Fibrin-targeted phase shift microbubbles for the treatment of microvascular obstruction.

Rationale: Microvascular obstruction (MVO) following percutaneous coronary intervention (PCI) is a common problem associated with adverse clinical outcomes. We are developing a novel treatment, termed sonoreperfusion (SRP), to restore microvascular patency. This entails using ultrasound-targeted microbubble cavitation (UTMC) of intravenously administered gas-filled lipid microbubbles (MBs) to dissolve obstructive microthrombi in the microvasculature. In our prior work, we used standard-sized lipid MBs. In the present study, to improve upon the efficiency and efficacy of SRP, we sought to determine the therapeutic efficacy of fibrin-targeted phase shift microbubbles (FTPSMBs) in achieving successful reperfusion of MVO. We hypothesized that owing to their much smaller size and affinity for thrombus, FTPSMBs would provide more effective dissolution of microthrombi when compared to that of the corresponding standard-sized lipid MBs. Methods: MVO in the rat hindlimb was created by direct injection of microthrombi into the left femoral artery. Definity MBs (Lantheus Medical Imaging) were infused through the jugular vein for contrast-enhanced ultrasound imaging (CEUS). A transducer was positioned vertically above the hindlimb for therapeutic US delivery during the concomitant administration of various therapeutic formulations, including (1) un-targeted MBs; (2) un-targeted phase shift microbubbles (PSMBs); (3) fibrin-targeted MB (FTMBs); and (4) fibrin-targeted PSMBs (FTPSMBs). CEUS cine loops with burst replenishment were obtained at baseline (BL), 10 min post-MVO, and after each of two successive 10-minute SRP treatment sessions (TX1, TX2) and analyzed (MATLAB). Results: In-vitro binding affinity assay showed increased fibrin binding peptide (FBP) affinity for the fibrin clots compared with the untargeted peptide (DK12). Similarly, in our in-vitro model of MVO, we observed a higher binding affinity of fluorescently labeled FTPSMBs with the porcine microthrombi compared to FTMBs, PSMBs, and MBs. Finally, in our hindlimb model, we found that UTMC with FTPSMBs yielded the greatest recovery of blood volume (dB) and flow rate (dB/sec) following MVO, compared to all other treatment groups. Conclusions: SRP with FTPSMBs achieves more rapid and complete reperfusion of MVO compared to FTMBs, PSMBs, and MBs. Studies to explore the underlying physical and molecular mechanisms are underway.

Dodecafluoropentane Emulsion as a Radiosensitizer in Glioblastoma Multiforme.

Purpose: Glioblastoma multiforme (GBM) is a hypoxic tumor resistant to radiotherapy. The purpose of this study was to assess the safety and efficacy of a novel oxygen therapeutic, dodecafluoropentane emulsion (DDFPe), in chemoradiation treatment of GBM. Experimental Design: In this multicenter phase Ib/II dose-escalation study, patients were administered DDFPe via intravenous infusion (0.05, 0.10, or 0.17 mL/kg) while breathing supplemental oxygen prior to each 2 Gy fraction of radiotherapy (30 fractions over 6 weeks). Patients also received standard-of-care chemotherapy [temozolomide (TMZ)]. Serial MRI scans were taken to monitor disease response. Adverse events were recorded and graded. TOLD (tissue oxygenation level-dependent) contrast MRI was obtained to validate modulation of tumor hypoxia. Results: Eleven patients were enrolled. DDFPe combined with radiotherapy and TMZ was well tolerated in most patients. Two patients developed delayed grade 3 radiation necrosis during dose escalation, one each at 0.1 and 0.17 mL/kg of DDFPe. Subsequent patients were treated at the 0.1 mL/kg dose level. Kaplan-Meier analysis showed a median overall survival of 19.4 months and a median progression-free survival of 9.6 months, which compares favorably to historical controls. Among 6 patients evaluable for TOLD MRI, a statistically significant reduction in tumor T1 was observed after DDFPe treatment. Conclusions: This trial, although small, showed that the use of DDFPe as a radiosensitizer in patients with GBM was generally safe and may provide a survival benefit. This is also the first time than TOLD MRI has shown reversal of tumor hypoxia in a clinical trial in patients. The recommended dose for phase II evaluation is 0.1 mL/kg DDFPe.Trial Registration: NCT02189109. Significance: This study shows that DDFPe can be safely administered to patients, and it is the first-in-human study to show reversal of hypoxia in GBM as measured by TOLD MRI. This strategy is being used in a larger phase II/III trial which will hopefully show a survival benefit by adding DDFPe during the course of fractionated radiation and concurrent chemotherapy.

