Combinations of MEK and PKC inhibitors for treating stroke

Abstract

The invention provides a MEK inhibitor for use in the treatment of stroke, wherein the MEK inhibitor is administered simultaneously, sequentially, or separately with a PKC inhibitor. The invention also provides a pharmaceutical composition comprising the MEK inhibitor and PKC inhibitor, a method of treating stroke comprising administration of a MEK inhibitor and a PKC inhibitor, and a kit of parts comprising a MEK inhibitor and a PKC inhibitor formulated for simultaneous and sequential use.

Description

[0001] COMBINATIONS OF MEK AND PKC INHIBITORS FOR TREATING STROKE

[0002] Field of the invention

[0003] The present invention relates to the treatment of stroke and associated conditions. In particular, the invention relates to MEK inhibitors and PKC inhibitors and their use as a medicament for the treatment of stroke and associated conditions.

[0004] Background of the invention

[0005] Stroke is a leading cause of death, neurological damage, and a major cause of longterm disabilities. Ischemic stroke, in which blood flow to the brain is stopped because of a blood clot, is the most common type of stroke, while haemorrhagic stroke is responsible for more deaths and disability. Subarachnoid haemorrhage (SAH) is caused by bleeding in the space that surrounds the brain. Most often, it occurs when a weak area in a blood vessel (aneurysm) on the surface of the brain bursts and leaks. The blood then builds up around the brain and inside the skull, increasing intracranial pressure on the brain. SAH is difficult to treat and can be fatal. It has been discovered that the damage is associated with early enhanced expression of contractile receptors via protein kinase induced activation (Edvinsson and Povlsen, J. Cereb. Blood Flow Metab. , 2011 , 31 (7), 1554-1571 ). The damage can be mitigated following early blockade of this pathway (Edvinsson and Krause, Transl. Stroke Res., 2024, DOI: 10.1007 / s12975-024-01234-z).

[0006] Patients who initially survive the damage, whether it is from a large blood clot or the rupture of an aneurysm, may experience a devastating turn for the worse days later and die or suffer permanent neurologic deficits (Macdonald, Nat. Rev. Neurol., 2014, 10, 44-58). This is attributed to secondary cerebral ischaemia (also called delayed cerebral ischemia (DCI)) following aneurysmal subarachnoid hemorrhage (aSAH), caused by vasospasm: constriction of blood vessels and restriction of blood flow in the brain. Blood supply to parts of the brain can become reduced to a dangerous extent, thereby disrupting the normal functions of the brain. After confirmation that the SAH was caused by a brain aneurysm, a surgical procedure to repair the affected blood vessel and prevent the aneurysm from bursting again may be recommended. This can be carried out using one of 2 main techniques: coiling and clipping. Clipping involves opening the skull to locate the aneurysm and placing clips around the neck of the aneurysm. Coiling involves advancing a catheter through the arteries that supply the brain via the groin (or other peripheral artery), and depositing platinum coils inside the aneurysm, causing a blood clot to form which limits blood flow, thereby limiting the amount of blood that can leak from the damaged aneurysm.

[0007] Secondary cerebral ischemia can be mitigated by pharmacological intervention with the calcium channel blocker nimodipine. The dosing of nimodipine recommended by the UK’s National Institute for Health and Care Excellence is 60 mg every 4 hours, to be started within 4 days of aneurysmal subarachnoid haemorrhage and continued for 21 days.

[0008] Nevertheless, current treatment options to restore and maintain blood flow and oxygen to protect the brain in the event of aSAH are limited. There remains a need for improved treatments for ischemic damage in the brain.

[0009] Summary of the invention

[0010] The invention provides a MEK inhibitor for use in the treatment of stroke, wherein the MEK inhibitor is administered simultaneously, sequentially, or separately with a PKC inhibitor.

[0011] The invention also provides a method of treating stroke, comprising administration of a MEK inhibitor and a PKC inhibitor to a patient, wherein the MEK inhibitor and the PKC inhibitor are administered simultaneously, sequentially, or separately.

[0012] The invention also provides a pharmaceutical composition comprising a MEK inhibitor and a PKC inhibitor.

[0013] The invention also provides a kit of parts comprising:

[0014] • a MEK inhibitor and

[0015] • a PKC inhibitor, wherein the MEK inhibitor and the PKC inhibitor are formulated for simultaneous or sequential use, and optionally instructions for use.

[0016] Brief description of the drawings

[0017] Figure 1 shows a comparison of ETB receptor behaviour in fresh basilar arteries and basilar arteries (BA) after incubation in organ culture for 48 hours.

[0018] Figure 2 shows effects of the MEK inhibitor, Trametinib and PKC inhibitor, RO- 317549, on pERK1 / 2 and NFkB phosphorylation in rat basilar arteries after 6 hours organ culture.

[0019] Figure 3 shows a comparison of the effects of the MEK inhibitor Trametinib and the PKC inhibitor RO-317549 on the S6C-mediated ETB receptor contraction of rat basilar arteries after 48 hours organ culture.

[0020] Figure 4 shows the effect of different ERK1 / 2 inhibitors on the S6c-mediated contraction of incubated rat basilar arteries for 48 hours.

[0021] Figure 5 shows the effect of NFkB inhibitors in the contractility of incubated basilar arteries (iBA) for 48 hours in organ culture.

[0022] Figure 6 shows the effect of time of inhibition and combination of inhibitors on S6c- mediated ETB receptor contraction of rat basilar arteries, at the start of incubation or after 6 hours incubation.

[0023] Detailed description

[0024] The invention provides a MEK inhibitor for use in the treatment of stroke, preferably subarachnoid haemorrhage (SAH), or global or focal ischemia, wherein the MEK inhibitor is administered simultaneously, sequentially, or separately with a PKC inhibitor. Treatment of stroke using MEK and PKC inhibitors

[0025] Endothelin (ET-1 ) has been linked to the detrimental outcome of stroke. Interestingly, experiments using clazosentan, an ETA receptor blocker, were reported to have a minor effect on large vessel vasospasm but did not result in any significant effect on outcome (Macdonald et al., Stroke, 2012, 43, 1463-1469). It has been reported that stroke induces elevated effect to exogenous ET-1 due to transcriptional upregulation of contractile ETB receptors (Edvinsson and Krause, Transl. Stroke Res., 2024, DOI: 10.1007 / s12975-024-01234-z). The upregulation of ETB receptors during cerebral ischemic stroke is closely linked to the mitogen-activated protein kinase / extracellular signal-regulated kinase (MEK-ERK1 / 2) signaling pathway, as evidenced by several studies (Edvinsson and Krause, Transl. Stroke Res., 2024, DOI: 10.1007 / s12975- 024-01234-z). A series of targeted protein kinase inhibitors revealed that both MEK and PKC signaling pathways were effective in the experimental organ culture model and has been effective in modulating the enhanced vasoconstrictor effects (Henriksson et al., BMC Neurosci., 2007, 8, 7; Henriksson et al., BMC Pharmacol.,

[0026] 2006, 6, 13; Henriksson et al., Exp. Brain Res., 2007, 178(4), 470-476; Henriksson et al., Stroke, 2003, 34(6), 1479-1483; Beg et al., J. Cereb. Blood Flow Metab.,

[0027] 2007, 27(1 ), 21 -32; Beg et al., J. Cereb. Blood Flow Metab., 2006, 26(6), 846-856). The MEK inhibitor Trametinib, for example, has been shown to counteract S6c- mediated ETB receptor vasoconstriction post-organ culture. Additionally, the involvement of the protein kinase C (PKC) pathway in ETB receptor upregulation post-organ culture in rat middle cerebral artery (MCA) has been highlighted, with protein and mRNA analyses linking this upregulation to phosphorylation events within the MAPKs, MEK-ERK1 / 2, and PKC pathways (Ansar and Edvinsson, Stroke,

[0028] 2008, 39(1 ), 185-190).

[0029] Despite these findings, the exact nature of the interaction between these pathways has not been fully revealed.

[0030] The current inventors have surprisingly found that, in contrast to previously held views (Edvinsson and Krause, Transl. Stroke Res., 2024, DOI: 10.1007 / s12975-024- 01234-z; Lyden, Stroke, 2021 , 52(9), 3033-3044), MEK1 / 2 and PKC operate via distinct and separate pathways rather than sequentially. The inventors have found that PKC does not significantly affect MEK1 / 2 phosphorylation, indicating parallel rather than consecutive pathways.

[0031] The inventors have found that administration of a MEK inhibitor and a PKC inhibitor in combination and at a lower dosage resulted in an inhibitory effect that was similar to the inhibitory effect shown by administering a higher dose of a MEK inhibitor alone. This particularly beneficial effect that is observed with the combination is not something that could have been expected and appears to be the result of potentiation. Studies of MEK and PKC inhibition in an organ culture model revealed that the combination of the minimum effective concentrations of a MEK inhibitor and a PKC inhibitor produced an increased (synergistic) effect, resembling the single effect of a higher dosage of the MEK inhibitor (shown in Example 3). As described below, this effect enables the MEK inhibitor to be used at a lower dosage which, in turn, reduces the risks of side effects from the administration of the drug.

