Classifi cation and Safety of Microbubble-Based Contrast Agents 3 1 Classification and Safety of Microbubble-Based Contrast Agents Emilio Quaia CONTENTS injection, which requires that microbubbles have a diameter smaller than 8–10 µm. 1.1 Introduction 3 Free air-filled microbubbles exhibited very lim- 1.2 Chemical Composition and Classification of Microbubble-Based Contrast Agents 3 ited persistence and efficacy, while aqueous solu- 1.2.1 Carbon Dioxide Microbubbles 5 tions, colloidal suspensions, and emulsions did not 1.2.2 Air-Filled Microbubbles 5 meet with the required efficacy and safety profile 1.2.4 Sulphur Hexafluoride-Filled Microbubbles 11 compatible with US. The physical and chemical 1.3 Pharmacokinetics and Clearance properties of the more recently introduced micro- of Microbubble-Based Contrast Agents 11 1.4 Orally Administered US Contrast Agents 12 bubble-based contrast agents are superior to those 1.5 Safety of Microbubble-Based Contrast Agents of the initial agents, and stabilized microbubbles in Humans 12 offer both excellent stability and safety profiles, as References 13 well as acceptable efficacy. Microbubble-based agents are injectable intra- venously and pass through the pulmonary capil- 1.1 lary bed after peripheral intravenous injection, Introduction since their diameter is below that of red blood cells (Fig. 1.1). Microbubble-based agents are isotonic The use of microbubble-based ultrasound (US) con- to human plasma and are eliminated through the trast agents is not a recent development in radiol- respiratory system. With the advent of the new-gen- ogy. The application of microbubbles to increase eration of perfluorocarbon or sulphur hexafluo- the backscattering of blood was firstly described ride-filled microbubbles, the duration of contrast in 1968 (Gramiak and Shah 1968) when contrast enhancement has increased up to several minutes phenomena in the aorta during cardiac catheter- which provides sufficient time for a complete study ization following injection of saline solution were of the vascular bed using slow bolus injections or observed. This was caused by air microbubbles pro- infusions. duced by cavitation during the injection of the solu- tion (Bove and Ziskin 1969; Kremkau et al. 1970). From that time on, enormous efforts were dedicated to developing clinically relevant microbubble-based 1.2 US contrast agents. Chemical Composition and Classification The first problem was the low stability of air-filled of Microbubble-Based Contrast Agents microbubbles in the peripheral circulation, and in the high-pressure environment of the left ventricle, Microbubble-based contrast agents may be defined which was progressively solved by the introduction as exogenous substances which can be adminis- of more stable bubbles covered by galactose-pal- tered, either in the bloodstream (Kabalnov et al. mitic acid or a phospholipid shell. The second prob- 1998a and 1998b) or in a cavity, to enhance ultra- lem was to make the microbubbles capable of pass- sonic backscattered signals (de Jong et al. 1992; ing through the lung circulation after intravenous Forsberg and Tao Shi 2001). Moreover, micro- bubbles which are prepared to be injected intrave- E. Quaia, MD nously must be distinguished from oral compounds Assistant Professor of Radiology, Department of Radiology, which are employed to remove the interposing bowel Cattinara Hospital, University of Trieste, Strada di Fiume 447, gas limiting the evaluation of organ parenchymas 34149 Trieste, Italy (Goldberg et al. 1994). 4 E. Quaia low diffusion coefficient. Microbubble-based con- trast agents are encapsulated (Fig. 1.2) or otherwise stabilized using a sugar matrix, such as galactose, or microspheres with albumin, lipids (Fig. 1.3), or polymers. The shell is also designed to reduce gas diffusion into the blood and may be stiff (e.g. denatured albumin) or more flexible (phospho- lipid), while the shell thickness may vary from 10 to 200 nm. Low-solubility and low-diffusibility gases, such as perfluorocarbons and sulphur hexafluoride gas (Fig. 1.3), have also been found to dramatically improve microbubble persistence in the peripheral circle. Microbubbles may be filled by air, perfluoro- carbon or sulphur hexafluoride inert gas. The ideal Fig. 1.1. Two-dimensional microscopic photo of SonoVue filling gas should be inert and should present a high (white arrows) microbubbles (20× magnifi cation; optical vapour pressure and the lowest solubility in blood. microscope) compared to red blood cells (black arrows). Air presents high solubility in blood, while perfluo- (Image courtesy of Peter JA Frinking, PhD, Bracco Research, Geneva, Switzerland) rocarbon and sulphur hexafluoride gases present a low diffusibility through the phospholipid layer and a low solubility