The impact of intravenous dodecafluoropentane on a murine model of acute lung injury.

INTRODUCTION: Intravenous oxygen therapeutics present an appealing option for improving arterial oxygenation in patients with acute hypoxemic respiratory failure, while limiting iatrogenic injury from conventional respiratory management. METHODS: We used an established two-hit murine model of acute lung injury (ARDS/VILI) to evaluate the effect of intravenous dodecafluoropentane (DDFPe) on oxygen saturation and bronchoalveolar lavage cell counts and protein levels. Twenty hours after challenge with intratracheal lipopolysaccharide, mice were intubated and ventilated with high tidal volumes (4 h) to produce acute lung injury. DDFPe (0.6 mL/kg) or saline was administered by IV bolus injection at the initiation of mechanical ventilation and again at 2 h. Oxygen saturation was measured every 15 min. Bronchoalveolar lavage was performed at the conclusion of the experiment. RESULTS: The two-hit ARDS/VILI model produced substantial inflammatory acute lung injury reflected by markedly increased bronchoalveolar lavage (BAL) cell counts compared to BAL cell counts in spontaneous breathing controls (5.29 ± 1.50 × 10-6 vs 0.74 ± 0.014 × 10-6 cells/mL) Similarly, BAL protein levels were markedly elevated in ARDS/VILI-challenged mice compared with spontaneous breathing controls (1109.27 ± 223.80 vs 129.6 ± 9.75 ng/mL). We fit a linear mixed effects model that showed a significant difference in oxygen saturation over time between DDFPe-treated mice and saline-treated mice, with separation starting after the 2-h injection. DDFPe-treated ARDS/VILI-challenged mice also exhibited significant reductions in BAL cell counts but not in BAL protein. CONCLUSION: DDFPe improves oxygen saturation in a murine model of ARDS/VILI injury with the potential for serving as an intravenous oxygen therapeutic.

Ultrasound-mediated gene delivery of factor VIII plasmids for hemophilia A gene therapy in mice.

Gene therapy offers great promises for a cure of hemophilia A resulting from factor VIII (FVIII) gene deficiency. We have developed and optimized a non-viral ultrasound-mediated gene delivery (UMGD) strategy. UMGD of reporter plasmids targeting mice livers achieved high levels of transgene expression predominantly in hepatocytes. Following UMGD of a plasmid encoding human FVIII driven by a hepatocyte-specific promoter/enhancer (pHP-hF8/N6) into the livers of hemophilia A mice, a partial phenotypic correction was achieved in treated mice. In order to achieve persistent and therapeutic FVIII gene expression, we adopted a plasmid (pHP-hF8-X10) encoding an FVIII variant with significantly increased FVIII secretion. By employing an optimized pulse-train ultrasound condition and immunomodulation, the treated hemophilia A mice achieved 25%-150% of FVIII gene expression on days 1-7 with very mild transient liver damage, as indicated by a small increase of transaminase levels that returned to normal within 3 days. Therapeutic levels of FVIII can be maintained persistently without the generation of inhibitors in mice. These results indicate that UMGD can significantly enhance the efficiency of plasmid DNA transfer into the liver. They also demonstrate the potential of this novel technology to safely and effectively treat hemophilia A.

Injectable oxygenation therapeutics: evaluating the oxygen delivery efficacy of artificial oxygen carriers and kosmotropes in vitro.

