Indirect Inhibitor Research Articles

Balancing thromboembolic risk against vitamin K antagonist-related bleeding and accelerated calcification: is fondaparinux the Holy Grail for end-stage renal disease patients with atrial fibrillation?

Patients with end-stage renal disease (ESRD) may classically present with highly contrasting derangements of haemostasis, ranging from haemorrhagic diathesis to increased thrombotic risk [1]. Unfortunately, the current guidelines on anticoagulant (AC) therapy do not fully cover the spectrum of thromboembolic conditions potentially affecting ESRD patients [2–4]. These guidelines recommend standard AC treatments for conditions, such as acute deep vein thrombosis, pulmonary embolism, mechanical heart valves and antiphospholipid syndrome, but appear much less clear-cut when dealing with another very common thromboembolic condition, such as atrial fibrillation (AF). In this regard, the value of conventional oral AC (OAC) therapy with vitamin K antagonists (VKA), such as warfarin, for ESRD patients with AF has been seriously challenged, except for those with prior cardioembolic ischaemic stroke [5]. Other recent data suggest some benefit for VKA use in lessadvanced stages of CKD, even though at the price of an increased risk of haemorrhagic complications [6]. Following the demonstration of the better efficacy/manageability compared with conventional OAC, a number of new AC drugs have been introduced that act by direct inhibition of either thrombin (dabigatran etexilate) or factor Xa (rivaroxaban and apixaban) [7]. All of these new drugs have the advantage of oral administration, without needing long-term monitoring; however, they are primarily excreted by the kidney, with a substantial renal clearance [8]. Thus, it is not surprising that renal impairment is one of the putative risk factors for bleeding complications. Unfortunately, a major problem is that renal patients, especially those with ESRD, have been often excluded from randomized controlled trials (RCTs) with these novel AC drugs [8]. Two major issues, specific to the clinical context of ESRD, render conventional OAC therapy even more challenging [9]: (i) the increased bleeding risk notoriously associated with declining kidney function; (ii) the recently discovered side-effect of VKA (the most commonly used conventional OAC drugs), consisting in the acceleration of medial and intimal vascular calcification, a strong and independent risk factor for cardiovascular complications and mortality [10]. Thus, alternatives to VKA with a sound safety profile are urgently required, especially for patients with the most advanced stages of chronic kidney disease (CKD). In this issue of Nephrol Dial Transplant., Speeckaert et al. [11] suggest that Fondaparinux, a parenteral indirect FXa inhibitor that shows a prolonged half-life in ESRD patients, thus allowing thrice weekly administration, could represent a possible therapeutic option in the conundrum of the prophylaxis against thromboembolic risk in ESRD with AF. In their observational pilot study on six ESRD patients undergoing a total of 459 treatments, Fondaparinux administered

Fondaparinux as an alternative to vitamin K antagonists in haemodialysis patients

Accelerated vascular calcification and increased risk of calciphylaxis can be a reason to restrict the use of vitamin K antagonists in dialysis patients. We describe the use of fondaparinux, a prototype indirect factor Xa inhibitor, as an alternative anticoagulant to coumarin derivatives in dialysis patients. In this case series, we included six chronic haemodialysis patients treated with vitamin K antagonists. Low-molecular-weight heparin given as anticoagulant during dialysis was replaced by fondaparinux. Anti-Xa activity was regularly measured pre- and postdialysis to adapt the dose of fondaparinux. Adequate continuous anticoagulation and circuit patency were registered by evaluating clotting in the bubble trap and dialyser membrane at the end of dialysis. Anticoagulation with fondaparinux at a starting dose of 2.5 mg resulted in an effective anticoagulation in the majority of dialysis sessions. Although median predialysis anti-Xa levels were significantly lower [0.36 IU/mL (0.30-0.42 IU/mL) (P < 0.0001)] than postdialysis levels [0.75 IU/mL (0.65-0.80 IU/mL)], predialysis anti-Xa levels were sufficient to limit the risk of thromboembolism. After an initial period of gradually increasing anti-Xa levels due to accumulation of fondaparinux, stable levels were achieved. Haemodialysis without clotting problems was possible in 96% of the sessions (clotting score ≤1), whereas two episodes (2/459 dialysis sessions) of major clotting were observed, defined as clotting of the extracorporeal circuit necessitating premature termination of the procedure. We demonstrated that fondaparinux is a valuable anticoagulant for patients dialysed with low-flux membranes in need of continuous anticoagulation.

