Low Molecular Weight Thrombin Inhibitors Research Articles

OC-017 International Multicentre Study Assessing the Effects of Anti-Thrombotic Use in Patients with Upper GI Bleeding

Introduction Anti-thrombotics (antiplatelets and anticoagulants; ATs) have been identified as risk factors for upper gastrointestinal bleeding (UGIB). However few international studies have evaluated their effect on patient outcome. We aimed to assess the effects of AT use on outcome in patients with high-risk UGIB requiring endoscopic therapy. Methods Patients presenting with UGIB who required endoscopic therapy at eight centres (Scotland, England, USA, Canada, Denmark, Italy, Singapore & New Zealand) were prospectively included over 12 months. Data recorded included the full Rockall score (FRS); AT use (Aspirin, Adenosine Diphosphate Receptor Inhibitors (ADP-RI), Vitamin-K Antagonists (VKA), Low Molecular Weight Heparin (LMWH), Thrombin inhibitors and Factor Xa inhibitors); endoscopic findings; blood transfusion; interventional radiology; surgery; rebleeding; 30 day mortality and length of hospital stay. Results Out of 3154 patients, 619 required endotherapy (44% for ulcer bleeding and 21% for varices). 187 (30%) patients were on aspirin, 61 (11%) ADP-RI, 57 (9%) VKA, 8 (1%) LMWH, 7 (1%) factor Xa-inhibitor and 1 patient a thrombin-inhibitor. 63 (11%) patients were treated with >1 type of AT. Patients treated with ATs were older (p Conclusion Patients with UGIB who require endoscopic therapy whilst on ATs do not experience a higher rate of rebleeding or mortality compared with UGIB patients who do not use ATs. We observed excess mortality in patients not taking ATs, which is likely due to the high rates of cirrhosis (40%) and variceal bleeding (33%) in these patients. Further studies are needed to clarify the risk of adverse outcome following UGIB in patients taking novel ATs. Disclosure of Interest None Declared

Improved Stability of Proline-Derived Direct Thrombin Inhibitors through Hydroxyl to Heterocycle Replacement.

Modification of the previously disclosed (S)-N-(2-(aminomethyl)-5-chlorobenzyl)-1-((R)-2-hydroxy-3,3-dimethylbutanoyl)pyrrolidine-2-carboxamide 2 by optimization of the P3 group afforded novel, low molecular weight thrombin inhibitors. Heterocycle replacement of the hydroxyl functional group helped maintain thrombin in vitro potency while improving the chemical stability and pharmacokinetic profile. These modifications led to the identification of compound 10, which showed excellent selectivity over related serine proteases as well as in vivo efficacy in the rat arteriovenous shunt. Compound 10 exhibited significantly improved chemical stability and pharmacokinetic properties over 2 and may be utilized as a structurally differentiated preclinical tool comparator to dabigatran etexilate (Pro-1) to interrogate the on- and off-target effects of oral direct thrombin inhibitors.

Lung fibroblast growth-stimulating activity of serine protease

We compared lung fibroblast growth-stimulating activity (FGA) of several serine proteases including thrombin in vitro, and examined the mechanism of FGA. FGA was measured by incorporation of 3H-thymidine into lung fibroblasts (IMR-90). The activities of the enzymes were measured by spectrofluorometric method with synthetic peptides specific for each enzyme, and these enzymes were added to the assay system for FGA at concentrations of 7 x 10(0)-7 x 10(5) unit/ml. Human thrombin, bovine trypsin and bovine alpha-chymotrypsin showed clear FGA, but that of alpha-chymotrypsin was lower than those of thrombin and trypsin. On the other hand, porcine pancreatic elastase and human neutrophil elastase did not show any FGA, and had a cytotoxic effect on fibroblasts. A specific low molecular-weight thrombin inhibitor, argatroban (MW. 562), inhibited not only the enzyme (peptidolytic) activity of thrombin, but also its FGA at the same concentration. These results suggest that serine proteases can be classified into at least two groups, showing FGA and cytotoxic activity, respectively, and that the FGA of the former group is mediated by their catalytic (enzymatic) action.

