WO2024050075A2

WO2024050075A2 - Methods and compositions for treatment of vascular obstruction

Info

Publication number: WO2024050075A2
Application number: PCT/US2023/031830
Authority: WO
Inventors: Evan C. Unger; Emmanuelle J. Meuillet
Original assignee: Microvascular Therapeutics, Llc
Priority date: 2022-09-01
Filing date: 2023-09-01
Publication date: 2024-03-07
Also published as: WO2024050075A3

Abstract

This invention provides novel phase-shift microbubbles (PSMBs) and methods of preparation and delivery thereof that are useful in treatment of vascular obstructions (e.g., deep vein thrombosis, pulmonary embolism, microvascular obstruction, perivascular obstruction).

Description

METHODS AND COMPOSITIONS FOR TREATMENT OF VASCULAR

OBSTRUCTION

Government Interest

[0001] This invention was made in part by support from the National Heart, Lung, and Blood

Institute of the National Institutes of Health under the award number 1R33HL156350-01. The

US Government has certain rights in this invention.

Priority Claims and Related Patent Applications

[0002] This application claims the benefit of priority from U.S. Provisional Application Serial No. 63/403,250, filed on September 1, 2022, the entire content of which is incorporated herein by reference for all purposes.

Technical Fields of the Invention

[0003] This invention relates to pharmaceutical compositions and methods of their preparation and diagnostic or therapeutic use. More particularly, the invention relates to phase- shift microbubbles (PSMB, also known in the art as nanodroplets), and emulsions thereof, that are useful in the detection and treatment of vascular obstructions (e.g., deep vein thrombosis, pulmonary embolism, microvascular obstruction, perivascular obstruction), as well as methods of preparation and use thereof.

Background of the Invention

[0004] Deep vein thrombosis (DVT)/pulmonary embolism (PE) affect as many as 900,000 people each year in the United States. It is estimated that 60,000-100,000 Americans die of DVT/PE each year. Between 10 to 30% of people with PE will die within one month of diagnosis. Sudden death is the first symptom in about 25% of people who have PE. Among people who have had DVT, one third to one half will have long-term complications (post- thrombotic syndrome) such as swelling, pain, discoloration, and scaling in the affected limb. Despite the use of anticoagulant therapy, the post-thrombotic syndrome develops within 2 years in approximately half of patients with proximal DVT. The post-thrombotic syndrome commonly causes chronic limb pain and swelling and can progress to cause major disability, leg ulcers, and impaired quality of life. Pharmacomechanical catheter-directed thrombolysis (PCDT) delivers a fibrinolytic drug into the thrombus with concomitant thrombus aspiration/agitation. The objective of pharmacomechanical thrombolysis is to remove thrombus using low-dose fibrinolysis and mechanical agitation, to reduce the risk of the post-thrombotic syndrome while minimizing the risk of bleeding. In a study of 691 patients with DVT randomized to anti- coagulation versus anti-coagulation plus PCDT there was no significant between-group difference in the percentage of patients with the post-thrombotic syndrome (47% with PCDT) versus 48% with anticoagulation alone. There was no significant difference in recurrent DVT over the 24-month follow-up. (Vedantham et al., 2017 NEngl J Med 377: 2240-2252).

[0005] Ultrasound has been deployed as a PCDT technique. The EKOSONICS™ ultrasound catheter is FDA approved and uses targeted ultrasonic waves in combination with clot-dissolving drugs in the treatment of PE. Ultrasonic waves delivered from the catheter improve the permeation of tPA into the clot. Reports have shown that microbubbles (MB) product enhance the rate of sonolysis with ultrasound. In vitro microbubble enhanced sonolysis studies have shown that clot lysis with this technique does not produce large fragments but rather dissolves clot into constituent components of fibrin, platelets and red blood cells. In a model of thrombus in the superior vena cava in pigs, microbubble enhanced sonothrombolysis was successful in dissolving the thrombus and pulmonary angiography showed no evidence of pulmonary emboli. (Kutty etal. 2012 PLoS One 7(12): e51453.)

[0006] The Microbubbles and UltraSound accelerated Thrombolysis (MUST Trial) for peripheral arterial occlusions phase II single-arm trial was performed to test the efficacy and safety of MB enhanced sonolysis in peripheral arterial occlusions. The MUST Trial demonstrated that the treatment was feasible and provided evidence of safety without side effects related to the microbubbles, as well as significant improvement in both arterial flow and pain scores. (Doelare et al. 2021 Ear J Vase End Surg 62(3) 463-468.)

[0007] Thrombi have variably porous structures composed of fibrin, platelets and red blood cells. While MB enhanced sonothrombolysis (SL) has shown promise in treating vascular thrombosis, MBs are micron-sized structures have limited penetration into thrombus structure and not easily permeate clots. These limitations imposed by the average fibrin spacing and MB size significantly restrain the therapeutic effect of MB.

[0008] An urgent need remains for improvement in both the MB compositions and delivery methodologies.

