Serum albumin targeted, pH-dependent magnetic resonance relaxation agents

Abstract

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.

Figure 1

Relaxivity vs pH at 37°C and 20 MHz for (a) 0.22 mM Gd-glu in the absence (circles) and presence of 0.79 mM HSA (triangles); (b) 0.21 mM Gd-bbu in the absence (circles) and presence of 0.69 mM HSA (triangles). Solid lines indicate fits to determine the pKa of the sulfonamide moiety.

Figure 2

(a) Relative scattering intensity (RSI) compared to pure water as a function of pH for 0.32 mM Gd-bbu in the absence of HSA. (b) Effect of added HSA on relative scattering intensity at pH 5, 0.20 mM Gd-bbu. Data in (a) and (b) are mean ± standard deviation for triplicate experiments; solid line indicates absence of aggregation. (c) DLS data for 0.20 mM Gd-bbu at pH 4.7; (d) DLS data for HSA in HEPES buffer at pH 5; (e) DLS data for 1.5:1 HSA: Gd-bbu at pH 5. All measurements were performed at 37°C.

Figure 3

Binding isotherm for Gd-bbu (37 °C, pH 5, 0.80 mM) to HSA (pH 5, from 0.05 to 1.05 mM in HEPES buffer) from the ultrafiltration experiment. Solid line is the best fit to a stoichiometric binding model.

Figure 4

(a) Relaxivity at 37°C, 0.47T, pH 5 for Gd-bbu (0.42 mM) as a function of added HSA. (b) Hydration number q of Eu-bbu at pH 5 as a function of added HSA. At [HSA]/[Eu-bbu]=0, the triangle indicates measurements made on a visible aggregate while the circle indicates measurements made on the supernatant.

Figure 5

Nuclear Magnetic Relaxation Dispersions (NMRD) of Gd-bbu: (a) 0.52 mM in absence of HSA at pH 8.5 (grey triangles) and pH 5 (black triangles), 37 °C; (b) 0.39 mM at pH 8.5 with 1.4 mM HSA at 5°C (grey circles) and 37°C (black circles); (c) 0.33 mM at pH 5 with 1.3 mM HSA at 5°C (grey squares) and 37°C (black squares). Solid lines in (b) and (c) are fits to the data, see text for model and Table 2 for parameters.

Scheme 1

Scheme 2