1 Classification and Safety of Microbubble-Based Contrast Agents Emilio Quaia

Home , Perflubron

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 intravenously 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 solution (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, microbubbles 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 contrast 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 (phospholipid), 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, perfluorocarbon 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 material such as a protein, lipid or polymer. The ideal microbubble contrast agents should be inert, intravenously 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 several 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 hepatocellular 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 composition (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 SH U454 Schering Galactose/air Filmix Cavcon Lipid/air Imavist (Imagent) AFO150 Imcor Pharmaceuticals Surfactant/perfluorohexane-air Levovist SH U508A Schering Galactose-palmitic acid/air Myomap AIP 201 Quadrant Healthcare Recombinant albumin/air Optison FS069 Amersham Health Inc., Perflutren protein-type A/perfluoro- Princeton, NJ butane Perflubron PFOB Alliance pharmaceuticals Perfluorooctyl bromide

Quantison Quadrant Healthcare Recombinant albumin/air Sonavista SH U563A Schering Polymer/air Sonazoid NC100100 Amersham Health Lipid/perfluorobutane SonoGen QW7437 Sonus Pharmaceuticals Surfactant/dodecafluoropentane SonoVue BR1 Bracco Phospholipid/sulphur hexafluoride a Withdrawn from the market 6 E. Quaia

Table 1.2. Ultrasound contrast agents for oral administration Table 1.3. Microbubble-based ultrasound contrast agents under development Trade name Manufacturer Formulation Code Name Manufacturer Formulation: shell/gas SonoRx ImaRx Pharmaceuticals Simethicone-coated cellulose AI-700 Acusphere Polymer/perfluorocarbon- Oralex Molecular Biosystems Polydextrose solution nitrogen BR14 Bracco Phospholipid/perfluorobutane BY 963 Byk-Gulden Lipid/air PESDA Porter Albumin/perfluorocarbon nous injection. This property of Echovist has been MP1550 Mallinckrodt Lipid/perfluorobutane employed to detect cardiac and extracardiac right-to- MP1950 Mallinckrodt Phospholipid/decafluorobutane left shunts that predispose to paradoxical embolism MP2211 Mallinckrodt Lipid/perfluorobutane (Droste et al. 2004a,b) with Doppler interrogation of MRX-408 ImaRx Pharma- Oligopeptide/perfluoropropane ceuticals the mean cerebral artery. The other use of Echovist SH U616A Schering Galactose/air is in hysterosalpinx contrast-sonography to assess tubal patency (Campbell et al. 1994).

1.2.2.1.2 Levovist

Levovist (SH U508 A; Schering AG, Berlin, Germany) was the first microbubble-based contrast agent approved in Europe and Canada for radiology applications, and nowadays is licensed for use in more than 60 countries worldwide. Levovist is available in vials of 2.5 and 4 g of galactose and 1 g of sterile powder (Fig. 1.4) contains 999 mg of galactose and 1 mg of palmitic acid. Concentrations of 200, 300, and 400 mg are reconsti- tuted by adding specific amounts of sterile water to the galactose powder followed by vigorous shaking of the vial. The 200-mg concentration is recommended for contrast-enhanced transcranial Doppler studies. The 300-mg concentration provides sufficient Doppler signal enhancement for most other applications. Both 300- and 400-mg concentrations can be used with non- Fig. 1.4. Package of the commercial agent Levovist (Schering, linear imaging sequences to enhance the echostructure Berlin, Germany) consisting in air-fi lled microbubbles covered of several organs (liver, kidney, heart). by galactose and palmitic acid shell. Sterile saline solution (A), When the sugar matrix dissolves within the lyophilisate powder (B), plastic vial (C) and syringe (D) plasma, the microbubbles are released and coated by a thin monolayer of palmitic acid (Frinking 1999). Levovist is characterized by air-filled microbubbles with a mean diameter of 2–3 µm and with 99% of microbubbles smaller than 7 µm covered by biodegradable galactose and palmitic acid shell. Palmitic acid is a fatty acid which increases the stability of the microbubbles to allow multiple recirculations. Microbubbles are stable enough to pass through capillary beds and produce systemic enhancement of Doppler signals for 1–5 min (Correas et al. 2001; Harvey et al. 2001). Microbubbles can be administered after a short resting period of 2 min. After blood pool clearance, Levovist has been shown to have a late hepatosplenic-specific parenchy- Fig. 1.5. Package of the commercial microbubble-based agents SonoVue (Bracco, Milan, Italy). Lyophilisate powder (A), plas- mal phase and can accumulate within the liver and the tic vial (B), piston of the syringe (C) and syringe fi lled with spleen up to 20 min after intravenous injection once sterile saline solution (D) Classifi cation and Safety of Microbubble-Based Contrast Agents 7

