Single-dose pharmacokinetics, pharmacodynamics and safety of AZD0837, a novel oral direct thrombin inhibitor, in young healthy male subjects.

OBJECTIVE: The novel oral anticoagulant AZD0837 is currently in clinical development for the prevention of stroke and systemic embolic events in patients with atrial fibrillation. AZD0837 is bioconverted to AR-H067637, a selective and reversible direct thrombin inhibitor. This first-time-in-man study (study code D1250C00001) investigated the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) of AZD0837. METHODS: Healthy Caucasian male volunteers (n = 44, age 20 - 39 y) were enrolled into this study of single oral escalating doses of AZD0837 given in solution (15 - 750 mg, n = 4 per dose). PD was assessed by ex vivo measurements of activated partial thromboplastin time (APTT), ecarin coagulation time (ECT), thrombin time (TT) and thrombin generation in plasma. RESULTS: AZD0837 was rapidly absorbed, with a mean oral bioavailability of 22 - 52%, and bioconverted to the active form, AR-H067637. In fasting subjects, maximum plasma concentrations (Cmax) for AR-H067637 occurred approximately 1 h post-dosing and declined with a mean half-life of 9.3 h. The Cmax and area under the curve for AR-H067637 showed a low to moderate inter-individual variability of 16% and 28%, respectively, and exhibited a slight deviation from dose-proportionality. AZD0837 produced a dose-dependent prolongation of APTT, ECT and TT, and decreased maximum free thrombin activity. AZD0837 was generally well tolerated. CONCLUSIONS: AZD0837 single oral doses (15 - 750 mg) are well tolerated in healthy male subjects and exhibit favorable PK properties and reproducible effects on ex vivo coagulation time variables that support further clinical development.

Increased activity of L-type Ca2+ channels exposed to serum from patients with type I diabetes.

Type I diabetes [insulin-dependent diabetes mellitus (IDDM)] is an autoimmune disease associated with the destruction of pancreatic beta cells. Serum from patients with IDDM increased L-type calcium channel activity of insulin-producing cells and of GH3 cells derived from a pituitary tumor. The subsequent increase in the concentration of free cytoplasmic Ca2+ ([Ca2+]i) was associated with DNA fragmentation typical of programmed cell death or apoptosis. These effects of the serum were prevented by adding a blocker of voltage-activated L-type Ca2+ channels. When the serum was depleted of immunoglobulin M (IgM), it no longer affected [Ca2+]i. An IgM-mediated increase in Ca2+ influx may thus be part of the autoimmune reaction associated with IDDM and contribute to the destruction of beta cells in vivo.

Fibrinolysis inhibitors in plaque stability: a morphological association of PAI-1 and TAFI in advanced carotid plaque.

Essentials Fibrinolysis inhibitors are localized in advanced atheroma by immunohistology of endarterectomies. Neovascular endothelium/neocapillaries show thrombin-activatable fibrinolysis inhibitor (TAFI). Macrophage areas show free plasminogen activator inhibitor (PAI-1), notably in the vulnerable part. Free PAI-1 and TAFI stabilize active plaque area by inhibition of fibrinolysis and inflammation. SUMMARY: Background Fibrinolysis plays an important role in destabilization of atherosclerotic plaques and is tightly regulated by specific inhibitors. Objective The fibrinolysis inhibitors plasminogen activator inhibitor type-1 (PAI-1) and thrombin-activatable fibrinolysis inhibitor (TAFI) were quantified and described in the morphological context of advanced carotid plaques American Heart Association VI-VIII to elucidate their role in plaque stability. Methods Immunohistochemistry in serial sections along the longitudinal axis of endarterectomies from patients with symptomatic carotid stenosis (n = 19) were studied using an antibody specific for free PAI-1 (I205), an antibody with high affinity for TAFI/TAFIa (CP17) and established antibodies for smooth muscle cells (α-actin), endothelial cells (von Willebrand factor [VWF]), macrophages (CD68) and platelets (CD42). Results PAI-1 and TAFI show a specific distribution in these advanced plaques with a maximum corresponding to the internal carotid artery (ICA). Free PAI-1 was mainly detected in macrophages and in intravascular thrombi, and TAFI in endothelial cells (ECs) but also macrophages. The one-way ANOVA analysis with Bonferroni's correction showed a significant increase of macrophages and ECs, TAFI and PAI-1 in areas with high neovascularization in endarterectomy sections corresponding to ICA. High Spearman factors for TAFI, PAI-1 and VWF indicate neovascularization as the main source of plasma proteins, transported by platelets into the atheroma (PAI-1) or expressed by ECs (TAFI). CD68 was highly associated with VWF, PAI-1 and especially TAFI, underlining the role of macrophages in fibrinolytic activity and inflammation. Conclusion The abundance of free PAI-1 and TAFI in the plaque may inhibit plasmin generation and thereby counteract plaque destabilization by fibrinolysis, cell migration and inflammation.