Club cell secretory protein improves survival in a murine obliterative bronchiolitis model

Club cell secretory protein (CCSP) is an indirect phospholipase A2 inhibitor with some immunosuppressive and antiproliferative properties that is expressed in bronchiolar Club cells. In our murine bone marrow transplant (BMT) model of obliterative bronchiolitis (OB), CCSP is diminished; however, its role is unknown. To determine the role of CCSP, B6 wild-type (WT) or CCSP-deficient (CCSP(-/-)) mice were lethally conditioned and given allogeneic bone marrow with a sublethal dose of allogeneic splenic T cells to induce OB. We found that CCSP(-/-) mice demonstrated a higher mortality following BMT-induced OB compared with WT mice. Mice were analyzed 60 days post-BMT for protein expression, pulmonary function, and histology. CCSP levels were reduced in WT mice with BMT-induced OB, and lower levels correlated to decreased lung compliance. CCSP(-/-) had a higher degree of injury and fibrosis as measured by hydroxy proline, along with an increased lung resistance and the inflammatory markers, leukotriene B4 and CXCL1. Replacement with recombinant intravenous CCSP partially reversed the weight loss and improved survival in the CCSP(-/-) mice. In addition, CCSP replacement improved histology and decreased inflammatory cells and markers. These findings indicate that CCSP has a regulatory role in OB and may have potential as a preventive therapy.

Differential effects of methoxyamine on doxorubicin cytotoxicity and genotoxicity in MDA-MB-231 human breast cancer cells

Pharmacological inhibition of DNA repair is a promising approach to increase the effectiveness of anticancer drugs. The chemotherapeutic drug doxorubicin (Dox) may act, in part, by causing oxidative DNA damage. The base excision repair (BER) pathway effects the repair of many DNA lesions induced by reactive oxygen species (ROS). Methoxyamine (MX) is an indirect inhibitor of apurinic/apyrimidinic endonuclease 1 (APE1), a multifunctional BER protein. We have evaluated the effects of MX on the cytotoxicity and genotoxicity of Dox in MDA-MB-231 metastatic breast cancer cells. MX has little effects on the viability and proliferation of Dox-treated cells. However, as assessed by the cytokinesis-block micronucleus assay (CBMN), MX caused a significant 1.4-fold increase (P<0.05) in the frequency of micronucleated binucleated cells induced by Dox, and also altered the distribution of the numbers of micronuclei. The fluorescence probe dihydroethidium (DHE) indicated little production of ROS by Dox. Overall, our results suggest differential outcomes for the inhibition of APE1 activity in breast cancer cells exposed to Dox, with a sensitizing effect observed for genotoxicity but not for cytotoxicity.

Pleiotropic effect of factor Xa inhibitor: fondaparinux inhibites ROS-induced cell proliferation and augments cardioprotective cytokine production in mouse cardiac-derived fibroblast

Purpose: Factor Xa inhibitors are anticoagulants to reduce in stroke and other thromboembolic events. Fondaparinux (FDP) is an approved indirect factor Xa inhibitor to prevent venous thromboembolism. It is known that the proliferation of cardiac fibroblasts leads the fibrosis of coronary vessels, which are deeply associated with the progression of coronary atherosclerosis, and cardiac remodeling to cause heart failure in clinical settings. However, the effect of FDP in cardiac fibroblasts for the development of atherosclerosis and cardiac remodeling is still remaining unclear. Therefore, we aim to evaluate the functional role of FDP in mouse cardiac fibroblasts stimulated by hydrogen peroxide (H2O2). Materials and methods: Confluent fibroblasts were pretreated with or without FDP (0.34 μg/ml) for 6 hours, and then stimulated by H2O2 in various times. Reactive oxygen species (ROS) were measured by DCFDA method. Cell proliferation was measured by MTT assay after 12 and 24 hours of H2O2 stimulation (25 μM). Interleukin (IL)-6, tumor necrosis factor (TNF)-α, and tissue growth factor (TGF)-β productions were evaluated by ELISA after 12, 24 and 48 hours of H2O2 stimulation (25 μM), respectively. Results: H2O2 stimulation by itself induced excessive ROS production and cell proliferation in mouse cardiac fibroblast. H2O2 (25 mM) induced cell proliferation was significantly decreased in FDP-pretreated cardiac fibroblasts compared to cells without FDP (86% decrease in 24 hours). Although H2O2 had no effect in TNF-α and TGF-β production, IL-6 production was significantly enhanced by FDP pretreatment (84% increase in 48 hours). Conclusion: These data suggest that the pleiotropic effects of factor Xa inhibitor may provide favorable effects on cardiovascular disease prevention, at least part of in vitro model.