a b

c d

Fig. 1.6a–d. Method of preparation of SonoVue: vial preparation. The piston of the syringe is screwed on the plastic support (a). The lyophilisate powder containing phospholipids is laid on the bottom of the bottle (b). The syringe is screwed on the plastic connection (c). The plug of the vial is removed (d)

it has disappeared from the blood pool (B lomley and Dittrich 1997). The mean diameter is 3.8 µm et al. 1998; Kitamura et al. 2002; Maruyama et al. with a standard deviation of 2.5 µm; however, the 2004). The underlying mechanism of the selective distribution of the microbubble population is quite late uptake of Levovist by hepatic and splenic paren- large. Albunex microbubbles are very sensitive to chyma is not fully understood. One possible explana- pressure changes and their half-life is very short tion is that the accumulation may be mediated by the (<1 min). After an intravenous peripheral injection, reticuloendothelial system (Hauff et al. 1997; Kono the microbubbles can pass through the pulmonary et al. 2002; Quaia et al. 2002), or that microbubbles capillary bed and reach the left ventricle. are entrapped in the liver sinusoids. 1.2.2.2.2 1.2.2.2 Quantison Air-Filled Microbubbles with Albumin Shell Quantison (Quadrant Ltd, Nottingham, UK) consists 1.2.2.2.1 of air-filled microbubbles encapsulated by a relatively Albunex thick (200–300 nm) and rigid shell of recombinant albumin. Imaging demonstrated that the liver was the Albunex (developed by Molecular Biosystems, organ with the highest uptake, with a mean uptake of San Diego, CA, USA; distributed by Mallinckrodt, 41.8% (SD 10.4%) of the administered dose 1 h follow- St Louis, Mo, USA) was the first transpulmonary ing intravenous administration (Perkins et al. 1997). microbubble-based agent which reached the market in 1993 but it is not longer in production. Albunex 1.2.2.2.3 is produced by sonicating 5% of human albumin to Myomap obtain air-filled microbubbles stabilized with a thin shell of human albumin of 3–5 µm. The microbubble Myomap (AIP 201, Quadrant Ltd, Nottingham, UK) concentration is 3–5×108 microspheres/ml (Killam consists of an air-filled microbubbles encapsulated 8 E. Quaia

ab c

def

Fig. 1.7a–f. Method of preparation of SonoVue: mixture of the powder with saline solution. The plastic connection is pressed on the bottle (a,b). Lyophilisate powder is mixed with water and the mixture has to be shaken for about 10 s (d,e). The obtained milky suspension of microbubbles has to remain at rest for about 1 min to promote macrobubbles breaking up (f) by recombinant albumin shell which is more than other microbubble enhancers, the microspheres of three times thicker (600–1000 nm) than the Quan- SH U563A circulate in the blood pool intact for up tison shell (Frinking 1999). The microbubble mean to 10 min after intravenous injection. The particles size is 10 µm (range 1.46–23.5 µm). are eventually taken up by the reticuloendothelial system during the late phase (Hauff et al. 1997; 1.2.2.3 Bauer et al. 1999), principally the Kupffer cells of Air-Filled Microbubbles with Cyanoacrylate Shell the liver, which gives them a diagnostic potential similar to that of microparticles of Iron oxide. 1.2.2.3.1 Sonavist 1.2.3 Perfluorocarbon-Filled Microbubbles Sonavist (SH U563A, Schering AG, Berlin, Ger- many) consists of air-filled microspheres with a 1.2.3.1 mean diameter of 2 µm (Bauer et al. 1999) pro- Perfluorochemical duced by emulsion polymerization. The shell of the microspheres is formed by a 100-nm thick layer of Perfluorochemicals (perflubron emulsion; Alliance a biodegradable n-butyl-2-cyanoacrylate polymer. Pharmaceutical Corporation, San Diego, USA) are The microspheres are pre-formed as a powdery sub- inert compounds with a low surface tension which stance which is suspended by shaking in physiologi- are immiscible with water and can be intravenously cal saline for a few seconds before injection. This injected if emulsified (Mattrey and Pelura 1997). suspension is isotonic and remains stable in the vial Perfluorochemicals accumulate in human tissues for several hours. Unlike free gas bubbles and most when inhaled, ingested or given intravenously. Classiﬁ cation and Safety of Microbubble-Based Contrast Agents 9

a b

c d e f

Fig. 1.8a-f. Method of preparation of SonoVue and microbubble injection. The milky microbubble suspension is withdrawn from the bottle (a) and still has to be gently shaken before injection to avoid microbubble sedimentation (b). A three-way stop-cock (c) should be employed and connected to a 16–18 Gauge intravenous cannula. The left arm is preferable since this does not hamper liver scanning during microbubble injection. The ﬁ rst port is connected to the intravenous cannula, the second to the syringe with microbubbles and the third to the syringe with saline solution (d). Needles should be avoided since microbubbles may be destroyed at the end of the needle during injection for the turbulence by Venturi phenomenon. Microbubbles are than injected (e) followed by saline ﬂ ush (f) to avoid microbubble persistence in the vial and in the vein