The aim of this paper was to utilise an existing in vitro setup to quantify the oxygen offloading capabilities of two different subsets of injectable oxygenation therapeutics: (1) artificial oxygen carriers (AOCs), which bind or dissolve oxygen and act as transport vectors, and (2) kosmotropes, which increase water hydrogen bonding and thereby decrease the resistance to oxygen movement caused by the blood plasma. Dodecafluoropentane emulsion (DDFPe) was chosen to represent the AOC subset while trans sodium crocetinate (TSC) was selected to represent the kosmotrope subset. PEG-Telomer-B (PTB), the surfactant utilised to encapsulate DDFP in emulsion form, was also tested to determine whether it affected the oxygen transport ability of DDFPe. The in vitro set-up was used to simulate a semi closed-loop circulatory system, in which oxygen could be delivered from the lungs to hypoxic tissues. Results of this study showed that (1) 0.5 ml of a PFC outperformed 6.25 ml of a kosmotrope in a controlled, in vitro setting and (2) that PTB and sucrose do not contribute to the overall oxygen transportation efficacy of DDFPe. These results could be therapeutically beneficial to ongoing and future pre-clinical and clinical studies involving various oxygenation agents.

Current status of targeted microbubbles in diagnostic molecular imaging of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is often associated with a poor prognosis due to silent onset, resistance to therapies, and rapid spreading. Most patients are ineligible for curable surgery as they present with advanced disease at the time of diagnosis. Present diagnostic methods relying on anatomical changes have various limitations including difficulty to discriminate between benign and malignant conditions, invasiveness, the ambiguity of imaging results, or the inability to detect molecular biomarkers of PDAC initiation and progression. Therefore, new imaging technologies with high sensitivity and specificity are critically needed for accurately detecting PDAC and noninvasively characterizing molecular features driving its pathogenesis. Contrast enhanced targeted ultrasound (CETUS) is an upcoming molecular imaging modality that specifically addresses these issues. Unlike anatomical imaging modalities such as CT and MRI, molecular imaging using CETUS is promising for early and accurate detection of PDAC. The use of molecularly targeted microbubbles that bind to neovascular targets can enhance the ultrasound signal specifically from malignant PDAC tissues. This review discusses the current state of diagnostic imaging modalities for pancreatic cancer and places a special focus on ultrasound targeted-microbubble technology together with its clinical translatability for PDAC detection.

Toward the Clinical Development and Validation of a Thy1-Targeted Ultrasound Contrast Agent for the Early Detection of Pancreatic Ductal Adenocarcinoma.

Early detection of pancreatic ductal adenocarcinoma (PDAC) represents the most significant step toward the treatment of this aggressive lethal disease. Previously, we engineered a preclinical Thy1-targeted microbubble (MBThy1) contrast agent that specifically recognizes Thy1 antigen overexpressed in the vasculature of murine PDAC tissues by ultrasound (US) imaging. In this study, we adopted a single-chain variable fragment (scFv) site-specific bioconjugation approach to construct clinically translatable MBThy1-scFv and test for its efficacy in vivo in murine PDAC imaging, and functionally evaluated the binding specificity of scFv ligand to human Thy1 in patient PDAC tissues ex vivo. MATERIALS AND METHODS: We recombinantly expressed the Thy1-scFv with a carboxy-terminus cysteine residue to facilitate its thioether conjugation to the PEGylated MBs presenting with maleimide functional groups. After the scFv-MB conjugations, we tested binding activity of the MBThy1-scFv to MS1 cells overexpressing human Thy1 (MS1Thy1) under liquid shear stress conditions in vitro using a flow chamber setup at 0.6 mL/min flow rate, corresponding to a wall shear stress rate of 100 seconds, similar to that in tumor capillaries. For in vivo Thy1 US molecular imaging, MBThy1-scFv was tested in the transgenic mouse model (C57BL/6J - Pdx1-Cre; KRas; Ink4a/Arf) of PDAC and in control mice (C57BL/6J) with L-arginine-induced pancreatitis or normal pancreas. To facilitate its clinical feasibility, we further produced Thy1-scFv without the bacterial fusion tags and confirmed its recognition of human Thy1 in cell lines by flow cytometry and in patient PDAC frozen tissue sections of different clinical grades by immunofluorescence staining. RESULTS: Under shear stress flow conditions in vitro, MBThy1-scFv bound to MS1Thy1 cells at significantly higher numbers (3.0 ± 0.8 MB/cell; P < 0.01) compared with MBNontargeted (0.5 ± 0.5 MB/cell). In vivo, MBThy1-scFv (5.3 ± 1.9 arbitrary units [a.u.]) but not the MBNontargeted (1.2 ± 1.0 a.u.) produced high US molecular imaging signal (4.4-fold vs MBNontargeted; n = 8; P < 0.01) in the transgenic mice with spontaneous PDAC tumors (2-6 mm). Imaging signal from mice with L-arginine-induced pancreatitis (n = 8) or normal pancreas (n = 3) were not significantly different between the two MB constructs and were significantly lower than PDAC Thy1 molecular signal. Clinical-grade scFv conjugated to Alexa Fluor 647 dye recognized MS1Thy1 cells but not the parental wild-type cells as evaluated by flow cytometry. More importantly, scFv showed highly specific binding to VEGFR2-positive vasculature and fibroblast-like stromal components surrounding the ducts of human PDAC tissues as evaluated by confocal microscopy. CONCLUSIONS: Our findings summarize the development and validation of a clinically relevant Thy1-targeted US contrast agent for the early detection of human PDAC by US molecular imaging.

