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 recent structure determination of the catalytic domain of tissue-type plasminogen activator (tPA) suggested residue Arg 174 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 Å 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 Asp 189 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 Arg 174 , which swings out to form the western edge of the S3 pocket. These data suggest that the side chain of Arg 174 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 60-kDa multidomain tissue-type plasminogen activator (tPA) 1 catalyzes the conversion of the zymogen plasminogen into the active enzyme plasmin, the rate-limiting step in the endogenous fibrinolytic cascade (1). Recombinant tPA is used therapeutically as a fibrinolytic agent in the treatment of acute myocardial infarction and pulmonary embolism. Its fibrin dependent activity has attracted particular interest as it allows targeting of enzymatic activity to its natural substrate plasminogen (2, 3).The recent structure determination of the catalytic domain of tPA (4) in complex with benzamidine (hereafter termed b-tPA) revealed a strong structural similarity to other trypsin-like serine proteases. 187 residues of tPA have topological equivalents in ␣-chymotrypsin, forming the basis for the chymotrypsinogen numbering of the tPA catalytic domain used in this report (4). Compared with chymotrypsin, tPA contains a deletion of three residues at the C terminus of the A-chain, two single residue deletions in the B-chain, and insertions at six different positions totaling 24 residues. Five of these insertions are noteworthy: one forms a helix, the "intermediate helix" (Pro 164 -Leu 171 ), 2 and four form surface loops, referred to as the 37-loop (Lys 36 -Arg 39 ), 60-loop (Phe 59 -His 62 ), 110-loop (Ser 110 -Cys 111 ), and 186-loop (Asp 185 -Ala 186G ) (see Fig. 1). Most of these surface loops cluster around the active site cleft and are involved in sp...

I ntoxication is a commonly encountered phenomenon. The etiology varies widely, and many different toxins can be involved. According to data from the Federal Statistical Office, 178 425 cases of poisoning were treated in German hospitals in 2016 (1, 2). A poison control center (PCC) is often consulted by medical and paramedical personnel (emergency rescue services, officebased physicians, hospital physicians, and pharmacists) or by members of the population for toxicological advice. Determination of the indication for treatment with activated char coal-also known as activated carbon-plays a major role in eliminating the toxic capability of a potentially hazardous substance. Activated charcoal was given in 0.89% of cases of poisoning in childhood registered in the USA in 2013. In that year, it was recommended in circa 50 000 patients across all age groups (3, 4). In Ger-Summary Background: In 2016, according to the German Federal Statistical Office, 178 425 cases of intoxication (poisoning) were treated in German hospitals. The poison control centers in the German-speaking countries gave advice in a total of 268 787 instances of poisoning in that year, and use of activated charcoal was recommended in 4.37% of cases. The application of activated charcoal plays a major role in both primary and secondary detoxification. This article serves as an overview of the mechanism of action, indications, contraindications, modes of application, and dosing of activated charcoal. Methods: This review is based on pertinent publications retrieved by a selective search in PubMed. The opinions of experts from the poison control centers in the German-speaking countries were considered in the interpretation of the data. Results: The administration of activated charcoal is indicated to treat moderately severe to life-threatening intoxication. It should be carried out as soon as possible, within the first hour of the ingestion; timed-release preparations can be given up to 6 hours after the ingestion. An important contraindication is impaired consciousness with the danger of aspiration in a patient whose airway has not yet been secured. Activated charcoal is ineffective or inadequately effective in cases of poisoning with acids or bases, alcohols, organic solvents, inorganic salts, or metals. The proper dosage consists of an amount that is 10 to 40 times as much as that of the intoxicating substance, or else 0.5-1 g/kg body weight in children or 50 g in adults. Repeated application is indicated for intoxications with agents that persist for a longer time in the stomach and for intoxications with timed-release drugs or drugs with a marked enterohepatic or entero-enteric circulation. The routine combination of activated charcoal with a laxative is not recommended. Conclusion: Even though intoxications are common, there is still no internationally valid guideline concerning the administration of activated charcoal. A precise analysis of the risks and benefits is needed for each administration, and a poison control center should be consulte...

Thrombin is the key enzyme in coagulation and its inhibitors are of therapeutic interest since they are potential anticoagulants. The most potent inhibitor of the benzamidine type is N alpha-(2-naphthylsulfonyl-glycyl)-4-amidinophenylalanine piperidide (NAPAP). However, NAPAP and other substances designed so far do not fulfill the pharmacological requirements. The goal of designing new compounds was to obtain potent inhibitors with improved pharmacokinetic properties, such as absorption after oral application and a sustained elimination. Novel derivatives of 3-amidinophenylalanine as key building block were synthesized. The amidino moiety and both the N alpha- and the C-terminal substituents were widely varied. Some of the newly synthesized compounds are potent inhibitors of thrombin and exert improved pharmacokinetic properties.

SummaryIn a thrombin generation test with continuous registration of thrombin activity in plasma we studied the ability of a variety of thrombin inhibitors of different type and mechanism of action to influence the activity of thrombin after activation of the coagulation system. Depending on the inhibitor, the peak of thrombin activity is delayed and/or reduced.By blocking the active site of generated thrombin inhibitors cause a concentration dependent reduction of the thrombin peak and inhibit feed-back reactions of thrombin resulting in a delay of thrombin generation. Highly potent synthetic active-site directed inhibitors (Ki ≤ 20 nM) reduce the thrombin activity formed in plasma after extrinsic or intrinsic activation with the same efficiency (IC50 0.1 - 0.6 μM) as hirudin. The delay and reduction of thrombin generation by inhibitors of the anion-binding exosite 1 of thrombin is only attributed to an inhibition of feed-back reactions of thrombin. For a 50% reduction of thrombin activity in plasma by this type of inhibitors relatively high concentrations were determined.