Preparation and comparative evaluation of ^99mTc‐HYNIC‐cNGR and ^99mTc‐HYNIC‐PEG₂‐cNGR as tumor‐targeting molecular imaging probes

Abstract

The tripeptide sequence asparagine-glycine-arginine (NGR) specifically recognizes aminopeptidase N (APN or CD13) receptors highly expressed on tumor cells and vasculature. Thus, NGR peptides can precisely deliver therapeutic and diagnostic compounds to CD13 expressing cancer sites. In this regard, 2 NGR peptide ligands, HYNIC-c(NGR) and HYNIC-PEG -c(NGR), were synthesized, radiolabeled with Tc, and evaluated in CD13-positive human fibrosarcoma HT-1080 tumor xenografts. The radiotracers, Tc-HYNIC-c(NGR) and Tc-HYNIC-PEG -c(NGR), could be prepared in approximately 95% radiochemical purity and exhibited excellent in vitro and in vivo stability. The radiotracers were hydrophilic in nature with log P values being -2.33 ± 0.05 and -2.61 ± 0.08. The uptake of 2 radiotracers Tc-HYNIC-c(NGR) and Tc-HYNIC-PEG -c(NGR) was similar in nude mice bearing human fibrosarcoma HT-1080 tumor xenografts, which was significantly reduced (P < .05) during blocking studies. The 2 radiotracers being hydrophilic cleared rapidly from blood, liver, and intestine and were excreted through renal pathway. The pharmacokinetics of Tc-labeled HYNIC peptide could not be modulated through introduction of PEG unit, thus posing a challenge for studies with other linkers towards enhanced tumor uptake and retention.

Design, synthesis, and comparative evaluation of 99mTc(CO)3‐labeled N‐terminal and C‐terminal modified asparagine–glycine–arginine peptide constructs

“…99m Tc‐labeled monomeric ( 99m Tc‐NGR1) and dimeric ( 99m Tc‐NGR2) NGR peptides were studied by Ma et al in HepG2 hepatoma xenografts hence cannot be directly compared . The dimer 99m Tc‐NGR2 however exhibited substantially high uptake in blood even at 4 hours p.i.. Our earlier studies with 99m Tc‐HYNIC‐c (NGR) and 99m Tc‐HYNIC‐PEG 2 ‐c (NGR) in human fibrosarcoma HT‐1080 tumor xenografts demonstrated lower tumor uptake but better tumor/blood and tumor/liver ratios …”

“…TNhe fully protected N‐terminal azide modified peptide H‐Lys(N 3 )‐Cys (Acm)‐Asn (Trt)‐Gly‐Arg (Pbf)‐Cys (Acm)‐Gly‐NH2 was assembled on Rink amide MBHA resin manually by standard Fmoc solid phase peptide synthesis . Coupling of each amino acid was accomplished by adding three‐fold excess of Fmoc‐amino acid as well as HATU along with six‐fold excess of N,N‐Diisopropylethylamine (DIPEA) in N,N‐Dimethylformamide (DMF) and shaking the reaction mixture for 120 minutes.…”

“…The CD13 receptors specifically recognize asparagine–glycine–arginine (NGR) motif containing peptides . Radiometalated NGR peptide constructs are hence being explored as molecular imaging probes for detection and monitoring of CD13 receptor‐expressing malignant sites . According to a literature report, carboxyl terminal of cyclic NGR [cCNGRC] peptide is a favorable position for tagging radioisotopes/drugs rather than N‐terminal that plays more important role in receptor binding .…”

“…Radiometalation of NGR peptides has been reported with 64 Cu, 68 Ga, and 99m Tc for diagnostic purpose . However, single‐photon emission computed tomography (SPECT) radioisotope, 99m Tc has wider coverage in nuclear medicine hospitals because of lower infrastructure and financial requirement in comparison with positron emission tomography (PET) isotopes, 64 Cu and 68 Ga .…”

Molecular Imaging of Aminopeptidase N in Cancer and Angiogenesis

“…The targeted 99m Tc-HYNIC-CLB-cCNGRC probe inhibited cancer cell growth, and its high hydrophilicity promoted rapid clearance from nontarget organs [ 57 ]. In an attempt to change the pharmokinectic profile, a PEG2 linker was added to the structure, but no change was observed [ 58 ]. A recent structural study showed that reporter groups attached to the Arg-Cys-COOH terminus of cCNGRC produced targeting agents with higher affinities for APN than analogues with reporter groups attached to the NH2-Cys-Asn terminus.…”