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Twenty-four-day-old immature and cyclic female rats in metoestrus were ovariectomized and bilaterally implanted with oestradiol benzoate (OB) and cholesterol at a ratio of 1:360 or with cholesterol alone in the medial preoptic area (MPOA), or, for control, the hypothalamic dorsomedial nucleus. Estimation of beta-endorphin immunoreactivity in the ventromedial-arcuate-median eminence region at 6 and 3 days after implantation, respectively, revealed a significantly higher concentration in rats implanted with OB in the MPOA as compared to those implanted with cholesterol. OB implants placed in the dorsomedial nuclei were ineffective in this regard. The hypothesis is put forward that inhibition of hypothalamic beta-endorphin release as probably resulting from the implantation of OB in the MPOA may be related to desensitization of the negative oestrogen feedback that is induced by similar preoptic oestrogen implantation.

ZusammenfassungDer Gerinnungsfaktor XIIIa (FXIIIa) stabilisiert die bei der Blutgerinnung entstehenden Fibringerinnsel und erhöht deren Widerstandsfähigkeit gegenüber Fibrinolyse. Darüber hinaus ist FXIIIa an anderen (patho)physiologischen Vorgängen, z. B. Wundheilung und Arteriosklerose, beteiligt. Für die Aufklärung der verschiedenen FXIIIa-Funktionen einschließlich ihrer pharmakologischen Beeinflussung könnten selektive Hemmstoffe ein wertvolles Hilfsmittel sein. Diese Arbeit gibt einen überblick über zurzeit bekannte Hemmstoffe von FXIIIa. Analoga der natürlichen FXIIIa-Substrate – dazu gehören glutaminhaltige Peptide und kleinmolekulare substituierte Alkylamine – werden als kompetitive Substrate in das Fibrinnetz eingebaut und verhindern die Quervernetzung. Natürliche, direkte Hemmstoffe von Faktor XIIIa wurden aus einer Blutegelspezies und verschiedenen Mikroorganismen isoliert. Der wirksamste bekannte Hemmstoff ist das Peptid Tridegin mit effektiven Konzentrationen im nanomolaren Bereich. Synthetische, kleinmolekulare Hemmstoffe binden meist kovalent an die SH-Gruppe von Cys314 im aktiven Zentrum von FXIIIa. Neben den relativ unspezifischen SH-Reagenzien wurden Azolderivate bzw. Azoliumsalze und verwandte Verbindungen als spezifische FXIIIa-Hemmstoffe beschrieben. Tierexperimentelle Untersuchungen zeigten, dass in Gegenwart eines FXIIIa-Hemmstoffs die Thrombolyse mit einem Plasminogenaktivator effektiver wird.

BM 06.022 is a t-PA deletion variant that is produced as inactive inclusion bodies in Escherichia coli and transformed into the native form by an in vitro refolding process. Until now, no X-ray and NMR structures of BM 06.022 were available. Therefore a detailed kinetic analysis of the hydrolysis of peptide substrates and of the inhibition by several benzamidine-derived inhibitors was carried out in order to assess that the active site region of the protease domain of BM 06.022 is correctly structured in comparison with t-PA. Our data reveal that the single-chain as well as the two-chain form of BM 06.022 and native t-PA are similar in catalytic and in inhibitor binding properties. This indicates that the active site and the highly complex rearrangement of t-PA upon cleavage of the Arg275-Ile276 bond are maintained in BM 06.022.

ProtacR, the single chain glycoprotein in the venom of Agkistrodon contortrix snake subspecies and related pit vipers has found broad application as a protein C activator in blood coagulation research and diagnosis. Inhibition studies were performed to further elucidate the nature of this serine proteinase. Benzamidine derivatives are poor inhibitors of ProtacR. Only a few 4-amidinoanilides of N alpha-substituted omega-phenyl-alpha-aminoalkylcarboxylic acids exert potent inhibitory activities. The active site of ProtacR appears to be accessible only for inhibitors with well-fitting structural features.

