A Manganese Alternative to Gadolinium for MRI Contrast

Abstract

Contrast-enhanced computed tomography (CT) and magnetic resonance imaging (MRI) are routinely used to diagnose soft tissue and vascular abnormalities. However, safety concerns limit the use of iodinated and gadolinium (Gd)-based CT and MRI contrast media in renally compromised patients. With an estimated 14% of the US population suffering from chronic kidney disease (CKD), contrast media compatible with renal impairment is sorely needed. We present the new manganese(II) complex [Mn(PyC3A)(H2O)](-) as a Gd alternative. [Mn(PyC3A)(H2O)](-) is among the most stable Mn(II) complexes at pH 7.4 (log KML = 11.40). In the presence of 25 mol equiv of Zn at pH 6.0, 37 °C, [Mn(PyC3A)(H2O)](-) is 20-fold more resistant to dissociation than [Gd(DTPA)(H2O)](2-). Relaxivity of [Mn(PyC3A)(H2O)](-) in blood plasma is comparable to commercial Gd contrast agents. Biodistribution analysis confirms that [Mn(PyC3A)(H2O)](-) clears via a mixed renal/hepatobiliary pathway with >99% elimination by 24 h. [Mn(PyC3A)(H2O)](-) was modified to form a bifunctional chelator and 4 chelates were conjugated to a fibrin-specific peptide to give Mn-FBP. Mn-FBP binds the soluble fibrin fragment DD(E) with Kd = 110 nM. Per Mn relaxivity of Mn-FBP is 4-fold greater than [Mn(PyC3A)(H2O)](-) and increases 60% in the presence of fibrin, consistent with binding. Mn-FBP provided equivalent thrombus enhancement to the state of the art Gd analogue, EP-2104R, in a rat model of arterial thrombosis. Mn metabolite analysis reveals no evidence of dechelation and the probe was >99% eliminated after 24 h. [Mn(PyC3A)(H2O)](-) is a lead development candidate for an imaging probe that is compatible with renally compromised patients.

Figure 1

(A) pH titration of PyC3A•TFA in the absence (L) and presence (ML) of 1 equiv MnCl₂ at 25 °C, I = 0.15 M NaCl. (B) Transmetallation of 1 mM [Gd(DTPA)(H₂O)]^2- (filled diamonds), [Mn(CDTA)(H₂O)]^2- (open circles) or [Mn(PyC3A)(H₂O)]^- filled circles) by 25 mM Zn²⁺ monitored by relaxivity change as a function of time in 50 mM pH 6.0 MES buffer, 37 °C, 1.4T. (C) H₂O tranverse relaxivity in the presence of [Mn(PyC3A)(H₂O)]^- as a function of temperature. Solid lines are fits to the data. (D) r₁ of [Mn(PyC3A)(H₂O)]^- measured in pH 7.4 Tris buffer, bovine blood plasma, and 4.5% wt/v bovine serum albumin (BSA) at 1.4T, 37 C°.

Figure 2

Coronal T₁-weighted MR images of a balb/C mouse acquired prior to (A), 3 min (B), and 25 min (C) following i.v. administration of 60 μmol/kg Na[Mn(PyC3A)(H₂O)]^-. Normalized signal-to-noise ratio (nSNR) as a function of time in the blood (D), kidney (E), and liver (F) following Na[Mn(PyC3A)(H₂O)]^- injection; N=4, error bars represent standard error of the mean; solid lines are monoexponential fits to the mean data.

Figure 3

Endogenous Mn concentration (nmol/g) in mouse tissues (grey bars, N=4, determined from naïve mice) and Mn tissue levels 24h following intravenous injection of 60 μmol/kg Na[Mn(PyC3A)(H₂O)] (black bars, N=4). Statistically elevated Mn levels (*, P<0.05) were observed only in the kidney and liver and together represent <0.05% of the injected dose per gram tissue.

Figure 4

(A) r₁ of Mn-FBP measured in pH 7.4 Tris buffer, bovine blood plasma, 4.5% wt/v BSA, human fibrinogen and human fibrin gel at 1.4T, 37 C°. The 60% r₁ increase in the presence of fibrin gel is the result of the reduction in Mn-FBP tumbling rate upon protein binding. (B) Fluorescence polarization anisotropy of fluorescein labeled fibrin binding peptide in DD(E) solution as a function of added Mn-FBP (filled circles) or EP2104R (open circles) to determine Ki to DD(E).

