P7C3-A20 Treatment Research Articles

P7C3-A20 treats traumatic brain injury in rats by inhibiting excessive autophagy and apoptosis.

Traumatic brain injury is a severe health problem leading to autophagy and apoptosis in the brain. 3,6-Dibromo-beta-fluoro-N-(3-methoxyphenyl)-9H-carbazole-9-propanamine (P7C3-A20) can be neuroprotective in various diseases, including ischemic stroke and neurodegenerative diseases. However, whether P7C3-A20 has a therapeutic effect on traumatic brain injury and its possible molecular mechanisms are unclear. Therefore, in the present study, we investigated the therapeutic effects of P7C3-A20 on traumatic brain injury and explored the putative underlying molecular mechanisms. We established a traumatic brain injury rat model using a modified weight drop method. P7C3-A20 or vehicle was injected intraperitoneally after traumatic brain injury. Severe neurological deficits were found in rats after traumatic brain injury, with deterioration in balance, walking function, and learning memory. Furthermore, hematoxylin and eosin staining showed significant neuronal cell damage, while terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining indicated a high rate of apoptosis. The presence of autolysosomes was observed using transmission electron microscope. P7C3-A20 treatment reversed these pathological features. Western blotting showed that P7C3-A20 treatment reduced microtubule-associated protein 1 light chain 3-II (LC3-II) autophagy protein, apoptosis-related proteins (namely, Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 [BNIP3], and Bcl-2 associated x protein [Bax]), and elevated ubiquitin-binding protein p62 (p62) autophagy protein expression. Thus, P7C3-A20 can treat traumatic brain injury in rats by inhibiting excessive autophagy and apoptosis.

P7C3-A20 Attenuates Microglial Inflammation and Brain Injury after ICH through Activating the NAD+/Sirt3 Pathway.

Intracerebral hemorrhage (ICH) is lethal but lacks effective therapies. Nicotinamide adenine dinucleotide (NAD+) is a central metabolite indispensable for a broader range of fundamental intracellular biological functions. Reduction of NAD+ usually occurs after acute brain insults, and supplementation of NAD+ has been proven neuroprotective. P7C3-A20 is a novel compound featuring its ability to facilitate the flux of NAD+. In this study, we sought to determine the potential therapeutic value of P7C3-A20 in ICH. In collagenase-induced ICH mouse models, we found that P7C3-A20 treatment could diminish lesion volume, reduce blood-brain barrier (BBB) damage, mitigate brain edema, attenuate neural apoptosis, and improve neurological outcomes after ICH. Further, RNA sequencing and subsequent experiments revealed that ICH-induced neuroinflammation and microglial proinflammatory activities were significantly suppressed following P7C3-A20 treatment. Mitochondrial damage is an important trigger of inflammatory response. We examined mitochondrial morphology and function and found that P7C3-A20 could attenuate OxyHb-induced impairment of mitochondrial dynamics and functions in vitro. Mechanistically, Sirt3, an NAD+-dependent deacetylase located in mitochondria, was then found to play a vital role in the protection of P7C3-A20 against mitochondrial damage and inflammatory response. In rescue experiments, P7C3-A20 failed to exert those protective effects in microglia-specific Sirt3 conditional knockout (CKO) mice. Finally, preclinical research revealed a correlation between the plasma NAD+ level and the neurological outcome in ICH patients. These results demonstrate that P7C3-A20 is a promising therapeutic agent for neuroinflammatory injury after ICH and exerts protective actions, at least partly, in a Sirt3-dependent manner.