Direct Thrombin Inhibitors and Factor Xa Inhibitors Can Influence the Diluted Prothrombin Time Used as the Initial Screen for Lupus Anticoagulant

Lupus anticoagulant (LA) is a heterogeneous group of antiphospholipid antibodies. Among others, diluted prothrombin time (dPT) is a sensitive screening test for LA; however, the interpretation of LA tests is difficult in patients treated with anticoagulants. The effect of different types of anticoagulants on the result of LA tests, particularly on dPT, has not been studied extensively. To determine whether the direct thrombin inhibitors lepirudin and argatroban and the predominantly factor Xa inhibitors enoxaparin, danaparoid, and fondaparinux could interfere with LA screening based on dPT. Each drug was added to normal and LA-positive plasmas in clinically relevant concentrations. Each sample was tested for dPT. Samples with factor Xa inhibitors were investigated before and after addition of heparinase. Mixing and confirmatory tests for LA were not performed. In the presence of lepirudin or argatroban, dPT increased notably and the dPT ratio exceeded the cutoff value even at subtherapeutic concentrations resulting in false positivity. With increasing factor Xa inhibitor concentrations, a linear increase of dPT ratios and false-positive results were also demonstrated. Although heparinase could almost completely neutralize the anti-Xa effect of all investigated factor Xa inhibitors, dPT ratio returned to the basal level only in case of enoxaparin. Here we provide evidence that both the direct thrombin and indirect factor Xa inhibitors influence dPT assay for LA, causing false positivity. This should be considered when interpreting LA results during anticoagulant therapy. However, dPT seems to be a reliable test for LA screening under enoxaparin therapy after neutralization by heparinase.

Up-regulation of Matrix Metalloproteinases-2 and -9 via an Erk1/2/NF-κB Pathway in Murine Mast Cells Infected with Toxoplasma gondii

Mast cells are key effectors in inflammation and contain proteinases that are released on activation. This study investigates associations between extracellular signal-regulated kinase (Erk)1/2, nuclear factor (NF)-κB, matrix metalloproteinase (MMP)-2 and MMP-9 in mast cells infected with Toxoplasma gondii tachyzoites. T. gondii infection led to increased mast cell degranulation. Phosphorylated (p)-Erk1/2 and p-NF-κB were increased significantly in mast cells infected with T. gondii. Pretreatment with the Erk kinase inhibitor PD98059 significantly decreased the expression of p-Erk1/2, p-NF-κB, MMP-2 and MMP-9. Treatment with MG132, an indirect NF-κB inhibitor, effectively reduced p-IκBα, p-NF-κB, MMP-2 and MMP-9 expression. Collectively, these data show that suppression of an Erk1/2/NF-κB signalling pathway caused a reduction in MMP-2 and -9 activities. Inhibiting this signalling pathway for MMP-2 and MMP-9 expression might offer a potential way to control early T. gondii infection. This pathway for the generation of MMP-2 and MMP-9 is important for mast cell secretionand the NF-κB/Erk1/2 signalling pathway may be key in MMP-2 and MMP-9 production in host defense against toxoplasmosis.

Proteomic analyses and identification of arginine methylated proteins differentially recognized by autosera from anti-Sm positive SLE patients

BackgroundAntibodies against spliceosome Sm proteins (anti-Sm autoantibodies) are specific to the autoimmune disease systemic lupus erythematosus (SLE). Anti-Sm autosera have been reported to specifically recognize Sm D1 and D3 with symmetric di-methylarginines (sDMA). We investigated if anti-Sm sera from local SLE patients can differentially recognize Sm proteins or any other proteins due to their methylation states.ResultsWe prepared HeLa cell proteins at normal or hypomethylation states (treated with an indirect methyltransferase inhibitor adenosine dialdehyde, AdOx). A few signals detected by the anti-Sm positive sera from typical SLE patients decreased consistently in the immunoblots of hypomethylated cell extracts. The differentially detected signals by one serum (Sm1) were pinpointed by two-dimensional electrophoresis and identified by mass spectrometry. Three identified proteins: splicing factor, proline- and glutamine-rich (SFPQ), heterogeneous nuclear ribonucleoprotein D-like (hnRNP DL) and cellular nucleic acid binding protein (CNBP) are known to contain methylarginines in their glycine and arginine rich (GAR) sequences. We showed that recombinant hnRNP DL and CNBP expressed in Escherichia coli can be detected by all anti-Sm positive sera we tested. As CNBP appeared to be differentially detected by the SLE sera in the pilot study, differential recognition of arginine methylated CNBP protein by the anti-Sm positive sera were further examined. Hypomethylated FLAG-CNBP protein immunopurified from AdOx-treated HeLa cells was less recognized by Sm1 compared to the CNBP protein expressed in untreated cells. Two of 20 other anti-Sm positive sera specifically differentiated the FLAG-CNBP protein expressed in HeLa cells due to the methylation. We also observed deferential recognition of methylated recombinant CNBP proteins expressed from E. coli by some of the autosera.ConclusionOur study showed that hnRNP DL and CNBP are novel antigens for SLE patients and the recognition of CNBP might be differentiated dependent on the level of arginine methylation.