[0032] Additionally, the inventors found that by administering a combination of a MEK inhibitor and a PKC inhibitor, a delay in administration of as much as 6 hours from the onset of stroke symptoms resulted in the same inhibitory effect as administering a MEK inhibitor alone immediately after stroke symptoms.

[0033] In an embodiment of the invention, the MEK inhibitor and the PKC inhibitor are administered simultaneously or sequentially. In a preferred embodiment, the PKC inhibitor is administered simultaneously with the MEK inhibitor.

[0034] In an embodiment of the invention, the MEK inhibitor is administered up to 24 hours after onset of a stroke. In a preferred embodiment, the MEK inhibitor is administered up to 12 hours after onset of a stroke, for example up to 6 hours after the onset of a stroke, or up to 3 hours after onset of a stroke. More preferably, the MEK inhibitor is administered less than 1 hour after onset of a stroke.

[0035] In an embodiment of the invention, the PKC inhibitor is administered up to 24 hours after onset of a stroke. In a preferred embodiment, the PKC inhibitor is administered up to 12 hours after onset of a stroke, for example up to 6 hours after the onset of a stroke, or up to 3 hours after onset of a stroke. More preferably, the PKC inhibitor is administered less than 1 hour after onset of a stroke.

[0036] In an embodiment of the invention, the MEK inhibitor and the PKC inhibitor are each administered, either simultaneously, sequentially, or separately, up to 24 hours of the onset of a stroke. Preferably, the MEK inhibitor and the PKC inhibitor are each administered up to 12 hours of the onset of a stroke, for example up to 6 hours after the onset of a stroke, or up to 3 hours of the onset of a stroke. More preferably, the MEK inhibitor and the PKC inhibitor are each administered up to 1 hour of the onset of a stroke.

[0037] In an especially preferred embodiment, the MEK inhibitor and the PKC inhibitor are administered simultaneously within 1 hour of the onset of stroke.

[0038] MEK inhibitors

[0039] The invention provides a MEK inhibitor, administered simultaneously, sequentially, or separately with a PKC inhibitor, for use in the treatment of stroke. A compound is preferably considered to be a MEK inhibitor if it has an ICso value at a MEK that is lower than 750nM, for example lower than 600nM, for example lower than 500mM. Preferably, a compound used in the current invention has an ICso at MEK 1 and / or MEK 2 that is lower than 250nM, for example lower than 100nM, for example lower than 50nM, for example lower than 10nM. Known MEK inhibitors (for example a MEK1 / 2 inhibitor) suitable for use in the invention, include, for example binimetinib, cobimetinib, selumetinib, trametinib, U0126, CI-1040, PD0325901 , MEK162, AZD8330, TAK-733, GDC-0623, refametinib, pimasertib, RO4987655, RO5126766, WX-554, HL-085, SL-327, imatinib, PD184352 (CI-1040), PD98059, BIX 02189, Bl- 847325, PD318088 or honokiol.

[0040] In an embodiment of the invention, the MEK inhibitor is selected from the group comprising binimetinib, cobimetinib, selumetinib and trametinib. In a more preferred embodiment, the MEK inhibitor is trametinib.

[0041] In an embodiment of the invention, the MEK inhibitor binds and inhibits the activity of MEK1 and / or MEK2. In a preferred embodiment, the MEK inhibitor binds and inhibits the activity of MEK1 and MEK2.

[0042] PKC inhibitors

[0043] The invention provides a PKC inhibitor, administered simultaneously, sequentially, or separately with a MEK inhibitor, for use in the treatment of stroke. A compound is preferably considered to be a PKC inhibitor if it has an IC50 value at a PKC that is lower than 750nM, for example lower than 600nM, for example lower than 500mM. Preferably, a compound used in the current invention has an IC50 at a PKC that is lower than 250nM, for example lower than 100nM, for example lower than 50nM, for example lower than 10nM.

[0044] In an embodiment of the invention, the PKC inhibitor is selected from the group comprising GO-6983, Enzastaurin, Staurosporine, GF 109203X (Bisindolylmaleimide I), Go6976, ZIP, LY 333531 hydrochloride (Ruboxistaurin), RO 31-8220 (for example as its mesylate salt), RO 32-0432 (for example as the hydrochloride salt), Sotrastaurin (AEB071 ), Rottierin, K252a, Baicalein, Quercetin, Luteolin, Bisindolylmaleimide II, Calphostin C, Chelerythrine chloride (CTC, chelerythrine), L- threo Dihydrosphingosine (Safingol), Melittin and PKC412 (Mldostaurin).

[0045] In an embodiment of the invention, the PKC inhibitor is selected from the group comprising RO-317549, RO-32-0432, and GO-6983.

[0046] In a more preferred embodiment, the PKC inhibitor is RO-317549 or RO-32-0432.

[0047] The PKC family includes several isoforms, divided into three subgroups: the conventional, the novel and the atypical PKCs depending on their structure and requirements for activation. In an embodiment of the invention, the PKC inhibitor binds and inhibits the activity of conventional PKC, for example PKCa, PKC[3I, PKCpil, PKCy and / or PKCS. In a preferred embodiment, the PKC inhibitor binds and inhibits the activity of PKCa and / or PKCb. In a particularly preferred embodiment, the PKC inhibitor binds and inhibits the activity of PKCb.

[0048] Stroke

[0049] Strokes can be classified into at least two major categories: ischemic and haemorrhagic. Ischemic strokes are caused by interruption of the blood supply to the brain, while haemorrhagic strokes result from the rupture of a blood vessel or an abnormal vascular structure. About 87% of strokes are ischemic, the rest being haemorrhagic. According to the present disclosure, a stroke may also include a transient ischemic attack (TIA) or can be the result of a heart stop or dramatic lowering of systemic blood pressure by other means, e.g. heart fibrillation.

[0050] In one embodiment of the invention, the stroke is selected from the group consisting of: ischemic stroke, haemorrhagic stroke, global ischemia, and / or transient ischemic attack.

[0051] In one embodiment of the invention, the stroke is selected from the group consisting of: global ischemia and focal ischemia. In a preferred embodiment, the stroke is focal ischemia.

[0052] A stroke may also occur as a complication of the removal of a blood clot in a major brain artery, either physically or with thrombolysis, which can trigger the activation in of the MEK / ERK and PKC pathways in the vessel walls. Administration of a MEK and a PKC inhibitor can reduce joint damage to the vessel walls, thereby reducing subsequent damage and ischemia.

[0053] In an embodiment of the invention, the stroke, or focal cerebral ischemia, results from the removal of a clot in a major brain artery, either physically or in conjunction with administration of a thrombolytic agent. Ischemic stroke

[0054] In an ischemic stroke, blood supply to part of the brain is decreased, leading to dysfunction of the brain tissue in that area. There are several main reasons why this might happen:

[0055] 1 . Thrombosis (obstruction of a blood vessel by a blood clot forming locally)

[0056] 2. Embolism (obstruction due to an embolus from elsewhere in the body),

[0057] 3. Systemic hypoperfusion (general decrease in blood supply, e.g., in shock)

[0058] 4. Cerebral venous sinus thrombosis,

[0059] 5. Increased intracranial pressure compromising venous outflow and reducing cerebral blood flow (CBF),

[0060] 6. Activation of vascular MEK / ERK and / or PKC pathways resulting in enhanced expression of contractile receptors, elevation of inflammatory pathways and blood-brain barrier (BBB) compromise.

[0061] But the stroke may also result from a sudden drop in blood pressure or heart stop, rupture of a cerebral artery or arteriole, or a combination thereof.

[0062] In one embodiment of the present disclosure, the ischemic stroke results from Traumatic Brain Injury (TBI) also known as an intracranial injury. For example, the ischemic stroke results from a concussion, also known as a mild traumatic brain injury (mTBI).

[0063] In one embodiment of the present disclosure, the ischemic stroke results from an embolism, thrombosis, systemic hypoperfusion, cerebral venous sinus thrombosis, a sudden drop in blood pressure or heart stop, rupture of a cerebral artery or arteriole, or a combination thereof.

[0064] In situations of a thrombosis in a cerebral artery, the treatment of the current invention may be especially helpful when local anti-thrombus medication is given (to dissolve the clot). In that situation, the use of a PKC inhibitor and a MEK inhibitor in accordance with the invention helps to recover the situation. That is especially the case if the treatment is given intravascularly (Arkelius et al., Scientific Reports, 2020, 10, 12191 ; Orset et al., Scientific Reports, 2021 , 11 , 11993).

[0065] In a preferred embodiment of the invention, the MEK inhibitor and the PKC inhibitor are administered to the subject prior to, concurrent with, or subsequent to administration of recombinant tissue-type plasminogen activator (rt-PA).