Perfluorooctyl bromide (perflubron) is a liquid ticles within each macrophage results in an aggre- perfluorocarbon emulsion with particle size rang- gate capable of reflecting US. ing from 0.06 to 0.25 µm which is composed of From the observation that liquid perfluorocar- carbon and bromine atoms. Perfluorooctyl bro- bon vapour in a contained space expanded the mide circulates in blood with a half-life of hours space by attracting air because of an osmotic gra- and is an effective microbubble-based contrast dient and differential partial pressure, the develop- agent which is also radiopaque at plain film X-ray ment of microbubbles that already contained per- and computed tomography. Perfluorochemical fluorocarbon vapour and surviving to circulation emulsions act as simple scatterers since they pres- was proposed (Mattrey et al. 1994; Schutt et al. ent a higher density (1.9 g/ml) and a lower acoustic 2003). velocity (600 m/s) than tissues with a difference in acoustic impedance of about 30% (Andre et al. 1.2.3.2 1990; Mattrey and Pelura 1997). Perfluorooctyl Perfluorocarbon-Filled Microbubbles bromide accumulates in the reticuloendothelial with a Phospholipid Shell cells and leaks from inflammatory or tumoral capil- laries into the interstitial space where the emulsion Perfluorocarbon-filled microbubbles are currently particles are phagocytosed by local macrophages the most important research field in microbubble- (Mattrey et al. 1982; Mattrey and Aguirre based agents development. There are at least five 2003). The accumulation of a large number of par- different perfluorocarbon-filled agents approved 10 E. Quaia for cardiac imaging in the US and at least three ment of Imavist is likely not related to Kupffer cell agents approved for non-cardiac imaging in Europe uptake, but rather to a mechanical slowdown within (Mattrey and Aguirre 2003). Development efforts liver sinusoids (Kono et al. 2002). have also focused on targeting liquid perfluorocarbon emulsion and microbubbles to clots and acti- 1.2.3.2.4 vated endothelial cells becoming visible only if Sonazoid attached to their target (Lanza et al. 1996). Sonazoid (NC100100, Amersham Health, Oslo, 1.2.3.2.1 Norway) (Marelli 1999) consists of lipid-coated BR14 microbubbles containing perfluorocarbon and within a well-defined size range (median diameter of approx- BR14 (Bracco Research, Geneva, Switzerland) is a imately 3 µm). Sonazoid is prepared as an easy-to- phospholipid-stabilized third-generation US con- use, non-toxic formulation. At electron microscopy, trast agent (Schneider et al. 1997) that produces Sonazoid was revealed to be exclusively internalized persistent contrast enhancement of tissue perfu- in Kupffer cells during late phase (Marelli 1999; sion. This persistent contrast enhancement has been Forsberg et al. 2002; Kindberg et al. 2003). attributed to its transient retention in the liver tissue and spleen microcirculation (Basilico et al. 2002; 1.2.3.3 Fisher et al. 2002). Perfluorocarbon-Filled Microbubbles with Albumin Shell 1.2.3.2.2 Definity Albumin is employed in perfluorocarbon-filled agents to further increase microbubble stability. Definity (MRX 115, DMP115, Bristol-Myers Squibb Medical Imaging, North Billerica, MA) contains 1.2.3.3.1 octafluoropropane (perflutren)-filled phospholipid Optison microbubbles coated with a single layer of phospholipids and with a mean diameter of 2.5 µm (Unger Optison (FS069; developed by Molecular Biosystems et al. 1997; Maruyama et al. 2000, 2003, 2004). The Inc., San Diego, CA, USA; distributed by Amersham microbubbles are formed after a 45-s mechanical Health Inc., Princeton, NJ) consists of perfluorobu- shaking and may be injected. The vial contains a tane (perflutren)-filled microbubbles coated by 15- clear, colourless, sterile, non-pyrogenic, hypertonic nm thick human albumin shell which are prepared liquid which, upon activation, provides a homoge- directly in solution. The vial contains a clear liquid neous, opaque, milky white injectable suspension of lower layer and a white upper layer that, after resus- perflutren lipid microspheres. pension by gentle mixing, provides a homogeneous, opaque, milky-white suspension for intravenous injec- 1.2.3.2.3 tion. This microbubble-based agent should be kept in Imavist or Imagent refrigerated and shaken before use. The mean diameter of the microbubbles ranges from 1.0 to 2.25 µm, Imagent (AFO-150; Imcor Pharmaceutical, San with 93% less than 10 µm, and the mean concentra- Diego, CA, USA) consists of a lipid-shell micro- tion ranges from 5 to 8×108 microspheres per mil- bubble containing perfluorohexane gas. The micro- lilitre. No immune reaction has been associated with spheres are composed by water-soluble structural the presence of the human serum albumin. Optison agents, surfactants, buffers and salts (Mattrey was recently approved in Europe, Canada, and the US and Pelura 1997). After reconstitution with sterile for cardiac applications in the case of inconclusive water, a suspension of perfluorohexane-filled micro- echocardiography to provide opacification of cardiac bubbles with a surfactant membrane is formed. The chambers and to improve left ventricular endocardial perfluorohexane presents a very low solubility in border delineation. The recommended dose varies blood and this improves microbubble stability. Like from 0.5 to 3.0 ml. Human albumin shell-covered Levovist, Sonavist and Sonazoid, Imavist showed a microbubbles have been shown to be captured and late hepato-specific phase 3–5 min after injection, phagocytosed by activated neutrophils while their suggesting a specific liver entrapment (Kono et al. acoustic properties for ultrasound are preserved 2002). However, the late liver parenchymal enhance- (Lindner et al. 2000). Classiﬁ cation and Safety of Microbubble-Based Contrast Agents 11

