Lodinated Particulate Suspensions

After bolus injection, water-soluble contrast media, routinely used in angiogrphy, are rapidly distributed throughout the intravascular space and diffuse across the capillary wall into the interstitial space. A brief time window exists for imaging the differential contrast between organs and adjacent tissues before nonspecific total body opacification occurs. In comparison, particulate contrast media are not hyperosmolar, remain longer in the intravascular space, and are specifically targeted to organs, such as blood pool, liver, and spleen (890). Particulate contrast media, when administered in the form of emulsions, liposomes, microcapsules, and nanocrystals, for example, exert no osmotic pressure and are rapidly taken up by the macrophages and the Kupffer cells in the reticuloendothelial (RE) system and accumulate in the liver and spleen. The RE system is saturable, and overflow from high doses will persist in the circulation. Par-ticulate agents produce high contrast between normal and pathologic tissues at a fraction of the dose required of conventional agents and maintain the density difference for CT imaging over prolonged periods of time. The adverse effects of the particulates, including the clearance, the effect of long-term retention, Kupffer cell activation, disturbances in'the microcirculation of the liver (891), and vacuoles in the RE cells (892), have yet to be further studied. Violante and Fisher (893) discussed the complexities and problems in designing and formulation of RE-selective contrast media. Later advances in producing liposomes and encapsulated vesicles entrapping water-soluble iodinated contrast media and other particulate suspensions are briefly mentioned below:

7.2.1 Liposomes. Liposomes are vesicles consisting of one or more bilayers of lipid encapsulating an aqueous core, carrying water-soluble contrast agents. The weight ratio of iodine to lipid (I/L ratio) is a measure of the amount of iodine per unit weight of lipid delivered to the target organ (894). For hepatic enhancement in CT this ratio needs to exceed 1:1 (895). The leakage and the I/L ratio of liposomes depend to a large extent on the lipid components, the microemulsification process, and the nature of water-soluble contrast agent. Methods for producing contrast-carrying liposomes may be categorized as (1) the microemulsification method using a high pressure microfluidizer (894,896, 897), (2)the de-hydration-rehydration method (891,898), and (3) the reverse-phase evaporation method (899,901).

Cheng et al. (894) produced liposomes by microemulsification through use of a high pressure microfluidizer from a contrast agent and a mixture of egg phosphatidylcholine, phosphatidylglycerol, and cholesterol in the molar ratio of 0.4:0.1:0.5. The liposomes carrying nonionic contrast agents have higher entrapment and weight ratio than those carrying ionic contrast agents; the decreasing order is as follows: iotrolan > metrizamide > io-hexol > iopamidol > ioxaglate > diatrizoate > iodipamide. For diatrizoate the mean I/L ratio was 0.062 ± 0.01 mg/mgand the entrapment value, 0.12 ± 0.02 L/mole. The weight ratio for nonionic contrast agents increased significantly in the liposomes with increasing initial concentrations of the contrast agent. The leakage rate of encapsulated diatrizoate was over 20%in physiological saline. Encapsulated iohexol gave greater and more prolonged opacification of the liver and spleen than plain iohexol. Maximum contrast was reached within 30 min after injection and maintained for more than 60 min. The results indicated that osmotic pressure, charge, viscosity, and lipophilicityof contrast media might influence the encapsulation process.

Leakage of water-soluble contrast agents from the encapsulated liposomes can be eliminated, using an interdigited lipid phase consisting of hydrogenated soy phosphatidylcho-line or distearoyl phosphatidylcholine to produce "interdigitation-fusion" (IF) vesicles. Janoff et al. (897) found that the IF vesicles carrying iotrolan, ioversol, ioparnidol, ioxaglate, and diatrizoate indeed have higher I/L ratios varying from 5.5 to 8.9, and vesicle sizes ranging from 1 to 5 jam in diameter. Leakage by incubation of the liposomes in uitro with fetal calf serum at 37°C for 4-24 h showed that iotrolan and ioxaglate liposomes retained almost all of the iodine, whereas iopamidol and diatrizoate liposomes lost about 50 and 80%of the encapsulated iodine, respectively. In dogs and rats, 1 h after injection at dose levels ranging from 25 to 250 mg I/kg, the liver retained an average of 93.7%of the injected 125I-labeled iotrolan IF-vesicles and the blood retained less than 5% of the dose (895). At 24 h the initial dose in the liver fell to about 10% and decreased thereafter with a half-life of about 6 h. By 115 days, 0.2% of the initial dose still remained in the liver. The high liver uptake was attributed to less lipid in the IF-vesicles so that the RE system was not prematurely saturated.

