The direct thrombin inhibitor melagatran and its oral prodrug H 376/95: intestinal absorption properties, biochemical and pharmacodynamic effects.

UNLABELLED: Suboptimal gastrointestinal absorption is a problem for many direct thrombin inhibitors. The studies presented herein describe the new oral direct thrombin inhibitor H 376/95, a prodrug with two protecting residues added to the direct thrombin inhibitor melagatran. Absorption properties in vitro: H 376/95 is uncharged at intestinal pH while melagatran is charged. H 376/95 is 170 times more lipophilic (octanol water partition coefficient) than melagatran. As a result, the permeability coefficient across cultured epithelial Caco-2 cells is 80 times higher for H 376/95 than for melagtran. Pharmacokinetic studies in healthy volunteers: H 376/95 is converted to melagatran in man. Oral bioavailability, measured as melagatran in plasma, is about 20% after oral administration of H 376/95, which is 2.7-5.5 times higher than after oral administration of melagatran. The variability in the area under the drug plasma concentration vs. time curve (AUC) is much smaller with oral H 376/95 (coefficient of variation 20%) than with oral melagatran (coefficient of variation 38%). Pharmacodynamic properties: H 376/95 is inactive towards human alpha-thrombin compared with melagatran [inhibition constant (K(i)) ratio, 185 times], a potential advantage for patients with silent gastrointestinal bleeding. In an experimental thrombosis model in the rat, oral H 376/95 was more effective than the subcutaneous low molecular weight heparin dalteparin in preventing thrombosis. CONCLUSION: By the use of the prodrug principle, H 376/95 endows the direct thrombin inhibitor melagatran with pharmacokinetic properties required for oral administration without compromising the promising pharmacodynamic properties of melagatran.

Effect of different types of thrombin inhibitors on thrombin/thrombomodulin modulated activation of protein C in vitro.

The objectives of this study were to investigate whether the affinity of thrombin for small-molecule, active site-directed thrombin inhibitors and substrates is affected by the presence of thrombomodulin (TM), and to what extent thrombin inhibitors inhibit TM-bound thrombin. Inhibition of human alpha-thrombin was studied in the presence and absence of solubilised rabbit lung TM in a buffer containing CaCl(2). TM inhibited thrombin-induced proteolysis of human fibrinogen with a dissociation constant (K(D)) of 4 nmol/l. With at least 16-fold molar excess of TM over thrombin the affinity of thrombin both for the small thrombin substrates (S-2366 and S-2238) and the reversible, active site-directed thrombin inhibitors (inogatran and melagatran) increased twofold. In contrast, the ability of hirudin to inhibit thrombin was reduced by TM, since hirudin competes with TM in binding to thrombin. The effect of thrombin inhibitors on protein C activation by thrombin bound to human kidney cells transfected with cDNA for human TM was also studied. The mean binding capacity of the transfected cells was approximately 320,000 quantified by flow cytometry with antibodies against TM. Hirudin, inogatran and melagatran inhibited the activation of protein C by thrombin complexed with cell-bound TM in a dose-dependent manner, with mean IC(50) values+/-S.D. of 4.4+/-0.8, 20.0+/-1.1 and 6.4+/-0.2 nmol/l, respectively. Antithrombin inhibited protein C activation with an IC(50) value of 290+/-10 nmol/l, which was enhanced fourfold (IC(50) 60 nmol/l) by the addition of heparin 0.5 U/ml. Heparin alone, up to a concentration of 1 U/ml, had no effect on the activation of protein C. Small direct thrombin inhibitors thus inhibited both free and TM-bound thrombin and therefore also inhibited the activation of protein C. Whether this will influence their clinical efficacy or safety versus heparin and warfarin, which also inhibit protein activation, respectively, lowers the concentration of protein C, remains to be studied in clinical trials.

Biosensor analysis of drug-target interactions: direct and competitive binding assays for investigation of interactions between thrombin and thrombin inhibitors.

