Calcitonin gene-related peptide regulates spinal microglial activation through the histone H3 lysine 27 trimethylation via enhancer of zeste homolog-2 in rats with neuropathic pain.

BACKGROUND: Calcitonin gene-related peptide (CGRP) as a mediator of microglial activation at the transcriptional level may facilitate nociceptive signaling. Trimethylation of H3 lysine 27 (H3K27me3) by enhancer of zeste homolog 2 (EZH2) is an epigenetic mark that regulates inflammatory-related gene expression after peripheral nerve injury. In this study, we explored the relationship between CGRP and H3K27me3 in microglial activation after nerve injury, and elucidated the underlying mechanisms in the pathogenesis of chronic neuropathic pain. METHODS: Microglial cells (BV2) were treated with CGRP and differentially enrichments of H3K27me3 on gene promoters were examined using ChIP-seq. A chronic constriction injury (CCI) rat model was used to evaluate the role of CGRP on microglial activation and EZH2/H3K27me3 signaling in CCI-induced neuropathic pain. RESULTS: Overexpressions of EZH2 and H3K27me3 were confirmed in spinal microglia of CCI rats by immunofluorescence. CGRP treatment induced the increased of H3K27me3 expression in the spinal dorsal horn and cultured microglial cells (BV2) through EZH2. ChIP-seq data indicated that CGRP significantly altered H3K27me3 enrichments on gene promoters in microglia following CGRP treatment, including 173 gaining H3K27me3 and 75 losing this mark, which mostly enriched in regulation of cell growth, phagosome, and inflammation. qRT-PCR verified expressions of representative candidate genes (TRAF3IP2, BCL2L11, ITGAM, DAB2, NLRP12, WNT3, ADAM10) and real-time cell analysis (RTCA) verified microglial proliferation. Additionally, CGRP treatment and CCI increased expressions of ITGAM, ADAM10, MCP-1, and CX3CR1, key mediators of microglial activation in spinal dorsal horn and cultured microglial cells. Such increased effects induced by CCI were suppressed by CGRP antagonist and EZH2 inhibitor, which were concurrently associated with the attenuated mechanical and thermal hyperalgesia in CCI rats. CONCLUSION: Our findings highly indicate that CGRP is implicated in the genesis of neuropathic pain through regulating microglial activation via EZH2-mediated H3K27me3 in the spinal dorsal horn.

Perampanel in real-world clinical care of patients with epilepsy: Interim analysis of a phase IV study.

Objective: To assess the retention rate, efficacy, safety, and dosing of perampanel administered to patients with epilepsy during routine clinical care in the retrospective phase IV, PROVE Study (NCT03208660). Methods: Exposure, efficacy, and safety data were obtained from the medical records of patients initiating perampanel after January 1, 2014, across 29 US study sites. The cutoff date for this interim analysis was October 10, 2018. The primary efficacy endpoint was retention rate. Secondary efficacy endpoints included median percent changes in seizure frequency, seizure-freedom rate, and overall investigator impression of seizure effect. Results: All enrolled patients (N = 1121) received perampanel. Mean (standard deviation [SD]) cumulative duration of exposure to perampanel was 16.6 (14.7) months; overall mean (SD) daily perampanel dose was 5.7 (2.7) mg. Perampanel uptitration occurred weekly (21.1%), biweekly (23.8%), every 3 weeks (1.5%), other (43.3%), and unknown (10.3%). Across the Safety Analysis Set (N = 1121), retention rate on perampanel at 24 months was 49.5% (n = 319/645).At 12 months, the median reduction in seizure frequency per 28 days from baseline in the small number of patients for whom data were available was 75.0% (n = 85), and 30/85 (35.3%) patients were seizure free. Based on investigator impression at the end of treatment, improvement, no change (ie, stable), or worsening of seizures was reported in 54.3%, 33.7%, and 12.0% of patients, respectively.Treatment-emergent adverse events occurred in 500 (44.6%) patients; the most common were dizziness (9.2%), aggression (5.4%), and irritability (4.5%). Serious treatment-emergent adverse events occurred in 32 (2.9%) patients. Significance: Favorable retention and sustained efficacy were demonstrated for ≥12 months following initiation of perampanel during routine clinical care in patients with epilepsy.

