title: Influence of biological assay conditions on stability assessment of radiometal-labelled peptides exemplified using a 177Lu-DOTA-minigastrin derivative.
abstract: BACKGROUND
  Lack of correlation between in vitro and in vivo stability is a general problem for the development of radiopeptides especially in the case of minigastrin derivatives for therapeutic applications. In this study, we compared the influence of experimental conditions on radiopeptide stability results in vitro using a model Minigastrin (MG) analogue labelled with Lu-177. Additionally, we attempted to characterize the main serum enzymatic cleavage sites by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) analysis.
  
  
  METHODS
  In vitro stability of a DOTA-minigastrin derivative ((177)Lu-DOTA-His-His-Glu-Ala-Tyr-Gly-Trp-NIe-Asp-Phe-NH(2)) was tested in serum, rat tissue homogenates and two different standardised enzymatic mixtures. Quantification of the metabolised radiopeptides at different time intervals was performed using reversed-phase high-performance liquid chromatography (RP-HPLC). Metabolites were characterised by MALDI-TOF-MS. Urine was collected after 15 min p.i. into the mice and compared with in vitro metabolites by RP-HPLC.
  
  
  RESULTS
  Faster degradation of the radiopeptide was found in blood in comparison with plasma and serum incubation and in components from rats faster than from human origin. Fast degradation was observed in kidney and liver homogenates as well as in standardised enzymatic mixtures, also revealing variations in the metabolic profile. In urine, no intact peptide was detected already 5 min post injection. MALDI-TOF-MS revealed major cleavage sites at the carboxy terminus of the peptide.
  
  
  CONCLUSIONS
  Very variable results may be found when different kind of incubation media for testing radiopeptide stabilities is used. Serum incubation studies may overestimate stability; therefore, results should be interpreted with care and combined with alternative in vitro and in vivo investigations.
authors.affiliation: Clinical Department of Nuclear Medicine, Medical University Innsbruck, A-6020, Innsbruck, Austria.
authors.forename: Meltem
authors.initials: M
authors.lastname: Ocak
authors.forename: Anna
authors.initials: A
authors.lastname: Helbok
authors.forename: Elisabeth
authors.initials: E
authors.lastname: von Guggenberg
authors.forename: Y
authors.initials: Y
authors.lastname: Ozsoy
authors.forename: Levent
authors.initials: L
authors.lastname: Kabasakal
authors.forename: Leopold
authors.initials: L
authors.lastname: Kremser
authors.forename: Clemens
authors.initials: C
authors.lastname: Decristoforo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001681:Biological Assay / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D005260:Female; D005755:Gastrins / Q000097:blood / Q000737:chemistry* / Q000378:metabolism; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry*; D006801:Humans; D007553:Isotope Labeling; D008187:Lutetium / Q000737:chemistry*; D051379:Mice; D008807:Mice, Inbred BALB C; D010447:Peptide Hydrolases / Q000378:metabolism; D055550:Protein Stability; D011868:Radioisotopes / Q000737:chemistry*; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014020:Tissue Extracts / Q000378:metabolism
chemical_list: D005755:Gastrins; D006573:Heterocyclic Compounds, 1-Ring; D011868:Radioisotopes; D014020:Tissue Extracts; C071349:1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid; C008530:minigastrin; D008187:Lutetium; D010447:Peptide Hydrolases