FIELD OF THE INVENTION

This invention relates to the use of a FAK-inhibitor and pharmaceutical formulations containing said FAK inhibitor, for the treatment of pulmonary arterial hypertension (PAH).

BACKGROUND TO THE INVENTION

Pulmonary arterial hypertension is a condition which is characterised by changes to the pulmonary vasculature (including thickening of the pulmonary vessel walls and vascular remodelling) leading to high resistance to blood flow through the lungs. This in turn results in increased stress on the heart and a compromised blood circulation through the whole body as a result of decreased cardiac output. This can severely impact a patient's ability to exercise or carry out normal daily activities. Symptoms may include dyspnoea (breathlessness), fatigue, dizziness, peripheral oedema, chest pain. Some of these symptoms are more prevalent during, and may therefore limit, physical activity. Diagnosis can be difficult, as these symptoms may also be present in more common respiratory disorders such as asthma and COPD. However, without appropriate treatment these symptoms become more severe over time and can severely limit the ability to carry out everyday activities. Eventually the condition can lead to right ventricular (RV) failure and death.

Pulmonary Arterial Hypertension (PAH) has been considered primarily a consequence of abnormal pulmonary vasoconstriction, but more recently it has been proposed that the disease may be caused mainly by pulmonary vascular remodelling with features analogous to carcinogenesis (Guignabert et al., Eur Respir Rev 2013; 22: 543-551).

The currently available PAH therapies are vasodilators and provide symptomatic support, which improves the quality of life, but do not fully address the underlying remodelling aspects of the disease. There is thus a significant unmet need for a specific disease modifying PAH therapy.

Paulin et al (Eur Respir J 2014; 43: 531-544) demonstrated that oral delivery of the FAK- inhibitor PF-573 228 inhibited rat monocrotaline induced PAH, and that in vitro, FAK was activated in PAH human lungs or pulmonary arterial smooth muscle cells (PASMC) when compared to those from healthy subjects.

Focal adhesion kinase (FAK, also known as Protein Tyrosine Kinase 2, PTK2), is one of the major proteins that regulate cell migration but its role in PAH is not fully understood.

WO2010/062578 describes pyrazolylaminopyridines that are said to inhibit Focal Adhesion Kinase (FAK), as well as compositions thereof. Said compounds are described as being useful for the treatment of cellular proliferation diseases, such as cancer, autoimmune disease, fungal disorders, arthritis, graft rejection, inflammatory bowel disease, proliferation induced after medical procedures, including, but not limited to, surgery, angioplasty, and the like. WO2010/062578 specifically describes inter alia the compound 2-[(5-chloro-2-{[3-methyl-l-(l-methylethyl)-l pyrazol-5-yl]amino}-4-pyridinyl)amino]-A ^£ -(methyloxy)benzamide.

SUMMARY OF THE INVENTION

In a first aspect the present invention provides the compound of structure (I) or a pharmaceutically acceptable salt thereof for use in the treatment of pulmonary arterial hypertension (PAH).

The compound of structure (I) is:

Structure (I)

Said compound may be named as:

2-[(5-Chloro-2-{[3-methyl-l-(l-methylethyl)-l pyrazol-5-yl]amino}-4-pyridinyl)amino]-A ^£ - (methyloxy)benzamide.

In a second aspect the present invention provides the use of the compound of structure (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the treatment of pulmonary arterial hypertension.

In a third aspect the present invention provides a method of treating pulmonary arterial hypertension, which method comprises administering a therapeutically effective amount of the compound of structure (I) or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

In a fourth aspect the present invention provides a pharmaceutical composition comprising the compound of structure (I) or a pharmaceutically acceptable salt thereof for use in the treatment of pulmonary arterial hypertension.

In a fifth aspect the present invention provides the use of a pharmaceutical composition comprising a compound of structure (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the treatment of pulmonary arterial hypertension.

In a sixth aspect the present invention provides a method of treating pulmonary arterial hypertension, which method comprises administering a therapeutically effective amount of the composition comprising a compound of structure (I) or a pharmaceutically acceptable salt thereof, to a subject in need thereof. In a seventh aspect the present invention provides a combination of the compound of Structure (I) or a pharmaceutically acceptable salt thereof with another therapeutic agent suitable for the treatment of PAH.

