Protein binding to lanthanide(III) complexes can reduce the water exchange rate at the lanthanide

The GdIII-based magnetic resonance imaging contrast agent MS-325 targets the blood protein serum albumin, resulting in an increased efficacy (relaxivity) as a relaxation agent. MS-325 showed different relaxivities when bound to serum albumin from different species, e.g., r1=30.5 mM-1 s-1 (rabbit) vs 46.3 mM-1 s-1 (human) at 35 degrees C and 0.47 T. To investigate the mechanism for this difference, surrogate complexes were prepared where the GdIII ion was replaced by other LnIII ions. Fluorescence lifetime measurements of the EuIII analogue indicated that the hydration number was q=1 and did not change when bound to either human, rat, rabbit, pig, or dog serum albumin. The YbIII analogue, YbL1, was prepared and characterized by 1H NMR. Line-shape analysis of the paramagnetic-shifted 1H NMR resonances in the presence of increasing amounts of human (HSA) or rabbit (RSA) serum albumin allowed estimation of the transverse relaxation rate, R2, of these resonances for the protein-bound YbL1. The rotational correlation time of YbL1 was calculated from R2, and the Yb-H distance and was tauR=8+/-1 ns when bound to HSA and 13+/-2 ns when bound to RSA. The water exchange rate at the DyIII analogue, DyL1, was determined from variable-temperature R2 measurements at 9.4 T when DyL1 was bound to either HSA or RSA. At 37 degrees C, water exchange at DyL1 was (31+/-5)x10(6) s-1 when bound to HSA but (3.8+/-0.2)x10(6) s-1 when bound to RSA. Slower water exchange upon RSA binding explains the differences in relaxivity observed. The approach of using surrogate lanthanides to identify specific molecular parameters influencing relaxivity is applicable to other protein-targeted GdIII contrast agents.     

Albumin binding, relaxivity, and water exchange kinetics of the diastereoisomers of MS-325, a gadolinium(III)-based magnetic resonance angiography contrast agent

The amphiphilic gadolinium complex MS-325 ((trisodium-{(2-(R)-[(4,4-diphenylcyclohexyl) phosphonooxymethyl] diethylenetriaminepentaacetato) (aquo)gadolinium(III)}) is a contrast agent for magnetic resonance angiography (MRA). MS-325 consists of two slowly interconverting diastereoisomers, A and B (65:35 ratio), which can be isolated at pH > 8.5 (TyeklAr, Z.; Dunham, S. U.; Midelfort, K.; Scott, D. M.; Sajiki, H.; Ong, K.; Lauffer, R. B.; Caravan, P.; McMurry, T. J. Inorg. Chem. 2007, 46, 6621-6631). MS-325 binds to human serum albumin (HSA) in plasma resulting in an extended plasma half-life, retention of the agent within the blood compartment, and an increased relaxation rate of water protons in plasma. Under physiological conditions (37 degrees C, pH 7.4, phosphate buffered saline (PBS), 4.5% HSA, 0.05 mM complex), there is no statistical difference in HSA affinity or relaxivity between the two isomers (A 88.6 +/- 0.6% bound, r1 = 42.0 +/- 1.0 mM(-1) s(-1) at 20 MHz; B 90.2 +/- 0.6% bound, r1 = 38.3 +/- 1.0 mM(-1) s(-1) at 20 MHz; errors represent 1 standard deviation). At lower temperatures, isomer A has a higher relaxivity than isomer B. The water exchange rates in the absence of HSA at 298 K, kA298 = 5.9 +/- 2.8 x 10(6) s(-1), kB298 = 3.2 +/- 1.8 x 10(6) s(-1), and heats of activation, DeltaHA = 56 +/- 8 kJ/mol, DeltaHB = 59 +/- 11 kJ/mol, were determined by variable-temperature 17O NMR at 7.05 T. Proton nuclear magnetic relaxation dispersion (NMRD) profiles were recorded over the frequency range of 0.01-50 MHz at 5, 15, 25, and 35 degrees C in a 4.5% HSA in PBS solution for each isomer (0.1 mM). Differences in the relaxivity in HSA between the two isomers could be attributed to the differing water exchange rates.     

