Cysteine Sulfoxides Enhance Steroid Hormone Production via Activation of the Protein Kinase A Pathway in Testis-Derived I-10 Tumor Cells.

Testosterone plays an important role in male sexual characteristics and maturation, and decreased testosterone levels increase the risk of several diseases. Recently, onion extract rich in cysteine sulfoxides, which are amino acids unique to onions, has been reported to alleviate age-related symptoms resulting from decreased testosterone levels in males. However, the mechanism underlying the suppression of low testosterone levels by cysteine sulfoxides has not been elucidated. In this study, we found that onion extract containing cysteine sulfoxides enhanced progesterone, a precursor of testosterone, in mouse testis-derived I-10 tumor cells. Furthermore, cysteine sulfoxides activated protein kinase A (PKA) and cyclic adenosine monophosphate response element-binding protein, which are key factors in steroidogenesis. These results suggest that cysteine sulfoxides enhance steroid hormone production via activation of the PKA signaling pathway.

An online dual-enzyme co-immobilized microreactor based on capillary electrophoresis for enzyme kinetics assays and screening of dual-target inhibitors against thrombin and factor Xa.

In this study, an online capillary electrophoresis (CE) based dual-enzyme (thrombin and factor Xa) co-immobilized microreactor (THR-FXa IMER) was constructed for studying enzyme kinetics and screening dual-target inhibitors against THR and FXa with the aid of the polydopamine/graphene oxide (PDA/GO) coating. Based on the developed THR-FXa IMER, the Michaelis-Menten constants (Km) of THR and FXa were calculated to be 187.26 and 48.80 µM, respectively. The inhibition constants (Ki) for two known inhibitors, argatroban and rivaroxaban, on THR and FXa were determined to be 14.73 and 0.41 nM, respectively. In addition, after 30 consecutive runs, the enzymes' activity was remained 98% of the initial immobilized activity for both THR and FXa, which shows that the constructed IMER has good stability and repeatability. Finally, the developed method was successfully applied to screen dual-target inhibitors against THR and FXa from 30 small molecular compounds. Among them, 10 compounds such as salvianolic acid C and epigallocatechin gallate (EGCG) have dual-enzyme inhibitory activity, and 2 compounds named saikosaponin A and oleuropein have single THR inhibitory activity, 5 compounds such as rosemary acid and salvianolic acid B have single FXa inhibitory activity. Finally, the molecular interactions between enzyme and potential inhibitors were further verified via the molecular docking, and a new compound with a theoretically good coagulation inhibition effect was designed by the scaffold hopping study. In summary, the developed THR-FXa IMER is a reliable method for screening THR and/or FXa inhibitors.

Effect of emicizumab on global coagulation assays for plasma supplemented with apixaban or argatroban.

Emicizumab is a bi-specific humanized monoclonal antibody mimicking the factor (F) VIII cofactor activity in mediating the activation of FX by FIXa. Recent observations showed that emicizumab when added to pooled normal plasma (PNP), hemophilic plasma or PNP added with unfractionated heparin is able to interfere with coagulation assays. To further explore the mechanisms of assay interference we investigated the effect of emicizumab on global coagulation assays for the PNP added with two direct oral anticoagulants, apixaban or argatroban. Aliquots of PNP were added with purified apixaban or argatroban at a concentration of 500 ng/mL and emicizumab at concentrations ranging from 0 to 100 µg/mL. Plasma samples were then tested for the activated partial thromboplastin time (APTT) and for thrombin generation (the latter for the apixaban plasma only). Emicizumab at a 25-50 µg/mL shortened the APTT of the PNP with or without apixaban or argatroban. The extent of correction was greater for the apixaban or argatroban plasma and amounted to 35% or 42%, respectively. The parameters of thrombin generation (lag-time and time-to-peak) for the PNP supplemented with apixaban were shortened by 30% or 25%, respectively and the endogenous thrombin potential and the peak-thrombin were marginally affected. Emicizumab attenuates in vitro the anticoagulant activity of the PNP induced by apixaban or argatroban as documented by the correction of prolonged APTT and velocity of thrombin generation (i.e., lag-time and time-to-peak). Whether the above effects have any relevance in vivo is unknown.

