb starts to grow in other places, such as in the ovaries and fallopian tubes. It can cause chronic pelvic pain, dysmenorrhea, period pain, pain during or after sex, premature birth, difficulty in conceiving, and in some cases, it can lead to infertility. Treatments include the use of painkillers, hormone medicine and contraceptives, and surgery.

W02012137012 indicates the involvement of the A2A receptor in endometrial cancer.

US 5877180 discloses a method of treating inflammatory disorders with A2A receptor agonists, and specifically recites the treatment of infertility from endometriosis.

Trapero et. al., in ‘Purinergic Signaling in Endometriosis-Associated Pain’ (Review), Int. J. Mol. Sci. 2020, 21 , 8512; doi:10.3390/ijms21228512, relate the A2A receptor-mediated anti-inflammatory actions of adenosine to analgesic effects that would be useful for the management of endometriosis-associated pain.

W02003000694 indicates the use of A2A receptor antagonists in the treatment of spontaneous infertility.

Pain

A2A antagonists are known to relieve pain. Vincenzi et. al., in ‘Targeting Adenosine Receptors: A Potential Pharmacological Avenue for Acute and Chronic Pain’, (Review) antagonists

relief of pain due to inflammation and peripheral neuropathy. Luongo et. al. , in Targeting metabotropic adenosine receptors for neuropathic pain: Focus on A2A’, Brain, Behavior, and Immunity, 69, 10, 1016/j.bbi, 2018.02.014, discuss A2AR selective blockade in decreasing surgical pain and A2AR inhibition in reducing neuropathic pain.

Neurogenerative Diseases

WO 2003063876 discloses A2A receptor antagonists in the treatment of movement disorders such as Parkinson’s disease.

US 5,484,920 describes compounds having a xanthine skeleton, and A2A receptor antagonist activity, as therapeutic agents for Parkinson’s disease.

Movement Disorders

W02004019949 discloses A2A receptor antagonists in the treatment of restless legs syndrome.

A2A receptor antagonists for the treatment of extra-pyramidal syndrome and other movement disorders, such as periodic leg movement in sleep, are described in US20060106040.

Immunosuppression and Cancer Therapy

The immunosuppressive activities of adenosine in cancer, and the development of adenosine inhibitors in this context, are discussed for example by Allard et. al., in Curr Opin Pharmacol. 2016 Aug;29:7-16.

There is evidence of the involvement of the adenosine pathway in modulating immune responses in cancer (Allard et al., Immunosuppressive activities of adenosine in cancer, Curr Opin Pharmacol., 2016 Aug, 29:7-16, and Sousa et al., Adenosine A1 and A2A Receptors in the Brain: Current Research and Their Role in Neurodegeneration, Molecules, 2017 Apr 23, 22(4):676). Cancer cells cause a loss of local oxygen supply in the surrounding tissue around the tumors, leading to the secretion of adenosine (a metabolite of ATP). Adenosine near the tumor microenvironment binds to the A2ARs on immune cells, activating the A2A signalling pathway and inactivating immune response function (Merighi et al., Targeting A3 and A2A adenosine receptors in the fight against cancer, Expert Opin Ther Targets, 2019 Aug, 23(8):669-678). Hence, inhibiting A2A receptors could offer a potential treatment strategy to promote immune response and thus supporting cancer therapy, for example as checkpoint inhibitors in such therapy, either alone or in combination with other therapeutic agents.

Psychological Disorders and Mental Health Conditions

W02009015236 discloses the compound 4-hydroxy-4- methyl-piperidine- 1 - carboxylic acid (4-methoxy-7-morpholin-4-yl-benzothiazol-2-yl)-amide, an A2A receptor antagonist, in the treatment of post-traumatic stress disorder.

W02006009698 describes the amelioration of addictive behaviour through the administration of an A2A receptor antagonist.

W02004108137 describes a method of treating an anxiety disorder selected from the group consisting of panic disorder, agoraphobia, obsessive-compulsive disorder, social phobia, post- traumatic stress disorder, and specific phobia, through administration of an A2A receptor antagonist.

Cardiovascular Conditions

WO9628163 discloses A2A receptor agonists for use increasing the contractile performance of a compromised myocardium in a mammal.

