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PubMed2006

Quantification of phosphatidylserine, phosphatidic acid and free fatty acids in an ultrasound contrast agent by normal-phase high-performance liquid chromatography with evaporative light scattering detection.

Sonazoid is a new contrast agent for ultrasound imaging. The product is an aqueous suspension of perfluorobutane microbubbles coated with phospholipids obtained from hydrogenated egg phosphatidylserine (H-EPS). A normal-phase high-performance liquid chromatographic (HPLC) method with evaporative light scattering detection was developed for quantification of free fatty acids, phosphatidylserine and phosphatidic acid in H-EPS and Sonazoid. Separation of the lipids was carried out on an HPLC diol column and a gradient of chloroform and methanol with 0.2% formic acid titrated to pH 7.5 with ammonia. The calibration standards contained stearic acid, distearoyl-phosphatidic acid (DSPA) and distearoyl-phosphatidylserine (DSPS) in the concentration range of 0.016-1.0mg/ml (0.4-25microg injected). The method was validated with a limit of quantification of the three lipids set to 0.4microg (approximately 20-60microM). The best fit of the three calibration curves were obtained when the logarithmic transformed theoretical lipid concentration was plotted against the logarithmic transformed area under the peak and fitted to a second order polynomial equation. Stearic acid, DSPA and DSPS were analysed with an intermediate precision ranging from 4.4% to 5.3% R.S.D. and they were extracted from an aqueous suspension with a recovery ranging from 103.3% to 113.3%. The sum of total phospholipid concentration determined in H-EPS ranged from 96.4% to 103.2% of the theoretical values. The lipids in the ultrasound product were quantitated with a repeatability ranging from 6.2% to 11.7% R.S.D.

Chromatography, High Pressure Liquid↗

PubMed2006

Synthesis of branched antisense oligonucleotides having multiple specificities. Treatment of hormone insensitive prostate cancer.

Antisense oligonucleotides (oligos) directed against transforming growth factor-alpha (TGF-alpha) and its binding site, the epidermal growth factor receptor (EGFR), have demonstrated in vitro and in vivo efficacy against both the PC-3 and LNCaP prostate tumor models. In an attempt to increase the efficiency of these oligos a new type of antisense compound called a bispecific oligo has been evaluated in vitro both alone and in combination with traditional chemotherapeutic agents. These bispecifics, which were first proposed in this journal in 2004, include binding sites for both TGF-alpha and EGFR along the same stretch of complementary DNA. Such bispecifics are able to deliver essentially two antisense activities in an equal molar ratio and can be directed against mRNA encoding proteins of different biochemical pathways. The first bispecifics were developed against two proteins regulating a single autocrine loop. Subsequent bispecifics have been developed which target both EGFR and the apoptosis regulating protein bcl-2. Bispecific activity of a single linear sequence oligo has already been shown to have efficacy. To further develop this multispecific approach, we now propose a branched antisense compound, again, having multiple binding site activities (to complementary sequenced mRNA). Active oligos would be attached to a fat soluble backbone which might enhance targeting and also intracellular entry, release and activity. Such a structure would also permit the customization of these branched forms to include oligos targeting specific proteins related to the growth of various tumor types. Problems associated with the development of antisense oligos have included both membrane solubility and specific targeting. By designing this branched form of antisense structure, multiple activities can be retained (added), solubility improved and delivery enhanced. Such a new formulation would include several antisense oligos covalently bound to and branching off from a lipid-like backbone. An elongated hydrocarbon chain would increase fat solubility and would permit oligo incorporation into nanoparticles or liposome derived delivery vehicles. Specific delivery of oligos could also be enhanced by the tendency of these nanoparticle or liposomal microbubbles to be disrupted under the influence of ultrasonic waves beamed at the targeted tissue.

Cell Line, Tumor↗

PubMed2006

Alternative explanations to the differences of femoral and brachial saline contrast injections for echocardiographic detection of patent foramen ovale.