[0066] Haemorrhagic stroke

[0067] There are at least two main types of haemorrhagic stroke:

[0068] • Intracerebral haemorrhage, which is bleeding within the brain itself (when an artery in the brain bursts, flooding the surrounding tissue with blood), due to either intraparenchymal haemorrhage (bleeding within the brain tissue) or intraventricular haemorrhage (bleeding within the brain's ventricular system).

[0069] • Subarachnoid haemorrhage (SAH), which is basically bleeding that occurs outside of the brain tissue but still within the skull, and precisely between the arachnoid mater and pia mater (the delicate innermost layer of the three layers of the meninges that surround the brain), usually due to the rupture of a cerebral artery or an arterial malformation.

[0070] The above two main types of haemorrhagic stroke are also two different forms of intracranial haemorrhage, which is the accumulation of blood anywhere within the cranial vault.

[0071] Haemorrhagic strokes may occur on the background of alterations to the blood vessels in the brain, such as cerebral amyloid angiopathy, cerebral arteriovenous malformation and an intracranial aneurysm, which can cause intraparenchymal or subarachnoid haemorrhage. In addition to neurological impairment, haemorrhagic strokes usually cause specific symptoms (for instance, subarachnoid haemorrhage classically causes a severe headache known as a thunderclap headache) or reveal evidence of a previous head injury.

[0072] In one embodiment of the invention, the MEK inhibitor and PKC inhibitor are provided for use in prevention or treatment of a stroke, which is a haemorrhagic stroke that results from intracerebral haemorrhage, subarachnoid haemorrhage, or a combination thereof.

[0073] In another embodiment, the intracerebral haemorrhage is intraparenchymal, intraventricular, or a combination thereof.

[0074] In another embodiment, the stroke results from subarachnoid haemorrhage.

[0075] Delayed cerebral ischemia (PCI)

[0076] Delayed cerebral ischemia may occur days after subarachnoid haemorrhage and represents a potentially treatable cause of morbidity for approximately one-third of those who survive the initial haemorrhage. While vasospasm has been traditionally linked to the development of cerebral ischemia several days after subarachnoid haemorrhage, emerging evidence reveals that delayed cerebral ischemia is part of a much more complicated post-subarachnoid haemorrhage syndrome. The development of delayed cerebral ischemia involves early arteriolar vasospasm with microthrombosis, perfusion mismatch and neurovascular uncoupling, spreading depolarizations, and inflammatory responses that begin at the time of the haemorrhage and evolve over time, culminating in cortical infarction.

[0077] In one embodiment, the MEK inhibitor and PKC inhibitor as defined herein are used in treatment or prevention of delayed cerebral ischemia (PCI). In one embodiment, the DCI presents with inflammation, oedema, delayed cerebral vasospasm (CVS), blood-brain barrier disruption and / or increase in contractile receptor expression, such as those for endothelin, angiotensin, serotonin and thromboxane or prostaglandins (Edvinsson and Krause, Transl. Stroke Res., 2024, DOI: 10.1007 / s12975-024-01234-z; Edvinsson and Povlsen, J. Cereb. Blood Flow Metab. , 2011 , 31 (7), 1554-1571 ) and recently also the P2Y6 receptor (Erdling et al. , Physiological Reports, 2022, 10(8), e15283).

[0078] Surgery and combination therapy

[0079] In one embodiment, the MEK inhibitor and the PKC inhibitor are administered to the subject without surgery prior to, concurrent with, or subsequent to the administration.

[0080] In one embodiment, the MEK inhibitor and the PKC inhibitor are administered to the subject prior to, concurrent with, or subsequent to thrombectomy.

[0081] In one embodiment, the MEK inhibitor and the PKC inhibitor are administered to the subject prior to, concurrent with, or subsequent to thrombolysis.

[0082] In one embodiment, the MEK inhibitor and the PKC inhibitor of the present disclosure are administered to the subject prior to, concurrent with, or subsequent to a surgical procedure selected from the group consisting of: coiling and clipping.

[0083] The procedure “coiling” or “endovascular coiling” is a procedure performed to block blood flow from an aneurysm (a weakened area in the wall of an artery). Endovascular coiling is a minimally invasive technique, which means an incision in the skull is not required to treat the brain aneurysm. Rather, a catheter is used to reach the aneurysm in the brain. During endovascular coiling, a catheter is passed through the groin up into the artery containing the aneurysm. Platinum coils are then released. The coils induce clotting (embolization) of the aneurysm and, in this way, prevent blood from getting into it.

[0084] The procedure “clipping” or “microsurgical clipping” is a technique that blocks the blood supply to an aneurysm using a metal clip. The procedure is well-known to a person of skill in the art.

[0085] In one embodiment, the MEK inhibitor and the PKC inhibitor are administered to the subject prior to, concurrent with, or subsequent to a neuroradiological procedure.

[0086] Most “neuroradiological procedures" or “interventional neuroradiology procedures” begin with insertion of a catheter into the femoral artery, which is a large artery located in the groin. The catheter, which is a long, flexible hollow tube, is threaded over a guide wire up into the aorta, the main artery supplying the body, and then into the neck vessel leading to the blocked brain artery. Images of the artery (also known as angiography) are then taken using a radiographic contrast dye similar to the one used for CT angiography. These pictures allow the interventional neuroradiologist to identify the site of occlusion and plan the intervention. A smaller catheter (micro catheter) is then placed through the initial catheter and past the occlusive clot.

[0087] There are two main approaches to clot removal: whole-clot retrieval (or thrombectomy) and clot aspiration. In the first technique, the clot retrieval device is placed through the micro catheter, and opened across the clot. The device which traps the clot is then removed. The second technique, clot aspiration, involves fragmentation and suction of the clot. This is performed using catheters larger than traditional micro catheters, which provide increased suction power. Thrombectomy and aspiration techniques are often used in combination. In addition to these mechanical approaches, many interventional neuroradiologists also use local TPA infusion into the clot to help dissolve it. When a clot is removed by an intravascular procedure, the artery wall can be affected. In that situation, the treatment of the current invention, can help to prevent possible damage to the local vasculature. That is especially the case if the treatment is given intravascularly.

[0088] In one embodiment of the present disclosure, the MEK inhibitor and the PKC inhibitor reduce or prevent reperfusion damage resulting from the neuroradiological procedure.

[0089] Reperfusion injury, sometimes called ischemia-reperfusion injury (IRI) or reoxygenation injury, is the tissue damage caused when blood supply returns to tissue (re- + perfusion) after a period of ischemia or lack of oxygen (anoxia or hypoxia). The absence of oxygen and nutrients from blood during the ischemic period creates a condition in which the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress rather than (or along with) restoration of normal function.

[0090] Terms and definitions

[0091] The terms “treatment” and “treating” as used herein refer to the management and care of a patient (or subject) for the purpose of combating a condition, disease, or disorder. The term is intended to include the full spectrum of treatments for a given condition from which the patient (or subject) is suffering. The patient (or subject) to be treated is preferably a mammal, in particular a human being. Treatment of animals, such as mice, rats, dogs, cats, horses, cows, sheep, pigs, monkeys and other primates is, however, also within the scope of the present context. The patients (or subjects) to be treated can be of various ages.

[0092] The term “global ischemia” as used herein refers to ischemia affecting a wider area of the brain and usually occurs when the blood supply to the brain has been drastically reduced or stopped. This is typically caused by a cardiac arrest. The term “focal ischemia” as used herein refers to ischemia confined to a specific area of the brain. It usually occurs when a blood clot has blocked an artery in the brain. Focal ischemia can be the result of a thrombus or embolus.

[0093] The term “traumatic brain injury” (TBI), also known as an intracranial injury, is an injury to the brain caused by an external force. TBI can be classified based on seventy, mechanism (closed or penetrating head injury) or other features (e.g., occurring in a specific location or over a widespread area). TBI can result in physical, cognitive, social, emotional, and behavioural symptoms, and outcomes can range from complete recovery to permanent disability or death.

[0094] Pharmaceutical compositions

[0095] In one embodiment the present invention relates a pharmaceutical composition comprising an effective amount of a MEK inhibitor, a PKC inhibitor, and optionally a further therapeutic agent.

[0096] While the MEK inhibitor and the PKC inhibitor as disclosed herein may be administered in the form of the bare chemical compounds, it is preferred to introduce the active ingredient, optionally in the form of a physiologically acceptable salt, in a pharmaceutical composition together with one or more adjuvants, excipients, carriers, buffers, diluents, and / or other customary pharmaceutical auxiliaries.

[0097] In one embodiment, the disclosure provides compositions comprising the MEK inhibitor and the PKC inhibitor as defined herein, or a pharmaceutically acceptable salt or derivative thereof, together with one or more pharmaceutically acceptable carriers therefor, and, optionally, other therapeutic and / or prophylactic ingredients know and used in the art. The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not harmful to the recipient thereof. In a further embodiment, the invention provides pharmaceutical compositions or compositions comprising more than one compound or prodrug for use according to the disclosure, such as two different compounds or prodrugs for use according to the disclosure.