When are Two Waters Worse Than One? Doubling the Hydration Number of a Gd–DTPA Derivative Decreases Relaxivity

The synthesis of a novel ligand, based on N-methyl-diethylenetriaminetetraacetate and containing a diphenylcyclohexyl serum albumin binding group (L1) is described and the coordination chemistry and biophysical properties of its Gd(III) complex Gd-L1 are reported. The Gd(III) complex of the diethylenetriaminepentaacetate analogue of the ligand described here (L2) is the MRI contrast agent MS-325. The effect of converting an acetate to a methyl group on metal-ligand stability, hydration number, water-exchange rate, relaxivity, and binding to the protein human serum albumin (HSA) is explored. The complex Gd-L1 has two coordinated water molecules in solution, that is, [Gd(L1)(H2O)2]2- as shown by D-band proton ENDOR spectroscopy and implied by 1H and 17O NMR relaxation rate measurements. The Gd-H(water) distance of the coordinated waters was found to be identical to that found for Gd-L2, 3.08 A. Loss of the acetate group destabilizes the Gd(III) complex by 1.7 log units (log K(ML) = 20.34) relative to the complex with L2. The affinity of Gd-L1 for HSA is essentially the same as that of Gd-L2. The water-exchange rate of the two coordinated waters on Gd-L1 (k(ex) = 4.4x10(5) s(-1)) is slowed by an order of magnitude relative to Gd-L2. As a result of this slow water-exchange rate, the observed proton relaxivity of Gd-L1 is much lower in a solution of HSA under physiological conditions (r1(obs) = 22.0 mM(-1) s(-1) for 0.1 mM Gd-L1 in 0.67 mM HSA, HEPES buffer, pH 7.4, 35 degrees C at 20 MHz) than that of Gd-L2 (r1(obs) = 41.5 mM(-1) s(-1)) measured under the same conditions. Despite having two exchangeable water molecules, slow water exchange limits the potential efficacy of Gd-L1 as an MRI contrast agent.     

The interaction of MS-325 with human serum albumin and its effect on proton relaxation rates

MS-325 is a novel blood pool contrast agent for magnetic resonance imaging currently undergoing clinical trials to assess blockage in arteries. MS-325 functions by binding to human serum albumin (HSA) in plasma. Binding to HSA serves to prolong plasma half-life, retain the agent in the blood pool, and increase the relaxation rate of water protons in plasma. Ultrafiltration studies with a 5 kDa molecular weight cutoff filter show that MS-325 binds to HSA with stepwise stoichiometric affinity constants (mM(-1)) of K(a1) = 11.0 +/- 2.7, K(a2) = 0.84 +/- 0.16, K(a3) = 0.26 +/- 0.14, and K(a4) = 0.43 +/- 0.24. Under the conditions 0.1 mM MS-325, 4.5% HSA, pH 7.4 (phosphate-buffered saline), and 37 degrees C, 88 +/- 2% of MS-325 is bound to albumin. Fluorescent probe displacement studies show that MS-325 can displace dansyl sarcosine and dansyl-L-asparagine from HSA with inhibition constants (K(i)) of 85 +/- 3 microM and 1500 +/- 850 microM, respectively; however, MS-325 is unable to displace warfarin. These results suggest that MS-325 binds primarily to site II on HSA. The relaxivity of MS-325 when bound to HSA is shown to be site dependent. The Eu(III) analogue of MS-325 is shown to contain one inner-sphere water molecule in the presence and in the absence of HSA. The synthesis of an MS-325 analogue, 5, containing no inner-sphere water molecules is described. Compound 5 is used to estimate the contribution to relaxivity from the outer-sphere water molecules surrounding MS-325. The high relaxivity of MS-325 bound to HSA is primarily because of a 60-100-fold increase in the rotational correlation time of the molecule upon binding (tau(R) = 10.1 +/- 2.6 ns bound vs 115 ps free). Analysis of the nuclear magnetic relaxation dispersion (T(1) and T(2)) profiles also suggests a decrease in the electronic relaxation rate (1/T(1e) at 20 MHz = 2.0 x 10(8) s(-1) bound vs 1.1 x 10(9) s(-1) free) and an increase in the inner-sphere water residency time (tau(m) = 170 +/- 40 ns bound vs 69 +/- 20 ns free).     