In carcinogenesis, proteinases play an important role in metastasis of solid malignant tumors. Aside from several matrix metalloproteinases and cathepsins, the urokinase-plasmin system seems to be one of the main players within the cell surface-associated proteolytic activity, facilitating tumor cell migration and invasion. Upon binding to a specific receptor, the enzymatic activity of urokinase is focused to the cell surface. The significance of the plasminogen activator system in malignancy is underlined by the finding of elevated levels of urokinase and its receptor in tumor tissues. Therefore, the possible use of urokinase inhibitors in the therapeutic treatment of cancer and metastasis has been the subject of intensive investigations. This review covers synthetic inhibitors of urokinase described up to December 2000, although the number of lead structures is still relatively small.

This contribution deals with the structure-based design and syntheses of the new serine protease inhibitors RA-1001 and RA-1002, which are analogues of the blue-green algae derived cyanopeptide aeruginosin 98-B. Both compounds inhibit thrombin with Ki values of 5.6 microM and 8.7 microM, respectively.

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.Intestinal permeability coefficients were studied using Caco-2 monolayers and a reversed-phase HPLC method for quantitation.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 ER3.4. 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.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.

Urokinase is a serine protease involved in cancer growth and metastasis. Here we present the first urokinase crystal structure in complex with reversible inhibitors at 2.1 and 2.6 A resolution. These inhibitor complex structures have been obtained from crystals of engineered urokinase type plasminogen activator designed to obtain a crystal form open for inhibitor soaking. The mutant C122S loses its flexible A-chain upon activation cleavage and crystallizes in the presence of benzamidine, which was later displaced by the desired inhibitor. This new soakable crystal form turned out to be of great value in the process of structure-based drug design. The evaluated binding mode of amiloride, and UKI-1D revealed a new subsite of the primary specificity pocket of urokinase that will be employed in the future ligand optimisation process.

Der Einsatz von Globaltests, die das Zusammenspiel der Faktoren des intrinsischen bzw. des extrinsischen Gerinnungsweges erfassen, ist bei der Untersuchung von erworbenen thrombophilen Risikosituation wie z. B. unter der Einnahme oraler Kontrazeptiva (OC) sehr wertvoll. Rotteveel et al. konnten 1993 zeigen [1], daβ das endogene Thrombinpotential ETP in Plasmaproben von Frauen, die orale Kontrazeptiva einnehmen im Vergleich zu Nichteinnehmerinnen signifikant erhoht ist. Rosing et al. fanden sogar Unterschiede im ETP bei der Einnahme oraler Kontrazeptiva der sog. 2. and 3. Generation. In einem APC-Resistenztest, der auf dem Thrombinbildungstest basiert, wurden ahnliche Ergebnisse erhalten [2].

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.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.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.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.

Die Hamostase zeichnet sich durch ein dynamisches Gleichgewicht zwischen prokoagulatorischen und fibrinolytischen Komponenten aus. Wichtigstes Bindeglied ist die Thrombinbildung und die durch Thrombin katalysierte Bildung von Fibrin aus Fibrinogen. Das nach extrinsischer oder intrinsischer Aktivierung gebildete Thrombin wird durch verschiedene Reaktionen inaktiviert, so das die Thrombusbildung auf den Ort des Bedarfs, die Gefaslasion, beschrankt wird. Verschiebungen in der Hamostase konnen zu hamorrhagischen oder thrombophilen Zustanden fuhren.

