Robert H. Sweitzer

Acoustically active lipospheres containing paclitaxel: A new therapeutic ultrasound contrast agent

RATIONALE AND OBJECTIVES Paclitaxel-carrying lipospheres (MRX-552) were developed and evaluated as a new ultrasound contrast agent for chemotherapeutic drug delivery. METHODS Paclitaxel was suspended in soybean oil and added to an aqueous suspension of phospholipids in vials. The headspace of the vials was replaced with perfluorobutane gas; the vials were sealed, and they were agitated at 4200 rpm on a shaking device. The resulting lipospheres containing paclitaxel were studied for concentration, size, acute toxicity in mice, and acoustic activity and drug release with ultrasound. Lipospheres containing sudan black dye were produced to demonstrate the acoustically active liposphere (AAL)-ultrasound release concept. RESULTS Acoustically active lipospheres containing paclitaxel had a mean particle count of approximately 1 x 10(9) particles per mL and a mean size of 2.9 microns. Acute toxicity studies in mice showed a 10-fold reduction in toxicity for paclitaxel in AALs compared with free paclitaxel. The AALs reflected ultrasound as a contrast agent. Increasing amounts of ultrasound energy selectively ruptured the AALs and released the paclitaxel. CONCLUSIONS Acoustically active lipospheres represent a new class of acoustically active drug delivery vehicles. Future studies will assess efficacy of AALs for ultrasound-mediated drug delivery.

Targeted-microbubble binding selectively to GPIIb IIIa receptors of platelet thrombi

Schumann PA, Christiansen JP, Quigley R, et al. Targeted-microbubble binding selectively to GPIIb IIIa receptors of platelet thrombi. Invest Radiol 2002;37:587–593. Rationale and Objectives.New targeted microbubbles directed to the GPIIb IIIa receptor have been developed. The objective was to determine whether targeting microbubbles to clots would enhance ultrasound imaging. Systematic studies were designed to determine whether in vitro methodology is an acceptable predictor of in vivo efficacy. Materials and Methods.Bioconjugate ligands were inserted into lipid-coated membranes of perfluorocarbon gas microbubbles and binding studies performed on activated platelets immobilized on cell culture plates. Targeted microbubble binding to clots in a flow through chamber was also assessed. Finally, microbubble binding studies on arteriolar and venular clots in a mouse cremasteric muscle model were conducted. Results.Binding studies on platelet-immobilized plates demonstrated an affinity for targeted microbubbles versus untargeted microbubbles. Semiquantitative light obscuration techniques helped to measure extent of targeted microbubble binding. Targeted microbubbles similarly bound to platelet clots in the flow model. Finally, studies in the mouse model confirmed binding of targeted microbubbles in both venules and arterioles. Conclusion.The use of receptor selective targeted microbubbles improved binding to vascular thrombi in both in vitro and in vivo settings.

Ultrasound enhances gene expression of liposomal transfection.

RATIONALE AND OBJECTIVES Cationic liposomes are under development as delivery agents for gene therapy. The authors studied the effect of ultrasound on gene expression in cell cultures during liposomal transfection experiments. METHODS Cationic liposomes of dipalmitoylethylphosphocholine and dioleoylphosphatidylethanolamine were used to transfect cultured HeLa, NIH/3T3, and C127I cells with the chloramphenicol acetyl transferase (CAT) gene. A cell viability assay was performed on cultured HeLa cells that were exposed to varying durations (5 seconds or 30 seconds) and intensities of 1 MHz continuous-wave therapeutic ultrasound after transfection, and gene expression was measured 48 hours later. RESULTS Cells survived 30 seconds or less at a power level of 0.5 watts/cm2 but died when exposed for 60 seconds or longer. Exposures of 5 seconds and 30 seconds of ultrasound resulted in significant increases in gene expression in all three cell types tested in this experiment. CONCLUSIONS Relatively low levels of ultrasound energy can be used to enhance gene expression from liposomal transfection. Additional experiments are needed to optimize this process and clarify the mechanisms involved.

Nanoparticle drug delivery system for intravenous delivery of topoisomerase inhibitors.

Camptothecin-based drugs, because of their poor solubility and labile lactone ring, pose challenges for drug delivery. The purpose of this research was to develop a nanoparticle delivery system for camptotheca alkaloids. After initial investigations SN-38 was selected as the candidate camptotheca alkaloid for further development. Nanoparticles comprising SN-38, phospholipids and polyethylene glycol were developed and studied in vitro and in vivo. The SN-38 formulations were stable in human serum albumin and high lactone concentrations were observed even after 3 h. In vivo studies in nude mice showed prolonged half-life of the active (lactone form) drug in whole blood and increased efficacy compared to Camptosar in a mouse xenograft tumor model.

Enhanced Visualization of Intravascular and Left Atrial Appendage Thrombus with the Use of a Thrombus-Targeting Ultrasonographic Contrast Agent (MRX-408A1): In Vivo Experimental Echocardiographic Studies

