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Item does not contain fulltext, Positron emission tomography (PET) is a molecular imaging modality that enables non-invasive visualization of tracer distribution and pharmacology. Recently, peptides with long half-lives allowed once-a-week dosing of glucagon-like peptide-1 receptor (GLP-1R) agonists with therapeutic applications in diabetes and obesity. PET imaging for such long-lived peptides is hindered by the typically used short-lived radionuclides. Zirconium-89 ((89)Zr) emerged as a promising PET radionuclide with a sufficiently long half-life to be applied for biodistribution studies of long-circulating biomolecules. A comparison between the biodistribution profiles obtained via (89)Zr-PET and the current standard, quantitative whole-body autoradiography (QWBA), will be valuable for the development of novel peptide drugs. We determined the PET biodistribution of a (89)Zr-labeled acylated peptide agonist of GLP-1R and compared it to the profile obtained by QWBA using analogous tritiated tracers for up to 1 week after administration. The plasma metabolic profile was obtained and identification was done for the tritiated tracers. We found that, at early time points, the biodistribution profiles agreed between PET and QWBA. At the latertime points, the (89)Zr tracer remained primarily trapped in the kidneys. The introduction of desferrioxamine (DFO) chelator reduced the peptide stability, and UPLC-MS analysis identified a circulating metabolite arising from DFO hydrolysis. Kidney accumulation of radiolabeled peptides and DFO metabolic instability may compromise biodistribution studies using (89)Zr-PET to support the development of new biopharmaceuticals. PET and QWBA biodistribution data correlated well during the absorption phase, but new and more stable (89)Zr chelators are needed for a more accurate description of the elimination phase.

Contains fulltext : 220838.pdf (Publisher’s version ) (Open Access), INTRODUCTION: Lately, zirconium-89 has shown great promise as a radionuclide for PET applications of long circulating biomolecules. Here, the design and synthesis of protracted and long-lived GLP-1 receptor agonists conjugated to desferrioxamine and labelled with zirconium-89 is presented with the purpose of studying their in vivo distribution by PET imaging. The labelled conjugates were evaluated and compared to a non-labelled GLP-1 receptor agonist in both in vitro and in vivo assays to certify that the modification did not significantly alter the peptides' structure or function. Finally, the zirconium-89 labelled peptides were employed in PET imaging, providing visual verification of their in vivo biodistribution. METHODS: The evaluation of the radiolabelled peptides and comparison to their non-labelled parent peptide was performed by in vitro assays measuring binding and agonistic potency to the GLP-1 receptor, physicochemical studies aiming at elucidating change in peptide structure upon bioconjugation and labelling as well as an in vivo food in-take study illustrating the compounds' pharmacodynamic properties. The biodistribution of the labelled GLP-1 analogues was determined by ex vivo biodistribution and in vivo PET imaging. RESULTS: The results indicate that it is surprisingly feasible to design and synthesize a protracted, zirconium-89 labelled GLP-1 receptor agonist without losing in vitro potency or affinity as compared to a non-labelled parent peptide. Physicochemical properties as well as pharmacodynamic properties are also maintained. The biodistribution in rats shows high accumulation of radiolabelled peptide in well-perfused organs such as the liver, kidney, heart and lungs. The PET imaging study confirmed the findings from the biodistribution study with a significant high uptake in kidneys and presence of activity in liver, heart and larger blood vessels. CONCLUSIONS AND ADVANCES IN KNOWLEDGE: This initial study indicates the potential to monitor t

Disorders in lipoprotein metabolism are critical in the etiology of several disease states such as coronary heart disease and atherosclerosis, Thus, there is considerable interest in the development of novel methods for the analysis of lipoprotein complexes. We report here a simple chromatographic method for the separation of highdensity lipoprotein, low-density lipoprotein, and very low-density lipoprotein from intact serum or plasma. The separation was achieved using a hydroxyapatite column and elution with pH 7.4 phosphate buffer with 100-muL injections of whole plasma. Coelution of HDL with plasma proteins such as albumin occurred, and this clearly limits quantitation of that species by HPLC peak integration. We also show, for the first time, the application of directly coupled HPLC H-1 NMR spectroscopy to confirm the identification of the three major lipoproteins, The full chromatographic run time was 90 min with stopped-now 600-MHz NMR spectra of each lipoprotein being collected using 128 scans, in 7 min. The H-1 NMR chemical shifts of lipid signals were identical to conventional NMR spectra of freshly prepared lipoprotein standards, confirming that the lipoproteins were not degraded by the HPLC separation and that their gross supramolecular organization was intact.

The on-line separation and structure elucidation of naphthodianthrones, flavonoids, and other constituents of an extract from Hypericum perforatum L, using high performance liquid chromatography (HPLC) coupled on-line with ultraviolet-visible, nuclear magnetic resonance (NMR), and mass spectrometry (MS) is described. A conventional reversed-phase HPLC system using ammonium acetate as the buffer substance in the eluent tvas used, and proton NMR spectra were obtained on a 500 MHz NMR instrument. The MS and MS/MS analyses were performed using negative electrospray ionization, In the present study, all of the major known constituents in extracts from Hypericum perforatum L, were identified, and two new substances which had not previously been reported as constituents of extracts of Hypericum perforatum L. were identified and their structures elucidated.