Jaclyn M. Chasse

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.

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.

RATIONALE AND OBJECTIVES: The donor atoms that bind to gadolinium in contrast agents influence inner-sphere water exchange and electronic relaxation, both of which determine observed relaxivity. The effect of these molecular parameters on relaxivity is greatest when the contrast agent is protein bound. We sought to determine an optimal donor atom set to yield high relaxivity compounds. METHODS: A total of 38 gadolinium-1,4,7,10-tetraazacyclo-dodecane-N,N',N'',N'''-tetraacetato derivatives were prepared and relaxivity was determined in the presence and absence of human serum albumin as a function of temperature and magnetic field. Each compound had a common albumin-binding group and differed only by substitution of different donor groups at one of the macrocycle nitrogens. Oxygen-17 isotope relaxometry at 7.05 T was performed to estimate water exchange rates. RESULTS: Changing a single donor atom resulted in changes in water exchange rates ranging across 3 orders of magnitude. Donor groups increased water exchange rate in the order: phosphonate ∼ phenolate > α-substituted acetate > acetate > hydroxamate ∼ sulfonamide > amide ∼ pyridyl ∼ imidazole. Relaxivites at 0.47 and 1.4 T, 37°C, ranged from 12.3 to 55.6 mM(-1)s(-1) and from 8.3 to 32.6 mM(-1)s(-1) respectively. Optimal relaxivities were observed when the donor group was an α-substituted acetate. Electronic relaxation was slowest for the acetate derivatives as well. CONCLUSIONS: Water exchange dynamics and relaxivity can be predictably tuned by choice of donor atoms.

We sought to determine whether there is a species dependence on plasma protein and serum album binding and/or relaxivity of the MR contrast agent MS-325.Equilibrium binding of MS-325 to plasma proteins or purified serum albumin was determined as a function of chelate concentration. T1 and T2 values were determined at 0.47 and 1.41 T, and NMRD profiles were measured to determine the changes in relaxivity over varying field strengths from 0.002 to 1.2 T.The binding of MS-325 to either animal plasma or serum albumin plateaus at chelate concentrations less than 0.1 mM with human, pig, and rabbit plasmas showing maximum binding. Human and pig plasmas show the greatest observed relaxivity enhancement in the presence of MS-325.MS-325 exhibits increased relaxivity in blood plasma as the result of plasma protein binding. Binding ranged from 64% to 91% and was species dependent: human > pig approximately rabbit > dog approximately rat approximately mouse.

EP-647 is a serum albumin-targeted magnetic resonance imaging contrast agent comprising a GdDTPA (DTPA, diethylenetriaminepentaacetate) chelate for magnetic resonance signal generation linked via a phosphodiester to a substituted biphenyl for albumin targeting. Albumin binding and relaxivity are higher than the benchmark magnetic resonance angiographic agent MS-325. EP-647 binds primarily to a unique site on serum albumin that is different from the MS-325 site and the binding sites of other drugs.