title: Identification of Dmt-D-Lys-Phe-Phe-OH as a highly antinociceptive tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20.
abstract: BACKGROUND
  Recently, we presented a novel compound (PK20, Dmt-D-Lys-Phe-Phe-Lys-Lys-Pro-Phe-Tle-Leu-OH) that targets single entity opioid and neurotensin pharmacophores. This endomorphin-2-like opioid peptide was introduced as a highly active analgesic because it elicited a strong dose- and time-dependent antinociceptive response when administered centrally and peripherally. Its pain-relieving activity was observed as rapidly as 5 min after drug injection. Such promising results led us to perform further studies, such as determining the resistance to enzymatic degradation, which resulted in obtaining a very stable opioid pharmacore PK20 metabolite.
  
  
  METHODS
  The synthesis of PK20 and its N-terminal tetrapeptide fragment has been accomplished using solid phase peptide chemistry. The biological stability of peptides has been measured in human serum and analyzed by HPLC/MS. Peptides were pharmacologically characterized in in vitro MOP and DOP receptor binding as well as [(35)S]GTPγS receptor binding assays. Antinociceptive properties of compounds were measured by in vivo assays in C57Bl6 mice after intravenous or intrathecal applications.
  
  
  RESULTS
  Dmt-D-Lys-Phe-Phe-OH (PK20M), an N-terminal tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20, is characterized by a long duration of action, as demonstrated by a preserved, long-lasting analgesic effect even 2 h post-injection (average % MPE = 69.33). In rat brain membranes, PK20M efficiently displaced both the MOP and DOP receptor selective radioprobes [(3)H]DAMGO and [(3)H]DIDI (pKi of 9.52 and 7.86, respectively) and potently stimulated [(35)S]GTPγS binding, proving full agonism at both receptor types. In the [(35)S]GTPγS assay, which measured the agonist-mediated G protein activation, PK20M together with PK20 and Met-enkephalin were potent stimulators of the regulatory G proteins. The relative affinities of PK20M for the μ and δ receptor subtypes revealed μ-receptor selectivity.
  
  
  CONCLUSIONS
  The novel MOP receptor selective metabolite has been shown to possess opioid subtype receptor selectivity, high potency, and effective analgesic activities as measured in various bioassays.
authors.affiliation: Mossakowski Medical Research Centre, Polish Academy of Sciences, Pawińskiego 5, PL 02-106 Warszawa, Poland. andrzej@lipkowski.org.
authors.forename: Patrycja
authors.initials: P
authors.lastname: Kleczkowska
authors.forename: Engin
authors.initials: E
authors.lastname: Bojnik
authors.forename: Anna
authors.initials: A
authors.lastname: Leśniak
authors.forename: Piotr
authors.initials: P
authors.lastname: Kosson
authors.forename: Isabelle
authors.initials: I
authors.lastname: Van den Eynde
authors.forename: Steven
authors.initials: S
authors.lastname: Ballet
authors.forename: Sandor
authors.initials: S
authors.lastname: Benyhe
authors.forename: Dirk
authors.initials: D
authors.lastname: Tourwé
authors.forename: Andrzej W
authors.initials: AW
authors.lastname: Lipkowski
mesh_terms: D000701:Analgesics, Opioid / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000187:drug effects; D004355:Drug Stability; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D009496:Neurotensin / Q000737:chemistry* / Q000378:metabolism; D018847:Opioid Peptides / Q000737:chemistry* / Q000378:metabolism; D010147:Pain Measurement / Q000187:drug effects*; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology*; D011869:Radioligand Assay; D051381:Rats; D017465:Receptors, Opioid, delta / Q000819:agonists*; D017450:Receptors, Opioid, mu / Q000819:agonists
chemical_list: D000701:Analgesics, Opioid; D018847:Opioid Peptides; D010455:Peptides; D017465:Receptors, Opioid, delta; D017450:Receptors, Opioid, mu; D009496:Neurotensin; D019204:GTP-Binding Proteins