BACKGROUND:: ( R,S)-ketamine has gained attention for its rapid-acting antidepressant actions in patients with treatment-resistant depression. However, widespread use of ketamine is limited by its side effects, abuse potential, and poor oral bioavailability. The ketamine metabolite, ( 2R,6R)-hydroxynorketamine, exerts rapid antidepressant effects, without ketamine's adverse effects and abuse potential, in rodents. METHODS:: We evaluated the oral bioavailability of ( 2R,6R)-hydroxynorketamine in three species (mice, rats, and dogs) and also evaluated five candidate prodrug modifications for their capacity to enhance the oral bioavailability of ( 2R,6R)-hydroxynorketamine in mice. Oral administration of ( 2R,6R)-hydroxynorketamine was assessed for adverse behavioral effects and for antidepressant efficacy in the mouse forced-swim and learned helplessness tests. RESULTS:: ( 2R,6R)-hydroxynorketamine had absolute bioavailability between 46-52% in mice, 42% in rats, and 58% in dogs. Compared to intraperitoneal injection in mice, the relative oral bioavailability of ( 2R,6R)-hydroxynorketamine was 62%, which was not improved by any of the candidate prodrugs tested. Following oral administration, ( 2R,6R)-hydroxynorketamine readily penetrated the brain, with brain to plasma ratios between 0.67-1.2 in mice and rats. Oral administration of ( 2R,6R)-hydroxynorketamine to mice did not alter locomotor activity or precipitate behaviors associated with discomfort, sickness, or stereotypy up to a dose of 450 mg/kg. Oral ( 2R,6R)-hydroxynorketamine reduced forced-swim test immobility time (15-150 mg/kg) and reversed learned helplessness (50-150 mg/kg) in mice. CONCLUSIONS:: These results demonstrate that ( 2R,6R)-hydroxynorketamine has favorable oral bioavailability in three species and exhibits antidepressant efficacy following oral administration in mice.

(R,S)-Ketamine produces rapid, robust, and sustained antidepressant effects in major depressive disorder. Specifically, its pharmacological efficacy in treatment refractory depression is considered a major breakthrough in the field. However, the mechanism of action of ketamine's rapid effect remains to be determined. In order to identify pathways that are responsible for ketamine's effect, a targeted metabolomic approach was carried out using a double-blind, placebo-controlled crossover design, with infusion order randomized with medication-free patients with treatment-resistant major depressive disorder (29 subjects) and healthy controls (25 subjects). The metabolomic profile of these subjects was characterized at multiple time points, and a comprehensive analysis was investigated between the following: MDD and healthy controls, treatment and placebo in both groups and the corresponding response to ketamine treatment. Ketamine treatment resulted in a general increase in circulating sphingomyelins, levels which were not correlated with response. Ketamine response resulted in more pronounced effects in the kynurenine pathway and the arginine pathway at 4 h post-infusion, where a larger decrease in circulating kynurenine levels and a larger increase in the bioavailability of arginine were observed in responders to ketamine treatment, suggesting possible mechanisms for response to ketamine treatment.

We have shown that antagomiR inhibition of miRNA miR-21 and miR-196b activity is sufficient to ablate MLL-AF9 leukemia stem cells (LSC) in vivo. Here, we used an shRNA screening approach to mimic miRNA activity on experimentally verified miR-196b targets to identify functionally important and therapeutically relevant pathways downstream of oncogenic miRNA in MLL-r AML. We found Cdkn1b (p27Kip1) is a direct miR-196b target whose repression enhanced an embryonic stem cell-like signature associated with decreased leukemia latency and increased numbers of leukemia stem cells in vivo. Conversely, elevation of p27Kip1 significantly reduced MLL-r leukemia self-renewal, promoted monocytic differentiation of leukemic blasts, and induced cell death. Antagonism of miR-196b activity or pharmacologic inhibition of the Cks1-Skp2-containing SCF E3-ubiquitin ligase complex increased p27Kip1 and inhibited human AML growth. This work illustrates that understanding oncogenic miRNA target pathways can identify actionable targets in leukemia.

