Pharmacological studies on the antithrombotic action of hirudin in experimental animals.

The pharmacodynamics and pharmacokinetics of hirudin were studied in dogs, rabbits and rats. Hirudin proved to be a well tolerated substance with low toxicity. After intravenous injection it was eliminated with a half time of 50 to 60 min. It was nearly completely excreted through the kidneys in biologically active form. The efficacy of hirudin in preventing venous thrombosis, vascular shunt occlusion and disseminated intravascular coagulation in rats was demonstrated.

Pharmacokinetics and anticoagulant effect of hirudin in man.

The pharmacokinetics and the effects on the haemostatic system of hirudin were assessed in six healthy subjects after single intravenous or subcutaneous dose (1000 AT-U/kg). When hirudin was given intravenously first-order elimination kinetics followed the initial distribution phase. The decline in plasma hirudin concentration was most adequately expressed by a biexponential equation describing a two compartment model. A mean elimination half-life of 0.84 hr and a mean volume of distribution of 12.9 l were calculated. After subcutaneous injection a low hirudin level (approximately 0.5 AT-U/ml) was maintained for a prolonged period of time. In the 24 hour-urine up to 50 per cent of the administered amount of hirudin was excreted in active form. Thrombin time, partial thromboplastin time and prothrombin time measured in plasma samples ex vivo were prolonged dependent on the hirudin plasma level. Platelet counts, fibrinogen level and the fibrinolytic system were unchanged. Bleeding time was prolonged twice at maximum. Subcutaneous or intravenous administration of pure hirudin was tolerated without side-effects.

On the isolation of the thrombin inhibitor hirudin.

A procedure for isolation of hirudin from crude preparations was described. By the use of ion exchange chromatography and affinity chromatography preparations were obtained with a specific activity of 10 to 15 antithrombin units/micrograms. Separation into several fractions with the same activity suggests the existence of isoinhibitors.

Inhibition of bovine and human thrombins by derivatives of benzamidine.

Thirty-four derivatives of benzamidine were tested for their inhibitory activities on bovine and human thrombins using the chromogenic peptide substrate N-D-Phe-Pip-Arg-pNA. The inhibition constants of small molecular size inhibitors of comparatively low affinity as well as those of tight binding inhibitors did not differ significantly with different species. Accordingly, the active site of bovine thrombin seems to correspond largely to that of human thrombin.

Cyclic amides of N alpha-arylsulfonylaminoacylated 4-amidinophenylalanine--tight binding inhibitors of thrombin.

Variation of the potent thrombin inhibitors derived from N alpha-arylsulfonyl-4-amidinophenylalanine was carried out by interposition of an omega-aminoalkylcarboxylic acid between the N alpha-arylsulfonyl residue and the 4-amidinophenylalanine part. The use of glycine as spacer renders the compounds tight binding inhibitors of thrombin. The Ki of the most potent inhibitor reaches the nmol/l range. The inhibitory effect is specifically directed against thrombin, the Ki values for inhibition of trypsin, plasmin and factor Xa are some orders of magnitude higher than those for thrombin inhibition.

[Production and characterization of a soluble ocrase-polyacrylamide preparation (author's transl)]. / Herstellung und Charakterisierung eines löslichen Ocrase-Polyacrylamidpräparates.

Using the carbodiimide procedure, ocrase, a proteinase isolated from Aspergillus ochraceus, has been bonded to a water-soluble acrylamide-acrylic acid copolymer and isolated by gel filtration. Binding to the high-molecular polymer had implications for the kinetics of the cleavage of the ethyl ester of benzoyl-L-arginine by this proteinase, and for the reaction of the latter with synthetic and natural inhibitors.

[Synthetic inhibitors of serine proteinases. Part 19: On the inhibitory effect of amidinobenzylidene derivatives of benzo-condensed cycloalkanones on trypsin, plasmin and thrombin (author's transl)]. / Synthetische Inhibitoren der Serinproteinasen. 19. Mitteilung: Uber die Hemmwirkung von Amidinobenzylidenderivaten benzokondensierter Cycloalkanone auf Trypsin, Plasmin und Thrombin.

