Local drug and gene delivery through microbubbles.

Ultrasound contrast agents (microbubbles) lower the threshold for cavitation by ultrasound energy. Ultrasound microbubbles may be used as cavitation nuclei for drug and gene delivery. By tailoring the physical properties of microbubbles and coating materials, drugs and genetic drugs can be incorporated into ultrasound contrast agents. As the microbubbles enter the region of insonation, the microbubbles cavitate, locally releasing the therapeutic agents. Cavitation also causes a local shockwave that improves cellular uptake of the therapeutic agent. As a result of the human genome project and continuing advances in molecular biology, many therapeutic genes have been discovered. In the cardiovascular system, gene therapy has the potential to improve myocardial vascularization and ameliorate congestive heart failure. For successful development of clinical gene therapy, however, effective gene delivery vectors are needed. Ultrasound contrast agents can be used to develop new, more effective vectors for gene delivery. Transthoracic ultrasound can be focused on the heart so that an intravenous injection of gene-bearing microbubbles will deliver genes relatively selectively to the myocardium. Using this technique, we have produced high levels of transgene expression in the insonated region of the myocardium. This new technology, using microbubbles and ultrasound for drug and gene delivery, merits further study and development.

Gene delivery using ultrasound contrast agents.

With the human genome product and continuing advances in molecular biology many therapeutic genes have been discovered. In the cardiovascular system, gene therapy has the potential to improve myocardial vascularization and ameliorate congestive heart failure. For successful development of clinical gene therapy, however, effective gene delivery vectors are needed. Ultrasound contrast agents can be used to develop new, more effective vectors for gene delivery. Ultrasound contrast agents lower the threshold for cavitation by ultrasound energy. Using physical properties of microbubbles and coating materials, genetic drugs have been incorporated into ultrasound contrast agents. Gene-bearing microbubbles can be injected IV and ultrasound energy applied to the target region. As the microbubbles enter the region of insonation, the microbubbles cavitate, locally releasing DNA. Cavitation also likely causes a local shockwave that improves cellular uptake of DNA. With transthoracic ultrasound, using commercially available diagnostic ultrasound system and an IV injection of gene-bearing microbubbles, high levels of transgene expression are observed in the insonated region of the myocardium. This new technology using microbubbles and ultrasound for gene delivery merits further study and development.

Advances in imaging of lymph flow disorders.

Conventional oil-contrast lymphography has long been the mainstay for lymphatic imaging. However, the emergence of computed tomography (CT) and magnetic resonance (MR) imaging has severely curtailed its use. Because of recent improvements and refinements, lymphangioscintigraphy now permits high-resolution imaging of peripheral lymphatic vessels and provides insight into lymph flow dynamics. It is indispensable for patients with known or suspected lymphatic circulatory disorders in confirming the diagnosis and delineating the pathogenesis and evolution of lymphedema. In addition, lymphangioscintigraphy helps evaluate lymphatic truncal anatomy and radiotracer transport. It can also be used to evaluate the efficacy of various treatment options designed to facilitate lymph flow or reduce lymph formation. The procedure is essentially noninvasive, can easily be repeated, and does not adversely affect the lymphatic vascular endothelium. MR imaging complements lymphangioscintigraphy in the monitoring and treatment of more complex lymphatic circulatory disorders, whereas CT facilitates catheter-guided percutaneous sclerosis or obliteration of specific lymphangiectasia or lymphangioma syndromes. Ultrasonography has proved useful in the setting of filariasis. Patients with a provisional diagnosis of peripheral lymphatic dysfunction or idiopathic edema should undergo diagnostic lymphangioscintigraphy and, in some cases, MR imaging to verify diagnostic accuracy, pinpoint the specific abnormality, and help guide subsequent therapy.

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.

Immunotherapy of advanced malignancy by direct gene transfer of an interleukin-2 DNA/DMRIE/DOPE lipid complex: phase I/II experience.