Ketamine, a racemic mixture consisting of (S)- and (R)-ketamine, has been in clinical use since 1970. Although best characterized for its dissociative anesthetic properties, ketamine also exerts analgesic, anti-inflammatory, and antidepressant actions. We provide a comprehensive review of these therapeutic uses, emphasizing drug dose, route of administration, and the time course of these effects. Dissociative, psychotomimetic, cognitive, and peripheral side effects associated with short-term or prolonged exposure, as well as recreational ketamine use, are also discussed. We further describe ketamine's pharmacokinetics, including its rapid and extensive metabolism to norketamine, dehydronorketamine, hydroxyketamine, and hydroxynorketamine (HNK) metabolites. Whereas the anesthetic and analgesic properties of ketamine are generally attributed to direct ketamine-induced inhibition of N-methyl-D-aspartate receptors, other putative lower-affinity pharmacological targets of ketamine include, but are not limited to, γ-amynobutyric acid (GABA), dopamine, serotonin, sigma, opioid, and cholinergic receptors, as well as voltage-gated sodium and hyperpolarization-activated cyclic nucleotide-gated channels. We examine the evidence supporting the relevance of these targets of ketamine and its metabolites to the clinical effects of the drug. Ketamine metabolites may have broader clinical relevance than was previously considered, given that HNK metabolites have antidepressant efficacy in preclinical studies. Overall, pharmacological target deconvolution of ketamine and its metabolites will provide insight critical to the development of new pharmacotherapies that possess the desirable clinical effects of ketamine, but limit undesirable side effects.

Background: A need to develop therapeutics to treat Ebola virus disease patients in remote and resource-challenged settings remains in the wake of the 2013-2016 epidemic in West Africa. Toward this goal, we screened drugs under consideration as treatment options and other drugs of interest, most being small molecules approved by the Food and Drug Administration. Drugs demonstrating in vitro antiviral activity were advanced for evaluation in combinations because of advantages often provided by drug cocktails. Methods: Drugs were screened for blockade of Ebola virus infection in cultured cells. Twelve drugs were tested in all (78 pair-wise) combinations, and 3 were tested in a subset of combinations. Results: Multiple synergistic drug pairs emerged, with the majority comprising 2 entry inhibitors. For the pairs of entry inhibitors studied, synergy was demonstrated at the level of virus entry into host cells. Highly synergistic pairs included aripiprazole/piperacetazine, sertraline/toremifene, sertraline/bepridil, and amodiaquine/clomiphene. Conclusions: Our study shows the feasibility of identifying pairs of approved drugs that synergistically block Ebola virus infection in cell cultures. We discuss our findings in terms of the theoretic ability of these or alternate combinations to reach therapeutic levels. Future research will assess selected combinations in small-animal models of Ebola virus disease.

During tumorigenesis, the high metabolic demand of cancer cells results in increased production of reactive oxygen species. To maintain oxidative homeostasis, tumor cells increase their antioxidant production through hyperactivation of the NRF2 pathway, which promotes tumor cell growth. Despite the extensive characterization of NRF2-driven metabolic rewiring, little is known about the metabolic liabilities generated by this reprogramming. Here, we show that activation of NRF2, in either mouse or human cancer cells, leads to increased dependency on exogenous glutamine through increased consumption of glutamate for glutathione synthesis and glutamate secretion by xc(-) antiporter system. Together, this limits glutamate availability for the tricarboxylic acid cycle and other biosynthetic reactions creating a metabolic bottleneck. Cancers with genetic or pharmacological activation of the NRF2 antioxidant pathway have a metabolic imbalance between supporting increased antioxidant capacity over central carbon metabolism, which can be therapeutically exploited.

