Potency, selectivity and prolonged binding of saxagliptin to DPP4: maintenance of DPP4 inhibition by saxagliptin in vitro and ex vivo when compared to a rapidly-dissociating DPP4 inhibitor

Abstract

BackgroundDipeptidylpeptidase 4 (DPP4) inhibitors have clinical benefit in patients with type 2 diabetes mellitus by increasing levels of glucose-lowering incretin hormones, such as glucagon-like peptide -1 (GLP-1), a peptide with a short half life that is secreted for approximately 1 hour following a meal. Since drugs with prolonged binding to their target have been shown to maximize pharmacodynamic effects while minimizing drug levels, we developed a time-dependent inhibitor that has a half-life for dissociation from DPP4 close to the duration of the first phase of GLP-1 release.ResultsSaxagliptin and its active metabolite (5-hydroxysaxagliptin) are potent inhibitors of human DPP4 with prolonged dissociation from its active site (Ki = 1.3 nM and 2.6 nM, t1/2 = 50 and 23 minutes respectively at 37°C). In comparison, both vildagliptin (3.5 minutes) and sitagliptin ( < 2 minutes) rapidly dissociated from DPP4 at 37°C. Saxagliptin and 5-hydroxysaxagliptin are selective for inhibition of DPP4 versus other DPP family members and a large panel of other proteases, and have similar potency and efficacy across multiple species.Inhibition of plasma DPP activity is used as a biomarker in animal models and clinical trials. However, most DPP4 inhibitors are competitive with substrate and rapidly dissociate from DPP4; therefore, the type of substrate, volume of addition and final concentration of substrate in these assays can change measured inhibition. We show that unlike a rapidly dissociating DPP4 inhibitor, inhibition of plasma DPP activity by saxagliptin and 5-hydroxysaxagliptin in an ex vivo assay was not dependent on substrate concentration when substrate was added rapidly because saxagliptin and 5-hydroxysaxagliptin dissociate slowly from DPP4, once bound. We also show that substrate concentration was important for rapidly dissociating DPP4 inhibitors.ConclusionsSaxagliptin and its active metabolite are potent, selective inhibitors of DPP4, with prolonged dissociation from its active site. They also demonstrate prolonged inhibition of plasma DPP4 ex vivo in animal models, which implies that saxagliptin and 5-hydroxysaxagliptin would continue to inhibit DPP4 during rapid increases in substrates in vivo.

TAK-063, a PDE10A Inhibitor with Balanced Activation of Direct and Indirect Pathways, Provides Potent Antipsychotic-Like Effects in Multiple Paradigms

“…In general, faster offrate enzyme inhibitors are more sensitive than slower offrate inhibitors to binding inhibition by enzyme substrates. For example, the faster off-rate dipeptidyl peptidase-4 inhibitor sitagliptin, but not the slower off-rate inhibitor saxagliptin, showed decreased inhibitory activity at higher concentrations of substrate compared with lower concentrations (Wang et al, 2012). Similarly, TAK-063 binding to PDE10A was more readily inhibited by lower concentrations of cyclic nucleotides than MP-10 ( Figure 4).…”

A [32P]NAD+-based method to identify and quantitate long residence time enoyl-acyl carrier protein reductase inhibitors

“…Residence times determined using the direct dissociation method developed here were compared with those measured using progress curve analysis, which is widely used method for analyzing time-dependent enzyme inhibition [11, 13, 36-38]. The direct dissociation method was first evaluated using a series of diphenyl ether inhibitors of the M. tuberculosis FabI (InhA).…”

“…Progress curve assays are robust and have been widely used to analyze time-dependent inhibition in systems such as FabI [11, 13, 36-38]. While thermodynamic and kinetic constants can be derived from progress curves yielding a detailed picture of the inhibition reaction coordinate [39], some caution should be exercised when applying this approach to highly potent compounds with very long residence times.…”

Clinical Pharmacokinetics and Pharmacodynamics of Saxagliptin, a Dipeptidyl Peptidase-4 Inhibitor

“…In vitro enzyme kinetic binding studies indicate that 5-hydroxy saxagliptin is approximately twofold less potent than parent saxagliptin with respect to DPP-4 inhibition at 37°C [2]. Thus, when the clinical pharmacology profile of saxagliptin is considered, particularly in situations in which the metabolite-toparent ratio is altered relative to a reference state or population (e.g.…”

“…Saxagliptin (Onglyza Ò ; AstraZeneca AB, Södertälje, Sweden, and AstraZeneca; Wilmington, DE, USA) is a highly potent, selective, reversible and competitive dipeptidyl peptidase (DPP)-4 inhibitor [1,2]. DPP-4 is an enzyme that selectively cleaves dipeptides from the N-terminus of oligopeptides with proline or alanine in the penultimate position [3].…”