The sensitivity of BIACORE technology is sufficient for detection and characterization of binding events involving low-molecular-weight compounds and their immobilized protein targets. The technology requires no labeling and provides information on the stability of the compound/target complex with a single injection of the compound. This is useful for qualifying hits obtained in a primary screen and in lead optimization. Although immobilized targets can be reused, the surface may slowly deteriorate, solvent effects can distort binding levels during injection of compounds, and some compounds may exhibit broad protein selectivity rather than target specificity. A reliable direct binding assay for compounds binding to immobilized thrombin using a combination of two reference surfaces, a dextran surface for subtraction and calibration of solvent effects and a protein surface for identification of compounds that tend to bind proteins, has been developed. Eleven compounds with known binding specificity to thrombin and 159 additional compounds were investigated. All compounds with known binding specificity were identified at 1 and 10 microM concentration. One additional compound was scored as positive. The direct binding assay compared favorably with two competitive assay formats, a surface competitive assay and a inhibitor in solution assay, that were examined in parallel.

Effects of melagatran, a new low-molecular-weight thrombin inhibitor, on thrombin and fibrinolytic enzymes.

Melagatran, a new, competitive and rapid inhibitor of thrombin with a molecular mass of 429 Da is described. Melagatran is well tolerated when administered in very high doses, and the oral bioavailability in the dog is relatively high. The aim of the study was to determine, in the preclinical setting, the degree of selectivity against the fibrinolytic system required for entering the clinical development phase. Melagatran was compared with two structurally similar thrombin inhibitors, inogatran and H 317/86. The potent inhibition of thrombin by melagatran was demonstrated by a low inhibition constant (Ki) for thrombin (0.002 micromol/l) and prolongation of clotting time to twice the control value in coagulation assays at low concentrations (0.010, 0.59 and 2.2 micromol/l for thrombin time, activated partial thromboplastin time and prothrombin time, respectively). Furthermore, thrombin-induced platelet aggregation was inhibited at the same concentration (IC50-value 0.002 micromol/l) as the Ki-value for thrombin. In two assays of global fibrinolysis, inhibition was observed at a concentration of 1.1 micromol/l in a euglobulin plasma fraction model, while no inhibition was observed at a concentration of < or = 10 micromol/l in a plasma model. In an in vivo model of endogenous fibrinolysis in the rat, inhibition of fibrinolysis was observed at > or = 1.0 micromol/l. In all assays, except the Ki-ratio determinations, the compounds could be graded with regard to selectivity against the fibrinolytic system: inogatran > melagatran > H 317/86. For melagatran, inhibition of fibrinolysis was not observed at concentrations below the upper limit of the proposed therapeutic plasma concentration interval (< 0.5 micromol/l). Thus, melagatran seems to have a sufficient selectivity against the fibrinolytic system, while H 317/86 was considered to be insufficient for clinical development.

In vitro effects of inogatran, a selective low molecular weight thrombin inhibitor.

The thrombin inhibitor inogatran is a synthetic peptidomimetic with a molecular weight of 439 dalton. In vitro studies have shown that inogatran is a classical competitive inhibitor of the active site of thrombin with a Ki of 15 x 10(-9) mol/l. Inogatran doubles the thrombin clotting time in human plasma at 20 x 10(-9) mol/l, APTT at 1.2 x 10(-6) mol/l, and prothrombin time at 4 x 10(-6) mol/l. The effects on rat and dog plasma are similar although slightly weaker. IC50 for inhibition of thrombin-induced aggregation of human platelets is 17 x 10(-9) mol/l. Inogatran has no effect on platelet aggregation induced by ADP or collagen. Up to a concentration of 10 x 10(-6) mol/l inogatran does not inhibit t-PA-induced fibrinolysis as seen in an ECLT system. Inogatran has good selectivity for thrombin as compared to several other serine proteases occurring in the blood. It is concluded that the properties of inogatran in vitro make the compound suitable for further studies in animals and man.

Thrombin inhibitors based on ketone derivatives of arginine and lysine.