Serum albumin targeted, pH-dependent magnetic resonance relaxation agents

The objective of this work was the synthesis of serum albumin targeted, Gd(III)-based magnetic resonance imaging (MRI) contrast agents exhibiting a strong pH-dependent relaxivity. Two new complexes (Gd-glu and Gd-bbu) were synthesized based on the DO3A macrocycle modified with three carboxyalkyl substituents?α to the three ring nitrogen atoms, and a biphenylsulfonamide arm. The sulfonamide nitrogen coordinates the Gd in a pH-dependent fashion, resulting in a decrease in the hydration state, q, as pH is increased and a resultant decrease in relaxivity (r(1)). In the absence of human serum albumin (HSA), r(1) increases from 2.0 to 6.0?mM(-1) s(-1) for Gd-glu and from 2.4 to 9.0?mM(-1) s(-1) for Gd-bbu from pH?5 to 8.5 at 37?°C, 0.47?T, respectively. These complexes (0.2?mM) are bound (>98.9?%) to HSA (0.69?mM) over the pH range 5-8.5. Binding to albumin increases the rotational correlation time and results in higher relaxivity. The r(1) increased 120?% (pH?5) and 550?% (pH?8.5) for Gd-glu and 42?% (pH?5) and 260?% (pH?8.5) for Gd-bbu. The increases in r(1) at pH?5 were unexpectedly low for a putative slow tumbling q=2 complex. The Gd-bbu system was investigated further. At pH?5, it binds in a stepwise fashion to HSA with dissociation constants K(d1)=0.65, K(d2)=18, K(d3)=1360?μM. The relaxivity at each binding site was constant. Luminescence lifetime titration experiments with the Eu(III) analogue revealed that the inner-sphere water ligands are displaced when the complex binds to HSA resulting in lower than expected r(1) at pH?5. Variable pH and temperature nuclear magnetic relaxation dispersion (NMRD) studies showed that the increased r(1) of the albumin-bound q=0 complexes is due to the presence of a nearby water molecule with a long residency time (1-2?ns). The distance between this water molecule and the Gd ion changes with pH resulting in albumin-bound pH-dependent relaxivity.     

Synthesis and physicochemical characterization of carbon backbone modified [Gd(TTDA)(H2O)]2- derivatives

The present study was designed to exploit optimum lipophilicity and high water-exchange rate (k(ex)) on low molecular weight Gd(III) complexes to generate high bound relaxivity (r(1)(b)), upon binding to the lipophilic site of human serum albumin (HSA). Two new carbon backbone modified TTDA (3,6,10-tri(carboxymethyl)-3,6,10-triazadodecanedioic acid) derivatives, CB-TTDA and Bz-CB-TTDA, were synthesized. The complexes [Gd(CB-TTDA)(H(2)O)](2-) and [Gd(Bz-CB-TTDA)(H(2)O)](2-) both display high stability constant (log K(GdL) = 20.28 and 20.09, respectively). Furthermore, CB-TTDA (log K(Gd/Zn) = 4.22) and Bz-CB-TTDA (log K(Gd/Zn) = 4.12) exhibit superior selectivity of Gd(III) against Zn(II) than those of TTDA (log K(Gd/Zn) = 2.93), EPTPA-bz-NO(2) (log K(Gd/Zn) = 3.19), and DTPA (log K(Gd/Zn) = 3.76). However, the stability constant values of [Gd(CB-TTDA)(H(2)O)](2-) and [Gd(Bz-CB-TTDA)(H(2)O)](2-) are lower than that of MS-325. The parameters that affect proton relaxivity have been determined in a combined variable temperature (17)O NMR and NMRD study. The water exchange rates are comparable for the two complexes, 232 × 10(6) s(-1) for [Gd(CB-TTDA)(H(2)O)](2-) and 271 × 10(6) s(-1) for [Gd(Bz-CB-TTDA)(H(2)O)](2-). They are higher than those of [Gd(TTDA)(H(2)O)](2-) (146 × 10(6) s(-1)), [Gd(DTPA)(H(2)O)](2-) (4.1 × 10(6) s(-1)), and MS-325 (6.1 × 10(6) s(-1)). Elevated stability and water exchange rate indicate that the presence of cyclobutyl on the carbon backbone imparts rigidity and steric constraint to [Gd(CB-TTDA)(H(2)O)](2-)and [Gd(Bz-CB-TTDA)(H(2)O)](2-). In addition, the major objective for selecting the cyclobutyl is to tune the lipophilicity of [Gd(Bz-CB-TTDA)(H(2)O)](2-). The binding affinity of [Gd(Bz-CB-TTDA)(H(2)O)](2-) to HSA was evaluated by ultrafiltration study across a membrane with a 30 kDa MW cutoff, and the first three stepwise binding constants were determined by fitting the data to a stoichiometric model. The binding association constants (K(A)) for [Gd(CB-TTDA)(H(2)O)](2-) and [Gd(Bz-CB-TTDA)(H(2)O)](2-) are 1.1 × 10(2) and 1.5 × 10(3), respectively. Although the K(A) value for [Gd(Bz-CB-TTDA)(H(2)O)](2-) is lower than that of MS-325 (K(A) = 3.0 × 10(4)), the r(1)(b) value, r(1)(b) = 66.7 mM(-1) s(-1) for [Gd(Bz-CB-TTDA)(H(2)O)](2-), is significantly higher than that of MS-325 (r(1)(b) = 47.0 mM(-1) s(-1)). As measured by the Zn(II) transmetalation process, the kinetic stabilities of [Gd(CB-TTDA)(H(2)O)](2-), [Gd(Bz-CB-TTDA)(H(2)O)](2-), and [Gd(DTPA)(H(2)O)](2-) are similar and are significantly higher than that of [Gd(DTPA-BMA)(H(2)O)](2-). High thermodynamic and kinetic stability and optimized lipophilicity of [Gd(CB-TTDA)(H(2)O)](2-) make it a favorable blood pool contrast agent for MRI.     