DESCRIPTION OF DRAWINGS/ FIGURES

FIG. 1 Represents the migration of human pulmonary arterial smooth muscle cells under the conditions of Experiment 1 wherein the membrane was coated only on the upper side (condition A). Cell index +/- SD, n=2. Key: Control no chemo attractant: Diamond. No chemoattractant, Compound A 5μΜ: Square. With chemo attractant (PDGF-BB+EGF): Circle. With chemo attractant (PDGF-BB+EGF) + Compound A 5μΜ: Triangle.

FIG. 2 Represents the migration of human pulmonary arterial smooth muscle cells under the conditions of Experiment 1 wherein the membrane was coated on both the upper side and lower side (condition B). Cell index +/- SD, n=2. Key:Control no chemo attractant: Diamond. No chemoattractant, Compound A 5μΜ: Square. With chemo attractant (PDGF-BB+EGF): Circle. With chemo attractant (PDGF-BB+EGF) + Compound A 5μΜ: Triangle.

FIG. 3 Analysis of figure 1 curves. Slopes were calculated for each individual points (2 per condition) between the time point 0:30:31 and 1:30:55

FIG. 4 Analysis of figure 2 curves. Slopes were calculated for each individual points (2 per condition) between the time point 0:30:31 and 1:30:55.

FIG. 5 Represents the migration of human pulmonary arterial smooth muscle cells under the conditions of Experiment 2. Cell index +/- SD, n=2. Key: Control no chemo attractant: diamond. With chemo attractant (PDGF-BB+EGF): circle. With chemo attractant (PDGF-BB+EGF) + Compound A 10μΜ: square. With chemo attractant (PDGF-BB+EGF) + Compound A 20μΜ: triangle.

FIG. 6 Analysis of figure 5 curves. Slopes were calculated for each individual points (2 per condition) between the time point 0:37:32 and 1:27:48.

FIG. 7 Represents the migration of human pulmonary arterial smooth muscle cells under the conditions of Experiment 2. Cell index n=2. Key: Black square, black circle, black triangle, black diamond, open circle, open square, open triangle : chemo attractant alone; chemo attractant + Compound A at 150nM, 310nM, 630nM, 1.25μΜ, 2.5μΜ and 5μΜ respectively.

FIG. 8 Dose response of Compound A on human pulmonary arterial smooth muscle cell migration. Cell index n=2.

FIG. 9 Represents the migration of human pulmonary arterial smooth muscle cells under the conditions of Experiment 3. Cell index n=2.Key: Black square, black circle, black triangle, black diamond, open circle, open square, open triangle : chemo attractant alone; chemo attractant + Compound A at 310nM, 630nM, 1.25μΜ, 2.5μΜ, 5μΜ, and 10μΜ respectively.

FIG. 10 Dose response of Compound A on HPASMC migration. Cell index, n=2. DETAILED DESCRIPTION OF THE INVENTION

STATEMENT OF THE INVENTION

In a first aspect the present invention provides the compound of structure (I) or a pharmaceutically acceptable salt thereof for use in the treatment of pulmonary arterial hypertension (PAH).

The compound of structure (I) viz:

-t S-Chloro- -itS-methyl-l- l-methylethy -l pyrazol-S-ynamino^-pyridiny amino]-/^ (methyloxy)benzamide; which may be depicted as:

Structure (I) and salts thereof, are described in WO2010/062578. WO2010/062578 also describes the preparation of the compound of structure (I), pharmaceutical formulations containing it and its use in therapy. The preparation of the monohydrochloride salt is also described in WO2010/062578 .

As used herein the term 'the compound of Structure (I)' may also refer to the compound in salt form, unless the context dictates otherwise.

We have carried out in vitro studies with the compound of structure (I), which indicate (a) that PTK2 mRNA coding for the FAK protein is strongly expressed in human pulmonary endothelial and smooth muscle cells and (b) that the compound of structure (I) is able to inhibit migration and/or adhesion of pulmonary smooth muscle cells.

As a prerequisite for a compound to be clinically effective in PAH, it is necessary for there to be expression of autoactivated, signalling-ready FAK, and also drug exposure in the relevant tissues. Uptake of the compound of structure (I) within the lung and/or heart can be identified by radio- imaging studies using a radio-labelled form of the compound of Structure (I), as described hereinafter.