Summary of the Invention

[0009] This invention relates to pharmaceutical compositions and methods of their preparation and delivery for therapeutic use. More particularly, the invention relates to compositions of PSMBs and delivery methodologies that are useful for improved treatment vascular obstruction over the existing therapies. More particularly, the invention relates to methods that comprise administration to a subject in need thereof a composition comprising PSMBs through peripheral IV or intra-arterial (via catheter) to the site of vascular thrombosis and administration of ultrasound over the region of thrombosis transcutaneously or via endovascular ultrasound catheter.

[0010] As demonstrated herein PSMBs permeate clots better than MBs for more effective sonothrombolysis (SL). PSMBs easily penetrated the thrombus and showed increased SL efficiency in the rat hindlimb model of microvascular obstruction (MVO).

[0011] The new generation of PSMBs disclosed herein are electrostatically neutral and are made by condensation of MBs. In the condensation process, the novel MB provide for superior condensation compared to available anionic MB - smaller PSMB with increased stability. The PSMBs are smaller diameter than microbubbles (e.g., 150-350 nm versus 1-3 microns for MBs). Because of their smaller diameter, PSMBs afford more effective sonolysis than MBs. Sonolysis with PSMBs may generate smaller particle size fragments than with MB. An in vitro study showed that PSMB enabled sonolysis of retracted, hard clots, which has generally not been achievable with MB sonolysis.

[0012] The present inventors tested both endovascular US (EKOSONIC Endovascular System) and transcutaneous US (GE Vivid E9) in combination with PSMB for SL in a porcine model of DBT. It was found that in combination with the EKOS Sonic Endovascular System PSMBs made with perfluoropropane completely dissolved occlusive DVT clots in a porcine model using 1/1000^th the tPA dose and l/10^th the treatment time compared to standard catheter administration of t-PA. Also shown was that transcutaneous ultrasound using a diagnostic transducer (GE ViviE9) produces similar SL effects in the presence and absence of t-PA. PSMB hold great potential as therapeutic agents for treating DVT either with transcutaneous or endovascular ultrasound and with or without tPA.

[0013] In one aspect, the invention generally relates to a method for treating vascular thrombosis or a related disease or condition, comprising: administering to a subject in need thereof, peripheral intravenously or via an intra-arterial catheter, to a site of vascular thrombosis, a composition comprising PSMBs, and administering ultrasound transcutaneously or endovascularly to the subject at a region having or near the site of vascular thrombosis.

[0014] In another aspect, the invention generally relates to a composition of PSMBs comprising condensed phase nanodroplets comprising one or more of sulfur hexafluoride, perfluoropropane, perfluorobutane and perfluoropentane, wherein the composition is a translucent suspension with PSMBs having a mean diameter in the range of about 150 nm to about 350 nm.

[0015] In yet another aspect, the invention generally relates a composition comprising PSMBs; one or more phospholipids; and one or more stabilizing agents selected from the group consisting of propylene, glycol, glycerol and sugars, in an aqueous solution/ suspension.

[0016] In yet another aspect, the invention generally relates a PSMB composition disclosed herein for use in treating vascular thrombosis or a related disease or condition.

Brief Description of the Drawings

[0017] FIG. 1. Pulmonary angiograms of post-tPA treatment (left) and post-PSMB treatment (right).

[0018] FIG. 2. Comparison between microbubbles (MBs) Vs. phase shift microbubbles (PSMBs). (A) Contrast-enhanced ultrasound images of rat hindlimb. (B) Peak plateau video intensity which reflects the vascular cross-sectional area and is directly proportional to blood volume (dB). (C) Flow rate (dB/sec). Data expressed as mean ± standard deviation (n=5 for MBs and n=6 for PSMBs). *p < 0.05, **p<0.001, ***p<0.0001.

Detailed Description of the Invention

[0019] The invention is based on the unexpected discovery of novel PSMBs compositions and delivery methodologies that are useful for treating vascular obstruction with improved treatment outcome over the existing therapies. PSMBs, through peripheral IV or intra-arterial (via catheter) to the site of vascular thrombosis combined with transcutaneous or endovascular administration of ultrasound lead to significantly improved treatment results than existing methods.

[0020] PSMBs are comprised of microbubbles wherein the gas inside the microbubbles are in the condensed state. Typically, MBs have diameters in the range of about 1-5 microns, whereas PSMBs have diameters in the much small range of about 150 nm to about 350 nm. Compared to MBs, the smaller diameters enable better permeation of PSMB into thrombus. The gas in the PSMBs may comprise one or more of sulfur hexafluoride, perfluoropropane, perfluorobutane and perfluoropentane, with perfluoropropane and perfluorobutane preferred. [0021] In one aspect, the invention generally relates to a method for treating vascular thrombosis or a related disease or condition, comprising: administering to a subject in need thereof, peripheral intravenously or via an intra-arterial catheter, to a site of vascular thrombosis, a composition comprising PSMBs, and administering ultrasound transcutaneously or endovascularly to the subject at a region having or near the site of vascular thrombosis.

