Mek inhibitor for treatment of stroke

Abstract

The present invention relates to MEK inhibitors for the treatment of stroke. The present invention relates to MEK inhibitors and compositions thereof for use in the treatment of stroke, in particular the treatment of subarachnoid hemorrhage (SAH).

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202610114696.3 (22) Application Date 2020.07.28 (30) Priority Data 19189069.8 2019.07.30 EP (62) Divisional Application Data 202080068488.5 2020.07.28 (71) Applicant Edwins Company Address Lund, Sweden (72) Inventor L. Edwinsson (74) Patent Agency Longtian Intellectual Property Agency Co., Ltd. 72003 Patent Attorney Yu Lei (51) Int.Cl. A61K 31 / 519 (2006.01) A61K 31 / 4422 (2006.01) A61K 31 / 506 (2006.01) A61P 9 / 10 (2006.01) (54) Title of Invention MEK Inhibitor for the Treatment of Stroke (57) Abstract This invention relates to MEK inhibitors for the treatment of stroke. This invention relates to MEK inhibitors and compositions thereof for the treatment of stroke, particularly for the treatment of subarachnoid hemorrhage (SAH). Claims 2 pages Description 33 pages Drawings 37 pages CN 121846102 A 2026.04.14 CN 1 21 84 61 02 A 1. Use of a MEK inhibitor of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of stroke in a subject, formula (I), wherein: R1 is a C1-C6 alkyl group, R2 is a C1-C6 alkyl group, and Ar is selected from aryl and heteroaryl groups. 2. Use of a MEK inhibitor of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for reducing or preventing reperfusion injury caused by neuroradiological procedures in a subject, formula (I), wherein: R1 is a C1-C6 alkyl group, R2 is a C1-C6 alkyl group, and Ar is selected from aryl and heteroaryl groups. 3. The use according to claim 1 or 2, wherein the MEK inhibitor has formula (II), or a pharmaceutically acceptable salt thereof. 4. The use according to claim 1, wherein the stroke is selected from ischemic stroke, hemorrhagic stroke, and transient ischemic attack. 5. The use according to claim 1, wherein the stroke is selected from global cerebral ischemia and focal ischemia. 6. The use according to claim 1, wherein the stroke is delayed cerebral ischemia (DCI). 7. The use according to claim 1 or 2, wherein the MEK inhibitor is formulated for oral, intrathecal, intraperitoneal, intraocular, intranasal, or intravenous administration. 8. The use according to claim 1 or 2, wherein the MEK inhibitor is formulated for intravenous administration.9. The use according to claim 1 or 2, wherein the subject is a human subject. 10. Use of a MEK inhibitor of formula (II) or a pharmaceutically acceptable salt thereof in combination with another drug in the preparation of a medicament for the prevention or treatment of stroke in a subject, formula (II), wherein the other drug is selected from: calcium channel blockers and endothelin receptor (ET) receptor blockers. 11. The use according to claim 10, wherein the calcium channel blocker is nimodipine. 12. The use according to claim 10, wherein the endothelin receptor (ET) receptor blocker is clarsentan. Claims 2 / 2 Page 3 CN 121846102 A MEK inhibitor for the treatment of stroke

[0001] This application is a divisional application of the application filed on July 28, 2020, with application number 202080068488.5 and entitled "MEK inhibitor for the treatment of stroke". Technical Field

[0002] This invention relates to MEK inhibitors and their use as medicines in the treatment of stroke and related conditions, including subarachnoid hemorrhage (SAH). Background Art

[0003] Stroke is the second leading cause of death worldwide and a leading cause of disability. Its incidence is increasing due to population aging. In addition, the incidence of stroke is increasing among young people, particularly in low- and middle-income countries. Ischemic stroke is the more common type of stroke, while hemorrhagic stroke causes more deaths and disability-adjusted life years lost. The incidence and mortality of stroke vary by country, geographic region, and ethnic group. Primarily in high-income countries, improvements in prevention, acute treatment, and neurorehabilitation have led to a significant reduction in the stroke burden over the past 30 years.

[0004] Aneurysmal subarachnoid hemorrhage (SAH) is a variant of hemorrhagic stroke, accounting for approximately 5% of all stroke events but with a mortality rate as high as 50%. Survivors often experience cognitive impairment and a decline in quality of life, making it a very debilitating condition. SAH (subarachnoid hemorrhage) is usually caused by the rupture of an aneurysm, leading to a rapid leak of arterial blood into the subarachnoid space, followed by a sharp increase in intracranial pressure (ICP) and a decrease in cerebral blood flow (CBF). This results in cerebral hypoxia and hypoglycemia, leading to cerebral ischemia and brain injury, often referred to as early brain injury. Delayed cerebral ischemia (DCI) is associated with secondary delayed brain injury and consists of various pathophysiological changes, including inflammation, edema, and disruption of the blood-brain barrier. DCI may be related to remodeling and narrowing of cerebral arteries, especially vascular hypersensitivity (often referred to as delayed cerebral vasospasm).Regarding vasospasm (CVS), there are currently very few treatment options available.

[0005] Therefore, vasoconstriction has become a focus of many clinical and preclinical studies attempting to prevent subsequent DCI. This includes recent attempts to modulate acute vasoconstriction, for example using endothelin receptor antagonists, including the specific endothelin A (ETA) and endothelin B (ETB) receptor antagonists clazosentan. Unfortunately, these attempts have not been successful.

[0006] Therefore, there is a need in the art to provide new and better stroke treatments to address the medical burden caused by various forms of stroke. Summary of the Invention

[0007] The inventors have surprisingly discovered that the MEK inhibitor trametinib and its analogues have shown superior efficacy compared to other effective MEK inhibitors in in vivo stroke models.

[0008] In a first aspect, a MEK inhibitor of formula (I) is provided for the prevention or treatment of stroke in a subject, (See specification 1 / 33 page 4 CN 121846102 A) of formula (I), or a pharmaceutically acceptable salt thereof, wherein: R1 is a C1-C6 alkyl group, such as methyl, R2 is a C1-C6 alkyl group, such as cyclopropyl, and Ar is selected from aryl, phenyl, and heteroaryl groups.

[0009] In a second aspect, the MEK inhibitor for use in this disclosure has formula (II), (II), or a pharmaceutically acceptable salt thereof.

[0010] In a third aspect, the use of the MEK inhibitor of formula (I) is provided for: a. reducing endothelin-1-induced contractility; b. reducing phenotypic changes in the function of relaxing endothelin B receptors towards a contractile phenotype; and / or c. improving neurological scores, which can be assessed by the subject's ability to rotate a bar after induced subarachnoid hemorrhage.

[0011] In a fourth aspect, a method is provided for treating a subject with stroke or reducing the risk of stroke in a subject, wherein the method comprises the steps of administering to a subject in need a MEK inhibitor of formula (I), (see page 2 / 33 of the specification, CN 121846102 A), or a pharmaceutically acceptable salt thereof, wherein: R1 is a C1-C6 alkyl group, such as methyl, R2 is a C1-C6 alkyl group, such as cyclopropyl, and Ar is selected from aryl, phenyl, and heteroaryl groups; thereby treating stroke or reducing the risk of stroke.

[0012] In a fifth aspect, a composition is provided which, separately or together, comprises a MEK inhibitor of formula (II), (II), or a pharmaceutically acceptable salt thereof, and additional medicaments.

[0013] Figures 1A-1C. Comparison of nine different MEK1 / 2 inhibitors (1 µL) after 48 hours of organ culture in the basilar artery.Inhibitory ability of M). This leads to phenotypic changes and upregulation of contractile ETB receptors in vascular smooth muscle cells. Figure 1A: Concentration-response curves for the ETB-specific agonist S6c after incubation with 1 µM of different MEK1 / 2 inhibitors. In fresh blood vessels, S6c causes cerebral vasodilation or has no effect, but after organ culture, a contractile phenotype appears and exhibits similar characteristics in different stroke models. Figure 1B: Maximum contraction at 60 mM K+ after incubation with 1 µM of different MEK1 / 2 inhibitors. No significant difference was found between the solvent (DMSO) and any antagonist. Figure 1C: Endothelial-induced dilatation was significantly increased in segments incubated with TAK-733, trametinib, and PD0325901 compared to the solvent (DMSO). Organ culture typically shows reduced endothelial function, but these inhibitors protected against this detrimental effect. Data are presented as mean ± SEM, and statistical analysis was performed using two-way ANOVA followed by Holm-Sidak multiple comparison test (page 3 / 33 of the instruction manual, CN 121846102 A). Compared with DMSO, P < 0.05, P < 0.01. n = 4–15.

[0014] Figures 2A–2C. Concentration-response curves of the selected high-efficiency MEK1 / 2 inhibitors. Concentration-response curves of the selected inhibitors are based on (Figure 2A) Emax of S6c, (Figure 2B) maximum contraction at 60 mM K+, and (Figure 2C) endothelial function as evaluated by adding 10–5 M carbachol to an artery pre-constricted with 3·10⁻⁷ M 5-HT. Data are presented as mean ± SEM, n = 4.

[0015] Figures 3A–3D. Effects of trametinib and PD0325901 on the pathways regulating vasomotor function after 48 hours of organ culture in the basilar artery. Arteries from OCs were pre-constricted with U46619 (1–3 × 10⁻⁷ M) or K+ (41 mM) in the presence of DMSO control (n = 5), 1 µM trametinib (n = 6), or PD0325901 (n = 5), and cumulative concentration-response curves were plotted by adding (Fig. 3A) carbachol (10⁻¹⁰–10⁻⁵ M), (Fig. 3B) SNP (10⁻¹¹–10⁻⁴ M), or (Fig. 3C) CGRP (10⁻¹²–10⁻⁷ M). (Fig. 3D) After organ culture with DMSO control (n = 15), 1 µM trametinib (n = 4), or PD0325901 (n = 4), cumulative concentration-response curves were plotted with Ca²⁺ (0.0125–3 mM) following ET-1 pre-constriction (10⁻⁷ M). The data are presented as mean ± SEM, and analyzed using two-way ANOVA followed by the Holm-Sidak multiple comparison test.Statistical results showed that P<0.05, P<0.01, and P<0.001. Geisser-Greenhouse sphericity correction was applied to normalized data.

[0016] Figures 4A-4B. Effects of trametinib and PD0325901 treatment on contractile response and endothelial function of 60 mM K+ after SAH and sham surgery in rats. Figure 4A: Emax, K+ (Nm-1) induced by 60 mM K+. Figure 4B: Endothelial function. Data included basilar arteries with a cutoff value below 2.0 mN for comparison of all arteries; sham surgery + solvent (n = 10), SAH (n = 11), SAH + solvent (n = 27), trametinib (n = 13), and PD0325901 (n = 12). The horizontal black dashed line shows the total mean for all n (n = 73). Data are presented as mean ± SEM, where “n” in this figure equals a single arterial segment. Statistical analysis was performed by one-way ANOVA followed by Holm-Sidak multiple comparison test. P < 0.05, P < 0.01.

[0017] Figures 5A-5B. Effects of trametinib and PD0325901 treatment on the contractile response of endothelin-1 after SAH or sham surgery in rats. Cumulative concentration-response curves of ET-1 in the basilar artery (10⁻¹⁴ - 10⁻⁷ M). For sham surgery + solvent (n = 5), SAH (n = 5), SAH + solvent (n = 12), SAH + trametinib (n = 6), and SAH + PD0325901 (n = 6), (Figure 5A) ET-1 (Nm⁻¹) and (Figure 5B) ET-1 (%) of ET-1max. Data are presented as mean ± SEM and statistically analyzed by two-way ANOVA followed by Holm-Sidak multiple comparison test. P = <0.05. ET-1max normalized data were corrected for sphericity using Geisser-Greenhouse method, and ET-1 curves were fitted with biphasic nonlinear regression curves.

[0018] Figure 6. Effect of trametinib and PD0325901 treatment on VDCC-independent ET-1-induced contraction in rats after SAH or sham surgery. The basilar artery was pre-constricted with ET-1 (10⁻⁷ M) and then cumulative concentration-response curves were plotted using Ca²⁺ (Nm⁻¹); sham surgery + solvent (n = 5), SAH (n = 5), SAH + solvent (n = 13), SAH + trametinib (n = 7), and SAH + PD0325901 (n = 6). Data are presented as mean ± SEM and statistically analyzed by two-way ANOVA followed by Holm-Sidak multiple comparison test. P = <0.05.

[0019] Figures 7A-7C. Effects of trametinib and PD0325901 treatment on neurological function.

[0020] In vivo test of rotating rod at 10 rpm: (Figure 7A) before SAH, (Figure 7B) 24 hours after SAH and (Figure 7C) 48 hours after SAH. Each animal was scored with 4 counts, i.e., 2 scores for each rotation to the left and right; low = failed on one attempt; high = passed on one attempt. Sham surgery + solvent (n = 5), SAH (n = 9), SAH + solvent (n = 14), SAH + trametinib (n = 7) and SAH + PD0325901 (n = 6). All animals were exposed to experimental SAH except for the sham surgery + solvent group. Statistical analysis was performed by Fischer exact test, two-sided, 95% CI. P < 0.05; P < 0.01; P < 0.001.

[0021] Figures 8A-8D. Effect of ip trametinib treatment after SAH surgery in rats. (Figure 8A) Emax (Nm⁻¹) of K⁺ induced by 60 mM K⁺ in SAH + ip solvent (n = 10) and ip trametinib (n = 12). Data are shown as mean ± SEM. In this figure, “n” equals a single arterial segment. (Figure 8B) Cumulative concentration-response curves of basilar artery ET⁻¹ (10⁻¹⁴-10⁻⁷ M) normalized to 60 mM K⁺ from animals treated with SAH + ip solvent (n = 5) or SAH + ip trametinib (n = 6). Data are shown as mean ± SEM and statistically analyzed by two-way ANOVA followed by Holm-Sidak multiple comparison test. P = <0.001. The ET-1 curve is a biphasic nonlinear regression curve fit. The right-hand image shows the rotating bar test for neurological function performed at 10 rpm at 24 hours post-SAH (Fig. 8C) and 48 hours post-SAH (Fig. 8D). Each animal was scored with four counts, i.e., two scores for each left and right rotation; low = failed on one attempt; high = passed on one attempt. Statistical analysis was performed using Fischer exact test, two-sided, 95% CI. P < 0.05.

[0022] Fig. 9. Table of intrathecal and intraperitoneal treatment protocols.

[0023] Fig. 10. Table of curve fitting and EC50 value comparison.

[0024] Fig. 11. Table of surgical data for intrathecal and intraperitoneal treatment.

[0025] Fig. 12. Table of data from the rotating bar test.

[0026] Fig. 13. Overview of the inhibitors used in this study.

[0027] Figure 14. Physiological parameters: experimentally induced SAH in female rats (intracerebral injection of 300 mg / L)Body weight, mean arterial blood pressure (MABP), pH, CO2 pressure (pCO2), and O2 pressure (pO2) of animals subjected to either autologous blood (µL autologous blood) or sham surgery (control). Values ​​are mean ± SEM, n = 16–18 rats per group.

