Novel benzimidazole phosphonates as potential inhibitors of protein prenylation.

Benzimidazole carboxyphosphonates and bisphosphonates have been prepared and evaluated for their activity as inhibitors of protein prenylation or isoprenoid biosynthesis. The nature of the phosphonate head group was found to dictate enzyme specificity. The lead carboxyphosphonate inhibits geranylgeranyl transferase II while its corresponding bisphosphonate analogue potently inhibits farnesyl diphosphate synthase. The most active inhibitors effectively disrupted protein prenylation in human multiple myeloma cells.

Combination of MEK Inhibitor and the JAK2-STAT3 Pathway Inhibition for the Therapy of Colon Cancer.

The study aimed to investigate the reason of HCT116 cell resistance to MEK inhibitor, and the combination treatment effects of MEK inhibitor AZD6244 and JAK2/STAT3 inhibitor AG490 on colon cancer in vitro and in vivo, including cell viability, apoptosis, and explore the partial mechanisms focused on AZD6244 promoted the activation of JAK2-STAT3 pathways. In vitro, we examined the HCT116 cell viability by CCK8, cell apoptosis by flow cytometry; Western blot measured p-ERK, p-JAK2, p-STAT3 and STAT3 expression. In vivo, nude mice were subcutaneously injected by HCT116 cells. The tumor volume and weight were detected. HCT116 cell resistance to MEK inhibitor AZD6244, which inhibited the activation of ERK and promoted the activation of JAK2-STAT3 signaling. The combination treatment of AZD6244 and AG490 significantly inhibited cell viability and induced cell apoptosis, and completely inhibited the activation of ERK and JAK2-STAT3 signaling. Combination treatment of AZD6244 and AG490 had a stronger effect than that of AZD6244 as a monotherapy in vitro and in vivo. The treatment of AZD6244 on K-Ras mutations HCT116 cells promoted the activation of JAK2/STAT3 signaling. JAK2/STAT3 inhibitor AG490 synergistically increases effects of AZD6244 on colon cancer in vitro and in vivo. Collectively, these results provide a rationale for combining inhibitors of the JAK/STAT pathway and MEK inhibitors to reduce the potential impact of drug resistance.

Design and Synthesis of Benzimidazoles As Novel Corticotropin-Releasing Factor 1 Receptor Antagonists.

Benzazole derivatives with a flexible aryl group bonded through a one-atom linker as a new scaffold for a corticotropin-releasing factor 1 (CRF1) receptor antagonist were designed, synthesized, and evaluated. We expected that structural diversity could be expanded beyond that of reported CRF1 receptor antagonists. In a structure-activity relationship study, 4-chloro-N(2)-(4-chloro-2-methoxy-6-methylphenyl)-1-methyl-N(7),N(7)-dipropyl-1H-benzimidazole-2,7-diamine 29g had the most potent binding activity against a human CRF1 receptor and the antagonistic activity (IC50 = 9.5 and 88 nM, respectively) without concerns regarding cytotoxicity at 30 µM. Potent CRF1 receptor-binding activity in brain in an ex vivo test and suppression of stress-induced activation of the hypothalamus-pituitary-adrenocortical (HPA) axis were also observed at 138 µmol/kg of compound 29g after oral administration in mice. Thus, the newly designed benzimidazole 29g showed in vivo CRF1 receptor antagonistic activity and good brain penetration, indicating that it is a promising lead for CRF1 receptor antagonist drug discovery research.

Idarucizumab for Dabigatran Reversal.

