Imaging efficacy of a hepatocyte-selective polyiodinated triglyceride for contrast-enhanced computed tomography.

RATIONALE AND OBJECTIVES: DHOG-LE is an injectable polyiodinated triglyceride lipid emulsion providing contrast enhancement of the liver in CT. Studies were conducted to characterize the imaging efficacy of various DHOG-LE formulations as a function of both the administered iodine dose and the formulation composition. MATERIALS AND METHODS: Four DHOG-LE preparations consisting of either 10, 20, 25, or 30% (w/v) total lipid were administered to anesthetized female Sprague Dawley rats as single intravenous bolus doses of 50, 100, 150, and/or 300 mg I/kg (n = 3 to 6 rats/formulation and dose). A 25% triolein lipid emulsion prepared without iodine was administered as a vehicle control at the highest dose volume (n = 6). Liver enhancement was evaluated as a function of time (0 to 24 hours) after administration of contrast by analyzing regions of interest from sequential body scans. RESULTS: At all dose levels, liver enhancement was observed after injection of each DHOG-LE formulation. Regardless of formulation composition, similar enhancement of the liver was noted when administered at an equivalent iodine dose. Liver enhancement increased proportionately with increasing iodine dose. Mean peak intensities for 50, 100, 150, and 300 mg I/kg doses were 78 HU (42% above baseline), 101 HU (84% above baseline), 125 HU (127% above baseline), and 195 HU (255% above baseline), respectively. Liver time-intensity profiles exhibited rapid uptake, prolonged enhancement up to 3 hours, and complete clearance of the majority of the formulations tested by 24 hours. Time and duration of peak intensities were also directly related to iodine dose. CONCLUSIONS: In the animal model tested, DHOG-LE imaging efficacy was directly related to iodine dose and was independent of formulation composition. Thus, administration of DHOG-LE in highly concentrated lipid preparations minimized administered dose volume and resulted in appreciable liver enhancement, even at the lowest dose of 50 mg I/kg.

Polyiodinated triglyceride lipid emulsions for use as hepatoselective contrast agents in CT: effects of physicochemical properties on biodistribution and imaging profiles.

RATIONALE AND OBJECTIVES: A novel lipid emulsion (LE) was developed for hepatoselective delivery of a polyiodinated triglyceride (ITG) with potential for use in CT. This work assessed the effects of mean particle size, total administered dose, and formulation composition on the in vivo biodistribution and imaging profiles of the ITG-LE in rats. METHODS: The concentration of radioactivity derived from intravenously administered 125I-ITG-LE was determined as a function of time after injection. CT imaging studies of the abdomen evaluated the extent of hepatic enhancement after administration of ITG-LE. RESULTS: Mean emulsion particle diameter and total administered dose exerted the greatest effect on ITG-LE biodistribution profiles. In the optimal delivery scenario, >70% of the administered dose localized to the liver 30 minutes after injection. Liver enhancement profiles in CT imaging studies were consistent with biodistribution profiles. CONCLUSIONS: These results suggest that an appropriately formulated and administered dose of ITG-LE provides tissue-selective localization of contrast material for use in CT.

CT depiction of experimental liver tumors: contrast enhancement with hepatocyte-selective iodinated triglyceride versus conventional techniques.

PURPOSE: To compare findings at computed tomography (CT) enhanced with a hepatocyte-selective contrast agent (iodinated triglyceride) and/or iohexol and at CT during arterial portography (CTAP). MATERIALS AND METHODS: Rabbit livers were directly inoculated with VX2 carcinoma. Results were compared for five helical CT examinations: unenhanced CT, iohexol-enhanced CT (600 mg iodine per kilogram of body weight [mg I/kg]), CTAP (with iohexol [600 mg I/kg]), triglyceride-enhanced CT (126 mg I/kg), and dual-contrast-enhanced CT (triglyceride plus iohexol [425 mg I/kg]). Attenuation of normal liver and tumor were compared with analysis of variance techniques and blinded reader evaluations. RESULTS: Normal liver attenuation was greatest at CTAP (127.3 HU +/- 5.3 [mean +/- standard error of the mean]), followed by dual-contrast-enhanced CT (112.4 HU +/- 1.2), iohexol-enhanced CT (97.9 HU +/- 2.2), triglyceride-enhanced CT (82.3 HU +/- 1.1), and unenhanced CT (54.9 HU +/- 1.8). Liver-to-lesion attenuation difference at triglyceride-enhanced CT was significantly greater than at iohexol-enhanced CT (P < .05), and attenuation differences at dual-contrast-enhanced CT were comparable to those at CTAP. Tumors did not enhance at triglyceride-enhanced CT, which increased conspicuity. Sensitivity values for lesion detection at dual-contrast-enhanced CT were greater than those at iohexol-enhanced CT or at CTAP (P < .05). CONCLUSION: At CT enhanced with triglyceride (especially when combined with iohexol), sensitivity values and liver-to-lesion attenuation differences were greater with lower iodine doses than with iohexol or at CTAP.