The compound of structure (I) may form pharmaceutically acceptable salts by treatment with a suitable acid. Suitable acids include pharmaceutically acceptable inorganic acids and organic acids. Representative pharmaceutically acceptable acids include hydrogen chloride, hydrogen bromide, nitric acid, sulfuric acid, sulfonic acid, phosphoric acid, acetic acid, hydroxyacetic acid, phenylacetic acid, propionic acid, butyric acid, valeric acid, maleic acid, acrylic acid, fumaric acid, malic acid, malonic acid, tartaric acid, citric acid, salicylic acid, benzoic acid, tannic acid, formic acid, stearic acid, lactic acid, ascorbic acid, />toluenesulfonic acid, oleic acid, and lauric acid. The compound of structure (I) may exist in a crystalline or noncrystalline form, or as a mixture thereof. The skilled artisan will appreciate that pharmaceutically acceptable solvates may be formed for crystalline compounds wherein solvent molecules are incorporated into the crystalline lattice during crystallization. The incorporated solvent molecules may be water molecules or non- aqueous such as ethanol, isopropanol, DMSO, acetic acid, ethanolamine, and ethyl acetate molecules. Crystalline lattice incorporated with water molecules are typically referred to as "hydrates." Hydrates include stoichiometric hydrates as well as compositions containing variable amounts of water.

In a second aspect the present invention provides the use of the compound of structure (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the treatment of pulmonary arterial hypertension.

In a third aspect the present invention provides a method of treating pulmonary arterial hypertension, which method comprises administering a therapeutically effective amount of the compound of structure (I) or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

In a fourth aspect the present invention provides a pharmaceutical composition comprising the compound of structure (I) or a pharmaceutically acceptable salt thereof for use in the treatment of pulmonary arterial hypertension.

In a fifth aspect the present invention provides the use of a pharmaceutical composition comprising the compound of structure (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for use in the treatment of pulmonary arterial hypertension.

In a sixth aspect the present invention provides a method of treating pulmonary arterial hypertension, which method comprises administering a therapeutically effective amount of a composition comprising the compound of structure (I) or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

In a seventh aspect the present invention provides a combination of the compound of Structure (I) or a pharmaceutically acceptable salt thereof with another therapeutic agent suitable for the treatment of pulmonary arterial hypertension. PHARMACEUTICAL COMPOSITIONS/ROUTES OF ADMINISTRATION/DOSAGES

While it is possible that, for use in therapy, the compound of structure (I), as well as salts, solvates and the like, may be administered as a neat preparation, i.e. no additional carrier, the more usual practice is to present the active ingredient confected with a carrier or diluent. Accordingly, the invention further provides a pharmaceutical composition, comprising the a compound of structure (I) or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients. The carrier(s), diluent(s) or excipient(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. In accordance with another aspect of the invention there is also provided a process for the preparation of a pharmaceutical formulation including admixing the compound of structure (I), or a pharmaceutically acceptable salt thereof , with one or more pharmaceutically acceptable carriers, diluents or excipients.

The compound of structure (I) and salts thereof may be formulated in any convenient and appropriate manner, including as described in WO2010/062578. Pharmaceutical compositions may for example be presented in unit dose forms containing a predetermined amount of active ingredient per unit dose. Such a unit may contain, for example, 0.5 mg to 3500mg, preferably 1 mg to 700 mg, more preferably 5 mg to 100 mg of the compound of the structure (I), depending on the condition being treated, the route of administration and the age, weight and condition of the patient. Preferred unit dosage compositions are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fraction thereof, of the active ingredient. Furthermore, such pharmaceutical compositions may be prepared by any of the methods well known in the pharmacy art.

Pharmaceutical compositions may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route, or by inhalation (including via the mouth or nose). Such compositions may be prepared by any method known in the art of pharmacy, for example by bringing into association the compound of structure (I) with one or more carrier(s) or excipient(s).

Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or nonaqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.

Capsules are made by preparing a powder mixture, as described above, and filling formed gelatin sheaths. Glidants and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate or solid polyethylene glycol can be added to the powder mixture before the filling operation. A disintegrating or solubilizing agent such as agar-agar, calcium carbonate or sodium carbonate can also be added to improve the availability of the medicament when the capsule is ingested.

Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Tablets are formulated, for example, by preparing a powder mixture, granulating or slugging, adding a lubricant and disintegrant and pressing into tablets. A powder mixture is prepared by mixing the compound, suitably comminuted, with a diluent or base as described above, and optionally, with a binder such as carboxymethylcellulose, an aliginate, gelatin, or polyvinyl pyrrolidone, a solution retardant such as paraffin, a resorption accelerator such as a quaternary salt and/or an absorption agent such as bentonite, kaolin or dicalcium phosphate. The powder mixture can be granulated by tablet forming dies by means of the addition of stearic acid, a stearate salt, talc or mineral oil. The lubricated mixture is then compressed into tablets. The compounds of the present invention can also be combined with a free flowing inert carrier and compressed into tablets directly without going through the granulating or slugging steps. A clear or opaque protective coating consisting of a sealing coat of shellac, a coating of sugar or polymeric material and a polish coating of wax can be provided. Dyestuffs can be added to these coatings to distinguish different unit dosages.

Oral fluids such as solution, syrups and elixirs can be prepared in dosage unit form so that a given quantity contains a predetermined amount of a compound of structure (I). Syrups can be prepared by dissolving the compound in a suitably flavored aqueous solution, while elixirs are prepared through the use of a non-toxic alcoholic vehicle. Suspensions can be formulated by dispersing the compound in a non-toxic vehicle. Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols and polyoxy ethylene sorbitol ethers, preservatives, flavor additive such as peppermint oil or natural sweeteners or saccharin or other artificial sweeteners, and the like can also be added.

Where appropriate, dosage unit pharmaceutical compositions for oral administration can be microencapsulated. The formulation can also be prepared to prolong or sustain the release as for example by coating or embedding particulate material in polymers, wax or the like.

Pharmaceutical formulations adapted for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The pharmaceutical compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

For administration by inhalation the compound of structure (I) or a pharmaceutically acceptable salt thereof may be formulated for example as a dry powder, an aerosol, a suspension, or a solution composition. Dry powder compositions for delivery to the lung by inhalation may comprise the compound of structure (I) or a pharmaceutically acceptable salt thereof as a finely divided powder together with one or more pharmaceutically-acceptable excipients as finely divided powders. Pharmaceutically-acceptable excipients particularly suited for use in dry powders are known to those skilled in the art and include lactose, starch, mannitol, and mono-, di-, and polysaccharides. The finely divided powder may be prepared by, for example, micronisation, milling or spray drying. Generally, the size-reduced (eg micronised) compound can be defined by a D ₅₀ value of about 1 to about 10 microns (for example as measured using laser diffraction).

The dry powder may be administered to the patient via a reservoir dry powder inhaler (RDPI) having a reservoir suitable for storing multiple (un-metered doses) of medicament in dry powder form. RDPIs typically include a means for metering each medicament dose from the reservoir to a delivery position. For example, the metering means may comprise a metering cup, which is movable from a first position where the cup may be filled with medicament from the reservoir to a second position where the metered medicament dose is made available to the patient for inhalation.

Alternatively, the dry powder may be presented in capsules (e.g. gelatin or plastic), cartridges, or blister packs for use in a multi-dose dry powder inhaler (MDPI). MDPIs are inhalers wherein the medicament is comprised within a multi-dose pack containing (or otherwise carrying) multiple defined doses (or parts thereof) of medicament. When the dry powder is presented as a blister pack, it comprises multiple blisters for containment of the medicament in dry powder form. The blisters are typically arranged in regular fashion for ease of release of the medicament therefrom. For example, the blisters may be arranged in a generally circular fashion on a disc-form blister pack, or the blisters may be elongate in form, for example comprising a strip or a tape.

Aerosols may be formed by suspending or dissolving a compound of structure (I) or a pharmaceutically acceptable salt thereof in a liquified propellant. Suitable propellants include halocarbons, hydrocarbons, and other liquified gases. Representative propellants include: trichlorofluoromethane (propellant 11), dichlorofluoromethane (propellant 12), dichlorotetrafluoroethane (propellant 114), tetrafluoroethane (HFA-134a), 1,1-difluoroethane (HFA- 152a), difluoromethane (HFA-32), pentafluoroethane (HFA-12), heptafluoropropane (HFA-227a), perfluoropropane, perfluorobutane, perfluoropentane, butane, isobutane, and pentane. Aerosols comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof will typically be administered to a patient via a metered dose inhaler (MDI). Such devices are known to those skilled in the art.