[0022] In certain embodiments, the PSMBs are administered to the subject peripheral intravenously.

[0023] In certain embodiments, the PSMBs are administered to the subject via an intra- arterial catheter.

[0024] In certain embodiments, ultrasound is administered transcutaneously to the subject at a region having or near the site of vascular thrombosis.

[0025] In certain embodiments, ultrasound is administered endovascularly to the subject at a region having or near the site of vascular thrombosis.

[0026] In certain embodiments, the PSMBs comprise one or more of sulfur hexafluoride, perfluoropropane, perfluorobutane and perfluoropentane in condensed phase.

[0027] In certain embodiments, the PSMBs comprise perfluoropropane in condensed phase.

[0028] In certain embodiments, the PSMBs comprise perfluorobutane in condensed phase.

[0029] In certain embodiments, PSMBs have diameters in the much small range of about 150 nm to about 350 nm (e.g., about 150 nm to about 300 nm, about 150 nm to about 250 nm, about 200 nm to about 350 nm, about 200 nm to about 300 nm).

[0030] In certain embodiments, administration of PSMBs is via a multi-side hole catheter. [0031] In certain embodiments, administration of PSMBs is via an end-hole catheter.

[0032] In certain embodiments, the catheter is positioned alongside or within a target clot.

[0033] In certain embodiments, the ultrasound administered is in the range from about 200

KHz to about 10 MHz (e.g., about 250 KHz to about 10 MHz, about 500 KHz to about 10 MHz, about 1 MHz to about 10 MHz, about 200 KHz to about 5 MHz, about 200 KHz to about 1 MHz, about 200 KHz to about 2 MHz, about 250 KHz to about 1 MHz). In certain embodiments, the ultrasound administered is in the range from about 250 KHz to about 2 MHz.

[0034] In certain embodiments, the method further comprises administering to the subject one or more lytic drugs, e.g., tissue plasminogen activator (tPA).

[0035] In certain embodiments, the one or more lytic drugs is in the same solution as the

PSMBs.

[0036] In certain embodiments, the lytic drug is tPA.

[0037] In certain embodiments, the vascular thrombosis or a related disease or condition is one or more selected from deep vein thrombosis (DVT), pulmonary emboli (PE), microvascular obstruction (MVO), peripheral artery occlusions (PAO), acute ischemic stroke (AIS) and acute ST-elevation myocardial infarction (STEMI).

[0038] In certain embodiments, the vascular thrombosis or a related disease or condition is DVT. In certain embodiments, the vascular thrombosis or a related disease or condition is PE. In certain embodiments, the vascular thrombosis or a related disease or condition is PAO. In certain embodiments, the vascular thrombosis or a related disease or condition is AIS. In certain embodiments, the vascular thrombosis or a related disease or condition is acute STEM!. In certain embodiments, the vascular thrombosis or a related disease or condition is MVO.

[0039] In another aspect, the invention generally relates to a composition of PSMBs comprising condensed phase nanodroplets comprising one or more of sulfur hexafluoride, perfluoropropane, perfluorobutane and perfluoropentane, wherein the composition is a translucent suspension with PSMBs having a mean diameter in the range of about 150 nm to about 350 nm.

[0040] In certain embodiments, the composition further comprises trehalose for increased stability and storage of the PSMBs.

[0041] In certain embodiments, the composition is characterized by gas chromatography for gas content in the PSMBs, by dynamic light scattering (DLS) for size and particle count. [0042] In yet another aspect, the invention generally relates a PSMB composition disclosed herein for use in treating vascular thrombosis or a related disease or condition.

[0043] In certain embodiments, the composition is suitable for peripheral intravenous administration or via an intra-arterial catheter administration to treat deep DVT, PE, PAO, MVO, AIS or acute STEMI.

[0044] In yet another aspect, the invention generally relates a composition comprising PSMBs; one or more phospholipids; and one or more stabilizing agents selected from the group consisting of propylene, glycol, glycerol and sugars, in an aqueous solution/suspension.

[0045] In certain embodiments of the composition, the sugar is one or more of trehalose, glucose, sucrose, maltose, sorbitol and erythritol.

[0046] In certain embodiments, the sugar is present at about 0.1% to about 3% w/vol (e.g., about 0.1% to about 2% w/vol, about 0.1% to about 1% w/vol, about 0.5% to about 3% w/vol, about 1% to about 3% w/vol, about 0.5% to about 2% w/vol, about 0.5% to about 1.5% w/vol).

[0047] In certain embodiments, the trehalose is present at about 0.1% to about 3% w/vol (e.g., about 0.1% to about 2% w/vol, about 0.1% to about 1% w/vol, about 0.5% to about 3% w/vol, about 1% to about 3% w/vol, about 0.5% to about 2% w/vol, about 0.5% to about 1.5% w/vol).

[0048] In certain embodiments, trehalose is present at about 0.5% to about 2% w/vol.

