Mutagenicity assessment of two potential impurities in preparations of 5-amino-2,4,6 triiodoisophthalic acid, a key intermediate in the synthesis of the iodinated contrast agent iopamidol.

5-Aminoisophthalic acid and 5-nitroisophthalic acid (5-NIPA) are potential impurities in preparations of 5-amino-2,4,6-triiodoisophthalic acid, which is a key intermediate in the synthesis of the iodinated contrast agent iopamidol. We have studied their mutagenicity in silico (quantitative structure-activity relationships, QSAR) and by the bacterial reverse mutation assay (Ames test). First, the compounds were screened with the tools Derek Nexus™ and Leadscope®. Both compounds were flagged as potentially mutagenic (class 3 under ICH M7). However, contrary to the in silico prediction, neither chemical was mutagenic in the Ames test (plate incorporation method) with or without S9 metabolic activation.

Evidence for in vivo macrophage mediated tumor uptake of paramagnetic/fluorescent liposomes.

Dysprosium (Dy)-loaded liposomes act as excellent T(2)-susceptibility agents at high magnetic field strength. The R(2)-enhancement increases with the size of the liposomes and the concentration of entrapped paramagnetic metal complexes. Neuro-2a tumor cells are readily labeled when Dy-loaded liposomes, suitably functionalized with glutamine residues (Gln), are added to the culture medium as glutamine receptors are highly expressed in such proliferating tumor cells. By using fluorescent liposomes doped with fluorescent dyes (either incorporated in the membrane or included in the inner cavity), confocal microscopy experiments showed that targeted liposomes are taken up much more avidly than non-targeted vesicles. In vivo studies showed that glutamine-functionalized and non-functionalized liposomes accumulate in the tumor region to a similar extent. Confocal images of the excised tumor showed extensive co-localization of liposomes and macrophages in both cases. It is suggested that the loss of tumor specificity, shown by Gln-functionalized liposomes in vivo, has to be associated with the efficient removal of liposomes operated by the RES (reticulo endoplasmatic system) or tumor associated macrophages.

Determination of water permeability of paramagnetic liposomes of interest in MRI field.

The water permeability of various liposome membranes has been determined at 298K by measuring the NMR longitudinal water proton relaxation rate of vesicles encapsulating the clinically approved Gd-HPDO3A complex (HPDO3A=10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid). Two basic formulations based on DPPC (dipalmitoylphosphatidylcholine) and POPC (palmitoyl-oleylphosphatidylcholine) phospholipids were selected and investigated. Furthermore, the permeability changes caused by the membrane incorporation of amphiphiles like cholesterol and/or metal complexes of interest for designing improved liposome-based MRI contrast agents, were also investigated. The incorporation of cholesterol and metal complexes bearing C18 saturated chains in POPC-based liposomes reduces the water diffusivity across the membrane bilayer. On the contrary, the incorporation of a macrocyclic metal complex bearing four C12 alkylic chains, one for each coordination arm of the ligand, considerably enhances the water permeability in DPPC-based liposomes. Finally, it is reported that the permeability of POPC-based bilayer is increased when the liposomes are subjected to an osmotic stress.

Lanthanide-loaded paramagnetic liposomes as switchable magnetically oriented nanovesicles.

Osmotically shrunken liposomes loaded with paramagnetic lanthanide(III) complexes orient in a static magnetic field according to the sign of their magnetic susceptibility anisotropy (Deltachi). The magnitude and sign of Deltachi are modulated by the magnetic properties of the Ln (III) ion, by the structural characteristics of the metal chelate, and by the stereochemical arrangement of the lipophilic substituents.

In vitro and in vivo magnetic resonance detection of tumor cells by targeting glutamine transporters with Gd-based probes.

The glutamine transporting system is up-regulated in tumor cells because cell proliferation requires the uptake of large quantities of glutamine. It has been found that the paramagnetic magnetic resonance imaging (MRI) reporter Gd-DOTAMA-C6-Gln, where the glutamine residue is covalently bound to the Gd chelate through a C6 spacer, accumulates in tumor cells both "in vitro" and "in vivo" experiments. The observation that the relaxivity of cellular pellets does not increase with the increase in the amounts of entrapped Gd chelate is taken as an indication that the internalization has occurred through receptor mediated endocytosis. The iv administration of Gd-DOTAMA-C6-Gln allowed the MRI visualization of tumor masses in A/J mice grafted with the murine neuroblastoma cell line Neuro-2a and in Her-2/neu transgenic mice developing multiple mammary carcinoma, respectively.

Molecular determinants in the transport of a bile acid-derived diagnostic agent in tumoral and nontumoral cell lines of human liver.

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.

Magnetic resonance imaging of tumor cells by targeting the amino acid transport system.

An early diagnosis of cancer is crucial in the battle against this disease and the in vivo visualization of tumors at cellular level is still the most challenging goal. In order to target tumor cells, we took into account their increased metabolism and amino acid nutrients or pseudo-nutrients, which are actively transported through the cell membrane, have been chosen as vectors for new MRI contrast agents. For this reason new gadolinium complexes conjugated to agmatine, arginine, and glutamine have been synthesized and studied.

In vitro and in vivo hepatic transport of the magnetic resonance imaging contrast agent B22956/1: role of MRP proteins.

The molecular mechanisms of the hepatic transport of B22956/1, a new gadolinium complex from the class of intravascular contrast agents for MRI, which undergoes extensive biliary elimination, were studied. Biliary and urinary elimination of B22956/1 were measured in normal and in mutant MRP2 lacking rats (TR(-)); cellular trafficking of the compound was assessed in wild and MRP1 or MRP2 transfected MDCKII cells. Eight hours after IV injection of B22956/1, 90+/-8% of the dose was recovered in the bile of normal rats. By contrast, in TR(-) rats, the biliary excretion was significantly lower (14+/-3%) while 55+/-9% of the compound was found in urine. In vitro, the cellular accumulation of B22956/1 was significantly lower in both MRP1 and MRP2 transfected cells as compared to wild type MDCKII cells, and the cellular efflux was prevented by the MRP inhibitor MK571, indicating the involvement of both MRP2 and MRP1 in the transport of B22956/1. Due to the distinct cellular localization of the proteins, MRP2 accounts for the biliary and urinary excretion of the compound, while MRP1 prevents cellular accumulation of the MRI agent. B22956/1 may be useful in clinical conditions where a defective biliary transport is present.