Identification of neuropeptide Y cleavage products in human blood to improve metabolic stability

Abstract

Regulatory, receptor‐binding peptides are considered as the agents of choice for diagnostic imaging and therapy of cancers, because their receptors are overexpressed in various human cancer cells. It has been recently indicated that there is a putative role of NPY in breast tumors. The expression of the two best‐investigated NPY receptor subtypes, Y1 and Y2, in breast tissue shows predominant occurrence of the Y1 receptor subtype in tumors, whereas Y2 receptors are found in nonproliferative tissue. To investigate the usefulness of NPY analogs for tumor diagnosis and therapy, we investigated the metabolic stability of receptor‐selective NPY analogs in human blood plasma. NPY analogs were synthesized by Fmoc/t‐Bu solid‐phase strategy. Prior to the cleavage of peptides from the resin, they were labeled with 5(6)‐carboxyfluorescein (CF) either at the N‐terminus or at the side chain of Lys4. For the metabolic stability study, the digestion of peptides was monitored by HPLC and the cleavage products were identified by MALDI‐ToF mass spectrometry. The data showed that full‐length [Phe7, Pro34]NPY analogs, which show high binding affinity to Y1 receptors are enzymatically more stable than centrally truncated analogs, which show high binding affinity to Y2 receptors. Furthermore, the N‐terminally CF‐labeled Y1 and Y2 receptor‐selective peptides were found to be enzymatically more resistant than their counterparts containing the CF label at Lys4 side chain. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 88: 182–189, 2007. This article was originally published online as an accepted preprint. The ‘Published Online’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

In vivo and in vitro degradation of peptide YY3–36 to inactive peptide YY3–34 in humans

“…Previous studies have shown that in vitro incubations of NPY in human plasma resulted in cleavage of the COOHterminal amino acid and dipeptide (1,22). We have demonstrated that PYY is significantly degraded to PYY in the pig and also shown that the liver is involved in the COOHterminal truncation (35).…”

“…When measuring PYY , it turned out that the plasma levels were comparable after PYY and PYY infusion. This discrepancy might be explained by the longer half-life of PYY , or it could be that the NH 2 -terminal structure influences COOH-terminal degradation, as has been suggested to be the case for in vitro degradation of NPY (22). PYY was not active on the Y2 receptor, and the COOH-terminal degradation is therefore likely to represent an inactivation and elimination step; however, it cannot at the current stage be excluded that PYY 3-34 exerts a physiological effect via a different receptor.…”

“…PYY and NPY are COOH terminally amidated and therefore considered relatively protected from COOH-terminal degradation. However, COOH-terminal degradation of NPY, rendering the peptide inactive on the Y1-and Y2-receptors, has been demonstrated in vitro (1,9,22,24). As the sequences of NPY and PYY are 70% homologous, we speculated that PYY might also be prone to COOH-terminal degradation.…”

Breast‐Cancer Diagnosis by Neuropeptide Y Analogues: From Synthesis to Clinical Application

“…[14] Time-dependent incubation showed halflives of 9.9 to 32 h; these values are in the same range or slightly reduced for the Y 1 receptor selective peptides compared to NPY (t 1/2 = 24.7 AE 1.4 h) (Figure 1 c). [15,16] For radiolabeling of the highly selective peptides 2 a and 2 b, an approved protocol [17][18][19] was used, yielding in 99m Tc V species, which are denoted 99m Tc(core) 3+ . Because the interaction between plasma proteins and peptides directly and indirectly affects different pharmacokinetic parameters, such as volume of distribution, metabolism and excretion of the drugs, and accordingly the dosage, [20] protein binding of 99m Tc(core) 3+ -labeled peptides 2 a and 2 b were studied in vitro in human blood at several time intervals and showed high stability of the formed 99m Tc(core) 3+ -peptide complex.…”

Targeted Tumor Diagnosis and Therapy with Peptide Hormones as Radiopharmaceuticals

“…Such a structure as the recently identified analog that contains two -cyclopropane residues might be a promising lead structure for the application of NPY in radiopharmacy. Further advances towards the application of NPY in radiopharmacy include selective labeling [165] as well as the investigated metabolic stability [166]. Further advances towards the application of NPY in radiopharmacy include selective labeling [165] as well as the investigated metabolic stability [166].…”