FOXM1 drives HPV+ HNSCC sensitivity to WEE1 inhibition.

Head and neck squamous cell carcinoma (HNSCC) associated with high-risk human papilloma virus (HPV) infection is a growing clinical problem. The WEE1 kinase inhibitor AZD1775 (WEE1i) overrides cell cycle checkpoints and is being studied in HNSCC regimens. We show that the HPV16 E6/E7 oncoproteins sensitize HNSCC cells to single-agent WEE1i treatment through activation of a FOXM1-CDK1 circuit that drives mitotic gene expression and DNA damage. An isogenic cell system indicated that E6 largely accounts for these phenotypes in ways that extend beyond p53 inactivation. A targeted genomic analysis implicated FOXM1 signaling downstream of E6/E7 expression and analyses of primary tumors and The Cancer Genome Atlas (TCGA) data revealed an activated FOXM1-directed promitotic transcriptional signature in HPV+ versus HPV- HNSCCs. Finally, we demonstrate the causality of FOXM1 in driving WEE1i sensitivity. These data suggest that elevated basal FOXM1 activity predisposes HPV+ HNSCC to WEE1i-induced toxicity and provide mechanistic insights into WEE1i and HPV+ HNSCC therapies.

Real-world utilization of andexanet alfa.

OBJECTIVE: In 2018, the FDA approved andexanet alfa for the reversal of life-threatening hemorrhages in patients anticoagulated with apixaban or rivaroxaban. Yet, cost-effective factor Xa inhibitor reversal remains controversial. The objective of this study was to describe real world utilization of andexanet alfa. METHODS: This was a retrospective case series of patients receiving andexanet alfa between July 28, 2018 and April 29, 2019 at a large academic health system. Baseline demographics, anticoagulant type and reversal, as well as brain imaging were collected. Primary endpoints were stability of hematoma for intracranial hemorrhage (ICH), and hemostatic effectiveness for patients undergoing surgical procedures. Secondary endpoints were thromboembolism and 30 day mortality. RESULTS: Of the 25 patients evaluated, 13 received andexanet alfa for ICH. Eleven of the 13 had follow-up imaging available and stability was observed in 90.9%. Three patients received andexanet alfa for reversal prior to surgical procedures, and 100% hemostatic effectiveness was achieved. Nine patients received andexanet alfa for reversal of extracranial bleeding, including gastrointestinal bleed (n=4). There were no thrombotic events in our cohort, and 30 day mortality was 24%. Sixty-four percent of patients would have met exclusion criteria for the ANNEXA-4 trial. CONCLUSION: This is the largest series to date describing real-world utilization of andexanet alfa. Our series showed hemostatic efficacy in 90.9% of patients with ICH, and 100% in patients undergoing surgical procedures. There were no thrombotic complications. Yet, larger and comparative studies are needed to clarify the optimal agent and patient selection for reversal of factor Xa inhibitors.

Long-Lasting In Vivo Effects of the Cannabinoid CB1 Antagonist AM6538.

AM6538 is a cannabinoid antagonist that binds CB1 receptors expressed in HEK-293 cells in a wash-resistant manner. The effects of AM6538 in live animals has not previously been established. We characterized the antagonist effects of AM6538 in male mice, using a warm-water tail-withdrawal assay, and in male squirrel monkeys trained to discriminate the CB1 agonist AM4054 from vehicle. The cannabinoid agonists WIN 55,212, Δ9-tetrahydrocannabinol (THC), and AM4054 all produced 100% maximum possible antinociceptive effects in mice following vehicle pretreatment. One-hour pretreatment with increasing doses of AM6538 (0.1-10 mg/kg) produced first rightward, then downward shifts of the agonist dose-effect functions. Rimonabant, 1-10 mg/kg, produced parallel rightward shifts of the AM4054 dose-effect functions, and baseline effects of AM4054 were nearly recovered within 24 hours following 10 mg/kg of rimonabant. In contrast, in mice treated with 10 mg/kg of AM6538, antagonism of THC or AM4054 lasted up to 7 days. AM6538 also antagonized the discriminative stimulus effects of AM4054 in squirrel monkeys in a dose-related manner, and the effects of 3.2 mg/kg of AM6538 endured for more than 7 days. The effective reduction in CB1 receptor reserve was used to calculate the relative efficacy (tau values) of WIN 55,212, THC, and AM4054 in mice and of AM4054 monkeys, with results indicating that THC has a lower efficacy than WIN 55,212 or AM4054 in mice. These results demonstrate that AM6538 is a long-acting CB antagonist in vivo, and further suggest that differences in CB efficacy can be revealed in behavioral assays following AM6538 treatment.