Abstract 628: A novel chemoresistance mechanism: HuR post-trancriptionally regulates WEE1, the mitotic inhibitor, upon DNA damage in pancreatic adenocarcinoma cells.

Abstract Introduction: We explored HuR's (an RNA binding protein) significance in the DNA damage response (DDR) in pancreatic ductal adenocarcinoma (PDA) cells. Experimental Procedures: We treated PDA cells with DNA damaging agent's mitomycin C (MMC), oxaliplatin, cisplatin, carboplatin, gemcitabine and a PARP inhibitor (ABT-888). HuR subcellular localization was assessed by immunoblotting. Additionally, we measured γH2AX foci, a marker of DNA damage, by immunofluorescence. Drug sensitivity assays were performed in isogenic PDA cells that differed in HuR expression (siRNA oligos against HuR, cDNAs encoding HuR for overexpression, and appropriate controls). A BrdU/PI pulse chase experiment monitored the cell cycle progression after treating cells with low (150nM) and high (1μM) doses of MMC. Ribonucleoprotein-immunoprecipitation (RIP) assays were used to isolate mRNAs bound to HuR to identify HuR targets in the DDR. Results: Enhanced-HuR cytoplasmic protein expression was observed in PDA cells exposed to all DNA damaging agents. Under high doses of certain DNA damaging agents (e.g., MMC) HuR was cleaved in a caspase-dependent manner. To assess HuR's role in the DDR, isogenic PDA cells lines manipulated with HuR levels were treated with MMC (150nM) for 2 hrs. γH2AX foci assay revealed a 3-fold increase in foci number with MMC treatment in the HuR depleted cells, as compared to the control cells, suggesting that DNA repair was delayed in the absence of HuR. Cell cycle analyses demonstrated that HuR-depleted PDA cells progressed through the G2/M checkpoint and into mitosis upon MMC treatment. A combinatorial approach with RIP-sequencing and an siRNA library screen for genes resistant to DNA damaging agents was performed, and WEE1, a mitotic inhibitor kinase, was identified as a novel HuR target. A separate RIP-qPCR experiment validated WEE1 as an HuR binder with 56-fold more expression upon MMC stress, compared to an IgG control-IP. WEE1 protein expression and Cdk1 (a target of the WEE1 kinase and G2/M phase regulator) phosphorylation was reduced in HuR depleted cells, validating HuR as an upstream post-transcriptional regulator of WEE1 and an indirect inhibitor of the cdk1-cyclin complex in the face of DNA damage. Conclusions: We demonstrate that HuR-deficiency allows PDA cells to enter the cell cycle more efficiently upon DNA damage. This event is dependent on HuR's regulation of WEE1 leading to activation of the CDK1-cyclin complex. These data support a model where HuR is integral to the G2/M checkpoint, forcing a cell cycle recovery response which is critical for PDA cells to avoid DNA damage. Therefore, HuR promotes survival of PDA cells through its acute activation of the DDR pathway. Disruption of HuR's molecular interaction with WEE1 represents a therapeutic opportunity to enhance clinically utilized chemotherapeutic DNA damaging agents for PDA. Citation Format: Shruti Lal, Richard A. Burkhart, Zoe Norris, Neil Beeharry, Vikram Bhattacharjee, Isidore Rigoutsos, Timothy Yen, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. A novel chemoresistance mechanism: HuR post-trancriptionally regulates WEE1, the mitotic inhibitor, upon DNA damage in pancreatic adenocarcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 628. doi:10.1158/1538-7445.AM2013-628

Hypoxia induced CA9 inhibitory targeting by two different sulfonamide derivatives including Acetazolamide in human Glioblastoma