1.2.3.4 density is 2×108 microbubbles per millilitre (mean Phase Shift Perfluorocarbon-Filled Microbubbles diameter 3 µm, 90% of the microbubbles <8 µm). The microbubbles are stabilized with several sur- Phase shift transition is a phenomenon in which the factants, such as polyethylene glycol, phospholipids material changes physical form, such as from liquid and palmitic acid, and are stable in the vial for a to gas (Correas et al. 1997). few hours (<6 h). However, after standing for more than 2 min buoyancy causes microbubbles to rise to 1.2.3.4.1 the surface and the vial has to be gently agitated in EchoGen a top-to-bottom manner to obtain a homogeneous suspension before intravenous injection (Fig. 1.8). EchoGen (QW3600; produced by Sonus Pharmaceu- SonoVue shows an elimination half-life of 6 min and ticals, Bothell, WA, USA) is a perflenapent liquid-in- more than 80% of the compound is exhaled through liquid emulsion which contains dodecafluoropen- the lungs in 11 min (Morel et al. 2000). tane liquid in the dispersed phase which shifts to Recently SonoVue obtained European approval a gas phase at body temperature forming micro- for both cardiac and liver applications. The advan- bubbles of 3–8 µm in diameter. Dodecafluoropen- tages of sulphur hexafluoride-filled compared to tane is a perfluorocarbon gas with a low boiling air-filled microbubbles is the high and prolonged point (28.5°C), low diffusibility, and low solubility in stability in the peripheral blood, due to the low solu- plasma. The emulsion contains particles with a mean bility of the gas and to stability of the phospholipids diameter of approximately 0.4 µm (Correas et al. shell, and the uniformity of the microbubble diam- 1997, 2001). Following intravenous administration, eter which improves the backscattering and har- microdroplets form a distribution of microbubbles monic behaviour at low acoustic power insonation of dodecafluoropentane with a mean diameter of (Gorce et al. 2000). 2–5 µm. The dodecafluoropentane microbubbles persist in solution much longer than similar sized microbubbles of air. The phase transition from liquid to gas state is achieved by producing a hypo- 1.3 baric pressure followed by an intense shock within Pharmacokinetics and Clearance the syringe immediately prior to administration or of Microbubble-Based Contrast Agents by injecting the emulsion as a bolus through a filter which induces a drop in pressure (Correas et al. After preparation of microbubble solution, it is 1997, 2001). EchoGen obtained a European approval always advisable to perform microbubble injection for cardiac indications but was withdrawn from the via a flexible venous indwelling cannula (Fig. 1.8) market in 2000 by Sonus Pharmaceuticals. of sufficiently large calibre (18 Gauge). An imme- diate after-injection flush of about 5–10 ml physiological saline solution is also advisable to wash 1.2.4 out microbubbles remaining in the cannula and in Sulphur Hexafluoride-Filled Microbubbles the proximal vein tract after injection. The use of a three-way tap open to all sides is recommended so 1.2.4.1 that the saline solution can be injected without delay SonoVue (Fig. 1.8). Microbubble-based contrast agents may be injected as a bolus or as a slow infusion. SonoVue (BR1, Bracco imaging, Milan, Italy) is a Bolus injection is simple to perform even though sulphur hexafluoride-filled microbubble contrast the increase in backscattering is brief. For bolus agent encapsulated by a flexible phospholipid shell injection, time-intensity curve exhibits a rapid first which is prepared as a lyophilisate powder (Fig. 1.5) pass followed by a slower washout and contrast (Schneider et al. 1995; Morel et al. 2000; C orreas enhancement exhibits a linear relation with the dose et al. 2000, 2001). A white, milky suspension of (Correas et al. 2000). The principal drawback of sulphur hexafluoride microbubbles is obtained by bolus injection is the possible presence of artefacts adding 5 ml of physiological saline (0.9% sodium during the high peak value of microbubbles. chloride) to the powder (25 mg), using standard In slow infusion microbubble administration clinical aseptic techniques, followed by hand agita- the enhancement is stable with a plateau-like pat- tion (Figs. 1.6, and 1.7). The obtained microbubble tern from 1 to 2 min from injection (Correas et al. 12 E. Quaia