The aerosol may contain additional pharmaceutically-acceptable excipients typically used with MDIs such as surfactants, lubricants, cosolvents and other excipients to improve the physical stability of the formulation, to improve valve performance, to improve solubility, or to improve taste. A therapeutically effective amount of the compound of structure (I) will depend upon a number of factors including, for example, the age and weight of the intended recipient, the precise condition requiring treatment and its severity, the nature of the formulation, and the route of administration, and will ultimately be at the discretion of the attendant prescribing the medication. However, an effective amount of the compound of structure (I) will generally be in the range of 0.001 to 100 mg/kg body weight of recipient per day, suitably in the range of .01 to 10 mg/kg body weight per day. For a 70kg adult the actual amount per day would suitably be from 7 to 700 mg and this amount may be given in a single dose per day or in a number (such as two, three, four, five or six) of sub-doses per day such that the total daily dose is the same. An effective amount of a salt or solvate, etc., may be determined as a proportion of the effective amount of the compound of structure (I) per se. It will be appreciated that a therapeutically effective amount should be both safe and effective.

COMBINATIONS

The compound of Structure (I) or its pharmaceutically acceptable salts can be combined with or co-administered with other therapeutic agents, including other agents for the treatment of

PAH. Such agents include vasodilators, eg epoprostenol (Flolan™), tadalafil (Adcirca™) or ambrisentan (Volibris™) By the term "co-administered" and derivatives thereof as used herein is meant either simultaneous administration or any manner of separate sequential administration of the compound of Structure (I) or a pharmaceutically acceptable salt thereof and a further active ingredient or ingredients, useful in the treatment of PAH. Co- administration may be separate, sequential or simultaneous. Furthermore, the compounds may be administered in the same or separate dosage forms, e.g. one compound may be administered by inhalation and another compound may be administered orally.

GENERAL SYNTHETIC ROUTES

The compound of structure (I) or its salts may be prepared as described in

WO2010/062578, eg in Examples 41(a) and 41(b).

ABBREVIATIONS

EGF - Epidermal growth factor

PDGF-BB - platelet derived growth factor, isoform BB

HPASMC - human pulmonary arterial smooth muscle cells

HPAEC - human pulmonary arterial endothelial cells BIOLOGICAL DATA

In the following experiments the compound of Structure (I) is used as the hydrochloride salt (2-[(5- chloro-2-{[3-methyl-l-(l-methylethyl)-l pyrazol-5-yl]amino}-4-pyridinyl)amino]-/V- (methyloxy)benzamide monohydrochloride) and is identified as Compound A

1- Studies of the mRNA expression of FAK in human pulmonary arterial smooth muscle cells (HPASMC) and human pulmonary arterial endothelial cells (HPAEC).

In order to demonstrate expression of the messenger RNA coding for the FAK protein (PTK2), we performed quantitative PCR on cells extracts. These cell extracts were from non- diseased tissue samples. We used 3 individual donors for primary human pulmonary arterial endothelial cell and 2 individual donors for primary human pulmonary artery smooth muscle cell. The results are presented in Table 1.

Table 1

hSAPDH (Ct) hActin (Ct) hcyclo (Ct) hB2M Ct) hPTK2 (Ct)

Mean SD Mean SD Mean SD Mean SD Mean SD

HPAEC donor 1 18.73 0.02 13,66 0, 14 19.66 0.09 19.33 0.04 22.82 0.12

HPAEC donor 2 18.64 0.09 18.59 0.13 19.79 0, 10 1B.77 0.34 22.80 0.07

HPAEC donor 3 18.22 0.03 18,10 0.08 19.34 0,09 1B.60 0.06 22.63 0.38

H PASMC donor 2 18.73 0.33 1122 0,09 20.41 0.24 20.01 0,11 23.74 0.19

H ASMC donor 3 18.08 0.04 17.99 0,08 19.90 0,08 19.59 0.15 23.04 0.09

As shown in Table 1 PTK2 mRNA seems to be strongly expressed in both cell types, and not donor related.