[0028] Figure 15. Contractile effects of 5-CT and ET-1 in cerebral arteries. Physiological parameters of the contractile response of the basilar artery (BA) and middle cerebral artery (MCA) to 5-CT (5HT1B / D agonist) and ET-1 (ETA / B agonist) in female rats 2 days after experimentally induced SAH (injection of 250 or 300 µL autologous blood) or sham surgery (control). Contractile response was characterized by the maximum contractile response (Emax) value (expressed as the percentage of contraction (K+ response) induced by 60 mM K+) and the negative logarithm of the molar concentration that produced the half-maximal contraction (pEC50). For biphasic concentration-contraction curves, Emax and pEC50 values ​​for each of the two phases are provided. Values ​​are mean ± SEM, n = number of rats.

[0029] Figure 16. Intracranial pressure and relative cerebral blood flow before, during and after surgery. Changes in intracranial pressure (ICP) and relative cerebral blood flow (rCBF) in female rats during surgery in experimentally induced SAH (300 µL autologous blood injection) or sham surgery (control). Values ​​are mean ± SEM, n = 16–18 rats per group.

[0030] Figures 17A–17C. Effects of ovariectomy on the vasoconstrictive response of the middle cerebral artery after transient middle cerebral artery occlusion. Figure 17A: Constriction induced by the selective endothelin B receptor agonist sarafotoxin (S6c). a: P < 0.01 compared to intact non-occlusion. b: Compared with oophorectomized (OVX) non-occluded, P < 0.01. Figure 17B: Contraction induced by the non-selective serotonin receptor agonist 5-carboxymethyltryptamine (5-CT). a: Complete non-occluded vs. occluded, P < 0.01. b: OVX non-occluded vs. occluded, P < 0.01. Figure 17C: Contraction induced by angiotensin II (Ang II) in the presence of angiotensin II receptor type 2 blockers (which induce angiotensin II receptor type 1 mediated responses). Contractions are expressed as the percentage of potassium-mediated maximum contraction (mean ± SEM). Experiments were performed in the presence of N-nitro-L-arginine methyl ester (100 µM) and indomethacin (10 µM), which block nitric oxide synthase and prostaglandin production, respectively. P < 0.05. MCA: Middle Cerebral Artery

[0031] Figures 18A-18C. Effects of estrogen replacement after oophorectomy on the middle cerebral artery after transient middle cerebral artery occlusion.Effects on vasoconstrictive response. Figure 18A: Contraction induced by the selective endothelin B receptor agonist vipertoxin 6c (S6c). Since there were no significant differences in responses between non-occluded arteries from different groups, the data were pooled and the mean is shown here. Figure 18B: Contraction induced by the non-selective serotonin receptor agonist 5-carboxymethyltryptamine (5-CT). Figure 18C: Contraction induced by angiotensin II (Ang II) in the presence of angiotensin II receptor type 2 blockers (which cause angiotensin II receptor type 1-mediated responses). Contraction is expressed as the percentage of potassium-mediated maximum contraction (mean ± SEM). Experiments were performed in the presence of N-nitro-L-arginine methyl ester (100 µM) and indomethacin (10 µM), which block nitric oxide synthase and prostaglandin production, respectively. P < 0.05, < 0.001. OVX: Ovariectomized, E: 17β-estradiol, P: Progesterone

[0032] Figure 19. Endothelin B receptor-mediated contraction of cultured middle cerebral arteries from ovariectomized females. Contraction induced in middle cerebral arteries subjected to 24-hour organ culture by the selective endothelin ETB receptor agonist keratoxin 6c (S6c). Comparison between middle cerebral arteries from intact females, ovariectomized (OVX), or OVX treated with 17β-estradiol (OVX+E). Contraction is expressed as the percentage of maximum potassium-mediated contraction (mean ± SEM).

[0033] Figure 20. Table: Comparison of Emax values ​​of contractile response in MCAs from intact females, ovariectomized females, and males after tMCAO. Maximum contractile response (Emax) induced by vipertoxin (S6c), 5-carboxymethylaminotryptamine (5-CT), and angiotensin II (Ang II) in occluded and non-occluded middle cerebral arteries isolated 48 hours after transient middle cerebral artery occlusion (tMCAO). Contraction is expressed as the percentage of potassium-mediated maximum contraction (mean ± SD). Intact: females with intact ovaries, OVX: females with ovariectomy. P < 0.05, compared with non-occluded. P < 0.01, compared with non-occluded. ns - No significant difference between occluded and non-occluded. a, b - Lower response than intact occlusion (P < 0.05). ns = No statistically significant difference compared with non-occluded.

[0034] Figures 21A-21C. In in vitro experiments, freshly isolated MCA (control) showed no contractile response to the ETB receptor agonist S6c. Figure 21A: After 48 hours of OC, S6c exhibits a strong contraction response in MCA incubated with the solvent.However, co-incubation with trametinib (GSK1120212) at 48 hours post-OC significantly inhibited S6c-induced contraction in a concentration-dependent manner. Figure 21B: Maximum contraction (Emax) induced by S6c in all groups. Figure 21C: 0.1 µM trametinib (GSK1120212) confirmed the inhibitory effect on increased ET-1-induced vasoconstriction.

[0035] Figures 22A-22C. In in vivo experiments, the effect of trametinib (described as GSK) treatment on increased ET-1-mediated vasoconstriction induced by SAH was investigated using two different treatment methods. 1 mM trametinib was administered intraperitoneally at 1 and 24 hours post-SAH (Figure 22A) or (Figure 22B) at 6 and 24 hours. (Figure 22C) Flow cytometry: The enhanced contractile response observed 6 hours after SAH treatment was validated by protein analysis using flow cytometry. The number of SMCs expressing the ETB receptor was significantly increased after SAH (solvent) compared to sham surgery (61.4% ± 10.2%; n=6) (74.2% ± 12.2%; n=7). Detailed Description

[0037] Terms and Definitions

[0038] As used herein, the term “treatment” refers to the management and care of a patient in response to a condition, disease, or symptom. This term is intended to encompass comprehensive treatment for a given condition that the patient is suffering from. Patients to be treated are preferably mammals, particularly humans. However, treatment of animals such as mice, rats, dogs, cats, horses, cattle, sheep, and pigs is also within the scope of this invention. Patients to be treated can be of different ages.

[0039] As used herein, the term “global ischemia” refers to ischemia affecting a relatively wide area of ​​the brain and typically occurs when blood supply to the brain has been drastically reduced or stopped. This is usually caused by cardiac arrest.

[0040] As used herein, the term “focal ischemia” refers to ischemia localized to a specific area of ​​the brain. It usually occurs when a blood clot blocks an artery in the brain. Focal ischemia may be the result of a thrombus or embolus.

[0041] The term “traumatic brain injury” (TBI), also known as intracranial injury, is brain injury caused by external force. TBI can be classified according to severity, mechanism (clogging or penetrating head injury) or other characteristics (e.g., occurring in a specific location or a wide area). TBI can lead to physical, cognitive, social, emotional, and behavioral symptoms, with consequences ranging from full recovery to permanent disability or death.

[0042] MEK inhibitors

[0043] In one embodiment, a MEK inhibitor of formula (I) is provided for the prevention or treatment of stroke in a subject, formula (I), or a pharmaceutically acceptable salt thereof, wherein: R1 is a C1-C6 alkyl, such as methyl, R2 is a C1-C6 alkyl, such as cyclopropyl, and Ar is selected from aryl and heteroaryl.

[0044] In one embodiment of this disclosure, a MEK inhibitor of formula (I) is provided, wherein R1 is a C1-C3 alkyl. In a preferred embodiment, R1 is a straight-chain C1-C3 alkyl. In a further preferred embodiment, R1 is methyl or ethyl. In the most preferred embodiment, R1 is methyl.

[0045] In one embodiment of this disclosure, a MEK inhibitor of formula (I) is provided, wherein R2 is a C2-C4 alkyl. In a further embodiment, R2 is a C3 or C4 cycloalkyl. In a preferred embodiment, R2 is cyclopropyl.

[0046] In one embodiment of this disclosure, a MEK inhibitor of formula (I) is provided, wherein Ar is phenyl or a substituted phenyl. In a further embodiment, Ar is a substituted phenyl. In a preferred embodiment, Ar is 2-fluoro-4-iodophenyl.

[0047] In one embodiment of this disclosure, a MEK inhibitor of formula (I) is provided, wherein R1 is a C1-C3 alkyl, R2 is a C2-C4 alkyl, and Ar is a substituted phenyl.

[0048] In a preferred embodiment of this disclosure, a MEK inhibitor of formula (I) is provided, wherein R1 is methyl or ethyl, R2 is a C3 or C4 cycloalkyl, and Ar is a substituted phenyl.

[0049] In one embodiment, a MEK inhibitor is provided for use as defined herein, wherein the MEK inhibitor has formula (II), (See page 7 / 33 of specification, 10 CN 121846102 A) Formula (II), or a pharmaceutically acceptable salt thereof.

[0050] In one embodiment, the MEK inhibitor of formula (I) is provided for use in the following ways: a. reducing endothelin-1-induced contractility; b. reducing increased contractile endothelin B receptor function; and / or c. improving neurological scores, which can be assessed by the subject's ability to rotate a rod after induced subarachnoid hemorrhage.

[0051] In one embodiment, a method for treating a subject with stroke or reducing the risk of stroke in a subject is provided, wherein the method comprises the step of administering to a subject in need a MEK inhibitor of formula (I), formula (I), or a pharmaceutically acceptable salt thereof, wherein: R1 is a C1-C6 alkyl group, such as methyl; R2 is a C1-C6 alkyl group, such as cyclopropyl; and Ar is selected from aryl, phenyl, and heteroaryl groups; thereby treating stroke or reducing the risk of stroke.

[0052] Substituents

[0053] "Alkyl" refers to a straight-chain, branched, or cyclic hydrocarbon chain group composed of carbon and hydrogen atoms, without unsaturation, and can be straight-chain or branched, substituted or unsubstituted. In some preferred embodiments, the alkyl group can consist of 1 to 12 carbon atoms, such as 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, etc., up to and including 12 carbon atoms. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butylisobutyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, and decyl. The alkyl moiety can be linked to the rest of the molecule by a single bond, such as methyl (Me), ethyl (Et), n-propyl (Pr), 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), and 3-methylhexyl. Unless otherwise specified in the specification, the alkyl group may optionally be replaced by one or more suitable substituents. The alkyl group may be monovalent, divalent, trivalent, or tetravalent, as appropriate to meet the valence requirements.

[0054] The term "alkylene" itself or as part of another substituent refers to a divalent group derived from the alkyl moiety, such as, but not limited to, -CH2CH2CH2CH2-.

[0055] "Cycloalkyl" refers to an alkyl group that specifically comprises a cyclic moiety. Exemplary cycloalkyl groups include, but are by no means limited to, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

[0056] "Substitution" means replacing a hydrogen atom from the parent moiety with another chemical group. Substituents considered include, but are not limited to: alkyl groups, such as C1-C6 alkyl groups; alkoxy groups, such as C1-C6 alkoxy groups; halogen atoms, such as -F, -Cl, -Br or -I; -CN; -NO; -NO2; -SO2H; -SO3H; -CO2H; hydroxyl; amino; mercapto; aryl; heteroaryl; or acyl.

[0057] "Aryl" refers to unsubstituted and substituted aryl groups.

[0058] "Heteroaryl" refers to unsubstituted and substituted heteroaryl groups.

[0059] Stroke Overview

[0060] Strokes can be divided into at least two major categories: ischemic and hemorrhagic. Ischemic strokes are caused by interruption of blood supply to the brain, while hemorrhagic strokes are caused by ruptured blood vessels or abnormal vascular structures. Approximately 87% of strokes are ischemic, and the remainder are hemorrhagic. According to this disclosure, stroke may also include transient ischemic attack (TIA) or may be the result of cardiac arrest or a sudden drop in systemic blood pressure caused by other means such as fibrillation.

[0061] In one embodiment of this disclosure, stroke is selected from ischemic stroke, hemorrhagic stroke, and transient ischemic attack.

[0062] In one embodiment of this disclosure, stroke is selected from: global cerebral ischemia and focal ischemia.

[0063] In one embodiment, a MEK inhibitor is administered to the subject before determining whether the subject has an acute ischemic stroke or a hemorrhagic stroke.

[0064] Ischemic stroke

[0065] In an ischemic stroke, the blood supply to a portion of the brain is reduced, resulting in dysfunction of the brain tissue in that area. There are four main causes of this: 1. Thrombosis (blockage of a blood vessel by the formation of a blood clot locally) 2. Embolism (blockage due to an embolus from another part of the body) 3. Systemic hypoperfusion (a general reduction in blood supply, such as in shock) 4. Cerebral venous sinus thrombosis.

[0066] However, stroke can also be caused by a sudden drop in blood pressure or cardiac arrest, rupture of a cerebral artery or arteriole, or a combination thereof.

[0067] In one embodiment of this disclosure, ischemic stroke is caused by traumatic brain injury (TBI), also known as intracranial injury.

[0068] In one embodiment of this disclosure, ischemic stroke is caused by embolism, thrombosis, systemic hypoperfusion, cerebral sinus thrombosis, sudden drop in blood pressure or cardiac arrest, rupture of cerebral arteries or arterioles, or a combination thereof.

[0069] Hemorrhagic Stroke

[0070] There are at least two main types of hemorrhagic stroke: "Intracerebral hemorrhage, which is essentially bleeding within the brain itself caused by bleeding within the brain parenchyma (bleeding within the brain tissue) or intraventricular hemorrhage (bleeding within the ventricular system) (when an artery in the brain ruptures and the surrounding tissues fill with blood).

[0071] Subarachnoid hemorrhage (SAH), which is essentially bleeding that occurs outside the brain tissue but still within the skull, usually due to rupture of a cerebral artery or arteriovenous malformation, specifically between the arachnoid mater and the pia mater (the fragile innermost of the three layers of meninges surrounding the brain).

[0072] The above two main types of hemorrhagic stroke are also two different forms of intracranial hemorrhage, i.e., blood pooling anywhere within the top of the skull.

[0073] Hemorrhagic stroke can occur in the context of vascular changes in the brain, such as cerebral amyloid angiopathy, arteriovenous malformations, and intracranial aneurysms, which can cause bleeding within the brain parenchyma or subarachnoid space.

[0074] In addition to nerve damage, hemorrhagic strokes typically cause specific symptoms (e.g., subarachnoid hemorrhage often results in a severe headache known as thunderclap headache) or show evidence of a previous head injury.

[0075] In one embodiment, a MEK inhibitor as defined herein is provided for the prevention or treatment of stroke, which isHemorrhagic stroke caused by intracerebral hemorrhage, subarachnoid hemorrhage, or a combination thereof.

[0076] In one embodiment, the intracerebral hemorrhage is intraparenchymal, intraventricular, or a combination thereof.