The recent structure determination of the catalytic domain of tissue-type plasminogen activator (tPA) suggested residue Arg174 could play a role in P3/P4 substrate specificity. Six synthetic chromogenic tPA substrates of the type R-Xaa-Gly-Arg-p-nitroanilide, in which R is an N-terminal protection group, were synthesized to test this property. Although changing the residue Xaa (in its L or D form) at position P3 from the hydrophobic Phe to an acidic residue, Asp or Glu, gave no improvement in catalytic efficiency, comparative analysis of the substrates indicated a preference for an acidic substituent occupying the S3 site when the S4 site contains a hydrophobic or basic moiety. The 2.9 A structure determination of the catalytic domain of human tPA in complex with the bis-benzamidine inhibitor 2, 7-bis-(4-amidinobenzylidene)-cycloheptan-1-one reveals a three-site interaction, salt bridge formation of the proximal amidino group of the inhibitor with Asp189 in the primary specificity pocket, extensive hydrophobic surface burial, and a weak electrostatic interaction between the distal amidino group of the inhibitor and two carbonyl oxygens of the protein. The latter position was previously occupied by the guanidino group of Arg174, which swings out to form the western edge of the S3 pocket. These data suggest that the side chain of Arg174 is flexible, and does not play a major role in the S4 specificity of tPA. On the other hand, this residue would modulate S3 specificity, and may be exploited to fine tune the specificity and selectivity of tPA substrates and inhibitors.

The x-ray crystal structure of recombinant leech-derived tryptase inhibitor (rLDTI) has been solved to a resolution of 1.9 A in complex with porcine trypsin. The nonclassical Kazal-type inhibitor exhibits the same overall architecture as that observed in solution and in rhodniin. The complex reveals structural aspects of the mast cell proteinase tryptase. The conformation of the binding region of rLDTI suggests that tryptase has a restricted active site cleft. The basic amino terminus of rLDTI, apparently flexible from previous NMR measurements, approaches the 148-loop of trypsin. This loop has an acidic equivalent in tryptase, suggesting that the basic amino terminus could make favorable electrostatic interactions with the tryptase molecule. A series of rLDTI variants constructed to probe this hypothesis confirmed that the amino-terminal Lys-Lys sequence plays a role in inhibition of human lung tryptase but not of trypsin or chymotrypsin. The location of such an acidic surface patch is in accordance with the known low molecular weight inhibitors of tryptase.

Besides guanidino compounds and amines structurally related to arginine and lysine, compounds with an amidino moiety are inhibitors of trypsin-like enzymes. However, in most cases ordinary benzamidine derivatives are not selective inhibitors. Relatively selective inhibitors of some enzymes were found among amino acids containing a benzamidine moiety at the side chain. For example, derivatives of phenyl-alpha-aminobutyric acid with one or two amidino moieties such as the 4'-amidinoanilide of N alpha-[4-amidinophenylsulfonyl]-phenyl-alpha- aminobutyric acid are potent and selective inhibitors of plasma kallikrein (Ki = 0.15 microM). Using N alpha-arylsulfonylated 3-amidinophenylalanine as the key building block, novel inhibitors of plasma kallikrein were developed. Several esters and amides of the nipecotic acid derivative were synthesized which inhibit plasma kallikrein competitively with Ki values between 0.1 and 1.0 microM. The compounds prolonged aPTT in micromolar concentration, indicating an inhibition of the intrinsic coagulation pathway.

In studies on the inhibition of activated protein C (APC) by benzamidine derivatives potent inhibitors of APC were found among anilides of 4-amidinophenyl-α-aminobutyric acid (K i = 0.58 μmol/1). Several bis-benzamidine derivatives containing a cycloalkanone linking bridge inhibit APC with K i values near the micromolar range. Potent and selective inhibitors of thrombin derived from 3- and 4-amidinophenylalanine do not inhibit APC. This is of great importance for further development of these inhibitors as potential anticoagulant drugs.