Effect of the NAMPT Activator P7C3-A20 on γ-Globin Expression in Baboon CD34+ Erythroid Cell Cultures

Increased levels of Fetal Hemoglobin (HbF) reduce the symptoms of sickle cell disease (SCD) and lengthen the life span of patients. New, more effective pharmacological agents that can be safely administered long term to increase HbF levels in SCD patients are highly sought. Expression of the γ-globin gene in adult erythroid cells is normally repressed by the recruitment of multi-protein co-repressor complexes to the γ-globin promoter by sequence-specific DNA binding proteins including BCL11A, LRF1 and TR2/TR4. Enzymes contained within these co-repressor complexes, such as DNMT1, LSD1, G9A, and HDACs, modify the chromatin surrounding the γ-globin promoter by catalyzing repressive epigenetic modifications to both histones and DNA. Small molecule pharmacological inhibitors of these enzymes are potent inducers of HbF in various in cell culture and animal models and in SCD patients, but the use of these drugs in patients has been hindered by their dose-dependent effects on hematopoietic differentiation. An alternative strategy to the use of these pharmacological inhibitors to increase HbF would be to employ pharmacological activators that increase the activity of proteins that positively promote γ-globin expression. Previous studies have shown that pharmacological activators of the Sirtuin 1 protein deacetylase increased γ-globin expression in cultured human CD34+ erythroid progenitor cell cultures (Dai et al; Am J Hematol 92:1177-1186, 2017). Because Sirtuin deacetylase activity is dependent upon nicotinamide adenine dinucleotide (NAD) as a co-factor, we tested the hypothesis that increased concentrations of nicotinamide, an NAD precursor, would also increase γ-globin expression. Baboon bone marrow derived CD34+ erythroid progenitor cells from 4 individual baboons were cultured on AFT024 monolayers for 14 days in the presence and absence of varying concentrations of nicotinamide. Globin chain expression was measured in cell lysates by high performance liquid chromatography (HPLC). Nicotinamide (500μM) appeared to increase γ-globin 2 fold (0.015±0.098 γ/γ+β) compared to untreated controls (0.072±0.04 γ/γ+β; n=4; p<0.08). Because the nicotinamide levels used in this experiments are higher than can be easily achieved by dietary supplementation, additional experiments were performed to test the effect of P7C3-A20, an allosteric activator of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD synthesis, on γ-globin expression. Addition of P7C3-A20 (2.5μM) to CD34+ erythroid progenitor cultures on d1, 4, 7, and 10 increased γ-globin 2.7 fold (0.247±0.10 γ/γ+β) compared to vehicle-treated controls (0.090±0.06 γ/γ+β; n=5; p<0.01). P7C3-A20 treatment did not affect cell viability or growth at concentration< 2.5μM and dose-response experiments showed increased γ-globin in cultures treated with submicromolar concentrations of the drug. Addition of P7C3-A20 to cultures on days 1 and 4 resulted in near maximal stimulation of γ-globin expression with lesser effects when the drug was added on later days (d4 and7 or d7 and 10) strongly suggesting that the drug targets cells at an early stage of differentiation. Additional experiments showed that the effect of P7C3-A20 (2.5μM) in combination with either the DNMT1 inhibitor decitabine (DAC) or the LSD1 inhibitor tranylcypromine (TCP) resulted in a greater than additive effects on γ-globin expression in the absence of cytotoxicity (Figure 1). In conclusion, the NAMPT activator P7C3-A20 increased γ-globin expression in baboon CD34+ erythroid progenitor cells with greater than additive effects in combination with DAC or TCP. P7C3-A20 has potent in vivo effects as a neuroprotective drug in mouse models and non-human primates. Therefore, the potential of this drug for in vivo HbF induction warrants further investigation. [Display omitted] DisclosuresSaunthararajah: EpiDestiny: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Maternal P7C3-A20 Treatment Protects Offspring from Neuropsychiatric Sequelae of Prenatal Stress.