[0077] In one embodiment, the stroke is caused by subarachnoid hemorrhage.

[0078] Delayed cerebral ischemia (DCI)

[0079] Delayed cerebral ischemia can occur several days after subarachnoid hemorrhage and is a potentially treatable cause of onset for approximately one-third of first-time hemorrhage survivors. While vasospasm has traditionally been associated with the development of cerebral ischemia several days after subarachnoid hemorrhage, emerging evidence suggests that delayed cerebral ischemia is part of a more complex post-subarachnoid hemorrhage syndrome. The development of delayed cerebral ischemia involves early small artery vasospasm, accompanied by microthrombus formation, perfusion mismatch and neurovascular uncoupling, diffuse depolarization and inflammatory response, which begin at the time of hemorrhage and develop over time, ultimately leading to cortical infarction.

[0080] In one embodiment, a MEK inhibitor as defined herein is used to treat or prevent delayed cerebral ischemia (DCI).

[0081] In one embodiment, DCI is characterized by inflammation, edema, delayed cerebral vasospasm (CVS), blood-brain barrier disruption and / or increased expression of contractile receptors, such as those targeting endothelin, angiotensin, serotonin and thromboxane or prostaglandins.

[0082] Surgery and Combination Therapy

[0083] In one embodiment, a MEK inhibitor is administered to a subject without surgery prior to, concurrent with or after administration.

[0084] In one embodiment, a MEK inhibitor is administered to a subject prior to, concurrent with or after thrombectomy.

[0085] In one embodiment, the MEK inhibitor is administered to the subject before, simultaneously with, or after thrombolysis.

[0086] In one embodiment, the MEK inhibitor of this disclosure is administered to the subject before, simultaneously with, or after a surgical procedure selected from coil embolization and clipping. Specification 10 / 33 pages 13 CN 121846102 A

[0087] The procedure “coil embolization” or “endovascular coil embolization” is a procedure performed to block blood flow from an aneurysm (a weakened area in the arterial wall). Endovascular coil embolization is a minimally invasive technique, meaning that a skull incision is not required to treat a cerebral aneurysm. Instead, a catheter is used to reach the aneurysm in the brain. During endovascular coil embolization, the catheter is inserted upward through the groin into the artery containing the aneurysm. Platinum coils are then released. The coils induce aneurysm coagulation (embolization), thereby preventing blood from entering it.

[0088] The procedure of “clamping” or “microsurgical clipping” is a technique that uses metal clips to block the blood supply to an aneurysm. This procedure is well known to those skilled in the art.

[0089] In one embodiment, a MEK inhibitor is administered to the subject before, during, or after the neuroradiological procedure.

[0090] Most “neuroradiological procedures” or “interventional neuroradiological procedures” begin with the insertion of a catheter into the femoral artery, a large artery located in the groin. The catheter is a long, flexible, hollow tube that passes through a guideline upwards into the aorta (i.e., the main artery that supplies blood to the body) and then into the neck vessels leading to the blocked cerebral artery. Images of the artery are then taken using a contrast dye similar to that used for CT angiography (also known as angiography). These images allow the interventional neuroradiologist to identify the site of occlusion and develop an interventional plan. A smaller catheter (microcatheter) is then placed through the initial catheter and through the occlusive clot.

[0091] There are two main methods of clot removal: whole-clot retrieval (or thrombectomy) and clot aspiration. In the first technique, a clot retrieval device is placed through a microcatheter and opened through the clot. The device that captures the clot is then removed. The second technique, clot aspiration, involves the fragmentation and aspiration of the clot. This is performed using a catheter that is larger than a conventional microcatheter, thus providing greater suction. Thrombotomy and aspiration techniques are often used in combination. In addition to these mechanical methods, many interventional neuroradiologists also use local TPA infusions into the clot to help dissolve it.

[0092] In one embodiment of this disclosure, MEK inhibitors reduce or prevent reperfusion injury caused by neuroradiological procedures.

[0093] Reperfusion injury, sometimes called ischemia-reperfusion injury (IRI) or reoxygenation injury, is tissue damage that occurs when blood supply to the tissue is restored (reperfusion) after a period of ischemia or hypoxia (hypoxia or low oxygen). The lack of oxygen and nutrients in the blood during ischemia leads to a condition in which the restoration of circulation results in inflammation and oxidative damage by inducing oxidative stress rather than (or simultaneously) restoring normal function.

[0094] In one embodiment, a composition is provided that comprises, separately or together, a MEK inhibitor of formula (II), formula (II), page 11 / 33 of specification 14 CN 121846102 A or a pharmaceutically acceptable salt thereof, and additional pharmaceuticals.

[0095] In one embodiment, the additional pharmaceuticals are selected from: calcium channel blockers such as nimodipine, and endothelin receptor (ET) receptor blockers such as clarsentan.

[0096] In one embodiment, the additional drug is selected from calcium channel blockers.

[0097] In one embodiment, the additional drug is selected from dihydropyridine, a subclass of calcium channel blockers.

[0098] In one embodiment, the additional drug is selected from dihydropyridines, such as amlodipine (Norvasc), aranidipine (Sapresta), azelnidipine (Calblock), barnidipine (HypoCa), benidipine (Coniel), cilnidipine (Atelec, Cinalong, Siscard), clovidipine (Cleviprex), efonidipine (Landel), felodipine (Plendil), isradipine (DynaCirc, Prescal), lacidipine (Motens, Lacipil), larcanidipine (Zanidip), and manidipine. (Calslot, Madipine), Nicardipine (Cardene, Carden SR), Nifedipine (Procardia, Adalat), Nivadipine (Nivadil), Nimodipine (Nimotop), Nisoldipine (Baymycard, Sular, Syscor), Nitrendipine (Cardif, Nitrepin, Baylotensin), Pranidipine (Acalas).

[0099] In one embodiment, a multi-component kit is provided, comprising: a MEK inhibitor as defined herein; and additional pharmaceuticals as defined herein; wherein the MEK inhibitor and additional pharmaceuticals are formulated for simultaneous or sequential use; and optional instructions for use.

[0100] Composition and Administration

[0101] In one embodiment, the present invention relates to a pharmaceutical composition comprising an effective amount of a MEK inhibitor and additional pharmaceuticals.

[0102] While the MEK inhibitors disclosed herein can be administered in the form of the original chemical compound, it is preferred that the active ingredient be introduced into the pharmaceutical composition, optionally in the form of a physiologically acceptable salt, together with one or more adjuvants, excipients, carriers, buffers, diluents, and / or other commonly used pharmaceutical adjuvants.

[0103] In one embodiment, the present disclosure provides compositions comprising a MEK inhibitor as defined herein or a pharmaceutically acceptable salt or derivative thereof, and one or more pharmaceutically acceptable carriers for use therein, and optionally other therapeutic and / or prophylactic ingredients known and used in the art. The carrier must be "acceptable" in the sense of compatibility with other components of the formulation and harmless to its recipient. In a further embodiment, the invention provides pharmaceutical compositions or compositions comprising more than one compound or prodrug for use according to the present disclosure, such as two different compounds or prodrugs for use according to the present disclosure.

[0104] The compositions of this disclosure may be those suitable for oral, rectal, bronchial, nasal, pulmonary, local (including oral and sublingual), transdermal, vaginal, or parenteral (including skin, subcutaneous, intramuscular, intraperitoneal, intravenous, intraarterial, intracerebral, and intraocular injection or infusion), or those suitable for inhalation or blowing (including powder and liquid aerosol) administration, or those administered via a sustained-release system. Suitable examples of sustained-release systems include a semi-permeable matrix of a solid hydrophobic polymer containing a compound of this disclosure, which may be in the form of a molded article, such as a membrane or microcapsule. Another suitable example is nanoparticles. Specification 12 / 33 pages 15 CN 121846102 A

[0105] In one embodiment, the MEK inhibitor for use as defined herein is administered orally, intrathecally, intraperitoneally, intraocularly, or intravenously.

[0106] In one embodiment, the MEK inhibitor for use as defined herein is administered intranasally.

[0107] In one embodiment, the MEK inhibitor is administered intravenously. In one embodiment, the MEK inhibitor is administered to the subject up to 6 hours after the stroke, for example, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, or up to 5 hours after the stroke. In one embodiment, treatment continues after the first dose of the MEK inhibitor following the stroke for up to 3 days.

[0108] In one embodiment, the MEK inhibitor is administered once or more daily for up to 3 days after the stroke.

[0109] In one embodiment, the MEK inhibitor is administered to the subject in combination with a neuroprotective agent. Treatment with the MEK inhibitor may be discontinued 1, 2, or 3 days after the stroke, while treatment with the neuroprotective agent continues. In one embodiment, neuroprotective treatment continues for one month or more months.

[0110] Subject

[0111] The subject according to this disclosure can be any subject who has or is about to have a stroke. Preferably, the subject is a human subject, such as a patient. In one embodiment, the subject is a human subject who has no history of stroke. In one embodiment, the human subject has previously had a stroke.

[0112] Item

[0113] 1. A MEK inhibitor of formula (I) for the prevention or treatment of stroke in a subject, formula (I), or a pharmaceutically acceptable salt thereof, wherein: R1 is a C1-C6 alkyl, such as methyl, R2 is a C1-C6 alkyl, such as cyclopropyl, and Ar is selected from aryl and heteroaryl.

[0114] 2. The MEK inhibitor according to any one of the preceding items, wherein R1 is a C1-C3 alkyl.

[0115] 3. The MEK inhibitor according to any one of the preceding items, wherein R1 is a straight-chain C1-C3 alkyl.

[0116] 4. The MEK inhibitor according to any one of the preceding items, wherein R1 is methyl or ethyl.

[0117] 5. The MEK inhibitor according to any one of the preceding items, wherein R1 is methyl.

[0118] 6. The MEK inhibitor according to any one of the preceding items, wherein R2 is C2-C4 alkyl.

[0119] 7. The MEK inhibitor according to any one of the preceding items, wherein R2 is C3 or C4 cycloalkyl.

[0120] 8. The MEK inhibitor according to any one of the preceding items, wherein R2 is cyclopropyl. Specification 13 / 33 pages 16 CN 121846102 A

[0121] 9. The MEK inhibitor according to any one of the preceding items, wherein Ar is phenyl or substituted phenyl.

[0122] 10. The MEK inhibitor according to any one of the preceding items, wherein Ar is substituted phenyl.

[0123] 11. The MEK inhibitor according to any one of the preceding items, wherein Ar is 2-fluoro-4-iodophenyl.

[0124] 12. The MEK inhibitor according to any one of the preceding items, wherein R1 is a C1-C3 alkyl, R2 is a C2-C4 alkyl, and Ar is a substituted phenyl.

[0125] 13. The MEK inhibitor according to any one of the preceding items, wherein R1 is methyl or ethyl, R2 is a C3 or C4 cycloalkyl, and Ar is a substituted phenyl.

[0126] 14. The MEK inhibitor for use according to any one of the preceding items, wherein the MEK inhibitor has formula (II), formula (II), or a pharmaceutically acceptable salt thereof.

[0127] 15. The MEK inhibitor for use according to any one of the preceding items, wherein the stroke is selected from: ischemic stroke, hemorrhagic stroke, and transient ischemic attack.

[0128] 16.17. A MEK inhibitor according to any of the preceding projects, wherein the stroke is selected from: global cerebral ischemia and focal ischemia.

[0129] 18. A MEK inhibitor according to any of the preceding projects, wherein the ischemic stroke is caused by embolism, thrombosis, systemic hypoperfusion, cerebral venous sinus thrombosis, sudden drop in blood pressure or cardiac arrest, rupture of cerebral arteries or arterioles or a combination thereof.

[0130] 19. A MEK inhibitor according to any of the preceding projects, wherein the hemorrhagic stroke is caused by intracerebral hemorrhage, subarachnoid hemorrhage or a combination thereof.

[0131] 20. A MEK inhibitor according to any of the preceding projects, wherein the intracerebral hemorrhage is intraparenchymal, intraventricular or a combination thereof.

[0132] 21. A MEK inhibitor according to any of the preceding projects, wherein the stroke is caused by subarachnoid hemorrhage.

[0133] 22. A MEK inhibitor according to any of the preceding projects, wherein the stroke is delayed cerebral ischemia (DCI).

[0134] 22. A MEK inhibitor for the use of any of the preceding items, wherein the stroke is caused by traumatic brain injury (TBI).

[0135] 23. A MEK inhibitor for the use of any of the preceding items, wherein the DCI manifests as inflammation, swelling, delayed cerebral vasospasm (CVS), blood-brain barrier disruption and / or increased expression of contractile receptors, such as those targeting endothelin, angiotensin, serotonin and thromboxane or prostaglandins.

[0136] 24. A MEK inhibitor for the use of any of the preceding items, wherein the MEK inhibitor is administered to the subject without the need for surgery before, during or after the administration.

[0137] 25. A MEK inhibitor for the use of any of the preceding items, wherein the MEK inhibitor is administered to the subject before, during or after thrombectomy.

[0138] 26. A MEK inhibitor for any of the preceding items, wherein the MEK inhibitor is administered to the subject before, simultaneously with, or after thrombolysis.

[0139] 27. A MEK inhibitor for any of the preceding items, wherein the MEK inhibitor is administered to the subject before, simultaneously with, or after a surgical procedure selected from coil embolization and clipping.

[0140] 28. A MEK inhibitor for any of the preceding items, wherein the MEK inhibitor is administered to the subject before, simultaneously with, or after a neuroradiological procedure.

[0141] 29. A MEK inhibitor for any of the preceding items, wherein the MEK inhibitor reduces or prevents...To prevent reperfusion injury caused by neuroradiological procedures.

[0142] 30. A MEK inhibitor for the use of any of the preceding items, wherein the MEK inhibitor is administered to the subject before determining whether the subject has an acute ischemic stroke or a hemorrhagic stroke.

[0143] 31. A MEK inhibitor for the use of any of the preceding items, wherein the MEK inhibitor is administered orally, intrathecally, intraperitoneally, intraocularly, or intravenously.

[0144] 32. A MEK inhibitor for the use of any of the preceding items, wherein the MEK inhibitor is administered intravenously.

[0145] 33. A MEK inhibitor for the use of any of the preceding items, wherein the MEK inhibitor is administered to the subject at most 6 hours after the stroke, for example, at most 1 hour, at most 2 hours, at most 3 hours, at most 4 hours, at most 5 hours after the stroke.

[0146] 34. A MEK inhibitor for the use of any of the preceding items, wherein the MEK inhibitor is administered once or more daily for up to 3 days after a stroke.

[0147] 35. A MEK inhibitor for the use of any of the preceding items, wherein the subject is a human subject.

[0148] 36. A MEK inhibitor of formula (I) as defined in any of the preceding items is used for: a. reducing endothelin-1-induced contractility; b. increasing endothelin B receptor function; and / or c. improving neurological scores, which can be assessed by the subject's ability to rotate a rod after induced subarachnoid hemorrhage.