Well-diffracting crystals of bovine epsilon-thrombin in complex with several "non-peptidic" benzamidine and arginine-based thrombin inhibitors have been obtained by co-crystallization. The 2.3 A crystal structures of three complexes formed either with NAPAP, 4-TAPAP, or MQPA, were solved by Patterson search methods and refined to crystallographic R-values of 0.167 to 0.178. The active-site environment of thrombin is only slightly affected by binding of the different inhibitors; in particular, the exposed "60-insertion loop" essentially maintains its typical projecting structure. The D-stereoisomer of NAPAP and the L-stereoisomer of MQPA bind to thrombin with very similar conformations, as previously inferred from their binding to bovine trypsin; the arginine side-chain of the latter inserts into the specificity pocket in a "non-canonical" manner. The L-stereoisomer of 4-TAPAP, whose binding geometry towards trypsin was only poorly defined, is bound to the thrombin active-site in a compact conformation. In contrast to NAPAP, the distal p-amidino/guanidino groups of 4-TAPAP and MQPA do not interact with the carboxylate group of Asp189 in the thrombin specificity pocket in a "symmetrical" twin N-twin O manner, but through "lateral" single N-twin O contacts; in contrast to the p-amidino group of 4-TAPAP, however, the guanidyl group of MQPA packs favourably in the pocket due to an elaborate hydrogen bond network, which includes two entrapped water molecules. These thrombin structures confirm previous conclusions of the important role of the intermolecular hydrogen bonds formed with Gly216, and of the good sterical fit of the terminal bulky hydrophobic inhibitor groups with the hydrophobic aryl binding site and the S2-cavity, respectively, for tight thrombin active site binding of these non-peptidic inhibitors. These accurate crystal structures are presumed to be excellent starting points for the design and the elaboration of improved antithrombotics.

Besides guanidino compounds and amines structurally related to arginine and lysine, compounds with other cationic groups are inhibitors of trypsin-like serine proteinases. Particularly aromatic ring structures with an amidino moiety have high affinity for these enzymes. In most cases ordinary benzamidine derivatives are no selective inhibitors, however, among derivatives of N alpha-arylsulfonyl-omega-amidinophenyl-alpha-amino-alkylcarboxylic acids selective competitive inhibitors of several enzymes were found. Amides of phenyl-alpha-aminobutyric acid containing an amidino moiety are inhibitors of plasma kallikrein. The p-amidinoanilide of 2-tosylamino-4-phenylbutyric acid inhibits selectively plasma kallikrein with a Ki of 0.70 mumol/l. In contrast, potent and selective inhibitors of glandular kallikrein were hardly found among benzamidines.

Besides guanidino compounds and amines structurally related to arginine and lysine, compounds with other cationic groups are inhibitors of trypsin-like serine proteinases. Particularly aromatic ring structures with an amidino moiety have high affinity for these enzymes. In most cases ordinary benzamidine derivatives are no selective inhibitors, however, among derivatives of N alpha-arylsulfonyl-omega-amidinophenyl-alpha-amino-alkylcarboxylic acids selective competitive inhibitors of several enzymes were found. Amides of phenyl-alpha-aminobutyric acid containing an amidino moiety are inhibitors of plasma kallikrein. The p-amidinoanilide of 2-tosylamino-4-phenylbutyric acid inhibits selectively plasma kallikrein with a Ki of 0.70 mumol/l. In contrast, potent and selective inhibitors of glandular kallikrein were hardly found among benzamidines.

Pharmacological profiling of recombinant hirudin (r-hirudin) has shown that this selective tight-binding thrombin inhibitor is a potent, well-tolerated anticoagulant. Clinical pharmacological studies were performed in human volunteers after single and repeated doses of 0.1–0.5 mg/kg. Generally, administration of r-hirudin was tolerated without side effects. Thrombin time and partial thromboplastin time were prolonged dependent on the r-hirudin level in plasma. Platelet counts, fibrinogen level and fibrinolytic system remained unchanged. Bleeding time was not prolonged. On intravenous injection, r-hirudin was rapidly distributed into the extracellular space and eliminated, with a dose-dependent half-life of 1–2 h (first-order kinetics). After subcutaneous administration, the rH level in blood reached plateau values within 60–120 min. The high recovery of unchanged r-hirudin in the urine identified renal excretion as the predominant route of r-hirudin clearance.