A self-assembled complex with a titanium(IV) catecholate core as a potential bimodal contrast agent

A ditopic chelating ligand (H(6)4) that bears catechol and diethylenetriamine-N,N,N',N',N'-pentaacetate (DTPA) has been designed and shown to specifically bind lanthanide(III) ions at the DTPA core ([Ln(H(2)4)(H(2)O)](-)) and further self-assemble with titanium(IV), thereby giving rise to the formation of a supramolecular metallostar complex with a lanthanide(III)-to-titanium(IV) ratio of 3:1, [(Ln4)(3)Ti(H(2)O)(3)](5-) (Ln=La, Eu, Gd). The efficacy of the metallostar complex as a potential bimodal optical/magnetic resonance imaging (MRI) agent has been evaluated. Nuclear magnetic relaxation dispersion (NMRD) measurements for the [(Gd4)(3)Ti(H(2)O)(3)](5-) complex have demonstrated an enhanced r(1) relaxivity that corresponds to 36.9 s(-1) mM(-1) per metallostar molecule at 20 MHz and 310 K, which is a result of a decreased tumbling rate. The ability of the complex to bind to human serum albumin (HSA) was also examined by relaxometric measurements. In addition, upon UV irradiation the [(Gd4)(3)Ti(H(2)O)(3)](5-) complex exhibits broad-band green emission in the range 400-750 nm with a maximum at 490 nm. Taking into account the high relaxivity and luminescence properties, the [(Gd4)(3)Ti(H(2)O)(3)](5-) complex is a good lead compound for the development of efficient bimodal contrast agents.     

Relaxometric and modelling studies of the binding of a lipophilic Gd-AAZTA complex to fatted and defatted human serum albumin

A new lipophilic gadolinium chelate consisting of a long aliphatic chain bound to the AAZTA coordination cage (Gd-AAZTAC17) has been synthesised. It possesses two coordinated water molecules (q=2) in fast exchange with the solvent (tau298(M) = 67 ns), which yields a relaxivity of 10.2 mM(-1) s(-1). At concentrations greater than 0.1 mM, it forms micelles (average diameter 5.5 nm) characterised by a relaxivity of approximately 30 mM(-1) s(-1) at 20 MHz and 298 K. The latter value appears to be "quenched" by magnetic interactions among the Gd(III) ions on the surface of the micelle that cause a decrease in the electronic relaxation time. A relaxivity of 41 mM(-1) s(-1) was recorded for this micellar system when 98 % of the Gd(III) ions were replaced by diamagnetic Y(III). Gd-AAZTAC17 exhibits a better affinity for fatted human serum albumin (HSA) than for defatted HSA, whereas the relaxivities of the supramolecular adducts are reversed. The relaxivity shown by Gd-AAZTAC17/defatted HSA ({r b(1) (20 MHz, 298 K)=84 mM(-1) s(-1)) is by far the highest relaxivity reported so far for non-covalent paramagnetic adducts with slow-moving substrates. As shown by molecular docking calculations, the gadolinium complex enters a hydrophobic pocket present in fatted HSA more extensively than the corresponding adduct with defatted HSA. Interestingly, no marked difference was observed in either the relaxation enhancement or the binding affinity between fatted and defatted HSA when the binding titrations were carried out at a Gd-AAZTAC17 concentration higher than its critical micellar concentration (cmc). This behaviour has been attributed to the formation of an association between the negatively charged micelle of the lipophilic metal complexes and the positive residues on the surface of the protein.     

Synthesis neutral rare earth complexes of diethylenetriamine-N,N'-bis(acetyl-isoniazid)-N,N',N'-triacetic acid as potential contrast enhancement agents for magnetic resonance imaging