In vitro model to compare the oxygen offloading behaviour of dodecafluoropentane emulsion (DDFPe).

The aim of this paper was to create an in vitro setup to quantify the oxygen offloading capabilities of dodecafluoropentane emulsion (DDFPe) in a hypoxic environment. Oxygen offloading from DDFPe was characterized under different gaseous environments and under pre-oxygenated conditions. Results of this study showed that (1) oxygen offloading is inversely related to the solubility of the selected sparging gas in saline and (2) both pre-oxygenated and simultaneously oxygenated DDFPe display similar magnitudes of oxygen transport. These results could be applicable to on-going and future studies involving a variety of hypoxic conditions where oxygen delivery might be therapeutically beneficial.

Imaging assessment of cardioprotection mediated by a dodecafluoropentane oxygen-carrier administered during myocardial infarction.

INTRODUCTION: The objective of this study was to investigate the cardioprotective effects of a dodecafluoropentane (DDFP)-based perfluorocarbon emulsion (DDFPe) as an artificial carrier for oxygen delivery to ischemic myocardium, using 99mTc-duramycin SPECT imaging. METHODS: Rat hearts with Ischemia-reperfusion (I/R) was prepared by coronary ligation for 45-min followed by reperfusion. The feasibility of 99mTc-duramycin in detecting myocardial I/R injury and its kinetic profile were first verified in the ischemic hearts with 2-h reperfusion (n = 6). DDFPe (0.6 mL/kg) was intravenously administered at 10 min after coronary ligation in fifteen rats and saline was given in thirteen rats as controls. 99mTc-duramycin SPECT images were acquired in the DDFPe-treated hearts and saline controls at 2-h (DDFPe-2 h, n = 7 and Saline-2 h, n = 6) or 24-h (DDFPe-24 h, n = 8 and Saline-24 h, n = 7) of reperfusion. RESULTS: SPECT images, showing "hot-spot" 99mTc-duramycin uptake in the ischemic myocardium, exhibited significantly lower radioactive retention and smaller hot-spot size in the DDFPe-2 h and DDFPe-24 h hearts compared to controls. The infarcts in the Saline-24 h hearts extended significantly relative to measurements in the Saline-2 h. The extension of infarct size did not reach a statistical difference between the DDFPe-2 h and DDFPe-24 h hearts. Ex vivo measurement of 99mTc-duramycin activity (%ID/g) was lower in the ischemic area of DDFPe-2 h and DDFPe-24 h than that of the Saline-2 h and Saline-24 h hearts (P < 0.05). The area of injured myocardium, delineated by the uptake of 99mTc-duramycin, extended more substantially outside the infarct zone in the controls. CONCLUSIONS: Significant reduction in myocardial I/R injury, as assessed by 99mTc-duramycin cell death imaging and histopathological analysis, was induced by DDFPe treatment after acute myocardial ischemia. 99mTc-duramycin imaging can reveal myocardial cell death in ischemic hearts and may provide a tool for the non-invasive assessment of cardioprotective interventions.

Dodecafluoropentane Emulsion (DDFPE) as a Resuscitation Fluid for Treatment of Hemorrhagic Shock and Traumatic Brain Injury: A Review.