WO201 1133696 relates to the use of A2A receptor antagonists for the treatment or inhibition of atherosclerotic lesions.

Da Silva et.al. report prevention of cardiac dysfunction due to hypertension associated with myocardial infarction using A2A receptor agonists: Drug Des Devel Ther. 2017 Mar 6;1 1 :553-562.

Wound Healing

The A2A receptor also plays a role in wound healing. A2A receptor stimulation has been found to promote wound healing, while inhibition of this target prevents/reduces dermal scarring (Jiang and Rinkevich, Int J Mol Sci. 2020 Jan 17;21 (2):617). Other experimental evidence suggests that inhibition of A2AR diminishes scarring while improving the healed wound's collagen composition and tensile strength (Perez-Aso et. al., FASEB J. 2012 Oct; 26(10):4254-63. doi: 10.1096/fj.12-209627. Epub 2012 Jul 5.).

Stenosis

A2A receptors are distributed in blood vessels and they mediate vasodilation, suggesting that activation of A2A receptor agonists can dilate blood vessels, particularly in clinical situations such as ischemic cardiac damage. WO9857651 discloses that selective stimulation of adenosine A2A receptors can reduce or prevent restenosis following revascularisation procedures.

Revascularisation procedures may be performed on, for example, coronary arteries, renal arteries, leg arteries and carotid arteries, and can include, for example, any of balloon angioplasty, insertion of a stent, insertion of a bypass graft, directional coronary atherectomy, percutaneous transluminal coronary angioplasty and excimer laser angioplasty.

Autoimmune Diseases

A2A receptors are known to play a role in the treatment of autoimmune diseases, as evidenced for example in Chhabra et. al., ‘The immunosuppressive role of adenosine A2A receptors in ischemia reperfusion injury and islet transplantation’, Curr Diabetes Rev. 2012 Nov;8(6):419-33 and Sun et. al., ‘In Vivo Positron Emission Tomography Imaging of Adenosine A2A Receptors’ Front Pharmacol, 2020 Nov 26;11 :599857.

Autoimmune diseases of interest include, for example, Hashimoto’s thyroiditis, Guillain-Barre syndrome, and pemphigus.

Ledent et. al., in ‘Thyroid expression of an A2 adenosine receptor transgene induces thyroid hyperplasia and hyperthyroidism’, The EMBO Journal vol .11 no.2 pp.537-542, 1992, demonstrate that A2A receptors are present in thyroid cells. Overexpression of A2A receptors in the thyroid was shown to induce spontaneous hyperthyroidism and thyroid enlargement.

W02003000694 indicates the use of A2A receptor antagonists in the treatment of both Hashimoto’s thyroiditis and pemphigus. CNS Disorders

WO 2011057199 discloses methods for treating CNS disorders including methods for increasing the blood brain barrier permeability in a subject through the administration of A1 and A2A receptor agonists.

Clinical applications of A2A receptor modulators are evident; WO 2012137012 cites (paragraph bridging pages 38 and 39) heart failure (such as acute decompensated heart failure and congestive heart failure); kidney failure (e.g. caused by heart failure); oedema; cancer (such as prostate, rectal, renal, ovarian, endometrial, thyroid, pancreatic, particularly breast, colon, bladder, brain, glia, melanoma, pineal gland and, more particularly, lung cancer (e.g. Lewis lung carcinoma)); diabetes; diarrhea; macular degeneration (such as macular degeneration caused by angiogenesis (e.g. retinal angiogenesis)); or, particularly (e.g. for disorders or conditions ameliorated by the inhibition of the A2A receptor), a disease of the central nervous system such as depression, a cognitive function disease, a neurodegenerative disease (such as Parkinson's disease, Huntington's disease, Alzheimer's disease, amyotrophic lateral sclerosis) and psychoses; an attention related disorder (such as attention deficit disorder (ADD) and attention deficit hyperactivity disorder (ADHD)); extra pyramidal syndrome (e.g. dystonia, akathisia, pseudoparkinsonism and tardive dyskinesia); a disorder of abnormal movement (such as restless leg syndrome (RLS) and periodic limb movement in sleep (PLMS)); cirrhosis; liver fibrosis; fatty liver; dermal fibrosis (e.g. in diseases such as scleroderma); a sleep disorder; stroke; brain injury and neuroinflammation (e.g. migraine or any disorder or condition caused by ischemia, stroke, head injury or CNS inflammation); and addictive behaviour.