Amidinobenzylidene derivatives of benzo-condensed cycloalkanones proved to be potent competitive inhibitors of thrombin and trypsin. On the contrary, the antiplasmin effect of these derivatives is considerably less marked. The conversion of 3-amidinochalcone to derivatives in which the carbonyl group is incorporated into a ring, does not lead to fundamental changes in the inhibitory effect on trypsin and thrombin, whereas the antiplasmin effect decreases. Compared to 4-amidinochalcone, the 2-(4-amidinobenzylidene) derivatives of indanone-(1) and tetralone-(1) exert a stronger inhibitory effect on trypsin and thrombin. The introduction of a hetero-atom to the cycloalkanone component affected the inhibitory effect on trypsin and thrombin but insignificantly. From these results it is concluded that also in amidinobenzylidene derivatives of benzo-condensed cycloalkanone derivatives, the carbonyl function shares in enzyme-inhibitor binding.

[Synthetic inhibitors of serine proteinases. 31. The inhibition of trypsin, plasmin and thrombin by isomeric compounds of N alpha -arylsulfonylated omega-amidinophenyl-alpha-aminoalkylcarboxylic acid amides]. / Synthetische Inhibitoren der Serinproteinasen. 31. Mitteilung: Uber die Hemmwirkung isomerer Verbindungen von N alpha-arylsulfonylierten omega-Amidinophenyl-alpha-aminoalkylcarbonsäureamiden gegenüber Trypsin, Plasmin und Thrombin.

Isomeric compounds of N alpha-arylsulfonylated amides of omega-amidinophenyl-alpha-aminoalkylcarboxylic acids--N alpha-amidinophenylsulfonylated amides or N alpha-arylsulfonylated amidino anilides of omega-phenyl-alpha-aminoalkylcarboxylic acids, respectively--possess weak antithrombin activity as compared to the derivatives of the basic structure. Thus, the assumption is corroborated that specific enzyme-inhibitor interactions account for the high antithrombin activity of certain derivatives of omega-amidinophenyl-alpha-aminoalkylcarboxylic acids. In contrast, amidinoanilides of N alpha-substituted omega-phenyl-alpha-aminoalkylcarboxylic acids are potent inhibitors of trypsin and plasmin, their inhibitory activity approaches that of primary amides of N alpha-substituted omega-amidinophenyl-alpha-aminoalkylcarboxylic acids.

Isolation and characterization of a thrombin inhibitor from the tick ixodes ricinus.

The secretion of the salivary glands of ticks, Ixodes ricinus, contains anticoagulant substances. Some years ago an antithromboplastin, ixodin, was described. The present paper refers the isolation and characterization of a second anticoagulant substance of the ticks named ixin. Ixin proved to be a relatively stable and specific thrombin inhibitor. The multi step procedure for isolation results in a 850-fold purification. The preparation obtained has a specific activity of 250 antithrombin units/mg. It is not homogeneous and still contaminated. The substance was assumed to be a miniprotein.

[Synthetic inhibitors of serine proteinases. Part 21: Studies on the structure-activity relationships in bisamidines (author's transl)]. / Synthetische Inhibitoren der Serinproteinasen. 21. Mitteilung: Untersuchungen über die Beziehungen zwischen Struktur und Wirkung bei Bisamidinen.

The antitrypsin, antiplasmin and antithrombin activities of bis(amidinobenzylidene)- and bis(amidinobenzyl)cycloalkanones are not markedly affected if the amidino group is substituted by an uncharged residue (H, OCH3, Cl, Br, NO2). In contrast, mono(amidinobenzylidene)cycloalkanones exhibit considerably lower inhibitory activities than the compounds of the abovementioned classes of substances. From the results obtained it is concluded that the second aromatic residue and not the second basic amidino group is decisive of the potent inhibitory action of the bisamidino derivatives.