Dodecafluoropentane emulsion (DDFPe) is a novel nanotechnology for oxygen delivery with therapeutic potential for hemorrhagic shock and/or traumatic brain injury (TBI). DDFPe demonstrates efficacy at smaller doses than previously tested perfluorocarbon oxygen therapeutics. This smaller dose potentially eliminates toxicities exhibited by previous oxygen therapeutics, whereas anti-inflammatory properties of DDFPe may alleviate damage from ischemia reperfusion injury. This minireview summarizes our progress in developing a battlefield-ready product to prevent combat death due to hemorrhagic shock and/or TBI. Preclinical studies, for both indications, show promising effects of DDFPe as a resuscitation fluid. DDFPe may become a part of the toolkit for tactical healthcare professionals in battlefield and domestic emergency medicine.

Overcoming tumor hypoxia as a barrier to radiotherapy, chemotherapy and immunotherapy in cancer treatment.

Hypoxia exists to some degree in most solid tumors due to inadequate oxygen delivery of the abnormal vasculature which cannot meet the demands of the rapidly proliferating cancer cells. The levels of oxygenation within the same tumor are highly variable from one area to another and can change over time. Tumor hypoxia is an important impediment to effective cancer therapy. In radiotherapy, the primary mechanism is the creation of reactive oxygen species; hypoxic tumors are therefore radiation resistant. A number of chemotherapeutic drugs have been shown to be less effective when exposed to a hypoxic environment which can lead to further disease progression. Hypoxia is also a potent barrier to effective immunotherapy in cancer treatment. Because of the recognition of hypoxia as an important barrier to cancer treatment, a variety of approaches have been undertaken to overcome or reverse tumor hypoxia. Such approaches have included breathing hyperbaric oxygen, artificial hemoglobins, allosteric hemoglobin modifiers, hypoxia activated prodrugs and fluorocarbons (FCs). These approaches have largely failed due to limited efficacy and/or adverse side effects. Oxygen therapeutics, based on liquid FCs, can potentially increase the oxygen-carrying capacity of the blood to reverse tumor hypoxia. Currently, at least two drugs are in clinical trials to reverse tumor hypoxia; one of these is designed to improve permeability of oxygen into the tumor tissue and the other is based upon a low boiling point FC that transports higher amounts of oxygen per gram than previously tested FCs.

Ultrasound Molecular Imaging of Atherosclerosis Using Small-Peptide Targeting Ligands Against Endothelial Markers of Inflammation and Oxidative Stress.

The aim of this study was to evaluate a panel of endothelium-targeted microbubble (MB) ultrasound contrast agents bearing small peptide ligands as a human-ready approach for molecular imaging of markers of high-risk atherosclerotic plaque. Small peptide ligands with established affinity for human P-selectin, VCAM-1, LOX-1 and von Willebrand factor (VWF) were conjugated to the surface of lipid-stabilized MBs. Contrast-enhanced ultrasound (CEUS) molecular imaging of the thoracic aorta was performed in wild-type and gene-targeted mice with advanced atherosclerosis (DKO). Histology was performed on carotid endarterectomy samples from patients undergoing surgery for unstable atherosclerosis to assess target expression in humans. In DKO mice, CEUS signal for all four targeted MBs was significantly higher than that for control MBs, and was three to sevenfold higher than in wild-type mice, with the highest signal achieved for VCAM-1 and VWF. All molecular targets were present on the patient plaque surface but expression was greatest for VCAM-1 and VWF. We conclude that ultrasound contrast agents bearing small peptide ligands feasible for human use can be targeted against endothelial cell adhesion molecules for inflammatory cells and platelets for imaging advanced atherosclerotic disease.

Endothelial vascular cell adhesion molecule 1 is a marker for high-risk carotid plaques and target for ultrasound molecular imaging.

