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MRX 501: A novel ultrasound contrast agent with therapeutic properties
MRX 501: A Novel Ultrasound Contrast Agent with Therapeutic Properties 1 Evan C. Unger, MD, Tom McCreery, MS, Robert Sweitzer, BS George Vielhauer, BS, GuanLi Wu, PhD, DeKang Shen, PhD David Yellowhair, MSEE2
Ultrasound is used for both therapeutic and diagnostic purposes. Microbubbles are under development as ultrasound contrast agents (1). Not only do microbubbles reflect ultrasound, they also absorb the energy from ultrasound. Microbubbles are known to decrease the threshold of energy for cavitation (2,3). Prior studies have suggested that microbubbles can be used as cavitation nuclei for procedures such as lithotripsy and to potentiate ultrasonic hyperthermia (4-6). Recent studies have shown that diagnostic levels of ultrasound can cause bubble destruction and that bubble destruction can be visualized with ultrasound (7). The objective of this study was to develop a microbubble-based drug delivery system for use with ultrasound (Fig 1). Such a drug delivery system should function as a contrast agent allowing drug delivery to be visualized with ultrasound imaging. Using ultrasound energy, it should be possible to rupture the microbubbles and mediate local drug release.
An in vitro setup was designed to evaluate bubble bursting with ultrasound. The setup consisted of a Plexiglas tank filled with degassed water at 37°C. Soundabsorbing rubber material was placed on the bottom of the
Acad Radio11998; 5(suppl 1):$247-$249 1From the Department of Radiology, University of Arizona, 1501 N Campbell Ave, Tucson, AZ 85724-5067 (E.C,U,); and ImaRx Pharmaceutical, Tucson, Ariz (E.C.U., T,M,, R.S,, G.V,, G.W,, D,S., D,Y,), Address reprint requests to E.C.U. 2 Current address: Hughes Missile Systems, Tucson, Ariz. © AUR, 1998
Figure 1. Representation of gas-filled liposome containing a hydropic or amphilic drug, which is incorporated into the membrane stabilizing the microbubble.
tank. A 10-mm polyethylene tube was entered and exited through the ends of the tank. One end of the tubing was connected to a peristaltic pump (Cole-Parmer Masterflex; Cole-Parmer, Chicago, Ill), and an injection port was positioned proximal to the entry point of the polyethylene tubing into the tank. The distal end of the tubing passed into a bucket (after exiting the tank). Contrast agent was injected into the septum to flow through the polyethylene tubing. Two transducers were immobilized with ring stands and positioned with their faces in the water within the tank. The first transducer was a therapeutic 1-MHz continuouswave ultrasound transducer (Rich-Mar model 25; RichMar, Enola, Okla). The second transducer was a 7.5-MHz
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Figure 2. Schematic diagram of chemical structure of dexamethasone pro-drug, 1,2dipalmitoyl-sn-glycerol-3-succinate-dexamethasone.
curvilinear probe (model 5200; Acoustic Imaging [AI], Tempe, Ariz). Aerosomes, MRX 115 (ImaRx Pharmaceutical, Tucson, Ariz), was injected into the septum to flow within the polyethylene tubing and visualized within the tubing by ultrasound with the AI machine. The AI machine was operated at intermediate power settings, and the focal zone was positioned on the polyethylene tubing. Simultaneous imaging and application of continuous-wave ultrasound was performed. In these experiments, the high-energy ultrasound was applied with the 1-MHz transducer upstream or proximal to the imaging transducer. The duty cycle was varied from between 10% to 100% and the power wattage from 0.5 W/cm 2 to 2.0 W/cm 2. The effect of the high-energy ultrasound on the microbubbles was determined by visualizing the signal within the tubing distal to the highenergy transducer. Images were recorded on videotape. Sudan Black (Sigma, St. Louis, Mo) was entrapped into Aerosomes microbubbles. Sudan Black (3.2 mg) was added to vials containing the Aerosomes lipids (lipid concentration = 1 mg/mL). The head space of the vials contained perfluorobutane. The sealed vials were shaken on a Wig-L-Bug dental amalgamator (Crescent Dental, Lyons, Ill) for 60 seconds at 2,800 RPM. The resulting vesicles were sized on an Accusizer (Model 270; Nicomps, Santa Barbara, Calif). Stability of the Sudan Black within the vesicles was determined by dialyzing the vesicles against normal saline with 1,000 MW cutoff dialysis tubing (Spectrum, Los Angeles, Calif) for 12 hours at room temperature.
