title: Oxidation of methionine - is it limiting the diagnostic properties of 99mTc-labeled Exendin-4, a Glucagon-Like Peptide-1 receptor agonist?
abstract: BACKGROUND
  Preliminary clinical evaluation of 99mTc-EDDA/HYNIC-Met14-Exendin-4 showed that the complex offers new diagnostic possibilities for insulinoma and MTC. Exendin-4 contains methionine at position 14 in the amino acid chain, which may be oxidized to methionine sulfoxide and, from the pharmaceutical point of view, the oxidized moiety becomes an undesired impurity in the final radioactive preparation. Therefore, the aim of this study was to investigate the influence of commonly used methods to eliminate the effect of methionine oxidation in peptides, i.e. the replacement of methionine by norleucine (Nle) and the addition of L-methionine, on the in vitro stability and the biodistribution.
  
  
  METHODS
  99mTc-EDDA/HYNIC-Met14-Exendin-4, 99mTc-EDDA/HYNIC-Nle14-Exendin-4, 99mTc-EDDA/HYNIC-Met14-Ex-endin-4 with the addition of L-methionine and an oxidized form of Exendin-4, i.e. 99mTc-EDDA/HYNIC-Met14(ox)-Exendin-4 were compared in vivo with 68Ga-NODAGA-Nle14-Exendin-4 in normal Wistar rats. The stability and lipophilicity were determined in vitro.
  
  
  RESULTS
  Biodistribution studies confirmed the specific uptake of all tested complexes in the GLP-1 positive organs: lungs, pancreas and stomach. The uptake of 99mTc-EDDA/HYNIC-Met14-Exendin-4 with the addition of L-methionine and for 68Ga-NODAGA-Nle14-Exendin-4 at 1h p.i. was around 2-fold higher than that of 99mTc-EDDA/HYNIC-Met14-Exendin-4 and 99mTc-EDDA/HYNIC-Nle14-Exendin-4.
  
  
  CONCLUSIONS
  Although the substitution of methionine by norleucine in the HYNIC-Exendin-4 did not result in improved bio-distribution, the use of L-methionine, as the excipient that inhibits the oxidation of methionine in the peptide chain resulted in higher lung/blood and stomach/blood uptake ratios. Our results confirmed that methionine at position 14 of amino acid chain of Exendin-4 plays an important role in the interaction with GLP-1 receptor positive tissue.
authors.forename: Barbara
authors.initials: B
authors.lastname: Janota
authors.forename: Urszula
authors.initials: U
authors.lastname: Karczmarczyk
authors.forename: Ewa
authors.initials: E
authors.lastname: Laszuk
authors.forename: Piotr
authors.initials: P
authors.lastname: Garnuszek
authors.affiliation: National Centre for Nuclear Research, Radioisotope Centre POLATOM, Otwock, Poland. r.mikolajczak@polatom.pl.
authors.forename: Renata
authors.initials: R
authors.lastname: Mikołajczak
mesh_terms: D000818:Animals; D004355:Drug Stability; D000077270:Exenatide; D057927:Hydrophobic and Hydrophilic Interactions; D007553:Isotope Labeling; D008715:Methionine / Q000737:chemistry*; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry* / Q000493:pharmacokinetics / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013667:Technetium / Q000737:chemistry*; D014018:Tissue Distribution; D014688:Venoms / Q000737:chemistry* / Q000493:pharmacokinetics / Q000494:pharmacology; D000097789:Glucagon-Like Peptide-1 Receptor Agonists
chemical_list: D010455:Peptides; D014688:Venoms; D013667:Technetium; D000077270:Exenatide; D008715:Methionine; D000097789:Glucagon-Like Peptide-1 Receptor Agonists
keywords: 68Ga-NODAGA-Nle14-Exendin-4; 99mTc-EDDA/HYNIC-Met14-Exendin-4; 99mTc-EDDA/HYNIC-Nle14-Exendin-4; GLP-1; L-methionine; methionine sulfoxide