The Interaction of MS-325 with Human Serum Albumin and Its Effect on Proton Relaxation Rates

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Abstract
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 Ka1 = 11.0 ± 2.7, Ka2 = 0.84 ± 0.16, Ka3 = 0.26 ± 0.14, and Ka4 = 0.43 ± 0.24. Under the conditions 0.1 mM MS-325, 4.5% HSA, pH 7.4 (phosphate-buffered saline), and 37 °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 (Ki) of 85 ± 3 μM and 1500 ± 850 μM, 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 (τR = 10.1 ± 2.6 ns bound vs 115 ps free). Analysis of the nuclear magnetic relaxation dispersion (T1 and T2) profiles also suggests a decrease in the electronic relaxation rate (1/T1e at 20 MHz = 2.0 × 108 s-1 bound vs 1.1 × 109 s-1 free) and an increase in the inner-sphere water residency time (τm = 170 ± 40 ns bound vs 69 ± 20 ns free).

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