2000). Slow infusion may be performed by a dedi- 1.4 cated automatic injector and it is mandatory in the Orally Administered US Contrast Agents quantitation of parenchymal perfusion since station- ary levels of microbubbles are necessary. Infusion US imaging of the abdomen often is compromised of microbubble-based agents is easily achieved and by artefacts due to adjacent bowel gas since the US allows the duration of enhancement to be increased beam is almost completely reflected when the acous- as long as desired. tic interface bowel gas–parenchyma is encountered. After intravenous injection microbubble-based Orally administered US contrast agents (Table 1.2) contrast agents present a pure intravascular distri- were introduced to reduce bowel gas interposition bution in the peripheral circle and are defined blood between the US beam and the parenchymal organs pool agents. After this preliminary vascular phase by filling the bowel lumen with a transonic solu- tissue specific agents are defined, some agents, such tion. Different attempts to decrease gas artefacts and as Levovist, Sonavist and Sonazoid, present a late improve US image quality were performed with poor hepatosplenic-specific phase (Hauff et al. 1997; results since a high inter-individual variability was Blomley et al. 1998; Bauer et al. 1999; Forsberg found. Recently, a simethicone-coated cellulose sus- et al. 1999, 2000, 2002; Quaia et al. 2002). This phe- pension, called SonoRx (ImaRx Pharmaceuticals, nomenon is not completely understood but is prob- Tucson/Bracco Princeton, NJ, USA) was introduced ably determined by the adherence and selective with improvement of the visualization of bowel and pooling of the microbubbles in the hepatic sinusoids abdominal anatomy with reduction of gas artefacts or by the selective uptake from the circulation by (Lund et al. 1992). phagocytic cells of the reticuloendothelial system in the liver and spleen (Walday et al. 1994; Forsberg et al. 1999, 2000, 2002; Hauff et al. 1997; Quaia et al. 2002). 1.5 Typically, the gas content is eliminated through Safety of Microbubble-Based Contrast the lungs while the stabilizing components are fil- Agents in Humans tered by the kidney and eliminated by the liver. Perfluorocarbons and sulphur hexafluoride are In humans, microbubbles showed an excellent safety inert gases which do not undergo metabolism in the profile with no specific renal, liver or cerebral tox- human body and are exhaled, such as air, via the icity (Correas et al. 2001). The adverse reactions lungs after a few minutes. Sulphur hexafluoride is are rare, usually transient, and of mild intensity eliminated for 40%–50% of the injected gas volume (Claudon et al. 2000; Correas et al. 2001). A tran- 2 min after intravenous injection while 80%–90% is sitory sensation of pain, warmth or cold and tissue eliminated in 11 min (Morel et al. 2000). irritation may occur in the vicinity of the injection The phospholipids of the shell enter in the site or along the draining vein during or immedi- normal metabolism. The galactose-based micro- ately after administration. Due to the hyperosmo- bubbles are quickly dissolved as a result of the larity of microbubble solution, a transitory aspecific concentration gradient. The galactose becomes irritation of vessels endothelium may be observed. dispersed in the extracellular space and is sub- Individual cases of dyspnea, chest pain, hypo- or jected to the glucose metabolism. Galactose is hypertension, nausea and vomiting, taste altera- stored primarily in the liver through the forma- tions, headache, vertigo, warm facial sensation, tion of galactose-1-phosphate, or is metabolised general flush and cutaneous eruptions have been ott orreas and broken down to CO2 after isomerisation to described (R 1999; C et al. 2001). glucose-1-phosphate. If the plasma galactose level Short-lasting tingling, a feeling of numbness, sen- exceeds about 50 mg/100 ml and, therefore, the sations of taste and dizziness have been reported. elimination rate of the liver, galactose is eliminated No hypersensitivity reactions to the administration via the kidneys. The elimination rate in patients of microbubble have so far been reported (Correas with liver disease is about one third lower than et al. 2001). Even though the impaired cardiopul- in healthy subjects, in whom the plasma galactose monary function – including congestive heart fail- level falls by 10% per minute. Total clearance is ure (New York Heart Association Class II–IV) with about 40% lower in patients with liver disease. or without pulmonary hypertension, moderate or Galactose has a half-life of about 10–11 min in severe chronic obstructive pulmonary disease, and adults and of about 7–9 min in children. patients with diffuse interstitial pulmonary fibrosis Classiﬁ cation and Safety of Microbubble-Based Contrast Agents 13