Drug Repositioning Inferred from E2F1-Coregulator Interactions Studies for the Prevention and Treatment of Metastatic Cancers.

Metastasis management remains a long-standing challenge. High abundance of E2F1 triggers tumor progression by developing protein-protein interactions (PPI) with coregulators that enhance its potential to activate a network of prometastatic transcriptional targets. Methods: To identify E2F1-coregulators, we integrated high-throughput Co-immunoprecipitation (IP)/mass spectometry, GST-pull-down assays, and structure modeling. Potential inhibitors of PPI discovered were found by bioinformatics-based pharmacophore modeling, and transcriptome profiling was conducted to screen for coregulated downstream targets. Expression and target gene regulation was validated using qRT-PCR, immunoblotting, chromatin IP, and luciferase assays. Finally, the impact of the E2F1-coregulator complex and its inhibiting drug on metastasis was investigated in vitro in different cancer entities and two mouse metastasis models. Results: We unveiled that E2F1 forms coactivator complexes with metastasis-associated protein 1 (MTA1) which, in turn, is directly upregulated by E2F1. The E2F1:MTA1 complex potentiates hyaluronan synthase 2 (HAS2) expression, increases hyaluronan production and promotes cell motility. Disruption of this prometastatic E2F1:MTA1 interaction reduces hyaluronan synthesis and infiltration of tumor-associated macrophages in the tumor microenvironment, thereby suppressing metastasis. We further demonstrate that E2F1:MTA1 assembly is abrogated by small-molecule, FDA-approved drugs. Treatment of E2F1/MTA1-positive, highly aggressive, circulating melanoma cells and orthotopic pancreatic tumors with argatroban prevents metastasis and cancer relapses in vivo through perturbation of the E2F1:MTA1/HAS2 axis. Conclusion: Our results propose argatroban as an innovative, E2F-coregulator-based, antimetastatic drug. Cancer patients with the infaust E2F1/MTA1/HAS2 signature will likely benefit from drug repositioning.

Phenolic fractions from nine Trifolium species modulate the coagulant properties of blood plasma in vitro without cytotoxicity towards blood cells.

OBJECTIVE: The study covers an evaluation of the influence of extracts (1-50 µg/ml), isolated from aerial parts of nine Trifolium L. species (i.e. T. alexandrinum, T. fragiferum, T. hybridum, T. incarnatum, T. pallidum, T. pratense, T. resupinatum var. majus, T. resupinatum var. resupinatum and T. scabrum) on haemostatic properties of blood plasma. METHODS: The clot formation and fibrinolysis assay (CFF), blood clotting times, the extrinsic and intrinsic coagulation pathway-dependent polymerization of plasma fibrin were measured. The effects of plant extracts on amidolytic activity of thrombin were also evaluated and compared with argatroban, an antithrombotic drug. Cytotoxicity was assessed in a model of blood platelets and as the viability of peripheral blood mononuclear cells. KEY FINDINGS: While no changes in blood clotting times or fibrinolytic properties of blood plasma were found, some fractions impaired the blood plasma coagulation induced by the intrinsic coagulation pathway. Reduction in the maximal velocity of fibrin polymerization was also observed in the clot formation and fibrinolysis assay. No cytotoxicity of Trifolium extracts towards the investigated cells was recorded. CONCLUSIONS: The most efficient anticoagulant activity in plasma was found for T. fragiferum and T. incarnatum extracts, while the T. alexandrinum fraction was the most effective inhibitor of thrombin amidolytic activity.

Randomized, Multicenter Trial of ARTSS-2 (Argatroban With Recombinant Tissue Plasminogen Activator for Acute Stroke).