[0149] 37. A method for treating a subject with stroke or reducing the risk of stroke in a subject, wherein the method comprises administering a MEK inhibitor of formula (I) to a subject in need, (See specification 15 / 33 pages 18 CN 121846102 A) Formula (I), or a pharmaceutically acceptable salt thereof, wherein: R1 is a C1-C6 alkyl group, such as methyl, R2 is a C1-C6 alkyl group, such as cyclopropyl, and Ar is selected from aryl, phenyl, and heteroaryl groups; thereby treating stroke or reducing the risk of stroke.

[0150] 38. A composition comprising, separately or together, a MEK inhibitor of formula (II), formula (II), or a pharmaceutically acceptable salt thereof, and additional pharmaceutical agents.

[0151] 39. The composition according to any one of the preceding items, wherein the additional pharmaceutical agent is selected from: calcium channel blockers such as nimodipine, and endothelin receptor (ET) receptor blockers such as clarsentan.

[0152] 40. A multi-component kit comprising: a MEK inhibitor as defined in any of the preceding items; and another drug as defined in any of the preceding items;MEK inhibitors and other drugs were formulated for simultaneous or sequential use; and optional instructions for use.

[0153] Examples

[0154] Example 1: Organ culture and concentration response curves for selected MEK1 / 2 inhibitors

[0155] Materials and methods

[0156] Feeding, housing and ethics instructions 16 / 33 pages 19 CN 121846102 A

[0157] 110 Sprague-Dawley rats (NTac:SD) obtained from Taconic (Denmark) were maintained in a 12 / 12 hour light / dark cycle (lighting started at 7 am) and housed at a constant temperature (22 ± 2 °C) and humidity (55 ± 10%) with free access to food and water. Rats were usually housed together in European standard cages (Type VI, with 123 cover) in groups of 2-6, and individually after surgical procedures (Type III, with 123 cover). Fifty-two male Sprague-Dawley rats (298–370 g) were used for the surgical procedure, with approval obtained from the Danish Animal Experimentation Inspectorate (License No. 2016-15-0201-00940). Animal work was conducted at the Glostrup Research Park in Rigshospitalet-Glostrup, Denmark.

[0158] Harvesting and organ culture of cerebral arteries (ex vivo model)

[0159] Rats were sedated with O2 / CO2 (30 / 70%) and euthanized by decapitation. The brains were gently removed and cooled in a cold oxygenated buffer solution of the following composition: 119 mM NaCl, 4.6 mM KCl, 1.5 mM CaCl2, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 15 mM NaHCO3, and 5.5 mM glucose; pH 7.4. The basilar artery (BA) was carefully dissected from the brain in physiological buffer solution and then either OC (in naïve animal) or directly mounted in a wire myograph (from surgically operated rats). The segments were incubated for 48 hours in DMEM supplemented with streptomycin and penicillin at 37°C in a humidified 5% CO2 / O2 atmosphere with an inhibitor or solvent (DMSO). The medium was changed after 24 hours.

[0160] Myograph – In Vitro Pharmacology (OC and In Vitro)

[0161] For contractility measurements, incubated BA (in vitro) and surgically operated BA (in vivo) were cut into segments and mounted on a pair of wires (40 μm) in a myograph bath. After 48 hours in the medium, the segments were mounted in the same manner.Arteries from OC. A filament was attached to a micrometer screw that allowed fine-tuning of the distance between the filaments to control vascular tone. A second filament was attached to a force displacement sensor paired with an analog-to-digital converter (AD Instruments, Oxford, UK).

[0162] These segments were equilibrated in a physiological buffer at pH 7.4 aerated with 95% O2 / 5% CO2 at a temperature of 37°C, and the filaments were separated for isometric pretension of 2 Nm⁻¹. The arterial segments were exposed to 60 mM K⁺ 2 or 3 times by exchanging the buffer with 60 mM K⁺ buffer. To maintain the same molar osmolar concentration, proportional amounts of Na⁺ were removed from the buffer. An absolute cutoff value of 2.0 mN K⁺max was set to include arterial segments from rats undergoing surgical manipulation. Endothelial function was evaluated by adding 5-HT (3.10⁻⁷ M) followed by carbachol (10⁻⁵ M). For arteries from OC, the experimental protocol was as follows: First, cumulative concentration-response curves were plotted for vipertoxin 6C (S6c, 10⁻¹⁴–10⁻⁷ M), followed by concentration-response curves for endothelin-1 (ET-1, 10⁻¹⁴–10⁻⁷ M). At the ET-1 peak (10⁻⁷ M), the buffer was changed to a Ca²⁺-free buffer containing 10⁻⁷ M ET-1 and nimodipine (an L-type voltage-dependent Ca²⁺ channel entry blocker, 10⁻⁷ M). Then, concentration-response curves for Ca²⁺ (0.0125–3 mM) were plotted. When studying vasodilation, arteries were pre-constricted with U46619 (1 × 10⁻⁷ - 3 × 10⁻⁷ M) or K⁺ (41 mM) and cumulative concentration-response curves were plotted by adding calcitonin gene-related peptide (CGRP, 10⁻¹² - 10⁻⁷ M), carbachol (10⁻¹⁰ - 10⁻⁵ M), or SNP (10⁻¹¹ - 10⁻⁴ M).

[0163] Two arterial segments were selected from each operated animal for the cumulative concentration-response curve of ET-1 (10⁻¹⁴ - 10⁻⁷ M) or the Ca²⁺ concentration-response curve (0.0125 - 3 mM) of ET-1 pre-constricted (10⁻⁷ M) in the presence of nimodipine (10⁻⁷ M). If only one curve was found above the cutoff value, the concentration-response curve for ET-1 was preferred. The segment exhibiting the highest K+ response typically corresponds to the concentration-response curve for ET-1. Concentration-response curves for Ca2+ were plotted by adding increased volume of CaCl2 (from a 125 mM stock solution) to a Ca2+-free buffer solution. The Ca2+-free buffer solution had a similar composition to the one described above, but at 1.5 mM...CaCl2 was replaced with 0.03 mM EDTA. Instructions 17 / 33 pages 20 CN 121846102 A

[0164] Statistics and Data Acquisition

[0165] Contractile response for each segment was adjusted for artery length and expressed as mN / mm (Nm⁻¹). If there was no significant difference in response to 60 mM K⁺, contractility data were shown as a percentage of the 60 mM K⁺max plateau response of a single vessel relative to baseline. To compare ET⁻¹ sensitivity, arteries were normalized to a percentage of ET⁻¹max for a single vessel. The curve was constrained to the maximum contraction when the artery reached maximum contraction before the final concentration was added. Relative log IC₅₀ / log EC₅₀ is the concentration corresponding to the median response between lower and higher plateau estimates. Emax is the maximum contraction in the concentration-response curve, and ET⁻¹max is the maximum contraction against ET⁻¹. Unless otherwise stated, all quantitative data are expressed as mean ± standard error of the mean (SEM).

[0166] K+ and endothelial-dependent responses were statistically compared using one-way ANOVA and Holm-Sidak multiple comparison tests (comparing all groups). Concentration-response curves were statistically compared using two-way repeated measures ANOVA and Holm-Sidak multiple comparison tests, with Geisser-Greenhouse sphericity correction applied to normalized ET-1max data. Competitive curve fitting was performed by comparing biphasic and nonlinear regression curve fits and log(agonist)-variable slope. For all ET-1 curves, the biphasic regression model was considered the best fit. The significance of neurological assessment scores was evaluated using the two-tailed Fischer exact test. Statistical analysis was performed using Graphpad 8.02 software, and significance p-values ​​were defined as: = p < 0.05, = p < 0.01, = p < 0.001.

[0167] Reagents

[0168] S6c was from PolyPeptide Group (Sweden), ET-1 was from Bachem (Germany), and CGRP was from Tocris (UK). Except for U0126, all MEK1 / 2 inhibitors were obtained from Selleckchem and dissolved in DMSO. U0126 monoethanolate (U120), DMSO (Sigma D2650), and all other chemicals were obtained from Sigma Aldrich.

[0169] Results

[0170] Rat basilar arteries (BAs) isolated from 588 rats (male Sprague Dawley rats, ~320 g) were cultured (OC), with each BA divided into 4 segments. Initially, a group of MEK1 / 2 inhibitors were tested at a concentration of 1 µM, which was just...The effect was significantly lower than the established threshold (10 µM) for the efficacy of U0126 used to date. Figure 1A shows the contraction induced by the highly specific ETB agonist S6c relative to 60 mM K+-induced contraction. Inhibitors were divided into four groups after initial screening. The groups are as follows: I) Ineffective in 1 µM solvent (DMSO) and U0126. II) Some effect: Binimetinib, Selumetinib, and RO5126766. III) Borderline effective: Refametinib. IV) Highly effective: Cobimetinib, TAK-733, Trametinib, and PD0325901. The latter group was further divided into two subgroups based on cell-free IC50 (Figure 9), as these two subgroups were indistinguishable from each other at 1 µM.

[0171] There was no significant difference in depolarization-induced contraction between arterial segments incubated with DMSO (solvent) or MEK1 / 2 inhibitors (Fig. 1B). Endothelial function of the arteries was also investigated. This was tested by applying 10⁻⁵ M carbachol to arteries pre-contracted with 3 × 10⁻⁷ M 5-HT. OC with either DMSO or U0126 was associated with poor endothelial response to carbachol (Fig. 1C). Interestingly, significant differences were found between some groups, which appeared to follow the same trend observed with S6c inhibition (Fig. 1A). The three MEK1 / 2 inhibitors TAK-733 (P = 0.0065), trametinib (P = 0.0026), and PD0325901 (P = 0.0474) showed significantly better endothelial function compared to the DMSO solvent.

[0172] The selected inhibitors were characterized. IC50 values ​​(based on S6c's Emax) of the six selected candidates were determined using the entire logarithmic concentration range from no inhibition of S6c-induced contraction to near-maximal inhibition (Figure 2A). Bimetinib (pIC50 5.17 ± 0.46) and RO5126766 (pIC50 5.68 ± 0.26) were the least potent inhibitors, consistent with the data in Figure 1. When analyzing the concentration-response curves of the inhibitors, it was clear that TAK-733 (pIC50 6.89 ± 0.31) and cobimetinib (pIC50 7.25 ± 0.51) were less effective than trametinib (pIC50 7.68 ± 0.32) and PD0325901 (pIC50 7.71 ± 0.29) (Figure 2A), which was not observed in Figure 1.

[0173] Furthermore, whether there was any effect on depolarization (60 mM K+)-induced arterial contractility was investigated (Figure 2B).No significant differences were observed between groups, and no obvious concentration-dependent effects were observed. Initial screening (Fig. 1C) showed interesting effects of MEK1 / 2 inhibitors on endothelial function. Fig. 2C shows the concentration-dependent effects of MEK1 / 2 inhibitors on endothelial function in response to carbacholine, exhibiting a similar trend to that observed for inhibitors in the concentration-response curves at the Emax of S6c (Fig. 2A).

[0174] Conclusion

[0175] It is known that cerebral arteries treated with 10 µM U0126 in 48 hours of OC show an inhibitory effect on ETB-specific S6c-induced contractility. In this disclosure, 1 µM U0126 did not show a significant effect, while 1 µM trametinib and PD0325901 almost completely inhibited the contractile response after 48 hours of organ culture (Fig. 1A). Data from OC experiments suggest a close association between MEK1 / 2 inhibition and functional upregulation of the ETB receptor after evaluation with the specific ETB agonist S6c. Cell-free IC50 values ​​(Fig. 13) were also closely correlated with IC50 values ​​for inhibiting ETB receptor upregulation (Fig. 2A). This supports the link between MEK1 / 2 inhibition and changes in functional receptors in cerebral arteries.

[0176] Therefore, by inhibiting signaling pathways including MEK1 / 2, functional upregulation of ex vivo ETB receptors can be completely prevented.

[0177] Example 2: Effects of effective MEK1 / 2 inhibitors on pathways regulating vasomotor function after 48-hour organ culture in basilar artery

[0178] Materials and Methods

[0179] Methods were performed as described in the preceding examples.

[0180] Results

[0181] Since arteries incubated with the most effective MEK1 / 2 inhibitors showed preservation of endothelial function, the possible reasons for this improvement were further investigated. Arteries treated with 1 µM trametinib or PD0325901 after OC were pre-constricted with the thromboxane A2 agonist U46619 (1 10⁻⁷ - 3 10⁻⁷ M) or K+ (41 mM). No significant differences in pre-constriction levels were observed between groups. In this new set of experiments, improved vasodilation in response to carbachol was confirmed (10⁻¹⁰ - 10⁻⁵ M, Figure 3A). This effect of carbachol may be due to improved endothelial NO release or changes in NO sensitivity in vascular smooth muscle cells (VSMCs). As shown in Figure 3B, there was a significant difference in Emax after the addition of the NO donor nitroprusside (SNP, 10⁻¹¹ – 10⁻⁴ M). This suggests that changes in cGMP and NO-sensitive signaling in VSMCs are the cause of increased epithelial function after incubation with MEK1 / 2 inhibitors. Since both cGMP and cAMP are important in regulating cerebral arterial vasodilation,The signaling pathways associated with cAMP production were also investigated. No difference in response to CGRP was observed (10⁻¹² - 10⁻⁷ M, Fig. 3C).

[0182] Cerebral arteries are known to exhibit VDCC (voltage-dependent calcium channel)-independent contraction after cerebral ischemia and SAH, which is absent in fresh arteries. To further characterize the changes in cellular pathways leading to enhanced contraction, concentration-response curves for Ca²⁺ were plotted by adding Ca²⁺ to BA pre-contracted with ET-1 in a Ca²⁺-free buffer containing 10⁻⁷ M nimodipine (an L-type VDCC inhibitor). Fig. 3D shows VDCC-independent contraction of BA, where only trametinib (P = 0.019) significantly prevented this increased VDCC-independent contraction.

[0183] Conclusion

[0184] VDCC is dominant in non-SAH cerebral arteries or in fresh arteries, which can be blocked by nimodipine (the standard treatment for SAH). In this disclosure, it was found that calcium channels changed during SAH and 48-hour OC. These manipulations resulted in an increase in the expression of VDCCs of an independent type, i.e., those that could not be blocked by standard treatment with nimodipine. Trametinib was found to significantly prevent this increased VDCC independent contraction, which (i) normalizes calcium channel expression and (ii) may make the subject fit for treatment with nimodipine.

[0185] Example 3: Comparison of Trametinib and PD0325901 in rats

[0186] Materials and Methods

[0187] Rat Subarachnoid Hemorrhage Model (In Vivo Model)

[0188] Rats were anesthetized and prepared for an intracisional infusion of autologous blood to simulate SAH. Sham-operated rats underwent the same procedure, omitting the 300 µL intracisional blood injection. At the end of the procedure, a PinPort (PNP3F22, Instech, USA) was placed at the end of the ICP catheter to provide access for intrathecal (it) treatment via a PinPort syringe (PNP-3M, Instech, US). Twenty-four hours post-operation, rats received a subcutaneous injection of carprofen (Norodyl, 5 mg / kg) (Scanvet, Denmark) for analgesia.