– is not a contraindication for the administration of spectral Doppler US. Levovist Renal Artery Stenosis Study microbubble-based agents (Kitzman and Wesley Group. Radiology 214:739-746 2000; Correas et al. 2001), the benefits must be Claudon M, Tranquart F, Evans AH et al (2002) Advances in Ultrasound. Eur Radiol 12(1):7-18 weighed very carefully against the risk in this clini- Correas JM, Kessler D, Worah D, Quay SC (1997) The first cal situation. phase shift ultrasound contrast agent: EchoGen. In: Gold- Recently, general guidelines for the safe employ- berg BB (ed) Ultrasound contrast agents. Dunitz, London, ment of microbubble-based contrast agents were pp 101-120 proposed (Claudon and Jager 2004; Albrecht Correas JM, Burns PN, Lai X, Qi X (2000) Infusion versus Bolus of an ultrasound contrast agent: in vivo dose-response et al. 2004). These guidelines include an initial measurements of BR1. Invest Radiol 35:72-79 general chapter describing the fundamentals of Correas JM, Bridal L, Lesavre A et al (2001) Ultrasound con- microbubble-based contrast agents, paying special trast agents: properties, principles of action, tolerance, attention to safety, and will be subject to changes and artifacts. Eur Radiol 11:1316-1328 that reflect future advances in scientific knowledge de Jong N, Hoff L, Skotland T, Bom N (1992) Absorption and scat- ter of encapsulated gas filled microspheres: theoretical con- within the rapidly evolving field of US technology siderations and some measurements. Ultrasonics 30:95-103 (Claudon et al. 2002). The main part of the present Droste DW, Lakemeier H, Ritter M et al (2004a) The identifi- text details the guidelines recommended for the cation of right-to-left shunts using contrast transcranial evaluation of liver lesions (Albrecht et al. 2004) Doppler ultrasound: performance and interpretation and, in the next future, guidelines will be directed modalities, and absence of a significant side difference of cardiac micro-emboli. Neurol Res 26:325-330 to the employment of microbubble-based agents in Droste DW, Schmidt-Rimpler C et al (2004b) Right-to-left- the kidneys. shunts detected by transesophageal echocardiography and transcranial Doppler sonography. Cerebrovasc Dis 17:191-196 Fisher NG, Christiansen JP, Leong-Poi H et al (2002) Myocar- dial and microcirculatory kinetics of BR14, a novel third- References generation intravenous ultrasound contrast agent. J Am Coll Cardiol 39:530-537 Albrecht T, Blomley M, Bolondi L et al (2004) Guidelines for Forsberg F, Tao Shi W (2001) Physics of contrast microbubbles. the use of contrast agents in ultrasound. Ultraschall Med In: Goldberg B, Raichlen JS, Forsberg F (eds) Ultrasound 25:249-256 contrast agents: basic principles and clinical applications. Andre M, Nelson T, Mattrey RF (1990) Physical and acousti- Dunitz, London, pp 15-23 cal properties of perfluorooctyl bromide, an ultrasound Forsberg F, Goldberg BB, Liu JB et al (1999) Tissue specific contrast agent. Invest Radiol 25:983-987 US contrast agent for evaluation of hepatic and splenic Basilico R, Blomley MJ, Cosgrove DO (2002) The first phase I parenchyma. Radiology 210:125-132 study of a novel ultrasound contrast agent (BR14): assess- Forsberg F, Liu JB, Merton DA et al (2000) Gray scale second ment of safety and efficacy in liver and kidneys. Acad harmonic imaging of acoustic emission signals improves Radiol 9 [Suppl 2]:S380-S381 detection of liver tumors in rabbits. J Ultrasound Med Bauer A, Blomley MJK, Leen E, Cosgrove D, Schlief R (1999) 19:557-563 Liver-specific imaging with SHU 563 A: diagnostic poten- Forsberg F, Piccoli CW, Liu JB et al (2002) Hepatic tumor detec- tial of a new class of ultrasound contrast media. Eur tion: MR imaging and conventional US versus pulse-inver- Radiol 9 [Suppl 3]:S349-S352 sion harmonic US of NC100100 during its reticuloendo- Blomley MJK, Albrecht T, Cosgrove DO et al (1998) Stimu- thelial system-specific phase. Radiology 222:824-829 lated acoustic emission in liver parenchyma with Levovist. Frinking PJA (1999) Ultrasound contrast agents: acoustic Lancet 351:568-569 characterization and diagnostic imaging. Optima Graf- Bove A, Ziskin M (1969) Ultrasonic detection of in vivo cavita- ische Communicatie, Rotterdam, The Netherlands tion and pressure effects of high speed injection through Goldberg BB, Liu JB, Forsberg (1994) Ultrasound contrast catheters. Invest Radiol 3:236-241 agents: a review. Ultrasound Med Biol 20:319-333 Campbell S, Bourne TH, Tan SL, Collins WP (1994) Hysterosal- Gorce JM, Arditi M, Schneider M (2000) Influence of bubble pingo contrast sonography (HyCoSy) and its future role size distribution on the echogenicity of ultrasound con- within the investigation of infertility in Europe. Ultra- trast agents: a study of SonoVue. Invest Radiol 35:661- sound Obstet Gynecol 4:245-253 671 Chatterjee D, Sarkar K (2003) A Newtonian rheological model Gramiak R, Shah PM (1968) Echocardiography of the aortic for the interface of microbubble contrast agents. Ultra- root. Invest Radiol 3:356-366 sound Med Biol 29:1749-1757 Harvey CJ, Blomley MJK, Eckersley RJ, Cosgrove DO (2001) Claudon M, Jager KA (2004) It is time to establish guidelines Developments in ultrasound contrast media. Eur Radiol for the use of ultrasound contrast agents. Ultraschall Med 11:675-689 25:247-248 Hauff P, Fritsch T, Reinhardt M et al (1997) Delineation of Claudon M, Plouin PF, Baxter G et al (2000) Renal arteries experimental liver tumors in rabbits by a new ultrasound in patients at risk of renal arterial stenosis: multicenter contrast agent and stimulated acoustic emission. Invest evaluation of the echo-enhancer SH U 508A at color and Radiol 32:94-99 14 E. Quaia

