A Novel Na+-K+-Cl- Cotransporter 1 Inhibitor STS66* Reduces Brain Damage in Mice After Ischemic Stroke

Abstract

Background and Purpose- Inhibition of brain NKCC1 (Na⁺-K⁺-Cl^- cotransporter 1) with bumetanide (BMT) is of interest in ischemic stroke therapy. However, its poor brain penetration limits the application. In this study, we investigated the efficacy of 2 novel NKCC1 inhibitors, a lipophilic BMT prodrug STS5 (2-(Dimethylamino)ethyl 3-(butylamino)-4-phenoxy-5-sulfamoyl-benzoate;hydrochloride) and a novel NKCC1 inhibitor STS66 (3-(Butylamino)-2-phenoxy-5-[(2,2,2-trifluoroethylamino)methyl]benzenesulfonamide), on reducing ischemic brain injury. Methods- Large-vessel transient ischemic stroke in normotensive C57BL/6J mice was induced with 50-min occlusion of the middle cerebral artery and reperfusion. Focal, permanent ischemic stroke in angiotensin II (Ang II)-induced hypertensive C57BL/6J mice was induced by permanent occlusion of distal branches of middle cerebral artery. A total of 206 mice were randomly assigned to receive vehicle DMSO, BMT, STS5, or STS66. Results- Poststroke BMT, STS5, or STS66 treatment significantly decreased infarct volume and cerebral swelling by ≈40% to 50% in normotensive mice after transient middle cerebral artery occlusion, but STS66-treated mice displayed better survival and sensorimotor functional recovery. STS5 treatment increased the mortality. Ang II-induced hypertensive mice exhibited increased phosphorylatory activation of SPAK (Ste20-related proline alanine-rich kinase) and NKCC1, as well as worsened infarct and neurological deficit after permanent distal middle cerebral artery occlusion. Conclusions- The novel NKCC1 inhibitor STS66 is superior to BMT and STS5 in reducing ischemic infarction, swelling, and neurological deficits in large-vessel transient ischemic stroke, as well as in permanent focal ischemic stroke with hypertension comorbidity.

Keywords: angiotensin II; bumetanide; hypertension; middle cerebral artery; prodrug.

Figure 1.. Efficacy of novel NKCC1 inhibitors in reducing ischemic infarction and cerebral edema in mice after t-MCAO.

(A) Experimental protocol in normotensive mice and representative TTC staining images. (B) Infarct and swelling. Data are mean ± SD, n = 6-15 (male, n = 3-9; female, n = 3-6), * p < 0.05. (C) Mice treated with drugs at an equimolar dose of 2 mg/kg BMT. Data are mean ± SD, n = 5-9 (male, n = 3-5; female, n = 2-4).

Figure 2.. Efficacy of novel NKCC1 inhibitors on post-stroke survival and neurological function in mice after t-MCAO.

(A) Changes of body weight and survival. (B) Neurological score, corner test, foot-fault test. (C) Adhesive removal and contact tests. Data are mean ± SD, n = 5-9 (male, n = 3-6; female, n = 2-3).

Figure 3.. Efficacy of novel NKCC1 inhibitors against permanent focal ischemic brain injury with hypertension comorbidity.

(A) Infarct and swelling. Data are mean ± SD. n = 4-8. (B) Representative NeuN, MAP2, and To-pro-3 staining in cortical peri-lesion areas from AngII Veh-, AngII BMT-, and AngII STS66-treated mice after 24 hr pd-MCAO. (C) Representative immunoblots and quantification. Data are mean ± SEM, n = 6.