A novel ligand, diethylenetriamine-N,N'-bis(acetyl-isoniazid)-N,N',N'-triacetic acid (H(3)L) has been synthesized from diethylene triamine pentaacetic acid (DTPA) and isoniazid. Ligand and its five neutral rare earth (RE=La, Sm, Eu, Gd, Tb) complexes holding promise of magnetic resonance imaging (MRI) were characterized on the basis of elemental analysis, molar conductivity, (1)H-NMR spectrum, FAB-MS, TG-DTA analysis and IR spectrum. The relaxivity (R(1)) of complexes and Gd(DTPA)(2-) used as a control were determined. The relaxivity of LaL, SmL, EuL, GdL, TbL and Gd(DTPA)(2-) were 0.14, 1.66, 3.14, 6.08, 2.79 and 4.34 l.mmol(-1).s(-1), respectively. The spin-lattice relaxivity of GdL was larger than that of Gd(DTPA)(2-). The relaxivity of GdL had also been investigated in human serum albumin (HSA) solution, the relaxivity of GdL was enhanced from 6.08 l.mmol(-1).s(-1) in water solution to 9.09 l.mmol(-1).s(-1) in HSA solution. In addition, thermodynamics stability constant of GdL complex was determined, the thermodynamic stability constant of GdL complex (K(GdL)=10(20.84)) was a few larger than that of Gd(DTPA)(2-) (K(Gd-DTPA)=10(20.73)). The results showed that complex of GdL may be a prospective MRI contrast agent with low osmotic pressure due to non-ion complex, high spin-lattice relaxivity, good stability and binding affinity for the serum protein.     

Kinetics and Mechanism of the Reaction between Serum Albumin and Auranofin (and Its Isopropyl Analogue) in Vitro

The first detailed kinetic analysis and mechanistic interpretation of the reactions between serum albumin and the second-generation gold drug Auranofin [Et(3)PAuSATg = (triethylphosphine)(2,3,4,6-tetra-O-acetyl-1-beta-D-glucopyranosato-S-) gold(I)] and its triisopropylphosphine analogue, iPr(3)PAuSATg, in vitro are reported. The reactions were investigated using Penefsky spun columns and NMR saturation transfer methods. Under the Penefsky chromatography conditions with 0.4-0.6 mM albumin and a wide range of Et(3)PAuSATg concentrations, the reaction is biphasic. The fast phase is apparently first order in albumin with a rate constant [k(1) = 3.4 +/- 0.3 x 10(-)(2) s(-)(1)] that decreases slightly in magnitude and becomes intermediate in order at low gold concentrations, [Et(3)PAuSATg] < [AlbSH]; it accounts for approximately 95% of the Au(I) that binds. A minor, slower step [k(2) = 2.3 +/- 0.3 x 10(-)(3) s(-)(1)), which accounts for only 5% of the reaction, is also first order with respect to albumin, and zero order with respect to auranofin. For iPr(3)PAuSATg, only the first step was observed, k(1) = (1.4 +/- 0.1) x 10(-)(2) s(-)(1), and is first order in albumin and independent of the iPr(3)PAuSATg concentration. (31)P-NMR saturation transfer experiments utilizing iPr(3)PAuSATg, under equilibrium conditions, yielded second-order rate constants for both the forward (1.2 x 10(2) M(-)(1) s(-)(1)) and the reverse (3.9 x 10(1) M(-)(1) s(-)(1)) directions. A multistep mechanism involving a conformationally altered albumin species was developed. Albumin domain IA opens with concomitant Cys-34 rearrangement, allowing facile gold binding and exchange, and then closes. In conjunction with the steady-state approximation, this mechanism accounts for the different reaction orders observed under the two set of conditions. The rate-determining conformational change of albumin governs the reaction as monitored by the Penefsky columns. Rapid second order exchange of R(3)PAuSATg at the exposed Cys-34 residue is observed under the NMR conditions. The mechanism predicts that under physiological conditions where [Et(3)PAuSATg] is 10-25 &mgr;M, the reaction will be second order and rapid with a rate constant of 8 +/- 2 x 10(2) M(-)(1) s(-)(1). The Penefsky spun columns revealed a previously unreported and novel binding mechanism, association of auranofin in the pocket of albumin-disulfide species, which was confirmed by Hummel-Dreyer gel chromatographic techniques under equilibrium conditions. This albumin-auranofin complex (AlbSSR-Et(3)PAuSATg) is weakly bound and readily dissociates during conventional gel exclusion chromatography.     

A new metallostar complex based on an aluminum(iii) 8-hydroxyquinoline core as a potential bimodal contrast agent