HIF-1α regulated genes are mainly responsible for tumour resistance to radiation- and chemo-therapy. Among these genes, carbonic anhydrase isoform IX (CA9) is highly over expressed in many types of cancer especially in high grade brain cancer like Glioblastoma (GBM). Inhibition of the enzymatic activity by application of specific chemical CA9 inhibitor sulphonamides (CAI) like Acetazolamide (Aza.), the new sulfonamide derivative carbonic anhydrase inhibitor (SU.D2) or indirect inhibitors like the HIF-1α inhibitor Chetomin or molecular inhibitors like CA9-siRNA are leading to an inhibition of the functional role of CA9 during tumorigenesis. Human GBM cells were treated with in vitro hypoxia (1, 6, or 24h at 0.1%, O2). Aza. application was at a range between 250 and 8000nM and the HIF-1α inhibitor Chetomin at a concentration range of 150–500nM. Cell culture plates were incubated for 24h under hypoxia (0.1% O2). Further, CA9-siRNA constructs were transiently transfected into GBM cells exposed to extreme hypoxic aeration conditions. CA9 protein expression level was detectable in a cell-type specific manner under normoxic conditions. Whereas U87-MG exhibited a strong aerobic expression, U251 and U373 displayed moderate and GaMG very weak normoxic CA9 protein bands. Aza. as well as SU.D2 displayed inhibitory characteristics to hypoxia induced CA9 expression in the four GBM cell lines for 24h of hypoxia (0.1% O2) at concentrations between 3500 and 8000nM, on both the protein and mRNA level. Parallel experiments using CA9-siRNA confirmed these results. Application of 150–500nM of the glycolysis inhibitor Chetomin under similar oxygenation conditions led to a sharply reduced expression of both CA IX protein and CA9 mRNA levels, indicating a clear glucose availability involvement for the hypoxic HIF-1α and CA9 expression in GBM cells. Hypoxia significantly influences the behaviour of human tumour cells by activation of genes involved in the adaptation to hypoxic stress. The main objective in malignant GBM therapy is either to eradicate the tumour or to convert it into a controlled, quiescent chronic disease. Aza., SU.D2, Chetomin or CA9-siRNA possesses functional CA9 inhibitory characteristics when applied against human cancers with hypoxic regions like GBM. They may be used as alternative or in conjunction with other direct inhibitors possessing similar functionality, thereby rendering them as potential optimal tools for the development of an optimized therapy in human brain cancer treatment.

Cullin-RING Ligases as Attractive Anti-cancer Targets

The ubiquitin-proteasome system (UPS) promotes the timely degradation of short-lived proteins with key regulatory roles in a vast array of biological processes, such as cell cycle progression, oncogenesis and genome integrity. Thus, abnormal regulation of UPS disrupts the protein homeostasis and causes many human diseases, particularly cancer. Indeed, the FDA approval of bortezomib, the first class of general proteasome inhibitor, for the treatment of multiple myeloma, demonstrated that the UPS can be an attractive anti-cancer target. However, normal cell toxicity associated with bortezomib, resulting from global inhibition of protein degradation, promotes the focus of drug discovery efforts on targeting enzymes upstream of the proteasome for better specificity. E3 ubiquitin ligases, particularly those known to be activated in human cancer, become an attractive choice. Cullin-RING Ligases (CRLs) with multiple components are the largest family of E3 ubiquitin ligases and are responsible for ubiquitination of ~20% of cellular proteins degraded through UPS. Activity of CRLs is dynamically regulated and requires the RING component and cullin neddylation. In this review, we will introduce the UPS and CRL E3s and discuss the biological processes regulated by each of eight CRLs through substrate degradation. We will further discuss how cullin neddylation controls CRL activity, and how CRLs are being validated as the attractive cancer targets by abrogating the RING component through genetic means and by inhibiting cullin neddylation via MLN4924, a small molecule indirect inhibitor of CRLs, currently in several Phase I clinical trials. Finally, we will discuss current efforts and future perspectives on the development of additional inhibitors of CRLs by targeting E2 and/or E3 of cullin neddylation and CRL-mediated ubiquitination as potential anti-cancer agents.

Induction of matrix metalloproteinase-2 and -9 via Erk1/2-NF-κB pathway in human astroglia infected with Toxoplasma gondii

Matrix metalloproteinase (MMP)-2 and MMP-9 can cleave fibronectin, allowing leukocyte migration to the site of Toxoplasma gondii infection during toxoplasmic encephalitis. The aim of this study was to investigate the association between extracellular signal-regulated kinase (Erk)1/2-nuclear factor (NF)-κB pathway and MMP-2/-9 expression in astroglia infected with T. gondii tachyzoite in vitro. Our results showed that phosphorylated (p)-Erk1/2 transiently increased 1h post-infection (PI) and p-NF-κB significantly increased from 1h PI to 12h PI in cell homogenates. NF-κB was bound directly to oligonucleotides containing putative NF-κB binding sites for the MMP-9 promoter. Additionally, expression of p-NF-κB, MMP-2, and MMP-9 was significantly decreased by MG132, an indirect NF-κB inhibitor. Treatment with PD98059, an Erk kinase inhibitor, efficiently reduced p-Erk1/2, p-NF-κB, MMP-2, and MMP-9 expression. These results suggest that suppression of the Erk1/2-NF-κB signaling pathway causes reductions in MMP-2 and MMP-9 activities in astroglia response to T. gondii infection. Thus, inhibiting this signaling intermediate involved in MMP-2 and MMP-9 expression may be a potential method for controlling inflammatory development of T. gondii-induced encephalitis.