Prolonged Scar-in-a-Jar: an in vitro screening tool for anti-fibrotic therapies using biomarkers of extracellular matrix synthesis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a rapidly progressing disease with challenging management. To find novel effective therapies, better preclinical models are needed for the screening of anti-fibrotic compounds. Activated fibroblasts drive fibrogenesis and are the main cells responsible for the accumulation of extracellular matrix (ECM). Here, a prolonged Scar-in-a-Jar assay was combined with clinically validated biochemical markers of ECM synthesis to evaluate ECM synthesis over time. To validate the model as a drug screening tool for novel anti-fibrotic compounds, two approved compounds for IPF, nintedanib and pirfenidone, and a compound in development, omipalisib, were tested. METHODS: Primary human lung fibroblasts from healthy donors were cultured for 12 days in the presence of ficoll and were stimulated with TGF-ß1 with or without treatment with an ALK5/TGF-ß1 receptor kinase inhibitor (ALK5i), nintedanib, pirfenidone or the mTOR/PI3K inhibitor omipalisib (GSK2126458). Biomarkers of ECM synthesis were evaluated over time in cell supernatants using ELISAs to assess type I, III, IV, V and VI collagen formation (PRO-C1, PRO-C3, PRO-C4, PRO-C5, PRO-C6), fibronectin (FBN-C) deposition and α-smooth muscle actin (α-SMA) expression. RESULTS: TGF-ß1 induced synthesis of PRO-C1, PRO-C6 and FBN-C as compared with unstimulated fibroblasts at all timepoints, while PRO-C3 and α-SMA levels were not elevated until day 8. Elevated biomarkers were reduced by suppressing TGF-ß1 signalling with ALK5i. Nintedanib and omipalisib were able to reduce all biomarkers induced by TGF-ß1 in a concentration dependent manner, while pirfenidone had no effect on α-SMA. CONCLUSIONS: TGF-ß1 stimulated synthesis of type I, III and VI collagen, fibronectin and α-SMA but not type IV or V collagen. Synthesis was increased over time, although temporal profiles differed, and was modulated pharmacologically by ALK5i, nintedanib, pirfenidone and omipalisib. This prolonged 12-day Scar-in-a-Jar assay utilising biochemical markers of ECM synthesis provides a useful screening tool for novel anti-fibrotic compounds.

Modeling medulloblastoma in vivo and with human cerebellar organoids.

Medulloblastoma (MB) is the most common malignant brain tumor in children and among the subtypes, Group 3 MB has the worst outcome. Here, we perform an in vivo, patient-specific screen leading to the identification of Otx2 and c-MYC as strong Group 3 MB inducers. We validated our findings in human cerebellar organoids where Otx2/c-MYC give rise to MB-like organoids harboring a DNA methylation signature that clusters with human Group 3 tumors. Furthermore, we show that SMARCA4 is able to reduce Otx2/c-MYC tumorigenic activity in vivo and in human cerebellar organoids while SMARCA4 T910M, a mutant form found in human MB patients, inhibits the wild-type protein function. Finally, treatment with Tazemetostat, a EZH2-specific inhibitor, reduces Otx2/c-MYC tumorigenesis in ex vivo culture and human cerebellar organoids. In conclusion, human cerebellar organoids can be efficiently used to understand the role of genes found altered in cancer patients and represent a reliable tool for developing personalized 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.

Activated prothrombin complex concentrate to reverse the factor Xa inhibitor (apixaban) effect before emergency surgery: a case series.

BACKGROUND: The lack of an antidote against factor Xa inhibitors in case of major bleeding or need for urgent surgery is a concern to clinicians. Guidelines on managing major bleeding in patients under anticoagulation with a factor Xa inhibitor suggest several hemostatic agents to reverse the effect, but there is no consensus regarding the choice of drug or appropriate dose. The ability of prothrombin complex concentrate, activated prothrombin complex concentrate, and recombinant factor VIIa to reverse the effect of factor Xa inhibitors has been evaluated in animal studies, in vitro studies, and healthy volunteers, but not yet in randomized clinical studies. CASE PRESENTATION: We report a consecutive case series of patients under factor Xa inhibitor (apixaban) treatment who received activated prothrombin complex concentrate to reverse the anticoagulation effect before emergency cardiovascular surgery. Patient 1, a 63-year-old white man, was operated with replacement of the aortic valve; patient 2, a 65-year-old white man, underwent heart transplantation; patient 3, a 68-year-old white man, was operated for acute type A aortic dissection. They all received activated prothrombin complex concentrate 25 IU/kg immediately before surgery. In two of the cases, the global coagulation assay thromboelastometry (ROTEM™) was performed before and after administering activated prothrombin complex concentrate. The ROTEM™ clotting time was reduced from 1900 seconds to 740 seconds and from 1482 to 807 seconds, respectively, after administering a dose of 25 IU/kg activated prothrombin complex concentrate. The apixaban concentration before reversal was within the range considered to be the therapeutic level in all cases. No bleeding complications occurred during surgery, but one case was complicated with bleeding postoperatively. No thromboembolic complications were observed during or after surgery. CONCLUSIONS: Activated prothrombin complex concentrate 25 IU/kg reversed the anticoagulation effect of apixaban effectively and safely before emergency cardiovascular surgery.

Reversal agents for oral anticoagulants.

For more than half a century, warfarin, a vitamin K antagonist, has been the anticoagulant of choice. However, direct oral anticoagulants are rapidly gaining in popularity, which poses the need for efficacious reversal agents. This review article summarizes the strategies and agents used to reverse oral anticoagulants.

[NOAC: Real-life data and the role of antidotes for the treatment of bleeding]. / NOAK: Real-life-Daten und Therapie von Blutungen mit Antidots.