in blood allowing a longer persis- The physical properties of microbubble-based tence in the bloodstream. The limited solubility in contrast agents are closely related to their gas con- blood determines an elevated vapour concentration tent and shell composition, besides the frequency in the microbubble relative to the surrounding blood of the US beam, the pulse repetition frequency and and establishes an osmotic gradient that opposes the employed acoustic power of insonation. At high the gas diffusion out of the bubble. The stability of a acoustic power, the microbubbles are disrupted microbubble in the peripheral circle is related to the releasing a large amount of acoustic energy rich in osmotic pressure of filling gas which counters the harmonic components. At low acoustic power, spe- sum of the Laplace pressure (surface tension) and cific pulse sequences (Meuwl et al. 2003; Shen and blood arterial pressure (Chatterjee and Sarkar Li 2003) driving the microbubbles to resonance are 2003). applied for real-time imaging, producing harmonic frequencies which may be selectively registered. Microbubble-based contrast agents (diameter 3–10 µm) are smaller than red blood cells (Fig. 1.1) and are composed by a shell of biocompatible mate- rial such as a protein, lipid or polymer. The ideal microbubble contrast agents should be inert, intra- venously injectable, by bolus or infusion, stable during cardiac and pulmonary passage, persisting within the blood pool or with a well-specified tissue distribution, provide a duration of effect comparable to that of the imaging examination, have a narrow distribution of bubble diameters and respond in a well-defined way to the peak pressure of the inci- dent US. Nowadays, only a few microbubble-based contrast agents have been approved for human use, even though this number may soon increase as sev- eral agents are currently undergoing the approval procedure. Two principal ways were developed to increase Fig. 1.2. Scanning-electron micrograph photo of SonoVue microbubble stability and persistence in the periph- microbubbles which represents the variability in microbubble eral circle: external bubble encapsulation with or diameter. (Image courtesy of Peter JA Frinking, PhD, Bracco without surfactants and selection of gases with Research, Geneva, Switzerland) Classifi cation and Safety of Microbubble-Based Contrast Agents 5 including mixing times, volume ratio of gas and liquid and the species of gas and liquid (Matsuda et al. 1998). Blood increases the surface tension and viscosity of solution with an increased stability and smaller diameter of microbubbles. Carbon dioxide microbubbles were employed to detect hepatocellu- lar carcinomas and to characterize focal liver lesions after injection through a catheter placed within the hepatic artery as for selective hepatic angiography (Kudo et al. 1992a,b). Carbon dioxide microbubbles are readily cleared from the lungs. Fig. 1.3. Scheme of a SonoVue microbubble with the periph- 1.2.2 eral phospholipids monolayer fi lled by sulphur hexafl uoride Air-Filled Microbubbles (SF6) gas. (Image courtesy of Peter JA Frinking, PhD, Bracco Research, Geneva, Switzerland) 1.2.2.1 The different microbubble-based contrast agents Air-Filled Microbubbles with a Galactose Shell (see also Wheatley 2001) are reported in Tables 1.1– 1.3. Different microbubble-based contrast agents 1.2.2.1.1 (Figs. 1.4 and 1.5) present different methods of Echovist preparation according to their different composi- tion (Figs. 1.6–1.8). Echovist (SH U454; Schering, Berlin, Germany) was the first microbubble-based contrast agent marketed in Europe in 1991. Echovist was approved for echo- 1.2.1 cardiography to opacify right heart cavities and to Carbon Dioxide Microbubbles detect cardiac shunts. The air-filled microbubbles are stabilized within a galactose matrix corresponding to Carbon dioxide microbubbles are prepared by vig- the peripheral shell. Even though the mean diameter orously mixing 10 ml of carbon dioxide, 10 ml of of these microbubbles is approximately 2 µm with heparinized normal saline and 5 ml of patient’s a relatively narrow size distribution (97% < 6 µm), blood (Kudo et al. 1992a,b). The size and the density Echovist stability is not sufficient to allow the micro- of gas microbubbles are affected by many factors, bubbles to cross the lungs after a peripheral intrave- Table 1.1. Microbubble-based ultrasound contrast agents for intravenous injection Trademark name Code name Manufacturer Formulation: shell/filling gas Albunex Mallinckrodt Human albumin/air Bisphere PB127 Point Biomedical Polymer bilayer – albumin/air Definity MRX-115 Bristol-Myers Squibb Phospholipid/perfluoropropane DMP-115 Echogena QW3600 Sonus Pharmaceutical Surfactantdodecafluoropentane Echovist