$248
In vivo studies were performed in mice (Balb/c, Harland Sprague Dawley, Indianapolis, Ind). The Sudan Black-containing Aerosomes were injected IV in the mice. There were four groups (three animals per group), control, no ultrasound, and ultrasound = 1 W/cm 2 and 2 W/cm 2 applied to the animal' s thigh muscle for a period of 60 seconds during infusion of the Sudan Black Aerosomes. A pro-drug of dexamethasone (DPGS-Dex) was synthesized (Fig 2). 1,2-Dipalmitoyl-sn-glycerol-3-succinate (DPGS) 0.32 g and dexamethasone (9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3,20-dione) 0.2 g and dimethylaminopyridine (DMAP) 10 mg were dissolved in chloroform, 30 mL, and added to a solution of dicyclohexyl carbodiimide (DCC) 0.11 g in chloroform, 10 mL, at 0°-5°C for 2 hours and stirred at room temperature overnight. 2% acetic acid was then added, stirred for 2 hours followed by isolation of the water phase (chemicals from Aldrich, Milwaukee, Wis). The organic phase was washed with 20 mL water and dried over anhydrous sodium sulfate. The organic solution was evaporated in a rotoevaporator, which resulted in a white residue. This residue was redissolved in acetonitrile. The small amount of precipitate was removed by filtration, and the solution was evaporated to dryness yielding 0.5 g of a white solid. The crude product was recrystallized from 10 mL methanol, resulting in a white crystal (m.p. 37°--40°). The activity of the pro-drug was compared to dexamethasone in an in vitro cytotoxicity assay in human multiple myeloma cells. DPGS-Dex was incorporated into the phospholipid vesicles at 15 and 30 mole % relative to the other lipids.
The vesicles were prepared as described to entrap perfluorobutane gas. The vesicles were sized and particle count determined as described. Retention of the pro-drug by the vesicles was determined by dialysis as described. The acoustic activity of the DPGS-Dex gas-filled vesicles was measured with a custom-built bench-top acoustic lab. The attenuation of the samples was measured as a function of pressure for vesicles with 15 and 30 mole % DPGS-Dex and compared to MRX 115. In vitro release of DPGS-Dex from the vesicles was assessed by sonicating the vesicles with a horn sonicator (Heat Systems Probe, Farmingdale, NY) at the lowest power setting for 15 seconds and measuring the amount of drug in the layer containing the gasfilled vesicles and the aqueous layer. In vitro study of the bioeffect of DPGS-Dex was assessed in cell culture in L1210, a mouse leukemic cell line (ATCC, Bethesda, Mr). DPGS-Dex and Dex were compared for cytotoxicity. An in vivo study was performed in a dog, anesthetized under general anesthesia with halothane, and instrumented with pulmonary and systemic arterial catheters. Contrast agents were injected at doses of 0.020 cc/kg as rapid IV boluses. Color Doppler and grayscale imaging was performed of the kidney, aorta, and IVC with a model 5200 Acoustic Imaging ultrasound scanner following intravenous injection of MRX 115 and MRX 501. Images were recorded on videotape.
RESULTS Studies with the flow-through phantom showed that high-energy ultrasound, 0.5 W/cm 2, or higher energies, with 100% duty cycle readily ruptured the microbubbles. The microbubbles containing Sudan Black had a mean size of about 2.5 microns and were stable to dialysis. The Sudan Black was retained with the microbubbles until the bubbles were popped with high-energy ultrasound. After exposure to high-energy ultrasound, the Sudan Black was released and equilibrated within the aqueous medium. The in vivo study with the Sudan Black Aerosomes showed increased deposition of Sudan Black in the muscle with ultrasound following IV injection. Significantly more Sudan Black was deposited in the insonated muscle than the contralateral side (P -- .0008), and the highest level was found with 2 W/cm 2. In vitro, the ability to initiate apoptosis of DPGS-Dex was identical to the parent compound dexamethasone. DPGS-Dex was incorporated into acoustically active microbubbles at 15 and 30 mole %, mean particle size was
about 2.5 microns. The pro-drug was retained by MRX 501 until the microbubbles were ruptured by ultrasound. The pressure stability of the pro-drug-containing gas-filled vesicles was only slightly less than MRX 115. Imaging in the dog showed slightly less efficacy than MRX 115 at an equivalent dose for gray-scale enhancement of the kidney but definite color Doppler enhancement of the renal parenchyma and up to 15 or 20 minutes of Doppler enhancement in the aorta and inferior vena cava.
