Neuroprotective efficacy of P7C3 compounds in primate hippocampus

Abstract

There is a critical need for translating basic science discoveries into new therapeutics for patients suffering from difficult to treat neuropsychiatric and neurodegenerative conditions. Previously, a target-agnostic in vivo screen in mice identified P7C3 aminopropyl carbazole as capable of enhancing the net magnitude of postnatal neurogenesis by protecting young neurons from death. Subsequently, neuroprotective efficacy of P7C3 compounds in a broad spectrum of preclinical rodent models has also been observed. An important next step in translating this work to patients is to determine whether P7C3 compounds exhibit similar efficacy in primates. Adult male rhesus monkeys received daily oral P7C3-A20 or vehicle for 38 weeks. During weeks 2-11, monkeys received weekly injection of 5'-bromo-2-deoxyuridine (BrdU) to label newborn cells, the majority of which would normally die over the following 27 weeks. BrdU+ cells were quantified using unbiased stereology. Separately in mice, the proneurogenic efficacy of P7C3-A20 was compared to that of NSI-189, a proneurogenic drug currently in clinical trials for patients with major depression. Orally-administered P7C3-A20 provided sustained plasma exposure, was well-tolerated, and elevated the survival of hippocampal BrdU+ cells in nonhuman primates without adverse central or peripheral tissue effects. In mice, NSI-189 was shown to be pro-proliferative, and P7C3-A20 elevated the net magnitude of hippocampal neurogenesis to a greater degree than NSI-189 through its distinct mechanism of promoting neuronal survival. This pilot study provides evidence that P7C3-A20 safely protects neurons in nonhuman primates, suggesting that the neuroprotective efficacy of P7C3 compounds is likely to translate to humans as well.

Fig. 1

Experimental timeline

Fig. 2. a P7C3-A20 compound levels were evaluated over a 24 h period on Days 1, 7, and 266, and the overall exposure measured (area under the curve: AUC) differed by no more than two-fold.

Compound levels varied from an average Cmax of 963 to 2320 ng/ml at around 4 h to a trough of 73–132 ng/ml at 24 h immediately prior to the next dose. Individual animal data from day 266 are presented in a. b P7C3-A20 levels were also measured once a month, 6 h after dosing, a time point close to the estimated maximal concentration point

Fig. 3. a Approximately 15 sections per animal evenly spaced 960 μm apart covering the rostral to caudal extent of the hippocampus were used to estimate the number of BrdU-labeled cells.

b Contours were outlined on Nissl-stained sections for anatomical reference. c Contours were drawn on BrdU immunohistochemically stained sections to measure volume and count the number of BrdU-labeled cells within the granule cell layer. d The total number of BrdU positive cells (left and right hemispheres) differed significantly (p = 0.029) between monkeys treated with P7C3A20 (mean = 3275, SD = 1847) and those that received the vehicle control (mean = 1095, SD = 368). However, median values fell just shy of the set significance value at 0.05 level (p = 0.056). e As predicted, the groups did not differ in bilateral GCL volume (p = 0.857) as a function of P7C3-A20 exposure

Fig. 4. Compounds were administered intraperitoneally at the daily dose indicated for 5 days, during which time mice were also dosed intraperitoneally daily with BrdU (50 mg kg^-1day^-1) to label newborn hippocampal neurons.

Representative micrographs are shown on the left, with quantified data on the right represented as mean +/- standard error of the mean (SEM). NSI-189-treated mice showed a nonsignificant increase in the number of BrdU+ cells that was statistically not different from vehicle. P7C3-A20-treated mice showed a statistically significant (p < .01) increase in the number of BrdU+ cells compared to vehicle-treated animals

Fig. 5. Efficacy on enhancing proliferation of hippocampal neural precursor cells and survival of young hippocampal neurons was compared between NSI-189 and P7C3-A20 at a dose of 10 mg kg⁻¹day⁻¹ administered intraperitoneally.

Representative micrographs of BrdU+ cells in the dentate gyrus are shown above, with quantified data displayed below as mean +/- standard error of the mean (SEM). Proliferation was assayed in animals that had received 3 days of daily P7C3-A20 or NSI-189, followed by a 1 h pulse of of BrdU (150 mg/kg ip) P7C3-A20 elicited no increase in cellular proliferation compared to the level seen in vehicle-treated animals. NSI-189 elevated proliferation of neural precursor cells by approximately 70% relative to vehicle (p < .001) and P7C3-A20-treated mice (p < 0.01). Neuroprotective efficacy for young hippocampal neurons was assayed in animals that received the same pulse of BrdU at the same time as initiation of 15 days of daily treatment with P7C3-A20 or NSI-189. P7C3-A20-treated animals showed a survival rate approximately three times greater than vehicle-treated (p < .0001) and NSI-189-treated (p < .001) animals, with migration of some cells out of the subgranular zone and into the dentate gyrus. Animals treated with NSI-189 showed a minor and non-significant trend towards increased survival, indicating that the proneurogenic efficacy of NSI-189 is likely confined to its pro-proliferative effect on cells. P7C3 compounds increase the net magnitude of hippocampal neurogenesis to a greater extent than NSI-189 by virtue of the unique ability of P7C3 compounds to protect neurons from cell death