A phase 1 study to evaluate the safety and pharmacokinetics of PQ912, a glutaminyl cyclase inhibitor, in healthy subjects.

INTRODUCTION: Pyroglutamate-amyloid-ß (pE-Aß) peptides are major components of Aß-oligomers and Aß-plaques, which are regarded as key culprits of Alzheimer's disease (AD) pathology. PQ912 is a competitive inhibitor of the enzyme glutaminyl cyclase (QC), essential for the formation of pE-Aß peptides. METHODS: A randomized, double-blind, placebo-controlled, single- and multiple-ascending oral dose study investigated the safety, pharmacokinetics, and pharmacodynamics of PQ912 in healthy nonelderly and elderly subjects. RESULTS: PQ912 was considered safe and well tolerated with dose-proportional pharmacokinetics up to doses of 200 mg. At higher doses up to 1800 mg, exposure was supraproportional and exposure in elderly subjects was approximately 1.5- to 2.1-fold higher. Exposure in cerebrospinal fluid (CSF) was approximately 20% of the unbound drug in plasma, and both serum and CSF QC activity was inhibited in a dose-related manner. DISCUSSION: This first-in-man study of a compound-targeting QC inhibition justifies further development of PQ912 for the treatment of AD.

Selective inhibition of dipeptidyl peptidase 4 by targeting a substrate-specific secondary binding site.

Dipeptidyl peptidase 4/CD26 (DP4) is a multifunctional serine protease liberating dipeptide from the N-terminus of (oligo)peptides which can modulate the activity of these peptides. The enzyme is involved in physiological processes such as blood glucose homeostasis and immune response. DP4 substrate specificity is characterized in detail using synthetic dipeptide derivatives. The specificity constant k(cat)/K(m) strongly depends on the amino acid in P1-position for proline, alanine, glycine and serine with 5.0 x 105 M⁻¹ s⁻¹, 1.8 x 104 M⁻¹ s⁻¹, 3.6 x 10² M⁻¹ s⁻¹, 1.1 x 10² M⁻¹ s⁻¹, respectively. By contrast, kinetic investigation of larger peptide substrates yields a different pattern. The specific activity of DP4 for neuropeptide Y (NPY) cleavage comprising a proline in P1-position is the same range as the k(cat)/K(m) values of NPY derivatives containing alanine or serine in P1-position with 4 x 105 M⁻¹ s⁻¹, 9.5 x 105 M⁻¹ s⁻¹ and 2.1 x 105 M⁻¹ s⁻¹, respectively. The proposed existence of an additional binding region outside the catalytic center is supported by measurements of peptide substrates with extended chain length. This 'secondary' binding site interaction depends on the amino acid sequence in P4'-P8'-position. Interactions with this binding site could be specifically blocked for substrates of the GRF/glucagon peptide family. By contrast, substrates not belonging to this peptide family and dipeptide derivative substrates that only bind to the catalytic center of DP4 were not inhibited. This more selective inhibition approach allows, for the first time, to distinguish between substrate families by substrate-discriminating inhibitors.

[Ser2]- and [SerP2] incretin analogs: comparison of dipeptidyl peptidase IV resistance and biological activities in vitro and in vivo.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP; also known as gastric inhibitory polypeptide) are incretin hormones that reduce postprandial glycemic excursions via enhancing insulin release but are rapidly inactivated by enzymatic N-terminal truncation. As such, efforts have been made to improve their plasma stability by synthetic modification or by inhibition of the responsible protease, dipeptidyl peptidase (DP) IV. Here we report a parallel comparison of synthetic GIP and GLP-1 with their Ser2- and Ser(P)2-substituted analogs, examining receptor binding and activation, metabolic stability, and biological effects in vivo. Both incretins and their Ser2-substituted analogs showed similar EC50s (0.16-0.52 nm) and IC50s (4.3-8.1 nm) at their respective cloned receptors. Although both phosphoserine 2-modified (Ser(PO3H2); Ser(P)) peptides were able to stimulate maximal cAMP production and fully displace receptor-bound tracer, they showed significantly right-shifted concentration-response curves and binding affinities. Ser2-substituted analogs were moderately resistant to DP IV cleavage, whereas [Ser(P)2]GIP and [Ser(P)2] GLP-1 showed complete resistance to purified DP IV. It was shown that the Ser(P) forms were dephosphorylated in serum and thus in vivo act as precursor forms of Ser2-substituted analogs. When injected subcutaneously into conscious Wistar rats, all peptides reduced glycemic excursions (rank potency: [Ser(P)2]incretins > or = [Ser2] incretins > native hormones). Insulin determinations indicated that the reductions in postprandial glycemia were at least in part insulin-mediated. Thus it has been shown that despite having low in vitro bioactivity using receptor-transfected cells, in vivo potency of [Ser(P)2] incretins was comparable with or greater than that of native or [Ser2]peptides. Hence, Ser(P)2-modified incretins present as novel glucose-lowering agents.

Structure-activity relationships of glucose-dependent insulinotropic polypeptide (GIP).

Six GIP(1-NH2) analogs were synthesized with modifications (de-protonation, N-methylation, reversed chirality, and substitution) at positions 1, 3, and 4 of the N-terminus, and additionally, a cyclized GIP derivative was synthesized. The relationship between altered structure to biological activity was assessed by measuring receptor binding affinity and ability to stimulate adenylyl cyclase in CHO-K1 cells transfected with the wild-type GIP receptor (wtGIPR). These structure-activity relationship studies demonstrate the importance of the GIP N-terminus and highlight structural constraints that can be introduced in GIP analogs. These analogs may be useful starting points for design of peptides with enhanced in vivo bioactivity.

Metformin effects on dipeptidylpeptidase IV degradation of glucagon-like peptide-1.

There is current interest in the use of inhibitors of dipeptidyl peptidase IV (DP IV) as therapeutic agents to normalize glycemic excursions in type 2 diabetic patients. Data indicating that metformin increases the circulating amount of active glucagon-like peptide-1 (GLP-1) in obese nondiabetic subjects have recently been presented, and it was proposed that metformin might act as a DP IV inhibitor. This possibility has been investigated directly using a number of in vitro methods. Studies were performed on DP IV enzyme from three sources: 20% human serum, purified porcine kidney DP IV, and recombinant human DP IV. Inhibition of DP IV hydrolysis of the substrate Gly-Pro-pNA by metformin was examined spectrophotometrically. Effects of metformin on GLP-1([7-36NH2]) degradation were assessed by mass spectrometry. In addition, surface plasmon resonance was used to establish whether or not metformin had any effect on GLP-1([7-36NH2]) or GLP-1([9-36NH2]) interaction with immobilized porcine or human DP IV. Metformin failed to alter the kinetics of Gly-Pro-pNA hydrolysis or GLP-1 degradation tested according to established methods. Surface plasmon resonance recordings indicated that both GLP-1([7-36NH2]) and GLP-1([9-36NH2]) show micromolar affinity (K(D)) for DP IV, but neither interaction was influenced by metformin. The results conclusively indicate that metformin does not act directly on DP IV, therefore alternative explanations for the purported effect of metformin on circulating active GLP-1 concentrations must be considered.