Patent foramen ovale (PFO) of the interatrial septum is a cardiac foetal remnant, which frequent persistence in adulthood has important implications in a variety of clinical conditions. Echographic diagnosis of PFO is based on detection of interatrial shunt by means of contrast microbubbles identification after venous injection of a first-generation echographic contrast agent. Current recommendations propose venous femoral injection of contrast for enhanced echographic detection of PFO instead of venous brachial administration, as femoral injection has been shown to have higher sensitivity for PFO detection. Inferior vena cava inflow directed toward interatrial septum has been considered the explanation for increased sensitivity of femoral delivery of contrast. In the present paper, it is hypothesised that the main determinants of these differences between injection sites are technical factors related to right atrial contrast opacification and proper transient right atrial pressure rise, rather than intraatrial flow streaming. Effects of inferior vena cava inflow stream, although significant during foetal life, would be negligible after birth. Rationale and evidence, basis for further research, and practical implications leading to a simpler and safer routine technique for echographic detection of PFO are presented and discussed.

Brachial Artery↗

PubMedDate not supplied

Assessment of myocardial perfusion with real-time myocardial contrast echocardiography: methodology and clinical applications.

Real-time myocardial contrast perfusion imaging (RTMCI) with echocardiography is a promising technique for evaluation of patients with known or suspected coronary artery disease. The technique is based on the utilization of small (<10 mum) microbubbles, which are capable of crossing the pulmonary circulation after intravenous injection. Unlike radioactive isotopes, which are taken actively or diffuse passively in the myocytes, myocardial contrast agents remain extracellularly in the capillaries and present a measure of the myocardial capillary blood volume and microvascular integrity. RTMCI has been shown to be a safe and feasible method for the assessment of myocardial perfusion at rest and with pharmacologic stress. Recent studies have shown the value of RTMCI with dobutamine stress in improving overall and regional detection of coronary artery disease and detecting of abnormalities at submaximal stress, therefore improving sensitivity in patients who are unable to achieve the target heart rate. The advantages of the technique include the ability to assess perfusion at bedside in one setting, simultaneous assessment of myocardial function, shorter imaging time, no need for ionizing irradiation, immediate availability of the results, and the ability to determine the ischemic threshold. Recent studies have shown that RTMCI improves the prognostic utility of standard dobutamine stress in addition to wall motion analysis. Patients with normal perfusion had a better outcome than those with normal wall motion. The combination of abnormal wall motion and perfusion identified patients at greatest risk of death and nonfatal myocardial infarction. Perfusion abnormalities were also shown to predict short-term cardiac events in patients presenting to the emergency department with chest pain and no ST-segment elevation. Refinement of imaging techniques is expected to improve the specificity of RTMCI, particularly in differentiating true perfusion defects from artifacts. This review will discuss the physiologic basis, methodology, clinical utility, and limitations of RTMCI in the assessment of patients with known or suspected coronary artery disease.

Computer Systems↗

PubMed2006

Overview of experimental studies of biological effects of medical ultrasound caused by gas body activation and inertial cavitation.

Ultrasound exposure can induce bioeffects in mammalian tissue by the nonthermal mechanism of gas body activation. Pre-existing bodies of gas may be activated even at low-pressure amplitudes. At higher-pressure amplitudes, violent cavitation activity with inertial collapse of microbubbles can be generated from latent nucleation sites or from the destabilization of gas bodies. Mechanical perturbation at the activation sites leads to biological effects on nearby cells and structures. Shockwave lithotripsy was the first medical ultrasound application for which significant cavitational bioeffects were demonstrated in mammalian tissues, including hemorrhage and injury in the kidney. Lithotripter shockwaves can also cause hemorrhage in lung and intestine by activation of pre-existing gas bodies in these tissues. Modern diagnostic ultrasound equipment develops pressure amplitudes sufficient for inertial cavitation, but the living body normally lacks suitable cavitation nuclei. Ultrasound contrast agents (UCAs) are suspensions of microscopic gas bodies created to enhance the echogenicity of blood. Ultrasound contrast agent gas bodies also provide nuclei for inertial cavitation. Bioeffects from contrast-aided diagnostic ultrasound depend on pressure amplitude, UCA dose, dosage delivery method and image timing parameters. Microvascular leakage, capillary rupture, cardiomyocyte killing, inflammatory cell infiltration, and premature ventricular contractions have been reported for myocardial contrast echocardiography with clinical ultrasound machines and clinically relevant agent doses in laboratory animals. Similar bioeffects have been reported in intestine, skeletal muscle, fat, lymph nodes and kidney. These microscale bioeffects could be induced unknowingly in diagnostic examinations; however, the medical significance of bioeffects of diagnostic ultrasound with contrast agents is not yet fully understood in relation to the clinical setting.