PURPOSE: We have completed a phase I study, followed by three phase I/II studies, in patients with metastatic melanoma, renal cell carcinoma (RCC), and sarcoma in order to evaluate the safety, toxicity, and antitumor activity of Leuvectin (Vical Inc, San Diego, CA), a gene transfer product containing a plasmid encoding human interleukin (IL)-2 formulated with the cationic lipid 1, 2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide/dioleyl-phosphatidyl-ethanolamine (DMRIE/DOPE) and administered intratumorally. PATIENTS AND METHODS: Twenty-four patients were treated in the phase I study. Leuvectin doses were 10 microg, 30 microg, or 300 microg weekly for 6 weeks. In three subsequent phase I/II studies, a total of 52 patients (18 with melanoma, 17 with RCC, and 17 with sarcoma) were treated with further escalating doses of Leuvectin: 300 microg twice a week for 3 weeks, 750 microg weekly for 6 weeks, and 1,500 microg weekly for 6 weeks. RESULTS: There were no drug-related grade 4 toxicities and only one grade 3 toxicity, but the majority of patients experienced mild constitutional symptoms after treatment. In the phase I/II studies, 45 patients were assessable for response (14 with RCC, 16 with melanoma, and 15 with sarcoma). Two patients with RCC and one with melanoma have achieved partial responses lasting from 16 to 19 months and continuing. In addition, two RCC, three melanoma, and six sarcoma patients had stable disease lasting from 3 to 18 months and continuing. The plasmid was detected by polymerase chain reaction assay in the posttreatment samples of 29 of 46 evaluated patients. Immunohistochemistry studies on serial biopsy specimens showed increased IL-2 expression and CD8(+) infiltration after treatment in the tumor samples of several patients (12 and 16, respectively). CONCLUSION: Direct intratumoral injection of Leuvectin is a safe and possibly effective immunotherapeutic approach in the treatment of certain tumor types.

Gadolinium-containing copolymeric chelates--a new potential MR contrast agent.

RATIONALE AND OBJECTIVES: To develop and partially characterize a new class of potential blood pool magnetic resonance (MR) contrast agents. METHODS: Various copolymeric chelates of gadolinium diethylenetriamine pentaacetic acid (Gd-DTPA) were prepared with differing molecular weights of polyethylene glycol (PEG) or polypropylene glycol (PPG) as linkers between the monomeric chelate units. Gadolinium content of the polymeric chelates was determined by atomic absorption spectra. Relaxivity of the polymeric chelates was measured at 1.5 Tesla and compared with Gadolinium DTPA. MR angiography (MRA) was performed in rabbits comparing Gd-DTPA with Gd copolymers. RESULTS: The gadolinium content of the copolymeric chelates ranged from 2.95 to 22.2% on weight basis. The molecular weight of the PEG linkers in the copolymers ranged from about 150 to about 3400. The r1 (1/T1, mM(-1) s(-1)) for Gd DTPA = 4.1. The r1 values for the different Gd-containing polymers ranged from 3.8 to 5.8, with the lowest r1 for the polymer prepared with the lowest-molecular-weight complex. The higher-molecular-weight complexes resulted in moderately higher relaxivity. MRA with Gd-copolymers, in rabbits, showed markedly greater vascular enhancement relative to an equivalent dose of Gd-DTPA. Vascular enhancement was much more sustained with the copolymeric agent and confined to vascular space; i.e. no appreciable background tissue enhancement--a reflection of distribution into extravascular fluid space--was observed. CONCLUSIONS: Relative to Gd-DTPA monomers, PEG-containing Gd DTPA polymeric complexes provided moderate increases in relaxivity but markedly greater efficacy during in vivo MRA. In vitro relaxivity studies of Gd-copolymers showed only an approximately 50% increase in r1 relaxivity compared with Gd-DTPA. The PEG-containing complex's lack of rigidity may have diminished the effect of spin diffusion on relaxation, thereby accounting for this modest increase. The greater efficacy of Gd-copolymers during in vivo MRA may reflect compartmentalization within the vascular space and possibly enhanced relaxation of the macromolecular copolymers in the blood. Gd-copolymers are promising agents that merit additional study.

Binding and lysing of blood clots using MRX-408.