Three recombinant variants of hirudin with the most evident difference in amino acid position 47 (Lys-47, Arg-47, Asn-47) were studied for their selectivity and affinity for the target enzyme thrombin in comparison to native hirudin. Native hirudin and the recombinant hirudins inhibit selectively the clotting enzyme thrombin. The affinity of native hirudin does not differ significantly from that of recombinant hirudin Lys-47 whereas a distinctly lower affinity for thrombin is found for recombinant hirudin Arg-47 and recombinant hirudin Asn-47. All hirudins investigated have the same potency to inhibit thrombin-induced coagulation. Changes in the affinity of hirudins for thrombin become evident from the inhibition of thrombin-induced platelet aggregation only.

Immature female rats were implanted with oestradiol benzoate or cholesterol in the medial preoptic area at different ages, and the inhibition of the ovariectomy-induced increase of LH secretion by s.c. injected oestradiol was investigated. Medial preoptic oestrogen implants reduced the inhibition of LH secretion in 4-week-old rats, but not in younger animals. Elevation of the circulating oestrogen concentration or suppression of the central nervous dopamine activity by daily injections of oestradiol and pimozide, respectively, from Day 26 to the day of vaginal opening, i.e. during the time when the mechanism of the oestrogen-induced desensitization of the negative oestrogen feedback matures, resulted in considerable diminution of the LH-inhibiting effect of oestradiol in ovariectomized adult females. In intact cyclic rats, both prepubertal treatments led to a significant increase of the average number of eggs per ovulation that was mainly caused by reduction of the number of animals with a low ovulation rate.

The B. moojeni thrombic protease, batroxobin, was acylated by 4-amidinophenyl benzoate at the active site serine hydroxyl. From the enzymatically inactive benzoyl-batroxobin, batroxobin is generated with a half-life of deacylation of about one hour. The clotting activity of benzoyl-batroxobin in plasma is recovered with deacylation. The effects of batroxobin and benzoyl-batroxobin were studied following intravenous injection in rats. Compared to defibrinogenation with batroxobin, that obtained with benzoyl-batroxobin was much retarded. Batroxobin caused microthrombosis initially, which did not develop upon injection of benzoyl-batroxobin in equivalent doses.

Recent studies have shown that oestrogen can induce desensitization to its own gonadotrophin-inhibiting effect in female rats by an action on the medial preoptic area (MPOA). Probably as a consequence of this action, sensitivity to the negative oestrogen feedback declines markedly between metoestrus and dioestrus of the 4-day ovarian cycle. To study this desensitization process in 5-day cyclic rats, females exhibiting regular 5-day vaginal cyclicity were ovariectomized on consecutive days of the cycle, injected with oestradiol benzoate (OB) or oil on the day of ovariectomy and autopsied 24 h after the injection. Estimation of the serum concentration of LH revealed that desensitization to negative oestrogen feedback occurred only between day 2 of dioestrus and pro-oestrus, i.e. 2 days later than in females with a 4-day cycle. In the latter animals, an injection of progesterone in metoestrus or early dioestrus, which induced lengthening of the ovarian cycle for 1 day, delayed the onset of desensitization to a degree similar to that found in spontaneously 5-day cyclic rats. In acutely ovariectomized females, progesterone implants placed in the MPOA, but not those located in the mediobasal hypothalamus, increased the LH-inhibiting effect of low doses of OB. The results suggest that the prolonged secretion of progesterone recorded in 5-day cyclic rats retards follicle maturation and delays the forthcoming ovulation by acting, at least partly, on the MPOA and antagonizing the desensitizing effect of oestrogen. In this way, inhibition of gonadotrophin secretion by oestrogen is enhanced and the increase in tonic LH secretion necessary for the completion of follicle maturation is retarded. J. Endocr. (1987) 114, 409–414

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.

