Gd-DOTA-G-NH(CH2)11CO-RSPAYYTAA-(CH2CH2O)8-R

GdPCA2

Zhang H.

Publication Details

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In vitro Rodents

Background

[PubMed]

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases localized at the cell surface or in extracellular compartments (1). MMPs degrade all components of the extracellular matrix (ECM) and are associated with a variety of pathological conditions such as wound healing, tissue remodeling, tumor angiogenesis, and embryo development. The active site of MMPs contains a catalytic domain coordinated by zinc to recognize motifs with a consensus sequence of PXX↓XHy, where ↓ represents the cleavage point and XHy represents a large hydrophobic residue (2). Excess MMP activity has been observed in conjunction with many diseases, including rheumatoid arthritis, osteoarthritis, autoimmune diseases, cardiovascular diseases, and cancer (3). For example, an overexpression of MMP subtype-2 (MMP-2 or gelatinase A), an enzyme that degrades type IV collagen and gelatin, is present in many human tumors (4). Thus, MMP has been an important therapeutic target for many years (5).

Gadolinium (Gd)-labeled DOTA-G-NH(CH2)11CO-RSPAYYTAA-(CH2CH2O)8-R (GdPCA2) is a proteinase-modulated contrast agent (PCA) for in vivo imaging of MMP-2 with magnetic resonance imaging (MRI) (6). GdPCA2 consists of four components: a peptide substrate (RSPAY↓YTAA) specific for MMP-2, a Gd-DOTA complex as an MRI probe, an alkyl chain of 12-carbon as a hydrophilic linker between the N-terminus of the peptide and the MRI probe, and an eight-unit polyethylene glycol (PEG8) chain linked to the C-terminus of the peptide to enhance the solubility of GdPCA2. The cleavage of GdPCA2 by MMP-2 produces a less soluble Gd3+-labeled fragment. Thus, the Gd species acts as a solubility switch specific for the enzyme MMP-2. GdPCA2 may have different pharmacokinetics than its cleaved product, which can be used to evaluate MMP-2 activity via dynamic MRI measurement.

Synthesis

[PubMed]

Lebel et al. briefly described the preparation of GdPCA2 (6). With standard protocols of solid-phase peptide synthesis, the N-terminus of the PCA2 peptide (RSPAYYTAA) was linked to the Gd-DOTA via a 12-carbon alkyl chain, and the C-terminus of the peptide was linked to an amino PEG8-Arg to yield GdPCA2.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

The cleavage efficacy (kcat/Km) of GdPCA2 was measured in vitro (6). Cleavage of GdPCA2 by MMP-2 produced a free amino group that was detectable with the addition of fluorescamine. The kcat/Km was 1.2 × 105 M-1s-1 for GdPCA2, which was slightly lower than the kcat/Km of 3.1 × 105 M-1s-1 for the PCA2 peptide but 37.5 times higher than the kcat/Km of 3.1 × 105 M-1s-1 for a scrambled GdPCA2 (GdPCA2-scrambled) that contained a scrambled peptide (SYPATAYA). The cleaved products were further examined with high-performance liquid chromatography and mass spectrometry; two fragments were found for GdPCA2 (specific cleavage) and four fragments for GdPCA2-scrambled (non-specific cleavage).

The T1 relaxivity of GdPCA2 and its cleaved product (cleaved-GdPCA2) was measured at 7 T (6). The value for the GdPCA2 was 2.06 ± 0.03 mM-1s-1 in H2O and 2.03 ± 0.03 mM-1s-1 in aqueous bovine albumin (BSA) (14 mg/ml), respectively. The values for the cleaved-GdPCA2 were slightly higher: 2.18 ± 0.03 mM-1s-1 in H2O and 2.19 ± 0.02 mM-1s-1 in aqueous BSA. In comparison, the T1 relaxivity of GdDTPA was 3.58 ± 0.10 mM-1s-1.

Animal Studies

Rodents

[PubMed]

Lebel et al. used GdPCA2 with dynamic contrast-enhanced MRI to examine the MMP-2 activity as a function of time (the pharmacokinetics) in vivo (6). Balb/c mice were implanted with two types of mammary carcinomas ~310 mm3 in volume: a wild-type MC7-L1 (WT) tumor that had overexpressed MMP-2 at the left hind limb and a knockdown MC7-L1 (KD) tumor that had suppressed MMP-2 (~51% lower) at the right hind limb. The tumor-bearing mice (n = 8) were injected with 2 μmol GdPCA2 via the caudal vein, and sequential T1-weighted images were collected at 7 T at a temporal resolution of 51 s. The signal in each voxel was converted into the relaxation rate difference (ΔR1) between the relaxation rate R1 and the precontrast relaxation rate R1,0. A rapid increase in ΔR1 was observed in the WT and KD tumors after the injection of GdPCA2, but the subsequent pharmacokinetics were different in the two types of tumors. In the WT tumor, ΔR1 remained constant 5–20 min after injection, then exhibited a second increase with a maximum at ~40 min. In the KD tumor, ΔR1 continued to decrease 5 min after injection. As a control, the tumor-bearing mice (n = 2) were injected with 2 μmol GdPCA2-scrambled and imaged with the same MRI protocols. A pharmacokinetics similar to that of the KD tumor after injection of GdPCA2 was observed in both the WT tumor and the KD tumor after injection of GdPCA2-scrambled. This result suggests that the GdPCA2 in WT tumors is different from the GdPCA2-scrambled in both tumors. In the WT tumor, the first increase in ΔR1 after injection of GdPCA2 was caused by the perfusion of GdPCA2 from the blood to the ECM, as seen in all tumors and/or injection with GdPCA2-scrambled; the second increase was attributed to the activation of enzyme MMP-2, which was only present in tumors overexpressing MMP-2 (WT type).

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.

References

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Overall C.M. , Kleifeld O. Tumour microenvironment - opinion: validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy. Nat Rev Cancer. 2006; 6 (3):227–39. [PubMed: 16498445]
6.
Lebel R. , Jastrzebska B. , Therriault H. , Cournoyer M.M. , McIntyre J.O. , Escher E. , Neugebauer W. , Paquette B. , Lepage M. Novel solubility-switchable MRI agent allows the noninvasive detection of matrix metalloproteinase-2 activity in vivo in a mouse model. Magn Reson Med. 2008; 60 (5):1056–65. [PubMed: 18956456]