BACKGROUND: Specific reversal agents for non-vitamin K antagonist oral anticoagulants are lacking. Idarucizumab, an antibody fragment, was developed to reverse the anticoagulant effects of dabigatran. METHODS: We undertook this prospective cohort study to determine the safety of 5 g of intravenous idarucizumab and its capacity to reverse the anticoagulant effects of dabigatran in patients who had serious bleeding (group A) or required an urgent procedure (group B). The primary end point was the maximum percentage reversal of the anticoagulant effect of dabigatran within 4 hours after the administration of idarucizumab, on the basis of the determination at a central laboratory of the dilute thrombin time or ecarin clotting time. A key secondary end point was the restoration of hemostasis. RESULTS: This interim analysis included 90 patients who received idarucizumab (51 patients in group A and 39 in group B). Among 68 patients with an elevated dilute thrombin time and 81 with an elevated ecarin clotting time at baseline, the median maximum percentage reversal was 100% (95% confidence interval, 100 to 100). Idarucizumab normalized the test results in 88 to 98% of the patients, an effect that was evident within minutes. Concentrations of unbound dabigatran remained below 20 ng per milliliter at 24 hours in 79% of the patients. Among 35 patients in group A who could be assessed, hemostasis, as determined by local investigators, was restored at a median of 11.4 hours. Among 36 patients in group B who underwent a procedure, normal intraoperative hemostasis was reported in 33, and mildly or moderately abnormal hemostasis was reported in 2 patients and 1 patient, respectively. One thrombotic event occurred within 72 hours after idarucizumab administration in a patient in whom anticoagulants had not been reinitiated. CONCLUSIONS: Idarucizumab completely reversed the anticoagulant effect of dabigatran within minutes. (Funded by Boehringer Ingelheim; RE-VERSE AD ClinicalTrials.gov number, NCT02104947.).

Lack of weight gain after angiotensin AT1 receptor blockade in diet-induced obesity is partly mediated by an angiotensin-(1-7)/Mas-dependent pathway.

BACKGROUND AND PURPOSE: Angiotensin AT1 receptor antagonists induce weight loss; however, the mechanism underlying this phenomenon is unknown. The Mas receptor agonist angiotensin-(1-7) is a metabolite of angiotensin I and of angiotensin II . As an agonist of Mas receptors, angiotensin-(1-7) has beneficial cardiovascular and metabolic effects. EXPERIMENTAL APPROACH: We investigated the anti-obesity effects of transgenically overexpressed angiotensin-(1-7) in rats. We secondly examined whether weight loss due to telmisartan (8 mg·kg(-1) ·d(-1) ) in diet-induced obese Sprague Dawley (SD) rats can be blocked when the animals were co-treated with the Mas receptor antagonist A779 (24 or 72 µg·kg(-1) ·d(-1) ). KEY RESULTS: In contrast to wild-type controls, transgenic rats overexpressing angiotensin-(1-7) had 1.) diminished body weight when they were regularly fed with chow; 2.) were protected from developing obesity although they were fed with cafeteria diet (CD); 3.) showed a reduced energy intake that was mainly related to a lower CD intake; 5.) remained responsive to leptin despite chronic CD feeding; 6.) had a higher, strain-dependent energy expenditure, and 7.) were protected from developing insulin resistance despite CD feeding. Telmisartan-induced weight loss in SD rats was partially antagonized after a high, but not a low dose of A779. CONCLUSIONS AND IMPLICATIONS: Angiotensin-(1-7) regulated food intake and body weight and contributed to the weight loss after AT1 receptor blockade. Angiotensin-(1-7)-like agonists may be drug candidates for treating obesity.

Idarucizumab Improves Outcome in Murine Brain Hemorrhage Related to Dabigatran.

Lack of specific antidotes is a major concern in intracerebral hemorrhage (ICH) related to direct anticoagulants including dabigatran (OAC-ICH). We examined the efficacy of idarucizumab, an antibody fragment binding to dabigatran, in a mouse model of OAC-ICH. Dabigatran etexilate (DE) dose-dependently prolonged diluted thrombin time and tail-vein bleeding time, which were reversed by idarucizumab. Pretreatment with DE increased intracerebral hematoma volume and cerebral hemoglobin content. Idarucizumab in equimolar dose prevented excess hematoma expansion for both DE doses. In more extensive ICH, idarucizumab significantly reduced mortality. Thus, idarucizumab prevents excess intracerebral hematoma formation in mice anticoagulated with dabigatran and reduces mortality.

