- Publikační typ
- abstrakt z konference MeSH
The synthetic analogs of regulatory peptides radiolabeled with adequate radionuclides are perspective tools in nuclear medicine. However, undesirable uptake and retention in the kidney limit their application. Specific in vitro methods are used to evaluate undesirable renal accumulation. Therefore, we investigated the usefulness of freshly isolated rat renal cells for evaluating renal cellular uptake of receptor-specific peptide analogs. Special attention was given to megalin as this transport system is an important contributor to the active renal uptake of the peptides. Freshly isolated renal cells were obtained from native rat kidneys by the collagenase method. Compounds with known accumulation in renal cells were used to verify the viability of cellular transport systems. Megalin expressions in isolated rat renal cells were compared to two other potential renal cell models by Western blotting. Specific tubular cell markers were used to confirm the presence of proximal tubular cells expressing megalin in isolated rat renal cell preparations by immunohistochemistry. Colocalization experiments on isolated rat kidney cells confirmed the presence of proximal tubular cells bearing megalin in preparations. The applicability of the method was tested by an accumulation study with several analogs of somatostatin and gastrin labeled with indium-111 or lutetium-177. Therefore, isolated rat renal cells may be an effective screening tool for in vitro analyses of renal uptake and comparative renal accumulation studies of radiolabeled peptides or other radiolabeled compounds with potential nephrotoxicity.
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
UNLABELLED: As angiogenesis plays a key role in tumor growth and metastasis, the angiogenic process has attracted scientific interest as a target for diagnostic and therapeutic agents. Factors influencing angiogenesis include the vascular endothelial growth factor (VEGF) family and the two associated receptor types (VEGFR-1 and VEGFR-2). VEGFR-1/-2 detection and quantification in cancer lesions are essential for tumor process management. As a result of the advantageous pharmacokinetics and image contrast, peptides radiolabeled with PET emitters have become interesting tools for the visualization of VEGFR-1/-2-positive tumors. In this study, we prepared 68Ga-labeled peptides containing 15 (peptide 1) and 23 (peptide 2) amino acids as new PET tracers for tumor angiogenic process imaging. METHODS: The peptides were conjugated with NODAGA-tris(t-Bu ester) and subsequently radiolabeled with [68Ga]Ga-chloride. The prepared [68Ga]Ga-NODAGA-peptide 1 and [68Ga]Ga-NODAGA-peptide 2 were tested for radiochemical purity and saline/plasma stability. Consequently, the binding affinity toward VEGFRs was assessed in vitro on human glioblastoma and kidney carcinoma cells. The found peptide receptor affinity was compared with the calculated values in the PROtein binDIng enerGY prediction (PRODIGY) server. Finally, the biodistribution study was performed on BALB/c female mice to reveal the basic pharmacokinetic behavior of radiopeptides. RESULTS: The in vitro affinity testing of [68Ga]Ga-NODAGA-peptides 1 and 2 showed retained receptor binding as characterized by equilibrium dissociation constant (KD) values in the range of 0.5-1.2 μM and inhibitory concentration 50% (IC50) values in the range of 3.0-5.6 μM. Better binding properties of peptide 2 to VEGFR-1/-2 were found in the PRODIGY server. The biodistribution study on mice showed remarkable accumulation of both peptides in the kidneys and urinary bladder with a short half-life after intravenous application. The in vitro plasma stability of [68Ga]Ga-NODAGA-peptide 2 was superior to that of [68Ga]Ga-NODAGA-peptide 1. CONCLUSIONS: The obtained results demonstrated a high radiolabeling yield with no need for purification and preserved binding potency of 68Ga-labeled peptides 1 and 2 toward VEGFRs in cancer cells. The peptide-receptor protein interaction assessed in protein-peptide docking determined the strongest interaction of peptide 2 with domain 2 of VEGFR-2 in addition to a more acceptable plasma stability (t1/2 = 120 min) than that for peptide 1. We found both radiolabeled peptides very potent in their receptor binding, which makes them suitable imaging agents. The rapid transition of the radiopeptides into the urinary tract indicates suitable pharmacokinetic characteristics.
- MeSH
- aminokyseliny MeSH
- chloridy MeSH
- estery MeSH
- heterocyklické sloučeniny monocyklické chemie MeSH
- lidé MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- peptidy chemie MeSH
- pozitronová emisní tomografie metody MeSH
- radioizotopy galia * chemie MeSH
- receptor 1 pro vaskulární endoteliální růstový faktor MeSH
- receptor 2 pro vaskulární endoteliální růstový faktor MeSH
- receptory vaskulárního endoteliálního růstového faktoru MeSH
- tkáňová distribuce MeSH
- vaskulární endoteliální růstový faktor A * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
Radioimmunoassay belongs to the analytical method enabling highly specific and sensitive quantification of molecules. The verification of the real-time radioimmunoassay technology usefulness for ligand-quality characteristics evaluation such as concentration, influence of radiolabeling on binding affinity and stability was estimated. The anti-epidermal growth factor receptor antibody 131 I-cetuximab was employed as the ligand antibody. The concentration of 131 I-cetuximab was derived from the shape of binding curves coming from the ligand-receptor interaction. The binding curves also allowed the estimation of 131 I-cetuximab binding affinity for different radiolabeling procedures (incubation times 1, 5, and 10 minutes) in stability testing up to 96 hours at 4°C. The stability testing also included comparative analysis by size exclusion high-performance liquid chromatography. The assessment of cetuximab concentrations using real-time method showed acceptable accordance between real and calculated values. The real-time method revealed that 1-minute radiolabeling proved to be the optimal incubation time for direct radioiodination of cetuximab. Stability testing showed the significant change in radioligand affinity by one order at the longest incubation times (72 and 96 hours). Characterization of stability and binding behavior of radiolabeled monoclonal antibodies by the verified real-time method before use in other assays may be employed to eliminate variability and suboptimal antibody performance.
- MeSH
- cetuximab chemie farmakologie MeSH
- lidé MeSH
- ligandy MeSH
- nádorové buněčné linie MeSH
- protinádorové látky imunologicky aktivní chemie farmakologie MeSH
- radiofarmaka chemie farmakologie MeSH
- radioimunoanalýza metody normy MeSH
- radioizotopy jodu chemie farmakologie MeSH
- radioligandová zkouška metody normy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