BACKGROUND: Molecular imaging of carotid plaque vulnerability to atheroembolic events is likely to lead to improvements in selection of patients for carotid endarterectomy (CEA). The aims of this study were to assess the relative value of endothelial inflammatory markers for this application and to develop molecular ultrasound contrast agents for their imaging. METHODS: Human CEA specimens were obtained prospectively from asymptomatic (30) and symptomatic (30) patients. Plaques were assessed by semiquantitative immunohistochemistry for vascular cell adhesion molecule 1 (VCAM-1), lectin-like oxidized low-density lipoprotein receptor 1, P-selectin, and von Willebrand factor. Established small peptide ligands to each of these targets were then synthesized and covalently conjugated to the surface of lipid-shelled microbubble ultrasound contrast agents, which were then evaluated in a flow chamber for binding kinetics to activated human aortic endothelial cells under variable shear conditions. RESULTS: Expression of VCAM-1 on the endothelium of CEA specimens from symptomatic patients was 2.4-fold greater than that from asymptomatic patients (P < .01). Expression was not significantly different between groups for P-selectin (P = .43), von Willebrand factor (P = .59), or lectin-like oxidized low-density lipoprotein receptor 1 (P = .99). Although most plaques from asymptomatic patients displayed low VCAM-1 expression, approximately one in five expressed high VCAM-1 similar to plaques from symptomatic patients. In vitro flow chamber experiments demonstrated that VCAM-1-targeted microbubbles bind cells that express VCAM-1, even under high-shear conditions that approximate those found in human carotid arteries, whereas binding efficiency was lower for the other agents. CONCLUSIONS: VCAM-1 displays significantly higher expression on high-risk (symptomatic) vs low-risk (asymptomatic) carotid plaques. Ultrasound contrast agents bearing ligands for VCAM-1 can sustain high-shear attachment and may be useful for identifying patients in whom more aggressive treatment is warranted.

Radiosensitization of Hs-766T Pancreatic Tumor Xenografts in Mice Dosed with Dodecafluoropentane Nano-Emulsion-Preliminary Findings.

Tumor hypoxia is an important mediator of radiation therapy resistance. We conducted a study to investigate whether an oxygen therapeutic based upon dodecafluoropentane (DDFP) nano-emulsion (NVX-108) could increase tumor PO2 in hypoxic tumors and improve radiation response. Pancreatic (Hs-766T) tumor xenografts were grown in the flanks of 29 SCID mice. Direct tumor PO2 measurements were performed in 9 mice treated with 0.3, 0.45 and 0.6 cc/kg NVX-108 (2% w/vol DDFP) in order to assess the dose dependent increase in tumor PO2. Twenty mice were randomized into 3 groups including control (no treatment), carbogen breathing treated with 12 Gy radiation, and carbogen breathing treated with 12 Gy radiation and NVX-108 (0.6 cc/kg NVX-108 administered as 30 minute IV infusion at time of radiation). Tumor volume was monitored to assess treatment efficacy. Results showed that tumor PO2 increased in NVX-108 treated mice up to 400% with the greatest effect seen at the highest dose of 0.6 cc/kg. Tumor growth was significantly reduced in both treatment groups relative to controls (p < 0.0001). The combination of carbogen, radiation, and NVX-108 demonstrated a 2-fold reduction in average tumor volume compared to carbogen plus radiation treatment (p = 0.01). Further study of NVX-108 as a radiation sensitizer is warranted.

Dodecafluoropentane emulsion elicits cardiac protection against myocardial infarction through an ATP-Sensitive K+ channel dependent mechanism.

PURPOSE: Dodecafluoropentane emulsion (DDFPe) is a perfluorocarbon with high oxygen dissolving, transport, and delivery capacity that may offer the potential to limit ischemic injury prior to clinical reperfusion. Here we investigated the cardiac protective potential of DDFPe in a mouse model of myocardial infarction. METHODS: Myocardial infarction was initiated by permanent ligation of the left anterior descending (LAD) coronary artery. Mice were administered vehicle or 5-hydroxydecanoate (5-HD) intravenously 10 min before LAD occlusion followed by a single intravenous administration of vehicle or DDFPe immediately after occlusion. Heart tissue and serum samples were collected 24 after LAD occlusion for measurement of infarct size and cardiac troponin I (cTnI) levels, respectively. RESULTS: DDFPe treatment reduced infarct size by approximately 72% (36.9 ± 4.2% for vehicle vs 10.4 ± 2.3% for DDFPe; p < 0.01; n = 6-8) at 24 h. Serum cTnI levels were similarly reduced by DDFPe (35.0 ± 4.6 ng/ml for vehicle vs 15.8 ± 1.6 ng/ml for DDFPe; p < 0.01; n = 6-8). Pretreatment with 5-HD, a mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) inhibitor, blocked the reduction in infarct size (29.2 ± 4.4% for 5-HD vs 35.4 ± 7.4% for 5-HD+DDFPe; p = 0.48; n = 6-8) and serum cTnI levels (27.4 ± 5.1 ng/ml for 5-HD vs 34.6 ± 5.3 ng/ml for 5-HD+DDFPe; p = 0.86; n = 6-8) by DDFPe. CONCLUSION: Our data indicate a cardiac protective role of DDFPe that persists beyond its retention time in the body and is dependent on mitoK(ATP), an important mediator of ischemic preconditioning induced cardiac protection.

