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).

The manganese(II) ion has many favorable properties that lead to its potential use as an MRI contrast agent: high spin number, long electronic relaxation time, labile water exchange. The present work describes the design, synthesis, and evaluation of a novel Mn(II) complex (MnL1) based on EDTA and also contains a moiety that noncovalently binds the complex to serum albumin, the same moiety used in the gadolinium based contrast agent MS-325. Ultrafiltration albumin binding measurements (0.1 mM, pH 7.4, 37 degrees C) indicated that the complex binds well to plasma proteins (rabbit: 96 +/- 2% bound, human: 93 +/- 2% bound), and most likely to serum albumin (rabbit: 89 +/- 2% bound, human 98 +/- 2% bound). Observed relaxivities (+/- 5%) of the complex were measured (20 MHz, 37 degrees C, 0.1 mM, pH 7.4) in HEPES buffer (r(1) = 5.8 mM(-)(1) s(-)(1)), rabbit plasma (r(1) = 51 mM(-)(1) s(-)(1)), human plasma (r(1) = 46 mM(-)(1) s(-)(1)), 4.5% rabbit serum albumin (r(1) = 47 mM(-)(1) s(-)(1)), and 4.5% human serum albumin (r(1) = 48 mM(-)(1) s(-)(1)). The water exchange rate was near optimal for an MRI contrast agent (k(298) = 2.3 +/- 0.9 x 10(8) s(-)(1)). Variable temperature NMRD profiles indicated that the high relaxivity was due to slow tumbling of the albumin-bound complex and fast exchange of the inner sphere water. The concept of a high relaxivity Mn(II)-based contrast agent was validated by imaging at 1.5 T. In a rabbit model of carotid artery injury, MnL1 clearly delineated both arteries and veins while also distinguishing between healthy tissue and regions of vessel damage.

Huang is an employee and shareholder of Theseus Pharmaceuticals and a former employee of ARIAD. Y. Hu is an employee of Takeda. F. Li is a former employee of ARIAD. M.T. Greenfield is a former employee of ARIAD. S.G. Zech is an employee and shareholder of Amgen Inc. and a former employee of ARIAD. B. Das is a former employee of ARIAD. N.I. Narasimhan is a former employee of ARIAD. T. Clackson is an employee and shareholder of Xilio Therapeutics and is a former employee of ARIAD. D. Dalgarno is an employee and shareholder of Theseus Pharmaceuticals and a former employee of ARIAD. W.C. Shakespeare is an employee and shareholder of Theseus Pharmaceuticals and a former employee of ARIAD. M. Fitzgerald is a former employee Research.