Sitagliptin elevates plasma and CSF incretin levels following oral administration to nonhuman primates: relevance for neurodegenerative disorders

Abstract

The endogenous incretins glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) possess neurotrophic, neuroprotective, and anti-neuroinflammatory actions. The dipeptidyl peptidase 4 (DPP-4) inhibitor sitagliptin reduces degradation of endogenous GLP-1 and GIP, and, thereby, extends the circulation of these protective peptides. The current nonhuman primate (NHP) study evaluates whether human translational sitagliptin doses can elevate systemic and central nervous system (CNS) levels of GLP-1/GIP in naive, non-lesioned NHPs, in line with our prior rodent studies that demonstrated sitagliptin efficacy in preclinical models of Parkinson's disease (PD). PD is an age-associated neurodegenerative disorder whose current treatment is inadequate. Repositioning of the well-tolerated and efficacious diabetes drug sitagliptin provides a rapid approach to add to the therapeutic armamentarium for PD. The pharmacokinetics and pharmacodynamics of 3 oral sitagliptin doses (5, 20, and 100 mg/kg), equivalent to the routine clinical dose, a tolerated higher clinical dose and a maximal dose in monkey, were evaluated. Peak plasma sitagliptin levels were aligned both with prior reports in humans administered equivalent doses and with those in rodents demonstrating reduction of PD associated neurodegeneration. Although CNS uptake of sitagliptin was low (cerebrospinal fluid (CSF)/plasma ratio 0.01), both plasma and CSF concentrations of GLP-1/GIP were elevated in line with efficacy in prior rodent PD studies. Additional cellular studies evaluating human SH-SY5Y and primary rat ventral mesencephalic cultures challenged with 6-hydroxydopamine, established cellular models of PD, demonstrated that joint treatment with GLP-1 + GIP mitigated cell death, particularly when combined with DPP-4 inhibition to maintain incretin levels. In conclusion, this study provides a supportive translational step towards the clinical evaluation of sitagliptin in PD and other neurodegenerative disorders for which aging, similarly, is the greatest risk factor.

Keywords: Dipeptidyl peptidase 4 (DPP-4); Glucagon-like peptide-1 (GLP-1); Glucose-dependent insulinotropic polypeptide (GIP); Nonhuman primate; Parkinson’s disease; Sitagliptin.

Fig. 1

Nonhuman primate protocol. Vehicle or one of three doses of sitagliptin (5, 20, or 100 mg/kg) was administered orally once daily for 6 consecutive days to rhesus monkeys (7–20 kg weight, mixed gender, 12 to 19 years of age, n = 5 to 7 per group) to attain steady-state conditions prior to a classical oral glucose load, which was administered on the “test day” (day 6) to initiate endogenous incretin (GLP-1 and GIP) release, since incretin release from L and K cells is stimulated by food presence within the gastrointestinal tract. Time-dependent plasma and CSF samples were obtained to permit measurement of (i) GIP and GLP-1 levels, (ii) sitagliptin levels, and (iii) DPP-4 activity. Notably, a 6-h liquid meal was administered to the sedated monkeys to preserve blood glucose levels since all animals were fasted overnight prior to experimental day 6. Drug/vehicle administration was initiated at the same time across all NHPs, and thus plasma and CSF collection times were the same across animals

Fig. 2

Plasma dose-dependent sitagliptin pharmacokinetics in nonhuman primates. Time-dependent plasma concentrations of sitagliptin following oral administration of one of three separate doses administered once daily (sitagliptin low 5 mg/kg, medium 20 mg/kg, high 100 mg/kg) were measured by LC/MS [43]. As noted, sitagliptin was administered at − 120 min (where zero represents the time when the oral glucose load was administered to initiate incretin release). Peak sitagliptin levels in plasma occurred at approx. 2 h post administration and were dose-dependent. All values are mean ± SEM

Fig. 3

CSF sitagliptin levels in nonhuman primates. CSF dose-dependent sitagliptin pharmacokinetics in NHPs and CSF/plasma concentration ratio were obtained at 3 and 5 h post sitagliptin oral administration. All values are mean ± SEM

