The influence of laser scribing on magnetic domain formation in grain oriented electrical steel visualized by directional neutron dark-field imaging.

The performance and degree of efficiency of transformers are directly determined by the bulk magnetic properties of grain oriented electrical steel laminations. The core losses can be improved by post manufacturing methods, so-called domain refinement techniques. All these methods induce mechanical or thermal stress that refines the domain structure. The most commonly used technique is laser scribing due to the no-contact nature and the ease of integration in existing production systems. Here we show how directional neutron dark-field imaging allows visualizing the impact of laser scribing on the bulk and supplementary domain structure. In particular, we investigate the domain formation during magnetization of samples depending on laser treatment parameters such as laser energy and line distances. The directional dark-field imaging findings were quantitatively interpreted in the context with global magnetic hysteresis measurements. Especially we exploit the orientation sensitivity in the dark-field images to distinguish between different domain structures alignment and their relation to the laser scribing process.

Pharmacokinetics of an emerging new class of anticoagulant/antithrombotic drugs. A review of small-molecule thrombin inhibitors.

Small-molecule direct thrombin inhibitors represent a new class of anticoagulants and are emerging as antithrombotic drugs with a range of indications. The tripeptide type or peptidomimetic compounds, including argatroban, efegatran, inogatran and napsagatran, hitherto clinically studied represent a first generation of thrombin inhibitors that are pharmacokinetically characterised by relatively rapid hepato-biliary clearance and short half-lives necessitating their administration as intravenous infusion. They are not orally bioavailable because of poor enteral absorption and presystemic hepatic extraction. Melagatran can be administered subcutaneously, and a prodrug form of melagatran, ximelagatran, is at present the only oral thrombin inhibitor available. Direct thrombin inhibitors produce predictable, stable and rapidly reversible anticoagulation measurable by common coagulation assays. Significant pharmacokinetic drug-drug interactions have not been reported. Possible pharmacodynamic interactions, in terms of prolongation of plasma clotting times, with other anticoagulant drugs must be taken into account when monitoring direct thrombin inhibitors using coagulation assays.

Structure-activity relationships of new NAPAP-analogs.

Several new analogs of the known thrombin inhibitor NAPAP were synthesized, in which the P2 glycine residue was substituted by natural and unnatural amino acids. The thrombin inhibitory potency was comparable to that of NAPAP. Several of the compounds had inhibition constants lower than 10 nM and a very high selectivity compared to trypsin, factor Xa and plasmin. In addition, analogs were prepared by alkylation of the N alpha-atom of the 4-amidinophenylalanine in P1 position, which showed a more than 10-fold lower thrombin inhibition. Furthermore, azaglycine was introduced instead of P2 glycine. For most of the inhibitors similar fast elimination rates were seen in rats after intravenous dosing, as found previously for NAPAP. Only some compounds, which contained a second basic group showed a slightly decreased cumulative biliary clearance.

Transport of peptidomimetic thrombin inhibitors with a 3-amidino-phenylalanine structure: permeability and efflux mechanism in monolayers of a human intestinal cell line (Caco-2).

PURPOSE: Peptidomimetic thrombin inhibitors derived from Nalpha-(2-naphthylsulfonyl)-3-amidino-phenylalanine with different basic and acidic substituents were investigated with respect to their intestinal transport behavior. METHODS: Intestinal permeability coefficients were studied using Caco-2 monolayers and a reversed-phase HPLC method for quantitation. RESULTS: Apparent permeability coefficients Papp of compounds with a free amidino group were in general low (<10 x 10(-8) cm/s) and independent of the structure of the amide part (C-terminus). Polarized efflux, however, was strongly affected by substituents in the amide moiety yielding the following efflux ratios (ER): methylpiperidide (1) (ER 45) > piperidine carboxylic acid methylester (ER 6-11) > piperidine carboxylic acids (ER 1.9-2.9) > piperazide (ER -0.17). Efflux of (1) was temperature-dependent, but independent of the enantiomeric configuration, accompanied by an increase in transepithelial electrical resistance (TEER), and could be reduced by P-gp inhibitors (PSC 833, Cremophor EL) but not by indomethacin. Replacement of the amidine group of (1) by aminomethyl-, amino-, and oxamidine- moieties drastically increased absorptive permeability (46-68 fold) with ER < 3.4. In contrast, the oxamidine with a C-terminal nipecotic acid residue (8) displayed also a temperature dependent efflux- without altering TEER (ER 22). This efflux was sensitive to PSC833/Cremophor EL and indomethacin. CONCLUSIONS: Basic and acidic residues of amidino-phenylalanine-derived thrombin inhibitors mediate affinity to intestinal efflux pumps. presumably P-gp and MRP. P-gp mediated efflux was related to a net positive charge and accompanied by an increased TEER. Among the methylpiperide (1) promoieties studied the oxamidino group seems to be very promising in overcoming both transport and efflux problems frequently encountered with peptidomimetics containing amidines.