A randomized, controlled, dose-guiding study of the oral direct thrombin inhibitor ximelagatran compared with standard therapy for the treatment of acute deep vein thrombosis: THRIVE I.

This randomized, controlled, multicentre study evaluated the efficacy and tolerability of the oral direct thrombin inhibitor ximelagatran, compared with a low-molecular-weight heparin (dalteparin) followed by warfarin, in the treatment of deep vein thrombosis (DVT) of the lower extremity. Patients with acute DVT received oral ximelagatran (24, 36, 48 or 60 mg twice daily) or dalteparin and warfarin for 2 weeks. Evaluation of paired venograms from 295 of 350 patients showed regression of the thrombus in 69% of patients treated with ximelagatran and 69% of patients treated with dalteparin and warfarin. Progression was observed in 8% and 3% of patients, respectively. Changes in thrombus size according to the Marder score were similar in all groups. Treatment discontinuation due to bleeding occurred in two patients receiving ximelagatran (24- and 36-mg groups) and in two patients receiving dalteparin and warfarin. Reduction in pain, edema and circumference of the affected leg was similar in all groups. Oral ximelagatran appears to be a promising alternative to current anticoagulant therapy to limit the progression of acute DVT, and it seems to possess a wide therapeutic window.

Acute antithrombotic effects of ximelagatran, an oral direct thrombin inhibitor, and r-hirudin in a human ex vivo model of arterial thrombosis.

BACKGROUND: Thrombin plays a major role in thrombus formation through activation of platelets and conversion of fibrinogen to fibrin. OBJECTIVES: To investigate the antithrombotic effects of the oral direct thrombin inhibitor (DTI) ximelagatran and the parenteral DTI r-hirudin in humans. SUBJECTS AND METHODS: Healthy male volunteers randomized into four parallel groups each with 15 subjects received either ximelagatran (20, 40 or 80 mg orally) or r-hirudin (0.4 mg kg-1 intravenous bolus + infusion of 0.15 mg kg-1 h-1 for 2 h and 0.075 mg kg-1 h-1 for 3 h). Antithrombotic effects were assessed as changes in total thrombus area (TTA) and total fibrin area (TFA) from baseline, using the Badimon perfusion chamber model at baseline and 2 h and 5 h after drug administration. RESULTS: Two hours postdosing, ximelagatran showed antithrombotic effects at both high and low shear rates (TTA% of mean baseline value +/- SEM was 76 +/- 13% and 71 +/- 17% [both P < 0.05] for the 20-mg dose, 85 +/- 11% [P > 0.05] and 62 +/- 15% [P < 0.05] for the 40-mg dose and 60 +/- 11% and 26 +/- 7% [both P < 0.05] for the 80-mg dose, respectively). r-Hirudin also showed a significant antithrombotic effect at high and low shear rates (76 +/- 11% [P = 0.05] and 57 +/- 17% [P < 0.05] of baseline values, 2 h postdosing, respectively). The inhibitory effects on TFA were similar to those on TTA. CONCLUSIONS: The oral DTI ximelagatran shows antithrombotic effects under both high and low shear conditions. The antithrombotic effect of 40-80 mg ximelagatran appeared comparable to that of parenterally administered r-hirudin, which has been previously demonstrated to be clinically effective in acute coronary syndromes.

Effect of melagatran on prothrombin time assays depends on the sensitivity of the thromboplastin and the final dilution of the plasma sample.