[0098] Compositions of the disclosure may be those suitable for oral, rectal, bronchial, nasal, pulmonal, topical (including buccal and sub-lingual), transdermal, vaginal or parenteral (including cutaneous, subcutaneous, intramuscular, intraperitoneal, intravenous, intraarterial, intracerebral, intraocular injection or infusion) administration, or those in a form suitable for administration by inhalation or insufflation, including powders and liquid aerosol administration, or by sustained release systems. Suitable examples of sustained release systems include semipermeable matrices of solid hydrophobic polymers containing the compound of the disclosure, which matrices may be in form of shaped articles, e.g. films or microcapsules. Another suitable example is nanoparticles.

[0099] Methods of administration

[0100] In one embodiment, the MEK inhibitor and the PKC inhibitor for use as defined herein is administered orally, intrathecally, intraperitoneally, intraocularly, intranasally, intravenously or by intracerebroventricular injection.

[0101] In one embodiment, the MEK inhibitor and the PKC inhibitor are administered intravenously. In one embodiment, the MEK inhibitor and the PKC inhibitor are administered to the subject up to 6 hours subsequent to the onset of the stroke, such as up to 1 hour, such as up to 2 hours, such as up to 3 hours, such as up to 4 hours, such as up to 5 hours subsequent to the onset of the stroke. In one embodiment, the treatment is continued past the first dose of MEK inhibitor and PKC inhibitor for up to 3 days subsequent to the onset of the stroke.

[0102] In one embodiment, the MEK inhibitor and the PKC inhibitor are administered one or more times daily for up to 3 days subsequent to the onset of the stroke. In one embodiment, the MEK inhibitor and the PKC inhibitor are administered to the subject in combination with a neuroprotective agent (Lyden, Stroke, 2021 , 52(9), 3033-3044; O’Collins et al., Ann. Neurol., 2006, 59(3), 467-477). Treatment of the subject by the MEK inhibitor and the PKC inhibitor may be discontinued 1 , 2 or 3 days subsequent to the onset of the stroke, while treatment with the neuroprotective agent is continued. In one embodiment, the neuroprotective treatment is continued for one or more months.

[0103] In an embodiment of the invention, a method of treating stroke is provided comprising: administration of a MEK inhibitor and a PKC inhibitor to a patient, wherein the MEK inhibitor and the PKC inhibitor are administered simultaneously, sequentially or separately.

[0104] In one embodiment, a kit of parts is provided comprising: a MEK inhibitor as defined herein; a PKC inhibitor as defined herein; and optionally a further medicament as defined herein; wherein the MEK inhibitor, the PKC inhibitor and, if present, the further medicament are formulated for simultaneous or sequential use; and optionally instructions for use.

[0105] The kit may also comprise additional components used in the administration of the compounds. For example, where one or both of the inhibitors are administered by injection, the kit may include a diluent for use in reconstituting one or both of the inhibitors. If both inhibitors are administered by injection, the kit may include more than one diluent, one for use in reconstituting each of the inhibitors. Dosing

[0106] The dose of MEK inhibitor that is administered depends on the particular efficacy of the MEK inhibitor that is selected for use, and the method of administration. For example, for oral administration, the MEK inhibitor may be used at a dose of 0.5 to 50 mg per administration, for example 0.5 to 20 mg, for example 0.5 to 10 mg. When Trametinib is used as the MEK inhibitor, it may be administered at a dose of 0.5 mg to 4.0 mg per administration. For example, 2.0 mg per administration.

[0107] When given intraperitoneally, the MEK inhibitor may be administered at a dose of from 1 mg / kg to 50 mg / kg, for example from 2 mg / kg to 40 mg / kg, for example from 3 mg / kg to 30 mg / kg. When given intravenously, the MEK inhibitor may be administered at a dose of from 0.2 mg / kg to 10 mg / kg, for example from 0.3 mg / kg to 7.5 mg / kg, for example from 0.5 mg / kg to 5 mg / kg.

[0108] The dose of PKC inhibitor that is administered depends on the particular efficacy of the PKC inhibitor that is selected for use, and the method of administration. For example, for oral administration, the PKC inhibitor may be used at a dose of 0.5 to 50 mg per administration, for example 0.5 to 20 mg, for example 0.5 to 10 mg. For example, the PKC inhibitor may be administered at a dose of 0.5 mg to 4.0 mg per administration. For example, 2.0 mg CC per administration.

[0109] When given intraperitoneally, the PKC inhibitor may be administered at a dose of from 1 mg / kg to 50 mg / kg, for example from 2 mg / kg to 40 mg / kg, for example from 3 mg / kg to 30 mg / kg. When given intravenously, the PKC inhibitor may be administered at a dose of from 0.2 mg / kg to 10 mg / kg, for example from 0.3 mg / kg to 7.5 mg / kg, for example from 0.5 mg / kg to 5 mg / kg.

[0110] Examples

[0111] Methods

[0112] Experimental design

[0113] Rat basilar arteries were cultured in serum-free Dulbecco's Modified Eagles Medium (DMEM) for 48 hours, in atmospheric air (~21 % O2) supplemented with 5% CO2. Various pathway-specific inhibitors were applied either individually or in combination. Vascular contractility was assessed using wire myography to measure responses to sarafotoxin (S6c), an ETB receptor agonist, and Western blot analysis was performed to confirm protein levels under different experimental conditions. Additionally, immunofluorescence was used to confirm the upregulation of ETB receptors in the experimental setup.

[0114] Animals

[0115] Male Sprague-Dawley rats, 270-330g (Taconic, Denmark / Sweden), were housed together in groups of 5 in Euro standard Type VI cages with 123-lid covers. They were maintained on a 12-hour light / dark cycle starting at 07:00, at a temperature of 22°C (± 2°C) and humidity of 55% (± 10%) in the Translational Research Centre, Rigshospitalet. Food and water were available ad libitum, the food consisting of standard chow and nuts. All animals were acclimatised for one week before euthanasia in accordance with the guidelines of the European Communities Council (86 / 609 / ECC). The procedure was approved by the Danish Animal Experimentation Inspectorate, and reported following the ARRIVE 2.0 guidelines (Percie du Serf et al., J. Cereb. Blood Flow Metab., 2020, 40(9), 1769-1777). The rats were sedated with a mixture of 70% CO2 and 30% O2, followed by euthanasia by decapitation.

[0116] Organ culture

[0117] The organ culture method employed is a method which shows a phenotypic alteration and upregulation of the endothelin ETB receptor resembling the phenomenon that occurs in vivo in stroke models (SAH and focal ischemia) in rodents and humans. Rat brains were excised immediately following euthanasia and placed in an ice-cold oxygenated sodium Krebs solution (Na+Krebs) consisting of: 119 mM NaCI, 15 mM NaHCOs, 4.6 mM KCI, 1.2 mM NaH2PO4, 1.2 mM MgCI2, 1.5 mM CaCI2, with 5.5 mM glucose. Basilar arteries (BAs) were dissected from the brain in a Petri dish with ice- cold oxygenated Na+Krebs buffer under a microscope and cut into 1.5-2 mm long cylindrical segments. Then the isolated basilar arterial segments (with one myograph wire inside) were cultured for 48 hours at 37° C in a humidified incubator containing atmospheric air (~21 % O2) supplemented with 5% CO2 in 2 mL of Dulbecco’s Modified Eagle’s medium (DMEM, Gibco #31966, Denmark) containing an antibiotic and antifungal mixture of penicillin and streptomycin. Specific inhibitors were added at time 0, or in certain cases, the media were changed after 6 hours of incubation with pure DMEM. The inhibitors used were:

[0118] • MEK inhibitor: trametinib GSK 1120212 (1 O-10— 10-6M, Selleck Chemicals, USA),

[0119] • PKC inhibitor: RO-317549 (10-8— 10-5M, Merck Life Science A / S, Denmark),

[0120] • ERK inhibitors: Ravoxertinib GDC-0994 (10-6M, Selleck Chemicals, USA), Temuterkib LY3214996 (10‘6M, Selleck Chemicals, USA) and Ulixertinib BVD- 523 (10-9-10-5M, Selleck Chemicals, USA),

[0121] • NF-KB inhibitor: BMS 345541 ab144822 (106-1 O’5M abeam, UK),

[0122] • dual AP-1 and NF-KB inhibitor: SP 100030 (1 O’6M, TOCRIS, UK)

[0123] • IKK[3 inhibitor: IMD 0354 ab144823 (10‘6M, abeam, UK).

[0124] The incubated segments were subsequently used for either Western blot and immunohistochemistry, or mounted in the myograph.