[0049] The Examples below describe certain exemplary embodiments of compounds prepared according to the disclosed invention. It will be appreciated that the following general methods, and other methods known to one of ordinary skill in the art, can be applied to compounds and subclasses and species thereof, as disclosed herein.

Examples

[0050] MBs used were an injectable phospholipid suspension containing 6.52 mg/mL of octafluoropropane (OFP) gas in the vial headspace. Each mL of the aqueous suspension contained a lipid blend of 0.045 mg DPPE, 0.401 mg DPPC, and 0.304 mg MPEG-5000-DPPE. Each mL of the aqueous suspension also contains 126.2 mg glycerin, 103.5 mg propylene glycol, 2.16 mg sodium phosphate dibasic heptahydrate, 2.34 mg sodium phosphate monobasic monohydrate, 1% w/vol USP trehalose and 4.87mg sodium chloride in Water for Injection. The pH was 6.2-6.8. The aqueous suspension of PSMB contained two endogenous phospholipids along with a pegylated phospholipid that, upon activation with the OFP gas, form micrometer-sized microspheres (microbubbles). The suspension was stabilized in a ternary solvent mixture of propylene glycol, glycerol, 1% w/vol trehalose and sterile water for injection which is buffered at pH 6.5 using sodium phosphate salts. For PSMB, the vials were activated using mechanical agitation. After agitation the sealed vial of microbubbles is placed in a freezer at -20 °C. The microbubble product was then withdrawn from the vial and subjected to pressure in the syringe. A 3-mL syringe was used to withdraw the product from a 1.3 mL volume in the syringe. A stopcock was placed on the end of the syringe and sealed. The product was subject to pressure by hand for about 5 minutes. After subjection to cold temperature and high pressure, PSMB appeared as a translucent suspension and the mean size of the PSMBs is between about 150-350 nanometers.

In-vitro Physical and Chemical Characterization

[0051] All the MBs and PSMBs parameters are summarized in Table X. The results from the particle sizing analysis showed that the diameter of the MBs ranged from 820 to 880 nm and the diameter of the PSMBs was -150 to 250 nm. All formulations have more than the concentration specification for perfluorocarbon gas in the vial headspace before activation, which is > 6.52 mg/mL. The concentration of each lipid and the total amount of the main lipids that form the shell are within the acceptance concentration range mentioned above. The pH of all formulations was within specifications (6.2 to 6.8). Finally, as expected, all formulations had a neutral net surface charge as evaluated by the zeta potential.

[0052] PSMB were made as follows: Formulated MB were incubated 3 min at -15°C-18°C, activated by agitation for 45 sec, incubated for 3 min at -15°C-18°C. Pressurized vials at 40 psi with N2, are incubated at -15°C-18°C for another 10 min to generate PSMB. The formulations were also prepared with and without sugars, glucose and trehalose. The results are shown in the Tables A1-A6 below.

Table Al

Concentration Lipid Content Zeta Potential

Type of Particle Size of OFP (mg/mL) (mV) bubbles (nm) (mg/mL) (%)

DPPE-MPEG-5K=

MB 880.00 ± 11.11 7.24 ± 0.21 0.256

Table A2

Table A3

Table A4

Table A5

Table A6

[0053J The above data show that PSMBs prepared from the formulation without sugar are much larger initially and rapidly dissipate. Glucose improves the PSMBs but the best effect is obtained with trehalose where the PSMBs retain the appropriate diameter after 24 hours.

[0054] The animal studies were performed at Synchrony Labs (Durham, North Carolina) under an approved protocol (IACUC 313-01-21). Yorkshire pigs (male and female, ~70kg) were anesthetized with isoflurane gas and ventilated on 100% O2 during the procedure. The animals were monitored for blood pressure, heart rate, EKG and pulse oximetry during the procedure.

Bilateral iliac vein occlusions were created by inflating a balloon catheter to occlude flow and injecting thrombin The clots were allowed to mature for at least 90 minutes and up to 4 hours prior to treatment. Angiography was performed to confirm clot position, size, and degree of occlusion.

[0055] One to four vials of PSMB were infused (administered in a 20 mL volume at a rate of

20 - 30 mL/hr) for a total treatment time ranging from 20-60 minutes. PSMB were infused in 3 animals using the multi-side hole Cragg McNamara catheter positioned alongside or within the clot with doses of tPA ranging from 0 to 2,000 micrograms of tPA. The EKOS studies were all done by infusing the PSMB through the sheath through which the EKOS catheter was placed. A GE Vivid E9 system was used for imaging and for sonolysis for the transcutaneous ultrasound experiments. Initial imaging was performed with a linear array 9L probe for localization and measurement of flow on Doppler. A Vivid E9 probe was used for sonolysis. The probe was operated in harmonic mode with a transmit frequency of 1.5 MHz and MI = 1.4 MPa at a frame rate of 46.2-54 Hz and depth = 6-12 cm for 60 minutes during infusion of MVT-101 administered intravascularly via syringe infusion pump. The tissue plasminogen activator (tPA) was included within the 20 mL diluted solution of PSMB.