Much attention is currently focused on inhibitors of thrombin as potential anticoagulants. We have previously reported thrombin inhibitors based on fragments of fibrinogen containing a ketomethylene isostere at P1-P'1. We now expand on these early findings by reporting on tripeptide based inhibitors of thrombin containing arginine or lysine ketones at the C-terminus. A large variety of such ketones have been studied and compared in their ability to increase the thrombin time in human plasma. In the case of arginine or lysine ketones the order of activity (i.e. decreasing IC50 TT) was: alkyl ketones < beta-ketoesters < difluoro beta-ketoamides < alkyloxymethyl ketones < fluoroketones. Lysine analogues were generally found to be ca. ten-fold less active than their arginine counterparts. However, in the case of alpha-ketoesters the lysine derivatives were superior to all the types of arginine ketones studied (including the arginine alpha-keto ester derived thrombin inhibitor). A mechanistic explanation of the relative inactivity of the arginine alpha-keto ester derivative is proposed. All the highly electrophilic ketones were found to be slow-binding with thrombin.

Urinary kallikrein excretion is low in malignant essential hypertension.

OBJECTIVE: To determine whether the urinary excretion of kallikrein is altered in patients with previously malignant hypertension. DESIGN: Twenty-two patients with malignant hypertension (fundus hypertonicus III or IV) in the Gothenburg area were studied over a 3-year period. After treatment had begun they were investigated for blood pressure control, family history of hypertension, renal function and urinary kallikrein and plasma prekallikrein concentrations. Twenty-two patients with treated non-malignant hypertension and 36 control subjects were investigated concomitantly. The two hypertensive groups were also separated into subgroups of essential and secondary hypertension. METHODS: Prekallikrein was activated and kallikrein determined by a spectrophotometric assay using a synthetic chromogenic substrate. Renal function was estimated by serum creatinine and 51Cr-ethylenediaminetetraacetic acid clearance. RESULTS: Prekallikrein levels tended to be elevated in all groups of hypertensive patients compared with controls whilst urinary kallikrein was significantly decreased in malignant hypertensives. The most pronounced suppression of urinary kallikrein was seen in the group of patients with essential malignant hypertension. The differences persisted when urinary kallikrein was related to the degree of renal impairment or when urinary volume was taken into account. There was no relation between family history of hypertension and low levels of urinary kallikrein. CONCLUSION: Decreased urinary kallikrein could indicate depressed activity in the renal kallikrein-kinin system, which may be associated with the initiation of essential malignant hypertension.

Thrombosis after hip replacement. Relationship to the fibrinolytic system.

Twenty-nine patients were operated on with the Charnley hip prosthesis. All the patients were given dextran 70 as thrombosis prophylaxis. Deep vein thrombosis (DVT) was diagnosed in 10 patients with the radioactive fibrinogen uptake test and phlebography. Variables of coagulation and fibrinolysis were studied before and after surgery. Tissue plasminogen activator (t-PA) activity in the plasma without venous occlusion decreased postoperatively, but there was no correlation with DVT. The t-PA activity in venous occlusion plasma was not reduced after surgery. Plasminogen activator inhibitor (PAI-1) levels were raised immediately postoperatively. There was a significant correlation between preoperative PAI-1 activity and development of postoperative DVT (P less than 0.05). Patients developing DVT had higher levels of PAI-1 postoperatively than patients not developing DVT. A defective fibrinolytic system, as defined by high PAI-1 activity, thus predisposed to postoperative DVT.

Determination of plasminogen activator inhibitor in plasma using t-PA and a chromogenic single-point poly-D-lysine stimulated assay.

A two stage method for determination of plasminogen activator inhibitor (PAI) activity in blood plasma is described. In the first stage, an excess of single-chain tissue plasminogen activator (t-PA) is added to plasma. In the second stage, the residual t-PA activity is determined with a plasminogen/chromogenic plasmin substrate assay utilizing poly-D-lysine as a t-PA stimulator. The method proved accurate since it correctly determined the PAI activity (range 0 to 110U/mL) in citrated plasma samples with levels established by time course analysis of t-PA inhibition and by titration with t-PA. Furthermore, correlation was excellent (r = 0.97) with the method of Chmielewska et al Thromb. Res. 31, 427-436, 1983. Plasma samples with increased platelet factor 4, indicative of platelet release, did not show increased levels of PAI activity.

Early activation of humoral proteolytic systems in patients with multiple trauma.