Prothrombin time (PT) assays are clotting methods that measure the activity of vitamin K-dependent coagulation factors (F) II, VII, and X. There are three main types of PT assays in general usage, namely the Quick assay, Owren's assay and PT dry chemistry test cards. PT assays were initially developed to monitor dose-adjustments of vitamin K antagonists such as warfarin. The aim of the present study was to investigate whether commercially available PT assays are suitable for evaluating the anticoagulant activity of direct thrombin inhibitors. Melagatran, a reversible direct thrombin inhibitor, was added to human plasma at concentrations ranging from 0.1 to 2.0 micromol/l. Seventeen different commercially available PT kits were used, including thirteen Quick reagents, two Owren reagents and two PT test cards. The sensitivity of the different reagents, expressed as the concentration of melagatran that doubled the prothrombin time (IC50) varied widely, with Thromboplastin S and Thromboplastin HS being the most sensitive (IC50 = 0.9 micromol/l). The reagents with apparently the lowest sensitivity were the two Owren reagents Nycotest PT and SPA 50 with an IC50 of 2.2 and 2.9 micromol/L, respectively. This is most likely due to a higher dilution of melagatran in these assays compared to the dilution in the Quick assays. The results were also dependent on the International Sensitivity Index (ISI) of each reagent. The concentration of melagatran that produced an International Normalized Ratio (INR) of 2 was calculated from dose-response curves for each assay, and these results revealed that reagents with a high ISI value gave an INR of 2 at much lower concentrations of melagatran (0.5-0.7 micromol/L) than those with an ISI-values around one (0.9-1.2 micromol/L). It was found that INR depends not only on the plasma concentration of melagatran, but also on the sensitivity of the PT reagent and on the final dilution of the plasma sample in the prothrombin time assay. Thus, since the same melagatran concentration can be associated with widely varying PT/INR results depending on the specific assay used it is concluded that PT assays and INR can not be used to monitor melagatran activity.

Factor V Leiden (G1691A) and prothrombin gene G20210A mutations as potential risk factors for venous thromboembolism after total hip or total knee replacement surgery.

Patients (n = 1600) from 12 European countries, scheduled for elective orthopaedic hip or knee surgery, were screened for Factor V Leiden and prothrombin gene G20210A mutations, found in 5.5% and 2.9% of the populations, respectively. All patients underwent prophylactic treatment with one of four doses of melagatran and ximelagatran or dalteparin, starting pre-operatively. Bilateral ascending venography was performed on study day 8-11. The patients were subsequently treated according to local routines and followed for 4-6 weeks postoperatively. The composite endpoint of screened deep vein thrombosis (DVT) and symptomatic pulmonary embolism (PE) during prophylaxis did not differ significantly between patients with or without these mutations. Symptomatic venous thromboembolism (VTE) during prophylaxis and follow-up (1.9%) was significantly over-represented among patients with the prothrombin gene G20210A mutation (p = 0.0002). A tendency towards increased risk of VTE was found with the Factor V Leiden mutation (p = 0.09). PE were few, but significantly over-represented in both the Factor V Leiden and prothrombin gene G20210A mutated patients (p = 0.03 and p = 0.05, respectively). However, since 90% of the patients with these genetic risk factors will not suffer a VTE event, a general pre-operative genotyping is, in our opinion, of questionable value.

Comparison of various D-dimer tests for the diagnosis of deep venous thrombosis.

Analyses of D-dimers in plasma are frequently used as diagnostic tools for deep venous thrombosis (DVT). Enzyme-linked immunosorbent assays (ELISAs) are considered to be the method of choice for quantitative assays, but are time consuming. Therefore, we have assessed plasma levels of D-dimers in patients with clinically suspected DVT using quantitative (Asserachrom D-Di ELISA and TintElize), semiquantitative (Minutex latex, D-Di latex, NycoCard D-Dimer) and qualitative (INSTANT.I.A) assays. Phlebography was used as the gold standard to verify or exclude the suspected diagnosis. We conclude that the fast assays, INSTANT.I.A and Minutex, have essentially the same negative predictive value [91% and 89%, respectively, using a cut-off value < 0.5 mg/l fibrinogen equivalent units (FEU)] for excluding DVT as the Asserachrom D-Di ELISA and TintElize tests (92%). The D-Di Latex assay had a negative predictive value of 82% (cut-off < 0.5 mg/l FEU) and turned out to be less useful in our material. The NycoCard D-dimer assay had a negative predictive value of 100% when using the cut-off value < 0.5 mg/l FEU, but this was substantially lower when the cut-off was changed to < or = 0.5 mg/l. Thus, we conclude that several fast tests offer a simpler and more rapid way of determining plasma levels of D-dimer than conventional ELISA methods without loss of clinical usefulness in excluding DVT.

Alpha 2-adrenoreceptor stimulation does not inhibit L-type calcium channels in mouse pancreatic beta-cells.