[0189] In vivo treatment regimens

[0190] The concentrations and doses of trametinib and PD0325901 used in this disclosure are based on in vitro data from this disclosure. All treatments were blinded throughout the study, and all treatment regimens can be found in Figure 9.

[0191] Intrathecal treatment

[0192] The intrathecal (it) treatment volume was estimated for approximately 90 µL of cerebrospinal fluid (CSF) volume (21), and the total dose was determined by...PinPort, placed in the ICP catheter within the cerebellomedullary cistern during the surgical procedure, was administered in three treatments (4 hours, 10 hours, and 24 hours). The first and third injections were performed while the rats were immobilized. Since the 10-hour postoperative treatment was administered by an investigator, the rats were briefly anesthetized with a mask using 3.5–4% isoflurane (maintained at 1.75–2%) at atmospheric / O2 (70% / 30%) to prevent sudden movement. Immediately postoperatively and along with the 10-hour and 24-hour treatments, the animals were subcutaneously given 2.5 ml of isotonic saline to prevent dehydration. All in vivo treatments were dissolved in Elliott's B (artificial CSF) solution containing 0.5% hydrogenated castor oil (cremophor) EL (Kolliphor EL): NaCl 125 mM, NaHCO3 23 mM, glucose 4 mM, MgSO4 1 mM, KCl 4 mM, CaCl2 1 mM, and Na2HPO4 1 mM.

[0193] Surgical Parameters – it Group

[0194] In all 41 rats, mean arterial blood pressure (MABP), pH, pCO2, pO2, ICP (138.4 ± 6.9 mmHg), and temperature were within acceptable physiological limits during surgery. In the SAH + solvent group, one postoperative death occurred 24 hours after SAH.

[0195] Results

[0196] Two of the most potent MEK1 / 2 inhibitors for SAH in rat models identified in the OC study were further investigated. Trametinib and PD0325901 showed the highest potency, meaning they could potentially be administered in smaller volumes than the current drug option U0126. After injecting 1 µM, 15 µL it into the rat's CSF volume (assumed to be 90 µL), the predicted diluted CSF concentration was approximately 10⁻⁷ M. This corresponds to the 75% inhibitory effect estimated in the in vitro OC study (Figure 2A).

[0197] Conclusion

[0198] Trametinib was found to have high potency, indicating that it can be used at a lower dose than the current drug choice, U0126. The preferred drug, U0126, was found to have similar beneficial effects in vivo, but due to its poor solubility, it could not be converted to systemic administration. Trametinib was found to have excellent solubility and potency, indicating that it can be used in a lower volume, allowing for systemic administration, while still showing favorable anti-SAH parameters.

[0199] Example 4: Effect of trametinib and PD0325901 treatment on contractile response and endothelial function of 60 mM K+

[0200] Materials and Methods Specification 20 / 33 pages 23 CN 121846102 A

[0201] The methods were performed as outlined in the preceding examples.

[0202] Results

[0203] Contractile response (Nm⁻¹) of all BAs to 60 mM K⁺ (including vessels below the cutoff value) showed a significantly higher contractile response in the SAH + it trametinib group (5.00 ± 0.29 Nm⁻¹) compared to both the sham surgery + it solvent group (3.30 ± 0.45 Nm⁻¹) and the SAH + it solvent group (3.45 ± 0.22 Nm⁻¹) (Fig. 4A). Individual segment lengths (range 0.9–1.2 mm; 1.0 ± 0.1 mm) did not differ significantly between groups. The SAH group had a slightly lower mean endothelial function compared to other groups, but this was not significant (SAH vs. it trametinib, P = 0.1382) (Fig. 4B).

[0204] Conclusion

[0205] The use of a MEK1 / 2 inhibitor with higher potency than U0126 allows for concentration-dependent preservation of apparent endothelial function (Fig. 2C). A similar pattern was observed for the functional upregulation of endothelial function and contractile ETB receptors (Fig. 2A). Therefore, the MEK1 / 2 pathway appears to be involved in disrupting endothelial and VSMC signaling in response to reduced blood flow through the arteries. This contrasts with neuronal vasodilatory signaling, exemplified by CGRP, where no changes were observed (Fig. 3C). Due to the relatively high variability, no significant changes in endothelial function were observed in animals treated in vivo with trametinib or PD0325901, although the mean values ​​in both groups were higher than in the SAH group and the SAH + it solvent group (Fig. 4B).

[0206] In contrast to the effects of trametinib or PD0325901 on ETB receptor contractility and apparent endothelial function, no concentration-dependent effect of the inhibitor on the K+ response after OC was observed (Fig. 2B). However, rats treated with it trametinib (but not it PD0325901) after experimental SAH showed a higher K+ response compared to those treated with it solvent (Fig. 4A). Blood vessels incubated with it-trametinib or it-PD0325901 exhibited a higher-order K+ response than those tested in the OC model (Figure 1B).

[0207] Example 5: Effect of it-trametinib and PD0325901 treatment on contractile response to ET-1

[0208] Materials and Methods

[0209] Methods were performed as outlined in the preceding examples.

[0210] Results

[0211] Due to the differences in K+ response, unnormalized data were initially used. BA from all treatment regimens (Figure 9) was compared by cumulative concentration-response curves of ET-1 (10⁻¹⁴ – 10⁻⁷ M). No significant differences were observed between the curves (Figure 1B).5A). Arterial ET-1 sensitivity was investigated by normalizing the curves to their own ET-1max. For ET-1max normalized data, the SAH + it trametinib group showed significantly reduced contraction at low ET-1 concentrations (10-12.5–10-11.0 M) compared to the SAH + solvent group (Fig. 5B). Competitive curve fitting was performed by comparing biphasic vs. four-parameter variable slope regression. For all groups, the biphasic regression model was considered the best curve fit (Fig. 10). The logEC50 (1) 95% confidence interval (-12.74 to -12.17) of the SAH group showed significantly higher sensitivity compared to the sham surgery + it solvent group (-11.91 to -10.07) and the SAH + it trametinib group (-11.04 to -9.936). In logEC50 (2), the sensitivity of the SAH + i.t. trametinib group (-8.964 to -8.862) was significantly lower than that of the SAH + i.t. solvent group (-9.228 to -9.050). The logEC50 (1) and logEC50 (2) values ​​were similar for the absolute curve (Nm-1) and the normalized curve of ET-1max (Figures 5A and 5B). All logEC50 (1) values, logEC50 (2) values, and 95% CI values ​​can be found in Figure 10.

[0212] Conclusion

[0213] SAH leads to increased sensitivity to ET-1, which is a hallmark of the disease. It was found that the sensitivity of SAH + i.t. trametinib to ET-1 was significantly lower than that of the SAH + i.t. solvent group. Therefore, trametinib can effectively prevent the detrimental upregulation of endothelin receptor after SAH.

[0214] Example 6: Effect of it-trametinib and PD0325901 treatment on VDCC-independent calcium ion contraction (Instructions for Use, Page 21 / 33, 24 CN 121846102 A)

[0215] Materials and Methods

[0216] The methods were performed as outlined in the preceding examples.

[0217] Results

[0218] To investigate whether in vivo treatment affected VDCC-independent contractility, cumulative concentration-response curves for Ca2+ (0.0125-3 mM) were plotted in BA pre-contracted with ET-1 (10⁻⁷ M) and in the presence of 10⁻⁷ M nimodipine. Compared with the sham surgery + i.t. solvent group (0.6 ± 1.2 Nm⁻¹), SAH + i.t. solvent group (1.2 ± 0.2 Nm⁻¹), and SAH + i.t. PD0325901 group (1.0 ± 0.3 Nm⁻¹), the SAH group (2.8 ± 0.5 Nm⁻¹) showed significantly higher nimodipine-insensitive ET-1max contraction, while SAH+ There was no significant difference between the trametinib group (1.8 ± 0.5 Nm⁻¹) and the control group (Figure 6).

[0219] Conclusion

[0220] SAH resulted in a higher degree of nimodipine-insensitive calcium channel response. Trametinib treatment normalized VDCC-independent contraction to a level similar to that of the control.

[0221] Example 7: Neurological assessment of the efficacy of trametinib and PD0325901 treatment

[0222] Materials and methods

[0223] Neurological assessment - Rotating bar test

[0224] Total sensorimotor function was evaluated using the rotating bar test, including baseline assessment one day prior to SAH induction. Briefly, movement through a rotating bar (45 mm in diameter and 150 cm in length) at 10 rpm was evaluated using a cage with the rats' own bedding material at the end (“smells like home”). The rats’ performance was scored according to the following definition: low, the animal could not pass through the bar without falling; high, the animal could pass through the entire bar without falling. All animals were trained to pass the bar before surgery. Each animal was scored twice for left and right rotations, both before SAH and on days 1 and 2 post-surgery, i.e., 4 counts per animal. Animals were graded by personnel unaware of the experimental groups. Data are presented as the percentage of high score counts / total score counts.

[0225] Results

[0226] The rotating bar test conducted in this paper is not a purely motor function test, as it does require prior training of the rats. In addition to learning aspects, the fact that the bar is rotating also makes motivation a factor for success. Therefore, successful scores involve memory, motivation, and attention. Rats were scored at three time points: before SAH, 24 hours and 48 hours post-SAH (data are presented as the percentage of high score counts / total score counts).

[0227] All rats scored 100% in the rotating bar test before surgery (Fig. 7A). Comparing 24 hours post-SAH with before SAH, all groups except the sham surgery + it solvent group showed neurological scoring deficits (Figs. 7A-C, Fig. 12). Compared with pre-SAH, rats after experimental SAH showed significantly worse neurological scores at 48 hours (previous score 100% to 48h score 75%, P = 0.0022). At the 48-hour endpoint, the neurological scores of rats in the SAH + i.t. trametinib group (48h score 96%), sham surgery + i.t. solvent group (48h score 100%), and SAH + i.t. solvent group (48h score 94%) were significantly higher than those in the SAH group (48h score 75%) (Figure 7C). See Figure 12 for all score percentages.

[0228] Conclusion

[0229] Improvement in neurological assessment scores (rotating bar test) was observed for trametinib, but for pharmacokinetics...Unstable PD0325901 was not observed, which supports the involvement of the MEK1 / 2 pathway in phenotypic regulation observed in VSMCs after SAH.

[0230] Example 8: Effect of ip trametinib treatment on contractile response to 60 mM K+, ET-1 and neurological assessments Specification 22 / 33 pages 25 CN 121846102 A

[0231] Materials and Methods

[0232] Intraperitoneal Treatment

[0233] For intraperitoneal treatment (ip), the compound was dissolved in a NaCl solution containing 10% hydrogenated castor oil EL (Kolliphor EL) and 10% PEG400, which was also used as a solvent. The total dose was administered as two treatments (6 hours and 24 hours). Immediately after surgery and along with the treatment, 2.5 ml of isotonic saline was administered subcutaneously to the animals to avoid dehydration.

[0234] Surgical Parameters – IP Group

[0235] In all 11 rats, mean arterial blood pressure (MABP), pH, pCO2, pO2, ICP (124.2 ± 6.3 mmHg), and temperature were within acceptable physiological limits during surgery.

[0236] Results

[0237] Starting with an it proof-of-concept study, trametinib was further tested using an IP injection treatment regimen. Animals were exposed to SAH and treated with IP injections of trametinib or a solvent at 6 and 24 hours post-SAH. Forty-eight hours after induction of experimental SAH, arterial dissection was performed using a filamentous kinesiometer. There were no significant differences in segment lengths (range 0.9–1.2 mm; 1.13 ± 0.02 mm) between SAH + IP solvent or SAH + IP trametinib. When comparing the SAH + ip trametinib group (3.03 ± 0.19 Nm⁻¹) with the SAH + ip solvent group (3.23 ± 0.27 Nm⁻¹), no difference was found in the contractile response (Nm⁻¹) of all BAs to 60 mM K⁺ (Fig. 8A). This contrasts with SAH + it treatment (Fig. 4A).

[0238] The SAH + ip trametinib group and the SAH + ip solvent group were compared by cumulative concentration-response curves of ET-1 (10⁻¹⁴ – 10⁻⁷ M). The contractility (at 10⁻⁹.5 M) of the SAH + ip trametinib group was significantly reduced compared with that of the SAH + ip solvent group. Competitive curve fitting was performed by comparing biphasic vs. four-parameter variable slope regression. For both groups, the biphasic regression model was considered the best curve fit, with logEC50 (1) values, logEC50 (2) values, and 95% CI values.See Figure 10. Neurological deficits in the same animals were evaluated by the rotating bar test (Figure 8 C / D). Rats were scored at two time points: 24 hours and 48 hours after SAH (data shown as percentage of high score count / total score count). At the 48-hour endpoint, rats receiving SAH + ip trametinib (100% score at 48h) scored significantly better than those receiving SAH + ip solvent (71% score at 48h) (p=0.0143). All score percentages are shown in Figure 12.

[0239] Conclusion

[0240] Compared with the SAH + ip solvent group, rats receiving SAH + ip trametinib showed significantly reduced contractility. In the rotating bar test, rats receiving SAH + ip trametinib scored significantly better than those receiving SAH + ip solvent.

[0241] Example 9: Effects of subacute subarachnoid hemorrhage in female rats

[0242] Materials and Methods

[0243] Animals

[0244] Female Sprague-Dawley rats (NTac:SD, Taconic, Denmark) were kept at a constant temperature (22 ± 2°C) and humidity (55 ± 10%) with a daily circadian rhythm of 12 hours of light / 12 hours of darkness, provided with standard food (Altromin, Scanbur, Denmark) and free access to water. Rats were usually housed together in European standard cages (Type VI, with 123 cover) in groups of 2–6 and individually after the surgical procedure (Type III, with 123 cover). All rats were acclimatized for 5–7 weeks prior to the experiment.

[0245] Vaginal smear-estrous cycle determination

[0246] Two weeks prior to the SAH surgery, vaginal smears were collected for microscopic characterization of the cell types present, and the estrous cycle of each rat was monitored daily. To minimize potential experimental variability due to fluctuations in estrogen levels, female rats in the proestrus phase were excluded from the study.

[0247] SAH experimental model instructions, 23 / 33 pages, 26 CN 121846102 A

[0248] All procedures were strictly performed in accordance with national laws and guidelines and were approved by the Danish Animal Laboratory Surveillance Agency (License No. 2016-15-0201-00940).