Practical synthesis of a peptidomimetic thrombin inhibitor

Compound 1 is a low molecular weight thrombin inhibitor developed for treatment of deep vein thrombosis and cardiovascular diseases. We herein report our efforts to develop a robust, efficient and reproducible process suitable for large-scale synthesis of compound 1.

Progress in the Design of Low Molecular Weight Thrombin Inhibitors

AbstractFor Abstract see ChemInform Abstract in Full Text.

Low Molecular Weight Thrombin Inhibitors with Excellent Potency, Metabolic Stability, and Oral Bioavailability.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Progress in the design of low molecular weight thrombin inhibitors.

Intravascular thrombosis and its complication, embolism, is a leading cause of morbidity and mortality throughout the world. Past few decades have seen a great deal of progress in the development of antithrombotic agents, though the current treatment options are limited to heparin, LMW heparins, and warfarin. Detailed understanding of the biochemical and biophysical mechanisms of activation and regulation of blood coagulation have helped in developing specific inhibitors of enzymes, especially thrombin, within the coagulation cascade. Thrombin plays a central role in the coagulation cascade and so has become the primary target for the development of antithrombotic drugs. The review covers the main pharmacological aspects of haemostasis and thrombosis and provides an update on low molecular weight thrombin inhibitors along with the limitations of the prevalent antithrombotic agents. Recent developments in small molecule inhibitors of Protease Activated Receptor-1 (PAR-1) which can be helpful for the treatment of thrombotic and vascular proliferative disorders, have also been discussed.

Low molecular weight thrombin inhibitors with excellent potency, metabolic stability, and oral bioavailability

Modification of lead compound 1 by reducing lipophilicity in the P3 group produced a series of low molecular weight thrombin inhibitors with excellent potency in functional assays, metabolic stability, and oral bioavailability. These modifications led to the identification of two optimized compounds, 14 and 16.

In vitro blood compatibility of polymeric biomaterials through covalent immobilization of an amidine derivative

We present a surface coating with anticoagulant characteristics showing significantly reduced coagulation activation. The synthesis of a monomeric conjugate containing a benzamidine moiety was carried out and its inhibitory activity against human thrombin, the key enzyme of the blood coagulation cascade, was determined using a chromogenic assay. Based on that, low-thrombogenic interfaces were prepared by covalent attachment of this low-molecular weight thrombin inhibitor on poly(octadecene- alt-maleic anhydride) copolymer thin films and characterized using ellipsometry, XPS and dynamic contact angle measurements. The in vitro hemocompatibility tests using freshly drawn human whole blood showed, in agreement with the SEM images, that a PO-MA film modified with a benzamidine moiety using a PEG spacer decreased the activation of coagulation, platelets and the complement system. The decreased protein adsorption, in addition to the specific inhibition of thrombin, effectively enhanced the short-term hemocompatibility characteristics.

New Cyanopeptide‐Derived Low Molecular Weight Inhibitors of Trypsin‐Like Serine Proteases.

AbstractFor Abstract see ChemInform Abstract in Full Text.

New cyanopeptide-derived low molecular weight thrombin inhibitors.

Thrombosis is the result of defective regulation of the hemostasis system. This cardiovascular disorder may lead to deep vein thrombosis, myocardial infarction, and stroke. The majority of current drug research is focused on finding inhibitors of thrombin - the global player in hemostasis. In our work, we emphasize investigation of the marine environment to yield new lead structures from marine organisms like blue-green algae (cyanobacteria). This article deals with the design, syntheses, and inhibition tests of new low molecular weight thrombin inhibitors utilizing cyanopeptides, the secondary metabolites of cyanobacteria with interesting biological activities, as new lead structures. Starting with aeruginosin 98-B (2) as a lead structure, we have developed and synthesized new, selective acting inhibitors of thrombin (RA-1001 and RA-1002), which are suitable targets for further structure-activity studies.

New cyanopeptide-derived low molecular weight inhibitors of trypsin-like serine proteases.