RATIONALE AND OBJECTIVES: A thrombus-specific ultrasound contrast agent, MRX-408, has been developed recently. This agent consists of phospholipid-coated microbubbles with a ligand capable of targeting the GPIIb/IIIa receptor, thereby allowing the microbubbles to bind with thrombi rich in activated platelets. In vitro and in vivo animal experiments have been conducted to examine imaging enhancement and sonothrombolysis using this agent compared with a nontargeted agent. METHODS: For clot binding, blood-smeared slides were incubated with microbubbles and examined under a light microscope. Change in backscatter signals from the blood clots after binding was examined by both an ultrasound scanner and two single-element transducers arranged in a transmitter-receiver pair. For clot lysis, either 1-MHz or 20-KHz ultrasound was used to enhance the lysing effects of MRX-408 with or without urokinase. RESULTS: Evidence of binding was demonstrated under a microscope. In vitro experiments showed that the "acoustic signature", or properties, of blood clots changed after binding. Clots became more echogenic and nonlinear. In vivo fundamental ultrasound imaging confirmed that as a result of binding, blood clots were more visible, the area of detection was improved, and shadowing behind clots was more noticeable. Under 1-MHz ultrasound and 30 minutes of treatment, lysis efficiency reached 34% with MRX-408, whereas there was no visible clot lysis with saline. CONCLUSION: The results of these preliminary studies show that as a contrast agent, MRX-408 enhanced clots under ultrasound imaging and facilitated sonothrombolysis with or without thrombolytic drugs.

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.

Accuracy of CT biopsy: laser guidance versus conventional freehand techniques.

RATIONALE AND OBJECTIVES: The purpose of this study was to determine whether laser-guided computed tomographic (CT) biopsy is more accurate than CT-guided biopsy with conventional freehand techniques. MATERIALS AND METHODS: Two independent operators performed an equal number of freehand and laser-guided needle passes at varying single and double angles (0 degree, 30 degrees, 60 degrees, 25 degrees/30 degrees, and 25 degrees/60 degrees) on targets within six pork and beef phantoms. A total of 180 biopsy passes were performed, and error distances of needle tip to target were tabulated. Data were analyzed by means of repeated measures analysis of variance (ANOVA) to compare the accuracy of laser guidance with freehand passes. ANOVA and correlation analysis were also used to confirm the relative equivalency of phantom targets and biopsy parameters. RESULTS: Overall, laser-guided passes were statistically significantly more accurate than freehand passes. Mean error with laser guidance was 5.01 mm (standard error [SE] = 0.41 mm), whereas mean error with freehand techniques was 10.58 mm (SE = 0.82 mm) (F = 52.0, df = 1.17, P = .0001). Ninety-three percent of laser-guided passes and 56% of freehand passes were within 1 cm of the intended target. Error increased for both laser-guided and freehand techniques with larger angles or double-angle biopsies, but the increases were greater with freehand technique. No statistically significant differences existed between the targets themselves or biopsy parameters for the two operators. CONCLUSION: Laser-guided CT biopsies were more accurate than freehand CT biopsies. Practical advantages of laser guidance over freehand CT biopsy methods may include decreased procedure times and reduced patient morbidity.

In vitro studies of a new thrombus-specific ultrasound contrast agent.

Ultrasound is used as a primary diagnostic technique for the detection of deep venous thrombosis. The purpose of this study is to describe the development of a new thrombus-specific ultrasound contrast agent: The linear hexapeptide (lysine-glutamine-alanine-glycine-aspartate-valine) was synthesized and coupled to a lipid moiety. The targeted lipid was then incorporated into the lipid blend for the contrast agent Aerosomes (ImaRx, Tucson, AZ, USA). The lipid blend was used to entrap perfluorobutane microbubbles. The microbubbles were sized and studied in vitro for acoustic stability, binding to blood clot, and ultrasound enhancement in vitro of blood clot. The results showed the mean size of the specific ultrasound contrast agent (MRX-408) was about 2.0 microm. The microbubbles appeared as smooth spherical structures. Microscopy showed that the targeted bubbles bound to blood clot whereas control, nontargeted bubbles did not bind to blood clot. In vitro acoustic study showed similar stability of the microbubbles compared with control microbubbles. The targeted microbubbles enhanced blood clot in vitro whereas nontargeted microbubbles did not enhance clot. Thus this promising new thrombus-specific ultrasound contrast agent could potentially improve detection of thrombosis by ultrasound and might be useful for distinguishing between new and old thrombosis. In vivo studies are in progress.

Potentials of a new laser guidance system for percutaneous musculoskeletal procedures.