Animals↗

PubMed2004

Development and characterization of a nano-scale contrast agent.

Agents injected parenterally must be less than approximately 8 microm diameter in order to traverse the capillaries in the pulmonary bed, but these agents remain in the vasculature until they are eliminated from the body by a variety of mechanisms. Targeting of cells outside the capillaries requires agent diameters of less than approximately 700 nm to enable escape through the larger-than-usual pores that have been noted in the leaky vasculature of a tumor. The objective of this study was to test the feasibility of creating a surfactant-stabilized nano-bubble with favorable acoustic properties, and identify the key parameters that influence size, yield and stability. Size distribution was characterized using laser light scattering. In vitro acoustic enhancement was assessed by generation of dose and time response curves. We previously developed a successful protocol to generate gas-filled microbubbles (containing perfluorocarbon, sulfur hexafluoride or air) with mean diameter of 1.5 microm, using sonication of carefully selected surfactant mixtures. This presentation describes generation of nano-bubbles with mean diameters ranging from 700 to 450 nm, depending on process variables. In all cases a centrifugation step was employed to separate the nano-sized particles. The in vitro dose response curves gave a maximum of 23-27 dB enhancement compared to buffer in the absence of agent, with the maximum enhancement and presence of shadowing at higher doses being dependent on the fabrication protocol. The effect of sonication time for solutions containing a mixture of the surfactants (Span 60 and Tween 80) was also tested, but was determined not to be an influencing factor. Future studies will involve development of a mathematical model characterizing the mean size as a function of centrifugal force, spin time and initial size distribution. Future work will also include imaging of tumor-bearing mice and measuring imaging potential in vivo in New Zealand white rabbits using power Doppler.

Contrast Media↗

PubMed2004

High-intensity focused ultrasound for the treatment of liver tumours.

High-intensity focused ultrasound (HIFU) has been investigated as a tool for the treatment of cancer for many decades, but is only now beginning to emerge as a potential alternative to conventional therapies. In recent years, clinical trials have evaluated the clinical efficacy of a number of devices worldwide. In Oxford, UK, we have been using the JC HIFU system (HAIFU Technology Company, Chongqing, PR China) in clinical trials since November 2002. This is the first report of its clinical use outside mainland China. The device is non-invasive, and employs an extracorporeal transducer operating at 0.8-1.6 MHz (aperture 12-15 cm, focal length 9-15 cm), operating clinically at Isp (free field) of 5-15 KWcm(-2). The aims of the trials are to evaluate the safety and performance of the device. Performance is being evaluated through two parallel protocols. One employs radiological assessment of response with the use of follow-up magnetic resonance imaging and microbubble-contrast ultrasound. In the other, histological assessment will be made following elective surgical resection of the HIFU treated tumours. Eleven patients with liver tumours have been treated with HIFU to date. Adverse events include transient pain and minor skin burns. Observed response from the various assessment modalities is discussed.

Contrast Media↗

PubMed2004

Perspectives in clinical uses of high-intensity focused ultrasound.

Focused ultrasound holds promise in a large number of therapeutic applications. It has long been known that high intensity focused ultrasound can kill tissue through coagulative necrosis. However, it is only in recent years that practical clinical applications are becoming possible, with the development of high power ultrasound phased arrays and noninvasive monitoring methods. These technologies, combined with more sophisticated treatment planning methods allow noninvasive focusing in areas such as the brain, that were once thought to be unreachable. Meanwhile, exciting investigations are underway in microbubble-enhanced heating which could significantly reduce treatment times. These developments have promoted an increase in the number of potential applications by providing valuable new tools for medical research. This paper provides an overview of the scientific and engineering advances that are allowing the growth in clinical focused ultrasound applications. It also discusses some of these prospective applications, including the treatment of brain disorders and targeted drug delivery.

Brain Diseases↗

PubMed2005

A study on the primary and secondary nucleation of ice by power ultrasound.