A ditopic DTPA monoamide derivative containing an 8-hydroxyquinoline moiety was synthesized and the corresponding gadolinium(iii) complex ([Gd(H5)(H(2)O)](-)) was prepared. After adding aluminum(iii), the 8-hydroxyquinoline part self-assembled into a heteropolymetallic triscomplex [(Gd5)(3)Al(H(2)O)(3)](3-). The magnetic and optical properties of this metallostar compound were investigated in order to classify it as a potential in vitro bimodal contrast agent. The proton nuclear magnetic relaxation dispersion measurements indicated that the relaxivity r(1) of [Gd(H5)(H(2)O)](-) and [(Gd5)(3)Al(H(2)O)(3)](3-) at 20 MHz and 310 K equaled 6.17 s(-1) mM(-1) and 10.9 s(-1) mM(-1) per Gd(iii) ion respectively. This corresponds to a relaxivity value of 32.7 s(-1) mM(-1) for the supramolecular complex containing three Gd(iii) ions. The high relaxivity value is prominently caused by an increase of the rotational tumbling time τ(R) by a factor of 2.7 and 5.5 respectively, in comparison with the commercially used MRI contrast agent Gd(iii)-DTPA (Magnevist?). Furthermore, upon UV irradiation, [(Gd5)(3)Al(H(2)O)(3)](3-) exposes green broad-band emission with a maximum at 543 nm. Regarding the high relaxivity and the photophysical properties of the [(Gd5)(3)Al(H(2)O)(3)](3-) metallostar compound, it can be considered as a lead compound for in vitro bimodal applications.     

Water Exchange and Rotational Dynamics of the Dimeric Gadolinium(III) Complex [BO{Gd(DO3A)(H(2)O)}(2)]: A Variable-Temperature and -Pressure (17)O NMR Study(1)

Rapid water exchange and slow rotation are essential for high relaxivity MRI contrast agents. A variable-temperature and -pressure (17)O NMR study at 14.1, 9.4, and 1.4 T has been performed on the dimeric BO(DO3A)(2), 2,11-dihydroxy-4,9-dioxa-1,12-bis[1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclododecyl]dodecane, complex of Gd(III). This complex is of relevance to MRI as an attempt to gain higher (1)H relaxivity by slowing down the rotation of the molecule compared to monomeric Gd(III) complexes used as contrast agents. From the (17)O NMR longitudinal and transverse relaxation rates and chemical shifts we determined the parameters characterizing water exchange kinetics and the rotational motion of the complex, both of which influence (1)H relaxivity. The rate constant and the activation enthalpy for the water exchange, k(ex) and DeltaH(), are (1.0 +/- 0.1) x 10(6) s(-)(1)and (30.0 +/- 0.2) kJ mol(-)(1), respectively, and the activation volume, DeltaV(), of the process is (+0.5 +/- 0.2) cm(3) mol(-)(1), indicating an interchange mechanism. The rotational correlation time becomes about three times longer compared to monomeric Gd(III) polyamino-polyacetate complexes studied so far: tau(R) = (250 +/- 5) ps, which results in an enhanced proton relaxivity by raising the correlation time for the paramagnetic interaction.     

Novel macrocyclic EuII complexes: fast water exchange related to an extreme M-O water distance

Eu(II) complexes are potential candidates for pO(2)-responsive contrast agents in magnetic resonance imaging. In this regard, we have characterized two novel macrocyclic Eu(II) chelates, [Eu(II)(DOTA)(H(2)O)](2-) and [Eu(II)(TETA)](2-) (H(4)DOTA=1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, H(4)TETA=1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid) in terms of redox and thermodynamic complex stability, proton relaxivity, water exchange, rotation and electron spin relaxation. Additionally, solid-state structures were determined for the Sr(II) analogues. They revealed no inner-sphere water in the TETA and one inner-sphere water molecule in the DOTA complex. This hydration pattern is retained in solution, as the (17)O chemical shifts and (1)H relaxation rates proved for the corresponding Eu(II) compounds. The thermodynamic complex stability, determined from the formal redox potential and by pH potentiometry, of [Eu(II)(DOTA)(H(2)O)](2-) (lg K(Eu(II))=16.75) is the highest among all known Eu(II) complexes, whereas the redox stabilities of both [Eu(II)(DOTA)(H(2)O)](2-) and [Eu(II)(TETA)](2-) are inferior to that of 18-membered macrocyclic Eu(II) chelates. Variable-temperature (17)O NMR, NMRD and EPR studies yielded the rates of water exchange, rotation and electron spin relaxation. Water exchange on [Eu(II)(DOTA)(H(2)O)](2-) is remarkably fast (k298(ex)=2.5 x 10(9) s(-1)). The near zero activation volume (DeltaV++ =+0.1+/-1.0 cm(3) mol(-1)), determined by variable-pressure (17)O NMR spectroscopy, points to an interchange mechanism. The fast water exchange can be related to the low charge density on Eu(II), to an unexpectedly long M-O(water) distance (2.85 A) and to the consequent interchange mechanism. Electron spin relaxation is considerably slower on [Eu(II)(DOTA)(H(2)O)](2-) than on the linear [Eu(II)(DTPA)(H(2)O)](3-) (H(5)DTPA=diethylenetriaminepentaacetic acid), and this difference is responsible for its 25 percent higher proton relaxivity (r(1)=4.32 mM(-1) s(-1) for [Eu(II)(DOTA)(H(2)O)](2-) versus 3.49 mM(-1) s(-1) for [Eu(II)(DTPA)(H(2)O)](3-); 20 MHz, 298 K).     