[0056] The EkoSonic catheter was advanced into the clot using 12-cm length acoustic model. PSMBs, 2 vials were prepared in 20 cc saline and infused through the sheath while ultrasound energy was applied through the catheter. Various conditions, tPA, no tPA, and no PSMB versus PSMB were tested, infusing the same volumes of saline as control.

[0057] The length and width of clots were measured. Post procedure, the percent resolution of the clot was estimated visually. The volume of the clot was also calculated by using the formula for calculation of a cylinder, length x radius² and the % resolution was calculated by:

(length x radius² pre) - (length x radius² post treatment).

[0058] Whole blood was drawn pre- and post-ultrasound treatments and was analyzed for hematology and chemistry parameters. Tables 11 and 12 summarize the results from these blood analyses.

[0059] Left Iliac Artery Occlusions example. The animal studies were performed at Synchrony Labs (Durham, North Carolina) under an approved protocol (IACUC 313-01-21). Yorkshire pigs were anesthetized with isoflurane gas and ventilated on 100% O2 during the procedure. The animals were monitored for blood pressure, heart rate, EKG and pulse oximetry during the procedure. Arterial occlusions were created by inflating a balloon catheter to occlude flow and injecting thrombin. X-ray fluoroscopy was used to introduce balloon catheterization and angiography was performed to visualize the arterial anatomy. The clots were allowed to mature for at least 90 minutes and up to 5 hours prior to treatment. Angiography was performed to confirm clot position, size, and degree of occlusion. [0060] For treatment of arterial clots, 1 to 4 vials of PSMB were infused (MVT-101, MVT- 101 -PFB, MVT-101 -FBP) at a rate of 20 - 30 mL/hr for a total treatment time ranging from 20- 60 minutes. Note MVT-101 refers to non-targeted PSMB with API = perfluoropropane. MVT- 101-PFB refers to non-targeted PSMB composed of perfluorobutane and MVT-101-FBP refers to PSMB composed of perfluoropropane but bearing targeting ligands directed to fibrin. PSMBs were infused with doses of tPA ranging from 0 to 2,000 micrograms in saline. During PSMB administration the clot was treated utilizing transcutaneous ultrasound. A Vivid E9 probe was used for sonolysis. The probe was operated in harmonic mode with a transmit frequency of 1.5- 1.7 MHz and MI= 1.2-1.4 MPa at a frame rate of 53.8- 54.1 Hz and a depth of 12 cm. The mean percent clot resolution for all treatments was 75.38% (SDEV %25.29). (Table 13)

[0061] Six pigs that had bilateral occlusive iliac DVT received four doses of PSMB that were infused (each administered in a 10 mL volume over 20 minutes each) for a total treatment time of 60 minutes. There was no significant change in blood pressure, heart rate, EKG or pulse oximetry during the treatment. Table 4 summarizes the size of the clots (volume in cm³) and the percent reduction in clot size. While only 4.23±8.06% (without 21.9; 1.4±2.4) was calculated with no tPA and no PSMB; the clot resolution increased to 85.12±7.33% in the presence of 2,000 pg tPA and 87.25±3.04 with 1,000 pg tPA both in combination with the PSMB. Blood samples were analyzed for general blood chemistry and comprehensive metabolic panel. All showed no significant effect of PSMB on the whole blood composition.

[0062] The data with the EKOS catheter were summarized in Tables III and are consistent with the data from transcutaneous ultrasound (Table 4). Ultrasound + PSMB still has efficacy without tPA while the EKOS catheter is not effective without tPA. The addition of PSMB appears to increase the rate of clot lysis by more than 10-fold. The data supports the safety and efficacy of PSMB in treatment of vascular thrombosis and suggest that catheter administration is more effective than peripheral i.v. administration.

[0063] The clot resolution was improved in Treatment Group 2 (PSMB only) in comparison to Treatment Group 4 (no PSMB, no tPA). The clot resolution was further improved in Treatment 1 (PSMB and tPA) in comparison to Treatment 2; Treatment Group 2 saw an average increase of approximately 23% in clot resolution (p=O.O33). Treatment Group 1 (PSMB and tPA) also saw a significant improvement over Treatment Group 3 (tPA only); Treatment Group 3 saw an average increase of approximately 49% in clot resolution (p=0.0017). There was no significant difference in performance between Treatment Group 2 (PSMB only) and Treatment Group 3 (tPA only); with a calculated p value of 0.077. The comparable efficacy seen in PSMB only and tPA only indicates that the PSMB provides an effective means by which to improve cavitation for the lysis of deep vein thrombi in patients who are not able to receive thrombolytic treatment.