Therapeutic ultrasound readily ruptures microbubbles. Spectrophotometrically visible dyes (eg, Sudan Black) and drugs (eg, a pro-drug of dexamethasone) can be incorporated into acoustically active carriers. Ultrasound can be used to rupture microbubbles containing drugs and thereby release these compounds from the carrier. Our preliminary in vivo study with mice shows how ultrasound increases the deposition in insonated tissue from a model drug, Sudan Black. We have synthesized a biologically active pro-drug of dexamethasone. The pro-drug is bound in MRX 501 to microbubbles and functions as a contrast agent. Ultrasound can be used to monitor and effect bubble rupture, and this should cause local delivery of dexamethasone with MRX 501. Presently, animal models are under development to explain the potential of local dexamethasone delivery with MRX 501. ~CKNOWLEDGMEN
The authors wish to thank Kristina Lg.rka for editorial assistance. !EFERI:NCE,c 1. Balen FG, Allen CM, Lees WR. Ultrasound contrast agents, Clin Radio11994; 49:77-82. 2. lemetti G, Ciuti P, Calligaris F, Francescutto A, Dezhkunov NV. Cavitation threshold d e p e n d e n c e on the rate of the transducer voltage variation. Ultrasonics 1996; 34:193-195. 3. Hynynen K. The threshold for thermally significant cavitation in dog's thigh muscle in vivo. Ultrasound Med Bio11991; 17:157-169. 4. Schlief. Ultrasound or shock wave work process and preparation for carrying out same. U.S. patent 5,380,411,1995. 5. Unger. Methods for providing localized therapeutic heat to biological tissues and fluids. U.S. patent 5,149,319, 1992, 6. Unger. Methods for providing localized therapeutic heat to biological tissues and fluids using gas filled Iiposomes, U,S. patent 5,209,720, 1993. 7, Walker KW, Sahn D. Ultrasound mediated destruction of contrast agents: effect of ultrasound intensity, exposure and frequency. Invest Radiol (in press).
$249
Ultrasound is used for both therapeutic and diagnostic purposes. Microbubbles are under development as ultrasound contrast agents (1). Not only do microbubbles reflect ultrasound, they also absorb the energy from ultrasound. Microbubbles are known to decrease the threshold of energy for cavitation (2,3). Prior studies have suggested that microbubbles can be used as cavitation nuclei for procedures such as lithotripsy and to potentiate ultrasonic hyperthermia (4-6). Recent studies have shown that diagnostic levels of ultrasound can cause bubble destruction and that bubble destruction can be visualized with ultrasound (7). The objective of this study was to develop a microbubble-based drug delivery system for use with ultrasound (Fig 1). Such a drug delivery system should function as a contrast agent allowing drug delivery to be visualized with ultrasound imaging. Using ultrasound energy, it should be possible to rupture the microbubbles and mediate local drug release.
An in vitro setup was designed to evaluate bubble bursting with ultrasound. The setup consisted of a Plexiglas tank filled with degassed water at 37°C. Soundabsorbing rubber material was placed on the bottom of the
Acad Radio11998; 5(suppl 1):$247-$249 1From the Department of Radiology, University of Arizona, 1501 N Campbell Ave, Tucson, AZ 85724-5067 (E.C,U,); and ImaRx Pharmaceutical, Tucson, Ariz (E.C.U., T,M,, R.S,, G.V,, G.W,, D,S., D,Y,), Address reprint requests to E.C.U. 2 Current address: Hughes Missile Systems, Tucson, Ariz. © AUR, 1998
Figure 1. Representation of gas-filled liposome containing a hydropic or amphilic drug, which is incorporated into the membrane stabilizing the microbubble.