Reversal of Direct Oral Anticoagulants: Current Status and Future Directions.

Direct oral anticoagulants (DOACs) are increasingly used for prevention and treatment of venous thromboembolism and for prevention of stroke in patients with nonvalvular atrial fibrillation. In phase III clinical trials that included more than 100,000 patients, the DOACs were at least as effective as vitamin K antagonists (VKAs) and were associated with less serious bleeding, particularly less intracranial bleeding. Real-world evidence supports these outcomes. Despite this, some physicians and patients are concerned about serious bleeding or emergencies unless specific reversal agents for the DOACs are available. However, in clinical trials performed without reversal agents, the outcome of major bleeds was similar or better in patients receiving DOACs than in those taking VKAs. Because of their short half-lives, supportive measures are sufficient to manage most bleeds in patients receiving DOACs. Anticoagulant reversal should only be considered with life-threatening bleeds, with bleeds that fail to respond to usual measures and in patients requiring urgent surgery. Idarucizumab is licensed for dabigatran reversal and andexanet alfa is likely to be soon licensed for reversal of rivaroxaban, apixaban, and edoxaban. To ensure responsible use of these agents, every hospital needs a bleeding management algorithm that identifies patients eligible for reversal and outlines appropriate dosing regimens.

Factor Eight Inhibitor Bypassing Agent (FEIBA) for Reversal of Target-Specific Oral Anticoagulants in Life-Threatening Intracranial Bleeding.

INTRODUCTION: As increasing number of patients present to emergency departments with life threatening hemorrhages, particularly intracranial hemorrhage on anticoagulation physicians must be cognizant of the limitations of the available reversal options. Based upon the available literature, our institution formulated a reversal algorithm for patients with life-threatening bleeding on factor Xa inhibitors by administering factor eight inhibitor bypassing agent (FEIBA) 20 units/kg. METHODS: A retrospective chart review was performed to include all patients who received FEIBA per institutional protocol. This case series excluded patients who received FEIBA for reversal of dabigatran. Pre and post FEIBA CT scans were compared for changes. Finally, patients were stratified by estimated mortality rates calculated based on pre-intervention characteristics via published risk models. RESULTS: Thirteen patients were initially included in this study yet two patients were excluded because they were on dabigatran. Fifty-five percent of patients demonstrated stable ICH on CT scan after FEIBA administration while thirty-six percent showed worsening scans. Two patients developed thrombotic events after FEIBA administration. DISCUSSION: FEIBA is a treatment option in patients on a TSOA with acute intracranial hemorrhage with evidence of at least partial pharmacologic reversal of their anticoagulation status. There does not appear to be any major risk of thromboembolic complications associated with FEIBA. Much larger study sizes will be necessary to establish statically significant clinical efficacy for FEIBA use in this patient population. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Emergency medicine physicians are first-line caretakers for patients with life threatening intracranial hemorrhages whether spontaneous or traumatic. FEIBA is a potentially safe option to reverse TSOA in this patient population.

Idarucizumab and factor Xa reversal agents: role in hospital guidelines and protocols.