However, only a small number of compounds that modulate the A2A receptor have reached the market. These include Adenosine for myocardial perfusion and Istradefylline (8-[(E)-2-(3,4-dimethoxyphenyl)ethenyl]-1 ,3-diethyl-7-methylpurine- 2, 6-dione) for the treatment of Parkinson’s disease, as an adjunct to levodopa and carbidopa.

Ebselen (CAS Registry Number 60940-34-3), 2-Phenyl-1 ,2-benzisoselenazol- 3(2/-/)-one, is an organoselenium compound with the following structure:

As reported by Azad et. al. in Mol Biol Rep. 2014 Aug;41 (8):4865-79, Ebselen mimics glutathione peroxidase activity, catalysing several reactions to protect cellular components from oxidative and free radical damage. It is classed an antioxidant, an anti-inflammatory agent, a cyclooxygenase inhibitor, a neuroprotective agent, and an anti-ulcer agent.

Ebselen is, or has been, the subject of clinical trials for a number of indications, including for use in patients with Covid-19, patients with Meniere’s Disease, and patients with Cystic Fibrosis.

Summary of the Invention

The present inventors have now found that Ebselen, 2-Phenyl-1 ,2- benzisoselenazol-3(2/-/)-one, is a modulator of the adenosine A2A receptor. As such, and in accordance with the present invention, Ebselen, 2-Phenyl-1 ,2- benzisoselenazol-3(2/-/)-one, or a pharmaceutically acceptable salts thereof, is indicated for use in therapy as a modulator of the A2A receptor.

The present invention therefore provides the compound Ebselen, 2-Phenyl-1 ,2- benzisoselenazol-3(2/-/)-one, or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of a disease or condition mediated by the adenosine A2A receptor.

The present invention also provides a pharmaceutical composition comprising the compound Ebselen, 2-Phenyl-1 ,2-benzisoselenazol-3(2/-/)-one, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, for use in the treatment or prevention of a disease or condition mediated by the adenosine A2A receptor.

In addition, the present invention provides a method of treating or preventing a disease or condition mediated by the adenosine A2A receptor, comprising administering to a patient in need thereof, a therapeutically effective amount of the compound Ebselen, 22-Phenyl-1 ,2-benzisoselenazol-3(2/-/)-one, or a pharmaceutically acceptable salt thereof.

Ebselen or a pharmaceutically salt thereof may be provided in the form of a pharmaceutical composition adapted for oral, intravenous, intradermal, subcutaneous, intramuscular, nasal, rectal, transdermal, transmucosal, intraperitoneal, or pulmonary administration, or by infusion, and may be used alone or in combination with another therapeutic agent

Detailed Description

Upon activation, A2A receptors bind to Gs protein at the intracellular site of the receptor, which activates adenylyl cyclase, inducing the rise of intracellular cAMP.

Specific binding of Ebselen to the A2A human receptor was demonstrated both in a radioligand binding assay, and in cell-based functional studies, as described herein.

The affinity of Ebselen for the human adenosine A2A receptor in transfected HEK- 293 (human embryonic kidney) cells was determined in the radioligand binding assay described in Example 1 herein. Ebselen was found to bind to, and have the potential to modulate the activity of, the A2A receptor.

Functional activity of Ebselen for the human adenosine A2A receptor in transfected HEK-293 cells was determined in the cAMP release assays described in Examples 2 and 3 herein. The results revealed Ebselen to be a partial agonist at the A2A receptor, and an antagonist of the A2A receptor, its action being concentration-dependent.

Thus, in accordance with the present invention, Ebselen is shown to be a modulator of the A2A receptor. As such, it is indicated in the treatment of or prevention of diseases or conditions mediated by the adenosine A2A receptor.

In accordance with a first aspect of the present invention, there is provided the compound Ebselen, 2-Phenyl-1 ,2-benzisoselenazol-3(2/-/)-one, or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of a disease or condition mediated by the adenosine A2A receptor.

References herein to ‘Ebselen’ include both the compound Ebselen and its pharmaceutically acceptable salts.