BACKGROUND AND PURPOSE: We conducted a randomized exploratory study to assess safety and the probability of a favorable outcome with adjunctive argatroban, a direct thrombin-inhibitor, administered to recombinant tissue-type plasminogen activator (r-tPA)-treated ischemic stroke patients. METHODS: Patients treated with standard-dose r-tPA, not receiving endovascular therapy, were randomized to receive no argatroban or argatroban (100 µg/kg bolus) followed by infusion of either 1 (low dose) or 3 µg/kg per minute (high dose) for 48 hours. Safety was incidence of symptomatic intracerebral hemorrhage. Probability of clinical benefit (modified Rankin Scale score 0-1 at 90 days) was estimated using a conservative Bayesian Poisson model (neutral prior probability centered at relative risk, 1.0 and 95% prior intervals, 0.33-3.0). RESULTS: Ninety patients were randomized: 29 to r-tPA alone, 30 to r-tPA+low-dose argatroban, and 31 to r-tPA+high-dose argatroban. Rates of symptomatic intracerebral hemorrhage were similar among control, low-dose, and high-dose arms: 3/29 (10%), 4/30 (13%), and 2/31 (7%), respectively. At 90 days, 6 (21%) r-tPA alone, 9 (30%) low-dose, and 10 (32%) high-dose patients were with modified Rankin Scale score 0 to 1. The relative risks (95% credible interval) for modified Rankin Scale score 0 to 1 with low, high, and either low or high dose argatroban were 1.17 (0.57-2.37), 1.27 (0.63-2.53), and 1.34 (0.68-2.76), respectively. The probability that adjunctive argatroban was superior to r-tPA alone was 67%, 74%, and 79% for low, high, and low or high dose, respectively. CONCLUSIONS: In patients treated with r-tPA, adjunctive argatroban was not associated with increased risk of symptomatic intracerebral hemorrhage and provides evidence that a definitive effectiveness trial is indicated. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique Identifier: NCT01464788.

Argatroban more effectively inhibits the thrombin activity in synovial fluid than naturally occurring thrombin inhibitors.

The purpose of this study was to clarify the precise effect of argatroban on the inhibition of cytokine secretion induced by thrombin on synovial cells. The efficiency of thrombin inactivation by thrombin inhibitors was evaluated in human synovial fluids (SFs). In SFs from 13 osteoarthritis (OA) and 11 rheumatoid arthritis (RA) patients, thrombin, Factor Xa (FXa), plasmin activity, IL-6, MMP-3, VEGF, and D-dimer concentrations were measured. Tissue factor (TF) activity or IL-6, MMP-3, and VEGF secretion of human synovial cells with or without thrombin and argatroban were measured. The efficiency of thrombin inactivation in SFs was compared for thrombin inhibitors: argatroban, antithrombin III (ATIII), or heparin cofactor II (HCII). In SFs, thrombin, FXa, plasmin, D-dimer, IL-6, and MMP-3 were significantly higher in RA than in OA. In synovial cell experiments, TNF-alpha and thrombin enhanced TF activity on the cell surface, and IL-6, MMP-3, and VEGF secretion were enhanced by thrombin. Increased TF activity, and IL-6, MMP-3, and VEGF secretion induced by thrombin were inhibited by argatroban. In SFs, argatroban inactivated thrombin more effectively than ATIII or HCII. Since thrombin plays an important role in the disease activity of OA and RA, it is a potential therapeutic molecular target. Argatroban was the most effective anticoagulant to inhibit thrombin activity in SF. Intra-articular injection is ideal administration because it can deliver high dose of argatroban without high risk of systematic complication.

A quantitative affinity-based technique for the identification of potential lead compounds.

Here, we describe the development and validation of a quantitative analytical method for rapid evaluation of protein-compound interactions. The method uses size-exclusion chromatography in a 96-well format with liquid chromatography/mass spectrometry (qSEC-LC/MS) by which the amount of a compound that was originally in complex with a target protein is determined as an indicator of the binding affinity. Proof of concept of this new analytical approach was performed using a thrombin-inhibitor model. The results showed that the qSEC-LC/MS could be developed into an effective affinity-based analytical technique, despite a few limitations such as difficulty in determining the K(d) value accurately.