Early experience using a new laser guidance device to assist CT-guided percutaneous musculoskeletal procedures is presented. We describe six cases, which demonstrate typical musculoskeletal applications of laser guidance. In our experience laser guidance for these procedures resulted in improved accuracy with no significant increase in biopsy time when a short learning period is considered. Other musculoskeletal procedures may benefit from laser guidance in preference to current standard CT-guided techniques, particularly when precision and accuracy are essential.

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.

Phase I clinical trials of MRX-115. A new ultrasound contrast agent.

RATIONALE AND OBJECTIVES: Stabilized microbubbles are under development as contrast agents for medical ultrasound. The authors report the results of Phase I clinical trials of a new ultrasound contrast agent based on lipidencapsulated perfluorocarbon gas microbubbles. METHODS: Lipids encapsulating perfluoropropane gas (Aerosomes MRX-115, ImaRx Pharmaceutical Corp., Tucson, AZ) were evaluated in Phase I clinical trials. Two separate studies were performed. The first was a single escalating-dose study (n = 30 subjects), and the second was a multiple-dose study (n = 18 subjects) with rechallenge in several subjects (n = 4) after 21 days. Echocardiographic examinations were performed before and after contrast agent for each test drug administration for both studies, with the exception of the rechallenge group. Doses tested in the single-dose study ranged from 0.005 mL/kg to 0.100 mL/kg body weight. In the multiple-dose study, five doses of 0.005 mL/ kg to 0.030 mL/kg (0.025-0.150 mL/kg total dose) were evaluated. Studies were single-masked, placebo-controlled, and safety assessment and adverse events were monitored. RESULTS: All doses in both studies were well tolerated with no treatment-related changes in safety measures for either study. Left ventricular cavity and myocardial enhancement were seen with all doses of MRX-115. CONCLUSIONS: MRX-115 is a promising new intravascular ultrasound contrast agent that was safe and well tolerated at the doses evaluated in these studies.

Ultrasonographic detection of testicular ischemia in a canine model using phospholipid coated microbubbles (MRX-115).

The purpose of this study was to determine if the sonographic contrast agent MRX-115 could improve the accuracy of Doppler ultrasonographic diagnosis of testicular ischemia. Testicular duplex ultrasonography was performed on six dogs before and after surgical ligation of the testicular artery, and before and after intravenous injection of MRX-115. Six radiologists blinded to experimental conditions rated the testicular blood flow. Receiver operating characteristics curves for conditions with and without administration of contrast agent were compared using Student's t-test for paired observations. Statistically significant differences were found both for the tests viewed individually (P = 0.003) and for the testes in comparison to the contralateral side (P = 0.0001). MRX-115 therefore is found to improve duplex sonographic evaluation of testicular ischemia.

Potentials of laser guidance system for percutaneous musculoskeletal procedures.

Early experience using a new laser guidance device to assist CT-guided percutaneous musculoskeletal procedures is presented. We describe six cases which demonstrate typical musculoskeletal applications of laser guidance. In our experience laser guidance for these procedures resulted in improved accuracy with no significant increase in biopsy time when a short learning period is considered. Other musculoskeletal procedures may benefit from laser guidance compared with current standard CT-guided techniques, particularly when precision and accuracy are essential.

Magnetic resonance imaging in coccidioidal arthritis.

OBJECTIVE: The authors assessed the MRI findings of appendicular coccidioidal arthritis. DESIGN: T1- and T2-weighted MR images of affected joints, both with and without intravenous gadopentetate dimeglumine, were performed in nine adult patients (ten studies) and evaluated by three masted readers, using a four-point certainty scale for: synovial abnormality, articular cartilage loss, subarticular bone loss, abnormal marrow signal, enhancement of osseous and articular structures, and assessment of disease activity. Findings were correlated with biopsy results or clinical course. RESULTS: Eight patients had active and one had inactive arthritis, involving the knee (five patients), ankle (two patients), and elbow (one patient). Synovial complex was the most common finding in active arthritis (P < 0.025). Cartilage and subarticular bone loss were seen 56% and 89% of patients with active disease, respectively. Abnormal marrow signal was uncommon (two patients). All cases showed synovial and/or osseus enhancement. CONCLUSIONS: MRI findings in coccidiodal arthritis are described. Enhancement of thickened synovium and erosions was seen after intravenous gadopentetate.