This paper deals with the design, syntheses, and inhibition tests of new low molecular weight thrombin inhibitors utilizing cyanopeptides, the secondary metabolites of cyanobacteria with interesting biological activities, as new lead structures. Starting with aeruginosin 98-B (1) as a lead structure, we have developed and synthesised new, selective acting inhibitors of serine proteases (RA-1005 and RA-1009, which are suitable targets for further structure-activity studies.

Novel bicyclic lactam inhibitors of thrombin: highly potent and selective inhibitors

The potency and selectivity of a previous series of low molecular weight thrombin inhibitors were improved through modifications of the P1 and P3 residues. Introduction of diphenyl substituted sulfonamides in the P3 moiety led to highly efficacious compounds. By correctly selecting the combination of P1 and P3 residues, high levels of potency, selectivity and in vivo efficacy were obtained.

Development and current applications of thrombin-specific inhibitors.

Thrombin-specific inhibitors directly diminish thrombin-induced coagulation and cellular activities without the side effects of heparin. Hirudin is the most potent natural thrombin-specific inhibitor. Recombinant hirudins (such as desirudin) have been shown to be effective in the treatment of heparin-induced thrombocytopenia (HIT) and in the prevention of thrombotic complications after hip or knee surgery. The application of recombinant hirudin has been limited mainly by hemorrhagic complications. Synthetic thrombin-specific inhibitors, including oligopeptides, tripeptides and non-peptide low molecular weight (LMW) thrombin inhibitors, have been designed according to their interactions with the active sites of thrombin. Bivalirudin (an anti-thrombin oligopeptide) has been approved for preventing thrombosis in unstable angina patients following angioplasty in adjunct to aspirin. Argotroban (a tripeptide thrombin inhibitor) has been used for the treatment of HIT, peripheral and cerebral thrombotic diseases. The benefit of using thrombin-specific inhibitors alone in acute myocardial infarction or unstable angina remains uncertain. A number of LMW thrombin-specific inhibitors have been developed. Some of them can be administrated orally, and cause less increase in bleeding time than other thrombin inhibitors. The efficacy, safety, stability and oral bioavailability of the thrombin inhibitors may be considerably improved through structural optimization. Most of the LMW thrombin inhibitors are currently being tested in animal models or at early stages of clinical trials. In this review, we will present an overview of recent advances in thrombin-specific inhibitors.

Anaesthesia in the fourth dimension. Is biological scaling relevant to veterinary anaesthesia?

Fundamental laws, such as gravity and thermodynamics, are in common use in the physical sciences but are less applicable to biology. However, although there are fewer biological laws relative to other disciplines, the ones that exist are of great importance, e.g. natural selection. Another example may be a new law of biological network design which explains observations on how metabolic rate changes as organisms change size. It has been known for some time that as animal size increases its metabolic rate falls ( Rubner, 1883 Rubner M Ueber den Einfluss der Körpergrösse auf Stoffund. Kraftwechsel Z Biol. 1883; 19: 535-562 Google Scholar ; Kleiber, 1932 Kleiber M Body size and metabolism. Hilgardia. 1932; 6: 315-353 Crossref Google Scholar ; Adolph, 1949 Adolph EF Quantitative relations in the physiological constitutions of mammals. Science. 1949; 109: 579-585 Crossref PubMed Scopus (368) Google Scholar ; Schmidt-Nielsen, 1984 Schmidt-Nielsen K Why Is Animal Size So Important? Cambridge University Press, Cambridge, UK1984: 56-74 Google Scholar ; Calabrese, 1991 Calabrese EJ Scaling: An attempt to find a common denominator. in: Principles of Animal Extrapolation Lewis. 1991: 449-527 Google Scholar ). This has developed from a physiological curiosity to a tool that is used extensively to predict drug effects across species ( Lave et al., 1999 Lave T Portmann R Schenker G Gianni A Guenzi A Girometta M-A Schmitt M Interspecies pharmacokinetic comparisons and allometric scaling of Napsagatran, a low molecular weight thrombin inhibitor. J Pharm Pharmacol. 1999; 51: 85-91 Crossref PubMed Scopus (55) Google Scholar ). The function of this article is to review the development of this concept, and to examine its application in veterinary anaesthesia.