Reduction of Dimethylarsinic Acid to the Highly Toxic Dimethylarsinous Acid by Rats and Rat Liver Cytosol

Dimethylarsinic acid (DMAs(V)), the major urinary metabolite of inorganic arsenic, is weakly cytotoxic, whereas its reduced form, dimethylarsinous acid (DMAs(III)), is highly toxic. Although glutathione S-transferase omega 1 (GSTO1) and arsenic methyltransferase have been shown or thought to catalyze DMAs(V) reduction, their role in DMAs(V) reduction in vivo, or in cell extracts is uncertain. Therefore, the reduction of DMAs(V) to DMAs(III) in rats and in rat liver cytosol was studied to better understand its mechanism. To assess DMAs(V) reduction in rats, a novel procedure was devised based on following the accumulation of red blood cell (RBC)-bound dimethylarsenic (DMAs), which represents DMAs(III), in the blood of DMAs(V)-injected anesthetized rats. These studies indicated that rats reduced DMAs(V) to DMAs(III) to a significant extent, as in 90 min 31% of the injected 50 μmol/kg DMAs(V) dose was converted to DMAs(III) that was sequestered by the circulating erythrocytes. Pretreatment of rats with glutathione (GSH) depletors (phorone or BSO) delayed the elimination of DMAs(V) and the accumulation of RBC-bound DMAs, whereas the indirect methyltransferase inhibitor periodate-oxidized adenosine was without effect. Assessment of DMAs(V)-reducing activity of rat liver cytosol revealed that reduction of DMAs(V) required cytosolic protein and GSH and was inhibited by thiol reagents, GSSG and dehydroascorbate. Although thioredoxin reductase (TRR) inhibitors (aurothioglucose and Sb(III)) inhibited cytosolic DMAs(V) reduction, recombinant rat TRR plus NADPH, alone or when added to the cytosol, failed to support DMAs(V) reduction. On ultrafiltration of the cytosol through a 3 kDa filter, the reducing activity in the retentate was lost but was largely restored by NADPH. Such experiments also suggested that the reducing enzyme was larger than 100 kDa and was not GSTO1. In summary, reduction of DMAs(V) to the highly toxic DMAs(III) in rats and rat liver cytosol is a GSH-dependent enzymatic process, yet its mechanism remains uncertain.

A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa

Inhibitors of coagulation factor Xa (fXa) have emerged as a new class of antithrombotics but lack effective antidotes for patients experiencing serious bleeding. We designed and expressed a modified form of fXa as an antidote for fXa inhibitors. This recombinant protein (r-Antidote, PRT064445) is catalytically inactive and lacks the membrane-binding γ-carboxyglutamic acid domain of native fXa but retains the ability of native fXa to bind direct fXa inhibitors as well as low molecular weight heparin-activated antithrombin III (ATIII). r-Antidote dose-dependently reversed the inhibition of fXa by direct fXa inhibitors and corrected the prolongation of ex vivo clotting times by such inhibitors. In rabbits treated with the direct fXa inhibitor rivaroxaban, r-Antidote restored hemostasis in a liver laceration model. The effect of r-Antidote was mediated by reducing plasma anti-fXa activity and the non-protein bound fraction of the fXa inhibitor in plasma. In rats, r-Antidote administration dose-dependently and completely corrected increases in blood loss resulting from ATIII-dependent anticoagulation by enoxaparin or fondaparinux. r-Antidote has the potential to be used as a universal antidote for a broad range of fXa inhibitors.