As expected with all antithrombotic agents, there is a risk of bleeding complications in patients receiving direct oral anticoagulants (DOACs) because of the DOAC itself, acute trauma, invasive procedures, or underlying comorbidities. For many bleeding events, a prudent course of action will be to withdraw the DOAC, then "wait and support" the patient, with the expectation that the bleeding event should resolve with time. Likewise, DOAC therapy may be interrupted ahead of a planned procedure, the stopping time being dependent on the agent involved and the patient's renal function. However, urgent reversal of anticoagulation is required in patients with serious or life-threatening bleeding or in those requiring urgent surgery or procedures. Novel specific reversal agents, either under development or recently approved, will need to be incorporated into local anticoagulation reversal protocols. For dabigatran-treated patients, idarucizumab recently has been approved for clinical use in cases of life-threatening or uncontrolled bleeding or when patients require emergency surgery or urgent procedures, both associated with a high risk of bleeding. As clinical experience with individual specific reversal agents grows, their roles in managing major bleeding events in DOAC-treated patients will become better defined. Future research, as well as ongoing use of idarucizumab, should help establish when it is appropriate to re-dose with idarucizumab, coadminister with prothrombin complex concentrates, or re-initiate DOAC after idarucizumab use. Ongoing trials should help identify the appropriate doses and expected durations of effect for andexanet alfa and ciraparantag, which are likely to vary depending on the individual oral anticoagulants.

Meloxicam inhibits fipronil-induced apoptosis via modulation of the oxidative stress and inflammatory response in SH-SY5Y cells.

Oxidative stress and inflammatory responses have been identified as key elements of neuronal cell apoptosis. In this study, we investigated the mechanisms by which inflammatory responses contribute to apoptosis in human neuroblastoma SH-SY5Y cells treated with fipronil (FPN). Based on the cytotoxic mechanism of FPN, we examined the neuroprotective effects of meloxicam against FPN-induced neuronal cell death. Treatment of SH-SY5Y cells with FPN induced apoptosis via activation of caspase-9 and -3, leading to nuclear condensation. In addition, FPN induced oxidative stress and increased expression of cyclooxygenase-2 (COX-2) and tumor necrosis factor-α (TNF-α) via inflammatory stimulation. Pretreatment of cells with meloxicam enhanced the viability of FPN-exposed cells through attenuation of oxidative stress and inflammatory response. FPN activated mitogen activated protein kinase (MAPK) and inhibitors of MAPK abolished FPN-induced COX-2 expression. Meloxicam also attenuated FPN-induced cell death by reducing MAPK-mediated pro-inflammatory factors. Furthermore, we observed both nuclear accumulation of p53 and enhanced levels of cytosolic p53 in a concentration-dependent manner after FPN treatment. Pretreatment of cells with meloxicam blocked the translocation of p53 from the cytosol to the nucleus. Together, these data suggest that meloxicam may exert anti-apoptotic effects against FPN-induced cytotoxicity by both attenuating oxidative stress and inhibiting the inflammatory cascade via inactivation of MAPK and p53 signaling.

[Perioperative coagulation management during ascending aorta replacement on apixaban]. / Perioperatives Gerinnungsmanagement bei Ersatz der Aorta ascendens unter Apixaban.

The direct oral anticoagulants (DOACs) present a valid therapeutic alternative to vitamin K antagonists in patients with non-valvular atrial fibrillation, for the prevention of venous thromboembolism, and for the treatment and prevention of the recurrence of pulmonary embolisms and deep vein thrombosis. Despite Idarucizumab as an antagonist of Dabigatran there are no other specific antidotes available yet. Therefore, perioperative coagulation management by DOACs is challenging in patients undergoing emergency surgical procedures with a high risk of bleeding complications. This case study describes the perioperative procedure during ascending aorta replacement after aortic dissection with apixaban administration.

[Treatment with inhibitors of new oral direct anticoagulants in patients with severe bleedings or urgent surgical procedures. The new dabigatran antidote: the place of idarucizumab in clinical practice]. / Teendok súlyos vérzés vagy sürgos sebészeti beavatkozás esetén direkt orális antikoaguláns gyógyszerrel kezeltekben. Az új dabigatran-antidótum: idarucizumab helye a klinikai gyakorlatban.

Only vitamin K antagonists could be applied as oral anticoagulants over the past six decades. Coumarols have narrow therapeutic range, and unpredictable anticoagulant effects are resulted by multiple drug interactions. Therefore, regular routine monitoring of the international normalized ratio is necessary. There are two groups of factor-specific anticoagulants: molecules with anti-FIIa (dabigatran) and anti-FXa (rivaroxaban, apixaban and edoxaban) effect. Author summarizes the most important clinical features of the new oral anticoagulants, their indications and the possibilities of laboratory controls. Bleedings are the most important side effects of anticoagulants. This review summarizes the current published evidences for new oral anticoagulants reversal (non-specific and specific) agents, especially in cases with severe acute bleedings or urgent surgery procedures. It reports on how to use inhibitors, the recommended doses and the most important clinical results. The review focuses on idarucizumab - already approved by the U.S. Food and Drug Administration and the European Medicines Agency - which has a key role as the first specific inhibitor of dabigatran.