[0249] SAH was induced in the same manner as in male rats. A mixture of hyponorm / midazolam (0.25 ml / kg) and a mixture of hyponorm (fentanyl citrate (0.16 mg / kg), fluanison (5.0 mg / kg) and midazolam (Hameln Pharma, Germany) (2.0 mg / kg) were administered subcutaneously (sc).Female Sprague-Dawley rats (230–300 g, 14–17 weeks old) were anesthetized by intraperitoneal (ip) administration of a mixture of ketamine (MSD Animal Health) (100 mg / ml) and xylazine (KVP Pharma, Germany) (20 mg / ml) in a 3:2 ratio, followed by cannulation and ventilation with 30% O2 and 70% atmospheric pressure. Blood samples (PaO2, PaCO2, and pH) were analyzed periodically using a blood gas analyzer (ABL80 FLEX, Radiometer, Denmark). Body temperature was maintained at 37°C ± 0.5°C using a regulated heating pad (TC-1000, CWE, Inc., PA, USA). MABP and ICP were measured continuously via catheters connected to pressure sensors and Powerlab units, inserted into the caudal artery and cerebellomedullary cistern, respectively, and recorded using LabChart software (both from AD Instruments, Oxford, UK). A laser Doppler flowmeter probe (Oxford Optronix, UK) was placed on the dura mater through holes drilled 4 mm anterior to the anterior fontanelle and 3 mm to the right of the midline on the skull (cooled by periodic saline flushing during the procedure). A 25G Spinocan® cannula (REF:4505905, B. Braun Melsungen AG, Germany) was stereotactically lowered at a final position immediately anterior to the optic chiasm through a second hole drilled 6.5 mm anterior to the anterior fontanelle on the midline. After equilibration for 10 minutes, 250 μL or 300 μL of blood was aspirated from the caudal catheter and manually injected through the cannula. The pressure and rate of blood injection were manually controlled to raise ICP to the upper range of the average MABP level in all animals (approximately 150 mmHg), while controlling the injection rate to produce an acute and prolonged drop in CBF. The rats were then maintained under anesthesia for an additional 30 minutes. At the end of the procedure, the tip of the ICP catheter was sealed with a PinPort (PNP3F22, Instech, USA) for later ICP measurement, and the tail catheter, needle, and laser Doppler probe were carefully removed, and the incision was closed. The rats were then awakened and extubated. At the end of the procedure and thereafter once daily, the rats received subcutaneous injections of carbofen (5 mg / kg, Scan Vet, Denmark) and 2.5 mL of isotonic saline. Sham-operated rats underwent the same procedure, except that the cannula did not drop and no blood was injected into the optic chiasm. The rats were kept in separate cages until euthanized by decapitation 2 days post-operation.

[0250] Experimental Group

[0251] In the preliminary study, subarachnoid hemorrhage (SAH) was induced in female rats by injecting 250 μL of autologous blood into the pre-cross cistern, and the contractile response of the basilar artery (BA) and middle cerebral artery (MCA) was evaluated by myokinesis (n = 12, 7 SAH rats and 5 sham-operated rats). In the actual study, female rats were injected with 300 μL of autologous blood. Sham-operated rats served as controls (n = 34, 18 SAH rats and 16 sham-operated rats). Rats were randomly assigned to either the SAH group or the sham-operated group. A total of 46 rats were used in this study.

[0252] Neurological tests

[0253] a) Rotating bar test

[0254] Total sensorimotor function was evaluated using the rotating bar test at different speeds (3 or 10 rpm). A cage with an entrance facing the bar was placed at one end of a bar (45 mm in diameter and 150 cm in length). The floor of the cage was covered with bedding material from the living cages of the rats being tested. Rats were scored according to the following criteria: 1 point, the animal could not maintain its balance on the bar and fell immediately; 2 points, the animal maintained its balance on the bar but had severe difficulty crossing it, moving < 30 cm; 3 points, the animal gripped the bar with its paws, did not reach the end of the bar, but managed to move > 30 cm; 4 points, the animal crossed the bar but gripped the bar with its paws and / or jumped with its hind legs; 5 points, the animal crossed the bar in a normal posture, but slipped > 3 times; 6 points, the animal crossed the bar perfectly, slipping < 3 times. All animals were trained before surgery until they reached a score of 5 or 6. On days 1 and 2 post-surgery, each animal was tested twice on a static bar and four times at each rotational speed, twice to the left and twice to the right.

[0255] b) Behavioral Observation

[0256] Rats were observed daily and their behavior was scored based on the following parameters collected: body temperature, body posture (low or bent back), eyes (closed, dry or bloody), fur (dirty or bristling), feces (dry or absent), rat noise (when handling rats), noise sensitivity (hyperactive), temperament (passive, aggressive), movement, balance, and ears (white). All 11 observations were scored as follows: normal state = 0, moderate state = 1, poor state = 2. The average score for each group was calculated for each day's observations.

[0257] Harvesting of Cerebral Arteries

[0258] Two days after the surgery, rats were decapitated under CO2 sedation. The brain was quickly removed and cooled in cold bicarbonate buffer solution. The basilar artery (BA) and middle cerebral artery (MCA) were carefully dissected from the brain. For contractility measurements, the BA and MCA were cut into cylindrical segments of 1–1.5 mm in length and mounted on a filamentous kinesiometer.

[0259] In vitro pharmacology

[0260] To measure the contractile response of cerebral arteries, isometric tension of isolated arterial segments was recorded using a kinesiometer (Danish Myograph Technology A / S, Denmark). The vascular segments were mounted on two 40 μm diameter stainless steel wires within the filamentous kinesiometer device. These segments were then immersed in a temperature-controlled bicarbonate buffer solution (37°C) with the following composition (mmol / L): NaCl 11g, NaHCO3 15g, KCl 4.6g, MgCl2 1.2g, NaH2PO4 1.2g, CaCl2 1.5g, and glucose 5.5g. The buffer was continuously aerated with O2 containing 5% CO2 to maintain pH 7.4. The vascular segments were stretched to the previously determined optimal pretension (2 mN) in a three-step process and then allowed to equilibrate at this tension for approximately 20–30 minutes. The vessel was then exposed to a bicarbonate buffer solution containing 60 mM K+, obtained by partially replacing 59.5 mmol / L NaCl with KCl in the aforementioned isotonic bicarbonate buffer solution. The K+-induced contractile response was used as a normalized reference value for the agonist-induced response and to evaluate depolarization-induced vasomotor capacity. Only BA and MCA values ​​with K+-induced responses >2 mN and >0.8 mN, respectively, were used for further evaluation. The presence of functional endothelium in the vessel segment was assessed by precontraction with serotonin (5-HT) (Sigma-Aldrich, H9523) (3 × 10⁻⁷ M) followed by relaxation with carbachol (Sigma-Aldrich, C4382) (10⁻⁵ M). The relaxation response to the cholinergic receptor agonist carbachol was considered an indicator of functional endothelium. Concentration-response curves were obtained by cumulative application of the natural ligand ET-1 (Bachem, 4040254) of the endothelin receptor (ETA / ETB) in the concentration range of 10⁻¹⁴ to 10⁻⁷ M. Similarly, concentration-response curves for 5-CT were obtained by cumulative application of the 5-HT1B / 5-HT1D agonist 5-carboxymethylaminotryptamine (5-CT) (Sigma-Aldrich, C117) in the concentration range of 10⁻¹² to 10⁻⁵ M.

[0261] Intracranial Pressure Measurement

[0262] ICP recordings were performed on days 1 and 2 postoperatively using a novel fluid-filled sealed PinPort system developed for continuous, real-time ICP measurements in rats. The sealed PinPort (PNP3F22, Instech, USA) of the cerebellomedullary cistern duct was connected to the pressure sensor via a fluid-filled tube with a PinPort syringe (PNP-3M, Instech, USA). Connect the pressure sensor to the power lab and use LabChart software (AD).ICP was recorded by Instruments, Oxford, UK. To obtain motion-free measurements, rats were sedated with 0.5 mL / kg midazolam (2.0 mg / kg) 15 minutes before ICP recording, followed by 15 minutes of ICP recording. The PinPort syringe was then removed and the rats were returned to the animal facility.

[0263] Assessment of Cerebral Edema

[0264] After decapitation, the brain was rapidly removed and placed in ice-cold bicarbonate buffer. The brain was divided into two hemispheres, excluding the cerebellum. Each hemisphere was further divided into the striatum, hippocampus, and cortex for determination of the regions where cerebral edema would form. Cerebral edema was assessed by comparing the wet-to-dry weight ratio (WDR). The tissue was weighed (wet weight (ww)) to within 0.1 mg using a balance. The dry weight (dw) of the brain was measured after heating the tissue at 110°C in a drying oven for 24 hours. The tissue water content was then calculated as a percentage of the brain water content using the following formula: (ww - dw / ww) × 100%.

[0265] Statistical and Data Analysis

[0266] Data are presented as mean ± standard error of mean (SEM), where n refers to the number of rats. For in vitro pharmacology studies, contractile response was expressed as the percentage of contraction induced by a maximum of 60 mM K+ relative to baseline. The Emax value represents the maximum contractile response induced by the agonist, while pEC50 is the negative logarithm of the drug concentration that elicits the half-maximal response. For biphasic responses, Emax1 and pEC50 1 describe the high-affinity phase, while Emax2 and pEC50 2 describe the low-affinity phase. Two-way ANOVA and Bonferroni post-hoc tests were used to analyze statistical differences between concentration-response curves, rotating rods, and ICP measurements. Unpaired t-tests were used to investigate statistical differences when comparing two different observation / time points. Statistical analysis and data presentation were performed using Graphpad 5.1 software. The significance p-values ​​were defined as: P = (< 0.05), P = (< 0.01), P = (< 0.001).

[0267] Results

[0268] Surgery, vaginal smears, and physiological parameters

[0269] All rats survived the study. Two rats were excluded from the study due to their hormonal status on the day of surgery (proestrus rats with high estrogen levels). Daily evaluation of vaginal smears confirmed that on the day of surgery, the remaining rats were randomly assigned between the sham-operated group and the SAH group relative to the cycle phase (metaestrus, diaestrus, estrus). Comparisons were made in the preliminary study (injection of 250 µL of autologous blood, data not shown) or the primary study (injection of 300 µL of blood, Figure 14).There were no significant differences in physiological parameters (weight, MABP, pH, pO2, or pCO2) between SAH rats and sham-operated rats. As a result of blood injection, cortical blood flow decreased to 16.24 ± 6% of resting flow in female rats receiving 300 µL of autologous blood (Fig. 15).

[0270] Preliminary study (female rats receiving 250 µL of blood)

[0271] Compared with sham-operated rats, female rats receiving 250 µL of autologous blood showed an increased portion of cerebral vasoconstriction 2 days after SAH.

[0272] There was no difference in vasoconstriction induced by depolarization with 60 mM K+-enriched buffer when comparing arterial segments (BA and MCA) from sham-operated rats and SAH-operated rats (Fig. 16). Similarly, there was no difference in endothelial-mediated dilation studied by acetylcholine-induced vasodilation in 5-HT pre-constricted vessels when comparing arterial segments (BA and MCA) from sham-operated and SAH rats. Previous studies have shown that arterial segments from male SAH-induced rats result in a leftward shift of the ET-1 concentration-contraction curve and a transition to a biphasic curve, while the ET-1 concentration-contraction curve from sham-operated male rats is S-shaped. The biphasic curves following SAH in male rats reflect the presence of contractile ETB receptors in the VSMC, in addition to the pre-existing contractile ETA receptors. In a preliminary study inducing SAH by injecting 250 µl of autologous blood into female rats, ET-1 induced S-shaped curves in both the BA and MCA segments from SAH-induced and sham-operated rats. However, compared to sham-operation, the BA and MCA segments from SAH-experienced female rats resulted in a leftward shift and a significantly increased sensitivity to ET-1 (Fig. 16). In the MCA segment from female SAH rats, a significant leftward shift of the 5-CT concentration-contraction curve was observed compared to sham-operation; therefore, there was no significant difference in 5-CT-induced contraction between SAH and sham-operated rats in the BA segment (Fig. 16).

[0273] Main Study (Female Rats Receiving 300 µL of Blood)

[0274] Preliminary studies have shown that SAH-treated female rats receiving 250 µL of blood intracerebrally induced increased vasoconstriction in cerebral arteries in response to ET-1 and 5-CT. However, due to the lack of transition to the biphasic curve in the ET-1-induced concentration-response curve, the high variability of the data, and the lack of difference in BA contraction induced by 5-CT compared to sham surgery and SAH, the increased volume of injected blood resulted in greater SAH-induced damage. Therefore, in the results presented below (main study), all SAH-treated rats received 300 µL of autologous blood intracerebrally.

[0275] Overall well-being and sensorimotor cognition decreased in female rats after SAH

[0276] To assess whether SAH induces neurological deficits in female rats, two different tests were used to observe overall well-being and a rotating bar test. As shown in Figure 1a, SAH resulted in a significant decrease in overall well-being scores on days 1 and 2 compared to sham surgery. Furthermore, female rats undergoing SAH exhibited significant balance and motor impairments when traversing the bar, whether without rotation or at two different rotation speeds. Sensorimotor deficits were significant in female rats on days 1 and 2 post-SAH compared to sham surgery.

[0277] Increased intracranial pressure in female rats during the subacute phase after SAH compared to sham surgery

[0278] In sham-operated rats, intracranial pressure was 4.0 ± 0.2 mmHg on the day of surgery, and there was no significant change in ICP on days 1 or 2 post-sham surgery (Figure 15). In rats prior to SAH, ICP was 4.1 ± 0.3 mmHg. The ICP then transiently increased to a mean of 149 ± 11.5 mmHg at SAH induction and 7.1 ± 0.8 mmHg 30 minutes after SAH (Figure 15). Compared with sham surgery, the mean ICP of female rats was significantly increased on both day 1 and day 2 after SAH.

[0279] In female SAH rats, the ICP of all rats increased on day 1 after surgery compared with pre-SAH levels, while on day 2, the ICP further increased (4 / 9 rats) or decreased (5 / 9 rats) relative to day 1 levels. However, in rats with decreased ICP on day 2 after SAH, the ICP was still increased compared with the pre-SAH recorded ICP in all but one of the 5 / 9 rats. In female sham-operated rats, ICP was slightly increased (5 / 9 rats) or remained at pre-operational levels on day 1 post-operation, and then on day 2, ICP was slightly decreased (2 / 9), remained at pre-SAH levels (3 / 9), or slightly increased (4 / 9) compared to day 1.

[0280] Cerebral vasoconstriction was increased 2 days post-SAH in female rats compared to sham-operated rats.

[0281] There was no significant difference in potassium-induced responses induced with 60 mM K+-enriched buffer between arterial segments (BA and MCA) from sham-operated and SAH animals (Fig. 16). Furthermore, there was no difference in endothelial-mediated dilation between arterial segments (BA and MCA) from sham-operated and SAH-operated rats.

[0282] The concentration-contraction curve of ET-1 for the BA segment from female SAH rats shifted to the left and began to transition to a biphasic curve. Compared to the BA segment from sham-operated rats, the BA segment from female SAH rats showed significantly increased sensitivity to ET-1 (Figure 16). The MCA segment from SAH-induced female rats also showed significant ET-1 contraction compared to sham-operated rats, which exhibited a biphasic curve.Significantly increased (Emax1 increased). In contrast to the ET-1 curve of the BA segment, the MCA curve did not shift to the left after SAH compared to sham surgery (Fig. 16).