Advances in the development of thrombin inhibitors.

Thromboembolic diseases are a major cause of morbidity and mortality, particularly in the Western world, which has stimulated enormous research efforts by the pharmaceutical industry to introduce new antithrombotic therapies. One strategy is the development of direct inhibitors of the serine protease thrombin, which holds a central position in the final steps of the blood coagulation cascade and in platelet activation. At present there is only limited clinical use of some parenteral preparations of thrombin inhibitors in acute situations, especially when the common antithrombotic drugs heparin, warfarin and aspirin are ineffective or associated with side effects. However, for use in prophylaxis of thrombotic diseases such inhibitors should be orally available, must be safe to avoid bleeding complications and should have an appropriate half-life, allowing once or twice daily dosing to maintain adequate antithrombotically effective blood levels. Details of several new and potent thrombin inhibitors have been published during the last years. For some of them oral bioavailability is claimed and they are effective in in vitro coagulation assays. However, most of them showed only limited efficacy in animal studies with respect to the doses administered. For that reason, effort is concentrated on the evaluation and optimisation of the overall physicochemical characteristics of the inhibitors in order to improve the pharmacokinetics and, thus, the development of promising drug candidates. Nevertheless, only careful clinical studies can give clear answers about the true therapeutical benefit of new developments in this field. This review summarises the current status of direct thrombin inhibitors which are already in clinical use and clinical development and gives an overview on recently published and promising new compounds.

New bivalent thrombin inhibitors with N(alpha)(methyl)arginine at the P1-position.

A series of bivalent thrombin inhibitors was synthesized, consisting of a d-phenylalanyl-prolyl-N(alpha)(methyl)arginyl active site blocking segment, a fibrinogen recognition exosite inhibitor part, and a peptidic linker connecting these fragments. The methylation of the P1 amino acid led to a moderate decrease in affinity compared with the unmethylated analog. In addition, it prevented the thrombin catalyzed proteolysis, independent of the P1' amino acid used. This is a significant advantage compared to the original hirulogs, which strictly require a proline as P1' amino acid to reduce the cleavage C-terminal to the arginyl residue. Several analogs were prepared by incorporation of different P1' amino acids found in natural thrombin substrates. The most potent inhibitor was I-11 [dCha-Pro-N(Me)Arg-Thr-(Gly)5-DYEPIPEEA-Cha-dGlu] with a Ki of 37 pM. I-11 is highly selective and no inhibition of the related serine proteases trypsin, factor Xa and plasmin was observed. The stability of I-11 in human plasma in vitro was strongly improved compared to hirulog-1. In addition, a significantly reduced plasma clearance of I-11 was observed after intravenous injection in rats. Results from molecular modeling suggest that a strong reorganization of the hydrogen bonds in the active site of thrombin may result in the proteolytic stability found in this inhibitor series.

[Deciduosis of the appendix. Differential diagnosis of acute appendicitis]. / Deciduose der Appendix. Zur Differentialdiagnose der akuten Appendicitis.

Deciduosis of the appendix is a rare cause of acute appendicitis in pregnancy. Ectopic decidual cells localized in the submesothelial stroma may represent a physiologic reaction of the pluripotent stromal cells to progestational hormonal stimulation. Less frequently, deciduosis is based on a preexisting extragenital endometriosis, visible in a localization other than strictly submesothelial, in residuals of cyclic proliferation and bleeding, and in endometrial glandular formations embedded in the decidual cells.

Design and evaluation of novel bivalent thrombin inhibitors based on amidinophenylalanines.