Figure 5

Molecular MR imaging of carotid artery thrombus in a rat model with Mn-FBP. Axial T₁-weighted images before (A, C) and 35 minutes after intravenous administration of Mn-FBP (B, D) at 1.5T. (C) and (D) are expanded regions from (A) and (B), respectively showing the common carotid arteries. Mn-FBP generates marked signal enhancement in the ipsilateral vessel (open arrow, D) after Mn-FBP injection, but not in contralateral vessel (filled arrow, D) or in the vessel prior to Mn-FBP injection (C). (E) Hematoxylin and Eosin stained sections of contralateral (left) and ipsilateral (right) carotid arteries showing occlusive thrombus in the injured vessel; scale bar = 300 μm. (F) and (G) show normalized signal-to-noise ratio (nSNR) of the thrombus (closed circles), contralateral vessel region (open circles), and muscle (closed diamonds) following administration of Mn-FBP and EP2104R, respectively, showing persistently enhanced thrombus with each probe and washout of signal from background tissue. (H) and (I) contrast-to-noise ratio (CNR) of thrombus-to-muscle (closed circles) and contralateral vessel region-to-muscle (open circles) following administration of Mn-FBP and EP2104R, respectively, showing large and persistently high CNR for the thrombus with each probe . N=4 for each probe, error bars represent standard error of the mean.

Figure 6

(A) Concentration of Mn or Gd in rat tissues (nmol metal per g tissue) 1.5h after intravenous administration of 40 μmol metal ion per kg body weight of Mn-FBP (black bars), EP2104R (grey bars), and MnCl₂ (blue bars); Mn levels are corrected for endogenous baseline metal content, N=4 per probe. (B) Concentration (nmol/g) of Mn in rat tissues at 24h following Mn-FBP administration (black bars, N=4) or in naïve rats (grey bars, N=4, showing endogenous Mn levels). Statistically significant differences (*, P<0.05) were observed in kidney and muscle and represent 0.37±0.14 and 0.02±0.02 percent of the injected dose per gram tissue, respectively. (C) LC-ICP-MS traces of plasma analyzed for Mn from blood drawn 2 min, 5 min, 15 min, 30 min, 1h, 2h, and 24h (front to back) after intraveneous Mn-FBP injection. Mn-FBP elutes at 8.5 min; no species arising from dechelated Mn are detected in the LC-ICP-MS traces. (D-F) Pharmacokinetic data (N=4) showing blood clearance of total Mn (D), Mn-FBP (E), and the metabolite eluting at 3.1 min (F), demonstrating rapid blood elimination of Mn-FBP and its metabolite. Error bars represent standard error of the mean; solid lines are biexponential fits to the mean data.

Scheme 1

Synthesis of [Mn(PyC3A)(H₂O)]^- (a) substoicheometric benzyl bromide, dioxane, RT; (b) 3 equiv. ^tbutyl-bromoacetate, 1 equiv. KI, DIPEA, DMF, RT; (c) 1 atm H₂ (g), 10% by wt. Pd/C, MeOH; (d) picolyl chloride, KI, DIPEA, DMF, RT; (e) 1:1 CH₂Cl₂:TFA, RT; (f) MnCl₂, pH 6.5.

Scheme 2

Synthesis of Mn-FBP. (a) N-bromosuccinimide, cat. benzoyl peroxide, CCl₄, reflux; (b) 5, KI, DIPEA, DMF, RT; (c) LiOH, 1:1 THF:H₂O, RT; (d) N,N′,-dicyclohexylcarbodiimine, N-hydroxysucciminide, THF, RT; (e) tetra-amine functionalized fibrin binding peptide, cat. DMAP, DIPEA, DMF, RT; (f) 91:3:3:3 TFA:methanesulfonic acid:1-dodecanethiol:H₂O, RT; (g) MnCl₂, pH 6.5. The structures of 8 and 9 are shown in Figure S7.