2- Studies of the migration of human pulmonary arterial smooth muscle cells (HPASMC)

In order to investigate the effect of the FAK inhibitor compounds on the capacity of lung smooth muscle cells to migrate toward a chemo attractant stimulus, we used label-free real time monitoring technology, CIM-Plate 16 from ACEA biosciences on an Xcellingence RTCA (real-time cell analyser) instrument. CIM-Plates 16 are devices used for performing cell invasion and cell migration assays on the RTCA DP (dual plate) Instrument.

Technology:

The CIM-Plate 16 comprises a plate cover, an upper chamber and a lower chamber. The upper chamber has 16 wells that are sealed at the bottom with a microporous polyethylene terephthalate (PET) membrane containing microfabricated gold electrode arrays on the bottom side of the membrane. The median pore size of this membrane is 8 μιτι. The lower chamber has 16 wells, each of which serves as a reservoir for media and any chemo attractant for the cells in corresponding upper chamber wells. Cells seeded in the upper chamber move through the microporous membrane into the lower chamber that contains a chemo attractant. Cells adhering to the microelectrode sensors affect the local ionic environment at the electrode/solution interface and lead to an increase in impedance, which is measured in real-time by the RTCA DP Instrument. The more cells are attached on the electrodes, the larger the increases in electrode impedance. Thus, electrode impedance, which is displayed as cell index (CI) values, can be used to monitor cell migration toward the PET membrane.

Results:

Experiment 1:

In this experiment, we aimed to address the effect of the FAK inhibitor on cell migration in two different conditions: In the first set (condition A), CIM-platel6 were coated with fibronectin only on the top of the PET membrane. In the second condition (condition B), both sides of the PET membranes were coated with fibronectin.

In each condition, 20000 HPASMC cells were seeded in the upper chamber. Lower chambers were filled with a growth media containing medium 231/ 0.5% FBS +/- chemo attractant cocktail (EGF 5ng/ml + PDGF-BB 5ng/ml). Wells without the chemo attractant cocktail were used as basal signal for analysis.

The compound of structure (I) (Compound A) was delivered in the upper chamber at the unique concentration of 5μΜ, before cell seeding. Impedance was measured as soon as possible after cell seeding.

The results are shown in Figs. 1-4 hereinafter.

Figure 1 below represents kinetic data of condition A. Figure 2 represents kinetic data of condition B.

Figure 1: HPASMC migration. Membrane were coated only on the upper side (condition A). Cell index +/- SD, n=2. Key: Control no chemo attractant: Diamond. No chemoattractant, Compound A 5μΜ: Square. With chemo attractant (PDGF-BB+EGF): Circle. With chemo attractant (PDGF-BB+EGF) + Compound A 5μΜ: Triangle.

Figure 2: HPASMC migration. Membrane were coated on both the upper side and lower side (condition B). Cell index +/- SD, n=2. Key: Control no chemo attractant: Diamond. No chemoattractant, Compound A 5μΜ: Square. With chemo attractant (PDGF-BB+EGF): Circle. With chemo attractant (PDGF-BB+EGF) + Compound A 5μΜ: Triangle.

Figure 3 and 4 are slope analyses of the curves presented figure 1 and 2 respectively. One may conclude that migration is stronger (more important) using the condition B, as shown by a stronger increased of the Cell Index (~1.5 CI in condition B vs. ~0.7 CI for condition A). This is in accordance with the fact that in condition B the membrane that the cells need to cross is coated on both side, allowing a better migration after cells crossed. When there is no chemo attractant (PDGF-BB+EGF) in the lower chamber, no migration could be evidenced, and the compound of structure (I) has little if any effect. On the contrary, on the strong chemo attractant induced migration, the compound of structure (I) at 5μΜ is able to inhibit migration (35% ± 16 inhibitions in condition A, 53% ± 13 migration inhibition in condition B).

Figure 3: Analysis of figure 1 curves. Slopes were calculated for each individual points (2 per condition) between the time point 0:30:31 and 1:30:55

Figure 4: Analysis of figure 2 curves. Slopes were calculated for each individual points (2 per condition) between the time point 0:30:31 and 1:30:55.