Krause et al. (899,900) studied the characterization of iopromide liposomes, produced from a lipid mixture of phosphatidyl choline, cholesterol, and stearic acid at a molar ratio of 4:5:1 by a dehydration-rehydration method. The liposome had an average diameter of 0.5 ±0.1 pm. Encapsulation efficiency was between 30 and 40%, with an iodine-lipid (I/L) ratio of approximately 1. The iopromide-car-rying liposomes had a leakage of 4.6%in rabbit plasma and 9.2% in human plasma in the 0- to 4-h period and a leakage of 9% in rabbit plasma and 20% in human plasma in the 0- to 24-h period. The LD„ value of iopromide liposomes was approximately 3 g 1/ kg in rat and mouse. The pharmacokinetic parameters of the liposomes in rat, such as total clearance and terminal half-life, were dose dependent. The terminal half-life in blood was increased from 0.8 h for a 250 mg I/kg dose to 2.9 h for 1000 mg I/kg dose, suggesting a longer circulation of liposomes after higher doses. When the liposomes were given to rats at a dose of 250 mg I/kg by mouth, recoveries (% of dose) from excretion in 0-72 h were approximately 93% (feces) and 3% (urine); by subcutaneous injection, recoveries in 0-48 h: 7% (feces), 61% (urine), and 31% (injectionsite); by intramuscular injection, recoveries in 0-72 h: 9% (feces),69% (urine),and 6% (injection site). In day 7, a total of 0.36% of the administered dose was recovered in organs and tissues. Thus, io-promide-carrying liposomes proved to be stable and highly tolerable vesicles for the targeting to specific organs in animal model. Fifteen minutes after intravenous injection of 100 mg rat blood showed the highest proportions of iodine, followed by the liver, the spleen, and the kidney. Significant proportions of iodine were found only in the liver and spleen, after total clearance from the blood and the kidneys. In the rabbits with VX2 carcinoma, the iopromide liposome showed strong contrast enhancement of the liver. In the dog, interdigital injection of iopromide liposomes enhanced the contrast of ipsilateral lymph nodes (899). Ioxaglate-carrying liposomes with a lipid component consisting of egg phosphatidylcholine alpha-tocopherol had a mean liposomal diameter of 220 nm and an encapsulation efficiency of 85% and at larger doses (^250 mg I/kg bw), showed a 10-fold greater enhancement for the spleen than for the liver and a sustained intravascular contrast enhancement of the aorta (902).

Petersein et al. (903) evaluated in healthy rabbits two new liposomal contrast agents aimed at the reticuloendothelial system for liver CT: BR2 and BR21 (from Bracco Research SA, Geneva, Switzerland). Both are suspensions of iomeprol-containing liposomes in an iomeprol solution; BR2 has twice the liposome concentration of BR21, that is, 40 vs. 20 mglipid/mL, at an iodine content of 260 vs. 320 mg I/mL, respectively. The liposomes are 0.4 mm unilamellar vesicles, made of a phospholipid bilayer surrounding an aqueous phase. The membranes consist of phospholip-ids (phosphatidyl choline and dipalmitoyl phosphatidic acid) and cholesterol in a 2:1 molar ratio. The authors studied the time course of contrast enhancement in liver CT with the liposomal contrast agents, by use of extracellular iomeprol at 300 mg I/mL as the control. In healthy rabbits, at doses of 1.5 mL/kg or greater, the two liposomal agents induce a significantly stronger and more prolonged enhancement of the liver than that of the extracellular iomeprol and provide a larger imaging window for optimizing CT examinations of the liver. Dick et al. (904) evaluated a new contrast agent, liposomal iodixanol (LI), made of a 1:1 mixture of 10% glucose and iodixanol (50% encapsulated iodixanol: 1 mL of that mixture contained 50 mg encapsulated iodine) for examinations of pyrogenic liver abscess in a rabbit model by CT. The experimental group of animals received the LI at a dose of 200 mg I/kg, and the control group received iopentol at a dose of 600 mg I/kg. Results showed that the LI exceeds the extracellular iopentol in overall abscess contrast and duration of the diagnostic interval, in that the liposomal io-gives higher hepatic vessel contrast and better abscess localization.

Activity of encapsulated liposomes is closely related to the method of preparation. Musu et al. (890) prepared large unilamellar vesicles (0.3-1 yx) carrying iopamidol from phosphatidylcholine and dipalmitoylphospha-tidic acid in a 9:1 ratio and iopamidol solution (300 mg I/mL) and extruded the vesicles through a polycarbonate membrane of different pore sizes (0.8-2.0 jll). Extrusion above the transition temperature (75°C) of the lipids reduced the average size and size distribution of the vesicles and increased their I/L ratio. Distribution studies of extruded and unextruded iopamidol-carrying liposomes in rats showed that extruded liposomes gave higher spleen uptake than did unextruded, whereas the liver uptake was comparable. Lung entrapment was significant with unextruded but almost eliminated with extruded liposomes.