Several different investigations have been carried out to study the primary and secondary nucleation of ice by sonocrystallisation. Firstly, the primary nucleation of discrete ice crystals in a supercooled sucrose solution has been observed. For increasing concentrations of sucrose solutions from 0 to 45 wt%, the nucleation temperature consistently occurs at a higher nucleation temperature in the presence of ultrasound. The nucleation temperature also increases as the power output and duty cycle of a commercial ultrasonic horn are increased. Snap shot images of the bubble clouds obtained from the ultrasonic horn also show that the number of bubbles appears to increase as the ultrasonic output is increased. This suggests that the nucleation of ice is related to the power output and number of cavitation bubbles. The effect of a single bubble on the sonocrystallisation of ice is discussed. High-speed movies (1120 fps) have shown that the crystallisation appears to occur in the immediate vicinity of the single bubble. In most cases, many crystals are observed and it is not known whether a single ice crystal is being fragmented by the bubble or whether many crystals are being initiated. The bubble appears to undergo a dancing regime, frequently splitting and rejoining and also emitting some small microbubbles. A study on the secondary nucleation of ice in sucrose solutions has been carried out using a unique ultrasonic cold stage device. Images taken using a microscope system show that the pre-existing ice dendrite crystals can be broken up into smaller fragments by an ultrasonic field. Cavitation bubbles appear to be important during the fragmentation process, possibly melting any ice crystals in their path. Flow patterns around cavitation bubbles have also been observed, and these may be responsible for the fragmentation of ice crystals.

Crystallization↗

PubMed2006

Study on the multiple scattering effects of ultrasound contrast agents.

In this paper, the multiple scattering of interacting encapsulated microbubbles in suspensions is calculated using two novel approaches--Kargl's effective medium approach and Ye and Ding's approach of 2nd-order correction. Two types of contrast agents with bubbles of different sizes and concentrations are chosen for our numerical simulations. One is Albunex, which is depicted by Church and has a size range of several microns, and the other is sodium laureate solution (before fractionation) described by Soetanto and Chan and has an average size of 35.5 microm. The numerical results from these two approaches are compared with that from the linear approximation. It is found that the multiple scattering effects on attenuation and dispersion of sound in suspensions are evident in the cases of high bubble volume fractions, basically greater than the order of 1 x 10(-4), and much more obvious for larger bubbles with average size of tens of microns.

Computer Simulation↗

PubMed2004

Impact of myocardial contrast echocardiography on vascular permeability: comparison of three different contrast agents.

Microvascular permeabilization, petechial hemorrhage and premature ventricular contractions (PVCs) have been demonstrated in an in vivo rat model of myocardial contrast echocardiography (MCE). The purpose of this study was to compare these effects for three US Food and Drug Administration (FDA)-approved ultrasound (US) contrast agents (US CA): Optison, Definity and Imagent. Evans blue dye, an indicator of microvascular permeability, and a contrast agent were injected IV in anesthetized rats suspended in a water bath to mimic scanning depths seen in clinical echocardiology. Diagnostic US B-mode scans with 1:4 end-systolic triggering were performed at 1.7 MHz using a cardiac phased-array scanhead to provide a short axis view of the left ventricle. To elicit readily measurable effects for comparisons, relatively high doses of the agent were used (50 to 500 microL kg(-1) for Optison, 25 to 200 microL kg(-1) for Imagent, 10 to 100 microL kg(-1) for Definity). Microvascular leakage was characterized by the area of Evans blue dye coloration on the hearts and by extraction of the dye from tissue samples. The number of petechia were counted on the epicardial surface of excised hearts. PVCs were counted from ECG traces recorded with the MCE images. Neither evidence of capillary leakage nor PVCs were seen in sham animals. Based on volume dose, Definity MCE produced more microvascular leakage, but there was no apparent difference between the three agents' microvascular damage potential, which increased linearly with dose at low doses, when expressed in terms of the number of stabilized microbubbles. Definity MCE resulted in fewer PVCs than the other agents. The effects increased strongly with peak rarefactional pressure amplitude, with apparent thresholds for petechiae at 0.4 MPa and for PVCs at about 1.0 MPa. These results should be of value for minimizing adverse potential in diagnosis and optimizing efficacy in therapeutic applications.

Albumins↗

PubMed2004

Ultrasound-induced cell membrane porosity.

