Methods for improved endovascular thrombectomy using 3,3′-diindolylmethane

#Provided herein are methods and compositions utilizing 3,3′-diindolylmethane to enhance mechanical thrombectomy during endovascular therapy for acute thrombosis.

#Thrombotic stroke is a leading cause of acute brain injury, death, and disability worldwide with increasing incidence as populations age. In the US, 87% of all strokes are Acute Ischemic Strokes (AIS). These account for about 690,000 AIS per year according to the Center for Disease Control and Prevention. Updates to the AIS guidelines released in 2019 now include endovascular therapy (EVT) using direct mechanical thrombectomy (MT) as a recommended intervention in addition to thrombolytic therapy with Tissue Plasminogen Activator (tPA) drugs. MT, as part of EVT using angiographically directed guidewires, catheters, and stent retrievers, is now credited with faster and more complete brain reperfusion for many patients. EVT of large vessel occluding thromboses (LVO) is now a standard of care and considered a mainstay of stroke treatment (Powers W J, et al., Guidelines for the Early Management of Patients With Acute Ischemic Stroke: 2019 Update to the 2018 Guidelines for the Early Management of Acute Ischemic Stroke: A Guideline for Healthcare Professionals From the American Heart Association/American Stroke Association, Stroke, 2019 December; 50(12):e344-e418). However, AIS patients treated with tPA and MT or MT alone still have uncertain outcomes that relate to resistance to tPA activity for lysing LVO thromboses, complete or partial failure of the MT procedure, and poor long-term outcome despite MT. In a meta-analysis of multiple studies, a lack of difference in outcome between MT alone and tPA plus MT treated patients in the setting of similar LVO showed that tPA is ineffective and insufficient as an adjunctive therapy for MT (Phan K et al., Endovascular Thrombectomy Alone versus Combined with Intravenous Thrombolysis, World Neurosurg., 2017 December; 108:850-858.e2.) Thus, a need for more effective adjunctive medical therapy for MT exists.

#Histologic evaluation of thrombi removed from patients during MT and clinical correlation has revealed that a higher percentage of white blood cells (WBCs) in the thrombus is associated with a greater number of device passes, an extended MT time, and less favorable recanalization. Neutrophils (PMNs) are a predominant WBC type present in thrombi as well as Neutrophil Extracellular Traps (NETs). Prior studies have demonstrated the role of PMNs and NETs in thrombus formation (Fuchs T, et al. Extracellular DNA traps promote thrombosis, Proc Natl Acad Sci USA, 2010 Sep. 7; 107(36):15880-5). NETs are web-like structures of DNA and proteins expelled from activated PMNs that ensnare pathogens as part of the innate or non-specific immune response. In the non-infectious setting of AIS, NET formation can lead to thrombus formation and clot propagation through NET interaction with platelets and atheromatous blood vessel walls. In forming NETs, PMNs execute a programmed process by which plasma membrane pores are formed, coiled nuclear proteins and DNA are uncoiled, and the nuclear contents are forcibly extruded into the extracellular space.

#FIGS. 1A-1D show extracellular DNA release in the presence of F Protein and DIM in a SMEDDS formulation (i.e., BR-9001, Example 1). FIGS. 1A and 1B show the time course of DNA release detected by Sytox Orange (representative of 2 independent experiments) as a proportion of DNA released compared to total release in the presence of saponin. 30veh=30 μM “equivalent” amount of SMEDDS excipients. FIGS. 1C and 1D show the time course of DNA release detected by MNase assay (at 4 h; n=3). Studies were conducted in the presence of F Protein (1 μg/mL) and SMEDDS DIM (1, 3, 10 and 30 μM)(FIGS. 1A and 1C); or LPS (10 μg/mL) and SMEDDS DIM (30 μM)(FIGS. 1B and 1D).

#FIG. 2 shows visualization of extracellular DNA by cell-impermeable Sytox Orange in the presence of DIM (30 μM). Neutrophils were treated with F Protein (1 μg/mL), LPS (10 μg/mL) and SMEDDS DIM or SMEDDS vehicle excipients alone in the absence of DIM for 4 h. Data is representative of 2 independent experiments.

#FIGS. 3A-3C show extracellular DNA release in the presence of F Protein and free DIM in DMSO. FIG. 3A shows representative time course of DNA release detected by Sytox Orange in the presence of F Protein (F; 1 μg/ml) and free DIM at 1, 3, 10, and 30 μM. FIG. 3B shows composite results of 7 independent Sytox Orange experiments depicted in FIG. 3A (mean+SEM) at 4 hrs. *P<0.05; **P<0.01 vs F Protein alone. NS, non-stimulated controls. FIG. 3C shows visualization of neutrophil DNA released in the presence of F Protein or LPS (10 μg/ml) in the presence and absence of free DIM.

#To facilitate understanding of the disclosure set forth herein, a number of terms are defined below.

#Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. All patents, applications, published applications and other publications are incorporated by reference in their entirety. In the event that there are a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

#The singular forms “a,” “an,” and “the” include plural references, unless the context clearly dictates otherwise.