After intravenous injection conventional liposomes are rapidly taken up by cells of the mononuclear phagocytic system (MPS). The inclusion of glycolipids and hydrophilic polymers in the liposome membrane modifies the surface characteristics to evade the MPS system and increases the liposomal circulation time (905). Sache et al. (906) prepared iopro-mide-containing liposomes by the continuous high pressure extrusion method, and used the liposomes without prior removal of unencap-sulated contrast agent for surface modification. The liposome membranes were made from soy phosphatidylcholine (SPC), cholesterol (CH), and soy phosphatidylglycerol (SPG) in a 6:3:1 (SPC/CH/SPG) molar ratio, and with the original lipid concentration at 120 mg/g total suspension. The liposome membranes were modified by inclusion of lipid derivatives of polyethylene glycol (PEG) by coating, simply carried out by mixing the preformed iopromide-containing liposomes with 5 mol % of either of the two coating agents (DSPE-PEG2000 or CHHS-PEG2000) for 16 h at room temperature with stirring. The DSPE-PEG coating increased the mean diameter of the vesicles to approximately 200 nm, probably attributable to aggregation and fusion of the vesicles, and decreased the zeta po tential of the negative surface charge. The stability of the unmodified and surface-modified iopromide liposomes in human plasma was determined by equilibrium dialysis of a liposome/plasma mixture against the respective plasma to be stable over a period of 6 h. The biodistribution of modified and unmodified iopromide liposomes was studied in rats, and no significant differences in blood concentration could be found 1 h after injection between different formulations at a dose of 250 mg I/kg body weight, corresponding to 500 mglipid/kg. Computed tomographic images were studied in rabbits. The unmodified and DSEP-PEG-modified liposomes displayed prolonged blood concentration with CT density differences above 70 HU units (aorta) for up to 20 min and proved to be useful for CT imaging, displaying favorable imaging properties.

Liposomes may also serve as a vehicle for carrying an oil-soluble contrast agent such as Ethiodol to the liver (907). To form Ethiodol liposomes, a chloroform solution of Ethiodol and a mixture of egg phosphatidylcholine and phosphatidic acid in a molar ratio of 7:1 and a chloroform solution of Ethiodol are mixed in a proportion of 3:2, followed by solvent evaporation and addition of water with stirring and sonication. The Ethiodol is contained within the liposomes, probably in the hydrophobic region. The liposomes have diameters of 0.015 to 0.2 ju, and show rapid liver uptake in the rabbits and long retention times. Unlike the normal liver tissue the tumor tissue in the rabbits with implanted VX2 carcinoma accumulates no Ethiodol. The dose of Ethiodol liposomes needed for contrast enhancement of the liver is about l/13th of that required of water-soluble diatrizoate.

Caride et al. (908)incorporated brominated phosphatidylcholine with or without cholesterol into brominated radiopaque liposomes. The liposomes were 1-5 ¡x in diameter and showed contrast enhancement of the liver in the dog 1 h after intravenous injection. A few hours after injection brominated liposomes were found inside the hepatocytes. Because the bromine atom is not as effective as the iodine atom in attenuating X-rays, a correspondingly large dose of the brominated liposomes had to be administered for imaging. No information on chemical characterization of brominated phosphatidylcholine or its fate and biotransformation was given.

Yang et al. (909) used poly(d,l-lactide), a polymer with molecular weight in the range of 5000-50,000 Da, for microcapsulation of ethyl esters of iopanoate and diatrizoate and Ethiodol by a solvent evaporation method. The microcapsules were about 1 ¡xmin diameter. Particles of this diameter or smaller can safely traverse the pulmonary capillary bed to be available for the liver and Kupffer cell phagocytosis. In vivo microscopic examination revealed the activity of Kupffer cells phagocytiz-ing the microcapsules. These particles were essentially macrophage-imaging agents. Microcapsules of all three contrast media increased the density of the liver in the rabbits. Maximum opacification of the liver parenchyma appeared 15-30 min after injection, leaving any focal hepatic lesions as defects.

7.2.2 Emulsions. Ethiodol may also be emulsified by mixing with a small amount of phospholipid as emulsifying agent (910). The oil droplets of size 2 to 3 microns in the emulsion are rapidly and efficiently taken up by the RE system of the liver. The imaging quality of ethiodol emulsions is comparable to that of ethiodol liposomes.

