ABSTRACT Encouraging results have been obtained with microbubble (MB) enhanced sonothrombolysis (SL) to treat vascular occlusions in acute ST elevation MI (STEMI), peripheral arterial occlusions (PAO) and ischemic stroke. However, there is no approved MB for these indications. Thrombi are porous structures composed variably of RBC’s, fibrin and platelets. MB are micron sized structures and probably too large to effectively permeate thrombi. Efforts to remove occlusive thrombi via thrombectomy, mechanical disruption, and/or biochemical dissolution have demonstrated efficacy; however, these are time consuming, show mixed results in improving clinical outcomes, and are accompanied by substantial risk of hemorrhagic complications. Efforts to enhance the safety and efficacy of thrombus removal have high potential clinical impact. Ultrasound (US) has been shown to disrupt thrombi and MB can locally amplify and accelerate US-enhanced thrombolysis at a lower energy level. A caveat to the use of MB stems from their size (1-3 microns), which may limit their access to the interior of the microthrombi responsible for MVO and PAO. Smaller acoustically active materials, including phase change nanodroplets (ND, ~100-200 nm), should more easily penetrate thrombus to increase sonothrombolytic efficiency and clinical efficacy. Microvascular Therapeutics (MVT) has developed a safe and more stable lipid- based MB (MVT-100) less likely to induce anaphylactoid reactions as compared to Definity® and is developing MVT-100 via the 505(b)(2) pathway. Moreover, MVT has subsequently made ND from MVT-100 (Patent, US 9,427,410B2; herein referred as MVT-101) and conjugated ND with a peptide ligand with high affinity for fibrin (FTND). Electron microscopy of fibrin clots shows that FTND permeate clot > non-targeted ND >> FTMB>MB. Preliminary studies performed in our laboratory as well as by our collaborators at the University of Pittsburgh Center for Ultrasound Molecular Imaging and Therapeutics in an in vitro model of MVO show that fibrin micro- clots are disrupted by US with ND >> MB. Finally, our international co-Investigator was able to show in a recent clinical study of SL in acute PAO that Definity® plus US shortened the time to reperfusion compared to standard lytic therapy along. In this R33 Catalyze Program, our combined groups propose to formulate and characterize fibrin targeted MB (FTMB) and ND (FTND) and evaluate their effectiveness in vivo in disrupting the microvascular thrombi in biologically relevant models reflective of MVO during acute myocardial infarction and PAO. After production of bioconjugate and FTND/FTMB, we will evaluate their binding to the fibrin target. We hypothesize that FTND will improve fibrin clot detection and achieve superior dissolution of thrombi upon US activation as compared to MB and FTMB. The overall goal of this program is to obtain in vivo results in both animal models for the usage of FTNDs and perform feasibility studies which will, if successful, support further preclinical development and IND-enabling studies of ND for either PAO or MVO therapeutic indication.

PROJECT NARRATIVE Encouraging results have been obtained with microbubble (MB) enhanced sonothrombolysis to treat vascular occlusions (MVO) in acute ST elevation MI, peripheral arterial occlusions (PAO) and ischemic stroke. However, there is no approved MB for these indications. Microvascular Therapeutics has developed novel Ultrasound- Stimulated Phase-Change Contrast Agents for sonothrombolysis. The overall goal of this CATALYZE R33 program is to obtain in vivo results for the usage of Fibrin Targeted Nanodroplets in models of PAO and MVO and to perform feasibility studies which will, if successful, support further preclinical development and IND- enabling studies of FTND for either PAO or MVO therapeutic indication.