Urokinase was acylated at the active site serine hydroxyl using p-amidinophenyl benzoate or p-nitrophenyl p'-guanidinobenzoate. The enzymatically inactive acyl-urokinase was reactivated in buffer (pH 7.5) or plasma at 37 degrees C with a half-life of 11 min (benzoyl-urokinase) or 10 h (p-guanidinobenzoyl-urokinase). Upon administration (50,000 IU/kg) to rabbits, urokinase was more rapidly eliminated than either acyl-enzyme. The results suggest that urokinase is eliminated via the binding to plasma inhibitors.

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.

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.

The pharmacological properties of a genetically engineered recombinant hirudin (r-hirudin) were studied in animal experiments. r-Hirudin proved to be a well tolerated substance. I.v. injection of up to 200 mg/kg did not lead to perceptible functional or morphological changes. There were no treatment-related effects on the cardiovascular system of dogs and rats after administration of up to 10 mg/kg. After long-term treatment (4 weeks, 1.0 mg/kg daily), no r-hirudin-related histopathological, haematological or biochemical changes could be found. Formation of antibodies was not detectable. Absorption, distribution, and elimination of r-hirudin were studied in dogs and rats. Pharmacokinetics could be best described by an open two-compartment model with first-order kinetics. After i.v. injection in dogs, r-hirudin is distributed into the extracellular space and eliminated through the kidneys in active form by glomerular filtration. After i.v. administration, a half-life of about 1 h was estimated; s.c. administration prolonged the apparent half-life. Pulmonary absorption was shown. Enteral absorption, placental transfer as well as transfer through the fetal integument were very low. r-Hirudin did not pass the blood-brain barrier. The efficacy of r-hirudin in preventing both venous and arterial thrombosis, vascular shunt occlusion or disseminated intravascular coagulation was demonstrated in rats. Depending on the dose, r-hirudin was able to prevent or reduce stasis-induced venous thrombosis, prolong the patency of an extracorporeal arteriovenous shunt, reduce the incidence of arterial thrombosis caused by vascular wall lesions as well as of microthrombosis induced by thrombin infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Among the inhibitors with a benzamidine moiety, 4-amidinophenyl esters of aromatic carboxylic acids that possess the structure of "inverse substrates" exhibit special kinetic characteristics. These compounds behave like substrates of trypsin-like enzymes, but acylation is much more rapid than deacylation. The result is accumulation of a comparatively stable acyl-enzyme, which may be easily isolated. By the use of 4-amidinophenyl esters acyl-derivatives of various trypsin-like enzymes can be prepared. In vivo, an acyl-enzyme behaves as an inert form of the enzyme and is protected from being inactivated by inhibitors. It is slowly reactivated in the circulation and has stimulated growing interest as a potential pro-drug.

The thrombin-like snake venom enzyme, batroxobin, was acylated by 4-amidinophenyl benzoate at the active site serine hydroxyl. From the enzymatically inactive benzoyl-batroxobin, batroxobin is generated with a half-life of deacylation of about 1 hour. The clotting activity of benzoyl-batroxobin in plasma is recovered with deacylation. The effect of batroxobin and benzoyl-batroxobin were studied following intravenous injection in rats. Compared to defibrinogenation with batroxobin, that obtained with benzoyl-batroxobin was much retarded. Batroxobin caused microthrombosis initially, which did not develop upon injection of benzoyl-batroxobin in equivalent doses.

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.

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.

To characterize the mode of action of heparin, the kinetic behaviour of the inhibition of thrombin, factor Xa, and plasmin by antithrombin III was studied in the presence and absence of heparin. Following the concentration dependence of inactivation, in both cases a linear dependence of the apparent first-order inactivation rate constant on the antithrombin III concentration was found. These results are explained by enzyme-heparin interactions. Thus, heparin is believed to act as an activator of the enzymes that makes them more susceptible to antithrombin III. Values of kinetic constants for the inactivation reaction of the several enzymes were determined. From these values it is concluded that heparin influences primarily the thrombin-antithrombin III interaction.