Diseases and conditions contemplated in all aspects of the present invention include all those mentioned above in the ‘background’ discussion, wherein references to the evidence of the role of the A2A receptor in the specified diseases are given.

In some embodiments, the diseases and conditions contemplated in all aspects of the present invention comprise bleeding disorders. For example, applications of Ebselen contemplated in the present invention include prophylaxis of bleeding disorders. In particular, Ebselen is contemplated in the treatment or prophylaxis of Haemophilia A, Haemophilia B, and von Willebrand’s disease. In some embodiments, Ebselen may be employed in combination with a recombinant Factor VIII, recombinant Factor IX and recombinant Factor XI. In some embodiments, Ebselen may be employed as an adjuvant treatment or in combination with a clotting factor such as one or more of Factor VIII, Factor IX and Factor XI. In some embodiments, Ebselen may be used as a replacement therapy in the treatment of bleeding and clotting disorders, either as regular treatments or for specific episodes such as during surgery.

In accordance with the present invention, Ebselen may also be used in diagnostic screens involved in bleeding disorders such as coagulation screens, coagulation factor assays, lupus acticoagulant testing, thrombophilia testing and platelet function testing.

In some embodiments, the diseases and conditions contemplated in all aspects of the present invention comprise diseases of the reproductive system. For example, applications of Ebselen contemplated in the present invention include infertility, endometriosis and the prevention of premature birth (often caused by endometrial disorders). As discussed above, A2A antagonists are known to relieve pain due to inflammation and peripheral neuropathy. Activation of A2AR by endogenous adenosine or exogenous agonists results in antinociception in case of inflammatory pain. As the present inventors have identified Ebselen as an A2AR modulator, it can be indicated for pain relief particularly in inflammatory conditions such as endometriosis.

Applications of Ebselen contemplated in some embodiments of the present invention, wherein Ebselen modulates the A2A receptor, are presented below:

- In dermal fibrosis and mechanisms of scarring or regeneration;

- In anti-scarring therapies and the promotion of tissue regeneration;

- In cancer therapy, for example as a checkpoint inhibitor;

- In treating bleeding disorders, for example wherein the bleeding indication is selected from the group consisting of Hemophilia A, Hemophilia B, Factor VIII deficiency, Factor XI deficiency, von Willebrand Disease, Glanzmann's Thrombasthenia, Bernard Soulier Syndrome, idiopathic thrombocytopenic purpura, and bleeding resulting from trauma;

- In treating patients diagnosed with post-traumatic stress disorder;

- In the treatment or prevention of Extrapyramidal Symptoms or Dystonia;

- To treat restless legs syndrome and related disorders;

- To treat periodic leg movement in sleep;

- In the treatment of fragile X syndrome;

- To treat olfactory disorders;

- As an agent to reduce or prevent stenosis that occurs following a revascularization procedure, for example performed on one or more of coronary arteries, renal arteries, leg arteries or carotid arteries and/or for example wherein the revascularization procedure is one of balloon angioplasty, insertion of a stent, insertion of a bypass graft, directional coronary atherectomy or percutaneous transluminal coronary angioplasty, or involves excimer laser angioplasty;

- In the treatment of autoimmune diseases such as Hashimoto's thyroiditis, Guillain-Barre syndrome, and pemphigus;

- In increasing the contractile performance of compromised myocardium;

- In improving cardiac function to increase exercise tolerance; - In the treatment of symptoms of menopause, hypercalcemia, hyperparathyroidism, Paget's disease, with corticosteroid treatment, in implantation and plastic surgery;

- For increasing the permeability of the blood-brain barrier for example in the treatment of brain disorders.

In accordance with a second aspect of the present invention, there is provided a pharmaceutical composition comprising the compound Ebselen, 2-Phenyl-1 ,2- benzisoselenazol-3(2H)-one, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, for use in the treatment or prevention of a disease or condition mediated by the adenosine A2A receptor.

The pharmaceutical composition may be adapted for example for oral, intravenous, intradermal, subcutaneous, intramuscular, nasal, rectal, transdermal, transmucosal, intraperitoneal, or pulmonary administration, or by infusion.

