Melpomeni Fani

[68Ga]NODAGA-RGD for imaging αvβ3 integrin expression

PurposeA molecular target involved in the angiogenic process is the αvβ3 integrin. It has been demonstrated in preclinical as well as in clinical studies that radiolabelled RGD peptides and positron emission tomography (PET) allow noninvasive monitoring of αvβ3 expression. Here we introduce a 68Ga-labelled NOTA-conjugated RGD peptide ([68Ga]NODAGA-RGD) and compare its imaging properties with [68Ga]DOTA-RGD using small animal PET.MethodsSynthesis of c(RGDfK(NODAGA)) was based on solid phase peptide synthesis protocols using the Fmoc strategy. The 68Ga labelling protocol was optimized concerning temperature, peptide concentration and reaction time. For in vitro characterization, partition coefficient, protein binding properties, serum stability, αvβ3 binding affinity and cell uptake were determined. To characterize the in vivo properties, biodistribution studies and microPET imaging were carried out. For both in vitro and in vivo evaluation, αvβ3-positive human melanoma M21 and αvβ3-negative M21-L cells were used.Results[68Ga]NODAGA-RGD can be produced within 5xa0min at room temperature with high radiochemical yield and purity (> 96%). In vitro evaluation showed high αvβ3 binding affinity (IC50u2009=u20094.7u2009±u20091.6xa0nM) and receptor-specific uptake. The radiotracer was stable in phosphate-buffered saline, pH 7.4, FeCl3 solution, and human serum. Protein-bound activity after 180xa0min incubation was found to be 12-fold lower than for [68Ga]DOTA-RGD. Biodistribution data 60xa0min post-injection confirmed receptor-specific tumour accumulation. The activity concentration of [68Ga]NODAGA-RGD was lower than [68Ga]DOTA-RGD in all organs and tissues investigated, leading to an improved tumour to blood ratio ([68Ga]NODAGA-RGD: 11, [68Ga]DOTA-RGD: 4). MicroPET imaging confirmed the improved imaging properties of [68Ga]NODAGA-RGD compared to [68Ga]DOTA-RGD.ConclusionThe introduced [68Ga]NODAGA-RGD combines easy accessibility with high stability and good imaging properties making it an interesting alternative to the 18F-labelled RGD peptides currently used for imaging αvβ3 expression.

Inflammatory neovascularization during graft-versus-host disease is regulated by αv integrin and miR-100

Acute graft-versus-host disease (GvHD) is a complex process involving endothelial damage and neovascularization. Better understanding of the pathophysiology of neovascularization during GvHD could help to target this process while leaving T-cell function intact. Under ischemic conditions, neovascularization is regulated by different micro RNAs (miRs), which potentially play a role in inflamed hypoxic GvHD target organs. We observed strong neovascularization in the murine inflamed intestinal tract (IT) during GvHD. Positron emission tomography imaging demonstrated abundant αvβ3 integrin expression within intestinal neovascularization areas. To interfere with neovascularization, we targeted αv integrin-expressing endothelial cells, which blocked their accumulation in the IT and reduced GvHD severity independent of immune reconstitution and graft-versus-tumor effects. Additionally, enhanced neovascularization and αv integrin expression correlated with GvHD severity in humans. Expression analysis of miRs in the inflamed IT of mice developing GvHD identified miR-100 as significantly downregulated. Inactivation of miR-100 enhanced GvHD indicating a protective role for miR-100 via blocking inflammatory neovascularization. Our data from the mouse model and patients indicate that inflammatory neovascularization is a central event during intestinal GvHD that can be inhibited by targeting αv integrin. We identify negative regulation of GvHD-related neovascularization by miR-100, which indicates common pathomechanistic features of GvHD and ischemia.