Reversal of dabigatran effects in models of thrombin generation and hemostasis by factor VIIa and prothrombin complex concentrate.

BACKGROUND: The oral thrombin inhibitor dabigatran has the drawbacks that it does not have a validated antidote. Data from animal studies and plasma coagulation assays suggest that prothrombin complex concentrate (PCC) or recombinant factor VIIa (FVIIa) might reverse dabigatran anticoagulation. METHODS: Cellular elements make a significant contribution to hemostasis. Our goals were to (1) test the hypothesis that both FVIIa and a 4-factor PCC improve parameters of thrombin generation in the presence of dabigatran in a cell-based model; and (2) determine whether results in a cell-based model correlate with hemostasis in vivo. RESULTS: PCC reversed dabigatran effects on the rate, peak, and total amount of thrombin but did not shorten the lag (n = 6 experiments in triplicate). By contrast, FVIIa shortened the lag, increased the rate and peak, but did not improve total thrombin (n = 6). Effects of PCC were seen at both therapeutic and markedly supratherapeutic dabigatran levels, whereas beneficial effects of FVIIa decreased as the dabigatran level increased. The PCC effect was reproduced by adding prothrombin, factor X, and factor IX. At therapeutic dabigatran levels, both PCC and FVIIa normalized hemostasis time in a mouse saphenous vein bleeding model. CONCLUSIONS: A cell-based model reflects the effects on thrombin generation of clinically relevant levels of FVIIa and PCC in the presence of dabigatran. Enhancing the rate of thrombin generation and peak thrombin level appear to correlate best with hemostasis in vivo. The ineffectiveness of FVIIa at supratherapeutic dabigatran levels may explain conflicting reports of its efficacy in dabigatran reversal.

Reversal agents in development for the new oral anticoagulants.

The new oral anticoagulants have many advantages over vitamin K antagonists, but they are still associated with a troublesome incidence of major bleeding. Additionally, the absence of a reversal agent for the new oral anticoagulants is a barrier to their more widespread use. Currently, there are 3 potential reversal agents in development: idarucizumab is a humanized murine monoclonal antibody fragment directed specifically at dabigatran; andexanet alfa is a recombinant modified decoy factor Xa that binds to factor Xa inhibitors; and PER977 is a small molecule that binds to factor Xa and IIa inhibitors and to heparin-based anticoagulants through charge interaction. These agents have undergone phase I clinical testing, appear to be well tolerated in healthy volunteers, and are effective in neutralizing their respective targets. All 3 are currently undergoing or entering into a phase II or III clinical study. This article reviews the available data for idarucizumab, andexanet alfa, and PER977.

Prothrombin complex concentrates and a specific antidote to dabigatran are effective ex-vivo in reversing the effects of dabigatran in an anticoagulation/liver trauma experimental model.

