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Contrast-enhanced magnetic resonance imaging (CE-MRI) is a valuable technique for the diagnosis of liver diseases. As gadocoletic acid trisodium salt (B22956/1), a new contrast agent showing high biliary excretion, may be potentially advantageous in hepatobiliary imaging, the aim of the study was to investigate the molecular mechanisms of hepatic transport of the B22956 ion in a cellular model of hepatic tumor. B22956 ion uptake was measured in tumoral (HepG2) and nontumoral (Chang liver) hepatic cell lines. Absolute quantitative real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) analyses, using cloned PCR products as standards, were performed on total RNA of both cell lines and normal liver to evaluate the transcription of 12 transport genes: SLCO1A2, SLCO2B1, SLCO1B1, SLCO3A1, SLCO4A1, SLCO1B3, SLC22A7, SLC22A8, SLC22A1, SLC10A1, SLC15A1, and SLC15A2. B22956 transport was more efficient in Chang liver than in HepG2 cells and was inhibited by cholecystokinin-8, a specific substrate of OATP1B3. Real-time RT-PCR analyses revealed different transcription profiles in the tumoral and nontumoral cell lines. Compared with normal liver, the expression of SLCO1B1, SLCO3A1, and SLCO1B3 was greatly repressed in HepG2 cells, whereas SLCO2B1, SLC22A7, and SLC22A8 expression was either maintained or increased. On the contrary, in Chang liver cells, SLC22A7 and SLC22A8 genes were undetectable, whereas the expression of SLCO3A1, SLCO4A1, and SLCO1B3 was similar to normal liver. Transport studies and gene expression analyses indicated that B22956 ion is a good substrate to the liver-specific OATP1B3, reported to be poorly expressed or absent in human liver tumors. Therefore, B22956 may be helpful in detecting hepatic neoplastic lesions by CE-MRI.

Rationale and Objectives: Inversion recovery, three-dimensional, gradient-recalled echo magnetic resonance coronary angiography (IR-3D-GRE-MRCA), performed after administration of an intravascular T1-relaxing agent with prolonged permanence in the blood, is one of the most promising approaches to noninvasive magnetic resonance imaging (MRI) of the coronaries. The aim of the present study was the evaluation of the physicochemical properties in solution, pharmacokinetics, elimination from the body, protein binding, and signal enhancement characteristics of gadocoletic acid trisodium salt (B22956/1), a candidate gadolinium-based MRI contrast agent for coronary angiography., Methods: The pharmacokinetics and elimination from the body of gadocoletate ion, the contrastographically active component of gadocoletic acid trisodium salt, was evaluated after intravenous administration in rats and monkeys, using for assays high-performance liquid chromatography, x-ray fluorescence, and inductively coupled plasma atomic emission spectrometry. The binding of the gadocoletate ion to animal and human serum albumin was studied by means of ultrafiltration. The imaging properties of blood outside coronary arteries after contrast agent administration were evaluated in cynomolgus monkeys (Macaca fascicularis) by measuring aortic signal-to-noise and contrast-to-noise ratios in lower body angiograms. The suitability of gadocoletic acid trisodium salt for achieving contrast-enhanced magnetic resonance coronary angiography (ceMRCA) was tested in Yucatan micropigs with an IR-3D-GRE sequence. All in vivo relaxation rate measurement and images were obtained using a 1.5 T Siemens Symphony scanner., Results: The fractional binding of gadocoletate ion at a concentration of 0.5 mM to serum albumin at the physiological concentration was 95%, 92%, 88%, and 86% for human, monkey, pig, and rat, respectively. In rats and monkeys, gadocoletate ion was excreted unmetabolized through the biliary and urinary routes. It was recovered with feces depending on the injected dose in percentages from 18% to 97%, providing evidence for a saturable biliary pathway. Plasma pharmacokinetics showed the complete elimination of gadocoletate ion within 24 hours after administration. In the monkey, the gadocoletate ion showed the pharmacokinetic behavior of a compound with partial vascular confinement and long plasma half-life, which may be ascribed to elevated binding to serum albumin. These properties manifested themselves in lower body angiograms with excellent image contrast between vessels and muscle. The slowly decaying aortic blood signal-to-noise and contrast-to-noise ratios over a 15-minute period is expected to allow 3-dimensional coronary angiography. The potential of gadocoletic acid trisodium salt for ceMRCA was also demonstrated in Yucatan micropigs. Elevated blood signal intensity and almost total myocardial signal suppression was maintained for almost 1 hour after administration, ie, for much longer than expected to be necessary for coronary angiography. During the whole period high resolution images of the right coronary artery could be obtained., Conclusions: On the basis of the pharmacokinetic profile and imaging characteristics, gadocoletic acid trisodium salt shows promise as a MR contrast agent for coronary angiography.

