Elsevier

Biochemical Pharmacology

Volume 77, Issue 2, 15 January 2009, Pages 228-237
Biochemical Pharmacology

In vivo profiling of DPP4 inhibitors reveals alterations in collagen metabolism and accumulation of an amyloid peptide in rat plasma

https://doi.org/10.1016/j.bcp.2008.09.032Get rights and content

Abstract

Dipeptidyl peptidase 4 (DPP4) inhibitors represent a novel class of oral anti-hyperglycemic agents. The complete pharmacological profile of these protease inhibitors remains unclear. In order to gain deeper insight into the in vivo effects caused by DPP4 inhibition, two different DPP4 inhibitors (vildagliptin and AB192) were analyzed using differential peptide display. Wistar rats were treated with the DPP4 inhibitors (0.3 mg kg−1; 1 mg kg−1 or 3 mg kg−1 body weight) and DPP4 activity was measured before and at the end of the experiment. One hour after compound administration, blood plasma samples were collected to generate peptide displays and to subsequently identify differentially regulated peptides. A dose-dependent decrease in blood plasma DPP4 activity was measured for both inhibitors. DPP4 inhibition influenced collagen metabolism leading to depletion of collagen derived peptides (e.g. collagen alpha 1 (III) 521–554) and accumulation of related N-terminally extended collagen derived peptides (e.g. collagen alpha 1 (III) 519–554). Furthermore, the intact amyloid rat BRI (1–23) peptide was detected in plasma following in vivo DPP4 inhibition. DPP4 catalyzed cleavage kinetics of the BRI peptide were determined in vitro. The kcat and Km for cleavage by DPP4 were 5.2 s−1 and 14 μM, respectively, resulting in a specificity constant kcat/Km of 0.36 × 106 s−1 M−1. Our results demonstrate that differential peptide analysis can be applied to monitor action of DPP4 inhibition in blood plasma. For the first time effects on basal collagen metabolism following DPP4 inhibition in vivo were demonstrated and the BRI amyloid peptide was identified as a novel DPP4 substrate.

Introduction

Dipeptidyl peptidase 4 (DPP4) inhibitors such as sitagliptin and vildagliptin represent a new class of antidiabetic agents that improve glycemic control by preventing glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) degradation. These intestinal peptides, also known as incretins, are postprandially secreted from enteroendocrine L- and K-cells and lead to a rise in insulin secretion. Preclinical [1], [2], [3], [4] and clinical studies [5], [6], [7], [8], [9], [10], [11] show that inhibition of DPP4 decreases blood glucose levels but long-term data on efficiency and safety are lacking. Several other peptides were identified in vitro as efficient DPP4 substrates, including chemokines (e.g. stromal cell derived factor 1 alpha or CXCL12, MDC or CCL22, and RANTES or CCL5), pancreatic polypeptide family members (e.g. neuropeptide Y), as well as diverse other substrates (e.g. endomorphin, kentsin). The in vivo relevance of these findings is not clear at the moment, but needs to be investigated [12], [13], [14], [15]. Blocking N-terminal truncation of peptides by DPP4 inhibition might lead to accumulation of the corresponding substrate and hence influence, for example, downstream mediated receptor signaling events. To evaluate the potential pharmacological effects of in vivo DPP4 inhibition independently of its glucose lowering properties and to identify physiological substrates of DPP4, we subcutaneously treated Wistar rats with two different DPP4 inhibitors – the experimental, irreversible inhibitor AB192, and the clinically used, reversible inhibitor vildagliptin – and evaluated endogenous blood plasma peptides using differential peptide display, which allows the identification of multiple changes in blood plasma composition on the peptide level (<15 kDa) using a combination of reverse phase HPLC, offline MALDI-MS and a bioinformatic data analysis. Signals of interest are sequenced and identified to elucidate DPP4 inhibitor related effects and evaluate pharmacological responses caused by therapeutic intervention. In contrast to in vitro experiments where inhibitors are tested usually against selected proteases or proteases against limited sets of possible substrates (peptide libraries), in vivo protease inhibitor profiling allows to test their action against all endogenous proteases and influences on natural substrates [16]. Similar to our approach, Yates et al. [17] used differential mass spectrometry for identifying substrates of DPP4 and aminopeptidase P2 in human plasma in vitro. Using this technique, we identified the BRI peptide as DPP4 substrate in rat plasma and were able to show for the first time the influence of DPP4 inhibition on the collagen metabolism in vivo.

Section snippets

Materials and methods

Reagents if not otherwise specified were purchased from Sigma–Aldrich, Munich, Germany.

DPP4 inhibition in vivo

One hour after the administration of DPP4 inhibitors (AB192 and vildagliptin), Wistar rats showed a dose-dependent decrease in blood plasma DPP4 activity reaching a maximal inhibition of approximately 80% at 3 mg kg−1 body weight as displayed in Fig. 1. The data reveal that vildagliptin inhibits DPP4 activity already by ∼50% at a dosage of 0.3 mg kg−1 body weight, whereas AB192 sufficiently inhibits DPP4 only in the higher dosage range (>1 mg kg−1 body weight). Similar results for vildagliptin were

Discussion

In the present study, we show that in vivo inhibition of DPP4 activity in rats using either the experimental irreversible DPP4 inhibitor AB192 or the clinically used inhibitor vildagliptin leads to pronounced effects on the collagen metabolism and accumulation of the so far unknown DPP4 substrate BRI in blood plasma. Thereby, vildagliptin appears to be the more potent inhibitor compared to AB192 by sufficiently inhibiting DPP4 activity already in the lower dosage range.

To the best of our

Acknowledgements

We gratefully acknowledge the superb technical assistance of Christina Grethe, Claudia Hubner, Michaela Brandt, Sigfried Zimmermann, Kerstin Kupke, and Vincent Hanebuth (all Digilab BioVisioN GmbH, Hannover, Germany). This study was supported by a grant from the Bundesministerium für Bildung und Forschung (PTJ-BIO/Wir/0313624). Part of this work was presented on the 2nd International Conference on Dipeptidyl Aminopeptidases, Magdeburg, Germany (2005). “Differential Peptide Display”,

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