Cardiovascular drug delivery with ultrasound and microbubbles.

Microbubbles lower the threshold for cavitation of ultrasound and have multiple potential therapeutic applications in the cardiovascular system. One of the first therapeutic applications to enter into clinical trials has been microbubble-enhanced sonothrombolysis. Trials were conducted in acute ischemic stroke and clinical trials are currently underway for sonothrombolysis in treatment of acute myocardial infarction. Microbubbles can be targeted to epitopes expressed on endothelial cells and thrombi by incorporating targeting ligands onto the surface of the microbubbles. Targeted microbubbles have applications as molecular imaging contrast agents and also for drug and gene delivery. A number of groups have shown that ultrasound with microbubbles can be used for gene delivery yielding robust gene expression in the target tissue. Work has progressed to primate studies showing delivery of therapeutic genes to generate islet cells in the pancreas to potentially cure diabetes. Microbubbles also hold potential as oxygen therapeutics and have shown promising results as a neuroprotectant in an ischemic stroke model. Regulatory considerations impact the successful clinical development of therapeutic applications of microbubbles with ultrasound. This paper briefly reviews the field and suggests avenues for further development.

Diagnostic ultrasound induced inertial cavitation to non-invasively restore coronary and microvascular flow in acute myocardial infarction.

Ultrasound induced cavitation has been explored as a method of dissolving intravascular and microvascular thrombi in acute myocardial infarction. The purpose of this study was to determine the type of cavitation required for success, and whether longer pulse duration therapeutic impulses (sustaining the duration of cavitation) could restore both microvascular and epicardial flow with this technique. Accordingly, in 36 hyperlipidemic atherosclerotic pigs, thrombotic occlusions were induced in the mid-left anterior descending artery. Pigs were then randomized to either a) ½ dose tissue plasminogen activator (0.5 mg/kg) alone; or same dose plasminogen activator and an intravenous microbubble infusion with either b) guided high mechanical index short pulse (2.0 MI; 5 usec) therapeutic ultrasound impulses; or c) guided 1.0 mechanical index long pulse (20 usec) impulses. Passive cavitation detectors indicated the high mechanical index impulses (both long and short pulse duration) induced inertial cavitation within the microvasculature. Epicardial recanalization rates following randomized treatments were highest in pigs treated with the long pulse duration therapeutic impulses (83% versus 59% for short pulse, and 49% for tissue plasminogen activator alone; p<0.05). Even without epicardial recanalization, however, early microvascular recovery occurred with both short and long pulse therapeutic impulses (p<0.005 compared to tissue plasminogen activator alone), and wall thickening improved within the risk area only in pigs treated with ultrasound and microbubbles. We conclude that although short pulse duration guided therapeutic impulses from a diagnostic transducer transiently improve microvascular flow, long pulse duration therapeutic impulses produce sustained epicardial and microvascular re-flow in acute myocardial infarction.

Control of solid tumor growth in mice using EGF receptor-targeted RNA replicase-based plasmid DNA.

AIM: Previously, it was shown that treatment of tumor-bearing mice with an RNA replicase-based plasmid that produces dsRNA when transfected into tumor cells significantly inhibited the tumor growth. In the present study, the feasibility of further improving the anti-tumor activity of the RNA replicase-based plasmid by targeting it into tumors cells was evaluated. MATERIAL & METHODS: An EGF-conjugated, polyethylene glycosylated cationic liposome was developed to deliver the RNA replicase-based plasmid, pSIN-ß, into EGF receptor-overexpressing human breast cancer cells (MDA-MB-468) in vitro and in vivo. RESULTS: Delivery of pSIN-ß using the EGF receptor-targeted liposome more effectively controlled the growth of MDA-MB-468 tumors (and human epidermoid carcinoma A431 tumors) in mice than using untargeted liposome. The pSIN-ß carried by the EGF receptor-targeted liposome caused the complete regression of MDA-MB-468 tumors in mice, probably due to the enhancement of its proapoptotic, antiproliferative and antiangiogenic activities. DISCUSSION: Tumor-targeted RNA replicase-based plasmids hold a strong potential in tumor therapy.