Neuroprotective effects of three different sizes nanochelating based nano complexes in MPP(+) induced neurotoxicity.

Parkinson's disease (PD) is the world's second most common dementia, which the drugs available for its treatment have not had effects beyond slowing the disease process. Recently nanotechnology has induced the chance for designing and manufacturing new medicines for neurodegenerative disease. It is demonstrated that by tuning the size of a nanoparticle, the physiological effect of the nanoparticle can be controlled. Using novel nanochelating technology, three nano complexes: Pas (150 nm), Paf (100 nm) and Pac (40 nm) were designed and in the present study their neuroprotective effects were evaluated in PC12 cells treated with 1-methyl-4-phenyl-pyridine ion (MPP (+)). PC12 cells were pre-treated with the Pas, Paf or Pac nano complexes, then they were subjected to 10 µM MPP (+). Subsequently, cell viability, intracellular free Calcium and reactive oxygen species (ROS) levels, mitochondrial membrane potential, catalase (CAT) and superoxide dismutase (SOD) activity, Glutathione (GSH) and malondialdehyde (MDA) levels and Caspase 3 expression were evaluated. All three nano complexes, especially Pac, were able to increase cell viability, SOD and CAT activity, decreased Caspase 3 expression and prevented the generation of ROS and the loss of mitochondrial membrane potential caused by MPP(+). Pre-treatment with Pac and Paf nano complexes lead to a decrease of intracellular free Calcium, but Pas nano complex could not decrease it. Only Pac nano complex decreased MDA levels and other nano complexes could not change this parameter compared to MPP(+) treated cells. Hence according to the results, all nanochelating based nano complexes induced neuroprotective effects in an experimental model of PD, but the smallest nano complex, Pac, showed the best results.

Soluble guanylate cyclase redox state under hypoxia or hypoxia/reoxygenation in isolated monkey coronary arteries.

Hypoxia or hypoxia/reoxygenation impairs nitric oxide (NO)-mediated relaxation through the increase in superoxide generation in monkey coronary arteries. Soluble guanylate cyclase (sGC), the target enzyme of NO, has been shown to change from the NO-sensitive reduced form to the NO-insensitive oxidized/heme-free form under substantial oxidative stress, so the present study investigated whether hypoxia or hypoxia/reoxygenation influences sGC redox equilibrium. In isolated monkey coronary arteries without endothelium, the relaxation caused by the sGC stimulator BAY 41-2272 (Emax: 93.3% ± 2.2%) was somewhat impaired under hypoxia (Emax: 86.3% ± 2.6%) or hypoxia/reoxygenation (Emax: 86.1% ± 3.2%), whereas that by the sGC activator BAY 60-2770 (Emax: 86.0% ± 3.2%) was significantly augmented under hypoxia (Emax: 94.4% ± 1.3%) or hypoxia/reoxygenation (Emax: 95.5% ± 1.1%). In addition, cGMP formation in response to BAY 41-2272 and BAY 60-2770 was inhibited and stimulated, respectively, under hypoxia or hypoxia/reoxygenation. The effects of hypoxia or hypoxia/reoxygenation on BAY 41-2272- and BAY 60-2770-induced vasorelaxation were completely canceled by the treatment with the superoxide dismutase mimetic tempol. These findings suggest that sGC redox equilibrium in the coronary artery is shifted towards the NO-insensitive form under hypoxia or hypoxia/reoxygenation and that superoxide seems to play an important role in this shift.

Novel thrombin and factor Xa inhibitors: challenges to reversal of their anticoagulation effects.