Physicochemical characterization of a synthetic lipid emulsion for hepatocyte-selective delivery of lipophilic compounds: application to polyiodinated triglycerides as contrast agents for computed tomography.

A synthetic lipid emulsion (LE) has been developed with physicochemical properties that closely resemble those of a specific class of naturally-occurring lipoproteins known as chylomicron remnants. The formulation has the potential to serve as a hepatocyte-selective delivery system for any lipophilic or amphipathic compounds that can be associated with the internal lipid phase of the emulsion. In the present studies, a lipophilic polyiodinated triglyceride (ITG) was successfully incorporated into the delivery vehicle to form a stable chylomicron-remnant-like emulsion capable of localizing material to the liver following intravenous injection. The preferred ITG-LE formulation was shown to have a mean particle diameter of less than 200 nm and a particle size stability profile in excess of 12 months. The viscosity, pH, and osmolality of the formulation also appeared favorable for safe and convenient intravenous injection. The particle size profile, chemical properties, and high degree of incorporation of ITG into the emulsion suggest that the ITG-LE formulation holds substantial promise as a hepatocyte-selective imaging agent for computed tomography of the liver. Biodistribution, elimination, and computed tomography (CT) imaging results in animals corroborated the hepatocyte-selective nature of the ITG-LE formulation.

Formulation of polyiodinated triglyceride analogues in a chylomicron remnant-like liver-selective delivery vehicle.

PURPOSE: A formulation methodology for the incorporation of polyiodinated triglyceride (ITG) analogues into a protein-free chylomicron remnant-like emulsion was developed to provide a vehicle for the selective hepatic delivery of these agents for contrast-enhanced X-ray computed tomography (CECT). METHODS: Triglyceride emulsions (10% w/v) were prepared at various processing pressures, temperatures and times with a Microfluidizer 110-S using different emulsion component proportions to establish processing and compositional parameters in order to afford stable ITG emulsions (ITG-LE) approaching 200 nm mean diameter. RESULTS: Preliminary data indicated that with a formulation composed of 2.4% dioleoyl PC with a cholesterol:DOPC mole ratio of 0.4 emulsified at 14,700 psi, 35 degrees C for 10 min routinely afforded ITG-LE in the desired size range. The elimination of salt and amino acid from the bulk phase enhanced the stability of the ITG-LE. Incorporation of cholesterol into the monolayer was of critical importance in generating a stable emulsion near the targeted size, with a C:DOPC mole ratio of 0.4 producing a size minimum relative to higher or lower C:DOPC values. CONCLUSIONS: The ITG analogues can be readily incorporated into stable remnant-like emulsions of relatively uniform particle size. Combination of the unique ITG contrast agent with the remnant-like delivery vehicle demonstrates a high degree of hepatic selectivity in biodistribution studies and offers significant potential for selective hepatic CECT.

Polyiodinated triglyceride analogs as potential computed tomography imaging agents for the liver.

A series of glyceryl 2-oleoyl 1,3-bis[omega-(3-amino-2,4,6-triiodophenyl)] alkanoates was synthesized, radioiodinated with iodine-125, emulsified, and evaluated for their ability to selectively localize in the liver for potential use as hepatographic agents in computed tomography. All seven analogs displayed rapid liver uptake wherein between 65 and 78% of the injected dose accumulated in the liver by 30 min. Liver values ranged from 46 to 93% 3 h after injection which corresponded to liver to blood ratios ranging from 21 to 450. Moreover, subsequent elimination of radioactivity from the liver was nearly linear with respect to alkyl chain length. Analogs with longer alkyl chain length were eliminated from the liver more rapidly than their shorter chain counterparts. Because of their biochemical similarities to naturally occurring triglycerides, these novel analogs may prove useful not only for high-resolution anatomic imaging of focal liver lesions, but also for evaluating a variety of diffuse diseases known to affect hepatic function and biochemistry.

alpha-Methylene and glycidic acid analogs of IPPA as potential myocardial imaging agents.

Using organozinc cross-coupling reactions, two radiolabeled analogs of 15-(p-iodophenyl) pentadecanoic acid (IPPA) have been designed and synthesized as potential scintigraphic imaging agents for the heart. Both 15-(4-iodophenyl)-tridecylglycidic acid and 15-(4-iodophenyl)-2-methylene pentadecanoic acid were synthesized and radioiodinated. In tissue biodistribution studies in rats, only the alpha-methylene derivative of IPPA displayed a consistently higher heart to blood ratio and a substantially lower degree of thyroid accumulation than did IPPA alone. With respect to a scintigraphic imaging efficacy, the alpha-methylene analog of IPPA and IPPA itself showed essentially equivalent cardiac imaging profiles in rabbits, with a slight extension in imaging time for the alpha-methylene analog of IPPA.

Site-directed mutagenesis of histidine residues involved in Cu(II) binding and reduction by sperm whale myoglobin.