[0125] Myography

[0126] For measurements of contractile responses of cerebral arteries, a myograph was used to record the isometric tension in segments of isolated arteries (Ansar et al., Am. J. Physiol. Heart Circ. Physiol., 2007, 293(6), H3750-H3758; Johansson et al., Eur. J. Pharmacol., 2019, 846, 109-118).

[0127] Artery segments were mounted on two 40 pm-diameter stainless steel wires on a Mulvany-Halpern wire myograph setup (Danish Myograph Technology A / S, Denmark) and were heated to 37°C, a temperature which was maintained during the experiments. The myographs were connected to a PowerLab Unit and responses were sampled in LabChart™ (ADInstruments, UK). To obtain optimal conditions for active tension development, BA segments were stretched to an optimal pretension (2.3 mN) in a three-step process as previously found optimal (Hdgestatt et al., Acta Physiol. Scand., 1983, 117(1 ), 49-61 ), after 20 minutes of equilibration. The segments were allowed to stabilize for 20-30 minutes, and kept at this standard tension during the entire period in the myograph. To ensure a stable pH of 7.40, all myograph baths were continuously aerated with 5% CO2 in 95% O2.

[0128] The vascular smooth muscle cell contractile function was confirmed by challenging the segments twice with a temperature-controlled 60 mM K+-Krebs solution (37°C) having the following composition: 59.5 mM NaCI, 15 mM NaHCOs, 60 mM KCI, 1 .2 mM MgCl2, 1.2 mM NaH2PO4, 1.5 mM CaCl2, with 5.5 mM glucose. Concentration-response curves were obtained by the cumulative application of sarafotoxin 6c (S6c, Bachem, Switzerland), a specific ligand for the ETB receptor in the concentration range of 10’13to 10’7M. All contractile responses are expressed as a percentage of the maximal contraction induced by the K+response.

[0129] Western blot

[0130] Western immunoblotting was performed for assessment of protein expression. Fresh and incubated basilar arterial (BAs) segments were immediately added to a RIPA lysis buffer (Apoteket, Region H, Denmark) and Laemmli SDS loading sample buffer (4x Laemmli Sample Buffer, Bio-Rad, cat. #1610747) heated to 95°C, for protein sample preparation (Ansar and Edvinsson, Stroke, 2008, 39, 185-190). The samples were frozen and subsequently stored at -20°C for use the next day, or -80°C for longer term storage. On the second day, the samples were sonicated and stored at -20°C or -80°C. For Western blotting, dithiothreitol reducing agent (DTT, Bio-Rad, cat. #1610610) was added to the samples (5%), and the samples were heated at 70°C for 10 minutes. 10 pL of the samples and 10 pL of SDS marker (Precision Plus Protein Kaleidoscope Prestained Protein Standards, Bio-Rad, cat. #1610375) were loaded on the gel (Mini- PROTEAN TGX Precast Gels, 4-20%, from Bio-Rad, cat. #456-1093). Samples were run in gels in parallel at the electrophoresis chamber with the following settings: 200V, 100 mA / gel for more than 1 hour. Proteins were transferred from the mini-gels in a 0.2 pm PVDF membrane (Trans-Blot Turbo Mini Transfer Pack, Bio-Rad, cat. #1704156) using the Trans-Blot Turbo cassette.

[0131] The membranes were then transferred to a rocking table at room temperature with Blocking Buffer (EveryBlot Blocking Buffer, Bio-Rad, cat. #12010020) for 10 minutes, and then the membranes were incubated with primary antibody dilutions overnight at 4°C (1 :1000 Mouse Anti-p44 I 42 MAP Kinase Monoclonal Antibody, Unconjugated, Clone L34F12 [Cell Signaling Technology, cat. #4696, lot: 29, RRID:AB_390780], 1 :1000 Phospho-p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) (D13.14.4E) XP® Rabbit mAb [Cell Signaling Technology, cat. #4370, Lot: 28, RRID:AB_2315112), 1 :1000 NF- KB p65 (L8F6) Mouse mAb [Cell Signaling Technology, cat. #6956, Lot: 10, RRID:AB_10828935], 1 :1000 Phospho-NF-KB p65 (Ser536) (93H1 ) Rabbit mAb [Cell Signaling Technology, cat. #3033, Lot: 19, RRID:AB_331284], GAPDH (D16H11 ) XP Rabbit mAb, [Cell Signaling Technology cat. #5174, Lot: 8, RRID:AB_10622025], 1 :1000 Actin, Smooth Muscle (1A4) Mouse Monoclonal Antibody [Cell Marque, cat. #202M-94, Lot: 0000243213, RRID:AB_1157937], 1 :1000 alpha smooth muscle Actin antibody (EPR5368) [Abeam, cat. #ab124964, Lot: GR303485-26, RRID:AB_11129103]).

[0132] The following day the membranes were placed on the rocking table for 10 minutes at room temperature, and then washed for 2 x 5 minutes with TBS-T buffer (10x Tris buffered saline, Bio-Rad, cat. #1706435, diluted in milliQ water with Tween20). The membranes were then transferred to secondary antibody dilution (1 :3000 Goat AntiRabbit IgG (H+ L)-HRP conjugate, [Bio-Rad, cat. #170-6515, RRID:AB_11125142], Goat Anti-Mouse IgG (HL)-HRP conjugate, [Bio-Rad, cat. #170-6516, RRID:AB_11125547]) for 1 hour at room temperature, then washed for 5 x 3 minutes with TBS-T buffer, and 1 x 5 minutes with distilled water. Bands were imaged using ECL (Clarity Western ECL Substrate: Clarity Western Peroxide Reagent and Clarity Western Luminol / Enhancer Reagent, Bio-Rad, cat. #1705060,) and imaged with Luminescent Image 219 Analyzer (LAS-4000, Fujifilm, Japan). Due to the small size of the vessel segments, protein concentration could not be measured separately. Therefore, normalisation to the housekeeping protein GAPDH was applied to account for loading variability, in accordance with standard practice. The stability of GAPDH was confirmed by quantifying GAPDH to alpha-smooth muscle actin. Protein expression was quantified using ImageJ.

[0133] Immunohistochemistry

[0134] Fresh and 48 h-incubated arterial segments (1 .5-2 mm) were fixed with 4% paraformaldehyde solution for 15 minutes. The arteries were cryoprotected by incubation in 10% sucrose (Merck, Germany) in Sorensen’s phosphate buffer (0.1 M NaH2PO4 and 0.1 M Na2HPO4) for 24-48 hours at 4°C. The arteries were covered with Tissue-Tek O.C.T. (Gibco) in cryomold squares, frozen in dry ice and kept at - 80°C overnight. The next day the blocks were sectioned at a thickness of 10 pm on a cryostat (Leica CM3050 S, Leica Microsystems, Germany) and mounted on SuperFrost slides (Hounisen, Germany).

[0135] The arterial sections were thawed at room temperature and then rehydrated and washed with 0.05% Tween20 diluted in PBS (PBS-T, Sigma) for 3 x 5 minutes. The sections were then permeabilized and blocked with blocking buffer (Triton X-100 (0.3%), bovine serum albumin (3%) and glycine (0.3 M) in PBS) for 20 minutes and incubated for 5 minutes in Ab diluent (Triton X-100 (0.1 %) and BSA (1 %) in PBS), followed by incubation with 1 :500 primary anti-endothelin B receptor / ETB antibody (Rb polyclonal ETB receptor antibody ab117529, abeam, RRID:AB_10902070) overnight in moisturized chambers. The following day, after the arteries were washed with the Ab diluent for 3 x 5 minutes, the secondary antibody (1 :500 Goat anti-Rabbit polyclonal IgG (H+L), Alexa Fluor 568, ab175471 , abeam, RRID:AB_2576207) was added and incubated for 1 hour at room temperature, in the dark to minimize loss of fluorescence. Excess secondary antibody was then washed with 0.05% Tween20 diluted in PBS for 3 x 5 minutes. Nuclei were stained with antifading medium (Vectashield, Vector Laboratories, USA) containing 4’,6-diamidino-2-phenylindole (DAPI). Immunoreactivity was visualized and photographed with a Nikon microscope.

[0136] Quantification of immunoreactivity was performed using Imaged software (NIH). The acquired TIFF images were imported into Imaged, and regions of interest were manually drawn around each artery, while carefully excluding any non-specific surrounding tissue or arterial branches. Mean fluorescence intensity and the area of each selected region were measured. The immunofluorescence intensity (arbitrary units, AU) was normalized by dividing the mean intensity by the corresponding area.

[0137] Data and statistical analysis

[0138] Data were analysed using GraphPad Prism software (GraphPad Software Inc., USA). The Emax(S6c) values refer to the maximum contraction calculated as a percentage of the contractile capacity of 60 mM K+, and pECso values refer to the negative logarithm of the molar concentration that produces the half-maximum contraction. Data are expressed as mean values ± SEM, and n refers to the number of arterial segments from the brains of different rats. For statistical analyses, t-test, and one-way and two-way AN OVA were performed, where p < 0.05 was considered significant. Post hoc tests were applied following ANOVA when the data were normally distributed when the variance was homogeneous. For statistics, Student’s t- test with and without a multiple correction (Holm-Sidak), repeated measures two-way ANOVA with Sidak’s post-test, or one-way ANOVA with Dunnet’s post-test, was used. The statistical approach used in each experiment is specified in the description of that figure / example. Basilar segments showing contraction of less than 1 mN in response to 60 mM K+were considered non-viable and excluded from the analysis.