[0283] In male rats, it has been demonstrated that the sensitivity of cerebral arteries to 5-CT is significantly increased after SAH compared to sham surgery. These curves show a leftward shift, which reflects the upregulation of 5-HT1B receptors. Compared to sham surgery, there was a significant increase in sensitivity to 5-CT-induced contraction in both the BA and MCA segments from female SAH rats. The concentration-response curves obtained from brain segments from female rats 2 days after SAH showed a leftward shift compared to sham surgery (Fig. 16).

[0284] Compared to sham surgery, the brain water content of female rats increased 2 days after SAH.

[0285] Edema formation in the striatum, cortex, and hippocampus was evaluated by calculating the brain water content in isolated brain regions. Compared with sham surgery, the percentage of cerebral water in the cortex of female rats was significantly increased 2 days after SAH (p < 0.0473). Compared with sham surgery, the percentage of cerebral water in the hippocampus of female rats tended to increase 2 days after SAH (P = 0.05). There was no difference in cerebral water content in the striatum between female rats with SAH and those with sham surgery (p < 0.2711). These results indicate that there is localized edema in the cortex and hippocampus of female rats 2 days after SAH compared with sham surgery. Specification 27 / 33 pages 30 CN 121846102 A

[0286] Conclusion

[0287] The neurological damage observed in female rats with SAH is similar to that observed in male rats after SAH, with decreased overall well-being and significantly reduced sensorimotor function. ICP was significantly increased on both day 1 and day 2 after SAH in female rats. The course of ICP changes in the first few days after SAH can be used to predict the severity of EBI and DCI. SAH in female rats leads to increased vasomotor activity towards ET-1 and 5-CT in cerebral arteries. Therefore, targeting vascular changes to prevent delayed neurological injury after SAH is a treatment strategy for both males and females.

[0288] Thus, preventing these sex-independent mechanisms provides a basis for a general treatment strategy for DCI after SAH.

[0289] Example 10: Effects of ovariectomy on vasomotor response of middle cerebral artery in female rats after focal cerebral ischemia

[0290] Materials and Methods

[0291] Ethics

[0292] The study design was approved by the Lund County Administrative Court (M178-11, M8-09). All procedures and animal treatments were performed in accordance with the guidelines of the Ethics Committee of Lund University. The study complied with the ARRIVE guidelines (Animal Studies: Reporting in vivo experiments).(Animals Research: Reporting in Vivo Experiments).

[0293] In vivo hormone therapy

[0294] Rats were ovariectomized by a supplier (Charles River, Ĺ Arbresle Cedex, France) and treated with subcutaneously implanted silicone capsules (1.57 mm ID x 3.18 mm OD, Dow Corning, Hemlock, MI, USA) containing progesterone (9 mm length) or 17β-estradiol (5 mm length) to restore hormone levels. Empty silicone capsules of appropriate length were used as placebos. Ovariectomy and capsule implantation were performed during the same time period. This protocol has been shown to produce 17β-estradiol and progesterone levels within the physiological range.

[0295] Uterine dry weight and serum 17β-estradiol were measured at euthanasia to verify the effectiveness of estrogen replacement. At the time of euthanasia, blood from the trunk or from cardiac puncture was collected in a plain tube and allowed to coagulate at room temperature for 40 minutes, then centrifuged at 2,000 xg for 12 minutes at 4°C. The supernatant was collected and stored in aliquots at -80°C until the time for 17β-estradiol determination (radioimmunoassay). The detection limit for 17β-estradiol in the radioimmunoassay was 11 pg / mL.

[0296] After 3 weeks of hormone treatment, compared with ovariectomized (OVX) animals, estrogen-treated ovariectomized rats (OVX+E) had lighter body weight (187 ± 4 g vs. 254 ± 8 g, P < 0.05) and higher uterine weight (98 ± 16 g vs. 19 ± 1 g, P < 0.05). Serum 17β-estradiol levels in OVX+E animals were within the physiological range (26 ± 2 pg / mL), while levels in ovariectomized animals were below the detection limit (< 11 pg / mL in all samples; p < 0.05).

[0297] Female rats with intact ovaries were included as controls (hereinafter referred to as “intact”). The estrous cycle of intact animals was monitored for three consecutive cycles using vaginal smears. The stage of the estrous cycle was determined by examining the cell type and amount of present cells according to an established method (Goldman et al., 2007, Develop Reprod Toxicol.). To eliminate the influence of fluctuations in hormone levels, intact rats used in the experiment underwent tMCAO at estrus day or estrus (when circulating estrogen and progesterone levels are lower compared to proestrus).

[0298] Transient unilateral middle cerebral artery occlusion

[0299] Female Wistar rats were subjected to transient unilateral middle cerebral artery occlusion (tMCAO) using endovascular occlusion technique (Stenman et al., 2002, Stroke) after 3 weeks of hormone therapy. Anesthesia was induced by N2O:O2 (70:30) containing 4.5% isoflurane (see instructions for page 28 / 33, CN 121846102 A) and maintained during the procedure by inhalation of N2O:O2 (70:30) containing 1.5-2.0% isoflurane. Mean arterial blood pressure, pCO2, pO2, pH, and plasma glucose were measured by a tail catheter (Radiometer; LabChart) before occlusion. Body temperature was maintained at +37°C during the procedure using a rectal thermometer connected to a temperature-controlled blanket. An incision was made in the midline of the neck to expose the right common carotid artery, internal carotid artery, and external carotid artery. The common carotid and external carotid arteries were permanently ligated with sutures, and an incision was made in the common carotid artery. A laser Doppler probe (Perimed, Järfä lla, Sweden) was fixed to the thinned skull in the region corresponding to the middle cerebral artery supply area (1 mm posterior to the anterior fontanelle, 6 mm to the right of the midline). A rubber-coated silicone monofilament (Doccol Corporation, Redlands, CA, USA) was inserted through the incision until the tip reached the entrance of the right middle cerebral artery (MCA). Occlusion was confirmed by a sudden decrease in cortical blood flow observed using laser Doppler monitoring. After securing the filament, the skin was sutured and anesthesia was discontinued. Two hours after occlusion, the rat was briefly re-anesthetized to remove the filament and allow reperfusion. A significant increase in blood flow, as shown by laser Doppler flowmetry, confirmed adequate reperfusion. The animal was allowed to recover for 48 hours post-surgery, with free access to food and water, and was then anesthetized with CO2 and decapitated. The brain was removed and immediately cooled in ice-cold bicarbonate buffer solution (see Drugs, Chemicals and Solutions for composition). The right (occluded) and left (non-occluded) middle cerebral arteries were dissected from the adhering tissue and used for myocardial studies.

[0300] Organ Culture

[0301] Ovaries were removed from 12-week-old female rats as described above, and silicone capsules containing 17β-estradiol (n = 6) were implanted. Rats treated with placebo after ovariectomy (n = 6) and intact rats (n = 6) were compared. Three weeks after ovariectomy and hormone capsule implantation, the animals were anesthetized with CO2 and decapitated. The brain was immediately removed and cooled in ice-cold bicarbonate buffer solution (see Drugs, Chemicals and Solutions for composition). The MCA was removed and immediately or in use supplemented with penicillin (100 U ml-1) and streptomycin (100 µg).Organs were cultured for 24 hours at +37°C in Dulbecco modified Eagle medium (DMEM; Gibco, Invitrogen, Carlsbad, CA, USA) with amphotericin B (0.25 µg mL⁻¹) and 0.25 µg mL⁻¹ in humidified air containing 5% CO₂. The study was performed using myographism.

[0302] In vitro pharmacology

[0303] The contractile properties of the middle cerebral artery were examined using a filamentous Mulvany-Halpern myograph (Danish Myo Technology A / S, Aarhus, Denmark) for recording isotropic tension. The artery was cut into cylindrical segments (2 mm) and mounted in the myograph via two parallel 40 µm filaments inserted through the lumen. The myograph bath contained 5 ml of +37°C bicarbonate buffer (see Drugs, Chemicals and Solutions for composition) and was continuously aerated with oxygen containing 5% carbon dioxide, resulting in a pH of 7.4. After a 20-minute equilibration period, the artery was stretched to 90% of its normal inner circumference using a micrometer screw attached to one of the wires, corresponding to the size of the artery under physiological conditions with a transmural pressure of 100 mm Hg. The other wire was attached to a force displacement sensor, which was connected to an analog-to-digital converter (AD Instruments, Chalgrove, UK). The results were recorded on a computer using a Power Lab unit (AD Instruments) and software LabChart (ADInstruments). After a normalization procedure, the artery was allowed to equilibrate under this tension for 20 minutes.

[0304] Contractile capacity was tested by switching the buffer to 5 ml of potassium-rich buffer (63.5 mM, see Drugs, Chemicals and Solutions). Maximum potassium-mediated contraction was used as a reference value for contractile capacity (=100%). To eliminate the influence of the endothelium, the production of nitric oxide and prostaglandins, which were present in the tissue bath throughout the experiment for arteries from an in vivo stroke model, were blocked, respectively, with 100 μM L-NG-nitroarginine methyl ester (L-NAME) and 10 μM indomethacin.

[0305] The serotonin receptor (5-HT) was evaluated by adding 5-carboxymethylaminotryptamine (5-CT, a non-selective 5-HT1 agonist) (Hansen-Schwartz) at concentrations ranging from 10⁻¹¹ to 10⁻⁵ M. Angiotensin II was evaluated by cumulatively applying angiotensin II at concentrations ranging from 10⁻¹² to 10⁻⁶ M. Thirty minutes before the experiment, the AT2 receptor antagonist PD123319 (page 29 / 33, CN 121846102 A, manual specification) was added to eliminate any AT2 receptor-mediated effects. Selective 5-HT receptor antagonists were added at concentrations ranging from 10⁻¹¹ to 10⁻⁷ M.Sex ETB receptor agonist vipertoxin 6c (S6c) (Alexis Biochemicals, Farmingdale, NY, USA).

[0306] Analysis and Statistics

[0307] The nonparametric Mann-Whitney test was used to compare the maximum contraction (Emax) of two groups, while the Kruskal-Wallis test followed by the Dunn multiple comparison test was used to compare three or more groups. Statistical analysis was considered significant if the p-value was less than 0.05. Results are expressed as mean ± SD, n = number of animals in the group.

[0308] Drugs and Solutions

[0309] Unless otherwise stated, all substances were purchased from Sigma-Aldrich (St. Louis, MO, USA). Bicarbonate buffer had the following composition: 119 mM NaCl, 15 mM NaHCO3, 4.6 mM KCl, 1.5 mM CaCl2, 1.2 mM NaH2PO4, 1.2 mM MgCl and 5.6 mM glucose. The bicarbonate buffer solution containing 63.5 mM K+ was obtained by partially exchanging NaCl for KCl in the above buffer solution.

[0310] Results

[0311] In vitro pharmacology

[0312] The maximum contractile response induced by high potassium (63.5 mM) was not significantly different between treatment groups, between arteries from the occluded and non-occluded hemispheres, or between fresh and cultured arteries (overall mean contraction was 4.2 mN). In each arterial segment, the maximum potassium-induced response was used as a reference for contractile capacity (= 100%) to compare changes in response.

[0313] Vasomotor response after tMCAO: Effect of ovariectomy

[0314] Female MCAs were examined with a filamentous kinesiometer 48 hours after cerebral ischemia. In arteries from the non-occluded side of the brain, as expected from previous studies, there was little to no contraction in response to the selective ETB receptor agonist S6c. Conversely, transiently occluded arteries showed significant contraction in response to S6c (Fig. 17A, Fig. 20). S6c-mediated contraction was significantly lower in the MCAs of ovariectomized females compared to intact females (8 ± 9% vs. 22 ± 16%, p < 0.05, respectively) (Fig. 17A, Fig. 20).

[0315] The concentration-response curves of the 5-hydroxytryptamine (5-HT) receptor agonist 5-CT were similar in non-occluded arteries from both intact and ovariectomized females (Fig. 17B, Fig. 20). The concentration-response curves in non-occluded arteries were biphasic, indicating that 5-CT acts on more than one type of contractile 5-HT receptor in the artery, as previously shown in male cerebral arteries.In intact female occluded arteries, 5-CT-mediated vasoconstriction was generally significantly lower than in unoccluded arteries, and the curves were monophasic, consistent with a single receptor subtype. Interestingly, arteries from ovariectomized animals showed almost no vasoconstrictive response to 5-CT (8 ± 11%), a significant difference from the response in intact females (27 ± 18%) (p < 0.01) (Fig. 17B, Fig. 20). Thus, ovariectomized rats showed a greater mean reduction in 5-CT-mediated response after tMCAO compared to intact females.

[0316] AT1 receptor-mediated vasoconstriction against angiotensin II (Ang II) was similar in both occluded and unoccluded arteries (Fig. 17C, Fig. 20). This finding is quite different from historical data from males, where AT1-mediated vasoconstriction was found to be relatively lower in unoccluded arteries compared to occluded arteries (Fig. 20). Ovariectomy did not affect the strong AT1 receptor-mediated contraction observed in both occluded and non-occluded arteries (Fig. 17C, Fig. 20).

[0317] Vasomotor response after tMCAO: Effects of 17β-estradiol and progesterone treatment

[0318] Ovariectomized rats were treated with 17β-estradiol, progesterone, or placebo for 3 weeks via an implanted capsule, followed by unilateral tMCAO. Generally, the maximal contractile response to S6c, Ang II, or 5-CT in both occluded and non-occluded arteries was unaffected by hormone treatment compared to arteries from placebo-treated ovariectomized rats (Fig. 18A-C). As shown in Fig. 17, ovariectomy resulted in significantly lower maximal contractile responses mediated by ETB- and 5-HT- receptors compared to those seen in intact females, while no difference was observed in the already strong AT1 receptor-mediated response. Therefore, maintaining physiological levels of progesterone or estrogen after ovariectomy is insufficient to prevent a reduction in the maximum contractile response to ETB and 5-HT receptor agonists.

[0319] Vasomotor response after organ culture in arteries from intact, ovariectomized, and 17β-estradiol-treated females

[0320] MCA segments from OVX, OVX+E, and intact female rats were studied in a filamentous kinesiometer immediately after isolation or 24 hours after organ culture. ETB receptor-mediated contraction was caused by cumulative application of S6c. No S6c-mediated contraction was observed in fresh arteries (fresh controls); however, strong contraction was observed in all cultured arteries with increasing S6c concentration, and this response was not different between the treatment groups (Fig. 19). Therefore, prior in vivo exposure to ovarian hormones does not affect the upregulation of ETB receptors in arteries during culture.

[0321] Conclusion

[0322] After I / R, endothelin BThe maximal contractile response mediated by the (ETB) receptor agonist vipertoxin 6c (S6c) was increased in female arteries, but significantly decreased in MCAs from ovariectomized females.