Troubleshooting the rabbit ferric chloride-induced arterial model of thrombosis to assess in vivo efficacy of antithrombotic drugs.

INTRODUCTION: The FeCl3-induced arterial model of thrombosis is one of the most widely used animal models to assess arterial efficacy of new antithrombotic drug candidates. This model is well-established in rodents but in a less extent in the rabbit. In this work, we present a methodology for a rabbit FeCl3-induced arterial model of thrombosis derived from our troubleshooting which allows the generation of reliable efficacy data for new antithrombotic drug candidates. METHODS: Rabbits were administered with heparin 4.5U/kg/min, argatroban 10µg/kg/min or saline by intravenous infusion. The blood flow was monitored using a Doppler flow probe. The time from the application of FeCl3 to the recorded zero blood flow was defined as the time to occlusion, with a maximum recording time of 60min post-FeCl3 application. After 30min of infusion, thrombosis was induced by wrapping a FeCl3-saturated filter paper around the carotid artery caudal to the flow probe. Animals were subject to exclusion criteria based on the visual aspect of the artery FeCl3-induced injury and based on changes in blood flow upon FeCl3 application. RESULTS: Following the application of FeCl3, a mean time to occlusion for saline, heparin and argatroban of 24.3±1.8, 52.5±4.8 and 53.5±4.5min was obtained, respectively. Mean time to occlusion for heparin and argatroban administered groups was significantly different when compared to the saline-treated group (p<0.05). These results for the test compounds represent approximately 80% of the maximum possible prolongation. DISCUSSION: The rabbit FeCl3-induced arterial model of thrombosis presented in this paper derived from our troubleshooting is sensitive and reproducible for the generation of accurate and reliable efficacy data in the assessment of new antithrombotic agents in preclinical drug development.

Autoantibody-mediated arthritis in the absence of C3 and activating Fcγ receptors: C5 is activated by the coagulation cascade.

INTRODUCTION: The effector functions of immunoglobulin G (IgG) are mediated by interaction of its Fc region with Fc receptors (FcγRs) and/or the complement system. The three main pathways of complement activation converge at C3. However, C3-independent pathways can activate C5 and other downstream complement components during IgG-initiated inflammatory responses. These C3-independent pathways of C5 activation are triggered by activating FcγRs in some systems or can be activated by factors of the coagulation cascade such as thrombin. Here we studied the interplay of C3, C5, and activating FcγRs in a model of spontaneous autoantibody-driven arthritis. METHODS: We utilized the K/BxN TCR transgenic mouse model of arthritis. We bred K/BxN mice bearing targeted or naturally-occurring mutations in one or more of the genes encoding complement components C3, C5, and FcRγ, the cytoplasmic signaling chain shared by the activating FcγRs. We measured arthritis development, the production of arthritogenic autoantibodies, T cell activation status and cytokine synthesis. In addition, we treated mice with anti-C5 monoclonal antibodies or with the thrombin inhibitor argatroban. RESULTS: We have previously shown that genetic deficiency of C5 protects K/BxN mice from the development of arthritis. We found here that C3-deficient K/BxN mice developed arthritis equivalent in severity to C3-sufficient animals. Arthritis also developed normally in K/BxN mice lacking both C3 and FcRγ, but could be ameliorated in these animals by treatment with anti-C5 monoclonal antibody or by treatment with argatroban. Production of arthritogenic autoantibodies, T cell activation, and T cell cytokine production were not affected by the absence of C3, C5, and/or FcRγ. CONCLUSIONS: In K/BxN mice, C5-dependent autoantibody-driven arthritis can occur in the genetic absence of both complement C3 and activating FcγRs. Our findings suggest that in this setting, thrombin activates C5 to provoke arthritis.

Ancillary approaches to plasminogen activators.