In accordance with a third aspect of the present invention, there is provided a method of treating or preventing a disease or condition mediated by the adenosine A2A receptor, comprising administering to a patient in need thereof, a therapeutically effective amount of the compound Ebselen, 2-Phenyl-1 ,2- benzisoselenazol-3(2/-/)-one, or a pharmaceutically acceptable salt thereof, for example in the form of a pharmaceutical composition in accordance with the second aspect of the present invention.

Figures

Figure 1 illustrates the agonist response of Ebselen (HT6094) vs control at the A2A receptor, in the GPCR cAMP assay described in Example 2 herein.

Figure 2 illustrates the antagonist response of Ebselen (HT6094) vs control at the A2A receptor, in the GPCR cAMP assay described in Example 2 herein.

Figure 3 is a dose-response curve for Ebselen (HT6094) vs control in the A2A Human Adenosine GPCR Cell-Based Antagonist cAMP Assay described in Example 3 herein. Examples

Example 1 - Radioligand binding assay

Purpose

To determine the affinity of Ebselen for the human adenosine A2A receptor in transfected HEK-293 (human embryonic kidney) cells.

Materials and Methods

1) Receptor Source: HEK-293 cells stably transfected with a plasmid encoding the human adenosine A2A receptor and were used to prepare membranes in modified Tris-HCI pH 7.4 (50 mM Tris-HCI, 10 mM MgCl2 and 2 Ul/ml ADA) buffer.

2) Cell membrane homogenates (40 pg protein) were incubated for 120 min at 22°C with 6 nM [^H]CGS 21680 in the absence or presence of Ebselen in 50 mM Tris-HCI (pH 7.4) buffer (*Luthin et al. 1995).

3) Nonspecific binding was determined in the presence of 10 pM NECA (5'-N- Ethylcarboxamidoadenosine).

4) Following incubation, the samples were filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% polyethylenimine.

5) After filtration, the fiber filters were rinsed several times with ice-cold 50 mM Tris-HCI using a 96-sample cell harvester (Unifilter, Packard).

6) The filters were dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).

7) Ebselen was screened at 10 pM. The radioactive ligand in the experiment was [3H]CGS 21680 with Kd 27 nM. (Ebselen was purchased from Cayman Chemical (Item# 70530, www.caymanchem.com) with 99% purity. Ebselen was dissolved in DMSO.) 8) The standard reference compound is NECA, which was tested in each experiment at several concentrations to obtain a competition curve from which its IC50 is calculated.

9) The assay was performed in 96 well plate format with 200pL volume. Ebselen was added at 100X stock dissolved in DMSO.

10)The results were expressed as a percent inhibition of the control radioligand specific binding (binding affinity of Ebeselen).

Results and Conclusions

Ebselen (HT 6094) binds to A2AR 102.58% at 10 pM concentration compared to NECA. 10 pM of Ebselen inhibits 102.58% of agonist radioligand, suggesting Ebselen binds to A2ARs and has the potential to modulate the activity of the A2A receptor. The results are presented in Table 1 below, in which the given values represent binding as a percentage increase above the basal binding and are the average values from two assays.

Table 1 :

(*Luthin DR, Olsson RA, Thompson RD, Sawmiller DR, Linden J. Characterization of two affinity states of adenosine A2a receptors with a new radioligand, 2-[2-(4- amino-3-[125l]iodophenyl)ethylamino]adenosine. Mol Pharmacol. 1995 Feb;47(2):307-13.) Example 2 - cAMP release assay

Purpose

To determine if Ebselen has any functional activity for the human adenosine A2A receptor in transfected HEK-293 (human embryonic kidney) cells.

Materials and Methods

Measurement of changes in cyclic adenosine monophosphate (cAMP) concentration is a widely used method to verify G-protein coupled receptor (GPCR) activation in the adenylyl cyclase pathway. Here, a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay was used to measure cAMP produced upon modulation of adenylyl cyclase activity by adenosine A2A receptors expressed in HEK-293 cells. The assay permits absolute quantification of cellular cAMP level, using radiometric readout.

1 ) Ebselen was purchased from Cayman Chemical (Item# 70530, www.caymanchem.com) with 99% purity. Ebselen was dissolved in DMSO for the assays.

2) The assay was performed in 96 well plate format with 200pL volume. Ebselen was added at 100X stock dissolved in DMSO.