[0064] Subsequently, all blood and other data were collected as follows: Baseline data was collected prior to the treatment with PSMBs. Data was then collected at the end of the study to see if the endpoints have been affected by the treatment. A complete table of the preclinical whole blood chemistry hematology and chemistry data is presented in Tables 11 and 12. The first set of preclinical safety data looked to understand and control the risk of pulmonary emboli via investigation of Pulmonary Artery Pressure (Table 4), Pulmonary Capillary Wedge Pressure (Table 5), and Pulmonary Angiogram (FIG. 1). All three endpoints can help ensure that a pulmonary embolism did not result from the treatment of the DVT. The preclinical safety data collected for Pulmonary Artery Pressure (PAP), Pulmonary Capillary Wedge Pressure (PCWP), and Pulmonary Angiogram did not indicate significant differences between the baseline and end of study results, indicating the absence of pulmonary emboli during treatment with PSMBs. The second set of preclinical safety data looked pulmonary function via Oxygen Saturation (Table 6) and Pulse Oximetry (Table 7). Again, no significant difference was seen between the baseline and end of study results, indicating maintained pulmonary function during treatment with PSMBs. The third set of preclinical safety data looked at the effect of treatment on hemodynamics via investigation of Mean Arterial Pressure (Table 8), Systolic Pressure (Table 9), and Diastolic Pressures (Table 10). All three endpoints can help ensure that adequate blood flow was maintained and that micro-clots were not produced or embolized over the course of treatment. The preclinical safety data collected for Mean Arterial Pressure, Systolic Pressure, and Diastolic Pressures did not indicate significant differences between the baseline and end of study results, indicating adequate blood flow was maintained and that micro-clots were not produced or embolized over the course of treatment.

[0065] Whole blood chemistry and most of the whole blood hematology analyses did not reveal any significant changes between baseline and post-treatment. The one indicator in the whole blood hematology analysis that changed significantly was the Neutrophil/Lymphocytes ratio (NLR), which is likely caused by induced acute DVT (Tables 11 and 12). [0066] While the preclinical efficacy data was completed, we acknowledge that this data was collected from an animal model with acute clot. To our knowledge, there is not an animal model that exists that has been able to reproducibly model chronic DVT; the current model for chronic DVT has a high mortality rate in pigs and cannot reliably illustrate the physiology of chronic DVT.

Table 1. PSMBs and transcutaneous ultrasound

Table 2. PSMBs and endovascular ultrasound (*Value was observed rather than calculated)

Table 3. Average Percent of Clot Resolution per Treatment Group with Endovascular

Ultrasound

Table 4. Pulmonary Artery Pressure (PAP) Before and After Treatment with PSMBs

Table 5. Pulmonary Capillary Wedge pressure (PCW) Before and After Treatment with PSMBs

Table 6. Oxygen Saturation (sO2) Before and After Treatment with PSMBs

Table 7. Pulse Oximetry (OX) Before and After Treatment with PSMBs

Table 8. Mean arterial pressure (MAP) Before and After Treatment with PSMBs

Table 9. Systolic pressure (SYS) Before and After Treatment with PSMBs

Table 10. Diastolic pressure (DIA) Before and After Treatment with PSMBs

Table 11. Whole Blood Hematology Analysis (n=5)

Table 12. Whole Blood Chemistry Analysis (n=5)

Table 13. Arterial Occlusions

Average Clot Resolution: 75.38 ±25.29

Myocardial Infarction Performed on Pig # 14:

[0067] A myocardial infarction (MI) was formed by taking a blood clot formed outside of the pig and injecting it directly into the left anterior descending coronary artery. The MI was treated utilizing transcutaneous ultrasound. Over the course of 30 minutes 1 vial of Fibrin targeted PSMBs (FBP-MVT-101) was administered in 10 mL of saline with .5 mg of TP A by syringe pump at a rate of 20 mL/hr into the left main coronary artery. During the administration of the PSMBs a Vivid E9 probe was used for sonolysis. The probe was operated in harmonic mode with a transmit frequency of 1.5 MHz and MI= 1.4 MPa at a frame rate of 54.1 Hz and a depth of 12 cm. Ultrasound was applied to the at-risk region of the myocardium across the intact chest. Arteriograms showed clearance of the clot with a small, distal obstruction.

Rodent Hindlimb Model of MVO

[0068] The Institutional Animal Care and Use Committee of the University of Pittsburgh approved all experimental protocols. Young Wistar male rats (Envigo Labs, Indianapolis, IN, USA) weighing 275 ± 25 g were anesthetized with 3% isoflurane and maintained with 2%. The right external jugular vein was cannulated with a polyethylene 10 tubing intravenous catheter for infusion of DEFINITY® for perfusion imaging. Polyethylene tubing was advanced from the right femoral artery into the abdominal aorta to administer microthrombi into the left hindlimb and subsequent therapeutic MBs or PSMBs infusion. The left hindlimb was shaved to allow for the positioning of the imaging and therapy transducers. A small animal’s vital signs continuously monitor the heart rate, respiratory rate, and oxygen saturation (MouseOx, Starr Life Science, Holliston, MA, USA).