Recent studies of ultrasound (US) methods for targeted drug delivery and nonviral gene transfection revealed new, advantageous possibilities. These studies utilized US contrast agents, commonly stabilized microbubbles, to facilitate delivery and suggested that US delivery resulted from cell sonoporation, the formation of temporary pores in the cell membrane induced by US. Using voltage clamp techniques, we obtained real-time measurements of sonoporation of single Xenopus oocyte in the presence of Optison trade mark, an agent consisting of albumin-shelled C(3)F(8) gas bubbles (mean diameter 3.2 microm). Ultrasound increased the transmembrane current as a direct result of decreased membrane resistance due to pore formation. We observed a distinct delay of sonoporation following US activation and characteristic stepwise increases of transmembrane current throughout US duration. We discovered that the resealing of cell membrane following US exposure required Ca(2+) entering the cell through US-induced pores.

Albumins↗

PubMed2005

Transcranial ultrasound angiography (T USA): a new approach for contrast specific imaging of intracranial arteries.

The goal was to develop an ultrasound contrast agent-specific imaging mode that offers an angiography-like view of the intracranial arteries and enables lower mechanical index MI settings compared to conventional transcranial duplex sonography. We studied 12 patients with transcranial ultrasound angiography (t USA) via the temporal bone window after an IV bolus injection of a perfluorocarbon-based microbubble contrast agent (Imagent). The aim was to display the intracranial vessel segments of the middle cerebral artery (M1, M2 and M3), the anterior cerebral artery (A1 and A2), the posterior cerebral artery (P1, P2 and P3) and the internal carotid artery (C1/2 and C3/4). t USA is a B-mode phase inversion imaging technique that uses wideband harmonic signals for image generation. We demonstrate, in this report, that t USA provides detailed anatomical display at native B-mode spatial resolution with fewer artifacts, yielding improved delineation of intracranial vessels that are in the 1- to 2-mm range.

Adolescent↗

PubMedDate not supplied

Approaches and methods in gene therapy for kidney disease.

Renal gene therapy may offer new strategies to treat diseases of native and transplanted kidneys. Several experimental techniques have been developed and employed using nonviral, viral, and cellular vectors. The most efficient vector for in vivo transfection appears to be adenovirus. Glomeruli, blood vessels, interstitial cells, and pyelum can be transfected with high efficiency. In addition, electroporation and microbubbles with ultrasound, both being enhanced naked plasmid techniques, offer good opportunities. Trapping of mesangial cells into the glomeruli as well as natural targeting of monocytes or macrophages to inflamed kidneys are elegant methods for site-specific delivery of genes. For gene therapy in kidney transplantation, hemagglutinating virus of Japan liposomes are efficient vectors for tubular transfection, whereas enhanced naked plasmid techniques are suitable for glomerular transfection. However, adenovirus offers the best opportunities in a renal transplantation setup because varying parameters of graft perfusion allows targeting of different cell types. In renal grafts, lymphocytes can be used for selective targeting to sites of inflammation. In conclusion, for both in vivo and ex vivo renal transfection, enhanced naked plasmids and adenovirus offer the best perspectives for effective clinical application. Moreover, the development of safer, nonimmunogenic vectors and the large-scale production could make clinical renal gene therapy a realistic possibility for the near future.

Animals↗

PubMed2000

Detection of angiographically significant coronary artery disease with accelerated intermittent imaging after intravenous administration of ultrasound contrast material.

BACKGROUND: Accelerated intermittent harmonic imaging (AII) is used to detect myocardial perfusion abnormalities after intravenous injection of ultrasound contrast medium. A low mechanical index and frame rates of 10 to 20 Hz are used to allow simultaneous wall motion analysis. The purpose of this study was to determine whether the myocardial contrast enhancement achieved with AII can be used to detect angiographically significant coronary artery disease during stress echocardiography. METHODS: We gave intravenous perfluorocarbon containing microbubbles to 45 patients (total of 270 regions) during dobutamine (n = 27) or exercise (n = 18) stress testing with AII. Quantitative angiography was performed on all patients after the stress echocardiograms were interpreted. RESULTS: Quantitative angiography showed >50% diameter stenosis of at least 1 vessel in 32 patients (total of 118 regions). There were visually evident contrast defects in 100 (85%) of these regions, and wall motion was abnormal in 64 (54%). Overall, there was agreement between regional perfusion and quantitative angiographic findings in 217 of the 270 regions (kappa = 0.61; 80% agreement). Agreement with findings at quantitative angiography was good for both dobutamine stress (kappa = 0.66; 83% agreement) and exercise (kappa = 0.53; 77% agreement). The greatest incremental benefit of AII versus wall motion was gained during dobutamine stress. The contrast studies depicted 90% of the regions supplied by a vessel with >50% stenosis, whereas wall motion depicted only 32% (P =.001). CONCLUSIONS: The results of this study indicated that accelerated intermittent perfusion imaging during stress echocardiography can improve the sensitivity of the study in detecting angiographically significant coronary artery disease, especially during dobutamine stress.