New Oral Anticoagulants: Comparative Pharmacology with Vitamin K Antagonists

New oral anticoagulants (OACs) that directly inhibit Factor Xa (FXa) or thrombin have been developed for the long-term prevention of thromboembolic disorders. These novel agents provide numerous benefits over older vitamin K antagonists (VKAs) due to major pharmacological differences. VKAs are economical and very well characterized, but have important limitations that can outweigh these advantages, such as slow onset of action, narrow therapeutic window and unpredictable anticoagulant effect. VKA-associated dietary precautions, monitoring and dosing adjustments to maintain international normalized ratio (INR) within therapeutic range, and bridging therapy, are inconvenient for patients, expensive, and may result in inappropriate use of VKA therapy. This may lead to increased bleeding risk or reduced anticoagulation and increased risk of thrombotic events. The new OACs have rapid onset of action, low potential for food and drug interactions, and predictable anticoagulant effect that removes the need for routine monitoring. FXa inhibitors, e.g. rivaroxaban and apixaban, are potent, oral direct inhibitors of prothrombinase-bound, clot-associated or free FXa. Both agents have a rapid onset of action, a wide therapeutic window, little or no interaction with food and other drugs, minimal inter-patient variability, and display similar pharmacokinetics in different patient populations. Since both are substrates, co-administration of rivaroxaban and apixaban with strong cytochrome P450 (CYP) 3A4 and permeability glycoprotein (P-gp) inhibitors and inducers can result in substantial changes in plasma concentrations due to altered clearance rates; consequently, their concomitant use is contraindicated and caution is required when used concomitantly with strong CYP3A4 and P-gp inducers. Although parenteral oral direct thrombin inhibitors (DTIs), such as argatroban and bivalirudin, have been on the market for years, DTIs such as dabigatran are novel synthetic thrombin antagonists. Dabigatran etexilate is a low-molecular-weight non-active pro-drug that is administered orally and converted rapidly to its active form, dabigatran--a potent, competitive and reversible DTI. Dabigatran has an advantage over the indirect thrombin inhibitors, unfractionated heparin and low-molecular-weight heparin, in that it inhibits free and fibrin-bound thrombin. The reversible binding of dabigatran may provide safer and more predictable anticoagulant treatment than seen with irreversible, non-covalent thrombin inhibitors, e.g. hirudin. Dabigatran shows a very low potential for drug-drug interactions. However, co-administration of dabigatran etexilate with other anticoagulants and antiplatelet agents can increase the bleeding risk. Although the new agents are pharmacologically better than VKAs--particularly in terms of fixed dosing, rapid onset of action, no INR monitoring and lower risk of drug interactions--there are some differences between them: the bioavailability of dabigatran is lower than rivaroxaban and apixaban, and so the dabigatran dosage required is higher; lower protein binding of dabigatran reduces the variability related to albuminaemia. The risk of metabolic drug-drug interactions also appears to differ between OACs: VKAs > rivaroxaban > apixaban > dabigatran. The convenience of the new OACs has translated into improvements in efficacy and safety as shown in phase III randomized trials. The new anticoagulants so far offer the greatest promise and opportunity for the replacement of VKAs.

The discovery of dabigatran etexilate.

Thromboembolic disease is a major cause of mortality and morbidity in the developed world and is caused by an excessive stimulation of coagulation. Thrombin is a key serine protease in the coagulation cascade and numerous efforts have been made to develop safe and effective orally active direct thrombin inhibitors (DTIs). Current anticoagulant therapy includes the use of indirect thrombin inhibitors (e.g., heparins, low-molecular-weight-heparins) and vitamin K antagonists such as warfarin. However there are several caveats in the clinical use of these agents including narrow therapeutic window, parenteral delivery, and food- and drug–drug interactions. Dabigatran is a synthetic, reversible DTI with high affinity and specificity for its target binding both free and clot-bound thrombin, and offers a favorable pharmacokinetic profile. Large randomized clinical trials have demonstrated that dabigatran provides comparable or superior thromboprophylaxis in multiple thromboembolic disease indications compared to standard of care. This minireview will highlight the discovery and development of dabigatran, the first in a class of new oral anticoagulant agents to be licensed worldwide for the prevention of thromboembolism in the setting of orthopedic surgery and stroke prevent in atrial fibrillation.

Small-molecule inhibitors of SREBP activation – potential for new treatment of metabolic disorders

Sterol regulatory element-binding proteins (SREBPs) are transcriptional factors that control lipid and cholesterol metabolism. Activation of SREBPs in response to a decrease in cellular sterols results in acceleration of the synthesis of fatty acids, triglycerides, and cholesterol. Aberrant SREBP activity has been linked to metabolic disease states, such as obesity, fatty liver, insulin resistance, hyperlipidemia, and atherosclerosis. Thus, inhibition of SREBP activation is a potential therapeutic approach to treating metabolic disorders. This review focuses on direct or indirect small-molecule inhibitors of SREBP activation.

Spinal Morphine but not Ziconotide or Gabapentin Analgesia is Affected by Alternative Splicing of Voltage-Gated Calcium Channel Cav2.2 Pre-mRNA

Presynaptic voltage-gated calcium CaV2.2 channels play a privileged role in spinal level sensitization following peripheral nerve injury. Direct and indirect inhibitors of CaV2.2 channel activity in spinal dorsal horn are analgesic in chronic pain states. CaV2.2 channels represent a family of splice isoforms that are expressed in different combinations according to cell-type. A pair of mutually exclusive exons in the CaV2.2 encoding Cacna1b gene, e37a and e37b, differentially influence morphine analgesia. In mice that lack exon e37a, which is enriched in nociceptors, the analgesic efficacy of intrathecal morphine against noxious thermal stimuli is reduced. Here we ask if sequences unique to e37a influence: the development of abnormal thermal and mechanical sensitivity associated with peripheral nerve injury; and the actions of two other classes of analgesics that owe part or all of their efficacy to CaV2.2 channel inhibition. We find that: i) the analgesic efficacy of morphine, but not ziconotide or gabapentin, is reduced in mice lacking e37a, ii) the induction and maintenance of behaviors associated with sensitization that accompany peripheral nerve injury, do not require e37a-specific sequence, iii) intrathecal morphine, but not ziconotide or gabapentin analgesia to thermal stimuli is significantly lower in wild-type mice after peripheral nerve injury, iv) the analgesic efficacy of ziconotide and gabapentin to mechanical stimuli is reduced following nerve injury, and iv) intrathecal morphine analgesia to thermal stimuli in mice lacking e37a is not further reduced by peripheral nerve injury. Our findings show that the analgesic action of morphine, but not ziconotide or gabapentin, to thermal stimuli is linked to which Cacna1b exon, e37a or e37b, is selected during alternative pre-mRNA splicing.