Ultrasound Imaging

[0069] A Sequoia 512 clinical US imaging system (Siemens, Mountain View, CA, USA) was used to measure hindlimb muscle perfusion using a contrast-specific mode (CPS 7 MHz, 15L8 probe, Siemens). With the rat in the right lateral decubitus position, the imaging transducer was positioned lateral to the muscle. Burst-replenishment (MI=1.9 for burst, 0.2 for imaging) contrast-enhanced US (CEUS) perfusion imaging was performed during continuous intravenous infusion of DEFINITY® via the internal jugular vein at 2 mL/h at defined points of the therapy protocol. The video compression curve, dynamic range, and system gain were kept constant throughout the study.

Contrast-Enhanced Ultrasound Perfusion Quantification

[0070] A region of interest (ROI) was selected on the CEUS images excluding the feeding vessels (microcirculation only), and the average video intensity in the ROI was quantified following the burst replenishment, up until the video intensity plateaued (typically < 30 s). It was previously reported by Wei et al. that the blood volume (A) and perfusion rate (AxB) could be estimated by fitting a mono-exponential function to the kinetics of video intensity (VI) using the equation: VI (t) = A(l-e-^Bt). (Wei et al. 1998 Circulation 97: 473-83) In the above equation, A is the maximal peak plateau video intensity, and AxB is the slope of the video intensity at t=0 and is consistent with the perfusion rate. A typical perfusion data and a fitted model have been previously described. (Yu etal. 2017 Theranostics 7: 3527-38; Pacella et al. 2015 Ultrasound Med Biol. 41: 456-64.) CEUS analysis was performed offline on the cine loops obtained in CPS mode using custom MATLAB (version R2021a, MathWorks) software.

Sonoreperfusion Therapy

[0071] With the rat in the right lateral decubitus position, the imaging transducer was positioned anterior to the muscle, and imaging proceeded in the longitudinal plane along the muscle’s midsection. Burst-replenishment imaging was performed as previously described. (Vymazal etal. 2009 Invest Radiol. 44: 697-704; Pacella et al. 2015 Ultrasound Med Biol. 41: 456-64; Villanueva etal. 2001 Circulation 103: 2624-30.) Therapeutic US was delivered with a flat single-element immersion transducer 12.7 mm in diameter (A303S, Olympus NDT, Center Valley, PA, USA), driven by an arbitrary function generator (AFG3252, Tektronix, Aliquippa, PA, USA) connected to a radiofrequency power amplifier (Model 250A250AM8, Amplifier Research). The therapy transducer was oriented directly over the lateral aspect of the biceps femoris, orthogonal to the imaging transducer, such that the muscle was in the near field of the transducer and within the treatment area, as confirmed by visualizing MBs/PSMBs destruction in the perfusion image immediately after delivery of a therapeutic pulse. The therapeutic US was delivered at 1 MHz, 1.5 MPa peak negative pressure, 5000 cycles, and 3-5 s pulse interval (duty cycle 0.167% or less) for each of two 10 min sessions. The US field was calibrated with a 200 pm capsule hydrophone (HGL-0200, Onda, Sunnyvale, CA, USA).

Concentration Comparisons

[0072] The concentration of perfluoropropane in MBs and PSMBs composed of perfluoropropane was measured via gas chromatography. The MBs were prepared by agitating the aqueous suspension of lipids with stabilizing media glycerol/propylene glycol with 1% trehalose in sealed 3 mL vials with 1.4 mL aqueous volume and 1.6 mL volume of perfluoropropane in the head space of the vial. The concentration of perfluoropropane in the headspace = 6.52 mg/ml. The concentration of perfluoropropane in the MBs after agitation = 1.222 +/- 0.115 mg/ml. PSMBs were prepared from the MBs by chilling the suspension of MBs to -20°C and then by applying pressure to the syringe or vial containing the MBs. The solution became clear or opalescent as the opaque, white suspension of MBs converted to PSMBs. The concentration of perfluoropropane in the PSMBs = 0.213 +/- 0.011 mg/ml, less than a fifth of the concentration of perfluoropropane in the MBs per ml. Yet equivalent volumes of PSMBs significantly outperformed MBs for sonothrombolysis, in terms of clot dissolution.

[0073] These results clearly demonstrated that on an equivalent amount of API, PSMBs are many-fold more effective than MBs in clot dissolution.

[0074] Applicant’s disclosure is described herein in preferred embodiments with reference to the Figures, in which like numbers represent the same or similar elements. Reference throughout this specification to “one embodiment,” “an embodiment,” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0075] The described features, structures, or characteristics of Applicant’s disclosure may be combined in any suitable manner in one or more embodiments. In the description herein, numerous specific details are recited to provide a thorough understanding of embodiments of the invention. One skilled in the relevant art will recognize, however, that Applicant’s composition and/or method may be practiced without one or more of the specific details, or with other methods, components, materials, and so forth. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the disclosure.

[0076] In this specification and the appended claims, the singular forms "a," "an, " and "the" include plural reference, unless the context clearly dictates otherwise.

[0077] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein can be modified by the term about.

[0078] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive.