3) DMSO containing Ebselen was incubated with HEK-293 Cells expressing A2ARs (1.25 x 10^A5/ml) in modified HBSS, 5 mM HEPES, 0.1% BSA, 30 pM Rolipram) pH 7.4 buffer at 37°C for 10 minutes.

4) After incubation with Ebselen, the reaction was evaluated for cAMP levels by TR-FRET. TR-FRET quantitation estimates cAMP accumulation (Gao et al. 2001 ; Poucher et al 1995).

Agonist stimulation

5) For the agonist stimulation assay, cells were assayed according to the protocol above. 10 pM of Ebselen was added in the stimulation buffer to activate A2ARs. The control activator was NECA (0.0012 pM).

6) Ebselen-induced cAMP increase relative to the 0.1 pM NECA control response indicated possible adenosine A2A receptor agonist activity. Antagonist stimulation

7) For the antagonist assay, cells were assayed according to the protocol above. 10 pM of Ebselen was added to evaluate the NECA induced cAMP. The control inhibitor was ZM 241385 (0.0095 pM).

8) Ebselen-induced inhibition of the 3 nM NECA-induced cAMP response indicated receptor antagonist activity.

Results and Conclusions

• Agonist stimulation assay:

In the absence of other ligands in the agonist cAMP assay, 10 pM of Ebselen activated the A2A receptor, eliciting a raise of 22.48% of intracellular cyclic AMP in comparison to NECA (EC50 is 0.00116pM) at 100%. These results are illustrated in Figure 1.

• Antagonist stimulation assay

The data shows Ebselen acts as a partial agonist in relatively higher concentrations when compared to the agonist activity of NECA. 10 pM of Ebselen also inhibited the NECA-induced cAMP response, suggesting its activity as an antagonist. In comparison to known antagonist ZM241385, Ebselen inhibited 90.74% of the activity of NECA-induced cAMP response. (Figure 2). Ebselen binds and activates A2AR but did not elicit a complete response. These results are illustrated in Figure 2.

Thus, Ebselen is a partial agonist at A2ARs. Ebselen binds to A2AR as determined by the binding studies, and it inhibited the action of the NECA (a potent agonist of A2ARs), suggesting it also acts as an antagonist. Thus, Ebselen modulates A2ARs, and its activity depends on its concentration and presence/absence of other ligands. Example 3 - Dose-Response Study

Purpose

To determine the ICso concentration of Ebselen for the human adenosine A2A receptor in transfected HEK-293 cells determined in a cAMP release assay. TR- FRET immunoassay was used to measure cAMP produced upon modulation of adenylyl cyclase activity by adenosine A2A receptors expressed in HEK-293 cells.

Materials and Methods

1) Ebselen was purchased from Cayman Chemical (Item# 70530, www.caymanchem.com) with 99% purity. Ebselen was dissolved in DMSO for the assays.

2) Ebselen was screened at 100, 30, 10, 3, 1 , 0.3, 0.1 , 0.03, 0.01 and 0.003 pM concentrations.

3) The control inhibitor ZM 241385 (0.014 pM IC50) and control activator NECA (0.0012 pM EC50) were used in the assay.

4) The assay was performed in 96 well plate format with 200pL volume. Ebselen were added at 100X stock dissolved in DMSO.

5) DMSO containing Ebselen was incubated with HEK-293 cells expressing A2A receptors (1.25 x 10^A5/ml) in modified HBSS, 5 mM HEPES, 0.1% BSA, 30 pM Rolipram) pH 7.4 buffer at 37°C for 10 minutes.

6) After incubation with Ebselen, the reaction was evaluated for cAMP levels by TR-FRET. TR-FRET quantitation estimates cAMP accumulation (**Gao et al. 2001 ; Poucher et al 1995).

7) Ebselen-induced inhibition of the 3 nM NECA-induced cAMP response indicates receptor antagonist activity. Results and Discussion

The results of this study are presented in Figure 3. Ebselen inhibits agonist (NECA) induced cAMP response, acting as an antagonist, with an ICso of 1 .11 pM. The data shows that Ebselen acts as a potent inhibitor of A2A activity.