Two bivalent thrombin inhibitors were synthesized, which consist of a benzamidine-based active-site-blocking segment, a fibrinogen recognition exosite inhibitor and a peptidic linker connecting these fragments. BZA-1 hirulog contains an Nalpha-(2-naphthylsulfonyl)-S-3-amidinophenylalanyl-is onipecotic acid residue connected via the carboxyl group to the linker segment. The active-site-directed moiety of BZA-2 hirulog [Nalpha-(2-naphthylsulfonyl-glutamyl)-R-4-amidinophenylal anyl-piperid ide] was coupled to the linker via the side chain of the glutamic acid. Both BZA-hirulogs contain almost identical linker-exo site inhibitor parts, except for the substitution of a glycine as the first linker residue in BZA-1 hirulog by a gamma-amino butyric acid in BZA-2 hirulog, thus increasing flexibility and linker length by two additional atoms. BZA-1 hirulog showed moderate potency (Ki = 0. 50 +/- 0.14 nM), while BZA-2 hirulog was characterized as a slow, tight binding inhibitor of thrombin (Ki = 0.29 +/- 0.08 pM). The stability in human plasma of both analogs was strongly improved compared with hirulog-1. For BZA-2 hirulog a significantly reduced plasma clearance was observed after intravenous injection in rats compared with BZA-1 hirulog and hirulog-1. The X-ray structure of the BZA-2 hirulog in complex with human alpha-thrombin was solved and confirmed the expected bivalent binding mode.

Design of benzamidine-type inhibitors of factor Xa.

A series of derivatives of rac-benzenesulfonyl-glycyl-phenylalanine or its ethyl ester with a combination of thioamido/amidino or amidino/amidino substituents in the benzene rings was synthesized as potential inhibitors of factor Xa (fXa). Among these, the racemic 4'-amidinobenzenesulfonyl-glycyl-4-amidinophenylalanine ethyl ester was found to exhibit the highest affinity for fXa despite the unfavored location of the amidino substituent in the para position. X-ray structural analysis of the trypsin complex with this bis-benzamidine compound revealed a retro-binding mode if compared to those of similar compounds, so far analyzed in complexes with trypsin or fXa. This noncanonical binding mode as well as its slow plasma clearance rates in rats, if compared to those of other benzamidine derivatives, suggests this compound as an interesting new lead structure for the design of fXa inhibitors.

Influence of indocyanine green on plasma disappearance and biliary excretion of a synthetic thrombin inhibitor of the 3-amidinophenyl-alanine piperazide-type in rats.

PURPOSE: The pharmacokinetics of a number of synthetic peptidomimetic thrombin inhibitors is determined by extensive hepatic elimination. The objective was to further characterize the disposition in vivo of Pefa 1023, a novel 3-amidinophenylalanine piperazide-type thrombin inhibitor, by influencing the hepatic handling with indocyanine green (ICG), which is actively taken up by the liver. METHODS: Pefa 1023 was administered intravenously to bile duct-cannulated rats, either alone or in combination with ICG. The concentrations of Pefa 1023 in blood plasma and bile were measured by a bioassay (thrombin clotting time), concentrations of indocyanine green were measured spectrophotometrically. RESULTS: ICG (10 mg/kg i.v. 15 min prior to or simultaneously with Pefa 1023) markedly influenced the plasma level and biliary excretion rate of the thrombin inhibitor Pefa 1023 given in a dose of 1 mg/kg i.v. The plasma level was more than twice that of the control, the maximum biliary excretion rate about one third and the fraction of dose excreted in bile about two thirds. CONCLUSIONS: The anionic dye ICG is able to interfere with the hepatic handling of a cationic, amidinophenylalanine piperazide-type thrombin inhibitor with the consequence of reduced hepatic clearance leading to higher plasma levels and lower biliary excretion of the latter.

Synthesis and structure-activity relationships of potent thrombin inhibitors: piperazides of 3-amidinophenylalanine.