Dodecafluoropentane emulsion decreases infarct volume in a rabbit ischemic stroke model.

PURPOSE: To assess the efficacy of dodecafluoropentane emulsion (DDFPe), a nanodroplet emulsion with significant oxygen transport potential, in decreasing infarct volume in an insoluble-emboli rabbit stroke model. MATERIALS AND METHODS: New Zealand White rabbits (N = 64; weight, 5.1 ± 0.50 kg) underwent angiography and received embolic spheres in occluded internal carotid artery branches. Rabbits were randomly assigned to groups in 4-hour and 7-hour studies. Four-hour groups included control (n = 7, embolized without treatment) and DDFPe treatment 30 minutes before stroke (n = 7), at stroke onset (n = 8), and 30 minutes (n = 5), 1 hour (n = 7), 2 hours (n = 5), or 3 hours after stroke (n = 6). Seven-hour groups included control (n = 6) and DDFPe at 1 hour (n = 8) and 6 hours after stroke (n = 5). DDFPe dose was a 2% weight/volume intravenous injection (0.6 mL/kg) repeated every 90 minutes as time allowed. After euthanasia, infarct volume was determined by vital stains on brain sections. RESULTS: At 4 hours, median infarct volume decreased for all DDFPe treatment times (pretreatment, 0.30% [P = .004]; onset, 0.20% [P = .004]; 30 min, 0.35% [P = .009]; 1 h, 0.30% [P = .01]; 2 h, 0.40% [P = .009]; and 3 h, 0.25% [P = .003]) compared with controls (3.20%). At 7 hours, median infarct volume decreased with treatment at 1 hour (0.25%; P = .007) but not at 6 hours (1.4%; P = .49) compared with controls (2.2%). CONCLUSIONS: Intravenous DDFPe in an animal model decreases infarct volumes and protects brain tissue from ischemia, justifying further investigation.

Coronary and microvascular thrombolysis with guided diagnostic ultrasound and microbubbles in acute ST segment elevation myocardial infarction.

BACKGROUND: During a microbubble infusion, guided high-mechanical index impulses from a diagnostic two-dimensional transducer improve microvascular recanalization in acute ST-segment elevation myocardial infarction. The purpose of this study was to further elucidate the mechanism of improved microvascular flow in normal and hyperlipidemic atherosclerotic pigs. METHODS: In 14 otherwise normal pigs, acute left anterior descending thrombotic coronary occlusions were created. Pigs subsequently received aspirin, heparin, and half-dose fibrinolytic agent (tenecteplase or tissue plasminogen activator), followed by randomization to either no additional treatment (group I) or a continuous infusion of nontargeted microbubbles and guided high-mechanical index impulses from a three-dimensional transducer (group II). Epicardial recanalization rates, ST-segment resolution, microsphere-derived myocardial blood flow, and ultimate infarct size using myocardial contrast echocardiography were compared. The same coronary thrombosis was created in a set of 12 hypercholesterolemic pigs, which were then treated with the same pharmacologic and ultrasound regimen (group III, n = 6) or the pharmacologic regimen alone (group IV, n = 6). RESULTS: Epicardial recanalization rates in groups I and II were the same (29%), but peri-infarct myocardial blood flow and ultimate infarct size improved after treatment in group II (P < .01 vs group I). In group III, epicardial recanalization was 100% (vs. 50% in group IV), and there were significant reductions in ultimate infarct size (P = .02 compared with group IV). CONCLUSIONS: Guided high-mechanical index impulses from a diagnostic transducer and nontargeted microbubbles improve peri-infarct microvascular flow in acute ST-segment elevation myocardial infarction, even when epicardial recanalization does not occur.