PURPOSE OF REVIEW: Warfarin has been the sole oral anticoagulant used in the management of thromboembolic disorders for over 60 years. Target-specific oral anticoagulants (TSOAs) have recently emerged as alternatives to warfarin, because they do not require laboratory monitoring. Nevertheless, with the rising use of TSOAs, there is growing concern among clinicians regarding management of bleeding in patients taking them. Unlike warfarin, there is no antidote or reversal agent for TSOAs. This review summarizes recent developments and attempts to provide a systematic approach to patients on TSOAs presenting with bleeding complications. RECENT FINDINGS: Currently, data involving clinical management of TSOAs are limited and primarily based on ex-vivo or animal models using hemostatic agents with uncertain implications in bleeding patients. There is a pressing need for randomized clinical trials evaluating the safety and efficacy of hemostatic agents. SUMMARY: Without evidence-based guidelines for TSOA management, appropriate patient care requires an understanding of TSOA pharmacology, their effect on coagulation tests and, hence, a correct interpretation of test results, as well as a systematic approach to bleeding complications.

p53 modulates the AMPK inhibitor compound C induced apoptosis in human skin cancer cells.

Compound C, a well-known inhibitor of the intracellular energy sensor AMP-activated protein kinase (AMPK), has been reported to cause apoptotic cell death in myeloma, breast cancer cells and glioma cells. In this study, we have demonstrated that compound C not only induced autophagy in all tested skin cancer cell lines but also caused more apoptosis in p53 wildtype skin cancer cells than in p53-mutant skin cancer cells. Compound C can induce upregulation, phosphorylation and nuclear translocalization of the p53 protein and upregulate expression of p53 target genes in wildtype p53-expressing skin basal cell carcinoma (BCC) cells. The changes of p53 status were dependent on DNA damage which was caused by compound C induced reactive oxygen species (ROS) generation and associated with activated ataxia-telangiectasia mutated (ATM) protein. Using the wildtype p53-expressing BCC cells versus stable p53-knockdown BCC sublines, we present evidence that p53-knockdown cancer cells were much less sensitive to compound C treatment with significant G2/M cell cycle arrest and attenuated the compound C-induced apoptosis but not autophagy. The compound C induced G2/M arrest in p53-knockdown BCC cells was associated with the sustained inactive Tyr15 phosphor-Cdc2 expression. Overall, our results established that compound C-induced apoptosis in skin cancer cells was dependent on the cell's p53 status.

Managing new oral anticoagulants in the intensive care unit.

Warfarin has been the mainstay of oral anticoagulation for more than half a century. Within the last several years, 2 new classes of oral anticoagulants have been introduced as potential alternatives to warfarin for certain indications. The oral direct thrombin inhibitor, dabigatran, and 2 factor Xa inhibitors, rivaroxaban and apixaban, are the newest agents approved for use in the United States. These agents have been studied in various areas including stroke prophylaxis in atrial fibrillation, prevention and treatment of venous thromboembolism, and for reduction of ischemic events following acute coronary syndromes. While these agents do not require routine monitoring of international normalized ratio, these agents may be more challenging to reverse than traditional warfarin therapy. The following review will focus on describing the areas where the new oral anticoagulant agents have been studied, the basic pharmacologic characteristics of each agent, and how to appropriately manage the reversal of these agents when indicated.

Activation of KCNN3/SK3/K(Ca)2.3 channels attenuates enhanced calcium influx and inflammatory cytokine production in activated microglia.