tank. A 10-mm polyethylene tube was entered and exited through the ends of the tank. One end of the tubing was connected to a peristaltic pump (Cole-Parmer Masterflex; Cole-Parmer, Chicago, Ill), and an injection port was positioned proximal to the entry point of the polyethylene tubing into the tank. The distal end of the tubing passed into a bucket (after exiting the tank). Contrast agent was injected into the septum to flow through the polyethylene tubing. Two transducers were immobilized with ring stands and positioned with their faces in the water within the tank. The first transducer was a therapeutic 1-MHz continuouswave ultrasound transducer (Rich-Mar model 25; RichMar, Enola, Okla). The second transducer was a 7.5-MHz
$247
Figure 2. Schematic diagram of chemical structure of dexamethasone pro-drug, 1,2dipalmitoyl-sn-glycerol-3-succinate-dexamethasone.
curvilinear probe (model 5200; Acoustic Imaging [AI], Tempe, Ariz). Aerosomes, MRX 115 (ImaRx Pharmaceutical, Tucson, Ariz), was injected into the septum to flow within the polyethylene tubing and visualized within the tubing by ultrasound with the AI machine. The AI machine was operated at intermediate power settings, and the focal zone was positioned on the polyethylene tubing. Simultaneous imaging and application of continuous-wave ultrasound was performed. In these experiments, the high-energy ultrasound was applied with the 1-MHz transducer upstream or proximal to the imaging transducer. The duty cycle was varied from between 10% to 100% and the power wattage from 0.5 W/cm 2 to 2.0 W/cm 2. The effect of the high-energy ultrasound on the microbubbles was determined by visualizing the signal within the tubing distal to the highenergy transducer. Images were recorded on videotape. Sudan Black (Sigma, St. Louis, Mo) was entrapped into Aerosomes microbubbles. Sudan Black (3.2 mg) was added to vials containing the Aerosomes lipids (lipid concentration = 1 mg/mL). The head space of the vials contained perfluorobutane. The sealed vials were shaken on a Wig-L-Bug dental amalgamator (Crescent Dental, Lyons, Ill) for 60 seconds at 2,800 RPM. The resulting vesicles were sized on an Accusizer (Model 270; Nicomps, Santa Barbara, Calif). Stability of the Sudan Black within the vesicles was determined by dialyzing the vesicles against normal saline with 1,000 MW cutoff dialysis tubing (Spectrum, Los Angeles, Calif) for 12 hours at room temperature.
$248
In vivo studies were performed in mice (Balb/c, Harland Sprague Dawley, Indianapolis, Ind). The Sudan Black-containing Aerosomes were injected IV in the mice. There were four groups (three animals per group), control, no ultrasound, and ultrasound = 1 W/cm 2 and 2 W/cm 2 applied to the animal' s thigh muscle for a period of 60 seconds during infusion of the Sudan Black Aerosomes. A pro-drug of dexamethasone (DPGS-Dex) was synthesized (Fig 2). 1,2-Dipalmitoyl-sn-glycerol-3-succinate (DPGS) 0.32 g and dexamethasone (9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-diene-3,20-dione) 0.2 g and dimethylaminopyridine (DMAP) 10 mg were dissolved in chloroform, 30 mL, and added to a solution of dicyclohexyl carbodiimide (DCC) 0.11 g in chloroform, 10 mL, at 0°-5°C for 2 hours and stirred at room temperature overnight. 2% acetic acid was then added, stirred for 2 hours followed by isolation of the water phase (chemicals from Aldrich, Milwaukee, Wis). The organic phase was washed with 20 mL water and dried over anhydrous sodium sulfate. The organic solution was evaporated in a rotoevaporator, which resulted in a white residue. This residue was redissolved in acetonitrile. The small amount of precipitate was removed by filtration, and the solution was evaporated to dryness yielding 0.5 g of a white solid. The crude product was recrystallized from 10 mL methanol, resulting in a white crystal (m.p. 37°--40°). The activity of the pro-drug was compared to dexamethasone in an in vitro cytotoxicity assay in human multiple myeloma cells. DPGS-Dex was incorporated into the phospholipid vesicles at 15 and 30 mole % relative to the other lipids.