High-grade gliomas (HGG) are a devastating group of cancers, and represent the leading cause of brain tumour-related death in both children and adults. Therapies aimed at mechanisms intrinsic to glioma cells have translated to only limited success; effective therapeutic strategies will need also to target elements of the tumour microenvironment that promote glioma progression. Neuronal activity promotes the growth of a range of molecularly and clinically distinct HGG types, including adult and paediatric glioblastoma (GBM), anaplastic oligodendroglioma, and diffuse intrinsic pontine glioma (DIPG). An important mechanism that mediates this neural regulation of brain cancer is activity-dependent cleavage and secretion of the synaptic adhesion molecule neuroligin-3 (NLGN3), which promotes glioma proliferation through the PI3K-mTOR pathway. However, the necessity of NLGN3 for glioma growth, the proteolytic mechanism of NLGN3 secretion, and the further molecular consequences of NLGN3 secretion in glioma cells remain unknown. Here we show that HGG growth depends on microenvironmental NLGN3, identify signalling cascades downstream of NLGN3 binding in glioma, and determine a therapeutically targetable mechanism of secretion. Patient-derived orthotopic xenografts of paediatric GBM, DIPG and adult GBM fail to grow in Nlgn3 knockout mice. NLGN3 stimulates several oncogenic pathways, such as early focal adhesion kinase activation upstream of PI3K-mTOR, and induces transcriptional changes that include upregulation of several synapse-related genes in glioma cells. NLGN3 is cleaved from both neurons and oligodendrocyte precursor cells via the ADAM10 sheddase. ADAM10 inhibitors prevent the release of NLGN3 into the tumour microenvironment and robustly block HGG xenograft growth. This work defines a promising strategy for targeting NLGN3 secretion, which could prove transformative for HGG therapy.

Ketamine is rapidly metabolized in the human body to a variety of metabolites, including the hydroxynorketamines. At least two hydroxynorketamines have significant antidepressant action in rodent models, with limited action against the N-methyl-d-aspartate (NMDA) receptor. The synthesis of 12 hydroxynorketamines and their binding affinity to the NMDA receptor is presented here.

Phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) is a lipid kinase involved in endosome maturation that emerged from a haploid genetic screen as being required for Ebola virus (EBOV) infection. Here we analyzed the effects of apilimod, a PIKfyve inhibitor that was reported to be well tolerated in humans in phase 2 clinical trials, for its effects on entry and infection of EBOV and Marburg virus (MARV). We first found that apilimod blocks infections by EBOV and MARV in Huh 7, Vero E6 and primary human macrophage cells, with notable potency in the macrophages (IC50, 10 nM). We next observed that similar doses of apilimod block EBOV-glycoprotein-virus like particle (VLP) entry and transcription-replication competent VLP infection, suggesting that the primary mode of action of apilimod is as an entry inhibitor, preventing release of the viral genome into the cytoplasm to initiate replication. After providing evidence that the anti-EBOV action of apilimod is via PIKfyve, we showed that it blocks trafficking of EBOV VLPs to endolysosomes containing Niemann-Pick C1 (NPC1), the intracellular receptor for EBOV. Concurrently apilimod caused VLPs to accumulate in early endosome antigen 1-positive endosomes. We did not detect any effects of apilimod on bulk endosome acidification, on the activity of cathepsins B and L, or on cholesterol export from endolysosomes. Hence by antagonizing PIKfyve, apilimod appears to block EBOV trafficking to its site of fusion and entry into the cytoplasm. Given the drug's observed anti-filoviral activity, relatively unexplored mechanism of entry inhibition, and reported tolerability in humans, we propose that apilimod be further explored as part of a therapeutic regimen to treat filoviral infections.

Major depressive disorder affects around 16 per cent of the world population at some point in their lives. Despite the availability of numerous monoaminergic-based antidepressants, most patients require several weeks, if not months, to respond to these treatments, and many patients never attain sustained remission of their symptoms. The non-competitive, glutamatergic NMDAR (N-methyl-d-aspartate receptor) antagonist (R,S)-ketamine exerts rapid and sustained antidepressant effects after a single dose in patients with depression, but its use is associated with undesirable side effects. Here we show that the metabolism of (R,S)-ketamine to (2S,6S;2R,6R)-hydroxynorketamine (HNK) is essential for its antidepressant effects, and that the (2R,6R)-HNK enantiomer exerts behavioural, electroencephalographic, electrophysiological and cellular antidepressant-related actions in mice. These antidepressant actions are independent of NMDAR inhibition but involve early and sustained activation of AMPARs (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors). We also establish that (2R,6R)-HNK lacks ketamine-related side effects. Our data implicate a novel mechanism underlying the antidepressant properties of (R,S)-ketamine and have relevance for the development of next-generation, rapid-acting antidepressants.