Edmund R. Marinelli

Tuftsin Binds Neuropilin-1 through a Sequence Similar to That Encoded by Exon 8 of Vascular Endothelial Growth Factor

Tuftsin, Thr-Lys-Pro-Arg (TKPR), is an immunostimulatory peptide with reported nervous system effects as well. We unexpectedly found that tuftsin and a higher affinity antagonist, TKPPR, bind selectively to neuropilin-1 and block vascular endothelial growth factor (VEGF) binding to that receptor. Dimeric and tetrameric forms of TKPPR had greatly increased affinity for neuropilin-1 based on competition binding experiments. On endothelial cells tetrameric TKPPR inhibited the VEGF165-induced autophosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) even though it did not directly inhibit VEGF binding to VEGFR-2. Homology between exon 8 of VEGF and TKPPR suggests that the sequence coded for by exon 8 may stabilize VEGF binding to neuropilin-1 to facilitate signaling through VEGFR-2. Given the overlap between processes involving neuropilin-1 and tuftsin, we propose that at least some of the previously reported effects of tuftsin are mediated through neuropilin-1.

A phospholipid-PEG2000 conjugate of a vascular endothelial growth factor receptor 2 (VEGFR2)-targeting heterodimer peptide for contrast-enhanced ultrasound imaging of angiogenesis.

The transition of a targeted ultrasound contrast agent from animal imaging to testing in clinical studies requires considerable chemical development. The nature of the construct changes from an agent that is chemically attached to microbubbles to one where the targeting group is coupled to a phospholipid, for direct incorporation to the bubble surface. We provide an efficient method to attach a heterodimeric peptide to a pegylated phospholipid and show that the resulting construct retains nanomolar affinity for its target, vascular endothelial growth factor receptor 2 (VEGFR2), for both the human (kinase insert domain-containing receptor - KDR) and the mouse (fetal liver kinase 1 - Flk-1) receptors. The purified phospholipid-PEG-peptide isolated from TFA-based eluents is not stable with respect to hydrolysis of the fatty ester moieties. This leads to the time-dependent formation of the lysophospholipid and the phosphoglycerylamide derived from the degradation of the product. Purification of the product using neutral eluent systems provides a stable product. Methods to prepare the lysophospholipid (hydrolysis product) are also included. Biacore binding data demonstrated the retention of binding of the lipopeptide to the KDR receptor. The phospholipid-PEG2000-peptide is smoothly incorporated into gas-filled microbubbles and provides imaging of angiogenesis in a rat tumor model.

Synthesis and evaluation of macrocyclic gadolinium chelates as hepatospecific MRI agents.

X-ray computed tomography (CT) is generally employed as the imaging modality of choice for the detection and staging of liver disease and focal hepatic lesions. However, the use of radiation and the relatively low resolution obtainable due to the lack of well-tolerated contrast agents that could specifically target the liver are disadvantageous in the clinic. Magnetic resonance imaging (MRI) offers high resolution and sensitivity, but general-purpose MRI contrast agents undergo almost exclusive renal and very little hepatobiliary excretion (1,2). Also problematic is that the rapid clearance of extracellular space (ECS) MRI agents from the extracellular space provides a short time window for imaging (3). An MRI contrast agent that provides sustained enhancement of normal hepatic tissue due to uptake by the hepatic parenchyma and increased relaxivity in hepatic tissue vs that in blood would be superior for differentiation of normal hepatic tissue from benign masses or metastatic lesions. Hepatic uptake and excretion of lipophilic compounds, particularly anions, is a high-flux process and has been exploited in the design of hepatobiliary radiographic and radiopharmaceutical imaging agents (4,5). We reasoned that it might be possible to employ such functionality in a compact, synthetically accessible design to obtain hepatospecific MRI contrast agents (6). The goal of this work was to prepare and evaluate the water solubility, biodistribution, hepatobiliary clearance, and acute tolerance of macrocyclic gadolinium chelates (Fig 1) as potential hepatospecific MRI agents. Secondary evaluation of the compound displaying the optimum combined set of properties would then be conducted, employing tissue relaxivity measurements and in vivo hepatobiliary MRI.