title: High metabolic in vivo stability and bioavailability of a palmitoylated ghrelin receptor ligand assessed by mass spectrometry.
abstract: The constitutive activity of the ghrelin receptor is of high physiological and pathophysiological relevance. In-depth structure-activity relationship studies revealed a palmitoylated ghrelin receptor ligand that displays an in vitro binding affinity in the low nanomolar range. Activity studies revealed inverse agonistic as well as antagonistic properties and in vitro metabolic analysis indicated a high stability in blood serum and liver homogenate. For metabolic testing in vivo, a combined approach of stable isotopic labeling and mass spectrometry-based analysis was established. Therefore, a heavy isotopic version of the peptide containing a (13)C-labeled palmitic acid was synthesized and a 1:1 ratio of a (12)C/(13)C-peptide mixture was injected into rats. Biological samples were analyzed by multiple reaction monitoring allowing simultaneous peptide detection and quantification. Measurements revealed a suitable bioavailability over 24h in rat serum and subsequent high-resolution mass spectrometry investigations showed only negligible degradation and slow body clearance. Hence, this method combination allowed the identification and evaluation of a highly potent and metabolically stable ghrelin receptor ligand in vivo.
authors.affiliation: Faculty of Biosciences, Pharmacy and Psychology, Institute of Biochemistry, Universität Leipzig, Brüderstraße 34, 04103 Leipzig, Germany.
authors.forename: Katja B
authors.initials: KB
authors.lastname: Kostelnik
authors.affiliation: Faculty of Biosciences, Pharmacy and Psychology, Institute of Biochemistry, Universität Leipzig, Brüderstraße 34, 04103 Leipzig, Germany.
authors.forename: Sylvia
authors.initials: S
authors.lastname: Els-Heindl
authors.affiliation: IFB AdiposityDiseases, Junior Research Group Animal Models, Universität Leipzig, Liebigstraße 21, 04103 Leipzig, Germany.
authors.forename: Nora
authors.initials: N
authors.lastname: Klöting
authors.affiliation: Faculty of Biosciences, Pharmacy and Psychology, Institute of Biochemistry, Universität Leipzig, Brüderstraße 34, 04103 Leipzig, Germany; Department of Metabolomics, Helmholtz Centre for Environmental Research-UFZ, Permoserstraße 15, 04318 Leipzig, Germany.
authors.forename: Sven
authors.initials: S
authors.lastname: Baumann
authors.affiliation: Department of Metabolomics, Helmholtz Centre for Environmental Research-UFZ, Permoserstraße 15, 04318 Leipzig, Germany; Department of Proteomics, Helmholtz Centre for Environmental Research-UFZ, Permoserstraße 15, 04318 Leipzig, Germany; Department of Biotechnology, Chemistry and Environmental Engineering, University of Aalborg, Denmark.
authors.forename: Martin
authors.initials: M
authors.lastname: von Bergen
authors.affiliation: Faculty of Biosciences, Pharmacy and Psychology, Institute of Biochemistry, Universität Leipzig, Brüderstraße 34, 04103 Leipzig, Germany. Electronic address: beck-sickinger@uni-leipzig.de.
authors.forename: Annette G
authors.initials: AG
authors.lastname: Beck-Sickinger
mesh_terms: D000818:Animals; D001682:Biological Availability; D019556:COS Cells; D002247:Carbon Isotopes; D002522:Chlorocebus aethiops; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004355:Drug Stability; D006801:Humans; D008024:Ligands; D054878:Lipoylation; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D019308:Palmitic Acid / Q000737:chemistry / Q000493:pharmacokinetics; D010455:Peptides / Q000097:blood / Q000737:chemistry / Q000493:pharmacokinetics*; D011917:Rats, Inbred Lew; D054440:Receptors, Ghrelin / Q000819:agonists / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D002247:Carbon Isotopes; D008024:Ligands; D010455:Peptides; D054440:Receptors, Ghrelin; D019308:Palmitic Acid
keywords: Ghrelin receptor; Inverse agonist; Mass spectrometry; Metabolic stability; Stable isotopic labeling