[0139] Example 1 : Upregulation of ETB receptors in organ culture and contractility following 48 hours organ culture

[0140] Rat basilar arteries (BAs), either freshly isolated or after 48 hours incubation in organ culture (OC), were used to identify the differences at the ETB receptor immunoreactivity (Figure 1A). For the incubated BAs, the ETB expression (Figure 1 B) was more pronounced (5.7 ± 0.4 AU) compared to the freshly isolated arteries (4.0 ± 0.2 AU, p = 0.008). Thus, immunohistochemistry showed a weak expression of ETB receptors in the endothelium and little in the vascular smooth muscle layer (VSM) (Figure 1 A). After organ culture for 24 hours, there was a clear expression of immunoreactivity in the VSM cells (Figure 1 A, arrows). This has been confirmed in previous studies using qPCR, Western blot, and flow cytometry (Spray et al., J. Mol. Neurosci., 2017, 61 (3), 396-411 ; Spray et al., Acta Physiol., 2017, 220(4), 417-431 ).

[0141] Secondly, the enhanced function of the ETB receptors in the experimental setup was examined using myography. In fresh arteries, sarafotoxin 6c (S6c), a specific ETB receptor agonist, does not induce contraction but dilatation in pre-contracted arteries (Hansen-Schwartz et al., Br. J. Pharmacol., 2002, 137(1 ), 118-126; Hansen- Schwartz et al., Front. Biosci., 2008, 13, 2160-2164; Vikman et al., J. Neurosurg., 2006, 105(3), 438-444) due to its expression on the endothelium. Organ culture has been shown to induce expression of ETB receptor mRNA expression in VSM to a contractile phenotype (Spray et al., J. Mol. Neurosci., 2017, 61 (3), 396-411 ). This expression was verified by immunohistochemistry (Figure 1A), and functionally S6c elicited a strong contraction (Figure 1 D). To examine if this was a general VSM contractility, we examined the effect of depolarization with 60 mM potassium (Figure 1 B). When stimulated with a depolarizing stimulus (60 mM K+), both the fresh BA (5.75 ± 0.6 mN) and the incubated BA (6.15 ± 0.8 mN) showed similar contractile responses (Figure 1 C).

[0142] Moreover, myography showed that after 48 hours of organ culture, S6c now induced a strong concentration-dependent contraction. While the fresh BA did not respond, the incubated BA did (Emax(S6c) = 116.1 ± 4.8 mN; Figure 1 D). S6c caused a sustained, concentration-dependent contraction of the incubated BA, as can be seen in the representative sample traces (Figure 1 E). This finding supports the upregulation of the ETB receptors in rat BA following 48 hours organ culture (OC), as has been shown previously using qPCR analysis and blockade with a specific ETB receptor antagonist (Spray et al., Acta Physiol., 2017, 220(4), 417-431 ; Henriksson et al., Exp. Brain Res., 2007, 178(4), 470-476).

[0143] Example 2: Inhibition ofMEK1 / 2 or PKC on protein expression of phosphorylation of ERK1 / 2 and NFkB

[0144] The upregulation of ETB receptor has previously been observed, but the underlying mechanisms have not previously been fully demonstrated. The protein expression of specific transcription blockers (the MEK1 / 2 inhibitor trametinib and the PKC inhibitor RO-317549) when present in the OC medium during incubation was examined (Figure 2). As has been demonstrated previously, the presence of trametinib prevented the occurrence of contractility in response to S6c (Christensen et al., Clin. Sci., 2019, 133(16), 1797-1811 ; Christensen et al., Drug Deliv., 2019, 26(1 ), 680- 688). However, the connection of these two pathways is not well studied. To investigate the acute phase of the upregulation, western blot analysis was performed on arterial segments after incubation in OC for 6 hours. Arterial segments were incubated with DMEM (control), 10’6M of trametinib or 10’5M of RO-317549.

[0145] The incubation of the control BA resulted in activation and phosphorylation of ERK (Figure 2A). The presence of the MEK1 / 2 inhibitor trametinib did not affect the total amount of the ERK (p = 0.9), however it significantly diminished the ERK1 / 2 phosphorylation (p = 0.02, Figure 2B). In contrast, the PKC inhibitor RO-317549 did not affect the phosphorylation of ERK1 / 2 compared with control (p = 0.9). Therefore, ERK1 / 2 phosphorylation was only prevented by the MEK inhibitor, whereas PKC inhibition did not affect pERK1 / 2 expression.

[0146] To ensure that the transcription factor activation can be affected by the PKC inhibitor, we examined one known transcription factor activated by PKC phosphorylation: the nuclear factor Kappa-B (NF-KB). In control BA, activation and phosphorylation of NF- KB was observed (Figure 2C). The total amount of NF-KB was not affected by the presence of RO-317549 (p = 0.8), however the pNF-KB expression was reduced compared to control (p = 0.03). In contrast, trametinib compared to control did not affect either the total (p = 0.7) or the pNF-KB (p = 0.16, Figure 2D). GAPDH was unaffected by the 6 hours of OC compared to alpha-smooth muscle actin (aSMA), a highly abundant protein in VSMCs (Figure 2E).

[0147] Example 3: Effect ofMEK1 / 2 and PKC inhibition on functional ETB receptor upregulation

[0148] To study a possible interplay between the MEK1 / 2 and PKC pathways, full concentration-response curves were generated for the specific ETB receptor agonist S6c to the inhibition by trametinib and RO-317549 in the OC model. To create a concentration-response evaluation of trametinib, the artery was incubated with varying concentrations (1 O’10— 10’6M) of trametinib, and the contractility was evaluated after 48 hours of OC with S6c. There was no significant effect of MEK1 / 2 inhibition on the general contractility, as examined using 60 mM K+(the level was set as 100% contraction relative to other contractile responses). S6c induced a strong concentration-dependent contraction of the BAs incubated with vehicle (DMSO) after 48 hours of incubation (Emax(S6c) = 94.7 ± 10.6%). The two lowest concentrations of trametinib did not affect the S6c contraction (10’1° M: Emax(S6c) = 108.8 ± 2%; and 10’9M: Emax(sec) = 96.9 ± 5.5%), whereas the first significant inhibitory effect was observed for the BAs incubated with 10’8M of trametinib (Emax(sec) = 72.8 ± 5.4%, p = 0.006). The inhibitory effect was further enhanced at 10’7M of trametinib (E max(S6c) — 7.1 ± 2.1 %), while no further inhibition was possible with 10’6M of trametinib (Emax(S6c) = 7 ± 3%) (Figure 3A). Hence, a concentration of 10’8M was the minimum single effective inhibitory concentration of trametinib (Figure 3B).

[0149] The subsequent experiment was designed to analyze the concentration-related inhibitory response to co-incubation of RO-317549 (10’8-10’5M). The general contractility induced by 60 mM K+was not affected by the PKC inhibitor. The data revealed a relationship between the increasing concentration of the inhibitor RO- 317549 in the 48-hour OC, and the reduction of the S6c-mediated contraction in the myography experiments (Figure 3C). In the BA incubated with vehicle, a strong contraction was observed (Emax(S6c) = 111 .4 ± 4.7%), while there was no significant inhibitory effect noted for the BAs incubated in the presence of 10’8M RO-317549 (Emax(S6c) = 108.4 ± 12.2%, p = 0.76) or 10-7M RO-317549 (Emax(S6c) = 110.6 ± 10%, p = 0.9). However, in the presence of a higher concentration of 10’6M RO-317549 in the OC of the incubated BAs (Emax(sec) = 106.1 ± 5%, p = 0.48), a significant reduction in contractility at 10’1° M S6c (mean = 2.34 ± 0.85%, p<0.001 ) and 10’9M S6c (mean = 65.7 ± 11 %, p<0.001 ) was observed. The shift in the contractility was also evident when comparing the logECso -9.141 (95% Cl: -9.27 to -9.03) of the BA incubated with 10’6M RO-317549 with the concentration-response curve to S6c of the BA incubated with vehicle (logECso -9.813 (95% Cl: -9.96 to -9.63)). 10’5M RO- 317549 in the OC eliminated the contractile effect to S6c completely (Emax(sec) = 0.46 ± 0.3 %, p<0.001 ). Hence, 10’6M RO-317549 is the minimal effective single concentration of RO-317549. Additionally, the inhibition profile of RO-317549 demonstrates a steeper decline in S6c contractility compared to trametinib, as the drop in S6c contractility occurs very sharply between 10’6and 10’5M of RO-317549, suggesting an “on / off”-like mechanism of action (Figure 3D).