[0323] In contrast, maximal contraction mediated by the 5-hydroxytryptamine receptor agonist 5-carboxymethylaminotryptamine (5-CT) decreased after I / R, with arteries from ovariectomized females showing a greater decrease in the maximal contractile response. Angiotensin II-induced contraction was not altered by any manipulation. Estrogen or progesterone supplementation in ovariectomized females did not alter the I / R-induced changes in ETB and 5-CT-induced vasoconstriction. Isolated MCAs subjected to organ culture showed an increase in ETB-mediated contraction. The response was similar in arteries cultured from intact, placebo-treated, and estrogen-treated ovariectomized females. These findings suggest that sex hormones do not directly affect the vasoconstrictor receptor alterations that occur after ischemic stroke; however, ovariectomy does affect the process.

[0324] Following tMCAO, ETB receptor upregulation was more pronounced in males than in females. In contrast, non-occluded female MCAs exhibited stronger contractile responses to 5-CT and Ang II than males: after tMCAO, 5-CT responses decreased in both sexes, and Ang II responses remained unchanged in females but increased in males. Thus, the behavior of vascular responses differed by sex (Fig. 20).

[0325] It was hypothesized that these male-female differences reflected the effects of sex hormones on contractile responses in cerebral arteries. Surprisingly, while maximum ETB-mediated contraction increased after tMCAO and organ culture, no effect of sex hormone replacement was observed after ovariectomy. Conversely, in female arteries following tMCAO, vasomotor responses to S6c and 5-CT were significantly lower than previously reported in males. Most interestingly, ovariectomy had a significant effect on vasomotor receptor responses after tMCAO, which was not reversed by estrogen or progesterone replacement. If so, this means that there are sex differences in the treatment of stroke effects, with females having an advantage over males.

[0326] Example 11: Systemic administration of MEK1 / 2 inhibitor trametinib (GSK1120212) after subarachnoid hemorrhage in rats - compared with U0126

[0327] Materials and Methods

[0328] Animals

[0329] Male Sprague-Dawley rats (300-350 g, Taconic, Denmark) were used. All procedures were strictly performed in accordance with national laws and guidelines and approved by the Danish Animal Laboratory Monitoring Board (2012-15-2934-389).

[0330] Subarachnoid hemorrhage in vivo

[0331] SAH was induced as described in detail previously (Povlsen et al, 2013, BMC Neurosci), differentThe method involves anesthetizing rats with a mixture of 2.5 mL·kg⁻¹ of hyponorm-midazolam (1:1:2) in sterile water and injecting 300 µl of blood into the optic chiasm.

[0332] Experimental Group

[0333] For this study, 32 rats underwent surgery. Animals were treated intraperitoneally with 250 μl / body weight of 1 mM trametinib solution (Selleckchem) diluted in 10% hydrogenated castor oil and 10% PEG400 NaCl solution to reach a final dose of 95 μg / body weight. Animals in the solvent and sham surgery groups were treated with 250 μl / body weight of 10% hydrogenated castor oil and 10% PEG400 NaCl solution. Treatment was administered intraperitoneally at 1 hour and 24 hours or 6 hours and 24 hours post-surgery. Animals were euthanized by CO2 anesthesia and decapitation 48 hours after surgery.

[0334] In vitro organ culture

[0335] The MCA was dissected from rats undergoing the first experiment and the segment (1.5 mm) was incubated for 48 hours in a humidified 5% CO2 atmosphere in Dulbecco modified Eagle medium containing L-glutamine (584 mg / L) supplemented with penicillin (100 U / ml) and streptomycin (100 mg / ml). Prior to incubation, four different concentrations of trametinib (5 µM, 1 µM, 0.1 µM or 0.03 µM) or 0.1% DMSO NaCl solution (solvent) were added.

[0336] Filament arteriography

[0337] The isometric tension in the segment (1.5 mm) of the isolated cerebral artery was recorded using a filament arteriograph. The vascular segments were subjected to an initial pretension of 2 mN / mm and pre-constricted with 63.5 mM K+ solution. Only the basilar artery (BA) with a K+-induced response of more than 2 mN and the middle cerebral artery (MCA) with a K+-induced response of more than 0.7 mN were used in the experiment. Concentration-response curves were obtained by cumulatively applying concentrations of the ETB receptor-specific agonist vipertoxin (S6c) (Alexis Biochemicals, USA) in the range of 10⁻¹² to 10⁻⁴ M and endothelin-1 (ET-1) (AnaSpec, USA) in the range of 10⁻¹⁴ to 10⁻⁷ M.

[0338] Intracellular flow cytometry

[0339] The MCA, BA, and Willis loop were collected from a rat. The inventors used two other protocols (Navone et al., 2013, Nat Protoc; van Beijnum et al., 2008, Nat Protoc) to measure the flow rate of the loop.Protoc proposed a new technique for isolating VSMCs from cerebral arteries. The tissue was mechanically destroyed (using a scalpel) and then enzymatically digested with highly purified collagenase I and collagenase II. The isolated cell suspension was fixed with 4% paraformaldehyde for 30 minutes, washed with PBS, and then permeabilized with 0.25% Triton X-100. The cells were resuspended in blocking buffer containing 5% donkey serum and double-stained overnight at 4°C with primary goat anti-SM22α (1:100, Abcam) or goat isotope control IgG (5 µg / mL, Abcam) and primary antibody rabbit anti-ETB (1:100, Abcam) or rabbit isotope control IgG (10 µg / mL, Abcam). The next day, the cell samples were incubated at room temperature for 2 hours (in the dark) with Alexa 488-labeled donkey anti-goat IgG (1:100, Jackson ImmunoResearch) and allophycocyanin (APC)-labeled donkey anti-rabbit IgG (1:100). Finally, the cell suspension was diluted with PBS to a final volume of 0.5 mL and then analyzed by fluorescence activated cell sorting (FACS) on a BD FACSVerse machine (BD Biosciences, USA). Fluorescence was induced using a 640 nm red laser. The proportion of SM22α-positive cells expressing ETB in each sample was calculated. Data were analyzed using BD FACSuite software.

[0340] Calculations and Statistics

[0341] Data are expressed as mean ± SEM, where n refers to the number of rats. Concentration-contraction curves were compared to two-dimensional ANOVA. For normalization, the K+-induced contraction response was set to 100%. Flow cytometry was analyzed using one-dimensional ANOVA. The significance level was set to p < 0.05.

[0342] Results

[0343] In all rats, physiological parameters and temperature were within acceptable limits during surgery, and there were no differences between groups. ICP increased from 5.4 mmHg to 116.8 mmHg, and cortical CBF decreased to 19% of resting flow (mean of all SAH agonists, page 32 / 33, CN 121846102 A).

[0344] In the initial in vitro experiments, freshly isolated MCA (control) showed no contractile response to the ETB receptor agonist S6c. After 48 hours of OC, S6c produced a strong contractile response in MCA incubated with the solvent. However, co-incubation with trametinib after 48 hours of OC significantly inhibited S6c-induced contraction in a concentration-dependent manner (Figure 21A). The maximum contraction (Emax) induced by S6c in all groups is shown in Figure 21B. Based on the above results, 0.1 µM trametinib confirmed the effect of increasing...The inhibitory effect of trametinib on ET-1-induced vasoconstriction was observed (Fig. 21C). Enhancement was observed in solvent-incubated MCA, with the ET-1 concentration-response curve shifting to the left and transitioning to a biphasic curve shape. Incubation of MCA in the presence of trametinib resulted in a rightward shift of the concentration-response curve, indicating complete blockade of the ETB receptor subtype response.

[0345] To confirm in vivo the effect of trametinib treatment on increased ET-1-mediated vasoconstriction induced by SAH, two different treatment methods were used; 1 mM trametinib was administered intraperitoneally at 1 and 24 hours after SAH (Fig. 22A) or 6 and 24 hours after SAH (Fig. 22B). An increased ET-1-induced concentration-response curve shifted to the left in BA after SAH, and was significantly inhibited by trametinib using both treatment methods. The enhanced vasoconstrictive response observed 6 hours after SAH treatment was validated by protein analysis using flow cytometry. Compared with sham surgery (61.4% ± 10.2%; n=6), there was a significant increase in SMCs expressing ETB receptors after SAH (solvent) (74.2% ± 12.2%; n=7). However, this increased protein expression was not significantly eliminated by trametinib with this specific treatment method (Fig. 22C).

[0346] Conclusion

[0347] The MEK1 / 2 inhibitor trametinib is an effective compound that can completely inhibit the increased ETB receptor-mediated contraction in cerebral arteries after OC in vitro. Trametinib can be administered systemically in vivo to rats, but still reduces the increased ET-1-mediated vasoconstriction in cerebral arteries 48 hours after SAH to the same extent as after intracisional administration.

[0348] Initial in vitro experiments showed that 0.1 µM trametinib can significantly inhibit the increased ETB receptor-mediated contraction induced by ET-1. In previous studies using the same in vitro settings, equivalent inhibition was achieved using 10 µM U0126. U0126 has previously been administered intraperitoneally in vivo in models of global and focal cerebral ischemia at a final concentration of 50 mM dissolved in 100% DMSO. However, intraperitoneal administration was never used in earlier studies of SAH; instead, U0126 (10 µM dissolved in 0.1% DMSO) was administered intracisionally at 6, 12, 24, and 36 hours post-SAH. In the current study, the potent and selective MEK1 / 2 inhibitor trametinib was dissolved in a non-DMSO solution (NaCl solution of hydrogenated castor oil / PEG400) and administered intraperitoneally to rats twice at a final concentration of 1 mM (1 and 24 hours or 6 and 24 hours post-SAH). Under these conditions, the results showed that trametinib had a positive effect on increased vasoconstriction in cerebral arteries 48 hours post-SAH.

[0349] In summary, the results indicate that the potent MEK1 / 2 inhibitor trametinib can be used for any therapeutic application to inhibit SAH.The subsequent increase in vasoconstriction. Instruction manual page 33 / 33, page 36, CN 121846102 A, Figure 1A; Instruction manual figure 1 / 37, page 37, CN 121846102 A, Figure 1B, Figure 1C; Instruction manual figure 2 / 37, page 38, CN 121846102 A, Figure 2A; Instruction manual figure 3 / 37, page 39, CN 121846102 A, Figure 2B; Instruction manual figure 4 / 37, page 40, CN 121846102 A, Figure 2C; Instruction manual figure 5 / 37, page 41, CN 121846102 A, Figure 3A; Instruction manual figure 6 / 37, page 42, CN 121846102 A, Figure 3B, Figure 3C; Instruction manual figure 7 / 37, page 43, CN 121846102 A, Figure 3D, Figure 4A; Instruction manual figure 8 / 37, page 44, CN 121846102 A, Figure 4B, Figure 5A; Instruction manual figure 9 / 37 Page 45 CN 121846102 A Figure 5B Instruction Manual Drawing 10 / 37 Page 46 CN 121846102 A Figure 6 Instruction Manual Drawing 11 / 37 Page 47 CN 121846102 A Figures 7A-7C Instruction Manual Drawing 12 / 37 Page 48 CN 121846102 A Figure 8A Figure 8B Instruction Manual Drawing 13 / 37 Page 49 CN 121846102 A Figure 8C Instruction Manual Drawing 14 / 37 Page 50 CN 121846102 A Figure 8D Instruction Manual Drawing 15 / 37 Page 51 CN 121846102 A Figure 9 Instruction Manual Drawing 16 / 37 Page 52 CN 121846102 A Figure 10 Instruction Manual Drawing 17 / 37 Page 53 CN 121846102 A Figure 11 Instruction Manual Drawing 18 / 37 Page 54 CN Figure 12 of 121846102 A is an illustration from the instruction manual, page 19 / 37, number 55. Figure 13 of 121846102 A is an illustration from the instruction manual, page 20 / 37, number 56. Figure 14 of 121846102 A is an illustration from the instruction manual, page 21 / 37, number 57. Figure 15 of 121846102 A is an illustration from the instruction manual, page 22 / 37, number 58. Figure 16 of 121846102 A is an illustration from the instruction manual, page 23 / 37, number 59.Figure 17A, Appendix to the Instruction Manual, Page 24 / 37, 60 CN 121846102 Figure 17B, Appendix to the Instruction Manual, Page 25 / 37, 61 CN 121846102 Figure 17C, Appendix to the Instruction Manual, Page 26 / 37, 62 CN 121846102 Figure 18A, Appendix to the Instruction Manual, Page 27 / 37, 63 CN 121846102 Figure 18B, Appendix to the Instruction Manual, Page 28 / 37, 64 CN 121846102 Figure 18C, Appendix to the Instruction Manual, Page 29 / 37, 65 CN 121846102 Figure 19, Appendix to the Instruction Manual, Page 30 / 37, 66 CN 121846102 Figure 20, Appendix to the Instruction Manual, Page 31 / 37, 67 CN 121846102 Figure 21A, Appendix to the Instruction Manual, Page 32 / 37, 68 CN 121846102 Figure 21B Figure 21C, page 33 / 37, CN 121846102 A, page 70, CN 121846102 A, page 72, CN 121846102 A, page 73, CN 121846102 A (Instruction Manual Figures) Abstract: The present invention relates to a MEK inhibitor for the treatment of stroke. The present invention relates to a MEK inhibitor and compositions thereof, for use in the treatment of stroke, in particular the treatment of subarachnoid haemorrhage (SAH).

Claims

1. Use of a MEK inhibitor of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention or treatment of stroke in a subject, Equation (I), in: R1 is a C1-C6 alkyl group. R2 is a C1-C6 alkyl group. Ar is selected from aryl and heteroaryl groups.

2. Use of a MEK inhibitor of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for reducing or preventing reperfusion injury in a subject caused by neuroradiological procedures. Equation (I), in: R1 is a C1-C6 alkyl group. R2 is a C1-C6 alkyl group. Ar is selected from aryl and heteroaryl groups.

3. The use according to claim 1 or 2, wherein the MEK inhibitor has formula (II), Equation (II), Or its pharmaceutically acceptable salt.

4. The use according to claim 1, wherein the stroke is selected from: ischemic stroke, hemorrhagic stroke and transient ischemic attack.

5. The use according to claim 1, wherein the stroke is selected from: global cerebral ischemia and focal ischemia.

6. The use according to claim 1, wherein the stroke is delayed cerebral ischemia (DCI).

7. The use according to claim 1 or 2, wherein the MEK inhibitor is formulated for oral, intrathecal, intraperitoneal, intraocular, intranasal or intravenous administration.

8. The use according to claim 1 or 2, wherein the MEK inhibitor is formulated for intravenous administration.

9. The use according to claim 1 or 2, wherein the subject is a human subject.

10. Use of a MEK inhibitor of formula (II) or a pharmaceutically acceptable salt thereof in combination with another drug in the preparation of a medicament for the prevention or treatment of stroke in a subject. Equation (II), The other drugs mentioned are selected from calcium channel blockers and endothelin receptor (ET) receptor blockers.

11. The use according to claim 10, wherein the calcium channel blocker is nimodipine.

12. The use according to claim 10, wherein the endothelin receptor (ET) receptor blocker is clasentan.

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