Microemulsions of a series of polyiodinated triglycerides (ITG), labeled with iodine-125 and processed in a microfluidizer, were investigated for their contrast enhancement of the liver and the tumor in normal and tumor-bearing (Walker 256) rats, in rabbits bearing VX2 carcinoma, and in normal dogs in CT (911). The mean particle diameter of micro-emulsions was less than 300 nm. These preparations were stable and autoclavable. Thirty minutes after intravenous injection, from 66 to 78% of the injected dose remained in the liver of the rats. After 3 h the liver still retained from 46% to 93% of the dose. At dose levels ranging from 20 to 70 mg I/kg, the increase in density was reported to be about 40 HU in the rats. In a female pig, the contrast enhancement within 1 h of injection was 90 HU. The liver uptake of ITGs was partially dependent on the formulation vehicle, but the metabolism and clearance from the liver were dependent on the chemical structure and the alkyl chain length (911).

7.2.3 Particulates. Particulates are prepared from insoluble derivatives of contrast agents, finely milled to uniform size, and suspended in water in the presence of small amounts of surfactants and stabilizers. These particles have sizes between 200 and 400 nm in diameter and are referred to as nanopar-ticles. Nanoparticles can be formulated as blood-pool and liver-spleen CT contrast agents for injection at concentrations of 15-20% (w/ v), corresponding to 89-118 mg I/mL. The insoluble ethyl esters of ionic contrast media in nanoparticles will be taken up by the RE system upon injection and hydrolyzed by esterases into ethanol and water-soluble ionic contrast agent and excreted. Rubin et al. (912) prepared nanoparticles from the water-insoluble diatrizoate ethyl ester and investigated the effect of different types of surfactants on contrast enhancement of aorta, liver, and spleen in the rabbits, and used iohexol as comparator. In the presence of a high molecular weight nonionic polymeric surfactant, the nanoparticles produced excellent and prolonged enhancement of aorta and vena cava, but in the presence of a low molecular weight anionic nonpolymeric surfactant, the nanoparticles markedly opacified liver and spleen. This difference in targeting to different organs was attributed to the shielding of the nanoparticles from opsonization by the high molecular weight nonionic polymeric surfactant, causing a decrease in the uptake by the RE system. Gazelle et al. (913) tested more than 50 insoluble derivatives of diatrizoic acid, iothalamic acid, urokonic acid (acetrizoic acid), and met-acid, formulated as nanocrystals for blood-pool and liver-spleen imaging. Most of these agents demonstrated either blood- or liver-dominant enhancement patterns in normal rabbits and rabbits with VX2 carcinoma. Some of the nanoparticles gave improved liver-to-lesion contrast compared to that of iohexol. The chemical identity of these insoluble derivatives was not divulged.

Violante et al. (914,915) prepared particles of iothalamate ethyl ester with a diameter of 2 ± 1 pm by injecting a solution of ethyl iothalamate in dimethylformamide at a rate of

3 mL/min into a 5% aqueous solution of polyvinylpyrrolidone (PVP), chilled to 0-2°C and circulated at a rate of 1000 mL/min. The particles precipitated out from solution as white suspensions after being warmed at 40°C for 30 min and were centrifuged at 3000# for 5 min, repeatedly washed with water, and resus-pended in saline for injection. These particles were stable in normal saline and showed no particle aggregation when mixed with rat, rabbit, dog, or human serum but formed aggregates up to 80 pm in diameter in plasma. It was found that fibrinogen interacts with the particles, forming aggregates. Aggregation was prevented by preincubating the particles in human serum albumin before introducing them into plasma. The authors also found that in solution, iothalamate has a higher intravenous LD50 value in mice than that of iodipam-ide (13-19 g/kg versus approximately 4 g/kg), whereas as nanoparticles, iothalamate ethyl ester has a lower intravenous LD50 by rapid injection in mice than that of iodipamide ethyl ester (550 mg I/kg versus 1200mgl/kg). Leeet al. (916) noted that in computed tomographic portography bolus injections of iodipamide ethyl ester particles were consistent in detecting all the lesions in pathologic liver in a canine model. This demonstrated that chole-graphic agents could be used to produce particulate contrast agents.

Li et al. (917, 918) synthesized ioxilan carbonate by reaction of ioxilan with carbonyldi-imidazole in dimethyl sulfoxide. The reaction is specific for nonionic contrast media, protecting all the hydroxyl groups in the molecule and rendering it water insoluble. The reaction is given on the following page.

The ioxilan carbonate particles were prepared as a contrast medium by solvent extract/ evaporation method. The preparation involved emulsification of a methylene chloride solution of the carbonate, removal of solvent, and washing and sizing the particles. The iodine content of the particles was 45%. The average diameter of the ioxilan carbonate particles was 1.1 pm, 95% of them ranging from 0.6 and 2.0 ¡xm. Electron microscopy showed the particle surface to be smooth, and the particles showed practically no aggregation when mixed with rat plasma. The LD50 value for the ioxilan carbonate particles was 1.4 g I/kg for ch3co conhch2chohch2oh I

ch3co

Carbonyldiimjdazole

XN'^|^^CONHCH2CH2OH

Dimethyl sulfoxide

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