Background

[PubMed]

Malignant cells that precede rapid cellular proliferation require an increasing supply of precursor amino acids to support protein and nucleotide biosynthesis (1). These substrates are translocated from the blood stream into cytoplasma (internalization) via highly efficient transporters that belong to a group of carrier proteins residing in the plasma membrane. After entering the cytoplasma, glutamine (Gln) is further transported into the mitochondria and hydrolyzed by phosphate-dependent glutaminase (2). As a main source of nitrogen, Gln circulates in the blood stream at 0.6–0.9 mmol/L and accumulates in some tissues in concentrations as high as 20 mmol/L (2). Tumors compete with surrounding tissues for nitrogen compounds, producing a negative nitrogen balance in host tissues and substantial nitrogen increase in tumors (3). Therefore, the evaluation of protein metabolism can be carried out via imaging of the Gln uptake in tumors (4, 5). Because the internalization of Gln is mediated by Na⁺-dependent amino acid transporters, attaching a small gadolinium (Gd) chelate to Gln should not interfere with this receptor-mediated endocytosis process (RME) (5, 6). Although receptors normally are found in concentrations of 10^-9 to 10^-13 mol/g of tissue (1 μM to 10^-4 μM), the RME mechanism recycles the receptor rapidly and results in a substantial ligand accumulation inside a cell in a short time (7). As an example, the ligands can be enriched to 10^-8 to 10^-9 mol/g in 1 h (7). For Gd chelates, this concentration meets the minimum requirement for magnetic resonance imaging (MRI) detection (8). The Gln transportation rate for tumors like hepatoma is 10–20 times faster than that for the healthy hepatocytes (1). Thus, the difference in Gln uptake can be used to distinguish between tumors and tissues via contrast agent–enhanced MRI (5).

Gadolinium 1-((11-S)-3,10-diaza-13-carboxamido-11-carboxy-2,9-dioxotridecyl)-N,N’,N”-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane (Gd-DOTAMA-C6-Gln) is a functionalized Gln in which the amino group is linked to a Gd-1,4,7,10-tetraazacyclododecane-N,N’,N”,N”’- tetraacetic monoamide (Gd-DOTA) through a linear aliphatic chain of six carbons (-C₆H₁₂-) (5). Gd-DOTA moiety has a high thermodynamic stability that protects against the in vivo release of Gd ions (9). The aliphatic chain serves as a spacer between the DOTA coordination cage and the Gln moiety to reduce the hindrance of the whole complex and improve the interaction between the Gln moiety and the receptor on the cell membrane (5). The conjugation of Gd chelate to the amino group sacrifices one of the four Gln recognition points on its transporting proteins and prevents the transporting protein from proceeding with the successive steps that bring the Gln into the mitochondria (5). Thus, the transporter may move the Gd-DOTAMA-C6-Gln to the clathrin-rich region, where the chelates are trapped in the endosomal vesicles. The entrapment can cause a quenching effect in the relaxation enhancement because the endosomal membrane forms barriers for water molecules to interact with the paramagnetic centers (5). The small size of DOTAMA-C6-Gln allows for easy extravasation and diffusion into the tissue to reach the surface of tumor cells.

Synthesis

[PubMed]

Crich et al. reported a detailed synthesis of Gd-DOTAMA-C6-Gln (5). DOTAMA-C6-Gln was obtained by a coupling reaction between the N-hydroxysuccinimide (NHS) activated ester of 1-(3-aza-8-carboxy-2-oxooctyl)-N,N’,N”-tris((1,1-dimethylethoxy)carbonylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAMA(tBuO)₃-(CH₂)₆-OH) and the unprotected L-glutamine in a homogeneous mixture of acetonitrile and a phosphate buffer (pH 8). After deprotection of the tert-butyl esters in the presence of trifluoroacetic acid and CH₂Cl₂ (1:1) at 50% yield, the purified ligand formed a complex with GdCl₃ (pH 6.5).

The precursor 1-(3-aza-8-carboxy-2-oxooctyl)-4,7,10-tris(1,1-dimethylethoxy)-1,4,7,10-tetraazacyclododecane NHS (DOTAMA(tBuO)₃-(CH₂)₆-NHS) was prepared in multiple steps. First, 6-aminohexanoic acid benzyl ester was reacted with bromoacetyl bromide in the presence of K₂CO₃ in acetonitrile to yield 7-aza-9-bromo-8-oxononanoic acid benzyl ester at 90% yield. This product was converted to 1-(3-aza-8-carboxy-2-oxooctyl)-N,N’,N”-tris((1,1-dimethylethoxy)carbonylmethyl)-1,4,7,10-tetraazacyclododecane benzyl ester at 96.5% yield by reacting it with 1,4,7,10-tetraazacyclododecane-N,N’,N”-tris-tert-butylester (DO3A-tris-tert-butylester) in the presence of K₂CO₃ in acetonitrile. Then, the ester was hydrogenated in methanol under catalyzation of 10% Pd/C to produce DOTAMA(tBuO)₃-(CH₂)₆-OH in 50% yield, which was further converted to DOTAMA-(CH₂)₆-NHS by reacting with NHS in CH₂Cl₂.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

The relaxation properties of water were examined with ¹H nuclear magnetic resonance (NMR) dispersion spectroscopy (NMRD) at different magnetic field strengths and with ¹⁷O NMR spectroscopy at different temperatures (5). The structural water residual time (τ_m) was found to be 1.5 μs at 25°C, and the electronic relaxation time (τ_v) and the rotation correlation time (τ_R) were 25 and 83 ps, respectively. The T₁ relaxivity was found to be 5.5 mM^-1s^-1 at 0.47 T and 25°C in aqueous solution. For hepatoma cellular pellets with internalized Gd-DOTAMA-C6-Gln, the T₁ relaxivity was measured as a function of Gd per cell, which exhibited a substantial deviation from the theoretical calculation. This indicated that the internalization of the chelates occurred through the entrapment into endosomal vesicles.

The uptake of Gd-DOTAMA-C6-Gln in cells was tested in healthy rat hepatocytes, rat hepatoma cells (HTC), rat glioma C6 cells, murine breast adenocarcinoma cells (TSA), and murine neuroblastoma cells (Neuro-2a) (5). Two to three million cells were incubated for 6 h at 37°C in Earl’s balanced salt solution (EBSS) containing Gd-DOTAMA-C6-Gln at 1.6 mM (three times higher than the glutamine physiological concentration). After incubation, the cells were washed three times and then destroyed. The Gd was released from the cells as free aquo-ions and quantified by inductively coupled plasma mass spectrometry (ICP-MS) to determine internalized Gd content (10). For all tumor cells, the internalized Gd was found to be >1 × 10^-8 mol Gd /mg cell protein. The uptake of Gd-DOTAMA-C6-Gln by HTC cells appeared to decrease markedly as the concentration of the free Gln added to the culture medium increased. This result also demonstrated that Gln residue present on the surface of Gd-DOTAMA-C6-Gln complex was the vehicle for the internalization through the amino transporting system. The Gd-DOTAMA-C6-Gln uptake for HTC cells was further compared with that for hepatocytes by in vitro MRI on a 7-T imager. After 4 h of incubation in 1.6 mM of Gd-DOTAMA-C6-Gln, 23 nmol of Gd/mg cell protein was found in HTC cells, which was four times higher than the 5.5 nmol of Gd/mg cell protein in the hepatocytes. This difference was also reflected in the T₁-weighted images on which a 45% increase in signal intensity was found for the HTC cells compared to the healthy hepatocytes.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

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