INTRODUCTION: New oral anticoagulants are effective alternatives to warfarin. However, no specific reversal agents are available for life-threatening bleeding or emergency surgery. Using a porcine model of trauma, this study assessed the ability of prothrombin complex concentrate (PCC), activated PCC (aPCC), recombinant FVIIa (rFVIIa) and a specific antidote to dabigatran (aDabi-Fab) to reverse the anticoagulant effects of dabigatran. METHODS: Dabigatran etexilate (DE) was given orally for 3 days (30 mg/kg bid) and intravenously on day 4 to achieve consistent, supratherapeutic concentrations of dabigatran. Blood samples were collected at baseline, after oral DE, after intravenous dabigatran, and 60 minutes post-injury. PCC (30 and 60 U/kg), aPCC (30 and 60 U/kg), rFVIIa (90 and 180 µg/kg) and antidote (60 and 120 mg/kg) were added to blood samples ex-vivo. Coagulation was assessed by thromboelastometry, global coagulation assays and diluted thrombin time. RESULTS: Plasma concentrations of dabigatran were 380 ± 106 ng/ml and 1423 ± 432 ng/ml after oral and intravenous administration, respectively, and all coagulation parameters were affected by dabigatran. Both PCCs and aDabi-Fab, but not rFVIIa, reversed the effects of dabigatran on thromboelastometry parameters and prothrombin time. In contrast, aPTT was only normalised by aDabi-Fab. Plasma concentration (activity) of dabigatran remained elevated after PCC and rFVIIa therapy, but was not measureable after aDabi-Fab. CONCLUSION: In conclusion, PCC and aPCC were effective in reducing the anticoagulant effects of dabigatran under different conditions, while aDabi-Fab fully corrected all coagulation measures and decreased the plasma concentration of dabigatran below the limit of detection. No significant effects were observed with rFVIIa.

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.

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.

Pharmacologic interventions for reversing the effects of oral anticoagulants.

PURPOSE: To describe the pharmacologic agents and strategies used for urgent reversal of warfarin and the target-specific oral anticoagulants dabigatran, rivaroxaban, and apixaban. SUMMARY: To reverse the anticoagulant effects of warfarin in patients who are bleeding or need surgery, exogenous vitamin K (phytonadione) may be used in combination with another, shorter-acting intervention, such as fresh frozen plasma (FFP), prothrombin complex concentrate (PCC), recombinant factor VIIa, or activated PCC (aPCC). Three-factor PCC contains factors II, IX, and X in an inactivated form, and four-factor PCC also includes factor VII in an inactivated form. No four-factor PCC products are available in the United States, but aPCC, which contains the same four factors with factor VII provided in an activated form, is available. The intervention depends on the International Normalized Ratio, presence of bleeding, and need for and timing of surgery. Research suggests that clotting factor concentrates are more effective than FFP alone for warfarin reversal. These products also may be useful for reversing the effects of target-specific oral anticoagulants, but limited efficacy and safety data are available to support their use. The risks and benefits associated with these products need to be weighed before their use for reversal of dabigatran, rivaroxaban, or apixaban. Additional clinical data are needed to clearly define the role of concentrated clotting factor products in reversal and to determine the optimal clotting factor concentrate product and dose for urgent reversal of oral anticoagulation. CONCLUSION: Phytonadione and clotting factor concentrates appear to have a role for reversal of warfarin, and limited evidence suggests that clotting factor concentrates could have a role in reversal of target-specific oral anticoagulants in an emergency situation.

Blocking direct inhibitor bleeding.

In this issue of Blood, Schiele et al report the development of a monoclonal antibody that reverses the anticoagulant effect of the direct thrombin inhibitor dabigatran.

A specific antidote for dabigatran: functional and structural characterization.

Dabigatran etexilate is a direct thrombin inhibitor and used widely as an anticoagulant for the prevention of stroke in patients with atrial fibrillation. However, anticoagulation therapy can be associated with an increased risk of bleeding. Here, we present data on the identification, humanization, and in vitro pharmacology of an antidote for dabigatran (aDabi-Fab). The X-ray crystal structure of dabigatran in complex with the antidote reveals many structural similarities of dabigatran recognition compared with thrombin. By a tighter network of interactions, the antidote achieves an affinity for dabigatran that is ~350 times stronger than its affinity for thrombin. Despite the structural similarities in the mode of dabigatran binding, the antidote does not bind known thrombin substrates and has no activity in coagulation tests or platelet aggregation. In addition we demonstrate that the antidote rapidly reversed the anticoagulant activity of dabigatran in vivo in a rat model of anticoagulation. This is the first report of a specific antidote for a next-generation anticoagulant that may become a valuable tool in patients who require emergency procedures.