Neuroprotective efficacy of P7C3 compounds in primate hippocampus

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.

Treatment with P7C3-A20. Daily oral administration of compound (10mg/kg, 0.5ml/kg) or vehicle control (0.5ml/kg) was implemented at 9am (+/-30min) for 266 consecutive days (38 weeks) by experienced animal care technicians. Animals were provided with food rewards at the time of oral administration, and there were no instances of noncompliance. hrs post treatment on Days 1, 7 and 266. Additional blood samples (8ml) were collected monthly at eight time points (Days 28,56,84,112,140,168,196,and 224). Blood samples were processed for plasma (PK samples) or both plasma and serum (Monthly samples), and stored at -80C. (iii) CBC/CHEM panels were run on Day 0 (pre-treatment), Day 7 and at each monthly blood sample. Detection of P7C3-A20 in plasma of monkeys was conducted by Abbvie Pharmaceuticals (North Chicago, IL) following a protocol previously developed for evaluation of P7C3-A20 in rodent samples (1). Compound was extracted from plasma samples or standards prepared by spiking blank rhesus monkey plasma with GLP grade P7C3-A20 by passage over a Phenomenex (Torrence, CA USA) Phree Phospholipid Removal 1 ml tube to remove phospholipids as well as proteins.
Briefly, plasma was loaded onto the column and volume of acetonitrile equal to 3-fold the volume of applied plasma and containing formic acid and an internal standard was added. The mixture was pipeted gently twice to mix. A vacuum was applied and the flow-through collected. An additional 3X volume of acetonitrile was applied to wash the column, and the flow through was then added to the original material. Compound levels in the resulting flow-through were monitored by LC-MS/MS. Pharmacokinetic parameters were determined using the noncompartmental tool in Phoenix WinNonlin (Certara Corp., Princeton, NJ).
Histology and toxicology analyses. Brain processing followed previously established protocols (2, 3) described briefly below. Monkeys were deeply anesthetized by intravenous injection of sodium pentobarbital (50 mg/kg i.v.; Fatal-Plus; Vortech Pharmaceuticals, Dearborn, MI), and then perfused transcardially with 1% paraformaldehyde in 0.1 M phosphate buffer (250 ml/min) for 2 min, followed by 4% paraformaldehyde (250 ml/min) for 10 min, and 4% paraformaldehyde (100 ml/min) for 50 min. Brains were extracted, placed in refrigerated 4% paraformaldehyde for 6 hours, and then cryoprotected in 10% glycerol / 2% DMSO for 24 hours, followed by 20% glycerol / 2% DMSO for 72 hours before being frozen in an isopentane bath chilled with an outer reservoir of dry ice and ethanol for a minimum of 45 minutes. Coronal sections were cut with a freezing microtome into six 30-μm series and one series at 60 μm (Microm HM 450). The 30-μm sections utilized in this study were collected in a tissue collection solution (TCS) consisting of 30% ethylene glycol, 25% glycerin in PB, and kept at -70°C until further processing. Following fixative perfusion, as described above, additional tissues were collected at necropsy and fixed by immersion in 4% formaldehyde solution for at least 24 hours. Tissues were trimmed and embedded in paraffin using standard histology procedures, and routine 6 um hematoxylin and eosin slides were prepared and identified by animal number but not treatment group. Bony tissues were decalcified in a commercially available decalcifier prior to embedding. The following tissues were qualitatively evaluated by a veterinary pathologist blind to experimental condition, including: eyes, lung, heart, aorta, tongue, spleen, liver, kidney, adrenal, thyroid, parathyroid, pancreas, stomach, testes, small intestine, large intestine, skeletal muscle, vesicular gland, spinal cord, peripheral nerve, prostate, salivary glands, gall bladder, bone marrow, epididymides, optic nerve, lymph nodes, mammary gland, larynx, skin, trachea, ureter, bone, joint and pituitary.
BrdU Immunohistochemistry. Approximately fifteen 30µm-thick sections per animal evenly spaced 960µm apart covering the entire rostral to caudal extent of the hippocampus was stained for BrdU using the freefloating method. An additional section from each case was prepared without primary antibody as a negative control. All washes and buffers were prepared in 0.1M phosphate buffered saline pH 7.4 and run at room temperature unless otherwise stated. 30µm thick sections were incubated in 2N HCl for 1 hour and then neutralized in 0.1M borate buffer, pH 8.5 for 15 minutes. Endogenous peroxidase activity was quenched with a 1% H 2 O 2 solution for 30 minutes. Non-specific protein cross reactivity was blocked for 1 hour using a 0.3% triton x-100, 5% normal rabbit serum, and 1% bovine serum albumin buffer. An avidin/biotin blocking kit (SP-

Rodent Studies
All rodent animal procedures were performed in accordance with the University of Iowa animal care committee's regulations. Animals were housed in temperature-controlled conditions, provided food and water ad libitum, and maintained on a 12 h light/dark cycle (7:00 A.M. to 7:00 P.M.). Male C57BL/6J mice were purchased from The Jackson Laboratory. We first compared P7C3-A20 to NSI-189 for ability to increase the net magnitude of hippocampal neurogenesis in a standard 5 day in vivo assay of BrdU-labeled cells in the dentate gyrus (4). Here, mice are socially isolated without any environmental enrichment for 2 weeks, and then subjected to daily administration of both the test proneurogenic compound and a daily dose of BrdU (50 mg/kg ip). We next compared P7C3-A20 to NSI-189 in standard assays of proliferation and survival of newborn hippocampal neurons in the mouse dentate gyrus. Proliferation was assayed by measuring the number of BrdU+ cells / mm 3 dentate gyrus 1 hour after a pulse of BrdU (150 mg/kg ip) in animals that had received 3 days of daily P7C3-A20 (10 mg/kg/d ip) or NSI-189 (10 mg/kg/d ip). Next, to assay neuroprotective efficacy in promoting survival of newborn hippocampal neurons, we measured the number of BrdU+ cells / mm 3 dentate gyrus 15 days after a pulse of BrdU (150 mg/kg ip), in animals that had received daily treatment with either P7C3-A20 (10 mg/kg/d ip) or NSI-189 (10 mg/kg/d ip) starting at the same time as the delivery of the BrdU pulse. After BrdU (Sigma-Aldrich) administration, mice were euthanized at the specified time points by transcardial perfusion with 4% paraformaldehyde at pH 7.4, and brains were then processed for immunohistochemical detection of incorporated BrdU in the hippocampus. Dissected brains were immersed in 4% paraformaldehyde overnight at 4°C, and then cryoprotected in sucrose before being sectioned into 40-μmthick free-floating sections. Unmasking of BrdU antigen was achieved through incubating tissue sections for 2 h in 50% formamide/2× saline-sodium citrate (SSC) at 65°C, followed by a 5 min wash in 2× SSC and subsequent incubation for 30 min in 2 M HCl at 37°C. Sections were processed for immunohistochemical staining with mouse monoclonal anti-BrdU (1:100, Roche). The number of BrdU+ cells in the entire dentate gyrus subgranular zone (SGZ) was quantified by counting BrdU+ cells within the SGZ and dentate gyrus in