Killam A, Dittrich HC (1997) Cardiac applications of Albunex behaviors of microbubble in the liver: time-related quan- and FS069. In: Goldberg BB (ed) Ultrasound contrast titative analysis of two ultrasound contrast agents, Levo- agents. Dunitz, London, pp 43-55 vist and Definity. Ultrasound Med Biol 30:1035-1040 Kindberg GM, Tolleshaug H, Roos N, Skotland T (2003) Hepatic Matsuda Y, Yabuuchi I, Ito T (1998) Properties of gas (CO2) clearance of Sonazoid perfluorobutane microbubbles by microbubbles made by hand agitation and it’s contrast Kupffer cells does not reduce the ability of liver to phago- enhancing effect. Nippon Rinsho 56:866-870 cytose or degrade albumin microspheres. Cell Tissue Res Mattrey RF, Aguirre D (2003) Advances in contrast media 312:49-54 research. Acad Radiol 10:1450-1460 Kitamura H, Kawasaki S, Nakajima K et al (2002) Correlation Mattrey RF, Pelura TJ (1997) Perfluorocarbon-based ultra- between microbubble contrast-enhanced color Doppler sound contrast agents. In: Goldberg BB (ed) Ultrasound sonography and immunostaining for Kupffer cells in assess- contrast agents. Dunitz, London, pp 83-99 ing the histopathologic grade of hepatocellular carcinoma: Mattrey RF, Scheible FW, Gosink BB et al (1982) Perfluorooc- preliminary results. J Clin Ultrasound 30:465-471 tyl bromide: a liver/spleen-specific and tumour-imaging Kitzman DW, Wesley DJ (2000) Safety assessment of perflena- ultrasound contrast material. Radiology 145:759-762 pent emulsion for echocardiographic contrast enhance- Mattrey RF, Wrigley R, Steinbach GC et al (1994) Gas emul- ment in patients with congestive heart failure or chronic sions as ultrasound contrast agents. Preliminary results in obstructive pulmonary disease. Am Heart J 139:1077- rabbits and dogs. Invest Radiol 29 [Suppl 2]:S139-S141 1080 Meuwl JY, Correas JM, Bleuzen A, Tranquart F (2003) Detec- Kabalnov A, Klein D, Pelura T et al (1998a) Dissolution of mul- tion modes of ultrasound contrast agents. J Radiol ticomponent microbubble in the blood stream: 1. Theory. 84:2013-2024 Ultrasound Med Biol 24:739-749 Morel DR, Schwieger I, Hohn L et al (2000) Human pharmaco- Kabalnov A, Bradley JA, Flam S et al (1998b) Dissolution of kinetics and safety evaluation of SonoVue™, a new contrast multicomponent microbubble in the blood stream: 2. agent for ultrasound imaging. Invest Radiol 35:80-85 Experiment. Ultrasound Med Biol 24:751-760 Perkins AC, Frier M, Hindle AJ et al (1997) Human biodis- Kono Y, Steinbach GC, Peterson T et al (2002) Mechanism of tribution of an ultrasound contrast agent (Quantison) parenchymal enhancement of the liver with a microbub- by radiolabelling and gamma scintigraphy. Br J Radiol ble-based US contrast medium: an intravital microscopy 70:603-611 study in rats. Radiology 224:253-257 Quaia E, Blomley MJK, Patel S et al (2002) Initial observations Kremkau FW, Gramiak R, Cartensen EL, Shah PM, Kramer H on the effect of irradiation on the liver-specific uptake of (1970) Ultrasonic detection of cavitation at catheter tips. Levovist. Eur J Radiol 41:192-199 AJR Am J Roentgenol 110:177-183 Rott HD (1999) Safety of ultrasonic contrast agents. European Kudo M, Tomita S, Tochio H et al (1992a) Sonography with Committee for Medical Ultrasound Safety. Eur J Ultra- intraarterial infusion of carbon dioxide microbubbles sound 9:195-197 (sonographic angiography): value in differential diagno- Schneider M, Arditi M, Barrau MB et al (1995) BR1 a new ultra- sis of hepatic tumors. Am J Roentgenol 158:65-74 sonographic contrast agent based on sulphur hexafluo- Kudo M, Tomita S, Tochio H et al (1992b) Small hepatocellular ride-filled microbubbles. Invest Radiol 30:451-457 carcinoma: diagnosis with US angiography with intraar- Schneider M, Broillet A, Bussat P et al (1997) Gray-scale liver terial CO2 microbubbles. Radiology 182:155-160 enhancement in VX2 tumor bearing rabbits using BR14, Lanza GM, Wallace KD, Scott MJ et al (1996) A novel site-tar- a new ultrasonographic contrast agent. Invest Radiol geted ultrasonic contrast agent with broad biomedical 32:410-417 application. Circulation 94:3334-3340 (erratum in Circu- Schutt EG, Klein DH, Mattrey RM, Riess JG (2003) Injectable lation 1997, 95:2458) microbubbles as contrast agents for diagnostic ultrasound Lindner JR, Dayton PA, Coggins MP et al (2000) Noninva- imaging: the key role of perfluorochemicals. Angew Chem sive imaging of inflammation by ultrasound detection of Int Ed Engl 42:3218-3235 phagocytosed microbubbles. Circulation 102:531-538 Shen CC, Li PC (2003) Pulse-inversion-based fundamental Lund PJ, Fritz TA, Unger EC et al (1992) Cellulose as a gastro- imaging for contrast detection. IEEE Trans Ultrason Fer- intestinal US contrast agent. Radiology 185:783-788 roelectr Freq Control 50:1124-1133 Marelli C (1999) Preliminary experience with NC100100, a new Unger E, Fritz T, McCreery T et al (1997) Lyposomes as myo- ultrasound contrast agent for intravenous injection. Eur cardial perfusion ultrasound contrast agents. In: Gold- Radiol 9 [Suppl 3]:S343-S346 berg BB (ed) Ultrasound contrast agents. Dunitz, London, Maruyama H, Matsutani S, Saisho H et al (2000) Grey-scale pp 57-74 contrast enhancement in rabbit liver with DMP115 at Walday P, Tolleshaug H, Gjoen T et al (1994) Biodistributions different acoustic power levels. Ultrasound Med Biol of air-filled albumin microspheres in rats and pigs. Bio- 26:1429-1438 chem J 199:437-443 Maruyama H, Matsutani S, Saisho H et al (2003) Extra-low Wheatley MA (2001) Composition of contrast microbubbles: acoustic power harmonic images of the liver with per- basic chemistry of encapsulated and surfactant-coated flutren. Novel imaging for real-time observation of liver bubbles. In: Goldberg B, Raichlen JS, Forsberg F (eds) perfusion. J Ultrasound Med 22:931-938 Ultrasound contrast agents: basic principles and clinical Maruyama H, Matsutani S, Saisho H et al (2004) Different applications. Dunitz, London, pp 3-11