Aims: Impaired embryonic cortical interneuron development from prenatal stress is linked to adult neuropsychiatric impairment, stemming in part from excessive generation of reactive oxygen species in the developing embryo. Unfortunately, there are no preventive medicines that mitigate the risk of prenatal stress to the embryo, as the underlying pathophysiologic mechanisms are poorly understood. Our goal was to interrogate the molecular basis of prenatal stress-mediated damage to the embryonic brain to identify a neuroprotective strategy. Results: Chronic prenatal stress in mice dysregulated nicotinamide adenine dinucleotide (NAD+) synthesis enzymes and cortical interneuron development in the embryonic brain, leading to axonal degeneration in the hippocampus, cognitive deficits, and depression-like behavior in adulthood. Offspring were protected from these deleterious effects by concurrent maternal administration of the NAD+-modulating agent P7C3-A20, which crossed the placenta to access the embryonic brain. Prenatal stress also produced axonal degeneration in the adult corpus callosum, which was not prevented by maternal P7C3-A20. Innovation: Prenatal stress dysregulates gene expression of NAD+-synthesis machinery and GABAergic interneuron development in the embryonic brain, which is associated with adult cognitive impairment and depression-like behavior. We establish a maternally directed treatment that protects offspring from these effects of prenatal stress. Conclusion: NAD+-synthesis machinery and GABAergic interneuron development are critical to proper embryonic brain development underlying postnatal neuropsychiatric functioning, and these systems are highly susceptible to prenatal stress. Pharmacologic stabilization of NAD+ in the stressed embryonic brain may provide a neuroprotective strategy that preserves normal embryonic development and protects offspring from neuropsychiatric impairment. Antioxid. Redox Signal. 35, 511-530.

P7C3-A20 treatment one year after TBI in mice repairs the blood–brain barrier, arrests chronic neurodegeneration, and restores cognition

Chronic neurodegeneration in survivors of traumatic brain injury (TBI) is a major cause of morbidity, with no effective therapies to mitigate this progressive and debilitating form of nerve cell death. Here, we report that pharmacologic restoration of the blood-brain barrier (BBB), 12 mo after murine TBI, is associated with arrested axonal neurodegeneration and cognitive recovery, benefits that persisted for months after treatment cessation. Recovery was achieved by 30 d of once-daily administration of P7C3-A20, a compound that stabilizes cellular energy levels. Four months after P7C3-A20, electron microscopy revealed full repair of TBI-induced breaks in cortical and hippocampal BBB endothelium. Immunohistochemical staining identified additional benefits of P7C3-A20, including restoration of normal BBB endothelium length, increased brain capillary pericyte density, increased expression of BBB tight junction proteins, reduced brain infiltration of immunoglobulin, and attenuated neuroinflammation. These changes were accompanied by cessation of TBI-induced chronic axonal degeneration. Specificity for P7C3-A20 action on the endothelium was confirmed by protection of cultured human brain microvascular endothelial cells from hydrogen peroxide-induced cell death, as well as preservation of BBB integrity in mice after exposure to toxic levels of lipopolysaccharide. P7C3-A20 also protected mice from BBB degradation after acute TBI. Collectively, our results provide insights into the pathophysiologic mechanisms behind chronic neurodegeneration after TBI, along with a putative treatment strategy. Because TBI increases the risks of other forms of neurodegeneration involving BBB deterioration (e.g., Alzheimer's disease, Parkinson's disease, vascular dementia, chronic traumatic encephalopathy), P7C3-A20 may have widespread clinical utility in the setting of neurodegenerative conditions.

Beneficial Effects of Delayed P7C3-A20 Treatment After Transient MCAO in Rats.

Despite ischemic stroke being the fifth leading cause of death in the USA, there are few therapeutic options available. We recently showed that the neuroprotective compound P7C3-A20 reduced brain atrophy, increased neurogenesis, and improved functional recovery when treatment was initiated immediately post-reperfusion after a 90-min middle cerebral artery occlusion (MCAO). In the present study, we investigated a more clinically relevant therapeutic window for P7C3-A20 treatment after ischemic stroke. MCAO rats were administered P7C3-A20 for 1week, beginning immediately or at a delayed point, 6h post-reperfusion. Delayed P7C3-A20 treatment significantly improved stroke-induced sensorimotor deficits in motor coordination and symmetry, as well as cognitive deficits in hippocampal-dependent spatial learning, memory retention, and working memory. In the cerebral cortex, delayed P7C3-A20 treatment significantly increased tissue sparing 7 weeks after stroke and reduced hemispheric infarct volumes 48 h after reperfusion. Despite no reduction in striatal infarct volumes acutely, there was a significant increase in spared tissue volume chronically. In the hippocampus, only immediately treated P7C3-A20 animals had a significant increase in tissue sparing compared to vehicle-treated stroke animals. This structural protection translated into minimal hippocampal-dependent behavioral improvements with delayed P7C3-A20 treatment. However, all rats treated with delayed P7C3-A20 demonstrated a significant improvement in both sensorimotor tasks compared to vehicle controls, suggesting a somatosensory-driven recovery. These results demonstrate that P7C3-A20 improves chronic functional and histopathological outcomes after ischemic stroke with an extended therapeutic window.