Magnetic Resonance Imaging is gaining a prominent role in the routine clinical investigation. To further improve this technique it is crucial that contrast agents are developed with more optimal organ specificity. This will not only result in a better diagnostic efficiency but also in a reduction of the amount of the agent administered. A combination of techniques has been employed to increase the target selectivity of the contrast agent and thereby the feasibility to visualize different organs. The organ targeting is based on the understanding of the mechanisms involved in the interaction of the agent with plasma proteins (albumin in particular) as well as the different membrane transporters involved in the uptake and in the excretion of the agent from the organ. The physicochemical properties of the contrast agents play a major role in the interaction with these various proteins. In this review we address the relationship between the structure of the contrast agents and their binding to different plasma proteins and membrane transporters in different organs, with special reference to the liver and kidney. The present and potentially future applications of these concepts in the clinical setting are also discussed.

A series of structurally different Gd(III) conjugates incorporating a bile acid moiety have been prepared. Polyaminopolycarboxylic ligands such as diethylenetriaminepentaacetic acid (DTPA) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA) have been selected as chelating subunit for the Gd(III) ion. Cholic acid, cholylglycine, and cholyltaurine have been incorporated as the bile acid moieties. In first generation conjugates the Gd(III) complex is linked to the carboxyl group of cholic acid. Second generation conjugates feature the attachment of the Gd(III) complex to the 3 position of the steroidic backbone of the bile acid. Finally, in third generation conjugates the Gd(III) complex is attached to the epsilon nitrogen atom of cholyllysine. The conjugates are eliminated through the biliary route to a various extent (7.5 to 77% in rats) according to their structural features. Among the most promising terms, a second generation conjugate in which the Gd(III) complex is linked to cholic acid through the 3alpha hydroxy group seems to enter hepatocytes using the Na(+)/taurocholate transporter. Noticeably, some of the second generation conjugates are characterized by very high tolerabilities (LD(50) up to 9.5 mmol/kg) after intravenous administration in mice.

Ytterbium and lutetium are well suited for optimized anomalous diffraction experiments using synchrotron radiation. Therefore, two lanthanide complexes Yb-HPDO3A and Lu-HPDO3A have been produced that are similar to the Gd-HPDO3A complex already known to give good derivative crystals. Derivative crystals of hen egg-white lysozyme were obtained by co-crystallization using 100 mM solutions of each lanthanide complex. De novo phasing has been carried out using single-wavelength anomalous diffraction on data sets collected on each derivative crystal at the L(III) absorption edge of the corresponding lanthanide (ff" = 28 e(-)). A third data set was collected on a Lu-HPDO3A derivative crystal at the Se K absorption edge with f"(Lu) = 10 e(-). The structures were refined and compared with the known structure of the Gd-HPDO3A lysozyme derivative. The quality of the experimental electron-density maps allows easy model building. With L(III) absorption edges at shorter wavelengths than the gadolinium absorption edge, lutetium and ytterbium, when chelated by a ligand such as HPDO3A, form lanthanide complexes that are especially interesting for synchrotron-radiation experiments in structural biology.

Seven gadolinium complexes are shown to be excellent compounds for the preparation of heavy-atom derivatives for macromolecular crystallography projects. De novo phasing has been carried out using single-wavelength anomalous diffraction (SAD) on a series of gadolinium-derivative crystals of two proteins: hen egg-white lysozyme and urate oxidase from Aspergillus flavus. Lysozyme derivative crystals were obtained by co-crystallizing the protein with the corresponding gadolinium complex at a concentration of 100 mM. Diffraction data were collected to a resolution of 1.7 A using Cu K(alpha) radiation from a rotating-anode generator, making use of the high anomalous signal of gadolinium at this wavelength. Urate oxidase derivative crystals were obtained by soaking native crystals in 100 mM gadolinium complex solutions. Diffraction data were collected to a resolution close to 3 A using X-rays at the Gd L(III) absorption edge, taking advantage of the sharp white line on that edge. For all urate oxidase derivative crystals and three of the lysozyme crystals, SAD phasing led to electron-density maps of very high quality, allowing unambiguous chain tracing. From this study, the binding effectiveness of the gadolinium complexes seems to be related to the nature of the precipitant used for crystallization. These gadolinium complexes represent a new class of high-phasing-power heavy-atom derivatives that may be used for high-throughput structure-determination projects.