Fig. 4

Time-dependent changes in GLP-1 and GIP levels following sitagliptin administration in nonhuman primates. Sitagliptin elevates plasma incretin levels in NHPs. Top: time- and dose-dependent levels of GLP-1. Bottom: similarly, of GIP in plasma following administration of vehicle or sitagliptin (5, 20, or 100 mg/kg P.O., at − 120 min), an oral glucose load (4 g/kg) at zero time and a liquid meal (6 h). Values are means. N.B. Zero time (0 min) is designated as the time of oral glucose administration to initiate incretin release, and sitagliptin was administered 120 min prior to this. A liquid meal was administered at 6 h to maintain the animals following an overnight fast

Fig. 5

Time-dependent changes in plasma DPP-4 activity following sitagliptin administration in nonhuman primates. Sitagliptin time- and dose-dependently reduces (i.e., inhibits) plasma DPP-4 activity (pmol/min). Values are mean ± SEM. DPP-4 activity changed < 5% across timepoints in animals administered vehicle alone (i.e., 0 mg sitagliptin)—not shown. N.B. The zero-time point (0 min) is designated as the time of glucose administration to initiate incretin release

Fig. 6

Time-dependent changes in incretin levels achieved in CSF following oral sitagliptin administration in nonhuman primates. Sitagliptin elevates CSF incretin levels in NHPs. Top: Time- and dose-dependent levels of GLP-1. Bottom: similarly, of GIP in CSF following oral administration of vehicle or sitagliptin (5, 20, or 100 mg/kg P.O.) at − 120 min, and a glucose load (4 g/kg) at zero time. The 1- and 3-h time points relate to the glucose load, and are at 3 and 5 h following sitagliptin dosing. Values are mean ± SEM (significant increase vs. vehicle *p < 0.05)

Fig. 7

Combined DPP-4 inhibition and incretins mitigate 6‐OHDA–mediated cell loss in cultured SH-SY5Y neuronal cells. (A) Representative photomicrographs indicate that challenge with 100 µM 6‐OHDA (left lower panel) reduced cell density as compared to the control (left upper panel) (scale bar = 100 µm). (A, right panels) Cell nuclei were stained with TOPRO‐3 following 24-h drug treatment/6-OHDA challenge. Cell density across 96‐well plates was measured by a LiCor Odyssey image system. Three representative TOPRO‐3 images were obtained from cells administered vehicle, 6‐OHDA, and 6‐OHDA + GLP-1 + DPP-4 inhibitor (DPP-4I: PF-000734,200, 1 µM). (B1) TOPRO‐3 fluorescence density in each well was quantified. Challenge with 6‐OHDA (100 µM, 2 h) significantly reduced cell density (p < 0.001). Administration of (B1) DPP-4I (1 µM), (B2) GLP-1 (10 nM), or (B3) GIP (10 nM) ameliorated 6‐OHDA‐mediated cell loss. (B3) GIP alone or co‐administration of (B4) DDP-4I and GLP-1 or (B5) DDP-4I and GIP fully protected against cell loss, as no difference was found between these groups and the vehicle control group. All values are mean ± SEM. * p < 0.05, one-way ANOVA on Rank + Dunn’s test

Fig. 8

GLP‐1 and GIP provide neuroprotection against 6‐OHDA-induced toxicity in primary ventral mesencephalic cultures that is further augmented by DPP-4 inhibition. Primary cultures prepared from rat VM (E15) were challenged with 6‐OHDA (100 µM) for 2 h initiated 10 min following administration of a DPP-4 inhibitor (DPP-4I: PF‐00734,200 1 µM) with/without GLP‐1 or GIP (10 nM). Cells were fixed for tyrosine hydroxylase (TH) immunostaining at 22 h after washing. Treatment with GLP‐1 or GIP provided a significant amelioration of 6-OHDA-mediated toxicity. Co‐administration of DPP-4I further reduced 6‐OHDA-induced loss of TH cells. All values are mean ± SEM. *p < 0.05, one‐way ANOVA. All data were normalized to the mean of TH density in the vehicle (control) group