Sperm whale myoglobin (Mb) reduces Cu(II) through a site-specific mechanism involving complexation by one or more surface histidine residues. Three mutants of Mb, derived from recombinant wild-type Mb, were designed in which surface histidine residues exhibiting strong Cu(II) binding were replaced with amino acids with comparatively poor metal binding characteristics. The kinetics of Cu(II)(Gly)2 reduction by native Mb, recombinant wild-type Mb, and the mutants were compared. Recombinant wild-type Mb reduced Cu(II) at a rate similar to that of native Mb. Two single mutations (His-48----Ala and His-116----Asp) decreased the rate by 31% and 7%, respectively, relative to wild-type Mb and decreased the rate by 38% and 16%, respectively, relative to native Mb. A double mutation (His-113----Ala, His-116----Asp) decreased the rate only slightly more than the single mutation at His-116. Previous NMR studies showed that His-113 exhibits the strongest Cu(II) binding of all surface histidines, but the present experiments suggest that it plays little or no role in the reduction of Cu(II) by Mb. His-48, located 12.7 A from the Fe(II)-heme, participates in one-third of the redox activity of the protein. His-116 appears to play a minor role in the overall redox activity of Mb, but its involvement shows that Mb has the ability to reduce Cu(II) through a histidine residue located more than 20 A from the Fe(II)-heme. These experiments demonstrate that electron transport from the Fe(II)-heme to site-specifically bound Cu(II) can be mediated through multiple pathways in sperm whale Mb.

Kinetics and mechanisms of reduction of Cu(II) and Fe(III) complexes by soybean leghemoglobin alpha.

The reduction of low-molecular-weight Cu(II) and Fe(III) complexes by soybean leghemoglobin alpha was characterized using both kinetic analysis and 1H-NMR experiments. Whereas Fe(III) (CN)6(3-) was reduced through an outer sphere transfer over the exposed heme edge, all other Cu(II) and Fe(III) complexes investigated were reduced via a site-specific binding of the metal to the protein. Reduction of all metal complexes was enhanced by decreasing pH while only Fe(III)NTA reduction kinetics were altered by changes in ionic strength. Rates of reduction for both Cu(II) and Fe(III) were also affected inversely by the effective binding constant of the metal chelate used. NMR data confirmed that both Cu(II)NTA and Fe(III)NTA were bound to specific sites on the protein. Cu(II) bound preferentially to distal His-61 and Fe(III) exerted its greatest effect on two surface lysine residues with epsilon proton resonances at 3.04 and 3.12 ppm. The Fe(III)NTA complex also had a mild but noticeable line broadening effect on the distal His-61 singlet resonance near 5.3 ppm. Like hemoglobin and myoglobin, leghemoglobin might function not only as an oxygen carrier, but also as a biological reductant for low-molecular-weight Cu(II) and Fe(III) complexes.

Determination of parameters effecting proton relaxation of hepatic and gallbladder biles in dogs.

The parameters which are important in causing changes in the T1 and T2 proton magnetic relaxation times of dogs bile were investigated. Three factors were found to be important in causing relaxation in bile: (i) total bile salt concentration; (ii) total protein concentration, and (iii) viscosity. The T1 and T2 values of hepatic and gallbladder biles were found to be independent of specific gravity, osmolarity and electrolyte concentrations. In vitro experiments were conducted with taurocholic acid, bovine serum albumin and porcine stomach mucin to examine the effects of intermolecular interactions on proton relaxation. Relative to each of the molecules alone, various combinations of the bile salt and proteins exhibit relaxation rates of 20 to 60% below theoretically expected values. This influence of in vitro molecular interactions on T1 and T2 is also likely to occur in hepatic and gallbladder biles in vivo. Thus, the effects of complex intermolecular interactions associated with the gallbladder microenvironment complicate but likely will not preclude direct assessment of physiologic data with magnetic resonance imaging.

Biodistribution of GdCl3 and Gd-DTPA and their influence on proton magnetic relaxation in rat tissues.

The biodistribution and relative molar effectiveness of the ionic (GdCl3) and chelated (Gd-DTPA) forms of gadolinium (Gd) to enhance proton relaxation rates in rat kidney, liver and spleen were evaluated. Rats were given intravenous injections of either GdCl3 (100 mumol/kg) or Gd-DTPA (178 mumol/kg). Gd-DTPA was primarily contained in the vascular compartment and was quickly accumulated in the kidney after injection with a relaxivity of 4.3 sec-1 (mumol/g kidney)-1. It was eliminated quickly from the body with only 2% of the injected dose remaining after 120 min. After GdCl3 injection, Gd was found primarily in liver and spleen. It accumulated continuously reaching 72% of the injected does in these two tissues after 120 min. Despite this continuous increase in tissue Gd concentration, the relaxation rates showed saturation in liver and spleen. The results suggest that after GdCl3 was injected it distributed either in a protein bound form that was effective at causing relaxation or in a colloid form that was not effective. The biodistribution of GdCl3 was such that it was determined by the phagocytic action of the recticuloendothelial system on a colloid. The biodistribution and tissue relaxivity of Gd-DTPA suggest it will be a useful vascular MRI contrast agent. However, the usefulness of GdCl3 as an MRI contrast agent is limited not only by its acute toxicity but also by its saturable effect on tissue relaxation rates. Consequently, GdCl3 has only a modest influence on tissue relaxivity.