[Synthetic inhibitors of serine proteinases. Part 26: Inhibition of trypsin, plasmin and thrombin by amides of N alpha-arylsulfonylated 2-amino-4-(4-amidino-phenyl) butyric acid and 2-amino-5-amidinophenyl valeric acids (author's transl)]. / Synthetische Inhibitoren der Serinproteinasen. 26. Mitteilung 2: Hemmung von Trypsin, Plasmin und Thrombin durch Amide N alpha-arylsulfonylierter 2-Amino-4-(4-amidinophenyl)buttersäure und 2-Amino-5-amidinophenylvaleriansäuren.

Among the derivatives of the N alpha-arylsulfonylated omega-amidinophenyl-alpha-aminoalkyl carboxylic acids, the primary amides of N alpha-arylsulfonylated 2-amino-4-(4-amidinophenyl) butyric acid proved to be compounds with strong antiplasmin and antitrypsin activity, they exert, however, only slight inhibitory effect on thrombin. So far, no benzamidine derivatives with strong inhibitory effects on plasmin but with weak antithrombin activity have been known. The secondary amides of the N alpha-arylsulfonylated 2-amino-4-(4-amidinophenyl)butyric acid possess slight inhibitory effects, the corresponding derivatives of 2-amino-5-amidinophenyl valeric acids, however, are potent inhibitors of thrombin.

[Synthetic serine proteinase inhibitors. 30. Synthesis of alpha-benzoylamino-4-amidinocinnamic acid and alpha-(4-amidinobenzoylamino) cinnamic acid amides, and their inhibitory effect on trypsin-like enzymes]. / Synthetische Inhibitoren der Serinproteinasen. 30. Mitteilung: Synthese von alpha-Benzoylamino-4-amidinozimtsäureamiden sowie alpha-(4-Amidinobenzoylamino)zimtsäureamiden und ihre Hemmwirkung gegenüber trypsinähnlichen Enzymen.

To test their inhibitory activity against enzymes, the authors synthetized the compounds named in the title. The synthesis started from the corresponding azlactones 1 and 9. Subsequent aminolysis led to the alpha-benzoyl-aminocinnamic acid amides 2 and 10 which contain a cyano function. The acid amides were converted, in different ways, to the desired amidine salts 4, 7, 12 and 15. The amidines obtained showed but little inhibitory activity against the serine proteinases thrombin, trypsin and plasmin.

[Synthetic serine protease inhibitors. 29. Synthesis of alpha-arylsulphonylamino-beta-(4-amidinophenyl)ethyl-chloromethylketones and their inhibitory activity against trypsin, plasmin and thrombin]. / Synthetische Inhibitoren von Serinproteinasen. 29. Mitteilung: Synthese von alpha-Arylsulfonylamino-beta-(4-amidinophenyl)ethyl-chlormethylketonen und deren Hemmwirkung gegenüber Trypsin, Plasmin und Thrombin.

The synthesis of alpha-arylsulphonylamino-beta-(4-amidinophenyl)ethyl-chloromethylketones, the structures of which correspond largely to those of the antiproteolytically very potent N alpha-arylsulphonylated 4-amidinophenylalaninamides, was realized starting from 4-cyanophenylalanine hydrochloride. After N alpha-arylsulphonylation this compound was converted via the acid chlorides by treatment with diazomethane into alpha-arylsulphonylamino-beta-(4-cyanophenyl)ethyl-diazomethylketones from which the corresponding chloromethylketones were afforded by treatment with concentrated hydrochloric acid. The conversion of the cyano function into the amidine function via the thiocarbamoyl and the thioimidic acid esters yielded the compounds named in the title. The substances produced no irreversible inhibition of the serine proteinases trypsin, plasmin and thrombin. Their competitive inhibitory effect was almost as potent as that of N alpha-arylsulphonylated 4-amidinophenylalanine ethylesters.

[Synthetic inhibitors of serine proteinases. Part 24: Inhibition of trypsin, plasmin and thrombin by cyclic amides of N alpha-arylsulfonylamidinophenyl-glycines (author's transl)]. / Synthetische Inhibitoren der Serinproteinasen. 24. Mitteilung: Hemmung von Trypsin, Plasmin und Thrombin durch cyclische Amide N alpha-arylsulfonylierter Amidinophenylglycine.