A two-component ligand system (1) containing 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) as the hosting unit for the lanthanide cations and an appended asymmetrically functionalized 1,10-phenanthroline (phen) as the chromophore was synthesized. The 1:1 complexes with Eu(3+), Gd(3+), Tb(3+), and Yb(3+) have been prepared and studied in aqueous solution. For Gd.1, a relaxivity value of 2.4 mM(-1) s(-1) has been measured at 20 MHz and 25 degrees C, which indicates that there are no water molecules in the first coordination sphere of the metal ion. The analysis of high resolution (1)H NMR spectra of Yb.1 supports this view and suggests the direct involvement of the phen moiety in the coordination of the metal ion. For Eu.1 and Tb.1, the absorption and luminescence spectra, the overall luminescence efficiencies, and the metal-centered (MC) lifetimes were obtained; coordination features were also determined by comparing luminescence properties in water and deuterated water. For Eu.1 and Tb.1, the overall emission sensitization (se) process in air-equilibrated water was found to be notably effective with phi(se) = 0.21 and 0.11, respectively. A detailed study of the steps originating from light absorption at the phen unit and leading to MC sensitized emission was performed.

The synthesis of DOTA tris(phenylmethyl) ester 2, a new monoreactive derivative of DOTA, is described. This versatile synthon can be easily coupled to compounds bearing an amino group and then deprotected to DOTA monoamide under mild and neutral conditions by catalytic hydrogenolysis. Accordingly, compound 2 has been used in the synthesis of a DOTA monoamide gadolinium complex containing two palmitic esters, which is a component of mixed micelles as MRI contrast agents.

Mixed micelles for MRA are multicomponent systems containing a phospholipid, a biocompatible non-ionic surfactant (e.g. Synperonic(R) F-108) and a lipophilic gadolinium complex. A variety of lipophilic gadolinium complexes were designed taking into account features such as: (i) nature of ligand (cyclic versus acyclic); (ii) lipophilic moiety; (iii) global charge of the complex; and (iv) nature of bond connecting the complex to the lipophilic moiety. All the lipophilic gadolinium complexes after formulation as mixed micelles show high relaxivities in water and in blood (rat). Mixed micelles containing gadolinium complexes bearing only one aliphatic chain cannot be used as MRA contrast agents because they have a high haemolytic effect. Furthermore, in rats they are quickly eliminated from the blood stream. These drawbacks are completely circumvented using gadolinium complexes bearing two aliphatic chains. Mixed micelles containing such complexes show high relaxivities, no haemolytic effect and long blood permanence. This makes them promising candidates as MRA contrast agents. However, elimination, which occurs exclusively through the liver, is not complete, even after 7 days. Complexes containing labile (e.g. ester) bonds between the lipophilic moieties and the chelate subunit are eliminated through both the liver and the kidneys. However, elimination is still not complete after 7 days.

The mechanism of excretion into bile of hepatospecific magnetic resonance imaging (MRI) contrast media employed labeled Gd-reagents EOB.DTPA, BOPTA, B 20790 (iopanoate-linked), and B 21690 (glycocholate-linked) for measurement in rat liver canalicular plasma membrane vesicles and yeast vacuoles. The presence of ATP gave threefold greater transport of B 20790 and B 21690 than of EOB.DTPA and BOPTA. In yeast vacuoles the ATP stimulatory effect was eightfold with B 20790 and fivefold greater for B 21690, whereas in YCF1- or YLLO115w-deleted yeast cells the transport was significantly reduced and absent from double mutants, YCF1 and YLLO15w. The transport was similar in wild-type and deletant cells for B 21690; taurocholate gave 85% inhibition. These data suggest that bilary secretion of structurally related MRI agents depend on molecular structure. The findings are suggestive as of possible value for clinical diagnosis of inherited hyperbilirubinemias and other liver disorders., (Copyright 2001 Academic Press.)