The effects of alpha 2-adrenergic stimulation on the Ca(2+)-current in mouse pancreatic beta-cells were investigated using the patch-clamp technique. When using the conventional whole-cell recording configuration (dialysis of cell interior with pipette solution), addition of adrenaline (1 microM) or the alpha 2-adrenergic agonist clonidine (5 microM) failed to reduce the Ca(2+)-current, irrespective of whether intracellular GTP (or GTP gamma S) was present or not and at both physiological (1.3 mM) and elevated (10.2 mM) Ca(2+)-concentrations. In fact, in the absence of added guanine nucleotides, the agonists tended to increase the Ca(2+)-current amplitude in the presence of the higher Ca(2+)-concentration. Ca(2+)-channel activation measured at 1.3 mM Ca2+ was not affected by clonidine. Half-maximal activation was observed at approximately -20 mV. In addition, when Ca(2+)-currents were recorded from intact beta-cells, using the perforated patch whole-cell configuration, clonidine (1 microM) also failed to detectably affect the Ca(2+)-current. It is therefore suggested that the inhibition of beta-cell electrical activity and insulin-secretion produced by alpha 2-adrenoreceptor stimulation does not result from suppression of the L-type Ca(2+)-current.

The oral direct thrombin inhibitor, ximelagatran, an alternative for anticoagulant treatment during the puerperium and lactation.

OBJECTIVE: To determine the excretion of the oral direct thrombin inhibitor (oral DTI), ximelagatran, and its active form, melagatran, in human milk, and to thus evaluate the potential exposure of breastfed infants to melagatran. DESIGN: An open, single dose, single centre study. SETTING: Department of Antenatal Care, Primary Health Care South Bohuslän and Institute for the Health of Women and Children, Göteborg University, Sweden. SAMPLE: Seven healthy Caucasian breastfeeding women who were at least two months postpartum were studied. METHODS: The concentrations of ximelagatran, its two intermediates, and melagatran were determined using liquid chromatography-mass spectrometry, with the limit of quantification of 2 nmol L(-1) for human milk and 10 nmol L(-1) for plasma concentrations. MAIN OUTCOME MEASURES: Concentrations of ximelagatran, its intermediates and melagatran were measured in breast milk over 72 hours, and in plasma over 12 hours, after a single oral 36 mg dose of ximelagatran. RESULTS: Neither ximelagatran nor its intermediates were detected in human breast milk. Only trace amounts of melagatran were detected. The mean cumulative amount of melagatran excreted into breast milk over the 72-hour period after dosing with oral ximelagatran was 0.00091% of the administered dose of ximelagatran. Ximelagatran was well tolerated, with no clinically relevant changes in laboratory variables or vital signs. CONCLUSIONS: Trace levels of melagatran are excreted in human breast milk following administration of the oral DTI ximelagatran. The exposure of breastfed infants to melagatran appears to be low and is therefore unlikely to be of clinical concern.

Glucose-stimulated insulin biosynthesis depends on insulin-stimulated insulin gene transcription.

Glucose stimulation of pancreatic beta-cells leads to insulin secretion as well as up-regulation of insulin biosynthesis. The acute elevation in pro-insulin levels is thought to be exclusively because of the activation of translation of pre-existing prepro-insulin mRNA. Glucose-stimulated insulin gene transcription is believed to be a long term effect and should therefore not contribute to the acute elevation in pro-insulin levels. We have recently shown that glucose activates insulin gene transcription within minutes and that secreted insulin is one of the key factors triggering this process in an autocrine manner. We now provide evidence that 50% of the glucose-stimulated, acute pro-insulin biosynthesis within 30 min results from up-regulated insulin gene transcription. Our data led us to propose that glucose elevates pro-insulin levels by stimulating both transcriptional and post-transcriptional/post-translational events to an equal extent. Whereas the stimulatory effect on transcription is mediated by insulin secreted in response to glucose, glucose directly stimulates the post-transcriptional/post-translational processes.

Activation by adrenaline of a low-conductance G protein-dependent K+ channel in mouse pancreatic B cells.

Insulin is produced and secreted by the B cells in the endocrine pancreas. In vivo, insulin secretion is under the control of a number of metabolic, neural and hormonal substances. It is now clear that stimulation of insulin release by fuel secretagogues, such as glucose, involves the closure of K+ channels that are sensitive to the intracellular ATP concentration (KATP channels). This leads to membrane depolarization and the generation of Ca2(+)-dependent action potentials. The mechanisms whereby hormones and neurotransmitters such as adrenaline, galanin and somatostatin, which are released by intraislet nerve endings and the pancreatic D cells, produce inhibition of insulin secretion are not clear. Here we show that adrenaline suppresses B-cell electrical activity (and thus insulin secretion) by a G protein-dependent mechanism, which culminates in the activation of a sulphonylurea-insensitive low-conductance K+ channel distinct from the KATP channel.