(**Gao Z, Li Z, Baker SP, Lasley RD, Meyer S, Elzein E, Palle V, Zablocki JA, Blackburn B, Belardinelli L. Novel short-acting A2A adenosine receptor agonists for coronary vasodilation: inverse relationship between affinity and duration of action of A2A agonists. J Pharmacol Exp Ther. 2001 Jul;298(1 ):209-18; Poucher SM, Keddie JR, Singh P, Stoggall SM, Caulkett PW, Jones G, Coll MG. The in vitro pharmacology of ZM 241385, a potent, non-xanthine A2a selective adenosine receptor antagonist. Br J Pharmacol. 1995 Jul;115(6):1096-102.)

Aspects and embodiments of the invention are defined in the following clauses:

1 . The compound Ebselen, 2-Phenyl-1 ,2-benzisoselenazol-3(2/-/)-one, or a pharmaceutically acceptable salt thereof, for use in the treatment or prevention of a disease or condition mediated by the adenosine A2A receptor.

2. A pharmaceutical composition comprising the compound Ebselen, 2- Phenyl-1 ,2-benzisoselenazol-3(2/-/)-one, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, for use in the treatment or prevention of a disease or condition mediated by the adenosine A2A receptor.

3. A method of treating or preventing a disease or condition mediated by the adenosine A2A receptor, comprising administering to a patient in need thereof, a therapeutically effective amount of the compound Ebselen, 2-Phenyl-1 ,2- benzisoselenazol-3(2H)-one, or a pharmaceutically acceptable salt thereof.

4. The compound for use as described in clause 1 , the composition for use as described in clause 2, or the method as described in clause 3, wherein the Ebselen or a pharmaceutically acceptable salt thereof treats or prevents said disease or condition by agonist action at the A2A receptor. 5. The compound for use as described in clause 1 , the composition for use as described in clause 2, or the method as described in clause 3, wherein the Ebselen or a pharmaceutically acceptable salt thereof treats or prevents said disease or condition by antagonist action at the A2A receptor.

6. The compound for use as described in clause 1 , the composition for use as described in clause 2, or the method as described in clause 3, wherein said disease or condition is selected from: stenosis, for example stenosis following a revascularisation procedure performed on, for example, coronary arteries, renal arteries, leg arteries and carotid arteries, for example involving any of balloon angioplasty, insertion of a stent, insertion of a bypass graft, directional coronary atherectomy, percutaneous transluminal coronary angioplasty and excimer laser angioplasty;

Hashimoto’s thyroiditis;

Guillain-Barre syndrome; pemphigus; cardiac inefficiency, for example for the improvement of contractile performance in a compromised myocardium or for the improvement of exercise tolerance; a brain disorder, for example for the improvement of the permeability of the bloodbrain barrier; symptoms of menopause; hypercalcaemia; hyperparathyroidism ;

Paget’s disease; inflammation following implantation or plastic surgery; and a condition treated with a corticosteroid.

7. The compound for use as described in clause 1 , the composition for use as described in clause 2, or the method as described in clause 3, wherein said disease or condition is selected from: dermal fibrosis; scarring; a bleeding disorder, for example Hemophilia A, Hemophilia B, Factor VIII deficiency, Factor XI deficiency, von Willebrand Disease, Glanzmann's Thrombasthenia, Bernard Soulier Syndrome, idiopathic thrombocytopenic purpura, or bleeding resulting from trauma; post-traumatic stress disorder; extrapyramidal symptoms; dystonia; restless legs syndrome; periodic leg movement in sleep; fragile X syndrome; and an olfactory disorder.

8. The compound for use as described in clause 1 , the composition for use as described in clause 2, or the method as described in clause 3, wherein said disease or condition is scarring.

9. The compound for use as described in clause 1 , the composition for use as described in clause 2, or the method as described in clause 3, wherein said disease or condition is dermal fibrosis.

10. The compound for use as described in clause 1 , the composition for use as described in clause 2, or the method as described in clause 3, wherein said disease or condition is a bleeding disorder selected from Hemophilia A and Hemophilia B.

Claims

2. The compound for use as claimed in claim 1 , wherein said disease or condition is a bleeding disorder.

3. The compound for use as claimed in claim 2, wherein the bleeding disorder is selected from Haemophilia A, Haemophilia B, and von Willebrand’s disease.

