Jörg Hauptmann

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.

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)

AbstractPurpose. Peptidomimetic thrombin inhibitors derived from Nα-(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 × 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.

Influence of structural variations in peptidomimetic 4-amidinophenylalanine-derived thrombin inhibitors on plasma clearance and biliary excretion in rats

AbstractPurpose. Systemic and hepato-biliary clearance of peptidomimetic thrombin inhibitors of the 4-amidinophenylalanine amide-type, derived from NAPAP (Nα-[2-naphthylsulfonyl-glycyl]-4-amidinophenylalanine-piperidide) by substituting Gly in P2 for natural and unnatural amino acids or by varying the C- and N-terminal moieties, resp., were investigated. Methods. Concentrations of the compounds administered as intravenous bolus injection at a dose of 1 mg/kg to bile duct-cannulated rats were determined in plasma and bile samples collected over 4 hours using reversed-phase HPLC. Results. NAPAP and the derivatives with additional charged groups are comparatively hydrophilic compounds. For NAPAP and most of the derivatives the biliary clearance accounted for a high percentage of the rapid systemic plasma clearance. Derivatives 2a-c with a second basic group in P2 position showed lower systemic and biliary clearance compared to NAPAP, whereas their cumulative biliary excretion after a period of 120 min was less affected. Bis-benzamidine derivatives 4a and 5 with the second amidino group in the N-terminal moiety had the lowest biliary clearance. Additional carboxylic groups reduced the systemic and biliary clearance only as free amidinophenylalanine carboxyl in 3a and 5. No influence compared to NAPAP was observed for 2d with a free carboxyl group in P2 position. Conclusions. The weak correlation of the log P values of the compounds with the clearance parameters indicates the influence of structural variations, especially of charged groups, in this series of compounds rather than overall lipophilicity on hepato-biliary elimination mediated by hepatocellular transporters.

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 Nα-atom of the 4-amidinophenyl-alanine in P1 position, which showed a more than 10-fold lower thrombin inhibition. Furthermore, aza-glycine 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.

Influence of Indocyanine Green on Plasma Disappearance and Biliary Excretion of a Synthetic Thrombin Inhibitor of the 3-Amidinophenyl-alanine Piperazide-type in Rats

AbstractPurpose. 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.