Coagulation, fibrinolytic, kallikrein, and complement systems were studied in 20 patients with multiple trauma. Three of four patients with a trauma score less than 10 on hospital arrival died, compared to one of 16 with a score over 10. Five patients developed disseminated intravascular coagulation. Signs of activated cascade systems were evident in most patients on hospital arrival. Changes were not related to trauma score, but patients with an arterial pressure below 110 mm Hg had significantly lower levels of antithrombin III and alpha 2-antiplasmin than those with higher BP. This study confirms that the cascade systems are activated very soon after multiple trauma.

A sensitive assay for tissue plasminogen activator activity in plasma, using adsorption on lysine-sepharose.

Tissue plasminogen activator (t-PA) in plasma was separated from inhibitors by adsorption on lysine-Sepharose. It was then determined indirectly by measuring the plasmin generated from plasminogen with poly-lysine as stimulator, in a chromogenic, parabolic rate assay. The reaction proceeded with tissue plasminogen activator and plasmin(ogen) adsorbed on the gel, and followed the kinetics described for similar parabolic rate assays in soluble systems. The assay was standardized against melanoma plasminogen activator (m-PA) and had the sensitivity range of 0.001-0.020 IU (4-80 pg). Anti-m-PA IgG quenched the activity generated in plasma on venous occlusion and part of the activity in pre-occlusion plasma. The method was sensitive to purified urokinase. The basic plasma values in resting normal individuals were: mean 0.08, range 0.01-0.26 X 10(3) IU/l (n = 19), and after 20 min of venous occlusion: mean 2.48, range 0.24-4.34 X 10(3) IU/l (n = 10). The assay correlates well with a fibrin plate method, r = 0.96.

alpha 2-Antiplasmin and alpha 2-macroglobulin--the main inhibitors of fibrinolysis--during the menstrual cycle, pregnancy, delivery, and treatment with oral contraceptives.

alpha 2-Antiplasmin and alpha 2-macroglobulin have been studied during the menstrual cycle, pregnancy and parturition in healthy women, and during use of various types of contraception in both healthy and diabetic women, and compared with a reference group of healthy men and women. alpha 2-Antiplasmin showed a slight sex difference, with higher values in women. The luteal phase of the menstrual cycle showed slightly higher values than the other phases. alpha 2-Antiplasmin increased during pregnancy, decreased (probably due to consumption) during labor and increased again in the puerperium. Treatment with neither combined contraceptive pills nor low dose progestogen pills gave any changes in alpha 2-antiplasmin. alpha 2-Macroglobulin showed low values during menstruation. The increase during pregnancy and treatment with combined contraceptive pills is in accordance with earlier findings. It is concluded that synthesis and metabolism of alpha 2-antiplasmin are under hormonal influence. The role of alpha 2-antiplasmin in the decreased fibrinolysis in pregnancy is discussed.

Natural protease inhibitors to fibrinolysis in liver diseases.

The newly discovered alpha 2-antiplasmin and some inhibitors of fibrinolysis were measured in 40 patients with liver diseases. alpha 2-antiplasmin was low in liver cirrhosis, but unchanged in cholestasis when compared with controls.

Determination of fast-acting plasmin inhibitor (alpha2-antiplasmin) in plasma from patients with tendency to thrombosis and increased fibrinolysis.

The physiologically important alpha2-antiplasmin has been measured by aid of a chromogenic tripepetide substrate. Low values in patients' plasmas are found in situations with increased fibrinolysis such as streptokinase therapy and liver cirrhosis, whereas high values are found postoperatively, postpartum and after an acute thrombosis.

Determination of a new rapid plasmin inhibitor in human blood by means of a plasmin specific tripeptide substrate.

A method for determination of antiplasmin activity is presented. Plasmin and plasma are incubated, and the remaining plasmin activity is measured spectrophotometrically by means of the plasmin specific tripeptide substrate H-d-Val-l-Leu-l-Lys-p-nitroanilide. The method is simple, rapid and easily automatized. By the immunoadsorption technique, and with the aid of purified substances it is shown that the measured activity is mainly due to a new antiplasmin [2,4] and possibly to some extent to alpha1-antitrypsin and C1-esterase inhibitor have no antiplasmin activity in the method. Heparin and epsilonaminocaproic acid interfered with the assay.