Pharmacokinetics and Pharmacodynamics of Melagatran, a Novel Synthetic LMW Thrombin Inhibitor, in Patients with Acute DVT

Forty-eight patients with acute proximal deep vein thrombosis (DVT) were randomised to intravenous infusions for 4 to 6 days with melagatran, a novel synthetic low molecular weight thrombin inhibitor, or unfractionated heparin adjusted by the activated partial thromboplastin time (APTT). The aim of the study was to investigate the pharmacokinetics, pharmacodynamics and the safety of melagatran therapy at three different doses. Steady-state plasma concentrations were rapidly achieved and maintained throughout the infusion period. The mean plasma concentrations in the low, medium and high dose groups were 0.17, 0.31 and 0.53 micromol/l, respectively. The prolongation of APTT was stable during the melagatran infusions and correlated to the plasma concentration. Phlebographically verified regression of thrombus size measured as decrease in Marder score was seen after 4 to 6 days in 8 of 12 patients, 6 of 12 patients and 5 of 11 patients in the low, medium and high dose groups of melagatran and in 5 of the heparin-treated patients. In the low dose group with melagatran, thrombus extension was seen in one patient. At the dose levels studied, melagatran was well tolerated with no clinically significant bleeding problems, suggesting that melagatran could safely be given to patients suffering from DVT.

Progress Towards the Discovery of Orally Active Thrombin Inhibitors

The serine protease thrombin is a key enzyme in the control of blood coagulation and displays numerous other effects on platelet, endothelial and smooth muscle cell function. The pre-eminence of thrombin in the coagulation cascade has made the enzyme a popular drug target in the search for more clinically acceptable and safe anti-coagulants. This concept has been particularly strengthened by the demonstration that direct inhibitors of thrombin such as Revasc are clinically effective. A number of low molecular weight thrombin inhibitors have now been described in the literature although to date because of their inherent low bioavailability compounds have been limited to the parenteral route of administration. The introduction of appropriate pharmacokinetic properties into these first generation thrombin inhibitors has been problematic despite intensive research in this area. However, the first pre clinical examples of direct thrombin inhibitors possessing good oral bioavailability is now emerging. This review updates current developments in the progress towards the discovery of orally available thrombin inhibitors and suggests that the first clinical validation of drugs from this field is imminent.

Heparin Is More Effective Than Inogatran, a Low-Molecular Weight Thrombin Inhibitor in Suppressing Ischemia and Recurrent Angina in Unstable Coronary Disease

Thrombin has been suggested as one of the main pharmacologic targets in unstable coronary syndromes. Electrocardiographic signs of ischemia during continuous monitoring convey prognostic information in these patients. This study assessed the anti-ischemic and clinical effects of the novel low-molecular weight thrombin inhibitor inogatran in patients with unstable angina and non–Q-wave infarction without persistent ST-segment elevation on hospital admission. Within 24 hours of the last episode of chest pain, 324 patients were randomized to 72 hours of treatment with inogatran or heparin. Continuous ST-segment analysis with computerized vectorcardiography was used to monitor ischemia for 24 hours. The occurrence of cardiac events during the first 7 days were studied and compared with ischemic episodes during the initial 24 hours. The heparin-treated patients had less episodes of ischemia (ST vector magnitude [ST-VM]: 1 ± 2.6 vs 2 ± 4.5, p <0.001 and ST change vector magnitude [STC-VM]: 3 ± 4.7 vs 6 ± 7.6, p <0.001) than the patients receiving inogatran. This was paralleled by a lower incidence of the combined end point of death, nonfatal infarction, refractory or recurrent angina during the first 7 days for the heparin-treated patients (35%) compared with the inogatran-treated patients (50%) (p <0.05). Patients who had episodes of ischemia in spite of anti-ischemic therapy were at increased risk of all events studied. Heparin is more effective than inogatran in suppressing myocardial ischemia and clinical events at short-term follow-up. Continuous ST-segment monitoring with vectorcardiography identifies nonresponders who are at an increased level of risk.