Acute ischemic stroke develops from an interruption in focal cerebral blood flow. In many cases, it is caused by an acute thromboembolism. Although systemic fibrinolytic therapy for acute ischemic stroke has been a significant breakthrough in the management of this disease, additional agents and methods that could improve or restore cerebral flow are necessary. Similarly to findings in acute myocardial infarction, combination pharmacotherapy has the potential to improve current thrombolytic treatment in acute ischemic stroke. In recent years, research efforts were directed toward various combination therapy with pharmacological and nonpharmacological methods. Several trials tested tissue plasminogen activator (t-PA) in combination with antiplateletes and anticoagulants. Combination of t-PA with nonpharmacological agents included sonothrombolysis (amplifying the thrombolytic effect), laser (neuro-recovery), hypothermia (cytoprotection and decreasing brain swelling), and blood flow augmentation (increasing residual flow and recruitment of collateral vessels). This paper will review ongoing clinical trials and safety of these promising combinatory treatments.

Glucose uptake stimulatory effect of 4-hydroxypipecolic acid by increased GLUT 4 translocation in skeletal muscle cells.

Peganum harmala Linn, commonly known as 'harmal' belonging to the family Zygophyllaceae, is one of the most important medicinal plants of India. In continuation of our drug development program on Indian medicinal plants we discovered antihyperglycemic activity in 4-hydroxypipecolic acid (4-HPA), isolated from the seed of P. harmala. Effect of 4-HPA on glucose uptake and glucose transporter-4 (GLUT-4) translocation was investigated in L6 skeletal muscle cell lines. Treatment with 4-HPA stimulated both glucose uptake and GLUT4 translocation from intracellular to cell surface in skeletal muscle cells in a concentration-dependent manner, which might be leading to antihyperglycemic effect.

Anti-diabetic and anti-oxidative effects of 4-hydroxypipecolic acid in C57BL/KsJ-db/db mice.

Hypoglycemic effect of ethanol extracts of Peganum harmala (commonly known as 'Harmal') seeds has been reported on normal and streptozotocin-induced diabetic rats. In the present study, the authors determine anti-diabetic and anti-oxidative properties of 4-hydroxypipecolic acid (4-HPA) isolated from seeds of P. harmala in C57BL/KsJ-db/db mice. Twelve week old male mice were administered 50 mg/kg body weight (4-HPA suspension were made in 1% gum acacia) for the period of 10 days, and a significant reduction in the fasting blood glucose, plasma triglycerides (TG), cholesterol, free fatty acid, low-density lipoprotein-cholesterol and a significant increase in high-density lipoprotein-cholesterol level was observed with respect to vehicle-treated db/db mice. The anti-oxidant activity of 4-hydroxypipecolic acid was studied in liver and kidney tissues by assessing malondialdehyde levels for lipid peroxidation and enzyme activity of catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD). Treatment of 4-HPA significantly lowered the lipid peroxidation in hepatic and renal tissue and increased the activity of CAT, GSH-Px and SOD in treated mice.

An optimized method to assess in vivo efficacy of antithrombotic drugs using optical coherence tomography and a modified Doppler flow system.

INTRODUCTION: Animal models of venous and arterial thrombosis are extremely useful to study the efficacy of antithrombotic agents. Variability in efficacy data is often observed in those preclinical studies. The goal of this study was to optimize the methodology for assessing antithrombotic drug efficacy by the use of optical coherence tomography (OCT) and a modified Doppler flow system in rat models of thrombosis. METHODS: Thrombus formation was assessed in both the rat venous and arterial ferric chloride (FeCl(3)) models of thrombosis. In the venous model, thrombus volume post-treatment was measured using OCT, and data were correlated against the thrombus weight. In the arterial model, the time to occlusion was measured using a Doppler flow probe connected to a perivascular flow module which allowed the reporting of dynamic blood flow data every 30s. Heparin (130 or 165U/kg), argatroban (4.5mg/kg), bivalirudin (1.3mg/kg) or saline were administered intravenously. RESULTS: In the venous model, for all treatment groups a strong linear correlation (R(2)=0.998) was observed between thrombus volume measured by OCT and thrombus weight. In the arterial model, using a high sampling rate of a dynamic blood flow using a modified Doppler flow system provided data accuracy and precision of the time to occlusion measurement. DISCUSSION: This study demonstrates that OCT is a powerful tool for the assessment of antithrombotic drug efficacy. Furthermore, it shows that a high Doppler sampling rates of dynamic blood flow leads to data accuracy and precision.

