Gadolinium-1,4,7,10-tetraazacyclododecane-N',N'',N''',N''''-tetraacetic acid-Pro-Leu-Ala-Leu-Lys-Arg-Asp-Arg

Gd-DOTA-PCA7

Leung K.

Publication Details

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Table

In vitro Rodents

Background

[PubMed]

Magnetic resonance imaging (MRI) maps information about tissues spatially and functionally. Protons (hydrogen nuclei) are widely used in imaging because of their abundance in water molecules. Water comprises ~80% of most soft tissue. The contrast of proton MRI depends primarily on the density of the nucleus (proton spins), the relaxation times of the nuclear magnetization (T1, longitudinal, and T2, transverse), the magnetic environment of the tissues, and the blood flow to the tissues. However, insufficient contrast between normal and diseased tissues requires the development of contrast agents. Most contrast agents affect the T1 and T2 relaxation times of the surrounding nuclei, mainly the protons of water. T2* is the spin–spin relaxation time composed of variations from molecular interactions and intrinsic magnetic heterogeneities of tissues in the magnetic field (1).

Extracellular matrix (ECM) adhesion molecules consist of a complex network of fibronectins, collagens, chondroitins, laminins, glycoproteins, heparin sulfate, tenascins, and proteoglycans that surround connective tissue cells, and they are mainly secreted by fibroblasts, chondroblasts, and osteoblasts (2). Cell substrate adhesion molecules are considered essential regulators of cell migration, differentiation, and tissue integrity and remodeling. These molecules play a role in inflammation and atherogenesis, but they also participate in the process of invasion and metastasis of malignant cells in the host tissue (3). Invasive tumor cells adhere to the ECM, which provides a matrix environment for permeation of tumor cells through the basal lamina and underlying interstitial stroma of the connective tissue. Overexpression of matrix metalloproteinases (MMPs) and other proteases by tumor cells allows intravasation of tumor cells into the circulatory system after degradation of the basement membrane and ECM (4). Several families of proteases are involved in atherogenesis, myocardial infarction, angiogenesis, and tumor invasion and metastasis (5-8). MMP-7, also known as matrilysin, is frequently overexpressed in human cancer tissues and is associated with cancer progression (9, 10). MMP-7 has been shown to play important roles not only in degradation of ECM proteins but also in the regulation of activation, degradation, and shedding of non-ECM proteins (11).

Gadolinium (Gd), a lanthanide metal ion with seven unpaired electrons, has been shown to be very effective in enhancing proton relaxation because of its high magnetic moment and water coordination (12, 13). Gd-Labeled diethylenetriamine pentaacetic acid (Gd-DTPA) was the first intravenous MRI contrast agent used clinically, and a number of similar Gd chelates have been developed in an effort to further improve clinical use. However, these low molecular weight Gd chelates have short blood and tissue retention times, which limit their use as imaging agents in the vasculature and cancer. Various macromolecular Gd complexes have demonstrated superior contrast enhancement for MRI of the vasculature and carcinomas (14-16); however, these Gd complexes cannot proceed into further clinical development because of high tissue accumulation and slow excretion of toxic Gd ions. Furthermore, they are largely nonspecific. An MMP-7 selective peptide, Pro-Leu-Ala-Leu-Lys-Arg-Asp-Arg (PLALKRDR), was found to be selectively cleaved by MMP-7 (17). Proteinase-modulated contrast agents (PCAs) are used with MRI to detect the activity of proteinases in vivo. The PCAs are based on the concept of a solubility switch, from hydrophilic to hydrophobic, which slows their efflux from the activity site. Gd-1,4,7,10-tetraazacyclododecane-N',N'',N''',N''''-tetraacetic acid-PLALKRDR (Gd-DOTA-PCA7-switch) was developed to detect MMP-7 activity in tumor-bearing mice.

Synthesis

[PubMed]

PCA7-switch, prepared with solid-phase synthesis, was coupled to DOTA with the use of DOTA-tri(t-butyl ester) (17). DOTA-PCA7-switch was isolated with column chromatography and incubated with 1.05-molar equivalents of GdCl3 for 24 h. Gd-DOTA-PCA7-switch was purified with high-performance liquid chromatography (HPLC) with a 61% yield. The mass of Gd-DOTA-PCA7-switch was confirmed with mass spectroscopy. Two control Gd-DOTA-PCAs were also prepared: PCA7-scrambled with an MMP-7–resistant peptide (RAKDRLLP), and PCA7-B with hydrophobic proteolytic cleavage products.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

PCA7-switch formed a more hydrophobic product after incubation in the presence of MMP-7 as determined with HPLC analysis (17). There was no change in T1 relaxation times and only 10% change in T2 relaxation times for PCA7-switch after MMP-7 cleavage. On the other hand, PCA7-B showed little difference in hydrophobicity as well as 35% change in T1 relaxation times and 75% change in T2 relaxation times after MMP-7 cleavage, possibly through aggregation after cleavage.

Animal Studies

Rodents

[PubMed]

Lepage et al. (17) used a 7-T MRI animal scanner to perform in vivo MRI in mice (n = 5) bearing human SW480 (MMP-7–negative) and SW480Mat (MMP-7–positive) colon cancer cells on the left and right hind limbs, respectively. Injection of Gd-DOTA-PCA7-switch (3.7 µmol/mouse) provided a steady enhancement in MRI contrast in the core of SW480Mat tumors within 60 min of injection, which continued up to 160 min. The rim of the tumor showed a marked clearance. All five mice injected with Gd-DOTA-PCA7-switch exhibited a significant difference in signal intensity between the MMP-7–positive and MMP-7–negative tumors (P < 0.01). On the other hand, the core of SW480 tumors showed a gradual decline in signal intensity after 60 min. Mice tested with Gd-DOTA-PCA7-scramble (2.3 µmol/mouse) and Gd-DTPA (10 µmol/mouse) showed much less pronounced differences between the two tumors. Pretreatment with GM6001 MMP-7 inhibitor (100 mg/kg per d) for 3 d in the mice bearing two tumors eliminated the difference in signal intensity between the two tumors by inhibiting the accumulation of the signal in the MMP-7–positive tumor. The washout was also faster in the MMP-7–positive tumors after GM6001 treatment.

Other Non-Primate Mammals

[PubMed]

No publication is currently available.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

[PubMed]

No publication is currently available.

NIH Support

P30 CA068485, R41 RR020835

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