Cyclic amides of n alpha-arylsulfonyl (3-amidinophenyl)glycine are potent inhibitors of the serine proteinase trypsin, plasmin and thrombin. The weak inhibitory activity of the corresponding 4-amidinophenylglycine derivatives cannot be explained in terms of the structure-activity relationships established for benzamidine derivatives. It might be caused by steric hindrance of enzyme-inhibitor interactions.

[Synthetic inhibitors of serine proteinases. Part 25: Inhibition of trypsin, plasmin and thrombin by amides of N alpha-substituted amidinophenylalanines and 3-amidinophenyl-3-aminopropionic acids (author's transl)]. / Synthetische Inhibitoren der Serinproteinasen. 25. Mittelung: Hemmung von Trypsin, Plasmin und Thrombin durch Amide N alpha-arylsulfonylierter Amidinophenylalanine und 3-Amidinophenyl-3-aminopropionsäuren.

Amides of N alpha-substituted 3-amidinophenylalanine are potent inhibitors of the serine proteinases trypsin, plasmin and thrombin. They belong to the most potent inhibitors of these enzymes of the benzamidine type. In contrast, amides of 4-amidinophenylalanine possess weak inhibitory activity towards trypsin and plasmin. The cyclic amides of this group, however, are potent thrombin inhibitors. These derivatives are the first benzamidines with specific antithrombin activity. The isomeric compounds of 3-amidinophenyl-3-aminopropionic acids possess weak inhibitory effects on trypsin, plasmin and thrombin.

[Synthetic inhibitors of serine proteinases. 32. Inhibition of trypsin, plasmin and thrombin by amides of N-alpha-substituted 4-amidinophenylalanine. Effect of various amino acids and blocking groups of the n-alpha residue on inhibitory activity]. / Synthetische Inhibitoren der Serinproteinasen. 32. Mitteilung2: Hemmung von Trypsin, Plasmin und Thrombin durch Amide des N alpha-substituierten 4-Amidinokphenylalanins. Einfluss verschiedener Aminosäuren und Schutzgruppen im N alpha-Rest auf die Inhibitoraktivität.

Cyclic amides of N alpha-arylsulfonylated 4-amidinophenylalanine are specific, highly potent inhibitors of thrombin. Introduction of amino acids between the arylsulfonyl blocking group and amino nitrogen influence particularly the antithrombin activity. By the use of glycine as spacer the compounds become tight binding thrombin inhibitors, while introduction of other omega-amino acids, Gly-Gly, L-Pro, Gly-L-Pro or L-Pro-Gly, reduces the specificity and potency of thrombin inhibition. Substitution of the arylsulfonyl blocking group for a heteroarylsulfonyl residue or an aryl residue causes a decrease in antithrombin activity, while substitution for a benzoyloxycarbonyl blocking group has only slight influence. It is concluded that the N alpha-moiety is of decisive importance for the antithrombin activity of derivatives of 4-amidinophenylalanine.

[Synthetic inhibitors of serine proteinases. 34. The effect of modification of the amidino function of N-alpha-substituted 4-amidinophenylalanine amides on inhibitory activity]. / Synthetische Inhibitoren der Serinproteinasen. 34. Mitteilung: Einfluss der Abwandlung der Amidinofunktion bei N alpha-substituierten 4-Amidino-phenylalaninamiden auf die Inhibitoraktivität.

Cyclic amides of N alpha-arylsulfonylated 4-amidinophenylalanine are selective inhibitors of thrombin. The exchange of the amidino function for an aminomethyl residue does not influence the selectivity and potency of their inhibitory activity. In contrast, the modification of the amidino function of the N alpha-arylsulfonylaminoacylated compound N alpha-(2-naphthylsulfonylglycyl)-4-amidinophenylalanine piperidide results in a drastic loss of inhibitory activity. Only the oxamidino derivative possesses considerable high affinity for thrombin. Obviously, in tight binding inhibitors of thrombin structural variation results in any case in a loss of inhibitory activity.