Thrombin inhibition by argatroban ameliorates early brain injury and improves neurological outcomes after experimental subarachnoid hemorrhage in rats.

BACKGROUND AND PURPOSE: We investigated the role of thrombin in early brain injury after subarachnoid hemorrhage (SAH). METHODS: The standard intravascular perforation model was used to produce experimental SAH in Sprague Dawley rats. Low-dose (0.3 mg/h) and high-dose (0.9 mg/h) argatroban, a direct thrombin inhibitor, were evaluated for effects on brain edema, blood-brain barrier (BBB) disruption, apoptotic cell death, inflammatory marker, and neurological outcomes after SAH. RESULTS: Both doses of argatroban attenuated BBB disruption; however, only high-dose was effective in lowering edema in all brain regions, reducing cell death, and inflammatory marker expression, and improving neurological outcomes. CONCLUSIONS: Thrombin inhibition by argatroban improves neurological outcomes and provides neuroprotection against acute events after SAH such as BBB disruption, brain edema, and cell death.

Inhibition of osteopontin dependent carcinogenesis.

Osteopontin is a molecule that promotes cellular proliferation as well as metastasis and plays a major role in the evolution of most systemic malignancies. Inhibition of carcinogenetic mechanisms that involve osteopontin will undoubtedly go a long way in controlling the growth of these tumors. A number of new molecules such as argatroban agelastatin A and selenium have been identified in this regard. For instance, Schulze et al. (in Breast Cancer Res Treat) have recently reported the successful utilization of argatroban for decreasing the growth of breast cancer tumor cells as well as their lymphatic metastasis. Similarly, anti osteopontin antibodies and antisense osteopontin RNA have been successfully used to inhibit tumor growth in prostate carcinomas and osteosarcomas, respectively. There is a clear and urgent need to expand the use of these molecules as well as to further identify other potent inhibitors of osteopontin mediated tumorigenesis.

Inhibition of thrombin generation in recalcified plasma.

Eight inhibitors of thrombin generation were compared in recalcified unfrozen plasma. Individual or pooled normal citrated plasma was supplemented on polystyrol flat-bottom wells (23 degrees C) with increasing concentrations of low-molecular-weight heparin, heparin, danaparoid, fondaparinux, hirudin, argatroban, corn trypsin inhibitor, or aprotinin. Thrombin was generated by addition of 5 microl fresh 250 mmol/l CaCl2 to 50 microl plasma in polystyrol flat-bottom wells and incubation for 20 min at 37 degrees C (recalcified coagulation activity assay). Arginine stopped hemostasis activation and then the generated thrombin activity was specifically quantified. The approximate 50% inhibitory concentrations of plasmatic anticoagulants for individual or pooled normal plasma are, respectively, 0.6 or 3.7 mIU/ml low-molecular-weight heparin, 0.3 or 1.6 mIU/ml heparin, 0.7 or 6.1 mU/ml danaparoid, 0.023 or 0.18 microg/ml fondaparinux, 75 or 230 pg/ml hirudin, 0.026 or 0.24 microg/ml argatroban, 1 or 2 U/ml corn trypsin inhibitor, and 2 or 4 KIU/ml aprotinin. The 50% inhibitory concentration values for corn trypsin inhibitor or aprotinin at plasmatic concentrations above 4-100 U/ml might increase pathologically the thrombin generation. The recalcified coagulation activity assay is a sensitive method to measure prothrombotic tendencies of blood or subtle concentrations of any plasmatic anticoagulant. It is suggested to analyze the individual patient's sensibility to certain plasmatic anticoagulants.

Differential effects of direct thrombin inhibitors and antithrombin-dependent anticoagulants on the dynamics of clot formation.