Antithrombotic and anticoagulant effects of direct factor Xa inhibitor darexaban in rat and rabbit models of venous thrombosis

The oral direct factor Xa inhibitor darexaban administered intraduodenally prevented venous thrombus formation in both rats and rabbits with no effect on bleeding. The indirect parenteral Factor Xa inhibitor fondaparinux exerted similar properties, only prolonging bleeding time at extremely high doses. In contrast, the thrombin inhibitor ximelagatran and low-molecular-weight heparin enoxaparin prolonged bleeding time at antithrombotic doses. Studies using human platelets showed darexaban glucuronide, a darexaban metabolite that predominantly determines darexaban antithrombotic effects in vivo, had no effect on platelet activation and aggregation, while heparin and enoxaparin activated platelets. Melagatran, heparin, and enoxaparin all inhibited thrombin-induced platelet aggregation at clinically relevant concentrations. Taken together, these results suggest that thrombin-inhibiting drugs may increase the risk of bleeding, while darexaban may have potential as an orally available antithrombotic agent with a wide therapeutic window.

PRT064445 but Not Recombinant Fviia Reverses Rivaroxaban Induced Anticoagulation As Measured by Reduction in Blood Loss in a Rabbit Liver Laceration Model

AbstractAbstract 3414We used a modified rabbit liver laceration model to demonstrate the effects of PRT064445, a recombinant fXa derivative, to reverse rivaroxaban induced anticoagulation as measured by reduction of blood loss, decrease of unbound fraction of rivaroxaban in plasma and relevant pharmacodynamic markers: anti-fXa activity, prothrombin (PT) and activated thromboplastin (aPTT) times. Recombinant fVIIa (rfVIIa) was tested in the same model for comparison.Anesthetized rabbits were administered vehicle or 1 mg/kg rivaroxaban via IV bolus injection. After 30 minutes to allow the rivaroxaban to biodistribute, vehicle, PRT064445 or rfVIIa was administered as a bolus injection followed by laceration of two liver lobes (5× each lobe: 1-cm long and 3-mm deep incisions with scalpel blade) and blood loss collected on pre-weighed gauze over 15 minutes. Blood loss due to rivaroxaban anticoagulation represented approximately 10% of the animal’s total blood volume. Total rivaroxaban concentration in plasma was measured by LC-MS/MS. Free fraction of rivaroxaban (non-protein, non-PRT064445 bound) was assessed using equilibrium dialysis method followed by LC-MS/MS quantitation. Anti-fXa activity was measured using a modified chromogenic LMW heparin kit. PT and aPTT was measured using commercial reagents (HemosIL). Anticoagulation by rivaroxaban (1.65 μM average plasma concentration at 30 min time point) resulted in 2.3-fold and 1.9-fold prolongation of PT and aPTT, respectively, and increased blood loss by 3.2-fold over vehicle. PRT064445 (75 mg/rabbit) administration reduced blood loss due to rivaroxaban anticoagulation by >85% and decreased peak anti-fXa activity by 98%, PT by 74%, aPTT by 66% and the free fraction of rivaroxaban in plasma from 26% to <0.5%. In contrast, rfVIIa (150 μg/kg) had no effect on blood loss but reversed PT by 85% and aPTT by 54%. PRT064445 and rfVIIa alone had no effect on blood loss, but rfVIIa decreased PT by 35%.These data demonstrate that PRT064445 can reduce blood loss due to rivaroxaban induced anticoagulation using a single bolus administration. Reduction of blood loss with PRT06445 correlated to the decrease in the free fraction of rivaroxaban in plasma as well as PD markers anti-fXa activity and PT, while rfVIIa decreased PT but had no effect on blood loss. These and our previous data with reversal of enoxaparin-induced anticoagulation in a rat tail transection model indicate that PRT064445 can be used as a universal antidote for both direct and indirect fXa inhibitors. Disclosures:Hollenbach:Portola Pharmaceuticals: Employment. Lu:Portola Pharmaceuticals: Employment. Tan:Portola Pharmaceuticals: Employment. Lee:Portola Pharmaceuticals: Employment. Athiwat:Portola Pharmaceuticals: Employment. Inagaki:Portola Pharmaceuticals: Employment. Sinha:Portola Pharmaceuticals Inc.: Employment.