Adult↗

PubMed1996

Myocardial contrast echocardiography: reliable, safe, and efficacious myocardial perfusion assessment after intravenous injections of a new echocardiographic contrast agent.

Reliable and reproducible myocardial opacification after intravenous administration of echocardiographic contrast agents has remained elusive. This study was performed to determine whether a new agent, FS069, a suspension of perfluoropropane-filled albumin microspheres (3.6 microns average microbubble size, concentration 8 x 8(8)/ml), could achieve safe and successful myocardial opacification in open-chest dogs. Seventeen dogs (group 1, n = 7, group 2, n = 10) underwent two-dimensional echocardiography before, during, and after the administration of intravenous FS069. Safety was evaluated by measuring arterial and pulmonary artery pressures, heart rate, blood gases, systolic function, myocardial blood flow, and postmortem analysis of myocardial viability by triphenyl-tetrazolium chloride staining. Efficacy to detect changes in regional myocardial perfusion was assessed by injecting FS069 at baseline, after sequential coronary occlusions and reperfusion, and during intravenous vasodilators with and without coronary occlusions. Results were compared with radiolabeled microspheres. FS069 was found to be safe and effective. In the absence of coronary occlusions, uniform myocardial opacification was observed in all dogs. A perfusion defect was observed in all dogs during coronary occlusions. Background-subtracted peak contrast intensity in the myocardium correctly identified regional myocardial blood flow changes and showed a significant correlation with radiolabeled microspheres (r = 0.65, p = 0.0001).

Albumins↗

PubMed1996

Detection of regional perfusion abnormalities during adenosine stress echocardiography with intravenous perfluorocarbon-exposed sonicated dextrose albumin.

Although perfluorocarbon-exposed sonicated dextrose albumin (PESDA) microbubbles produce myocardial contrast after intravenous injection, it is unknown whether their use can accurately identify myocardial blood-flow abnormalities during stress echocardiography. Accordingly, we compared the background-subtracted peak myocardial videointensity (PMVI) after intravenous injections of PESDA before and during adenosine stress (100 to 140 units/kg/min) in 10 open-chest dogs with angiographically significant left circumflex artery disease. The ratios of PMVI in the ischemic region compared with the adjacent normal left anterior descending perfusion bed were measured, as were wall-thickening and coronary-flow ratios. In the dogs with a >50% diameter stenosis, there was a decrease in PMVI ratio during adenosine stress by >0.20 in 9, whereas wall-thickening ratios decreased in only 5. PMVI in the ischemic zone increased by <1.5 units during adenosine infusion in 8 of 10 dogs, whereas it increased by >1.5 units in 8 of 1O adjacent normal zones. We conclude that regional myocardial-perfusion abnormalities can be detected with intravenous PESDA during adenosine stress echocardiography.

Adenosine↗

PubMed1999

Clinical applications of transpulmonary contrast echocardiography.

BACKGROUND: The recent development of new fluorocarbon-based echocardiographic contrast agents that are capable of opacification of the left-sided cardiac chambers after intravenous injection is a major new advance in diagnostic cardiac imaging. METHODS AND RESULTS: This is a review article focusing on these novel contrast agents, new echocardiographic imaging techniques to optimize their efficacy, and their clinical applications. Specific clinical applications of these agents are (1) enhancement of endocardial border definition to improve assessment of regional and global left ventricular function, (2) myocardial perfusion imaging by intravenous contrast echocardiography, (3) augmentation of spectral and color flow Doppler images, and (4) tissue-specific targeting of microbubbles for delivery of therapeutic agents. CONCLUSIONS: New intravenous contrast agents offer the possibility to assess myocardial perfusion echocardiographically. It is also possible to use these agents for delivery of therapeutic agents, including gene therapy.

Albumins↗