[0150] Based on the data from the Western blot experiments above, and the difference in kinetics in the S6c inhibition when comparing MEK1 / 2 and PKC inhibition, it was hypothesized that RO-317549 could potentiate the inhibitory effect of trametinib. We therefore combined the minimum effective concentrations of RO-317549 (10-6M) and trametinib (10’8M). As observed in sample traces (Figure 3E), these two inhibitors worked together, as the combination of 10’6M RO-317549 and 10’8M trametinib resembled the single effect of 10’7M trametinib, i.e. in the presence of a PKC inhibitor, a lower dose of trametinib showed the same effect as a higher dose when applied alone. The hypothesis was further tested with a concentrationresponse relation study of trametinib (10’1° M-10’6M) in the presence of 10’6M RO- 317549 (Figure 3F). The data showed a 10-fold shift in the logECso for S6c-induced contractility (E max(S6c) values) with single concentrations of trametinib having a logECso of -7.8 (95% Cl: -7.93 to -7.63) compared to the combination with 10’6M RO-317549 having a logECso of -8.72 (95% Cl: -8.85 to -8.53). Combining 10’9M trametinib with 10’6M RO-317549 resulted in a Emax(S6c) of 74.9 ± 4.2%, which is similar (p = 0.8) to that of 10’8M trametinib (Emax(sec) = 72.8 ± 5.4%). Moreover, the combination of 10’8M trametinib with 10’6M RO-317549 resulted to an Emax(S6c) Of 7.1 ± 1.8%, mimicking the single effect of 10’7M trametinib (Emax(S6c) = 7.1 ± 2.1 %, p = 1 ). These results support the hypothesis that the PKC inhibitor RO-317549 has an increased effect to the MEK1 / 2 inhibition by trametinib in the OC model, when evaluated by S6c-induced contraction in myography experiments.

[0151] Example 4: Effect of different ERK inhibitors

[0152] Based on the Western blot data above (Figure 2), the MEK1 / 2 inhibitor trametinib was able to inhibit ERK1 / 2 activation / phosphorylation. Since the initial studies were performed in the OC model, newly developed ERK inhibitors have become available. The available ERK1 / 2 inhibitors were therefore evaluated in the OC model. We incubated BA with ravoxertinib (GDC-0994), temuterkib (LY3214996) or ulixertinib (BVD-523), all at 10’6M, and evaluated the contractility to S6c after 48 hours in the myograph (Figure 4A).

[0153] The BAs incubated with vehicle showed induction of strong contractility by S6c (Emax(S6c) = 112.1 ± 5.9%). The presence of the ERK1 / 2 inhibitor ravoxertinib in the OC resulted in an Emax(S6c) of 46.8 ± 10%, significantly different from the vehicle (p<0.001 ), which was similar to the inhibitory effect induced by the presence of the ERK1 / 2 inhibitor temuterkib (Emax(S6c) = 56.2 ± 22%, p = 0.49). However, the BA incubated with the ERK1 / 2 inhibitor ulixertinib resulted in an Emax(sec) of only 13.9 ± 7%, which was significantly different not only from the control (p < 0.001 ), but also from ravoxertinib (p < 0.01 ) and temuterkib (p = 0.02). A full concentration-response curve was obtained for ulixertinib (10-9M-10’5M). S6c induced a contraction of BAs incubated with vehicle (Emax(S6c) = 112.1 ± 6%, Figure 4B). The three lowest concentrations did not affect the S6c-induced contraction (10’9M, Emax(S6c) = 116.3 ± 6%, p = 0.6; 1 Q-8M, Emax(S6c) = 120 ± 5%, p = 0.3; 10’7M, Emax(sec) = 100.4 ± 4%, p = 0.2). Incubation of BAs with 10’6M of ulixertinib resulted in a significant reduction of the contractility (Emax(sec) = 26.1 ± 14%, p < 0.001 ). The highest concentration (10’5M) of ulixertinib also eliminated the contractility effect of S6c (Emax(sec) = 13.9 ± 7%, p < 0.001 ), but was not significantly different from 10’6M ulixertinib (p = 0.5). However, the 10’5M concentration affected the overall contractility, with the 60 mM K+resulting in mean = 0.08 ± 0.02 mN (p < 0.01 from vehicle). Of note, the concentration curve for the Emax(sec) values of ulixertinib with logECso of -6.520 (95% Cl: -6.855 to -6.191 ) was significantly different from the Emax(sec) concentration curve of trametinib, which was -7.8 (95% Cl: -7.93 to -7.63).

[0154] To further investigate a possible interplay and feedback between the MEK / ERK1 / 2 and PKC pathways, the ERK1 / 2 inhibitor ulixertinib was combined with the MEK1 / 2 inhibitor trametinib or the PKC inhibitor RO-317549 (Figure 4D). The single effect of 10’6M RO-317549 (Emax(S6c) = 106.1 ± 5%) was significantly different from the combination of 10’6M RO-317549 and 10’7M ulixertinib (Emax(S6c) = 55.9 ± 11 %, p < 0.0001 ). Moreover, the single effect of 10’8M trametinib (Emax(sec) = 72.8 ± 5%) was significantly different from the combination of 10’8M trametinib and 10’7M ulixertinib (Emax(sec) = 43.5 ± 10%, p = 0.02). The combination of 10’6M RO-317549 and 10’7M ulixertinib was not significantly different from the combination of 10’8M trametinib and 10’7M ulixertinib (p = 0.42). These results showed that the ERK1 / 2 inhibitor caused an increased effect for both the PKC and MEK1 / 2 pathways.

[0155] Example 5: Effect of the NF-KB inhibitor BMS 345541

[0156] The above experiments suggested that adding a downstream inhibitor of the MEK1 / 2 pathway increased the effectiveness, and further Western blot data suggested that RO-317549 was linked to the activation of NF-KB. BAS were incubated with different NF-KB inhibitors, IMD 0354, SP100030 and BMS 345541 , all at 10’6M, and the S6c contractility after 48 hours of OC was evaluated and compared to vehicle (Figure 5A). The BA incubated with vehicle resulted in Emax(S6c) of 100.5 ± 7%. 10’6M of IMD 0354 had an inhibitory effect (Emax(S6c) = 66.7 ± 9%, p = 0.06), but greatly reduced the overall contractility to 60 mM K+(0.47 ± 0.2 mN). The presence of SP100030 (10’6M), a dual inhibitor of AP-1 and NF-kB, in the OC resulted in Emax(sec) of 106.5 ± 3%, which was not significantly different from the vehicle (p = 0.7). For the BA incubated with BMS 345541 , an IkB / IKK specific inhibitor, no effect was observed at 10’6M concentration (Emax(sec) = 93 ± 10%, p = 0.6). A higher concentration of BMS 345541 (10’5M) eliminated the contraction to S6c (Emax(sec) = 3.3 ± 3%, p < 0.001 , Figure 5B).

[0157] To investigate a possible interplay, 10’6M RO-317549 and / or 10’8M trametinib were combined with 10’6M BMS 345541 . It was observed that the single effect of 10’6M RO-317549 (Emax(sec) = 106.1 ± 5%) was significantly different (p = 0.02) from the combination of 10’6M RO-317549 with 10’6M BMS 345541 (Emax(S6c) = 68.7 ± 9%). Further, the single effect of 10’8M trametinib (Emax(S6c) = 72.8 ± 5%) was also significantly different (p = 0.002) from the combination of 10’8M trametinib with the 10’6M BMS 345541 (Emax(S6c) = 20.8 ± 6%), similar to what was observed for the ERK1 / 2 inhibitor. Comparison of the two combinations showed that the difference between them was 47.97 ± 12% (p = 0.004), indicating that the effect of trametinib is significantly more potent than that of RO-317549.

[0158] Example 6: Effect of inhibition time

[0159] It is known that time is an important factor in the activation of the signaling pathways. To investigate the order of activation in the above pathways, the inhibitors used in the previous experiments were added either early, i.e. at the start of the incubation at time 0, or delayed, i.e. after the arteries had been incubated in OC with DMEM for 6 hours (Figure 6A). The 6-hour time point was selected both based on the Western blot data, and reflecting the clinical situation in embolic stroke, where currently approved treatments such as thrombolysis or thrombectomy are not administered beyond this window (Powers et al., Stroke, 2015, 46(10), 3020-3035; Rehani et al., Neurohospitalist, 2020, 10(1 ), 29-37). S6c induced a contraction of the incubated BA both when the vehicle was added at time 0 (Emax(S6c) = 94.7 ± 11 %) or delayed 6 hours (Emax(S6c) = 77 ± 12%, p = 0.4). 10’7M Trametinib at time 0 had a significant effect compared to vehicle at time 0 ( Emax(S6c) = 7.1 ± 2%, p = 0.003). When 10’7M trametinib was added at 6 hours, its inhibitory action was significantly reduced (Emax(sec) = 51.3 ± 9%, p = 0.003). Moreover, this inhibitory effect was not significantly different from the vehicle at 6 hours (p = 0.1 ) indicating that the MEK1 / 2 inhibitor is not effective if added at the delayed time point of 6 hours. The data from time 0 shown in Figure 6 is from previous experiments.