New anticoagulants, including the direct thrombin inhibitors (DTIs) and fondaparinux, are increasingly replacing unfractionated heparin and enoxaparin. We examined the effects of argatroban (n = 60), bivalirudin (n = 44), heparin (n = 14), enoxaparin (n = 22), and fondaparinux (n = 24) on clot formation utilizing thromboelastography. Blood samples containing anticoagulants at clinically relevant concentrations were prepared ex vivo and analyzed using kaolin or tissue factor activation. Thromboelastography parameters of clot initiation (R), clot propagation (K and angle), clot rigidity (maximum amplitude) and clot elasticity (G) were compared between anticoagulants. Thromboelastography was also performed on blood from eight patients receiving anticoagulants. Each anticoagulant exerted significant concentration-dependent effects on R, K and angle. Only heparin, enoxaparin, and fondaparinux significantly affected maximum amplitude and G. Significant differences existed for all parameters between heparin and each anticoagulant and between fondaparinux and each DTI (P < 0.001), and for angle, maximum amplitude, and G between enoxaparin and each DTI (P < 0.008). Thromboelastography responses in ex-vivo samples and patient samples were comparable. In conclusion, whereas argatroban, bivalirudin, heparin, enoxaparin and fondaparinux each delay clot formation, the DTIs do not alter clot rigidity or elasticity. The reduced bleeding reported with DTIs versus heparin may relate to the fact that clots form with normal rigidity and elasticity.

Synthesis and pharmacochemical study of novel polyfunctional molecules combining anti-inflammatory, antioxidant, and hypocholesterolemic properties.

We have designed and synthesized a series of novel molecules having a residue of a classical NSAID and an antioxidant moiety, both attached through amide bonds to a known nootropic structure, an L-proline, trans-4-hydroxy-L-proline or DL-pipecolinic acid residue. The compounds were found to retain anti-inflammatory and antioxidant activities, to acquire hypocholesterolemic action, and to possess a greatly reduced gastrointestinal toxicity. The novel molecules could find useful applications, among others, in slowing the progression or delaying the onset of neurodegenerative diseases.

Antithrombotic potential of new direct thrombin inhibitors built on the azaphenylalanine scaffold in two rat venous thrombosis models.

The antithrombotic potential of new direct thrombin inhibitors built on the azaphenylalanine scaffold (LK-732, LK-639 and LK-731) and their amidoxime prodrugs (LK-658, LK-633 and LK-730) was studied in comparison to argatroban and nadroparin in two rat models of venous thrombosis, induced either by complete stasis combined with hypercoagulability (model 1) or by partial stasis combined with vessel injury (model 2). In initial experiments LK-732 was established as the most promising antithrombotic of the LK inhibitors and as such was further tested. In model 1, intravenous bolus administration of LK-732 produced a dose-dependent inhibition of thrombus formation with an ID50 value of 1.3 mg/kg. This ID50 value was approximately four times higher than the ID50 value of argatroban (0.3 mg/kg; p=0.011). However, in model 2, LK-732 and argatroban decreased thrombus weight by 50% at similar ID50 values (3.8 mg/kg vs 3.0 mg/kg, respectively; p=0.726). The ex vivo anticoagulant effect of LK-732 was substantially weaker compared to argatroban at doses that produced comparable antithrombotic effects. After subcutaneous administration, in vivo thrombus weight reduction of LK inhibitors (10 mg/kg) ranged between 22 to 48%. However, their oral antithrombotic effect at a dose of 30 mg/kg was rather low. LK amidoxime prodrugs failed to produce a substantial antithrombotic effect after subcutaneous (10 mg/kg) as well as after oral administration (30 mg/kg). In conclusion, thrombin inhibitors built on the azaphenylalanine scaffold represent a new group of intravenously effective antithrombotics. However, optimisation of the oral antithrombotic effect of amidoxime prodrug LK-658 of the lead inhibitor LK-732 is required for justifying further development of these inhibitors.