The neuroprotective compound P7C3-A20 promotes neurogenesis and improves cognitive function after ischemic stroke

Ischemic stroke is a devastating condition with few therapeutic interventions available. The neuroprotective compound P7C3-A20 inhibits mature neuronal cell death while also increasing the net magnitude of postnatal neurogenesis in models of neurodegeneration and acute injury. P7C3 compounds enhance flux of nicotinamide adenine dinucleotide (NAD) in mammalian cells, a proposed therapeutic approach to treating cerebral ischemia. The effectiveness of P7C3-A20 treatment on chronic histopathological and behavioral outcomes and neurogenesis after ischemic stroke has not previously been established. Here, a transient middle cerebral artery occlusion in rats was followed by twice daily injection of P7C3-A20 or vehicle for 7days. P7C3-A20-treated rats performed significantly better than vehicle-treated controls in sensorimotor cylinder and grid-walk tasks, and in a chronic test of spatial learning and memory. These behavioral improvements with P7C3-A20 treatment were correlated with significantly decreased cortical and hippocampal atrophy, and associated with increased neurogenesis in the subventricular zone and hippocampal dentate gyrus subgranular zone. Furthermore, cerebral ischemia significantly reduced NAD in the cortex but P7C3-A20 treatment restored NAD to sham levels. Thus, P7C3-A20 treatment mitigates neurodegeneration and augments repair in the brain after focal ischemia, which translates into chronic behavioral improvement. This suggests a new therapeutic approach of using P7C3 compounds to safely augment NAD and thereby promote two independent processes critical to protecting the brain from ischemic stroke: mature neuron survival and postnatal neurogenesis throughout the post-ischemic brain.

Neuroprotective Efficacy of an Aminopropyl Carbazole Derivative P7C3-A20 in Ischemic Stroke.

NAMPT is a novel therapeutic target of ischemic stroke. The aim of this study was to investigate the effect of a potential NAMPT activator, P7C3-A20, an aminopropyl carbazole derivative, on ischemic stroke. In vitro study, neuron protection effect of P7C3-A20 was investigated by co-incubation with primary neurons subjected to oxygen-glucose deprivation (OGD) or oxygen-glucose deprivation/reperfusion (OGD/R) injury. In vivo experiment, P7C3-A20 was administrated in middle cerebral artery occlusion (MCAO) rats and infarct volume was examined. Lastly, the brain tissue nicotinamide adenine dinucleotide (NAD) levels were detected in P7C3-A20 treated normal or MCAO mice. Cell viability, morphology, and Tuj-1 staining confirmed the neuroprotective effect of P7C3-A20 in OGD or OGD/R model. P7C3-A20 administration significantly reduced cerebral infarction in MCAO rats. Moreover, brain NAD levels were elevated both in normal and MCAO mice after P7C3-A20 treatment. P7C3-A20 has neuroprotective effect in cerebral ischemia. The study contributes to the development of NAMPT activators against ischemic stroke and expands the horizon of the neuroprotective effect of aminopropyl carbazole chemicals.