[0160] 10’5M RO-317549 at time 0 eliminated the S6c-mediated contractile effect compared to the vehicle at time 0 (Emax(S6c) = 0.46 ± 0.3%, p < 0.001 ). When added at 6 hours, 10’5M RO-317549 resulted in Emax(S6c) of 7 ± 0.8%, which was significantly different from that at time 0 (p < 0.001 ), even though it was still effective compared to the vehicle at 6 hours (p < 0.001 ). These data showed that the PKC inhibition is not very time sensitive in the O-to-6-hour window, i.e. it worked well at the different time points studied.

[0161] Furthermore, 10’5M BMS 345541 at time 0 resulted in inhibition of the S6c-mediated contractile effect (Emax(sec) = -0.2 ± 2%, p < 0.001 ). The 6 hours delayed time did not affect the inhibitory ability of 10’5M BMS 345541 (Emax(S6c) =4.8 ± 3%, p = 0.3) compared to time 0, indicating that the effectiveness is not time dependent, similarly to PKC inhibition. The S6c-mediated contractility was also inhibited by 10’6M u I ixertin ib at time 0 (Emax(sec) = 22.9 ± 9%, p < 0.001 ) compared to vehicle, and as well as at 6 hours delayed (Emax(S6c) = 38.7 ± 11 %) with no significant difference from the time 0 (p = 0.3).

[0162] Combinations of the single lowest effective concentrations of trametinib and RO- 317549 were studied to determine whether they could have potential inhibitory effect when added at 6 hours (Figure 6B). 10’8M Trametinib at time 0 resulted in E max(S6c) of 71 .4 ± 6%, which was significantly different from the vehicle (p = 0.047). When added at 6 hours, 10’8M trametinib resulted in an Emax(sec) of 82.4 ± 9%, which was not significantly different (p = 0.3) from time 0 or from the vehicle at 6 hours (p = 0.7), indicating that the single effect of 10’8M trametinib was not effective at 6 hours. At time 0, 10’6M RO-317549 resulted in Emax(S6c) of 106.1 ± 5% which was not significantly different from the vehicle (p = 0.4). The previous significant change at this concentration was observed at 10’10and 10’9M S6c, making it the minimum single effective concentration of RO-317549. At 6 hours, there was still no effect of a single dose of 10’6M RO-317549 compared to time 0 (Emax(S6c) = 90 ± 7%, p = 0.1 ). Similarly to previous results, the combination of 10’8M trametinib and 10’6M RO- 317549 at time 0 resulted in Emax(sec) of 4.1 ± 0.6%, which was significantly different from the vehicle (p < 0.001 ). The same combination when added at 6 hours strongly inhibited the S6c-mediated contraction (Emax(sec) = 10.8 ± 4%), a result which was not different than that at time 0 (p = 0.1 ). Interestingly, the loss of inhibition observed with trametinib when added at 6 hours delay can be compensated for by adding a PKC inhibitor, as the inhibition of the S6c-mediated contraction by the combination was robust compared to the vehicle (p = 0.002), suggesting a potential feedback activation by PKC.

[0163] The above examples support the conclusion that MEK and PKC function as parallel and distinct pathways rather than in sequence, and that MEK-ERK1 / 2 and PKC pathways act independently and contribute separately to ETB receptor upregulation under conditions of flow cessation.

[0164] The effects observed with combined MEK and PKC pathway inhibition indicate therapeutic potential for addressing ETB receptor-mediated vascular pathologies associated with ischemia caused by flow cessation. Furthermore, the effectiveness of delayed PKC inhibition indicates potential relevance for therapeutic intervention for ischemia beyond the acute phase.

Claims

Claims1 . A MEK inhibitor for use in the treatment of stroke, wherein the MEK inhibitor is administered simultaneously, sequentially, or separately with a PKC inhibitor.

2. The compound for use as claimed in claim 1 , wherein the PKC inhibitor is administered simultaneously with the MEK inhibitor.

3. The compound for use as claimed in claim 1 or claim 2, wherein the MEK inhibitor is administered up to 24 hours after onset of a stroke.

4. The compound for use as claimed in any of the previous claims, wherein the PKC inhibitor is administered up to 24 hours after onset of a stroke.

5. The compound for use as claimed in any of the previous claims, wherein the MEK inhibitor is selected from the group consisting of binimetinib, cobimetinib, selumetinib, U0126 and trametinib.

6. The compound for use as claimed in claim 5, wherein the MEK inhibitor is trametinib.

7. The compound for use as claimed in any of the previous claims, wherein the PKC inhibitor is selected from the group consisting of RO-317549, RO-32- 0432, and GO-6983.

8. The compound for use as claimed in claim 7, wherein the PKC inhibitor is RO- 317549.

9. The compound for use as claimed in any of the previous claims, wherein the stroke is selected from the group comprising: ischemic stroke, haemorrhagic stroke, and transient ischemic attack.

10. The compound for use as claimed in any of claims 1 to 8, wherein the stroke is selected from the group comprising global ischemia and focal ischemia.11 . The compound for use as claimed in claim 10, wherein the stroke is an ischemic stroke resulting from an embolism, thrombosis, systemic hypoperfusion, cerebral venous sinus thrombosis, a sudden drop in blood pressure or heart stop, rupture of a cerebral artery or arteriole, or a combination thereof.

12. The compound for use as claimed in claim 10, wherein the ischemic stroke results from traumatic brain injury (TBI).

13. The compound for use as claimed in claim 9, wherein the stroke is a haemorrhagic stroke resulting from intracerebral haemorrhage, subarachnoid haemorrhage, or a combination thereof.

14. The compound for use as claimed in claim 13, wherein the stroke is intracerebral haemorrhage that is intraparenchymal, intraventricular, or a combination thereof.

15. The compound for use as claimed in claim 13, wherein the stroke is a delayed cerebral ischemia (DCI), for example wherein the DCI presents with inflammation, oedema, delayed cerebral vasospasm (CVS), blood-brain barrier disruption and / or increase in contractile receptor expression, such as those for endothelin, angiotensin, serotonin, P2Y6 and thromboxane or prostaglandins.

16. The compound for use as claimed in any of the previous claims, wherein the MEK inhibitor and the PKC inhibitor are administered to the subject without surgery prior to, concurrent with, or subsequent to the administration.

17. The compound for use as claimed in any of claims 1 to 15, wherein the MEK inhibitor and the PKC inhibitor are administered to the subject prior to, concurrent with, or subsequent to thrombectomy.

18. The compound for use as claimed in any of claims 1 to 15, wherein the MEK inhibitor and the PKC inhibitor are administered to the subject prior to, concurrent with, or subsequent to thrombolysis.

19. The compound for use as claimed in any of claims 1 to 15, wherein the MEK inhibitor and the PKC inhibitor are administered to the subject prior to, concurrent with, or subsequent to a surgical procedure selected from the group consisting of coiling and clipping.

20. The compound for use as claimed in any of claims 1 to 15, wherein the MEK inhibitor and the PKC inhibitor are administered to the subject prior to, concurrent with, or subsequent to a neuroradiological procedure.21 . The compound for use as claimed in claim 20, wherein the MEK inhibitor and the PKC inhibitor reduces or prevents reperfusion damage resulting from the neuroradiological procedure.

22. A pharmaceutical composition comprising a MEK inhibitor, a PKC inhibitor, a pharmaceutically acceptable carrier, and optionally a further therapeutic agent.

23. The pharmaceutical composition as claimed in claim 22, wherein the further therapeutic agent is nimodipine or clazosentan.

24. The compound for use as claimed in claims 1 to 21 , wherein the MEK inhibitor and / or the PKC inhibitor is administered orally, intrathecally, intraperitoneally, intraocularly, intranasally, or intravenously, by infusion, or by intracerebroventricular injection, for example wherein the MEK inhibitor and / or the PKC inhibitor is administered intravenously.

25. The pharmaceutical composition as claimed in claim 22 or claim 23, wherein the pharmaceutical composition is formulated for administration orally, intrathecally, intraperitoneally, intraocularly, intranasally, or intravenously, byinfusion, or by intracerebroventricular injection, for example wherein the pharmaceutical composition is formulated for administration intravenously.

26. A method of treating stroke, comprising: administration of a MEK inhibitor and a PKC inhibitor to a patient, wherein the MEK inhibitor and the PKC inhibitor are administered simultaneously, sequentially, or separately.

27. A kit of parts comprising: a MEK inhibitor, a PKC inhibitor, and optionally instructions for use, wherein the MEK inhibitor and the PKC inhibitor are formulated for simultaneous or sequential use.

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