EP-2104R: a fibrin-specific gadolinium-Based MRI contrast agent for detection of thrombus

Thrombus (blood clot) is implicated in a number of life threatening diseases, e.g., heart attack, stroke, pulmonary embolism. EP-2104R is an MRI contrast agent designed to detect thrombus by binding to the protein fibrin, present in all thrombi. EP-2104R comprises an 11 amino acid peptide derivatized with 2 GdDOTA-like moieties at both the C- and N-terminus of the peptide (4 Gd in total). EP-2104R was synthesized by a mixture of solid phase and solution techniques. The La(III) analogue was characterized by and 1D and 2D NMR spectroscopy and was found to have the expected structure. EP-2104R was found to be significantly more inert to Gd(III) loss than commercial contrast agents. At the most extreme conditions tested (pH 3, 60 degrees C, 96 hrs), less than 10% of Gd was removed from EP-2104R by a challenge with a DTPA based ligand, while the commercial contrast agents equilibrated within minutes to hours. EP-2104R binds equally to two sites on human fibrin (Kd = 1.7 +/- 0.5 microM) and has a similar affinity to mouse, rat, rabbit, pig, and dog fibrin. EP-2104R has excellent specificity for fibrin over fibrinogen (over 100-fold) and for fibrin over serum albumin (over 1000-fold). The relaxivity of EP-2104R bound to fibrin at 37 degrees C and 1.4 T was 71.4 mM(-1) s(-1) per molecule of EP-2104R (17.4 per Gd), about 25 times higher than that of GdDOTA measured under the same conditions. Strong fibrin binding, fibrin selectivity, and high molecular relaxivity enable EP-2104R to detect blood clots in vivo.     

Facile synthesis of non-ionic dimeric molecular resonance imaging contrast agent: its relaxation and luminescence studies

A bis-polyazamacrocycle, 10'-bis(acetamido)ethane-bis[1,4,7-tri(carboxymethane)-1,4,7,10-tetraazacyclododecane] (DO3A-AME-DO3A) was synthesized for application in magnetic resonance imaging. The efficacy of DO3A-AME-DO3A as non ionic magnetic contrast agent was tested by performing relaxometric studies on its gadolinium complex. The longitudinal relaxivity, r(1) and transverse relaxivity, r(2) values were found to be 5.84 mM(-1)s(-1) and 6.82 mM(-1)s(-1), per Gd(III) at pH 7.0, 37 °C. The luminescence properties of europium complex of DO3A-AME-DO3A were investigated in aqueous medium. The lifetime of Eu(2)-DO3A-AME-DO3A in water was found to be 0.786 ms. Emission and luminescence lifetime measurements on the europium complex of DO3A-AME-DO3A gives a hydration number of q = 1.9. The reaction enthalpy and entropy were found to be, ΔH(0) = -(6.2 ± 2) kJ mol(-1), ΔS(0) = - (1.8 ± 0.4) kJ mol(-1)K(-1), and K(Eu)(298) = (1.8 ± 0.1).     

Synthesis and relaxivity studies of a tetranuclear gadolinium(III) complex of DO3A as a contrast-enhancing agent for MRI

A tetranuclear gadolinium(III) complex, [Gd4(H2O)8], of DO3A appended onto the pentaerythrityl framework was synthesized to improve the water proton relaxivity for MRI application. The longitudinal relaxivity of [Gd4(H2O)8] is 28.13 mM-1 s-1 (24 MHz, 35+/-0.1 degrees C, pH 5.6) which is 5.86 times higher than that of [Gd(DO3A)(H2O)2]. The relaxivity is based on "molecular" relaxivity of the tetramer and the r1p value is "7 per Gd". The high relaxivity of the tetramer is the result of the decrease in the rotational correlation (tauR) and the presence of eight inner-sphere water molecules (q=8). The complex exhibits pH-dependent longitudinal relaxivity, and the high relaxivity both at low and high pH (r1p=28.13 mM-1 s-1 at pH 5.6 and 16.52 mM-1 s-1 at pH 9.5) indicates that it could be used as a pH-responsive MRI contrast agent. The transverse relaxivity of the tetramer is 129.97 mM-1 s-1 (24 MHz, 35+/-0.1 degrees C, pH 5.6), and the r2p/r1p ratio of 4.6 shows that it could be used as a T2-weighted contrast agent.     