Abstract P1-15-07: The neuroprotective aminopropyl carbazole, P7C3-A20, prevents paclitaxel-induced pain

Abstract Paclitaxel (PTX), a microtubule-targeting anticancer agent, produces a debilitating peripheral neuropathy that is accompanied by neuropathic pain. Currently, there are only marginally effective therapeutic interventions available. Consequently, patients are forced to reduce or discontinue life-saving chemotherapy to cope with the pain. Recently, a newly identified agent, P7C3-A20, was found to be protective in several models of neurodegeneration, including Parkinson's disease and traumatic brain injury. Given that PTX triggers progressive degeneration of peripheral afferent neurons, this study was performed to evaluate the potential neuroprotective efficacy of P7C3-A20 in rodent models of PTX-induced peripheral neuropathy. P7C3-A20 (10 mg/kg) or vehicle (Cremophor EL/DMSO/5% Dextrose; 1:1:3) was administered intraperitoneally (i.p.) to Sprague-Dawley rats (250-300 g) everyday over a 28-day experimental paradigm. Following two days of treatment with P7C3-A20 or vehicle, rats also received 3 injections of PTX (11.7 mg/kg, i.p.) or vehicle (Cremophor EL/DMSO/5% Dextrose; i.p.) administered every other day. Treatment with P7C3-A20 did not alter body weights or leukocyte counts in control or PTX-treated rats. PTX treatment increased sensitivity to mechanical and cold stimulation (allodynia) of the hindpaw, evidence of peripheral neuropathy. Notably, P7C3-A20 treatment prevented the development of allodynia in response to PTX. Immunohistochemical analysis of paw biopsies indicated that P7C3-A20 prevented PTX-mediated degeneration of terminal nerve endings. Collectively, these data suggest that P7C3-A30 prevented the neurotoxic effects of PTX on peripheral sensory neurons. A xenograft tumor model of triple negative breast cancer was then used to determine whether P7C3-A20 diminished the antitumoral efficacy of PTX. MDA-MB-231 tumors were bilaterally implanted into flanks of female athymic nude mice and allowed to grow for 3 weeks prior to treatment. Using the same treatment protocol as the rats, tumor-bearing mice received daily treatment with P7C3-A20 (20 mg/kg, i.p.) or vehicle for 16 days. P7C3-A20 did not alter PTX-mediated inhibition of tumor growth. Furthermore, P7C3-A20 prevented PTX-induced mechanical allodynia in the mice. Taken together, this work indicates that P7C3-A20 prevents PTX-induced neuropathic damage without diminishing its antitumoral efficacy. P7C3-A20 may be an exciting new candidate to prevent peripheral neuropathy in patients undergoing cancer treatment with PTX. Citation Format: LoCoco PM, Risinger AL, Mooberry SL, Berg KA, Clarke WP. The neuroprotective aminopropyl carbazole, P7C3-A20, prevents paclitaxel-induced pain. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-15-07.

Neuroprotective Efficacy of a Proneurogenic Compound after Traumatic Brain Injury

Traumatic brain injury (TBI) is characterized by histopathological damage and long-term sensorimotor and cognitive dysfunction. Recent studies have reported the discovery of the P7C3 class of aminopropyl carbazole agents with potent neuroprotective properties for both newborn neural precursor cells in the adult hippocampus and mature neurons in other regions of the central nervous system. This study tested, for the first time, whether the highly active P7C3-A20 compound would be neuroprotective, promote hippocampal neurogenesis, and improve functional outcomes after experimental TBI. Sprague-Dawley rats subjected to moderate fluid percussion brain injury were evaluated for quantitative immunohistochemical and behavioral changes after trauma. P7C3-A20 (10 mg/kg) or vehicle was initiated intraperitoneally 30 min postsurgery and twice per day every day thereafter for 7 days. Administration of P7C3-A20 significantly reduced overall contusion volume, preserved vulnerable anti-neuronal nuclei (NeuN)-positive pericontusional cortical neurons, and improved sensorimotor function 1 week after trauma. P7C3-A20 treatment also significantly increased both bromodeoxyuridine (BrdU)- and doublecortin (DCX)-positive cells within the subgranular zone of the ipsilateral dentate gyrus 1 week after TBI. Five weeks after TBI, animals treated with P7C3-A20 showed significantly increased BrdU/NeuN double-labeled neurons and improved cognitive function in the Morris water maze, compared to TBI-control animals. These results suggest that P7C3-A20 is neuroprotective and promotes endogenous reparative strategies after TBI. We propose that the chemical scaffold represented by P7C3-A20 provides a basis for optimizing and advancing new pharmacological agents for protecting patients against the early and chronic consequences of TBI.