4. The compound for use as claimed in claim 1 or claim 2, wherein said use is in combination with a clotting factor, for example Factor VIII, Factor IX, Factor XI, recombinant Factor VIII, recombinant Factor IX or recombinant Factor XI.

5. The compound for use as claimed in claim 2, wherein said use is as a replacement therapy in regular treatment or during surgery.

6. The compound for use as claimed in claim 1 , wherein said disease or condition is a disease of the reproductive system.

7. The compound for use as claimed in claim 6, wherein said disease or condition is an endometrial disease.

8. The compound for use as claimed in claim 6 or claim 7, wherein said disease or condition is selected from infertility, endometriosis, and premature birth risk.

9. The compound for use as claimed in claim 1 , wherein in the disease or condition is pain, for example inflammatory pain in endometriosis.

10. A pharmaceutical composition comprising the compound Ebselen, 2- Phenyl-1 ,2-benzisoselenazol-3(2H)-one, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, for use in the treatment or prevention of a disease or condition mediated by the adenosine A2A receptor.

11 . The pharmaceutical composition for use as claimed in claim 10, wherein the disease or condition mediated by the adenosine A2A receptor is a disease or condition according to any one of claims 2-9.

12. A method of treating or preventing a disease or condition mediated by the adenosine A2A receptor, comprising administering to a patient in need thereof, a therapeutically effective amount of the compound Ebselen, 2-Phenyl-1 ,2- benzisoselenazol-3(2H)-one, or a pharmaceutically acceptable salt thereof.

13. The method as claimed in claim 12, wherein the disease or condition mediated by the adenosine A2A receptor is a disease or condition according to any one of claims 2-9.

14. The compound for use as claimed in claim 1 , the composition for use as claimed in claim 10, or the method as claimed in claim 12, wherein the Ebselen or a pharmaceutically acceptable salt thereof treats or prevents said disease or condition by agonist action at the A2A receptor.

15. The compound for use as claimed in claim 1 , the composition for use as claimed in claim 10, or the method as claimed in claim 12, wherein the Ebselen or a pharmaceutically acceptable salt thereof treats or prevents said disease or condition by antagonist action at the A2A receptor.

16. The compound for use as claimed in claim 1 , the composition for use as claimed in claim 10, or the method as claimed in claim 12, wherein said disease or condition is selected from: stenosis, for example stenosis following a revascularisation procedure performed on, for example, coronary arteries, renal arteries, leg arteries and carotid arteries, for example involving any of balloon angioplasty, insertion of a stent, insertion of a bypass graft, directional coronary atherectomy, percutaneous transluminal coronary angioplasty and excimer laser angioplasty;

Hashimoto’s thyroiditis;

Guillain-Barre syndrome; pemphigus;; cardiac inefficiency, for example for the improvement of contractile performance in a compromised myocardium or for the improvement of exercise tolerance; a brain disorder, for example for the improvement of the permeability of the bloodbrain barrier; symptoms of menopause; hypercalcaemia; hyperparathyroidism ;

17. The compound for use as claimed in claim 1 , the composition for use as claimed in claim 10, or the method as claimed in claim 12, wherein said disease or condition is selected from: dermal fibrosis; scarring; a bleeding disorderfor example Hemophilia A, Hemophilia B, Factor VIII deficiency, Factor XI deficiency, von Willebrand Disease, Glanzmann's Thrombasthenia, Bernard Soulier Syndrome, idiopathic thrombocytopenic purpura, or bleeding resulting from trauma; post-traumatic stress disorder; extrapyramidal symptoms; dystonia; restless legs syndrome; periodic leg movement in sleep; fragile X syndrome; and an olfactory disorder.

18. The compound for use as claimed in claim 1 , the composition for use as claimed in claim 10, or the method as claimed in claim 12, wherein said disease or condition is scarring.

19. The compound for use as claimed in claim 1 , the composition for use as claimed in claim 10, or the method as claimed in claim 12, wherein said disease or condition is dermal fibrosis.

20. The compound for use as claimed in claim 1 , the composition for use as claimed in claim 10, or the method as claimed in claim 12, wherein said disease or condition is a bleeding disorder selected from Hemophilia A and Hemophilia B.