In neurons, small-conductance calcium-activated potassium (KCNN/SK/K(Ca)2) channels maintain calcium homeostasis after N-methyl-D-aspartate (NMDA) receptor activation, thereby preventing excitotoxic neuronal death. So far, little is known about the function of KCNN/SK/K(Ca)2 channels in non-neuronal cells, such as microglial cells. In this study, we addressed the question whether KCNN/SK/K(Ca)2 channels activation affected inflammatory responses of primary mouse microglial cells upon lipopolysaccharide (LPS) stimulation. We found that N-cyclohexyl-N-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidinamine (CyPPA), a positive pharmacological activator of KCNN/SK/K(Ca)2 channels, significantly reduced LPS-stimulated activation of microglia in a concentration-dependent manner. The general KCNN/SK/K(Ca)2 channel blocker apamin reverted these effects of CyPPA on microglial proliferation. Since calcium plays a central role in microglial activation, we further addressed whether KCNN/SK/K(Ca)2 channel activation affected the changes of intracellular calcium levels, [Ca(2+)](i), in microglial cells. Our data show that LPS-induced elevation of [Ca(2+)](i) was attenuated following activation of KCNN2/3/K(Ca)2.2/K(Ca)2.3 channels by CyPPA. Furthermore, CyPPA reduced downstream events including tumor necrosis factor alpha and interleukin 6 cytokine production and nitric oxide release in activated microglia. Further, we applied specific peptide inhibitors of the KCNN/SK/K(Ca)2 channel subtypes to identify which particular channel subtype mediated the observed anti-inflammatory effects. Only inhibitory peptides targeting KCNN3/SK3/K(Ca)2.3 channels, but not KCNN2/SK2/K(Ca)2.2 channel inhibition, reversed the CyPPA-effects on LPS-induced microglial proliferation. These findings revealed that KCNN3/SK3/K(Ca)2.3 channels can modulate the LPS-induced inflammatory responses in microglial cells. Thus, KCNN3/SK3/K(Ca)2.3 channels may serve as a therapeutic target for reducing microglial activity and related inflammatory responses in the central nervous system.

Structural basis for CARM1 inhibition by indole and pyrazole inhibitors.

CARM1 (co-activator-associated arginine methyltransferase 1) is a PRMT (protein arginine N-methyltransferase) family member that catalyses the transfer of methyl groups from SAM (S-adenosylmethionine) to the side chain of specific arginine residues of substrate proteins. This post-translational modification of proteins regulates a variety of transcriptional events and other cellular processes. Moreover, CARM1 is a potential oncological target due to its multiple roles in transcription activation by nuclear hormone receptors and other transcription factors such as p53. Here, we present crystal structures of the CARM1 catalytic domain in complex with cofactors [SAH (S-adenosyl-L-homocysteine) or SNF (sinefungin)] and indole or pyazole inhibitors. Analysis of the structures reveals that the inhibitors bind in the arginine-binding cavity and the surrounding pocket that exists at the interface between the N- and C-terminal domains. In addition, we show using ITC (isothermal titration calorimetry) that the inhibitors bind to the CARM1 catalytic domain only in the presence of the cofactor SAH. Furthermore, sequence differences for select residues that interact with the inhibitors may be responsible for the CARM1 selectivity against PRMT1 and PRMT3. Together, the structural and biophysical information should aid in the design of both potent and specific inhibitors of CARM1.

CB1 receptor antagonist AM4113 reverts the effects of cannabidiol on cue and stress-induced reinstatement of cocaine-seeking behaviour in mice.

No pharmacological treatments are yet approved for patients with cocaine use disorders. Cannabidiol, a constituent of the C. sativa plant has shown promising results in rodent models of drug addiction. However, the specific effects and mechanisms of action of cannabidiol in rodent operant models of extinction-based abstinence and drug-seeking relapse remain unclear. Cannabidiol (10 and 20 mg/kg, i.p.) was injected during extinction training to male CD-1 mice previously trained to self-administer cocaine (0.75 mg/kg/infusion). Then, we evaluated the reinstatement of cocaine seeking induced by cues and stressful stimuli (footshock). We found that cannabidiol (10 and 20 mg/kg) did not modulate extinction learning. After cannabidiol 20 mg/kg treatment, increased levels of CB1 receptor protein were found in the prelimbic and orbitofrontal regions of the prefrontal cortex, and in the ventral striatum; an effect paralleled by a reduction of striatal ∆FosB accumulation and an increment of GluR2 AMPA receptor subunits. Furthermore, cue-induced reinstatement of cocaine seeking was attenuated by cannabidiol. Unexpectedly, cannabidiol 20 mg/kg facilitated stress-induced restoration of cocaine-seeking behaviour. To ascertain the participation of CB1 receptors in these behavioural changes, we administered the CB1 antagonist AM4113 (5 mg/kg) before each reinstatement session. Both, the attenuation of cue-induced reinstatement and the facilitation of stress-induced reestablishment were abolished by AM4113 in cannabidiol 20 mg/kg-treated mice. Our results reveal a series of complex CB1-related changes induced by cannabidiol with a varying impact on the reinstatement of cocaine-seeking behaviour that could limit its therapeutic applications.