The vesicles were prepared as described to entrap perfluorobutane gas. The vesicles were sized and particle count determined as described. Retention of the pro-drug by the vesicles was determined by dialysis as described. The acoustic activity of the DPGS-Dex gas-filled vesicles was measured with a custom-built bench-top acoustic lab. The attenuation of the samples was measured as a function of pressure for vesicles with 15 and 30 mole % DPGS-Dex and compared to MRX 115. In vitro release of DPGS-Dex from the vesicles was assessed by sonicating the vesicles with a horn sonicator (Heat Systems Probe, Farmingdale, NY) at the lowest power setting for 15 seconds and measuring the amount of drug in the layer containing the gasfilled vesicles and the aqueous layer. In vitro study of the bioeffect of DPGS-Dex was assessed in cell culture in L1210, a mouse leukemic cell line (ATCC, Bethesda, Mr). DPGS-Dex and Dex were compared for cytotoxicity. An in vivo study was performed in a dog, anesthetized under general anesthesia with halothane, and instrumented with pulmonary and systemic arterial catheters. Contrast agents were injected at doses of 0.020 cc/kg as rapid IV boluses. Color Doppler and grayscale imaging was performed of the kidney, aorta, and IVC with a model 5200 Acoustic Imaging ultrasound scanner following intravenous injection of MRX 115 and MRX 501. Images were recorded on videotape.
RESULTS Studies with the flow-through phantom showed that high-energy ultrasound, 0.5 W/cm 2, or higher energies, with 100% duty cycle readily ruptured the microbubbles. The microbubbles containing Sudan Black had a mean size of about 2.5 microns and were stable to dialysis. The Sudan Black was retained with the microbubbles until the bubbles were popped with high-energy ultrasound. After exposure to high-energy ultrasound, the Sudan Black was released and equilibrated within the aqueous medium. The in vivo study with the Sudan Black Aerosomes showed increased deposition of Sudan Black in the muscle with ultrasound following IV injection. Significantly more Sudan Black was deposited in the insonated muscle than the contralateral side (P -- .0008), and the highest level was found with 2 W/cm 2. In vitro, the ability to initiate apoptosis of DPGS-Dex was identical to the parent compound dexamethasone. DPGS-Dex was incorporated into acoustically active microbubbles at 15 and 30 mole %, mean particle size was
about 2.5 microns. The pro-drug was retained by MRX 501 until the microbubbles were ruptured by ultrasound. The pressure stability of the pro-drug-containing gas-filled vesicles was only slightly less than MRX 115. Imaging in the dog showed slightly less efficacy than MRX 115 at an equivalent dose for gray-scale enhancement of the kidney but definite color Doppler enhancement of the renal parenchyma and up to 15 or 20 minutes of Doppler enhancement in the aorta and inferior vena cava.
Therapeutic ultrasound readily ruptures microbubbles. Spectrophotometrically visible dyes (eg, Sudan Black) and drugs (eg, a pro-drug of dexamethasone) can be incorporated into acoustically active carriers. Ultrasound can be used to rupture microbubbles containing drugs and thereby release these compounds from the carrier. Our preliminary in vivo study with mice shows how ultrasound increases the deposition in insonated tissue from a model drug, Sudan Black. We have synthesized a biologically active pro-drug of dexamethasone. The pro-drug is bound in MRX 501 to microbubbles and functions as a contrast agent. Ultrasound can be used to monitor and effect bubble rupture, and this should cause local delivery of dexamethasone with MRX 501. Presently, animal models are under development to explain the potential of local dexamethasone delivery with MRX 501. ~CKNOWLEDGMEN
The authors wish to thank Kristina Lg.rka for editorial assistance. !EFERI:NCE,c 1. Balen FG, Allen CM, Lees WR. Ultrasound contrast agents, Clin Radio11994; 49:77-82. 2. lemetti G, Ciuti P, Calligaris F, Francescutto A, Dezhkunov NV. Cavitation threshold d e p e n d e n c e on the rate of the transducer voltage variation. Ultrasonics 1996; 34:193-195. 3. Hynynen K. The threshold for thermally significant cavitation in dog's thigh muscle in vivo. Ultrasound Med Bio11991; 17:157-169. 4. Schlief. Ultrasound or shock wave work process and preparation for carrying out same. U.S. patent 5,380,411,1995. 5. Unger. Methods for providing localized therapeutic heat to biological tissues and fluids. U.S. patent 5,149,319, 1992, 6. Unger. Methods for providing localized therapeutic heat to biological tissues and fluids using gas filled Iiposomes, U,S. patent 5,209,720, 1993. 7, Walker KW, Sahn D. Ultrasound mediated destruction of contrast agents: effect of ultrasound intensity, exposure and frequency. Invest Radiol (in press).
$249