The non-vitamin K antagonists (NOAC) are an integral component of our antithrombotic prevention and therapy. For four of the NOAC, their non-inferiority or even superiority versus vitamin K antagonists (VKA) has been proven. Thus, the management of special patient cohorts or the management of active bleeding complications is a focus of current discussion.In addition to prospective trials, numerous retrospective analyses of health insurers or public health provider data have been analyzed and published as "real life" or "real-world evidence" data. In almost all data sets the results of the NOAC approval trials were confirmed, demonstrating their non-inferiority or even superiority versus VKA. Attempts to compare the various NOAC with each other must be viewed critically since the real-world evidence (RWE) analysis provides very divergent results depending on the cohorts analyzed. Thus, a substantial prescriber-bias must be taken into account and never be excluded.In order to improve the management of bleeding complications, NOAC antidotes were developed. While the factors Xa antidote, andexanet alpha, a modified coagulation factor deleted of an intrinsic activity, will not be available before 2018, the dabigatran antidote idarucizumab is already in clinical use. Idarucizumab, a monoclonal antibody fragment directed against dabigatran, is able to completely antagonize the effect of dabigatran within minutes. Therefore, the drug has the potential to terminate life-threatening bleeding complications earlier and make emergency surgical or interventional procedures possible without an elevated bleeding risk.

Clinical implications of reversal agents for direct oral anticoagulants.

Direct oral anticoagulants (DOACs) are effective in preventing and treating venous thromboembolism, and preventing stroke in atrial fibrillation. Until recently, there has been no specific reversal agent for DOACs. Now, a specific antidote for the direct thrombin inhibitor, dabigatran has been approved for use, and antidotes for factor Xa inhibitors (rivaroxaban, apixaban and edoxaban) are being developed. We review the evidence for currently used and emerging reversal strategies, and discuss possible clinical implications, including increased prescription of DOACs, use of DOACs in clinical situations previously felt to pose too great a risk of bleeding, and use of reversal agents beyond currently approved indications.

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.

Optimizing the safety of treatment for venous thromboembolism in the era of direct oral anticoagulants.

Direct oral anticoagulants (DOACs) are rapidly replacing vitamin K antagonists (VKAs) for treatment of venous thromboembolism (VTE). The DOACs include dabigatran, which inhibits thrombin, and rivaroxaban, apixaban, and edoxaban, which inhibit factor Xa. When compared with conventional VTE treatment consisting of a parenteral anticoagulant followed by a VKA, the DOACs were equally effective for prevention of recurrence, but were associated with less bleeding. With similar efficacy, better safety, and the convenience of fixed dosing without the need for routine coagulation monitoring, guidelines now recommend DOACs over VKAs for VTE treatment in patients without active cancer. Nonetheless, measures are needed to optimize the safety of DOACs. Focusing on these measures, this paper summarizes the results of phase III trials evaluating DOACs for VTE treatment; identifies which VTE patients are or are not candidates for DOACs; provides guidance on how to choose among DOACs; lists the licensed dosing information for DOACs; discusses the optimal treatment duration for VTE; describes periprocedural management of DOACs in patients requiring surgery or intervention; and finally, reviews the management of bleeding, including the role for specific reversal agents.

[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.

Bleeding management in patients with new oral anticoagulants.

New oral anticoagulants (NOACs) have been developed in recent years and are increasingly used in clinical practice. Dabigatran is a direct thrombin (factor II) inhibitor while rivaroxaban, apixaban and edoxaban are direct inhibitors of factor Xa. The European Medicines Agency (EMA) currently approves these NOACs for different clinical uses. NOACs do not require routine monitoring of coagulation although an assessment of anticoagulation activity in these patients may be required in different conditions. NOACs show a similar or lower incidence of bleeding compared with conventional therapies in phase III trials. In case of bleeding, non-specific reversal strategies are available while specific reversal agents are the subject of ongoing trials. The role of this review is to summarize the current knowledge on NOCAs focusing on bleeding management in the perioperative period.

Novel oral anticoagulants in plastic surgery.

Novel oral anticoagulants (NOACs) have emerged as a good alternative to warfarin in the prevention of stroke for patients with atrial fibrillation. NOAC use is increasing rapidly; therefore, greater understanding of their use in the perioperative period is important for optimal care. Studies and reviews that reported on the use of NOACs were identified, with particular focus on the perioperative period. PubMed was searched for relevant articles published between January 2000 and August 2015. The inevitable rise in the use of NOACs such as rivaroxaban (Xarelto™), apixaban (Eliquis™), edoxaban (Lixiana™) and dabigatran (Pradaxa™) may present a simplified approach to perioperative anticoagulant management due to fewer drug interactions, rapidity of onset of action and relatively short half-lives. Coagulation status, however, cannot reliably be monitored and no antidotes are currently available. When planning for discontinuation of NOACs, special consideration of renal function is required. Advice regarding the management of bleeding complications is provided for consideration in emergency surgery. In extreme circumstances, haemodialysis may be considered for bleeding with the use of dabigatran. NOACs will increasingly affect operative planning in plastic surgery. In order to reduce the incidence of complications associated with anticoagulation, the management of NOACs in the perioperative period requires knowledge of the time of last dose, renal function and the bleeding risk of the planned procedure. Consideration of these factors will allow appropriate interpretation of the current guidelines.