Echocardiographic evaluation for the recognition of intravascular and left atrial appendage thrombus remains a difficult problem. A thrombus-specific ultrasonographic contrast agent has the potential for an alternative approach for their delineation. The aim of this study was to investigate the usefulness of thrombus-specific contrast agent MRX-408A1 for the detection of acute experimentally created intravascular and intracardiac thrombus. In the first study, we created inferior vena cava thrombus in 9 dogs. With the use of fundamental 2-dimensional echocardiography imaging, we recorded images of the inferior vena cava thrombus at baseline (n = 9), with the thrombus-specific contrast agent MRX-408A1 (n = 9), and with nonspecific contrast agent MRX-113 (n = 6). In the second study, we created a left atrial appendage thrombus in 8 dogs. We imaged left atrial appendage thrombus at baseline and during MRX-113 and MRX-408A1 infusion. Thrombus was successfully created in all dogs in study 1 and in 6 of 8 dogs in study 2. MRX-408A1 produced a visually apparent increase in ultrasonographic contrast enhancement of the thrombus in all cases in which thrombus was found on autopsy. In both studies, MRX-408A1 increased the videointensity of the thrombus significantly compared with baseline images and images obtained during MRX-113 infusion. The size of the visually detectable thrombus on the image was also significantly larger during MRX-408A1 infusion than at baseline and during MRX-113 infusion. These data provide in vivo demonstration of the efficacy of a thrombus-specific contrast agent, MRX-408A1, in the detection of acute intravascular and intracardiac thrombus. It has the potential to improve the diagnostic accuracy of ultrasonography for the detection of acute thrombi at various cardiovascular sites in the clinical setting.

Biomedical implications of a thrombus-specific US contrast agent

We have developed a novel thrombus-specific ultrasound (US) contrast agent, MRX-408. MRX-408 has been shown to enhance thrombi in the left ventricular apex, left atrial appendage, peripheral veins, and peripheral arteries (1). This enhancement has been observed in fundamental and harmonic imaging modes. The targeting moiety used in MRX-408 is specific to activated platelets. It has a primary affinity to the GPIIbIIIa receptor (2). The GPIIbIIIa receptor is also a target in antithrombotic therapeutics such as abciximab (ReoPro; Centocor, Malvern, Pa) (3). Due to the potential antithrombotic effect of blocking the GPIIbIIIa receptor, it is essential that we demonstrate that MRX-408 can enhance the clots without causing an antithrombotic effect in compromised patients. The first assay that is used to evaluate the effect of the targeting moiety on platelets is in vivo platelet aggregation (4). In these assays, whole blood is collected from normal human volunteers and centrifuged to prepare platelet-rich plasma. This material is used in the aggregation studies. The targeting moiety was tested as a free molecule (Fig 1) and in the microbubble form (Fig 2) and was found not to affect platelet aggregation. Administration of 100 times the human dose equivalent resulted in 44%–58% inhibition of platelet aggregation. Platelet aggregation was then carried out during imaging studies in a canine model. Multiple large doses of MRX-408, for a total dose of 100 L/kg, were administered. No change was seen from the initial measurements taken before the first injection (Fig 3). Bleeding time was also assayed in the canine model. No change was seen from initial measurements taken before the first injection. MRX-408 enhances cardiac and peripheral thrombi without having a pharmacologic effect on platelet aggregation or bleeding time. This provides a large safety index for a diagnostic agent. Acad Radiol 2002; 9(suppl 1):S56–S57

Therapeutic applications of microbubbles

While microbubbles have been developed for diagnosis as ultrasound contrast agents, the greatest potential of these agents may lie in therapy. Microbubbles have extensive applications for treating vascular thrombosis, drug and gene delivery. Targeted microbubbles containing ligands for cell or site‐specific recognition have been developed. These agents function like ‘‘smart bubbles’’ to home in on their targets. One such agent, MRX‐408, a microbubble bearing targeting ligands directed to the activated receptor of platelets has undergone extensive pre‐clinical testing. Recent studies comparing Sonothrombolysis with MRX‐408 and nontargeted microbubbles show significantly more clot lysis with targeted as compared to nontargeted microbubbles. The targeted bubbles effectively concentrate cavitation nuclei within the clot. Additional targeted microbubbles for drug and gene delivery are also under development.

Gene activation and gene delivery with ultrasound

Transfection experiments were performed in vitro in cell culture and in vivo in fish and mice using cationic liposomes with several different reporter genes. The effect of continuous‐wave 1‐MHz ultrasound on cell viability, cell permeability, and gene expression was assessed. RT‐PCR was used to monitor gene expression of cell repair genes in insonated cells. Acoustically active halocarbon‐containing liposomes were prepared for gene delivery. Ultrasound increased cell permeability and gene expression following liposomal transfection. RT‐PCR showed upregulation of repair of a number of cell repair genes after ultrasound exposure. The acoustically active gene delivery vehicles provided highly efficient transfection both with and without ultrasound in vitro and in vivo. Following IV administration of halocarbon‐containing liposomes/gene complexes, preferential gene expression was observed in the insonated tissue. Ultrasound has potentially useful applications for targeting and enhancing gene therapy.

Gas‐filled liposomes as ultrasound contrast agents for blood pool, thrombus‐specific and therapeutic applications

Our group has developed technology for stabilizing microbubbles with phospholipid coatings (Aerosomes®‐ImaRx Pharmaceutical Corp.). The first agent (MRX‐115) is based upon lipid‐coated microspheres filled with perfluoropropane gas and is in phase III clinical trials (October, 1997) for radiology and cardiology applications. Myocardial perfusion studies show the potential for the agent to detect ischemia in patients with myocardial infarcts. Targeting ligands have been covalently bound to lipids and incorporated into the stabilizing shells on the microbubbles. The first targeted agent planned for clinical trials is MRX‐408 (perfluorobutane gas, linear hexapeptide‐RGD analog). In vitro studies show enhanced visualization of thrombi, and in vivo studies in dogs with arterial and venous thrombi show selective enhancement, even in animals injected with heparin. In vitro studies have been performed to test sonothrombolysis comparing MRX‐115 to MRX‐408 with and without urokinase. The targeted contrast agent MRX‐...