Synthesis and characterization of a hypoxia-sensitive MRI probe

Tissue hypoxia occurs in pathologic conditions, such as cancer, ischemic heart disease and stroke when oxygen demand is greater than oxygen supply. An imaging method that can differentiate hypoxic versus normoxic tissue could have an immediate impact on therapy choices. In this work, the gadolinium(III) complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) with a 2-nitroimidazole attached to one carboxyl group via an amide linkage was prepared, characterized and tested as a hypoxia-sensitive MRI agent. A control complex, Gd(DO3A-monobutylamide), was also prepared in order to test whether the nitroimidazole side-chain alters either the water proton T(1) relaxivity or the thermodynamic stability of the complex. The stabilities of these complexes were lower than that of Gd(DOTA)(-) as expected for mono-amide derivatives. The water proton T(1) relaxivity (r(1)), bound water residence lifetime (τ(M)) and rotational correlation time (τ(R)) of both complexes was determined by relaxivity measurements, variable temperature (17) O?NMR spectroscopy and proton nuclear magnetic relaxation dispersion (NMRD) studies. The resulting parameters (r(1) =6.38?mM(-1) s(-1) at 20?MHz, τ(M) =0.71?μs, τ(R) =141?ps) determined for the nitroimidazole derivative closely parallel to those of other Gd(DO3A-monoamide) complexes of similar molecular size. In vitro MR imaging experiments with 9L rat glioma cells maintained under nitrogen (hypoxic) versus oxygen (normoxic) gas showed that both agents enter cells but only the nitroimidazole derivative was trapped in cells maintained under N(2) as evidenced by an approximately twofold decrease in T(1) measured for hypoxic cells versus normoxic cells exposed to this agent. These results suggest that the nitroimidazole derivative might serve as a molecular reporter for discriminating hypoxic versus normoxic tissues by MRI.     

Basic design and synthesis of sulfonated contrast agents for magnetic resonance imaging based on gadolinium complexes.

Magnetic resonance angiography (MRA) is an imaging method to examine blood vessels based on the magnetic resonance imaging (MRI) technique. For this purpose, blood pool contrast agents have been developed to selectively increase the signal intensity of the intravascular lumen for improvement of the contrast-to-noise ratio in MR images. Here, we describe the design and the syntheses of six novel sulfonated contrast agents (KMR-Sulfo1 - 6), their chemical properties and their in vivo applications. In this study, we investigated the lipophilicity and the hydrophilicity of a gadolinium complex using a convenient two-step synthesis route, with the goal of prolonging the plasma half-life by binding mainly to human serum albumin. We confirmed that KMR-Sulfo5 fulfilled the requirements as a blood pool contrast agent: it showed a sufficient relaxivity r(1) of 5.9 mM(-1) s(-1), a long plasma half-life of 25.7 min and complete elimination from the body within 12 h after the administration.     

A benzene-core trinuclear GdIII complex: towards the optimization of relaxivity for MRI contrast agent applications at high magnetic field

A novel ligand, H(12)L, based on a trimethylbenzene core bearing three methylenediethylenetriamine-N,N,N',N'-tetraacetate moieties (-CH(2)DTTA(4-)) for Gd(3+) chelation has been synthesized, and its trinuclear Gd(3+) complex [Gd(3)L(H(2)O)(6)](3-) investigated with respect to MRI contrast agent applications. A multiple-field, variable-temperature (17)O NMR and proton relaxivity study on [Gd(3)L(H(2)O)(6)](3-) yielded the parameters characterizing water exchange and rotational dynamics. On the basis of the (17)O chemical shifts, bishydration of Gd(3+) could be evidenced. The water exchange rate, k(ex)(298)=9.0+/-3.0 s(-1) is around twice as high as k(ex)(298) of the commercial [Gd(DTPA)(H(2)O)](2-) and comparable to those on analogous Gd(3+)-DTTA chelates. Despite the relatively small size of the complex, the rotational dynamics had to be described with the Lipari-Szabo approach, by separating global and local motions. The difference between the local and global rotational correlation times, tau(lO)(298)=170+/-10 ps and tau(gO)(298)=540+/-100 ps respectively, shows that [Gd(3)L(H(2)O)(6)](3-) is not fully rigid; its flexibility originates from the CH(2) linker between the benzene core and the poly(amino carboxylate) moiety. As a consequence of the two inner-sphere water molecules per Gd(3+), their close to optimal exchange rate and the appropriate size and limited flexibility of the molecule, [Gd(3)L(H(2)O)(6)](3-) has remarkable proton relaxivities when compared with commercial contrast agents, particularly at high magnetic fields (r(1)=21.6, 17.0 and 10.7 mM(-1)s(-1) at 60, 200 and 400 MHz respectively, at 25 degrees C; r(1) is the paramagnetic enhancement of the longitudinal water proton relaxation rate, referred to 1 mM concentration of Gd(3+)).