The mechanisms were investigated for the hepatic transport of 4 different gadolinium complexes used as contrast agents for magnetic resonance imaging (MRI). In basolateral rat hepatocyte plasma membrane vesicles, Gd-DTPA uptake was indistinguishable from non-specific binding to vesicles; Gd-BOPTA and Gd-EOB-DTPA entered plasma membrane vesicles following a linear, concentration-dependent mechanism up to 1.5 mM of substrate. By contrast, Gd-B 20790 uptake followed a saturative kinetic with an apparent Km of 92 +/- 15 microM and a Vmax of 143 +/- 42 pmol/mg prot/15 sec, and it occurred into an osmotic-sensitive space. Sulfobromophthalein ant taurocholate, but not unconjugated bilirubin inhibited the uptake rate of Gd-B 20790 but not that of the other three compounds. Injection into Xenopus laevis oocytes of 5 ng of human OATP cRNA resulted, after 3 days, in a >/=2-fold stimulation (p < 0.001) of transport of Gd-B 20790 but not of Gd-BOPTA or Gd-EOB-DTPA. Collectively, these data indicate that the hepatic uptake of the MRI contrast agent Gd-B 20790 is a carrier-mediated mechanism operated by OATP while MRI compounds with other chemical structures enter the hepatocyte by other mechanisms., (Copyright 1999 Academic Press.)

Bisalkylation of suitably protected L-glutamic acid and L-lysine derivatives with tert-butyl N-(2-bromoethyl)iminodiacetate 2, followed by deprotection of the omega functional group affords N, N-bis[2-[bis[2-(1, 1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-glutamic acid 1-(1, 1-dimethylethyl) ester 4 and N2,N2-bis[2-[bis[2-(1, 1-dimethylethoxy)-2-oxoethyl]amino]ethyl]-L-lysine 1,1-dimethylethyl ester 7. Such compounds feature a carboxylic or an amino group, respectively, which are available for conjugation with a suitable partner via formation of an amide bond. The conjugates, which can be prepared in this way, contain a chelating subunit in which all five acetic residues of DTPA are available for the complexation of metal ions. Direct bisalkylation of glycine with 2 promptly gives N, N-bis[2-[bis[2-(1,1-dimethylethoxy)-2-oxoethyl]amino]ethyl]glycine 11. The latter allows to achieve conjugates in which the central acetic group of DTPA is selectively converted into an acetamide.

A series of gadolinium complexes conjugated to bile acids was prepared and investigated as possible hepatospecific MR imaging contrast agents. In the design of such compounds, features such as the nature of the bile acid, the site of conjugation on the bile acid skeleton, and the global charge of the conjugate were taken into account. Relaxivity measurements carried out in human serum indicate interaction of the conjugates with human serum proteins; even small structural variations significantly affect relaxivity in human serum. Pharmacokinetic data (biliary elimination in the range of 18.4-45.6%) show that bile acids can be used as address moieties to transport gadolinium complexes through hepatocytes. For a homogeneous series of compounds, differing only in the bile acid residue conjugated, it was unexpectedly found that cholic acid is twice as efficient an address moiety as cholylglycine or cholyltaurine. Preliminary results show that none of the conjugates is transported through the basolateral membrane of hepatocytes by the Na+/taurocholate carrier.

The relaxation properties of Gd(DOTP)5- (1, 4, 7, 10-tetra-azacyclododecane- N,N',N'',N'''-tetrakis(methylenephosphonic acid)) have been investigated as a function of pH, temperature, concentration, and magnetic field strength. We have found that the complex has one exchangeable water molecule in its inner coordination sphere, at a distance of 3.26 A from the metal ion, and it does not form oligomers in solution in the concentration range 0.2 to 10 mM. The possible presence of two species in solution with an average fractional hydration number is also taken into accounts. The NMRD profiles were recorded at 5 degrees C, 25 degrees C, and 35 degrees C and quantitatively analyzed in terms of the paramagnetic relaxation equations. Interestingly the addition to a solution of the Gd(III)-complex of nitrogen bases results in a marked relaxation enhancement, which shows a strong pH dependence with a maximum around pH = 9. The relaxivity gain has been shown to depend on outer-sphere effects originating from multiple electrostatic interactions between the anionic complex and the organic cations that bring the exchangeable protons of the substrate molecules in to close proximity with the paramagnetic center. High resolution NMR relaxation data for N-methyl-D(-)-glucamine suggest that the hydroxyl group on the beta-carbon plays a role in stabilizing the interaction, presumably through a hydrogen bond with an uncoordinated oxygen atom of the complex.