Thrombin is the key enzyme in the blood coagulation system, and inhibitors of its proteolytic activity are of therapeutic interest since they are potential anticoagulants. The most potent inhibitor of the benzamidine type is N alpha-[(2-naphthylsulfonyl)glycyl]-4-amidinophenylalanylpiperid ide (NAPAP). However, NAPAP and other benzamidine derivatives do not show favorable pharmacological properties; above all, they have very low systemic bioavailability after oral administration. The goal of designing new compounds was to obtain potent inhibitors with improved pharmacokinetic properties. Piperazide derivatives of 3-amidinophenylalanine as the key building block were synthesized. The piperazine moiety opened the possibility to introduce quite different substituents on the second nitrogen using common synthetic procedures. Some of the newly synthesized compounds are potent inhibitors of thrombin and offer an approach to study structure-function relationships for inhibition of thrombin and related enzymes and for the improvement of their pharmacokinetic properties.

The fibrinolytic activity of staphylokinase mutants in the fibrin plate assay.

The fibrinolytic activity of the new plasminogen activator, recombinant staphylokinase, was compared to that of streptokinase and tissue-type plasminogen activator in the fibrin plate assay. The pattern of fibrinolysis by staphylokinase on fibrin plates differs from the other plasminogen activators. A number of mutants of staphylokinase with various amino acids in position 26 substituted for methionine in wild-type staphylokinase were compared with respect to their fibrinolytic potencies. Only the mutants with cysteine or leucine in this position have a fibrinolytic activity comparable to wild-type staphylokinase. The results on the fibrinolytic activities in the fibrin plate assay correlate with those of a plasmin generation assay, the latter is, however, less sensitive.

Differential effects of staphylokinase, streptokinase and tissue-type plasminogen activator on the lysis of retracted human plasma clots and fibrinolytic plasma parameters in vitro.

A novel plasma clot lysis system was used to compare the fibrinolytic characteristics of staphylokinase, streptokinase and tissue-type plasminogen activator. 125I-fibrinogen-labelled human plasma clots were formed on needles and mechanically compressed after spontaneous retraction. This model is relatively resistant to lysis and differentiates between fibrin-specific and non-fibrin-specific plasminogen activators. The novel plasminogen activator, recombinant staphylokinase, produced high rates of clot lysis without markedly influencing fibrinogen, plasminogen and alpha 2-antiplasmin in the plasma containing the clots. At equimolar concentrations, streptokinase markedly depleted these parameters in plasma despite low clot lysis rates. Tissue-type plasminogen activator showed relatively high lysis rates at low concentrations, but at higher concentrations, plasminogen depletion caused a decrease in clot lysis. Staphylokinase can be characterised as a fibrin-specific and plasminogen-saving fibrinolytic agent with a high clot lysis potential.

Anticoagulant potential of synthetic and recombinant inhibitors of factor Xa and thrombin in vitro.

The anticoagulant effects of thrombin inhibitors in vitro and ex vivo are well characterized. Inhibitors of factor Xa might also be effective as anticoagulants. We studied several synthetic, low-molecular-weight inhibitors with varying affinity to thrombin and factor Xa using three in vitro clot-based assays representative of the effects of thrombin added or generated via the intrinsic or extrinsic coagulation pathways. The recombinant selective thrombin and factor Xa inhibitors, hirudin and antistasin, were studied for comparison. The thrombin time assay is a measure of the antithrombin potential of a given inhibitor, while the activated partial thromboplastin time is influenced by inhibition of thrombin and/or of factor Xa in a similar manner. The prothrombin time assay was more sensitive to inhibition of factor Xa, as evidenced by the potent and selective inhibitor antistasin as well as for less potent and less selective synthetic inhibitors of factor Xa. Direct synthetic or recombinant inhibitors of factor Xa with Ki values in the submicromolar range were potent anticoagulants, approaching or exceeding the potency of hirudin in prolonging the prothrombin time and the activated partial thromboplastin time in vitro.

Degradation of a benzamidine-type synthetic inhibitor of coagulation enzymes in plasma of various species.

The synthetic coagulation enzyme inhibitor N alpha-tosylglycyl-3-amidinophenylalanine methyl ester is anticoagulantly active in vitro in concentrations corresponding rather to its thrombin inhibiting activity than to its anti-factor Xa activity. The anticoagulant effect is not diminished by incubation in human plasma in vitro. In guinea pig and rat plasma, however, the inhibitor is hydrolysed, at different rates, yielding the corresponding N alpha-tosylglycyl-3-amidinophenylalanine, a less potent inhibitor of thrombin and factor Xa.