Coronary thrombosis/thrombolysis in pigs: effects of heparin, asa, and the thrombin inhibitor inogatran

The aim of the present study was to develop a coronary thrombolysis model using the copper coil technique in closed-chest pigs. The first goal (protocol I) was to obtain a reproducible size of myocardial infarction by controlling the coronary occlusion period, a prerequisite for evaluation of myocardioprotective interventions. The second goal (protocol II) was to study if thrombin and platelet aggregation inhibitors influence the rate of thrombolysis, the degree of reocclusion, and the time of coronary patency when added to a thrombolytic regimen (recombinant tissue-type plasminogen activator, rt-PA). Coronary thrombosis was produced by insertion of a thrombogenic copper coil into the LAD of 40 anesthetized pigs. The animals were divided into six groups as follows: Protocol I, group 1: Open-chest, lysis initiated with intracoronary rt-PA (50 mg) concomitant with intravenous heparin and acetylsalicylic acid (ASA) ( n = 6). Group 2: Closed-chest, lysis initiated with intracoronary rt-PA concomitant with intravenous heparin and ASA ( n = 10). Protocol II, group 3: Closed-chest, lysis initiated with intravenous rt-PA ( n = 6). Group 4: Closed-chest, lysis initiated with intravenous rt-PA concomitant with heparin ( n = 6). Group 5: Closed-chest, lysis initiated with intravenous rt-PA concomitant with inogatran, a low molecular weight thrombin inhibitor ( n = 6). Group 6: Closed-chest, lysis initiated with intravenous rt-PA immediately after intravenous administration of ASA ( n = 6). Protocol 1; Reperfusion was achieved in all closed- and open-chest pigs. The time to thrombolysis was 5 ± 1.6 and 6 ± 3.0 min (mean ± SD) for closed- and open-chest pigs, respectively. Reocclusions were rare (one in group 1). The size of the ischemic myocardial area was 21 ± 11% of the left ventricular area in group 1 and 22 ± 6% in group 2. The corresponding values for infarct size as a proportion of the ischemic area were 58 ± 10% and 68 ± 14%, respectively. The closed-chest model was subsequently used to study the effect of the thrombin and platelet aggregation inhibitors (inogatran, heparin, and ASA) as conjunctive agents to rt-PA-induced thrombolysis (groups 3–6). To mimic its clinical use, rt-PA was administered intravenously. Time to lysis after rt-PA only (group 3) was 33 ± 24 min. Concomitant treatment with heparin (group 4), inogatran (group 5), and ASA (group 6) did not significantly influence time to lysis. All adjunctive compounds did, however, prolong the time to reocclusion, which occurred in 100%, 75%, 67%, and 20% of the animals in groups 3, 4, 5, and 6. Thus, concomitant treatment with heparin and inogatran did not shorten time to lysis or reduce the reocclusion rate, and ASA turned out to be the only effective adjunct to rt-PA, significantly reducing both time to and frequency of reocclusion ( p < 0.05). Conclusion: The described closed-chest pig model was feasible as regards the induction and lysis of a thrombus in the left coronary artery, giving reproducible areas of myocardial ischemia and infarction. This model was useful for the evaluation of pharmacological interventions in the thrombolysis process.

Melagatran, a Low Molecular Weight Thrombin Inhibitor, Counteracts Endotoxin-induced Haemodynamic and Renal Dysfunctions in the Pig

Coagulation and fibrinolysis are crucial in septic shock and inhibition of thrombin may be beneficial in this circumstance. Since porcine endotoxaemia has been found to replicate severe septic shock, a low molecular weight thrombin inhibitor, melagatran, was infused during the first 3 out of 6 h of endotoxaemia in pigs. Plasma creatinine (p <0.01) and urinary output (p <0.05) were less affected in the melagtran + endotoxin group (n=6) as compared to endotoxaemic controls (n=9). The left ventricular stroke work index, systemic vascular resistance index and oxygen extraction were all less affected (p <0.05) by endotoxin during the infusion of melagatran. The plasma concentration of melagatran declined with an apparent plasma half-life of 5 h as soon as the infusion was stopped. APTT, however, continued to increase after the infusion of melagatran had stopped and reached a maximum of 113 s at 5 h (baseline 17 s). APTT in endotoxaemic control pigs reached a maximum of 22 s. Thus, melagatran may counteract some consequences of endotoxaemia.