Experiment 2:

In this experiment, we aimed to address the effect of Compound A on cell migration using two high concentrations of the compound of structure (I). As previously, 20000 HPASM cells were seeded in the upper chamber. Lower chambers were filled with a growth media containing medium 231/ 0.5% FBS +/- chemo attractant cocktail (EGF 5ng/ml + PDGF-BB 5ng/ml). Wells without the chemo attractant cocktail were used as basal signal for analysis. The migration membrane was fibronectin coated in both sides. The compound of structure (I) was delivered in the upper chamber at the concentration of 10 μΜ or 20 μΜ, before cell seeding. Impedance was measured as soon as possible after cell seeding, (figure 5 and 6).

Figure 5: HPASMC migration. Cell index +/- SD, n=2. Key: Control no chemo attractant: diamond. With chemo attractant (PDGF-BB+EGF): circle. With chemo attractant (PDGF-BB+EGF) + Compound A 10μΜ: square. With chemo attractant (PDGF-BB+EGF) + Compound A 20μΜ: triangle.

Figure 6: Analysis of figure 5 curves. Slopes were calculated for each individual points (2 per condition) between the time point 0:37:32 and 1:27:48.

As shown in Figs 5 and 6,on the strong chemo attractant induced migration, the compound of structure (I) at 10 μΜ is able to inhibit migration by 59% ± 5 and by 65% ± 0.3 when using Compound A at 20μΜ.

In this experiment, we also used an independent CIM-platel6 to test dose response of Compound A. The protocol was the same than described above and concentration of the compound of structure (I) ranged from 156nM to 5μΜ. (figure 7).

Figure 7: HPASMC migration. Cell index n=2. Key: Black square, black circle, black triangle, black diamond, open circle, open square, open triangle : chemo attractant alone; chemo attractant + Compound A at 150nM, 310nM, 630nM, 1.25μΜ, 2.5μΜ, 5μΜ, respectively.

Analysis of the migration curves of figure 7 was performed (figure 8). Compound A demonstrate an IC50 of 1.75μΜ in this experiment.

Figure 8: Dose response of Compound A on HPASMC migration. Cell index n=2.

Experiment 3:

In this experiment, we aimed to repeat the dose response of Compound A on inhibition of HPASMC migration seen in experiment 2. We used the same protocol but amended the concentrations used. Curves of migration are shown in figure 9 and analysis in figure 10

Figure 9: HPASMC migration. Cell index n=2. Key: Black square, black circle, black triangle, black diamond, open circle, open square, open triangle : chemo attractant alone; chemo attractant + Compound A at 310nM, 630nM, 1.25μΜ, 2.5μΜ, 5μΜ, ΙΟμΜ, respectively.

Figure 10: Dose response of Compound A on HPASMC migration. Cell index, n=2.

In this particular experiment, Compound A demonstrated an IC50 of ~500nM (=518nM) on the inhibition of migration of HPASMC.

Experiment 4 - PET Imaging study using Compound A

This PET imaging study using a microdose of "C-radiolabelled Compound A is designed to enable identification of the cardiopulmonary sites where there is both expression of autoactivated, signalling-ready, FAK and drug exposure.

Objective(s)/ Endpoint(s)

Overall Design

• This will be a cross-sectional study exploring the uptake of [ Q- Compound A in PAH

patients and healthy age and gender-matched controls.

• This is a non-randomised, open label study where all volunteers will receive one microdose of the radiolabeled compound.

Treatment Arms and Duration

• The total duration of the study for each subject will be approximately 2 months from

screening to follow-up. The screening period will be followed by 1 scanning day and the follow-up visit. • All subjects will receive one microdose of [ Q- Compound A. The maximum amount of radioactivity injected during the -PET scan will be 500 MBq and maximum mass of [ ⁿ C]- Compound A administered will be <^g.

• Subject completion for the primary endpoint is defined as a subject who completes the PET- CT scan visit.

Type and Number of Subjects

• Up to 12 healthy subjects and 12 PAH subjects will be enrolled to provide sufficient PET data to quantify the uptake and distribution of [ Q- Compound A.

• If subjects prematurely discontinue the study, additional subjects may be enrolled as

replacment subjects at the discretion of the Investigator/GSK Medical Monitor

STUDY TREATMENT

Investigational Product and Other Study Treatment

The term 'study treatment' is used herein to describe any combination of products received by the subject as per the study design. Study treatment may therefore refer to the individual study treatments or the combination of those study treatments. In this study, the 'study treatment' is considered a microdose.

preparation of radio-pharmaceutical products to be administered to humans.

RESULTS