T3D-959: A Multi-Faceted Disease Remedial Drug Candidate for the Treatment of Alzheimer's Disease

Abstract

Background: T3D-959, a dual PPAR-δ/PPAR γ nuclear receptor agonist and former diabetes drug candidate, has been repositioned as an Alzheimer's disease (AD)-modifying therapy.

Objective: This study examines the effectiveness and mechanisms of T3D-959's therapeutic effects using in vivo and ex vivo rat models of sporadic AD.

Methods: A sporadic AD model was generated by intracerebral (i.c.) administration of streptozotocin (STZ). Control and i.c. STZ treated rats were gavaged with saline or T3D-959 (0.3 to 3.0 mg/kg/day) for 28 days. Spatial learning and memory were evaluated using the Morris water maze test. Frontal lobe slice cultures generated 24 hours after i.c. STZ or vehicle were used to study early effects of T3D-959 (0.5-1.0 μM) on viability and molecular markers of AD.

Results: T3D-959 significantly improved spatial learning and memory in i.c STZ-treated rats. Mechanistically, T3D-959 significantly improved culture viability and brain morphology, reduced levels of oxidative stress and Aβ, and normalized expression of phospho-tau, choline acetyltransferase, and myelin-associated glycoprotein. Protective effects occurred even at the lowest tested dose of T3D-959.

Conclusions: Pre-clinical proof of concept has been demonstrated that T3D-959 can improve multiple pathologies of AD resulting in significant improvements in cognitive function and molecular and biochemical indices of neurodegeneration. These results support the theses that (1) effective disease modification in AD can be achieved by targeting relevant nuclear receptors, and (2) treating AD as a metabolic disease has the potential to be disease remedial. A Phase 2a trial of T3D-959 in mild-to-moderate AD patients has been initiated (ClinicalTrials.gov identifier NCT02560753).

Keywords: Alzheimer’s disease; PPAR agonist; Streptozotocin; T3D-959; Type 3 diabetes; amyloid; slice culture; spatial learning and memory.

Figures

Fig. 1

Intracerebral (i.c.) streptozotocin (STZ) impairs spatial learning and memory. Long Evans rats were administered i.c. STZ or normal saline (Control). On 4 consecutive days (Days 24–27 post-treatment), rats were subjected to MWM tests with 3 trials per day (see Materials and Methods). Latency to reach and land on the platform in the maze was measured. Graphs depict calculated area-under-curve results. Inter-group comparisons were made using ANOVA with the Tukey post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ξ0.05 < p < 0.10 (Trend effect).

Fig. 2

T3D-959 rescue of i.c. STZ-mediated impairment of spatial learning and memory. Long Evans rats were administered i.c. STZ or normal saline (Control) and gavaged with a single daily dose of T3D-959. MWM tests were performed over 4 consecutive days with 3 trials per day. Area-under-curve calculations were used as measures of cumulative performance over the 3 daily trials. Graphs depict (A–D) Day 1-Day 4 results from control (i.c. saline), i.c. STZ, and i.c. STZ+0.3 or 1 mg/kg/day T3D-959 with interventions begun 1 or 7 days after the i.c. STZ administration. E) Results of one-way ANOVA tests for inter-group comparisons on each trial day. F) Symbols for significant differences from control+Vehicle or i.c. STV+Vehicle.

Fig. 3

T3D-959 dose and time delay post i.c. STZ effects of spatial learning and memory demonstrated using the MWM test. Long Evans rats were administered i.c. STZ or normal saline (Control) and gavaged with a single daily dose of (A,E) 0.3, (B,F) 0.7, (C,E) 1.0, or (D,H) 3.0 mg/kg of T3D-959 beginning (A–D) 1 day or (E–H) 7 days after the i.c. injections. On 4 consecutive days (Days 24–27 post-treatment), rats were subjected to MWM tests with 3 trials per day (see Materials and Methods). Latencies to reach and land on the platform in the maze were measured with video-tracking. Graphs depict mean ± S.E.M. corresponding to the area-under-curve results (cumulative performance). Inter-group comparisons were made using Two-Way Row Means (Trial day) ANOVA tests (see Table 2) with the Holm-Sidak post hoc test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig. 4

T3D-959 Prevents neurodegeneration. Frontal lobe slice cultures were generated from rats i.c. injected with (B, D) normal saline (control) or (C, E) STZ 24 h earlier. Cultures were treated with (B,C) vehicle or (D,E) 0.5 µM T3D-959 for 72 h from the same day of culture. A) Cytotoxicity was evaluated using the G6PD release assay (higher levels indicate increased cytotoxicity). B–E) Representative slice cultures were formalin-fixed paraffin embedded to generate H&E stained histological sections. Note reduced cell density in the frontal lobe culture section from the vehicle-treated i.c. STZ group (C) relative to the corresponding control (B) (encircled regions). T3D-959 co-treatment improved tissue integrity and cellular morphology (nuclei are more basophilic, larger and better organized in D and E relative to B and C) in both control and STZ cultures. T3D-959 mediated increased cell survival is clearly evident in the STZ groups (compare encircled regions in B and E).

Fig. 5

Effects of T3D-959 on neuronal and glial cell protein expression in i.c. STZ treated rats: Frontal lobe slice cultures were generated from control or i.c. STZ-treated rats. The cultures were treated with vehicle (normal saline) or 0.5 µM T3D-959 for 72 h. Protein homogenates of the cultures were used to measure immunoreactivity to (A) myelin-associated glycoprotein 1 (MAG-1), (B) glial fibrillary acidic protein (GFAP), (C) choline acetyltransferase (ChAT), and (D) acetylcholinesterase (AChE) by duplex ELISA with results normalized to RPLPO (see Materials and Methods). Results were analyzed by two-way ANOVA (Table 2). Post hoc significance tests determined the specific inter-group differences as shown in the panels. *p < 0.05; ***p < 0.001; ****p < 0.0001.

Fig. 6

Effects of T3D-959 on AD biochemical markers: Frontal lobe slice cultures were generated from control or i.c. STZ-treated rats. The cultures were treated with vehicle (normal saline) or 0.5 µM T3D-959 for 72 h. Protein homogenates of the cultures were used to measure immunoreactivity to (A) Tau, (B) S396+T205-pTau, (C) AβPP-Aβ, and (D) ubiquitin were measured by duplex ELISA with results normalized to RPLPO (see Materials and Methods). Results were analyzed by two-way ANOVA (Table 2). Post hoc significance tests determined the specific inter-group differences as shown in the panels. *p < 0.05; ***p < 0.001; ****p < 0.0001.