[0079] The term “comprising”, when used to define compositions and methods, is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. The term “consisting essentially of’, when used to define compositions and methods, shall mean that the compositions and methods include the recited elements and exclude other elements of any essential significance to the compositions and methods. For example, “consisting essentially of’ refers to administration of the pharmacologically active agents expressly recited and excludes pharmacologically active agents not expressly recited. The term consisting essentially of does not exclude pharmacologically inactive or inert agents, e.g., pharmaceutically acceptable excipients, carriers or diluents. The term “consisting of’, when used to define compositions and methods, shall mean excluding trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.

[0080] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. Methods recited herein may be carried out in any order that is logically possible, in addition to a particular order disclosed. Incorporation by Reference

[0081] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made in this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material explicitly set forth herein is only incorporated to the extent that no conflict arises between that incorporated material and the present disclosure material. In the event of a conflict, the conflict is to be resolved in favor of the present disclosure as the preferred disclosure.

Equivalents

[0082] The representative examples are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples and the references to the scientific and patent literature included herein. The examples contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

Want is Claimed is: CLAIMS

1. A method for treating vascular thrombosis or a related disease or condition, comprising: administering to a subject in need thereof, peripheral intravenously or via an intra- arterial catheter, to a site of vascular thrombosis, a composition comprising phase shift microbubbles (PSMBs), and administering ultrasound transcutaneously or endovascularly to the subject at a region having or near the site of vascular thrombosis.

2. The method of claim 1, wherein the PSMBs are administered to the subject peripheral intravenously.

3. The method of claim 1, wherein the PSMBs are administered to the subject via an intra- arterial catheter.

4. The method of any one of claims 1-3, wherein ultrasound is administered transcutaneously to the subject at a region having or near the site of vascular thrombosis.

5. The method of any one of claims 1-3, wherein ultrasound is administered endovascularly to the subject at a region having or near the site of vascular thrombosis.

6. The method of any one of claims 1-5, wherein in the PSMBs comprise one or more of sulfur hexafluoride, perfluoropropane, perfluorobutane and perfluoropentane in condensed phase.

7. The method of claim 6, wherein the PSMBs comprise perfluoropropane in condensed phase.

8. The method of claim 6, wherein the PSMBs comprise perfluorobutane in condensed phase.

9. The method of claim 3, wherein administration of PSMBs is via a multi-side hole catheter.

10. The method of claim 3, wherein administration of PSMBs is via an end-hole catheter.

11. The method of claim 9 or 10, wherein the catheter is positioned alongside or within a target clot.

12. The method of any one of claims 1-11, wherein the ultrasound administered is in the range from about 200 KHz to about 10 MHz.

13. The method of claim 12, wherein the ultrasound administered is in the range from about

250 KHz to about 2 MHz.

14. The method of any one of claims 1-13, further comprising administering to the subject one or more lytic drugs.

15. The method of claim 14, wherein the lytic drug is tissue plasminogen activator (tPA).

16. The method of claim 14 or 15, wherein the one or more lytic drugs is in the same solution as the PSMBs.

17. The method of any one of claims 1-16, wherein the vascular thrombosis or a related disease or condition is one or more selected from deep vein thrombosis (DVT), pulmonary emboli (PE), peripheral artery occlusions (PAO), acute ischemic stroke (AIS) and acute ST-elevation myocardial infarction (STEMI).

18. The method of claim 17, wherein the vascular thrombosis or a related disease or condition is DVT.

19. The method of claim 17, wherein the vascular thrombosis or a related disease or condition is PE.

20. The method of claim 17, wherein the vascular thrombosis or a related disease or condition is PAO.

21. The method of claim 17, wherein the vascular thrombosis or a related disease or condition is AIS.

22. The method of claim 17, wherein the vascular thrombosis or a related disease or condition is acute STEM!

23. A composition of phase shift microbubbles (PSMBs) comprising condensed phase nanodroplets comprising one or more of sulfur hexafluoride, perfluoropropane, perfluorobutane and perfluoropentane, wherein the composition is a translucent suspension with PSMBs having a mean diameter in the range of about 150 nm to about 250 nm.

24. The composition of claim 23, further comprising one or more stabilizing agents.

25. The composition of claim 23 or 24 for use in treating vascular thrombosis or a related disease or condition.

26. The composition of claim 25, for use in treating deep vein thrombosis (DVT), pulmonary emboli (PE), peripheral artery occlusions (PAO), acute ischemic stroke (AIS) or acute ST-elevation myocardial infarction (STEM!).

27. A composition comprising phase shift microbubbles (PSMBs); one or more phospholipids; and one or more stabilizing agents selected from the group consisting of propylene, glycol, glycerol and sugars, in an aqueous solution/ suspension.

28. The composition of claim 27, wherein the sugar is one or more of trehalose, glucose, sucrose, maltose, sorbitol and erythritol.

29. The composition of claim 28, wherein the sugar is present at about 0.1% to about 3% w/vol.

30. The composition of claim 28 or 29, wherein the trehalose is present at about 0.1% to about 3% w/vol.

31. The composition of claim 30, wherein trehalose is present at about 0.5% to about 2% w/vol.